5610417 IN-VITRO REAL-TIME OBSERVATION OF MODERATE SICKLING KINETICS IN SICKLE ERYTHROCYTES TREATED BY VOXELOTOR UNDER MONOTONIC AND CYCLIC HYPOXIA

Abstract

Background: Hypoxia-induced sickle hemoglobin (HbS) polymerization in sickle red blood cells (sRBCs) is the root cause of the symptoms and complications in sickle cell disease (SCD). In microcirculation, timing is critical for sRBCs to pass through narrow capillaries before they become sickled under hypoxia and rigid enough to get trapped. Hence, the delay time in cell sickling due to HbS polymerization is likely a key parameter predictive of the SCD severity. Recent studies have also shown the delay time is shortened due to repeated hypoxia cycles. Voxelotor is a known hemoglobin oxygen-affinity modulator, which has been proven to effectively inhibit HbS polymerization and favorably modify SCD outcomes. However, there has been no direct in-situ observation of the dose-dependent anti-sickling effect of voxelotor treatment under monotonic hypoxia or multiple hypoxia cycles. Aims: 1. We will test the anti-sickling effect of voxelotor on sRBCs by measuring their dose-dependent hypoxia-induced sickling kinetics using our microfluidics assay. 2. We will further test the efficacy of voxelotor on the cumulative impairment to sRBCs from repetitive sickling cycles. Methods: Blood samples from patients with homozygous SCD were studied. Sample specimens used for this study are restricted to patients that were not in crisis and patients with transfusions 30 days or longer prior to blood draw. Details about the microfluidic device and related methods can be found elsewhere [1-3]. Single deoxygenation-reoxygenation cycle or multiple deoxygenation-reoxygenation cycles is created by switching between two gas supplies with 2% and 20% O2 at predetermined time intervals [3]. Results: Figure 1A-B shows significant decrease in sickled fraction with the increase of voxelotor modification percentage from 0% to 30%, 60% and 100%, which are consistent among patients taking hydroxyurea (HU ON) or not taking hydroxyurea (HU OFF). Secondly, we have investigated the efficacy of voxelotor (30% modification) on sickling kinetics progression after 20 repeated deoxygenation-reoxygenation cycles (each cycle includes 60s of deoxygenation and 30s of oxygenation). From the measurements from 4 patient samples, we have confirmed that the sickling kinetics of sickle cells (vehicle, 0% modification) become progressively faster with subsequent deoxygenation-oxygenation cycles, which is quantified by the reduced delay time and higher sickled fraction versus deoxygenation time. Figure 1C-D shows that the treatment of voxelotor (30% modification) has significant efficacy in mitigating the progressively accelerated sickling of sRBCs due to repeated deoxygenation-oxygenation cycles in vitro. The sickled fraction measured at t = 20s in the 20th cycle decreases from 53%±9% for vehicle samples (0% modification) to 21%±11% for voxelotor treated samples (30% modification) (p < 0.05). Similarly, the sickled fraction measured at t = 60s in the 20th cycle decreases from 64%±11% (0% modification) to 30%±16% (30% modification) (p < 0.05).Summary-Conclusion: Voxelotor significantly slows down the rate of sickling and reduces the sickled fraction of sRBCs under monotonic hypoxia with dose-dependency. Furthermore, voxelotor is effective for mitigating the cumulative impairment to sRBCs from repetitive sickling events. Funding statement: This study has been supported by Global Blood Therapeutics Inc. Y.Q. and M.D. acknowledge partial support from NIH R01HL158102.