WO2016170015A1

WO2016170015A1 - Epoxy derivatives of ion chelating ligands, their polymeric conjugates and medical uses thereof

Info

Publication number: WO2016170015A1
Application number: PCT/EP2016/058811
Authority: WO
Inventors: Alessandro Sannino; Vincenzo Maria DE BENEDICTIS; Christian DEMITRI
Original assignee: Komplexis S.R.L
Priority date: 2015-04-20
Filing date: 2016-04-20
Publication date: 2016-10-27
Also published as: GB201506663D0

Abstract

Epoxy derivatives of ion chelating ligands, method for production and use thereof. Methods for producing polymers covalently linked with ion chelating ligands and their use as drug delivery and ion removal agents.

Description

Epoxy derivatives of ion chelating ligands, their polymeric

conjugates and medical uses thereof

Background

Ion chelator agents are commonly used for in-vivo control of ionic concentrations and detoxification of excess metals. Metal ions play an important role in biological systems. Cells utilize metal ions for a wide variety of functions, such as regulating enzyme activity, protein structure, cellular signalling, as catalysts, as templates for polymer formation and as regulatory elements for gene transcription. Metal ions can also have a deleterious effect when present in excess of bodily requirements or capacity to excrete. Various medical conditions, such as diseased renal function, genitourinary disease, cancer, congestive heart failure, and or the treatment of these condition can lead to or predispose patients to ion imbalances, for example in the form of hyperkalemia, hypernatremia, hypercalcemia, hypermagnesia, hypersideremia, hyperzincemia and hypercopperemia.

Potassium is the major cation inside animal cells, while sodium is the major cation outside animal cells. The concentration differences between these charged particles causes a difference in electric potential between the inside and outside of cells, known as the membrane potential. The balance between potassium and sodium is maintained by ion transporters in the cell membrane. Patients afflicted with an increased level of potassium (hyperkalemia) may exhibit a variety of symptoms ranging from malaise, palpitations, muscle weakness and, in severe cases, cardiac arrhythmias. Patients afflicted with increased levels of sodium (hypernatremia) may exhibit a variety of symptoms including, lethargy, weakness, irritability, edema and in severe cases, seizures and coma.

Iron has several vital functions in the body. It serves as a carrier of oxygen to the tissues from the lungs by red blood cell haemoglobin, as a transport medium for electrons within cells, and as an integrated part of important enzyme systems in various tissues. Most of the iron in the body is present in the erythrocytes as haemoglobin, a molecule composed of four units, each containing one heme group and one protein chain. The structure of haemoglobin allows it to be fully loaded with oxygen in the lungs and partially unloaded in the tissues (e.g., in the muscles) . Several iron- containing enzymes, the cytochromes, also have one heme group and one globin protein chain. These enzymes act as electron carriers within the cell and their structures do not permit reversible loading and unloading of oxygen. Their role in the oxidative metabolism is to transfer energy within the cell and specifically in the mitochondria. Other key functions for the iron-containing enzymes (e.g., cytochrome P450) include the synthesis of steroid hormones and bile acids; detoxification of foreign substances in the liver; and signal controlling in some neurotransmitters, such as the dopamine and serotonin systems in the brain. Iron is reversibly stored within the liver as ferritin and hemosiderin whereas it is transported between different compartments in the body by the protein transferrin.

Iron is not actively excreted from the body in urine or in the intestines. Iron is only lost with cells from the skin and the interior surfaces of the body-intestines, urinary tract, and airways. The body has three unique mechanisms for maintaining iron balance and preventing iron deficiency and iron overload. The first is the continuous reutilisation of iron from catabolised erythrocytes in the body. Uptake and distribution of iron in the body is regulated by the synthesis of transferrin receptors on the cell surface. This system for internal iron transport not only controls the rate of flow of iron to different tissues according to their needs but also effectively prevents the appearance of free iron and the formation of free radicals in the circulation. The second mechanism is the access of the specific storage protein, ferritin, which can store and release iron to meet excessive iron demands. This iron reservoir is especially important in the third trimester of pregnancy. The third mechanism involves the regulation of absorption of iron from the intestines, with an increased iron absorption in the presence of decreasing body iron stores and a decreased iron absorption when iron stores increase. Iron absorption decreases until an equilibrium is established between absorption and requirements. Iron balance (absorption equals losses) may be present not only in normal subjects but also during iron deficiency and iron overload .

Inorganic phosphates, including monophosphate (P0₄ ^3~) , diphosphate (P₂0₇ ^4~) , or triphosphate (P₃Oi₀ ^5~) and phosphate containing metabolites, in particular adenosine monophosphate (AMP) , adenosine diphosphate (ADP) and adenosine triphosphate (ATP) play an essential role in many biological processes, spanning from metabolism to biosynthesis, gene regulation, signal transduction and muscle contraction. Furthermore, phosphate ions are constituent parts of two universally found biopolymers, DNA and RNA, as well as membrane lipids (phospholipids) .

Patients afflicted with an increased level of phosphates, even severe hyperphosphatemia are often asymptomatic. Morbidity In patients with this condition is more commonly associated with an underlying disease than with increased phosphate values. Calcium levels are lowered, due to precipitation of phosphate with the calcium in tissues and patients occasionally report hypocalcemic symptoms, such as muscle cramps, tetany, and perioral numbness or tingling, other symptoms include bone and joint pain, pruritus, and rash. More commonly, patients report symptoms related to the underlying hyperphosphatemia, such as fatigue, shortness of breath, anorexia, nausea, vomiting and sleep disturbances.

A large number of natural and synthetic materials are known to selectively or non-selectively bind to or chelate metal ions. Chemical compounds that bind to a particular ion selectively are called generally ionophore, in case of iron are specifically called siderophres. Natural ionophores have been obtained from the cells of various microorganisms, and include (Beauvericin (Ca²⁺, Ba²⁺) , Calcimycine (or A23187 Ca²⁺) , Enniatin (NH₄ ⁺) , Gramicidin A (H⁺, Na⁺, K⁺) , Ionomycin (Ca²⁺) , Monensin (Na⁺, H⁺) , Nigericin (K⁺, H⁺, Pb²⁺) Nonactin (NH₄ ⁺) , Salinomycin (K⁺) , Valinomycin (K⁺) , Ferrichrome (Fe ) , Rhodotorulic acid (Fe ) , Coprogen (Fe ) , Deferoxamine (Fe ) , Enterobactin (Fe³⁺) .

Most prominent synthetic ionophores are based on crown ethers or calixarenes, but these have shown limited use as in-vivo treatments.

In general, a useful property for metal ion chelating is the ability to discriminate a selected metal ion in the presence of other interferent metal ions. For example discrimination of K+ in the presence of other metal ions like Na+ is particularly useful for certain biological or environmental applications. Moreover, it is essential that the materials be effective in aqueous solutions, in pH values over the physiological range (pH 6-8) and sensitive to ion concentrations in the physiological range.

United States Patent 6818626 Bl discloses chelator-containing systems for use in biologic systems. More particularly, chelators and polychelators are utilized in the delivery of molecules, polymers, nucleic acids and genes to animal cells. At least one chelator such as crown ether is attached to a polymer and then associated with another polymer such as DNA. An ion is added to the mixture thereby forming condensed DNA. In condensed form and in complex with the chelator, DNA can be delivered to a cell. In the present disclosure however, the crown ether chelator is attached to a high molecular weight polymer to avoid the penetration into animal cells .

United States Patent 5217998 discloses a method for scavenging free iron or aluminium in physiological fluids involving a provision in such fluids of a soluble polymer substrate having chelator immobilized thereon. Compounds comprise polysaccharides or proteins having a deferoxamine moiety thereon. The deferoxamine is stabilized in a manner which reduces its toxicity and increases its in-vivo vascular retention time. The deferoxamine is stabilized by covalent bonding to a pharmaceutically-acceptable polymer so that the deferoxamine substantially retains its ability to complex ferric and aluminium ions. For in-vivo use, e.g. in the blood stream, the deferoxamine is preferably bound to a water-soluble biopolymer such as a polysaccharide or a protein. When intended for the in-vitro complexation of ferric ion, e.g. from an extracorporeal stream of blood, deferoxamine may sometimes be bound to a biologically-inert, water-insoluble polymeric support such as cellulose, agarose or a cross-linked dextran. Such a compound can be used for the treatment of iron overload, as well as to inhibit cell damage from oxidation/ reduction reactions .

Patent Application WO9015610A1 discloses a composition for removing or inactivating harmful components including virus from blood or other extracellular liquids. Said composition comprises a crown ether compound substituted on a polysaccharide, preferably dextran. The composition may be soluble in water, preferably for intravenous application, or insoluble for extracorporeal application.

