Clinical trial NCT02888171
Conditions: Chronic Kidney Disease Iron Deficiency Anemia
Sponsor: University of Alabama at Birmingham
Start date: 2016-09-01; Completion date: 2019-03-30
Enrollment: 60 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The main objective of the study is to compare the impact of oral ferric citrate compared to standard of care oral ferrous sulfate on serum iron, percent transferrin saturation, ferritin, hepcidin and hemoglobin levels in individuals with moderate to severe chronic kidney disease (CKD) and absolute iron deficiency.

Detailed description:
Ferric citrate is an FDA-approved oral phosphorus binder that has been shown to be effective in reducing serum phosphorus and fibroblast growth factor 23 (FGF23) concentrations and increasing iron stores and hemoglobin in individuals with non-dialysis-dependent CKD who have iron-deficiency anemia. This may prove to be advantageous in individuals with pre-dialysis CKD who require iron supplementation for iron-deficiency anemia. This is because ferric citrate may not only restore iron stores in individuals who are iron deficient, but by lowering FGF23 concentrations, ferric citrate may increase local and systemic concentrations of 1,25-dihydroxyvitamin D, a powerful inhibitor of hepcidin synthesis, potentially attenuating the increase in hepcidin following oral iron supplementation. When compared to standard iron supplementation therapies (e.g., oral ferrous sulfate) that powerfully stimulate hepcidin secretion, this may then allow for greater iron bioavailability by increasing iron absorption in the gut while also reducing the degree of iron sequestration in reticuloendothelial system stores. However, little is known about the comparative effectiveness of treatment with oral ferric citrate vs. oral ferrous sulfate (currently the standard of care) in increasing iron stores and hemoglobin in iron-deficient CKD patients. If ferric citrate is shown to not only improve overall iron status, but also partially mitigate the long-term effects of iron supplementation on hepcidin secretion by increasing endogenously produced 1,25-dihydroxyvitamin D, this may indicate that ferric citrate can provide superior short- and long-term effects on iron-restricted erythropoiesis in CKD as compared to the current standard of care. The main objectives of the study are to compare the impact of ferric citrate compared to standard of care ferrous sulfate on serum iron, percent transferrin saturation (TSAT), ferritin, hemoglobin and hepcidin concentrations in individuals with moderate to severe CKD and absolute iron deficiency.

Arms: arm 1: Participants randomized to the ferric citrate arm will receive 2 grams of ferric citrate three times a day with each meal. arm 2: Participants randomized to the ferrous sulfate arm will receive 325 mg of ferrous sulfate three times a day
Interventions: intervention 1: ferric citrate intervention 2: ferrous sulfate
Intervention descriptions: intervention 1: Participants randomized to the ferric citrate arm will take 2 grams of ferric citrate three times a day with meals. intervention 2: Participants randomized to the ferrous sulfate arm will take 325 mg of ferrous sulfate three times a day.

Protocol documents:
Prot_SAP_000.pdf: 
 
 
Clinical Study Protocol 
 
Impact of ferric citrate vs ferrous sulfate on iron parameters and hemoglobin in 
individuals with moderate to severe chronic kidney disease (CKD) with iron deficiency 
 
