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Drug Metabolism and Disposition Fast Forward
First published on May 31, 2007; DOI: 10.1124/dmd.107.014746

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Received for publication January 31, 2007.
Revised May 29, 2007.
Accepted for publication May 30, 2007.

IN VITRO METABOLIC STUDY OF TEMSIROLIMUS: PREPARATION, ISOLATION, AND IDENTIFICATION OF THE METABOLITES

Ping Cai 1*, Rushung Tsao 1, Mark E. Ruppen 1

1 Wyeth Research

* Address correspondence to: E-mail: caip{at}wyeth.com

Abstract

The in vitro metabolism of temsirolimus, {rapamycin-42-[2,2-bis-(hydroxymethyl)]-propionate}, an antineoplastic agent, was studied using human liver microsomes as well as recombinant human P450s, namely CYP-3A4, 1A2, 2A6, 2C8, 2C9, 2C19, and 2E1. Fifteen metabolites were detected by liquid chromatography-tandem mass spectrometry. CYP3A4 was identified as the main enzyme responsible for the metabolism of the compound. Incubation of temsirolimus with recombinant CYP3A4 produced most of the metabolites detected from incubation with human liver microsomes, which was used for large-scale preparation of the metabolites. By silica gel chromatography followed by semi-preparative reverse-phase HPLC, individual metabolites were separated and purified for structural elucidation and bioactivity studies. The minor metabolites were mainly the hydroxylated or demethylated macrolide ring-opened temsirolimus derivatives identified by both positive and negative MS and MS/MS spectroscopic methods. Since these compounds were unstable and only present in trace amounts, no further investigations were conducted. Six major metabolites were identified as 36-hydroxyl temsirolimus (M8), 35-hydroxyl temsirolimus (M9), 11-hydroxyl temsirolimus with opened hemi-ketal ring (M10 and M11), N-oxide temsirolimus (M12), and 32-O-desmethyl temsirolimus (M13) using combined LC-MS, MS/MS, MS/MS/MS, and NMR techniques. Compared to the parent compound, these metabolites showed dramatically decreased activity against LNCaP cellular proliferation.


Key words: anticancer agents, CYP3A, HPLC, human CYP enzymes, metabolite identification


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