Long-term safety and efficacy of ferric citrate in phosphate-lowering and iron-repletion effects among patients with on hemodialysis: A multicenter, open-label, Phase IV trial.

BACKGROUND: We explored the long-term safety and efficacy of ferric citrate in hemodialysis patients in Taiwan, and further evaluated the iron repletion effect and change of iron parameters by different baseline groups.
METHODS: This was a 12-month, Phase IV, multicenter, open-label study. The initial dose of ferric citrate was administered by patients' clinical condition and further adjusted to maintain serum phosphorus at 3.5-5.5 mg/dL. The primary endpoint was to assess the safety profiles of ferric citrate. The secondary endpoints were to evaluate the efficacy by the time-course changes and the number of subjects who achieved the target range of serum phosphorus.
RESULTS: A total of 202 patients were enrolled. No apparent or unexpected safety concerns were observed. The most common treatment-emergent adverse events were gastrointestinal-related with discolored feces (41.6%). Serum phosphorus was well controlled, with a mean dose of 3.35±1.49 g/day, ranging from 1.5 to 6.0 g/day. Iron parameters were significantly improved. The change from baseline of ferritin and TSAT were 227.17 ng/mL and 7.53%, respectively (p-trend<0.001), and the increase started to slow down after 3-6 months of treatment. In addition, the increase trend was found only in patients with lower baseline level of ferritin (≤500 ng/mL) and TSAT (<30%).
CONCLUSIONS: Ferric citrate is an effective phosphate binder with favorable safety profile in ESRD patients. The iron-repletion by ferric citrate is effective, and the increase is limited in patients with a higher baseline. In addition to controlling hyperphosphatemia, ferric citrate also shows additional benefits in the treatment of renal anemia. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT03256838; 12/04/2017.

Introduction

Hyperphosphatemia and anemia are the two most frequent complications encountered in end-stage renal disease (ESRD) patients. Hyperphosphatemia is associated with an increased risk of anemia, mineral and bone disorders, and cardiovascular morbidity and mortality [1-3]. Hemodialysis and peritoneal dialysis are standard modalities for ESRD patients. However, dialysis patients still suffer from hyperphosphatemia due to the average intake of phosphorus in the gastrointestinal tract as high as 900 mg/day [4, 5]. Traditional phosphate binders are used to reduce phosphorus absorption in the gastrointestinal tract, which may lead to adverse results such as hypercalcemia, systemic toxicity, gastroduodenal lesions, or lower adherence rates due to heavy pill burden and inconvenient formulations [6]. As kidney function declines in chronic kidney disease (CKD) progression, the erythropoietin production decreases, and it exacerbates anemic condition due to lowering of iron level. With its additional benefits of iron supplementing for renal anemia, iron-based phosphate binder has become a popular choice for CKD patients [7, 8].

Ferric citrate is safe and efficacious in the management of hyperphosphatemia and anemia in both non-dialysis [9-15] and dialysis-dependent [16-18] CKD patients. All studies showed consistent serum phosphorus control with good tolerability. The most common adverse events (AEs) were feces discoloration and diarrhea. Although ferric iron was traditionally deemed as a non-absorbable form of iron, the long-term studies of ferric citrate showed the ferric iron from the citric chelated structure could still be utilized to improve iron panel and hemoglobin level, and further reduced the need for erythropoiesis-stimulating agents (ESA) and intravenous iron in hemodialysis patients [19-23]. The available long-term data can only be found in the US and Japanese populations which represented two extreme ferritin groups, 887 ng/ml and 73 ng/ml respectively [24, 25]. No study has ever evaluated the long-term effect for other ESRD patient groups with a medium level of ferritin (300~500 ng/mL) such as Taiwanese population. This Phase IV trial designed as observational and non-interventional study evaluated the safety, tolerability, and effectiveness in real-world settings in which concomitant treatments (intravenous iron and ESAs) were not limited. By further analyzing the change of ferritin and transferrin saturation (TSAT) with different baseline levels, this study allows understanding of ferric citrate’s iron absorption/utilization and its effects on anemia improvement in ESRD patients with medium level of ferritin, its impacts on existing anemia treatments and long-term safety profile in the clinical practice.

Materials and methods

Study design

This study is a multicenter, open-label, Phase IV study to evaluate the long-term safety and efficacy of ferric citrate in subjects with ESRD on hemodialysis. The study was conducted in accordance with the Declaration of Helsinki, and all participants provided written informed consent. The study protocol was approved by the Institutional Review Board the participating centers (Shin Kong Wu Ho-Su Memorial Hospital, #20170101C; Taichung Veterans General Hospital, #SC16169B; Kaohsiung Chang Gung Memorial Hospital, #201700047A3; Keelung Chang Gung Memorial Hospital, #201700047A3; Far Eastern Memorial Hospital, #105086-I; Taipei Medical University Hospital, #N201612068; Kaohsiung Medical University Chung-Ho Memorial Hospital, #KMUHIRB-F(I)-20170004; National Taiwan University Hospital, #201701034MSA; and Taipei Veterans General Hospital, #2017-02-003AU). Written informed consent was obtained from each participant. All procedures performed in the present study were in accordance with Declaration of Helsinki. The study was registered on ClinicalTrials.gov (NCT03256838; 12/04/2017), and the clinical study protocol was shown in S1 Appendix. All participants received the standard care for hemodialysis according to routine hospital practice, except for pharmacological interventions for hyperphosphatasemia. There was no washout period between previous medications/therapies and this study medication. Ferric citrate (500 mg/capsule containing 105 mg of ferric iron) was taken with meals for 12 months. For subjects whose previous phosphate binder doses were equivalent to less than and greater than 4.5 g/day of calcium-based phosphate binder, the initial doses of ferric citrate were 3 g/day and 4.5 g/day, respectively. During the study, the dose of ferric citrate was further titrated by the investigators to achieve the goal of maintaining serum phosphorus levels between 3.5 and 5.5 mg/dL, with a maximum dose of 12 g/day.

The study period included an enrollment visit (baseline, M0), routine monthly visits during the 12-month treatment period (M1, M2, M3, etc.), and a follow-up visit one month after the end of treatment (EOT). EOT was defined as the final treatment visit or early termination of treatment. In addition to physical examination, vital signs, essential hematology, and biochemistry tests were performed at each visit. The use of concomitant medications such as vitamin D, intravenous iron preparations, or ESA was not limited during the study.

Subject population

From April 2017 to September 2019, a total of 224 subjects were screened for eligibility. The inclusion criteria included (1) age > 18 years and provision of written informed consent; (2) ESRD patients undergoing hemodialysis 3 times/week, and necessary to receive medication for hyperphosphatemia; (3) serum ferritin < 1,000 ng/mL and TSAT < 50% at the time of enrollment; and (4) women of child-bearing potential were willing to use contraception during the study period. Patients who met any of the following criteria were excluded from this study. (1) Patients had any known contraindication to ferric citrate, including but not limited to allergy to ferric citrate; hypophosphatemia; hemochromatosis or iron overload syndrome; and active severe gastrointestinal disease. (2) Patients underwent parathyroidectomy or percutaneous ethanol injection therapy within 3 months prior to enrollment visit or patients had serum calcium < 7 mg/dL at the enrollment visit. (3) Patients participated in another interventional study within 30 days prior to the enrollment. (4) The female patients were currently pregnant or breastfeeding. (5) Patients with unstable medical conditions or psychiatric conditions and were not suitable for this study based on the investigator’s judgment.

