SUCCESSES AND PITFALLS IN ORPHAN DRUG DEVELOPMENT FOR SICKLE CELL DISEASE.

Sickle cell disease (SCD) is a hereditary red cell disorder with large global burden problem. In the United States (US) and Europe, medicines may qualify for orphan designation (OD), a regulatory status that provides incentives to boost development. We evaluated the development of new therapies for SCD using data for OD granted in the US and Europe over the last two decades (2000-2021). We analyzed their characteristics, pathophysiological targets, trends, and OD sponsors. We then investigated the approval outcomes, including the phase success rate and reasons for discontinuation across different variables. We identified 57 OD for SCD: 43 (75.4%) small molecules, 32 (56.1%) for oral administration, and 36 (63.1%) for chronic use to prevent SCD complications. At the end of the study (2021) development of 34/57 ODs was completed. Four OD were approved with a success rate of 11.8%. Products targeting upstream causative events of SCD pathophysiology had a 1.8 higher success rate compared to products targeting disease consequences. Large companies showed a fourfold higher success rate compared to small-medium enterprises. Failures in clinical development were mainly seen in Phase 3 for a lack of efficacy on vaso-occlusive crisis as the primary study endpoint, likely related to variable definitions and heterogeneity of pain scoring and treatment. Both advances in SCD knowledge and regulatory incentives paved the way for new therapies for SCD. Our finding of high failure rates in late-stage clinical development signals the need for better early-stage predictive models, also in the context of meaningful clinical endpoints.

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).

Reducing transfusion utilization for children with sickle cell anemia in sub-Saharan Africa with hydroxyurea: Analysis from the phase I/II REACH trial.

Children with sickle cell anemia (SCA) in Africa frequently require transfusions for SCA complications. Despite limited blood supplies, strategies to reduce their transfusion needs have not been widely evaluated or implemented. We analyzed transfusion utilization in children with SCA before and during hydroxyurea treatment. REACH (Realizing Effectiveness Across Continents with Hydroxyurea, NCT01966731) is a longitudinal Phase I/II trial of hydroxyurea in children with SCA from Angola, Democratic Republic of Congo, Kenya, and Uganda. After enrollment, children had a two-month pre-treatment screening period followed by 6 months of fixed-dose hydroxyurea (15-20 mg/kg/day), 18 months of dose escalation, and then stable dosing at maximum tolerated dose (MTD). Characteristics associated with transfusions were analyzed with univariate and multivariable models. Transfusion incidence rate ratios (IRR) across treatment periods were calculated. Among 635 enrolled children with 4124 person-years of observation, 258 participants (40.4%) received 545 transfusions. The transfusion rate per 100 person-years was 43.2 before hydroxyurea, 21.7 on fixed-dose, 14.5 during dose escalation, and 10.8 on MTD. During MTD, transfusion incidence was reduced by 75% compared to pre-treatment (IRR 0.25, 95% confidence interval [CI] 0.18-0.35, p < .0001), and by 50% compared to fixed dose (IRR 0.50, 95% CI 0.39-0.63, p < .0001). Hydroxyurea at MTD decreases transfusion utilization in African children with SCA. If widely implemented, universal testing and hydroxyurea treatment at MTD could potentially prevent 21% of all pediatric transfusions administered in sub-Saharan Africa. Increasing hydroxyurea access for SCA should decrease the transfusion burden and increase the overall blood supply.

Hydroxyurea reduces infections in children with sickle cell anemia in Uganda.

ABSTRACT: After starting hydroxyurea treatment, Ugandan children with sickle cell anemia had 60% fewer severe or invasive infections, including malaria, bacteremia, respiratory tract infections, and gastroenteritis, than before starting hydroxyurea treatment (incidence rate ratio, 0.40 [95% confidence interval, 0.29-0.54]; P < .001).

Screening for haemoglobin disorders: One size may not fit all.

