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.

Randomized, placebo-controlled, single-ascending-dose study of BMS-791325, a hepatitis C virus (HCV) NS5B polymerase inhibitor, in HCV genotype 1 infection.

BMS-791325 is a nonnucleoside inhibitor of hepatitis C virus (HCV) NS5B polymerase with low-nanomolar potency against genotypes 1a (50% effective concentration [EC50], 3 nM) and 1b (EC50, 7 nM) in vitro. BMS-791325 safety, pharmacokinetics, and antiviral activity were evaluated in a double-blind, placebo-controlled, single-ascending-dose study in 24 patients (interferon naive and experienced) with chronic HCV genotype 1 infection, randomized (5:1) to receive a single dose of BMS-791325 (100, 300, 600, or 900 mg) or placebo. The prevalence and phenotype of HCV variants at baseline and specific posttreatment time points were assessed. Antiviral activity was observed in all cohorts, with a mean HCV RNA decline of ≈2.5 log10 copies/ml observed 24 h after a single 300-mg dose. Mean plasma half-life among cohorts was 7 to 9 h; individual 24-hour levels exceeded the protein-adjusted EC90 for genotype 1 at all doses. BMS-791325 was generally well tolerated, with no serious adverse events or discontinuations. Enrichment for resistance variants was not observed at 100 to 600 mg. At 900 mg, variants (P495L/S) associated with BMS-791325 resistance in vitro were transiently observed in one patient, concurrent with an observed HCV RNA decline of 3.4 log10 IU/ml, but were replaced with wild type by 48 h. Single doses of BMS-791325 were well tolerated; demonstrated rapid, substantial, and exposure-related antiviral activity; displayed dose-related increases in exposure; and showed viral kinetic and pharmacokinetic profiles supportive of once- or twice-daily dosing. These results support its further development in combination with other direct-acting antivirals for HCV genotype 1 infection. (This trial has been registered at ClinicalTrials.gov under registration no. NCT00664625.).

Daclatasvir plus sofosbuvir for previously treated or untreated chronic HCV infection.

BACKGROUND: All-oral combination therapy is desirable for patients with chronic hepatitis C virus (HCV) infection. We evaluated daclatasvir (an HCV NS5A replication complex inhibitor) plus sofosbuvir (a nucleotide analogue HCV NS5B polymerase inhibitor) in patients infected with HCV genotype 1, 2, or 3. METHODS: In this open-label study, we initially randomly assigned 44 previously untreated patients with HCV genotype 1 infection and 44 patients infected with HCV genotype 2 or 3 to daclatasvir at a dose of 60 mg orally once daily plus sofosbuvir at a dose of 400 mg orally once daily, with or without ribavirin, for 24 weeks. The study was expanded to include 123 additional patients with genotype 1 infection who were randomly assigned to daclatasvir plus sofosbuvir, with or without ribavirin, for 12 weeks (82 previously untreated patients) or 24 weeks (41 patients who had previous virologic failure with telaprevir or boceprevir plus peginterferon alfa-ribavirin). The primary end point was a sustained virologic response (an HCV RNA level of <25 IU per milliliter) at week 12 after the end of therapy. RESULTS: Overall, 211 patients received treatment. Among patients with genotype 1 infection, 98% of 126 previously untreated patients and 98% of 41 patients who did not have a sustained virologic response with HCV protease inhibitors had a sustained virologic response at week 12 after the end of therapy. A total of 92% of 26 patients with genotype 2 infection and 89% of 18 patients with genotype 3 infection had a sustained virologic response at week 12. High rates of sustained virologic response at week 12 were observed among patients with HCV subtypes 1a and 1b (98% and 100%, respectively) and those with CC and non-CC IL28B genotypes (93% and 98%, respectively), as well as among patients who received ribavirin and those who did not (94% and 98%, respectively). The most common adverse events were fatigue, headache, and nausea. CONCLUSIONS: Once-daily oral daclatasvir plus sofosbuvir was associated with high rates of sustained virologic response among patients infected with HCV genotype 1, 2, or 3, including patients with no response to prior therapy with telaprevir or boceprevir. (Funded by Bristol-Myers Squibb and Pharmasset (Gilead); A1444040 ClinicalTrials.gov number, NCT01359644.).

