The Role of CYPs and Transporters in the Biotransformation and Transport of the Anti-hepatitis C Antiviral Agents Asunaprevir, Daclatasvir, and Beclabuvir: Impact of Liver Disease, Race and Drug-drug Interactions on Safety and Efficacy.

Asunaprevir, daclatasvir, and beclabuvir are direct-acting antiviral agents used in the treatment of patients infected with hepatitis C genotype 1b. This article reviews the biotransformation and disposition of these drugs in relation to the safety and efficacy of therapy. CYP3A4 and 3A5 catalyze the oxidative biotransformation of the drugs, while P-glycoprotein mediates their efflux from tissues. Asunaprevir is also a substrate for the influx transporters OATP1B1 and OATP2B1 and the efflux transporter MRP2, while beclabuvir is also a substrate for the efflux transporter BCRP. Liver disease decreases the expression of CYPs and transporters that mediate drug metabolism and disposition. Serum asunaprevir concentrations, but not those of daclatasvir or beclabuvir, are increased in patients with severe liver disease, which may produce toxicity. Pharmacogenomic variation in CYPs and transporters also has the potential to disrupt therapy with asunaprevir, daclatasvir and beclabuvir; some variants are more prevalent in certain racial groups. Pharmacokinetic drug-drug interactions, especially where asunaprevir, daclatasvir, and beclabuvir are victim drugs, are mediated by coadministered rifampicin, ketoconazole and ritonavir, and are attributable to inhibition and/or induction of CYPs and transporters. Conversely, there is also evidence that asunaprevir, daclatasvir and beclabuvir are perpetrators of drug interactions with coadministered rosuvastatin and dextromethorphan. Together, liver disease, pharmacogenomic variation and drug-drug interactions may disrupt therapy with asunaprevir, daclatasvir and beclabuvir due to the impaired function of important CYPs and transporters.

Real-world long-term analysis of daclatasvir plus asunaprevir in patients with hepatitis C virus infection.

Background and Aim: This study aimed to evaluate the long-term clinical course of patients achieving a sustained virologic response (SVR) with daclatasvir plus asunaprevir (DCV/ASV) therapy. Methods: A total of 911 patients who achieved SVR with DCV/ASV were assessed. To evaluate pretreatment factors contributing to hepatocellular carcinoma (HCC) after SVR, univariate and multivariate analyses were performed in all patients, in those with preexisting HCC, and in those without preexisting HCC. We selected a low-risk group of HCC cases after SVR. Finally, we evaluated liver function after achieving SVR. Results: In multivariable analyses, male sex, older age, patients with a history of HCC treatment, excess alcohol use, lower albumin, and low platelet count remained significant in the overall group; male sex and low albumin remained significant in patients with a history of HCC treatment; and male sex, older age, excess alcohol use, low platelet count, high alpha-fetoprotein (AFP), and high des-γ-carboxy prothrombin (DCP) remained significant in those without a history of HCC treatment. Patients who had not received treatment for HCC, females, those under 70 years of age, and those with platelet count ≥13 (×104/µL), AFP <6 ng/mL, and DCP <23 mAU/mL were at low risk of HCC. The process of liver function improvement was different according to the factors. Conclusions: The incidence rate of HCC, risk factors associated with HCC, group with very low risk of developing HCC, and the clinical course in a real-world long-term study were evaluated.

A Reversible Chemogenetic Switch for Chimeric Antigen Receptor T†Cells.

As a revolutionary cancer treatment, the chimeric antigen receptor (CAR) T cell therapy suffers from complications such as cytokine release syndromes and T cell exhaustion. Their mitigation desires controllable activation of CAR-T cells that is achievable through regulatory display of CARs. By embedding the hepatitis C virus NS3 protease (HCV-NS3) between the single-chain variable fragment (scFv) and the hinge domain, we showed that the display of anti-CD19 scFv on CAR-T cells was positively correlated to the presence of a clinical HCV-NS3 inhibitor asunaprevir (ASV). This novel CAR design that allows the display of anti-CD19 scFv in the presence of ASV and its removal in the absence of ASV creates a practically reversible chemical switch. We demonstrated that the intact CAR on T cells can be repeatedly turned on and off by controlling the presence of ASV in a dose dependent manner both in vitro and in vivo, which enables delicate modulation of CAR-T activation during cancer treatment.

