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Gene Transfer of Cocaine Hydrolase Suppresses Cardiovascular Responses to Cocaine in Rats

Department of Molecular Pharmacology, Mayo Clinic, Rochester, Minnesota (Y.G., E.A., N.S., S.B.); Eli Lilly-Applied Molecular Evolution, San Diego, California (J.D.P., J.D.W.); and Institute of Psychology, Chinese Academy of Science, Beijing, China (N.S.)

We previously found that injection of a cocaine hydrolase (CocE) engineered from human butyrylcholinesterase will transiently accelerate cocaine metabolism in rats while reducing physiological and behavioral responses. To investigate more extended therapeutic effects, CocE cDNA was incorporated into a replication-incompetent type-5 adenoviral vector with a cytomegalovirus promoter. In rats dosed with this agent (2.2 x 10⁹ plaque-forming units), the time course of expression was characterized by reverse transcription polymerase chain reaction for CocE mRNA and by radiometric assay for enzyme activity. Liver and plasma showed comparable expression, beginning 2 days after vector administration and peaking between 5 and 7 days. Plasma CocE content was up to 100 mU/ml, with total cocaine hydrolyzing activity 3000-fold greater than in "empty vector" or untreated controls. This level of expression approximated that found immediately after i.v. injection of purified hydrolase, 3 mg/kg, a dose that shortened cocaine halflife and blunted cardiovascular effects. Sucrose density gradient analysis showed that 96% of the circulating CocE activity was associated with tetrameric enzyme forms, expected to be stable in vivo. Consistent with this expectation, CocE from vector-treated rats showed a plasma t_1/2 of 33 h when reinjected into naive rats. Transduction of another mutant butyrylcholinesterase, Applied Molecular Evolution mutant 359 (AME₃₅₉), caused plasma cocaine hydrolase activity to rise 50,000-fold. At the point of peak AME₃₅₉ expression, cocaine was cleared from the blood too rapidly for accurate measurement, and pressor responses to the injection of drug were greatly impaired.

Address correspondence to: Dr. S. Brimijoin, Department of Molecular Pharmacology, Mayo Clinic, 200 First St. S.W., Rochester MN 55905. E-mail: brimijoi{at}mayo.edu

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