Abstract

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 Δ⁴ bond in the steroid A-ring. Kinetic analysis of tirilazad reduction in human liver microsomes revealed that kinetically distinct type 1 and type 2 5α-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 5α-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 primarilyvia CYP3A4 metabolism. Therefore, concurrent administration of therapeutic agents that modulate 5α-reductase type 1 or CYP3A activity are anticipated to affect the pharmacokinetics of PNU-89678.