Pyruvate Kinase Activity Research Articles

Effects of soil drought on cottonseed kernel carbohydrate metabolism and kernel biomass accumulation

Cottonseed is the main coproduct of cotton production. The carbohydrate metabolism provides carbon substrate for the accumulation of cottonseed kernel biomass which was the basis of cottonseed kernel development. However, the responses of drought stress on carbohydrate metabolism in kernels are still unclear. To address this, two cotton cultivars (Dexiamian 1 and Yuzaomian 9110) were cultivated under three water treatments including soil relative water content (SRWC) at (75 ± 5)% (control), (60 ± 5)% (mild drought) and (45 ± 5)% (severe drought) to investigate the effects of soil drought on cottonseed kernel carbohydrate metabolism and kernel biomass accumulation. Results suggested that drought restrained the accumulation of cottonseed kernel biomass which eventually decreased cottonseed kernel biomass at maturity. In detail, the down-regulation of sucrose phosphate synthase (SPS) activity led to the inhibition of sucrose synthesis, while the up-regulation of invertase (INV) promoted the sucrose decomposite, which reduced the sucrose content eventually under drought. Though hexose content was increased, phosphoenolpyruvic acid (PEP) content was decreased under drought by downregulating 6-phosphofructokinase (PFK) and pyruvate kinase (PK) activities, which hindered the conversion of hexose to PEP. The large decrease of sucrose and PEP contents hindered the accumulation of kernel biomass. The related substances contents and enzyme activities in carbohydrate metabolism of Yuzaomian 9110 were more susceptible to drought stress than Dexiamian 1.

Dark fermentative hydrogen production and transcriptional analysis of genes involved in the unicellular halotolerant cyanobacterium Aphanothece halophytica under nitrogen and potassium deprivation.

The unicellular halotolerant cyanobacterium Aphanothece halophytica is known as a potential hydrogen (H2) producer. This study aimed to investigate the enhancement of H2 production under nutrient deprivation. The results showed that nitrogen and potassium deprivation induced dark fermentative H2 production by A. halophytica, while no differences in H2 production were found under sulfur and phosphorus deprivation. In addition, deprivation of nitrogen and potassium resulted in the highest H2 production in A. halophytica due to the stimulation of hydrogenase activity. The effect of adaptation time under nitrogen and potassium deprivation on H2 production was investigated. The results showed that the highest H2 accumulation of 1,261.96 ± 96.99µmol H2 g dry wt-1 and maximum hydrogenase activity of 179.39 ± 8.18µmol H2 g dry wt-1 min-1 were obtained from A. halophytica cells adapted in the nitrogen- and potassium-deprived BG11 medium supplemented with Turk Island salt solution (BG110-K) for 48h. An increase in hydrogenase activity was attributed to the decreased O2 concentration in the system, due to a reduction of photosynthetic O2 evolution rate and a promotion of dark respiration rate. Moreover, nitrogen and potassium deprivation stimulated glycogen accumulation and decreased specific activity of pyruvate kinase. Transcriptional analysis of genes involved in H2 metabolism using RNA-seq confirmed the above results. Several genes involved in glycogen biosynthesis (glgA, glgB, and glgP) were upregulated under both nitrogen and potassium deprivation, but genes regulating enzymes in the glycolytic pathway were downregulated, especially pyk encoding pyruvate kinase. Interestingly, genes involved in the oxidative pentose phosphate pathway (OPP) were upregulated. Thus, OPP became the favored pathway for glycogen catabolism and the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which resulted in an increase in H2 production under dark anaerobic condition in both nitrogen- and potassium-deprived cells.

Alpha-ketoglutarate partially alleviates effects of high-fat high-fructose diet in mouse muscle.

Consumption of high-calorie diets leads to excessive accumulation of storage lipids in adipose tissue. Metabolic changes occur not only in adipose tissue but in other tissues, too, such as liver, heart, muscle, and brain. This study aimed to explore the effects of high-fat high-fructose diet (HFFD) alone and in the combination with alpha-ketoglutarate (AKG), a well-known cellular metabolite, on energy metabolism in the skeletal muscle of C57BL/6J mice. Five-month-old male mice were divided into four groups - the control one fed a standard diet (10 % kcal fat), HFFD group fed a high-fat high-fructose diet (45 % kcal fat, 15 % kcal fructose), AKG group fed a standard diet with 1 % sodium AKG in drinking water, and HFFD + AKG group fed HFFD and water with 1 % sodium AKG. The dietary regimens lasted 8 weeks. Mice fed HFFD had higher levels of storage triacylglycerides, lower levels of glycogen, and total water-soluble protein, and higher activities of key glycolytic enzymes, namely hexokinase, phosphofructokinase, and pyruvate kinase, as compared with the control group. The results suggest that muscles of HFFD mice may suffer from lipotoxicity. In HFFD + AKG mice, levels of the metabolites and activities of glycolytic enzymes did not differ from the respective values in the control group, except for the activity of pyruvate kinase, which was significantly lower in HFFD + AKG group compared with the control. Thus, metabolic changes in mouse skeletal muscles, caused by HFFD, were alleviated by AKG, indicating a protective role of AKG regarding lipotoxicity.

CARBOHYDRATES METABOLISM IN THE BLOOD OF RATS WITH IMPAIRED GLUCOSE TOLERANCE UNDER LONG TERM MELATONIN INJECTIONS.

The aim: To determine the influence of melatonin on the glucose level and content of malondialdehyde, activities of pyruvate kinase and glucose-6-phosphate dehydrogenase enzymes in the blood; histochemical features of glycogen distribution in liver of rats with impaired glucose tolerance. Materials and methods: Diabetes in rats was induced by intra-abdominal injection of a 5% solution of alloxan monohydrate at the rate of 170 mg/kg of body weight. Four days after animals were divided into rats with impaired glucose tolerance and melatonin-group with impaired glucose tolerance (5 mg/ kg «Sigma» USA, daily and intraperitoneal for 42 days starting from 5th day). Impaired glucose tolerance was determined by measurement of glucose profiles - fasting <5.6 mmol/l; postprandial (2h post-load) 7.8 - 11.0 mmol/l. Histochemical examination of the liver was performed according to the standard method of PAS-reaction staining. Statistical analysis was performed using Statistica 10 StatSoft Inc. Results: Pyruvate kinase activity in erythrocytes and optical density of glycogen in hepatocytes of animals with impaired glucose tolerance decreased on 18% and 11%, activity of glucose-6-phosphate dehydrogenase and content of malondialdehyde increased on 35% and 23%, respectively compared with the control. We have reached the recovery of the pyruvate kinase and normalization of glucose-6-phosphate dehydrogenase activities, malondialdehyde levels, glucose profiles in the blood as well as glycogen distribution in the liver caused by melatonin injections. Conclusions: We have determined that long term melatonin injections did better glucose tolerance in rats.

THE IMPACT OF HORMONE-VITAMIN COMPLEX ON FUNCTIONAL ACTIVITY OF THE MUSCLE TISSUE OF DESCENDANTS OF IRRADIATED ANIMALS.

