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Guo Y.,Merck And Co. | Little R.J.,University of Michigan | McConnell D.S.,University of Michigan
Epidemiology | Year: 2012

Background: Covariate measurement error is common in epidemiologic studies. Current methods for correcting measurement error with information from external calibration samples are insufficient to provide valid adjusted inferences. We consider the problem of estimating the regression of an outcome Y on covariates X and Z, where Y and Z are observed, X is unobserved, but a variable W that measures X with error is observed. Information about measurement error is provided in an external calibration sample where data on X and W (but not Y and Z) are recorded. Methods: We describe a method that uses summary statistics from the calibration sample to create multiple imputations of the missing values of X in the regression sample, so that the regression coefficients of Y on X and Z and associated standard errors can be estimated using simple multiple imputation combining rules, yielding valid statistical inferences under the assumption of a multivariate normal distribution. Results: The proposed method is shown by simulation to provide better inferences than existing methods, namely the naive method, classical calibration, and regression calibration, particularly for correction for bias and achieving nominal confidence levels. We also illustrate our method with an example using linear regression to examine the relation between serum reproductive hormone concentrations and bone mineral density loss in midlife women in the Michigan Bone Health and Metabolism Study. Conclusions: Existing methods fail to adjust appropriately for bias due to measurement error in the regression setting, particularly when measurement error is substantial. The proposed method corrects this deficiency. © 2011 by Lippincott Williams & Wilkins.

Zakrison T.L.,University of Toronto | Hille D.A.,Merck And Co. | Namias N.,University of Miami
Surgical Infections | Year: 2012

Background: Complicated intra-abdominal infections (cIAI) are a common problem in surgical practice. The effect of body mass index (BMI) on the outcome is poorly understood. We compared the association of BMI and type of antibiotic therapy for cIAI described in a previously published trial of ertapenem vs. piperacillin-tazobactam (Namias N, Solomkin JS, Jensen EH, et al. Randomized, multicenter, double-blind study of efficacy, safety, and tolerability of intravenous ertapenem versus piperacillin/tazobactam in treatment of complicated intra-abdominal infections in hospitalized adults. Surg Infect 2007;8:15-28). Methods: A post-hoc analysis was performed using data obtained from the published study. The effect of BMI and type of antibiotic used for therapy were calculated for clinically favorable outcomes at early follow-up assessment (EFA). Results: The 231 patients who were microbiologically evaluable at EFA were stratified by BMI (<30 or ≥30 kg/m 2). Twelve patients were excluded because of missing BMI data, leaving 219 patients for analysis. There were some differences in baseline characteristics between patients with a BMI <30 kg/m 2, including the source of intra-abdominal infection (more appendicitis in BMI <30 group; p=0.01) and gender (more men in the BMI <30 group; p=0.03). There was no difference in cure rates between the groups (82.9% for BMI <30 kg/m 2 vs. 74.5% for those with BMI ≥30 kg/m 2; 8% difference in proportions, 95% confidence interval [CI]-5%, 25%). There was an 80% favorable clinical response to ertapenem in the BMI <30 group compared with an 81% favorable rate in the BMI ≥30 group (-1% difference in proportions; 95% CI-22%, 19%). This compared with an 86% favorable response rate to piperacillin-tazobactam in the BMI <30 group vs. a 65% favorable clinical response rate in the BMI ≥30 group (21% difference in proportions; 95% CI-1%, 47%). Conclusions: There was no difference in the cure rate of patients with cIAI in the BMI <30 and BMI ≥30 kg/m 2 groups. There were no statistically significant differences in the likelihood of response to an antibiotic regimen. However, there was a nominally 21% lower cure rate in the high BMI group receiving piperacillin-tazobactam (86% vs. 65%; 21% difference in proportions; 95% CI-1%, 47%), whereas there was only a 1% difference in the cure rate between BMI groups in the patients receiving ertapenem. This difference may be related to gender and etiology of infection. Although limited by the small number of high BMI patients and post-hoc methodology, these results merit consideration of the design of future prospective antibiotic trials to include stratification for BMI and consideration of the effect of BMI on pharmacokinetics and pharmacodynamics. © 2012, Mary Ann Liebert, Inc.

Andersen A.N.,Copenhagen University | Witjes H.,Biostatistics and Research Decision science | Gordon K.,Merck And Co. | Mannaerts B.,Global Clinical Research
Human Reproduction | Year: 2011

Background: Prediction of ovarian response prior to the first controlled ovarian stimulation (COS) cycle is useful in determining the optimal starting dose of recombinant FSH (rFSH). However, potentially predictive factors may be subject to inter-cycle variability and many patients are pre-treated with oral contraceptives (OC) for scheduling purposes. Our objective was to determine predictive factors of ovarian response for patients undergoing COS with rFSH in a gonadotrophin-releasing hormone antagonist protocol and to determine the inter-cycle variability of these factors.Methods: In this multinational trial, 442 patients were randomized to receive either OC treatment or no treatment prior to their first COS cycle. For candidate predictive factors, patient characteristics were collected at screening, and endocrine and sonographic data were collected during the early follicular phase of the two subsequent cycles. A treatment regimen of 200 IU rFSH and 0.25 mg ganirelix was applied during the second cycle. Predictive factors of ovarian response and of too low (<6 oocytes) or too high (>18 oocytes) ovarian responses were determined using stepwise linear regression and stepwise logistic regression, respectively.Results: Anti-Mllerian hormone (AMH) and basal FSH were statistically significant predictors of the number of oocytes retrieved and of an excessive ovarian response. For low ovarian response, AMH was the only significant predictive factor. In the non-OC group, the predictive value was higher than in the OC group and higher at the early follicular phase of the stimulation cycle than of the previous cycle. The inter-cycle variation for AMH was low compared with the inter-cycle variation of other hormones. Between the two groups, there were no differences in the number or quality of embryos obtained or transferred, but the implantation rate was significantly lower in the OC group (24.1 versus 30.1, P 0.03), resulting in an ongoing pregnancy rate of 26.3 compared with 35.7 in the non-OC group (P 0.05). Conclusions: The best predictive model of ovarian response was in the non-OC group and included both AMH and basal FSH determined at the early follicular phase of the stimulation cycle. In the proceeding cycle, AMH alone had sufficient predictive value since it was not affected by inter-cycle variability or OC pretreatment.Clinical trial identifier: NCT00778999. © 2011 The Author. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

Rocha B.A.,Merck And Co.
Behavioural Pharmacology | Year: 2013

Identifying the abuse potential of drug products in the premarketing and postmarketing environment has been a critical component in the implementation of drug abuse control laws worldwide. In the US, the Controlled Substances Act of 1970 (CSA) is a comprehensive federal law enacted to prevent the abuse or diversion of substances with abuse liability or addiction potential (for present purposes, these terms are used interchangeably). Under the jurisdiction of the Drug Enforcement Administration, the law applies to the manufacture and distribution of narcotics and other drug substances with potential of abuse. The CSA classifies substances with abuse potential into schedules I-V based on the substance's risk of diversion or abuse, and thus provides a legal framework for the assessment of abuse liability of New Molecular Entities. When the Food and Drug Administration reviews the safety and efficacy of a New Drug Application it also determines whether the drug has potential for abuse, and if so, will begin the process to schedule the drug under the CSA. As the assessment of abuse potential is a critical component of a marketing application, pharmaceutical companies (sponsors) bear the responsibility of generating a comprehensive preclinical and clinical data package for regulators to review and make decisions on labeling and the corresponding postmarketing surveillance. Recent regulatory guidelines adopted in the European Union (EU) (2006), Canada (2007), and USA (2010) provide recommendations to sponsors on preclinical and clinical methodologies for the assessment of abuse potential. This paper reviews the legal framework of the assessment of abuse liability and scheduling of controlled substances in the USA and describes the current global regulatory environment and the challenges that sponsors and regulators face when assessing abuse liability of New Molecular Entities, from the early stages of development through the late stages, review, and approval. © 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Chandarlapaty S.,Sloan Kettering Cancer Center | Sawai A.,Sloan Kettering Cancer Center | Scaltriti M.,University of Barcelona | Rodrik-Outmezguine V.,Sloan Kettering Cancer Center | And 5 more authors.
Cancer Cell | Year: 2011

Activation of the PI3K-AKT pathway in tumors is modulated by negative feedback, including mTORC1-mediated inhibition of upstream signaling. We now show that AKT inhibition induces the expression and phosphorylation of multiple receptor tyrosine kinases (RTKs). In a wide spectrum of tumor types, inhibition of AKT induces a conserved set of RTKs, including HER3, IGF-1R, and insulin receptor. This is in part due to mTORC1 inhibition and in part secondary to a FOXO-dependent activation of receptor expression. PI3K-AKT inhibitors relieve this feedback and activate RTK signaling; this may attenuate their antitumor activity. Consistent with this model, we find that, in tumors in which AKT suppresses HER3 expression, combined inhibition of AKT and HER kinase activity is more effective than either alone. © 2011 Elsevier Inc.

Shahly V.,Harvard University | Berglund P.A.,University of Michigan | Coulouvrat C.,Sanofi S.A. | Fitzgerald T.,Merck And Co. | And 6 more authors.
Archives of General Psychiatry | Year: 2012

Context: Insomnia is a common and seriously impairing condition that often goes unrecognized. Objectives: To examine associations of broadly defined insomnia (ie, meeting inclusion criteria for a diagnosis from International Statistical Classification of Diseases, 10th Revision , DSM-IV , or Research Diagnostic Criteria/International Classification of Sleep Disorders, Second Edition) with costly workplace accidents and errors after excluding other chronic conditions among workers in the America Insomnia Survey (AIS). Design/Setting: A national cross-sectional telephone survey (65.0% cooperation rate) of commercially insured health plan members selected from the more than 34 million in the HealthCore Integrated Research Database. Participants : Four thousand nine hundred ninetyone employed AIS respondents. Main Outcome Measures: Costly workplace accidents or errors in the 12 months before the AIS interview were assessed with one question about workplace accidents "that either caused damage or work disruption with a value of $500 or more" and another about other mistakes "that cost your company $500 or more." Results: Current insomnia with duration of at least 12 months was assessed with the Brief Insomnia Questionnaire, a validated (area under the receiver operating characteristic curve, 0.86 compared with diagnoses based on blinded clinical reappraisal interviews), fully structured diagnostic interview. Eighteen other chronic conditions were assessed with medical/pharmacy claims records and validated self-report scales. Insomnia had a significant odds ratio with workplace accidents and/or errors controlled for other chronic conditions (1.4). The odds ratio did not vary significantly with respondent age, sex, educational level, or comorbidity. The average costs of insomnia-related accidents and errors ($32 062) were significantly higher than those of other accidents and errors ($21 914). Simulations estimated that insomnia was associated with 7.2% of all costly workplace accidents and errors and 23.7% of all the costs of these incidents. These proportions are higher than for any other chronic condition, with annualized US population projections of 274 000 costly insomnia-related workplace accidents and errors having a combined value of US $31.1 billion. Conclusion: Effectiveness trials are needed to determine whether expanded screening, outreach, and treatment of workers with insomnia would yield a positive return on investment for employers. ©2012 American Medical Association. All rights reserved.

Weiss T.W.,Merck And Co. | Zimet G.D.,Indiana University | Rosenthal S.L.,Columbia University | Brenneman S.K.,i3 Innovus | And 2 more authors.
Journal of Adolescent Health | Year: 2010

Purpose: We assessed U.S. physicians' attitudes and perceptions regarding potential human papillomavirus (HPV) vaccination of males. Methods: We surveyed a random sample of 2,714 pediatricians and family practitioners identified in administrative claims of a U.S. health plan as HPV vaccinators of females; 595 pediatricians and 499 family practitioners participated. Results: Most physicians would recommend HPV vaccination to males aged 11-12 (63.9%), 13-18 (93.4%), and 19-26 (92.7%) years. Physicians agreed that males should be vaccinated to prevent them from getting genital and anal warts (52.9% strongly and 36.0% somewhat) and to protect females from cervical cancer (75.3% strongly and 20.8% somewhat). Physicians agreed that an HPV vaccine recommendation for males would increase opportunities to discuss sexual health with adolescent male patients (58.7% strongly, 35.3% somewhat). Most did not strongly agree (15.4% strongly, 45.4% somewhat) that parents of adolescent male patients would be interested in HPV vaccination for males, that a gender-neutral HPV vaccine recommendation would increase acceptance by adolescent females and their parents (19.6% strongly, 42.0% somewhat), or that a gender-neutral recommendation would improve current female vaccination rates (10.4% strongly, 26.0% somewhat). Conclusions: Physicians who currently vaccinate females against HPV supported the concept of vaccinating males for its benefits for both sexes. They agreed that a gender-neutral HPV vaccination recommendation would be appropriate with regard to public health and believed that it would increase opportunities for sexual health discussions, but were less sure that such a recommendation would change patient or parental attitudes toward HPV vaccination or improve current HPV vaccination efforts. © 2010 Society for Adolescent Health and Medicine.

Allen-Ramey F.C.,Merck And Co. | Gupta S.,Health Outcomes Practice | DiBonaventura M.C.,Health Outcomes Practice
International Journal of COPD | Year: 2012

Background: Recent literature has suggested that emphysema and chronic bronchitis, traditionally considered to be entities overlapping within chronic obstructive pulmonary disease (COPD), may be distinct disorders. Few studies have examined the differences in patient characteristics and health outcomes between these conditions. This study examined whether COPD phenotypes represent distinct patient populations, in a large nationally representative US sample. Methods: Data were obtained from the 2010 US National Health and Wellness Survey (NHWS). NHWS respondents (n = 75,000) were categorized as a COPD phenotype based on their self-reported diagnosis of COPD only (n = 970), emphysema only (n = 399), or chronic bronchitis only (n = 2071). Phenotypes were compared on demographics, health characteristics, treatment patterns, health outcomes, work productivity, and resource use. Variables were compared using Chi-square and analysis of variance tests for categorical and continuous outcomes, respectively. Health outcomes were also examined using regression modeling, controlling for demographic and health characteristic covariates. Results: Patients with chronic bronchitis were significantly younger (51.38 years versus 63.24 years for COPD versus 63.30 years for emphysema, P < 0.05) and more likely to be employed (46.98% versus 23.81% for COPD versus 28.33% for emphysema, P < 0.05). Relative to the other phenotypes, patients with chronic bronchitis were also significantly more likely to be female, nonwhite, and to exercise currently (all P < 0.05), and were significantly less likely to be a current or former smoker (P < 0.05). Controlling for demographic and health characteristics, patients self-identified as having COPD only reported significantly worse physical quality of life (adjusted mean 36.69) and health utilities (adjusted mean 0.65) and significantly more absenteeism (adjusted mean 7.08%), presenteeism (adjusted mean 30.73%), overall work impairment (adjusted mean 34.06%), and activity impairment (adjusted mean 46.59%) than the other phenotypes (all P < 0.05). Conclusion: These results suggest considerable heterogeneity among different COPD phenotypes with respect to demographics, health characteristics, disease characteristics, treatment patterns, and health outcomes. Research aimed at understanding the differences in patient characteristics and disease presentation of these phenotypes could be used to guide treatment recommendations. © 2012 Allen-Ramey et al, publisher and licensee Dove Medical Press Ltd.

El Khoury A.C.,Merck And Co. | Vietri J.,Kantar Health | Prajapati G.,AllSource PPS
Digestive Diseases and Sciences | Year: 2012

Background: Hepatitis C virus (HCV) infection is widespread and associated with high economic costs and reduced quality of life, but the impact of untreated HCV infection on patient outcome is not well understood. Aims: To estimate the impact of untreated HCV infection on work productivity, daily activity, healthcare use, economic costs, and health-related quality of life (HRQoL). Methods: Respondents to the 2010 US National Health and Wellness Survey (n = 75,000) reporting physician diagnosis of HCV infection but not current or previous treatment (patients) were matched to respondents without HCV infection (controls) by use of propensity scores. Those reporting infection with hepatitis B virus (HBV) or human immunodeficiency virus (HIV) were excluded. Selfreported work impairment, activity impairment, healthcare resource use, and HRQoL were compared between patients and controls. Indirect and direct costs were estimated. Results: A total of 306 patients met inclusion criteria. Patients were more impaired at work than controls, with overall work impairment of 26 % versus 15 %, respectively (P<0.001), mostly because of presenteeism in both groups. Annual productivity losses were estimated at $10,316 per employed patient compared with $5,469 per control (P<0.001). Patients used more healthcare, with all-cause healthcare costs estimated at $22,818 per patient annually, compared with $15,362 per control (P<0.001). HRQoL and activity impairment were also worse among patients than controls. Conclusions: Untreated HCV infection is associated with substantial economic costs to society, through loss of productivity and increased use of healthcare resources, and with impaired well-being of the patient. © Springer Science+Business Media, LLC 2012.

Patients in large clinical trials and in studies employing large observational databases report many different adverse events, most of which will not have been anticipated at the outset. Conventional hypothesis testing of between group differences for each adverse event can be problematic: Lack of significance does not mean lack of risk, the tests usually are not adjusted for multiplicity, and the data determine which hypotheses are tested. This article describes a Bayesian screening approach that does not test hypotheses, is self-adjusting for multiplicity, provides a direct assessment of the likelihood of no material drug-event association, and quantifies the strength of the observed association. The criteria for assessing drug-event associations can be determined by clinical or regulatory considerations. In contrast to conventional approaches, the diagnostic properties of this new approach can be evaluated analytically. Application of the method to findings from a vaccine trial yields results similar to those found by methods using a false discovery rate argument or a hierarchical Bayes approach.© Merck & Co., Inc.

Mather K.J.,Indiana University | Steinberg H.O.,Merck And Co. | Baron A.D.,Elcelyx Therapeutics
Journal of Clinical Investigation | Year: 2013

Insulin resistance is typically defined as a reduced ability of insulin to induce glucose uptake by target tissues such as fat and skeletal muscle cells. It accompanies several disease states, including obesity, type 2 diabetes, hepatitis C, and polycystic ovary syndrome, and is a primary feature of metabolic syndrome. Outside of its effects on blood glucose levels, insulin resistance is also associated with a 2- to 3-fold increased risk of cardiovascular mortality. In 1996, Alain Baron, Helmut Steinberg, and colleagues demonstrated that insulin resistance is associated with endothelial dysfunction. This seminal observation led to significant advances in our understanding of insulin's action in health and disease.

Popescu I.,Fundeni Clinical Institute | Ramesh M.K.,Bangalore Medical College and Research Institute | Lipka J.,Astrazeneca | Lipka J.,Cerexa Inc. | And 2 more authors.
Journal of Antimicrobial Chemotherapy | Year: 2013

Objectives: Avibactam, a novel non-β-lactam β-lactamase inhibitor, restores the in vitro activity of ceftazidime against class A, C and some class D β-lactamase-producing pathogens, including those commonly associated with complicated intra-abdominal infections (cIAIs). This randomized, active-controlled, double-blind, Phase II trial (NCT00752219) aimed to evaluate the safety and efficacy of ceftazidime/avibactam plus metronidazole compared with meropenem in hospitalized patients with cIAI. Methods: Adults with confirmed cIAI requiring surgical intervention and antibiotics were randomized 1: 1 to receive intravenously either (i) 2000 mg of ceftazidime plus 500 mg of avibactam plus a separate infusion of 500 mg of metronidazole or (ii) 1000 mg of meropenem plus placebo every 8 h for a minimum of 5 days and a maximum of 14 days. The primary efficacy endpoint was the clinical response in microbiologically evaluable (ME) patients at the test-of-cure (TOC) visit 2 weeks after the last dose of study therapy. Results: Overall, 101 patients received ceftazidime/avibactam plus metronidazole; 102 received meropenem. The median duration of treatment was 6.0 and 6.5 days, respectively. Favourable clinical response at the TOC visit in the ME population was observed in 91.2% (62/68) and 93.4% (71/76) of patients in the ceftazidime/avibactam plus metronidazole and meropenem groups, respectively (observed difference: -2.2%; 95% CI: -20.4%, 12.2%). The incidence of treatment-emergent adverse events was similar for ceftazidime/avibactam plus metronidazole (64.4%) and meropenem (57.8%). Conclusions: Ceftazidime/avibactam plus metronidazole was effective and generally well tolerated in patients with cIAI, with a favourable clinical response rate in the ME population of >90%, similar to that of meropenem. © The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

Accurate prediction of the extent of mechanism-based CYP3A inhibition is critical in determining the timing of clinical drug interaction studies in drug development. To evaluate the prediction accuracy of the static and Simcyp time-based approaches, 54 clinical drug interactions involving mechanism-based CYP3A inhibitors were predicted using both methods. The Simcyp time-based approach generated better prediction when 0.03 h-1 was used as the hepatic CYP3A enzyme degradation rate constant (kdeg) value. Of the predictions 87 and 55% had an error less than 2 and 0.5, respectively, relative to the observed values, compared with 57 and 20%, respectively, when the Simcyp default kdeg value of 0.0077 h-1 was used. Accuracy improvement using the kdeg value of 0.03 over 0.0077 h-1 was most evident for trials with observed magnitude of interaction greater than 2-fold; predictions with an error less than 0.5 relative to clinical observations increased from 8 to 48%. For the static approach, 76 and 35% of the predictions had an error less than 2 and 0.5, respectively. Both methods generated good predictions for weak and moderate inhibitors. The prediction accuracy could be affected by our knowledge of disposition of a substrate compound, in vitro inactivation parameter estimates, and the ability of Simcyp to accurately simulate the pharmacokinetics of inhibitors. Nonetheless, both the Simcyp and static approaches are useful tools for assessing the drug-drug interaction potential of a mechanism-based CYP3A inhibitor, especially when human pharmacokinetics of the inhibitor is known and 0.03 h-1 is used as the hepatic CYP3A kdeg value. Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics.

Qiao Y.,Arthritis and Tissue Degeneration Program and Genomics Center | Giannopoulou E.G.,New York Medical College | Chan C.H.,Arthritis and Tissue Degeneration Program and Genomics Center | Park S.-H.,Arthritis and Tissue Degeneration Program and Genomics Center | And 9 more authors.
Immunity | Year: 2013

Synergistic activation of inflammatory cytokine genes by interferon-γ (IFN-γ) and Toll-like receptor (TLR) signaling is important for innate immunity and inflammatory disease pathogenesis. Enhancement of TLR signaling, a previously proposed mechanism, is insufficient to explain strong synergistic activation of cytokine production in human macrophages. Rather, we found that IFN-γ induced sustained occupancy of transcription factors STAT1, IRF-1, and associated histone acetylation at promoters and enhancers at the TNF, IL6, and IL12B loci. This priming of chromatin did not activate transcription but greatly increased and prolonged recruitment of TLR4-induced transcription factors and RNA polymerase II to gene promoters and enhancers. Priming sensitized cytokine transcription to suppression by Jak inhibitors. Genome-wide analysis revealed pervasive priming of regulatory elements by IFN-γ and linked coordinate priming of promoters and enhancers with synergistic induction of transcription. Our results provide a synergy mechanism whereby IFN-γ creates a primed chromatin environment to augment TLR-induced gene transcription. © 2013 Elsevier Inc.

Margolis D.M.,University of North Carolina at Chapel Hill | Hazuda D.J.,Merck And Co.
Current Opinion in HIV and AIDS | Year: 2013

PURPOSE OF REVIEW: A serious effort has begun to develop therapies that may be capable of eradicating established HIV infection in man. Because of the biological complexity of HIV infection that persists despite potent antiretroviral therapy, it is widely believed that if such therapies can be developed they will involve complex, multimodality approaches. We highlight some of the recent studies in this effort. RECENT FINDINGS: An inhibitor of histone deacetylase has been demonstrated to disrupt latency in man, and new histone deacetylase inhibitors have been identified. Other potential targets, such as histone methyltransferase, protein kinase C, and BRD4, have been recently studied. Model systems, both in primary cells and in animal models, are beginning to be validated. In the clinic, immune-based therapies to aid in the clearance of persistent infection are also being tested. SUMMARY: It is too early to know what combination eradication therapies for HIV infection will look like in the future, but candidate therapies and model systems to perform preclinical validation are beginning to take shape. © 2013 Wolters Kluwer Health | Lippincott Williams &Wilkins.

Bar-Magen T.,McGill University | Sloan R.D.,McGill University | Donahue D.A.,McGill University | Kuhl B.D.,McGill University | And 5 more authors.
Journal of Virology | Year: 2010

MK-2048 represents a prototype second-generation integrase strand transfer inhibitor (INSTI) developed with the goal of retaining activity against viruses containing mutations associated with resistance to first-generation INSTIs, raltegravir (RAL) and elvitegravir (EVG). Here, we report the identification of mutations (G118R and E138K) which confer resistance to MK-2048 and not to RAL or EVG. These mutations were selected in vitro and confirmed by site-specific mutagenesis. G118R, which appeared first in cell culture, conferred low levels of resistance to MK-2048. G118R also reduced viral replication capacity to approximately 1% that of the isogenic wild-type (wt) virus. The subsequent selection of E138K partially restored replication capacity to ≈13% of wt levels and increased resistance to MK-2048 to ≈8-fold. Viruses containing G118R and E138K remained largely susceptible to both RAL and EVG, suggesting a unique interaction between this second-generation INSTI and the enzyme may be defined by these residues as a potential basis for the increased intrinsic affinity and longer "off" rate of MK-2048. In silico structural analysis suggests that the introduction of a positively charged arginine at position 118, near the catalytic amino acid 116, might decrease Mg2+ binding, compromising enzyme function and thus leading to the significant reduction in both integration and viral replication capacity observed with these mutations. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Stead D.,University of York | Carbone G.,University of York | O'Brien P.,University of York | Campos K.R.,Merck And Co. | And 2 more authors.
Journal of the American Chemical Society | Year: 2010

The high yielding asymmetric deprotonation trapping of N-Boc piperidine is successfully realized using s-BuLi and a (+)-sparteine surrogate. Monitoring of the lithiation by in situ React IR allowed the direct observation of a prelithiation complex.

Nam K.,Vanderbilt University | O'Neal R.L.,Vanderbilt University | Coffey R.J.,Vanderbilt University | Finke P.E.,Merck And Co. | And 3 more authors.
Gut | Year: 2012

Objective: Metaplastic lineages in the oxyntic mucosa of the stomach are critical preneoplastic precursors of gastric cancer. Recent studies have demonstrated that spasmolytic polypeptide-expressing metaplasia (SPEM) in the mouse oxyntic mucosa arises from transdifferentiation of mature gastric chief cells. Other investigations of intestinal progenitor cells have shown that cells demonstrating transcriptional activity for leucine-rich repeat containing G-protein-coupled receptor 5 (Lgr5) in the intestine, colon and gastric antrum function as adult stem cells. We have now investigated whether cells demonstrating Lgr5 transcriptional activity in the oxyntic mucosa of mice might be responsible for development of metaplasia. Design: Lgr5-EGFP-IRES-Cre ERT2/+;Rosa26R mice were used to examine the distribution of Lgr5 transcriptionally active cells in the normal oxyntic mucosa as well as after treatment with DMP-777 or L-635 to induce acute SPEM. Lineage mapping was performed to determine if Lgr5-expressing cells gave rise to SPEM. Results: Cells expressing transcriptional activity for Lgr5 in the oxyntic mucosa were present as scattered rare cells only along the lesser curvature of the stomach. These cells also stained for markers of chief cells (intrinsic factor and pepsinogen) but never showed any staining for proliferative markers (Ki-67). In Lgr5-EGFP-IRES- CreERT2/+;Rosa26R mice induced with tamoxifen, treatment with either DMP-777 or L-635 to induce acute oxyntic atrophy caused induction of SPEM, but no lineage mapping into SPEM from Lgr5-expressing cells was observed. Conclusion: The results indicate that, while chief cells with Lgr5 transcriptional activity are present along the lesser curvature of the gastric oxyntic mucosa, they are not responsible for production of metaplasia.

Staab J.F.,Johns Hopkins University | Kahn J.N.,Merck And Co. | Kahn J.N.,Rutgers University | Marr K.A.,Johns Hopkins University
Antimicrobial Agents and Chemotherapy | Year: 2010

The recently described species Aspergillus lentulus exhibits differential and reduced susceptibilities to echinocandins and other antifungal drugs in vitro. A. lentulus isolates overall are less susceptible to caspofungin, although they maintain susceptibility to anidulafungin and micafungin. Mutations or polymorphisms in fks, the gene encoding the catalytic subunit of β-1,3-glucan synthase, are known to confer decreased susceptibility to echinocandins in Candida spp. and Aspergillus fumigatus. The analysis of the A. lentulus fks sequence did not reveal a polymorphism at any of the known hot-spot regions of the gene. Caspofungin and micafungin kinetic inhibition profiles of the A. lentulus glucan synthase were comparable to those from susceptible A. fumigatus enzymes. Although the basal cell wall chitin levels in A. lentulus averaged 60% of those in A. fumigatus, echinocandin treatment promoted the increase of cell wall chitin in both organisms, indicating that A. lentulus displays a compensatory chitin response similar to that of A. fumigatus. The data suggest that differential echinocandin susceptibilities in A. lentulus are independent of the echinocandin target, Fksp, and they emphasize the potential that the drugs' capacity to inhibit the target enzyme is unequal at the cellular level. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Krawczyk C.M.,University of Pennsylvania | Krawczyk C.M.,Merck And Co. | Holowka T.,University of Pennsylvania | Sun J.,University of Pennsylvania | And 10 more authors.
Blood | Year: 2010

Dendritic cells (DCs) are key regulators of innate and acquired immunity. The maturation of DCs is directed by signal transduction events downstream of toll-like receptors (TLRs) and other pattern recognition receptors. Here, we demonstrate that, in mouse DCs, TLR agonists stimulate a profound metabolic transition to aerobic glycolysis, similar to the Warburg metabolism displayed by cancer cells. This metabolic switch depends on the phosphatidyl inositol 3′-kinase/Akt pathway, is antagonized by the adenosine monophosphate (AMP)-activated protein kinase (AMPK), and is required for DC maturation. The metabolic switch induced by DC activation is antagonized by the antiinflammatory cytokine interleukin-10. Our data pinpoint TLR-mediated metabolic conversion as essential for DC maturation and function and reveal it as a potential target for intervention in the control of excessive inflammation and inappropriately regulated immune responses. © 2010 by The American Society of Hematology.

