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Recent Articles in PLoS Medicine / Public Libary of Science

Montefiori D, Sattentau Q, Flores J, Esparza J, Mascola J
Antibody-Based HIV-1 Vaccines: Recent Developments and Future Directions.
PLoS Med. 2007 Dec 1;4(12):e348. [Abstract/Link to Full Text]

HIV Treatment Proceeds as Prevention Research Confounds.
PLoS Med. 2007 Dec 1;4(12):e347. [Abstract/Link to Full Text]

G�tte M
Should We Include Connection Domain Mutations of HIV-1 Reverse Transcriptase in HIV Resistance Testing.
PLoS Med. 2007 Dec 1;4(12):e346. [Abstract/Link to Full Text]

Sch�pbach J, Gebhardt MD, Tomasik Z, Niederhauser C, Yerly S, B�rgisser P, Matter L, Gorgievski M, Dubs R, Schultze D, Steffen I, Andreutti C, Martinetti G, G�ntert B, Staub R, Daneel S, Vernazza P
Assessment of Recent HIV-1 Infection by a Line Immunoassay for HIV-1/2 Confirmation.
PLoS Med. 2007 Dec 1;4(12):e343.
BACKGROUND: Knowledge of the number of recent HIV infections is important for epidemiologic surveillance. Over the past decade approaches have been developed to estimate this number by testing HIV-seropositive specimens with assays that discriminate the lower concentration and avidity of HIV antibodies in early infection. We have investigated whether this "recency" information can also be gained from an HIV confirmatory assay. METHODS AND FINDINGS: The ability of a line immunoassay (INNO-LIA HIV I/II Score, Innogenetics) to distinguish recent from older HIV-1 infection was evaluated in comparison with the Calypte HIV-1 BED Incidence enzyme immunoassay (BED-EIA). Both tests were conducted prospectively in all HIV infections newly diagnosed in Switzerland from July 2005 to June 2006. Clinical and laboratory information indicative of recent or older infection was obtained from physicians at the time of HIV diagnosis and used as the reference standard. BED-EIA and various recency algorithms utilizing the antibody reaction to INNO-LIA's five HIV-1 antigen bands were evaluated by logistic regression analysis. A total of 765 HIV-1 infections, 748 (97.8%) with complete test results, were newly diagnosed during the study. A negative or indeterminate HIV antibody assay at diagnosis, symptoms of primary HIV infection, or a negative HIV test during the past 12 mo classified 195 infections (26.1%) as recent (</= 12 mo). Symptoms of CDC stages B or C classified 161 infections as older (21.5%), and 392 patients with no symptoms remained unclassified. BED-EIA ruled 65% of the 195 recent infections as recent and 80% of the 161 older infections as older. Two INNO-LIA algorithms showed 50% and 40% sensitivity combined with 95% and 99% specificity, respectively. Estimation of recent infection in the entire study population, based on actual results of the three tests and adjusted for a test's sensitivity and specificity, yielded 37% for BED-EIA compared to 35% and 33% for the two INNO-LIA algorithms. Window-based estimation with BED-EIA yielded 41% (95% confidence interval 36%-46%). CONCLUSIONS: Recency information can be extracted from INNO-LIA-based confirmatory testing at no additional costs. This method should improve epidemiologic surveillance in countries that routinely use INNO-LIA for HIV confirmation. [Abstract/Link to Full Text]

Brou H, Djohan G, Becquet R, Allou G, Ekouevi DK, Viho I, Leroy V, Desgr�es-du-Lo� A
When Do HIV-Infected Women Disclose Their HIV Status to Their Male Partner and Why? A Study in a PMTCT Programme, Abidjan.
PLoS Med. 2007 Dec 1;4(12):e342.
BACKGROUND: In Africa, women tested for HIV during antenatal care are counselled to share with their partner their HIV test result and to encourage partners to undertake HIV testing. We investigate, among women tested for HIV within a prevention of mother-to-child transmission of HIV (PMTCT) programme, the key moments for disclosure of their own HIV status to their partner and the impact on partner HIV testing. METHODS AND FINDINGS: Within the Ditrame Plus PMTCT project in Abidjan, 546 HIV-positive and 393 HIV-negative women were tested during pregnancy and followed-up for two years after delivery. Circumstances, frequency, and determinants of disclosure to the male partner were estimated according to HIV status. The determinants of partner HIV testing were identified according to women's HIV status. During the two-year follow-up, disclosure to the partner was reported by 96.7% of the HIV-negative women, compared to 46.2% of HIV-positive women (chi(2) = 265.2, degrees of freedom [df] = 1, p < 0.001). Among HIV-infected women, privileged circumstances for disclosure were just before delivery, during early weaning (at 4 mo to prevent HIV postnatal transmission), or upon resumption of sexual activity. Formula feeding by HIV-infected women increased the probability of disclosure (adjusted odds ratio 1.54, 95% confidence interval 1.04-2.27, Wald test = 4.649, df = 1, p = 0.031), whereas household factors such as having a co-spouse or living with family reduced the probability of disclosure. The proportion of male partners tested for HIV was 23.1% among HIV-positive women and 14.8% among HIV-negative women (chi(2) = 10.04, df = 1, p = 0.002). Partners of HIV-positive women who were informed of their wife's HIV status were more likely to undertake HIV testing than those not informed (37.7% versus 10.5%, chi(2) = 56.36, df = 1, p < 0.001). CONCLUSIONS: In PMTCT programmes, specific psychosocial counselling and support should be provided to women during the key moments of disclosure of HIV status to their partners (end of pregnancy, weaning, and resumption of sexual activity). This support could contribute to improving women's adherence to the advice given to prevent postnatal and sexual HIV transmission. [Abstract/Link to Full Text]

Baral S, Sifakis F, Cleghorn F, Beyrer C
Elevated Risk for HIV Infection among Men Who Have Sex with Men in Low- and Middle-Income Countries 2000-2006: A Systematic Review.
PLoS Med. 2007 Dec 1;4(12):e339.
BACKGROUND: Recent reports of high HIV infection rates among men who have sex with men (MSM) from Asia, Africa, Latin America, and the former Soviet Union (FSU) suggest high levels of HIV transmission among MSM in low- and middle-income countries. To investigate the global epidemic of HIV among MSM and the relationship of MSM outbreaks to general populations, we conducted a comprehensive review of HIV studies among MSM in low- and middle-income countries and performed a meta-analysis of reported MSM and reproductive-age adult HIV prevalence data. METHODS AND FINDINGS: A comprehensive review of the literature was conducted using systematic methodology. Data regarding HIV prevalence and total sample size was sequestered from each of the studies that met inclusion criteria and aggregate values for each country were calculated. Pooled odds ratio (OR) estimates were stratified by factors including HIV prevalence of the country, Joint United Nations Programme on HIV/AIDS (UNAIDS)-classified level of HIV epidemic, geographic region, and whether or not injection drug users (IDUs) played a significant role in given epidemic. Pooled ORs were stratified by prevalence level; very low-prevalence countries had an overall MSM OR of 58.4 (95% CI 56.3-60.6); low-prevalence countries, 14.4 (95% CI 13.8-14.9); and medium- to high-prevalence countries, 9.6 (95% CI 9.0-10.2). Significant differences in ORs for HIV infection among MSM in were seen when comparing low- and middle-income countries; low-income countries had an OR of 7.8 (95% CI 7.2-8.4), whereas middle-income countries had an OR of 23.4 (95% CI 22.8-24.0). Stratifying the pooled ORs by whether the country had a substantial component of IDU spread resulted in an OR of 12.8 (95% CI 12.3-13.4) in countries where IDU transmission was prevalent, and 24.4 (95% CI 23.7-25.2) where it was not. By region, the OR for MSM in the Americas was 33.3 (95% CI 32.3-34.2); 18.7 (95% CI 17.7-19.7) for Asia; 3.8 (95% CI 3.3-4.3) for Africa; and 1.3 (95% CI 1.1-1.6) for the low- and middle-income countries of Europe. CONCLUSIONS: MSM have a markedly greater risk of being infected with HIV compared with general population samples from low- and middle-income countries in the Americas, Asia, and Africa. ORs for HIV infection in MSM are elevated across prevalence levels by country and decrease as general population prevalence increases, but remain 9-fold higher in medium-high prevalence settings. MSM from low- and middle-income countries are in urgent need of prevention and care, and appear to be both understudied and underserved. [Abstract/Link to Full Text]

Yap SH, Sheen CW, Fahey J, Zanin M, Tyssen D, Lima VD, Wynhoven B, Kuiper M, Sluis-Cremer N, Harrigan PR, Tachedjian G
N348I in the Connection Domain of HIV-1 Reverse Transcriptase Confers Zidovudine and Nevirapine Resistance.
PLoS Med. 2007 Dec 1;4(12):e335.
BACKGROUND: The catalytically active 66-kDa subunit of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) consists of DNA polymerase, connection, and ribonuclease H (RNase H) domains. Almost all known RT inhibitor resistance mutations identified to date map to the polymerase domain of the enzyme. However, the connection and RNase H domains are not routinely analysed in clinical samples and none of the genotyping assays available for patient management sequence the entire RT coding region. The British Columbia Centre for Excellence in HIV/AIDS (the Centre) genotypes clinical isolates up to codon 400 in RT, and our retrospective statistical analyses of the Centre's database have identified an N348I mutation in the RT connection domain in treatment-experienced individuals. The objective of this multidisciplinary study was to establish the in vivo relevance of this mutation and its role in drug resistance. METHODS AND FINDINGS: The prevalence of N348I in clinical isolates, the time taken for it to emerge under selective drug pressure, and its association with changes in viral load, specific drug treatment, and known drug resistance mutations was analysed from genotypes, viral loads, and treatment histories from the Centre's database. N348I increased in prevalence from below 1% in 368 treatment-na�ve individuals to 12.1% in 1,009 treatment-experienced patients (p = 7.7 x 10(-12)). N348I appeared early in therapy and was highly associated with thymidine analogue mutations (TAMs) M41L and T215Y/F (p < 0.001), the lamivudine resistance mutations M184V/I (p < 0.001), and non-nucleoside RTI (NNRTI) resistance mutations K103N and Y181C/I (p < 0.001). The association with TAMs and NNRTI resistance mutations was consistent with the selection of N348I in patients treated with regimens that included both zidovudine and nevirapine (odds ratio 2.62, 95% confidence interval 1.43-4.81). The appearance of N348I was associated with a significant increase in viral load (p < 0.001), which was as large as the viral load increases observed for any of the TAMs. However, this analysis did not account for the simultaneous selection of other RT or protease inhibitor resistance mutations on viral load. To delineate the role of this mutation in RT inhibitor resistance, N348I was introduced into HIV-1 molecular clones containing different genetic backbones. N348I decreased zidovudine susceptibility 2- to 4-fold in the context of wild-type HIV-1 or when combined with TAMs. N348I also decreased susceptibility to nevirapine (7.4-fold) and efavirenz (2.5-fold) and significantly potentiated resistance to these drugs when combined with K103N. Biochemical analyses of recombinant RT containing N348I provide supporting evidence for the role of this mutation in zidovudine and NNRTI resistance and give some insight into the molecular mechanism of resistance. CONCLUSIONS: This study provides the first in vivo evidence that treatment with RT inhibitors can select a mutation (i.e., N348I) outside the polymerase domain of the HIV-1 RT that confers dual-class resistance. Its emergence, which can happen early during therapy, may significantly impact on a patient's response to antiretroviral therapies containing zidovudine and nevirapine. This study also provides compelling evidence for investigating the role of other mutations in the connection and RNase H domains in virological failure. [Abstract/Link to Full Text]

Heiden D, Ford N, Wilson D, Rodriguez WR, Margolis T, Janssens B, Bedelu M, Tun N, Goemaere E, Saranchuk P, Sabapathy K, Smithuis F, Luyirika E, Drew WL
Cytomegalovirus Retinitis: The Neglected Disease of the AIDS Pandemic.
PLoS Med. 2007 Dec 1;4(12):e334. [Abstract/Link to Full Text]

How can we draw the line between clinical care and medical research.
PLoS Med. 2007 Nov 27;4(11):e340. [Abstract/Link to Full Text]

Raschetti R, Albanese E, Vanacore N, Maggini M
Cholinesterase inhibitors in mild cognitive impairment: a systematic review of randomised trials.
PLoS Med. 2007 Nov 27;4(11):e338.
BACKGROUND: Mild cognitive impairment (MCI) refers to a transitional zone between normal ageing and dementia. Despite the uncertainty regarding the definition of MCI as a clinical entity, clinical trials have been conducted in the attempt to study the role of cholinesterase inhibitors (ChEIs) currently approved for symptomatic treatment of mild to moderate Alzheimer disease (AD), in preventing progression from MCI to AD. The objective of this review is to assess the effects of ChEIs (donepezil, rivastigmine, and galantamine) in delaying the conversion from MCI to Alzheimer disease or dementia. METHODS AND FINDINGS: The terms "donepezil", "rivastigmine", "galantamine", and "mild cognitive impairment" and their variants, synonyms, and acronyms were used as search terms in four electronic databases (MEDLINE, EMBASE, Cochrane, PsycINFO) and three registers: the Cochrane Collaboration Trial Register, Current Controlled Trials, and ClinicalTrials.gov. Published and unpublished studies were included if they were randomized clinical trials published (or described) in English and conducted among persons who had received a diagnosis of MCI and/or abnormal memory function documented by a neuropsychological assessment. A standardized data extraction form was used. The reporting quality was assessed using the Jadad scale. Three published and five unpublished trials met the inclusion criteria (three on donepezil, two on rivastigmine, and three on galantamine). Enrolment criteria differed among the trials, so the study populations were not homogeneous. The duration of the trials ranged from 24 wk to 3 y. No significant differences emerged in the probability of conversion from MCI to AD or dementia between the treated groups and the placebo groups. The rate of conversion ranged from 13% (over 2 y) to 25% (over 3 y) among treated patients, and from 18% (over 2 y) to 28% (over 3 y) among those in the placebo groups. Only for two studies was it possible to derive point estimates of the relative risk of conversion: 0.85 (95% confidence interval 0.64-1.12), and 0.84 (0.57-1.25). Statistically significant differences emerged for three secondary end points. However, when adjusting for multiple comparisons, only one difference remained significant (i.e., the rate of atrophy in the whole brain). CONCLUSIONS: The use of ChEIs in MCI was not associated with any delay in the onset of AD or dementia. Moreover, the safety profile showed that the risks associated with ChEIs are not negligible. The uncertainty regarding MCI as a clinical entity raises the question as to the scientific validity of these trials. [Abstract/Link to Full Text]

Longini IM, Nizam A, Ali M, Yunus M, Shenvi N, Clemens JD
Controlling endemic cholera with oral vaccines.
PLoS Med. 2007 Nov 27;4(11):e336.
BACKGROUND: Although advances in rehydration therapy have made cholera a treatable disease with low case-fatality in settings with appropriate medical care, cholera continues to impose considerable mortality in the world's most impoverished populations. Internationally licensed, killed whole-cell based oral cholera vaccines (OCVs) have been available for over a decade, but have not been used for the control of cholera. Recently, these vaccines were shown to confer significant levels of herd protection, suggesting that the protective potential of these vaccines has been underestimated and that these vaccines may be highly effective in cholera control when deployed in mass immunization programs. We used a large-scale stochastic simulation model to investigate the possibility of controlling endemic cholera with OCVs. METHODS AND FINDINGS: We construct a large-scale, stochastic cholera transmission model of Matlab, Bangladesh. We find that cholera transmission could be controlled in endemic areas with 50% coverage with OCVs. At this level of coverage, the model predicts that there would be an 89% (95% confidence interval [CI] 72%-98%) reduction in cholera cases among the unvaccinated, and a 93% (95% CI 82%-99%) reduction overall in the entire population. Even a more modest coverage of 30% would result in a 76% (95% CI 44%-95%) reduction in cholera incidence for the population area covered. For populations that have less natural immunity than the population of Matlab, 70% coverage would probably be necessary for cholera control, i.e., an annual incidence rate of < or = 1 case per 1,000 people in the population. CONCLUSIONS: Endemic cholera could be reduced to an annual incidence rate of < or = 1 case per 1,000 people in endemic areas with biennial vaccination with OCVs if coverage could reach 50%-70% depending on the level of prior immunity in the population. These vaccination efforts could be targeted with careful use of ecological data. [Abstract/Link to Full Text]

von Seidlein L
Vaccines for cholera control: does herd immunity play a role.
PLoS Med. 2007 Nov 27;4(11):e331. [Abstract/Link to Full Text]

Bj�rklund P, Akerstr�m G, Westin G
An LRP5 receptor with internal deletion in hyperparathyroid tumors with implications for deregulated WNT/beta-catenin signaling.
PLoS Med. 2007 Nov 27;4(11):e328.
BACKGROUND: Hyperparathyroidism (HPT) is a common endocrine disorder with incompletely understood etiology, characterized by enlarged hyperactive parathyroid glands and increased serum concentrations of parathyroid hormone and ionized calcium. We have recently reported activation of the Wnt signaling pathway by accumulation of beta-catenin in all analyzed parathyroid tumors from patients with primary HPT (pHPT) and in hyperplastic parathyroid glands from patients with uremia secondary to HPT (sHPT). Mechanisms that may account for this activation have not been identified, except for a few cases of beta-catenin (CTNNB1) stabilizing mutation in pHPT tumors. METHODS AND FINDINGS: Reverse transcription PCR and Western blot analysis showed expression of an aberrantly spliced internally truncated WNT coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) in 32 out of 37 pHPT tumors (86%) and 20 out of 20 sHPT tumors (100%). Stabilizing mutation of CTNNB1 and expression of the internally truncated LRP5 receptor was mutually exclusive. Expression of the truncated LRP5 receptor was required to maintain the nonphosphorylated active beta-catenin level, transcription activity of beta-catenin, MYC expression, parathyroid cell growth in vitro, and parathyroid tumor growth in a xenograft severe combined immunodeficiency (SCID) mouse model. WNT3 ligand and the internally truncated LRP5 receptor strongly activated transcription, and the internally truncated LRP5 receptor was insensitive to inhibition by DKK1. CONCLUSIONS: The internally truncated LRP5 receptor is strongly implicated in deregulated activation of the WNT/beta-catenin signaling pathway in hyperparathyroid tumors, and presents a potential target for therapeutic intervention. [Abstract/Link to Full Text]

Henderson GE, Churchill LR, Davis AM, Easter MM, Grady C, Joffe S, Kass N, King NM, Lidz CW, Miller FG, Nelson DK, Peppercorn J, Rothschild BB, Sankar P, Wilfond BS, Zimmer CR
Clinical trials and medical care: defining the therapeutic misconception.
PLoS Med. 2007 Nov 27;4(11):e324. [Abstract/Link to Full Text]

Levine M, Adida B, Mandl K, Kohane I, Halamka J
What are the benefits and risks of fitting patients with radiofrequency identification devices.
PLoS Med. 2007 Nov 27;4(11):e322.
Background to the debate: In 2004, the United States Food and Drug Administration approved a radiofrequency identification (RFID) device that is implanted under the skin of the upper arm of patients and that stores the patient's medical identifier. When a scanner is passed over the device, the identifier is displayed on the screen of an RFID reader. An authorized health professional can then use the identifier to access the patient's clinical information, which is stored in a separate, secure database. Such RFID devices may have many medical benefits--such as expediting identification of patients and retrieval of their medical records. But critics of the technology have raised several concerns, including the risk of the patient's identifying information being used for nonmedical purposes. [Abstract/Link to Full Text]

Byass P
Who needs cause-of-death data?
PLoS Med. 2007 Nov 20;4(11):e333. [Abstract/Link to Full Text]

Garrett L, Fidler DP
Sharing H5N1 viruses to stop a global influenza pandemic.
PLoS Med. 2007 Nov 20;4(11):e330. [Abstract/Link to Full Text]

Murray CJ, Lopez AD, Feehan DM, Peter ST, Yang G
Validation of the symptom pattern method for analyzing verbal autopsy data.
PLoS Med. 2007 Nov 20;4(11):e327.
BACKGROUND: Cause of death data are a critical input to formulating good public health policy. In the absence of reliable vital registration data, information collected after death from household members, called verbal autopsy (VA), is commonly used to study causes of death. VA data are usually analyzed by physician-coded verbal autopsy (PCVA). PCVA is expensive and its comparability across regions is questionable. Nearly all validation studies of PCVA have allowed physicians access to information collected from the household members' recall of medical records or contact with health services, thus exaggerating accuracy of PCVA in communities where few deaths had any interaction with the health system. In this study we develop and validate a statistical strategy for analyzing VA data that overcomes the limitations of PCVA. METHODS AND FINDINGS: We propose and validate a method that combines the advantages of methods proposed by King and Lu, and Byass, which we term the symptom pattern (SP) method. The SP method uses two sources of VA data. First, it requires a dataset for which we know the true cause of death, but which need not be representative of the population of interest; this dataset might come from deaths that occur in a hospital. The SP method can then be applied to a second VA sample that is representative of the population of interest. From the hospital data we compute the properties of each symptom; that is, the probability of responding yes to each symptom, given the true cause of death. These symptom properties allow us first to estimate the population-level cause-specific mortality fractions (CSMFs), and to then use the CSMFs as an input in assigning a cause of death to each individual VA response. Finally, we use our individual cause-of-death assignments to refine our population-level CSMF estimates. The results from applying our method to data collected in China are promising. At the population level, SP estimates the CSMFs with 16% average relative error and 0.7% average absolute error, while PCVA results in 27% average relative error and 1.1% average absolute error. At the individual level, SP assigns the correct cause of death in 83% of the cases, while PCVA does so for 69% of the cases. We also compare the results of SP and PCVA when both methods have restricted access to the information from the medical record recall section of the VA instrument. At the population level, without medical record recall, the SP method estimates the CSMFs with 14% average relative error and 0.6% average absolute error, while PCVA results in 70% average relative error and 3.2% average absolute error. For individual estimates without medical record recall, SP assigns the correct cause of death in 78% of cases, while PCVA does so for 38% of cases. CONCLUSIONS: Our results from the data collected in China suggest that the SP method outperforms PCVA, both at the population and especially at the individual level. Further study is needed on additional VA datasets in order to continue validation of the method, and to understand how the symptom properties vary as a function of culture, language, and other factors. Our results also suggest that PCVA relies heavily on household recall of medical records and related information, limiting its applicability in low-resource settings. SP does not require that additional information to adequately estimate causes of death. [Abstract/Link to Full Text]

Murray CJ, Lopez AD, Barofsky JT, Bryson-Cahn C, Lozano R
Estimating population cause-specific mortality fractions from in-hospital mortality: validation of a new method.
PLoS Med. 2007 Nov 20;4(11):e326.
BACKGROUND: Cause-of-death data for many developing countries are not available. Information on deaths in hospital by cause is available in many low- and middle-income countries but is not a representative sample of deaths in the population. We propose a method to estimate population cause-specific mortality fractions (CSMFs) using data already collected in many middle-income and some low-income developing nations, yet rarely used: in-hospital death records. METHODS AND FINDINGS: For a given cause of death, a community's hospital deaths are equal to total community deaths multiplied by the proportion of deaths occurring in hospital. If we can estimate the proportion dying in hospital, we can estimate the proportion dying in the population using deaths in hospital. We propose to estimate the proportion of deaths for an age, sex, and cause group that die in hospital from the subset of the population where vital registration systems function or from another population. We evaluated our method using nearly complete vital registration (VR) data from Mexico 1998-2005, which records whether a death occurred in a hospital. In this validation test, we used 45 disease categories. We validated our method in two ways: nationally and between communities. First, we investigated how the method's accuracy changes as we decrease the amount of Mexican VR used to estimate the proportion of each age, sex, and cause group dying in hospital. Decreasing VR data used for this first step from 100% to 9% produces only a 12% maximum relative error between estimated and true CSMFs. Even if Mexico collected full VR information only in its capital city with 9% of its population, our estimation method would produce an average relative error in CSMFs across the 45 causes of just over 10%. Second, we used VR data for the capital zone (Distrito Federal and Estado de Mexico) and estimated CSMFs for the three lowest-development states. Our estimation method gave an average relative error of 20%, 23%, and 31% for Guerrero, Chiapas, and Oaxaca, respectively. CONCLUSIONS: Where accurate International Classification of Diseases (ICD)-coded cause-of-death data are available for deaths in hospital and for VR covering a subset of the population, we demonstrated that population CSMFs can be estimated with low average error. In addition, we showed in the case of Mexico that this method can substantially reduce error from biased hospital data, even when applied to areas with widely different levels of development. For countries with ICD-coded deaths in hospital, this method potentially allows the use of existing data to inform health policy. [Abstract/Link to Full Text]

Kai J, Beavan J, Faull C, Dodson L, Gill P, Beighton A
Professional uncertainty and disempowerment responding to ethnic diversity in health care: a qualitative study.
PLoS Med. 2007 Nov 13;4(11):e323.
BACKGROUND: While ethnic disparities in health and health care are increasing, evidence on how to enhance quality of care and reduce inequalities remains limited. Despite growth in the scope and application of guidelines on "cultural competence," remarkably little is known about how practising health professionals experience and perceive their work with patients from diverse ethnic communities. Using cancer care as a clinical context, we aimed to explore this with a range of health professionals to inform interventions to enhance quality of care. METHODS AND FINDINGS: We conducted a qualitative study involving 18 focus groups with a purposeful sample of 106 health professionals of differing disciplines, in primary and secondary care settings, working with patient populations of varying ethnic diversity in the Midlands of the UK. Data were analysed by constant comparison and we undertook processes for validation of analysis. We found that, as they sought to offer appropriate care, health professionals wrestled with considerable uncertainty and apprehension in responding to the needs of patients of ethnicities different from their own. They emphasised their perceived ignorance about cultural difference and were anxious about being culturally inappropriate, causing affront, or appearing discriminatory or racist. Professionals' ability to think and act flexibly or creatively faltered. Although trying to do their best, professionals' uncertainty was disempowering, creating a disabling hesitancy and inertia in their practice. Most professionals sought and applied a knowledge-based cultural expertise approach to patients, though some identified the risk of engendering stereotypical expectations of patients. Professionals' uncertainty and disempowerment had the potential to perpetuate each other, to the detriment of patient care. CONCLUSIONS: This study suggests potential mechanisms by which health professionals may inadvertently contribute to ethnic disparities in health care. It identifies critical opportunities to empower health professionals to respond more effectively. Interventions should help professionals acknowledge their uncertainty and its potential to create inertia in their practice. A shift away from a cultural expertise model toward a greater focus on each patient as an individual may help. [Abstract/Link to Full Text]

Roussilhon C, Oeuvray C, M�ller-Graf C, Tall A, Rogier C, Trape JF, Theisen M, Balde A, P�rignon JL, Druilhe P
Long-term clinical protection from falciparum malaria is strongly associated with IgG3 antibodies to merozoite surface protein 3.
PLoS Med. 2007 Nov 13;4(11):e320.
BACKGROUND: Surrogate markers of protective immunity to malaria in humans are needed to rationalize malaria vaccine discovery and development. In an effort to identify such markers, and thereby provide a clue to the complex equation malaria vaccine development is facing, we investigated the relationship between protection acquired through exposure in the field with naturally occurring immune responses (i.e., induced by the parasite) to molecules that are considered as valuable vaccine candidates. METHODS AND FINDINGS: We analyzed, under comparative conditions, the antibody responses of each of six isotypes to five leading malaria vaccine candidates in relation to protection acquired by exposure to natural challenges in 217 of the 247 inhabitants of the African village of Dielmo, Senegal (96 children and 121 older adolescents and adults). The status of susceptibility or resistance to malaria was determined by active case detection performed daily by medical doctors over 6 y from a unique follow-up study of this village. Of the 30 immune responses measured, only one, antibodies of the IgG3 isotype directed to merozoite surface protein 3 (MSP3), was strongly associated with clinical protection against malaria in all age groups, i.e., independently of age. This immunological parameter had a higher statistical significance than the sickle cell trait, the strongest factor of protection known against Plasmodium falciparum. A single determination of antibody was significantly associated with the clinical outcome over six consecutive years in children submitted to massive natural parasite challenges by mosquitoes (over three parasite inoculations per week). Finally, the target epitopes of these antibodies were found to be fully conserved. CONCLUSIONS: Since anti-MSP3 IgG3 antibodies can naturally develop along with protection against P. falciparum infection in young children, our results provide the encouraging indication that these antibodies should be possible to elicit by vaccination early in life. Since these antibodies have been found to achieve parasite killing under in vitro and in vivo conditions, and since they can be readily elicited by immunisation in na�ve volunteers, our immunoepidemiological findings support the further development of MSP3-based vaccine formulations. [Abstract/Link to Full Text]

Daniels K, Swartz L
Understanding health care workers' anxieties in a diversifying world.
PLoS Med. 2007 Nov 13;4(11):e319. [Abstract/Link to Full Text]

Nunn AS, Fonseca EM, Bastos FI, Gruskin S, Salomon JA
Evolution of antiretroviral drug costs in Brazil in the context of free and universal access to AIDS treatment.
PLoS Med. 2007 Nov 13;4(11):e305.
BACKGROUND: Little is known about the long-term drug costs associated with treating AIDS in developing countries. Brazil's AIDS treatment program has been cited widely as the developing world's largest and most successful AIDS treatment program. The program guarantees free access to highly active antiretroviral therapy (HAART) for all people living with HIV/AIDS in need of treatment. Brazil produces non-patented generic antiretroviral drugs (ARVs), procures many patented ARVs with negotiated price reductions, and recently issued a compulsory license to import one patented ARV. In this study, we investigate the drivers of recent ARV cost trends in Brazil through analysis of drug-specific prices and expenditures between 2001 and 2005. METHODS AND FINDINGS: We compared Brazil's ARV prices to those in other low- and middle-income countries. We analyzed trends in drug expenditures for HAART in Brazil from 2001 to 2005 on the basis of cost data disaggregated by each ARV purchased by the Brazilian program. We decomposed the overall changes in expenditures to compare the relative impacts of changes in drug prices and drug purchase quantities. We also estimated the excess costs attributable to the difference between prices for generics in Brazil and the lowest global prices for these drugs. Finally, we estimated the savings attributable to Brazil's reduced prices for patented drugs. Negotiated drug prices in Brazil are lowest for patented ARVs for which generic competition is emerging. In recent years, the prices for efavirenz and lopinavir-ritonavir (lopinavir/r) have been lower in Brazil than in other middle-income countries. In contrast, the price of tenofovir is US$200 higher per patient per year than that reported in other middle-income countries. Despite precipitous price declines for four patented ARVs, total Brazilian drug expenditures doubled, to reach US$414 million in 2005. We find that the major driver of cost increases was increased purchase quantities of six specific drugs: patented lopinavir/r, efavirenz, tenofovir, atazanavir, enfuvirtide, and a locally produced generic, fixed-dose combination of zidovudine and lamivudine (AZT/3TC). Because prices declined for many of the patented drugs that constitute the largest share of drug costs, nearly the entire increase in overall drug expenditures between 2001 and 2005 is attributable to increases in drug quantities. Had all drug quantities been held constant from 2001 until 2005 (or for those drugs entering treatment guidelines after 2001, held constant between the year of introduction and 2005), total costs would have increased by only an estimated US$7 million. We estimate that in the absence of price declines for patented drugs, Brazil would have spent a cumulative total of US$2 billion on drugs for HAART between 2001 and 2005, implying a savings of US$1.2 billion from price declines. Finally, in comparing Brazilian prices for locally produced generic ARVs to the lowest international prices meeting global pharmaceutical quality standards, we find that current prices for Brazil's locally produced generics are generally much higher than corresponding global prices, and note that these prices have risen in Brazil while declining globally. We estimate the excess costs of Brazil's locally produced generics totaled US$110 million from 2001 to 2005. CONCLUSIONS: Despite Brazil's more costly generic ARVs, the net result of ARV price changes has been a cost savings of approximately US$1 billion since 2001. HAART costs have nevertheless risen steeply as Brazil has scaled up treatment. These trends may foreshadow future AIDS treatment cost trends in other developing countries as more people start treatment, AIDS patients live longer and move from first-line to second and third-line treatment, AIDS treatment becomes more complex, generic competition emerges, and newer patented drugs become available. The specific application of the Brazilian model to other countries will depend, however, on the strength of their health systems, intellectual property regulations, epidemiological profiles, AIDS treatment guidelines, and differing capacities to produce drugs locally. [Abstract/Link to Full Text]

Bhattarai A, Ali AS, Kachur SP, M�rtensson A, Abbas AK, Khatib R, Al-Mafazy AW, Ramsan M, Rotllant G, Gerstenmaier JF, Molteni F, Abdulla S, Montgomery SM, Kaneko A, Bj�rkman A
Impact of artemisinin-based combination therapy and insecticide-treated nets on malaria burden in zanzibar.
PLoS Med. 2007 Nov 6;4(11):e309.
BACKGROUND: The Roll Back Malaria strategy recommends a combination of interventions for malaria control. Zanzibar implemented artemisinin-based combination therapy (ACT) for uncomplicated malaria in late 2003 and long-lasting insecticidal nets (LLINs) from early 2006. ACT is provided free of charge to all malaria patients, while LLINs are distributed free to children under age 5 y ("under five") and pregnant women. We investigated temporal trends in Plasmodium falciparum prevalence and malaria-related health parameters following the implementation of these two malaria control interventions in Zanzibar. METHODS AND FINDINGS: Cross-sectional clinical and parasitological surveys in children under the age of 14 y were conducted in North A District in May 2003, 2005, and 2006. Survey data were analyzed in a logistic regression model and adjusted for complex sampling design and potential confounders. Records from all 13 public health facilities in North A District were analyzed for malaria-related outpatient visits and admissions. Mortality and demographic data were obtained from District Commissioner's Office. P. falciparum prevalence decreased in children under five between 2003 and 2006; using 2003 as the reference year, odds ratios (ORs) and 95% confidence intervals (CIs) were, for 2005, 0.55 (0.28-1.08), and for 2006, 0.03 (0.00-0.27); p for trend < 0.001. Between 2002 and 2005 crude under-five, infant (under age 1 y), and child (aged 1-4 y) mortality decreased by 52%, 33%, and 71%, respectively. Similarly, malaria-related admissions, blood transfusions, and malaria-attributed mortality decreased significantly by 77%, 67% and 75%, respectively, between 2002 and 2005 in children under five. Climatic conditions favorable for malaria transmission persisted throughout the observational period. CONCLUSIONS: Following deployment of ACT in Zanzibar 2003, malaria-associated morbidity and mortality decreased dramatically within two years. Additional distribution of LLINs in early 2006 resulted in a 10-fold reduction of malaria parasite prevalence. The results indicate that the Millennium Development Goals of reducing mortality in children under five and alleviating the burden of malaria are achievable in tropical Africa with high coverage of combined malaria control interventions. [Abstract/Link to Full Text]

Schluger N, Karunakara U, Lienhardt C, Nyirenda T, Chaisson R
Building clinical trials capacity for tuberculosis drugs in high-burden countries.
PLoS Med. 2007 Nov 6;4(11):e302. [Abstract/Link to Full Text]

Casenghi M, Cole ST, Nathan CF
New approaches to filling the gap in tuberculosis drug discovery.
PLoS Med. 2007 Nov 6;4(11):e293. [Abstract/Link to Full Text]

Mitnick CD, Castro KG, Harrington M, Sacks LV, Burman W
Randomized trials to optimize treatment of multidrug-resistant tuberculosis.
PLoS Med. 2007 Nov 6;4(11):e292. [Abstract/Link to Full Text]

Mellinghoff I
Why do cancer cells become "addicted" to oncogenic epidermal growth factor receptor?
PLoS Med. 2007 Oct 30;4(10):1620-2. [Abstract/Link to Full Text]

Beyrer C, Masenior N
The US Anti-Prostitution Pledge: Authors' Reply.
PLoS Med. 2007 Oct 30;4(10):e318. [Abstract/Link to Full Text]

Cragg MS, Kuroda J, Puthalakath H, Huang DC, Strasser A
Gefitinib-induced killing of NSCLC cell lines expressing mutant EGFR requires BIM and can be enhanced by BH3 mimetics.
PLoS Med. 2007 Oct 30;4(10):1681-89; discussion 1690.
BACKGROUND: The epidermal growth factor receptor (EGFR) plays a critical role in the control of cellular proliferation, differentiation, and survival. Abnormalities in EGF-EGFR signaling, such as mutations that render the EGFR hyperactive or cause overexpression of the wild-type receptor, have been found in a broad range of cancers, including carcinomas of the lung, breast, and colon. EGFR inhibitors such as gefitinib have proven successful in the treatment of certain cancers, particularly non-small cell lung cancers (NSCLCs) harboring activating mutations within the EGFR gene, but the molecular mechanisms leading to tumor regression remain unknown. Therefore, we wished to delineate these mechanisms. METHODS AND FINDINGS: We performed biochemical and genetic studies to investigate the mechanisms by which inhibitors of EGFR tyrosine kinase activity, such as gefitinib, inhibit the growth of human NSCLCs. We found that gefitinib triggered intrinsic (also called "mitochondrial") apoptosis signaling, involving the activation of BAX and mitochondrial release of cytochrome c, ultimately unleashing the caspase cascade. Gefitinib caused a rapid increase in the level of the proapoptotic BH3-only protein BIM (also called BCL2-like 11) through both transcriptional and post-translational mechanisms. Experiments with pharmacological inhibitors indicated that blockade of MEK-ERK1/2 (mitogen-activated protein kinase kinase-extracellular signal-regulated protein kinase 1/2) signaling, but not blockade of PI3K (phosphatidylinositol 3-kinase), JNK (c-Jun N-terminal kinase or mitogen-activated protein kinase 8), or AKT (protein kinase B), was critical for BIM activation. Using RNA interference, we demonstrated that BIM is essential for gefitinib-induced killing of NSCLC cells. Moreover, we found that gefitinib-induced apoptosis is enhanced by addition of the BH3 mimetic ABT-737. CONCLUSIONS: Inhibitors of the EGFR tyrosine kinase have proven useful in the therapy of certain cancers, in particular NSCLCs possessing activating mutations in the EGFR kinase domain, but the mechanisms of tumor cell killing are still unclear. In this paper, we demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR-MEK-ERK signaling cascade is critical for BIM activation. Moreover, we demonstrate that addition of a BH3 mimetic significantly enhances killing of NSCLC cells by the EGFR tyrosine kinase inhibitor gefitinib. It appears likely that this approach represents a paradigm shared by many, and perhaps all, oncogenic tyrosine kinases and suggests a powerful new strategy for cancer therapy. [Abstract/Link to Full Text]

Recent Articles in BMC Medicine

Hermens ML, van Hout HP, Terluin B, Ader HJ, Penninx BW, van Marwijk HW, Bosmans JE, van Dyck R, de Haan M
Clinical effectiveness of usual care with or without antidepressant medication for primary care patients with minor or mild-major depression: a randomized equivalence trial.
BMC Med. 2007 Dec 7;5(1):36.
ABSTRACT: BACKGROUND: Minor and mild-major depression are highly prevalent in primary care. There is insufficient evidence for the effectiveness of antidepressants in the treatment of minor and mild-major depression. We compared the effectiveness of usual primary care treatment, with or without antidepressants, in minor and mild-major depression. METHOD: A pragmatic patient-randomized equivalence trial with 52 weeks follow-up in The Netherlands. 59 Primary Care Physicians (PCPs) recruited and treated 181 adult patients with minor or mild-major depression. Patients were randomized to 4 consultations within three months of usual care plus antidepressants (UCandAD) or usual care alone (UCnoAD). The Montgomery Asberg Depression Rating Scale (MADRS) was used to assess changes in severity of depressive symptoms. The predefined equivalence margin was set at 5 points. Multilevel analysis was used to analyze the data. Secondary outcome measures were the Short-Form 36 (SF-36), and the Client Satisfaction Questionnaire (CSQ-8). RESULTS: Patients received on average 3.0 (SD 1.4) 15-minute consultations within three months with (n=85) or without paroxetine (n=96). Equivalence of UCandAD and UCnoAD was demonstrated in the intention-to-treat analyses as well as the per-protocol analysis after 6 weeks, but not at 13, 26, and 52 weeks follow-up. No statistical differences in effectiveness between treatment groups were found in the intention-to-treat analysis. No differences in the physical and mental functioning (SF-36) were found between the treatment groups. Patients allocated to UCandAD were slightly more satisfied with their treatment at 13 weeks follow-up (but not at 52 weeks follow-up) than patients allocated to UCnoAD. Preliminary analyses suggested that subgroups like patients with mild-major (instead of a minor) depression may benefit from antidepressant treatment. Patients who were assigned to their preferred treatment (in particular to UCnoAD) were more often compliant and had better clinical outcomes. CONCLUSION: UCandAD was as effective as UCnoAD over the first 6 weeks, but not at 13, 26, and 52 weeks. However, superiority of either treatment could not be demonstrated either. The question whether antidepressants add any clinical effect to usual care remains unresolved. We recommend future studies to look for subgroups of patients who may benefit from antidepressants. Trial registration: Dutch Trial Registry ISRCN03007807. [Abstract/Link to Full Text]

Dendukuri N, Chiu K, Brophy JM
Validity of Electron Beam Computed Tomography for Coronary Artery Disease: A Systematic Review and Meta-analysis.
BMC Med. 2007 Nov 25;5(1):35.
ABSTRACT: BACKGROUND: Electron beam computed tomography (EBCT) is a method for measuring coronary calcification and has been promoted as a possible non-invasive screening/diagnostic tool for coronary artery disease (CAD). Our objective was to carry out a systematic review and meta-analysis of electron beam computed tomography (EBCT) for screening of asymptomatic patients and diagnosis of symptomatic patients for coronary artery disease (CAD). METHODS: Studies were identified from the PUBMED, MEDLINE, EMBASE, Current Contents, INAHTA and Cochrane Collaboration databases. We identified studies published in English evaluating EBCT using: 1) a prospective design among asymptomatic patients where CAD was measured in terms of clinical outcomes (e.g. myocardial infarction, death, revascularization), 2) a cross-sectional design among symptomatic patients where CAD was measured by coronary angiography. We compared the risk of CAD in EBCT score categories defined as: Low (0-10), Moderate (11-400) and High (>400). A hierarchical meta-analysis was used to pool risk ratios comparing categories across studies. RESULTS: We identified 9 studies of asymptomatic patients and 10 studies of symptomatic patients. In both types of studies, we found variability in EBCT category distribution and risk of CAD within categories. For studies of asymptomatic patients we estimated the following risk ratios (95% credible intervals): Moderate vs. Low: 3.5 (2.4, 5.1) and High vs. Low: 9.9 (5.3, 17.6). Similar results were obtained for studies of symptomatic patients. Ratios comparing risk of no CAD among symptomatic patients were: Moderate vs. Low: 0.5 (0.3, 0.8) and High vs. Low: 0.12 (0.05, 0.2). CONCLUSIONS: Increasing EBCT scores indicate higher risk for CAD in both asymptomatic and symptomatic patients. In general, asymptomatic patients with EBCT scores in the High category can perhaps be considered for preventive medical therapy and risk factor modification. Symptomatic patients with EBCT scores in the Low category can perhaps, at least temporarily, avoid invasive coronary angiography. However, the non-uniform quality of studies and the lack of availability of individual-level data preclude the extension of our results to individual patients. [Abstract/Link to Full Text]

Colizza V, Barrat A, Barthelemy M, Vespignani A
Predictability and epidemic pathways in global outbreaks of infectious diseases: the SARS case study.
BMC Med. 2007 Nov 21;5(1):34.
ABSTRACT: BACKGROUND: The global spread of the severe acute respiratory syndrome (SARS) epidemic has clearly shown the importance of considering the long-range transportation networks in the understanding of emerging diseases outbreaks. The introduction of extensive transportation data sets is therefore an important step in order to develop epidemic models endowed with realism. METHODS: We develop a general stochastic meta-population model that incorporates actual travel and census data among 3 100 urban areas in 220 countries. The model allows probabilistic predictions on the likelihood of country outbreaks and their magnitude. The level of predictability offered by the model can be quantitatively analyzed and related to the appearance of robust epidemic pathways that represent the most probable routes for the spread of the disease. RESULTS: In order to assess the predictive power of the model, the case study of the global spread of SARS is considered. The disease parameter values and initial conditions used in the model are evaluated from empirical data for Hong Kong. The outbreak likelihood for specific countries is evaluated along with the emerging epidemic pathways. Simulation results are in agreement with the empirical data of the SARS worldwide epidemic. CONCLUSIONS: The presented computational approach shows that the integration of long-range mobility and demographic data provides epidemic models with a predictive power that can be consistently tested and theoretically motivated. This computational strategy can be therefore considered as a general tool in the analysis and forecast of the global spreading of emerging diseases and in the definition of containment policies aimed at reducing the effects of potentially catastrophic outbreaks. [Abstract/Link to Full Text]

Prentice LM, Klausen C, Kalloger S, Kobel M, McKinney S, Santos JL, Kenney C, Mehl E, Gilks CB, Leung P, Swenerton K, Huntsman DG, Aparicio SA
Kisspeptin and GPR54 immunoreactivity in a cohort of 518 patients defines favourable prognosis and clear cell subtype in ovarian carcinoma.
BMC Med. 2007 Nov 15;5(1):33.
ABSTRACT: BACKGROUND: Kisspeptins and their G-protein coupled receptor, GPR54 are required for GnRH release and have been associated with anti-metastatic tumour cell behaviour in model systems. The latter may suggest that their overexpression would be associated with a better prognosis in cancer. However, kisspeptin/GPR54 interactions (autocrine, paracrine, and/or endocrine) could also impact tumour behaviour in a negative manner. Here, for the first time, we associate the immunoreactivity of the kisspeptin/GPR54 ligand-receptor pair with favourable prognosis in a large cohort of ovarian carcinomas. METHODS: Immunohistochemical analysis for kisspeptin and GPR54 was performed on a tissue microarray (TMA) consisting of 518 early stage ovarian carcinomas, all with linked clinical outcome data. The TMA was scored using a staining intensity scale of 0 (negative), +1 (mild-moderate), and +2 (strong). Strong staining cases were considered either kisspeptin or GPR54 positive and designated as 1, while all other cases were considered negative and designated 0. All statistical analysis was conducted using two-sided tests and a p-value equal to or less than 0.05 was considered significant. RESULTS: Kisspeptin and GPR54 immunoreactive cases show a favourable prognosis in univariable disease specific survival (p=0.0023, p=0.0092), as well as in overall survival (p=0.0006, p=0.0002). Furthermore, kisspeptin is an independent marker for favourable prognosis as determined by multivariable disease specific (p=0.0046) and overall survival analysis (p=0.0170), while GPR54 is an independent marker for overall survival only (p=0.0303). Both kisspeptin positive and GPR54 positive cases are strongly associated with the ovarian carcinoma clear cell subtype (p<0.0001, p<0.0001), and GPR54 is significantly associated with favourable prognosis in overall survival within the clear cell subtype (p=0.0102). CONCLUSIONS: Kisspeptin and GPR54 immunoreactivity are significantly associated with favourable prognosis in both disease specific and overall survival, as well as being significantly associated with the clear cell ovarian carcinoma subtype, thereby creating the first independent prognostic biomarkers specific for ovarian clear cell carcinomas. [Abstract/Link to Full Text]

Gonzales DA, Norsworthy KJ, Kern SJ, Banks S, Sieving PC, Star RA, Natanson C, Danner RL
A Meta-analysis of N-acetylcysteine in Contrast-induced Nephrotoxicity: Unsupervised Clustering to Resolve Heterogeneity.
BMC Med. 2007 Nov 14;5(1):32.
ABSTRACT: BACKGROUND: Meta-analyses of N-acetylcysteine (NAC) for preventing contrast-induced nephrotoxicity (CIN) have led to disparate conclusions. Here we examine and attempt to resolve the heterogeneity evident among these trials. METHODS: Two reviewers independently extracted and graded the data. Limiting studies to randomized, controlled trials with adequate outcome data yielded 22 reports with 2746 patients. RESULTS: Significant heterogeneity was detected among these trials (I2 = 37%; p = 0.04). Meta-regression analysis failed to identify significant sources of heterogeneity. A modified L'Abbe plot that substituted groupwise changes in serum creatinine for nephrotoxicity rates, followed by model-based, unsupervised clustering resolved trials into two distinct, significantly different (p < 0.0001) and homogeneous populations (I2 = 0 and p > 0.5, for both). Cluster 1 studies (n = 18; 2445 patients) showed no benefit (relative risk (RR) = 0.87; 95% confidence interval (CI) 0.68-1.12, p = 0.28), while cluster 2 studies (n = 4; 301 patients) indicated that NAC was highly beneficial (RR = 0.15; 95% CI 0.07-0.33, p < 0.0001). Benefit in cluster 2 was unexpectedly associated with NAC-induced decreases in creatinine from baseline (p = 0.07). Cluster 2 studies were relatively early, small and of lower quality compared with cluster 1 studies (p = 0.01 for the three factors combined). Dialysis use across all studies (five control, eight treatment; p = 0.42) did not suggest that NAC is beneficial. CONCLUSION: This meta-analysis does not support the efficacy of NAC to prevent CIN. [Abstract/Link to Full Text]

Macphail C, Pettifor AE, Pascoe S, Rees HV
Contraception use and pregnancy among 15-24 year old South African women: a nationally representative cross-sectional survey.
BMC Med. 2007 Oct 28;5(1):31.
ABSTRACT: BACKGROUND: Adolescent reproductive health has not continued to receive the attention it deserves since the start of the HIV epidemic. In South Africa, high numbers of adolescent women report pregnancies that are unwanted and yet few have accessed available termination of pregnancy services. Enabling contraception use is vital for meeting the goals of HIV prevention. METHODS: A nationally representative survey of South African 15-24 year olds was undertaken. Participants completed a questionnaire on sexual behaviour and provided an oral fluid sample for HIV testing. Analysis of the data was restricted to women (n = 6 217), particularly those who reported being sexual active in the last 12 months (n = 3 618) and was conducted using svy methods in the program STATA 8.0 to take account of sampling methods. Univariate and multivariate analyses were conducted to explore factors associated with contraceptive use. RESULTS: Two thirds of all women reported having ever been sexually active and among these 87% were sexually active in the past 12 months. Among women who reported currently being sexually active, 52.2% reported using contraceptives. There was evidence of association between contraceptive use and being employed or a student (vs unemployed); fewer sex partners; type of last sex partner; having talked to last partner about condom use and having ever been pregnant. CONCLUSIONS: Specific emphasis must be placed on encouraging young women to use contraceptive methods that offer protection against pregnancy and STIs/HIV. Our consistent finding of a relationship between discussing condom use with partners and condom use indicates the importance of involvement of male partners in women's contraceptive decisions. [Abstract/Link to Full Text]

Ioannidis JP, Patsopoulos NA, Kavvoura FK, Tatsioni A, Evangelou E, Kouri I, Contopoulos-Ioannidis DG, Liberopoulos G
International ranking systems for universities and institutions: a critical appraisal.
BMC Med. 2007 Oct 25;5(1):30.
ABSTRACT: BACKGROUND: Ranking of universities and institutions has attracted wide attention recently. Several systems have been proposed that attempt to rank academic institutions worldwide. METHODS: We review the two most publicly visible ranking systems, the Shanghai Jiao Tong University 'Academic Ranking of World Universities' and the Times Higher Education Supplement 'World University Rankings' and also briefly review other ranking systems that use different criteria. We assess the construct validity for educational and research excellence and the measurement validity of each of the proposed ranking criteria, and try to identify generic challenges in international ranking of universities and institutions. RESULTS: None of the reviewed criteria for international ranking seems to have very good construct validity for both educational and research excellence, and most don't have very good construct validity even for just one of these two aspects of excellence. Measurement error for many items is also considerable or is not possible to determine due to lack of publication of the relevant data and methodology details. The concordance between the 2006 rankings by Shanghai and Times is modest at best, with only 133 universities shared in their top 200 lists. The examination of the existing international ranking systems suggests that generic challenges include adjustment for institutional size, definition of institutions, implications of average measurements of excellence versus measurements of extremes, adjustments for scientific field, time frame of measurement and allocation of credit for excellence. CONCLUSION: Naive lists of international institutional rankings that do not address these fundamental challenges with transparent methods are misleading and should be abandoned. We make some suggestions on how focused and standardized evaluations of excellence could be improved and placed in proper context. [Abstract/Link to Full Text]

Yerman T, Gan WQ, Sin DD
The influence of gender on the effects of aspirin in preventing myocardial infarction.
BMC Med. 2007;529.
BACKGROUND: There is considerable variation in the effect of aspirin therapy reducing the risk of myocardial infarction (MI). Gender could be a potential explanatory factor for the variability. We conducted a systematic review and meta-analysis to determine whether gender mix might play a role in explaining the large variation of aspirin efficacy across primary and secondary MI prevention trials. METHODS: Randomized placebo-controlled clinical trials that examined the efficacy of aspirin therapy on MI were identified by using the PUBMED database (1966 to October 2006). Weighted linear regression technique was used to determine the relationship between log-transformed relative risk (RR) of MI and the percentage of male participants in each trial. The reciprocal of the standard error of the RR in each trial (1/SE) was used as the weight. RESULTS: A total of 23 trials (n = 113 494 participants) were identified. Overall, compared with placebo, aspirin reduced the risk of non-fatal MI (RR = 0.72, 95% confidence interval (CI) 0.64-0.81, p < 0.001) but not of fatal MI (RR = 0.88, 95% CI 0.75-1.03, p = 0.120). A total of 27% of the variation in the non-fatal MI results could be accounted for by considering the gender mix of the trials (p = 0.017). Trials that recruited predominantly men demonstrated the largest risk reduction in non-fatal MI (RR = 0.62, 95% CI 0.54-0.71), while trials that contained predominately women failed to demonstrate a significant risk reduction in non-fatal MI (RR = 0.87, 95% CI 0.71-1.06). CONCLUSION: Gender accounts for a substantial proportion of the variability in the efficacy of aspirin in reducing MI rates across these trials, and supports the notion that women might be less responsive to aspirin than men. [Abstract/Link to Full Text]

Kawano H, Komaba S, Kanamori T, Kaneda Y
A new therapy for highly effective tumor eradication using HVJ-E combined with chemotherapy.
BMC Med. 2007;528.
BACKGROUND: Inactivated HVJ (hemagglutinating virus of Japan; Sendai virus) particles (HVJ envelope vector; HVJ-E can incorporate and deliver plasmid DNA, siRNA, antibody and peptide and anti-cancer drugs to cells both in vitro and in vivo. We attempted to eradicate tumors derived from mouse colon cancer cells, CT26, by combining bleomycin (BLM)-incorporated HVJ-E (HVJ-E/BLM) with cisplatin (CDDP) administration. METHODS: CT-26 tumor mass was intradermally established in Balb/c mice. HVJ-E/BLM was directly injected into the tumor mass with or without intraperitoneal administration of CDDP. The anti-tumor effect was evaluated by measuring tumor size and cytotoxic T cell activity against CT26. Re-challenge of tumor cells to treated mice was performed 10 days or 8 months after the initial tumor inoculation. RESULTS: We found that three intratumoral injections of HVJ-E/BLM along with a single intraperitoneal administration of CDDP eradicated CT26 tumors with more than 75% efficiency. When tumor cells were intradermally re-injected on day 10 after the initial tumor inoculation, tumors on both sides disappeared in most of the mice that received the combination therapy of HVJ-E/BLM and CDDP. Eight months after the initial tumor eradication, surviving mice were re-challenged with CT26 cells. The re-challenged tumors were rejected in all of the surviving mice treated with the combination therapy. Cytotoxic T lymphocytes specific for CT26 were generated in these surviving mice. CONCLUSION: Combination therapy consisting of HVJ-E and chemotherapy completely eradicated the tumor, and generated anti-tumor immunity. The combination therapy could therefore be a promising new strategy for cancer therapy. [Abstract/Link to Full Text]

Zhang Y, Koukounari A, Kabatereine N, Fleming F, Kazibwe F, Tukahebwa E, Stothard JR, Webster JP, Fenwick A
Parasitological impact of 2-year preventive chemotherapy on schistosomiasis and soil-transmitted helminthiasis in Uganda.
BMC Med. 2007;527.
BACKGROUND: Schistosomiasis and soil-transmitted helminthiasis (STH) are among the neglected tropical diseases in Africa. A national control program for these diseases was initiated in Uganda during March 2003. Annual treatment with praziquantel and albendazole was given to schoolchildren in endemic areas and to adults in selected communities where local prevalence of Schistosoma mansoni in schoolchildren was high. METHODS: The impact of the treatment program was monitored through cohorts of schoolchildren and adults. Their infection status with S. mansoni and STH was determined by parasitological examinations at baseline and at annual follow-ups. The prevalence and intensity of S. mansoni and STH before and after treatment were analyzed. RESULTS: Two rounds of treatment significantly reduced the prevalence of S. mansoni infection in schoolchildren across three regions in the country from 33.4-49.3% to 9.7-29.6%, and intensity of infection from 105.7-386.8 eggs per gram of faeces (epg) to 11.6-84.1 epg. The prevalence of hookworm infection was reduced from 41.2-57.9% to 5.5-16.1%, and intensity of infection from 186.9-416.8 epg to 3.7-36.9 epg. The proportion of children with heavy S. mansoni infection was significantly reduced from 15% (95% CI 13.4-16.8%) to 2.3% (95% CI 1.6-3.0%). In adults, significant reduction in the prevalence and intensity of S. mansoni and hookworm infections was also observed. More importantly, the prevalence and intensity of both S. mansoni and hookworm infections in the cohorts of newly-recruited 6-year-olds who had never previously received treatment decreased significantly over 2 years: 34.9% (95% CI 31.9-37.8%) to 22.6% (95% CI 19.9-25.2%) and 171.1 epg (95% CI 141.5-200.7) to 72.0 epg (95% CI 50.9-93.1) for S. mansoni; and 48.4% (95% CI 45.4-51.5) to 15.9% (95% CI 13.6-18.2) and 232.7 epg (95% CI 188.4-276.9) to 51.4 epg (95% CI 33.4-69.5) for hookworms, suggesting a general decline in environmental transmission levels. CONCLUSION: Annual anthelminthic treatment delivered to schoolchildren and to adults at high risk in Uganda can significantly reduce the prevalence and intensity of infection for schistosomiasis and STH, and potentially also significantly reduce levels of environmental transmission of infection. [Abstract/Link to Full Text]

Garelick AI, Gross SR, Richardson I, von der Tann M, Bland J, Hale R
Which doctors and with what problems contact a specialist service for doctors? A cross sectional investigation.
BMC Med. 2007;526.
BACKGROUND: In the United Kingdom, specialist treatment and intervention services for doctors are underdeveloped. The MedNet programme, created in 1997 and funded by the London Deanery, aims to fill this gap by providing a self-referral, face-to-face, psychotherapeutic assessment service for doctors in London and South-East England. MedNet was designed to be a low-threshold service, targeting doctors without formal psychiatric problems. The aim of this study was to delineate the characteristics of doctors utilising the service, to describe their psychological morbidity, and to determine if early intervention is achieved. METHODS: A cross-sectional study including all consecutive self-referred doctors (n = 121, 50% male) presenting in 2002-2004 was conducted. Measures included standardised and bespoke questionnaires both self-report and clinician completed. The multi-dimensional evaluation included: demographics, CORE (CORE-OM, CORE-Workplace and CORE-A) an instrument designed to evaluate the psychological difficulties of patients referred to outpatient services, Brief Symptom Inventory to quantify caseness and formal psychiatric illness, and Maslach Burnout Inventory. RESULTS: The most prevalent presenting problems included depression, anxiety, interpersonal, self-esteem and work-related issues. However, only 9% of the cohort were identified as severely distressed psychiatrically using this measure. In approximately 50% of the sample, problems first presented in the preceding year. About 25% were on sick leave at the time of consultation, while 50% took little or no leave in the prior 12 months. A total of 42% were considered to be at some risk of suicide, with more than 25% considered to have a moderate to severe risk. There were no significant gender differences in type of morbidity, severity or days off sick. CONCLUSION: Doctors displayed high levels of distress as reflected in the significant proportion of those who were at some risk of suicide; however, low rates of severe psychiatric illness were detected. These findings suggest that MedNet clients represent both ends of the spectrum of severity, enabling early clinical engagement for a significant proportion of cases that is of importance both in terms of personal health and protecting patient care, and providing a timely intervention for those who are at risk, a group for whom rapid intervention services are in need and an area that requires further investigation in the UK. [Abstract/Link to Full Text]

Perrin MC, Terry MB, Kleinhaus K, Deutsch L, Yanetz R, Tiram E, Calderon R, Friedlander Y, Paltiel O, Harlap S
Gestational diabetes as a risk factor for pancreatic cancer: a prospective cohort study.
BMC Med. 2007;525.
BACKGROUND: Diabetes is known to be associated with cancer of the pancreas, though there is some debate as to whether it is a cause or a consequence of the disease. We investigated the incidence of pancreatic cancer in a cohort of 37926 Israeli women followed for 28-40 years for whom information on diabetes had been collected at the time they gave birth, in 1964-1976, in Jerusalem. There were 54 cases of pancreatic cancer ascertained from the Israel Cancer Registry during follow-up. METHODS: We used Cox proportional hazards models to adjust for age at baseline and explore effects of other risk factors, including ethnic groups, preeclampsia, birth order and birth weight of offspring. RESULTS: We observed no cases of pancreatic cancer in the women with insulin dependent diabetes; however, there were five cases in the women with gestational diabetes. The interval between the record of diabetes in pregnancy and the diagnosis of pancreatic cancer ranged from 14-35 years. Women with a history of gestational diabetes showed a relative risk of pancreatic cancer of 7.1 (95% confidence interval, 2.8-18.0). CONCLUSION: We conclude that gestational diabetes is strongly related to the risk of cancer of the pancreas in women in this population, and that gestational diabetes can precede cancer diagnosis by many years. [Abstract/Link to Full Text]

Perry HN, McDonnell SM, Alemu W, Nsubuga P, Chungong S, Otten MW, Lusamba-dikassa PS, Thacker SB
Planning an integrated disease surveillance and response system: a matrix of skills and activities.
BMC Med. 2007;524.
BACKGROUND: The threat of a global influenza pandemic and the adoption of the World Health Organization (WHO) International Health Regulations (2005) highlight the value of well-coordinated, functional disease surveillance systems. The resulting demand for timely information challenges public health leaders to design, develop and implement efficient, flexible and comprehensive systems that integrate staff, resources, and information systems to conduct infectious disease surveillance and response. To understand what resources an integrated disease surveillance and response system would require, we analyzed surveillance requirements for 19 priority infectious diseases targeted for an integrated disease surveillance and response strategy in the WHO African region. METHODS: We conducted a systematic task analysis to identify and standardize surveillance objectives, surveillance case definitions, action thresholds, and recommendations for 19 priority infectious diseases. We grouped the findings according to surveillance and response functions and related them to community, health facility, district, national and international levels. RESULTS: The outcome of our analysis is a matrix of generic skills and activities essential for an integrated system. We documented how planners used the matrix to assist in finding gaps in current systems, prioritizing plans of action, clarifying indicators for monitoring progress, and developing instructional goals for applied epidemiology and in-service training programs. CONCLUSION: The matrix for Integrated Disease Surveillance and Response (IDSR) in the African region made clear the linkage between public health surveillance functions and participation across all levels of national health systems. The matrix framework is adaptable to requirements for new programs and strategies. This framework makes explicit the essential tasks and activities that are required for strengthening or expanding existing surveillance systems that will be able to adapt to current and emerging public health threats. [Abstract/Link to Full Text]

Furler J, Harris E, Harris M, Naccarella L, Young D, Snowdon T
Health inequalities, physician citizens and professional medical associations: an Australian case study.
BMC Med. 2007;523.
BACKGROUND: As socioeconomic health inequalities persist and widen, the health effects of adversity are a constant presence in the daily work of physicians. Gruen and colleagues suggest that, in responding to important population health issues such as this, defining those areas of professional obligation in contrast to professional aspiration should be on the basis of evidence and feasibility. Drawing this line between obligation and aspiration is a part of the work of professional medical colleges and associations, and in doing so they must respond to members as well as a range of other interest groups. Our aim was to explore the usefulness of Gruen's model of physician responsibility in defining how professional medical colleges and associations should lead the profession in responding to socioeconomic health inequalities. METHODS: We report a case study of how the Royal Australian College of General Practitioners is responding to the issue of health inequalities through its work. We undertook a consultation (80 interviews with stakeholders internal and external to the College and two focus groups with general practitioners) and program and policy review of core programs of College interest and responsibility: general practitioner training and setting of practice standards, as well as its work in public advocacy. RESULTS: Some strategies within each of these College program areas were seen as legitimate professional obligations in responding to socioeconomic health inequality. However, other strategies, while potentially professional obligations within Gruen's model, were nevertheless contested. The key difference between these lay in different moral orientations. Actions where agreement existed were based on an ethos of care and compassion. Actions that were contested were based on an ethos of justice and human rights. CONCLUSION: Colleges and professional medical associations have a role in explicitly leading a debate about values, engaging both external stakeholder and practicing member constituencies. This is an important and necessary step in defining an agreed role for the profession in addressing health inequalities. [Abstract/Link to Full Text]

Briollais L, Wang Y, Rajendram I, Onay V, Shi E, Knight J, Ozcelik H
Methodological issues in detecting gene-gene interactions in breast cancer susceptibility: a population-based study in Ontario.
BMC Med. 2007;522.
BACKGROUND: There is growing evidence that gene-gene interactions are ubiquitous in determining the susceptibility to common human diseases. The investigation of such gene-gene interactions presents new statistical challenges for studies with relatively small sample sizes as the number of potential interactions in the genome can be large. Breast cancer provides a useful paradigm to study genetically complex diseases because commonly occurring single nucleotide polymorphisms (SNPs) may additively or synergistically disturb the system-wide communication of the cellular processes leading to cancer development. METHODS: In this study, we systematically studied SNP-SNP interactions among 19 SNPs from 18 key genes involved in major cancer pathways in a sample of 398 breast cancer cases and 372 controls from Ontario. We discuss the methodological issues associated with the detection of SNP-SNP interactions in this dataset by applying and comparing three commonly used methods: the logistic regression model, classification and regression trees (CART), and the multifactor dimensionality reduction (MDR) method. RESULTS: Our analyses show evidence for several simple (two-way) and complex (multi-way) SNP-SNP interactions associated with breast cancer. For example, all three methods identified XPD-[Lys751Gln]*IL10-[G(-1082)A] as the most significant two-way interaction. CART and MDR identified the same critical SNPs participating in complex interactions. Our results suggest that the use of multiple statistical approaches (or an integrated approach) rather than a single methodology could be the best strategy to elucidate complex gene interactions that have generally very different patterns. CONCLUSION: The strategy used here has the potential to identify complex biological relationships among breast cancer genes and processes. This will lead to the discovery of novel biological information, which will improve breast cancer risk management. [Abstract/Link to Full Text]

Rechel B, McKee M
The effects of dictatorship on health: the case of Turkmenistan.
BMC Med. 2007;521.
BACKGROUND: There is a health crisis in Turkmenistan similar to, but more severe than, in other Central Asian countries. This paper asks whether the health crisis in Turkmenistan is attributable to the consequences of the dictatorship under president Niyazov, who died in 2006. METHODS: The basis for this paper was a series of semi-structured in-depth interviews with key informants complemented by an iterative search of internet sites, initially published as a report in April 2005, and subsequently updated with feedback on the report as well as a comprehensive search of secondary information sources and databases. RESULTS: This paper describes in depth three areas in which the dictatorship in Turkmenistan had a negative impact on population health: the regime's policy of secrecy and denial, which sees the "solution" to health care problems in concealment rather than prevention; its complicity in the trafficking of drugs from Afghanistan; and the neglect of its health care system. CONCLUSION: The paper concludes that dictatorship has contributed to the health crisis facing Turkmenistan. One of the first tests of the new regime will be whether it can address this crisis. [Abstract/Link to Full Text]

Wolozin B, Wang SW, Li NC, Lee A, Lee TA, Kazis LE
Simvastatin is associated with a reduced incidence of dementia and Parkinson's disease.
BMC Med. 2007;520.
BACKGROUND: Statins are a class of medications that reduce cholesterol by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Whether statins can benefit patients with dementia remains unclear because of conflicting results. We hypothesized that some of the confusion in the literature might arise from differences in efficacy of different statins. We used a large database to compare the action of several different statins to investigate whether some statins might be differentially associated with a reduction in the incidence of dementia and Parkinson's disease. METHODS: We analyzed data from the decision support system of the US Veterans Affairs database, which contains diagnostic, medication and demographic information on 4.5 million subjects. The association of lovastatin, simvastatin and atorvastatin with dementia was examined with Cox proportional hazard models for subjects taking statins compared with subjects taking cardiovascular medications other than statins, after adjusting for covariates associated with dementia or Parkinson's disease. RESULTS: We observed that simvastatin is associated with a significant reduction in the incidence of dementia in subjects > or =65 years, using any of three models. The first model incorporated adjustment for age, the second model included adjusted for three known risk factors for dementia, hypertension, cardiovascular disease or diabetes, and the third model incorporated adjustment for the Charlson index, which is an index that provides a broad assessment of chronic disease. Data were obtained for over 700,000 subjects taking simvastatin and over 50,000 subjects taking atorvastatin who were aged >64 years. Using model 3, the hazard ratio for incident dementia for simvastatin and atorvastatin are 0.46 (CI 0.44-0.48, p < 0.0001) and 0.91 (CI 0.80-1.02, p = 0.11), respectively. Lovastatin was not associated with a reduction in the incidence of dementia. Simvastatin also exhibited a reduced hazard ratio for newly acquired Parkinson's disease (HR 0.51, CI 0.4-0.55, p < 0.0001). CONCLUSION: Simvastatin is associated with a strong reduction in the incidence of dementia and Parkinson's disease, whereas atorvastatin is associated with a modest reduction in incident dementia and Parkinson's disease, which shows only a trend towards significance. [Abstract/Link to Full Text]

Ertresvg JM, Stovner LJ, Kvavik LE, Johnsen HJ, Zwart JA, Helde G, Bovim G
Migraine aura or transient ischemic attacks? A five-year follow-up case-control study of women with transient central nervous system disorders in pregnancy.
BMC Med. 2007;519.
BACKGROUND: Migraine aura may be difficult to differentiate from transient ischemic attacks and other transient neurological disorders in pregnant women. The aims of the present study were to investigate and diagnose all pregnant women with transient neurological disorders of suspected central nervous system origin, and to compare this group with a control group of pregnant women with regard to vascular risk factors and prognosis. METHODS: During a 28 month period, 41 patients were detected with transient neurological symptoms during pregnancy. These were studied in detail with thorough clinical and laboratory investigations in order to make a certain diagnosis and to evaluate whether the episodes might be of a vascular nature. For comparison, the same investigations were performed in 41 pregnant controls. To assess the prognosis, both patients and controls were followed with questionnaires every year for five years. RESULTS: Migraine with aura was the most common cause of symptoms during pregnancy, occurring in 34 patients, while 2 were diagnosed with stroke, 2 with carpal tunnel syndrome, 1 with partial epilepsy, 1 with multiple sclerosis and 1 with presyncope. Patients had more headache before pregnancy than controls, but the average levels of vascular risk factors were similar. None of the patients or the controls reported cerebrovascular episodes during the five-year follow-up. CONCLUSION: The diagnosis of migraine aura was difficult because for many patients it was their first ever attack and headache tended to be absent or of non-migraineous type. The aura features were more complex, with several aura symptoms and a higher prevalence of sensory and dysphasic aura than usual. Gradually developing aura symptoms, or different aura symptoms occurring in succession as described in the International Classification of Headache Disorders, seem to be useful for differentiating aura from other transient disorders. A meticulous history and clinical neurological examination are more useful than routine supplementary investigations for cerebrovascular disease. The five-year follow-up clearly indicates that migraine with aura in pregnancy usually has a good prognosis with regard to cerebrovascular events. [Abstract/Link to Full Text]

Hinkle RT, Lefever FR, Dolan ET, Reichart DL, Dietrich JA, Gropp KE, Thacker RI, Demuth JP, Stevens PJ, Qu XA, Varbanov AR, Wang F, Isfort RJ
Corticortophin releasing factor 2 receptor agonist treatment significantly slows disease progression in mdx mice.
BMC Med. 2007;518.
BACKGROUND: Duchenne muscular dystrophy results from mutation of the dystrophin gene, causing skeletal and cardiac muscle loss of function. The mdx mouse model of Duchenne muscular dystrophy is widely utilized to evaluate the potential of therapeutic regimens to modulate the loss of skeletal muscle function associated with dystrophin mutation. Importantly, progressive loss of diaphragm function is the most consistent striated muscle effect observed in the mdx mouse model, which is the same as in patients suffering from Duchenne muscular dystrophy. METHODS: Using the mdx mouse model, we have evaluated the effect that corticotrophin releasing factor 2 receptor (CRF2R) agonist treatment has on diaphragm function, morphology and gene expression. RESULTS: We have observed that treatment with the potent CRF2R-selective agonist PG-873637 prevents the progressive loss of diaphragm specific force observed during aging of mdx mice. In addition, the combination of PG-873637 with glucocorticoids not only prevents the loss of diaphragm specific force over time, but also results in recovery of specific force. Pathological analysis of CRF2R agonist-treated diaphragm muscle demonstrates that treatment reduces fibrosis, immune cell infiltration, and muscle architectural disruption. Gene expression analysis of CRF2R-treated diaphragm muscle showed multiple gene expression changes including globally decreased immune cell-related gene expression, decreased extracellular matrix gene expression, increased metabolism-related gene expression, and, surprisingly, modulation of circadian rhythm gene expression. CONCLUSION: Together, these data demonstrate that CRF2R activation can prevent the progressive degeneration of diaphragm muscle associated with dystrophin gene mutation. [Abstract/Link to Full Text]

Ramos-Nino ME, MacLean CD, Littenberg B
Association between cancer prevalence and use of thiazolidinediones: results from the Vermont Diabetes Information System.
BMC Med. 2007;517.
BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) have emerged as important drug targets for diabetes. Drugs that activate PPARgamma, such as the thiazolidinediones (TZDs), are widely used for treatment of Type 2 diabetes mellitus. PPARgamma signaling could also play an anti-neoplastic role in several in vitro models, although conflicting results are reported from in vivo models. The effects of TZDs on cancer risk in humans needs to be resolved as these drugs are prescribed for long periods of time in patients with diabetes. METHODS: A total of 1003 subjects in community practice settings were interviewed at home at the time of enrolment into the Vermont Diabetes Information System, a clinical decision support program. Patients self-reported their personal and clinical characteristics, including any history of malignancy. Laboratory data were obtained directly from the clinical laboratory and current medications were obtained by direct observation of medication containers. We performed a cross-sectional analysis of the interviewed subjects to assess a possible association between cancer diagnosis and the use of TZDs. RESULTS: In a multivariate logistic regression model, a diagnosis of cancer was significantly associated with TZD use, even after correcting for potential confounders including other oral anti-diabetic agents (sulfonylureas and biguanides), age, glycosylated hemoglobin A1C, body mass index, cigarette smoking, high comorbidity, and number of prescription medications (odds ratio = 1.59, P = 0.04). This association was particularly strong among patients using rosiglitazone (OR = 1.89, P = 0.02), and among women (OR = 2.07, P = 0.01). CONCLUSION: These data suggest an association between TZD use and cancer in patients with diabetes. Further studies are required to determine if this association is causal. [Abstract/Link to Full Text]

Wilson DP, Blower S
How far will we need to go to reach HIV-infected people in rural South Africa?
BMC Med. 2007;516.
BACKGROUND: The South African Government has outlined detailed plans for antiretroviral (ART) rollout in KwaZulu-Natal Province, but has not created a plan to address treatment accessibility in rural areas in KwaZulu-Natal. Here, we calculate the distance that People Living With HIV/AIDS (PLWHA) in rural areas in KwaZulu-Natal would have to travel to receive ART. Specifically, we address the health policy question 'How far will we need to go to reach PLWHA in rural KwaZulu-Natal?'. METHODS: We developed a model to quantify treatment accessibility in rural areas; the model incorporates heterogeneity in spatial location of HCFs and patient population. We defined treatment accessibility in terms of the number of PLWHA that have access to an HCF. We modeled the treatment-accessibility region (i.e. catchment area) around an HCF by using a two-dimensional function, and assumed that treatment accessibility decreases as distance from an HCF increases. Specifically, we used a distance-discounting measure of ART accessibility based upon a modified form of a two-dimensional gravity-type model. We calculated the effect on treatment accessibility of: (1) distance from an HCF, and (2) the number of HCFs. RESULTS: In rural areas in KwaZulu-Natal even substantially increasing the size of a small catchment area (e.g. from 1 km to 20 km) around an HCF would have a negligible impact (~2%) on increasing treatment accessibility. The percentage of PLWHA who can receive ART in rural areas in this province could be as low as ~16%. Even if individuals were willing (and able) to travel 50 km to receive ART, only ~50% of those in need would be able to access treatment. Surprisingly, we show that increasing the number of available HCFs for ART distribution ~ threefold does not lead to a threefold increase in treatment accessibility in rural KwaZulu-Natal. CONCLUSION: Our results show that many PLWHA in rural KwaZulu-Natal are unlikely to have access to ART, and that the impact of an additional 37 HCFs on treatment accessibility in rural areas would be less substantial than might be expected. There is a great length to go before we will be able to reach many PLWHA in rural areas in South Africa, and specifically in KwaZulu-Natal. [Abstract/Link to Full Text]

Kane B, Luz S, O'Briain DS, McDermott R
Multidisciplinary team meetings and their impact on workflow in radiology and pathology departments.
BMC Med. 2007;515.
BACKGROUND: The development of multidisciplinary team meetings (MDTMs) for radiology and pathology is a burgeoning area that increasingly impacts on work processes in both of these departments. The aim of this study was to examine work processes and quantify the time demands on radiologists and pathologists associated with MDTM practices at a large teaching hospital. The observations reported in this paper reflect a general trend affecting hospitals and our conclusions will have relevance for others implementing clinical practice guidelines. METHODS: For one month, all work related to clinical meetings between pathology and radiology with clinical staff was documented and later analysed. RESULTS: The number of meetings to which pathology and radiology contribute at a large university teaching hospital, ranges from two to eight per day, excluding grand rounds, and amounts to approximately 50 meetings per month for each department. For one month, over 300 h were spent by pathologists and radiologists on 81 meetings, where almost 1000 patients were discussed. For each meeting hour, there were, on average, 2.4 pathology hours and 2 radiology hours spent in preparation. Two to three meetings per week are conducted over a teleconferencing link. Average meeting time is 1 h. Preparation time per meeting ranges from 0.3 to 6 h for pathology, and 0.5 to 4 for radiology. The review process in preparation for meetings improves internal quality standards. Materials produced externally (for example imaging) can amount to almost 50% of the material to be reviewed on a single patient. The number of meetings per month has increased by 50% over the past two years. Further increase is expected in both the numbers and duration of meetings when scheduling issues are resolved. A changing trend in the management of referred patients with the development of MDTMs and the introduction of teleconferencing was noted. CONCLUSION: Difficulties are being experienced by pathology and radiology departments participating fully in several multidisciplinary teams. Time spent at meetings, and in preparation for MDTMs is significant. Issues of timing and the coordination of materials to be reviewed are sometimes irreconcilable. The exchange of patient materials with outside institutions is a cause for concern when full data are not made available in a timely fashion. The process of preparation for meetings is having a positive influence on quality, but more resources are needed in pathology and radiology to realise the full benefits of multidisciplinary team working. [Abstract/Link to Full Text]

Bachmann LM, Gutzwiller FS, Puhan MA, Steurer J, Steurer-Stey C, Gigerenzer G
Do citizens have minimum medical knowledge? A survey.
BMC Med. 2007;514.
BACKGROUND: Experts defined a "minimum medical knowledge" (MMK) that people need for understanding typical signs and/or risk factors of four relevant clinical conditions: myocardial infarction, stroke, chronic obstructive pulmonary disease and HIV/AIDS. We tested to what degree Swiss adult citizens satisfy this criterion for MMK and whether people with medical experience have acquired better knowledge than those without. METHODS: Questionnaire interview in a Swiss urban area with 185 Swiss citizens (median age 29 years, interquartile range 23 to 49, 52% male). We obtained context information on age, gender, highest educational level, (para)medical background and specific health experience with one of the conditions in the social surrounding. We calculated the proportion of MMK and examined whether citizens with medical background (personal or professional) would perform better compared to other groups. RESULTS: No single citizen reached the full MMK (100%). The mean MMK was as low as 32% and the range was 0-72%. Surprisingly, multivariable analysis showed that participants with a university degree (n = 84; beta (95% CI) +3.7% MMK (0.4-7.1) p = 0.03), (para)medical background (n = 34; +6.2% MMK (2.0-10.4), p = 0.004) and personal illness experience (n = 96; +4.9% MMK (1.5-8.2), p = 0.004) had only a moderately higher MMK than those without, while age and sex had no effect on the level of MMK. Interaction between university degree and clinical experience (personal or professional) showed no effect suggesting that higher education lacks synergistic effect. CONCLUSION: This sample of Swiss citizens did not know more than a third of the MMK. We found little difference within groups with medical experience (personal or professional), suggesting that there is a consistent and dramatic lack of knowledge in the general public about the typical signs and risk factors of relevant clinical conditions. [Abstract/Link to Full Text]

Kuffner EK, Green JL, Bogdan GM, Knox PC, Palmer RB, Heard K, Slattery JT, Dart RC
The effect of acetaminophen (four grams a day for three consecutive days) on hepatic tests in alcoholic patients--a multicenter randomized study.
BMC Med. 2007;513.
BACKGROUND: Hepatic failure has been associated with reported therapeutic use of acetaminophen by alcoholic patients. The highest risk period for alcoholic patients is immediately after discontinuation of alcohol intake. This period exhibits the largest increase in CYP2E1 induction and lowest glutathione levels. Our hypothesis was that common liver tests would be unaffected by administration of the maximum recommended daily dosage of acetaminophen for 3 consecutive days to newly-abstinent alcoholic subjects. METHODS: Adult alcoholic subjects entering two alcohol detoxification centers were enrolled in a prospective double-blind, randomized, placebo-controlled trial. Subjects were randomized to acetaminophen, 4 g/day, or placebo for 3 consecutive days. The study had 95% probability of detecting a 15 IU/L difference in serum ALT. RESULTS: A total of 443 subjects were enrolled: 308 (258 completed) received acetaminophen and 135 subjects (114 completed) received placebo. Study groups did not differ in demographics, alcohol consumption, nutritional status or baseline laboratory assessments. The peak mean ALT activity was 57 +/- 45 IU/L and 55 +/- 48 IU/L in the acetaminophen and placebo groups, respectively. Subgroup analyses for subjects presenting with an elevated ALT, subjects fulfilling a diagnosis of alcoholic hepatitis and subjects attaining a peak ALT greater than 200 IU/L showed no statistical difference between the acetaminophen and control groups. The one participant developing an increased international normalized ratio was in the placebo group. CONCLUSION: Alcoholic patients treated with the maximum recommended daily dose of acetaminophen for 3 consecutive days did not develop increases in serum transaminase or other measures of liver injury. Treatment of pain or fever for 3 days with acetaminophen appears safe in newly-abstinent alcoholic patients, such as those presenting for acute medical care. [Abstract/Link to Full Text]

J�rgensen KJ, Klahn A, G�tzsche PC
Are benefits and harms in mammography screening given equal attention in scientific articles? A cross-sectional study.
BMC Med. 2007;512.
BACKGROUND: The CONSORT statement specifies the need for a balanced presentation of both benefits and harms of medical interventions in trial reports. However, invitations to screening and newspaper articles often emphasize benefits and downplay or omit harms, and it is known that scientific articles can be influenced by conflicts of interest. We wanted to determine if a similar imbalance occurs in scientific articles on mammography screening and if it is related to author affiliation. METHODS: We searched PubMed in April 2005 for articles on mammography screening that mentioned a benefit or a harm and that were published in 2004 in English. Data extraction was performed by three independent investigators, two unblinded and one blinded for article contents, and author names and affiliation, as appropriate. The extracted data were compared and discrepancies resolved by two investigators in a combined analysis. We defined three groups of authors: (1) authors in specialties unrelated to mammography screening, (2) authors in screening-affiliated specialties (radiology or breast cancer surgery) who were not working with screening, or authors funded by cancer charities, and (3) authors (at least one) working directly with mammography screening programmes. We used a data extraction sheet with 17 items described as important benefits and harms in the 2002 WHO/IARC-report on breast cancer screening. RESULTS: We identified 854 articles, and 143 were eligible for the study. Most were original research. Benefits were mentioned more often than harms (96% vs 62%, P < 0.001). Fifty-five (38%) articles mentioned only benefits, whereas seven (5%) mentioned only harms (P < 0.001). Overdiagnosis was mentioned in 35 articles (24%), but was more often downplayed or rejected in articles that had authors working with screening, (6/15; 40%) compared with authors affiliated by specialty or funding (1/6; 17%), or authors unrelated with screening (1/14; 7%) (P = 0.03). Benefits in terms of reduced breast cancer mortality were mentioned in 109 (76%) articles, and was more often provided as a relative risk reduction than an absolute risk reduction, where quantified (45 articles (31%) versus 6 articles (3%) (P < 0.001)). CONCLUSION: Scientific articles tend to emphasize the major benefits of mammography screening over its major harms. This imbalance is related to the authors' affiliation. [Abstract/Link to Full Text]

Schmitt HJ, Booy R, Aston R, Van Damme P, Schumacher RF, Campins M, Rodrigo C, Heikkinen T, Weil-Olivier C, Finn A, Olc�n P, Fedson D, Peltola H
How to optimise the coverage rate of infant and adult immunisations in Europe.
BMC Med. 2007;511.
BACKGROUND: Although vaccination has been proved to be a safe, efficacious, and cost-effective intervention, immunisation rates remain suboptimal in many European countries, resulting in poor control of many vaccine-preventable diseases. DISCUSSION: The Summit of Independent European Vaccination Experts focused on the perception of vaccines and vaccination by the general public and healthcare professionals and discussed ways to improve vaccine uptake in Europe. Despite the substantial impact and importance of the media, healthcare professionals were identified as the main advocates for vaccination and the most important source of information about vaccines for the general public. Healthcare professionals should receive more support for their own education on vaccinology, have rapid access to up-to-date information on vaccines, and have easy access to consultation with experts regarding vaccination-related problems. Vaccine information systems should be set up to facilitate promotion of vaccination. SUMMARY: Every opportunity to administer vaccines should be used, and active reminder systems should be set up. A European vaccine awareness week should be established. [Abstract/Link to Full Text]

