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.
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