United States Patent US20120107381A1 discloses a powder formulation of a potassium-binding active agent. The potassium-binding active agent comprises a crown ether, a crown-ether like molecule, or a potassium-binding polymer that comprises a crosslinked aliphatic carboxylic polymer. The author claims that these pharmaceutical compositions are useful to bind potassium in the gastrointestinal tract. The crown ethers are cited in the description of the patent, but no covalent chemical bond was formed between potassium-binding polymer (e.g. crosslinked aliphatic carboxylic polymer) and potassium-binding active agent (e.g. crown ethers) .

United States Patent US20130189216A1 discloses pharmaceutical compositions comprising a polyol and a salt of a crosslinked cation exchange polymer, with the polyol present in an amount sufficient to reduce the release of fluoride ion from the cation exchange polymer during storage. The author claims that these pharmaceutical compositions are useful to bind potassium in the gastrointestinal tract .

United States Patent US20130259949A1 discloses compositions of matter, including pharmaceutical compositions of core-shell particle for removing monovalent cation from the gastrointestinal tract of a mammal and methods for treating abnormally elevated monovalent cation, such as abnormally elevated serum potassium ion (e.g., hyperkalemia) or abnormally elevated serum sodium ion (e.g., hypertension) . The core component has a net negative charge under physiological conditions (to provide the capacity for binding monovalent cation) and the shell polymer has a net positive charge under physiological conditions, the core and shell components are significantly attracted to each other and, as a result, only monovalent cations rather than divalent cations can penetrate to the core and bind. The shell component comprises a crosslinked polymer

(e.g., hydrophilic polymer or polyvinylic polymer, such as polyvinylamine polymer or polyalkyleneimine polymer such as polyethyleneimine) . The core component comprises an organic material

(polyacrylic acid polymers, polyhaloacrylic acid polymers, polystyrenic polymers, polysulfonic polymers and polystyrenesulfonate polymers) or an inorganic material (ceramics, microporous and mesoporous zeolites) with capacity for binding monovalent cation.

United States Patent US20130272992A1 discloses compositions and methods for the removal of potassium ions from the gastro-intestinal tract using a potassium binding polymer. Disclosed is a core of potassium binding polymer, optionally surrounded by a shell, which may be a crown ether, and are used to decrease the passage of sodium, magnesium, calcium and other interfering molecules to the core and as a result, increase the in-vivo potassium binding capacity of a core polymer. Crown ethers are cited in the description of the patent, but not used in the examples, moreover no covalently chemical bond was formed between potassium-binding polymer (e.g. core crosslinked -fluoroacrylate polymer) and crown ethers used as shell materials.

WO2013106086A1 discloses crosslinked cation-binding polymers comprising monomers containing carboxylic acid groups and methods of preparation and using these polymers to treat various diseases or disorders . United States Patent US20130195975A1 discloses a method and material for removing water from the intestinal tract of patients suffering from fluid overload states. The method involves directly delivering, by orally administering, a non-systemic, non-toxic, non-digestible, fluid absorbing polymer to the intestinal tract where it absorbs fluid as it passes therethrough and is subsequently excreted. Applicable polymers include polyelectrolyte, carboxylate containing polymers such as polyacrylates , non-polyelectrolyte compounds or non-ionic polymers such as polyacrylamide gels, polyvinyl alcohol gels, and polyurethane gels.

United States Patent 7989617B2 discloses chromophoric or fluorescent derivatives of crown ethers for applications as optical indicators and sensors for the detection, discrimination and quantification of metal cations in a biological environment, and also permits to monitoring and quantifying the level of these ions, in particular sodium in living cells. Exemplary compounds include crown ethers of the type shown below, and their conjugates:

140

These crown ethers were conjugated with aminodextran to resolve problems such as binding ion chelators to intracellular proteins, and non-selective sequestration in intracellular vesicles due to the relatively small size of crown ethers that altered the chelator's metal binding properties. The compounds described have not been used for medical purposes, or in the treatment of any disease conditions.

United States Patent US5865994 discloses compositions useful for performing improved liquid chromatography. The invention relates to an improved chromatographic composition and method for performing cation-exchange chromatography, where attached to a synthetic resin support particles employed therein are both (1) standard ionic cation-exchange functional groups such as sulfonates, carboxylates and/or phosphonates and (2) non-ionic crown ether-based functional groups, thereby providing a bifunctional stationary phase which provides unique separation characteristics and selectivity for numerous cationic species including alkali metals, alkaline-earth metals, ammonia, amines, and the like. The method is employed to separate a trace amount of one cation (i.e. potassium) from a large excess concentration of another cation (i.e. sodium or ammonium respectively) . The compounds described have not been used for medical purposes, or in the treatment of any disease conditions.

United States Patent US4256859 discloses the synthesis of certain substituted crown polyethers .

United States Patent US5393777 discloses compositions useful for removing ferric iron from aqueous liquids using novel siderophore for deferration therapy and other related applications. Representative siderophores include phenolate compounds such as "agrobactin" from Agrobacterium tumefaciens and "pseudobactin" from Pseudomonas, and hydroxamates such as " schizokinin" from Bacillus megaterium and ferrioxamines from Actinomyces.

Summary of the invention

Treatment of diseases or disorders associated with ion imbalances may employ the use of metal chelating compounds to restore in-vivo ion balance. However the administration of these compounds as drugs is not straightforward, and can be improved by the use of drug delivery conjugates. The administration of ion chelation agent drugs can be improved by conjugation with polymeric materials. The conjugation further improves the ability to remove the ions from the body as the polymers can be readily excreted having absorbed the ions. The compounds described herein are improved ion chelating delivery and removal compounds for therapeutic use due to the covalent attachment of the ion chelating moiety to a polymer. For these reasons, these metal chelating materials find utility in controlling physiological levels of electrolytes in-vivo. The polymeric ion chelators may be excreted without undergoing digestion, thereby selectively removing certain ions from the body.

This invention particularly relates to epoxy derivatives of ion chelating ligands. This invention includes covalently linking epoxy derivatives of ion chelating ligands to polymeric materials. Also disclosed are covalently linked derivatives of ion chelating ligands to polymers for the improved in-vivo delivery of the ion chelating ligands. The compounds may be used for the selective removal of ions. Active chelating agents chemically and covalently linked with bioavailable polymeric compounds can bind selectively many electrolytes in gastro-intestinal tract. Provided are injectable or orally active chelating agents chemically and covalently functionalized with bioavailable polymeric compounds that bind selectively many electrolytes including physiological relevant levels of metal cations such as potassium K⁺, sodium Na⁺, calcium Ca²⁺, magnesium Mg²⁺, zinc Zn²⁺, silver Ag⁺, lead Pb²⁺, mercury Hg²⁺, iron Fe²⁺ and Fe³⁺, copper Cu⁺ and Cu²⁺ and inorganic anions such as phosphates. The present disclosure also relates to methods of preparation of the ion chelating agents or compositions, formulations, and or dosage forms containing the ion chelating agents and polymers, and methods of using the polymers or compositions, formulations, and or dosage forms containing the ion chelating agents and polymers to treat various diseases or disorders .

Detailed description Described herein is a drug delivery product composition comprising a polymer and a covalently attached ion chelating agent for therapeutic use. Described herein is a drug delivery product composition comprising a water soluble polymer and a covalently attached ion chelating agent. The covalent attachment can be formed of one or more bonds. Depending on the size of the polymer, each polymer molecule may have one or more ion chelating ligands covalently attached. The polymer may have one ion chelating ligand per polymer chain, or may have two or more per chain. For large polymer chains, there may be 10, 100 or more ion chelating ligands per chain. The polymers may be straight chains, branched chains or cross-linked chains. The polymeric material may be a hydrogel polymer .

Described herein are epoxy derivatives of ion chelating ligands. Said chelating ligands are able to selectively bind ions by complexation, chelation, ion exchange, chemical trapping, physical trapping, electrostatic interaction or a combination thereof.

The ion to be chelated may be a cation, including potassium K , sodium Na⁺, calcium Ca²⁺, magnesium Mg²⁺, zinc Zn²⁺, silver Ag⁺, lead Pb²⁺, mercury Hg²⁺, iron Fe²⁺ and Fe³⁺, copper Cu⁺ and Cu²⁺.

Alternatively the ion may be an anion. The ion may be an inorganic phosphate, including monophosphate (P0₄ ^3~) , diphosphate (P₂0₇ ^4~) , or triphosphate (Ρ₃Οι₀ ^5~) and phosphate containing metabolites, in particular adenosine monophosphate (AMP) , adenosine diphosphate (ADP) and adenosine triphosphate (ATP) .

