Midazolam Limited Sampling Strategy With a Population Pharmacokinetic Approach to Simultaneously Estimate Cytochrome P450 (CYP) 3A Constitutive, Inhibition, and Induction/Activation Conditions in Healthy Adults.

We have previously described a midazolam limited sampling strategy employing a population pharmacokinetic (PK) approach to estimate constitutive cytochrome P450 (CYP) 3A activity. This study evaluated expansion of this approach to estimate CYP3A constitutive, inhibitory, and induction activities. Midazolam concentrations (n = 4441) from adults (n = 152) were obtained from previous studies after single, oral, or intravenous administration with intensive sample collection. Data were fit to a 2-compartment population PK model that incorporated CYP3A conditions as covariates for clearance (CL), volume of distribution, and bioavailability (F). Limited sampling models using single- or 2-time point concentrations were compared with full PK profiles using the empiric Bayesian post hoc estimations of midazolam area under the plasma concentration-time curve derived from the population PK model. Ketoconazole, rifampin, and pleconaril were significant covariates of CL, while ketoconazole, rifampin, and grapefruit juice were significant covariates for F. Typical midazolam CL and F estimates were 32.9 L/h and 0.31 for the constituent state, while the ratio of inducer/inhibitor for midazolam CL and CL/F for the induced/inhibited (rifampin/ketoconazole) states were 14.2 and 85.3. Upon comparison to the population PK model, the majority of evaluated single- and 2-time point limited sampling models estimated area under the plasma concentration-time curve had unacceptable r2 and/or unacceptable bias and precision. Exclusively during CYP3A inhibitory conditions, the 4- and 6-hour limited sampling model had acceptable limits of r2 , bias, and precision. Consequently, development of a single- or 2-time point midazolam limited sampling model for general, widespread use to simultaneously evaluate various CYP3A conditions remains elusive.

Midazolam Single Time Point Concentrations to Estimate Exposure and Cytochrome P450 (CYP) 3A Constitutive Activity Utilizing Limited Sampling Strategy With a Population Pharmacokinetic Approach.

Midazolam is the preferred probe to phenotype cytochrome P450 (CYP) 3A activity. This study evaluated a single-concentration, midazolam limited sampling strategy utilizing a population pharmacokinetic (PK) approach to estimate area under the curve, and thus CYP3A activity. Midazolam concentrations from adults during CYP3A constitutive conditions were obtained from previous studies after single, oral or intravenous administration. Population PK modeling was conducted by nonlinear mixed-effects modeling. Potential covariates of clearance, volume of distribution, and bioavailability were evaluated. A limited sampling model at 1, 2, 4, or 6 hours was selected and fitted with post hoc estimation with the final population PK model. Preset criterion for the limited sampling model selection was a coefficient of determination ≥0.9. Bias and precision were also evaluated. The studies provided 2122 observations from 152 healthy adults. Midazolam concentrations were adequately described by a two-compartment model with first order absorption. Age and sex were significant covariates of central volume (V2 ) and were retained in the final model. An estimate (interindividual variability) of midazolam clearance was 32.5 L/hr (52.9%), covariate of central volume was 67 L (39.1%), and oral bioavailability was 0.33 (45.5%). The final population parameter estimates were within the 95% confidence intervals and were similar to the median bootstrap estimates. Upon comparison to the population PK model, the 4-hour limited sampling model estimated area under the curve had an acceptable coefficient of determination and acceptable bias and precision limits. A 4-hour, but not the 1-, 2-, and 6-hour, single concentration accurately estimated midazolam area under the curve during constitutive CYP3A conditions in healthy adults.

A call to incorporate systems theory and human factors into the existing investigation of harm in clinical research involving healthcare products.

