Toward highly sensitive and reproducible LC-MS/MS analysis of MK-8591 phosphorylated anabolites in human peripheral blood mononuclear cells.

Aim: MK-8591 (EFdA), a novel anti-HIV nucleoside analog, is converted to mono-, di- and tri-phosphates (MK-8591-MP, MK-8591-DP and MK-8591-TP) intracellularly, among which MK-8591-TP is the active pharmacological form. An ultrasensitive LC-MS/MS assay was required to measure MK-8591-DP and MK-8591-TP levels in human peripheral blood mononuclear cells (PBMCs). Sensitivity and reproducibility were major bottlenecks in these analyses. Materials and methods: Human PBMCs were isolated from blood and lysed with 70/30 methanol/RPMI-1640. An LC-MS/MS method was developed to simultaneously quantify MK-8591-DP and MK-8581-TP in PBMC lysates. Results: Low flow LC and dimethyl sulfoxide mediated signal enhancement enabled an extreme sensitivity with limit of quantitation at 0.1 ng/ml. Assay accuracy was 92.5-106% and precision was 0.7-12.1% for a linear curve range of 0.1-40 ng/ml. Matrix variability and interference liability were comprehensively evaluated. Conclusion: Our study findings and steps taken in addressing clinical sample issues help understand and overcome the challenges facing intracellular nucleotide analog analysis.

Microdosing Cocktail Assay Development for Drug-Drug Interaction Studies.

Methodology for analysis of a microdosing drug cocktail designed to evaluate the contribution of drug transporters and drug metabolizing enzymes to disposition was developed using liquid chromatography-mass spectrometry-based detection. Fast and sensitive methods were developed and qualified for the quantification of statins (pitavastatin, pitavastain lactone, rosuvastatin, atorvastatin, 2-hydroxy, and 4-hydroxy atorvastatin), midazolam, and dabigatran in human plasma. Chromatographic separation was accomplished using reversed-phase liquid chromatography or hydrophilic interaction liquid chromatography with gradient elution and detection by tandem mass spectrometry in the positive ionization mode using electrospray ionization. The lower limit of quantitation (LLOQ) for the statins assay was 1 pg/mL for the 6 analytes with a linear range from 1 to 1000 pg/mL processing 250 µL plasma sample. The midazolam assay LLOQ was 0.5 pg/mL with a linear range of 0.5 to 1000 pg/mL. For the dabigatran assay, the LLOQ was 10 pg/mL with a linear range of 10 to 5000 pg/mL processing 100 µL plasma sample. The intraday and interday precision and accuracy of the assays were within acceptable ranges, and the assays were successfully applied to support a study where a microdose cocktail was dosed to healthy human subjects for simultaneous assessment of clinical drug-drug interactions mediated by major drug transporters and CYP3A.

Intrapulmonary Pharmacokinetics of Relebactam, a Novel ß-Lactamase Inhibitor, Dosed in Combination with Imipenem-Cilastatin in Healthy Subjects.

This phase I study assessed the intrapulmonary pharmacokinetic profiles of relebactam (MK-7655), a novel ß-lactamase inhibitor, and imipenem. Sixteen healthy subjects received 250 mg relebactam with 500 mg imipenem-cilastatin, given intravenously every 6 h for 5 doses, and were randomized to bronchoscopy/bronchoalveolar lavage at 0.5, 1, 1.5, or 3 h after the last dose (4 subjects per time point). Both drugs penetrated the epithelial lining fluid (ELF) to a similar degree, with the profiles being similar in shape to the corresponding plasma profiles and with the apparent terminal half-lives in plasma and ELF being 1.2 and 1.3 h, respectively, for relebactam and 1.0 h in both compartments for imipenem. The exposure (area under the concentration-time curve from time zero to infinity) in ELF relative to that in plasma was 54% for relebactam and 55% for imipenem, after adjusting for protein binding. ELF penetration for relebactam was further analyzed by fitting the data to a two-compartment pharmacokinetic model to capture its behavior in plasma, with a partitioning coefficient capturing its behavior in the lung compartment. In this model, the time-invariant partition coefficient for relebactam was found to be 55%, based on free drug levels. These results support the clinical evaluation of relebactam with imipenem-cilastatin for the treatment of bacterial pneumonia.

Pooled population pharmacokinetic model of imipenem in plasma and the lung epithelial lining fluid.

AIMS: Several clinical trials have confirmed the therapeutic benefit of imipenem for treatment of lung infections. There is however no knowledge of the penetration of imipenem into the lung epithelial lining fluid (ELF), the site of action relevant for lung infections. Furthermore, although the plasma pharmacokinetics (PK) of imipenem has been widely studied, most studies have been based on selected patient groups. The aim of this analysis was to characterize imipenem plasma PK across populations and to quantify imipenem ELF penetration. METHODS: A population model for imipenem plasma PK was developed using data obtained from healthy volunteers, elderly subjects and subjects with renal impairment, in order to identify predictors for inter-individual variability (IIV) of imipenem PK. Subsequently, a clinical study which measured plasma and ELF concentrations of imipenem was included in order to quantify lung penetration. RESULTS: A two compartmental model best described the plasma PK of imipenem. Creatinine clearance and body weight were included as subject characteristics predictive for IIV on clearance. Typical estimates for clearance, central and peripheral volume, and inter-compartmental clearance were 11.5 l h(-1) , 9.37 l, 6.41 l, 13.7 l h(-1) , respectively (relative standard error (RSE) <8%). The distribution of imipenem into ELF was described using a time-independent penetration coefficient of 0.44 (RSE 14%). CONCLUSION: The identified lung penetration coefficient confirms the clinical relevance of imipenem for treatment of lung infections, while the population PK model provided insights into predictors of IIV for imipenem PK and may be of relevance to support dose optimization in various subject groups.