Said ion chelating ligands may be linear or cyclic compounds. The ion chelating agent may be a macrocyclic compound of 12 atoms or greater. Suitable macrocyclic compounds include crown ethers, cyclic alkylamino (azacrowns), porphyrins, calixarines or siderophores . The particular size of the macrocyclic compound may be chosen depending on the choice of ion to be chelated.

Ion chelating ligands useful for potassium removal include cr ethers, calixarenes, carboxyl derivatives or a combination thereof Crown ethers are cyclic chemical compounds that consist of a ring containing several ether groups, The most common crown ethers are oligomers of ethylene oxide, the repeating unit being ethyleneoxy (shown below) :

12-crown-4 15-crown-5 18-crown-6

Crown ethers strongly bind (chelate) certain cations, forming complexes. The oxygen atoms are well situated to coordinate with a cation located at the interior of the ring, whereas the exterior of the ring is hydrophobic. For example 12-crown-4 strongly and selectively bind lithium ions, 15-crown-5 strongly and selectively binds sodium ions, 18-crown-6 strongly and selectively binds potassium ions.

Calixarenes are characterised by a three-dimensional basket, cup or bucket shape. A calixarene is a macrocycle or cyclic oligomer based on a hydroxyalkylation product of a phenol and an aldehyde, as shown below .

Calixarenes are characterised by a wide upper rim and a narrow lower rim and a central annulus. With phenol as a starting material the 4 hydroxyl groups are intrannular on the lower rim. The 4 hydroxyl groups interact by hydrogen bonding and stabilize the cone conformation. This conformation is in dynamic equilibrium with the other conformations. Conformations can be locked in place with proper substituents replacing the hydroxyl groups which increase the rotational barrier.

Calixarenes are efficient sodium ionophores and are applied as such in chemical sensors. With the right chemistry these molecules exhibit great selectivity towards other cations. Calixarenes are used in commercial applications as sodium selective electrodes for the measurement of sodium levels in blood. Calixarenes also form complexes with cadmium, lead, lanthanides and actinides and also form exo-calix ammonium salts with aliphatic amines such as piperidine .

Ion chelating ligands useful for iron (either as Fe²⁺ or Fe³⁺) removal include siderophore, in particular ferrichrome, enterobactin, coprogen, deferoxamine, carboxyl derivatives or a combination thereof.

Siderophores are small, high-affinity iron chelating compounds secreted by microorganisms such as bacteria, fungi and grasses. They are amongst the strongest soluble Fe3+ binding agents known.

Siderophores form a stable, hexadentate, octahedral complex preferentially with Fe 3+ compared to other naturally occurring abundant metal ions, although if there are less than six donor atoms water can also coordinate. The most effective siderophores are those that have three bidentate ligands per molecule, forming a hexadentate complex and causing a smaller entropic change than that caused by chelating a single ferric ion with separate ligands. Fe³⁺ ion is a hard Lewis acid, preferring hard Lewis bases such as anionic or neutral oxygen to coordinate with. Microbes usually release the iron from the siderophore by reduction to Fe²⁺ which has little affinity to these ligands.

Siderophores are usually classified by the ligands used to chelate the ferric iron. The major groups of siderophores include the catecholates (phenolates ) , hydroxamates and carboxylates (e.g. derivatives of citric acid) . Citric acid can also act as a siderophore. The wide variety of siderophores may be due to evolutionary pressures placed on microbes to produce structurally different siderophores which cannot be transported by other microbes' specific active transport systems, or in the case of pathogens deactivated by the host organism.

Suitable siderophores include, but are not limited to, ferrichrome, enterobactin, coprogen and deferoxamine.

Ferrichrome

ńeferoxamine

Citric acid derivatives

Ion chelating ligands useful for phosphate removal include amines, porphyrins, sapphyrins or expanded porphyrins, azacro n ethers or a combination thereof.

The ion chelating ligand may be a porphyrin or expanded porphyrin.

where n is 1 or 2. The porphyrin or expanded porphyrin (sapphyrin) may be further substituted. The further substitution may allow covalent attachment of the polymer chains. Disclosed is a porphyrin or expanded porphyrin (sapphyrin) suitable for covalent attachment to a water soluble polymer, the porphyrin or expanded porphyrin (sapphyrin) having a reactive group X, wherein X is selected from NH₂, OH, CHO, epoxide, an activated carboxylate, NCO .

The ion chelating ligand may be a porphyrin or expanded porphyrin.

where n is 1 or 2 and Y is a linker selected from CH₂-0-CH₂, CH₂CH₂ 0-CH₂, CH₂-NH-CH₂ and CH₂COOCH₂.

Sapphyrins are expanded porphyrin systems, the core of sapphyrin is expanded relative to that of porphyrin with an additional pyrrole, inserted between a meso-carbon and an R-pyrrolic position. Porphyrins and related tetrapyrrolic macrocycles serve a variety of critical roles in living systems and are present in such functionally disparate systems as photosynthetic reaction centers, coenzyme B_i2, hemoglobin, myoglobin, cytochromes, peroxidases, and catalyases for example. Macrocyclic sapphiryns possesses unique characteristics that the porphyrins do not possess, such as anion binding .

Azacrowns are cyclic alkylamino compounds. Azacrowns are crown ethers wherein one or more of the oxygen atoms are replaced with nitrogen atoms . The ring may be of different sizes, as shown below:

12-crown-4 15-crown-5 18-crown-6

Azacrown compounds bind anions such as phosphate. The smaller rings bind smaller monophosphate ions, whereas larger rings bind diphosphate and triphosphate ions. The ring structures may contain a mixture of nitrogen and oxygen atoms. The rings may be of type

where each X is independently selected from N or 0.

This invention relates to epoxy derivatives of chelating ligands. Said epoxy functionalisation of the chelating ligands may allow covalent attachment of polymer chains.

Disclosed is a crown ether suitable for covalent attachment to a water soluble polymer, the crown ether having a reactive group X, wherein X is selected from NH₂, OH, CHO, epoxide, an activated carboxylate, NCO .

The crown ether/amine may have a nucleophilic group attached. The crown ether/amine may have a hydroxyl or amino group attached. The crown ether/amine may have an electrophilic group attached.

The crown ether/amine may be

Disclosed are chelating agents functionalised with an epoxide group. Disclosed is a method of covalently labelling a polymer using a labelling agent with an epoxide group. The labelling agent may be a crown ether or a crown amine. The term labelling agent refers to a moiety having an ion chelating agent and a reactive group for attachment to the polymer. The crown ether/amine may be

where each X is independently selected from N or 0, n is 1, 2 or 3 and Y is a linker selected from CH₂-0-CH₂, CH₂-NH-CH₂ and CH₂COOCH₂.

The chelating agents may be functionalised with one or more epoxide groups to obtain mono-functional, bi-functional or multifunctional chelating agents .

The crown ether/amine may be

where each X is independently selected from N or 0, n is 1, 2 or 3, m is 1 to 12 and Y is a linker selected from CH₂-0-CH CH₂-NH-CH 2

The crown ether may be :

In particular, the chelating agents may be functionalised with two epoxide groups to obtain bi-functional chelating agents.

The crown ether/amine may be

where each X is independently selected from N or 0 and Y is a linker selected from CH₂-0-CH₂, CH₂-NH-CH₂ and CH₂COOCH₂.

This invention also relates to polymeric materials and their use as delivery systems for selective removal of ions. The composition accordingly may be such that the ion chelating agents chelates metal ions . The chelated metal ions may remain bound to the polymer conjugates in-vivo such that the ions can be excreted. Excretion of excess levels of metal ions allows the control of physiological levels of electrolytes, which is useful in the treatment of a variety of diseases, particularly kidney dysfunctions. The metal may be selected from potassium, sodium, calcium, magnesium, zinc, silver, lead, mercury, iron or copper.

Alternatively the ion may be an anion. The ion may be an inorganic phosphate. The phosphate may be a monophosphate (P0₄ ^3~) , diphosphate (P₂0₇ ^4") , or triphosphate (P₃Oi₀ ^5") .

Described herein are methods of covalently linking derivatives of ion chelating ligands to polymeric materials. Preferably, the derivatives of ion chelating ligands are epoxy derivatives of ion chelating ligands. The covalent attachment can be formed of one or more bonds. The polymer conjugate may have a degree of substitution from about 1% to about 100%, more specifically from about 10% to about 90%, more specifically from about 50% to about 70%, more specifically from about 70% to about 90%. Depending on the size of the polymer, each polymer molecule may have one or more ion chelating ligands covalently attached. The polymer may have one ion chelating ligand per polymer chain, or may have two or more per chain. For large polymer chains, there may be 10, 100 or more ion chelating ligands per chain. The polymers may be straight chains, branched chains or cross-linked chains. The polymeric material may be a hydrogel polymer.