The aim: To determine the hormone-vitamin complex impact on the terminal links of glycolysis, the tricarboxylic acids cycle, and the initial stage of glucone¬ogenesis in the muscle tissue in descendants of irradiated animals. Materials and methods: Pyruvate kinase, lactate dehydrogenase, malate dehydrogenase, NADP-dependent malate dehydrogenasee and phosphoenolpyruvate carboxykinase activities, the content of lactate, pyruvate, malate and oxaloacetate were determined in the blood, myocardium and thigh muscles of 66 rats after exposure to ionizing gamma-radiation. Rats were injected by a hormone-vitamin complex which efficacy was determined using the abovementioned indexes. Results: Hormone-vitamin complex administration to descendants of irradiated animals exposed to 1.0 Gy results to pyruvate kinase activity increase in the myocardium and skeletal muscles of descendants from animals irradiated by 0.5 Gy and exposed to 1.0 Gy irradiation. Blood serum pyruvate kinase activity in descendants from animals irradiated by 1.0 Gy and exposed to 1.0 Gy radiation after the pharmacological correction was higher compared with the same index before pharmacological correction. The lactate dehydrogenase activity in the myocardium, skeletal muscles and blood in descendants born from animals irradiated by maximal dose exposed to 1.0 Gy radiation was less in these tissues after pharmacological correction. Conclusions: The hormone-vitamin complex use in the descendants of irradiated animals led to muscle tissue energy resources improvement. Our data are the experimental background for theoriginal hormone-vitamin complex efficacy further evaluation in the aspect of vital organs and body systems functional activity restoration under the influence of ionizing radiation.

β-Cyclodextrins alter the energy metabolism-related enzyme activities in rats.

Although widely used in medicine, separation technology, and other fields, the effects of cyclodextrins on the activities of phosphoryl transfer enzymes have not been previously evaluated. In vivo studies evaluated the function of cyclodextrins as active compounds. Despite the use of cyclodextrins as active compounds, the effects of cyclodextrins on hepatic and renal tissues remain to be fully elucidated. The primary objective of this study was to evaluate the effects of β- cyclodextrins, methyl-β-cyclodextrin (M-β- cyclodextrins), and (2-hydroxypropyl)-β-cyclodextrin (HP-β-cyclodextrins) on enzyme activities regulating the maintenance of energy homeostasis in the kidney and liver tissues in relation to toxicity. Serum levels of liver and kidney markers were measured, and oxidative stress parameters were assessed. After 60-day treatments, we observed that the administration of β-cyclodextrins and M-β-cyclodextrins inhibited the hepatic activity of pyruvate kinase, an irreversible enzyme within the glycolytic pathway. Additionally, administration of HP-β-cyclodextrins inhibited creatine kinase activity and increased the total sulfhydryl content in kidneys. Here, we demonstrated for the first time that β-cyclodextrins, M-β-cyclodextrins, and HP-β-cyclodextrins cause bioenergetic dysfunction in renal and hepatic tissues. These findings suggest that understanding the balance between cyclodextrins' efficacy and adverse effects is essential for better accepting their use in medicine.

Novel Specific Pyruvate Kinase M2 Inhibitor, Compound 3h, Induces Apoptosis and Autophagy through Suppressing Akt/mTOR Signaling Pathway in LNCaP Cells

Pyruvate kinase M2 (PKM2) is a key enzyme involved in the regulation of glycolysis. Although PKM2 is overexpressed in various tumor tissues, its functional role in cancer chemotherapy remains unexplored. In this study, we investigated the anticancer activity of a new PKM2 inhibitor, compound 3h, through the cell metabolism and associated signaling pathways in prostate cancer cells. To evaluate the molecular basis of specific PKM2 inhibitors, the interactions of compounds 3h and 3K with the PKM2 protein were assessed via molecular docking. We found that, compared to compound 3K, compound 3h exhibited a higher binding affinity for PKM2. Moreover, compound 3h significantly inhibited the pyruvate kinase activity and PKM2 expression. Cytotoxicity and colony formation assays revealed the potent anticancer activity of compound 3h against LNCaP cells. Compound 3h significantly increased the apoptotic and autophagic cell death in LNCaP cells. In addition, compound 3h induced AMPK activation along with the inhibition of the mTOR/p70S6K pathway. Furthermore, compound 3h significantly inhibited glycolysis and mitochondrial respiration, as determined by analyzing the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) production. Our results revealed that compound 3h caused apoptotic and autophagic cell death in LNCaP cells by inhibiting cancer cell metabolism. Therefore, blocking glycolytic pathways using specific PKM2 inhibitors can target cancer cell metabolism in PKM2-overexpressed prostate cancer cells.

Triptolide inhibits intrahepatic cholangiocarcinoma growth by suppressing glycolysis via the AKT/mTOR pathway

BackgroundHigh levels of glycolysis supply large quantities of energy and biological macromolecular raw materials for cell proliferation. Triptolide (TP) is a kind of epoxy diterpene lactone extracted from the roots, flowers, leaves, or grains of the Celastraceae plant, Tripterygium wilfordii. TP has multiple biological activities, including anti-inflammatory, immunologic suppression, and anti-cancer effects. Nevertheless, it is little known regarding its anti-intrahepatic cholangiocarcinoma (ICC) growth, and the mechanism still require exploration. PurposeThis research explored the effect of TP on ICC growth and investigated whether TP inhibits glycolysis via the AKT/mTOR pathway. MethodsCell proliferation was analyzed by Cell Counting Kit-8 (CCK-8), clonogenic assay, and flow cytometry. The underlying molecular mechanism was identified by determining glucose consumption, ATP production, lactate production, hexokinase (HK) and pyruvate kinase (PK) activity, and Western blot analysis. A rapid ICC model of AKT/YapS127A oncogene coactivation in mice was used to clarify the effect of TP treatment on tumor growth and glycolysis. ResultsThe results showed that TP treatment significantly inhibited ICC cell proliferation and glycolysis in a dose- and time-dependent manner(P < 0.05). Further analysis suggested that TP suppressed ICC cell glycolysis by targeting AKT/mTOR signaling. Additionally, we found that TP inhibits tumor growth and glycolysis in AKT/YapS127A mice(P < 0.05). ConclusionTaken together, we revealed that TP suppressed ICC growth by suppressing glycolysis via the AKT/mTOR pathway and may provide a potential therapeutic target for ICC treatment.

Linc-UROD stabilizes ENO1 and PKM to strengthen glycolysis, proliferation and migration of pancreatic cancer cells

Pancreatic cancer (PC) is a fatal malignancy, threatening human health in worldwide. Long non-coding RNAs (lncRNAs) have been acknowledged to be essential regulators in various biological processes of human cancers. However, the role of some novel lncRNAs in PC remain to be explored. In this study, we focused on the function and molecular mechanism of a novel lncRNA linc-UROD (also named TCONS_00002016 or XLOC_000166) in PC. The expression of linc-UROD was found to be upregulated in PC cells. The results of loss-of-function assays demonstrated that linc-UROD knockdown suppressed cell proliferation and migration, induced cell cycle G0/G1 arrest, and accelerated apoptosis of PC cells. Through mechanistic experiments, we found that IGF2BP3 stabilized linc-UROD through METTL3-mediated m6A modification. In addition, linc-UROD enhances the stability of ENO1 and PKM through interacting with them to inhibit ubiquitination. Detection on glucose consumption, pyruvate kinase activity and lactate production indicated that linc-UROD accelerated glycolysis of PC cells through PKM/ENO1-mediated pathway. To summarize, linc-UROD stabilized by IGF2BP3/METTL3 contributes to glycolysis and malignant phenotype of PC cells by stabilizing ENO1 and PKM. The findings suggest that linc-UROD may be a novel therapeutic target for PC patients.

