This Phase 2, open-label, non-randomized, multicenter, dose-finding study (clinicaltrials.gov NCT02410213) was conducted at eight sites in Poland and two in Russia; sites were either large hospitals or a specialist outpatient clinic.

The study was conducted in accordance with the Declaration of Helsinki, all applicable local and state regulations, and International Council for Harmonisation guidelines. The protocol, amendments to the protocol, and the informed consent form were reviewed and approved by institutional review boards at each study site prior to initiation of the study. Ethical approval for the eight study sites in Poland was provided by the Bioethics Committee at the Medical University in Lublin, Lublin, Poland. For the two study sites in the Russian Federation, ethical approval was provided by the Ethics Committee at State Budgetary Educational Institution of Higher Professional Education (SBEI HPE), Ryazan State Medical University named after academician I.P. Pavlov, Ministry of Public Health of the Russian Federation, Ryazan, Russian Federation or the Ethics Committee at SBEI HPE, Saint-Petersburg State Pediatric Medical University, Ministry of Healthcare of the Russian Federation, St Petersburg, Russian Federation.

Informed consent was obtained from all participants, and a written consent form was signed by the participant and/or their legal representative in their native language prior to study participation. A Data Safety Monitoring Board (DSMB) provided oversight.

Boys and girls eligible for enrollment at study sites in Poland were 1–17 years of age and those in Russia were 6–17 years of age. At screening, eligible patients had a hemoglobin concentration <11 g/dL and a transferrin saturation (TSAT) <20%. Therapy with an erythropoietin-stimulating agent was permitted, provided that the dose had been stable for more than 8 weeks prior to screening and that no changes in the dose or product were anticipated for the duration of the trial.

Patients were excluded with a ferritin level >300 ng/mL at screening, with a body mass index (BMI) ≤5th percentile for age, or, for individuals who were enrolled while 1 year of age, with body weight <12 kg. Other exclusion criteria included active infection; anemia for reasons other than iron deficiency; receipt of immunosuppressive therapy, other than steroid therapy, that could exacerbate anemia; receipt of IV iron and/or a blood transfusion within 4 weeks prior to screening; history of acquired iron overload, hemochromatosis, or other iron accumulation disorder; severe diseases of the liver or the hematopoietic or cardiovascular system; CKD treated with hemodialysis; and/or evidence of infection with human immunodeficiency virus, or hepatitis B or C viruses with evidence of active hepatitis.

Patients who met the selection criteria entered a ≤14-day screening period to assess eligibility. Eligible patients were enrolled sequentially in one of two dose groups in which they received a single IV dose of FCM (Vifor Pharma Ltd., St. Gallen, Switzerland) (either 7.5 mg/kg or 15 mg/kg, with a maximum total dose of 750 mg). Enrollment in the group to receive the higher dose of FCM (15 mg/kg) began after all patients in the lower dose group (FCM 7.5 mg/kg) had received study drug and been observed for 4 weeks post-dose with no safety concerns as determined by the DSMB on the basis of an interim analysis of the FCM 7.5 mg/kg cohort.

FCM was administered as an undiluted solution at a rate of 100 mg/min. Doses less than 100 mg were administered as a slow undiluted IV push injection within 1 min. All patients were followed for 35 days. After infusion of FCM on Day 0, patients returned to the clinic at 24, 48, and 72 h post-dose, and subsequently on study Days 14, 28, and 35 for safety, PK, and PD assessments.

Safety was assessed by the incidence of treatment-emergent adverse events (TEAEs), serious TEAEs, and mean changes from baseline in clinical laboratory values at each scheduled visit. Blood for clinical laboratory values was collected at screening (up to Day −14), at 72 h post-dose, and on Days 14, 28, and 35. Medical Dictionary for Regulatory Activities (MedDRA) version 17.0 was used to classify all TEAEs by system organ class and preferred term; severity was classified with National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0.

Blood samples were collected on Day −1 at 8 AM, 12 PM, and 8 PM to characterize each patient’s baseline iron status, pre-dose on Day 0, and then at 1, 2, 6, 12, 24, 48, and 72 h post-dose. Whole blood samples were collected in 5 mL evacuated red-top blood collection tubes (no anticoagulant) and were allowed to clot while standing at room temperatures and then centrifuged at 2000g. Serum samples (minimum volume 800 µL) were then transferred to cryovials, frozen at –20 °C and shipped on dry ice for analysis (Butterworth Laboratories Limited, Teddington, Middlesex, UK). Serum samples were analyzed for iron content using inductively coupled plasma-mass spectrometry. The assay was validated over the range of 1–1000 µg/mL and does not distinguish between endogenous serum iron and that derived from the FCM infusion.

PK parameters were determined for each patient. The primary PK parameters for iron were maximum serum concentration (Cmax), time to Cmax (Tmax), area under the serum concentration-time curve (AUC) from time zero to the last sampling time with a quantifiable concentration (AUC0-last), AUC from time zero extrapolated to infinity (AUC0-inf), and elimination half-life (t½), with baseline values subtracted from all measured samples.

Secondary PK parameters for iron were mean residence time, apparent serum clearance (CL), and apparent volume of distribution (Vd), where Vd refers to the apparent volume of distribution associated with the terminal phase calculated as dose of iron in mg/(λz × AUC0-inf).

PK analyses were conducted by ICON plc (Reading, UK) using Phoenix WinNonlin v6.3 (Certara Corp, St Louis, MO). PK parameters for baseline-corrected total serum iron were calculated using noncompartmental analysis (WinNonlin Model 200–202 for IV dosing). Actual elapsed time from the start of the infusion and actual dose amounts were used in calculations. Concentrations determined to be below the limit of quantitation were assigned a value of zero before achievement of Cmax and were treated as missing values after achievement of Cmax.

PD assessments included serum ferritin, serum transferrin, serum hemoglobin, reticulocyte count, and TSAT. Blood samples for all PD assessments were collected at screening (up to 14 days before treatment Day 0), at 72 h post-dose, and on Days 14, 28, and 35. The change from baseline in ferritin, hemoglobin, and TSAT at each scheduled visit; and the change from baseline to the highest post-dose value in serum ferritin, transferrin, hemoglobin, reticulocyte count, and TSAT were summarized.

Sample size determination followed practical, feasibility, and empirical considerations for a Phase 2 dose-finding study. The planned enrollment was 32 patients with 16 patients in each treatment group equally distributed by age (eight patients aged 1–6 years and eight aged >6–17 years).

The safety population included all patients who received FCM; the PK population included all patients in the safety population who had at least one measurable concentration of FCM; the PD population included all patients in the safety population who had at least one PD assessment. Baseline was defined as Day 0; if data on Data 0 were missing or not captured, the screening value was used.

No hypothesis testing was planned for this study; only descriptive, summary statistics were planned for assessment of dosing and safety. Statistical programming and analyses were performed using SAS v9.1.3 (SAS Institute Inc., Cary, NC).