Cross-linked polymers and their use in methods of binding cations

Abstract

Disclosed are methods of binding cations using cross-linked polymers, cross-linked polymers suitable for binding cations and compositions and devices comprising the same, methods of producing cross-linked polymers, and methods of treating a pathology associated with an aberrant presence of cations, such as Wilson's disease, which is characterized by an aberrant presence of copper ions.

Description

[0001] CROSS-LINKED POLYMERS AND THEIR USE IN METHODS OF BINDING CATIONS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to methods of binding cations and of treating pathologies associated with an aberrant presence of cations. In particular, the invention relates to crosslinked polymers suitable for binding cations, to methods of producing such polymers, and to methods of binding cations, such as copper ions, using cross-linked polymers. Furthermore, the invention relates to methods of treating diseases associated with an aberrant presence of cations, such as Wilson's disease, which is characterized by an aberrant presence of copper ions.

[0004] BACKGROUND OF THE INVENTION

[0005] Wilson's disease is a rare disease with ~30,000 patients in the western world. Patients with Wilson's disease cannot remove extra copper from their bodies, and the copper accumulates in their organs, causing severe damage. Untreated, the average life expectancy is app. 40 years. The current treatment of Wilson's disease is a medicament comprising a copper- binder, such as Trientine, which binds copper in a complex and is excreted via the kidneys, as well as a low-copper diet. Trientine works systemically and can have serious side-effects which are aggravated by the fact that the treatment is life-long. The same is true for the low- copper diet.

[0006] The present inventors have previously developed Molecular Imprinted Polymers (MIPs) for treatment of another rare disease in which a component of a normal diet is toxic for the patient, namely phenylketonuria (PKU). In a patient with PKU, phenylalanine (L-Phe) from the diet accumulates in the blood and brain, which can lead to irreversible brain damage in children and young adults and to classical symptoms of poisoning in older adults. The majority of PKU patients currently does not have an effective medical treatment available. Instead, these patients have to eat a very restrictive and problematic diet for their whole lives. There are two marketed drugs against PKU, but these are only effective for ~25% of the patients. The MIP product developed by the present inventors is a cross-linked polymer comprising methacrylic acid (MAA) and l,4-bis(acryloyl)piperazine (DAP). The MIP is polymerized in the presence of a template molecule, which gives the MIP its molecular imprinting. For example, polymerization in the presence of L-Phe results in a MIP which can bind with high affinity to L-Phe, and when administered orally to a PKU patient such a MIP can then bind to the L-Phe present in the gastro-intestinal (GI) tract of the patient, which was ingested via the food, and the MIP-Phe complexes are then excreted via the faeces, thereby removing the L-Phe from the GI tract of the patient before it reaches the patient's blood and becomes toxic (see, e.g., WO 2019 / 002535).

[0007] SUMMARY OF THE INVENTION

[0008] While working on developing their MIP drug against PKU, it was surprisingly discovered by the present inventors that the cross-linked polymer constituting their MIP binds very well to copper ions.

[0009] As described in Example 1, the inventors labelled their Phe-imprinted MIP (called Phelimin) with the radioactive copper isotope54Cu in order to measure its distribution in different organs of experimental animals after oral administration by PET scanning. As expected, they found that Phelimin passed through the GI tract without entering the blood and becoming systemic, with time ending up in the faeces (Example 1, Figure 2). However, the stability with which the54Cu2+bound to the MIP was unexpected, and it remained bound for at least 18 hours. The stability of the MIP-Cu2+complex was later confirmed in vitro, as it was shown that the copper ions remained bound to the MIP, even in the presence of relevant competitors (Example 1, Figure 3).

[0010] The inventors realized that this discovery enables the use of such cross-linked polymers in methods of binding cations, including copper ions, in vitro and in vivo. Importantly, it enables the in vivo binding of copper ions and thus can be used to treat Wilson's disease.

[0011] The inventors therefore made further investigations (as described in Example 2). They demonstrated that polymers comprising DAP and MAA in different ratios and polymers that had been produced under different pH conditions also bound to copper ions (Example 2, Figure 4A and 4B), and for one of these polymers they showed that the binding capacity for Cu2+was so high that it is realistic that the polymer can bind sufficient amounts of dietary copper that is part of a daily intake (Example 2, Figure 5). Furthermore, for one of the polymers they showed that a relatively low concentration of the polymer binds so much copper that the dose to be given to a Wilson's patient is in a realistic and comfortable range, and that it does not matter if the polymer is in suspension or in dry form (Example 2, Figure 6). The inventors also showed for one of the polymers that it binds to Cu2+at the pH of the intestines, where copper gets absorbed (Example 2, Figure 7), and for a different polymer they showed that the binding to Cu2+is not disturbed by the presence of a large excess of Zn2+or Ca2+(Example 2, Figure 8), indicating that the polymer will bind sufficiently to Cu2+in the presence of potential competitors in the intestine. Interestingly, the inventors tested a range of other polymers, including polymers comprising DAP and (vinylbenzyl)trimethylammonium chloride (VBTA) but no MAA, and these polymers bound to Cu2+as well (Example 2, Figure 9).

[0012] As such, the inventors have shown that a range of cross-linked polymers bind to Cu2+, and they have provided convincing data suggesting that these cross-linked polymers could be well-suited for use in treating Wilson's disease.

[0013] Furthermore, as described above, for Phelimin it has already been shown that it is confined to the GI tract and thus is not present systemically after oral administration. In contrast, Trientine, a current drug for Wilson's disease, is present systemically and thus affects all organs. One of Trientine's side-effects is nausea, which is of course problematic for patients who have to take a drug every day for their whole lives. It is expected that a drug based on a cross-linked polymer as described herein, which is non-systemic, is made of an inert, synthetic material, and which does not get degraded on its way through the Gl-tract (which has also been demonstrated for the Phe-binding MIP Phelimin) could be a superior drug to those already available for treatment of Wilson's disease, especially due to the expectation that its non-systemic nature causes insignificant side-effects.

[0014] So, in a first aspect the present invention relates to a method of binding cations, the method comprising a step of: contacting a composition comprising cations with a cross-linked polymer comprising :

[0015] - a first monomer comprising at least one vinyl group and i. at least one carboxyl group or II. at least one benzyl group, and

[0016] - a second monomer which is l,4-bis(acryloyl)piperazine (DAP), thereby binding cations of the composition.

[0017] In a second aspect, the invention relates to a cross-linked polymer comprising :

[0018] - a first monomer selected from: (vinylbenzyl)trimethylammonium chloride (VBTA), acrylic acid, itaconic acid, and 3-Butene-l,2,3-tricarboxylic acid, preferably VBTA, and

[0019] - a second monomer which is l,4-bis(acryloyl)piperazine (DAP).

[0020] In a third aspect, the invention relates to a composition comprising the cross-linked polymer as defined in the first aspect of the invention or embodiments thereof or according to the second aspect of the invention.

[0021] In a fourth aspect, the invention relates to a method for producing a cross-linked polymer, the method comprising a step of: polymerizing a mixture comprising

[0022] - a first monomer comprising at least one vinyl group and i. at least one carboxylic acid or II. at least one benzyl group, and

[0023] - a second monomer which is l,4-bis(acryloyl)piperazine (DAP), and optionally

[0024] - a template molecule, in the presence of a catalyst, so as to obtain the cross-linked polymer, which is optionally attached to, enclosing, and / or covalently bound to, the template molecule.

[0025] In a fifth aspect, the invention relates to a method of treating a pathology associated with an aberrant presence of cations in a subject in need thereof, the method comprising administering to the subject the cross-linked polymer as defined in the first aspect of the invention or embodiments thereof or according to the second aspect of the invention or embodiments thereof or the composition according to the third aspect of the invention or embodiments thereof.

[0026] In a 6thaspect, the invention relates to the cross-linked polymer according to the second aspect of the invention or embodiments thereof or the composition according to the third aspect of the invention or embodiments thereof, insofar as it relates to the cross-linked polymer according to the second aspect of the invention, for use as a medicament.

[0027] In a 7thaspect, the invention relates to the cross-linked polymer as defined in the first aspect of the invention or embodiments thereof or according to the second aspect of the invention or embodiments thereof or the composition according to the third aspect of the invention or embodiments thereof, for use in a method of treating a pathology associated with an aberrant presence of cations.

[0028] In an 8thaspect, the invention relates to a method of treating a pathology associated with an aberrant presence of cations in a subject in need thereof, the method comprising contacting a bodily fluid, such as blood, derived from said subject with the cross-linked polymer as defined in the first aspect of the invention or embodiments thereof or according to the second aspect of the invention or embodiments thereof ex vivo, and returning the resulting cation-reduced bodily fluid to said subject.

[0029] In a 9thaspect, the invention relates to the cross-linked polymer as defined in the first aspect of the invention or embodiments thereof or according to the second aspect of the invention or embodiments thereof, for use in a method of treating a pathology associated with an aberrant presence of cations in a subject in need thereof, the method comprising contacting a bodily fluid, such as blood, derived from said subject with the cross-linked polymer ex vivo, and returning the resulting cation-reduced bodily fluid to said subject.