Safety and efficacy assessments

The safety endpoint was to evaluate the safety profile, including treatment-emergent adverse events (TEAEs), clinical laboratory evaluations, vital sign measurements, physical examination, and 12 lead electrocardiograms. The efficacy was evaluated by the time-dependent change in serum phosphorus levels and the proportion of achieving the target range of serum phosphorus (3.5–5.5 mg/dL) throughout the treatment. Additional exploratory study endpoints included serum calcium, iron, ferritin, TSAT, total iron binding capacity (TIBC), hemoglobin, intact parathyroid hormone (iPTH), and the dose change of intravenous iron and ESA.

All subjects who received at least one dose of study medication were evaluated as the safety population. The full analysis set (FAS) was defined as the participants who satisfied all eligibility criteria, took at least one dose of study medication, and had at least one post-treatment evaluation for efficacy.

Statistical analyses

Continuous data were expressed as mean and standard deviation (SD), and categorical data were expressed in numbers and percentages in the analysis of baseline characteristics and TEAEs. The values of serum phosphorus, iron-, anemia-, and bone-mineral-related parameters were expressed as mean and standard error (SE). P-trend was calculated by applying a general linear model for all continuous variables. P-value was used to compare the M0 and EOT by paired t-test. Statistical analyses were performed by SPSS software version 22.0 (IBM, Armonk, NY, USA), and a two-sided p<0.05 was considered statistically significant.

Results

Participant characteristics

A total of 224 subjects were screened for eligibility, of which 202 subjects were enrolled and 117 subjects (57.9%) completed all the scheduled ferric citrate treatment for 12 months (Fig 1). All 202 subjects, received at least one dose of study medication, were included in the safety population, and 197 patients were evaluated as FAS. The baseline demographic characteristics of the safety population are shown in Table 1. The study group comprised 53.5% male, and the mean age was 60.9 ± 9.8 years, with a mean bodyweight of 64.9 ± 14.1 kg. The mean duration of hemodialysis was 8.2 ± 6.6 years. Patients undergoing hemodialysis for > 5 years were 59.9%. The main causes of renal disease were diabetic nephropathy (33.7%) and hypertensive nephrosclerosis (25.7%). The most common comorbidities were hypertension (69.8%), diabetes mellitus (40.6%), and hyperlipidemia (31.2%). There were 94.1% subjects who received ESA, 56.9% who received intravenous iron, and 52.5% who received vitamin D before the study. Calcium-based phosphate binder was the most common prior to the study medication (82.7%), and other prior use of phosphate binders included aluminum-based (19.3%), sevelamer-based (7.4%), lanthanum-based (7.9%), and ferric citrate-based binders (5.9%).

Fig 1

Flow diagram of subjects by completion status.

Table 1

Demographic and baseline characteristics of study population (N = 202).

Baseline characteristics
Age (year)		60.9 ± 9.8
Gender
	Male	108	(53.5%)
	Female	94	(46.5%)
Body weight (kg)		64.9 ± 14.1
Age at diagnosis of ESRD (year)		49.7 ± 12.0
History of hemodialysis (year)		8.2 ± 6.6
History of hemodialysis
	≤ 1 year	10	(5.0%)
	1–5 years	71	(35.1%)
	5–10 years	52	(25.7%)
	>10 years	69	(34.2%)
Primary etiology
	Diabetes nephropathy	68	(33.7%)
	Hypertensive nephrosclerosis	52	(25.7%)
Comorbidity a
	Hypertension	141	(69.8%)
	Diabetes mellitus	82	(40.6%)
	Hyperlipidemia	63	(31.2%)
Prior use of medication a,*
	ESA	190	(94.1%)
	Intravenous iron	115	(56.9%)
	Vitamin D	106	(52.5%)
Prior use of phosphate binder a,*
	Calcium-based binders	167	(82.7%)
	Aluminum-based binders	39	(19.3%)
	Sevelamer-based binders	15	(7.4%)
	Lanthanum-based binders	16	(7.9%)
	Ferric citrate-based binders	12	(5.9%)

Continuous variables were presented as mean ± standard deviation, and categorical variables were presented as number and percentage.

a Multiple entries allowed.

* Within 3 months before to the study.

Abbreviations: ESRD, end-stage renal disease; ESA, erythropoiesis stimulating agents.

Safety

The safety was evaluated based on the physical examinations, changes in biochemical laboratory parameters, and profile of TEAEs. No clinically significant fluctuations in physical examinations and laboratory parameters including aluminum, sodium, potassium, albumin, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were observed during the study (S1 Table).

There were no drug-related deaths during the study period. The most common TEAEs with an incidence rate ≥ 20% reported by System Organ Class (SOC) were gastrointestinal disorders, infections and infestations, injury, poisoning, and procedural complications, respiratory, thoracic and mediastinal disorders, musculoskeletal and connective tissue disorders, and skin and subcutaneous tissue disorders (Table 2). Discolored feces (41.6%) was the most common TEAE. Other common TEAEs with an incidence rate ≥ 10% reported by preferred term were cough, diarrhea, and constipation. Most of these TEAEs were mild in severity. The most frequently reported serious TEAEs, including pneumonia (5.9%), acute myocardial infarction (3.0%), cardiac failure congestive, and coronary artery disease (2.0% each, respectively) (Table 3). None of the serious TEAEs were suspected to be related to ferric citrate treatment. S2 Table showed the most common drug-related TEAEs with discolored feces (41.1%) in the majority. Most TEAEs that led to drug discontinuation were mild to moderate, mainly related to gastrointestinal disorders (S3 Table). Overall, none of the serious TEAEs led to drug discontinuation, suggesting that ferric citrate is safe for ESRD patients.

Table 2

TEAEs with incidence rate ≥ 20% by SOC (N = 202).

TEAEs by SOC and PT		N	(%)
Gastrointestinal disorders
	Discolored feces	84	(41.6%)
	Diarrhea	28	(13.9%)
	Constipation	26	(12.9%)
	Abdominal pain	18	(8.9%)
	Abdominal distension	13	(6.4%)
	Gastroesophageal reflux disease	10	(5.0%)
Infections and infestations
	Upper respiratory tract infection	18	(8.9%)
	Pneumonia	14	(6.9%)
	Viral upper respiratory tract infection	14	(6.9%)
Injury, poisoning and procedural complications
	Arteriovenous fistula site complication	20	(9.9%)
	Shunt malfunction	13	(6.4%)
Respiratory, thoracic and mediastinal disorders
	Cough	33	(16.3%)
Musculoskeletal and connective tissue disorders
	Pain in extremity	14	(6.9%)
	Back pain	11	(5.4%)
	Musculoskeletal pain	10	(5.0%)
Skin and subcutaneous tissue disorders
	Pruritus	19	(9.4%)

Data was presented as number and percentage, and the N was based on the number of patients experiencing ≥ 1 TEAE, not the number of events.

Abbreviations: TEAEs, treatment-emergent adverse events; SOC, system organ class; PT, preferred term.

Table 3

Most common serious TEAEs with incidence rate >1% (N = 202).