Sheikh L, Johnston S, Thangaratinam S, Kilby MD, Khan KS
A review of the methodological features of systematic reviews in maternal medicine.
BMC Med. 2007;510.
BACKGROUND: In maternal medicine, research evidence is scattered making it difficult to access information for clinical decision making. Systematic reviews of good methodological quality are essential to provide valid inferences and to produce usable evidence summaries to guide management. This review assesses the methodological features of existing systematic reviews in maternal medicine, comparing Cochrane and non-Cochrane reviews in maternal medicine. METHODS: Medline, Embase, Database of Reviews of Effectiveness (DARE) and Cochrane Database of Systematic Reviews (CDSR) were searched for relevant reviews published between 2001 and 2006. We selected those reviews in which a minimum of two databases were searched and the primary outcome was related to the maternal condition. The selected reviews were assessed for information on framing of question, literature search and methods of review. RESULTS: Out of 2846 citations, 68 reviews were selected. Among these, 39 (57%) were Cochrane reviews. Most of the reviews (50/68, 74%) evaluated therapeutic interventions. Overall, 54/68 (79%) addressed a focussed question. Although 64/68 (94%) reviews had a detailed search description, only 17/68 (25%) searched without language restriction. 32/68 (47%) attempted to include unpublished data and 11/68 (16%) assessed for the risk of missing studies quantitatively. The reviews had deficiencies in the assessment of validity of studies and exploration for heterogeneity. When compared to Cochrane reviews, other reviews were significantly inferior in specifying questions (OR 20.3, 95% CI 1.1-381.3, p = 0.04), framing focussed questions (OR 30.9, 95% CI 3.7- 256.2, p = 0.001), use of unpublished data (OR 5.6, 95% CI 1.9-16.4, p = 0.002), assessment for heterogeneity (OR 38.1, 95%CI 2.1, 688.2, p = 0.01) and use of meta-analyses (OR 3.7, 95% CI 1.3-10.8, p = 0.02). CONCLUSION: This study identifies areas which have a strong influence on maternal morbidity and mortality but lack good quality systematic reviews. Overall quality of the existing systematic reviews was variable. Cochrane reviews were of better quality as compared to other reviews. There is a need for good quality systematic reviews to inform practice in maternal medicine. [Abstract/Link to Full Text]

Vieira JC, Cooper PJ, Lovato R, Mancero T, Rivera J, Proa�o R, L�pez AA, Guderian RH, Guzm�n JR
Impact of long-term treatment of onchocerciasis with ivermectin in Ecuador: potential for elimination of infection.
BMC Med. 2007;59.
BACKGROUND: Onchocerciasis is a leading cause of blindness worldwide, hence elimination of the infection is an important health priority. Community-based treatment programs with ivermectin form the basis of control programs for the disease in Latin America. The long-term administration of ivermectin could eliminate Onchocerca volvulus infection from endemic areas in Latin America. METHODS: A strategy of annual to twice-annual treatments with ivermectin has been used for onchocerciasis in endemic communities in Ecuador for up to 14 years. The impact of ivermectin treatment on ocular morbidity, and O. volvulus infection and transmission was monitored in seven sentinel communities. RESULTS: Over the period 1990-2003, high rates of treatment coverage of the eligible population were maintained in endemic communities (mean 85.2% per treatment round). Ivermectin reduced the prevalence of anterior segment disease of the eye to 0% in sentinel communities and had a major impact on the prevalence and transmission of infection, with possible elimination of infection in some foci. CONCLUSION: The distribution of ivermectin in endemic communities in Ecuador might have eliminated ocular morbidity and significant progress has been made towards elimination of the infection. A strategy of more frequent treatments with ivermectin may be required in communities where the infection persists to achieve the objective of elimination of the infection from Ecuador. The elimination of the infection from an endemic country in Latin America would be a major public health achievement and could stimulate the implementation of elimination strategies in other endemic countries. [Abstract/Link to Full Text]

Dewhurst NG, McManus C, Mollon J, Dacre JE, Vale AJ
Performance in the MRCP(UK) Examination 2003-4: analysis of pass rates of UK graduates in relation to self-declared ethnicity and gender.
BMC Med. 2007;58.
BACKGROUND: Male students and students from ethnic minorities have been reported to under-perform in undergraduate medical examinations. We examined the effects of ethnicity and gender on pass rates in UK medical graduates sitting the Membership of the Royal Colleges of Physicians in the United Kingdom [MRCP(UK)] Examination in 2003-4. METHODS: Pass rates for each part of the examination were analysed for differences between graduate groupings based on self-declared ethnicity and gender. RESULTS: All candidates declared their gender, and 84-90% declared their ethnicity. In all three parts of the examination, white candidates performed better than other ethnic groups (P < 0.001). In the MRCP(UK) Part 1 and Part 2 Written Examinations, there was no significant difference in pass rate between male and female graduates, nor was there any interaction between gender and ethnicity. In the Part 2 Clinical Examination (Practical Assessment of Clinical Examination Skills, PACES), women performed better than did men (P < 0.001). Non-white men performed more poorly than expected, relative to white men or non-white women. Analysis of individual station marks showed significant interaction between candidate and examiner ethnicity for performance on communication skills (P = 0.011), but not on clinical skills (P = 0.176). Analysis of overall average marks showed no interaction between candidate gender and the number of assessments made by female examiners (P = 0.151). CONCLUSION: The cause of these differences is most likely to be multifactorial, but cannot be readily explained in terms of previous educational experience or differential performance on particular parts of the examination. Potential examiner prejudice, significant only in the cases where there were two non-white examiners and the candidate was non-white, might indicate different cultural interpretations of the judgements being made. [Abstract/Link to Full Text]

Brown H, Hofmeyr GJ, Nikodem VC, Smith H, Garner P
Promoting childbirth companions in South Africa: a randomised pilot study.
BMC Med. 2007;57.
BACKGROUND: Most women delivering in South African State Maternity Hospitals do not have a childbirth companion; in addition, the quality of care could be better, and at times women are treated inhumanely. We piloted a multi-faceted intervention to encourage uptake of childbirth companions in state hospitals, and hypothesised that lay carers would improve the behaviour of health professionals. METHODS: We conducted a pilot randomised controlled trial of an intervention to promote childbirth companions in hospital deliveries. We promoted evidence-based information for maternity staff at 10 hospitals through access to the World Health Organization Reproductive Health Library (RHL), computer hardware and training to all ten hospitals. We surveyed 200 women at each site, measuring companionship, and indicators of good obstetric practice and humanity of care. Five hospitals were then randomly allocated to receive an educational intervention to promote childbirth companions, and we surveyed all hospitals again at eight months through a repeat survey of postnatal women. Changes in median values between intervention and control hospitals were examined. RESULTS: At baseline, the majority of hospitals did not allow a companion, or access to food or fluids. A third of women were given an episiotomy. Some women were shouted at (17.7%, N = 2085), and a few reported being slapped or struck (4.3%, N = 2080). Despite an initial positive response from staff to the childbirth companion intervention, we detected no difference between intervention and control hospitals in relation to whether a companion was allowed by nursing staff, good obstetric practice or humanity of care. CONCLUSION: The quality and humanity of care in these state hospitals needs to improve. Introducing childbirth companions was more difficult than we anticipated, particularly in under-resourced health care systems with frequent staff changes. We were unable to determine whether the presence of a lay carer impacted on the humanity of care provided by health professionals. Trial registration: Current Controlled Trials ISRCTN33728802. [Abstract/Link to Full Text]

Recent Articles in BMC Complementary and Alternative Medicine

Hagins M, Moore W, Rundle A
Does practicing hatha yoga satisfy recommendations for intensity of physical activity which improves and maintains health and cardiovascular fitness?
BMC Complement Altern Med. 2007 Nov 30;7(1):40.
ABSTRACT: BACKGROUND: Little is known about the metabolic and heart rate responses to a typical hatha yoga session. The purposes of this study were to determine 1) whether a typical yoga practice using various postures meets the current recommendations for levels of physical activity required to improve and maintain health and cardiovascular fitness; 2) to determine the reliability of metabolic costs of yoga across sessions; 3) to compare the metabolic costs of yoga practice to those of treadmill walking. METHODS: In this observational study, 20 intermediate-to-advanced level yoga practitioners, age 31.4 +/- 8.3 years, performed an exercise routine inside a human respiratory chamber (indirect calorimeter) while wearing heart rate monitors. The exercise routine consisted of 30 minutes of sitting, 56 minutes of beginner-level hatha yoga administered by video, and 10 minutes of treadmill walking at 3.2 and 4.8 kph each. Measures were mean oxygen consumption (VO2), heart rate (HR), percentage predicted maximal heart rate (%MHR), metabolic equivalents (METs), and energy expenditure (kcal). Seven subjects repeated the protocol so that measurement reliability could be established. RESULTS: Mean values across the entire yoga session for VO2, HR, %MHR, METs, and energy/min were 0.6 L/kg/min; 93.2 beats/min; 49.4 %; 2.5; and 3.2 kcal/min; respectively. Results of the ICCs (2,1) for mean values across the entire yoga session for kcal, METs, and %MHR were 0.979 and 0.973, and 0.865, respectively. CONCLUSIONS: Metabolic costs of yoga averaged across the entire session represent low levels of physical activity, are similar to walking on a treadmill at 3.2 kph, and do not meet recommendations for levels of physical activity for improving or maintaining health or cardiovascular fitness. Yoga practice incorporating sun salutation postures exceeding the minimum bout of 10 minutes may contribute some portion of sufficiently intense physical activity to improve cardio-respiratory fitness in unfit or sedentary individuals. The measurement of energy expenditure across yoga sessions is highly reliable. [Abstract/Link to Full Text]

Gardiner P, Kemper KJ, Legedza A, Phillips RS
Factors associated with herb and dietary supplement use by young adults in the United States.
BMC Complement Altern Med. 2007 Nov 30;7(1):39.
ABSTRACT: BACKGROUND: Little is known about the association between use of herbs and dietary supplements (HDS) and lifestyle/behavior factors young adults in the US. METHODS: Analyzing the 2002 National Health Interview Survey (NHIS), we examined the patterns of HDS (excluding vitamins/minerals) use among young adults in the United States using descriptive statistics and logistic regression. RESULTS: In our sample of 18 to 30 year olds (n=6666), 26% were current smokers, 24% were moderate/ heavy drinkers, 43% had high physical activity, and 54% and 76% use prescription and over the counter (OTC) medications respectively. Non-vitamin, non-mineral HDS was used by 17% of the overall sample in the last 12 months. In the multivariable analysis, the lifestyle and behavioral factors associated with HDS use include: current smoking (odds ratio 1.41 95% CI [1.16-1.72]); being a former smoker (1.50 [1.15-1.95]); moderate/heavy alcohol use (2.02 [1.53-2.65]); high physical activity levels (2.45 [1.98-3.03]); and prescription medication use (1.51 [1.26-1.81]). Among HDS users, only 24% discussed their use with a health care professional. CONCLUSIONS: Nearly one in five young adults report using non-vitamin/non-mineral HDS. The associations between health risk (smoking drinking), health protective (exercise) and illness behaviors (use of prescription medications), suggest the need for additional investigations to explain these complex relationships. [Abstract/Link to Full Text]

Wapf V, Busato A
Patients motives for choosing a physician: comparison between conventional and complementary medicine in Swiss primary care.
BMC Complement Altern Med. 2007 Nov 16;7(1):38.
ABSTRACT: BACKGROUND: The study is part of a nationwide evaluation of complementary and alternative medicine (CAM) in primary care in Switzerland. The Objective was to identify patients' expectations and reasons governing the choice of complementary medicine compared with conventional primary care (CONV). METHODS: The data were derived from the PEK study (Programm Evaluation Komplementarmedizin), which was conducted in 2002-2003 with 7879 adult patients and parents of 1291 underage patients, seeking either complementary (CAM) or conventional (CONV) primary care. The study was performed as a cross-sectional survey. The respondents were asked to document their (or their children's) self-perceived health status, reasons governing their choice, and treatment expectations. Physicians were practicing conventional medicine and/or complementary methods (homeopathy, anthroposophic medicine, neural therapy, and traditional Chinese medicine). Reasons governing the choice of physician were evaluated on the basis of a three-part classification (physician-related, procedure-related, and pragmatic/other reasons) Results and Discussion Patients seeing CAM physicians tend to be younger and more often female. CAM patients referred to procedure-related reasons more frequently, whereas pragmatic reasons dominated among CONV patients. CAM respondents expected fewer adverse side effects compared to conventional care patients. CONCLUSIONS: The majority of alternative medicine users appear to have chosen CAM mainly because they wish to undergo a certain procedure; additional reasons include desire for more comprehensive treatment, and expectation of fewer side-effects. [Abstract/Link to Full Text]

Flegal KE, Kishiyama S, Zajdel D, Haas M, Oken BS
Adherence to yoga and exercise interventions in a 6-month clinical trial.
BMC Complement Altern Med. 2007 Nov 9;7(1):37.
ABSTRACT: BACKGROUND: To determine factors that predict adherence to a mind-body intervention in a randomized trial. METHODS: We analyzed adherence data from a 3-arm trial involving 135 generally healthy seniors 65-85 years of age randomized to a 6-month intervention consisting of: an Iyengar yoga class with home practice, an exercise class with home practice, or a wait-list control group. Outcome measures included cognitive function, mood, fatigue, anxiety, health-related quality of life, and physical measures. Adherence to the intervention was obtained by class attendance and biweekly home practice logs. RESULTS: The drop-out rate was 13%. Among the completers of the two active interventions, average yoga class attendance was 77% and home practice occurred 64% of all days. Average exercise class attendance was 69% and home exercise occurred 54% of all days. There were no clear effects of adherence on the significant study outcomes (quality of life and physical measures). Class attendance was significantly correlated with baseline measures of depression, fatigue, and physical components of health-related quality of life. Significant differences in baseline measures were also found between study completers and drop-outs in the active interventions. Adherence was not related to age, gender, or education level. CONCLUSIONS: Healthy seniors have good attendance at classes with a physically active intervention. Home practice takes place over half of the time. Decreased adherence to a potentially beneficial intervention has the potential to decrease the effect of the intervention in a clinical trial because subjects who might sustain the greatest benefit will receive a lower dose of the intervention and subjects with higher adherence rates may be functioning closer to maximum ability before the intervention. Strategies to maximize adherence among subjects at greater risk for low adherence will be important for future trials, especially complementary treatments requiring greater effort than simple pill-taking. [Abstract/Link to Full Text]

Prasad S, Kashyap RS, Deopujari JY, Purohit HJ, Taori GM, Daginawala HF
Effect of Fagonia Arabica (Dhamasa) on in vitro thrombolysis.
BMC Complement Altern Med. 2007 Nov 6;7(1):36.
ABSTRACT: BACKGROUND: Atherothrombotic diseases such as myocardial or cerebral infarction are serious consequences of the thrombus formed in blood vessels. Thrombolytic agents are used to dissolve the already formed clots in the blood vessels however; these drugs have certain limitations which cause serious and sometimes fatal consequences. Herbal preparations are used since ancient times for the treatment of several diseases. Herbs and its components possessing antithrombotic activity have been reported earlier, however, herbs that could be used for thrombolysis has not been reported so far. This study's aim was to investigate whether herbal preparations (aqueous extract) possess thrombolytic activity or not. METHODS: An in vitro thrombolytic model was used to check the clot lysis effect of the six aqueous herbal extracts viz., Tinospora cordifolia, Rubia cordifolia, Hemidesmus indicus, Glycyrrhiza glabra Linn, Fagonia Arabica and Bacopa monnieri Linn along with Streptokinase as a positive control and water as a negative control. RESULTS: Using an in vitro thrombolytic model, Tinospora cordifolia, Rubia cordifolia, Hemidesmus indicus, Glycyrrhiza glabra Linn, Fagonia Arabica and Bacopa monnieri Linn showed 19.3%, 14.5%, 20.3%, 17.8%, 75.6% and 41.8% clot lysis respectively. Among the herbs studied Fagonia arabica showed significant % of clot lysis (75.6%) with reference to Streptokinase (86.2%). CONCLUSION: Through our study it was found that Dhamasa possess thrombolytic property that could lyse blood clots in vitro, however, in vivo clot dissolving property and active component(s) of Dhamasa for clot lysis are yet to find out. Once found Dhamasa could be incorporated as a thrombolytic agent for the improvement of the patients suffering from Atherothrombotic diseases. [Abstract/Link to Full Text]

Tam LS, Leung PC, Li TK, Zhang L, Li EK
Acupuncture in the treatment of rheumatoid arthritis: a double-blind controlled pilot study.
BMC Complement Altern Med. 2007 Nov 3;7(1):35.
ABSTRACT: BACKGROUND: In planning a randomized controlled trial of acupuncture, we conducted a pilot study using validated outcome measures to assess the feasibility of the protocol, and to obtain preliminary data on efficacy and tolerability of 3 different forms of acupuncture treatment as an adjunct for the treatment of chronic pain in patients with Rheumatoid arthritis (RA). METHODS: The study employs a randomized, prospective, double-blind, placebo-controlled trial to evaluate the effect of electroacupuncture (EA), traditional Chinese acupuncture (TCA) and sham acupuncture (Sham) in patients with RA. All patients received 20 sessions over a period of 10 weeks. Six acupuncture points were chosen. Primary outcome is the changes in the pain score. Secondary outcomes included the changes in the ACR core disease measures, DAS 28 score and the number of patients who achieved ACR 20 at week 10. RESULTS: From 80 eligible patients, 36 patients with mean age of 58 +/- 10 years and disease duration of 9.3 +/- 6.4 years were recruited. Twelve patients were randomized to each group. Twelve, 10 and 7 patients from the EA, TCA and Sham group respectively completed the study at 20 weeks (p<0.03); all except one of the premature dropouts were due to lack of efficacy. At week 10, the pain score remained unchanged in all 3 groups. The number of tender joints was significantly reduced for the EA and TCA groups. Physician's global score was significantly reduced for the EA group and patient's global score was significantly reduced for the TCA group. All the outcomes except patient's global score remained unchanged in the Sham group. CONCLUSION: This pilot study has allowed a number of recommendations to be made to facilitate the design of a large-scale trial, which in turn will help to clarify the existing evidence base on acupuncture for RA. Trial registration: ClinicalTrials.gov NCT00404443. [Abstract/Link to Full Text]

Mehta K, Gala J, Bhasale S, Naik S, Modak M, Thakur H, Deo N, Miller MJ
Comparison of glucosamine sulfate and a polyherbal supplement for the relief of osteoarthritis of the knee: a randomized controlled trial [ISRCTN25438351].
BMC Complement Altern Med. 2007;734.
BACKGROUND: The efficacy and safety of a dietary supplement derived from South American botanicals was compared to glucosamine sulfate in osteoarthritis subjects in a Mumbai-based multi-center, randomized, double-blind study. METHODS: Subjects (n = 95) were screened and randomized to receive glucosamine sulfate (n = 47, 1500 mg/day) or reparagen (n = 48, 1800 mg/day), a polyherbal consisting of 300 mg of vincaria (Uncaria guianensis) and 1500 mg of RNI 249 (Lepidium meyenii) administered orally, twice daily. Primary efficacy variable was response rate based on a 20% improvement in WOMAC pain scores. Additional outcomes were WOMAC scores for pain, stiffness and function, visual analog score (VAS) for pain, with assessments at 1, 2, 4, 6 and 8 weeks. Tolerability, investigator and subject global assessments and rescue medication consumption (paracetamol) were measured together with safety assessments including vital signs and laboratory based assays. RESULTS: Subject randomization was effective: age, gender and disease status distribution was similar in both groups. The response rates (20% reduction in WOMAC pain) were substantial for both glucosamine (89%) and reparagen (94%) and supported by investigator and subject assessments. Using related criteria response rates to reparagen were favorable when compared to glucosamine. Compared to baseline both treatments showed significant benefits in WOMAC and VAS outcomes within one week (P < 0.05), with a similar, progressive improvement over the course of the 8 week treatment protocol (45-62% reduction in WOMAC or VAS scores). Tolerability was excellent, no serious adverse events were noted and safety parameters were unchanged. Rescue medication use was significantly lower in the reparagen group (p < 0.01) at each assessment period. Serum IGF-1 levels were unaltered by treatments. CONCLUSION: Both reparagen and glucosamine sulfate produced substantial improvements in pain, stiffness and function in subjects with osteoarthritis. Response rates were high and the safety profile was excellent, with significantly less rescue medication use with reparagen. Reparagen represents a new natural productive alternative in the management of joint health. TRIAL REGISTRATION: Current Controlled Trials ISRCTN25438351. [Abstract/Link to Full Text]

Hui KK, Nixon EE, Vangel MG, Liu J, Marina O, Napadow V, Hodge SM, Rosen BR, Makris N, Kennedy DN
Characterization of the "Deqi" Response in Acupuncture.
BMC Complement Altern Med. 2007 Oct 31;7(1):33.
ABSTRACT: BACKGROUND: Acupuncture stimulation elicits deqi, a composite of unique sensations that is essential for clinical efficacy according to traditional Chinese medicine (TCM). There is lack of adequate experimental data to indicate what sensations comprise deqi, their prevalence and intensity, their relationship to acupoints, how they compare with conventional somatosensory or noxious response. The objective of this study is to provide scientific evidence on these issues and to characterize the nature of the deqi phenomenon in terms of the prevalence of sensations as well as the uniqueness of the sensations underlying the deqi experience. METHODS: Manual acupuncture was performed at LI4, ST36 and LV3 on the extremities in randomized order during fMRI in 42 acupuncture naive healthy adult volunteers. Non-invasive tactile stimulation was delivered to the acupoints by gentle tapping with a von Frey monofilament prior to acupuncture to serve as a sensory control. At the end of each procedure, the subject was asked if each of the sensations listed in a questionnaire or any other sensations occurred during stimulation, and if present to rate its intensity on a numerical scale of 1-10. Statistical analysis including paired t-test, analysis of variance, Spearman's correlation and Fisher's exact test were performed to compare responses between acupuncture and sensory stimulation. RESULTS: The deqi response was elicited in 71% of the acupuncture procedures compared with 24% for tactile stimulation when thresholded at a minimum total score of 3 for all the sensations. The frequency and intensity of individual sensations were significantly higher in acupuncture. Among the sensations typically associated with deqi, aching, soreness and pressure were most common, followed by tingling, numbness, dull pain, heaviness, warmth, fullness and coolness. Sharp pain of brief duration that occurred in occasional subjects was regarded as inadvertent noxious stimulation. The most significant differences in the deqi sensations between acupuncture and tactile stimulation control were observed with aching, soreness, pressure and dull pain. Consistent with its prominent role in TCM, LI4 showed the most prominent response, the largest number of sensations as well as the most marked difference in the frequency and intensity of aching, soreness and dull pain between acupuncture and tactile stimulation control. Interestingly, the dull pain generally preceded or occurred in the absence of sharp pain in contrast to reports in the pain literature. An approach to summarize a sensation profile, called the deqi composite, is proposed and applied to explain differences in deqi among acupoints. CONCLUSION: The complex pattern of sensations in the deqi response suggests involvement of a wide spectrum of myelinated and unmyelinated nerve fibers, particularly the slower conducting fibers in the tendinomuscular layers. The study provides scientific data on the characteristics of the 'deqi' response in acupuncture and its association with distinct nerve fibers. The findings are clinically relevant and consistent with modern concepts in neurophysiology. They can provide a foundation for future studies on the deqi phenomenon. [Abstract/Link to Full Text]

Talcott JA, Clark JA, Lee IP
Measuring Perceived Effects of Drinking an Extract of Basidiomycetes Agaricus blazei Murill: A Survey of Japanese Consumers with Cancer.
BMC Complement Altern Med. 2007 Oct 29;7(1):32.
ABSTRACT: BACKGROUND: To survey cancer patients who consume an extract of the Basidiomycetes Agaricus blazei Murill mushroom (Sen-Sei-Ro) to measure their self-assessment of its effects and to develop an instrument for use in future randomized trials. METHODS: We designed, translated and mailed a survey to 2,346 Japanese consumers of Sen-Sei-Ro self-designated as cancer patients. The survey assessed consumer demographics, cancer history, Sen-Sei-Ro consumption, and its perceived effects. We performed exploratory psychometric analyses to identify distinct, multi-item scales that could summarize perceptions of effects. RESULTS: We received completed questionnaires from 782 (33%) of the sampled Sen-Sei-Ro consumers with a cancer history. Respondents represented a broad range of cancer patients familiar with Sen-Sei-Ro. Nearly all had begun consumption after their cancer diagnosis. These consumers expressed consistently positive views, though not extremely so, with more benefit reported for more abstract benefits such as emotional and physical well-being than relief of specific symptoms. We identified two conceptually and empirically distinct and internally consistent summary scales measuring Sen-Sei-Ro consumers' perceptions of its effects, Relief of Symptoms and Functional Well-being (Cronbach's alpha: Relief of Symptoms, alpha =.74; Functional Well-Being, alpha =.91). CONCLUSIONS: Respondents to our survey of Sen-Sei-Ro consumers with cancer reported favorable perceived effects from its use. Our instrument, when further validated, may be a useful outcome in trials assessing this and other complementary and alternative medicine (CAM) substances in cancer patients. [Abstract/Link to Full Text]

Takakura N, Yajima H
A double-blind placebo needle for acupuncture research.
BMC Complement Altern Med. 2007 Oct 10;7(1):31.
ABSTRACT: BACKGROUND: Placebo needles that can mask acupuncture practitioners to the type of needle used have been considered almost impossible to develop until now. METHODS: We designed a double-blind non-penetrating placebo needle, the needle tip of which simply presses against the skin, and a matched penetrating needle. The needles are encased inside an opaque guide tube and the appearance and feel of the pair are designed to be indistinguishable. To validate the masking effect for the practitioner, 10 acupuncturists each applied 23 non-penetrating needles and 17 penetrating needles to the large intestine-4 point. After removing each needle, they judged whether the needle was penetrating, non-penetrating or unidentifiable. For the validation of patient masking, an acupuncturist randomly applied a non-penetrating/penetrating needle pair to the bilateral Sanjiao-5 points in 60 volunteers. When both applications were completed, we asked them to write down anything that they noticed regarding the needle application and associated sensations. RESULTS: The mean [SD] of correct/unidentifiable/incorrect answers given by the 10 acupuncturists were 17.0 [4.1]/6.4 [3.6]/16.6 [3.0], respectively. Regarding patient masking, none of the subjects commented in the questionnaire that they had received a non-penetrating needle. Of 60 penetrating and 60 non-penetrating needle applications, 48 (80.0%) and 25 (41.7%) applications elicited skin penetration sensation and 48 (80.0%) and 20 (33.3%) applications elicited de qi, respectively. CONCLUSIONS: These needles have the potential to mask both practitioners and patients from the type of needle used in acupuncture research. [Abstract/Link to Full Text]

Amira OC, Okubadejo NU
Frequency of complementary and alternative medicine utilization in hypertensive patients attending an urban tertiary care centre in Nigeria.
BMC Complement Altern Med. 2007;730.
BACKGROUND: To study the frequency and pattern of use of complementary and alternative medicine (CAM) in patients with essential hypertension attending a tertiary hypertension clinic. METHODS: Two hundred and twenty-five consecutive hypertensive patients attending the hypertension clinic of the Lagos University Teaching Hospital over a 3-month period were interviewed. Socio-demographic data, duration of hypertension, clinic attendance, current blood pressure, and compliance to conventional medications was documented. CAM utilization was explored using both structured and open-ended questions. RESULTS: There were 90 (40%) male and 135 (60%) female patients with mean age +/- SD overall was 55.1 +/- 12.4 years. 88 (39.1%) of the respondents used CAM. Herbal products were the most commonly used CAM type. Amongst the CAM users, the most common herbal product used was garlic (69.3%). Others were native herbs (25%), ginger (23.9%), bitter leaf (Vernonia amygdalina) (9.1%), and aloe vera (4.5%). 2.5% used spiritual therapy. There was no difference in the clinical characteristics, socio-economic status, and blood pressure control of CAM users and non-users. Patients who utilized CAM had higher BMI compared with those who did not, but the difference was not statistically significant (mean BMI +/- SD of 29.1 +/- 5.6 vs 27.1 +/- 5.9 kg/m2; P = 0.05). CONCLUSION: A significant proportion of hypertensive patients attending our tertiary facility and receiving conventional treatment also use CAM therapies. Clinicians need to be aware of this practice, understand the rationale for this health-seeking behaviour, proactively enquire about their use, and counsel patients regarding the potential of some of the therapies for adverse reactions and drug interactions. [Abstract/Link to Full Text]

Fernandes NP, Lagishetty CV, Panda VS, Naik SR
An experimental evaluation of the antidiabetic and antilipidemic properties of a standardized Momordica charantia fruit extract.
BMC Complement Altern Med. 2007;729.
BACKGROUND: The MCE, Momordica charantia fruit extract Linn. (Cucurbitaceae) have been documented to elicit hypoglycemic activity on various occasions. However, due to lack of standardization of these extracts, their efficacy remains questionable. The present study was undertaken by selecting a well standardised MCE. This study reports hypoglycemic and antilipidemic activities of MCE employing relevant animal models and in vitro methods. METHODS: Diabetes was induced in Wistar rats by a s.c., subcutaneous injection of alloxan monohydrate (100 mg/kg) in acetate buffer (pH 4.5). MCE and glibenclamide were administered orally to alloxan diabetic rats at doses of 150 mg/kg, 300 mg/kg & 600 mg/kg, and 4 mg/kg respectively for 30 days, blood was withdrawn for glucose determination on 0, 7, 14, 21 and 30th days. On the 31st day, overnight fasted rats were sacrificed and blood was collected for various biochemical estimations including glycosylated haemoglobin, mean blood glucose, serum insulin, cholesterol, triglcerides, protein and glycogen content of liver. The hemidiaphragms and livers were also isolated, carefully excised and placed immediately in ice cooled perfusion solution and processed to study the glucose uptake/transfer processes. Hypolipidemic activity in old obese rats was evaluated by treating two groups with MCE (150 mg/kg & 300 mg/kg) orally for 30 days and determining total cholesterol, triglyceride and HDL-CH, LDL-CH and VLDL-CH levels from serum samples. RESULTS: Subchronic study of MCE in alloxan induced diabetic rats showed significant antihyperglycemic activity by lowering blood glucose and GHb%, percent glycosylated haemoglobin. Pattern of glucose tolerance curve was also altered significantly. MCE treatment enhanced uptake of glucose by hemidiaphragm and inhibited glycogenolysis in liver slices in vitro. A significant reduction in the serum cholesterol and glyceride levels of obese rats following MCE treatment was also observed. CONCLUSION: Our experimental findings with respect to the mechanism of action of MCE in alloxan diabetic rats suggest that it enhances insulin secretion by the islets of Langerhans, reduces glycogenesis in liver tissue, enhances peripheral glucose utilisation and increases serum protein levels. Furthermore, MCE treatment restores the altered histological architecture of the islets of Langerhans. Hence, the biochemical, pharmacological and histopathological profiles of MCE clearly indicate its potential antidiabetic activity and other beneficial effects in amelioration of diabetes associated complications. Further, an evaluation of its antilipidemic activity in old obese rats demonstrated significant lowering of cholesterol and triglyceride levels while elevating HDL-cholesterol levels. Also, the extract lowered serum lipids in alloxan diabetic rats, suggesting its usefulness in controlling metabolic alterations associated with diabetes. [Abstract/Link to Full Text]

Ezeome ER, Anarado AN
Use of complementary and alternative medicine by cancer patients at the University of Nigeria Teaching Hospital, Enugu, Nigeria.
BMC Complement Altern Med. 2007;728.
BACKGROUND: The use of Complementary and Alternative Medicine (CAM) by cancer patients is very common and varies between populations. The referenced English literature has no local study from Africa on this subject. This study was conducted to define the prevalence, pattern of use, and factors influencing the use of CAM by cancer patients at the University of Nigeria Teaching Hospital Enugu (UNTH-E), Nigeria METHOD: Face-to-face interviews using semi-structured questionnaire were used to determine the use of CAM by cancer patients. All consenting cancer patients were interviewed as they presented at the core surgical units of the UNTH- E, from June 2003 to September 2005. RESULTS: 160 patients were interviewed; 68 (42.5%) were males and 94 (57.5%) were females. Ages ranged from 13-86 years. Breast, urogenital system, gastrointestinal system, and soft tissue cancers predominated. One hundred and four patients (65.0%) have used CAM at some time during their current cancer illness; 56 (35.0%) patients have not used any form of CAM. There were more females than males among the non-CAM users. The use of CAM was not affected by age, marital status, level of education, religious affiliation, or socioeconomic status. The most frequently used CAMs were herbs (51.9%), faith/prayer healing (49.4%), aloe vera (23.1%), Forever Living Products (16.3%), medicinal tea (14.4%), and Blackstone (12.5%). Over 23% of those who used CAM were satisfied, but 68.3% were disappointed. Most users (67.3%) did not see any benefit from the CAM, but 25% could describe some specific benefits. More than 21% of users reported various unwanted effects. While 86.5% of CAM users will use orthodox medicine instead of CAM in the future, 9.6% will use the two together to help each other. Most users (79.8%) will not repeat CAM or recommend its use for cancer. The majority of patients (55.8%) did not mention their use of CAM to their doctors - mostly because the doctor did not ask. CONCLUSION: CAM use is common among cancer patients in Nigeria. Most users do not obtain the expected benefits, and adverse events are not uncommon. Every clinician in the field of oncology should ask his/her patients about the use of CAM; this knowledge will enable them to better counsel the patients. [Abstract/Link to Full Text]

Li A, Zhang RX, Wang Y, Zhang H, Ren K, Berman BM, Tan M, Lao L
Corticosterone mediates electroacupuncture-produced anti-edema in a rat model of inflammation.
BMC Complement Altern Med. 2007;727.
BACKGROUND: Electroacupuncture (EA) has been reported to produce anti-edema and anti-hyperalgesia effects on inflammatory disease. However, the mechanisms are not clear. The present study investigated the biochemical mechanisms of EA anti-inflammation in a rat model. METHODS: Three experiments were conducted on male Sprague-Dawley rats (n = 7-8/per group). Inflammation was induced by injecting complete Freund's adjuvant (CFA) subcutaneously into the plantar surface of one hind paw. Experiment 1 measured plasma corticosterone (CORT) levels to see if EA regulates CORT secretion. Experiment 2 studied the effects of the adrenal gland on the therapeutic actions of EA using adrenalectomy (ADX) rats. Experiment 3 determined whether a prototypical glucocorticoid receptor antagonist, RU486, affects EA anti-edema. EA treatment, 10 Hz at 3 mA and 0.1 ms pulse width, was given twice, for 20 min each, once immediately after CFA administration and again 2 h post-CFA. Plasma CORT levels, paw thickness, indicative of the intensity of inflammation, and paw withdrawal latency (PWL) were measured 2 h and 5 h after the CFA injection. RESULTS: EA significantly increased plasma corticosterone levels 2 h (5 folds) and 5 h (10 folds) after CFA administration compared to sham EA control, but EA alone in naive rats and CFA alone did not induce significant increases in corticosterone. Adrenalectomy blocked EA-produced anti-edema, but not EA anti-hyperalgesia. RU486 (15 mul, 15 mug/mul), a prototypical glucocorticoid receptor antagonist, also prevented EA anti-edema. CONCLUSION: The data demonstrate that EA activates the adrenals to increase plasma corticosterone levels and suppress edema and suggest that EA effects differ in healthy subjects and in those with pathologies. [Abstract/Link to Full Text]

Nassiri-Asl M, Shariati-Rad S, Zamansoltani F
Anticonvulsant effects of aerial parts of Passiflora incarnata extract in mice: involvement of benzodiazepine and opioid receptors.
BMC Complement Altern Med. 2007;726.
BACKGROUND: Passion flower (Passiflora incarnata) is used in traditional medicine of Europe and South America to treat anxiety, insomnia and seizure. Recently, it has shown antianxiety and sedative effects in human. METHODS: In this study, anticonvulsant effects of hydro- alcoholic extract of Passiflora, Pasipay, were examined by using pentylentetrazole model (PTZ) on mice. Pasipay, diazepam, and normal saline were injected intraperitoneally at the doses 0.4-0.05 mg/kg, 0.5-1 mg/kg and 10 ml/kg respectively 30 minutes before PTZ (90 mg/kg, i.p). The time taken before the onset of clonic convulsions, the duration of colonic convulsions, and the percentage of seizure and mortality protection were recorded. For investigating the mechanism of Pasipay, flumazenil (2 mg/kg, i.p) and naloxone (5 mg/kg, i.p) were also injected 5 minutes before Pasipay. RESULTS: An ED50 value of Pasipay in the PTZ model was 0.23 mg/kg (%95 CL: 0.156, 0.342). Pasipay at the dose of 0.4 mg/kg prolonged the onset time of seizure and decreased the duration of seizures compared to saline group (p < 0.001). At the dose of 0.4 mg/kg, seizure and mortality protection percent were 100%. Flumazenil and naloxone could suppress anticonvulsant effects of Pasipay. CONCLUSION: It seems that Pasipay could be useful for treatment absence seizure and these effects may be related to effect of it on GABAergic and opioid systems. More studies are needed in order to investigate its exact mechanism. [Abstract/Link to Full Text]

Evans M, Shaw A, Thompson EA, Falk S, Turton P, Thompson T, Sharp D
Decisions to use complementary and alternative medicine (CAM) by male cancer patients: information-seeking roles and types of evidence used.
BMC Complement Altern Med. 2007;725.
BACKGROUND: Complementary and Alternative Medicine (CAM) is increasingly popular with cancer patients and yet information provision or discussion about CAM by health professionals remains low. Previous research suggests that patients may fear clinicians' 'disapproval' if they raise the subject of CAM, and turn to other sources to acquire information about CAM. However, little empirical research has been conducted into how cancer patients acquire, and, more importantly evaluate CAM information before deciding which CAM therapies to try. METHODS: Qualitative study, comprising semi-structured interviews with 43 male cancer patients of varying ages, cancer type and stage of illness, 34 of whom had used CAM. They were recruited from a range of NHS and non-NHS settings in Bristol, England. RESULTS: As a result of the lack of CAM information from health professionals, men in this study became either 'pro-active seekers' or 'passive recipients' of such information. Their main information resource was the 'lay referral' network of family, friends and acquaintances, especially females. 'Traditional' information sources, including books, magazines, leaflets and the media were popular, more so in fact than the internet. Views on the internet ranged from enthusiasm or healthy scepticism through to caution or disinterest. CAM information was generally regarded as 'empowering' as it broadened treatment and self-care options. A minority of participants were information averse fearing additional choices that might disrupt their fragile ability to cope. There was general consensus that CAM information should be available via the NHS, to give it a 'stamp of approval', which combined with guidance from informed health professionals, could help patients to make 'guided' choices. However, a small minority of these men valued the independence of CAM from the NHS and deliberately sought 'alternative' information sources and treatment options. Men were selective in identifying particular therapies to use and sceptical about others, basing their choices on forms of 'evidence' that were personally meaningful: personal stories of individuals who had been helped by CAM; the long history and enduring popularity of some therapies; the plausibility of the mechanism of action; a belief or trust in individual therapies or their providers; scientific evidence. Scientific evidence ranked low in the men's personal decision-making about CAM, while it was recognised as important for NHS support for CAM. CONCLUSION: These male cancer patients valued the support and guidance of 'trusted individuals' in making choices about CAM. Trusted health professionals could also play a significant role in helping patients to make informed choices. Any such dialogue must, however, acknowledge the different standards of evidence used by patients and clinicians to evaluate the benefits or otherwise of CAM therapies. Such open communication could help to foster an environment of mutual trust where patients are encouraged to discuss their interest in CAM, rather than perpetuate covert, undisclosed use of CAM with its attendant potential hazards. [Abstract/Link to Full Text]

Okoli CO, Akah PA, Okoli AS
Potentials of leaves of Aspilia africana (Compositae) in wound care: an experimental evaluation.
BMC Complement Altern Med. 2007;724.
BACKGROUND: The potentials of the leaves of the haemorrhage plant, Aspilia africana C. D Adams (Compositae) in wound care was evaluated using experimental models. A. africana, which is widespread in Africa, is used in traditional medicine to stop bleeding from wounds, clean the surfaces of sores, in the treatment of rheumatic pains, bee and scorpion stings and for removal of opacities and foreign bodies from the eyes. The present study was undertaken to evaluate the potentials for use of leaves of this plant in wound care. METHODS: The effect of the methanol extract (ME) and the hexane (HF) and methanol (MF) fractions (obtained by cold maceration and graded solvent extraction respectively) on bleeding/clotting time of fresh experimentally-induced wounds in rats, coagulation time of whole rat blood, growth of microbial wound contaminants and rate of healing of experimentally-induced wounds in rats were studied as well as the acute toxicity and lethality (LD50) of the methanol extract and phytochemical analysis of the extract and fractions. RESULTS: The extract and fractions significantly (P < 0.05) reduced bleeding/clotting time in rats and decreased coagulation time of whole rat blood in order of magnitude of effect: MF>ME>HF. Also, the extract and fractions caused varying degrees of inhibition of the growth of clinical isolates of Pseudomonas fluorescens and Staphylococcus aureus, as well as typed strains of Ps. aeruginosa (ATCC 10145) and Staph. aureus (ATCC 12600), and reduced epithelialisation period of wounds experimentally-induced in rats. Acute toxicity and lethality (LD50) test in mice established an i.p LD50 of 894 mg/kg for the methanol extract (ME). Phytochemical analysis revealed the presence of alkaloids, saponins, tannins, flavonoids, resins, sterols, terpenoids and carbohydrates. CONCLUSION: The leaves of A. africana possess constituents capable of arresting wound bleeding, inhibiting the growth of microbial wound contaminants and accelerating wound healing which suggest good potentials for use in wound care. [Abstract/Link to Full Text]

Hosseinzadeh H, Hosseini A, Nassiri-Asl M, Sadeghnia HR
Effect of Salvia leriifolia Benth. root extracts on ischemia-reperfusion in rat skeletal muscle.
BMC Complement Altern Med. 2007;723.
BACKGROUND: Salvia leriifolia have been shown to decrease ischemia-reperfusion (I/R) injury in brain tissues. In this study, the effects of S. leriifolia aqueous and ethanolic extracts were evaluated on an animal model of I/R injury in the rat hind limb. METHODS: Ischemia was induced using free-flap surgery in skeletal muscle. The aqueous and ethanolic extracts of S. leriifolia (100, 200 and 400 mg/kg) root and normal saline (10 ml/kg) were administered intraperitoneally 1 h prior reperfusion. During preischemia, ischemia and reperfusion conditions the electromyographic (EMG) potentials in the muscles were recorded. The markers of oxidative stress including thiobarbituric acid reactive substances (TBARS), total sulfhydryl (SH) groups and antioxidant capacity of muscle (using FRAP assay) were measured. RESULTS: In peripheral ischemia, the average peak-to-peak amplitude during ischemic-reperfusion was found to be significantly larger in extracts groups in comparison with control group. Following extracts administration, the total SH contents and antioxidant capacity were elevated in muscle flap. The MDA level was also declined significantly in test groups. CONCLUSION: It is concluded that S. leriifolia root extracts have some protective effects on different markers of oxidative damage in muscle tissue injury caused by lower limb ischemia-reperfusion. [Abstract/Link to Full Text]

King M, Chatelain K, Farris D, Jensen D, Pickup J, Swapp A, O'Malley S, Kingsley K
Oral squamous cell carcinoma proliferative phenotype is modulated by proanthocyanidins: a potential prevention and treatment alternative for oral cancer.
BMC Complement Altern Med. 2007;722.
BACKGROUND: Despite the recently reported drop in the overall death rate from cancer, the estimated survival rate and number of deaths from oral cancer remain virtually unchanged. Early detection efforts, in combination with strategies for prevention and risk-reduction, have the potential to dramatically improve clinical outcomes. The identification of non-toxic, effective treatments, including complementary and alternative therapies, is critical if the survival rate is to be improved. Epidemiologic studies have suggested a protective effect from certain plant-derived foods and extracts; however, it has been difficult to isolate and identify the compounds most responsible for these observations. The primary purpose of this study was to investigate the response of human oral squamous cell carcinoma (OSCC) to proanthocyanidin (PAC), a plant-derived compound that may inhibit the progression of several other cancers. METHODS: Using a series of in vitro assays, we sought to quantify the effects of PAC on OSCC, cervical carcinoma, and non-cancerous cell lines, specifically the effects of PAC on cell proliferation. Recent data suggest that infection with the human papillomavirus (HPV) may also modulate the proliferative potential of OSCC; therefore, we also measured the effects of PAC administration on HPV-transfected OSCC proliferation. RESULTS: Our results demonstrated that PAC administration was sufficient to significantly suppress cellular proliferation of OSCC in a dose-dependent manner. In addition, the increased proliferation of OSCC after transfection with HPV 16 was reduced by the administration of PAC, as was the proliferation of the cervical cancer and non-cancerous cell lines tested. Our results also provide preliminary evidence that PAC administration may induce apoptosis in cervical and oral cancer cell lines, while acting merely to suppress proliferation of the normal cell line control. CONCLUSION: These results signify that PAC may be a compelling candidate for testing in both animal and human models. Furthermore, these data provide adequate justification for elucidating the divergent mechanisms of PAC-induced proliferation, inhibition, and apoptosis among these and other cell lines. [Abstract/Link to Full Text]

Kemper KJ, Gardiner P, Woods C
Changes in use of herbs and dietary supplements (HDS) among clinicians enrolled in an online curriculum.
BMC Complement Altern Med. 2007;721.
BACKGROUND: Little is known about clinicians' use of herbs and dietary supplements (HDS), how their personal HDS use changes with time and training, and how changes in their personal use affect their confidence or communication with patients about HDS. METHODS: We conducted a prospective cohort study of clinicians before and after an on-line curriculum about HDS in winter-spring, 2005. RESULTS: Of the 569 clinicians who completed surveys both at baseline and after the course, 25% were male and the average age was 42 years old; 88% used HDS before and after the course. The average number of supplements used fell slightly from 6.2 at baseline to 5.8 after the course (P < 0.01). The most commonly used supplements at baseline were: multivitamins (65%), calcium (42%), B vitamins (34%), vitamin C (34%), green tea (27%), fish oil (27%) and vitamin E (25%). Use of fish oil increased to 30% after the course (P = 0.01). Use of supplements traditionally used to treat colds decreased: vitamin C (34% to 27%), zinc (13% to 10%), and echinacea (7% to 5%, P < 0.05 for all three). Changes in personal HDS use were not associated with significant changes in confidence or communication with patients. CONCLUSION: Many clinicians use HDS personally; use changes seasonally and to a small extent with professional education. Professional use of HDS is dynamic and seasonal. Additional research is needed to understand the impact of personal use on professional attitudes and behavior in populations with lower baseline uses of HDS. [Abstract/Link to Full Text]

Lawson BR, Belkowski SM, Whitesides JF, Davis P, Lawson JW
Immunomodulation of murine collagen-induced arthritis by N, N-dimethylglycine and a preparation of Perna canaliculus.
BMC Complement Altern Med. 2007;720.
BACKGROUND: Rheumatoid arthritis (RA) and its accepted animal model, murine collagen-induced arthritis (CIA), are classic autoimmune inflammatory diseases which require proinflammatory cytokine production for pathogenesis. We and others have previously used N, N-dimethylglycine (DMG) and extracts from the New Zealand green-lipped mussel Perna canaliculus (Perna) as potent immunomodulators to modify ongoing immune and/or inflammatory responses. METHODS: In our initial studies, we treated lipopolysaccahride (LPS) stimulated THP-1 monocytes in vitro with increasing concentrations of Perna extract or DMG. Additionally, we treated rat peripheral blood neutrophils with increasing concentrations of Perna extract and measured superoxide burst. In subsequent in vivo experiments, CIA was induced by administration of type II collagen; rats were prophylactically treated with either Perna or DMG, and then followed for disease severity. Finally, to test whether Perna and/or DMG could block or inhibit an ongoing pathologic disease process, we induced CIA in mice and treated them therapeutically with either of the two immunomodulators. RESULTS: Following LPS stimulation of THP-1 monocytes, we observed dose-dependent reductions in TNF-alpha and IL-12p40 production in Perna treated cultures. DMG treatment, however, showed significant increases in both of these cytokines in the range of 0.001-1 microM. We also demonstrate that in vitro neutrophil superoxide burst activity is dose-dependently reduced in the presence of Perna. Significant reductions in disease incidence, onset, and severity of CIA in rats were noted following prophylactic treatment with either of the two immunomodulators. More importantly, amelioration of mouse CIA was observed following therapeutic administration of Perna. In contrast, DMG appeared to have little effect in mice and may act in a species-specific manner. CONCLUSION: These data suggest that Perna, and perhaps DMG, may be useful supplements to the treatment of RA in humans. [Abstract/Link to Full Text]

Penza M, Montani C, Jeremic M, Mazzoleni G, Hsiao WL, Marra M, Sharma H, Di Lorenzo D
MAK-4 and -5 supplemented diet inhibits liver carcinogenesis in mice.
BMC Complement Altern Med. 2007;719.
BACKGROUND: Maharishi Amrit Kalash (MAK) is an herbal formulation composed of two herbal mixtures, MAK-4 and MAK-5. These preparations are part of a natural health care system from India, known as Maharishi Ayur-Veda. MAK-4 and MAK-5 are each composed of different herbs and are said to have maximum benefit when used in combination. This investigation evaluated the cancer inhibiting effects of MAK-4 and MAK-5, in vitro and in vivo. METHODS: In vitro assays: Aqueous extracts of MAK-4 and MAK-5 were tested for effects on ras induced cell transformation in the Rat 6 cell line assessed by focus formation assay. In vivo assays: Urethane-treated mice were put on a standard pellet diet or a diet supplemented with MAK-4, MAK-5 or both. At 36 weeks, livers were examined for tumors, sera for oxygen radical absorbance capacity (ORAC), and liver homogenates for enzyme activities of glutathione peroxidase (GPX), glutathione-S-transferase (GST), and NAD(P)H: quinone reductase (QR). Liver fragments of MAK-fed mice were analyzed for connexin (cx) protein expression. RESULTS: MAK-5 and a combination of MAK-5 plus MAK-4, inhibited ras-induced cell transformation. In MAK-4, MAK-5 and MAK4+5-treated mice we observed a 35%, 27% and 46% reduction in the development of urethane-induced liver nodules respectively. MAK-4 and MAK4+5-treated mice had a significantly higher ORAC value (P < 0.05) compared to controls (200.2 +/- 33.7 and 191.6 +/- 32.2 vs. 152.2 +/- 15.7 ORAC units, respectively). The urethane-treated MAK-4, MAK-5 and MAK4+5-fed mice had significantly higher activities of liver cytosolic enzymes compared to the urethane-treated controls and to untreated mice: GPX(0.23 +/- 0.08, 0.21 +/- 0.05, 0.25 +/- 0.04, 0.20 +/- 0.05, 0.21 +/- 0.03 U/mg protein, respectively), GST (2.0 +/- 0.4, 2.0 +/- 0.6, 2.1 +/- 0.3, 1.7 +/- 0.2, 1.7 +/- 0.2 U/mg protein, respectively) and QR (0.13 +/- 0.02, 0.12 +/- 0.06, 0.15 +/- 0.03, 0.1 +/- 0.04, 0.11 +/- 0.03 U/mg protein, respectively). Livers of MAK-treated mice showed a time-dependent increased expression of cx32. CONCLUSION: Our results show that a MAK-supplemented diet inhibits liver carcinogenesis in urethane-treated mice. The prevention of excessive oxidative damage and the up-regulation of connexin expression are two of the possible effects of these products. [Abstract/Link to Full Text]

Sawni A, Thomas R
Pediatricians' attitudes, experience and referral patterns regarding Complementary/Alternative Medicine: a national survey.
BMC Complement Altern Med. 2007;718.
BACKGROUND: To assess pediatricians' attitudes toward & practice of Complementary/Alternative Medicine (CAM) including their knowledge, experience, & referral patterns for CAM therapies. METHODS: An anonymous, self-report, 27-item questionnaire was mailed nationally to fellows of the American Academy of Pediatrics in July 2004.648 of 3500 pediatricians' surveyed responded (18%). RESULTS: The median age ranged from 46-59 yrs; 52% female, 81% Caucasian, 71% generalists, & 85% trained in the US. Over 96% of pediatricians' responding believed their patients were using CAM. Discussions of CAM use were initiated by the family (70%) & only 37% of pediatricians asked about CAM use as part of routine medical history. Majority (84%) said more CME courses should be offered on CAM and 71% said they would consider referring patients to CAM practitioners. Medical conditions referred for CAM included; chronic problems (headaches, pain management, asthma, backaches) (86%), diseases with no known cure (55.5%) or failure of conventional therapies (56%), behavioral problems (49%), & psychiatric disorders (47%). American born, US medical school graduates, general pediatricians, & pediatricians who ask/talk about CAM were most likely to believe their patients used CAM (P < 0.01). CONCLUSION: Pediatricians' have a positive attitude towards CAM. Majority believe that their patients are using CAM, that asking about CAM should be part of routine medical history, would consider referring to a CAM practitioner and want more education on CAM. [Abstract/Link to Full Text]

Maha N, Shaw A
Academic doctors' views of complementary and alternative medicine (CAM) and its role within the NHS: an exploratory qualitative study.
BMC Complement Altern Med. 2007;717.
BACKGROUND: There has been a marked increase in the use of complementary and alternative medicine (CAM) in the UK population in recent years. Surveys of doctors' perspectives on CAM have identified a variety of views and potential information needs. While these are useful for describing the proportions of doctors who hold particular attitudes towards CAM, they are less helpful for understanding why. In addition, while the views of non-academic doctors have begun to be studied, the perspective and rationales of academic doctors remains under-researched. It seems important to investigate the views of those with a research-orientation, given the emphasis on the need for more scientific evidence in recent debates on CAM. METHODS: This exploratory study used qualitative methods to explore academic doctors' views of CAM and the rationales they provided for their views. A purposeful sampling strategy was used to identify doctors with a dual clinical and academic role in the Bristol area, with an anticipated variety of views on CAM. Semi-structured interviews were conducted with nine doctors. The data were analysed thematically, drawing on the Framework Approach. RESULTS: The doctors expressed a spectrum of views on CAM, falling into three broad groups: the 'enthusiasts', the 'sceptics' and the 'undecided'. Scepticism or uncertainty about the value of CAM was prominent, except among those practising a form of CAM. A variety of rationales underpinned their perspectives on CAM, a key recurring rationale being their perspective on the scientific evidence base. The main themes arising included: the role of doctors' professional experiences of conventional medicine and CAM in shaping their attitudes towards CAM, doctor-patient communication about CAM and patient disclosure, whether there is a need for training and education in CAM for doctors, a hierarchy of acceptability of CAM and the nature of evidence; and the role of CAM within the NHS. CONCLUSION: Despite the caution or scepticism towards CAM expressed by doctors in this study, more open doctor-patient communication about CAM may enable doctors' potential concerns about CAM to be addressed, or at least enhance their knowledge of what treatments or therapies their patients are using. Offering CAM to patients may serve to enhance patients' treatment choices and even increase doctors' fulfilment in their practice. However, given the recurring concerns about lack of scientific evidence expressed by the doctors in this study, perceptions of the evidence base may remain a significant barrier to greater integration of CAM within the NHS. [Abstract/Link to Full Text]

Smith TC, Ryan MA, Smith B, Reed RJ, Riddle JR, Gumbs GR, Gray GC
Complementary and alternative medicine use among US Navy and Marine Corps personnel.
BMC Complement Altern Med. 2007;716.
BACKGROUND: Recently, numerous studies have revealed an increase in complementary and alternative medicine (CAM) use in US civilian populations. In contrast, few studies have examined CAM use within military populations, which have ready access to conventional medicine. Currently, the prevalence and impact of CAM use in US military populations remains unknown. METHODS: To investigate CAM use in US Navy and Marine Corps personnel, the authors surveyed a stratified random sample of 5,000 active duty and Reserve/National Guard members between December 2000 and July 2002. Chi-square tests and multivariable logistic regression were used to assess univariate associations and adjusted odds of CAM use in this population. RESULTS AND DISCUSSION: Of 3,683 service members contacted, 1,446 (39.3%) returned a questionnaire and 1,305 gave complete demographic and survey data suitable for study. Among respondents, more than 37% reported using at least one CAM therapy during the past year. Herbal therapies were among the most commonly reported (15.9%). Most respondents (69.8%) reported their health as being very good or excellent. Modeling revealed that CAM use was most common among personnel who were women, white, and officers. Higher levels of recent physical pain and lower levels of satisfaction with conventional medical care were significantly associated with increased odds of reporting CAM use. CONCLUSION: These data suggest that CAM use is prevalent in the US military and consistent with patterns in other US civilian populations. Because there is much to be learned about CAM use along with allopathic therapy, US military medical professionals should record CAM therapies when collecting medical history data. [Abstract/Link to Full Text]

Fleming S, Rabago DP, Mundt MP, Fleming MF
CAM therapies among primary care patients using opioid therapy for chronic pain.
BMC Complement Altern Med. 2007;715.
BACKGROUND: Complementary and alternative medicine (CAM) is an increasingly common therapy used to treat chronic pain syndromes. However; there is limited information on the utilization and efficacy of CAM therapy in primary care patients receiving long-term opioid therapy. METHOD: A survey of CAM therapy was conducted with a systematic sample of 908 primary care patients receiving opioids as a primary treatment method for chronic pain. Subjects completed a questionnaire designed to assess utilization, efficacy and costs of CAM therapies in this population. RESULTS: Patients were treated for a variety of pain problems including low back pain (38.4%), headaches (9.9%), and knee pain (6.5%); the average duration of pain was 16 years. The median morphine equivalent opioid dose was 41 mg/day, and the mean dose was 92 mg/day. Forty-four percent of the sample reported CAM therapy use in the past 12 months. Therapies utilized included massage therapy (27.3%, n = 248), chiropractic treatment (17.8%, n = 162), acupuncture (7.6%, n = 69), yoga (6.1%, n = 55), herbs and supplements (6.8%, n = 62), and prolotherapy (5.9%, n = 54). CAM utilization was significantly related to age female gender, pain severity income pain diagnosis of neck and upper back pain, and illicit drug use. Medical insurance covered chiropractic treatment (81.8%) and prolotherapy (87.7%), whereas patients primarily paid for other CAM therapies. Over half the sample reported that one or more of the CAM therapies were helpful. CONCLUSION: This study suggests CAM therapy is widely used by patients receiving opioids for chronic pain. Whether opioids can be reduced by introducing such therapies remains to be studied. [Abstract/Link to Full Text]

Eggenschwiler J, von Balthazar L, Stritt B, Pruntsch D, Ramos M, Urech K, Rist L, Sim�es-W�st AP, Viviani A
Mistletoe lectin is not the only cytotoxic component in fermented preparations of Viscum album from white fir (Abies pectinata).
BMC Complement Altern Med. 2007;714.
BACKGROUND: Preparations of mistletoe (Viscum album) are the form of cancer treatment that is most frequently used in the complementary medicine. Previous work has shown that these preparations are able to exert cytotoxic effects on carcinoma cells, the extent of which might be influenced by the host tree species and by the content of mistletoe lectin. METHODS: Using colorimetric assays, we have now compared the cytotoxic effects of Viscum album preparations (VAPs) obtained from mistletoe growing on oak (Quercus robur and Q. petraea, VAP-Qu), apple tree (Malus domestica,, VAP-M), pine (Pinus sylvestris, VAP-P) or white fir (Abies pectinata, VAP-A), on the in vitro growth of breast and bladder carcinoma cell lines. While MFM-223, KPL-1, MCF-7 and HCC-1937 were the breast carcinoma cell lines chosen, the panel of tested bladder carcinoma cells comprised the T-24, TCC-SUP, UM-UC-3 and J-82 cell lines. RESULTS: Each of the VAPs inhibited cell growth, but the extent of this inhibition differed with the preparation and with the cell line. The concentrations of VAP-Qu, VAP-M and VAP-A which led to a 50 % reduction of cell growth (IC50) varied between 0.6 and 0.03 mg/ml. Higher concentrations of VAP-P were required to obtain a comparable effect. Purified mistletoe lectin I (MLI) led to an inhibition of breast carcinoma cell growth at concentrations lower than those of VAPs, but the sensitivity towards purified MLI did not parallel that towards VAPs. Bladder carcinoma cells were in most cases more sensitive to VAPs treatment than breast carcinoma cells. The total mistletoe lectin content was very high in VAP-Qu (54 ng/mg extract), intermediate in VAP-M (25 ng/mg extract), and very low in VAP-P (1.3 ng/mg extract) and in VAP-A (1 ng/mg extract). As to be expected from the low content of mistletoe lectin, VAP-P led to relatively weak cytotoxic effects. Most remarkably, however, the lectin-poor VAP-A revealed a cytotoxic effect comparable to, or even stronger than, that of the lectin-rich VAP-Qu, on all tested bladder and breast carcinoma cell lines. CONCLUSION: The results suggest the existence of cytotoxic components other than mistletoe lectin in VAP-A and reveal an unexpected potential of this preparation for the treatment of breast and bladder cancer. [Abstract/Link to Full Text]

Nilforoushzadeh MA, Jaffary F, Moradi S, Derakhshan R, Haftbaradaran E
Effect of topical honey application along with intralesional injection of glucantime in the treatment of cutaneous leishmaniasis.
BMC Complement Altern Med. 2007;713.
BACKGROUND: Leishmaniasis is an endemic disease in Iran. Although many treatments have been suggested for this disease, there hasn't been an effective and safe treatment yet. Regarding the healing effect of honey in the chronic ulcers and its reported therapeutic effect in cutaneous leishmaniasis, we performed a study to better evaluate the efficacy of honey in cutaneous leishmaniasis and its final scar. METHODS: In a prospective clinical trial, 100 patients with confirmed cutaneous leishmaniasis were selected and randomized into 2 groups. Group A were treated with topical honey twice daily along with intralesional injection of glucantime once weekly until complete healing of the ulcer or for maximum of 6 weeks. Group B were treated with intralesional injection of glucantime alone until complete healing of the ulcer or for a maximum of 6 weeks, too. The patients were followed for 4 months. The collected data were analyzed statistically using statistical tests including Chi-square, Mann Whitney and Kaplan-Mayer tests. RESULTS: In this study, 45 patients that had cutaneous leishmaniasis were treated with intralesional glucantime alone and 45 patients were treated with topical honey and glucantime. Ten patients left out the study. In the glucantime alone treated group, 32 patients (71.1%) had complete cure whereas in the group treated with both glucantime & topical honey, 23 patients (51.1%) achieved complete cure. This difference was significant statistically (p = 0.04). CONCLUSION: Further studies to better clarify the efficacy of honey in cutaneous leishmaniasis is needed. We suggest that in another study, the efficacy of honey with standardized level of antibacterial activity is evaluated against cutaneous leishmaniasis. [Abstract/Link to Full Text]

Jones JF, Maloney EM, Boneva RS, Jones AB, Reeves WC
Complementary and alternative medical therapy utilization by people with chronic fatiguing illnesses in the United States.
BMC Complement Altern Med. 2007;712.
BACKGROUND: Chronic fatiguing illnesses, including chronic fatigue syndrome (CFS), pose a diagnostic and therapeutic challenge. Previous clinical reports addressed the utilization of health care provided to patients with CFS by a variety of practitioners with other than allopathic training, but did not examine the spectrum of complementary and alternative medicine (CAM) therapies used. This study was designed to measure CAM therapy use by persons with fatiguing illnesses in the United States population. METHODS: During a random-digit dialing survey to estimate the prevalence of CFS-like illness in urban and rural populations from different geographic regions of the United States, we queried the utilization of CAM including manipulation or body-based therapies, alternative medical systems, mind-body, biologically-based, and energy modalities. RESULTS: Four hundred forty fatigued and 444 non-fatigued persons from 2,728 households completed screening. Fatigued subjects included 53 persons with prolonged fatigue, 338 with chronic fatigue, and 49 with CFS-like illness. Mind-body therapy (primarily personal prayer and prayer by others) was the most frequently used CAM across all groups. Among women, there was a significant trend of increasing overall CAM use across all subgroups (p-trend = 0.003). All categories of CAM use were associated with significantly poorer physical health scores, and all but one (alternative medicine systems) were associated with significantly poorer mental health scores. People with CFS-like illness were significantly more likely to use body-based therapy (chiropractic and massage) than non-fatigued participants (OR = 2.52, CI = 1.32, 4.82). Use of body-based therapies increased significantly in a linear trend across subgroups of non-fatigued, prolonged fatigued, chronic fatigued, and CFS-like subjects (p-trend = 0.002). People with chronic fatigue were also significantly more likely to use body-based therapy (OR = 1.52, CI = 1.07, 2.16) and mind-body (excluding prayer) therapy than non-fatigued participants (OR = 1.73, CI = 1.20 - 2.48). CONCLUSION: Utilization of CAM was common in fatiguing illnesses, and was largely accounted for by the presence of underlying conditions and poor physical and mental health. Compared to non-fatigued persons, those with CFS-like illness or chronic fatigue were most likely to use body-based and mind-body therapies. These observations have important implications for provider education programs and development of intervention strategies for CFS. [Abstract/Link to Full Text]

Sinha M, Manna P, Sil PC
Amelioration of galactosamine-induced nephrotoxicity by a protein isolated from the leaves of the herb, Cajanus indicus L.
BMC Complement Altern Med. 2007;711.
BACKGROUND: Galactosamine (GalN), an established experimental toxin, mainly causes liver injury via the generation of free radicals and depletion of UTP nucleotides. Renal failure is often associated with end stage liver damage. GalN intoxication also induces renal dysfunction in connection with hepatic disorders. Present study was designed to find out the effect of a protein isolated from the leaves of the herb Cajanus indicus against GalN induced renal damage. METHODS: Both preventive as well as curative effect of the protein was investigated in the study. GalN was administered intraperitoneally at a dose of 800 mg/kg body weight for 3 days pre and post to protein treatment at an intraperitoneal dose of 2 mg/kg body weight for 4 days. The activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione-S-transferase (GST), levels of cellular metabolites, reduced glutathione (GSH), total thiols, oxidized glutathione (GSSG) and lipid peroxidation end products were determined to estimate the status of the antioxidative defense system. In addition, serum creatinine and urea nitrogen (UN) levels were also measured as a marker of nephrotoxicity. RESULTS: Results showed that GalN treatment significantly increased the serum creatinine and UN levels compared to the normal group of mice. The extent of lipid peroxidation and the level of GSSG were also enhanced by the GalN intoxication whereas the activities of antioxidant enzymes SOD, CAT, GR and GST as well as the levels of total thiols and GSH were decreased in the kidney tissue homogenates. Protein treatment both prior and post to the toxin administration successfully altered the effects in the experimental mice. CONCLUSION: Our study revealed that GalN caused a severe oxidative insult in the kidney. Protein treatment both pre and post to the GalN intoxication could protect the kidney tissue against GalN induced oxidative stress. As GalN induced severe hepatotoxicity followed by renal failure, the protective role of the protein against GalN induced renal damages is likely to be an indirect effect. Since the protein possess hepatoprotective activity, it may first ameliorate GalN-induced liver damage and consequently the renal disorders are reduced. To the best of our knowledge, this is probably the first report describing GalN-induced oxidative stress in renal damages and the protective role of a plant protein molecule against it. [Abstract/Link to Full Text]

Recent Articles in Evidence-based Complementary and Alternative Medicine

Rocca G, Dioni F, Rocca N, Oliveri F, Brunetto MR, Bonino F
Thermal Care of Functional Dyspepsia Based on Bicarbonate-Sulphate-Calcium Water: A Sequential Clinical Trial.
Evid Based Complement Alternat Med. 2007 Sep;4(3):381-391.
Drug treatment of functional dyspepsia is often unsatisfactory. We assessed the efficacy of a bicarbonate-sulphate-calcium thermal water cycle of 12 days, in patients with functional dyspepsia. Patients with functional dyspepsia were sent by their general practitioners to 12 days of treatment with thermal water, 200-400 ml in the morning, at temperature of 33 degrees C (91.4 F) and were evaluated on a strict intention to treat basis. Four efficacy endpoints were analyzed as follows: (i) reduction of the global symptoms score, (ii) reduction of intensity to a level not interfering with everyday activities, (iii) specific efficacy on ulcer-like or dysmotility-like dyspepsia and (iv) esophageal or abdominal-associated symptoms. Statistical significance was reached for all three primary outcomes after the first 29 consecutive patients. Thermal water reduced the global symptom score, reduced intensity of symptoms to a level not interfering with everyday activity, but was unable to completely suppress all symptoms. A parallel effect emerged for ulcer-like and dyspepsia-like subgroups. The effect on heartburn and abdominal symptoms was not significant, suggesting a specific effect of the water on the gastric and duodenal wall. The Roma II criteria identify a natural kind of dyspepsia that improves with thermal water. Ulcer-like and dysmotility-like are not therapeutically distinguishable subgroups. Patients with dominant esophageal or abdominal symptoms should receive a different therapy. Sequential methods are very effective for the evaluation of traditional care practices and should be considered preliminary and integrative to randomized controlled trials in this context. [Abstract/Link to Full Text]

Seely D, Singh R
Adaptogenic potential of a polyherbal natural health product: report on a longitudinal clinical trial.
Evid Based Complement Alternat Med. 2007 Sep;4(3):375-80.
Stress is a risk factor for a number of diseases and is an important predictor of health in general. Herbal medicines have been used as adaptogens to regulate and improve the stress response and there is evidence to support the use of herbal medicines for this purpose. We conducted an open-label longitudinal study on the natural health product, OCTA(c), a compound mixture of eight herbs, to determine its effects on perceptions of stress. Eighteen participants were enrolled in the study and were followed over a period of 3 months. Primary endpoints included scores from four validated questionnaires (SF-36v2, PSS, STAI and BDI-II), serum DHEA, ALT, AST and creatinine all measured at 12 weeks. Seventeen patients completed the study. Except for the physical summary score of the SF36 questionnaire, all the subjective scores indicated a highly significant (P < 0.0001) improvement in the participants' ability to cope with stress. No adverse effects were reported and there was no evidence of damage to the liver or kidney based on serum markers. Initial evidence for this polyherbal compound supports its potential as an effective 'adaptogenic' aid in dealing with stress. Further research using a randomized controlled design is necessary to confirm the findings from this pilot study. [Abstract/Link to Full Text]

Tsao JC, Meldrum M, Kim SC, Jacob MC, Zeltzer LK
Treatment Preferences for CAM in Children with Chronic Pain.
Evid Based Complement Alternat Med. 2007 Sep;4(3):367-74.
CAM therapies have become increasingly popular in pediatric populations. Yet, little is known about children's preferences for CAM. This study examined treatment preferences in chronic pediatric pain patients offered a choice of CAM therapies for their pain. Participants were 129 children (94 girls) (mean age = 14.5 years +/- 2.4; range = 8-18 years) presenting at a multidisciplinary, tertiary clinic specializing in pediatric chronic pain. Bivariate and multivariate analyses were used to examine the relationships between CAM treatment preferences and patient's sociodemographic and clinical characteristics, as well as their self-reported level of functioning. Over 60% of patients elected to try at least one CAM approach for pain. The most popular CAM therapies were biofeedback, yoga and hypnosis; the least popular were art therapy and energy healing, with craniosacral, acupuncture and massage being intermediate. Patients with a diagnosis of fibromyalgia (80%) were the most likely to try CAM versus those with other pain diagnoses. In multivariate analyses, pain duration emerged as a significant predictor of CAM preferences. For mind-based approaches (i.e. hypnosis, biofeedback and art therapy), pain duration and limitations in family activities were both significant predictors. When given a choice of CAM therapies, this sample of children with chronic pain, irrespective of pain diagnosis, preferred non-invasive approaches that enhanced relaxation and increased somatic control. Longer duration of pain and greater impairment in functioning, particularly during family activities increased the likelihood that such patients agreed to engage in CAM treatments, especially those that were categorized as mind-based modalities. [Abstract/Link to Full Text]

Tiemann P, Toelg M, Ramos F MH
Administration of Ratanhia-based herbal oral care products for the prophylaxis of oral mucositis in cancer chemotherapy patients: a clinical trial.
Evid Based Complement Alternat Med. 2007 Sep;4(3):361-6.
Oral complications are a common side effect of cancer chemotherapy, as antineoplastic agents affect both the immune system and the oral mucosa. This study demonstrates preventive and therapeutic effects of dental treatment and regular use of Weleda Ratanhia-Mundwasser((R)) (herbal mouthwash) and Weleda Pflanzen-Zahngel((R)) (herbal toothgel) on oral mucositis during chemotherapy. Thirty-two female patients with breast cancer starting on chemotherapy were evaluated in this study. Plaque index, gingival index, degree of mucositis and 10 single symptoms were monitored once weekly for four consecutive weeks. After four weeks, plaque and gingival indexes were slightly decreased compared to baseline values. The degree of mucositis was increased by one grade in 15.6 % of the patients and over 70 % remained without symptoms. On the whole, single symptoms decreased from day 7 since beginning of chemotherapy to day 28. Mucositis symptoms were moderate in severity, and the results indicate a positive influence of using Weleda Ratanhia-Mundwasser and Weleda Pflanzen-Zahngel. Further studies might be promising. [Abstract/Link to Full Text]

Vieira JR, de Souza IA, do Nascimento SC, Leite SP
Indigofera suffruticosa: An Alternative Anticancer Therapy.
Evid Based Complement Alternat Med. 2007 Sep;4(3):355-359.
Indigofera suffruticosa Mill (Fabeceae) occurs in the Northeast countryside and has intensive popular use in the treatment of infectious, inflammatory and other processes. The main aim of the present work was to investigate the cytotoxic and antitumor effects of aqueous extracts of leaves of I. suffruticosa obtained by infusion and maceration as well as to evaluate the toxicological properties. Aqueous extracts did not exhibit cytotoxicity against HEp-2 (human epidermoid cancer cell) cell lines by MTT method. From the aqueous extract by infusion, the toxicological assay showed low order of toxicity. The antitumor effect of aqueous extracts by infusion (64.53%) and maceration (62.62%) against sarcoma 180 in mice at a dose of 50 mg kg(-1) (intraperitoneally), based on low order of toxicity was comparable to the control group, which showed 100% development. Considering the low order of toxicity and that it is highly effective in inhibiting growth of solid tumors, the aqueous extracts of leaves of I. suffruticosa may be used as an alternative anticancer agent. [Abstract/Link to Full Text]

Mahmoudabadi AZ, Dabbagh MA, Fouladi Z
In Vitro Anti-Candida Activity of Zataria multiflora Boiss.
Evid Based Complement Alternat Med. 2007 Sep;4(3):351-353.
Zataria multiflora Boiss known as Avishan Shirazi (in Iran) is one of the valuable Iranian medicinal plants. The aim of study was to evaluate anti-Candida activity of Z. multiflora against different species of Candida in vitro. Anti-Candida activity of the aqueous, ethanolic and methanolic maceration extract of the aerial parts of Z. multiflora Boiss was studied in vitro. Anti-Candida activity against Candida species was done using serial dilutions of extracts in Sabouraud's dextrose agar. Minimal inhibitory concentration (MIC) of the methanolic and ethanolic extracts was 70.7 and 127 mg l(-1), respectively. Aqueous extract showed no remarkable activity against Candida species. We conclude that methanolic extract of the aerial parts of Z. multiflora Boiss has more anti-Candida effect at 70.7 mg l(-1) compared to ethanolic extract 127 mg l(-1). In addition, the isolates of Candida parapsilosis were more susceptible to methanolic extract than other tested species. [Abstract/Link to Full Text]

Kumar M, Samarth R, Kumar M, Selvan SR, Saharan B, Kumar A
Protective Effect of Adhatoda vascia Nees Against Radiation-Induced Damage at Cellular, Biochemical and Chromosomal Levels in Swiss Albino Mice.
Evid Based Complement Alternat Med. 2007 Sep;4(3):343-50.
Extract of Adhatoda vasica (L) Nees leaves has been used for treatment of various diseases and disorders in Ayurved and Unani medicine. Modulatory effect of ethanolic extract of A. vasica (L) Nees against radiation-induced changes in terms of histological alterations in testis, reduced glutathione (GSH), lipid peroxidation (LPO), acid and alkaline phosphatases levels, and chromosomal alterations in Swiss albino mice was studied at various post-irradiation intervals between 1 and 30 days. Mice exposed to 8 Gy radiation showed radiation-induced sickness including marked changes in histology of testis and chromosomal aberrations in bone marrow cells with 100% mortality within 22 days. When ethanolic leaf extract of A. vasica was given orally at a dose of 800 mg kg(-1) body weight per mouse for 15 consecutive days and then exposed to radiation, death of Adhatoda-pretreated irradiated mice was reduced to 70% at 30 days. The radiation dose reduction factor was 1.43. There was significantly lesser degree of damage to testis tissue architecture and various cell populations including spermatogonia, spermatids and Leydig cells. Correspondingly, a significant decrease in the LPO and an increase in the GSH levels were observed in testis and liver of Adhatoda-pretreated irradiated mice. Similarly, a significant decrease in level of acid phosphatase and increase in level of alkaline phosphatase were observed. Adhatoda pretreatment significantly prevented radiation-induced chromosomal damage in bone marrow cells. The study suggests that Adhatoda plant extract has significant radioprotective effects on testis that warrants further mechanistic studies aimed at identifying the role of major ingredients in the extract. [Abstract/Link to Full Text]

Nishida S, Satoh H
Vascular pharmacology of mokuboito (mu-fang-yi-tang) and its constituents on the smooth muscle and the endothelium in rat aorta.
Evid Based Complement Alternat Med. 2007 Sep;4(3):335-41.
Pharmacological actions of Mokuboito and its constituents (Sinomenium acutum and sinomenine) on rat aorta were examined. Mokuboito and S. acutum at lower concentrations (0.03-1 mg ml(-1)) contracted the non-loaded aorta, but at higher concentrations (1-3 mg ml(-1)), reversed to dilate it. The vasoconstriction was blocked by phentolamine (10 muM). Sinomenine failed to exhibit the vasoconstriction. On the other hand, Mokuboito and S. acutum dilated the NE (5 muM)-induced vasoconstriction: at 3 mg ml(-1), by 98.9 +/- 2.5% (n = 6, P < 0.01) and 97.0 +/- 4.8% (n = 6, P < 0.01). Vasorelaxation induced by Mokuboito and S. acutum was attenuated by indomethacin, L-NMMA and nicardipine. Propranolol decreased the vasorelaxation induced by Mokuboito, but not by S. acutum. Sinomenine also relaxed the constriction and at 100 muM, by 68.8 +/- 5.1% (n = 7, P < 0.01). This vasorelaxation was attenuated by indomethacin, L-NMMA and nicardipine, and also by propranolol. Therefore, these results indicate that Mokuboito and its constituents exert both vasodilating actions mediated by endothelium-dependent mechanisms (PGI(2) and NO from endothelium) and by endothelium-independent mechanisms (Ca(2+) influx control on smooth muscle cells). Simultaneously, Mokuboito and S. acutum cause the vasoconstrictions mediated through alpha-adrenoceptor stimulation, but not sinomenine. Also, Mokuboito and sinomenine possess beta-adrenoreceptor stimulating action, but not S. acutum. [Abstract/Link to Full Text]

Chun SC, Jee SY, Lee SG, Park SJ, Lee JR, Kim SC
Anti-Inflammatory Activity of the Methanol Extract of Moutan Cortex in LPS-Activated Raw264.7 Cells.
Evid Based Complement Alternat Med. 2007 Sep;4(3):327-33.
Moutan Cortex (MCE) has been used in traditional medicine to remove heat from the blood, promote blood circulation and alleviate blood stasis. This study was conducted to evaluate the effects of MCE on regulatory mechanisms of cytokines and nitric oxide (NO) involved in immunological activity of Raw264.7 cells. Cells were pretreated with methanolic extracts of MCE, and further cultured for an appropriate time after lipopolyssacharide (LPS) addition. During the entire experimental period, 0.1 and 0.3 mg ml(-1) of MCE had no cytotoxicity. In these concentrations, MCE inhibited the production of NO and prostaglandin E(2) (PGE(2)), the expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) and phosphorylated inhibitor of kappaBalpha (p-IkappaBalpha), and the activation of nuclear factor kappaB (NF-kappaB). MCE also reduced the concentration of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in the Raw264.7 cells that were activated by LPS. These results demonstrate that MCE has anti-inflammatory effects through the inhibition of iNOS and COX-2 expression by suppressing the phosphorylation of I-kappaBalpha and the activation of NF-kappaB. [Abstract/Link to Full Text]

Essa MM, Subramanian P
Hibiscus sabdariffa Affects Ammonium Chloride-Induced Hyperammonemic Rats.
Evid Based Complement Alternat Med. 2007 Sep;4(3):321-5.
Hibiscus sabdariffa (HS) is an edible medicinal plant, indigenous to India, China and Thailand and is used in Ayurveda and traditional medicine. Alcoholic extract of HS leaves (HSEt) was studied for its anti-hyperammonemic and antioxidant effects in brain tissues of ammonium chloride-induced hyperammonemic rats. Oral administration of HSEt (250 mg kg(-1) body weight) significantly normalizes the levels of ammonia, urea, uric acid, creatinine and non-protein nitrogen in the blood. HSEt significantly reduced brain levels of lipid peroxidation products such as thiobarbituric acid and reactive substances (TBARS) and hydroperoxides (HP). However, the administered extract significantly increased the levels of antioxidants such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and reduced glutathione (GSH) in brain tissues of hyperammonemic rats. This investigation demonstrates significant anti-hyperammonemic and antioxidant activity of HS. [Abstract/Link to Full Text]

Emami SA, Asili J, Mohagheghi Z, Hassanzadeh MK
Antioxidant activity of leaves and fruits of Iranian conifers.
Evid Based Complement Alternat Med. 2007 Sep;4(3):313-9.
Cupressus semipervirens var. horizontalis, Cupressus semipervirens var. semipervirens, Cupressus semipervirens cv. Cereifeormis, Juniperus communis subsp. hemisphaerica, Juniperus excelsa subsp. excelsa, Juniperus excelsa subsp. polycarpos, Juniperus foetidissima, Juniperus oblonga, Juniperus sabina, Platycladus orientalis and Taxus baccata are Iranian conifers. The antioxidant activity of leaves and fruits of these 11 different taxons were evaluated. The leaves of both male and female, and fruits of these plants were collected from different areas of the country. Methanol extract of leaves and fruits of these taxons were prepared. Antioxidant activity of each extracts was measured using two different tests of the ferric thiocyanate method and thiobarbituric acid. Results indicated that the methanol extracts of leaves, of male and female, and fruits of all these species (27 samples) possessed antioxidant activity when tested with both methods. The antioxidant activity was then compared with those of alpha-tocopherol (a natural antioxidant) and butylated hydroxytoluene (a synthetic antioxidant). Methanol extract of fruits of C. semipervirens cv. Cereifeormis showed the highest antioxidant activity while the methanol extract of leaves of C. semipervirens var. semipervirens possessed the lowest antioxidant activity. However, our finding showed that most of the tested extracts were showing strong antioxidant activity even higher than alpha-tocopherol. [Abstract/Link to Full Text]

Wiart C
Goniothalamus species: a source of drugs for the treatment of cancers and bacterial infections?
Evid Based Complement Alternat Med. 2007 Sep;4(3):299-311.
Irrespective of the presence of cytotoxic acetogenins and styryl-lactones in the genus Goniothalamus, only 22 species in the genus Goniothalamus, out of 160 species (13.7%) have so far been investigated. In an effort to promote further research on the genus Goniothalamus which could represent a source of drugs for the treatment of cancers and bacterial infections, this work offers a broad analysis of current knowledge on Goniothalamus species. Therefore, it includes (i) taxonomy (ii) botanical description (iii) traditional medicinal uses and (iv) phytochemical and pharmacological studies. We discuss the molecular mechanisms of actions of acetogenins and styryl-lactones, with some emphasis on the possible involvement of protein kinase, Bax and TRAIL receptors in the cytotoxic effects of styryl-lactones. We also report (v) the growth inhibition of several nosocomial bacteria by Goniothalamus. scortechinii. The crude methanol extract of G. scortechinii showed a good and broad spectrum of antibacterial activity against both Gram-negative and Gram-positive bacteria. [Abstract/Link to Full Text]

Kavoussi B
Chinese medicine: a cognitive and epistemological review*.
Evid Based Complement Alternat Med. 2007 Sep;4(3):293-8.
In spite of the common belief that Chinese natural philosophy and medicine have a unique frame of reference completely foreign to the West, this article argues that they in fact have significant cognitive and epistemic similarities with certain esoteric health beliefs of pre-Christian Europe. From the standpoint of Cognitive Science, Chinese Medicine appears as a proto-scientific system of health observances and practices based on a symptomological classification of disease using two elementary dynamical-processes pattern categorization schemas: a hierarchical and combinatorial inhibiting-activating model (Yin-Yang), and a non-hierarchical and associative five-parameter semantic network (5-Elements/Agents). The concept-map of the five-parameter model amounts to a pentagram, a commonly found geomantic and spell casting sigil in a number of pre-Christian health and safety beliefs in Europe, to include the Pythagorean cult of Hygieia, and the Old Religion of Northern Europe. This non-hierarchical pattern-recognition archetype/prototype was hypothetically added to the pre-existing hierarchical one to form a hybrid nosology that can accommodate for a change in disease perceptions. The selection of five parameters rather than another number might be due to a numerological association between the integer five, the golden ratio, the geometry of the pentagram and the belief in health and wholeness arising from cosmic or divine harmony. In any case, this body of purely empirical knowledge is nowadays widely flourishing in the US and in Europe as an alternative to Western Medicine and with the claim of being a unique, independent and comprehensive medical system, when in reality it is structurally-and perhaps historically-related to the health and safety beliefs of pre-Christian Europe; and without the prospect for an epistemological rupture, it will remain built upon rudimentary cognitive modalities, ancient metaphysics, and a symptomological view of disease. [Abstract/Link to Full Text]

