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Preclinical Characterization of TMC435350, a novel macrocyclic inhibitor of the HCV NS3/4A serine protease
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Reported by Jules Levin
14th International Symposium on Hepatitis C Virus & Related Viruses, Glasgow, UK, 9-13 September 2007
Simmen K.1, Lenz O.1, Lin T.-I.1, Fanning G.1, Raboisson P.1, de Kock H.1, Vendeville S.1, Broeckaert F.1, Van 't Klooster G.1, Rosenquist A.2, Vrang L.2, Nilsson M.2, Edlund M.2, Samuelsson B.2
1Tibotec, Mechelen, Belgium, 2Medivir, Huddinge, Sweden
Abstract
Small molecule HCV protease inhibitors have been shown to decrease HCV viremia in clinical trials, either as monotherapy or in addition to pegylated interferon-_ (PEG-IFN-_) and ribavirin therapy. Their contribution to the sustained clearance of HCV from patients is currently being evaluated. We have developed a potent NS3/4A serine protease inhibitor and present here preclinical
data in support of its potential benefit in the treatment of HCV patients. A series of novel macrocyclic analogues were synthesized and profiled using genotype 1 based enzymatic NS3/4A protease assays and the cellular replicon model. TMC435350 was identified as a potent and specific inhibitor of HCV replication in genotype 1 replicon cells with an EC50 value of 8 nM and a selectivity index
(SI) of > 2,000 (Displayed minimal cytotoxicity in tested human cell lines). In the biochemical protease assay, Ki values of 0.5 and 0.4 nM were obtained for subtypes 1a (H77) and 1b (con1b) enzymes, respectively. Specificity was confirmed using i) a panel of human proteases, ii) extensive profiling against a representative panel of RNA and DNA viruses and iii) cytotoxicity testing in multiple cell lines. Combining IFN-_ with TMC435350 in the replicon generated synergistic activity in reducing HCV RNA, and suppressed the emergence of drug resistant replicon colonies. In 9-day assays the combination of TMC435350 and IFN-_ decreased replicon RNA by > 4 log10. In pharmacokinetic and safety pharmacology studies TMC435350 displayed a drug-like profile. In conclusion, TMC435350 is a novel potent and specific HCV NS3/4A inhibitor, and has recently entered clinical trials.
AUTHOR CONCLUSIONS
TMC435350:
-- has potent anti-HCV enzymatic and cellular activity.
-- demonstrates good selectivity to other viruses and human proteases.
-- displays synergy in combination with IFN-_ in decreasing HCV replication and resistance generation.
-- is a drug-like small molecule as shown in pharmacokinetic and preclinical safety studies.
TMC435350 is currently being evaluated in phase I clinical trials.
Huh7-Luc-ET cells were treated for 3 days with TMC435350 in combination with IFN-_. The relationship between inhibitory activities of TMC435350 and IFN-_ was explored using CalcuSyn (Biosoft, Ferguson, MO). CI values of <1, =1, and >1 indicate synergy, an additive effect, or antagonism, respectively.
In the replicon model, combination of TMC435350 with IFN-_ resulted in:
-- > 4 log10 reduction of HCV RNA in 9 days.
-- synergy of TMC435350 with IFN-_.
-- increased suppression of drug resistant replicon colonies.
-- Good systemic oral bioavailability.
-- Plasma exposure: C8h-plasma > EC99; liver exposure C24h-liver > EC99.
Preclinical safety
-- Negative in a battery of genotoxicity assays.
-- Favourable profile in in vitro and in vivo safety pharmacology
assays (CNS, cardiovascular, pulmonary functions).
TMC435350 was identified as a potent inhibitor of HCV from a series of macrocyclic analogues in enzymatic assays using genotype 1a and 1b HCV NS3/4A proteases and cellular replicon models. Selectivity and cytoxicity were further characterized, and studies with IFN-_ in the replicon demonstrated the
potential of this combination. Preclinical studies support the selection of TMC435350 for clinical development.
RESULTS
- Potent inhibitor of both genotype 1a and 1b HCV NS3/4A proteases.
- Inhibition values on all host proteases tested were over 1,000 times higher than those for NS3/4A protease.
HCV cellular activity and selectivity
- Potent and highly specific HCV inhibitor (EC50 = 8 nM).
- Displayed minimal cytotoxicity in tested human cell lines (SI>2,000).
Dose-dependent inhibition of HCV replication was monitored with genotype 1b Huh7-Luc-ET (kindly provided by R. Bartenschlager as HCV con1b derived bicistronic replicon clone ET), Huh7-con1b and Huh7-SG1a replicon cells (kindly provided by Apath L.L.C. as con1b and H77 derived replicons) using luciferase reporter read-out, immunological blotting and quantitative RNA detection by real-time PCR.
References
(1) Lin, K. et al. 2004. AAC 48: 4784
(2) Mo, H. et al. 2005. AAC 49: 4305
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