New Phosphate Binding Agents: Ferric Compounds

J Am Soc Nephrol 10: 1274–1280, 1999 New Phosphate Binding Agents: Ferric Compounds

CHEN H. HSU, SANJEEVKUMAR R. PATEL, and ERIC W. YOUNG Nephrology Division, Department of Internal Medicine, University of Michigan Medical School, VA Medical Center, Ann Arbor, Michigan.

Abstract. Several prior studies suggest that ferric compounds 0.058), 77.0 mg/d for the ferric citrate-treated group (P ϭ bind dietary phosphate and possess clinical potential as phos- 0.057), and 62.5 mg/d for the ferric chloride-treated group phate binding agents. Therefore, this study was conducted to (P Ͻ 0.002). Urinary phosphate excretion fell, sometimes to an measure the effect of several ferric compounds on intestinal even greater extent than did intestinal absorption, yielding no phosphate binding and absorption. Balance studies lasting 2 to net reduction in phosphate balance in these growing, young 4 wk were performed in normal and azotemic (achieved by animals with relatively preserved renal function. Calcium bal- subtotal nephrectomy) rats maintained on a 1.02% phosphorus ance was largely unaffected by the ferric compounds. There diet supplemented with ferric salts (formulated to 0.95% Fe) or were trends toward decreased serum phosphorus and parathy- no ferric salt (control). In rats with normal renal function roid hormone concentrations and increased iron and hematocrit (average creatinine clearance, 4.0 ml/min per kg), the average in the ferric-treated azotemic groups. All tested ferric com- net intestinal absorption of phosphate over all balance periods pounds were well tolerated, but animal growth was stunted in was 103.3 mg/d for the control group versus 84.7 mg/d for the the ferric chloride animals compared with the control group. ferric citrate group (P Ͻ 0.005). In the azotemic rats (average Phosphate binding was estimated at 85 to 180 mg per gram of creatinine clearance, 3.3 ml/min per kg), the average net intes- elemental iron, which is comparable to other phosphate bind- tinal absorption of phosphate over all balance periods was ing agents. Ferric salts decrease net intestinal phosphate ab- significantly lower for the three ferric groups than the control sorption and hold promise for the treatment of phosphate groups (P Յ 0.02): 95.3 mg/d for the control group versus 75.6 retention in patients with renal failure. mg/d for the ferric ammonium citrate-treated group (P ϭ

