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Nutrients ◽  
2022 ◽  
Vol 14 (2) ◽  
pp. 236
Author(s):  
Kamila Płoszczyca ◽  
Małgorzata Chalimoniuk ◽  
Iwona Przybylska ◽  
Miłosz Czuba

The aim of this study was to evaluate the effects of sodium phosphate (SP) supplementation on aerobic capacity in hypoxia. Twenty-four trained male cyclists received SP (50 mg·kg−1 of FFM/day) or placebo for six days in a randomized, crossover study, with a three-week washout period between supplementation phases. Before and after each supplementation phase, the subjects performed an incremental exercise test to exhaustion in hypoxia (FiO2 = 16%). Additionally, the levels of 2,3-diphosphoglycerate (2,3-DPG), hypoxia-inducible factor 1 alpha (HIF-1α), inorganic phosphate (Pi), calcium (Ca), parathyroid hormone (PTH) and acid-base balance were determined. The results showed that phosphate loading significantly increased the Pi level by 9.0%, whereas 2,3-DPG levels, hemoglobin oxygen affinity, buffering capacity and myocardial efficiency remained unchanged. The aerobic capacity in hypoxia was not improved following SP. Additionally, our data revealed high inter-individual variability in response to SP. Therefore, the participants were grouped as Responders and Non-Responders. In the Responders, a significant increase in aerobic performance in the range of 3–5% was observed. In conclusion, SP supplementation is not an ergogenic aid for aerobic capacity in hypoxia. However, in certain individuals, some benefits can be expected, but mainly in athletes with less training-induced central and/or peripheral adaptation.


Molecules ◽  
2021 ◽  
Vol 26 (24) ◽  
pp. 7571
Author(s):  
Petr Sedivy ◽  
Tereza Dusilova ◽  
Milan Hajek ◽  
Martin Burian ◽  
Martin Krššák ◽  
...  

Most in vivo 31P MR studies are realized on 3T MR systems that provide sufficient signal intensity for prominent phosphorus metabolites. The identification of these metabolites in the in vivo spectra is performed by comparing their chemical shifts with the chemical shifts measured in vitro on high-field NMR spectrometers. To approach in vivo conditions at 3T, a set of phantoms with defined metabolite solutions were measured in a 3T whole-body MR system at 7.0 and 7.5 pH, at 37 °C. A free induction decay (FID) sequence with and without 1H decoupling was used. Chemical shifts were obtained of phosphoenolpyruvate (PEP), phosphatidylcholine (PtdC), phosphocholine (PC), phosphoethanolamine (PE), glycerophosphocholine (GPC), glycerophosphoetanolamine (GPE), uridine diphosphoglucose (UDPG), glucose-6-phosphate (G6P), glucose-1-phosphate (G1P), 2,3-diphosphoglycerate (2,3-DPG), nicotinamide adenine dinucleotide (NADH and NAD+), phosphocreatine (PCr), adenosine triphosphate (ATP), adenosine diphosphate (ADP), and inorganic phosphate (Pi). The measured chemical shifts were used to construct a basis set of 31P MR spectra for the evaluation of 31P in vivo spectra of muscle and the liver using LCModel software (linear combination model). Prior knowledge was successfully employed in the analysis of previously acquired in vivo data.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2029-2029
Author(s):  
Minke A.E. Rab ◽  
Myrthe J. Van Dijk ◽  
Jennifer Bos ◽  
Brigitte A. van Oirschot ◽  
Johan Gerrits ◽  
...  

Abstract Background: Sickle cell disease (SCD) is a monogenetic red blood disorder that is characterized by hemolytic anemia and vaso-occlusive crises. Among the many factors that contribute to disease pathophysiology is stiffening and sickling of red blood cells (RBC), which is the direct result of the formation of abnormal hemoglobin S. Sickling is one of the core factors that cause vaso-occlusion and sickling is modulated by glycolytic intermediates such as 2,3-diphosphoglycerate (2,3-DPG) and ATP. Previously we showed that red blood cell pyruvate kinase (PKR), the key regulatory enzyme of glycolysis, is impaired in SCD and that ex vivo treatment with mitapivat, an allosteric activator of PKR, increased enzymatic activity and thermostability, reduced 2,3-DPG levels, decreased p50, and subsequently reduced sickling (Rab et al, Blood 2021). Currently, mitapivat is in phase 1 and phase 2 trials for SCD (#NCT04000165 and EudraCT#2019-003438). Aims: Recently, AG-946, a next-generation activator of PKR has been developed. Here we investigate the pharmacodynamic effects of AG-946 in ex vivo treatment of RBC from SCD patients in comparison with mitapivat. Methods: Buffy coat depleted whole blood obtained from five patients with SCD was incubated for 20-24 hrs in absence or presence of mitapivat (100 mM) or AG-946 (1 mM, 5 mM, 50 mM). After ex vivo treatment, enzymatic activities of PKR and PK-thermostability was measured. Glycolytic intermediates ATP