GBT1118, a voxelotor analog, protects red blood cells from damage during severe hypoxia

Abstract

A lack of objective metrics in Sickle Cell Disease (SCD) makes it difficult to assess individual patient therapy options or assess the effects of therapy. This is further complicated by mechanisms of action involving multiple interconnected effects, that combine to relieve SCD symptoms. In 2019, based on the increase in hemoglobin concentration observed in the HOPE trial, the Food and Drug Administration approved voxelotor (Oxbryta®, Global Blood Therapeutics) for SCD patients 12 years and older. The main mechanism of action for voxelotor was increased hemoglobin-oxygen affinity, but other mechanisms may apply. In this study, we assessed the effect of GBT1118, an Oxbryta analog, on hypoxia-induced lethal and sub-hemolytic red blood cell (RBC) membrane damage using RBC Mechanical Fragility (MF), a metric of existing membrane damage and prospective hemolysis. RBC MF was measured non-invasively using a proprietary system comprising an electromagnetic bead mill and fiberoptic spectrophotometry detection. Three cycles of severe hypoxia (<5% oxygenated hemoglobin) with follow-up reoxygenation resulted in a significant increase in RBC MF for all SCD (Hb-S >60%) samples. Supplementation with GBT1118 caused no significant changes in pre-hypoxia RBC MF. However, following GBT1118 treatment, cell stability showed significantly less degradation, as evidenced by a significantly smaller RBC MF increase after three cycles of hypoxia-reoxygenation. These findings indicate that GBT1118 prevents hypoxia-induced membrane damage in sickled RBC, in part by alternative mechanisms not associated with induced changes in hemoglobin-oxygen affinity.

Keywords: Sickle cell disease; erythrocyte; hypoxia; mechanical fragility; polymerization; voxelotor.

Figure 1

Absorption spectra of a blood sample normoxic before hypoxia (---), deoxygenated (―), and at normoxia after sample reoxygenation (––) not supplemented (A) and supplemented (B) with GBT1118. Note that pre-deoxygenation and reoxygenated spectra, (observed absorption maxima at 543 and 577 nm), are essentially overlapping, and clearly different from deoxygenated Hb spectrum (with the observed maximum at 555 nm).

Figure 2

Contribution of cell fractions with different MF to RBC lysis for SCD Hb-S subjects (ν) and normal (Hb-A) donors (ν). Fractions are represented by incremental increase in hemolysis over 1 minute of consecutive stress application (FR_MFI). Significance is shown between the fractions from recruited Hb-S subjects and normal donors with P<0.05 (***), ns, no significance. Error bars are ±1 SD.

Figure 3

Changes in RBC induce hemolysis resulting from 3 cycles of hypoxia as compared to the control sample without GBT1118 supplementation for SCD subjects (A) and for normal donors (B). Shown are changes in total induced hemolysis (Hem₁₀) after 10 minutes of stress application with error bars are ± SD on the four measurements, and relative contribution of induced hemolysis fractions as a percent of total induced hemolysis in each sample. The difference for each of i RBC hemolyzed fraction is expressed in terms of the magnitude of induced hemolysis over the consecutive 30 second durations normalized to the total hemolysis in the sample, and was calculated according to the formula 2: Where HemC_i⁰ and HemC_i³ are incremental increases in hemolysis associated with i fraction in a sample before and after 3 cycles of hypoxia. HemC⁰₁₀ and HemC³₁₀ are the values of total hemolysis in the sample achieved over the 10 minutes of stress application before and after the hypoxia.

Figure 4

Changes in RBC mechanical fragility for SCD subjects (A) and normal donors (B) as shown by changes in the most labile fraction (MFI_1), and in the cumulative induced RBC MF index for 10 minutes of stress application (MFI_10). Shown is the difference between averaged MFI values before and after 3 cycles of hypoxia with (+) and without (-) supplementation with GBT1118. ↔ denotes statistical significance of hypoxia-induced changes compared to the pre-hypoxia normoxic baseline, and ***P<0.05) and *0.05<P<0.1 denote statistical significance of the differences of hypoxia-induced MF changes in samples with and without GBT1118. ns, no significance.

Figure 5

Changes in RBC-induced hemolysis as a result of incubation with GBT1118 after 3 cycles of hypoxia as compared to control sample without GBT1118 supplementation for SCD subjects (A) and for normal donors (B) with error bars representing ± SD on the three measurements. Shown are changes in total induced hemolysis after 10 minutes of stress application. Also shown are relative contributions of induced hemolysis fractions as a percent of total induced hemolysis in the four individual SCD and four normal donor samples. The difference for each of i RBC hemolyzed fraction is expressed in terms of the magnitude of induced hemolysis over the consecutive 1-minute durations normalized to the total hemolysis in the sample, and was calculated according to the formula 3: Where HemT_i⁰ and HemT_i³ are incremental increases in hemolysis associated with i fraction in a sample supplemented (Treated) with GBT1118 before and after 3 cycles of hypoxia. HemT⁰₁₀ and HemT³₁₀ are the values of total hemolysis in treated samples achieved over the 10 minutes of stress application. Other values as in Figure 3.