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

Intracellular Effects of the Hepatitis C Virus Nucleoside Polymerase Inhibitor RO5855 (Mericitabine Parent) and Ribavirin in Combination

Updated information and services can be found at: http://aac.asm.org/content/58/5/2614 These include: SUPPLEMENTAL MATERIAL REFERENCES

CONTENT ALERTS

Supplemental material This article cites 54 articles, 16 of which can be accessed free at: http://aac.asm.org/content/58/5/2614#ref-list-1 Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more»

Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/

Downloaded from http://aac.asm.org/ on September 11, 2014 by Univ of Idaho

H. Ma, S. Le Pogam, S. Fletcher, F. Hinojosa-Kirschenbaum, H. Javanbakht, J.-M. Yan, W.-R. Jiang, N. Inocencio, K. Klumpp and I. Nájera Antimicrob. Agents Chemother. 2014, 58(5):2614. DOI: 10.1128/AAC.02250-13. Published Ahead of Print 18 February 2014.

Intracellular Effects of the Hepatitis C Virus Nucleoside Polymerase Inhibitor RO5855 (Mericitabine Parent) and Ribavirin in Combination H. Ma,a S. Le Pogam,b S. Fletcher,b* F. Hinojosa-Kirschenbaum,b* H. Javanbakht,b J.-M. Yan,b W.-R. Jiang,c* N. Inocencio,c* K. Klumpp,b* I. Nájerab* Genentech, Inc., South San Francisco, California, USAa; Hoffmann-La Roche, Inc., Nutley, New Jersey, USAb; Hoffmann-La Roche, Inc., Palo Alto, California, USAc

T

he treatment paradigm for chronic hepatitis C virus (HCV) infection is evolving rapidly (1). The first direct-acting antiviral agents (DAAs) for HCV, i.e., the HCV NS3/4A protease inhibitors boceprevir and telaprevir, were approved in 2011 for patients infected with HCV genotype 1 (G1) virus (1). These agents significantly increase sustained virological response (SVR) rates in patients with G1 infection but must be administered in combination with pegylated alpha interferon plus ribavirin (triple therapy) (2– 7). Triple therapy with boceprevir or telaprevir also increases the adverse event burden on patients and is associated with complex drug-drug interactions that are potentially difficult to manage (2–7). When protease inhibitors were evaluated as monotherapy, resistant variants emerged rapidly and control of viral replication was lost within days (8). Moreover, when telaprevir was evaluated in combination with pegylated alpha-2a interferon but without ribavirin, treatment failure rates were unacceptably high (9). Thus, the combination of pegylated alpha interferon plus ribavirin currently remains an essential component of approved protease inhibitor-based combination regimens (1). In order to further improve convenience, safety, and SVR rates in G1-infected patients and to extend the benefits of combination DAA regimens to patients infected with other HCV genotypes, additional classes of DAAs are required, especially to achieve the goal of interferonfree regimens. Novel agents from several DAA classes are in development, and recent data suggest that high SVR rates can be achieved with interferon-free DAA combination regimens (10–14). Although pegylated interferon may not be a necessary component of future combination regimens, ribavirin appears to remain an essential

2614

aac.asm.org

component of many but not all interferon-free DAA regimens (10–12, 14–17). Mericitabine (RG7128) is the di-isobutyl ester prodrug of the cytidine nucleoside analog RO5855 (␤-D-2=-deoxy-2=-fluoro-2=C-methylcytidine). RO5855 is a highly selective inhibitor of the HCV NS5B RNA-dependent RNA polymerase that has activity against all HCV genotypes and a high barrier to resistance (18– 20). Mericitabine has been well tolerated when administered for up to 24 weeks in phase II clinical trials (21, 22). After oral administration, mericitabine is rapidly absorbed and converted to RO5855 in plasma (23). RO5855 is taken up by cells and phosphorylated to form active CTP (RO5855 triphosphate [RO5855TP]) and UTP (RO2433-TP) metabolites (24, 25). The UTP metabolite is almost as potent as the CTP metabolite in vitro against the HCV polymerase; however, the phosphorylated uridine me-

Antimicrobial Agents and Chemotherapy

Received 30 October 2013 Returned for modification 13 November 2013 Accepted 13 February 2014 Published ahead of print 18 February 2014 Address correspondence to H. Ma, [email protected], or I. Nájera, [email protected]. * Present address: S. Fletcher, Gilead Sciences, Foster City, California, USA; F. Hinojosa-Kirschenbaum, Gilead Sciences, Foster City, California, USA; N. Inocencio, Gilead Sciences, Foster City, California, USA; W.-R. Jiang, Pfizer, Inc., South San Francisco, California, USA; K. Klumpp, Novira Therapeutics, Inc., Doylestown, Pennsylvania, USA; I. Nájera, Hoffmann-La Roche, Ltd., Shanghai, China. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.02250-13. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.02250-13

p. 2614 –2625

May 2014 Volume 58 Number 5


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 drugdrug 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 ␮M 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.

Compatibility of Mericitabine and Ribavirin

MATERIALS AND METHODS Cell lines and culture conditions. A Huh7 hepatoma cell line was obtained from the American Type Culture Collection (41). An interferoncured Huh7 cell line was obtained from R. Bartenschlager (42). A Huh7derived cell line (2209-23) that contains stable, autonomously replicating, subgenomic G1b HCV RNA was established at Roche, as previously described (43). Briefly, this cell line harbors a dicistronic HCV replicon, of which the first open reading frame expresses the Renilla luciferase and the second open reading frame expresses HCV NS3, 4A, 4B, 5A, and 5B of the G1b Con1 sequence. The Huh7 cell lines were cultured as previously described (43). Freshly plated human hepatocytes or hepatocyte suspensions from more than three different donors were obtained from CellzDirect, Inc. (Invitrogen, Carlsbad, CA). The human hepatocytes were plated in sixwell collagen-coated plates (BD Biosciences, San Jose, CA) at 1.5 ⫻ 106 cells per well, using the medium and supplements obtained from the suppliers. The medium was replaced after 4 h of cell attachment and every 24 h during incubation. Cells were maintained at 37°C in a humidified 5% CO2 atmosphere. Cryopreserved human peripheral blood mononuclear cells (PBMCs) were obtained from StemCell Technologies, Inc. (Vancouver, British Columbia, Canada) and incubated in a commercial medium (RPMI 1640


