A pharmacokinetic-pharmacodynamic analysis of l-glutamine for the treatment of sickle cell disease: Implications for understanding the mechanism of action and evaluating response to therapy.

The mechanisms of action of l-glutamine for the treatment of sickle cell disease (SCD) are not well understood and there are no validated clinical biomarkers to assess response. We conducted a three-week, dose-ascending trial of glutamine and measured the pharmacokinetic (PK) exposure parameters, peak concentration (Cmax) and area under the curve (AUC). We used a panel of biomarkers to investigate the pharmacodynamics (PD) of glutamine and studied PK-PD relationships. There was no plasma accumulation of glutamine, glutamate, arginine or other amino acids over time, but modestly improved arginine bioavailability was observed. In standard analysis by dose levels over time, there were no measurable effects on blood counts, viscosity, ektacytometry or reactive oxygen species (ROS). In PK-PD analysis, however, higher glutamine exposure (Cmax or AUC) was associated with increased whole blood viscosity and cellular dehydration, yet also with higher haemoglobin concentration, increased haematocrit-to-viscosity ratio, decreased reticulocyte ROS, improved RBC deformability and decreased point of sickling. This novel PK-PD analysis identified biomarkers reflecting the positive and negative effects of glutamine, helping to elucidate its mechanisms of action in SCD. PK-optimized dosing to achieve glutamine exposure (AUC or Cmax) that is associated with salutary biological effects should be studied to support its therapeutic use.

Multicenter, phase 1 study of etavopivat (FT-4202) treatment for up to 12 weeks in patients with sickle cell disease.

ABSTRACT: Etavopivat is an investigational, once daily, oral, selective erythrocyte pyruvate kinase (PKR) activator. A multicenter, randomized, placebo-controlled, double-blind, 3-part, phase 1 study was conducted to characterize the safety and clinical activity of etavopivat. Thirty-six patients with sickle cell disease (SCD) were enrolled into 4 cohorts: 1 single-dose, 2 multiple ascending doses, and 1 open-label (OL). In the OL cohort, 15 patients (median age 33.0 years [range, 17-55]) received 400 mg etavopivat once daily for 12 weeks; 14 patients completed treatment. Consistent with the mechanism of PKR activation, increases in adenosine triphosphate and decreases in 2,3-diphosphoglycerate were observed and sustained over 12 weeks' treatment. This translated clinically to an increase in hemoglobin (Hb; mean maximal increase 1.6 g/dL [range, 0.8-2.8]), with >1 g/dL increase in 11 (73%) patients during treatment. In addition, the oxygen tension at which Hb is 50% saturated was reduced (P = .0007) with a concomitant shift in point of sickling (P = .0034) to lower oxygen tension in oxygen-gradient ektacytometry. Hemolysis markers (absolute reticulocyte count, indirect bilirubin, and lactate dehydrogenase) decreased from baseline, along with matrix metalloproteinase-9 and erythropoietin. In the OL cohort, adverse events (AEs) were mostly grade 1/2, consistent with underlying SCD; 5 patients had serious AEs. Vaso-occlusive pain episode was the most common treatment-emergent AE (n = 7) in the OL cohort. In this, to our knowledge, the first study of etavopivat in SCD, 400 mg once daily for 12 weeks was well tolerated, resulting in rapid and sustained increases in Hb, improved red blood cell physiology, and decreased hemolysis. This trial was registered at www.ClinicalTrials.gov as #NCT03815695.

Rapid measurement of hemoglobin-oxygen dissociation by leveraging Bohr effect and Soret band bathochromic shift.

