Sofosbuvir and Ribavirin Liver Pharmacokinetics in Patients Infected with Hepatitis C Virus

Abstract

Sofosbuvir and ribavirin exert their anti-hepatitis C virus (anti-HCV) activity following metabolic activation in the liver. However, intrahepatic concentrations of the pharmacologically active nucleotide metabolites in humans are poorly characterized due to the inaccessibility of tissue and technical challenges with measuring nucleotide levels. A clinical study assessing the efficacy of sofosbuvir and ribavirin administered prior to liver transplantation to prevent HCV recurrence provided a unique opportunity to quantify nucleotide concentrations in human liver. We analyzed nucleotides using high-performance liquid chromatography coupled to tandem mass spectrometry in liver tissue from 30 HCV-infected patients with hepatocellular carcinoma who were administered sofosbuvir (400 mg/day) and ribavirin (1,000 to 1,200 mg/day) for 3 to 52 weeks prior to liver transplantation. Median total hepatic metabolite concentrations (the sum of nucleoside and mono-, di-, and triphosphates) were 77.1 μM for sofosbuvir and 361 μM for ribavirin in patients on therapy at the time of transplantation. Ribavirin and sofosbuvir efficiently loaded the liver, with total hepatic metabolite concentrations exceeding maximal levels in plasma by approximately 30-fold. Ribavirin metabolite levels suggest that its monophosphate is in great excess of its inhibition constant for IMP dehydrogenase and that its triphosphate is approaching the binding constant for incorporation by the HCV NS5B RNA-dependent RNA polymerase. In accordance with the potent antiviral activity of sofosbuvir, these results demonstrate that the liver triphosphate levels achieved following sofosbuvir administration greatly exceed the inhibition constant for HCV NS5B. In conclusion, this study expands the quantitative understanding of the pharmacology of sofosbuvir and ribavirin by establishing efficient hepatic delivery in the clinic. (This study has been registered at ClinicalTrials.gov under identifier NCT01559844.).

Keywords: direct-acting antiviral; mass spectrometry; nucleoside analog; pharmacokinetics; prodrug; transplantation.

FIG 1

Monitored plasma and intrahepatic metabolites. After entry into the cell, sofosbuvir is cleaved by carboxylesterase 1 (CES1) to form metabolite X. Subsequent cleavage of the phosphoramidate bond results in the formation of GS-331007 monophosphate (MP), which can be dephosphorylated to form GS-331007 or sequentially phosphorylated to form the pharmacologically active metabolite GS-331007 triphosphate (TP). Ribavirin is primarily taken up into hepatocytes by nucleoside transporters and is rapidly phosphorylated to form its pharmacologically active nucleotide metabolites.

FIG 2

LC–MS-MS chromatograms for sofosbuvir metabolites. Shown is the separation of intracellular metabolites GS-331007 (a), GS-331007 monophosphate (MP) (b), metabolite X (c), GS-331007 diphosphate (DP) (d), GS-331007 triphosphate (TP) (e), and the internal standard chloro-ATP (f) using reverse-phase ion-pairing chromatography and negative-mode MS-MS analysis. The retention times and mass-to-charge ratios (m/z) for the parent and daughter ions of each analyte are indicated. A liver sample from a representative patient was analyzed.

FIG 3

Median concentrations of sofosbuvir and ribavirin metabolites in human liver sections after 3 to 52 weeks of treatment with sofosbuvir and ribavirin. Concentrations of each individual metabolite and total metabolites are shown for sofosbuvir (a and c, respectively) and ribavirin (b and d, respectively). Results for patients who had been off therapy for more than 5 days (□), those with detectable HCV at the time of transplantation (△), those who relapsed posttransplantation (○), and those who had pTVR (●) are indicated. Results for one subject who had been off therapy for 5 days and also had detectable HCV at the time of transplantation are indicated by asterisks. The five patients with the lowest levels of sofosbuvir-related metabolites stopped therapy ≥3 weeks before transplantation. Lines indicate median concentrations of sofosbuvir (70.3 μM; range, 0.382 to 252 μM) and ribavirin (329 μM; range, 10.1 to 1,800 μM) in the 30 patients.

FIG 4

Endogenous adenosine nucleotide concentrations in human liver sections. ATP concentrations (median, 360 μM) and the ratio of ATP to the sum of AMP and ADP (0.2) were approximately 10-fold lower than those reported in the literature (3,100 μM and 2, respectively) (23), suggesting postcollection dephosphorylation of the samples. Lines represent the median concentrations in liver sections collected from 30 patients.

FIG 5

In vitro metabolism of sofosbuvir (left) and ribavirin (right) in primary human hepatocytes. Following a 2-h pulse incubation, the drug-containing medium was removed and was replaced with fresh medium without drug. Incubation with sofosbuvir led to the rapid appearance of metabolite X, which egressed following removal of the drug from the medium, allowing more-persistent exposure to the pharmacologically active triphosphate. Ribavirin was concentrated in cells and rapidly metabolized to its triphosphate metabolite. At 24 h, the triphosphate metabolites accounted for approximately 70% of the total intracellular metabolites for both sofosbuvir and ribavirin. Based on the observation of dose linearity, incubations of 1, 5, and 10 μM sofosbuvir were dose-normalized to 1 μM. The pulse incubation concentration for ribavirin was 100 μM. Results are means ± standard deviations for cells from 14 (sofosbuvir) or 2 (ribavirin) donors, with results at each time point taken from duplicate incubations.