Serious TEAEs by PT		N	(%)
	Pneumonia	12	(5.9%)
	Acute myocardial infarction	6	(3.0%)
	Cardiac failure congestive	4	(2.0%)
	Coronary artery disease	4	(2.0%)
	Acute pulmonary edema	3	(1.5%)
	Angina unstable	3	(1.5%)
	Cellulitis	3	(1.5%)
	Hyperparathyroidism	3	(1.5%)
	Pulmonary oedema	3	(1.5%)

Data was presented as number and percentage, and the N was based on the number of patients experiencing ≥1 TEAE, not the number of events. Abbreviations: TEAEs, treatment-emergent adverse events; PT, preferred term.

Efficacy

Serum phosphorus level was well controlled and maintained throughout the study. The mean serum phosphorus was 5.38 mg/dL at M0 and 5.19 mg/dL at EOT with a significant overall decreasing trend from M0 to EOT (P-trend = 0.003) (Table 4). There was a slight increase in the serum phosphorus level in the first month of study due to the monthly titration design from a low starting dose without washout (Fig 2A). However, the subsequent serum phosphorus levels were stably maintained within the target range of 3.5–5.5 mg/dL with an achievement rate of 64.4% at M6 and 53.6% at EOT.

Table 4

Changes in serum phosphorus, hemoglobin, iron-related parameters, calcium and iPTH (N = 197).

	M0	M3	M6	M9	EOT	P-trend
Serum phosphorus (mg/dL)	5.38 ± 0.09	5.16 ± 0.10	5.16 ± 0.11	5.12 ± 0.11	5.19 ± 0.11	0.003
Hemoglobin (g/dL)	10.65 ± 0.09	11.15 ± 0.12	11.11 ± 0.12	11.06 ± 0.12	11.03 ± 0.12	<0.001
Serum iron (μg/dL)	64.48 ± 1.57	80.36 ± 2.46	86.02 ± 3.09	81.93 ± 3.34	75.64 ± 2.03	<0.001
Ferritin (ng/mL)	353.77 ± 16.00	481.49 ± 28.47	588.41 ± 26.70	619.98 ± 26.70	581.37 ± 27.91	<0.001
TSAT (%)	24.11 ± 0.87	33.07 ± 1.36	34.17 ± 1.75	33.23 ± 1.85	32.36 ± 1.24	<0.001
TIBC (μg/dL)	238.59 ± 3.42	216.96 ± 3.23	219.65 ± 4.03	212.57 ± 3.26	212.07 ± 2.93	<0.001
Serum calcium (mg/dL)	9.57 ± 0.06	9.51 ± 0.07	9.46 ± 0.07	9.35 ± 0.08	9.32 ± 0.06	0.02
iPTH (ng/L)	453.25 ± 32.36	486.75 ± 36.05	575.77 ± 39.26	613.78 ± 48.76	555.37 ± 35.41	0.03

Data was presented as mean ± standard error. P-trend values were based on variable containing mean value.

Abbreviations: M0, baseline; M3, 3 months; M6, 6 months; M9, 9 months; EOT, end of treatment; TSAT, transferrin saturation; TIBC, total iron-binding capacity; iPTH, intact parathyroid hormone.

Fig 2

The impact of ferric citrate treatment on the changes of (A) serum phosphorus, (B) hemoglobin and serum iron, (C) TSAT, ferritin, and TIBC and (D) iPTH and calcium from M0 to EOT. Abbreviations: TSAT, transferrin saturation; TIBC, total iron binding capacity; iPTH, intact parathyroid hormone; M0, baseline; EOT, end-of-treatment. The values were expressed as mean and standard error (SE).

With overall treatment adherence of 83.02%, patients achieved the efficacy target with a mean daily dose of 3.35 ± 1.49 g/day. A majority (> 90%) of the patients received a dose of under 6 g/day and > 80% of patients received a dose ranging between 1.5~6 g/day.

Changes of hematological and biochemical laboratory parameters

Hemoglobin and serum iron levels increased significantly and reached the highest levels after 3 and 6 months of ferric citrate treatment, respectively (Fig 2B, P-trend < 0.001). Serum ferritin and TSAT level increased gradually while TIBC showed a decreased trend, and the changes reached a plateau at M3 and maintained till the EOT (Fig 2C, P-trend < 0.001). The calcium level gradually decreases (P-trend = 0.02) and the iPTH levels gradually increased (P-trend = 0.03), as shown in Fig 2D.

Changes in demand for intravenous iron and ESA administration

The required dose of intravenous iron was gradually decreased from 0.63 mg/day at the first quarter (1–3 months) to 0.16 mg/day in the fourth quarter (9–12 months), showing a 74.6% reduction over the 12-month observation (S1 Fig). In addition, the proportion of ESRD patients requiring intravenous iron administration was gradually decreased from 23.3% to 6.5%. Although the proportion of ESRD patients requiring ESA treatment has not changed obviously, the required ESA dose was decreased from 597.2 IU/day at the first quarter to 465/1 IU/day in the fourth quarter, showing a 22.1% reduction over the 12 months of ferric citrate treatment.

Changes of ferritin and TSAT by baseline level

A post hoc subset analysis was performed to stratify ferritin (<300 ng/mL, 300–500 ng/mL, >500 ng/mL) and TSAT (<30%, ≥30%) by baseline levels. The results shown in Fig 3 indicated that the increase was greater in subjects with lower baseline level for ferritin <300 ng/mL and 300–500 ng/mL, which changed from 144.53 ± 8.91 ng/mL to 437.42 ± 37.17 ng/mL and 397.55 ± 7.92 ng/mL to 623.22 ± 39.98 ng/mL respectively (P-trend < 0.001) and TSAT <30%, which changed from 18.45 ± 0.83% to 29.35 ± 1.56% (P-trend < 0.001), however did not alter significantly in higher baseline subsets for ferritin >500 ng/mL and TSAT ≥30% which changed from 650.14 ± 17.42 ng/mL to 765.17 ± 63.31 ng/mL (P-trend = 0.502) and 37.36 ± 0.64% to 38.74 ± 1.75% (P-trend = 0.08) respectively.

Fig 3

Changes in (A) ferritin and (B) TSAT from M0 to EOT according to different baselines of ferritin (<300 ng/mL, 300–500 ng/mL, >500 ng/mL) and TSAT (<30%, ≥30%). The trend of continuous data was calculated by a linear regression. Abbreviations: TSAT, transferrin saturation; M0, baseline; EOT, end-of-treatment. The values were expressed as mean and standard error (SE).

Effect of vitamin D concomitant treatment on iPTH level

Despite serum phosphorus levels was controlled during the study period, iPTH levels gradually increased. The use of the concomitant vitamin D was further analyzed (S4 Table) and it was found no significant change in iPTH level for subjects prescribed with vitamin D treatment (P = 0.189). For subjects without vitamin D treatment, the iPTH level was significantly increased from 323.82 ± 40.98 to 472.94 ± 47.74 ng/L (P<0.001).

Discussion

This is a multicenter, open-label, Phase IV study to evaluate the long-term safety and efficacy of ferric citrate for phosphate-lowing effect in ESRD patients on maintenance hemodialysis. Predictably, ferric citrate, at a mean daily dose of 3.35 ± 1.49 g/day, was well-tolerated during the 12-month treatment and efficaciously controlled serum phosphorus levels (3.5–5.5 mg/dL). Furthermore, the levels of serum iron, ferritin, and TSAT, as well as hemoglobin significantly increased during the treatment, while the level of TIBC decreased. Although the intravenous iron and ESAs were not limited in this study, the demands for these concomitant medications were reduced. No drug-related death or serious TEAEs were observed during 12-month treatment. No new or unexpected safety concerns were observed. The common TEAEs occurred in the gastrointestinal tract. Discolored feces, diarrhea, and constipation were the most common gastrointestinal disorders. The results of this study indicated that ferric citrate is a safe and effective phosphate binder to control hyperphosphatemia with good tolerability, and effectively to improve iron utility and stores in real-world settings.

