CYTOCHROME P450 DEPENDENT METABOLISM OF DRUGS IN BRAIN

描述(摘自申请者的摘要)：大脑的一个角色 细胞色素P450在治疗药物代谢中的作用一直被怀疑 大脑微粒子首次被证明能够激活致癌物和 神经毒剂。脑部对异物的清除率低 然而，微粒体降低了人们对大脑作为大脑器官的热情 解毒。我们实验室克隆了6个新的细胞色素P450亚型 从一个脑内表达文库，即CYP2D18、CYP3A9、CYP4F1、CYP4F4， 细胞色素P4F5和细胞色素P4F6。利用这些新克隆的两种异构体，CYP2D18和 CYP4F6在经P450还原酶重组的COS M6细胞裂解物中的表达 和脂质，我们能够提供直接证据证明这些大脑亚型 能够催化AN的脱甲基化和10-羟基化 抗抑郁药丙咪嗪。此拨款申请请求支持 定义对用作治疗药物的药物的催化活性 脑部疾病-氯丙嗪、氟哌啶醇、乙硫胺、苯妥英钠和 丙咪嗪-由新克隆的表达重组蛋白展示 脑内P450的亚型有：细胞色素P450、细胞色素P450、细胞色素P4D18、细胞色素P3A9、细胞色素P4FJ、细胞色素P4F4、细胞色素P4FS和细胞色素P4F6。我们 将通过定义Km来确定这些异构体的催化能力 以及具有每种形式的每种衬底的Vmax值。此外，我们将定义 两种底物对异构体的催化能力，即初级底物 底物和也是底物的产品，例如 丙咪嗪到地地帕明再到二甲基丙咪嗪。我们将获得费率 此A-to B-to C序列的常量，并确定真实乘积 通过定义哪个是最好的来实现整个反应序列的周转 底物和哪种化合物是真正的产品。我们还将定义 新的P450亚型在脑中的区域定位。到时候我们会的 使用此信息在特定位置放置微透析探头 并定义这些治疗药物的局部原位代谢。 这种体外和体内检测的结合将建立一个 评估脑细胞色素P450亚型潜力的明确基础 在体内代谢用于治疗脑部疾病的药物。

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.