TW202502392A - A functionalized polymer for use in a method for treating radioactive contamination - Google Patents

Abstract

The present disclosure relates to methods for treating radioactive contamination, in particular internal radioactive contamination, said method comprising orally administering, in a subject at risk or suspected of radioactive contamination, an efficient amount of a functionalized polymer, wherein said functionalized polymer is a specific statistic chitosan polymer wherein a part of the monomeric units are functionalized with a chelating moiety which complexes said radionuclide.

Description

Functionalized polymers for use in methods of treating radioactive contamination

The present invention relates to a method for treating radioactive contamination, particularly internal radioactive contamination, comprising orally administering an effective amount of a functionalized polymer to a subject who is at risk of or suspected of being radioactively contaminated, wherein the functionalized polymer is a specific statistical chitosan polymer, a portion of the monomer units of which are functionalized with a chelating portion that complexes with radionuclides.

In the last few decades, unfortunately, there have been more and more situations that could lead to the spread of radioactive isotopes in densely populated areas, including the use of nuclear weapons in the context of terrorism or war or incidents at nuclear facilities such as Fukushima or Chernobyl (Rump A. et al. , 2018, Mil. Med. Res. , 5 , 27). Nuclear explosions can lead to the release of more than 400 radioactive isotopes, 40 of which are essential for human life due to their long radioactive half-life and their high concentrations in organs (Vergara VB et al. , 2021, Nutrients , 13 , 2545), which emphasizes the importance of developing a decorporating agent that can show efficiency against large amounts of isotopes.

Of these isotopes, four are of particular concern: cesium-137 (t _1/2 = 30.17 y), cobalt-60 (t _1/2 = 5.27 y), strontium-90 (t _1/2 = 28.9 y), and uranium-238 (t _1/2 = 4.47.10 ⁹ y) [2]. Brambilla et al. recently reviewed a list of radionuclides that are more likely to be found in bombs and represent a wide range of species ( Brambilla S. et al. , 2023, J. Environ. Radioact. , 263 , 107166).

During a nuclear incident, internal contamination may occur through different routes including ingestion, inhalation, skin absorption, or open wounds (Bodin L. et al. , 2021, J. Radiol. Prot. , 41 , S427-S437).

Unfortunately, to date, only three compounds (only one for prophylaxis) have been accepted by the FDA, and they are limited to treatments with iodine (KI for prophylactic thyroid protection in 1978), cesium (Prussian blue in 2003), and terbium and aluminum (Zn-DTPA or Ca-DTPA in 2004). Unfortunately, DTPA has poor solubility and bioavailability and can only be administered intravenously (iv) or by nebulization. In addition, compared with many other endogenous cations, the chelation constant for U(VI) is relatively low (log K = 16) (Li Y. et al. , 2023, J. Inorg. Biochem. , 238 , 112034; Wang X. et al ., 2019, Nature Com. , 10 , 2570). In addition, it is rapidly excreted after intravenous injection and toxicity issues preclude its use in a prophylactic manner. Compared with oral formulations, intravenous injection limits its use in large-scale population treatment situations (Cassatt DR et al. , 2008, Radiat. Res . 170 , 540-548). If contamination occurs, different studies have shown the importance of starting treatment as soon as possible (emergency treatment) (Rump A. et al. , 2021, Mil. Med. Res. , 8 , 3). Simple administration is required, and the use of prophylactic drugs may be better if it is to protect people who are about to be contaminated or soldiers and/or workers traveling to contaminated areas.

Recently, a new orally administrable biopolymer (Mex-CD1) was proposed for treating heavy metal (lead and cadmium) contamination in food (Howard JA et al. , 2023, Sci. Rep. , 13 , 2215). This polymer is based on a chitosan backbone grafted with DOTAGA (Natuzzi M. et al. , 2021, Sci. Rep. , 11 , 11948).

WO2019/122790 further discloses a medical device that can be inserted into the body to maintain metal balance to achieve therapeutic purposes, which includes a chelating part for extracting metals.

WO2022/023677 describes a statistical polysaccharide having a weight average molecular weight between 100 kDa and 1000 kDa and its use in a dialysis process to capture at least one metal during magnetic resonance imaging (MRI), in brachytherapy or in a food labeling process to prevent counterfeiting.

Therefore, there remains a need to devise a safe and effective treatment method to prevent radioactive contamination, particularly internal contamination. More specifically, to the knowledge of the inventors, there are no known oral medications for the prophylactic treatment of subjects at risk of exposure to radioactive contaminants that will prevent the absorption of said radioactive contaminants and thus reduce the toxicity associated with internal contamination. There are also no known oral medications for the prophylactic treatment of subjects at risk of exposure to radioactive contaminants that further reduce the toxicity associated with external contamination through their ROS scavenging capabilities.

The inventors have now demonstrated that certain specific polymers, when orally administered to mice, are capable of at least reducing the toxicity associated with oral exposure to radionuclides, in particular the toxicity associated with internal contamination with uranium radioisotopes. More generally, such specific polymers can be used to treat or protect subjects from internal and/or external contamination.

Summary of the invention

The present invention relates to a functionalized polymer for use in a method for treating radioactive contamination, the method comprising orally administering an effective amount of a functionalized polymer to a subject at risk of or suspected of being radioactively contaminated, wherein the functionalized polymer is a functionalized statistical chitosan of formula (I) having a weight average molecular weight between 100 kDa and 1000 kDa: (I) wherein each Rc is a chelating moiety, each Z is independently a linker that is a single bond or a hydrocarbon chain containing 1 to 12 carbon atoms, the hydrocarbon chain being linear or branched and optionally containing one or more unsaturations and one or more impurity atoms preferably selected from nitrogen, oxygen, sulfur, halogens, x is between 0.005 and 0.7, preferably between 0.05 and 0.7, y is between 0.005 and 0.5, preferably between 0.01 and 0.2, the ratio of y/x is greater than or equal to 0.01, preferably greater than or equal to 0.02, and the sum of x + y is greater than or equal to 0.15, preferably greater than or equal to 0.30.

In a specific embodiment, the functionalized polymer is a functionalized statistical chitosan of formula (II): (II) wherein _Rc1 is a chelating moiety, and _Rc2 is another chelating moiety or a radical oxygen species (ROS) scavenging moiety, _Z1 and _Z2 are linkers that are single bonds or hydrocarbon chains containing 1 to 12 carbon atoms, and _Z1 and Z2 are ₂ are the same or different, the hydrocarbon chain is linear or branched and optionally contains one or more unsaturation and one or more impurity atoms preferably selected from nitrogen, oxygen, sulfur, halogens, x is between 0.005 and 0.7, preferably between 0.05 and 0.7, more preferably between 0.2 and 0.6, y=z+w, and y is between 0.01 and 0.7, preferably between 0.05 and 0.3, the ratio of y/x is greater than or equal to 0.05, preferably greater than or equal to 0.15, and the sum of x+y is greater than or equal to 0.15, preferably greater than or equal to 0.30, more preferably greater than or equal to 0.35.

In a specific embodiment, the functionalized statistical chitosan has the following formula (IV): (IV) wherein x is between 0.2 and 0.6, more preferably between 0.25 and 0.4, typically about 0.3, and y=z+w, and y is between 0.05 and 0.3, more preferably between 0.1 and 0.2, typically about 0.12.

Preferred examples of the functionalized polymer according to the present invention include but are not limited to the functionalized polymer of the following formula (III): (III) wherein x is between 0.25 and 0.4, typically about 0.3, and y is between 0.05 and 0.2, typically about 0.07, or, preferred examples of the functionalized polymer according to the present invention include but are not limited to functionalized polymers of formula (V) or formula (VI): (V) wherein x is from 0.2 to 0.6, more preferably from 0.25 to 0.4, usually about 0.3, and y=z+w is from 0.05 to 0.3, more preferably from 0.1 to 0.2, usually about 0.12, (VI) wherein x is between 0.2 and 0.6, more preferably between 0.25 and 0.4, typically about 0.3, and y=z+w, and y is between 0.05 and 0.3, more preferably between 0.1 and 0.2, typically about 0.12.

Definition

The terms used in this article are defined below with their meanings.

As used herein, the term "about" or "approximately" means that the following numerical value may vary by ± 20%, preferably ± 10%, more preferably ± 5%, even more preferably ± 2%, even more preferably ± 1%.

Unless otherwise defined, herein, "%" means weight percent (wt% or w/w%).

As used herein, unless otherwise indicated, the terms "treating" or "treatment" mean reversing, alleviating, inhibiting the progression of, or preventing, alleviating, or reversing, alleviating, inhibiting the progression of, or preventing one or more symptoms of, a disorder or condition to which the term applies. More preferably, in the context of the present invention, "treating" may include reducing toxicity associated with exposure to radioactive contaminants, such as by inhalation or ingestion, wound contamination, or percutaneous absorption. In specific embodiments, the term "preventing" means at least significantly reducing toxicity associated with exposure to radioactive contaminants, wherein the treatment is administered orally prior to exposure to the radioactive contaminant.

As used herein in the context of the methods of the present invention, the term "radioactive contaminants," also referred to as "radioactive materials" or "radioactive contaminants," refers to atoms and cations of radionuclides that are toxic to humans. For example, radioactive materials may be spread throughout an area following an industrial nuclear reactor accident or the use of a radioactive diffusion device (also known as a "dirt bomb") or nuclear weapon in a war zone.

Examples of such radioactive contaminants may be cesium-137, cesium-134, strontium-89, strontium-90, yttrium-90, ruthenium-103, ruthenium-106, ruthenium-106, zirconium-95, barium-140, rhenium-140, cobalt-60, cobalt-58, iron-55, manganese-54, zinc-65, silver-110m, aluminum-241, uranium-239, uranium-238, uranium-240, uranium-234, uranium-235, uranium-238, radium-226, Caliberium-252, thorium-244 and thorium-232, especially in the event of an accident at a nuclear power plant (https://www.irsn.fr/savoir-comprendre/crise/principaux-radionucleides-rejetes-cas-daccident-affectant-centrale#.Y_XaVyaZPEY).

Examples of such radioactive contaminants may be aluminum-241, cesium-137, strontium-90, yttrium-90, pelargonium-238, pelargonium-239, pelargonium-240, pelargonium-241, particularly in the case of nuclear weapons contamination (McClellan, RO Health Effects of Nuclear Weapons and Releases of Radioactive Materials. In Handbook of Toxicology of Chemical Warfare Agents ; Elsevier, 2020; pp 707–743. https://doi.org/10.1016/B978-0-12-819090-6.00043-X.).

In the case of contamination from a radioactive dispersion device (or "bomb"), examples of such radioactive contaminants may be proton-201, strontium-90, cobalt-60, cesium-137, iridium-192, radium-226, and aluminum-241 (Brambilla, S.; Nelson, MA; Brown, MJ 2023, Journal of Environmental Radioactivity 2023 , 263, 107166).