The polymers may be cross-linked chains. The chains of polymers may be cross-linked using cross-linking agents. Crosslinking of the polymers is optional. Cross-linking of the polymers improves mechanical properties of the materials including elasticity and resistance to degradation. Any cross-linker known in the art may be used. In particular, butandiol diglycidylether (BDDE) may be used.

The polymer may be any hydrogel polymer. Hydrogel copolymers are capable of absorbing water and are hydrophilic in nature. The polymer may contain amino, hydroxyl, or carboxylate groups. The polymers may be cross-linked chains. The polymer may be a polysaccharide. The polysaccharide may be chitosan or cellulose or derivatives thereof. The polymer may be chitosan. The polymer may be cellulose. The polymer may be a cellulose derivative. The polymer may be pullulan or a derivative thereof. The polymer may be alginate or a derivative thereof. The polymer may be agarose or a derivative thereof. The polymer may be mannan or a derivative thereof. The polymer may be glucomannan or a derivative thereof. The polymer may be acemannan or a derivative thereof. The polymer may be hyaluronate or a derivative thereof. The polymer may be polyvinyl alcohol (PVA) , polyacrylic acid, polyacrylate , polyacrylamide, polyallylamine or polyethylene glycol (PEG) or derivatives thereof. The polymer may be made of a mixture of different polymer types within the same composition.

Chitosan, a β-(1-4) linked 2-amino, 2-deoxy, -D-glucan, is the only amino polysaccharide distributed in large amounts in nature.

Chitosan is the deacetylated derivative of chitin, the most abundant natural polymer on earth after cellulose, this biopolymer is synthesized by an enormous number of living organisms, such as marine crustaceans, shrimp and crabs and some fungi. Chitosan, is biodegradable, biocompatible and exhibits bioadhesive characteristics, it is a linear polycationic polysaccharide in acidic medium (pKa 6.5) .

Recently, chitosan has attracted much interest in the biomedical industry because of its excellent biodegradability, biocompatibility, antimicrobial activity, and accelerated wound- healing properties. Because of its unique polymeric cationic character, net negatively charged compounds such as DNA, glycosaminoglycans , and some proteins can bind to chitosan without the use of harsh and denaturing organic solvents. Therefore, chitosan has been extensively examined in the pharmaceutical industry for its potential use in the development of a controlled release implant system and it has been widely used in industries including wastewater treatment, food, and cosmetics.

Carboxymethylcellulose and other cellulose derivatives (i.e. hydroxylethyl cellulose) are water soluble, biocompatible and bioresorbable semi-synthesized polysaccharides.

The safety of commercially available carboxymethylcellulose having high purity has been identified and approved by the Food and Drug Administration (FDA) for incorporation into many products. Carboxymethylcellulose is able to react with various polymers by way of electrostatic interaction, ionic cross-linking, hydrogen bonding, Van der Waals interactions, and physical interpenetration . Because of its safety, convenience and diversity of physico-chemical properties, carboxymethylcellulose has demonstrated applications in the pharmaceutical, food and cosmetic industries. Carboxymethylcellulose is one type of carboxypolysaccharide (CPS) . CPSs have also been used in the manufacture of implantable polymers. CPSs are polymers made of saccharide monomers in which some of the hydroxyl (—OH) groups are replaced with carboxyl groups (-COOH or - COO^~) . Thus, CPSs such as CMC have some hydroxyl groups and some carboxyl groups present. Carboxylation can permit ionic interaction within a polymer chain or can permit interaction between polymer chains, thereby forming a gel. Such gels have been used for a variety of applications, including implantable medical polymers. Poly (ethylene glycol) (PEG) and its functionalized derivatives, are an effective biomaterials . PEG is a water-soluble, hydrophilic polymer that has been widely explored for biomedical applications due to its non-toxic, minimal immunogenicity and anti-protein fouling properties. As the US Food and Drug Administration (FDA) has approved this polymer for use in drug and cosmetic applications.

PEG exhibits a very low toxicity and immunogenicity compared with other biopolymers, for this reason is used in mono or bis- functionalized form in a large number of reactions called pegilatons .

Polyvinyl alcohol (PVA) is widely used as principal component of biomedical devices. PVA is a highly polar polymer obtained through hydrolysis from polyvinyl acetate. The properties of the polymer depend on the degree of polymerization and the percentage of hydrolysis. Depending upon its application, PVA with partial or total hydrolysis can be obtained and in pharmaceutical applications is used with a most elevating hydrolysis degree because of vinylacetate toxicity. In aqueous solution, PVA is used as an agent which increases the viscosity of injectable solutions, as an ophthalmic lubricating agent and substitute of vitreous body. Solutions of PVA have been proposed as substitutes of blood plasma since, at concentration of the 2-3% in physiological salt solution, PVA has a viscosity and an osmotic pressure comparable to human plasma. PVA membranes have been developed as artificial pancreas and for haemodialysis .

PVA is only minimally absorbed following oral administration and possesses a low order of acute oral toxicity. The safety of PVA is documented by a number of dietary toxicity studies, there was no evidence of toxicity in either a 90-day or 2- generation reproductive toxicity study in rats, or in-vitro and in-vivo genotoxicity assays studies, at the highest dose levels tested of 5000 mg/kg bw/day. PVA is neither mutagenic nor genotoxic. There is no evidence to indicate that PVA has carcinogenic activity. Controlled human studies are limited, but there is a history of use of PVA for several different applications. In particular, PVA is commonly used in film coating formulations for pharmaceutical tablets and capsules in Europe, Japan, and the United States. There is no evidence that such use has resulted in any adverse effects in humans .

The composition or polymer conjugate can be prepared using any particular covalent linkage. Suitable covalent linkages include amides, esters, amines or ethers. The covalent linkage can be prepared using suitable nucleophiles and electrophiles . The nucleophile can be on the ion chelating ligand or the polymer. Similarly the electrophile can be on the ion chelating ligand or the polymer. Suitable nucleophiles include amines. The amines may be primary amines (NH₂) or secondary amines (NHR) where R is an alkyl substituent. Suitable nucleophiles include hydroxyl groups.

Suitable electrophilies include aldehydes (CHO) , epoxides, isocyanates (NCO) , acid chlorides (COC1) or any other type of activated carboxylate. The term activated carboxylate includes species of type C(=0)OX and C(=0)NY where OX and NY symbolises leaving groups allowing the formations of amides or esters . Activated carboxylates can be made using carbodiimide reagents such as EDC or DCC . Activated carboxylates includes succinimide esters such as NHS esters.

Preferably the conjugation chemistry can be carried out in water. Preferably the conjugation chemistry can be carried out without the addition of further catalysts or reagents such that the conjugates can be immediately used as therapeutics.

Preferably, the invention relates to a method of attaching a polymer to an ion chelating ligand by reacting an ion chelating ligand having an epoxy group with a polymer. Said polymer may have a hydroxyl, carboxy or amino group.

The ion chelating species includes crown ethers and crown amines . The crown ethers/amines may be optionally further substituted. The further substitution may allow covalent attachment of the polymer chains. Disclosed is a crown ether suitable for covalent attachment to a water soluble polymer, the crown ether having a reactive group X, wherein X is selected from NH₂, OH, CHO, epoxide, an activated carboxylate, NCO .

The crown ether/amine may be

where each X is independently selected from N or 0 and the ring is further substituted.

Disclosed is a method of covalently labelling a polymer using a labelling agent with an epoxide group. The labelling agent may be a crown ether or a crown amine. The term labelling agent refers to a moiety having an ion chelating agent and a reactive group for attachment to the polymer.

The crown ether/amine may be

Alternatively, the crown ether/amine may be

where each X is independently selected from N or 0, n is 1, 2 or 3, m is 1 to 12 and Y is a linker selected from CH₂-0-CH₂, CH₂-NH-CH₂ and CH₂COOCH₂.

The crown ether/amine may be

The ion chelating ligand may chelate iron, either as Fe or Fe . The chelating compound may be a siderophore. Siderophores are small, high-affinity iron chelating compounds secreted by microorganisms such as bacteria, fungi and grasses. They are amongst the strongest soluble Fe³⁺ binding agents known. Siderophores form a stable, hexadentate, octahedral complex preferentially with Fe³⁺ compared to other naturally occurring abundant metal ions, although if there are less than six donor atoms water can also coordinate. The most effective siderophores are those that have three bidentate ligands per molecule, forming a hexadentate complex and causing a smaller entropic change than that caused by chelating a single ferric ion with separate ligands. Fe³⁺ ion is a hard Lewis acid, preferring hard Lewis bases such as anionic or neutral oxygen to coordinate with. Microbes usually release the iron from the siderophore by reduction to Fe²⁺ which has little affinity to these ligands.

