Safety, pharmacokinetics, and antiviral activity of the capsid inhibitor AB-506 from Phase 1 studies in healthy subjects and those with hepatitis B.

AB-506 is a potent, pan-genotypic small molecule capsid inhibitor that inhibits hepatitis B virus (HBV) pregenomic RNA encapsidation. We assessed the safety, pharmacokinetics, and antiviral activity of AB-506 in two randomized, double-blinded Phase 1 studies in healthy subjects (HS) and subjects with chronic HBV infection (CHB). Single ascending and multiple doses of AB-506 or placebo (30-1000 mg or 400 mg daily for 10 days) were assessed in HS. AB-506 or placebo was assessed at either 160 mg or 400 mg daily for 28 days in subjects with CHB. A second follow-up study examined AB-506 or placebo at 400 mg daily for 28 days in 14 Caucasian and 14 East-Asian HS. Twenty-eight days of AB-506 at 160 mg and 400 mg produced mean HBV-DNA declines from baseline of 2.1 log10 IU/ml and 2.8 log10 IU/ml, respectively. Four subjects with CHB (all Asian) had Grade 4 alanine aminotransferase (ALT) elevations (2 at each dose) as HBV DNA was declining; three events led to treatment discontinuation. In the second follow-up study, 2 Asian HS had serious transaminitis events leading to treatment and study termination. No subjects had bilirubin elevations or signs of hepatic decompensation. Conclusion: AB-506 demonstrated mean HBV-DNA declines of >2 log10 ; however, transient but severe ALT flares were observed in 4 Asian subjects with CHB. In the follow-up study in HS, 2 additional Asian HS had Grade 4 flares, suggesting that AB-506 hepatotoxicity contributed to the ALT elevations. The AB-506 development program was terminated because of these findings.

Population Pharmacokinetic Analysis of Asunaprevir in Subjects with Hepatitis C Virus Infection.

INTRODUCTION: Asunaprevir (ASV) is a potent, pangenotypic, twice-daily hepatitis C virus (HCV) NS3 inhibitor indicated for the treatment of chronic HCV infection. METHODS: A population pharmacokinetic (PPK) model was developed using pooled ASV concentration data from 1239 HCV-infected subjects who received ASV either as part of the DUAL regimen with daclatasvir or as part of the QUAD regimen with daclatasvir and peg-interferon/ribavirin. RESULTS: A two-compartment model with first-order elimination from the central compartment, an induction effect on clearance, and an absorption model consisted of zero-order release followed by first-order absorption adequately described ASV PK after oral administration. A typical value for ASV clearance (CL/F) was 50.8 L/h, increasing by 43% after 2 days to a CL/F of 72.5 L/h at steady-state, likely due to auto-induction of cytochrome P450 3A4 (CYP3A4). Factors indicative of hepatic function were identified as key influential covariates on ASV exposures. Subjects with cirrhosis had an 84% increase in ASV area under the concentration time curve (AUC) and subjects with baseline aspartate aminotransferase (AST) above 78 IU/L had a 58% increase in area under the concentration time curve (AUC). Asians subjects had a 46% higher steady-state AUC relative to White/Caucasian subjects. Other significant covariates were formulation, age, and gender. CONCLUSION: The current PPK model provided a parsimonious description of ASV concentration data in HCV-infected subjects. Key covariates identified in the model help explain the observed variability in ASV exposures and may guide clinical use of the drug. FUNDING: Bristol-Myers Squibb.

Daclatasvir: A Review of Preclinical and Clinical Pharmacokinetics.

