Development of an in vitro mouse genetic reference platform to improve preclinical drug safety assessment

项目摘要/摘要 许多药物在实现安全性问题之前都要经过临床前和早期临床研究， 患者面临风险，并在药物开发过程中造成瓶颈。我们的长远目标是 研究目的是改进药物安全检测中的人体风险评估。遗传易感性是一个重要的 目前临床前毒理学模型中未显示的药物不良反应的特征。因此，我们 在临床前安全性研究中受控纳入遗传多样性的假设将得到改善 对人类药物不良反应的预测和理解。此外，具体的识别 导致毒性易感性的基因和途径将使我们能够更好地理解 临床前毒理学结果和患者反应之间的关系。 我们以前已经证明了协同交叉(CC)小鼠群体在建立毒性模型方面的作用 需要遗传易感因素的反应。目前，CC方法需要大量的活体研究 既耗时又昂贵，而且范围有限。我们正在开发一种新型的体外CC平台 含有从CC系分离的原代细胞，并在多孔板上培养，以允许多个 浓度、治疗方案和终点将在一次实验中跨重复井进行分析。 我们的平台将能够快速和经济有效地识别与治疗相关的基因相互作用 在药物开发的所有阶段都有药物不良反应。 我们正在开始用培养的CC肝细胞进行平台开发。这将支持最初对药物的关注- 诱发性肝损伤是导致临床停药的主要不良反应之一 开发计划和从市场上撤回批准的药物，以及我们在这方面有很好的- 成熟的专业知识。该平台将包括从CC系分离并培养的冷冻保存的肝细胞 在3D球体中，这将增加体外模型的生理相关性，同时减少数量 细胞(和动物)的总体需求。我们将评估体外CC平台在筛选新药方面的应用 在改进人类首例最大安全起始剂量的估计方面，DILI责任和援助的候选人 临床试验(目标1)；提供对DILI机制的新理解，并告知精确医学风险 改善患者安全和降低药物开发成本的缓解战略(目标2)；以及验证 因果联系，并告知促成药物反应的遗传因素的物种差异(目标3)。 总而言之，我们提议的研究将改进临床前药物安全筛选并支持鉴定 导致人类药物毒性的遗传风险因素、机制和物种间差异。 总而言之，这些见解将进一步降低患者伤害的可能性和药物开发的成本。

PROJECT SUMMARY/ABSTRACT Many drugs pass through preclinical and early clinical studies before safety concerns are realized, putting patients at risk and creating a bottleneck in the drug development process. The long-term objective of our research is to improve human risk assessment in drug safety testing. Genetic susceptibility is an important feature of adverse drug reactions not currently represented in preclinical toxicology models. Therefore, we hypothesize that controlled incorporation of genetic diversity in preclinical safety studies would improve prediction and understanding of adverse drug reactions in humans. Furthermore, the identification of specific genes and pathways contributing to toxicity susceptibility would allow us to better understand the relationship between preclinical toxicology findings and patient response. We have previously demonstrated the utility of the Collaborative Cross (CC) mouse population to model toxicity responses that require genetic susceptibility factors. Currently, the CC approach requires large in vivo studies that are time consuming, expensive, and limited in scope. We are developing a novel in vitro CC platform containing primary cells isolated from CC lines and cultured on multi-well plates to allow for multiple concentrations, treatment regimens, and endpoints to be assayed across replicate wells in a single experiment. Our platform will enable the rapid and cost-effective identification of gene-by-treatment interactions associated with adverse drug response at all stages of drug development. We are beginning platform development with cultured CC hepatocytes. This will support an initial focus on drug- induced liver injury (DILI), which is one of the main adverse responses leading to the termination of clinical drug development programs and withdrawal of approved drugs from the market, and an area in which we have well- established expertise. The platform will include cryopreserved hepatocytes isolated from CC lines and cultured in 3D spheroids which will increase the physiological relevance of the in vitro model while decreasing the number of cells (and animals) needed overall. We will evaluate the utility of the in vitro CC platform to screen new drug candidates for DILI liability and aid in the improved estimation of maximum safe starting dose for first-in-human clinical trials (Aim 1); provide new understanding of the mechanisms of DILI and inform precision medicine risk mitigation strategies to improve patient safety and reduce the cost of drug development (Aim 2); and validate causal associations and inform species differences in genetic factors contributing to drug response (Aim 3). Collectively, our proposed research will improve preclinical drug safety screening and support the identification of genetic risk factors, mechanisms, and interspecies differences contributing to drug toxicity in humans. Together, these insights will further reduce the potential for patient harm and the cost of drug development.