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Effect on HCV Replication by Combinations of Direct Acting Antivirals Including NS5A Inhibitor Daclatasvir
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Antimicrob. Agents Chemother
Lenore A. Pelosi,1* Stacey Voss, 1 Mengping Liu, 1 Min Gao, 1 Julie A. Lemm1*
Affiliation(s): 1 Department of Virology, Bristol-Myers Squibb Research & Development, 5
Research Parkway, Wallingford, Connecticut, USA, 06492
published online ahead of print on 30 July 2012
Taken as a whole, the results from multiple experiments demonstrate that combinations of these inhibitors display mixed additivity and/or synergy at the 50%, 75% and 90% effective dose levels of drug. Importantly, no antagonistic effects were observed with any of these combinations, nor was any increase in cytotoxicity observed
ABSTRACT
Three Hepatitis C virus (HCV) inhibitors, asunaprevir (ASV, BMS-650032), daclatasvir (DCV, BMS-790052), and BMS-791325, each targeting a different non-structural protein of the virus (NS3, NS5A, and NS5B, respectively), have independently demonstrated encouraging pre-clinical profiles and are currently undergoing clinical evaluation. Since drug-resistant variants have rapidly developed to monotherapy with almost all direct-acting antiviral agents (DAAs) for HCV, the need for combination therapies to effectively eradicate the virus from infected patients is clear. These studies demonstrate the additive/synergistic effects on replicon inhibition and clearance of combining NS3 protease or NS5B RNA polymerase inhibitors with the first-in-class, NS5A replication complex inhibitor daclatasvir (DCV), and reveal new resistance pathways for combinations of two small molecule inhibitors that differ from those that develop during monotherapy. The results suggest that under a specific selective pressure, a balance must be reached in the fitness costs of substitutions in one target gene when substitutions are also present in another target gene. Further synergies and additional novel resistance substitutions were observed during triple combination treatment relative to dual-drug therapy indicating that in combination, HCV inhibitors can exert cross-target influences on resistance development. Enhanced synergies in replicon inhibition and a reduced frequency of resistance together lend strong support to the utility of combinations of DAAs for the treatment of HCV, and the identification of altered resistance profiles during combination treatment provides useful information for monitoring resistance in the clinic.
INTRODUCTION
Hepatitis C virus (HCV) is a positive-stranded RNA virus in the Flaviviridae family of enveloped virions, which affects an estimated 170 million people worldwide and is the major cause of chronic hepatitis. Currently, approximately 50% of patients infected with genotype 1, the most prevalent form of the virus, fail to achieve a sustained reduction in viral load with pegylated interferon alpha plus ribavirin (alfa/ RBV) (56, 54, 58). A substantial fraction (20%) of chronically infected patients develop serious progressive liver disease, including cirrhosis or hepatocellular carcinoma. Alfa/RBV treatment is associated with a high incidence (>30%) of adverse effects, some of which are of sufficient severity to cause patients to discontinue therapy (58). Despite the recent approval of wo new direct acting antiviral agents (DAAs), boceprevir and telaprevir, for use in combination with alfa/ RBV (18,49), their use may be limited by poor efficacy in some patient populations, inconvenient 3-times daily dosing of the DAA, and association with side effects including anemia, rash, and gastro-intestinal effects, in addition to the well documented spectrum of adverse effects associated with alfa/RBV. Although addition of these DAAs to the standard of care for HCV represents a significant improvement in patient therapy, there is still an unmet medical need for new agents and more tolerable treatment regimens for newly diagnosed patients and those failing current therapies.
The 9.6 kb HCV genome encodes a polyprotein of about 3000 amino acids via translation of a single, uninterrupted open reading frame. The polyprotein is cleaved co- and post77 translationally in infected cells by cellular and virus-encoded proteases, to produce a multi component replication complex (8, 34). The serine protease encoded by the N-terminal region of NS3 is thought to be responsible for all downstream cis and trans proteolytic cleavages (9, 17). NS5A possesses no known enzymatic activity, but exists in different states of phosphorylation, and influences multiple functions at various stages of the viral replication cycle (43, 61). It has been shown to interact with an extensive array of host proteins, and to play a role in IFN resistance (38, 42). NS5B is the RNA-dependent RNA polymerase responsible for replication of HCV RNA (1, 4).
The essential roles of non-structural proteins NS3-NS5 in viral replication render each an attractive target for antiviral intervention (2). Clinical proof-of-concept has been achieved for a number of DAAs targeting some of these proteins, including the serine protease activity of NS3 (11, 88 16, 26, 32, 33, 47, 55) and the RNA-dependent RNA polymerase activity of NS5B (60, 21, 27, 48). More recently, daclatasvir (DCV) (Table 1) was the first NS5A replication complex inhibitor to show proof-of-concept in the clinic, demonstrating in early clinical testing the potential for this class of inhibitor to become a valuable component of an all-oral treatment regimen for HCV (15).
The high turnover rate and error-prone nature of the HCV RNA polymerase contribute to the production of potentially resistant viral quasi-species. In practice, resistance has emerged to all small molecule inhibitors of HCV tested as monotherapy except some nucleoside/nucleotide NS5B inhibitors. Resistance mutations have been identified both in vitro and in vivo upon treatment with nearly all inhibitors of HCV serine protease, NS5A, or allosteric RNA polymerase inhibitors advanced to date (3, 20, 23, 24, 25, 31, 37, 39, 53, 54, 59, 63, 65), with good correlation observed between resistance emergence in the replicon system and in vivo . Recent literature indicates that treatment with combinations of non-cross-resistant inhibitors not only improves antiviral activity during treatment, but also suppresses the post-treatment viral rebound often associated with monotherapy (20, 22, 27). To achieve a sustained viral response (SVR), it will be essential to use combination therapies similar to those that have recently been explored in replicon (5, 10, 30), animal models (46), and patients (14, 53) as a viable approach to improving the efficacy, tolerability, and compliance issues associated with current therapies. In this report, the effects of a combination approach to HCV therapy have been studied in the HCV replicon system using two- and three-drug combinations that include NS5A replication complex inhibitor (DCV), NS3 protease inhibitor asunaprevir (ASV), and non-nucleoside NS5B RNA polymerase inhibitor BMS-791325.
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