BIOTRANSFORMATION OF TIRILAZAD IN HUMAN .1. CYTOCHROME-P450 3A-MEDIATED HYDROXYLATION OF TIRILAZAD MESYLATE IN HUMAN LIVER-MICROSOMES

Tirilazad mesylate (Freedox), a potent inhibitor of membrane lipid per oxidation in vitro, is under clinical development for the treatment of subarachnoid hemorrhage. In humans, tirilazad is cleared almost exclu sively via hepatic elimination. Characterization of three major micros omal metabolites of tirilazad by mass spectrometry indicated that hydr oxylation had occurred in the pyrrolidine ring(s) and at the 6 beta-po sition of the steroid domain. A role for CYP3A4 in the formation of th e three major metabolites (tirilazad hydroxylase activity) was establi shed in human liver microsomal preparations: 1) Tirilazad hydroxylatio n was potently inhibited by troleandomycin and ketoconazole, specific inhibitors of CYP3A4. 2) The rates of tirilazad hydroxylation within a population of 14 human livers displayed a 9-fold interindividual vari ation and a significant correlation (r(2) = .95) between tirilazad hyd roxylation and testosterone 6 beta-hydroxylation. 3) Kinetic analysis of tirilazad hydroxylase activity in three human livers resulted in an apparent K-m of 2.12, 1.68 and 1.66 mu M, and V-max = 0.85, 0.44 and 3.45 (nmol/mg protein/min) for HL14, HL17 and HL21, respectively. In a ddition, an apparent K-m of 2.07 mu M was established for tirilazad hy droxylation in a cDNA-expressed CYP3A4 microsomal system. Collectively , these data indicate that the metabolic clearance of tirilazad in hum ans is catalyzed primarily by CYP3A4 and provide an insight into facto rs (i.e., age, sex, drug-drug interactions) that modulate the metaboli c clearance of tirilazad in vivo.