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.

Cytokinesis failure in RhoA-deficient mouse erythroblasts involves actomyosin and midbody dysregulation and triggers p53 activation.

RhoA GTPase has been shown in vitro in cell lines and in vivo in nonmammalian organisms to regulate cell division, particularly during cytokinesis and abscission, when 2 daughter cells partition through coordinated actomyosin and microtubule machineries. To investigate the role of this GTPase in the rapidly proliferating mammalian erythroid lineage, we developed a mouse model with erythroid-specific deletion of RhoA. This model was proved embryonic lethal as a result of severe anemia by embryonic day 16.5 (E16.5). The primitive red blood cells were enlarged, poikilocytic, and frequently multinucleated, but were able to sustain life despite experiencing cytokinesis failure. In contrast, definitive erythropoiesis failed and the mice died by E16.5, with profound reduction of maturing erythroblast populations within the fetal liver. RhoA was required to activate myosin-regulatory light chain and localized at the site of the midbody formation in dividing wild-type erythroblasts. Cytokinesis failure caused by RhoA deficiency resulted in p53 activation and p21-transcriptional upregulation with associated cell-cycle arrest, increased DNA damage, and cell death. Our findings demonstrate the role of RhoA as a critical regulator for efficient erythroblast proliferation and the p53 pathway as a powerful quality control mechanism in erythropoiesis.

Gene targeting RhoA reveals its essential role in coordinating mitochondrial function and thymocyte development.

Thymocyte development is regulated by complex signaling pathways. How these signaling cascades are coordinated remains elusive. RhoA of the Rho family small GTPases plays an important role in actin cytoskeleton organization, cell adhesion, migration, proliferation, and survival. Nonetheless, the physiological function of RhoA in thymocyte development is not clear. By characterizing a conditional gene targeting mouse model bearing T cell deletion of RhoA, we show that RhoA critically regulates thymocyte development by coordinating multiple developmental events. RhoA gene disruption caused a strong developmental block at the pre-TCR checkpoint and during positive selection. Ablation of RhoA led to reduced DNA synthesis in CD4(-)CD8(-), CD4(+)CD8(-), and CD4(-)CD8(+) thymocytes but not in CD4(+)CD8(+) thymocytes. Instead, RhoA-deficient CD4(+)CD8(+) thymocytes showed an impaired mitosis. Furthermore, we found that abrogation of RhoA led to an increased apoptosis in all thymocyte subpopulations. Importantly, we show that the increased apoptosis was resulted from reduced pre-TCR expression and increased production of reactive oxygen species (ROS), which may be because of an enhanced mitochondrial function, as manifested by increased oxidative phosphorylation, glycolysis, mitochondrial membrane potential, and mitochondrial biogenesis in RhoA-deficient thymocytes. Restoration of pre-TCR expression or treatment of RhoA-deficient mice with a ROS scavenger N-acetylcysteine partially restored thymocyte development. These results suggest that RhoA is required for thymocyte development and indicate, to our knowledge, for the first time that fine-tuning of ROS production by RhoA, through a delicate control of metabolic circuit, may contribute to thymopoiesis.

Erythrocyte NADPH oxidase activity modulated by Rac GTPases, PKC, and plasma cytokines contributes to oxidative stress in sickle cell disease.

Chronic inflammation has emerged as an important pathogenic mechanism in sickle cell disease (SCD). One component of this inflammatory response is oxidant stress mediated by reactive oxygen species (ROS) generated by leukocytes, endothelial cells, plasma enzymes, and sickle red blood cells (RBC). Sickle RBC ROS generation has been attributed to sickle hemoglobin auto-oxidation and Fenton chemistry reactions catalyzed by denatured heme moieties bound to the RBC membrane. In this study, we demonstrate that a significant part of ROS production in sickle cells is mediated enzymatically by NADPH oxidase, which is regulated by protein kinase C, Rac GTPase, and intracellular Ca(2+) signaling within the sickle RBC. Moreover, plasma from patients with SCD and isolated cytokines, such as transforming growth factor ß1 and endothelin-1, enhance RBC NADPH oxidase activity and increase ROS generation. ROS-mediated damage to RBC membrane components is known to contribute to erythrocyte rigidity and fragility in SCD. Erythrocyte ROS generation, hemolysis, vaso-occlusion, and the inflammatory response to tissue damage may therefore act in a positive-feedback loop to drive the pathophysiology of sickle cell disease. These findings suggest a novel pathogenic mechanism in SCD and may offer new therapeutic targets to counteract inflammation and RBC rigidity and fragility in SCD.