[0030] Finally, in a 10thaspect, the invention relates to a device suitable for binding cations of a composition, said device comprising a solid phase having the cross-linked polymer as defined in the first aspect of the invention or embodiments or according to the second aspect of the invention or embodiments thereof anchored to it.

[0031] Further aspects of the invention

[0032] It is apparent that cross-linked polymers other than the ones described in the lst-10thaspects of the invention may share properties with those polymers which make them just as good binders of cations, including copper ions. Such a cross-linked polymer may, e.g., be a polymer comprising MAA and a nitrogen-containing monomer, such as nitrogen-containing monomer comprising at least 2 vinyl groups, more generally.

[0033] Thus, in an 11thaspect, the invention relates to a method of binding cations, the method comprising a step of: contacting a composition comprising cations with a cross-linked polymer comprising :

[0034] - a first monomer comprising at least one vinyl group and i. at least one carboxyl group or

[0035] II. at least one benzyl group, and

[0036] - a second monomer which is a nitrogen-containing monomer, thereby binding cations of the composition.

[0037] In some embodiments, the second monomer comprises at least 2 vinyl groups.

[0038] In some embodiments, the first monomer is methacrylic acid (MAA).

[0039] In a 12thaspect, the invention relates to a method of treating a pathology associated with an aberrant presence of cations in a subject in need thereof, the method comprising administering to the subject a cross-linked polymer as described in the 11thaspect of the invention.

[0040] In a 13thaspect, the invention relates to a method of treating a pathology associated with an aberrant presence of cations in a subject in need thereof, the method comprising contacting a bodily fluid, such as blood, derived from said subject with a cross-linked polymer as described in the 11thaspect of the invention ex vivo, and returning the resulting cation- reduced bodily fluid to said subject. In a 14thaspect, the invention relates to a device suitable for binding cations of a composition, said device comprising a solid phase having the cross-linked polymer as described in the 11thaspect of the invention anchored to it.

[0041] A cross-linked polymer binding to cations may be useful in other ways than in isolating / removing cations. For example, a cross-linked polymer which is easily labelled with cations may be used for diagnostic or therapeutic purposes, e.g., if the cross-linked polymer is targeted against a specific target and the cation has useful properties in terms of its detectability and / or therapeutic effect. Suitable cations include those that are detectable by magnetic resonance imaging (MRI) and radioactive cations detectable by nuclear imaging techniques such as, e.g., positron emission tomography (PET). These include, without limitation,54Cu. Cations suitable for therapeutic purposes include radioactive cations that can deliver a therapeutic dose of radiation to cancer cells when complexed with a cross-linked polymer binding a target molecule associated with the cancer cells.

[0042] Thus, in a 15thaspect, the invention relates to a complex comprising : a) a cross-linked polymer comprising :

[0043] - a first monomer comprising at least one vinyl group and i. at least one carboxyl group or II. at least one benzyl group, and

[0044] - a second monomer which is a nitrogen-containing monomer, and b) a cation, optionally for use as a medicament or imaging agent.

[0045] In some embodiments, the second monomer comprises at least 2 vinyl groups.

[0046] In some embodiments, the first monomer is methacrylic acid (MAA).

[0047] In a 16thaspect, the invention relates to a complex comprising : a) a cross-linked polymer comprising :

[0048] - a first monomer comprising at least one vinyl group and i. at least one carboxyl group or II. at least one benzyl group, and

[0049] - a second monomer which is l,4-bis(acryloyl)piperazine (DAP), and b) a cation, optionally for use as a medicament or imaging agent.

[0050] In some embodiments, the first monomer is methacrylic acid (MAA).

[0051] In some embodiments of the 15thand 16thaspects, the cation is selected from a cation of any one of the following isotopes:43Sc,44Sc,47Sc,51Cr,52mMn,55Co,57Co,58Co,52Fe,55Ni,57Ni, preferably54Cu.

[0052] In some embodiments of the 15thand 16thaspects, the cation is an aluminium fluoride ion, such as AIF2+, optionally [18F]-AIF2+.

[0053] LEGENDS TO THE FIGURES

[0054] Fig. 1 : Recovery of54Cu (tracer) in various organs and faeces at various time points after dosing of mice with free54CuCI2by oral gavage. The dissected organs, blood, urine or faeces were placed in tubes and counted in a HIDEX gamma counter. The counted values were corrected back to the starting point using the half-life value of54Cu (Tl / 2 = 12.7 h) and corrected for the volume-fraction that was counted in the HIDEX. Finally, the fractions for the individual organ of the value of the dosed value were calculated in %.

[0055] Fig. 2 : Recovery of54Cu (tracer) in various organs and faeces at various time points after dosing of mice with54Cu complexed to Phelimin (54Cu-Phelimin) by oral gavage. The dissected organs, blood, urine or faeces were placed in tubes and counted in a HIDEX gamma counter. The counted values were corrected back to the starting point using the half-life value of54Cu (Tl / 2 = 12.7 h) and corrected for the volume-fraction that was counted in the HIDEX. Finally, the fractions for the individual organ of the value of the dosed value were calculated in %. The missing counts in the faeces from the 9 hours mouse can be explained by low degree of peristaltic movements in that individual mouse. Approx. 85% of the54Cu activity of the 9 hours mouse is found in the caecum and colon. The low recovery of54Cu activity in the 3 hours mouse, approx. 50%, is explained by the fact that, by mistake, no faeces was collected for that mouse.

[0056] Fig. 3 :54Cu2+-Phelimin complex stability at low pH and in the presence of various competitors. lOx concentrated simulated gastro-intestinal fluid (SGF, pH 3.5) or acetate buffer (pH 5.0) was added to54Cu2+-Phelimin complex samples, and after 15 min the samples were centrifuged and a sample of the supernatant was analysed in a HIDEX gamma counter. The counts in the supernatant represent the54Cu not bound to the Phelimin, and from that the percentage of54Cu that remained bound to the Phelimin was calculated. Fig. 4: Binding of the three different polymer batches S231, S242 and S248 (see Table 1) to copper ions. A) Percentage of copper ions bound to each polymer at three different Cu2+concentrations as indicated. B) Amount of copper ions bound to each polymer at three different Cu2+concentrations as indicated, shown as pg Cu2+ / g polymer.

[0057] Fig. 5: Amount of copper ions bound to the polymer batch S242 (see Table 1) at six different Cu2+concentrations as indicated.

[0058] Fig. 6: Percentage of copper ions bound to the polymer batch S242 (see Table 1) at six different polymer concentrations as indicated. Black spheres indicate binding of the polymer in dried form, whereas light grey spheres indicate binding of the polymer in suspension.

[0059] Fig. 7: Percentage of copper ions bound to the polymer batch S242 (see Table 1) at varying pH as indicated.

[0060] Fig. 8: Percentage of copper ions bound to the polymer batch S248 (see Table 1) in the presence of a 50x (0.1 M) or lOOx (0.2 M) excess of Ca2+or Zn2+, as indicated.

[0061] Fig. 9: Binding of the five different polymer batches S238, S245, S249, S250 and S254 (see Table 1) to copper ions. The graph shows the percentage of copper ions bound to each polymer at three different Cu2+concentrations as indicated.

[0062] DETAILED DISCLOSURE OF THE INVENTION

[0063] Definitions

[0064] A "cation", as used herein, has its normal meaning in the art, i.e. a positively charged ion. The term includes atoms and molecules which have a net positive charge due to having more protons than electrons. In preferred embodiments as described below, the cation is a metal ion, more preferably a copper ion.

[0065] A "cross-linked polymer", "polymer" or "cross-linked polymer particle" ("particle" in short), as used herein, is the product of polymerization of at least two different monomers, optionally in the presence of a template molecule, as well as the product of any subsequent production steps, such as fragmentation and any other steps described herein, performed on the polymerization product. Such particles may be molecular imprinted polymers (MIPs; defined below) or non-imprinted polymers (NIPs; defined below). A "molecular imprinted polymer" (MIP) is a cross-linked polymer as described above comprising cavities (or voids) that at least in part correspond to one or more template molecules that have been incorporated in a monomer matrix including cross-linking monomers prior to polymerization. The resulting polymer after polymerization includes a number of cavities which correspond in shape to the template molecule. Typically, the MIP is comminuted (fragmented, micronized) into small particles, thereby facilitating removal of template and leaving partial cavities open for interaction with a target molecule which resembles or is identical to the template molecule.

[0066] A "non-imprinted polymer" (NIP) is a cross-linked polymer as described above which, in contrast to a MIP, does not comprise cavities (or voids) corresponding to one or more template molecules, since the polymerization of the NIP has occurred in the absence of a template molecule.

[0067] It will be understood that the cross-linked polymer particles employed in the present invention are insoluble molecules / entities even though they may appear stable in suspension if sufficiently small. The particles are especially suitable as pharmaceutical for use in the gastrointestinal tract since their insolubility limits or prevents their passage into the body (e.g. into circulation) from the gastrointestinal tract. In other words, when administered orally, the particles used in the present invention will substantially remain confined to the gastrointestinal tract until they are disposed off in the faeces.

[0068] A "monomer", as used herein, has its normal meaning in the art, i.e. a molecule which can react with other, identical and / or different, molecules to form a polymer comprising many, identical and / or different, chemically connected monomers.