In healthy individuals, phosphate is primarily eliminated by the retention, potentially exacerbating soft-tissue mineralization kidneys, which effectively regulate phosphate balance and the and organ dysfunction (6–10). blood phosphate concentration. Phosphate excretion is com- The problems associated with aluminum and calcium salts promised in patients with renal failure, resulting in systemic illustrate the need for new phosphate binding agents. Candidate phosphate retention and hyperphosphatemia. Phosphate reten- agents must be effective, safe, well tolerated, and relatively tion is a major toxic complication of renal failure, potentially inexpensive. Ferric compounds potentially fulfill these criteria. contributing to soft tissue mineral deposition, progression of Several human (11,12) and animal (13–16) studies from many renal disease, and secondary hyperparathyroidism (1–5). years ago strongly indicate that ferric compounds can bind Restriction of phosphate intake can potentially prevent hy- dietary phosphate and dramatically alter phosphate metabo- perparathyroidism and other toxic manifestations of phosphate lism. retention (2–5). However, dietary phosphate restriction alone is In view of these early reports, we performed studies in normal and azotemic rats designed to explore the efficacy and often insufficient to control hyperphosphatemia. Consequently, tolerability of ferric compounds as phosphate binders. Ferric most patients are treated with orally administered aluminum or citrate, ferric ammonium citrate, and ferric chloride were in- calcium salts that bind dietary phosphate and facilitate fecal vestigated. Our primary goal was to determine whether these elimination rather than intestinal absorption. Long-term use of compounds could bind dietary phosphate. We also examined aluminum compounds can cause bone and other toxicities; other aspects of mineral metabolism and animal viability. accordingly, calcium salts have become the phosphate binding agents of choice. However, calcium therapy can be compli- Materials and Methods cated by the development of hypercalemia and net calcium Phosphate Binding Effect of Ferric Citrate in Normal Rats Normal male Sprague Dawley rats (n ϭ 6) were fed a standard rat Received March 4, 1997. Accepted December 15, 1998. diet containing 1.02% phosphate (P) and 0.95% calcium (Ca) (ICN Correspondence to Dr. Chen H. Hsu, 3914 Taubman Center, Nephrology Biomedicals, Cleveland, OH) for 2 wk. The dietary phosphate content Division, University Hospital, Ann Arbor, MI 48109-0364. Phone: 313-936- was verified in our laboratory. An additional six normal male rats 9480; Fax: 313-936-9621; E-mail: [email protected] were fed the same diet supplemented with 4% ferric citrate for 2 wk. 1046-6673/1006-1274 All animals were housed in individual metabolic cages with a food Journal of the American Society of Nephrology container attached to the outside. Powdered food was used to prevent Copyright © 1999 by the American Society of Nephrology contamination of urine and stool. The animal was able to reach the J Am Soc Nephrol 10: 1274–1280, 1999 New Phosphate Binding Agents: Ferric Compounds 1275 food container but unable to bring food into the cage. Body weight, Table 1. Average weight, urinary creatinine excretion, and food consumption, urine output, and stool excretion were monitored blood measurements in rats with normal renal daily for 4 d per week for 2 wk. The daily stool and urine measure- functiona ments for each 4-d observation period were pooled and expressed as the average per day. Blood (1.5 ml) was taken from the tail vein of Parameter Control Ferric Citrate nonfasted rats in the morning (8 a.m. to 10 a.m.) each week for measurement of plasma phosphorus and creatinine. At the end of the Weight (g) study, blood was obtained from the aorta of anesthetized animals (also week 1 274 Ϯ 3 280 Ϯ 3 from 8 a.m. to 10 a.m.) for measurement of parathyroid hormone week 2 313 Ϯ 5 318 Ϯ 4 (PTH), calcitriol, and iron concentrations. Urine creatinine (mg/d) week 1 8.7 Ϯ 0.4 9.0 Ϯ 0.8 Phosphate Binding Effect of Ferric Compounds in Rats week 2 10.0 Ϯ 0.6 9.4 Ϯ 0.6 with Renal Failure Creatinine clearance (ml/min per kg) The phosphate binding effect of ferric compounds was also studied week 1 4.3 Ϯ 0.3 4.3 Ϯ 0.4 in Sprague Dawley rats with renal failure, achieved by subtotal week 2 3.9 Ϯ 0.2 3.7 Ϯ 0.2 nephrectomy. Two-thirds of one kidney was surgically removed by Blood values (week 2) arterial ligation, and the other kidney was removed through a flank Ϯ Ϯ incision 3 d later. The control group was fed the standard powdered rat PTH (pg/ml) 16 4163 Ϯ Ϯ diet containing 1.02% phosphate and 0.95% calcium. The other three calcitriol (pg/ml) 84 2822 groups of animals were fed the standard diet supplemented with one iron (␮g/ml) 1.8 Ϯ 0.2 1.7 Ϯ 0.1 of the following ferric compounds (Sigma Chemical Co., St. Louis, hematocrit (%) 49 Ϯ 148Ϯ 1 MO): 5% ferric ammonium citrate (molecular weight approximately a Ϯ 325 to 330 kD, 16.5 to 18.5% elemental Fe3ϩ), 4% ferric citrate Values are given as mean 1 SEM. PTH, parathyroid ⅐ hormone. (FeC6H5O7, 245 kD), or 4.4% ferric chloride (FeCl3 6H2O, 270 kD). Each ferric-containing diet was formulated to contain approximately 0.95% elemental iron. Body weight, food consumption, urine output, and stool excretion were monitored daily for 4 d per week over the 4-wk study period using the same techniques described for the rats with normal renal function. Daily measurements from each 4-d balance period were averaged for each week of the study. Blood was taken once weekly for measurement of the plasma phosphorus and creatinine concentrations and at the end of the study for measurement of PTH, calcitriol, iron concentration, and hematocrit. Analytical Methods All phosphate determinations were measured and expressed as phosphorus. Stools were ashed at 800°C in a muffled furnace for 30 min, and phosphorus was extracted with 10% perchloric acid over- night before phosphorus measurement. Phosphorus and creatinine were measured as described previously (17). Plasma calcitriol was measured in duplicate according to the methods of Reinhardt et al. (18) and Hollis (19). The interassay coefficients of variation were 7.0% for the low control (20 pg/ml, n ϭ 12) and 4.1% for the high control (100 pg/ml, n ϭ 12). The intraassay coefficients of variation were 5.4% for the low control (n ϭ 6) and 4.7% for the high control. Calcitriol recovery averaged 65%. PTH was measured by immunora- diometric assay, using a rat PTH assay kit (Nichols Institute, Capist- rano, CA). The plasma iron concentration was measured using a Figure 1. Phosphate (left side) and calcium (right side) metabolism in commercial assay kit (Sigma Chemical Co.). rats with normal renal function. Average measurements shown for the 1- and 2-wk balance periods. Black bars indicate control animals and Statistical Analyses stippled bars indicate ferric citrate-treated animals. Data are shown as mean Ϯ 1 SEM. Statistical analyses were performed using repeated measures ANOVA for the rats with normal renal function. Data from the azotemic animals were analyzed using a mixed model to better accommodate repeated measurements, miss- ance in the two diet groups (control, ferric citrate) over the ing data values (see below), and random effects (20). 2-wk study (Table 1). Fecal phosphate excretion was higher in the ferric citrate group than in the control group with no Results difference in phosphate intake (data not shown). Consequently, Phosphate Binding Effect of Ferric Citrate in net intestinal absorption of phosphate was lower in the ferric Normal Rats citrate group than in the control group (Figure 1, left panels; There were no significant differences in body weight, P Ͻ 0.005 for treatment effect, time effect not significant). growth rate, urinary creatinine excretion, and creatinine clear- Approximately 85 mg of phosphate (measured as phosphorus) 1276 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 1274–1280, 1999