As used herein, the term "internal contamination" refers to the accumulation of radioactive contaminants in target organs within the body of a human subject. Internal contamination may occur when the radioactive contaminants are ingested, inhaled, or absorbed through the skin or from contaminated wounds.

As used herein, the term "external contamination" refers to the toxicity that occurs when radioactive contaminants in the form of dust, powder, or liquid come into contact with human skin, hair, or clothing. Contact with radioactive contaminants is external to the human body. External contamination produces reactive oxygen species in the body, which may cause DNA damage and related diseases, such as cancer. Toxicity associated with external contamination can be addressed by reactive oxygen species (ROS) scavengers.

As used herein, the term "ROS scavenger" refers to a compound that is capable of reacting with reactive oxygen species and other reactive free radicals. Such compounds are typically thiol-containing molecules, polyphenols, or vitamins. Examples of ROS scavenger compounds include, but are not limited to, amifostine, WR-1065, lipoic acid, genistein, cilantro, N-acetylcysteine, cysteine, cysteamine, vitamin A, β-carotene, vitamin C, recilisib sodium.

As used herein, the term "effective amount" or "therapeutically effective amount" of a compound refers to an amount of a compound that will elicit a biological or medical response in a subject, such as improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing symptoms. In a specific embodiment, such a therapeutically effective amount is sufficient to prevent toxicity associated with radioactive contaminants, such as toxicity associated with irradiation of the subject's body after internal contamination with the radioactive contaminants.

The terms "patient", "subject", "individual", etc. are used interchangeably herein and refer to human beings. In some embodiments, patients, subjects, or individuals in need of treatment include those who have been exposed to radioactive contaminants or are suspected of having been exposed to radioactive contaminants. In some embodiments, patients, subjects, or individuals in need of treatment include those who are at risk of exposure to radioactive contaminants.

Functionalized polymers according to the present invention

The functionalized polymer used in the treatment method according to the present invention is a functionalized chitosan having a statistical macromolecular structure with a weight average molecular weight between 100 kDa and 1000 kDa of formula (I): (I) wherein each Rc is a chelating moiety, each Z is independently a linker that is a single bond or a hydrocarbon chain containing 1 to 12 carbon atoms, the hydrocarbon chain being linear or branched and optionally containing one or more unsaturations, and may contain one or more impurity atoms preferably selected from nitrogen, oxygen, sulfur, halogens, x is between 0.005 and 0.7, preferably between 0.05 and 0.7, y is between 0.01 and 0.7, preferably between 0.05 and 0.2, the ratio of y/x is greater than or equal to 0.05, preferably greater than or equal to 0.15, and x + The sum of y is greater than or equal to 0.15, preferably greater than or equal to 0.30, and more preferably greater than or equal to 0.35.

As used herein, the term "chelating portion" or "chelating agent" or "chelating moiety" is used to define a chemical structure or moiety that exhibits a relatively high affinity for certain elements and exhibits at least two coordination sites. The affinity enables the chelating agent to chelate radioactive contaminants, i.e., radionuclides, and more specifically, radionuclides selected from the group consisting of manganese-54, iron-55, cobalt-58, cobalt-60, zinc-65, strontium-89, strontium-90, yttrium-90, zirconium-95, ruthenium-103, ruthenium-106, thorium-106, silver-110m, cesium-112, thorium-113, thorium-114, thorium-115, thorium-116, thorium-117, thorium-118, thorium-119, thorium-120, thorium-121, thorium-123, thorium-124, thorium-125, thorium-127, thorium-128, thorium-129, thorium-130, thorium-131, thorium-132, thorium-133, thorium-134, thorium-135, thorium-136, thorium-137, thorium-138, thorium-139, thorium-139, thorium-137 -134, cesium-137, barium-140, rhenium-140, iridium-192, proton-201, radium-226, thorium-232, uranium-234, uranium-235, uranium-238, uromadium-238, uromadium-239, uromadium-240, uromadium-241, aluminum-241, ruthenium-244, and californium-252.

In specific embodiments, the chelating moiety exhibits a high chelating affinity for radioactive isotopes of uranium, thrombium, proton, cesium, strontium, iridium, and cobalt, or mixtures thereof.

The main function of chelators is to "neutralize" the radioactive contaminants by maintaining chelation with these metals and preventing them from reacting and interacting with other biological materials. Chelators can present a clamp-like structure or moiety with two or more opposing parts formed by chemical groups with negative charges (sulfhydryl, keto, carboxyl, hydroxyl, etc.) or neutral charges (amine groups) in the biological environment. These groups are spaced accordingly to allow the metal ions to be comfortably accommodated within their structure.

As used herein, the term "independent" for the Rc moiety (i.e., the chelating moiety) means that each Rc of the functionalized statistical chitosan of formula (I) can be a different or the same chelating moiety. For example, each Rc is the same, that is, the entire functionalized statistical chitosan has only one Rc, or the entire functionalized statistical chitosan can have more than one Rc, that is, two, three, four, five, or even n different Rcs. They are all independently selected from the group with the chelating moiety. The same applies to the Z-linker, there can be multiple Z-linkers, and they can be the same or different from each other.

In one embodiment, in formula (I), x is between 0.005 and 0.6; y is between 0.1 and 0.9; the ratio of y/x is greater than 0.16; and the sum of x + y is greater than 0.30.

In one embodiment, the functionalized statistical chitosan of the present invention has a complexation constant of at least 10 ¹⁵ for the d or f transition element.

In one embodiment, the functionalized statistical chitosan of formula (I) is the functionalized statistical chitosan of formula (II): (II) wherein _Rc1 is a chelating moiety, and _Rc2 is another chelating moiety or a ROS scavenging moiety, _Z1 and _Z2 are linkers that are single bonds or hydrocarbon chains containing 1 to 12 carbon atoms, and _Z1 and Z2 are ₂ are the same or different, the hydrocarbon chain is linear or branched and optionally contains one or more unsaturation and one or more impurity atoms preferably selected from nitrogen, oxygen, sulfur, halogens, x is between 0.005 and 0.7, preferably between 0.05 and 0.7, more preferably between 0.2 and 0.6, y=z+w, and y is between 0.01 and 0.7, preferably between 0.05 and 0.3, the ratio of y/x is greater than or equal to 0.05, preferably greater than or equal to 0.15, and the sum of x+y is greater than or equal to 0.15, preferably greater than or equal to 0.30, more preferably greater than or equal to 0.35. In another embodiment, in Formula (II), x is between 0.005 and 0.6; y is between 0.1 and 0.9; the ratio of y/x is greater than 0.3; and z is between 0.5 and 1.

Mode (I) And (II) The polymer is preferably Rc part (Rc , Rc ₁ , Rc ₂ )

As used herein, the term "Rc moiety" refers to the Rc moiety of formula (I), and the term " _Rc1 and _Rc2 moieties" refers to _Rc1 and _Rc2 of formula (II) when _Rc2 is present. According to the present invention, the _Rc1 group is a chelating moiety and the _Rc2 group is a chelating moiety or a ROS scavenging moiety. In other words, the Rc and _Rc1 moieties can chelate one or more metals by forming a complex; alternatively, _Rc2 can also chelate one or more metals by forming a complex, or _Rc2 is a ROS scavenging moiety.

In a specific embodiment, each of the Rc, _Rc1 and optional _Rc2 moieties may contain more than two coordination positions. Preferably, the coordination position is a nitrogen or oxygen atom. Advantageously, each of the Rc, _Rc1 and optional _Rc2 moieties contains 4 to 8 coordination positions, more preferably 6 to 8 coordination positions, and each of the Rc, _Rc1 and optional _Rc2 moieties more preferably contains 6 coordination positions.

As used herein, the term "coordination site" refers to a single functional group capable of complexing a radionuclide. For example, an amine functional group represents a coordination site by forming a coordination bond between a nitrogen atom and a metal, and an isohydroxyoxime functional group also represents a coordination site by forming a coordination bond between the oxygen of the carbonyl unit and by forming a covalent bond with the oxygen of the N-oxide unit, thereby forming a five-membered ring.

In one embodiment, with respect to the functionalized succinic acid of formula (I), each Rc moiety is independently selected from DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (1,4,7-triazacyclononane-1-pentanedioic acid-4,7-diacetic acid), DOTAGA (2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)pentanedioic acid), DOTAM (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraazacyclododecane-2-yl)pentanedioic acid), N OTAM (1,4,7-tetra(aminomethyl)-1,4,7-triazacyclononane), DOTP (1,4,7,10-tetra(methylenephosphonic acid)), NOTP (1,4,7-tetra(methylenephosphonic acid)-1,4,7-triazacyclononane), TETA (1,4,8,11-tetra(aminomethyl)-cyclotetradecane-N,N',N'',N'''-tetraacetic acid), TETAM (1,4,8,11-tetra(aminomethyl)-cyclotetradecane-N,N',N'',N'''-tetra(aminomethyl)), DTPA (diethylenetriaminepentaacetic acid), Bz-DFO (benzyldeferroic acid The Rc group is preferably selected from the group consisting of DOTAGA, Bz-DFO, DFO, DOTAM and DTPA, and the Rc group is more preferably DOTAGA.

In another embodiment, with respect to the functionalized statistical chitosan of formula (II), _Rc1 and _Rc2 are independently selected from the group consisting of DOTA, NOTA, NODAGA, DOTAGA, DOTAM, NOTAM, DOTP, NOTP, TETA, TETAM, DTPA, Bz-DFO and DFO, preferably selected from the group consisting of DOTAGA, Bz-DFO, DFO, DOTAM and DTPA.

In one embodiment, the chelating moiety is selected from the group consisting of:

In one embodiment, with respect to the functionalized statistical chitosan of formula (I), the Rc group is DOTAGA, preferably, z/y=1.

In another embodiment, with respect to the functionalized statistical chitosan of formula (II), the _Rc1 group is DOTAGA and the _Rc2 group is Bz-DFO. In one embodiment, with respect to the functionalized statistical chitosan of formula (II), _Rc2 is a ROS scavenging moiety independently selected from the group consisting of 4-thiobutylamidine, amifostine, WR-1065, lipoic acid, genistein, cilantro, N-acetylcysteine, cysteine, cysteamine, vitamin A, β-carotene, vitamin C, vitamin E, and rexib.

In a specific embodiment of the functionalized statistical chitosan of formula (II), _Rc1 is a chelating moiety (e.g., a DOTA or DOTAGA moiety), and _Rc2 is selected from the following ROS scavenger moieties:

An example of a functionalized statistical chitosan of formula (II) is formula (V): (V) wherein x is between 0.2 and 0.6, more preferably between 0.25 and 0.4, typically about 0.3, and y=z+w, and y is between 0.05 and 0.3, more preferably between 0.1 and 0.2, typically about 0.12.

