DEVELOPMENT OF A MODEL TO PREDICT DRUG HEPATOTOXICITY

描述（由申请人提供）： 肝毒性是导致批准药物退出临床使用的最常见单一不良事件。药物性肝损伤是急性肝功能衰竭的主要原因，通常是致命的。本研究计划的总体目标是开发一种新的体外模型来预测药物及其衍生代谢物的肝毒性。药物诱导的肝毒性的潜在机制多种多样且知之甚少。最近的证据支持这一假设，即肝小管胆盐输出泵（Bsep）的抑制，导致胆汁酸在肝细胞内蓄积和随后的毒性，是药物诱导的肝损伤的重要机制。“测试化合物”[已知的肝毒性药物（波生坦、曲格列酮、利托那韦、甲氨蝶呤、对乙酰氨基酚）和阴性对照（他莫昔芬）]以及六种NCI化合物将在新的体外培养的大鼠肝细胞模型中测定，以测试药物/代谢物的Bsep抑制与这些药物的肝毒性潜力相关的假设。在每组实验中，将在含有标准或高浓度（以维持CYP 450活性）地塞米松的培养基中培养的体外培养大鼠肝细胞暴露于供试化合物，以分别评估在不存在和存在生成的代谢产物的情况下母体药物的相对肝毒性。在体外培养的大鼠肝细胞中，将在急性暴露期间不足以产生明显细胞毒性的浓度下测定Bsep药物抑制的IC 50，以避免全身细胞毒性对Bsep功能的混淆效应。随后，将确定培养物中随时间推移在IC 50（或如果未观察到特定化合物的Bsep抑制，则细胞毒性的最高无作用水平）下的药物作用，以定义长期暴露的结果（例如，细胞毒性、胆汁酸或磷脂浓度升高、转运蛋白表达改变）。三明治培养的肝细胞是研究药物诱导的肝毒性的一种令人兴奋的工具，因为该模型保持了肝细胞的极性、形态、肝脏特异性功能（包括代谢活性），并允许直接进入肝细胞和邻近的胆管室。在该模型中，肝转运蛋白表达并随时间定位于正确的膜结构域。重要的是，可以在该系统中直接评估运输功能。使用来自毒理学研究中通常使用的动物种属的肝细胞（例如，狗、猴）以及人的体外模型是该新型体外模型的特别有吸引力的特征，并且将在随后的R33应用中在该模型系统的放大阶段期间进行开发。

DESCRIPTION (provided by applicant): Hepatotoxicity has been the most common single adverse event resulting in removal of approved drugs from clinical use. Drug-induced liver injury is a leading cause of acute liver failure, and often is fatal. The overall goal of this research program is to develop a novel in vitro model to predict hepatotoxicity of drugs and derived metabolites. The mechanisms underlying drug-induced hepatotoxicity are varied and poorly understood. Recent evidence supports the hypothesis that inhibition of the hepatic canalicular bile salt export pump (Bsep), resulting in bile acid accumulation within the hepatocyte and subsequent toxicity, is an important mechanism of drug-induced liver injury. "Test Compounds" [known hepatotoxic drugs (bosentan, troglitazone, ritonavir, methotrexate, acetaminophen) and a negative control (tamoxifen)], as well as six NCI compounds, will be assayed in a novel sandwich-cultured rat hepatocyte model to test the hypothesis that Bsep inhibition by drugs/metabolites correlates with the hepatotoxic potential of these drugs. In each set of experiments, sandwich-cultured rat hepatocytes cultured in medium containing either standard or high (to maintain CYP450 activity) dexamethasone concentrations will be exposed to test compounds to assess the relative hepatotoxicity of the parent drug in the absence and presence of generated metabolites, respectively. The IC50 for drug inhibition of Bsep will be determined at concentrations insufficient to produce overt cytotoxicity during acute exposure in sandwich-cultured rat hepatocytes to avoid confounding effects of generalized cell toxicity on Bsep function. Subsequently, drug effects at the IC50 (or highest no-effect level for cytotoxicity if Bsep inhibition is not observed for a particular compound) over time in culture will be determined to define the outcome of prolonged exposure (e.g., cytotoxicity, elevated bile acid or phospholipid concentrations, altered transport protein expression). Sandwich-cultured hepatocytes represent an exciting tool for studying drug-induced hepatotoxicity because this model maintains hepatocyte polarity, morphology, liver-specific functions including metabolic activity, and allows direct access to the hepatocyte and adjacent biliary compartment. Hepatic transport proteins are expressed and localize to the correct membrane domain over time in this model. Importantly, transport function can be assessed directly in this system. Use of hepatocytes from animal species typically employed in toxicology studies (e.g., dog, monkey) as well as from humans is a particularly attractive feature of this novel in vitro model, and will be developed during the scale-up phase of this model system in a subsequent R33 application.