Nonabsorbable Iron Binding Polymers Prevent Dietary Iron Absorption

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Nonabsorbable Iron Binding Polymers Prevent Dietary Iron Absorption for the Treatment of Iron Overload Jian Qian,†,§ Bradley P. Sullivan,†,§ Samuel J. Peterson,† and Cory Berkland*,†,‡ †

Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, United States Department of Chemical and Petroleum Engineering, The University of Kansas, Lawrence, Kansas 66045, United States

‡

S Supporting Information *

ABSTRACT: Chronic iron overload is a serious condition that develops as a consequence of long-term accumulation of iron, eventually overwhelming iron storage systems and causing oxidative stress and subsequent organ damage. Current pharmaceuticals used to treat iron overload typically suffer from toxicities leading to relatively high rates of adverse events. To address this need, we designed a new class of nonabsorbable iron binding polymers (IBPs) that bind and sequester iron within the gastrointestinal (GI) tract. IBPs were synthesized by cross-linking polyallylamine containing various amounts of conjugated 2,3-dihydroxybenzoic acid (DHBA). In vitro studies indicated that IBPs possessed high affinity, substantial binding capacity, and excellent selectivity toward iron. Moreover, in vivo studies demonstrated that IBPs showed no signs of side effects in mice and increased fecal iron excretion when compared to a similar dose of cross-linked polyallylamine. IBPs are a novel, nonabsorbed oral therapeutic agent that may ultimately prevent iron absorption as a safe alternative to iron chelation therapies for patients with hemochromatosis or other iron overload diseases.

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peroxides leading to free radicals, which are highly reactive toward body tissues.7,8 If left unchecked, this state of iron overload can lead to a myriad of health problems, including diabetes, cardiomyopathy, and liver cirrhosis.9 The current standard of care for patients without chronic transfusional-dependent iron overload is routine phlebotomy. Unfortunately, some patients poorly tolerate plebotomy, and it is contraindicated in patients with blood disorders requiring chronic transfusion therapy.8,10 For these patients, iron chelation therapy is a life-saving and life-prolonging option. Currently, there are three FDA-approved therapeutics to treat chronic hemochromatosis: deferoxamine (DFO), deferiprone (DFP), and deferasirox (DFX).10 Unfortunately, all three of these therapeutics suffer from significant side effects that limit dose and have challenges associated with long-term patient compliance.10 DFO therapy was reported to be orally inactive and has a narrow therapeutic window. Additionally, patients given DFO commonly report injection site pain and/or reactions at the site of administration. Although both DFX and DFP are administered orally, both of these drugs are associated with relatively high levels of adverse events and toxicities. Furthermore, the Institute for Safe Medical Practices labeled DFX as the second drug on the list of “Most frequently suspected drugs in reported patient deaths” compiled in 2009.11 Improving drug safety and long-term patient compliance would

ron is a trace element found in nearly all living organisms and is essential for many biological processes.1 Most mammals, including humans, possess highly regulated systems for iron uptake, storage, and efficient recycling with limited capacity to excrete excess iron.2 In general, the main mechanisms for iron excretion are through hemorrhage, desquamation of the epidermal and intestinal mucosal layer(s), and perspiration.3 Consequently, the iron content in the human body is generally linked to the rate of iron absorption that takes place in the gastrointestinal (GI) tract by protein-mediated mechanisms.4 Normally, the daily iron absorption from food is well regulated by a tightly controlled hormonal system to compensate for the loss of iron through the aforementioned excretion pathways to limit excess accumulation of iron in the body.4,5 Unfortunately, several patient populations are predisposed to iron overload such as patients with genetic conditions that induce primary hemochromatosis and patients that require chronic blood transfusions, a complication of therapeutic intervention to treat genetic blood disorders such as sickle cell anemia and beta-thalassemia.6 In individuals with hereditary primary hemochromatosis, dietary iron absorption can also be altered, which leads to accumulation of iron that exceeds the rate of elimination.6 Generally, the body has a large capacity to store excess iron; however, long-term accumulation of iron can overwhelm these storage systems and iron transport proteins, leading to high levels of free “labile” iron in the blood.7 High levels of free labile iron in the blood have been shown to increase the generation of © XXXX American Chemical Society