Follow-up after Direct-acting Antiviral Treatment for Chronic Hepatitis C Virus Infection: Most Patients Are Followed Appropriately.

Objective Chronic hepatitis C virus (HCV) infection carries a residual risk of hepatocarcinogenesis even after viral elimination, so appropriate follow-up is necessary. The present study investigated the current hospital visits and hepatocarcinogenesis status of patients who received daclatasvir plus asunaprevir treatment (DCV+ASV) to determine whether or not appropriate follow-up was being performed. Methods We retrospectively analyzed hepatocarcinogenesis, the overall survival, and the length of hospital visits in 442 patients who applied for the medical expense subsidy system for viral hepatitis and received DCV+ASV treatment in Gunma Prefecture between October 2014 and December 2015. This also included 61 patients who had a history of hepatocellular carcinoma (HCC). Results Among 442 patients, 388 achieved a sustained viral response (SVR) by DCV+ASV therapy (87.8%), and 95.9% achieved an SVR if additional treatment was included. HCC was found in 75 cases (17.0%). A history of HCC, the FIB-4 index and the treatment effect SVR were determined to be factors affecting the incidence of HCC. Regarding the follow-up rate, 89.9% of patients continued to regularly visit the hospital after 5 years of treatment. However, patients ≤60 years old had significantly lower persistence rates than older patients. The persistence rate of hospital visits to the same institution was 67.7% over a 5-year period, which was significantly better in small and medium-sized institutions than in large, specialized institutions (71.7% vs. 63.9%, p=0.039). Conclusion Patients with direct-acting antiviral treatment generally received adequate follow-up, but younger patients had a slightly higher rate of follow-up interruption and were considered to need support.

Asunaprevir, a Potent Hepatitis C Virus Protease Inhibitor, Blocks SARS-CoV-2 Propagation.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has become a global health concern. Various SARS-CoV-2 vaccines have been developed and are being used for vaccination worldwide. However, no therapeutic agents against coronavirus disease 2019 (COVID-19) have been developed so far; therefore, new therapeutic agents are urgently needed. In the present study, we evaluated several hepatitis C virus direct-acting antivirals as potential candidates for drug repurposing against COVID-19. Theses include asunaprevir (a protease inhibitor), daclatasvir (an NS5A inhibitor), and sofosbuvir (an RNA polymerase inhibitor). We found that asunaprevir, but not sofosbuvir and daclatasvir, markedly inhibited SARS-CoV-2-induced cytopathic effects in Vero E6 cells. Both RNA and protein levels of SARS-CoV-2 were significantly decreased by treatment with asunaprevir. Moreover, asunaprevir profoundly decreased virion release from SARS-CoV-2-infected cells. A pseudoparticle entry assay revealed that asunaprevir blocked SARS-CoV-2 infection at the binding step of the viral life cycle. Furthermore, asunaprevir inhibited SARS-CoV-2 propagation in human lung Calu-3 cells. Collectively, we found that asunaprevir displays broad-spectrum antiviral activity and therefore might be worth developing as a new drug repurposing candidate for COVID-19.

Non-alcoholic fatty liver disease is a risk factor for occurrence of hepatocellular carcinoma after sustained virologic response in chronic hepatitis C patients: A prospective four-years follow-up study.

