MECHANISMS OF DRUG INTERACTIONS AND REACTIVE METABOLITES

本申请中描述的研究的长期目标 是在分子水平上理解相互作用的机制 在生物转化为有毒代谢物的药物和其他 调节毒性的药物。对涉及到的因素的了解 活性代谢物的产生和处置对 药物的安全使用，并可提供对生化和化学的见解 形成有毒代谢物的途径。特别是在这一点上 细胞色素P450的研究、激活和诱导机制 催化广泛使用的止痛药/退烧药的氧化， 对乙酰氨基酚，将被调查。这些研究的基本原理是 对乙酰氨基酚会导致危及生命的肝坏死 P450氧化产物N-乙酰基-对苯二酚亚胺的初级介导 扑热息痛的代谢物，以及与扑热息痛一起使用的其他药物 增加这种有毒代谢物的形成速度。 第一，细胞色素P3A2介导的对乙酰氨基酚的激活机制 将检查咖啡因的氧化作用。抗P450抗体的制备 还原酶和细胞色素b5，以及针对CYP3A2的定点突变体 用来确定哪些氨基酸残基参与了 激活过程。突变地点的选择将基于 发现与CYP3A2同源约90%的CYP3A1未被激活 尤其是咖啡因。 第二，抗结核药物诱导细胞色素P450_2E_1的机制 药物异烟肼将通过光谱和动力学研究进行研究 脱脂蛋白、全蛋白和信使核糖核酸的稳定性。的时间进程 异烟肼与对乙酰氨基酚的药物相互作用及其更具特异性 这两种药物的底物，氯唑沙宗将进行详细的检测。 人类和大鼠，开发一种药物相互作用的模型，这种药物可能 稳定细胞色素P450以防降解。 第三，假设新型胃酸抗分泌药物， 奥美拉唑，增加N-乙酰-对苯二酚的形成清除量 亚胺由扑热息痛通过在人体内诱导产生的细胞色素P1A2，将被测试。 奥美拉唑治疗及预处理对机体代谢的影响 对乙酰氨基酚和咖啡因3-去甲基化的影响 反应，将在人类志愿者身上确定。 最后，催化对乙酰氨基酚氧化的主要人类亚型 对有毒的苯二酚亚胺和无毒的儿茶酚代谢物，3- 羟基对乙酰氨基酚，将被鉴定。核磁共振顺磁弛豫 然后将使用方法来确定两者的形成率 产物与对乙酰氨基酚在活性阶段的取向有关 确定了P450亚型的位置。

The long-term objectives of the research described in this application are to understand, at the molecular level, the mechanisms of interaction between drugs that are biotransformed to toxic metabolites and other drugs which modulate the toxicity. Knowledge of factors involved in the generation and disposition of reactive metabolites is important to the safe use of drugs, and can provide insights into biochemical and chemical pathways that form toxic metabolites. Specifically in this investigation, mechanisms of activation and induction of cytochrome P450s that catalyze the oxidation of the widely used analgesic/antipyretic, acetaminophen, will be investigated. The rationale for these studies is that acetaminophen can cause life-threatening hepatic necrosis which is primary mediated by N-acetyl-p-benzoquinone imine, a P450 oxidative metabolite of acetaminophen, and other drugs used with acetaminophen may increase the formation rate of this toxic metabolite. First, the mechanism of activation of CYP3A2-mediated acetaminophen oxidation by caffeine will be examined. Antibodies prepared to P450 reductase and cytochrome b5, and site-directed mutants to CYP3A2 will be used to determine which amino acid residues are involved in the activation process. Sites for mutation will be chosen based on the finding that CYP3A1, which is ~90% homologous to CYP3A2, is not activated significantly by caffeine. Secondly, the mechanism of induction of CYP2E1 by the antituberculosis drug, isoniazid, will be investigated by spectral and kinetic studies of the stability of apoprotein, holoprotein, and mRNA. The time-course of drug interactions of isoniazid with acetaminophen and the more specific CYP2E1 substrate, chlorzoxazone, will be examined in detail in both humans and rats, to develop a model for interactions of drug that may stabilize cytochromes P450 against degradation. Third, the hypothesis that the new gastric acid antisecretory drug, omeprazole, increases the formation clearance of N-acetyl-p-benzoquinone imine from acetaminophen in humans by inducing CYP1A2, will be tested. The effects of omeprazole treatment and pretreatment on the metabolism of acetaminophen and on the 3-demethylation of caffeine, a CYP1A2 probe reaction, will be determined in human volunteers. Finally, the major human isoforms that catalyze acetaminophen oxidation to the toxic quinone imine and the non-toxic catechol metabolite, 3- hydroxyacetaminophen, will be identified. NMR paramagnetic relaxation methods will then be used to determine if formation rates of the two products correlate with the orientation of acetaminophen at the active site of the P450 isoforms identified.