Design of Polymeric Iron Chelators for Treating Iron Overload in

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Design of Polymeric Iron Chelators for Treating Iron Overload in Cooley's Anemia A N T H O N Y WINSTON, JAMES ROSTHAUSER, DAVID FAIR, JAMSHED BAPASOLA, and WEERASAK LERDTHUSNEE West Virginia University, Department of Chemistry, Morgantown, WV 26506

Iron chelating polymers based on hydroxamic acid and catechol groups have been synthesized for use in iron chelation therapy for treating iron overload in Cooley's Anemia. A mouse screen for in vivo activity of iron chelators has shown that poly(N-methacryloyl-ß-alanine hydroxamic acid) (P-11) i s effective and about equal to the standard drug desferrioxamine. The 11-atom spacing between hydroxamic acid groups is about optimum for intramolecular iron chelation and good solubility of the iron complex. A similar polymer, poly(N-methacryloylhydroxamic acid) (P-3), which has only 3 atoms between hydroxamic acids, is also active, but also rather toxic, a result that can be attributed to the tendency of P-3 to form insoluble intermolecular crosslinked complexes. A polymer bearing catechol groups is weekly active in removing iron in the mouse screen. Improvement may be possible with better control of the spacing of the catechol groups.

Cooley's Anemia is a genetic disorder relatively rare in the United States, but found more often among peoples of the Mediterranean area and within a band extending through India and southeast Asia (1). In addition to severe anemia, the disease causes bone malformation and general deterioration and distruetion of the v i t a l organs leading eventually to death. The disease is characterized by an inability of the body to synthesize adequate amounts of the chain of hemoglobin. The usual and really the only treatment is to administer blood transfusions on a regular basis throughout the lifetime of the individual. However, such a continual whole blood transfusion raises the iron level to the point where iron deposits form in the

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l i v e r , spleen, bone, heart, and other organs. Heart failure in the teens and early 20 s is usually the final outcome (2). In order to retard or prevent this accumulation of iron, a powerful iron chelator, desferrioxamine-B, is administered either by injection or by intravenous infusion. This chelator is capable of forming a very stable complex with iron and then carrying the iron out of the body with the urine or stool. Iron chelation therapy greatly improves the well-being of the patient and extends the lifetime of the patient appreciably (3). Desferrioxamine-B (DFB) (I) is produced in a fermentation process by Ciba Pharmaceutical Co. and is supplied as the methane sulfonate salt under the name Desferal. Although there are many excellent iron chelators known, DFB is the only one that not only works in humans, but is available in sufficient amounts to supply the need for iron chelation therapy. The iron chelating a b i l i t y of DFB is due to the hydroxamic acid, a functional group that possesses a natural a f f i n i t y for i r o n ( l l l ) . Also inherent in this structure is a powerful chelate effect due to the 9 atom spacing between hydroxamic acid groups, which permits three neighboring hydroxamic acids to f i t the octahedral coordination sphere of the iron(III) without severe steric strain. The chelate effect of DFB causes the formation constant to be greater than that of the monomeric acetohydroxamic acid by two orders of magnitude, Table 1. Other naturally occurring iron chelators, called siderochromes, include various modifications of DFB, ferrichrome (II), rhodotorulic acid (III) and enterobactin (IV), a l l of which possess high formation constants for iron, due in part to powerful chelate effects, Table 1.


f

Table 1. Formation Constants of Iron Chelators Reference Chelator log K Acetohydroxamic Acid 28.2 (4) Desferrioxamine-B 30.6 (4) (4) De s ferr ioxamine-E 32.5 (4) Desferrichrome 29.1 (5) Rhodotorulic Acid (RA) 62.3 Enterobactin 52 (6) Fe (RA) a

a

2

3

Although DFB therapy is effective in removing large quantities of iron rapidly, there are some drawbacks in i t s use. One of these is the short plasma residence time, about 30 minutes, which causes a significant reduction in the efficiency of DFB to remove iron. To counteract this rapid plasma clearance, the chelator is often administered by means of a portable pump that is worn by the patient and which continuously administers a controlled amount of the drug (7). On the other hand, the pump

Carraher and Gebelein; Biological Activities of Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1982.