Received: December 12, 2016 Accepted: March 15, 2017

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DOI: 10.1021/acsmacrolett.6b00945 ACS Macro Lett. 2017, 6, 350−353

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ACS Macro Letters greatly benefit patients suffering from iron overload diseases such as hereditary hemochromatosis. One possible safe intervention is to prevent iron accumulation after chelation therapy or phlebotomy has restored systemic iron to safe levels. Absorption of dietary iron from the GI tract may be inhibited by dosing iron-chelating molecules that are not absorbed and thus act locally within the gastrointestinal system. Nonabsorbable, cross-linked polymeric polymers, which selectively sequester and remove undesirable ionic species from the GI tract, have been successfully developed for the treatment of other diseases.12 For example, Cholestyramine and Welchol are commonly used nonabsorbable cross-linked polymers that sequester bile acids locally in the gastrointestinal lumen.12d Additionally, sevelamer hydrochloride (Renagel), a cross-linked poly(allylamine hydrochloride) (PAH) polymer, is an FDA-approved phosphatebinding drug used to treat hyperphosphatemia in patients with chronic kidney disease.12a The success of these polymeric therapeutics inspired us to design cross-linked iron binding polymers (IBPs) that could selectively sequester and remove dietary iron from the GI tract. Polymeric iron chelators have traditionally been designed for systemic administration, often with the goal of prolonging chelator half-life in the blood.13 Injected polymeric chelators, however, still expose the entire circulatory system, suggesting the overall toxicity may be similar to small-molecule iron chelators. Conversely, oral administration of nonabsorbed polymeric iron chelators may limit toxicity by remaining in the gastrointestinal tract and chelating dietary iron to prevent iron accumulation. Nonabsorbed iron binding polymers must possess strong affinity, high binding capacity, and excellent selectivity toward iron, even when exposed to the complex environment of the GI tract. In addition, the molecule should be able to bind iron rapidly and relatively irreversibly to form nontoxic, inert complexes that are not absorbed into the body. Thus, we designed an iron binding polymer based on these parameters utilizing cross-linked polyallylamine (PAH) with conjugated 2,3-dihydroxybenzoic acid (DHBA) and tested the iron binding capacity of the resultant cross-linked polymers both in vitro and in vivo to assess the feasibility as an nonabsorbed iron binding therapeutic. The PAH cross-linking and DHBA conjugation were conducted in a single synthetic step to create IBPs. DHBA conjugation was controlled by adjusting the NHS-activated DHBA/polymer feed ratios. Several IBPs with various DHBA content (5−30% of total amines) but the same cross-linking density (∼1%) were prepared via this one-step strategy. DHBA conjugation ratios (Table 1) were determined by 1H NMR analysis. As the DHBA content increased, the swelling ratios decreased, suggesting that the cross-linked polymer gel became more hydrophobic as DHBA conjugation increased. When incubated with Fe3+ solution, all the IBPs exhibited dark color indicating chelation with Fe3+, while the cross-linked PAH gel (G0) did not show any color change (Figure 1E). Next, we evaluated the iron binding capacity of each test IBP, which describes the maximum iron adsorbed by the polymer. In order to reach the maximum iron chelation, all the samples were incubated in a Fe3+ solution for 1 week. The theoretical and experimental iron binding capacities of the IBPs with various DHBA contents were determined (Figure 2A). As the DHBA content increased, the experimental iron binding capacities also increased for IBPs with low DHBA conjugation

Table 1. Characterization of Iron Binding Polymers swelling index sample

cross-linking densitya

feed DHBA/ amineb

found DHBA/ aminec

pH 7.4

pH 6.5

G0 G5 G10 G15 G20 G25 G30

0.01 0.01 0.01 0.01 0.01 0.01 0.01

0 0.05 0.10 0.15 0.20 0.25 0.30

0 0.0366 0.0715 0.123 0.154 0.188 0.232

28.2 22.7 15.4 10.8 8.1 7.5 7.4

31.1 24.8 17.3 12.3 8.9 7.9 7.8

a Feed molar ratio of cross-linker to total amines. bFeed molar ratio of DHBA to total amines. cFound molar ratio of DHBA to total amines by HPLC analysis.