Pfaller M.A.,University of Iowa | Diekema D.J.,University of Iowa | Andes D.,University of Wisconsin - Madison | Arendrup M.C.,Statens Serum Institute | And 4 more authors.
Drug Resistance Updates | Year: 2011

The CLSI established clinical breakpoints (CBPs) for caspofungin (CSF), micafungin (MCF) and anidulafungin (ANF) versus Candida. The same CBP (susceptible (S): MIC ≤ 2 mcg/ml; non-S: MIC > 2 mcg/ml) was applied to all echinocandins and species. More data now allow reassessment of these CBPs. We examined cases of echinocandin failure where both MICs and fks mutations were assessed; wild type (WT) MICs and epidemiological cutoff values (ECVs) for a large Candida collection; molecular analysis of fks hotspots for Candida with known MICs; and pharmacokinetic and pharmacodynamic (PK/PD) data. We applied these findings to propose new species-specific CBPs for echinocandins and Candida. Of 18 candidiasis cases refractory to echinocandins and with fks mutations, 28% (CSF), 58% (ANF) and 66% (MCF) had MICs in the S category using CBP of ≤2 mcg/ml, while 0-8% would be S using CBP of ≤0.25 mcg/ml. WT MIC distributions revealed ECV ranges of 0.03-0.25 mcg/ml for all major species except C. parapsilosis (1-4 mcg/ml) and C. guilliermondii (4-16 mcg/ml). Among Candida tested for fks mutations, only 15.7-45.1% of 51 mutants were detected using the CBP for NS of >2 mcg/ml. In contrast, a cutoff of >0.25 mcg/ml for C. albicans, C. tropicalis, C. krusei, and C. dubliniensis detected 85.6% (MCF) to 95.2% (CSF) of 21 mutant strains. Likewise, a cutoff of >0.12 mcg/ml for ANF and CSF and of >0.06 mcg/ml for MCF detected 93% (ANF) to 97% (CSF, MCF) of 30 mutant strains of C. glabrata. These data, combined with PK/PD considerations, support CBPs of ≤0.25 mcg/ml (S), 0.5 mcg/ml (I), ≥1 (R) for CSF/MCF/ANF and C. albicans, C. tropicalis and C. krusei and ≤2 mcg/ml (S), 4 mcg/ml (I), and ≥8 mcg/ml (R) for these agents and C. parapsilosis. The CBPs for ANF and CSF and C. glabrata are ≤0.12 mcg/ml (S), 0.25 mcg/ml (I), and ≥0.5 mcg/ml (R), whereas those for MCF are ≤0.06 mcg/ml (S), 0.12 mcg/ml (I), and ≥0.25 mcg/ml (R). New, species-specific CBPs for Candida and the echinocandins are more sensitive to detect emerging resistance associated with fks mutations, and better able to predict risk for clinical failure. © 2011 Elsevier Ltd. All rights reserved.

Bednar B.,Merck And Co. | Ntziachristos V.,Helmholtz Center Munich
Current Pharmaceutical Biotechnology | Year: 2012

Optical imaging has seen significant developments over the past decade as an investigational tool for in-vivo visualization of cellular and sub-cellular events. With the recent addition of optoacoustic (photoacoustic) methods, in particular multi-spectral opto-acoustic tomography (MSOT), to the already rich armamentarium of photonic methods the capacity of optical molecular imaging across scales has widened significantly. MSOT brings unique features into optical imaging, namely high resolution optical imaging over several millimeters to centimeters of tissue depth and the ability to simultaneously resolve multiple tissue molecules and extrinsically administered optical or optoacoustic agents with physiological or molecular specificity. Here, we discuss the implications of utilizing MSOT in the context of drug discovery and review suitable optoacoustic agents against disease and drug efficacy biomarkers. The combination of existing knowledge on generating optical targeted contrast, with the high resolution deep tissue visualization offered by MSOT, allows for the development of next-generation biological optical imaging and corresponding drug discovery applications. © 2012 Bentham Science Publishers.

Faith M.S.,University of Pennsylvania | Butryn M.,Drexel University | Wadden T.A.,University of Pennsylvania | Fabricatore A.,University of Pennsylvania | And 2 more authors.
Obesity Reviews | Year: 2011

Obesity may lead to depression or be one of its consequences. We reviewed population-based studies in order to, first, identify the most commonly used research methods, and, second, to evaluate the strength of evidence for prospective associations among obesity and depression. We examined 25 studies, of which 10 tested 'obesity-to-depression' pathways, and 15 tested 'depression-to-obesity' pathways. Descriptive statistics summarized the frequency with which various measurements, designs and data analytic strategies were used. We tallied the number of studies that reported any vs. no statistically significant associations, and report on effect sizes, identified moderating variables within reports, and sought common findings across studies. Results indicated considerable methodological heterogeneity in the literature. Depression was assessed by clinical interview in 44% of studies, weight and height were directly measured in 32%, and only 12% used both. In total, 80% of the studies reported significant obesity-to-depression associations, with odds ratios generally in the range of 1.0 to 2.0, while only 53% of the studies reported significant depression-to-obesity associations. Sex was a common moderating variable. Thus, there was good evidence that obesity is prospectively associated with increased depression, with less consistent evidence that depression leads to obesity. Recommendations for future research regarding study samples, measurement and data analysis are provided. © 2011 The Authors. obesity reviews © 2011 International Association for the Study of Obesity.

McGraw T.,Merck And Co.
Clinical and Experimental Gastroenterology | Year: 2016

Purpose: To evaluate the safety and tolerability of aqueous solution concentrate (ASC) of polyethylene glycol (PEG) 3350 in patients with functional constipation. Patients and methods: The patients who met Rome III diagnostic criteria for functional constipation were randomized in this multicenter, randomized, placebo-controlled, single-blind study to receive once daily dose of PEG 3350 (17 g) ASC or placebo solution for 14 days. The study comprised a screening period (visit 1), endoscopy procedure (visits 2 and 3), and followup telephone calls 30 days post-treatment. Safety end points included adverse events (AEs), clinical laboratory evaluations, vital signs, and others. The primary end points were the proportion of patients with abnormalities of the oral and esophageal mucosa, detected by visual and endoscopic examination of the oral cavity and esophagus, respectively, compared with placebo. A secondary objective was to compare the safety and tolerability of ASC by evaluating AEs or adverse drug reactions. Results: A total of 65 patients were enrolled in this study, 31 were randomized to PEG 3350 ASC and 34 were randomized to placebo, of which 62 patients completed the study. No patients in either group showed abnormalities in inflammation of the oral mucosa during visit 2 (before treatment) or visit 3 (after treatment). Fewer abnormalities of the esophageal mucosa were observed in the PEG 3350 ASC group than in the placebo group on visit 3, with no significant difference in the proportion of abnormalities between the treatment groups. Overall, 40 treatment-emergent AEs were observed in 48.4% of patients treated with PEG 3350 ASC, and 41 treatment-emergent AEs were observed in 55.9% of patients treated with placebo-nonsignificant difference of -7.5% (95% CI: -21.3, 6.3) between treatment groups. No serious AEs or deaths were reported, and no patient discontinued because of an AE. Conclusion: PEG 3350 ASC is safe and well tolerated in patients with functional constipation (NCT01885104). © 2016 McGraw.

Feng D.,Merck And Co. | Tierney L.,University of Iowa
Journal of Statistical Software | Year: 2011

This paper presents an R package for magnetic resonance imaging (MRI) tissue classification. The methods include using normal mixture models, hidden Markov normal mixture models, and a higher resolution hidden Markov normal mixture model fitted by various optimization algorithms and by a Bayesian Markov chain Monte Carlo (MCMC) method. Functions to obtain initial values of parameters of normal mixture models and spatial parameters are provided. Supported input formats are ANALYZE, NIfTI, and a raw byte format. The function slices3d in misc3d is used for visualizing data and results. Various performance evaluation indices are provided to evaluate classification results. To improve performance, table lookup methods are used in several places, and vectorized computation taking advantage of conditional independence properties are used. Some computations are performed by C code, and OpenMP is used to parallelize key loops in the C code.

Mandl J.N.,U.S. National Institutes of Health | Mandl J.N.,McGill University | Ahmed R.,Emory University | Barreiro L.B.,University of Montreal | And 4 more authors.
Cell | Year: 2015

Zoonotic viruses, such as HIV, Ebola virus, coronaviruses, influenza A viruses, hantaviruses, or henipaviruses, can result in profound pathology in humans. In contrast, populations of the reservoir hosts of zoonotic pathogens often appear to tolerate these infections with little evidence of disease. Why are viruses more dangerous in one species than another? Immunological studies investigating quantitative and qualitative differences in the host-virus equilibrium in animal reservoirs will be key to answering this question, informing new approaches for treating and preventing zoonotic diseases. Integrating an understanding of host immune responses with epidemiological, ecological, and evolutionary insights into viral emergence will shed light on mechanisms that minimize fitness costs associated with viral infection, facilitate transmission to other hosts, and underlie the association of specific reservoir hosts with multiple emerging viruses. Reservoir host studies provide a rich opportunity for elucidating fundamental immunological processes and their underlying genetic basis, in the context of distinct physiological and metabolic constraints that contribute to host resistance and disease tolerance. © 2015 Elsevier Inc. All rights reserved.

Rolan P.,University of Adelaide | Sun H.,Novartis | MacLeod C.,Merck And Co. | Bracken K.,Novartis | Evans T.G.,Novartis
Clinical Pharmacology and Therapeutics | Year: 2011

Peptide deformylase (PDF) inhibitors represent a potential new class of antibiotics targeting a large number of bacterial species. We studied the pharmacokinetics and safety of LBM415, a novel PDF inhibitor, administered as a single oral dose at 100-3,000mg in the fasted state and at 1,000mg in the fed state in healthy volunteers. LBM415 was then administered at dosages ranging from 100mg q.d. to 1,000mg t.i.d. for 11 days. Dose-proportional pharmacokinetics was observed, with a peak plasma concentration (C max) of 17.85 5.96νg/ml at 1,000mg b.i.d. (the projected therapeutic dose) and an area under the concentration-time curve (AUC) 0-24h of 36.83 10.36νg/mlh. The half-life, as determined after a 1,000-mg single dose, was 2.18 0.61h. The compound was well tolerated at low doses, but at the highest dose, 1,000mg t.i.d., reversible cyanosis and low oxygen saturation, attributable to methemoglobinemia, were detected on day 11. Oxygen saturation was as low as 88% in one subject on day 11. © 2011 american society for Clinical Pharmacology and therapeutics.

Capen R.,Merck And Co.
International Journal of Biostatistics | Year: 2011

It is often necessary to compare two measurement methods in medicine and other experimental sciences. This problem covers a broad range of data with applications arising from many different fields. The Bland-Altman method has been a favorite method for concordance assessment. However, the Bland-Altman approach creates a problem of interpretation for many applications when a mixture of fixed bias, proportional bias and/or proportional error occurs. In this paper, an improved Bland-Altman method is proposed to handle more complicated scenarios in practice. This new approach includes Bland-Altman's approach as its special case. We evaluate concordance by defining an agreement interval for each individual paired observation and assessing the overall concordance. The proposed interval approach is very informative and offers many advantages over existing approaches. Data sets are used to demonstrate the advantages of the new method. © 2011 Berkeley Electronic Press. All rights reserved.

Sherlock J.P.,University of Birmingham | Sherlock J.P.,University of Oxford | Buckley C.D.,University of Birmingham | Cua D.J.,Merck And Co.
Molecular Immunology | Year: 2014

The spondyloarthropathies represent highly enigmatic conditions and although their clinical features, anatomical distribution of disease and genetic predisposing factors have been known for some time, a unified concept of the basic pathobiology underlying these illnesses has remained undefined. Recently progress has been made because numerous independent studies have converged upon IL-23 as a central cytokine in spondyloarthropathy and the mechanism and sites of action of this cytokine have now become much clearer. These findings enable the rational design of therapeutic strategies which it is hoped will profoundly modify the progression of these diseases. We will review the anatomical sites affected and the evidence for the importance of IL-23 in these conditions, before drawing these lines of investigation together to propose a model for the unified understanding of spondyloarthropathy. © 2013 Elsevier Ltd.

Gibertini M.,INC Research | Nations K.R.,Merck And Co. | Whitaker J.A.,INC Research
International Clinical Psychopharmacology | Year: 2012

The high failure rate of antidepressant trials has spurred exploration of the factors that affect trial sensitivity. In the current analysis, Food and Drug Administration antidepressant drug registration trial data compiled by Turner et al. is extended to include the most recently approved antidepressants. The expanded dataset is examined to further establish the likely population effect size (ES) for monoaminergic antidepressants and to demonstrate the relationship between observed ES and sample size in trials on compounds with proven efficacy. Results indicate that the overall underlying ES for antidepressants is approximately 0.30, and that the variability in observed ES across trials is related to the sample size of the trial. The current data provide a unique real-world illustration of an often underappreciated statistical truism: that small N trials are more likely to mislead than to inform, and that by aligning sample size to the population ES, risks of both erroneously high and low effects are minimized. The results in the current study make this abstract concept concrete and will help drug developers arrive at informed gate decisions with greater confidence and fewer risks, improving the odds of success for future antidepressant trials. © 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Zhang W.,UBS Equities | Zhu J.,Merck And Co. | Schadt E.E.,Sage Bionetworks | Schadt E.E.,Pacific Biosciences | Liu J.S.,Harvard University
PLoS Computational Biology | Year: 2010

Studies of the relationship between DNA variation and gene expression variation, often referred to as "expression quantitative trait loci (eQTL) mapping", have been conducted in many species and resulted in many significant findings. Because of the large number of genes and genetic markers in such analyses, it is extremely challenging to discover how a small number of eQTLs interact with each other to affect mRNA expression levels for a set of co-regulated genes. We present a Bayesian method to facilitate the task, in which co-expressed genes mapped to a common set of markers are treated as a module characterized by latent indicator variables. A Markov chain Monte Carlo algorithm is designed to search simultaneously for the module genes and their linked markers. We show by simulations that this method is more powerful for detecting true eQTLs and their target genes than traditional QTL mapping methods. We applied the procedure to a data set consisting of gene expression and genotypes for 112 segregants of S. cerevisiae. Our method identified modules containing genes mapped to previously reported eQTL hot spots, and dissected these large eQTL hot spots into several modules corresponding to possibly different biological functions or primary and secondary responses to regulatory perturbations. In addition, we identified nine modules associated with pairs of eQTLs, of which two have been previously reported. We demonstrated that one of the novel modules containing many daughter-cell expressed genes is regulated by AMN1 and BPH1. In conclusion, the Bayesian partition method which simultaneously considers all traits and all markers is more powerful for detecting both pleiotropic and epistatic effects based on both simulated and empirical data. © 2010 Zhang et al.

Frederick C.B.,Merck And Co. | Obach R.S.,Pfizer
Clinical Pharmacology and Therapeutics | Year: 2010

To address questions relating to the safety assessment of circulating drug metabolites in humans, the US Food and Drug Administration (FDA) and the International Conference on Harmonisation (ICH) have recently issued regulatory guidances1,2 that effectively require metabolite profiling in humans during early clinical development of a candidate drug. The clinical metabolite profiling results may require separate safety assessment studies in humans of stable circulating metabolites if such metabolites are not formed in sufficient concentrations in the plasma during nonclinical safety assessment of the parent drug. © 2009 American Society for clinical Pharmacology and Therapeutics.

Wang F.,Yale University | Simen A.,Yale University | Simen A.,Merck And Co. | Arias A.,Yale University | And 2 more authors.
Human Genetics | Year: 2013

Alcohol dependence (AD) is a common neuropsychiatric disorder with high heritability. A number of studies have analyzed the association between the Taq1A polymorphism (located in the gene cluster ANKK1/DRD2) and AD. In the present study, we conducted a large-scale meta-analysis to confirm the association between the Taq1A polymorphism and the risk for AD in over 18,000 subjects included in 61 case-control studies that were published up to August 2012. Our meta-analysis demonstrated both allelic and genotypic association between the Taq1A polymorphism and AD susceptibility [allelic: P(Z) = 1.1 × 10-5, OR = 1.19; genotypic: P(Z) = 3.2 × 10 -5, OR = 1.24]. The association remained significant after adjustment for publication bias using the trim and fill method. Sensitivity analysis showed that the effect size of the Taq1A polymorphism on AD risk was moderate and not influenced by any individual study. The pooled odds ratio from published studies decreased with the year of publication, but stabilized after the year 2001. Subgroup analysis indicated that publication bias could be influenced by racial ancestry. In summary, this large-scale meta-analysis confirmed the association between the Taq1A polymorphism and AD. Future studies are required to investigate the functional significance of the ANKK1/DRD2 Taq1A polymorphism in AD. © 2012 Springer-Verlag Berlin Heidelberg.

Narayanan M.,Seattle Genetics | Narayanan M.,Merck And Co. | Vetta A.,McGill University | Schadt E.E.,Seattle Genetics | And 2 more authors.
PLoS Computational Biology | Year: 2010

Many genome-wide datasets are routinely generated to study different aspects of biological systems, but integrating them to obtain a coherent view of the underlying biology remains a challenge. We propose simultaneous clustering of multiple networks as a framework to integrate large-scale datasets on the interactions among and activities of cellular components. Specifically, we develop an algorithm JointCluster that finds sets of genes that cluster well in multiple networks of interest, such as coexpression networks summarizing correlations among the expression profiles of genes and physical networks describing protein-protein and protein-DNA interactions among genes or gene-products. Our algorithm provides an efficient solution to a well-defined problem of jointly clustering networks, using techniques that permit certain theoretical guarantees on the quality of the detected clustering relative to the optimal clustering. These guarantees coupled with an effective scaling heuristic and the flexibility to handle multiple heterogeneous networks make our method JointCluster an advance over earlier approaches. Simulation results showed JointCluster to be more robust than alternate methods in recovering clusters implanted in networks with high false positive rates. In systematic evaluation of JointCluster and some earlier approaches for combined analysis of the yeast physical network and two gene expression datasets under glucose and ethanol growth conditions, JointCluster discovers clusters that are more consistently enriched for various reference classes capturing different aspects of yeast biology or yield better coverage of the analysed genes. These robust clusters, which are supported across multiple genomic datasets and diverse reference classes, agree with known biology of yeast under these growth conditions, elucidate the genetic control of coordinated transcription, and enable functional predictions for a number of uncharacterized genes. © 2010 Narayanan et al.

Yawn B.P.,Olmsted Medical Center | Wollan P.C.,Olmsted Medical Center | Kurland M.J.,Olmsted Medical Center | St Sauver J.L.,Mayo Medical School | Saddier P.,Merck And Co.
Mayo Clinic Proceedings | Year: 2011

OBJECTIVE: To present population-based estimates of herpes zoster (HZ) recurrence rates among adults. PATIENTS AND METHODS: To identify recurrent cases of HZ, we reviewed the medical records (through December 31, 2007) of all Olmsted County, Minnesota, residents aged 22 years or older who had an incident case of HZ between January 1, 1996, and December 31, 2001. Kaplan-Meier curves and Cox regression models were used to describe recurrences by age, immune status, and presence of prolonged pain at the time of the incident HZ episode. RESULTS: Of the 1669 persons with a medically documented episode of HZ, 95 had 105 recurrences (8 persons with >1 recurrence) by December 31, 2007, an average follow-up of 7.3 years. The Kaplan-Meier estimate of the recurrence rate at 8 years was 6.2%. With a maximum follow-up of 12 years, the time between HZ episodes in the same person varied from 96 days to 10 years. Recurrences were significantly more likely in persons with zoster-associated pain of 30 days or longer at the initial episode (hazard ratio, 2.80; 95% confidence interval, 1.84-4.27; P<.001) and in immunocompromised individuals (hazard ratio, 2.35; 95% confidence interval, 1.35-4.08; P=.006). Women and anyone aged 50 years or older at the index episode also had a greater likelihood of recurrence. CONCLUSION: Rates of HZ recurrence appear to be comparable to rates of first HZ occurrence in immunocompetent individuals, suggesting that recurrence is sufficiently common to warrant investigation of vaccine prevention in this group. © 2011 Mayo Foundation for Medical Education and Research.

Allen C.,Merck And Co.
Pharmaceutical Medicine | Year: 2016

Reviewing promotional materials for pharmaceutical products is important to help ensure their high quality and allow recipients to be well-informed about benefits and risks. This article provides an overview of control mechanisms that can influence the overall quality of the product combined with practical advice, based on personal US and international experience. US case reports are used to illustrate examples of promotional violations, and the consequences of government enforcement. © 2016, Springer International Publishing Switzerland.

Berger R.,Sharp Corporation | Berger R.,Merck And Co. | Berger W.E.,Allergy and Asthma Associates of Southern California
Allergy and Asthma Proceedings | Year: 2013

Inhaled corticosteroids administered by dry powder inhaler (DPI) or metered-dose inhaler are potent anti-inflammatory agents recommended for management of persistent asthma. Respiratory pharmacotherapy is unique in that proper delivery of the medication can be as important as the medication that is delivered. This study was designed to assess the mass median aerodynamic diameter (MMAD) of mometasone furoate (MF) particles from the 100- and 200-microgram/inhalation strength Twisthhaler (Merck and Co., Inc., Whitehouse Station, NJ) and to examine clinical effects of MF-DPI on midexpiratory flow. The MMAD with 100- and 200-microgram Twisthaler inhalers was analyzed using an Anderson cascade impactor. Clinical trial data for MF-DPI administered q.d. in the evening (P.M.) in adults and adolescents aged ≥12 years, as well as children aged 4-11 years, were examined post hoc for changes in forced expiratory flow between 25 and 75% of vital capacity (FEF25-75). The average MMAD of the 100-microgram strength Twisthaler (n = 24) was 2.0 micrometers; the average MMAD of the 200-microgram strength Twisthaler (n = 24) was 2.2 micrometers. In adults and adolescents (n = 1149), significant improvements in FEF25-75 occurred with MF-DPI administered q.d. P.M. versus placebo (p < 0.05). In children (n = 296), FEF25-75 improved significantly with MF-DPI at 100 microgram q.d. P.M. versus placebo at day 4 and every visit thereafter (p < 0.05). In vitro study suggests that the particle size of MF is optimal (̃2 micrometers) for efficient lung deposition when administered via the Twisthaler. Furthermore, randomized, controlled trials provide clinical evidence that MF-DPI q.d. treatment improves small airway function in patients with mild persistent or moderate asthma. Copyright © 2013, OceanSide Publications, Inc., U.S.A.

Welch C.J.,Merck And Co. | Hawkins J.M.,Pfizer | Tom J.,Bristol Myers Squibb
Organic Process Research and Development | Year: 2014

As the pharmaceutical industry continues to explore new ways to stimulate innovation, reduce costs, and streamline operations, the idea of joining forces in cross-pharma collaborations on the development of new and valuable precompetitive technologies becomes increasingly attractive. Before industry-wide precompetitive collaboration can become truly commonplace and more effective, improved approaches for creating and maintaining collaborations between competitors are needed. A recent Council for Chemical Research workshop at the University of Pennsylvania on precompetitive collaborations on enabling chemical and chemical engineering technologies for the pharmaceutical industry revealed widespread interest among participants from industry, academia and government to augment and improve current methods. While it is important not to underestimate the challenges of multiparty collaborations, the value coming from pooling resources and combining intellectual input provides strong incentive to improve and foster precompetitive collaborations. Here we summarize key points from this discussion and propose a new hybrid model for improved collaborative development of new enabling technologies for the pharmaceutical industry. © 2014 American Chemical Society.

Klimas M.,Merck And Co.
JACC: Cardiovascular Imaging | Year: 2013

Objectives This study hypothesized that 1H magnetic resonance spectroscopy (1H-MRS) can identify carotid plaque cholesteryl ester in vivo in humans. Background Liquid phase cholesteryl ester comprises a major fraction of atherosclerotic plaque, and its abundance is associated with plaque rupture and atherothrombosis. A noninvasive imaging technique to detect liquid cholesteryl ester that has been applied ex vivo is now demonstrated in vivo. Methods 1H-MRS scans were obtained of carotid plaques of 35 subjects at 3.0 T. Turbo spin echo, black blood, T1-weighted images were acquired for localization. Spectra were acquired using a 2-dimensional point resolved spectroscopy sequence: repetition time/echo time = 1,100/30 ms, 5-mm slice thickness, 8 × 8-cm field of view, 16 × 16 matrix size, and 13-min acquisition time. Saturation bands were placed around the artery. Resonance of methylene protons and allylic methylene protons were assigned to 1.2 ppm and 2.0 ppm. The 2.0:1.2 ppm ratio was calculated to reflect the ratio of the fatty acid composition of plaque cholesteryl ester to that of triglycerides of perivascular tissue. We obtained spectra of lipid standards as a reference. Results Our 1H-MRS data showed typical spectra of cholesteryl ester mixed with triglycerides, with intense resonance from methylene (1.2 ppm) and allylic methylene (2.0 ppm) protons. The average 2.0:1.2 ppm ratio was 0.10 ± 0.03. The 2.0:1.2 ppm ratio correlated with the plaque tissue volume to perivascular tissue volume ratio (Spearman rho = 0.55, p = 0.02), suggesting that more 1H-MRS signal was obtained from cholesteryl ester when the 1H-MRS voxel comprised more plaque tissue. Repeat 1H-MRS scans in 4 subjects showed an intraclass correlation coefficient of 0.92 (95% prediction intervals: 0.40 to 0.99), indicating good reproducibility. Seventeen of the 35 1H-MRS spectra were of adequate quality for analysis. Conclusions In vivo image-guided 1H-MRS for detection of liquid phase cholesteryl ester in carotid atherosclerotic plaques in humans is feasible. © 2013 by the American College of Cardiology Foundation Published by Elsevierinc .

Tynebor R.M.,Merck And Co.
Bioorganic & medicinal chemistry letters | Year: 2012

This manuscript concludes the Structure Activity Relationship (SAR) on the pyridazinone scaffold and identifies a compound with subnanomolar p38α activity and 24h coverage in the rat arthritis efficacy model. Copyright © 2012 Elsevier Ltd. All rights reserved.

For a clinical trial incorporating a group sequential test that allows early stopping for efficacy or futility (GSTEF), the primary hypothesis concerns efficacy. However, the type II error probability of the tests of efficacy is neither specified nor known. The type II error probability of a GSTEF is partitioned into the sum of its component type II error probabilities of futility and efficacy. This partitioning provides transparency, allowing researchers flexibility to set these component error probabilities directly and to know the impact on the total type II error probability and vice versa. This transparency and flexibility should improve the application of GSTEF to clinical trials. © 2013 Copyright Taylor and Francis Group, LLC.

Merck consolidated discovery stage bioanalytical functions into the Department of Pharmacokinetics, Pharmacodynamics & Drug Metabolism in 2007. Since then procedures and equipment used to provide important quantitative data to project teams have been harmonized and in many cases standardized. This approach has enabled movement of work across the network of laboratories and has resulted in a lean, flexible and efficient organization. The overall goal was to reduce time and resources spent on routine activities while creating time to perform research in new areas and technologies to support future scientific needs. The current state of discovery bioanalysis at Merck is discussed, including hardware and software platforms, workflow procedures and performance metrics. Examples of improved processes will be discussed for compound tuning, LC method development, analytical acceptance criteria, automated sample preparation, sample analysis platforms, data processing and data reporting.

Xie H.,Central South University | Xie H.,Southern Medical University | Xie H.,Johns Hopkins Hospital | Xie H.,Merck And Co. | Xie H.,Johns Hopkins University
Nature medicine | Year: 2014

Osteogenesis during bone modeling and remodeling is coupled with angiogenesis. A recent study showed that a specific vessel subtype, strongly positive for CD31 and endomucin (CD31(hi)Emcn(hi)), couples angiogenesis and osteogenesis. Here, we found that platelet-derived growth factor-BB (PDGF-BB) secreted by preosteoclasts induces CD31(hi)Emcn(hi) vessel formation during bone modeling and remodeling. Mice with depletion of PDGF-BB in the tartrate-resistant acid phosphatase-positive cell lineage show significantly lower trabecular and cortical bone mass, serum and bone marrow PDGF-BB concentrations, and fewer CD31(hi)Emcn(hi) vessels compared to wild-type mice. In the ovariectomy (OVX)-induced osteoporotic mouse model, serum and bone marrow levels of PDGF-BB and numbers of CD31(hi)Emcn(hi) vessels are significantly lower compared to sham-operated controls. Treatment with exogenous PDGF-BB or inhibition of cathepsin K to increase the number of preosteoclasts, and thus the endogenous levels of PDGF-BB, increases CD31(hi)Emcn(hi) vessel number and stimulates bone formation in OVX mice. Thus, pharmacotherapies that increase PDGF-BB secretion from preosteoclasts offer a new therapeutic target for treating osteoporosis by promoting angiogenesis and thus bone formation.

Herlogsson L.,Linkoping University | Crispin X.,Linkoping University | Tierney S.,Merck And Co. | Berggren M.,Linkoping University
Advanced Materials | Year: 2011

Organic complementary inverters and ring oscillators based on polyelectrolyte-gated thin-film transistors are demonstrated. Detrimental electrochemical doping is suppressed by using polyanionic and polycationic gate insulators in the p- and n-channel transistors, respectively. The circuits operate at supply voltages between 0.2 V and 1.5 V, have a static power consumption of less than 2.5 nW per logic gate and show propagation delays down to 0.26 ms per stage. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Kim S.-G.,University of Pittsburgh | Harel N.,University of Minnesota | Jin T.,University of Pittsburgh | Kim T.,University of Pittsburgh | And 2 more authors.
NMR in Biomedicine | Year: 2013

The cerebral blood volume (CBV) is a crucial physiological indicator of tissue viability and vascular reactivity. Thus, noninvasive CBV mapping has been of great interest. For this, ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, including monocrystalline iron oxide nanoparticles, can be used as long-half-life, intravascular susceptibility agents of CBV MRI measurements. Moreover, CBV-weighted functional MRI (fMRI) with USPIO nanoparticles provides enhanced sensitivity, reduced large vessel contribution and improved spatial specificity relative to conventional blood oxygenation level-dependent fMRI, and measures a single physiological parameter that is easily interpretable. We review the physiochemical and magnetic properties, and pharmacokinetics, of USPIO nanoparticles in brief. We then extensively discuss quantifications of baseline CBV, vessel size index and functional CBV change. We also provide reviews of dose-dependent sensitivity, vascular filter function, specificity, characteristics and impulse response function of CBV fMRI. Examples of CBV fMRI specificity at the laminar and columnar resolution are provided. Finally, we briefly review the application of CBV measurements to functional and pharmacological studies in animals. Overall, the use of USPIO nanoparticles can determine baseline CBV and its changes induced by functional activity and pharmacological interventions. © 2012 John Wiley & Sons, Ltd.

