Oral ferroportin inhibitor VIT‐2763: First‐in‐human, phase 1 study in healthy volunteers

Abstract Restriction of iron availability by ferroportin inhibition is a novel approach to treating non‐transfusion‐dependent thalassemia (β‐thalassemia intermedia). This first‐in‐human, Phase I study (https://www.clinicaltrialsregister.eu; EudraCT no. 2017‐003395‐31) assessed the safety, tolerability, pharmacokinetics and pharmacodynamics of single‐ and multiple‐ascending doses (SAD and MAD) of the oral ferroportin inhibitor, VIT‐2763, in healthy volunteers. Participants received VIT‐2763 5/15/60/120/240 mg or placebo in the SAD phase and VIT‐2763 60/120 mg once daily, VIT‐2763 60/120 mg twice daily, or placebo for 7 days in the MAD phase. Seventy‐two participants completed treatment. VIT‐2763 was well tolerated and demonstrated a similar safety profile to the placebo. There were no serious or severe adverse events, or discontinuations due to adverse events. VIT‐2763 absorption was relatively fast, with detectable levels 15 to 30 minutes post‐dose. Following multiple dosing there was no apparent change in absorption and accumulation was minimal. Mean elimination half‐life was 1.9 to 5.3 hours following single dosing, and 2.1 to 3.8 hours on Day 1 and 2.6 to 5.3 hours on Day 7, following repeated dosing. There was a temporary decrease in mean serum iron levels with VIT‐2763 single doses ≥60 mg and all multiple doses; mean calculated transferrin saturation (only assessed following multiple dosing) also temporarily decreased. A shift in mean serum hepcidin peaks followed administration of all iron‐lowering doses of VIT‐2763. This effect was less pronounced after 7 days of multiple dosing (aside from with 120 mg once daily). These results support the initiation of clinical studies in patients with non‐transfusion‐dependent thalassemia and documented iron overload due to ineffective erythropoiesis.


| INTRODUCTION
Iron is essential for multiple cellular processes, with large amounts being used in the production of hemoglobin during erythropoiesis. Thus, erythropoietic demand for iron is a primary regulator of iron absorption. [1][2][3] Ineffective erythropoiesis can lead to excessive iron absorption in certain inherited hemoglobin disorders, such as thalassemia and other conditions associated with variations in hemoglobin structure. The two main forms of thalassemia, αand β-thalassemia, are caused by defects in αand β-hemoglobin chain synthesis, respectively. 4 In β-thalassemia, partial or complete loss of β-globin synthesis, due to mutations in the β-globin gene, results in a relative excess of α-chains and the formation of α-globin-heme complexes on red blood cell (RBC) membranes. 1,5 The severity of symptoms, which are generally related to anemia and/or iron overload, depends on the extent to which β-globin production is diminished and the degree of αand β-chain imbalance. 4,5 Phenotypes of thalassemia and their severity can be characterized according to dependence on blood transfusions. Patients are conventionally described as having transfusion-dependent thalassemia (TDT; β-thalassemia major) when they have a life-long requirement for regular transfusions, which results in iron overload. 6,7 Chronic transfusions are not essential for those with non-transfusion-dependent thalassemia (NTDT; β-thalassemia intermedia) to survive, but for limited periods of time, infrequent or sometimes repeated transfusions might be necessary. 6,8,9 In NTDT, ineffective erythropoiesis leads to chronic hemolytic anemia and subsequent hypoxia. This drives compensatory elevated levels of erythropoiesis and suppressed synthesis of the iron-regulatory hormone, hepcidin. Consequently, intestinal iron absorption is increased, recycled iron from the reticuloendothelial system is released, macrophage iron is depleted, and portal and hepatocyte iron loading is increased. 1,5,[10][11][12][13] Subsequently, the amount of released iron exceeds the binding capacity of transferrin and nontransferrin-bound iron (NTBI) is formed in the circulation. 14,15 Due to its ability to generate reactive-oxygen species, accumulation of unbound iron in tissues can be toxic, resulting in tissue injury (including trophic skin changes and leg ulcers), organ dysfunction (including liver disease, heart failure and endocrinopathies), and death. 1,7,15,16 The clinical management of β-thalassemia generally involves blood transfusions, iron chelation, and treatments to alleviate symptoms of iron overload. 17,18 Potentially curative treatments include bone marrow transplant in selected patients, 17,18 and gene therapies that aim to partially, if not fully, correct the disorder. [19][20][21][22][23] The gene therapy, lentiglobin, which acts via the transplantation of lentiviral-transduced hematopoietic stem cells with increased production of β-globin, 21,23,24 was approved for the treatment of TDT in the EU in June 24, 2019. Other emerging therapies are based on animal model evidence that restriction of iron to the erythron can improve ineffective erythropoiesis by decreasing the formation of RBC membrane-associated α-globin aggregates, and subsequent reactive-oxygen species-mediated oxidative stress and apoptosis. [25][26][27] One such therapy, VIT-2763, is a novel, smallmolecule oral inhibitor of ferroportin. Ferroportin is the only known iron transporter in mammals and is mainly expressed in tissues of iron absorption (duodenum), recycling and storage (liver, spleen and mononuclear phagocyte system), mediating the transfer of iron into the blood. 3,[28][29][30][31] In vitro, VIT-2763 inhibited cellular iron efflux with a potency comparable to that of hepcidin, the natural inhibitor of ferroportin, and in preclinical studies, VIT-2763 demonstrated good oral bioavailability. 32 In 14-day toxicity studies in Wistar rats, the "no observed adverse effect level" was >600 mg/kg; in longer-term studies, no dose-limiting toxicity was observed. Dose-limiting effects in rodents were related to the pharmacology of VIT-2763 (restricted iron uptake) and resulting iron deficiency anemia and subsequent secondary effects thereof. 32 In a mouse model of NTDT, VIT-2763 decreased serum iron levels, ameliorated anemia and improved ineffective erythropoiesis. 32 Thus, limiting iron availability for erythropoiesis via ferroportin inhibition is a potential approach to treating NTDT. 33 The aim of this first-in-human, Phase I study (EudraCT no. 2017-003395-31) was to assess the safety and tolerability, and to determine the pharmacokinetics (PK) and pharmacodynamics (PD), of single-and multiple-ascending oral doses of VIT-2763 in healthy volunteers.