Included herein are methods of preparing a compound as described herein, the method comprising reacting a water soluble polymer with an ion chelating agent to form a covalent bond between them. Optionally the conjugation chemistry can be carried out in presence of water. Optionally the conjugation chemistry can be carried out in the absence of any solvent. Preferably the conjugation chemistry can be carried out without the addition of further catalysts or reagents such that the conjugates can be immediately used as therapeutics. The conjugation reaction can be carried out by freeze drying a mixture of the polymer and the reactive ligand and the heating the freeze dried material to initiate the covalent bond forming reactions .

The method can be carried out when either the polymer or the ion chelating agent contains a reactive group. The reactive group can be CHO, epoxide, an activated carboxylate, NCO. The activated carboxylate can be COC1 or an active ester. The active ester can be made using a carbodiimide reagent such as EDC.

Suitable compounds and methods of the invention are outlined below

Activating agent/

Reactive groups Reactive groups

Catalyst Final

on on ion binding

(removed after products polymer/monomer agent

reaction)

Cellulose-CH₂- Azomethine

Crown-CH₂-NH₂ TEMPO/NaC10/NaBr

OH-^CHO (Shiff base) Cellulose-COOH

COEDC

EDC/NHS,

Cellulose-COOH

Cro n-CH₂-OH SOCl₂, Ester

-►coci

Benzoyl Chloride.

Cellulose-COOH

COCI

Cellulose-COOH

COEDC

EDC/NHS,

Cellulose-COOH

Cro n-CH₂-NH₂ SOCl₂, Amide

-►coci

Benzoyl Chloride.

Cellulose-COOH

COCI

Cro n-CH₂-OH-^ Azomethine

Chitosan-NH₂ CHO TEMPO/NaC10/NaBr (Schiff base )

Cellulose-CH₂-

Crown-CH₂- OH Acid/Base Ether

Epoxide

PVA-OH

Crown-CH₂-

Chitosan-NH₂ Acid/Base Amine

Epoxide

Cro n-COOH COEDC

EDC/NHS,

Cro n-COOH -►

Chitosan-NH₂ SOCl₂, Amide

COCI

Benzoyl Chloride.

Crown-COOH COCI

Crown-COOH COEDC

Cellulose-CH₂- Crown-COOH -►

OH Ester

COCI

PVA-OH

Crown-COOH COCI

Cellulose-CH₂- OH Crown-NCO Urethanes

PVA-OH

Chitosan-NH₂ Crown-NCO Ureas

Cellulose-CH₂- OH Crown-CH₂-OH Citric Acid Bi-Ester

PVA-OH

CH₂=CH-COCl Crown-CH₂-OH Ester (as monomer for Crown-CH₂-NH₂ Amide following

acrylic

polymerization)

Functionalization of activated polymers with ionophores/siderophores or of polymers with activated ionophores/ siderophores can performed in either a homogeneous or heterogeneous phase. In a homogeneous phase during the linking process of polymer, the crown ether is distributed into the bulk of polymer. In a heterogeneous phase, after linking of the polymer, the crown ether is distributed only on the surface of polymer. A manufacture method for producing linked and functionalized polymer in homogeneous phase comprises the steps of:

1. Prepare a polymer solution in an appropriate solvent (solution A) ;

2. Prepare a linking agent solution in an appropriate solvent, typically a bi-functional or multifunctional linking agent in high concentration, or use pure linking agent (Solution B) ;

3. Prepare an optional activator or catalyst solution in a appropriate solvent (solution C) ;

4. Mixing the solutions in an appropriate order, depending of type of reaction;

5. Increase the temperature from 50 to 80 °C and stirring from hours to days to allow linking and functionalization reactions and to obtain functionalized polymer in hydrogel form.

6. Purifying the functionalized polymer to remove the excess of unreacted agents. The step of purifying the linked polymers may be by dialysis or washing with water or a buffer solution.

A manufacture method for producing linked and functionalized polymer in homogeneous phase comprises the steps of:

1. Preparing the following solutions:

a. a polymer solution in an appropriate solvent (Solution A) ;

b. a chelating agent solution in an appropriate solvent, typically a mono-functional, bi-functional or multifunctional chelating agent in high concentration, or use pure chelating agent (Solution B) ;

c. an optional activator or catalyst solution in an appropriate solvent (Solution C)

d. an optional cross-linking agent solution in an appropriate solvent, typically a bi-functional or multifunctional linking agent in high concentration, or use pure linking agent (Solution D) ;

Mixing the solutions in an appropriate order, depending on the type of reaction;

Controlling the temperature and stirring from hours to days to allow functionalization and linking reactions to obtain functionalized polymer.

Purifying the functionalized polymer to remove the excess of unreacted agents. The step of purifying the linked polymers may be by dialysis or washing with water or a buffer solution. of this method includes the following possibilities:

Mixing solutions A and B;

Mixing solutions A and B and C;

Mixing solutions A, B and D;

Mixing solutions A, B, C and D;

Mixing solutions A and B in a first step, and adding solution D to the product of the first step in a second step;

Mixing solutions A, B and C in a first step, and adding solution D to the product of the first step in a second step; Mixing solutions A and B in a first step, and adding solutions C and D to the product of the first step in a second step;

Mixing solutions A, B and C in a first step, and adding solutions C and D to the product of the first step in a second step;

Mixing solutions A and D in a first step, and adding solution B to the product of the first step in a second step;

Mixing solutions A, C and D in a first step, and adding solution B to the product of the first step in a second step; Mixing solutions A and D in a first step, and adding solutions B and C to the product of the first step in a second step; ii . Mixing solutions A, C and D in a first step, and adding solutions B and C to the product of the first step in a second step . Reactions in homogeneous phase have the advantage of being efficient, with high yields, and the products obtained are homogeneously linked.

Regarding the purification step, the above mentioned dialysis or washing cannot remove all of the residual linking agent, especially that which has a bi-functional bonding-state end and hence a free- state end which may not be linked to the polymer. Therefore, the polymer may contain residual reactive linking agents, which may result in undesired side effects in-vivo.

Additionally, the polymer needs few days to wash and purify the gel, which is under almost neutral conditions and may be polluted by microorganisms. It is important that the purifying step is done under sterile conditions to avoid the introduction of bacterial contamination.

Whilst the purification can be carried out by dialysis, the manufacture method for purifying linked polymer by dialysis or washing suffers from the following difficulties:

1. Refining is not easily scaled up in industry.

2. The polymer requires sterile conditions otherwise contaminants are easily integrated into a final product.

3. When the polymer is a hydrogel with low degree of cross- linking, it swells significantly, so is difficult to be washed and may be lost easily during washing. Similarly, when a cross-linked polysaccharide is hydrogel with high degree of cross-linking swells insignificantly, and the cross-linking agent is hard to be removed.

It is therefore advantageous if the method can be carried out without the presence of solution C. If the synthesis is carried out without the activator or catalyst, then the purification step can be eliminated, and the functionalized product can be used directly without purification.

A manufacture method for producing linked and functionalized polymer in a heterogeneous phase comprises the steps of:

1. Drying a polymer in order to obtain porous material with high surface area.

2. Immersing the obtained dry material in a solution of the functionalizing agent, typically an activated crown ether as previously shown.

3. Control the temperature and stirring from hours to days to allow the functionalization reaction and to obtain a surface functionalized polymer.

4. Optionally purifying the functionalized polymer to remove the excess of unreacted agent. The step of purifying the functionalized polymer may be by dialysis or washing with water or a buffer solution.

Step 1 could be performed either with a freeze-drying process, static or convection oven or spray dryer.

Functionalization reactions in heterogeneous phase have the advantage that only surface is functionalized, and other advantages such a facile purification of final polymer, but they have some disadvantages due to poor contact surface between polymer and functionalizing agent, for example: low efficiency, low yields, and the products obtained are heterogeneously functionalized.

An objective of the present disclosure is to provide a method for producing linked polymer in accordance with the present invention comprises linking one or more polymers at low temperature between 0 to 30 °C, for a reaction time between 1 to 48 hours under mild acidic or basic conditions with a linking agent to form a linked polymer. The polymer can be selected from the group consisting of chitosan, cellulose and cellulose derivatives, pullulans, alginate, agarose, mannan, glucomannan, acemannan, hyaluronate or derivatives or mixtures thereof. The polymer can be selected from the group consisting of polyvinyl alcohol (PVA) , polyacrylic acid, polyacrylate, polyacrylamide, polyallylamine or derivatives or mixtures thereof. The reaction can be carried out without any activation agents or catalysts. Preferably, the reaction is carried out at pH 6.0 to 8.0. More preferably, the reaction is carried out in unbuffered water.