Bhardwaj S.P.,University of Minnesota | Bhardwaj S.P.,Merck And Co. | Suryanarayanan R.,University of Minnesota
Pharmaceutical Research | Year: 2013

Purpose: The work aims at investigating the correlation of water sorption potential with different measures of molecular mobility in an annealed amorphous model compound (trehalose). Methods: Amorphous trehalose, prepared by freeze-drying, was annealed at 100 C (17 C < T g) for up to 120 h. Global molecular mobility was studied using a broadband dielectric spectrometer in the frequency range of 106-10-2 Hz. Enthalpic recovery was measured by differential scanning calorimetry and water sorption profiles were obtained using an automated vapor sorption balance. Results: As a function of annealing time, there was an increase, both in average α-relaxation time and enthalpic recovery and a decrease in the amount of sorbed water. A strong linear correlation was observed between the water sorption potential and the dielectric relaxation time, indicating a common underlying mechanism of the effect of annealing time on these properties. Enthalpic recovery, which is widely used as a measure of structural relaxation, did not correlate well with the extent of water sorption. Conclusions: The α-relaxation time can be used as a predictor of the water sorption potential of amorphous trehalose. It will be of interest and value to develop such predictive models for other amorphous compounds of pharmaceutical interest. © 2012 Springer Science+Business Media New York.

Zhao M.,University of East Anglia | Barker S.A.,University of East Anglia | Belton P.S.,University of East Anglia | McGregor C.,Merck And Co. | Craig D.Q.M.,University of East Anglia
European Journal of Pharmaceutics and Biopharmaceutics | Year: 2012

The aim of the study was to prepare molecular dispersions of a physically highly unstable amorphous drug, paracetamol (acetaminophen with a Tg of ca. 25 °C) via co-spray drying with a variety of polymers. Solid dispersions at a range of drug loadings (10-90%w/w) using hydroxypropyl methylcellulose/acetate succinate (HPMC/HPMC AS), polyvinylpyrrolidone (PVP) and copovidone were produced and characterised by modulated temperature differential scanning calorimetry (MTDSC), thermogravimetric analysis (TGA), X-ray powder diffraction (XRPD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). PVP-based polymers showed a greater tendency than the HPMC-based group to generate temperature-stable dispersions. In particular, copovidone (Plasdone® S-630) was found to be the most effective of the polymers studied and could formulate molecular dispersions at drug loadings up to and including 40%w/w. However, no evidence for direct drug-polymer interactions was found for such systems as a possible stabilising mechanism. The expected relationship of a higher Tg of the polymer leading to greater stabilisation was not observed, while there was an inverse relationship between viscosity grade and amorphous phase generation. The study has therefore shown that temperature-stable amorphous dispersions of a low Tg drug may be prepared by co-spray drying, particularly using PVP-based polymers. © 2012 Elsevier B.V. All rights reserved.

Sun S.,Cold Spring Harbor Laboratory | Sun S.,University of California at San Diego | Zhang Z.,Cold Spring Harbor Laboratory | Zhang Z.,Merck And Co. | And 3 more authors.
RNA | Year: 2012

RBFOX1 and RBFOX2 are alternative splicing factors that are predominantly expressed in the brain and skeletal muscle. They specifically bind the RNA element UGCAUG, and regulate alternative splicing positively or negatively in a position-dependent manner. The molecular basis for the position dependence of these and other splicing factors on alternative splicing of their targets is not known. We explored the mechanisms of RBFOX splicing activation and repression using an MS2-tethering assay. We found that the Ala/Tyr/Gly-rich C-terminal domain is sufficient for exon activation when tethered to the downstream intron, whereas both the C-terminal domain and the central RRM are required for exon repression when tethered to the upstream intron. Using immunoprecipitation and mass spectrometry, we identified hnRNP H1, RALY, and TFG as proteins that specifically interact with the C-terminal domain of RBFOX1 and RBFOX2. RNA interference experiments showed that hnRNP H1 and TFG modulate the splicing activity of RBFOX1/2, whereas RALY had no effect. However, TFG is localized in the cytoplasm, and likely modulates alternative splicing indirectly. Published by Cold Spring Harbor Laboratory Press. Copyright © 2012 RNA Society.

Severyn B.,Merck And Co.
ACS chemical biology | Year: 2011

Combination therapies that enhance efficacy or permit reduced dosages to be administered have seen great success in a variety of therapeutic applications. More fundamentally, the discovery of epistatic pathway interactions not only informs pharmacologic intervention but can be used to better understand the underlying biological system. There is, however, no systematic and efficient method to identify interacting activities as candidates for combination therapy and, in particular, to identify those with synergistic activities. We devised a pooled, self-deconvoluting screening paradigm for the efficient comprehensive interrogation of all pairs of compounds in 1000-compound libraries. We demonstrate the power of the method to recover established synergistic interactions between compounds. We then applied this approach to a cell-based screen for anti-inflammatory activities using an assay for lipopolysaccharide/interferon-induced acute phase response of a monocytic cell line. The described method, which is >20 times as efficient as a naïve approach, was used to test all pairs of 1027 bioactive compounds for interleukin-6 suppression, yielding 11 pairs of compounds that show synergy. These 11 pairs all represent the same two activities: β-adrenergic receptor agonists and phosphodiesterase-4 inhibitors. These activities both act through cyclic AMP elevation and are known to be anti-inflammatory alone and to synergize in combination. Thus we show proof of concept for a robust, efficient technique for the identification of synergistic combinations. Such a tool can enable qualitatively new scales of pharmacological research and chemical genetics.

Liao J.J.Z.,Merck And Co.
Pharmaceutical Statistics | Year: 2010

It is often necessary to compare two measurement methods in medicine and other experimental sciences. This problem covers a broad range of data. Many authors have explored ways of assessing the agreement of two sets of measurements. However, there has been relatively little attention to the problem of determining sample size for designing an agreement study. In this paper, a method using the interval approach for concordance is proposed to calculate sample size in conducting an agreement study. The philosophy behind this is that the concordance is satisfied when no more than the prespecified k discordances are found for a reasonable large sample size n since it is much easier to define a discordance pair. The goal here is to find such a reasonable large sample size n. The sample size calculation is based on two rates: the discordance rate and tolerance probability, which in turn can be used to quantify an agreement study. The proposed approach is demonstrated through a real data set. Copyright © 2009 John Wiley & Sons, Ltd.

Martin G.E.,Merck And Co.
Journal of Heterocyclic Chemistry | Year: 2012

Posaconazole is a structurally complex triazole antifungal agent that, by virtue of its structural complexity, provides a good test molecule for the evaluation of NMR structure elucidation methodologies. Although GHMBC and related long-range 1H- 13C heteronuclear shift correlation techniques are extremely powerful, at the same time, when dealing with unknowns, they can be problematic in that there is no way to readily differentiate adjacent ( 2 J CH) correlations from longer range correlations, e.g., 3J CH and nJ CH, n > 3. The 1,1-ADEQUATE experiment, in contrast, provides unequivocal experimental access to adjacent carbon-carbon correlation information, albeit with a sensitivity penalty, as the experiment involves an adjacent 13C- 13C out-and-back magnetization transfer. In part, the sensitivity penalty can be overcome by using unsymmetrical indirect covariance or general indirect covariance processing methods. The application of these methods through the coprocessing of multiplicity-edited GHSQC and 1,1-ADEQUATE data to generate an HSQC-ADEQUATE correlation plot is demonstrated for posaconazole. © 2012 Hetero Corporation.

Okin P.M.,Cornell University | Bang C.N.,Cornell University | Wachtell K.,Gentofte Hospital | Hille D.A.,Merck And Co. | And 4 more authors.
Circulation: Arrhythmia and Electrophysiology | Year: 2013

Background-Prevalent atrial fibrillation (AF) is associated with a higher sudden cardiac death (SCD) rate in some populations, and incident AF predicts increased mortality risk in the general population and after myocardial infarction. However, the relationship of SCD to new-onset AF is unclear. Methods and Results-The relationship of SCD to new-onset AF was evaluated in 8831 hypertensive patients with electrocardiographic left ventricular hypertrophy with no history of AF, in sinus rhythm on their baseline electrocardiogram, randomly assigned to losartan- or atenolol-based treatment. During 4.7±1.1 years mean follow-up, new-onset AF occurred in 701 patients (7.9%) and SCD in 151 patients (1.7%). In univariate Cox analyses, new-onset AF was associated with a > 4-fold higher risk of SCD (hazard ratio, 4.69; 95% CI interval, 2.96-7.45; P<0.001). In multivariate Cox analyses adjusting for age, sex, race, diabetes mellitus, history of heart failure, myocardial infarction, ischemic heart disease, stroke, smoking, serum high-density lipoprotein cholesterol, creatinine, glucose, and urine albumin/creatinine ratio as standard risk factors, and for incident myocardial infarction, in-treatment use of digoxin, systolic and diastolic pressure, heart rate, QRS duration, Cornell voltage-duration product, and Sokolow-Lyon voltage left ventricular hypertrophy treated as time-varying covariates, new-onset AF remained associated with a > 3-fold increased risk of SCD (hazard ratio, 3.13; 95% confidence interval, 1.87-5.24; P<0.001). Conclusions-Development of new-onset AF identifies hypertensive patients at increased risk of SCD. © 2013 American Heart Association, Inc.

Murthi A.,Ohio State University | Murthi A.,National Institute of Allergy and Infectious Diseases | Shaheen H.H.,Ohio State University | Shaheen H.H.,Merck And Co. | And 5 more authors.
Molecular Biology of the Cell | Year: 2010

tRNAs in yeast and vertebrate cells move bidirectionally and reversibly between the nucleus and the cytoplasm. We investigated roles of members of the β-importin family in tRNA subcellular dynamics. Retrograde import of tRNA into the nucleus is dependent, directly or indirectly, upon Mtr10. tRNA nuclear export utilizes at least two members of the β-importin family. The β-importins involved in nuclear export have shared and exclusive functions. Los1 functions in both the tRNA primary export and the tRNA reexport processes. Msn5 is unable to export tRNAs in the primary round of export if the tRNAs are encoded by intron-containing genes, and for these tRNAs Msn5 functions primarily in their reexport to the cytoplasm. The data support a model in which tRNA retrograde import to the nucleus is a constitutive process; in contrast, reexport of the imported tRNAs back to the cytoplasm is regulated by the availability of nutrients to cells and by tRNA aminoacylation in the nucleus. Finally, we implicate Tef1, the yeast orthologue of translation elongation factor eEF1A, in the tRNA reexport process and show that its subcellular distribution between the nucleus and cytoplasm is dependent upon Mtr10 and Msn5. © 2010 by The American Society for Cell Biology.

McFarland K.N.,University of Florida | Huizenga M.N.,Massachusetts General Hospital | Darnell S.B.,Massachusetts General Hospital | Sangrey G.R.,Massachusetts General Hospital | And 4 more authors.
Human Molecular Genetics | Year: 2014

Transcriptional dysregulation has been proposed to play amajor role in the pathology of Huntington's disease (HD). However, the mechanisms that cause selective downregulation of target genes remain unknown. Previous studies have shown that mutant huntingtin (Htt) protein interacts with a number of transcription factors thereby altering transcription. Here we report that Htt directly interacts with methyl-CpG binding protein 2 (MeCP2) in mouse and cellular models of HD using complimentary biochemical and Fluorescent Lifetime Imaging to measure Fö rster Resonance Energy Transfer approaches. Htt-MeCP2 interactions are enhanced in the presence of the expanded polyglutamine (polyQ) tract and are stronger in the nucleus compared with the cytoplasm. Furthermore, we find increased binding of MeCP2 to the promoter of brain-derived neurotrophic factor (BDNF), a gene that is downregulated in HD, in the presence of mutant Htt. Finally, decreasing MeCP2 levels in mutant Htt-expressing cells using siRNA increases BDNF levels, suggesting that MeCP2 downregulates BDNF expression in HD. Taken together, these findings suggest that aberrant interactions between Htt and MeCP2 contribute to transcriptional dysregulation in HD. © The Author 2013. Published by Oxford University Press. All rights reserved.

We previously reported the development of a human monoclonal antibody (CS-D7, IgG(1)) with specificity and affinity for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus. CS-D7 mediates opsonophagocytic killing in vitro and protection in a murine sepsis model. In light of recent data indicating that IsdB specific T cells (CD4+, Th17), not Ab, mediate protection after vaccination with IsdB, it is important to investigate the mechanism of protection mediated by CS-D7. The mAb was examined to determine if it blocked heme binding to IsdB in vitro. The mAb was not found to have heme blocking activity, nor did it prevent bacterial growth under in vivo conditions, in an implanted growth chamber. To assess the role of the mAb Fc a point mutation was introduced at aa 297 (CS-D7·N297A). This point mutation removes Fc effector functions. In vitro analysis of the mutein confirmed that it lacked measurable binding to FcγR, and that it did not fix complement. The mutein had dramatically reduced in vitro opsonic OP activity compared to CS-D7. Nonetheless, the mutein conferred protection equivalent to the wild type mAb in the murine sepsis model. Both wild type and mutein mAbs were efficacious in FcγR deletion mice (including both FcγRII(-/-) mice and FcγRIII(-/-) mice), indicating that these receptors were not essential for mAb mediated protection in vivo. Protection mediated by CS-D7 was lost in Balb/c mice depleted of C3 with cobra venom factor (CFV), was lost in mice depleted of superoxide dismutase (SOD) in P47phox deletion mice, and as previously reported, was absent in SCID mice (Joshi et al., 2012). Enhanced clearance of S. aureus in the liver of CS-D7 treated mice and enhanced production of IFN-γ, but not of IL17, may play a role in the mechanism of protection mediated by the mAb. CS-D7 apparently mediates survival in challenged mice through a mechanism involving complement, phagocytes, and lymphocytes, but which does not depend on interaction with FcγR, or on blocking heme uptake.

Berlin M.,Merck And Co.
Expert Opinion on Therapeutic Patents | Year: 2010

Importance of the field: The glucocorticoid receptor plays a number of fundamental roles in human physiology. Glucocorticosteroids are the ultimate anti-inflammatory and immunosuppressive agents highly efficacious in the treatment of serious diseases, but also associated with serious side effects. Improvement in the therapeutic profiles of drugs, acting at the glucocorticoid receptor, is highly desired and may potentially arise from the separation of their gene transactivating and gene transrepressing properties. Areas covered in this review: The review summarizes progress towards novel glucocorticoid drug candidates as indicated by the patent applications over the last 2 years (2008 2009). A brief discussion of glucocorticoid receptor biology and previous drug candidates is included. What the reader will gain: The understanding of the structural scope and biological profiles of the glucocorticoid receptor modulators, currently in preclinical and clinical development, based on the review of ∼ 180 composition-of-matter and method-of-use patent applications. Take home message: The information on the good chemotypical diversity of glucocorticoid receptor modulators needs to be supplemented by the clinical data presumably, soon to become available to allow a look into a possible improvement in therapeutic index over the classic glucocorticosteroids. © 2010 Informa UK. Ltd.

Biotransformation of chemically stable compounds to reactive metabolites that can bind covalently to macromolecules (such as proteins and DNA) is considered an undesirable property of drug candidates. Due to the possible link, which has not yet been conclusively demonstrated, between reactive metabolites and adverse drug reactions, screening for metabolic activation of lead compounds through in vitro chemical trapping experiments has become an integral part of the drug discovery process in many laboratories. In this review, we provide an overview of the recent advances in the application of high-resolution MS. These advances facilitated the development of accurate-mass-based data mining tools for high-throughput screening of reactive drug metabolites in drug discovery. © 2012 Future Science Ltd.

Thome J.J.C.,Columbia University | Yudanin N.,Columbia University | Ohmura Y.,Columbia University | Kubota M.,Columbia University | And 7 more authors.
Cell | Year: 2014

Mechanisms for human memory T cell differentiation and maintenance have largely been inferred from studies of peripheral blood, though the majority of T cells are found in lymphoid and mucosal sites. We present here a multidimensional, quantitative analysis of human T cell compartmentalization and maintenance over six decades of life in blood, lymphoid, and mucosal tissues obtained from 56 individual organ donors. Our results reveal that the distribution and tissue residence of naive, central, and effector memory, and terminal effector subsets is contingent on both their differentiation state and tissue localization. Moreover, T cell homeostasis driven by cytokine or TCR-mediated signals is different in CD4+ or CD8+ T cell lineages, varies with their differentiation stage and tissue localization, and cannot be inferred from blood. Our data provide an unprecedented spatial and temporal map of human T cell compartmentalization and maintenance, supporting distinct pathways for human T cell fate determination and homeostasis. © 2014 Elsevier Inc.

Mohan P.,Childrens National Medical Center | Barton B.A.,University of Massachusetts Medical School | Narkewicz M.R.,Aurora University | Molleston J.P.,Indiana University | And 5 more authors.
Hepatology | Year: 2013

Clinical and histologic progression of liver disease in untreated children with chronic hepatitis C virus (HCV) infection is poorly documented. The aim of this retrospective study was to characterize changes in liver histology over time in a cohort of HCV-infected children who had more than one liver biopsy separated by over 1 year. Forty-four untreated children without concurrent liver diseases, who had repeat liver biopsies at eight U.S.-based medical centers, were included. Biopsies were scored by a single pathologist for inflammation, fibrosis, and steatosis and were correlated with demographic data including age at biopsy, time from infection to biopsies, and laboratory values such as serum alanine aminotransferase (ALT). Mode of transmission was vertical in 25 (57%) and from transfusions in 17 children (39%). Genotype 1 was present in 30/35 (84%) children. The mean age at first and final biopsy was 8.6 and 14.5 years, respectively, and the mean interval between biopsies was 5.8 ± 3.5 years. Duration of infection to biopsy was 7.7 and 13.5 years, respectively. Laboratory values did not change significantly between the biopsies. Inflammation was minimal in about 50% at both timepoints. Fibrosis was absent in 16% in both biopsies, limited to portal/periportal in 73% in the first biopsy, and 64% in the final biopsy. Between the two biopsies, the proportion of patients with bridging fibrosis/cirrhosis increased from 11% to 20% (P = 0.005). Conclusion: Although in aggregate this cohort did not show significant histologic progression of liver disease over 5 years, 29.5% (n = 13) of children showed an increase in severity of fibrosis. These findings may have long-term implications for the timing of follow-up biopsies and treatment decisions. © 2013 by the American Association for the Study of Liver Diseases.

LaFace D.,Merck And Co. | Talmadge J.,University of Nebraska Medical Center
International Immunopharmacology | Year: 2011

Recent investigations of regulatory myeloid cell mobilization and amplification in response to cancer and chronic inflammation associated with infectious disease and autoimmunity have provided numerous insights into the suppressive mechanisms, pathobiology and potential pharmaceutical modulation of MDSC. Controversies have emerged as hematology investigators, have suggested that regulatory myeloid cells, including MDSC merely reflect normal physiological responses to inflammatory and growth factor stimuli. A recurring theme at the recent "International Immunopharmacology Conference on Regulatory Myeloid cells" held in Arlington, Virginia, October 21-24, 2010, focused on efforts to more clearly elucidate molecular features of MDSC from the immature myeloid cells observed in the circulation in response to acute infection and inflammatory responses. Evidence for the potential contribution of regulatory myeloid cells to pathologic processes and sequelae associated with numerous inflammatory and malignant diseases also suggested that these cells may represent a significant deviation from the normal physiologic responses to acute inflammatory stimuli. The relevance of these research efforts was evident from the discussions by numerous clinical investigations regarding their efforts to modulate MDSC in cancer patients to augment immunotherapeutic treatments. © 2011 Elsevier B.V. All rights reserved.

Nti-Gyabaah J.,Merck And Co. | Gbewonyo K.,BioResources International Inc. | Chiew Y.C.,Rutgers University
Journal of Chemical and Engineering Data | Year: 2010

The solubility of artemisnin in 12 different organic solvents, methanol, ethanol, butanol, acetone, ethyl acetate, isopropyl acetate, acetonitrile, hexane, heptane, 2-butanone (MEK), methyl tert-butyl ether (MTBE), and toluene, as well as in three binary solvent mixtures of ethyl acetate + ethanol, ethyl acetate + acetone, and ethanol + acetone, within the temperature range of (284.10 and 323.15) K, is obtained. The solubility data were fitted to the Two-Liquid-Non-Random (NRTL) activity coefficient equation to obtain the model binary interaction parameters, which were used to predict the solubility of artemisinin in ethyl acetate and acetone or ethanol binary solvent mixtures. The predicted values compared favorably with the experimental data. © 2010 American Chemical Society.

Scapin G.,Merck And Co.
Acta Crystallographica Section D: Biological Crystallography | Year: 2013

The 'phase problem' in crystallography results from the inability to directly measure the phases of individual diffracted X-ray waves. While intensities are directly measured during data collection, phases must be obtained by other means. Several phasing methods are available (MIR, SAR, MAD, SAD and MR) and they all rely on the premise that phase information can be obtained if the positions of marker atoms in the unknown crystal structure are known. This paper is dedicated to the most popular phasing method, molecular replacement (MR), and represents a personal overview of the development, use and requirements of the methodology. The first description of noncrystallographic symmetry as a tool for structure determination was explained by Rossmann and Blow [Rossmann & Blow (1962), Acta Cryst. 15, 24-31]. The term 'molecular replacement' was introduced as the name of a book in which the early papers were collected and briefly reviewed [Rossmann (1972), The Molecular Replacement Method. New York: Gordon & Breach]. Several programs have evolved from the original concept to allow faster and more sophisticated searches, including six-dimensional searches and brute-force approaches. While careful selection of the resolution range for the search and the quality of the data will greatly influence the outcome, the correct choice of the search model is probably still the main criterion to guarantee success in solving a structure using MR. Two of the main parameters used to define the 'best' search model are sequence identity (25% or more) and structural similarity. Another parameter that may often be undervalued is the quality of the probe: there is clearly a relationship between the quality and the correctness of the chosen probe and its usefulness as a search model. Efforts should be made by all structural biologists to ensure that their deposited structures, which are potential search probes for future systems, are of the best possible quality.

Stauffer M.E.,SCRIBCO | Fan T.,Merck And Co.
PLoS ONE | Year: 2014

Anemia is one of the many complications of chronic kidney disease (CKD). However, the current prevalence of anemia in CKD patients in the United States is not known. Data from the National Health and Nutrition Examination Survey (NHANES) in 2007-2008 and 2009-2010 were used to determine the prevalence of anemia in subjects with CKD. The analysis was limited to adults aged >18 who participated in both the interview and exam components of the survey. Three outcomes were assessed: the prevalence of CKD, the prevalence of anemia in subjects with CKD, and the self-reported treatment of anemia. CKD was classified into 5 stages based on the glomerular filtration rate and evidence of kidney damage, in accordance with the guidelines of the National Kidney Foundation. Anemia was defined as serum hemoglobin levels ≤12 g/dL in women and ≤13 g/dL in men. We found that an estimated 14.0% of the US adult population had CKD in 2007-2010. Anemia was twice as prevalent in people with CKD (15.4%) as in the general population (7.6%). The prevalence of anemia increased with stage of CKD, from 8.4% at stage 1 to 53.4% at stage 5. A total of 22.8% of CKD patients with anemia reported being treated for anemia within the previous 3 months-14.6% of patients at CKD stages 1-2 and 26.4% of patients at stages 3-4. These results update our knowledge of the prevalence and treatment of anemia in CKD in the United States. © 2014 Stauffer, Fan.

Seghieri M.,University of Pisa | Rebelos E.,University of Pisa | Gastaldelli A.,CNR Institute of Clinical Physiology | Astiarraga B.D.,University of Pisa | And 7 more authors.
Diabetologia | Year: 2013

Aims/hypothesis: Glucagon-like peptide-1 (GLP-1) lowers glucose levels by potentiating glucose-induced insulin secretion and inhibiting glucagon release. The question of whether GLP-1 exerts direct effects on the liver, independently of the hormonal changes, is controversial. We tested whether an exogenous GLP-1 infusion, designed to achieve physiological postprandial levels, directly affects endogenous glucose production (EGP) under conditions mimicking the fasting state in diabetes. Methods: In 14 healthy volunteers, we applied the pancreatic clamp technique, whereby plasma insulin and glucagon levels are clamped using somatostatin and hormone replacement. The clamp was applied in paired, 4 h experiments, during which saline (control) or GLP-1(7-37)amide (0.4 pmol min-1 kg-1) was infused. Results: During the control study, plasma insulin and glucagon were maintained at basal levels and plasma C-peptide was suppressed, such that plasma glucose rose to a plateau of ∼10.5 mmol/l and tracer-determined EGP increased by ∼60%. During GLP-1 infusion at matched plasma glucose levels, the rise of EGP from baseline was fully prevented. Lipolysis (as indexed by NEFA concentrations and tracer-determined glycerol rate of appearance) and substrate utilisation (by indirect calorimetry) were similar between control and GLP-1 infusion. Conclusions/interpretation: GLP-1 inhibits EGP under conditions where plasma insulin and glucagon are not allowed to change and glucose concentrations are matched, indicating either a direct effect on hepatocytes or neurally mediated inhibition. © 2012 Springer-Verlag Berlin Heidelberg.

Goals of work A number of prognostic factors have been identified as risk factors for chemotherapy-induced emesis. This post-hoc analysis addressed whether: (1) these prognostic factors can identify a low-risk group for whom ondansetron plus dexamethasone alone provide a high level of protection (≥80% no emesis); (2) the NK1 receptor antagonist aprepitant improves antiemetic outcome regardless of emetic risk. Patients and methods Breast cancer patients in a phase III double-blind, placebo-controlled trial were randomized to antiemetic regimens including ondansetron and dexamethasone, or aprepitant, ondansetron, and dexamethasone. Multivariate logistic regression models were used to assess the impact on emesis (but not nausea) of the regimen with aprepitant, and previously reported risk factors, including age (<55 and =55 years), ethanol use (0-4 or =5 drinks/week), history of pregnancy-related morning sickness, and history of motion sickness, using a modified intent-to-treat approach. Results Treatment with aprepitant (P<0.0001), older age (P=0.006), ethanol use (P=0.0048), and no history of morning sickness (P=0.0007) were all significantly associated with reduced likelihood of emesis. The proportion of patients with one, two, or three risk factors who remained emesis free was significantly higher with the aprepitantcontaining regimen than with the active control (70.2-82.8% vs. 38.6-66.4%, respectively). Conclusions Aprepitant markedly improved control of emesis in patients with one or more risk factors. This analysis did not support using risk factors for modifying the antiemetic approach. A low-risk group with zero risk factors for whom aprepitant provided little benefit was of questionable clinical utility, since they comprised less than 3% of patients. © 2011 Springer-Verlag.

Objective: Compare the efficacy of 2 NRTIs combined with raltegravir (RAL), efavirenz (EFV), or protease inhibitors (PI) in the management of antiretroviral-naïve HIV adult patients.Methods: By means of a systematic literature view, 7 randomized controlled trials were identified: 2 RAL vs EFV trials; 1 ritonavir-boosted lopinavir (LPV/RTV) vs EFV trial; 1 ritonavir-boosted atazanavir (ATV/RTV) vs LPV/RTV trial; 1 ritonavir-boosted darunavir (DRV/RTV) vs LPV/RTV trial; 1 ritonavir-boosted fosamprenavir (FPV/RTV) vs LPV/RTV trial; and 1 FPV/RTV vs ATV/RTV trial. Endpoints concerned virological suppression and immunologic efficacy. Trials were analyzed with Bayesian mixed treatment comparison meta-analysis.Results: For up to 24 weeks of treatment, a PI-based regimen resulted in a lower proportion of patients with virological response than an EFV-based regimen, whereas RAL seems more efficacious than EFV up to at least 12 weeks. After 48 weeks, the odds ratio (OR) of virological suppression with RAL relative to EFV was 1.34 (95% credible interval [CrI], 0.87-2.07). ORs for PIs relative to EFV varied from 0.68 (0.41-1.07) with LPV/RTV to 0.99 (0.52-1.84) with DRV/RTV. RAL demonstrated a greater improvement in CD4+ T cell counts than EFV at 48 weeks. The PI regimens showed all similar improvements relative to EFV.Conclusion: Based on available RCTs, the fastest virological suppression is expected with RAL followed by EFV and PIs. Over time, RAL appears to be at least as good as PI and EFV regimens. CD4+ cell recovery seems the greatest with LPV/RTV, DRV/RTV, and RAL. Given the limited number of RCTs, additional studies are recommended. © 2011 Thomas Land Publishers, Inc.

