Evaluation of anti-fibrotic and anti-inflammatory semi-synthetic oxysterol, Oxy210, as a therapeutic drug candidate for non-alcoholic steatohepatitis

摘要 病理性组织纤维化，由对急性或慢性损伤的不受控制的伤口愈合反应所推动，是一种慢性损伤。 包括肾、肺和肝在内的多个器官出现严重问题。肝纤维化是一个主要的健康问题 导致患病人群发病和死亡。肝纤维化发生在各种 病理学包括慢性酒精暴露，非酒精性脂肪肝（NAFLD），可导致非酒精性肝病。 酒精性脂肪性肝炎（NASH）和病毒感染，并可导致肝硬化和肝细胞癌。一 许多分子机制已被仔细研究，以确定肝纤维化的根本原因。其中， 突出的研究指出，异常的Hedgehog（Hh）和转化生长因子-β（TGF-β）信号传导是导致肿瘤细胞凋亡的主要原因。 肝纤维化发病机制的潜在因素，因此被广泛研究。越来越多的证据 还表明Toll样受体（TLR）在介导肝细胞对炎症反应中的作用， 内源性因子和细菌产物存在于NAFLD和NASH的肝脏中。 在MAX BioPharma，我们已经确定了作为Hh通路调节剂的半合成氧化固醇， 激动剂和拮抗剂。我们发现氧化固醇Hh通路拮抗剂也具有有效的抑制作用 对成纤维细胞和原代人肝星状细胞（HSC）中TGF-β信号传导的影响。的支持下 一个SBIR阶段1从NIDDK的赠款，我们最近发表了关于抗NASH性能的研究结果， 氧固醇类似物Oxy 210，其导致了本申请。Oxy 210具有抗纤维化和抗 通过抑制Hh、TGF-β和TLR信号传导介导的炎症特性。Oxy 210诱导 在NASH的人源化高脂血症小鼠模型ApoE*3-Leiden.CETP小鼠中， 高脂肪西方饮食，通过抑制肝脏脂质沉积、炎性细胞因子表达 和纤维化，与减少的肝细胞凋亡和改善的循环ALT水平有关。基于 Oxy 210的这些特性，以及其良好的药代动力学和安全性特征、口服利用度， 可扩展性，在目前的SBIR第2阶段应用中，我们建议继续检查疾病 在ApoE*3-Leiden.CETP小鼠中，与Oxy 210在逆转已经建立的疾病中的修饰作用相比， 我们发表的报告显示，在疾病开始时给予Oxy 210。我们建议确定 通过进行代谢试验，了解Oxy 210抗NASH特性的分子和细胞机制 在有或没有Oxy 210治疗的NASH小鼠的肝脏中进行RNA测序研究。我们建议执行 非GLP体外和体内安全性研究，将为未来IND启用GLP提供重要信息 我们预期将Oxy 210作为靶向治疗的一流新药候选药物提交IND申请所需的研究 纳什NASH是一个巨大的未满足的医疗需求，因为没有FDA批准的疗法，留下了大量的 全球人群易患严重肝衰竭和需要肝移植。

ABSTRACT Pathologic tissue fibrosis, impelled by uncontrolled wound healing responses to acute or chronic injury is a significant problem in many organs including kidney, lung, and liver. Liver fibrosis is a major health problem that causes morbidity and mortality in the affected patient population. Liver fibrosis occurs in various pathologies including chronic alcohol exposure, non-alcoholic fatty liver disease (NAFLD) that can lead to non- alcoholic steatohepatitis (NASH), and viral infection, and can lead to cirrhosis and hepatocellular carcinoma. A number of molecular mechanisms have been scrutinized to identify the root causes of liver fibrosis. Among these, prominent studies point to aberrant Hedgehog (Hh) and transforming growth factor-β (TGF-β) signaling as prime factors underlying the pathogenesis of liver fibrosis, and hence extensively investigated. Accumulating evidence also suggests the role of Toll-Like Receptors (TLRs) in mediating the inflammatory responses of liver cells to endogenous factors and bacterial products present in the liver in NAFLD and NASH. At MAX BioPharma, we have identified semi-synthetic oxysterols that act as Hh pathway modulators, both as agonists and antagonists. We found oxysterol Hh pathway antagonists that also have potent inhibitory effects on TGF-β signaling in fibroblastic cells and in primary human hepatic stellate cells (HSC). With support from an SBIR Phase 1 grant from NIDDK, we recently published findings about the anti-NASH properties of an oxysterol analogue, Oxy210, that has led to the present application. Oxy210 has anti-fibrotic as well as anti- inflammatory properties that are mediated through inhibition of Hh, TGF-β and TLR signaling. Oxy210 induced robust inhibitory effects in a humanized hyperlipidemic mouse model of NASH, ApoE*3-Leiden.CETP mice on a high fat Western Diet, evidenced by inhibition of hepatic lipid deposition, inflammatory cytokine expression and fibrosis, associated with reduced hepatic cell apoptosis and improved circulating ALT levels. Based on these properties of Oxy210, in addition to its favorable pharmacokinetic and safety profiles, oral availability, and scalability, in the present SBIR Phase 2 application we propose to continue the examination of the disease modifying effects of Oxy210 in ApoE*3-Leiden.CETP mice in reversing already established disease in contrast to our published report when Oxy210 was administered at the initiation of the disease. We propose to identify the molecular and cellular mechanisms of anti-NASH properties of Oxy210 by performing metabolic tests as well as RNA-sequencing studies in livers of NASH mice with or without Oxy210 treatment. We propose to perform non-GLP in vitro and in vivo safety studies that will provide essential information for future IND-enabling GLP studies required in our anticipated IND filing for Oxy210 as a first-in-class new drug candidate for targeting NASH. NASH is a huge unmet medical need since there are no FDA approved therapies, leaving a large global population susceptible to severe liver failure and the need for liver transplantation.