Elbasvir is an investigational NS5A inhibitor with in vitro activity against multiple HCV genotypes. Antiviral activity of elbasvir was measured in replicons derived from wild-type or resistant variants of genotypes 1a, 1b, and 3. The barrier to resistance was assessed by the number of resistant colonies selected by exposure to various elbasvir concentrations. In a phase 1b dose-escalating study, virologic responses were determined in 48 noncirrhotic adult men with chronic genotype 1 or 3 infections randomized to placebo or elbasvir from 5 to 50 mg (genotype 1) or 10 to 100 mg (genotype 3) once daily for 5 days. The NS5A gene was sequenced from plasma specimens obtained before, during, and after treatment. Elbasvir suppressed the emergence of resistanceassociated variants (RAVs) in vitro in a dose-dependent manner. Variants selected by exposure to high elbasvir concentrations typically encoded multiple amino acid substitutions (most commonly involving loci 30, 31, and 93), conferring high-level elbasvir resistance. In the monotherapy study, patients with genotype 1b had greater reductions in HCV RNA levels than patients with genotype 1a at all elbasvir doses; responses in patients with genotype 3 were generally less pronounced than for genotype 1, particularly at lower elbasvir doses. M28T, Q30R, L31V, and Y93H in genotype 1a, L31V and Y93H in genotype 1b, and A30K, L31F, and Y93H in genotype 3 were the predominant RAVs selected by elbasvir monotherapy. Virologic findings in patients were consistent with the preclinical observations. NS5A-RAVs emerged most often at amino acid positions 28, 30, 31, and 93 in both the laboratory and clinical trial. (The MK-8742 P002 trial has been registered at ClinicalTrials.gov under identifier NCT01532973.)

Cholesteryl ester transfer protein (CETP) is a hydrophobic plasma protein that promotes the bidirectional transfer of cholesteryl esters (CE) and triglycerides (TG) between and among HDL particles and atherogenic apolipoprotein B-containing (ApoB-containing) lipoproteins, including the predominantly TG-rich VLDL, intermediate-density lipoprotein (IDL), and LDL particles (1-3). Genetic deficiency of CETP is associated with elevated HDL cholesterol (HDL-C) and reduced LDL-C (1), and common variants at the CETP locus are associated with HDL-C and LDL-C in inverse directions (3). Pharmacologic inhibition of CETP activity in humans raises HDL-C levels and generally reduces LDL-C levels (4-7).The mechanism by which CETP inhibition reduces LDL-C remains unknown. A study of ApoB kinetics during administration of the CETP inhibitor torcetrapib (120 mg), with or without atorvastatin (ATV), to subjects with dyslipidemia (8) suggested that in dyslipidemic subjects, torcetrapib monotherapy reduced LDL ApoB by increasing the fractional catabolic rate (FCR) and that torcetrapib administered with ATV may have reduced production of LDL ApoB. However, none of these changes were statistically significant. Thus, the study was underpowered for detecting changes in many of the ApoB kinetic parameters and led to no firm conclusions regarding the mechanisms responsible for the lowering of ApoB.BACKGROUND. Individuals treated with the cholesteryl ester transfer protein (CETP) inhibitor anacetrapib exhibit a reduction in both LDL cholesterol and apolipoprotein B (ApoB) in response to monotherapy or combination therapy with a statin. It is not clear how anacetrapib exerts these effects; therefore, the goal of this study was to determine the kinetic mechanism responsible for the reduction in LDL and ApoB in response to anacetrapib. METHODS.We performed a trial of the effects of anacetrapib on ApoB kinetics. Mildly hypercholesterolemic subjects were randomized to background treatment of either placebo (n = 10) or 20 mg atorvastatin (ATV) (n = 29) for 4 weeks. All subjects then added 100 mg anacetrapib to background treatment for 8 weeks. Following each study period, subjects underwent a metabolic study to determine the LDL-ApoB-100 and proprotein convertase subtilisin/kexin type 9 (PCSK9) production rate (PR) and fractional catabolic rate (FCR). RESULTS.Anacetrapib markedly reduced the LDL-ApoB-100 pool size (PS) in both the placebo and ATV groups. These changes in PS resulted from substantial increases in LDL-ApoB-100 FCRs in both groups. Anacetrapib had no effect on LDL-ApoB-100 PRs in either treatment group. Moreover, there were no changes in the PCSK9 PS, FCR, or PR in either group. Anacetrapib treatment was associated with considerable increases in the LDL triglyceride/cholesterol ratio and LDL size by NMR. CONCLUSION.These data indicate that anacetrapib, given alone or in combination with a statin, reduces LDL-ApoB-100 levels by increasing the rate of ApoB-100 fractional clearance. TRIAL REGISTRATION. ClinicalTrials.gov NCT00990808.

Relebactam is a novel class A and C β-lactamase inhibitor that is being developed in combination with imipenem-cilastatin for the treatment of serious infections with Gram-negative bacteria. Here we report on two phase 1 randomized, double-blind, placebo-controlled pharmacokinetics, safety, and tolerability studies of relebactam administered with or without imipenem-cilastatin to healthy participants: (i) a single-dose (25 to 1,150 mg) and multiple-dose (50 to 625 mg every 6 h [q6h] for 7 to 14 days) escalation study with men and (ii) a single-dose (125 mg) study with women and elderly individuals.

