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Sickle cell anemia (SCA) results from an abnormal sickle hemoglobin (HbS). HbS polymerizes upon deoxygenation, resulting in red blood cell (RBC) sickling and membrane damage that cause vaso-occlusions and hemolysis. Sickle RBCs contain less adenosine triphosphate and more 2,3-diphosphoglycerate than normal RBCs, which allosterically reduces hemoglobin (Hb) oxygen (O2) affinity (ie, increases the partial pressure of oxygen at which hemoglobin is 50% saturated with oxygen [P50]), potentiating HbS polymerization. Herein, we tested the effect of investigational agent FT-4202, an RBC pyruvate kinase (PKR) activator, on RBC sickling and membrane damage by administering it to Berkeley SCA mice. Two-week oral FT-4202 administration was well tolerated, decreasing HbS P50 to levels similar to HbA and demonstrating beneficial biological effects. In FT-4202–treated animals, there was reduced sickling in vivo, demonstrated by fewer irreversibly sickled cells, and improved RBC deformability, assessed at varying shear stress. Controlled deoxygenation followed by reoxygenation of RBCs obtained from the blood of FT-4202–treated mice showed a shift in the point of sickling to a lower partial pressure of oxygen (pO2). This led to a nearly 30% increase in RBC survival and a 1.7g/dL increase in Hb level in the FT-4202–treated SCA mice. Overall, our results in SCA mice suggest that FT-4202 might be a potentially useful oral antisickling agent that warrants investigation in patients with SCA.

Hydroxyurea (hydroxycarbamide) is an effective treatment for sickle cell anaemia (SCA), but clinical responses depend primarily upon the degree of fetal haemoglobin (HbF) induction and the heterogeneity of HbF expression across erythrocytes. The number and characteristics of HbF-containing cells (F-cells) are not assessed by traditional HbF measurements. Conventional hydroxyurea dosing (e.g. fixed doses or low starting doses with stepwise escalation) produces a moderate heterocellular HbF induction, but haemolysis and clinical complications continue. Robust, pancellular HbF induction is needed to minimise or fully inhibit polymerisation of sickle haemoglobin. We treated children with hydroxyurea using an individualised, pharmacokinetics-guided regimen starting at predicted maximum tolerated dose (MTD). We observed sustained HbF induction (mean >30%) for up to 6 years, which was not dependent on genetic determinants of HbF expression. Nearly 70% of patients had ≥80% F-cells (near-pancellular), and almost half had ≥90% F-cells (pancellular). The mean HbF/F-cell content was ~12 pg. Earlier age of initiation and better medication adherence were associated with high F-cell responses. In summary, early initiation of hydroxyurea using pharmacokinetics-guided starting doses at predicted MTD can achieve sustained near-pancellular or pancellular HbF expression and should be considered an achievable goal for children with SCA treated with hydroxyurea at optimal doses. Clinical trial registration number: NCT02286154 (clinicaltrials.gov).

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.

Introduction: Hydroxyurea has well-described clinical and laboratory benefits in sickle cell anemia (SCA). Traditionally, hemoglobin concentration (Hb) and fetal hemoglobin (HbF) are used to assess treatment response, but these aggregate parameters do not fully capture the range of erythrocyte abnormalities in SCA. Dense red blood cells (DRBCs) are defined as RBCs with density >1.11 g/mL as measured by density-gradient fractionation methods, which are tedious and require technical expertise. This subpopulation of dehydrated RBCs has been associated with increased SCA severity but has not been extensively studied in children. Here we describe the utility of rapid, automated DRBC quantitation and show that the percent of DRBCs (%DRBCs) is a robust biomarker of hydroxyurea response in children with SCA. Methods: The Therapeutic Response Evaluation and Adherence Trial (TREAT, NCT02286154) was a single-center prospective study that demonstrated individualized, pharmacokinetic (PK)-guided hydroxyurea dosing in young patients (mean age 12.1 months at initiation) results in robust and sustained HbF levels >30-40% for most adherent patients. The longitudinal follow-up phase of TREAT aims to comprehensively evaluate hematologic parameters and organ function to demonstrate the long-term benefits of this treatment strategy. During clinic visits (every 3-6 months), TREAT participants had complete blood counts (CBCs) with the ADVIA® 2120i system. This automated analyzer quantifies hyperchromic RBCs within seconds by directly measuring cell hemoglobin concentration, where DRBCs are known to correspond to RBCs with a measured MCHC >41 g/dL. Data were analyzed at baseline and after at least 6 months of hydroxyurea therapy (M≥6). α- and β-globin genotypes were determined for all patients. Results: Thirty-three TREAT participants had ADVIA data available for analysis (Table). All had homozygous SCA except one with sickle-β0-thalasemia; 17 (51.5%) were male. The median age at hydroxyurea initiation was 10.3 months (mean 26.1 months; range 6 months - 17 years). The median duration of hydroxyurea therapy was 27.7 months (mean 31.5). At baseline, median %DRBCs was 2.1%. Baseline %DRBCs were directly correlated with age at initiation of hydroxyurea (p=0.022) and inversely correlated with baseline HbF (p=0.001). After initiation of hydroxyurea (M≥6), there was a 47.6% reduction in %DRBCs (p=0.001). Median %DRBCs at M≥6 was directly correlated with absolute reticulocyte (ARC) (p=0.002), absolute neutrophil (ANC) (p=0.003) and platelet counts (p Conclusions: DRBCs can be rapidly quantified without the tedium of classical density-gradient fractionation methods using the automated ADVIA 2120i hematology analyzer, which directly measures hyperchromic RBCs. These data from TREAT demonstrate the feasibility of serial %DRBC measurements in clinical trials and clinical practice. TREAT participants had low baseline DRBCs compared to untreated adult SCA patients (reported mean ≈12%), reflecting the young age of these patients. Nevertheless, there was a significant decline in %DRBCs with treatment that correlated strongly with the known laboratory benefits of hydroxyurea. In conclusion, automated DRBC measurements are a robust biomarker that can be incorporated in a panel of laboratory measurements of response to hydroxyurea (and severity of SCA). A larger, multicenter study of PK-guided hydroxyurea therapy (HOPS, NCT03789591) is currently underway in 11 US sickle cell centers and will provide generalizable data on DRBC responses with optimal hydroxyurea therapy. Disclosures Malik: Aruvant Sciences, Forma Therapeutics, Inc.: Consultancy; Aruvant Sciences, CSL Behring: Patents & Royalties. Kalfa:Agios Pharmaceuticals, Inc: Consultancy, Research Funding; Forma Therapeutics, Inc: Research Funding.

