Gamma-ketoaldehyde scavengers for alcoholic liver disease

描述(申请人提供)：长期饮酒过量是全球疾病负担的第三大风险因素，也是肝脏疾病和癌症的常见共病。虽然戒酒是治疗的基石，但人们对确定酒精性肝病(ALD)的其他治疗干预措施很感兴趣。由于对酒精特异性代谢反应和肝脏病理生理学之间关系的不完全理解，这些进展变得复杂起来。作为ALD的标志，促炎状态的诱导似乎依赖于活性氧(ROS)、蛋白质加合物的形成以及先天性和获得性免疫反应之间的不利相互作用。在这项研究的第一阶段，我们假设高活性化合物的形成，称为γ-酮醛(γ-KA)，主要由非酶自由基催化的脂质过氧化，其次是环氧合酶(COX)的活性，在酒精性肝损伤中是至关重要的。γ-KAS与蛋白质赖氨基残基反应非常迅速，形成稳定的内酰胺加合物，从而引发特异性免疫反应。这些γ-KA蛋白加合物的形成导致免疫原性、炎症和终末器官损伤。因此，使用选择性的γ-KA清除剂应能阻断致病性炎症反应，从而改善ALD。越来越多的证据表明，ROS、脂质过氧化和免疫反应之间的相互作用在ALD的发病机制中起着重要作用。我们的合作者(LJ Roberts，MD)已经确定水杨胺是一种快速和优先与γ-Kas反应的试剂，以防止它们加成到细胞蛋白质和其他胺中。该方案的目的是验证肝脏γ-KA清除将减少肝损伤(ALT)并对ALD的先天免疫反应和获得性免疫反应有不同影响的假说。我们将在急性(2天)和慢性(25天)乙醇喂养的小鼠模型上，确定口服水杨胺对减轻酒精性肝损伤和免疫反应的影响。在研究1中，我们将检测水杨胺对肝功能(丙氨酸氨基转移酶、天门冬氨酸氨基转移酶、甘油三酯)以及肝脏和循环中γ-KA、抗γ-KA抗体效价以及肝单核细胞浸润的量效关系。我们预计水杨胺将显著减少蛋白质加合物的形成并改善肝功能，其方式与降低的获得性免疫反应一致，包括T和B细胞增殖、核因子κB激活和TLR4信号。在研究2中，我们将使用相同的2天和25天乙醇喂养的小鼠模型来诱导肝损伤，但将在初级乙醇注射后给予水杨胺(以剂量依赖的方式)，然后监测与研究1相同的终点。如果这些研究表明水杨胺可以减少酒精性肝损伤和/或对免疫反应有不同的影响，第二阶段研究将被提议在随后的SBIR拨款中进一步完善水杨胺的作用机制，以用于产品开发和监管批准。

DESCRIPTION (provided by applicant): Chronic alcohol overconsumption is the third largest risk factor for disease burden worldwide and is a frequent comorbidity of liver disease and cancer. While abstinence is a cornerstone of treatment, there is considerable interest in identifying other therapeutic interventions for alcoholic liver disease (ALD). Such advances are complicated by an incomplete understanding of the relationship between alcohol-specific metabolic responses and liver pathophysiology. The induction of a pro-inflammatory state, hallmark of ALD, appears to depend upon the unfavorable interplay among reactive oxygen species (ROS), protein adduct formation, and both the innate and adaptive immune responses. In this SBIR Phase I project, we hypothesize that the formation of highly reactive compounds, termed γ-ketoaldehydes (γ-KA), formed primarily by non-enzymatic free radical catalyzed lipid peroxidation and secondarily by cyclooxygenases (COX) activity, is crucial for ethanol-induced liver injury. γ-KAs react very rapidly with protein lysyl residues to form stable lactam adducts which elicit specific immune reactions. The formation of these γ-KA-protein adducts lead to immunogenicity, inflammation and end-organ damage. Hence, the use of selective scavengers of γ-KAs should lead to interruption of the pathogenic inflammatory responses leading to improvements in ALD. It has become increasingly evident that the interplay among ROS, lipid peroxidation and immune responses is important in the pathogenesis of ALD. Our collaborator (LJ Roberts, MD) has identified Salicylamine as an agent that rapidly and preferentially reacts with γ-KAs to prevent their adduction to cellular proteins and other amines. The goal of this proposal is to test the hypothesis that hepatic γ-KA scavenging will reduce liver injury (ALT) and have differential effects on innate and adaptive immune responses in ALD. We will determine the effects of orally administered Salicylamine on reducing the severity of alcohol liver injury an on immune responses in acute (2 day) and chronic (25 day) ethanol-fed mouse models. In study 1, we will examine the dose-response effects of Salicylamine on liver function (ALT, AST, triglyceride), as well as hepatic and circulating γ-KAs, anti-γ-KA antibody titers as well as livr monocyte infiltration when Salicylamine is administered concurrent with ethanol. We anticipate Salicylamine will significantly reduce protein adduction formation and improve liver function in a manner that is concordant with diminished adaptive immune responses including T and B cell proliferation, NFκB activation and TLR4 signaling. In study 2 we will employ the same 2 day and 25 day ethanol-fed mouse models to induce liver injury, but will administer Salicylamine (in a dose-dependent manner) after the primary ethanol administration then monitor the same endpoints as in study 1. If these studies demonstrate that Salicylamine reduces alcohol liver injury and/or have a differential effect on immune responses, Phase II studies will be proposed to further refine the mechanisms of action of Salicylamine desired for product development and regulatory approvals in a subsequent SBIR grant.