Vol. 31, Issue 1, 122-132, January 2003

Metabolism of (S)-5,6-Difluoro-4-cyclopropylethynyl-4-trifluoromethyl-3, 4-dihydro-2(1H)-quinazolinone, a Non-Nucleoside Reverse Transcriptase Inhibitor, in Human Liver Microsomes. Metabolic Activation and Enzyme Kinetics

Hao Chen, Weiqi Chen, Liang-Shang Gan, and Abdul E. Mutlib

Metabolism and Pharmacokinetics Section, Bristol-Myers Squibb Pharma Company, Wilmington, Delaware

(S)-5, 6-Difluoro-4-cyclopropylethynyl-4-trifluoromethyl-3, 4-dihydro- 2-(1H)-quinazolinone (DPC 963), a specific non-nucleoside inhibitor of human immunodeficiency virus-1 reverse transcriptase, is primarily metabolized in humans to the glucuronide conjugate of 8-OH DPC 963 (M8). Electrospray ionization-liquid chromatography/mass spectrometry analyses of urine from subjects dosed with DPC 963 also revealed the presence of other minor metabolites including glucuronide conjugate of 6-OH DPC 963 (M7). An oxidative defluorination pathway involving a putative p-benzoquinone imine capable of being reduced to the hydroquinone (M7) is postulated. The formation of the benzoquinone imine [detected as a glutathione (GSH) adduct, M5] was primarily carried out by CYP3A4, whereas M8 was formed mainly by the polymorphic CYP2B6. The kinetic studies with human liver microsomes showed that the apparent K_m and V_max values for the formation of M5 were 65.8 µM and 25.6 pmol/min/mg of protein, respectively. The formation of M8 showed K_m and V_max values of 15.1 µM and 22.9 pmol/min/mg of protein, respectively. The microsomal studies also revealed the occurrence of a possible oxirene intermediate that was trapped as GSH adducts M3 and M4. It was demonstrated, for the first time, that CYP3A4 was capable of directly oxidizing the triple bond of the cyclopropyl ethynyl group to an unstable oxirene. The apparent K_m and V_max values for the formation of an oxirene (detected as the GSH adduct M3) were 1.9 mM and 10.2 pmol/min/mg of protein, respectively. These results suggest that CYP2B6 has a higher affinity than CYP3A4 toward DPC 963. This consequently leads to greater levels of CYP2B6-catalyzed product, M8, than CYP3A4-mediated bioactivation of DPC 963 to benzoquinone imine or oxirene intermediates.