The two most common genotypes causing sickle cell disease (SCD) are homozygous bsbs producing hemoglobin S (HbSS), and compound heterozygous bsbc producing HbS and HbC (HbSC). HbS polymerizes in both, when deoxygenated. Patients with HbSC have distinct hematologic characteristics such as higher Hb levels but lower fetal hemoglobin (HbF); increased corpuscular Hb concentration, due to pronounced red blood cell (RBC) K+ loss and dehydration, contributes to HbS polymerization. While hydroxyurea (HU) as a HbF-inducing agent clearly improves HbSS phenotype, targeting of other pathogenetic mechanisms in SCD may offer additional benefit in both HbSS and HbSC patients. The RBC metabolism depends on anaerobic glycolysis for ATP production. Key RBC functions are intricately linked to metabolism: hemoglobin-oxygen (Hb-O2) affinity is modulated by 2,3-diphosphoglycerate (2,3-DPG), RBC membrane health depends on adequate synthesis of ATP, and heme-iron is maintained in the functional reduced state by the synthesis of antioxidants, such as GSH, supplying NADH to methemoglobin reductase. Erythrocyte pyruvate kinase (PKR) performs a rate limiting step in RBC glycolysis, converting phosphoenolpyruvate to pyruvate and generating ATP. Insufficient PKR activity due to any of a variety of causes, including excessive energy demand, can alter the abundance of metabolic products and intermediates, including ATP and 2,3-DPG, adversely affecting RBC function. We assessed baseline 2,3-DPG and ATP levels in whole blood of patients with HbSS (N=18) by LC/MS analysis. As well-reported, hemoglobin (Hb) levels were significantly decreased in patients with HbSS compared to healthy volunteers (9.68 ± 0.32 g/dL vs 14.88 ± 0.27 g/dL, p Whole blood samples in K2EDTA were collected from donors with confirmed HbSS or HbSC disease in accordance with an approved IRB protocol in Cincinnati Children's Hospital. % HbF and % F-cells were measured for each sample. Packed RBCs were washed, resuspended to 20% v/v (20% hematocrit), and then incubated for 4 hr at 37oC with 20 μM FT-4202 or equivalent volume of vehicle control DMSO. P50 representing the partial pressure of oxygen at which Hb is 50% saturated with oxygen, was determined using non-linear regression analysis of hemoglobin-oxygen equilibrium curves obtained on a HEMOX Analyzer (TCS Scientific). RBC deformability as a function of oxygen pressure, or Oxygenscan, was measured using Lorrca® (RR Mechatronics) (Rab et al., 2019, Am J Hematol). Point of Sickling (PoS) was calculated as the pO2 at which RBC deformability is decreased by 5% of maximum value during deoxygenation. The P50 and PoS values in FT-4202 versus DMSO-treated HbSC or HbSS RBCs from patients treated with or without HU were analyzed by paired Wilcoxon test. FT-4202 treatment significantly decreased P50 in both HU-treated and not HU-treated HbSS RBCs, as well as in HbSC RBCs, as detailed in Table below showing mean ± se (*p These data suggest that increasing flux through the glycolytic pathway by FT-4202 treatment may offer dual beneficial effects of increased Hb-O2 affinity and membrane flexibility in HbSC as well as in HbSS RBCs irrespective of HU use. Figure Disclosures Fulzele: FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics. Guichard:FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics; AstraZeneca: Other: Shareholder. Kalfa:Agios Pharmaceuticals, Inc: Consultancy, Research Funding; Forma Therapeutics, Inc: Research Funding.

