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`JOURNAL OF VIROLOGY, Mar. 2007, p. 3005–3008
`0022-538X/07/$08.00⫹0 doi:10.1128/JVI.02083-06
`Copyright © 2007, American Society for Microbiology. All Rights Reserved.
`
`Vol. 81, No. 6
`
`Synergy of Small Molecular Inhibitors of Hepatitis C Virus Replication
`Directed at Multiple Viral Targets䌤
`David L. Wyles,1* Kelly A. Kaihara,1 Florin Vaida,2 and Robert T. Schooley1
`Department of Medicine, Division of Infectious Diseases,1 and Department of Family and Preventive Medicine,2
`University of California, San Diego, La Jolla, California
`
`Received 22 September 2006/Accepted 10 December 2006
`
`Chronic hepatitis C virus (HCV) infection is a significant worldwide health problem with limited therapeutic
`options. A number of novel, small molecular inhibitors of HCV replication are now entering early clinical trials
`in humans. Resistance to small molecular inhibitors is likely to be a significant hurdle to their use in patients.
`A systematic assessment of combinations of interferon and/or novel anti-hepatitis C virus agents from several
`different mechanistic classes was performed in vitro. Combinations of inhibitors with different mechanisms of
`action consistently demonstrated more synergy than did compounds with similar mechanisms of action. These
`results suggest that combinations of inhibitors with different mechanisms of action should be prioritized for
`assessment in clinical trials for chronic hepatitis C virus infection.
`
`Chronic hepatitis C virus (HCV) infection is a major world-
`wide health problem; in the United States, an estimated 3
`million persons are chronically infected (4). Estimates of the
`health care burden of chronic HCV infection predict a drastic
`increase in hospitalizations and medical costs related to com-
`plications such as cirrhosis and hepatocellular carcinoma over
`the next 1 to 2 decades (3). Effective and better-tolerated
`therapy for HCV could effectively stem this tide (7).
`Current interferon-based therapy for chronic HCV infection
`results in sustained responses in roughly 55% of patients and is
`accompanied by significant toxicity. Genotype 1 HCV, the
`most prevalent genotype in the United States, responds less
`well to therapy with pegylated interferon plus ribavirin, with
`response rates of 42 to 46% (11, 23). These limitations have
`spurred an intense drug discovery effort, resulting in a number
`of promising compounds (8).
`Hepatitis C virus replication takes place in the cytoplasm,
`with the replication complex being tightly associated with lipid
`membranes (1). Key components of the replication complex
`include several promising antiviral
`targets,
`including the
`NS3/4A protease and the NS5B RNA-dependent RNA poly-
`merase. A number of candidate protease inhibitors (PIs) which
`have excellent potency in vitro have been developed (2, 17, 20);
`several of these compounds have also been evaluated in phase
`I/II trials, with encouraging results (15, 16, 29, 36). Resistance
`to this class of inhibitors has been described, with some muta-
`tions conferring cross-resistance to several compounds (17, 18,
`21, 34, 35).
`The NS5B RNA polymerase is also essential for viral repli-
`cation, and a number of nucleoside inhibitors and nonnucleo-
`side inhibitors (NNIs) of the HCV polymerase with potent
`activity in vitro and in early clinical trials have been described
`(5, 12, 13, 27, 30). Resistance to both nucleoside and non-
`nucleoside inhibitors in vitro has been described (22, 24, 26).
`
`* Corresponding author. Mailing address: 9500 Gilman Drive, MC
`0711, La Jolla, CA 92093. Phone: (858) 822-1779. Fax: (858) 822-5362.
`E-mail: dwyles@ucsd.edu.
`䌤 Published ahead of print on 20 December 2006.
`
`We have assessed a number of combinations of HCV inhibitors
`with several molecular targets currently in development, using
`an HCV genotype 1 replicon-based luciferase reporter system.
`Replicon constructs. The BM4-5 replicon is a subgenomic
`HCV genotype 1b replicon which contains a deletion of a
`serine in NS5A and has been previously described (14). The
`firefly luciferase gene was inserted in the BM4-5 replicon, in a
`manner previously described (33), to create a luciferase/neo-
`mycin phosphotransferase fusion protein (FEO) and the rep-
`licon (BM4-5 FEO). Briefly, the Photinus pyralis luciferase
`gene was amplified using primers coding for the AscI restric-
`tion site. Following amplification, both the BM4-5 plasmid and
`luciferase PCR product were restriction digested with AscI.
`Ligation was then carried out to insert the luciferase gene in
`phase with the neomycin phosphotransferase gene, creating
`the desired BM4-5 FEO replicon. The sequence of the repli-
`con was verified by DNA sequencing.
