Intracellular effects of the Hepatitis C virus nucleoside polymerase inhibitor RO5855 (Mericitabine Parent) and Ribavirin in combination

Abstract

Mericitabine (RG7128) is the prodrug of a highly selective cytidine nucleoside analog inhibitor (RO5855) of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. This study evaluated the effects of combining RO5855 and ribavirin on HCV replication in the HCV subgenomic replicon by using two drug-drug interaction models. The effects of RO5855 and ribavirin on the intracellular metabolism of each compound, on interferon-stimulated gene (ISG) expression, and on the viability of hepatocyte-derived cells were also investigated. RO5855 and ribavirin had additive inhibitory activities against HCV subgenomic replicon replication in drug-drug interaction analyses. RO5855 did not affect the uptake or phosphorylation of ribavirin in primary human hepatocytes, human peripheral blood mononuclear cells, or genotype 1b (G1b) replicon cells. Similarly, ribavirin did not affect the concentrations of intracellular species derived from RO5855 in primary human hepatocytes or the formation of the triphosphorylated metabolites of RO5855. Ribavirin at concentrations of >40 μM significantly reduced the viability of primary hepatocytes but not of Huh7, the G1b replicon, or interferon-cured Huh7 cells. RO5855 alone or with ribavirin did not significantly alter the viability of Huh7 or G1b replicon cells, and it did not significantly affect the viability of primary hepatocytes when it was administered alone. The viability of primary hepatocytes was reduced when they were incubated with RO5855 and ribavirin, similar to the effects of ribavirin alone. RO5855 alone or with ribavirin had no effect on ISG mRNA levels in any of the cells tested. In conclusion, RO5855 did not show any unfavorable interactions with ribavirin in human hepatocytes or an HCV subgenomic replicon system.

FIG 1

Interactions between RO5855 and ribavirin in HCV replicon cells, shown as a Prichard's model synergy plot (A), Greco's model interaction plot (B), or Prichard's model synergy plot for cell viability (C). Graphs for A and B were plotted with an average of seven experiments, and the graph for C was plotted with an average of five experiments.

FIG 2

Effects of RO5855 on total intracellular species concentrations (A, C, and E) and on ribavirin triphosphate concentrations (B, D, and F) in primary human hepatocytes (A and B), peripheral blood mononuclear cells (C and D), and subgenomic replicon cells (E and F). Data are expressed as mean ± standard deviation (SD) values from three donors.

FIG 3

Effects of ribavirin on levels of total intracellular species and unphosphorylated RO5855 and RO2433 parent compounds (A) and on the formation of RO5855-triphosphate and RO2433-triphosphate (B) in primary human hepatocytes. Data are expressed as mean ± SD values from three donors, with the exception of values for 2 and 25 μM (derived from two donors).

FIG 4

Effects of ribavirin on the viability of primary human hepatocytes, Huh7 cells, G1b replicon cells, and interferon-cured Huh7 cells, evaluated in parallel with the assessment of effects on interferon-stimulated gene expression. Mean ± SD values from three or more independent experiments are plotted. Bonferroni-corrected P values for comparison with the phosphate-buffered saline control were as follows: ***, P < 0.001.

FIG 5

Effects of RO5855, with and without ribavirin, on the viability of Huh7 cells (A), G1b replicon cells (B), and primary human hepatocytes (C). Mean ± SD values from three or more independent experiments are plotted. Bonferroni-corrected P values for comparison with controls were as follows: **, P < 0.01; ***, P < 0.001.

FIG 6

Effects of RO5855, with and without ribavirin, on the expression of mRNA for interferon-stimulated gene 15 (ISG15) (A to C), signal transducer and activator of transcription 1 (STAT1) (D to F), interferon regulatory factor 7 (IRF7) (G to I), and IRF9 (J to L) in Huh7 cells (A, D, G, and J), G1b replicon cells (B, E, H, and K), and primary human hepatocytes (C, F, I, and L). Transcript levels normalized to 18S rRNA levels were expressed as fold changes relative to the no-compound control. The mean ± SD values of three or more independent replicates are plotted. Bonferroni-corrected P values for comparison with control were as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 7

Effects of ribavirin on the expression of interferon-stimulated gene 15 (ISG15) (A), interferon regulatory factor 7 (IRF7) (B), interferon regulatory factor 9 (IRF9) (C), and signal transducer and activator of transcription 1 (STAT1) (D) mRNAs. Transcript levels normalized to 18S rRNA levels were expressed as fold changes relative to controls (dotted lines). The mean ± SD values of three or more independent replicates are plotted. Bonferroni-corrected P values for comparison with control were as follows: **, P < 0.01; ***, P < 0.001. Ribavirin concentrations were as follows: 10 μg/ml = 41 μM, 30 μg/ml = 123 μM, and 100 μg/ml = 410 μM.