BACKGROUND AND AIM: The incidence of hepatocellular carcinoma (HCC) decreases significantly in chronic hepatitis C (CHC) patients with sustained virologic response (SVR) after pegylated-interferon plus ribavirin (PR) or direct-acting antiviral (DAAs) therapy. We follow-up a single cohort of CHC patients to identify risk factors associated with HCC development post-SVR. METHOD: CHC patients with SVR in Beijing/Hong Kong were followed up at 12-24 weekly intervals with surveillance for HCC by ultrasonography and alpha-fetoprotein (AFP). Multivariate Cox proportional hazards regression analysis was used to explore factors associated with HCC occurrence. RESULTS: Between October 2015 and May 2017, SVR was observed in 519 and 817 CHC patients after DAAs and PR therapy respectively. After a median post -SVR follow-up of 48 months, HCC developed in 54 (4.4%) SVR subjects. By adjusted Cox analysis, older age (≥55 years) [HR 2.4, 95% CI (1.3-4.3)], non-alcoholic fatty liver diseases [HR 2.4, 95%CI (1.3-4.2), higher AFP level (≥20 ng/ml) [HR 3.4, 95%CI (2.0-5.8)], higher liver stiffness measurement (≥14.6 kPa) [HR 4.2, 95%CI (2.3-7.6)], diabetes mellitus [HR 4.2, 95%CI (2.4-7.4)] at pre-treatment were associated with HCC occurrence. HCC patients in the DAAs induced SVR group had a higher prevalence of NAFLD as compared with those in the PR induced SVR group, 62% (18/29) vs 28% (7/25), p = 0.026. A nomogram formulated with the above six independent variables had a Concordance-Index of 0.835 (95% CI 0.783-0.866). CONCLUSION: Underlying NAFLD is associated with increased incidence of HCC in chronic HCV patients post-SVR, particularly in those treated with DAA.

Retreatment of Chronic Hepatitis C Failed to Daclatasvir Plus Asunaprevir by Other Direct-acting Antivirals.

The pegylated interferon plus ribavirin combination therapy has been used as the primary treatment for chronic hepatitis C (CHC) but fails to produce a sustained viral response (SVR) in many patients. In recent years, the treatment of CHC has been rapidly changing because of the introduction of direct-acting antivirals (DAAs), which have a high cure rate. However, retreatment of patients after failure of the first DAA therapy is difficult. We report two rare cases of CHC that showed acquired SVR with other DAA combinations after failure to daclatasvir and asunaprevir.

Features of resistance-associated substitutions after failure of multiple direct-acting antiviral regimens for hepatitis C.

BACKGROUND & AIMS: We aimed to clarify the features of resistance-associated substitutions (RASs) after failure of multiple interferon (IFN)-free regimens in HCV genotype 1b infections. METHODS: A total of 1,193 patients with HCV for whom direct-acting antiviral (DAA) treatment had failed were enrolled from 67 institutions in Japan. The RASs in non-structural protein (NS)3, NS5A, and NS5B were determined by population sequencing. RESULTS: Failure of 1, 2, and 3 regimens was observed in 1,101; 80; and 12 patients, respectively. Among patients with failure of 1 regimen, Y56H and D168V in NS3 were more frequently detected after failure of paritaprevir, whereas D168E was more frequently detected after failure of regimens including asunaprevir. R30H and L31-RAS in NS5A were frequently detected after failure of regimens including daclatasvir. The prevalence of Y93-RAS was high irrespective of the regimen. S282T RAS in NS5B was detected in 3.9% of ledipasvir/sofosbuvir failures. The prevalence of D168-RAS increased significantly according to the number of failed regimens (p <0.01), which was similar to that seen with L31-RAS and Y93-RAS. The prevalence of patients with RASs in either NS3 or NS5A, or in both, increased significantly with increasing numbers of failed regimens. The P32del, which is unique to patients for whom DAA had failed, was linked to the absence of Y93H, the presence of L31F, and previous exposure to IFN plus protease inhibitor regimens. CONCLUSIONS: Failure of multiple DAA regimens can lead to the generation of multiple RASs in the NS3 and NS5A regions of the HCV 1b genome. These mutations contribute to viral resistance to multiple treatment regimens and, therefore, should be considered during decision making for treatment of chronic HCV. LAY SUMMARY: Resistance-associated substitutions (RAS) in the genome of the hepatitis C virus are 1 of the major causes for failed treatment. We investigated RASs after failure of various treatments for chronic hepatitis C, and found that more complicated RASs accumulated in the viral genome with successive failed treatments. The highly resistant P32del RAS at NS5A region was uniquely found in patients for whom DAA treatments had failed, and was linked to the presence and absence of specific RASs.

Favorable outcome of retreatment by direct-acting antivirals for hepatitis C patients with daclatasvir plus asunaprevir combination therapy failure.

