Hemoglobin Modifiers for Sickle Cell Disease Therapy

描述(申请人提供)：我们的目标是设计和建立治疗镰状细胞病(SCD)的新型药物，即抑制最初的镰状血红蛋白(HBS)的药物 聚合反应和随后的病理生理学。当脱氧时，HBS聚合成长的、坚硬的和不溶的纤维，导致红细胞(RBC)呈镰刀状，这一过程因HBS对氧气的异常低亲和力而恶化，导致氧气过早释放。根据我们的初步数据、其他人的研究以及最近完成的先导化合物5-HMF(HB的变构效应物，AEH)的I/II期临床测试结果，我们假设AEHs不仅可以防止HBs聚合，还可以减轻几种与继发性疾病相关的病理事件，包括炎症、氧化应激/损伤、红细胞溶血和疼痛。我们也有初步证据表明，我们的下一代AEH(Inn系列和TD系列)显示出更强的效力和更长的体外作用时间。这些AEH通过一种新的作用机制发挥作用，即除了增加Hb对氧的亲和力外，还破坏HBS聚合物接触的稳定性；提供积极的协同效应。 我们建议通过进一步研究Inn-和TD-系列的候选药物以及5-HMF和Inn-312的衍生物的药理性质来验证我们的假设，重点是次级SCD途径，以及使用我们的模型系统来解决潜在的HBS聚合问题。具体目标为：1.新型血红蛋白变构效应物(AEHs)的设计合成。我们将对母体化合物进行修饰，合成药效更强、半衰期更长的衍生物。我们还将根据需要合成Prod地毯，以保护活性醛功能部分不受醛脱氢酶(ALDH)介导的代谢的影响。2.研究新型AEHs的体外功能、抗蚁活性和细胞毒活性。我们将研究AEHs的体外蚂蚁镰刀/功能活性(RBC镰刀试验、P50分析、HBs溶解度和Hb加合物形成)，并仔细监测不良反应。3.测定SCD Berkeley小鼠体内/体外PK/PD的性质、结合和代谢，评价AEHs的临床前疗效。我们将证明血清白蛋白结合和/或ALDH在红细胞或肝细胞胞浆中的代谢不太可能对体内药理活性产生不利影响。我们将使用SCD转基因小鼠的Berkeley小鼠模型来证明AEHs显示出强大的药理作用，提高小鼠的短期和长期存活率。我们还将研究其潜在的有益作用，例如，改善溶血、炎症、内皮损伤，以及完全逆转在该模型中观察到的SCD病理生理。4.确定AEHs与Hb之间的原子相互作用。X射线结晶学将被用来验证我们的假设，即AEH的效力直接取决于它们将Hb与其吡啶取代基结合到Hb分子表面的能力。这些结构将提供有价值的见解，帮助指导合理的修饰，以获得更好的药理特性。

DESCRIPTION (provided by applicant): Our goal is to design and establish novel therapeutic agents for sickle cell disease (SCD), namely drugs that inhibit the initial sickle hemoglobin (HbS) polymerization and the subsequent pathophysiology. When deoxygenated, HbS polymerizes into long, rigid, and insoluble fibers causing red blood cells (RBCs) to sickle, a process worsened by the unusual low affinity of HbS for oxygen, resulting in premature release of oxygen. Based on several evidence--our preliminary data, studies by others, and the results of a recently completed phase I/II clinical testing of our lead compound, 5-HMF (an allosteric effector of Hb, AEH)-we hypothesize that AEHs, not only prevent HbS polymerization, but also mitigates several secondary sickling related pathological events that include inflammation, oxidative stress/damage, RBC hemolysis, and pain. We also have preliminary evidence that our next generation AEHs (INN- and TD-series) exhibit enhanced potency and improved in-vitro duration of action. These AEHs act via a novel mechanism of action, i.e., destabilize HbS polymer contacts, in addition to increasing Hb affinity for oxygen; providing positive synergistic effects. We propose to test our hypothesis by further investigating candidate drugs from the INN- and TD-series, as well as derivatives of 5-HMF and INN-312 for their pharmacologic properties, focusing on the secondary SCD pathways, as well as the underlying HbS polymerization problem using our model systems. The specific aims are: 1. Design and synthesis of novel allosteric effectors of hemoglobin (AEHs). We will modify our parent compounds and synthesize derivatives with enhanced efficacy and prolonged half-lives. We will also synthesize prod rugs to protect the active aldehyde functional moiety from aldehyde dehydrogenase (ALDH)- mediated metabolism as necessary. 2. Investigate in-vitro functional, ant sickling, and cytotoxicity activities of novel AEHs. We will investigate the AEHs for their in-vitro ant sickling/functional activities (RBC sickling tests, P50 analyses, HbS solubility, and Hb adduct formation), and carefully monitor for adverse effects. 3. Determine in-vivo/in-vitro PK/PD properties, binding and metabolism and evaluate preclinical efficacy of AEHs in SCD Berkeley mice. We will show that serum albumin binding and/or metabolism by ALDH in RBC or hepatic cytosol are not likely to adversely affect in-vivo pharmacologic activity. We will demonstrate using a Berkeley mouse model of SCD transgenic mice that AEHs show potent pharmacologic effects, increase short- and long-term survival rates of mice. We will also study their potential beneficial effects, e.g. amelioration of hemolysis, inflammation, endothelial damage, and overall reversal of the SCD pathophysiology observed in this model. 4. Determine the atomic interactions between AEHs and Hb. X-ray crystallography will be used to validate our hypothesis that AEH potency is directly dependent upon their abilities to bind Hb with their pyridyl substituents toward the surface of the Hb molecule. The structures would provide valuable insight to help guide rational modifications for better pharmacologic properties.