Etavopivat, a Pyruvate Kinase Activator in Red Blood Cells, for the Treatment of Sickle Cell Disease

Etavopivat is an investigational, oral, small molecule activator of erythrocyte pyruvate kinase (PKR) in development for the treatment of sickle cell disease (SCD) and other hemoglobinopathies. PKR activation is proposed to ameliorate the sickling of SCD red blood cells (RBC) through multiple mechanisms, including reduction of 2,3-diphosphoglycerate (2,3-DPG), which consequently increases hemoglobin (Hb)–oxygen affinity; increased binding of oxygen reduces HbS polymerization and sickling. In addition, PKR activation increases adenosine triphosphate (ATP) produced via glycolytic flux, which helps preserve membrane integrity and RBC deformability. We evaluated the pharmacodynamic response to etavopivat in non-human primates (NHP) and in healthy human subjects, and the effects in RBC from patients with SCD after ex vivo treatment with etavopivat. A single dose of etavopivat decreased 2,3-DPG in NHP and healthy subjects. Hb–oxygen affinity was significantly increased in healthy subjects after 24 hours. Following daily dosing of etavopivat over 5 consecutive days in NHP, ATP was increased by 38% from baseline. Etavopivat increased Hb–oxygen affinity and reduced sickling in RBC collected from SCD patients with either HbSS or HbSC disease. Collectively, these results demonstrate the ability of etavopivat to decrease 2,3-DPG and increase ATP, resulting in increased Hb–oxygen affinity and improved sickle RBC function. Etavopivat is currently being evaluated in clinical trials for the treatment of SCD. Here, we describe the cellular effects of etavopivat (FT-4202), a potent, selective, orally bioavailable PKR activator (Ericsson et al., 2020). Using oral dosing studies in non-human primates (NHP) and healthy subjects, we demonstrate that etavopivat decreases 2,3-DPG and increases ATP in RBC. We also present the results of a series of pharmacodynamic (PD) assays after in vivo treatment of healthy RBCs and ex vivo treatment of sickle RBCs with etavopivat supporting the relevance of these biomarkers when evaluating response to this investigational agent. This work demonstrates the translational value of measurements adopted in preclinical and early clinical development, which are indicative of RBC health. We present target engagement data (single 700-mg dose) from the first-in-human clinical trial that provides a platform of evidence supporting further clinical development of etavopivat for the treatment of SCD. Samples were oxygenated using compressed air. Measurement using the HEMOX Analyzer (TCS Scientific) started at an oxygen pressure of 145 mmHg following replacement of air with nitrogen and was stopped automatically at an oxygen pressure of 1.9 mmHg. Results were analyzed using the HEMOX Analyzer Software; non-linear regression analysis was performed to obtain the P 50 value.


SIGNIFICANCE STATEMENT
Etavopivat-a small molecule activator of the glycolytic enzyme erythrocyte pyruvate kinasedecreased 2,3-diphosphoglycerate in red blood cells (RBC) from non-human primates and healthy subjects and significantly increased hemoglobin (Hb)-oxygen affinity in healthy subjects. Using ex vivo RBC from donors with sickle cell disease (SCD) (HbSS or HbSC genotype), etavopivat increased Hb-oxygen affinity and reduced sickling under deoxygenation.
Etavopivat shows promise as a treatment for SCD, that potentially might reduce vaso-occlusion and improve anemia.

