Pulmonary fibrosis is often triggered by an epithelial injury resulting in the formation of fibrotic lesions in the lung, which progress to impair gas exchange and ultimately cause death. Recent clinical trials using drugs that target either inflammation or a specific molecule have failed, suggesting that multiple pathways and cellular processes need to be attenuated for effective reversal of established and progressive fibrosis. Although activation of MAPK and PI3K pathways have been detected in human fibrotic lung samples, the therapeutic benefits of in vivo modulation of the MAPK and PI3K pathways in combination are unknown. Overexpression of TGFα in the lung epithelium of transgenic mice results in the formation of fibrotic lesions similar to those found in human pulmonary fibrosis, and previous work from our group shows that inhibitors of either the MAPK or PI3K pathway can alter the progression of fibrosis. In this study, we sought to determine whether simultaneous inhibition of the MAPK and PI3K signaling pathways is a more effective therapeutic strategy for established and progressive pulmonary fibrosis. Our results showed that inhibiting both pathways had additive effects compared to inhibiting either pathway alone in reducing fibrotic burden, including reducing lung weight, pleural thickness, and total collagen in the lungs of TGFα mice. This study demonstrates that inhibiting MEK and PI3K in combination abolishes proliferative changes associated with fibrosis and myfibroblast accumulation and thus may serve as a therapeutic option in the treatment of human fibrotic lung disease where these pathways play a role.

Sickle cell anemia (SCA) results from an abnormal sickle hemoglobin (HbS). HbS polymerizes upon deoxygenation, resulting in red blood cell (RBC) sickling and membrane damage that cause vaso-occlusions and hemolysis. Sickle RBCs contain less adenosine triphosphate and more 2,3-diphosphoglycerate than normal RBCs, which allosterically reduces hemoglobin (Hb) oxygen (O2) affinity (ie, increases the partial pressure of oxygen at which hemoglobin is 50% saturated with oxygen [P50]), potentiating HbS polymerization. Herein, we tested the effect of investigational agent FT-4202, an RBC pyruvate kinase (PKR) activator, on RBC sickling and membrane damage by administering it to Berkeley SCA mice. Two-week oral FT-4202 administration was well tolerated, decreasing HbS P50 to levels similar to HbA and demonstrating beneficial biological effects. In FT-4202–treated animals, there was reduced sickling in vivo, demonstrated by fewer irreversibly sickled cells, and improved RBC deformability, assessed at varying shear stress. Controlled deoxygenation followed by reoxygenation of RBCs obtained from the blood of FT-4202–treated mice showed a shift in the point of sickling to a lower partial pressure of oxygen (pO2). This led to a nearly 30% increase in RBC survival and a 1.7g/dL increase in Hb level in the FT-4202–treated SCA mice. Overall, our results in SCA mice suggest that FT-4202 might be a potentially useful oral antisickling agent that warrants investigation in patients with SCA.