[0069] "Micronization" or "fragmentation" denotes the process of sequestering cross-linked polymer particles into smaller particles. Any method suitable for this purpose may be used.

[0070] When discussing sizes of cross-linked polymer particles herein, e.g. particles being smaller or larger than a given length X (e.g. 63 pm), is meant that the size of the particles are such that they are capable or incapable of passing through sieves having a defined cut-off, i.e. diameter of the holes in the sieve X - the particles that pass through are "smaller than" X, and the particles that are retained are "larger than" X. In other words, a molecule may in theory be larger than 63 pm along an axis, but still capable of passing through a 63 pm sieve: in such a case, the molecule is said to be smaller than 63 pm.

[0071] A "target molecule" of a MIP is used herein to describe any molecule or molecular motif which specifically binds to the MIP in one or more of its imprinted binding cavities, i.e. typically (but not always) the molecule, or a derivative of the molecule, which was used to imprint the MIP. The cations, which are disclosed herein to bind stably to MIPs, may thus be considered target molecules for a MIP which has been imprinted with the cations, or with similar molecules, and thus bind specifically in binding cavities of the MIP, whereas cations that bind to the MIP but not in its binding cavities, and which MIP may have been imprinted with a totally unrelated molecule, are not described as "target molecules" herein.

[0072] A "template molecule" is normally identical to the target molecule, but may also be a mimic thereof ( / .e. a molecule having at least in part an identical 3D structure and profile which matches that of the target molecule - a mimic may for instance be constituted by a fragment of the target molecule or by a larger molecule of which the intended target is an important part). The template serves as the "generator" of the voids in the MIP structure which subsequently are to be able to bind the target molecule.

[0073] As described, the cross-linked polymer may be "attached to, enclosing, and / or covalently bound to" a template molecule. This should be understood as a relatively strong interaction between the polymer and the template molecule, being it that they are directly covalently bound to each other or that the template is simply "trapped" (or "enclosed") within the binding cavity of the polymer.

[0074] A "phenylalanine-derivative where a phenylalanine motif is exposed" denotes a template molecule comprising a benzyl group, a phenyl ring or the benzyl group or phenyl ring combined with either a carboxylic group or an amino group. Therefore, any peptide containing phenylalanine (preferably di- or tripeptides) can be used as such a template molecule.

[0075] A "pathology", as used herein, also called a medical condition, is a condition which involves signs and / or symptoms. It may be a temporary condition, such as an intoxication, or it may be a disease, such as a chronic disease.

[0076] A "solid phase" is in the present context any material which may be used to anchor a capture agent, such as a cross-linked polymer or a MIP, by means of covalent or non-covalent binding. Hence, any material (plastic polymers, sugars, metals, glass, silica, rubber, etc.) which is conventionally used in the preparation of chromatographic materials may serve as the solid phase. The solid phase material may contain suitable functional groups which allow coupling of the capture agent to the material in question. Such derivatized materials are known to the person of skill in the art of chromatographic purification of proteins and other macromolecules. Hence, the solid phase may be in the form of fibers (preferably hollow), a chromatography matrix, beads (preferably those that may be separated by electromagnetic means) or any other suitable form.

[0077] Specific embodiments of the invention

[0078] 1staspect of the invention and embodiments thereof

[0079] The first aspect of the present invention relates to a method of binding cations, the method comprising a step of: contacting a composition comprising cations with a cross-linked polymer comprising :

[0080] - a first monomer comprising at least one vinyl group and i. at least one carboxyl group or

[0081] II. at least one benzyl group, and

[0082] - a second monomer which is l,4-bis(acryloyl)piperazine (DAP), thereby binding cations of the composition.

[0083] In some embodiments, the binding is performed in vitro or ex vivo.

[0084] In some embodiments, the binding is performed in vivo.

[0085] Examples of embodiments where the binding is performed in vitro may be isolation and / or extraction of cations from a composition. In such embodiments, the cross-linked polymer may be comprised within a purification column.

[0086] In some embodiments, the cations are divalent cations. In further embodiments, the divalent cations are selected from any one or more of: Cu2+, Ca2+, Mg2+, Fe2+, Mn2+, Ni2+, Pb2+, and Zn2+.

[0087] In some embodiments, the cations are metal ions. In further embodiments, the metal ions are selected from any one or more of: Cu2+, Ca2+, Mg2+, Fe2+, Mn2+, Ni2+, Pb2+, Zn2+, Li+, Na+, K+, Al3+, and Fe3+.

[0088] In preferred embodiments, the cations are divalent metal ions. In further embodiments, the divalent metal ions are selected from any one or more of: Cu2+, Ca2+, Mg2+, Fe2+, Mn2+, Ni2+, Pb2+, and Zn2+.

[0089] In preferred embodiments, the cations are copper ions, such as Cu2+. The cross-linked polymer

[0090] The normal binding of a target molecule by a MIP occurs in a binding pocket, which binding pocket has been formed by molecular imprinting, usually with the target molecule itself as template molecule, during the production of the MIP. Such binding is a specific binding between the MIP and its target, akin to the binding between an antibody and its target (see, e.g., WO 2007 / 095949, WO 2011 / 033021, WO 2013 / 127433, and WO 2019 / 002535, the contents of which are all incorporated herein by reference). Without being bound by theory, in contrast to this normal binding of a MIP to its target, the binding of copper ions disclosed herein may occur independently of the molecular imprinting of the MIP. As such, the binding of copper ions may occur due to properties inherent in the cross-linked polymer material constituting the MIP. This interpretation is indeed supported by the finding presented in Example 2 (Figure 9), that S254, a batch which was polymerized in the absence of a template molecule, binds to Cu2+.

[0091] As used herein, the term "cross-linked polymer" should be understood as a broad term comprising both MIPs (i.e., cross-linked polymer particles comprising a specific binding pocket due to having been imprinted with a template molecule) and non-imprinted polymers (NIPs; i.e., cross-linked polymer particles which have not been imprinted with a template molecule).

[0092] As disclosed herein, an example of a cross-linked polymer discovered to bind stably to copper ions is the cross-linked polymer constituting the Phe-binding MIP described in WO 2019 / 002535. It comprises 2-methylprop-2-enoic acid, also termed methacrylic acid (MAA), and l,4-bis(acryloyl)piperazine (DAP). Since the copper ion-binding may not necessarily be due to specific binding in the binding pocket of the MIP, other cations may, e.g. due to their positive charge, bind to this cross-linked polymer as well. This is supported by the results presented in Example 1 that Phelimin bound to [18F]-AIF2+. In some embodiments, the first monomer is selected from methacrylic acid (MAA), acrylic acid, itaconic acid, 3-Butene-l,2,3- tricarboxylic acid, and (vinylbenzyl)trimethylammonium chloride (VBTA), preferably MAA.

[0093] In some embodiments, the cross-linked polymer further comprises a third monomer, which monomer is different from the first monomer. In further embodiments, the third monomer is selected from methacrylic acid (MAA), acrylic acid, itaconic acid, 3-Butene-l,2,3-tricarboxylic acid, and (vinylbenzyl)trimethylammonium chloride (VBTA).

[0094] In preferred embodiments, the first monomer is MAA. In other preferred embodiments, the first monomer is VBTA. In other preferred embodiments, the first monomer is MAA, and the third monomer is VBTA. In some embodiments, the molar ratio of DAP:the first monomer (mol / mol) in the crosslinked polymer is in the range between 1:4 and 30: 1. In some embodiments, the molar ratio of DAP:the first monomer in the cross-linked polymer is selected from: about 1 :3, about 1 :2, about 1 : 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9 : 1, about 10: 1, about 11 : 1, about 12: 1, about 13: 1, about 14: 1, about 15: 1, about 16: 1, about 17: 1, about 18: 1, about 19: 1, about 20: 1, about 21 : 1, about 22: 1, about 23: 1, about 24: 1, about 25: 1, about 26: 1, about 27: 1, about 28: 1, and about 29: 1.

[0095] In some embodiments, the first monomer is MAA, and the molar ratio of DAP: MAA is in the range between 1 :2 and 14: 1.

[0096] In the experiment shown in Example 2, Figure 4A, a polymer batch with a relatively low ratio of DAP:MAA of 1 : 1 (S242) bound way better to copper ions than batches with a DAP:MAA ratio of 13.5: 1 (S231 and S248). Although in this case the three batches were not entirely comparable, it could suggest that a high relative amount of MAA is preferable.

[0097] Therefore, in preferred embodiments, the first monomer is MAA, and the molar ratio of DAP:MAA is in the range between 1 :2 and 9 : 1, such as a ratio selected from about 1 : 1, about 3: 1, and about 8: 1.

[0098] In some embodiments, the cross-linked polymer has a capacity to bind at least 8 mg Cu2+per g polymer, such as at least 10, such as at least 20, such as at least 30, such as at least 40, and such as at least 45, mg Cu2+per g polymer.

[0099] The copper ion binding capacity may be determined using the assay described in Example 2. In short, the polymer is incubated in the presence of Cu2+at a pre-determined concentration. The suspension is then spun down, and EDTA is added to the supernatant, which is run on an HPLC using a method that detects the [Cu(EDTA)]2-complex. A standard curve is used to calculate the amount of copper that was in solution, i.e. the amount of copper that had not bound to the polymer. Based on that it can be calculated how much copper bound to the polymer.