This study showed consistent efficacy and safety results with previous studies including a placebo-controlled study in Taiwanese [26], long-term studies in Japanese [18, 27], and a 52-week active-controlled followed by a 4-week placebo-controlled study in the United States and Israel [16]. In a previous 8-week study of hemodialysis patients in Taiwan [26], daily dose of 4–6 g was well-tolerated. After 8 weeks of treatment, serum phosphorus declined significantly, while ferritin levels increased significantly. In addition to confirming long-term safety of efficacy, this present study further revealed the different iron repletion effects of ferric citrate in ESRD patients with high, medium and low baseline ferritin levels. As expected, that the bodyweight, size, and diet habits were similar in Asia, the average dose used to maintain serum phosphorus target was similar at around 3 g/day in Taiwanese and Japanese patients. However, the baseline ferritin and TSAT levels in the Japanese ESRD population were much lower [25], and that of Taiwanese ESRD patients are generally maintained at ferritin level of 300−500 ng/mL and TSAT of 30%−50% which are similar to the anemia management suggested in other Asian countries and Europe [28]. This study provided a practical direction to physicians to use ferric citrate in patients with a medium level of ferritin and TSAT.

The treatment of ferric citrate improved iron utility and stores. Pergola et al. [15] and Komatsu et al. [14] recently reported that ferric citrate is also safe and effective to treat iron deficiency anemia with and without nondialysis-dependent CKD, respectively. Long-term use of ferric citrate in nondialysis-dependent CKD has no apparent detrimental effect on kidney functions. In this present study, the ingested iron is absorbed for hematopoiesis as reflected from an average increase in hemoglobin level around 0.5 g/dL in ESRD patients, and the hemoglobin level was maintained until the end of the study, despite the reduced uses of intravenous iron and ESA. The increase of iron storage was saturated when TSAT reached a plateau level and the rate of increase in ferritin slowed down after 6 months of treatment. Subset analysis of ferritin and TSAT showed that the increase was greater in patients with lower baseline levels but did not alter significantly in higher baseline subsets (ferritin >500ng/mL, TSAT ≥ 30%). Unlike traditional intravenous iron injections, oral ferric citrate is under endogenous regulatory control in the gastrointestinal tract. The regulation from hepcidin and membrane proteins on the small intestine mucosal cell such as DcytB (Ferrireductase), DMT1 (divalent metal transporter1), and Fpn1 (Ferroportin1) tightly control iron’s uptake and mobilization into the blood stream [29]. This mechanism allows iron absorption as needed, and lowers the chance of excessive absorption when the iron stores in the body are sufficient.

As ferric citrate is simultaneously improving the iron stores and hemoglobin level, the dose and demand for intravenous iron and ESA in ESRD patients are reduced accordingly. Anemia is frequently encountered in CKD patients and prevalence rate increases along with CKD progression. As kidney function declines, it negatively affects erythropoietin production and iron absorption, therefore leading to chronic renal anemia. Kidney Disease: Improving Global Outcomes (KDIGO; https://kdigo.org/guidelines/) proposed the clinical guidelines for the management of anemia and iron supplementation in CKD patients [22]. However, the use of high doses of ESA is known to be related with an increased risk of stroke [30, 31], hypertension [32], vascular access thrombosis [33], and cardiovascular disease [20, 34–37]. High doses of ESA may also lead to functional iron deficiency in CKD and ESRD patients. Similarly, intravenous iron administration is also associated with risk of thrombosis, infection, allergic and non-allergic reactions, and cardiovascular death [19, 21, 23]. Intravenous iron aggravated oxidative stress, increased atherogenesis and cardiovascular toxicity, and increased the tendency for infections [38, 39]. It is worth noting that in this study, ferric citrate gradually reduced the demand (72.1% reduction) and the dose (74.6% reduction) of intravenous iron in ESRD patients. The ESA dose was also reduced by 22.1% after 12-month of ferric citrate treatment, while the demand for ESA was not decreased dramatically. This dose reduction effect was also reported by Umanath et al. while hemoglobin was stably maintained [17]. In the pharmacoeconomic aspect, fewer intravenous injections may reduce the shortage of nurses, overall medical expenses, and the risks of side effects such as infections and allergies. However, the benefits in iron repletion and reduction of concurrent anemia treatments were only observed in ferric citrate but not in other iron-based binders. In a 52-week, Phase III study by Covic et al. [40], both sevelamer carbonate and sucroferric oxyhydroxide did not show an increase in iron stores and a reduction in the dose or need for ESA and intravenous iron [11, 17].

In this study, iPTH levels increased despite a significant reduction in serum phosphorus and calcium levels. As secondary hyperparathyroidism is also a common complication in CKD patients, reducing dietary phosphorus uptake along with the use of vitamin D analogs and calcimimetics is usually recommended by KDIGO and other guidelines. In a retrospective study by Yoshida et al. [18], more than 93% of patients concomitantly prescribed vitamin D during the 36-month observation period and no significant change in iPTH was found with ferric citrate treatment. In our present study, only 50.3% of patients in this study concomitantly prescribed vitamin D (S4 Table), suggesting that vitamin D may play a role in controlling the iPTH levels in ESRD patients. Further prospective study is required to clarify this issue.

This study presented long-term clinical experience for up to one year in real-world setting, and some limitations were acknowledged. First, there was a lack of intervention, and it was limited with bias by the missing values resulting from patients who did not attend all scheduled visits. Second, 5.9% of patients had taken ferric citrate prior to the study. These patients may have a lower risk of adverse events during the study period, which may increase bias. Third, the safety and efficacy analyses are limited to 12 months of treatment; therefore, the conclusions of this study can only be limited within this time frame. Nonetheless, this Phase IV study proves that 12-month ferric citrate treatment is safe and effective in the control of serum phosphorus in hemodialysis patients in Taiwan with the additional benefit of iron-repletion.

Conclusion

Long-term ferric citrate treatment is safe and well-tolerated for ESRD patients with hyperphosphatemia, with a dose range of 1.5–6.0 g/day in Taiwanese population. Ferric citrate can simultaneously replete iron, increase hemoglobin levels, and reduce the dose and/or demand of ESA and intravenous iron. There is no obvious safety concern during 12-month ferric citrate treatment. In routine clinical practice, the dosage of ferric citrate can be adjusted based on serum phosphorous levels and iron-related parameters. Overall, ferric citrate as a phosphate binder has shown promising iron-replenishing benefit for the long-term treatment of hyperphosphatemia in ESRD patients and well-tolerated.

CONSORT 2010 checklist of information to include when reporting a randomised trial*.

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TREND statement checklist.

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Changes of clinical laboratory parameters during the treatment period.

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Most common drug-related TEAEs with incidence rate > 1%.

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Most common TEAEs leading to drug discontinuation with incidence rate >1%.

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Changes of iPTH levels after stratified by concomitant vitamin D treatment.

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Ferric citrate reduces the need for intravenous iron and ESA.

(A) Intravenous iron dosage and administration. (B) ESA dosage and use.

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Clinical study protocol.

