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Hepatitis C virus resistance to new antivirals
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Resistance Workshop Review: Part 7
Mark Mascolini
Years from now--perhaps not too many years from now--today's standard regimen for hepatitis C virus (HCV) infection--will seem pre-Oslerian in its bluntness and toxicity. Pegylated interferon plus ribavirin is a good step forward from standard interferon monotherapy, but the therapeutic course is lengthy, the drugs can be tough on psyche and soma, and they don't work very well for people with HCV genotype 1.
Partly because the new antiretroviral age arrived with the dawn of HIV protease inhibitors (PIs), work on inhibitors of HCV protease stirred early interest. But the Resistance Workshop reflected strides in walking HCV polymerase inhibitors toward big clinical trials. The news ranged from mixed to (perhaps) pretty good.
Resistance to HCV polymerase inhibitors
The structure of HCV polymerase resembles the human paw--at least to the visually imaginative--with one piece suggesting the palm, another the fingers, and another the thumb. Isabel Najera and a team at Roche Palo Alto found that combining anti-HCV drugs that inhibit both thumb site and palm site polymerase drew forth resistance-conferring mutations in 98% of cloned replicons--the cell culture system used to study HCV replication (1). Almost two thirds of the replicons--64% of them--picked up mutations making HCV resistant to both thumb-binding inhibitors and palm-binding inhibitors.
Exposing replicons to a thumb inhibitor alone egged forth L419M, M423T/I, and I482L mutations in the thumb domain. Monotherapy with a palm inhibitor elicited different substitutions, N411S and M414T or M414L. Single mutations from this list caused high-level resistance to either thumb or palm polymerase inhibitors.
The good news--insofar as this culture system can reckon--is that single mutants resistant to one type of polymerase inhibitor remained susceptible to the other type. Double mutants, but less often triple mutants, made HCV resistant to both types of inhibitors. According to results of molecular cloning studies, 98% of replicons contained amino acid substitutions conferring resistance to one type of inhibitor, and 64% of replicons had mutations conferring resistance to both.
Abbott's Akhteruzzaman Molla reported that single mutations can render HCV 33- to 962-fold resistant to the company's candidate polymerase inhibitor, A-848837 (2). But as in the Roche study, virus resistant to A-848837 was not cross-resistant to another polymerase inhibitor. Nor was A-848837-resistant virus cross-resistant to interferon, with which it may well be combined. The Abbott compound was additive or synergistic with an HCV PI and with interferon.
A-848837 looks potent against HCV genotype 1b in the replicon cell system, at 50% effective concentrations of 4 nM in the absence of human serum and 62 nM in 40% human serum. That could be good news for HCV-1b-infected people, who typically get less antiviral oomph from interferon and ribavirin than people with genotype 2 or 3. Abbott's polymerase inhibitor, in contrast, has no activity against HCV genotypes 2, 3, and 4.
Exposing replicon cells to high concentrations of A-848837 provoked emergence of single polymerase gene mutations including I392F, M414T, Y448H, G554D, and S556G, which made HCV 4- to 255-fold resistant to the Abbott agent. All of these single mutants retained susceptibility to another HCV polymerase inhibitor (thiophene-2-carboxylic acid), to an HCV PI, and to interferon. Teamed with interferon, A-848837 cleared HCV in the replicon system despite emergence of G554D and Y448H.
HCV polymerase inhibitor looks good in chimps
Probably the most inspiring HCV polymerase study came from David Olsen and Merck coworkers, who reported that their candidate in this class choked off HCV replication in chimps at least as well as HCV PIs did in earlier studies (3). But the hefty HCV load plunges seen in this study must be interpreted cautiously because the experiments involved only 2 animals.
Earlier work suggested that nucleoside and nonnucleoside HCV polymerase inhibitors could not knock down viral replication as well as HCV PIs. But the Merck purine nucleoside analog changed that trend. Olsen planned to study several chimpanzees, but for various reasons ended up with only 2. Disappointed, the Merck team decided nonetheless to proceed with their study.
