CYTOCHROME P450 DEPENDENT METABOLISM OF DRUGS IN BRAIN

描述（改编自申请人摘要）：大脑的作用 细胞色素P450在治疗药物代谢中的作用一直受到怀疑， 脑微粒体首次被证明能够激活致癌物质， 神经毒剂 大脑对外源性物质的清除率低 然而，微粒体降低了人们对大脑作为一种器官的热情。 解毒 我们实验室克隆了6种新型细胞色素P450亚型 从脑cDNA表达文库，即CYP2D18、CYP3A9、CYP4F1、CYP4F4， CYP4F5和CYP4F6 使用这些新克隆的亚型中的2种，CYP 2D18和 CYP4F6，在用P450还原酶复溶的COS M6细胞裂解物中表达 和脂质，我们能够提供直接的证据表明，这些大脑亚型 能够催化脱甲基和10-羟基化的一个 抗抑郁药丙咪嗪 此补助金申请要求支持 定义对用作治疗剂的药物的催化活性， 脑疾病-氯丙嗪、氟哌啶醇、乙琥胺、苯妥英和 丙咪嗪-表现为新克隆的 脑P450亚型CYP2D18、CYP3A9、CYP4F1、CYP4F4、CYP4FS和CYP4F6。 我们 将通过定义Km来确定这些异构体的催化能力 以及每种形式的每种基板的Vmax值。 此外，我们将定义 异构体与2种底物的催化能力，即主要的 基底和也是基底的产品，例如 丙咪嗪到地昔帕明到去二甲基丙咪嗪。 我们将获得率 常数为这个A-到B-到C序列，并确定真正的产品 通过定义哪一个是最好的， 底物和哪种化合物是真正的产物。 我们还将定义 新的P450亚型在脑中的区域定位。 然后我们将 使用该信息将用于微透析的探针放置在特定位置 并确定这些治疗药物的局部原位代谢。 这种体外和体内测定的组合将建立一个 评价脑细胞色素P450亚型潜力的明确基础 来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.