AIM: In patients with hepatitis C virus, treatment failure of daclatasvir plus asunaprevir combination therapy (DCV + ASV) seems to become intractable due to the induction of resistance-associated substitutions. This study aimed to investigate the outcomes of retreatment with direct-acting antivirals (DAAs) in patients with DCV + ASV therapy failure, as well as changes in drug resistance mutations. METHODS: We retrospectively analyzed 44 patients re-treated with DAAs after DCV + ASV failure between December 2015 and April 2018. All patients were analyzed for amino acid substitutions, and additional treatment regimens were selected based on the results and current treatment guidelines. RESULTS: The sustained virological response rate with second-line treatment was 81.8% (36/44), and relapse occurred in five of 16 patients who received sofosbuvir/ledipasvir and three of seven patients who received DCV/ASV/beclabuvir. Third- and fourth-line treatments were also tried in relapsed cases, and the overall sustained virological response rates were 90.9% (40/44) and 93.2% (41/44), respectively. A high rate of viral clearance was eventually observed. Before second-line treatment, the prevalence of mutations in the NS5A and NS3/4A regions was 100% (44/44) and 86.4% (38/44), respectively. There was no significant increase in the number of amino acid substitutions in patients for whom second-line treatment failed. CONCLUSIONS: Amino acid substitutions were frequently observed in patients with DCV + ASV failure, but most patients achieved a sustained virological response after retreatment with DAAs. Although the spread of drug-resistant viruses due to unsuccessful DAA treatment was a matter of concern, most cases of DCV + ASV failure were overcome with additional treatment.

A case with acute-on-chronic liver failure receiving liver transplantation during daclatasvir and asunaprevir therapy in chronic hepatitis C patient.

We report a case with hepatitis C virus genotype 1 b liver cirrhosis who received liver transplantation because of acute-on-chronic liver failure during daclatasvir (DSV) and asunaprevir (ASV) combination therapy. To our knowledge, this is the first case received liver transplantation during DSV + ASV therapy. Therefore, clinicians should pay particular attention to the possibility of acute liver failure during DSV and ASV combination therapy.

Durability of Sustained Virologic Response and Improvement of Fibrosis Markers after Daclatasvir and Asunaprevir Treatment in Genotype 1b Hepatitis C Virus-Infected Patients: a Real Life and Multicenter Study.

BACKGROUND: The long-term data with direct acting antiviral agents were rare. This study investigated the durability of a sustained virologic response (SVR) and the improvement of fibrosis after daclatasvir and asunaprevir (DCV/ASV) treatment in genotype 1b (GT1b) hepatitis C virus (HCV)-infected patients. METHODS: A total of 288 HCV GT1b patients without baseline non-structural 5A (NS5A) resistance-associated substitution (RAS) treated with DCV/ASV were enrolled. Virologic response was measured at 12 weeks and 1 year after treatment completion. In cirrhotic patients, liver function, aspartate transaminase to platelet ratio index (APRI), FIB-4 index, fibrosis index (FI), and liver stiffness measurement (LSM) at baseline and 1 year after treatment completion were evaluated. RESULTS: SVR12 was obtained in 278 patients (96.5%). Six patients who checked NS5A RAS after treatment failure were RAS positive. Only one patient showed no durability of SVR. In cirrhotic patients who achieved SVR12 (n = 59), the changes of albumin (3.8 [2.2-4.7] to 4.3 [2.4-4.9] g/dL; P < 0.001), platelet count (99 [40-329] to 118 [40-399] × 10³/mm³; P < 0.001), APRI (1.8 [0.1-14.8] to 0.6 [0.1-4.8]; P < 0.001), FIB-4 index (5.45 [0.6-32.8] to 3.3 [0.4-12.2]; P < 0.001), FI (5.5 [0.6-32.8] to 3.3 [0.4-12.2]; P < 0.001), and LSM (17.2 [5.3-48.0] to 11.2 [3.7-28.1] kPa; P = 0.001) between baseline and 1 year after treatment completion were observed. CONCLUSION: DCV/ASV treatment for HCV GT1b infected patients without RAS achieved high SVR rates and showed durable SVR. Cirrhotic patients who achieved SVR12 showed the improvement of liver function and fibrosis markers.