medium; Invitrogen, Carlsbad, CA) at 37°C in a humidified 5% CO2 atmosphere. Prior to assays, cells were incubated for a minimum of 48 h in interleukin-2-containing medium. For the gene expression studies, a suspension of human hepatocytes was centrifuged at 228 ⫻ g and then resuspended in Williams’ medium E without phenol red, supplemented with primary hepatocyte culture incubation supplements. Analysis of drug-drug interactions using G1b subgenomic replicon. The effects of the combination of ribavirin with RO5855, sofosbuvir (GS7977), or recombinant interferon alfa-2a on G1b replicon replication and replicon cell viability were examined in vitro. Sofosbuvir was chosen as a control because it is a nucleoside inhibitor of the HCV polymerase and because, in common with mericitabine, RO2433-TP is the active species. Cells were plated on a 96-well plate (BD Biosciences, San Jose, CA), at a density of 5,000 cells/well, in Dulbecco’s modified Eagle medium (DMEM) (with Glutamax-I; Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA) and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA). Cells were allowed to equilibrate for 24 h at 37°C and 5% CO2 before the test compounds were added. Ribavirin (MP Biochemicals, Solon, OH) was titrated (from 50 to 1.56 ␮M, in 2-fold serial dilutions) in a checkerboard fashion with either RO5855 (from 50 to 0.02 ␮M, in 3-fold serial dilutions), sofosbuvir (from 10 to 0.005 ␮M, in 3-fold serial dilutions), or interferon alfa-2a (from 30 to 0.01 IU/ml, in 3-fold serial dilutions). Each experiment was performed in duplicate and was performed independently ⱖ3 times. The Renilla luciferase reporter signal was assessed 72 h after the addition of compounds, as previously described (43). Mean values of percent inhibition of replicon replication were generated from all experiments and were used in the two in vitro drug-drug interaction models (MacSynergy II model and Greco’s model [Interact]) (44–46). Cell viability testing was performed in parallel and under the same conditions as the HCV replicon assay, using the WST-1 cell proliferation assay (Roche Diagnostics, Indianapolis, IN, USA), as previously described (43). Assessment of cell viability was performed 72 h after compound addition, according to the manufacturer’s recommendations. The 50% cytotoxic concentration (CC50) values for single compounds were defined as the concentrations required to reduce cell viability by 50% in comparison with untreated control values. The effects of the combination of RO5855 or sofosbuvir with ribavirin on cell viability were determined using Prichard’s model-based synergy (MacSynergy II model; see below); mean values for percent inhibition generated from all experiments were used in the in vitro drug-drug interaction model. MacSynergy II. The MacSynergy II program allows the three-dimensional examination of drug interactions using all data points generated from the checkerboard combination of two compounds, with the Bliss independence model (46). The volumes of synergy or antagonism are graphed in three dimensions and represent the relative quantity of synergism/additivity or antagonism per change in the two drug concentrations. If the 95% confidence interval (CI) limits do not overlap the theoretical additive surface, then the interaction between the two drugs differs significantly from additive. Values of synergy or antagonism volume under 25 ␮M2 % are considered insignificant, those between 25 ␮M2 % and 50 ␮M2 % indicate minor synergy or antagonism, those between 50 ␮M2 % and 100 ␮M2 % indicate moderate synergy or antagonism, and those over 100 ␮M2 % indicate significant synergy or antagonism. Values of maximal percent synergy or antagonism below 25% are not considered significant. Values between 25% and 50% are considered to be of moderate significance. Values above 50% are considered to be highly significant. Greco’s model (Interact). The Interact program was developed by the research biostatistics group at Roche (Palo Alto, CA) using SAS software. This program uses the concentration-response surface approach described by Greco and colleagues (45). A seven-parameter nonlinear model is fitted to all experimental data with unweighted least-squares nonlinear regression. Synergy is indicated when the drug interaction parameter (␣) is positive, with the 95% CI not including 0, and the maximal percent

aac.asm.org 2615


tabolite in primary human hepatocytes is formed primarily by deamination of the CMP metabolite (RO5855 monophosphate [RO5855-MP]) (24). As noted above, ribavirin remains an important component of therapy for chronic hepatitis C. Ribavirin (1-␤-D-ribofuranosyl1H-1,2,4-triazole-3-carboxamide) is a nucleoside analog with an unnatural base moiety. Ribavirin administered as monotherapy produces modest reductions in HCV RNA levels (26), but ribavirin used in combination with pegylated alpha interferon or with pegylated alpha interferon and an HCV NS3/4A protease inhibitor significantly increases SVR rates, primarily by preventing relapse after the completion of treatment (9, 27, 28). The precise mechanism of action by which ribavirin exerts its antiviral activity is uncertain. The drug is a weak inhibitor of the HCV polymerase and may act as a mutagen after being incorporated into nascent HCV RNA strands (29–33). Ribavirin also depletes intracellular guanine reserves, which are required for the initiation of HCV genome replication, through inhibition of IMP dehydrogenase (34). Recently, ribavirin has been shown to enhance the binding of signal transducer and activator of transcription 1 (STAT1) to DNA and to upregulate certain interferonstimulated genes (ISGs) in vitro and in patients receiving treatment for chronic hepatitis C (35, 36). Being a nucleoside analog that undergoes intracellular phosphorylation, ribavirin has the potential to interfere with the metabolism of other drugs that require phosphorylation to become active. For example, ribavirin reduces the in vitro phosphorylation of pyrimidine analogs used to treat HIV (37–39) and was shown to antagonize the in vitro anti-HCV activity of the HCV nucleoside analog polymerase inhibitor valopicitabine in a human hepatoblastoma cell line (Huh6) (40). To address the potential for ribavirin to interact with HCV polymerase inhibitors such as RO5855, a series of in vitro studies was designed to determine the effects of the combination of RO5855 and ribavirin on the intracellular metabolism of either agent, on ISG expression, and on the viability of a panel of hepatocyte-derived cells. The effect of the combination on HCV replication in the HCV subgenomic replicon was also evaluated using two drug-drug interaction models.

Ma et al.

TABLE 1 Prichard’s model-based synergy and antagonism volumes and maximal percent inhibition for the pairwise checkerboard combinations 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.

synergy is greater than 10%. Antagonism is indicated when ␣ is negative, with the 95% CI not including 0, and the maximal percent antagonism is less than ⫺10%. No interaction is indicated when the 95% CI of ␣ includes 0. Effects of RO5855 on ribavirin metabolism and of ribavirin on RO5855 metabolism. The uptake and metabolism of radiolabeled ribavirin by human PBMCs from three donors, primary human hepatocytes from three donors, and subgenomic replicon cells were characterized in the presence or absence of RO5855. The effects of RO5855 on ribavirin

2616

aac.asm.org

Drug 1

Drug 2

Synergy volume (␮M2 %)

RO5855 Sofosbuvir Alpha-2a interferon

Ribavirin Ribavirin Ribavirin

0 0 0

Combination

Antagonism volume (␮M2 %)

Maximal synergy or antagonism range (%)

Assessmenta

⫺33.91 ⫺6.51 ⫺14.75

⫺6.39 to 0 ⫺5.28 to 0 ⫺1.65 to 0

Additive Additive Additive

a Assessments follow the guidance in the MacSynergy II software manual. Values of synergy or antagonism volumes under 25 ␮M2 % are considered insignificant. Values between 25 ␮M2 % and 50 ␮M2 % are considered to indicate minor synergy or antagonism, values between 50 ␮M2 % and 100 ␮M2 % indicate moderate synergy or antagonism, and values over 100 ␮M2 % indicate significant synergy. Maximal percent of synergy or antagonism values below 25% are not considered significant, values between 25% and 50% are considered of moderate significance, and values above 50% are considered of high significance. The mean percent inhibition values from 4 to 7 experiments (n ⫽ 7 for RO5855 plus ribavirin and alpha-2a interferon plus ribavirin and n ⫽ 4 for sofosbuvir plus ribavirin) were used to generate the assessments.



uptake and phosphorylation were determined by incubating PBMCs with 5 or 10 ␮M [3H]ribavirin in the absence or presence of RO5855 at concentrations up to 100 ␮M. Similarly, the uptake and metabolism of radiolabeled [3H]RO5855 by primary human hepatocytes from three donors were characterized in the presence or absence of ribavirin. Hepatocytes were incubated with 2 ␮M [3H]RO5855 (10 ␮Ci/ml) alone or in combination with 2, 10, 25, 50, or 250 ␮M unlabeled ribavirin. In these experiments, duplicate cell samples were harvested 24 h after the addition of radiolabeled RO5855 with or without unlabeled ribavirin and 48 h after the addition of radiolabeled ribavirin with or without RO5855. The viable cell numbers for the untreated cell control were counted at the end of the experiment by using the trypan blue exclusion method, as previously described (24). The preparation and reconstitution of cellular extracts for the analysis of intracellular metabolites were conducted as previously described (24). The intracellular species of ribavirin and RO5855 were identified by comparing the retention times of peaks in the radiochromatogram with the retention times of nonradioactive reference standards detected by UV absorption at 212 and 270 nm, respectively. A 25-␮l extract from each of the reconstituted samples was added to 5 ml of Ultima-Flow AP cocktail (PerkinElmer, Waltham, MA, USA) and counted using a Beckman LS 6500 liquid scintillation counter, to determine the total radioactivity. Counts, in disintegrations per minute (dpm) per 25 ␮l, were expressed as counts per dpm per million cells, based on the cell count for each experiment. Using the conversion factor of 1 ␮Ci ⫽ 2.22 ⫻ 106 dpm, counts were expressed in ␮Ci per million cells. Such values were then converted to pmol per million cells based on the concentration and specific activity of the radiolabeled compound. Effects of ribavirin and RO5855, alone and in combination, on ISG expression and cell viability. The transcriptional responses to ribavirin and to ribavirin combined with RO5855 were evaluated in primary human hepatocytes and in various human hepatoma cell lines by quantitative reverse transcription (qRT)-PCR measurements of selected interferon-stimulated genes. Primary hepatocytes were plated on 96-well collagen-coated plates, at a density of 50,000 cells per well, in Williams’ medium E without phenol red, with supplements supplied by the vendor. Huh7 and Huh-7-interferon-cured cells were plated on 96-well plates, also at a density of 50,000 cells per well, in DMEM supplemented with 5% FBS. Cells were allowed to equilibrate for at least 4 h at 37°C in 5% CO2 before the test compounds were added for 24 h before harvesting for analyses. Ribavirin concentrations (0, 41, 123, and 410 ␮M, i.e., 0, 10, 30, and 100 ␮g/ml, respectively) were selected to match those in a study that evaluated the transcriptional responses to this compound (35). The RO5855 concentrations evaluated ranged from 0.05 to 100 ␮M. A no-