Figure 1. Synthesis and in vitro iron loading of iron binding polymers. (A) Schematic of iron binding polymer synthesis. G25 polymer (B) dehydrated and (C) hydrated with dH2O or (D) hydrated with dH2O containing 0.5 mM FeCl3. (E) G0 and (F) G25 polymers hydrated with dH2O containing 0.5 mM FeCl3.

Figure 2. Iron binding characteristics of iron binding polymers. (A) Theoretical (dashed line) and experimentally determined (solid line) iron binding capacity for experimental polymers. (B) Hydrated G25 polymer was incubated in FeSSIF containing 5 mM FeCl3 at 37 °C for 4 h. Iron levels in the supernatant were determined and used to calculate the percent of iron bound to the polymer. (C) Experimentally determined binding constants of test polymers. (D) Test polymers were incubated in the presence of iron (Fe), copper (Cu), zinc (Zn), manganese (Mn), nickel (Ni), and calcium (Ca) for 5 days, and the levels of polymer-bound metal were then determined.

(5−15%) and reached a plateau (20−30%) around 20 mg Fe/g polymer. For all the samples tested, only the IBPs with low DHBA content (G5 and G10) achieved the theoretical iron sequestration capacities based on conjugation density. The decreased swelling index (increased hydrophobicity) of the IBPs at higher DHBA conjugation ratios probably limited Fe3+ 351

DOI: 10.1021/acsmacrolett.6b00945 ACS Macro Lett. 2017, 6, 350−353

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others. Selectivity to iron is especially important for the treatment of iron overload due to the presence of other dietary metals that are present in the intestinal juice. Poor selectivity of chelating moieties could reduce the absorption of essential dietary metal ions such as Cu2+, Zn2+, Ca2+, Mn2+, or Ni2+. The influence of other metals on the sequestration of Fe3+ by the IBPs was investigated using a multimetal system. The concentration of each metal was fixed at 0.4 mM, and the metal/polymer ratio was fixed at 0.2 mMol per gram of polymer. All the samples absorbed almost 100% of the iron present in the media, while the absorption of other essential metals was substantially lower, demonstrating high selectivity for iron (Figure 2D). We next evaluated whether IBPs could function in vivo. To test the feasibility of IBPs to bind iron in the gut, we selected G25 as it had the highest iron binding capacity of all the polymers tested. After a 2 week iron loading period, mice were switched to a low iron diet or a low iron diet that contained G0 or G25 and were fed ad libitum for an additional 2 week period. We note that even the low iron diet exposes mice to ∼2 mg/ kg/day, which is much higher than an average daily iron consumption for humans (∼0.1−0.5 mg/kg/day). Similar to in vitro experiments, levels of iron in the fecal materials were higher in mice fed the G25 IBP compared to mice fed G0 polymer and to mice fed the low iron diet (Figure 3A).

