Efficacy and safety of early‐start deferiprone in infants and young children with transfusion‐dependent beta thalassemia: Evidence for iron shuttling to transferrin in a randomized, double‐blind, placebo‐controlled, clinical trial (START)

Children with transfusion‐dependent thalassemia (TDT) require regular blood transfusions that, without iron‐chelation therapy, lead to iron‐overload toxicities. Current practice delays chelation therapy (late‐start) until reaching iron overload (serum ferritin ≥1000 μg/L) to minimize risks of iron‐depletion. Deferiprone's distinct pharmacological properties, including iron‐shuttling to transferrin, may reduce risks of iron depletion during mild‐to‐moderate iron loads and iron overload/toxicity in children with TDT. The early‐start deferiprone (START) study evaluated the efficacy/safety of early‐start deferiprone in infants/young children with TDT. Sixty‐four infants/children recently diagnosed with beta‐thalassemia and serum ferritin (SF) between 200 and 600 μg/L were randomly assigned 1:1 to receive deferiprone or placebo for 12 months or until reaching SF‐threshold (≥1000 μg/L at two consecutive visits). Deferiprone was initiated at 25 mg/kg/day and increased to 50 mg/kg/day; some recipients' dosages increased to 75 mg/kg/day based on iron levels. The primary endpoint was the proportion of patients ≥SF‐threshold by month 12. Monthly transferrin saturation (TSAT) assessment evaluated iron‐shuttling. At baseline, there was no significant difference in mean age (deferiprone: 3.03 years, placebo: 2.63 years), SF (deferiprone: 513.8 μg/L, placebo: 451.7 μg/L), or TSAT (deferiprone: 47.98%, placebo: 43.43%) between groups. At month 12, there was no significant difference in growth or adverse event (AE) rates between groups. No deferiprone‐treated patients were iron‐depleted. At month 12, 66% of patients receiving deferiprone remained below SF threshold versus 39% of placebo (p = .045). Deferiprone‐treated patients showed higher TSAT levels and reached ≥60% TSAT threshold faster. Early‐start deferiprone was well‐tolerated, not associated with iron depletion, and efficacious in reducing iron overload in infants/children with TDT. TSAT results provide the first clinical evidence of deferiprone shuttling iron to transferrin.