Oxygen (O2) binds to hemoglobin (Hb) in the lungs and is then released (dissociated) in the tissues. The Bohr effect is a physiological mechanism that governs the affinity of Hb for O2 based on pH, where a lower pH results in a lower Hb-O2 affinity and higher Hb-O2 dissociation. Hb-O2 affinity and dissociation are crucial for maintaining aerobic metabolism in cells and tissues. Despite its vital role in human physiology, Hb-O2 dissociation measurement is underutilized in basic research and in clinical laboratories, primarily due to the technical complexity and limited throughput of existing methods. We present a rapid Hb-O2 dissociation measurement approach by leveraging the Bohr effect and detecting the optical shift in the Soret band that corresponds to the light absorption by the heme group in Hb. This new method reduces Hb-O2 dissociation measurement time from hours to minutes. We show that Hb deoxygenation can be accelerated chemically at the optimal pH of 6.9. We show that time and pH-controlled deoxygenation of Hb results in rapid and distinct conformational changes in its tertiary structure. These molecular conformational changes are manifested as significant, detectable shifts in Hb's optical absorption spectrum, particularly in the characteristic Soret band (414 nm). We extensively validated the method by testing human blood samples containing normal Hb and Hb variants. We show that rapid Hb-O2 dissociation can be used to screen for and detect Hb-O2 affinity disorders and to evaluate the function and efficacy of Hb-modifying therapies. The ubiquity of optical absorption spectrophotometers positions this approach as an accessible, rapid, and accurate Hb-O2 dissociation measurement method for basic research and clinical use. We anticipate this method's broad adoption will democratize the diagnosis and prognosis of Hb disorders, such as sickle cell disease. Further, this method has the potential to transform the research and development of new targeted and genome-editing-based therapies that aim to modify or improve Hb-O2 affinity.

Diagnosis and management of pyruvate kinase deficiency: international expert guidelines.

Pyruvate kinase (PK) deficiency is the most common cause of chronic congenital non-spherocytic haemolytic anaemia worldwide, with an estimated prevalence of one in 100 000 to one in 300 000 people. PK deficiency results in chronic haemolytic anaemia, with wide ranging and serious consequences affecting health, quality of life, and mortality. The goal of the International Guidelines for the Diagnosis and Management of Pyruvate Kinase Deficiency was to develop evidence-based guidelines for the clinical care of patients with PK deficiency. These clinical guidelines were developed by use of GRADE methodology and the AGREE II framework. Experts were invited after consideration of area of expertise, scholarly contributions in PK deficiency, and country of practice for global representation. The expert panel included 29 expert physicians (including adult and paediatric haematologists and other subspecialists), geneticists, laboratory specialists, nurses, a guidelines methodologist, patients with PK deficiency, and caregivers from ten countries. Five key topic areas were identified, the panel prioritised key questions, and a systematic literature search was done to generate evidence summaries that were used in the development of draft recommendations. The expert panel then met in person to finalise and vote on recommendations according to a structured consensus procedure. Agreement of greater than or equal to 67% among the expert panel was required for inclusion of a recommendation in the final guideline. The expert panel agreed on 31 total recommendations across five key topics: diagnosis and genetics, monitoring and management of chronic complications, standard management of anaemia, targeted and advanced therapies, and special populations. These new guidelines should facilitate best practices and evidence-based PK deficiency care into clinical practice.

Loss of TBC1D2B causes a progressive neurological disorder with gingival overgrowth.

Biallelic loss-of-function variants in TBC1D2B have been reported in five subjects with cognitive impairment and seizures with or without gingival overgrowth. TBC1D2B belongs to the family of Tre2-Bub2-Cdc16 (TBC)-domain containing RAB-specific GTPase activating proteins (TBC/RABGAPs). Here, we report five new subjects with biallelic TBC1D2B variants, including two siblings, and delineate the molecular and clinical features in the ten subjects known to date. One of the newly reported subjects was compound heterozygous for the TBC1D2B variants c.2584C>T; p.(Arg862Cys) and c.2758C>T; p.(Arg920*). In subject-derived fibroblasts, TBC1D2B mRNA level was similar to control cells, while the TBC1D2B protein amount was reduced by about half. In one of two siblings with a novel c.360+1G>T splice site variant, TBC1D2B transcript analysis revealed aberrantly spliced mRNAs and a drastically reduced TBC1D2B mRNA level in leukocytes. The molecular spectrum included 12 different TBC1D2B variants: seven nonsense, three frameshifts, one splice site, and one missense variant. Out of ten subjects, three had fibrous dysplasia of the mandible, two of which were diagnosed as cherubism. Most subjects developed gingival overgrowth. Half of the subjects had developmental delay. Seizures occurred in 80% of the subjects. Six subjects showed a progressive disease with mental deterioration. Brain imaging revealed cerebral and/or cerebellar atrophy with or without lateral ventricle dilatation. The TBC1D2B disorder is a progressive neurological disease with gingival overgrowth and abnormal mandible morphology. As TBC1D2B has been shown to positively regulate autophagy, defects in autophagy and the endolysosomal system could be associated with neuronal dysfunction and the neurodegenerative disease in the affected individuals.