This is a joint statement from individual pharmacology and pharmaceutical professionals acting in their own capacity, including members of the Alliance for Clinical Research Excellence and Safety (ACRES) and the International Society of Pharmacovigilance (ISoP). By building on the extensive pharmacological and regulatory investigations that already take place, we are calling for a fuller and more robust systems-based approach to the independent investigation of clinical research when serious incidents of harm occur, starting with first-in-human clinical trials. To complement existing activities and regulations, we propose an additional approach blending evidence derived from both pharmacological and organizational science, which addresses human factors and transparency, to enhance organizational learning and continuous improvement. As happens with investigations in other sectors of society, such as the chemical and aviation sector, this systems approach should be seen as an additional way to understand how problems occur and how they might be prevented in the future. We believe that repetition of potentially preventable and adverse outcomes during clinical research, by failing to identify and act upon all systematic vulnerabilities, is a situation that needs urgent change. As we will discuss further on, approaches based on applying systems theory and human factors are much more likely to improve objectivity and transparency, leading to better system design.

Comprehensive review of visual defects reported with topiramate.

OBJECTIVE: The objective of this study was to analyze clinical patterns of visual field defects (VFDs) reported with topiramate treatment and assess possible mechanism of action (MOA) for antiepileptic drug (AED) associated VFDs. METHODS: A comprehensive topiramate database review included preclinical data, sponsor's clinical trials database, postmarketing spontaneous reports, and medical literature. All treatment-emergent adverse events (TEAEs) suggestive of retinal dysfunction/damage were summarized. Relative risk (RR) was computed from topiramate double-blind, placebo-controlled trials (DBPCTs) data. RESULTS: Preclinical studies and medical literature review suggested that despite sharing gamma-aminobutyric acid (GABA)-ergic MOA with other AEDs, topiramate treatment was not associated with VFDs. TEAEs suggestive of retinal dysfunction/damage were observed in 0.3%-0.7% of adults and pediatric patients with topiramate (N=4,679) versus ≤0.1% with placebo (N=1,834) in DBPCTs for approved indications (epilepsy and migraine prophylaxis); open-label trials (OLTs) and DBPCTs for investigational indications had similar incidence. Overall, 88% TEAEs were mild or moderate in severity. Serious TEAEs were very rare (DBPCTs: 0%; OLTs: ≤0.1%), and most were not treatment limiting, and resolved. The most common visual TEAEs (approved indications) were VFD, scotoma, and optic atrophy. The incidence of TEAEs in DBPCTs (approved and investigational indications) was higher in topiramate-treated (N=9,169) versus placebo-treated patients (N=5,023; 0.36% vs 0.24%), but the RR versus placebo-treated patients was not significant (RR: 1.51 [95% confidence interval: 0.78, 2.91]). CONCLUSION: VFDs do not appear to be a class effect for AEDs with GABA-ergic MOA. The RR for VFDs is not significantly different between topiramate and placebo treatment.

Hot Button Protocol and Operational Issues Between Sponsors and Sites in Clinical Pharmacology Studies: A Moderated Forum Session.

This paper summarizes a discussion that took place at the 52nd Annual DIA Meeting in Philadelphia, PA, on June 30, 2016, titled "Hot-Button Protocol and Operational Issues between Sponsors and Sites in Clinical Pharmacology Studies." The symposium was a moderated panel of phase 1 clinical research experts representing the sponsor, and investigational sites. Conference attendees of similar experience joined in the discussion after commentary by each panelist. The learning objectives of the symposium were (1) to recognize issues that can provoke sponsor/site conflict or diminish conduct efficiency when they arise in the course of preparing to conduct or execution of phase 1 clinical studies, (2) to discuss how to handle such issues with counterparts when they arise and describe ways to negotiate and formulate a successful resolution. Sponsors and sites both have challenges in executing clinical trials on time and within budget. Both need to set and maintain realistic expectations and communicate with honesty, transparency, and timeliness. Achieving this goal will advance the more important take-away message, that developing new drugs requires sound execution of clinical trials.

Safety in FIH Trials: A Summary of the Symposium "Fatal Drug Trial in Phase 1: Understanding Risk, Subject Safety, Timelines, and Cost".

