Characterization of a Novel Selective Cytochrome P450 3A5 Substrate

描述（由申请人提供）：人们普遍认为，当其中一种药物干扰参与药物代谢的关键酶（主要是细胞色素P450家族中的酶）的活性时，服用多种药物的患者容易发生药代动力学药物相互作用。在过去的十年里，FDA通常要求制药公司进行临床试验，以确定这种药物-药物相互作用的结果。一些药物如酮康唑已被证明能抑制单个P450酶的活性；而其他药物如利福平或奥美拉唑则会诱导肝脏中P450水平的升高。这些相互作用可能需要调整剂量，以避免药物浓度升高和潜在的毒性增加或药物水平不足而无效。现有的体外工具可以合理地预测可能引起大多数常见的基于代谢的药物-药物相互作用的化合物。然而，目前还没有工具来区分主要的CYP3A酶，CYP3A4和CYP3A5。这两种酶比其他p450酶参与更多药物的代谢。CYP3A4/5活性的改变可能会产生严重的后果，通过这一途径代谢的药物通常会有警告：不要与葡萄柚汁、酮康唑、圣约翰疣和许多其他CYP3A抑制剂或诱导剂一起服用。CYP3A5可能是造成处方药代谢个体间和种族间差异的最重要的遗传因素。具有至少一个活性CYP3A5*1等位基因的个体表达大量的功能性CYP3A5，而CYP3A5*3*3导致剪接变异，导致CYP3A5表达最少。大约60%的非裔美国人和30%的白种人至少有一个*1等位基因，并且CYP3A5和CYP3A4的表达水平大致相当。CYP3A4和CYP3A5活性在生物学相关样本中无法明确区分。目前，几乎所有被批准的化合物的CYP3A抑制数据都是通过CYP3A测定混合人肝微粒体的睾酮羟基化或咪达唑仑羟基化产生的。虽然这两种底物都被CYP3A4和CYP3A5代谢，但由此产生的抑制常数主要反映CYP3A4，因为多个肝微粒体供体池中CYP3A4的含量大约是CYP3A5的5至6倍。我们已经确定了CYP3A5的第一个高选择性底物，并提出建立一个选择性CYP3A5测定能够定量CYP3A5活性和抑制在肝微粒体孵育。

DESCRIPTION (provided by applicant): It is widely accepted that patients on multiple medications are susceptible to pharmacokinetic drug-drug interactions when one of the drugs interferes with the activity of key enzymes involved in drug metabolism, chiefly enzymes in the cytochrome P450 family. In the past decade the FDA has commonly required companies to conduct clinical trials to determine the result of such drug-drug interactions. Some drugs such as ketoconazole have been shown to inhibit the activity of individual P450 enzymes; while other drugs such as rifampicin or omeprazole induce expression of higher P450 levels in the liver. These interactions can require dose adjustments to avoid having elevated drug concentrations and potentially increasing toxicities or having insufficient drug levels to be efficacious. Existing in vitro tools can reasonably predict compounds that are likely to cause most of the common metabolism-based drug-drug interactions. However, tools are not currently available to distinguish between the major CYP3A enzymes, CYP3A4 and CYP3A5. These two enzymes are implicated in the metabolism of more pharmaceuticals than any other P450s. Alteration of CYP3A4/5 activity can have serious consequences and drugs that are metabolized through this pathway often come with warnings: not to take with grapefruit juice, ketoconazole, St. Johns Wart, and numerous other CYP3A inhibitors or inducers. CYP3A5 may be the most important genetic contributor to interindividual and interracial differences in the metabolism of prescription drugs. Individuals with at least one gene for the active CYP3A5*1 allele express large amounts of functional CYP3A5, whereas CYP3A5*3*3 causes a splice variant that results in minimal CYP3A5 expression. Approximately 60% of African Americans and 30% of Caucasians have at least one *1 allele and express roughly equivalent levels of CYP3A5 and CYP3A4. CYP3A4 and CYP3A5 activity cannot be clearly differentiated in biologically relevant samples. Currently, the data for CYP3A inhibition of almost every approved compound is generated using a CYP3A assay measuring testosterone hydroxylation or midazolam hydroxylation by pooled human liver microsomes. While both of these substrates are metabolized by CYP3A4 and CYP3A5, the resulting inhibition constants primarily reflect CYP3A4 because multiple donor pools of hepatic microsomes contain approximately five to six times more CYP3A4 than CYP3A5. We have identified the first highly selective substrate of CYP3A5 and propose to build a selective CYP3A5 assay capable of quantifying CYP3A5 activity and inhibition in hepatic microsomal incubations.