A Population Pharmacokinetic Analysis of L-Glutamine Exposure in Patients with Sickle Cell Disease: Evaluation of Dose and Food Effects.

BACKGROUND AND OBJECTIVE: L-Glutamine is a treatment for children and adults with sickle cell disease. A comprehensive evaluation of the pharmacokinetics of L-glutamine in sickle cell disease has not been conducted. We aimed to assess the effects of long-term dosing, multiple dose levels, and food intake on L-glutamine exposure in patients with sickle cell disease compared to normal participants. METHODS: We conducted an open-label dose-ascending trial of L-glutamine in pediatric and adult participants with sickle cell disease (N = 8) and adult healthy volunteers (N = 4), providing a total of 400 plasma L-glutamine concentrations. Each participant received three ascending oral doses (0.1 and 0.3 g/kg twice daily and 0.6 g/kg once daily) over 3 weeks. Plasma L-glutamine concentrations were quantified using ion exchange chromatography. Both a non-compartmental pharmacokinetic analysis and a population pharmacokinetic analysis were performed. RESULTS: L-glutamine had rapid absorption and elimination, and there was no significant change in the baseline (pre-dose) L-glutamine concentration throughout the study, indicating no drug accumulation. Pharmacokinetics was best described by a one-compartment model with first-order kinetics. The dose-normalized peak concentration decreased with dose escalation, indicating the capacity-limited non-linear pharmacokinetics of oral L-glutamine. A covariate analysis showed that baseline L-glutamine concentrations correlated negatively with glutamine clearance, whereas dose positively correlated with volume of distribution. Food intake did not significantly affect glutamine clearance, indicating that L-glutamine can be taken with or without food. CONCLUSIONS: We report the first pharmacokinetic study of multiple-dose, long-term oral L-glutamine therapy and the first population pharmacokinetic analysis of L-glutamine for sickle cell disease. These findings may permit optimized dosing of L-glutamine for patients with sickle cell disease to maximize treatment benefits. CLINICAL TRIAL REGISTRATION: This trial is registered at ClinicalTrials.gov (NCT04684381).

Red cell abnormalities characterized by ektacytometry in children with cholestasis.

BACKGROUND: Spur-cell anemia sometimes accompanies cholestasis. We postulated that even in the absence of spur-cells, cholestasis might alter red blood cell (RBC) osmotic fragility and deformability. Therefore, we assessed these RBC measures by ektacytometry in pediatric patients. METHODS: We conducted a single center, prospective, cross-sectional investigation of RBC membrane characteristics by ektacytometry in pediatric patients with intra- and extrahepatic cholestasis followed at Cincinnati Children's Hospital Medical Center. We measured red cell membrane fragility and deformability in 17 patients with cholestasis and 17 age-matched controls without cholestasis. RESULTS: Patients with cholestasis had decreased RBC osmotic fragility compared to controls, with a significant left shift in Omin, indicating increased RBC surface-to-volume ratio. One showed spur cell morphology. However, the other 16 had no spurring, indicating that ektacytometry is a sensitive method to detect RBC membrane abnormalities. Left shift of Omin positively correlated with serum conjugated bilirubin levels and even more negatively with serum vitamin E concentration. CONCLUSIONS: This study suggests that subclinical red blood cell membrane abnormalities exist in most pediatric patients with cholestasis, increasing risk for hemolysis when subjected to oxidative stress. Hence minimizing pro-oxidants exposure and maximizing antioxidant exposure is advisable for this group. GOV IDENTIFIER: NCT05582447 https://clinicaltrials.gov/ct2/show/NCT05582447?cond=Cholestasis&cntry=US&state=US%3AOH&city=Cincinnati&draw=2&rank=2 . IMPACT: Spur cell anemia due to decreased red cell osmotic fragility and decreased deformability has been reported among patients with cholestasis. Ektacytometry is a reliable, reproducible method to measure red cell osmotic fragility and deformability. Few data describe red cell osmotic fragility or deformability in patients with cholestasis who may or may not have spur cell anemia. Ektacytometry shows that red cell osmotic fragility and deformability are decreased in many children with cholestasis even when spur cell anemia has not yet occurred. Factors associated with decreased osmotic fragility include elevated serum bilirubin, elevated serum bile acids, and decreased serum vitamin E.

