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Reaction Phenotyping
反应表现型研究评估的影响enzyme isoform-specific inhibitors on the disappearance of a parent test article incubated in pooled human liver microsomes. Incubation with recombinant individual drug metabolizing enzyme isoforms provides confirmation of enzyme activities. Assessing the enzymes responsible for metabolizing a test article can highlight potential liabilities such as metabolism routes catalyzed by single enzymes or by polymorphically-expressed enzymes.
法规considerations for reaction phenotyping studies
The investigation of CYP enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6 and CYP3A) is recommended as an initial investigation to better understand the clearance pathways of the drug according to the drug-drug interaction (DDI) guidelines. If these major CYP enzymes are not involved in the metabolism of the drug, then other Phase I and Phase II enzymes should be evaluated including:
- CYP酶（CYP2A6，CYP2J2，CYP4F2和CYP2E1）
- 包括醛氧化酶（AO），羧基酶（CES），单胺氧化酶（MAO），黄嘌呤单氧化酶（FMO），黄嘌呤氧化酶（XO）和醇/醛脱氢酶（ADH / ALDH）的非CYP酶（ADH / ALDH）
- Conjugative enzymes including UDP glucuronosyl transferases (UGT) and sulfotransferases (SULTs)
反应表现型提供有限公司mation regarding the fraction metabolized of test articles and can be used to assess victim DDI potential, inform clinical study design, predict individual variability in pharmacokinetics and evaluate the impact of genetic polymorphism.
方法
- Test system: Pooled human hepatic microsomes and recombinant individually expressed human enzymes with required cofactors
- CYP isoforms: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. (Other CYPs, UGTs and drug metabolizing enzymes are also available).
- Isoform-specific chemical inhibitors and recombinant enzymes are used for confirmation.
用磷酸盐缓冲液中的微粒体预孵育测试制品至少5分钟。通过添加NADPH或其他合适的辅助因子来启动测试制品的代谢。通过加入有机溶剂终止孵育，然后使用液相色谱 - 质谱（LC-MS）分析用于试验制品和/或其代谢物的样品。
Phase I - Optimization of In Vitro Incubation Conditions
The test article is incubated in triplicates with a range of microsome concentrations (typically 0.1 – 1 mg protein/mL) for four time points up to 60 minutes in the presence of NADPH or other appropriate cofactor.
在不存在辅因子的情况下进行控制孵育。生产线性速率的微粒体浓度和时间点的孵育条件用于定义后续实验。
Phase II - Kinetic Analysis of Test Article Metabolism
The rates of how quickly the test article disappears are monitored in the incubation samples with at least eight concentrations of the test article in triplicates under optimized conditions. Data are analyzed using Michaelis-Menten curve fitting, and kinetic parameters Km和V.最大限度are determined for the test article.
III期 - 使用CYP特异性化学抑制剂的抑制分析
测试制品以浓度
IV期 - 使用重组人CYPS的代谢
测试制品以浓度
可交付成果
These assays will identify enzymes responsible for and the extent of metabolism of the test article in vitro. Kinetics of the test article disappearance and/or metabolite formation are determined. In vitro metabolism assessment identifies the major enzymes involved and alerts you to potential in vivo clearance mechanism liabilities prior to first-in-human testing.