Disposition into Adipose Tissue Determines Accumulation and Elimination Kinetics of the Cholesteryl Ester Transfer Protein Inhibitor Anacetrapib in Mice.

The cholesteryl ester transfer protein (CETP) inhibitor anacetrapib exhibits a long terminal half-life (t½) in humans; however, the dispositional mechanisms that lead to this long t½ are still being elucidated. As it is hypothesized that disposition into adipose tissue and binding to CETP might play a role, we sought to delineate the relative importance of these factors using a preclinical animal model. A multiple-dose pharmacokinetic study was conducted in C57BL6 wild-type (WT) lean, WT diet-induced obese (DIO), natural flanking region (NFR) CETP-transgenic lean, and NFR-DIO mice. Mice were dosed orally with 10 mg/kg anacetrapib daily for 42 days. Drug concentrations in blood, brown and white adipose tissue, liver, and brain were measured up to 35 weeks postdose. During dosing, a 3- to 9-fold accumulation in 72-hour postdose blood concentrations of anacetrapib was observed. Drug concentrations in white adipose tissue accumulated ∼20- to 40-fold, whereas 10- to 17-fold accumulation occurred in brown adipose and approximately 4-fold in liver. Brain levels were very low (<0.1 µM), and a trend of accumulation was not seen. The presence of CETP as well as adiposity seems to play a role in determining the blood concentrations of anacetrapib. The highest blood concentrations were observed in NFR DIO mice, whereas the lowest concentrations were seen in WT lean mice. In adipose and liver tissue, higher concentrations were seen in DIO mice, irrespective of the presence of CETP. This finding suggests that white adipose tissue serves as a potential depot and that disposition into adipose tissue governs the long-term kinetics of anacetrapib in vivo.

Bioavailability of alendronate and vitamin D(3) in an alendronate/vitamin D(3) combination tablet.

These studies were designed to demonstrate that the alendronate (ALN) component of an ALN/vitamin D(3) combination tablet was bioequivalent to the 70-mg ALN tablet and that the pharmacokinetic parameters of vitamin D(3) were similar with or without ALN. These were open-label, randomized, 2-part, 2-period, crossover studies. In part I, participants received either a single combination tablet or ALN 70 mg. In part II, participants received either a single combination tablet or vitamin D(3) alone. Results from part I showed that the geometric mean ratio (GMR) for total urinary excretion of ALN for both studies fell within the prespecified bioequivalence bounds. Results from part II showed that the pharmacokinetic profiles of vitamin D(3) with or without ALN were also similar. The combination tablets are bioequivalent to the ALN 70-mg tablet with respect to ALN bioavailability. The bioavailability of vitamin D(3) is similar in the combination tablets and when administered alone. No serious adverse experiences were reported.

Short-acting T-type calcium channel antagonists significantly modify sleep architecture in rodents.

A novel phenyl acetamide series of short-acting T-type calcium channel antagonists has been identified and evaluated using in vitro and in vivo assays. Heterocycle substitutions of the 4-position of the phenyl acetamides afforded potent and selective antagonists that exhibited desired short plasma half-lives across preclinical species. Lead compound TTA-A8 emerged as a compound with excellent in vivo efficacy as indicated by its significant modulation of rat sleep architecture in an EEG telemetry model, favorable pharmacokinetic properties, and excellent preclinical safety. TTA-A8 recently progressed into human clinical trials, and in line with our predictions, preliminary studies (n = 12) with a 20 mg oral dose afforded a high C max of 1.82 ± 0.274 µM with an apparent terminal half-life of 3.0 ± 1.1 h.

Single-dose pharmacokinetics and pharmacodynamics of anacetrapib, a potent cholesteryl ester transfer protein (CETP) inhibitor, in healthy subjects.

