The anti-hepatitis C virus nucleotide prodrug GS-6620 employs a double-prodrug approach, with L-alanine-isopropyl ester and phenol moieties attached to the 5=-phosphate that release the nucleoside monophosphate in hepatocytes and a 3=-isobutyryl ester added to improve permeability and oral bioavailability. Consistent with the stability found in intestinal homogenates, following oral administration, intact prodrug levels in blood plasma were the highest in dogs, followed by monkeys, and then were the lowest in hamsters. In contrast, liver levels of the triphosphate metabolite at the equivalent surface area-adjusted doses were highest in hamsters, followed by in dogs and monkeys. Studies in isolated primary hepatocytes suggest that relatively poor oral absorption in hamsters and monkeys was compensated for by relatively efficient hepatocyte activation. As intestinal absorption was found to be critical to the effectiveness of GS-6620 in nonclinical species, stomach pH, formulation, and food effect studies were completed in dogs. Consistent with in vitro absorption studies in Caco-2 cells, the absorption of GS-6620 was found to be complex and highly dependent on concentration. Higher rates of metabolism were observed at lower concentrations that were unable to saturate intestinal efflux transporters. In first-in-human clinical trials, the oral administration of GS-6620 resulted in poor plasma exposure relative to that observed in dogs and in large pharmacokinetic and pharmacodynamic variabilities. While a double-prodrug approach, including a 3=-isobutyryl ester, provided higher intrinsic intestinal permeability, this substitution appeared to be a metabolic liability, resulting in extensive intestinal metabolism and relatively poor oral absorption in humans.A pproximately 170 million people worldwide are infected by the hepatitis C virus (HCV), and nearly 2% of the U.S. population is estimated to have HCV (1). HCV is a major health issue since approximately 15 to 35% of those who are chronically infected will develop cirrhosis and 1 to 3% will progress to hepatocellular carcinoma over 30 years (2). Currently, the recently approved protease inhibitors (boceprevir and telaprevir) are used in combination with pegylated interferon alpha plus ribavirin (PEG-RBV) for the treatment of chronic HCV infection. While these treatments show improved response rates and the potential for shorter duration of treatment over PEG-RBV alone, they lack efficacy against genotypes other than genotype 1, require thricedaily dosing, and cause additional side effects from the new directacting antivirals on top of the already-challenging tolerability profile of PEG-RBV. Therefore, there is a need for more potent anti-HCV compounds with improved clinical efficacy and greater tolerability in order to more broadly address the unmet medical needs of those with chronic HCV infection. Combinations of antiviral agents targeting viral proteins essential to HCV replication have the potential to achieve increased clinical efficacy across HCV genotypes, ...