Effects of dietary creatine levels on the growth, muscle energy metabolism and meat quality of spotted seabass (Lateolabrax maculatus) fed low-fishmeal diets

The study was performed to evaluate the effects of dietary creatine (CR) levels on the growth, muscle energy metabolism and meat quality of spotted seabass (Lateolabrax maculatus) fed low-fishmeal diets. The experimental diets were formulated with 15% fishmeal, and contained five creatine levels of 0.03%, 0.07%, 0.12%, 0.21% and 0.39%. Spotted seabass (initial body weight: 4.38 ± 0.06 g) were randomly allocated into 15 tanks (20 fish per tank) and fed twice daily for 8 weeks. With increasing dietary CR levels, the final body weight (FBW) and weight gain (WG) significantly increased, and the peak occurred in the 0.21% group. Second-order polynomial regression analysis showed that the optimum CR supplementation level based on WG was 0.25% for spotted seabass. With the increase in dietary CR levels, anaerobic glycolysis (the activities of lactic dehydrogenase and pyruvate kinase) in fish muscle was significantly reduced. The energy reserve (the content of ATP and activity of creatine kinase) was significantly increased to promote the pH value at 48 h postmortem with increasing CR levels. Water holding capacity increased as dietary CR levels increasing from 0.03% to 0.39%. The mRNA expression of myog, myod, myf5, mef2c, mstn, capn1 and capn2 was upregulated, but the mRNA expression of cast was downregulated with increasing CR levels from 0.03% to 0.39%. In conclusion, dietary CR supplementation promoted the growth performance of spotted seabass fed low-fishmeal diets, and the optimum creatine level was 0.25%. Dietary CR supplementation increased energy reserve and deceased anaerobic glycolysis at 48 h postmortem and enhanced meat quality.

Effects of Pyruvate Kinase Activators on Red Blood Cell Rheology, Sickling and Senescence in Sickle Cell Disease

Introduction: Pyruvate Kinase Activators (PKA) are a new class of pharmacological agents that enhance pyruvate kinase (PKR) activity and glycolytic flux in red blood cells (RBC), resulting in increased intracellular adenosine triphosphate (ATP) and decreased 2,3-diphosphoglycerate (2,3-DPG) contents. In patients with sickle cell disease (SCD), decreased PKR activity and thermostability result in decreased ATP/2,3-DPG ratio, which promotes HbS polymerization and sickling. Previous ex-vivo studies have shown that AG-348 (mitapivat) could represent a therapeutic option for those patients by restoring PK activity and thermostability and by increasing RBC deformability and the ATP/2,3-DPG ratio, thus reducing the propensity of RBC to sickle under deoxygenation (1). Recently, a new PKA molecule has been developed (AG-946) but its effects on RBC deformability and sickling have not been fully studied (2). Moreover, the effects of the two molecules in a context of enhanced oxidative stress - a situation frequently encountered in SCD - have not been investigated. Materials and Methods: Blood samples from non-transfused adults and children with SCD (3 to 53 years-old) were collected in EDTA tubes. HbF levels ranged from 1.3 to 47.1%. Washed RBC were resuspended in phosphate buffer saline after buffy-coat removal and incubated for 4 hours at 37°C, with or without 0.4 mM of cumene hydroperoxide (CH), a molecule that causes a considerable oxidative stress to the cells. Three different incubation conditions (AG-348 at 50 µM, AG-946 at 5 µM and a vehicle control) were systematically compared for the following ex-vivo experiments on RBC: (i) determination of the partial pressure of oxygen at which the hemoglobin is 50% saturated with oxygen (p50) with the Hemox® device; (ii) RBC deformability at normoxia (EImax and SS1/2 parameters), in hypoxia (EImin) and the partial pressure of oxygen at which sickling starts during deoxygenation (PoS; Point of Sickling) by ektacytometry; (iii) analyses of RBC senescence markers (i.e. Phosphatidylserine externalization, reactive oxygen species, intra-cellular calcium (Ca2+) and membrane CD47) by flow cytometry. One-way ANOVA with the Newman-Keuls method was performed for multiple comparisons and the significant level was set at 0.05. Results: A significant decrease of the p50 and PoS values was observed for both AG-molecules, with and without CH, compared to the control condition (Fig. 1-A). Without CH, both AG-molecules decreased SS1/2 (i.e., the shear stress for half-maximal deformation of RBCs) and AG-946 increased EImin and EImax. With CH, the two molecules still decreased the PoS compared to the control condition and a trend through an increase of EImin was observed with the AG-946 molecule. A slight increase of intra-cellular Ca2+ with the two AG-molecules (trend for AG-946) was observed in absence of oxidative stress (Fig. 2). The other RBC senescence markers were not modified, regardless of the incubation condition. Conclusion: Our results show that both PKAs increase the oxygen affinity and reduce the sickling propensity of RBCs from patients with SCD in vitro, in the presence or absence of enhanced oxidative stress. The observed small increase of intracellular Ca2+ upon drug treatment may be a result of increased overall cellular metabolic activity, as a consequence of PKR activation. Interestingly, the increased intracellular Ca2+ is not accompanied by enhanced RBC senescence. Overall, these data support testing PKA in prospective clinical trials for the reduction of vaso-occlusive crisis in patients with SCD. Bibliography: (1): Decreased activity and stability of pyruvate kinase in sickle cell disease: a novel target for mitapivat therapy. Rab MAE, Bos J, van Oirschot BA, van Straaten S, Kosinski PA, Chubukov V, Kim H, Mangus H, Schutgens REG, Pasterkamp G, Dang L, Kung C, van Beers EJ, van Wijk R.Blood. 2021 PMID: 33690814. (2): Pharmacodynamic effects of AG-946, a highly potent next-generation activator of pyruvate kinase, in ex vivo treatment of red blood cells from sickle cell disease patients. Rab, MAE, Van Dijk MJ, Van Oirschot BA, Gerrits J Kosinski PA, Kung C, Jans JJM, Van Beers EJ, Dang L and Van Wijk R. 2021 American Society of Hematology Congress, Abstract 113. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Untargeted Metabolomics on Dried Blood Spots of Patients with Sickle Cell Disease Treated with the Pyruvate Kinase Activator Mitapivat

