Abstract
Cocaine toxicity is a widespread problem in the United States, responsible for more than 500,000 emergency department visits a year. There is currently no U.S. Food and Drug Administration-approved pharmacotherapy to directly treat cocaine toxicity. To this end, we have developed a mutant bacterial cocaine esterase (DM-CocE), which has been previously shown to rapidly hydrolyze cocaine into inert metabolites, preventing and reversing toxicity with limited immunogenic potential. Herein we describe the ability of DM-CocE to hydrolyze the active cocaine metabolites norcocaine and cocaethylene and its inability to hydrolyze benzoylecgonine. DM-CocE hydrolyzes norcocaine and cocaethylene with 58 and 45% of its catalytic efficiency for cocaine in vitro as measured by a spectrophotometric assay. We have developed a mass spectrometry method to simultaneously detect cocaine, benzoylecgonine, norcocaine, and ecgonine methyl ester to quantify the effect of DM-CocE on normal cocaine metabolism in vivo. DM-CocE administered to rats 10 min after a convulsant dose of cocaine alters the normal metabolism of cocaine, rapidly decreasing circulating levels of cocaine and norcocaine while increasing ecgonine methyl ester formation. Benzoylecgonine was not hydrolyzed in vivo, but circulating concentrations were reduced, suggesting that DM-CocE may bind and sequester this metabolite. These findings suggest that DM-CocE may reduce cocaine toxicity by eliminating active and toxic metabolites along with the parent cocaine molecule.
Footnotes
This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grants DA028086-01, DA025100, DA021416]; the National Institutes of Health National Institute of General Medical Sciences [Grant GM007767]; and Reckitt Benckiser.
J.H.W. and R.K.S. have received funding from and have been consultants to Reckitt Benckiser Pharmaceuticals. R.L.B., D.N., J.H.W., and R.K.S. are inventors on a patent for DM-CocE and other cocaine esterases: Landry D, MacDonald J, Stojanovic MN, Sunahara RK, Narasimhan D, Woods JH, Tesmer JJG, and Brim RL (2008), inventors; The Trustees of Columbia University in the City of New York, The Regents of the University of Michigan, et al., assignees. Thermostabilization of proteins. World patent WO2009009669. 2008 Oct 7.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.074534.
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ABBREVIATIONS:
- CocE
- cocaine esterase
- DM-CocE
- T172R/G173Q-CocE
- LC
- liquid chromatography
- MS/MS
- tandem mass spectroscopy
- PBS
- phosphate-buffered saline
- rcf
- relative centrifugal force
- ACN
- acetonitrile
- HPLC
- high-performance liquid chromatography
- SRM
- single reaction monitoring.
- Received June 27, 2011.
- Accepted September 1, 2011.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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