Ohnishi ST
Ki: a key to transform the century of death to the century of life.
Evid Based Complement Alternat Med. 2007 Sep;4(3):287-92.
This is my response to the commentary written by Mr James Flowers with the title of 'What is Qi?' in the issue 4 of Vol.3 (2006) of eCAM. I will explain my opinions regarding the importance of Ki research, philosophical aspects of Ki and a possible role of Ki now and in the future. [Abstract/Link to Full Text]

Boon H, Macpherson H, Fleishman S, Grimsgaard S, Koithan M, Norheim AJ, Walach H
Evaluating Complex Healthcare Systems: A Critique of Four Approaches.
Evid Based Complement Alternat Med. 2007 Sep;4(3):279-285.
The purpose of this paper is to bring clarity to the emerging conceptual and methodological literature that focuses on understanding and evaluating complex or 'whole' systems of healthcare. An international working group reviewed literature from interdisciplinary or interprofessional groups describing approaches to the evaluation of complex systems of healthcare. The following four key approaches were identified: a framework from the MRC (UK), whole systems research, whole medical systems research described by NCCAM (USA) and a model from NAFKAM (Norway). Main areas of congruence include acknowledgment of the inherent complexity of many healthcare interventions and the need to find new ways to evaluate these; the need to describe and understand the components of complex interventions in context (as they are actually practiced); the necessity of using mixed methods including randomized clinical trials (RCTs) (explanatory and pragmatic) and qualitative approaches; the perceived benefits of a multidisciplinary team approach to research; and the understanding that methodological developments in this field can be applied to both complementary and alternative medicine (CAM) as well as conventional therapies. In contrast, the approaches differ in the following ways: terminology used, the extent to which the approach attempts to be applicable to both CAM and conventional medical interventions; the prioritization of research questions (in order of what should be done first) especially with respect to how the 'definitive' RCT fits into the process of assessing complex healthcare systems; and the need for a staged approach. There appears to be a growing international understanding of the need for a new perspective on assessing complex healthcare systems. [Abstract/Link to Full Text]

Cooper EL
Colony collapse disorder may affect complementary and alternative medicine.
Evid Based Complement Alternat Med. 2007 Sep;4(3):275-7. [Abstract/Link to Full Text]

Mitsumoto Y
Mitochondrial Nutrition as a Strategy for Neuroprotection in Parkinson's Disease-Research Focus in the Department of Alternative Medicine and Experimental Therapeutics at Hokuriku University.
Evid Based Complement Alternat Med. 2007 Jun;4(2):263-265. [Abstract/Link to Full Text]

Laeeque H, Boon H, Kachan N, Cohen JC, D'Cruz J
The Canadian Natural Health Products (NHP) Regulations: Industry Compliance Motivations.
Evid Based Complement Alternat Med. 2007 Jun;4(2):257-62.
This qualitative study explores corporations' motivations to comply with new natural health products (NHP) Regulations in Canada. Interviews were conducted with representatives from 20 Canadian NHP companies. Findings show that the rationale for compliance differs for large compared to small and medium-sized enterprises (SMEs). Large firms are motivated to comply with the regulations because of the deterrent fear of negative media coverage, social motivations, ability to comply and maintaining a competitive market advantage. In contrast, SMEs are motivated to comply due to the deterrent fear of legal prosecution and a sense of duty. [Abstract/Link to Full Text]

Shmueli A, Shuval J
Are users of complementary and alternative medicine sicker than non-users?
Evid Based Complement Alternat Med. 2007 Jun;4(2):251-5.
Higher utilization of complementary and alternative medicine (CAM), both in cross-sections and over time, is commonly related to better socioeconomic status and to increased dissatisfaction with conventional medicine and its values. Little is known about health differences between users and non-users of CAM. The objective of the paper is to explore the difference in health measured by the SF-36 instrument between users and non-users of CAM, and to estimate the relative importance of the SF-36 health domains scales to the likelihood of consulting CAM providers. Interviews were used to collect information from a sample of 2000 persons in 1993 and 2500 persons in 2000, representing the Israeli Jewish urban population aged 45-75 in those years. Bivariate and logistic regression analyses were used to explore the above associations. The results show that while users of CAM enjoy higher socioeconomic status and younger age, they tend to report worse health than non-users on the eight SF-36 health domains scales in both years. However, controlling for personal characteristics, lower scores on the bodily pain, role-emotional and vitality scales are related to greater likelihood of CAM use in 2000. In 1993, no scale had a significant adjusted association with the use of CAM. The conclusions are that CAM users tend to report worse health. With CAM becoming a mainstream, though somewhat luxurious, medical practice, pain and affective-emotional distress are the main drivers of CAM use. [Abstract/Link to Full Text]

Lonsdale D
Three case reports to illustrate clinical applications in the use of erythrocyte transketolase.
Evid Based Complement Alternat Med. 2007 Jun;4(2):247-50.
Non-caloric nutrients (NCN) are extremely numerous and it is more than obvious that they work in a team relationship. These vitally important interactions are, for the most part, poorly understood. These brief case reports illustrate this in the therapeutic use of thiamin in a clinical setting. The initially abnormal erythrocyte transketolase activity (TKA) and/or the thiamin pyrophosphate effect (TPPE), indicating intracellular cofactor deficiency, usually improves with thiamin administration. Biochemical correction of the abnormality is, however, invariably dependent on the provision of other NCN, especially magnesium. In two patients reported here, this correction required several infusions containing magnesium and other NCN administered intravenously. In a third patient, hemoconcentration associated with an abnormal TPPE was normalized after administration of nutrients that included thiamin and magnesium. [Abstract/Link to Full Text]

Tai CJ, Chang CP, Huang CY, Chien LY
Efficacy of sanfujiu to treat allergies: patient outcomes at 1 year after treatment.
Evid Based Complement Alternat Med. 2007 Jun;4(2):241-6.
Sanfujiu is a treatment method of applying herbal paste onto the acupoints Fengmen and Feishu during the three hottest days of summer to treat patients with allergies. The objectives of this study were to determine the treatment efficacy at 1 year after the Sanfujiu treatment, and examine variations in the perceived efficacy of Sanfujiu among different subgroups, based on the patients' ages, diagnoses and number of reactive symptoms immediately after the treatment. We enrolled 105 patients who completed Sanfujiu treatment at a medical university hospital in Taipei as the subjects. One year after treatment, trained interviewers conducted telephone interviews with the patients. Approximately 60% of them perceived the treatment as being effective at 1 year later, which was higher than that at 1 week after treatment (45.7%). Younger subjects (<19 years of age) and patients with asthma were more likely to report the treatment as being effective. Patients who had more reactive symptoms after the third Sanfujiu treatment were more likely to report the treatment as being effective. The results demonstrated that Sanfujiu was moderately effective, as perceived by patients in Taiwan, in treating their allergic symptoms. [Abstract/Link to Full Text]

Schiff E, Gurgevich S, Caspi O
Potential Synergism between Hypnosis and Acupuncture-Is the Whole More Than the Sum of Its Parts?
Evid Based Complement Alternat Med. 2007 Jun;4(2):233-240.
Both hypnosis and acupuncture have gained credibility over the years in their effectiveness for treating various health conditions. Currently, each of these treatments is administered in distinct settings and separate times. That is, even if patients receive both treatments as part of a multidimensional therapeutic program, they would typically receive them separately rather than simultaneously at the same session. This separation however might be undesirable since, at least theoretically, hypnosis and acupuncture could potentially augment each other if administered concomitantly. In this article we outline the rationale for this hypothesis and discuss the potential ramifications of its implementation. [Abstract/Link to Full Text]

Tsuyoshi Ohnishi S, Nishino K, Uchiyama S, Ohnishi T, Yamaguchi M
Ki-energy (Life-energy) Stimulates Osteoblastic Cells and Inhibits the Formation of Osteoclast-like Cells in Bone Cell Culture Models.
Evid Based Complement Alternat Med. 2007 Jun;4(2):225-232.
Some practitioners of the Nishino Breathing Method (NBM) were found to have a higher bone density than the average values of age- and gender-matched non-practitioners. Using bone cell culture models, we investigated a possible mechanism behind this observation. For the study of bone mineralization, we performed the following two experiments using cultured osteoblastic MC3T3-E1 cells: (i) Kozo Nishino, a Japanese Ki expert, sent Ki-energy to the cells once for 5 or 10 min after they were seeded in culture dishes in the presence of 10% fetal bovine serum (FBS). They were incubated for 72 h and the cells were counted. The number in the dish with 10-min Ki-exposure was significantly greater than that in the control (P < 0.01 with n = 8). We performed a reverse transcription-polymerase chain reaction (RT-PCR) study using these cells, but the mRNA expressions did not change significantly. (ii) After cells were incubated for 72 h without Ki-exposure (in the presence of FBS), they were further cultured for 48 h (in the absence of FBS) to promote differentiation. At the beginning of the second culture stage, Ki was applied once for 10 min. After 48 h, RT-PCR was performed. The mRNA expressions which are related to bone mineralization, such as Runx2, alpha1(I) collagen, alkaline phosphatase and osteocalcin, increased significantly (P < 0.05 and n = 4 for all). For the bone resorption study, we used mouse marrow cultures, which can form osteoclast-like cells in the presence of (1-34) parathyroid hormone (PTH), and stimulate resorption. We exposed these cells to Ki-energy twice for the duration of 5 or 10 min on day 0 and day 4. On day 7, the cells were counted. The number of osteoclast-like cells in dishes with Ki exposure was significantly smaller than those in control dishes (P < 0.05 with n = 5). The difference between 5-min exposure and 10-min exposure was not statistically significant. All of our data suggest that the Ki-effect on osteoporosis should be further explored. [Abstract/Link to Full Text]

Lippiello L
Collagen Synthesis in Tenocytes, Ligament Cells and Chondrocytes Exposed to a Combination of Glucosamine HCl and Chondroitin Sulfate.
Evid Based Complement Alternat Med. 2007 Jun;4(2):219-24.
Clinical testing of the nutraceuticals glucosamine (glcN) and chondroitin sulfate (CS) has shown efficacy in providing relief from symptoms in osteoarthritic patients. In vitro and in vivo studies support existence of a synergistic relationship upregulating synthetic activity in chondrocytes. A combination of glcN and CS may also be useful as adjunct therapy in sports-related injuries if similar upregulation of collagen synthesis is elicited in accessory ligament and tendon joint tissue. Collagen and non-collagenous protein (NCP) synthesis in cultures of bovine tenocytes, ligament cells and chondrocytes exposed to glcN + CS were assayed by uptake of radiolabeled proline into collagenase-sensitive material. Assay of radiolabel in hydroxyproline (a specific marker for collagen synthesis) following HPLC isolation confirmed the specificity of the metabolic effect. Synthesis of total collagenase-sensitive material was maximally upregulated at physiologically obtainable doses of glcN + CS. Tissue response followed the sequence ligament cells (+69%) > chondrocytes (+56%) > tenocytes (+22%). Labeled hydroxyproline increased by 132% in ligament cells, 27% in tenocytes and 49% in epitendon cells after a 48 h exposure to 5 mug ml(-1) glcN + 4 mug ml(-1) CS. Low dose combinations of glcN and CS effectively stimulate in vitro collagen and NCP synthesis by ligament cells, tenocytes and chondrocytes. Hence, therapeutic use following accessory joint tissue trauma may help augment repair processes. [Abstract/Link to Full Text]

Lenon GB, Li CG, Xue CC, Thien FC, Story DF
Inhibition of release of vasoactive and inflammatory mediators in airway and vascular tissues and macrophages by a chinese herbal medicine formula for allergic rhinitis.
Evid Based Complement Alternat Med. 2007 Jun;4(2):209-17.
Herbal therapies are being used increasingly for the treatment of allergic rhinitis. The aim of this study was to investigate the possible pharmacological actions and cellular targets of a Chinese herbal formula (RCM-101), which was previously shown to be effective in reducing seasonal allergic rhinitis symptoms in a randomized, placebo-controlled clinical trial. Rat and guinea pig isolated tissues (trachea and aorta) were used to study the effects of RCM-101 on responses to various mediators. Production of leukotriene B(4) in porcine neutrophils and of prostaglandin E(2) and nitric oxide (NO) in Raw 264.7 cells were also measured. In rat and guinea pig tracheal preparations, RCM-101 inhibited contractile responses to compound 48/80 but not those to histamine (guinea pig preparations) or serotonin (rat preparations). Contractile responses of guinea pig tracheal preparations to carbachol and leukotriene C(4,) and relaxant responses to substance P and prostaglandin E(2) were not affected by RCM-101. In rat aortic preparations, precontracted with phenylephrine, endothelium-dependent relaxant responses to acetylcholine and endothelium-independent relaxant responses to sodium nitroprusside were not affected by RCM-101. However, RCM-101 inhibited relaxations to l-arginine in endothelium-denuded rat aortic preparations, which had been pre-incubated with lipopolysaccharide. RCM-101 did not affect leukotriene B(4) formation in isolated porcine neutrophils, induced by the calcium ionophore A23187; however, it inhibited prostaglandin E(2) and NO production in lipopolysaccharide-stimulated murine macrophages (Raw 264.7 cells).The findings indicate that RCM-101 may have multiple inhibitory actions on the release and/or synthesis of inflammatory mediators involved in allergic rhinitis. [Abstract/Link to Full Text]

Xia R, Huang P, Shao GM
Nourishing Yin and Promoting Blood Circulation of TCM to Treat Hemorheologic Disorder Induced by Diabetes Mellitus in Rats.
Evid Based Complement Alternat Med. 2007 Jun;4(2):203-207.
Diabetes mellitus, DM, is commonly accompanied with various stages of hemorheologic disturbances that are the main causes of the development of chronic DM. In this study, simple Chinese material medica [yang-yin jiang-tang preparation (YYJT)] was given to alloxan-induced DM rats and analyzed to compare the changes of fasting blood glucose (FBG), fasting insulin (FINS), hemorheologic parameters and insulin-like growth factor II (IGF-II) before and after administration. The results suggested that YYJT can significantly downregulate FBG (P < 0.005), improve insulin resistance and beta-cell secretion (P < 0.05), decrease whole blood viscosity at low and high shear rates, gathering of blood index test (GIT) and fibrinogen (FIB) (P < 0.05), and enlarge the function of IGF-II (P < 0.05). We concluded that YYJT could prevent and treat hemorheologic disorder in DM rats by means of reducing glucose, improving insulin resistance and elevating IGF-II. [Abstract/Link to Full Text]

Wang L, Muxin G, Nishida H, Shirakawa C, Sato S, Konishi T
Psychological Stress-Induced Oxidative Stress as a Model of Sub-Healthy Condition and the Effect of TCM.
Evid Based Complement Alternat Med. 2007 Jun;4(2):195-202.
Distress-mediated tissue oxidative stress was examined as a model of sub-healthy condition defined in traditional Chinese medicine theory. Mice were subjected to psychologically stressful conditions by whiskers removal. Under this condition, spontaneous locomotive activity was significantly enhanced in the dark (P < 0.05 versus the control mice in three different movements), and granulocytes/lymphocytes balance shifted to granulocytes. At the same time, peroxynitrite level in blood plasma increased to approximately 180% from that of the control mice at 6 h after removal of the whiskers (P < 0.01), and was maintained even after 12 h. Both protein carbonyl formation and lipid peroxidation were significantly increased under this condition in brain, heart, liver and spleen at 6 h after removal of whiskers (P < 0.05 or P < 0.01), and these levels were maximized after 12 h (increased to 120-160%, P < 0.05 or P < 0.01). The oxidative tissue injuries observed at 12 h after the removal of the whiskers were effectively prevented by two traditional Chinese medicine formula: Shengmai San (SMS) and Ling Gui Zhu Gan Tang (LGZGT), when administered for 5 days before the removal of the whiskers. Therefore, this stress model is considered useful in assessing the preventive potential of antioxidants and antioxidant-based herbal mixtures in treating the pathophysiology associated with psychological or emotional distress. [Abstract/Link to Full Text]

Hall Z, Luu T, Moore D, Yount G
Radiation response of cultured human cells is unaffected by johrei.
Evid Based Complement Alternat Med. 2007 Jun;4(2):191-4.
Johrei has been credited with healing thousands from radiation wounds after the Hiroshima and Nagasaki bombs in 1945. This alternative medical therapy is becoming increasingly popular in the United States, as are other Energy Medicine modalities that purport to influence a universal healing energy. Human brain cells were cultured and exposed to increasing doses of ionizing radiation. Experienced Johrei practitioners directed healing intentionality toward the cells for 30 min from a distance of 20 cm and the fate of the cells was observed by computerized time-lapse microscopy. Cell death and cell divisions were tallied every 30 min before, during and after Johrei treatment for a total of 22.5 h. An equal number of control experiments were conducted in which cells were irradiated but did not receive Johrei treatment. Samples were assigned to treatment conditions randomly and data analysis was conducted in a blinded fashion. Radiation exposure decreased the rate of cell division (cell cycle arrest) in a dose-dependent manner. Division rates were estimated for each 30 min and averaged over 8 independent experiments (4 control and 4 with Johrei treatment) for each of 4 doses of X-rays (0, 2, 4 and 8 Gy). Because few cell deaths were observed, pooled data from the entire observation period were used to estimate death rates. Analysis of variance did not reveal any significant differences on division rate or death rate between treatment groups. Only radiation dose was statistically significant. We found no indication that the radiation response of cultured cells is affected by Johrei treatment. [Abstract/Link to Full Text]

Salvioli S, Sikora E, Cooper EL, Franceschi C
Curcumin in Cell Death Processes: A Challenge for CAM of Age-Related Pathologies.
Evid Based Complement Alternat Med. 2007 Jun;4(2):181-190.
Curcumin, the yellow pigment from the rhizoma of Curcuma longa, is a widely studied phytochemical which has a variety of biological activities: anti-inflammatory and anti-oxidative. In this review we discuss the biological mechanisms and possible clinical effects of curcumin treatment on cancer therapy, and neurodegenerative diseases such as Alzheimer's Disease, with particular attention to the cell death processes induced by curcumin. Since oxidative stress and inflammation are major determinants of the aging process, we also argue that curcumin can have a more general effect that slows down the rate of aging. Finally, the effects of curcumin can be described as xenohormetic, since it activates a sort of stress response in mammalian cells. [Abstract/Link to Full Text]

Tsao JC
Effectiveness of Massage Therapy for Chronic, Non-malignant Pain: A Review.
Evid Based Complement Alternat Med. 2007 Jun;4(2):165-79.
Previous reviews of massage therapy for chronic, non-malignant pain have focused on discrete pain conditions. This article aims to provide a broad overview of the literature on the effectiveness of massage for a variety of chronic, non-malignant pain complaints to identify gaps in the research and to inform future clinical trials. Computerized databases were searched for relevant studies including prior reviews and primary trials of massage therapy for chronic, non-malignant pain. Existing research provides fairly robust support for the analgesic effects of massage for non-specific low back pain, but only moderate support for such effects on shoulder pain and headache pain. There is only modest, preliminary support for massage in the treatment of fibromyalgia, mixed chronic pain conditions, neck pain and carpal tunnel syndrome. Thus, research to date provides varying levels of evidence for the benefits of massage therapy for different chronic pain conditions. Future studies should employ rigorous study designs and include follow-up assessments for additional quantification of the longer-term effects of massage on chronic pain. [Abstract/Link to Full Text]

Recent Articles in Journal of Negative Results in Biomedicine

Cotignola J, Roy P, Patel A, Ishill N, Shah S, Houghton A, Coit D, Halpern A, Busam K, Berwick M, Orlow I
Functional polymorphisms in the promoter regions of MMP2 and MMP3 are not associated with melanoma progression.
J Negat Results Biomed. 2007 Oct 24;6(1):9.
ABSTRACT: BACKGROUND: The matrix metalloproteinases (MMPs) are enzymes that cleave various components of the extracellular matrix (ECM) and basement membranes. MMPs are expressed in melanocytes and their overexpression has been linked to tumor development, progression and metastasis. At the genetic level, the following functional promoter polymorphisms are known to modify the gene transcription: -1306 C/T and -735 C/T in the MMP2 gene, and -1171 5A/6A in the MMP3 gene. Functional polymorphisms in MMP genes' promoter regions may modulate the risk for melanoma progression. METHODS: We evaluated MMP2 and MMP3 germline polymorphisms in a group of 1002 melanoma patients using PCR-based methods, including fragment size analysis and melting temperature profiles. Two-sided Chi-Square, Cochran-Armitage tests for trend, Fisher's exact tests, and Kendall's Tau tests were performed to evaluate the associations between genotype and various clinical and epidemiologic factors. Multivariate analyses were conducted using logistic regression, adjusting for known melanoma confounders such as age, sex, phenotypic index, moles, freckles, and race. Survival estimates were computed using the Kaplan-Meier method and differences in survival were assessed using the log rank test. RESULTS: All genotypes were in Hardy-Weinberg equilibrium. After adjustment for age, sex and phenotypic characteristics of melanoma risk, no significant associations were identified with the clinical, pathological, and epidemiological variables studied. The melting profile for MMP2 -735 C/T identified a new change in one sample. A new PCR-amplification followed by direct sequencing confirmed a heterozygote G to A substitution at position -729. CONCLUSIONS: This study does not provide strong evidence for further investigation into the role of the MMP2 and MMP3 variants in melanoma progression. [Abstract/Link to Full Text]

McNally MA, Baek RC, Avila RL, Seyfried TN, Strichartz GR, Kirschner DA
Peripheral nervous system manifestations in a Sandhoff disease mouse model: nerve conduction, myelin structure, lipid analysis.
J Negat Results Biomed. 2007;68.
BACKGROUND: Sandhoff disease is an inherited lysosomal storage disease caused by a mutation in the gene for the beta-subunit (Hexb gene) of beta-hexosaminidase A (alphabeta) and B (beta beta). The beta-subunit together with the GM2 activator protein catabolize ganglioside GM2. This enzyme deficiency results in GM2 accumulation primarily in the central nervous system. To investigate how abnormal GM2 catabolism affects the peripheral nervous system in a mouse model of Sandhoff disease (Hexb-/-), we examined the electrophysiology of dissected sciatic nerves, structure of central and peripheral myelin, and lipid composition of the peripheral nervous system. RESULTS: We detected no significant difference in signal impulse conduction velocity or any consistent change in the frequency-dependent conduction slowing and failure between freshly dissected sciatic nerves from the Hexb+/- and Hexb-/- mice. The low-angle x-ray diffraction patterns from freshly dissected sciatic and optic nerves of Hexb+/- and Hexb-/- mice showed normal myelin periods; however, Hexb-/- mice displayed a approximately 10% decrease in the relative amount of compact optic nerve myelin, which is consistent with the previously established reduction in myelin-enriched lipids (cerebrosides and sulfatides) in brains of Hexb-/- mice. Finally, analysis of lipid composition revealed that GM2 content was present in the sciatic nerve of the Hexb-/- mice (undetectable in Hexb+/-). CONCLUSION: Our findings demonstrate the absence of significant functional, structural, or compositional abnormalities in the peripheral nervous system of the murine model for Sandhoff disease, but do show the potential value of integrating multiple techniques to evaluate myelin structure and function in nervous system disorders. [Abstract/Link to Full Text]

Kapoor B, Dunlop C, Wynn-Jones C, Fryer AA, Strange RC, Maffulli N
Vitamin D and oestrogen receptor polymorphisms in developmental dysplasia of the hip and primary protrusio acetabuli--a preliminary study.
J Negat Results Biomed. 2007;67.
We investigated the association of developmental dysplasia of the hip (DDH) and primary protrusion acetabuli (PPA) with Vitamin D receptor polymorphisms Taq I and Fok I and oestrogen receptor polymorphisms Pvu II and Xba I. 45 patients with DDH and 20 patients with PPA were included in the study. Healthy controls (n = 101) aged 18-60 years were recruited from the same geographical area. The control subjects had a normal acetabular morphology based on a recent pelvic radiograph performed for an unrelated cause. DNA was obtained from all the subjects from peripheral blood. Genotype frequencies were compared in the three groups. The relationship between the genotype and morphology of the hip joint, severity of the disease, age at onset of disease and gender were examined. The oestrogen receptor Xba I wild-type genotype (XX, compared with Xx and xx combined) was more common in the DDH group (55.8%) than controls (37.9%), though this just failed to achieve statistical significance (p = 0.053, odds ratio = 2.1, 95% CI = 0.9-4.6). In the DDH group, homozygosity for the mutant Taq I Vitamin D receptor t allele was associated with higher acetabular index (Mann-Whitney U-test, p = 0.03). Pvu II pp oestrogen receptor genotype was associated with low centre edge angle (p = 0.07). This study suggests a possible correlation between gene polymorphism in the oestrogen and vitamin D receptors and susceptibility to, and severity of DDH. The Taq I vitamin D receptor polymorphisms may be associated with abnormal acetabular morphology leading to DDH while the Xba I oestrogen receptor XX genotype may be associated with increased risk of developing DDH. No such correlations were found in the group with PPA. [Abstract/Link to Full Text]

Hanley B
Variance in multiplex suspension array assays: carryover of microspheres between sample wells.
J Negat Results Biomed. 2007;66.
BACKGROUND: This study was undertaken because of the accidental observation that a sample of 60+ beads was obtained by the instrument from a completely dry, unused well in a 96 well plate. Others have observed unexplained outliers in replicated wells. The problem was first observed on an older instrument, and replicated on a new instrument. METHODS AND RESULTS: Data is presented from two instruments using a multiple blank following well experiment that shows a surprising amount of carryover that has an unexpected nature. When it occurs, it does not necessarily decline from one well to the next. There appears to be two types of carryover, one that is small, predictable and declines consistently, and another which is potentially very large, unpredictable, and does not decline. The former can be compensated for or ignored. The latter cannot be addressed without using multiple replicated samples or an intraplex method. CONCLUSION: This problem has significance for analysis of results obtained with suspended microarray instruments. A special notation is made that biostatisticians need to be made aware of these results before experiments are undertaken and data generated for them to analyze. The problem can be handled by enough replicated samples, or an intraplex method. The applicability of these results to oligonucleotide based assays is unknown. [Abstract/Link to Full Text]

Sim�es PD, Ramos T
Human pluripotent embryonal carcinoma NTERA2 cl.D1 cells maintain their typical morphology in an angiomyogenic medium.
J Negat Results Biomed. 2007;65.
BACKGROUND: Pluripotent embryonal carcinomas are good potential models, to study, "in vitro," the mechanisms that control differentiation during embryogenesis. The NTERA2cl.D1 (NT2/D1) cell line is a well known system of ectodermal differentiation. Retinoic acid (RA) induces a dorsal pattern of differentiation (essentially neurons) and bone morphogenetic protein (BMP) or hexamethylenebisacetamide (HMBA) induces a more ventral (epidermal) pattern of differentiation. However, whether these human cells could give rise to mesoderm derivatives as their counterpart in mouse remained elusive. We analyzed the morphological characteristics and transcriptional activation of genes pertinent in cardiac muscle and endothelium differentiation, during the growth of NT2/D1 cells in an inductive angiomyogenic medium with or without Bone Morphogenetic Protein 2 (BMP2). RESULTS: Our experiments showed that NT2/D1 maintains their typical actin organization in angiomyogenic medium. Although the beta myosin heavy chain gene was never detected, all the other 15 genes analyzed maintained their expression throughout the time course of the experiment. Among them were early and late cardiac, endothelial, neuronal and teratocarcinoma genes. CONCLUSION: Our results suggest that despite the NT2/D1 cells natural tendency to differentiate into neuroectodermal lineages, they can activate genes of mesodermal lineages. Therefore, we believe that these pluripotent cells might still be a good model to study biological development of mesodermal derivatives, provided the right culture conditions are met. [Abstract/Link to Full Text]

Jerjes W, Madland G, Feinmann C, El Maaytah M, Kumar M, Hopper C, Upile T, Newman S
Psycho-education programme for temporomandibular disorders: a pilot study.
J Negat Results Biomed. 2007;64.
BACKGROUND: Temporomandibular disorders (TMDs) are by far the most predominant condition affecting the temporomandibular joint (TMJ), however many patients have mild self-limiting symptoms and should not be referred for specialist care.The aim of this pilot study was to develop a simple, cost-effective management programme for TMDs using CD-ROM. 41 patients (age 18-70) participated in this study, patients were divided into three groups: the 1st group were involved in an attention placebo CD-ROM (contain anatomical information about the temporomandibular system), the 2nd group received information on CD-ROM designed to increase their control and self efficacy, while the 3rd group received the same programme of the 2nd group added to it an introduction to self-relaxing techniques followed by audio tape of progressive muscle relaxation exercises. Each of the groups was asked to complete a number of questionnaires on the day of initial consultation and six weeks afterwards. RESULTS: The two experimental groups (2nd & 3rd) were equally effective in reducing pain, disability and distress, and both were more effective than the attention placebo group (1st), however the experimental groups appeared to have improved at follow-up relative to the placebo-group in terms of disability, pain and depressed mood. CONCLUSION: This pilot study demonstrates the feasibility and acceptability of the design. A full, randomized, controlled trial is required to confirm the efficacy of the interventions developed here. [Abstract/Link to Full Text]

Laessoe U, Hoeck HC, Simonsen O, Sinkjaer T, Voigt M
Fall risk in an active elderly population--can it be assessed?
J Negat Results Biomed. 2007;62.
BACKGROUND: Falls amongst elderly people are often associated with fractures. Training of balance and physical performance can reduce fall risk; however, it remains a challenge to identify individuals at increased risk of falling to whom this training should be offered. It is believed that fall risk can be assessed by testing balance performance. In this study a test battery of physiological parameters related to balance and falls was designed to address fall risk in a community dwelling elderly population. RESULTS: Ninety-four elderly males and females between 70 and 80 years of age were included in a one year follow-up study. A fall incidence of 15% was reported. The test battery scores were not different between the fallers and non-fallers. Test scores were, however, related to self-reported health. In spite of inclusion of dynamic tests, the test battery had low fall prediction rates, with a sensitivity and specificity of 50% and 43% respectively. CONCLUSION: Individuals with poor balance were identified but falls were not predicted by this test battery. Physiological balance characteristics can apparently not be used in isolation as adequate indicators of fall risk in this population of community dwelling elderly. Falling is a complex phenomenon of multifactorial origin. The crucial factor in relation to fall risk is the redundancy of balance capacity against the balance demands of the individuals levels of fall-risky lifestyle and behavior. This calls for an approach to fall risk assessment in which the physiological performance is evaluated in relation to the activity profile of the individual. [Abstract/Link to Full Text]

Guajardo-Salinas GE, Carvajal JA, Gaytan-Ramos AA, Arroyo L, L�pez-Reyes AG, Islas JF, Cano BG, Arroyo-Curr�s N, D�valos A, Madrid G, Moreno-Cuevas JE
Effects of bone marrow cell transplant on thyroid function in an I131-induced low T4 and elevated TSH rat model.
J Negat Results Biomed. 2007;61.
BACKGROUND: We developed a study using low dose radioactive iodine creating an animal model of transient elevation of thyroid stimulating hormone (TSH). Male derived bone marrow cells were transplanted to asses their effect on thyroid function and their capability to repair the thyroid parenchyma. RESULTS: At 40 an 80 days after I131 treatment, the study groups TSH and T4 serum values both increased and decreased significantly respectively compared to the negative control group. Eight weeks after cell transplantation, neither TSH nor T4 showed a significant difference in any group. The mean number of SRY gene copies found in group I (Left Intracardiac Transplant) was 523.3 and those in group II (Intrathyroid Transplant) were only 73. Group III (No Transplant) and IV had no copies. Group I presented a partial restore of the histological pattern of rat thyroid with approximately 20%-30% of normal-sized follicles. Group II did not show any histological differences compared to group III (Positive control). CONCLUSION: Both a significant increase of TSH and decrease of T4 can be induced as early as day 40 after a low dose of I131 in rats. Restore of normal thyroid function can be spontaneously achieved after using a low dose RAI in a rat model. The use of BM derived cells did not affect the re-establishment of thyroid function and might help restore the normal architecture after treatment with RAI. [Abstract/Link to Full Text]

Lippmann T, Pasternack SM, Kraczyk B, Dudek SE, Dekomien G
Indirect exclusion of four candidate genes for generalized progressive retinal atrophy in several breeds of dogs.
J Negat Results Biomed. 2006;519.
BACKGROUND: Generalized progressive retinal atrophy (gPRA) is a hereditary ocular disorder with progressive photoreceptor degeneration in dogs. Four retina-specific genes, ATP binding cassette transporter retina (ABCA4), connexin 36 (CX36), c-mer tyrosin kinase receptor (MERTK) and photoreceptor cell retinol dehydrogenase (RDH12) were investigated in order to identify mutations leading to autosomal recessive (ar) gPRA in 29 breeds of dogs. RESULTS: Mutation screening was performed initially by PCR and single strand conformation polymorphism (SSCP) analysis, representing a simple method with comparatively high reliability for identification of sequence variations in many samples. Conspicuous banding patterns were analyzed via sequence analyses in order to detect the underlying nucleotide variations. No pathogenetically relevant mutations were detected in the genes ABCA4, CX36, MERTK and RDH12 in 71 affected dogs of 29 breeds. Yet 30 new sequence variations were identified, both, in the coding regions and intronic sequences. Many of the sequence variations were in heterozygous state in affected dogs. CONCLUSION: Based on the ar transmittance of gPRA in the breeds investigated, informative sequence variations provide evidence allowing indirect exclusion of pathogenetic mutations in the genes ABCA4 (for 9 breeds), CX36 (for 12 breeds), MERTK (for all 29 breeds) and RDH12 (for 9 breeds). [Abstract/Link to Full Text]

Freathy RM, Mitchell SM, Knight B, Shields B, Weedon MN, Hattersley AT, Frayling TM
A study of association between common variation in the growth hormone-chorionic somatomammotropin hormone gene cluster and adult fasting insulin in a UK Caucasian population.
J Negat Results Biomed. 2006;518.
BACKGROUND: Reduced growth during infancy is associated with adult insulin resistance. In a UK Caucasian cohort, the CSH1.01 microsatellite polymorphism in the growth hormone-chorionic somatomammotropin hormone gene cluster was recently associated with increases in adult fasting insulin of approximately 23 pmol/l for TT homozygote males compared to D1D1 or D2D2 homozygotes (P = 0.001 and 0.009; n = 206 and 92, respectively), but not for females. TT males additionally had a 547-g lower weight at 1 year (n = 270; P = 0.008) than D2D2 males. We sought to replicate these data in healthy UK Caucasian subjects. We genotyped 1396 subjects (fathers, mothers and children) from a consecutive birth study for the CSH1.01 marker and analysed genotypes for association with 1-year weight in boys and fasting insulin in fathers. RESULTS: We found no evidence for association of CSH1.01 genotype with adult male fasting insulin concentrations (TT/D1D1 P = 0.38; TT/D2D2 P = 0.18) or weight at 1 year in boys (TT/D1D1 P = 0.76; TT/D2D2 P = 0.85). For fasting insulin, our data can exclude the previously observed effect sizes as the 95 % confidence intervals for the differences observed in our study exclude increases in fasting insulin of 9.0 and 12.6 pmol/l for TT relative to D1D1 and D2D2 homozygotes, respectively. Whilst we have fewer data on boys' 1-year weight than the original study, our data can exclude a reduction in 1-year weight greater than 557 g for TT relative to D2D2 homozygotes. CONCLUSION: We have not found association of the CSH1.01 genotype with fasting insulin or weight at 1 year. We conclude that the original study is likely to have over-estimated the effect size for fasting insulin, or that the difference in results reflects the younger age of subjects in this study relative to those in the previous study. [Abstract/Link to Full Text]

Chaouachi K
A critique of the WHO TobReg's "Advisory Note" report entitled: "Waterpipe tobacco smoking: health effects, research needs and recommended actions by regulators".
J Negat Results Biomed. 2006;517.
BACKGROUND AND AIM: The World Health Organisation Study Group on Tobacco Product Regulation (TobReg) has issued in 2005 an "Advisory Note" entitled: "Waterpipe Tobacco Smoking: Health Effects, Research Needs and Recommended Actions by Regulators". "Waterpipe" smoking is now considered a global public health threat and the corresponding artefact is actually known in the world under three main terms: hookah, narghile and shisha. This important report, the first ever prepared by WHO on the subject, poses two major problems. On one hand, its bibliographical references dismiss world chief relevant studies. On the other, it contains a certain number of errors of many orders: biomedical, sociological, anthropological and historical. The purpose of the present study is to highlight, one by one, where these weaknesses and errors lie and show how this official report can be considerably improved. RESULTS: We realise that widely advertised early anthropological studies were not taken into consideration whereas they shed a substantial light on this peculiar form of smoking and help understanding its high complexity. As for concrete errors to be found in this report, they deal with the chemistry of smoke, health-related effects, smoking patterns, description and history of the artefact and its use, gender and underage use aspects, prevention and research needs in this field. CONCLUSION: The scientific credibility of an international expert report may be at stake if its recommendations do not rely on sound objective research findings and a comprehensive review of the existing literature. The critical comments in this study will certainly help improve the present WHO report. [Abstract/Link to Full Text]

Wagenaar DA, Pine J, Potter SM
Searching for plasticity in dissociated cortical cultures on multi-electrode arrays.
J Negat Results Biomed. 2006;516.
We attempted to induce functional plasticity in dense cultures of cortical cells using stimulation through extracellular electrodes embedded in the culture dish substrate (multi-electrode arrays, or MEAs). We looked for plasticity expressed in changes in spontaneous burst patterns, and in array-wide response patterns to electrical stimuli, following several induction protocols related to those used in the literature, as well as some novel ones. Experiments were performed with spontaneous culture-wide bursting suppressed by either distributed electrical stimulation or by elevated extracellular magnesium concentrations as well as with spontaneous bursting untreated. Changes concomitant with induction were no larger in magnitude than changes that occurred spontaneously, except in one novel protocol in which spontaneous bursts were quieted using elevated extracellular magnesium concentrations. [Abstract/Link to Full Text]

Landi S, Gemignani F, Bottari F, Gioia-Patricola L, Guino E, Cambray M, Biondo S, Capella G, Boldrini L, Canzian F, Moreno V
Polymorphisms within inflammatory genes and colorectal cancer.
J Negat Results Biomed. 2006;515.
BACKGROUND: Chronic inflammation is a risk factor for colorectal cancer and polymorphisms in the inflammatory genes could modulate the levels of inflammation. We have investigated ten single nucleotide polymorphisms (SNPs) in the following inflammation-related genes: TLR4 (Asp299Gly), CD14 (-260 T>C), MCP1 (-2518 A>G), IL12A (+7506 A>T, +8707 A>G, +9177 T>A, +9508 G>A), NOS2A (+524T>C), TNF (-857C>T), and PTGS1 (V444I) in 377 colorectal (CRC) cancer cases and 326 controls from Barcelona (Spain). RESULTS: There was no statistically significant association between the SNPs investigated and colorectal cancer risk. CONCLUSION: The lack of association may show that the inflammatory genes selected for this study are not involved in the carcinogenic process of colorectum. Alternatively, the negative results may derive from no particular biological effect of the analysed polymorphisms in relation to CRC. Otherwise, the eventual biological effect is so little to go undetected, unless analysing a much larger sample size. [Abstract/Link to Full Text]

Patel JV, Cummings DE, Girod JP, Mascarenhas AV, Hughes EA, Gupta M, Lip GY, Reddy S, Brotman DJ
Role of metabolically active hormones in the insulin resistance associated with short-term glucocorticoid treatment.
J Negat Results Biomed. 2006;514.
BACKGROUND: The mechanisms by which glucocorticoid therapy promotes obesity and insulin resistance are incompletely characterized. Modulations of the metabolically active hormones, tumour necrosis factor alpha (TNF alpha), ghrelin, leptin and adiponectin are all implicated in the development of these cardiovascular risk factors. Little is known about the effects of short-term glucocorticoid treatment on levels of these hormones. RESEARCH METHODS AND PROCEDURES: Using a blinded, placebo-controlled approach, we randomised 25 healthy men (mean (SD) age: 24.2 (5.4) years) to 5 days of treatment with either placebo or oral dexamethasone 3 mg twice daily. Fasting plasma TNFalpha, ghrelin, leptin and adiponectin were measured before and after treatment. RESULTS: Mean changes in all hormones were no different between treatment arms, despite dexamethasone-related increases in body weight, blood pressure, HDL cholesterol and insulin. Changes in calculated indices of insulin sensitivity (HOMA-S, insulin sensitivity index) were strongly related to dexamethasone treatment (p < 0.001). DISCUSSION: Our data do not support a role for TNF alpha, ghrelin, leptin or adiponectin in the insulin resistance associated with short-term glucocorticoid treatment. [Abstract/Link to Full Text]

O'Gorman DB, Wu Y, Seney S, Zhu RD, Gan BS
Wnt expression is not correlated with beta-catenin dysregulation in Dupuytren's Disease.
J Negat Results Biomed. 2006;513.
BACKGROUND: Dupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in finger flexion contractures. Increased cellular beta-catenin levels have been identified as characteristic of this disease. As Wnts are the most widely recognized upstream regulators of cellular beta-catenin accumulation, we have examined Wnt gene expression in surgical specimens and in DD-derived primary cell cultures grown in two-dimensional monolayer culture or in three-dimensional FPCL collagen lattice cultures. RESULTS: The Wnt expression profile of patient-matched DD and unaffected control palmar fascia tissue was determined by a variety of complimentary methods; Affymetrix Microarray analysis, specific Wnt and degenerative primer-based Reverse Transcriptase (RT)-PCR, and Real Time PCR. Microarray analysis identified 13 Wnts associated with DD and control tissues. Degenerate Wnt RT-PCR analysis identified Wnts 10b and 11, and to a lesser extent 5a and 9a, as the major Wnt family members expressed in our patient samples. Competitive RT-PCR analysis identified significant differences between the levels of expression of Wnts 9a, 10b and 11 in tissue samples and in primary cell cultures grown as monolayer or in FPCL, where the mRNA levels in tissue > FPCL cultures > monolayer cultures. Real Time PCR data confirmed the down-regulation of Wnt 11 mRNA in DD while Wnt 10b, the most frequently isolated Wnt in DD and control palmar fascia, displayed widely variable expression between the methods of analysis. CONCLUSION: These data indicate that changes in Wnt expression per se are unlikely to be the cause of the observed dysregulation of beta-catenin expression in DD. [Abstract/Link to Full Text]

Vehmas T, Solovieva S, Leino-Arjas P
Radiographic 2D:4D index in females: no relation to anthropometric, behavioural, nutritional, health-related, occupational or fertility variables.
J Negat Results Biomed. 2006;512.
BACKGROUND: The ratio of index finger to ring finger length (2D:4D index) may be an indicator of gonadal hormone exposure, because the differentiation of gonads, fingers and toes is influenced by the same HOXA and HOHD genes. Some previous studies have found significant associations between the 2D:4D index and sexual, psychological or behavioural variables. We studied the usability of the radiographic 2D:4D index as a potential predictor of several features in a large female sample. METHODS: 271 female dentists and 219 teachers (age 45 - 63 years) had their hands radiographed and their right 2nd and 4th fingers measured from the base of the bony proximal phalanxes to the tip of the distal phalanxes to define the radiographic 2D:4D index. The study subjects were classified into two distinctly separate clusters (by using cluster analysis with the K-means algorithm) in each of the following dimensions: anthropometric (including four items), behavioural (five items), nutritional (five items), health-related (seven items), occupational (Karasek job control and job demand scores) and fertility (four items). RESULTS: The radiographic 2D:4D index ranged from 0.845 to 0.981 (mean 0.925, SD 0.021). The intraclass correlation between three radiographers' measurements (31 cases) was 0.971. No differences concerning the 2D:4D index were found between clusters 1 and 2 in any studied dimension, nor did any of the items in clusters have relations with the 2D:4D index when tested separately with bivariate tests. CONCLUSION: Despite the ideal set-up of the measuring possibilities in a relatively large radiographic material the variables currently studied were not dependent on the length of finger bones. It can therefore be questioned whether any real associations between the bony 2D:4D index in adult life and (direct or indirect) hormone dependent effects exist. There may be a publication bias explaining that mostly positive findings have been the previously reported. However, the associations of the 2D:4D index with various features, if present, may be related to the soft parts of fingers rather than to the length of bones. [Abstract/Link to Full Text]

Weickert MO, Reimann M, Otto B, Hall WL, Vafeiadou K, Hallund J, Ferrari M, Talbot D, Branca F, B�gel S, Williams CM, Zunft HJ, Koebnick C
Soy isoflavones increase preprandial peptide YY (PYY), but have no effect on ghrelin and body weight in healthy postmenopausal women.
J Negat Results Biomed. 2006;511.
BACKGROUND: Soy isoflavones show structural and functional similarities to estradiol. Available data indicate that estradiol and estradiol-like components may interact with gut "satiety hormones" such as peptide YY (PYY) and ghrelin, and thus influence body weight. In a randomized, double-blind, placebo-controlled, cross-over trial with 34 healthy postmenopausal women (59 +/- 6 years, BMI: 24.7 +/- 2.8 kg/m2), isoflavone-enriched cereal bars (50 mg isoflavones/day; genistein to daidzein ratio 2:1) or non-isoflavone-enriched control bars were consumed for 8 weeks (wash-out period: 8-weeks). Seventeen of the subjects were classified as equol producers. Plasma concentrations of ghrelin and PYY, as well as energy intake and body weight were measured at baseline and after four and eight weeks of each intervention arm. RESULTS: Body weight increased in both treatment periods (isoflavone: 0.40 +/- 0.94 kg, P < 0.001; placebo: 0.66 +/- 0.87 kg, P = 0.018), with no significant difference between treatments. No significant differences in energy intake were observed (P = 0.634). PYY significantly increased during isoflavone treatment (51 +/- 2 pmol/L vs. 55 +/- 2 pmol/L), but not during placebo (52 +/- 3 pmol/L vs. 50 +/- 2 pmol/L), (P = 0.010 for treatment differences, independent of equol production). Baseline plasma ghrelin was significantly lower in equol producers (110 +/- 16 pmol/L) than in equol non-producers (162 +/- 17 pmol/L; P = 0.025). CONCLUSION: Soy isoflavone supplementation for eight weeks did not significantly reduce energy intake or body weight, even though plasma PYY increased during isoflavone treatment. Ghrelin remained unaffected by isoflavone treatment. A larger and more rigorous appetite experiment might detect smaller differences in energy intake after isoflavone consumption. However, the results of the present study do not indicate that increased PYY has a major role in the regulation of body weight, at least in healthy postmenopausal women. [Abstract/Link to Full Text]

Meirhaeghe A, Thomas S, Ancot F, Cottel D, Arveiler D, Ferri�res J, Amouyel P
Study of the impact of perilipin polymorphisms in a French population.
J Negat Results Biomed. 2006;510.
BACKGROUND: Perilipins are proteins localized at the surface of the lipid droplet in adipocytes, steroid-producing cells and ruptured atherosclerotic plaques playing a role in the regulation of triglyceride deposition and mobilization. We investigated whether perilipin gene polymorphisms were associated with obesity, type 2 diabetes, and their related variables (anthropometric variables, plasma leptin, lipids, glucose and insulin concentrations) in a cross-sectional random sample of 1120 French men and women aged 35 to 65 years old, including 227 obese (BMI >or= 30 kg/m2) and 275 type 2 diabetes subjects. RESULTS: Among 7 perilipin polymorphisms tested, only 2 (rs4578621 and rs894160) of them were frequent enough to be fully investigated and we genotyped the sample using the PCR-RFLP method. No significant associations could be found between any of these polymorphisms and the studied phenotypes. CONCLUSION: The rs4578621 and rs894160 polymorphisms of the perilipin gene are not major genetic determinants of obesity and type 2 diabetes-related phenotypes in a random sample of French men and women. [Abstract/Link to Full Text]

Villacorta H, Bortolotto LA, Arteaga E, Mady C
Aortic distensibility measured by pulse-wave velocity is not modified in patients with Chagas' disease.
J Negat Results Biomed. 2006;59.
BACKGROUND: Experimental studies demonstrate that infection with trypanosoma cruzi causes vasculitis. The inflammatory lesion process could hypothetically lead to decreased distensibility of large and small arteries in advanced Chagas' disease. We tested this hypothesis. METHODS AND RESULTS: We evaluated carotid-femoral pulse-wave velocity (PWV) in 53 Chagas' disease patients compared with 31 healthy volunteers (control group). The 53 patients were classified into 3 groups: 1) 16 with indeterminate form of Chagas' disease; 2) 18 with Chagas' disease, electrocardiographic abnormalities, and normal systolic function; 3) 19 with Chagas' disease, systolic dysfunction, and mild-to-moderate congestive heart failure. No difference was noted between the 4 groups regarding carotid-femoral PWV (8.4 +/- 1.1 vs 8.2 +/- 1.5 vs 8.2 +/- 1.4 vs 8.7 +/- 1.6 m/s, P = 0.6) or pulse pressure (39.5 +/- 7.6 vs 39.3 +/- 8.1 vs 39.5 +/- 7.4 vs 39.7 +/- 6.9 mm Hg, P = 0.9). A positive, significant, similar correlation occurred between PWV and age in patients with Chagas' disease (r = 0.42, P = 0.002), in controls (r = 0.48, P = 0.006), and also between PWV and systolic blood pressure in both groups (patients with Chagas' disease, r = 0.38, P = 0.005; healthy subjects, r = 0.36, P = 0.043). CONCLUSION: Carotid femoral pulse-wave velocity is not modified in patients with Chagas' disease, suggesting that elastic properties of large arteries are not affected in this disorder. [Abstract/Link to Full Text]

Berger I, Stahl S, Rychkova N, Felbor U
VEGF receptors on PC12 cells mediate transient activation of ERK1/2 and Akt: comparison of nerve growth factor and vascular endothelial growth factor.
J Negat Results Biomed. 2006;58.
Vascular endothelial growth factor (VEGF) and endostatin are angiogenic and anti-angiogenic molecules, respectively, that have been implicated in neurogenesis and neuronal survival. Using alkaline phosphatase fusion proteins, we show that the PC12 neuronal cell line contains cell membrane receptors for VEGF but not for endostatin and the collagen XV endostatin homologue. Immunocytochemistry confirmed that proliferating and differentiated PC12 cells express VEGF receptors 1, 2 and neuropilin-1. While no functional effects of VEGF on PC12 cell proliferation and differentiation could be observed, a slight VEGF-induced reduction of caspase-3 activity in differentiated apoptotic PC12 cells was paralleled by transient activation of ERK1/2 and Akt. In direct comparison, nerve growth factor proved to be a strikingly more potent neuroprotective agent than VEGF. [Abstract/Link to Full Text]

Conforti FL, Sprovieri T, Mazzei R, Ungaro C, Tessitore A, Tedeschi G, Patitucci A, Magariello A, Gabriele A, Labella V, Simone IL, Majorana G, Monsurr� MR, Valentino P, Muglia M, Quattrone A
Sporadic ALS is not associated with VAPB gene mutations in Southern Italy.
J Negat Results Biomed. 2006;57.
Mutations in the Cu/Zn superoxide dismutase (Sod1) gene have been reported to cause adult-onset autosomal dominant Amyotrophic Lateral Sclerosis (FALS). In sporadic cases (SALS) de novo mutations in the Sod1 gene have occasionally been observed. The recent finding of a mutation in the VAMP/synaptobrevin-associated membrane protein B (VAPB) gene as the cause of amyotrophic lateral sclerosis (ALS8), prompted us to investigate the entire coding region of this gene in SALS patients. One hundred twenty-five unrelated patients with adult-onset ALS and 150 healthy sex-age-matched subjects with the same genetic background were analyzed. Genetic analysis for all exons of the VAPB gene by DHPLC revealed 5 variant profiles in 83 out of 125 SALS patients. Direct sequencing of these PCR products revealed 3 nucleotide substitutions. Two of these were found within intron 3 of the gene, harbouring 4 variant DHPLC profiles. The third nucleotide variation (Asp130Glu) was the only substitution present in the coding region of the VAPB gene, and it occurred within exon 4. It was found in three patients out of 125. The frequency of the detected exon variation in the VAPB gene was not significantly different between patients and controls. In conclusion, our study suggests that VAPB mutations are not a common cause of adult-onset SALS. [Abstract/Link to Full Text]

Reynolds PJ, Fan W, Andresen MC
Capsaicin-resistant arterial baroreceptors.
J Negat Results Biomed. 2006;56.
BACKGROUND: Aortic baroreceptors (BRs) comprise a class of cranial afferents arising from major arteries closest to the heart whose axons form the aortic depressor nerve. BRs are mechanoreceptors that are largely devoted to cardiovascular autonomic reflexes. Such cranial afferents have either lightly myelinated (A-type) or non-myelinated (C-type) axons and share remarkable cellular similarities to spinal primary afferent neurons. Our goal was to test whether vanilloid receptor (TRPV1) agonists, capsaicin (CAP) and resiniferatoxin (RTX), altered the pressure-discharge properties of peripheral aortic BRs. RESULTS: Periaxonal application of 1 microM CAP decreased the amplitude of the C-wave in the compound action potential conducting at <1 m/sec along the aortic depressor nerve. 10 microM CAP eliminated the C-wave while leaving intact the A-wave conducting in the A-delta range (<12 m/sec). These whole nerve results suggest that TRPV1 receptors are expressed along the axons of C- but not A-conducting BR axons. In an aortic arch--aortic nerve preparation, intralumenal perfusion with 1 microM CAP had no effect on the pressure-discharge relations of regularly discharging, single fiber BRs (A-type)--including the pressure threshold, sensitivity, frequency at threshold, or maximum discharge frequency (n = 8, p > 0.50) but completely inhibited discharge of an irregularly discharging BR (C-type). CAP at high concentrations (10-100 microM) depressed BR sensitivity in regularly discharging BRs, an effect attributed to non-specific actions. RTX (< or = 10 microM) did not affect the discharge properties of regularly discharging BRs (n = 7, p > 0.18). A CAP-sensitive BR had significantly lower discharge regularity expressed as the coefficient of variation than the CAP-resistant fibers (p < 0.002). CONCLUSION: We conclude that functional TRPV1 channels are present in C-type but not A-type (A-delta) myelinated aortic arch BRs. CAP has nonspecific inhibitory actions that are unlikely to be related to TRV1 binding since such effects were absent with the highly specific TRPV1 agonist RTX. Thus, CAP must be used with caution at very high concentrations. [Abstract/Link to Full Text]

Lee PL, West C, Crain K, Wang L
Genetic polymorphisms and susceptibility to lung disease.
J Negat Results Biomed. 2006;55.
Susceptibility to infection by bacterium such as Bacillus anthracis has a genetic basis in mice and may also have a genetic basis in humans. In the limited human cases of inhalation anthrax, studies suggest that not all individuals exposed to anthrax spores were infected, but rather, individuals with underlying lung disease, particularly asthma, sarcoidosis and tuberculosis, might be more susceptible. In this study, we determined if polymorphisms in genes important in innate immunity are associated with increased susceptibility to infectious and non-infectious lung diseases, particularly tuberculosis and sarcoidosis, respectively, and therefore might be a risk factor for inhalation anthrax. Examination of 45 non-synonymous polymorphisms in ten genes: p47phox (NCF1), p67phox (NCF2), p40phox (NCF4), p22phox (CYBA), gp91phox (CYBB), DUOX1, DUOX2, TLR2, TLR9 and alpha 1-antitrypsin (AAT) in a cohort of 95 lung disease individuals and 95 control individuals did not show an association of these polymorphisms with increased susceptibility to lung disease. [Abstract/Link to Full Text]

Burel A, Mouchel T, Odent S, Tiker F, Knebelmann B, Pellerin I, Guerrier D
Role of HOXA7 to HOXA13 and PBX1 genes in various forms of MRKH syndrome (congenital absence of uterus and vagina).
J Negat Results Biomed. 2006;54.
The Mayer-Rokitansky-K�ster-Hauser (MRKH) syndrome refers to the congenital absence or severe hypoplasia of the female genital tract, often described as uterovaginal aplasia which is the prime feature of the syndrome. It is the second cause of primary amenorrhea after gonadal dysgenesis and occurs in approximately 1 in 4500 women. Aetiology of this syndrome remains poorly understood. Frequent association of other malformations with the MRKH syndrome, involving kidneys, skeleton and ears, suggests the involvement of major developmental genes such as those of the HOX family. Indeed mammalian HOX genes are well known for their crucial role during embryogenesis, particularly in axial skeleton, hindbrain and limb development. More recently, their involvement in organogenesis has been demonstrated notably during urogenital differentiation. Although null mutations of HOX genes in animal models do not lead to MRKH-like phenotypes, dominant mutations in their coding sequences or aberrant expression due to mutated regulatory regions could well account for it. Sequence analysis of coding regions of HOX candidate genes and of PBX1, a likely HOX cofactor during M�llerian duct differentiation and kidney morphogenesis, did not reveal any mutation in patients showing various forms of MRKH syndrome. This tends to show that HOX genes are not involved in MRKH syndrome. However it does not exclude that other mechanisms leading to HOX dysfunction may account for the syndrome. [Abstract/Link to Full Text]

Manna I, Valentino P, La Russa A, Condino F, Nistic� R, Liguori M, Clodomiro A, Andreoli V, Pirritano D, Cittadella R, Quattrone A
Genetic variation in the myeloperoxidase gene and cognitive impairment in multiple sclerosis.
J Negat Results Biomed. 2006;53.
There is evidence that multiple sclerosis (MS) may associated with cognitive impairment in 25 to 40% of cases. The gene encoding myeloperoxidase (MPO) is involved in molecular pathways leading to beta-amyloid deposition. We investigated a functional biallelic (G/A) polymorphism in the promoter region (-463) of the MPO gene in 465 patients affected by MS, divided into 204 cognitively normal and 261 impaired. We did not find significant differences in allele or genotype distributions between impaired and preserved MS patients. Our findings suggest that MPO polymorphism is not a risk factor for cognitive impairment in MS. [Abstract/Link to Full Text]

Brauner JS, Clausell N
Neurohumoral, immunoinflammatory and cardiovascular profile of patients with severe tetanus: a prospective study.
J Negat Results Biomed. 2006;52.
INTRODUCTION: Autonomic disturbances in tetanus are traditionally associated with adrenergic variations and/or cardiac dysfunction, based on case report data. The objective of this study was to measure catecholamines, (TNF)-alpha and troponin T relative to and left ventricular ejection fraction (LVEF) in patients with severe tetanus. METHODS: This prospective study was carried out at two general Intensive Care Units and included 21 patients consecutively admitted with severe tetanus. Catecholamines (dopamine, norepinephrine, epinephrine and total catecholamines), tumor necrosis factor (TNF)-alpha and LVEF were assessed during the first week of autonomic instability and following tetanus recovery. Troponin T was measured during autonomic instability only. RESULTS: Mean age of patients was 46 +/- 17 years, median Acute Physiology and Chronic Health Evaluation II (APACHE II) score was 8 (range 1-23). All patients had both blood pressure and heart rate instability. Two patients were recuperated from cardiac arrest. Intensive Care Unit mortality was 14% (3 cases). No increase in total catecholamines or in TNF-alpha levels was observed during autonomic instability or in the recovery period. Six patients had troponin T > 0.01 ng/ml and six had > 0.1 ng/ml. Mean LVEF was similar during autonomic instability and after tetanus recovery, 67 +/- 7% and 65 +/- 7%, respectively. Troponin T levels correlated with pressoric instability during autonomic instability. CONCLUSION: Our study demonstrated that in patients with severe tetanus no significant increased levels of catecholamines or TNF-alpha or evidence of cardiac systolic dysfunction was observed either during autonomic instability or in the recovery period. Elevated values of troponin T detected during autonomic instability were not associated with left ventricular dysfunction. Our data do not support the hypothesis that autonomic disturbances in tetanus are associated with adrenergic variations or cardiac dysfunction. [Abstract/Link to Full Text]

Guerrier D, Mouchel T, Pasquier L, Pellerin I
The Mayer-Rokitansky-K�ster-Hauser syndrome (congenital absence of uterus and vagina)--phenotypic manifestations and genetic approaches.
J Negat Results Biomed. 2006;51.
The Mayer-Rokitansky-K�ster-Hauser (MRKH) syndrome affects at least 1 out of 4500 women and has for a long time been considered as a sporadic anomaly. Congenital absence of upper vagina and uterus is the prime feature of the disease which, in addition, is often found associated with unilateral renal agenesis or adysplasia as well as skeletal malformations (MURCS association). The phenotypic manifestations of MRKH overlap various other syndromes or associations and thus require accurate delineation. Since MRKH manifests itself in males, the term GRES syndrome (Genital, Renal, Ear, Skeletal) might be more appropriate when applied to both sexes. The MRKH syndrome, when described in familial aggregates, seems to be transmitted as an autosomal dominant trait with an incomplete degree of penetrance and variable expressivity. This suggests the involvement of either mutations in a major developmental gene or a limited chromosomal deletion. Until recently progress in understanding the genetics of MRKH syndrome has been slow, however, now HOX genes have been shown to play key roles in body patterning and organogenesis, and in particular during genital tract development. Expression and/or function defects of one or several HOX genes may account for this syndrome. [Abstract/Link to Full Text]

Hansen W, Saft C, Andrich J, M�ller T, Wieczorek S, Epplen JT, Arning L
Failure to confirm influence of methyltetrahydrofolate reductase (MTHFR) polymorphisms on age at onset of Huntington disease.
J Negat Results Biomed. 2005;412.
BACKGROUND: Huntington disease (HD) is a fully penetrant, autosomal dominantly inherited disorder associated with abnormal expansions of a stretch of perfect CAG repeats in the 5' part of the IT15 gene. The number of repeat units is highly predictive for the age at onset (AO) of the disorder. But AO is only modestly correlated with repeat length when intermediate HD expansions are considered. Recently, suggestive association has been reported between a single nucleotide polymorphism (SNP; rs1801131, also known as A1298C) in the methyltetrahydrofolate reductase (MTHFR) gene and AO of HD. 5,10-MTHFR is a key enzyme in the folate metabolism, diverting metabolites toward methylation reactions or nucleotide synthesis. Using part of a previously established study cohort plus additional patients and appropriate statistical methods, we reinvestigated two polymorphisms in the MTHFR gene, C677T and A1298C, as well as their association with AO in 167 HD patients. RESULTS: There was no statistically significant impact on AO for HD patients, neither of MTHFR SNPs nor of the combinations thereof. CONCLUSION: Contrary to previously described evidence the A1298C polymorphism in the MTHFR gene does not appear to modulate AO of HD patients. [Abstract/Link to Full Text]

Wolski KM, Haller E, Cameron DF
Cortactin and phagocytosis in isolated Sertoli cells.
J Negat Results Biomed. 2005;411.
BACKGROUND: Cortactin, an actin binding protein, has been associated with Sertoli cell ectoplasmic specializations in vivo, based on its immunolocalization around the heads of elongated spermatids, but not previously identified in isolated Sertoli cells. In an in vitro model of Sertoli cell-spermatid binding, cortactin was identified around debris and dead germ cells. Based on this observation, we hypothesized that this actin binding protein may be associated with a non-junction-related physiological function, such as phagocytosis. The purpose of this study was to identify the presence and distribution of cortactin in isolated rat Sertoli cells active in phagocytic activity following the addition of 0.8 microm latex beads. RESULTS: Sertoli cell monocultures were incubated with or without follicle stimulating hormone (FSH; 0.1 microg/ml) in the presence or absence of cytochalasin D (2 microM), as an actin disrupter. Cortactin was identified by standard immunostaining with anti-cortactin, clone 4F11 (Upstate) after incubation times of 15 min, 2 hr, and 24 hr with or without beads. Cells exposed to no hormone and no beads appeared to have a ubiquitous distribution of cortactin throughout the cytoplasm. In the presence of cytochalasin D, cortactin immunostaining was punctate and distributed in a pattern similar to that reported for actin in cells exposed to cytochalasin D. Sertoli cells not exposed to FSH, but activated with beads, did not show cortactin immunostaining around the phagocytized beads at any of the time periods. FSH exposure did not alter the distribution of cortactin within Sertoli cells, even when phagocytic activity was upregulated by the presence of beads. CONCLUSION: Results of this study suggest cortactin is not associated with peripheralized actin at junctional or phagocytic sites. Further studies are necessary to clarify the role of cortactin in Sertoli cells. [Abstract/Link to Full Text]

Schlitter AM, Kurz M, Larsen JP, Woitalla D, Mueller T, Epplen JT, Dekomien G
Exclusion of PINK1 as candidate gene for the late-onset form of Parkinson's disease in two European populations.
J Negat Results Biomed. 2005;410.
BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disorder. Recently, mutations in the PINK1 (PARK6) gene were shown to rarely cause autosomal-recessively transmitted, early-onset parkinsonism. In order to evaluate whether PINK1 contributes to the risk of common late-onset PD we analysed PINK1 sequence variations. A German (85 patients) and a Norwegian cohort (90 patients) suffering from late-onset PD were screened for mutations and single nucleotide polymorphisms (SNPs) in the PINK1 gene. Both cohorts consist of well-characterized patients presenting a positive family history of PD in approximately 17%. Investigations were performed by single strand conformation polymorphism (SSCP), denaturating high performance liquid chromatography (DHPLC) and sequencing analyses. SNP frequencies were compared by the chi2 test. RESULTS: Several common SNPs were identified in our cohorts, including a recently identified coding variant (Q115L) in exon 1. Genotyping of the Q115L variation did not reveal significant frequency differences between patients and controls. Pathogenic mutations in the PINK1 gene were not identified, neither in the German nor in the Norwegian cohort. CONCLUSION: Sequence variation in the PINK1 gene appears to play a marginal quantitative role in the pathogenesis of the late-onset form of PD, in German and Norwegian cohorts, if at all. [Abstract/Link to Full Text]

Recent Articles in The Canadian Journal of Clinical Pharmacology

Fetal Alcohol Canadian Expertise Face Research Roundtable TA
POSTER COMPETITION ABSTRACTS September 7, 2007 Winnipeg, Manitoba.
Can J Clin Pharmacol. 2007;14(3):e313-e321.
Poster competition abstracts presented at the 8th Annual Fetal Alcohol Canadian Expertise (FACE) Research Roundtable, held in Winnipeg, Manitoba on Septemeber 7, 2007. [Abstract/Link to Full Text]

McFarlane A, Rajani H
Rural FASD diagnostic services model: Lakeland Centre for Fetal Alcohol Spectrum Disorder.
Can J Clin Pharmacol. 2007;14(3):e301-6.
There are few FASD multi-disciplinary diagnostic teams in rural regions of Canada. Families often have difficulty accessing their services because of the lack of clinics available in Canada and the distance rural residents must travel to access one. Since its grass-roots beginning in 1995, the Lakeland Centre for FASD in north eastern Alberta has developed a community-based FASD diagnostic services model that utilizes the resources available in local communities and enhances the support to individuals and families living far away from urban centers. The article describes the history of the Lakeland Centre for FASD relative to the development of the model and diagnostic process used to diagnose children and adults. Rural adaptations to similar urban models are discussed. Critical elements to rural, in-kind services are also discussed along with ongoing challenges. Acknowledging the change in terminology over the years covered by this article, the term FASD (Fetal Alcohol Spectrum Disorder) is used throughout. [Abstract/Link to Full Text]

Cohen-Kerem R, Bar-Oz B, Nulman I, Papaioannou VA, Koren G
Hearing in children with fetal alcohol spectrum disorder (FASD).
Can J Clin Pharmacol. 2007;14(3):e307-12.
BACKGROUND: Alcohol is the most prevalent human teratogen affected by early exposure of the fetus. Although not listed as a major part of the fetal alcohol spectrum disorder (FASD), different texts list hearing loss as a characteristic of the FASD, based on several small studies. OBJECTIVE: To characterize hearing in children with FASD, diagnosed in the Motherisk Program in Toronto. DESIGN: Cross sectional cohort study. SETTING: Academic referral center. PATIENTS: Children 4-16 years of age that met criteria for FASD, with no other known risk factor for sensorineural hearing loss. A consecutive sample of 41 children (13 girls, mean age 8.9 +/-3 years) was collected. Intervention: Physical examination, audiometry and tympanometry. Outcome measures: External and middle ear pathology on physical examination, pure tone average (PTA), speech reception threshold (SRT), discrimination and tympanometry. Results were compared to reference values in the normal population. Hearing loss equal or greater than 16dB hearing-level in each frequency tested was considered to be clinically significant. RESULTS: A total of 5 (11.2%) of children had hearing loss of at least 16dB hearing-level, mostly unilateral. SRT was within the normal range in 40 (98%) of children with FASD and discrimination was normal in all children. None had auricular or external canal dysmorphology. 14.7% of the children had frequent episodes of acute otitis media.Middle ear effusion was detected in 8 ears (9.8%). CONCLUSIONS: The prevalence of mild sensorineural hearing loss in children diagnosed with FASD (16dB hearing-level or greater) was not higher than expected in this age group. However, because children with FASD are academically and behaviorally challenged, early detection of hearing loss and early intervention is warranted. [Abstract/Link to Full Text]

Kao RL, Kelly LM
Fatal exertional heat stroke in a patient receiving zuclopenthixol, quetiapine and benztropine.
Can J Clin Pharmacol. 2007;14(3):e322-5.
OBJECTIVE: To report a case of fatal exertional heat stroke associated with the use of zuclopenthixol, quetiapine and benztropine. CASE SUMMARY: A 36-year-old male with a history of schizophrenia and bipolar disease was working as a roofer during the third day of a heat wave. His medications included zuclopenthixol, quetiapine, benztropine, carbamazepine and levothyroxine. He developed loss of consciousness late in the day and presented to hospital with a Glasgow Coma Scale 3 and a rectal temperature of 42.2 degrees C. He progressed to severe multiple organ dysfunction and asystole, and expired the following morning. Neuroleptic and anticholinergic agents have long been associated with heat alteration, but there are few reports involving the newer antipsychotic agents. Physicians and pharmacists should ensure that appropriate counseling is given to patients receiving these medications regarding early recognition of signs and symptoms and prompt treatment of heat related illness and heat stroke. [Abstract/Link to Full Text]

Bradette M, Wawer AR, Balshaw R, Kelly S, Barbeau M, Sambrook R
Characteristics, diagnostic and symptom profile of patients receiving tegaserod in routine clinical practice in Canada.
Can J Clin Pharmacol. 2007;14(3):e291-300.
OBJECTIVE: This study was designed to assess the diagnostic and symptom profile of patients receiving tegaserod in routine clinical practice, and to identify their demographic characteristics, as well as the association between these characteristics and diagnosis. METHODS: This prospective, observational study collected data from physicians on the symptoms and/or diagnosis, age range and gender for patients to whom they prescribed tegaserod. Details of the physician characteristics included whether they were a family physician or a specialist, and the region of Canada in which their practice was located. RESULTS: A total of 500 patients were enrolled at 85 sites in Canada. The majority (85%) of the patients were enrolled by family physicians, and the remainder by community-based specialists. The patients were predominantly female (87%) and the highest percentages were in the 35-44 (23%) and 45-54 (25%) age groups. Nearly all patients (96%) were prescribed tegaserod on the basis of both symptoms and diagnosis. The most frequently reported symptoms were abdominal pain and/or discomfort (87%), bloating (80%) and constipation (75%). Most patients (57%) presented with all three of these symptoms. Constipation-predominant Irritable Bowel Syndrome (IBS-C) was the most common diagnosis (55%), followed by IBS alternating between constipation and diarrhea (IBS-A) (23%). Based on this, 67% of patients were given tegaserod strictly according to the label, although it was appropriately prescribed to 87%. CONCLUSIONS: In Canada, tegaserod is prescribed to patients with symptoms of abdominal pain and/or discomfort, bloating and constipation. Most of them will also have a diagnosis of either IBS-C or IBS. It is generally being prescribed appropriately. [Abstract/Link to Full Text]

McCarthy L, Dolovich L, Haq M, Thabane L, Kaczorowski J
Frequency of risk factors that potentially increase harm from medications in older adults receiving primary care.
Can J Clin Pharmacol. 2007;14(3):e283-90.
BACKGROUND: Many circumstances elevate patients, especially older adults, risk for drug-related morbidity and misadventures. Understanding the frequency of these situations can help with the design of initiatives to address or alter these circumstances with the aim of reducing medication therapy-related concerns and associated expenditures. OBJECTIVE: To describe the frequency of circumstances that may place older adults at higher risk for drug-related morbidity and misadventures in a large sample of elderly patients visiting family medicine clinics. METHODS: Elderly adults at 7 family medicine practices across Ontario self-completed the 10-item Medication Risk Questionnaire (MRQ). RESULTS: Surveys were completed by 907 patients, with a mean age of 72.4 (SD 10.7) years and a mean number of 4.8 medical conditions (SD 2.3; min-max: 0-14). Many subjects were taking multiple medications (mean 6.9 (SD 3.8; min-max: 0-21)) and over 90% of respondents reported at least one indicator that potentially increases their risk of drug-related morbidity. CONCLUSION: Number of medications, number of medical conditions and number of daily medication doses were the most frequently observed risks for medication-related issues in this large sample of elderly patients visiting family medicine clinics. [Abstract/Link to Full Text]

Lum EY, Sharpe HM, Nilsson C, Andrews EM, Tsuyuki RT, Mayers I, Cowie RL
Urban and rural differences in the management of asthma amongst primary care physicians in Alberta.
Can J Clin Pharmacol. 2007;14(3):e275-82.
BACKGROUND: Inconsistencies in rural and urban health care exist; however, little has been done to evaluate the potential differences in asthma management. OBJECTIVE: To compare asthma management in rural versus urban primary care physician practices. METHODS: Forty-two of 136 consenting primary care physicians were randomly selected for chart review. The charts of 3072 patients diagnosed with asthma based on the ICD-9 Classification of Diseases were reviewed. RESULTS: Standards of asthma care were compared between rural and urban primary care physicians. 2671 patients (87%) were cared for by urban physicians and 401 patients (13%) by rural physicians. Greater proportions of male and pediatric patients were found in the rural group. Rural patients made more emergency department or hospital visits than urban patients. Rural physicians performed more pulmonary function tests and made more referrals to other healthcare specialists. Urban patients had more asthma symptoms and triggers documented and used peak flow monitoring more often. Urban physicians provided more asthma education and prescribed more oral corticosteroids and antibiotics. Overall, rates of referral, use of spirometry and use of written action plans were low globally. CONCLUSIONS: Our study indicates that the management of asthma in the rural settings is comparable to that of urban settings. Improvements in the areas of pulmonary function testing, asthma education and use of written action plans are necessary in both settings. [Abstract/Link to Full Text]

Caswell M, Thompson WO, Kanapka JA, Galt DJ
The time course and effect on serum electrolytes of oral sodium phosphates solution in healthy male and female volunteers.
Can J Clin Pharmacol. 2007;14(3):e260-74.
BACKGROUND: Although oral sodium phosphates solution is used extensively for bowel preparation, the pharmacokinetic profile of 2 x 45 mL oral sodium phosphates solution has not been reported. OBJECTIVES: The primary objective of this study was to evaluate the time course and degree of electrolyte shifts in two age groups and two gender groups following administration of oral sodium phosphates solution. Secondary objectives included evaluation of electrocardiograms, postural blood pressure, standard serum chemistry and hematology panels, and adverse experiences. METHODS: Twenty-four healthy adult volunteers were divided equally into groups based on age and gender. Each received 2 x 45 mL oral sodium phosphates solution at 7:00pm (hour 0) and at 7:00am (hour 12). Serum electrolytes were measured at sixteen different time points. RESULTS: Mean serum phosphate concentrations exceeded the upper normal limit within 1 hour following the first dose, peaking at 3 hours (6.26 mg/dL; p < 0.0001) before decreasing. Following the second dose at 12 hours, mean serum phosphate concentrations peaked at 14 hours (6.86 mg/dL; p < 0.0001), before decreasing to normal limits by hour 24. Mean serum sodium, potassium, and calcium concentrations fluctuated within the normal range. However, serum sodium concentrations peaked at 1 hour following the second dose of phosphate, showing a statistically significant (p < 0.0001) increase of 2.4% from baseline to 144.8 mmol/L. No clinically significant changes in ECG were observed. Mean reductions in supine and standing systolic blood pressure were not associated with postural change. No subject had postural decreases in systolic blood pressure of greater than 20 mmHg. CONCLUSIONS: Administration of 2 x 45 mL oral sodium phosphates solution 12 hours apart with proper hydration caused transient serum electrolyte shifts, which were clinically insignificant and resolved within 24 hours. [Abstract/Link to Full Text]

The 3rd Sickkids Foundation forum on complementary and alternative health care and paediatrics.
Can J Clin Pharmacol. 2007;14(2):e87-e102.
The 3rd Sickkids Foundation forum on complementary and alternative health care and paediatrics. May 4, 2007 - Toronto Hosted by: Sickkids Foundation. [Abstract/Link to Full Text]

Nair KM, Levine MA, Lohfeld LH, Gerstein HC
"I take what I think works for me": a qualitative study to explore patient perception of diabetes treatment benefits and risks.
Can J Clin Pharmacol. 2007;14(2):e251-9.
BACKGROUND: Diabetes is impacting more and more people each year. A key aspect of disease management is patient adherence to prescribed treatments. Treatment adherence is influenced by many factors, including the understanding of a treatment's benefits and risks. OBJECTIVE: This study sought to describe the experience of benefit and risk assessment for people with type 2 diabetes when making treatment decisions. METHODS: This study utilized qualitative research methods. Individual interviews were conducted using a semi-structured interview guide. Both purposeful and theoretical sampling was used. A grounded theory approach was employed to facilitate data collection and analysis. RESULTS: The 18 study participants were on varying treatment regimens for diabetes (diet therapy, oral medications, and insulin). Many people felt that they had not received enough information about the benefits and risks of treatment at the point of decision-making and later sought this information on their own. Participants did not seem to consciously assess treatment benefits and risks when treatments were prescribed or suggested, but rather continued to make decisions after the clinical encounter by means of experimentation or experience with treatments. In general, benefits and risks were conceptualized very broadly, and some people were not able to verbally articulate their perceptions of treatment benefits and risks. CONCLUSION: Patients' assessment of treatment benefits and risks is an ongoing, often unconscious process that requires continuous interaction with the health care system. Access to information and an opportunity to discuss treatment options with health care providers are important to people with diabetes when making treatment decisions. [Abstract/Link to Full Text]

Lynd LD, Goeree R, Crowther MA, O'Brien BJ
A probabilistic cost-effectiveness analysis of enoxaparin versus unfractionated heparin for the prophylaxis of deep-vein thrombosis following major trauma.
Can J Clin Pharmacol. 2007;14(2):e215-26.
BACKGROUND: In the absence of major contraindications, treatment guidelines recommend that, following a major traumatic event, all patients receive low molecular weight heparin (e.g. enoxaparin) as thromboprophylaxis for the prevention of deep vein thrombosis (DVT). OBJECTIVE: To estimate the incremental cost-effectiveness of enoxaparin versus low dose unfractionated heparin (UH) for the prophylaxis of DVT following major trauma. METHODS: Using probabilistic decision-analytic modeling, we estimated the incremental cost-effectiveness of enoxaparin versus unfractionated heparin for the prophylaxis of DVT following moderate to severe trauma (injury severity score > or = 9) over a life-time time horizon from the perspective of the health care payer. Cost effectiveness was calculated based on both the incremental cost (DeltaC) per DVT averted and the DeltaC per life year gained (LYG). RESULTS: The incremental cost of enoxaparin relative to UH was C$90, and the incremental effectiveness was 0.085 DVTs averted and -0.13 LYG. This resulted in an incremental cost-effectiveness ratio of C$1,059 per DVT averted, and the conclusion that UH is the dominant strategy in terms of LYG. In addition to the probabilistic analysis, one-way and two-way sensitivity analysis revealed that the model was most sensitive to variation in the discount rate (3% - 7%), but that UH remained the dominant strategy in terms of life years independent of the parameter estimates. CONCLUSIONS: Although enoxaparin appears to be a cost-effective alternative when considering the intermediate endpoint of DVTs averted, it may be dominated by UH in terms of LYG due to the higher incidence of major bleeds in patients receiving enoxaparin versus UH. [Abstract/Link to Full Text]

4th Canadian Therapeutics Congress - The virtuous circle: Therapeutics from molecule to patient to p.
Can J Clin Pharmacol. 2007;14(2):e104-204.
Presentations of abstracts from Canadian Association for Population Therapeutics (CAPT), Canadian College of Clinical Pharmacy (CCCP) and Canadian Society for Clinical Pharmacology(CSCP) 4th Canadian Therapeutics Congress May 27-30,2007 Halifax, Nova Scotia. [Abstract/Link to Full Text]

Chen N, Aleksa K, Woodland C, Rieder M, Koren G
Prevention of ifosfamide nephrotoxicity by N-acetylcysteine: clinical pharmacokinetic considerations.
Can J Clin Pharmacol. 2007;14(2):e246-50.
BACKGROUND: Ifosfamide, which is routinely given to treat a variety of solid tumours in children, causes serious nephrotoxicity in treated children. Previous in vitro studies have shown that depletion of intracellular glutathione can enhance ifosfamide nephrotoxicity. Presently, there is no therapeutic agent that can prevent ifosfamide nephrotoxicity. We have recently shown that N-acetylcysteine (NAC) at 0.4 mM prevents ifosfamide-induced nephrotoxicity in vitro. However, this in vitro concentration of NAC needed to be compared to those used in human pharmacokinetic studies since the in vitro pharmacological effect of a compound is achieved at concentrations exceeding those used in clinical. OBJECTIVE: The aim of the present study was to verify whether the in vitro concentration of NAC, which was found to protect renal cells from ifosfamide-induced damages, is comparable to the currently used clinical concentrations. METHODS: A systematic literature review of all published papers reporting on the pharmacokinetics of NAC in humans was conducted. RESULTS: The steady state concentrations of NAC administered intravenously to humans ranged from 0.04 mM to 0.9 mM and the urine concentration of NAC was 2 mM. CONCLUSION: This suggests that the concentration chosen for in vitro studies is well within the range of clinical levels. [Abstract/Link to Full Text]

Chiang TH, Walt JG, McMahon JP, Mansfield JE, Simonyi S
Real-world utilization patterns of cyclosporine ophthalmic emulsion 0.05% within managed care.
Can J Clin Pharmacol. 2007;14(2):e240-5.
BACKGROUND: Cyclosporine 0.05% ophthalmic emulsion (Restasis) is a treatment for dry eye disease. OBJECTIVES: To examine patients' cyclosporine 0.05% utilization patterns by analyzing prescription fill data. METHODS: A retrospective analysis with a large de-identified longitudinal patient database was conducted. Participants in the study had 1 prescription fill for cyclosporine 0.05% during a 3-month "enrollment" period from January 1 to March 31, 2004, and at least 1 refill within the following 12 months. Continuing patients had at least 1 cyclosporine 0.05% prescription fill, and new patients had none during 12 months prior to the "enrollment" period. Daily, monthly, and annual utilizations were assessed. RESULTS: 38,164 patients met the inclusion criteria. The majority of patients were female (82%), 50 years or older (77%), and new to therapy (59%). The FDA-recommended use is 2 vials daily (2 trays/month, each tray containing 32 vials) to receive the prescribed dosage of 1 drop in each eye twice daily. Prescription refill patterns demonstrated 73% of patients used 1 tray/month; similarly, 80% of the patients used 11 trays or less per year. Daily utilization differed between continuing and new patients. New patients had a bimodal use pattern. Over 30% were using > or = 1.75 vials/day and approximately 55% were using 0.25 to 1.25 vials/day. The majority of continuing patients (approximately 80%), however, used 0.25 to 1.25 vials/day. CONCLUSIONS: Most patients used about 1 vial per day, less than the labeled 2 per day. The cost to managed care for cyclosporine 0.05% ophthalmic emulsion may be less than anticipated. Key words: Cyclosporine 0.05% ophthalmic emulsion, longitudinal patient database, cost, utilization. [Abstract/Link to Full Text]

L�vy E, Agbokou C, Ferreri F, Chouinard G, Margolese HC
Topiramate-induced weight loss in schizophrenia: a retrospective case series study.
Can J Clin Pharmacol. 2007;14(2):e234-9.
OBJECTIVE: Atypical antipsychotics have been associated with weight gain. This study examines the efficacy of adjunctive topiramate in patients with schizophrenia and schizoaffective disorder with antipsychotic-induced weight gain. METHODS: A 2-year retrospective case analysis was performed in all 300 patients of the outpatient Special Follow-up Clinic for chronic schizophrenia and related psychoses at the Allan Memorial Institute, McGill University Health Centre (Montreal, Canada), a tertiary care University teaching hospital. RESULTS: 10 patients met study inclusion criteria. Mean daily topiramate dose was 197.5 mg (A+/-77) (range, 125-400 mg). Topiramate produced continued weight loss throughout the study duration without tolerance. Patients treated for 6 months and more had significantly higher Body Mass Index (BMI) differences than those treated for shorter durations (BMI-d6 months=-4.7A+/-2.4; BMI-d2 months=-3.2A+/-2.3; P=0.015). BMI changes were similar across genders. CONCLUSION: This study supports topiramate use to target weight loss in stable overweight schizophrenic patients as a potential therapy that requires further investigation. [Abstract/Link to Full Text]

Cohen J, Adams S, Patten S
No association found between patients receiving isotretinoin for acne and the development of depression in a Canadian prospective cohort.
Can J Clin Pharmacol. 2007;14(2):e227-33.
BACKGROUND: There has been concern that the use of isotretinoin to treat acne may lead to depression. To date, research has not conclusively determined if this concern is warranted when contemplating the use of isotretinoin. OBJECTIVE: This study investigated the impact of isotretinoin use for patients with acne on mood status. The hypothesis was that an association exists between the use of isotretinoin and the development of depression, aside from acne severity. METHODS: We studied the relationship between isotretinoin and depression using a prospective, controlled, cohort design. The study was conducted in a community dermatology clinic. The exposed cohort consisted of consenting patients who were initiating isotretinoin treatment for acne. Patients were either treated with isotretinoin (Acutane�) therapy (study group) (N=100) or by oral (N=41) or topical acne therapy (control group) (N=59). The Center for Epidemiologic Studies Depression scale and the Zung Depression Status Inventory were used to assess depression both at baseline and after 2 months of prescribed use of isotretinoin or a control medication (topical or oral antibiotics). RESULTS: There was no correlation between isotretinoin use and the development of depression, based on either the Centre for Epidemiologic Studies Depression scale (Fisher�s exact test, P=0.497) or Zung Depression Status Inventory (ANOVA; F=1.4, P=0.2). CONCLUSION: Isotretinoin does not appear to be associated with the development of depression. Thus, denying patients with significant acne an effective medication for fear of developing depression may not be indicated at this point in time. [Abstract/Link to Full Text]