| Participants
Healthy male or female volunteers aged between 18 and 65 years were eligible. Exclusion criteria comprised any history of iron storage diseases; history or clinical findings of iron utilization disorders; known hemoglobinopathy; use of intravenous iron therapy, erythropoietin stimulating agent therapy and/or blood transfusions in the previous 3 months, and/or excessive oral iron or oral iron-containing products in the previous 4 weeks. A complete list of inclusion and exclusion criteria is provided in Table S1. Participants were admitted on Day −1 and received VIT-2763 or placebo the following day (Day 1 in the SAD phase) or for the next 7 days (Day 1 to Day 7 in the MAD phase). Participants remained hospitalized for assessments up to and including Day 4 or Day 10 in the SAD and MAD phases, respectively, returning for a final study assessment 3 days later. Participants were assigned a randomization number prior to dosing, according to a code generated by the Biostatistics Department of PRA Health Sciences. In the SAD phase, sequential, ascending cohorts received single oral doses of VIT-2763 (5, 15, 60, 120 or 240 mg) or matching placebo. Sentinel dosing was used, whereby the first two participants in each SAD cohort were randomized to receive either VIT-2763 or placebo. Dosing of the remaining participants at the same dose level occurred at least 48 hours later, following a safety evaluation of the sentinel participants. Escalation to the next dose level occurred following data review by the Safety Review Committee. In the MAD phase, sequential, ascending cohorts received multiple oral doses of VIT-2763 (60 or 120 mg, once daily

| Study design
[QD] or twice daily [BID]) or matching placebo for 7 days. Dosing in the MAD phase followed an assessment of data from the first four cohorts of the SAD phase. Subsequent multiple-dose cohorts were initiated once data from the preceding SAD cohort had been evaluated, along with cumulative data from the preceding multiple-dose cohorts. Starting and maximum doses, which were based on preclinical toxicology studies and physiologically based PK/PD modeling and simulation, were in accordance with European Medicines Agency (EMA) guidelines. 34 If a predefined safety threshold was reached (C max ≥3805 ng/mL; equivalent to the no observed effect level in dogs) dosing at that level or higher would not continue.