An example of a functionalized statistical chitosan of formula (II) is formula (VI): (VI) wherein x is between 0.2 and 0.6, more preferably between 0.25 and 0.4, typically about 0.3, and y=z+w, and y is between 0.05 and 0.3, more preferably between 0.1 and 0.2, typically about 0.12.

Mode (I) And (II) The polymer is preferably Z Connector (Z , Z ₁ , Z ₂ )

As used herein, the term "Z linker" refers to the Z linker of formula (I), and the term " _Z1 and _Z2 linkers" refers to the _Z1 and _Z2 linkers of formula (II) when the _Z2 linker is present.

The choice of the Z, Z ₁ , Z ₂ linkers in formula (I) and (II) depends essentially on the Rc, Rc ₁ , Rc ₂ moieties and the metal to be chelated. In fact, especially because of stearic acid, the Rc, Rc ₁ , Rc ₂ moieties can be more or less close to the 6-membered ring of the nitrogen of the glucosamine unit.

In formula (I), each Z is independently a single bond or a linker of a hydrocarbon chain containing 1 to 12 carbon atoms, which is linear or branched and optionally contains one or more unsaturations and one or more heteroatoms preferably selected from nitrogen, oxygen, sulfur, and halogens.

In one embodiment, in formula (I), each Z is independently selected from the group consisting of a single bond, a straight or branched alkyl chain having 1 to 12 carbon atoms, and a straight or branched alkenyl chain having 2 to 12 carbon atoms, wherein the alkyl and alkenyl chains may be substituted by one or more C6-C10 aryl groups and/or one or more heteroatoms or selected from -O-, -S-, -C(O)-, -NR'-, -C(O)NR'-, -NR'-C(O)-, -NR'-C( The alkyl and alkenyl chains may be substituted by one or more groups selected from the group consisting of halogen, -OR', -COOR', -SR', and -NR'2, and each R' is independently H or a C1-C6 alkyl group.

Advantageously, in formula (I), each Z is independently selected from the group consisting of a single bond and a straight or branched alkyl chain having 1 to 12 carbon atoms, wherein the alkyl chain may be interrupted by one or more C6-C10 aromatic groups and/or one or more heteroatoms or groups selected from the group consisting of -O-, -S-, -C(O)-, -NR'-, -C(O)NR'-, -NR'-C(O)-, -C(S)NR'-, -NR'-C(S)-NR', and each R' is independently H or a C1-C6 alkyl group.

In one embodiment, each Z is an alkyl chain having 1 to 12 carbon atoms.

In another embodiment, each Z is polyethylene glycol (PEG).

Advantageously, in formula (II), Z ₁ and Z ₂ are single bonds or hydrocarbon chains containing 1 to 12 carbon atoms, which are linear or branched and may have one or more unsaturations and one or more heteroatoms preferably selected from nitrogen, oxygen, sulfur, halogens.

In one embodiment, in formula (II), _Z1 and _Z2 are independently selected from the group consisting of a single bond, a straight or branched alkyl chain having 1 to 12 carbon atoms, and a straight or branched alkenyl chain having 2 to 12 carbon atoms, wherein the alkyl and alkenyl chains may be substituted by one or more C6-C10 aryl groups and/or one or more heteroatoms or selected from -O-, -S-, -C(O)-, -NR'-, -C(O)NR'-, -NR'-C(O)-, -NR'-C( The alkyl and alkenyl chains may be substituted by one or more groups selected from the group consisting of halogen, -OR', -COOR', -SR', and -NR'2, and each R' is independently H or a C1-C6 alkyl group.

In one embodiment, in formula (II), _Z1 and _Z2 are independently selected from the group consisting of a single bond and a straight or branched alkyl chain having 1 to 12 carbon atoms, wherein the alkyl chain may be inserted by one or more C6-C10 aromatic groups and/or one or more heteroatoms or groups selected from the group consisting of -O-, -S-, -C(O)-, -NR'-, -C(O)NR'-, -NR'-C(O)-, -C(S)NR'-, -NR'-C(S)-NR', and each R' is independently H or a C1-C6 alkyl group.

In one embodiment, _Z1 and/or _Z2 is an alkyl chain having 1 to 12 carbon atoms.

In another specific embodiment, Z ₁ and/or Z ₂ are polyethylene glycol (PEG).

Mode (I) And (II) The functionalization statistics of chitosan show the optimal monomer unit arrangement

The functionalized statistical chitosan of formula (I) used in the method of the present invention comprises three different monomer units, namely N-acetyl glucosamine type A unit, glucosamine type B uni and glucosamine type C unit, which are functionalized with a chelating moiety (Rc type) linked to the nitrogen of glucosamine via a linker (Z type).

Functionalized statistical chitosan is a statistical polymer. In other words, the order of the individual A-type, B-type, and C-type monomer units is random.

The functionalized statistical chitosan of formula (II) of the present invention comprises four different monomer units, namely, an A-type N-acetylglucosamine unit, a B-type glucosamine unit and two C-type glucosamine units (i.e., C1 and C2 glucosamine units), wherein the units are functionalized by a chelating portion ( _Rc1 or _Rc2 type) connected to the nitrogen of glucosamine via a linker ( _Z1 or _Z2 type).

The functionalized statistical chitosan of formula (II) is a statistical polymer. In other words, the order of the various A-type, B-type, C1-type, and C2-type monomer units is random.

In formula (I) and formula (II), x represents the ratio of A units, and x is between 0.005 and 0.7, preferably between 0.05 and 0.7, more preferably between 0.2 and 0.6, even more preferably between 0.25 and 0.4, and usually about 0.3. In one embodiment, x is between 0.025 and 0.075, more preferably between 0.04 and 0.06, and usually about 0.05.

In formula (I) and formula (II), y represents the proportion of C-type units and y is between 0.01 and 0.7, preferably between 0.05 and 0.2. In one embodiment, y is between 0.03 and 0.2, preferably between 0.05 and 0.1, even more preferably between 0.07 and 0.08, typically about 0.072. In another embodiment, y is between 0.05 and 0.3, more preferably between 0.1 and 0.2, typically about 0.15 or 0.12.

In another embodiment, when _Rc1 and _Rc2 of formula (II) are independently selected from the group consisting of DOTA, NOTA, NODAGA, DOTAGA, DOTAM, NOTAM, DOTP, NOTP, TETA, TETAM, DTPA, Bz-DFO and DFO, preferably selected from the group consisting of DOTAGA, Bz-DFO, DFO, DOTAM and DTPA, and y is between 0.05 and 0.3, more preferably between 0.1 and 0.2, typically about 0.12, _Rc1 is between 0.06 and 0.08, typically about 0.07, and _Rc2 is between 0.04 and 0.06, typically about 0.05.

The remaining monomer units in formula (I) and formula (II) are B units. Therefore, in formula (I) and formula (II), the ratio of B units is equal to 1-x-y.

According to the present invention, in formula (I) and formula (II), the ratio y/x is greater than or equal to 0.05, preferably greater than or equal to 0.15. In fact, the effectiveness of the functionalized statistical chitosan is determined by the number of chelated sites, which is directly related to the amount of metal required, for example, to reduce local inflammation caused by and/or induced by metal imbalance and to reduce oxidative stress in subjects in need thereof.

In order to be effective when administered to a subject in need thereof in the form of a solution, the functionalized statistical chitosan must be soluble at physiological pH (i.e., pH between 4.8 and 8). To this end, the sum of x + y may be greater than or equal to 0.15, preferably greater than or equal to 0.30, and more preferably greater than or equal to 0.35.

The combination of the specific ratio between the amount of A units and the amount of C units and the sum of the ratio of A units and the ratio of C units allows for sufficient chelation and solubility to allow the functionalized statistical chitosan to be used in the treatment of diseases caused by local inflammation caused by metal imbalance and increased oxidative stress in subjects in need thereof.

According to the present invention, z/y is 0.5 to 1. In other words, the C-type unit may exclusively be a unit having Z ₁ as a linker and having Rc ₁ as a group having a chelating moiety.

In one embodiment, the functionalized statistical chitosan is selected from the following functionalized statistical chitosan: a functionalized statistical chitosan of formula (II), wherein z/y=1, _Rc1 is DOTAGA and _Z1 is a single bond; a functionalized statistical chitosan of formula (II), wherein z/y=1, _Rc1 is DTPA and _Z1 is a single bond; and a functionalized statistical chitosan of formula (II), wherein 0.5≤z/y＜1, _Rc1 is DOTAGA and _Z1 is a single bond, and Rc2 is Bz-DFO and Z1 is a single bond. ₂ is selected from the group consisting of a single bond and a straight or branched alkyl chain having 1 to 12 carbon atoms, wherein the alkyl chain may be inserted by one or more C6-C10 aryl groups and/or one or more heteroatoms or groups selected from the group consisting of -O-, -S-, -C(O)-, -NR'-, -C(O)NR'-, -NR'-C(O)-, -C(S)NR'-, -NR'-C(S)-NR', and each R' is independently H or a C1-C6 alkyl group.

Formula for the treatment method of the present invention (III) Or (IV) The preferred functionalized polymer

In a more specific embodiment, the functionalized statistical chitosan has the following formula (III): (III) wherein x is between 0.25 and 0.4, typically about 0.3, and y is between 0.05 and 0.2, typically about 0.07.

In another embodiment, the functionalized statistical chitosan has the formula (III), wherein x is between 0.025 and 0.075, more preferably between 0.04 and 0.06, typically about 0.05, and y is between 0.05 and 0.3, more preferably between 0.1 and 0.2, typically about 0.15.

In certain embodiments, the weight average molecular weight of the functionalized polymer is between 100 kDa and 1000 kDa, preferably between 150 kDa and 750 kDa, more preferably between 200 kDa and 500 kDa, even more preferably between 250 kDa and 400 kDa, and even more preferably about 300 kDa.

In one embodiment, the functionalized statistical chitosan is soluble in aqueous solution at physiological pH (i.e., pH between 4.8 and 8) and corresponds to the formula: (DS _DOTAGA (%) + 3.5) * (DA (%) + 8) ≥ 150, wherein DS is the degree of substitution of DOTAGA, and DA is the degree of acetylation of the functionalized statistical chitosan.

In another embodiment, the functionalized statistical chitosan has the following formula (IV): (IV) wherein x is between 0.2 and 0.6, more preferably between 0.25 and 0.4, typically about 0.3, and y=z+w, and y is between 0.05 and 0.3, more preferably between 0.1 and 0.2, typically about 0.12.