Daclatasvir is a first-in-class, highly selective, hepatitis C virus, non-structural protein 5a polymerase replication complex inhibitor with picomolar potency and broad genotypic coverage in vitro. Daclatasvir undergoes rapid absorption, with a time to reach maximum plasma concentration of 1-2 h and an elimination half-life of ~ 10 to 14 h observed in single-ascending dose studies. Steady state was achieved by day 4 in multiple-ascending dose studies. Daclatasvir can be administered without regard to food or pH modifiers. Daclatasvir exposure is similar between healthy subjects and subjects infected with hepatitis C virus. Intrinsic factors such as age, race, or sex do not impact daclatasvir exposure. No dose adjustment is necessary for patients with any degree of hepatic or renal impairment. Daclatasvir has low-to-moderate clearance with the predominant route of elimination via cytochrome P450 3A4-mediated metabolism and P-glycoprotein excretion and intestinal secretion. Renal clearance is a minor route of elimination for daclatasvir. As a result, the dose of daclatasvir should be reduced from 60 to 30 mg once daily when co-administered with strong inhibitors of cytochrome P450 3A4. No dose adjustment is required when daclatasvir is co-administered with moderate inhibitors of cytochrome P450 3A4. The dose of daclatasvir should be increased from 60 to 90 mg once daily when co-administered with moderate inducers of cytochrome P450 3A4. Co-administration of daclatasvir with strong inducers of cytochrome P450 3A4 is contraindicated. Concurrent medications with inhibitory effects on P-glycoprotein without concurrent inhibition of cytochrome P450 3A4 are unlikely to cause marked changes in daclatasvir exposure, as the clearance of daclatasvir is through both cytochrome P450 3A4 and P-glycoprotein. The potential for daclatasvir to affect the pharmacokinetics of concomitantly administered drugs that are substrates of the cytochrome P450 enzyme system is low. In vitro, daclatasvir is a weak-to-moderate inhibitor of transporters including organic cation transporter 1, P-glycoprotein, organic transporting polypeptide 1B1, organic transporting polypeptide 1B3, and breast cancer resistance protein, although in clinical studies, daclatasvir has not altered the pharmacokinetics of concomitantly administered drugs that are substrates of these transporters to an appreciable degree, except for rosuvastatin. In summary, daclatasvir is a hepatitis C virus, non-structural protein 5a-selective inhibitor with a well-characterized pharmacokinetic profile that forms part of potent and well-tolerated all-oral treatment regimens for chronic hepatitis C virus infection.

Effects of a Fixed-Dose Co-Formulation of Daclatasvir, Asunaprevir, and Beclabuvir on the Pharmacokinetics of a Cocktail of Cytochrome P450 and Drug Transporter Substrates in Healthy Subjects.

BACKGROUND: A fixed-dose combination of daclatasvir (DCV; hepatitis C virus NS5A inhibitor), asunaprevir (ASV; non-structural protein 3 inhibitor), and beclabuvir (BCV; non-structural protein 5B inhibitor) is approved in Japan for hepatitis C virus genotype 1. OBJECTIVE: The objective of this study was to assess the combination's drug-drug interaction potential in vivo using a validated cocktail of eight cytochrome P450 (CYP) and transporter probes. METHODS: We conducted an open-label single-sequence study in healthy adults (n = 20) given single-dose caffeine (CYP1A2 substrate), metoprolol (CYP2D6), flurbiprofen (CYP2C9), montelukast (CYP2C8), omeprazole (CYP2C19), midazolam (CYP3A4), digoxin (P-glycoprotein), and pravastatin (organic anion-transporting polypeptide), alone or with steady-state twice-daily DCV/ASV/BCV 30/200/75 mg (with or without additional BCV 75 mg to adjust for higher exposure in hepatitis C virus infection). RESULTS: Daclatasvir/asunaprevir/beclabuvir did not affect CYP1A2, CYP2C8, or CYP2C9; the probe maximum observed concentration and area under the concentration-time curve extrapolated to infinite time geometric mean ratios and 90% confidence intervals were all within the 0.8-1.25 bioequivalence range. Beclabuvir showed moderate dose-dependent CYP2C19 induction; omeprazole maximum observed concentration and area under the concentration-time curve from 0 to the last quantifiable concentration were lower with additional BCV [geometric mean ratio 0.36 (90% confidence interval 0.23-0.55) and 0.34 (0.25-0.46), respectively] than without [0.57 (0.42-0.78), 0.48 (0.39-0.59)]. Weak-to-moderate CYP3A4 induction was observed, plus weak CYP2D6, P-glycoprotein, and organic anion-transporting polypeptide inhibition [maximum observed concentration and area under the concentration-time curve extrapolated to infinite time without additional BCV: midazolam 0.57 (0.50-0.65), 0.53 (0.47-0.60); metoprolol 1.40 (1.20-1.64), 1.71 (1.49-1.97); digoxin 1.23 (1.12-1.35), 1.23 (1.17-1.29); pravastatin 2.01 (1.63-2.47), 1.68 (1.43-1.97)]. CONCLUSIONS: No dose adjustments with DCV/ASV/BCV are indicated for CYP1A2, CYP2C8, CYP2C9, or P-glycoprotein substrates. CYP3A4, CYP2D6, and OATP substrates should be co-administered with caution. Co-administration with agents solely metabolized by CYP2C19 is not recommended.

Pharmacokinetics and Pharmacodynamics of Atazanavir in HIV-1-Infected Children Treated With Atazanavir Powder and Ritonavir: Combined Analysis of the PRINCE-1 and -2 Studies.