Table 2. Effect of ferric compounds on phosphorus absorption

Average Net ⌬ P Phosphorus Binding Capacity Ferric Compound Absorption versus Control Diet (mg/d) mg P/g Compound mg P/g Fe3ϩ

Normal renal function ferric citrate 18.6 19.1 84.8 Azotemic ferric ammonium citrate 19.9 17.7 101.1 ferric citrate 18.6 19.8 87.9 ferric chloride 33.0 36.9 181.2

Table 3. Blood measurements on azotemic rats taken at the end of week 4a

Ferric Parameter Control Ammonium Ferric Ferric Citrate Citrate Chloride

PTH (pg/ml) 112.9 Ϯ 57 31.2 Ϯ 8.1 28.0 Ϯ 4.1 22.4 Ϯ 3.3 Calcitriol (pg/ml) 54.2 Ϯ 2.8 56.1 Ϯ 2.2 55.5 Ϯ 1.6 45.9 Ϯ 3.8 Iron (␮g/ml) 1.26 Ϯ 0.09 1.57 Ϯ 0.21 2.10 Ϯ 0.29b 1.70 Ϯ 0.09 Hematocrit (%) 40.0 Ϯ 4.4 44.7 Ϯ 1.0c 43.3 Ϯ 0.6d 44.8 Ϯ 0.6b

a Values are given as mean Ϯ 1 SEM. b P Ͻ 0.005 versus control. c P Ͻ 0.01 versus control. d P Ͻ 0.05 versus control.

was bound per gram of elemental iron (Table 2). Urinary Among the azotemic group, the ferric ammonium citrate- phosphate excretion was also lower in the ferric citrate animals and ferric citrate-treated animals grew at the same rate as the compared with control (Figure 1, P Յ 0.0001 for treatment control animals (Figure 2, top panel). However, the growth rate effect). The decline in urinary phosphate excretion exceeded was slower for the animals treated with the ferric chloride diet the reduction in intestinal absorption, yielding a mild increase compared to the control animals (Figure 2), with no significant in net phosphate balance in the ferric citrate group (Figure 1, difference in food intake (not shown). The average urinary P Ͻ 0.001 for treatment effect). Serum phosphorous concen- creatinine excretion was also lower in the ferric chloride group tration was not different between the two groups (Figure 1). relative to the other three groups over the course of the exper- Calcium metabolism was not significantly different in the two iment (Figure 2, middle panel). Creatinine clearance was com- groups except for a slightly lower (P Ͻ 0.05) urinary calcium parable and stable across all four treatments (Figure 2, bottom excretion in the ferric citrate group (Figure 1, right panels). panel). Two animals, one in the ferric ammonium citrate group There were no significant differences in PTH, calcitriol, iron, and the other in the ferric citrate group, developed an unknown and hematocrit (Table 1). acute illness on day 22 (fourth week) and were sacrificed. Figure 3 shows phosphate metabolism for the four experi- Phosphate Binding Effect of Ferric Compounds in mental groups for each of the four weekly balance periods. Azotemic Rats Fecal phosphate excretion was higher in the three ferric-treated The creatinine clearance was approximately 23% lower in groups than in controls in the face of comparable phosphate the 5/6 nephrectomy group (azotemic) compared to the group intake (data not shown). Consequently, the net intestinal ab- with normal renal function: 3.3 versus 4.3 mg/min per kg at sorption of phosphate was lower in the ferric-treated animals week 1 and 3.3 versus 3.8 ml/min per kg at week 2 (Table 1, than in the control group throughout the experiment (Figure 3, Figure 1, P Ͻ 0.0005 for overall effect of partial nephrectomy). top panel, P Յ 0.02 for overall group differences, P ϭ 0.058 Compared to the group with normal renal function, the for ferric ammonium citrate versus control, P ϭ 0.057 for azotemic animals also had higher serum concentrations of PTH ferric citrate versus control, P Ͻ 0.002 for ferric chloride (Tables 1 and 3, P ϭ 0.06) and phosphorus (Figure 1 and versus control, no significant time effect). From the differences Figure 3, lower panels, P Յ 0.0001) and lower levels of in phosphate absorption, the estimated binding capacity of the calcitriol (Tables 1 and 3, P Յ 0.0001) and hematocrit (Tables ferric compounds ranged from 88 to 181 mg of phosphate per 1 and 3, P Յ 0.0001). Thus, the overall laboratory profile of the gram of elemental iron (Table 2). The ferric compounds were partially nephrectomized animals was consistent with mild also associated with a reduction in urinary phosphate excretion azotemia. (Figure 3, second panel, P Յ 0.0001 for overall group effect J Am Soc Nephrol 10: 1274–1280, 1999 New Phosphate Binding Agents: Ferric Compounds 1277

