Gene transfer of cocaine hydrolase suppresses cardiovascular responses to cocaine in rats
Gene transfer of cocaine hydrolase suppresses cardiovascular responses to cocaine in rats
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DOI：
10.1124/mol.104.006924
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发表时间：
2005-01-01
影响因子：
3.6
通讯作者：
Brimijoin, S
中科院分区：
文献类型：
--
作者：
Gao, Y;Atanasova, E;Brimijoin, S
我们之前发现，注射由人丁酰胆碱酯酶改造的可卡因水解酶（CocE）会暂时加速大鼠的可卡因代谢，同时减少生理和行为反应。为了研究更广泛的治疗效果，CocE cDNA 被整合到带有巨细胞病毒启动子的无复制能力的 5 型腺病毒载体中。在服用该试剂（2.2 x 10(9) 噬菌斑形成单位）的大鼠中，通过 CocE mRNA 的逆转录聚合酶链反应和酶活性的放射测定来表征表达的时间过程。肝脏和血浆显示出相当的表达，在载体施用后2天开始，并在5至7天之间达到峰值。血浆 CocE 含量高达 100 mU/ml，总可卡因水解活性比“空载体”或未处理对照高 3000 倍。这种表达水平近似于静脉注射后立即发现的表达水平。注射纯化水解酶，3 毫克/公斤，该剂量可缩短可卡因半衰期并减弱心血管影响。蔗糖密度梯度分析表明，96% 的循环 CocE 活性与四聚体酶形式相关，预计在体内稳定。与这一预期一致，来自载体处理的大鼠的 CocE 在重新注射到初始大鼠体内时显示出 33 小时的血浆 t(1/2)。另一种丁酰胆碱酯酶突变体应用分子进化突变体 359 (AME(359)) 的转导导致血浆可卡因水解酶活性升高 50,000 倍。在 AME(359) 表达峰值时，可卡因从血液中清除的速度太快，无法进行准确测量，并且对注射药物的升压反应也大大受损。
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 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 half-life 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.