and 2,3-DPG were measured using LC-MS/MS. Hemoglobin oxygen affinity (p50) was measured with the Hemox Analyzer. RBC sickling was analyzed with the oxygenscan, a newly developed method that characterizes individual sickling behavior by oxygen gradient ektacytometry. Individual tendency to sickle is reflected by Point-of-Sickling (PoS) that indicates the specific pO 2 at which RBCs start to sickle during deoxygenation under shear stress. Results: PKR activity was increased compared to vehicle (DMSO) to a similar extent in presence of both mitapivat and AG-946 (Figure 1A). In addition, PKR thermostability was significantly increased compared to vehicle (mean 22%, SD 6%) in samples treated with mitapivat 100 mM (mean 78%, SD 11%), as well as AG-946 5 mM (mean 66%, SD 23%), and AG-946 50 mM (mean 95%, SD 17%, Figure 1B). The glycolytic intermediate 2,3-DPG decreased after incubation with both mitapivat and AG-946 (Figure 1C), which was further illustrated by the improved ATP/2,3-DPG ratio (Figure 1D). In line with these latter results p50 decreased significantly after incubation with mitapivat 100 mM (mean 95%, SD 2%), as well as AG-946 1 mM (mean 96%, SD 2%), AG-946 5 mM (mean 94%, SD 2%), and AG-946 50 mM (mean 95%, SD 3%, Figure 1E). The improved metabolic status and p50 was accompanied by a decreased PoS compared to vehicle in RBCs treated with mitapivat or AG-946, indicating reduced RBC sickling tendency in vitro (Figure 1F). Conclusion: Ex vivo treatment of SCD RBCs with the next-generation PKR activator AG-946 activates and stabilizes PK, decreases 2,3-DPG levels, improves the ATP/2,3-DPG ratio, improves p50 and lowers the PoS. These beneficial effects are similar to ex vivo treatment with mitapivat but, importantly, are obtained at much lower concentrations. Therefore, AG-946 may be a potent activator of PKR in SCD. Taken together, these results are the first in an ex vivo model to demonstrate that the next-generation PK activator AG-946 has a similar favorable pharmacodynamic profile to mitapivat with enhanced PK-stabilizing properties and, hence, represents a potential novel therapeutic option in addition to mitapivat for the treatment of SCD and other hemolytic anemias. Figure 1 Figure 1. Disclosures Rab: Axcella Health: Research Funding; Agios Pharmaceuticals: Research Funding. Van Dijk: Axcella Health: Research Funding; Agios Pharmaceuticals: Research Funding. Kosinski: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Kung: Agios Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Van Beers: Pfizer: Research Funding; RR Mechatronics: Research Funding; Novartis: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Dang: Agios Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Wijk: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Axcella health: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3109-3109
Author(s):  
Joanna Howard ◽  
Kevin H.M. Kuo ◽  
Abdulafeez Oluyadi ◽  
Hui Shao ◽  
Susan Morris ◽  
...  

Abstract Background: Sickle cell disease (SCD) is a life-threatening, hereditary hemoglobin (Hb) disorder characterized by chronic hemolytic anemia, pain, end-organ damage, and poor quality of life. The key pathology is red blood cell (RBC) sickling due to polymerization of deoxygenated sickle Hb (HbS), which can be exacerbated by increased levels of the glycolytic metabolite 2,3-diphosphoglycerate (2,3-DPG), and decreased ATP. Sickled RBCs are rigid, not deformable, and fragile, resulting in vaso-occlusion triggering pain and chronic hemolysis. SCD treatment options are limited, with an unmet need for safe and effective therapies to improve anemia and reduce pain. Mitapivat is an investigational, first-in-class, oral, small-molecule allosteric activator of the RBC-specific form of pyruvate kinase (PKR), a key enzyme in glycolysis. Activation of wild-type PKR decreases 2,3-DPG and increases ATP, which may reduce HbS polymerization, RBC sickling, and hemolysis in SCD. Data from the Phase 1 National Institutes of Health multiple ascending dose study of up to 100 mg mitapivat twice daily (BID) in SCD (NCT04000165) showed that mitapivat was safe and tolerable, and was associated with a dose-dependent increase in ATP and decrease in 2,3-DPG, in addition to improvements in anemia and hemolytic markers. Based on these results, a Phase 2/3 study investigating the safety and efficacy of mitapivat in patients (pts) with SCD is planned. Methods: This Phase 2/3, double-blind, randomized, placebo-controlled, multicenter study aims to evaluate the efficacy and safety of mitapivat in pts with SCD. Eligible: pts ≥ 16 years of age with documented SCD (HbSS, HbSC, HbSβ 0/HbSβ + thalassemia, other SCD variants), 2-10 sickle cell pain crises (SCPCs; acute pain needing medical contact, acute