Species Dependent Metabolism of a Covalent nsP2 Protease Inhibitor with in Vivo Anti-alphaviral Activity

Abstract

RA-0002034 (1) is a potent covalent inhibitor targeting the alphavirus nsP2 cysteine protease. The species-dependent pharmacokinetics and metabolism of 1 were investigated to evaluate its therapeutic potential. Pharmacokinetic profiling revealed rapid clearance in mice, predominantly mediated by glutathione S-transferase (GST)-catalyzed conjugation. This metabolic liability contrasted with slower clearance observed in human hepatocytes and preclinical species such as rats, dogs, and monkeys. Cross-species studies confirmed the dominance of GST-driven metabolism in mice, whereas oxidative pathways were more pronounced in dogs. Despite rapid systemic clearance, 1 achieved antiviral efficacy in mice, reducing CHIKV viral loads in multiple tissues. Initial estimations of human hepatic clearance and half-life extrapolated from animal data indicate that b.i.d. dosing of 1 will be possible to maintain concentrations sufficient for antiviral activity in humans. These cross-species pharmacokinetic and metabolism studies support the continued evaluation of 1 as a promising anti-alphaviral therapeutic.

Figure 1.

nsP2pro inhibitor RA-0002034 (1) and cyclic isomer 2

Figure 2.

GST-catalyzed GSH conjugation of 1. GSH (100x), phosphate buffer pH 7.4, mouse/human GST (10,000 ng/mL), 30 °C

Figure 3.

Metabolites of 1 in mice. (A) LC-MS of pooled plasma with identification of 18 metabolites of 1 (parent, P). X-axis represents total ion count. (B) Structural assignment of the metabolites (M1–M18) from MS fragmentation. Molecular ions are shown in parentheses. (C) Summary of the major sites of metabolism of 1 in mice.

Figure 4.

Clearance of 1 in mice. Male C57BL/6 mice dosed with 1 (30 mg/kg p.o.) in the presence of EA (10 or 30 mg/kg p.o.) or 1-ABT (100 mg/kg p.o.). EA was dosed twice at −6 and −2 h. 1-ABT was dosed once at −2 h. All data are the average from three mice.

Figure 5.

In vivo antiviral efficacy of 1. (A) Plasma concentration of 1 in C57BL/6 mice (n = 3) dosed p.o. at 100 mg/kg t.i.d. Black arrows indicate the time of dosing. Solid circles are the total drug level. Open circles are the calculated unbound concentrations. The dotted line indicates the EC₉₀ for inhibition of CHIKV replication (56 ng/mL). (B) Protocol for the mouse efficacy study. Black arrows indicate the time of dosing of 1 in mice (n = 5) at 100 mg/kg p.o. The open arrow indicates the time of inoculation with CHIKV (10³ PFU) into the left foot. Viral load was determined 8 h after the last dose. (C) Virus levels in 5 tissues and serum for individual mice (symbols) and group average (grey bars). The dotted line indicates the lower limit of quantification. Significance between 1 and vehicle (V) treated mice by t-test is indicated. ns = non-significant.

Figure 6.

Metabolites of 1 in Primary Hepatocytes. Mo, mouse; R, rat; D, dog; Mn, monkey; H, human.

Figure 7.

Cross-species Plasma Profile of 1. Oral dosing of 1 at 30 mg/kg in four species from Tables 1 and 6. Dotted line indicates the EC₉₀ for inhibition of CHIKV replication.