Human TLR8 induces inflammatory bone marrow erythromyeloblastic islands and anemia in SLE-prone mice.

Anemia commonly occurs in systemic lupus erythematosus, a disease characterized by innate immune activation by nucleic acids. Overactivation of cytoplasmic sensors by self-DNA or RNA can cause erythroid cell death, while sparing other hematopoietic cell lineages. Whereas chronic inflammation is involved in this mechanism, less is known about the impact of systemic lupus erythematosus on the BM erythropoietic niche. We discovered that expression of the endosomal ssRNA sensor human TLR8 induces fatal anemia in Sle1.Yaa lupus mice. We observed that anemia was associated with a decrease in erythromyeloblastic islands and a block in differentiation at the CFU-E to proerythroblast transition in the BM. Single-cell RNAseq analyses of isolated BM erythromyeloblastic islands from human TLR8-expressing mice revealed that genes associated with essential central macrophage functions including adhesion and provision of nutrients were down-regulated. Although compensatory stress erythropoiesis occurred in the spleen, red blood cell half-life decreased because of hemophagocytosis. These data implicate the endosomal RNA sensor TLR8 as an additional innate receptor whose overactivation causes acquired failure of erythropoiesis via myeloid cell dysregulation.

Crosstalk between terminal erythropoiesis and granulopoiesis within their common niche: the erythromyeloblastic island.

PURPOSE OF REVIEW: The identity of the erythroblastic island (EBI) macrophage (Mϕ) has been under investigation for decades since it was recognized as the first hematopoietic niche 'nursing' terminal erythropoiesis. This review will focus on the current insights to the characteristics and the role of the EBI Mϕ balancing terminal erythropoiesis and granulopoiesis. RECENT FINDINGS: While the EBI has long been known as the niche for erythroid precursors, significant advancements in biology research technologies, including optimization of EBI enrichment protocols, single-cell ribonucleic acid sequencing, and imaging flow cytometry, have recently revealed that granulocytic precursors co-exist in this niche, termed erythromyeloblastic island (EMBI). More importantly, the balance noted at baseline between terminal granulopoiesis and erythropoiesis within EBIs/EMBIs is altered with diseases affecting hematopoiesis, such as stress erythropoiesis and inflammatory conditions causing anemia of inflammation. The role of the EMBI niche has yet to be fully investigated mechanistically, however, a notable degree of transcriptional and cell surface marker heterogeneity has been identified for the EMBI Mϕ, implicating its plasticity and diverse function. SUMMARY: Terminal erythropoiesis and granulopoiesis are regulated within the EMBI. Investigations of their balance within this niche in health and disease may reveal new targets for treatment of diseases of terminal hematopoiesis.

Navigating the marrow sea towards erythromyeloblastic islands under normal and inflammatory conditions.

PURPOSE OF REVIEW: Terminal erythroid differentiation occurs in specialized niches called erythroblastic islands. Since their discovery in 1958, these niches have been described as a central macrophage surrounded by differentiating erythroblasts. Here, we review the recent advances made in the characterization of these islands and the role they could play in anaemia of inflammation. RECENT FINDINGS: The utilization of multispectral imaging flow cytometry (flow cytometry with microscopy) has enabled for a more precise characterization of the niche that revealed the presence of maturing granulocytes in close contact with the central macrophage. These erythromyeloblastic islands (EMBIs) can adapt depending on the peripheral needs. Indeed, during inflammation wherein inflammatory cytokines limit erythropoiesis and promote granulopoiesis, EMBIs present altered structures with increased maturing granulocytes and decreased erythroid precursors. SUMMARY: Regulation of the structure and function of the EMBI in the bone marrow emerges as a potential player in the pathophysiology of acute and chronic inflammation and its associated anaemia.

