CYTOCHROME P450 DEPENDENT METABOLISM OF DRUGS IN BRAIN

大脑中细胞色素 P450 依赖性药物代谢

基本信息

批准号：
6186508
负责人：
HENRY W STROBEL
金额：
$ 25.9万
依托单位：
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-04-01 至 2003-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6186508
关键词：
brain metabolism chlorpromazine cytochrome P450 drug metabolism enzyme activity enzyme substrate haloperidol imipramine immunocytochemistry in situ hybridization laboratory rat microdialysis phenytoin protein isoforms pyrrolidinediones recombinant proteins

项目摘要

DESCRIPTION (adapted from applicant's abstract): A role for brain cytochromes P450 in metabolism of therapeutic drugs has been suspected since brain microsomes were first shown to be able to activate carcinogens and neurotoxic agents. The low rate of clearance of xenobiotics by brain microsomes, however, diminished enthusiasm for brain as an organ of detoxification. Our laboratory has cloned 6 novel cytochrome P450 isoforms from a brain cDNA expression library namely CYP2D18, CYP3A9, CYP4F1, CYP4F4, CYP4F5 and CYP4F6. Using 2 of these newly cloned isoforms, CYP2D18 and CYP4F6, expressed in COS M6 cell lysates reconstituted with P450 reductase and lipid, we were able to provide direct evidence that these brain isoforms were able to catalyze the demethylation and 10-hydroxylation of an antidepressant, imipramine. This grant application requests support to define the catalytic activity toward drugs used as therapeutic agents for brain diseases - chlorpromazine, haloperidol, ethosuximide, phenytoin and imipramine - exhibited by expressed recombinant proteins of the newly cloned brain P450 isoforms CYP2D18, CYP3A9, CYP4FJ, CYP4F4, CYP4FS and CYP4F6. We will determine the catalytic ability of these isoforms by defining the Km and Vmax values for each substrate with each form. Moreover, we will define the catalytic ability of the isoforms with 2 substrates, that is the primary substrate and the product which is also a substrate such as is the case for imipramine to desipramine to didesmethyl imipramine. We will obtain rate constants for this A - to B - to C sequence and determine the true product turnover for the full reaction sequence by defining which is the best substrate and which compound is the true product. We will also define the regional localization in the brain of the new P450 isoforms. We will then use this information to place probes for microdialysis in specific locations in the brain and define local in situ metabolism of these therapeutic drugs. This combination of in vitro and in vivo determinations will establish a clear basis for evaluating the potential of brain cytochrome P450 isoforms to metabolize in situ drugs used to treat brain diseases.

描述（改编自申请人的摘要）：大脑的角色自从怀疑治疗药物的代谢中的细胞色素P450从那以后一直怀疑首先证明脑微粒体能够激活致癌物和神经毒性剂。大脑清除异生物学的率低然而，微粒体降低了对大脑的热情解毒。我们的实验室克隆了6个新型细胞色素P450同工型来自脑cDNA表达式库，即CYP2D18，CYP3A9，CYP4F1，CYP4F4， CYP4F5和CYP4F6。使用这些新克隆的同工型中的2个，CYP2D18和 CYP4F6，在COS M6细胞裂解物中表达，用P450还原酶重构和脂质，我们能够直接证明这些大脑同工型能够催化脱甲基和10-羟基化抗抑郁药，丙咪胺。本授予申请请求支持定义对用作治疗剂的药物的催化活性脑部疾病 - 氯丙嗪，氟哌啶醇，乙糖酰亚胺，苯妥英和丙咪嗪 - 由新克隆的表达重组蛋白表现出来 Brain P450同工型CYP2D18，CYP3A9，CYP4FJ，CYP4F4，CYP4FS和CYP4F6。我们将通过定义KM来确定这些同工型的催化能力每个形式的每个基板的VMAX值。而且，我们将定义同工型具有2个底物的催化能力，即主要基材和也是基材的产品，例如丙咪嗪将甲丙胺胺甲基丙氨酸胺。我们将获得费率 a -to b-到c序列的常数并确定真实产物通过定义最好的反应序列的营业额底物和哪种化合物是真正的产品。我们还将定义新的P450同工型大脑中的区域定位。然后我们会使用此信息将微透析的探针放在特定位置在大脑中，定义了这些治疗药物的局部原位代谢。体外和体内确定的这种组合将建立评估脑细胞色素P450同工型的潜力的明确基础代谢用于治疗脑部疾病的原位药物。