Stam W.B.,MAPI Group | Jansen J.P.,MAPI Group | Taylor S.D.,Merck And Co.
Open Rheumatology Journal | Year: 2012

Objective: To compare the efficacy of etoricoxib, lumiracoxib, celecoxib, non-selective (ns) NSAIDs and acetaminophen in the treatment of osteoarthritis (OA) Methods: Randomized placebo controlled trials investigating the effects of acetaminophen 4000mg, diclofenac 150mg, naproxen 1000mg, ibuprofen 2400mg, celecoxib 100-400mg, lumiracoxib 100-400mg, and etoricoxib 30-60mg with treatment duration of at least two weeks were identified with a systematic literature search. The endpoints of interest were pain, physical function and patient global assessment of disease status (PGADS). Pain and physical function reported on different scales (VAS or LIKERT) were translated into effect sizes (ES). An ES 0.2 - 0.5 was defined as a "small" treatment effect, whereas ES of 0.5 - 0.8 and > 0.8 were defined as "moderate" and "large", respectively. A negative effect indicated superior effects of the treatment group compared to the control group. Results of all trials were analyzed simultaneously with a Bayesian mixed treatment comparison. Results: There is a >95% probability that etoricoxib (30 or 60mg) shows the greatest improvement in pain and physical function of all interventions compared. ESs of etoricoxib 30mg relative to placebo, celecoxib 200mg, ibuprofen 2400mg, and diclofenac 150mg were -0.66 (95% Credible Interval -0.83; -0.49), -0.32 (-0.50; -0.14), -0.25 (-0.53; 0.03), and -0.17 (-0.41; 0.08), respectively. Regarding physical functioning, ESs of etoricoxib 30mg relative to placebo, celecoxib 200mg, ibuprofen 2400mg, and diclofenac 150mg were -0.61 (-0.76; -0.46), -0.27 (-0.43; -0.10), -0.20 (-0.47; 0.07), and -0.09 (-0.33; 0.14) respectively. The greatest improvements in PGADS were expected with either etoricoxib or diclofenac. Conclusion: The current study estimated the efficacy of acetaminophen, nsNSAIDs, and COX-2 selective NSAIDs in OA and found that etoricoxib 30 mg is likely to result in the greatest improvements in pain and physical function. Differences in PGADS between interventions were smaller. © Stam et al.

Bigal M.E.,Merck And Co. | Bigal M.E.,Yeshiva University | Rapoport A.M.,University of California at Los Angeles
Current Pain and Headache Reports | Year: 2012

Obesity may be the greatest epidemic of modern times. It leads to diabetes and heart disease and shortens lifespan. Although not a risk factor for migraine, it is associated with an increased frequency and intensity of migraine. Obesity is also comorbid with chronic daily headache and is a major risk factor for chronification of episodic migraine in adults and children. Although obesity is not a factor in the effectiveness of migraine treatment, it does increase the peripheral and central events in migraine, ultimately increasing the neurologic potential for migraine. Although evidence suggests that obesity is a modifiable risk factor for migraine progression, it is unknown if weight loss is related to decrease in headache frequency. Recent surgical results suggest that this is true. We suggest all possible effective techniques aimed at weight loss be undertaken for migraineurs, especially obese migraineurs, and that carefully monitoring weight changes should be routinely done as part of their migraine care. © 2011 Springer Science+Business Media, LLC.

Perez-Trujillo M.,Autonomous University of Barcelona | Castanar L.,Autonomous University of Barcelona | Monteagudo E.,Autonomous University of Barcelona | Kuhn L.T.,European Neuroscience Institute Gottingen ENI G | And 4 more authors.
Chemical Communications | Year: 2014

NMR enantiodifferentiation studies are greatly improved by the simultaneous determination of 1H and 13C chemical shift differences through the analysis of highly resolved cross-peaks in spectral aliased pure shift (SAPS) HSQC spectra. This journal is © the Partner Organisations 2014.

Lu-Yao G.L.,The New School | Lu-Yao G.L.,Dean and Betty Gallo Prostate Cancer Center | Albertsen P.C.,University of Connecticut Health Center | Moore D.F.,The New School | And 6 more authors.
JAMA Internal Medicine | Year: 2014

Importance One in 6 American men will be diagnosed as having prostate cancer during their lifetime. Although there are no data to support the use of primary androgen-deprivation therapy (ADT) for early-stage prostate cancer, primary ADT has been widely used for localized prostate cancer, especially among older patients. OBJECTIVE To determine the long-term survival impact of primary ADT in older men with localized (T1/T2) prostate cancer. Design, Setting, and Participants Thiswas a population-based cohort study of 66 717 Medicare patients 66 years or older diagnosed from 1992 through 2009 who received no definitive local therapy within 180 days of prostate cancer diagnosis. The study was conducted in predefined US geographical areas covered by the Surveillance, Epidemiology, and End Results (SEER) Program. Instrumental variable analysis was used to assess the impact of primary ADT and control for potential biases associated with unmeasured confounding variables. The instrumental variable comprised combined health services areas with various usage rates of primary ADT. The analysis compared survival outcomes in the top tertile areas with those in the bottom tertile areas. Main Outcomes and Measures Prostate cancer-specific survival and overall survival. Results With a median follow-up of 110 months, primary ADT was not associated with improved 15-year overall or prostate cancer-specific survival following the diagnosis of localized prostate cancer. Among patients with moderately differentiated cancers, the 15-year overall survival was 20.0%in areas with high primary ADT use vs 20.8%in areas with low use (difference: 95%CI, -2.2%to 0.4%), and the 15-year prostate cancer survival was 90.6%in both high- and low-use areas (difference: 95%CI, -1.1%to 1.2%). Among patients with poorly differentiated cancers, the 15-year cancer-specific survival was 78.6%in high-use areas vs 78.5%, in low-use areas (difference: 95%CI, -1.8%to 2.4%), and the 15-year overall survival was 8.6%in high-use areas vs 9.2%in low-use areas (difference: 95%CI, -1.5%to 0.4%). Conclusions and Relevance Primary ADT is not associated with improved long-term overall or disease-specific survival for men with localized prostate cancer. Primary ADT should be used only to palliate symptoms of disease or prevent imminent symptoms associated with disease progression. © 2014 American Medical Association. All rights reserved.

Ter Meulen J.,Merck And Co.
Infectious Disease Clinics of North America | Year: 2011

Of the more than 20 monoclonal antibodies (mAbs) generated to combat infectious diseases (ID) that are in clinical development in 2011, most are in phase 1 or 2 and are directed against either viruses or bacterial toxins. Several high-profile anti-ID mAbs have recently failed in clinical trials. Despite the advancement in recombinant engineering technologies, anti-ID mAbs have yet to deliver on their promise as " magic bullets," especially against nosocomial infections. A paradigm shift in favor of developing mAb combinations, which act synergistically with each other or with small molecule drugs, may be required to move the field forward. © 2011 Elsevier Inc.

Aragon E.,Barcelona Institute for Research in Biomedicine | Goerner N.,Barcelona Institute for Research in Biomedicine | Goerner N.,Biocrates Life Sciences | Xi Q.,Sloan Kettering Cancer Center | And 7 more authors.
Structure | Year: 2012

Transforming growth factor (TGF)-β and BMP signaling is mediated by Smads 1-5 (R-Smads and Co-Smads) and inhibited by Smad7, a major hub of regulation of TGF-β and BMP receptors by negative feedback and antagonistic signals. The transcription coactivator YAP and the E3 ubiquitin ligases Smurf1/2 and Nedd4L target R-Smads for activation or degradation, respectively. Pairs of WW domain in these regulators bind PY motifs and adjacent CDK/MAPK and GSK3 phosphorylation sites in R-Smads in a selective and regulated manner. In contrast, here we show that Smad7 binds YAP, Smurf1, Smurf2, and Nedd4L constitutively, the binding involving a PY motif in Smad7 and no phosphorylation. We also provide a structural basis for how regulators that use WW domain pairs for selective interactions with R-Smads, resort to one single versatile WW domain for binding Smad7 to centralize regulation in the TGF-β and BMP pathways. © 2012 Elsevier Ltd.

Hill A.,Evotec | Mesens N.,Janssen Pharmaceutical | Steemans M.,Janssen Pharmaceutical | Xu J.J.,Merck And Co. | Aleo M.D.,Pfizer
Drug Metabolism Reviews | Year: 2012

Drug-induced liver injury (DILI) is a major cause of attrition during both the early and later stages of the drug development and marketing process. Reducing or eliminating drug-induced severe liver injury, especially those that lead to liver transplants or death, would be tremendously beneficial for patients. Therefore, developing new pharmaceuticals that have the highest margins and attributes of hepatic safety would be a great accomplishment. Given the current low productivity of pharmaceutical companies and the high costs of bringing new medicines to market, any early screening assay(s) to identify and eliminate pharmaceuticals with the potential to cause severe liver injury in humans would be of economic value as well. The present review discusses the background, proof-of-concept, and validation studies associated with high-content screening (HCS) by two major pharmaceutical companies (Pfizer Inc and Jansen Pharmaceutical Companies of Johnson & Johnson) for detecting compounds with the potential to cause human DILI. These HCS assays use fluorescent-based markers of cell injury in either human hepatocytes or HepG2 cells. In collaboration with Evotec, an independent contract lab, these two companies also independently evaluated larval zebrafish as an early-stage in vivo screen for hepatotoxicity in independently conducted, blinded assessments. Details about this model species, the need for bioanalysis, and, specifically, the outcome of the phenotypic-based zebrafish screens are presented. Comparing outcomes in zebrafish against both HCS assays suggests an enhanced detection for hepatotoxicants of most DILI concern when used in combination with each other, based on the U.S. Food and Drug Administration DILI classification list. © 2012 Informa Healthcare USA, Inc.

Shaw P.M.,Merck And Co. | Patterson S.D.,Amgen Inc.
Journal of the National Cancer Institute - Monographs | Year: 2011

To gain insights into human biology and pathobiology, ready access to banked human tissue samples that encompass a representative cross section of the population is required. For optimal use, the banked human tissue needs to be appropriately consented, collected, annotated, and stored. If any of these elements are missing, the studies using these samples are compromised. These elements are critical whether the research is for academic or pharmaceutical industry purposes. An additional temporal element that adds enormous value to such banked samples is treatment and outcome information from the people who donated the tissue. To achieve these aims, many different groups have to work effectively together, not least of which are the individuals who donate their tissue with appropriate consent. Such research is unlikely to benefit the donors but others who succumb to the same disease. The development of a large accessible human tissue bank resource (National Cancer Institute's Cancer HUman Biobank [caHUB]) that provides an ongoing supply of human tissue for all working toward the common goal of understanding human health and disease has a number of advantages. These include, but are not limited to, access to a broad cross section of healthy and diseased populations beyond what individual collections may achieve for understanding disease pathobiology, therapeutic target discovery, as well as a source of material for diagnostic assay validation. Models will need to be developed to enable fair access to caHUB under terms that enable appropriate intellectual property protection and ultimate data sharing to ensure that the biobank successfully distributes samples to a broad range of researchers. © The Author 2011. Published by Oxford University Press. All rights reserved.

Williamson R.T.,Merck And Co. | Buevich A.V.,Merck | Martin G.E.,Merck
Organic Letters | Year: 2012

A relatively unexplored and unexploited means of establishing molecular structure, stereochemistry, and probing vicinal bond angles is through the use of long-range 13C-13C coupling constants. The measurement of these multifunctional, diagnostic 3JCC couplings has not been reported on sample amounts viable for the practicing organic chemist. A generalized protocol for the measurement of 1JCC and 3JCC couplings using a 4.6 mg sample of strychnine as a model compound is described, and the utility of DFT calculations for the prediction of these useful molecular descriptors and the congruence of the calculated and experimental data is demonstrated. © 2012 American Chemical Society.

Russell M.R.,Childrens Hospital of Philadelphia | Levin K.,Childrens Hospital of Philadelphia | Rader J.,Childrens Hospital of Philadelphia | Belcastro L.,Childrens Hospital of Philadelphia | And 9 more authors.
Cancer Research | Year: 2013

Neuroblastoma is uniquely sensitive to single-agent inhibition of the DNA damage checkpoint kinase Chk1, leading us to examine downstream effectors of this pathway and identify mitotic regulator Wee1 as an additional therapeutic target in this disease. Wee1 was overexpressed in both neuroblastoma cell lines and high-risk patient tumors. Genetic or pharmacologic abrogation of Wee1 signaling results in marked cytotoxicity in 10 of 11 neuroblastoma cell lines with a median IC50 of 300 nmol/L for the Wee1-selective small-molecule inhibitor MK- 1775. Murine tumor lines derived from mice that were either heterozygous or homozygous for MycN were particularly sensitive to single-agent inhibition of Wee1 (IC50s of 160 and 62 nmol/L, respectively). Simultaneous pharmacologic inhibition of Chk1 and Wee1 acted in a synergistic fashion to further impede neuroblastoma cell growth in vitro, in a manner greater than the individual inhibitors either alone or combined with chemotherapy. Combination Chk1 and Wee1 inhibition also revealed in vivo efficacy in neuroblastoma xenografts. Taken together, our results show that neuroblastoma cells depend on Wee1 activity for growth and that inhibition of this kinase may serve as a therapeutic for patients with neuroblastoma. © 2012 AACR.

Kumar P.,University of Utah | Kumar P.,Merck And Co. | Thakur A.,University of Utah | Hong X.,University of California at Los Angeles | And 2 more authors.
Journal of the American Chemical Society | Year: 2014

A Ni/N-heterocyclic carbene catalyst couples diynes to the C(α)-C(β) double bond of tropone, a type of reaction that is unprecedented for metal-catalyzed cycloadditions with aromatic tropone. Many different diynes were efficiently coupled to afford [5-6-7] fused tricyclic products, while [5-7-6] fused tricyclic compounds were obtained as minor byproducts in a few cases. The reaction has broad substrate scope and tolerates a wide range of functional groups, and excellent regioselectivity is found with unsymmetrical diynes. Theoretical calculations show that the apparent enone cycloaddition occurs through a distinctive 8π insertion of tropone. The initial intramolecular oxidative cyclization of diyne produces the nickelacyclopentadiene intermediate. This intermediate undergoes an 8π insertion of tropone, and subsequent reductive elimination generates the [5-6-7] fused tricyclic product. This initial product undergoes two competing isomerizations, leading to the observed [5-6-7] and [5-7-6] fused tricyclic products. © 2014 American Chemical Society.

Shen H.C.,Merck And Co.
Expert Opinion on Therapeutic Patents | Year: 2010

Importance of the field: Soluble epoxide hydrolase (sEH) inhibitors have been shown to effectively increase the levels of epoxyeicosatrienoic acids and reduce the levels of dihydroxyeicosatrienoic acids, which may be translated to therapeutic potentials for multiple disease indications. It has been claimed that sEH inhibitors can be used to treat hypertension, diabetes, stroke, dyslipidemia, pain, immunological disorders, eye diseases, neurological diseases and other indications. Areas covered in this review: A comprehensive synopsis of patent literature on sEH inhibitors is provided. What the reader will gain: A total of more than 100 patent publications describing multiple classes of sEH inhibitors are analyzed. These include amides, ureas, thioamides, thioureas, carbamates, acyl hydrazones, chalcone oxdies, etc. In addition to selected in vitro and in vivo data of representative sEH inhibitors, a wide range of proposed applications of sEH inhibitors are also summarized. Take home message: Several sEH inhibitors with potent in vitro and in vivo target inhibition appear promising, including one Phase II clinical candidate. The clinical evaluation will be critical to assess the proclaimed therapeutic utility of sEH inhibition. © 2010 Informa UK. Ltd.

Hargreaves R.J.,Merck And Co.
ACS Medicinal Chemistry Letters | Year: 2012

In the neuroscience landscape, there is no condition with higher unmet medical and societal need than Alzheimer's disease (AD). There are significant opportunities to improve upon symptomatic treatments in AD, and as yet, there are no treatments to modify (slow, stop, or prevent) underlying disease progression. Our goals are to discover new symptomatic AD therapies with improved efficacy and longevity; to complete definitive studies that refute or prove the amyloid hypothesis, potentially opening multiple avenues to new therapeutic modalities; and to initiate tests of novel mechanisms that can prevent tau pathology and neurodegeneration. It's a critical time in the testing of novel AD therapeutics-let's hope we succeed. © 2012 American Chemical Society.

Cook J.L.,University of Missouri | Kuroki K.,University of Missouri | Visco D.,Merck And Co. | Pelletier J.-P.,University of Montreal | And 2 more authors.
Osteoarthritis and Cartilage | Year: 2010

The dog is a common model for study of osteoarthritis (OA). Subjective histologic scoring systems have often served as the reference standard for presence and severity of OA. However, these scoring systems have perceived shortcomings. The system developed for this report attempts to address these shortcomings by providing a standardized methodology for global assessment of the joint, versatility and the potential for relative weighting of pathology, allowing for comparison among time points, studies, and centers, and critical analysis of the system's reliability. The proposed system for assessment of canine tissues appears to provide an effective method for global assessment of articular pathology in OA. The system is versatile, comprehensive, and reliable and appears to have advantages over conventional scoring systems. © 2010 Osteoarthritis Research Society International.

Young T.A.,University of Sheffield | Rowen D.,University of Sheffield | Norquist J.,Merck And Co. | Brazier J.E.,University of Sheffield
Quality of Life Research | Year: 2010

Purpose: Condition-specific measures may not always have independent items, yet existing techniques of developing health state utility values from these measures are inappropriate when items are not independent. This study develops methods for deriving and valuing health states for a condition-specific preference-based measure where items are not independent. Methods: The analysis has three stages: firstly, Rasch analysis is used to develop a health state classification system from the Flushing Symptoms Questionnaire (FSQ) that is amenable to valuation and to identify a set of health states for valuation. Secondly, a valuation survey of the health states using time-trade-off (TTO) methods is conducted to elicit health state values. Finally, regression models are applied to map the relationship between mean TTO values and Rasch logit values. The model is then used to estimate health state values for all possible health states. Results: Rasch models were fitted to 1,270 responders to the FSQ and a series of 16 health states were identified for the valuation exercise. An ordinary least squares model best described the relationship between mean TTO values and Rasch logit values (R2 = 0.958; root mean square error = 0.042). Conclusions: This study demonstrates how health state utility values can be mapped onto Rasch logit values in order to value all states defined by the FSQ, a condition-specific measure where items are not independent. This should significantly enhance research in this field.

Lane S.W.,Harvard University | Lane S.W.,University of Queensland | Gilliland D.G.,Harvard University | Gilliland D.G.,Merck And Co.
Seminars in Cancer Biology | Year: 2010

Leukemia stem cells (LSC) reside within a hierarchy of malignant hematopoiesis and possess the ability to instigate, maintain and serially propagate leukemia in vivo, while retaining the capacity to differentiate into committed progeny that lack these properties. In most cases, LSC appear to share immunophenotypic characteristics with committed hematopoietic progenitors, however have pathologically enhanced self-renewal, mediated through the activation of certain cellular pathways. The presence of a LSC that solely possesses the ability to initiate and sustain leukemia has implications for the treatment of patients with this disease. In this review, we will discuss these issues as well as some of the recent controversies regarding LSC frequency and alternative theories of leukemogenesis. © 2009.

Carr R.D.,Novo Nordisk AS | Carr R.D.,Merck And Co. | Larsen M.O.,Novo Nordisk AS | Jelic K.,Novo Nordisk AS | And 5 more authors.
Journal of Clinical Endocrinology and Metabolism | Year: 2010

Context: Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are cleaved by dipeptidyl peptidase-4 (DPP-4); plasma activity of DPP-4 may be increased in obesity. The impact of this increase on incretin hormone secretion and metabolism is not known. Objective: The aim of the study was to assess incretin hormone secretion and degradation in lean and obese nondiabetic subjects. Design, Settings, and Participants: We studied the ingestion of a mixed meal (560 kcal) or oral glucose (2 g/kg) in healthy lean (n=12; body mass index, 20-25 kg/m2) or obese (n=13; body mass index, 30-35 kg/m2) males at a University Clinical Research Unit. Main Outcome Measures: We measured the area under the curve of plasma intact (i) and total (t) GIP and GLP-1 after meal ingestion and oral glucose. Results: Plasma DPP-4 activity was higher in the obese subjects (38.5 ± 3.0 vs. 26.7 ± 1.6 mmol/ min • μl; P=0.002). Although GIP secretion (AUC tGIP) was not reduced in obese subjects after meal ingestion or oral glucose, AUCiGIP was lower in obese subjects (8.5 ± 0.6 vs. 12.7 ± 0.9 nmol/liter x 300 min; P<0.001) after meal ingestion. GLP-1 secretion (AUCtGLP-1) was reduced in obese subjects after both meal ingestion (7.3±0.9 vs. 10.0±0.6 nmol/liter x 300 min; P=0.022) and oral glucose (6.6 ± 0.8 vs. 9.6 ± 1.1 nmol/liter x 180 min; P = 0.035). iGLP-1 was reduced in parallel to tGLP-1. Conclusions: 1) Release and degradation of the two incretin hormones show dissociated changes in obesity: GLP-1 but not GIP secretion is lower after meal ingestion and oral glucose, whereas GIP but not GLP-1 metabolism is increased after meal ingestion. 2) Increased plasma DPP-4 activity in obesity is not associated with a generalized augmented incretin hormone metabolism. Copyright © 2010 by The Endocrine Society.

We have previously shown that IsdB, a conserved protein expressed by Staphylococcus aureus, induces a robust antibody response which correlates with protection in a murine challenge model. Here we investigate the role of cellular immunity in IsdB mediated protection using lymphocyte deficient SCID mice. As opposed to WT CB-17 mice the CB-17 SCID mice were not protected against a lethal challenge of S. aureus after active and passive immunizations with IsdB. Adoptive transfer of in vitro isolated lymphocyte subsets revealed that reconstituting mice with IsdB specific CD3+ or CD4+ T-cells conferred antigen specific protection while CD8(+) T-cells, CD19(+) B-cells and plasma cells (CD138(high)B220(int)CD19(lo)) alone were not protective. A combination of CD3(+) T-cells plus CD19(+) B-cells conferred protection in CB-17 SCID mice, whereas bovine serum albumin (BSA) immune lymphocytes did not confer protection. Active immunization experiments indicated that IsdB immunized Jh mice (B-cell deficient) were protected against lethal challenge, while nude (T-cell deficient) mice were not. In vitro assays indicated that isolated IsdB specific splenocytes from immunized mice produced abundant IL-17A, much less IFN-γ and no detectable IL-4. IL-23 deficient mice were not protected from a lethal challenge by IsdB vaccination, pointing to a critical role for CD4(+) Th17 in IsdB-mediated vaccination. Neutralizing IL-17A, but not IL-22 in vivo significantly increased mortality in IsdB immunized mice; whereas, neutralizing IFN-γ did not alter IsdB-mediated protection. These findings suggest that IL-17A producing Th17 cells play an essential role in IsdB vaccine-mediated defense against invasive S. aureus infection in mice.

Wang L.,Merck And Co. | Plump A.,Sanofi S.A. | Ringel M.,Boston Consulting Group
Drug Discovery Today | Year: 2015

The pharmaceutical industry continues to face fundamental challenges because of issues with research and development (R&D) productivity and rising customer expectations. To lower R&D costs, move beyond me-too therapies, and create more transformative portfolios, pharmaceutical companies are actively capitalizing on external innovation through precompetitive collaboration with academia, cultivation of biotech start-ups, and proactive licensing and acquisitions. Here, we review the varying innovation strategies used by pharmaceutical companies, compare and contrast these models, and identify the trends in external innovation. We also discuss factors that influence these external innovation models and propose a preliminary set of metrics that could be used as leading indicators of success. © 2014 Elsevier Ltd. All rights reserved.

Pischon T.,Molecular Epidemiology Group | Hu F.B.,Harvard University | Girman C.J.,Merck And Co. | Rifai N.,Harvard University | And 3 more authors.
Atherosclerosis | Year: 2011

Objective: To examine prospectively the association of total and high molecular weight (HMW) adiponectin, and HMW/total adiponectin ratio with risk of incident coronary heart disease (CHD) in women, and to examine to what extent adjustment for potentially intermediary variables would explain this association. Methods and results: Among 30,111 women from the Nurses' Health Study, 468 women developed non-fatal myocardial infarction or fatal CHD during 14 years of follow-up. Using risk set sampling, controls were selected 2:1 matched on age, smoking, and date of blood draw. Adjusted for matching factors, parental history of myocardial infarction, hormone replacement therapy, alcohol consumption, physical activity, body mass index, hypertension, and low-density lipoprotein cholesterol levels, the relative risk in the highest versus lowest quintile was 0.50 (95%-CI 0.33-0.75; p trend = 0.001) for total adiponectin, 0.53 (95%-CI 0.35-0.80; p trend = 0.004) for HMW adiponectin, and 0.63 (95%-CI 0.43-0.93; p trend = 0.03) for HMW/total adiponectin ratio. After adjustment for diabetes, HDL-cholesterol, HbA1c, and CRP these associations were attenuated and no longer significant (RRs, 0.84; 95%-CI 0.53-1.33; p trend = 0.62; 0.95; 95%-CI 0.60-1.52; p trend = 0.98; 0.97; 95%-CI 0.64-1.47; p trend = 0.80). Conclusions: High levels of total and HMW adiponectin, and HMW/total adiponectin ratio are associated with a lower risk of CHD among women. HMW adiponectin and HMW/total adiponectin ratio are not more closely related to risk than total adiponectin. These associations are largely mediated by parameters related to glucose and lipid metabolism and inflammation, especially HDL-cholesterol levels. © 2011 Elsevier Ireland Ltd.

Kumar V.,University of Washington | Prasad B.,University of Washington | Patilea G.,University of Washington | Gupta A.,Astrazeneca | And 4 more authors.
Drug Metabolism and Disposition | Year: 2015

To predict transporter-mediated drug disposition using physiologically based pharmacokinetic models, one approach is to measure transport activity and relate it to protein expression levels in cell lines (overexpressing the transporter) and then scale these to via in vitro to in vivo extrapolation (IVIVE). This approach makes two major assumptions. First, that the expression of the transporter is predominantly in the plasma membrane. Second, that there is a linear correlation between expression level and activity of the transporter protein. The present study was conducted to test these two assumptions. We evaluated two commercially available kits that claimed to separate plasma membrane from other cell membranes. The Qiagen Qproteome kit yielded very little protein in the fraction purported to be the plasma membrane. The Abcam Phase Separation kit enriched the plasma membrane but did not separate it from other intracellular membranes. For the Abcam method, the expression level of organic anion-transporting polypeptides (OATP) 1B1/2B1 and breast cancer resistance protein (BCRP) proteins in all subcellular fractions isolated from cells or human liver tissue tracked that of Na+-K+ ATPase. Assuming that Na+-K+ ATPase is predominantly located in the plasma membrane, these data suggest that the transporters measured are also primarily located in the plasma membrane. Using short hairpin RNA, we created clones of cell lines with varying degrees of OATP1B1 or BCRP expression level. In these clones, transport activity of OATP1B1 or BCRP was highly correlated with protein expression level (r2 > 0.9). These data support the use of transporter expression level data and activity data from transporter overexpressing cell lines for IVIVE of transporter-mediated disposition of drugs. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Mach H.,Merck And Co. | Middaugh C.R.,University of Kansas
Journal of Pharmaceutical Sciences | Year: 2011

Ubiquitous ultraviolet absorption spectroscopy, despite the availability of more sophisticated techniques, remains an indispensable tool that can give an initial insight into the concentration and aggregation state of protein samples. The high degree of reproducibility afforded by diode-array spectrophotometers, combined with their powerful vendor-supplied algorithms, presents an opportunity for improving the accuracy and throughput for their use in pharmaceutical development. In this review, factors important to optimal utilization of the technique, as applied to the development of monoclonal antibodies, are discussed, and specific methodologies are described. In particular, techniques to probe the intrinsic structural properties of proteins, and their behavior under a wide variety of conditions, through the application of second-derivative spectroscopy combined with advanced computational treatments are presented. The information contained in this review is specifically directed to practitioners of the technique in contemporary research and development settings. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association.

Leigh K.E.,Harvard University | Sharma M.,Harvard University | Mansueto M.S.,Harvard University | Mansueto M.S.,Merck And Co. | And 6 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2015

Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50. Here, we present an NMR-determined solution-state structure of the murine CMV homolog of UL50 (M50; residues 1-168) with a strikingly intricate protein fold that is matched by no other known protein folds in its entirety. Using NMR methods, we mapped the interaction of M50 with a highly conserved UL53-derived peptide, corresponding to a segment that is required for heterodimerization. The UL53 peptide binding site mapped onto an M50 surface groove, which harbors a large cavity. Point mutations of UL50 residues corresponding to surface residues in the characterized M50 heterodimerization interface substantially decreased UL50-UL53 binding in vitro, eliminated UL50-UL53 colocalization, prevented disruption of nuclear lamina, and halted productive virus replication in HCMV-infected cells. Our results provide detailed structural information on a key protein-protein interaction involved in nuclear egress and suggest that NEC subunit interactions can be an attractive drug target. © 2015, National Academy of Sciences. All rights reserved.

Lopez D.,Autonomous University of Madrid | Garcia-Calvo M.,Merck And Co. | Smith G.L.,Imperial College London | Del Val M.,Autonomous University of Madrid
Journal of Immunology | Year: 2010

CD8+ cytotoxic T lymphocytes recognize infected cells in which MHC class I molecules present pathogen-derived peptides that have been processed mainly by proteasomes. Many infections induce a set of proteases, the caspases involved in apoptosis or inflammation. In this study, we report that processing and presentation of a short vaccinia virus-encoded Ag can take place also by a nonproteasomal pathway, which was blocked in infected cells with chemical inhibitors of caspases. By cleaving at noncanonical sites, at least two caspases generated antigenic peptides recognized by T lymphocytes. The sites and the peptidic products were partially overlapping but different to those used and produced by proteasomes in vitro. Antigenic natural peptides produced in infected cells by either pathway were quantitatively and qualitatively similar. Finally, coexpression of the natural vaccinia virus protein B13, which is an inhibitor of caspases and apoptosis, impaired Ag presentation by the caspase pathway in infected cells. These data support the hypothesis that numerous cellular proteolytic systems, including those induced during infection, such as caspases involved in apoptosis or in inflammation, contribute to the repertoire of presented peptides, thereby facilitating immunosurveillance. Copyright © 2010 by The American Association of Immunologists, Inc.

Quinlan M.,Novartis | Stroup W.,University of Nebraska - Lincoln | Schwenke J.,Applied Research Consultants | Christopher D.,Merck And Co.
Journal of Biopharmaceutical Statistics | Year: 2013

The goal of shelf life estimation is to determine the storage time during which the entire product meets specification with acceptably high probability. The estimated shelf life should be "applicable to all future batches" (ICH Q1E, International Conference on Harmonization, 2003b). There is compelling evidence of issues with the International Conference on Harmonization (ICH) guidelines for shelf life estimation. Issues include fixed batch effects, poolability tests, and confidence intervals for the mean. Two conclusions from evaluating the ICH procedure are that batch effects should be random and that focus should be on a quantile. A procedure is needed that combines random batches with the ICH objective of estimating the minimum batch shelf life. © Taylor & Francis Group, LLC.

