Sickle cell disease molecular pathogenesis and drug therapy

镰状细胞病分子发病机制及药物治疗

基本信息

批准号：
10924926
负责人：
William Eaton
金额：
$ 194.29万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10924926
关键词：
Abnormal Hemoglobins Adenosine Triphosphate Antisickling Agents Ants Binding Biological Assay Blood Cell Volumes Characteristics Chronic Clinical Trials Data Analyses Disease Dose Drug Kinetics Erythrocytes Fetal Hemoglobin Fiber Foundations Frequencies Funding Hemoglobin Hemoglobin concentration result Hemolysis Human Individual Libraries Measurement Methods Microscope Molecular Conformation Molecular Disease Monitor Mus Nitrogen Oral Organ Oxygen Pain Pathogenesis Patients Pharmaceutical Preparations Pharmacodynamics Pharmacotherapy Polymers Pyruvate Kinase Safety Sample Size Serum Sickle Cell Sickle Cell Anemia Sickle Hemoglobin Structure System Testing Therapeutic Therapeutic Agents Time Transgenic Organisms United States Food and Drug Administration Whole Blood diphosphoglycerate drug repurposing high throughput screening hydroxyurea inhibitor machine learning algorithm molecular drug target mutant nanomolar novel therapeutics phase 1 study polymerization screening sickling small molecule targeted treatment therapeutically effective

项目摘要

1. Screening for new drugs. Although the molecular target for drug therapy of sickle cell disease has been known since Linus Pauling's discovery that sickle cell anemia is a molecular disease over 70 years ago, only 2 ant-sickling drugs have been approved by the United States Food and Drug Administration. One is hydroxyurea. Hydroxyurea reduces chronic organ damage and the frequency of pain crises. It does so by increasing the synthesis of fetal hemoglobin, which dilutes the abnormal hemoglobin S, markedly slowing its polymerization to form the fibers that distort (sickle) and make the red cells inflexible. This drug is, however, only partially successful in reducing the frequency of pain crises and the chronic organ damage characteristic of the disease. A second, controversial drug, voxelotor, was approved in November 2019 by the FDA. It acts by preferentially binding to the R quaternary conformation, which cannot polymerize. There is a modest increase in hemoglobin levels for patients taking this drug, but there is no evidence yet that it decreases organ damage or reduces the frequency of pain crises. The search for additional and more effective therapeutic agents has been severely hampered by the lack of a rapid and sensitive assay for inhibition of sickling. A truly high throughput screen has been developed using a sophisticated automated microscope system (Biotek Lionheart) based on nitrogen deoxygenation of red cells in a 384 well plate format and a robust machine learning algorithm to determine the time at which an individual cell sickles. The assay has been used to screen the 12,657 compound library of compounds that have been tested in humans from the Scripps Institute funded by the Gates Foundation at 10 micromolar. Inhibitors from this screen were investigated at 9 concentrations from 1 nanomolar to 10 micromolar to determine the concentrations at which they are effective. So far, we have discovered 106 compounds from this library that inhibit sickling to a degree that is potentially therapeutic. Once we know the concentrations of these "hits" that are found in the serum of humans, information that has been challenging to determine, we can determine which compounds are candidates should be further investigated in the detail that is necessary to begin clinical trials. To compare with serum concentrations will require measurements on whole blood instead of the highly diluted blood (1:1000) used in the assay. In a parallel, project measurements of oxygen binding and cell volume are being made to determine whether the mechanism of anti-sickling is switching the quaternary structure from the polymerizing T conformation to the non-polymerizing R conformation or from the decrease in intracellular hemoglobin concentration from ana increase in cell volume. Knowing the mechanism will inform us on what drugs can be taken together because they do not compete for the same target. 2. Clinical trials with Mitapivat. Increased intracellular 2,3- diphosphoglycerate (2,3-DPG) stabilizes fibers and promotes sickling, while decreased intracellular adenosine triphosphate (ATP) levels can lead to hemolysis. Mitapivat (AG-348) is an oral, small molecule, allosteric activator capable of activating both mutant and wild type red cell pyruvate kinase (PKR), thus decreasing 2,3-DPG and increasing ATP levels in red cells and potentially acting as an anti-sickling agent. The safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of multiple ascending doses of mitapivat were assessed in subjects with SCD in a Phase I study. Mitapivat demonstrated an acceptable safety profile across the tested dose levels in 8 subjects with SCD. Analyses of data show promising evidence of efficacy in terms of Hb increase from baseline with concomitant decreases in hemolytic markers. The accompanying changes in metabolites and sickling studies are consistent with the proposed mechanism of the drug. The study is ongoing with a planned sample size of 15 subjects completing 6-8 weeks of treatment.

