Safety and efficacy of voxelotor in pediatric patients with sickle cell disease aged 4 to 11 years.

BACKGROUND: Sickle cell disease (SCD) is a devastating, multisystemic disorder that affects millions of people worldwide. The earliest clinical manifestations of SCD can affect infants as young as 6 months of age, and pediatric patients are at risk for acute and life-threatening complications. Early intervention with treatments that target the underlying pathophysiological mechanism of SCD, sickle hemoglobin (HbS) polymerization, are expected to slow disease progression and circumvent disease-associated morbidity and mortality. PROCEDURE: The HOPE-KIDS 1 trial (NCT02850406) is an ongoing four-part, phase 2a, open-label, single- and multiple-dose study to evaluate the pharmacokinetics, efficacy, and safety of voxelotor-a first-in-class HbS polymerization inhibitor-in patients aged 6 months to 17 years with SCD. Initial findings from a cohort of 45 patients aged 4 to 11 years who received voxelotor treatment for up to 48 weeks are reported. RESULTS: Hemoglobin (Hb) response, defined as a >1.0 g/dl increase from baseline, was achieved at week 24 by 47% (n = 16/34) of patients with Hb measurements at baseline and week 24. At week 24, 35% (n = 12/34) and 21% (n = 7/34) of patients had a >1.5 g/dl increase and a >2.0 g/dl increase from baseline in Hb concentration, respectively. Concurrent improvements in hemolytic markers were observed. Voxelotor was well tolerated in this young cohort, with no newly emerging safety signals. CONCLUSIONS: Based on its mechanism as an HbS polymerization inhibitor, voxelotor improves Hb levels and markers of hemolysis and has the potential to mitigate SCD-related complications; these results support its use in patients aged ≥4 years.

Controlled trial of transfusions for silent cerebral infarcts in sickle cell anemia.

BACKGROUND: Silent cerebral infarcts are the most common neurologic injury in children with sickle cell anemia and are associated with the recurrence of an infarct (stroke or silent cerebral infarct). We tested the hypothesis that the incidence of the recurrence of an infarct would be lower among children who underwent regular blood-transfusion therapy than among those who received standard care. METHODS: In this randomized, single-blind clinical trial, we randomly assigned children with sickle cell anemia to receive regular blood transfusions (transfusion group) or standard care (observation group). Participants were between 5 and 15 years of age, with no history of stroke and with one or more silent cerebral infarcts on magnetic resonance imaging and a neurologic examination showing no abnormalities corresponding to these lesions. The primary end point was the recurrence of an infarct, defined as a stroke or a new or enlarged silent cerebral infarct. RESULTS: A total of 196 children (mean age, 10 years) were randomly assigned to the observation or transfusion group and were followed for a median of 3 years. In the transfusion group, 6 of 99 children (6%) had an end-point event (1 had a stroke, and 5 had new or enlarged silent cerebral infarcts). In the observation group, 14 of 97 children (14%) had an end-point event (7 had strokes, and 7 had new or enlarged silent cerebral infarcts). The incidence of the primary end point in the transfusion and observation groups was 2.0 and 4.8 events, respectively, per 100 years at risk, corresponding to an incidence rate ratio of 0.41 (95% confidence interval, 0.12 to 0.99; P=0.04). CONCLUSIONS: Regular blood-transfusion therapy significantly reduced the incidence of the recurrence of cerebral infarct in children with sickle cell anemia. (Funded by the National Institute of Neurological Disorders and Stroke and others; Silent Cerebral Infarct Multi-Center Clinical Trial ClinicalTrials.gov number, NCT00072761, and Current Controlled Trials number, ISRCTN52713285.).

Silent cerebral infarction, income, and grade retention among students with sickle cell anemia.

