HypoxyStat, a small-molecule form of hypoxia therapy that increases oxygen-hemoglobin affinity

Abstract

We have previously demonstrated that chronic inhaled hypoxia is remarkably therapeutic in the premier animal model of mitochondrial Leigh syndrome, the Ndufs4 knockout (KO) mouse. Subsequent work has extended this finding to additional mitochondrial diseases and more common conditions. However, challenges inherent to gas-based therapies have hindered the rapid translation of our findings to the clinic. Here, we tested a small molecule (hereafter termed HypoxyStat) that increases the binding affinity of hemoglobin for oxygen, thereby decreasing oxygen offloading to tissues. Daily oral dosing of HypoxyStat caused systemic hypoxia in mice breathing normoxic (21% O₂) air. When administered prior to disease onset, this treatment dramatically extended the lifespan of Ndufs4 KO mice and rescued additional aspects of disease, including behavior, body weight, neuropathology, and body temperature. HypoxyStat was also able to reverse disease at a very late stage, thereby serving as a clinically tractable form of hypoxia therapy.

Keywords: Leigh syndrome; hemoglobin; hyperoxia; hypoxia; mitochondrial disease; oxygen; red blood cells; therapy.

Figure 1.. HypoxyStat binds hemoglobin and increases oxygen-binding affinity

(A) Schematic of left-shifting concept. Increased Hb-O₂ binding affinity decreases the release or “offloading” of oxygen to tissues, resulting in relative tissue hypoxia. (B) Structures of GBT-440 and HypoxyStat. (C) Known structure of GBT-440 bound to Hb (adapted from Metcalf et al.) and modeled structure of HypoxyStat bound to Hb. (D) Oxygen-hemoglobin dissociation curve of red blood cells incubated with vehicle, 10 mM GBT-440, or 10 mM HypoxyStat for 1 h. Traces are representative of triplicate experiments. See also Figure S1.

Figure 2.. GBT-440 is unable to rescue Ndufs4 KO survival, body temperature, or behavior

(A) PK/PD data for GBT-440 vs. other candidate small-molecule left-shifters, including HypoxyStat. (B) Kaplan-Meier survival curve of mice treated daily with vehicle vs. GBT-440 at the maximum-tolerated dose (chronic 400 mg/kg after 1 week of adaptation at 200 mg/kg). (C) Body temperature of KO mice at post-natal day ~P55 treated with vehicle vs. GBT-440 does not show a significant rescue. (D and E) (D) Time on accelerating rotarod or (E) spontaneous movement during open-field test similarly do not show a significant rescue of Ndufs4 KO mice by maximum-tolerated dose of GBT-440. #p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ANOVA.

Figure 3.. HypoxyStat administration results in tissue hypoxia comparable with inhaled hypoxia

(A) Left panel: blood and plasma pharmacokinetic profile of HypoxyStat following single oral administration in C57BL/6 mice (10 mg/kg in 0.5% HPMC). Right panel: blood levels of HypoxyStat following 12-day administration in C57BL/6 mice (20, 60, 200, and 600 mg/kg, 3 times per week in 0.5% HPMC). (B) Pharmacokinetic modeling of HypoxyStat at various schedules and doses. (C and D) (C) Plasma Epo concentration (n = 3+) and (D) hematocrit (n = 5+) following administration of HypoxyStat at 600 mg/kg, 3 times per week, in 0.5% HPMC for 12 days. (E–H) (E) Hematocrit, (F) plasma Epo, (G) blood glucose, and (H) five hypoxia-induced transcripts in the brain measured post daily administration of vehicle or HypoxyStat (400 mg/kg) or initiation of continuous 11% O₂ breathing. This was done after 2 days (plasma Epo, HIF targets) or after 2 weeks (hematocrit, blood glucose). p values relative to vehicle. HypoxyStat and inhaled hypoxia have similar effect sizes, whereas GBT-440 is less effective. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ANOVA. See also Figures S2 and S3.

Figure 4.. HypoxyStat improves survival and outcomes of Ndufs4 KO model of Leigh syndrome

(A) Kaplan-Meier survival curve of KO mice given vehicle vs. HypoxyStat (every 48 h or every 24 h, light blue and dark blue, respectively). The optimized dosing results in a 3–4× lifespan extension of KO mice. *p < 0.001 by Mantel-Cox test for 24 h dosing relative to vehicle and p < 0.01 for 48 h dosing. n = 6 (vehicle), n = 9 (48 h HypoxyStat), and n = 9 (24 h HypoxyStat). (B) Body weight (mean ± SD) of same cohort of mice as (A). (C) Representative neuropathology images of Iba1 staining in WT and KO mice +/− HypoxyStat or vehicle starting at P30. (D) Representative spontaneous movement traces of P50 mice from 48 h dosing (24 h dosing data in Figure S5). (E) Quantification of body temperature, time on accelerating rotating rotarod, and open-field movement in the same experimental groups. #p < 0.1,*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ANOVA. See also Figures S4, S5, and S6.

Figure 5.. HypoxyStat reverses disease in Ndufs4 KO mice

(A) Kaplan-Meier Survival curve of KO mice treated at a very late stage of disease (once a subset of KO mice have already started dying). p < 0.05 by Mantel-Cox test. (B) Body weight (mean ± SD) of KO mice before and after daily treatment with HypoxyStat. (C and D) (C) Neuropathology; (D) body temperature, rotarod, and open-field data for KO mice before (P50) or after initiation of HypoxyStat at late stages of disease (P80). **p < 0.01, unpaired t test.