chest syndrome, priapism, hepatic or splenic sequestration) in the prior 12 months, and an Hb level of 5.5-10.5 g/dL. If taking hydroxyurea (HU), the dose must be stable for ≥ 90 days before starting study drug. Not eligible: pts receiving regularly scheduled blood transfusions, with severe kidney disease or hepatobiliary disorders, currently receiving treatment with SCD therapies (excluding HU) or who have received gene therapy, bone marrow or stem cell transplantation. In the double-blind Phase 2 part of the study, 69 pts will be randomized (1:1:1) to receive 50 mg mitapivat, 100 mg mitapivat, or placebo BID for 12 weeks. The primary objective of the Phase 2 part of the study is to determine the recommended Phase 3 dose of mitapivat by evaluating anemia and safety vs placebo via the following endpoints: Hb response, defined as a ≥ 1.0 g/dL increase in average Hb concentration over Weeks 10-12 compared with baseline; and type, severity, and relationship of adverse events (AEs) and serious AEs (SAEs). In the double-blind Phase 3 part of the study, 198 pts who did not participate in the Phase 2 study will be randomized (2:1) to receive the selected Phase 3 dose of mitapivat or placebo, BID, for 52 weeks, stratified by the number of SCPCs in the prior year (< 5, ≥ 5) and by HU use. The primary objectives of the Phase 3 study are to determine the effect of mitapivat vs placebo on anemia in pts with SCD, measured by Hb response, defined as a ≥ 1.0 g/dL increase in average Hb concentration over Weeks 24-52 compared with baseline, and to determine the effect of mitapivat vs placebo on SCPC, measured by annualized rate of SCPCs. Key secondary endpoints include change over Weeks 24-52 compared with baseline in the following: average Hb concentration, indirect bilirubin, percent reticulocyte, and Patient-Reported Outcomes Measurement Information System ® (PROMIS) fatigue 13a Short Form scores; annualized frequency of hospitalizations for SCPC. Other secondary objectives include evaluation of effect on additional markers of hemolysis and erythropoiesis, additional clinical efficacy measures related to SCPC, additional patient-reported measures of fatigue and pain, physical activity, safety, and pharmacokinetics/pharmacodynamics of mitapivat. Pts who complete the double-blind period of either the Phase 2 or Phase 3 part of the study will be eligible to receive mitapivat for an additional 216 weeks in the open-label extension period. Results: Not yet available Conclusion: This Phase 2/3 study will investigate the efficacy and safety of the pyruvate kinase activator mitapivat in pts ≥ 16 years of age with SCD and enrollment will begin in 2021. Figure 1 Figure 1. Disclosures Howard: Resonance Health: Honoraria; Novartis: Consultancy, Honoraria; Bluebird Bio: Research Funding; Forma Therapeutics: Consultancy; Agios Pharmaceuticals: Consultancy; Novo Nordisk: Consultancy; Global Blood Therapeutics: Consultancy; Imara: Consultancy, Honoraria. Kuo: Bluebird Bio: Consultancy; Apellis: Consultancy; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Pfizer: Consultancy, Research Funding; Alexion: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Bioverativ: Membership on an entity's Board of Directors or advisory committees. Oluyadi: Agios Pharmaceuticals: Current Employment, Current holder of stock options in a privately-held company. Shao: Agios Pharmaceuticals: Current Employment, Current holder of stock options in a privately-held company. Morris: Agios Pharmaceuticals: Current Employment, Current holder of stock options in a privately-held company. Zaidi: Agios Pharmaceuticals: Current Employment. Van Beers: RR Mechatronics: Research Funding; Novartis: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 8-8
Author(s):  
Theodosia A. Kalfa ◽  
Marilyn J. Telen ◽  
Santosh L. Saraf ◽  
R. Clark Clark Brown ◽  
Katie Giger Seu ◽  
...  

Abstract The hallmark of sickle cell disease (SCD) is polymerization of deoxygenated hemoglobin S (HbS), resulting in red blood cell (RBC) sickling, oxidative and membrane damage, hemolysis, vaso-occlusion, and end-organ damage. Exacerbating the pathogenesis of SCD, the sickle RBC (sRBC) has increased 2,3-DPG levels with decreased oxygen (O 2) affinity (increased P 50) and decreased ATP. Etavopivat, a small molecule activator of erythrocyte pyruvate kinase (PKR), increases PKR activity, resulting in decreased 2,3-DPG levels and increased ATP levels in RBCs. In a Phase 1 study in patients with SCD [NCT03815695], etavopivat significantly improved anemia and hemolysis after 2 weeks of treatment (Brown et al. Blood 2020). To evaluate the potential of etavopivat to reduce vaso-occlusive crises, exploratory studies were conducted to characterize the sRBC specific (intrinsic) and