Background Sickle cell disease (SCD) is one of the most common inherited red blood cell (RBC) disorders. The disease is characterized by polymerization of sickle hemoglobin (HbS) upon deoxygenation resulting in, among other things, chronic life-threatening complications. The pathophysiology of SCD is multifactorial and highly complex. Novel anti-sickling agents under development for SCD include oral, small molecule pyruvate kinase activators such as mitapivat (AG-348). Mitapivat could reduce HbS polymerization by decreasing the level of the glycolytic intermediate metabolite 2,3-diphosphoglycerate (2,3-DPG), thereby increasing the oxygen affinity of hemoglobin, and the level of adenosine triphosphate (ATP) to increase cellular energy. We investigated, as an exploratory outcome, changes in untargeted metabolomics on dried blood spots (DBS) of patients with SCD treated with mitapivat in the 8-week Dose Finding Period of an investigator-initiated, open label, phase 2 study (EudraCT 2019-003438-18, ESTIMATE study). Methods Untargeted metabolomics was performed by direct-infusion high-resolution mass spectrometry on DBS of patients ≥16 years with SCD (HbSS, HbS/β0 or HbS/β+-thalassemia) collected at baseline and at completion of the 8-week Dose Finding Period. In this period, patients received initial dosing of 20 mg mitapivat twice daily with a maximum of two sequential dose escalations to 50 mg and 100 mg twice daily every two weeks depending on safety. DBS samples obtained from healthy adults were used to calculate Z-scores. Data analyses were conducted in MetaboAnalyst (v5.0) without data filtering or normalization. Distinct metabolic profiles between baseline samples of patients with SCD and healthy controls were identified by multivariate principal component analysis and partial least square discriminant analysis, as well as independent two-sample t-tests with equal group variance. False Discovery Rate (FDR) adjusted p-values were used to correct for multiple testing. A significance level of 0.05 was applied. Distinctive increased or decreased metabolites were also analyzed at treatment week 8 and compared to baseline in the cohort of patients with SCD. Additional analyses were performed in Graphpad Prism (v9.3.0) including paired two-sample t-tests for individual Z-scores of features. Results In total, 1907 unique metabolite features were identified in DBS of 9 patients with SCD (n=7 HbSS, n=1 HbS/β0, n=1 HbS/β+-thalassemia; median age 22 years (range 16-59 years); 6/9 (67%) female) and 29 healthy controls (median age 38 years (range 25-65 years); 21/29 (72%) female). One HbSS patient and one healthy control were determined as outliers and excluded from final analysis. A total of 55 metabolites were significantly different between patient samples at baseline and healthy control samples. The top 35 are shown in a heatmap (Figure 1) and include increased acyl carnitines, (derivatives of) polyamines, purines and pyrimidines, and decreased carbohydrates and benzenoids. Patient samples showed an increase in 8/55 metabolites, of which 5 towards normalization, after 8-week treatment with mitapivat (unadjusted p-value <0.05), and no significant decreases (Table 1). In patients a significant increase was seen in 2/1907 unique metabolite features after 8 weeks of treatment with mitapivat compared to baseline: butenylcarnitine, which is an acyl carnitine, and inosinic acid, or inosine-5-monophosphate (5'-IMP), which is a purine nucleotide (FDR-adjusted p-value <0.05; data not shown). Conclusion Patients with SCD showed a distinct metabolic profile compared to healthy controls. The distinctions were determined by metabolites produced by RBC-related pathways including the glycolytic and purinergic signaling pathway that mediate hypoxic metabolic reprogramming, carnitine derivatives, involved in turnover and repair of the RBC membrane, and polyamines, that probably stabilize the RBC membrane (as reported from other hereditary hemolytic anemias). Moreover, after 8 weeks of treatment with mitapivat showed, again, changes in an acyl carnitine and purine nucleotide compared to baseline. We conclude that the distinct metabolic profile from healthy controls is a promising starting point to further unravel the underlying pathophysiology of SCD and to characterize the endogenous and exogenous effects of pyruvate kinase activators and other novel therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evaluation of Red Blood Cell Metabolism in Patients with Low-Risk Myelodysplastic Syndrome (LR-MDS): A Proof-of-Concept Study

Low risk myelodysplastic syndromes (MDS) show high clinical heterogeneity ranging from mild anemia with nearly normal performance status to life-threatening cytopenias, frequent hospitalization, and disability. Anemia occurs in 80%-85% of patients and therapeutic strategies are still very limited mainly consisting of of erythropoiesis stimulating agents and red blood cell (RBC) transfusion support. Starting from the working hypothesis that the defective maturation of hematopoietic precursors may dampen the "functionality" of MDS-RBCs, we aimed at evaluating RBC metabolic pathway in anemic patients with LR-MDS. We focused on the glycolytic pathway, which is the prominent metabolic cascade in RBCs and is currently targeted with allosteric stimulators of pyruvate kinase (PK) enzyme in clinical trials of PK deficiency, thalassemia, and sickle cell diseases, which are also marked by ineffective erythropoiesis like MDS. Sixty-six consecutive anemic patients (i.e. Hb<11 g/dL) diagnosed with MDS according to WHO 2016 classification that met IPSS-R criteria for very low, low, or intermediate-risk MDS were enrolled in the study. All patients were sampled for peripheral blood at least 4 weeks after the last RBC unit to reduce possible blood donor contamination. The determination of activities of the glycolytic RBC enzymes pyruvate kinase (PK) and hexokinase (HK) (the terminal and initial steps of the glycolytic pathway), and of ATP levels was performed by standard spectrophotometric method according to Beutler et al, 1984. 2,3 DPG determination was tested by AssayGenie Kit in a subgroup of patients. Ektacytometric analysis by LoRRca MaxSis (Laser-Assisted Optical Rotational Cell Analyzer, Mechatronics, NL) was also performed in a subgroup of patients. As shown in table 1, patients were mainly elderly males, and mostly belonged to IPSS low (78%) and IPSS-R very low/low groups (85%). Median levels of PK activity were 13.7 IU/gHb, with a wide range (7.5-35.9) and were reduced in 23 cases (35%; normal reference range 11.9-16.7 IU/gHb); median values of HK activity were 2.3 (0.8-8.1), increased in 60 patients (91%; normal reference range 0.78-1.32 IU/gHb). This resulted in an abnormally reduced PK/HK ratio (median 5.45, range 2-15) in 63 subjects (95%; (normal reference range calculated on 167 normal subjects: 11.55 - 17.65). To asses if impaired glycolysis may result in altered RBC deformability or in lack of energy, ATP levels, 2,3 DPG, and ektacytometric curve were evaluated. Median ATP levels were 3.4 mcM/gHb (1.3-6.5), reduced in 33/56 tested patients (59%; normal reference range 3.6-4.9 mcM/gHb), of whom 32 had simultaneous reduction of PK/HK activity ratio (figure 1), and 11 decreased PK activity. Concerning 2,3 DPG levels preliminarily analysed in a subgroup of patients, they were close to the upper limit of the normal range (median, 389 nM/gHb, 318-639). Osmoscan, performed in 10 patients did not show altered RBC deformability. Preliminary data show decreased glycolytic activity in RBCs from patients with LR-MDS highlighted by reduced PK activity in more than 1/3 of cases, and increased HK activity in almost all subjects. This results in reduced RBC ATP content in up to 2/3 of patients (possibly due to reduced production or to increased consumption) indicating an overall lack of energy in MDS-RBCs. The latter may be an interesting target for allosteric PK stimulators and warrants further investigations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Feasibility of Near-Infrared Spectroscopy for Monitoring Hemodynamic Changes in Patients with Sickle Cell Disease Treated with Mitapivat