Real-world virological efficacy and safety of daclatasvir/asunaprevir/beclabuvir in patients with chronic hepatitis C virus genotype 1 infection in Japan.

BACKGROUND: The virological efficacy and safety of the direct-acting antiviral (DAA) regimen consisting of daclatasvir, asunaprevir, and beclabuvir (DCV/ASV/BCV) for patients chronically infected with hepatitis C virus (HCV) genotype 1 have not been previously evaluated in Japanese real-world settings. METHODS: In a Japanese nationwide multicenter study, the rate of sustained virologic response (SVR) and safety were analyzed in 91 patients who started the DCV/ASV/BCV regimen between November 2016 and July 2017. SVR rates were compared based on baseline patient characteristics. RESULTS: More than 60% of patients had a history of failure to achieve SVR with interferon (IFN)-free DAA therapy. Overall, 50 of 91 patients (54.9%) achieved SVR. Multivariate analysis identified a history of failure with IFN-free DAA therapy and pretreatment HCV RNA levels as factors significantly associated with treatment failure. Whereas the SVR rate in patients without a history of IFN-free DAA therapy was 91.7% (33 of 36 patients), it was only 30.9% (17 of 55 patients) among patients with a history of IFN-free DAA therapy. The rate of discontinuation due to an adverse event was 4.4%. CONCLUSIONS: Many patients treated with the DCV/ASV/BCV regimen have a history of a failure to achieve SVR with previous IFN-free DAA therapy. SVR rate was not as high as that in pre-approval clinical trial of this regimen in IFN-free DAA-naïve patients. In addition, most patients with a history of failure with IFN-free DAA therapy, particularly the DCV/ASV regimen, showed resistance to this regimen.

Low susceptibility of asunaprevir towards R155K and D168A point mutations in HCV NS3/4A protease: A molecular dynamics simulation.

Hepatitis C has become an important health problem that requires expensive treatment and leads to liver tumorigenesis. Hepatitis C virus (HCV), which is the main cause of hepatitis C, has a high mutation rate due to the lack of proofreading activity of the RNA polymerase enzyme. The NS3/4A serine protease is an important target for anti-HCV drug discovery and development because of its crucial role in the cleavage of the polypeptides involved in viral replication. In the present study, all-atom molecular dynamics simulation was performed to elucidate the effect of the single point mutations R155K and D168A in the HCV genotype 1 NS3/4A protease on the structural dynamics, molecular interactions and susceptibility of asunaprevir (ASV), a second-generation NS3/4A protease inhibitor. Principal component analysis indicated that these two mutations converted the direction of motion of residues 123, 155 and 168 in the binding pocket to significantly point outwards from ASV, resulting in a loss of the hydrogen bond network of residues R123···R155···D168. The free energy calculations based on different semiempirical QM/MM-GBSA methods revealed that the binding affinity of ASV with the two mutant forms of the NS3/4A protease was significantly decreased in the order of wild-type < R155K < D168A. This work provided useful structural information regarding the atomistic understanding of acquired drug resistance against ASV caused by the R155K and D168A mutations.

Exposure-Response Analysis for Efficacy of Daclatasvir, Asunaprevir, and Beclabuvir Combinations in HCV-Infected Patients.

The combination regimen of daclatasvir, asunaprevir, and beclabuvir (3DAA regimen) was developed as a fixed-dose combination for the treatment of hepatitis C virus (HCV) infection in Japan. The objectives of this analysis were to characterize the relationship between drug exposure and sustained virologic response at posttreatment week 12 (SVR12) in HCV-infected subjects and to evaluate the impact of demographic covariates and clinical factors on the exposure-response (E-R) relationship. The E-R efficacy analysis was performed with data from phase 2 and phase 3 studies in HCV-infected subjects treated with the 3DAA regimen. The relationship between the probability of achieving SVR12 and exposure to daclatasvir, asunaprevir, and beclabuvir was described using a logistic regression model and included assessments of the potential covariate effects. The impacts of the covariates on the rate of SVR12 and interactions of covariates with the individual drug effects were tested. The final model for SVR12 included effects of non-genotype-1a status, resistance-associated NS5A-Q30 substitution in genotype-1a subjects, and baseline RNA level on the intercept, and effect of prior peg-interferon failure on the beclabuvir slope. Sex, race, age, weight, fibrosis score, alanine transaminase, and cirrhosis status had no statistically significant impact on the rate of SVR12. The individual E-R relationships with each drug, were relatively flat, and the effects of exposure were not significant. With the exception of the NS5A-Q30 substitution in genotype-1a subjects, statistically significant covariate effects had little impact on SVR12 rates. Overall, the E-R model was developed that captured the high SVR12 rates and the effect of covariates for the 3DAA regimen in HCV-infected patients.