systemic effects of PKR activation. Patients with SCD received once daily etavopivat 300 or 600 mg for 2 weeks or 400 mg for up to 12 weeks. Peripheral blood was collected prior to treatment (ie, baseline), on treatment and 7-28 days post treatment. Studies to assess the sRBC intrinsic effects of PKR activation included evaluation of RBC parameters and reticulocyte counts (ADVIA ®), membrane deformability (Lorrca ®), enzyme function studies, and membrane markers by flow cytometry. Studies to assess the systemic effects of PKR activation included markers of coagulation, inflammation, and hypoxia in the 12-week cohort only. As of May 24, 2021, 15 patients who completed the 2-week dose cohorts and 7 patients treated in the 12-week dose cohort were evaluable for this analysis. The intrinsic effects of etavopivat on the sRBCs of patients receiving 2 weeks of treatment are summarized in Table 1. Etavopivat significantly increased Hb and reduced reticulocytes, including immature reticulocytes (CD71 +), suggesting that an etavopivat-mediated increase in sRBC lifespan is accompanied by a decrease in erythropoietic stress. In addition, etavopivat reduced 2,3-DPG levels thereby increasing HbS O 2 affinity (decreased P 50) resulting in a significant shift in the point of sickling (PoS) to a lower partial O 2 pressure. The deformability (EI max) of the sRBCs as measured by oxygenscan and osmoscan was significantly improved with etavopivat treatment, consistent with reduced membrane damage due to decreased HbS polymerization and improved membrane repair enabled by increased ATP production, collectively improving the health of the sRBC membrane. This improvement in membrane health was further supported by a significant reduction in the external expression of phosphatidylserine (PS) following etavopivat treatment. Finally, etavopivat significantly improved enzymatic activity not only of PKR but also superoxide dismutase and glutathione reductase, enzymes involved in reducing oxidative stress in sRBCs. This suggests that etavopivat-treated sRBCs may have an improved ability to inhibit and repair damage caused by reactive O 2 species, thereby improving overall sRBC health and function. Initial results on the effect of etavopivat on systemic biomarkers that are commonly elevated in SCD are shown in Table 2. In patients receiving etavopivat 400 mg once daily for up to 12 weeks, tumor necrosis factor-a and prothrombin 1.2, as systemic markers of inflammation and hypercoagulability, respectively, showed a significant decrease compared with baseline. Furthermore, a trend towards reduced erythropoietin levels suggests that etavopivat treatment may reduce tissue hypoxia. Patients with SCD treated with etavopivat for at least 2 weeks demonstrated a significant increase in RBC membrane deformability and improved antioxidant capacity that resulted in increased sRBC survival and decreased anemia. The reduced reticulocyte count and lowered PS surface membrane expression suggest that etavopivat-treated sRBC may have reduced adhesive properties and may thus be less likely to promote vaso-occlusion. Initial studies evaluating the downstream effects of up to 12 weeks of etavopivat treatment once daily provided evidence for a reduction in markers of inflammation and hypercoagulability, with improved O 2 delivery capacity. These initial results suggest that the multimodal effects of decreased 2,3-DPG and increased ATP by PKR activation with etavopivat may have an impact on both the anemia and vaso-occlusive events that characterize SCD. Figure 1 Figure 1. Disclosures Kalfa: Agios Pharmaceuticals, Inc.: Other: Steering Committee, Research Funding; FORMA Therapeutics, Inc: Research Funding. Telen: GlycoMimetics, Inc.: Consultancy; Novartis, Inc.: Other: Data Safety Monitoring Board; Forma Therapeutics, Inc.: Consultancy, Research Funding; CSL Behring, Inc.: Research Funding; Doris Duke Charitable Foundation: Research Funding; National Institutes of Health: Research Funding. Saraf: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding. Brown: Novo Nordisk: Consultancy; Imara: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Pfizer: Research Funding; Forma Therapeutics: Research Funding. Larkin: Forma Therapeutics, Inc.: Research Funding. Ribadeneira: Forma Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Schroeder: Forma Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Wu: Forma Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Kelly: Forma Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Kuypers: Forma Therapeutics, Inc.: Research Funding.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2047-2047
Author(s):  
Myrthe J. van Dijk ◽  
Minke A.E. Rab ◽  
Anita W. Rijneveld ◽  
Erfan Nur ◽  
Marije Bartels ◽  
...  