NCT02888171 
 
Edition No. 
06/14/18 
 


 
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Clinical Protocol 
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1 
Backgroun
d and 
Rationale 
Disturbances in iron homeostasis and bone/mineral metabolism are common complications of 
chronic kidney disease (CKD) that lead to a number of adverse outcomes including anemia, 
bone disease and cardiovascular mortality.1 In clinical practice, disorders of iron homeostasis 
and bone/mineral metabolism are considered distinct entities and are treated separately. 
However, emerging evidence suggests that hormones regulating bone/mineral metabolism and 
iron homeostasis interact with each other on multiple levels, and that taking account of these 
interactions will improve the treatment of both disorders of iron homeostasis and disturbances in 
bone/mineral metabolism. 
Iron plays a crucial role in the physiology of oxygen transport. Excess iron can lead to oxidative 
stress, while its deficiency can lead to anemia and poor oxygen delivery. Tight control of iron 
metabolism is required for normal physiological function. Since humans lack the ability to 
actively excrete excess iron, iron absorption is tightly controlled at the proximal small intestine 
and within the reticuloendothelial system.2 Hepcidin is the master regulator of iron metabolism.3 
Hepcidin regulates iron metabolism by binding to the cell-surface iron transporter, ferroportin, 
resulting in its internalization and lysosomal degradation.4 This effectively blocks iron absorption 
in the gut and iron release from macrophage and hepatocytes resulting in decreased iron 
availability for erythropoiesis.4,5 States of increased iron demand such as iron deficiency lead to 
a fall in hepcidin levels and thus increased iron absorption.3,6 Alternatively, in states of 
inflammation (such as CKD) or iron overload, hepcidin concentrations are elevated leading to 
decreased iron availability for erythropoiesis.3,6 
Iron deficiency is highly prevalent in CKD and is a major mechanism underlying anemia of 
CKD.7,8 It can be broadly classified as either absolute—in which total iron stores are depleted—
or functional, in which iron stores are sufficient, but unable to be mobilized from the 
reticuloendothelial system (RES) for erythropoiesis. Hepcidin concentrations are elevated in 
CKD and are a key mechanism underlying anemia of CKD.1 Experimental data show that 
reducing hepcidin improves anemia of CKD,9-11 suggesting that interventions that mitigate the 
rise in hepcidin can help to reduce the prevalence and severity of anemia in CKD patients. 
Individuals with CKD respond poorly to oral iron relative to individuals without kidney disease.  
One major reason for this is that iron supplementation stimulates hepcidin secretion in CKD 
patients, who already have constitutively elevated hepcidin concentrations, further exacerbating 
iron-restricted erythropoiesis. For this reason, mitigating the rise in hepcidin with iron 
supplementation in individuals with CKD could be highly advantageous in that it would allow for 
better iron bioavailability (improvement in serum iron parameters) by enhancing gut iron 
absorption while at the same time attenuating iron sequestration within the RES.  In addition to 
treating anemia of iron deficiency, this could enhance responsiveness to erythropoietin 
stimulating agents which require iron mobilization from the RES for maximum efficacy.3  
Pharmaceutical agents that can both increase iron stores and mitigate the rise in hepcidin 
secretion following iron supplementation are not currently available. 
Low vitamin D concentrations are independently associated with increased prevalence and 
severity of anemia.12-15 The proposed mechanisms by which vitamin D influences erythropoiesis 
have largely focused on the inhibitory effects of vitamin D on inflammation and its stimulatory 
effects on erythropoiesis.16 However, recent data suggest vitamin D may also impact 
erythropoiesis by modulating iron homeostasis via regulation of hepcidin synthesis. Bacchetta et 
al17 showed that 1,25-dihydroxyvitamin D directly inhibits hepcidin expression by binding to a 
vitamin D response element in the gene encoding hepcidin (HAMP). Further, in a small study 


 
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healthy volunteers, treatment with a single oral dose of ergocalciferol (100,000 IU) reduced 
hepcidin by 34% within 24 hours, which was sustained at 72 hours.17 Another study showed that 
nutritional vitamin D supplementation was associated with reduced hepcidin concentration in 
individuals with stage 3 or 4 CKD.18 Together, these data indicate that vitamin D is a potent 
inhibitor of hepcidin expression and increasing 1,25-dihydroxyvitamin D can lower hepcidin 
concentrations in individuals with CKD. 
An important mechanism underlying the high prevalence of 1,25-dihydroxyvitamin D in CKD is 
elevated fibroblast growth factor 23 (FGF23) concentrations. FGF23 is a hormone secreted by 
bone cells that regulates vitamin D and phosphorus homeostasis.19 FGF23 enhances urinary 
phosphorus excretion and decreases the activation of vitamin D by inhibiting 1-alpha-
hydroxylase, the enzyme which converts 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D. 
Elevated circulating concentrations of FGF23 are extremely common in CKD, likely as a 
physiological adaptation to help maintain serum phosphorus in the normal range by enhancing 
urinary phosphorus excretion and reducing gut absorption of phosphorus via inactivation of 
vitamin D.19  While this is important for maintaining phosphorus balance in the short term, this 
has long-term consequences such as progressive reductions in the synthesis of 1,25-
dihyroxyvitamin D.   
Ferric citrate effectively binds dietary phosphorus while 
improving iron status and hemoglobin in individuals with 
CKD.20  In addition, ferric citrate has been shown to reduce 
FGF23 concentrations in individuals with pre-dialysis CKD.20 
Based upon these data, we hypothesize that in individuals 
with moderate to severe CKD, as compared to standard oral 
iron formulations used to treat iron deficiency anemia, 
treatment with ferric citrate will more effectively improve iron 
parameters than ferrous sulfate, in part by mitigating the rise 
in hepcidin concentrations following iron repletion by reducing 
FGF23 concentrations and enhancing vitamin D activation. 
Thus, ferric citrate will be shown to be unique in that it can 
improve iron parameters and hemoglobin while at the same 
time mitigating iron-induced stimulation of hepcidin secretion, 
providing superior short- and long-term effects on iron-
restricted erythropoiesis in CKD (Figure 1). 
 