[0100] In some embodiments, the cross-linked polymer is in the form of particles having an average size in the range of 50 nm to 63 pm. In some embodiments, the average size is selected from: about 100 nm, about 200 nm, about 250 nm, about 500 nm, about 750 nm, about 1 pm, about 2 pm, about 3 pm, about 4 pm, about 5 pm, about 10 pm, about 20 pm, about 30 pm, about 40 pm, about 50 pm, and about 60 pm. A size in the range of 50 nm to 63 pm, as defined above, is typically to be preferred for MIPs having a target they need to bind inside a binding pocket. However, the inventors have discovered that, in the case of copper ion binding, the cross-linked polymer particles may be larger than 63 pm, indeed much larger, without it having a negative influence on copper ion binding.

[0101] That the particles may be large is advantageous because, as will be described in more detail below, this saves a step in the production of the cross-linked polymer, as no "micronization" step is required. Thus, a simpler and faster process may be used to produce the cross-linked polymer for this purpose than for other purposes.

[0102] Therefore, in some embodiments, the cross-linked polymer is in the form of particles and / or aggregates of particles, and the particles or the aggregates of particles have a size above 63 pm, such as above 100 pm, such as above 200 pm, and such as above 500 pm. As described above, the Phe-binding MIP described in WO 2019 / 002535 consists of a cross-linked polymer comprising MAA and DAP. Furthermore, it comprises a molecularly imprinted binding pocket for binding to Phe.

[0103] In contrast to the Phe-binding MIP, the cross-linked polymer according to aspects of the present invention may not necessarily be molecularly imprinted, i.e., it may not necessarily be a MIP. As described above, the cation binding of the cross-linked polymer is expected to occur independently of a molecularly imprinted binding pocket. However, as can be learned from Example 1, the presence of a binding pocket, even a binding pocket for binding to a different molecule (such as Phe), is not an obstacle to cation binding, and it is possible that the presence of such a binding pocket could even enhance the cation (such as copper ion) binding to the cross-linked polymer, e.g., due to negative charges and / or a hydrophilic environment in the binding pocket.

[0104] Thus, in some embodiments, the cross-linked polymer has been polymerized in the absence of a template molecule.

[0105] In other embodiments, the cross-linked polymer has been polymerized in the presence of a template molecule.

[0106] The template molecule may be L-Phe or an L-Phe derivative exposing a phenylalanine motif, such as Gly-Phe. It can be speculated that positively charged templates may form binding pockets containing negative charges and / or a hydrophilic environment which may increase the binding of cations such as copper ions to cross-linked polymers.

[0107] As such, in some embodiments, the template molecule may be a positively charged molecule.

[0108] Surprisingly, the inventors have discovered that polymers that were treated with a high pH (such as a pH between 6 and 8) during their production had increased Cu2+binding (see last paragraph of Example 2). This treatment has the effect that the template molecule which was used to imprint the polymer is not washed out of the polymer, and thus the template stays bound to or enclosed within the polymer. Surprisingly, this may be what is responsible for the increased Cu2+binding of polymers treated with a high pH during their production.

[0109] Therefore, in preferred embodiments, the cross-linked polymer is attached to, enclosing, and / or covalently bound to, a template molecule, such as the template molecule L-Phe or an L-Phe derivative exposing a phenylalanine motif, such as Gly-Phe.

[0110] There is also the possibility that the binding of a cross-linked polymer to cations may be improved by imprinting the cross-linked polymer with cations, such as with copper ions, thus making a cation-binding, such as a copper ion-binding, MIP.

[0111] Thus, in some embodiments in which the cross-linked polymer has been polymerized in the presence of a template molecule, the template molecule is a cation. In some embodiments, the cation is a divalent cation. In some embodiments, the cation is a copper ion.

[0112] 2ndaspect of the invention and embodiments thereof

[0113] The second aspect of the invention relates to a cross-linked polymer comprising :

[0114] - a first monomer selected from: (vinylbenzyl)trimethylammonium chloride (VBTA), acrylic acid, itaconic acid, and 3-Butene-l,2,3-tricarboxylic acid, preferably VBTA, and

[0115] - a second monomer which is l,4-bis(acryloyl)piperazine (DAP).

[0116] In some embodiments, the cross-linked polymer is as defined in the first aspect of the invention or embodiments thereof. 3rdaspect of the invention and embodiments thereof

[0117] The third aspect of the invention relates to a composition comprising the cross-linked polymer as defined in the first aspect of the invention or embodiments thereof.

[0118] In some embodiments, the composition is a dry composition, such as a powder, capsule or a tablet.

[0119] It is advantageous that the polymer may be dosed in dry form, as it is simple and convenient.

[0120] In other embodiments, the composition comprises a solvent, such as water, and the crosslinked polymer is suspended in the solvent.

[0121] Process for producing the cross-linked polymer

[0122] The cross-linked polymer according to aspects of the present invention may be produced by various methods. Examples of preparation methods for producing a Phe-binding MIP are disclosed in WO 2019 / 002535.

[0123] 4thaspect of the invention and embodiments thereof

[0124] The fourth aspect of the invention relates to a method for producing a cross-linked polymer, the method comprising a step of: polymerizing a mixture comprising

[0125] - a first monomer comprising at least one vinyl group and i. at least one carboxylic acid or II. at least one benzyl group, and

[0126] - a second monomer which is l,4-bis(acryloyl)piperazine (DAP), and optionally

[0127] - a template molecule, in the presence of a catalyst, so as to obtain the cross-linked polymer, which is optionally attached to, enclosing, and / or covalently bound to, the template molecule.

[0128] In some embodiments, the polymerization occurs at a pH between 6 and 8.

[0129] As described above, performing the polymerization at a pH between 6 and 8 has the surprising effect that the polymer has increased Cu2+binding. In cases where a template is included in the polymerization mixture, the high pH may have the effect that the template stays bound to or enclosed within the polymer, and this attached template may affect the copper ion binding, perhaps due to the presence of additional carboxylic acid groups from the template which affect the charge. However, the inventors have discovered that an increased pH during polymerization may have a positive effect on Cu2+binding in itself, also in the absence of a template molecule.

[0130] In further embodiments, the first monomer is selected from methacrylic acid (MAA), acrylic acid, itaconic acid, 3-Butene-l,2,3-tricarboxylic acid, and (vinylbenzyl)trimethylammonium chloride (VBTA).

[0131] In preferred embodiments, the first monomer is VBTA.

[0132] In other preferred embodiments, the first monomer is MAA, and the polymerization occurs at a pH between 6 and 8. In a further embodiment, the mixture comprises a template molecule, which is Gly-Phe.

[0133] In some embodiments, the mixture further comprises a third monomer, which monomer is different from the first monomer, optionally wherein the third monomer is selected from methacrylic acid (MAA), acrylic acid, itaconic acid, 3-Butene-l,2,3-tricarboxylic acid, and (vinylbenzyl)trimethylammonium chloride (VBTA).

[0134] In preferred embodiments, the first monomer is MAA, and the third monomer is VBTA.

[0135] In some embodiments, the molar ratio of DAP:the first monomer in the mixture is in the range between 1:4 and 30: 1. In some embodiments, the molar ratio of DAP:the first monomer (mol / mol) in the cross-linked polymer is selected from: about 1 :3, about 1 :2, about 1 : 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9 : 1, about 10: 1, about 11 : 1, about 12: 1, about 13: 1, about 14: 1, about 15: 1, about 16: 1, about 17: 1, about 18: 1, about 19: 1, about 20: 1, about 21 : 1, about 22: 1, about 23: 1, about 24: 1, about 25: 1, about 26: 1, about 27: 1, about 28: 1, and about 29: 1.

[0136] In some embodiments, the first monomer is MAA, and the molar ratio of DAP: MAA is in the range between 1 :2 and 14: 1, more preferably in the range between 1 :2 and 9: 1, such as a ratio selected from about 1 :1, about 3: 1, and about 8: 1.

[0137] MIPs that are produced with the aim of binding a target in their binding cavity generally have some limitations of access to the binding cavity due to their size. For this reason, production of these particles has often included a micronization step after the polymerization, where large particles have been fragmented into smaller particles. The inventors have discovered that for the purpose of binding cations, such as copper ions, it is not necessary to perform a micronization step, because it does not matter if the particles are large. This may be because the copper ions are not dependent on binding in the binding cavity, and therefore it is not a problem if large particles have a poor access to their binding cavity (if they have any). That a micronization step is not required is a clear advantage, as it provides for a faster and simpler production method.

[0138] However, in some embodiments, the method does comprise a micronization step. In cases where it matters how large the polymers are, micronization will typically be performed when the particle sizes after polymerization do not include a sufficient amount of cross-linked polymer particles having particle sizes smaller than a certain size, such as 63 pm.

[0139] As such, in some embodiments, the method comprises a step b) (the polymerization step may be called step a)) of fragmenting the cross-linked polymer obtained from the polymerization step to obtain a first fragmented polymer and collecting the cross-linked polymer particles having particle sizes smaller than 63 pm.

