STEREOSPECIFIC SULFATION OF CHIRAL DRUGS IN HUMANS

本提案的目的是确定以下化合物的立体化学： 手性酚类药物，特别是拟交感神经药硫酸酯缀合 胺药物，由人酚磺基转移酶（PST），酶 机制及其对处置的对映体的影响 这些药物。 这类药物（异丙肾上腺素，沙丁胺醇， 特布他林）作为外消旋混合物使用，尽管药理学上 各个对映异构体的性质差别很大。 基于 根据初步观察，我们提出了一个假设， 这些药物的磺基缀合是对映选择性的，组织和同工酶 的对映异构体的可用性的一个关键决定因素 这些药物在其多个作用部位。 这一假设将是 使用组织细胞溶胶，分离的PST同工酶， 培养的细胞系和体内。 在目标1中，我们将建立动力学 (Km，Vmax）的硫酸盐缀合的影响。 药物，特别是人体肠道和肝脏，但也 血小板和其他组织。 我们将使用细胞质作为PST源 以35 S标记的PAPS为共底物， 形成的完整硫酸盐缀合物的方法学。 在目标2中， 分离和纯化它们胞质PST同工酶，首先从人 肠和肝脏然后是其他组织。 我们将描述 对映体硫酸化的单个同工酶特异性 拟交感神经胺类药物 抗PST抗体 同工酶将用于它们的表征和定量， 各种组织。 在目标3中，将建立人肠 和肝细胞系作为立体选择性的简单和可重复的模型 系统前硫酸盐结合，口服的关键决定因素 这些药物的对映体的生物利用度。 在目标4中， 检查立体选择性硫酸酯缀合对 拟交感神经胺药物在人体内的药代动力学， 评估我们在人类胞质溶胶中发现的临床意义， 纯化的PST同工酶和细胞系。 具体而言，拟议的研究 将确定磺基共轭中的立体选择性对 手性药物在其作用部位的可用性。 更一般地说， 这些研究应有助于更好地了解分布情况， 人类PSTs的性质和功能，并为研究 它们的结合/催化位点。

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.