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 bal- ance 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).