| INTRODUCTION
Iron overload is the main risk factor for morbidity and mortality in patients with transfusion-dependent thalassemia unless treated with life-long iron chelation therapies such as deferiprone, deferasirox, or deferoxamine. 1 Current clinical practice delays chelation therapy in newly diagnosed patients until they reach the iron overload criterion of serum ferritin (SF) levels ≥1000 μg/L or > 10 transfusions. 2,3 This late-start strategy was thought to minimize toxicity risks of iron depletion, observed during early-start studies with deferoxamine. [4][5][6] Unfortunately, iron accumulation in organs (e.g., heart, 7,8 liver, 7,8 or pituitary gland 9 ) during late-start therapy commonly leads to iron overload in transfusion-dependent children and potentially increased toxicities later in life. 1,2 Initial studies suggested that early-start deferoxamine at high doses (50-80 mg/kg/day) may cause iron depletion and stunted growth in young children, 6 contributing to no iron chelators currently being approved for use in children younger than 2 years of age. [10][11][12] Late-start deferiprone has been found to be safe and efficacious in adults and older children, 13,14 and has distinct pharmacological properties compared with other clinical iron chelators that may facilitate "iron shuttling" to transferrin and reduce the risks of early-start chelation. 15 This iron shuttling may reduce the risks associated with both late-start iron overload and early-start iron depletion. As iron overload remains common in young transfusiondependent children, when to begin iron chelation therapy remains an important consideration.
Deferiprone has greater tissue permeability, indicated by a higher volume of distribution (V D = 90.3) 16 than deferasirox (V D = 14.4), 17 likely allowing it to better reduce iron load in organs.
Second, deferiprone has a lower iron affinity (pFe 3+ = 19.3) 15 than deferoxamine (pFe 3+ = 26.6), 15 deferasirox (pFe 3+ = 23.5), 18 and transferrin (pFe 3+ = 22.3), 15 which may reduce the risk of iron depletion from biological stores. In vitro and in vivo models suggest that, due to its lower affinity for iron, deferiprone can shuttle excess iron to unbound transferrin and, thus, increase transferrin saturation (TSAT). 15 Given that deferiprone has a lower affinity for iron than transferrin and deferoxamine and deferasirox have a higher affinity for iron than transferrin, deferiprone may cause less chelation of iron-bound transferrin that is necessary for normal iron metabolism. 4,19 This iron shuttling by deferiprone may explain the low risk of iron depletion even when there is no systemic iron overload, 20 such as in young children recently diagnosed with thalassemia. A recent randomized, nonplacebo-controlled pilot study showed that earlystart deferiprone treatment could safely delay iron accumulation without inducing toxicity. 21 To further evaluate early-start deferiprone in young children diagnosed with transfusion-dependent thalassemia, we designed a randomized, double-blind, placebo-controlled clinical trial (START; NCT03591575). The primary objective of START was to evaluate the efficacy and safety of early-start deferiprone to prevent/reduce transfusional iron overload. An additional endpoint was the evaluation of TSAT, which could indicate the ability of deferiprone to shuttle iron to transferrin. times the upper limit of normal or creatinine levels >2 times the upper limit of normal); or a history of neutropenia (absolute neutrophil count <1.5 Â 10 9 /L).
The study design and treatment regimen are shown in supplemental 2. Patients and providers were double-blinded to treatment assignment until the end of the study. Patients were randomly assigned 1:1 to receive either a deferiprone oral solution or placebo for 12 months. Patients in the deferiprone arm began with 25 mg/kg/day deferiprone, divided across three times a day dosing (TID; Ferriprox ® , Oral Solution, Chiesi, Canada). After 2 weeks, the dose was increased to 50 mg/kg/day deferiprone (TID) for all patients unless otherwise noted. If a patient had SF levels between 800 μg/L and 1000 μg/L, or SF levels increased for three consecutive visits, the dose was increased to 75 mg/kg/day (TID). Treatment was interrupted if a patient displayed confirmed mild neutropenia with an absolute neutrophil count (ANC) ≥ 1.0 Â 10 9 /L and <1.5 Â 10 9 /L. Treatment was re-initiated after ANC levels normalized or the event was determined not to indicate neutropenia. If either moderate neutropenia (ANC < 1.0 Â 10 9 /L) or agranulocytosis (ANC < 0.5 Â 10 9 /L) was confirmed, the patient was withdrawn from the study. Otherwise, treatment continued for 12 months or until patients reached the SF threshold (≥1000 μg/L at two consecutive visits). Once patients reached the SF threshold, patients were considered to have completed the study and transferred to standard chelation therapy for ethical reasons.

| Efficacy
To determine the efficacy of early-start deferiprone compared to placebo, the primary endpoint was the proportion of patients with an iron load lower than the current threshold for initiation of chelation therapy. The SF threshold in the current study was defined as SF levels ≥ 1000 μg/L at two consecutive visits by Month 12, aligning with thalassemia management guidelines. 2,3 Secondary efficacy endpoints were: The time to reach the SF iron overload threshold, the proportion of patients below the SF threshold at each month by imputation, and the mean SF or TSAT at each month.

| Iron shuttling
The ability of deferiprone to shuttle iron to transferrin was evaluated in the placebo and deferiprone groups using: (1) the proportion of patients above the TSAT threshold (defined as ≥ 60% TSAT) at each month; (2) the mean TSAT; and (3) the time to reach ≥ 60% TSAT using a Kaplan-Meier analysis. Labile plasma iron (LPI; a component of non-transferrin-bound iron) was measured at each visit. However, many LPI values were below the lower limit of quantitation and had to be treated as missing, which reduced the number of analyzable results. Therefore, LPI results were not considered evaluable for drawing conclusions.