Accurate laboratory screening for sickle cell disease and other haemoglobin disorders is expanding worldwide. Two new reports describe different methods and strategies for screening in Mali and Denmark, respectively, and their encouraging results suggest that countries should tailor their screening programmes according to local needs, resources and opportunities. Commentary on: Guindo et al. Potential for a large-scale newborn screening strategy for sickle cell disease in Mali: a comparative diagnostic performance study of two rapid diagnostic tests (SickleScan® and HemotypeSC®) on cord blood. Br J Haematol 2024;204:337-345 and Gravholt et al. The Danish national haemoglobinopathy screening programme: report from 16 years of screening in a low-prevalence, non-endemic region. Br J Haematol 2024;204:329-336.

Rationale, Development, and Validation of HdxSim, a Clinical Decision Support Tool for Model-Informed Precision Dosing of Hydroxyurea for Children with Sickle Cell Anemia.

Hydroxyurea treatment for children with sickle cell anemia (SCA) is effective and life-saving. Stepwise escalation to maximum tolerated dose (MTD) provides optimal benefits, but is logistically challenging and time-consuming, especially in low-income countries where most people with SCA live. Model-informed precision dosing (MIPD) of hydroxyurea expedites MTD determination and improves outcomes compared with trial-and-error dose adjustments. HdxSim, a user-friendly, online, clinical decision support tool was developed to facilitate hydroxyurea MIPD and evaluated using real-world pharmacokinetic (PK) data. First-dose hydroxyurea PK profiles were analyzed from two clinical trial datasets (Hydroxyurea Study of Long-Term Effects (HUSTLE), NCT00305175 and Therapeutic Response Evaluation and Adherence Trial (TREAT), NCT02286154). Areas under the concentration-time curve (AUC) estimated by HdxSim were compared with those determined using traditional trapezoidal methodology and PK software (MWPharm-DOS). The doses predicted by HdxSim and MWPharm-DOS were compared with the observed clinical MTD. For HUSTLE participants, HdxSim accurately estimated hydroxyurea AUC compared with the trapezoidal method, with < 20% variance. The average (mean ± SD) AUC for TREAT participants estimated with HdxSim (68.6 ± 18.0 mg*hour/L) was lower than MWPharm-DOS (78.6 ± 20.7 mg*hour/L, P = 0.012), but the average recommended doses were not different (425 vs. 423 mg/day, P = 0.97). Moreover, HdxSim was non-inferior to MWPharm-DOS at predicting clinical MTD (absolute difference 3.9 ± 5.8 vs. 4.9 ± 8.2 mg/kg/day, P = 0.19). HdxSim accurately estimates hydroxyurea exposure and is noninferior to traditional PK approaches at predicting the clinical hydroxyurea MTD. Hydroxyurea dosing based on target exposure leads to improved outcomes in children with SCA, and has the potential to make PK-guided hydroxyurea dosing more accessible to this neglected population globally.

Prospective identification of variables as outcomes for treatment (PIVOT): study protocol for a randomised, placebo-controlled trial of hydroxyurea for Ghanaian children and adults with haemoglobin SC disease.

BACKGROUND: Haemoglobin SC (HbSC) is a common form of sickle cell disease (SCD), especially among individuals of West African ancestry. Persons with HbSC disease suffer from the same clinical complications and reduced quality of life that affect those with sickle cell anaemia (HbSS/Sß0). Retrospective anecdotal data suggest short-term safety and benefits of hydroxyurea for treating HbSC, yet rigorous prospective data are lacking regarding optimal dosing, clinical and laboratory effects, long-term safety and benefits, and appropriate endpoints to monitor. Prospective Investigation of Variables as Outcomes for Treatment (PIVOT) was designed with three aims: (1) to measure the toxicities of hydroxyurea treatment on laboratory parameters, (2) to assess the effects of hydroxyurea treatment on sickle-related clinical and laboratory parameters, and (3) to identify study endpoints suitable for a future definitive phase III trial of hydroxyurea treatment of HbSC disease. METHODS: PIVOT is a randomised, placebo-controlled, double blind clinical trial of hydroxyurea. Approximately 120 children and 120 adults ages 5-50 years with HbSC disease will be enrolled, screened for 2 months, and then randomised 1:1 to once-daily oral hydroxyurea or placebo. Study treatment will be prescribed initially at 20 ± 5 mg/kg/day with an opportunity to escalate the dose twice over the first 6 months. After 12 months of blinded study treatment, all participants will be offered open-label hydroxyurea for up to 4 years. Safety outcomes include treatment-related cytopenias, whole blood viscosity, and adverse events. Efficacy outcomes include a variety of laboratory and clinical parameters over the first 12 months of randomised treatment, including changes in haemoglobin and fetal haemoglobin, intracranial arterial velocities measured by transcranial Doppler ultrasound, cerebral oxygenation using near infrared spectrometry, spleen volume and kidney size by ultrasound, proteinuria, and retinal imaging. Exploratory outcomes include functional erythrocyte analyses with ektacytometry for red blood cell deformability and point-of-sickling, patient-reported outcomes using the PROMIS questionnaire, and 6-min walk test. DISCUSSION: For children and adults with HbSC disease, PIVOT will determine the safety of hydroxyurea and identify measurable changes in laboratory and clinical parameters, suitable for future prospective testing in a definitive multi-centre phase III clinical trial. TRIAL REGISTRATION: PACTR, PACTR202108893981080. Registered 24 August 2021, https://pactr.samrc.ac.za.