Mode (I) And (II) Synthesis of functionalized polymers

The polymers of formula (I) and (II) (including polymers of formula (III) and (IV)) can be synthesized using the methods disclosed in patent WO 2022/023677 and reference Natuzzi, M., Grange, C., Gréa, T. et al. Feasibility study and direct extraction of endogenous free metallic cations combining hemodialysis and chelating polymer. Sci Rep 11, 19948 (2021 ).

Functionalized polymer drug compositions

The functionalized polymer is used in a form formulated with one or more pharmaceutically acceptable excipients or administered orally.

As used herein, the term "pharmaceutically acceptable excipient" refers to an inactive substance added with a drug and is a part of a formulation mixture. Pharmaceutically acceptable excipients include fillers, solvents, diluents, carriers, adjuvants, dispensing agents, and sensing agents, delivery agents, such as preservatives, disintegrants, wetting agents, emulsifiers, suspending agents, thickeners, sweeteners, flavoring agents, fragrances, antibacterial agents, bactericides, lubricants, long-acting control agents, antioxidants, and glidants. Their selection and appropriate proportion depend on the properties, administration method, and dosage.

Any suitable excipient known to those of ordinary skill in the art for use in pharmaceutical compositions may be used in the compositions described herein.

In one embodiment, the pharmaceutical composition may be in a liquid form suitable for oral administration, such as an aqueous solution of the functionalized polymer. In another embodiment, the pharmaceutical composition may be a solid dosage form suitable for oral administration. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In a preferred embodiment, the pharmaceutical composition is a capsule or a tablet.

In one embodiment, the release of the contents of the capsule or tablet can be immediate release or modified release, such as delayed release, targeted release or extended release. In a preferred embodiment, the solid dosage form is an immediate release dosage form.

A second object of the present invention relates to an oral formulation comprising the functionalized polymer of the present invention and one or more pharmaceutically acceptable excipients.

In one embodiment, the pharmaceutically acceptable excipient comprises a filler, a disintegrant, a lubricant, and a glidant.

Filler

Fillers (also called diluents, dilutants, or thinners) are substances added to a drug substance to make it suitable for oral administration (e.g., capsules, tablets). Fillers themselves should not cause any pharmacological reactions in humans. Examples of fillers include mannitol, microcrystalline cellulose, lactose monohydrate, anhydrous lactose, corn starch, xylitol, sorbitol, sucrose, dicalcium phosphate, maltodextrin, and gelatin.

Disintegrants

Disintegrants are added to oral solid dosage forms to aid in their deagglomeration. The function of a disintegrant is to cause the solid dosage form to disintegrate rapidly upon contact with moisture. Disintegration is generally considered the first step in the dissolution process. Examples of disintegrants include modified starches such as sodium glycolate, sodium carboxymethyl starch, and pregelatinized starches, cross-linked polymers such as cross-linked polyvinyl pyrrolidone (crospovidone) or cross-linked sodium carboxymethyl cellulose (crospovidone sodium), and calcium silicate.

Lubricant

Lubricants are substances used in tablet and gel formulations to reduce friction. Lubricants facilitate the extrusion of tablets from the matrix, thus preventing scratches on its surface. Lubricants can be divided into two categories based on their properties: a) fats and fat-like substances; b) powdered substances. Powdered substances are more suitable than fat-like substances because fat-like substances affect the solubility and chemical stability of tablets. Powdered lubricants are made by grinding granules into powder. They provide a constant rate of mass outflow from the hopper into the matrix for tableting, thereby ensuring the accuracy and consistency of the amount of the raw drug substance.

Examples of lubricants include magnesium stearate, hydrogenated castor oil, glyceryl behenate, calcium stearate, zinc stearate, mineral oil, silicone oil, sodium lauryl sulfate, L-leucine, and sodium stearyl fumarate.

Slip Agent

Glidants are mixed with the formulation to enhance the flow characteristics of the tablet core blend material. During the early stages of compression, the glidant is mixed into the particle arrangement of the tablet powder mixture to improve flow and uniformity within the film cavity of the tablet press. Glidants promote the flow of tablet particles by reducing the friction between the particles. The effect of the glidant on the flow of the particles depends on the particle size and shape of the particles and the glidant. Above a certain concentration, the glidant will actually function to inhibit flow. In tablet manufacturing, glidants are usually added before compression. Examples of glidants include colloidal silica, starch, magnesium stearate and talc.

Any suitable excipient for pharmaceutical compositions known to those of ordinary skill in the art may be used in the compositions described herein.

In a specific embodiment, the unit dose of the functionalized polymer is between 0.1 mg and 500 mg, preferably between 1 mg and 100 mg, such as between 2 mg and 10 mg, such as about 5 mg.

How to use

The functionalized polymers and drug compositions thereof as described in the previous section can be used as drugs in a method for treating radioactive contamination, the method comprising oral administration to a subject at risk of or suspected of being radioactively contaminated.

In a specific embodiment, the present invention relates to a method for treating radioactive contamination, the method comprising orally administering an effective amount of a functionalized polymer to a subject at risk of or suspected of being radioactively contaminated, wherein the functionalized polymer is a functionalized statistical chitosan of formula (I) having a weight average molecular weight between 100 kDa and 1000 kDa: (I) wherein each Rc is a chelating moiety, each Z is independently a linker that is a single bond or a hydrocarbon chain containing 1 to 12 carbon atoms, the hydrocarbon chain being linear or branched and optionally containing one or more unsaturations and one or more impurity atoms preferably selected from nitrogen, oxygen, sulfur, halogens, x is between 0.005 and 0.7, preferably between 0.05 and 0.7, y is between 0.005 and 0.5, preferably between 0.01 and 0.2, the ratio of y/x is greater than or equal to 0.01, preferably greater than or equal to 0.02, and the sum of x + y is greater than or equal to 0.15, preferably greater than or equal to 0.30.

In a specific embodiment, the present invention relates to a method for preparing a drug for treating radioactive contamination in a subject using a functionalized polymer, wherein the functionalized polymer is a functionalized statistical chitosan of formula (I) having a weight average molecular weight between 100 kDa and 1000 kDa: (I) wherein each Rc is a chelating moiety, each Z is independently a linker that is a single bond or a hydrocarbon chain containing 1 to 12 carbon atoms, the hydrocarbon chain being linear or branched and optionally containing one or more unsaturations and one or more impurity atoms preferably selected from nitrogen, oxygen, sulfur, halogens, x is between 0.005 and 0.7, preferably between 0.05 and 0.7, y is between 0.005 and 0.5, preferably between 0.01 and 0.2, the ratio of y/x is greater than or equal to 0.01, preferably greater than or equal to 0.02, and the sum of x + y is greater than or equal to 0.15, preferably greater than or equal to 0.30.

Any functionalized polymer as described in the previous section, in particular functionalized polymers of formula (I), formula (II), formula (III), formula (IV), and formula (V), can be advantageously selected for use in the treatment methods disclosed herein.

In a specific embodiment, the subject is a mammal at risk of or suspected of being radioactively contaminated, such as a human subject.

For example, radioactive contamination may result from an accident involving a nuclear reactor, an industrial source, or a medical source. Radioactive contamination may also result from criminal or terrorist activity. Radioactive contamination may result from the spread of radioactive material over an area (usually a combat zone) by a "radioactive dispersion device" ("dump") using conventional (non-nuclear) explosives.

Thus, “at-risk” subjects could include military personnel in combat zones, rescue workers, or any subject who is susceptible to being in a potentially contaminated area.

Therefore, "suspected" subjects may include subjects who have been in contaminated areas or suspected contaminated areas.

In a specific embodiment, a subject suitable for the disclosed treatment method is a human subject at risk of or suspected of being contaminated with radioactive substances, wherein the radioactive substances are selected from manganese-54, iron-55, cobalt-58, cobalt-60, zinc-65, strontium-89, strontium-90, yttrium-90, zirconium-95, ruthenium-103, ruthenium-106, niobium-106, ruthenium-107, ruthenium-108, ruthenium-109, ruthenium-110, ruthenium-111, ruthenium-112, ruthenium-113, ruthenium-114, ruthenium-115, ruthenium-116, ruthenium-117, ruthenium-118, ruthenium-119, ruthenium-121, ruthenium-123, ruthenium-124, ruthenium-125, ruthenium-126, ruthenium-127, ruthenium-128, ruthenium-129, ruthenium-130, ruthenium-131, ruthenium-132, ruthenium-133, ruthenium-134, ruthenium-135, ruthenium-136, ruthenium-137, ruthenium-138, ruthenium-139, ruthenium-139 Radioactive contamination from the radionuclides of 110m, 134c, 137c, 140b, 140r, 192i, 201m, 226r, 232m, 234u, 235u, 238u, 238b, 239u, 240u, 241u, 241a, 244b and 252c.

In a specific embodiment, a subject suitable for the disclosed treatment method is a human subject at risk of or suspected of being radioactively contaminated by radionuclides selected from the group consisting of radioisotopes of uranium, thorium, molybdenum, cesium, strontium, iridium and cobalt, or mixtures thereof.

In specific embodiments, subjects suitable for the disclosed treatment methods are human subjects at risk or suspected of internal radioactive contamination. Internal contamination may occur through inhalation of radioactive contaminants, ingestion, or through contaminated wounds. Indeed, without wishing to be bound by any theory, the inventors believe that the functionalized polymers used herein advantageously act locally by retaining a portion of the ingested radionuclides in the gastrointestinal tract, such as by chelation, thereby preventing them from crossing the intestinal barrier, such as after accidental radioactive contamination (usually from nuclear reactors, industrial sources, or medical sources), criminal or terrorist activities, or in war zones, before they are assimilated. Thus, the fact that the polymer does not cross the intestinal barrier allows for local action in the gastrointestinal tract and avoids the usual adverse effects of the presence of chelated moieties in the systemic compartment as observed in prior art treatments. Furthermore, it is well known that even by inhalation, a portion of the radionuclides may cross the gastrointestinal barrier and then be captured by the functionalized polymer. Finally, by limiting the passage of the intestinal barrier, it is also possible to adequately excrete any radionuclides that have been internalized, for example by other routes such as the inhalation route, and avoid or reduce their accumulation in the target organs.

Thus, in one embodiment, an effective amount is an amount of the functionalized polymer administered orally that reduces or prevents gastrointestinal absorption of the radioactive contaminant and thereby reduces internal contamination by ingestion or inhalation.

In specific embodiments, for example, the effective amount of the functionalized polymer used in the treatment method reduces the absorption of the radioactive contaminant by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100% in a subject exposed to the radioactive contaminant compared to a subject not treated with the functionalized polymer of the present invention and similarly exposed to the radioactive contaminant.