1.1 
Study Rationale 
Ferric citrate is an FDA-approved oral phosphorus binder that has been shown to be effective in 
reducing serum phosphorus and FGF23 concentrations and increasing iron stores and 
hemoglobin in individuals with non-dialysis-dependent CKD who have iron-deficiency anemia.20  
This may prove to be advantageous in individuals with pre-dialysis CKD who require iron 
supplementation for iron-deficiency anemia.  This is because ferric citrate may not only restore 
iron stores in individuals who are iron deficient, but by lowering FGF23 concentrations, ferric 
citrate may increase local and systemic concentrations of 1,25-dihydroxyvitamin D, a powerful 
inhibitor of hepcidin synthesis, potentially attenuating the increase in hepcidin following oral iron 
supplementation. When compared to standard iron supplementation therapies (e.g., oral ferrous 
sulfate) that powerfully stimulate hepcidin secretion, this may then allow for greater iron 
bioavailability by increasing iron absorption in the gut while also reducing the degree of iron 
sequestration in RES stores.  However, little is known about the comparative effectiveness of 
treatment with oral ferric citrate vs. oral ferrous sulfate (currently the standard of care) in 
increasing iron stores and hemoglobin in iron-deficient CKD patients.  If ferric citrate is shown to 
not only improve overall iron status, but also partially mitigate the long-term effects of iron 
Figure 1 


 
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supplementation on hepcidin secretion by increasing endogenously produced 1,25-
dihydroxyvitamin D, this may indicate that ferric citrate can provide superior short- and long-term 
effects on iron-restricted erythropoiesis in CKD as compared to the current standard of care. 
2 
Study 
Objective 
and 
Hypotheses 
The main objectives of the study are to compare the impact of ferric citrate compared to 
standard of care ferrous sulfate on serum iron, percent transferrin saturation (TSAT), ferritin, 
hemoglobin and hepcidin concentrations in individuals with moderate to severe CKD and 
absolute iron deficiency.  We will test the following hypotheses: 
Primary Hypothesis: In patients with moderate to severe CKD not requiring dialysis (estimated 
glomerular filtration rate 15-45 ml/min/1.73m2) with absolute iron deficiency (serum ferritin 
≤300ng/ml and TSAT ≤ 30%), treatment with ferric citrate will lead to greater improvements in 
markers of iron bioavailability (serum iron, ferritin, transferrin saturation) than treatment with 
ferrous sulfate.  
Secondary Hypotheses: In patients with moderate to severe CKD not requiring dialysis 
(estimated glomerular filtration rate 15-45 ml/min/1.73m2) with absolute iron deficiency (serum 
ferritin ≤300ng/ml and TSAT ≤ 30%), as compared to treatment with ferrous sulfate for 12 
weeks, treatment with ferric citrate for twelve weeks will result in (1) a greater rise in hemoglobin 
concentrations and (2) a lower rise in serum hepcidin concentrations.  
3 
Investigatio
nal Plan 
(Figure) 
 
 


 
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3.1 
General overview 
• 
Randomized, open-label study of ferric citrate vs. ferrous sulfate in patients with 
moderate to severe CKD (eGFR 15 – 45 ml/min/1.73m2). 
• 
Participants will be recruited from the outpatient nephrology clinics at the University of 
Alabama at Birmingham (UAB). 
• 
Written informed consent will be obtained at the screening visit after the study has been 
explained in full. 
• 
Participants taking oral iron supplements at the time of the screening visit will undergo a 
four week wash-out period prior to beginning study medications. 
• 
After eligibility is confirmed following the screening visit (and wash-out period if 
necessary), participants will be randomly assigned 1:1 to take ferric citrate at a dose of 2 
grams three times a day with meals (n=30) or ferrous sulfate at a dose of 325 mg by 
mouth three times a day between meals (n=30) for a total of twelve weeks. 
• 
Fasting blood and urine samples will be collected at baseline and two, six and 12 weeks 
after baseline for measurement of study outcome variables. Data for the outcome 
variables as well as safety will be collected at these time points. 
• 
The study will end at 12 weeks, a final blood and urine sample will be drawn and 
intervention will be stopped. 
 