average hematocrit level than the control group, suggesting some degree of intestinal iron absorption (Table 3).

Discussion Phosphate retention is a common problem in patients with renal failure. In the early stages of renal disease, the plasma phosphorous concentration is usually maintained in the normal range, presumably due to hyperparathyroidism, which stimulates urinary phosphate excretion. However, the plasma phosphate concentration may not accurately reflect total body phosphate content (21), and these patients are still prone to net phosphate retention. Overt hyperphosphatemia often develops when the GFR falls below approximately 20 ml/min. For end-stage renal disease patients, the estimated phosphate absorption is 4200 mg/wk (assuming dietary intake of 1000 mg/d and a fractional absorption of 60%) (22), and the average phosphate removal by hemodialysis is 3171 mg/wk (23). Thus, in the absence of treatment with binders, phosphate accumu- lates in the body at a rate of approximately 150 mg/d. Phos- phate retention may be exacerbated in patients who are treated with calcitriol (22). In many patients with renal failure, phosphate retention cannot be controlled with dietary phosphate restriction or dialysis (23), necessitating the use of phosphate binding agents. Calcium compounds have been preferentially used to limit phosphate absorption. However, most patients with end-stage renal disease are in positive calcium balance because they lack a reliable route of calcium excretion (10). The use of calcium salts as phosphate binders further disposes patients to hyper- Figure 2. Body weight, urinary creatinine excretion, and creatinine calcemia and, in association with phosphate retention, poten- clearance in azotemic rats. Measurements shown corresponding to tially contributes to soft tissue mineralization and organ dys- balance periods at weeks 1, 2, 3, and 4. Treatment groups: control, function. The problems associated with calcium and aluminum thick gray line; ferric ammonium citrate, closed squares; ferric citrate, compounds highlight the pressing need for alternative phos- open triangles; ferric chloride, closed circles. phate binding agents for treatment of patients with renal failure. Ferric compounds possess heretofore under-appreciated promise as phosphate binding agents. In 1941, Liu et al. and for individual paired contrasts with control, no significant reported that ferric ammonium citrate caused hypophos- time effect) that exceeded the fall in intestinal absorption, phatemia in the one patient who received the drug (11). Sub- ϭ yielding a nonsignificant (P 0.27) increase in net balance sequently, Liu and Chu used ferric ammonium citrate (6 to 12 (Figure 3, third panel). The serum phosphorus concentration g/d) to successfully control hyperphosphatemia in two patients tended to be lower in the ferric-treated animals than the control with chronic renal failure (12). The iron compound was well group but the effect was not statistically significant (Figure 3, tolerated except for occasional diarrhea. Animal experiments bottom panel). also suggested that ferric salts, such as aluminum, can pro- Figure 4 shows calcium metabolism for the four groups. The foundly affect phosphate metabolism. Ferric salts were found week 3 results, showing negative calcium absorption and bal- to dramatically reduce bone ash, plasma phosphate, urinary ance, suggest anomalous measurements. Calcium metabolism phosphate excretion, and bone phosphorus in guinea pigs and was not significantly different among groups except for a rabbits (13). Ferric compounds also induced phosphate deple- slightly higher (P Յ 0.0001) urinary calcium excretion in the tion in growing rats as manifest by growth retardation, hy- ferric-treated groups. pophosphatemia, decreased bone ash, decreased total body Among the azotemic animals, there was a nonsignificant calcium, and decreased total body phosphorus (14,15). Ferric trend toward lower PTH concentrations in the ferric-treated salts also produced severe rickets and hypophosphatemia in groups compared to the control animals (Table 3). There were chicks after 3 wk of treatment (16). In the current study, we no significant group differences in the serum calcitriol concen- sought to extend these prior observations by directly measuring tration. The plasma concentration of iron was higher in the the effect of ferric compounds on intestinal phosphate absorp- ferric-treated animals than in controls, significantly so for the tion in normal and azotemic animals. ferric citrate group. All three ferric-treated groups had a higher We found that ferric compounds effectively bound dietary 1278 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 1274–1280, 1999

