In vitro metabolism and drug interaction potential of a new highly potent anti-cytomegalovirus molecule, CMV423 (2-chloro 3-pyridine 3-yl 5,6,7,8-tetrahydroindolizine I-carboxamide)

Abstract

Aims: To identify the enzymes involved in the metabolism of CMV423, a new anticytomegalovirus molecule, to evaluate its in vitro clearance and to investigate its potential involvement in drug/drug interactions that might occur in the clinic.

Methods: The enzymes involved in and the kinetics of CMV423 biotransformation were determined using pools of human liver subcellular fractions and heterologously expressed human cytochromes P450 (CYP) and FMO. The effect of CMV423 on CYP probe activities as well as on indinavir and AZT metabolism was determined, and 26 drugs were tested for their potential to inhibit or activate CMV423 metabolism.

Results: CMV423 was oxidized by CYP and not by FMO or cytosolic enzymes. The Km values for 8-hydroxylation to rac-RPR 127025, an active metabolite, and subsequent ketone formation by human liver microsomes were 44 +/- 13 microM and 47 +/- 11 microM, respectively, with corresponding Vmax/Km ratios of 14 and 4 microl min(-1) nmol(-1) P450. Inhibition with selective CYP inhibitors indicated that CYP1A2 was the main isoform involved, with some participation from CYP3A. Expressed human CYP1A1, 1A2, 2C9, 3A4 and 2C8 catalysed rac-RPR 127025 formation with Km values of < 10 microM, 50 +/- 21 microM, 55 +/- 19 microM, circa 282 +/- 61 microM and circa 1450 microM, respectively. CYP1B1, 2A6, 2B6, 2C19, 2D6, 2E1 or 3A5 did not catalyse the reaction to any detectable extent. CYP1A1 and 3A4 also catalysed ketone formation from rac-RPR 127025. In human liver microsomes, CMV423 at 1 and 10 microM inhibited CYP1A2 activity up to 31% and 63%, respectively, CYP3A4 activity up to 40% (10 microM) and CYP2C9 activity by 35% (1 and 10 microM). No effect was observed on CYP2A6, 2D6 and 2E1 activities. CMV423 had no effect on indinavir and AZT metabolism. Amongst 26 drugs tested, none inhibited CMV423 metabolism in vitro at therapeutic concentrations.

Conclusions: CMV423 is mainly metabolized by CYP1A2 and 3A4. Its metabolism should not be saturable at the targeted therapeutic concentrations range (Cmax < 1 microM). CMV423 will probably affect CYP1A2 and 1A1 activities in vivo to some extent, but no other drug-drug interactions are expected.

Figure 1

H.p.l.c. profiles of CMV423 biotransformation in human liver microsomes. Analysis of the supernatants obtained after incubating 10 µm CMV423 (b) or 10 µmrac-RPR 127025 (a) for 10 min with human liver microsomes (1 mg ml⁻¹) in the presence of NADPH (1 mm).

Figure 2

Rate vs substrate concentration plots of CMV423 and rac-RPR 127025 metabolism in microsomes from human liver and from yeast expressing human CYPs. Human liver microsomes (HLM): 0.75 mg ml⁻¹, pool 1. Expressed CYPs: 200 pmol ml⁻¹, except CYPA1 100 pmol ml⁻¹. DMSO: 0.5%, except CYP3A4 1.9%. The data were fitted by non linear regression. Inserts: Eadie Hofstee plots.

Figure 3

The metabolism of CMV423 by human liver. The formation of rac-RPR 127025 and RPR 12830 are the major routes. Rank order of quantitative involvement of individual CYPs: bold>regulars>italic text.