Abstract Background Sickle cell disease (SCD) is one of the most common and devastating inherited blood disorders characterized by a single nucleotide mutation in the beta-globin chain leading to the production of mutant hemoglobin S (HbS). HbS polymerizes upon deoxygenation causing red blood cells (RBC) to sickle which results in extremely painful episodes of vaso-occlusive crisis (VOC), severe hemolytic anemia, chronic multiorgan failure and a reduced life span. An important metabolic feature directly associated with RBC sickling is increased intracellular levels of the glycolytic intermediate 2,3-diphosphyglycerate (2,3-DPG) which promotes deoxygenation by lowering the oxygen affinity of hemoglobin (Hb). Pyruvate kinase R (PKR) is a key enzyme in RBC metabolism, generating adenosine triphosphate (ATP) to maintain energy homeostasis, membrane integrity and deformability, and modulates 2,3-DPG levels. Mitapivat (AG-348) is an oral, small molecule allosteric activator of PKR and shows promise as an anti-sickling agent in addition to its effect in PK deficiency and thalassemia. The safety and efficacy of mitapivat in subjects with SCD was evaluated in the dose finding period of this ongoing phase 2 study. Methods The ESTIMATE study is a phase 2, open-label study in which subjects ≥16 years with SCD (HbSS, HbS/β0, HbS/β+) with a baseline hemoglobin >6.1 g/dL and ≤11.1 g/dL, no chronic transfusion and adequate organ function were eligible. In the 8-week Dose Finding Period, initial dosing of mitapivat was 20 mg twice daily (BID). Subjects received a maximum of two sequential dose escalations of mitapivat (i.e. from 20 mg BID to 50 mg BID and 100 mg BID) depending on safety. The primary endpoints were safety, evaluated by frequency and severity of adverse events (AEs), and efficacy of mitapivat on RBC sickling. RBC sickling was evaluated by change in Point of Sickling (PoS), the pO2 at which sickling occurs as measured by oxygen gradient ektacytrometry on the Lorrca (RR Mechatronics). Secondary endpoints included changes in hematological parameters, levels of 2,3-DPG and ATP, Hb-oxygen affinity (p50) and surrogate markers of organ damage and mortality. Subjects who safely tolerated mitapivat and showed evidence of clinical improvement, were eligible to continue a 52-week follow-up period (Fixed Dose Extension Period). Results Six subjects have been enrolled as of September 2020 and completed the Dose Finding Period. All had homozygous HbSS except one patient who had HbS/β0-thalassemia. Baseline characteristics were: median age of 36 years (range 20-59 years), 4 (66.7%) were female and 5 (83.3%) were on stable-dose hydroxyurea. All subjects received dose escalation to a maximum dose of 100 mg BID. No serious adverse events (SAEs) occurred. Adverse events (AEs) were mild and often transient, with the most common treatment emergent AEs: transaminase increase (n=3 [50.0%], Grade 1), gastrointestinal disorders including dyspepsia, diarrhea and abdominal discomfort (n=3 [50.0%], Grade 1) and headache (n=2 [33.3%], Grade 1). One VOC occurred without hospital admission and did not require dose reduction or discontinuation. Table 1 summarizes the anti-sickling effect as well as the hematological and biochemical response to mitapivat treatment. Sickling occurred at lower pO2 levels in all 6 patients during the Dose Finding Period reflected by a significant decrease in treatment week 8 mean PoS compared to baseline. 5/6 subjects (83.3%) achieved a Hb increase of ≥1 g/dL during this period, which was accompanied by a decrease in hemolytic markers. Consistent with activation of PKR, 2,3-DPG levels decreased and ATP levels increased. Additional results including biomarker data will be presented. Conclusion Mitapivat demonstrated an adequate safety profile during the 8-week Dose Finding Period in patients with SCD. The data show promising efficacy in terms of a decrease in the pO2 at which RBCs start to sickle, as well as increase in Hb from baseline and a concomitant decrease in markers reflecting hemolysis. The observed changes in 2,3-DPG and ATP levels are consistent with the proposed mechanism of the drug. The study is ongoing and further data including follow-up data, patient-reported outcomes, PKR activity and thermostability will be reported at a later stage. Figure 1 Figure 1. Disclosures van Dijk: Agios Pharmaceuticals: Research Funding; Axcella Health: Research Funding. Rab: Axcella Health: Research Funding; Agios Pharmaceuticals: Research Funding. Rijneveld: Servier: Research Funding; Amgen: Research Funding. Nur: Celgene: Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Roche: Speakers Bureau. Schutgens: CSL Behring: Research Funding; Novo Nordisk: Research Funding; OctaPharma: Research Funding; Pfizer: Research Funding; Shire/Takeda: Research Funding; Bayer: Research Funding. Wijk: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Axcella health: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van Beers: Pfizer: Research Funding; Novartis: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; RR Mechatronics: Research Funding.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2043-2043
Author(s):  
Varsha Iyer ◽  
Elizabeth Merica ◽  
Sebastien Ronseaux ◽  
Tressa Gamache ◽  
Nancy J. Mulrow ◽  
...  