BACKGROUND: Two clinical studies (PRINCE-1 and -2) in HIV-1-infected children assessed the safety, efficacy and pharmacokinetics of dual nucleos(t)ide reverse transcriptase inhibitor background therapy plus once-daily atazanavir (ATV) powder formulation boosted with ritonavir (ATV + RTV). Here, we present a combined analysis of ATV pharmacokinetics and pharmacodynamics across these studies. METHODS: Intensive 24-hour pharmacokinetic profiles at steady state compared ATV exposures (area under the concentration-time curve in one dosing interval) in 5 ATV + RTV baseline weight-band dosing categories, with historic data in adults receiving ATV + RTV 300/100 mg capsules. Repeated ATV Ctrough measurements over 48 weeks explored relationships between ATV composite Ctrough quartiles (CCQs) with virologic efficacy and key safety parameters. RESULTS: Of 146 children included in this combined analysis, 49.3% were male, 56.8% were Black/African American and 62.3% were antiretroviral experienced. Proportions with HIV-1 RNA <50 copies/mL at week 48 were 13/32, 24/32, 19/32 and 13/28 in the lowest through highest ATV CCQs, respectively. Mean changes from baseline in total bilirubin at week 48 were +0.3, +0.5, +0.6 and +1.0 mg/dL in the lowest through highest ATV CCQs, respectively. Corresponding proportions with adverse events of hyperbilirubinemia by week 48 were 1/36, 4/36, 5/36 and 13/35, respectively. Changes from baseline in total amylase or electrocardiogram parameters and adverse events of diarrhea did not vary by ATV CCQs. CONCLUSIONS: Weight-band dosing of ATV + RTV plus optimized dual nucleos(t)ide reverse transcriptase inhibitors in young HIV-1-infected children achieved similar ATV exposure to that in adults; no unexpected safety findings occurred, and with the exception of lower virologic suppression in the lowest ATV CCQ, there was no apparent trend in virologic suppression across ATV CCQs.

Asunaprevir: An HCV Protease Inhibitor With Preferential Liver Distribution.

Asunaprevir is an inhibitor of the hepatitis C virus (HCV) NS3/4A protease, demonstrating efficacy in clinical studies in patients infected with HCV genotype 1 or 4, with either peginterferon/ribavirin or combinations of direct-acting antivirals. Because of preferential distribution of asunaprevir to the liver via organic anion-transporting polypeptide (OATP)-mediated transport, asunaprevir demonstrates high apparent oral clearance and very low plasma concentrations. Asunaprevir plasma concentrations are markedly increased by single-dose rifampin (an OATP inhibitor) and in subjects with moderate to severe hepatic impairment. In addition, modestly higher plasma concentrations of asunaprevir have been noted in subjects infected with HCV relative to healthy subjects and in Asian subjects relative to whites. At the marketed dose, infrequent hepatic transaminase abnormalities were poorly predicted by plasma concentrations. For a compound with these characteristics, hepatic concentrations may have provided an improved understanding of the in vivo pharmacokinetic and pharmacodynamic data to support decision making during development.

Efficacy and safety of daclatasvir plus pegylated-interferon alfa 2a and ribavirin in previously untreated HCV subjects coinfected with HIV and HCV genotype-1: a Phase III, open-label study.

BACKGROUND: Daclatasvir (DCV) is a potent, pangenotypic, hepatitis C virus (HCV) non-structural protein 5A inhibitor with low potential for drug interactions with antiretroviral therapy (ART). We evaluated the safety and efficacy of DCV plus peginterferon alfa-2a/ribavirin (PegIFN/RBV) in HIV-1/HCV genotype-1-coinfected patients. METHODS: AI444043 (NCT01471574), an open-label, Phase III, single-arm, response-guided treatment (RGT) study included 301 patients. They received DCV doses of 30, 60 or 90 mg once daily (depending on concomitant ART), plus weight-based RBV (<75 kg, 1000 mg/day; or ≥75 kg, 1200 mg/day), and once-weekly PegIFN 180 µg, for 24 weeks. If required by RGT, PegIFN/RBV without DCV was extended for an additional 24 weeks of therapy. The primary endpoint was the proportion of patients with sustained virologic response at post-treatment Week 12 (SVR12). RESULTS: Overall, 224 (74%) patients achieved SVR12 and the lower bound of the 95% confidence interval was higher than the historic SVR rate with PegIFN/RBV alone (70 vs. 29%). Most common adverse events (AEs) were fatigue, neutropenia, anemia, asthenia and headache. On-treatment serious AEs occurred in 24/301 (8%) patients; 18/301 (6%) discontinued treatment due to AE. CONCLUSIONS: DCV + PegIFN/RBV led to sustained HCV virologic response in the majority of HIV-1-HCV-coinfected patients, regardless of concomitant ART. HIV control was not compromised and no new safety signals were identified. This study supports DCV use in HIV-1-HCV-coinfected patients, while allowing the vast majority of patients to remain on their existing ART regimen.

