Human Brain CYP P450s and Psychoactive Drug Metabolism

描述（由申请人提供）：使用原位杂交和免疫组织化学检测技术，已经证明在肝脏中表达的许多细胞色素P450也在人类和大鼠脑中表达。此外，使用人类和大鼠脑微粒体，药物和甾体化合物的P450依赖性代谢已被证明，表明脑细胞色素P450具有催化活性。最近，一种独特的CYP2D形式已被确定为仅在20例尸体供体中的9例中表达。在Neuro 2A细胞中克隆和表达的这种CYP2D形式具有独特的催化活性，将可待因专门转化为吗啡，从而不仅证明了独特的脑特异性定位，而且还证明了特异性功能活性。 该提案将集中于这种和其他P450形式在人脑中独特表达的表达的定义。这一目标将通过定义脑中这些形式的区域分布作为性别、年龄和种族的函数以及通过定义这些表达的纯化P450对临床上用于治疗脑疾病的一组神经活性精神活性药物的催化能力来实现。 药物小组将包括抗抑郁药氟西汀和丙咪嗪，精神安定药氯丙嗪和氟哌啶醇，抗焦虑药阿普唑仑，抗多动症药哌醋甲酯，镇痛药乙基吗啡和苯丙胺。我们将确定这些药物在人脑微粒体中的催化活性。我们将使用从脑区域分离的RNA以及通过原位杂交技术的QRTPCR来定义这些新形式的区域表达。我们将在Neuro 2A、COS 7、E.大肠杆菌和酵母细胞，以评估表达的蛋白质的催化活性与脑微粒体相比。我们将研究所表达的独特蛋白质与野生型蛋白质的精神活性底物结合。 这些不同水平的方法将使我们能够定义在大脑中独特表达的DP450形式，并评估它们在代谢精神活性药物中的功能作用。

DESCRIPTION (provided by applicant): A number of cytochromes P450 expressed in liver have also been shown to be expressed in human and rat brain using techniques of in situ hybridization and immunohistochemical detection. Moreover, using human and rat brain microsomes, the P450-dependent metabolism of drugs and stercids has been demonstrated showing that brain cytochromes P450 are catalytically active. Recently a unique CYP2D form has been identified as expressed only in the brain of 9 out of 20 cadaveric donors examined. This CYP2D form, cloned and expressed in Neuro 2A cells, has a unique catalytic activity, converting codeine exclusively to morphine, thereby demonstrating not only unique brain specific localization, but also, a specific functional activity. This proposal will focus on the definition of expression of this and other P450 forms uniquely expressed in human brain. This objective will be accomplished by defining the regional distribution of such forms in brain as a function of gender, age and ethnicity as well as by definition of the catalytic capacities of these expressed purified P450s towards a panel of neuroactive psychoactive drugs clinically utilized to treat brain disorders. The drug panel will include the antidepressants fluoxetine and imipramine the neuroleptics chlorpromazine and haloperidol, the antianxiety drug alprazolam, the antihyperactivity drug methylphenidate, the analgesic ethylmorphine and the amphetamine benzphetamine. We will define the catalytic activities of these drugs in human brain microsomes. We will define the regional expression of these novel forms using QRTPCR with RNA isolated from brain regions as well as by in situ hybridization techniques. We will express the cloned unique forms in heterologous expression systems such as Neuro 2A, COS 7, E. coli and yeast cells in order to assess the catalytic activities of the expressed protein in comparison with brain microsomes. We will examine psychoactive substrate binding of the expressed unique proteins versus wild type forms. These approaches at various levels will enable us to define CYP P450 forms expressed uniquely in brain and assess their functional roles in metabolizing psychoactive drugs.