| Safety
Adverse events (AEs) were collected from the screening visit until 30 days after last treatment administration. An AE was considered the appearance or worsening of any undesirable sign, symptom, or medical condition after starting a study drug, even if the event is not considered related to the drug. A serious adverse event (SAE) was considered an AE that was fatal or life-threatening, resulted in persistent or significant disability/incapacity, necessitated inpatient hospitalization or prolonged existing hospitalization, constituted a congenital anomaly/birth defect, or was otherwise medically significant. AEs considered to be at least possibly related to the study product were identified as adverse drug reactions (ADRs). The study protocol defined agranulocytosis as ANC < 0.5 Â 10 9 /L, though this commonly reflects severe neutropenia because agranulocytosis is often defined as ANC < 0.1 Â 10 9 /L. 22 Neutropenia was defined as a confirmed ANC ≥ 0.5 Â 10 9 /L but <1.5 Â 10 9 /L. Additional parameters to measure safety included creatinine and prolactin levels, in addition to growth. For growth measures, the change from baseline to Month 12 was compared between treatment groups using Z-score values for the child's height/weight at their age.
The rate of change in height was assessed using regression analysis.

| Statistical analyses
All analyses were performed using the statistical software SAS Windows 9.4 (Cary, NC, USA). A power analysis indicated that a sample size of 64 provided >90% power to detect a statistically significant difference (α = 0.05) between treatment groups in the primary endpoint. A t-test was used to compare two means between the deferiprone and placebo groups. The Fisher's exact test was used to compare two proportions between the deferiprone and placebo groups.
Significance thresholds were set at p < .05 unless otherwise noted. The primary analysis population was the intent-to-treat (ITT) population, consisting of randomly assigned patients receiving ≥ 1 treatment and post-baseline measurement for efficacy variables. The safety population included all patients who received at least 1 dose of treatment.
The proportion of patients who had not reached the SF and TSAT thresholds at each month was compared between the deferiprone and placebo groups using the Fisher's exact test. The time to reach the SF level ≥ 1000 μg/L or TSAT ≥60% threshold was compared between treatment groups using the Kaplan-Meier log-rank test. A p < .05 was considered statistically significant. The analyses did not consider individuals who withdrew before Month 12 and whose results might have made a difference had they remained in the study. Accordingly, post hoc analyses were conducted using imputed data to estimate SF values that would likely have been seen at later time points in patients who terminated (whether because they had reached the SF threshold or for any other reason) had they continued.
There were no statistically significant differences between treatment groups in baseline demographics or the clinical measures of SF levels and TSAT (Table 1). The overall mean ± standard deviation (SD) for age was 2.83 ± 2.08 years with a range of 0.61-9.0 years. Most patients were white (n = 55; 86%) and male (n = 41; 64%).
Adherence to treatment (% dosage taken) was not significantly different between groups, with a mean ± SD of 95% ± 14% for deferiprone and 93% ± 10% for placebo (p = .48). Six patients receiving deferiprone withdrew from the study (four due to parent request, two due to AEs) and one patient receiving a placebo withdrew (parent request) before reaching 12 months of treatment or the SF threshold (defined as SF levels ≥1000 μL at two consecutive visits). There were 23 protocol deviations deemed major with the most being noncompliance (n = 16) and assessment deviations (n = 5). Two other major protocol deviations were a violation of inclusion criteria (n = 1) and mistaken early termination (n = 1).

Transfusional iron burden was similar between groups.
Mean ± SD transfusional iron input was not significantly different between deferiprone (0.30 ± 0.24 mg/kg/day) and placebo were mistakenly escalated to 75 mg/kg/day deferiprone before meeting the requisite criteria. They were returned to 50 mg/kg/day deferiprone before re-escalation and maintenance at 75 mg/kg/day deferiprone, per protocol. Six patients withdrew from the study before escalating from 50 mg/kg/day deferiprone. More patients had SF levels >1000 μg/L at study completion than at initiation in both the deferiprone and placebo groups (supplemental 5). At completion, more placebo-treated patients had SF ranges >1000 μg/L. The difference in SF ranges between deferiprone and placebo was not significant at baseline ( p = .38) but trended toward significance at study completion ( p = .069).  Similarly, the mean % TSAT values were significantly higher in the deferiprone group at months 3, 4, 6, 9, and 11 (all p < .05; Figure 1D).