Tran YB, Frial T, Miller PS
Statin's cost-effectiveness: a Canadian analysis of commonly prescribed generic and brand name statins.
Can J Clin Pharmacol. 2007;14(2):e205-14.
BACKGROUND: Generic statins may be considered as a compelling treatment option for managing dyslipidemia, due to their reduced cost, compared to their brand name equivalent. However, further assessment is needed to determine whether using a particular generic statin is more cost-effective relative to other brand-name statins. OBJECTIVE: The purpose of this study is to compare the cost-effectiveness of the most commonly prescribed statins in Canada with respect to 1) lowering low-density lipoprotein cholesterol level (LDL-C) and 2) achieving National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) LDL-C goal. METHODS: The study was conducted from the perspective of Canadian payers over a 1-year time horizon. Clinical data were obtained from the STELLAR trial (n=2268) in which patients received fixed doses of rosuvastatin, atorvastatin, simvastatin and pravastatin. Brand and generic drug costs were based on wholesale acquisition costs. Relative cost-effectiveness was assessed using the net monetary benefit approach (NMB), which allows probabilistic cost-effectiveness comparison of the various treatment options over a wide range of willingness-to-pay (WTP) values for a unit of clinical effect. RESULTS: Rosuvastatin 10mg was the most cost-effective statin over the largest range of WTP values. Pravastatin 10mg was cost-effective when the clinical outcomes had little or no monetary value. Rosuvastatin 20 mg was more cost-effective at the highest end of the WTP spectrum. CONCLUSION: The result of this analysis provides evidence that prescribing generic statins in Canada does not necessarily translate into the most cost-effective option for treating dyslipidemia; especially as the monetary value of 1% decrease in LDL-C or patients achieving NCEP ATP III target increases. [Abstract/Link to Full Text]

Koren G
Complementary and alternative medicine for children--time to enter the evidence-based era.
Can J Clin Pharmacol. 2007;14(2):e103.
Children receive natural health products and other forms of complementary and alternative medicine that lack evidence of safety and efficacy. The Toronto Hospital for Sick Children has established a national research support program to address this knowledge gap. On May 4, 2007, the Toronto Scientific forum presents cutting edge research in this area. [Abstract/Link to Full Text]

Koren G, Nulman I
Critical appraisal rounds: a new training model for clinical pharmacologists.
Can J Clin Pharmacol. 2007;14(1):e81-5.
A new training model for critical appraisal in therapeutics is presented. Postgraduate trainees in Clinical Pharmacology are assigned to critically appraise scientific papers that faculty members were asked to review by Journals. The manuscripts were discussed in group rounds, allowing teaching of all aspects of therapeutics research. Formal evaluation of this model by trainees revealed high marks for "critical thinking", "learning study design", and "how to write/not to write". [Abstract/Link to Full Text]

Kozyrskyj AL, Raymond C, Racher A
Use of celecoxib immediately post marketing in Canada: acute or chronic pain?
Can J Clin Pharmacol. 2007;14(1):e70-80.
OBJECTIVES: The diffusion of innovations theory suggests that early users of innovations influence others. This study was undertaken to apply the diffusion of innovations theory to the prescribing of celecoxib and to determine if prescriber and patient characteristics differed amongst early use of celecoxib for acute pain versus chronic musculoskeletal conditions. METHODS: Using Manitoba�s population-based prescription and health care databases, diffusion time from market availability to first prescription for celecoxib was determined for each prescriber. The diffusion of prescribing curves for celecoxib in acute pain versus chronic musculoskeletal conditions were compared. Separately for acute and chronic conditions, the likelihood of being an early or late prescriber or user of celecoxib was determined according to physician factors (specialty and place of training) and patient demographics. This multivariate analysis was completed using polytomous logistic regression, with majority prescribers as the reference. RESULTS: The use of celecoxib for chronic musculoskeletal conditions demonstrated faster diffusion than for acute pain. The majority of early use of celecoxib was for chronic conditions; however 36% of first prescriptions were for acute pain, including the treatment of back pain and injuries. Early prescribers of celecoxib for acute pain were more likely than majority prescribers to be general practitioners (OR = 2.24, 95%CI: 1.53-3.29) and have hospital affiliations (OR=1.54, 95%CI: 1.04-2.27). Early users of celecoxib for chronic conditions were less likely to be low income (OR=0.56, 95%CI: 0.35-0.91). CONCLUSIONS: Immediately after market release in Canada, celecoxib was commonly prescribed for the treatment of acute pain; these prescriptions were associated with general practitioners and hospital affiliation status. [Abstract/Link to Full Text]

Goodwin J, Rieder S, Rieder MJ, Matsui D
Counseling regarding pregnancy--related drug exposures by family physicians in Ontario.
Can J Clin Pharmacol. 2007;14(1):e58-69.
BACKGROUND: Family physicians may play a significant role in providing information to their patients on the effects of medication exposure during pregnancy. Women must receive accurate information, as unrealistic perception of teratogenic risk may lead to inadequate treatment of maternal disease or termination of otherwise wanted pregnancies. OBJECTIVES: To collect data on the current practices of family physicians in providing information regarding pregnancy-related drug exposures, in particular, their confidence in providing counseling and their sources of information. METHODS: A mailed survey was sent to a random sample of family physicians in Ontario. Outcome measures included the proportion of family physicians that feel confident in providing counseling regarding drugs in pregnancy, most common resources, barriers to counseling and preferences for future educational programs. RESULTS: Of the 756 surveys, 400 (53%) were returned, 265 (66%) by practicing physicians caring for women of childbearing age. Most (80.3%) felt confident in providing counseling, though a majority (56%) stated that available sources of information are not adequate. The most commonly consulted source was the Motherisk Program (62%). Lack of evidence-based information was cited as the major barrier. CONCLUSIONS: Although family physicians were confident in providing counseling to pregnant patients with regards to drug use, more than one-half thought that the available sources of information are not adequate. The dissemination of more evidence-based information in this field is needed. [Abstract/Link to Full Text]

Carleton BC, Smith MA, Gelin MN, Heathcote SC
Paediatric adverse drug reaction reporting: understanding and future directions.
Can J Clin Pharmacol. 2007;14(1):e45-57.
BACKGROUND: Severe adverse drug reactions (ADRs) are an important cause of childhood morbidity and mortality. 95% of ADRs are likely not reported, less than 25% of marketed drugs can be advertised as safe and effective in children; yet over 50% of Canadian children receive prescription drugs annually. OBJECTIVES: To increase understanding of reported ADRs in Canadian children. METHODS: A retrospective analysis of 1193 suspected ADRs reported to Health Canada (January 1998 - May 2002). These data were a paediatric subset of the Canadian Adverse Drug Reaction Information System database. RESULTS: 58.6% of ADRs were for children over 13 years. 61% of reports were defined by Health Canada as serious. Case outcomes include: death (n=41) and recovered with sequelae (n=14). 4 reports of interacting drugs had fatal outcomes. Drugs most frequently cited include: isotretinoin (n=56), paroxetine (n=42), methylphenidate (n=41), amoxicillin (n=40), and valproic acid (n=32). Most frequent reaction descriptors include: psychiatric disorders (isotretinoin and paroxetine) and nervous system disorders (valproic acid, bupropion and carbamazepine). Causal links between suspected ADRs and clinical outcomes have not been established. CONCLUSIONS: Current ADR reporting is insufficient to improve patient safety. More detailed reporting, including case outcomes, is needed. Mandatory ADR reporting is unlikely to improve underreporting. Trained surveillance personnel located in major health centres and solely dedicated to ADR reporting may provide a more accurate determination of ADRs in Canadian children. [Abstract/Link to Full Text]

Du W, Levine M, Wang L, Zhang Y, Yi C, Wang H, Wang X, Xie H, Xu J, Jin H, Wang T, Huang G, Wu Y
Building a structured monitoring and evaluating system of postmarketing drug use in Shanghai.
Can J Clin Pharmacol. 2007;14(1):e40-4.
In order to understand a drug's full profile in the post-marketing environment, information is needed regarding utilization patterns, beneficial effects, ADRs and economic value. China, the most populated country in the world, has the largest number of people who are taking medications. To begin to appreciate the impact of these medications, a multifunctional evaluation and surveillance system was developed, the Shanghai Drug Monitoring and Evaluative System (SDMES). Set up by the Shanghai Center for Adverse Drug Reaction Monitoring in 2001, the SDMES contains three databases: a population health data base of middle aged and elderly persons; hospital patient medical records; and a spontaneous ADR reporting database. Each person has a unique identification and Medicare number, which permits record-linkage within and between these three databases. After more than three years in development, the population health database has comprehensive data for more than 320,000 residents. The hospital database has two years of inpatient medical records from five major hospitals, and will be increasing to 10 hospitals in 2007. The spontaneous reporting ADR database has collected 20,205 cases since 2001 from approximately 295 sources, including hospitals, pharmaceutical companies, drug wholesalers and pharmacies. The SDMES has the potential to become an important national and international pharmacoepidemiology resource for drug evaluation. [Abstract/Link to Full Text]

Tomalin A
Drugs used in pregnancy: the regulatory process.
Can J Clin Pharmacol. 2007;14(1):e5-9. [Abstract/Link to Full Text]

Benegbi M
45 years later...where do we stand?
Can J Clin Pharmacol. 2007;14(1):e37-9. [Abstract/Link to Full Text]

Moretti M
Collection and analysis of drug safety data in pregnancy.
Can J Clin Pharmacol. 2007;14(1):e34-6. [Abstract/Link to Full Text]

Farine D
Challenges in drug use near term and during delivery.
Can J Clin Pharmacol. 2007;14(1):e29-33. [Abstract/Link to Full Text]

Koren G, MacLeod S, Davis D
Drugs in pregnancy: acknowledging challenges--finding solutions.
Can J Clin Pharmacol. 2007;14(1):e2-4. [Abstract/Link to Full Text]

Finkelstein Y, Rezvani M, Garcia-Bournissen F, Nurmohamed L, Koren G
Inactive pharmaceutical ingredients : implications for pregnancy.
Can J Clin Pharmacol. 2007;14(1):e17-28. [Abstract/Link to Full Text]

Koren G
The way women perceive teratogenic risk.
Can J Clin Pharmacol. 2007;14(1):e10-6. [Abstract/Link to Full Text]

Recent Articles in Journal of Pharmacy & Pharmaceutical Sciences

Roger SD, Mikhail A
Biosimilars: opportunity or cause for concern?
J Pharm Pharm Sci. 2007;10(3):405-10.
Biopharmaceuticals are drug products containing biotechnology-derived proteins as active substances, and have revolutionised the treatment of many diseases. A number of biopharmaceutical patents are due to expire in the next few years, or have already expired. The subsequent production of follow-on products, or 'biosimilars' has aroused interest within the pharmaceutical industry as biosimilar manufacturers strive to obtain part of an already large and rapidly-growing market. The potential opportunity for price reductions versus the originator biopharmaceuticals remains to be determined, as the advantage of a slightly cheaper price may be outweighed by the hypothetical increased risk of side-effects from biosimilar molecules that are not exact copies of their originators. This review focuses on the issues surrounding biosimilars, including manufacturing, quality control, clinical efficacy and side effects, and how government and industry regulations are evolving to deal with these topics. [Abstract/Link to Full Text]

Magalh�es PO, Lopes AM, Mazzola PG, Rangel-Yagui C, Penna TC, Pessoa A
Methods of endotoxin removal from biological preparations: a review.
J Pharm Pharm Sci. 2007;10(3):388-404.
PURPOSE: Endotoxins, also called lipopolysaccharides (LPS), are major contaminants found in commercially available proteins or biologically active substances, which often complicate study of the biological effects of the main ingredient. The presence of small amounts of endotoxin in recombinant protein preparations can cause side effects in host organism such as endotoxin shock, tissue injury, and even death. Due to these reactions, it is essential to remove endotoxins from drugs, injectables, and other biological and pharmaceutical products. An overview of this subject is provided by this article. METHODS: An extensive review of literature with regard to methods for removal of endotoxin from biotechnological preparations was carried out. RESULTS: A short history of endotoxin is presented first. This is followed by a review of chemical and physical properties of endotoxin and its pathophysiological effects when the body is exposed to LPS excessively or systemically. The techniques of endotoxin determination and interaction of endotoxin with proteins is also presented, taking into consideration the established techniques as well as the state of the art technology in this field. A review of techniques of endotoxin removal from biotechnological preparations is described, emphasizing how endotoxin removal can be carried out in an economical way based on a number of processes discussed in the literature (e.g., adsorption, two-phase partitioning, ultrafiltration and chromatography). Different methods are mentioned with relatively high protein recoveries; however, special attention is given to two-phase aqueous micellar systems, which are valuable tools for endotoxin removal from pharmaceutical proteins on a small scale because they provide a mild environment for biological materials. CONCLUSIONS: Efficient and cost-effective removal of endotoxins from pharmaceutical and biotechnology preparations is challenging. Despite development of novel methods, such as the two-phase aqueous micellar systems, in recent years, more research is needed in this field. [Abstract/Link to Full Text]

Rajaraman G, Wang G, Smith HJ, Gong Y, Burczynski FJ
Effect of diltiazem isomers and thiamine on piglet liver microsomal peroxidation using dichlorofluorescein.
J Pharm Pharm Sci. 2007;10(3):380-7.
PURPOSE: We investigated a potential hepatoprotective role of d-cis diltiazem, l-cis diltiazem, thiamine and the combination d-cis diltiazem and thiamine against lipid peroxidation in a piglet liver microsomal model. A modified in vitro dichlorofluorescein assay was developed to assess the extent of peroxidative damage induced by reactive oxygen species in the piglet liver microsomal fraction. METHODS: Microsomal membrane fraction, obtained from 3 week old female piglets, was treated with either the biologically vasoactive d-cis diltiazem or the non-vasoactive stereoisomer l-cis diltiazem (5-1000 microM) for 1 hour at 37 degrees C followed by one hour incubation with the free radical generator AAPH (2,2'-azobis-(2-amidinopropane) dihydrochloride; 1 mM) to initiate lipid peroxidation. In a separate study, piglet liver microsomes were pre-treated with d-cis diltiazem (50 or 500 microM) and thiamine (10-100 microM) to assess the antioxidant activity of the combination. RESULTS: A dose dependant inhibition of membrane lipid peroxidation was observed with d-cis diltiazem (p<0.05) but not with l-cis diltiazem, suggesting that diltiazem is stereospecific in protecting against microsomal lipid peroxidation. Combining diltiazem with thiamine further protected microsomes against lipid peroxidation compared to use of individual drugs. CONCLUSION: We conclude that diltiazem and the combination of diltiazem and thiamine offers a hepatoprotective effect against free radicals. [Abstract/Link to Full Text]

Zakeri-Milani P, Valizadeh H, Tajerzadeh H, Azarmi Y, Islambolchilar Z, Barzegar S, Barzegar-Jalali M
Predicting human intestinal permeability using single-pass intestinal perfusion in rat.
J Pharm Pharm Sci. 2007;10(3):368-79.
PURPOSE: The aim of the study was the prediction of human intestinal permeability and fraction absorbed of oral dose using single-pass intestinal perfusion technique (SPIP) in rats. METHODS: Permeability coefficients in anaesthetized rats were determined for 14 compounds. Drug solution in phosphate buffered saline (PBS) was perfused through a ingle-pass intestinal perfusion (SPIP) with flow rate of 0.21 ml/min and samples were taken from outlet tubing at different time points up to 90 min. Phenol red was used as a non-absorbable marker to correct water flux through the segment. Drug concentrations in samples were determined using HPLC and permeability coefficients (Peff) were calculated. RESULTS: The examined compounds demonstrated approximately 12.5 fold difference in magnitude for rat permeability coefficients among themselves. These values were compared with published data for human intestinal permeability, and a strong correlation was found between Peff (rat) and Peff (human); (Peff (human) = 11.04 Peff (rat) - 0.0003; R2= 0.93, P<0.0001). Subsequently the fraction dose absorbed in human (Fa) was estimated and predicted after oral dosing considering Fa(human)=1-e - 38450Peff(rat) (R2= 0.91, P<0.0001). CONCLUSIONS: Considering the high correlation of rat Peff values with those of human we conclude that the SPIP could be utilized with precision to predict the human intestinal permeability. It may also be used as a reliable technique to predict the fraction of dose absorbed following oral administration of drug in solution or regular release dosage form in human. [Abstract/Link to Full Text]

Gupta U, Agashe HB, Jain NK
Polypropylene imine dendrimer mediated solubility enhancement: effect of pH and functional groups of hydrophobes.
J Pharm Pharm Sci. 2007;10(3):358-67.
Dendrimers today are known for their three dimensional, monodispersed, highly branched, macromolecular nano-scopic architecture with number of reactive end groups. Dendrimers have been reported to act as solubilizing agents to host both hydrophilic and hydrophobic drugs. The present study was performed to investigate the effect of pH on poly(propylene) imine dendrimers (5.0G) mediated solubility enhancement of hydrophobes differing in functional groups (pKa). Weakly basic, (famotidine, -NH2 functional group; pKa 7.1), weakly acidic (indomethacin, -COOH functional group; pKa 4.5) and amphoteric (amphotericin B, -COOH and -NH2 functional groups; pKa 5.7 and 10.0) hydrophobes were selected for the study. The experiment was carried out at pH 4.0, 7.4 and 10.0. The solubility of all the drugs was enhanced at pH 7.4 and 10.0 but not at pH 4.0. The drug-dendrimer complexes followed 1:1 stoichiometry (AL type of curve) and were characterized for stability of complex, complexation efficiency and thermodynamic properties. Thermodynamic properties were utilized to elucidate the mechanism behind dendrimer mediated solubility enhancement. The data suggested that hydrophobic and electrostatic interactions were responsible for solubility enhancement. Conclusively, PPI dendrimers were found useful in solubility enhancement of not only acidic and basic but also amphoteric drugs, their solubilization ability was clearly regulated by pH and chemical nature of drug. [Abstract/Link to Full Text]

Wu J, Lu Y, Lee A, Pan X, Yang X, Zhao X, Lee RJ
Reversal of multidrug resistance by transferrin-conjugated liposomes co-encapsulating doxorubicin and verapamil.
J Pharm Pharm Sci. 2007;10(3):350-7.
PURPOSE: Liposomes co-encapsulating doxorubicin (DOX) and verapamil (VER), and conjugated to transferrin (Tf-L-DOX/VER) were synthesized and evaluated in K562 leukemia cells. The design of this formulation was aimed at selective targeting of tumor cells, reducing cardiotoxicity of DOX and VER, as well as overcoming P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) phenotype. METHODS: The liposomes were prepared by polycarbonate membrane extrusion, followed by pH-gradient driven remote loading and Tf conjugation. Kinetics of in vitro release of DOX and VER from liposomes was determined by measuring changes in the concentration of encapsulated drugs. Uptake of Tf-conjugated liposomes by K562 cells was evaluated by fluorescence microscopy and by fluorometry. Cytotoxicities of various formulations of DOX were determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolum bromide (MTT) assay. RESULTS: Efficiencies for liposomal loading of DOX and VER were 95% and 70%, respectively. The mean particle diameter for the liposomes was approximately 110nm. Rates of release for DOX and VER were similar in singly-loaded and co-loaded liposomes. Tf-L-DOX/VER showed efficient uptake by the TfR+ K562 cells. In DOX-resistant K562 cells (K562/DOX), Tf-L-DOX/VER showed 5.2 and 2.8 times greater cytotoxicity (IC50 = 4.18 muM) than non-targeted liposomes (L-DOX/VER) (IC50 = 21.7 muM) and Tf-targeted liposomes loaded with DOX alone (Tf-L-DOX) (IC50 = 11.5 muM), respectively. CONCLUSIONS: The combination of TfR targeting and co-encapsulation of DOX and VER was highly effective in overcoming drug resistance in K562 leukemia cells. [Abstract/Link to Full Text]

Uskokovi?-Markovi? S, Milenkovi? M, Topi? A, Kotur-Stevuljevi? J, Stefanovi? A, Anti?-Stankovi? J
Protective effects of tungstophosphoric acid and sodium tungstate on chemically induced liver necrosis in wistar rats.
J Pharm Pharm Sci. 2007;10(3):340-9.
PURPOSE: Many chemical compounds and infectious agents such as viruses induce liver damage like necrosis or fulminant hepatic failure which is sometimes difficult to manage by medical therapies. The induced liver necrosis by carbon tetrachloride (CCl4) and thioacetamide (TAA) are exemplary models for experimental liver necrosis caused by oxygen free radicals. The aim of this study was to investigate the effects of tungstophosphoric acid (TPA) and sodium tungstate (ST) on liver injury induced by CCl4 or TAA. METHODS: Hepatoprotective effects of TPA and ST on acute liver necrosis, chemically induced, were evaluated by the activity of serum enzymes (alkaline phosphatase, alanine transaminase and aspartate transaminase), oxidative stress parameters (activity of xanthine oxidase, concentrations of malondialdehyde and production of superoxide anion), antioxidative defence markers (concentration of reduced glutathione), and histopathology in Wistar rats. Liver necrosis was induced by administering a single intraperitoneal (i.p.) injection of CCl4 (1.0 ml/kg b.wt. of 80% CCl4 in corn oil) or a single i.p. injection of TAA (400 mg/kg b.w. dissolved in normal saline). TPA and ST were administrated to rats orally for 7 weeks (50 mg/kg b.wt.) prior to induction of liver necrosis. RESULTS: Induced liver necrosis caused significant elevation of activity of liver enzymes, parameters of oxidative stress and marked changes in histopathology, like necrosis of hepatocytes, hepatocyte degeneration and infiltration of inflammatory cells. In TPA and ST pretreated rats histopathological changes were almost absent, serum enzymes and oxidative stress parameters were decreased, while at the same time the concentration of reduced gluthathione was increased. CONCLUSION: The present findings suggest that treatment with TPA and ST for 7 weeks could be useful for the prevention of hepatic injury in rats. [Abstract/Link to Full Text]

Kobayashi M, Kagawa T, Takano R, Itagaki S, Hirano T, Iseki K
Effect of medium pH on the cytotoxicity of hydrophilic statins.
J Pharm Pharm Sci. 2007;10(3):332-9.
PURPOSE: The aim of this study was to examine the mechanism of pravastatin- and rosuvastatin-induced cytotoxicity and the relationship between pravastatin- and rosuvastatin-induced cytotoxicity and medium pH using human prototypic embryonal rhabdomyosarcoma cell line (RD) and rat myoblast cell line (L6) as a model of in vitro skeletal muscle. METHODS: Statin-induced reduction of cell viability and apoptosis was measured by 3-(4,5-dimethylthiazol-2-yl)2,5 -diphenyl tetrazolium bromide (MTT) assay and caspase assay. Intracellular accumulation of statins was determined using an HPLC system. RESULTS: Rosuvastatin cytotoxicity, reduction of cell viability, morphological changes and caspase activation at acidic pH (pH 6.8) were significantly greater than those at neutral pH (pH 7.4). Rosuvastatin accumulation at acidic pH was greater than that at pH 7.4. On the other hand, medium pH had no effect on pravastatin accumulation. CONCLUSIONS: Rosuvastatin cytotoxicity at acidic pH is associated with increasing intracellular accumulation of rosuvastatin. On the other hand, medium pH had no effect on cytotoxicity of pravastatin. [Abstract/Link to Full Text]

Sachs-Barrable K, Thamboo A, Lee SD, Wasan KM
Lipid excipients Peceol and Gelucire 44/14 decrease P-glycoprotein mediated efflux of rhodamine 123 partially due to modifying P-glycoprotein protein expression within Caco-2 cells.
J Pharm Pharm Sci. 2007;10(3):319-31.
PURPOSE: The objective of this study was to determine the influence of two lipid excipients, Peceol(c) and Gelucire(c) 44/14 on P-glycoprotein (Pgp) activity and protein expression in human colon adenocarcinoma cells (Caco-2). Lipid excipients are increasingly used as drug delivery systems for hydrophobic drugs to increase their bioavailability by overcoming the barrier of low absorption. This study will probe a novel mechanism by which lipid excipients reduce Pgp-mediated efflux and thereby increase bioavailability of orally administered therapeutics. METHODS: Non-cytotoxic concentrations of Peceol(c) and Gelucire(c) 44/14 were determined for 24-hour treatments of Caco-2 cells using integrity of the cell membranes and mitochondrial respiration as markers. Pgp activity after treatment with non-cytotoxic concentrations of Peceol(c) and Gelucire(c) 44/14 was measured with a fluorescent Pgp substrate, rhodamine 123 (Rh123). The activity of Pgp was ascertained by measuring accumulation and the directional flux of Rh123 using the Transwell(c) semi-permeable cell culture support system. To assess the effect of Peceol(c) and Gelucire(c) 44/14 on Pgp protein expression, Western blotting with a specific Pgp antibody was performed. RESULTS. The two assays for cytotoxicity were in agreement and showed that concentrations of less than 0.5% (v/v) Peceol(c) and less than 0.02% (w/v) Gelucire(c) 44/14 were not toxic to Caco-2 cells. Rh123 accumulation was increased up to 3-fold in cells treated with sub-toxic concentrations of the excipients. The flux of Rh123 across the cell monolayer was unaffected by treatment in the absorptive (apical to basolateral) direction but the efflux transport was reduced after treatment with Peceol(c), Gelucire(c) 44/14 or the positive control , 100microM verapamil. Some of the reduction in Pgp efflux activity can be explained by the reduction in protein expression after treatment with the lipid excipients; treatment with 0.25% (v/v) and 0.5% (v/v) Peceol(c) reduced Pgp protein levels to 62.4% and 68.4% of the control respectively while Gelucire(c) 44/14 treatments of 0.01% (w/v) and 0.02% (w/v) reduced Pgp to 64.5% and 51.8% respectively. CONCLUSION: In this study we utilized established methodologies to assess the inhibitory effect of the excipients on the Pgp-mediated efflux of the probe, Rh123 and tested the hypothesis that long-term treatment of Caco-2 cells with the lipid excipients, Peceol(c) and Gelucire(c) 44/14, decreased Pgp protein expression. The results suggest a new mechanism which may contribute to the improved bioavailability seen for drugs formulated with lipid-based excipients. [Abstract/Link to Full Text]

Hugnet C, Lespine A, Alvinerie M
Multiple oral dosing of ketoconazole increases dog exposure to ivermectin.
J Pharm Pharm Sci. 2007;10(3):311-8.
PURPOSE: The parasiticide ivermectin and the antimicrobial drug ketoconazole are macrolides that interact with P-glycoprotein. We investigated the effects of ketoconazole at a clinical dose on the pharmacokinetics of ivermectin, a CYP3A substrate with low hepatic clearance. METHODS: Beagle dogs received a single subcutaneous injection of ivermectin at 0.05 mg/kg alone (n=6) or in combination with a daily oral dose of ketoconazole 10 mg/kg over 5 days before and after ivermectin administration (n=6). The plasma kinetics of ivermectin and its metabolite were followed over 15 days by HPLC analysis. RESULTS: Co-administered ketoconazole induced a higher plasma concentration and longer residence time of ivermectin in dogs, leading to a substantial increase in the overall exposure of the animal to the drug. Ketoconazole does not interfere with the production of the ivermectin metabolite but it may rather inhibit the elimination of the parental drug by interfering with P-gp transport. CONCLUSIONS: Multiple oral dosing of ketoconazole dramatically altered the pharmacokinetics of ivermectin in dogs leading to an increase in systemic exposure to the drug. Neurotoxicity of ivermectin means that inhibition of the P-gp function at the blood-brain barrier during polytherapy using P-gp inhibitors must be taken into consideration. [Abstract/Link to Full Text]

Foster DR, Zheng X
Cephalexin inhibits N-formylated peptide transport and intestinal hyperpermeability in Caco2 cells.
J Pharm Pharm Sci. 2007;10(3):299-310.
PURPOSE: Intestinal barrier integrity is diminished in critical illness and inflammatory bowel disease. Bacterial-derived N-formylated peptides, absorbed by the intestinal oligopeptide transporter, hPEPT1, are involved in the pathogenesis of disease-induced intestinal barrier dysfunction, via stimulation of polymorphonuclear leukocyte (PMN) migration. The purpose of this study was to determine if the hPEPT1 substrate, cephalexin, inhibits the absorption of the N-formylated peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine ("fMLP"), thereby preventing hyperpermeability in Caco2 cells. METHODS: Caco2 monolayers were grown on permeable supports. fMLP (0.1 microM) was added to apical chambers with and without cephalexin (5 and 10 mM), and fMLP effective permeability was calculated. To determine the ability of cephalexin to attenuate intestinal dysfunction, Caco2 cells were co-cultured with human PMN's in the presence of fMLP, cephalexin, and inflammatory cytokines. Monolayer integrity was assessed by measuring mannitol permeability. RESULTS: Cephalexin 10 mM significantly reduced fMLP permeability (p=0.007). Monolayer integrity (as indicated mannitol permeability) was decreased in cultures treated with inflammatory cytokines and fMLP, an effect that was attenuated by cephalexin (p<0.01). CONCLUSION: Cephalexin inhibits fMLP transport across cultured intestinal monolayers, and partially attenuates PMN-induced intestinal hyperpermeability. The use of pharmacologic hPEPT1 substrates may represent a novel means of preserving intestinal barrier integrity. [Abstract/Link to Full Text]

Junyaprasert VB, Boonme P, Songkro S, Krauel K, Rades T
Transdermal delivery of hydrophobic and hydrophilic local anesthetics from o/w and w/o Brij 97-based microemulsions.
J Pharm Pharm Sci. 2007;10(3):288-98.
PURPOSE: To characterize the physicochemical properties of drug-loaded oil-in-water (o/w) and water-in-oil (w/o) Brij 97-based microemulsions in comparison to their blank counterparts and to investigate the influence of microemulsion type on in vitro skin permeation of model hydrophobic drugs and their hydrophilic salts. METHODS: The microemulsion systems were composed of isopropyl palmitate (IPP), water and a 2:1 w/w mixture of Brij 97 and 1-butanol. The samples were characterized by visual appearance, pH, refractive index, electrical conductivity, viscosity and determination of the state of water and IPP in the formulations using differential scanning calorimetry (DSC). Transdermal flux of lidocaine, tetracaine, dibucaine and their respective hydrochloride salts through heat-separated human epidermis was investigated in vitro using modified Franz diffusion cells. RESULTS: The physicochemical properties of drug-loaded microemulsions and their blank counterparts were generally similar; however, slight changes in some physicochemical properties (apparent pH and conductivity) were observed due to the intrinsic properties of the drugs. The o/w microemulsions resulted in the highest flux of lidocaine, tetracaine and dibucaine as compared to the other formulations with in the same group of drugs. CONCLUSIONS: The characterization results showed that incorporation of the model drugs into the microemulsions did not change the microemulsion type. The permeation data exhibited that the nature of the microemulsions was a crucial parameter for transdermal drug delivery. The o/w microemulsions containing hydrophobic drugs provided the highest skin permeation enhancement. In addition, skin permeation was depended on the molecular weight of the model drugs. [Abstract/Link to Full Text]

Bhandari KH, Newa M, Yoon SI, Kim JS, Kim DD, Kim JA, Yoo BK, Woo JS, Lyoo WS, Choi JY, Lim HT, Lee JH, Choi HG, Yong CS
Evaluation of skin permeation and accumulation profiles of ketorolac fatty esters.
J Pharm Pharm Sci. 2007;10(3):278-87.
PURPOSE: Classic penetration enhancement/retardation methods for improved dermal drug delivery primarily focus on co-applied chemicals aided alterations in skin accumulation/permeation profile, and in many cases, this has been achieved by compromising the systemic absorption/toxicities of penetrant/enhancer/retarder. In this study, higher dermal accumulation without systemic absorption of ketorolac and its fatty esters (esters) will be achieved by synthesizing lipophilic fatty ester soft prodrugs of ketorolac. METHODS: Ketorolac decenoate (C10:1), dodecenoate (C12:1) and palmitoleate (C16:1) were synthesized and evaluated for their lipophilicity, enzymatic hydrolysis, chemical stabilities, and skin permeation and accumulation profiles using the combination of common permeation enhancing techniques such as the use of lipophilic receptor solution, enhancer pretreatment of skins, removal of stratum corneum and delipidization of skins etc. RESULTS: Esters were highly lipophilic, chemically stable, enzymatically unstable in hairless mouse skin/liver homogenates and impermeable into the receptor solution. CONCLUSION: Higher dermal accumulation, absence of skin permeation, relative enzymatic stability in whole skins during permeation study and the pharmaceutical stability of esters could delineate a preliminary possibility for designing safer dermal agents with minimum potential for systemic absorption without the co-application of permeation enhancers or retarders. [Abstract/Link to Full Text]

Jouyban A, Soltanpour Sh, Soltani S, Chan HK, Acree WE
Solubility prediction of drugs in water-cosolvent mixtures using Abraham solvation parameters.
J Pharm Pharm Sci. 2007;10(3):263-77.
PURPOSE: To provide predictive cosolvency models, the Abraham solvation parameters of solutes and the solvent coefficients were combined with the Jouyban-Acree and the log-linear models. These models require two and one solubility data points to predict the solubility of drugs in water-cosolvent mixtures. Ab initio prediction methods also were employed and the results were discussed. METHOD: The Jouyban-Acree model constants were correlated with variables derived from the Abraham solvation parameters of solutes and the solvent coefficients to present quantitative structure property relationship (QSPR) models. The calculated model constants using the QSPR models were used to predict the solubility in water-cosolvent mixtures. The mean percentage deviation (MPD), average absolute error (AAE) and root mean square error (RMSE) criteria were calculated to show the accuracy of the predictions. RESULTS: The overall MPD (+/-SD) of the proposed method employing solubility data in mono-solvents, i.e. two data points for each set, was 18.5+/-12.0 which indicates an acceptable prediction error from the practical point of view. The best cosolvency model employing aqueous solubility data was produced overall MPD of 75.2+/-72.6. The overall MPD of the proposed ab initio method was 74.9+/-19.3%. The models produced the same accuracy pattern considering MPD, AAE and RMSE criteria. CONCLUSION: The proposed model employing two solubility data points for each set produced acceptable prediction error (>18%) and could be recommended for practical applications in pharmaceutical industry. MPD, AAE and RMSE criteria produced similar results considering various models. However, MPD criterion was preferred since its numerical values could be compared with experimental relative standard deviations for repeated experiments. [Abstract/Link to Full Text]

Guan LP, Jin QH, Tian GR, Chai KY, Quan ZS
Synthesis of some quinoline-2(1H)-one and 1, 2, 4 - triazolo [ 4 , 3 -a ] quinoline derivatives as potent anticonvulsants.
J Pharm Pharm Sci. 2007;10(3):254-62.
PURPOSE: A new series of substituted quinoline-2(1H)-one and 1,2,4-triazolo[4,3-a]-quinoline derivatives were designed and synthesized to meet the structural requirements essential for anticonvulsant properties. METHODS: 4-substituted-phenyl-3,4-dihydro-2(1H)-quinolines, 5-substituted-phenyl-4,5-dihydro-1,2,4-triazolo[4,3a]quinolines and 5-substituted-phenyl-4,5-dihydro-1,2,4-triazolo-[4,3-a]quinoline-1-(2H)-ones derivatives were synthesized using 3-substituted-phenyl-N-phenyl-acrylamide as a starting material. Their anticonvulsant activity were evaluated by maximal electroshock (MES) test, subcutaneous pentylenetetrazol (scPTZ) test, and their neurotoxic effects were determined by the rotarod neurotoxicity test. RESULTS: The compounds 4-substitued-phenyl-3,4-dihydro-2(1H)-quinolines (2a-f) had increased anticonvulsant effects compared to the parental compounds. The compounds 5-substituted-phenyl-4,5-dihydro-1,2,4-triazolo[4,3-a]quinolines (3a-f) had significantly increased anticonvulsant activity compared to 2a-f. However, the compounds 5-substituted-phenyl-4,5-dihydro-1,2,4-triazolo[4,3-a]quinoline-1(2H)-ones(4a-f), exhibited no anticonvulsant effects even under a high dose of 300 mg/kg. CONCLUSIONS: The triazole, but not the triazolone, modified series showed stronger anticonvulsant effects than the parent compounds. Among them, compound (3f), 5-(p-fluorophenyl)-4,5-dihydro-1,2,4-triazolo[4,3-a]quinoline, showed the strongest anticonvulsant effect with ED50 of 27.4mg/kg and 22.0mg/kg in the anti-MES and anti-PTZ test, respectively. [Abstract/Link to Full Text]

Prasad V, Fetscher S, Baum RP
Changing role of somatostatin receptor targeted drugs in NET: Nuclear Medicine's view.
J Pharm Pharm Sci. 2007;10(2):321s-337s. [Abstract/Link to Full Text]

Patt M, Solbach C, W�llner U, Blocher A, Stahlschmidt A, G�ndisch D, Kovar KA, Machulla HJ
Synthetic approaches and bio-distribution studies of [11C]methyl-phenidate.
J Pharm Pharm Sci. 2007;10(2):312s-320s.
PURPOSE: The purpose of this study was a) to present a facilitated method for the preparation and workup of [11C]d-threo-methylphenidate ([11C]d-threo-MP) (a ligand that was shown to bind selectively to the presynaptic dopaminergic transporters) from [11C]methyliodide ([11C]CH3I), b) to demonstrate that the ligand can as well be produced by an alternative labeling method employing [11C]diazomethane as the labeling agent and c) to present biodistribution data for this tracer obtained in rats. METHODS: 11C-labeling with [11C]CH3I was performed using either [d-threo-1-(2-nitrophenylsulfanyl)piperidin-2-yl]phenyl-acetic acid (d-threo-N-NPS-ritalinic acid) under addition of sodium hydroxide as base or the previously prepared sodium salt of d-threo-N-NPS-ritalinic acid. The two approaches were compared with regard to radiochemical yield and purification procedures needed in order to obtain a sufficiently pure tracer solution for human use. For the alternative reaction pathway using [11C]diazo-methane as the labeling agent the reaction was performed with d-threo-N-NPS-ritalinic acid. The biodistribution of [11C]d-threo-MP was determined in rats at 5, 10 and 30 min post injection of the tracer. RESULTS: The application of the sodium salt of d-threo-N-NPS-ritalinic acid as precursor resulted in higher radiochemical yields than the use of the free acid under basic conditions, the yields were 20 +/- 8% and 6 +/- 3%, respectively for the final isolated product (based on [11C]CH3I starting activity). The alternative labeling approach by means of [11C]diazomethane as the labeling agent was demonstrated to give radiochemical yields of 76 +/- 8% (based on [11C]diazomethane starting activity, determined by HPLC analysis of the crude reaction mixture before final work-up) within shorter process times. Based on [11C]methane starting activity both approaches result in similar yields (17% and 15%, respectively) Biodistribution studies in rats revealed a low blood activity (0.09% injected dose/g (% ID/g)) at 5 min post injection (p.i.), as well as a relatively high liver uptake (15.9% ID at 30 min) compared to a lower kidney uptake (3.2% ID at 30 min). Brain uptake was 0.9% ID/g already 5 and 10 min p.i.. CONCLUSIONS: The application of the sodium salt of d-threo-N-NPS-ritalinic acid as precursor for the radiosynthesis of [11C]d-threo-MP reduces the amount of [11C]methanol formed from the reaction of [11C]CH3I with sodium hydroxide, that is added to generate the carboxylic anion of d-threo-N-NPS-ritalinic acid needed for labeling with [11C]CH3I. The purification process could be simplified (omission of one solid phase extraction step), resulting in an easily automated process for the production of the tracer. The preparation of [11C]d-threo-MP by means of [11C]diazomethane as the labeling agent appears to be an interesting alternative to the [11C]CH3I methods because of shorter overall process times and high labeling yields. Biodistribution data show a rapid extraction of the tracer from the blood pool. Tracer excretion seems to take place predominantly via the hepatic pathway since liver uptake at 30 min was considerably higher than kidney uptake. [11C]d-threo-MP exhibits a rapid and sufficiently high brain uptake in rats. [Abstract/Link to Full Text]

Mirzayans R, Andrais B, Scott A, Tessier A, Murray D
A sensitive assay for the evaluation of cytotoxicity and its pharmacologic modulation in human solid tumor-derived cell lines exposed to cancer-therapeutic agents.
J Pharm Pharm Sci. 2007;10(2):298s-311s.
PURPOSE: Reliable in vitro cytotoxicity assays are essential for determining the responses of human normal and cancer-derived cells to therapeutic agents and also for the identification and pre-clinical evaluation of new drugs capable of selectively augmenting the susceptibility of cancer cells to conventional therapies. The clonogenic survival assay is considered as the "gold standard" in this regard because it measures the sum of all modes of cell death, encompassing both early and late events such as delayed growth arrest. In this assay, however, the impact of cell-to-cell communication is disregarded because the cells are plated out at very low densities. In addition, here we provide evidence that human breast cancer cell lines cannot be reliably evaluated by clonogenic assays. We developed a novel long-term, High Density Survival (HDS), assay that circumvents the various intrinsic shortcomings of the conventional cytotoxicity assays. METHODS: In the HDS assay, the cells are maintained at a high density for 24 h prior to, and for 24 h after, exposure to a DNA-damaging agent to facilitate intercellular communication. After a carefully scheduled subculturing for approximately 7 days, cultures are assessed for the extent of growth. RESULTS: The degree of radiosensitivity and cisplatin sensitivity evaluated by the HDS assay in human cancer cells was comparable to that measured by the clonogenic assay. Pharmacological inhibitors of CaMKII and/or PI3K signaling elicited a greater degree of radiosensitization when determined by the HDS assay than the clonogenic assay. In all these experiments, there was no relationship between the degree of cytotoxicity measured by the clonogenic survival and viability assays. In the HDS assay, all seven human breast cancer cell lines that we tested exhibited a high degree of radioresistance. CONCLUSIONS: The novel HDS assay appears to be a powerful tool for evaluating cancer cell responses to therapeutic agents under conditions which incorporate some aspects of intercellular communication. [Abstract/Link to Full Text]

Graham K, Wang Q, Garcia Boy R, Eisenhut M, Haberkorn U, Mier W
Synthesis and evaluation of intercalating somatostatin receptor binding peptide conjugates for endoradiotherapy.
J Pharm Pharm Sci. 2007;10(2):286s-297s.
PURPOSE: Intercalators, planar aromatic compounds, are able to interact with DNA by sandwiching themselves between the stacked bases at right angles to the long axis of the helix. Under certain circumstances, Auger-electron-emitting radionuclides can be extremely radiotoxic and produce extensive DNA damage. Auger electron-emitting radioisotopes, are known to be highly cytotoxic when localized in cell nuclei due to highly localized energy deposition by low energy Auger electrons. In addition binding to the DNA might increase the retention in the receptor expressing tissues. METHODS: In order to exploit the cytotoxic potential of intercalator-Auger-emitter conjugates, bis-benzimidazole dyes, Hoechst 33258 and 33342, were linked to a somatostatin receptor affine carrier peptide. For this purpose a bis-benzimidazole intercalating moiety was prepared using variations on the literature methods. The intercalating moieties were coupled under normal SPPS conditions to the carrier peptide, Tyr3-octreotate. To attach the chelating agent (DOTA) to the intercalating moiety, a free amine derivative was prepared and coupled in solution to DOTA tris-t-butyl ester. The resulting chelator-intercalator conjugate was then coupled to a Tyr3-octreotate carrying resin using SPPS. RESULTS: The peptide conjugates were obtained in good yields after HPLC chromatography. The cellular uptake of the novel conjugates was determined using fluorescence microscopy. All intercalator-peptide conjugates revealed somatostatin receptor binding affinities in the nanomolar range. CONCLUSIONS: The novel chelator-intercalator derivatives of the somatostatin receptor binding Tyr3-octreotate introduce a new scope to the range of tracers for therapeutic purposes. [Abstract/Link to Full Text]

Walte A, Sriyapureddy SS, Korkmaz Z, Krull D, Bolte O, Hofmann M, Meyer GJ, Knapp WH
Preparation and evaluation of 211At labelled antineoplastic antibodies.
J Pharm Pharm Sci. 2007;10(2):277s-285s.
PURPOSE: The objective of this study was to determine and verify the stability of 211At-labelled antibodies under physiological conditions and their specific cell-binding capacity for selected epitopes, in order to evaluate the potential of 211At for alpha-radioimmunotherapy. METHODS: 211At was produced at the department's cyclotron and was linked via the intermediate 3-211At-succinimidyl-benzoate (SAB) to the antineoplastic antibodies rituximab, gemtuzumab and gemtuzumab ozogamicin. The stability of the labelled antibodies was determined in serum for 21 h. Cell-binding experiments on HL-60 and CI-1 cells included kinetic, saturation and competitive binding studies. For comparison the binding to antigen-negative cells was determined. The binding specificity and affinity and the IC50-values were evaluated. RESULTS: A consistent yield of 30% and a specific activity of 3 MBq/nmol was obtained. The stability of 211At-antibodies in murine serum exceeded 85% at 37 degrees C. Cell-binding to antigen-positive cells was >25%, while binding to antigen-negative cells did not exceed the unspecific binding and was smaller than 1%. IC50 values ranged between 2 and 11 nmol/L. CONCLUSIONS: A routine preparation of 211At-labelled antibodies was established and the stability of the 211At-labelled antibodies under physiologic conditions was verified. Apparently, labelling of antibodies with 211At by the method described does not compromise the affinity and specificity to the respective epitopes. [Abstract/Link to Full Text]

Solbach C, Patt M, Reimold M, Blocher A, Dohmen BM, Bares R, Zeller KP, Machulla HJ
[11C]Vinblastine syntheses and preliminary imaging in cancer patients.
J Pharm Pharm Sci. 2007;10(2):266s-276s.
PURPOSE: The primary aim of this work was to establish a radiolabeling procedure of vinblastine, a vinca alkaloid widely used in chemotherapy, with the positron-emitter carbon-11 for application in positron-emission-tomography (PET) studies in cancer patients. The optimized reaction conditions were transferred to an automated radiosynthesizer system for the preparation of [11C]vinblastine under GMP conditions for human use. We report about the whole body activity distribution after injection of [11C]vinblastine as well as the pharmacokinetic behavior in selected organs and the tumor in two patients that were investigated with [11C]vinblastine PET before chemotherapy. METHODS: For carbon-11 labeling of vinblastine the reaction conditions were determined with respect to the two possible labeling precursors (i.e. [11C]methyl iodide and [11C]diazomethane), solvent, reaction temperature and reaction time. Both, [11C]diazomethane and [11C]methyl iodide were tested as labeling precursors with the corresponding demethyl compound of vinblastine, i.e. the vinblastine acid and the potassium salt of vinblastine acid. Two patients with renal carcinoma underwent [11C]vinblastine PET before chemotherapy. One patient underwent a second scan during infusion of unlabeled vinblastine at a therapeutic dose. RESULTS: Best results for the labeling procedure were found when methylation was carried out at 100 degrees C within 20 min using 2 mg/mL of the potassium salt of vinblastine acid in DMSO and [11C]methyl iodide as labeling precursor. Based on [11C]methyl iodide starting activity a radiochemical yield of up 53 % [11C]vinblastine was achieved. In addition, the synthesis was transferred to a remotely controlled module for routine GMP conform production for human use. In large scale production runs up to 1 GBq of [11C]vinblastine was obtained ready for injection within 45 min after EOB. In one patient, whole body PET scans 40 min after injection of 112 MBq [11C]vinblastine showed a focally increased [11C]vinblastine uptake and [11C]vinblastine metabolite uptake, respectively in the known metastases, along with a slow but continuous washout during the measurement interval (0-60 min p.i.). Another patient showed no focally increased [11C]vinblastine uptake and [11C]vinblastine metabolite uptake in the tumor, where radioactivity concentration was comparable to that in the blood. In this patient, a second PET scan during infusion of unlabeled vinblastine revealed similar kinetics with a trend towards delayed hepatic metabolism and higher blood and tumor concentrations. Whereas this patient showed a partial response to chemotherapy, the first patient did not, hypothetically due to the observed vinblastine washout from the tumor. CONCLUSIONS: The carbon-11 labeling of vinblastine using [11C]methyl iodide is superior to the method using [11C]diazomethane. A well working automated radiosynthesis was established for the production of [11C]vinblastine for PET-investigations in cancer patients. The individual pharmacokinetic behavior of the chemo-therapeutic agent to the tumor can be assessed with PET, thus, can be considered to be a realistic approach for individualized chemotherapy. [Abstract/Link to Full Text]

Kumar P, Mercer J, Doerkson C, Tonkin K, McEwan AJ
Clinical production, stability studies and PET imaging with 16-alpha-[18F]fluoroestradiol ([18F]FES) in ER positive breast cancer patients.
J Pharm Pharm Sci. 2007;10(2):256s-265s.
PURPOSE: 18F-Fluoroestradiol [18F]FES has emerged as a valuable PET tracer to predict the response to hormone therapy in recurrent or metastatic breast cancer patients. A clinically acceptable product requires a rapid reliable synthesis and must be demonstrated to maintain chemical stability and receptor specific uptake during patient studies. [18F]FES then becomes a dependable tracer for the evaluation and management of breast cancer patients. METHODS: An improved automated radiosynthesis of [18F]FES was developed. Stability studies of the injectible form of [18F]FES were performed up to 24 h after dose formulation under normal storage conditions. A comparative FES/FDG PET imaging in ER+ breast cancer patients is reported. RESULTS: The improved synthesis procedure utilizes fewer hydrolysis steps and a single high performance liquid column chromatography (HPLC) purification of the labeled mixture affording [18F]FES in good yield with high radiochemical purity (>99%). Stability studies with purified [18F]FES in saline/ethanol (85:15 v/v) indicated no radiolytic or chemical degradation of this radiopharmaceutical when stored for 24 h at 20-24 degrees C. Positron Emission Tomography (PET) studies with [18F]FES and [18F]FDG in estrogen receptor positive (ER+) breast cancer patients indicated that while FDG accumulation was seen in all metabolically hyperactive sites, the uptake of FES clearly delineated the ER+ tissues regions. CONCLUSIONS: An improved automated synthesis of [18F]FES has been developed and the integrity of this product has been validated by long term stability studies and clinical PET imaging studies in ER+ breast cancer patients. A lack of concordance between FES and FDG uptake in a patient with metastatic breast cancer suggests specificity of the FES for tumors expressing estrogen receptors. [Abstract/Link to Full Text]

Guthrie JW, Ryu JH, Le XC, Wiebe LI
Characterization of a cyclodextrinoligonucleotide complex by capillary electrophoresis using laser-induced fluorescence.
J Pharm Pharm Sci. 2007;10(2):246-55.
PURPOSE: Cyclodextrins (CDs) have been identified as a viable alternative to viral vectors for use in therapeutic applications. Here, the stability of the complex formed between the a multiply charged, cationic, fully substituted heptakis-(6-amino-2-galactosyl)cyclodextrin (BCDX12) with a multiply charged 12-mer hexachlorofluorescein tagged arabinopolynucleotide (Hex-PAH) have been evaluated. METHODS: The stability of complexes of Hex-PAH and BCD-X12 was studied with respect to mole ratio (1:1, 1:2, and 1:5 Hex-PAH:BCD-X12), pH, buffer concentration, temperature, and agitation using capillary electrophoresis with laser induced fluorescence detection (CE/LIF). Two neutral CDs and an additional cationic CD were also tested under the same analytical conditions to determine their ability to form complexes. RESULTS: Hex-PAH:BCDX12 complexes at mole ratios of 1:2 were stable in 10 mM (160 mM total borate concentration) sodium tetraborate buffer at pH 7.5 and at temperatures of 4 degrees C and 25 degrees C over 48 hours. However, the Hex-PAH:BCD-X12 complex was less stable at 37 degrees C and at higher buffer concentrations and pH values. Strong vortex mixing prior to analysis was found to disrupt the complex. Of the four CDs tested for their ability to complex with Hex-PAH, only BCDX12 formed stable complexes with Hex-PAH under the test conditions. CONCLUSIONS: Capillary electrophoresis was found to be well suited to test the stability of cyclodextrin-nucleotide complexes. CE/LIF indicated that only a single Hex- PAH:BCD-X12 complex was formed at all formulation ratios, and that the complexes were electrophoretically identical to each other, and increasing the molar ratio beyond 1:2 did not contribute measurably to complex stability. Storage temperature and agitation conditions were found to influence complex stability. Since no stable complexes were formed with neutral cyclodextrins, the results support the hypothesis of a 'charge associated' complex rather than an inclusion complex, although inclusion complexes cannot be excluded on the basis of these studies. [Abstract/Link to Full Text]

Emami S, Kumar P, Yang J, Kresolic Z, Paproski R, Cass C, McEwan AJ, Wiebe LI
Synthesis, transportability and hypoxiaselective binding of 1-beta-D-(5-Deoxy-5-fluororibofuranosyl)-2-nitroimidazole (beta-5-FAZR), a configurational isomer of the clinical hypoxia marker, FAZA.
J Pharm Pharm Sci. 2007;10(2):237-45.
PURPOSE: Cellular uptake of most azomycin-based radiosensitizers depends on perfusion and diffusion, rather than on active transport. In medical imaging using radioisotopically labeled azomycin nucleosides, image contrast depends on rapid diffusion from normoxic tissues and rapid renal clearance from the central compartment. [18F]FAZA [1-alpha-D-(5-deoxy-18F]fluoroarabinofuranosyl)-2-nitroimidazole], an azomycin nucleoside currently under clinical evaluation as a marker of tissue hypoxia in medical centers world wide, provides high contrast but its uptake is diffusion dependent and therefore low. 1-D-(5-Fluoro-5-deoxyribofuranosyl)-2-nitroimidazole 6 (beta-5-FAZR), a Beta-ribose analog of FAZA, has now been developed to exploit transport across cell membranes to improve absolute uptake in hypoxic regions and high contrast. METHODS: Beta-5-FAZR was synthesized by classical sugar base coupling followed by regioselective fluorination. In radiosensitization of hypoxic and normoxic to 60Co x-rays was determined relative to known radiosensitizers. The relative abilities of five human nucleoside transporters (hENT1/2, hCNT1/2/3 to bind the radiosensitizers were determined by quantifying their inhibition of uridine transport by recombinant transporters produced in yeast. RESULTS: Beta-5-FAZR was synthesized in 44 percent yield. Beta-5-FAZR had moderate radiosensitization effect on human HCT116/100 colorectal carcinoma (OER 1.8). Beta-5-FAZR was a weak inhibitor of uridine transport relative to nonfluorinated 1-beta-D-(ribofuranosyl)-2-nitroimidazole (beta-AZR). CONCLUSION: Facile synthesis of beta-5-FAZR was achieved and its activity as a radiosensitizer was confirmed. Substitution of C-5 hydroxyl by fluorine in the ribose moiety greatly reduced interaction with hENT1/2 and hCNT1/2 and moderately reduced interaction with hCNT3 relative to thymidine and beta-AZR. [Abstract/Link to Full Text]

Yeung PK, Ferguson C, Jarrar A, King B, Li ML
Development and validation of a sensitive and specific HPLC assay of cladribine for pharmacokinetics studies in rats.
J Pharm Pharm Sci. 2007;10(2):231-6.
PURPOSE: To develop and validate a sensitive and specific HPLC assay for cladribine (CdA) in plasma for pharmacokinetic studies in rats. METHODS: CdA and the internal standard AZT were purchased from Sigma-Aldrich Chem. The HPLC system consisted of a Shimadzu LC-9A pump, a 3 im, 250 x 2.0 mm I.D. high speed C18 column (Jupitertrade mark), preceded by a 5 im 4 4 mm I.D. C18 guard column (Licrocarttrade mark), an Agilent Model 1050 UV-VIS detector and a 3395 Integrator. The mobile phase was made up of 0.01M KH2PO4 (pH 5): methanol: acetonitrile 90:5:5). The system was operated at ambient temperature with a flow rate of 0.3 mL/min, and UV wavelength at 265 nm, and an operating pressure of ca. 1.56 kpsi. Extraction of cladribine and AZT from plasma was achieved by solid phase extraction using 100 mg/mL C18 SPE columns Extra-septrade mark). The assay was validated for sensitivity, precision, specificity and application for pharmacokinetic study in rats. RESULTS: Under these conditions, the average retention times of CdA and AZT were 13.5 and 21 min, respectively, and recoveries were between 80 - 95%. Standard curve constructed from plasma standards was linear from 0.1 ug/mL to 1 ug/mL with regression coefficient (r2) 0.99 or greater. Sensitivity assessed by on column injection was < 1 ng. Using a 50-uL plasma sample size, the mean intra assay variations 0.1 ug/mL were 7%, and inter assay variations over a period of 3 months for 5 separate batches were less than 20%. The assay was used to study a single dose pharmacokinetic study of CdA in rats after a 2 mg/kg subcutaneous injection. CONCLUSION: The described HPLC assay has adequate sensitivity and specificity to study pharmacokinetics of CdA in rats, and could be adapted also to clinical pharmacokinetic studies. [Abstract/Link to Full Text]

Wang D, Molavi O, Lutsiak ME, Elamanchili P, Kwon GS, Samuel J
Poly(D,L-lactic-co-glycolic acid) microsphere delivery of adenovirus for vaccination.
J Pharm Pharm Sci. 2007;10(2):217-30.
PURPOSE: To study the effect of encapsulation of recombinant adenovirus type 5 encoding Beta-galactosidase (Ad5-Betagal) in poly (D,Llactic-co-glycolic acid) (PLGA) microspheres on viral delivery to professional antigen presenting cells (APCs) in vitro, viral dissemination in vivo, and induction of protective immune responses in vivo. METHODS: PLGA microspheres containing Ad5-alphagal were prepared by a double emulsion solvent evaporation method. Encapsulation efficiency, in vitro release profile, in vitro cellular uptake and in vivo biodistribution of Ad5-alphagal loaded PLGA microspheres were determined using 125I-labeled Ad5-alphagal (125I-Ad5-alphagal). To evaluate the potential of PLGA microsphere delivery of Ad-alphagal for induction of antigen specific immune responses in vivo, Balb/c mice were immunized with the subcutaneous injection of the formulations then splenocytes of the immunized mice were assayed for cytotoxic T lymphocyte (CTL) activity against a variety of target cells in a 51Cr-release assay. Anti-alphagal antibody responses were assessed in the sera of the immunized mice by enzyme linked immunosorbent assay (ELISA). The effect of encapsulated Ad5-alphagal immunization on protection against a tumor challenge was tested in a murine artificial metastatic lung tumor model with alphagalexpressing tumor cells, CT26.CL25. RESULTS: PLGA microspheres encapsulated Ad5-alphagal with 24.8 +/- 1.4 % encapsulation efficiency and 11.4 +/- 3.6 % of the encapsulated virus retained the functional activity. In vitro release study showed slow release (15% in 11 days) of the virus from the microspheres. PLGA microsphere delivery of Ad5-alphagal resulted in enhanced uptake of the virus by APCs with an increase in the transgene expression in vitro. Administration of the virus in the encapsulated form resulted in substantially decreased viral dissemination to remote organs and tissues as compared to the free virus. Encapsulated virus were capable of eliciting antigen specific CTL as well as antibody responses against alphagal and induced protective immune responses against lethal tumor challenge at a significantly lower infectious viral dose as compared to the free virus. CONCLUSION: PLGA microsphere with Ad5-alphagal enhances the delivery of virus to APCs with reduced viral dissemination in other organs and induces protective antigen specific immune responses against viral encoded transgene. [Abstract/Link to Full Text]

Seki K, Nishijima K, Kuge Y, Tamaki N, Wiebe LI, Ohkura K
A novel and efficient synthesis of [2-11C]5-fluorouracil for prognosis of cancer chemotherapy.
J Pharm Pharm Sci. 2007;10(2):212-6.
PURPOSE: In order to facilitate the use of the PET-based Strauss test for 5-FU sensitivity, a rapid and facile synthesis of [2-11C]5-fluorouracil ([2-11C]5-FU), based on = [11C]phosgene ([11C]COCl2), is reported. METHOD: The key intermediate (E)-beta- benzoylamino-Alpha-fluoroacrylamide (1) and [11C]phosgene was submitted to cyclocondensation to give [2-11C]5-fluorouracil. RESULTS: [2-11C]5-Fluorouracil was synthesized in 17 min with high (25%) radiochemical yield. CONCLUSION: The present study provides a rapid, simple, and efficient synthesis of [2-11C]5-FU, that would serve as a useful prognostic PET tracer for 5-FU chemotherapy. [Abstract/Link to Full Text]

Reischl G, Dorow DS, Cullinane C, Katsifis A, Roselt P, Binns D, Hicks RJ
Imaging of tumor hypoxia with [124I]IAZA in comparison with [18F]FMISO and [18F]FAZA--first small animal PET results.
J Pharm Pharm Sci. 2007;10(2):203-11.
PURPOSE: This study was performed to compare the 2-nitroimidazole derivatives [124I]IAZA, [18F]FAZA and well known [18F]FMISO in visualization of tumor hypoxia in a mouse model of human cancer using small animal PET. METHODS: PET imaging of female Balb/c nude mice bearing A431 tumors on a Phillips Mosaic small animal PET scanner was performed 3 h p.i. for all three tracers. Mice injected with [124I]IAZA were scanned again after 24 h and 48 h. In addition to the mice breathing air, in the case of [18F]FAZA and [124I]IAZA a second group of mice for each tracer was kept in an atmosphere of carbogen gas (5% of CO2 + 95 % of O2; from 1 h before to 3 h after injection) to evaluate the oxygenation dependency on uptake (all experiments n = 4). After the final PET scan animals were sacrificed and biodistribution was studied. RESULTS: Mice injected with [18F]FAZA displayed significantly higher tumor-to background (T/B) ratios (5.19 +/- 0.73) compared to those injected with [18F]FMISO (3.98 +/- 0.66; P $lt; 0.05) or [124I]IAZA (2.06 +/- 0.26; P $lt; 0.001) 3 h p.i. Carbogen breathing mice showed lower ratios ([18F]FAZA: 4.06 +/- 0.59; [124I]IAZA: 2.02 +/- 0.36). The T/B ratios increased for [124I]IAZA with time (24 h: 3.83 +/- 0.61; 48 h: 4.20 +/- 0.80), but after these late time points the absolute whole body activity was very low, as could be seen from the biodistribution data (< 0.1 %ID/g for each investigated organ) and ratios were still lower than for [18F]FAZA 3 h p.i. Due to de-iodination uptake in thyroid was high. Biodistribution data were in good agreement with the PET results. CONCLUSIONS: [18F]FAZA showed superior biokinetics compared to [18F]FMISO and [124I]IAZA in this study. Imaging at later time points that are not possible with the short lived 18F labeled tracers resulted in no advantage for [124I]IAZA, i. e. tumor to normal tissue ratios could not be improved. [Abstract/Link to Full Text]

Mercer JR
Molecular imaging agents for clinical positron emission tomography in oncology other than fluorodeoxyglucose (FDG): applications, limitations and potential.
J Pharm Pharm Sci. 2007;10(2):180-202.
Recent efforts in radiopharmaceutical design for positron emission tomography (PET) imaging in clinical oncology have provided a library of tracers that have the potential to contribute to individualizing cancer patient management. These tracers can provide PET images that reveal aspects of the fundamental underlying biochemistry in the tumor before and during treatment. For a number of these PET tracers the cellular processing has been well described and they are now generally referred to as molecular imaging agents. Despite their recognized value in clinical oncology these tracers have not yet obtained widespread acceptance and are not generally available at centers performing PET scans. There are a number of barriers and challenges to the widespread use of these PET tracers that include; a limited clinical demand, challenges presented by the chemistry and formulation for clinical acceptability, the short physical half life of the PET radionuclides, regulatory issues and the overall costs associated with PET radiopharmaceutical production. In addition the interpretation of the PET images requires a clear understanding of the biochemical processes involved at the cellular level. A concerted effort is required among stakeholders including clinicians, scientists, industry and governmental agencies if the potential of these agents in clinical oncology is to be realized. [Abstract/Link to Full Text]

Marsh RE, Riauka TA, McQuarrie SA
Use of a simulated annealing algorithm to fit compartmental models with an application to fractal pharmacokinetics.
J Pharm Pharm Sci. 2007;10(2):168-79.
Increasingly, fractals are being incorporated into pharmacokinetic models to describe transport and chemical kinetic processes occurring in confined and heterogeneous spaces. However, fractal compartmental models lead to differential equations with power-law time-dependent kinetic rate coefficients that currently are not accommodated by common commercial software programs. This paper describes a parameter optimization method for fitting individual pharmacokinetic curves based on a simulated annealing (SA) algorithm, which always converged towards the global minimum and was independent of the initial parameter values and parameter bounds. In a comparison using a classical compartmental model, similar fits by the Gauss-Newton and Nelder-Mead simplex algorithms required stringent initial estimates and ranges for the model parameters. The SA algorithm is ideal for fitting a wide variety of pharmacokinetic models to clinical data, especially those for which there is weak prior knowledge of the parameter values, such as the fractal models. [Abstract/Link to Full Text]

Recent Articles in Journal of Drug Targeting

Motlekar NA, Fasano A, Wachtel MS, Youan BB
Zonula occludens toxin synthetic peptide derivative AT1002 enhances in vitro and in vivo intestinal absorption of low molecular weight heparin.
J Drug Target. 2006 Jun;14(5):321-9.
Zonula occludens toxin (Zot) is an enterotoxin obtained from the bacterium vibrio cholerae that has been shown to reversibly and safely open the tight junctions and enhance paracellular transport. AT1002 is a novel synthetic hexapeptide derived from Zot. The hypothesis to be tested in this study is that AT1002 enhances the oral absorption of ardeparin, a low molecular weight heparin (LMWH). To test this hypothesis, drug transport through Caco-2 cell monolayers was monitored in the presence and absence of AT1002. Regional permeability studies using rat intestine were performed. Cell viability in the presence of various concentrations of enhancer was determined. The absorption of ardeparin after oral administration in rats was measured by anti-factor Xa assay. Furthermore, the eventual mucosal and epithelial damage was histologically evaluated. Higher ardeparin permeability (approximately 2-fold) compared to control was observed in the presence of 0.025% of AT1002. Regional permeability studies revealed that the permeability of ardeparin across the duodenal membrane was improved by the AT1002. Cell viability studies showed no significant cytotoxicity below 0.0028% of AT1002. In the presence of 100 microg/kg of AT1002, ardeparin oral bioavailability was significantly increased (F(relative/s.c) approximately 20.5%). Furthermore, AT1002 at a dose of 100 microg/kg did not induce any observable morphological damage on gastrointestinal (GI) tissues in vivo. These in vivo and in vitro results suggest that the co-administration of LMWH with AT1002 may be a useful delivery strategy to increase its permeability and hence oral absorption. [Abstract/Link to Full Text]

Motlekar NA, Srivenugopal KS, Wachtel MS, Youan BB
Oral delivery of low-molecular-weight heparin using sodium caprate as absorption enhancer reaches therapeutic levels.
J Drug Target. 2005 Dec;13(10):573-83.
The primary objective of this study was to evaluate sodium caprate as an oral penetration enhancer for low molecular weight heparin (LMWH), ardeparin. In vitro studies using Caco-2 cell monolayer indicated that 0.0625% of sodium caprate gave approximately 2-fold enhancement of ardeparin compared to negative control with almost 100% cell survival as evaluated by MTT cytotoxicity assay. In vivo studies in rats with ardeparin (1,200 IU/kg) and sodium caprate (100 mg/kg) led to a relative bioavailability of 27% with plasma anti-factor Xa levels within the therapeutic range (>0.2 IU/ml). Moreover, under these conditions, histological examination provided evidence that there was no damage to the gastrointestinal wall. Regional permeability studies using rat intestine indicated the colon as the region of maximum permeation. These results suggest that, at the dose administered, sodium caprate acts as a relatively safe and efficient absorption enhancer in the quest for alternatives for the oral delivery of LMWH. [Abstract/Link to Full Text]

Wang J, Mongayt D, Torchilin VP
Polymeric micelles for delivery of poorly soluble drugs: preparation and anticancer activity in vitro of paclitaxel incorporated into mixed micelles based on poly(ethylene glycol)-lipid conjugate and positively charged lipids.
J Drug Target. 2005 Jan;13(1):73-80.
Paclitaxel-loaded mixed polymeric micelles consisting of poly(ethylene glycol)-distearoyl phosphoethanolamine conjugates (PEG-PE), solid triglycerides (ST), and cationic Lipofectin lipids (LL) have been prepared. Micelles with the optimized composition (PEG-PE/ST/LL/paclitaxel = 12/12/2/1 by weight) had an average micelle size of about 100 nm, and zeta-potential of about -6 mV. Micelles were stable and did not release paclitaxel when stored at 4 degree C in the darkness (just 2.9% of paclitaxel have been lost after 4 months with the particle size remaining unchanged). The release of paclitaxel from such micelles at room temperature was also insignificant. However, at 37 degree C, approx. 16% of paclitaxel was released from PEG-PE/ST/LL/paclitaxel micelles in 72 h, probably, because of phase transition in the ST-containing micelle core. In vitro anticancer effects of PEG-PE/ST/LL/paclitaxel and control micelles were evaluated using human mammary adenocarcinoma (BT-20) and human ovarian carcinoma (A2780) cell lines. Paclitaxel in PEG-PE/ST/LL micelles demonstrated the maximum anti-cancer activity. Cellular uptake of fluorescently-labeled paclitaxel-containing micelles by BT-20 cells was investigated using a fluorescence microscopy. It seems that PEG-PE/ST/LL micelles, unlike micelles without the LL component, could escape from endosomes and enter the cytoplasm of BT-20 cancer cells thus increasing the anticancer efficiency of the micellar paclitaxel. [Abstract/Link to Full Text]

Kaul G, Amiji M
Biodistribution and targeting potential of poly(ethylene glycol)-modified gelatin nanoparticles in subcutaneous murine tumor model.
J Drug Target. 2004;12(9-10):585-91.
PURPOSE: In order to develop a safe and effective systemically-administered biodegradable nanoparticle delivery system for solid tumors, the comparative biodistribution profiles of gelatin and poly(ethylene-glycol)(PEG)-modified (PEGylated) gelatin nanoparticles was examined in subcutaneous Lewis lung carcinoma (LLC)-bearing female C57BL/6J mice. METHODS: Type-B gelatin and PEGylated gelatin nanoparticles were radiolabeled ((125)I) for the in vivo biodistribution studies after intravenous (i.v.) administration through the tail vein in LLC-bearing mice. At various time intervals, the mice were sacrificed and blood, tumor, and major organs harvested for analysis of radioactivity corresponding to the localization of the nanoparticles. Percent recovered dose was determined and normalized to the weight of the fluid or tissue sample. Non-compartmental pharmacokinetic analysis was performed to determine the long-circulating property and preferential tumor targeting potential of PEGylated gelatin nanoparticles in vivo. RESULTS: From the radioactivity in plasma and various organs collected, it was evident that the majority of PEGylated nanoparticles were present either in the blood pool or taken up by the tumor mass and liver. For instance, after 3 h, the concentrations of PEGylated gelatin nanoparticles was almost 2-fold higher in the blood pool than the control gelatin nanoparticles. PEGylated gelatin nanoparticles remained in the blood pool for a longer period of time due to the steric repulsion effect of the PEG chains as compared to the gelatin nanoparticles. In addition, approximately 4-5% of the recovered dose of PEGylated gelatin nanoparticles was present in the tumor mass for up to 12 h. The plasma and the tumor half-lives, the mean residence time, and the area-under-the-curve of the PEGylated gelatin nanoparticles were significantly higher than those for the gelatin nanoparticles. CONCLUSIONS: The results of this study show that PEGylated gelatin nanoparticles do possess long circulating properties and can preferentially distribute in the tumor mass after systemic delivery. [Abstract/Link to Full Text]

Recent Articles in Drug Metabolism and Pharmacokinetics

Aueviriyavit S, Furihata T, Morimoto K, Kobayashi K, Chiba K
Hepatocyte nuclear factor 1 alpha and 4 alpha are factors involved in interindividual variability in the expression of UGT1A6 and UGT1A9 but not UGT1A1, UGT1A3 and UGT1A4 mRNA in human livers.
Drug Metab Pharmacokinet. 2007 Oct;22(5):391-8.
UDP-glucuronosyltransferases (UGTs) catalyze phase-II biotransformation reaction of a variety of substances. Among the UGT1A isoforms, UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9 are predominantly expressed in the liver. Interindividual variability in expression of these isoforms would cause interindividual differences in drug response, toxicity and cancer susceptibility. In the present study, we investigated the interindividual variability in UGT1A mRNA expression and whether hepatocyte nuclear factor 1alpha (HNF1alpha) and HNF4alpha were factors responsible for their variability in human livers. The amounts of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, HNF1alpha and HNF4alpha mRNA in 18 human livers were measured by quantitative real-time polymerase chain reaction. The largest and smallest interindividual differences in expression levels were observed in UGT1A1 (8.6-fold) and UGT1A4 (2.5-fold) mRNA, respectively. The amounts of HNF1alpha and HNF4alpha mRNA were strongly correlated with the amount of UGT1A9 mRNA and moderately correlated with that of UGT1A6 mRNA, whereas no significant correlation was found with the amounts of UGT1A1, UGT1A3 and UGT1A4 mRNA. Our results suggest that HNF1alpha and HNF4alpha are the factors involved in the interindividual variability of UGT1A6 and UGT1A9 mRNA expression. Further studies of other transcription factors are needed to clarify the factor(s) determining the interindividual variations in UGT1A1, UGT1A3 and UGT1A4 mRNA expression. [Abstract/Link to Full Text]

Chiba M, Itagaki S, Kobayashi M, Hirano T, Iseki K
Characterization of hepatobiliary organic anion transporters in Long-Evans Cinnamon rats.
Drug Metab Pharmacokinet. 2007 Oct;22(5):387-90.
The liver plays important roles in the detoxification of xenobiotics. Hepatobiliary transporters contribute to hepatic uptake and efflux processes of xenobiotecs. Expressions of these transporters may be modulated under the condition of hepatic failure. Long-Evans Cinnamon (LEC) rats provide a pertinent model for basic and clinical studies on hepatitis. However, only a few reports describing the properties of hepatobiliary transporters in LEC rats have appeared in the literature. We investigated the expression levels of hepatobiliary transporters in LEC rats by real-time RT-PCR. We found that hepatic expressions of three sinusoidal organic anion transporters, Ntcp, Oatp1a1 and Oatp1a4, were decreased in LEC rats. However, no significant difference of the expressions of Mrp2 and Bsep, organic anion transporters located on canalicular membrane, were found between Wistar rats and LEC rats. [Abstract/Link to Full Text]

Honda M, Toyoda W, Shimizu T, Horiuchi I, Kayano Y, Taguchi M, Nozawa T, Inoue H, Hashimoto Y
UGT2B7*3 did not affect the pharmacokinetics of R- and S-carvedilol in healthy Japanese.
Drug Metab Pharmacokinet. 2007 Oct;22(5):382-6.
We previously investigated the pharmacokinetics of R- and S-carvedilol in 54 healthy Japanese subjects, and reported that the oral clearance (CL/F) and apparent volume of distribution (V/F) of both enantiomers in subjects with the CYP2D6*10 allele were significantly lower than those in subjects without the CYP2D6*10 allele. In the present study, we examined the genotype of UGT2B7 in these 54 subjects, and investigated the effect of UGT2B7*3 on the pharmacokinetics of R- and S-carvedilol. Forty-three subjects did not have the UGT2B7*3 allele, and 11 subjects had one UGT2B7*3 allele. CL/F and V/F values of R- and S-carvedilol in the subjects with one UGT2B7*3 allele were similar to those without the UGT2B7*3 allele, indicating that the UGT2B7*3 allele did not significantly affect the systemic clearance (CL) and bioavailability (F) of the two enantiomers. [Abstract/Link to Full Text]

Fukumori S, Murata T, Taguchi M, Hashimoto Y
Rapid and drastic induction of CYP3A4 mRNA expression via vitamin D receptor in human intestinal LS180 cells.
Drug Metab Pharmacokinet. 2007 Oct;22(5):377-81.
The aim of this study was to evaluate the usefulness of human intestinal LS180 cells for studying the induction of CYP3A4 mRNA expression via vitamin D receptor (VDR). CYP3A4 mRNA expression in LS180 cells treated with 100 nM 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) for 6 and 24 h was approximately 80- and 500-fold higher than the control, respectively. A protein kinase (PK) inhibitor (staurosporine), c-jun N-terminal kinase (JNK) pathway inhibitor (curcumin), and JNK inhibitor (SP600125) attenuated 1alpha,25(OH)(2)D(3)-induced CYP3A4 mRNA expression, suggesting that the PK-JNK pathway contributed to the rapid and drastic induction of CYP3A4 expression via VDR in LS180 cells. The ability of CYP3A4 mRNA induction in LS180 cells was highly dependent on the site and number of vitamin D(3) and D(2) hydroxylation. In addition, short-time (6 h) treatment of LS180 cells with cytotoxic secondary bile acids, lithocholic acid (LCA) and 3-keto-LCA also significantly induced the mRNA expression of CYP3A4. LS180 cells may be useful to quickly investigate the CYP3A4-inducing effect of drugs, xenobiotics, and/or endogenous substrates in the intestinal epithelia. [Abstract/Link to Full Text]

Ishii Y, Iwanaga M, Nishimura Y, Takeda S, Ikushiro S, Nagata K, Yamazoe Y, Mackenzie PI, Yamada H
Protein-protein interactions between rat hepatic cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs): evidence for the functionally active UGT in P450-UGT complex.
Drug Metab Pharmacokinet. 2007 Oct;22(5):367-76.
The interaction between cytochrome P450s (CYP, P450) and UDP-glucuronosyltransferases (UGTs) was studied by co-immunoprecipitation. P450 isoform-selective antibody was used as a probe to co-precipitate UGTs with the P450s from solubilized rat liver microsomes. Antibodies toward CYP3A2, CYP2B2, CYP2C11/13 and CYP1A2 co-precipitated UGTs with corresponding P450s. However, calnexin, a type-I membrane protein, in the endoplasmic reticulum was not co-precipitated by anti-P450 antibodies. UGT activity toward 4-methylumbelliferone was detected in all co-precipitates, suggesting that UGT in the complex with P450s is functionally active. Repeated washing of co-immunoprecipitates revealed differences among P450 isoforms with regard to the affinity for UGT. Larger amounts of UGT1A1 and UGT1A6, compared with UGT2B1, were washed out from UGTs-CYP2C11/13 co-precipitates, whereas UGT-CYP3A2 and UGT-CYP2Bs complexes were resistant to thorough washing. Thus, CYP2C11/13 could associate with UGTs, but the affinity is assumed to be weaker than that of CYP2B/3As. These results suggest that there is isoform specificity in the interaction between P450s and UGTs. [Abstract/Link to Full Text]

Kubo M, Koue T, Maune H, Fukuda T, Azuma J
Pharmacokinetics of aripiprazole, a new antipsychotic, following oral dosing in healthy adult Japanese volunteers: influence of CYP2D6 polymorphism.
Drug Metab Pharmacokinet. 2007 Oct;22(5):358-66.
We investigated the pharmacokinetics (PK) of aripiprazole, a newly developed antipsychotic, and its active metabolite in healthy Japanese, and the influence of CYP2D6 polymorphism on the PK of aripiprazole. Following a single oral 6 mg dose, the mean C(max), t(max), and t(1/2, z) (terminal phase half life) of aripiprazole were 31.0 ng/mL, 3.6 hr, and 61.0 hr, respectively. The t(1/2, z) in CYP2D6 IM subjects (75.2 hr) was significantly (p<0.01) longer than that in CYP2D6 EM subjects (45.8 hr), and the systemic clearance of IM subjects was approximately 60% that of EM subjects. The PK in one subject with the CYP2D6*41 homozygote was similar to that of IM subjects. In repeated oral administration, plasma concentrations of aripiprazole and active metabolite both reached a steady state by Day 14. The half-life of aripiprazole following repeated administration was similar to that following single administration, suggesting that pharmacokinetics was constant during 14-day administration. Our investigations revealed that there is no clear ethnic difference between Japanese and Western subjects in terms of mean plasma PK, while the CYP2D6*10 allele distinctive to Asian populations influences the PK of aripiprazole. Moreover, our observations suggest that the CYP2D6*41 allele significantly affects drug-metabolizing activity. [Abstract/Link to Full Text]

Yagi Y, Aoki M, Iguchi M, Shibasaki S, Kurosawa T, Kato Y, Tsuji A
Transporter-mediated hepatic uptake of ulifloxacin, an active metabolite of a prodrug-type new quinolone antibiotic prulifloxacin, in rats.
Drug Metab Pharmacokinet. 2007 Oct;22(5):350-7.
Prulifloxacin (PUFX) is a prodrug-type new quinolone antibiotic and immediately converted to an active metabolite, ulifloxacin (UFX). It has been previously reported that UFX is highly excreted into the bile, although the hepatic uptake process of UFX has not been investigated yet. In this study, we attempted to characterize the mechanism of hepatic uptake of UFX in rats. The hepatic uptake in vivo was evaluated by integration plot analysis. Furthermore, the uptake of [(14)C]-UFX by isolated rat hepatocytes was measured, and the effects of several transporter inhibitors and other quinolone antibiotics on the uptake were examined. The hepatic uptake clearance of UFX (1 mg/kg) was calculated to be 37.7 mL/min/kg, which was larger than those at doses of 5 and 25 mg/kg and was decreased by co-administration of cyclosporine A (CysA; 30 mg/kg). The uptake of [(14)C]-UFX by isolated rat hepatocytes linearly increased up to 1 min and also inhibited by CysA. Other quinolone antibiotics inhibited the [(14)C]-UFX uptake in a concentration-dependent manner, whereas taurocholate and estrone-3-sulfate partially inhibited the [(14)C]-UFX uptake. These results demonstrate that a carrier-mediated transport system which is common to the quinolone antibiotics is involved in the uptake of UFX in the rat liver. [Abstract/Link to Full Text]

Obach RS, Margolis JM, Logman MJ
In vitro metabolism of CP-122,721 ((2S,3S)-2-phenyl-3-[(5-trifluoromethoxy-2-methoxy)benzylamino]piperidine), a non-peptide antagonist of the substance P receptor.
Drug Metab Pharmacokinet. 2007 Oct;22(5):336-49.
The metabolism of CP-122,721, a neurokinin-1 antagonist, has been examined in vitro using hepatic microsomes from human and animal species, and recombinant heterologously expressed P450 enzymes. Metabolism occurs primarily via O-demethylation and N-dealkylation reactions. In human liver microsomes, O-demethylation was shown to be catalyzed by CYP2D6 with a low K(M) value. N-dealkyation was shown to be catalyzed primarily by CYP3A4. When scaled to in vivo, in vitro intrinsic clearance data yielded a reasonable correlation across species. CP-122,721 was shown to be metabolized by parallel pathways to 5-trifluoromethoxysalicylic acid, which had been observed as a major circulating metabolite in humans after oral administration of CP-122,721. The involvement of CYP1A2, CYP3A4, and MAO-B was demonstrated in the pathways leading to 5-trifluoromethoxysalicylic acid. The O-desmethyl metabolite of CP-122,721 was shown to undergo a P450 catalyzed O-detrifluoromethylation reaction yielding a p-hydroquinone metabolite. The reaction was shown to be catalyzed by CYP3A4. Incubation under (18)O(2) yielded the hydroquinone containing O-18, consistent with this reaction occurring via an ispo substitution mechanism. Combined, these findings provide a comprehensive understanding of the metabolism of this new agent. [Abstract/Link to Full Text]

Iwasaki K
Metabolism of tacrolimus (FK506) and recent topics in clinical pharmacokinetics.
Drug Metab Pharmacokinet. 2007 Oct;22(5):328-35.
Tacrolimus (FK506), an immunosuppressive drug, is co-medicated with multiple drugs under clinical conditions. Tacrolimus is highly lipophilic and is excreted from the body after receiving extensive metabolism. Due to its narrow therapeutic window following organ transplantation, tacrolimus requires therapeutic drug monitoring by an enzyme immunoassay using the monoclonal antibody raised against tacrolimus. Therefore, metabolism studies including drug-drug interaction and metabolite identification studies are essential for the efficient development and clinically optimal usage of this drug. Tacrolimus was metabolized by the cytochrome P450 (CYP) 3A subfamily. Metabolic drug-drug interaction studies were conducted to provide information regarding the optimal usage of tacrolimus, and its metabolism was inhibited by known CYP3A inhibitors such as ketoconazole, cyclosporine A, and nifedipine. Recent reports on clinical pharmacokinetics indicate that dose levels of tacrolimus need to be adjusted in transplant patients with CYP3A5 polymorphism. [Abstract/Link to Full Text]

Sudo K
Microdosing for reduction of the time and resources for drug development.
Drug Metab Pharmacokinet. 2007 Oct;22(5):327. [Abstract/Link to Full Text]

Hart SN, Li Y, Nakamoto K, Wesselman C, Zhong XB
Novel SNPs in cytochrome P450 oxidoreductase.
Drug Metab Pharmacokinet. 2007 Aug;22(4):322-6.
Cytochrome P450 oxidoreductase (POR) is the single flavoprotein which donates electrons to the microsomal cytochrome P450 enzymes for oxidation of their substrates. In this study, we sequenced all 15 exons and the surrounding intronic sequences of POR in 100 human liver samples to identify novel and confirm known genetic polymorphisms in POR. Thirty-four single nucleotide polymorphisms (SNPs) were identified including 9 in the coding exons (5 synonymous and 4 nonsynonymous), 20 in the intronic regions, and 5 in the 3'-UTR. Of these, 9 were novel SNPs, including three nonsynonymous SNPs, SNH313003 (817733G>C; K49N), SNH313020 (848661C>A; L420M), and SNH313029 (849577T>C; L577P) with minor allele frequencies of 0.005, 0.045, and 0.020, respectively. We also confirmed a previously reported non-synonymous SNP rs1057868 (A503V) as well as five synonymous SNPs (G5G, T29T, P129P, S485S, and S572S) all with allele frequencies similar to those previously reported. Structurally, these polymorphisms occur in different regions: SNH313003 (K49N) in the amino-terminal tail, SNH313020 (L420M) in the connecting domain, SNH313029 (L577P) in the NADPH-binding domain, and rs1057868 (A503V) in the FAD binding domain. [Abstract/Link to Full Text]

