title: In vitro metabolism of tirilazad mesylate in male and female rats. Contribution of cytochrome P4502C11 and delta 4-5 alpha-reductase.
abstract: Tirilazad mesylate, a potent inhibitor of membrane lipid peroxidation in vitro, is under clinical development for the treatment of subarachnoid hemorrhage and head injury. In rat, tirilazad seems to be highly extracted and is cleared almost exclusively via hepatic elimination. The biotransformation of tirilazad has been investigated in liver microsomal preparations from adult male and female Sprague-Dawley rats. Tirilazad metabolism in male rat liver microsomes resulted in the formation of two primary metabolites: M1 and M2. In incubations with female rat liver microsomes, M2 was the only primary metabolite detected. Structural characterization of M1 and M2 by mass spectrometry demonstrated that M2 was formed by reduction of the delta 4-double bond in the steroid A-ring, whereas M1 arose from oxidative desaturation of one pyrrolidine ring. Further structural analysis of M2 by proton NMR demonstrated 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, CYP2C11 and 3-oxo-5 alpha-steroid:NADP+ delta 4-oxidoreductase (5 alpha-reductase) were identified as responsible for the formation of M1 and M2, respectively. The formation of M1 was inhibited by testosterone, nicotine, cimetidine, and anti-CYP2C11 IgG. The formation of M2 was inhibited by finasteride, a potent inhibitor of 5 alpha-reductase. Kinetic analysis of CYP2C11-mediated M1 formation in male rat liver microsomal incubations revealed that M1 formation occurred through a low-affinity/low-capacity process (KM = 16.67 microM, Vmax = 0.978 nmol/mg microsomal protein/min); the formation of M2 was mediated by 5 alpha-reductase in a high-affinity/low-capacity process (KM = 3.07 microM, Vmax = 1.06 nmol/mg microsomal protein/min). In contrast, the formation of M2 in female rat liver microsomes was mediated by 5 alpha-reductase in a high-affinity/high-capacity process (KM = 2.72 microM, Vmax = 4.11 nmol/mg microsomal protein/min). Comparison of calculated intrinsic formation clearances (Vmax/KM) for M1 and M2 indicated that the female rat possessed a greater in vitro metabolic capacity for tirilazad biotransformation than the male rat. Therefore, the clearance of tirilazad mesylate in the rat is mediated primarily by rat liver 5 alpha-reductase, and the capacity in the female rat is 5-fold the capacity in the male. These observations correlate with documented differences in 5 alpha-reductase activity and predict a gender difference in tirilazad hepatic clearance in vivo.
authors.affiliation: Upjohn Laboratories, Kalamazoo, MI 49001, USA.
authors.forename: L C
authors.initials: LC
authors.lastname: Wienkers
authors.forename: R C
authors.initials: RC
authors.lastname: Steenwyk
authors.forename: S A
authors.initials: SA
authors.lastname: Mizsak
authors.forename: P G
authors.initials: PG
authors.lastname: Pearson
mesh_terms: D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000378:metabolism*; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001711:Biotransformation; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D008054:Lipid Peroxides / Q000378:metabolism* / Q000493:pharmacokinetics; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008657:Metabolic Clearance Rate; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D015394:Molecular Structure; D011281:Pregnatrienes / Q000378:metabolism* / Q000493:pharmacokinetics; D011522:Protons; D051381:Rats; D017207:Rats, Sprague-Dawley; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000378:metabolism*
chemical_list: D008054:Lipid Peroxides; D011281:Pregnatrienes; D011522:Protons; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C450279:CYP2C11 protein, rat; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase; C053355:tirilazad