Introduction
Sickle cell disease (SCD) is an autosomal recessive blood disorder caused by a single point mutation in the beta globin gene resulting in the expression of sickle hemoglobin (HbS).
HbS tends to polymerize when deoxygenated in the low oxygen tension of capillaries, or even in arterioles. HbS polymerization alters red blood cell (RBC) morphology and negatively affects their function (Sundd et al., 2019). The two most common SCD genotypes are due to homozygosity for the beta-S mutation (p.Glu6Val) (HbSS), or compound heterozygosity for beta-S and beta-C (p.Glu6Lys) (HbSC) (da Guarda et al., 2020). The formation of cytoplasmic HbS polymers causes the RBC to adopt a rigid, sickle-like shape that is the defining characteristic of SCD (Kato et al., 2018). Sickled RBC display abnormal rheological properties, aggregate, and can lead to microcapillary blockage causing tissue hypoxia, which is experienced as a painful vaso-occlusive crisis (VOC). Repeated HbS polymerization and depolymerization with changing oxygen pressure, as well as oxidative damage, alters the RBC membrane, ultimately leading to a significantly shortened RBC lifespan, i.e., hemolysis (Sundd et al., 2019).
Despite increased reticulocyte production, this decreased RBC lifespan results in anemia.
The physiologic metabolic response of RBC to anemia is to increase 2,3-DPG, thereby promoting release and offloading of oxygen from hemoglobin (Hb) to tissues. (Charache et al.,7 2007), resulting in the sudden, severe, and painful VOC episodes. VOC is the leading diagnosis associated with emergency department visits for patients with SCD (Yusuf et al., 2010); upwards of 40% of such emergency department visits can result in hospital admission (Lanzkron et al., 2010). VOCs also lead to secondary complications, such as acute chest syndrome, that cause end-organ damage and premature death in patients with SCD (Novelli and Gladwin, 2016).
Pyruvate kinase-R (PKR) is the RBC-expressed isoform of the key glycolytic enzyme pyruvate kinase. PKR catalyzes the last and rate-limiting step of glycolysis from phosphoenolpyruvate to pyruvate while generating adenosine triphosphate (ATP) from adenosine diphosphate. In patients with pyruvate kinase deficiency, decreased PKR activity results in anemia (Zanella et al., 2005), while activation of PKR improves RBC survival and Hb levels (Grace et al., 2019). We hypothesized that PKR activation above normal levels would alter several steps of the glycolytic pathway utilizing glucose to generate pyruvate. Increased PKR activity would be expected to decrease production of 2,3-DPG and increase production of ATP within RBC (Koralkova et al., 2014;Bianchi et al., 2019). PKR is therefore a logical target to activate for treatment of SCD as a decrease in 2,3-DPG levels would increase the affinity of Hb for oxygen, lowering the potential for HbS polymerization and RBC sickling.
Furthermore, an increase in ATP synthesis can enhance overall RBC function and health. dual beneficial effect of addressing the chronic anemia associated with SCD and reducing the incidence of VOC.
Here, we describe the cellular effects of etavopivat (FT-4202), a potent, selective, orally bioavailable PKR activator (Ericsson et al., 2020). Using oral dosing studies in non-human primates (NHP) and healthy subjects, we demonstrate that etavopivat decreases 2,3-DPG and increases ATP in RBC. We also present the results of a series of pharmacodynamic (PD) assays after in vivo treatment of healthy RBCs and ex vivo treatment of sickle RBCs with etavopivat supporting the relevance of these biomarkers when evaluating response to this investigational agent. This work demonstrates the translational value of measurements adopted in preclinical and early clinical development, which are indicative of RBC health. We present target engagement data (single 700-mg dose) from the first-in-human clinical trial that provides a platform of evidence supporting further clinical development of etavopivat for the treatment of SCD.

