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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Catalytic Antibodies that Hydrolyze (−)-Cocaine Obtained by a High-Throughput Procedure

John R. Cashman, Clifford E. Berkman and Gail E. Underiner
Journal of Pharmacology and Experimental Therapeutics June 2000, 293 (3) 952-961;
John R. Cashman
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Clifford E. Berkman
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Gail E. Underiner
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Abstract

Antibodies to a 2β-carboxamido-2β-phosphonate transition-state analog of (−)-cocaine benzoate ester hydrolysis were elicited in mice. A large number of hybridoma cell lines were propagated, and the catalytic activity of culture fluid was determined with a high-throughput photometric assay using cocaine benzoyl thioester as substrate. Binding avidity of the hybridoma supernatants to the phosphonate hapten was also determined. The initial rate constants for cocaine benzoyl thioester hydrolysis and binding avidity for a large number of hybridoma supernatants elicited to the phosphonate hapten did not always correlate. The lack of correlation of substrate hydrolysis with the binding affinity of 70 catalytic antibodies was also observed for (−)-cocaine hydrolysis using derivatization and HPLC analysis of methyl ecgonine as meta-nitrococaine. Thekcat values for cocaine benzoyl thioester hydrolysis by monoclonal antibodies 3, 5, and 12 were 38, 4.2, and 0.6 min−1, respectively. For monoclonal antibody 5, the selectivity ratios (Ki value divided by theKm value for the hydrolysis of cocaine benzoyl thioester) with ecgonine benzoyl ester, ecgonine methyl ester, norcocaine, and ecgonine were 101, 25, 9.4, and 4, respectively. Three active esterolytic monoclonal antibodies identified with the high-throughput assay procedure were examined in detail for their ability to hydrolyze (−)-cocaine. The kcatvalues for the hydrolysis of (−)-cocaine with monoclonal antibodies 3, 5, and 12 were 6.6, 0.4, and 0.1 min−1, respectively. Hydrolysis of (−)-cocaine by monoclonal antibody 3 approached thekcat value for that of human butyrylcholinesterase. Cocaine esterolytic catalytic antibodies that approach or exceed the catalytic efficiency of human butyrylcholinesterase may represent a new pharmacological intervention approach to the treatment of cocaine abuse, and the high-throughput process described here represents an advance in the effort to develop clinically useful antibodies.

Footnotes

  • Send reprint requests to: John R. Cashman, Ph.D., Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA 92121. E-mail: ledcash{at}AOL.com

  • ↵1 This work was supported by National Institute on Drug Abuse, National Institutes of Health, Grants DA08531 and DA11547.

  • ↵2 Current address: Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132.

  • ↵3 Current address: Process Development, Cauldron, 383 Phoenixville Pike, Malvern, PA 19355.

  • Abbreviations:
    BuChE
    butyrylcholinesterase
    KLH
    keyhole limpet hemocyanin
    ELISA
    enzyme-linked immunoassay
    ELS
    evaporative light scattering
    DTNB
    5,5′-dithiobis(2-nitrobenzoic acid)
    mAb
    monoclonal antibody
    vis
    visible
    • Received November 23, 1999.
    • Accepted February 8, 2000.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 293 (3)
Journal of Pharmacology and Experimental Therapeutics
Vol. 293, Issue 3
1 Jun 2000
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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Catalytic Antibodies that Hydrolyze (−)-Cocaine Obtained by a High-Throughput Procedure

John R. Cashman, Clifford E. Berkman and Gail E. Underiner
Journal of Pharmacology and Experimental Therapeutics June 1, 2000, 293 (3) 952-961;

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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Catalytic Antibodies that Hydrolyze (−)-Cocaine Obtained by a High-Throughput Procedure

John R. Cashman, Clifford E. Berkman and Gail E. Underiner
Journal of Pharmacology and Experimental Therapeutics June 1, 2000, 293 (3) 952-961;
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