An Endogenous Metabolic Modulator Composition Improves Hydration Status and Decreases Adhesion Properties of Red Blood Cells from Patients with Sickle Cell Disease

Background Sickle cell disease (SCD) is a group of hemoglobinopathies caused by point mutations in the β-globin gene leading to abnormal red blood cells (RBCs). There are various forms of SCD; sickle cell anemia (SCA) is characterized by homozygosity for hemoglobin (Hb) S (HbSS) whereas HbSC disease results from compound heterozygosity for HbS and HbC. HbC does not lead to RBC sickling, but induces loss of intracellular K⁺ and H₂O causing RBC dehydration. Similarly, in HbSS RBCs, through dehydration, an increase of the intracellular concentration of poorly soluble HbS enhances polymerization upon deoxygenation, causing RBCs to become rigid and to sickle. Until recently, the only approved therapy for SCD was hydroxyurea (HU). HU increases fetal Hb (HbF) levels and improves RBC hydration and deformability. For decades, amino acids have been explored as potential treatment to restore the disrupted pathophysiological pathways in SCD. RCitNacQCarLKHVS, an Endogenous Metabolic Modulator (EMM) composition of ten different amino acids and derivatives, was designed to target and support RBC metabolism. In this study, we investigated the effect of ex vivo treatment of this EMM on RBC properties and function in SCA and HbSC.

Methods Blood of 12 different adult SCD patients (9 HbSS [of whom 6 were on chronic RBC exchange (RBCX)], 3 HbSC) and 2 healthy controls was collected. Whole blood was incubated at standardized hematocrit (20%, patient plasma) in the presence or absence of different RCitNacQCarLKHVS concentrations for 4h at 37°C. Routine hematological parameters were measured using the Cell-Dyn Sapphire (Abbott Diagnostics). Percentage of total dense RBCs (%DRBCs) was analyzed from 10/12 patients (4 HbSS on RBCX, 3 HbSS not on RBCX, 3 HbSC) using the Advia 120/2120 hematology analyzer (Siemens Healthcare Diagnostics). Point of sickling (PoS), RBC deformability (EImax), and hydration status (Ohyper) were assessed by oxygen gradient ektacytometry and osmotic gradient ektacytometry, respectively, in 12/12 patients using the Lorrca (RR Mechatronics). Blood viscosity was measured in 6/12 patients (3 HbSS not on RBCX and 3 HbSC) at 37°C using a cone-plate viscometer (Brookfield). The hematocrit-to-viscosity ratio (HVR) was calculated as follows: hematocrit/blood viscosity at 300 s^-1. RBC adhesion to laminin (BioLamina) was assessed in 4/12 patients (3 HbSS not on RBCX, 1 HbSC) using a microfluidic device (IBIDI μ-Slide VI 0.4). Statistical analysis was performed in Graphpad Prism v9.3.0 using the Friedman test.

Results Ex vivo treatment of blood of SCD patients with RCitNacQCarLKHVS showed a significant dose-dependent increase in Ohyper as measured by osmotic gradient ektacytometry (Figure 1), indicating an improvement of RBC hydration status of SCD RBCs. This was accompanied by a significant increase in mean corpuscular volume (MCV) and reduction in mean corpuscular Hb concentration (MCHC). RBCs of healthy controls showed a similar trend in Ohyper, MCV and MCHC. In addition, %DRBCs showed a significant dose-dependent decrease in both HbSS and HbSC RBCs as a result of RCitNacQCarLKHVS treatment. Oxygen gradient ektacytometry showed a significant increase in maximum deformability (EImax), but no significant change in point of sickling. The HVR did not significantly change during dose-response experiments. Median RBC adhesion to laminin decreased as a result of ex vivo treatment with RCitNacQCarLKHVS (Figure 2).

Conclusion Ex vivo treatment with RCitNacQCarLKHVS demonstrated a significant dose-dependent increase in RBC hydration (Ohyper, MCV, MCHC and %DRBCs), deformability (EImax) and decreased adhesion properties in SCD RBCs. Dehydrated and dense RBCs with reduced deformability and increased adhesion are more prone to hemolysis, and associated with vaso-occlusive complications such as priapism, skin ulcers, and nephropathy. Results from this study therefore suggest benefit of EMM in modulating clinically-relevant rheological properties such as hydration state and adhesion of SCD RBCs in both SCA and HbSC. The fact that HVR did not change may represent an additional therapeutic benefit, especially for HbSC patients who have, in general, higher blood viscosity compared to HbSS patients. Moreover, it would be worthwhile to investigate if the concomitant use of select amino acid combinations with HU or other anti-sickling agents has an additional beneficial effect in SCD.

van Dijk:Agios Pharmaceuticals, Inc.: Research Funding; Axcella Therapeutics: Research Funding. Traets:Agios Pharmaceuticals, Inc.: Research Funding. de Wilde:Agios Pharmaceuticals, Inc.: Research Funding. Koziel:Axcella Therapeutics: Current Employment. Wani:Axcella Therapeutics: Ended employment in the past 24 months. van Beers:Sobi: Research Funding; RR Mechatronics: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Agios Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Global Blood Therapeutics: Consultancy; Sanofi: Consultancy. Wijk:Agios Pharmaceuticals, Inc.: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; RR Mechatronics: Research Funding; Axcella Therapeutics: Research Funding. Rab:Global Blood Therapeutics: Research Funding; Agios Pharmaceuticals, Inc.: Consultancy, Research Funding; Axcella Therapeutics: Research Funding; RR Mechatronics: Research Funding.