Randomized trial of daclatasvir and asunaprevir with or without PegIFN/RBV for hepatitis C virus genotype 1 null responders.

BACKGROUND & AIMS: Patients with chronic hepatitis C virus (HCV) infection and prior null response (<2 log HCV RNA decline after ⩾ 12 weeks of PegIFN/RBV) have limited options. We evaluated daclatasvir plus once- or twice-daily asunaprevir in non-cirrhotic genotype 1 null responders. METHODS: In this randomized, phase 2a, open-label, 24-week treatment study, 101 patients received daclatasvir (60 mg) once-daily. In addition, 38 genotype 1b patients received asunaprevir (200mg) twice- (DUAL A1) or once-daily (DUAL A2); 36 genotype 1a and 5 genotype 1b patients received asunaprevir twice- (QUAD B1) or once-daily (QUAD B2) plus PegIFN/RBV; and 18 genotype 1a and 4 genotype 1b patients received asunaprevir twice-daily plus ribavirin (TRIPLE B3). The primary endpoint was undetectable HCV RNA 12 weeks post-treatment (sustained virologic response, SVR12). RESULTS: Across all groups, mean HCV RNA was ⩾ 6 log IU/ml, and 99% of patients had a non-CC IL28B genotype. SVR12 rates were 78% (A1), 65% (A2), 95% (B1), and 95% (B2). In B3, most genotype 1a patients experienced virologic breakthrough. The most common adverse events were headache, diarrhea, and asthenia. Grade 3-4 aminotransferase elevations were infrequent and not treatment-limiting. CONCLUSIONS: In genotype 1 null responders, daclatasvir plus twice-daily asunaprevir DUAL therapy is effective for most genotype 1b patients, and daclatasvir, asunaprevir, and PegIFN/RBV QUAD therapy is effective for nearly all genotype 1a and 1b patients; but neither DUAL nor TRIPLE therapy is effective for genotype 1a patients. Interferon-free regimens including daclatasvir and twice-daily asunaprevir for genotype 1 null responders should be tailored to subtype.

Efficacy of an interferon- and ribavirin-free regimen of daclatasvir, asunaprevir, and BMS-791325 in treatment-naive patients with HCV genotype 1 infection.

BACKGROUND & AIMS: The combination of peginterferon and ribavirin with telaprevir or boceprevir is the standard treatment of hepatitis C virus (HCV) genotype 1 infection. However, these drugs are not well tolerated because of their side effects and suboptimal virologic responses. In a phase 2a, open-label study, we examined the safety and efficacy of an interferon-free, ribavirin-free regimen of direct-acting antivirals, comprising daclatasvir (an NS5A replication complex inhibitor), asunaprevir (an NS3 protease inhibitor), and BMS-791325 (a non-nucleoside NS5B inhibitor), in patients with chronic HCV infection. METHODS: We analyzed data from 66 treatment-naive patients with HCV genotype 1 infection without cirrhosis who were assigned randomly to groups given daclatasvir (60 mg, once daily), asunaprevir (200 mg, twice daily), and BMS-791325 (75 or 150 mg, twice daily) for 12 or 24 weeks. The primary end point was an HCV-RNA level less than 25 IU/mL at 12 weeks after treatment (sustained virologic response at 12 weeks [SVR12]). RESULTS: In 64 patients, HCV-RNA levels were less than 25 IU/mL by week 4 of treatment (including 48 of 49 patients with HCV genotype 1a infection and 45 of 46 patients with the non-CC interleukin 28B genotype). Sixty-one patients (92%) achieved SVR12, based on a modified intention-to-treat analysis. Virologic responses were similar between 12 and 24 weeks of treatment. During the study, 2 patients experienced viral breakthrough and 1 patient relapsed. There were no grade 3-4 increases in levels of alanine or aspartate aminotransferases or bilirubin; there were no deaths or discontinuations resulting from serious adverse events or adverse events related to the treatment regimen. The most common adverse events were headache, asthenia, and gastrointestinal symptoms. CONCLUSIONS: In a phase 2a study, the all-oral, interferon-free, and ribavirin-free regimen of daclatasvir, asunaprevir, and BMS-791325 was well tolerated and achieved high rates of SVR12 in patients with HCV genotype 1 infection. Further studies of this regimen are warranted. ClinicalTrials.gov, number NCT01455090.

