Received for publication August 22, 2007.
Revised November 20, 2007.
Accepted for publication November 21, 2007.

Taxane's substituents at C3' affect its regioselective metabolism -Different in vitro metabolism of cephalomannine and paclitaxel

Abstract

To investigate how taxane's substituents at C3' affect its metabolism, we compared the metabolism of cephalomannine and paclitaxel, a pair of analogue which differ slightly at C3' position. After cephalomannine was incubated with human liver microsomes in a NADPH-generating system, two mono-hydroxylated metabolites (M-1, M-2) were detected by LC/MS/MS. C4'' (M-1) and C6 (M-2) were proposed as the possible hydroxylation sites, and the structure of M-1 was confirmed by ¹HNMR. Chemical inhibition studies and assays with recombinant human CYPs indicated that 4''-hydroxycephalomannine was generated predominantly by CYP3A4 and 6-hydroxycephalomannine by CYP2C8. The overall biotransformation rate between paclitaxel and cephalomannine differed slightly (184 versus 145 pmol/min/mg), but the average ratio of metabolites hydroxylated at C13 side chain to C6 for paclitaxel and cephalomannine varied significantly (15:85 versus 64:36) in five human liver samples. Compared with paclitaxel, the major hydroxylation site transferred from C6 to C4'', and the main metabolizing CYP changed from CYP2C8 to CYP3A4 for cephalomannine. In the incubation system with rat or minipig liver microsomes, only 4''-hydroxycephalomannine was detected, and its formation was inhibited by CYP3A inhibitors. Molecular docking by Autodock suggested that cephalomannine adopted an orientation in favor of 4''-hydroxylation, while paclitaxel adopted an orientation favoring 3'-p-hydroxylation. Kinetic studies showed that CYP3A4 catalyzed cephalomannine more efficiently than paclitaxel due to an increased V_m. Our results demonstrate that relative minor modification of taxane at C3’ have major consequence on the metabolism.

Key words: anticancer agents, cytochrome P450 catalyzed oxidations, human CYP enzymes, ligand docking, liver microsomes, mass spectrometry, metabolite identification

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