In Vivo Studies
Procedures in Cynomolgus Monkeys. The pharmacokinetics (PK) and PD of etavopivat were assessed in male cynomolgus monkeys. The decision to use male cynomolgus monkeys was based on availability only. There were no PK/PD gender differences observed during preclinical multiple-dose safety studies. Animals (AlphaGenesis, Inc., SC, USA) were of Chinese origin, in good health (non-naïve), and housed, cared for, and acclimated to study procedures in compliance with the testing facility Institutional Animal Care and Use Committee (IACUC) Guidelines and Standard Operating Procedure (Biomere, MA, USA). NHP experiments were approved by the IACUC at Biomere, MA and conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).
Single-Dose Plasma PK and PD of Etavopivat in Healthy Subjects. Key aspects of the firstin-human study of etavopivat are summarized below. The study was performed in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines and was approved by the relevant institutional review board. Subjects were healthy volunteers aged 18 to 60 years inclusive and provided written informed consent.
The single-ascending dose portion of the study was a phase 1, single-center, randomized, placebo-controlled, double-blind dose escalation study to assess the safety, tolerability, PK, and PD following a single oral dose of etavopivat in healthy subjects (ClinicalTrials.gov identifier: NCT03815695). Placebo and etavopivat were administered orally to subjects after overnight This article has not been copyedited and formatted. The final version may differ from this version. ng/ml, respectively. 2,3-DPG and ATP in whole blood were measured using a separate LC-MS/MS method as described below. Hb-oxygen affinity and values for the dissolved oxygen pressure (pO 2 ) at which Hb is 50% saturated with oxygen (P 50 ) were measured in whole blood by generating oxygen equilibrium curves as described below. Non-compartmental PK analysis of etavopivat was conducted using Phoenix WinNonlin ® (Certara, Princeton, NJ). Clara, CA) and a Thermo Scientific CTC-PAL autosampler (Thermo Fisher Scientific, Waltham, MA). Analyst ® 1.6.2 (Sciex) and Aria MX software (Thermo Fisher Scientific) were used for instrument control, data acquisition, and data analysis. Calibration standards (25.0-1500 µg/ml), quality controls, and study samples were thawed on wet ice and vortex-mixed for approximately 2 minutes before being pipetted. Because ATP and 2,3-DPG are endogenous molecules present in whole blood at high levels, the low, middle, and high quality control calibration standards were prepared in deionized water (surrogate matrix). Whole blood samples (15 µl) were spiked with stable isotope-labeled internal standard ( 13 C 10 , 15 N 5 -ATP, and D 3 -2,3-DPG), processed by protein precipitation extraction, and analyzed using ZIC ® -pHILIC separation with Turbo Ion Spray ® MS/MS detection. Negative (M-H) − ions for ATP and 2,3-DPG and their respective internal standards, 13 C 10 , 15 N 5 -ATP, and D 3 -2, 3-DPG, were monitored in multiple reaction monitoring mode. Analyte to internal standard peak area ratios for the standards were used to create a quadratic calibration curve using 1/x 2 weighted least-squares regression analysis. For both analytes, the overall accuracy of the method was within ±10.5% (% relative error), and the intra-and inter-assay precision percent coefficient of variation (%CV) were less than 7%. Samples were oxygenated using compressed air. Measurement using the HEMOX Analyzer (TCS Scientific) started at an oxygen pressure of 145 mmHg following replacement of air with nitrogen and was stopped automatically at an oxygen pressure of 1.9 mmHg. Results were analyzed using the HEMOX Analyzer Software; non-linear regression analysis was performed to obtain the P 50 value.

Oxygenscan and PoS Calculations. Oxygen gradient ektacytometry (oxygenscan) using a Laser
Optical Rotational Red Cell Analyzer (Lorrca ® ) (RR Mechatronics, Zwaag, The Netherlands) (Rab et al., 2019;Sadaf et al., 2021) was performed to evaluate changes in the deformability of RBC with or without previous etavopivat treatment as a function of oxygen pressure under steady shear stress. 200×10 6 RBC from patients with SCD treated ex vivo with DMSO or etavopivat, as detailed above, were suspended in 5 ml of Oxy Iso (osmolality 282-286 mOsm/kg, pH 7.35-7.45) at room temperature. The RBC suspension was injected into a rotating wall cylinder maintained at 37°C with a fixed shear stress of 30 pascal. Deoxygenation and reoxygenation of the RBC suspension was achieved by gradual entry of nitrogen gas and ambient air, respectively. One oxygenscan consisted of approximately 80 measurements of the elongation index (EI) during one round of deoxygenation (1300 seconds) and was followed by reoxygenation (280 seconds). Oxygen saturation in the RBC suspension was calculated every 20 seconds based on quenching of the signal using a luminophore oxygen-sensor. Ambient air pO 2 in the cup of the device started at approximately 150 mmHg and was gradually lowered to below 20 mmHg of oxygen. The PoS in oxygenscan is calculated as the pO 2 (mmHg) at which the EI This article has not been copyedited and formatted. The final version may differ from this version. falls below 5% of the maximum EI (EImax) during deoxygenation and indicates the oxygen pressure at which the polymerization of HbS starts to show effects on RBC deformability.

Statistics
Statistical analysis was performed using Prism, Version 8.4 (GraphPad Software, San Diego, CA). Paired T-test or Wilcoxon tests were used as appropriate. A P value less than 0.05 was considered statistically significant for all tests. All preclinical experiments should be considered exploratory. The sample size for the phase 1 study was considered adequate to evaluate the safety, tolerability, and PK/PD of etavopivat; no formal power calculations were performed.

Etavopivat activates PKR in Healthy Human RBC in vitro
The activation of wild type PKR by etavopivat in mature human RBC in vivo was evaluated in purified RBC. Wild type PKR was activated in a concentration-dependent manner. The calculated mean maximum activation was 89% and the half maximal effective concentration (EC 50 ) was 113 nM.