Figure 2 


 
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3.2 
Study Population 
3.2.1 Participant Recruitment 
Participants will be recruited via several different methods: 
• 
Outpatient clinic visits: Study staff will pre-screen patients coming in for their scheduled 
appointments at the Kirklin Clinic (TKC), the major outpatient nephrology clinic at UAB.  
• 
Advertisements: Advertisements will be posted in local newspapers and online 
resources to recruit individuals with a history of kidney disease from the city of 
Birmingham and surrounding communities. Study flyers will be posted around the UAB 
hospital and outpatient clinic locations. These strategies have been successful in 
recruiting participants with CKD in randomized, controlled studies of CKD patients at our 
center. 
3.2.2 Inclusion Criteria 
• 
18 years of age or older 
• 
Moderate to severe kidney disease not requiring renal replacement therapy (eGFR 15-
45 ml/min/1.73m2 as determined by the CKD-EPI formula). 
• 
Absolute iron deficiency as defined as a transferrin saturation ≤ 30% and a ferritin ≤ 300 
ng/ml. 
 
3.2.3 Exclusion Criteria 
• 
Hemoglobin concentrations > 13 g/dL 
• 
Severe anemia defined as a hemoglobin < 8.0 g/dL for males or a hemoglobin <7.0 g/dL 
for females.  
• 
Known disorder of iron homeostasis (e.g., hemochromatosis) 
• 
Known gastrointestinal disorder (irritable bowel disease, inflammatory bowel disease) 
• 
Known or biochemical evidence of liver disease (ALT/AST or bilirubin > 3x normal) 
• 
Serum phosphorus concentrations < 3.0 mg/dL 
• 
Any known cause of anemia other than iron deficiency or CKD (e.g., sickle cell anemia) 
• 
Symptomatic gastrointestinal bleeding within 12 weeks prior to the screening visit. 
• 
Subjects receiving any form of renal replacement therapy including hemodialysis, 
peritoneal dialysis, or renal transplant. 
• 
Pregnancy or lactation in female participants 
• 
Life expectancy less than 6 months 
• 
Receipt of erythropoiesis stimulating agents within 4 weeks of screening. 
• 
Receipt of intravenous iron therapy within 8 weeks of screening. 
• 
Blood transfusion within 4 weeks of screening 
• 
Known allergies or severe adverse reactions to previous oral iron therapy 
• 
Current use of oral phosphorus binders 
• 
Current use of an active vitamin D analog 
• 
Concomitant participant in another clinical study with investigational medicinal products. 
 
3.2.4 Concurrent use of oral iron supplements 
Potential participants taking oral iron (either as an over-the-counter supplement or prescribed 
medication) will not be excluded but will be required to stop the iron for at least 4 weeks prior to 
entering the study. 