Population Pharmacokinetic Analysis of Daclatasvir in Subjects with Chronic Hepatitis C Virus Infection.

BACKGROUND AND OBJECTIVE: Daclatasvir is a potent, pangenotypic once-daily hepatitis C virus (HCV) NS5A inhibitor that is approved for the treatment of chronic HCV infection. The objective of this analysis was to characterize the pharmacokinetics of daclatasvir in subjects with chronic HCV infection. METHODS: A population pharmacokinetic (PPK) model was developed to evaluate effects of covariates on daclatasvir pharmacokinetics in subjects with chronic HCV infection (n = 2149 from 11 studies). All significant demographic, laboratory, prognostic and treatment covariates (p < 0.05) from univariate screening were included in the full model. The final model was reached by backward elimination (p < 0.001) and simulations were performed to further evaluate the effects of covariates on daclatasvir exposures. The plasma pharmacokinetics of daclatasvir was described by a two-compartment model with linear elimination. Absorption was modeled as a zero-order release followed by a first-order absorption into the central compartment. RESULTS: The typical value of apparent clearance (CL/F) was 5.7 L/h (1.58% relative standard error [RSE]) and of apparent volume of the central compartment (V c/F) was 58.6 L (2.00% RSE). Modest inter-individual variability was estimated for CL/F (35.1%) and V c/F (29.5%). Statistically significant covariates in the final model were sex, race, virus genotype, baseline creatinine clearance, and alanine aminotransferase (ALT) on CL/F and sex, race, and body weight on V c/F. Covariate effects demonstrated a 30% higher area under the plasma concentration-time curve at steady state (AUCss) in female subjects; effects of all other covariates were <16%. CONCLUSIONS: The model adequately described the daclatasvir pharmacokinetics and estimated relatively small covariate effects. Considering the exposure range for the therapeutic dose of daclatasvir 60 mg once daily and the favorable safety profile, the small difference in exposures due to these covariates is not considered clinically relevant.

A Review of Daclatasvir Drug-Drug Interactions.

The treatment of hepatitis C virus (HCV) infection has been revolutionized in recent years by the development of direct-acting antiviral regimens that do not contain peginterferon (pegIFN) and/or ribavirin (RBV). While direct-acting antiviral-based regimens have been shown to be greatly superior to pegIFN/RBV-based regimens in terms of efficacy and safety, they have a greater susceptibility to drug-drug interactions (DDIs). Daclatasvir (DCV)-the benchmark pangenotypic nonstructural protein 5A inhibitor-has been shown to be efficacious and generally well tolerated in partnership with other HCV direct-acting antivirals, including sofosbuvir, asunaprevir (ASV), and ASV plus beclabuvir. DCV may be the object of a DDI via the induction or inhibition of cytochrome P450 (CYP) 3A4 and/or P-glycoprotein (P-gp) by the concomitant medication, or the precipitant of a DDI via DCV-based induction/inhibition of CYP 3A4 or inhibition of P-gp, organic anion transporting polypeptide 1B1/B3, and/or breast cancer resistance protein. This article presents an overview of the drug interaction studies conducted during the clinical development of DCV, the findings of these studies that led to the guidance on concomitant medication use and dosage along with any required DCV dose modifications, and the use of the known metabolic pathway of DCV to guide concomitant dosing where direct drug-drug studies have not been conducted. The robust characterization of the DCV clinical pharmacology program has demonstrated that DCV has few or no clinically relevant DDIs with medications with which it is likely to be co-administered, and the majority of DDIs that do occur can be predicted and easily managed. FUNDING: Bristol-Myers Squibb.

No clinically significant pharmacokinetic interactions between dolutegravir and daclatasvir in healthy adult subjects.