TABLE 2 Greco’s model-based ␣ factors for the pairwise checkerboard combinations Combination Drug 1

Drug 2

␣ (standard error [95% CI])

Range of synergy or antagonism (%)

Assessmenta

RO5855 Sofosbuvir Alpha-2a interferon

Ribavirin Ribavirin Ribavirin

⫺0.13 (0.023 [⫺0.18 to ⫺0.089]) ⫺0.023 (0.059 [⫺0.14 to 0.092]) ⫺0.008 (0.06 [⫺0.12 to 0.10])

⫺2.57 to ⫺0.01 ⫺0.33 to ⫺0.0005 ⫺0.75 to 0.083

Additive Additive Additive

a Synergy is indicated when the drug interaction parameter (␣) is positive, with the 95% CI not including 0, and the maximal percent synergy is greater than 10%. Antagonism is indicated when ␣ is negative, with the 95% CI not including 0, and the maximal percent antagonism is less than ⫺10%. No interaction is indicated when the 95% CI of ␣ includes 0. The mean percent inhibition values from 4 to 7 experiments (n ⫽ 7 for RO5855 plus ribavirin and alpha-2a interferon plus ribavirin and n ⫽ 4 for sofosbuvir plus ribavirin) were used to generate the assessments.

RESULTS

Inhibition of HCV replicon replication by RO5855 and ribavirin. We conducted in vitro experiments to determine the effects of combining RO5855 and ribavirin on HCV replicon replication. The inhibitory activities (50% effective concentration [EC50] and EC90) of each compound, together with the percent inhibition observed with the maximal concentrations in the cell viability assay, were first determined (see Table S2 in the supplemental material). The inhibitory activities observed in the replicon assays


were in agreement with previously published data, and all concentration ranges chosen for the drug-drug interaction analyses were below the cytotoxic concentrations for the compounds (47–49). In the Prichard model-based synergy plot for the combination of RO5855 and ribavirin, the sum of the antagonism volumes determined for each concentration was calculated as ⫺33.91 ␮M2 (95% confidence limit [CL]), with a maximal percent antagonism range of ⫺6.39% to 0%. While the antagonism volume exceeded the threshold of ⫺25 ␮M2, the maximal percent antagonism was low and well within the range of ⫺20% to 20%, indicating nonsignificance. The combination of RO5855 and ribavirin therefore met the definition of additivity in this model (Fig. 1A and Table 1). In the Greco model-based analysis, the calculated alpha factor was ⫺0.13, with a 95% CI of ⫺0.18 to ⫺0.089. The maximal percent antagonism ranged from ⫺2.51% to ⫺0.01% (Fig. 1B and Table 2). The combination of ribavirin and RO5855 met the definition of additivity in this model, as the maximal percent antagonism did not exceed 10%. The effect of the combination of RO5855 and ribavirin on cell viability was also determined using Prichard’s model-based synergy (Fig. 1C). The combination met the definition of additivity, as maximal percent antagonism or synergy did not extend outside the range of ⫺20% to 20% (⫺3.75% to 0%). Similar to the results obtained with RO5855 and ribavirin, the combination of sofosbuvir (a phosphoramidate derivative of 2=deoxy-2=-␣-fluoro-␤-C-methyluridine-5=-monophosphate) and ribavirin was found to be additive in both drug-drug interaction models (Tables 1 and 2). No effect of the combination on cell viability was observed (data not shown). As a control, alpha-2a interferon in combination with ribavirin was found to be additive (Tables 1 and 2), with no effect on cell viability (data not shown). The maximal percent inhibition of cell viability in the combination experiments was seen at the maximal concentration of each compound (50 ␮M for RO5855 and ribavirin and 10 ␮M for sofosbuvir) and was not significantly greater than that observed with ribavirin alone (see Table S2 in the supplemental material). Effects of RO5855 on ribavirin metabolism and of ribavirin on RO5855 metabolism. As both ribavirin and RO5855 require phosphorylation by cellular kinases to exert their pharmacological effects, we conducted experiments to determine the potential for mutual interference in the uptake and intracellular phosphorylation of the two compounds. At concentrations up to 100 ␮M, RO5855 did not affect the uptake or phosphorylation of 5 ␮M ribavirin in primary human hepatocytes (Fig. 2A and B), of 5 or 10 ␮M ribavirin in human PBMCs (Fig. 2C and D), or of 5 ␮M ribavirin in subgenomic replicon cells (Fig. 2E and F). Consistent with the additive inhibition of HCV replicon replication by the

aac.asm.org 2617


treatment control and a phosphate-buffered saline (PBS) control (PBS at a concentration of 5.1%, to match the highest compound concentration) were included on each plate as negative controls. Recombinant alpha-2a interferon (311 IU/ml) was included as a positive control for the induction of ISG expression. Cell viability was assessed in parallel with the effects of ribavirin and RO5855, alone and in combination, on ISG expression under the same assay conditions, using the CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI, USA) and the manufacturers’ procedures. The inhibition of cell viability was calculated as the percent reduction in comparison with the untreated control values, and CC50 values were defined as the compound concentrations required to reduce cell viability by 50%, in comparison with the untreated control values. Total RNA extraction and qRT-PCR analysis. qRT-PCR was performed to measure the expression of select genes. Total cellular RNA was isolated from the primary hepatocytes and hepatoma cell lines 24 h after the addition of compounds, by using the PerfectPure RNA 96 cell kit or CellVac kit according to the manufacturer’s instructions (5 Prime, Inc., Gaithersburg, MD). The RNA was then reverse transcribed (Transcriptor first-strand cDNA synthesis kit; Roche Applied Science, Indianapolis, IN) using a random hexamer as the primer. qRT-PCR analysis was performed on an Applied Biosystems 7900 system (Foster City, CA) using EagleTaq (Roche Applied Science, Indianapolis, IN, USA), primers, and 6-carboxyfluorescein (FAM)-6-carboxytetramethylrhodamine (TAMRA), FAMIowa Black, or FAM-ZEN-Iowa Black dually labeled probes (IDT, Coralville, IA). Primers and probes were designed against 18S rRNA and interferon regulatory factor 7 (IRF7), IRF9, ISG15, and STAT1 mRNAs (see Table S1 in the supplemental material). Expression levels for each ISG were quantitated and normalized to 18S rRNA levels. These ISGs were selected because they have been reported to be transcriptionally responsive to ribavirin (36). Statistical analysis. Statistical significance was determined by 2-way analysis of variance (ANOVA) with the Bonferroni correction for multiple testing. P values of ⬍0.05 were considered to indicate statistically significant differences. A consequence of expressing data relative to the no-treatment control was removal of variability in the latter population (i.e., all values were set to 100% or 1). Therefore, for statistical analysis, comparisons were made to the PBS control. Statistical analyses were performed using GraphPad Prism version 5.03 (GraphPad Software, Inc., La Jolla, CA). Plotted data were displayed as means and standard deviations.

Ma et al.

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.

combination of ribavirin and RO5855 observed in the drug-drug interaction experiments (described above), RO5855 at concentrations up to 100 ␮M did not inhibit the uptake or phosphorylation of 5 or 10 ␮M ribavirin in subgenomic replicon cells. Conversely, the addition of ribavirin at concentrations up to 250 ␮M did not affect the concentrations of the total intracellular species derived from the incubation of 2 ␮M radiolabeled RO5855 in primary human hepatocytes obtained from three different donors (Fig. 3A). The results were consistent across cells derived from the three donors.