Sustained and Boosted Antibody Responses in Breast Milk After Maternal SARS-CoV-2 Vaccination.

Background: Pregnant and lactating women were not included in the initial large vaccine clinical trials for SARS-CoV-2 (COVID) infection. Delineating the antibody titers in serum and breast milk of lactating women is important to determine the safety and benefits of vaccination in this special population. Objective: To investigate COVID vaccinations in breastfeeding dyads and effects on lactation, the Antibody Detection of Vaccine-Induced Secretory Effects trial (ADVISE) prospectively evaluated anti-COVID antibodies in serum and breast milk after initial paired and booster vaccines. Methods: This is a prospective longitudinal surveillance cohort study of lactating women. Eligibility criteria included ≥18 years of age, currently lactating, and at enrollment either received COVID vaccination within the past 60 days or planning vaccination within 60 days. Results: Among 63 lactating mothers, COVID vaccination led to breast milk secretory IgA (sIgA) and IgG antibodies with consistent viral neutralizing activity. Milk sIgA titers increased further after second vaccination and were prolonged after a third booster dose, including women with extended breastfeeding beyond 12 months. Milk IgG antibody titers were higher and more sustained than sIgA. Antibody titers were not associated with individual dyad characteristics or vaccine manufacturer. Vaccine-induced antibodies from milk were not detected in infant circulation. Conclusions and Relevance: Maternal COVID vaccination during lactation is well tolerated and generates sustained and boosted antibody responses in breast milk. COVID-specific sIgA and IgG antibodies with neutralizing activity are found in breast milk, including boosted mothers who continue breastfeeding beyond 12 months. These data support universal COVID vaccinations for all lactating mothers, including booster immunizations during extended breastfeeding (NCT04895475).

Cost-Effectiveness of Hydroxyurea for Sickle Cell Anemia in a Low-Income African Setting: A Model-Based Evaluation of Two Dosing Regimens.

BACKGROUND AND OBJECTIVE: The disease burden of sickle cell anemia (SCA) in sub-Saharan African (SSA) countries is substantial, with many children dying without an established diagnosis or proper treatment. The global burden of SCA is increasing each year, making therapeutic intervention a high priority. Hydroxyurea is the only disease-modifying therapy with proven feasibility and efficacy suitable for SSA; however, no one has quantified the health economic implications of its use. Therefore, from the perspective of the health care provider, we estimated the incremental cost-effectiveness of hydroxyurea as a fixed-dose regimen or maximum tolerated dose (MTD) regimen, versus SCA care without hydroxyurea. METHODS: We estimated the cost of providing outpatient treatment at a pediatric sickle cell clinic in Kampala, Uganda. These estimates were used in a discrete-event simulation model to project mean costs (2021 US$), disability-adjusted life years (DALYs), and consumption of blood products per patient (450 mL units), for patients between 9 months and 18 years of age. We calculated cost-effectiveness as the ratio of incremental costs over incremental DALYs averted, discounted at 3% annually. To test the robustness of our findings, and the impact of uncertainty, we conducted probabilistic and one-way sensitivity analyses, scenario analysis, and price threshold analyses. RESULTS: Hydroxyurea treatment averted an expected 1.37 DALYs and saved US$ 191 per patient if administered at the MTD, compared with SCA care without hydroxyurea. In comparison, hydroxyurea at a fixed dose averted 0.80 DALYs per patient at an incremental cost of US$ 2. The MTD strategy saved 11.2 (95% CI 11.1-11.4) units of blood per patient, compared with 9.1 (95% CI 9.0-9.2) units of blood per patient at the fixed-dose alternative. CONCLUSIONS: Hydroxyurea at MTD is likely to improve quality of life and reduce the consumption of blood products for children with SCA living in Uganda. Compared with a fixed dose regimen, treatment dosing at MTD is likely to be a cost-effective treatment for SCA, using realistic ranges of hydroxyurea costs that are relevant across SSA. Compared with no use of the drug, hydroxyurea could lead to substantial net savings per patient, while reducing the disease morbidity and mortality and increasing quality of life.