 
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3.2.4.1 Concurrent use of other medications 
All concurrent medications will be recorded in study participants. If participants are taking oral  
calcium supplements or nutritional vitamin D analog, they will be allowed to continue to take 
them as long as the dose does not change during the course of the study. 
3.3 
Justification of selection criteria 
The selection criteria were chosen to focus enrollment on individuals with moderate to severe 
CKD (eGFR 15 – 45 ml/min/1.73m2) who are iron deficient and (1) would not be exposed to 
particular risks from the study drug and (2) would not have conditions that would impact the 
aims of the study.  The reason why we chose this cut-off for eGFR is that we wanted to enroll 
individuals who would benefit the most from an intervention that not only provided supplemental 
iron but also could potentially help to attenuate the rise in hepcidin following iron 
supplementation by reducing FGF23 concentrations.  While it is possible that CKD patients with 
a higher eGFR would also benefit from using ferric citrate to increase iron stores, these 
individuals would likely have milder forms of iron deficiency and/or mildly increased FGF23 
concentrations, making it more difficult to ascertain whether a therapy that combined iron 
repletion with phosphorus lowering would more effectively improve iron bioavailability than 
individuals with more severe forms of kidney disease.  We chose not to include individuals with 
an eGFR < 15 ml/min/1.73m2 because in our experience, it would be very challenging to find 
individuals at this low an eGFR who would be eligible for enrollment given our exclusion 
criteria—particularly avoidance of ESA exposure—within the period of time for recruitment 
proposed for this study (2 years).  Furthermore, these individuals are more likely to be 
hospitalized and/or progress to ESRD requiring dialysis, which would increase the drop-out rate 
and require us to either extend the recruitment period or enroll a larger sample size than 
currently planned. 
3.4 
Study Schedule (Figure 2) 
3.4.1 Screening Visit 
Written, informed consent will be obtained by study staff during the screening visit after the 
study has been explained in full.  Participants will be instructed to come to the screening visit 
after at least an 8-hour fast.  Participants who provide consent will have height and weight 
obtained.  Age, gender and race/ethnicity data will be recorded. Blood samples will be drawn to 
measure screening labs to ensure eligibility.  In addition, female subjects of child-bearing age (≤ 
55 years) will have a rapid urine test for beta-HCG performed to rule out pregnancy.  Eligibility 
to continue with the study will be determined after review of screening tests. If any screening 
laboratory values are found to disqualify the participant, he or she will be informed of the 
findings, and will be asked to follow-up with his or her primary care physician or nephrologist for 
further evaluation if necessary (e.g., for a very low hemoglobin level). 
Participants found not to be eligible following the screening visit and lab testing will be allowed 
to re-screen at a later time provided that the major reason for screening failure can be 
reasonably expected to be resolved within six months of the original screening visit (for 
example, a CKD patient who received IV iron three weeks ago, but who will refrain from IV iron 
within the next six months may be eligible for re-screening at a later date). 
3.4.2 Washout Period 
Participants who are currently receiving oral iron ferrous sulfate (or other oral iron preparations) 
will undergo washout of these medications for four weeks.  
3.4.3 Visit 1 (Day zero) 
After eligibility is confirmed in the screening visit and following the washout period if necessary, 
participants will come to the UAB Clinical Research Unit (CRU) in the morning after an overnight 
fast for visit 1(Day 0).  During this visit, participants will undergo fasting blood collection to 


 
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establish baseline measurements for primary outcome variables (iron, TIBC, ferritin), secondary 
outcome variables (hemoglobin, hepcidin, FGF23) and safety measures (eGFR, calcium, 
phosphorus).  At this time subjects will also be randomly assigned to a treatment arm (ferric 
citrate vs. ferrous sulfate). 
3.4.3.1 Randomization (Visit 1) 
All eligible participants will be randomized by the research pharmacist in a 1:1 fashion into one 
of two treatment arms: 
• 
Ferrous sulfate at a dose of 325 mg by mouth three times a day between meals 
• 
Ferric citrate at a dose of 2 grams three times a day with meals 
3.4.4 Visit 2 (week 2) 
• 
Participants will come to the UAB CRU, where participants will undergo vital sign 
measurement and fasting blood collection for measurement of study outcome variables 
and safety measures. 
• 
Pills remaining in the study medication bottles of each participant will be counted to 
ensure compliance and accountability for discrepancies in study drug use will be 
performed. 
• 
Participants will be asked questions about intercurrent medical events and changes in 
clinical condition from the last visit.  Events that meet the definition of an adverse event 
(see section 3.9.2) will be documented.   
3.4.5 Visit 3 (week 6) 
• 
Participants will come to the UAB CRU, where participants will undergo vital sign 
measurement and fasting blood collection for measurement of study outcome variables 
and safety measures. 
• 
Pills remaining in the study medication bottles of each participant will be counted to 
ensure compliance and accountability for discrepancies in study drug use will be 
performed. 
• 
Participants will be asked questions about intercurrent medical events and changes in 
clinical condition from the last visit.  Events that meet the definition of an adverse event 
(see section 3.9.2) will be documented. 
3.4.6 Visit 4 (week 12) 
• 
Participants will come to the UAB CRU, where participants will undergo vital sign 
measurement and fasting blood collection for measurement of study outcome variables 
and safety measures. 
• 
Pills remaining in the study medication bottles of each participant will be counted to 
ensure compliance and accountability for discrepancies in study drug use will be 
performed. 
• 
Participants will be asked questions about intercurrent medical events and changes in 
clinical condition from the last visit.  Events that meet the definition of an adverse event 
(see section 3.9.2) will be documented.  At the end of this visit, the study will be 
completed. 
3.5 
Study Drug Dispensing and Logistics 
3.5.1 Study drug dispensing 
The UAB research pharmacy will be responsible for storage of all study drugs in a secure 
facility.  They will also be responsible for procurement of ferrous sulfate from commercial 