A cross-section of clinical research professionals convened at the June 2016 Drug Information Association annual meeting in Philadelphia to discuss and critically analyze the first-in-human (FIH) clinical trial conducted by a French CRO with BIA 10-2474 (BIA) under development for pain relief by Bial-Portela & Ca., S.A., that resulted in 6 healthy volunteers hospitalized with serious adverse events, which resulted in 1 death. This paper summarizes the background, presentations, and discussion points of Symposium no. 107 in an effort to share the learnings of our symposium with others who conduct studies. Novel investigational products studied in phase 1 clinical trials propose a heightened risk of adverse events that may not be foreseeable when relying on animal studies to project outcomes in humans. Novelty of molecular structure, drug class, mechanism of action, proper dose selection, pharmacokinetics, and therapeutic window are all contributing factors that heighten risks when transitioning from animal to human trials. The potential for catastrophic events confronts every sponsor/investigator in clinical trials. Minimizing risks to subjects is an essential ethical and scientific mandate for all those involved in the clinical trials. A complex matrix of planning, conducting, and communicating preclinical and clinical observations need to be considered carefully by the Sponsor, Investigator and others during the planning, execution, and interpretation of FIH studies, in order to promote participant safety and study data integrity. Suggestions may be applied to FIH studies, which may provide a new or improved way to address complex system and prevent or mitigate situations such as what occurred with the Bial FIH trial, where seemingly a number of issues coincided in a "perfect storm" and the system failed to sound a warning or detect an issue before a life was tragically lost.

Structured Risk Assessment for First-in-Human Studies.

We describe a structured risk assessment and risk mitigation process that is currently used to evaluate proposed first-in human (FiH) studies. This process balances the inherent risks of an FiH study with maximal protection of subjects. Risk assessment should consider all available data, carefully identifying aspects that may lead to risk for healthy subjects. A structured risk assessment avoids omissions and promotes consistency. Such a risk assessment should be performed for Investigational Products as well as for challenge agents and study procedures. Careful risk assessment recognizes gaps of knowledge and emphasizes that FiH studies are tolerability, not toxicity, studies.

Is uveitis associated with topiramate use? A cumulative review.

Occasional reports of uveitis following topiramate use necessitated an investigation of relevant cases from safety databases and published biomedical literature. Data mining of the Food and Drug Administration Adverse Event Reporting System and cumulative review of cases from the global safety database (sponsor database) and published literature were conducted to assess association between topiramate use and uveitis. The Food and Drug Administration Adverse Event Reporting System search identified disproportional reporting of uveitis (n=23) and related terms (choroidal detachment, n=25; iridocyclitis, n=17). The postmarketing reporting frequency of uveitis and related events from the global safety database and based on an estimated topiramate exposure of 11,185,740 person-years from launch to April 2015 was 0.38 per 100,000 person-years and assigned as very rare. A total of 14 potential uveitis cases were identified from the cumulative review. Seven of these 14 cases were complicated by inadequate documentation, appearance of uveitic signs following drug withdrawal, or concurrent use of other sulfonamides. In acute angle-closure glaucoma and uveal effusions cases, insufficient evidence for underlying inflammation suggested that uveitis was not a component. Only seven of 14 cases were well documented, potentially topiramate-associated uveitis cases. Uveitis may occur in the setting of topiramate use only in very rare instances. Current evidence did not reveal a dose- or duration-dependent relationship between uveitis and topiramate use.

Inhibition of prostacyclin and thromboxane biosynthesis in healthy volunteers by single and multiple doses of acetaminophen and indomethacin.

This double-blind, randomized crossover study assessed the effect of acetaminophen (1000 mg every 8 hours) versus indomethacin (50 mg every 8 hours) versus placebo on cyclooxygenase enzymes (COX-1 and COX-2). Urinary excretion of 2,3-dinor-6-keto-PGF1α, (prostacyclin metabolite, PGI-M; COX-2 inhibition) and 11-dehydro thromboxane B2 (thromboxane metabolite, Tx-M; COX-1 inhibition) were measured after 1 dose and 5 days of dosing. Peak inhibition of urinary metabolite excretion across 8 hours following dosing was the primary end point. Mean PGI-M excretion was 33.7%, 55.9%, and 64.6% on day 1 and 49.4%, 65.1%, and 80.3% on day 5 (placebo, acetaminophen, and indomethacin, respectively). Acetaminophen and indomethacin inhibited PGI-M excretion following single and multiple doses (P = .004 vs placebo). PGI-M excretion inhibition after 1 dose was similar for indomethacin and acetaminophen, but significantly greater with indomethacin after multiple doses (P = .006). Mean Tx-M excretion was 16.2%, 45.2%, and 86.6% on day 1 and 46.2%, 58.4%, and 92.6% on day 5 (placebo, acetaminophen, and indomethacin, respectively). Tx-M excretion inhibition following 1 dose was reduced by acetaminophen (P ≤ .003). Indomethacin reduced Tx-M excretion significantly more than acetaminophen and placebo after single and multiple doses (P ≤ .001). Acetaminophen and indomethacin inhibited COX-1 and COX-2 following a single dose, but acetaminophen was a less potent COX-1 inhibitor than indomethacin.