Figure 4. Calcium metabolism in azotemic animals for balance studies at each of 4 wk. Treatment groups: control, black; ferric ammonium citrate, wavy; ferric citrate, stippled; ferric chloride, cross-hatched.

Figure 3. Phosphate metabolism in azotemic animals for balance studies at each of 4 wk. Treatment groups: control, black; ferric failure including phosphate retention, iron deficiency, and ane- ammonium citrate, wavy; ferric citrate, stippled; ferric chloride, cross- mia. The ferric compounds had little direct effect on calcium hatched. balance (Figures 1 and 4). In general, the ferric compounds used in this study appeared to be well tolerated. Food intake was comparable in all groups phosphate and decreased intestinal phosphate absorption in of animals. Body weight, growth, and urinary creatinine ex- both normal and azotemic rats (Figures 1 and 3). There was cretion (partly a marker of muscle mass) were similar among also a commensurate decrease in urinary phosphate excretion, the control, ferric citrate, and ferric ammonium citrate groups presumably as an adaptive response to the diversion of dietary (Figure 2). However, growth and creatinine excretion were phosphate to the stool in these young growing rats. The ferric lower in the animals treated with ferric chloride. Two azotemic compounds did not decrease phosphate balance because the animals treated with different ferric compounds became ill decline in urinary excretion exceeded the decline in net intes- during the fourth week of the study, but a connection with the tinal absorption. The failure to reduce net phosphate balance treatment seems doubtful. probably reflects the fact that the rats were in a rapid growth Although our study has demonstrated that ferric compounds phase and the degree of renal dysfunction in the azotemic can effectively bind dietary phosphate, long-term safety has animals was mild. Animals with more advanced renal failure not been fully addressed. Toxicity in humans has been ob- would probably be less able to maintain phosphate balance by served in association with ingestion of large quantities of iron, altering renal excretion. The study achieved its central objec- particularly in children (24,25). Acute iron poisoning is usually tive of demonstrating that ferric compounds can bind dietary associated with blood iron concentrations above 5 ␮g/ml (26), phosphate and reduce intestinal absorption, consistent with much higher than the levels seen in this study (Table 3). The earlier reports. PTH levels, which were elevated in the manifestations of iron toxicity, as reported in patients and azotemic animals, also tended to decrease in the ferric-treated experimental models, can include gastrointestinal hemorrhage, groups. In addition, there was evidence for increased iron hepatic injury, coagulation defects, metabolic acidosis, shock, absorption and a corresponding increment in hematocrit among bone deposition, hemosiderosis, hypoparathyroidism, nephro- the animals treated with ferric compounds. Thus, ferric com- toxicity, and depressed myocardial contractility (27–31). For pounds could potentially ameliorate several problems of renal ferric ammonium citrate, the LD50 was 1.75 g/kg for guinea J Am Soc Nephrol 10: 1274–1280, 1999 New Phosphate Binding Agents: Ferric Compounds 1279 pigs, 2.8 g/kg for rabbits, and 5.0 g/kg for mice (32). However, 6. McDonald SJ, Clarkson EM, De Wardener HE: The effect of a the iron was administered in the fasting state by gastric lavage, large intake of calcium citrate in normal subjects and patients which would increase iron absorption and the risk of intestinal with chronic renal failure. Clin Sci 26: 27–39, 1964 hemorrhage (33,34). 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