Children with sickle cell anemia have a higher-than-expected prevalence of poor educational attainment. We test two key hypotheses about educational attainment among students with sickle cell anemia, as measured by grade retention and use of special education services: (1) lower household per capita income is associated with lower educational attainment; (2) the presence of a silent cerebral infarct is associated with lower educational attainment. We conducted a multicenter, cross-sectional study of cases from 22 U.S. sites included in the Silent Infarct Transfusion Trial. During screening, parents completed a questionnaire that included sociodemographic information and details of their child's academic status. Of 835 students, 670 were evaluable; 536 had data on all covariates and were used for analysis. The students' mean age was 9.4 years (range: 5-15) with 52.2% male; 17.5% of students were retained one grade level and 18.3% received special education services. A multiple variable logistic regression model identified that lower household per capita income (odds ratio [OR] of quartile 1 = 6.36, OR of quartile 2 = 4.7, OR of quartile 3 = 3.87; P = 0.001 for linear trend), age (OR = 1.3; P < 0.001), and male gender (OR, 2.2; P = 0.001) were associated with grade retention; silent cerebral infarct (P = 0.31) and painful episodes (P = 0.60) were not. Among students with sickle cell anemia, household per capita income is associated with grade retention, whereas the presence of a silent cerebral infarct is not. Future educational interventions will need to address both the medical and socioeconomic issues that affect students with sickle cell anemia.

Practice patterns of stroke screening and hydroxyurea use in children with sickle cell disease: a survey of health care providers.

Incidence of stroke in sickle cell disease (SCD) has declined with the use of transcranial Doppler ultrasound and chronic transfusion therapy. There is little information regarding their use in genotypes other than HbSS and HbSß. Silent cerebral infarcts (SCIs) have been identified by magnetic resonance imaging (MRI) in SCD patients and it is believed that these may increase the risk of overt stroke. No evidence-based guidelines exist regarding MRI screening for SCIs. Hydroxyurea is a standard therapy in patients with history of acute chest syndrome and severe, recurrent, SCD-associated pain episodes, but has not been established for use with other sickle-associated morbidities. A total of 102 institutions received a survey (with 62 responses) to assess the use of transcranial Doppler ultrasound for stroke screening, use of screening MRI for SCIs, and institutional patterns for prescribing hydroxyurea. Nineteen percent of institutions screen genotypes other than HbSS and HbSß, and 24% use MRI to screen for SCIs. Twenty-six percent of institutions prescribed hydroxyurea in patient found to have SCIs. Results indicate significant variation in stroke screening and hydroxyurea use often correlating with clinic size, number of physician providers, and geographic location. There are currently no evidence-based guidelines to support many of these practices.

Oxidant-mediated modification of the cellular thiols is sufficient for arginase activation in cultured cells.

Increased arginase activity in the vasculature has been implicated in the regulation of nitric oxide (NO) homeostasis, leading to the development of vascular disease and the promotion of tumor cell growth. Recently, we showed that cysteine, in the presence of iron, promotes arginase activity by driving the Fenton reaction. In the present report, we showed that induction of oxidative stress in erythroleukemic cells with the thiol-specific oxidant, diamide, led to an increase in arginase activity by 42% (P = 0.02; vs. control). By using specific antibodies, it was demonstrated that this increase correlated with an increase in arginase-1 levels in the cells and with corresponding decreases in glutathione and protein thiol levels. Treatment of cells with aurothiomalate (ATM), a protein thiol-complexing agent, diminished the activity of arginase and arginase-1 levels by 19.5 and 35.2%, respectively (vs. control) and significantly decreased both glutathione and protein thiol levels, further implicating the thiol redox system in the cellular activation of arginase. Furthermore, diamide significantly altered the kinetics of arginase, resulting in the doubling of its V(max) (vs. control). Our presented data demonstrate, for the first time that the intracellular arginase activation is may be enhanced in part, via a cellular thiol-mediated mechanism.

Associated risk factors for silent cerebral infarcts in sickle cell anemia: low baseline hemoglobin, sex, and relative high systolic blood pressure.