BACKGROUND: Daclatasvir (DCV) is an NS5A replication complex inhibitor recently approved for chronic hepatitis C virus treatment. METHODS: To assess drug interactions between the HIV integrase strand transfer inhibitor dolutegravir (DTG) and DCV, subjects were randomized into 1 of 2 sequences in an open-label, 3-period, crossover study. Subjects received either DTG 50 mg once daily or DCV 60 mg once daily for 5 days in periods 1 and 2 and DTG 50 mg plus DCV 60 mg once daily for 5 days in period 3, with no washout between periods 2 and 3. Between periods 1 and 2, there was a washout period of at least 7 days. RESULTS: The geometric least-squares mean ratios (90 % confidence intervals) of DCV area under the concentration-time curve over a dosing interval (AUC0-τ), maximum observed concentration (Cmax), and concentration at the end of the dosing interval (Cτ) were 0.978 (0.831-1.15), 1.03 (0.843-1.25), and 1.06 (0.876-1.29), respectively, when DCV was administered with DTG compared with DCV alone. Compared with DTG alone, coadministration of DTG with DCV increased DTG AUC0-τ, Cmax, and Cτ by approximately 33, 29, and 45 %, respectively. CONCLUSIONS: DCV plasma exposure was not meaningfully affected by DTG. Coadministration of DTG with DCV resulted in slight increases in DTG AUC0-τ, Cmax, and Cτ. Accumulated safety and tolerability data in humans receiving DTG to date suggests this effect is not considered clinically significant. DTG and DCV can be coadministered without dose adjustment. TRIAL REGISTRATION: Registered on March 6, 2014 with ClinicalTrials.gov; registration number: NCT02082808 and as Study ID: 201102 on the ViiV Clinical Study Registry.

Characterization of ADME properties of [(14)C]asunaprevir (BMS-650032) in humans.

1. Asunaprevir (ASV, BMS-650032), a highly selective and potent NS3 protease inhibitor, is currently under development for the treatment of chronic hepatic C virus infection. This study describes in vivo biotransformation in humans and the identification of metabolic enzymes of ASV. 2. Following a single oral dose of [(14)C]ASV to humans, the majority of radioactivity (>73% of the dose) was excreted in feces with <1% of the dose recovered in urine. Drug-related radioactivity readily appeared in circulation and the plasma radioactivity was mainly attributed to ASV. A few minor metabolites were observed in human plasma and are not expected to contribute to the pharmacological activity because of low levels. The area under the curve (AUC) values of each circulating metabolite in humans were well below their levels in animals used in the long-term toxicological studies. In bile and feces, intact ASV was a prominent radioactive peak suggesting that both metabolism and direct excretion played important roles in ASV clearance. 3. The primary metabolic pathways of ASV were hydroxylation, sulfonamide hydrolysis and the loss of isoquinoline. In vitro studies with human cDNA expressed CYP enzymes and with human liver microsomes (HLM) in the presence of selective chemical inhibitors demonstrated that ASV was primarily catalyzed by CYP3A4 and CYP3A5.

A highly sensitive and selective LC-MS/MS method to quantify asunaprevir, an HCV NS3 protease inhibitor, in human plasma in support of pharmacokinetic studies.

Asunaprevir (BMS-650032) is a selective hepatitis C virus (HCV) NS3 protease inhibitor with potent activity against HCV genotypes 1, 4, 5 and 6. It has been developed in conjunction with direct-acting antiviral agents, in interferon- and ribavirin-free regimen, to improve existing therapies for HCV infection. To support the pharmacokinetic analyses in asunaprevir clinical studies, we have developed and validated a highly sensitive and robust LC-MS/MS method to quantify asunaprevir in human EDTA plasma with an LLOQ of 0.05ng/mL, which was a 20-fold sensitivity improvement over a previously reported assay for asunaprevir. A deuterated labeled [D9]-asunaprevir was used as the internal standard (IS). The analyte and the IS were extracted using a semi-automated liquid-liquid extraction (LLE) at pH 7 with methyl-t-butyl ether (MTBE) in a 96-well plate containing 10µL of 10% CHAPS as the surfactant to prevent non-specific binding issue. Chromatographic separation was achieved on a Genesis C8 column (2.1×50mm, 4µm) with a gradient elution using 0.1% formic acid in water as mobile phase A and a mixture of methanol: acetone: formic acid (95:5:0.1; v/v/v) as the mobile phase B. Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 748→648 for asunaprevir and m/z 757→649 for [D9]-asunaprevir,and a collision energy of 30 electron Volts (eV). The assay was validated over a standard curve range from 0.05 to 50ng/mL for asunaprevir in human plasma. The intra- and inter assay precisions were within 7.1% CV, and the % deviation was within 5.5% of their nominal concentrations. This assay has been successfully applied to multiple clinical studies with excellent assay ruggedness and reproducibility.

Daclatasvir + asunaprevir + beclabuvir ± ribavirin for chronic HCV genotype 1-infected treatment-naive patients.