[0140] Optionally, the method may also comprise a step c) of washing and drying the cross-linked polymer. This step may be performed after step b), but if no micronization is performed, it may be performed right after step a).

[0141] Thus, in some embodiments, the method comprises a step of washing and drying the crosslinked polymer obtained from the polymerization step, optionally wherein the drying is performed by freeze-drying, spray drying, vacuum drying or by drying in an oven.

[0142] The steps a)-c) (with b) and c) being optional) will lead to cross-linked polymer particles (including MIPs) according to the present invention. As described above, whether the crosslinked polymer is a MIP or a NIP is not essential for the present purpose.

[0143] The method may comprise a few more steps after step c), such as a step d) in which the polymer fraction obtained from step a), b) or c) is further fragmented, resulting in a second fragmented polymer. The purpose of this step is to make the cross-linked polymer particles smaller, as it will result in particles of a size of no more than app. 250 nm. This may not necessarily be relevant for the present purpose.

[0144] The method may further comprise a step e) of subjecting the polymer fraction obtained from step a), b) or c) or the second polymer fraction obtained from step d) to affinity chromatography, preferentially using a cation of interest, such as a copper ion, as the binding target in a chromatographic matrix. Such a step may be performed for a cross-linked polymer which is a MIP, such as a MIP imprinted with a cation, such as a copper ion. But it may also be performed with a cross-linked polymer which is not a MIP (has no molecular imprinting). The cross-linked polymer particles which bind to the chromatography matrix will then be collected.

[0145] The exact polymerization method can vary. Some examples are given in WO 2019 / 002535 including bulk polymerization followed by a fragmentation step and a reverse phase emulsion polymerization method, optionally also followed by a fragmentation step.

[0146] The catalyst is typically selected from those that catalyse polyacrylamide gel polymerization in aqueous media. Useful catalysts are tetramethylethylenediamine (TEMED), dimethylpiperazine and 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH). TEMED and AAPH are preferred catalysts.

[0147] Thus, in some embodiments, the catalyst is selected from TEMED, dimethylpiperazine and AAPH, preferably from TEMED and AAPH.

[0148] Some catalysts, like TEMED and dimethylpiperazine, have to be used together with a strong oxidizing agent. AAPH is itself an oxidizing agent and thus does not have to be used with another oxidizing agent.

[0149] Thus, in some embodiments, the polymerization of the mixture occurs in the presence of an oxidizing agent.

[0150] Preferred oxidizing agents include ammonium persulfate (APS), potassium persulfate, sodium persulfate (SPS), and sodium thiosulfate. AAPH is used as a catalyst and as an oxidizing agent when the pH of the polymerizing mixture is above 5.

[0151] Thus, in some embodiments, the oxidizing agent is selected from APS, potassium persulfate, SPS, sodium thiosulfate, and AAPH, preferably from APS, SPS and AAPH.

[0152] The fragmentation method utilised in step b) may be any method for downsizing of MIPs known in the art: for instance, the fragmentation in step b) can comprise grinding, milling, explosion, hammering, ball milling, cryo grinding, or collision homogenisation, as well as any combination of these methods.

[0153] In order to ensure that the cross-linked polymer particles obtained in step b) are smaller than 63 pm, standard metal sieves with defined cut-offs can be used. For instance, particles having sizes in the 25-63 pm range can be attained by utilising sieves with a 63 pm cut-off and collecting the particles that are able to pass through, and subsequently using sieves with a 25 pm cut-off where the material retained is used in subsequent steps. However, since smaller particles can be subjected to the subsequent steps, step b) need not isolate particles of any defined minimum size.

[0154] The optional washing step c) is performed at alternating pH with an organic solvent, as disclosed in WO 2019 / 002535. This step is used to remove oil and, when the cross-linked polymer is a MIP, it may also effect that accessible bound template is effectively removed. One convenient way of washing cross-linked polymers is to pack them in a column such as an HPLC column, thereby facilitating washing under elevated pressure with a solvent passing through the HPLC column. Alternatively, the particles can be washed by tangential flow filtration (TFF).

[0155] Subsequent step d) (the further fragmentation of cross-linked polymer particles) is conveniently carried out in a ball mill or bead mill but as for step b) the exact micronization method employed in inessential for the result to be achieved, as long as the method is capable of providing a sufficient fragmentation. Hence, the methods recited above as convenient in step b) are all relevant for the purposes of step d).

[0156] After the production process, the cross-linked polymer particles can be isolated / collected. This may be done by suspending the particles in a solvent (preferably an aqueous solvent such as water). They may also be subsequently incubated in an ultrasound bath (which can in its own right effect micronization), followed by centrifugation and isolation of the supernatant, which contains the smaller particles.

[0157] 5th-7thaspects of the invention and embodiments thereof

[0158] The 5thaspect of the invention relates to a method of treating a pathology associated with an aberrant presence of cations in a subject in need thereof, the method comprising administering to the subject the cross-linked polymer as defined in the first aspect of the invention or embodiments thereof or according to the second aspect of the invention or embodiments thereof or the composition according to the third aspect of the invention or embodiments thereof.

[0159] The 6thaspect of the invention relates to the cross-linked polymer according to the second aspect of the invention or embodiments thereof or the composition according to the third aspect of the invention or embodiments thereof, insofar as it relates to the cross-linked polymer according to the second aspect of the invention, for use as a medicament.

[0160] The 7thaspect of the invention relates to the cross-linked polymer as defined in the first aspect of the invention or embodiments thereof or according to the second aspect of the invention or embodiments thereof or the composition according to the third aspect of the invention or embodiments thereof, for use in a method of treating a pathology associated with an aberrant presence of cations.

[0161] As described above and demonstrated in Example 1, a large advantage of using cross-linked polymer particles such as MIPs for treatment of pathologies, such as diseases, in which components in food cause problems and should be removed is that these particles are confined to the GI tract, where they can bind to the molecules that should be removed, and thus do not cause unwanted systemic effects. Thus, in some embodiments, the cross-linked polymer is provided non-systemically. In preferred embodiments, the cross-linked polymer is administered orally.

[0162] Some general considerations about in vivo treatment with MIPs are disclosed in WO 2011 / 033021 (hereby incorporated herein in its entirety).

[0163] The dosage regimen will depend on the cross-linked polymer composition and its exact capacity for binding of the relevant disease-provoking cation, the amount of symptomprovoking cation to be removed by means of the treatment and the constitution and age of the individual to be treated. The skilled artisan will be able to determine the relevant dosage parameters on a case-by-case basis.

[0164] In preferred embodiments, the pathology is Wilson's disease. As described above, the ability of the cross-linked polymer according to the present invention to stably bind to copper ions makes it suitable for use in a method of treating Wilson's disease in a subject.

[0165] It should be possible to control the level of copper in persons suffering from Wilson's disease by administering a feasible dosage per standard meal of the cross-linked polymer according to the invention. Such a dosage may, e.g., be in the range of 0.1 g to 10 g per meal, such as a dosage selected from: about 0.15 g, about 0.20 g, about 0.25 g, about 0.30 g, about 0.35 g, about 0.40 g, about 0.45 g, about 0.50 g, about 1 g, about 2 g, about 3 g, about 4 g, about 5 g, about 6 g, about 7 g, about 8 g, and about 9 g, per meal. As described in Example 2, Experiment 2, the S242 batch (see Table 1) had a high enough copper binding capacity that a dose of 250-500 mg polymer per meal is estimated to be sufficient.

[0166] With respect to formulation, the cross-linked polymer particles according to the present invention can be included in suspended or dry form in any convenient form, typically for oral administration.

[0167] That the cross-linked polymer particles may be formulated in either suspended or dry form is supported by Example 2, Experiment 3, where it was shown that there was no difference between how well polymers in the two different forms bound to Cu2+(Figure 6).

[0168] In certain embodiments, the particles are simply suspended in water or formulated in a tablet, capsule or powder, optionally with addition of state-of-the art additives to improve taste (for oral compositions), colour, smell, consistency / texture, release, distribution, etc. It is also possible to integrate the particles in foodstuffs and drinks which are prepared by simple admixture.

[0169] 8thand 9thaspects of the invention and embodiments thereof

[0170] The 8thaspect of the invention relates to a method of treating a pathology associated with an aberrant presence of cations in a subject in need thereof, the method comprising contacting a bodily fluid, such as blood, derived from said subject with the cross-linked polymer as defined in the first aspect of the invention or embodiments thereof or according to the second aspect of the invention or embodiments thereof ex vivo, and returning the resulting cation-reduced bodily fluid to said subject.

[0171] The 9thaspect of the invention relates to the cross-linked polymer as defined in the first aspect of the invention or embodiments thereof or according to the second aspect of the invention or embodiments thereof, for use in a method of treating a pathology associated with an aberrant presence of cations in a subject in need thereof, the method comprising contacting a bodily fluid, such as blood, derived from said subject with the cross-linked polymer ex vivo, and returning the resulting cation-reduced bodily fluid to said subject.

[0172] This method may, e.g., be a method of performing plasmapheresis with the purpose of removing cations from the blood of a patient suffering from a pathology associated with an aberrant presence of cations. In preferred embodiments, the pathology is Wilson's disease. Plasmapheresis could be useful in a situation in which a Wilson's patient, or a non-Wilson's patient, suffers from acute copper intoxication.