2618

aac.asm.org

The formation of RO5855-TP and RO2433-TP, the active triphosphorylated metabolites derived from RO5855, was not affected by the addition of ribavirin at concentrations up to 50 ␮M (Fig. 3B). At a ribavirin concentration of 250 ␮M, the concentration of RO2433-TP formed with a 2 ␮M extracellular concentration of RO5855 was reduced by a mean of 54% in the three donors, whereas the concentration of RO5855-TP was increased by a mean of 169%. This apparent reciprocal effect on the two triphosphate species may indicate inhibition of the deamination pathway in the presence of high ribavirin concentrations, because



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


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 phosphatebuffered saline control were as follows: ⴱⴱⴱ, P ⬍ 0.001.

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 ␮M (derived from two donors).

RO2433-TP is formed in hepatocytes by deamination of RO5855MP. However, triphosphate quantification under high-ribavirin conditions was associated with relatively high data variability across replicates, which may have been caused by ribavirin-associated cytotoxicity in human hepatocytes at concentrations above 40 ␮M. Further studies would therefore be needed to determine if high concentrations of ribavirin or ribavirin metabolites can interfere with RO5855-MP deamination. Effects of ribavirin, with and without RO5855, on cell viability. We investigated the effects of RO5855 in combination with ribavirin on the viability of primary human hepatocytes, Huh7 cells, interferon-cured Huh7 cells, and the G1b replicon cell line. Ribavirin significantly reduced the viability of primary human hepatocytes after 24 h of exposure at all concentrations tested (41 ␮M, 123 ␮M, and 410 ␮M) and reduced hepatocyte viability in a dose-dependent manner, by up to 65% ⫾ 10% at 410 ␮M (Fig. 4), as measured with the CellTiter-Glo luminescent cell viability assay. In contrast, ribavirin did not have a significant impact on the viability of Huh7 cells and G1b replicon cells (as observed in the drug-drug interaction experiments described above) or interferon-cured Huh7 cells, under the same assay conditions, at any of the concentrations tested (Fig. 4). Under the same assay conditions as described above, RO5855


at concentrations up to and including 100 ␮M, alone or in combination with ribavirin, did not significantly alter the viability of Huh7 cells (Fig. 5A) or G1b replicon cells (Fig. 5B). When administered alone, RO5855 did not significantly alter the viability of primary human hepatocytes (Fig. 5C). When RO5855 was combined with ribavirin, however, a reduction in the viability of primary human hepatocytes was observed (Fig. 5C), similar to the extent observed with ribavirin alone (Fig. 4). Effects of RO5855 and ribavirin, alone and in combination, on ISG expression. At concentrations up to and including 100 ␮M, RO5855 alone did not alter the mRNA levels of ISG15 (Fig. 6A to C), STAT1 (Fig. 6D to F), IRF7 (Fig. 6G to I), or IRF9 (Fig. 6J to L) in any of the cells tested. Consistent with these observations, the combination of RO5855 with ribavirin showed levels of expression of ISG15 (Fig. 6A to C), STAT1 (Fig. 6D to F), IRF7 (Fig. 6G to I), and IRF9 (Fig. 6J to L) similar to those seen with ribavirin alone. Next, we evaluated the effects of ribavirin and RO5855, alone and in combination, on the expression of ISGs that were previously reported to be responsive to ribavirin (36). There was apparent ribavirin dose-dependent induction of ISG mRNA in Huh7, Huh7-derived HCV replicon cells, and interferon-cured replicon cells (Fig. 7A). ISG15 mRNA induction ranged from 2.0to 2.7-fold at the highest concentration of ribavirin (100 ␮g/ml, 410 ␮M) in Huh7 cells. The induction of ISG15 mRNA by ribavirin in Huh7-derived HCV replicon cells (2209-23 cells) ranged from 0.9- to 2.1-fold across all experiments and that in interferoncured replicon cells ranged from 1.2- to 1.8-fold at the highest ribavirin concentration. The induction of ISG15 transcription in Huh7 cells by ribavirin at both 123 ␮M and 410 ␮M reached statistical significance by 2-way ANOVA with the Bonferroni correction (P ⬍ 0.05), whereas the inductions in HCV replicon cells and interferon-cured replicon cells did not reach statistical significance (Fig. 7A). Additional linear regression and analysis of covariance (ANCOVA) of slopes and intercepts showed statistical significance of ISG15 mRNA induction in Huh7 and HCV repli-

aac.asm.org 2619


FIG 4 Effects of ribavirin on the viability of primary human hepatocytes,

Ma et al.

con cells, while the trend of increasing mRNA induction did not reach statistical significance in interferon-cured replicon cells. ISG15 mRNA induction levels in primary human hepatocytes were not significant and ranged from 0.42- to 1.4-fold at the highest ribavirin concentration (Fig. 7A). In addition, ribavirin did not significantly induce the expression of IRF7 (Fig. 7B) or IRF9 (Fig. 7C) in any of the cell types tested. Ribavirin did not significantly alter STAT1 mRNA levels in Huh7 cells, G1b replicon cells, or interferon-cured Huh7 cells at all concentrations up to 410 ␮M, and ribavirin did not significantly alter STAT1 mRNA levels in primary hepatocytes at up to 123 ␮M (Fig. 7D). The highest concentration of ribavirin (410 ␮M) slightly decreased STAT1 mRNA levels in primary hepatocytes (Fig. 7D). No conclusions regarding the biological significance of the observed reduction of STAT1 expression with 410 ␮M ribavirin can be made, due to the reduced viability of primary hepatocytes exposed to this high concentration of ribavirin (Fig. 4). DISCUSSION

Ribavirin is an essential component of the approved dual- and triple-therapy combination regimens for treatment of chronic hepatitis C (1, 50). Although it is ineffective as monotherapy (38), ribavirin significantly increases SVR rates by preventing relapse, through an unknown mechanism of action (9, 27, 28). Of note, ribavirin also increases SVR rates and prevents relapse when included in diverse combination DAA regimens, including regimens that contain the nucleotide analog sofosbuvir (11, 12, 15–17, 51). As both ribavirin and RO5855 need to be phosphorylated by cellular kinases to exert their pharmacological effects (24, 52), we conducted in vitro experiments to determine the effects of the two drugs in combination on HCV replicon replication and cell viability and on their mutual interference in the uptake and intracellular phosphorylation of each other. Drug-drug interaction studies using the G1b subgenomic replicon system with a range of concentrations of RO5855 and ribavirin showed consistently that the combination of these two drugs is additive, according to two drug-drug interaction models. In addition, the combination of RO5855 and ribavirin did not have a

2620

aac.asm.org

significant effect on replicon cell viability when evaluated over 3 days. Overall, the results presented here are in agreement with previously published in vitro data on the effects of the combination of an anti-HCV nucleoside analog with ribavirin on the HCV replicon in Huh7 cells. For example, the combinations of sofosbuvir and ribavirin (53) and IDX-184 and ribavirin (54) have been reported to be weakly synergistic, and the combination of VX-135 and ribavirin has been reported to have additive activity (55). These findings are in contrast to those of Coelmont et al. (40), who showed with Prichard’s model that ribavirin had an antagonistic effect on the anti-HCV activity of 2=-C-methylcytidine, the pharmacologically active metabolite of valopicitabine, in Huh6 cells containing a subgenomic G1b HCV replicon. It is unclear whether their observations were mediated by interference with the intracellular metabolism of 2=-C-methylcytidine or by another unidentified mechanism in this particular cell line, which has been shown to differ from Huh7 in critical aspects necessary for the maintenance of replicon replication (56). Ribavirin was cytotoxic ex vivo to primary human hepatocytes from all donors (n ⫽ 5). The effect was dose dependent over the concentration range of 41 to 410 ␮g/ml, with marked cytotoxicity at 123 and 410 ␮M (30 and 100 ␮g/ml, respectively). In contrast, RO5855 was not cytotoxic to primary human hepatocytes at concentrations up to 100 ␮M in any of the cell lines evaluated and did not augment the cytotoxicity of ribavirin in human hepatocytes when the two drugs were combined. RO5855 did not affect the uptake or phosphorylation of ribavirin in human hepatocytes, PBMCs, or HCV replicon cells at concentrations up to 100 ␮M. Conversely, ribavirin did not affect the uptake or phosphorylation of RO5855 in primary human hepatocytes. There were no changes in the amounts of total intracellular species when cells were incubated with radiolabeled RO5855 alone or in combination with ribavirin at concentrations of up to 50 ␮M. Moreover, the levels of the active triphosphorylated metabolites of mericitabine (RO5855-TP and RO2433-TP) in primary human hepatocytes were not altered by the presence of ribavirin at up to 50 ␮M. Surprisingly, we observed a 54% mean reduction in the concentrations of the UTP analog RO2433-TP in the presence of 250 ␮M extracellular ribavirin in primary human



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: ⴱⴱ, P ⬍ 0.01; ⴱⴱⴱ, P ⬍ 0.001.