Abstract Background: Glycolysis is the primary source of ATP in red blood cells (RBCs), which is critical for maintaining RBC health. Pyruvate kinase red cell isoform (PKR) catalyzes the final step of glycolysis to generate ATP. Enhancing ATP production via PKR activation is under investigation as a potential therapeutic approach in hemolytic anemias. Increased levels of the glycolytic metabolite 2,3-diphosphoglycerate (2,3-DPG) in sickle cell disease (SCD) decreases hemoglobin oxygen affinity and results in increased RBC sickling. PKR activation has been shown to reduce 2,3-DPG. In previous clinical studies in pyruvate kinase deficiency, thalassemia and SCD, treatment with mitapivat, a PKR activator, led to improvements in hemoglobin and markers of hemolysis. AG-946 is an investigational, next-generation, oral small molecule activator of wild-type and mutant PKR isoforms, with high potency. Modelling from preclinical studies suggest that AG-946 is likely to have a pharmacokinetic (PK) profile that allows for once daily (QD) dosing and long duration of pharmacodynamic (PD) effects in humans. Here we report preliminary blinded results from an ongoing study assessing the safety, tolerability, PK and PD of AG-946 in healthy volunteers (NCT04536792). Methods: In this phase 1, randomized, double-blind, placebo (P)-controlled study, single ascending oral doses (SAD) or multiple ascending oral doses (MAD) of AG-946 were administered under fasting conditions to healthy men and women (18-55 years of age) in sequential cohorts. In SAD (4 cohorts of 8 subjects each) and in MAD (2 cohorts of 8 subjects each) subjects were planned to be randomized to receive either AG-946 (n=6) or P (n=2). The dose levels studied so far are 1, 3, 10, and 30 mg in SAD and 1 mg QD and 2 mg QD for 14 days in MAD. Safety assessments included vital signs, physical exams, electrocardiograms, clinical laboratory parameters and adverse events (AEs). Serial blood samples were drawn for PK and PD (2,3-DPG; ATP) assessments at regular intervals throughout the study period. Results: As of June 02, 2021, 39 (median age 33 years; n = 33 male) subjects in SAD and 17 (median age 36 years; all male) subjects in MAD received AG-946 or P. There were 6 (SAD, n = 5; MAD, n = 1) early discontinuations; all were unrelated to study treatment. In SAD, 4/39 (10.3%) subjects experienced ≥ 1 treatment-emergent AE (TEAE); all TEAEs were assessed as mild (Grade [Gr] 1). In MAD 4/17 (23.5%) subjects experienced ≥ 1 TEAE; the majority of the TEAEs were mild (Gr 1), with 1 subject experiencing a serious AE (Gr 2) of exercise-induced rhabdomyolysis 14 days after last dose, considered unrelated to study treatment. All other AEs in SAD and MAD were also considered unrelated to study treatment. In both SAD and MAD, AG-946 exhibited rapid absorption with median T max (time to maximum concentration) ranging from 0.5 to 1 hour. Following SAD, dose-normalized AG-946 exposures (AUC and C max [area under the curve and maximum concentration observed]) increased with increasing AG-946 doses, suggesting a greater than dose proportional increase in exposure over the tested dose range. Following MAD, AG-946 exposures were higher on Day 14 compared with Day 1, with mean accumulation ratios based on AUC of 3.6 at 1 mg QD and 3.3 at 2 mg QD, and mean accumulation ratios based on C max of 1.95 at 1 mg QD and 1.6 at 2 mg QD. In both SAD and MAD, an increase in AG-946 dose was associated with a decrease in 2,3-DPG concentrations (Figure 1), and an increase in ATP concentrations (Figure 2). The PD changes were sustained up to 168 hours after a single dose and > 7 days after the last day of multiple QD dosing, consistent with the slow off-rate from PKR. As expected, no clinically significant changes in Hb have been observed in the SAD or MAD cohorts, to date. Conclusions: AG-946, a highly potent PKR activator, was well tolerated in healthy volunteers following single dose administrations up to 30 mg and multiple 14-day dosing with 1 mg QD and 2 mg QD. The PK profile of AG-946 supports QD dosing, and is accompanied by sustained dose-dependent increases in ATP and decreases in 2,3-DPG, consistent with activation of the glycolytic pathway. Enrollment into additional SAD and MAD cohorts is ongoing and will be followed by an open-label phase in subjects with SCD. Figure 1 Figure 1. Disclosures Iyer: Novartis: Current equity holder in publicly-traded company; Agios Pharmaceuticals: Current Employment, Current holder of stock options in a privately-held company. Ronseaux: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Gamache: Agios Pharmaceuticals: Current Employment. Callaghan: Agios Pharmaceuticals: Current Employment.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 10-10
Author(s):  
Julia Z. Xu ◽  
Anna Conrey ◽  
Ingrid Frey ◽  
Eveline Gwaabe ◽  
Laurel A Menapace ◽  
...  

