IN VITRO STUDIES OF METABOLISM, ABSORPTION, AND TOXICITY

货车Breeman的项目将通过评估活性植物化合物的体外和体内肝脏代谢、毒性、药代动力学和血脑屏障通透性，与该植物中心内的所有其他项目和核心相互作用。这些信息将有助于为妇女健康提供安全有效的植物膳食补充剂的合理依据。在具体目标1中，将使用人和大鼠肝微粒体和肝细胞在体外研究Farnsworth和博尔顿的项目和分析核心中鉴别的活性化合物的代谢。将鉴定负责形成这些代谢物的人酶的特异性同工酶，并确定这些化合物或其代谢物是否抑制这些酶。这些研究将表明这些化合物引起植物-药物相互作用的潜力。在具体目标2中，本实验室开发的超滤LC-MS-MS分析将用于确定植物提取物中化合物的任何亲电和可能有毒的代谢产物是否可以 在肝脏中形成。这些研究将有助于预测高剂量的毒性。此外，将开发一种基于蛋白Keap 1烷基化和随后破坏Keap 1-Nrf 2复合物的新测定法作为补充测定法。由于Keap 1-Nrf 2复合物的破坏会导致多种II期解毒酶的上调，因此暴露于低水平的与Keap 1结合的植物化合物可能实际上会产生保护性反应。在具体目标3中，将使用以下方法测定博尔顿项目中确定的具有潜在CNS效应的植物提取物中的化合物的血脑屏障通透性： 牛脑上皮细胞单层模型系统。最后，在具体目标4中，将在大鼠和健康绝经后妇女的I期临床试验中研究活性化合物的体内代谢、生物利用度和药代动力学。这些研究将提供关于啤酒花中活性成分的安全性和药代动力学的信息。总的来说，这些代谢、代谢活化、渗透性、生物利用度和药代动力学的体外和体内研究将有助于设计这些植物膳食补充剂的安全性和有效性的未来II期和III期临床试验。

Project by van Breeman will interact with all the other Projects and Cores within this Botanical Center by evaluating in vitro and in vivo the hepatic metabolism, toxicity, pharmacokinetics, and blood-brain barrier permeability of active botanical compounds. This information will help provide a rational basis for the safe and effective use of botanical dietary supplements for women's health. In Specific Aim 1, the metabolism of active compounds identified in Projects by Farnsworth and Bolton and Analytical Core will be investigated in vitro using human and rat liver microsomes and hepatocytes. The specific isozymes of the human enzymes that are responsible for the formation of these metabolites will be identified and whether or not these compounds or their metabolites inhibit these enzymes will be determined. These studies will indicate the potential of these compounds to cause botanical-drug interactions. In Specific Aim 2, an ultrafiltration LC-MS-MS assay developed in this laboratory will be used to determine whether any electrophilic and possibly toxic metabolites of compounds in the botanical extracts can be formed in the liver. These studies will help predict toxicity at high doses. In addition, a new assay based on alkylation of the protein Keap1 and subsequent disruption of the Keap1-Nrf2 complex will be developed as a complementary assay. Since disruption of the Keap1-Nrf2 complex produces up-regulation of a variety of Phase II detoxification enzymes, exposure to low levels of botanical compounds that bind to Keap1 might actually produce a protective response. In Specific Aim 3, compounds in botanical extracts that are determined in Project by Bolton to have potential CNS effects will be assayed for blood-brain barrier permeability using a bovine brain epithelial cell monolayer model system. Finally in Specific Aim 4, the in vivo metabolism, bioavailability, and pharmacokinetics of active compounds will be investigated in rats and in a Phase I clinical trial in healthy post-menopausal women. These studies will provide information regarding the safety and pharmacokinetics of the active constituents in hops. Overall, these in vitro and in vivo studies of metabolism, metabolic activation, permeability, bioavailability, and pharmacokinetics will facilitate the design of future Phase II and III clinical trials of the safety and efficacy of these botanical dietary supplements.