In a specific embodiment, for example, compared to not being treated with the functionalized polymer of the present invention and similarly exposed to a metal selected from the group consisting of manganese-54, iron-55, cobalt-58, cobalt-60, zinc-65, strontium-89, strontium-90, yttrium-90, zirconium-95, ruthenium-103, ruthenium-106, ruthenium-106, silver-110m, cesium-134, cesium-137, barium-140, yttrium-140, iridium-192, ruthenium-201, radium-226, thorium-232, The effective amount of the functionalized polymer used in the method of treating a subject exposed to radionuclides of uranium-234, uranium-235, uranium-238, uranium-238, uranium-239, uranium-240, uranium-241, aluminum-241, proton-244, and californium-252 reduces the uptake of the radionuclides by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% in the subject exposed to the radionuclides.

In a specific embodiment, the functionalized polymer reduces the amount of a metal selected from the group consisting of cesium-137, cesium-134, strontium-89, strontium-90, yttrium-90, ruthenium-103, ruthenium-106, ruthenium-106, zirconium-95, barium-140, rhenium-140, cobalt-60, cobalt-58, iron-55, manganese-54, zinc-65, silver-110m, Absorption of radionuclides from the group consisting of aluminum-241, uranium-239, uranium-238, uranium-239, uranium-240, uranium-234, uranium-235, uranium-238, radium-226, californium-252, uranium-244 and thorium-232, in subjects exposed to external and/or internal contamination as a result of an accident at a nuclear power plant. (https://www.irsn.fr/savoir-comprendre/crise/principaux-radionucleides-rejetes-cas-daccident-affectant-centrale#.Y_XaVyaZPEY)

In specific embodiments, the functionalized polymer reduces the uptake of radionuclides selected from the group consisting of aluminum-241, cesium-137, strontium-90, yttrium-90, thallium-238, thallium-239, thallium-240, and thallium-241, wherein the subject is particularly in the context of contamination caused by nuclear weapons.

In specific embodiments, the functionalized polymer reduces the uptake of radionuclides selected from the group consisting of protons-201, strontium-90, cobalt-60, cesium-137, iridium-192, radium-226, and aluminum-241, wherein the subject is exposed to external and/or internal contamination caused by a radioactive dispersal device (or "bomb").

In more specific embodiments, the functionalized polymer reduces the absorption of a uranium isotope and the subject is exposed to radioactivity of the uranium isotope, such as due to a nuclear accident or the use of a radiation dispersal device (also known as a "bomb").

One skilled in the art would consider various factors including the type and severity of the internal contamination (or risk of internal contamination), the patient, etc. to determine a specific dosing regimen, such as the appropriate dose of the functionalized polymer, the appropriate duration, and the frequency of dosing.

A typical daily dose may be 0.1 mg to 500 mg, preferably 1 mg to 100 mg, more preferably 2 mg to 10 mg, even more preferably about 5 mg, preferably functionalized statistical amount of chitosan.

In a specific embodiment, the functionalized polymer is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before the first exposure to the radioactive contaminant. Preferably, the functionalized polymer is administered at least 12 hours before the first exposure to the radioactive contaminant.

In a specific embodiment, the functionalized polymer is administered to the subject once, twice, or three times daily, as needed, for example, for at least 3, 4, 5, 6, or 7 days after exposure to radioactive contamination.

Functionalized polymers can be administered in combination with one or more additional drugs, including, for example: Free chelators to accelerate elimination by renal excretion, such as Ca-DTPA or Zn-DTPA, blockers and diluents to reduce uptake by target tissues, such as potassium iodide (KI) for radioactive iodine, mobilizing agents, i.e. compounds that enhance and increase the natural turnover process of radioactive contaminants and accelerate their release from tissues, including propylthiouracil, ammonium chloride, diuretics, expectorants and inhalants, parathyroid extract and corticosteroids or other drugs.

As used herein, "combination" refers to a fixed combination in the form of a dosage unit, or a combined administration, wherein the compounds of the present invention and the combination partner (e.g., another drug as described above) can be administered independently at the same time or separately at time intervals, particularly when these time intervals allow the combination partner to show a synergistic effect, such as a synergistic effect. Single components can be packaged in a kit or packaged separately. Single ingredients can be packaged in a kit or packaged separately. One or two ingredients (e.g., powders or liquids) can be reconstituted or diluted to the desired dose before administration. The terms "co-administration" or "combination administration" and the like used herein are intended to cover the administration of selected combination partners to a single subject (e.g., a patient) in need thereof, and are intended to include treatment regimens in which the agents do not necessarily have to be administered by the same route of administration or at the same time.

Specific embodiments

The specific embodiments of the method of use disclosed herein are described as follows:

Example 1: A functionalized polymer for use in a method for treating radioactive contamination, the method comprising orally administering an effective amount of a functionalized polymer to a subject at risk of or suspected of being radioactively contaminated, wherein the functionalized polymer is a functionalized statistical chitosan of formula (I) having a weight average molecular weight between 100 kDa and 1000 kDa: (I) wherein each Rc is a chelating moiety, each Z is independently a linker that is a single bond or a hydrocarbon chain containing 1 to 12 carbon atoms, the hydrocarbon chain being linear or branched and optionally containing one or more unsaturations and one or more impurity atoms preferably selected from nitrogen, oxygen, sulfur, halogens, x is between 0.005 and 0.7, preferably between 0.05 and 0.7, y is between 0.005 and 0.5, preferably between 0.01 and 0.2, the ratio of y/x is greater than or equal to 0.01, preferably greater than or equal to 0.02, and the sum of x + y is greater than or equal to 0.15, preferably greater than or equal to 0.30.

Example 2: The functionalized polymer used in Example 1, wherein the functionalized polymer is a functionalized statistical chitosan of formula (II): (II) wherein _Rc1 is a chelating moiety, and _Rc2 is another chelating moiety or a ROS scavenging moiety, _Z1 and _Z2 are linkers that are single bonds or hydrocarbon chains containing 1 to 12 carbon atoms, and _Z1 and Z2 are ₂ are the same or different, the hydrocarbon chain is linear or branched and optionally contains one or more unsaturation and one or more impurity atoms preferably selected from nitrogen, oxygen, sulfur, halogens, x is between 0.005 and 0.7, preferably between 0.05 and 0.7, more preferably between 0.2 and 0.6, y=z+w, and y is between 0.01 and 0.7, preferably between 0.05 and 0.3, the ratio of y/x is greater than or equal to 0.05, preferably greater than or equal to 0.15, and the sum of x+y is greater than or equal to 0.15, preferably greater than or equal to 0.30, more preferably greater than or equal to 0.35.

Example 3: A functionalized polymer of formula (II) used according to Example 2, wherein x is between 0.005 and 0.6; y is between 0.1 and 0.9; the ratio y/x is greater than 0.3; and z is between 0.5 and 1.

Example 4: A functionalized polymer of formula (II) used according to Example 2 or 3, wherein _Rc2 is a ROS scavenging moiety independently selected from the group consisting of 4-thiobutylamidine, amifostine, WR-1065, lipoic acid, genistein, cilantro, N-acetylcysteine, cysteine, cysteamine, vitamin A, β-carotene, vitamin C, vitamin E, and rexib.

Embodiment 5: The functionalized polymer of formula (II) used according to any one of embodiments 2 to 4, wherein the functionalized polymer is a functionalized statistical chitosan of formula (V) or formula (VI): (V) wherein x is between 0.2 and 0.6, more preferably between 0.25 and 0.4, typically about 0.3, and y=z+w, and y is between 0.05 and 0.3, more preferably between 0.1 and 0.2, typically about 0.12. (VI) wherein x is between 0.2 and 0.6, more preferably between 0.25 and 0.4, typically about 0.3, and y=z+w, and y is between 0.05 and 0.3, more preferably between 0.1 and 0.2, typically about 0.12.

Example 6: A functionalized polymer used according to any one of Examples 1 to 5 to prevent internal contamination by radioactive pollutants.

Example 7: The functionalized polymer used according to any one of Examples 1 to 6, further serving as a reactive oxygen species (ROS) scavenger to reduce toxicity associated with external radioactive contamination.

Embodiment 8: The functionalized polymer used according to any one of embodiments 1 to 7, wherein the radioactive contaminant is selected from aluminum-241, californium-252, cesium-141, cobalt-60, uranium-232, uranium-233, uranium-234, uranium-235, uranium-236, uranium-238, uranium-238, uranium-239, uranium-240, uranium-241, uranium-242, uranium-243, uranium-244, uranium-245, uranium-246, uranium-247, uranium-248, uranium-249, uranium-250, uranium-251, uranium-252, uranium-253, uranium-254, uranium-255, uranium-256, uranium-257, uranium-258, uranium-259, uranium-261, uranium-263, uranium-264, uranium-265, uranium-266, uranium-267, uranium-268, uranium-269, uranium-270, uranium-271, uranium-272, uranium-273, uranium-274, uranium-275, uranium-276, uranium-277, uranium-278, uranium-279, uranium-280, uranium-281, uranium-283, uranium-28 The radionuclides are: proton-239, proton-240, proton-241, strontium-89, strontium-90, cesium-135, cesium-137, thorium-144, zirconium-93, zirconium-95, ruthenium-106, technetium-99, tin-126, iridium-192, proton-210, radium-226, thorium-232 and proton-201.

Embodiment 9: The functionalized polymer used according to Embodiment 8, wherein the radioactive contaminant is a radionuclide selected from the group consisting of radioisotopes of uranium, thorium, molybdenum, cesium, strontium, iridium and cobalt, or a mixture thereof.

Embodiment 10: The functionalized polymer used according to any one of embodiments 1 to 9, wherein oral administration of the functionalized polymer reduces or prevents gastrointestinal absorption of the radioactive contaminant.

Embodiment 11: The functionalized polymer used according to any one of embodiments 1 to 10, wherein the chelating portion is selected from the group consisting of DOTA, NOTA, NODAGA, DOTAGA, DOTAM, NOTAM, DOTP, NOTP, TETA, TETAM, DTPA, Bz-DFO, DFO and mixtures thereof, preferably selected from the group consisting of DOTAGA, Bz-DFO, DFO, DOTAM, DTPA and mixtures thereof.

Embodiment 12: The functionalized polymer used according to any one of embodiments 1 to 11, wherein the weight average molecular weight of the functionalized statistical chitosan is between 200 kDa and 500 kDa.

Example 13: The functionalized polymer used according to any one of Examples 1 and Examples 6 to 12 has the following formula (III): (III) wherein x is between 0.25 and 0.4, typically about 0.3, and y is between 0.05 and 0.2, typically about 0.07.

Embodiment 14: The functionalized polymer used according to any one of embodiments 1 to 13, wherein the functionalized polymer is administered to the subject in a unit dose of between 0.1 mg and 500 mg, preferably between 1 mg and 100 mg, more preferably between 2 mg and 10 mg, and even more preferably about 5 mg.

Embodiment 15: The functionalized polymer for use according to any one of embodiments 1 to 14, wherein the functionalized polymer is administered once, twice or three times a day.