[0173] 10thaspect of the invention and embodiments thereof

[0174] The 10thaspect of the invention relates to a device suitable for binding cations of a composition, said device comprising a solid phase having the cross-linked polymer as defined in the first aspect of the invention or embodiments thereof or according to the second aspect of the invention or embodiments thereof anchored to it.

[0175] In some embodiments, the device is a chromatography matrix.

[0176] In some embodiments, the solid phase is as described in any part of the definition of "solid phase" above.

[0177] EXAMPLE 1

[0178] Phelimin binds cations including copper ions

[0179] As described above, when working with their Phe-imprinted MIPs called Phelimin (as described in, e.g., WO 2019 / 002535), the inventors discovered that these MIPs bind very well to copper ions. This example describes these initial experiments performed with Phelimin.

[0180] Biodistribution study

[0181] The purpose of this study was to test the biodistribution of Phelimin after oral administration to mice. Phelimin is cross-linked polymers of methacrylic acid (MAA) and 1,4- bis(acryloyl)piperazine (DAP), which are imprinted with Phe. In order to measure its distribution in different organs of the animals by PET scanning, Phelimin was labelled with the radioactive copper isotope54Cu as the ion54Cu2+.

[0182] 54Cu was purchased as54CuCI2salt and dissolved in 0.05 M NaCH3COO, 0.13 M NaCI, and adjusted to pH 4-5 with 0.1 M HCI. The total volume was 10 ml. The54Cu-Phelimin complex was prepared by mixing the54Cu solution 1 : 1 with a suspension of 15 mg Phelimin and incubating for 30 min before first dosing.

[0183] Either PD10 G25 MidiTrap columns, Nanosep 3K spinfilter or ultracentrifugation was used for product purification and / or determination of product radiochemical purity.54Cu-Phelimin was obtained in >98% radiochemical purity and 50% radiochemical yield, at pH 5.5 - 6.0.

[0184] Mice were administered either free54CuCI2or 0.2 - 5 MBq54Cu-Phelimin complex by oral gavage. They were each dosed with 100 pl, and the individual time points were noted for later correction of the counting values. A reference sample of 100 pl of the free54Cu or54Cu- Phelimin was counted as well. The mice were standard Swiss that were individually placed in metabolic cages after dosing. They had free access to standard food and water during the experiment.

[0185] At various time points after dosing of the mice, as indicated in Figures 1 and 2, blood, faeces, and urine samples were collected, and the mice were euthanized and their organs dissected. The dissected organs, blood, urine and faeces were placed in tubes and counted in a HIDEX gamma counter. The counted values were corrected back to the starting point using the halflife value of54Cu (Tl / 2 = 12.7 h) and corrected for the volume-fraction that was counted in the HIDEX. Finally, the fractions for the individual organ of the value of the dosed value were calculated in %.

[0186] Figure 1 shows the tracer recovery (54Cu) in different organs at different time-points after oral administration of free54CuCI2, whereas Figure 2 shows the same but for administration of the54Cu-Phelimin complex.

[0187] It is apparent from Figure 1 that the majority of the free54Cu had ended up in the faeces after 8 hours, however a relatively large proportion, approx. 40%, could still be found in organs of the animal 8 hours after dosing. Only a very small amount of54Cu seemed to be systemic, maximum 5%, and only in the liver.

[0188] It can be seen from Figure 2 that similar results were obtained for the54Cu-Phelimin complex. Like for the free54Cu, only a maximum of 5% of the54Cu-Phelimin complexes appeared to be present systemically, and they were only found in the liver. And like for the free54Cu, the vast majority of the54Cu-Phelimin complexes were found in the faeces after 8 hours, with a tendency to higher54Cu recovery in the faeces when the54Cu was complexed to Phelimin. In addition to labeling with54Cu, it was also attempted to label Phelimin withnC and18F. Attempts to label Phelimin withnC proved elusive, whereas Phelimin was successfully labeled with18F.

[0189] For the labeling attempt withnC, 15 mg Phelimin (size 50 - 200 nm) dissolved in aqueous / organic solvent mixtures with auxiliary base present was reacted withnC-methyl iodide, to convert residual acid functionality to the corresponding ester, followed by sizeexclusion chromatography for product purification.

[0190] For the labeling with18F,18F-aluminium-fluoride was mixed with 15 mg Phelimin in aqueous sodium acetate buffer at varying pH. Either PD10 G25 MidiTrap columns, Nanosep 3K spinfilter or ultracentrifugation was used for product purification and / or determination of product radiochemical purity. Optimum conditions for18F-labeling were pH 4.5, yet radiochemical yield was only 5-10% and >90% purity.

[0191] The18F-labeling resulted in complexes of the aluminium-fluoride ion [18F]-AIF2+and Phelimin, which was administered to mice (in a dose of 2-25 MBq) and could be imaged for up to 18 hours in mice. MicroPET imaging showed primarily distribution of radioactivity to GI tract and intestines, although bones were also clearly visible.

[0192] Thus, Phelimin formed very stable complexes with54Cu2+and [18F]-AIF2+that survived the travel through the GI tract. However,18[F]-AIF-Phelimin showed some defluorination.

[0193] 64Cu-Phelimin complex stability study

[0194] To test the stability of the54Cu-Phelimin complex it was exposed to either lower pH or various competitors in vitro. lOx concentrated simulated gastro-intestinal fluid (SGF, pH 3.5) or acetate buffer (pH 5.0), or various competitors as indicated in Figure 3, were added to54Cu2+-Phelimin complex samples and incubated for 15 min. The samples were then centrifuged to isolate the Phelimin (and complexed54Cu) in the pellet, and the supernatants were analysed in the HIDEX gamma counter and compared to a reference sample containing a Phelimin suspension ("MIP in water" in Figure 3).

[0195] In case the change in pH, or the presence of a competitor, would de-complex some54Cu from Phelimin, the54Cu would be present in the supernatant. The counts in the supernatant represent the54Cu not bound to the Phelimin, and from that the percentage of54Cu that remained bound to the Phelimin compared to the "MIP in water" control was calculated. As can be seen in Figure 3, all or almost all of the54Cu remained bound to the Phelimin after the various treatments. One exception is the treatment with Trientine (as described above, Trientine is a drug for Wilson's disease). The sample incubated with Trientine retained app. 90% of its54Cu-binding, which is still a very high percentage.

[0196] This study indicates that the complex formation of54Cu2+to Phelimin is very stable.

[0197] EXAMPLE 2

[0198] Different cross-linked polymers bind copper ions

[0199] After the discovery described in Example 1 that Phelimin binds copper ions, the inventors investigated other types of cross-linked polymers for their ability to bind copper ions and further characterized different aspects of this binding. These experiments will be described in this example.

[0200] Overview of cross-linked polymers

[0201] The composition and characteristics of the various polymers tested in this example are set out in Table 1 :

[0202] Table 1. DAP = l,4-bis(acryloyl)piperazine, MAA = methacrylic acid, VBTA =

[0203] (vinylbenzyl)trimethylammonium chloride.

[0204] From Table 1, the composition of the various cross-linked polymers can be seen but also some characteristics of their production method. For example, S231 was produced with a high pH (pH 6-8) during polymerization, and S248 was produced with a high pH (6-8) in the water phase. By "high pH during polymerization" is meant a process in which the sodium hydroxide was added not to the water phase but to the homogenized droplets in oil. As such, it is unlikely that the pH was high in all the water droplets formed. By "high pH in water phase" is meant a process in which the pH was deliberately increased in the water phase and measured, hence all the water later homogenized to water droplets in oil had a high pH.

[0205] Production of the cross-linked polymers

[0206] Cross-linked polymers according to the present invention may be produced using the general method described under the description of the 4thaspect of the invention above.

[0207] The cross-linked polymers tested in this example were produced using the following method:

[0208] Cithrol DPHS (0.0135 kg) was dissolved in Isopar M (1.678 kg) at 40-50°C followed by cooling to 10-20°C.

[0209] 1,4-Bis(acryloyl(piperazine (see amount in Table 3), methacrylic acid (see amount in Table 3) and Gly-L-Phe (see amount in Table 3) were dissolved in purified water (0.225 kg) at app. 40°C followed by cooling to ambient temperature. An oxidizing agent (see amount in Table 3; AAPH is both an oxidizing agent and a catalyst) was added, and this mixture was filtered into the solution of Cithrol DPHS in Isopar M followed by cooling to 10-14°C. The mixture was circulated through a homogenizer for 18 min using a jacket temperature of 5°C on both reactor and homogenizer. If the mixture did not already contain a catalyst (in the form of AAPH), a catalyst was added. The mixture was heated. Between 55°C and 80°C polymerization started (exothermic), and the resulting suspension was stirred further ~80 min.

[0210] The suspension was washed with ethanol (75 L) followed by purified water (50 kg). The suspension was then concentrated to a final solid content of 10-20 % (w / w).

[0211] The reagents used are listed in Table 2:

[0212]

[0213] Table 2.