Uno S, Kawase A, Tsuji A, Tanino T, Iwaki M
Decreased intestinal CYP3A and P-glycoprotein activities in rats with adjuvant arthritis.
Drug Metab Pharmacokinet. 2007 Aug;22(4):313-21.
Adjuvant-induced arthritis (AA) rats have been used as an animal model for rheumatoid arthritis. Several studies have shown that the pharmacokinetics of a number of drugs are altered in AA rats. We investigated the effects of AA on the barrier functions of the intestine using a rat model. Intestinal CYP3A activities (midazolam 1'-hydroxylation and 7-benzyloxy-4-(trifluoromethyl)-coumarin 7-hydroxylation) in AA rats were significantly decreased compared with those in normal rats, with marked decrease observed in the upper segment of intestine. Intestinal P-glycoprotein (P-gp) activity at upper segment was also significantly decreased in AA rats to 60% of that in normal rats, and the other segments (middle and lower) of intestine also exhibited tendencies toward decrease in P-gp activity. This decrease was supported by the finding that levels of mdr1a mRNA and P-gp protein were decreased in AA rats. No significant differences were observed in intestinal paracellular and transcellular permeability between AA and normal rats. These results suggest that intestinal CYP3A and P-gp activities are decreased in AA rats, and that the pharmacokinetics and bioavailabilities of drugs whose membrane permeation is limited by intestinal CYP3A and/or P-gp may be altered in rheumatic diseases. [Abstract/Link to Full Text]

Kimoto E, Seki S, Itagaki S, Matsuura M, Kobayashi M, Hirano T, Goto Y, Tadano K, Iseki K
Efflux transport of N-monodesethylamiodarone by the human intestinal cell-line Caco-2 cells.
Drug Metab Pharmacokinet. 2007 Aug;22(4):307-12.
Amiodarone (AMD) is a benzofurane derivative with class III antiarrhythmic activity that is effective in controlling intractable cardiac arrhythmias. One of the most common and serious drug interactions in clinical practice is the interaction between digoxin and an antiarrhythmic agent. It has been reported that AMD and N-monodesethylamiodarone (DEA), the active metabolite of AMD, inhibit the P-glycoprotein (P-gp/MDR1)-mediated digoxin transport. However, the intestinal transport processes of AMD and DEA have not been fully revealed. In this study, we focused on the intestinal transport mechanism of DEA and characterized the intestinal transport of DEA using Caco-2 cells. Basal-to-apical transport of DEA by Caco-2 cells was greater than apical-to-basal transport. The relationship between concentration and basal-to-apical flux rate appeared to approach saturation. The uptake of DEA by Caco-2 cells was increased in the presence of typical ATP-depletion compounds and thyroid hormones. On the other hand, substrates for P-gp, multidrug resistance-associated proteins (MRPs/ABCCs) and breast cancer resistance protein (BCRP/ABCG2) had no effect on the efflux of DEA. These results suggest that an ATP-binding cassette (ABC) transporter, which is different from P-gp, MRPs and BCRP, mediates the efflux of DEA across the apical membrane in Caco-2 cells and that thyroid hormone inhibits this transporter. [Abstract/Link to Full Text]

Rashidi MR, Beedham C, Smith JS, Davaran S
In vitro study of 6-mercaptopurine oxidation catalysed by aldehyde oxidase and xanthine oxidase.
Drug Metab Pharmacokinet. 2007 Aug;22(4):299-306.
In spite of over 40 years of clinical use of 6-mercaptopurine, many aspects of complex pharmacology and metabolism of this drug remain unclear. It is thought that 6-mercaptopurine is oxidized to 6-thiouric acid through 6-thioxanthine or 8-oxo-6-mercaptopurine by one of two molybdenum hydroxylases, xanthine oxidase (XO), however, the role of other molybdenum hydroxylase, aldehyde oxidase (AO), in the oxidation of 6-mercaptopurine and possible interactions of AO substrates and inhibitors has not been investigated in more details. In the present study, the role of AO and XO in the oxidation of 6- mercaptopurine has been investigated. 6-mercaptopurine was incubated with bovine milk xanthine oxidase or partially purified guinea pig liver molybdenum hydroxylase fractions in the absence and presence of XO and AO inhibitor/substrates, and the reactions were monitored by spectrophotometric and HPLC methods. According to the results obtained from the inhibition studies, it is more likely that 6- mercaptopurine is oxidized to 6-thiouric acid via 6-thioxanthine rather than 8-oxo-6-mercaptopurine. The first step which is the rate limiting step is catalyzed solely by XO, whereas both XO and AO are involved in the oxidation of 6-thioxanthine to 6-thiouric acid. [Abstract/Link to Full Text]

Kamiyama Y, Matsubara T, Yoshinari K, Nagata K, Kamimura H, Yamazoe Y
Role of human hepatocyte nuclear factor 4alpha in the expression of drug-metabolizing enzymes and transporters in human hepatocytes assessed by use of small interfering RNA.
Drug Metab Pharmacokinet. 2007 Aug;22(4):287-98.
Hepatocyte nuclear factor 4alpha (HNF4alpha) is an important transcription factor in hepatic gene expression. Here, we have investigated the role of HNF4alpha in the expression of drug-metabolizing enzymes and transporters in human hepatocytes using an adenovirus expressing human HNF4alpha-small interfering RNA (hHNF4alpha-siRNA). The hHNF4alpha-siRNA effectively reduced the mRNA and nuclear protein levels of hHNF4alpha in a concentration-dependent manner. The hHNF4alpha-siRNA also decreased the mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, UGT1A1, UGT1A9, SULT2A1, ABCB1, ABCB11, ABCC2, OATP1B1 and OCT1, as well as those of PXR and CAR. To discern the role of these nuclear receptors, we co-infected hepatocytes with hHNF4alpha-siRNA and PXR- or CAR-expressing adenovirus. The hHNF4alpha-siRNA-induced reductions of the enzyme and transporter mRNA levels were not restored except CYP2B6 mRNA levels, which were returned to the control level by overexpressing CAR. Furthermore, although hHNF4alpha-siRNA did not significantly affect the fold-induction of CYP2B6, CYP2C8, CYP2C9, or CYP3A4 mRNA levels following treatment with CYP inducers, the levels in hHNF4alpha-suppressed cells fell significantly compared to the control. These results suggest that HNF4alpha plays a dominant role in the expression of drug-metabolizing enzymes and transporters in human hepatocytes, and that HNF4alpha expression levels is a possible determinant for inter-individual variations in the expression of these enzymes and transporters. [Abstract/Link to Full Text]

Kojima K, Nagata K, Matsubara T, Yamazoe Y
Broad but distinct role of pregnane x receptor on the expression of individual cytochrome p450s in human hepatocytes.
Drug Metab Pharmacokinet. 2007 Aug;22(4):276-86.
In the present study, we have utilized a target selective human pregnane X receptor-siRNA (hPXR-siRNA)-adenovirus expression system to examine the contribution of hPXR on the gene regulation of drug-metabolizing P450s in human hepatocytes. Introduction of the hPXR-siRNA adenoviral vector reduced the level of PXR mRNA. After infection with Ad hPXR-siRNA, the basal and ligand-activated CYP2A6, CYP2C8, CYP3A4 and CYP3A5 mRNA levels were decreased significantly in dose-dependent manners, whereas CYP2B6, CYP2C9 and CYP2C19 mRNA levels were moderately influenced after infection with Ad hPXR-siRNA. These data suggest the distinct PXR influences on the regulation of these genes. The expression of CYP1A2 and CYP2D6 mRNA were not affected by the introduction of hPXR-siRNA, suggesting that PXR plays no functional role in the expression of either of these genes. This is the first report to compare simultaneously the relative contribution of hPXR on the expression of nine forms of P450 in primary cultured human hepatocytes. Mutual sharing among nuclear receptors of their binding cis-elements becomes clear now. Thus, the present method using the combination of adenovirus-mediated hPXR-siRNA expression and human hepatocytes may offer clear information on the relative role of nuclear receptors such as hPXR on the expression of drug metabolizing genes. [Abstract/Link to Full Text]

Kamiyama E, Yoshigae Y, Kasuya A, Takei M, Kurihara A, Ikeda T
Inhibitory effects of angiotensin receptor blockers on CYP2C9 activity in human liver microsomes.
Drug Metab Pharmacokinet. 2007 Aug;22(4):267-75.
We investigated the inhibitory effects of the angiotensin receptor blockers (ARBs), candesartan, irbesartan, losartan, losartan active metabolite (EXP-3174), olmesartan, telmisartan and valsartan (0.3-300 microM), on the CYP2C9 activity in human liver microsomes using (S)-(-)-warfarin as a typical CYP2C9 substrate. Except for olmesartan and valsartan, these ARBs inhibited the activity of 7-hydroxylation of (S)-(-)-warfarin with IC50 values of 39.5-116 microM. Of six synthetic derivatives of olmesartan, five compounds which possess either alkyl groups or a chloro group at the same position as that of the hydroxyisopropyl group in olmesartan inhibited CYP2C9 activity with IC50 values of 21.7-161 microM. Olmesartan and the olmesartan analogue, RNH-6272, both having a hydroxyisopropyl group, showed no inhibition, indicating that the hydrophilicity of this group greatly contributes to the lack of CYP2C9 inhibition by these two compounds. A three-dimensional model for docking between EXP-3174 and CYP2C9 indicated that the chloro group of EXP-3174 is oriented to a hydrophobic pocket in the CYP2C9 active site, indicating that the lipophilicity of the group present in ARBs at the position corresponding to that of the hydroxyisopropyl group in olmesartan is important in inhibiting CYP2C9 activity. [Abstract/Link to Full Text]

Kose N, Asashima T, Muta M, Iizasa H, Sai Y, Terasaki T, Nakashima E
Altered expression of basement membrane-related molecules in rat brain pericyte, endothelial, and astrocyte cell lines after transforming growth factor-beta1 treatment.
Drug Metab Pharmacokinet. 2007 Aug;22(4):255-66.
The basement membrane at the blood-brain barrier (BBB) plays important roles in maintaining the structure and function of capillary vessels. The BBB is constructed from endothelial cells, astrocytes and pericytes, but their interactions in the formation or maintenance of basement membrane have not been established. Transforming growth factor-beta1 (TGF-beta1) is known to increase fibronectin in brain capillary basement membrane with deposition of beta-amyloid. We previously reported that the mRNA level of alpha-smooth muscle actin in a brain capillary pericyte cell line TR-PCT1 was increased by treatment with TGF-beta1. In this study, expression of mRNAs encoding basement membrane-related molecules in TR-PCT1, a rat endothelial cell line TR-BBB13, and a type 2 astrocyte cell line TR-AST4 was evaluated by RT-PCR. The effects of TGF-beta1 on expression of basement membrane-related genes in these cell lines were also examined. Fibronectin, MMP-9, tPA, TIMP-1, and PAI-l in TR-PCT1 were higher than in TR-BBB13 and TR-AST4. In TR-PCT1 treated with TGF-beta1, collagen type IV, PAI-1, and MMP-9 were increased, and TIMP-2 was reduced. The change in PAI-1 mRNA was faster than those in MMP-9, TIMP-2, collagen type IV mRNAs. These results suggest that pericytes may be key cells in the maintenance of the basement membrane at the BBB. [Abstract/Link to Full Text]

Sugano K, Okazaki A, Sugimoto S, Tavornvipas S, Omura A, Mano T
Solubility and dissolution profile assessment in drug discovery.
Drug Metab Pharmacokinet. 2007 Aug;22(4):225-54.
The purposes of the review are to: a) Provide a comprehensible introduction of the-state-of-the-art sciences of solubility and dissolution, b) introduce typical technologies to assess solubility and dissolution, and c) propose the best practice strategy. The theories of solubility and dissolution required in drug discovery were reviewed especially from the view point of oral absorption. The physiological conditions in the gastrointestinal fluid in humans and animals were then briefly summarized. Technologies to assess solubility and dissolution in drug discovery were then introduced. Recently, these technologies have been improved by the laboratory automation and computational technologies. Finally, the strategies to apply these technologies for a drug discovery project were discussed. [Abstract/Link to Full Text]

Pucci L, Geppetti A, Maggini V, Lucchesi D, Maria Rossi A, Longo V
CYP1A2 F21L and F186L polymorphisms in an Italian population sample.
Drug Metab Pharmacokinet. 2007 Jun;22(3):220-2.
P450 cytochromes (CYPs) enzymes play a major role in variability of drug response and cancer susceptibility. In particular, up to 60-fold interindividual variation has been detected in the activity of CYP1A2, which is involved in the metabolism of caffeine, several drugs and various toxic and carcinogenic compounds. Aim of this study is to assess the frequency of CYP1A2 F21L and F186L polymorphisms (formerly CYP1A2(*)2 and (*)11 alleles), up to now found in Asiatic populations only. These variants were absent in 500 Italian healthy subjects. Therefore it can be suggested that the variation of CYP1A2-dependent metabolism in the Caucasian population is not related to these two CYP1A2 polymorphisms. Thus, this study supports the view that ethnicity is a relevant factor to be carefully considered in pharmacogenetic studies. [Abstract/Link to Full Text]

Fukushima-Uesaka H, Saito Y, Maekawa K, Kamatani N, Kajio H, Kuzuya N, Noda M, Yasuda K, Sawada J
Genetic variations and haplotype structures of transcriptional factor Nrf2 and its cytosolic reservoir protein Keap1 in Japanese.
Drug Metab Pharmacokinet. 2007 Jun;22(3):212-9.
Transcriptional factor Nrf2 and its cytosolic reservoir protein Keap1 play important roles in induction of the expression of genes for xenobiotic metabolism and disposition, many of which are involved in protection from oxidative stress. In this study, 5 NFE2L2 (encoding Nrf2) and 6 KEAP1 exons and their flanking introns were comprehensively screened for genetic variations in 84 Japanese subjects. As for NFE2L2, 14 genetic variations were found, including 9 novel ones: 7 were located in the 5'-flanking region, 1 in the 5'-untranslated region (5'-UTR), 3 (1 synonymous and 2 nonsynonymous) in the coding exons, 1 in the intron, and 2 in the 3'-UTR. Two novel nonsynonymous variations, 697C>T (Pro233Ser) and 1094G>T (Ser365Ile), were heterozygously found with allele frequencies of 0.012 and 0.006, respectively. Regarding KEAP1, 18 genetic variations were detected, including 13 novel ones: 2 were located in the 5'-flanking region, 4 in the coding exons (4 synonymous), 5 in the introns, 4 in the 3'-UTR, and 3 in the 3'-flanking region. Based on the linkage disequilibrium (LD) profiles, both genes were analyzed as single LD blocks, where 14 (NFE2L2) and 18 (KEAP1) haplotypes were inferred. Six (NFE2L2) and 5 (KEAP1) haplotypes were relatively prevalent (>or=0.03 frequencies) and accounted for >or=88% of the inferred haplotypes. Haplotype-tagging variations of each gene were identified to capture these prevalent haplotypes. These data would be fundamental and useful information for pharmacogenetic studies on Nrf2-regulated genes for xenobiotic metabolism and disposition. [Abstract/Link to Full Text]

Furubayashi T, Inoue D, Kamaguchi A, Higashi Y, Sakane T
Influence of formulation viscosity on drug absorption following nasal application in rats.
Drug Metab Pharmacokinet. 2007 Jun;22(3):206-11.
The aim of this research is to clarify the influence of the viscosity of the nasal formulation on in vivo nasal drug absorption and its mechanism using an in vitro Caco-2 system. The drug solution was made viscous by the addition of dextran (Dex). The disappearance of FITC-labeled Dextran (FD, a marker of the dosing solution) applied with control solution followed monoexponential kinetics, while FD applied with Dex solution showed biexponential elimination. The mean residence time of FD in the nasal cavity was increased with the increase in Dex concentration. The nasal absorption of acyclovir was similar in the formulation with low viscosity, increased in the formulation with moderate viscosity and markedly decreased in the formulation with high viscosity. The result from the normal Caco-2 transport study could not explain the relation of in vivo drug absorption with viscosity, while the modified Caco-2 system provided data partly reflecting the change in in vivo absorption in rats. In conclusion, the residence of the applied solution in the nasal cavity was enhanced by the addition of Dex in a viscosity-dependent manner. Moderate viscosity of the dosing solution improved the in vivo nasal absorption of acyclovir, while higher viscosity decreased it. [Abstract/Link to Full Text]

Sugiyama Y, Mimura N, Kuwabara T, Kobayashi H, Ushiki J, Fuse E
Effect of benidipine on simvastatin metabolism in human liver microsomes.
Drug Metab Pharmacokinet. 2007 Jun;22(3):199-205.
Benidipine, which is a calcium channel blocker that has clinical advantages in the treatment of hypertension, is metabolized by CYP3A4 in humans. The effect of benidipine on the metabolism of simvastatin by human liver microsomes was investigated in order to predict the potential of in vivo drug-drug interactions between benidipine and other substrates of CYP3A4. The results were compared with data generated with azelnidipine, which is also metabolized by CYP3A4. Both benidipine and azelnidipine inhibited simvastatin metabolism in vitro in a concentration-dependent manner. Assuming competitive inhibition, the K(i) values based on the unbound concentrations, were calculated to be 0.846 and 0.0181 microM for benidipine and azelnidipine, respectively. If simvastatin (10 mg) and benidipine (8 mg, the clinically recommended highest dose) were to be administered concomitantly, the ratio of the areas under the concentration-time curves of simvastatin with and without benidipine (AUC((+I))/AUC) was predicted to be 1.01. On the other hand, if simvastatin (10 mg) and azelnidipine (8 mg) were co-administered, the AUC((+I))/AUC for simvastatin was predicted to be 1.72, which is close to the observed value (1.9) in healthy volunteers. These data suggest that benidipine is unlikely to cause a drug interaction by inhibiting CYP3A4 activity in the liver. [Abstract/Link to Full Text]

Fujimoto N, Inoue K, Ohgusu Y, Hayashi Y, Yuasa H
Enhanced uptake of glycerol by butyrate treatment in HCT-15 human colon cancer cell line.
Drug Metab Pharmacokinet. 2007 Jun;22(3):195-8.
The HCT-15 human colon cancer cell line has a Na(+)-dependent carrier-mediated transport system for the uptake of glycerol. A similar transport system has been suggested to be present also in the small intestine and is of interest with regard to its role in the absorption of glycerol and possibly some structurally related compounds. To help clarifying functional characteristics of such glycerol transport systems, we examined the effect of butyrate, an agent known to facilitate the differentiation of cells, on glycerol uptake in HCT-15 cells. The uptake of glycerol (0.4 microM) was found to be about 5-fold greater in HCT-15 cells pretreated with butyrate (2 mM) for 24 h than in those untreated. The increase in the uptake by the butyrate treatment was due to an increase in the maximum transport rate. The effect of butyrate was almost completely suppressed when actinomycin D, an inhibitor of gene transcription, and cycloheximide, an inhibitor of protein synthesis, were added to the medium during the butyrate treatment. These results support the suggestion that a specific carrier protein is involved in glycerol uptake by HCT-15 cells and the carrier protein is one of those inducible by butyrate-induced cell differentiation. [Abstract/Link to Full Text]

Ariyoshi N, Shimizu Y, Kobayashi Y, Nakamura H, Nakasa H, Nakazawa K, Ishii I, Kitada M
Identification and partial characterization of a novel CYP2C9 splicing variant encoding a protein lacking eight amino acid residues.
Drug Metab Pharmacokinet. 2007 Jun;22(3):187-94.
CYP2C9 is known as an enzyme responsible for the metabolism of various clinically important drugs. Recently, we cloned a cDNA corresponding to a CYP2C9 splicing variant (SV), which seemed to have an open reading frame of a protein with 482 amino acid residues. To investigate whether or not the SV can be translated as a functionally active protein, we expressed the CYP2C9SV in insect cells, and spectrophotometric and enzymatic properties were characterized. The CYP2C9SV protein showed a typical reduced CO-difference spectrum, indicating that the translated protein binds a heme moiety. However, CYP2C9SV did not metabolize tolbutamide or diclofenac at all, suggesting that the SV protein appeared to lack the ability to catalyze reactions mediated by CYP2C9. Although the CYP2C9SV mRNA was detected in all human liver samples examined in this study by real-time PCR, the level was generally low, ranging between 0.7 and 9.6% of the normal CYP2C9 mRNA. These results suggest that the CYP2C9SV protein is unlikely to contribute to CYP2C9 activities, although it appears to be expressed in most individuals. [Abstract/Link to Full Text]

Nishimura M, Koeda A, Suganuma Y, Suzuki E, Shimizu T, Nakayama M, Satoh T, Narimatsu S, Naito S
Comparison of inducibility of CYP1A and CYP3A mRNAs by prototypical inducers in primary cultures of human, cynomolgus monkey, and rat hepatocytes.
Drug Metab Pharmacokinet. 2007 Jun;22(3):178-86.
This study was conducted to investigate the effects of treatment with the prototypical inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) on the mRNA levels of drug-metabolizing enzymes in primary cultures of cryopreserved human, cynomolgus monkey, and rat hepatocytes. Analysis was performed by quantitative real-time RT-PCR using primers and TaqMan probes. Treatment with Ome substantially increased the mRNA levels of both CYP1A1 and CYP1A2 in human hepatocytes, but increased only the mRNA level of CYP1A1 in monkey hepatocytes, whereas it had no marked effect on the mRNA levels of CYP1A1 or CYP1A2 in rat hepatocytes. Treatment with Rif or Dex did not markedly affect the mRNA level of CYP1A in any of the hepatocyte cultures under the conditions used. All three inducers increased the mRNA level of CYP3A8 in monkey hepatocytes (in the order Rif>Dex>or=Ome), and a similar profile was observed for the mRNA level of CYP3A4 in human hepatocytes, but the potency of induction was markedly attenuated. In contrast, only Dex substantially increased the mRNA level of CYP3A1 in rat hepatocytes, with Rif and Ome showing no effects. These results indicate that the molecular mechanisms responsible for the regulation of CYP1A2 genes differ between humans and cynomolgus monkeys, although the regulatory mechanisms for CYP1A1 and CYP3A genes are similar. [Abstract/Link to Full Text]

Sato S, Nakajima M, Honda A, Konishi T, Miyazaki H
Pharmacokinetics of theophylline in Guinea pig tears.
Drug Metab Pharmacokinet. 2007 Jun;22(3):169-77.
It is well recognized that the theophylline (TP) concentration in human tears correlates well with the free TP concentration in human plasma. However this correlation was found only in a very narrow range of concentrations of TP, and pharmacokinetic analysis of TP in tears has not been carried out for a wide range of concentrations of TP. The aims of this investigation were to develop a simple kinetic model for TP in guinea pig plasma (total [Cf+b] and free [Cf]), cerebrospinal fluid (CSF) [C](CSF) and tears [C](T), and to examine whether [Cf], [Cf+b] and [C](CSF) can be predicted from [C](T) using the resulting kinetic parameters. [Cf+b], [Cf], [C](CSF) and [C](T) were determined by GC/EI-SIM following bolus i.v. injection of TP in doses of 10, 50 and 100 mg/kg into guinea pigs. The wide range of concentrations of [Cf+b] could be quantitatively described by a two-compartment model with non-linear elimination kinetics and individual volume distribution of TP at each dose. [C](T) and [C](CSF) were analyzed using passive diffusion models with and without the pH-partition theory, respectively. The value of [Cf] could be predicted from the value of [C](T). Thus, the measurement of [C](T) which can be collected non-invasively would be a useful method for the therapeutic drug monitoring of TP. [Abstract/Link to Full Text]

Inoue S, Ohtani H, Tsujimoto M, Hori S, Sawada Y
Development of a pharmacokinetic model to optimize the dosage regimen of TS-1, a combination preparation of tegafur, gimeracil and oteracil potassium.
Drug Metab Pharmacokinet. 2007 Jun;22(3):162-8.
BACKGROUND: TS-1 is a combination preparation of tegafur, a prodrug of 5-fluorouracil (5-FU), with gimeracil, a potent inhibitor of dihydropyrimidine dehydrogenase (DPD), which mediates the inactivation of 5-FU. UFT is a combination preparation of tegafur with uracil, which also inhibits DPD, though less potently; UFT has a higher content of tegafur than that in TS-1. We aimed to develop a pharmacokinetic model to describe the kinetics of tegafur and 5-FU after the administration of TS-1 and UFT. METHODS: We developed a model incorporating the inhibition of DPD by gimeracil and uracil, and fitted the model to the observed kinetics of tegafur and 5-FU after the administration of TS-1 and UFT. Then, we simulated the plasma 5-FU profiles in patients with renal dysfunction and those after replacement of TS-1 with UFT and compared them with the observed profiles. RESULTS: The developed model could appropriately describe the plasma concentration profiles of 5-FU and tegafur after the administration of TS-1 in patients with normal and impaired renal function. CONCLUSION: The developed model may be useful to optimize the dosage regimen of TS-1 under various clinical conditions. [Abstract/Link to Full Text]

Li X, Bratton S, Radominska-Pandya A
Human UGT1A8 and UGT1A10 mRNA are expressed in primary human hepatocytes.
Drug Metab Pharmacokinet. 2007 Jun;22(3):152-61.
It is widely believed that the UGT1A isoforms, UGT1A8 and -1A10, are expressed exclusively in extrahepatic tissues. In this work, human primary hepatocytes from six donors were analyzed for UGT1A8 and -1A10 mRNA expression by semi-quantitative RT-PCR. New primers to amplify UGT1A8 mRNA were designed and found to differ from those previously published. We demonstrated that UGT1A8 and -1A10 mRNA are expressed in hepatocytes. Although basal UGT mRNA levels were detected in untreated hepatocytes, significant up-regulation of the levels of mRNA for these isoforms were seen after treatment with 3-methylcholanthrene (3-MC) and rifampicin (Rif). RT-PCR products for all UGTs were sequenced and unambiguously identified as matching the corresponding cDNA. The discovery of these isoforms in hepatocytes is a novel discovery and will stimulate studies on the potential role for these isoforms in hepatic detoxification. [Abstract/Link to Full Text]

Kawakami S, Hashida M
Targeted delivery systems of small interfering RNA by systemic administration.
Drug Metab Pharmacokinet. 2007 Jun;22(3):142-51.
RNA interference (RNAi) is induced by 21-25 nucleotide, double-stranded small interfering RNA (siRNA), which is incorporated into the RNAi-induced silencing complex (RISC) and is a guide for cleavage of the complementary target mRNA in the cytoplasm. There are many obstacles to in vivo delivery of siRNAs, such as degradation by enzymes in blood, interaction with blood components and non-specific uptake by the cells, which govern biodistribution in the body. In order to achieve the knockdown by siRNAs in vivo, many delivery systems of siRNAs based on physical and pharmaceutical approaches have been proposed. In addition, the immune responses of siRNA must be taken into account when considering the application of siRNAs to in vivo therapy. This review focuses on recent reports about delivery systems and immune responses of siRNAs. [Abstract/Link to Full Text]

Recent Articles in Journal of Pharmacological Sciences

Hamabe W, Maeda T, Kiguchi N, Yamamoto C, Tokuyama S, Kishioka S
Negative Relationship Between Morphine Analgesia and P-Glycoprotein Expression Levels in the Brain.
J Pharmacol Sci. 2007 Dec 8;
It is known that opioid analgesics given systemically have limited distribution into the brain because of their interaction with P-glycoprotein (P-gp), an ATP-dependent efflux pump acting at the blood-brain barrier (BBB). We previously found that morphine and fentanyl showed higher analgesic potencies in P-gp-deficient mice compared with those in wild-type mice, suggesting that their analgesic effects are considerably dependent on P-gp expression. In this study, we focused on individual differences in the analgesic effectiveness of morphine, in cortical P-gp expression, and in basal P-gp ATPase activity in male ICR mice. We found that there were 3- to 10-fold differences between the magnitude of morphine analgesia (3 mg/kg, s.c.; tail-pinch method) in mice. Furthermore, there was a significant negative correlation between morphine's analgesic effects and individual P-gp expression in the cortex as estimated by western blot analysis. In addition, basal P-gp ATPase activities in isolated membrane preparations of brain capillary endothelial cells (BCECs) were negatively correlated with the magnitude of the analgesic effect of morphine. These results indicate that the individual differences in morphine analgesia may be due to some functional or quantitative differences in individual P-gp in BCECs, acting at the BBB. [Abstract/Link to Full Text]

Ito S, Mori T, Namiki M, Suzuki T, Sawaguchi T
Complicated Interaction Between Psychostimulants and Morphine in Expression of Phenotype of Behavior in the Dopaminergic System of BALB/c Mice.
J Pharmacol Sci. 2007 Dec 1;
It is believed that BALB/c mice appear to be less sensitive to the locomotor effects of abused drugs compared to other strains, and several behaviors induced by abused drugs depend on genetic factors. The present study was designed to investigate the effects of the interaction between psychostimulants and morphine on behavior in BALB/c mice. Morphine and cocaine induced hyperlocomotion and hypolocomotion, respectively, while methamphetamine did not affect locomotor activity and high doses of methamphetamine significantly increased self-injurious behavior. Cocaine or methamphetamine increased the effects of morphine on locomotor behavior. Haloperidol (a dopamine-receptor antagonist) attenuated the hyperlocomotion induced by the combination of cocaine or methamphetamine plus morphine. These results indicate that the synergistic effects of methamphetamine or cocaine and morphine on locomotor activity are mediated through enhancement of the dopaminergic system and that combinations of psychostimulants and morphine enhance the locomotor activity in BALB/c mice. On the other hand, morphine completely attenuated methamphetamine-induced self-injurious behavior. Furthermore, a low dose (0.01 mg/kg) of haloperidol significantly increased the effects of methamphetamine and morphine on the locomotor activity. Hyperlocomotion induced by psychostimulants is mediated by the mesolimbic dopaminergic system, whereas stereotyped behaviors is mediated by the nigrostriatal dopaminergic system. Our findings suggest that balances of the activation of dopaminergic neurons (between mesolimbic and nigrostriatal systems) may play an important role to engender corresponding behavioral outcomes in BALB/c mice. [Abstract/Link to Full Text]

Nakao T, Shiota M, Tatemoto Y, Izumi Y, Iwao H
Pravastatin Induces Rat Aortic Endothelial Cell Proliferation and Migration via Activation of PI3K/Akt/mTOR/p70 S6 Kinase Signaling.
J Pharmacol Sci. 2007 Dec 1;
The HMG-CoA reductase inhibitors (statins) have been shown to exert several vascular protective effects that are not related to changes in cholesterol profile, and these effects of statins are partly caused by the activation of angiogenesis. Endothelial cell (EC) proliferation and migration are crucial events for angiogenesis and statins are known to enhance these events. However, the molecular mechanism by which statins promote EC proliferation and migration is not fully understood. In this study, we show Akt and its downstream target mammalian target of rapamycin (mTOR) play an important role in pravastatin-induced EC proliferation and migration. We found that pravastatin significantly enhanced the proliferation and migration of rat aortic endothelial cells (rAECs). The addition of pravastatin to rAECs resulted in rapid phosphorylation of Akt and p70 S6 kinase (p70S6K). LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), blocked both Akt and p70S6K phosphorylation, whereas rapamycin, a specific inhibitor of mTOR, suppressed only p70S6K phosphorylation induced by pravastatin. Furthermore, both LY294002 and rapamycin inhibited pravastatin-induced rAEC proliferation and migration. Taken together, our findings indicate that pravastatin activates PI3K/Akt/mTOR/p70S6K signaling in this sequential manner and this pathway contributes to pravastatin-induced rAEC proliferation and migration. [Abstract/Link to Full Text]

Nishida H, Ichikawa H, Konishi T
Shengmai-san Enhances Antioxidant Potential in C2C12 Myoblasts Through the Induction of Intracellular Glutathione Peroxidase.
J Pharmacol Sci. 2007 Dec 1;
Cellular and tissue injury associated with reactive oxygen species (ROS) has been reported in many kinds of disorders. While the antioxidant enzymes play critical roles in inhibiting the ROS-mediated injury, glutathione peroxidase (GPx) is scavenging hydroperoxides including H(2)O(2). We previously reported that Shengmai-san (SMS), a traditional Chinese medicine, prevented ischemia/reperfusion injury of the brain and other organs in rats. To clarify the effect of SMS on intracellular responses of muscle cells against oxidative stress, C2C12 myoblasts were subjected to H(2)O(2) abuse. SMS pre-incubation prevented the decreasing cell viability after H(2)O(2) treatment. The accumulations of cellular protein carbonyl associated with apoptotic cell death were also inhibited by the SMS pre-incubation prior to oxidative damage induction. At the same time, enhanced activity, protein, and mRNA expression levels of GPx were observed in cells pre-incubated with SMS prior to H(2)O(2) abuse. Moreover, intracellular GSH was subsequently decreased after H(2)O(2) treatment. These findings suggest that SMS improved the antioxidant capacity against acute oxidative stress through the constitutive enhancement of GPx expression in C2C12 myoblasts. Because of its antioxidative property, SMS might be useful not only for the oxidative damage associated diseases but also for the transplantation of myoblasts into muscular dystrophy patients. [Abstract/Link to Full Text]

Egashira N, Koushi E, Mishima K, Iwasaki K, Oishi R, Fujiwara M
DOI Inhibits Delta(9)-Tetrahydrocannabinol-Induced Catalepsy-Like Immobilization in Mice.
J Pharmacol Sci. 2007 Dec 1;
The effect of the serotonin 5-HT(2A/2C)-receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) on Delta(9)-tetrahydrocannabinol (THC)-induced catalepsy-like immobilization was studied in mice. DOI (0.3 and 1 mg/kg, i.p.) significantly inhibited the catalepsy-like immobilization induced by THC (10 mg/kg, i.p.). In contrast, the selective 5-HT(2C)-receptor agonist 8,9-dichloro-2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5(6H)-one (WAY 161503) had no effect on this catalepsy-like immobilization. Moreover, the 5-HT(2A)-receptor antagonist ketanserin (0.3 mg/kg, i.p.) reversed the inhibition of THC-induced catalepsy-like immobilization caused by DOI (1 mg/kg), whereas the selective 5-HT(2C)-receptor antagonist 6-chloro-2,3-dihydro-5-methyl-N-[6-(2-methyl-3-pyridinyl)oxyl]-3-pyridinyl]-1H-indole-1-carboxyamide (SB 242084) did not affect this inhibitory effect of DOI. On the other hand, ketanserin (0.3 and 1 mg/kg, i.p.) enhanced the catalepsy-like immobilization induced by THC (6 mg/kg, i.p.). Thus, on the basis of these results, it appears that 5-HT(2A)-receptor mechanisms might be responsible for the inhibitory effect of DOI on THC-induced catalepsy-like immobilization. [Abstract/Link to Full Text]

Brug�s L, Gesztelyi R, Zsuga J, Cseppento A, Benko I, Galajda Z, De�k G, Sipka S, Roszer T, Kov�cs P, Szilasi M, Edes I, Szentmikl�si AJ
Modulation of adenosine-induced responses in the Guinea-pig trachea during long-term caffeine treatment: possible role of epithelium.
J Pharmacol Sci. 2007 Nov;105(3):279-90.
The responses to adenosine were studied on isolated, methacholine-precontracted tracheal strips of guinea pigs in the course of long-term caffeine or solvent treatment. Guinea pigs were fed caffeine for 10 weeks (average serum caffeine concentration: 39.1 +/- 3.9 muM). In epithelium-intact tracheal preparations (EITPs), sensititization to adenosine-induced relaxation (AIR) developed. It attained a maximum in week 1 of caffeine treatment, and then its level diminished and disappeared completely by weeks 4 - 6. In epithelium-denuded tracheal preparations (EDTPs), an increase in the sensitivity to adenosine was observed from week 1 to week 10 (a 4 - 6-fold reduction in EC(50)). Use of a coaxial bioassay system confirmed the role of epithelium in this process. The enhancement of the AIR of the EITPs was not modified by inhibitors of cyclooxygenase and lipoxygenase. Following depletion of the neuropeptides by acute capsaicin pretreatment, the AIR of the EITPs was strongly enhanced after caffeine treatment for 6 weeks. In chronically caffeine-treated EITPs, the inhibition of neutral endopeptidase led to dramatic reduction of the AIR. On the basis of the results by inhibiting nitric oxide synthase, it can be supposed that nitric oxide released from EITPs of long-lasting caffeine-treated animals operated as a constrictor agent. Our results show that chronic caffeine treatment gives rise to an initial sensitization to adenosine of the EITPs, this being followed by the development of a specific adaptive process in the epithelial cells, which counterbalances the increased tracheal sensitivity to adenosine. [Abstract/Link to Full Text]

Mizutani N, Nabe T, Ohtani Y, Han HY, Fujii M, Yoshino S, Hirayama T, Kohno S
Polycyclic aromatic hydrocarbons aggravate antigen-induced nasal blockage in experimental allergic rhinitis.
J Pharmacol Sci. 2007 Nov;105(3):291-7.
It has been hypothesized that air pollution has played a role in the increase in allergy prevalence. However, it remains unclear what exact roles are played by polycyclic aromatic hydrocarbons (PAHs), which are encountered in the environment in the form of air pollution, in allergic rhinitis. Thus, we examined whether benzo(a)pyrene (BaP) and 1-nitropyrene (1-NP), representative PAHs, aggravate allergic rhinitis symptoms, using a guinea-pig model. Sensitized animals were repeatedly challenged by inhalation of Japanese cedar pollen once a week. BaP or 1-NP was daily and intranasally administered for 2 weeks (short-term treatment) or for 22 weeks from the time before the sensitization period (long-term treatment). The short-term treatment affected neither nasal blockage nor sneezing induced by antigen. In contrast, the long-term treatment aggravated the antigen-induced nasal blockage that was induced 7 weeks after the start of the treatment with BaP or 1-NP. This aggravation continued during the intranasal treatment with PAH. However, neither sneezing nor Cry j 1-specific IgE antibody production was affected even by the long-term treatment. In conclusion, the long-term treatment with BaP and 1-NP can aggravate allergic rhinitis. The mechanisms underlying this aggravation are not associated with production of Cry j 1-specific IgE. [Abstract/Link to Full Text]

Tang HB, Li YS, Nakata Y
The Release of Substance P From Cultured Dorsal Root Ganglion Neurons Requires the Non-neuronal Cells Around These Neurons.
J Pharmacol Sci. 2007 Nov;105(3):264-71.
Substance P is known to be released from dorsal root ganglion (DRG) neurons, but the possible involvement of non-neuronal cells in the process of substance P release is not known. During the culture period, the number of surviving DRG neurons, the total substance P content from the culture medium and DRG cells, and the total protein of DRG cells were monitored. Both the number of surviving neurons and the total substance P content decreased in a time-dependent manner, whereas the total protein synthesis was increased. The localization of substance P in small-to-medium-sized neurons was further confirmed by immunocytochemistry. Finally, the substance P release levels from the neuronal and non-neuronal enrichments were investigated by radioimmunoassay. Both partially purified DRG neurons and non-neuronal cells exhibited a weaker substance P release response to capsaicin or KCl, relative to unpurified DRG cells. The total substance P content from the partially purified DRG neurons was almost the same as that from the unpurified DRG cells, but much more than that from the partially purified DRG non-neuronal cells. These findings suggest that substance P is released from DRG neurons, and this process should require the coexistence of neurons and non-neuronal cells. [Abstract/Link to Full Text]

Toda A, Okabe M, Yoshida T, Nikaido T
The potential of amniotic membrane/amnion-derived cells for regeneration of various tissues.
J Pharmacol Sci. 2007 Nov;105(3):215-28.
Regenerative medicine is a new field based on the use of stem cells to generate biological substitutes and improve tissue functions, restoring damaged tissue with high proliferability and differentiability. It is of interest as a potential alternative to complicated tissue/organ transplantation. Recently, amnion-derived cells have been reported to have multipotent differentiation ability, and these cells have attracted attention as a cell source for cell-transplantation therapy. The amnion possesses considerable advantageous characteristics: the isolated cells can differentiate into all three germ layers; they have low immunogenicity and anti-inflammatory functions; and they do not require the sacrifice of human embryos for their isolation, thus avoiding the current controversies associated with the use of human embryonic stem cells. Moreover, we developed human amniotic cell-sheets using a novel culture surface coated with a noncytotoxic, temperature-responsive elastic protein-based polymer. We also generated a "hyper-dry-amnion", which has already been applied clinically in the ophthalmological field. Compared to cryopreserved fresh amnion, "hyper-dry-amnion" is easy to handle and has started to bring good results to patients. These materials from the amnion are also expected to open a new field in tissue engineering. Thus, amnion, which had been discarded after parturition, has started to be appreciated as an attractive material in the field of regenerative medicine. In this review, the most recent and relevant clinical and experimental data about the use of amniotic membrane and cells derived from it are described. [Abstract/Link to Full Text]

Nishida A, Reien Y, Ogura T, Uemura H, Tamagawa M, Yabana H, Nakaya H
Effects of azimilide on the muscarinic acetylcholine receptor-operated k(+) current and experimental atrial fibrillation in Guinea-pig hearts.
J Pharmacol Sci. 2007 Nov;105(3):229-39.
Effects of azimilide, a class III antiarrhythmic drug, on the acetylcholine (ACh) receptor-operated K(+) current (I(K.ACh)) and the delayed rectifier K(+) current (I(K)) were examined in guinea-pig atrial cells using patch-clamp techniques. Effects of azimilide on experimental atrial fibrillation (AF) were also examined in isolated guinea-pig hearts. In single atrial myocytes, azimilide inhibited both the rapid (I(Kr)) and slow component of I(K) (I(Ks)). Azimilide inhibited the I(K.ACh) induced by carbachol (CCh, 1 muM), adenosine (10 muM), and intracellular loading of GTPgammaS (100 muM) in a concentration-dependent manner. The IC(50) values of azimilide for inhibiting the CCh-, adenosine-, and GTPgammaS-induced I(K.ACh) were 1.25, 29.1, and 20.9 muM, respectively, suggesting that azimilide inhibits I(K.ACh) mainly by blocking the muscarinic receptors. Azimilide concentration-dependently (0.3 - 10 muM) prolonged the action potential duration (APD) in the absence and presence of muscarinic stimulation. In isolated hearts, perfusion of CCh shortened the duration of the monophasic action potential (MAP) and effective refractory period (ERP) of the left atrium and lowered the atrial fibrillation threshold (AFT). Addition of azimilide inhibited the induction of AF by prolonging the duration of MAP and ERP. The I(K.ACh) inhibition by azimilide may at least in part contribute to the effectiveness to prevent parasympathetic-type AF. [Abstract/Link to Full Text]

Hamada K, Yoshida M, Isayama H, Yagi Y, Kanazashi S, Kashihara Y, Takeuchi K, Yamaguchi I
Possible Involvement of Endogenous 5-HT in Aggravation of Cerulein-Induced Acute Pancreatitis in Mice.
J Pharmacol Sci. 2007 Nov;105(3):240-50.
The aim of the present study was to elucidate the pathogenic role of endogenous 5-HT in pancreatitis. Injections of cerulein at hourly intervals caused edematous pancreatitis in mice characterized by hyperenzymemia and histological alterations. While the cerulein-induced hyperenzymemia was attenuated in mice pretreated with p-CPA, a 5-HT depletor, it was exaggerated by the preferential 5-HT(2A) agonist (DOI), but not by the preferential 5-HT(2B) agonist (BW723C86) or the preferential 5-HT(2C) agonist (mCPP). Selective 5-HT(2A) antagonists (risperidone, spiperone, ketanserin, AMI-193, and MDL 11,939) dose-dependently attenuated the hyperenzymemia; and their potency order, excepting that of ketanserin which has considerable affinity at the 5-HT(2C) receptor as well, paralleled their reported pK(i) values at the 5-HT(2A) receptor. Selective 5-HT(2B) (SB204741) and 5-HT(2C) (SB242084) antagonists hardly affected the hyperenzymemia. Although the non-selective 5-HT(2A)/(2B)/(2C) antagonists (metergoline, ritanserin, and methysergide) dose-dependently attenuated the hyperenzymemia, they were relatively less potent compared to their high pK(i) values at the 5-HT(2A) receptor. In another set of experiments, risperidone, but not SB204741 and SB242084, dose-dependently reversed the cerulein-induced histological alteration of the pancreas (inflammatory cell infiltration). These results suggest that endogenously released 5-HT activates 5-HT(2A) receptors to aggravate cerulein-induced pancreatitis. We propose that selective 5-HT(2A) antagonists may provide a new therapy for acute pancreatitis. [Abstract/Link to Full Text]

Han HY, Nabe T, Mizutani N, Fujii M, Terada T, Takenaka H, Kohno S
Nasal Blockage Induced by Oral Administration of Non-steroidal Anti-inflammatory Drugs in a Guinea-Pig Model of Allergic Rhinitis.
J Pharmacol Sci. 2007 Nov;105(3):251-7.
To elucidate the mechanisms underlying nasal symptoms in patients with aspirin hypersensitivity, we evaluated the effects of orally administered non-steroidal anti-inflammatory drugs (NSAIDs) on the nasal patency of guinea pigs with cedar pollen-induced chronic allergic rhinitis. Indomethacin (10 mg/kg) administered 1 h before a pollen challenge amplified the antigen-induced nasal blockage. More interestingly, even in the absence of the pollen challenge, indomethacin induced nasal blockage at 30 min at 4 h after administration. However, indomethacin-induced nasal blockage was not provoked in non-sensitized animals. Another NSAID, diclofenac (30 mg/kg), also evoked nasal blockage, but unexpectedly, aspirin (500 mg/kg) did not affect nasal patency. Indomethacin-induced nasal blockage was unaffected by a cysteinyl leukotriene receptor (CysLT(1) receptor) antagonist, pranlukast (30 mg/kg, p.o.), or by prostaglandin E(2) (10(-3) M, intranasal), suggesting that the nasal blockage may not be due to hyperproduction of cysteinyl leukotrienes or inhibition of prostaglandin E(2) production. These results indicate that the indomethacin-induced nasal blockage may not be an identical phenomena to airway symptoms in aspirin hypersensitivity patients. However, because chronic nasal inflammation is indispensable for the development of nasal blockage, indomethacin-induced nasal blockage may become a clue to elucidate new mechanisms underlying hypersensitivity to NSAIDs. [Abstract/Link to Full Text]

Morita M, Yoshizaki K, Nakane A, Kudo Y
Inhibitory Effect of the Phosphoinositide 3-Kinase Inhibitor LY294002 on Muscarinic Acetylcholine Receptor-Induced Calcium Entry in PC12h Cells.
J Pharmacol Sci. 2007 Nov;105(3):258-63.
Phosphoinositide-3 kinase (PI3K) and phospholipase C (PLC) utilize the same phosphoinositides as substrates to produce different signaling molecules. These enzymes are activated by a similar set of cell signaling mechanisms, i.e., tyrosine kinases and G proteins, and affect common cell functions, including proliferation, motility, and intracellular trafficking. Despite these similarities, the interplay between these enzymes is not well understood. To address this issue, the effects of the PI3K inhibitor LY294002 on carbachol-induced calcium increase in PC12h cells were examined. As carbachol stimulates both Gq- and Gi-coupled muscarinic acetylcholine receptors (mAChRs), PI3K and PLC are activated simultaneously in this protocol. LY294002 was found to reduce the carbachol-induced calcium increase, and the reduction was attributed to suppression of calcium entry. As LY294002 did not affect either carbachol-induced calcium release or calcium entry induced by calcium store depletion, this agent was found to suppress calcium entry directly activated by mAChRs. Although PI3K was supposed to compete for substrates with PLC, the PI3K inhibitor did not enhance PLC-dependent cellular responses. As LY294002 was still effective by treating cells after carbachol stimulation, it is likely that this agent blocks the calcium entry channels directly. [Abstract/Link to Full Text]

Miyata S, Shimoi T, Hirano S, Yamada N, Hata Y, Yoshikawa N, Ohsawa M, Kamei J
Effects of serotonergic anxiolytics on the freezing behavior in the elevated open-platform test in mice.
J Pharmacol Sci. 2007 Nov;105(3):272-8.
Freezing behavior is thought to be a sign of fear in animals. We examined whether the freezing behavior during the elevated open-platform stress, which is a psychological stressor without painful stimulus, is modulated by serotonergic neurotransmission and would be a useful marker for screening anxiolytic and/or antidepressant. Male ICR mice (6 - 8-week-old) were individually placed on an elevated open-platform and the duration of freezing behavior of mouse was measured for 10 min. Fluoxetine and citalopram, selective serotonin (5-HT) reuptake inhibitors, markedly decreased the duration of freezing. Fenfluramine, a 5-HT releaser, and 8-OH-DPAT, a potent 5-HT(1A)-receptor agonist, also significantly decreased the duration of freezing. In contrast, the 5-HT-synthesis inhibitor p-chlorophenylalanine significantly increased the duration of freezing. Diazepam, a benzodiazepine anxiolytic, had no effect on the duration of freezing at doses having no effect on locomotor activity. Imipramine and clomipramine, tricyclic antidepressants, also did not affect the duration of freezing. Reboxetine, a selective noradrenaline reuptake inhibitor, significantly increased the duration of freezing. These results indicate that the activation of serotonergic neurotransmission attenuates the fear-related behavior in the elevated open-platform test, while the activation of noradrenergic neurotransmission increases the fear-related behavior. In addition, this test is convenient for assaying anxiolytic drugs that affect serotonergic neurotransmission. [Abstract/Link to Full Text]

Jin YR, Han XH, Lee JJ, Lim Y, Kim TJ, Yoo HS, Hong JT, Lee CK, Yun YP
Antiplatelet effect of NQ12: a possible mechanism through the arachidonic acid cascade.
J Pharmacol Sci. 2007 Oct;105(2):193-200.
NQ12, an antithrombotic agent, has been reported to display a potent antiplatelet activity. This study was undertaken to reveal the effect of NQ12 on rabbit platelet aggregation and signal transduction involved in the arachidonic acid (AA) cascade. NQ12 concentration-dependently suppressed collagen-, AA-, and U46619-induced rabbit platelet aggregation, with IC(50) values of 0.71 +/- 0.2, 0.82 +/- 0.3, and 0.45 +/- 0.1 microM, respectively. In addition, the concentration-response curve of U46619 was shifted to the right after NQ12 treatment, indicating an antagonism on thromboxane (TX) A(2) receptors. The collagen-stimulated AA liberation was inhibited by NQ12 in the same pattern as its inhibition of platelet aggregation. Further study revealed that NQ12 potently suppressed AA-mediated TXA(2) formation, but had no effect on the PGD(2) production, indicating an inhibitory effect on TXA(2) synthase, which was supported by a TXA(2) synthase activity assay indicating that NQ12 concentration-dependently inhibited TXA(2) formation converted from PGH(2). On the other hand, the AA-stimulated 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) formation was also suppressed by NQ12. Taken together, these results suggest that NQ12 has a potential to inhibit TXA(2) synthase activity and TXA(2) receptors, and it can modulate AA liberation as well as 12-HETE formation in platelets. This may be a convincing mechanism for the antithrombotic action of NQ12. [Abstract/Link to Full Text]

Kim DR, Rah SH, Sohn JH, Yeh BI, Ko CM, Park JS, Kim MJ, Lee JW, Kong ID
Calcium mobilization by activation of M(3)/M(5) muscarinic receptors in the human retinoblastoma.
J Pharmacol Sci. 2007 Oct;105(2):184-92.
Activation of muscarinic acetylcholine receptors (mAChR) is one of the most important signal transduction pathways in the human body. In this study, we investigated the role of mAChR activation in relation to its subtypes in human retinoblastoma cell-lines (WERI-Rb-1) using Ca(2+) measurement, real-time PCR, and Western Blot techniques. Acetylcholine (ACh) produced prominent [Ca(2+)](i) transients in a repeated manner in WERI-Rb-1 cells. The maximal amplitude of the [Ca(2+)](i) transient was almost completely suppressed by 97.3 +/- 0.8% after atropine (1 microM) pretreatment. Similar suppressions were noted after pretreatments with thapsigargin (1 microM), an ER Ca(2+)-ATPase (SERCA) inhibitor, whereas the ACh-induced [Ca(2+)](i) transient was not affected even in the absence of extracellular calcium. U-73122 (1 microM), a PLC inhibitor, and xestospongin C (2 microM), an IP(3)-receptor antagonist, elicited 11.5 +/- 2.9% and 17.8 +/- 1.9% suppressions, respectively. The 50% inhibitory concentration of (IC(50)) values for blockade of a 100 microM ACh response by pirenzepine and 4-DAMP were 315.8 and 9.1 nM, respectively. Moreover, both M(3) and M(5) mAChRs were prominent in quantitative real-time-PCR. Taken together, the M(3)/M(5) subtypes appear to be the major contributor, leading to intracellular calcium mobilization from the internal store via an IP(3)-dependent pathway in the undifferentiated retinoblastoma cells. [Abstract/Link to Full Text]

Ge X, Low B, Liang M, Fu J
Angiotensin II directly triggers endothelial exocytosis via protein kinase C-dependent protein kinase D2 activation.
J Pharmacol Sci. 2007 Oct;105(2):168-76.
Angiotensin II (AII) has been reported to induce leukocyte adhesion to endothelium through up-regulation of P-selectin surface expression. However, the underlying molecular and cellular mechanisms remain unknown. P-selectin is stored in Weibel-Palade bodies (WPBs), large secretory granules, in endothelial cells. In this study, we examined the role of protein kinase D (PKD), a newly identified regulator of protein transport, in AII-induced WPB exocytosis and the resultant P-selectin surface expression. We demonstrated that PKD2 was rapidly activated by AII in endothelial cells through phosphorylation of the activation loop at Ser744/748. AII-induced PKD2 activation correlated with increased P-selectin surface expression. Furthermore, AII-regulated PKD2 activation is protein kinase C (PKC) alpha-dependent. Importantly, knock-down of either PKD2 or PKCalpha expression inhibited AII-mediated P-selectin surface expression and monocyte adhesion. Our findings provide the first evidence that stimulation of P-selectin surface expression via PKCalpha-dependent PKD2 activation could be an important mechanism in the early onset of AII-initiated endothelial adhesiveness. [Abstract/Link to Full Text]

Nakajima A, Wada K
Life style-related diseases of the digestive system: from molecular mechanisms to therapeutic strategies: preface.
J Pharmacol Sci. 2007 Oct;105(2):127-8. [Abstract/Link to Full Text]

Takahashi H, Yoneda K, Tomimoto A, Endo H, Fujisawa T, Iida H, Mawatari H, Nozaki Y, Ikeda T, Akiyama T, Yoneda M, Inamori M, Abe Y, Saito S, Nakajima A, Nakagama H
Life style-related diseases of the digestive system: colorectal cancer as a life style-related disease: from carcinogenesis to medical treatment.
J Pharmacol Sci. 2007 Oct;105(2):129-32.
Life style-related diseases are associated with an increased risk of colorectal cancer (CRC). Recently, an association has been demonstrated between obesity and CRC. CRC has been associated with markers of insulin or glucose control, and insulin resistance might be the unifying mechanism by which several risk factors affect colorectal carcinogenesis. We evaluated the association between the number of aberrant crypt foci (ACF) and obesity, insulin resistance, hyperlipidemia, and other factors of life style-related disease. As a result, age, body mass index (BMI), waist circumference, and visceral fat obesity were significantly associated with the number of ACF. These results suggest that visceral fat obesity is an important target for CRC prevention. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily and is highly expressed in CRC. PPARgamma ligand administration for 1 to 8 months significantly reduced the number of ACF in human subjects. PPARgamma ligand is a promising candidate as a chemopreventive agent. Further investigation is needed to elucidate these mechanisms. [Abstract/Link to Full Text]

Wada K, Sakamoto H, Nishikawa K, Sakuma S, Nakajima A, Fujimoto Y, Kamisaki Y
Life style-related diseases of the digestive system: endocrine disruptors stimulate lipid accumulation in target cells related to metabolic syndrome.
J Pharmacol Sci. 2007 Oct;105(2):133-7.
Many reports indicated that endocrine disruptors (EDs) affect several hormonal functions in various living things. Here, we show the effect of EDs on lipid accumulation in target cells involved in the onset of metabolic syndrome. Treatment with nonylphenol and bisphenol A, typical EDs, stimulated the accumulation of triacylglycerol in differentiated adipocytes from 3T3-L1, preadipocytes, in time- and concentration-dependent manners. Up-regulation of gene expressions involved in lipid metabolism and metabolic syndrome were observed in adipocytes treated with EDs. Similarly, stimulatory effects of EDs were also observed on the human hepatoma cell line HuH-7. These observations indicate that exposure to EDs stimulates the lipid accumulation in target cells involved in the metabolic syndrome and may cause the dysfunction of those cells, resulting in induction of metabolic syndrome. [Abstract/Link to Full Text]

Saito S, Heller T, Yoneda M, Takahashi H, Nakajima A, Liang JT
Lifestyle-related diseases of the digestive system: a new in vitro model of hepatitis C virion production: application of basic research on hepatitis C virus to clinical medicine.
J Pharmacol Sci. 2007 Oct;105(2):138-44.
The hepatitis C virus (HCV) is an enveloped virus with a single positive-strand RNA genome of about 9.6 kb. It is a major cause of liver disease worldwide. Clear understanding of the viral life cycle has been hampered by the lack of a robust cell culture system. While the development of the HCV replicon system was a major breakthrough, infectious virions could not be produced with the replicon system. Recently, several groups have reported producing HCV virions using in vitro systems. One of these is a replicon system, but with the special genotype 2a strain JFH-1. Another is a DNA transfection system, with the construct containing the cDNA of the known infectious HCV genotype 1b flanked by two ribozymes. The development of these models further extends the repertoire of tools available for the study of HCV biology, and in particular, they may help to elucidate the molecular details of hepatitis C viral assembly and release. This review discusses the progression of experimental strategies related to HCV and how these strategies may be applied to clinical medicine. [Abstract/Link to Full Text]

Ikeda M, Kato N
Life style-related diseases of the digestive system: cell culture system for the screening of anti-hepatitis C virus (HCV) reagents: suppression of HCV replication by statins and synergistic action with interferon.
J Pharmacol Sci. 2007 Oct;105(2):145-50.
Hepatitis C virus (HCV) infection causes chronic hepatitis and leads to liver fibrosis and hepatocellular carcinoma. Pegylated-interferon and ribavirin is the current standard therapy for chronic hepatitis C. However, the therapy is only effective in 50% of the patients. To overcome this problem, we recently developed the HCV cell culture system (OR6 system) for the screening of anti-HCV reagents. In this OR6 system, the luciferase gene was introduced into the upstream portion of the HCV genome to facilitate the monitoring of HCV RNA replication. Recently lipid metabolism is reported to be involved in HCV RNA replication. Cholesterol and sphingolipid are the major components in lipid rafts, which seem to be the scaffold for HCV RNA replication. Statins inhibit cholesterol biosynthesis and also have the pleiotropic effects by the inhibition of prenylation. We demonstrated different anti-HCV effects of statins (atorvastatin, simvastatin, fluvastatin, lovastatin, and pitavastatin) using the OR6 system. Surprisingly, in contrast to the other statins, pravastatin exhibited no anti-HCV effect. Furthermore, statins enhanced the anti-HCV effect of interferon in combination. Statins may be a promising candidate for the adjuvant in interferon therapy and may improve the efficiency of the current interferon and ribavirin therapy. [Abstract/Link to Full Text]

Yoneda M, Endo H, Nozaki Y, Tomimoto A, Fujisawa T, Fujita K, Yoneda K, Takahashi H, Saito S, Iwasaki T, Yamamoto S, Tsutsumi S, Aburatani H, Wada K, Hotta K, Nakajima A
Life style-related diseases of the digestive system: gene expression in nonalcoholic steatohepatitis patients and treatment strategies.
J Pharmacol Sci. 2007 Oct;105(2):151-6.
Nonalcoholic steatohepatitis (NASH) is a subset of nonalcoholic fatty liver disease (NAFLD) and sometimes progresses to cirrhosis and liver failure. We analyzed the expression profiles of approximately 50,000 genes and biological pathways in NASH patients in comparison with simple steatosis patients by using the analytical technique of GSEA (Gene Set Enrichment Analysis) by DNA microarrays. Although expressions of various genes were altered, GSEA showed clearly lower expression of nuclear receptors, including the peroxisome proliferator-activated receptor gamma (PPARgamma) pathway. In a preliminary study we therefore investigated the therapeutic effect of low-dose pioglitazone (15 mg/day per body for 24 weeks), a synthetic ligand for PPARgamma, in 12 NASH patients. A decrease in aminotransferase (ALT) values to within the normal range was observed in 7 (58.3%) of the patients, and because the dose of pioglitazone was lower than that ordinarily used, no side effects, such as fatigue, lower extremity edema, or weight gain, were observed. In conclusion, the results confirmed involvement of the PPARgamma pathway in NASH and the therapeutic utility of a PPARgamma ligand. [Abstract/Link to Full Text]

Ueno Y, Ohmi T, Yamamoto M, Kato N, Moriguchi Y, Kojima M, Shimozono R, Suzuki S, Matsuura T, Eda H
Orally-administered caspase inhibitor PF-03491390 is retained in the liver for prolonged periods with low systemic exposure, exerting a hepatoprotective effect against alpha-fas-induced liver injury in a mouse model.
J Pharmacol Sci. 2007 Oct;105(2):201-5.
In a mouse model of alpha-Fas-induced acute liver injury, the orally-administered caspase inhibitor PF-03491390 (formerly named IDN-6556) was retained in the liver for prolonged periods with a low systemic exposure. Reductions in the elevated plasma levels of alanine aminotransferase (ALT) revealed that the retention of PF-03491390 in the liver exerted a hepatoprotective effect, even when pre-administered to mice 4 h before alpha-Fas insult. Prolonged retention of PF-03491390 in the liver after oral administration has the benefit of low systemic exposure, making this a beneficial agent for the treatment of liver diseases. [Abstract/Link to Full Text]

Toga T, Kohmura Y, Kawatsu R
The 5-HT(4) agonists cisapride, mosapride, and CJ-033466, a Novel potent compound, exhibit different human ether-a-go-go-related gene (hERG)-blocking activities.
J Pharmacol Sci. 2007 Oct;105(2):207-10.
The blocking effect of three 5-HT(4) agonists, cisapride, mosapride, and the newly discovered CJ-033466 on the human ether-a-go-go-related gene (hERG) channel was studied using a whole cell patch-clamp technique in HEK293 cells. Cisapride was found to be the most potent of the hERG blockers. CJ-033466 had the widest safety margin between its hERG blocking activity and 5-HT(4) agonism among the tested compounds. This suggests a lower clinical risk of cardiac arrhythmia in CJ-033466 compared with the other 2 agonists. Therefore, CJ-033466 has the potential to be a drug with higher therapeutic efficacy and less cardiac risk than both cisapride and mosapride. [Abstract/Link to Full Text]

Egashira N, Hayakawa K, Osajima M, Mishima K, Iwasaki K, Oishi R, Fujiwara M
Involvement of GABA(A) receptors in the neuroprotective effect of theanine on focal cerebral ischemia in mice.
J Pharmacol Sci. 2007 Oct;105(2):211-4.
We investigated the involvement of gamma-aminobutyric acid(A) (GABA(A)) receptors in the neuroprotective effect of gamma-glutamylethylamide (theanine), a component of Japanese green tea, following a 4-h middle cerebral artery (MCA) occlusion in mice. Theanine (1 mg/kg) reduced the size of the cerebral infarct and alterations of NeuN, GFAP, and Iba 1 expression levels at 24 h after MCA occlusion. This neuroprotective effect of theanine was prevented by bicuculline (GABA(A)-receptor antagonist, 10 mg/kg) but not 3-mercaptopropionic acid (glutamate decarboxylase inhibitor). These results suggest that the neuroprotective effect of theanine is mediated, at least in part, by GABA(A) receptors. [Abstract/Link to Full Text]

Saiki R, Okazaki M, Iwai S, Kumai T, Kobayashi S, Oguchi K
Effects of pioglitazone on increases in visceral fat accumulation and oxidative stress in spontaneously hypertensive hyperlipidemic rats fed a high-fat diet and sucrose solution.
J Pharmacol Sci. 2007 Oct;105(2):157-67.
We examined oxidative stress and metabolic characteristics of the spontaneously hypertensive hyperlipidemic rat (SHHR) when it was fed a high-fat diet and sucrose solution (HFDS) after N(G)-nitro-L-arginine methyl ester ingestion to develop a rat model of metabolic syndrome. This study was carried out to assess the effects of pioglitazone on levels of lipid peroxide (LPO), Cu,Zn superoxide dismutase (Cu,Zn-SOD), catalase (CAT), glutathione peroxidase (GPx), and non-esterified fatty acids (NEFA) in the plasma and liver tissue in HFDS-SHHR compared with Sprague-Dawley rats (SD). In the HFDS-treated groups, levels of LPO, CAT, GPx, and NEFA were elevated and levels of Cu,Zn-SOD were reduced in the plasma and liver tissue, with a marked accumulation of visceral fat. The changes induced by HFDS feeding were severe in the SHHR model that had essential hypertension and hyperlipidemia, when compared with SD that did not have these essential risk factors. Subcutaneous injection of 10 mg/kg per day of pioglitazone for 2 months significantly restored levels of LPO, CAT, GPx, Cu,Zn-SOD, and NEFA in the HFDS-SHHR group, and visceral fat accumulation was reduced. These results suggest that HFDS-SHHR is a suitable model of metabolic syndrome and that pioglitazone treatment can improve oxidative dysregulation in this rat model. [Abstract/Link to Full Text]

Shibasaki M, Katsura M, Kurokawa K, Torigoe F, Ohkuma S
Regional differences of L-type high voltage-gated calcium channel subunit expression in the mouse brain after chronic morphine treatment.
J Pharmacol Sci. 2007 Oct;105(2):177-83.
As functional changes in L-type high voltage-gated calcium channels (HVCCs) are recognized to be one of the major neurochemical modifications occurring in brains of animals with morphine physical dependence, this study attempts to examine whether regional difference in the expressions of HVCC subunits are produced in the brains under such pathological conditions. Scatchard analysis of [(3)H]PN200-110 binding showed increased Bmax values in the cerebral cortex and the mesolimbic region including the nucleus accumbence, which are brain regions participating in the development of morphine physical dependence, but not in the cerebellum. In the former two brain regions, alpha1C and alpha1D subunits of L-type HVCCs and alpha2/delta1 subunit increased, although decreases of alpha1B and alpha2/delta1 subunits were observed in the cerebellum. A single dose of morphine did not change the expression of any of the HVCC subunits. These results indicate that the increased L-type HVCC subunits in the cerebral cortex and mesolimbic region participate in the development of morphine physical dependence. [Abstract/Link to Full Text]

Nakajima A, Yamakuni T, Haraguchi M, Omae N, Song SY, Kato C, Nakagawasai O, Tadano T, Yokosuka A, Mimaki Y, Sashida Y, Ohizumi Y
Nobiletin, a citrus flavonoid that improves memory impairment, rescues bulbectomy-induced cholinergic neurodegeneration in mice.
J Pharmacol Sci. 2007 Sep;105(1):122-6.
We have recently reported that nobiletin, a citrus flavonoid, improves impaired memory in olfactory-bulbectomized (OBX) mice, which have been widely utilized as a useful paradigm that shares some major clinical features of Alzheimer's disease. Here, we examined the effects of nobiletin on OBX-induced cholinergic neurodegeneration in mice. OBX mice showed reduced acetylcholinesterase (AChE) staining and choline acetyltransferase (ChAT) expression in the hippocampus. An 11-day administration of nobiletin rescued OBX-induced decrease in the density of AChE-staining and ChAT expression in the hippocampus. These results suggest that nobiletin rescues OBX-induced cholinergic neurodegeneration, accompanied by improvement of impaired memory in OBX mice. [Abstract/Link to Full Text]

Dengiz GO, Odabasoglu F, Halici Z, Cadirci E, Suleyman H
Gastroprotective and antioxidant effects of montelukast on indomethacin-induced gastric ulcer in rats.
J Pharmacol Sci. 2007 Sep;105(1):94-102.
Montelukast, a selective reversible cysteinyl leukotriene D(4)-receptor (LTD(4) receptor) antagonist, is used in the treatment of asthma. We have investigated alterations in the glutathione (GSH) and activity levels of antioxidative enzymes [superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and glutathione reductase (GR)] and myeloperoxidase (MPO), as markers of the ulceration process following oral administration of montelukast, lansoprazole, famotidine, and ranitidine, respectively, in rats with indomethacin-induced ulcers. In the present study, we found that 1) montelukast, lansoprazole, famotidine, and ranitidine all reduced the development of indomethacin-induced gastric damage, with this reduction occurring at a greater magnitude for montelukast, famotidine, and lansoprazole than for ranitidine; 2) montelukast and ranitidine both alleviated increases in the activity levels of CAT and GST enzymes resulting from gastric injury; 3) montelukast and ranitidine both ameliorated depressions in the GSH and activity levels of SOD and GR enzymes caused by indomethacin administration; and 4) all doses of montelukast, lansoprazole, and ranitidine decreased amplification of MPO activity resulting from induced gastric injuries. These results suggest that the gastroprotective effects of montelukast on indomethacin-induced ulcerations can be attributed to its ameliorating effect on oxidative damage and MPO activity. [Abstract/Link to Full Text]

Recent Articles in Alternative Medicine Review

L-tyrosine - monograph.
Altern Med Rev. 2007 Dec;12(4):364-8.
L-tyrosine is a conditionally essential amino acid because under normal conditions the body synthesizes sufficient quantities from phenylalanine. For those with phenylketonuria, however, a severe deficiency in the enzyme phenylalanine hydroxylase prevents conversion of phenylalanine to tyrosine, making tyrosine an essential amino acid for this population. Tyrosine is incorporated into proteins of all life forms and is a precursor for synthesis of thyroxin, melanin, and the neurotransmitters dopamine and norepinephrine. Food sources of tyrosine include fish, soy products, poultry, eggs, dairy products, lima beans, almonds, peanuts, sesame seeds, pumpkin seeds, wheat germ, oats, avocados, and bananas. Clinical conditions for which tyrosine supplementation may be of therapeutic benefit include depression, hypertension, stress, cognitive function and memory, Parkinson's disease, phenylketonuria, and narcolepsy. [Abstract/Link to Full Text]

Momordica charantia (bitter melon) - Monograph.
Altern Med Rev. 2007 Dec;12(4):360-3.
Momordica charantia (MC), a member of the Cucurbitaceae family, is known as bitter melon, bitter gourd, balsam pear, karela, and pare. It grows in tropical areas of the Amazon, East Africa, Asia, India, South America, and the Caribbean and is used traditionally as both food and medicine. The plant is a climbing perennial with elongated fruit that resembles a warty gourd or cucumber. The unripe fruit is white or green in color and has a bitter taste that becomes more pronounced as the fruit ripens. The seeds, fruit, leaves, and root of the plant have been used in traditional medicine for microbial infections, sluggish digestion and intestinal gas, menstrual stimulation, wound healing, inflammation, fever reduction, hypertension, and as a laxative and emetic. Clinical conditions for which M. charantia extracts (primarily from the fruit) are currently being used include diabetes, dyslipidemia, microbial infections, and potentially as a cytotoxic agent for certain types of cancer. [Abstract/Link to Full Text]

Poulin Y, Bissonnette R, Juneau C, Cantin K, Drouln R, Poubelle PE
XP-828L in the treatment of mild to moderate psoriasis: randomized, double-blind, placebo-controlled study.
Altern Med Rev. 2007 Dec;12(4):352-359.
Background: XP-828L, a protein extract obtained from sweet whey, has demonstrated potential benefit for the treatment of mild to moderate psoriasis in an open-label study. Objective: To study in a randomized, double-blind, placebo-controlled study the safety and efficacy of XP-828L in the treatment of mild to moderate psoriasis. Design: XP-828L 5 g/d (group A, n=42) or placebo (group B, n=42) was given orally for 56 days followed by XP-828L 5 g/d in group A and by XP-828L 10 g/d in group B for an additional 56 days. Results: Patients receiving XP-828L 5 g/d for 56 days had an improved Physician's Global Assessment (PGA) score compared with patients under placebo (p less than 0.05). Considering the data of group A only, the PGA score improved from day 1 to day 56 (p less than 0.01); the Psoriasis Area and Severity Index score improved as well, but to a lesser extent (p less than 0.05). Conclusion: Oral administration of 5 g/d XP-828L compared with a placebo significantly improved the PGA score of patients with mild to moderate psoriasis. [Abstract/Link to Full Text]

Carlson DA, Smith AR, Fischer SJ, Young KL, Packer L
The plasma pharmacokinetics of R-(+)-lipoic acid administered as sodium R-(+)-lipoate to healthy human subjects.
Altern Med Rev. 2007 Dec;12(4):343-351.
BACKGROUND: The racemic mixture, RS-(+/-)-alpha-lipoic acid (rac-LA) has been utilized clinically and in a variety of disease models. Rac-LA and the natural form, R-lipoic acid (RLA), are widely available as nutritional supplements, marketed as antioxidants. Rac-LA sodium salt (NaLA) or rac-LA potassium salt (KLA) has been used to improve the aqueous solubility of LA. STUDY RATIONALE: Several in vitro and animal models of aging and age-related diseases have demonstrated efficacy for the oral solutions of LA salts in normalizing age-related changes to those of young animals. Other models and studies have demonstrated the superiority of RLA, the naturally occurring isomer over rac-LA. Despite this, RLA pharmacokinetics (PK) is not fully characterized in humans, and it is unknown whether the concentrations utilized in animal models can be achieved in vivo. Due to its tendency to polymerize, RLA is relatively unstable and suffers poor aqueous solubility, leading to poor absorption and low bioavailability. A preliminary study demonstrated the stability and bioavailability were improved by converting RLA to its sodium salt (NaRLA) and pre-dissolving it in water. The current study extends earlier findings from this laboratory and presents PK data for the 600-mg oral dosing of 12 healthy adult subjects given NaRLA. In addition, the effect of three consecutive doses was tested on a single subject relative to a one-time dosing in the same subject to determine whether plasma maximum concentration (Cmax) and the area under the plasma concentration versus time curve (AUC) values were comparable to those in animal studies and those achievable via intravenous infusions in humans. METHODS: Plasma RLA was separated from protein by a modification of a published method. Standard curves were generated from spiking known concentrations of RLA dissolved in ethanol and diluted in a phosphate-buffered saline (PBS) into each individual's baseline plasma to account for inter-individual differences in protein binding and to prevent denaturing of plasma proteins. Plasma RLA content was determined by the percent recovery using high-performance liquid chromatography (electrochemical/coulometric detection) (HPLC/ECD). RESULTS: As anticipated from the preliminary study, NaRLA is less prone to polymerization, completely soluble in water, and displays significantly higher Cmax and AUC values and decreased time to maximum concentration (Tmax) and T1/2 values than RLA or rac-LA. In order to significantly extend Cmax and AUC, it is possible to administer three 600-mg RLA doses (as NaRLA) at 15-minute intervals to achieve plasma concentrations similar to those from a slow (20-minute) infusion of LA. This is the first study to report negligible unbound RLA even at the highest achievable plasma concentrations. [Abstract/Link to Full Text]

Waterman E, Lockwood B
Active components and clinical applications of olive oil.
Altern Med Rev. 2007 Dec;12(4):331-42.
The olive tree, Olea europaea, is native to the Mediterranean basin and parts of Asia Minor. The fruit and compression-extracted oil have a wide range of therapeutic and culinary applications. Olive oil also constitutes a major component of the "Mediterranean diet." The chief active components of olive oil include oleic acid, phenolic constituents, and squalene. The main phenolics include hydroxytyrosol, tyrosol, and oleuropein, which occur in highest levels in virgin olive oil and have demonstrated antioxidant activity. Antioxidants are believed to be responsible for a number of olive oil's biological activities. Oleic acid, a monounsaturated fatty acid, has shown activity in cancer prevention, while squalene has also been identified as having anticancer effects. Olive oil consumption has benefit for colon and breast cancer prevention. The oil has been widely studied for its effects on coronary heart disease (CHD), specifically for its ability to reduce blood pressure and low-density lipoprotein (LDL) cholesterol. Antimicrobial activity of hydroxytyrosol, tyrosol, and oleuropein has been demonstrated against several strains of bacteria implicated in intestinal and respiratory infections. Although the majority of research has been conducted on the oil, consumption of whole olives might also confer health benefits. [Abstract/Link to Full Text]

Traub M, Marshall K
Psoriasis - pathophysiology, conventional, and alternative approaches to treatment.
Altern Med Rev. 2007 Dec;12(4):319-30.
Psoriasis is a common T-cell-mediated immune disorder characterized by circumscribed, red, thickened plaques with an overlying silver-white scale. It occurs worldwide, although the incidence is lower in warmer, sunnier climates. The primary cause of psoriasis is unknown. During an active disease state, an underlying inflammatory mechanism is frequently involved. Many conventional treatments focus on suppressing symptoms associated with psoriasis and have significant side effects. This article reviews several of the researched natural approaches to psoriasis treatment, while addressing its underlying cause. [Abstract/Link to Full Text]

Lamson DW
The roles of complementary medicine in oncology.
Altern Med Rev. 2007 Dec;12(4):313-8.
Cancer is a major health problem and to most of the public it is mysterious and frightening. Advanced cancer has a poor track record of cure. Along with referral to an oncologist, many persons with a medical diagnosis of malignancy request help from a naturopathic physician or other complementary/alternative medicine (CAM) practitioner. There are many variables to the situation: the cancer type and location, the stage of development, the grade of malignancy, and the person's age, personal orientation about medical care, psychology, and more. The point of the present discussion is to begin formation of a framework for decisions about the degree and kind of medical support offered by CAM practitioners to individuals diagnosed with cancer. [Abstract/Link to Full Text]

L-lysine. Monograph.
Altern Med Rev. 2007 Jun;12(2):169-72. [Abstract/Link to Full Text]

Coenzyme Q10. Monograph.
Altern Med Rev. 2007 Jun;12(2):159-68. [Abstract/Link to Full Text]

Chappell LT
Should EDTA chelation therapy be used instead of long-term clopidogrel plus aspirin to treat patients at risk from drug-eluting stents?
Altern Med Rev. 2007 Jun;12(2):152-8.
The recently discovered increased risk of blood clots, leading to myocardial infarction and sudden death beginning six months after medicated stents are implanted in patients following percutaneous transluminal coronary angioplasty (PTCA), has left cardiologists pondering what course of action to take. The purpose of adding implanted medication to a stent is to prevent thrombin accumulation and restenosis. However, these stents may increase, rather than decrease, the risk. Although long-term treatment with clopidogrel bisulfate (Plavix) plus aspirin for at least 12 months has been suggested as a preventive treatment, there is no evidence from randomized, controlled trials that this treatment is effective for more than six months. Clopidogrel also increases the risk of major bleeding episodes. The author served as the primary investigator for a study that showed cardiovascular patients treated with EDTA chelation therapy had a lower rate of subsequent cardiac events, including myocardial infarction and death, than those treated with cardiac medications, PTCA, or coronary artery bypass graft (CABG). The data also indicated chelation therapy might be effective in preventing thrombosis and cardiac events from stent implantation. There is evidence EDTA chelation therapy might prevent hypercoagulability resulting from the placement of stents, although not specifically medicated stents. Based on the limited data currently available, intravenous EDTA may be safe and effective for treating patients who have implanted medicated stents. Prospective clinical trials are needed, and EDTA should be included in those trials. [Abstract/Link to Full Text]

Friel PN
EEG biofeedback in the treatment of attention deficit hyperactivity disorder.
Altern Med Rev. 2007 Jun;12(2):146-51.
Electroencephalogram (EEG) biofeedback, also known as neurofeedback, is a promising alternative treatment for patients with attention deficit/hyperactivity disorder (AD/HD). EEG biofeedback therapy rewards scalp EEG frequencies that are associated with relaxed attention, and suppresses frequencies associated with under- or over-arousal. In large-scale clinical trials, the efficacy of EEG biofeedback for AD/HD is comparable to that of stimulant medications. Many different EEG biofeedback protocols for AD/HD are available. Single-channel protocols developed by Lubar and interhemispheric protocols developed by the Othmers are widely practiced and supported by large-scale clinical studies. [Abstract/Link to Full Text]

McCormick RK
Osteoporosis: integrating biomarkers and other diagnostic correlates into the management of bone fragility.
Altern Med Rev. 2007 Jun;12(2):113-45.
Bone health, characterized by its mass, density, and micro-architectural qualities, is maintained by a balanced system of remodeling. The lack of these qualities, caused by an uncoupling of the remodeling process, leads to bone fragility and an increased risk for fracture. The prime regulator of bone remodeling is the RANK/RANKL/OPG system. The common origin of both bone and immune stem cells is the key to understanding this system and its relationship to the transcription factor nuclear factor kappaB in bone loss and inflammation. Via this coupled osteo-immune relationship, a catabolic environment from heightened proinflammatory cytokine expression and/or a chronic antigen-induced activation of the immune system can initiate a switch-like diversion of osteoprogenitor-cell differentiation away from monocyte-macrophage and osteoblast cell formation and toward osteoclast and adipocyte formation. This disruption in bone homeostasis leads to increased fragility. Dietary and specific nutrient interventions can reduce inflammation and limit this diversion. Common laboratory biomarkers can be used to assess changes in body metabolism that affect bone health. This literature review offers practical information for applying effective strategic nutrition to fracture-risk individuals while monitoring metabolic change through serial testing of biomarkers. As examples, the clinician may recommend vitamin K and potassium to reduce hypercalciuria, _-lipoic acid and N-acetylcysteine to reduce the bone resorption marker N-telopeptide (N-Tx), and dehydroepiandrosterone (DHEA), whey, and milk basic protein (the basic protein fraction of whey) to increase insulin-like growth factor-1 (IGF-1) and create a more anabolic profile. [Abstract/Link to Full Text]

Patrick LR
Restless legs syndrome: pathophysiology and the role of iron and folate.
Altern Med Rev. 2007 Jun;12(2):101-12.
Restless Legs Syndrome (RLS) is a common movement disorder characterized by a circadian variation in symptoms involving an urge to move the limbs, usually the legs. Pregnant women, patients with end-stage renal disease or iron-deficiency anemia, and children with attention deficit hyperactivity disorder (AD/HD) have a significantly higher prevalence of RLS. The classic presentation includes the onset or worsening of symptoms when at rest and the circadian pattern of exacerbation of symptoms at night. These symptoms reflect a circadian fluctuation of dopamine in the substantia nigra. Patients with RLS have lower levels of dopamine in the substantia nigra and respond to iron administration. Iron, as a cofactor in dopamine production, plays a central role in the etiology of RLS. Folic acid administration has also been shown to alleviate the symptoms of RLS and may play a role in the treatment of primary (familial) RLS. [Abstract/Link to Full Text]

Biotin.
Altern Med Rev. 2007 Mar;12(1):73-8. [Abstract/Link to Full Text]

Centella asiatica.
Altern Med Rev. 2007 Mar;12(1):69-72. [Abstract/Link to Full Text]

Konno S
Effect of various natural products on growth of bladder cancer cells: two promising mushroom extracts.
Altern Med Rev. 2007 Mar;12(1):63-8.
Despite the availability of several therapeutic options, a safer and more effective modality is urgently needed for treatment of bladder cancer. Specific immunotherapy is effective, but severe side effects limit its clinical use and underscore the need for unconventional therapies using less toxic substances. Many natural substances are touted for their medicinal aspects and side effect profiles, and some of these have been well characterized for their biological and medicinal properties. Accordingly, the effects on bladder cancer cells in vitro were investigated. Eight commercially available natural products were tested for possible effects on the growth of human bladder cancer T24 cells. This study demonstrated that two mushroom extracts, GD- and PL-fractions, induced a significant (>90 percent) growth reduction in 72 hours, whereas the remaining six products had no effect. Interestingly, non-toxic concentrations of the GD- or PL-fractions, when combined with a non-toxic concentration of vitamin C, became highly cytotoxic, resulting in >90-percent cell death. Thus, vitamin C appears to act synergistically with these fractions to potentiate their bioactivity (cytotoxicity). No other products tested demonstrated such a synergistic potentiation with vitamin C. The present study indicates that GD- and PL-fractions appear to have the most potent cytotoxic effect on human bladder cancer T24 cells. It is thus plausible that these substances could be used, solely or combined with conventional modalities, for the treatment of superficial bladder cancer. [Abstract/Link to Full Text]

Kelly GS
Body temperature variability (Part 2): masking influences of body temperature variability and a review of body temperature variability in disease.
Altern Med Rev. 2007 Mar;12(1):49-62.
This is the second of a two-part review on body temperature variability. Part 1 discussed historical and modern findings on average body temperatures. It also discussed endogenous sources of temperature variability, including variations caused by site of measurement; circadian, menstrual, and annual biological rhythms; fitness; and aging. Part 2 reviews the effects of exogenous masking agents - external factors in the environment, diet, or lifestyle that can be a significant source of body temperature variability. Body temperature variability findings in disease states are also reviewed. [Abstract/Link to Full Text]

Roxas M, Jurenka J
Colds and influenza: a review of diagnosis and conventional, botanical, and nutritional considerations.
Altern Med Rev. 2007 Mar;12(1):25-48.
The common cold is the leading cause of doctor visits in the United States and annually results in 189 million lost school days. In the course of one year the U.S. population contracts approximately 1 billion colds. Influenza infection is still a leading cause of morbidity and mortality, accounting for 20-25 million doctor visits and 36,000 deaths per year in the United States. Conventional therapies for colds and flu focus primarily on temporary symptom relief and include over-the-counter antipyretics, anti-inflammatories, and decongestants. Treatment for influenza also includes prescription antiviral agents and vaccines for prevention. This article reviews the common cold and influenza viruses, presents the conventional treatment options, and highlights select botanicals (Echinacea spp., Sambucus nigra, larch arabinogalactan, Astragalus membranaceous, Baptisia tinctoria, Allium sativa, Panax quinquefolium, Eleutherococcus senticosus, Andrographis paniculata, olive leaf extract, and Isatis tinctoria) and nutritional considerations (vitamins A and C, zinc, high lactoferrin whey protein, N-acetylcysteine, and DHEA) that may help in the prevention and treatment of these conditions. [Abstract/Link to Full Text]