Seeburger J.L.,Merck And Co.
Cephalalgia : an international journal of headache | Year: 2011

The study was carried out to assess the efficacy and tolerability of rizatriptan orally disintegrating tablet (ODT) for treating acute migraine in patients who are non-responders to sumatriptan. Many migraineurs report dissatisfaction with sumatriptan efficacy. It is unclear whether sumatriptan 100mg non-responders will respond to other triptans. This was a randomized, placebo-controlled, double-blind study in adults with >1-year history of ICHD-II (International Classification of Headache Disorders, second edition) migraine who reported that they generally do not respond to sumatriptan (≥50% unsatisfactory response). In the baseline phase, participants treated a single moderate/severe migraine attack with open-label generic sumatriptan 100mg. Those who continued to experience moderate/severe pain at two hours post-dose were eligible to enter the double-blind treatment phase, during which participants treated three migraine attacks in crossover fashion (two with rizatriptan 10-mg ODT, one with placebo) after being randomly assigned to one of three treatment sequences (1:1:1 ratio). The primary endpoint was two-hour pain relief. A total of 102 (94%) participants treated at least one study migraine. Pain relief at two hours was significantly greater with rizatriptan compared with placebo (51% vs. 20%, p<.001). Response rates also favored rizatriptan on two-hour pain freedom (22% vs. 12%, p=.013) as well as 24-hour sustained pain relief (38% vs. 14%, p<.001) and sustained pain freedom (20% vs. 11%, p=.036). Treatment was generally well tolerated. Rizatriptan 10-mg ODT was superior to placebo at providing two-hour pain relief and two-hour pain freedom in the treatment of acute migraine in those who do not respond to sumatriptan 100mg. Rizatriptan was generally well tolerated in this population.

Burstein R.,Beth Israel Deaconess Medical Center | Burstein R.,Harvard University | Jakubowski M.,Beth Israel Deaconess Medical Center | Garcia-Nicas E.,Beth Israel Deaconess Medical Center | And 5 more authors.
Annals of Neurology | Year: 2010

Objective: Focal somatic pain can evolve into widespread hypersensitivity to nonpainful and painful skin stimuli (allodynia and hyperalgesia, respectively). We hypothesized that transformation of headache into whole-body allodynia/ hyperalgesia during a migraine attack is mediated by sensitization of thalamic neurons that process converging sensory impulses from the cranial meninges and extracephalic skin. Methods: Extracephalic allodynia was assessed using single unit recording of thalamic trigeminovascular neurons in rats and contrast analysis of blood oxygenation level-dependent (BOLD) signals registered in functional magnetic resonance imaging (fMRI) scans of patients exhibiting extracephalic allodynia. Results: Sensory neurons in the rat posterior thalamus that were activated and sensitized by chemical stimulation of the cranial dura exhibited long-lasting hyperexcitability to innocuous (brush, pressure) and noxious (pinch, heat) stimulation of the paws. Innocuous, extracephalic skin stimuli that did not produce neuronal firing at baseline (eg, brush) became as effective as noxious stimuli (eg, pinch) in eliciting large bouts of neuronal firing after sensitization was established. In migraine patients, fMRI assessment of BOLD signals showed that brush and heat stimulation at the skin of the dorsum of the hand produced larger BOLD responses in the posterior thalamus of subjects undergoing a migraine attack with extracephalic allodynia than the corresponding responses registered when the same patients were free of migraine and allodynia. Interpretation: We propose that the spreading of multimodal allodynia and hyperalgesia beyond the locus of migraine headache is mediated by sensitized thalamic neurons that process nociceptive information from the cranial meninges together with sensory information from the skin of the scalp, face, body, and limbs. © 2010 American Neurological Association.

Ye J.,Merck And Co.
Biotechnology progress | Year: 2010

During early preclinical development of therapeutic proteins, representative materials are often required for process development, such as for pharmacokinetic/pharmacodynamic studies in animals, formulation design, and analytical assay development. To rapidly generate large amounts of representative materials, transient transfection is commonly used. Because of the typical low yields with transient transfection, especially in CHO cells, here we describe an alternative strategy using stable transfection pool technology. Using stable transfection pools, gram quantities of monoclonal antibody (Mab) can be generated within 2 months post-transfection. Expression levels for monoclonal antibodies can be achieved ranging from 100 mg/L to over 1000 mg/L. This methodology was successfully scaled up to a 200 L scale using disposable bioreactor technology for ease of rapid implementation. When fluorescence-activated cell sorting was implemented to enrich the transfection pools for high producers, the productivity could be improved by about three-fold. We also found that an optimal production time window exists to achieve the highest yield because the transfection pools were not stable and productivity generally decreased over length in culture. The introduction of Universal chromatin-opening elements elements into the expression vectors led to significant productivity improvement. The glycan distribution of the Mab product generated from the stable transfection pools was comparable to that from the clonal stable cell lines. © 2010 American Institute of Chemical Engineers

Rintoul G.L.,Simon Fraser University | Reynolds I.J.,Merck And Co.
Biochimica et Biophysica Acta - Molecular Basis of Disease | Year: 2010

Alterations in mitochondrial function may have a central role in the pathogenesis of many neurodegenerative diseases. The study of mitochondrial dysfunction has typically focused on ATP generation, calcium homeostasis and the production of reactive oxygen species. However, there is a growing appreciation of the dynamic nature of mitochondria within cells. Mitochondria are highly motile organelles, and also constantly undergo fission and fusion. This raises the possibility that impairment of mitochondrial dynamics could contribute to the pathogenesis of neuronal injury. In this review we describe the mechanisms that govern mitochondrial movement, fission and fusion. The key proteins that are involved in mitochondrial fission and fusion have also been linked to some inherited neurological diseases, including autosomal dominant optic atrophy and Charcot-Marie-Tooth disease 2A. We will discuss the evidence that altered movement, fission and fusion are associated with impaired neuronal viability. There is a growing collection of literature that links impaired mitochondrial dynamics to a number of disease models. Additionally, the concept that the failure to deliver a functional mitochondrion to the appropriate site within a neuron could contribute to neuronal dysfunction provides an attractive framework for understanding the mechanisms underlying neurologic disease. However, it remains difficult to clearly establish that altered mitochondrial dynamics clearly represent a cause of neuronal dysfunction. © 2009 Elsevier B.V. All rights reserved.

Ayer T.,University of Wisconsin - Madison | Chhatwal J.,Merck And Co. | Alagoz O.,University of Wisconsin - Madison | Kahn Jr. C.E.,Medical College of Wisconsin | And 2 more authors.
Radiographics | Year: 2010

Computer models in medical diagnosis are being developed to help physicians differentiate between healthy patients and patients with disease. These models can aid in successful decision making by allowing calculation of disease likelihood on the basis of known patient characteristics and clinical test results. Two of the most frequently used computer models in clinical risk estimation are logistic regression and an artificial neural network. A study was conducted to review and compare these two models, elucidate the advantages and disadvantages of each, and provide criteria for model selection. The two models were used for estimation of breast cancer risk on the basis of mammographic descriptors and demographic risk factors. Although they demonstrated similar performance, the two models have unique characteristics-strengths as well as limitations-that must be considered and may prove complementary in contributing to improved clinical decision making. © RSNA, 2009.

Ovacik A.M.,Merck And Co.
Mathematical Biosciences | Year: 2015

Monoclonal antibodies (mAbs) are large glycoproteins that recognize and remove/neutralize a specific target. Inflammation and inflammatory diseases are often treated with mAb-based therapeutics. Mathematical modeling is widely used in development of mAbs. Bioinformatics and structural modeling is used for humanization of mAbs and PK/PD modeling is extensively used in preclinical and clinical development. The objective of this commentary is to introduce systems biology-based modeling that can accelerate and improve development of mAbs. © 2014 Elsevier Inc..

Young R.M.,University of Pennsylvania | Ackerman D.,University of Pennsylvania | Quinn Z.L.,University of Pennsylvania | Quinn Z.L.,Howard Hughes Medical Institute | And 14 more authors.
Genes and Development | Year: 2013

Solid tumors exhibit heterogeneous microenvironments, often characterized by limiting concentrations of oxygen (O2), glucose, and other nutrients. How oncogenic mutations alter stress response pathways, metabolism, and cell survival in the face of these challenges is incompletely understood. Here we report that constitutive mammalian target of rapamycin complex 1 (mTORC1) activity renders hypoxic cells dependent on exogenous desaturated lipids, as levels of de novo synthesized unsaturated fatty acids are reduced under low O2. Specifically, we demonstrate that hypoxic Tsc2-/- (tuberous sclerosis complex 2-/-) cells deprived of serum lipids exhibit a magnified unfolded protein response (UPR) but fail to appropriately expand their endoplasmic reticulum (ER), leading to inositol-requiring protein-1 (IRE1)-dependent cell death that can be reversed by the addition of unsaturated lipids. UPR activation and apoptosis were also detected in Tsc2-deficient kidney tumors. Importantly, we observed this phenotype in multiple human cancer cell lines and suggest that cells committed to unregulated growth within ischemic tumor microenvironments are unable to balance lipid and protein synthesis due to a critical limitation in desaturated lipids. © 2013 by Cold Spring Harbor Laboratory Press.

Lee S.,University of Pittsburgh | Nolte H.,Merck And Co. | Benninger M.S.,Cleveland Clinic
American Journal of Rhinology and Allergy | Year: 2015

Background: Allergen immunotherapy (AIT) has been in practice for more than 100 years. However, research in novel routes and delivery methods of immunotherapy to treat allergic rhinitis (AR) and conjunctivitis has only recently occurred in the United States, where the predominant form of AIT provided is largely via a subcutaneous immunotherapy (SCIT) route. AIT may prevent new sensitizations, improve symptoms, decrease medication usage, and prevent allergic asthma. Although AIT is the only potentially curative treatment for AR, access and adherence continue to be problematic. Only a fraction of eligible patients actually undergo treatment, and attrition rates are high. An obvious limitation of SCIT includes the requirement of regular injections to be provided in the physician's office due to the potential for anaphylaxis. Sublingual immunotherapy (SLIT) for home administration has been investigated as a potential alternative to address this limitation of SCIT. Methods: A literature review was performed including the current findings from randomized clinical trials and meta-analyses with a discussion of the most recent evidence for the efficacy, safety, and dosing of allergen SLIT. Results: The current data suggest that SLIT is effective for treatment of seasonal allergies, can potentially prevent asthma, and has a favorable safety profile. Head-to-head studies, however, are few, and comparative effectiveness still remains to be answered. Optimal treatment algorithms for SLIT have not yet been established, with wide variation in dosage selection and schedules. Similarly to SCIT, only a few allergens such as ragweed and grass pollen have been found to be effective in large clinical trials. Conclusion: Recent data indicate that SLIT is an effective treatment modality for seasonal AR, improve quality of life, and can potentially prevent asthma but head-to head studies comparing SLIT to SCIT are needed. Copyright © 2015, OceanSide Publications, Inc., U.S.A.

Hsieh Y.,Merck And Co.
Current Drug Discovery Technologies | Year: 2010

Hydrophilic interaction chromatography (HILIC) offers a difference in selectivity as compared to traditional normal phase and reversed-phase liquid chromatography and therefore has great potential for the separation of a variety of pharmaceuticals. This review is devoted to summarizing HILIC coupled to tandem mass spectrometric (MS/MS) methods for the rapid, sensitive and accurate determinations of small molecules employed for supporting drug discovery. The perspectives and challenges on performing HILIC-MS/MS assays are also presented. © 2010 Bentham Science Publishers Ltd.

Junker B.,Merck And Co.
BioPharm International | Year: 2012

the aim of this paper is to describe a methodology for developing a per product qualitative and semi-quantitative business case for application of quality-by-design (QbD) for a biopharmaceutical product. Previous authors have not frequently approached the business case topic on a per product basis. Instead, they have examined benefits across a portfolio for a company or aggregated across the pharmaceutical industry as a whole (1). In contrast, this effort focuses on a per product basis of business benefit estimate.

Katlama C.,University Pierre and Marie Curie | Deeks S.G.,University of California at San Francisco | Autran B.,University Pierre and Marie Curie | Martinez-Picado J.,Autonomous University of Barcelona | And 8 more authors.
The Lancet | Year: 2013

Antiretroviral therapy for HIV infection needs lifelong access and strict adherence to regimens that are both expensive and associated with toxic effects. A curative intervention will be needed to fully stop the epidemic. The failure to eradicate HIV infection during long-term antiretroviral therapy shows the intrinsic stability of the viral genome in latently infected CD4T cells and other cells, and possibly a sustained low-level viral replication. Heterogeneity in latently infected cell populations and homoeostatic proliferation of infected cells might affect the dynamics of virus production and persistence. Despite potent antiretroviral therapy, chronic immune activation, inflammation, and immune dysfunction persist, and are likely to have important effects on the size and distribution of the viral reservoir. The inability of the immune system to recognise cells harbouring latent virus and to eliminate cells actively producing virus is the biggest challenge to finding a cure. We look at new approaches to unravelling the complex virus-host interactions that lead to persistent infection and latency, and discuss the rationale for combination of novel treatment strategies with available antiretroviral treatment options to cure HIV. Copyright © 2013 Elsevier B.V.

Jiang G.,University of Utah | Jiang G.,Merck And Co. | Madan D.,Echelon | Prestwich G.D.,University of Utah
Bioorganic and Medicinal Chemistry Letters | Year: 2011

Autotaxin (ATX) is an attractive target for the anticancer therapeutics that inhibits angiogenesis, invasion and migration. ATX is an extracellular lysophospholipase D that hydrolyzes lysophosphatidylcholine to form the bioactive lipid lysophosphatidic acid. The aromatic phosphonate S32826 was the first described nanomolar inhibitor of ATX. However, the tridecylamide substituent on aromatic ring contributed to its poor solubility and bioavailability, severely limiting its utility in vivo. c Log P calculations revealed that the lipophilicity of S32826 could be lowered by shortening its hydrophobic chain and by introducing substituents alpha to the phosphonate. Herein, we describe the synthesis of a small set of α-substituted phosphonate analogs of S32826, and we show that shortening the chain and adding α-halo or α-hydroxy substituents increased solubility; however, ATX inhibition was reduced by most substitutions. An optimal compound was identified for examination of biological effects of ATX inhibition in vivo. © 2011 Elsevier Ltd. All rights reserved.

Vander Cruyssen B.,OLV Hospital Aalst and SJK Hospital | Vastesaeger N.,Merck And Co. | Collantes-Estevez E.,University of Cordoba, Spain
Current Opinion in Rheumatology | Year: 2013

PURPOSE OF REVIEW: Hip disease occurs in about one-third of patients with ankylosing spondylitis (AS) and can often be disabling, necessitating total hip replacement in young adults. There have been recent articles on a number of aspects of this problem, including the epidemiology and pathology. The most recent studies on diagnosis, prognosis and therapeutic management are reviewed here. RECENT FINDINGS: Several large studies have evaluated the prevalence and outcome of hip involvement in AS. Hip involvement can be diagnosed clinically, radiologically, by MRI or by ultrasonography. These examinations highlight different aspects of hip disease in AS. Hip disease is more prevalent in patients with a younger disease onset and seems to be associated with more severe axial disease. Antitumour necrosis factor (TNF) agents are helpful for pain relief and improvement of function in patients with active axial and active hip disease. However, it is not clear whether this treatment option can prevent progression of structural damage. In case of end-stage hip disease, total hip replacement should be considered. SUMMARY: In patients with AS, the hips should be routinely assessed, at least by clinical examination. Anti-TNF therapy should be considered in patients with NSAID-resistant active axial disease who have concomitant hip disease. © 2013 Wolters Kluwer Health / Lippincott Williams & Wilkins.

Proportion differences are often used to estimate and test treatment effects in clinical trials with binary outcomes. In order to adjust for other covariates or intra-subject correlation among repeated measures, logistic regression or longitudinal data analysis models such as generalized estimating equation or generalized linear mixed models may be used for the analyses. However, these analysis models are often based on the logit link which results in parameter estimates and comparisons in the log-odds ratio scale rather than in the proportion difference scale. A two-step method is proposed in the literature to approximate the calculation of confidence intervals for the proportion difference using a concept of effective sample sizes. However, the performance of this two-step method has not been investigated in their paper. On this note, we examine the properties of the two-step method and propose an adjustment to the effective sample size formula based on Bayesian information theory. Simulations are conducted to evaluate the performance and to show that the modified effective sample size improves the coverage property of the confidence intervals. © 2012 John Wiley & Sons, Ltd.

He W.,Merck And Co. | Cao X.,University of Minnesota | Xu L.,Sanofi S.A.
Statistics in Medicine | Year: 2012

The evaluation of clinical proof of concept, optimal dose selection, and phase III probability of success has traditionally been conducted by a subjective and qualitative assessment of the efficacy and safety data. This, in part, was responsible for the numerous failed phase III programs in the past. The need to utilize more quantitative approaches to assess efficacy and safety profiles has never been greater. In this paper, we propose a framework that incorporates efficacy and safety data simultaneously for the joint evaluation of clinical proof of concept, optimal dose selection, and phase III probability of success. Simulation studies were conducted to evaluate the properties of our proposed methods. The proposed approach was applied to two real clinical studies. On the basis of the true outcome of the two clinical studies, the assessment based on our proposed approach suggested a reasonable path forward for both clinical programs. © 2011 John Wiley & Sons, Ltd.

Coldham I.,University of Sheffield | Robinson S.P.,University of Sheffield | Baxter C.A.,Merck And Co.
Synlett | Year: 2012

Homoallylic amines were prepared by asymmetric deprotonation of benzylamines using n-BuLi and (-)-sparteine to give chiral organolithiums to which were added prenyl bromide. Removal of the Boc protecting group gave anilines that were found to undergo Brønsted acid catalyzed or iodine-mediated cyclization to give aryl-substituted pyrrolidine heterocyclic products with high enantioselectivity. © Georg Thieme Verlag Stuttgart. New York.

Jia T.,University of Pennsylvania | Bellomo A.,University of Pennsylvania | Baina K.E.,University of Pennsylvania | Dreher S.D.,Merck And Co. | Walsh P.J.,University of Pennsylvania
Journal of the American Chemical Society | Year: 2013

The palladium-catalyzed α-arylation of unactivated sulfoxides has been developed. The weakly acidic α-protons of sulfoxides are reversibly deprotonated by LiOtBu, and a palladium phosphine complex facilitates the arylation. A variety of aryl methyl sulfoxides were coupled with aryl bromides. More challenging coupling partners, such as alkyl methyl sulfoxides (including dimethyl sulfoxide) and aryl chlorides proved to be suitable under the optimized conditions. This method was utilized to synthesize bioactive benzyl sulfoxide intermediates. © 2013 American Chemical Society.

Weyn-Vanhentenryck S.M.,Columbia University | Mele A.,Howard Hughes Medical Institute | Yan Q.,Columbia University | Sun S.,Cold Spring Harbor Laboratory | And 12 more authors.
Cell Reports | Year: 2014

The RNA binding proteins Rbfox1/2/3 regulate alternative splicing in the nervous system, and disruption of Rbfox1 has been implicated in autism. However, comprehensive identification of functional Rbfox targets has been challenging. Here, we perform HITS-CLIP for all three Rbfox family members in order to globally map, at a single-nucleotide resolution, their invivo RNA interaction sites in the mouse brain. We find that the two guanines in the Rbfox binding motif UGCAUG are critical for protein-RNA interactions and crosslinking. Using integrative modeling, these interaction sites, combined with additional datasets, define 1,059 direct Rbfox targetalternative splicing events. Over half of the quantifiable targets show dynamic changes during brain development. Of particular interest are 111 events from 48 candidate autism-susceptibility genes, including syndromic autism genes Shank3, Cacna1c, and Tsc2. Alteration of Rbfox targets in some autistic brains is correlated with downregulation of all three Rbfox proteins, supporting the potential clinical relevance of the splicing-regulatory network. © 2014 The Authors.

Erickson J.C.,Merck And Co.
CONTINUUM Lifelong Learning in Neurology | Year: 2010

Headache is a common symptomafter traumatic head injury and is a frequent feature of the postconcussive syndrome. A variety of headache subtypes can be precipitated by head trauma, although posttraumatic headaches most often resemble migraine or tension-type headache. A lack of clinical trials limits evidence-based treatment recommendations for both acute and chronic posttraumatic headaches. However, numerous pharmacologic and nonpharmacologic interventions can be used to successfully manage posttraumatic headaches. This article reviews the classification, epidemiology, prognosis, and pathophysiology of headaches after head trauma and provides a practical clinical approach for evaluating and treating patients with posttraumatic headaches. Copyright © 2010 Lippincott Williams & Wilkins.

Bergstrom T.,Uppsala University | Holmqvist K.,Merck And Co. | Tararuk T.,Uppsala University | Johansson S.,Uppsala University | Forsberg-Nilsson K.,Uppsala University
Biochimica et Biophysica Acta - General Subjects | Year: 2014

Background It is becoming increasingly apparent that the extracellular matrix acts as an important regulator of the neural stem niche. Previously we found that neural stem and progenitor cells (NSPCs) derived from the early postnatal subventricular zone of mice adhere to a collagen/hyaluronan hydrogel, whereas NSPCs from the adult and embryonic brain do not. Methods To examine the specific adhesive properties of young stem cells in more detail, NSPCs isolated from embryonic, postnatal day 6 (P6), and adult mouse brains were cultured on collagen I. Results Early postnatal NSPCs formed paxillin-positive focal adhesions on collagen I, and these adhesions could be prevented by an antibody that blocked integrin β1. Furthermore, we found the corresponding integrin alpha subunits α2 and α11 levels to be highest at the postnatal stage. Gene ontology analysis of differentially expressed genes showed higher expression of transcripts involved in vasculature development and morphogenesis in P6 stem cells, compared to adult. Conclusions The ability to interact with the extracellular matrix differs between postnatal and adult NSPCs. General significance Our observations that the specific adhesive properties of early postnatal NSPCs, which are lost in the adult brain, can be ascribed to the integrin subunits expressed by the former furthering our understanding of the developing neurogenic niche. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. © 2014 Published by Elsevier B.V.

Gibson T.B.,Thomson Reuters | Mahoney J.,Florida Health Care Coalition | Ranghell K.,Florida Health Care Coalition | Cherney B.J.,Florida Health Care Coalition | McElwee N.,Merck And Co.
Health Affairs | Year: 2011

We evaluated the effects of implementing a value-based insurance design program for patients with diabetes in two groups within a single firm. One group participated in disease management; the other did not. We matched members of the two groups to similar enrollees within the company that did not offer the value-based program. We found that participation in both value-based insurance design and disease management resulted in sustained improvement over time. Use of diabetes medications increased 6.5 percent over three years. Adherence to diabetes medical guidelines also increased, producing a return on investment of $1.33 saved for every dollar spent during a three-year follow-up period. ©2011 Project HOPE - The People-to-People Health Foundation, Inc.

Sheridan R.P.,Merck And Co.
Journal of Chemical Information and Modeling | Year: 2015

In QSAR, a statistical model is generated from a training set of molecules (represented by chemical descriptors) and their biological activities (an "activity model"). The aim of the field of domain applicability (DA) is to estimate the uncertainty of prediction of a specific molecule on a specific activity model. A number of DA metrics have been proposed in the literature for this purpose. A quantitative model of the prediction uncertainty (an "error model") can be built using one or more of these metrics. A previous publication from our laboratory (Sheridan, R. P. J. Chem. Inf. Model. 2013, 53, 2837 -2850) suggested that QSAR methods such as random forest could be used to build error models by fitting unsigned prediction errors against DA metrics. The QSAR paradigm contains two useful techniques: descriptor importance can determine which DA metrics are most useful, and cross-validation can be used to tell which subset of DA metrics is sufficient to estimate the unsigned errors. Previously we studied 10 large, diverse data sets and seven DA metrics. For those data sets for which it is possible to build a significant error model from those seven metrics, only two metrics were sufficient to account for almost all of the information in the error model. These were TREE-SD (the variation of prediction among random forest trees) and PREDICTED (the predicted activity itself). In this paper we show that when data sets are less diverse, as for example in QSAR models of molecules in a single chemical series, these two DA metrics become less important in explaining prediction error, and the DA metric SIMILARITYNEAREST1 (the similarity of the molecule being predicted to the closest training set compound) becomes more important. Our recommendation is that when the mean pairwise similarity (measured with the Carhart AP descriptor and the Dice similarity index) within a QSAR training set is less than 0.5, one can use only TREE-SD, PREDICTED to form the error model, but otherwise one should use TREE-SD, PREDICTED, SIMILARITYNEAREST1. (Figure Presented) © 2015 American Chemical Society.

Edvinsson L.,Lund University | Eftekhari S.,Lund University | Salvatore C.A.,Merck And Co. | Warfvinge K.,Lund University
Molecular and Cellular Neuroscience | Year: 2011

Clinical and experimental results have revealed a fundamental role of calcitonin gene-related peptide (CGRP) in primary headaches. CGRP is widely expressed in neurons both in the central nervous system (CNS) and in peripheral sensory nerves. In the CNS there is a wide distribution of CGRP-containing neurons with the highest levels seen in striatum, amygdale and cerebellum. Moreover, in acute attacks of migraine there is evidence of cerebellar activation. To understand the role of CGRP, antibodies towards the CGRP receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein type 1 (RAMP1) have been developed. In the present study we therefore examined immunohistochemically the distribution of CGRP and its receptor components in the cerebellum.CGRP immunoreactivity was only found intracellularly in the cerebellar Purkinje cell bodies, whereas CLR and RAMP1 were detected on the surface of the Purkinje cell bodies and in their processes. The elaborate dendritic tree of Purkinje cell fibers was distinctly visualized with the RAMP1 antibody. In addition, profoundly stained fibers spanning from the molecular layer into the medulla was observed with the RAMP1 antibody. Judged from the high density of immunoreactive cells expressing CGRP, RAMP1 or CLR, and from the double staining of CGRP and RAMP1 it is likely that most, if not all, Purkinje cells express both the peptide and the receptor components. Double staining with RAMP1 and the glial cell markers glial fibrillary acidic protein (GFAP) and S-100 revealed an almost identical staining pattern of the antibodies in the area of the cell body surfaces. However, as judged by confocal microscopy, no double staining was present. Instead, it was discovered that the glial cells tightly surrounded the Purkinje cells which easily could be interpreted as co-localization in the epifluorescence microscope.Our observations demonstrate that there is a rich expression of CGRP and CGRP receptor elements in the cerebellum which points towards a functional role of CGRP in cerebellar Purkinje cells. Recent advances in the biology of the cerebellum indicate that there may be a role in nociception; hence a target of the recently discovered CGRP receptor antagonists that have demonstrated improvement in migraine pain and associated symptoms could be cerebellar CGRP receptors. © 2010 Elsevier Inc.

Lockley W.J.S.,University of Surrey | Hesk D.,Merck And Co.
Journal of Labelled Compounds and Radiopharmaceuticals | Year: 2010

Metal-catalysed hydrogen isotope exchange has been widely used in the preparation of deuterium- and tritium-labelled compounds. This review details the development and utility of homogeneous and heterogeneous rhodium and ruthenium catalysts for the preparation of deuterium- and tritium-labelled compounds by hydrogen isotope exchange methodology. Copyright © 2010 John Wiley & Sons, Ltd.

A method for the diastereoselective synthesis of tetrahydroquinolines via a palladium-catalyzed Suzuki terminated Heck reaction is described. The reaction provides access to tetrahydroquinolines containing both quaternary and tertiary stereocenters. Ligand effects, a rationale for the high level of diastereoselectivity, and a mechanistic hypothesis are discussed. © 2012 Elsevier Ltd. All rights reserved.

Metz A.E.,University of Pennsylvania | Berritt S.,University of Pennsylvania | Dreher S.D.,Merck And Co. | Kozlowski M.C.,University of Pennsylvania
Organic Letters | Year: 2012

Palladium-catalyzed cross-coupling conditions were developed that efficiently afford 2-aryl-2-nitroacetates from aryl bromides and the very acidic nitroacetates. © 2012 American Chemical Society.

Guan S.,Merck And Co.
Journal of Biopharmaceutical Statistics | Year: 2012

The main purpose of a Phase I trial of a new antitumor agent is to determine the appropriate dosing regimen and characterize the safety profile of a new molecular or monoclonal antibody. Phase II cancer clinical trials are conducted to assess the efficacy of a new anticancer therapy and to determine whether it has sufficient activity against a specific type of tumor to warrant further development. In this paper, commonly used statistical designs, based on either frequentist approaches or Bayesian methods, for Phase I and Phase II cancer clinical trials are reviewed and discussed. Future directions of designing more efficient trial are explored. © 2012 Taylor and Francis Group, LLC.

Van Der Meer P.,University of Groningen | Postmus D.,University of Groningen | Ponikowski P.,Klinika Kardiologii | Cleland J.G.,University of Hull | And 10 more authors.
Journal of the American College of Cardiology | Year: 2013

Objectives: The study sought to investigate the clinical correlates and prognostic role of anemia and changes in hemoglobin in patients hospitalized for acute decompensated heart failure (AHF). Background: Anemia is related to a poor outcome in patients with heart failure. In addition, an increase in hemoglobin during hospitalization might be a sign of effective decongestion and therefore related to improved outcome. Methods: This is a post hoc analysis of the PROTECT (Placebo-Controlled Randomized Study of the Selective Adenosine A1 Receptor Antagonist Rolofylline for Patients Hospitalized with Acute Decompensated Heart Failure and Volume Overload to Assess Treatment Effect on Congestion and Renal Function) study in 1,969 patients with AHF and mild to moderate impaired renal function. Hemoglobin levels were measured daily for the first 4 days and at day 7. The endpoint was 180-day all-cause mortality. Results: Anemia at baseline was observed in 50.3% of the patients. During follow-up, 359 patients (18.2%) died. Hemoglobin increased in 69.1% and was associated with a better renal function at baseline and more weight loss, but was associated with a deterioration of renal function (p = 0.01), whereas total dose diuretics was lower in patients with hemoconcentration (p < 0.01). Interaction analysis showed that greater weight loss and better baseline renal function were associated with a more rapid increase in hemoglobin concentration (p < 0.01 for both). The absolute change in hemoglobin (g/dl) independently predicted outcome (hazard ratio: 0.66; 95% confidence interval: 0.51 to 0.86; p = 0.002), whereas baseline hemoglobin levels did not. Conclusions: Patients with AHF and preserved renal function are decongested better, as shown by an increase in hemoglobin. A rapid increase in hemoglobin during the first week is independently associated with a favorable outcome, despite a slight decrease in renal function. © 2013 American College of Cardiology Foundation.

