Vol. 287, Issue 2, 583-590, November 1998

Biotransformation of Tirilazad in Human: 3. Tirilazad A-Ring Reduction by Human Liver Microsomal 5-Reductase Type 1 and Type 2

Larry C. Wienkers, Rick C. Steenwyk, Michael J. Hauer, Joseph C. Fleishaker and Paul G. Pearson

Drug Metabolism and Disposition Research (L.C.W., R.C.S., M.J.H., P.G.P.) and Clinical Pharmacokinetics (J.C.F., P.G.P.), Pharmacia & Upjohn, Kalamazoo, Michigan

Tirilazad mesylate (FREEDOX), a potent inhibitor of membrane lipid peroxidation in vitro, is under clinical development for the treatment of subarachnoid hemorrhage. In humans, tirilazad is cleared almost exclusively via hepatic elimination with a medium-to-high extraction ratio. In human liver microsomal preparations, tirilazad is biotransformed to multiple oxidative products and one reduced, pharmacologically active metabolite, U-89678. Characterization of the reduced metabolite by mass spectrometry and cochromatography with an authentic standard demonstrated that U-89678 was formed via stereoselective reduction of the ⁴ bond in the steroid A-ring. Kinetic analysis of tirilazad reduction in human liver microsomes revealed that kinetically distinct type 1 and type 2 5-reductase enzymes were responsible for U-89678 formation; the apparent K_M values for type 2 and type 1 were ~15 and ~0.5 µM, respectively. Based on pH dependence and finasteride inhibition studies, it was inferred that 5-reductase type 1 was the high affinity/low capacity microsomal reductase that contributed to tirilazad clearance in vivo. In addition, a role for CYP3A4 in the metabolism of U-89678 was established using cDNA expressed CYP3A4 and correlation studies comparing U-89678 consumption with cytochrome P450 activities across a population of human liver microsomes. Collectively, these data suggest that formation of U-89678, a circulating pharmacologically active metabolite, contributes to the total metabolic elimination of tirilazad in humans and that clearance of U-89678 is mediated primarily via CYP3A4 metabolism. Therefore, concurrent administration of therapeutic agents that modulate 5-reductase type 1 or CYP3A activity are anticipated to affect the pharmacokinetics of PNU-89678.