Background: Hydroxyurea is the standard of care for children with sickle cell anemia (SCA). We have developed and prospectively validated a population pharmacokinetics (PK)-based dosing model to initiate hydroxyurea at a personalized dose that achieves maximum tolerated dose (MTD) quickly and produces a more robust HbF response than traditional weight-based dosing. HbF is distributed unequally across RBCs, and most mature RBCs have no detectable HbF. Genetic traits and hydroxyurea increase the number of HbF-containing RBCs. Measurement of HbF alone reflects an average of the HbF content across all RBCs in a lysate, so the varying, cell-specific compartmentalization of HbF across RBCs is not evaluated. In contrast, flow cytometry can be used to detect HbF-containing RBCs (F-cells) and non-F-cells, and the amount of HbF per F-cell (F/F-cell) can also be quantified. Prior studies indicate that an F/F-cell of 10 pg should completely prevent sickling. Methods:We studied participants in the Therapeutic Response Evaluation and Adherence Trial (TREAT, ClinicalTrials.gov NCT02286154), which is a prospective study with the primary objective of developing and evaluating a population PK model to predict the MTD of hydroxyurea for individualized dosing. Individuals with genotype-confirmed HbSS or sickle-β0-thalassemia (both classified here as SCA) were enrolled; none had gene-deletion HPFH. We recently developed a multiparametric flow cytometric assay (F-cell analysis) to measure the amount and distribution of HbF in all RBCs and in immature (CD71+) reticulocytes. The total number of HbF-containing RBCs (F-cells) and HbF-containing immature reticulocytes (F-retics) were enumerated. Mean concentration of HbF per F-cell (F/F-cell) was calculated from F-cell number, Hb F quantitation by capillary zone electrophoresis, and automated blood counts (RBC count and hemoglobin concentration). Results:To date, 36 TREAT patients have had F-cell analysis with a mean age of 5.6 years (median 4.1; range 1.2-21.1). Mean age at start of hydroxyurea was 3.2 years (median 1.0; range 0.5-19.5) with a mean duration of hydroxyurea therapy of 2.4 years (median 2.1; range 0.5-4.1). Mean dose of hydroxyurea at the time of F-cell analysis was 27.0 mg/kg (median 27.6; range 17.8-38.6). HbF induction was robust with a mean HbF level of 30.8% (median 32; range 7.7-52.6). HbF level had a curvilinear association with F-cell fraction (Figure A). Mean total F-cell fraction (percent of all RBCs that are F-cells) was 82.2% (median 89.8; range 33.6-99.0); 24/36 (67%) had >80% F-cells, and 17/36 (47%) had >90% F-cells, indicating the ability to achieve a near pancellular distribution of HbF with PK-guided hydroxyurea therapy in a significant fraction of patients, especially those with high adherence (Figure B). The mean F/F-cell was 11.9 pg (median 11.5; range 5.1-19.4; Figure C), and 26/36 (72%) had an mean F/F-cell ≥10 pg, an amount of HbF in an individual RBC that completely prevents sickling (mean F/F-cell does not reflect the variance of F/F-cell across the RBC population). All patients who had a HbF level ≥30% (22/36, 61%) had mean F/F-cell ≥10 pg (Figure D). Of those with total F-cells ≥80%, 22/24 (91%) had mean F/F-cell ≥10 pg. As expected, higher total F-cells and mean F/F-cell were associated with higher hemoglobin concentration and lower reticulocyte counts. Mean F/F-cell >16 pg was associated with a mean hemoglobin >10 g/dL and mean absolute reticulocyte count F-retics), indicating the difference between F-cell production (F-retics) and peripheral survival advantage of F-cells. For F-cells >80%, however, the difference between F-cell and F-retic fraction decreased rapidly, consistent with little non-F-cell production by the bone marrow. Discussion: PK-guided dosing of hydroxyurea at MTD can achieve near-pancellular HbF expression in a large fraction of patients with sickle cell anemia. Our data suggest that an F-cell fraction of 80% and a mean F/F-cell of 10 pg should be the minimum goals for hydroxyurea therapy to minimize or prevent sickling, hemolysis, and anemia. These same minimum goals should apply to any HbF-inducing therapy, including genetic therapies, and F-cell analysis should be included in the standard laboratory analysis for these treatments. Figure Disclosures Quinn: Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Other: Research Support. Ware:Bristol Myers Squibb: Other: Research Drug Donation; Addmedica: Other: Research Drug Donation; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Nova Laboratories: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: DSMB.