PI-05: PHARMACODYNAMIC EFFECTS OF AG-946, A HIGHLY POTENT NOVEL ACTIVATOR OF PYRUVATE KINASE, IN EX VIVO TREATMENT OF RED BLOOD CELLS FROM SICKLE CELL DISEASE PATIENTS

Abstract

Purpose: Sickle cell disease (SCD) is a monogenetic red blood cell (RBC) disorder that is characterized by hemolytic anemia and vaso-occlusive crises. Among the many factors that contribute to disease pathophysiology is stiffening and sickling of RBCs, which is the direct result of the polymerization of abnormal hemoglobin S. Sickling is modulated by glycolytic intermediates such as 2,3-diphosphoglycerate (2,3-DPG) and ATP. Previously we showed that RBC pyruvate kinase (PKR), the key regulatory enzyme of glycolysis, is impaired in SCD and that ex-vivo treatment with mitapivat, an allosteric activator of PKR, increased enzymatic activity and thermostability, reduced 2,3-DPG levels, decreased p50, and subsequently reduced sickling (Rab et al, Blood 2021). Currently, mitapivat is being investigated in phase 1, phase 2, and phase 2/3 trials in patients with SCD (#NCT04000165, EudraCT#2019-003438-18, and NCT05031780). Recently, AG-946, a novel PK activator, has been developed. Here we investigate the pharmacodynamic effects of AG-946 in ex vivo treatment of RBC from SCD patients in comparison with mitapivat. Materials and methods: Buffy coat depleted whole blood obtained from five SCD patients was incubated for 20-24 hours in absence or presence of mitapivat (100mM) or AG-946 (1mM, 5mM, 50mM). After ex-vivo treatment, enzymatic activities of PKR and PKR-thermostability were measured. Glycolytic intermediates ATP and 2,3-DPG were measured using LC-MS/MS. Hemoglobin oxygen affinity (p50) was measured and RBC sickling was analyzed with oxygen gradient ektacytometry, a method that characterizes individual sickling behavior. Individual tendency to sickle is reflected by Point-of-Sickling (PoS) that indicates the specific pO2 at which RBCs start to sickle during deoxygenation under shear stress. Results: PKR activity was increased compared to vehicle (DMSO) to a similar extent in the presence of mitapivat or AG-946 (Figure 1A). In addition, PKR thermostability was significantly increased compared to vehicle (mean 22%, SD 6%) in samples treated with mitapivat 100 mM (mean 78%, SD 11%), as well as AG-946 5mM (mean 66%, SD 23%), and AG-946 50mM (mean 95%, SD 17%, Figure 1B). After incubation with mitapivat or AG-946, 2,3-DPG decreased (Figure 1C), which was further illustrated by the improved ATP/2,3-DPG ratio (Figure 1D). Accordingly, p50 decreased significantly after incubation with mitapivat 100mM (mean 95%, SD 2%), as well as AG-946 1mM (mean 96%, SD 2%), AG-946 5mM (mean 94%, SD 2%), and AG-946 50mM (mean 95%, SD 3%, Figure 1E). The improved metabolic status and p50 was accompanied by a decreased PoS in RBCs treated with mitapivat or AG-946, indicating reduced RBC sickling tendency (Figure 1F). Conclusion: Ex vivo treatment of SCD RBCs with the novel PK activator AG-946 activates and stabilizes PKR, decreases 2,3-DPG levels, improves the ATP/2,3-DPG ratio, improves p50 and lowers the PoS. These beneficial effects are similar to ex-vivo treatment with mitapivat but, importantly, are obtained at much lower concentrations. Taken together, these results are the first in an ex-vivo model to demonstrate that the novel PK activator AG-946 has a similar favorable pharmacodynamic profile to mitapivat with enhanced PKR-stabilizing properties and, hence, might represent a potential novel therapeutic option in addition to mitapivat for the treatment of SCD.Pharmacodynamic effects of AG-946 in ex vivo treatment of red blood cells (RBC) from sickle cell disease (SCD) patients in comparison with mitapivat. Buffy coat depleted whole blood was incubated for 20-24 hrs in absence or presence of mitapivat (100?M, P. KOSINSKI declares a conflict of interest: Stock shareholder: employee and stock holder of Agios Pharmaceuticals, Inc