Abstract
Cocaine abuse and toxicity remain widespread problems in the United States. Currently cocaine toxicity is treated only symptomatically, because there is no Food and Drug Administration-approved pharmacotherapy for this indication. To address the unmet need, a stabilized mutant of bacterial cocaine esterase [T172R/G173Q-CocE (DM-CocE)], which hydrolyzes cocaine into inactive metabolites and has low immunogenic potential, has been developed and previously tested in animal models of cocaine toxicity. Here, we document the rapid cocaine hydrolysis by low doses of DM-CocE in vitro and in vivo, as well as the pharmacokinetics and distribution of the DM-CocE protein in rats. DM-CocE at 50.5 μg/kg effectively eliminated 4 mg/kg cocaine within 2 min in both male and female rats as measured by mass spectrometry. We expanded on these findings by using a pharmacologically relevant dose of DM-CocE (0.32 mg/kg) in rats and monkeys to hydrolyze convulsant doses of cocaine. DM-CocE reduced cocaine to below detection limits rapidly after injection; however, elimination of DM-CocE resulted in peripheral cocaine redistribution by 30 to 60 min. Elimination of DM-CocE was quantified by using [35S] labeling of the enzyme and was found to have a half-life of 2.1 h in rats. Minor urinary output of DM-CocE was also observed. Immunohistochemistry, Western blotting, and radiography all were used to elucidate the mechanism of DM-CocE elimination, rapid proteolysis, and recycling of amino acids into all tissues. This rapid elimination of DM-CocE is a desirable property of a therapeutic for cocaine toxicity and should reduce the likelihood of immunogenic or adverse reactions as DM-CocE moves toward clinical use.
Footnotes
This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grants DA028086-01, DA025100, DA021416, DA023213]; the National Institutes of Health National Institute of General Medical Sciences [Grant GM007767]; and Reckitt Benckiser.
J.H.W. and R.K.S. have been consultants to Reckitt Benckiser Pharmaceuticals (Richmond, VA). R.B., D.N., M.-C.K., J.H.W., and R.K.S. are inventors on patent number PCT/US2008/069659 for DM-CocE.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- CocE
- cocaine esterase
- DM-CocE
- T172R/G173Q-CocE
- PAGE
- polyacrylamide gel electrophoresis
- PS
- phenyl-Sepharose
- LB
- lysogeny broth
- TBS
- Tris-HCl-buffered saline
- LS
- lima bean trypsin inhibitor
- Ni-NTA
- nickel-nitriloacetic acid
- LC
- liquid chromatography
- MS
- mass spectrometry
- PBS
- phosphate-buffered saline
- SRM
- single reaction monitoring
- PVDF
- polyvinylidene difluoride
- AUC
- area under the curve
- PEG
- polyethylene glycol
- DIC
- differential interference contrast
- TXR
- Texas Red.
- Received August 5, 2011.
- Accepted October 6, 2011.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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