Both animals had been infected for decades, one with HCV genotype 1a and one with genotype 3a. Pretreatment HCV loads stood around 3 million copies/mL, levels similar to those seen in humans with untreated HCV infection. Intravenous doses of 0.2 mg/kg a day for 7 days trimmed HCV loads modestly by 0.6 to 0.8 log copies/mL in the HCV-1a-infected chimp and by 1.4 log in the HCV-3a-infected chimp.
Then Merck upped the dose. A single intravenous pulse of 2 mg/kg sliced viral loads 2.6 to 3 log copies/mL. After 5 days of dosing, viral loads plunged 5.7 log copies/mL, below a 20 IU/mL limit of quantitation. HCV loads rebounded after treatment stopped, and an S282T resistance mutation emerged transiently in one of the animals.
Work detailed by Matthias Gotte from Montreal's McGill University suggested that purine nucleoside analogs, like the Merck candidate, may hold a resistance advantage over pyrimidine nucleosides (4). Gotte, an expert on the fine tunings of resistance to anti-HIV nucleosides, found that one of the same mechanisms explaining resistance to nucleoside chain terminators of HIV-1 reverse transcriptase--phosphorolytic excision--also affects nonobligate chain terminators with activity against HCV.
Nucleosides prevent HCV or HIV replication by latching onto--and preventing further growth of--the viral DNA chain resulting from reverse transcription. Excision literally cuts the nucleoside analog off the chain and allows its link-by-link growth to resume. Gotte's experiments showed that excision of a pyrimidine analog was much more efficient than excision of a purine analog.
Combining HCV polymerase and protease inhibitors
David Wyles and colleagues from the University of California, San Diego, devised an HCV-1 replicon system they used to demonstrate synergy between HCV PIs and polymerase inhibitors, and between those classes and interferon-alfa (5).
Wyles doused cultured human liver cells for 48 house with three PIs, two polymerase inhibitors, and interferon--alone and in various combinations. By themselves the PIs had 50% inhibitory concentrations (IC50s) ranging from 5 to 160 nM, while individual polymerase inhibitors had IC50s of 200 to 1200 nM. Teaming two PIs yielded additive ant-HCV activity, as did double polymerase inhibitors. Combining a PI with a polymerase inhibitor yielded synergistic antiviral activity. Two of the PIs and one polymerase inhibitor also proved synergistic with interferon-alfa. None of these combinations looked cytotoxic in Wyles' system.
If results like these hold true in humans, eventual development of one or more HCV PIs and polymerase inhibitors could pave the way to triple regimens including pegylated interferon. But trials of such alluring combinations are not around the corner.
Mark Mascolini writes about HIV (and sometimes HCV) infection
(mailmark@ptd.net).
References
1. Le Pogam S, Kang H, Harris SF, et al. Selection and characterization of hepatitis C virus replicon variants dually resistant to thumb and palm binding non-nucleoside polymerase inhibitors. XV International HIV Drug Resistance Workshop. June 13-17, 2006. Sitges. Abstract 3.
2. Molla A, Lu L, Krishnan P, et al. Selection and characterization of mutations conferring resistance to a HCV RNA dependent RNA polymerase inhibitor in vitro. XV International HIV Drug Resistance Workshop. June 13-17, 2006. Sitges. Abstract 4.
3. Olsen DB, Carroll SS, Davies M-El, et al. Robust suppression of viral replication in HCV infected chimpanzees by a nucleoside inhibitor of the NS5B polymerase. XV International HIV Drug Resistance Workshop. June 13-17, 2006. Sitges. Abstract 5.
4. Deval J, D'Abramo CM, Gotte M. Selective excision of non-obligate chain-terminators by the hepatitis C virus NS5B polymerase. XV International HIV Drug Resistance Workshop. June 13-17, 2006. Sitges. Abstract 1.
5. Wyles DL, Kaihara K, Schooley RT. Synergy of small molecular inhibitors of hepatitis C virus replication directed at different viral targets. XV International HIV Drug Resistance Workshop. June 13-17, 2006. Sitges. Abstract 2.
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