 
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sources (ferric citrate will be shipped to the research pharmacy from the study sponsor).  The 
research pharmacy will also be responsible for participant randomization and dispensation of 
study drug in appropriately marked containers.  Written instructions on how study drugs are to 
be taken will be provided to all participants, and will be reinforced by face-to-face contact with 
study staff at the baseline and all follow-up visits.   
3.6 
Compliance assessment 
Study drug will be provided to participants at the baseline visit (visit 1), visit 2 and visit 3. Study 
staff will maintain a study drug dispensing log to document drug dispending during the entire 
study.  Participants will be instructed to return the study drug bottles including unused study 
drug at each follow-up visit.  Any discrepancies between the remaining study drug in the bottles 
and the number that should be remaining will be discussed with the study participants at the 
time of the visits and an explanation must be documented.  Participants will also be required to 
inform study personnel of intentional or inadvertent failure to take a study drug according to the 
written schedule between study visits. 
3.7 
Protocol for Handling of Withdrawals 
3.7.1 Discontinuation of the study drug 
Participants who discontinue the study drug will be expected to continue in the follow-up period 
and complete all study-specific visits.  If a participant is unable to attend a follow-up study visit, 
contact by telephone is permitted to determine the reason why they are not able to attend a 
follow-up study visit and determine whether these assessments can be rescheduled. 
 
The study drug can be discontinued if: 
• 
The participant becomes pregnant 
• 
Any investigational drug other than the study drug is used 
• 
Any suspected drug-related adverse events or serious adverse events occur 
• 
If any of the exclusion criteria apply during treatment (e.g., receipt of kidney transplant) 
• 
A significant violation of the study protocol occurs. 
 
Importantly, study drugs can be temporarily discontinued for reasons as outlined in section 
3.9.1.1 below.   
3.7.2 Discontinuation from the study 
Participants can be withdrawn from the study at any time if they specifically ask to withdraw or if, 
in the opinion of the study PI, continuation of the study would be harmful to the participant.  In 
addition, a participant can be withdrawn at any time by the PI if he or she repeatedly violates 
study protocol. 
Participants withdrawn prior to the final scheduled visit will be asked to attend a close-out visit in 
which they provide blood and urine samples (for final analyses), and complete documentation 
related to the development of any adverse events. 
If the withdrawal is due to an adverse event, participants will be followed until the event resolves 
or it is shown that the adverse event was not due to the study medication.   
Randomized participants who withdraw prematurely will not be replaced.  A participant who 
withdraws after signing informed consent but before randomization can be replaced. 
3.8 
Justification for interventions 
The initial starting dose of ferrous sulfate (325 mg po tid) was chosen because this is currently 
the standard of care as outlined in the KDIGO guidelines for iron deficient individuals with CKD. 
The starting dose of ferric citrate needed to balance the requirement to provide sufficient 
lowering of dietary phosphorus absorption to effect a decrease in FGF23 concentrations (and 