Hydroxyurea pharmacokinetics and precision dosing in low-resource settings.

Introduction: Hydroxyurea is effective disease-modifying treatment for sickle cell anemia (SCA). Escalation to maximum tolerated dose (MTD) achieves superior benefits without additional toxicities, but requires dose adjustments with serial monitoring. Pharmacokinetic (PK)-guided dosing can predict a personalized optimal dose, which approximates MTD and requires fewer clinical visits, laboratory assessments, and dose adjustments. However, PK-guided dosing requires complex analytical techniques unavailable in low-resource settings. Simplified hydroxyurea PK analysis could optimize dosing and increase access to treatment. Methods: Concentrated stock solutions of reagents for chemical detection of serum hydroxyurea using HPLC were prepared and stored at -80C. On the day of analysis, hydroxyurea was serially diluted in human serum, then spiked with N-methylurea as an internal standard and analyzed using two commercial HPLC machines: 1) standard benchtop Agilent with 449 nm detector and 5 micron C18 column; and 2) portable PolyLC with 415 nm detector and 3.5 micron C18 column. After validation in the United States, the portable HPLC and chemicals were transported to Tanzania. Results: A calibration curve using hydroxyurea 2-fold dilutions ranging from 0 to 1000 µM was plotted against the hydroxyurea:N-methylurea ratio. In the United States, both HPLC systems yielded calibration curves with R2 > 0.99. Hydroxyurea prepared at known concentrations confirmed accuracy and precision within 10%-20% of the actual values. Both HPLC systems measured hydroxyurea with <10% variance from the prepared concentrations, and paired analysis of samples on both machines documented <15% variance. Serial measurements of 300 and 100 µM concentrations using the PolyLC system were precise with 2.5% coefficient of variance. After transport to Tanzania with setup and training, the modified PolyLC HPLC system produced similar calibration curves with R2 > 0.99. Conclusion: Increasing access to hydroxyurea for people with SCA requires an approach that eases financial and logistical barriers while optimizing safety and benefits, especially in low-resource settings. We successfully modified a portable HPLC instrument to quantify hydroxyurea, validated its precision and accuracy, and confirmed capacity building and knowledge transfer to Tanzania. HPLC measurement of serum hydroxyurea is now feasible in low-resource settings using available laboratory infrastructure. PK-guided dosing of hydroxyurea will be tested prospectively to achieve optimal treatment responses.

Evaluation of association of anti-PEG antibodies with anaphylaxis after mRNA COVID-19 vaccination.

