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采购商之一，并开发了分离、冷冻保存和分离原代肝细胞再生应用的专有技术。当Vesta为我们的研究项目处理细胞时，收集了大量剩余的成熟肝细胞，[这些肝细胞可供学术，临床和制药研究人员使用。原代人肝细胞是制药公司用于测试新型候选药物的吸收、分布、代谢、排泄（ADME）和毒性（T）特性的有价值和关键的模型。然而，随着美国肥胖率的增加，器官采购组织[用于研究目的]提供的不可移植器官中有很高比例是脂肪变性的（脂肪），这给潜在的测试化合物的毒性增加了一个不可控制的可变层。[As因此，制药公司不愿意利用脂肪变性肝细胞，并且被迫使用非生理动物和肿瘤细胞模型用于药物开发。过去几年的研究表明，许多靶向脂质生物合成、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，尚未探索将这些方法应用于原代人肝细胞以产生标准化供应的非脂肪变性肝细胞的可行性。] Vesta在评估这些技术方面具有独特的优势，因为我们可以持续供应脂肪变性肝脏和大量冷冻保存的脂肪变性肝细胞。我们建议单独或联合使用胆汁酸、PPAR激动剂和胆碱来降低人肝细胞中的脂肪变性水平，这些药物在动物和人体研究中已显示出逆转脂肪变性。我们将记录清除脂质的最佳条件、脂质清除的时间过程、清除的稳定性以及“无脂质”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妨碍其在药物发现应用中的使用。我们的总体目标是确定清除肝细胞中过量脂质的最小“侵入性”方案，以解决学术、临床和制药应用中原代人肝细胞严重短缺的问题。公共卫生相关性： 在“清除肝内脂质以辅助毒性测试”的申请中，研究旨在检查靶向已知调节脂质（脂肪）代谢的三种途径的可行性，目的是降低从捐赠肝脏中分离的人肝细胞中的脂肪水平。Vesta Therapeutics是美国不可移植肝脏的主要采购商之一，并开发了从这些肝脏中分离和保存原代肝细胞的专有技术。目前，制药公司不愿意利用具有高水平脂肪的肝细胞，这给测试化合物的毒性增加了一个无法控制的可变层。设计从新鲜分离或冷冻保存的肝细胞中消除脂肪的策略将为充分利用每年捐赠的有限数量的肝脏提供非常有价值的技术。因此，如果成功的话，这种“无脂肪”肝细胞的来源可以使许多公共卫生目标受益。

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.