Etavopivat Modulates 2,3-DPG and ATP in Cynomolgus Monkeys
Following the administration of a single 50-mg/kg dose of etavopivat in NHP, the etavopivat plasma concentration reached its maximal value (C max ) between 1-and 2-hours post-dose, and 2,3-DPG declined to 47% of the baseline concentration between 12-and 24-hours post-dose (P=0.04) (Fig. 1), which approaches the theoretical maximum calculated by 2,3-DPG PK/PD This article has not been copyedited and formatted. The final version may differ from this version. modeling (Text S1). Following five consecutive daily doses of etavopivat at lower dose levels (3, 8, and 22 mg/kg), the maximum mean decrease in the concentration of 2,3-DPG occurred 12 hours post-dose on day 1 ( Fig. 2A), ~12 hours post-etavopivat C max (Fig. 2B). The mean concentrations of 2,3-DPG observed on day 5 were comparable to those observed on day 1 ( Fig.   2A), with mean decreases from baseline of 19% (P=0.09), 25% (P=0.02), and 36% (P=0.002), respectively, 12h after the last dose on day 5.
Due to the temporal delay between etavopivat plasma concentration and 2,3-DPG modulation, direct plots between etavopivat PK and 2,3-DPG response are not meaningful. An indirect PK/PD model, previously applied to describe 2,3-DPG PK/PD relationships (Kha et al., 2015) and described in the supplementary section (Text S1), was used to quantitate the exposure-response relationship. This analysis estimates the maximal 2,3-DPG decrease from baseline in NHP with etavopivat is approximately 50%, where the plasma concentration to achieve half of this maximum effect at PK/PD steady state (RC 50 ), a 25% decrease in 2,3-DPG, is ~18 ng/mL (34 nM) from equation 4. Primary PK/PD parameters in NHP are summarized in Table S1.  Table S2. Day 5 percent changes of 2,3-DPG and ATP, along with exposure (Day 5 AUC 0-24 ) are summarized in Fig. 3.

Affinity and Reduced PoS
The ability of etavopivat to increase Hb-oxygen affinity in healthy subjects treated with a single dose (Fig. 4A) is indicative of the potential to mitigate the rate and extent of RBC sickling in patients with SCD. We tested this possibility ex vivo, using whole blood from donors with  (Table S3). In the HbSS group, P 50 (mean ± S.D.) was reduced to 24.8 ± 1.82 mmHg after 4-hour incubation with etavopivat compared with 26.1 ± 1.99 mmHg in the vehicle-treated group (P=0.0002) (Fig. 4B).
For the HbSC group, P 50 was reduced to 24.8 ± 1.58 mmHg after incubation with etavopivat compared with 26.3 ± 1.24 mmHg in the vehicle-treated group (P=0.0313) (Fig. 4C). Etavopivat either HbSS or HbSC (Fig. 4D, E) suggesting an improved oxygen-carrying capacity of Hb under conditions of decreased oxygen pressure.

Human PK of Etavopivat
Following a single oral dose of 700 mg in healthy subjects (one female, five male), etavopivat was rapidly absorbed and median time to maximum concentration was approximately 0.5 hours. After reaching peak concentrations, mean concentrations of etavopivat declined in a multi-exponential manner (Fig. 6). The estimated elimination t 1/2 was approximately 13 hours.

Etavopivat Decreases 2,3-DPG and Increases Hb-Oxygen Affinity in Healthy Subjects
The PD profile following a single 700-mg dose of etavopivat in healthy subjects was comparable with a single 50-mg/kg dose in NHP. The maximum inhibition of 2,3-DPG was observed 24-hours post-dose, lagging behind the etavopivat C max , which was observed approximately 0.5 hours post-dose. In healthy subjects, concentrations of 2,3-DPG were collected for 7 days following a single 700 mg dose; maximal reductions of 2,3-DPG (mean 49%; p=0.0313) were durable, lasted 48-hours post-dose, and returned to baseline by day 7 (Fig.   6). Changes in whole blood concentrations of ATP following a single dose were not significant.
Following a single 700-mg dose in healthy subjects, etavopivat was also shown to increase Hboxygen affinity (or decrease P 50 ) by 4.85 mmHg at 24 hours, when the greatest decrease in 2,3-DPG was observed (Table S3). 2,3-DPG and P 50 were correlated positively, providing a PKR activation-mediated PD response to etavopivat that increased Hb-oxygen affinity (Fig. 7).

