Abstract
Antitumor prodrug irinotecan is used for a variety of malignancies such as colorectal cancer. It is hydrolyzed to the metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), which exerts its antineoplastic effect. Several human and rodent carboxylesterases are shown to hydrolyze irinotecan, but the overall activity varies from enzyme to enzyme. This report describes a novel mouse liver and kidney carboxylesterase (M-LK) that is highly active toward this prodrug. Northern analyses demonstrated that M-LK was abundantly expressed in the liver and kidney and slightly in the intestine and lung. Lysates from M-LK transfected cells exhibited a markedly higher activity on irinotecan hydrolysis than lysates from the cells transfected with mouse triacylglycerol hydrolase (TGH) (6.9 versus 1.3 pmol/mg/min). Based on the immunostaining intensity with purified rat hydrolase A, M-LK had a specific activity of 173 pmol/mg/min, which ranked it as one of the most efficient esterases known to hydrolyze irinotecan. A chimeric carboxylesterase and its wild-type enzyme (e.g., M-LKn and M-LK), sharing three quarters of the entire sequence from the N-terminus, exhibited the same substrate preference toward irinotecan and two other substrates, suggesting that the N-terminal sequence determines substrate selectivity. M-LK transfected cells manifested more severe cytotoxicity than TGH transfected cells upon being exposed to irinotecan. Topoisomerase I inhibitors such as irinotecan represent a promising class of anticancer drugs. Identification of M-LK as an efficient carboxylesterase to activate irinotecan provides additional sequence information to locate residues involved in irinotecan hydrolysis and thus facilitates the design of new analogs.
Footnotes
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This work was partially supported by a Grant ES07965 from the National Institute of Environmental Health Sciences and a New Investigator Award from the American Association of Colleges of Pharmacy.
- Abbreviations used are::
- Irinotecan
- 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11)
- HCE
- human carboxylesterase
- rCE
- rabbit carboxylesterase
- M-LK
- mouse liver and kidney carboxylesterase
- TGH
- triacylglycerol hydrolase
- HPLC
- high performance liquid chromatography
- LDH
- lactate dehydrogenase
- HS
- anti-hydrolase S antibody
- 4PP
- 4-piperidino-piperidine
- SN-38
- 7-ethyl-10-hydroxycamptothecin
- Received August 7, 2002.
- Accepted September 24, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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