CSL112 Increases Plasma Cholesterol Efflux Capacity and Reduces Erythrocyte Membrane Cholesterol Content in Blood Samples from Patients with Sickle Cell Disease

Background: Lipid homeostasis is abnormal in sickle cell disease (SCD) and contributes to elevated erythrocyte cholesterol content which has been implicated in sickling. The highly oxidative environment in blood from patients with SCD alters high-density lipoprotein (HDL) composition and impairs HDL particle remodeling. Specifically, it negatively impacts on the capacity of HDL particles to mediate apolipoprotein A-I (apoA-I) exchange and protect against inflammation. Low apoA-I levels and altered composition of HDL have been well-documented at steady state, associated with high risk features of SCD, including endothelial dysfunction and risk of pulmonary hypertension. The low levels and activity of apoA-I fall even further during vaso-occlusive events in SCD. This diminished pool of apoA-I activity may mediate reduced capacity of HDL to extract cholesterol from erythrocyte membranes and increase risk of vascular dysfunction.

Purpose: In this study we aimed to determine whether plasma derived apoA-I (CSL112; apolipoprotein A-I (human)) could elevate the cholesterol efflux capacity (CEC) of plasma from SCD patients and thereby reduce erythrocyte membrane cholesterol content.

Methods: All SCD subjects (n=14) had confirmed HbSS genotype except one who was with HbS/β⁰-thalassemia, healthy control subjects (n=13) were not race-matched. CSL112 was incubated with fresh blood derived from control subjects and SCD patients for 6 hours at 37°C. The cholesterol content of ghost membranes of isolated erythrocytes was measured. HDL particle size distribution was assessed by native gel electrophoresis followed by western blotting. The ex vivo capacity of apoB-depleted plasma to efflux cholesterol was determined using [³H]cholesterol-loaded RAW264.7 macrophages.

Results: CEC of SCD plasma was attenuated (75.9 ± 7.4%, n=14) compared to controls (100%, n=13), with a predominant decrease of ATP binding cassette transporter A1 (ABCA1)-dependent CEC (77.7 ± 9.2% vs 100%, respectively). Consistently, SCD plasma showed a trend towards lower levels of pre-β1 HDL (78.5 ± 6.5%) as compared to controls (100 %). Incubation with CSL112 raised plasma levels of small HDL species and pre-β1 HDL, resulting in a more than 3.5 fold increase of CEC in both patients and controls. Treatment of SCD blood with CSL112 reduced erythrocyte cholesterol content to a greater extent compared to control blood (% reduction: 12.8% ± 3.1% in SCD, 6.3% ± 5.4% in controls).

Conclusion: Our data suggest that reduced levels of lipid-poor pre-β1 HDL in SCD plasma reflect impaired HDL particle remodeling and account for a decreased CEC. CSL112 may acutely generate HDL species which optimally support ABCA1-dependent cholesterol efflux and thereby improve HDL function, normalize erythrocyte membrane cholesterol content and correct the imbalance in cellular cholesterol homeostasis in SCD patients. ApoA-I as a potential therapeutic could have implications in the pathobiologic understanding and future clinical management of vaso-occlusive crisis and of chronic vascualopathy such as pulmonary hypertension.

Didichenko:CSL Behring AG: Current Employment, Research Funding. Navdaev:CSL Behring AG: Current Employment, Research Funding. Saxenhofer:CSL Behring AG: Current Employment, Research Funding. Graeter:CSL Behring AG: Current Employment, Research Funding. Schaub:CSL Behring AG: Current Employment, Research Funding. Rovo:CSL Behring: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria; Alexion: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; OrPhaSwiss GmbH: Membership on an entity's Board of Directors or advisory committees; Swedish Orphan Biovitrum AG: Membership on an entity's Board of Directors or advisory committees. Kato:CSL Behring: Current Employment, Current equity holder in publicly-traded company, Research Funding. Kingwell:CSL Limited: Current Employment, Current equity holder in publicly-traded company, Research Funding.