Hills F.A.,Middlesex University | Mehmet H.,Merck And Co. | Sullivan M.H.,Imperial College London
European Journal of Obstetrics Gynecology and Reproductive Biology | Year: 2012

Objective: This study aimed to determine the effects of insulin-like growth factors (IGF-I and IGF-II), heparin, aspirin and vitamin C on the proliferation and apoptosis of human villous cytotrophoblast from first trimester and term placentae. Study design: Villous cytotrophoblast cells were isolated from uncomplicated first trimester (n = 12) and term placental tissues (n = 12) using negative immunoselection with an antibody to HLA class I antigens. Cells were incubated with IGF-I, IGF-II, heparin, aspirin and vitamin C either alone, or in combination with either TNF-α/IFN-γ or staurosporine. Proliferation was determined by measurement of Ki67 expression using immunocytochemistry. Trophoblast apoptosis was determined by TUNEL staining. Finally RT-PCR was carried out to identify IGF-binding insulin receptor isoforms. Data were expressed as means ± SEM. One way analysis of variance (ANOVA) with Bonferroni correction was used to determine if differences between groups were statistically significant. Results: Following negative immunoselection >98% of cells were positively stained for cytokeratin 7, a marker for cytotrophoblasts, and <1% were vimentin positive. First trimester and term trophoblasts underwent spontaneous apoptosis which was inhibited by approximately 50% in the presence of IGF-II or heparin. Apoptosis was significantly increased following incubation with a combination of TNF-α and IFN-γ or staurosporine. Apoptosis was decreased to basal levels following coincubation with IGF-II or heparin. Incubation with IGFs or heparin resulted in a small, but significant increase in Ki67 expression. Insulin receptor isoform A, which binds IGF-II with high affinity, was present in all trophoblast samples tested. Conclusion: These results suggest that heparin and IGF-II, but not IGF-I are important regulators of villous cytotrophoblast survival in early and late pregnancy. © 2012 Elsevier Ireland Ltd. All rights reserved.

Chen H.-C.,Academia Sinica, Taiwan | Chen H.-C.,National Taiwan University | Schiffman M.,U.S. National Institutes of Health | Lin C.-Y.,Taipei Medical University | And 10 more authors.
Journal of the National Cancer Institute | Year: 2011

Background:Human papillomavirus (HPV) persistence is the pivotal event in cervical carcinogenesis. We followed a large-scale community-based cohort for 16 years to investigate the role of genotype-specific HPV persistence in predicting cervical cancer including invasive and in situ carcinoma. Methods:At the baseline examination in 1991-1992, 11 923 participants (aged 30-65 years) consented to HPV testing and cytology; 6923 participants were reexamined in 1993-1995. For HPV testing, we used a polymerase chain reaction-based assay that detected 39 HPV types. Women who developed cervical cancer were identified from cancer and death registries. Cumulative risks for developing cervical cancer among infected and persistently infected women were calculated by the Kaplan-Meier method. Results:Of 10 123 women who were initially cytologically normal, 68 developed cervical cancer. The 16-year cumulative risks of subsequent cervical cancer for women with HPV16, HPV58 (without HPV16), or other carcinogenic HPV types (without HPV16 or HPV58) were 13.5%, 10.3%, and 4.0%, respectively, compared with 0.26% for HPV-negative women. Women with type-specific persistence of any carcinogenic HPV had greatly increased risk compared with women who were HPV-negative at both visits (hazard ratio = 75.4, 95% confidence interval = 31.8 to 178.9). The cumulative cervical cancer risks following persistent carcinogenic HPV infections increased with age: The risks were 5.5%, 14.4%, and 18.1% for women aged 30-44 years, 45-54 years, and 55 years and older, respectively. However, newly acquired infections were associated with a low risk of cervical cancer regardless of age. Conclusions:HPV negativity was associated with a very low long-term risk of cervical cancer. Persistent detection of HPV among cytologically normal women greatly increased risk. Thus, it is useful to perform repeated HPV testing following an initial positive test. © The Author 2011. Published by Oxford University Press. All rights reserved.

Abrahamson M.J.,Georgia Institute of Technology | Vazquez-Figueroa E.,Georgia Institute of Technology | Woodall N.B.,Georgia Institute of Technology | Moore J.C.,Merck And Co. | Bommarius A.S.,Georgia Institute of Technology
Angewandte Chemie - International Edition | Year: 2012

A leucine dehydrogenase has been successfully altered through several rounds of protein engineering to an enantioselective amine dehydrogenase. Instead of the wild-type α-keto acid, the new amine dehydrogenase now accepts the analogous ketone, methyl isobutyl ketone (MIBK), which corresponds to exchange of the carboxy group by a methyl group to produce chiral (R)-1,3-dimethylbutylamine. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Burashnikov A.,Masonic Medical Research Laboratory | Pourrier M.,Cardiome Pharma | Gibson J.K.,AAKVSL Pharma Consulting LLC. | Lynch J.J.,Merck And Co. | Antzelevitch C.,Masonic Medical Research Laboratory
Circulation: Arrhythmia and Electrophysiology | Year: 2012

Background-Several clinical trials have shown that vernakalant is effective in terminating recent onset atrial fibrillation (AF). The electrophysiological actions of vernakalant are not fully understood. Methods and Results-Here we report the results of a blinded study comparing the in vitro canine atrial electrophysiological effects of vernakalant, ranolazine, and dl-sotalol. Action potential durations (APD 50,75,90), effective refractory period (ERP), post repolarization refractoriness (PRR), maximum rate of rise of the action potential (AP) upstroke (V max), diastolic threshold of excitation (DTE), conduction time (CT), and the shortest S1-S1 permitting 1:1 activation (S1-S1) were measured using standard stimulation and microelectrode recording techniques in isolated normal, non-remodeled canine arterially perfused left atrial preparations. Vernakalant caused variable but slight prolongation of APD 90 (P=not significant), but significant prolongation of APD 50 at 30 μmol/L and rapid rates. In contrast, ranolazine and dl-sotalol produced consistent concentration- and reverse rate-dependent prolongation of APD 90. Vernakalant and ranolazine caused rate-dependent, whereas dl-sotalol caused reverse rate-dependent, prolongation of ERP. Significant rate-dependent PRR developed with vernakalant and ranolazine, but not with dl-sotalol. Other sodium channel-mediated parameters (ie, V max, CT, DTE, and S1-S1) also were depressed significantly by vernakalant and ranolazine, but not by dl-sotalol. Only vernakalant elevated AP plateau voltage, consistent with blockade of ultrarapid delayed rectified potassium current and transient outward potassium current. Conclusions-In isolated canine left atria, the effects of vernakalant and ranolazine were characterized by use-dependent inhibition of sodium channel-mediated parameters, and those of dl-sotalol by reverse rate-dependent prolongation of APD 90 and ERP. This suggests that during the rapid activation rates of AF, the INa blocking action of the mixed ion channel blocker vernakalant takes prominence. This mechanism may explain vernakalant's anti-AF efficacy. © 2012 American Heart Association, Inc.

In randomized clinical trials, measurements are often collected on each subject at a baseline visit and several post-randomization time points. The longitudinal analysis of covariance in which the postbaseline values form the response vector and the baseline value is treated as a covariate can be used to evaluate the treatment differences at the postbaseline time points. Liang and Zeger (2000, Sankhyā: The Indian Journal of Statistics, Series B 62, 134-148) propose a constrained longitudinal data analysis in which the baseline value is included in the response vector together with the postbaseline values and a constraint of a common baseline mean across treatment groups is imposed on the model as a result of randomization. If the baseline value is subject to missingness, the constrained longitudinal data analysis is shown to be more efficient for estimating the treatment differences at postbaseline time points than the longitudinal analysis of covariance. The efficiency gain increases with the number of subjects missing baseline and the number of subjects missing all postbaseline values, and, for the pre-post design, decreases with the absolute correlation between baseline and postbaseline values. © 2009, The International Biometric Society.

Nalls M.A.,U.S. National Institute on Aging | Escott-Price V.,University of Cardiff | Williams N.M.,University of Cardiff | Lubbe S.,University College London | And 3 more authors.
Movement Disorders | Year: 2015

Background: Recent genomewide association study meta-analyses have identified 28 loci associated with risk of Parkinson's disease (PD). We sought to investigate whether these genetic risk factors are associated with PD age at onset. Methods: Genetic risk scores from these loci were calculated for 6,249 cases. Linear regression tested associations between cumulative genetic risk and PD age at onset. Results: Increasing genetic risk scores were associated with earlier age at onset (beta=-0.10, P=2.92 × 10-8, adjusted r2=0.27). Single standard deviation increase in genetic risk score is associated with 37.44 d earlier age at onset. Highest genetic risk was found at 31 to 60 y, onset slightly below average age at onset (AAO). Conclusions: Common genetic risk factors have a small but consistent association with AAO in PD. © 2015 International Parkinson and Movement Disorder Society.

Su B.,CAS Beijing National Laboratory for Molecular | Wei J.-B.,CAS Beijing National Laboratory for Molecular | Wu W.-L.,Merck And Co. | Shi Z.-J.,CAS Beijing National Laboratory for Molecular
ChemCatChem | Year: 2015

In the context of transition-metal-catalyzed C-H functionalization, directing-group strategy was developed for the improvement of chemical reactivity and selectivity. Recently, to avoid the inherent limitations of traditional mono-role directing groups, a dual-role oxidizing-directing-group strategy was developed, in which the directing group acts both as directing group and oxidant. Herein, we report a multirole directing group, which possesses multiple reactive sites, exhibits unique reactivity and selectivity, and leads to four different types of products from a single starting material through rhodium-catalyzed C-H activation/alkyne annulation reactions. The excellent product diversity and regio- and redox selectivity were well controlled by the tuning of solvents and oxidants. Chemical multitasking: A novel N-N bond containing a multirole directing group is developed, which enables the synthesis of four different products selectively from the same starting material through RhIII catalysis. Key features include ready availability, multiple reactive sites, high redox selectivity and controllability, and multifarious transformations toward important scaffolds and structural diversification. Phth=phthaloyl; DCE=1,2-dichloroethane. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Patel U.D.,Duke University | Greiner M.A.,Duke University | Fonarow G.C.,University of California at Los Angeles | Phatak H.,Merck And Co. | And 2 more authors.
American Heart Journal | Year: 2010

Background: Kidney disease is common among patients with heart failure, but relationships between worsening renal function (WRF) and outcomes after hospitalization for heart failure are poorly understood, especially among patients with preserved systolic function. We examined associations between WRF and 30-day readmission, mortality, and costs among Medicare beneficiaries hospitalized with heart failure. Methods: We linked data from a clinical heart failure registry to Medicare inpatient claims for patients ≥65 years old hospitalized with heart failure. We defined WRF as a change in serum creatinine ≥0.3 mg/dL from admission to discharge. Main outcome measures were readmission and mortality at 30 days after hospitalization and total inpatient costs. Results: Among 20,063 patients hospitalized with heart failure, WRF was common (17.8%) and more likely among patients with higher baseline comorbidity and more impaired renal function. In unadjusted analyses, WRF was associated with similar subsequent mean inpatient costs ($3,255 vs $3,277, P = .2) but higher readmission (21.8% vs 20.6%, P = .01) and mortality (10.0% vs 7.2%, P < .001). The differences persisted after adjustment for baseline patient and hospital characteristics (hazard of readmission 1.10 [95% CI 1.02-1.18], hazard of mortality 1.53 [95% CI 1.34-1.75]). Associations of WRF with readmission and mortality were similar between patients with reduced and preserved systolic function. Conclusions: Worsening renal function during hospitalization for heart failure is an independent predictor of early readmission and mortality in patients with reduced and preserved systolic function. © 2010, Mosby, Inc. All rights reserved.

Reddy M.R.,University of Hyderabad | Reddy M.R.,RR Laboratories Inc. | Singh U.C.,AM Technology | Erion M.D.,Merck And Co.
Journal of the American Chemical Society | Year: 2011

Standard molecular mechanics (MM) force fields predict a nearly linear decrease in hydration free energy with each successive addition of a methyl group to ammonia or acetamide, whereas a nonadditive relationship is observed experimentally. In contrast, the non-additive hydration behavior is reproduced directly using a quantum mechanics (QM)/MM-based free-energy perturbation (FEP) method wherein the solute partial atomic charges are updated at every window. Decomposing the free energies into electrostatic and van der Waals contributions and comparing the results with the corresponding free energies obtained using a conventional FEP method and a QM/MM method wherein the charges are not updated suggests that inaccuracies in the electrostatic free energies are the primary reason for the inability of the conventional FEP method to predict the experimental findings. The QM/MM-based FEP method was subsequently used to evaluate inhibitors of the diabetes drug target fructose-1,6-bisphosphatase adenosine 5′-monophosphate and 6-methylamino purine riboside 5′-monophosphate. The predicted relative binding free energy was consistent with the experimental findings, whereas the relative binding free energy predicted using the conventional FEP method differed from the experimental finding by an amount consistent with the overestimated relative solvation free energies calculated for alkylamines. Accordingly, the QM/MM-based FEP method offers potential advantages over conventional FEP methods, including greater accuracy and reduced user input. Moreover, since drug candidates often contain either functionality that is inadequately treated by MM (e.g., simple alkylamines and alkylamides) or new molecular scaffolds that require time-consuming development of MM parameters, these advantages could enable future automation of FEP calculations as well as greatly increase the use and impact of FEP calculations in drug discovery. © 2011 American Chemical Society.

Lu K.,Merck And Co. | Jiang L.,Kendle International Inc. | Tsiatis A.A.,North Carolina State University
Biometrics | Year: 2010

Often a binary variable is generated by dichotomizing an underlying continuous variable measured at a specific time point according to a prespecified threshold value. In the event that the underlying continuous measurements are from a longitudinal study, one can use the repeated-measures model to impute missing data on responder status as a result of subject dropout and apply the logistic regression model on the observed or otherwise imputed responder status. Standard Bayesian multiple imputation techniques (Rubin, 1987, in Multiple Imputation for Nonresponse in Surveys) that draw the parameters for the imputation model from the posterior distribution and construct the variance of parameter estimates for the analysis model as a combination of within- and between-imputation variances are found to be conservative. The frequentist multiple imputation approach that fixes the parameters for the imputation model at the maximum likelihood estimates and construct the variance of parameter estimates for the analysis model using the results of Robins and Wang (2000, Biometrika 87, 113-124) is shown to be more efficient. We propose to apply (Kenward and Roger, 1997, Biometrics 53, 983-997) degrees of freedom to account for the uncertainty associated with variance-covariance parameter estimates for the repeated measures model. © 2010, The International Biometric Society.

Burghardt A.J.,University of California at San Francisco | Kazakia G.J.,University of California at San Francisco | Sode M.,University of California at San Francisco | Sode M.,University of California at Berkeley | And 4 more authors.
Journal of Bone and Mineral Research | Year: 2010

The goal of this study was to characterize longitudinal changes in bone microarchitecture and function in women treated with an established antifracture therapeutic. In this double-blind, placebo-controlled pilot study, 53 early postmenopausal women with low bone density (age=56±4 years; femoral neck T-score=-1.5±0.6) were monitored by high-resolution peripheral quantitative computed tomography (HR-pQCT) for 24 months following randomization to alendronate (ALN) or placebo (PBO) treatment groups. Subjects underwent annual HR-pQCT imaging of the distal radius and tibia, dual-energy X-ray absorptiometry (DXA), and determination of biochemical markers of bone turnover (BSAP and uNTx). In addition to bone density and microarchitecture assessment, regional analysis, cortical porosity quantification, and micro-finite-element analysis were performed. After 24 months of treatment, at the distal tibia but not the radius, HR-pQCT measures showed significant improvements over baseline in the ALN group, particularly densitometric measures in the cortical and trabecular compartments and endocortical geometry (cortical thickness and area, medullary area) (p<.05). Cortical volumetric bone mineral density (vBMD) in the tibia alone showed a significant difference between treatment groups after 24 months (p<.05); however, regionally, significant differences in Tb.vBMD, Tb.N, and Ct.Th were found for the lateral quadrant of the radius (p<.05). Spearman correlation analysis revealed that the biomechanical response to ALN in the radius and tibia was specifically associated with changes in trabecular microarchitecture (|ρ|=0.51 to 0.80, p<.05), whereas PBO progression of bone loss was associated with a broad range of changes in density, geometry, and microarchitecture (|ρ|=0.56 to 0.89, p<.05). Baseline cortical geometry and porosity measures best predicted ALN-induced change in biomechanics at both sites (ρ > 0.48, p<.05). These findings suggest a more pronounced response to ALN in the tibia than in the radius, driven by trabecular and endocortical changes. © 2010 American Society for Bone and Mineral Research.

Kyle A.F.,University of Oxford | Jakubec P.,University of Oxford | Cockfield D.M.,University of Manchester | Cleator E.,Merck And Co. | And 2 more authors.
Chemical Communications | Year: 2011

A highly diastereoselective bifunctional organocatalyst controlled Michael addition, a nitro-Mannich/lactamization cascade, a furan N-acyliminium cyclisation, a sequential alkyne RCM/syn-reduction and an alkene RCM has allowed a 19 step, highly stereoselective synthesis of (-)-nakadomarin A. © The Royal Society of Chemistry 2011.

Yang F.,University of Washington | Babak T.,Merck And Co. | Shendure J.,University of Washington | Disteche C.M.,University of Washington
Genome Research | Year: 2010

X inactivation equalizes the dosage of gene expression between the sexes, but some genes escape silencing and are thus expressed from both alleles in females. To survey X inactivation and escape in mouse, we performed RNA sequencing in Mus musculus 3 Mus spretus cells with complete skewing of X inactivation, relying on expression of single nucleotide polymorphisms to discriminate allelic origin. Thirteen of 393 (3.3%) mouse genes had significant expression from the inactive X, including eight novel escape genes. We estimate that mice have significantly fewer escape genes compared with humans. Furthermore, escape genes did not cluster in mouse, unlike the large escape domains in human, suggesting that expression is controlled at the level of individual genes. Our findings are consistent with the striking differences in phenotypes between female mice and women with a single X chromosome - a near normal phenotype in mice versus Turner syndrome and multiple abnormalities in humans. We found that escape genes are marked by the absence of trimethylation at lysine 27 of histone H3, a chromatin modification associated with genes subject to X inactivation. Furthermore, this epigenetic mark is developmentally regulated for some mouse genes. © 2010 by Cold Spring Harbor Laboratory Press.

McGrew G.I.,University of Pennsylvania | Stanciu C.,University of Pennsylvania | Zhang J.,University of Pennsylvania | Carroll P.J.,University of Pennsylvania | And 2 more authors.
Angewandte Chemie - International Edition | Year: 2012

Ligand in a haystack: The first catalytic asymmetric cross-coupling of benzyllithiums α to tertiary amines using [Cr(CO)3] activation of benzylic C sp 3-H bonds is described. The stabilized organolithium undergoes Pd-catalyzed coupling with aryl triflates (ArOTf) by a novel dynamic kinetic resolution to yield enantioenriched diarylmethylamines. The chiral ligand for this reaction was identified using high-throughput experimentation with 192 ligands. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Kudryashova E.,University of California at Los Angeles | Struyk A.,Merck And Co. | Mokhonova E.,University of California at Los Angeles | Spencer M.J.,University of California at Los Angeles
Human Molecular Genetics | Year: 2011

Mutations in tripartite motif protein 32 (TRIM32) are responsible for several hereditary disorders that include limb girdle muscular dystrophy type 2H (LGMD2H), sarcotubular myopathy (STM) and Bardet Biedl syndrome. Most LGMD2H mutations in TRIM32 are clustered in the NHL β-propeller domain at the C-terminus and are predicted to interfere with homodimerization. To get insight into TRIM32's role in the pathogenesis of LGMD2H and to create an accurate model of disease, we have generated a knock-in mouse (T32KI) carrying the c.1465G > A (p.D489N) mutation in murine Trim32 corresponding to the human LGMD2H/STM pathogenic mutation c.1459G > A (p.D487N). Our data indicate that T32KI mice have both a myopathic and a neurogenic phenotype, very similar to the one described in the Trim32-null mice that we created previously. Analysis of Trim32 gene expression in T32KI mice revealed normal mRNA levels, but a severe reduction in mutant TRIM32 (D489N) at the protein level. Our results suggest that the D489N pathogenic mutation destabilizes the protein, leading to its degradation, and results in the same mild myopathic and neurogenic phenotype as that found in Trim32-null mice. Thus, one potential mechanism of LGMD2H might be destabilization of mutated TRIM32 protein leading to a null phenotype. © The Author 2011. Published by Oxford University Press. All rights reserved.

Warner A.W.,Merck And Co.
The journal of investigative dermatology. Symposium proceedings / the Society for Investigative Dermatology, Inc. [and] European Society for Dermatological Research | Year: 2013

Alopecia areata, alopecia totalis, and alopecia universalis likely represent a constellation of related diseases with similar, yet distinct heritability markers. There is currently no known curative therapy that works universally for all patients. Pharmacogenomic research enables the pharmaceutical industry to understand variability of patient responses to drugs during clinical drug development and during post-marketing surveillance. Understanding the genetic basis for patient response/non-response can enable the development of individualized therapies for those patients with an inherited basis for altered response to drug therapy. There are multiple examples of drugs that now contain a recommendation for genetic testing before dosing in their drug labels, directing clinicians to obtain genetic information for each individual patient in order to help direct drug therapy.

Kendrick J.,University of Colorado at Denver | Shlipak M.G.,University of California at San Francisco | Targher G.,University of Verona | Cook T.,Merck And Co. | And 2 more authors.
American Journal of Kidney Diseases | Year: 2010

Background: Chronic kidney disease (CKD) is associated with an increased risk of incident cardiovascular disease (CVD); however, the role of statins for the primary prevention of acute cardiovascular events in patients with CKD and the effect of statins on kidney function loss in persons without prevalent CVD have not been studied. Study Design: Post hoc analysis of the Air Force/Texas Coronary Atherosclerosis Prevention Study. Setting & Participants: Multicenter, randomized, double-blind, placebo-controlled trial of 5,608 men and 997 women without CVD randomly assigned to treatment with lovastatin or placebo. Intervention: Placebo or lovastatin, 20 mg/d. Outcomes & Measurements: First major acute cardiovascular event in participants with mild CKD and kidney function loss in persons with or without CKD. Estimated glomerular filtration rate was calculated using the 4-variable Modification of Diet in Renal Disease Study equation. Results: At baseline, mean estimated glomerular filtration rate in participants with CKD (n = 304) was 53.0 ± 6.0 mL/min/1.73 m2. After an average follow-up of 5.3 ± 0.8 years, the incidence of a fatal and nonfatal CVD event was lower in participants with CKD receiving lovastatin than in those receiving placebo (adjusted relative risk [RR], 0.31; 95% CI, 0.13-0.72; P = 0.01). Tests for interaction suggested that the benefit of lovastatin was independent of the presence of CKD. Lovastatin did not reduce the annualized mean decrease in estimated glomerular filtration rate (-1.3 ± 0.07 vs -1.4 ± 0.07 mL/min/1.73 m2/y, respectively; P = 0.1) or the frequency of a ≥ 25% decrease in kidney function (adjusted RR, 1.10; 95% CI, 0.96-1.28; P = 0.2) or incident CKD (adjusted RR, 1.04; 95% CI, 0.86-1.27; P = 0.6). Limitations: Unable to determine the cause and duration of kidney disease, and information regarding proteinuria was not available. Conclusions: Lovastatin is effective for the primary prevention of CVD in patients with CKD, but is not effective in decreasing kidney function loss in persons with no CVD. © 2009 National Kidney Foundation, Inc.

Cotero V.E.,The New School | Zhang B.B.,Merck And Co. | Routh V.H.,The New School
Journal of Neuroendocrinology | Year: 2010

Obesity and type 2 diabetes mellitus (T2DM) are associated with dysfunctional insulin signalling and impaired central glucose sensing. Glucose sensing neurones reside in key areas of the brain involved in glucose and energy homeostasis (e.g. ventromedial hypothalamus; VMH). We have recently shown that insulin attenuates the ability of glucose-excited (GE) neurones to sense decreased glucose. We hypothesise that this effect of insulin on VMH GE neurones is impaired during T2DM when insulin signalling is dysfunctional. To test our hypotheses, we used whole cell patch clamp recording techniques to evaluate the effects of insulin on VMH GE neurones in brain slices from wild-type and diabetic (db/db) mice. The effects of decreasing glucose from 2.5 to 0.1 mm on VMH GE neurones were similar in wild-type and db/db mice. However, decreasing glucose from 2.5 to 0.5 mm decreased the action potential frequency, membrane potential and input resistance of VMH GE neurones to a significantly greater extent in db/db versus wild-type mice. Furthermore, insulin (5 nm) blunted the effects of decreased glucose in wild-type, but not db/db mice. These differences in both glucose and insulin sensitivity between wild-type and db/db mice were completely ameliorated by the insulin sensitiser, Compound 2 (300 nm). These data are consistent with our hypothesis that impaired insulin signalling in T2DM sensitises VMH GE neurones to decreased glucose. © 2010 The Authors. Journal Compilation © 2010 Blackwell Publishing Ltd.

Ornetti P.,University of Burgundy | Ornetti P.,French Institute of Health and Medical Research | Ornetti P.,University of Paris Descartes | Dougados M.,University of Paris Descartes | And 4 more authors.
Annals of the Rheumatic Diseases | Year: 2011

Objectives: (1) To investigate the psychometric properties of a patient-reported numerical rating scale (NRS) for evaluating functional disability in osteoarthritis (OA), in comparison with the WOMAC function scale and with a physician-reported function NRS; (2) to estimate the patient acceptable symptomatic state (PASS) and the minimal clinically important improvement (MCII) values for treatment with non-steroidal anti-inflammatory drugs (NSAIDs). Methods: Data were extracted from a prospective multicentre study involving 1186 patients with knee or hip OA. The psychometric properties assessed were feasibility: percentage of responses, floor and ceiling effects; construct validity by examining the correlations with classically used OA outcomes measures; responsiveness by comparing the results of before and 1 month after treatment with NSAIDs using standardised response mean (SRM) and effect size (ES). The MCII and PASS values of each function scale were calculated by an anchoring method. Results: No floor or ceiling effect was observed. High correlations were observed as expected between the patient NRS and WOMAC function, pain visual analogue scale and patient global assessment. The responsiveness was moderate to large, with SRM and ES ranging from 0.6 (hip OA) to 0.9 (knee OA) and higher than that of the WOMAC function scale. The PASS was close to 3 for the NRS scales. The MCII appears to be the change that makes the OA functional disability decrease from baseline to the PASS. Conclusion: The patient-reported NRS demonstrated good psychometric properties, similar to the WOMAC function scale and can be regarded as a promising tool in therapeutic evaluation and decision-making in OA.

Wehby G.L.,University of Iowa | Ullrich F.,University of Iowa | Xie Y.,Merck And Co.
Medical Care | Year: 2012

Background: Previous studies of very low birth weight (VLBW) hospital volume effects on in-hospital mortality have used standard risk-adjusted models that only account for observable confounders but not for self-selection bias due to unobservable confounders. Objective: To assess the effects of hospital volume of VLBW infants on in-hospital mortality while explicitly accounting for unobservable confounders and self-selection bias using an instrumental variables (IV) model. Methods: The sample includes 4553 VLBW infants born in 63 hospitals in 2000-2004 in New Jersey. We use IV analysis with the differences between the patient's distances to the nearest low (<50 VLBW infants annually), moderate (51-100 infants annually), and high (>100 VLBW infants annually)-volume hospitals as instruments. We evaluate several volume measures and adjusted for observable infant and hospital characteristics. Results: We find beneficial volume effects on survival that are significantly underestimated in classic risk-adjusted models, under which low and moderate volumes compared with high volumes increase mortality odds by 1.8 and 1.88 times, respectively (risk ratios of 1.4 and 1.5, respectively). However, using the IV approach, we find that low and moderate volumes increase mortality odds by 5.42 and 3.51 times, respectively (risk ratios of 2.76 and 2.21, respectively). These findings suggest unobservable confounders that increase the selection of infants at a greater mortality risk into higher-volume hospitals. Conclusions: Accounting for unobserved self-selection bias reveals large survival benefits from delivering and treating VLBW infants at high-volume hospitals. This supports policies regionalizing the delivery and care for pregnancies at risk for VLBW at high-volume hospitals. © 2012 by Lippincott Williams & Wilkins.