Gaby AR
Natural approaches to epilepsy.
Altern Med Rev. 2007 Mar;12(1):9-24.
This article reviews research on the use of diet, nutritional supplements, and hormones in the treatment of epilepsy. Potentially beneficial dietary interventions include identifying and treating blood glucose dysregulation, identifying and avoiding allergenic foods, and avoiding suspected triggering agents such as alcohol, aspartame, and monosodium glutamate. The ketogenic diet may be considered for severe, treatment-resistant cases. The Atkins diet (very low in carbohydrates) is a less restrictive type of ketogenic diet that may be effective in some cases. Nutrients that may reduce seizure frequency include vitamin B6, magnesium, vitamin E, manganese, taurine, dimethylglycine, and omega-3 fatty acids. Administration of thiamine may improve cognitive function in patients with epilepsy. Supplementation with folic acid, vitamin B6, biotin, vitamin D, and L-carnitine may be needed to prevent or treat deficiencies resulting from the use of anticonvulsant drugs. Vitamin K1 has been recommended near the end of pregnancy for women taking anticonvulsants. Melatonin may reduce seizure frequency in some cases, and progesterone may be useful for women with cyclic exacerbations of seizures. In most cases, nutritional therapy is not a substitute for anticonvulsant medications. However, in selected cases, depending on the effectiveness of the interventions, dosage reductions or discontinuation of medications may be possible. [Abstract/Link to Full Text]

Benfotiamine. Monograph.
Altern Med Rev. 2006 Sep;11(3):238-42. [Abstract/Link to Full Text]

Alpha-lipoic acid. Monograph.
Altern Med Rev. 2006 Sep;11(3):232-7. [Abstract/Link to Full Text]

Cherniak EP
Biofeedback and other therapies for the treatment of urinary incontinence in the elderly.
Altern Med Rev. 2006 Sep;11(3):224-31.
Alternative therapies hold potential promise for the treatment of urinary incontinence in the elderly. Assessment and comparisons of the efficacies of such therapies have been hindered by a lack of standardized definitions of urinary incontinence in the study populations, lack of standardization of treatment protocols, inadequate sample sizes, and lack of blinding and appropriate controls. Biofeedback has been the most extensively studied therapy and may provide appropriate adjunctive or primary therapy for select individuals. Other potential therapies, such as acupuncture, hypnosis, and herbal therapies, have not been sufficiently examined to make definitive recommendations. [Abstract/Link to Full Text]

Moskowitz D
A comprehensive review of the safety and efficacy of bioidentical hormones for the management of menopause and related health risks.
Altern Med Rev. 2006 Sep;11(3):208-23.
Numerous forms of estrogens and progestins are utilized for the treatment of menopausal complaints and associated conditions that occur temporally. Although known to be different with respect to molecular structure, receptor affinity, metabolism, and other physiological traits, most have been treated as if they were clinically identical. The majority of these hormone preparations, commonly referred to as hormone replacement therapy (HRT), should perhaps be more aptly referred to as hormone substitution therapy, as most of the therapies utilized do not exactly match those produced in the body. Research indicates these synthetic hormones vary clinically in safety and efficacy. As such, women and their physicians have, in increasing numbers, been opting for the use of bioidentical hormones; i.e., those that match the structure and function of hormones produced in the body. With greater utilization and research surrounding bioidentical hormones, the differences can now begin to be fully assessed and appreciated. This article reviews the disparities between synthetic and bioidentical estrogens and progestins/progesterone with respect to safety and efficacy; special attention is devoted to clinical outcomes in the breast, endometrium, bone, cardiovascular system, and brain. The studies reviewed suggest bioidentical progesterone does not have a negative effect on blood lipids or vasculature as do many synthetic progestins, and may carry less risk with respect to breast cancer incidence. Studies of both bioidentical estrogens and progesterone suggest a reduced risk of blood clots compared to non-bioidentical preparations. Bioidentical hormone preparations have demonstrated effectiveness in addressing menopausal symptoms. The author advocates for continued research on bioidentical hormones and concludes there is currently sufficient evidence to support their preferred use over that of their synthetic cousins. [Abstract/Link to Full Text]

Helms S, Miller A
Natural treatment of chronic rhinosinusitis.
Altern Med Rev. 2006 Sep;11(3):196-207.
Chronic rhinosinusitis (CRS) is one of the most common long-term illnesses in the United States, affecting approximately 14 percent of the population. CRS is a challenging condition to treat, partly due to its multifaceted, poorly understood pathophysiology. Treatment goals include maintaining open drainage and decreasing inflammation while improving tissue integrity and limiting causative factors. This review covers the etiology, pathology, and diagnosis of CRS, as well as mainstream and alternative treatments. Discussion of alternative therapeutics includes nutrients and botanicals (ascorbic acid, bromelain, N-acetylcysteine, quercetin, undecylenic acid, and Urtica dioica and other herbal medicines) and procedures (nasal irrigation and naso-sympatico treatments). The influences of diet and air quality on CRS are also discussed. [Abstract/Link to Full Text]

Gaby AR
Natural remedies for scleroderma.
Altern Med Rev. 2006 Sep;11(3):188-95.
Scleroderma is an autoimmune disease of the connective tissue characterized by fibrosis and thickening of various tissues. It can be limited to the skin or affect multiple organs, and its course ranges from slowly to rapidly progressive. Penicillamine, glucocorticoids, and other drugs are used to treat scleroderma, but none of these treatments has a high degree of efficacy. This article reviews several promising natural treatments for scleroderma, including para-aminobenzoic acid, vitamin E, vitamin D, evening primrose oil, estriol, N-acetylcysteine, bromelain, and an avocado/soybean extract. [Abstract/Link to Full Text]

Chondroitin sulfates. Monograph.
Altern Med Rev. 2006 Dec;11(4):338-43. [Abstract/Link to Full Text]

5-methyltetrahydrofolate. Monograph.
Altern Med Rev. 2006 Dec;11(4):330-7. [Abstract/Link to Full Text]

Head KA
Peripheral neuropathy: pathogenic mechanisms and alternative therapies.
Altern Med Rev. 2006 Dec;11(4):294-329.
Peripheral neuropathy (PN), associated with diabetes, neurotoxic chemotherapy, human immunodeficiency virus (HIV)/antiretroviral drugs, alcoholism, nutrient deficiencies, heavy metal toxicity, and other etiologies, results in significant morbidity. Conventional pain medications primarily mask symptoms and have significant side effects and addiction profiles. However, a widening body of research indicates alternative medicine may offer significant benefit to this patient population. Alpha-lipoic acid, acetyl-L-carnitine, benfotiamine, methylcobalamin, and topical capsaicin are among the most well-researched alternative options for the treatment of PN. Other potential nutrient or botanical therapies include vitamin E, glutathione, folate, pyridoxine, biotin, myo-inositol, omega-3 and -6 fatty acids, L-arginine, L-glutamine, taurine, N-acetylcysteine, zinc, magnesium, chromium, and St. John's wort. In the realm of physical medicine, acupuncture, magnetic therapy, and yoga have been found to provide benefit. New cutting-edge conventional therapies, including dual-action peptides, may also hold promise. [Abstract/Link to Full Text]

Kelly G
Body temperature variability (Part 1): a review of the history of body temperature and its variability due to site selection, biological rhythms, fitness, and aging.
Altern Med Rev. 2006 Dec;11(4):278-93.
Body temperature is a complex, non-linear data point, subject to many sources of internal and external variation. While these sources of variation significantly complicate interpretation of temperature data, disregarding knowledge in favor of oversimplifying complex issues would represent a significant departure from practicing evidence-based medicine. Part 1 of this review outlines the historical work of Wunderlich on temperature and the origins of the concept that a healthy normal temperature is 98.6 degrees F (37.0 degrees C). Wunderlich's findings and methodology are reviewed and his results are contrasted with findings from modern clinical thermometry. Endogenous sources of temperature variability, including variations caused by site of measurement, circadian, menstrual, and annual biological rhythms, fitness, and aging are discussed. Part 2 will review the effects of exogenous masking agents - external factors in the environment, diet, or lifestyle that can influence body temperature, as well as temperature findings in disease states. [Abstract/Link to Full Text]

Winters M
Ancient medicine, modern use: Withania somnifera and its potential role in integrative oncology.
Altern Med Rev. 2006 Dec;11(4):269-77.
Withania somnifera Dunal, commonly known as ashwagandha, has been used for centuries in Ayurvedic medicine to increase longevity and vitality. Western research supports its polypharmaceutical use, confirming antioxidant, anti-inflammatory, immune-modulating, and antistress properties in the whole plant extract and several separate constituents. This article reviews the literature pertaining to Withania somnifera and its botanical constituents as antitumor agents and in conjunction with radiation and chemotherapy treatment. Following a search of MEDLINE and EBSCO databases, it can be concluded that Withania somnifera reduces tumor cell proliferation while increasing overall animal survival time. Furthermore, it has been shown to enhance the effectiveness of radiation therapy while potentially mitigating undesirable side effects. Withania somnifera also reduces the side effects of chemotherapeutic agents cyclophosphamide and paclitaxel without interfering with the tumor-reducing actions of the drugs. These effects have been demonstrated in vitro on human cancer cell lines, and in vivo on animal subjects, but there have been no human trials to date. Given its broad spectrum of cytotoxic and tumor-sensitizing actions, Withania somnifera presents itself as a novel complementary therapy for integrative oncology care. [Abstract/Link to Full Text]

Recent Articles in Journal of Physiology and Pharmacology

Bonior J, Jaworek J, Kot M, Konturek SJ, Pawlik WW
Endotoxemia in the infant rats modulates HSP60 protein level in the pancreatic acinar cells.
J Physiol Pharmacol. 2007 Aug;58 Suppl 3189-98.
Lipopolysaccharide (endotoxin, LPS) is responsible for septic shock and multiorgan failure, but pretreatment of the rats with low doses of LPS reduced pancreatic damage produced by caerulein-induced pancreatitis (CIP). In spite of this observations the effects of LPS and caerulein on pro-apoptotic HSP60 and Bax protein expression in the pancreatic acinar cells has not been examined yet. The aim of this study was to assess the effects of endotoxemia induced in the early period of life on the pro-apoptotic nuclear HSP60 and mitochondrial Bax protein expressions detected in the pancreas of adult animals. Newborn rats (25 g) were injected with endotoxin (Escherichia coli) for 5 consecutive days, at the total doses of 25, 50 or 75 mg/kg. Control animals received injections of physiological saline. Two months later the pancreatic acinar cells were isolated from all above groups of rats and subjected to caerulein over stimulation (10(-8)M). Total nuclear HSP60 and mitochondrial Bax protein expression were isolated for Western blot and co-immunoprecipitation studies. High levels of pro-apoptotic nuclear HSP60 and mitochondrial Bax protein has been observed in the pancreatic acinar cells under basal conditions. Pretreatment of newborn rats with LPS failed to affect significantly the HSP60 and Bax protein levels in the pancreatic acini isolated from the same animals 2 months later, as compared to the control group. Caerulein stimulation significantly reduced the level of these proteins. Pretreatment of suckling rats with LPS (at the total doses of 25, 50 or 75 mg/kg) reversed above caerulein-induced suppression of pro-apoptotic nuclear HSP60 and mitochondrial Bax protein levels in the pancreatic acini obtained from adult rats. We conclude that pretreatment of suckling rats with LPS reversed the suppression of pro-apoptotic HSP60 and Bax protein levels produced by caerulein overstimulation in the pancreatic acini. This mechanism could take a part in the LPS-induced protection of the pancreatic tissue against acute damage. [Abstract/Link to Full Text]

Leja-Szpak A, Jaworek J, Szklarczyk J, Konturek SJ, Pawlik WW
Melatonin stimulates HSP27 phosphorylation in human pancreatic carcinoma cells (PANC-1).
J Physiol Pharmacol. 2007 Aug;58 Suppl 3177-88.
Heat shock protein 27 (HSP27) is a cytoprotective chaperone, activated by stressful stimuli. HSP27 modulates aggregation and degradation of many proteins. Recent evidence suggests that HSP27 could be involved in the progression of tumor growth and in the development of resistance of various tumors to chemo- and radiotherapy. It has been reported that melatonin protects pancreatic cells and various tissues against inflammatory damage. Previous experimental studies have shown that melatonin stimulates pancreatic enzyme secretion and improves the outcome of experimental pancreatitis. To investigate whether melatonin could affect HSP27 protein level in human pancreatic carcinoma cells (PANC-1). PANC-1 cells were incubated in the standard medium DMEM supplemented with 10% fetal bovine serum at 37 degrees C with 5% CO(2) and humidified atmosphere under basal conditions or in the presence of decreasing doses of melatonin (10(-6) - 10(-12)M). Control experiments were performed with the vehicle only (0,1% DMSO) without melatonin. After 24 h and 48 h the cells were harvested, the cytoplasmic and nuclear proteins were isolated for western blot and immunoblotting studies. Incubation of the PANC-1 cells with melatonin resulted in the stimulation both cytoplasmic and nuclear nonphosphorylated HSP27 protein levels after 24 h of incubation, however, above pools of nonphosphorylated chaperone protein levels were strongly diminished after subsequent 24 h. These changes were accompanied by marked rise of nuclear phosphorylated HSP27. The significant increase of this nuclear protein was observed after 48h of incubation. Conclusion: Melatonin stimulates phosphorylation of HSP27 in human pancreatic carcinoma cells (PANC-1). [Abstract/Link to Full Text]

Pajak B, Orzechowski A
Sodium butyrate-dependent sensitization of human colon adenocarcinoma COLO 205 cells to TNF-alpha-induced apoptosis.
J Physiol Pharmacol. 2007 Aug;58 Suppl 3163-76.
COLO 205 colon adenocarcinoma cells are highly resistant to extrinsic apoptosis induced by immunomodulatory cytokines. One of the antiapoptotic mechanisms is the expression of cFLIP protein, which inhibits TNF-alpha-induced cell death. The use of metabolic inhibitors, such as sodium butyrate (NaBt), the potent repressor of histone deacetylase, sensitizes tumor cells to TNF-alpha-mediated apoptosis. The Western-blot analysis revealed that in COLO 205 cells the susceptibility to apoptogenic stimuli results from time-dependent reduction in cFLIP(L) protein assembled with DISC complex. At the same time, the level of transmembrane TNF-alpha receptor 1 (TNF-R1) was elevated which is consistent with the exaggerated rate of cell death. Since preincubation of COLO 205 cells with N-acetyl-L-cysteine (NAC), or sodium ascorbate (ASC) did not protect cells from combined NaBt- and TNF-alpha-induced apoptosis, we concluded that deletion of cancer cells is not evoked by oxidative stress. Our results suggest that the combination of TNF-alpha with NaBt targets antiapoptotic protein(s) and may provide efficient and non-toxic treatment of colon cancer. [Abstract/Link to Full Text]

Kwiecie? S, Pawlik MW, Sliwowski Z, Kwiecie? N, Brzozowski T, Pawlik WW, Konturek SJ
Involvement of sensory afferent fibers and lipid peroxidation in the pathogenesis of stress-induced gastric mucosa damage.
J Physiol Pharmacol. 2007 Aug;58 Suppl 3149-62.
Ablation of sensory nerves impairs healing of gastric ulcers, but the role of free radicals in the healing process has been little studied. The aim of our present investigations was to determine the participation of reactive oxygen species (ROS) in sensory nerve activity during WRS. Experiments were carried out on male Wistar rats and the number of gastric lesions was measured by planimetry. Colorimetric assays were used to determine gastric mucosal levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), as well as superoxide dismutase (SOD) activity. We found that capsaicin-inactivation of sensory nerves resulted in magnification of gastric mucosal damage induced by the WRS. In this process, oxidative stress occurs, as reflected by an increase of MDA and 4-HNE tissue concentrations (an index of lipid peroxidation), and a decrease of SOD activity, could play an important role. Pentoxyfilline-induced gastroprotection and hyperemia depends upon attenuation of the oxidative stress. This protection and hyperemia were, at least in part, attenuated by ASA. Afferent sensory fibers participate in the pathogenesis of ulcers. Lipid peroxidation plays an important role in this process. [Abstract/Link to Full Text]

Konturek PC, Burnat G, Hahn EG
Inhibition of Barret's adenocarcinoma cell growth by simvastatin: involvement of COX-2 and apoptosis-related proteins.
J Physiol Pharmacol. 2007 Aug;58 Suppl 3141-8.
Recent studies demonstrated that inhibitors of 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG-CoAR), called "statins", independently of their well known plasma cholesterol lowering effect, exert favourable influence on a diverse range of physiologic processes including endothelial function, oxidant stress and antitumor effect. A number of epidemiological studies demonstrated that statins may have protective effect against cancer. The role of statins in the prevention and therapy of Barrett's adenocarcinoma (BA) has not been investigated so far. The aim of the present study was to analyze: 1) the impact of HMG-CoAR inhibitor, simvastatin, on human BA cell growth and 2) effect of simvastatin on apoptosis related proteins Bax/Bcl-2 and cyclooxygenase-2. BA cells (OE-19 cells) were incubated with simvastatin (1-30 muM). MTT assay was used to determine the antiproliferative effects. The expression of COX-2, Bax and Bcl-2 was analyzed at mRNA and protein level by quantitative RT-PCR and immunoblot. MTT assay demonstrated a significant dose-dependent inhibition of OE-19 cell growth by simvastatin, which also caused a significant reduction in Bcl-2 expression and an increase in Bax expression. In OE-19 cells, the COX-2 expression was detected and significantly increased by the addition of TNFalpha into the medium, however, this effect was significantly attenuated by simvastatin. Our in vitro data demonstrate that statins possess anticancerogenic properties possibly due to the induction of apoptosis and inhibition of COX-2. Clinical trial are necessary to prove the beneficial effects of statins on cancerogenesis in Barrett's esophagus. [Abstract/Link to Full Text]

Mazur M, Furga?a A, Jab?o?ski K, Madroszkiewicz D, Cie?ko-Michalska I, Bugajski A, Thor PJ
Dysfunction of the autonomic nervous system activity is responsible for gastric myoelectric disturbances in the irritable bowel syndrome patients.
J Physiol Pharmacol. 2007 Aug;58 Suppl 3131-9.
The pathogenesis of the irritable bowel syndrome (IBS) is still unsolved. Lately most attention has been focused on visceral hypersensitivity related to dysfunction of the autonomic nervous system (ANS). The aim of this study was to evaluate changes in the ANS activity and gastric motility in constipation-predominant IBS patients using the heart rate variability (HRV) and gastric myoelectric activity (EGG) recording. 23 patients (45+/-13 yrs) matching Manning criteria and 30 healthy volunteers (47+/-5 yrs) participated in the study. EGG and HRV in fasted and fed subjects with fasted serum catecholamine levels were measured in both groups. Fasting IBS pts showed gastric dysrrhythmia (29+/-14% vs. 11+/-7%), DP was 128.860 +/- 112.000 vs. 46.000+/- 23.200muV(2), DF 2.37+/-0.8 vs. 2.9+/-0.2cpm. Feeding (300 kcal) improved dysrrhythmia to 20+/-13% vs. 8+/-5%, DP decreased to 74.500+/-57.720 vs. 165.600+/-89.000muV(2) and DF increased to 2.53+/-0.7 vs. 3.2+/-0.3cpm. In fasted and fed IBS pts SWC (channels 3-4) was about 60+/-11 vs. 84+/-8% and 68+/-14 vs. 92+/-8% respectively. In IBS pts resting HRV parameters were lower (LF - 650.3 vs. 811.6 ms(2); HF - 508.8 vs. 854.6 ms(2)); with higher LF/HF ratio in IBS patients (1.52 vs. 1.2). The serum fasting level of adrenaline and noradrenaline in IBS pts were higher 1.28+/-0.06 vs. 0.65+/-0.05 nmol/L, and 3.54+/-1.2 vs. 2.89+/- 08 nmol/L, p<0.05 respectively. Increased sympathetic drive in IBS pts reflected by high catecholamine levels and LH/HF ratio is responsible for gastric dysrrhythmias and low DF and coupling. Meal has negligible effect on EGG parameters improvement. The ANS dysfunction observed in IBS patients is most probably responsible for disturbances in gastric myoelectric activity presented as gastric dysrrhythmias resulting in gastric emptying delay and dyspeptic symptoms. [Abstract/Link to Full Text]

Kapica M, Zabielska M, Puzio I, Jankowska A, Kato I, Kuwahara A, Zabielski R
Obestatin stimulates the secretion of pancreatic juice enzymes through a vagal pathway in anaesthetized rats - preliminary results.
J Physiol Pharmacol. 2007 Aug;58 Suppl 3123-30.
Obestatin is a 23 amino acid peptide derived from the preproghrelin precursor, and originally purified from the rat stomach mucosa. It was shown that obestatin may counteract the effects of its sister peptide, ghrelin, on food intake and gastrointestinal motility but the other roles in controlling the gastrointestinal function remain unknown. The aim of the present study was to determine the influence of exogenous obestatin on the secretion of pancreatic juice. In anesthetized male Wistar rats the external jugular vein was catheterized, and the common biliary-pancreatic duct was cannulated with polyethylene tubing for collection of pancreatic-biliary juice (P-BJ). Obestatin boluses (30, 100 and 300 nmol/kg b. wt.) were injected intravenously or intraduodenally every 30 min. Obestatin was also administered in vagotomized (subdiaphragmatic vagotomy) rats. In the examined rats, obestatin intravenous and intraduodenal boluses did not affect the P-BJ volume. On the other hand, obestatin boluses increased the protein output and trypsin activity. Vagotomy abolished the effects of exogenous obestatin administration. In conclusion, the present study demonstrates for the first time that exogenous obestatin may stimulate the secretion of pancreatic juice enzymes. The effect is dose-dependent and requires intact vagal supply. [Abstract/Link to Full Text]

Strza?kowski AK, Godlewski MM, Hallay N, Kulasek G, Gajewski Z, Zabielski R
The effect of supplementing sow with bioactive substances on neonatal small intestinal epithelium.
J Physiol Pharmacol. 2007 Aug;58 Suppl 3115-22.
Development of the small intestinal epithelium in early postnatal period has a significant influence on pig's survival rate and further productivity. The aim of this research was to verify whether the diet supplementation of pregnant and lactating sow with a blend of bioactive substances (flax seed, rapeseed, linden inflorescence, taurine, L-carnitine and tocopherol acetate) had an effect on the development of intestinal epithelium in their offspring. The doses of bioactive substances were calculated to meet the demands for optimal supply of the pig fetuses and newborns. Pig neonates from two groups of sows, control and supplemented, were sacrificed at the day 1, 2, 4, 7 and 14 of life. The samples taken from mid-jejunum were evaluated for mitosis (Ki67), apoptosis (active caspase 3), autophagy (MAP I LC3), and DNA damage (p53). Increase of mitotic index was noticed at day 1, 4 and 7 for supplemented group when compared to the control. Reduction of apoptotic index was observed at day 2 as compared to control. A tendency toward elevated autophagy was observed during the first 2-4 postnatal days in both groups. p53 expression was significantly lower in supplemented group as compared to control. Overall, the mitosis to programmed cell death ratio was increased and the maturation of epithelial cells quickened. We suppose that the supplementation of pregnant and lactating sow diet with bioactive substances enhanced maturation of the small intestinal epithelium in their offspring during the early postnatal period. [Abstract/Link to Full Text]

Godlewski MM, Hallay N, Bier?a JB, Zabielski R
Molecular mechanism of programmed cell death in the gut epithelium of neonatal piglets.
J Physiol Pharmacol. 2007 Aug;58 Suppl 397-113.
In the intestinal mucosa of pig, calf and rat neonates, we observed the cells die in the packets which suggests involvement of some paracrine factors. The death signal was transferred via tissue continuum as well as across the gut lumen, and the involvement of TGF-beta1 and TNFalpha was demonstrated. Present study aimed to clarify the molecular mechanisms of programmed cell death in the mucosa of the small intestine of pig neonates. Groups (packets) of cells and the neighboring cells underwent apoptosis, and expressed an enhanced TGF-RII. In the dying cells the death signal promoted via TGF-RII was associated with enhanced expression of active caspase 8, TGF-beta1, TNFalpha and Bid. Quantitative study showed that high expression of TGF-beta1 was positively correlated with expression of BID and negatively with BCL-2, illustrating the transmission of signal from TGF-RII through SMAD cascade and RunX protein. We hypothesize that TGF-beta1 sensitizes the enterocytes for TNFalpha signaling and both cytokines control the apoptosis process in the gut epithelium. Intensive mitosis triggers many errors in DNA replication, and the role of p53 is to detect them and promote either repair or apoptosis. During first days of live all damaged cells were directed towards apoptosis while at day 7 at least some of them were repaired. Autophagy, the second form of programmed cell death, was recognized by its key marker MAP I LC3. Our data showed the colocalization of MAP I LC3 with active caspase 3 thus suggesting a coexistence between these two forms of cell death, at least in the early postnatal life. [Abstract/Link to Full Text]

Skrzypek T, Valverde Piedra J, Skrzypek H, Kazimierczak W, Biernat M, Zabielski R
Gradual disappearance of vacuolated enterocytes in the small intestine of neonatal piglets.
J Physiol Pharmacol. 2007 Aug;58 Suppl 387-95.
The unique feature of enterocytes in newborn mammals is the presence of an apical canalicular system (ACS) leading to production of large vacuoles, important for colostral macromolecule uptake. The vacuolated fetal-type enterocytes (VFE) enable transfer of colostral and milk proteins from the intestinal lumen across the epithelium without loosing their biological activity. First VFE are observed in the pig and lamb fetuses in the second trimester of pregnancy, located at the upper part of villi in the proximal region of the fetal small intestine and subsequently in the middle and distal regions. After birth the VFE are replaced with enterocytes lacking ACS. The present study aimed to investigate the depletion of VFE in the small intestine in the sow reared pig neonates during the first postnatal weeks using scanning electron microscopy (SEM). The SEM analysis demonstrated the gradual disappearance of vacuolated enterocytes in time. VFE remained in the jejunum for a few days after birth, whereas in the duodenum single VFE were present only at birth. In the proximal jejunum, the VFE were localized in the upper part of the villi, and disappeared until the day 3 of life. VFE were present in the mid and distal jejunum, and diminished gradually until day 14 of life. By the day 21 of life, the vacuolated cells were not observed neither in the jejunum nor ileum. In conclusion, morphology analysis of pig small intestinal mucosa suggests that replacement of fetal type vacuolated enterocytes is resumed within 21 days after birth. [Abstract/Link to Full Text]

Ch?opecka M, Dziekan N, Mendel M, Baka?a A, Ma?dyk J, Wiechetek M
Evaluation of the time-stability of an alternative research model based on isolated rat gastrointestinal strips.
J Physiol Pharmacol. 2007 Aug;58 Suppl 373-86.
Experiments were performed on rat isolated gastrointestinal (GI) strips. The study was carried out in isotonic conditions during a long-term (5-8 h) incubation in modified Krebs-Henseleit solution, (M K-HS) at 37 degrees C. The reaction of GI muscle strips to acetylcholine (ACh) and histological images of the tissues strips were studied. Additionally, spontaneous motor activity of the jejunum strips was evaluated. The obtained results indicate that as well the spontaneous motility as the response of jejunum muscle strips to acetylcholine and the histological structure of the preparations changed throughout the incubation time. It suggests low time-stability of jejunum strips model during long-term experiment. In contrast, the response of stomach (corpus and fundus) strips to ACh application did not change significantly during 6 h incubation. Moreover, the histological structure of the stomach preparations was maintained unaffected for a long incubation time. Thus, the isolated stomach strips can be described as an alternative experimental model of high time-stability during long-term experiments. [Abstract/Link to Full Text]

Trojniar W, Pluci?ska K, Ignatowska-Jankowska B, Jankowski M
Damage to the nucleus accumbens shell but not core impairs ventral tegmental area stimulation-induced feeding.
J Physiol Pharmacol. 2007 Aug;58 Suppl 363-71.
Food intake is regulated not only by homeostatic requirements but also by emotional factors (e.g. palatability of food, alleviation of emotional tension etc.). The nucleus accumbens (Acb) is a part of the mesolimbic dopaminergic system which is responsible for a positive emotional aspect of various homeostasis-relevant stimuli. In the present work, we tested the Acb involvement in feeding behaviour using an experimental paradigm specifically designed to assess motivational vs motor aspect of food ingestion. In rats, feeding was evoked by electrical stimulation of the midbrain ventral tegmental area (a somatodendritic region of mesolimbic system) and assessed quantitatively with the use of the latency to feed/stimulation frequency curve-shift paradigm before and after electrolytic lesion of Acb. An impairment of stimulation-induced feeding manifesting as an elevation of the reaction threshold and a rightward, parallel shift of the stimulation frequency/reaction latency curve in the range of frequency which is sensitive to motivational aspects of food occurred after lesions localized mainly in the Acb shell. The lesions situated mainly in the Acb core were ineffective. The results obtained indicate that the Acb shell connected with the limbic system but not the motor-related Acb core affects motivational aspects of feeding behaviour. [Abstract/Link to Full Text]

Sikora A, Grzesiuk E
Heat shock response in gastrointestinal tract.
J Physiol Pharmacol. 2007 Aug;58 Suppl 343-62.
Heat shock response is one of the defense mechanisms common to eukaryotic and prokaryotic cells. The highly conserved and ubiquitous heat shock proteins (HSPs) are essential for cell survival during stress. Stress tolerance, i.e., adaptation of cells to stress conditions, is a characteristic feature of heat shock response. The lumen of the gastrointestinal tract is an external environment to the body. Epithelium of the digestive tract is exposed to various stress factors inducing the heat shock response, e.g., bacteria and their toxins, food borne chemical compounds, drugs and diet deficiencies. Other factors like plant lecitins, glutamine or short fatty acids are mild stressors and can modulate the heat shock response in cells. All these factors are presumed to influence the normal microflora that is an integral part of the digestive tract. This review is focused on the induction/modulation of heat shock proteins expression in the epithelium of the gastrointestinal tract by various factors, on the protective role of HSPs and mechanisms leading to stress protection inside the gut. Heat shock response is one of the key mechanisms of maintenance of gastrointestinal tract homeostasis. It is involved in pathogenic bacteria adaptation to life in the digestive tract, especially in colony formation and in their role in infectious processes. [Abstract/Link to Full Text]

Celi?ski K, Cichoz-Lach H
Therapeutic endoscopy in gastroenterology.
J Physiol Pharmacol. 2007 Aug;58 Suppl 333-41.
The role of therapeutic endoscopy in current gastroenterology is very important. Therapuetic endoscopy is useful in treatment of gastrointestinal bleeding. Endoscopic control of gastrointestinal bleeding includes the following procedures of haemostasis techniques: photocoagulation, electrocoagulation, thermocoagulation and injection method. Owing to these procedures mortality has significantly decreased. Endoscopic hemostasis eliminates the risk of surgery, is less expensive and better tolerated by patients. Colonoscopic polypectomy is a widely used technique. By removal of polyps the incidence of colon cancer can be decreased. The "hot biopsy" forceps can be used to excise polyps of up to 6 mm. Larger polyps can be removed safely by snare electrocautery and retrieved for histologic study. Endoscopic retrograde cholangiopancreatography has a therapeutic application designed to cut the sphincter of Oddi fibers of the distal common bile duct, what is indicated currently in choledocholithiasis and papillary stenosis with ascending cholangitis, acute gallstone pancreatitis. Endoscopic sphincterotomy in now an established procedure that is indicated in patients with common bile duct calculi. Endoscopic decompression of the biliary tree - dilatation benign structures of the biliary tree with baloon catheters and placement an internal endoprothesis allows the nonoperative decompression and significant palliation for patients with obstructing tumors. [Abstract/Link to Full Text]

Cichoz-Lach H, Celi?nski K
Modern methods of endoscopic diagnosis of gastrointestinal tract.
J Physiol Pharmacol. 2007 Aug;58 Suppl 321-31.
The last years brought the substantial development of new diagnostic procedures of gastrointestinal tract. High resolution endoscopy and magnifying endoscopy both give the detailed picture of the mucosal surface of esophagus, stomach, duodenum and large bowel. They are very useful in diagnosis precancer states or early cancer. The use of the biopsy forceps and cytology brush allows histologic confirmation of endoscopic impression. The new methods, which allow complete diagnosis of small bowel are double balloon enteroscopy and capsule endoscopy. They are recommended in case of Crohn's disease, identification of bleeding source, detection of stenoses and neoplastic changes in the small intestine, diagnosis of chronic diarrhea and detection of visceral diseases or malabsorption syndromes. Only double balloon enteroscopy allows to take the biopsy as well as to make endoscopic therapy. Chromoendoscopy involves topical application of stains to the mucosa on endoscopy in order to delineate and better characterize specific findings. This technique is thought to be helpful for both the diagnosis of early cancer for evaluation of non-neoplastic diseases. Endoscopic ultrasonography connecting endoscopic and ultrasonographic techniques is applied to diagnose the intramural abnormalities. [Abstract/Link to Full Text]

Czesnikiewicz-Guzik M, Konturek SJ, Loster B, Wisniewska G, Majewski S
Melatonin and its role in oxidative stress related diseases of oral cavity.
J Physiol Pharmacol. 2007 Aug;58 Suppl 35-19.
The role of the oral cavity in the pathogenesis of diseases of various systems such as the gastro-intestinal tract (GIT), cardiovascular and immune systems has been recently evaluated. While initially the oral cavity was considered to be mainly a source of various bacteria, their toxins and antigens, recent studies showed that it may also be a location of oxidative stress and periodontal inflammation. Accordingly, this review focuses on the involvement of melatonin (MT) in oxidative stress diseases of oral cavity as well as on potential therapeutic implications of MT in dental disorders, especially in periodontal inflammation. MT is generated and released by pineal gland and by entero-endocrine (EE) cells located in the GIT. The pattern of MT secretion from the pineal gland is controlled by an endogenous circadian timing system that conveys information about the light/dark cycle to various organs of the body, thereby organizing its seasonal and circadian rhythms. The secretion of MT from the EE cells of GIT is related mainly to feeding periods. MT is a non-toxic highly lipophilic indole, and this feature facilitates its penetration through cell membranes and its compartments. However, the most important effect of MT seems to result from its potent antioxidant, immuno-modulatory, protective and anti-cancer properties. It stimulates synthesis of type I collagen fibers and promotes bone formation. Thus, MT could be used therapeutically for instance, locally, in the oral cavity damage of mechanical, bacterial, fungal or viral origin, in post-surgical wounds caused by tooth extractions and other oral surgeries and, in helping bone formation in various auto-immunological disorders such as Sjorgen syndrome, in periodontal diseases, and in oral cancers. [Abstract/Link to Full Text]

Jawie? J, Gajda M, Olszanecki R, Korbut R
BAY x 1005 attenuates atherosclerosis in apoE/LDLR - double knockout mice.
J Physiol Pharmacol. 2007 Sep;58(3):583-8.
Recently, we have shown that MK-886 - an inhibitor of five lipoxygenase activating protein (FLAP) inhibits atherosclerosis in apolipoprotein E / LDL receptor - double knockout mice. We, therefore, wanted to find out if other FLAP inhibitor - BAYx1005 given at a dose of 1.88 mg per 100 mg of body weight per day during 16 weeks, could also attenuate atherogenesis. In apoE/LDLR - DKO mouse model BAYx1005 inhibited atherogenesis, measured both by "en face" method (23.84 +/- 2.7% vs. 15.16 +/- 1.4%) and "cross-section" method (497236 +/- 31516 microm(2) vs. 278107 +/- 21824 microm(2)). This is the first report that shows the effect of BAYx1005 on atherogenesis in gene-targeted mice. [Abstract/Link to Full Text]

Smolinska N, Siawrys G, Kaminski T, Przala J
Leptin gene and protein expression in the trophoblast and uterine tissues during early pregnancy and the oestrous cycle of pigs.
J Physiol Pharmacol. 2007 Sep;58(3):563-81.
Leptin is a 16-kDa protein hormone encoded by the obese (ob) gene and acts on receptors in the hypothalamus to regulate food intake and energy balance. The identification of leptin and its receptor mRNAs and proteins in human and mouse endometrium and placental trophoblast has attracted attention to the potential role of leptin in implantation. Thus, the aim of this study was to compare the expression levels of porcine leptin mRNA and protein in endometrium and myometrium during mid- and late-luteal phases of the oestrous cycle (days 10 - 12 and 14 - 16) as well as during two stages of pregnancy respondent to the beginning (days 14 - 16) and the end (days 30 - 32) of the implantation process, and in trophoblast during both periods of pregnancy. Leptin gene and protein expression in myometrium, and leptin mRNA expression in endometrium was more pronounced in the mid- and late-luteal phases of the cycle in comparison to studied periods of pregnancy, whereas leptin protein concentration in endometrium was either enhanced on days 30 - 32 of pregnancy in relation to days 14 - 16 of the cycle or there were no changes between pregnancy and luteal phase of the cycle. On days 30 - 32 of pregnancy, expression of the leptin gene in the endometrium, and of the leptin gene and protein in the myometrium was more pronounced in comparison to the earlier stage of pregnancy. Moreover, leptin gene expression in porcine trophoblast increased during the beginning of the implantation process compared to days 30 - 32 of pregnancy, while the protein concentration decreased on days 14 - 16 of pregnancy. In conclusion, the finding of leptin gene and protein expression in porcine endometrium, myometrium and trophoblast indicates that locally synthesised leptin can participate in the control of pig reproduction. The fluctuation of the hormone concentration during pregnancy and changes in its level between pregnancy and the oestrous cycle may indicate leptin's involvement in the implantation process. [Abstract/Link to Full Text]

Soszynski D, Chelminiak M
Intracerebroventricular injection of neuronal and inducible nitric oxide synthase inhibitors attenuates fever due to LPS in rats.
J Physiol Pharmacol. 2007 Sep;58(3):551-61.
Nitric oxide (NO) has been shown to be an important mediator of febrile response to lipopolisaccharide (LPS). To clarify the role of different isoforms of NO synthase (NOS) in febrile response to immune challenge, effects of selective iNOS and nNOS inhibitors on fever to LPS were examined in freely moving biotelemetered rats. Vinyl-L-NIO (N(5) - (1-Imino-3-butenyl) - ornithine (vL-NIO), a neuronal nitric oxide synthase (nNOS) inhibitor, and aminoguanidine hydrochloride, an inducible nitric oxide synthase (iNOS) inhibitor, were injected intracerebroventricularly at a dose of 10 microg/rat just before intraperitoneal injection of LPS at a dose of 50 microg/kg. Both inhibitors injected at a selected doses had no effect on normal day-time body temperature (T(b)) and normal night-time T(b). vinyl-L-NIO and aminoguanidine injected intracerebroventricularly at a dose of 10 microg/animal suppressed the LPS-induced fever in rats. The fever index calculated for rats pretreated with v-LNIO or with aminoguanidine and injected with LPS was reduced by 43% and 72%, respectively, compared to that calculated for water-pretreated and LPS-injected rats. Whereas vL-NIO partly attenuated both phases of febrile rise in T(b), administration of aminoguanidine into the brain completely prevented fever induced by LPS. These data indicate that activation of iNOS inside the brain is not only responsible for triggering but also for maintaining of LPS-induced fever in rats. It is, therefore, reasonable to hypothesize that, activation of iNOS inside the brain is more important in fever development than activation of nNOS. [Abstract/Link to Full Text]

Goraca A, J�zefowicz-Okonkwo G
Protective effects of early treatment with lipoic acid in LPS-induced lung injury in rats.
J Physiol Pharmacol. 2007 Sep;58(3):541-9.
A lipopolysaccharide (LPS) stimulates the synthesis and releases several metabolites from phagocytes which can lead to an endotoxic shock characterized by multiple organ injury with the earliest to occur in the lungs. Among LPS-induced metabolites, reactive oxygen species are considered to play a crucial pathogenetic role in the lung damage. In this study, the effect of early administration of an antioxidant, alpha-lipoic acid (LA), on pulmonary lipid peroxidation, lung hydrogen peroxide (H(2)O(2)) concentration, and lung sulfhydryl group content was evaluated in rats with endotoxic shock induced by administration of LPS (Escherichia coli 026:B6, 30 mg/kg, i.v.). In addition, lung edema was assessed with wet-to-dry lung weight (W/D) ratio. Animals were treated intravenously with normal saline or LA 60 mg/kg or 100 mg/kg 30 min after LPS injection. After a 5 h observation, animals were killed and the lungs were isolated for measurements. Injection of LPS alone resulted in the development of shock and oxidative stress, the latter indicated by a significant increase in the lung thiobarbituric acid reacting substances (TBARS) and H(2)O(2) concentrations, and a decrease in the lung sulfhydryl group content. The increase in the W/D ratio after the LPS challenge indicated the development of lung edema in response to LPS. Administration of LA after the LPS challenge resulted in an increase in the sulfhydryl group content and a decrease in TBARS and H202 concentration in the lungs as compared with the LPS group. An insignificant decrease in the W/D ratio was observed in rats treated with either dose of LA. These results indicate that the LPS-induced oxidative lung injury in endotoxic rats can be attenuated by early treatment with LA. Administration of LA could be a useful adjunct to conventional approach in the management of septic shock. [Abstract/Link to Full Text]

Bujak-Gizycka B, Madej J, Wo?kow PP, Olszanecki R, Drabik L, Rutowski J, Korbut R
Measurement of angiotensin metabolites in organ bath and cell culture experiments by liquid chromatography - electrospray ionization - mass spectrometry (LC-ESI-MS).
J Physiol Pharmacol. 2007 Sep;58(3):529-40.
The metabolism of renin-angiotensin system (RAS) is more complicated than previously expected and understanding the biological phenomena regulated by variety of angiotensin metabolites requires their precise and possibly comprehensive quantitation. Physiological concentrations of angiotensins (Ang) in biological fluids are low, therefore their accurate measurements require very sensitive and specific analytical methods. In this study we developed an accurate and reproducible method of quantitation of angiotensin metabolites through coupling of liquid chromatography and electrospray ionization - mass spectrometry (LC-ESI-MS). With this method main angiotensin metabolites (Ang I, II, III, IV, 1-9, 1-7, 1-5) can be reliably measured in organ bath of rat tissues (aorta, renal artery, periaortal adipose tissue) and in medium of cultured endothelial cells (EA.hy926), exposed to Ang I for 15 minutes, in the absence or in the presence of angiotensin converting enzyme inhibitor, perindoprilat. Presented LC-ESI-MS method proved to be a quick and reliable solution to comprehensive analysis of angiotensin metabolism in biological samples. [Abstract/Link to Full Text]

Mogielnicki A, Kramkowski K, Pietrzak L, Buczko W
N-methylnicotinamide inhibits arterial thrombosis in hypertensive rats.
J Physiol Pharmacol. 2007 Sep;58(3):515-27.
There are few findings indicating that nicotinamide may potentially influence intravascular thrombosis. Interestingly, N-methylnicotinamide, one of the metabolites of nicotinamide - could be more potent than its parent compound. In the present study we have investigated the influence of N-methylnicotinamide on arterial thrombosis in normotensive and renovascular hypertensive rats. The contribution of platelets, coagulation and fibrinolytic systems in the mode of N-methylnicotinamide action was also determined. Furthermore, we examined the role of nitric oxide/prostacyclin in the mechanisms of N-methylnicotinamide action. N-methylnicotinamide, but not nicotinamide, administered intravenously into renovascular hypertensive rats developing electrically induced arterial thrombosis caused dose-dependent decrease of thrombus weight, collagen-induced platelet aggregation and plasma antigen/activity of plasminogen activator inhibitor - 1, without changing of occlusion time, routine coagulation parameters and plasma activity of tissue plasminogen activator. Indomethacin - an inhibitor of prostacyclin synthesis, completely abolished the antithrombotic and antiplatelet effect of N-methylnicotinamide, and the plasma level of 6-keto-PGF(1alpha) , prostacyclin metabolite, increased simultaneously with the inhibition of thrombus formation. Our study shows that N-methylnicotinamide via production/release of prostacyclin inhibits arterial thrombosis development. The antithrombotic effect of N-methylnicotinamide is accompanied by platelet inhibition and enhanced fibrinolysis, due to the decrease production of plasminogen activator inhibitor - 1. [Abstract/Link to Full Text]

Jantzen F, K�neman S, Wolff B, Barth S, Staudt A, Kroemer HK, Dahm JB, Felix SB, Landsberger M
Isoprenoid depletion by statins antagonizes cytokine-induced down-regulation of endothelial nitric oxide expression and increases NO synthase activity in human umbilical vein endothelial cells.
J Physiol Pharmacol. 2007 Sep;58(3):503-14.
Endothelial dysfunction and atherosclerosis are associated with an inflammation-induced decrease in endothelial nitric oxide synthase (eNOS) expression. Based on the differences between hydrophobic and hydrophilic statins in their reduction of cardiac events, we analyzed the effects of rosuvastatin and cerivastatin on eNOS and inducible NO synthase (iNOS) expression and NOS activity in TNF-alpha-stimulated human umbilical vein endothelial cells (HUVEC). Both statins reversed down-regulation of eNOS mRNA and protein expression by inhibiting HMG-CoA reductase and isoprenoid synthesis. Cerivastatin tended to a more pronounced effect on eNOS expression compared to rosuvastatin. NOS activity - measured by conversion of [(3)H]-L-arginine to [(3)H]-L-citrulline - was enhanced under treatment with both drugs due to inhibition of HMG-CoA reductase. Statin-treatment reduced iNOS mRNA expression under normal conditions, but had no relevant effects on iNOS mRNA expression in cytokine-treated cells. Rosuvastatin and cerivastatin reverse the detrimental effects of TNF-alpha-induced down-regulation in eNOS protein expression and increase NO synthase activity by inhibiting HMG-CoA reductase and subsequent blocking of isoprenoid synthesis. These results provide evidence that statins have beneficial effects by increasing eNOS expression and activity during the atherosclerotic process. [Abstract/Link to Full Text]

Bernatowa I, Csizmadiova Z, Kopincova J, Puzserova A
Vascular function and nitric oxide production in chronic social-stress-exposed rats with various family history of hypertension.
J Physiol Pharmacol. 2007 Sep;58(3):487-501.
The study investigated the effect of chronic crowding stress on vascular function and nitric oxide (NO) production in rats with various family history of hypertension. Wistar (W), wBHR (offspring of W dams and spontaneously hypertensive sires), sBHR (offspring of spontaneously hypertensive dams and W sires) and spontaneously hypertensive rats (SHR) were used. Twelve-week-old males were divided into the control or crowded group for eight weeks. Basal blood pressure (BP, determined by tail-cuff plethysmography) of W, wBHR, sBHR and SHR rats was 112 +/- 3, 129 +/- 2, 135 +/- 2 and 187 +/- 3 mmHg, respectively. Crowding increased BP and reduced aortic NO synthase activity only in sBHR and SHR rats, without alterations in hypothalamic NO production. Acetylcholine-induced vasorelaxation of the femoral artery of stress-exposed rats was improved in W, unaltered in wBHR and sBHR and reduced in SHR. Crowding reduced serotonin-induced vasoconstriction in W and wBHR rats but had no effect in sBHR and SHR rats. In conclusion, the results suggest that crowded offspring of normotensive mothers were able to modify their vascular function in order to maintain BP at normal levels. On the other hand, offspring of hypertensive mothers were unable of effective adaptation of vascular function in stressful conditions resulting in gradual development of hypertension. [Abstract/Link to Full Text]

Zubrzycka M, Janecka A
Effect of galanin on substance P- and vasoactive intestinal polypeptide-induced nociceptive trigemino-hypoglossal reflex in rats.
J Physiol Pharmacol. 2007 Sep;58(3):479-86.
Substance P (SP), vasoactive intestinal polypeptide (VIP) and galanin (GAL), present in primary sensory neurons, are involved in transmission of nociceptive signaling from the peripheral to central nervous system. In this study we investigated the effect of GAL on SP-induced or VIP-induced evoked tongue jerks (ETJ) in response to noxious tooth pulp stimulation during perfusion of the cerebral ventricles with SP or VIP solutions. The experiments were carried out on rats under chloralose anesthesia. It was shown that both, SP and VIP, perfused through the cerebral ventricles enhanced the ETJ amplitude as compared with control, but the effect produced by SP was stronger. The intracerebroventricular perfusion of GAL 5 minutes before SP caused a dose-dependent inhibition of SP-induced ETJ, whereas GAL perfused through the cerebral ventricles 5 minutes before VIP did not reduce the excitatory effect of VIP on ETJ. These results indicate that the antinociceptive effect of GAL perfused through the cerebral ventricles, tested on the trigemino-hypoglossal reflex in rats, is specifically mediated by the SP-ergic system. [Abstract/Link to Full Text]

Kasacka I, Majewski M
An immunohistochemical study of endocrine cells in the stomach of hypertensive rats.
J Physiol Pharmacol. 2007 Sep;58(3):469-78.
Essential hypertension is a complex disease with both genetic and environmental determinants. The effect of spontaneous hypertension on the distribution and occurrence of somatostatin-, gastrin- and serotonin-immunoreactive cells in the fundus and pylorus of the rat stomach was examined by immunohistochemistry. The animals were killed by decapitation at 4 and 16 weeks of age (5 control rats and 5 hypertensive rats). Endocrine cells generally increase in number in hypertensive rats as compared to control rats. However, the detailed responses of endocrine cells to hypertension depend on the cell type, region of gastric mucosa and age of animals. The present results suggest that hypertension has an influence on the intrinsic regulatory system by endocrine cells control in the rat stomach. [Abstract/Link to Full Text]

Holma R, Salmenper� P, Virtanen I, Vapaatalo H, Korpela R
Prophylactic potential of montelukast against mild colitis induced by dextran sulphate sodium in rats.
J Physiol Pharmacol. 2007 Sep;58(3):455-67.
Cysteinyl leukotrienes play a part in inflammatory processes such as inflammatory bowel diseases. The present study aimed to evaluate the effects of the cys-LT-1 receptor antagonist montelukast on a mild colitis model in rats. Colitis was induced by administrating 4% dextran sulphate sodium (DSS, MW 45,000) in drinking water for 9 days. Montelukast (10 mg/kg/day) or vehicle was given by gastric gavage once daily simultaneously with DSS administration. A healthy control group receiving water as drinking fluid and vehicle by gastric gavage was included. Body weight loss, consistency of faeces (loose/diarrhoea) and occult blood in the faeces/ gross bleeding were assessed on days 6 - 9. After sacrifice, the following were assessed: colonic histology, the expression of inducible nitric oxide synthase, macrophage/monocyte marker ED1, cyclooxygenase-1 and cyclooxygenase-2, as well as the production of leukotriene B(4) and E(4), prostaglandin E(2), its metabolite bicyclic-prostaglandin E(2) and thromboxane B(2) in the colonic tissue incubation in vitro. Rats receiving DSS exhibited bloody diarrhoea from day 6 onwards. Montelukast significantly reduced the occult blood in the faeces/ gross bleeding, maintained normal body weight gain and tended to decrease the ratio of leukotriene B(4)/ prostaglandin E(2) production in the colon in vitro. The results indicate that montelukast has some potential to ameliorate mild experimental colitis induced by DSS. [Abstract/Link to Full Text]

Daniluk J, Dabrowski A
The effect of concomitant stimulation with cholecystokinin and epidermal growth factor on extracellular signal-regulated kinase (ERK) activity in pancreatic acinar cells.
J Physiol Pharmacol. 2007 Sep;58(3):441-53.
The transmission of extracellular proliferation and differentiation signals into their intracellular targets is mediated by a signaling cascade culminating in mitogen-activated protein kinase (MAPK) also known as ERK. In pancreatic acinar cells both cholecystokinin (CCK) and epidermal growth factor (EGF) are known to stimulate ERK. Regulatory interactions among individual receptor-coupled signaling cascades are critically important for establishing cellular responses in the face of multiple stimuli. The aim of our study was to evaluate the effect of concomitant stimulation of G protein-coupled receptors (GPCR) and EGF receptors on ERK activity in isolated pancreatic acinar cells. ERK activity was determined by means of Western-blotting, with the use of the antibody which recognizes active, tyrosine-phosphorylated kinase (pY-ERK). pY-ERK level was strongly elevated by 10 nM CCK-8, 100 microM carbachol (CAR), or 100 nM EGF. The addition of EGF to 60 min-lasting incubations of acini with CCK-8 or CAR caused abrupt decrease of pY-ERK level to 56 and 59% of control, respectively. Similar phenomenon was observed when short stimulation with CCK-8 or CAR was superimposed on the effect of EGF. After the addition of EGF to acini incubated previously with phorbol ester TPA, strong decrease in pY-ERK level was also observed. In conclusion, in pancreatic acinar cells, concomitant stimulation with CCK or CAR and EGF has strong inhibitory effect on ERK cascade. This inhibitory cross-talk may be mediated, at least partially, by protein kinase C (PKC). These mutual inhibitory interactions demonstrate novel mechanism for integration of multiple signals generated by activation of G-protein-coupled and growth factor receptors in pancreatic acinar cells. [Abstract/Link to Full Text]

Granados MP, Salido GM, Pariente JA, Gonz�les A
Modulation of CCK-8-evoked intracellular Ca2+ waves by hydrogen peroxide in mouse pancreatic acinar cells.
J Physiol Pharmacol. 2007 Sep;58(3):423-40.
In the present study we have employed single cell imaging analysis to monitor the propagation of cholecystokinin-evoked Ca(2+) waves in mouse pancreatic acinar cells. Stimulation of cells with 1 nM CCK-8 led to an initial Ca(2+) release at the luminal cell pole and subsequent spreading of the Ca(2+) signal towards the basolateral membrane in the form of a Ca(2+) wave. Inhibition of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) activity by 1 microM thapsigargin, preincubation in the presence of 100 microM H(2)O(2) or inhibition of PKC with either 5 microM Ro31-8220 or 3 microM GF-109203-X all led to a faster propagation of CCK-8-induced Ca(2+) signals. The propagation of CCK-8-evoked Ca(2+) signals was slowed down by activation of PKC with 1 microM PMA, and preincubation of cells in the presence of H(2)O(2) counteracted the effect of PKC inhibition. The protonophore FCCP (100 nM) and the inhibitor of the mitochondrial Ca(2+)-uniporter Ru360 (10 microM) led to an increase in the propagation rate of CCK-8-evoked Ca(2+) waves. Finally, depolymerisation of actin cytoskeleton with cytochalasin D (10 microM) led to a faster propagation of CCK-8-evoked Ca(2+) signals. Stabilization of actin cytoskeleton with jasplakinolide (10 microM) did not induce significant changes on CCK-8-evoked Ca(2+) waves. Preincubation of cells in the presence of H(2)O(2) counteracted the effect of cytochalasin D on CCK-8-evoked Ca(2+) wave propagation. Our results suggest that spreading of cytosolic Ca(2+) waves evoked by CCK-8 can be modulated by low levels of oxidants acting on multiple Ca(2+)-handling mechanisms. [Abstract/Link to Full Text]

Nishio H, Terashima S, Nakashima M, Aihara E, Takeuchi K
Involvement of prostaglandin E receptor EP3 subtype and prostacyclin IP receptor in decreased acid response in damaged stomach.
J Physiol Pharmacol. 2007 Sep;58(3):407-21.
We investigated the roles of cyclooxygenase (COX) isozymes and prostaglandin E (PGE) receptor EP1 and EP3 subtypes or prostacyclin IP receptors in the decrease in acid secretion in the damaged mouse stomach. Male C57/BL6 mice, both wild type and animals lacking EP1, EP3, or IP receptors, were used after 18 h of fasting. Under urethane anesthesia, the stomach was mounted on an ex-vivo chamber and perfused with saline, and acid secretion as well as transmucosal potential difference (PD) was measured before and after exposure to 20 mM taurocholate Na (TC) for 20 min. Indomethacin, SC-560 or rofecoxib was given i.d. 30 min before TC. Mucosal exposure to TC in wild-type mice caused a reduction in PD, followed by decrease in acid secretion. Indomethacin attenuated the decrease in acid secretion after exposure to TC in wild-type mice, an effect mimicked by SC-560 but not rofecoxib, yet none of these drugs affected the decrease in PD. An altered acid response after exposure to TC was similarly observed in EP1 (-/-) mice but mitigated in mice lacking either EP3 or IP receptors, although a decrease in PD was observed in all groups. Furthermore, the decreased acid response was also attenuated by prior administration of the EP3- but not EP1- antagonist. Mucosal levels of PGE(2) and 6-keto PGF(1a) increased after exposure to TC in all groups of mice. In conclusion, the decrease in acid secretion in the damaged stomach is mediated by endogenous PGs derived from COX-1, through PGE(2)/EP3 receptors and prostacyclin/IP receptors. [Abstract/Link to Full Text]

Recent Articles in Polish Journal of Pharmacology and Pharmacy

Abstracts of the 8th Conference on Progress in Etiopathogenesis of Seizures. Lublin, Poland, 21 November 2003.
Pol J Pharmacol. 2004 Jan-Feb;56(1):145-53. [Abstract/Link to Full Text]

Samochowiec-Donocik E, Koraszewska-Matuszewska B
Influence of beta-adrenergic antagonists on tear secretion in children.
Pol J Pharmacol. 2004 Nov-Dec;56(6):871-3.
The aim of the study was to compare the results of tear film volume, conjunctival and corneal state of children eyes both treated with beta-blocker and healthy ones. We have examined 40 eyes of 20 children at the age from 7 to 17 years. Group I - 20 glaucomatous eyes treated with 0.5% timolol twice daily during at least 12 months. Group II - 20 eyes of control age-matched group. Schirmer I test without anesthesia and lissamine green staining was performed to evaluate conjunctival and corneal surface. The values of Schirmer I test were: group I from 12 to 24 mm, the mean 17.06 +/- 1.78 mm and group II from 16 to 35 mm, the mean 29.3 +/- 2.67, p = 0.000004. Keratoepitheliopathy was observed in 4 eyes (20%) of group I and was rated as 3rd and 6th degree of Franck classification. In the group II, there were only single staining points classified as 1st degree noticed in 2 eyes (10%), p = 0.37. The long-term treatment with timolol causes a decrease in tear secretion in children, which can further generate keratoepiteliopathy. Therefore, we suggest application of the artificial tears in these patients. [Abstract/Link to Full Text]

Wiero?ska JM, Stachowicz K, K?odzi?ska A, Rmia?owska M, Pilc A
Intraamygdaloid administration of BIBO 3304 increases water intake and extends anxiolytic effects.
Pol J Pharmacol. 2004 Nov-Dec;56(6):867-70.
The present study was designed to evaluate the effects of BIBO 3304 in the Vogel's conflict drinking test and in the water intake test in non-deprived rats after injection of the drug into the basolateral nucleus of the amygdaloid complex. BIBO 3304 was given at the doses of 25, 100 and 200 pmol/0.5 microl/site. We investigated also the effect of 5-hydroxytryptophan (5-HTP), given intraperitoneally at a dose of 20 mg/kg, which was used as a positive control in the water intake test. Water consumption was measured 1, 2, 4, 6 and 24 h after drug administration. We found that water intake was increased both after 5-HTP and BIBO 3304 administration. [Abstract/Link to Full Text]

Pa?ucha A, Bra?ski P, Pilc A
Selective mGlu5 receptor antagonist MTEP attenuates naloxone-induced morphine with-drawal symptoms.
Pol J Pharmacol. 2004 Nov-Dec;56(6):863-6.
Several lines of evidence suggest a crucial involvement of glutamate in the mechanism of drug addiction. The involvement of group I mGlu receptors in the mechanism of addiction has also been proposed. Given the recent discovery of selective and brain penetrable mGlu5 receptor antagonists, the effects of 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP) were evaluated in the naloxone-precipitated morphine withdrawal model. Experiments were performed on male C57BL/6J (20-25 g) mice. Mice were rendered morphine-dependent and withdrawal was precipitated with naloxone. Two hours and 15 min after the last dose of morphine, mice were injected with a mGlu5 receptor antagonist. MTEP (1-10 mg/kg) in a dose-dependent manner inhibited the naloxone-induced symptoms of morphine withdrawal in morphine-dependent mice, remaining without any effect on the locomotor activity of mice. The data suggest that selective mGlu5 receptor antagonists may play a role in the therapy of drug-dependence states. [Abstract/Link to Full Text]

Haduch A, W�jcikowski J, Daniel WA
Effects of chronic treatment with classic and newer antidepressants and neuroleptics on the activity and level of CYP2D in the rat brain.
Pol J Pharmacol. 2004 Nov-Dec;56(6):857-62.
The aim of the present work was to study the effect of chronic treatment with pharmacological doses of selected antidepressants (imipramine, mirtazapine) and neuroleptics (thioridazine, risperidone) on the activity and level of CYP2D in the rat brain. Our previous studies carried out on the liver showed that after chronic treatment with psychotropics, the activity of CYP2D was significantly decreased by imipramine, thioridazine and risperidone, but increased by mirtazapine. Our preliminary results suggest that the same may happen in the brain, where similar tendencies in changes in CYP2D activity were observed. Imipramine, thioridazine and risperidone diminished, while mirtazapine tended to accelerate the rate of ethylmorphine O-deethylation, a specific reaction for measurement of CYP2D activity. In the case of thioridazine, the observed decrease in the enzyme activity was the most pronounced and statistically significant. The level of brain CYP2D4 was not substantially changed by the prolonged administration of the investigated drugs (imipramine 136.3 +/- 14.9%, thioridazine 121.9 +/- 3.5%, risperidone 113.5 +/- 7.8%, mirtazapine 80.3 +/- 1.5% of the control), and did not correspond positively with the measured CYP2D activity. This may imply that the observed changes in the CYP2D activity were not caused by the involvement of those psychotropics in the regulation of CYP2D4. In conclusion, our preliminary results suggest that the effects of prolonged treatment with antidepressants and neuroleptics on the activity of CYP2D found in our previous study in the liver also occur in the brain, which may have an impact on the pharmacological and clinical profile of those drugs. [Abstract/Link to Full Text]

Rog�z Z, Skuza G, W�jcikowski J, Daniel WA, Wr�bel A, Dudek D, Zieba A
Effect of metyrapone supplementation on imipramine therapy in patients with treatment-resistant unipolar depression.
Pol J Pharmacol. 2004 Nov-Dec;56(6):849-55.
The paper describes the effect of metyrapone supplementation on imipramine therapy in patients (with treatment-resistant unipolar depression) who fulfilled DSM IV criteria for major depression. Nine patients were enrolled to the study on the basis of history of their illness and therapy. Following 2 weeks of washout period, the patients were treated with imipramine twice daily (100 mg/day) for 6 weeks, and then metyrapone was introduced (twice daily, 500 mg/day), and administered jointly with imipramine for further 6 weeks. Hamilton Depression Rating Scale (HDRS) and Beck Depression Inventory (BDI) were used to assess efficacy of antidepressant therapy. Imipramine changed neither HDRS nor BDI score after 6 weeks of treatment when compared with baseline (before treatment). Metyrapone supplementation significantly reduced both HDRS and BDI scores after 6-week supplementation. Moreover, pharmacokinetic data indicate that metyrapone did not influence significantly the plasma concentration of imipramine and its metabolite, desipramine in the patients during joint treatment with metyrapone and imipramine, what suggests the lack of pharmacokinetic interaction. This preliminary study is the first demonstration of the benefit of metyrapone supplementation in imipramine therapy of treatment-resistant unipolar depression and suggests that a change in the level of neurotransmitters, hormones and immunological parameters, which are disturbed in depression, may contribute to the mechanism of the action of this drug. [Abstract/Link to Full Text]

Kossakowski J, Raszkiewicz A, Bugno R, Bojarski AJ
Introduction of a new complex imide system into the structure of LCAPs. The synthesis and a 5-HT1A, 5-HT2A and D2 receptor binding study.
Pol J Pharmacol. 2004 Nov-Dec;56(6):843-8.
A series of 17 long-chain arylpiperazines containing bulky, complex imide systems (5,8-dimethyl-3b,9-epoxy-(3a,4,5,6,7,8,9,9a)-octahydro-1H-benzo[e]isoindole-1,3(2H)-dione or 4,9-diphenyl-4,9-epoxy-3a,4,9,9a-tetra-hydro-1H-benzo[f]isoindole-1,3(2H)-dione) was synthesized and evaluated for their affinity for serotonin 5-HT1A, 5-HT2A and dopamine D2 receptors. Most of the new compounds showed moderate activity at 5-HT1A binding sites (Ki = 100-492 nM), and two derivatives were found to have marked affinity for the 5-HT2A receptor subtype. None of the tested compounds displayed appreciable binding to dopamine D2 receptors Structure-activity relationships were discussed in respect to an arylpiperazine fragment, whereas the comparison of different imide terminals enabled determination of the size of a hydrophobic pocket (approximately 300 A3) within the 5-HT1A receptor. [Abstract/Link to Full Text]

Belowski D, Kowalski J, Madej A, Herman ZS
Influence of antidepressant drugs on macrophage cytotoxic activity in rats.
Pol J Pharmacol. 2004 Nov-Dec;56(6):837-42.
The aim of the study was to evaluate the in vivo and in vitro effects of antidepressant drugs on cytotoxic activity of rat spleen macrophages. In the in vivo experiment, rats were injected subcutaneously with two different doses (2 or 10 mg/kg) of desipramine, fluvoxamine and fluoxetine. The drugs were given once, for 2, 4 or 8 weeks. In the in vitro experiment, spleen macrophages were cultured with three different concentrations of desipramine (3.75, 0.75, or 0.075 mM), fluvoxamine (3.14, 0.62, or 0.062 mM), and fluoxetine (3.23, 0.64, or 0.064 mM) for 72 h. The cytotoxic activity of macrophages was evaluated by measuring the lysis of ((51)Cr) chromate-labelled P-815 target cells. In the in vivo experiment, a single dose of fluvoxamine (2 and 10 mg/kg) and fluoxetine (10 mg/kg) significantly decreased macrophage cytotoxic activity. Fluvoxamine (2 and 10 mg/kg), fluoxetine (10 mg/kg) and desipramine (10 mg/kg) administrated for 14 days also decreased macrophage cytotoxic activity. Twenty-eight day treatment with desipramine (2 and 10 mg/kg) decreased macrophage cytotoxic activity. Desipramine, fluvoxamine and fluoxetine given for 56 days did not affect macrophage cytotoxic activity. In the in vitro experiment, antidepressant drugs did not affect the cytotoxic activity of macrophages. The results of the study indicate that the effects of antidepressant drugs on macrophage cytotoxic activity depend on the drug type, dose and duration of the treatment. [Abstract/Link to Full Text]

Kreiner G, Bielawski A, Zelek-Molik A, Kowalska M, Nalepa I
Chronic treatment with citalopram does not affect the expression of alpha1-adrenergic receptor (alpha1-AR) subtypes.
Pol J Pharmacol. 2004 Nov-Dec;56(6):831-6.
We previously reported that chronic treatment with imipramine and electroconvulsive shock up-regulate the density and alpha1A-adrenergic receptor (alpha1A-AR) mRNA level in the rat prefrontal cortex, while the expression of the alpha1B subtype was unchanged. The present study examined whether repeatedly given citalopram, a selective serotonin reuptake inhibitor, induces any changes in the expression of alpha1A and alpha1B subtypes of alpha1-AR. The receptors density was assessed in the rat cerebral cortex by [3H]prazosin binding while the expression of alpha1A and alpha1B receptors' mRNA was measured in the rat prefrontal cortex by Northern blot analysis or competitive reverse transcription and polymerase chain reaction (RT-PCR), respectively. We did not find any changes in alpha1A- and alpha1B-AR density or mRNA expression in the investigated rat brain structures of citalopram-treated rats. Thus, it seems that up-regulation of alpha1A-AR subtype is characteristic only of those antidepressant agents in which a noradrenergic component is involved in their pharmacological mechanism of action. [Abstract/Link to Full Text]

Grabowska M, Schlegel-Zawadzka M, Papp M, Nowak G
Effect of imipramine treatment on plasma dopamine beta-hydroxylase activity in chronic mild stress in rats.
Pol J Pharmacol. 2004 Nov-Dec;56(6):825-9.
Dopamine beta-hydroxylase (DBH) which catalyzes conversion of dopamine into noradrenaline, may be a good blood marker of unipolar depression. Therefore, we studied the effect of classic antidepressant drug imipramine (10 mg/kg ip) on activity of this enzyme in plasma of rats subjected to chronic mild stress (CMS), the model of anhedonia. CMS induced reductions in DBH activity by the second day and 5th week of stress duration. Imipramine treatment minimized these CMS-induced reductions. The data indicate that, similarly to human depression, CMS also affects DBH activity, and, moreover, the CMS-induced alterations are normalized by imipramine treatment. [Abstract/Link to Full Text]

Rliwi?ski L, Janiec W, Pytlik M, Folwarczna J, Kaczmarczyk-Sedlak I, Pytlik W, Cegie?a U, Nowi?ska B
Effect of administration of alendronate sodium and retinol on the mechanical properties of the femur in ovariectomized rats.
Pol J Pharmacol. 2004 Nov-Dec;56(6):817-24.
Alendronate sodium, an aminobisphosphonate with potent antiresorptive activity, is used in the treatment of postmenopausal osteoporosis. Retinol, as a component of multivitamin preparations, is frequently used especially by elderly people. There are no reports on the interaction of alendronate sodium and retinol. The aim of the present study was to investigate the effect of administration of alendronate sodium and retinol on mechanical properties of the femoral bone in bilaterally ovariectomized rats. The experiments were carried out on 3-month-old Wistar rats, divided into 7 groups: I - sham-operated control rats, II - ovariectomized control rats, III - ovariectomy + alendronate sodium 3 mg/kg po, IV - ovariectomy + retinol 700 IU/kg po, V - ovariectomy + retinol 3500 IU/kg po, VI - ovariectomy + alendronate sodium 3 mg/kg po + retinol 700 IU/kg po, VII - ovariectomy + alendronate sodium 3 mg/kg po + retinol 3500 IU/kg po. The drugs were administered to the rats daily by oral gavage for 28 days. Body mass gain, bone mass, bone mineral content and calcium content in the femur and L-4 vertebra and mechanical properties of the whole femur (extrinsic stiffness, ultimate load, breaking load, deformation caused by the ultimate load) and the neck of the femur (load at fracture), were examined. Bilateral ovariectomy induced osteopenic changes in the rat skeletal system. Alendronate sodium (3 mg/kg po) counteracted the development of osteopenia induced by ovariectomy. Retinol at both used doses unfavorably affected the examined bone parameters of ovariectomized rats. Retinol administered with alendronate sodium lessened the preventive action of alendronate on the development of osteopenic changes in the skeletal system of ovariectomized rats. [Abstract/Link to Full Text]

Cegie?a U, Pytlik M, Janiec W
Changes and their regression in the osseous system in rats after administering a cytostatic drug inhibiting tumor cell division in the phase of DNA synthesis.
Pol J Pharmacol. 2004 Nov-Dec;56(6):805-16.
Chemotherapeutic drugs may disturb the bone tissue metabolism and cause osteopenia, however, the pathomechanism of the damaging effect of cytostatics on this tissue has not been well recognized so far. The detrimental effect may result from a direct cytotoxic action of these drugs on cells remodeling the bone, or on osteogenic cells present in the bone and in the bone marrow, or may be the result of hormonal disorder caused by impaired function of gonads. The aim of this study was to investigate the in vivo effect of 5-fluorouracil (5-FU), a cytostatic agent which inhibits tumor cell division in the phase of DNA synthesis, on the bone remodeling in rats and to examine whether the period of 4 weeks was sufficient for regression of changes elicited by administering 5-FU. Changes in the bone tissue following administration of 5-FU and their regression were evaluated by assessing macrometric and histomorphometric parameters as well as of mechanical properties of the femur. The tests were carried out on male Wistar rats. 5-FU was administered at the doses: 30 mg/kg per os (po) daily for 5 days every 2 weeks; 15 mg/kg im daily for 5 days every 2 weeks; 65 mg/kg im once weekly. Changes in the osseous tissue were examined 4 weeks after the first dose of 5-FU administration. Regression of the changes was examined 8 weeks after the first dose of 5-FU administration (the 5-FU was not administered between 30th and 57th day after the first dose of 5-FU administration). As a result of our research, it was established that 5-FU disturbed the bone remodeling processes in rats, mostly by impairing the process of new bone matrix synthesis, which leads to impaired mineralization process and decreased mechanical endurance of the femur. It was also established that the period of 4 weeks was not sufficient for regression of the changes in the osseous tissue caused by 5-FU administration. [Abstract/Link to Full Text]

S�leyman H, Demircan B, G��er F, Halici Z, Hacim�ft�o?lu A
Role of adrenal gland hormones in the mechanism of antiulcer action of nimesulide and ranitidine.
Pol J Pharmacol. 2004 Nov-Dec;56(6):799-804.
In the present study, we investigated whether the antiulcer effects of nimesulide (100 mg kg(-1) and ranitidine (150 mg kg(-1) were dependent on the adrenal cortex hormones. The antiulcer effects of nimesulide and ranitidine were examined in the indomethacin-induced gastric ulcer model in rats (first experiment). The mean ulcer areas in the control and ranitidine-treated groups were 11.1 +/- 3.18, 1.4 +/- 1.11 mm2, respectively. There was not any gastric damage in nimesulide-treated group. The mean ulcer area of control group (second experiment) administered metyrapone and indomethacin was 11.8 +/- 9.9, and it measured 2.0 +/- 1.41 mm2 in ranitidine-given group, while gastric damage was not observed in nimesulide-administered group. In adrenalectomized and indomethacin-treated rats (third experiment), the mean ulcer area was 17.9 +/- 11.5 mm2 in the nimesulide group, gastric ulcer was not seen in ranitidine group. In adrenalectomized rats (fourth experiment), the mean ulcer areas were 29 +/- 14.3, 23 +/- 11.2 and 1.3 +/- 2.4 mm2 in control group given indomethacin, only nimesulide or indomethacin + ranitidine, respectively. The obtained results indicated that adrenal cortex hormones played a role in antiulcer effect of nimesulide, but not ranitidine. [Abstract/Link to Full Text]

Kuzner J, Drevensek G, Gersak B, Budihna M
Hypoxic and pharmacological preconditioning preserves vasomotor response of porcine coronary artery.
Pol J Pharmacol. 2004 Nov-Dec;56(6):789-97.
Vasomotor response of the coronary artery depends on both endothelial and smooth muscle cells. Response is altered by hypoxia-reoxygenation-induced damages. Hypoxic preconditioning and pharmacological preconditioning as well can prevent these alterations. We compared the effectiveness of both types of preconditioning against hypoxia-reoxygenation-induced changes in vasomotor response of the isolated artery. Porcine arterial rings (3-4 mm wide) were cut from the left anterior descending porcine coronary artery and placed in Krebs-Henseleit solution. In order to obtain control response of the arteries, we contracted arterial rings with 20 mM KCl before ("standard contraction") and after 60-min hypoxia and 30-min reoxygenation. In other groups, nitric oxide-synthase and cyclooxygenase were inhibited. Then, the rings were pre-contracted with U46619 and relaxed by cumulative addition of the substance P. Contractions and relaxations of non-preconditioned and hypoxically or pharmacologically preconditioned rings were compared. Hypoxic preconditioning was performed by two periods of 5-min hypoxia and 10-min reoxygenation. For pharmacological preconditioning, we used application of adenosine, adrenaline, acetylcholine and angiotensin II. Analysis was performed with one-way ANOVA, followed by Dunnett's Multiple Comparison Test. After hypoxia-reoxygenation, in non-preconditioned rings KCl-induced contractions were significantly increased compared to standard contraction. Relaxations of hypoxically and pharmacologically preconditioned rings (expressed as percentages of U46619-induced pre-contraction) were significantly decreased (p < 0.01) compared to hypoxic but not to normoxic rings. Hypoxic and pharmacological preconditioning may preserve contraction and endothelium-dependent relaxation of porcine coronary artery after long-lasting hypoxia-reoxygenation. [Abstract/Link to Full Text]

Okopie? B, Krysiak R, Madej A, Belowski D, Zieli?ski M, Kowalski J, Herman ZS
Effect of simvastatin and fluvastatin on plasma fibrinogen levels in patients with primary hypercholesterolemia.
Pol J Pharmacol. 2004 Nov-Dec;56(6):781-7.
The aim of the study was to compare the effect of treatment with two different statins on plasma fibrinogen levels in patients with primary isolated hypercholesterolemia. Sixty three patients enrolled into the study were randomly divided into two groups, treated with simvastatin (20 mg/d) or fluvastatin (40 mg/d), respectively. Plasma lipid profile and fibrinogen levels were measured after 4 and 12 weeks of the therapy. Both drugs decreased total and LDL cholesterol and apoprotein B levels. Simvastatin additionally reduced triglyceride levels. After 4 weeks of treatment both drugs tended to increase plasma fibrinogen levels, while after 12 weeks fibrinogen level was significantly increased in the simvastatin-treated patients. The effect on fibrinogen did not correlate with their lipid-lowering potential, gender and was similar in patients positive and negative for anti-Helicobacter pylori or anti-Chlamydia pneumoniae antibodies. Our results support the findings about a relatively weak effect of statin therapy on plasma fibrinogen level and partially explain contradictory results of previous studies. [Abstract/Link to Full Text]

S�leyman H, Demircan B, Karag�z Y, Ozta?an N, S�leyman B
Anti-inflammatory effects of selective COX-2 inhibitors.
Pol J Pharmacol. 2004 Nov-Dec;56(6):775-80.
In this study, effects of rofecoxib, celecoxib, nimesulide on the acute phase of inflammation were studied in the carrageenan-induced paw edema model and their influence on the chronic phase of inflammation was evaluated in the cotton pellet granuloma tests. Additionally, effects of these drugs on capillary vascular permeability were examined in the hyaluronidase test and were compared with that of indomethacin (nonselective COX inhibitor). The results of the study demonstrated that rofecoxib, celecoxib, nimesulide, indomethacin at a dose of 10 mg kg(-1) reduced the volume of paw edema by 40.6% (p < 0.05), 21.6% (p < 0.05), 20.3% (p < 0.05), 64.0% (p < 0.05), respectively. Anti-proliferative effect of rofecoxib was of 29%, while those of celecoxib and nimesulide were of 13.5 and 21.2%, respectively. Indomethacin had an anti-proliferative effect of 44.2%. When the drugs were given at a dose of 25 mg kg(-1) rofecoxib, celecoxib, nimesulide reduced carrageenan-induced paw edema by 50.6% (p < 0.004), 27.9% (p < 0.004) and 33.0% (p < 0.004), respectively. Positive control, indomethacin, reduced the paw edema by 86.1% (p < 0.004). As a result, indomethacin, rofecoxib, celecoxib, nimesulide significantly inhibited both acute and chronic inflammation. While indomethacin, celecoxib, nimesulide significantly reduced capillary vascular permeability, the effect of rofecoxib was insignificant. We could not clarify this observation. Further studies are required to enlighten this effect of rofecoxib. [Abstract/Link to Full Text]

Kozlovski VI, Vdovichenko VP, Chlopicki S, Malchik SS, Praliyev KD, Zcilkibayev OT
Antiarrhythmic profile and endothelial action of novel decahydroquinoline derivatives.
Pol J Pharmacol. 2004 Nov-Dec;56(6):767-74.
We tested antiarrhythmic and endothelial action of novel decahydroquinoline derivatives. Antiarrhythmic activity was analyzed using models of aconitine-, calcium chloride-, and adrenaline-induced arrhythmias in rats. Potency to induce nitric oxide (NO)-dependent coronary vasodilation was assessed in isolated guinea pig heart perfused according to Langendorff technique. Among 15 novel decahydroquinoline derivatives (D1-15), four of them displayed antiarrhythmic activity (D12-D15). D12-D15 compounds were more active in the model of aconitine-induced arrhythmias than in calcium chloride-induced arrhythmias and were inactive in the model of adrenaline-induced arrhythmias. Profile of antiarrhythmic activity of D12-D15 compounds was similar to that of quinidine and procainamide. Interestingly, in the isolated guinea pig heart D14 and D15 (10(-5) M) induced coronary vasodilation, that was mediated by endothelium-derived NO. In conclusion, novel decahydroquinoline derivatives described here (D12-D15) show antiarrhythmic activity typical of antiarrhythmic drugs of class I. Importantly, some of these compounds (D14, D15) release NO from coronary endothelium, which may provide an additional therapeutic benefit. [Abstract/Link to Full Text]

Ch?o?-Rzepa G, Paw?owski M, Zygmunt M, Filipek B, Maciag D
Synthesis and cardiovascular activity of new 8-alkylamino-1,3-dimethyl-7-(2-hydroxy-3- piperazinopropyl)-3,7-dihydro-1H-purine-2,6-diones.
Pol J Pharmacol. 2004 Nov-Dec;56(6):755-66.
7-{2-Hydroxy-3-[4-(2-phenoxyethyl)-piperazinyl-1-yl]-propyl}-1,3-di-methyl-3,7-dihydro-1H-purine-2,6-dione dihydrochloride (2), and several of its 8-alkylamino substituted derivatives (11-17) were synthesized and tested for electrocardiographic, antiarrhythmic and hypotensive activity. Also their alpha(1)- and alpha(2)-adrenoreceptor affinities were determined. It was found that compound 2, and its analogue 15 with 8-(2-morpholin-4-yl-ethylamino) substituent displayed a strong prophylactic antiarrhythmic activity in experimentally induced arrhythmia (LD50/ED50 = 54.9 and 55.0, respectively). The hypotensive activity was observed for 8-benzylamino (11) or 8-(pyridin-2-yl-methylamino) (12) analogues. All the new derivatives (11-17) and 2 showed a weak affinity for alpha1-(Ki = 0.225-1.400 microM) and alpha2-(Ki = 0.152-4.299 microM) receptors. [Abstract/Link to Full Text]

Paluchowska MH, Charakchieva-Minol S, Tatarczy?ska E, K?odzi?ska A, Stachowicz K, Chojnacka-W�jcik E
New 4-[omega-(diarylmethylamino)alkyl]- and 4-[omega-(diarylmethoxy)alkyl]-1-arylpiperazines as selective 5-HT1A/5-HT2A receptor ligands with differentiated in vivo activity.
Pol J Pharmacol. 2004 Nov-Dec;56(6):743-54.
Two series of novel 4-ethyl- or 4-propyl-1-arylpiperazines (5-12) with the 4,4'-disubstituted diphenylmethylamino (series a) or the diphenylmethoxy (series b) terminal fragment were synthesized and evaluated for their binding affinity at 5-HT1A and 5-HT2A receptors. The influence of the introduction of 4-methyl, 4-chloro or 4-fluoro substituents at both phenyl rings of that terminal moiety on in vitro and in vivo 5-HT1A receptor activity of those modified compounds was discussed. Compounds 5a, 6a, 9a-12a, 5b, 6b, 9b, 11b and 12b displayed high to fairly high affinity for 5-HT1A receptors (Ki = 2.4-72 nM). Compounds of both series showed low or very low 5-HT2A receptor affinity (Ki = 155-5400 nM). Amines 5a, 6a, 11a, and their ether analogs 5b, 6b and 11b, also possessed high or moderate alpha(1)-adrenoceptor affinity (K(i) = 6-104 nM). The functional activity of compounds 5a, 6a, 9a-12a, 5b, 8b, 9b, 11b and 12b was tested in vivo in the commonly used animal models. The majority of those ligands behaved like 5-HT1A receptor antagonists, their influence on the pre- and/or postsynaptic sites being diverse, though. They exhibited characteristics of partial agonists of postsynaptic 5-HT1A receptors (11a), of weak antagonists of pre- and postsynaptic sites (12a, 9b), of antagonists of presynaptic (5a) or of antagonists of postsynaptic 5-HT(1A) receptors (9a, 10a, 5b, 8b, 11b and 12b) while, 6a was devoid of functional activity at those receptors. The above findings indicate that introduction of 4-methyl, 4-chloro or 4-fluoro substituents to the diphenylmethyl part of the 1-(2-methoxyphenyl)piperazines tested in vivo may modify their 5-HT1A receptor functional activity. [Abstract/Link to Full Text]

Rog�z Z, Dziedzicka-Wasylewska M, Daniel WA, W�jcikowski J, Dudek D, Wr�bel A, Zieba A
Effects of joint administration of imipramine and amantadine in patients with drug-resistant unipolar depression.
Pol J Pharmacol. 2004 Nov-Dec;56(6):735-42.
The paper describes the effect of amantadine (AMA) supplementation on imipramine (IMI) therapy in patients (with treatment-resistant unipolar depression) who fulfilled DSM IV criteria for major depression. Twelve patients were enrolled to the study on the basis of history of their illness and therapy. Following 2 weeks of washout period, the patients were treated with IMI twice daily (100-150 mg/day) for 6 weeks, and then AMA was introduced (twice daily, 100-150 mg/day) and administered jointly with IMI for further 6 weeks. Thereafter, AMA was withdrawn, and the patients were treated with IMI alone for 2 weeks. Hamilton Depression Rating Scale (HDRS) and Beck Depression Inventory (BDI) were used to assess efficacy of antidepressant therapy. IMI changed neither HDRS nor BDI score after 3 or 6 weeks of treatment when compared with washout (before treatment). AMA supplementation significantly reduced both HDRS and BDI scores after 3- or 6-week supplementation. AMA augmentation of IMI treatment was beneficial and lasted even after AMA withdrawal. Moreover, pharmacokinetic data indicate that AMA did not influence significantly the plasma concentration of the IMI and its metabolite, desipramine, in the patients during joint treatment with AMA and IMI, what suggests the lack of pharmacokinetic interaction. These results suggest that joint therapy with IMI and AMA may be successful in the treatment-resistant unipolar depression. [Abstract/Link to Full Text]

Patsenka A, Antkiewicz-Michaluk L
Inhibition of rodent brain monoamine oxidase and tyrosine hydroxylase by endogenous compounds - 1,2,3,4-tetrahydro-isoquinoline alkaloids.
Pol J Pharmacol. 2004 Nov-Dec;56(6):727-34.
Four different noncatecholic and one catecholic tetrahydroisoquinolines (TIQs), cyclic condensation derivatives of beta-phenylethylamine and dopamine with aldehydes or keto acids, were examined for the inhibition of rat and mouse brain monoamine oxidase (MAO) and rat striatum tyrosine hydroxylase (TH) activity. Simple noncatecholic TIQs were found to act as moderate (TIQ, N-methyl-TIQ, 1-methyl-TIQ) or weak (1-benzyl-TIQ), MAO B and MAO A inhibitors. 1-Methyl-TIQ inhibited more potently MAO-A than MAO-B; the similar but more modest effect was exerted by salsolinol. Only salsolinol markedly inhibited TH activity, being competitive with the enzyme biopterin cofactor. The inhibition of MAO and TH by TIQs is discussed in relation to their ability to regulate monoamine metabolism. [Abstract/Link to Full Text]