Limited sampling strategy of partial area under the concentration-time curves to estimate midazolam systemic clearance for cytochrome P450 3A phenotyping.

OBJECTIVE: Intravenous (IV) midazolam is the preferred cytochrome P450 (CYP) 3A probe for phenotyping, with systemic clearance (CL) estimating hepatic CYP3A activity. A limited sampling strategy was conducted to determine whether partial area under the concentration-time curves (AUCs) could reliably estimate midazolam systemic CL during conditions of CYP3A baseline activity, inhibition, and induction/activation. METHODS: Midazolam plasma concentrations during CYP3A baseline (n = 93), inhibition (n = 40), and induction/activation (n = 33) were obtained from 7 studies in healthy adults. Noncompartmental analysis determined observed CL (CL(obs)) and partial AUCs. Linear regression equations were derived from partial AUCs to estimate CL (CL(pred)) during CYP3A baseline, inhibition, and induction/activation. Preestablished criterion for linear regression analysis was r(2) ≥ 0.9. CL(pred) was compared with CL(obs), and relative bias and precision were assessed using percent mean prediction error and percent mean absolute error. RESULTS: During CYP3A baseline and inhibition, all evaluated partial AUCs failed to meet criterion of r(2) ≥ 0.9 and/or percent mean absolute error <15%. During CYP3A induction/activation, equations derived from partial AUCs from 0 to 1 hour (AUC0-1), 0 to 2 hours (AUC0-2), and 0 to 4 hours (AUC0-4) were acceptable, with good precision and minimal bias. These equations provided the same conclusions regarding equivalency testing compared with intense sampling. CONCLUSIONS: During CYP3A induction/activation, but not baseline or inhibition, midazolam partial AUC0-1, AUC0-2, and AUC0-4 reliably estimated systemic CL and consequently hepatic CYP3A activity in healthy adults.

Investigators' Experience With Expedited Safety Reports Prior to the FDA's Final IND Safety Reporting Rule.

Prior to enactment of the final investigational new drug application (IND) safety reporting rule, an attempt was made to document the effort expended at investigative sites in processing IND safety reports from sponsors and to assess the effect of these expedited reports on trial conduct. Investigators were asked to (1) prospectively document time to process IND safety reports and (2) retrospectively review safety reports from a previous 3-month period, documenting resultant actions. In this limited sample, sites spent a median of 0.25 hours per report at a median cost of US$22. Few expedited safety reports were retrospectively said to have changed study conduct or informed consent. However, a low response rate and the concentration of clinical sites in a single therapeutic area preclude generalizing these results. The authors discuss the challenges in gaining investigators' cooperation to evaluate the impact of regulatory requirements. Better methods to facilitate this type of research will enrich the scientific basis of future clinical trial regulation and guidance.

Detection and localization of bile duct leaks after cholecystectomy using Gd-EOB-DTPA-enhanced MR cholangiography: retrospective study of 16 patients.

OBJECTIVE: To determine if gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance (MR) cholangiography can detect and localize bile duct leaks in postcholecystectomy patients. MATERIAL AND METHODS: Four blinded independent radiologists performed a retrospective review of 16 consecutive patients who underwent MR cholangiography with intravenous Gd-EOB-DTPA for the evaluation of possible biliary leak. Image quality, ductal opacification, and presence and location of any bile leak were evaluated. An independent observer determined the criterion standard using a consensus of all chart, clinical, and imaging findings. RESULTS: All 6 bile leaks confirmed at endoscopic retrograde cholangiopancreatography were diagnosed by all reviewers (sensitivity, 100%). Of the 10 patients with no leak, only one reader incorrectly diagnosed a bile leak in a single case (specificity, 98%). The accuracy for detection of the site of leak with Gd-EOB-DTPA-enhanced MR cholangiography was 80%. CONCLUSION: Gadolinium-EOB-DTPA-enhanced MR can detect bile leaks with a high sensitivity and specificity.