 
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thereby help attenuate the rise in hepcidin) while at the same time providing sufficient iron 
supplementation to treat iron deficiency anemia.  While a starting dose of 1 gram of ferric citrate 
three times a day was considered, in the seminal study comparing the efficacy of ferric citrate 
vs. placebo in pre-dialysis CKD patients,20 the average final dose of ferric citrate needed to 
achieve adequate phosphorus reduction was 5.1 grams per day.  Therefore, we chose 2 grams 
three times per day with meals to provide sufficient phosphorus binding effect to lower FGF23 
while at the same time providing adequate iron supplementation to compare against ferrous 
sulfate. 
3.9 
Procedures and Evaluations 
3.9.1 Assessment of Safety 
3.9.1.1 Risks and Discomforts 
Phlebotomy 
Subjects will experience the usual discomfort caused by phlebotomy.  The total amount of blood 
to be collected for the entire study period will be ~150 ml, or ~ ½ cup, less than the volume of 
blood collected during a single blood donation (500 ml or 2 cups).  We will exclude subjects with 
hemoglobin levels < 7 g/dl for women and < 8 g/dl for men during their screening visit.   
Severe hypophosphatemia 
Ferrous citrate is an oral phosphorus binder.  It is possible that participants taking the 
prescribed ferrous citrate dose (2 grams three times a day with meals) may develop low serum 
phosphorus concentrations. In a prior study of 72 individuals with moderate CKD not requiring 
dialysis being treated with a higher cumulative dose of ferric citrate (5.1 grams/day) than 
proposed in the current study, there were no events of symptomatic hypophosphatemia, and 
only two instances of serum phosphorus concentrations falling below 2.5 mg/dL (which resolved 
on their own).  Therefore, the likelihood of severe hypophosphatemia occurring is low, 
particularly since we will exclude individuals with serum phosphorus concentrations less than 
3.0 mg/dL on screening labs.  Nonetheless, serum phosphorus concentrations will be monitored 
through the study.  Study drug titration will occur as follows: 
• 
If serum phosphorus concentrations fall to between 2.5 and 3.0 mg/dL, participants will 
be asked to reduce the dose of ferric citrate to 1 tablet three times a day with meals. 
• 
If serum phosphorus concentrations fall below 2.5 mg/dL, participants will be asked to 
hold the study drug until serum phosphorus concentrations are above 3.0 mg/dL, and 
then re-start the study medication at 1 tablet three times a day with meals.   
• 
If serum phosphorus concentrations remain below 3.0 mg/dL despite reduction in study 
drug dose, the participant will be asked to stop ferric citrate and follow-up blood draws 
will be obtained two weeks after to ensure that the phosphorus concentration recover to 
above 3.0 mg/dL. 
Elevated iron concentrations: Increases in markers of iron metabolism are anticipated in 
participants taking either oral ferrous sulfate or ferric citrate.  In order to ensure that iron marker 
concentrations do not increase too high during the course of intervention, both iron saturation 
(TSAT) and ferritin will be monitored throughout the intervention period. If TSAT rises above 
65% or ferritin rises above 1000 ng/ml at any time point the lab results will be repeated within 48 
hours.  If the results remain elevated, the participant will be asked to stop the study medication 
but will continue to be followed for the duration of the study visits. 
Gastrointestinal upset: It is possible that study participants can develop gastrointestinal upset 
(dyspepsia, constipation, diarrhea) during the study duration.  If this occurs, the following will 
happen: 


 
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• 
If the participant was randomized to the ferrous sulfate arm, he or she will be asked to 
reduce the dose of ferrous sulfate to twice a day.  If one week after this reduction he or 
she continues to experience gastrointestinal upset, he or she will be asked to reduce the 
dose of ferrous sulfate to once a day. If one week after this reduction he or she 
continues to experience gastrointestinal upset, he or she will be withdrawn from the 
study. 
• 
If the participant was randomized to the ferric citrate arm, they will be asked to reduce 
the dose of ferric citrate to 1 gram by mouth three times a day with meals.  If he or she 
continues to experience gastrointestinal upset one week after they make this change, he 
or she will be withdrawn from the study. 
3.9.2 Adverse events 
Any adverse medical event (sign/symptom/laboratory results) occurring in a research participant 
will be captured in standardized forms at each follow-up study visit.  Adverse events will be 
coded using the Medical Dictionary for Regulatory Activities (MedDRA) and will be grouped by 
SMQs.   
A serious adverse event includes any event that results in a death, is life-threatening, results in 
an inpatient hospitalization, results in a significant disability, or in any other serious injury or 
prolongs the hospitalization as judged by the PI.    
3.9.2.1 Documentation of adverse events 
Each adverse event will be documented according to the following criteria: 
• 
Intensity 
o Mild: transient in nature, minor interference in activities of daily living 
o Moderate: serious impediment to normal daily activities 
o Severe: prevents normal activities 
• 
Relationship with study drug exposure 
o The PI will determine whether the adverse event was likely or unlikely related to 
study drug exposure.  If the latter, the most likely alternative explanation for the 
adverse event will be documented in case report forms. 
• 
Action taken 
o The study staff will document whether any action was taken to resolve the 
adverse event. 
3.9.3 Adverse event reporting 
The principal investigator will report any adverse events to the IRB within the required time 
frame for reporting as outlined below: 
• 
For serious adverse events, the principal investigator will report the events to the IRB 
immediately (within 24 hours of event) by telephone, fax or email. The principal 
investigator will then submit a full written report using the IRB Adverse Event Form within 
10 working days or 14 calendar days.   
• 
For mild to moderate adverse events that are unexpected, but are thought to be related 
to the intervention, the principal investigator will report the events to the IRB in writing 
using the IRB Adverse Event Form within 20 working days or 30 calendar days. 