The properties of the polymer can be optimised by the degree of cross linking in the polymer chains. The method of the invention is applicable to all polymers with suitable activation, and provides tunable mechanical, swelling degree, degradation stability of final product depending on the level of polymer cross linking.

The method of the invention provides a tunable substitution degree of metal chelating agent along the polymer chains and therefore tunable affinity towards ion of the final polymer therapeutic product. The chemical process provides high yields, high efficiency, high homogeneity, with a low use of linking agent, and without depolymerization of substrate during the linking reaction.

Described herein is a drug delivery product composition comprising a polymer and a covalently attached ion chelating agent for therapeutic use. Described herein is a drug delivery product composition comprising a water soluble polymer and a covalently attached ion chelating agent. The covalent attachment may be derived from the opening of an epoxide on the ion chelating agent, and may therefore comprise a hydroxyl group.

Compounds described herein can be used therapeutically, in particular to control physiological levels of electrolytes. Compounds may be used in the treatment of hyperkalemia, hypernatremia, hypercalcemia, hypermagnesia, hypersiderremia, hyperzincemia, hypercopperemia and hyperphosphatemia. The polymeric compounds may be administered orally. The compounds may be insoluble and indigestible such that the polymeric carriers are passed through the digestive system intact and are excreted once the ions have been chelated. In such a way, ions may be removed from the body. The compounds can be formulated as edible powders or administered as solutions .

Compounds described herein can be used in the manufacture of medicaments. The medicaments can be used to control physiological levels of electrolytes. Compounds may be used in the treatment of hyperkalemia, hypernatremia, hypercalcemia, hypermagnesia, hypersiderremia, hyperzincemia, hypercopperemia and hyperphosphatemia. The polymeric compounds may be administered orally. The compounds may be insoluble and indigestible such that the polymeric carriers are passed through the digestive system intact and are excreted once the ions have been chelated. In such a way, ions may be removed from the body. The compounds can be formulated as edible powders or administered as solutions.

While it is possible for the delivery conjugates to be administered alone it may be preferable to present them as pharmaceutical formulations. The formulations of the invention, both for veterinary and for human use, comprise at least one active ingredient, together with one or more acceptable carriers and optionally other therapeutic ingredients. The carrier (s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and physiologically innocuous to the recipient thereof.

The term "pharmaceutical formulation" in the context of this invention means a composition comprising an active agent and comprising additionally one or more pharmaceutically acceptable carriers. The composition may further contain ingredients selected from, for example, diluents, adjuvants, excipients, vehicles, preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents including liposomes or nanoparticulates , sweetening agents, flavouring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispersing agents, depending on the nature of the mode of administration and dosage forms. The compositions may take the form, for example, of tablets, dragees, powders, elixirs, syrups, liquid preparations including suspensions, sprays, inhalants, tablets, lozenges, emulsions, solutions, cachets, granules, capsules and suppositories, as well as liquid preparations for injections, including liposome preparations.

The formulations include but are not limited to those suitable for the administration routes described herein. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally are found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.), herein incorporated by reference in its entirety. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxymethylcellulose, stearic acid and the like. The pH of the formulations ranges from about 3 to about 11, but is ordinarily about 7 to 10. Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in- water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be administered as a bolus, electuary or paste. Tablets may optionally be coated or scored and optionally are formulated so as to provide slow or controlled release of the active ingredient. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable. These excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate; granulating and disintegrating agents, such as maize starch, or alginic acid; binding agents, such as cellulose, microcrystalline cellulose, starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.

Dispersible powders and granules of the invention suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, for example sweetening, flavouring and colouring agents, may also be present .

The pharmaceutical compositions of the invention may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned herein. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1 , 3-butane-diol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides . In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables.

Other objectives, advantages and novel features of the invention will become more apparent from the following detailed description when taken in conjunction with the accompanying examples, figures and tables.

Figure 1 FT-IR Spectra of compounds. Signals at 3062, 3004, 912, 856 and 785 cm^-1 in compound C indicates effective functionalization of compound A with compound B.

Figure 2 FT-IR Spectra of obtained compounds. Signal at 1750 cm ¹ in compound F indicates new ester bond according to reaction scheme 2. Presence of signals at 1456, 1351 and 1252 cm^-1 in compounds E and F indicates functionalization with compound C. Disappearing of signals at 912, 856 and 785 cm^-1 in compounds E and F indicates effective epoxy ring opening of compound C and subsequent functionalization of compounds D with compound C.

Figure 3 Indirect measurement of bounded cations by CMC-18C6. The figure shows that the polymeric conjugated material rapidly binds potassium ions, but have limited binding of sodium ions, and are thus suitable for therapeutic use to remove potassium ions from the gut .

Example 1

Synthesis of Glycidylether-2-Hydroxymethyl-18-crown-6 (Compound C)

2-Hydroxymethyl 18-crown-6 (Compound A), (441.0 mg, 1.5 mmol) was dissolved in 50 mL of anhydrous tetrahydrofuran and dried by azeotropic distillation of the solvent. The hydroxyl end group of 2- hydroxymethyl 18-crown-6 was then converted into sodium alkoxide by reaction with sodium hydride (48 mg, 2 mmol) at 30°C for 2 h. Epichlorohydrin (Compound B) , (185 mg, 2.0 mmole) was added to the solution and reacted at 40°C for 6 h. The glycidylether of 2- hydroxymethyl 18-crown-6 (Compound C) was precipitated in diethyl ether, filtered off, washed with diethyl ether, dried, and redissolved in dichloromethane (200 mL) . The dichloromethane solution was extracted twice with water, followed by drying over anhydrous magnesium sulfate, filtration, and elimination of dichloromethane .

2-Hydroxymethyl- 18-crown-6 Epichlorohydrin Glycidylether-2-Hydroxymethyl- 18- Compound A Compound B Compound C

Scheme reaction 1: Synthesis of Glycidylether-2-Hydroxymethyl-18- crown- 6 (Compound C) .

FT-IR Spectra of compounds is shown in Figure 1. Signals at 3062, 3004, 912, 856 and 785 cm-1 in compound C indicates effective functionalization of compound A with compound B.

Example 2

Synthesis of Glycidylether-2-Hydroxymethyl-18-crown-6 (Compound C) via ring closure of 2-Chlorohydrin methyl 18-crown-6

Step A) Synthesis of 2-Chlorohydrin methyl 18-crown-6 (Compound CI) . 2-Hydroxymethyl 18-crown-6 (Compound A) (441.0 mg, 1.5 mmole) is placed in a 3-necked flask equipped with a nitrogen inlet, a stirrer, a thermometer and a condenser. The Lewis acid catalyst, Boron trifluoride BF₃ or Lanthanum (III) trifluoromethanesulfonate (1.0 μτηοΐ), is added and the mixture is stirred, with warming, until the catalyst dissolves. The mixture is heated to 120°-130°C, and epichlorohydrin (Compound B) (185 mg, 2.0 mmole) is added, over about 1 hour, cooling when necessary. After a further 2 hours at 130°C, the epoxide value of the mixture is zero.

Compound CI

Step B) Ring Closure to produce Compound C

Ring closure was carried out by adding NaOH 50% aqueous solution (83.3 mg, 2.08 mmole), at 40°C over about 1 hour. After stirring for a further hour, the aqueous solution containing glycidylether-2- Hydroxymethyl 18-crown-6 (Compound C) and sodium chloride was heated to 60 °C, under vacuum, to remove the added water and unreacted epichlorohydrin .

1. For immediate use, compound C and NaCl were dissolved in water and added to polymer substrate for functionalization reaction. 2. For stocking, the dried compound C was then redissolved in dichloromethane (200 mL) and centrifugated and filtered to remove the sodium chloride. The dichloromethane solution was extracted twice with water, followed by drying over anhydrous magnesium sulfate, filtration, and elimination of dichloromethane.

Step C) Glycidylation of Residual Hydroxyl Groups Some of the material prepared in step B) (116 mg = 0,30 mmole-OH) is placed into a 3-necked round-bottomed flask, equipped for azeotropic distillation, having a reverse Dean & Stark water trap. Epichlorohydrin (98.1 g, 1.06 mole) is added, together with tetramethylammonium chloride catalyst (0.95 g as a 50% aqueous solution), and the mixture is heated to 55°-60° C, under vacuum, to remove the added water. Sodium hydroxide (15.5 g, 0.39 mole, as a 50% aqueous solution) is added, dropwise, over about 3 hours, with continuous separation of the formed and added water in the trap, maintaining the temperature at 55°-60° C. At the end of the addition process, the reaction is allowed to go to completion for 3.5 hours. The product is filtered and stripped at 100° C, under vacuum, to give a material with epoxide value of 6.91 mmol.g^-1 and a total chlorine content of 1.1%.

