Research Project
6494379
DESCRIPTION (Adapted from applicant's abstract): Cytochromes P-450 (P-450), a family of heme proteins, are major drug metabolizing enzymes. Although liver is the primary organ involved in drug metabolism, it is increasingly recognized that metabolism of drugs, in situ, in the target organ plays a vital role in the pharmacological regulation of drug action. The objective of this application is to determine the capability of human brain P-450 to metabolize psychoactive drugs, characterize and localize the multiple forms of P-450 involved therein. Our long term goal is to identify the role of cerebral metabolism in the pharmacological action of these drugs. The immediate specific aims are: Aim 1. Identification of specific form of P-450 involved in the metabolism of psychoactive drugs in human brain by studying (a) P-450-mediated biotransformation of psychoactive drugs, (b) the effect of inhibitors and antibody to isoforms of P-450, on these biotransformation and (c) the relative quantitation of various P-450 forms by immunoblotting, using microsomes prepared from human brain regions such as prefrontal cortex, striatum, thalamus and midbrain. Aim 2-Purification and characterization of various forms of P-450 from human brain cortex, N-terminal sequencing and reconstitution of activity in vivo. Aim 3. Immunocytochemical localization of P-450 forms in human brain using antisera to hepatic and purified brain P-450s. Rat brain microsomes would be used for standardizing the assay of P-450-mediated activities using psychoactive drugs as substrates, for purification of inducible forms of brain P-450 and generation of polyclonal antisera. Human brain tissue (from males and females, obtained at autopsy) would be used for characterization, including identification of possible gender-related differences. All human brain tissue would be subjected to RFLP and allele-specific amplification by PCR to detect genetic polymorphism of P-4502D6. Future plans include cloning of brain P-450 (using oligonucleotides corresponding to the N-terminal sequences of purified brain P-450s for screening cDNA libraries), sequencing and expression of the P-450 isoforms. Human brain P-450 may influence pharmacological modulation of drugs acting on CNS; it is therefore important to identify P-450 mediated biotransformation of psychoactive drugs and characterize the enzymes mediating such reactions.
