Drug Metab Dispos. 1995 Mar;23(3):383-92.  

In vitro metabolism of tirilazad mesylate in male and female rats. Contribution
of cytochrome P4502C11 and delta 4-5 alpha-reductase.

Wienkers LC, Steenwyk RC, Mizsak SA, Pearson PG.

Upjohn Laboratories, Kalamazoo, MI 49001, USA.

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