Sickle cell disease molecular pathogenesis and drug therapy

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Linked publications & trials

Abstract

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 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, somewhat 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 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 concentration 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 pf 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. 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. 3. Voxelotor. HPLC and integrated mass spectrometric analysis collectively confirmed that voxelotor labeling was specific to the N-terminus thereby ruling out other potential ligand binding sites. The results from this optimized analytical approach allowed detection of a stable -specific GBT440 adduct in patient hemolysate in a dose dependent manner. The results and methods presented could therefore potentially help therapeutic monitoring of voxelotor in patients taking this drug. A therapeutic trial of Voxelotor in an untransfusable sickle cell anemia patient did not increase hemoglobin. However, hemoglobin occupancy failed to reach the 25%-30% percent modification required to be therapeutic in sickle cell patients with less severe anemia. A theoretical study comparing oxygen delivery with and without voxelotor shows that voxelotor does not improve oxygen delivery, consistent with clinical trials that show no decrease in crisis frequency.