Background: Continuous inflammation and vasculopathy underlies the pathology of sickle cell disease (SCD). Therapeutics intervening the different stages in the pathophysiological cascade - polymerization of hemoglobin S (HbS) and "sickling" of the red blood cells (RBCs), vaso-occlusion and inflammation- are under development. Sickled RBCs initiate vaso-occlusion and have altered biomechanical properties that impact the rheology and hemodynamics in patients with SCD. There is an unmet need for robust, compact technologies that can non-invasively characterize the microvascular hemodynamics in SCD. Such a tool would be a valuable adjunct for clinicians both for broadening understanding of as well as monitoring therapeutic changes. However current clinical approaches can be time- and resource- intensive which limit their utility. Near-infrared spectroscopy (NIRS) is a non-invasive, optical technique for quantifying microvascular hemodynamics including hemoglobin concentration, tissue oxygen saturation, and blood flow (Cerussi et al, 2005). Here, we evaluate the sensitivity of NIRS to hemodynamic changes in patients with SCD undergoing treatment with Mitapivat (AG-348), a pyruvate kinase activator under clinical development to determine how well NIRS parameters compare with standard hematology markers. Methods: We studied patients currently enrolled in a Phase I/II clinical trial (ClinicalTrials.gov, NCT04610866) that evaluates the long-term safety and tolerability of Mitapivat in the SCD population. We performed tissue hemodynamic assessments by affixing NIRS probes to the forearm and forehead of the subjects at baseline (prior to drug initiation) and over the course of three months of treatment with Mitapivat totaling five measurement visits per patient. The optical hemodynamic assessment consisted of resting baseline measurements followed by a brachial cuff occlusion and a recovery period. We acquired optical measures of hemodynamic markers, such as the concentration of oxy- and deoxy- hemoglobin and tissue oxygen saturation (StO2). Additionally, we calculated measures of vascular reactivity by analyzing the post-occlusion reactive hyperemic response. These metrics are compared for each patient across time to determine if there are any significant changes compared to baseline. Optical metrics were compared against changes in blood hemoglobin (Hb) level, an expected therapeutic effect of Mitapivat (Xu et al, 2022). Nine patients with complete NIRS data at baseline and spanning 3 months of Mitapivat were analyzed. Results: We calculated optical hemodynamic metrics for all 45 measurement visits; one visit was excluded due to poor NIRS data quality. Figure 1A shows a typical measurement setup with NIRS probes acquiring simultaneous measurements from the forehead and forearm, which reflect tissue hemodynamics in the prefrontal cortex and skeletal muscle respectively. Figure 1B compares the mean Hb level, mean forehead StO2, and mean forearm StO2 across the first three months of treatment. On average, forehead StO2 acquired by NIRS trended similarly to the Hb level acquired by blood draws over the first three months. We observed that 7/9 patients exhibited a sustained increase in blood Hb compared to baseline; the mean Hb increase for all patients compared to baseline was 1.2 g/dL at the end of three months. Similarly, forehead StO2 measured by NIRS also showed a similar, sustained increase. The mean forehead StO2 increase after three months compared to baseline was 4.0%. Discussion: We have demonstrated that NIRS could be a promising adjunct for non-invasively monitoring hemodynamics in patients with SCD undergoing treatment with Mitapivat. NIRS can provide objective measures of tissue composition and metabolism and vascular reactivity which can be performed in a simple, non-invasive measurement. Future analysis will include analyzing optical metrics of vascular reactivity and blood flow. While this initial cohort is small, the preliminary results are encouraging and warrant continued study into NIRS as a tool for monitoring hemodynamic changes promoted by other therapies for SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

AG-946 Normalizes Glycolysis and Improves Red Cell Indices in a Humanized Sickle Cell Mouse Model

We have previously demonstrated that enzymes of the glycolysis pathway and deoxy hemoglobin (deoxyHb) reversibly bind to the cytoplasmic domain of Band 3 (CdB3). For these studies we generated 3 transgenic mice in which the endogenous mouse Band 3 was replaced with: i) human wild type CdB3 (WT); ii) human CdB3 lacking the deoxyHb binding site (Hb-); iii) human CdB3 with high-affinity binding site for deoxyHb (Hb+). In the humanized WT strain, glycolytic enzymes (including fructose-1,6-biphosphate [FBP], dihydroxyacetone phosphate/ glyceraldehyde-3-phosphate [G3P] and 3-phosphoglycerate/2-phosphoglycerate [PG]) bind to CdB3 in oxygenated conditions and are displaced by the binding of deoxyHb in deoxygenated conditions. Erythrocytes from the mutant lines were insensitive to oxygen concentration (Chu et al. Blood 128, 2016, Zheng et al. JBC 294, 2019, Zhou et al. Sci. Adv. 5, 2019), showing that CdB3 constitutes a molecular switch regulating assembly of glycolytic enzymes on the erythrocyte membrane based on the oxygenation state. To study if this mechanism plays a role in Sickle Cell Disease (SCD), we crossed the humanized Band 3 mouse strains to the Townes SCD mouse model. -Hb_SS mice had a significantly higher percentage of sickled cells and a higher rate of sickling compared to WT_SS animals. ++Hb_SS mice showed a decrease in the percentage of sickled cells and the rate of sickling. We hypothesized that the inability of the glycolytic enzymes to reversibly bind to CdB3 in the -Hb mice inhibited glycolysis. To test this hypothesis, we analyzed a panel of 28 cellular metabolites in each genotype (WT_AA, _AS, and _SS; -Hb_AA, _AS, _SS; and ++Hb_AA, _AS, _SS) using an API 4500 triple quadrupole mass spectrometer (AB Sciex), with a polymeric amino column (apHera by Supelco) with stable isotope spike in controls allowing the absolute quantification of each metabolite. Consistent with the constitutive binding of the terminal glycolytic enzymes to CdB3 in -Hb erythrocytes, glycolysis was inhibited, as evidenced by significant accumulation of the intermediates at the top of the glycolysis pathway, including FBP, G3P and PG (p values all <0.01) compared to WT cells. In the ++Hb mutant where the terminal glycolytic enzymes are constitutively displaced from CdB3, significantly lower levels of FBP, G3P and PG compared to WT cells were observed (p values all <0.01). AG-946 is a novel small-molecule activator of erythrocyte pyruvate kinase that mediates the final step of glycolysis yielding 2 ATP and 2 pyruvate molecules per glucose molecule. We hypothesized that treatment of the -Hb-SS animals treated with AG-946 would reduce the levels of the glycolytic intermediates by accelerating the terminal stage of glycolysis. We designed a pilot study in which -Hb-SS mice were fed control chow for 4 weeks and then randomly separated into a group receiving control chow and a group receiving AG-946 at an approximate dose of 10 mg/kg/day. After 8 weeks on study, blood was collected from treated and untreated animals for analysis of complete blood counts, plasma levels of AG-946, the levels of 28 metabolites, intracellular 2,3-DPG, and the rate and degree of sickling. AG946 was well tolerated, all mice in both arms of the study gained weight over the 8 weeks. At the end of the study, the plasma levels of AG946 in the treated animals ranged from 736 to 2281 nM. In the treated animals we observed a complete normalization of the levels of the glycolytic intermediates and 2,3-DPG while the levels in the untreated group were unchanged. Treated animals showed significant increases in the red blood cell counts (from 4.6 + 0.52 in the control group to 7.07 + 0.21 1012/L in the treated group), hematocrit (from 24.7 + 1.8% to 33 + 1.8%) and hemoglobin (6.9 + 0.8 to 8.9 + 0.3 g/dL; p values all <0.016). We observed significant decreases in the MCV (53.5 + 2.3 to 46.6 + 1.1) and MCH (14.9 + 0.05 to 12.6 + 0.25; p values all <0.01). These differences did not correlate with the plasma levels of AG-946 indicating that levels of 736 nM are sufficient to correct the block in glycolysis and improve the red cell indices. No differences were observed in MCHC or WBC, or in the rate or degree of sickling. We conclude that AG-946 treatment effectively enhances glycolysis in humanized Band 3/SS mice. The normalization of glycolytic intermediates is accompanied by increases in critical red cell indices (RBC, HCT, Hb, and MCV). AG946 may be an effective treatment for sickle cell disease.