Population Pharmacokinetic Analysis of Daclatasvir, Asunaprevir, and Beclabuvir Combination in HCV-Infected Subjects.

A fixed-dose combination of daclatasvir (pangenotypic NS5A inhibitor), asunaprevir (NS3/4A protease inhibitor), and beclabuvir (nonnucleoside NS5B inhibitor) was approved for hepatitis C virus treatment in Japan. The objectives of the analyses were to develop the daclatasvir, asunaprevir, and beclabuvir population pharmacokinetic models for the combination regimen. First, an original population pharmacokinetic model was developed using the data in non-Japanese hepatitis C virus-infected subjects. The model was subsequently updated after a phase 3 study in Japanese hepatitis C virus-infected subjects was available. A total of 11,382, 11,300, and 10,728 pharmacokinetic records from 1,228 subjects were included for daclatasvir, asunaprevir, and beclabuvir in the updated model, respectively. Daclatasvir and beclabuvir pharmacokinetics (PK) were described by a 1-compartment model with linear elimination and asunaprevir PK was described by 2-compartment model with linear elimination. Cirrhosis, baseline, and time-varying ALT were significant covariates on asunaprevir apparent oral clearance. Asian subjects had greater asunaprevir and beclabuvir exposures than white subjects. The effects of all covariates on daclatasvir PK were modest and not considered clinically significant. With the exception of race on asunaprevir and beclabuvir PK, no other parameters for daclatasvir, asunaprevir and beclabuvir population PK models were meaningfully impacted during the refinement with Japanese subjects.

Efficacy and Safety of Daclatasvir and Asunaprevir in Patients with Hepatitis C Virus Genotype 1b Infection on Hemodialysis.

Background/Aims: We evaluated the efficacy and safety of daclatasvir (DCV) and asunaprevir (ASV) in patients with chronic hepatitis C virus (HCV) infection on hemodialysis. Methods;: We performed a single-arm, multicenter prospective study. Twenty-one chronic hemodialysis patients with HCV infection were prospectively enrolled from February 2016 to April 2017. We evaluated the virological responses at weeks 4, 12, and 24 (end of treatment [EOT]) and the sustained virological response at 12 weeks after the EOT (SVR12). The tolerability and safety of the drugs were also assessed. Results: None of the 20 patients had the NS5A resistance-associated variant (NS5A RAV), and one patient was indeterminate for the NS5A RAV. Seventeen patients (80%) completed the 24 weeks of treatment with DCV and ASV. Four patients discontinued the study prior to week 12. In an intention-to-treat analysis, the SVR12 was 76.1%. In a per-protocol analysis, patients who completed DCV and ASV treatment achieved an SVR12 of 100%. DCV and ASV were well tolerated by the majority of patients. Three patients discontinued treatment due to adverse events (AEs) including dizziness, dyspnea, and neutropenia. The patient with indeterminate NS5A RAV showed viral breakthrough and discontinued treatment. Conclusions: DCV and ASV combination therapy in chronic hemodialysis patients with HCV infection achieved a high SVR12 rate with few AEs. To maximize the SVR12 rate, it is important to identify candidates by baseline RAV testing. Close monitoring of the safety and tolerability of DCV and ASV may be necessary in HCV-infected patients on hemodialysis. (ClinicalTrials.gov ID NCT02580474).

Safety Exposure-Response Analysis for Daclatasvir, Asunaprevir, and Beclabuvir Combinations in HCV-Infected Subjects.