Erythroblastic islands foster granulopoiesis in parallel to terminal erythropoiesis.

The erythroblastic island (EBI), composed of a central macrophage surrounded by maturing erythroblasts, is the erythroid precursor niche. Despite numerous studies, its precise composition is still unclear. Using multispectral imaging flow cytometry, in vitro island reconstitution, and single-cell RNA sequencing of adult mouse bone marrow (BM) EBI-component cells enriched by gradient sedimentation, we present evidence that the CD11b+ cells present in the EBIs are neutrophil precursors specifically associated with BM EBI macrophages, indicating that erythro-(myelo)-blastic islands are a site for terminal granulopoiesis and erythropoiesis. We further demonstrate that the balance between these dominant and terminal differentiation programs is dynamically regulated within this BM niche by pathophysiological states that favor granulopoiesis during anemia of inflammation and favor erythropoiesis after erythropoietin stimulation. Finally, by molecular profiling, we reveal the heterogeneity of EBI macrophages by cellular indexing of transcriptome and epitope sequencing of mouse BM EBIs at baseline and after erythropoietin stimulation in vivo and provide a searchable online viewer of these data characterizing the macrophage subsets serving as hematopoietic niches. Taken together, our findings demonstrate that EBIs serve a dual role as niches for terminal erythropoiesis and granulopoiesis and the central macrophages adapt to optimize production of red blood cells or neutrophils.

Autism-associated chromatin remodeler CHD8 regulates erythroblast cytokinesis and fine-tunes the balance of Rho GTPase signaling.

CHD8 is an ATP-dependent chromatin-remodeling factor whose monoallelic mutation defines a subtype of autism spectrum disorders (ASDs). Previous work found that CHD8 is required for the maintenance of hematopoiesis by integrating ATM-P53-mediated survival of hematopoietic stem/progenitor cells (HSPCs). Here, by using Chd8F/FMx1-Cre combined with a Trp53F/F mouse model that suppresses apoptosis of Chd8-/- HSPCs, we identify CHD8 as an essential regulator of erythroid differentiation. Chd8-/-P53-/- mice exhibited severe anemia conforming to congenital dyserythropoietic anemia (CDA) phenotypes. Loss of CHD8 leads to drastically decreased numbers of orthochromatic erythroblasts and increased binucleated and multinucleated basophilic erythroblasts with a cytokinesis failure in erythroblasts. CHD8 binds directly to the gene bodies of multiple Rho GTPase signaling genes in erythroblasts, and loss of CHD8 results in their dysregulated expression, leading to decreased RhoA and increased Rac1 and Cdc42 activities. Our study shows that autism-associated CHD8 is essential for erythroblast cytokinesis.

Rapid degradation of protein tyrosine phosphatase 1B in sickle cells: Possible contribution to sickle cell membrane weakening.

Although both protein tyrosine phosphatases and kinases are constitutively active in healthy human red blood cells (RBCs), the preponderance of phosphatase activities maintains the membrane proteins in a predominantly unphosphorylated state. We report here that unlike healthy RBCs, proteins in sickle cells are heavily tyrosine phosphorylated, raising the question regarding the mechanism underpinning this tyrosine phosphorylation. Upon investigating possible causes, we observe that protein tyrosine phosphatase 1B (PTP1B), the major erythrocyte tyrosine phosphatase, is largely digested to a lower molecular weight fragment in sickle cells. We further find that the resulting truncated form of PTP1B is significantly less active than its intact counterpart, probably accounting for the intense tyrosine phosphorylation of Band 3 in sickle erythrocytes. Because this tyrosine phosphorylation of Band 3 promotes erythrocyte membrane weakening that causes release of both membrane vesicles and cell free hemoglobin that in turn initiates vaso-occlusive events, we conclude that cleavage of PTP1B could contribute to the symptoms of sickle cell disease. We further posit that methods to inhibit proteolysis of PTP1B could mitigate symptoms of the disease.