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26 Oct 2021

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Additional Editor Comments (if provided):

This study needs a deep revision from the authors to be considered fro publication. The reviewers have distinct approaches and analysis, all of the very relevant. The reviewer 1 has raised concerns about the relevance and the design, while the reviewer has made very important questions about the statistical analysis. The reviewer 3 has focused in the improvement of the discussion on the explanations for your results.So, may decision is "major revision "

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Reviewers' comments:

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Lee et al. examined the long-term safety and efficacy of ferric citrate in patients undergoing hemodialysis. They showed that ferric citrate efficaciously and safely decreased serum phosphate levels in a prospective, multi-center, open-label, one-year study.

There are a lot of previous studies showing that ferric citrate efficaciously and safely decreased serum phosphate levels. However, many of them have not been cited in this manuscript. The novelty of the present study is questionable.

As described in the Methods section, 5.9% of patients had taken ferric citrate prior to the study. These patients were very low risk of adverse events in the study period. The study has been biased on this point.

In the Discussion section, the authors described that “the safety and efficacy analyses are limited to 12 months of treatment; therefore, the conclusions of this study can only be limited within this time frame” as a limitation of this study. Regarding this point, a 3-year retrospective study (Yoshida et al., Int Urol Nephrol, 2021) has been reported recently.

Please correct the style of references.

Multiple names of adverse events have been written using the capital letters. What do they mean?

Reviewer #2: Phosphate binders and iron supplementation are a part of the basic treatment of HD patients. Phosphate binders bind phosphorus in the gut and prevent their absorption.

Iron based phosphate binders are a new line of treatment. In HD patients iron deficiency is usually treated by intravenous route since oral route in HD patients is not efficacious and poorly tolerated.

At the present time there are 2 classes of iron-based phosphate binders: sucroferric oxhydroxide and ferric citrate. Both are effective in the treatment of hyperphosphatemia.

Sucroferric oxhydroxid is poorly absorbed in the gut functioning as a real binder, while ferric citrate is absorbed in the gut also improving iron parameters. Ferric citrate shows a dual functionality controlling serum phosphate and increasing ferritin levels and transferrin saturation. This effect on iron parameters is not observed with sucroferric oxhydroxide.

This study is a 12-month phase IV, multicenter, open-label study that has enrolled 202 patients. It has verified that ferric citrate is effective as phosphate binder but also improved iron parameters (ferritin and TSAT). This effect was only found in patients with low Ferritin levels (≤500 ng/mL) an TSAT (<30%) at baseline.

We have at present 2 iron-based phosphate binders with different properties regarding iron supplementation that may be taken in consideration based on the iron state of the patient.

Ferric citrate is an interesting option for treating both hyperphosphatemia and anemia. It improved hemoglobin levels, TSAT and ferritin with a reduced need for erythropoiesis stimulating agents and intravenous iron. Diarrhea was the most common adverse reaction.

Phosphate binding is a chronic treatment. Is there a need for surveillance of iron overload in patients treated with Ferric Citrate?

What are the mechanisms that avoid iron absorption with sucoferric oxhydroxide?

What are the mechanisms that explain the amelioration of iron parameters only in the patients with low ferritin levels and low transferrin saturation?

Reviewer #3: The primary objective of this 12-month, Phase IV, multicenter, open-label study is to explore the long-term safety and efficacy of ferric citrate in hemodialysis patients in Taiwan, and further evaluate the iron repletion effect and change of iron parameters by different baseline groups. Although this is an interesting study, there are several major trial design and statistical concerns.

Critiques

1. The Abstract should only report the primary and secondary pre-determined endpoints in the protocol or https://clinicaltrials.gov/ct2/show/NCT03256838, i.e., number of subjects with treatment-emergent adverse events (TEAEs), percentage of subjects with treatment-emergent adverse events (TEAEs), and serum phosphorus. Please clearly state which of these are the primary and secondary endpoints. The rest of the analyses should be exploratory, and the results from these analyses should not be included in the Abstract.

2. Please provide either the power analysis or precision analysis in the revised manuscript. In the power analysis, please clearly specify the hypothesis, type I error (one or two-sided), study power, clinical significance level with justification, and the statistical method for determining the study sample size.

3. The authors used the paired t-test to examine the trend effect. Please use the mixed effects model to examine the trend effect because each subject has multiple measurements. The mixed effects model should also adjust for confounding variables. Please provide a detailed multivariable data analysis plan in the revised manuscript that includes (1) model assumptions checking, (2) model performance evaluation, (3) the strategy of handling non-linear terms, (4) the methods of analyzing the missing data, and (5) the method of analyzing interaction terms.

4. All the conclusions, e.g., “the iron-repletion by ferric citrate is effective and gradual, and the increase is limited in patients with a higher baseline,” should be supported by statistical tests with 95% confidence intervals.

5. The authors should conduct a sensitivity analysis for 117 subjects (57.9%) who had completed all the scheduled ferric citrate treatment for 12 months.

6. The authors should discuss the similarities or differences in the results and baseline distributions between this Phase IV trial and previously published Phase III trials.

7. All the tables should include the sample size.

8. Please add the original trial protocol to the Supplemental Information section.

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Reviewer #1: No

Reviewer #2: Yes: Teresa Adragao

Reviewer #3: No

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10 Dec 2021

Response letter to PONE-D-21-25691

Reviewer #1: Lee et al. examined the long-term safety and efficacy of ferric citrate in patients undergoing hemodialysis. They showed that ferric citrate efficaciously and safely decreased serum phosphate levels in a prospective, multi-center, open-label, one-year study.

1. There are a lot of previous studies showing that ferric citrate efficaciously and safely decreased serum phosphate levels. However, many of them have not been cited in this manuscript. The novelty of the present study is questionable.

Response: The clinical effects of ferric citrate in patients with renal disease are received significant attention, with evidence from a number of clinical trials and retrospective studies. In addition to the previous studies mentioned in our article studying ferric citrate for the treatment of hyerpohsphoatemia, Tadashi Yoshida investigated the efficacy and safety of ferric citrate in 33 hemodialysis patients (Int Urol Nephrol. 2021; Jul 15). Some recent research are studying ferric citrate for the treatment of iron deficiency anemia. Pergola et al. recently reported the efficacy and safety of a 12-month ferric citrate treatment in non-dialysis-dependent chronic kidney disease (CKD) (Am J Nephrol. 2021;52(7):572-581). The phase 3 non-inferiority study by Komatsu et al. explored the safety and efficacy of ferric citrate in Japanese patients with iron deficiency anemia (Int J Hematol. 2021 Jul;114(1):8-17). Although these studies continue to confirm that ferric citrate is an effective phosphate binder in different patient groups, the pharmacologic effect of iron repletion on different basal ferritin and TSAT levels are inadequate.

The previous studies with long term results were from Japanese and US population. However, the iron status of Japanese and US dialysis patients represents two extreme iron status groups. In Japan, the average serum ferritin level was 73 ng/ml while it was 887 ng/ml in the US. There are no studies to evaluate its long-term effect in other ESRD patient groups of most of other countries (such as European, Chinese or Korean populations) having ferritin (300~500 ng/ml) at medium level. Therefore, the remaining questions to be explored is what is the suitable practice for ferric citrate in balancing its phosphate binding and iron supplement effect for the majority of the patients around the world. Our study conducted in Taiwan provides real world data to this unknown question. The results of our study revealed the clinical values for of ferric citrate in ESRD patients with medium (300-500 ng/mL) ferritin levels.

