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Clinical Trial
. 2005 Jun;49(6):2267-75.
doi: 10.1128/AAC.49.6.2267-2275.2005.

In vitro antiviral activity and single-dose pharmacokinetics in humans of a novel, orally bioavailable inhibitor of human rhinovirus 3C protease

Amy K Patick  1 , Mary A Brothers, Fausto Maldonado, Susan Binford, Oscar Maldonado, Shella Fuhrman, Annkatrin Petersen, George J Smith 3rd, Leora S Zalman, Leigh Ann Burns-Naas, Jonathan Q Tran
Affiliations
Clinical Trial

In vitro antiviral activity and single-dose pharmacokinetics in humans of a novel, orally bioavailable inhibitor of human rhinovirus 3C protease

Amy K Patick et al. Antimicrob Agents Chemother. 2005 Jun.

Abstract

(E)-(S)-4-((S)-2-{3-[(5-methyl-isoxazole-3-carbonyl)-amino]-2-oxo-2H-pyridin-1-yl}-pent-4-ynoylamino)-5-((S)-2-oxo-pyrrolidin-3-yl)-pent-2-enoic acid ethyl ester (Compound 1) is a novel, irreversible inhibitor of human rhinovirus (HRV) 3C protease {inactivation rate constant (Kobs/[I]) of 223,000 M-1s-1}. In cell-based assays, Compound 1 was active against all HRV serotypes (35 of 35), HRV clinical isolates (5 of 5), and related picornaviruses (8 of 8) tested with mean 50% effective concentration (EC50) values of 50 nM (range, 14 to 122 nM), 77 nM (range, 72 to 89 nM), and 75 nM (range, 7 to 249 nM), respectively. Compound 1 inhibited HRV 3C-mediated polyprotein processing in infected cells in a concentration-dependent manner, providing direct confirmation that the cell-based antiviral activity is due to inhibition of 3C protease. In vitro and in vivo nonclinical safety studies showed Compound 1 to be without adverse effects at maximum achievable doses. Single oral doses of Compound 1 up to 2,000 mg in healthy volunteers were found to be safe and well tolerated in a phase I-ascending, single-dose study. Compound 1 estimated free observed maximum concentration in plasma (Cmax) for 500-, 1,000-, and 2,000-mg doses were higher than the protein binding-corrected EC50 required to inhibit 80% of the HRV serotypes tested. Treatment of HRV 52-infected cells with one to five 2-h pulses of 150 nM Compound 1 (corresponding to the Cmax at the 500-mg dose) was sufficient to effect a significant reduction in viral replication. These experiments highlight Compound 1 as a potent, orally bioavailable, irreversible inhibitor of HRV 3C protease and provide data that suggest that Cmax rather than the Cmin might be the key variable predicting clinical efficacy.

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Figures

FIG. 1.
FIG. 1.
Chemical structure of (E)-(S)-4-((S)-2-{3-[(5-methyl-isoxazole-3-carbonyl)-amino]-2-oxo-2H-pyridin-1-yl}-pent-4-ynoylamino)-5-((S)-2-oxo-pyrrolidin-3-yl)-pent-2-enoic acid ethyl ester (Compound 1).
FIG. 2.
FIG. 2.
In vitro antiviral activity of Compound 1 against HRV serotypes. EC50 and EC90 values of Compound 1 for 35 serotypes were determined by measuring XTT dye reduction following 1 to 5 days of infection of H1-HeLa cells as described in Materials and Methods.
FIG. 3.
FIG. 3.
Inhibition of HRV 14 3C-mediated proteolytic processing by Compound 1. SDS solubilized lysates were prepared from uninfected H1-HeLa cells (cell ctrl) or infected cells treated with Compound 1, pleconaril, rupintrivir, or medium only (virus ctrl). Equal amounts of protein were analyzed by polyacrylamide gel electrophoresis (PAGE) as described in Materials and Methods except for cell control (0.1×), which represents 1/10th the amount of uninfected cell lysate protein analyzed. P2-P3, P1, VPO, 2C, and VP1 designate HRV-specific polypeptides.
FIG. 4.
FIG. 4.
Profiles of mean Compound 1 (total drug) concentrations in plasma after oral administration of ascending single doses under fed conditions (six subjects for each dose).
FIG. 5.
FIG. 5.
Profiles of mean Compound 1 (estimated free drug) concentrations in plasma after oral administration of ascending single doses under fed conditions (six subjects for each dose) relative to the tissue culture binding-corrected EC50 value required to inhibit 80% of HRV serotypes.
FIG. 6.
FIG. 6.
In vitro pulse experiments of Compound 1. Antiviral activity was determined by measuring XTT dye reduction following 53 h of infection with HRV 52 using the CPE inhibition assay as described in Materials and Methods. Infected cells were treated with 2 nM (n = 24), 150 nM Compound 1 (n = 24), or 2 nM Compound 1 and subsequently treated with one, three, four, or five 2-h pulses of 150 nM Compound 1 (n = 6). Data is expressed as percent inhibition (± standard error) utilizing the mock infected cell control and compound-free virus control as the 100% and 0% virus inhibition values, respectively. Significant reductions in viral replication due to treatment with pulses of 150 nM as compared to treatment with 2 nM alone were determined as described in Materials and Methods, with resulting P values of 0.011 (*), 2.4 × 10−7 (**), 1.5 × 10−7 (***), and 6.6 × 10−6 (****).
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