The most common form of neurologic injury in sickle cell anemia (SCA) is silent cerebral infarction (SCI). In the Silent Cerebral Infarct Multi-Center Clinical Trial, we sought to identify risk factors associated with SCI. In this cross-sectional study, we evaluated the clinical history and baseline laboratory values and performed magnetic resonance imaging of the brain in participants with SCA (HbSS or HbSß° thalassemia) between the ages of 5 and 15 years with no history of overt stroke or seizures. Neuroradiology and neurology committees adjudicated the presence of SCI. SCIs were diagnosed in 30.8% (251 of 814) participants who completed all evaluations and had valid data on all prespecified demographic and clinical covariates. The mean age of the participants was 9.1 years, with 413 males (50.7%). In a multivariable logistic regression analysis, lower baseline hemoglobin concentration (P < .001), higher baseline systolic blood pressure (P = .018), and male sex (P = .030) were statistically significantly associated with an increased risk of an SCI. Hemoglobin concentration and systolic blood pressure are risk factors for SCI in children with SCA and may be therapeutic targets for decreasing the risk of SCI. This study is registered at www.clinicaltrials.gov as #NCT00072761.

Transfusional iron overload in children with sickle cell anemia on chronic transfusion therapy for secondary stroke prevention.

Chronic transfusion reduces the risk of recurrent stroke in children with sickle cell anemia (SCA) but leads to iron loading. Management of transfusional iron overload in SCA has been reported as suboptimal [1], but studies characterizing monitoring and treatment practices for iron overload in children with SCA, particularly in recent years with the expansion of chelator options, are lacking. We investigated the degree of iron loading and treatment practices of 161 children with SCA receiving transfusions for a history of stroke who participated in the Stroke with Transfusions Changing to Hydroxyurea (SWiTCH) trial. Data obtained during screening, including past and entry liver iron concentration (LIC) measurements, ferritin values, and chelation were analyzed. The mean age at enrollment was 12.9 ± 4 years and the mean duration of transfusion was 7 ± 3.8 years. Baseline LIC (median 12.94 mg/g dw) and serum ferritin (median 3,164 ng/mL) were elevated. Chelation therapy was initiated after a mean of 2.6 years of transfusions. At study entry, 137 were receiving chelation, most of whom (90%) were receiving deferasirox. This study underscores the need for better monitoring of iron burden with timely treatment adjustments in chronically transfused children with SCA.

The impact of parenteral narcotic choice in the development of acute chest syndrome in sickle cell disease.

BACKGROUND: Acute chest syndrome (ACS) represents a serious morbidity and often fatal complication in patients with sickle cell disease. Painful episodes which require hospitalization are most often treated with opioids, which may then influence the development of ACS. Nalbuphine is a parenteral opioid which effectively treats pain and may cause less ACS. PROCEDURE: This retrospective chart review documented 988 admissions for painful episodes at 2 institutions and recorded the incidence of ACS and opioid used. RESULTS: At the Children's Hospital in St Louis, Missouri, the incidence of ACS in patients treated with morphine alone was 10.8% versus at the Children's Mercy Hospital in Kansas City, Missouri, the incidence was 2.1% for patients treated solely with nalbuphine. CONCLUSIONS: When nalbuphine is used alone as the single parenteral opioid agent to treat painful episodes in patients with sickle cell disease, the incidence of ACS is less than when compared with other opioids used to treat pain.

Silent cerebral infarcts occur despite regular blood transfusion therapy after first strokes in children with sickle cell disease.