Embodiment 16: The functionalized polymer used according to any one of embodiments 1 to 15, wherein the functionalized polymer is administered at least 6 hours before at least 12 hours of the first exposure to the radioactive contaminant.

Embodiment 17: The functionalized polymer for use according to any one of embodiments 1 to 16, wherein the subject will, is or has been exposed to radioactive contamination due to an accident involving a nuclear reactor.

Embodiment 18: A functionalized polymer for use according to any one of embodiments 1 to 17, wherein the subject will, is or has been exposed to radioactive contamination because the subject was present in an area during or after an incident involving a nuclear reactor.

Embodiment 19: The functionalized polymer for use according to any one of embodiments 1 to 18, wherein the subject is at risk of exposure to radioactive contamination due to being present in a combat zone during or after the use of a nuclear explosion or a radioactive dispersal device.

The present invention is further described in detail below with reference to embodiments, but the present invention is not limited thereto.

Example

Synthesis of the functionalized polymer of the present invention:

MEX-CD1 ( Chitosan as a polymer and DOTAGA As a chelating part )

In the first step, 60 g of chitosan, 4 L of ultrapure water, and 45 mL of glacial acetic acid were added to a 10 L reactor and stirred at pH 4.5 ± 0.5 for 16 hours. A light yellow solution was observed.

In the second step, 1.2 L of 1,2-propylene glycol was added to the light yellow solution obtained from the first step and stirred for 1 hour. 600 mL of a solution of 14 mL of acetic anhydride in 1,2-propylene glycol was added slowly over 10 minutes to obtain uniform acetylation along the polymer chains. The medium was maintained and stirred for 4 hours.

The acetylation rate can be determined by elemental analysis. The unacetylated monomer of the polymer (monomer B) presents a molar mass of 161.2 gmol ^-1 (C ₆ NO ₄ H ₁₁ ), while the acetylated monomer of the polymer (monomer A) presents a molar mass of 203.2 gmol ^-1 (C ₈ NO ₅ H ₁₃ ). The elemental analysis of the polysaccharide obtained after the second step is as follows: C 39.22%; H 7.55%; N 6.77%, corresponding to an acetylation rate of 29% confirmed by ¹ H NMR (x=0.29).

In the third step, 2 L of the solution obtained after the second step are placed under stirring. 120 g of DOTAGA anhydride are added and stirred for 16 hours. After the end of this reaction, the solution is diluted 10 times in ultrapure water and purified by tangential purification using a 100 kDa membrane. After the first dilution step, which ends with a volume of 16 L, the solution is filtered with 480 L of 0.1 M acetic acid, maintaining a constant volume of 16 L, and then filtered with 320 L of ultrapure water. This purification ends with reconcentration to a volume of 8 L. HPLC-UV can verify whether unreacted DOTAGA has been successfully removed from the solution. The solution was then filtered using a nylon filter (0.4 um) at a concentration of 10 g/L before freeze drying.

¹ H NMR can confirm the percentage of DOTAGA(y) functionalized monomers on a polymer of known acetylation percentage x.

The amount of grafted DOTAGA was confirmed by spectrophotometric UV dosage at 295 nm. MEX-CD1 contained 0.345 mmol of DOTAGA per gram of polymer.

The molar fraction x of the N-acetylglucosamine repeating unit measured by ¹ H-NMR was x=0.29, the molar fraction y of the DOTAGA grafted repeating unit measured by ¹ H-NMR was y=0.075, and the copper dosage was measured by HLPC-SEC.

MEX-DTPA ( Chitosan as a polymer and DTPA As a chelating part )

In the first step, 1 g of chitosan, 66 mL of ultrapure water, and 0.84 mL of glacial acetic acid were added and stirred at pH 4.5 ± 0.5 for 16 hours. A light yellow solution was observed.

In the second step, 20 mL of 1,2-propylene glycol was added to the light yellow solution obtained from the first step and stirred for 1 hour. 30 mL of a solution of 0.704 mL of acetic anhydride in 1,2-propylene glycol was added slowly over 10 minutes to obtain uniform acetylation along the polymer chains. The medium was maintained and stirred for 4 hours.

In the third step, 0.8 g of DTPA dianhydride is added to the previous solution and stirred for 16 hours. After the end of this reaction, the solution is diluted 10 times in ultrapure water and purified by tangential purification using a 100 kDa membrane. After the first step of dilution, which ends with a volume of 1 L, the solution is filtered with 5 L of 0.1 M acetic acid, maintaining a constant volume of 1 L, and then filtered with 5 L of ultrapure water. This purification ends with reconcentration to a volume of ~100 mL. HPLC-UV can verify whether the unreacted DTPA has been successfully removed from the solution.

The amount of grafted DTPA was confirmed by spectrophotometric UV quantification at 295 nm. MEX-DTPA contained 0.21 mmol of DTPA per gram of polymer.

The molar fraction x of the N-acetylglucosamine repeating unit measured by ¹ H-NMR was x=0.58, and the molar fraction y of the DTPA-grafted repeating unit measured by ¹ H-NMR was y=0.045.

The MEX-DTPA synthesized herein can be used in the processing methods described in the present invention.

MEX-DOTAM ( Chitosan as a polymer and DOTAM As a chelating part )

In the first step, 1 g of chitosan, 66 mL of ultrapure water, and 0.84 mL of glacial acetic acid were added and stirred at pH 4.5 ± 0.5 for 16 hours. A light yellow solution was observed.

In the second step, 20 mL of 1,2-propylene glycol was added to the light yellow solution obtained from the first step and stirred for 1 hour. 30 mL of a solution of 0.704 mL of acetic anhydride in 1,2-propylene glycol was added slowly over 10 minutes to obtain uniform acetylation along the polymer chains. The medium was maintained and stirred for 4 hours. This solution was then purified by tangential purification using a 100 kDa membrane and 7 L of ultrapure water to remove the solvent and re-concentrate to ~100 mL. The pH of this solution was raised to 7 ± 0.1 using NaOH. 8 mL of DMSO was added to the solution to form an environment of 5% DMSO in water and stirred for 1 hour.

In the third step, 11 mL of a 100 g/L solution of DOTAM NHS ester in DMSO are added within 10 minutes. The solution is then diluted two-fold with ultrapure water and stirred for 16 hours. At the end of this reaction, the solution is diluted 10-fold in ultrapure water and purified by tangential purification using a 100 kDa membrane. After the first dilution step, which ends with a volume of 1 L, the solution is filtered with 5 L of 0.1 M acetic acid, maintaining a constant volume of 1 L, and then filtered with 5 L of ultrapure water. This purification ends with reconcentration to a volume of ~100 mL. HPLC-UV can verify whether unreacted DOTAM has been successfully removed from the solution.

The amount of grafted DOTAM was confirmed by spectrophotometric UV dosing at 295 nm. MEX-DOTAM contained 0.049 mmol of DOTAM per gram of polymer.

The molar fraction x of the N-acetylglucosamine repeating unit measured by ¹ H-NMR was x=0.58, and the molar fraction y of the DOTAM-grafted repeating unit measured by ¹ H-NMR was y=0.0097.

The MEX-DOTAM synthesized herein can be used in the treatment methods described in the present invention.

MEX-CD-TBA ( Chitosan as a polymer, DOTAGA As a chelating moiety and 4- Thiobutylamidine as the substituted part )

In a 1 L round bottom flask, 750 mg of MEX-CD1 (synthesized as above) was dissolved in 500 mL of ultrapure water and stirred for 2 hours. 50 mL of this solution was transferred to a 100 mL round bottom flask, and the pH was adjusted with 10 ^-1 M sodium hydroxide solution. 40 mg of 2-iminothiolane was added to the solution and stirred at room temperature for 48 hours. The mixture was then purified by tangential filtration with 50 mL of 5 mM hydrochloric acid using a 100 kDa cassette at a flow rate of 150 mL/min, and then the volume was reduced to 30 mL; the volume was then increased to 100 mL and reduced to 30 mL twice. The same operation was performed once more using 5 mM and 1% NaCl solution. Finally, the same operation was performed twice using MilliQ water. The polymer was freeze-dried for further use.

The obtained polymer has the following structure:

Example 1 : Biodistribution of oral MEX-CD1 in mice

Materials and Methods

Solution preparation:

Solution 1: 10 g/L MEX-CD1

Freeze-dried MEX-CD1 was dissolved in ultrapure water at a concentration of 10 g/L.

Solution 2: 5 g/L MEX-CD1 + 3.5 g/L NaCl

Dissolve 3.5 g/L NaCl in ultrapure water. Then dissolve freeze-dried MEX-CD1 in this solution to a final concentration of 5 g/L. Sterilize this solution at 121 ºC for 20 minutes.

Solution A: Concentrated saline solution for in vitro laboratory testing

Ultrapure water contains 45 g/L glucose, 263 g/L NaCl, 3.35 g/L KCl, 6.24 g/L CaCl ₂ , 2.14 g/L MgCl ₂ , 10.8 g/L acetic acid

Solution B: 0.1 M acetate buffer, pH 4.6

The acetate buffer solution contained 11.4 mL of acetic acid (MS grade), 15.4 g of ammonium acetate, and 2 L of ultrapure water.

Experiment Guide (protocol) :

Guide 1 : High Pressure Liquid Chromatography-Mass Spectrometry (HPLC-MS)

The eluent used in this method was acetate buffer (solution B, prepared with MS grade certified product), the injection volume was 10 uL, and the flow rate was fixed at 4 mL/min. The column used was SEC Polysep GFC-P 4000 series. The HPLC was coupled to an inductively coupled plasma mass spectrometer (ICP-MS) to detect the selected isotopes.

Guide 2 : Vivaspin Ultrafiltration Experiment

Use a 20 mL Vivaspin centrifuge tube with an inner membrane of 30 kDa. Perform centrifugation at 4000 rpm for 10-30 minutes each time. After each centrifugation, collect the solution that passes through the membrane for each sample, which is referred to as the undernatanr. Ideally, ~0.5 mL of supernatant will remain in one centrifugation and collect this supernatant for ICP-MS analysis.

Samples containing the original solution, supernatant and lower supernatant were analyzed by ICP-MS to determine the concentration of the metal of interest in each solution. The samples were diluted (at least) 10 times with 1% HNO _3. An internal standard (Indium) was added to each sample to a final concentration of 2 ppb In. The analysis was performed in Kinetic Energy Discrimination (KED) mode or in Standard mode, which introduces a flux of Helium in the chamber. Additional analyses were performed for other more sensitive elements when analyzed in KED, but most samples were run in Standard mode: 208Pb, 206Pb, 111Cd, 112Cd, 114Cd.