The combination regimen of daclatasvir, asunaprevir, and beclabuvir has been developed for the treatment of hepatitis C virus infection. The objectives of this analysis were to characterize the relationship between the exposures of the daclatasvir, asunaprevir, and beclabuvir regimen and liver-related laboratory elevations (Grade 3 or 4 alanine aminotransferase [ALT] and total bilirubin [Tbili]), and to evaluate the impact of selected covariates on the exposure-response relationships. The exposure-response analysis was performed with data from 1 phase 2 and 3 phase 3 studies in hepatitis C virus-infected subjects. The probability of liver-related laboratory elevations were modeled using linear logistic regression. Selected covariates were tested using a forward-addition and backward-elimination approach. The final model for ALT elevation included Asian race, body weight in non-Asian subjects, and asunaprevir exposure. The final model for Tbili elevation included Asian race, fibrosis score (F0-F3 or F4) and asupanprevir exposure. Asian subjects had greater the Grade 3 or 4 ALT and Tbili elevation rates than non-Asians. The Grade 3 or 4 ALT elevation rate increased with decreasing body weight in non-Asian subjects. Subjects with F4 fibrosis score had a higher rate of Grade 3 or 4 Tbili elevation compared to subjects with F0 to F3 fibrosis score. Higher asunaprevir exposure was associated with increases in Grade 3 or 4 ALT and Tbili elevation rates; however, the impact on the ALT elevation was not clinically relevant and the effect on Tbili elevation was smaller than the other significant covariates.

HCV genotype 1-6 NS3 residue 80 substitutions impact protease inhibitor activity and promote viral escape.

BACKGROUND & AIMS: Protease inhibitors (PIs) are of central importance in the treatment of patients with chronic hepatitis C virus (HCV) infection. HCV NS3 protease (NS3P) position 80 displays polymorphisms associated with resistance to the PI simeprevir for HCV genotype 1a. We investigated the effects of position-80-substitutions on fitness and PI-resistance for HCV genotypes 1-6, and analyzed evolutionary mechanisms underlying viral escape mediated by pre-existing Q80K. METHODS: The fitness of infectious NS3P recombinants of HCV genotypes 1-6, with engineered position-80-substitutions, was studied by comparison of viral spread kinetics in Huh-7.5 cells in culture. Median effective concentration (EC50) and fold resistance for PIs simeprevir, asunaprevir, paritaprevir, grazoprevir, glecaprevir and voxilaprevir were determined in short-term treatment assays. Viral escape was studied by long-term treatment of genotype 1a recombinants with simeprevir, grazoprevir, glecaprevir and voxilaprevir and of genotype 3a recombinants with glecaprevir and voxilaprevir, next generation sequencing, NS3P substitution linkage and haplotype analysis. RESULTS: Among tested PIs, only glecaprevir and voxilaprevir showed pan-genotypic activity against the original genotype 1-6 culture viruses. Variants with position-80-substitutions were all viable, but fitness depended on the specific substitution and the HCV isolate. Q80K conferred resistance to simeprevir across genotypes but had only minor effects on the activity of the remaining PIs. For genotype 1a, pre-existing Q80K mediated accelerated escape from simeprevir, grazoprevir and to a lesser extent glecaprevir, but not voxilaprevir. For genotype 3a, Q80K mediated accelerated escape from glecaprevir and voxilaprevir. Escape was mediated by rapid and genotype-, PI- and PI-concentration-dependent co-selection of clinically relevant resistance associated substitutions. CONCLUSIONS: Position-80-substitutions had relatively low fitness cost and the potential to promote HCV escape from clinically relevant PIs in vitro, despite having a minor impact on results in classical short-term resistance assays. LAY SUMMARY: Among all clinically relevant hepatitis C virus protease inhibitors, voxilaprevir and glecaprevir showed the highest and most uniform activity against cell culture infectious hepatitis C virus with genotype 1-6 proteases. Naturally occurring amino acid changes at protease position 80 had low fitness cost and influenced sensitivity to simeprevir, but not to other protease inhibitors in short-term treatment assays. Nevertheless, the pre-existing change Q80K had the potential to promote viral escape from protease inhibitors during long-term treatment by rapid co-selection of additional resistance changes, detected by next generation sequencing.