BACKGROUND: The mechanism for anaphylaxis following mRNA COVID-19 vaccination has been widely debated; understanding this serious adverse event is important for future vaccines of similar design. A mechanism proposed is type I hypersensitivity (i.e., IgE-mediated mast cell degranulation) to polyethylene glycol (PEG). Using an assay that, uniquely, had been previously assessed in patients with anaphylaxis to PEG, our objective was to compare anti-PEG IgE in serum from mRNA COVID-19 vaccine anaphylaxis case-patients and persons vaccinated without allergic reactions. Secondarily, we compared anti-PEG IgG and IgM to assess alternative mechanisms. METHODS: Selected anaphylaxis case-patients reported to U.S. Vaccine Adverse Event Reporting System December 14, 2020-March 25, 2021 were invited to provide a serum sample. mRNA COVID-19 vaccine study participants with residual serum and no allergic reaction post-vaccination ("controls") were frequency matched to cases 3:1 on vaccine and dose number, sex and 10-year age category. Anti-PEG IgE was measured using a dual cytometric bead assay (DCBA). Anti-PEG IgG and IgM were measured using two different assays: DCBA and a PEGylated-polystyrene bead assay. Laboratorians were blinded to case/control status. RESULTS: All 20 case-patients were women; 17 had anaphylaxis after dose 1, 3 after dose 2. Thirteen (65 %) were hospitalized and 7 (35 %) were intubated. Time from vaccination to serum collection was longer for case-patients vs controls (post-dose 1: median 105 vs 21 days). Among Moderna recipients, anti-PEG IgE was detected in 1 of 10 (10 %) case-patients vs 8 of 30 (27 %) controls (p = 0.40); among Pfizer-BioNTech recipients, it was detected in 0 of 10 case-patients (0 %) vs 1 of 30 (3 %) controls (p >n 0.99). Anti-PEG IgE quantitative signals followed this same pattern. Neither anti-PEG IgG nor IgM was associated with case status with both assay formats. CONCLUSION: Our results support that anti-PEG IgE is not a predominant mechanism for anaphylaxis post-mRNA COVID-19 vaccination.

Simultaneous adjunctive treatment of malaria and its coevolved genetic disorder sickle cell anemia.

Effective treatments for genetic disorders that coevolved with pathogens require simultaneous betterment of both conditions. Hydroxyurea (HU) offers safe and efficacious treatment for sickle cell anemia (SCA) by reducing clinical complications, transfusions, and death rates. Despite concerns that the HU treatment for SCA would increase infection risk by the human malaria Plasmodium falciparum, (the genetic driver of the sickle mutation), HU instead reduced clinical malaria. We used physiologically relevant drug exposures that mimic in vivo pharmacokinetics in humans. Under these conditions, we showed that HU and other ribonucleotide reductase (RNR) inhibitors have significant, intrinsic killing activity in vitro against schizont stages of P falciparum in both normal and sickle red blood cells. Long-term in vitro selection with HU increased the expression of Pfrnr genes but showed a low risk of eliciting stably resistant parasites or compromising the potency of current antimalarial drugs. Additive activity devoid of antagonism by HU was observed with a wide spectrum of commonly used antimalarial treatments. These data endorse broad, safe, and long-term use of HU for SCA in malaria-endemic countries and provide a novel biological model for the treatment of a genetic disorder with simultaneous, adjunct therapy of a life-threatening infection needed in a global health setting.

Hydroxyurea treatment for sickle cell anemia during pregnancy and lactation: Current evidence and knowledge gaps.

Sickle cell anemia (SCA) is a life-threatening genetic condition contributing to high-risk pregnancies affecting both the mother and fetus. With improved management of children with SCA, this life-threatening hematological disorder has evolved into a chronic disease of adults, and consequently parenthood has now become a possible and important life goal for many patients. Providing continuous management with healthy red blood cell function and avoiding SCA-associated complications, such as pain crises, acute chest syndrome, and stroke, are crucial for a healthy pregnancy. Despite its excellent safety profile in non-pregnant adults and children, and based on theoretical concerns derived from data using animal models and supraphysiological dosing, hydroxyurea is currently contraindicated for pregnant and lactating women with SCA. Clinical experience of hydroxyurea use during pregnancy is increasingly reported, however, and has shown inconsistent results of fetal or infant adverse effects. How the hydroxyurea exposure level may correlate with pregnancy outcomes is still unclear. Accordingly, efforts should be made to systemically evaluate exposure and safety of hydroxyurea treatment during pregnancy and lactation. Novel approaches such as physiologically based pharmacokinetic (PBPK) modeling, coupled with the ex vivo human placental cotyledon perfusion assay, provide opportunities to understand hydroxyurea exposure not only in pregnant women but also in the developing fetus. Combined with animal data, research using these approaches might be able to define safe and effective hydroxyurea dosing regimens for pregnant and lactating women with SCA, when the benefits of continuing hydroxyurea treatment likely outweigh the risks of non-treatment, by avoiding substantial morbidity and even mortality for both mothers and infants.