Talarek S, Fidecka S
Involvement of nitricoxidergic system in the hypnotic effects of benzodiazepines in mice.
Pol J Pharmacol. 2004 Nov-Dec;56(6):719-26.
The influence of nitric oxide (NO) on hypnotic activity of diazepam, chlordiazepoxide and clonazepam was studied in mice. Administration of both non-selective NO synthase inhibitors: N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-nitro-L-arginine (L-NOARG) and selective NO synthase inhibitor 7-nitroindazole (7-NI) resulted in significant increase in the duration of diazepam-, chlordiazepoxide- and clonazepam-induced sleep. The effects of co-administration of the examined inhibitors with benzodiazepines were not changed by L-arginine, a substrate for NO formation. Administration of L-arginine alone had no effect on the duration of sleep induced by benzodiazepines. Methylene blue, the guanyl cyclase inhibitor, was able to increase the duration of benzodiazepine-induced sleep. These findings suggest that the cGMP/NO system may participate in hypnotic effects of benzodiazepines. [Abstract/Link to Full Text]

Rmia?owska M, Wiero?ska JM, Bra?ski P, Obuchowicz E, Pilc A
MPEP, mGlu5 receptor antagonist, regulates NPYmRNA expression in hippocampal and amygdalar neurons.
Pol J Pharmacol. 2004 Nov-Dec;56(6):709-18.
Our earlier studies showed that the metabotropic glutamate receptor 5 (mGluR5) antagonist, MPEP, could regulate neuropeptide Y (NPY) neurons in the amygdala, influencing both peptide expression and its antianxiety effects. Two brain structures are particularly engaged in a regulation of anxiety, namely the amygdala and also the hippocampus. They both belong to the limbic system and contain NPY neurons and mGlu5 receptors. Therefore, in the present study, we examined the effect of MPEP on NPY and NPYmRNA expression in the amygdala and the hippocampus of the rat brain. NPY expression was studied by immunohistochemical method, and radioimmunoassay, and the NPY synthesis was examined using NPYmRNA in situ hybridization. Immunohistochemical localization of mGluR5 was also carried out. It was found that MPEP given 3 times every 8 h potently decreased NPYmRNA expression 30 min after the last dose in both those structures (to 8-20% of the control level). After single MPEP treatment, we did not observe any changes in NPYmRNA level in the hippocampus, and its decrease in the amygdala 6 h after MPEP administration. The obtained results suggest a positive regulatory control of NPY synthesis by mGlu5 receptors in hippocampal and amygdalar neurons. [Abstract/Link to Full Text]

W�jcikowski J
Potential role of the brain dopaminergic system in the regulation of cytochrome P-450 expression.
Pol J Pharmacol. 2004 Nov-Dec;56(6):701-8.
Although there are no literature data, which would clearly describe the role of catecholaminergic systems (including dopaminergic system) in the expression of the liver cytochrome P-450, there are well documented reports on physiological regulation of cytochrome P-450 by endogenous hormones (glucocorticoids, sex hormones, thyroid hormones, growth hormone, insulin) and by the immune system (cytokines), and all these factors remain under central nervous system control. Therefore, one can expect that dysfunction of catecholaminergic systems may lead to significant changes in activities of particular cytochrome P-450 isoenzymes by altering levels of endogenous hormones. The mechanism underlying dopamine-induced regulation of the liver cytochrome P-450 expression seems not to be direct, but rather mediated by pituitary hormones and cytokines. [Abstract/Link to Full Text]

Kajta M
Apoptosis in the central nervous system: Mechanisms and protective strategies.
Pol J Pharmacol. 2004 Nov-Dec;56(6):689-700.
Naturally occurring neuronal cell death, which is essential for normal development, tissue homeostasis and as a defence against pathogens, is generally considered to be apoptotic. Apoptosis, called also a programmed cell death, though the meanings of these terms are not identical, is a common phenomenon induced either by specific insults mediated through so-called "death receptors" (external pathway) or by non-specific insults leading to reduction of mitochondrial potential (internal pathway). At the molecular level, apoptosis is regulated by the activation of the caspase cascade, which depends either upon the participation of mitochondria and the activation of procaspase-9 or on the interaction of a death receptor with its ligand and subsequent activation of procaspase-8. Considering various mechanisms of programmed cell death, either during neural development or neurodegeneration, finding the most suitable and effective protective strategy needs evaluation of risk-to-benefit ratio. Unlike malignant tissues, neuronal cells have to be protected, but only if the protection does not disturb natural cell elimination. Defining molecular mechanisms is, therefore, the necessary step preceding the adaptation of new protective and then, possibly, therapeutic strategies. At present, the most promising are discoveries related to caspases, JNK, and GSK-3beta and their crucial role in stress-dependent and spontaneous apoptosis. Inhibitors of caspases and JNKs succeed in defending cells against kainate-, beta-amyloid- or MPTP-induced neuronal cell death. Among protective agents those, which easily cross the blood-brain barrier and exert profound neuroprotective action without side effects, seem to be the most important. [Abstract/Link to Full Text]

Ma?kowiak M, Chocyk A, Markowicz-Kula K, Wedzony K
Neurogenesis in the adult brain.
Pol J Pharmacol. 2004 Nov-Dec;56(6):673-87.
Neurogenesis is a process that involves cell proliferation, migration and differentiation. Adult neurogenesis has been discovered by Altman in the mid 1960s. It is known now that neurogenesis occurs in two main neurogenic areas of the adult mammalian brain: the olfactory bulb and the hippocampal dentate gyrus, although other brain regions, such as cortex or substantia nigra cannot be excluded. The rate of neurogenesis can be regulated in a positive and negative manner by several factors like, age, growth factors, hormones, environmental or pharmacological stimuli. Functional significance of adult neurogenesis is still under investigation, however, several evidences suggest involvement of newly generated neurons in cognitive processes. There are also several findings indicating that the impairment of adult neurogenesis may be involved in the pathophysiology of some brain diseases, like depression, epilepsy, ischemia or neurodegenerative disorders. It appears that alterations in the rate of neurogenesis may have important functional and therapeutic implications. [Abstract/Link to Full Text]

Dziedzicka-Wasylewska M
Brain dopamine receptors--research perspectives and potential sites of regulation.
Pol J Pharmacol. 2004 Nov-Dec;56(6):659-71.
Dopamine mediates its biological actions via at least five distinct G protein-coupled receptors. Recently, significant progress has been made in the understanding of the molecular and cellular consequences of activation of the different dopamine receptors. Not only their anatomical localization has been revisited, what implicates new insights into well known D1/D2 receptor synergism, but also evidence has been provided that dopamine receptors can physically interact with each other as well as with other neurotransmitter receptors. Dopamine receptors are subject to phosphorylation by G protein-coupled receptor kinases, the specificity of which has started to be revealed. Some progress has been made in elucidating the functional significance of polymorphisms observed in genes encoding dopamine receptor. All these new findings need to be appreciated by the psychopharmacologists in order to better understand the behavioral consequences of activation of dopamine receptors. [Abstract/Link to Full Text]

Abstracts of the XVth International Congress of the Polish Pharmacological Society. Poznan, Poland, September 12-14, 2004.
Pol J Pharmacol. 2004 Sep;56 Suppl1-259. [Abstract/Link to Full Text]

Zambrzycka A
Aging decreases phosphatidylinositol-4,5-bisphosphate level but has no effect on activities of phosphoinositide kinases.
Pol J Pharmacol. 2004 Sep-Oct;56(5):651-4.
Phosphoinositides play an important role in cell signaling and cytoskeleton function. In this study, we investigate the effect of brain aging on phosphatidylinositol-4-monophosphate (PI(4)P) and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) synthesis by PI 4-kinase (PI4K) and PI(4)P 5-kinase (PIP5K) in cortical synaptic plasma membranes in the presence of endogenous and exogenous substrates and gamma-[(32)P]ATP. Our data demonstrated that aging decreases PI(4,5)P(2) formation by 40% in the presence of endogenous substrate, but had no effect on PI4K and PIP5K activities. One can hypothesize that aging decreases PI(4,5)P(2) level probably by enhancement of polyphosphoinositides degradation. Alteration of PI(4,5)P(2) concentration may disturb cellular signaling and cytoskeleton dynamics. [Abstract/Link to Full Text]

Chalimoniuk M, Stepie? A
Influence of the therapy with pergolide mesylate plus L-DOPA and with L-DOPA alone on serum cGMP level in PD patients.
Pol J Pharmacol. 2004 Sep-Oct;56(5):647-50.
The aim of our study was to investigate serum cGMP level in patients treated with L-DOPA alone and L-DOPA with pergolide mesylate (PM) and to compare the obtained values with age-matched healthy subjects. PD patients treated with L-DOPA or with PM + L-DOPA had significantly higher cGMP levels in serum as compared to control. Moreover, cGMP level was significantly higher after treatment with PM + L-DOPA then L-DOPA alone. We did not observed significant differences in UPDRS scores between both PD groups. Our data suggest that changes in serum cGMP level in PD patients are related with PM + L-DOPA or L-DOPA alone therapy. [Abstract/Link to Full Text]

Recent Articles in Acta Pharmacologica Sinica

Shen LL, Liu GX, Tang Y
Molecular docking and 3D-QSAR studies of 2-substituted 1-indanone derivatives as acetylcholinesterase inhibitors.
Acta Pharmacol Sin. 2007 Dec;28(12):2053-63.
Aim: To explore the binding mode of 2-substituted 1-indanone derivatives with acetylcholinesterase (AChE) and provide hints for the future design of new derivatives with higher potency and specificity. Methods: The GOLD-docking conformations of the compounds in the active site of the enzyme were used in subsequent studies. The highly reliable and predictive three-dimensional quantitative structure-activity relationship (3D-QSAR) models were achieved by comparative molecular field analysis (CoMFA) and comparative molecular similarity analysis (CoMSIA) methods. The predictive capabilities of the models were validated by an external test set. Moreover, the stabilities of the 3D-QSAR models were verified by the leave-4-out cross-validation method. Results: The CoMFA and CoMSIA models were constructed successfully with a good cross-validated coefficient (q(2)) and a non-cross-validated coefficient (r(2)). The q(2)and r(2)obtained from the leave-1-out cross validation method were 0.784 and 0.974 in the CoMFA model and 0.736 and 0.947 in the CoMSIA model, respectively. The coefficient isocontour maps obtained from these models were compatible with the geometrical and physicochemical properties of AChE. Conclusion: The contour map demonstrated that the binding affinity could be enhanced when the small protonated nitrogen moiety was replaced by a more hydrophobic and bulky group with a highly partial positive charge. The present study provides a better understanding of the interaction between the inhibitors and AChE, which is helpful for the discovery of new compounds with more potency and selective activity. [Abstract/Link to Full Text]

Li XH, Zou HJ, Wu AH, Ye YL, Shen JH
Structure-based drug design of a novel family of chalcones as PPARalpha agonists: virtual screening, synthesis, and biological activities in vitro.
Acta Pharmacol Sin. 2007 Dec;28(12):2040-52.
Aim: To design and synthesize a novel class of peroxisome proliferator-activated receptors (PPAR)alpha agonists, which is obtained by the combination of the classical fibrate phead groupq, a linker with appropriate length and a chalcone. Methods: Thirty seven compounds were designed and identified employing the virtual screening approach. Six compounds were then selected for synthesis and bioassay according to the virtual screening results, structural similarity, and synthetic complexity. Results: Six new compounds (4b and 4d-h) were synthesized and bioassayed. All were found to be potent PPARalpha agonists, compound 4 h being the most prominent with a 50% effective concentration value of 0.06 mumol/L. Conclusion: This study provides a promising novel family of chalcones with a potential hypolipidemic effect. [Abstract/Link to Full Text]

Liu Q, Zhang YY, Lu HL, Li QY, Zhou CH, Wang MW
Rhodanine derivatives as novel peroxisome proliferator-activated receptor gamma agonists.
Acta Pharmacol Sin. 2007 Dec;28(12):2033-9.
Aim: To characterize the in vitro bioactivities of rhodanine derivatives as novel peroxisome proliferator-activated receptor (PPAR) gamma modulators, based on a hit (SH00012671) identified during high-throughput screening (HTS) of a diverse synthetic compound library, and to preliminarily elucidate the structure-activity relationship of this class of PPARgamma agonists. Methods: Full-length PPARgamma and retinoid X receptor alpha (RXRalpha), biotinylated PPAR response element (PPRE), [3H]BRL49653 (rosiglitazone), and streptavidin-coated FlashPlate or microbeads were used to measure the receptor-binding properties of various compounds based on the scintillation proximity assay (SPA) technology. A recombinant PPRE vector was transiently cotransfected with PPARgamma and RXRalpha plasmids into the African green monkey kidney (CV-1) cells, and the effects of BRL49653 and test compounds on transcription mediated by PPARgamma were determined by examining luciferase (reporter) responses. 3T3-L1 cells were employed to determine whether the compounds facilitated adipogenesis upon PPARgamma activation. Results: Of the 16 000 samples screened with the SPA method, only 1 compound (SH00012671) displayed a similar binding affinity (Ki=186.7 nmol/L) to PPARgamma as BRL49653, but it was inactive in the cell-based assays. A series of rhodanine derivatives were synthesized based on the core structure of SH00012671 and 8 of them showed agonist activities in both cotransfection and pre-adipocyte differentiation assays. To reduce intrinsic cytotoxicities, the sulphur on the rhodanine was changed to oxygen. This alteration led to a decrease in receptor-binding affinities while modified analogues generally maintained agonist efficacies in the cell-based assays. Of the analogues studied, compound 31 exhibited about 70% the efficacy exerted by BRL49653 in both cotransfection and pre-adipocyte differentiation assays. Conclusion: Through minor chemical modifications on the core structure of the initial HTS hit, SH00012671 was transformed to possess both molecular (PPARgamma binding) and cellular (adipogenesis) activities. The rhodanine derivatives reported here may represent a new scaffold in further understanding the molecular mechanism of agonism at PPARgamma. [Abstract/Link to Full Text]

Chen YC, Chen KT
Novel selective inhibitors of hydroxyxanthone derivatives for human cyclooxygenase-2.
Acta Pharmacol Sin. 2007 Dec;28(12):2027-32.
Aim: To screen the selective inhibitors for human cyclooxygenase-2 ((h)COX-2) utilizing molecular simulation. Methods: Eight xanthone derivatives, compounds A-H, were employed by the structure-based research methodology. Resveratrol and NS-398 were selected as the control compounds for COX-1 and COX-2, respectively. The docking results were scored and the interaction energies of the complexes were calculated by CHARMm forcefield. Results: NS-398 could not dock into the active site of COX-1. However, resveratrol, the specific selective compound for COX-1, gained lower interaction energy while docked in COX-1. The lower interaction energies were investigated, while compound B and F were docked into the catalytic sites of COX-1 and COX-2, respectively. Compound A, 1,3,6,7-tetrahydroxyxanthone, revealed high inhibitory potency to both COX-1 and COX-2. Conclusion: The conformations of the docking would influence the values of interaction energies. The hydrogen bond could also increase the stabi-lity of the whole complex, which might suggest that compound B had a suitable conformation in the tunnel-like active site of COX-1. Compound F, a potent agent for COX-2, revealed a strong hydrogen bond with Ser516 in human COX-2 to form a stable complex. [Abstract/Link to Full Text]

Meng XX, Wan JQ, Jing M, Zhao SG, Cai W, Liu EZ
Specific targeting of gliomas with multifunctional superparamagnetic iron oxide nanoparticle optical and magnetic resonance imaging contrast agents.
Acta Pharmacol Sin. 2007 Dec;28(12):2019-26.
Aim: To determine whether glioma cells can be specifically and efficiently targeted by superparamagnetic iron oxide nanoparticle (SPIO)-fluorescein isothiocyanate (FITC)-chlorotoxin (SPIOFC) that is detectable by magnetic resonance imaging (MRI) and optical imaging. Methods: SPIOFC was synthesized by conjugating SPIO with FITC and chlorotoxin. Glioma cells (human U251-MG and rat C6) were cultured with SPIOFC and SPIOF (SPIO-FITC), respectively. Neural cells were treated with SPIOFC as the control for SPIOFC-targeted glioma cells. The internalization of SPIOFC by glioma cells was assessed by MRI and was quantified using inductively-coupled plasma emission spectroscopy. The optical imaging ability of SPIOFC was evaluated by confocal laser scanning microscopy. Results: Iron per cell of U251 (72.5+/-1.8 pg) and C6 (74.9+/-2.2 pg) cells cultured with SPIOFC were significantly more than those of U251 (6.6+/-1.0 pg) and C6 (7.1+/-0.8 pg) cells incubated with SPIOF. The T2 signal intensity of U251 and C6 cells cultured with SPIOFC (233.6+/-25.9 and 211.4+/-17.2, respectively) were substantially lower than those of U251 and C6 cells incubated with SPIOF (2275.3+/-268.6 and 2342.7+/-222.4, respectively). Moreover, there were significant differences in iron per cell and T2 signal intensity between SPIOFC-treated neural cells (1.3+/-0.3; 2533.6+/-199.2) and SPIOFC-treated glioma cells. SPIOFC internalized by glioma cells exhibited green fluorescence by confocal laser scanning microscopy. Conclusion: SPIOFC is suitable for the specific and efficient targeting of glioma cells. MRI and optical imaging in conjunction with SPIOFC can differentiate glioma cells from normal brain tissue cells. [Abstract/Link to Full Text]

Sun K, Wang Q, Huang XH, Zhen MC, Li W, Zhang LJ
Establishment of multiplexed, microsphere-based flow cytometric assay for multiple human tumor markers.
Acta Pharmacol Sin. 2007 Dec;28(12):2011-8.
Aim: The multiplexed, microsphere-based flow cytometric assay (MFCA) for multiple human tumor markers was established for the early screening and detection of suspected cancer patients. Methods: Covalent coupling of capture antibodies directed against their respective tumor markers to fluorescent microspheres was performed by following the protocols recommended by a commercial corporation with some modifications. The coupling efficiency and cross-reactivity were identified by the Luminex 100 system and associated software. The standard curve was constructed by using serial dilution of recombinant tumor marker standards and was validated by comparison with ELISA for quantifying the tumor markers in serum samples. Results: The identifications revealed that the coupling procedures were successful without non-specific cross-reactivity and the standard curve was highly efficient. However, it was necessary to ensure the quality control of the coupling process since slight variations in the coupling procedures could profoundly affect the density of capture reagents coupled to the microspheres and consequently adversely affect the assay precision. In addition to its multi-analyte capability, the MFCA system had definite advantages, such as higher reproducibility, greater dynamic range of measurement, and considerably less preparation time and labor over the conventional pgold standardq, which was the ELISA. Conclusion: The successful establishment of the MFCA system for the simultaneous detection of multiple tumor markers will provide the foundation for the further study of clinical applications. [Abstract/Link to Full Text]

Zhang XY, Li TT, Liu XJ
Detecting robust gene signature through integrated analysis of multiple types of high-throughput data in liver cancer.
Acta Pharmacol Sin. 2007 Dec;28(12):2005-10.
Aim: To investigate the robust gene signature in liver cancer, we applied an integrated approach to perform a joint analysis of a highly diverse collection of liver cancer genome-wide datasets, including genomic alterations and transcription profiles. Methods: 1-class Significance Analysis of Microarrays coupled with ranking score method were used to identify the robust gene signature in liver tumor tissue. Results: In total, 1 625 051 gene expression measurements from 16 public microarrays, 2 pairs of serial analyses of gene expression experiments, and 252 loss of heterozygosity reports obtained from 568 publications were used in this integrated study. The resulting robust gene signatures included 90 genes, which may be of great importance to liver cancer research. A system assessment analysis revealed that our integrative method had an accuracy of 92% and a correlation coefficient value of 0.88. Conclusion: The system assessment results indicated that our method had the ability of integrating the datasets from various types of sources, and eliciting more accurate results, as can be very useful in the study of liver cancer. [Abstract/Link to Full Text]

Pan QW, Zhong SY, Liu BS, Liu J, Cai R, Wang YG, Liu XY, Qian C
Enhanced sensitivity of hepatocellular carcinoma cells to chemotherapy with a Smac-armed oncolytic adenovirus.
Acta Pharmacol Sin. 2007 Dec;28(12):1996-2004.
Aim: The aim of the present study was to further improve the therapeutic effects for human hepatocellular carcinoma (HCC) and reduce the damage in normal cells using a novel chemo-gene-virotherapeutic strategy. Methods: An oncolytic adenoviral vector (ZD55) similar to the typical oncolytic adenovirus ONYX-015, with a deletion of E1B-55K gene, was employed to express the second mitochondria-derived activator of caspases (Smac) protein by constructing a recombinant virus ZD55-Smac. The enhanced cytotoxicity of the combined treatment of ZD55-Smac with cisplatin or 5-fluorouracil (5-FU) was evaluated in several HCC cell lines. Moreover, the negative effects on normal cells have been tested in human normal liver cell lines L-02 and QSG-7701 cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and apoptotic cell staining. Results: According to our observation, ZD55-Smac is superior to ONYX-015 in sensitizing chemotherapy, ZD55-Smac used in conjunction with chemotherapy was found to exhibit obviously enhanced cytotoxicity in HCC cells, yet significantly abolished the negative toxicity in normal cells by utilizing the tumor selective replication vector and reducing the dosage. Conclusion: This chemo-gene-virotherapeutic (cisplatin or 5-FU+ZD55-Smac) strategy is superior to the conventional chemo-gene or chemo-viro approach. [Abstract/Link to Full Text]

Guo JM, Xiao BX, Liu Q, Zhang S, Liu DH, Gong ZH
Anticancer effect of aloe-emodin on cervical cancer cells involves G2/M arrest and induction of differentiation.
Acta Pharmacol Sin. 2007 Dec;28(12):1991-5.
Aim: The aim of this study was to investigate the effects of aloe-emodin, a natural compound from the root and rhizome of Rheum palmatum, on the growth of human cervical cancer cells, HeLa. Methods: HeLa cells were treated with various concentrations of aloe-emodin for 1-5 d, and cell growth was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. The long-term growth effect was investigated by crystal violet assay. The distributions of the cell cycle and apoptosis were analyzed by flow cytometry. The alkaline phosphatase (ALP) activity was analyzed by a chemical analyzer. Finally, Western blotting was used to indicate the abundant changes of protein kinase C (PKC), c-myc, cyclins, cyclin-dependent kinases (CDK), and proliferating cell nuclear antigen (PCNA). Results: Aloe-emodin inhibited the growth of HeLa cells in a dose-dependent manner at concentrations ranging between 2.5 and 40 mumol/L. The flow cytometric analysis showed that HeLa cells were arrested at the G2/M phase. This effect was associated with the decrease in cyclin A and CDK2, and the increase in cyclin B1 and CDK1. More importantly, the ALP activity was found to be increased by aloe-emodin treatment, and accompanied by the inhibition of PCNA expression. In addition, aloe-emodin suppressed the expression of PKCalpha and c-myc. Conclusion: These findings provide a possible mechanistic explanation for the growth inhibitory effect of aloe-emodin on HeLa, which includes cell cycle arrest and inducing differentiation. [Abstract/Link to Full Text]

Guan TJ, Qin FJ, Du JH, Geng L, Zhang YY, Li M
AICAR inhibits proliferation and induced S-phase arrest, and promotes apoptosis in CaSki cells.
Acta Pharmacol Sin. 2007 Dec;28(12):1984-90.
Aim: The aim of the present study was to determine the effect of 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) on proliferation, cell cycle, and apoptosis in the human epithelial cervical cancer cell line CaSki cells. Methods: Cell count and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were used to determine cell proliferation and viability. Hoechst 33258 staining was conducted to distinguish the apoptotic cells. Cell cycle and Annexin-V/propidium iodide staining were analyzed by fluorescence-activated cell sorting (FACS). A Western blot assay was used to evaluate the expression of AKT (also known as protein kinase B), mammalian target of rapamycin (mTOR), p53, and extracellular signal-regulated kinase (ERK). Results: AICAR (500 mumol/L) significantly inhibi-ted the proliferation of CaSki cells treated for 24, 48, and 72 h as determined by cell count. The cells at the G1 and G2 phases were dramatically decreased while cells at the S phase were increased in response to AICAR treatment for 24, 48, and 72 h. The MTT assay showed less viable cells and Hoechst fluorescent staining showed more apoptotic cells upon AICAR stimulation. The results of the Annexin-V staining demonstrated a time-dependent increase of apoptosis in cells treated with AICAR for 24, 36, and 48 h. Furthermore, AICAR activated caspase-3 in a time-dependent manner. It was also found that AICAR inhibited the phosphorylation of AKT and mTOR, which are important kinases regulating cell growth and survival. AICAR stimulation obviously increased the expression of the tumor suppressor p53 and the phosphorylation of ERK. Conclusion: AICAR inhibited proliferation and induced S phase arrest and promoted apoptosis in CaSki cells, which might be mediated by the downregulation of the AKT/mTOR pathway and the upregulation of the p53/ERK pathway. [Abstract/Link to Full Text]

Yu JH, Cui Q, Jiang YY, Yang W, Tashiro S, Onodera S, Ikejima T
Pseudolaric acid B induces apoptosis, senescence, and mitotic arrest in human breast cancer MCF-7.
Acta Pharmacol Sin. 2007 Dec;28(12):1975-83.
Aim: The aim of the present study was to investigate the inhibitory effect of pseudolaric acid B (PAB) on human breast cancer MCF-7 cells. Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, morphological changes, acridine orange staining, and agarose gel electrophoresis were applied to detect apoptosis. The percentage of apoptotic and necrotic cells was calculated by the lactate dehydrogenase activity-based cytotoxicity assay; senescence associated (SA)-beta-galactosidase activity was detected to evaluate senescence; flow cytometric analysis of propidium iodide staining was carried out to investigate the distribution of cell cycle, and the protein expression was examined by Western blot analysis. Results: During apoptosis, the half maximal inhibitory concentration IC(50)was 3.4 and 1.35 mumol/L at 36 and 48 h after PAB treatment, respectively. The MCF-7 cells exposed to PAB showed typical characteristics of apoptosis, including the morphological changes and DNA fragmentation. The MCF-7 cells treated with 4 mumol/L PAB for 36 h underwent apoptosis, but not necrosis. The apoptosis induced by PAB was independent of the death receptor pathway. The senescent cells became larger and flatter, and the SA-beta-galactosidase staining was positive. PAB induced obvious mitotic arrest and it preceded apoptosis and senescence. The expressions of p21 and p53 was upregulated with PAB treatment, and cyclin B1 was upregulated and transported from the cytoplasm to nuclei, and sustained stable levels. Conclusion: PAB induced mitotic arrest in the MCF-7 cells and inhibited proliferation through apoptosis and senescence. The apoptosis was independent of the death receptor pathway. [Abstract/Link to Full Text]

Wang H, Tan SS, Wang XY, Liu DH, Yu CS, Bai ZL, He DL, Zhao J
Silencing livin gene by siRNA leads to apoptosis induction, cell cycle arrest, and proliferation inhibition in malignant melanoma LiBr cells.
Acta Pharmacol Sin. 2007 Dec;28(12):1968-74.
Aim: The aim of the present study was to investigate the effects of silencing the livin gene by small interfering RNA (siRNA) on the expression of livin and the effects on apoptosis, cell cycle, and proliferation in human malignant melanoma LiBr cells. Methods: Three chemically-synthetic siRNA duplexes targeting livin were transiently transfected into the LiBr cells, and the effects on livin expression were detected both at the mRNA level by real-time RT-PCR and at the protein level by Western blotting. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, flow cytometric analysis, and the expression of procaspase-3 and activated caspase-3 analysis by Western blotting. Cell cycle was analyzed by flow cytometry. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Results: One of the 3 designed siRNA could effectively knock down the livin expression both at the mRNA and protein levels in dose- and time-dependent manners; 100 nmol/L with maximum downregulation on mRNA at 48 h, and on the protein at 72 h after transfection. Silencing livin could significantly induce apoptosis, arrest cell cycle at the G0/G1 phase, and inhibit proliferation in LiBr cells. Meanwhile, caspase-3 was activated. Conclusion: The livin gene could serve as a potential molecular target for gene therapy by siRNA for malignant melanoma. [Abstract/Link to Full Text]

Jiao Y, Wang HC, Fan SJ
Growth suppression and radiosensitivity increase by HMGB1 in breast cancer.
Acta Pharmacol Sin. 2007 Dec;28(12):1957-67.
Aim: HMGB1 (high-mobility group box-1) is a nuclear protein containing a consensus RB (retinoblastoma)-binding LXCXE motif. In this study, we studied the potential association of HMGB1 and RB and the in vitro and in vivo activities of HMGB1 in human breast cancer cells. Methods: The protein-protein interaction was determined by immunoprecipitation-Western blotting and glutathione-S-transferase capture assays; cell growth and radiosensitivity were examined by cell counts, MTT assay, and clonogenic assay; cell cycle progression and apoptosis were evaluated using flow cytometry; and the antitumor activity of HMGB1 was examined with tumor xenografts in nude mice. Results: HMGB1 was associated with RB via a LXCXE motif-dependent mechanism. HMGB1 enhanced the ability of RB for E2F and cyclin A transcription repression. The increased expression of HMGB1 conferred an altered phenotypes characterized by the suppression of cell growth; G1 arrest and apoptosis was induced in MCF-7 cells containing the wild-type retinoblastoma (Rb) gene, but showed no activities in BT-549 cells containing the Rb gene deletion. The HMGB1-induced apoptosis accompanied by caspase 3 activation and PARP (poly(ADP-ribose)polymerase) cleavage. HMGB1 elevated the radiosensitivity of breast cancer cells in both the MCF-7 and BT-549 cell lines. The enhanced expression of HMGB1 caused a suppression of growth of MCF-7 tumor xenografts in nude mice, while LXCXE-defective HMGB1 completely lost antitumor growth activity. Conclusion: HMGB1 functions as a tumor suppressor and radiosensitizer in breast cancer. A HMGB1-RB interaction is critical for the HMGB1-mediated transcriptional repression, cell growth inhibition, G1 cell cycle arrest, apoptosis induction, and tumor growth suppression, but is not required for radiosensitization. Therefore, it may be possible to design new therapies for the treatment of breast cancer that exert their effects by modulating the HMGB1 and RB regulatory pathway and HMGB1-related gene therapy. [Abstract/Link to Full Text]

Mao XQ, Wu Y, Wu K, Liu M, Zhang JF, Zou F, Ou-Yang JP
Astragalus polysaccharide reduces hepatic endoplasmic reticulum stress and restores glucose homeostasis in a diabetic KKAy mouse model.
Acta Pharmacol Sin. 2007 Dec;28(12):1947-56.
Aim: To examine the potential effects of Astragalus polysaccharide (APS) on hepatic endoplasmic reticulum (ER) stress in vivo and in vitro and its link with hypoglycemia activity, thus establishing the mechanism underlying the hypoglycemic action of APS. Methods: The obese and type 2 diabetic KKAy mouse model, which is the yellow offspring of the KK mice expressed Ay gene (700 mg/kg/d, 8 weeks) and a high glucose-induced HepG2 cell model (200 mug/mL, 24 h) were treated with APS. The oral glucose tolerance test was measured to reflex insulin sensitivity with the calculated homeostasis model assessment (HOMA-IR) index. XBP1 (XhoI site-binding protein 1) transcription and splicing, an indicator of ER stress, was analyzed by RT-PCR and real-time PCR. The expression and activation of glycogen synthase kinase 3 beta (GSK3beta), an insulin signaling protein, was measured by Western blotting. Results: APS can alleviate ER stress in cultured cells in vivo. The hyperglycemia status, systemic insulin sensitivity, fatty liver disease, and insulin action in the liver of diabetic mice were partly normalized or improved in response to APS administration. Conclusion: Our results indicate that APS enables insulin-sensitizing and hypoglycemic activity at least in part by enhancing the adaptive capacity of the ER, which can further promote insulin signal transduction. Thus, APS has promising application in the treatment of type 2 diabetes. [Abstract/Link to Full Text]

Shi YH, Zhao S, Wang C, Li Y, Duan HJ
Fluvastatin inhibits activation of JAK and STAT proteins in diabetic rat glomeruli and mesangial cells under high glucose conditions.
Acta Pharmacol Sin. 2007 Dec;28(12):1938-46.
Aim: The aim of the present study was to further elucidate the mechanism of the protective role of fluvastatin on diabetic nephropathy. Methods: Streptozotocin-induced diabetic rats were treated daily with fluvastatin (4 mg/kg body weight) by gavage. The animals were killed 4 weeks later and urine and blood samples were collected. The kidney tissues were removed and subjected to the following experiments. Rat glomerular mesangial cells (GMC) were cultured under normal glucose (5.5 mmol/L), high glucose (HG, 30 mmol/L), HG+AG490 (10 mumol/L), or HG with fluvastatin (1 mumol/L). Glomeruli or the GMC lysate was immunoprecipitated and/or immunoblotted with antibodies against Janus kinase 2 (JAK2), SH2-domain containing tyrosine phosphatase-1 (SHP-1), phosphospecific SHP-2, and signal transducer and activators of transcription (STAT), respectively. Transforming growth factor-beta (TGF-beta1) mRNA was measured by RT-PCR. The protein synthesis of TGF-beta1 and fibronectin in the culture medium of GMC was detected by ELISA. Results: The phosphorylation levels of JAK2, STAT1, STAT3, and SHP-2 increased significantly, and SHP-1 phosphorylation was reduced in glomeruli of diabetic rats. Treatment with fluvastatin reduced phosphorylation levels of JAK2, STAT1, STAT3, and SHP-2 in glomeruli of diabetic rats, but it had no effect on the dephosphorylation of SHP-1. The exposure of GMC to 30 mmol/L glucose caused the activation of JAK2, STAT1, STAT3, and SHP-2. It upregulated TGF-beta1 expression and increased protein synthesis of fibronectin. These high glucose-induced changes were suppressed by fluvastatin, as well as AG490, a JAK2 inhibitor. Conclusion: The regulation of the phosphorylation of JAK/STAT by fluvastatin may be responsible for its renal protective effects on diabetic nephropathy. [Abstract/Link to Full Text]

Min J, Shang CZ, Chen YJ, Zhang L, Liu L, Deng XG, Yang M, Chen DP, Cao J, Song EW, Chen JS
Selective enrichment of hepatocytes from mouse embryonic stem cells with a culture system containing cholestatic serum.
Acta Pharmacol Sin. 2007 Dec;28(12):1931-7.
Aim: There is increasing evidence indicating that embryonic stem (ES) cells are capable of differentiating into hepatocyte-like cells in vitro. However, it is necessary to improve the differentiation efficiency so as to promote the clinical application. Here, we report an efficient culture system to support hepatocyte differentiation from ES cells by utilizing cholestatic serum. Methods: One week after the induction of E14 mouse ES cells into hepatocytes with sodium butyrate, cholestatic serum was added into the culture system at various concentrations and hepatocyte-like cells were induced to proliferate. The morphological and phenotypic markers of hepatocytes were characterized using light microscopy, immunocytochemistry, and RT-PCR, respectively. The function of glycogen storage of the differentiated cells was detected by Periodic acid-Schiff (PAS) reaction, and the ratio of hepatic differentiation was determined by counting the albumin and PAS-positive cells. Results: In the presence of conditional selective medium containing cholestatic serum, numerous epithelial cells resembling hepatocytes were observed. The RT-PCR analysis showed that undifferentiated ES cells did not express any hepatic-specific markers; however, in the presence of sodium butyrate and conditional selective medium containing cholestatic serum, hepatic differentiation markers were detected. Immunofluorescence staining showed that those ES-derived hepatocytes were alpha-fetoprotein, albumin, and cytokeratin 18 positive, with the ability of storing glycogen. Further determination of the hepatic differentiation ratio showed that the application of cholestatic serum efficiently enriched ES-derived hepatocyte-like cells by inducing lineage differentiation and enhancing lineage proliferation. Conclusion: The conditional selective medium containing cholestatic serum is optimal to selectively enrich hepatocyte-like cells from mixed differentiated ES cells, which may provide a novel method to improve the hepatic differentiation ratio of ES cells. [Abstract/Link to Full Text]

Fang S, Qiu YD, Mao L, Shi XL, Yu DC, Ding YT
Differentiation of embryoid-body cells derived from embryonic stem cells into hepatocytes in alginate microbeads in vitro.
Acta Pharmacol Sin. 2007 Dec;28(12):1924-30.
Aim: Embryonic stem (ES) cells are being widely investigated as a promising source of hepatocytes with their proliferative, renewable, and pluripotent capacities. However, controlled and scalable ES cell differentiation culture into functional hepatocytes is challenging. In this study, we examined the differentiating potential of embryoid-body cells derived from ES cells into hepatocytes in alginate microbeads containing exogenous growth factors in vitro. Methods: Embryoid bodies were formed from ES cells by suspension methods. Embryoid bodies cultured for 5 d were treated with trypsin-EDTA. The disaggregated cells were encapsulated in alginate microbeads and stimulated with exogenous growth factors to induce hepatic differentiation. In the course of cell differentiation, cell morphology and viability were observed, and the expression patterns of some genes of the hepatocyte were confirmed by RT-PCR. An immunofluorescence analysis revealed the expression of albumin (ALB) and cytokeratin-18 (CK18). Hepatocyte functional assays were confirmed by the secretion of ALB and urea. Results: We showed that embryoid-body cells could maintain cell viability in alginate microbeads in vitro. We also found that directed differentiated cells expressed several hepatocyte genes including alpha-fetoprotein (AFP), ALB, Cyp7a1, CK18, transthyretin (TTR) and tyrosine aminotransferase (TAT) and produced ALB and urea in alginate microbeads. The directed differentiated cells expressed ALB and CK18 proteins on d 14. However, embryoid-body cells could not form hepatocytes without exogenous growth factors in alginate microbeads. Conclusion: The differentiation of embryoid-body cells into hepatocytes containing exogenous growth factors in alginate microbeads gives rise to functional hepatocytes and may develop scalable stem cell differentiation strategies for bioartificial livers and hepatocyte transplantation. [Abstract/Link to Full Text]

He JK, Yu SD, Zhu HJ, Wu JC, Qin ZH
Triptolide inhibits NF-kappaB activation and reduces injury of donor lung induced by ischemia/reperfusion.
Acta Pharmacol Sin. 2007 Dec;28(12):1919-23.
Aim: To investigate the protective effect of triptolide (TRI) on ischemia/reperfusion-induced injury of transplanted rabbit lungs and to investigate the mechanisms underlying the actions of TRI. Methods: We established the rabbit lung transplantation model and studied lung injury induced by ischemia/reperfusion and the inhibitory effect of TRI on NF-kappaB. The severity of lung injury was determined by a gradual decline in PvO2, the degree of lung edema, the increase in the myeloperoxidase (MPO) activity, and the ultrastructural changes of transplanted lungs. The activation of NF-kappaB was measured by immunohistochemistry. The increase in intercellular adhesion molecule-1 (ICAM-1), which is the target gene of NF-kappaB, was evaluated by ELISA. Results: After reperfusion, there was a gradual decline in the PvO2 level in the control group (group I). The level of PvO2 in the group treated with lipopolysaccharide (group II) was significantly decreased, whereas that of the group treated with TRI (group III) was markedly improved (P<0.01). In group III, the activity of MPO was downregulated, and the pulmonary edema did not become severe and the ultrastructure of the donor lung remained normal. The activity of NF-kappaB and the expression of ICAM-1 was significantly increased in the donor lungs. TRI blocked NF-kappaB activation and ICAM-1 expression. Conclusion: The effects of TRI on reducing injury to donor lungs induced by ischemia/reperfusion may possibly be mediated by inhibiting the activity of NF-kappaB and the expression of the NF-kappaB target gene ICAM-1. Thus, TRI could be used in lung transplantations for improving the function of donor lungs. [Abstract/Link to Full Text]

Tan Y, Tang Q, Hu BR, Xiang JZ
Antioxidant properties of berberine on cultured rabbit corpus cavernosum smooth muscle cells injured by hydrogen peroxide.
Acta Pharmacol Sin. 2007 Dec;28(12):1914-8.
Aim: To investigate the antioxidant properties of berberine (Ber) on corpus cavenosum smooth muscle cells (CCSMC) in penile erectile dysfunction. Methods: We examined the effects of Ber on cultured rabbit CCSMC damaged by hydrogen peroxide (H2O2) through examining cell viability by methyl thiazolyl tetrazolium assay and assessing the level of malondialdehyde (MDA), superoxide dismutase (SOD) activity, nitric oxide (NO) products, and lactate dehydrogenase (LDH) release in cells after stimulation with H2O2. Results: Treatment with 1 mmol/L H2O2 significantly decreased the cell viability, NO products, and SOD activity of CCSMC from 100% to 48.57%+/-4.1% (P<0.01), 66.8+/-16.3 to 6.7+/-2.1 mumol/L (P<0.01), and 49.5+/-1.8 to 30.1+/-2.6 U/mL (P<0.01), respectively, and increased LDH release and MDA content from 497.6+/-69.5 to 1100.5+/-56.3 U/L (P<0.01) and 3.7+/-1.3 to 78.4+/-2.9 nmol/mg protein (P<0.01), respectively. However, treatment with different concentrations of Ber (10-1000 mumol/L) inhibited the damaging effects of H2O2, with increased cell viability (P<0.05 or P<0.01), NO production (P<0.01), and SOD activity (P<0.01) and decreased LDH release and MDA content (both P<0.01). Conclusion: Ber could produce its antioxidant action on oxidative stress-induced cultured CCSMC. These effects may be of benefit in the prevention of penile erectile dysfunction. [Abstract/Link to Full Text]

Liu DH, Yuan HY, Cao CY, Gao ZP, Zhu BY, Huang HL, Liao DF
Heat shock protein 90 acts as a molecular chaperone in late-phase activation of extracellular signal-regulated kinase 1/2 stimulated by oxidative stress in vascular smooth muscle cells.
Acta Pharmacol Sin. 2007 Dec;28(12):1907-13.
Aim: To investigate whether cytosolic heat shock protein 90 (HSP90) acts as a molecular chaperone on the activated extracellular signal-regulated kinase 1/2 (ERK1/2) and cell proliferation stimulated by reactive oxygen species (ROS) in rat vascular smooth muscle cells (VSMC). Methods: VSMC were exposed to 1 mumol/L LY83583 (6-anilinoquinoline-5,8-quinolinedione, producer of ROS) for 120 min in the presence or absence of 5 mumol/L geldanamycin, a specific inhibitor of HSP90. Then the total, soluble, and insoluble proteins of the cells were collected. HSP90, ERK1/2, and phosphor-ERK1/2 in the cell lysate were measured by Western blotting. The interaction of HSP90 and phosphor-ERK1/2 was analyzed by immunoprecipitation assay, and the nuclear phosphor-ERK1/2 was measured by Western blotting and immunofluorescence. Cell proliferation was tested by cell counting and 3-(4,5-dimethylthiazol-2-y1)-3,5-di-phenyltetrazolium bromide (MTT). Results: The cytosolic HSP90 of VSMC was upregulated by LY83583 in a time-dependent manner with the peak at 120 min, which is consistent with the late peak of phosphor-ERK1/2. Immunoprecipitation and Western blotting analyses showed that LY83583 increased the interaction of HSP90 with phosphor-ERK1/2, the phosphor-ERK1/2 level, and the soluble phosphor-ERK1/2 level by 1.8-, 2.5-, and 2.9-fold, respectively. In contrast, the insoluble phosphor-ERK1/2 of VSMC was decreased. Interestingly, LY83583 treatment promoted the nuclear phosphor-ERK1/2 by 7.6-fold as confirmed by Western blotting and immunofluorescence assays. Furthermore, cell counting and the MTT assay showed that LY83583 stimulated VSMC proliferation with the increased expression of HSP90 and levels of soluble and nuclear phosphor-ERK1/2. Pretreatment of geldanamycin antagonized the effect of LY83583. Conclusion: HSP90 could mediate the oxidative stress-stimulated, late-phase activation of ERK1/2 and VSMC proliferation by promoting the ERK1/2 phosphorylation, the association of itself with phosphor-ERK1/2, and the solubility and nuclear translocation of phosphor-ERK1/2. [Abstract/Link to Full Text]

Wang T, Hou LB, Liu ZJ, Wang Y, Chen CL, Xiao X, Wang DW
Intramuscular delivery of rAAV-mediated kallikrein gene reduces hypertension and prevents cardiovascular injuries in model rats.
Acta Pharmacol Sin. 2007 Dec;28(12):1898-906.
Aim: The overexpression of the human tissue kallikrein (HK) gene can reduce blood pressure and ameliorate the secondary syndromes associated with hypertension in animal models. The current study was designed to investigate hypotensive effect of intramuscular delivery of HK gene. Methods: We generated an recombinant adeno-associated virus (rAAV) vector expressing human tissue kallikrein under the control of a cytomegalovirus promoter and administered the rAAV-HK vector to a spontaneously hypertensive rat model at a dose of 1x1010 virons/rat through intramuscular injection. Results: A persistent, high-level expression of HK post-gene delivery was confirmed by ELISA. The systolic blood pressure in the rats receiving rAAV-LacZ and saline increased from 171.3 mmHg to 182.3 mmHg 28 weekso post injection. In contrast, the delivery of the HK gene by AAV vectors attenuated the increase of the systolic blood pressure in the treated group. The systolic blood pressure was only slightly lowered (from a level of 174 mmHg to 170.5 mmHg) post-vector administration. The difference in blood pressure between the treated group and the control groups is statistically significant at 12.6 mmHg. The hypotensive effect of rAAV-HK persisted until the end of the testing period. In addition, a significant amelioration of cardiovascular hypertrophy, renal injury, and collagen depositions in the rAAV-HK-treated animals were also observed. Conclusion: All the effects are comparable with those of intravenous delivery. Therefore, the intramuscular administration of rAAV-HK may be used in gene therapy for hypertension. [Abstract/Link to Full Text]

Yu Y, Chen XQ, Cui YY, Hu GY
Electrophysiological actions of cyclosporin A and tacrolimus on rat hippocampal CA1 pyramidal neurons.
Acta Pharmacol Sin. 2007 Dec;28(12):1891-7.
Aim: The aim of the present study was to investigate the electrophysiological actions of cyclosporin A (CsA) and tacrolimus (FK506) on neurons in the brain, and to elucidate the relevant mechanisms. Methods: Whole-cell current-clamp recording was made in CA1 pyramidal neurons in rat hippocampal slices; whole-cell voltage-clamp recording was made in dissociated hippocampal CA1 pyramidal neurons of rats. Results: CsA (100 mumol/L) and FK506 (50 mumol/L) did not significantly alter the passive electrical properties of hippocampal CA1 pyramidal neurons, but slowed down the repolarizing phase of the action potential. CsA (10-100 mumol/L) selectively inhibited the delayed rectifier K+ current (IK) in a concentration-dependent manner. CsA did not affect the kinetic properties of IK. Intracellular dialysis of CsA (100 mumol/L) had no effect on IK. The inhibition of IK by CsA (100 mumol/L) persisted under the low Ca2+ conditions that blocked the basal activity of calcineurin. Conclusion: CsA exerted calcineurin-independent inhibition on the IK in rat hippocampal pyramidal neurons. Taken together with our previous finding with FK506, it is conceivable that the spike broadening caused by the immunosuppressant drugs is due to direct inhibition on the IK. [Abstract/Link to Full Text]

Jin Y, Yan EZ, Fan Y, Guo XL, Zhao YJ, Zong ZH, Liu Z
Neuroprotection by sodium ferulate against glutamate-induced apoptosis is mediated by ERK and PI3 kinase pathways.
Acta Pharmacol Sin. 2007 Dec;28(12):1881-90.
Aim: To investigate whether sodium ferulate (SF) can protect cortical neurons from glutamate-induced neurotoxicity and the mechanisms responsible for this protection. Methods: Cultured cortical neurons were incubated with 50 mumol/L glutamate for either 30 min or 24 h, with or without pre-incubation with SF (100, 200, and 500 mumol/L, respectively). LY294002, wortmannin, PD98059, and U0126 were added respectively to the cells 1 h prior to SF treatment. After incubation with glutamate for 24 h, neuronal apoptosis was quantified by scoring the percentage of cells with apoptotic nuclear morphology after Hoechst 33258 staining. After incubation with glutamate for either 30 min or 24 h, cellular extracts were prepared for Western blotting of active caspase-3, poly (ADP-ribose) polymerase (PARP), mu-calpain, Bcl-2, phospho-Akt, phosphorylated ribosomal protein S6 protein kinase (p70S6K), phospho-mitogen-activated protein kinase kinase (MEK1/2) and phosphorylated extracellular signal-regulated kinase (ERK) 1/2. Results: SF reduced glutamate-evoked apoptotic morphology, active caspase-3 protein expression, and PARP cleavage and inhibited the glutamate-induced upregulation of the mu-calpain protein level. The inhibition of the phosphatidylinositol 3-kinase (PI3K) and the MEK/ERK1/2 pathways partly abrogated the protective effect of SF against glutamate-induced neuronal apoptosis. SF prevented the glutamate-induced decrease in the activity of the PI3K/Akt/p70S6K and the MEK/ERK1/2 pathways. Moreover, incubation of cortical neurons with SF for 30 min inhibited the reduction of the Bcl-2 expression induced by glutamate. Conclusion: The results indicate that PI3K/Akt/p70S6K and the MEK/ERK signaling pathways play important roles in the protective effect of SF against glutamate toxicity in cortical neurons. [Abstract/Link to Full Text]

Ning W, Xu SJ, Chiang H, Xu ZP, Zhou SY, Yang W, Luo JH
Effects of GSM 1800 MHz on dendritic development of cultured hippocampal neurons.
Acta Pharmacol Sin. 2007 Dec;28(12):1873-80.
Aim: To evaluate the effects of global system for mobile communications (GSM) 1800 MHz microwaves on dendritic filopodia, dendritic arborization, and spine maturation during development in cultured hippocampal neurons in rats. Methods: The cultured hippocampal neurons were exposed to GSM 1800 MHz microwaves with 2.4 and 0.8 W/kg, respectively, for 15 min each day from 6 days in vitro (DIV6) to DIV14. The subtle structures of dendrites were displayed by transfection with farnesylated enhanced green fluorescent protein (F-GFP) and GFP-actin on DIV5 into the hippocampal neurons. Results: There was a significant decrease in the density and mobility of dendritic filopodia at DIV8 and in the density of mature spines at DIV14 in the neurons exposed to GSM 1800 MHz microwaves with 2.4 W/kg. In addition, the average length of dendrites per neuron at DIV10 and DIV14 was decreased, while the dendritic arborization was unaltered in these neurons. However, there were no significant changes found in the neurons exposed to the GSM 1800 MHz microwaves with 0.8 W/kg. Conclusion: These data indicate that the chronic exposure to 2.4 W/kg GSM 1800 MHz microwaves during the early developmental stage may affect dendritic development and the formation of excitatory synapses of hippocampal neurons in culture. [Abstract/Link to Full Text]

Qin ZH, Tao LY, Chen X
Dual roles of NF-kappaB in cell survival and implications of NF-kappaB inhibitors in neuroprotective therapy.
Acta Pharmacol Sin. 2007 Dec;28(12):1859-72.
NF-kappaB is a well-characterized transcription factor with multiple physiological and pathological functions. NF-kappaB plays important roles in the development and maturation of lymphoids, regulation of immune and inflammatory response, and cell death and survival. The influence of NF-kappaB on cell survival could be protective or destructive, depending on types, developmental stages of cells, and pathological conditions. The complexity of NF-kappaB in cell death and survival derives from its multiple roles in regulating the expression of a broad array of genes involved in promoting cell death and survival. The activation of NF-kappaB has been found in many neurological disorders, but its actual roles in pathogenesis are still being debated. Many compounds with neuroprotective actions are strongly associated with the inhibition of NF-kappaB, leading to speculation that blocking the pathological activation of NF-kappaB could offer neuroprotective effects in certain neurodegenerative conditions. This paper reviews the recent developments in understanding the dual roles of NF-kappaB in cell death and survival and explores its possible usefulness in treating neurological diseases. This paper will summarize the genes regulated by NF-kappaB that are involved in cell death and survival to elucidate why NF-kappaB promotes cell survival in some conditions while facilitating cell death in other conditions. This paper will also focus on the effects of various NF-kappaB inhibitors on neuroprotection in certain pathological conditions to speculate if NF-kappaB is a potential target for neuroprotective therapy. [Abstract/Link to Full Text]

Hu
Antitumor effects of Chi-Shen extract from Salvia miltiorrhiza and Paeoniae radix on human hepatocellular carcinoma cells.
Acta Pharmacol Sin. 2007 Oct;28(10):1705. [Abstract/Link to Full Text]

JQ, Iwashita N, Du H, Wang YT, Wang YY, Zhao M, Wang J, Watada H, Kawamori R
Angiotensin II receptor blocker provides pancreatic beta-cell protection independent of blood pressure lowering in diabetic db/db mice.
Acta Pharmacol Sin. 2007 Oct;28(10):1705. [Abstract/Link to Full Text]

Qin XQ, Yuan Y, Liu CS, Wang QY, Shen X, Yang BC
Preparation of liposomal brucine and its pharmaceutical/pharmacodynamic characterization.
Acta Pharmacol Sin. 2007 Nov;28(11):1851-8.
AIM: To prepare a novel transdermal preparation of liposomal brucine (LB) and investigate its pharmaceutical/pharmacodynamic characterization. METHODS: LB was prepared by a modified ethanol-dripping method. Its drug encapsulation efficiency (EE), particle size, in vitro release, and skin permeation were studied. Furthermore, a safety evaluation and pharmacodynamic analysis of LB, including acute dermal toxicity, skin irritation, and analgesic and anti-inflammatory effects were investigated. RESULTS: the EE of LB was 72% and the mean particle size of the liposomes was 55.4 nm. The in vitro release profile indicated that less than 68% of the encapsulated brucine was released in 10 h. A skin permeation study showed that compared with the free brucine, LB exhibited higher cumulative drug permeation through the skin and lower drug accumulation in skin tissue, indicative of an obvious promotion of skin permeation with liposomal encapsulation. The acute dermal LD50 of LB was greater than 100 mg/kg (brucine content) and skin irritation tests revealed that LB had no irritation to both integrity and broken skin. A pharmacodynamic evaluation of LB was performed by xylene-induced mouse ear edema test and acetic acid-induced writhing test at the dosage of 1.5, 3, and 6 mg/kg, respectively. The results showed that anti-inflammatory activities and analgesic effects of brucine encapsulated were significantly higher than that of the free brucine (P<0.01). Moreover, LB maintained a remarkably longer antiinflammatory and analgesic duration. CONCLUSION: It can be proposed that LB prepared here could represent a safe, effective and promising transdermal formulation for analgesic and anti-inflammatory effects. [Abstract/Link to Full Text]

Huang MT, Chen ZX, Wei B, Zhang B, Wang CH, Huang MH, Liu R, Tang CW
Preoperative growth inhibition of human gastric adenocarcinoma treated with a combination of celecoxib and octreotide.
Acta Pharmacol Sin. 2007 Nov;28(11):1842-50.
AIM: To gain insight into the histopathological responses and molecular targets in the inhibition of growth of human gastric cancer treated with celecoxib (a cyclooxygenase [COX]-2 inhibitor) combined with octreotide. METHODS: Seventy five patients with gastric cancer undergoing curative gastrectomy or extended resection were randomly divided into 3 groups. The apoptosis of tumor cells was measured by terminal deoxynucleotide transferase-mediated dUTP nick endlabeling (TUNEL) assay. Gastric cancer microvessel density (MVD) and the expression of COX-2 were evaluated by immunohistochemical staining. The expression of somatostatin receptor (SSTR)-2 was detected with the biomolecular interaction analysis system. The transcription of non-steroidal anti-inflammatory drug-activated gene (NAG)-1 was measured by RT-PCR. RESULTS: Compared with the control and celecoxib groups, more necrosis in the combination group was observed. The apoptotic rate in the combination group (7.06%+/-0.67%) was significantly higher than that in the control group (6.23%+/-1.29%, P<0.05). The MVD decreased considerably in the combination group. The upregulation of NAG-1 was displayed both in the celecoxib and combination groups. The positive rate of SSTR-2 in gastric cancers treated with celecoxib (48%) was significantly higher than that of control group (12%) after surgery (P<0.05). CONCLUSION: Celecoxib combined with octreotide significantly promoted necrosis in gastric adenocarcinoma through the induction of apoptosis and the reduction of MVD. NAG-1 and SSTR-2 might be the molecular targets for celecoxib or octreotide. [Abstract/Link to Full Text]

Yuan XW, Zhu XF, Huang XF, Sheng PY, He AS, Yang ZB, Deng R, Feng GK, Liao WM
Interferon-alpha enhances sensitivity of human osteosarcoma U2OS cells to doxorubicin by p53-dependent apoptosis.
Acta Pharmacol Sin. 2007 Nov;28(11):1835-41.
AIM: To determine whether interferon-alpha(IFNalpha) can enhance doxorubicin sensitivity in osteosarcoma cells and its molecular mechanism. METHODS: Cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was studied using Flow cytometry analysis, Hoechst33258 staining, DNA fragmentation assay, as well as the activation of caspase-3 and poly (ADP-ribose) polymerase. Protein expression was detected by Western blotting. The dependence of p53 was determined using p53-siRNA transfection. RESULTS: IFNalpha increased doxorubicin-induced cytotoxicity to a much greater degree through apoptosis in human osteosarcoma p53-wild U2OS cells, but not p53-mutant MG63 cells. IFNalpha markedly upregulated p53, Bax, Mdm2, and p21, downregulated Bcl-2, and activated caspase-3 and PARP cleavage in response to doxorubicin in U2OS cells. Moreover, the siRNA-mediated silencing of p53 significantly reduced the IFNalpha/doxorubicin combination-induced cytotoxicity and PARP cleavage. CONCLUSION: IFNalpha enhances the sensitivity of human osteosarcoma U2OS cells to doxorubicin by p53-dependent apoptosis. The proper combination with IFNalpha and conventional chemotherapeutic agents may be a rational strategy for improving the treatment of osteosarcoma with functional p53. [Abstract/Link to Full Text]

Recent Articles in Biological & Pharmaceutical Bulletin

Hirose K, Onishi H, Sasatsu M, Takeshita K, Kouzuma K, Isowa K, Machida Y
In vivo evaluation of Kumazasa extract and chitosan films containing the extract against deep skin ulcer model in rats.
Biol Pharm Bull. 2007 Dec;30(12):2406-11.
Kumazasa extract (KE) and its whole solid component (EXT)-containing chitosan films produced by drying in air and lyophilization, abbreviated to ND and FD films, respectively, were examined for efficacy and healing features using a deep skin ulcer model in rats. Their effects were compared with those of clinically available dosage forms, Beschitin W, Geben cream and U-PASTA. KE alone exhibited a better effect as compared with other preparations, and FD films also more effective than control in the early stage. Histological analysis showed that KE alone reduced necrosis rapidly and accelerated granulation. ND films delayed healing rate as compared with control. FD films showed histological features between control and KE alone, but tended to delay healing rate in the later period. Thus, reduction rate of wound area and histological features suggested that KE alone should be excellent for the promotion of wound healing. Although FD films were less effective than KE alone, they were superior as to usability such as changing the preparation. [Abstract/Link to Full Text]

Hirata N, Tokunaga M, Naruto S, Iinuma M, Matsuda H
Testosterone 5alpha-reductase inhibitory active constituents of Piper nigrum leaf.
Biol Pharm Bull. 2007 Dec;30(12):2402-5.
Previously we reported that Piper nigrum leaf extract showed a potent stimulation effect on melanogenesis and that (-)-cubebin (1) and (-)-3,4-dimethoxy-3,4-desmethylenedioxycubebin (2) were isolated as active constituents. As a part of our continuous studies on Piper species for the development of cosmetic hair-care agents, testosterone 5alpha-reductase inhibitory activity of aqueous ethanolic extracts obtained from several different parts of six Piper species, namely Piper nigrum, P. methysticum, P. betle, P. kadsura, P. longum, and P. cubeba, were examined. Among them, the extracts of P. nigrum leaf, P. nigrum fruit and P. cubeba fruit showed potent inhibitory activity. Activity-guided fractionation of P. nigrum leaf extract led to the isolation of 1 and 2. Fruits of P. cubeba contain 1 as a major lignan, thus inhibitory activity of the fruit may be attributable to 1. As a result of further assay on other known constituents of the cited Piper species, it was found that piperine, a major alkaloid amide of P. nigrum fruit, showed potent inhibitory activity, thus a part of the inhibitory activity of P. nigrum fruit may depend on piperine. The 5alpha-reductase inhibitory activities of 1 and piperine were found for the first time. In addition, the P. nigrum leaf extract showed in vivo anti-androgenic activity using the hair regrowth assay in testosterone sensitive male C57Black/6CrSlc strain mice. [Abstract/Link to Full Text]

Sugimoto Y, Tagawa N, Kobayashi Y, Hotta Y, Yamada J
Effects of the serotonin and noradrenaline reuptake inhibitor (SNRI) milnacipran on marble burying behavior in mice.
Biol Pharm Bull. 2007 Dec;30(12):2399-401.
The effects of milnacipran, a serotonin and noradrenaline reuptake inhibitor (SNRI) on the obsessive compulsive disorder (OCD) model, marble burying behavior, were investigated in mice. Milnacipran above the dosage of 10 mg/kg inhibited marble burying behavior significantly in mice as similar to fluvoxamine. Milnacipran inhibiting marble burying behavior did not affect locomotor activity. These results suggest that milnacipran can inhibit marble burying behavior and that milacipran may be useful for OCD therapy. [Abstract/Link to Full Text]

Lee ER, Kang YJ, Choi HY, Kang GH, Kim JH, Kim BW, Han YS, Nah SY, Paik HD, Park YS, Cho SG
Induction of apoptotic cell death by synthetic naringenin derivatives in human lung epithelial carcinoma A549 cells.
Biol Pharm Bull. 2007 Dec;30(12):2394-8.
Although flavonoids, which are both qualitatively and quantitatively one of the largest groups of natural products, exhibit a variety of beneficial health effects, the exact molecular mechanism of the cellular activities is still not fully explained and there currently exists a lack of evidence for any relationship between the structure-activity relationship and apoptosis-inducing activity. In order to determine the importance of the OH group or substitution of the 5 or carbon-7 in the diphenylpropane skeleton of flavonoids, we originally synthesized several modified naringenin derivatives, including 7-O-benzyl naringenin (KUF-1) and 7-O-(MeO-L-Leu-D-Pro-carbonylmethyl) naringenin (KUF-7). Treatment with KUF-1 or KUF-7 resulted in significant apoptosis-inducing effects concomitant with chromatin condensation, caspase activation, and intracellular ROS production. Our data indicate that originally synthesized naringenin derivatives, KUF-1 and KUF-7 differentially regulate the apoptosis of A549 cells via intracellular ROS production coupled with the concomitant activation of the caspase cascade signaling pathway, thereby implying that hydroxylation or substitution at Carbon-7 is critical for the apoptosis-inducing activity of flavonoids. [Abstract/Link to Full Text]

Takao K, Ozawa T, Shibata S, Wada M, Sugita Y, Shirahata A, Samejima K
Formation of spermidine or norspermidine from synthetic diacetylpolyamines by acetylpolyamine oxidase in cultured cells.
Biol Pharm Bull. 2007 Dec;30(12):2389-93.
Prodrugs that can readily release polyamine into cells without the problem of generating cytotoxic compound by serum amine oxidase would be extremely useful for elucidation of polyamine function. As linear polyamines with acetamide groups on both sides are thought to be stable in the presence of serum amine oxidase and produce polyamines by the catalytic reaction of acetylpolyamine oxidase (PAO), a series of diacetyltetraamines, diacetylpentaamines and diacetylhexaamines was prepared as prodrugs and tested for substrate activity against PAO, partially purified from rat liver. Of the compounds, N(1),N(15)-diacetyl-1,15-diamino-4,8,12-triazapentadecane (DA3333) and N(1),N(16)-diacetyl-1,16-diamino-4,8,13-triazahexadecane (DA3343) were found to be stable in culture medium containing newborn bovine serum, and to produce reasonable amounts of norspermidine and spermidine, respectively. DA3333 and DA3343 were then applied to 1-aminooxy-3-aminopropane (AOAP)-treated HTC cells with depleted putrescine and spermidine, and arrested growth. Cell growth recovered with DA3333 and DA3343, but growth rate was reduced in cells with added DA3333 compared with growth rates in cells with added DA3343 and control cells untreated with AOAP. Significant amounts of norspermidine and spermidine were found in cells with added DA3333 and DA3343, respectively. These results show the potential use of diacetylpolyamines in introducing polyamines into cells. [Abstract/Link to Full Text]

Tamai T, Murota I, Maruyama K, Baba T, Toyama T, Watanabe N, Kudo N, Kawashima Y
Effects of supplemented diacylglycerol rich in docosahexaenoic acid on serum triacylglycerol in a diet-induced hyperlipidemic model of rats are essentially equivalent to those of triacylglycerol rich in docosahexaenoic acid.
Biol Pharm Bull. 2007 Dec;30(12):2381-8.
Effects of supplemented docosahexaenoic acid (DHA), given as diacylglycerol (DG) rich in DHA (DHA-DG), triacylglycerol (TG) rich in DHA (DHA-TG) or fish oil concentrate (DHA-70), on the serum concentration of TG and its bioavailability in the rats with diet-induced hyperlipidemia were studied. Hypertriglyceridemia was induced by feeding male Wistar rats a semi-purified diet that contained 5% corn oil and 50% sucrose by weight. In addition to the feeding of dietary corn oil, the rats received DHA intragastrically at a dose of 500 mg/kg body weight once a day for 28 d and the control rats were given olive oil. The serum concentration of TG in the rats that received DHA-DG was significantly lower than in the control rats. However, there were no significant differences in diet intake, energy intake, body weight gain, visceral fat mass or fecal excretion of total fatty acids among the four groups. The amounts of DHA excreted into the feces of the three groups of rats that received DHA were approximately 0.4% of the DHA administered. The extent of the decreases induced by DHA-DG in the serum level of TG was almost the same as those induced by DHA-TG and DHA-70. The administration of DHA, regardless of the differences in molecular structure, did not affect the hepatic contents of TG or phospholipid. The administration of DHA-DG considerably increased the proportions of DHA and eicosapentaenoic acid (EPA) while decreasing the proportion of arachidonic acid in hepatic lipids, and as a result in the lipids in serum and erythrocytes, to the same extents as did DHA-TG and DHA-70. These results suggest that the hypotriglyceridemic effects and bioavailability of DHA when supplemented in the form of DG are essentially equivalent to those of DHA-TG and DHA-70. [Abstract/Link to Full Text]

Li C, Taneda S, Suzuki AK, Furuta C, Watanabe G, Taya K
Effects of 3-methyl-4-nitrophenol on the suppression of adrenocortical function in immature male rats.
Biol Pharm Bull. 2007 Dec;30(12):2376-80.
In previous studies, we found that 3-methyl-4-nitrophenol (4-nitro-m-cresol; PNMC) isolated from diesel exhaust particles, and also a degradation product of the insecticide fenitrothion, exhibited testicular toxicity in the male of both immature rat and adult Japanese quail. It is well established that a functional relationship exists between the gonads and adrenals. The present study investigates the effect of PNMC on the adrenocortical functions of immature male rats. We subcutaneously injected 28-d-old rats with PNMC (1, 10 or 100 mg/kg) daily for 5 d. The adrenal glands weights significantly decreased in rats treated with 10 or 100 mg/kg PNMC. Plasma concentrations of adrenocorticotropic hormone (ACTH) were significantly increased in animals treated with 100 mg/kg PNMC. In contrast, plasma concentrations of corticosterone were significantly decreased in all PNMC-treated groups, and plasma concentrations of progesterone were significantly decreased in rats treated with 10 or 100 mg/kg PNMC. To investigate the direct effects of PNMC on the secretion of ACTH from the anterior pituitary gland, and on the secretion of corticosterone from the adrenal, we exposed cultured primary anterior pituitary and adrenal cells to PNMC (10(-8), 10(-7), 10(-6), or 10(-5 m)) for 24 h. PNMC did not change basal levels of ACTH released from cultured anterior pituitary cells. However, PNMC significantly inhibited ACTH-stimulated production of corticosterone and progesterone from cultured adrenal cells. These results clearly show that PNMC has a direct effect on the adrenal gland to reduce corticosterone secretion, and the associated increase in plasma ACTH is probably due decreased negative feedback regulation by corticosterone. [Abstract/Link to Full Text]

Nakamura T, Nozu K, Iijima K, Yoshikawa N, Moriya Y, Yamamori M, Kako A, Matsuo M, Sakurai A, Okamura N, Ishikawa T, Okumura K, Sakaeda T
Association of cumulative cyclosporine dose with its irreversible nephrotoxicity in Japanese patients with pediatric-onset autoimmune diseases.
Biol Pharm Bull. 2007 Dec;30(12):2371-5.
Cyclosporine (CsA)-induced nephrotoxicity can become a major obstacle to continuous use. The aim of this study was to optimize CsA dose to avoid its irreversible nephrotoxicity. Twenty-three Japanese patients with pediatric-onset systemic lupus erythematosus or idiopathic nephrotic syndrome, who were maintained in a stable condition by oral dosing of CsA microemulsion, were enrolled in this study. The patients were stratified into 3 groups; those with no, reversible, and irreversible nephrotoxicity, according to periodically performed renal pathohistological examinations. A higher concentration of CsA in blood (p=0.002-0.011) and a longer duration of CsA treatment (p=0.002) were risk factors for irreversible nephrotoxicity, and the cumulative CsA dose, the product of the maintenance dose and duration of CsA treatment, was predictive of nephrotoxicity (p=0.036). The maximum target blood concentration at 2 h post-dose, C(2), to avoid CsA-induced irreversible nephrotoxicity was 700 ng/ml, although the cumulative CsA dose of 4850 mg/kg would result in a 50% probability of nephrotoxicity. [Abstract/Link to Full Text]

Shiose Y, Ochi Y, Kuga H, Yamashita F, Hashida M
Relationship between drug release of DE-310, macromolecular prodrug of DX-8951f, and cathepsins activity in several tumors.
Biol Pharm Bull. 2007 Dec;30(12):2365-70.
DE-310 is composed of the topoisomerase-I inhibitor DX-8951 (exatecan) and carboxymethyldextran polyalcohol (CM-Dex-PA) carrier, which are covalently linked via peptidyl spacer (Gly-Gly-Phe-Gly). In this study, we investigated relationship between the cathepsin activity and the drug release of DE-310 by use of human liver origin cathepsin (B, L and H) and tumor cells (murine tumor cells (Meth A and M5076), and human tumor cells (HCT116, A549, PC-12, T98G, and HL-60)). Preliminary studies indicated that human liver cathepsin B produced Glycyl DX-8951 (G-DX-8951) from DE-310 more preferentially than DX-8951, whereas human liver cathepsin L produced DX-8951 preferentially. Release of drugs from DE-310 and cathepsin activities were measured in tumor cell types. The release of both DX-8951 and G-DX-8951 from DE-310 correlated well with cathepsin B activity of tumor cells. The release of DX-8951 was weakly, but not significantly, correlated with cathepsin L activity. In M5076 (high cathepsin activity) or Meth A (low cathepsin activity) xenograft models, the levels of DX-8951 and G-DX-8951 in M5076 were higher than in Meth A after single intravenous administration of DE-310. Our findings suggest that cathepsin B is primarily responsible for drug release from DE-310 in tumor. [Abstract/Link to Full Text]

Nakamura T, Yamada M, Teshima M, Nakashima M, To H, Ichikawa N, Sasaki H
Electrophysiological characterization of tight junctional pathway of rabbit cornea treated with ophthalmic ingredients.
Biol Pharm Bull. 2007 Dec;30(12):2360-4.
The purpose of this study was to investigate the continuous and real-time influence of ophthalmic ingredients on rabbit cornea by monitoring electrophysiological characteristics. The tight junctional permeabilities of FITC-dextran 4,400 (FD-4) was also determined through the cornea in the presence of ophthalmic ingredients. Intact cornea showed approximately one k-ohmxcm(2) of transepithelial electrical resistance (TEER) and extremely low permeability of FD-4. The ophthalmic ingredients used in the present study were benzalkonium chloride (BK; 0.002%, 0.01%, 0.05%), ethylenediaminetetraacetic acid (EDTA; 0.5%), capric acid (C10; 0.25%), saponin (SP; 0.1%), taurocholic acid (TA; 1.0%) and sodium dodecyl sulfate (SDS; 0.01%). They were previously reported to be effective on corneal penetrations of various drugs at those concentrations without severe toxicity. These ingredients decreased TEER and increased corneal permeability of FD-4. BK reduced TEER in a concentration-dependent manner. There was a significant correlation (gamma=0.860) between the permeability coefficient (Papp) of FD-4 and conductance (Gm), which is the reciprocal value of TEER. It was also indicated that Papp and Gm have a relationship with the corneal cytotoxicity of the ingredients. In conclusion, an electrophysiological method using isolated cornea was very useful to determine the continuous and real-time influence of ophthalmic ingredients on the cornea. In this method, electrophysiological conductance must be able to predict corneal tight junction permeability and the corneal cytotoxicity of ingredients. [Abstract/Link to Full Text]

Kang K, Lee HJ, Kim CY, Lee SB, Tunsag J, Batsuren D, Nho CW
The chemopreventive effects of Saussurea salicifolia through induction of apoptosis and phase II detoxification enzyme.
Biol Pharm Bull. 2007 Dec;30(12):2352-9.
The ethanol extract of the aerial part of the Mongolian medicinal plant Saussurea salicifolia induced a dose-dependent cell growth inhibition in both human gastric adenocarcinoma AGS cells and mouse hepatoma Hepa 1c1c7 cells (IC(50)=30.22 and 116.96 mug/ml), respectively. The extract induced an apoptosis in AGS cells inference from the externalization of the phosphatidylserine, the increase of the sub G0/G1 content (%) and the apoptotic morphological changes including membrane blebbing, the formation of apoptotic bodies and chromatin condensation. In order to identify active substances causing the apoptosis, we further isolated major compounds present in Saussurea salicifolia and 7 compounds were isolated including a sesquiterpene lactone, cynaropicrin, 3 lignans (trachelogenin, matairesinol and arctigenin) and 3 lignan glycosides (tracheloside, matairesinoside and arctiin). In general the lignan aglycones were more cytotoxic than their lignan glycosides in both AGS cells and Hepa 1c1c7 cells. Cynaropicrin not only showed the most potent cytotoxicity among the 7 major compounds but also it induced an apoptosis and a weak G2/M arrest in AGS cells. Arctigenin had the second-best cytotoxicity among 7 major compounds, and induced an apoptosis. In order to evaluate the induction of the phase II detoxification enzyme, we measured the induction of quinone reductase activity of the extract, fractions and compounds in Hepa 1c1c7 cells. The ethyl acetate fraction and arctigenin showed the strongest cancer chemopreventive activity (chemoprevention index=9.88 and 7.57, respectively). These data suggest that the extract as well as the lignan compounds (especially arctigenin) originated from Saussurea salicifolia may be served as potential cancer chemopreventive agents for prevention or treatment of human cancers. [Abstract/Link to Full Text]

Kim JB, Han AR, Park EY, Kim JY, Cho W, Lee J, Seo EK, Lee KT
Inhibition of LPS-induced iNOS, COX-2 and cytokines expression by poncirin through the NF-kappaB inactivation in RAW 264.7 macrophage cells.
Biol Pharm Bull. 2007 Dec;30(12):2345-51.
We previously reported that poncirin, a flavanone glycoside isolated from the EtOAc extract of the dried immature fruits of Poncirus trifoliata, is an anti-inflammatory compound that inhibits PGE(2) and IL-6 production. The present work was undertaken to investigate the molecular actions of poncirin in RAW 264.7 macrophage cell line. Poncirin reduced lipopolysaccharide (LPS)-induced protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and the mRNA expressions of iNOS, COX-2, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in a concentration-dependent manner, as determined by Western blotting and RT-PCR, respectively. Furthermore, poncirin inhibited the LPS-induced DNA binding activity of nuclear factor-kappaB (NF-kappaB). Moreover, this effect was accompanied by a parallel reduction in IkappaB-alpha degradation and phosphorylation that in by nuclear translocations of p50 and p65 NF-kappaB subunits. Taken together, our data indicate that anti-inflammatory properties of poncirin might be the result from the inhibition iNOS, COX-2, TNF-alpha and IL-6 expression via the down-regulation of NF-kappaB binding activity. [Abstract/Link to Full Text]

Cho HJ, Shon YH, Nam KS
Ginkgolide C inhibits platelet aggregation in cAMP- and cGMP-dependent manner by activating MMP-9.
Biol Pharm Bull. 2007 Dec;30(12):2340-4.
In this report, we investigated the effect of ginkgolide C (GC) from Ginkgo biloba leaves in collagen (10 mug/ml)-stimulated platelet aggregation. It has been known that matrix metalloproteinase-9 (MMP-9) is released from human platelets, and that it significantly inhibited platelet aggregation stimulated by collagen. Zymographic analysis confirmed that pro-MMP-9 (92-kDa) was activated by GC to form an activated MMP-9 (86-kDa) on gelatinolytic activities. And then, GC dose-dependently inhibited platelet aggregation, intracellular Ca(2+) mobilization, and thromboxane A(2) (TXA(2)) formation in collagen-stimulated platelets. In addition, GC significantly increased the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which have an anti-platelet function in both resting and collagen-stimulated platelets. Therefore, we demonstrate that the inhibitory effect of GC on platelet aggregation might be involved into the following pathways. GC may increase intracellular cAMP and cGMP production and MMP-9 activity, inhibit intracellular Ca(2+) mobilization and TXA(2) production, thereby leading to inhibition of platelet aggregation. These results strongly indicate that GC is a potent inhibitor of collagen-stimulated platelet aggregation. It may be a suitable tool for a negative regulator during platelet activation. [Abstract/Link to Full Text]

Samanta S, Alam SM, Basu S, Maji T, Roy DK, Jha T
Chemoimmunotherapeutic approach to prolonged survival time in combination with immunization and glutamic Acid derivatives with antitumor activity in tumor-bearing mice.
Biol Pharm Bull. 2007 Dec;30(12):2334-9.
Cancer is one of the major causes of death. For cancer, the general conventional treatment and standard of care for clinical oncology remains surgery followed by radiation and/or systemic chemotherapy as deemed appropriate based on the clinical findings. Chemoimmunotherapy is an approach to treat cancer where chemotherapy is given along with immunotherapy. Chemoimmunotherapy may be useful to enhance survival time in cancer by improve immunity of the patients. This approach may enhance the therapeutic efficacy. A comparative study was done to assess the therapeutic efficacy of the whole cell vaccine and the tumor extract with or without combination chemotherapy with the synthesized glutamine and glutamic acid derivatives and analogs as well as the standard drug etoposide against Ehrlich Ascites Carcinoma (EAC) cells in Swiss Albino mice. The study showed promising results with the compound 5-N-n-hexyl-2-(4-iso-butylbenzenesulphonyl)glutamine. The compound when combined with the whole cell vaccine as well as the tumor extract increases the survival time and the therapeutic efficacy which is comparable with that of standard drug etoposide. [Abstract/Link to Full Text]

Nonaka R, Nagai F, Ogata A, Satoh K
In vitro screening of psychoactive drugs by [(35)S]GTPgammaS binding in rat brain membranes.
Biol Pharm Bull. 2007 Dec;30(12):2328-33.
We constructed a reproducible, simple, and small-scale determination method of the psychoactive drugs that acted directly on the monoamine receptor by measuring the activation of [(35)S]guanosine-5'-O-(3-thio)-triphosphate binding to guanine nucleotide-binding proteins (G proteins). This method can simultaneously measure the effects of three monoamines, namely dopamine (DA), serotonin (5-HT), and norepinephrine (NE), in rat brain membranes using a 96-well microplate. Activation of D(1) and D(2) receptors in striatal membranes by DA as well as 5-HT and NEalpha(2) receptors in cortical membranes could be measured. Of 12 tested phenethylamines, 2,5-dimethoxy-4-chlorophenethylamine (2C-C), 2,5-dimethoxy-4-ethylphenethylamine (2C-E), and 2,5-dimethoxy-4-iodophenethylamine (2C-I) stimulated G protein binding. The other phenethylamines did not affect G protein binding. All 7 tryptamines tested stimulated G protein binding with the following rank order of potency; 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT)>5-methoxy-N,N-diallyltryptamine (5-MeO-DALT)>5-methoxy-alpha-methyltryptamine (5-MeO-AMT)>or=5-methoxy-N,N-methylisopropyltryptamine (5-MeO-MIPT)>5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT)>N,N-dipropyltryptamine (DPT)>or=alpha-methyltryptamine (AMT). This assay system was able to designate psychoactive drugs as prohibited substances in accordance with criteria set forth by the Tokyo Metropolitan government. [Abstract/Link to Full Text]

Okutsu H, Noguchi Y, Ohtake A, Suzuki M, Sato S, Sasamata M
Effects of intravenously and orally administered solifenacin succinate (YM905) on carbachol-induced intravesical pressure elevation and salivary secretion in mice.
Biol Pharm Bull. 2007 Dec;30(12):2324-7.
Solifenacin succinate is a novel muscarinic receptor antagonist used for the treatment of overactive bladder (OAB). We investigated the effects of solifenacin by oral and intravenous administration on carbachol (CCh)-induced intravesical pressure (IVP) elevation and compared its efficacy with that on CCh-induced salivary secretion in anesthetized mice. Additionally, we also investigated the change in effects between single and repeated oral administration of solifenacin on CCh-induced IVP elevation. Results showed that intravenous administration of solifenacin dose-dependently inhibited the IVP elevation and salivary secretion. The ratio of bladder response to salivary response (ratio of ID(50) values) was 2.1. Oral administration of solifenacin (0.3-30 mg/kg) also inhibited CCh-induced IVP elevation and salivary secretion. Although inhibition of these responses by solifenacin (10, 30 mg/kg) was comparable at early time points (0.5 and 1 h after administration at 10 mg/kg and 0.5 to 2 h after administration at 30 mg/kg), inhibition of CCh-induced IVP elevation was stronger at later time points (2 to 8 h after administration at 10 mg/kg and 4 to 24 h after administration at 30 mg/kg). No significant difference in ID(50) values for IVP elevation was observed between single and repeated (11 d) oral administration of solifenacin (1-30 mg/kg), suggesting no change in efficacy on chronic administration. In conclusion, intravenous and oral solifenacin inhibits CCh-induced IVP elevation more potently than salivary secretion. These results provide further evidence for the clinical use of solifenacin as a promising therapeutic drug for OAB with a low incidence of dry mouth. [Abstract/Link to Full Text]

Koga N, Matsuo M, Ohta C, Haraguchi K, Matsuoka M, Kato Y, Ishii T, Yano M, Ohta H
Comparative study on nobiletin metabolism with liver microsomes from rats, Guinea pigs and hamsters and rat cytochrome p450.
Biol Pharm Bull. 2007 Dec;30(12):2317-23.
In vitro metabolism of nobiletin, a polymethoxy-flavonoid abundantly present in citrus peels, was studied using liver microsomes of rats, hamsters and guinea pigs and ten cDNA-expressed rat cytochrome P450 (P450). The effects of P450 inducers on nobiletin metabolism were also investigated. Aerobical incubation with NADPH and animal liver microsomes transformed nobiletin to five metabolites, M-1, M-2, M-3, M-4 and M-5. From LC-MS and (1)H-NMR data and a time-course study, these were assumed to be 4'-hydroxy (OH)-, 7-OH-, 6-OH-, 3',4'-diOH- and 6,7-diOH-metabolites, respectively. Pretreatment of animals with phenobarbital increased M-2 and M-3 to about 2-fold that in untreated animals. Pretreatment with 3-methylcholanthrene (MC) resulted in remarkable increases of both M-1 and M-4 (3 to 9-fold that of untreated). Males had 2-3 times higher M-2 and M-3 formation activities in rats, and for M-2 in hamsters than did females. Immunoinhibition study using antiserum against P450 revealed the involvement of hamster CYP1A2 in the formation of M-1 and M-4 in hamster liver. Of ten rat P450s, CYP2C11, CYP3A1, CYP3A2 and CYP2D1 had high activities for the formation of M-1, M-2 and M-3. Another P450s (CYP1A1, CYP2C12 and CYP1A2) also showed activity for the formation of M-1. Only CYP1A1 produced 3',4'-diOH-metabolites (M-4). However, CYP2A1, CYP2B1 and CYP2E1 had no activity for nobiletin. These results suggested that constitutive P450s such as CYP2C11, CYP2D1, CYP3A1, CYP3A2 and CYP2C12 are responsible for the demethylation at the 6-, 7-, 3'- and 4'-positions; whereas, MC-inducible P450s, CYP1A1 and CYP1A2, preferentially catalyzed demethylation at the 3'-and 4'-positions. [Abstract/Link to Full Text]

Nameda S, Miura NN, Adachi Y, Ohno N
Lincomycin protects mice from septic shock in beta-glucan-indomethacin model.
Biol Pharm Bull. 2007 Dec;30(12):2312-6.
We have developed a septic shock model in mice by sequential administration of beta-glucan, a biological response modifier, and indomethacin (IND), a nonsteroidal anti-inflammatory drug. Lethality was significantly related to the translocation of gut flora to various organs and mal-adjustment of the cytokine network. In the present study, we have examined the effect of antibiotics on this model to further clarify meanings of microbial flora. Schizophyllan (SPG), antitumor beta-glucan for clinical use, obtained from the culture filtrate of Schizophyllum commune, was used to induce sepsis. Lincomycin (LCM), imipenem (IPM), cilastatine (CS), and ampicillin (ABPC) were used for antibiotics treatment. The survival rate of SPG/IND-treated mice was significantly increased by administering LCM or ABPC/IPM/CS, and the effect was more significant by LCM. In in vitro spleen cell culture, LCM decreased proinflammatory cytokine production. Moreover, prednisolone, immune suppresser treatment improved survival of SPG/IND-treated mice. These findings suggest that LCM is an effective antibiotic in this endogenous septic model by modulating gut microbial flora and, at least a part, by regulating cytokine production of leukocytes. [Abstract/Link to Full Text]