[0214] The specific amounts of the reagents that were used for the different polymer batches are shown in Table 3:

[0215] Table 3. Experiment 1 : Copper binding capacity of 3 different polymers

[0216] The purpose of this experiment was to test three polymer batches for their copper (Cu2+) binding capacity; S231 (solid load : 19.9 g / g), S242 (solid load: 13.3 g / g) and S248 (solid load : 19.1 g / g). By "solid load" is meant the concentration of dry polymer (g) per suspension (g). All three polymers are made of DAP and MAA, and Gly-Phe was used as a template. However, whereas S231 and S248 contain a molar excess of DAP, S242 contains equal molar amounts of DAP and MAA, and furthermore, S231 and S48 were produced under high pH conditions (either during the polymerization or afterwards, which has the effect that the Gly- Phe template was not washed out after the polymerization and thus remained bound to the polymer (see also Table 1)). Suspensions of these different polymers were tested at different concentrations of copper to determine a maximum binding capacity [pg copper / g polymer].

[0217] Method :

[0218] Copper-gluconate stock solution (D-Gluconic acid Copper(II) salt, 0.1 M), MQ water, acetate buffer (1 M, pH 5) and polymer suspension was mixed in a 100 mL blue cap bottle. The pH of the mixtures was measured and adjusted to pH 4.9-5.1. In this and the later described copper binding experiments, the pH was generally adjusted using sodium hydroxide (NaOH, 0.5 M) or hydrochloric acid (HCI, 0.5 M). The mixtures were stirred overnight (approximately 18 hours) using a magnetic bar and magnetic stirring.

[0219] On the following day, a sample of 2 mL was taken from each sample and centrifuged for 30 min at 13400 rpm (2212 RCF) using an Eppendorf miniSpin centrifuge. After centrifugation, the supernatant was collected and filtered through a 0.22 pm syringe filter. 1 mL of the filtered supernatant was pipetted into an HPLC vial (Hounisen) and an amount of 50 mM Ethylenediaminetetraacetic acid (EDTA) was added to the vial. The amount of EDTA added was calculated as having 1.5 times moles compared to the amount of copper moles in each 1 mL sample if none of the copper binds the polymer. E.g. 54 pL 50 mM EDTA (2.7 pmol) was added to sample A containing 1.8 pmol Cu2+in the HPLC vial. The samples were afterwards run on an HPLC using a method that detects the [Cu(EDTA)]2-complex at 280 nm. A standard curve was used to calculate the amount of copper that was in solution, i.e. the amount of copper that had not bound to the polymer. The blank sample was used as a negative control and corresponded to the amount of copper in solution if nothing binds to the polymer.

[0220] The experimental conditions are shown in Table 4:

[0221] Table 4.

[0222] Results:

[0223] The results are shown in Table 5 and Figure 4.

[0224] Tab e 5. All three batches bound copper. The more copper in the mixture, the lower percentage of the copper was bound to the polymer. However, batch S242 seemed to bind way better than batches S231 and S248 at each copper concentration (Figure 4A). This increased binding could be due to a higher amount of MAA in S242 as compared to S231 and S248, due to the 1 : 1 ratio of DAP: MAA, in contrast to the 13.5: 1 ratio in S231 and S248. A maximum capacity was reached for batch S231 at ~8,700 pg Cu2+ / g polymer and for batch S248 at ~8,500 pg Cu2+ / g polymer. As for batch S242, a maximum had not been reached at a concentration of 9 mM copper, where the polymer bound ~36,500 pg Cu2+ / g polymer (Figure 4B). The maximum binding capacity of S242 was further tested in the experiment described next.

[0225] Experiment 2: Copper binding capacity of batch S242

[0226] The purpose of this experiment was to further test the copper (Cu2+) binding capacity of the polymer batch S242 (solid load : 13.3 g / g) at 6 different copper concentrations from 0.9 mM to 15 mM. The method was as described above.

[0227] The experimental conditions are shown in Table 6:

[0228] Table 6.

[0229] Results:

[0230] The results are shown in Table 7 and Figure 5.

[0231] Table 7.

[0232] S242 still did not reach a maximum at a concentration of 15 mM copper (Figure 5), where the polymer was found to bind ~47,900 pg Cu2+ / g polymer (Table 7). Almost 50 mg Cu2+bound per g polymer gives an amount of copper where it is realistic that the polymer can bind sufficient amounts of dietary copper that is part of a daily intake.

[0233] Experiment 3: Copper binding at varying polymer concentration

[0234] The purpose of this experiment was to investigate how well a batch binds copper when varying the polymer concentration at one specific copper concentration. The copper concentration (0.315 mM) used was lower than previously tested to simulate the copper concentration (from the diet) in the stomach and intestines. The polymer concentration was tested at 7 different concentrations from 250 pg / L to 9000 pg / L and should give an idea about how much polymer is needed to bind an amount corresponding to copper intake in one meal.

[0235] It should be noted that the incubation time for this experiment was lowered to 2 hours to also simulate the environment in the stomach / intestines better. Otherwise, the method was as described above.

[0236] The experimental conditions are shown in Table 8:

[0237] Table 8.

[0238] Results:

[0239] The results are shown in Table 9 and Figure 6.

[0240] Table 9.

[0241] As can be seen from Figure 6 (light grey spheres), 80% of the copper was bound to the polymer in suspension at a polymer concentration of 9 g / L. It is expected that a clinically relevant dose of polymer would be able to bind 1000-1500 pg of copper for the treatment of Wilson's disease. Samples D and E that contained 0.257 g and 0.415 g of polymer, respectively, were able to bind 1170 pg Cu2+and 1434 pg Cu2+, respectively (Tables 8 and 9). This indicates that a relevant dose for the treatment of Wilson's disease could be 250-500 mg of dried polymer particles, which potentially could be given as a tablet or a capsule.

[0242] Polymers in suspension vs dried polymer:

[0243] The same experiment as above was done with dried polymer. The exact same parameters were used except that the polymer suspension was dried in an oven at 60°C overnight and added to the mixtures as dried powder. Particle size was measured on the dried powder to approximately 80% of the particles being 100 pm on average and 20% around 2 pm, which indicates aggregation during drying in the oven.

[0244] There was no difference between the two forms - the dried powder bound as well as the polymer in suspension (Figure 6, black spheres). Therefore, it seems likely that the polymer can be formulated as tablets or capsules for the treatment of Wilson's disease. Experiment 4: Copper binding as a function of pH

[0245] The purpose of this experiment was to test the copper (Cu2+) binding of the polymer batch S242 (solid load : 13.3 g / g) as a function of pH, in a range from pH 2 to pH 6.

[0246] The method was as described above, except that the pH was adjusted to a value between 2 and 6 using sodium hydroxide (NaOH, 0.5 M) or hydrochloric acid (HCI 0.5 M).

[0247] The experimental conditions are shown in Table 10:

[0248] Table 10.

[0249] Results:

[0250] The results are shown in Table 11 and Figure 7.

[0251] Table 11.

[0252] The amount of Cu2+bound to the polymer increased with increasing pH, with no binding below pH 3 and max binding from pH 5-6 (Figure 7). Copper binding was not tested above pH 6.5, as copper precipitates as Cu(OH)2above pH 6.5. The results indicate that the polymer will not bind copper in the stomach, as the pH is low in the stomach, but will bind copper well in the intestines, where copper gets absorbed.

[0253] Experiment 5: Copper binding - inhibition with Zn2+and Ca2+in excess of 50 and 100 times

[0254] The purpose of this experiment was to test the polymer batch S248 for copper (Cu2+) binding with Zn2+and Ca2+in excess of 50 and 100 times to investigate if copper still binds with other divalent cations in the mixture.

[0255] Method :

[0256] Copper chloride stock solution (CuCI2, 0.1 M), zink chloride (ZnCI2, IM), calcium chloride (CaCI2*2H2O, IM), MQ water, acetate buffer (1 M, pH 5) and polymer suspension were mixed in a 100 mL blue cap bottle. The pH of the mixtures was measured and adjusted to pH 4.9- 5.1 using sodium hydroxide (NaOH, 1 M).

[0257] The rest of the procedure was as described above.

[0258] The experimental conditions are shown in Table 12:

[0259] Table 12.

[0260] Results:

[0261] The results are shown in Table 13 and Figure 8.

[0262] Table 13. As can be seen from Figure 8, there was barely any inhibitory effect of zink or calcium at a concentration of 50 (100 mM) or 100 (200 mM) times higher than the copper concentration (1.8 mM). Only copper binding was measured, so it is unknown whether some of the zink or calcium also bound to the polymer at the same time as the copper. However, it is important that specificity for copper is high as this increases the likelihood of binding to copper in the intestines, as there will likely also be potential competitors in the intestinal media together with food. Experiment 6: Copper binding of 5 further polymers

[0263] The purpose of this experiment was to test the copper (Cu2+) binding capacity of five further polymer batches; S238, S245, S249, S250 and S254. S238 and S254 are both made of DAP and MAA, but whereas S238 contains a large molar excess of DAP of 13: 1, S254 contains a smaller molar excess of DAP of 3: 1. Furthermore, while S238 was polymerized in the presence of the Gly-Phe template, S254 was polymerized in the absence of template. S245 and S250 have the same composition. They are made of DAP, MAA and VBTA, with Gly-Phe as template. S249 contains DAP and VBTA, and Gly-Phe was used as template (also see Table 1). Suspensions of these different polymers were tested at three different concentrations of copper; 1.8 mM, 4.5 mM and 9 mM.

