Screening for a Drug for Sickle Cell Disease

镰状细胞病药物的筛选

• 批准号: 9148756
• 负责人: William Eaton
• 金额: $ 132.09万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 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/9148756
• 关键词: Abnormal Hemoglobins; Binding; Biological Assay; Cells; Characteristics; Chronic; Clinical Trials; Dependence; Disease; Erythrocytes; FDA approved; Fetal Hemoglobin; Fiber; Frequencies; Hemoglobin; Hemolysis; Human; Libraries; Measurement; Measures; Methods; Microcirculation; Molecular Target; Organ; Osmolar Concentration; Oxygen; Pain; Pharmaceutical Preparations; Pharmacotherapy; Physiological; Plasma Proteins; Population; Preclinical Drug Evaluation; Probability; Serum; Severity of illness; Sickle Cell Anemia; Sickle Hemoglobin; Swelling; Testing; Therapeutic Agents; Time; United States Food and Drug Administration; base; drug testing; hydroxyurea; polymerization; screening; sickling

Although the molecular target for drug therapy of sickle cell disease has been known for over 50 years, the only drug approved by the United States Food and Drug Administration is hydroxyurea. Hydroxyurea reduces the probability of vaso-occlusion 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. The search for additional and more effective therapeutic agents has been severely hampered by the lack of a sensitive assay for inhibition of sickling. We have developed a method to rapidly, accurately and sensitively test for anti-sickling activity in large populations of human red blood cells by measuring the distribution of sickling times. The first drug screen was carried out on the 2,000 compound library of Microsource Discovery, which contains 800 of the approximately 1,500 of unique compounds contained in the approximatelyy 3,500 FDA-approved drugs. The initial screen was carried out under hyperosmolar conditions. A second screen under iso-osmolar (i.e. physiological) conditions showed time-dependent effects for the most promising compounds. With technical improvements we now can make measurements at physiological osmolarity as a function of both time and concentration, and are re-screening a library of approximately 1,000 FDA approved compounds. The most promising compounds so far are those that reduce intracellular hemoglobin concentration by swelling red cells, taking advantage of the enormous concentration dependence of the delay time. We are also developing (1) assays to assess the effect of drug binding to plasma proteins, (ii) to test drugs on fractionated SS cells, and (iii) and very high thru-put assay for cell swelling. If the delay time is increased significantly at serum concentrations found for an FDA-approved compound, then, in principle, only two simple additional tests are required before clinical trials could begin, namely the hemolysis test and an oxygen binding curve to insure that the oxygen delivery function of the treated red cells is intact. Both of the assays are now fully developed.

尽管镰状细胞疾病的药物治疗的分子靶标已闻名已有50多年了，但美国食品药品监督管理局批准的唯一药物是羟基脲。羟基脲通过增加胎儿血红蛋白的合成来降低血管封闭的可能性，胎儿血红蛋白的合成稀释了异常的血红蛋白S，显着减慢了其聚合以形成扭曲（镰状）的纤维，并使红细胞变为衰弱。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。由于缺乏抑制疾病的敏感测定法，寻找其他更有效的治疗剂受到了严重阻碍。我们已经开发了一种方法，可以通过测量疾病时间的分布来快速，准确，敏感地测试大量人类红细胞中的抗裂缝活性。 第一个药物筛选是在Microsource Discovery的2,000个化合物库上进行的，其中包含大约3,500 FDA批准药物中约1,500种独特化合物中的800个。初始屏幕是在高渗条件下进行的。 在异摩尔（即生理）条件下的第二个屏幕显示最有前途的化合物的时间依赖性效果。 通过技术的改进，我们现在可以在生理渗透压下进行测量，这是时间和浓度的函数，并重新筛选了大约1,000次FDA批准化合物的库。 到目前为止，最有希望的化合物是那些利用延迟时间的巨大浓度依赖性来降低细胞内血红蛋白浓度的化合物。 我们还开发了（1）测定法，以评估药物与血浆蛋白的结合，（ii）在分离的SS细胞上测试药物，以及（iii）和非常高的Thru-Put测定法对细胞肿胀进行测定。 如果在FDA批准化合物的血清浓度下显着增加了延迟时间，则原则上，在临床试验开始之前，只需要进行两次简单的额外测试，即溶血测试和氧结合曲线以确保处理后红细胞的氧递送功能是完整的。 现在，这两个测定法都是完全开发的。