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

Abstract

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 ϫ 10 9 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 (AME 359 ), caused plasma cocaine hydrolase activity to rise 50,000-fold. At the point of peak AME 359 expression, cocaine was cleared from the blood too rapidly for accurate measurement, and pressor responses to the injection of drug were greatly impaired.

Adenovirus-Transduced Human Butyrylcholinesterase in Mouse Blood Functions as a Bioscavenger of Chemical Warfare Nerve Agents

“…Gao et al (2005) also reported expression of very high levels of two mutant Hu BChEs with cocaine hydrolase activity in rats after a single injection of Ad containing the genes for these enzymes. In this study, we report on the biochemical properties of rHu BChE expressed by Ad-containing the full-length and truncated Hu BChE gene.…”

Gene-Delivered Butyrylcholinesterase Is Prophylactic against the Toxicity of Chemical Warfare Nerve Agents and Organophosphorus Compounds

“…Although the peak expression levels were phenomenally high on day 4, the drop soon after peak was equally phenomenal. A similar pattern of expression, with peak on days 4 to 7 and Ͼ90% drop by day 15, was reported for CocE expression mediated by adenoviral vectors (Gao et al, 2005). This pattern of expression is apparently common with adenoviral vectors and is a reflection of immunological reactions, be it humoral or T-cell mediated (Dai et al, 1995;Yang et al, 1995).…”

“…An engineered form of BChE, cocaine hydrolase (CocE), has been studied for expression in rats using adenovirus system (Gao et al, 2005). A direct comparison between our study and the CocE study is not possible because the enzyme assays used are distinctly different.…”

“…In contrast, full-length human BChE expressed by adenovirus in mice yielded dimers, tetramers, and monomers in equal proportion at an expression level of 177 U/ml (Chilukuri et al, 2009). In addition, CocE, a doubly mutated (A328W/Y332A) version of human BChE (Sun et al, 2002), when expressed by adenoviral means in rats, was predominantly (96%) tetrameric in circulation (Gao et al, 2005).…”

Identification of Human Butyrylcholinesterase Organophosphate-Resistant Variants through a Novel Mammalian Enzyme Functional Screen

“…This method therefore met all of the conditions required for an efficient screening system. Successful expression of active hBChE with an adenoviral vector has been reported previously (Gao et al, 2005), and we optimized the use of an adenovirus system to develop the functional screening approach reported herein.…”