Example 3

Solvent free large-scale synthesis of Glycidylether-2-Hydroxymethyl- 18-crown-6 (Compound C)

A 5 liter 5-neck flask equipped with an inlet and outlet for inert gas (nitrogen) , was fitted with a reflux condenser, long stem thermometer dipping into the reaction flask, a 500 ml tap funnel, a sealed paddle stirrer and an inverted pattern Dean-Stark water- separator tube. The flask was charged with 2-Hydroxymethyl 18-crown- 6 (Compound A), (441.0 g, 1.5 mol) , dissolved directly in 1.2 L of epichlorohydrin (Compound B) , (1.39 kg, 15 mol) . The mixture was gently stirred at the temperature of 115° C and sparged with nitrogen gas. 60 mL of 50% aqueous solution sodium hydroxide (1.5 mol) was slowly added over about 16 hours, (about 2 drops per min) to avoid any sudden exothermic reaction. Water was periodically removed from the Dean-Stark tube. The reaction was left to run until no more water was being formed as a layer above the organic phase in the Dean-Stark tube. The reaction was left to cool and stand overnight under nitrogen stream. After cooling, the mixture was filtered through a sintered glass funnel to remove the solid sodium chloride and the filtrate containing compound C was collected. The filtrate was placed in a rotary evaporator and the excess epichlorohydrin was evaporated, re-collected and distilled for next re-uses, the yield of compound C was 95% in weight with an epoxy equivalent of 60 mol.g^-1.

Example 4

Carboxymethyl cellulose functionalization with glycidylether-2- hydroxymethyl-18-crown-6 (Compound E) with basic catalyst (ether linkage) .

Carboxymethyl cellulose (Compound D) was grafted with glycidylether- 2-hydroxymethyl 18-crown-6 (Compound C) obtained from example 1 or 2 with a basic catalyst by an ether linkage. Carboxymethyl cellulose food grade (2 g, 8.2 mmol of carboxylate moiety) was dissolved in 80 ml of 0.5 M of NaOH water solution at room temperature under moderate stirring for 6 h. Purified glycidylether-2-hydroxymethyl 18-crown-6 (2 g, 5.7 mmol) was dissolved in 20 ml of distilled water at room temperature under moderate stirring for 1 h. The glycidylether-2-hydroxymethyl 18-crown-6 aqueous solution was added dropwise to carboxymethyl cellulose aqueous solution, the mixture was kept at 30 °C and gently mixed for 1 hour, after mixing the mixture was kept in a controlled bath for 8 hours to complete reaction.

The mixture was washed with 0.5 M of HC1 solution to neutralize excess of NaOH, freeze dried for 24 h to obtain a porous material.

Example 5

Carboxymethyl cellulose functionalization with glycidylether-2- hydroxymethyl-18-crown-6 (Compound F) with acid catalyst (ester linkage) .

Carboxymethyl cellulose (Compound D) was grafted with glycidylether- 2-hydroxymethyl 18-crown-6 (Compound C) obtained from example 1 or 2 with a basic catalyst by an ether linkage. Carboxymethyl cellulose food grade (2 g, 8.2 mmol of carboxylate moiety) was dissolved in 80 ml of 0.5 M of HC1 water solution at room temperature under moderate stirring for 6 h. Purified glycidylether-2-hydroxymethyl 18-crown-6 (2 g, 5.7 mmol) was dissolved in 20 ml of distilled water at room temperature under moderate stirring for 1 h. The glycidylether-2- hydroxymethyl 18-crown-6 aqueous solution was added dropwise to carboxymethyl cellulose aqueous solution, the mixture was kept at 30 °C and gently mixed for 1 hour, after mixing the mixture was kept in a controlled bath for 8 hours to complete reaction.

The mixture was washed with 0.5 M of NaOH solution to neutralize excess of HC1, freeze dried for 24 h to obtain a porous material.

CarboxyMethyl Cellulose Sodiu

Compound D

Carb e

Scheme reaction 2: Carboxymethyl cellulose functionalization with glycidylether-2-hydroxymethyl-18-crown-6 in basic and acid environment .

Example 6

Carboxymethyl cellulose functionalization with glycidylether-2- hydroxymethyl-18-crown-6 (CMC-18C6) catalyst free (ester linkage) .

Compound F

Compound C attached to a carbohydrate polymer

Carboxymethyl cellulose (Compound D) was grafted with glycidylether- 2-hydroxymethyl 18-crown-6 (Compound C) obtained from example 1 or 2. Carboxymethyl cellulose food grade (2 g, 8.2 mmol of carboxylate moiety) was dissolved in 80 ml of distilled water at room temperature under moderate stirring for 6 h. Purified glycidylether- 2-hydroxymethyl 18-crown-6 (2 g, 5.7 mmol) was dissolved in 20 ml of distilled water at room temperature under moderate stirring for 1 h. The glycidylether-2-hydroxymethyl 18-crown-6 aqueous solution was added dropwise to carboxymethyl cellulose aqueous solution, and the mixture was heated to 50 °C and mixed for 1 hour.

The mixture was freeze dried for 24 h to obtain a porous material, and placed in an oven at 90 °C for 2 h to complete the grafting reaction with epoxy ring opening.

FT-IR Spectra of obtained compounds is shown in Figure 2. Signal at 1750 cm-1 in compound F indicates new ester bond according to reaction scheme 2. Presence of signals at 1456, 1351 and 1252 cm-1 in compounds E and F indicates functionalization with compound C. Disappearing of signals at 912, 856 and 785 cm-1 in compounds E and F indicates effective epoxy ring opening of compound C and subsequent functionalization of compounds D with compound C.

Example 7

Solvent free large-scale synthesis of Dilycidylether-2 , 11- Dihydroxymethyl-18-crown-6 (Compound G)

A 5 liter 5-neck flask equipped with an inlet and outlet for inert gas (nitrogen) , was fitted with a reflux condenser, long stem thermometer dipping into the reaction flask, a 500 ml tap funnel, a sealed paddle stirrer and an inverted pattern Dean-Stark water- separator tube. The flask was charged with 2 , 11-Dihydroxymethyl 18- crown-6, (486.4 g, 1.5 mol), dissolved directly in 1.2 L of epichlorohydrin (Compound B) , (1.39 kg, 15 mol) . The mixture was gentle stirred at the temperature of 115° C and sparged with nitrogen gas. 120 mL of 50% aqueous solution sodium hydroxide (1.5 mol) was slow added in about 16 hours, (about 2 drops per min) to avoid any sudden exothermic reaction. Water was periodically removed from the Dean-Stark tube. The reaction runs until no more water was forming as a layer above the organic phase in the Dean-Stark tube. The reaction was left to cool and stand overnight under nitrogen stream. After cooled, the mixture was filtered through sintered glass funnel to remove the solid sodium chloride and the filtrate containing compound C was collected. The filtrate was placed in a rotary evaporator and the excess epichlorohydrin was evaporated recollected and distilled for next re-uses, the yield of compound G was about 90% in weight with an epoxy equivalent of 110 mol g^-1.

2,11-Dihydroxymethyl-18-crown-6 Epichlorohydrin Digly cidylether-2,11-Dihydroxymethyl-18- Compound B Compound G Example 8

Carboxymethyl cellulose functionalized and crosslinked with

diglycidylether-2 , ll-dihydroxymethyl-18-crown-6 (Compound H) with basic catalyst (ether linkage) .

Carboxymethyl cellulose was crosslinked with diglycidylether-2, 11- dihydroxymethyl 18-crown-6 obtained in the same way as described in example 1 or 2 starting by 2 , 11-dihydroxymethyl 18-crown-6, with basic catalyst by an ether linkage. Carboxymethyl cellulose food grade (2 g, 8.2 mmol of carboxylate moiety) was dissolved in 80 ml of 0.5 M of NaOH water solution at room temperature under moderate stirring for 6 h. Purified diglycidylether-2, 11-dihydroxymethyl 18- crown-6 (2 g, 4.6 mmol) was dissolved in 20 ml of distilled water at room temperature under moderate stirring for 1 h. The

diglycidylether, 11-dihydroxymethyl 18-crown-6 aqueous solution was added dropwise to carboxymethyl cellulose aqueous solution, the mixture was kept at 30 °C and gently mixed for 1 hours, after mixing the mixture was kept in a controlled bath for 8 hours to complete reaction .

Compound H

Example 9

Carboxymethyl cellulose crosslinked with BDDE (butandiol diglycidylether) with basic catalyst and subsequently functionalized with glycidylether-2-hydroxymethyl-18-crown-6 (Compound I) .