Activating Pyruvate Kinase Improves Red Blood Cell Integrity By Reducing Band3 Tyrosine Phosphorylation

Background: The scientific rationale for mitapivat (AG-348) as an anti-sickling therapy is that it enhances red cell pyruvate kinase (PKR) activity and glycolytic flux, leading to a reduction of intracellular 2,3-diphosphoglycerate (2,3-DPG) and a concomitant increase in adenosine triphosphate (ATP). Reducing 2,3-DPG destabilizes hemoglobin S (HbS) fibers, while increasing ATP improves red blood cell (RBC) integrity and cellular hydration. Mitapivat increased ATP and decreased 2,3-DPG levels in a dose-dependent manner in this Phase 1 study (Xu et al, 2022). The Hb increase was sustained after stopping mitapivat until end of study (EOS) at 4 weeks, suggesting an improvement in RBC survival. However, pathways modulated by mitapivat that may contribute to sickle RBC survival remain largely unknown. Mitapivat has been shown to improve oxidative stress by increasing GSH/GSSG ratio (Matte et al 2021). In SCD, elevated oxidative stress inhibits RBC tyrosine (Tyr) phosphatases that normally maintain the RBC Band3 (Bd3) tyrosines in an unphosphorylated state. Tyr-phosphorylation of Bd3 destabilizes membrane integrity and reduces RBC deformability (Noomuna et al, 2020). We hypothesize that PKR activators, such as mitapivat and AG-946 (a next-generation activator of PKR), improve the membrane integrity via regulating Tyr-phosphorylation of Bd3. Methods: Of the 16 patients in the Phase1 study, frozen whole blood samples were evaluable in 15, from which RBC ghosts (membranes) at the different timepoints (baseline and following 2 weeks of treatment at each dose level) were isolated. Tyr-phosphorylation of Bd3 was analyzed by Western blotting using a specific phospho-Tyr Bd3 antibody. Ex-vivo experiments were conducted by treating RBCs from different HbSS patients with different concentrations (1 µM, 3 µM, 10 µM, and 30 µM) of mitapivat or AG-946, for 4 hours at 37°C. To investigate the effect of mitapivat on kinases and phosphatases, HbSS RBCs were treated with different concentrations of mitapivat in a combination or without Syk kinase inhibitor-fostamatinib (MedChemExpress). We measured the effect of mitapivat on ATP production in-vitro by treating HbSS RBCs with 30 µM mitapivat for 2h, 4h, 6h, and 8h. The ATP level was detected using CellTiter-Glo® 2.0 Cell Viability Assay (Promega). Meanwhile, the same samples were used to explore the effect of mitapivat on the expression of intact protein tyrosine phosphatase 1B (PTP1B) on the RBC membrane. Results: A dose-dependent decrease in mean Bd3 Tyr-phosphorylation of 20%, 34%, 42%, and 42%, from baseline was observed in the 15 subjects, corresponding to ascending BID dose levels of 2 weeks mitapivat 5 mg, 20 mg, 50 mg, 100 mg, respectively; followed by a return to near baseline by EOS (Figure 1A). Consistent with the in-vivo data, ex-vivo treated HbSS RBCs also showed a dose-dependent decrease in Bd3 Tyr-phosphorylation after either mitapivat or AG-946 treatment (Figure 1B, C). Interestingly, combination of mitapivat with fostamatinib showed an enhanced effect on the reduction of Bd3 Tyr-phosphorylation compared with single drug treatment, which indicated that mitapivat may affect the activity of PTP1B (Figure 1D). Since the activity of PTP1B could be regulated by ATP, we then investigated the effect of mitapivat on ATP level. The ATP level was elevated after 6 hours' mitapivat treatment, even after correction for cell number (Figure 1E). Concomitantly, the intact PTP1B associated with cell membrane also increased after 6 hours of mitapivat treatment (Figure 1F), which might explain the de-phosphorylation of Bd3. Conclusion: A recent study showed that PTP1B is the major phosphatase that maintains Bd3 tyrosine in an unphosphorylated state (Noomuna et al, 2022). PTP1B could be cleaved into an inactive form by calpain, which needs to be activated by calcium. The balance of calcium in RBC relies on the activity of the plasma membrane Ca2+ ATPase (PCMA) pump, which is fueled by ATP. Our results suggest that in enhancing PKR activity, mitapivat leads to an increase in intracellular ATP which increases activity of the PCMA pump, and an increased efflux of intracellular calcium. A reduction in intracellular Ca2+ reduces the activity of calpain, and thereby suppresses calpain's cleavage of phosphatase-PTP1B, resulting in reduction of Bd3 Tyr-phosphorylation. ATP increase is thus a key mechanism of mitapivat's beneficial effect on the hemoglobin increase in SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Fetal Hemoglobin Induced Improvement in Relative Age of Sickle Red Blood Cells Determined through Glycated Hemoglobin Assessment By Flow Cytometry