Downloaded from http://aac.asm.org/ on September 11, 2014 by Univ of Idaho 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: ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01; ⴱⴱⴱ, P ⬍ 0.001.


aac.asm.org 2621

Ma et al.

hepatocytes from two donors, which was mirrored by a 169% mean increase in the concentrations of the CTP analog RO5855TP. The changes in RO5855-TP and RO24533-TP concentrations with exposure to 250 ␮M extracellular ribavirin could be due to assay variability with cytotoxic ribavirin concentrations or may indicate reduced cytidylate deamination in the presence of very high ribavirin concentrations. As both the CTP and UTP analogs are potent inhibitors of HCV polymerase (24), the biological consequences of this shift are likely to be small. Further studies could determine whether human dCMP deaminase is sensitive to inhibition by ribavirin or ribavirin phosphates. Ribavirin was shown previously to influence ISG expression in Huh7.5.1 cells (36). In the present study, ribavirin dose-dependent increases were apparent in Huh7, HCV replicon, and interferon-cured replicon cells. Ribavirin significantly increased ISG15 mRNA levels in Huh7 and HCV replicon cells, with induction levels ranging from 0.9- to 2.7-fold across cell lines and in several independent experiments at the highest ribavirin concentration tested (410 ␮M, 100 ␮g/ml). In contrast, ribavirin showed no significant induction of ISG15 mRNA in primary human hepatocytes or interferon-cured replicon cells. However, the measurements in hepatocytes were confounded by significant cytotoxicity of ribavirin at 123 and 410 ␮M (30 and 100 ␮g/ml, respectively),

2622

aac.asm.org

the highest doses used in the experiments. Ribavirin did not induce significant changes in IRF7, IRF9, or STAT1 mRNA levels in any of the cell types tested. RO5855, when applied alone or in combination with ribavirin, had no effect on gene expression levels in any of the cell lines. The effects of ribavirin on ISG induction in our experiments differed from those reported by Thomas et al. (36), despite the use of similar concentrations of ribavirin. Thomas et al. (36) reported that ribavirin induced not only ISG15 but also IRF7 and IRF9 in Huh7.5.1 cells. The reasons for this difference are not immediately obvious; however, Thomas et al. (36) used Huh7.5 cells in their in vitro experiments, which are defective in the retinoic acid-inducible gene-1 pathway, in contrast to the Huh7 and replicon cells used in our studies. Moreover, Thomas et al. (36) examined the impact of ribavirin on ISG expression in liver biopsy specimens obtained from patients treated with pegylated interferon with or without ribavirin, whereas we conducted ex vivo experiments with freshly isolated primary human hepatocytes. Mericitabine is an attractive component in combination regimens for the treatment of chronic hepatitis C. It has pangenotypic activity and a high barrier to resistance (18, 19). Importantly, mericitabine has been well tolerated in clinical studies to date and did not exacerbate ribavirin-associated anemia (21, 22). The re-



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: ⴱⴱ, P ⬍ 0.01; ⴱⴱⴱ, P ⬍ 0.001. Ribavirin concentrations were as follows: 10 ␮g/ml ⫽ 41 ␮M, 30 ␮g/ml ⫽ 123 ␮M, and 100 ␮g/ml ⫽ 410 ␮M.


ACKNOWLEDGMENTS This work was supported by Hoffmann-La Roche, Inc. Support for thirdparty writing assistance for the manuscript was provided by F. Hoffmann-La Roche Ltd.

REFERENCES 1. Ghany MG, Nelson DR, Strader DB, Thomas DL, Seeff LB. 2011. An update on treatment of genotype 1 chronic hepatitis C virus infection: 2011 practice guideline by the American Association for the Study of Liver Diseases. Hepatology 54:1433–1444. http://dx.doi.org/10.1002/hep.24641. 2. Bacon BR, Gordon SC, Lawitz E, Marcellin P, Vierling JM, Zeuzem S, Poordad F, Goodman ZD, Sings HL, Boparai N, Burroughs M, Brass CA, Albrecht JK, Esteban R. 2011. Boceprevir for previously treated chronic HCV genotype 1 infection. N. Engl. J. Med. 364:1207–1217. http: //dx.doi.org/10.1056/NEJMoa1009482. 3. Flamm SL, Lawitz E, Jacobson I, Bourliere M, Hezode C, Vierling JM, Bacon BR, Niederau C, Sherman M, Goteti V, Sings HL, Barnard RO, Howe JA, Pedicone LD, Burroughs MH, Brass CA, Albrecht JK, Poordad F. 2013. Boceprevir with peginterferon alfa-2a-ribavirin is effective for previously treated chronic hepatitis C genotype 1 infection. Clin. Gastroenterol. Hepatol. 11:81– 87. http://dx.doi.org/10.1016/j.cgh.2012.10 .006. 4. Jacobson IM, McHutchison JG, Dusheiko G, Di Bisceglie AM, Reddy KR, Bzowej NH, Marcellin P, Muir AJ, Ferenci P, Flisiak R, George J, Rizzetto M, Shouval D, Sola R, Terg RA, Yoshida EM, Adda N, Bengtsson L, Sankoh AJ, Kieffer TL, George S, Kauffman RS, Zeuzem S. 2011. Telaprevir for previously untreated chronic hepatitis C virus infection. N. Engl. J. Med. 364:2405–2416. http://dx.doi.org/10.1056 /NEJMoa1012912. 5. Poordad F, McCone J, Jr, Bacon BR, Bruno S, Manns MP, Sulkowski MS, Jacobson IM, Reddy KR, Goodman ZD, Boparai N, DiNubile MJ, Sniukiene V, Brass CA, Albrecht JK, Bronowicki JP. 2011. Boceprevir for untreated chronic HCV genotype 1 infection. N. Engl. J. Med. 364: 1195–1206. http://dx.doi.org/10.1056/NEJMoa1010494. 6. Sherman KE, Flamm SL, Afdhal NH, Nelson DR, Sulkowski MS, Everson GT, Fried MW, Adler M, Reesink HW, Martin M, Sankoh AJ, Adda N, Kauffman RS, George S, Wright CI, Poordad F. 2011. Response-guided telaprevir combination treatment for hepatitis C virus infection. N. Engl. J. Med. 365:1014 –1024. http://dx.doi.org/10.1056/NEJ Moa1014463.