Safety, Pharmacokinetics, and Pharmacodynamics of Etavopivat (FT-4202), an Allosteric Activator of Pyruvate Kinase-R, in Healthy Adults: A Randomized, Placebo-Controlled, Double-Blind, First-in-Human Phase 1 Trial.

Etavopivat (FT-4202) is an orally administered, small-molecule allosteric activator of erythrocyte pyruvate kinase-R (PKR) in clinical development for the treatment of sickle cell disease and other hemoglobin disorders. This randomized, placebo-controlled, double-blind, first-in-human combination single-ascending dose and multiple-ascending dose phase 1 trial (NCT03815695) evaluated the safety and pharmacokinetics/pharmacodynamics of etavopivat in 90 healthy adult subjects. In 4 single-ascending dose cohorts, 8 participants were randomized 3:1 to a single oral dose of either etavopivat (n = 6) or placebo (n = 2). In four 14-day multiple-ascending dose cohorts, 12 participants were randomized 3:1 to 14 days of etavopivat (n = 9) or placebo (n = 3). In these studies, most treatment-emergent adverse events were of mild severity (grade 1) and none led to study discontinuation. Etavopivat exhibited a linear and time-independent pharmacokinetic profile (at doses ≤400 mg) and elicited the expected pharmacodynamic effects of PKR activation (decreased 2,3-diphosphoglycerate and increased adenosine triphosphate) and evidence of improved hemoglobin-oxygen affinity. In addition, pharmacodynamic responses were durable with effects continuing for 48 to 72 hours after the last dose, thereby supporting once-daily dosing. Food appeared to have no clinically meaningful effects on etavopivat exposure, thus facilitating administration with or without food. In conclusion, the evaluation of etavopivat in healthy subjects demonstrated proof of mechanism (PKR activation) without significant adverse events. This study also allowed for the selection of dose levels, projected to have an acceptable safety profile and provide therapeutic benefit, for evaluation in future trials in patients with sickle cell disease.

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

Etavopivat is an investigational, oral, small molecule activator of erythrocyte pyruvate kinase (PKR) in development for the treatment of sickle cell disease (SCD) and other hemoglobinopathies. PKR activation is proposed to ameliorate the sickling of SCD red blood cells (RBCs) through multiple mechanisms, including reduction of 2,3-diphosphoglycerate (2,3-DPG), which consequently increases hemoglobin (Hb)-oxygen affinity; increased binding of oxygen reduces sickle hemoglobin polymerization and sickling. In addition, PKR activation increases adenosine triphosphate (ATP) produced via glycolytic flux, which helps preserve membrane integrity and RBC deformability. We evaluated the pharmacodynamic response to etavopivat in nonhuman primates (NHPs) and in healthy human subjects and evaluated the effects in RBCs from patients with SCD after ex vivo treatment with etavopivat. A single dose of etavopivat decreased 2,3-DPG in NHPs and healthy subjects. Hb-oxygen affinity was significantly increased in healthy subjects after 24 hours. After daily dosing of etavopivat over 5 consecutive days in NHPs, ATP was increased by 38% from baseline. Etavopivat increased Hb-oxygen affinity and reduced sickling in RBCs collected from patients with SCD with either homozygous hemoglobin S or hemoglobin S and C disease. Collectively, these results demonstrate the ability of etavopivat to decrease 2,3-DPG and increase ATP, resulting in increased Hb-oxygen affinity and improved sickle RBC function. Etavopivat is currently being evaluated in clinical trials for the treatment of SCD. SIGNIFICANCE STATEMENT: Etavopivat, a small molecule activator of the glycolytic enzyme erythrocyte pyruvate kinase, decreased 2,3-diphosphoglycerate in red blood cells (RBCs) from nonhuman primates and healthy subjects and significantly increased hemoglobin (Hb)-oxygen affinity in healthy subjects. Using ex vivo RBCs from donors with sickle cell disease (SCD) (homozygous hemoglobin S or hemoglobin S and C genotype), etavopivat increased Hb-oxygen affinity and reduced sickling under deoxygenation. Etavopivat shows promise as a treatment for SCD that could potentially reduce vaso-occlusion and improve anemia.