BACKGROUND AND AIMS: This phase-2b study examined the safety and efficacy of an all-oral, interferon-free combination of the NS5A replication complex inhibitor daclatasvir (DCV), the NS3 protease inhibitor asunaprevir (ASV), and the nonnucleoside NS5B polymerase inhibitor beclabuvir (BCV) with or without ribavirin in patients with HCV genotype (GT) 1 infection. METHODS: A total of 187 patients received 12 weeks of DCV 30 mg BID plus ASV 200 mg BID and BCV 150 mg BID (n = 86) or 75 mg BID with (n = 21) or without (n = 80) weight-based ribavirin BID. The primary endpoint was HCV RNA <25 IU/ml at post-treatment week 12 (SVR12). RESULTS: Overall, 90% of patients (169/187) in the combined treatment groups achieved SVR on or after post-treatment week 12. SVR rates were similar across subgroups (by mITT analysis), i.e. patients with cirrhosis (88%, 14/16), HCV GT-1a (90%, 137/155), and IL28B non-CC genotype (90%, 115/128). There were no drug-related serious AEs or grade 4 AEs. The most frequently reported AEs were headache, diarrhoea, fatigue and nausea. Addition of ribavirin to DCV+ASV+BCV was associated with decreased haemoglobin, compared with DCV+ASV+BCV alone. There were six grade 3/4 laboratory abnormalities noted, all unrelated to the study drugs. Viral breakthrough occurred in 2.5-4.8% of patients across groups and appeared unrelated to BCV dose or ribavirin inclusion. CONCLUSIONS: Results support phase 3 evaluation of a twice-daily, fixed-dose formulation of this DCV+ASV+BCV regimen with or without ribavirin in HCV GT-1-infected patients.

Asunaprevir: A Review of Preclinical and Clinical Pharmacokinetics and Drug-Drug Interactions.

Asunaprevir is a tripeptidic acylsulfonamide inhibitor of the hepatitis C virus (HCV) NS3/4A protease. Asunaprevir undergoes rapid absorption, with a time to reach maximum plasma concentration (T max) of 2-4 h and an elimination half-life (t ½) of ≈15-20 h observed in single-ascending dose studies. Steady state was achieved by day 7 in multiple-ascending dose studies. The large food effect observed with earlier formulations was mitigated by the soft-gel capsule. Asunaprevir demonstrates high apparent oral clearance and minimal renal elimination, and is eliminated primarily via cytochrome P450 (CYP) 3A4-mediated hepatic oxidative metabolism and faecal excretion. Highly preferential distribution of asunaprevir to the liver occurs via organic anion-transporting polypeptide (OATP)-mediated transport and results in low plasma concentrations. The condition of the liver affects disposition, as higher asunaprevir plasma exposures are observed in patients infected with HCV and/or hepatic impairment. Japanese patients also have higher exposure relative to North American/European patients, but comparable safety at the registrational dose. Asunaprevir has a low potential to perpetrate drug-drug interactions via CYP3A4, P-glycoprotein and OATP, but is a moderate CYP2D6 inhibitor; concomitant drugs that are substrates of CYP2D6 or P-glycoprotein and have a narrow therapeutic index should be used with care. Asunaprevir plasma exposure is strongly affected by inhibitors of OATP transport. No clinically significant interactions were observed between asunaprevir and daclatasvir or daclatasvir/beclabuvir. Asunaprevir has a complex pharmacokinetic profile and forms part of potent and well-tolerated all-oral regimens for the treatment of chronic HCV infection.

Multiplexed LC-MS/MS method for the simultaneous quantitation of three novel hepatitis C antivirals, daclatasvir, asunaprevir, and beclabuvir in human plasma.

Dual or triple combination regimens of novel hepatitis C direct-acting antivirals (DAA, daclatasvir, asunaprevir, or beclabuvir) provide high sustained virological response rates and reduced frequency of resistance compared to clinical monotherapy. To support pharmacokinetic (PK) assessments in clinical studies, a multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitation of daclatasvir, asunaprevir, beclabuvir (BMS-791325) and its active metabolite (BMS-794712) in human plasma was developed and validated. Human plasma samples were extracted with methyl-t-butyl ether followed by an LC-MS/MS analysis, which was conducted in a multiple reaction monitoring (MRM) mode. The lower limits of quantitation (LLOQ) were 1 ng/mL for daclatasvir, asunaprevir, and BMS-794712, and 2 ng/mL for beclabuvir. Intra-run precision (≤4.5% CV), inter-run precision (≤2.9% CV), and accuracy (±5.3% deviation) based on different concentration levels (low, geometric mean, mid and high) of the quality control samples (QCs) provided evidence of the methods accuracy and precision. Selectivity and matrix effect on LC-MS/MS detection, stability in plasma, and potential interference of coadministered drugs (ribavirin and interferon) were all evaluated and the results were acceptable. Method reproducibility was demonstrated by the reanalysis of a portion of study samples. The cross-validation results for QCs demonstrated the equivalency between this method and two single-analyte methods which were previously validated for quantitation of daclatasvir in human plasma. This approach of using a multiplexed LC-MS/MS method for the simultaneous quantitation of three DAAs is time- and cost-effective, and can maintain good data quality in sample analysis.