access to the polymer particle interior, leading to a lower than expected actual iron binding capacity. Interestingly, although IBPs share a similar polymer backbone (polyallylamine) with Renagel, they did not show strong phosphate binding. The phosphate binding capacity of G25 was only about 0.4 mg/g, which is much lower than Renagel (150 mg/g).14 The low phosphate binding capacity may be due to the low swelling index of the G25 IBP. In order to investigate the influence of hydrophobicity on the iron binding capacity, G5 and G10 IBPs were modified with various amounts of 2,3-dimethoxybenzoic acid (DMOBA) which is a hydrophobic analogue of DHBA but does not chelate iron. Both G5 and G10 IBPs showed a dramatic decrease in the swelling index and iron binding capacity as the amount of DMOBA conjugated to IBPs increased (Table S1 and Figure S2). For example, as DMOBA conjugation increased from 0 to 25% of the amines on PAH, the iron binding capacity of G5 decreased by 82.3%. Increasing the amount of DMOBA on IBPs reduced the swelling index, thus limiting Fe3+ access to the cross-linked polymer particle interior. In further support of this conclusion, the influence of cross-linking density on iron binding capacity of G25 was also investigated (Figure S3). Iron binding capacity decreased as the cross-linking density increased, again suggesting the permeability of the cross-linked polymer particles is important when attempting to maximize iron binding capacity. For the purposes of iron binding in the gastrointestinal tract, the kinetics of iron binding is also critical for evaluating the practical applicability of the IBPs as there is a limited window where the material must act in the gut prior to dietary iron absorption in the small intestine. The iron binding kinetics were investigated in fed state simulated intestinal fluid (FeSSIF). The experimental IBPs showed rapid iron binding, with more than 50% of the iron bound in less than 5 min and extending to near 100% by 30 min (Figure 2B), a rapid kinetic course that may outcompete the kinetics of iron absorption beginning in the duodenum. In addition to speed being a critical determinant for inhibiting iron absorption in the small intestine, the iron stability constant, which represents the strength of chelation between iron and the IBPs, is also a critical parameter for iron chelating materials, especially since there are other high affinity iron binding compounds and proteins that aid in iron uptake. The conventional iron stability constant was determined by a ligand competition method using EDTA as the competition ligand in PBS. Importantly, all the polymers with various DHBA contents showed significantly higher iron stability constants than EDTA (Figure 2C). The G5 sample had the highest conventional stability constant (31.3), which indicated that the iron affinity of G5 polymer is 106.7 times stronger than EDTA (Log stability constant is 25.1). All the other IBPs tested showed at least 102 times higher iron affinity than EDTA. As the DHBA content increased from 5 to 30%, the conventional stability constants of IBPs decreased from 31.8 to 27.4. It should be noted that, theoretically, all the IBPs should have almost the same stability constant since the intrinsic stability constant of the DHBA groups in different samples was the same; however, the decreased swelling index (increased hydrophobicity) of the polymers as DHBA content increased may have hindered Fe3+ diffusion into the polymer, hence reducing the apparent iron stability constants. Selectivity, one of the important features of metal-chelating polymers, is the ability to specifically bind the target metal over

Figure 3. Orally dosed iron binding polymer increases fecal concentration of iron in mice. Wild-type C57Bl/6 mice were fed a high iron diet for 2 weeks. Mice were then fed a low iron diet or a low iron diet containing G0 or G25 (5% by weight) ad libitum for 2 weeks. (A) Fecal iron concentration was determined on samples obtained from the last 10 days of study. (B) Total food consumption and (C) percent weight gain was determined for the 2-week period post dietary switch. *Indicates statistically significant from low iron diet control fed mice, p < 0.05. n = 8−10 mice per group. #Indicates statistically significant from mice fed a low iron diet containing G0, p < 0.05. n = 10 mice per group.

Moreover, our data (Figure S4) showed that all the iron in the consumed food was eliminated, which indicated that G25 can chelate iron in food and prevent its absorption from the GI tract. Food consumption (iron intake) was consistent across all groups (Figure 3B). Mice given the G25 IBP continued gaining weight after the dietary switch, but this weight gain was significantly lower than the weight gain of mice fed the low iron diet. Interestingly, mice given the diet containing G0 lost weight (Figure 3C) after the dietary switch. Overall, the data confirmed that oral dosing of our novel IBPs can increase fecal elimination of iron even when consuming a low iron diet. 352