The study population of our study, Taiwanese ESRD population, provides an interesting study angle to evaluate pharmacology effect of a CKD drug product for other Asian countries. Firstly, Taiwan has highest ESRD prevalence rate in the world for many years, and secondly, the dialysis program is fully reimbursed by its healthcare budget. That means 100% coverage with sophisticated management and monitoring system to have a comprehensive view on drug products’ true response. In Taiwan, the iron contents are maintained at a ferritin level of 300−500 ng/mL and TSAT of 30%−50%, which is similar to 2004 European guidance of 200-500 ng/ml and 30%-40% for ferritin and TSAT, respectively. As the diet habits are similar across China, Korea, Taiwan, and several Asian countries, we hope this study could provide a practical direction for ferric citrate’s use in managing both hyperphosphatemia and anemia, the two most prevalent complications in dialysis patients. To facilitate physicians’ decision and prediction on the pharmacological reactions, we further conduct several sub-group analyses by different baseline characteristics after administration of ferric citrate. For example, the changes of ferritin and TSAT in patients with different baseline levels that is commonly used in evaluating iron supplementation. The changes of serum phosphorus in patients with different level to provide more optimized starting dose and lower pill burden.

As this is a Phase IV study to evaluate the effect in the real-world settings, the non-limiting use on the ESAs and IV iron actually provide more insight on the impact of anemia management. We believe that our study may bring useful information to answer following questions (1) While managing serum phosphorus in the target range, could the use of ferric citrate also benefit iron stores in these majority of ESRD patients having mid rang ferritin level? (2) What’s the impact on the existed anemia treatments such as ESAs and IV iron uses? (3) What is the long-term safety of this drug product?

Note:

According to UK renal registry 20th annual report, the median ferritin in HD patients was 410 mg/dL. [1]

In a prospective observational study of Korean Clinical Research Center (CRC) for End-Stage Renal Disease (ESRD), which included dialysis patients enrolled from September 1, 2008, at 31 centers in Korea.. The mean ferritin of these Korean patients is 275.5 ng/mL [2]

[1] Rhodri Pyart et al. UK Renal Registry 20th Annual Report: Chapter 7 Haemoglobin, Ferritin and Erythropoietin in UK Adult Dialysis Patients in 2016: National and Centre-specific Analyses. Nephron. 2018;139(suppl1):165–190. doi: 10.1159/000490965

[2] Kwon O et al. Clinical Research Center for End-Stage Renal Disease (CRC- ESRD) Investigators. The Korean Clinical Research Center for End-Stage Renal Disease Study Validates the Association of Hemoglobin and Erythropoiesis-Stimulating Agent Dose with Mortality in Hemodialysis Patients. PLoS One. 2015 Oct 9;10(10): e0140241. doi: 10.1371/journal.pone.0140241.

2. As described in the Methods section, 5.9% of patients had taken ferric citrate prior to the study. These patients were very low risk of adverse events in the study period. The study has been biased on this point.

Response: We agree with the reviewer’s concern of bias of the 5.9% of patients with a history of using ferric citrate. Thus, this bias concern was added in the limitation section of the revised manuscript (Line 378, Page 16).

3. In the Discussion section, the authors described that “the safety and efficacy analyses are limited to 12 months of treatment; therefore, the conclusions of this study can only be limited within this time frame” as a limitation of this study. Regarding this point, a 3-year retrospective study (Yoshida et al., Int Urol Nephrol, 2021) has been reported recently.

Response: The retrospective study by Yoshida et al. reported the 3-year safety and efficacy of ferric citrate in 33 hemodialysis patients. Although our study has the limitation of 1-year follow-up, our prospective study still shows different clinical values. First and foremost, based on the inclusion criteria of Yoshida’s study, a total of 33 patients received ferric citrate for "at least" 1 month. In addition, patients in that study were co-treated with other phosphate binders that the prescribed dose was smaller than the doses used in other clinical studies. In contrast, in our study, a total of 117 patients completed 12 months of ferric citrate treatment as sole treatment for hyperphosphatemia. Second, the baseline ferritin levels of the Japanese patients in the Yoshida’s study (34 ng/mL) was much more lower and significantly different from patients of most of the world. The Yoshida et al study can only provide conclusion on efficacy and safety in limited patients with a low baseline ferritin level.

Ferric citrate has dual pharmacological effect on both serum phosphate and iron state. When we use ferric citrate to control serum phosphate level, the impact on the iron state should also be taken into consideration. Serum ferritin is an indicator of body iron stores. As the baseline iron storage is a factor associated with regulation of iron absorption. It is necessary to understand more the effect of ferric citrate in patients with different iron storage level to further explore the impact on the anemia management.

The baseline ferritin level of our study (353 ng/mL) is much similar to patients of most of the world. Thus, the results of this study can provide more insight on the impact of anemia management in patient with ferritin at medium level. In addition, the post hoc analysis results of this study can help us understand the iron repletion effect of ferric citrate in patients with low (<300 ng/mL), medium (300-500 ng/mL) and high (>500 ng/mL) basal ferritin levels. In addition, it provides a real-world clinical basis for doctors to predicate the ferritin levels during the treatment of ESRD patients with ferric citrate.

4. Please correct the style of references.

Response: Thanks to the referee for pointing out this mistake. In the revised manuscript, the style of references was modified to meet the standard of PLoS One.

5. Multiple names of adverse events have been written using the capital letters. What do they mean?

Response: We just keep the original terms of the adverse events of the trial protocol, and have no other meanings that we want to emphasize. To avoid unintentional confusion, we amended the capital letters of adverse events in the revised manuscript.

Reviewer #2:

This study is a 12-month phase IV, multicenter, open-label study that has enrolled 202 patients. It has verified that ferric citrate is effective as phosphate binder but also improved iron parameters (ferritin and TSAT). This effect was only found in patients with low Ferritin levels (≤500 ng/mL) an TSAT (<30%) at baseline.

1. Phosphate binders and iron supplementation are a part of the basic treatment of HD patients. Phosphate binders bind phosphorus in the gut and prevent their absorption. Iron based phosphate binders are a new line of treatment. In HD patients iron deficiency is usually treated by intravenous route since oral route in HD patients is not efficacious and poorly tolerated. At the present time there are 2 classes of iron-based phosphate binders: sucroferric oxhydroxide and ferric citrate. Both are effective in the treatment of hyperphosphatemia. Sucroferric oxhydroxid is poorly absorbed in the gut functioning as a real binder, while ferric citrate is absorbed in the gut also improving iron parameters. Ferric citrate shows a dual functionality controlling serum phosphate and increasing ferritin levels and transferrin saturation. This effect on iron parameters is not observed with sucroferric oxhydroxide. We have at present 2 iron-based phosphate binders with different properties regarding iron supplementation that may be taken in consideration based on the iron state of the patient. Ferric citrate is an interesting option for treating both hyperphosphatemia and anemia. It improved hemoglobin levels, TSAT and ferritin with a reduced need for erythropoiesis stimulating agents and intravenous iron. Diarrhea was the most common adverse reaction. Phosphate binding is a chronic treatment. Is there a need for surveillance of iron overload in patients treated with Ferric Citrate?