Sensitive and accurate liquid chromatography-tandem mass spectrometry methods for quantitative determination of a novel hepatitis C NS5B inhibitor BMS-791325 and its active metabolite in human plasma and urine.

BMS-791325 is a novel hepatitis C NS5B inhibitor which is currently in clinical development. To support pharmacokinetic (PK) assessments, sensitive, accurate, precise, and reproducible liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed and validated for the quantitation of BMS-791325 and its active N-demethyl metabolite (BMS-794712) in human plasma and urine. Plasma and urine samples were extracted with methyl-t-butyl ether followed by an LC-MS/MS analysis which was conducted in a multiple reaction monitoring (MRM) mode for the simultaneous detection of the two analytes in human plasma (0.1-50 ng/mL) and in human urine (5-2500 ng/mL). Intra-run precision (3.0% R.S.D.), inter-run precision (5.3% R.S.D.), and accuracy (±4.7% deviation) from plasma and urine quality control samples provide evidence of the methods accuracy and precision. Selectivity, stability in matrices, extraction recovery, matrix effect on LC-MS detection, and interference of coadministered drugs (famotidine and ritonavir) were all acceptable. Reproducibility of the plasma method was demonstrated by reanalysis of a portion of study samples. The results of cross-validations demonstrated the equivalency of two methods validated in two labs. The plasma method was applied to the analysis of several thousand clinical study samples for PK evaluations of the drug in normal healthy subjects and in patients. The urine method was used in the first in human study to evaluate renal clearance and urinary recovery.

The effect of hepatic impairment on the pharmacokinetics of asunaprevir, an HCV NS3 protease inhibitor.

BACKGROUND: It is necessary to evaluate the impact of hepatic impairment on the pharmacokinetic profile of direct-acting antiviral agents for the treatment of HCV infection. METHODS: In this open-label, parallel group, multiple-dose study subjects (aged 18-70 years with a body mass index <35 kg/m(2)) with mild (n=6), moderate (n=6) and severe hepatic impairment (n=4) received asunaprevir 200 mg twice daily; healthy subjects (n=12) were matched (age, weight, gender) 1:1 to the first 4 subjects in each hepatic impairment group to act as controls. Pharmacokinetic sampling and analyses were performed on days 1 and 7 of dosing. Pharmacokinetic parameters were derived by non-compartmental methods. Geometric mean ratios (GMRs) and 90% CIs were used to assess the impact of hepatic impairment on the pharmacokinetics of asunaprevir, relative to healthy matched controls. RESULTS: Compared with healthy subjects, mild hepatic impairment did not result in meaningful alterations in asunaprevir exposure (day 7 maximal plasma concentration [Cmax] GMR: 0.58 [90% CI 0.35, 0.98]; area under the plasma concentration-time curve in one dosing interval [AUCtau] GMR: 0.79 [90% CI 0.55, 1.15]); clinically significant increases in asunaprevir exposure were observed in subjects with moderate (Cmax GMR: 5.03 [90% CI 2.99, 8.47]; AUCtau GMR: 9.83 [90% CI 6.76, 14.28]) and severe hepatic impairment (Cmax GMR: 22.92 [90% CI 12.57, 41.81]; AUCtau GMR: 32.08 [90% CI 20.84, 49.40]). Correlation between increased asunaprevir exposure and all individual components of the Child-Pugh classification system was observed in subjects with moderate and severe hepatic impairment. CONCLUSIONS: Mild hepatic impairment does not meaningfully affect the pharmacokinetic profile of asunaprevir. The dosing of asunaprevir in patients with moderate-to-severe hepatic impairment is not recommended. Clinicaltrials.gov identifier NCT01019070.

Quantitative determination of free/bound atazanavir via high-throughput equilibrium dialysis and LC-MS/MS, and the application in ex vivo samples.