Daclatasvir and asunaprevir combination therapy for patients with chronic hepatitis C virus genotype 1b infection in real world.

BACKGROUND/AIMS: Previous studies have reported a high rate of sustained virologic response (SVR) and a low rate of serious adverse events with the use of daclatasvir (DCV) and asunaprevir (ASV) combination therapy. We evaluated the efficacy and safety of DCV and ASV combination therapy for patients with chronic hepatitis C virus (HCV) genotype 1b infection in real world. METHODS: We enrolled 278 patients (184 treatment-naïve patients) from five hospitals in Daegu and Gyeongsangbuk-do. We evaluated the rates of rapid virologic response (RVR), end-of-treatment response (ETR), and SVR at 12 weeks after completion of treatment (SVR12). Furthermore, we investigated the rate of adverse events and predictive factors of SVR12 failure. RESULTS: The mean age of patients was 59.5 ± 10.6 years, and 140 patients (50.2%) were men. Seventy-seven patients had cirrhosis. Baseline information regarding nonstructural protein 5A (NS5A) sequences was available in 268 patients. Six patients presented with pretreatment NS5A resistance-associated variants. The RVR and the ETR rates were 96.6% (258/267) and 95.2% (223/232), respectively. The overall SVR12 rate was 91.6% (197/215). Adverse events occurred in 17 patients (7.9%). Six patients discontinued treatment because of liver enzyme elevation (n = 4) and severe nausea (n = 2). Among these, four achieved SVR12. Other adverse events observed were fatigue, headache, diarrhea, dizziness, loss of appetite, skin rash, and dyspnea. Univariate analysis did not show significant predictive factors of SVR12 failure. CONCLUSION: DCV and ASV combination therapy showed high rates of RVR, ETR, and SVR12 in chronic HCV genotype 1b-infected patients in real world and was well tolerated without serious adverse events.

Durability of Sustained Virologic Response and Improvement of Fibrosis Markers after Daclatasvir and Asunaprevir Treatment in Genotype 1b Hepatitis C Virus-Infected Patients: a Real Life and Multicenter Study

BACKGROUND: The long-term data with direct acting antiviral agents were rare. This study investigated the durability of a sustained virologic response (SVR) and the improvement of fibrosis after daclatasvir and asunaprevir (DCV/ASV) treatment in genotype 1b (GT1b) hepatitis C virus (HCV)-infected patients. METHODS: A total of 288 HCV GT1b patients without baseline non-structural 5A (NS5A) resistance-associated substitution (RAS) treated with DCV/ASV were enrolled. Virologic response was measured at 12 weeks and 1 year after treatment completion. In cirrhotic patients, liver function, aspartate transaminase to platelet ratio index (APRI), FIB-4 index, fibrosis index (FI), and liver stiffness measurement (LSM) at baseline and 1 year after treatment completion were evaluated. RESULTS: SVR12 was obtained in 278 patients (96.5%). Six patients who checked NS5A RAS after treatment failure were RAS positive. Only one patient showed no durability of SVR. In cirrhotic patients who achieved SVR12 (n = 59), the changes of albumin (3.8 [2.2–4.7] to 4.3 [2.4–4.9] g/dL; P < 0.001), platelet count (99 [40–329] to 118 [40–399] × 103/mm3; P < 0.001), APRI (1.8 [0.1–14.8] to 0.6 [0.1–4.8]; P < 0.001), FIB-4 index (5.45 [0.6–32.8] to 3.3 [0.4–12.2]; P < 0.001), FI (5.5 [0.6–32.8] to 3.3 [0.4–12.2]; P < 0.001), and LSM (17.2 [5.3–48.0] to 11.2 [3.7–28.1] kPa; P = 0.001) between baseline and 1 year after treatment completion were observed. CONCLUSION: DCV/ASV treatment for HCV GT1b infected patients without RAS achieved high SVR rates and showed durable SVR. Cirrhotic patients who achieved SVR12 showed the improvement of liver function and fibrosis markers.