Mitapivat-Induced Improvements in RBC Deformability and Membrane Integrity in Patients with Sickle Cell Disease Are Sustained during Extended Therapy

The ability of red blood cells (RBCs) to deform is a key factor in the pathophysiology of sickle cell disease (SCD). Deformability measurements of RBCs via ektacytometry are increasingly used as biomarkers of therapeutic efficacy in anti-sickling therapies, including mitapivat, a pyruvate kinase (PK) activator. It has been shown that mitapivat therapy in SCD improves RBC deformability in response to shear stress, osmotic pressure, and decreasing oxygen tension, likely via bolstered membrane integrity. Indeed, analysis of RBC samples from HbSS patients treated with mitapivat in a Phase 1 study showed a significant dose-dependent reduction in tyrosine phosphorylation RBC band 3 (Tyr-p-bd3) with increased membrane-associated ankyrin-1 and intact (active) protein tyrosine phosphatase 1B (PTP1B). Here, we assess sustainability of mitapivat-induced changes in RBC deformability, correlate these changes with those on Tyr-p-bd3 and how they impact the hematologic parameters.

METHODS

We studied 15 HbSS patients (aged 25-57 years; 10 males) currently enrolled under protocol NCT04610866 evaluating long-term safety and tolerability of mitapivat in SCD patients. All patients started mitapivat at 50 mg twice daily (BID), escalating to 100 mg BID after 4 wks; dose adjustments were performed for safety and tolerability, per PI discretion. Fresh whole blood in EDTA tubes was obtained and processed at baseline (V1, prior to drug initiation) and longitudinal time points up to 2 years (V12).

Laser-Optical Rotational Red Cell Analyzer (LORRCA, RR Mechatronics, Netherlands) assays were performed on fresh blood following standard procedures. The Elongation Index (EI) measures the cells' ability to deform under different shear stress, continuous osmolality and oxygen pressures. RBC membranes were isolated from frozen whole blood and subjected to Western blotting analyses for Tyr-p-bd3, ankyrin-1 and intact (active) PTP1B and quantified by densitometry. For all assays, we analyzed the %change at each timepoint from baseline for each subject, and then derived the mean %change for each timepoint for all subjects. Significance testing was derived by Wilcox signed rank test. Correlations assessments were based on nonparametric Spearman correlation, with all analyses conducted using R (v4.2.3).

RESULTS

Mitapivat therapy increased elongation indices (EIs) across all shear stress levels as early as 2 wks, e.g., at shear stress 3 Pa and 30 Pa (mean% ± SD) 30.3 ± 51.7, P=0.001; and 22.2 ± 40.4, P=0.005, respectively. These improvements were sustained with extended therapy although the changes were not significant: 18.3 ± 66.8 at 3 Pa P=0.919, and 12.7 ± 56.9 at 30 Pa, P>0.999, respectively at 2 yrs. The O hyper (Osmoscan) was significantly increased after 12 weeks (3.2 ± 6.9; P=0.036) of treatment and remained significant (3.6 ± 8.3; P= 0.048) after 52 weeks of treatment. Reductions in Point of Sickling (PoS) by Oxygenscan, were observed at 12 (-16.8 ± 13.0; P=0.007) and 52 (-20.4 ± 15.5; P=0.015) wks of treatment.

Mitapivat therapy significantly reduced RBC Tyr-p-bd3, accompanied by a significant increase in membrane-associated ankyrin-1 and intact PTP1B, within 2 wks of exposure and sustained improvements throughout 2 yrs of therapy. The %changes in Tyr-p-bd3 and ankyrin-1 and PTP1B were significantly (P<0.0001) negatively correlated and that between ankyrin1 and PTP1B, positively correlated.

RBC Deformability, as measured by LORRCA assays, was negatively correlated with LDH (=-0.603 at EI=3 Pa, P<0.0001), and total bilirubin (=-0.412 at EI=3 Pa), P<0.0001. Improved RBC deformability negatively correlated with %change in Tyr-p-bd3, with the association increasing as shear stress increases (= -0.228 at 3 Pa, P = 0.005). Tyr-p-bd3 was also inversely associated with EI max ( = -0.404, P=0.144), and EI at PoS ( = -0.404, P<0.0001) for the Osmoscan and Oxygenscan assays respectively, indicating improved sickling kinetics.

CONCLUSION

Mitapivat-induced improvements in RBC deformability in SCD patients is rapid and sustained throughout extended therapy, accompanied by decreased hemolysis. Improvements in deformability were accompanied by significant reduction in Tyr-p-bd3, a critical determinant of RBC integrity. Our findings confirm that activating PK in SCD improves RBC deformability, a key mechanism is the reduction in Tyr-p-bd3, leading to an increased interaction with the anchoring protein, ankyrin.

Yates:Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Wind-Rotolo:Agios: Current Employment, Current equity holder in publicly-traded company.