Pharmacokinetics of the NS3 Protease Inhibitor, Asunaprevir (ASV, BMS-650032), in Phase I Studies in Subjects With or Without Chronic Hepatitis C.

Asunaprevir (BMS-650032, ASV) is a potent, selective hepatitis C virus (HCV) NS3 protease inhibitor in clinical evaluation for chronic hepatitis C treatment. ASV pharmacokinetics were evaluated in four single- and multiple-ascending-dose studies in healthy subjects or subjects with HCV genotype 1 infection and in human mass balance and food-effect studies. Median Tmax was 2-4 hours. Although T½ was 14-23 hours, oral clearance was high (302-668 L/h at doses ≥100 mg). Steady state was achieved by Day 7. The ASV dose-exposure relationship was disproportionate at doses <200 mg but largely proportional for Cmax and AUC at clinically relevant doses of 200-600 mg (capsule). Following multiple doses, the accumulation index for AUC[TAU] and Cmin was lower at doses ≥200 mg, suggestive of possible auto-induction. ASV exposure increased when administered as a solution (vs. suspension) or with a high-fat meal; the effect was greater for Cmax than AUC, suggesting a saturable first-pass process, the mechanism of which remains to be defined. The apparent complexities of ASV pharmacokinetics will be further explored; nevertheless, data from these studies and antiviral activity in phase 2a/2b studies support further development. Clinical studies are ongoing with ASV in combination with other antivirals.

Resistance analysis of the hepatitis C virus NS3 protease inhibitor asunaprevir.

Asunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) NS3 protease inhibitor demonstrating efficacy in alfa interferon-sparing, direct-acting antiviral dual-combination regimens (together with the NS5A replication complex inhibitor daclatasvir) in patients chronically infected with HCV genotype 1b. Here, we describe a comprehensive in vitro genotypic and phenotypic analysis of asunaprevir-associated resistance against genotypes 1a and 1b using HCV replicons and patient samples obtained from clinical studies of short-term asunaprevir monotherapy. During genotype 1a resistance selection using HCV replicons, the primary NS3 protease substitutions identified were R155K, D168G, and I170T, which conferred low- to moderate-level asunaprevir resistance (5- to 21-fold) in transient-transfection susceptibility assays. For genotype 1b, a higher level of asunaprevir-associated resistance was observed at the same selection pressures, ranging from 170- to 400-fold relative to the wild-type control. The primary NS3 protease substitutions identified occurred predominantly at amino acid residue D168 (D168A/G/H/V/Y) and were associated with high-level asunaprevir resistance (16- to 280-fold) and impaired replication capacity. In asunaprevir single-ascending-dose and 3-day multiple-ascending-dose studies in HCV genotype 1a- or 1b-infected patients, the predominant pre-existing NS3 baseline polymorphism was NS3-Q80K. This substitution impacted initial virologic response rates in a single-ascending-dose study, but its effects after multiple doses were more ambiguous. Interestingly, for patient NS3 protease sequences containing Q80 and those containing K80, susceptibilities to asunaprevir were comparable when tested in an enzyme assay. No resistance-associated variants emerged in these clinical studies that significantly impacted susceptibility to asunaprevir. Importantly, asunaprevir-resistant replicons remained susceptible to an NS5A replication complex inhibitor, consistent with a role for asunaprevir in combination therapies.

Single- and multiple-ascending-dose studies of the NS3 protease inhibitor asunaprevir in subjects with or without chronic hepatitis C.

Hepatitis C virus (HCV) protease inhibitors combined with pegylated alfa interferon-ribavirin have demonstrated improved efficacy compared with pegylated alfa interferon-ribavirin alone for the treatment of chronic hepatitis C. Asunaprevir (BMS-650032), a novel HCV NS3 protease inhibitor in clinical development, was evaluated for safety, antiviral activity, and resistance in four double-blind, placebo-controlled, sequential-panel, single- and multiple-ascending-dose (SAD and MAD) studies in healthy subjects or subjects with chronic HCV genotype 1 infection. In SAD studies, subjects (healthy or with chronic HCV infection) were randomized to receive asunaprevir in dose groups of 10 to 1,200 mg or a placebo. In MAD studies, healthy subjects were randomized to receive asunaprevir in dose groups of 10 to 600 mg twice daily or a placebo for 14 days; subjects with HCV infection received asunaprevir in dose groups of 200 to 600 mg twice daily, or a placebo, for 3 days. Across all four studies, headache and diarrhea were the most frequent adverse events in asunaprevir recipients. Asunaprevir at doses of 200 to 600 mg resulted in rapid HCV RNA decreases from the baseline; maximal mean changes in HCV RNA over time were 2.7 and 3.5 log(10) IU/ml in the SAD and MAD studies, respectively. No enrichment of signature asunaprevir-resistant viral variants was detected. In conclusion, the novel NS3 protease inhibitor asunaprevir, when administered at single or multiple doses of 200 to 600 mg twice daily, is generally well tolerated, achieving rapid and substantial decreases in HCV RNA levels in subjects chronically infected with genotype 1 HCV.