In vivo molecular imaging of HER2 expression in a rat model of Barrett's esophagus adenocarcinoma.

Human epidermal growth factor receptor 2 (HER2) is involved in the malignant progression of several human cancers, including esophageal adenocarcinoma (EAC). The purpose of this study was to evaluate HER2 overexpression and to explore the feasibility of confocal laser endomicroscopy for in vivo molecular imaging of HER2 status in an animal model of Barrett's-related EAC. Rats underwent esophagojejunostomy with gastric preservation. At 30 weeks post-surgery, the esophagus of 46 rats was studied; endoscopic and histological findings were correlated with HER2 immunofluorescence on excised biopsies and gross specimens. At this age, 23/46 rats developed Barrett's esophagus (BE), and 6/46 had cancer (four EAC and two squamous cell carcinomas). A significant overexpression of HER2 was observed in esophageal adenocarcinoma compared with normal squamous esophagus (9.4-fold) and BE (6.0-fold). AKT and its phosphorylated form were also overexpressed in cancer areas. Molecular imaging was performed at 80 weeks post-surgery in four rats after tail injection of fluorescent-labeled anti-HER2 antibody. At this age, 3/4 rats developed advance adenocarcinoma and showed in vivo overexpression of HER2 by molecular confocal laser endomicroscopy with heterogeneous distribution within cancer; no HER2 signal was observed in normal or Barrett's tissues. Therefore, HER2 overexpression is a typical feature of the surgical induced model of EAC that can be easily quantified in vivo using an innovative mini-invasive approach including confocal endomicroscopy; this approach may avoid limits of histological evaluation of HER2 status on 'blinded' biopsies.

Signaling and cytoskeletal requirements in erythroblast enucleation.

To understand the role of cytoskeleton and membrane signaling molecules in erythroblast enucleation, we developed a novel analysis protocol of multiparameter high-speed cell imaging in flow. This protocol enabled us to observe F-actin and phosphorylated myosin regulatory light chain (pMRLC) assembled into a contractile actomyosin ring (CAR) between nascent reticulocyte and nucleus, in a population of enucleating erythroblasts. CAR formation and subsequent enucleation were not affected in murine erythroblasts with genetic deletion of Rac1 and Rac2 GTPases because of compensation by Rac3. Pharmacologic inhibition or genetic deletion of all Rac GTPases altered the distribution of F-actin and pMRLC and inhibited enucleation. Erythroblasts treated with NSC23766, cytochalasin-D, colchicine, ML7, or filipin that inhibited Rac activity, actin or tubulin polymerization, MRLC phosphorylation, or lipid raft assembly, respectively, exhibited decreased enucleation efficiency, as quantified by flow cytometry. As assessed by high-speed flow-imaging analysis, colchicine inhibited erythroblast polarization, implicating microtubules during the preparatory stage of enucleation, whereas NSC23766 led to absence of lipid raft assembly in the reticulocyte-pyrenocyte border. In conclusion, enucleation is a multistep process that resembles cytokinesis, requiring establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring, and coalescence of lipid rafts between reticulocyte and pyrenocyte.

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.

Automated Oxygen Gradient Ektacytometry: A Novel Biomarker in Sickle Cell Anemia.

Sickle cell anemia (SCA) is a hereditary hemoglobinopathy with a variable phenotype. There is no single biomarker that adequately predicts disease severity and can be used to monitor treatment response in patients in clinical trials and clinical care. The use of clinical outcomes, such as vaso-occlusive crises (VOC), requires long and expensive studies, sometimes with inconclusive results. To address these limitations, there are several biomarkers under study to improve the ability to predict complications and assess treatment response in both clinical and research settings. Oxygen gradient ektacytometry, also called as oxygenscan, is an assay that measures the effects of deoxygenation and reoxygenation on red blood cell (RBC) deformability and is gaining popularity in SCA research, because it captures the dynamic sickling capacity of a patient's RBCs as they are subjected to an oxygen gradient under steady shear stress. We describe here the oxygenscan methodology and evaluate the correlation between oxygenscan parameters and more well-known biomarkers of SCA such as fetal hemoglobin (HbF), F-cells, and dense red blood cells (DRBCs). Our data indicate that the oxygenscan curve is affected by all these parameters and the result incorporates the effects of %HbF, %F-cells, RBC hydration, and RBC membrane deformability.