Children with sickle cell disease (SCD) and strokes receive blood transfusion therapy for secondary stroke prevention; despite this, approximately 20% experience second overt strokes. Given this rate of second overt strokes and the clinical significance of silent cerebral infarcts, we tested the hypothesis that silent cerebral infarcts occur among children with SCD being transfused for secondary stroke prevention. A prospective cohort enrolled children with SCD and overt strokes at 7 academic centers. Magnetic resonance imaging and magnetic resonance angiography of the brain were scheduled approximately every 1 to 2 years; studies were reviewed by a panel of neuroradiologists. Eligibility criteria included regularly scheduled blood transfusion therapy. Forty children were included; mean pretransfusion hemoglobin S concentration was 29%. Progressive cerebral infarcts occurred in 45% (18 of 40 children) while receiving chronic blood transfusion therapy; 7 had second overt strokes and 11 had new silent cerebral infarcts. Worsening cerebral vasculopathy was associated with new cerebral infarction (overt or silent; relative risk = 12.7; 95% confidence interval, 2.65-60.5, P = .001). Children with SCD and overt strokes receiving regular blood transfusion therapy experience silent cerebral infarcts at a higher rate than previously recognized. Additional therapies are needed for secondary stroke prevention in children with SCD.

Mutation in erythroid specific transcription factor KLF1 causes Hereditary Spherocytosis in the Nan hemolytic anemia mouse model.

KLF1 regulates definitive erythropoiesis of red blood cells by facilitating transcription through high affinity binding to CACCC elements within its erythroid specific target genes including those encoding erythrocyte membrane skeleton (EMS) proteins. Deficiencies of EMS proteins in humans lead to the hemolytic anemia Hereditary Spherocytosis (HS) which includes a subpopulation with no known genetic defect. Here we report that a mutation, E339D, in the second zinc finger domain of KLF1 is responsible for HS in the mouse model Nan. The causative nature of this mutation was verified with an allelic test cross between Nan/+ and heterozygous Klf1(+/-) knockout mice. Homology modeling predicted Nan KLF1 binds CACCC elements more tightly, suggesting that Nan KLF1 is a competitive inhibitor of wild-type KLF1. This is the first association of a KLF1 mutation with a disease state in adult mammals and also presents the possibility of being another causative gene for HS in humans.

Serum ferritin level changes in children with sickle cell disease on chronic blood transfusion are nonlinear and are associated with iron load and liver injury.

Chronic blood transfusion is increasingly indicated in patients with sickle cell disease. Measuring resulting iron overload remains a challenge. Children without viral hepatitis enrolled in 2 trials for stroke prevention were examined for iron overload (STOP and STOP2; n = 271). Most received desferrioxamine chelation. Serum ferritin (SF) changes appeared nonlinear compared with prechelation estimated transfusion iron load (TIL) or with liver iron concentrations (LICs). Averaged correlation coefficient between SF and TIL (patients/observations, 26 of 164) was r = 0.70; between SF and LIC (patients/observations, 33 of 47) was r = 0.55. In mixed models, SF was associated with LIC (P = .006), alanine transaminase (P = .025), and weight (P = .026). Most patients with SF between 750 and 1500 ng/mL had a TIL between 25 and 100 mg/kg (72.8% +/- 5.9%; patients/observations, 24 of 50) or an LIC between 2.5 and 10 mg/g dry liver weight (75% +/- 0%; patients/observations, 8 of 9). Most patients with SF of 3000 ng/mL or greater had a TIL of 100 mg/kg or greater (95.3% +/- 6.7%; patients/observations, 7 of 16) or an LIC of 10 mg/g dry liver weight or greater (87.7% +/- 4.3%; patients/observations, 11 of 18). Although SF changes are nonlinear, levels less than 1500 ng/mL indicated mostly acceptable iron overload; levels of 3000 ng/mL or greater were specific for significant iron overload and were associated with liver injury. However, to determine accurately iron overload in patients with intermediately elevated SF levels, other methods are required. These trials are registered at www.clinicaltrials.gov as #NCT00000592 and #NCT00006182.

Growth inhibitory and differentiation effects of chloroquine and its analogue on human leukemic cells potentiate fetal hemoglobin production by targeting the polyamine pathway.