Hydroxyurea with dose escalation for primary stroke risk reduction in children with sickle cell anaemia in Tanzania (SPHERE): an open-label, phase 2 trial.

BACKGROUND: Transcranial Doppler screening with chronic transfusions reduces stroke risk in children with sickle cell anaemia but is not feasible in low-resource settings. Hydroxyurea is an alternative treatment to decrease stroke risk. We aimed to estimate stroke risk in children with sickle cell anaemia in Tanzania and to determine the efficacy of hydroxyurea to decrease and prevent stroke. METHODS: We did an open-label, phase 2 trial (SPHERE) at Bugando Medical Centre, Mwanza, Tanzania. Children aged 2-16 years with a diagnosis of sickle cell anaemia confirmed by haemoglobin electrophoresis were eligible for enrolment. Participants had transcranial Doppler ultrasound screening by a local examiner. Participants with elevated Doppler velocities, either conditional (170-199 cm/s) or abnormal (≥200 cm/s), received oral hydroxyurea starting at 20 mg/kg once daily and escalated every 8 weeks by 5 mg/kg per day to the maximum tolerated dose. Participants with normal Doppler velocities (<170 cm/s) received usual care from the sickle cell anaemia clinic and were rescreened after 12 months to determine whether they qualified for treatment on trial. The primary endpoint was change in transcranial Doppler velocity from the baseline visit to after 12 months of hydroxyurea treatment, analysed in all patients who had paired baseline and follow-up measurements collected after 12 months of treatment. Safety was analysed in the per-protocol population (all participants who received study treatment). This study is registered with ClinicalTrials.gov, NCT03948867. FINDINGS: Between April 24, 2019, and April 9, 2020, 202 children were enrolled and had transcranial Doppler screening. Sickle cell anaemia was confirmed by DNA-based testing in 196 participants (mean age 6·8 years [SD 3·5], 103 [53%] were female, and 93 [47%] were male). At the baseline screening, 47 (24%) of 196 participants had elevated transcranial Doppler velocities (43 [22%] conditional, four [2%] abnormal); 45 initiated hydroxyurea at a mean dose of 20·2 mg/kg per day (SD 1·4) with escalation to a mean dose of 27·4 mg/kg per day (5·1) after 12 months. Treatment response was analysed after 12 months (± 1 month; median 11 months, IQR 11-12) and 24 months (±3 months; median 22 months, 22-22). Transcranial Doppler velocities decreased to a mean of 149 cm/s (SD 27) compared with 182 cm/s (12) at baseline, which was significantly lower than baseline (p<0·0001), with an average decline of 35 cm/s (SD 23) after 12 months of treatment in 42 participants with paired results available at baseline and 12 months. No clinical strokes occurred, and 35 (83%) of 42 participants reverted to normal transcranial Doppler velocities. Clinical adverse events were mild, and dose-limiting toxicities were uncommon. The most common grade 3 adverse events were malaria (12 [29%] episodes in 45 patients) and sepsis (13 [32%] episodes). There were three serious adverse events, none of which were treatment-related, and no treatment-related deaths occurred. INTERPRETATION: Children with sickle cell anaemia in Tanzania have a high baseline stroke risk. Hydroxyurea at the maximum tolerated dose significantly lowers transcranial Doppler velocities and reduces primary stroke risk. Transcranial Doppler screening plus hydroxyurea at the maximum tolerated dose is an effective stroke prevention strategy, supporting wider hydroxyurea access for patients with sickle cell anaemia across sub-Saharan Africa. FUNDING: American Society of Hematology, National Institutes of Health, Cincinnati Children's Research Foundation.

Zinc for infection prevention in children with sickle cell anemia: a randomized double-blind placebo-controlled trial.