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diseases, orphan drugs and orphan patients. World J. Methodol. 2014, 4, 163−188. (12) (a) Perry, C. M.; Plosker, G. L. Sevelamer carbonate: A review in hyperphosphataemia in adults with chronic kidney disease. Drugs 2014, 74, 771−792. (b) Moore, R. B.; Crane, C. A.; Frantz, I. D., Jr. Effect of cholestyramine on the fecal excretion of intravenously administered cholesterol-4-14C and its degradation products in a hypercholesterolemic patient. J. Clin. Invest. 1968, 47, 1664−1671. (c) Qian, J.; Sullivan, B. P.; Berkland, C. pH-Responsive micelle sequestrant polymers inhibit fat absorption. Biomacromolecules 2015, 16, 2340−2346. (d) Out, C.; Groen, A. K.; Brufau, G. Bile acid sequestrants: more than simple resins. Curr. Opin. Lipidol. 2012, 23, 43−55. (13) (a) Hamilton, J. L.; Imran ul-haq, M.; Abbina, S.; Kalathottukaren, M. T.; Lai, B. F. L.; Hatef, A.; Unniappan, S.; Kizhakkedathu, J. N. In vivo efficacy, toxicity and biodistribution of ultra-long circulating desferrioxamine based polymeric iron chelator. Biomaterials 2016, 102, 58−71. (b) Imran ul-haq, M.; Hamilton, J. L.; Lai, B. F. L.; Shenoi, R. A.; Horte, S.; Constantinescu, I.; Leitch, H. A.; Kizhakkedathu, J. N. Design of long circulating nontoxic dendritic polymers for the removal of iron in vivo. ACS Nano 2013, 7, 10704− 10716. (c) Polomoscanik, S. C.; Cannon, C. P.; Neenan, T. X.; Holmes-Farley, S. R.; Mandeville, W. H.; Dhal, P. K. Hydroxamic acidcontaining hydrogels for nonabsorbed iron chelation therapy: synthesis, characterization, and biological evaluation. Biomacromolecules 2005, 6, 2946−2953. (d) Zhou, T.; Kong, X. L.; Liu, Z. D.; Liu, D. Y.; Hider, R. C. Synthesis and iron(III)-chelating properties of novel 3-hydroxypyridin-4-one hexadentate ligand-containing copolymers. Biomacromolecules 2008, 9, 1372−1380. (e) Zhou, T.; Neubert, H.; Liu, D. Y.; Liu, Z. D.; Ma, Y. M.; Kong, X. L.; Luo, W.; Mark, S.; Hider, R. C. Iron binding dendrimers: A novel approach for the treatment of haemochromatosis. J. Med. Chem. 2006, 49, 4171−4182. (f) Zhou, T.; Winkelmann, G.; Dai, Z.-Y.; Hider, R. C. Design of clinically useful macromolecular iron chelators. J. Pharm. Pharmacol. 2011, 63, 893−903. (14) Rosenbaum, D. P.; Holmes-Farley, S. R.; Mandeville, W. H.; Pitruzzello, M.; Goldberg, D. I. Effect of RenaGel, a non-absorbable, cross-linked, polymeric phosphate binder, on urinary phosphorus excretion in rats. Nephrol., Dial., Transplant. 1997, 12, 961−964.

In this study, we synthesized and evaluated the performance of nonabsorbed IBPs. We found that conjugating 2,3-DHBA to cross-linked polyallylamine yielded polymers with extremely high iron stability constants, high binding capacity, and selectivity for iron over other dietary metals. These features would limit the dose required while selectively preventing the absorption of iron by sequestering iron in the gut. Most importantly, the addition of IBPs substantially increased the fecal concentrations of iron when compared to a similar dose of the control polymer. Overall, the data suggested that the IBPs may be a novel agent to prevent iron accumulation in patients with hemochromatosis or other iron overload diseases.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.6b00945. Experimental details (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jian Qian: 0000-0003-0609-7045 Cory Berkland: 0000-0002-9346-938X Author Contributions §

J.Q. and B.P.S. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This funding was supported by the University of Kansas Institute for Advancing Medical Innovation. REFERENCES

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DOI: 10.1021/acsmacrolett.6b00945 ACS Macro Lett. 2017, 6, 350−353