Response: This is a very good question. Patients who chronically receive transfusions or use iron-containing medications or supplements (including administered via either iv or oral route) are at risk of iron accumulation and are subject to surveillance of iron overload. Although iron is an essential mineral, excessive iron is harmful for human health. As the iron loading continues, the dedicated iron-binding protein, transferrin, becomes saturated. Excessive iron may damage tissues by catalyzing the formation of reactive oxygen species (ROS) that attach cellular membranes, proteins and DNA, leading to organ damage (Clin J Oncol Nurs. 2009 Oct;13:511-7). Thus, the clinical consequences of iron overload may lead to liver damage, cardiac disease and endocrine disorders. Therefore, it is necessary to assess and monitor the iron status of the patients. On the other hand, since anemia is a common complications of hemodialysis patients and intravenous iron administration is common management of ESRD patients, anemia-related tests (including iron parameters) are routinely performed and monitored in dialysis centers. Therefore, the surveillance of iron overload in patients treated with ferric citrate can be done easily.

2. What are the mechanisms that avoid iron absorption with sucoferric oxhydroxide?

Response: Sucroferric oxyhydroxide is another iron-based phosphate binder recently also approved for the treatment of hyperphosphataemia. Based on the study by Mario Cozzolino et al., sucroferric oxyhydroxide exhibits a low iron-releasing property in the physiological pH range of the gastrointestinal tract. Therefore, the low iron release properties of sucroferric oxyhydroxide support the clinical feature of minimal iron absorption, rather than being caused by avoiding iron absorption.

Reference: Preclinical Pharmacokinetics, Pharmacodynamics and Safety of Sucroferric Oxyhydroxide. Current Drug Metabolism, 2014, 15, 953-965.

3. What are the mechanisms that explain the amelioration of iron parameters only in the patients with low ferritin levels and low transferrin saturation?

Response: The absorption of iron from the intestine into the blood is tightly regulated by iron regulatory proteins and hepcidin/ferroportin system. Hepcidin, a small circulating regulatory hormone peptide, is responsible for regulating the iron homeostasis. Ferroportin is an iron exporter located on the basolateral surface of intestinal enterocytes and macrophages. Hepcidin can bind to ferroportin and induces its internalization and degradation, resulting in cellular iron retention and decreased iron export into circulation. On the other hand, hepcidin levels is negatively regulated by plasma iron concentration and iron stores in the liver (similar to the regulation of glucose by insulin).

When the body is iron deficient, hepcidin concentrations are low, thereby favoring iron absorption and delivery to the plasma from storage sites; but when the body is iron replete, a higher hepcidin concentration reduces iron absorption and impairs iron release from stores. For patients with relatively low iron storage (ferritin < 500 ng/mL or TSAT< 30% in this study), the body system would be more favor to intestinal iron absorption than those patients with a higher baseline iron storage and it leads to increase serum ferritin levels and TAST level. In contrast, for patients with higher iron storage (ferritin > 500 ng/mL or TSAT>30% in this study), intestinal iron absorption is limited due to systemic iron homeostasis mechanism. We described this in the discussion section of revised manuscript as follows (Line 311-317): Unlike traditional intravenous iron injections, oral ferric citrate is under endogenous regulatory control in the gastrointestinal tract. The regulation from hepcidin and membrane proteins on the small intestine mucosal cell such as DcytB (Ferrireductase), DMT1 (divalent metal transporter1), and Fpn1 (Ferroportin1) tightly control iron’s uptake and mobilization into the blood stream. This mechanism allows iron absorption as needed, and lowers the chance of excessive absorption when the iron stores in the body are sufficient.

Reviewer #3: The primary objective of this 12-month, Phase IV, multicenter, open-label study is to explore the long-term safety and efficacy of ferric citrate in hemodialysis patients in Taiwan, and further evaluate the iron repletion effect and change of iron parameters by different baseline groups. Although this is an interesting study, there are several major trial design and statistical concerns.

1. The Abstract should only report the primary and secondary pre-determined endpoints in the protocol or https://clinicaltrials.gov/ct2/show/NCT03256838, i.e., number of subjects with treatment-emergent adverse events (TEAEs), percentage of subjects with treatment-emergent adverse events (TEAEs), and serum phosphorus. Please clearly state which of these are the primary and secondary endpoints. The rest of the analyses should be exploratory, and the results from these analyses should not be included in the Abstract.

Response: Thanks for the reviewer’s comments. The primary endpoint was to assess the safety profiles of ferric citrate. The secondary endpoints were to evaluate the efficacy by the time-course changes of serum phosphorus levels and the number of subjects who achieved the target range of serum phosphorus. The primary and secondary endpoints were added in the Abstract of the revised manuscript.

On the other hand, although exploratory outcome measures were not main reason we conducted this study, the results are still very important, especially in the aspect of iron repletion. This is because anemia is the most common complication in ESRD patients. Thus, we removed the description of hemoglobin, aluminum, ALT and AST in the abstract section, but hope to keep the description of result of iron improvement.

2. Please provide either the power analysis or precision analysis in the revised manuscript. In the power analysis, please clearly specify the hypothesis, type I error (one or two-sided), study power, clinical significance level with justification, and the statistical method for determining the study sample size.

Response: Thanks for the reviewer’s suggestion. Since this is a single-arm Phase-IV study, and the major purpose is to collect the real-world effectiveness of ferric citrate as evaluated in an observational, non-interventional trial in a naturalistic setting. In fact, safety profile for a minimum drug exposure in 100 evaluable patients for 6 months and 1 year are required by the Taiwan regulatory authority. Thus, recruiting 200 subjects should be sufficient to collect a 1-year safety profile of at least 100 patients, and the final sample size of 200 subjects is driven by a post-approval commitment to the regulatory authority.

However, our post-power analysis also supports that the sample size of 200 participants is large enough for this study. We performed the post hoc power analysis to evaluate whether the sample size was sufficient to detect time trend of study outcomes across 13 measurements among hemodialysis patients receiving ferric citrate treatment. The power analysis was based on repeated ANOVA with one group (i.e., all patients received ferric citrate medication) and 13 measurements. The significance level was 0.05 and total sample size was 197. The variance explained by the time effect was 2.48 and the error variance was 1.00. The Greenhouse-Geisser Epsilon was 0.84 for nonsphericity correction. The estimated power was higher than 0.99, indicating that the sample size as well as parameters assumed and given statistics were sufficient to detect the time effect of serum phosphorus across 13 measurements.

3. The authors used the paired t-test to examine the trend effect. Please use the mixed effects model to examine the trend effect because each subject has multiple measurements. The mixed effects model should also adjust for confounding variables. Please provide a detailed multivariable data analysis plan in the revised manuscript that includes (1) model assumptions checking, (2) model performance evaluation, (3) the strategy of handling non-linear terms, (4) the methods of analyzing the missing data, and (5) the method of analyzing interaction terms.

Response: Thanks for the reviewer’s comment. According to reviewer’s suggestion, mixed effects model for primary outcomes were performed to deal with within-subject correlation given patients being followed repeatedly. Statistical assumption, model specifications, and main results were presented.

First, we checked if laboratory tests followed normal distribution by using Kolmogorov-Smirnov test, a method to examine normal assumption. If normal assumption was violated, linear models would not be appropriate for estimation of efficacy of ferric citrate. The results showed that all laboratory measures violated normal assumption, except for hemoglobin. Therefore, all measures were log transformed for subsequent regression analyses, which was a common method of transforming skewed data to conform to normality. After log transformation, the distribution of all laboratory tests, but not TSAT and ferritin, were normally distributed. As a result, all linear regression models were performed by using transformed data.