Evaluation of partial area under the concentration time curve to estimate midazolam apparent oral clearance for cytochrome P450 3A phenotyping.

BACKGROUND: Midazolam apparent oral clearance (CLORAL) is used to estimate intestinal and hepatic cytochrome P450 (CYP) 3A activity. A limited sampling approach was performed to access a midazolam partial area under the concentration time curve (AUC) to estimate CLORAL. METHODS: Midazolam plasma concentrations from healthy adults were obtained during CYP3A baseline (n=116), inhibition (n=75), and induction or activation (n=66) from seven published studies. Observed CLORAL and partial AUCs of AUC0-2, AUC0-4, AUC0-6, AUC1-2, AUC1-4, AUC2-4, and AUC2-6 were determined by noncompartmental analysis. Subject data were randomly divided into a training set and a validation set. Linear regression equations, derived from partial AUCs, were developed from training set data. Predicted CLORAL was determined from these equations from validation set data. Preset criterion was a coefficient of determination (r2) greater than or equal to 0.9. Bias and precision were evaluated by relative percent mean prediction error (%MPE) and relative percent mean absolute error (%MAE). RESULTS: During CYP3A baseline conditions, all of the evaluated CLORAL equations had unacceptable r2 (range: 0.34-0.86). During CYP3A inhibition, all of the evaluated CLORAL equations had unacceptable %MAE. Acceptable r2, %MPE, and %MAE were observed during CYP3A induction/activation with AUC0-4 (r2=0.99, %MPE=3.9, %MAE=12.5) and AUC1-4 (r2=0.99, %MPE=6%, %MAE=11.1%). The same equations also predicted the extent of CYP3A induction as a lack of equivalence was observed with AUC0-4 and AUC1-4. CONCLUSIONS: Midazolam partial AUCs were unable to estimate CYP3A activity during the evaluated baseline and inhibitory conditions. Midazolam CLORAL utilizing a partial AUC0-4 and AUC1-4 was able to estimate CYP3A induction with rifampin and Ginkgo biloba extract.

MK-0448, a specific Kv1.5 inhibitor: safety, pharmacokinetics, and pharmacodynamic electrophysiology in experimental animal models and humans.

BACKGROUND: We evaluated the viability of I(Kur) as a target for maintenance of sinus rhythm in patients with a history of atrial fibrillation through the testing of MK-0448, a novel I(Kur) inhibitor. METHODS AND RESULTS: In vitro MK-0448 studies demonstrated strong inhibition of I(Kur) with minimal off-target activity. In vivo MK-0448 studies in normal anesthetized dogs demonstrated significant prolongation of the atrial refractory period compared with vehicle controls without affecting the ventricular refractory period. In studies of a conscious dog heart failure model, sustained atrial fibrillation was terminated with bolus intravenous MK-0448 doses of 0.03 and 0.1 mg/kg. These data led to a 2-part first-in-human study: Part I evaluated safety and pharmacokinetics, and part II was an invasive electrophysiological study in healthy subjects. MK-0448 was well-tolerated with mild adverse experiences, most commonly irritation at the injection site. During the electrophysiological study, ascending doses of MK-0448 were administered, but no increases in atrial or ventricular refractoriness were detected, despite achieving plasma concentrations in excess of 2 µmol/L. Follow-up studies in normal anesthetized dogs designed to assess the influence of autonomic tone demonstrated that prolongation of atrial refractoriness with MK-0448 was markedly attenuated in the presence of vagal nerve simulation, suggesting that the effects of I(Kur) blockade on atrial repolarization may be negated by enhanced parasympathetic neural tone. CONCLUSIONS: The contribution of I(Kur) to human atrial electrophysiology is less prominent than in preclinical models and therefore is likely to be of limited therapeutic value for the prevention of atrial fibrillation.