Calibration curves for lead and cadmium were constructed over the range of 0.01 to 10 ppb to allow the system to convert the counts per second recorded for each sample into concentrations in ppb.

Eight mice were orally dosed with 0.2 mL of solution 4. Two mice were sacrificed at each time point (1, 2, 4, 24 h). Fluorescence studies were performed using a CCD camera with the following parameters: 2 sets of flashlights for excitation at 633 nm and 470 nm, 2 sets of filters at 680 ± 20 nm and 520 ± 20 nm, followed by digestion of organs in nitric acid for gadoid quantification using ICPMS.

result

Biodistribution characterization by fluorescence

After 1, 2, 4, and 24 hours, no fluorescence was observed in the kidney, liver, brain, spleen, heart, urine, lung, bone, skin, blood, and muscle at any time point. After 1, 2, and 4 hours, fluorescence was observed only in the stomach, intestines, and colon. A small degree of autofluorescence was seen in the stomach of control mice.

Fluorescence results showed that MEX-CD1 was only present in the stomach, intestines, and colon (digestive tract) (Table 1). organ Sacrifice time The range of fluorescence intensity of cyanine 5.5 (x1000) Stomach 1 hour 817.8 – 1210.7 2 hours 350.6 – 1301.3 4 hours 575.3 – 652.8 24 hours 66.0 – 990.4 Control group 278.3 Intestine 1 hour 311.2 – 314.0 2 hours 485.4 – 584.4 4 hours 66.9 – 516.3 24 hours 0.3 – 178.4 Control group 6.4 Colon 1 hour 3.5 - 76.4 2 hours 154.0 – 203.0 4 hours 167.2 – 615.8 24 hours 96.6 – 126.0 Control group 184.9

Some autofluorescence was observed in the stomachs of control mice, which explains the residual fluorescence in both stomachs after 24 hours.

by ICP-MS Quantitative biodistribution

As shown in Figure 1, there were two experimental mice at each time point, so the average gadolinium percentage was taken. It was then plotted with the standard deviation in Figure 1. The trend shows that within the first hour, most of the MEX-CD1 was found in the intestine, and at 24 hours, all of the MEX-CD1 was excreted from the digestive system of the mice.

Other organs were evaluated, all of which had gadolinium concentrations less than 1% of the administered drug and were therefore considered zero.

Therefore, the oral medication MEX-CD1 It remains in the digestive tract, does not cross the intestinal membrane and enters the blood, and is completely excreted after one day.

Example 2 :

Materials and Methods

Solution preparation:

Solution 1 : 10 g/L MEX-CD1

Freeze-dried MEX-CD1 was dissolved in ultrapure water at a concentration of 10 g/L.

Solution 2 : 10 g/L chitosan

Chitosan (pharmaceutical grade and animal origin; weight and number average molar mass w = 2.583*10 ⁵ g/mol, Mn = 1.323*10 ⁵ g/mol, respectively; degree of acetylation = 6 ± 0.5%) was dissolved in ultrapure water and acetic acid (pH = 4.6) at a concentration of 10 g/L.

Solution 3 : 10 g/L sterilized MEX-CD1

Dissolve freeze-dried MEX-CD1 in ultrapure water at a concentration of 10 g/L. Sterilize the solution at 121 ºC for 20 minutes.

Solution 4 : 3.5 g/L NaCl for sterilization

Dissolve 3.5 g/L NaCl in ultrapure water. Then dissolve freeze-dried MEX-CD1 in this solution to a final concentration of 5 g/L. Sterilize this solution at 121 ºC for 20 minutes.

Solution A : 0.1 M acetate buffer, pH 4.6

The acetate buffer solution contained 11.4 mL of acetic acid (MS grade), 15.4 g of ammonium acetate (MS grade), and 2 L of ultrapure water.

Experiment Guide:

Guide 1 : High Pressure Liquid Chromatography-Mass Spectrometry (HPLC-ICP-MS)

The eluent used in this method was acetate buffer (solution A, prepared with MS grade certified product), the injection volume was 10 uL, and the flow rate was fixed at 4 mL/min. The column used was SEC Polysep GFC-P 4000 series. The HPLC was coupled to an inductively coupled plasma mass spectrometer (ICP-MS) to detect the selected isotopes.

Guide 2 : Vivaspin Ultrafiltration Experiment

Use a 20 mL Vivaspin centrifuge tube with an inner membrane of 50 kDa. Perform centrifugation at 4000 rpm for 10-30 minutes each time. After each centrifugation, collect the solution that passes through the membrane for each sample (hereinafter referred to as the supernatant), and refill the centrifuge tube with the original solution. Repeat this cycle until all the original solution passes through the membrane. Ideally, ~1 mL of supernatant will remain and be collected for ICP-MS analysis.

Samples containing the original solution, supernatant, and lower supernatant were analyzed by ICP-MS to determine the concentration of the metal of interest in each solution. The lower supernatant and original solution of the sample were diluted 10 ⁴ times with 1% HNO ₃ , and the supernatant of the sample was diluted 10 ⁴ times with 1% HNO _3. An internal standard (indium) was added to each sample to a final concentration of 2 ppb of In. The analysis was performed in standard mode.

Calibration curves for Cs, Ir, Th, Tl, Sr, and U in the range of 0.001 to 0.2 ppb were constructed to enable the system to convert the counts per second recorded for each sample into concentrations in ppb.

1. Oral administration of drugs in mice exposed to uranium MEX-CD1

This study was conducted in C57Bl/6 young male mice. Mice were gavaged daily for 7 days with solution 3, a dose of 50 mg/kg of MEX-CD1. On the 4th day, the uranium salt UO ₂ (NO ₃ ) ₂ *6H ₂ O was orally administered in a saline solution, a dose of 50 mg/kg of pure uranium. The total dose of uranium was given in two doses, 4 hours apart. There were three control groups:

1) Mice in which all test substances were replaced by solution 4 ( healthy control group );

2) Mice treated with MEX-CD1 and Solution 4 instead of uranium salt ( MEX-CD1 control group - to exclude any MEX-CD1 toxicity);

3) Mice in which MEX-CD1 was replaced with uranium salt and solution 4 ( positive control group - mice poisoned with uranium, not treated).

All animals were sacrificed on day 8, and blood, kidneys, and spleen were collected for uranium detection, biochemical, and hematological studies. During the study, mice were observed and weighed daily.

result

Daily medication MEX-CD1 and / Or uranium mice 8 Weight change within a day

There were no significant changes in body weight between the MEX-CD1 and healthy controls (Figure 2), supporting the innocuous nature of MEX-CD1. For both groups given uranium, weight loss due to uranium toxicity was confirmed; however, MEX-CD1 appeared to accelerate weight recovery in mice, as observed in the last three days of the study (Figure 3).

Oral medication MEX-CD1 and / Uranium levels in blood and tissues of mice fed with

As suggested by Bing, D.; Jing-Wei, H.; Lian, D.; Liang, D. Biodistribution of Uranium in Mice and Influencing Factor Preliminary Discussion. Asian J. Chem . 2014 , 26 (13), 3932–3936. https://doi.org/10.14233/ajchem.2014.16043, no uranium was detected in the blood on day 4 after dosing (study day 8). However, uranium accumulated in the kidney and spleen, and MEX-CD1 partially (in the kidney) or completely (in the spleen) prevented uranium accumulation in these organs (Figure 4). These results show a reduction in uranium content in both the kidney and spleen (the reduction was more pronounced in the spleen) after administration of MEX-CD1, which is the first sign that MEX-CD1 is able to reduce uranium toxicity in the body. The partial preservation of uranium in the kidney can be explained by the fact that uranium accumulates primarily and in large quantities in this organ.

Daily medication MEX-CD1 and / Or uranium mice 8 The hematological status of the queen

Some hematological parameters of mice treated with saline showed acute uranium poisoning (see Figure 5); these changes are indicators of inflammatory processes. For all these hematological parameters, there were no changes in the group that only received MEX-CD1 (MEX-CD1 control group) compared to the healthy control group, thus providing evidence that this treatment had no negative effects on the hematological parameters. In addition, the above figure shows in particular that after oral administration of MEX-CD1, the white blood cell (WBC) and platelet (PLT) counts returned to normal levels.

Daily medication MEX-CD1 and / Or uranium mice 8 Creatinine level after 24 hours

Serum creatinine levels are an indicator of renal function; acute uranium poisoning is associated with elevated creatinine levels in plasma (Vicente-Vicente, L.; Quiros, Y.; Pérez-Barriocanal, F.; López-Novoa, JM; López-Hernández, FJ; Morales, AI Nephrotoxicity of Uranium: Pathophysiological, Diagnostic and Therapeutic Perspectives. Toxicological Sciences 2010 , 118 (2), 324–347. https://doi.org/10.1093/toxsci/kfq178). The results shown in Figure 6 show that creatinine significantly returned to normal levels after administration of MEX-CD1.

2. Detection of in vitro chelation of cobalt and thorium chelated by MEX-CD1 using HPLC-SEC-ICP-MS

result

Solution A In MEX-CD1 Cobalt chelate

The following 13 samples were prepared using a metal mixture of Co, Th, Tl, Cs, Sr, Ir, U at a concentration of 140 ppb or 2800 ppb (in 1% HNO ₃ ) prepared from metal standards purchased from SCP Science, Solution 1 or Solution 2, and Solution A and analyzed by HPLC-MS according to Guide 1. Sample polymer Metal concentration (ppb) 0 1 g/L MEX-CD1 0 1 0.25 2 0.5 3 2.5 4 5 5 12.5 6 25 7 37.5 8 50 9 62.5 10 75 11 87.5 12 100 19 1 g/L chitosan 25 20 37.5 twenty one 50 twenty two 75 twenty three 87.5 twenty four 100

The chromatogram of MEX-CD1 showed a linear increase in the frontal area (specifically corresponding to the polymer) with the cobalt concentration at 15 min (data not shown). In contrast, the chromatograms of chitosan and metal solutions did not show any peaks at this retention time (data not shown).

This demonstrates the ability of MEX-CD1 to chelate cobalt at a concentration of at least 25 ppb at pH 4.5.

Solution A In MEX-CD1 Chelated

The following nine samples were prepared using a 5 ppb Th solution (in 1% HNO ₃ ) prepared with a 1000 ppm metal standard (SCP Science), Solution 1, and Solution A and analyzed by HPLC-MS according to Guide 1. Sample polymer Thion concentration (ppb) 55 1 g/L MEX-CD1 50 56 75 57 100 58 125 59 150 60 175 61 200 62 250 63 500

The results showed that the convex area (especially corresponding to the polymer) increased linearly with the thrombium concentration at 15 minutes (data not shown), and the slope was interrupted after 200 ppb. This proved that MEX-CD1 (1 g/L) could chelate thrombium at a concentration of at least 50 ppb at pH 4.6 (data not shown).