Discussion
Advancements in disease-modifying therapies are urgently needed for patients with SCD. Targeting PKR activation to reduce the concentration of 2,3-DPG is based on the ratelimiting role that PKR plays in regulating glycolysis in RBC. A decrease in 2,3-DPG with PKR activation has been demonstrated in preclinical studies, in healthy subjects, and in patients with PKR deficiency after treatment (Chubukov et al., 2016;Kung et al., 2017;Yang et al., 2019).
Decreases in 2,3-DPG in both NHP and humans were delayed relative to peak plasma concentrations of etavopivat. More specifically, in NHP, maximum decreases in whole blood 2,3-DPG were observed between 12-and 24-hours post-dose, sometime after the mean C max was achieved, typically 1-hour post-dose. A comparable observation was made following a single 700-mg dose in healthy subjects. Notably, the maximal decrease in 2,3-DPG in healthy subjects treated with a single 700 mg dose of etavopivat was durable for 48 hours before slowly returning to baseline by day 7. Following a single dose, maximum decreases in 2,3-DPG in NHP dosed with 50 mg/kg and healthy human subjects treated with 700 mg was 47% and 49%, respectively, consistent with comparable unbound exposure at those dose levels (2342 and 2099 ng/ml*h for NHP and humans, respectively). Mitigating 2,3-DPG has been proposed as a means to reduce HbS polymerization in patients with SCD (Eaton and Bunn, 2017). Basal concentrations of 2,3-DPG are higher in patients with SCD compared with healthy controls (Charache et al., 1970;Castro, 1980;Ould Amar et al., 1996), and depletion of 2,3-DPG has been shown to reduce the tendency for RBC sickling in patients with SCD (Poillon et al., 1995).
2,3-DPG strongly influences the binding of oxygen to Hb; by binding within the central cavity of the Hb tetramer, 2,3-DPG causes allosteric changes, and reduces the molecule's affinity to oxygen (MacDonald, 1977). Increased Hb-oxygen affinity (decreased P 50 ) with a mean delta P 50 of 4.85 mmHg was observed in healthy subjects following the administration of a single 700-This article has not been copyedited and formatted. The final version may differ from this version. In addition to the reduction in 2,3-DPG and associated improvement in Hb-oxygen affinity, the increased glycolytic flux induced by PKR activation is expected to increase RBC function and health by increasing ATP and reducing oxidative stress. Although no meaningful elevation in ATP was observed following a single dose in monkeys or healthy subjects, ATP concentrations were elevated by as much as 38% (P=0.004) relative to day 1 in NHP following 5 consecutive days of QD doses of etavopivat. In a recent study in Berkeley sickle cell anemia mice, ATP levels were increased (P<0.01) following two weeks of treatment with etavopivat, corresponding with etavopivat plasma levels of 7702 ± 796 ng/mL (Shrestha et al., 2021). ATP plays an essential role in maintenance of RBC membrane-cytoskeletal integrity (Betz et al., 2009), membrane repair via activation of flippase to re-internalize phosphatidyl serine to the inner leaflet of the RBC membrane (Soupene and Kuypers, 2006;Weiss et al., 2012), cell hydration (Gallagher, 2017), maintenance of ionic (Mg 2+ and Ca 2+ ) gradients (Ortiz et al. 1990;Raftos et al., 1999;Rivera et al., 2005), RBC deformability (Weed et al., 1969;McMahon, 2019), and protection from oxidative damage (Banerjee and Kuypers, 2004). Therefore, This article has not been copyedited and formatted. The final version may differ from this version. increasing ATP concentrations is likely to have broad beneficial effects for patients with SCD.
Moreover, in the current study, ex vivo treatment with etavopivat significantly improved the PoS in the RBC from donors with the HbSS genotype, likely due to a reduction in 2,3-DPG. These dual effects of decreased 2,3-DPG and increased ATP suggest that etavopivat has the potential to reduce RBC sickling, hemolysis, anemia, and consequently, VOC.