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is often psychologically unacceptable, and frequent injections throughout the day are painful and not very practical. For these reasons a program designed to synthesize new iron chelators is being supported by the Cooley's Anemia Foundation of New York and the National Institute of A r t h r i t i s , Metabolism and Digestive Diseases of the National Institutes of Health. The object is to devise structures that not only remove iron from the body but also possess properties that provide a better mode of administration. A definite advantage would be achieved i f the new drug could be given orally, the most convenient route. Also, i f the plasma survival time could be extended, the efficiency of drug u t i l i z a t i o n would be increased, and thus the frequency of treatment and quantity of drug required would be reduced. Advances have been made in the area of designing and synthesizing chemical structures for binding iron tightly and s p e c i f i cally, and possessing the appropriate solubility characteristics and chemical s t a b i l i t y for drug use. On the other hand, there is no assurance that any such compound, no matter how carefully designed, w i l l actually work in vivo. Although the compound must, of course, have a high a f f i n i t y for iron, there is no simple relation between this property and the a b i l i t y of the compound to remove excess iron from living systems (8). Adding to the complexity of the problem is the idea that there are several iron pools open to attack by an iron chelator, such as transferrin, f e r r i t i n and iron in transit. However, knowledge as to which of these pools is available to an iron chelator such as DFB is not understood. Also, there is evidence that the rat and mouse screens, used to provide a preliminary evaluation of the drug, do not always provide a measure of the behavior of the drug in humans. For example, rhodotorulic acid, which appeared promising on the basis of animal tests, nevertheless produced painful reactions when used in humans (9). Clearly there is much to be learned about iron transport, the manner in which iron chelators really work, and the effect of structure on adverse physiological reactions. Design of Iron Chelators The design of new iron chelators has been directed largely toward mimicking the naturally occurring siderochromes such as desferrioxamine and enterobactin. Three functional groups are under serious investigation, hydroxamic acids, catechols, and phenols. These functional groups have been incorporated into a variety of structures in order to produce compounds having exceptionally high stability constants for iron, close to, or higher than that of desferrioxamine. Unfortunately, a high iron binding constant does not assure high activity in vivo (8), and thus many compounds w i l l have to be prepared and screened biologically in order to find a satisfactory balance of chemical and physiological properties.


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Several powerful iron chelators, some recently synthesized specifically for the Cooley's Anemia program, are being tested to ascertain their potential as drugs for use in iron chelation therapy for treating iron overload in Cooley's Anemia. Some of the more promising ones include:


Ethylenediamine-JI,N'-bis(2-hydroxyphenylacetic acid) (EDHPA) (V) (8) Derivatives of N,N'-bis(2-hydroxybenzyl)ethylenediamine-4l,JJ'diacetic acid (HBED) (VI) (8), 1,3,5

-N,N',N"-tris(2,3-dihydroxybenzoyl)triaminomethylbenzene~(VII) (10,11),

l,5,9-N,N'N"-tris(2,3-dihydroxybenzoyl)triazacyclotridecane (VIII) (10) polyOl-methacryloyl-6-alaninehydroxamic (U).

acid) (P-ll) (IX)

A l l of these compounds have exceptionally high binding constants for iron. Polymeric Iron Chelators The structure of the hydroxamic acid polymer P - l l was designed to maximize the formation constant for iron chelation. The formation constants of a series of such polymers is directly related to the magnitude of the chelate effect, which i s , in turn, a function of the side chain length, Table I I . A maximum chelate effect appears at a spacing of 11 atoms. A shorter spacing, 9 atoms, (P-9) i s insufficient to permit three neighboring hydroxamic acids to f i t about a single iron without strain, hence the lower value of log K. With increasing side chain length, P-13 and P-15, the chelate effect decreases with the increasing degrees of freedom and a grandual decrease in log K results (12). Table I I . Formation Constants of Iron Complexes of Hydroxamic Acid Polymers (12) Polymer n log K P-9 1 28.6 P-ll 2 29.7 P-13 3 29.3 P-15 29.0 Code number indicates number of atoms separating hydroxamic acid groups. ^Numbers refer to structure IX. Spacer is glycylglycine. 3

b

Fe

c

a

c


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IX


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Another hydroxamic acid polymer, polyOf-methylacryloylhydroxamic acid) (P-3) (X) i n which the hydroxamic acid units are separated by only three atoms was similarly prepared. Because of the short side chains and closely spaced hydroxamic acid groups, P-3 tends to cross link and precipitate on complexation with iron. In cases such as this, the groups are too closely spaced to give soluble intramolecular complexes with iron.