Heyse J.,Merck And Co.
Statistical Methods in Medical Research | Year: 2015

A question of interest in many vaccine clinical development programmes is whether vaccine-induced serum antibody level can be used as a correlate of vaccine efficacy; that is, whether serum antibody levels induced by a candidate vaccine can reliably predict the risk of breakthrough disease. Traditionally, analyses to answer this question have been based on modelling the incidence of breakthrough disease as a function of antibody level, among vaccinated subjects in clinical trials. The Proportion of Similar Response (PSR) method will be described and explored, and compared to the Receive Operator Characteristics (ROC) curve as a graphical tool and the area under the ROC (AUROC) as a summary measure in the context of evaluating correlates of protection. A way to use PSR analysis as complementary to Youden's index as a simple and elegant method to determine the discriminatory ability of a test and to set an optimal threshold value will be presented. In addition, the relationships among PSR and other measures of overlap and discrimination will be described. An example based on a clinical trial from a development programme for a vaccine against human papillomavirus (HPV) will be presented. © The Author(s) 2011 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Foraker R.E.,Ohio State University | Rose K.M.,SRA International, Inc. | Suchindran C.M.,University of North Carolina at Chapel Hill | Chang P.P.,University of North Carolina at Chapel Hill | And 2 more authors.
Circulation: Heart Failure | Year: 2011

Background-Among patients with heart failure (HF), early readmission or death and repeat hospitalizations may be indicators of poor disease management or more severe disease. Methods and Results-We assessed the association of neighborhood median household income (nINC) and Medicaid status with rehospitalization or death in the Atherosclerosis Risk in Communities cohort study (1987 to 2004) after an incident HF hospitalization in the context of individual socioeconomic status and evaluated the relationship for modification by demographic and comorbidity factors. We used generalized linear Poisson mixed models to estimate rehospitalization rate ratios and 95% CIs and Cox regression to estimate hazard ratios (HRs) and 95% CIs of rehospitalization or death. In models controlling for race and study community, sex, age at HF diagnosis, body mass index, hypertension, educational attainment, alcohol use, and smoking, patients with a high burden of comorbidity who were living in low-nINC areas at baseline had an elevated hazard of all-cause rehospitalization (HR, 1.40; 95% CI, 1.10 to 1.77), death (HR, 1.36; 95% CI, 1.02 to 1.80), and rehospitalization or death (HR, 1.36; 95% CI, 1.08 to 1.70) as well as increased rates of hospitalization compared to those with a high burden of comorbidity living in high-nINC areas. Medicaid recipients with a low level of comorbidity had an increased hazard of all-cause rehospitalization (HR, 1.19; 95% CI, 1.05 to 1.36) and rehospitalization or death (HR, 1.21; 95% CI, 1.07 to 1.37) and a higher rate of repeat hospitalizations compared to non-Medicaid recipients. Conclusions-Comorbidity burden appears to influence the association among nINC, Medicaid status, and rehospitalization and death in patients with HF. © 2011 American Heart Association, Inc.

Schonherr H.,Columbia University | Cernak T.,Merck And Co.
Angewandte Chemie - International Edition | Year: 2013

The methyl group is one of the most commonly occurring carbon fragments in small-molecule drugs. This simplest alkyl fragment appears in more than 67 % of the top-selling drugs of 2011 and can modulate both the biological and physical properties of a molecule. This Review focuses on so-called magic methyl effects on binding potency, where the seemingly mundane change of C-H to C-Me improves the IC50 value of a drug candidate more than 100-fold. This discussion is followed by a survey of recent advances in synthetic chemistry that allow the direct methylation of C(sp2)-H and C(sp3)-H bonds. It is our hope that the relevance of the meager methyl group to drug discovery as presented herein will inspire reports on new C-H methylation reactions. It′s a kind of magic: The methyl group is one of the most prominent functional groups in bioactive small molecules and appears in more than 67 % of the top-selling drugs. This Review highlights examples of the magic methyl effect, whereby the installation of a single methyl group boosts potency by more than two orders of magnitude. New C-H activation reactions are required to facilitate the direct introduction of methyl groups. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Troup G.M.,Merck And Co. | Georgakis C.,Tufts University
Computers and Chemical Engineering | Year: 2013

The purpose of this paper is to provide a summary of the current state of the application of process systems engineering tools in the pharmaceutical industry. In this paper, we present the compiled results of an industrial questionnaire submitted to pharmaceutical industry professionals. The topics covered in the questionnaire include process analytics, process monitoring, plant-wide information systems, unit operation modeling, quality control, and process optimization. A futuristic view of what process systems engineering tools will enable the pharmaceutical industry will be also be presented. While the industry is regularly using the traditional Design of Experiments approach to identify key parameters and to define control spaces, these approaches result in passive control strategies that do not attempt to compensate for disturbances. Special new approaches are needed for batch processes due to their essential dependence on time-varying conditions. Lastly, we briefly describe a novel data driven modeling approach, called Design of Dynamic Experiments that enables the optimization of batch processes with respect to time-varying conditions through an example of a simulated chemical reaction process. Many more approaches of this type are needed for the calculation of the design and control spaces of the process, and the effective design of feedback systems. © 2012 Elsevier Ltd.

Elbasha E.H.,Merck And Co. | Chhatwal J.,University of Houston
PharmacoEconomics | Year: 2015

Purpose: Previous research using numerical methods suggested that use of a cohort-based model instead of an individual-based model can result in significant heterogeneity bias. However, the direction of the bias is not known a priori. We characterized mathematically the conditions that lead to upward or downward bias. Method: We used a standard three-state disease progression model to evaluate the cost effectiveness of a hypothetical intervention. We solved the model analytically and derived expressions for life expectancy, discounted quality-adjusted life years (QALYs), discounted lifetime costs and incremental net monetary benefits (INMB). An outcome was calculated using the mean of the input under the cohort-based approach and the whole input distribution for all persons under the individual-based approach. We investigated the impact of heterogeneity on outcomes by varying one parameter at a time while keeping all others constant. We evaluated the curvature of outcome functions and used Jensen’s inequality to determine the direction of the bias. Results: Both life expectancy and QALYs were underestimated by the cohort-based approach. If there was heterogeneity only in disease progression, total costs were overestimated, whereas QALYs gained, incremental costs and INMB were under- or overestimated, depending on the progression rate. INMB was underestimated when only efficacy was heterogeneous. Both approaches yielded the same outcome when the heterogeneity was only in cost or utilities. Conclusion: A cohort-based approach that does not adjust for heterogeneity underestimates life expectancy and may underestimate or overestimate other outcomes. Characterizing the bias is useful for comparative assessment of models and informing decision making. © 2015, Springer International Publishing Switzerland.

Peng F.,Columbia University | Peng F.,Merck And Co. | Danishefsky S.J.,Columbia University | Danishefsky S.J.,Sloan Kettering Institute for Cancer Research
Journal of the American Chemical Society | Year: 2012

The total synthesis of racemic maoecrystal V has been accomplished. Key steps include an intramolecular Diels-Alder cyclization to rapidly construct the core system from simple starting materials and the creation of the A-C ring trans-fusion through intramolecular delivery of a hydrogen to the hindered β-face of the ring system. © 2012 American Chemical Society.

Sparrowe D.,Imperial College London | Sparrowe D.,Merck And Co. | Baklar M.,Imperial College London | Stingelin N.,Imperial College London
Organic Electronics: physics, materials, applications | Year: 2010

By blending poly(3-hexylthiophene) with poly(vinylidene fluoride) and with careful solvent selection, field-effect transistors are fabricated from solution with deposition temperatures suitable for use in low-cost roll-to-roll manufacturing techniques. A percolation threshold of only -3 wt.% P3HT is demonstrated, with field-effect mobilities of 0.04 cm2/ Vs obtained in such PVDF-rich blend devices being equal to neat P3HT field-effect transistors. We illustrate the potential of this material system by the fabrication of working fieldeffect transistor devices, wherein the semiconductor blend is deposited via flexo-printing. Flexible polyethylene naphthalate substrates and an organic gate dielectric were utilized for this purpose. © 2010 Elsevier B.V. All rights reserved.

Molander G.A.,University of Pennsylvania | Trice S.L.J.,University of Pennsylvania | Dreher S.D.,Merck And Co.
Journal of the American Chemical Society | Year: 2010

Although much current research focuses on developing new boron reagents and identifying robust catalytic systems for the cross-coupling of these reagents, the fundamental preparations of the nucleophilic partners (i.e., boronic acids and derivatives) has been studied to a lesser extent. Most current methods to access boronic acids are indirect and require harsh conditions or expensive reagents. A simple and efficient palladium-catalyzed, direct synthesis of arylboronic acids from the corresponding aryl chlorides using an underutilized reagent, tetrahydroxydiboron B 2(OH) 4, is reported. To ensure preservation of the carbon-boron bond, the boronic acids were efficiently converted to the trifluoroborate derivatives in good to excellent yields without the use of a workup or isolation. Further, the intermediate boronic acids can be easily converted to a wide range of useful boronates. Finally, a two-step, one-pot method was developed to couple two aryl chlorides efficiently in a Suzuki-Miyaura-type reaction. © 2010 American Chemical Society.

Burlina F.,CNRS Biomolecules Laboratory | Papageorgiou G.,National Insitiute for Medical Research | Morris C.,National Insitiute for Medical Research | White P.D.,Merck And Co. | Offer J.,National Insitiute for Medical Research
Chemical Science | Year: 2014

The progress of total chemical protein synthesis has been hampered by difficulties in preparing peptide thioesters by standard Fmoc peptide synthesis. The amino acid, α-methylcysteine, sited at the C-terminus of a peptide can substitute for a thioester in peptide ligation reactions. C-terminal α-methylcysteine is fully compatible with Fmoc peptide synthesis and its use in ligation is very simple and robust. Its potential is demonstrated with the synthesis of model proteins. © 2014 The Royal Society of Chemistry.

Aims/hypothesis: The aim of this work was to compare treatment intensification strategies based on orally administered vs injectable incretin-based antihyperglycaemic agents in patients with type 2 diabetes mellitus on metformin monotherapy. Methods: In a 26 week, open-label study, 653 patients (baseline HbA1c = 8.2% [66 mmol/mol]) were randomised at 111 sites in 21 countries in a 1:1 ratio to a strategy using oral agents (starting with sitagliptin 100 mg/day) or a strategy using the injectable drug liraglutide starting at a dose of 0.6 mg/day, up-titrated to 1.2 mg/day after 1 week. The following patients with type 2 diabetes mellitus were recruited for the study: those aged 18-79 years, on a stable dose of metformin monotherapy ≥1,500 mg/day for ≥12 weeks, with an HbA1c ≥7.0% (53 mmol/mol) and ≤11.0% (97 mmol/mol) and a fasting fingerstick glucose (FFG) <15 mmol/l (<270 mg/dl) at the randomisation visit, deemed capable by the investigator of using a Victoza pen injection device (containing 6 mg/ml liraglutide; Novo Nordisk, Bagsværd, Denmark). Women taking part in the study agreed to remain abstinent or use an acceptable method of birth control during the study. Randomisation was performed via a computer-generated allocation schedule using an interactive voice response system. After 12 weeks, patients on sitagliptin with HbA1c ≥ 7.0% (53 mmol/mol) and fasting glucose >6.1 mmol/l had their treatment intensified with glimepiride; patients on liraglutide with HbA1c ≥ 7.0% (53 mmol/mol) had the dose up-titrated to 1.8 mg/day. The primary analysis assessed whether the strategy using oral drugs was non-inferior to that using an injectable drug regarding HbA1c change from baseline at week 26 using a per-protocol (PP) population and a non-inferiority margin of 0.4%. Results: In the PP population (522 patients included: oral strategy, n = 269; injectable strategy, n = 253) antihyperglycaemic therapy was intensified at week 12 in 50.2% and 28.5%, respectively. HbA1c decreased over 26 weeks in both treatment strategy groups, with a larger initial reduction at week 12 in the injectable strategy group. The LS mean change in HbA1c at week 26 was -1.3% (95% CI -1.4, -1.2) in the oral strategy group and -1.4% (95% CI -1.5, -1.3) in the injectable strategy group; the study met the non-inferiority criterion. Both treatment regimens were generally well tolerated; hypoglycaemia was reported more often with the oral strategy, while nausea, vomiting, diarrhoea and abdominal pain were reported more often with the injectable strategy. Conclusions/interpretation: An oral, incretin-based treatment strategy with sitagliptin and, if needed, glimepiride may be a good approach in many patients with type 2 diabetes mellitus for managing inadequate glycaemic control on metformin monotherapy, as compared with an injectable treatment strategy with liraglutide. The oral and injectable strategies had similar effects on HbA 1c and had good overall tolerability. Trial registration ClinicalTrials.gov NCT01296412 Funding The study was sponsored by Merck Sharp & Dohme Corp., a subsidiary of Merck and Co., Inc., Whitehouse Station, NJ, USA. © 2013 Springer-Verlag Berlin Heidelberg.

Lin H.V.,Columbia University | Lin H.V.,Merck And Co. | Ren H.,Columbia University | Samuel V.T.,Yale University | And 4 more authors.
Diabetes | Year: 2011

OBJECTIVE - Impaired insulin-dependent glucose disposal in muscle and fat is a harbinger of type 2 diabetes, but murine models of selective insulin resistance at these two sites are conspicuous by their failure to cause hyperglycemia. A defining feature of muscle and fat vis-à-vis insulin signaling is that they both express the insulin-sensitive glucose transporter Glut4. We hypothesized that diabetes is the result of impaired insulin signaling in all Glut4-expressing tissues. RESEARCH DESIGN AND METHODS - To test the hypothesis, we generated mice lacking insulin receptors at these sites ("GIRKO" mice), including muscle, fat, and a subset of Glut4-positive neurons scattered throughout the central nervous system. RESULTS - GIRKO mice develop diabetes with high frequency because of reduced glucose uptake in peripheral organs, excessive hepatic glucose production, and β-cell failure. CONCLUSIONS - The conceptual advance of the present findings lies in the identification of a tissue constellation that melds cell-autonomous mechanisms of insulin resistance (in muscle/fat) with cell-nonautonomous mechanisms (in liver and β-cell) to cause overt diabetes. The data are consistent with the identification of Glut4 neurons as a distinct neuroanatomic entity with a likely metabolic role. © 2011 by the American Diabetes Association.

Roemer T.,Merck And Co. | Boone C.,University of Toronto
Nature Chemical Biology | Year: 2013

Here, we review the 'target-centric' genomic strategy to antimicrobial discovery and share our perspective on identification, validation and prioritization of potential antimicrobial drug targets in the context of emerging chemical biology, genomics and phenotypic screening strategies. We propose that coupling the dual processes of antimicrobial small-molecule screening and target identification in a whole-cell context is essential to empirically annotate 'druggable' targets and advance early stage antimicrobial discovery. We also advocate a systems-level approach to annotating synthetic-lethal genetic interactions comprehensively within yeast and bacteria models. The resulting genetic interaction networks provide a landscape to rationally predict and exploit drug synergy between cognate inhibitors. We posit that synergistic combination agents provide an important and largely unexploited strategy to 'repurpose' existing chemical space and simultaneously address issues of potency, spectrum, toxicity and drug resistance in early stages of antimicrobial drug discovery. © 2013 Nature America, Inc. All rights reserved.

Chu H.,University of North Carolina at Chapel Hill | Guo H.,Clinical Translational Science Institute | Zhou Y.,Merck And Co.
Medical Decision Making | Year: 2010

Bivariate random effect models are currently one of the main methods recommended to synthesize diagnostic test accuracy studies. However, only the logit transformation on sensitivity and specificity has been previously considered in the literature. In this article, the authors consider a bivariate generalized linear mixed model to jointly model the sensitivities and specificities, and they discuss the estimation of the summary receiver operating characteristic curve (ROC) and the area under the ROC curve (AUC). As the special cases of this model, the authors discuss the commonly used logit, probit, and complementary log-log transformations. To evaluate the impact of misspecification of the link functions on the estimation, they present 2 case studies and a set of simulation studies. Their study suggests that point estimation of the median sensitivity and specificity and AUC is relatively robust to the misspecification of the link functions. However, the misspecification of link functions has a noticeable impact on the standard error estimation and the 95% confidence interval coverage, which emphasizes the importance of choosing an appropriate link function to make statistical inference.

Thomas D.M.,Montclair State University | Schoeller D.A.,University of Wisconsin - Madison | Redman L.A.,Pennington Biomedical Research Center | Martin C.K.,Pennington Biomedical Research Center | And 2 more authors.
American Journal of Clinical Nutrition | Year: 2010

Background: Energy intake (EI) during weight loss is difficult and costly to measure accurately. Objective: The objective was to develop and validate a computational energy balance differential equation model to determine individual EI during weight loss. Design: An algorithm was developed to quantify EI during weight loss based on a validated one-dimensional model for weight change. By using data from a 24-wk calorie-restriction study, we tested the validity of the EI model against 2 criterion measures: 1) EI quantified through food provision from weeks 0-4 and 4-12 and 2) EI quantified through changes in body energy stores [measured with dual-energy X-ray absorptiometry (DXA)] and energy expenditure [measured with doubly labeled water (DLW)] from weeks 4-12 and 12-24. Results: Compared with food provision, the mean (6SD) model errors were 41 ± 118 kcal/d and -22 ± 230 kcal/d from weeks 0-4 and 4-12, respectively. Compared with EI measured with DXA and DLW, the model errors were -71 ± 272 kcal/d and -48 ± 226 kcal/d from weeks 4-12 and 12-24, respectively. In every comparison, the mean error was never significantly different from zero (P values > 0.10). Furthermore, Bland and Altman analysis indicated that error variance did not differ significantly over amounts of EI (P values > 0.26). Almost all individual participants' values were within CI limits. Conclusion: The validity of the newly developed EI model was supported by experimental observations and can be used to determine an individual participant's EI during weight loss. © 2010 American Society for Nutrition.

Lavoie B.,University of Vermont | Balemba O.B.,University of Vermont | Godfrey C.,University of California at San Francisco | Watson C.A.,University of Vermont | And 4 more authors.
Journal of Physiology | Year: 2010

Hydrophobic bile salts are thought to contribute to the disruption of gallbladder smooth muscle (GBSM) function that occurs in gallstone disease, but their mechanism of action is unknown. The current study was undertaken to determine how hydrophobic bile salts interact with GBSM, and how they reduce GBSM activity. The effect of hydrophobic bile salts on the activity of GBSM was measured by intracellular recording and calcium imaging using wholemount preparations from guinea pig and mouse gallbladder. RT-PCR and immunohistochemistry were used to evaluate expression of the G protein-coupled bile acid receptor, GPBAR1. Application of tauro-chenodeoxycholate (CDC, 50-100 μm) to in situ GBSM rapidly reduced spontaneous Ca 2+ flashes and action potentials, and caused a membrane hyperpolarization. Immunoreactivity and transcript for GPBAR1 were detected in gallbladder muscularis. The GPBAR1 agonist, tauro-lithocholic acid (LCA, 10 μm) mimicked the effect of CDC on GBSM. The actions of LCA were blocked by the protein kinase A (PKA) inhibitor, KT5720 (0.5-1.0 μm) and the K ATP channel blocker, glibenclamide (10 μm). Furthermore, LCA failed to disrupt GBSM activity in Gpbar1 -/- mice. The findings of this study indicate that hydrophobic bile salts activate GPBAR1 on GBSM, and this leads to activation of the cyclic AMP-PKA pathway, and ultimately the opening of K ATP channels, thus hyperpolarizing the membrane and decreasing GBSM activity. This inhibitory effect of hydrophobic bile salt activation of GPBAR1 could be a contributing factor in the manifestation of gallstone disease. © 2010 The Authors. Journal compilation © 2010 The Physiological Society.

Schmink J.R.,University of Pennsylvania | Tudge M.T.,Merck And Co.
Tetrahedron Letters | Year: 2013

A palladium-catalyzed cross coupling of nitrogen bearing heterocyclic chloromethyl derivatives with aryl and heteroaryl boronic acids has been developed. In almost all cases, highly efficient cross-couplings were observed at ambient temperature, mitigating unwanted thermally induced side-reactions. The comprehensive substrate scope and respectable yields highlight the synthetic utility of this reaction. © 2012 Elsevier Ltd. All rights reserved.

Sun S.,Cold Spring Harbor Laboratory | Sun S.,State University of New York at Stony Brook | Zhang Z.,Merck And Co. | Sinha R.,Cold Spring Harbor Laboratory | And 3 more authors.
Nature Structural and Molecular Biology | Year: 2010

SF2/ASF is a prototypical serine-and arginine-rich protein, with important roles in splicing and other aspects of mRNA metabolism. Splicing factor, arginine/serine-rich 1 (SFRS1), the gene encoding SF2/ASF, is a potent proto-oncogene with abnormal expression in many tumors. We found that SF2/ASF negatively autoregulates its expression to maintain homeostatic levels. We characterized six alternatively spliced SF2/ASF mRNA isoforms: the major isoform encodes full-length protein, whereas the others are either retained in the nucleus or degraded by nonsense-mediated mRNA decay. Unproductive splicing accounts for only part of the autoregulation, which occurs primarily at the translational level. The effect is specific to SF2/ASF and requires RNA recognition motif 2 (RRM2). The ultraconserved 3′ untranslated region (UTR) is necessary and sufficient for downregulation. SF2/ASF overexpression shifts the distribution of target mRNA toward monoribosomes, and translational repression is partly independent of Dicer and a 5′ cap. Thus, multiple post-transcriptional and translational mechanisms are involved in fine-tuning the expression of SF2/ASF. © 2010 Nature America, Inc. All rights reserved.

El Khoury A.C.,Merck And Co. | Wallace C.,Center for Disease Analysis | Klimack W.K.,Center for Disease Analysis | Razavi H.,Center for Disease Analysis
Journal of Medical Economics | Year: 2012

Background: Globally, hepatitis C virus (HCV) infects ∼3 of the population. The objective of this study was to review published work and determine the direct medical costs for diseases associated with HCV infection globally, with the exception of the US. Methods: A systematic literature search was conducted to identify studies reporting the costs of hepatitis C sequelae between January 1990 and January 2011. Over 400 references were identified, of which 45 were pertinent. The costs were compiled, converted to US dollars, and adjusted to 2010 costs using the medical component of the consumer price index. Results: The median cost of liver transplants was estimated at $139,070 ($15,430-$443,700), refractory ascites at $16,740 ($8990-$35,940), hepatocellular carcinoma (HCC) at $15,310 ($3370-$84,710), decompensated cirrhosis at $14,660 ($3810-$48,360), variceal hemorrhage at $12,190 ($3550-$46,120), hepatic encephalopathy at $9180 ($5370-$50,120), diuretic sensitive ascites at $3400 ($1320-$7470), compensated cirrhosis at $820 ($50-$2890), and chronic hepatitis C at $280 ($90-$1860). The variation among studies was mainly due to the methodology used to assess cost, local cost and government reimbursement, and country-specific treatment protocols. Limitations: All costs were adjusted to 2010 US dollars using the US medical component of the consumer price index (CPI) which may not reflect the change in medical costs in other countries. In addition, the costs, in the local currency were converted to US dollars in the year of the study. However, medical expenses may not vary with exchange rate, leading to artificial variations. Finally, there was no assessment of the quality of individual studies, which resulted in the same weighting to all studies. Conclusions: Hepatitis C imposes a high economic burden globally. Knowing the burden of HCV sequelae is useful for policy decisions as well as serving as a basis for determining the value of HCV screening and treatment. © 2012 Informa UK Ltd.

MacH H.,Merck And Co. | Arvinte T.,University of Geneva | Arvinte T.,Therapeomic Inc.
European Journal of Pharmaceutics and Biopharmaceutics | Year: 2011

As the share of therapeutic proteins in the arsenal of modern medicine continue increasing, relatively little progress has been made in the development of analytical methods that would address specific needs encountered during the development of these new drugs. Consequently, the researchers resort to adaptation of existing instrumentation to meet the demands of rigorous bioprocess and formulation development. In this report, we present a number of such adaptations as well as new instruments that allow efficient and precise measurement of critical parameters throughout the development stage. The techniques include use of atomic force microscopy to visualize proteinacious sub-visible particles, use of extrinsic fluorescent dyes to visualize protein aggregates, particle tracking analysis, determination of the concentration of monoclonal antibodies by the analysis of second-derivative UV spectra, flow cytometry for the determination of subvisible particle counts, high-throughput fluorescence spectroscopy to study phase separation phenomena, an adaptation of a high-pressure liquid chromatography (HPLC) system for the measurement of solution viscosity and a variable-speed streamlined analytical ultracentrifugation method. An ex vivo model for understanding the factors that affect bioavailability after subcutaneous injections is also described. Most of these approaches allow not only a more precise insight into the nature of the formulated proteins, but also offer increased throughput while minimizing sample requirements. © 2011 Elsevier B.V. All rights reserved.

Leiser S.C.,Lundbeck Research United States Inc. | Dunlop J.,Pfizer | Bowlby M.R.,Merck And Co. | Devilbiss D.M.,University of Wisconsin - Madison
Biochemical Pharmacology | Year: 2011

Electroencephalography (EEG) and related methodologies offer the promise of predicting the likelihood that novel therapies and compounds will exhibit clinical efficacy early in preclinical development. These analyses, including quantitative EEG (e.g. brain mapping) and evoked/event-related potentials (EP/ERP), can provide a physiological endpoint that may be used to facilitate drug discovery, optimize lead or candidate compound selection, as well as afford patient stratification and Go/No-Go decisions in clinical trials. Currently, the degree to which these different methodologies hold promise for translatability between preclinical models and the clinic have not been well summarized. To address this need, we review well-established and emerging EEG analytic approaches that are currently being integrated into drug discovery programs throughout preclinical development and clinical research. Furthermore, we present the use of EEG in the drug development process in the context of a number of major central nervous system disorders including Alzheimer's disease, schizophrenia, depression, attention deficit hyperactivity disorder, and pain. Lastly, we discuss the requirements necessary to consider EEG technologies as a biomarker. Many of these analyses show considerable translatability between species and are used to predict clinical efficacy from preclinical data. Nonetheless, the next challenge faced is the selection and validation of EEG endpoints that provide a set of robust and translatable biomarkers bridging preclinical and clinical programs. © 2010 Elsevier Inc.

Chu H.,University of North Carolina at Chapel Hill | Zhou Y.,Merck And Co. | Cole S.R.,University of North Carolina at Chapel Hill | Ibrahim J.G.,University of North Carolina at Chapel Hill
Statistics in Medicine | Year: 2010

To evaluate the probabilities of a disease state, ideally all subjects in a study should be diagnosed by a definitive diagnostic or gold standard test. However, since definitive diagnostic tests are often invasive and expensive, it is generally unethical to apply them to subjects whose screening tests are negative. In this article, we consider latent class models for screening studies with two imperfect binary diagnostic tests and a definitive categorical disease status measured only for those with at least one positive screening test. Specifically, we discuss a conditional-independent and three homogeneous conditional-dependent latent class models and assess the impact of misspecification of the dependence structure on the estimation of disease category probabilities using frequentist and Bayesian approaches. Interestingly, the three homogeneous-dependent models can provide identical goodness-of-fit but substantively different estimates for a given study. However, the parametric form of the assumed dependence structure itself is not 'testable' from the data, and thus the dependence structure modeling considered here can only be viewed as a sensitivity analysis concerning a more complicated non-identifiable model potentially involving a heterogeneous dependence structure. Furthermore, we discuss Bayesian model averaging together with its limitations as an alternative way to partially address this particularly challenging problem. The methods are applied to two cancer screening studies, and simulations are conducted to evaluate the performance of these methods. In summary, further research is needed to reduce the impact of model misspecification on the estimation of disease prevalence in such settings. Copyright © 2010 John Wiley & Sons, Ltd.

Wang F.T.,Optumlnsight Epidemiology | Mast T.C.,Merck And Co. | Glass R.J.,Optumlnsight Epidemiology | Loughlin J.,Optumlnsight Epidemiology | And 2 more authors.
Pediatric Infectious Disease Journal | Year: 2013

Background: Effectiveness of the pentavalent rotavirus vaccine (RV5) after administration of the complete (3 dose) regimen has been demonstrated in a real-world setting. This study assessed the effectiveness of RV5 following partial completion of the 3-dose regimen. Methods: Using a large national health insurance claims database, 2 cohorts of infants (those who received RV5 and a concurrent group who received diphtheria-tetanus-acellular pertussis, but not RV5) were followed through the 2007 and 2008 rotavirus seasons (January 1 to May 31) to identify cases of rotavirus gastroenteritis and all-cause gastroenteritis resulting in medical care encounters. Vaccine effectiveness following the first and the second RV5 doses was estimated by quantifying reductions in hospitalizations, emergency department (ED) and physician office visits. Results: A first RV5 dose was received by 42,306 infants whereas 28,417 infants in the concurrent comparison group received a first diphtheria-tetanus- acellular pertussis dose; 43,704 infants received a second RV5 dose, and 31,810 infants received a second diphtheria-tetanus-acellular pertussis dose. One dose of RV5 was associated with 88% effectiveness against rotavirus gastroenteritis hospitalizations and ED visits and 44% effectiveness against all-cause gastroenteritis hospitalizations and ED visits. A 2-dose regimen of RV5 was associated with 94% effectiveness against rotavirus gastroenteritis hospitalizations and ED visits and 40% effectiveness against all-cause gastroenteritis hospitalizations and ED visits. Conclusion: The RV5 vaccine exhibits effectiveness against rotavirus gastroenteritis even before completing the full 3-dose regimen. These results are of particular relevance when considering the benefits of a partially completed rotavirus vaccine series. Copyright © 2013 by Lippincott Williams & Wilkins.

Montano R.,Norris Cotton Cancer Center | Chung I.,Duksung Womens University | Garner K.M.,Norris Cotton Cancer Center | Parry D.,Merck And Co. | Eastman A.,Norris Cotton Cancer Center
Molecular Cancer Therapeutics | Year: 2012

Many anticancer agents damage DNA and arrest cell-cycle progression primarily in S or G 2 phase of the cell cycle. Previous studies with the topoisomerase I inhibitor SN38 have shown the efficacy of the Chk1 inhibitor UCN-01 to overcome this arrest and induce mitotic catastrophe. UCN-01 was limited in clinical trials by unfavorable pharmacokinetics. SCH900776 is a novel and more selective Chk1 inhibitor that potently inhibits Chk1 and abrogates cell-cycle arrest induced by SN38. Like UCN-01, abrogation of SN38-induced arrest enhances the rate of cell death but does not increase overall cell death. In contrast, SCH900776 reduced the growth-inhibitory concentration of hydroxyurea by 20- to 70-fold. A similar magnitude of sensitization was observed with cytarabine. A 5- to 10-fold sensitization occurred with gemcitabine, but no sensitization occurred with cisplatin, 5-fluorouracil, or 6-thioguanine. Sensitization occurred at hydroxyurea concentrations that marginally slowed DNA replication without apparent activation of Chk1, but this led to dependence on Chk1 that increased with time. For example, when added 18 hours after hydroxyurea, SCH900776 induced DNA double-strand breaks consistent with rapid collapse of replication forks. In addition, some cell lines were highly sensitive to SCH900776 alone, and these cells required lower concentrations of SCH900776 to sensitize them to hydroxyurea. We conclude that some tumors may be very sensitive to the combination of SCH900776 and hydroxyurea. Delayed administration of SCH900776 may be more effective than concurrent treatment. SCH900776 is currently in phase I clinical trials, and these results provide the rationale and schedule for future clinical trials. ©2011 AACR.

