SCY-635, a Novel Nonimmunosuppressive Analog of Cyclosporine That Exhibits Potent Inhibition of Hepatitis C Virus RNA Replication In Vitro

SCY-635 is a novel nonimmunosuppressive cyclosporine-based analog that exhibits potent suppression of hepatitis C virus (HCV) replication in vitro. SCY-635 inhibited the peptidyl prolyl isomerase activity of cyclophilin A at nanomolar concentrations but showed no detectable inhibition of calcineurin phosphatase activity at concentrations up to 2 μM. Metabolic studies indicated that SCY-635 did not induce the major cytochrome P450 enzymes 1A2, 2B6, and 3A4. SCY-635 was a weak inhibitor and a poor substrate for P-glycoprotein. Functional assays with stimulated Jurkat cells and stimulated human peripheral blood mononuclear cells indicated that SCY-635 is a weaker inhibitor of interleukin-2 secretion than cyclosporine. A series of two-drug combination studies was performed in vitro. SCY-635 exhibited synergistic antiviral activity with alpha interferon 2b and additive antiviral activity with ribavirin. SCY-635 was shown to be orally bioavailable in multiple animal species and produced blood and liver concentrations of parent drug that exceeded the 50% effective dose determined in the bicistronic con1b-derived replicon assay. These results suggest that SCY-635 warrants further investigation as a novel therapeutic agent for the treatment of individuals who are chronically infected with HCV.Hepatitis C virus (HCV) is a member of the Flaviviridae family, which comprises three distinct genera, including the flaviviruses (such as yellow fever virus, dengue virus, West Nile virus, and Japanese encephalitis virus), the pestiviruses (bovine viral diarrhea virus and classical swine fever virus), and the hepaciviruses (of which HCV is the only member) (16). HCV is highly polymorphic, and current taxonomic schemes recognize six major genotypes and several subtypes. Although no strict relationship exists between the genotype and the severity of HCV disease or the clinical outcome, numerous clinical studies indicate that patients who are infected with genotype 1 viruses are less responsive to antiviral therapy than individuals who are infected with genotypes 2 through 6 (10, 11). Chronic infection with HCV now represents a major global health problem, with approximately 170 million people worldwide being infected (26). The current standard of care for chronic hepatitis C virus infection involves treatment for up to 1 year with combination chemotherapy of pegylated alpha interferon coadministered with ribavirin. At this time, there are no approved drugs specifically indicated for the treatment of patients who do not respond to first-line therapy. Complete clearance of the virus is achieved in approximately 50% of all HCV-infected patients who initiate therapy (10, 11), and the response rates are related to viral factors (the genotype and the viral load), as well as multiple host factors (the presence of liver fibrosis, cirrhosis, ethnicity, coinfection with HIV type 1 [HIV-1], alcohol consumption, and metabolic disorders).At this time, the combined action of interferon and ribavirin against HCV infection is poorly understood. The exogenous administration of type 1 alpha interferon confers a nonspecific antiviral state which is characterized by the induction of a broad array of interferon-stimulated genes (ISGs). The principal actions of the ISGs are to block the initiation of viral protein synthesis and to decrease the stability of viral RNA, as well as to stimulate both the adaptive and the innate immune responses to infection (6). Clinically, interferon (most notably, its pegylated derivatives) has been demonstrated to induce multi-log-unit declines in the levels of plasma viremia. Ribavirin undergoes intracellular phosphorylation to its mono-, di-, and triphosphate derivatives. Ribavirin triphosphate is a low-affinity inhibitor of the viral NS5B polymerase and a substrate for incorporation into nascent genomic RNA. The utilization of ribavirin triphosphate as a substrate for RNA synthesis may ultimately inhibit viral RNA replication through error catastrophe. Ribavirin monophosphate competitively inhibits IMP dehydrogenase, which could deplete intracellular GTP levels, further augmenting the inhibitory effects of ribavirin triphosphate (6). Studies of ribavirin monotherapy indicate that it results in the transient and modest suppression of plasma viremia in some but not all patients (20); however, its greatest treatment benefit may be in suppressing the rebound of viremia following the completion of combination therapy with pegylated interferon. Treatment with pegylated alpha interferon and ribavirin is associated with a wide range of severe toxicities, including neuropsychiatric events, bone marrow toxicities, endocrine disorders, cardiovascular events, and anemia (25). In addition, ribavirin is teratogenic in multiple animal species. These observations underscore the need to discover and develop potent and specific antiviral agents that can augment the clinical anti-HCV activity of the current standard of care without increasing toxicity. Ultimately, it is envisioned that highly specific orally bioavailable antiviral agents could form the cornerstones of new treatment regimens that do not contain interferon and ribavirin.Several groups have confirmed that the replication of subgenomic and full-length HCV genomes depends on the expression of the host protein cyclophilin A (CyPA) (4, 14, 34). The levels of HCV RNA replication and viral protein production were reduced up to 1,000-fold in cell lines harboring the stable knockdown of CyPA expression. The knockdown of cyclophilin B, C, and D expression had no effect on HCV-specific RNA replication or protein production. The inhibition of replication was rescued by the overexpression of a wild-type CyPA escape mutant; however, the overexpression of a mutant CyPA containing the H126Q substitution (which abolishes peptidyl prolyl isomerase [PPIase] activity) did not rescue replication. These results further suggest that HCV RNA replication requires PPIase catalytic activity. The in vitro anti-HCV activity of cyclosporine (CsA) has been reported, thus confirming that the cyclophilins are potential drug targets (28). The potential clinical utility of cyclosporine-based inhibitors has been suggested by the introduction of nonimmunosuppressive analogs of CsA that retain cyclophilin binding activity. NIM 811 and Debio-025 contain modifications at the three sarcosine and the four methyl leucine residues. Both compounds inhibit HCV-specific RNA replication in vitro (17, 19). Clinical studies indicate that Debio-025 suppresses HCV plasma viremia when it is given as monotherapy for 14 days to patients coinfected with HIV-1 and HCV and when it is given as a component of a two-drug combination regimen with pegylated interferon for 28 days to HCV-monoinfected patients (8, 9).This report describes the preclinical profile of SCY-635, a 3,4-disubstituted nonimmunosuppressive CsA analog that exhibits potent suppression of HCV RNA replication in vitro. SCY-635 is a reversible, nanomolar inhibitor of the PPIase activity expressed by CyPA. SCY-635 is orally bioavailable in multiple species and distributes extensively to hepatocytes. Metabolic studies indicate that the administration of SCY-635 is unlikely to result in adverse pharmacological interactions. Two-drug synergy studies indicate that SCY-635 exhibits additive to synergistic antiviral activity when it is tested in vitro with alpha interferon 2b (IFNα-2b) or ribavirin without increasing cell cytotoxicity. These results are consistent with the recent observation of the potent clinical antiviral activity of SCY-635 (13) and suggest that further clinical studies assessing the safety and antiviral activity of SCY-635 in combination with interferon and ribavirin are warranted.