AIM: The determination of drug-protein binding is important in the pharmaceutical development process because of the impact of protein binding on both the pharmacokinetics and pharmacodynamics of drugs. Equilibrium dialysis is the preferred method to measure the free drug fraction because it is considered to be more accurate. The throughput of equilibrium dialysis has recently been improved by implementing a 96-well format plate. Results/methodology: This manuscript illustrates the successful application of a 96-well rapid equilibrium dialysis (RED) device in the determination of atazanavir plasma-protein binding. DISCUSSION/CONCLUSION: This RED method of measuring free fraction was successfully validated and then applied to the analysis of clinical plasma samples taken from HIV-infected pregnant women administered atazanavir. Combined with LC-MS/MS detection, the 96-well format equilibrium dialysis device was suitable for measuring the free and bound concentration of pharmaceutical molecules in a high-throughput mode.

The pharmacokinetics of daclatasvir and asunaprevir administered in combination in studies in healthy subjects and patients infected with hepatitis C virus.

BACKGROUND AND OBJECTIVES: The combination of direct-acting antiviral agents in patients with chronic hepatitis C virus (HCV) infection has demonstrated clinical benefit; however, evaluation of potential drug-drug interactions is required prior to therapy. METHODS: An open-label study assessed the pharmacokinetics and tolerability of the HCV NS5A replication complex inhibitor daclatasvir and the HCV NS3 protease inhibitor asunaprevir when co-administered in healthy subjects. Daclatasvir 60 mg once daily and asunaprevir 600 mg twice daily were dosed for 7 days alone followed by combination dosing for 14 days at 30 mg once daily and 200 mg twice daily, respectively. Further assessments were provided comparing exposures from the current study with those from studies in HCV-infected patients receiving either the same or higher doses of daclatasvir or asunaprevir administered alone or together. RESULTS: Dose-normalized daclatasvir and asunaprevir morning exposures were comparable with control in healthy subjects, with geometric mean area under the concentration-time curve ratios of 1.202 (90 % CI 1.113-1.298) and 0.868 (90 % CI 0.726-1.038), respectively. In HCV patients daclatasvir and asunaprevir exposures were largely comparable, when administered together or alone. CONCLUSIONS: Additional data support the conclusion that there is no clinically meaningful interaction between daclatasvir and asunaprevir in either healthy subjects or HCV-infected patients, including those also receiving peginterferon-α/ribavirin, and that the combination of daclatasvir 60 mg once daily and asunaprevir 200 mg twice daily is generally well-tolerated.

Randomized trial of asunaprevir plus peginterferon alfa and ribavirin for previously untreated genotype 1 or 4 chronic hepatitis C.

BACKGROUND & AIMS: Asunaprevir is a selective HCV NS3 protease inhibitor, active against genotypes 1, 4, 5, and 6 in vitro. We evaluated asunaprevir plus peginterferon alfa-2a/ribavirin (PegIFNα/RBV) for genotype 1 and 4 chronic HCV. METHODS: In this phase 2b, double-blind, placebo-controlled study, treatment-naive adults with genotype 1 (n=213) or 4 (n=25) were randomly assigned (3:1) to asunaprevir 200mg or placebo twice daily plus PegIFNα/RBV. Asunaprevir recipients, achieving protocol-defined response (HCV-RNA below quantification limit at week 4 and undetectable at week 10), were rerandomized at week 12 to continue asunaprevir-based triple therapy or receive placebo plus PegIFNα/RBV for weeks 13-24. Patients without protocol-defined response (PDR) and placebo recipients continued PegIFNα/RBV through week 48. Co-primary end points were undetectable HCV-RNA at week 4 and 12 (eRVR) and 24 weeks posttreatment (SVR24). RESULTS: Most patients were male (64.3%), white (83.6%), and had non-CC IL28B genotypes (71.3%). Among genotype 1 patients, eRVR rates (asunaprevir vs. placebo) were 67% (80% CI 62, 72) vs. 6% (80% CI 2, 10); corresponding SVR24 rates were 64% (80% CI 59, 68) vs. 44% (80% CI 36, 53). SVR24 among genotype 4 patients was 89% (asunaprevir) vs. 43% (placebo). Rates of rash and haematologic adverse events were similar between treatment groups. Five asunaprevir-treated patients had grade 4 alanine aminotransferase elevations that resolved following discontinuation (n=4) or with continued dosing (n=1). CONCLUSIONS: Addition of asunaprevir to PegIFNα/RBV in treatment-naive genotype 1- or 4-infected patients improves response rates and is well tolerated, with aminotransferase elevations that were manageable with appropriate monitoring. ClinicalTrials.gov ID: NCT01030432.