c=o i N-OH I CH, X Hydroxamic Acid Polymer P - l l has been prepared in two molecular weight varieties, [r] 1 MF-H 0 "* °' dl/g ( P - l l ) . The formation constant reported in Table II i s for the lower molecular weight sample. Although the formation constant of the higher molecular weight material has not been determined, i t is expected that there would not be much d i f f e r ence between the two. A polymer (P-DHB) (XI) based on catechol, the active functional group of enterobactin, was recently synthesized by the reaction of polyvinyl amine with the ethyl ester of 2,3-dihydroxybenzoic acid (DHB). Only about one third of the amine groups was found to be substituted with DHB units. The formation constant of the iron(IIl) complex (log K • 40) is the same as that reported for the simple dimethyl amide of DHB and so there does not appear to be any appreciable chelate effect. 2

D

,

2

4

( H P

1 1 )

a n d

2

XI


8 7

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Bioassay of Polymeric Iron Chelators The iron chelator screen consists essentially of treating iron overloaded mice with the iron chelator by daily I.P. injections over a period of 7 days. Urine and stools are collected daily. At the end of the week, the mice are sacrificed and the livers and spleens are collected. The samples are then reduced to a suitable form and analyzed for iron by atomic absorption. An active chelator should produce increased iron levels in the urine and stool and decreased iron in the liver and spleen. Polymers P - l l (both molecular weight forms) P-3 and P-DHB were subjected to the mouse screen. The results are shown in Table III and compared with the drug DFB, which is used as a standard for comparison. Overall, P - l l and HP-11 are judged to be about equal in potency to the standard DFB and except for lower fecal output occasionally observed at higher dose levels, both appear to be completely non-toxic. In the case of P-3, i t is suggested that the extreme toxicity and high mortality is due to the tendency of P-3 to cross-link and precipitate on iron chelation. Such an event occurring in the blood stream would have obvious deleterious effects. Since the structural features of P-3 are similar to P - l l i t is d i f f i c u l t to account for the toxicity on any basis other than the spacing of the groups. Regardless of the exact cause of the observed toxicity, i t i s clear that changes in spacing of the chelating groups profoundly affect both chemical and physiological activity. P-DHB shows some weak activity in the mouse screen and gives some hope to the idea that a better placement of the DHB groups may result in a more positive biological outlook. Potential Advantage of Polymeric Iron Chelators Although the synthetic work on polymeric iron chelators has been directed primarily toward achieving strong chelate effects by appropriate spacing of functional groups, there is another means by which polymers might be of some distinct advantage over smaller molecules. And that concerns the residence time of the chelator in the plasma. As mentioned above, DFB is almost completely cleared from the plasma in 30 minutes, an effect which lowers the efficiency of DFB considerably. Although we do not know the direct cause of the rapid clearance of DFB, two possib i l i t i e s have been suggested. Either the uncomplexed form is being degraded by metabolic processes, or the chelator is simply being lost by normal diffusion out through the membranes. Although degradation i s a likely path, nevertheless, there is evidence to show that the iron complex of DFB is eliminated intact. If the loss of DFB is by simple diffusion, a high molecular weight analog of DFB, polymer P - l l for example, might diffuse out less rapidly, increasing the active lifetime in the


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Table I I I . Results of Mouse Bioassay of Polymeric Iron Chelators


Treatment Drug

3

Iron Content, % change

Dose mg/kg

Spleen

Liver

Urin

Design of Polymeric Iron Chelators for Treating Iron Overload in

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