IN-VITRO METABOLISM OF TIRILAZAD MESYLATE IN MALE AND FEMALE RATS - CONTRIBUTION OF CYTOCHROME P4502C11 AND DELTA(4)-5-ALPHA-REDUCTASE

Tirilazad mesylate, a potent inhibitor of membrane lipid peroxidation in vitro, is under clinical development for the treatment of subarachn oid hemorrhage and head injury. In rat, tirilazad seems to be highly e xtracted and is cleared almost exclusively via hepatic elimination. Th e biotransformation of tirilazad has been investigated in liver micros omal preparations from adult male and female Sprague-Dawley rats. Tiri lazad metabolism in male rat liver microsomes resulted in the formatio n of two primary metabolites: M1 and M2. In incubations with female ra t liver microsomes, M2 was the only primary metabolite detected. Struc tural characterization of M1 and M2 by mass spectrometry demonstrated that M2 was formed by reduction of the Delta(4)-double bond in the ste roid A-ring, whereas M1 arose from oxidative desaturation of one pyrro lidine ring. Further structural analysis of M2 by proton NMR demonstra ted that reduction at C-5 had occurred by addition of hydrogen in the alpha-configuration, Using metabolic probes and antibodies specific to individual hepatic microsomal enzymes, CYPSC11 and 3-oxo-5 alpha-ster oid:NADP(+)Delta(4)-oxidoreductase (5 alpha-reductase) were identified as responsible for the formation of M1 and M2, respectively, The form ation of M1 was inhibited by testosterone, nicotine, cimetidine, and a nti-CYP2C11 IgG, The formation of M2 was inhibited by finasteride, a p otent inhibitor of 5 alpha-reductase. Kinetic analysis of CYP2C11-medi ated M1 formation in male rat liver microsomal incubations revealed th at M1 formation occurred through a low-affinity/low-capacity process ( K-M = 16.67 mu M, V-max = 0.978 nmol/mg microsomal protein/min); the f ormation of M2 was mediated by 5 alpha-reductase in a high-affinity/lo w-capacity process (K-M = 3.07 mu M, V-max = 1.06 nmol/mg microsomal p rotein/min). In contrast, the formation of M2 in female rat liver micr osomes was mediated by 5 alpha-reductase in a high-affinity/high-capac ity process (K-M = 2.72 mu M, V-max = 4.11 nmol/mg microsomal protein/ min). Comparison of calculated intrinsic formation clearances (V-max/K -M) for M1 and M2 indicated that the female rat possessed a greater in vitro metabolic capacity for tirilazad biotransformation than the mal e rat, Therefore, the clearance of tirilazad mesylate in the rat is me diated primarily by rat liver 5 alpha-reductase, and the capacity in t he female rat is 5-fold the capacity in the male. These observations c orrelate with documented differences in 5 alpha-reductase activity and predict a gender difference in tirilazad hepatic clearance in vivo.