Identification and characterization of efavirenz metabolites by liquid chromatography/mass spectrometry and high field NMR: Species differences in the metabolism of efavirenz

Efavirenz (Sustiva, Fig. 1) is a potent and specific inhibitor of HIV-1 rev erse transcriptase approved for the treatment of HIV infection. To examine the potential differences in the metabolism among species, liquid chromatog raphy/mass spectrometry profiles of efavirenz metabolites in urine of rats, guinea pigs, hamsters, cynomolgus monkeys, and humans were obtained and co mpared. The metabolites of efavirenz were isolated, and structures were det ermined unequivocally by mass spectral and NMR analyses. Efavirenz was meta bolized extensively by all the species as evidenced by the excretion of non e or trace quantities of parent compound in urine. Significant species diff erences in the metabolism of efavirenz were observed. The major metabolite excreted in the urine of all species was the O-glucuronide conjugate (M1) o f the 8-hydroxylated metabolite. Efavirenz was also metabolized by direct c onjugation with glucuronic acid, forming the N-glucuronide (M2) in all five species. The sulfate conjugate of 8-OH efavirenz (M3) was found in the uri ne of rats and cynomolgus monkeys but not in humans. In addition to the aro matic ring-hydroxylated products, metabolites with a hydroxylated cycloprop ane ring (at C14) were also isolated. GSH-related products of efavirenz wer e identified in rats and guinea pigs. The cysteinylglycine adduct (M10), fo rmed from the GSH adduct (M9), was found in significant quantities in only rat and guinea pig urine and was not detected in other species. In vitro me tabolism studies were conducted to show that the GSH adduct was produced fr om the cyclopropanol intermediate (M11) in the presence of only rat liver a nd kidney subcellular fractions and was not formed by similar preparations from humans or cynomolgus monkeys. These studies indicated the existence of a specific glutathione-S-transferase in rats capable of metabolizing the c yclopropanol metabolite (M11) to the GSH adduct, M9. The biotransformation pathways of efavirenz in different species were proposed based on some of t he in vitro results.