 
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• 
For mild to moderate adverse events that are both unexpected and are thought to be 
unrelated to the intervention, the principal investigator will summarize the events in a 
progress report for the annual continuing review.  
• 
For mild to moderate and expected adverse events, the principal investigator will 
summarize the events in a progress report for the annual continuing review. 
The principal investigator will also report all serious adverse events that occur 2 weeks after 
active study participation and are thought to be related to the study intervention.  
4 
Statistical 
Analyses 
4.1 
Primary outcome  
The primary outcome variables for this analysis will be transferrin saturation (TSAT) and ferritin 
concentrations.   
4.2 
Secondary outcome variables 
The secondary outcome variables will include hemoglobin, serum hepcidin, serum 
erythroferrone, serum phosphorus and plasma FGF23 concentrations. Serum hemoglobin, 
phosphorus and plasma FGF23 concentrations will be measured in all participants at all visits 
depending on available samples.  Serum hepcidin and erythroferrone will be examined in a 
subset of participants (n=30) with samples available at the baseline visit, week 2 and week 6 or 
12.   
4.3 
Statistical Plan 
To compare the baseline characteristics between the two groups, student’s t-test or chi square 
test will be used for parametric and non-parametric variables respectively. Subjects’ baseline 
values will serve as the reference for comparison with changes post-intervention.  Longitudinal 
changes in the primary outcome variables will be examined using repeated measures linear 
mixed models.  In these models, time and treatment group will be the fixed variables and 
participants will be modeled as random terms. We will first test for a time by treatment group 
interaction to determine whether the change in the outcome variables over time differs by 
treatment group assignment.  If we detect a statistically significant interaction, we will stratify the 
analysis by treatment group and test the main effect of time in stratified models.  If we do not 
detect a significant interaction, we will remove the interaction term from the model and will test 
for the main effects of time and treatment group.  If we find any differences in baseline 
characteristics despite randomization, we will adjust for these covariates in secondary models.  
We will analyze data using an intention-to-treat principle such that all randomized participants 
who received at least one dose of study medication and had at least one post-baseline 
assessment will be included to assess efficacy.   
 
4.4 
Sample Size Calculation: 
The sample size estimation is powered to detect a significant difference in the change in ferritin 
in response to oral ferric citrate vs ferrous sulfate challenge. In a prior study, treatment with 
ferric citrate for 12 weeks raised serum ferritin by 77.5 [95%CI 56.2-98.7] as compared to 
placebo.  To detect as small as a 20% lower rise in ferritin in individuals taking ferrous sulfate 
vs. ferric citrate over a similar period of intervention, we would need recruit 60 individuals overall 
(30 per arm) assuming a type I error of 0.05 and a power of 80%.  This will also provide ample 
power to detect at least a 20% difference in the change in TSAT as well. While we also plan on 
examining changes in hepcidin in this protocol, we did not specifically power the analysis for this 
outcome variable as it is a secondary outcome.  
 


 
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A prior randomized, controlled study of patients with stage 3-5 CKD (eGFR 10-59 
ml/min/1.73m2) showed that treatment with oral ferrous sulfate (325 mg po bid) resulted in a 
mean increase in hemoglobin of 0.2 ± 0.9 g/dL (Agarwal R et al, Am J Nephrol 26:445-454, 
2006).  If we assume that treatment with ferric citrate for 12 weeks will increase hemoglobin by 
0.6 ± 0.4 mg/dL (Block et al, Am J Kidney Dis 65(5):728-736), the current sample size of 60 
participants overall will provide ~60% power to detect a mean difference in hemoglobin increase 
of 0.4 ± 0.7  between the two groups.  
 
 
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