LONG-TERM RHEOLOGICAL BENEFITS OF GBT021601 IMPROVE PATHOLOGICAL ANGIOGENESIS IN THE SICKLE MOUSE BONE MARROW AND PREVENTS DEVELOPMENT OF HYPERALGESIA

In sickle cell disease (SCD), the abnormal sickle hemoglobin (HbS) on deoxygenation, polymerizes causing repeated vaso-occlusive episodes and hypoxic injury leading to significant morbidity and mortality. Acute pain is the single most cause of hospitalization, but by adulthood, 55% develop chronic pain. Hypoxia-induced angiogenesis is hyperinflammatory and linked to chronic pain in other chronic pain disorders; pathologic angiogenesis is seen in the sickle mouse bone that is reversible with chronic transfusions. Currently there are no approved oral therapies targeting pathologic angiogenesis and chronic pain in SCD. Chronic pain can be due to allodynia, where ordinary stimuli induce pain response vs hyperalgesia where pain response is exaggerated with painful stimuli. The von Frey Filament test is a validated method to assess hyperalgesia in the sickle mouse model. Evaluate the impact of a novel oral second generation HbS stabilizer, GBT210601, on the bone marrow vasculature, angiogenesis, and pain in the Townes sickle mouse model. 8-week-old Townes HbSS mice were fed with chow containing either 0.2% or 0.4% of GBT021601, compared to control chow. After 12-15 weeks, blood count was measured from peripheral blood using an Element HT5 (HESKA) hematology analyzer. Bone marrow was harvested by centrifugation and apoptosis assessed by AnnexinV staining by flow cytometry. Femoral bone vasculature was assessed using 3D confocal microscopy staining for Sca-1 and immunoblot and ELISA was performed on plasma for proangiogenic markers VCAM-1, Ang-1, Ang-2, and VEGF. Pain phenotype was assessed by manual Von Frey method, grip strength, and sensitivity to hot and cold plate at 1 week and 12 weeks of treatment of 601 therapy. GBT021601 increased Hb in a dose dependent manner (0.4%: 16.5 g/dL, p < 0.001; 0.2%: 13.4 g/dL, p = 0.03; vs control 8.9 g/dL), compared to control mice. GBT021601 reduced markers of hypoxia and angiogenesis VCAM-1 and Ang-1 (p < 0.05) by western blot and VCAM-1 and Ang-2 by ELISA (p < 0.05). Arterial marker SCA-1 on confocal microscopy compared to controls was significantly lower in bone marrow treated with 601 vs controls, appearing more similar to HbAA bone marrow (3.11% vs 5.13%, p = 0.04). Whole bone marrow cells had significantly lower percent apoptotic cells compared to controls (4.4% vs 7.2%, p = 0.003). von Frey and grip strength testing was similar between HbSS mice on control and 601 chow. There was significant increase in paw withdrawal