Clearing intrahepatic lipids to aid in toxicity testing

描述（由申请人提供）：Vesta Therapeutics是美国不可移植人类肝脏的主要供应商之一，并开发了分离的原代肝细胞的分离、冷冻保存和再生应用的专有技术。在Vesta为我们的研究项目处理细胞的过程中，我们收集了大量多余的成熟肝细胞，供学术、临床和制药研究人员使用。原代人肝细胞是制药公司用来测试新型候选药物的吸收、分布、代谢、排泄（ADME）和毒性(T)特性的重要模型。然而，随着美国肥胖率的上升，器官采购组织（为研究目的）提供的非移植器官中有很大一部分是脂肪变性的，这给潜在测试化合物的毒性增加了一层不可控制的可变性。因此，制药公司不愿意使用脂肪变性肝细胞，而被迫使用非生理性动物和肿瘤细胞模型进行药物开发。过去几年的研究表明，许多针对脂质生物合成、氧化或肝内输出的因素可以逆转动物和人类患者的脂肪变性。然而，将这些方法应用于原代人肝细胞以产生非脂肪变性肝细胞的标准化供应的可行性尚未得到探讨。鉴于我们持续的脂肪变性肝供应和广泛的脂肪变性肝细胞冷冻储备，Vesta在评估这些技术方面具有独特的优势。我们建议使用胆汁酸、PPAR激动剂和胆碱单独或联合使用来降低人肝细胞中的脂肪变性水平，这些药物在动物和人类研究中已被证明可以逆转脂肪变性。我们将记录清除脂质的最佳条件，清除脂质的时间过程，清除的稳定性以及“无脂”肝细胞的功能特征。在拟议研究的后期阶段，我们将应用这些方案从含有脂肪变性水平的肝脏中分离肝细胞，这些脂肪变性水平通常阻碍了它们在药物发现应用中的应用。我们的总体目标是确定最小的“侵入性”方案来清除过量的肝细胞脂质，以解决学术、临床和制药应用中原发性人肝细胞的严重短缺。公共卫生相关性：

DESCRIPTION (provided by applicant): Vesta Therapeutics is one of the leading procurers of non-transplantable human livers in the United States and has developed proprietary technologies for the isolation, cryopreservation, and regenerative application of isolated primary hepatocytes. As Vesta processes cells for our research programs, a significant number of surplus mature hepatocytes are collected, [which are made available to academic, clinical and pharmaceutical researchers. Primary human hepatocytes are valuable and crucial models used by pharmaceutical companies to test the absorption, distribution, metabolism, excretion (ADME) and toxicity (T) properties of novel drug candidates. However,] with the increasing rates of obesity in the United States, a high percentage of the non-transplantable organs offered by Organ Procurement Organizations [for research purposes] are steatotic (fatty), which adds an uncontrollable layer of variability to the toxicity of potential test compounds. [As a consequence, pharmaceutical companies are reluctant to utilize steatotic hepatocytes, and are forced to use non-physiological animal and tumor cell models for drug development.] Research over the last few years has shown that a number of factors that target lipid biosynthesis, oxidation, or intrahepatic export can reverse steatosis in animals and in human patients. [However, the feasibility of applying these methodologies to primary human hepatocytes for the purpose of generating a standardized supply of non-steatotic hepatocytes has not been explored.] Vesta is uniquely positioned to assess these technologies given our consistent supply of steatotic livers and our extensive cryopreserved stocks of steatotic hepatocytes. We propose to decrease steatosis levels in human hepatocytes using bile acids, PPAR agonists, and choline singly or in combination, which have been shown to reverse steatosis in animal and human studies. We will document the optimal conditions to clear lipids, the time course of lipid clearing, the stability of clearance, and the functional characteristics of "lipid-free" hepatocytes. In the latter stages of the proposed studies, we will apply these protocols to isolate hepatocytes from livers containing levels of steatosis that typically preclude their use in drug discovery applications. Our overall goal is to determine the least "invasive" protocol to clear hepatocytes of excess lipids to address the acute shortage of primary human hepatocytes for academic, clinical, and pharmaceutical applications. PUBLIC HEALTH RELEVANCE: In the application "Clearing intrahepatic lipids to aid in toxicity testing," studies are aimed at examining the feasibility of targeting three pathways known to regulate lipid (fat) metabolism with the aim of reducing the fat levels in human hepatocytes isolated from donated livers. Vesta Therapeutics is one of the leading procurers of non-transplantable livers in the United States and has developed proprietary technologies for the isolation and preservation of primary hepatocytes from these livers. Currently, pharmaceutical companies are reluctant to utilize hepatocytes with high levels of fat, which add an uncontrollable layer of variability to the toxicity of test compounds. Devising strategies to eliminate fat from freshly isolated or cryopreserved hepatocytes would provide extremely valuable technologies for the full utilization of limited number of livers donated each year. Thus, if successful, this source of "fat-free" hepatocytes could benefit many goals of public health.