Carboxymethyl cellulose (Compound D) was firstly crosslinked with BDDE, with basic catalyst by an ether linkage. Carboxymethyl cellulose food grade (2 g, 8.2 mmol of carboxylate moiety) was dissolved in 80 ml of 0.5 M of NaOH water solution at room temperature under moderate stirring for 6 h. Purified BDDE (1 g, 4.9 mmol) was dissolved in 20 ml of distilled water at room temperature under moderate stirring for 1 h. The BDDE aqueous solution was added dropwise to carboxymethyl cellulose aqueous solution, the mixture was kept at 30°C and gently mixed for 1 hour, after mixing the mixture was kept in a controlled bath for 8 hours to complete reaction .

BDDE crosslinked carboxymethyl cellulose was subsequently grafted with glycidylether-2-hydroxymethyl 18-crown-6 (Compound C) obtained from example 1 or 2 with basic catalyst by an ether linkage. Purified glycidylether-2-hydroxymethyl 18-crown-6 (2 g, 5.7 mmol) was dissolved in 20 ml of distilled water at room temperature under moderate stirring for 1 hour. The glycidylether-2-hydroxymethyl 18- crown-6 aqueous solution was added dropwise to BDDE crosslinked carboxymethyl cellulose hydrogel, the mixture was kept at 30 °C and gently mixed for 1 hour, after mixing the mixture was kept in a controlled bath for 8 hours to complete reaction.

Example 10

Carboxymethyl cellulose functionalization with glycidylether-2- hydroxymethyl-18-crown-6 with basic catalyst (ether linkage) and subsequently crosslinked with BDDE (Compound I) .

Carboxymethyl cellulose (Compound D) was firstly grafted with glycidylether-2-hydroxymethyl 18-crown-6 (Compound C) obtained from example 1 or 2 with basic catalyst by an ether linkage. Carboxymethyl cellulose food grade (2 g, 8.2 mmol of carboxylate moiety) was dissolved in 80 ml of 0.5 M of NaOH water solution at room temperature under moderate stirring for 6 hours. Purified glycidylether-2-hydroxymethyl 18-crown-6 (2 g, 5.7 mmol) was dissolved in 20 ml of distilled water at room temperature under moderate stirring for 1 h. The glycidylether-2-hydroxymethyl 18- crown-6 aqueous solution was added dropwise to carboxymethyl cellulose aqueous solution, the mixture was kept at 30 °C and gently mixed for 1 hour, after mixing the mixture was kept in a controlled bath for 8 hours to complete reaction. Purified BDDE (1 g, 4 . 9 mmol) was dissolved in 2 0 ml of distilled water at room temperature under moderate stirring for 1 h. The BDDE aqueous solution was added dropwise to functionalized carboxymethyl cellulose (compound E) aqueous solution. The mixture was kept at 30 °C and gently mixed for 1 hour, after mixing the mixture was kept in a controlled bath for 8 hours to complete reaction.

The mixture was washed with 0 . 5 M of HC1 solution to neutralize excess of NaOH, freeze dried for 2 4 h to obtain a porous material.

Compound I

In-Vitro Assays

The carboxymethyl cellulose functionalized with glycidylether- 2 hydroxymethyl-18-crown-6 in form of porous material and in th compositions of the invention are characterized by various features such as the capability of binding a particular cation (e.g. potassium ion over sodium ion) , selectivity, and persistence o binding cation over the time. These features of th materials/compositions can be determined using in-vitro assay protocols that mimic or are representative of inorganic ion concentrations and may include components which model other species (than inorganic ions) which are commonly found in the gastrointestinal tract, and especially of the lower intestine and/or of the colon.

A first assay, referred here as GI Assay No.l, is a competitive assay involving potassium ions and sodium ions at equal molar concentrations selected to be generally typical and representative of the concentrations seen in various regions of the intestinal tract. This first assay consists essentially of incubating the porous material at a concentration of 2 g/1 (1 g of material have a theoretical bound capacity of 1,7 meq of potassium) in an assay solution. The assay solution consists of 55 meq KC1, 55 mM NaCl and a buffer, 50 mM 2-morpholinoethanesulfonic acid monohydrate, at a pH of 6.5 and a temperature of 37°C. The composition was incubated for 24 h with agitation. The cations bound to the material were measured, directly and indirectly over time. Direct measurement of bound cations was performed by recovering the material and analyzing the ion content thereof, for example, by releasing bound cations by hydrolizing material with hot acid or base, and measuring the released cations. Indirect measurement of bound cations was performed by determining the change in ion concentration of the assay solution. Measurements of cation concentration was performed using ion chromatography or ICP-MS techniques .

Figure 3 shows that the polymeric conjugated material rapidly binds potassium ions, but have limited binding of sodium ions, and are thus suitable for therapeutic use to remove potassium ions from the gut .

Claims

Claims :

1. An ion chelating ligand having at least one epoxy group.

2. The ion chelating ligand of claim 1 wherein the ion chelating ligands selectively binds ions by complexation, chelation, ion exchange, chemical trapping, physical trapping, electrostatic interaction or a combination thereof.

3. The ion chelating ligand of claim 1 or claim 2 wherein the ion chelating ligands are linear or cyclic compounds.

4. The ion chelating ligand of claims 1, 2 or 3 wherein the ion chelating ligands is a macrocyclic compound of 12 atoms or greater .

5. The ion chelating ligand of any one previous claim wherein the ions are metal cations selected from potassium K⁺, sodium Na⁺, calcium Ca²⁺, magnesium Mg²⁺, zinc Zn²⁺, silver Ag⁺, lead Pb²⁺, mercury Hg²⁺, iron Fe²⁺ and Fe³⁺, copper Cu⁺ and Cu²⁺.

6. The ion chelating ligand of claim 5 wherein the ion chelating ligands are crown ethers, calixarenes, carboxyl derivatives or a combination thereof useful for potassium removal.

7. The ligand of claim 6 which is 18-crown-6 having at least one epoxy group.

8. The ligand of claim 6 which is

9. The ion chelating ligand of claim 5 wherein the ion chelating ligands are Ferrichrome, Enterobactin, Coprogen, Deferoxamine, carboxyl derivatives or a combination thereof useful for iron removal .

The ion chelating ligand of any one of claims 1, 2 or 3 wherein the ions are inorganic phosphates including monophosphate (P0₄ ^3~) , diphosphate (P₂0₇ ^4~) , or triphosphate (P₃0io^5~) and phosphate containing metabolites, in particular adenosine monophosphate (AMP) , adenosine diphosphate (ADP) and adenosine triphosphate (ATP) .

The ion chelating ligand of claim 10 wherein the ion chelating ligands are amines, porphyrins, sapphyrins or expanded porphyrins, azacrown ethers or a combination thereof useful for phosphate removal.

The ion chelating ligand of any previous claims wherein the epoxy group is used to covalently attach a polymer.

A method of attaching a polymer to an ion chelating ligand by reacting an ion chelating ligand having an epoxy group with a polymer having a hydroxy, carboxy or amino group.

The method according to claim 13 wherein the reaction is carried out at pH 6.0 to 8.0.

The method according to claim 14 wherein the method is carried out in unbuffered water.

The polymer conjugate produced by the method of claim 13.

The method of claims 13 to 15 or the polymer conjugate of claim 16 wherein the polymer is a polysaccharide.

The method or polymer conjugate of claim 17 wherein the polysaccharide is selected from chitosan, cellulose, alginates, mannans, glucomannans , acemannans, hyaluronates , or derivatives thereof.

The method of claims 13 to 15 or the polymer conjugate of claim 16 wherein the polymer is selected from polyvinyl alcohol (PVA) , polyacrylic acid, polyacrylate, polyacrylamide, polyallylamine, polyethylene glycol (PEG) or derivatives thereof .

The polymer conjugate according to claim 17 wherein the ion chelating ligand is attached via an ether, ester or amine linkage . The polymer conjugate according to any one of claims 14-20 wherein the functionalized polymer with ion chelating ligands has a degree of substitution from about 1% to about 100% , more specifically from about 10% to about 90%, more specifically from about 50% to about 70%, more specifically from about 70% to about 90%.

A composition according to any one of claims 16-21 comprising a polymer and covalently attached ion chelating ligands for therapeutic use.

A composition according to claim 22 comprising a polymer and covalently attached ion chelating ligands in the form of a porous powder.

A method to control physiological levels of electrolytes in a subject in need thereof, the method comprising administering a composition according to claim 22.

The method according to claim 24 wherein the administration is by injection.

The method according to claim 25 wherein the administration is oral .

A composition according to any one of claims 16-23 for use in the treatment of hyperkalemia, hypernatremia, hypercalcemia, hypermagnesia, hypersiderremia, hyperzincemia hypercopperemia and hyperphosphatemia.