AIMS: Anacetrapib is an orally active and potent inhibitor of CETP in development for the treatment of dyslipidaemia. These studies endeavoured to establish the safety, tolerability, pharmacokinetics and pharmacodynamics of rising single doses of anacetrapib, administered in fasted or fed conditions, and to preliminarily assess the effect of food, age, gender and obesity on the single-dose pharmacokinetics and pharmacodynamics of anacetrapib. METHODS: Safety, tolerability, anacetrapib concentrations and CETP activity were evaluated. RESULTS: Anacetrapib was rapidly absorbed, with peak concentrations occurring at approximately 4 h post-dose and an apparent terminal half-life ranging from approximately 9 to 62 h in the fasted state and from approximately 42 to approximately 83 h in the fed state. Plasma AUC and C(max) appeared to increase in a less than approximately dose-dependent manner in the fasted state, with an apparent plateau in absorption at higher doses. Single doses of anacetrapib markedly and dose-dependently inhibited serum CETP activity with peak effects of approximately 90% inhibition at t(max) and approximately 58% inhibition at 24 h post-dose. An E(max) model best described the plasma anacetrapib concentration vs CETP activity relationship with an EC(50) of approximately 22 nm. Food increased exposure to anacetrapib; up to approximately two-three-fold with a low-fat meal and by up to approximately six-eight fold with a high-fat meal. Anacetrapib pharmacokinetics and pharmacodynamics were similar in elderly vs young adults, women vs men, and obese vs non-obese young adults. Anacetrapib was well tolerated and was not associated with any meaningful increase in blood pressure. CONCLUSIONS: Whereas food increased exposure to anacetrapib significantly, age, gender and obese status did not meaningfully influence anacetrapib pharmacokinetics and pharmacodynamics.

Effect of the cholesteryl ester transfer protein inhibitor, anacetrapib, on lipoproteins in patients with dyslipidaemia and on 24-h ambulatory blood pressure in healthy individuals: two double-blind, randomised placebo-controlled phase I studies.

BACKGROUND: The inhibition of cholesteryl ester transfer protein (CETP) is considered a potential new mechanism for treatment of dyslipidaemia. Anacetrapib (MK-0859) is a CETP inhibitor currently under development. We aimed to assess anacetrapib's effects as monotherapy on low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) and on 24-h ambulatory blood pressure. METHODS: We did two double-blind, randomised, placebo-controlled phase I studies. In the first study, 50 patients with dyslipidaemia (LDL-C 100-190 mg/dL; 40 active, 10 placebo) aged 18-75 years received anacetrapib doses of 0, 10, 40, 150, or 300 mg orally once a day with a meal for 28 days. Standard lipid and lipoprotein monitoring, safety monitoring, and anacetrapib concentrations for pharmacokinetics were done. In the second study, 22 healthy participants aged 45-75 years received either 150 mg of anacetrapib once a day or matching placebo with a meal for 10 days in each crossover period, in a randomised sequence, with at least a 14-day washout between the treatment periods. Continuous 24-h ambulatory blood pressure monitoring was done on day -1 and day 10 of each treatment period in this study. The primary or secondary endpoints of safety and tolerability were assessed in both studies by monitoring clinical adverse experiences, physical examinations, vital signs, 12-lead electrocardiogram, and laboratory safety. Analysis was per protocol. These trials are registered with ClinicalTrials.gov, number NCT00565292 and NCT00565006. FINDINGS: In the dyslipidaemia study, one patient withdrew consent and one was excluded from the data analysis for HDL-C and LDL-C because complete pre-dose measurements were not available. Anacetrapib produced dose-dependent lipid-altering effects with peak lipid-altering effects of 129% (mean 51.1 [SD 3.8]-114.9 [7.9] mg/dL) increase in HDL-C and a 38% (138.2 [11.4]-77.6 [7.9] mg/dL) decrease in LDL-C in patients with dyslipidaemia. In the 24-h ambulatory blood pressure study in healthy individuals, least squares difference between anacetrapib and placebo groups on day 10 were 0.60 (90% CI -1.54 to 2.74; p=0.634) mm Hg for systolic blood pressure and 0.47 (90% CI -0.90 to 1.84; p=0.561) mm Hg for diastolic blood pressure. INTERPRETATION: Anacetrapib seems to exhibit HDL-C increases greater than those seen with other investigational drugs in this class and LDL-C lowering effects similar to statins. Despite greater lipid-altering effects relative to other members of this class, anacetrapib seems not to increase blood pressure, suggesting that potent CETP inhibition by itself might not lead to increased blood pressure.

Cyclobenzaprine pharmacokinetics, including the effects of age, gender, and hepatic insufficiency.

The pharmacokinetics and bioavailability of cyclobenzaprine, a widely used muscle relaxant, were investigated in four clinical studies, and the effects of age, gender, and hepatic insufficiency were characterized. Cyclobenzaprine plasma clearance was 689 ml/min, and the bioavailability of a 5 mg oral dose was 0.55. Following oral doses of 2.5 to 10 mg tid in healthy young subjects, cyclobenzaprine pharmacokinetics were linear, and plasma concentrations generally increased proportional to dose. There was about a fourfold accumulation of the drug in plasma on multiple dosing, corresponding to an effective half-life of 18 hours. Steady-state plasma concentrations of cyclobenzaprine in elderly subjects were twice as high as in young subjects following oral doses of 5 mg tid. Steady-state plasma concentration also appeared to be up to twofold higher in subjects with mild hepatic insufficiency compared to healthy controls. The magnitude of any difference in steady-state plasma concentration between males and females appears to be small relative to intersubject variability. A reduction in dose or dosing frequency should be considered in the elderly and in patients with liver disease.