Abstract Background. Hemoglobin S (HbS) polymerization causes red cell sickling, hemolysis, and vaso-occlusion, key pathological features of sickle cell disease (SCD). Mitapivat (AG-348) has potential as an oral anti-sickling agent in SCD via increasing glycolytic activity, which reduces intracellular levels of 2,3-diphosphoglycerate (2,3-DPG) in parallel with increasing adenosine triphosphate (ATP). Reducing 2,3-DPG decreases HbS polymerization, while increasing ATP improves red cell membrane integrity. Here, we report the complete results of our single-center Phase 1 study of multiple ascending doses of mitapivat in subjects with SCD. Methods. We enrolled adult subjects (age ≥ 18 years) with confirmed SCD (HbSS) and baseline Hb ≥ 7 g/dL; with no recent transfusions, erythropoietin therapy, or changes in SCD-specific therapies including hydroxyurea (HU) and L-glutamine. Subjects received either 3 or 4 ascending dose levels of mitapivat (5 mg BID, 20 mg BID, 50 mg BID, 100 mg BID) for 2 weeks' duration each, followed by a 12-15 day drug taper. Safety and tolerability were assessed by frequency and severity of adverse events (AEs) and changes in hemoglobin (Hb) level and hemolytic markers. For each dose level, pharmacokinetics (PK), pharmacodynamics (PD; 2,3-DPG and ATP levels), and markers of oxygen (O 2) affinity (p50) and HbS polymerization (t50) were assessed pre-dose, post-dose, at end of taper, and at end of study. p50 is the partial pressure of O 2 at which 50% of the hemes in the Hb molecule have O 2 bound; t50 is the time at which 50% of erythrocytes are sickled in response to gradual deoxygenation with nitrogen to a final O 2 partial pressure of 38 torr. Results. Out of 17 subjects enrolled, 16 escalated to 50 mg BID. One subject, withdrawn 3 days after starting the study for a pre-existing pulmonary embolus, was not evaluable for response. After a protocol amendment, 9/10 eligible subjects completed the 100 mg BID dose level; 1 subject self-discontinued treatment after completing 3 dose levels. Mean age of the 17 subjects was 39 years (range 23-55 years); 11 were male, and 12 were on HU. Mitapivat was well tolerated; the most commonly reported drug-related AEs were insomnia (n=6 subjects, Grades 1-2), arthralgia (n=3, Grades 1-2), and hypertension (n=3, Grades 1-3). Six serious AEs (SAEs) were reported in 6/17 subjects, including 4 vaso-occlusive crises (VOCs), 1 non-VOC-related pain, and 1 pre-existing pulmonary embolism; 2/6 SAEs were deemed possibly drug-related. Of the 4 VOCs, 2 occurred during drug taper and were possibly drug-related, and 2 occurred during the 28-day safety follow up post-treatment in the setting of known VOC triggers. In 16 evaluable subjects, a dose-dependent decrease in mean 2,3-DPG levels and increase in mean ATP levels were consistently observed, followed by a return to near baseline by end of study (Figure 1A-B). There was a mean decrease in p50 and increase in t50 (Figure 1D-E), indicating increased oxygen affinity and slower sickling, respectively. The mean Hb increase at the 50 mg BID dose level was 1.2 g/dL (range -0.3-2.9 g/dL; Figure 1C). Over half (9/16, 56.3%) of subjects achieved a Hb response, defined as a ≥ 1 g/dL increase in Hb at any dose level compared to baseline. Subjects also experienced a mean reduction in the hemolytic markers of lactate dehydrogenase, total serum bilirubin, absolute reticulocyte count, and aspartate aminotransferase during the dose escalation period (Figure 1F-I), though responses were variable. Mean corpuscular volume (MCV) and HbF levels remained relatively stable throughout the study, supporting the notion that hydroxyurea exposure remained stable throughout the treatment period. Conclusion. During a 6-8 week treatment period, mitapivat demonstrated an acceptable safety and tolerability profile at multiple ascending dose levels in subjects with SCD. Mitapivat improved anemia, reduced markers of hemolysis, decreased 2,3-DPG and increased ATP levels, improved oxygen affinity, and decreased sickling rate, signaling its potential to improve clinically meaningful outcomes in SCD. Long-term disease modifying effects of mitapivat treatment in SCD are being evaluated in an ongoing extension study (ClinicalTrials.gov NCT04610866). Figure 1 Figure 1. Disclosures Iyer: Novartis: Current equity holder in publicly-traded company; Agios Pharmaceuticals: Current Employment, Current holder of stock options in a privately-held company. Mangus: Agios Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company; Bristol-Myers Squibb: Current equity holder in publicly-traded company. Kung: Agios Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Dang: Agios Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Kosinski: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Hawkins: Bristol-Myers Squibb: Current equity holder in publicly-traded company; Agios: Current equity holder in publicly-traded company.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 953-953
Author(s):  
Xunde Wang ◽  
Mickias Tegegn ◽  
Fayaz Seifuddin ◽  
Stephan Menzel ◽  
Hamel Patel ◽  
...  

