Human Brain CYP P450s and Psychoactive Drug Metabolism

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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.