OHMORI Shigeru Chiba University Hospital, Division of Pharmacy, Associate Professor, 医学部・付属病院薬剤部, 助教授 (70169069)
HOSOKAWA Masakiyo Chiba University, Faculty of Pharmaceutical Sciences, Research Associate, 薬学部, 助手 (70181500)
|Budget Amount *help
¥7,000,000 (Direct Cost : ¥7,000,000)
Fiscal Year 1994 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1993 : ¥6,000,000 (Direct Cost : ¥6,000,000)
Drugs and naturally occurring products are metabolized and detoxified by various drug metabolizing enzymes. Recently, gene technology has been progressively developed and these techniques are used for the studies on the metabolism and toxicity of drugs. In this regard, we tried to incorporate the genes of the drug metabolizing enzymes into the cells to express their enzymatic functions. The drug metabolizing enxyme which we used in these studies are carboxylesterases, glutathione S-transferase a dn cytochrome P-450, and we had carried out the cDNA cloning of these enzymes. We had extracted and purified the RNA from rats, mice, monkeys and human livers, and synthesized cDNA of each enzyme. Then, we prepared the libraries using ramda gt 11, ramda gt 10 and ramda gt Zap as the expression vectors. Next, we have used the respective antibodies against each enzyme as the probes to screen the cDNA libraries, and determine the DNA sequences of the positive clones of each enzyme. As the results of these experiments, we had six cDNA clones of the drug metabolizing enzymes mentioned above. Among these cDNA,we have expressed the cDNA of esterases using mammalian cells. In fact, we treated the cDNAs of liver esterases of rat RL1 and RH1 and mouse MH1 with pCR3 as an expression vector, and incorporated the genes of these esterases into COS 7 cells. As the consequence of these experiments, the activities of esterase isozymes were expressed. The novel mammalian cells possessing the genes of several drug metabolizing enzymes we have developed here are extremely useful for new drug development in future.