Preliminary study of two antiviral agents for hepatitis C genotype 1.

BACKGROUND: Patients with chronic hepatitis C virus (HCV) infection who have not had a response to therapy with peginterferon and ribavirin may benefit from the addition of multiple direct-acting antiviral agents to their treatment regimen. METHODS: This open-label, phase 2a study included an exploratory cohort of 21 patients with chronic HCV genotype 1 infection who had not had a response to previous therapy (i.e., had not had ≥2 log(10) decline in HCV RNA after ≥12 weeks of treatment with peginterferon and ribavirin). We randomly assigned patients to receive the NS5A replication complex inhibitor daclatasvir (60 mg once daily) and the NS3 protease inhibitor asunaprevir (600 mg twice daily) alone (group A, 11 patients) or in combination with peginterferon alfa-2a and ribavirin (group B, 10 patients) for 24 weeks. The primary end point was the percentage of patients with a sustained virologic response 12 weeks after the end of the treatment period. RESULTS: A total of 4 patients in group A (36%; 2 of 9 with HCV genotype 1a and 2 of 2 with genotype 1b) had a sustained virologic response at 12 weeks after treatment and also at 24 weeks after treatment.. Six patients (all with HCV genotype 1a) had viral breakthrough while receiving therapy, and resistance mutations to both antiviral agents were found in all cases; 1 patient had a viral response at the end of treatment but had a relapse after the treatment period. All 10 patients in group B had a sustained virologic response at 12 weeks after treatment, and 9 had a sustained virologic response at 24 weeks after treatment. Diarrhea was the most common adverse event in both groups. Six patients had transient elevations of alanine aminotransferase levels to more than 3 times the upper limit of the normal range. CONCLUSIONS: This preliminary study involving patients with HCV genotype 1 infection who had not had a response to prior therapy showed that a sustained virologic response can be achieved with two direct-acting antiviral agents only. In addition, a high rate of sustained virologic response was achieved when the two direct-acting antiviral agents were combined with peginterferon alfa-2a and ribavirin. (Funded by Bristol-Myers Squibb; ClinicalTrials.gov number, NCT01012895.).

A cell-based model of HCV-negative-strand RNA replication utilizing a chimeric hepatitis C virus/reporter RNA template.

The inability of hepatitis C virus (HCV) to replicate in cell culture has hindered the discovery of antiviral agents against this virus. One of the biggest challenges has been to find a model that allows one to easily and accurately quantify the level of HCV RNA replication that is occurring inside the cell. In an attempt to solve this problem, we have created a plasmid pMJ050 that encodes a chimeric 'HCV-like' RNA that can act as a reporter for HCV RNA replication. This RNA consists of an antisense copy of the firefly luciferase sequence flanked by the 5' and 3' untranslated regions of the negative strand of the HCV RNA. If, in cells that contain functional HCV proteins, the chimeric RNA is recognized as a substrate for the viral RNA-dependent RNA polymerase, the chimeric RNA will be transcribed into the complementary strand. This RNA has a 5' HCV internal ribosome entry site and the luciferase sequence in the coding orientation, allowing translation of the RNA into biologically active luciferase. When pMJ050 was transfected into a cell line that is stably transfected with a cDNA copy of the HCV 1b genome, luciferase was produced in a manner that was dependent upon the presence of at least a functional HCV RNA-dependent RNA polymerase. In addition, we constructed a cell line, 293B4alpha that constitutively produced luciferase in response to the presence of functional HCV proteins. This system permits the accurate determination of the level of HCV RNA replication by the quantification of luciferase.