7. Zeuzem S, Andreone P, Pol S, Lawitz E, Diago M, Roberts S, Focaccia R, Younossi Z, Foster GR, Horban A, Ferenci P, Nevens F, Mullhaupt B, Pockros P, Terg R, Shouval D, van Hoek B, Weiland O, Van Heeswijk R, De Meyer S, Luo D, Boogaerts G, Polo R, Picchio G, Beumont M. 2011. Telaprevir for retreatment of HCV infection. N. Engl. J. Med. 364:2417–2428. http://dx.doi.org/10.1056/NEJMoa1013086. 8. Sarrazin C, Kieffer TL, Bartels D, Hanzelka B, Muh U, Welker M, Wincheringer D, Zhou Y, Chu HM, Lin C, Weegink C, Reesink H, Zeuzem S, Kwong AD. 2007. Dynamic hepatitis C virus genotypic and phenotypic changes in patients treated with the protease inhibitor telaprevir. Gastroenterology 132:1767–1777. http://dx.doi.org/10.1053/j.gastro .2007.02.037. 9. Hezode C, Forestier N, Dusheiko G, Ferenci P, Pol S, Goeser T, Bronowicki JP, Bourliere M, Gharakhanian S, Bengtsson L, McNair L, George S, Kieffer T, Kwong A, Kauffman RS, Alam J, Pawlotsky JM, Zeuzem S. 2009. Telaprevir and peginterferon with or without ribavirin for chronic HCV infection. N. Engl. J. Med. 360:1839 –1850. http://dx.doi .org/10.1056/NEJMoa0807650. 10. Everson GT, Sims KD, Rodriguez-Torres M, Hezode C, Lawitz E, Bourliere M, Loustaud-Ratti V, Rustgi V, Schwartz H, Tatum H, Marcellin P, Pol S, Thuluvath PJ, Eley T, Wang X, Huang SP, McPhee F, Wind-Rotolo M, Chung E, Pasquinelli C, Grasela DM, Gardiner DF. 2014. Efficacy of an interferon- and ribavirin-free regimen of daclatasvir, asunaprevir, and BMS-791325 in treatment-naive patients with HCV genotype 1 infection. Gastroenterology 146:420 – 429. http://dx.doi.org/10 .1053/j.gastro.2013.10.057. 11. Gane EJ, Stedman CA, Hyland RH, Ding X, Svarovskaia E, Symonds WT, Hindes RG, Berrey MM. 2013. Nucleotide polymerase inhibitor sofosbuvir plus ribavirin for hepatitis C. N. Engl. J. Med. 368:34 – 44. http: //dx.doi.org/10.1056/NEJMoa1208953. 12. Kowdley KV, Lawitz E, Poordad F, Cohen DE, Nelson D, Zeuzem S, Everson GT, Kwo P, Foster GR, Sulkowski M, Xie W, Larsen L, Khatri A, Podsadecki T, Bernstein B. 2013. Safety and efficacy of interferon-free regimens of ABT-450/r, ABT-267, ABT-333 ⫹/⫺ ribavirin in patients with chronic HCV GT1 infection: results from the AVIATOR study. J. Hepatol. 58(Suppl):S2. http://dx.doi.org/10.1016/S0168-8278(13)60005-7. 13. Sulkowski MS, Gardiner DF, Rodriguez-Torres M, Reddy KR, Hassainen T, Jacobson I, Lawitz E, Lok AS, Hinestrasa F, Thuluvath PJ, Schwartz H, Nelson DR, Everson GT, Eley T, Wind-Rotolo M, Huang S-P, Gao M, McPhee F, Hernandez D, Sherman D, Hindes R, Symonds W, Pasquinelli C, Grasela DM. 2013. Sustained virologic response with daclatasvir plus sofosbuvir ⫹/⫺ ribavirin (RBV) in chronic HCV genotype (GT) 1-infected patients who previously failed telaprevir or boceprevir (BOC). J. Hepatol. 58(Suppl):S570. http://dx.doi.org/10.1016/S0168 -8278(13)61416-6. 14. Sulkowski MS, Gardiner DF, Rodriguez-Torres M, Reddy KR, Hassainen T, Jacobson IM, Lawitz E, Lok AS, Hinestrasa F, Thuluvath PJ, Schwartz H, Nelson DR, Eley T, Wind-Rotolo M, Huang S-P, Gao M, McPhee F, Sherman D, Hindes R, Symonds WT, Pasquinelli C, Grasela DM. 2012. High rate of sustained virologic response with the all-oral combination of daclatasvir (NS5A inhibitor) plus sofosbuvir (nucleotide NS5B inhibitor), with or without ribavirin, in treatment-naive patients chronically infected with HCV genotype 1, 2, or 3, abstr LB-2. Abstr. 63rd Ann. Meet. Am. Assoc. Study Liver Dis., Boston, MA, 9 to 12 November 2012. 15. Jacobson IM, Sulkowski MS, Gane EJ, Koziel MJ, DeSouza C, Kieffer TL, Penney MS, Zhang EZ, George S, Kauffman RS, Nelson DR, DiBisceglie AM. 2012. VX-222, telaprevir and ribavirin in treatmentnaive patients with genotype 1 chronic hepatitis C: results of the ZENITH Study interferon-free regimen. Hepatology 56(Suppl):308A. http://dx.doi .org/10.1002/hep.26040. 16. Jacobson IM, Gordon SC, Kowdley KV, Yoshida EM, Rodriguez-Torres M, Sulkowski MS, Shiffman ML, Lawitz E, Everson G, Bennett M, Schiff E, Al-Assi MT, Subramanian GM, An D, Lin M, McNally J, Brainard D, Symonds WT, McHutchison JG, Patel K, Feld J, Pianko S, Nelson DR. 2013. Sofosbuvir for hepatitis C genotype 2 or 3 in patients without treatment options. N. Engl. J. Med. 368:1867–1877. http://dx.doi.org/10.1056 /NEJMoa1214854. 17. Zeuzem S, Soriano V, Asselah T, Bronowicki JP, Lohse AW, Mullhaupt B, Schuchmann M, Bourliere M, Buti M, Roberts SK, Gane EJ, Stern JO, Vinisko R, Kukolj G, Gallivan JP, Bocher WO, Mensa FJ. 2013. Faldaprevir and deleobuvir for HCV genotype 1 infection. N. Engl. J. Med. 369:630 – 639. http://dx.doi.org/10.1056/NEJMoa1213557.

aac.asm.org 2623


sults of these in vitro studies are also consistent with the recently completed JUMP-C study, which showed that the combination of mericitabine plus pegylated alpha-2a interferon-ribavirin significantly increased SVR rates above those achieved with placebo plus pegylated alpha-2a interferon-ribavirin in treatment-naive patients infected with G1 or G4 (21). Consistent with the findings in the JUMP-C study, mericitabine improved SVR rates in previously treated patients when it was added to ritonavir-boosted danoprevir (HCV protease inhibitor) plus pegylated alpha-2a interferon-ribavirin (46). In patients with chronic hepatitis C who were treated with mericitabine at 500 or 1,000 mg twice daily plus pegylated alpha-2a interferon-ribavirin, the mean trough plasma concentrations of RO5855 at steady state were 1.2 ␮g/ml (4.6 ␮M) and 2.0 ␮g/ml (7.7 ␮M), respectively, and the mean trough concentrations of ribavirin at steady state were 2.4 (9.8 ␮M) and 2.1 ␮g/ml (8.6 ␮M), respectively. Thus, the ranges of concentrations used in the experiments reported in the present study encompass clinically relevant serum concentration ranges for ribavirin and RO5855. The absence of intracellular drug-drug interactions between RO5855 and ribavirin, as demonstrated in studies reported here, and the absence of pharmacokinetic drug-drug interactions between these two drugs, as reported elsewhere (57), support the use of combinations of these two compounds in clinical studies for the treatment of HCV infection.

Ma et al.

2624

aac.asm.org

32. Maag D, Castro C, Hong Z, Cameron CE. 2001. Hepatitis C virus RNA-dependent RNA polymerase (NS5B) as a mediator of the antiviral activity of ribavirin. J. Biol. Chem. 276:46094 – 46098. http://dx.doi.org/10 .1074/jbc.C100349200. 33. Vo NV, Young KC, Lai MM. 2003. Mutagenic and inhibitory effects of ribavirin on hepatitis C virus RNA polymerase. Biochemistry 42:10462– 10471. http://dx.doi.org/10.1021/bi0344681. 34. Prosise GL, Wu JZ, Luecke H. 2002. Crystal structure of Tritrichomonas foetus inosine monophosphate dehydrogenase in complex with the inhibitor ribavirin monophosphate reveals a catalysis-dependent ion-binding site. J. Biol. Chem. 277:50654 –50659. http://dx.doi.org/10.1074/jbc .M208330200. 35. Feld JJ, Nanda S, Huang Y, Chen W, Cam M, Pusek SN, Schweigler LM, Theodore D, Zacks SL, Liang TJ, Fried MW. 2007. Hepatic gene expression during treatment with peginterferon and ribavirin: identifying molecular pathways for treatment response. Hepatology 46:1548 –1563. http: //dx.doi.org/10.1002/hep.21853. 36. Thomas E, Feld JJ, Li Q, Hu Z, Fried MW, Liang TJ. 2011. Ribavirin potentiates interferon action by augmenting interferon-stimulated gene induction in hepatitis C virus cell culture models. Hepatology 53:32– 41. http://dx.doi.org/10.1002/hep.23985. 37. Baba M, Pauwels R, Balzarini J, Herdewijn P, De Clercq E, Desmyter J. 1987. Ribavirin antagonizes inhibitory effects of pyrimidine 2=,3=dideoxynucleosides but enhances inhibitory effects of purine 2=,3=dideoxynucleosides on replication of human immunodeficiency virus in vitro. Antimicrob. Agents Chemother. 31:1613–1617. http://dx.doi.org /10.1128/AAC.31.10.1613. 38. Hoggard PG, Kewn S, Barry MG, Khoo SH, Back DJ. 1997. Effects of drugs on 2=,3=-dideoxy-2=,3=-didehydrothymidine phosphorylation in vitro. Antimicrob. Agents Chemother. 41:1231–1236. 39. Vogt MW, Hartshorn KL, Furman PA, Chou TC, Fyfe JA, Coleman LA, Crumpacker C, Schooley RT, Hirsch MS. 1987. Ribavirin antagonizes the effect of azidothymidine on HIV replication. Science 235:1376 –1379. http://dx.doi.org/10.1126/science.2435003. 40. Coelmont L, Paeshuyse J, Windisch MP, De CE, Bartenschlager R, Neyts J. 2006. Ribavirin antagonizes the in vitro anti-hepatitis C virus activity of 2=-C-methylcytidine, the active component of valopicitabine. Antimicrob. Agents Chemother. 50:3444 –3446. http://dx.doi.org/10 .1128/AAC.00372-06. 41. Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J. 1982. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res. 42:3858 –3863. 42. Lohmann V, Hoffmann S, Herian U, Penin F, Bartenschlager R. 2003. Viral and cellular determinants of hepatitis C virus RNA replication in cell culture. J. Virol. 77:3007–3019. http://dx.doi.org/10.1128/JVI.77.5.3007 -3019.2003. 43. Klumpp K, Leveque V, LePogam S, Ma H, Jiang WR, Kang H, Granycome C, Singer M, Laxton C, Hang JQ, Sarma K, Smith DB, Heindl D, Hobbs CJ, Merrett JH, Symons J, Cammack N, Martin JA, Devos R, Najera I. 2006. The novel nucleoside analog R1479 (4=azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture. J. Biol. Chem. 281:3793– 3799. http://dx.doi.org/10.1074/jbc.M510195200. 44. Craig J, Duncan I, Whittaker L, Roberts N. 1990. Antiviral synergy between inhibitors of HIV proteinase and reverse transcriptase. Antivir. Chem. Chemother. 4:161–166. 45. Greco WR, Park HS, Rustum YM. 1990. Application of a new approach for the quantitation of drug synergism to the combination of cisdiamminedichloroplatinum and 1-␤-D-arabinofuranosylcytosine. Cancer Res. 50:5318 –5327. 46. Prichard MN, Shipman C, Jr. 1990. A three-dimensional model to analyze drug-drug interactions. Antiviral Res. 14:181–205. http://dx.doi.org /10.1016/0166-3542(90)90001-N. 47. Ali S, Leveque V, Le Pogam S, Ma H, Philipp F, Inocencio N, Smith M, Alker A, Kang H, Najera I, Klumpp K, Symons J, Cammack N, Jiang WR. 2008. Selected replicon variants with low-level in vitro resistance to the hepatitis C virus NS5B polymerase inhibitor PSI-6130 lack crossresistance with R1479. Antimicrob. Agents Chemother. 52:4356 – 4369. http://dx.doi.org/10.1128/AAC.00444-08. 48. Bassit L, Grier J, Bennett M, Schinazi RF. 2008. Combinations of 2=-C-methylcytidine analogues with interferon-alpha2b and triple combination with ribavirin in the hepatitis C virus replicon system. Antivir. Chem. Chemother. 19:25–31.



18. Leveque V, Fung A, Le Pogam S, Kang H, Harris S, Cammack N, Najera I, Klumpp K. 2009. Nucleoside analog R7128, a prodrug of PSI-6130, shows inhibition potency across HCV genotypes 1-6 in vitro, poster P-215. Abstr. 16th Int. Symp. Hepatitis C Virus Relat. Viruses, 3 to 7 October, 2009. 19. Pawlotsky JM, Najera I, Jacobson I. 2012. Resistance to mericitabine, a nucleoside analogue inhibitor of HCV RNA-dependent RNA polymerase. Antivir. Ther. 17:411– 423. http://dx.doi.org/10.3851/IMP2088. 20. Stuyver LJ, McBrayer TR, Tharnish PM, Clark J, Hollecker L, Lostia S, Nachman T, Grier J, Bennett MA, Xie MY, Schinazi RF, Morrey JD, Julander JL, Furman PA, Otto MJ. 2006. Inhibition of hepatitis C replicon RNA synthesis by beta-D-2=-deoxy-2=-fluoro-2=-C-methylcytidine: a specific inhibitor of hepatitis C virus replication. Antivir. Chem. Chemother. 17:79 – 87. 21. Pockros PJ, Jensen D, Tsai N, Taylor R, Ramji A, Cooper C, Dickson R, Tice A, Kulkarni R, Vierling JM, Lou MM, Chen YC, Najera I, Thommes J. 2013. JUMP-C: a randomized trial of mericitabine plus pegylated interferon alpha-2a/ribavirin for 24 weeks in treatment-naive HCV genotype 1/4 patients. Hepatology 58:514 –523. http://dx.doi.org/10.1002/hep .26275. 22. Wedemeyer H, Jensen D, Herring R, Jr, Ferenci P, Ma MM, Zeuzem S, Rodriguez-Torres M, Bzowej N, Pockros P, Vierling J, Ipe D, Munson ML, Chen YC, Najera I, Thommes J. 2013. PROPEL: a randomized trial of mericitabine plus peginterferon alpha-2a/ribavirin therapy in treatment-naive HCV genotype 1/4 patients. Hepatology 58:524 –537. http: //dx.doi.org/10.1002/hep.26274. 23. Haznedar J, Hisoire G, Masjedizadeh M, Washington C. 2011. [14C]RG7128 is extensively converted to the cytidine nucleoside analog PSI6130: ADME results following a single oral dose to healthy male subjects. Clin. Pharmacol. Ther. 89(Suppl):S55. http://dx.doi.org/10.1038/clpt .2010.332. 24. Ma H, Jiang WR, Robledo N, Leveque V, Ali S, Lara-Jaime T, Masjedizadeh M, Smith DB, Cammack N, Klumpp K, Symons J. 2007. Characterization of the metabolic activation of hepatitis C virus nucleoside inhibitor ␤- D -2=-deoxy-2=-fluoro-2=-C-methylcytidine (PSI-6130) and identification of a novel active 5=-triphosphate species. J.Biol.Chem.282:29812–29820.http://dx.doi.org/10.1074/jbc.M70527 4200. 25. Murakami E, Niu C, Bao H, Micolochick Steuer HM, Whitaker T, Nachman T, Sofia MA, Wang P, Otto MJ, Furman PA. 2008. The mechanism of action of ␤-D-2=-deoxy-2=-fluoro-2=-C-methylcytidine involves a second metabolic pathway leading to ␤-D-2=-deoxy-2=-fluoro-2=C-methyluridine 5=-triphosphate, a potent inhibitor of the hepatitis C virus RNA-dependent RNA polymerase. Antimicrob. Agents Chemother. 52:458 – 464. http://dx.doi.org/10.1128/AAC.01184-07. 26. Di Bisceglie AM, Conjeevaram HS, Fried MW, Sallie R, Park Y, Yurdaydin C, Swain M, Kleiner DE, Mahaney K, Hoofnagle JH. 1995. Ribavirin as therapy for chronic hepatitis C: a randomized, double-blind, placebo-controlled trial. Ann. Intern. Med. 123:897–903. 27. Bronowicki JP, Ouzan D, Asselah T, Desmorat H, Zarski JP, Foucher J, Bourliere M, Renou C, Tran A, Melin P, Hezode C, Chevalier M, Bouvier-Alias M, Chevaliez S, Montestruc F, Lonjon-Domanec I, Pawlotsky JM. 2006. Effect of ribavirin in genotype 1 patients with hepatitis C responding to pegylated interferon alfa-2a plus ribavirin. Gastroenterology 131:1040 –1048. http://dx.doi.org/10.1053/j.gastro .2006.07.022. 28. Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Goncales FL, Jr, Haussinger D, Diago M, Carosi G, Dhumeaux D, Craxi A, Lin A, Hoffman J, Yu J. 2002. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N. Engl. J. Med. 347:975–982. http://dx.doi.org /10.1056/NEJMoa020047. 29. Crotty S, Cameron CE, Andino R. 2001. RNA virus error catastrophe: direct molecular test by using ribavirin. Proc. Natl. Acad. Sci. U. S. A. 98:6895– 6900. http://dx.doi.org/10.1073/pnas.111085598. 30. Crotty S, Maag D, Arnold JJ, Zhong W, Lau JY, Hong Z, Andino R, Cameron CE. 2000. The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen. Nat. Med. 6:1375–1379. http://dx.doi.org/10 .1038/82191. 31. Hofmann WP, Polta A, Herrmann E, Mihm U, Kronenberger B, Sonntag T, Lohmann V, Schonberger B, Zeuzem S, Sarrazin C. 2007. Mutagenic effect of ribavirin on hepatitis C nonstructural 5B quasispecies in vitro and during antiviral therapy. Gastroenterology 132:921–930. http: //dx.doi.org/10.1053/j.gastro.2006.12.005.



54. Lallos L, LaColla M, Serra I, Bilello J, Soubasakos M, Li B, Panzo R, Gillum J, Bonin A, Seifer M, Standring DN. 2007. Combination of IDX184, a nucleotide prodrug polymerase inhibitor, with other classes of HCV inhibitors is additive to synergistic in the HCV replicon in vitro. Hepatology 50(Suppl):267A. http://dx.doi.org/10.1002/hep.23299. 55. Tan H, Kang H, May C, Jiang M, Reagan D, Deval J, Dyatkina N, Wang G, Kieffer TL, Rijnbrand R, Beigelman L, Smith D, Blatt L, Symons J. 2012. Analysis of ALS-2200, a novel potent nucleotide analog, combination drug interactions in the hepatitis C virus (HCV) subgenomic replicon system. Hepatology 56(Suppl):1072A. 56. Windisch MP, Frese M, Kaul A, Trippler M, Lohmann V, Bartenschlager R. 2005. Dissecting the interferon-induced inhibition of hepatitis C virus replication by using a novel host cell line. J. Virol. 79:13778 – 13793. http://dx.doi.org/10.1128/JVI.79.21.13778-13793.2005. 57. Chen Y-C, Bernaards C, Kulkarni R, Moreira S, Zhu Y, Chan A, Badman E, Ackrill A, Thommes J, Smith PF. Evaluation of the pharmacokinetics and pharmacodynamics of the HCV polymerase inhibitor mericitabine from the phase 2 JUMP-C and PROPEL studies. Brit. J. Clin. Pharmacol., in press.

aac.asm.org 2625


49. Lam AM, Espiritu C, Bansal S, Micolochick Steuer HM, Niu C, Zennou V, Keilman M, Zhu Y, Lan S, Otto MJ, Furman PA. 2012. Genotype and subtype profiling of PSI-7977 as a nucleotide inhibitor of hepatitis C virus. Antimicrob. Agents Chemother. 56:3359 –3368. http://dx.doi.org/10.112 8/AAC.00054-12. 50. Ghany MG, Strader DB, Thomas DL, Seeff LB. 2009. Diagnosis, management, and treatment of hepatitis C: an update. Hepatology 49:1335– 1374. http://dx.doi.org/10.1002/hep.22759. 51. Gane EJ, Pockros P, Zeuzem S, Marcellin P, Shikhman A, Bernaards C, Yetzer ES, Shulman N, Tong X, Najera I, Bertasso A, Hammond J, Stancic S. 2012. Interferon-free treatment with a combination of mericitabine and danoprevir/r with or without ribavirin in treatment-naive HCV genotype 1-infected patients. J. Hepatol. 56(Suppl 2):S555–S556. http://dx.doi.org/10.1016/S0168-8278(12)61423-8. 52. Parker WB. 2005. Metabolism and antiviral activity of ribavirin. Virus Res. 107:165–171. http://dx.doi.org/10.1016/j.virusres.2004.11.006. 53. Hebner C, Lee Y-J, Han B, Chiu S, Tian Y, Pagratis N, Miller M, Mo H. 2012. In vitro pan-genotypic and combination activity of sofosbuvir (GS7977) in stable replicon cell lines. Hepatology 56(Suppl):1066A. http://dx .doi.org/10.1002/hep.26040.

The effect of mild to moderate renal impairment on the pharmacokinetics of the nucleoside analog hepatitis C virus polymerase inhibitor mericitabine.

protease inhibitor combination in severe hepatitis C virus-associated mixed cryoglobulinemia vasculitis.

Discovery and early development of TMC647055, a non-nucleoside inhibitor of the hepatitis C virus NS5B polymerase.

Combination therapy with pegylated interferon plus ribavirin in the treatment of hepatitis C virus-related thrombocytopenia.

Mechanism of inhibition for BMS-791325, a novel non-nucleoside inhibitor of hepatitis C virus NS5B polymerase.

Novel 4-thiazolidinones as non-nucleoside inhibitors of hepatitis C virus NS5B RNA-dependent RNA polymerase.

Pharmacokinetic, Pharmacodynamic, and Drug-Interaction Profile of the Hepatitis C Virus NS5B Polymerase Inhibitor Sofosbuvir.

In vitro activity and resistance profile of dasabuvir, a nonnucleoside hepatitis C virus polymerase inhibitor.

The Intracellular Cholesterol Transport Inhibitor U18666A Inhibits the Exosome-Dependent Release of Mature Hepatitis C Virus.

Patterns of longitudinal change in hepatitis C virus neutralization titers correlate with the outcome of peginterferon and ribavirin combination therapy.

Mutation in the Hepatitis E Virus Polymerase and Outcome of Ribavirin Therapy.

Hepatitis C Virus-Coinfected Patients.

Plasma and intracellular ribavirin concentrations are not significantly altered by abacavir in hepatitis C virus-infected patients.

Preclinical characterization of BMS-791325, an allosteric inhibitor of hepatitis C Virus NS5B polymerase.

Effect of Hepatitis C Virus Genotype 1b Core and NS5A Mutations on Response to Peginterferon Plus Ribavirin Combination Therapy.

Daclatasvir-sofosbuvir combination therapy with or without ribavirin for hepatitis C virus infection: from the clinical trials to real life.

Discovery and preclinical characterization of the cyclopropylindolobenzazepine BMS-791325, a potent allosteric inhibitor of the hepatitis C virus NS5B polymerase.

Hepatitis C virus polymerase-polymerase contact interface: significance for virus replication and antiviral design.

hepatitis C virus-coinfected liver transplant recipients.

Effects of ITPA polymorphism on decrease of hemoglobin during simeprevir, peg-interferon, and ribavirin combination treatment for chronic hepatitis C.

Alisporivir plus ribavirin, interferon free or in combination with pegylated interferon, for hepatitis C virus genotype 2 or 3 infection.

Synthesis of ribavirin 2'-Me-C-nucleoside analogues.

Ribavirin revisited in the era of direct-acting antiviral therapy for hepatitis C virus infection.

Inhibition of hepatitis C virus replication by GS-6620, a potent C-nucleoside monophosphate prodrug.