| Adverse events
Twenty-nine (91%) patients receiving deferiprone reported 174 AEs and 29 patients (91%) receiving a placebo reported 160 AEs (Table 2). There were no significant differences in the rate of AEs between treatment groups. There were no AEs associated with iron depletion or liver enzymes that were deemed related to treatment.
Overall, 11 patients (34%) experienced at least 1 ADR, defined as an AE at least possibly related to treatment ( Table 2). Of these, in the deferiprone group, one patient (3%) developed one event of agranulocytosis (ANC < 0.5 Â 10 9 /L), and four patients (13%) had four events of neutropenia (three mild severity, one moderate severity). In the placebo group, one patient (3%) developed one event of neutropenia. All cases of neutropenia were rated as SAEs, irrespective of ANC value or clinical significance; ANC counts ranged from 0.79 Â 10 9 /L to 6.02 Â 10 9 /L. Five patients receiving deferiprone and two patients receiving a placebo experienced SAEs (   Combined "abdominal pain" and "lower abdominal pain" terms. d For the patients who experienced neutropenia, neutrophil counts ranged from 0.79 to 6.02 Â 10 9 /L. e The lowest neutrophil count during the agranulocytosis episode was 0.2 Â 10 9 /L. The episode lasted for 16 days, and the patient did not report infection.
f ADRs included AEs that were considered to be at least possibly related to the study treatment. g Decreased neutrophil count was defined as a single occurrence of absolute neutrophil count below 1.5 Â 10 9 /L with a normal neutrophil count at the next assessment within 3 days.
There were no significant differences in mean creatinine levels between treatment groups at any time point (all p > .05; supplemental 7).
Additionally, there were no significant differences in mean predose prolactin levels between the deferiprone and placebo groups at baseline or Month 12, but there was a transient increase in prolactin levels The study's primary endpoint (patients reaching the SF threshold) was met, with significantly more placebo-treated than deferiprone- This study also demonstrated higher TSAT levels in patients receiving deferiprone than in patients receiving a placebo, which, to the authors' knowledge, is the first human evidence of deferiprone shuttling iron to transferrin. Cellular and animal models show deferiprone can shuttle excess iron to unbound transferrin 23 to increase TSAT, which may explain our results as shown in the proposed model (Figure 2). Deferiprone's ability to relocate iron from labile cellular pools to transferrin 23 is likely due to its lower iron affinity (pFe 3+ = 19.3) than transferrin (pFe 3+ = 22.3). 15 Our TSAT results provide clinical evidence that deferiprone can shuttle iron from intracellular pools to transferrin before reaching iron overload, which is consistent with deferiprone's low risk of chelation-induced iron depletion in mild to moderate iron overload. 4,20,24 Further, deferiprone's greater tissue permeability (i.e., it has a higher apparent volume 16 than deferasirox, 25 and deferoxamine is not orally bioavailable) supports its ability to chelate iron from organs and cellular stores (supplemental 9).
In the current study, the deferiprone group experienced a more rapid and frequent increase in TSAT levels than the placebo group.
These results differ from a previous nonplacebo-controlled, early-start deferiprone pilot study, where TSAT was found to increase more slowly in deferiprone-treated patients than in patients not receiving chelation treatment. 21 The differences may be because in the current study, patients had mean hemoglobin levels <90 g/L at most time points, considerably lower than the deferiprone early-start pilot study that had mean hemoglobin levels above 100 g/L at every time point. 21 Hemoglobin levels <90 g/L are associated with increased erythropoiesis, leading to high iron utilization, which could explain the low TSAT levels in the pilot study. 26,27 Differences in TSAT approached significance at months 1 and 2 before reaching significance in month 3, likely due to the natural increase in TSAT levels of the placebo group. The early-start deferiprone pilot study was limited by not being placebocontrolled, using a subtherapeutic dose, and having minimal population diversity. 21 The current study addresses those limitations, with both studies supporting that early-start deferiprone can delay the progression of iron overload based on maintenance (or delayed increase) of SF levels. 21 The low pre-transfusion hemoglobin levels observed in the current study were likely associated with increased erythropoiesis and high iron utilization and may explain the maintenance of mean TSAT <75% during the trial. It is possible that the slightly and not statistically significant higher hemoglobin levels and rate of transfusions in the deferiprone group at various times during the trial could have impacted the difference in TSAT. However, the lower TSAT in the placebo group, even at times this group had higher hemoglobin levels than the deferiprone group (baseline, months 2, 9, and 12), suggests this was not the case.