Technical aspects in endoscopic biopsy of lesions in esophageal pemphigus vulgaris.

BACKGROUND AND AIMS: Aim of this study is to compare a specific kind of biopsy forceps to a traditional one in providing an adequate specimen of esophageal pemphigus vulgaris lesions that includes the basement membrane for definitive diagnosis. PATIENTS AND METHODS: Prospective, randomized, blind, single-center study. We performed upper endoscopy with biopsy in 32 patients divided into two groups of 16 each: in group A with a commercially available standard biopsy forceps while in group B with a commercially available rocking biopsy forceps. Hundred-ninety-six biopsy specimens from both groups were blindly evaluated by the same pathologist. RESULTS: In group A 18.8% of biopsy specimens were adequate (basement membrane included). In group B 87.5% of biopsy specimens were adequate. The presence of the entire thickness of the mucosa was significantly higher in group B compared to group A. All parameters typically taken into account by pathologist for diagnosis of esophageal pemphigus vulgaris were significantly improved in group B. CONCLUSIONS: The biopsy forceps used in group B permits a rocking motion of the tip on contact with the mucosa, produces a deeper full-thickness mucosal sample up to the basement membrane and assists in the evaluation of histologic features of esophageal pemphigus vulgaris.

The role of upper endoscopy in identifying oesophageal involvement in patients with oral pemphigus vulgaris.

BACKGROUND AND AIMS: The involvement of oesophagus in pemphigus vulgaris is still debated. The aims of this study were to evaluate the prevalence of oesophageal involvement and the gastro-duodenal mucosa appearance before and after high-dose corticosteroid therapy in a group of patients with oral pemphigus vulgaris. METHODS: We prospectively studied 28 consecutive patients with oral pemphigus by oesophageal symptom standardised questionnaire, upper gastro-intestinal endoscopy, exfoliative cytology and histological biopsy. After clinical remission, all patients underwent new endoscopy. RESULTS: The prevalence of oesophageal symptoms was 57.1%. Endoscopic examination revealed oesophageal involvement with different degrees of severity in 67.8% of patients. After corticosteroid therapy, endoscopy showed normal oesophageal-gastro-duodenal mucosa. No examination-related exacerbations of the oesophageal lesions were seen. CONCLUSIONS: The upper gastro-intestinal endoscopic examination, in oral pemphigus vulgaris patients with oesophageal symptoms, is safe in skilled hands technique and a useful diagnostic tool prior to starting therapy.

[Personal experience with the endoscopic treatment of pancreatic pseudocysts. Long-term results and analysis of prognostic factors]. / Esperienza personale nel trattamento per via endoscopica delle pseudocisti pancreatiche. Risultati a lungo termine ed analisi dei fattori prognostici.

BACKGROUND: The aim of this study was to assess the safety and utility of endoscopic treatment of pancreatic pseudocysts. Prognostic factors for the outcome of endoscopic drainage were assessed in a prospective analysis. METHODS: Forty-nine consecutive symptomatic patients were included in the study. Transmural drainage was used in 30 patients and transpapillary drainage in 19 patients. RESULTS: Successful drainage was achieved in 27/30 (90%) of patients after transmural drainage and in 16/19 (84.2%) patients after transpapillary drainage. Twelve (24.5%) patients had complications; 2 patients had bleeding, 2 had mild pancreatitis, 8 had cyst infection, in relation to the presence of necrosis (5 patients) or stent clogging (3 patients). Nine patients (20.9%) had recurrence of pseudocyst. Endoscopic drainage was a definitive treatment in 37 out of 49 (75.5%) patients (median follow-up: 25.9 months). Presence of necrosis was the only significant prognostic factor for infectious complication. CONCLUSIONS: Endoscopic drainage provides a successful and safe minimally invasive approach to the management of pancreatic pseudocysts.

Identification of a murine erythroblast subpopulation enriched in enucleating events by multi-spectral imaging flow cytometry.

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation. We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis. We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation. Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry. Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters "aspect ratio" of a cell in the bright-field channel that aids in the recognition of elongated cells and "delta centroid XY Ter119/Draq5" that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate. The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.