Naturally Occurring Agent for Alcohol Liver Diseases

描述(申请人提供)：慢性重度酒精摄入对哺乳动物肝脏有重大不良影响，即诱导肝细胞损伤和损伤后再生障碍。对于一个专注于酒精性肝病的药物开发项目，在分子水平上了解对肝细胞增殖反应产生不利影响的细胞信号转导通路的潜在变化以及对肝脏再生至关重要的生存机制是至关重要的。这一STTR第一阶段和第二阶段快速通道应用的目标是开发一种自然产生的药物，即水飞蓟宾(NPI-MK-001)，作为酒精性肝脏问题的肝脏保护剂。根据我们的初步研究，NPI-MK-001是从水飞蓟种子中分离得到的生物活性成分，水飞蓟是目前研究最多的治疗肝病的药用植物。本实验拟在体外培养的大鼠肝细胞上研究NPIMK-001对慢性酒精摄入所致肝损伤的细胞保护作用。我们还将通过分析与肝细胞DNA合成相关的特异性生长因子信号转导通路的影响，来评估NPI-MK-001在体内肝修复过程中的治疗潜力。我们的具体目标是：目的1-确定NPI-MK-001对慢性酒精喂养低氧所致肝细胞损伤的体外细胞保护作用；目的2-探讨NPI-MK-001在体内对慢性酒精喂养大鼠和IRS-1转基因小鼠肝修复相关生长因子信号转导通路的作用。在第一阶段，我们将专注于NPI-MK-001的标准化，这是进行体外和体内评估的先决条件。我们建议(1) 对水飞蓟种子的收集、加工、提取和污染控制(杀虫剂、除草剂和化肥)进行田间检查；(2)优化NPI-MK-001定性和定量分析的高效液相分析条件；(3)分离纯化足够数量的NPI-MK-001及其非对映异构体(水飞蓟宾A和B)，用于化学标记，并进行体内外评估；以及(4)建立一种经济高效的NPI-MK-001大规模纯化的分离程序。在第二阶段，我们建议：(1)通过体外和体内模型评估NPI-MK-001及其非对映异构体(水飞蓟宾A和水飞蓟宾B)的相对效力；(2)按照AIMS 1和2的规定，研究NPI-MK-001的作用机制；(3)合成放射性标记的NPIMK-001并评估其生物利用度和药代动力学；(4)根据FDA指南对NPI-MK-001和相应的安慰剂进行标准化；(5)获得有关突变和急慢性毒性的数据；(6)对标准化的NPI-MK-001进行稳定性测试；以及(7)建立GMP条件下标准NPI-MK-001和安慰剂的包囊方案。 天然药物国际公司是一家老牌的天然产品实验室，它组建了一支优秀的联合团队，以确保NPI-MK-001的质量和纯度符合FDA的严格要求。STTR项目成功的关键不仅在于团队成员在第一阶段和第二阶段开展拟议研究方面的非凡经验，还在于支持NPI-MK-001进一步发展的杰出业务发展计划，包括在第三阶段提交DMF和IND。

DESCRIPTION (provided by applicant): Chronic heavy ethanol consumption has major adverse effects on the mammalian liver, namely the induction of hepatocellular damage and impairment of regeneration following injury. For a medication development project focusing on alcohol liver diseases, it is essential to understand, at the molecular level, potential alterations in cellular signal transduction pathways that adversely affect the hepatocyte proliferative response, as well as those survival mechanisms critical for liver regeneration. The objective of this STTR Phase I and II fast track application is to develop a naturally occurring agent, namely Silybin (NPI-MK-001), as a hepatoprotective agent for alcohol liver problems. Based on our preliminary studies, NPI-MK-001 is the bioactive component isolated from the seed of Milk thistle (Silybum marianum), the best-studied medicinal plant for the treatment of liver disease. We plan to study the cytoprotective effect of NPIMK-001 on liver injury produced by chronic ethanol consumption in rat hepatocyte culture. We will also evaluate the therapeutic potential of NPI-MK-001 on the hepatic repair process in vivo by analyzing effects on specific growth factor signal transduction cascades associated with hepatocyte DNA synthesis. Our specific aims are: Aim #1 - Determine the in vitro cytoprotective effects of NPI-MK-001 on hepatocyte injury caused by chronic ethanol feeding+ hypoxia; Aim #2 - Explore the in vivo action of NPI-MK-001 on the IRS-1 mediated growth factor signal transduction pathway associated with liver repair in chronic ethanol-fed rats and IRS-1 transgenic mice. In Phase I, we will focus on the standardization of NPI-MK-001, a prerequisite for in vitro and in vivo evaluation. We propose to (1) conduct field inspection of the collection, processing, extraction, and contamination control (insecticides, herbicides, and chemical fertilizers) of the seed of Milk Thistle; (2) optimize the analytical HPLC conditions for qualitative and quantitative analysis of NPI-MK-001; (3) isolate and purify a sufficient quantity of NPI-MK-001 and its diasteric isomers (Silybin A and B) for chemical markers, and for in vitro and in vivo evaluation; and (4) develop a cost-effective separation procedure for large scale purification of NPI-MK-001. In Phase II, we propose to: (1) evaluate relative potency of NPI-MK-001and its diasteric isomers (Silybin A and Silybin B) by in vitro and in vivo models; (2) investigate the mechanism of action of NPI-MK-001 as specified in Aims 1 and 2; (3) Synthesize radio-labeled NPIMK-001 and assess the bioavailability and pharmcokinetics; (4) standardize NPI-MK-001 and corresponding placebo according to FDA guidelines; (5) obtain data on mutagenesis and acute and chronic toxicity; (6) conduct stability testing of the standardized NPI-MK-001; and (7) establish protocols for the capsulation of standardized NPI-MK-001 and placebo under GMP conditions. Natural Pharmacia International, Inc, an established natural products laboratory, has assembled an excellent consortial team to assure that the quality and purity of NPI-MK-001would meet the stringent FDA requirements. The key to the success of this STTR program is not merely the exceptional experience of the team members in carry out the proposed studies in Phases I and II, but also the outstanding business development plan which supports the further development of NPI-MK-001 including filing of a DMF and an IND in Phase III.