This phase I study assessed the intrapulmonary pharmacokinetic profiles of relebactam (MK-7655), a novel β-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.

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Objective Anacetrapib, an inhibitor of cholesteryl ester transfer protein (CETP) activity, increases plasma concentrations of HDL-C, apoA-I, apoA-II, and CETP. The mechanisms responsible for these treatment-related increases in apolipoproteins and plasma CETP are unknown. We performed a randomized, placebo-controlled, double-blind, fixed-sequence study to examine the effects of anacetrapib on the metabolism of HDL apoA-I and apoA-II and plasma CETP. Approach and Results Twenty-nine participants received atorvastatin 20mg/day plus placebo for four weeks, followed by atorvastatin plus anacetrapib 100 mg/day for 8 weeks (ATV-ANA). Ten participants received double placebo for four weeks followed by placebo plus anacetrapib for 8 weeks (PBO-ANA). At the end of each treatment, we examined the kinetics of HDL apoA-I, HDL apoA-II and plasma CETP after D3-leucine administration as well as 2D gel analysis of HDL subspecies. In the combined ATV-ANA and PBO-ANA groups, anacetrapib treatment increased plasma HDL-C (63.0%, P < 0.001) and apoA-I levels (29.5%, P < 0.001). These increases were associated with reductions in HDL apoA-I fractional clearance rate (FCR) (18.2%, P = 0.002) without changes in production rate (PR). Although the apoA-II levels increased by 12.6% (P < 0.001), we could not discern significant changes in either apoA-II FCR or PR. CETP levels increased 102% (P < 0.001) on anacetrapib due to a significant reduction in the FCR of CETP (57.6%, P < 0.001) with no change in CETP PR. Conclusion Anacetrapib treatment increases HDL apoA-I and CETP levels by decreasing the fractional clearance rate of each protein. Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT00990808

Cholesteryl ester transfer protein (CETP) mediates the transfer of HDL cholesteryl esters for triglyceride (TG) in VLDL/LDL. CETP inhibition, with anacetrapib, increases HDL-cholesterol, reduces LDL-cholesterol, and lowers TG levels. This study describes the mechanisms responsible for TG lowering by examining the kinetics of VLDL-TG, apoC-II, apoC-III, and apoE. Mildly hypercholesterolemic subjects were randomized to either placebo (N = 10) or atorvastatin 20 mg/qd (N = 29) for 4 weeks (period 1) followed by 8 weeks of anacetrapib, 100 mg/qd (period 2). Following each period, subjects underwent stable isotope metabolic studies to determine the fractional catabolic rates (FCRs) and production rates (PRs) of VLDL-TG and plasma apoC-II, apoC-III, and apoE. Anacetrapib reduced the VLDL-TG pool on a statin background due to an increased VLDL-TG FCR (29%; P = 0.002). Despite an increased VLDL-TG FCR following anacetrapib monotherapy (41%; P = 0.11), the VLDL-TG pool was unchanged due to an increase in the VLDL-TG PR (39%; P = 0.014). apoC-II, apoC-III, and apoE pool sizes increased following anacetrapib; however, the mechanisms responsible for these changes differed by treatment group. Anacetrapib increased the VLDL-TG FCR by enhancing the lipolytic potential of VLDL, which lowered the VLDL-TG pool on atorvastatin background. There was no change in the VLDL-TG pool in subjects treated with anacetrapib monotherapy due to an accompanying increase in the VLDL-TG PR.

RATIONALE Apolipoprotein(a) [apo(a)] is the defining protein component of lipoprotein(a) [Lp(a)], an independent risk factor for cardiovascular disease. The regulation of Lp(a) levels in blood is poorly understood in part due to technical challenges in measuring Lp(a) kinetics. Improvements in the ability to readily and reliably measure the kinetics of apo (a) using a stable isotope labeled tracer is expected to facilitate studies of the role of Lp(a) in cardiovascular disease. Since investigators typically determine the isotopic labeling of protein-bound amino acids following acid-catalyzed hydrolysis of a protein of interest [e.g., apo(a)], studies of protein synthesis require extensive protein purification which limits throughput and often requires large sample volumes. We aimed to develop a rapid and efficient method for studying apo(a) kinetics that is suitable for use in studies involving human subjects. METHODS Microfluidic device and tandem mass spectrometry were used to quantify the incorporation of [2H3]-leucine tracer into protein-derived peptides. RESULTS We demonstrated that it is feasible to quantify the incorporation of [2H3]-leucine tracer into a proteolytic peptide from the non-kringle repeat region of apo(a) in human subjects. Specific attention was directed toward optimizing the multiple reaction monitoring (MRM) transitions, mass spectrometer settings, and chromatography (i.e., critical parameters that affect the sensitivity and reproducibility of isotopic enrichment measurements). The results demonstrated significant advantages with the use of a microfluidic device technology for studying apo(a) kinetics, including enhanced sensitivity relative to conventional micro-flow chromatography, a virtually drift-free elution profile, and a stable and robust electrospray. CONCLUSIONS The technological advances described herein enabled the implementation of a novel method for studying the kinetics of apo(a) in human subjects infused with [2H3]-leucine.