Pharmacologic interventions in clinical practice or development for the treatment of SCD include approaches to increase fetal Hb or Hb oxygenation in order to decrease Hb polymerization, and to decrease vascular adhesion. The current standard of care for most patients with SCD is hydroxyurea (HU), which reduces sickling by increasing fetal Hb in RBC and reduces painful episodes of VOC by ~50%; however, the effectiveness of HU can be confounded by poor patient compliance and discontinuation rates can be high (Brandow and Panepinto, 2010;Agrawal et al., 2014;Shah et al., 2019). In addition, while HU is broadly efficacious, response to treatment can be variable, and a proportion of patients (10-20%) are non-responsive (Steinberg et al., 1997;Ma et al., 2007;Steinberg, 2008). Furthermore, HU is a myelosuppressive agent that may cause neutropenia and thrombocytopenia and thus requires routine blood monitoring for side effects (Agrawal et al., 2014). While HU has been used to treat SCD for over 30 years, there remains a need for improved disease management, more pharmacological options, and the possibility of combination therapies to target different aspects of SCD pathology. Crizanlizumab was recently approved to reduce the frequency of VOC in adults and pediatric patients aged 16 years and older with SCD. However, no improvement in hemolysis, anemia, or markers of inflammation have been observed (Ataga et al., 2017;Blair, 2020a). These data suggest that although targeting endothelial adhesion may decrease the incidence and severity of VOC, these agents are unlikely to address the anemia or chronic tissue damage associated with SCD. Drugs This article has not been copyedited and formatted. The final version may differ from this version.
JPET Fast Forward. Published on January 14, 2022 as DOI: 10.1124/jpet.121.000743 at ASPET Journals on January 20, 2022 jpet.aspetjournals.org Downloaded from that increase Hb-oxygen affinity, such as the recently approved agent voxelotor, directly interfere with HbS polymerization by maintaining a higher proportion of Hb in oxygenated state (Vichinsky et al., 2019;Blair, 2020b). Although the clinical safety profile for voxelotor to date has been positive, there is a recognized risk that excessive oxygen affinity could cause hypoxia by preventing release of oxygen into tissues, for which routine neurovascular monitoring has been recommended (Hebbel and Hedlund, 2017). Activation of PKR follows a physiological pathway that increases Hb-oxygen affinity by decreasing 2,3-DPG and is likely to counteract the pathophysiology of SCD while enabling oxygen release in hypoxic tissues. As a result of the combination of decreased 2,3-DPG and increased ATP, it is proposed that targeting PKR activation may have a broad and significant impact on SCD, both in HbSS and HbSC, by improving the membrane integrity of RBC.
In conclusion, by virtue of its ability to promote activity in the glycolytic pathway, etavopivat is hypothesized to have unique, beneficial, disease-modifying effects on SCD. As the enzyme that catalyzes the last step of glycolysis, PKR underpins reactions that directly impact the metabolic health and primary functions of RBC. Etavopivat has two key effects: first, it decreases 2,3-DPG, which can reduce HbS polymerization and potentially attenuate clinical sequela of vaso-occlusion in SCD, and second, it increases ATP, which provides metabolic resources to support membrane integrity and protect against the loss of RBC deformability, and potentially to mitigate hemolysis in patients with SCD. The PD response to etavopivat in relation to PKR was demonstrated pre-clinically and confirmed in the first-in-human trial, providing valuable evidence of target modulation, which supports the rationale for subsequent clinical trials. Furthermore, the preliminary safety profile of etavopivat supports its advancement to This article has not been copyedited and formatted. The final version may differ from this version. JPET Fast Forward. Published on January 14, 2022as DOI: 10.1124 at ASPET Journals on January 20, 2022 jpet.aspetjournals.org Downloaded from additional clinical trials to determine the risk/benefit profile of PKR activation in SCD. Clinical trials of etavopivat in patients with SCD are currently in progress.

Unnumbered Footnotes
This study was funded by Forma Therapeutics, Inc., Watertown, MA.
Pharmacokinetic data from healthy subjects following a single dose of etavopivat was previously presented at the European Hematology Association Annual Congress, 2020(Estepp JH et al, Hemasphere, 2020.    Hb, hemoglobin; HbSC, hemoglobin SC; HbSS, hemoglobin SS; HS, healthy subjects; P 50 , the dissolved oxygen pressure at which Hb-oxygen-binding is 50% saturated; RBC, red blood cells; SAD, single-ascending dose; SCD, sickle cell disease.   oxygen affinity (a decrease in P 50 ) that was positively correlated with decreases in the mean blood concentrations of 2,3-DPG.
2,3-DPG, 2,3-diphosphoglycerate; Hb, hemoglobin; P 50 , the partial pressure of dissolved oxygen at which Hb is 50% saturated with oxygen. This article has not been copyedited and formatted. The final version may differ from this version.