| Endpoints and assessments
Primary endpoints were safety related and comprised the incidence of adverse events (AEs) and serious AEs, which were coded according to Medical Dictionary for Regulatory Activities code (version 21.0), changes from baseline in vital signs, clinical laboratory tests (hematology, biochemistry, coagulation, urinalysis), twelve-lead electrocardiogram (ECG), cardiac telemetry, and physical examinations. The severity of AEs and relationship of AE to study drug were also assessed. Secondary endpoints were VIT-2763 PK and PD. A complete list of endpoints is included in Table S2 and a summary of all assessments and related time points is included in Figure S1.

| Statistical analysis
As this was a first-in-human study of VIT-2763, no formal sample-size calculation was performed. The PK and PD analyses were performed on the full analysis set (FAS), comprising all participants randomized to treatment, who received ≥1 complete dose and had ≥1 post-baseline PK and PD assessment. The PK and PD analyses were repeated using the per-protocol set, comprising all participants who fulfilled FAS criteria and had no major protocol violations. The safety set comprised all randomized participants who had received ≥1 dose of study medication. Safety, PK and PD statistical analyses were descriptive. Continuous variables were presented as mean, SD, SE or median, range and 95% confidence interval, as appropriate; categorical and ordinal variables were presented as frequencies and percentages. Results are presented for each VIT-2763 dose cohort; data for participants receiving placebo were pooled across cohorts. All statistical analyses were performed using SAS Version 9.3 or higher and with WinNonlin Version 6.3 or higher. There was no imputation of missing values, except missing start and end times of AEs, and PK concentrations below lower limit of quantification (LLOQ).

| Data-sharing statement
The key aspects of the study protocol and fully anonymized data were submitted to the EudraCT clinical trial registry at https://www. clinicaltrialsregister.eu at the end of October 2019. Detailed inclusion criteria, study endpoints, the most common AEs and all reported AEs are included in this publication.

| Participant disposition and baseline characteristics
A total of 72 participants were randomized and completed treatment.
The number of participants assigned to each dose level in the SAD and MAD cohorts is shown in Figure S2. Demographic variables and baseline characteristics of the treatment cohorts in the SAD and MAD phases are presented in Table 1. A higher proportion of participants were female than male (66% and 71% of participants in the overall SAD and MAD cohorts, respectively), and the treatment cohorts were slightly imbalanced in terms of proportions of female to male participants. Median age ranged from 21.5 to 29.0 years in SAD cohorts, and from 22.5 to 33.5 years in the MAD cohorts and mean body mass index (BMI) ranged from 21.9 to 25.3 kg/m 2 in the SAD cohorts, and from 21.8 to 24.6 kg/m 2 in MAD cohorts. Despite differences in the proportions of female to male participants, mean baseline levels of serum iron, calculated transferrin saturation, transferrin and hemoglobin were generally comparable across treatment cohorts in both the SAD and MAD phases; during the MAD phase, median (min, max) values for hemoglobin were similar between treatment cohorts.  (Table S5). Of the drug-related AEs, one case of decreased blood iron in the MAD phase was considered to be probably/likely related to the study drug and all others were deemed as possibly related to the study drug. All AEs in the SAD phase and the majority (93/96) of AEs in the MAD phase were mild in severity (Table S5). Moderate AEs occurred in three participants (in the MAD phase); there were no AEs of severe or life-threatening severity.

| Safety and tolerability
The majority of AEs were transient and resolved without sequelae; at follow-up, three AEs in the SAD phase and four AEs in the MAD phase, all of which were mild in severity, had not yet resolved. There were no clinically relevant changes from baseline in laboratory-assessed safety parameters, vital signs, ECG results or physical examination.