Abstract Background: Acute pain, the most prominent complication of sickle cell disease (SCD), results from vasoocclusion triggered by sickling of deoxygenated red blood cells (RBCs). A key factor influencing HbS oxygenation is the intracellular concentration of 2,3- diphosphoglycerate (2,3-DPG). 2,3-DPG, an intermediate substrate in the glycolytic pathway, decreases oxygen binding and stabilizes the deoxygenated hemoglobin. Pyruvate kinase (gene PKLR, protein PKR) is a rate-limiting enzyme in glycolysis; variants in PKLR may affect PKR activity, 2,3-DPG levels in RBCs, subsequent frequency of sickling and acute pain episodes (APE). There is thus a strong biological basis for exploring PKLR as a candidate gene affecting acute pain in SCD. Methods: The study population for genetic association consists of 2 cohorts: 1) 242 adults with HbSS from King's College Hospital (KCH), London, UK, with complete hospitalisation records over 10 years (2004-2013 inclusive) as the "discovery" cohort; 2) 977 children with HbSS or HbSb 0 thalassemia from the Silent Infarct Transfusion (SIT) trial, with a 3-year history of severe vasoocclusive pain based on hospitalization, as the "validation" cohort. Both studies were approved by the local Institutional Review Boards at KCH and Vanderbilt University Medical Center, respectively. An independent cohort comprises 52 adults with SCD enrolled under 3 protocols - NCT00011648, NCT00081523, and NCT03685721 - approved by the NHLBI Review Board (NIH), for evaluation of imbalance in allele expression. Genome scan for the KCH cohort was performed using llumina's Infinium "MEGA" chip (1.7m markers). The SIT DNA samples were genotyped using Illumina HumanHap650Y array 5 (661K markers) or Illumina Infinium HumanOmni1-Quad array (1.1m markers). The results were quality controlled followed by genotype imputation based on the 1000 Genomes Project phase 3 data. An annualised "hospitalisation rate" as a measure of pain incidence rate, was calculated by dividing the number of hospital admissions for severe acute pain by the number of years of observation for KCH and SIT cohorts (Fig A). We performed association analysis with common SNPs at PKLR locus using data from our genome-wide SNP set and a linear mixed modelling approach incorporating a genetic relatedness matrix to take account of relatedness, plus sex and age as fixed covariates. We corrected for multiple testing after quantifying the linkage disequilibrium (LD) within PKLR and used this to calculate appropriate significance levels. For the PKLR region and hospitalisation rate, the modified significance level was p<0.001268 for the discovery (KCH) cohort. For the allele expression assays, a synonymous variant, rs1052176 (R596R), in exon 11 of PKLR acted as a marker of relative expression levels of the 2 alleles of the gene. Allele specific expression was carried using the Bio-Rad digital droplet PCR system. Results: 7 of 47 variants evaluated in PKLR were associated with hospitalization rate (LnLnHospRate) in the discovery cohort: intron 4 - rs071053, and intron 2 - rs8177970, rs116244351, rs114455416, rs12741350, rs3020781, and rs8177964). All 7 were validated in Fisher's meta-analyses of the KCH and the SIT cohorts using p<0.0071 as threshold to correct for multiple testing (Fig B). We examined the pairwise LD between PKLR variants, and found all the intron 2 variants in tight LD, while R596R belongs to another LD block (Fig C). 52 SCD individuals had the R596R variant, of which 29 were heterozygous and 23 homozygous for the intron 2 haplotype associated with APE in SCD. We performed a Wilcoxon rank sum test and compared the variation in PKLR expression between the 2 alleles in subjects homozygous and heterozygous for the wildtype intron 2 haplotype, using genomic DNA as internal control for each subject. The results reveal a significant deviation from the expected expression ratio in those heterozygous for the intron 2 haplotype (mean 0.2073, +/- SD 0.0135) when compared with to those without the variant (mean 0.1239, +/- SD 0.0682), p=0.0297 (Fig D). Conclusion: Intronic variants of PKLR are associated with hospitalization rate for acute pain episodes in adults and children with SCD. We show that the intronic variants are likely to influence acute pain by affecting expression of the PKLR gene using allele-specific expression analyses, although the causal variant is unclear. These results support PKLR as a genetic modifier of SCD. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.


2021 ◽  
pp. 096032712110255
Author(s):  
HC Nwankwo ◽  
AA Idowu ◽  
A Muhammad ◽  
AD Waziri ◽  
YS Abubakar ◽  
...  

Sickle cell disease (SCD) treatment and management remain a challenging puzzle especially among developing Nations. Chrysin’s sickling-suppressive properties in human sickle (SS) erythrocytes in addition to its effect on AA-genotype erythrocytes were evaluated. Sickling was induced (76%) with 2% sodium metabisulphite at 3 h. Chrysin prevented (81.19%) the sickling and reversed same (84.63%) with strong IC50s (0.0257 µM and 0.00275 µM, respectively). The levels of oxygenated haemoglobin in the two groups (before and after induction approaches) were similar but significantly (P < 0.05) higher than that of SS erythrocytes (the ‘induced’ control), with chrysin-treated AA-genotype showing no effects relative to the untreated. The level of deoxygenated haemoglobin in the ‘induced’ control group was significantly (P < 0.05) higher than those of the chrysin-treated SS erythrocytes. Normal and chrysin-untreated erythrocytes (AA-untreated) were significantly more resistant to osmotic fragility than the SS-untreated. However, treatment with chrysin significantly reduced the osmotic fragility of the cells relative to the untreated cells. Furthermore, chrysin treatment significantly lowers the high level of 2,3-diphosphoglycerate (2,3-DPG) observed in the sickle erythrocytes, with no effects on AA-genotype erythrocytes. Based on functional chemistry, chrysin treatment alters the functional groups in favour of its antisickling effects judging from the observed bends and shifts. From metabolomics analysis, it was observed that chrysin treatment favors fatty acid alkyl monoesters (FAMEs) production with concomitant shutting down-effects on selenocompound metabolism. Thus, sickling-suppressive effects of chrysin could potentially be associated with modulation of oxygenated and deoxygenated haemglobin via alteration of human sickle erythrocyte’s functional chemistry and metabolic pathways implicated in SCD crisis.


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