1。筛查新药。尽管自Linus Pauling发现镰状细胞贫血是70年前的分子疾病以来，已经知道了镰状细胞疾病的药物治疗的分子靶标，但美国食品药品监督管理局仅批准了2种蚂蚁踢药物。一个是羟基脲。羟基脲可减少慢性器官损伤和疼痛危机的频率。它通过增加胎儿血红蛋白的合成来做到这一点，从而稀释异常的血红蛋白S，显着减慢其聚合以形成扭曲（镰刀）并使红细胞不灵活的纤维。但是，这种药物仅在降低疼痛危机的频率和慢性器官损伤的特征方面仅部分成功。 FDA在2019年11月批准了第二种有争议的药物，素素。它是通过优先结合的第四纪构象来起作用的，该构象无法聚合。服用该药物的患者血红蛋白水平有所增加，但尚无证据表明它会减少器官损害或降低疼痛危机的频率。由于缺乏抑制疾病的快速和敏感测定法，寻找其他更有效的治疗剂受到了严重阻碍。使用复杂的自动显微镜系统（Biotek Lionheart）开发了一个真正的高吞吐量屏幕，该系统基于384井板格式的红细胞的氮脱氧化和强大的机器学习算法，以确定单个细胞小虫的时间。该测定法已用于筛选12,657个化合物库，这些化合物库是在Scripps Institute在Gates Foundation资助的10 Micrololar资助的人类中进行了测试的。研究了该筛选的抑制剂，以从1个纳摩尔到10个微摩尔的9个浓度进行研究，以确定它们的有效浓度。到目前为止，我们已经发现了该图书馆的106种化合物，这些化合物抑制了可恶的程度，具有潜在的治疗性。一旦我们知道在人类血清中发现的这些“命中”的浓度，即确定的信息，我们就可以确定哪些化合物是候选者，应在开始临床试验的必要详细信息中进一步研究。与血清浓度进行比较，需要在测定中使用的全血，而不是高度稀释的血液（1：1000）。在平行的情况下，正在制定氧结合和细胞体积的项目测量值，以确定抗踢的机理是将第四纪结构从聚合T构象转换为非聚合R构象还是从细胞体积中增加的细胞内血红蛋白浓度的降低。知道该机制将告知我们可以一起使用哪些药物，因为它们没有争夺相同的目标。 2。临床试验。细胞内2,3-二磷酸甘油酸（2,3-DPG）稳定纤维并促进疾病，而细胞内三磷酸腺苷（ATP）水平降低会导致溶血。 Mitapivat（AG-348）是一种口服，小分子的变构激活剂，能够激活突变体和野生型红细胞丙酮酸激酶（PKR），因此降低了2,3-DPG并降低了红细胞中的ATP水平，并且潜在地充当抗弹药剂。在I期研究中，在患有SCD的受试者中评估了多种升级剂量的Mitapivat的安全性，耐受性，药代动力学（PK）和药效学（PD）。 Mitapivat在8位患有SCD的受试者中表现出在测试剂量水平上的可接受的安全性。数据分析表明，从基线增加的HB增加，溶血标志物的伴随降低方面有令人鼓舞的证据。随附的代谢产物和可恶研究的变化与该药物的拟议机制一致。这项研究正在进行中，计划的样本量为15名受试者完成治疗6-8周。