A Novel Composition of Endogenous Metabolic Modulators, AXA4010, Impacts Adhesion, Inflammation and RBC Membrane Deformability in Preclinical Models of Sickle Cell Disease
Authors: Hamill Michael , Carroll Sean , Wani Revati , Russell Matthew , Steuert Zoe , Nitzel Arianna , Downey Andrew , Cokol Murat , Afeyan Raffi , Marukian Svetlana , Tramontin Tony 
Published: February 2020

Background: In sickle cell disease (SCD), red blood cell (RBC) sickling propagates hemolysis, inflammation, vascular and endothelial injury with associated disruption in metabolic pathways including nitric oxide depletion, oxidative stress and an increased anabolic and energetic demand due to high RBC turnover. These metabolic functions highlight the role of amino acid biology in blood health and diseases such as SCD, which has motivated numerous individual amino acid (AA) based clinical studies. Here, we measured AA profiles of SCD plasma samples and examined the impact of combinations of AAs and metabolites on preclinical models of vascular adhesion, inflammation, and RBC function. This combined work serves as the basis for investigational product AXA4010, a composition of 10 distinct AAs and metabolites designed to target and support RBC, inflammatory, endothelial and vascular health, with the potential to impact the multifaceted pathology of SCD.
Methods: Plasma AA profiles of SCD adults (n=38) and healthy adults (n=40) were measured by LC-MS analysis. Plasma AA profiles from SCD adults informed the design of a defined "SCD medium" used for culturing primary human lung microvascular endothelial cells (HLMVEC) as a preclinical SCD model of adhesion and inflammation. HLMVEC cells cultured in "SCD medium" were co-treated with TNFα and either AXA4010, components of AXA4010, or individual constituent AAs/metabolites, and adhesion, inflammation, and cell migration pathways were characterized using functional assays. Healthy RBCs were treated ex vivo with AXA4010 or individual constituents and stressed using tert-Butyl hydroperoxide (t-BuOOH) treatment, RBC deformability was characterized by ektacytometry (LORRCA, RR Mechatronics).
Results: When compared to age-match controls, plasma AA profiles of SCD adults had significantly reduced arginine (-36%, p = 1.8e-4), leucine (-38%, p = 3.6e-6), valine (-36%, p = 2.8e-8), lysine (-38%, p = 6.2e-9), and histidine (-23%, p = 2.8e-4). In "SCD medium" cultured HLMVECs treated with TNFα, we observed AXA4010 reduced markers of adhesion (↓VCAM-1, ↓ICAM-1), inflammation (↓IL-6), and cell migration (↓MCP-1) in a dose dependent manner. In RBCs, we observed AXA4010 protected against t-BuOOH induced loss of RBC deformability. Deconvolution of AXA4010 and subset components revealed unexpected combinatorial interactions and potentiating effects of the constituents within the composition.
Conclusion: We combined human plasma AA profiling and combinatorial cell-based experimentation to design AXA4010. In primary human cell systems with AXA4010 we observed improved adhesion, inflammation, and RBC morphology markers, which are associated with blood health and SCD pathogenesis, in a composition specific manner. These results warrant clinical investigation of AXA4010, which we have now initiated in a cohort of SCD subjects to investigate safety, tolerability and impact on blood physiology.

Disclosures
Hamill: Axcella Health: Employment. Carroll:Axcella Health: Employment. Wani:Axcella Health: Employment. Russell:Axcella Health: Employment. Steuert:Axcella Health: Employment. Nitzel:Axcella Health: Employment. Downey:Axcella Health: Employment. Cokol:Axcella Health: Employment. Afeyan:Axcella Health: Employment. Marukian:Axcella Health: Employment. Tramontin:Axcella Health: Employment.