Elevated arginase activity has been implicated in several pathological conditions in sickle cell disease (SCD) and other inflammatory disorders. Recently, we showed that chloroquine (CQ), an anti-malarial and anti-rheumatoid drug, displays a competitive mode of inhibition on sickle erythrocyte arginase. However, the effects of CQ and its analogue, hydroxychloroquine (HCQ) on erythroid differentiation leading to induced fetal hemoglobin (Hb F) production is unknown. In the present study, we obtained evidence of the anti-proliferative and differentiation effects of CQ and HCQ at pharmacologically attainable concentrations. This differentiation effect was linked to a dose-dependent inhibition of arginase activity and induced hemoglobinization, as Hb F synthesis was increased by 3.4- and 3.2-fold for CQ or HCQ, respectively. Treatment of K562 cells with lipopolysaccharide (LPS) or 8-bromo-cAMP (Br-cAMP) failed to reverse the inhibitory effects of CQ or HCQ on arginase activity. Indeed, the combination of Br-cAMP with CQ in LPS-treated cells resulted in a significant enhancement of Hb F and total hemoglobin production. Further, we showed that CQ or HCQ maximally stimulated intracellular cGMP levels by 6.6- and 3.0-fold at 6 and 3h, respectively, as demonstrated by immunosorbent assay. However, co-treatment of K562 cells with CQ or HCQ in the presence of inhibitors of sGC-PKG-pathways reduced Hb F stimulation, suggesting the possible involvement of the sGC-PKG pathway. This is the first evidence demonstrating the capacity of anti-rheumatoid drugs to modulate the arginine-pathway and result in the enhancement of Hb F production, and thus may provide a paradigm for targeted therapy of hemoglobinopathies and other inflammation-related disorders.

Cysteine-iron promotes arginase activity by driving the Fenton reaction.

Impairment of nitric oxide bioavailability secondary to increased arginase activity and overproduction of reactive oxygen species (ROS) is thought to be a major cause of vascular complications in sickle cell disease (SCD). However, the role of ROS in the induction of arginase activity is unknown. This study investigated whether the mechanism of arginase activation involves the ROS produced during oxidative stress. Our study reveals that cysteine-iron dose-dependently stimulated arginase activity with a corresponding increase in (.)OH radical formation. The ()OH radicals produced were significantly inhibited by salicylic acid derivatives and superoxide dismutase. Surprisingly, the inhibition of (.)OH radicals parallels the inhibition of arginase activity, thus suggesting the role of cysteine-iron in the stimulation of arginase via the Fenton reaction. This is the first evidence demonstrating the participation of (.)OH radicals in the stimulation of arginase activity, and thus provides novel avenues for therapeutic modalities in hemoglobinopathies and other inflammation-mediated diseases.

A colorimetric microplate assay method for high-throughput analysis of arginase activity in vitro.

Several analytical methods have been developed for the determination of arginase (l-arginine amidinohydrolase) activity in physiological samples. These methods are limited by the considerable effort and time required to obtain reliable and reproducible measurements. Here we describe a simple high-throughput colorimetric assay for the determination of arginase activity based on the ornithine-ninhydrin reaction. This method is an improvement over the original single cuvette assay developed by Chinard in that no boiling step is required. The turnaround time has been reduced, with improved precision and reproducibility. The method was extended to the determination of arginase activity in human leukemic (K562) cells and sickle erythrocytes. We believe that the method will find applications for routine analysis as well as for characterizing the action of novel and potent inhibitors on arginase activity.

In vitro evidence of the inhibitory capacity of chloroquine on arginase activity in sickle erythrocytes.

