BIOTRANSFORMATION OF TIRILAZAD IN HUMAN - 3 - TIRILAZAD A-RING REDUCTION BY HUMAN LIVER MICROSOMAL 5-ALPHA-REDUCTASE TYPE-1 AND TYPE-2

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 with a medium-to-high extraction ratio. In human liver microsomal preparations, tirilazad is biotransformed t o multiple oxidative products and one reduced, pharmacologically activ e metabolite, U-89678. Characterization of the reduced metabolite by m ass spectrometry and cochromatography with an authentic standard demon strated that U-89678 was formed via stereoselective reduction of the D elta(4) bond in the steroid A-ring. Kinetic analysis of tirilazad redu ction in human liver microsomes revealed that kinetically distinct typ e 1 and type 2 5 alpha-reductase enzymes were responsible for U-89678 formation; the apparent K-M values for type 2 and type 1 were similar to 15 and similar to 0.5 mu M, respectively; Based on pH dependence an d finasteride inhibition studies, it was inferred that 5 alpha-reducta se type 1 was the high affinity/low capacity microsomal reductase that contributed to tirilazad clearance in vivo. In addition, a role for C YP3A4 in the metabolism of U-89678 was established using cDNA expresse d CYP3A4 and correlation studies comparing U-89678 consumption with cy tochrome P450 activities across a population of human liver microsomes . Collectively, these data suggest that formation of U-89678, a circul ating pharmacologically active metabolite, contributes to the total me tabolic elimination of tirilazad in humans and that clearance of U-896 78 is mediated primarily via CYP3A4 metabolism. Therefore, concurrent administration of therapeutic agents that modulate 5 alpha- reductase type 1 or CYP3A activity are anticipated to affect the pharmacokinetic s of PNU-89678.