Second, to evaluate the time trend of study outcomes, non-linear relationship was assessed by adding quadratic term in the mixed effects model. If the quadratic term is statistically significant, there exists a non-linear trend (e.g., a U-shape or an inverted U-shape). Table 1 shows that there were non-linear trends for hemoglobin, serum iron, ferritin, TSAT, and iPTH (all inverted U-shape across time). However, given that the magnitude of coefficients of quadratic form for other lab measures is not obvious despite statistical significance, linear trend would be reported for all study outcomes.

Table 1. Testing for non-linear trend of study outcomes

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Third, multivariate mixed effects analyses were also performed with adjustment for study sites, age, sex, and body weight at baseline. Slope was used to represent the time trend of study outcomes; a positive value indicates an increasing trend while a negative value indicates a decreasing trend. The estimate slopes of time trend for study outcomes performed by multivariate mixed effects regression model were presented in Table 2, adjustment for study sites, age, sex, and body weight. Serum phosphorus (slope=-0.004; 95% CI=-0.0076, 0.0002; p=0.042), TIBC (slope=-0.008; 95% CI=-0.0098, -0.0066; p<0.001), and serum calcium (slope=-0.002; 95% CI=-0.0029, -0.0009; p<0.001) had decreasing trend after baseline. There was no linear trend for hemoglobin (slope=0.001; 95% CI=-0.0011,0.0021; p=0.542). Other measures increased across time (i.e., serum iron, ferritin, TSAT, and iPTH) (Table 2).

Table 2. Multivariate mixed effects regression analysis for study outcomes

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Forth, to assess the influence of missing data due to incomplete follow-ups, we performed a sensitivity analysis by including patients with complete follow-ups, that is, patients who completed 13 visits in the trial. The results were presented in Table 3. The estimated mean and standard error for each laboratory measure at each visit did not change by excluding patients without complete follow-ups (Table 3). Sensitivity analysis for linear trend estimation was also done by including patients with complete follow-ups. The magnitude of estimates for slopes and standard errors was similar to those in our primary analyses. Time trend for serum phosphorus became statistical insignificance (slope= -0.003; 95% CI=-0.0071,0.0011; p=0.157) (Table 4). These results suggested that our main results were not influenced by the missingness due to incomplete follow-ups.

On the other hand, we did not assess group-by-time interactions due to one-arm study design in the current study.

Table 3. Sensitivity analysis for changes in serum phosphorus, hemoglobin, iron-related parameters, calcium and iPTH in patients with complete follow-up (n=117)

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Table 4. Multivariate mixed effects regression analysis for study outcomes in patients with complete follow-up (n=117)

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4. All the conclusions, e.g., “the iron-repletion by ferric citrate is effective and gradual, and the increase is limited in patients with a higher baseline,” should be supported by statistical tests with 95% confidence intervals.

Response: Regarding the reviewer’s concern, linear trend estimation was performed to assess the time trend of the 3 ferritin levels. The results also support our previous statistical analysis, showing that ferritin < 300 ng/ mL (95% CI=0.0342, 0.0970) and ferritin 300-500 ng/mL (95% CI=0.0128, 0.0354) had the same significant increase trend after baseline (P<0.001). In addition, the linear trend was not significant over time for ferritin > 500 ng/mL (95% CI=-0.0086, 0.00228; P=0.374).

[Based on the regulations of PLOS ONE, only text can be filled in this part. Please download the detailed response letter that we uploaded to PLOS ONE]

5. The authors should conduct a sensitivity analysis for 117 subjects (57.9%) who had completed all the scheduled ferric citrate treatment for 12 months.

Response: Thanks for the reviewer’s comment. To assess the influence of missing data due to incomplete follow-ups, sensitivity analysis was performed by including patients with complete follow-ups, that is, patients who completed 13 visits in the trial. After excluding patients without complete follow-ups, the mean and standard error of each laboratory measure at each visit did not change much (below table 3).

Table 3. Sensitivity analysis for changes in serum phosphorus, hemoglobin, iron-related parameters, calcium and iPTH in patients with complete follow-up (n=117)

[Based on the regulations of PLOS ONE, only text can be filled in this part. Please download the detailed response letter that we uploaded to PLOS ONE]

Sensitivity analysis for linear trend estimation was also done by including patients with complete follow-ups. The magnitude of estimates for slopes and standard errors was similar to those in our primary analyses. Only the time trend for serum phosphorus became statistical insignificance. These results suggested that serum phosphorus did slightly influence by the missingness due to incomplete follow-ups.

Table 4. Multivariate mixed effects regression analysis for study outcomes in patients with complete follow-up (n=117)

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6. The authors should discuss the similarities or differences in the results and baseline distributions between this Phase IV trial and previously published Phase III trials.

Response: Thanks for the reviewer’s comment. In our previous Phase III trial in Taiwanese, a total of 147 ESRD patients received fixed dose of ferric citrate 4 g/day or 6 g/day for 8 weeks (J Nephrol. 2015 Feb;28(1):105-13. doi: 10.1007/s40620-014-0108-6. Epub 2014 May 20). The results demonstrated the effectiveness at a daily dose of 4-6 g/day and both doses were well-tolerated in ESRD patients during the 8-week study period. The most common adverse events were gastrointestinal disorder, mostly discolored feces and diarrhea. After 8 weeks of treatment, serum phosphorus declined significantly, while ferritin levels increased significantly. The current Phase IV study further showed the long-term safety and efficacy of ferric citrate at a dose range of 1.5-6.0 g/day. The serum phosphorus was sustainably controlled in long term, while the increase of iron storage showed a trend of plateau after 3-6 months of treatment. In addition, the current Phase IV study further revealed the different iron repletion effects of ferric citrate in ESRD patients with high, medium and low baseline ferritin levels. Furthermore, our real-world effectiveness of ferric citrate as evaluated in an observational, non-interventional trial in a naturalistic setting showed that long-term use of ferric citrate did greatly reduced the need for erythropoiesis-stimulating agents (ESA) and intravenous iron.

7. All the tables should include the sample size.

Response: Thanks for the reviewer’s suggestion. The sample size was added to each tables of the revised manuscript.

8. Please add the original trial protocol to the Supplemental Information section.

Response: The original protocol of this trial was uploaded as the supplementary information.

Submitted filename: Response Letter.docx

Click here for additional data file.

16 Feb 2022

Long-term safety and efficacy of ferric citrate in phosphate-lowering and iron-repletion effects among patients with on hemodialysis: a multicenter, open-label, Phase IV trial

PONE-D-21-25691R1

Dear Dr.  Der-Cherng Tarng

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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After rereading the manuscript and based on the unanimous decision of the reviewers, my decision is "Accept"

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

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Reviewer #1: (No Response)

Reviewer #2: The authors have answered to my questions in a satisfactory way. Ferric citrate is an interesting option for treating both hyperphosphatemia and anemia.

Reviewer #3: The authors have responded well to the statistical issues raised in the previous review. There is no further statistical concern about this revised manuscript.

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Reviewer #1: No

Reviewer #2: Yes: Teresa Adragao

Reviewer #3: No

22 Feb 2022

PONE-D-21-25691R1

Long-term safety and efficacy of ferric citrate in phosphate-lowering and iron-repletion effects among patients with on hemodialysis: a multicenter, open-label, Phase IV trial

Dear Dr. Tarng:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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