Girling P.R.,Durham University | Kiyoi T.,Merck And Co. | Whiting A.,Durham University
Organic and Biomolecular Chemistry | Year: 2011

A review into the aza-Diels-Alder reaction, mainly concentrating on literature examples that form piperidin-4-ones from the reaction of imines and electron rich dienes or enones, either through a Lewis acidic/Brønsted acid approach or through the use of an organocatalyst. This review questions whether the mechanism of the aza-Diels-Alder reaction is step wise as opposed to concerted when using oxygenated dienes. © 2011 The Royal Society of Chemistry.

Terebetski J.L.,Rutgers University | Terebetski J.L.,Merck And Co. | Michniak-Kohn B.,Rutgers University
International Journal of Pharmaceutics | Year: 2014

The combination of a highly soluble salt form of a drug with a polymeric precipitation inhibitor has the potential to prolong drug supersaturation even following salt disproportionation. In this study, dissolution profiles of ibuprofen sodium in the presence of various cellulosic polymers, including hydroxypropyl methylcellulose (HPMC), methylcellulose (MC), and hydroxypropyl cellulose (HPC), were examined in order to assess degree and duration of supersaturation. In addition, the roles that the polymers played in altering drug solubility, media viscosity, physical form, and particle morphology were also assessed. A deep dive into the mechanisms of supersaturation revealed that intermolecular hydrogen bonding between ibuprofen and HPMC was driving supersaturation through nucleation inhibition and crystal growth modification. Polymer viscosity was proposed as the primary factor prolonging supersaturation of ibuprofen in the presence of MC, while mechanisms other than hydrogen bonding were likely to be attributed to supersaturation with the most hydrophobic polymer evaluated, HPC. Overall, the study suggested that induction of intermolecular interactions between ibuprofen and HPMC were more effective at inhibiting nucleation and maintaining prolonged supersaturation than physical modulation of solution properties, such as viscosity. © 2014 Published by Elsevier B.V.

Metzger M.J.,Rutgers University | Metzger M.J.,Merck And Co. | Glasser B.J.,Rutgers University
Powder Technology | Year: 2013

Size reduction is an essential operation across many industries. Despite its prevalence, the efficiency of the conversion of applied force to the creation of new surfaces is extremely poor, none worse than the commonly encountered ball mill. However, tuning of operational parameters may be a means to increase the low efficiency levels, if the proper connection between operating conditions and breakage conditions can be determined. Described here is the implementation of the Bonded Particle Model (BPM) within the Discrete Element Method (DEM) framework to analyze the breakage of bonded agglomerates within a batch ball mill. The effect of agglomerate bond strength (σ-max), grinding media diameter (dm), grinding media fill level (J) and drum rotation rate (φc) on breakage and flow is analyzed using the Attainable Region (AR) approach. It is found that agglomerate strength and grinding media diameter affect breakage significantly, whereas grinding media fill level has a minor affect. Drum rotation rate has potential as a control parameter for fine tuning of the breakage behavior. The majority of breakage occurs near the mill shell, rather than at the point of impact between the media and material and the extent of material breakage has a significant influence on the material flow within the mill. Our work demonstrates that computational simulation is a powerful tool for analyzing the flow and breakage in ball mills and can aid in decision-making for more efficient operation. © 2012 Elsevier B.V.

Junker B.,Merck And Co.
Biotechnology and Bioengineering | Year: 2010

The subject matter of manuscripts by industrial authors has primarily focused on elements with perceived commercial or regulatory significance. Once published, this information interacted and ultimately influenced manuscripts from authors of other affiliations, creating the rapid advancements that culminated in the current multi-billion dollar worldwide biotechnology industry. This paper discusses trends in "solely industrial" articles published in the specific journal of Biotechnology and Bioengineering over the past five decades of this journal's lifetime. "Solely industrial" articles were defined as papers in which all the authors were affiliated with industry. Data were gathered concerning "solely industrial" article distribution and frequency, authoring companies, subject classification, and category distribution. Selected articles and their impact were related to current and past technology milestones as well as associated challenges. Suggestions for areas of greater emphasis to influence the number and subject matter of "solely industrial" articles for the journal's sixth decade were offered for consideration. © 2009 Wiley Periodicals, Inc.

Nelson H.,National Jewish Health | Cartier S.,Optum BurlingtonON | Allen-Ramey F.,Merck And Co. | Lawton S.,ALK Horsholm | Calderon M.A.,Imperial College London
Journal of Allergy and Clinical Immunology: In Practice | Year: 2015

Background: Subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT) have been shown to effectively treat grass pollen allergies, although direct comparisons are sparse. Objective: To estimate the relative efficacy of SLIT tablets compared with SCIT and SLIT drops in commercially available products though network meta-analysis. Methods: A literature search of MEDLINE, Embase, and Cochrane Library publications. Randomized, double-blind clinical trials of SCIT, SLIT drops, and SLIT tablets for grass pollen were included. Bayesian network meta-analyses estimated the standardized mean difference (SMD) across 3 immunotherapy modalities on allergic rhinoconjunctivitis symptom and medication score data from publications or received from authors. Both fixed and random effects models were investigated. Results: Thirty-seven studies were included in meta-analyses for symptom scores and 31 studies for medication scores. In the random effects model, SCIT and SLIT tablets were significantly different from placebo for symptom scores: SMDs (95% CI) of-0.32 (-0.45 to-0.18) and-0.32 (-0.41 to-0.23), respectively. No significant difference was identified for SLIT drops compared with placebo (SMD,-0.17;-0.37 to 0.04). For medication scores, significant differences compared with placebo were observed for SCIT (SMD,-0.33; 95% CI,-0.52 to-0.13), SLIT tablets (SMD,-0.23; 95% CI,-0.29 to-0.17), and SLIT drops (SMD,-0.44; 95% CI,-0.83 to-0.06). Network meta-analysis revealed no significant differences in SMDs (95% credible interval) for symptom scores (0.0145 [-0.19 to 0.23]) or medication scores (0.133 [-0.31 to 0.57]) between SLIT tablets and SCIT, or for symptom scores (-0.175 [-0.37 to 0.02]) and medication scores (0.188 [-0.18 to 0.56]) between SLIT tablets and SLIT drops. Conclusions: The comparisons for grass pollen immunotherapy products commercialized in at least 1 country indicate comparable reductions in allergic rhinoconjunctivitis symptoms and supplemental medication use for SLIT tablets and SCIT in the first pollen season. © 2014 American Academy of Allergy, Asthma & Immunology.

LoRusso P.M.,Barbara Ann Karmanos Cancer Institute | Canetta R.,Bristol Myers Squibb | Wagner J.A.,Merck And Co. | Balogh E.P.,Institute of Medicine | And 3 more authors.
Clinical Cancer Research | Year: 2012

Cancer cells contain multiple genetic changes in cell signaling pathways that drive abnormal cell survival, proliferation, invasion, and metastasis. Unfortunately, patients treated with single agents inhibiting only one of these pathways - even if showing an initial response - often develop resistance with subsequent relapse or progression of their cancer, typically via the activation of an alternative uninhibited pathway. Combination therapies offer the potential for inhibiting multiple targets and pathways simultaneously to more effectively kill cancer cells and prevent or delay the emergence of drug resistance. However, there are many unique challenges to developing combination therapies, including devising and applying appropriate preclinical tests and clinical trial designs, prioritizing which combination therapies to test, avoiding overlapping toxicity of multiple agents, and overcoming legal, cultural, and regulatory barriers that impede collaboration among multiple companies, organizations, and/or institutions. More effective strategies to efficiently develop combination cancer therapies are urgently needed. Thus, the Institute of Medicine's National Cancer Policy Forum recently convened a workshop with the goal of identifying barriers that may be impeding the development of combination investigational cancer therapies, as well as potential solutions to overcome those barriers, improve collaboration, and ultimately accelerate the development of promising combinations of investigational cancer therapies. ©2012 AACR.

Marshall C.L.,Pennsylvania State University | Rajniak P.,Merck And Co. | Matsoukas T.,Pennsylvania State University
Powder Technology | Year: 2013

Fluid bed granulation is a complex, multi-phase, multi-component unit operation often used in the pharmaceutical industry. Various attributes such as particle morphology, size, porosity, wettability, and binder viscosity play key roles in determining the overall size and composition of the granules that are produced. In this paper, we examine the interaction between binder addition and particle morphology and its impact on granule growth. A multi-dimensional population balance model that tracks the evolution of granule size and binder content distribution is used. A constant-number Monte Carlo algorithm that has been adapted to accommodate continuous processes such as the continuous addition of binder to a system is employed to solve the population balance. Simulation results are then compared with experimental results. © 2012 Elsevier B.V.

Eberhart A.J.,University of Manchester | Imbriglio J.E.,Merck And Co. | Procter D.J.,University of Manchester
Organic Letters | Year: 2011

Aryl and heteroaryl sulfoxides undergo ortho allylation upon treatment with Tf 2O and allylsilanes. The method complements the use of sulfoxides to direct ortho-metalation and reaction with electrophiles as it allows allylic carbon nucleophiles to be added ortho to the directing group in a metal-free process. The versatile sulfide adducts can be selectively manipulated using various methods including Kumada-Corriu cross-coupling of the organosulfanyl group. © 2011 American Chemical Society.

Liao G.,Beth Israel Deaconess Medical Center | Detre C.,Beth Israel Deaconess Medical Center | Berger S.B.,Beth Israel Deaconess Medical Center | Engel P.,University of Barcelona | And 4 more authors.
Gastroenterology | Year: 2012

Background & Aims: The glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR; also called TNFRSF18 or CD357) regulates the T cellmediated immune response and is present on surfaces of regulatory T (Treg) cells and activated CD4 + T cells. We investigated the roles of GITR in the development of colitis in mice. Methods: Chronic enterocolitis was induced by the transfer of wild-type or GITR -/- CD4 + T cells to GITR -/- × Rag -/- or Rag -/- mice. We determined the severity of colitis by using the disease activity index; measured levels of inflammatory cytokines, T cells, and dendritic cells; and performed histologic analysis of colon samples. Results: Transfer of nonfractionated CD4 + cells from wild-type or GITR -/- donors induced colitis in GITR -/- × Rag -/- but not in Rag -/- mice. Among mice with transfer-induced colitis, the percentage of Treg and T-helper (Th) 17 cells was reduced but that of Th1 cells increased. Treg cells failed to prevent colitis in GITR -/- × Rag -/- recipients; this was not the result of aberrant function of GITR -/- Treg or T effector cells but resulted from an imbalance between the numbers of tolerogenic CD103 + and PDCA1 + plasmacytoid dendritic cells in GITR -/- mice. This imbalance impaired Treg cell development and expanded the Th1 population in GITR -/- × Rag -/- mice following transfer of nonfractionated CD4 + cells. Conclusions: GITR is not required on the surface of Treg and T effector cells to induce colitis in mice; interactions between GITR and its ligand are not required for induction of colitis. GITR instead appears to control dendritic cell and monocyte development; in its absence, mice develop aggravated chronic enterocolitis via an imbalance of colitogenic Th1 cells and Treg cells. © 2012 AGA Institute.

Huang X.,Merck And Co. | Chen D.Y.-K.,Seoul National University
ChemMedChem | Year: 2012

In recent years, combination therapy has received growing popularity as a powerful therapeutic tactic for the treatment of diseases. The justifications and benefits of combination therapy are far-reaching, including but not limited to addressing unmet medical needs such as cancer, malaria, and HIV/AIDS, improved clinical efficacy and safety with reduced dosage of a single medication, understanding the underlying science of the disease, alleviating pharmaco-economic impacts, and better drug life-cycle management. Using the ezetimibe/simvastatin combination therapy as a case study, a comprehensive overview of the successful discovery and development of the single-pill combination, Vytorin, is presented in this review. A cursory introduction to combination therapy and the rationale for the ezetimibe/simvastatin combination for the treatment of hyperlipidemia provides an instructive entry point. The discovery and mode of action of simvastatin and ezetimibe monotherapies set the scene for a detailed discussion on the discovery and development of Vytorin, with emphasis on bioequivalency studies, clinical efficacy and safety profile studies, and the economic consequences of the single-pill combination therapy. Large-scale outcome clinical trials are also discussed to demonstrate the long-term effects of Vytorin. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Wischke C.,University of Michigan | Wischke C.,Research Center Geesthacht GmbH | Zhang Y.,University of Michigan | Mittal S.,Merck And Co. | Schwendeman S.P.,University of Michigan
Pharmaceutical Research | Year: 2010

Purpose: Although efficient in vitro, fenretinide has not been successful clinically for either of the targeted indications-cancer prevention and dry age-related macular degeneration-because of various issues, such as low oral bioavailability. Therefore, controlled release carriers for parenteral delivery of fenretinide were developed. Methods: After examining the solubility profile of fenretinide, the drug was encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles at 20% drug loading by an s/o/w methodology as well as into in situ-forming PLGA implants. The carrier morphology and drug release kinetics in an elevated polysorbate 80-containing release medium were studied. Results: Preformulation studies revealed increased fenretinide solubility in various PLGA solvents including N-methylpyrrolidone (NMP) and 1:9 v/v methanol:methylene chloride. Co-solvent emulsion methods resulted in low encapsulation efficiency. With a s/o/w method, fenretinide release rates from injectable microparticles were adjusted by the o-phase concentration of end-capped PLGA, the drug particle size, and the particle porosity. In situ implants from non-capped PLGA in NMP exhibited a continuous release of ~70% drug over 1 month. Conclusions: Injectable carriers for fenretinide were successfully prepared, exhibiting excellent drug stability. Based on the in vitro release properties of the different carriers, the preferred injection sites and in vivo release rates will be determined in future preclinical studies. © 2010 Springer Science+Business Media, LLC.

Zhang X.D.,Merck And Co.
Journal of Biomolecular Screening | Year: 2010

In most genome-scale RNA interference (RNAi) screens, the ultimate goal is to select siRNAs with a large inhibition or activation effect. The selection of hits typically requires statistical control of 2 errors: false positives and false negatives. Traditional methods of controlling false positives and false negatives do not take into account the important feature in RNAi screens: many small-interfering RNAs (siRNAs) may have very small but real nonzero average effects on the measured response and thus cannot allow us to effectively control false positives and false negatives. To address for deficiencies in the application of traditional approaches in RNAi screening, the author proposes a new method for controlling false positives and false negatives in RNAi high-throughput screens. The false negatives are statistically controlled through a false-negative rate (FNR) or false nondiscovery rate (FNDR). FNR is the proportion of false negatives among all siRNAs examined, whereas FNDR is the proportion of false negatives among declared nonhits. The author also proposes new concepts, q*-value and p*-value, to control FNR and FNDR, respectively. The proposed method should have broad utility for hit selection in which one needs to control both false discovery and false nondiscovery rates in genome-scale RNAi screens in a robust manner. © 2010 Society for Laboratory Automation and Screening.

Background - Acute heart failure is a common reason for admission, and outcome is often poor. Improved prognostic risk stratification may assist in the design of future trials and in patient management. Using data from a large randomized trial, we explored the prognostic value of clinical variables, measured at hospital admission for acute heart failure, to determine whether a few selected variables were inferior to an extended data set. Methods and Results - The prognostic model included 37 clinical characteristics collected at baseline in PROTECT, a study comparing rolofylline and placebo in 2033 patients admitted with acute heart failure. Prespecified outcomes at 30 days were death or rehospitalization for any reason; death or rehospitalization for cardiovascular or renal reasons; and, at both 30 and 180 days, all-cause mortality. No variable had a c-index >0.70, and few had values >0.60; c-indices were lower for composite outcomes than for mortality. Blood urea was generally the strongest single predictor. Eighteen variables contributed independent prognostic information, but a reduced model using only 8 items (age, previous heart failure hospitalization, peripheral edema, systolic blood pressure, serum sodium, urea, creatinine, and albumin) performed similarly. For prediction of all-cause mortality at 180 days, the model c-index using all variables was 0.72 and for the simplified model, also 0.72. Conclusions - A few simple clinical variables measured on admission in patients with acute heart failure predict a variety of adverse outcomes with accuracy similar to more complex models. However, predictive models were of only moderate accuracy, especially for outcomes that included nonfatal events. Better methods of risk stratification are required. © 2013 American Heart Association, Inc.

Hayashi N.,Merck And Co.
Japanese Journal of Clinical Pharmacology and Therapeutics | Year: 2010

To demonstrate the usefulness of the techniques used in PK/PD analysis as well as Modeling and Simulation with clinical data (pharmacometrics), the following 3 examples are described : 1) modeling based on the data from healthy volunteers administered erythropoietin, and simulation to predict both route/dosage/effect relationship and maintenance dose distribution in renal anemia patients ; 2) simultaneous population PK/PD analysis of the anti-IgE antibody drug, omalizumab, using plasma concentrations of the drug, free IgE and drug-IgE complex, and model validation using simulation ; 3) population PK and PK/PD analyses of sildenafil using peak oxygen consumption in pediatric patients with pulmonary arterial hypertension, and simulation using both models to predict the optimal dosage in pediatric patient population. In addition, the background and current situation of pharmacometrics in overseas countries are introduced.

Derti A.,Harvard University | Derti A.,Merck And Co. | Cenik C.,Harvard University | Kraft P.,Harvard University | Roth F.P.,Harvard University
PLoS Genetics | Year: 2010

The major histocompatibility complex (MHC) of immunity genes has been reported to influence mate choice in vertebrates, and a recent study presented genetic evidence for this effect in humans. Specifically, greater dissimilarity at the MHC locus was reported for European-American mates (parents in HapMap Phase 2 trios) than for non-mates. Here we show that the results depend on a few extreme data points, are not robust to conservative changes in the analysis procedure, and cannot be reproduced in an equivalent but independent set of European-American mates. Although some evidence suggests an avoidance of extreme MHC similarity between mates, rather than a preference for dissimilarity, limited sample sizes preclude a rigorous investigation. In summary, fine-scale molecular-genetic data do not conclusively support the hypothesis that mate selection in humans is influenced by the MHC locus. © 2010 Derti et al.

Artz N.S.,University of Wisconsin - Madison | Hines C.D.G.,Merck And Co. | Brunner S.T.,University of Wisconsin - Madison | Agni R.M.,University of Wisconsin - Madison | And 4 more authors.
Investigative Radiology | Year: 2012

OBJECTIVE: The aim of this study was to compare dual-energy computed tomography (DECT) and magnetic resonance imaging (MRI) for fat quantification using tissue triglyceride concentration and histology as references in an animal model of hepatic steatosis. MATERIALS AND METHODS: This animal study was approved by our institution's Research Animal Resource Center. After validation of DECT and MRI using a phantom consisting of different triglyceride concentrations, a leptin-deficient obese mouse model (ob/ob) was used for this study. Twenty mice were divided into 3 groups based on expected levels of hepatic steatosis: low (n = 6), medium (n = 7), and high (n = 7) fat. After MRI at 3 T, a DECT scan was immediately performed. The caudate lobe of the liver was harvested and analyzed for triglyceride concentration using a colorimetric assay. The left lateral lobe was also extracted for histology. Magnetic resonance imaging fat-fraction (FF) and DECT measurements (attenuation, fat density, and effective atomic number) were compared with triglycerides and histology. RESULTS: Phantom results demonstrated excellent correlation between triglyceride content and each of the MRI and DECT measurements (r ≥ 0.96, P ≤ 0.003). In vivo, however, excellent triglyceride correlation was observed only with attenuation (r = 0.89, P < 0.001) and MRI-FF (r = 0.92, P < 0.001). Strong correlation existed between attenuation and MRI-FF (r = 0.86, P < 0.001). Nonlinear correlation with histology was also excellent for attenuation and MRI-FF. CONCLUSIONS: Dual-energy computed tomography (CT) data generated by the current Gemstone Spectral Imaging analysis tool do not improve the accuracy of fat quantification in the liver beyond what CT attenuation can already provide. Furthermore, MRI may provide an excellent reference standard for liver fat quantification when validating new CT or DECT methods in human subjects. Copyright © 2012 Lippincott Williams &Wilkins.

Xu X.,Scripps Research Institute | Shi Y.,Scripps Research Institute | Zhang H.-M.,Florida State University | Zhang H.-M.,Merck And Co. | And 5 more authors.
Nature Communications | Year: 2012

New domains were progressively added to cytoplasmic aminoacyl transfer RNA (tRNA) synthetases during evolution. One example is the UNE-S domain, appended to seryl-tRNA synthetase (SerRS) in species that developed closed circulatory systems. Here we show using solution and crystal structure analyses and in vitro and in vivo functional studies that UNE-S harbours a robust nuclear localization signal (NLS) directing SerRS to the nucleus where it attenuates vascular endothelial growth factor A expression. We also show that SerRS mutants previously linked to vasculature abnormalities either deleted the NLS or have the NLS sequestered in an alternative conformation. A structure-based second-site mutation, designed to release the sequestered NLS, restored normal vasculature. Thus, the essential function of SerRS in vascular development depends on UNE-S. These results are the first to show an essential role for a tRNA synthetase-associated appended domain at the organism level, and suggest that acquisition of UNE-S has a role in the establishment of the closed circulatory systems of vertebrates. © 2012 Macmillan Publishers Limited. All rights reserved.

Lonskaya I.,Georgetown University | Hebron M.L.,Georgetown University | Desforges N.M.,Georgetown University | Schachter J.B.,Merck And Co. | Moussa C.E.-H.,Georgetown University
Journal of Molecular Medicine | Year: 2014

Alzheimer's disease (AD) is a neurodegenerative disorder associated with amyloid accumulation and autophagic changes. Parkin is an E3 ubiquitin ligase involved in proteasomal and autophagic clearance. We previously demonstrated decreased parkin solubility and interaction with the key autophagy enzyme beclin-1 in AD, but tyrosine kinase inhibition restored parkin-beclin-1 interaction. In the current studies, we determined the mechanisms of nilotinib-induced parkin-beclin-1 interaction, which leads to amyloid clearance. Nilotinib increased endogenous parkin levels and ubiquitination, which may enhance parkin recycling via the proteasome, leading to increased activity and interaction with beclin-1. Parkin solubility was decreased and autophagy was altered in amyloid expressing mice, suggesting that amyloid stress affects parkin stability, leading to failure of protein clearance via the lysosome. Isolation of autophagic vacuoles revealed amyloid and parkin accumulation in autophagic compartments but nilotinib decreased insoluble parkin levels and facilitated amyloid deposition into lysosomes in wild type, but not parkin -/- mice, further underscoring an essential role for endogenous parkin in amyloid clearance. These results suggest that nilotinib boosts the autophagic machinery, leading to increased level of endogenous parkin that undergoes ubiquitination and interacts with beclin-1 to facilitate amyloid clearance. These data suggest that nilotinib-mediated autophagic changes may trigger parkin response via increased protein levels, providing a therapeutic strategy to reduce Aβ and Tau in AD. Key message: Parkin solubility (stability) is decreased in AD and APP transgenic mice. Nilotinib-induced autophagic changes increase endogenous parkin level. Increased parkin level leads to ubiquitination and proteasomal recycling. Re-cycling decreases insoluble parkin and increases parkin-beclin-1 interaction. Beclin-1-parkin interaction enhances amyloid clearance. © 2013 Springer-Verlag Berlin Heidelberg.

Background to the debate: The human rights responsibilities of drug companies have been considered for years by nongovernmental organizations, but were most sharply defined in a report by the UN Special Rapporteur on the right to health, submitted to the United Nations General Assembly in August 2008. The "Human Rights Guidelines for Pharmaceutical Companies in relation to Access to Medicines" include responsibilities for transparency, management, monitoring and accountability, pricing, and ethical marketing, and against lobbying for more protection in intellectual property laws, applying for patents for trivial modifications of existing medicines, inappropriate drug promotion, and excessive pricing. Two years after the release of the Guidelines, the PLoS Medicine Debate asks whether drug companies are living up to their human rights responsibilities. Sofia Gruskin and Zyde Raad from the Harvard School of Public Health say more assessment is needed of such responsibilities; Geralyn Ritter, Vice President of Global Public Policy and Corporate Responsibility at Merck & Co. argues that multiple stakeholders could do more to help States deliver the right to health; and Paul Hunt and Rajat Khosla introduce Mr. Hunt's work as the UN Special Rapporteur on the right to the highest attainable standard of health, regarding the human rights responsibilities of pharmaceutical companies and access to medicines. © 2010 Geralyn S. Ritter.

Rustandi R.R.,Merck And Co.
Methods in Molecular Biology | Year: 2013

Hydrophobic interaction chromatography (HIC) is one of many separation techniques that can be used to analyze proteins. The separation mechanism is based on the adsorption of the hydrophobic region of the protein to the hydrophobic ligands attached to the column in the presence of high salt. The proteins are then eluted by descending salt concentration. Here we describe the use of this HIC technique to evaluate the hydrophobicity of different monoclonal antibodies (mAbs) and to separate different heterogeneities that occur in mAb. © Springer Science+Business Media New York 2013.

Chen Y.H.J.,Merck And Co. | DeMets D.L.,University of Wisconsin - Madison | Lan K.K.G.,J and J Pharmaceutical Research and Development L.L.C.
Statistics in Medicine | Year: 2010

A scenario not uncommon at the end of a Phase II clinical development is that although choices are narrowed down to two to three doses, the project team cannot make a recommendation of one single dose for the Phase III confirmatory study based upon the available data. Several 'drop-the-loser' designs to monitor multiple doses of an experimental treatment compared with a control in a pivotal Phase III study are considered. Ineffective and/or toxic doses compared with the control may be dropped at the interim analyses as the study continues, and when the accumulated data have demonstrated convincing efficacy and an acceptable safety profile for one dose, the corresponding dose or the study may be stopped to make the experimental treatment available to patients. A decision to drop a toxic dose is usually based upon a comprehensive review of all the available safety data and also a risk/benefit assessment. For dropping ineffective doses, a non-binding futility boundary may be used as guidance. The desired futility boundary can be derived by using an appropriate combination of risk level (i.e. error rate for accepting null hypothesis when the dose is truly efficacious) and spending strategy (dropping a dose aggressively in early analyses versus late). For establishing convincing evidence of the treatment efficacy, three methods for calculating the efficacy boundary are discussed: the Joint Monitoring (JM) approach, the Marginal Monitoring method with Bonferroni correction (MMB), and the Marginal Monitoring method with Adjustment for correlation (MMA). The JM approach requires intensive computation especially when there are several doses and multiple interim analyses. The marginal monitoring methods are computationally more attractive and also more flexible since each dose is monitored separately by its own alpha-spending function. The JM and MMB methods control the false positive rate. The MMA method tends to protect the false positive rate and is more powerful than the Bonferroni-based MMB method. The MMA method offers a practical and flexible solution when there are several doses and multiple interim looks. Copyright © 2010 John Wiley & Sons, Ltd.

Josten M.,University of Bonn | Reif M.,University of Bonn | Szekat C.,University of Bonn | Al-Sabti N.,University of Bonn | And 6 more authors.
Journal of Clinical Microbiology | Year: 2013

Nosocomial infections involving epidemic methicillin-resistant Staphylococcus aureus (MRSA) strains are a serious problem in many countries. In order to analyze outbreaks, the infectious isolates have to be typed; however, most molecular methods are expensive or labor-intensive. Here, we evaluated matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) of cell extracts for the molecular characterization of S. aureus strains. The peak patterns of 401 MRSA and methicillin-susceptible S. aureus (MSSA) strains, including clinical and laboratory strains, were analyzed. Database searches indicated the peptides that were represented by the corresponding peaks in the spectra. The identities of the peptides were confirmed by the sequencing of mutants, the expression of antisense RNA fragments that resulted in the knockdown of the peptide of interest and the concomitant loss of the signal, or tandem MALDI-TOF MS (MALDI-TOF/TOF MS). It was shown that the signals derive mainly from stress proteins and ribosomal proteins. Peak shifts that differentiate the main S. aureus clonal complexes CC5, CC22, CC8, CC45, CC30, and CC1 correlate to point mutations in the respective genes. Retrospective typing of an MRSA outbreak showed that it is possible to differentiate unrelated MSSA, MRSA, and borderline resistant S. aureus (BORSA) strains isolated from health care workers. In conclusion, this method allows for the detection of the epidemic lineages of S. aureus during species identification by MALDI-TOF MS analysis. Copyright © 2013, American Society for Microbiology. All Rights Reserved.

The use of sulfate additives such as H2SO4 greatly increases the reactivity of palladium catalysts for the cyanation of aryl and heteroaryl chlorides and renders them more robust toward adventitious air. Using this method, a wide variety of aromatic and heteroaromatic nitriles were prepared in high yield. © 2010 Elsevier Ltd. All rights reserved.

Ahn E.-Y.,University of California at San Diego | DeKelver R.,University of California at San Diego | Lo M.-C.,University of California at San Diego | Nguyen T.,University of California at San Diego | And 6 more authors.
Molecular Cell | Year: 2011

It has been suspected that cell-cycle progression might be functionally coupled with RNA processing. However, little is known about the role of the precise splicing control in cell-cycle progression. Here, we report that SON, a large Ser/Arg (SR)-related protein, is a splicing cofactor contributing to efficient splicing of cell-cycle regulators. Downregulation of SON leads to severe impairment of spindle pole separation, microtubule dynamics, and genome integrity. These molecular defects result from inadequate RNA splicing of a specific set of cell-cycle-related genes that possess weak splice sites. Furthermore, we show that SON facilitates the interaction of SR proteins with RNA polymerase II and other key spliceosome components, suggesting its function in efficient cotranscriptional RNA processing. These results reveal a mechanism for controlling cell-cycle progression through SON-dependent constitutive splicing at suboptimal splice sites, with strong implications for its role in cancer and other human diseases. © 2011 Elsevier Inc.

Shentu Y.,Rutgers University | Shentu Y.,Merck And Co. | Xie M.,Rutgers University