Introduction: Sickle cell anemia (SCA) results from a mutant β-globin gene that produces abnormal hemoglobin S (HbS). HbS polymerizes upon deoxygenation, resulting in red blood cell (RBC) sickling and membrane damage, leading to vaso-occlusions and hemolysis. Additionally, sickle RBCs contain less ATP and more 2,3-diphosphoglycerate (2,3-DPG) than normal RBCs; 2,3,DPG allosterically reduces hemoglobin (Hb) oxygen (O2)-affinity [i.e. increases P50], promoting faster unloading of O2, which potentiates HbS polymerization and RBC sickling. FT-4202, a selective and orally bioavailable allosteric activator of RBC pyruvate kinase (PKR), decreases 2,3-DPG and increases ATP in normal human RBCs (Blood, 2019, 134, Supplement 1:616). We hypothesized that oral administration of FT-4202 to SCA mice will increase HbS O2-affinity, and thereby decrease RBC sickling and membrane damage. Methods: Berkeley SCA mice were given 500-1000 mg/kg/day FT-4202 in chow (FT-4202 group) or control chow (control group) in 4 cohorts for 2 weeks (total 17-18 mice/group). In all cohorts, the health status, weight, and average chow consumption of each mouse was determined 3 times/week. Three cohorts were injected with sulfo-NHS-biotin 1 week into treatment (10-11 mice/group), and RBC survival assessed over the next week with serial micro-bleeds while on treatment. The 4th cohort was only bled at 2 week time-point to obtain P50 (Hemox Analyzer) and Hb levels (Hemavet). At experiment termination, all cohorts were terminally bled to determine (a) RBC levels of 2,3-DPG and ATP, (c) plasma levels of FT-4202 by LC-MS/MS, (d) the proportion of irreversibly sickled RBC (ISC) on blood smears (Image-J analysis), (e) the kinetics of experimentally-induced sickling (Lorrca®Oxygenscan) and (f) membrane deformability (Lorrca®Ektacytometry). Results: SCA mice on FT-4202 consumed a similar amount of food, and had similar weights and survival, compared to SCA mice on control chow throughout the 2-week period. As hypothesized, HbS O2 affinity increased, reflected by a decrease in P50 from 29.6 ± 0.62 mmHg (mean ± SEM) in the control group to 27.6 ± 0.58 mmHg in the FT-4202 group (p<0.03). Determinations of 2,3-DPG, ATP and FT-4202 are ongoing and will be presented. As expected, this increased HbS O2-affinity in the FT-4202 group reduced RBC sickling and membrane damage. At 2 weeks, the proportion of ISCs on blood smears was reduced in the FT-4202 group to 2.4 ± 0.3% vs. 5.9 ± 1.4% in the control group (p<0.02). The sickle RBC half-life increased to 1.8 ± 0.07 days in FT-4202 group vs. 1.4 ± 0.1 days in the control group, a 28% increase in RBC survival (p<0.01, Figure 1A). Hence, Hb levels in the FT-4202 group increased from 9.1 ± 0.2 g/dL before treatment, to 10.8 ± 0.3 g/dL 2 weeks after treatment (p<0.001), while Hb levels in the control group remained unchanged (Figure 1B). The reticulocytes remained unchanged in both groups before and after treatment. When sickle RBCs were de-oxygenated from an ambient pO2 of ~150 mmHg to a pO2 of 10-15 mmHg, followed by their re-oxygenation to ambient pO2 at a constant shear stress of 30 Pa (Oxygenscan), the point of sickling (PoS; pO2 level when the EI becomes 95% of the EI at ambient O2) decreased on average from 37% pO2 in the control group, to 30% pO2 in the FT-4202 group (p<0.002, Figure 1C), with a significantly improved Elongation Index at the point of minimum pO2 (EImin), (p<0.05). Next, RBC membrane deformability was measured under ambient pO2 (normoxic conditions), but varying shear stress after the de-oxygenation/re-oxygenation cycle on the Oxygenscan. Sickle RBCs from the FT-4202 group were significantly more deformable [i.e. had a higher Elongation Index (EI)] compared to control sickle RBCs (p<0.01, Figure 1D), as shear stress increased to ≥3 Pa, demonstrating that FT-4202 sickle RBCs sustained significantly less membrane damage following sickling and un-sickling. Conclusion: A 2-week oral FT-4202 administration was well tolerated by SCA mice and demonstrated beneficial biological effects: improved RBC membrane deformability and sickling parameters, with a shift in the PoS to lower pO2, and increased RBC survival and Hb levels. A parallel human phase-I study in healthy subjects and sickle cell disease patients to assess the safety and PK/PD of FT-4202 is ongoing (NCT03815695). Overall, our results suggest that FT-4202 can be a potentially useful orally available agent with significant anti-sickling effect. Disclosures Drake: Forma Therapeutics: Other: Shareholder of Forma Therapeutics. Fulzele:FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics. Guichard:FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics; AstraZeneca: Other: Shareholder. Malik:Aruvant Sciences, Forma Therapeutics, Inc.: Consultancy; Aruvant Sciences, CSL Behring: Patents & Royalties.