[0264] The method was as described above.

[0265] The experimental conditions are shown in Table 14:

[0266] Table 14.

[0267] Results: The results are shown in Table 15 and Figure 9.

[0268] Table 15.

[0269] As can be seen from Figure 9, the fraction of copper that bound to the polymer decreased with increasing concentration of copper. All tested batches bound copper. Batches S245 and S250 bound better than the other batches at each copper concentration. As described above,

[0270] S245 and S250 contain DAP, VBTA and MAA. However, the polymer containing only DAP and VBTA and no MAA (S249) also bound copper, demonstrating that MAA is not required for copper (Cu2+) binding. Though, the binding was much better when the polymer contained MAA (much more Cu2+was bound per g polymer; Tables 14 and 15). Additionally, that S254, which was polymerized in the absence of a template molecule, binds to Cu2+, supports the hypothesis that a binding pocket is not necessary for the binding of cross-linked polymers to Cu2+.

[0271] As can be seen from Table 1, S238 and S248 have the same composition, except that S248 was treated with a high pH in the water phase. When comparing the binding data for S248 from Figure 4A with the binding data for S238 from Figure 9, it can be seen that S248 binds much better to Cu2+than S238 (56% vs. 21% Cu2+bound at a 1.8 M Cu2+-concentration). What happens when the cross-linked polymer is treated with a high pH (such as a pH between 6 and 8) is that the template molecule which was used to imprint the polymer (in this case Gly-Phe) is not washed out of the polymer, and thus the template stays bound to or entrapped inside the polymer. The present data thus suggests that it is an advantage to have a template molecule bound to the polymer, because it gives an increased Cu2+binding.

Claims

CLAIMS1. A method of binding cations, the method comprising a step of: contacting a composition comprising cations with a cross-linked polymer comprising :- a first monomer comprising at least one vinyl group and i. at least one carboxyl group or II. at least one benzyl group, and- a second monomer which is l,4-bis(acryloyl)piperazine (DAP), thereby binding cations of the composition.

2. The method according to claim 1, wherein the first monomer is selected from methacrylic acid (MAA), acrylic acid, itaconic acid, 3-Butene-l,2,3-tricarboxylic acid, and (vinylbenzyl)trimethylammonium chloride (VBTA), preferably wherein the first monomer is MAA.

3. The method according to claim 1 or 2, wherein the cross-linked polymer further comprises a third monomer, which monomer is different from the first monomer, optionally wherein the third monomer is selected from methacrylic acid (MAA), acrylic acid, itaconic acid, 3-Butene- 1,2,3-tricarboxylic acid, and (vinylbenzyl)trimethylammonium chloride (VBTA).

4. The method according to any one of the preceding claims, wherein the binding is performed in vitro or ex vivo.

5. The method according to any one of claims 1-3, wherein the binding is performed in vivo.

6. The method according to any one of the preceding claims, wherein the molar ratio of DAP:the first monomer (mol / mol) in the cross-linked polymer is in the range between 1 :4 and 30: 1.

7. The method according to claim 6, wherein the molar ratio of DAP:the first monomer in the cross-linked polymer is selected from: about 1 :3, about 1 :2, about 1 : 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9: 1, about 10: 1, about 11 : 1, about 12: 1, about 13: 1, about 14: 1, about 15: 1, about 16: 1, about 17: 1, about 18: 1, about 19: 1, about 20: 1, about 21 : 1, about 22: 1, about 23: 1, about 24: 1, about 25: 1, about 26: 1, about 27: 1, about 28: 1, and about 29: 1.

8. The method according to claim 6, wherein the first monomer is MAA, and wherein the molar ratio of DAP: MAA is in the range between 1 :2 and 14: 1, more preferably in the range between 1 :2 and 9: 1, such as a ratio selected from about 1 : 1, about 3: 1, and about 8: 1.

9. The method according to any one of the preceding claims, wherein the cations are divalent cations.

10. The method according to any one of the preceding claims, wherein the cations are copper ions.

11. The method according to any one of claims 1-4 and 6-10, wherein the cross-linked polymer is comprised within a purification column.

12. The method according to any one of the preceding claims, wherein the cross-linked polymer is in the form of particles having an average size in the range of 50 nm to 63 pm.

13. The method according to claim 12, wherein the average size is selected from: about 100 nm, about 200 nm, about 250 nm, about 500 nm, about 750 nm, about 1 pm, about 2 pm, about 3 pm, about 4 pm, about 5 pm, about 10 pm, about 20 pm, about 30 pm, about 40 pm, about 50 pm, and about 60 pm.

14. The method according to any one of claims 1-11, wherein the cross-linked polymer is in the form of particles and / or aggregates of particles, and wherein the particles or the aggregates of particles have a size above 63 pm, such as above 100 pm, such as above 200 pm, and such as above 500 pm.

15. The method according to any one of the preceding claims, wherein the cross-linked polymer has been polymerized in the absence of a template molecule.

16. The method according to any one of claims 1-14, wherein the cross-linked polymer has been polymerized in the presence of a template molecule.

17. The method according to claim 16, wherein the template molecule is L-Phe or an L-Phe derivative exposing a phenylalanine motif, such as Gly-Phe.

18. The method according to any one of claims 1-14 and 16-17, wherein the cross-linked polymer is attached to, enclosing, and / or covalently bound to, a template molecule, such as the template molecule L-Phe or an L-Phe derivative exposing a phenylalanine motif, such as Gly-Phe.

19. A cross-linked polymer comprising :- a first monomer selected from: (vinylbenzyl)trimethylammonium chloride (VBTA), acrylic acid, itaconic acid, and 3-Butene-l,2,3-tricarboxylic acid, preferably VBTA, and- a second monomer which is l,4-bis(acryloyl)piperazine (DAP).

20. The cross-linked polymer according to claim 19, wherein the cross-linked polymer is as defined in any one of claims 3, 6-7, and 11-18.

21. A composition comprising the cross-linked polymer as defined in any one of claims 1-3, 6-8, and 12-20.

22. The composition according to claim 21, wherein the composition is a dry composition, such as a powder, capsule or a tablet.

23. The composition according to claim 21, comprising a solvent, such as water, and wherein the cross-linked polymer is suspended in the solvent.

24. A method for producing a cross-linked polymer, the method comprising a step of: polymerizing a mixture comprising- a first monomer comprising at least one vinyl group and i. at least one carboxylic acid or II. at least one benzyl group, and- a second monomer which is l,4-bis(acryloyl)piperazine (DAP), and optionally- a template molecule, in the presence of a catalyst, so as to obtain the cross-linked polymer, which is optionally attached to, enclosing, and / or covalently bound to, the template molecule.

25. The method according to claim 24, wherein the polymerization occurs at a pH between 6 and 8.

26. The method according to claim 24 or 25, wherein the first monomer is selected from methacrylic acid (MAA), acrylic acid, itaconic acid, 3-Butene-l,2,3-tricarboxylic acid, and (vinylbenzyl)trimethylammonium chloride (VBTA).

27. The method according to any one of claims 24-26, comprising a step of washing and drying the cross-linked polymer obtained from the polymerization step, optionally wherein the drying is performed by freeze-drying, spray drying, vacuum drying or by drying in an oven.

28. A method of treating a pathology associated with an aberrant presence of cations in a subject in need thereof, the method comprising administering to the subject the cross-linked polymer as defined in any one of claims 1-3, 6-8, and 12-20, or the composition according to any one of claims 21-23.

29. The cross-linked polymer according to claim 19 or 20 or the composition according to any one of claims 21-23, insofar as they relate to claim 19 or 20, for use as a medicament.

30. The cross-linked polymer defined in any one of claims 1-3, 6-8, and 12-20, or the composition according to any one of claims 21-23, for use in a method of treating a pathology associated with an aberrant presence of cations.

31. A method of treating a pathology associated with an aberrant presence of cations in a subject in need thereof, the method comprising contacting a bodily fluid, such as blood, derived from said subject with the cross-linked polymer as defined in any one of claims 1-3, 6-8, and 12-20 ex vivo, and returning the resulting cation-reduced bodily fluid to said subject.

32. The cross-linked polymer as defined in any one of claims 1-3, 6-8, and 12-20 for use in a method of treating a pathology associated with an aberrant presence of cations in a subject in need thereof, the method comprising contacting a bodily fluid, such as blood, derived from said subject with the cross-linked polymer ex vivo, and returning the resulting cation-reduced bodily fluid to said subject.

33. The method according to claim 28 or the cross-linked polymer or composition for the use according to claim 29 or 30, wherein the cross-linked polymer is administered orally.

34. The method according to claim 28 or 31 or the cross-linked polymer or composition for the use according to any one of claims 29, 30 and 32, wherein the pathology is Wilson's disease.

35. A device suitable for binding cations of a composition, said device comprising a solid phase having the cross-linked polymer as defined in any one of claims 1-3, 6-8, and 12-20 anchored to it.

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