DEVELOPMENT OF A MODEL TO PREDICT DRUG HEPATOTOXICITY

描述(由申请人提供)： 肝毒性一直是导致批准药物退出临床使用的最常见的单一不良事件。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。这项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。药物引起肝毒性的机制多种多样，且知之甚少。最近的证据支持这一假说，抑制肝小管胆盐输出泵(BSEP)，导致胆汁酸在肝细胞内积聚，从而导致毒性，是药物性肝损伤的重要机制。“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)]以及六种非编码化合物将在一种新的夹心培养大鼠肝细胞模型中进行检测，以检验药物/代谢物对BSEP的抑制与这些药物的肝毒性潜力相关的假设。在每组实验中，夹心培养的大鼠肝细胞在含有标准或高地塞米松浓度(以保持细胞色素P450活性)的培养液中培养，将分别暴露于测试化合物中，以评估母药在没有和存在产生的代谢物的情况下的相对肝毒性。BSEP的药物抑制IC50将被确定为在三明治培养的大鼠肝细胞急性暴露期间不足以产生明显细胞毒性的浓度，以避免普遍的细胞毒性对BSEP功能的混杂影响。随后，随着时间的推移，将确定在培养中处于IC50的药物效应(如果没有观察到特定化合物对BSEP的抑制，则为细胞毒性的最高无效应水平)，以确定长期暴露的结果(例如，细胞毒性、胆汁酸或磷脂浓度升高、运输蛋白表达改变)。三明治培养的肝细胞是研究药物诱导的肝毒性的一个令人兴奋的工具，因为该模型保持了肝细胞的极性、形态、肝脏特异的功能，包括代谢活动，并允许直接进入肝细胞和邻近的胆管间隔。在这个模型中，随着时间的推移，肝脏转运蛋白被表达并定位到正确的膜域。重要的是，运输功能可以在这个系统中直接进行评估。使用通常用于毒理学研究的动物物种(例如，狗、猴子)以及来自人类的肝细胞是这一新型体外模型的一个特别吸引人的特征，并将在随后的R33应用中开发该模型系统的放大阶段。

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.