| Pharmacokinetic parameters
In the SAD cohorts, the initial oral absorption of VIT-2763 was relatively fast, with detectable levels observed in most participants 15 to 30 minutes post-dose. Across the VIT-2763 cohorts in the SAD phase, the median T max ranged from 0.50 to 3.00 hours and the geometric mean elimination T 1/2 was 1.88 to 5.33 hours (Table 2). For the majority of participants who received single doses of ≥15 mg, a second peak or plateauing in the concentration─time profile occurred 3 to 4 hours post-dose ( Figure 1A). Elimination was monophasic following     Therefore, no participants in the study received higher doses.
As in the SAD phase, a second peak in the concentration─time profiles was seen 3 to 4 hours post-dose on Day 1 and 7 in the MAD cohorts ( Figure 1B,C). No significant accumulation was observed after

| Pharmacodynamic parameters
There was a temporary decrease in mean serum iron levels following the higher single doses of VIT-2763 (60, 120 and 240 mg; Figure 2A  . Abbreviations: BID, twice daily; LLOQ, lower limit of quantification; PK, pharmacokinetic; QD, once daily. Morning doses of VIT-2763 were administered after 10 hours of overnight fast. In the BID dosing cohorts, the evening dose was administered approximately 12 hours later, before dinner but after PK sampling. Fewer blood samples were taken following the evening dose compared with the morning dose due to logistical reasons

| DISCUSSION
The results from this first-in-human study demonstrate that VIT-2763, at single oral doses up to 240 mg, or multiple oral doses up to 120 mg twice daily, was well tolerated in healthy volunteers. There were no serious or severe AEs, or discontinuations due to AEs, and no discernible effects on any other safety parameters. After administration, VIT-2763 was rapidly absorbed, showed no evidence of accumulation upon repeated dosing, and was associated with a decrease in serum iron levels. Together, these findings support the potential use of VIT-2763 as a treatment for NTDT and other indications in which iron metabolism is involved. no corresponding decreases were observed with placebo. This ironlowering PD effect is consistent with observations in a preclinical mouse model of NTDT and in wild-type mice. 32 In the current study, VIT-2763 (all multiple-dose levels) was also found to reduce transferrin saturation, an important finding since detectable levels of NTBI typically occur when transferrin saturation exceeds 75%. 35 Hepatocytes and cardiomyocytes rapidly take up NTBI, which is thought to be responsible for toxicity to the liver and heart, the most critical organs damaged by iron-loading disorders. 36,37 The PD effects of VIT-2763 observed in the current study, in terms of reduced serum iron and transferrin saturation, suggest that VIT-2763, via iron restriction, has the potential to improve erythropoiesis and anemia in patients with ineffective erythropoiesis, such as in NTDT, sickle cell disease and myelodysplastic syndrome. Also, VIT-2763 may be able to reduce excessive iron absorption in hereditary hemochromatosis, and reduce excessive erythropoiesis in polycythemia vera. Of note, data from the current study indicate that VIT-2763 dosing impacts the kinetics of hepcidin, shifting peak levels to an earlier time point, compared with the placebo group. This finding is surprising given that preclinical data show that in healthy mice, administration of VIT-2763 leads to a rapid lowering of hepcidin transcription in the liver as a feedback response to declining blood iron levels (unpublished data). Clearly, the discrepancy between preclinical and clinical findings cannot easily be explained with the limited data from this Phase I study and warrants further investigation. The shift in peak levels was less pronounced after 7 days of multiple dosing, especially in the twice daily cohorts, where there was a near complete lack of hepcidin peak. No changes were observed in other hematologic biomarkers, which is as expected in a healthy population and over the short study period (only 1 week of dosing). The gene therapy lentiglobin acts to increase hemoglobin levels and may provide a curative treatment for β-thalassemia. However, the therapy is currently only approved for selected patients with TDT (those 12 years of age or over, who do not have a β0/β0 genotype, and for whom hematopoetic stem cell transplantation is appropriate but a suitable donor is lacking). 24   Strengths of the current study include the wide range of safety, PK and PD endpoints assessed, both following single and multiple dosing which will support and inform the design of future trials. A limitation of the study is that blood samples for PK and PD analysis were not taken between 12 and 24 hours post-dose, which may have impacted some observations, particularly with regards to serum iron in the twice daily dosing cohorts. Another potential limitation is that plasma VIT-2763 PK parameters were calculated using non-compartmental analysis; compartmental analytic techniques may have produced different results.

| CONCLUSION
When VIT-2763, an oral ferroportin inhibitor, was administered at single oral doses up to 240 mg, or multiple oral doses up to 120 mg twice daily, it had a comparable safety profile to the placebo, and was well tol-

AUTHOR CONTRIBUTIONS
All authors contributed to the research, conception and design of the study, data analysis and interpretation. All authors reviewed and revised drafts of the manuscript and approved the final draft for submission.