Double-blind crossover study to assess potential differences in cytochrome P450 3A4 activity in healthy subjects receiving ondansetron plus dexamethasone, with and without aprepitant.

PURPOSE: Because glucocorticoids and the neurokinin-1 receptor antagonist aprepitant influence CYP3A4 activity, this study assessed whether aprepitant added to a 5-HT(3) antagonist and glucocorticoid would affect CYP3A4 induction. METHODS: In this double-blind, 2-period crossover study, 12 subjects were randomized to receive a triple regimen (oral aprepitant [A] 125 mg, intravenous ondansetron [O] 32 mg, and oral dexamethasone [D] 12 mg day 1; A 80 mg and D 8 mg days 2-3; D 8 mg day 4) in 1 of 2 periods, and a dual regimen (O 32 mg and D 20 mg day 1; D 8 mg bid days 2-4); the D dose was adjusted to account for known dexamethasone/aprepitant interaction. Oral (2 mg) and intravenous (1 mg) stable isotope ((13)C(5) (15)N(1))-labeled midazolam were simultaneously given as probes on days -1, 6, 8, 15, and 22 of each period. If the a priori 90% confidence interval for the day 6 geometric mean oral midazolam AUC(0-∞) ratio (triple/dual regimen) of fold-change from baseline was above 0.5, it would be concluded that there was no clinically meaningful between-regimen difference in CYP3A4 activity. RESULTS: Day 6 oral midazolam AUC(0-∞) geometric mean fold-change from baseline was 0.84 (0.30-1.58 with A, 0.46-1.69 without A). The ratio of geometric mean oral midazolam AUC(0-∞) fold-changes was 1.00 (90% confidence interval 0.80, 1.25). CONCLUSIONS: Aprepitant plus a 5-HT(3) antagonist and dexamethasone is unlikely to have a significant additional inductive effect on CYP3A4 activity beyond that of the dual regimen.

Safety and immunogenicity of a novel Staphylococcus aureus vaccine: results from the first study of the vaccine dose range in humans.

Merck V710 is a novel vaccine containing the conserved Staphylococcus aureus iron surface determinant B shown to be protective in animal models. A phase I, multicenter, double-blind study of the dose range was conducted to assess the immunogenicity and safety of an adjuvanted liquid formulation of V710. A total of 124 adults (18 to 55 years of age) were randomized 1:1:1:1 to receive one 0.5-ml intramuscular injection of V710 (5 µg, 30 µg, or 90 µg) or saline placebo. A positive immune response was defined as at least a 2-fold increase in IsdB-specific IgG levels from baseline levels. Local and systemic adverse events were assessed for 5 and 14 days, respectively, following vaccination. Positive immune responses were detected in 12 (67%) of the 18 subjects in the groups receiving 30 and 90 µg V710 tested at day 10. At day 14, a significantly greater proportion of subjects manifested a positive immune response with higher geometric mean concentrations in the V710 30-µg (86%; geometric mean concentration of 116 µg/ml) and 90-µg (87%; geometric mean concentration of 131 µg/ml) dose groups than in the V710 5-µg (29%; geometric mean concentration of 51 µg/ml) or placebo (4%; geometric mean concentration of 23 µg/ml) groups. Immune responses were durable through day 84. Subjects <40 and ≥40 years of age had comparable immune responses. The most common adverse events were injection-site pain, nausea, fatigue, and headache, usually of mild intensity. No immediate reactions or serious adverse events were reported. In this first study of V710 in humans, a single 30-µg or 90-µg dose was more immunogenic than the 5-µg dose or placebo. Immune responses were evident by 10 to 14 days after vaccination in most responders.

Blastomycosis presenting as multiple splenic abscesses: Case report and review of the literature.

