Oral administration of FT-4202 improves RBC parameters in the BERK mouse model. 2,3-DPG (A) and ATP (B) concentration in blood of BERK mice fed with control or FT-4202 chow for 2 weeks. (A-B) n = 17 mice per group. (C) P₅₀ curve/oxygen equilibrium curve (OEC) showing the HbS P₅₀ level in the blood of BERK assessed after 2 weeks of FT-4202 vs control chow administration. The oxygen dissociation curves are plotted using the average values of partial pressure of oxygen (pO₂) and the percentage of oxygen saturation at each point of 4 control BERK mice (red line) and 4 FT-4202 BERK mice (blue line). Dashed arrows mark the P₅₀ values for each group. (D) P₅₀ levels. (C-D) n = 4 mice per group. (E) Representative peripheral blood smears showing ISCs (black arrow) stained with modified Wright's stain; scale bar, 10 µM. (F) Irreversibly sickled cells (ISCs) quantification in control and FT-4202 BERK mice. Images of the blood smears were taken using a Nikon Eclipse Ti inverted microscope, and 10 diffesrent high magnification fields per mouse were quantified for ISCs using ImageJ analysis software. ISCs were identified as elongated RBCs with a longitudinal-to-transverse diameter ratio of >2.0. The percentage of ISCs was calculated as the number of ISCs normalized to the total number of red blood cells; n = 9 control BERK mice; n = 7 FT-4202 BERK mice. (A-F) *P < .05; **P < .01; unpaired Student t test. (G-I) Assessment of RBC sickling kinetics and membrane deformability in control BERK vs FT-4202 BERK mice after 2 weeks of treatment, using Lorrca Oxygenscan. (G) RBCs from FT-4202 and control BERK mice were subjected to controlled deoxygenation from ambient pO₂ (normoxia) to a pO₂ of <15 mm Hg followed by reoxygenation to normoxia. At fixed shear stress of 30 Pa, RBC deformability (EI) was measured across an O₂ gradient from normoxia to hypoxia (G). The highest (maximum) EI is seen at ambient pO₂ (EI_max). As pO₂ decreases, HbS polymerization ensues, making the RBCs stiff/rigid, and reducing their EI. The point of sickling (PoS) is the pO₂ when the EI drops to 95% of the EI_max. A further precipitous decline in EI occurs during the rapid HbS polymerization phase, resulting in extremely rigid RBCs with a minimum EI (EI_min) at pO₂ <20 mm Hg. RBCs are then reoxygenated back and EI was measured. The graph represents an average (Avg) EI (n = 13 control BERK mice, shown in red; n = 12 FT-4202 BERK, shown in blue). PoS is denoted by the black filled circles in each group. (H-I) RBC membrane deformability (EI) measured under normoxia, across a gradient of shear stress between 0.3 and 30 Pa. Because HbS polymerization does not occur under normoxia, this assay measures membrane damage reflected in reduced deformability. Deformability before deoxygenation (H) and after 1 cycle of deoxygenation/reoxygenation (I) on the Lorcca Oxygenscan. Here, in both instances, measurements of EI under normoxia ensured assessment of membrane damage without the confounding effect of HbS polymerization/sickling, but assessed the membrane damage before and after 1 cycle of experimentally induced sickling. Comparison at the different individual shear-stress levels were made using unpaired Student t tests: *P < .05; **P < .01; ***P < .001. Comparisons at overall shear stress of the entire deformability curve were made using 2-way analysis of variance (ANOVA): **P < .01. Curves in panel H were plotted using mean plus or minus standard error of the mean (SEM), EI of n = 14 control BERK mice (depicted in red) and n = 14 FT-4202 BERK mice (depicted in blue).