The objective of this research is to elucidate the mechanism or mechanisms by which substituted amphetamines, such as methylenedioxyamphetamine (MDA), rho-choloroamphetamine (pCA) and methylenedioxymethamphetamine (MDMA) cause a long term reduction in brain serotonin (5HT) levels and cause lesions in certain brain regions. These compounds are members of the phenylisopropylamine class of stimulants and hallucinogens that have a long history of abuse. The appearance of the methylenedioxy compounds as abused substances has caused concern because of their potential for toxicity and the lack of understanding of the detailed pharmacology. The research is centered about a model in which the drugs are metabolized either peripherally or in the brain to substance that concentrate in serotonin neurons and generate reactive electrophilic compounds and/or toxic oxygen radicals. The approach utilizes both whole animal and cell culture as the biological media and a number of chemical methods for analysis of changes in cellular properties and in the drug itself. In vivo studies investigation the metabolic N-demethylenation to catecholamines, the N-demethylation of MDMA, and the O-derivatization of catecholamine metabolites are proposed using GC/MS to analyze plasma and brain tissue extracts. The effects of heavy isotope substitution on metabolically active centers on toxicity will also be assessed as an approach to determining the metabolic pathway for toxicity. The neurochemical changes will be monitored in the whole animal by microdialysis and HPLC methodology. The data from in vivo experiments will be analyzed by pharmacokinetic modelling techniques to assess the relationship between drug levels and pharmacological response. Previous studies had demonstrated that a glutathione (GSH) adduct of a metabolite was formed in vitro and its identity and conditions for its formation will be determined. A neuroblastoma based cell line (NG108-15) has been shown to be sensitive to the toxic actions of some of the phenylisopropylamine derivatives. This line will be used to study the formation of this adduct and the biochemical sequence of toxin-induced cell death. The results of these experiments and the relevant methodology will then be applied to studies on the brains of treated animals to assess the validity of the in vitro experiments. The studies utilized techniques in pharmacokinetics, cell culture and general analytical chemistry to elucidate mechanisms.
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