Kiyota A, Matsushita T, Ueoka R
Induction and high density culture of human hepatoblasts from fetal hepatocytes with suppressing transformation.
Biol Pharm Bull. 2007 Dec;30(12):2308-11.
It is well known that it would be important to cultivate human hepatocytes of about 10(10) cells at a high cell density, about 1 x 10(7) cells/cm(3), in the bioreactor for the development of bioartificial liver. However, since primary human hepatocytes lack an ability to proliferate in vitro, it is essential to establish a culture method for the proliferation of normal human hepatic stem cells as a cell source. In this study, it was found that human hepatoblasts, a kind of hepatic stem cells, were induced from human fetal hepatocytes while keeping the ability of proliferation by the treatment of 1mM sodium butyrate (SB) for 12 d of culture. The transformation of hepatoblasts was evaluated by abnormal prothrombin (PIVKA-II) assay, which is a clinical marker for hepatocellular carcinoma. The PIVKA-II production rate of the cells was suppressed to the normal level under 1 mM SB. The cells including hepatoblasts under 1 mM SB attached to the porous hydroxyapatite carriers and proliferated to a high cell density of about 1 x 10(7) cells/cm(3) in the carriers. The liver-specific function, cytochrome P450 3A4 activity (4.2 pmol/mg protein/min) of the cells in the carriers under 1 mM SB was comparable to that of primary human hepatocytes. Ammonia metabolizing activity (0.21 micromol/10(6) cells/h) of the cells was also comparable to that of porcine hepatocytes used in the bioartificial liver. The PIVKA-II production rate of the cells in the carrier was suppressed to the normal level. These results suggested that induction of human hepatoblasts from fetal hepatocytes by the treatment of 1mM SB and proliferation of the cells at a high cell density using hydroxyapatite carriers should be one of the more promising culture methods for bioartificial liver developments. [Abstract/Link to Full Text]

Katanasaka Y, Asai T, Naitou H, Ohashi N, Oku N
Proteomic characterization of angiogenic endothelial cells stimulated with cancer cell-conditioned medium.
Biol Pharm Bull. 2007 Dec;30(12):2300-7.
To characterize the protein expression profiles and identify the molecules associated with tumor angiogenesis, the cellular proteins of human umbilical vein endothelial cells (HUVECs) in response to cancer cell-conditioned medium (CM) prepared from HT1080 human fibrosarcoma cells were analyzed using fluorescence-labeled 2D gel-based proteomics. Most differentially expressed proteins in HT1080-CM-stimulated cells were found to be downregulated (88%) rather than upregulated (12%) based on statistical analysis of protein spot signals. Additionally, we examined the effects of vascular endothelial cell growth factor (VEGF), a proangiogenic factor, on cellular protein expression. In contrast, most differentially expressed proteins were found to be upregulated (59%) rather than downregulated (41%) in VEGF-stimulated HUVECs. Comparative analyses of 29 and 35 protein species identified in CM-stimulated and VEGF-stimulated HUVECs, respectively, revealed the remarkable differences between these two stimulations. Only four proteins were differentially expressed by both treatments: annexin A2, enolase 1, and T-plastin (downregulated by CM but upregulated by VEGF), and RAN (downregulated by both CM and VEGF). These findings provide new information regarding the regulation of protein expression associated with tumor angiogenesis. [Abstract/Link to Full Text]

Ootsuka S, Asami S, Sasaki T, Yoshida Y, Nemoto N, Shichino H, Chin M, Mugishima H, Suzuki T
Analyses of novel prognostic factors in neuroblastoma patients.
Biol Pharm Bull. 2007 Dec;30(12):2294-9.
Neuroblastoma (NB) is the most common malignant solid tumor in childhood. There are well-recognized prognostic factors in NB such as age at diagnosis, organ of origin, stages, MYCN gene amplification, and expression of H-ras, trkA and survivin. Moreover, we investigated the expression of vascular endothelial growth factor (VEGF), tyrosine hydroxylase (TH), p53, stem cell factor (SCF) and c-kit of its receptor with quantitative real-time polymerase chain reaction (PCR) in 22 NBs and 4 other tumors (one malignant lymphoma, one malignant teratoma, and 2 rhabdomyosarcomas) samples. The correlation between patients' prognoses and the expression of TH or c-kit was newly recognized, particularly the good prognosis in patients in whom c-kit highly expressed and the poor prognosis contrarily associated with low or no expression, although the SCF of its ligand had no relationship with patient prognosis. It is possible that tumors without c-kit expression can not react with SCF (via the autocrine or paracrine system) and remain immature. It may be that this is a new critical clinical event in NB patients. [Abstract/Link to Full Text]

Chang HJ, Yoon G, Park JS, Kim MH, Baek MK, Kim NH, Shin BA, Ahn BW, Cheon SH, Jung YD
Induction of apoptosis by the licochalcone E in endothelial cells via modulation of NF-kappaB and Bcl-2 Family.
Biol Pharm Bull. 2007 Dec;30(12):2290-3.
Licochalcones have a variety of biological properties including anti-tumor, anti-parasitic and anti-bacterial activities. Recently, a new retrochalcone (licochalcone E, Lico-E) was isolated from the roots of Glycyrrhiza inflata (Chem. Pharm. Bull., 53, 2005, Yoon et al.) by cytotoxicity-guided fractionation. This study examined whether or not Lico-E-induced endothelial cell death occurs through apoptosis, and investigated molecular mechanisms involved in this process. Lico-E was found to suppress ECV304 cell growth and induce apoptosis. The induction of apoptosis by Lico-E was confirmed by the ladder-patterned DNA fragmentation, the presence of cleaved and condensed nuclear chromatin and the increased number of annexin V-positive cells. Lico-E could effectively inhibit the constitutive NF-kappaB activation, as revealed by the electrophoretic mobility shift assay and NF-kappaB-dependent luciferase reporter study. In addition, the Lico-E treatment caused a change in the Bax/Bcl-2 ratio that favored apoptosis. These results suggest that Lico-E induces endothelial cell apoptosis by modulating NF-kappaB and the Bcl-2 family. [Abstract/Link to Full Text]

Azuma Y, Miura K, Higai K, Matsumoto K
Protein O-N-acetylglucosaminylation modulates promoter activities of cyclic AMP response element and activator protein 1 and enhances E-selectin expression on HuH-7 human hepatoma cells.
Biol Pharm Bull. 2007 Dec;30(12):2284-9.
High glucose accelerates O-N-acetylglucosaminylation (O-GlcNAcylation) of proteins and causes diabetic complications. In the present study, we found that treatment of HuH-7 human hepatoma cells with high glucose or the protein O-N-acetylglucosaminidase (O-GlcNAcase) inhibitor O-(2-acetoamide-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) increased the cell surface expression of E-selectin. A dual luciferase reporter assay indicated that high glucose and PUGNAc suppressed promoter activities of the cyclic AMP response element (CRE) and enhanced those of activator protein 1 (AP-1). Enhanced CRE promoter activities in HuH-7 cells treated with dibutyryl cAMP or co-transfected with a protein kinase A expression vector pFC-PKA that enhances the phosphorylation of CRE binding protein (CREB) were suppressed by PUGNAc. In contrast, PUGNAc further increased the enhanced AP-1 promoter activity in cells transfected with a mitogen-activated protein kinase kinase kinase expression vector pFC-MEKK that enhances c-Jun phosphorylation. Immuno-blotting using an anti-O-GlcNAc antibody revealed that high glucose and PUGNAc accelerated protein O-GlcNAcylation and that there were substantial differences in the O-GlcNAcylated proteins in the cytoplasmic and nuclear fractions. In addition, PUGNAc increased the nuclear import of O-GlcNAcylated CREB. These results suggest that protein O-GlcNAcylation modulates the promoter activities of E-selectin gene, suppression of CRE and enhancement of AP-1, and enhances E-selectin protein expression on hepatocytes. [Abstract/Link to Full Text]

Liu F, Xie ZH, Cai GP, Jiang YY
The effect of survivin on multidrug resistance mediated by P-glycoprotein in MCF-7 and its adriamycin resistant cells.
Biol Pharm Bull. 2007 Dec;30(12):2279-83.
Although anticancer chemotherapeutic drugs have been designed to inhibit the growth of tumor cells, chemotherapy frequently fails due to the development of multidrug resistance (MDR). In this paper, the effect of survivin on multidrug resistance mediated by P-glycoprotein (Pgp) was investigated in breast cancer cells. Overexpression of survivin in MCF-7 cells transfected with survivin expression vector pEGFP/survivin results in decreasing sensitivity to anticancer drugs and activation of Pgp to export drug out of cells. Down regulation of survivin in MCF-7/adriamycin (ADR) transfected with RNAi directed against survivin vector psh1/survivin could increase the drug accumulation in cells by inhibiting Pgp. Downregulation of the expression of the Pgp with the specific inhibitor verapamil could markedly suppress the survivin mRNA expression, whereas the reverse impact was not observed. Survivin might modulate the turnover of Pgp or transport by Pgp in cells, which result in anti-apoptosis and drug resistance. Our results suggest that survivin might play a key role in MDR in the presence of Pgp, and this might represent a novel strategy for modulating MDR in cancer cells. [Abstract/Link to Full Text]

Yin DK, Yao WB, Gao XD
Chemically modified heparin inhibits mesangial cell proliferation induced by high glucose through interfering with the cell cycle.
Biol Pharm Bull. 2007 Dec;30(12):2274-8.
The aims of this study were to investigate whether chemically modified non-anticoagulation heparin derivate (Periodate-Oxidized/Borohydride-Reduced modified heparin (OR-heparin)) can inhibit high glucose-induced human mesangial cell proliferation and its influence on the cell cycle. OR-heparin with low anticoagulation activity inhibited high glucose-induced early proliferation in a dose-dependent manner. OR-heparin released high glucose-arrested mesangial cells at G(1) phase, and dose-dependently increased S phase. OR-heparin also inhibited high glucose-activated ERK1/2 phosphorylation, induced p27(Kip1) expression, and suppressed reactive oxygen species (ROS) accumulation in a dose-dependent manner. Our results suggest that OR-heparin releases high glucose-arrested cells on G(1) phase and inhibits high glucose-induced mesangial cell proliferation through blocking ERK1/2 phosphorylation and delaying S phase progression, which may be in correlation with OR-heparin suppressing ROS accumulation. [Abstract/Link to Full Text]

Kamaraj S, Vinodhkumar R, Anandakumar P, Jagan S, Ramakrishnan G, Devaki T
The effects of quercetin on antioxidant status and tumor markers in the lung and serum of mice treated with benzo(a)pyrene.
Biol Pharm Bull. 2007 Dec;30(12):2268-73.
Chemoprevention has emerged as a very effective preventive measure against carcinogenesis. Several bioactive compounds present in fruits and vegetables have revealed their cancer curative potential on benzo(a)pyrene (B(a)P) induced carcinogenesis. In the present study, the efficacy of quercetin on the level of lipid peroxides, activities of antioxidant enzymes and tumor marker enzymes in B(a)P induced experimental lung carcinogenesis in Swiss albino mice was assessed. In lung cancer bearing animals there was an increase in lung weight, lipid peroxidation and marker enzymes such as aryl hydrocarbon hydroxylase, gamma glutamyl transpeptidase, 5'-nucleotidase, lactate dehydrogenase and adenosine deaminase with subsequent decrease in body weight and antioxidant enzymes-superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, glutathione reductase, reduced glutathione, vitamin E and vitamin C. Quercetin supplementation (25 mg/kg body weight) attenuated all these alterations, which indicates the anticancer effect that was further confirmed by histopathological analysis. Overall, the above data shows that the anticancer effect of quercetin is more pronounced when used as an chemopreventive agent rather than as a chemotherapeutic agent against B(a)P induced lung carcinogenesis. [Abstract/Link to Full Text]

Morita H, Mizuuchi Y, Abe T, Kohno T, Noguchi H, Abe I
Cloning and functional analysis of a novel aldo-keto reductase from Aloe arborescens.
Biol Pharm Bull. 2007 Dec;30(12):2262-7.
A novel aldo-keto reductase (AKR) was cloned and sequenced from roots of Aloe arborescens by a combination of RT-PCR using degenerate primers based on the conserved sequences of plant polyketide reductases (PKRs) and cDNA library screening by oligonucleotide hybridization. A. arborescens AKR share similarities with known plant AKRs (40-66% amino acid sequence identity), maintaining most of the active-site residues conserved in the AKR superfamily enzymes. Interestingly, despite the sequence similarity with PKRs, recombinant enzyme expressed in Escherichia coli did not exhibit any detectable PKR activities. Instead, A. arborescens AKR catalyzed NADPH-dependent reduction of various carbonyl compounds including benzaldehyde and DL-glyceraldehyde. Finally, a homology model on the basis of the crystal structure of Hordeum vulgare AKR predicted the active-site architecture of the enzyme. [Abstract/Link to Full Text]

Coban TA, Beydemir S, G�l�in I, Ekinci D
Morphine inhibits erythrocyte carbonic anhydrase in vitro and in vivo.
Biol Pharm Bull. 2007 Dec;30(12):2257-61.
Morphine is implicated in diverse functions, from development to immune modulation in the central and peripheral nervous systems. At the present time, morphine is one of the most effective antinociceptive agents used to manage pain. It has been used extensively in the clinical management of pain due to its potent analgesic effect. In this study, the in vitro and in vivo inhibitory effects of morphine on erythrocyte carbonic anhydrase (CA) were investigated. Human erythrocyte isoenzymes, HCA-I and HCA-II, were purified by Sepharose-4B affinity chromatography column with a yield of 66.95 and 62.82%, a specific activity of 3892.3 and 11663.2 EU/mg proteins with 745.1 and 2232.6-fold purification of each isoenzyme, respectively. To determine enzyme purity, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. In vitro inhibition of erythrocyte HCA-I and HCA-II by morphine using the CO(2)-hydratase enzyme gave IC(50) values 4.50 x 10(-5) M (r(2): 0.954) and 9.23 x 10(-5) M (r(2): 0.996), respectively. CA activity was significantly attenuated in vivo in Spraque-Dawley rats for up to 3 h (p<0.001) following intraperitoneal administration of morphine. In conclusion, morphine inhibited CA activity both in vitro and in vivo. [Abstract/Link to Full Text]

Obi R, Tohda M, Zhao Q, Obi N, Hori H, Murakami Y, Goto H, Shimada Y, Ochiai H, Matsumoto K
Chotosan enhances macrophage colony-stimulating factor mRNA expression in the ischemic rat brain and C6Bu-1 glioma cells.
Biol Pharm Bull. 2007 Dec;30(12):2250-6.
Macrophage colony stimulating factor (M-CSF) is a cytokine which has been recently reported to have a neuroprotective effect on ischemic rat brain. In this study, we investigated the effect of chotosan, an oriental medicine, which has been clinically demonstrated to be effective for the treatment of vascular dementia, on M-CSF gene expression in rats with permanent occlusion of bilateral common carotid arteries (P2VO) in vivo and in a C6Bu-1 glioma cell line in vitro. The expression level of M-CSF mRNA in the cerebral cortices of P2VO rats was significantly higher than that in the cerebral cortices of sham-operated animals. Repeated treatment of P2VO rats with chotosan (75 mg/kg per day) for 4 d after P2VO significantly increased the expression level of M-CSF mRNA in the cortex but it had no effect on the expression of beta-actin, granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF) mRNAs. Moreover, the present in vitro studies revealed that chotosan treatment (10-100 mug/ml) of C6Bu-1 glioma cells dose-dependently enhanced M-CSF mRNA expression without affecting the expression of G-CSF, GM-CSF, and inducible nitric oxide synthase mRNAs. The effect of chotosan was reversed by Ro 31-8220 (1 muM), a selective protein kinase C (PKC) inhibitor, but not by H-89 (10 muM), a selective protein kinase A (PKA) inhibitor. These findings suggest that the upregulatory effect of chotosan on M-CSF mRNA expression involves PKC and may play an important role in the anti-vascular dementia action of this formula. [Abstract/Link to Full Text]

Go EK, Jung KJ, Kim JM, Lim H, Lim HK, Yu BP, Chung HY
Betaine modulates age-related NF-kappaB by thiol-enhancing action.
Biol Pharm Bull. 2007 Dec;30(12):2244-9.
Depletion of glutathione levels and perturbations in redox status are considered to play a crucial role in aging and chronic inflammatory processes through the activation of redox sensitive transcription factors, including nuclear factor-kappaB (NF-kappaB). In the current study, we assessed the regulatory action of dietary betaine in the suppression of NF-kappaB by comparing kidney tissue from old, betaine-supplemented rats or non-betaine-supplemented rats (age 21 months) and 7 month-old rats. In addition, cultured HEK 293T cells were utilized for the molecular assessment of betaine's restorative ability of redox status when treating cells with potent glutathione (GSH)-depleting agents. Results showed that in old rats a short-term feeding (10 d) with betaine attenuated the age-related decrease in thiol levels, increase in reactive species and TNFalpha expression via NF-kappaB activation, compared to the young controls. These findings were verified in the cell-cultured system. Further investigations found that redox imbalance due to thiol depletion caused increased NF-kappaB activation, and cyclooxygenase (COX)-2 and TNFalpha levels, both of which were suppressed by betaine treatment. Based on both in vivo and in vitro data, we concluded that betaine exerts its efficacy by maintaining thiol status in the regulation of COX-2 and TNFalpha via NF-kappaB activation during aging. [Abstract/Link to Full Text]

Recent Articles in Chemical & Pharmaceutical Bulletin (Tokyo)

Hayashi K, Kujime E, Katayama H, Sano S, Nagao Y
Effects of magnesium salts and amines on the stereoselectivity in the imine aldol reaction.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1773-5.
In the imine aldol reactions of 1 with aromatic aldehydes using magnesium salts in the presence of amines, the threo/erythro ratios of products increased in the order Mg(ClO4)2>MgI2>MgBr2>MgCl2>Mg(OTf)2 and N,N,N',N'-tetramethylethylenediamine (TMEDA)>Et3N. This increase in the threo/erythro ratios of products was estimated to be caused by a retro-imine aldol reaction under thermodynamic control. [Abstract/Link to Full Text]

Kusano R, Tanaka T, Matsuo Y, Kouno I
Structures of epicatechin gallate trimer and tetramer produced by enzymatic oxidation.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1768-72.
During black tea production, catechins and their galloyl esters are enzymatically oxidized to generate a complex mixture of black tea polyphenols. The role of galloyl ester groups in this process has yet to be determined. Enzymatic oxidation of epicatechin 3-O-gallate (1) yielded two new oxidation products, theaflavate C and bistheaflavate A, along with theaflavate A (2), a known dimer of 1 generated by coupling of the B-ring with the galloyl group. Theaflavate C is a trimer of 1 and possesses two benzotropolone moieties generated by the oxidative coupling of the galloyl groups with the catechol B-rings. Bistheaflavate A was found to be a tetramer produced by intermolecular coupling of two benzotropolone moieties of 2. From the structures of the products, it was deduced that oxidative coupling of galloyl groups resulted in extension of the molecular size of the products in catechin oxidation. [Abstract/Link to Full Text]

Sasaki H
Synthesis of 2,2'-Bis(3,6,9-triazanonyl)-4,4'-bithiazole and related compounds as new DNA cleavage agents.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1762-7.
Two new bithiazole derivatives, 2,2'-bis(3,6,9-triazanonyl)- and 2,2'-bis(3,7,11-triazaundecyl)-4,4'-bithiazoles (3a, b), were readily synthesized in six steps using the corresponding dialkylenetriamine as starting materials. Under physiological conditions, 5.0 microM 3a exhibited significant DNA cleavage activity in the presence of Co(II), whereas even at 50 micriM, 3b exhibited no DNA cleavage activity. Furthermore, it was demonstrated that 3a forms a 1 : 2 complex with Co(II) ions, whereas 3b does not. These conclusions were based on measurements of stoichiometries of the bithiazole-cobalt complexes obtained by the Job continuous variation method. In contrast, 3a, which contains diethylenetriamine moieties, showed decreased affinity for Calf Thymus (CT) DNA compared with that of 3b, which contains dipropylenetriamine moieties. These findings indicate that the structure of the two aminoalkyl side chains attached at the 2- and 2'-positions of the 4,4'-bithiazole ring significantly influence the formation of cobalt complexes, and affects the compound's ability to cleave DNA as well as its affinity for double-stranded DNA. [Abstract/Link to Full Text]

Kurosu M, Katayama S, Shibuya H, Kitagawa I
A study of the calcium complex of a glucosylceramide, soya-cerebroside II.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1758-61.
In order to study calcium ion complex of soya-cerebroside II (1), an ionophoretic glucosylceramide isolated from soybean, C8-cerebroside (3) and 3,3'',6''-trideoxy-C8-cerebroside (4) are designed and synthesized. On the basis of extensive 1H-NMR studies in the presence of Ca2+ and a continuous variation method via (1)H-NMR, soya-cerebroside II is suggested to form a calcium complex with 1/Ca2+ ratio of 1 : 1. Soya-cerebroside II serves as a tridentate chelating ligand for Ca2+; the amide carbonyl, C2'-hydroxy, and C2''-hydroxy oxygens are responsible for the Ca2+ binding. Soya-cerebroside II is structurally analogous to a neural glucosylceramide. Thus, the accumulated neural glucosylceramide inside of endoplasmic reticulum (ER) membrane may serve as an endogenous Ca2+-binding and -transport molecule (ionophore) that result in mobilization of Ca2+ from intracellular calcium stores. [Abstract/Link to Full Text]

Xu H, Jian KZ, Guan Q, Ye F, Lv M
Antifungal activity of some diaryl ethers.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1755-7.
Several diaryl ethers were synthesized and tested in vitro against seven phytopathogenic fungi, namely Fusarium graminearum, Alternaria alternate, Helminthosporium sorokinianum, Pyricularia oryzae, Fusarium oxysporum f. sp. vasinfectum, Fusarium oxysporum f. sp. cucumarinum and Alternaria brassicae. Compared to a commercial agricultural fungicide, hymexazol, especially compounds a, b, e, g and k were found to be more effective at 50 mug/ml against F. graminearum, F. oxysporum f. sp. vasinfectum and F. oxysporum f. sp. cucumarinum. Meantime, some structure-activity relationships were also observed. [Abstract/Link to Full Text]

Noguchi-Yachide T, Miyachi H, Aoyama H, Aoyama A, Makishima M, Hashimoto Y
Structural development of liver X receptor (LXR) antagonists derived from thalidomide-related glucosidase inhibitors.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1750-4.
Following our previous discovery of LXR antagonistic activity of 2'-substituted phenylphthalimides derived from thalidomide-related glucosidase inhibitors, structure-activity studies and further structural development led to 5-chloro-N-2'-n-pentylphenyl-1,3-dithiophthalimide (5CPPSS-50), with IC50 values of about 10 and 13 microM for LXRalpha and LXRbeta, respectively. [Abstract/Link to Full Text]

Hasegawa T, Ishibashi M, Takata T, Takano F, Ohta T
Cytotoxic fatty acid from Pleurocybella porrigens.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1748-9.
The new conjugated ketonic fatty acid, porrigenic acid (1), was isolated as a cytotoxic constituent of Pleurocybella porrigens. The structure of 1 was elucidated using spectroscopic methods including 1D and 2D NMR and MS. The absolute stereochemistry of 1 was determined by application of the exciton chirality method. Compound 1 exhibited cytotoxic activity against myeloma THP-1 cells, but did not show any significant toxicity against B16F1 melanoma. This is the first report of the isolation and structural elucidation of the new cytotoxic constituent porrigenic acid (1) from the edible mushroom P. porrigens. [Abstract/Link to Full Text]

Yu HW, Li BG, Li GY, Li CS, Fang DM, Zhang GL
Abietane lactones and iridoids from Goldfussia yunnanensis.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1744-7.
Two new abietane diterpene lactones (1--2), three new abietane diterpene lactone glycosides (3--5) and a new iridoid glycoside (6), together with five known compounds, were isolated from the aerial parts of Goldfussia yunnanensis. The new compounds were determined to be 18-hydroxyhelioscopinolide A (1), 18-oxohelioscopinolide A (2), 18-hydroxy-3-O-beta-D-glucopyranosylhelioscopinolide A (3), 3-O-beta-D-glucopyranosylhelioscopinolide A (4), 3-O-beta-D-galactopyranosylhelioscopinolide A (5), and 6-O-trans-cinnamoyl E-harpagoside (6) on the basis of spectral data and chemical evidence. [Abstract/Link to Full Text]

Chiang CC, Mouscadet JF, Tsai HJ, Liu CT, Hsu LY
Synthesis and HIV-1 integrase inhibition of novel bis- or tetra-coumarin analogues.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1740-3.
Present studies were undertaken on the preparation of synthetic analogues of bis- or tetra-coumarins and their activity against HIV-1 integrase (HIV-1 IN). Among these coumarin analogues, compounds 14, 16 and 18 were found to be potent molecules against HIV-1 IN at IC50 values of 0.96, 0.58, and 0.49 microM, respectively. The results provided a tool for guiding the further design of more potent antiviral agents and for predicting the affinity of related compounds. [Abstract/Link to Full Text]

Bang SC, Seo HH, Yun HY, Jung SH
Facile synthesis of trisaccharide moiety corresponding to antitumor activity in triterpenoid saponins isolated from Pullsatilla roots.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1734-9.
A trisaccharide found in triterpenoid saponins isolated from Pullsatilla roots appears as an important promoiety for the enhancement of anticancer activity of their aglycones. Thus a facile synthetic method for a trisaccharide moiety, allyl-2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->2)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->4)]-3-O-benzoyl-beta-L-arabinopyranoside (3), has been firstly developed through the regio- and stereoselective glycosylations from arabinose in total 16% yield via route 2 (eight steps). In this synthetic procedure, the protection of anomeric -OH of L-arabinose with equatorially oriented allyl group unlike with the axial 4-methoxybenzyl protecting group well promoted glycosyl bond formation between alpha-L-rhamnopyranosyl trichloroacetimidate and 2-OH of arabinose. As expected, the synthesized trisaccharide moiety 3 has no cytotoxicity (ED50: >100 microM) against three human cancer cell lines (A-549, SK-OV-3, and SK-MEL-2), respectively. [Abstract/Link to Full Text]

Takahashi Y, Yamada M, Kubota T, Fromont J, Kobayashi J
Metachromins R--T, new sesquiterpenoids from marine sponge Spongia sp.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1731-3.
Three new sesquiterpenoids, metachromins R--T (1--3), have been isolated from an Okinawan marine sponge Spongia sp. The structures and stereochemistry of 1--3 were elucidated on the basis of the spectroscopic data. Metachromins S (2) and T (3) showed modest cytotoxicity. [Abstract/Link to Full Text]

Okimura K, Ohki K, Sato Y, Ohnishi K, Sakura N
Semi-synthesis of polymyxin B (2-10) and colistin (2-10) analogs employing the Trichloroethoxycarbonyl (Troc) group for side chain protection of alpha,gamma-diaminobutyric acid residues.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1724-30.
Improved strategies for the chemical conversion of natural polymyxin B and colistin to their N-terminal analogs are reported. First, the protection of the side chains of five L-alpha,gamma-diaminobutyric acid (Dab) residues in natural polymyxin B and colistin was achieved with trichloroethoxycarbonyl (Troc), then the resulting pentakis(N gamma-Troc)-polymyxin B and pentakis(N gamma)Troc)-colistin were treated with trifluoroacetic acid (TFA) : methanesulfonic acid (MSA) : dimethylformamide (DMF) : H2O (10 : 30 : 55 : 5) at 40 degrees C in order to remove N alpha-alkanoyl-Dab(Troc)-OH selectively. The new key compounds, tetrakis(N gamma-Troc)-polymyxin B (2-10) and tetrakis(N gamma-Troc)-colistin (2-10), were obtained in 19% and 15% yields, respectively, which is higher than previous reports using trifluoroacetyl (Tfa) for tetrakis(N gamma-Tfa)-polymyxin B (2-10) and tetrakis(N gamma-Tfa)-colistin (2-10), respectively. Acylation of tetrakis(N gamma-Troc)-polymyxin B (2-10) and tetrakis(N gamma-Troc)-colistin (2-10) with various hydrophobic acids bearing aliphatic or aromatic ring structures, followed by the deprotection of Troc by Zn in AcOH, produced polymyxin B (2-10) and colistin (2-10) analogs which were used for structure-activity relationship studies. It was found that cyclohexylbutanoyl-, 4-biphenylacetyl-, and 1-adamantaneacetyl-polymyxin B (2-10) showed potent antimicrobial activity equal to that of polymyxin B against three Gram-negative bacterial strains. The lipopolysacharide (LPS) binding activity of cyclohexylbutanoyl-, 4-biphenylacetyl-, and cyclododecanecarbonyl-polymyxin B (2-10) increased greatly in comparison with that of polymyxin B (2-10). The various N alpha-acylated polymyxin B (2-10) analogs showed slightly higher antimicrobial and LPS binding activities than the corresponding N alpha-acylated colistin (2-10) analogs. [Abstract/Link to Full Text]

Chao CH, Huang HC, Wu YC, Lu CK, Dai CF, Sheu JH
Glycolipids from the formosan soft coral Lobophytum crassum.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1720-3.
Three glycolipids (1--3), possessing a sugar moiety at C-2 of glycerol ether, have been isolated from the Formosan soft coral Lobophytum crassum. Their structures were elucidated by spectroscopic methods, particularly in 1D- and 2D-NMR experiments. The absolute configurations on the sugar portion and lipid aglycon of 1--3 were determined by methanolysis, chemical transformation and the application of Mosher's method on 1 and 3. Compounds 1--3 exhibited weak cytotoxic activities. [Abstract/Link to Full Text]

Arida AI, Al-Tabakha MM, Hamoury HA
Improving the high variable bioavailability of griseofulvin by SEDDS.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1713-9.
To enhance the dissolution and oral absorption of poorly water-soluble griseofulvin (GF), self-emulsifying drug delivery system (SEDDS) composed of oil, surfactant and cosurfactant for oral administration of griseofulvin was formulated, and its physicochemical properties and pharmacokinetic parameters were evaluated. The solubility of griseofulvin was further improved by the addition of hydrochloric acid. Droplet size of griseofulvin emulsion was kept constant both in simulated gastric fluid without pepsin and simulated intestinal fluid throughout 12 weeks incubation period. Griseofulvin in the SEDDS rapidly dissolved in different dissolution media. This was not the case for the commercial GRIS-PEG tablets. In different fed diet groups, AUC 0-->24 h, Cp max, and T max of griseofulvin after oral administration of SEDDS in rats were comparable to those after oral dose of GRIS-PEG tablet. Although, in fed lipidic diet group, the mean AUC and Cp max after oral administration of GRIS-PEG in rats were 1.28 and 1.15 fold higher, respectively, compared with those of SEDDS, these have not shown to be significantly different. These results demonstrate that the SEDDS of griseofulvin composed of Capmul GMO-50, Poloxamer and Myvacet 9-45 greatly enhanced the dissolution of griseofulvin (without ultramicronisation). However, food intake effect on the bioavailability of griseofulvin has remained. Thus, this system may provide a useful dosage form for oral water-insoluble drugs which have problems in their dissolution. [Abstract/Link to Full Text]

Nagamine T, Inomata K, Endo Y
Enantioselective intramolecular aldol reaction mediated by a combination of L-amino Acid and Br�nsted Acid to construct a bicyclic enedione containing a 7-membered ring.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1710-2.
The enantioselectivity of the intramolecular asymmetric aldol reaction of 1,3-cycloheptanedione bearing a C-2 methyl substituent, mediated by a series of combinations of L-amino acid and Br�nsted acid, was examined in detail. [Abstract/Link to Full Text]

Padmavathi V, Venkata Subbaiah DR, Mahesh K, Radha Lakshmi T
Synthesis and bioassay of amino-pyrazolone, amino-isoxazolone and amino-pyrimidinone derivatives.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1704-9.
Novel amino-pyrazolone, amino-isoxazolone and amino-pyrimidinone derivatives were prepared from ethyl 4-phenylsulfonyl-2-(2'-phenylsulfonylethyl)-2-cyanobutyrate (1), ethyl 4-arylsulfonyl-3-aryl-2-cyanobutyrate (7) and ethyl 4-arylmethylsulfonyl-3-aryl-2-cyanobutyrate (8). The lead molecules have been tested for their antimicrobial activity and antioxidant property. [Abstract/Link to Full Text]

Kato H, Kino T, Yamamoto F, Kaneshiro T, Mukai T, Maeda M
Ascorbate analogs for use in medical imaging: synthesis and radical scavenging activity of 5-O-(4'-iodobenzyl)-L-ascorbic acid.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1700-3.
As part of our program to develop potential imaging agents for ascorbate bioactivity in the brain, 5-O-(4'-iodobenzyl)-L-ascorbic acid was prepared through a seven-step sequence which involved C5-O-alkylation with p-iodobenzyl bromide in the presence of Ag2O and CaSO4 as the key step, starting from L-ascorbic acid. The scavenging activity of the p-iodobenzylated analog against 2,2-diphenyl-1-picrylhyrazyl (DPPH) radical was almost the same as that of L-ascorbic acid itself. [Abstract/Link to Full Text]

Djurdjevi? P, Joksovi? L, Jeli? R, Djurdjevi? A, Stankov MJ
Solution equilibria between aluminum(III) ion and some fluoroquinolone family members. Spectroscopic and potentiometric study.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1689-99.
Complex formation between aluminum(III) ion and fluoroquinolone antibacterials-either moxifloxacin (4th generation antibiotic) or fleroxacin (2nd generation antibiotic) were studied in aqueous solutions without and in the presence of sodium dodecylsulfate (SDS). The investigations were performed by glass electrode potentiometric (ionic medium: 0.1 mol/dm(3) LiCl, 298 K), UV spectrophotometric, multinuclear (1H and 13C) magnetic resonance and ESI-MS measurements. The experimental data were consistent with the formation of Al(HL)L2+, Al(HL)3+ AlL2+, Al(OH)L+ and Al(OH)2L complexes in the pH interval ca. 3-8 and up to 5 : 1 ligand to metal mole ratio with range of Al3+ concentrations between ca. 0.025 to 1.0 mmol/dm3. The binary complex, AlL2+ is fairly stable (log beta(1,0,1) ca. 11.0) and its stability increases in the presence of SDS. At higher concentration ratios of ligands to aluminum, up to 5 : 1, the complex Al(HL)L2+ is formed with rather high overall stability constant (log beta(1,1,2) ca. 24.0). The ESI-MS data generally, confirmed the derived model, and the formation of the complex with ligand to metal ratio 2 : 1. NMR measurements indicate that both ligands utilize 4-carbonyl and carboxyl oxygens as donor atoms. The presence of surface active substance, SDS, favors the formation of the complex in which the ligand is protonated, i.e. Al(HL) and its maximum formation is shifted toward milder acidic region (pH ca. 4). The aluminum-quinolone complexes may affect the bio-distribution of both, quinolone and/or aluminum ion upon concomitant ingestion of aluminum-based antacids or phosphate binders and fluoroquinolones. [Abstract/Link to Full Text]

Kanamitsu N, Osaki T, Itsuji Y, Yoshimura M, Tsujimoto H, Soga M
Novel water-soluble sedative-hypnotic agents: isoindolin-1-one derivatives.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1682-8.
We developed new intravenous sedative-hypnotic compounds with the isoindolin-1-one skeleton focusing on the water-soluble property and in vivo safety. We synthesized approximately 170 derivatives and evaluated their hypnotic effects by intravenous administration of the compounds to mice. A series of the 2-phenyl-3-[2-(4-methyl-1-piperazinyl)-2-oxoethyl]isoindolin-1-one analogs, 3(-), 5(-), 27(-), and 47(-) [JM-1232(-)], showed potent sedative-hypnotic activity with good water solubility and a wide safety margin. The hypnotic doses (HD50s) of these 4 compounds when administered to mice were 2.35, 1.90, 2.17, and 3.12 mg/kg, respectively, and the lethal doses (LD50s) were 88.67, 64.69, >120, and >120 mg/kg, respectively. The therapeutic indexes (LD50/HD50) were 37.73, 34.05, >55.30, and >38.46, respectively. Among these compound, 47(-) [JM-1232(-)] is being considered as the most potential candidate for clinical trials in humans. [Abstract/Link to Full Text]

Tian XY, Wang YH, Liu HY, Yu SS, Fang WS
On the chemical constituents of Dipsacus asper.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1677-81.
Bioassay-guided fractionation of 95% EtOH extract from the roots of Dipsacus asper lead to the isolation of some phenolic acids (caffeic acid, 2,6-dihydroxycinnamic acid, vanillic acid, 2'-O-caffeoyl-D-glucopyranoside ester, and caffeoylquinic acid) as the major active components, and five new iridoid glucoside dimers (1-5) and one new iridoid glucoside monomer (6), other known iridoid glycosides loganin, cantleyoside, triplostoside A, lisianthioside, 6'-O-beta-D-apiofuranosyl sweroside, as well as triterpenoids oleanic acid and akebiasaponin D. The structures of new compounds 1-6 were determined as dipsanosides C (1), D (2), E (3), F (4), G (5), and 3'-O-beta-D-glucopyranosyl sweroside (6) by spectroscopic, including 1D and 2D NMR techniques, and chemical methods. [Abstract/Link to Full Text]

Ito H, Iwasaki J, Sato Y, Aoyagi M, Iguchi K, Yamori T
Marine diterpenoids with a briarane skeleton from the Okinawan soft coral Pachyclavularia violacea.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1671-6.
Five new briarane-type diterpenoids, pachyclavulides E (5), F (6), G (7), H (8) and I (9), were isolated from the Okinawan soft coral Pachyclavularia violacea. The structures of these compounds were elucidated based on the results of spectroscopic analysis. Compound 5 showed a weak growth-inhibitory activity in vitro toward cancer cells. [Abstract/Link to Full Text]

El-Enany N, El-Sherbiny D, Belal F
Spectrophotometric, spectrofluorometric and HPLC determination of desloratadine in dosage forms and human plasma.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1662-70.
Four sensitive, simple and specific methods were developed for the determination of desloratadine (DSL), a new antihistaminic drug in pharmaceutical preparations and biological fluids. Methods I and II are based on coupling DSL with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in borate buffer of pH 7.6 where a yellow colored reaction product was obtained and measured spectrophotometrically at 485 nm (Method I). The same product could be measured spectrofluorometrically at 538 nm after excitation at 480 nm (Method II). Methods III and IV, on the other hand, involved derivatization of DSL with 2,4-dinitrofluorobenzene (DNFB) in borate buffer of pH 9.0 producing a yellow colored product that absorbs maximally at 375 nm (Method III). The same derivative was determined after separation adopting HPLC (Method IV). The separation was performed on a column packed with cyanopropyl bonded stationary phase equilibrated with a mobile phase composed of acetonitrile-water (60 : 40, v/v) at a flow rate of 1.0 ml min(-1) with UV detection at 375 nm. The calibration curves were linear over the concentration ranges of 0.5-6, 0.02-0.4, 1-10 and 1-30 microg ml(-1) for Methods I, II, III and IV, respectively. The lower detection limits (LOD) were 0.112, 0.004, 0.172 and 0.290 microg ml(-1), respectively, for the four methods. The limits of quantification (LOQ) were 0.340, 0.012, 0.522 and 0.890 microg ml(-1) for Methods I, II, III and IV, respectively. The proposed methods were applied to the determination of desloratadine in its tablets and the results were in agreement with those obtained using a reference method. Furthermore, the spectrofluorometric method (Method II) was extended to the in-vitro determination of the drug in spiked human plasma, with a mean percentage recovery (n=4) of 99.7+/-3.54. Interference arising from endogenous amino acids has been overcome using solid phase extraction. The proposed methods are highly specific for determination of DSL in the presence of the parent drug loratadine. A proposal for the reaction pathways is postulated. [Abstract/Link to Full Text]

Misiuk W, Tykocka A
Sensitive extractive spectrophotometric methods for the determination of nortriptyline hydrochloride in pharmaceutical formulations.
Chem Pharm Bull (Tokyo). 2007 Dec;55(12):1655-61.
Two simple, sensitive and rapid extractive spectrophotometric methods have been developed for the assay of the antidepressant drug nortriptyline (NOR) hydrochloride in pure form and in different dosage forms. The methods involve the formation of colored ion-pairs between the drug and the complex of niobium(V)-thiocyanate (Nb-SCN) or iron(III)-thiocyanate (Fe-SCN) followed by their extraction with butanol or a mixture of butanol and chloroform and quantitative determination at 360 nm and 490 nm, using Nb-SCN and Fe-SCN, respectively. The experimental conditions were optimized to obtain the maximum colour intensity. The methods permit the determination of nortriptyline over a concentration range of 15-100 microg/ml and 5-24 microg/ml with the detection limit of 0.84 microg/ml and 0.32 microg/ml, using Nb-SCN and Fe-SCN, respectively. The proposed methods are applicable for the assay of the investigated drug in different dosage forms and the results are in good agreement with those obtained by the official and HPLC methods. No interference was observed from common excipients present in pharmaceutical formulations. The proposed procedures were applied to determine the amount of nortriptyline hydrochloride as active ingredient in the presence of its degradation product, dibenzosuberone. The extractive spectrophotometric methods can also be used to determine the amount of nortriptyline hydrochloride in tablets after its solid phase extraction (SPE). [Abstract/Link to Full Text]

Yasui E, Wada M, Takamura N
New entry for synthesis of N-acylhydrazones, pyridazinones, and 1,3,4-oxadiazin-6-ones from alpha-amino acid esters.
Chem Pharm Bull (Tokyo). 2007 Nov;55(11):1652-4.
Versatile electrophiles N-acylhydrazones are synthesized via diazotization, reduction, and acylation of alpha-amino acid esters. Reduction of diazo esters with L-selectride or tributylphosphine affords the corresponding hydrazones in good yields. Both reducing agents give anti-hydrazones as the major product although the reactivity of each reductant is slightly different. The resulting hydrazones are acylated to give N-acylhydrazones, which are subjected to further reactions to give 1,3,4-oxadiazin-6-ones that serve as useful synthetic intermediates for the Diels-Alder reaction. [Abstract/Link to Full Text]

Inagaki M, Shiizaki M, Hiwatashi T, Miyamoto T, Higuchi R
Constituents of Crinoidea. 5. Isolation and structure of a new glycosyl inositolphosphoceramide-type ganglioside from the feather star Comanthina schlegeli.
Chem Pharm Bull (Tokyo). 2007 Nov;55(11):1649-51.
A new glycosyl inositolphosphoceramide-type ganglioside, CSP2, was obtained from the polar lipid fraction of the chloroform/methanol extract of the feather star Comanthina schlegeli together with a known same type of ganglioside CJP2. The structure of this ganglioside has been determined on the basis of chemical and spectroscopic evidence to be 9-O-methyl-(N-acetyl-alpha-D-neuraminosyl)-(2-->3)-inositolphosphoceramide, which contains C(16)-sphingosine and C(22:0)-, C(24:0)-fatty acid as major component. This is the first report on the isolation and structural elucidation of a glycosyl inositolphosphoceramide-type ganglioside possessing N-acetyl-neuraminic acid (NeuAc) residue. [Abstract/Link to Full Text]

Pongcharoen W, Rukachaisirikul V, Isaka M, Sriklung K
Cytotoxic metabolites from the wood-decayed fungus Xylaria sp. BCC 9653.
Chem Pharm Bull (Tokyo). 2007 Nov;55(11):1647-8.
Chemical investigation of the wood-decayed fungus Xylaria sp. BCC 9653 has led to the isolation of a new methyl aminobenzoate (1) together with eleven known compounds. The structures were established by analysis of spectroscopic data. Cytochalasin D (2), one of the known metabolites, exhibited potent cytotoxicity against African green monkey kidney fibroblast (Vero) cells with an IC(50) value of 0.19 microM. [Abstract/Link to Full Text]

Uekusa Y, Sugimoto N, Sato K, Yun YS, Kunugi A, Yamazaki T, Tanamoto K
Neocrocin A: a novel crocetin glycoside with a unique system for binding sugars isolated from gardenia yellow.
Chem Pharm Bull (Tokyo). 2007 Nov;55(11):1643-6.
A novel crocetin glycosyl ester, neocrocin A (2), was isolated from gardenia yellow. The structure of 2 was elucidated as that of an all-trans-crocetin beta-D-gentiobiosyl beta-D-glucopyranosyl-(1-->6)-D-2-deoxy-glucopyranos-2-yl diester based on chemical and spectral data. The findings provide evidence that the binding system of crocetin glycosides is not limited to the anomeric position. [Abstract/Link to Full Text]

Wabo HK, Kouam SF, Krohn K, Hussain H, Tala MF, Tane P, Ree T, Hu Q, Schulz B
Prenylated anthraquinones and other constituents from the seeds of Vismia laurentii.
Chem Pharm Bull (Tokyo). 2007 Nov;55(11):1640-2.
Two new prenylated anthraquinones, laurenquinone A (1) and B (2) were isolated from the seeds of Vismia laurentii together with four known compounds; xanthone V(1) (3), physcion (4), 3-geranyloxyemodin anthrone (5) and friedelin (6). The structures of the new metabolites were determined with the help of spectroscopic data including extensive 2D-NMR spectroscopy. The known compounds were identified by comparison of their physical and spectroscopic data with those reported in the literature. Compounds 1, 4 and 5 exhibited moderate algicidal activity against Chlorella fusca and 3 showed moderate activity against the gram-positive bacterium Bacillus megaterium. [Abstract/Link to Full Text]

da Silva MA, Oliveira AP, Sannomiya M, Sano PT, Varanda EA, Vilegas W, dos Santos LC
Flavonoids and a naphthopyranone from Eriocaulon ligulatum and their mutagenic activity.
Chem Pharm Bull (Tokyo). 2007 Nov;55(11):1635-9.
A new acylated flavonoid, 6,4'-dimethoxyquercetin-3-O-beta-D-6''[3,4,5-trihydroxy (E)-cinnamoyl]glucopyranoside, and a naphthopyranone dimer, named eriocauline, together with 2 other known flavonoids, 6-methoxyapigenin-7-O-beta-D-glucopyranoside and 6-methoxyapigenin-7-O-beta-D-allopyranoside, have been isolated from the capitulae of Eriocaulon ligulatum. The compounds were identified using spectroscopic methods (HR-ESI-MS, and 1-D and 2-D NMR). The methanol extract exhibited mutagenic activity in the Salmonella/microsome assay, in strains TA100, TA97a and TA102 and for dichloromethane extract tested in strain TA98. [Abstract/Link to Full Text]

Kim MS, Kim JS, Hwang SJ
The effect of sodium alginate on physical and dissolution properties of Surelease-matrix pellets prepared by a novel pelletizer.
Chem Pharm Bull (Tokyo). 2007 Nov;55(11):1631-4.
The aim of this study was to investigate the effect of sodium alginate on the physical and dissolution properties of Surelease-matrix pellets prepared by a novel pelletizer-equipped piston extruder and double-arm counter-rotating rollers. The mean values of the shape factor (e(R)) and the aspect ratio of Surelease-matrix pellets were 0.615-0.625 and 1.06-1.070, respectively, indicating good sphericity of the pellets. The drug release rate increased as the amount of sodium alginate increased due to hydration, swelling, and erosion within the Surelease-matrix pellets. In addition, the porosity of pellets also increased with increasing sodium alginate content. The results of this study show that sodium alginate has a greater effect on the drug release rate than the drug release mechanism within the Surelease-matrix for sparingly water-soluble drug, such as tamsulosin hydrochloride. [Abstract/Link to Full Text]

Recent Articles in The Journal of Experimental Medicine

Bashyam H
CTLA-4: From conflict to clinic.
J Exp Med. 2007 Jun 11;204(6):1243.
CTLA-4 was first identified in 1991 as a second receptor for the T cell costimulation ligand B7. Uncertainties about its biological function plagued the early years after its discovery until 1995, when it was confirmed to be an inhibitor of T cell responses. CTLA-4 has since scored in the clinic as a target for antitumor therapy and as a soluble inhibitor of autoimmunity. [Abstract/Link to Full Text]

Cyrklaff M, Kudryashev M, Leis A, Leonard K, Baumeister W, Menard R, Meissner M, Frischknecht F
Cryoelectron tomography reveals periodic material at the inner side of subpellicular microtubules in apicomplexan parasites.
J Exp Med. 2007 Jun 11;204(6):1281-7.
Microtubules are dynamic cytoskeletal structures important for cell division, polarity, and motility and are therefore major targets for anticancer and antiparasite drugs. In the invasive forms of apicomplexan parasites, which are highly polarized and often motile cells, exceptionally stable subpellicular microtubules determine the shape of the parasite, and serve as tracks for vesicle transport. We used cryoelectron tomography to image cytoplasmic structures in three dimensions within intact, rapidly frozen Plasmodium sporozoites. This approach revealed microtubule walls that are extended at the luminal side by an additional 3 nm compared to microtubules of mammalian cells. Fourier analysis revealed an 8-nm longitudinal periodicity of the luminal constituent, suggesting the presence of a molecule interacting with tubulin dimers. In silico generation and analysis of microtubule models confirmed this unexpected topology. Microtubules from extracted sporozoites and Toxoplasma gondii tachyzoites showed a similar density distribution, suggesting that the putative protein is conserved among Apicomplexa and serves to stabilize microtubules. [Abstract/Link to Full Text]

Atkinson JP, Goodship TH
Complement factor H and the hemolytic uremic syndrome.
J Exp Med. 2007 Jun 11;204(6):1245-8.
Immune recognition is coupled to powerful proinflammatory effector pathways that must be tightly regulated. The ancient alternative pathway of complement activation is one such proinflammatory pathway. Genetic susceptibility factors have been identified in both regulators and activating components of the alternative pathway that are associated with thrombotic microangiopathies, glomerulonephritides, and chronic conditions featuring debris deposition. These observations indicate that excessive alternative pathway activation promotes thrombosis in the microvasculature and tissue damage during debris accumulation. Intriguingly, distinct genetic changes in factor H (FH), a key regulator of the alternative pathway, are associated with hemolytic uremic syndrome (HUS), membranoproliferative glomerulonephritis (dense deposit disease), or age-related macular degeneration (AMD). A mouse model of HUS designed to mirror human mutations in FH has now been developed, providing new understanding of the molecular pathogenesis of complement-related endothelial disorders. [Abstract/Link to Full Text]

Katschke KJ, Helmy KY, Steffek M, Xi H, Yin J, Lee WP, Gribling P, Barck KH, Carano RA, Taylor RE, Rangell L, Diehl L, Hass PE, Wiesmann C, van Lookerenb Campagne M
A novel inhibitor of the alternative pathway of complement reverses inflammation and bone destruction in experimental arthritis.
J Exp Med. 2007 Jun 11;204(6):1319-25.
Complement is an important component of the innate and adaptive immune response, yet complement split products generated through activation of each of the three complement pathways (classical, alternative, and lectin) can cause inflammation and tissue destruction. Previous studies have shown that complement activation through the alternative, but not classical, pathway is required to initiate antibody-induced arthritis in mice, but it is unclear if the alternative pathway (AP) plays a role in established disease. Previously, we have shown that human complement receptor of the immunoglobulin superfamily (CRIg) is a selective inhibitor of the AP of complement. Here, we present the crystal structure of murine CRIg and, using mutants, provide evidence that the structural requirements for inhibition of the AP are conserved in human and mouse. A soluble form of CRIg reversed inflammation and bone loss in two experimental models of arthritis by inhibiting the AP of complement in the joint. Our data indicate that the AP of complement is not only required for disease induction, but also disease progression. The extracellular domain of CRIg thus provides a novel tool to study the effects of inhibiting the AP of complement in established disease and constitutes a promising therapeutic with selectivity for a single complement pathway. [Abstract/Link to Full Text]

Nolte MA, Leibundgut-Landmann S, Joffre O, Reis e Sousa C
Dendritic cell quiescence during systemic inflammation driven by LPS stimulation of radioresistant cells in vivo.
J Exp Med. 2007 Jun 11;204(6):1487-501.
Dendritic cell (DC) activation is a prerequisite for T cell priming. During infection, activation can ensue from signaling via pattern-recognition receptors after contact with pathogens or infected cells. Alternatively, it has been proposed that DCs can be activated indirectly by signals produced by infected tissues. To address the contribution of tissue-derived signals, we measured DC activation in a model in which radioresistant cells can or cannot respond to lipopolysaccharide (LPS). We report that recognition of LPS by the radioresistant compartment is sufficient to induce local and systemic inflammation characterized by high circulating levels of tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta, IL-6, and CC chemokine ligand 2. However, this is not sufficient to activate DCs, whether measured by migration, gene expression, phenotypic, or functional criteria, or to render DC refractory to subsequent stimulation with CpG-containing DNA. Similarly, acute or chronic exposure to proinflammatory cytokines such as TNF-alpha +/- interferon alpha/beta has marginal effects on DC phenotype in vivo when compared with LPS. In addition, DC activation and migration induced by LPS is unimpaired when radioresistant cells cannot respond to the stimulus. Thus, inflammatory mediators originating from nonhematopoietic tissues and from radioresistant hematopoietic cells are neither sufficient nor required for DC activation in vivo. [Abstract/Link to Full Text]

Sather BD, Treuting P, Perdue N, Miazgowicz M, Fontenot JD, Rudensky AY, Campbell DJ
Altering the distribution of Foxp3(+) regulatory T cells results in tissue-specific inflammatory disease.
J Exp Med. 2007 Jun 11;204(6):1335-47.
CD4(+)Foxp3(+) regulatory T cells (T reg) are essential for maintaining self-tolerance, but their functional mechanisms and sites of action in vivo are poorly defined. We examined the homing receptor expression and tissue distribution of T reg cells in the steady state and determined whether altering their distribution by removal of a single chemokine receptor impairs their ability to maintain tissue-specific peripheral tolerance. We found that T reg cells are distributed throughout all nonlymphoid tissues tested, and are particularly prevalent in the skin, where they express a unique CCR4(+)CD103(hi) phenotype. T reg cell expression of CCR4 and CD103 is induced by antigen-driven activation within subcutaneous lymph nodes, and accumulation of T reg cells in the skin and lung airways is impaired in the absence of CCR4 expression. Mice with a complete loss of CCR4 in the T reg cell compartment develop lymphocytic infiltration and severe inflammatory disease in the skin and lungs, accompanied by peripheral lymphadenopathy and increased differentiation of skin-tropic CD4(+)Foxp3(+) T cells. Thus, selectively altering T reg cell distribution in vivo leads to the development of tissue-specific inflammatory disease. [Abstract/Link to Full Text]

Reiley WW, Jin W, Lee AJ, Wright A, Wu X, Tewalt EF, Leonard TO, Norbury CC, Fitzpatrick L, Zhang M, Sun SC
Deubiquitinating enzyme CYLD negatively regulates the ubiquitin-dependent kinase Tak1 and prevents abnormal T cell responses.
J Exp Med. 2007 Jun 11;204(6):1475-85.
The deubiquitinating enzyme CYLD has recently been implicated in the regulation of signal transduction, but its physiological function and mechanism of action are still elusive. In this study, we show that CYLD plays a pivotal role in regulating T cell activation and homeostasis. T cells derived from Cyld knockout mice display a hyperresponsive phenotype and mediate the spontaneous development of intestinal inflammation. Interestingly, CYLD targets a ubiquitin-dependent kinase, transforming growth factor-beta-activated kinase 1 (Tak1), and inhibits its ubiquitination and autoactivation. Cyld-deficient T cells exhibit constitutively active Tak1 and its downstream kinases c-Jun N-terminal kinase and IkappaB kinase beta. These results emphasize a critical role for CYLD in preventing spontaneous activation of the Tak1 axis of T cell signaling and, thereby, maintaining normal T cell function. [Abstract/Link to Full Text]

Delano MJ, Scumpia PO, Weinstein JS, Coco D, Nagaraj S, Kelly-Scumpia KM, O'Malley KA, Wynn JL, Antonenko S, Al-Quran SZ, Swan R, Chung CS, Atkinson MA, Ramphal R, Gabrilovich DI, Reeves WH, Ayala A, Phillips J, Laface D, Heyworth PG, Clare-Salzler M, Moldawer LL
MyD88-dependent expansion of an immature GR-1(+)CD11b(+) population induces T cell suppression and Th2 polarization in sepsis.
J Exp Med. 2007 Jun 11;204(6):1463-74.
Polymicrobial sepsis alters the adaptive immune response and induces T cell suppression and Th2 immune polarization. We identify a GR-1(+)CD11b(+) population whose numbers dramatically increase and remain elevated in the spleen, lymph nodes, and bone marrow during polymicrobial sepsis. Phenotypically, these cells are heterogeneous, immature, predominantly myeloid progenitors that express interleukin 10 and several other cytokines and chemokines. Splenic GR-1(+) cells effectively suppress antigen-specific CD8(+) T cell interferon (IFN) gamma production but only modestly suppress antigen-specific and nonspecific CD4(+) T cell proliferation. GR-1(+) cell depletion in vivo prevents both the sepsis-induced augmentation of Th2 cell-dependent and depression of Th1 cell-dependent antibody production. Signaling through MyD88, but not Toll-like receptor 4, TIR domain-containing adaptor-inducing IFN-beta, or the IFN-alpha/beta receptor, is required for complete GR-1(+)CD11b(+) expansion. GR-1(+)CD11b(+) cells contribute to sepsis-induced T cell suppression and preferential Th2 polarization. [Abstract/Link to Full Text]

Yuan Q, Bromley SK, Means TK, Jones KJ, Hayashi F, Bhan AK, Luster AD
CCR4-dependent regulatory T cell function in inflammatory bowel disease.
J Exp Med. 2007 Jun 11;204(6):1327-34.
Inflammatory bowel disease (IBD) is an idiopathic inflammatory disease of the intestine. CD4(+) T lymphocytes play an important role in both initiating and regulating intestinal inflammatory immune responses. CD4(+)CD25(+)CD45RB(low) regulatory T (T reg) cells are capable of preventing the development of colitis in a mouse model of IBD. The precise mechanism of T reg cell-mediated prevention of colitis in this model is unclear, and the role of chemokine receptors in the trafficking and function of T reg cells in this model has not been determined. We examined the role of the chemokine receptor CCR4 in in vivo trafficking and suppressive function of T reg cells in a mouse adoptive transfer model of IBD. CCR4-deficient T reg cells failed to accumulate in the mesenteric lymph nodes (MLNs) at early time points (2-5 d) after adoptive transfer, resulting in a failure to suppress the generation of pathogenic T cells and the development of colitis. Moreover, although CCR4-deficent T cells had equivalent in vitro suppressive activity and accumulated in MLNs at later time points (42-56 d), they were unable to suppress colitis. Our study demonstrates that CCR4 plays an important role in T reg cell trafficking in LNs and that this is critical for T reg cell suppressive function in vivo. [Abstract/Link to Full Text]

Campbell JA, Trossman DS, Yokoyama WM, Carayannopoulos LN
Zoonotic orthopoxviruses encode a high-affinity antagonist of NKG2D.
J Exp Med. 2007 Jun 11;204(6):1311-7.
NK and T lymphocytes express both activating and inhibiting receptors for various members of the major histocompatibility complex class I superfamily (MHCISF). To evade immunologic cytotoxicity, many viruses interfere with the function of these receptors, generally by altering the displayed profile of MHCISF proteins on host cells. Using a structurally constrained hidden Markov model, we discovered an orthopoxvirus protein, itself distantly class I-like, that acts as a competitive antagonist of the NKG2D activating receptor. This orthopoxvirus MHC class I-like protein (OMCP) is conserved among cowpox and monkeypox viruses, secreted by infected cells, and bound with high affinity by NKG2D of rodents and humans (K(D) approximately 30 and 0.2 nM, respectively). OMCP blocks recognition of host-encoded ligands and inhibits NKG2D-dependent killing by NK cells. This finding represents a novel mechanism for viral interference with NKG2D and sheds light on intercellular recognition events underlying innate immunity against emerging orthopoxviruses. [Abstract/Link to Full Text]

Stary G, Bangert C, Tauber M, Strohal R, Kopp T, Stingl G
Tumoricidal activity of TLR7/8-activated inflammatory dendritic cells.
J Exp Med. 2007 Jun 11;204(6):1441-51.
Imiquimod (IMQ), a synthetic agonist to Toll-like receptor (TLR) 7, is being successfully used for the treatment of certain skin neoplasms, but the exact mechanisms by which this compound induces tumor regression are not yet understood. While treating basal cell carcinoma (BCC) patients with topical IMQ, we detected, by immunohistochemistry, sizable numbers of both myeloid dendritic cells (mDCs) and plasmacytoid DCs (pDCs) within the inflammatory infiltrate. Surprisingly, peritumoral mDCs stained positive for perforin and granzyme B, whereas infiltrating pDCs expressed tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The biological relevance of this observation can be deduced from our further findings that peripheral blood-derived CD11c(+) mDCs acquired antiperforin and anti-granzyme B reactivity upon TLR7/8 stimulation and could use these molecules to effectively lyse major histocompatibility complex (MHC) class I(lo) cancer cell lines. The same activation protocol led pDCs to kill MHC class I-bearing Jurkat cells in a TRAIL-dependent fashion. While suggesting that mDCs and pDCs are directly involved in the IMQ-induced destruction of BCC lesions, our data also add a new facet to the functional spectrum of DCs, ascribing to them a major role not only in the initiation but also in the effector phase of the immune response. [Abstract/Link to Full Text]

Wirzenius M, Tammela T, Uutela M, He Y, Odorisio T, Zambruno G, Nagy JA, Dvorak HF, Yl�-Herttuala S, Shibuya M, Alitalo K
Distinct vascular endothelial growth factor signals for lymphatic vessel enlargement and sprouting.
J Exp Med. 2007 Jun 11;204(6):1431-40.
Lymphatic vessel growth, or lymphangiogenesis, is regulated by vascular endothelial growth factor-C (VEGF-C) and -D via VEGF receptor 3 (VEGFR-3). Recent studies suggest that VEGF, which does not bind to VEGFR-3, can also induce lymphangiogenesis through unknown mechanisms. To dissect the receptor pathway that triggers VEGFR-3-independent lymphangiogenesis, we used both transgenic and adenoviral overexpression of placenta growth factor (PlGF) and VEGF-E, which are specific activators of VEGFR-1 and -2, respectively. Unlike PlGF, VEGF-E induced circumferential lymphatic vessel hyperplasia, but essentially no new vessel sprouting, when transduced into mouse skin via adenoviral vectors. This effect was not inhibited by blocking VEGF-C and -D. Postnatal lymphatic hyperplasia, without increased density of lymphatic vessels, was also detected in transgenic mice expressing VEGF-E in the skin, but not in mice expressing PlGF. Surprisingly, VEGF-E induced lymphatic hyperplasia postnatally, and it did not rescue the loss of lymphatic vessels in transgenic embryos where VEGF-C and VEGF-D were blocked. Our data suggests that VEGFR-2 signals promote lymphatic vessel enlargement, but unlike in the blood vessels, are not involved in vessel sprouting to generate new lymphatic vessels in vivo. [Abstract/Link to Full Text]

�rsted DD, Bojesen SE, Tybjaerg-Hansen A, Nordestgaard BG
Tumor suppressor p53 Arg72Pro polymorphism and longevity, cancer survival, and risk of cancer in the general population.
J Exp Med. 2007 Jun 11;204(6):1295-301.
p53 is an important tumor suppressor, normally preventing cancer development via apoptosis. A genomic Arg72Pro substitution in the p53 protein has important influence on cell death via apoptosis, which could be beneficial. We therefore tested the hypotheses that this polymorphism influences longevity, survival after a cancer diagnosis, and risk of cancer in the general population. We examined a cohort of 9,219 participants ages 20-95 from the Danish general population with 100% follow-up. The overall 12-yr survival was increased in p53 Arg/Pro heterozygotes with 3% (P = 0.003) and in Pro/Pro homozygotes with 6% (P = 0.002) versus Arg/Arg homozygotes, corresponding to an increase in median survival of 3 yr for Pro/Pro versus Arg/Arg homozygotes. We also demonstrated an increased survival after the development of cancer, or even after the development of other life-threatening diseases, for Pro/Pro versus Arg/Arg homozygotes. The Arg72Pro substitution did not associate with decreased risk of cancer. In conclusion, in this large cohort from the general population, we show that a well-known functional single nucleotide polymorphism in the tumor suppressor p53 protein leads to increased longevity, but not to decreased risk of cancer. The increased longevity may be due to increased survival after a diagnosis of cancer or other life-threatening diseases. [Abstract/Link to Full Text]

Van Nguyen T, Puebla-Osorio N, Pang H, Dujka ME, Zhu C
DNA damage-induced cellular senescence is sufficient to suppress tumorigenesis: a mouse model.
J Exp Med. 2007 Jun 11;204(6):1453-61.
Tumor suppressor p53-dependent apoptosis is critical in suppressing tumorigenesis. Previously, we reported that DNA double-strand breaks (DSBs) at the V(D)J recombination loci induced genomic instability in the developing lymphocytes of nonhomologous end-joining (NHEJ)-deficient, p53-deficient mice, which led to rapid lymphomagenesis. To test the ability of p53-dependent cell cycle arrest to suppress tumorigenesis in the absence of apoptosis in vivo, we crossbred NHEJ-deficient mice into a mutant p53R172P background; these mice have defects in apoptosis induction, but not cell cycle arrest. These double-mutant mice survived longer than NHEJ/p53 double-null mice and, remarkably, were completely tumor free. We detected accumulation of aberrant V(D)J recombination-related DSBs at the T cell receptor (TCR) locus, and high expression levels of both mutant p53 and cell cycle checkpoint protein p21, but not the apoptotic protein p53-upregulated modulator of apoptosis. In addition, a substantial number of senescent cells were observed among both thymocytes and bone marrow cells. Cytogenetic studies revealed euploidy and limited chromosomal breaks in these lymphoid cells. The results indicate that precursor lymphocytes, which normally possess a high proliferation potential, are able to withdraw from the cell cycle and undergo senescence in response to the persistence of DSBs in a p53-p21-dependent pathway; this is sufficient to inhibit oncogenic chromosomal abnormality and suppress tumorigenesis. [Abstract/Link to Full Text]

Precopio ML, Betts MR, Parrino J, Price DA, Gostick E, Ambrozak DR, Asher TE, Douek DC, Harari A, Pantaleo G, Bailer R, Graham BS, Roederer M, Koup RA
Immunization with vaccinia virus induces polyfunctional and phenotypically distinctive CD8(+) T cell responses.
J Exp Med. 2007 Jun 11;204(6):1405-16.
Vaccinia virus immunization provides lifelong protection against smallpox, but the mechanisms of this exquisite protection are unknown. We used polychromatic flow cytometry to characterize the functional and phenotypic profile of CD8(+) T cells induced by vaccinia virus immunization in a comparative vaccine trial of modified vaccinia virus Ankara (MVA) versus Dryvax immunization in which protection was assessed against subsequent Dryvax challenge. Vaccinia virus-specific CD8(+) T cells induced by both MVA and Dryvax were highly polyfunctional; they degranulated and produced interferon gamma, interleukin 2, macrophage inflammatory protein 1beta, and tumor necrosis factor alpha after antigenic stimulation. Responding CD8(+) T cells exhibited an unusual phenotype (CD45RO(-)CD27(intermediate)). The unique phenotype and high degree of polyfunctionality induced by vaccinia virus also extended to inserted HIV gene products of recombinant NYVAC. This quality of the CD8(+) T cell response may be at least partially responsible for the profound efficacy of these vaccines in protection against smallpox and serves as a benchmark against which other vaccines can be evaluated. [Abstract/Link to Full Text]

Hormones and breast cancer: controlling the danger within.
J Exp Med. 2007 Apr 16;204(4):699. [Abstract/Link to Full Text]

HUGGINS C, BRIZIARELLI G, SUTTON H
Rapid induction of mammary carcinoma in the rat and the influence of hormones on the tumors.
J Exp Med. 1959 Jan 1;109(1):25-42. [Abstract/Link to Full Text]

Pickering MC, de Jorge EG, Martinez-Barricarte R, Recalde S, Garcia-Layana A, Rose KL, Moss J, Walport MJ, Cook HT, de C�rdoba SR, Botto M
Spontaneous hemolytic uremic syndrome triggered by complement factor H lacking surface recognition domains.
J Exp Med. 2007 Jun 11;204(6):1249-56.
Factor H (FH) is an abundant serum glycoprotein that regulates the alternative pathway of complement-preventing uncontrolled plasma C3 activation and nonspecific damage to host tissues. Age-related macular degeneration (AMD), atypical hemolytic uremic syndrome (aHUS), and membranoproliferative glomerulonephritis type II (MPGN2) are associated with polymorphisms or mutations in the FH gene (Cfh), suggesting the existence of a genotype-phenotype relationship. Although AMD and MPGN2 share pathological similarities with the accumulation of complement-containing debris within the eye and kidney, respectively, aHUS is characterized by renal endothelial injury. This pathological distinction was reflected in our Cfh association analysis, which demonstrated that although AMD and MPGN2 share a Cfh at-risk haplotype, the haplotype for aHUS was unique. FH-deficient mice have uncontrolled plasma C3 activation and spontaneously develop MPGN2 but not aHUS. We show that these mice, transgenically expressing a mouse FH protein functionally equivalent to aHUS-associated human FH mutants, regulate C3 activation in plasma and spontaneously develop aHUS but not MPGN2. These animals represent the first model of aHUS and provide in vivo evidence that effective plasma C3 regulation and the defective control of complement activation on renal endothelium are the critical events in the molecular pathogenesis of FH-associated aHUS. [Abstract/Link to Full Text]

Coutanceau E, Decalf J, Martino A, Babon A, Winter N, Cole ST, Albert ML, Demangel C
Selective suppression of dendritic cell functions by Mycobacterium ulcerans toxin mycolactone.
J Exp Med. 2007 Jun 11;204(6):1395-403.
Mycolactone is a polyketide toxin produced by Mycobacterium ulcerans (Mu), the causative agent of the skin disease Buruli ulcer (BU). Surprisingly, infected tissues lack inflammatory infiltrates. Structural similarities between mycolactone and immunosuppressive agents led us to investigate the immunomodulatory properties of mycolactone on dendritic cells (DCs), the key initiators and regulators of immune responses. At noncytotoxic concentrations, phenotypic and functional maturation of both mouse and human DCs was inhibited by mycolactone. Notably, mycolactone blocked the emigration of mouse-skin DCs to draining lymph nodes, as well as their maturation in vivo. In human peripheral blood-derived DCs, mycolactone inhibited the ability to activate allogeneic T cell priming and to produce inflammatory molecules. Interestingly, production of the cytokines interleukin (IL) 12, tumor necrosis factor alpha, and IL-6 was only marginally affected, whereas production of the chemokines macrophage inflammatory protein (MIP) 1alpha, MIP-1beta, regulated on activation, normal T cell expressed and secreted, interferon gamma-inducible protein 10, and monocyte chemoattractant protein 1 was abolished at nanomolar concentrations. Importantly, mycolactone endogenously expressed by Mu mediated similar inhibitory effects on beta-chemokine production by DCs. In accordance with the histopathological features of BUs, our results suggest that bacterial production of mycolactone may limit both the initiation of primary immune responses and the recruitment of inflammatory cells to the infection site. Moreover, they highlight a potential interest in mycolactone as a novel immunosuppressive agent. [Abstract/Link to Full Text]

Tsai KJ, Tsai YC, Shen CK
G-CSF rescues the memory impairment of animal models of Alzheimer's disease.
J Exp Med. 2007 Jun 11;204(6):1273-80.
Most of the current clinical treatments for Alzheimer's disease (AD) are largely symptomatic and can have serious side effects. We have tested the feasibility of using the granulocyte colony-stimulating factor (G-CSF), which is known to mobilize hematopoietic stem cells (HSCs) from the bone marrow into the peripheral blood, as a therapeutic agent for AD. Subcutaneous administration of G-CSF into two different beta-amyloid (Abeta)-induced AD mouse models substantially rescued their cognitive/memory functions. The rescue was accompanied by the accumulation of 5-bromo-2'deoxyuridine-positive HSCs, as well as local neurogenesis surrounding the Abeta aggregates. Furthermore, the level of acetylcholine in the brains of Tg2576 mice was considerably enhanced upon G-CSF treatment. We suggest that G-CSF, a drug already extensively used for treating chemotherapy-induced neutropenia, should be pursued as a novel, noninvasive therapeutic agent for the treatment of AD. [Abstract/Link to Full Text]

Kearley J, McMillan SJ, Lloyd CM
Th2-driven, allergen-induced airway inflammation is reduced after treatment with anti-Tim-3 antibody in vivo.
J Exp Med. 2007 Jun 11;204(6):1289-94.
T cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) is a surface molecule that is preferentially expressed on activated Th1 cells in comparison to Th2 cells. Blockade of Tim-3 has been shown to enhance Th1-driven pathology in vivo, suggesting that blockade of Tim-3 may improve the development of Th2-associated responses such as allergy. To examine the effects of Tim-3 blockade on the Th2 response in vivo, we administered anti-Tim-3 antibody during pulmonary inflammation induced by transfer of ovalbumin (OVA)-reactive Th2 cells, and subsequent aerosol challenge with OVA. In this model, anti-Tim-3 antibody treatment before each airway challenge significantly reduced airway hyperreactivity, with a concomitant decrease in eosinophils and Th2 cells in the lung. We examined Th1 and Th2 cytokine levels in the lung after allergen challenge and found that pulmonary expression of the Th2 cytokine IL-5 was significantly reduced, whereas IFN-gamma levels were significantly increased by anti-Tim-3 antibody treatment. Thus, blocking Tim-3 function has a beneficial effect during pulmonary inflammation by skewing the Th2 response toward that of a Th1 type, suggesting an important role for Tim-3 in the regulation of allergic disease. [Abstract/Link to Full Text]

An G, Wei B, Xia B, McDaniel JM, Ju T, Cummings RD, Braun J, Xia L
Increased susceptibility to colitis and colorectal tumors in mice lacking core 3-derived O-glycans.
J Exp Med. 2007 Jun 11;204(6):1417-29.
Altered intestinal O-glycan expression has been observed in patients with ulcerative colitis and colorectal cancer, but the role of this alteration in the etiology of these diseases is unknown. O-glycans in mucin core proteins are the predominant components of the intestinal mucus, which comprises part of the intestinal mucosal barrier. Core 3-derived O-glycans, which are one of the major types of O-glycans, are primarily expressed in the colon. To investigate the biological function of core 3-derived O-glycans, we engineered mice lacking core 3 beta1,3-N-acetylglucosaminyltransferase (C3GnT), an enzyme predicted to be important in the synthesis of core 3-derived O-glycans. Disruption of the C3GnT gene eliminated core 3-derived O-glycans. C3GnT-deficient mice displayed a discrete, colon-specific reduction in Muc2 protein and increased permeability of the intestinal barrier. Moreover, these mice were highly susceptible to experimental triggers of colitis and colorectal adenocarcinoma. These data reveal a requirement for core 3-derived O-glycans in resistance to colonic disease. [Abstract/Link to Full Text]

Nguyen M, Pace AJ, Koller BH
Mice lacking NKCC1 are protected from development of bacteremia and hypothermic sepsis secondary to bacterial pneumonia.
J Exp Med. 2007 Jun 11;204(6):1383-93.
The contribution of the Na(+)-K(+)-Cl(-) transporter (NKCC1) to fluid in ion transport and fluid secretion in the lung and in other secretory epithelia has been well established. Far less is known concerning the role of this cotransporter in the physiological response of the pulmonary system during acute inflammation. Here we show that mice lacking this transporter are protected against hypothermic sepsis and bacteremia developing as a result of Klebsiella pneumoniae infection in the lung. In contrast, this protection was not observed in NKCC1(-/-) mice with K. pneumoniae-induced peritonitis. Although overall recruitment of cells to the lungs was not altered, the number of cells present in the airways was increased in the NKCC1(-/-) animals. Despite this robust inflammatory response, the increase in vascular permeability observed in this acute inflammatory model was attenuated in the NKCC1(-/-) animals. Our studies suggest that NKCC1 plays a unique and untoward unrecognized role in acute inflammatory responses in the lung and that specific inhibition of this NKCC isoform could be beneficial in treatment of sepsis. [Abstract/Link to Full Text]

Dhodapkar KM, Banerjee D, Connolly J, Kukreja A, Matayeva E, Veri MC, Ravetch JV, Steinman RM, Dhodapkar MV
Selective blockade of the inhibitory Fcgamma receptor (FcgammaRIIB) in human dendritic cells and monocytes induces a type I interferon response program.
J Exp Med. 2007 Jun 11;204(6):1359-69.
The ability of dendritic cells (DCs) to activate immunity is linked to their maturation status. In prior studies, we have shown that selective antibody-mediated blockade of inhibitory FcgammaRIIB receptor on human DCs in the presence of activating immunoglobulin (Ig) ligands leads to DC maturation and enhanced immunity to antibody-coated tumor cells. We show that Fcgamma receptor (FcgammaR)-mediated activation of human monocytes and monocyte-derived DCs is associated with a distinct gene expression pattern, including several inflammation-associated chemokines, as well as type 1 interferon (IFN) response genes, including the activation of signal transducer and activator of transcription 1 (STAT1). FcgammaR-mediated STAT1 activation is rapid and requires activating FcgammaRs. However, this IFN response is observed without a detectable increase in the expression of type I IFNs themselves or the need to add exogenous IFNs. Induction of IFN response genes plays an important role in FcgammaR-mediated effects on DCs, as suppression of STAT1 by RNA interference inhibited FcgammaR-mediated DC maturation. These data suggest that the balance of activating/inhibitory FcgammaRs may regulate IFN signaling in myeloid cells. Manipulation of FcgammaR balance on DCs and monocytes may provide a novel approach to regulating IFN-mediated pathways in autoimmunity and human cancer. [Abstract/Link to Full Text]

Deaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, Chen JF, Enjyoji K, Linden J, Oukka M, Kuchroo VK, Strom TB, Robson SC
Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression.
J Exp Med. 2007 Jun 11;204(6):1257-65.
The study of T regulatory cells (T reg cells) has been limited by the lack of specific surface markers and an inability to define mechanisms of suppression. We show that the expression of CD39/ENTPD1 in concert with CD73/ecto-5'-nucleotidase distinguishes CD4(+)/CD25(+)/Foxp3(+) T reg cells from other T cells. These ectoenzymes generate pericellular adenosine from extracellular nucleotides. The coordinated expression of CD39/CD73 on T reg cells and the adenosine A2A receptor on activated T effector cells generates immunosuppressive loops, indicating roles in the inhibitory function of T reg cells. Consequently, T reg cells from Cd39-null mice show impaired suppressive properties in vitro and fail to block allograft rejection in vivo. We conclude that CD39 and CD73 are surface markers of T reg cells that impart a specific biochemical signature characterized by adenosine generation that has functional relevance for cellular immunoregulation. [Abstract/Link to Full Text]

Rossi SW, Kim MY, Leibbrandt A, Parnell SM, Jenkinson WE, Glanville SH, McConnell FM, Scott HS, Penninger JM, Jenkinson EJ, Lane PJ, Anderson G
RANK signals from CD4(+)3(-) inducer cells regulate development of Aire-expressing epithelial cells in the thymic medulla.
J Exp Med. 2007 Jun 11;204(6):1267-72.
Aire-expressing medullary thymic epithelial cells (mTECs) play a key role in preventing autoimmunity by expressing tissue-restricted antigens to help purge the emerging T cell receptor repertoire of self-reactive specificities. Here we demonstrate a novel role for a CD4(+)3(-) inducer cell population, previously linked to development of organized secondary lymphoid structures and maintenance of T cell memory in the functional regulation of Aire-mediated promiscuous gene expression in the thymus. CD4(+)3(-) cells are closely associated with mTECs in adult thymus, and in fetal thymus their appearance is temporally linked with the appearance of Aire(+) mTECs. We show that RANKL signals from this cell promote the maturation of RANK-expressing CD80(-)Aire(-) mTEC progenitors into CD80(+)Aire(+) mTECs, and that transplantation of RANK-deficient thymic stroma into immunodeficient hosts induces autoimmunity. Collectively, our data reveal cellular and molecular mechanisms leading to the generation of Aire(+) mTECs and highlight a previously unrecognized role for CD4(+)3(-)RANKL(+) inducer cells in intrathymic self-tolerance. [Abstract/Link to Full Text]

Bopp T, Becker C, Klein M, Klein-Hessling S, Palmetshofer A, Serfling E, Heib V, Becker M, Kubach J, Schmitt S, Stoll S, Schild H, Staege MS, Stassen M, Jonuleit H, Schmitt E
Cyclic adenosine monophosphate is a key component of regulatory T cell-mediated suppression.
J Exp Med. 2007 Jun 11;204(6):1303-10.
Naturally occurring regulatory T cells (T reg cells) are a thymus-derived subset of T cells, which are crucial for the maintenance of peripheral tolerance by controlling potentially autoreactive T cells. However, the underlying molecular mechanisms of this strictly cell contact-dependent process are still elusive. Here we show that naturally occurring T reg cells harbor high levels of cyclic adenosine monophosphate (cAMP). This second messenger is known to be a potent inhibitor of proliferation and interleukin 2 synthesis in T cells. Upon coactivation with naturally occurring T reg cells the cAMP content of responder T cells is also strongly increased. Furthermore, we demonstrate that naturally occurring T reg cells and conventional T cells communicate via cell contact-dependent gap junction formation. The suppressive activity of naturally occurring T reg cells is abolished by a cAMP antagonist as well as by a gap junction inhibitor, which blocks the cell contact-dependent transfer of cAMP to responder T cells. Accordingly, our results suggest that cAMP is crucial for naturally occurring T reg cell-mediated suppression and traverses membranes via gap junctions. Hence, naturally occurring T reg cells unexpectedly may control the immune regulatory network by a well-known mechanism based on the intercellular transport of cAMP via gap junctions. [Abstract/Link to Full Text]

Johansson C, Wetzel JD, He J, Mikacenic C, Dermody TS, Kelsall BL
Type I interferons produced by hematopoietic cells protect mice against lethal infection by mammalian reovirus.
J Exp Med. 2007 Jun 11;204(6):1349-58.
We defined the function of type I interferons (IFNs) in defense against reovirus strain type 1 Lang (T1L), which is a double-stranded RNA virus that infects Peyer's patches (PPs) after peroral inoculation of mice. T1L induced expression of mRNA for IFN-alpha, IFN-beta, and Mx-1 in PPs and caused localized intestinal infection that was cleared in 10 d. In contrast, T1L produced fatal systemic infection in IFNalphaR1 knockout (KO) mice with extensive cell loss in lymphoid tissues and necrosis of the intestinal mucosa. Studies of bone-marrow chimeric mice indicated an essential role for hematopoietic cells in IFN-dependent viral clearance. Dendritic cells (DCs), including conventional DCs (cDCs), were the major source of type I IFNs in PPs of reovirus-infected mice, whereas all cell types expressed the antiviral protein Mx-1. Neither NK cells nor signaling via Toll-like receptor 3 or MyD88 were essential for viral clearance. These data demonstrate a requirement for type I IFNs in the control of an intestinal viral infection and indicate that cDCs are a significant source of type I IFN production in vivo. Therefore, innate immunity in PPs is an essential component of host defense that limits systemic spread of pathogens that infect the intestinal mucosa. [Abstract/Link to Full Text]

Huang CY, Sharma GG, Walker LM, Bassing CH, Pandita TK, Sleckman BP
Defects in coding joint formation in vivo in developing ATM-deficient B and T lymphocytes.
J Exp Med. 2007 Jun 11;204(6):1371-81.
Ataxia-telangiectasia mutated (ATM)-deficient lymphocytes exhibit defects in coding joint formation during V(D)J recombination in vitro. Similar defects in vivo should affect both T and B cell development, yet the lymphoid phenotypes of ATM deficiency are more pronounced in the T cell compartment. In this regard, ATM-deficient mice exhibit a preferential T lymphopenia and have an increased incidence of nontransformed and transformed T cells with T cell receptor alpha/delta locus translocations. We demonstrate that there is an increase in the accumulation of unrepaired coding ends during different steps of antigen receptor gene assembly at both the immunoglobulin and T cell receptor loci in developing ATM-deficient B and T lymphocytes. Furthermore, we show that the frequency of ATM-deficient alphabeta T cells with translocations involving the T cell receptor alpha/delta locus is directly related to the number of T cell receptor alpha rearrangements that these cells can make during development. Collectively, these findings demonstrate that ATM deficiency leads to broad defects in coding joint formation in developing B and T lymphocytes in vivo, and they provide a potential molecular explanation as to why the developmental impact of these defects could be more pronounced in the T cell compartment. [Abstract/Link to Full Text]

Difilippantonio S, Celeste A, Kruhlak MJ, Lee Y, Difilippantonio MJ, Feigenbaum L, Jackson SP, McKinnon PJ, Nussenzweig A
Distinct domains in Nbs1 regulate irradiation-induced checkpoints and apoptosis.
J Exp Med. 2007 May 14;204(5):1003-11.
The chromosomal instability syndromes Nijmegen breakage syndrome (NBS) and ataxia telangiectasia (AT) share many overlapping phenotypes, including cancer predisposition, radiation sensitivity, cell-cycle checkpoint defects, immunodeficiency, and gonadal dysfunction. The NBS protein Nbs1 is not only a downstream target of AT mutated (ATM) kinase but also acts upstream, promoting optimal ATM activation, ATM recruitment to breaks, and ATM accessibility to substrates. By reconstituting Nbs1 knockout mice with bacterial artificial chromosomes, we have assessed the contribution of distinct regions of Nbs1 to the ATM-dependent DNA damage response. We find that T cell and oocyte development, as well as DNA damage-induced G2/M and S phase checkpoint arrest and radiation survival are dependent on the N-terminal forkhead-associated domain, but not on the principal residues phosphorylated by ATM (S278 and S343) or on the evolutionarily conserved C-terminal region of Nbs1. However, the C-terminal region regulates irradiation-induced apoptosis. These studies provide insight into the complex interplay between Nbs1 and ATM in the DNA damage response. [Abstract/Link to Full Text]