Data from small clinical trials in the United States and India suggest zinc supplementation reduces infection in adolescents and adults with sickle cell anemia (SCA), but no studies of zinc supplementation for infection prevention have been conducted in children with SCA living in Africa. We conducted a randomized double-blind placebo-controlled trial to assess zinc supplementation for prevention of severe or invasive infections in Ugandan children 1.00-4.99 years with SCA. Of 252 enrolled participants, 124 were assigned zinc (10 mg) and 126 assigned placebo once daily for 12 months. The primary outcome was incidence of protocol-defined severe or invasive infections. Infection incidence did not differ between treatment arms (282 vs. 270 severe or invasive infections per 100 person-years, respectively, incidence rate ratio of 1.04 [95% confidence interval (CI), 0.81, 1.32, p=0.78]), adjusting for hydroxyurea treatment. There was also no difference between treatment arms in incidence of serious adverse events or SCA-related events. Children receiving zinc had increased serum levels after 12-months, but at study exit, 41% remained zinc deficient (<65 µg/dL). In post-hoc analysis, occurrence of stroke or death was lower in the zinc treatment arm (adjusted hazard ratio (95% CI), 0.22 (0.05, 1.00); p=0.05). Daily 10 mg zinc supplementation for 12 months did not prevent severe or invasive infections in Ugandan children with SCA, but many supplemented children remained zinc deficient. Optimal zinc dosing and the role of zinc in preventing stroke or death in SCA warrant further investigation. This trial was registered at clinicaltrials.gov as #NCT03528434.

Lack of hydroxyurea-associated mutagenesis in pediatric sickle cell disease patients.

Hydroxyurea is approved for treating children and adults with sickle cell anemia (SCA). Despite its proven efficacy, concerns remain about its mutagenic and carcinogenic potential that hamper its widespread use. Cell culture- and animal-based investigations indicate that hydroxyurea's genotoxic effects are due to indirect clastogenicity in select cell types when high dose and time thresholds are exceeded (reviewed by Ware & Dertinger, 2021). The current study extends these preclinical observations to pediatric patients receiving hydroxyurea for treatment of SCA. First, proof-of-principle experiments with testicular cancer patients exposed to a cisplatin-based regimen validated the ability of flow cytometric blood-based micronucleated reticulocyte (MN-RET) and PIG-A mutant reticulocyte (MUT RET) assays to detect clastogenicity and gene mutations, respectively. Second, these biomarkers were measured in a cross-sectional study with 26 SCA patients receiving hydroxyurea and 13 SCA patients without exposure. Finally, a prospective study was conducted with 10 SCA patients using pretreatment blood samples and after 6 or 12 months of therapy. Cancer patients exposed to cisplatin exhibited increased MN-RET within days of exposure, while the MUT RET endpoint required more time to reach maximal levels. In SCA patients, hydroxyurea induced MN-RET in both the cross-sectional and prospective studies. However, no evidence of PIG-A gene mutation was found in hydroxyurea-treated children, despite the fact that the two assays use the same rapidly-dividing, highly-exposed cell type. Collectively, these results reinforce the complementary nature of MN-RET and MUT RET biomarkers, and indicate that hydroxyurea can be clastogenic but was not mutagenic in young patients with SCA.

Genome-wide association study of early ischaemic stroke risk in Brazilian individuals with sickle cell disease implicates ADAMTS2 and CDK18 and uncovers novel loci.

Ischaemic stroke is a common complication of sickle cell disease (SCD) and without intervention can affect 11% of children with SCD before the age of 20. Within the Trans-Omics for Precision Medicine (TOPMed), a genome-wide association study (GWAS) of ischaemic stroke was performed on 1333 individuals with SCD from Brazil (178 cases, 1155 controls). Via a novel Cox proportional-hazards analysis, we searched for variants associated with ischaemic stroke occurring at younger ages. Variants at genome-wide significance (p < 5 × 10-8 ) include two near genes previously linked to non-SCD early-onset stroke (<65 years): ADAMTS2 (rs147625068, p = 3.70 × 10-9 ) and CDK18 (rs12144136, p = 2.38 × 10-9 ). Meta-analysis, which included the independent SCD cohorts Walk-PHaSST and PUSH, exhibited consistent association for variants rs1209987 near gene TBC1D32 (p = 3.36 × 10-10 ), rs188599171 near CUX1 (p = 5.89 × 10-11 ), rs77900855 near BTG1 (p = 4.66 × 10-8 ), and rs141674494 near VPS13C (1.68 × 10-9 ). Findings from this study support a multivariant model of early ischaemic stroke risk and possibly a shared genetic architecture between SCD individuals and non-SCD individuals younger than 65 years.