In START, the deferiprone and placebo groups showed a similar safety profile with no significant differences between rates of AEs, SAEs, or ADRs (all p > .05). Deferiprone showed high tolerability, indicated by a high adherence and low dropout rate, whereas deferoxamine can have poor compliance. 28 No patients reported AEs involving increased liver enzymes, a common AE observed with iron chelators, including deferiprone. 14 In contrast, the deferasirox film-coated tablet has been reported to have an increased frequency of AEs in younger patients, including high liver enzyme activity. 29 Furthermore, the frequent AEs in children treated with deferasirox contributed to high dropout rates, with all patients aged 2-6 years discontinuing. 29 Agranulocytosis and the less severe neutropenia-are the most concerning safety issues associated with iron chelators. The rates of agranulocytosis and neutropenia observed in START were low and consistent with rates seen in other age groups receiving deferiprone. 30 Another safety concern of iron chelator treatment is impaired renal function, which has been observed in patients receiving deferasirox. 31 Deferiprone-treated patients in the present study did not show impaired renal function as measured by creatinine levels. Furthermore, no patients reported AEs associated with hyperprolactinemia despite the transient increase in prolactin levels post-deferiprone administration, consistent with findings in healthy deferiprone-treated adults. 32 The elevated prolactin levels may be related to the known regulatory effect of iron on prolactin production. 33 The AEs in both START and the deferiprone early-start pilot study 21 match previously reported AEs for deferiprone, which include gastrointestinal symptoms (nausea, abdominal pain, and vomiting), neutropenia, and agranulocytosis. 2 Early-start deferoxamine initiated after the second or third transfusion was associated with significant toxicity and slower growth, mainly due to excessive iron depletion from natural stores. 5,6,34 In contrast, early-start deferiprone did not cause any observable changes in growth rate. This may be due to deferiprone's lower affinity for iron compared with deferoxamine, 18 as prior studies showed deferiprone reduces the risk of chelation-induced iron depletion during localized or mild-to-moderate iron overload. 20,24 Weight and height Z-scores were not significantly different between deferiprone-treated and placebo-treated groups. As commonly seen in a population with thalassemia, patients in this study were shorter and weighed less than general population averages. 14 12 and may not be easily implemented in young children. In adults, we previously reported that continuation of deferiprone and infrequent neutrophil monitoring was not associated with prolonged neutropenia or agranulocytosis progression. 35 The START study showed that deferiprone can be safely initiated in infants and young children with transfusion-dependent beta-thalassemia with SF levels between 200 and 600 μg/L, which is below the current F I G U R E 2 Proposed iron-shuttling mechanism to explain increased TSAT in deferiprone-treated patients. Deferiprone may "shuttle" iron from free iron pools in the bloodstream and cellular compartments to transferrin. Deferiprone-bound iron can then relocate iron to unbound transferrin, which has a higher affinity for iron than for deferiprone. Because deferoxamine/deferasirox have a higher affinity for iron than transferrin, they may be more likely to deplete iron from biological stores in "early-start" therapy. a  provided the first clinical evidence supporting the ability of deferiprone to transfer iron to transferrin. Importantly, we did not observe signs of iron depletion or slowed growth in the early-start deferiprone group, unlike previous observations reported with early-start deferoxamine therapy. 6 These data suggest that early-start deferiprone may reduce the increased risk of iron overload toxicities later in life. 36 Early-start deferiprone was efficacious at maintaining low iron levels; it was generally well-tolerated with a safety profile comparable to deferiprone treatment in older patients.
A plain-language summary of this study is available as supplemental material (supplemental 10: Deferiprone for iron overload in young children or infants who need blood transfusions to treat thalassemia).

AUTHOR CONTRIBUTIONS
Mohsen S. Elalfy, Fernando Tricta, and Caroline Fradette provided substantial contributions to the conception and design of the work. All authors provided substantial contributions to the acquisition, analysis, and interpretation of data for this analysis. All authors contributed to drafting/revising the work and provided final approval of the version to be published. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.