School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom.
School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom
Cold Spring Harb Protoc. 2020 Nov 2;2020(11):pdb.prot106096. doi: 10.1101/pdb.prot106096.
Failure to predict drug-induced toxicity reactions is a major problem contributing to a high attrition rate and tremendous cost in drug development. Drug screening in embryos is high-throughput relative to screening in rodents, potentially making them ideal for this use. embryos have been used as a toxicity model in the frog embryo teratogenesis assay on (FETAX) for the early stages of drug safety evaluation. We previously developed compound-screening methods using embryos and believe they could be used for in vitro drug-induced toxicity safety assessment before expensive preclinical trials in mammals. Specifically, embryos could help predict drug-induced hepatotoxicity and consequently aid lead candidate prioritization. Here we present methods, which we have modified for use on embryos, to help measure the potential for a drug to induce liver toxicity. One such method examines the release of the liver-specific microRNA (miRNA) miR-122 from the liver into the vasculature as a result of hepatocellular damage, which could be due to drug-induced acute liver injury. Paracetamol, a known hepatotoxin at high doses, can be used as a positive control. We previously showed that some of the phenotypes of mammalian paracetamol overdose are reflected in embryos. Consequently, we have also included here a method that measures the concentration of free glutathione (GSH), which is an indicator of paracetamol-induced liver injury. These methods can be used as part of a panel of protocols to help predict the hepatoxicity of a drug at an early stage in drug development.
未能预测药物诱导的毒性反应是导致药物开发高淘汰率和巨大成本的主要问题。胚胎中的药物筛选相对于啮齿动物中的筛选具有高通量的特点，这使得它们非常适合这种用途。胚胎已被用作青蛙胚胎致畸分析 (FETAX) 中的毒性模型，用于药物安全性评估的早期阶段。我们之前开发了使用胚胎的化合物筛选方法，并且相信它们可以在昂贵的哺乳动物临床前试验之前用于体外药物诱导的毒性安全性评估。具体来说，胚胎可以帮助预测药物诱导的肝毒性，并有助于确定候选药物的优先级。在这里，我们介绍了经过修改后可用于胚胎的方法，以帮助测量药物引起肝毒性的潜力。其中一种方法是检查由于肝细胞损伤导致肝脏特异性 microRNA (miRNA) miR-122 从肝脏释放到脉管系统，这可能是由于药物引起的急性肝损伤。扑热息痛是一种已知的高剂量肝毒素，可以用作阳性对照。我们之前表明，哺乳动物扑热息痛过量的一些表型反映在胚胎中。因此，我们还在这里包括了一种测量游离谷胱甘肽 (GSH) 浓度的方法，GSH 是扑热息痛诱导的肝损伤的指标。这些方法可以作为一组方案的一部分，用于在药物开发的早期阶段帮助预测药物的肝毒性。