Increased levels of erythrocyte arginase activity are believed to play a key role in the pathogenesis of sickle cell disease (SCD). Because increased arginase activity has been implicated in the exacerbation of pulmonary hypertension in SCD, this enzyme is considered an important therapeutic target for identifying new drugs to treat and manage SCD and other inflammatory disorders. Although chloroquine (CQ) is prescribed as an anti-malarial and anti-rheumatoid drug, the mechanism of its anti-inflammatory activity is largely unknown. The present study found that CQ inhibited arginase in a dose-dependent manner, and also displayed a linear competitive inhibition on sickle erythrocyte arginase. The apparent K(M) values in the presence of inhibitor were considerably reduced at both physiological and slightly acidic pHs. Slope replots of the double reciprocal plots at pH 6.8 and 7.4 also indicated simple competitive inhibitory mechanism of CQ, and Ki values for CQ were within micromolar levels. To our knowledge, this is the first example of an anti-malarial and anti-inflammatory agent displaying competitive inhibition kinetics on arginase. The outcome of this study may provide a template for the rational design of specific agents either alone or in combination with CQ for the inhibition of arginase activity.

Exchange blood transfusion compared with simple transfusion for first overt stroke is associated with a lower risk of subsequent stroke: a retrospective cohort study of 137 children with sickle cell anemia.

A retrospective cohort study of children with sickle cell anemia (SCA) and strokes was used to test the hypothesis that exchange transfusion at the time of stroke presentation more effectively prevents second strokes than does simple transfusion. Children receiving simple transfusion had a 5-fold greater relative risk (95% confidence interval = 1.3 to 18.6) of second stroke than those receiving exchange transfusion.

Disseminated nontuberculous mycobacterial infections in sickle cell anemia patients.

Nontuberculous mycobacteria (NTM) are ubiquitous in nature and have been implicated in skin/soft-tissue, pulmonary, middle ear, bone, and surgical/traumatic wound infections. Disseminated disease occurs infrequently and almost exclusively in the immunocompromised. We describe the first 2 reported cases of disseminated Mycobacterium fortuitum infection in teenagers with sickle hemoglobinopathy. Both had central venous catheters (CVCs), frequent admissions for vaso-occlusive painful episode and received hydroxyurea. Diagnosis was confirmed by multiple positive blood cultures and pulmonary dissemination occurred in both. Both had successful treatment after CVC removal and combination drug therapy. Positive cultures persisted in 1 patient due to drug resistance emphasizing the need for accurate susceptibility data. NTM infection should be added to the list of pathogens in sickle cell patients with CVCs and fever. Investigation for disseminated disease should be undertaken based on clinical signs and symptoms. Although some routine blood culture systems can identify NTM, specific mycobacterial blood culture is optimal. Removal of involved CVCs is essential and treatment of NTM must be guided by susceptibilities. As dissemination almost always occurs in those with impaired cellular immunity, human immunodeficiency virus testing should be performed. Hydroxyurea may be a risk factor for dissemination and needs further evaluation.

Modulation of erythrocyte arginase activity in sickle cell disease patients during hydroxyurea therapy.

An elevated erythrocyte arginase activity with a corresponding decrease in nitric oxide (NO) level has been implicated in the pathophysiology of sickle cell disease (SCD). Recent studies have shown that hydroxyurea (HU) increases the production of NO, which increases the soluble guanylate cyclase activity and fetal haemoglobin (HbF) synthesis. To study the effects of HU on the arginase and nitric oxide synthase (NOS) activities in SCD patients, we compared levels of arginase activity and NO metabolites in red blood cells and plasma, respectively, from 23 patients with SCD (HbSS) receiving HU therapy, with those of 12 SCD patients not receiving HU treatment. Patients on HU therapy showed significantly lower arginase activity than that of HbSS patients not on HU therapy (1.36+/-0.2 U/10(8) cells vs. 3.31+/-0.29 U/10(8) cells). NOS activity was higher in patients on HU therapy than in untreated patients (0.72+/-0.4 nmol/ml/min vs. 0.35+/-0.15 nmol/ml/min, P<0.05). Among the HU-treated patients, the decreased level of arginase activity correlated (r=0.71) with HbF level as well as the mean corpuscular haemoglobin content. These data suggest that one of the beneficial effects of HU in vivo may involve the regulation of arginase activity and a concomitant induction of NOS activity, which may lead to an increased production of NO. The outcome of this study may lead to the development of improved NO-based treatments for SCD.