A 31-year-old Canadian Aboriginal man from northwestern Ontario presented with left upper quadrant pain and a tender left upper quadrant mass. Evaluation with a computed tomography scan showed multiple lesions within the spleen, a collection between the splenic tip and splenic flexure of the colon, and several small adrenal lesions. Computed tomographic-guided needle biopsy showed necrotizing granulomatous inflammation and multinucleated giant cells. Gomori's methenamine silver stain showed broad-based budding yeast consistent with Blastomyces dermatitidis. Abdominal symptoms resolved after two months of oral itraconazole. Multiple splenic abscesses are a rare presentation of blastomycosis and should be considered in the differential diagnosis of left upper quadrant abdominal pain in a patient with a history of travel or residence in a region endemic for B dermatitidis.

Diurnal variation of the human adipose transcriptome and the link to metabolic disease.

BACKGROUND: Circadian (diurnal) rhythm is an integral part of the physiology of the body; specifically, sleep, feeding behavior and metabolism are tightly linked to the light-dark cycle dictated by earth's rotation. METHODS: The present study examines the effect of diurnal rhythm on gene expression in the subcutaneous adipose tissue of overweight to mildly obese, healthy individuals. In this well-controlled clinical study, adipose biopsies were taken in the morning, afternoon and evening from individuals in three study arms: treatment with the weight loss drug sibutramine/fasted, placebo/fed and placebo/fasted. RESULTS: The results indicated that diurnal rhythm was the most significant driver of gene expression variation in the human adipose tissue, with at least 25% of the genes having had significant changes in their expression levels during the course of the day. The mRNA expression levels of core clock genes at a specific time of day were consistent across multiple subjects on different days in all three arms, indicating robust diurnal regulation irrespective of potential confounding factors. The genes essential for energy metabolism and tissue physiology were part of the diurnal signature. We hypothesize that the diurnal transition of the expression of energy metabolism genes reflects the shift in the adipose tissue from an energy-expending state in the morning to an energy-storing state in the evening. Consistent with this hypothesis, the diurnal transition was delayed by fasting and treatment with sibutramine. Finally, an in silico comparison of the diurnal signature with data from the publicly-available Connectivity Map demonstrated a significant association with transcripts that were repressed by mTOR inhibitors, suggesting a possible link between mTOR signaling, diurnal gene expression and metabolic regulation. CONCLUSION: Diurnal rhythm plays an important role in the physiology and regulation of energy metabolism in the adipose tissue and should be considered in the selection of novel targets for the treatment of obesity and other metabolic disorders.

Minimal effects of ritonavir and efavirenz on the pharmacokinetics of raltegravir.

Raltegravir is a novel human immunodeficiency virus type 1 (HIV-1) integrase strand transfer inhibitor with potent in vitro activity against HIV-1 (95% inhibitory concentration = 31 nM in 50% human serum). The possible effects of ritonavir and efavirenz on raltegravir pharmacokinetics were separately examined. Two clinical studies of healthy subjects were conducted: for ritonavir plus raltegravir, period 1, 400 mg raltegravir; period 2, 100 mg ritonavir every 12 h for 16 days with 400 mg raltegravir on day 14; for efavirenz plus raltegravir, period 1, 400 mg raltegravir; period 2, 600 mg efavirenz once daily for 14 days with 400 mg raltegravir on day 12. In the presence of ritonavir, raltegravir pharmacokinetics were weakly affected: the plasma concentration at 12 h (C(12 h)) geometric mean ratio (GMR) (90% confidence interval [CI]) was 0.99 (0.70, 1.40), area under the concentration-time curve from zero to infinity (AUC(0-infinity)) was 0.84 (0.70, 1.01), and maximum concentration of drug in serum (C(max)) was 0.76 (0.55, 1.04). In the presence of efavirenz, raltegravir pharmacokinetics were moderately to weakly reduced: C(12 h) GMR (90% CI) was 0.79 (0.49, 1.28); AUC(0-infinity) was 0.64 (0.52, 0.80); and C(max) was 0.64 (0.41, 0.98). There were no substantial differences in the time to maximum concentration of drug in plasma or the half-life. Plasma concentrations of raltegravir were not substantially affected by ritonavir. Though plasma concentrations of raltegravir were moderately to weakly reduced by efavirenz, the degree of this reduction was not clinically meaningful. No dose adjustment is required for raltegravir with coadministration with ritonavir or efavirenz.