3. use Vivaspin and ICP-MS In different pH Down MEX-CD1 Detection of in vitro capture of strontium, caesium, iridium, barium, thorium, and uranium

Using 1000 ppb Th, Tl, Cs, Sr or U metal standards (in 5% _HNO3 ) or 1000 ppb Ir metal standards (in 10% HCl) purchased from SCP Science and Solution 1, prepare the 9 samples listed in Table 2 below.

Table 2: Sample Metal concentration (ppm) MEX-CD1 concentration (g/L) pH 1 2 0 1 2 2 0 5 3 2 1 1 4 2 1 2 5 2 1 3 6 2 1 4 7 2 1 5 8 2 1 6 9 2 1 7

The pH was adjusted using 1 M NaOH and 1 M HCl. The samples were analyzed experimentally according to Guide 2. Table 3 below summarizes the mass percentage of each metal captured by the polymer.

Table 3: Percentage of metal captured Sample pH Cs(I) Ir(III) Th(IV) Tl(I) Sr(II) U(VI) Metal only 1 2% 5% 6% 2% 2% 3% Metal only 5 4% 7% 100% 2% 0% 4% 1 g/L MEX-CD1 + 2 ppm metals 1 9% twenty two% 57% 11% 0% 16% 1 g/L MEX-CD1 + 2 ppm metals 2 5% 20% 93% 5% -4% 11% 1 g/L MEX-CD1 + 2 ppm metals 3 2% 14% 96% 2% 12% 19% 1 g/L MEX-CD1 + 2 ppm metals 4 19% 30% 97% 19% 29% 59% 1 g/L MEX-CD1 + 2 ppm metals 5 14% 28% 94% 16% 69% 70% 1 g/L MEX-CD1 + 2 ppm metals 6 twenty two% 28% 95% 29% 93% 83% 1 g/L MEX-CD1 + 2 ppm metals 7 51% 54% 97% 56% 95% 93%

This percentage (% _capture ) is calculated using the metal mass m _UN in the supernatant and the initial metal mass _mi in the solution as shown below.

Since the volume of the supernatant is significantly reduced, its measurement is not as accurate as that of the sub-supernatant. In addition, the metal content in the supernatant may be biased by polymer adhesion to the membrane; therefore, the most reliable results are those of the analysis of the sub-supernatant (even though the sub-supernatant generally follows the same trend). For all metals analyzed, the metal content in the sub-supernatant decreased significantly at pH 7, and even at lower pH, this was also the case for Sr, U, and Th.

Figure 7 shows the percentage of the amount of metals captured by MEX-CD1 obtained by analyzing the supernatant (see previous Table 3). It shows that this percentage increases with pH for all metals (Cs, Th, Tl, Ir, Sr, U), which demonstrates the efficacy of MEX-CD1 in capturing these metals significantly and preventing them from passing through the membrane.

Example 3 : Protecting intestinal cells from reactive oxygen species through MEX-CD1

In this study, the caco2 cell line was used as a model of the intestinal epithelial barrier. Cells were cultured in MEM medium with 20% fetal bovine serum. Cells were incubated at 37°C and passaged every 5 days. Confluence was measured every 6 hours for three days using the CellCyte X ^TM live cell imaging device from CYTENA. Cell death was assessed using the same device and a propidium iodide (PI) fluorescent probe (Exc=580-598 nm; Em=612-680 nm).

1. MEX-CD1 right caco2 Cell toxicity studies

Cells were treated with increasing doses of MEX-CD1 (0.5 g/L, 1 g/L, 2 g/L, 5 g/L in culture medium) in 96-well plates. Negative (untreated) and positive (with oxaliplatin) controls were also performed (data not shown for positive controls). At least 6 replicates were performed for each condition. The death index (an indicator of cell death; cell index = PI/confluency) was calculated.

Figure 8 shows the evolution of the mortality index within 78 hours after treatment.

MEX-CD1 was not toxic below a concentration of 2 g/L; significant mortality occurred at 5 g/L. Based on these results, a concentration of 2 g/L was determined as the limiting dose for toxicity for further studies.

2. By MEX-CD1 protect caco2 Cells are protected from _{H2O2 ​} Study on the effects of induced oxidative stress

Caco2 cells were incubated under the following four different conditions: untreated, treated with 2 g/L MEX-CD1, treated with 1 mM H ₂ O ₂ , and treated with 2 g/L MEX-CD1 and 1 mM H ₂ O ₂ .

Figure 9 shows the mortality index for all these conditions.

It showed that after administration of 2 g/L of MEX-CD1, the death index of H ₂ O ₂ -treated cells decreased significantly, indicating that MEX-CD1 has great potential in protecting cells from oxidative stress.

without

Figure 1: Average percentage of Gd administered intra-digestively over 24 hours (Example 1).

Figure 2: Body weight changes of all four groups of mice over 8 days in the study (Example 2.1)

Figure 3: Changes in body weight (ΔBWt) of mice before (left) and after (right) administration of uranium on day 4 of the study (Example 2.1). *p<0.05 compared with each group.

Figure 4: Uranium levels in blood and tissues of mice on day 4 after oral administration of uranium (50 mg/kg), where MexCD1 (50 mg/kg) was administered daily starting 3 days before uranium exposure and continued for 7 days (Example 2.1). Detection thresholds: blood – 0.03 mg/L, kidney – 0.05 ug/g, spleen – 0.15 ug/g; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5: The counts of all white blood cells (WBC) and their separated subpopulations (lymphocytes-LYM, monocytes and eosinophils-MID, granulocytes-GRAN) in mice as indicators of platelet maturity, the count of platelets (PLT) and their mean volume (MPV) (Example 5.1). Compared with each group, *p<0.05, **p<0.01.

Figure 6: Final serum creatinine level in mice (Example 2.1). *p<0.05 compared with each group.

Figure 7: Estimated percentage of the amount of metal captured by MEX-CD1 (% chelated = (m _OG -m _UN )/m _OG ) (Example 2.3).

Fig. 8: Evolution of the death index (=PI/confluency) of caco2 cells after administration of different doses of MEX-CD1 (0 g/L, 0.5 g/L, 1 g/L, 2 g/L, 5 g/L, respectively).

Figure 9: Evolution of the death index (=PI/confluence) of caco2 cells after no drug administration, administration of 2 g/L MEX-CD1 or 1 mM H ₂ O ₂ , or simultaneous administration of 2 g/L MEX-CD1 and 1 mM H ₂ O ₂ .

Claims (13)

A functionalized polymer for use in a method for treating radioactive contamination, the method comprising orally administering an effective amount of the functionalized polymer to a subject at risk of or suspected of being radioactively contaminated, wherein the functionalized polymer is a functionalized statistical chitosan of formula (I) having a weight average molecular weight between 100 kDa and 1000 kDa: (I) wherein each Rc is a chelating moiety, each Z is independently a linker that is a single bond or a hydrocarbon chain containing 1 to 12 carbon atoms, the hydrocarbon chain being linear or branched and optionally containing one or more unsaturations and one or more impurity atoms preferably selected from nitrogen, oxygen, sulfur, halogens, x is between 0.005 and 0.7, preferably between 0.05 and 0.7, y is between 0.005 and 0.5, preferably between 0.01 and 0.2, the ratio of y/x is greater than or equal to 0.01, preferably greater than or equal to 0.02, and the sum of x + y is greater than or equal to 0.15, preferably greater than or equal to 0.30. The functionalized polymer as claimed in claim 1, wherein the functionalized polymer is a functionalized statistical chitosan of formula (II): (II) wherein _Rc1 is a chelating moiety, _Rc2 is another chelating moiety or a radical oxygen species (ROS) scavenging moiety, _Z1 and _Z2 are linkers that are single bonds or hydrocarbon chains containing 1 to 12 carbon atoms, and _Z1 and Z2 are ₂ are the same or different, the hydrocarbon chain is linear or branched and optionally contains one or more unsaturation and one or more impurity atoms preferably selected from nitrogen, oxygen, sulfur, halogens, x is between 0.005 and 0.7, preferably between 0.05 and 0.7, more preferably between 0.2 and 0.6, y=z+w, and y is between 0.01 and 0.7, preferably between 0.05 and 0.3, the ratio of y/x is greater than or equal to 0.05, preferably greater than or equal to 0.15, and the sum of x+y is greater than or equal to 0.15, preferably greater than or equal to 0.30, more preferably greater than or equal to 0.35. The functionalized polymer of claim 2, wherein x is between 0.005 and 0.6, y is between 0.1 and 0.9, the ratio of y/x is greater than 0.3, and z is between 0.5 and 1. A functionalized polymer as described in claim 2 or 3, wherein _Rc2 is a ROS scavenging portion independently selected from the group consisting of 4-thiobutylamidine, amifostine, WR-1065, lipoic acid, genistein, cilantro, N-acetylcysteine, cysteine, cysteamine, vitamin A, β-carotene, vitamin C, vitamin E, and recilisib. The functionalized polymer as described in claim 2, wherein the functionalized polymer is a functionalized statistical chitosan of formula (V) or formula (VI): (V) wherein x is between 0.2 and 0.6, more preferably between 0.25 and 0.4, typically about 0.3, and y=z+w, and y is between 0.05 and 0.3, more preferably between 0.1 and 0.2, typically about 0.12, (VI) wherein x is between 0.2 and 0.6, more preferably between 0.25 and 0.4, typically about 0.3, and y=z+w, and y is between 0.05 and 0.3, more preferably between 0.1 and 0.2, typically about 0.12. The functionalized polymer as described in claim 1 is used to prevent internal contamination by a radioactive contaminant. The functionalized polymer of claim 1 is used as a reactive oxygen species (ROS) scavenger for reducing toxicity associated with external radioactive contamination. The functionalized polymer as described in claim 1, wherein the radioactive contaminant is a radionuclide selected from the group consisting of radioactive isotopes of uranium, thorium, molybdenum, cesium, strontium, iridium and cobalt, or a mixture thereof. The functionalized polymer as described in claim 1 has the following formula (III): (III) wherein x is between 0.25 and 0.4, typically about 0.3, and y is between 0.05 and 0.2, typically about 0.07. The functionalized polymer as described in claim 1, wherein the functionalized polymer is administered at least 6 hours, preferably at least 12 hours, before the first exposure to the radioactive contaminant. A functionalized polymer as described in claim 1, wherein the subject will, is, or has been exposed to radioactive contamination due to an accident involving a nuclear reactor. A functionalized polymer as described in claim 1, wherein the subject will, is, or has been exposed to radioactive contamination because the subject was present in an area during or after an incident involving a nuclear reactor. The functionalized polymer of claim 1, wherein the subject is at risk of exposure to radioactive contamination due to presence in a combat zone during or after use of a nuclear explosion or radiological dispersion device.