In Vitro Metabolic Study of Temsirolimus: Preparation, Isolation, and Identification of the Metabolites
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 cytochrome P450s, namely CYP3A4, 1A2, 2A6, 2C8, 2C9, 2C19, and 2E1. Fifteen metabolites were detected by liquid...
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Published in | Drug metabolism and disposition Vol. 35; no. 9; pp. 1554 - 1563 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
Bethesda, MD
American Society for Pharmacology and Experimental Therapeutics
01.09.2007
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Subjects | |
Online Access | Get full text |
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Summary: | 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 cytochrome P450s, namely CYP3A4, 1A2, 2A6, 2C8, 2C9, 2C19,
and 2E1. Fifteen metabolites were detected by liquid chromatography (LC)-tandem mass spectrometry (MS/MS or MS/MS/MS). 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 semipreparative reverse-phase high-performance liquid
chromatography, individual metabolites were separated and purified for structural elucidation and bioactivity studies. The
minor metabolites (peaks 1-7) were identified as hydroxylated or desmethylated macrolide ring-opened temsirolimus derivatives
by both positive and negative mass spectrometry (MS) and MS/MS spectroscopic methods. Because 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 an opened hemiketal 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 with the parent compound,
these metabolites showed dramatically decreased activity against LNCaP cellular proliferation. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0090-9556 1521-009X |
DOI: | 10.1124/dmd.107.014746 |