Introduction: Sickle cell disease (SCD) is characterized by acute vaso-occlusive events and chronic hemolytic anemia, originating from the polymerization of the abnormal hemoglobin S (HbS) under low oxygen tension. Hemolysis is defined as an imbalance between the rate of red blood cell (RBC) production and destruction. It results in an overall reduced lifespan of RBCs, as they are prematurely removed from the circulation. While the normal RBC lifespan is approximatively 120 days, those of SCD is only a sixth of it (Hebbel, 2011). Fetal Hb (HbF) is the main clinical modulator of SCD severity by inhibiting HbS polymerization. Its protective effect relies on its quantity and distribution among the RBC population. However, HbF is often heterogeneously distributed in RBC from SCD patients. Among the numerous improvements triggered by HbF, a significant increase in RBC lifespan has been measured in high-HbF containing cells, as compared to low-HbF containing cells (Franco et al., 2006). As they age, normal RBCs exhibit physiological changes, such as dehydration, a decrease of the intracellular enzymes activities (pyruvate kinase and hexokinase) and an increase in Hb concentration. Glycated Hb (the most abundant being HbA1c form) also increases, as RBC age. The glycation of Hb is a non-enzymatical reaction that is affected by the plasmatic concentration of glucose and the time RBCs last in the circulation. In non-diabetic subjects, the Hb glycation only depends on the RBC lifespan. To date, current methods to assess RBC lifespan are invasive and time consuming, requiring infusion of autologous labeled RBCs and long follow-up. Objectives: This present work aims at 1/ evaluating the RBC relative age of SCD patients using glycated Hb measured by flow cytometry and 2/ estimating the HbF-induced increase in RBC lifespan of SCD patients. Methods: Glycated Hb was measured and compared between five healthy donors (AA), seven non-diabetic and non-transfused homozygous SCD patients (SS) and five non-diabetic, transfused SS patients. Total glycated Hb was measured by reference biochemical methods, cation-exchange high-pressure liquid chromatography or rapid glycohaemoglobin immunoassay (DCA test) used for AA-RBC and SS-RBC, respectively. The single cell glycation was assessed by flow cytometry using mouse anti-human HbA1c antibody. HbA, HbS and HbF were assessed by flow cytometry using anti βA-globin, anti βS-globin and anti g-globin antibodies, respectively. Results: The glycated Hb was significantly lower in SS patients than in healthy donors (glycated HbS =3.7%, glycated HbA =5.3%, p=0.0025 by DCA and HPLC respectively, and by flow cytometry p=0.0061). In transfused SS patients, results showed that glycated Hb was lower in autologous SS-RBCs than in AA-RBCs by a factor of four (P= 0.008), thus confirming the characterized reduced lifespan of SS RBCs. Glycated Hb levels were measured in RBC sub-populations based on their HbF level (high HbF or low HbF subfractions) in non-transfused SS patients. Glycated Hb level was lower in low HbF RBCs, as compared to high HbF RBCs (P=0.0023). This difference was not detected in healthy donors, thereby indicating that HbF has an effect on relative age only in SCD patients. Conclusion: Single cell glycated Hb assessed by flow cytometry allows to simultaneously estimate the relative age of different RBC sub-populations. This new tool is noninvasive and requires only one single time point measurement. It should also be considered to evaluate the efficacy of treatments that aim at improving hemolysis. Figure. Glycated Hb levels assessed by HPLC and DCA biochemical methods, and flow cytometry in different participant groups and RBC subpopulations. Results were expressed as median, percentage or mean fluorescence intensity (MFI) when appropriate. Glycated Hb level was compared between two groups using a nonparametric Mann-Whitney t-test. (A) Total glycated Hb measured by HPLC in AA healthy donors (n=5) and by DCA test in SS patients (n=7). (B) Single cell glycated Hb measured by flow cytometry in SS RBCs (n=7), compared to AA healthy donors (n=4). (C) Single cell glycated Hb measured in low HbF RBCs (n=7) and high HbF RBCs (purple, n=7) subpopulations in SS patients and AA healthy donors (n=4). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mitapivat Improves Markers of Erythropoietic Activity in Long-Term Study of Adults with Alpha- or Beta-Non-Transfusion-Dependent Thalassemia

Background: The thalassemias comprise a group of hemoglobinopathies characterized by ineffective erythropoiesis and hemolysis, which lead to chronic anemia and associated complications. Thalassemic red blood cells (RBCs) do not have sufficient levels of adenosine triphosphate (ATP) to meet their increased energy demands. Mitapivat is an allosteric activator of pyruvate kinase (PK), a key enzyme that regulates the final step of glycolysis responsible for ATP production in RBCs. We previously reported primary results of an open-label phase 2 study (NCT03692052) of mitapivat in pts with α- or β-non-transfusion-dependent thalassemia (NTDT) showing 80.0% (16/20) of pts achieved a hemoglobin (Hb) response of ≥1.0 g/dL increase from baseline at ≥1 assessments between Weeks (Wk) 4-12, inclusive. The increase in Hb was sustained, with a mean Hb (standard deviation) increase of 1.7 g/dL (0.5) at Wk 72; hemolysis markers were also improved. This analysis (data cutoff 27Mar22) evaluated the effect of mitapivat on markers of erythropoietic activity through Wk 72, along with Hb and markers of hemolysis and iron homeostasis. Methods: Eligibility criteria: ≥18 years (y) of age, known medical history of α- or β-thalassemia, Hb concentration ≤10.0 g/dL, and ≤5 RBC units transfused in the prior 24 wk and none in the 8 wk prior to study drug. Pts started mitapivat 50 mg twice daily (BID) and escalated to 100 mg BID based on individual safety and Hb assessments. Pts who completed the 24-wk core period and achieved a Hb response or a delayed Hb response (after Wk 12) were eligible to continue to the extension period (at Wk 24 dose) if they had no ongoing grade ≥3 treatment-emergent adverse events related to study drug. Study visits during the extension occur every 12 wk for up to 3 y. Assessments collected within 8 wk (56 days) after a transfusion were excluded from baseline derivation and analysis. Results: Nineteen of 20 pts completed the core period, 17 entered the extension period; 16 pts (4 with α- and 12 with β-thalassemia) were ongoing at the data cutoff date. At baseline, pts had biomarker levels consistent with ineffective erythropoiesis and hemolysis (Table). Changes from baseline showed a durable increase in Hb with a median (Q1, Q3) of 1.3 g/dL (0.60, 1.90) at Wk 12 and 1.2 g/dL (-0.03, 2.2) at Wk 72 (Table). Concurrently, there were trends for decreases in medians for erythropoietin, erythroferrone, soluble transferrin receptor, indirect bilirubin, and reticulocyte/erythrocyte fraction that were maintained over time. Median hepcidin levels remained relatively stable. Conclusions: These results indicate that mitapivat's mechanism ameliorates multiple aspects of the complex pathophysiology underlying α- and β-NTDT, including ineffective erythropoiesis, hemolysis, and anemia. The sustained long-term effects of mitapivat may offer a novel disease-modifying approach. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Long-Term Improvements in Patient-Reported Outcomes in Patients with Pyruvate Kinase Deficiency Treated with Mitapivat

