Patricia Jumes

Anacetrapib lowers LDL by increasing ApoB clearance in mildly hypercholesterolemic subjects

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. FUNDING Merck & Co. Inc., Kenilworth, New Jersey, USA. Additional support for instrumentation was obtained from the National Center for Advancing Translational Sciences (UL1TR000003 and UL1TR000040).

Susceptibilities of Genotype 1a, 1b, and 3 Hepatitis C Virus Variants to the NS5A Inhibitor Elbasvir

ABSTRACT 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 resistance-associated 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.)

Measurement of apo(a) kinetics in human subjects using a microfluidic device with tandem mass spectrometry

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 [(2)H3]-leucine tracer into protein-derived peptides. RESULTS We demonstrated that it is feasible to quantify the incorporation of [(2)H3]-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 [(2)H3]-leucine.

Effects of extended release niacin/laropiprant, laropiprant, extended release niacin and placebo on platelet aggregation and bleeding time in healthy subjects

Laropiprant (LRPT) has been shown to reduce flushing symptoms induced by niacin and has been combined with niacin for treatment of dyslipidemia. LRPT, a potent PGD2 receptor (DP1) antagonist that also has modest activity at the thromboxane receptor (TP), may have the potential to alter platelet function either by enhancing platelet reactivity through DP1 antagonism or by inhibiting platelet aggregation through TP antagonism. Studies of platelet aggregation ex vivo and bleeding time have shown that LRPT, at therapeutic doses, does not produce clinically meaningful alterations in platelet function. The present study was conducted to assess platelet reactivity to LRPT using methods that increase the sensitivity to detect changes in platelet responsiveness to collagen and ADP. The responsiveness of platelets was quantified by determining the EC50 of collagen to induce platelet aggregation ex vivo. At 24 hours post-dose on Day 7, the responsiveness of platelets to collagen-induced aggregation was similar following daily treatment with extended-release niacin (ERN) 2 g/LRPT 40 mg or ERN 2 g. At 2 hours post-dose on Day 7, the EC50 for collagen-induced platelet aggregation was approximately two-fold higher in the presence of LRPT, consistent with a small, transient inhibition of platelet responsiveness to collagen. There was no clinical difference between treatments for bleeding time, suggesting that this small effect on collagen EC50 does not result in a clinically meaningful alteration of platelet function in vivo. The results of this highly sensitive method demonstrate that LRPT does not enhance platelet reactivity when given alone or with ERN.

Cholesteryl Ester Transfer Protein Inhibition With Anacetrapib Decreases Fractional Clearance Rates of High-Density Lipoprotein Apolipoprotein A-I and Plasma Cholesteryl Ester Transfer Protein

Objective—Anacetrapib (ANA), an inhibitor of cholesteryl ester transfer protein (CETP) activity, increases plasma concentrations of high-density lipoprotein cholesterol (HDL-C), apolipoprotein A-I (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 (PBO)-controlled, double-blind, fixed-sequence study to examine the effects of ANA on the metabolism of HDL apoA-I and apoA-II and plasma CETP. Approach and Results—Twenty-nine participants received atorvastatin (ATV) 20 mg/d plus PBO for 4 weeks, followed by ATV plus ANA 100 mg/d for 8 weeks (ATV-ANA). Ten participants received double PBO for 4 weeks followed by PBO plus ANA 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, ANA 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 (18.2%; P=0.002) without changes in production rate. Although the apoA-II levels increased by 12.6% (P<0.001), we could not discern significant changes in either apoA-II fractional clearance rate or production rate. CETP levels increased 102% (P<0.001) on ANA because of a significant reduction in the fractional clearance rate of CETP (57.6%, P<0.001) with no change in CETP production rate. Conclusions—ANA treatment increases HDL apoA-I and CETP levels by decreasing the fractional clearance rate of each protein.

CETP (Cholesteryl Ester Transfer Protein) Inhibition With Anacetrapib Decreases Production of Lipoprotein(a) in Mildly Hypercholesterolemic Subjects

Objective— Lp(a) [lipoprotein (a)] is composed of apoB (apolipoprotein B) and apo(a) [apolipoprotein (a)] and is an independent risk factor for cardiovascular disease and aortic stenosis. In clinical trials, anacetrapib, a CETP (cholesteryl ester transfer protein) inhibitor, causes significant reductions in plasma Lp(a) levels. We conducted an exploratory study to examine the mechanism for Lp(a) lowering by anacetrapib. Approach and Results— We enrolled 39 participants in a fixed-sequence, double-blind study of the effects of anacetrapib on the metabolism of apoB and high-density lipoproteins. Twenty-nine patients were randomized to atorvastatin 20 mg/d, plus placebo for 4 weeks, and then atorvastatin plus anacetrapib (100 mg/d) for 8 weeks. The other 10 subjects were randomized to double placebo for 4 weeks followed by placebo plus anacetrapib for 8 weeks. We examined the mechanisms of Lp(a) lowering in a subset of 12 subjects having both Lp(a) levels >20 nmol/L and more than a 15% reduction in Lp(a) by the end of anacetrapib treatment. We performed stable isotope kinetic studies using 2H3-leucine at the end of each treatment to measure apo(a) fractional catabolic rate and production rate. Median baseline Lp(a) levels were 21.5 nmol/L (interquartile range, 9.9–108.1 nmol/L) in the complete cohort (39 subjects) and 52.9 nmol/L (interquartile range, 38.4–121.3 nmol/L) in the subset selected for kinetic studies. Anacetrapib treatment lowered Lp(a) by 34.1% (P⩽0.001) and 39.6% in the complete and subset cohort, respectively. The decreases in Lp(a) levels were because of a 41% reduction in the apo(a) production rate, with no effects on apo(a) fractional catabolic rate. Conclusions— Anacetrapib reduces Lp(a) levels by decreasing its production. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00990808.

Effects of CETP Inhibition with Anacetrapib on Metabolism of VLDL TG and Plasma Apolipoproteins C-II, C-III, and E

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.

Safety and efficacy of the fixed-dose combination regimen of MK-3682/grazoprevir/ruzasvir in cirrhotic or non-cirrhotic patients with chronic HCV GT1 infection who previously failed a direct-acting antiviral regimen (C-SURGE)

193 • HCV NS3/4A protease inhibitor • 50 mg per tablet • HCV NS5A next-generation inhibitor • 30 mg per tablet • HCV NS5B polymerase nucleotide inhibitor • 225 mg per tablet Ruzasvir (MK-8408) Grazoprevir (MK-5172) MK-3682 C-SURGE: MK-3682/Grazoprevir/Ruzasvir • MK3 is a three-drug regimen formulated into a fixed-dose combination tablet. The regimen is given as two tablets, once-daily, without regard to food • Triplet also called MK-3682B Lawitz E, et al. 67th AASLD; Boston, MA; November 11-15, 2016; Abst. 110. C-SURGE: Study Design • This multicenter, open-label trial randomized 94 HCV GT1infected patients who relapsed after a regimen of LDV/SOF or EBR/GZR (randomized 1:1; stratified by GT1a/1b and cirrhosis) MK-3682 + GZR + RZR + RBV† (16 weeks), n=45 MK-3682 + GZR + RZR (24 weeks), n=49 TW8 TW16 TW24 FW12 D1 SVR12 1° Endpoint Wyles D, et al. 67th AASLD; Boston, MA; November 11-15, 2016; Abst. 193. C-SURGE: Study Demographics Demographics 16 Weeks + RBV, n=44* 24 Weeks without RBV, n=49 Overall GT1 N=93* Male, n (%) 37 (84) 43 (88) 80 (86) Age, median years, (range) 61.0 (33 to 70) 60.0 (25 to 71) 60.0 (25 to 71) Race, White, n (%) 31 (71) 37 (76) 68 (73) HCV Genotype 1a, n (%) 40 (90) 40 (82) 80 (86) Non-cirrhotic, n (%) Cirrhotic, n (%) 25 (57) 19 (43) 29 (6) 20 (41) 54 (58) 39 (42) NS5A RAVs at baseline, n (%)† NS3 RAVs at baseline, n (%)‡ 32 (79) 25 (57) 46 (94) 35 (71) 78 (84) 60 (65) Baseline HCV RNA >800,00 IU/mL, n (%) 35 (80) 44 (90) 79 (85) Median baseline HCV RNA (log10 IU/mL) 6.5 6.4 6.5 Previously failed: 12 – 24 weeks of LDV/SOF 8 weeks of LDV/SOF 12 weeks of EBR/GZR 26 (59) 9 (21) 9 (21) 31 (63) 5 (10) 13 (27) 57 (61) 14 (15) 22 (24) RAVs detected by next generation sequencing performed with a 15% sensitivity threshold. * Does not include 1 patient in the 16 week + RBV arm who withdrew prior to beginning treatment. Cirrhosis = Liver biopsy at any time showing cirrhosis, Fibroscan result of >12,5kPa within 12 months of enrollment, or Fibrotest >0.75 and APRI >2 at time of enrollment † NS5A RAVs = any change from wild-type at 4 positions (28, 30, 31, or 93) ‡ NS3 RAVs = any change from wild-type at 14 positions ( 36, 54, 55, 56, 80, 107, 122, 132, 155, 156, 158, 168, 170, or 175) Wyles D, et al. 67th AASLD; Boston, MA; November 11-15, 2016; Abst. 193. C-SURGE: Results – Efficacy (Full Analysis Set) Wyles D, et al. 67th AASLD; Boston, MA; November 11-15, 2016; Abst. 193. 91 98 98 92 100 100 0 10 20 30 40 50 60 70 80 90 100 TW4 SVR4 SVR8 Pe rc en t o f P at ie nt s w ith H C V R N A <1 5 IU /m L 16 Weeks + RBV 24 Weeks without RBV C-SURGE: Efficacy (mFAS*) TW =treatment week; SVR4 = % of patients with HCV RNA <15 IU/mL at 4 weeks after end of treatment *Excludes 1 patient from the 16-week + RBV arm who withdrew after receiving 3 doses of study medication 93 100 100 92 100 100 0 10 20 30 40 50 60 70 80 90 100 TW4 SVR4 SVR8 Pe rc en ta ge o f P at ie nt s w ith H C V R N A <1 5 IU /m L 16 Weeks + RBV 24 Weeks without RBV 40 43 45 49 43 43 38 38 43 43 30 30 Wyles D, et al. 67th AASLD; Boston, MA; November 11-15, 2016; Abst. 193. C-SURGE: No Impact of Baseline NS5A or NS3 RAVs on SVR4 (Resistance Analysis Population) 100% 100% 100% 100% 100% 100% 100% 100% 0% 20% 40% 60% 80% 100% 16 Weeks + RBV 24 Weeks 16 Weeks + RBV 24 Weeks SV R 4 Patients without RAVs Patients with RAVs 35 35 3 3 28 28 10 10 31 31 12 12 24 24 19 19 Wyles D, et al. 67th AASLD; Boston, MA; November 11-15, 2016; Abst. 193. No RAVS 12/43 28% RAVs 31/43 72% No RAVS 19/43 44% RAVs 24/43 56% No RAVS 10/38 26% RAVs 28/38 74% PR EV A LE N C E NS5A NS3 16 Weeks + RBV 16 Weeks + RBV 24 Weeks 24 Weeks

Pharmacokinetics, Safety, and Tolerability of Phentermine in Healthy Participants Receiving Taranabant, a Novel Cannabinoid‐1 Receptor (CB1R) Inverse Agonist

This study assessed the potential pharmacokinetic interaction and safety/tolerability of taranabant and phentermine coadministration. This was a randomized, double‐blind, 3‐panel, fixed‐sequence study in healthy participants. Panels A, B, and C evaluated the safety/tolerability of phentermine 15 mg coadministered with taranabant 0.5, 1, and 2 mg for 7 days (panel A) and 28 days (panels B and C). In panels A and C, phentermine 15 mg was administered both with (7 days, panel A; 28 days, panel C) and without (7 days) taranabant 0.5 mg or 2 mg to evaluate pharmacokinetics. The primary endpoint was phentermine AUC0–24 h in panels A and C. Secondary endpoints were changes from baseline in blood pressure and heart rate for all panels. The geometric mean ratios and 90% confidence intervals for phentermine AUC0–24 h in the presence/absence of taranabant 0.5 mg and 2 mg were 1.08 (0.99, 1.17) and 1.04 (0.98, 1.10), respectively. No significant differences in blood pressure and heart rate were observed with any treatment versus placebo. Coadministration of taranabant 0.5 mg, 1 mg, and 2 mg with phentermine was well tolerated with no pharmacokinetic interaction and did not result in meaningful changes in blood pressure or heart rate versus placebo.

Grazoprevir, Ruzasvir, and Uprifosbuvir for HCV After NS5A Treatment Failure

People with hepatitis C virus (HCV) infection who have failed treatment with an all‐oral regimen represent a challenging treatment population. The present studies evaluated the safety and efficacy of grazoprevir, ruzasvir, and uprifosbuvir, with or without ribavirin, in participants who had failed an NS5A inhibitor‐containing regimen. C‐SURGE (PN‐3682‐021) and C‐CREST Part C (PN‐3682‐011 and ‐012) were open‐label, multicenter studies. Participants who had previously relapsed following an NS5A inhibitor–containing all‐oral regimen were retreated with grazoprevir 100 mg, ruzasvir 60 mg, and uprifosbuvir 450 mg alone for 24 weeks or with ribavirin for 16 weeks. The primary efficacy endpoint was sustained virologic response (HCV RNA below the limit of quantitation [<15 IU/mL]) 12 weeks after treatment completion (SVR12). In C‐SURGE, SVR12 was achieved by 49/49 (100%) and 43/44 (98%) genotype (GT)1 participants in the 24‐week no ribavirin arm and the 16‐week plus ribavirin arm (lost to follow‐up, n = 1), respectively. In C‐CREST Part C, SVR12 was achieved by 23/24 (96%) participants treated for 16 weeks with ribavirin (GT1, 2/2 [100%]; GT2, 13/14 [93%]; GT3, 8/8 [100%]). One participant with GT2 infection discontinued study medication after a single dose of grazoprevir, ruzasvir, and uprifosbuvir plus ribavirin due to serious adverse events of vomiting and tachycardia. The presence of baseline resistance‐associated substitutions had no impact on SVR12. No participant who completed treatment in either study experienced virologic failure. Conclusion: Grazoprevir, ruzasvir, and uprifosbuvir, with or without ribavirin, for 16 or 24 weeks was safe and highly effective in participants with HCV infection who had previously failed NS5A inhibitor–containing therapy. (Hepatology 2017;66:1794–1804)