`Cell culture. Human hepatoma Huh-7.5.1 cells (a kind gift
`from Francis Chisari, Scripps Research Institute, La Jolla, CA)
`and BM4-5 FEO cells stably expressing the BM4-5 FEO rep-
`licon were grown at 37°C and 5% CO2 in Dulbecco’s modifi-
`cation of Eagle’s medium supplemented with 2 mM L-glu-
`tamine, 100 units/ml penicillin, 100 ␮g/ml streptomycin, and
`10% fetal bovine serum. BM4-5 FEO cells were additionally
`grown in the presence of 500 ␮g/ml of G-418.
`Transfection and clone selection. The BM4-5 FEO plasmid
`was linearized with ScaI. In vitro transcription (Megascript;
`Ambion) was carried out according to the manufacturer’s in-
`struction to yield BM4-5 FEO RNA. Transfection was per-
`formed as previously described (32). Four hundred microliters
`of a Huh-7.5.1 cell suspension (107 cells/ml) was placed in a
`0.4-cm cuvette with 10 ␮g of BM4-5 FEO RNA. The mixture
`was electroporated (Bio-Rad Gene Pulser) at 270 V and 975
`␮F and transferred to a 10-cm tissue culture dish. G-418 at 500
`␮g/ml was added at 24 h, and the medium was changed every
`3 to 4 days. Individual G-418-resistant colonies were visible
`within 2 to 3 weeks. Individual colonies were harvested and
`expanded for characterization of luciferase expression.
`Gilead 2003
`I-MAK v. Gilead
`IPR2018-00211
`
`3005
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`

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`3006
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`NOTES
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`J. VIROL.
`
`TABLE 1. Activities of different small molecular inhibitors in the
`BM4-5 replicon
`
`Compound
`
`Structure
`
`HCV BM4-5
`replicon
`IC50 (nM)a
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`a The IC50 is the average ⫾ standard error of mean of the results from at least
`three independent experiments.
`
`The IC50 for each of the individual compounds is listed in
`Table 1. CI50, CI70, and CI90 refer to the combination index at
`the IC50, IC70, and IC90, respectively, of each drug. All com-
`pounds tested were additive (CI50 and CI70) or mildly syner-
`gistic (CI90) with alpha interferon. Antagonism was not dem-
`onstrated for any combination of small molecular inhibitors,
`including compounds targeting the same viral protein. Signif-
`icantly more synergy was demonstrated between compounds in
`the group combining two small molecular inhibitors targeting
`the same viral enzyme (in this case NS3 protease) than be-
`tween the group of compounds combined with alpha interferon
`at both the CI70 (P ⫽ 0.043) and CI90 (P ⫽ 0.017) levels. There
`was no significant difference between the groups at the CI50
`level (P ⫽ 0.108) (Fig. 1). Similarly, the group consisting of two
`inhibitors with different viral targets showed significantly lower
`combination indices than either of the other two groups, i.e.,
`compounds with interferon (P ⬍ 0.001 at all levels) or com-
`pounds with same mechanism of action (P ⫽ 0.038 and 0.037
`at the CI50 and CI70 levels, respectively). The comparison of
`CI90s between small molecular inhibitors with the same and
`different viral targets showed a trend toward a lower combina-
`tion index in the group with two compounds with different viral
`targets (P ⫽ 0.056 at the CI90 level) (Fig. 1). None of the
`compounds or combinations showed cytotoxicity at the con-
`centrations tested in the activity and synergy studies (data not
`shown).
`Small molecular inhibitors of the HCV protease and poly-
`
`Luciferase compound assay. BM4-5 FEO cells were seeded
`into 96-well plates at a density of 10,000 cells per well in 100 ␮l
`medium. After allowing 4 h for attachment, compounds were
`added to wells at the specified concentrations. All conditions
`were run in triplicate. Cells and compounds were incubated for
`48 h. The luciferase assay (Bright-Glo; Promega) was carried
`out according to the manufacturer’s instructions. Luciferase
`activity was determined using a microplate luminometer (Ve-
`ritas microplate luminometer; Turner Biosystems). The rela-
`tive light units (RLU) for each condition were reported as the
`mean ⫾ the standard error of the mean for the three wells.
`Compounds tested. Compounds tested included two pep-
`tidomimetic HCV PIs, BILN 2061 (16) and a Vertex PI (19)
`(Vicki Sato, Vertex Pharmaceuticals, Cambridge, MA); a
`GlaxoSmithKline trans-lactam PI active-site mimic (2) (Karen
`Romines, GlaxoSmithKline, Research Triangle Park, NC); one
`nucleoside analog HCV RNA-dependent RNA polymerase
`inhibitor (RdRpI), 2⬘-C-methyladenosine (10) (William Lee,
`Gilead Sciences, Foster City, CA); one nonnucleoside GSK
`benzo-thiadiazine RNA polymerase inhibitor directed at the
`“thumb” region of the polymerase (Karen Romines, Glaxo-
`SmithKline) (9); and alpha interferon (Interferon-␣A; Sigma-
`Aldrich).
`The 50% inhibitory concentration (IC50) of each compound
`was determined independently and used to set the range of
`concentrations used for the synergy experiments. Each com-
`pound was tested singly and in combination at two twofold
`serial dilutions above and below the IC50. The ratio of the two
`compounds tested remained fixed across the dosing range.
`Potential cytotoxicity of individual compounds and all combi-
`nations was assessed using a luminescent ATP-based cell via-
`bility assay (Cell Titer-Glo; Promega).
`Data analysis. Determinations of compound interactions
`were based on the median-effect principle and the multiple
`drug effect equation as described by Chou and Talalay (6).
`Combination indices (CIs) were determined using Calcusyn
`(Biosoft) for each experiment at the IC50, IC70, and IC90 levels.
`In total, 15 combinations were evaluated with from three to
`five replicates per condition; this yielded a total of 61 data
`points per CI level analyzed. A CI of ⬍0.9 was considered
`synergistic, a CI of ⱖ0.9 or ⱕ1.1 was considered additive, and
`a CI of ⬎1.1 was deemed antagonistic.
`Statistical analysis. At each of the three inhibitory concen-
`trations evaluated (IC50, IC70, and IC90) the CIs in the three
`synergy groups were compared using a linear mixed-effects
`model allowing for different means in the three synergy groups
`and random effects for the individual drug combinations. The
`random effects were not significant (likelihood ratio test), in-
`dicating no statistical difference in CI values between the an-
`tiviral compound combinations in the same synergy group. The
`CI replicates were further compared between synergy groups
`by using the Wilcoxon rank test.
`Synergy of small molecular inhibitors. Transfection of Huh-
`7.5.1 cells with BM4-5 FEO RNA yielded numerous (⬎50)
`G-418-resistant clones. Individual clones were expanded and
`assessed using the luciferase assay to determine the individual
`clones with highest RLU per cell. Four clones yielded from
`30,000 to 50,000 RLU per 10,000 cells at 48 h (data not
`shown); these clones were expanded and used for all subse-
`quent studies.
`
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`NOTES
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`FIG. 1. CI50s, CI70s, and CI90s for the compound combinations evaluated. Dotted lines at combination index values of 0.9 and 1.1 indicate the
`boundaries of an additive interaction. The P values displayed (ⴱ) are for analyses at the CI70 level.
`
`merase show antiviral activity in our genotype 1 replicon sys-
`tem. Most importantly, no combination of small molecular
`inhibitors of HCV replication demonstrated antagonism in our
`system, including those with the same mechanism of action or
`viral target. Combinations of inhibitors targeting different viral
`proteins (PI-RdRpI or PI-NNI) or with different mechanisms
`of
`inhibiting the same viral protein (RdRpI-NNI) were
`strongly synergistic and had significantly lower combination
`indices than the other two groups. Combinations targeting the
`same site within a viral protein showed lesser degrees of syn-
`ergy or were additive, but they still possessed significantly
`lower combination indices than the group composed of the
`same compounds with alpha interferon. It is important to re-
`member that the definition of synergy as a CI of less than 0.9
`is an arbitrary distinction (along a continuum) and thus does
`not preclude two inhibitors which occupy the same site from
`being “synergistic” according to a CI of ⬍0.9. Additionally,
`metabolic interactions between compounds or the impact of
`divergent resistance pathways on different compounds may
`also affect the appearance of drug-drug interactions as assessed
`by the combination index.
`HCV, like human immunodeficiency virus type 1, possesses
`an error-prone RNA polymerase, and it replicates to levels 10-
`to 100-fold higher than those of human immunodeficiency
`virus type 1 in chronically infected individuals (25, 28). These
`characteristics suggest that selection of drug-resistant viral
`variants will be a challenge to the use of small molecular
`inhibitors. In fact, resistance to these compounds both in vitro
`and in vivo has already been described (17, 24, 26, 31, 34).
`Synergistic combinations of HCV inhibitors may produce
`
`greater viral load decreases in vivo and could potentially delay
`the appearance of multiply drug-resistant virus. This system
`provides a useful approach for the in vitro testing of antiviral
`combinations in anticipation of rationally designed clinical
`studies of combination chemotherapy directed at HCV. Our
`results support the evaluation of combinations of small molec-
`ular inhibitors in human clinical trials and further suggest that
`combinations with different mechanisms of action may be par-
`ticularly attractive.
`
`This work was funded in part by a 2005 developmental grant from
`the UC San Diego Center for AIDS Research, an NIH-funded pro-
`gram (no. 5P30 AI-36214).
`
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