Background: Pyruvate kinase (PK) deficiency is a rare, hereditary hemolytic anemia caused by a single gene defect encoding the red blood cell (RBC)-specific form of PK. This condition is associated with acute and long-term complications, as well as a spectrum of signs and symptoms including jaundice, fatigue, and dyspnea, that have a profound, wide-ranging impact on health-related quality of life (HRQoL). Patients (pts) report physical limitations, negatively impacted social functioning, reduced self-esteem, and concerns for the future. Mitapivat, a first-in-class, oral, allosteric activator of PK, has demonstrated improvement in hemoglobin, hemolysis, and transfusion burden in pts with PK deficiency, and is approved by the US Food and Drug Administration for the treatment of hemolytic anemia in adults with PK deficiency. In addition, improvements in patient-reported outcomes (PROs) (measured by disease-specific PRO instruments: the PK deficiency diary [PKDD] and the PK deficiency impact assessment [PKDIA]) were observed in two global, phase 3 clinicals trials among adults with PK deficiency treated with mitapivat who were not regularly (ACTIVATE; NCT03548220) or regularly transfused (ACTIVATE-T; NCT03559699). Here, we describe PKDD and PKDIA outcomes up to Week (Wk) 84 of the long-term extension (LTE) study (NCT03853798) of mitapivat in pts with PK deficiency previously enrolled in ACTIVATE and ACTIVATE-T. Methods: In the placebo (PBO)-controlled ACTIVATE trial, pts were randomized 1:1 to receive mitapivat (5/20/50 mg twice daily) or PBO for 24 wks. In the open-label, single-arm ACTIVATE-T trial, pts were treated with mitapivat for 40 wks. Pts who completed either trial were eligible to continue in the LTE, where all pts received mitapivat. Pts from ACTIVATE were categorized into either the mitapivat-to-mitapivat arm (M/M) or the PBO-to-mitapivat arm (P/M). Pts from ACTIVATE-T continued to receive mitapivat (M). Two PK deficiency-specific PRO instruments were used: the PKDD is a 7-item measure of the core signs and symptoms of PK deficiency, and the PKDIA is a 14-item measure of the impacts of PK deficiency on pts' HRQoL. In the ACTIVATE/LTE and ACTIVATE-T/LTE analyses, changes from baseline (BL; defined as the last complete assessment before start of study treatment in ACTIVATE/ACTIVATE-T) in PKDD and PKDIA mean scores were assessed at scheduled visits up to Wk 84 of the LTE for each of the 3 treatment arms. For both PRO instruments, a lower score represents a lower disease burden, and the minimal clinically important change is defined as a reduction of 4.2 and 5.5 in PKDD and PKDIA scores, respectively. Changes in PKDD and PKDIA scores were summarized descriptively using mean and SD. Results: In total, 80 pts were included in the ACTIVATE/LTE analysis (M/M=40; P/M=40) and 27 pts were included in the ACTIVATE-T/LTE analysis. At Wk 84 of the LTE study (ie, Wk 108 for ACTIVATE pts; Wk 124 for ACTIVATE-T pts), mean (SD) change from BL in PKDD score was ‒7.19 (6.740) for M/M pts, ‒4.58 (5.758) for P/M pts, and ‒3.94 (14.087) for M pts; mean (SD) change from BL in PKDIA score was ‒6.3 (7.12) for M/M pts, ‒6.3 (9.09) for P/M pts, and ‒11.5 (11.53) for M pts. Notably, P/M pts experienced improvements in PKDD and PKDIA mean scores after starting mitapivat in the LTE that were consistent with initial improvements seen in the mitapivat-treated group in the ACTIVATE trial. Across both PRO instruments, improvements among mitapivat-treated pts were sustained over time in the LTE (Figures 1A and 1B). Conclusions: Treatment with mitapivat was associated with long-term, durable, and clinically meaningful improvements in signs, symptoms, and functional impacts based on disease-specific PRO instruments, irrespective of transfusion status. These results suggest that by improving HRQoL, treatment with mitapivat may provide meaningful pt-centric benefits. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Pyruvate Kinase Activator Mitapivat Improves Red Blood Cell Deformability and Sickling Kinetics in Adult Patients with Sickle Cell Disease

Background: A hallmark of sickle cell disease (SCD) is compromised red blood cell (RBC) deformability. Polymerization of hemoglobin S (HbS) underlies this "sickling" which results in altered biomechanical properties that impact rheology and hemodynamics (Barabino et al, 2010). There have been exciting clinical developments in anti-sickling therapies - including enhancement of red cell pyruvate kinase (PKR) modality. Importantly, deformability measurements of RBCs via ektacytometry are increasingly used to aid the assessment of their therapeutic efficacy. Here we employed the Laser assisted Optical Rotational Red Cell Analyzer (LORRCA, RR Mechatronics), which permits the evaluation of RBC deformability as a function of shear stress, osmotic pressure, and oxygen tension, to evaluate the changes in RBC deformability in subjects undergoing treatment with mitapivat (AG-348), a PKR activator under clinical development for sickle cell disease. We analyzed changes in deformability measurements of RBCs in HbSS subjects after 4 weeks and 12 weeks of mitapivat therapy and compared them to t50 - a single parameter measure of sickling kinetics (Dunkelberger et al, 2018). Methods: We studied subjects currently enrolled under IRB approved clinical trial NCT04610866 that evaluates the long-term safety and tolerability of mitapivat in SCD patients. Fifteen subjects (aged 25 - 57 years; 5 females, 10 males; African/African-American) were evaluated, though not all endpoints were available for every patient across all assays. Fresh whole blood (collected in EDTA tubes) was obtained at baseline (prior to drug initiation), after 4 weeks and after 12 weeks of therapy. Cells were counted and diluted in polyvinylpyrrolidone (PVP) solution before undergoing same-day LORRCA assays. The Elongation Index (EI) is a measure of the cells' ability to undergo deformation at different physiologically relevant conditions. EI was assessed at increasing shear stresses of .3 Pascals (Pa), .95 Pa, 3 Pa, 9.49 Pa, and 30 Pa (elongation assay). Parameters derived under continuous osmotic changes (0-600mOsm/kg - osmoscan) include O min which informs on RBC fragility, O Hyper which informs on hydration state and corresponds to 50% of the maximum deformability in the hypertonic region, EI max (osmo) which reveals maximum deformability and informs on RBC cytoskeletal mechanics, and O Max which gives the osmolality at which EI Max is achieved. Point of Sickling (POS) gives the oxygen tension at which the cells start to sickle during nitrogen facilitated deoxygenation of the sample (oxyscan). These parameters of deformability are generated by LORRCA software. For all assays, we analyzed the % change from baseline after 4 weeks and 12 weeks of therapy (Wilcoxon signed rank test, R v1.4.1106) and compared these changes to those in t50 (Spearman correlation test, Prism v9) (Figure 1). Results: Mitapivat therapy resulted in increased elongation indices across all shear stress levels, with significance reached at shears of .3 Pa, .95 Pa, and 3 Pa (respective mean % changes of 78.5, 181.8, and 23.0 (4 weeks) and 101.2, 212.9, and 30.1 (12 weeks)) suggesting improved deformability of RBCs. Likewise, significant increases in O hyper (mean % changes of 2.8 and 3.4) and osmo EI Max (mean % changes 7.9 and 9.5) respectively, indicate improvements in RBC hydration and cytoskeletal dynamics in response to changing osmotic pressures. Furthermore, a significant decrease in the average point of sickling was noted at both 4 weeks and 12 weeks (-20.15% and -16.8%, respectively), suggesting RBCs were susceptible to sickling at partial pressures of oxygen lower than those observed before therapy. Conclusion: Altogether, these findings indicate that mitapivat therapy results in improved ability of RBCs to withstand deformational changes in response to shear stress, osmotic pressure, and decreasing oxygen tension, likely via bolstered membrane integrity. Interestingly, when compared to t50 values, significant correlation was observed at all levels of shear stress and at the maximal deformability (EI max) achieved during both the osmoscan and oxyscan assays. Acknowledgement: This study is part of a Cooperative Research and Development Agreement (CRADA) with AGIOS Pharmaceuticals, Inc., Cambridge, MA, United States Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal