STEREOSPECIFIC SULFATION OF CHIRAL DRUGS IN HUMANS

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The objective of this proposal is to determine the stereochemistry of sulfate conjugation of chiral phenolic drugs, most notably sympathomimetic amine drugs, by the human phenolsulfotransferases (PSTs), the enzymatic mechanisms involved and its impact on the disposition of the enantiomers of these drugs. Most drugs of this class (isoproterenol, salbutamol, terbutaline) are used as racemic mixtures, although the pharmacologic properties of the individual enantiomers differ greatly. Based on preliminary observations, we have developed the hypothesis that sulfoconjugation of these drugs is enantioselective, tissue and isoenzyme specific and a key determinant of the availability of the enantiomers of these drugs at their multiple sites of action. This hypothesis will be tested directly in humans using tissue cytosols, isolated PST isoenzymes, cultured cell lines and in vivo. In Aim 1 we will establish the kinetics (Km, Vmax) of sulfate conjugation of the individual enantiomers of these drugs, in particular by the human intestine and liver, but also by platelets and other tissues. We will use the cytosol as the PST source with 35S-labeled PAPS as the cosubstrate and stereospecific analytical methodology for the intact sulfate conjugates formed. In Aim 2 we will isolate and purify they cytosolic PST isoenzymes, first from human intestine and liver and then from other tissues. We will characterize the specificity of the individual isoenzymes for sulfation of the enantiomers of the sympathomimetic amine drugs. Antibodies raised against the PST isoenzymes will be used in their characterization and quantitation in various tissues. In Aim 3 w will establish the utility of human intestinal and hepatic cell lines as simple and reproducible models of stereoselective presystemic sulfate conjugation, the key determinant of the oral bioavailability of the enantiomers of these drugs. In aim 4 we will examine the impact of stereoselective sulfate conjugation on the pharmacokinetics of sympathomimetic amine drugs in vivo in man in order to evaluate the clinical implications of our findings with human cytosol, purified PST isoenzymes and cell lines. Specifically, the proposed studies will determine the effect of stereoselectivity in sulfoconjugation on the availability of chiral drugs at their sites of action. More generally, these studies should lead to an improved understanding of the distribution, nature and function of human PSTs as well as provide a basis for studies of their binding/catalytic sites.