Polysaccharides containing soluble chelating groups at physiological pH and their use

Abstract

The present invention relates to statistical polysaccharides of the following formula I having a mass average molecular weight of 100 kDa to 1000 kDa: [C1] JPEG2023535504000009.jpg46170 wherein each Rc independently represents a group comprising a chelating agent, each Z independently represents a linking group which may be a single bond or a hydrocarbon-based chain comprising 1 to 12 carbon atoms, said chain may be linear or branched and may contain one or more unsaturations and may contain one or more heteroatoms preferably selected from the nitrogen, oxygen, sulfur and halogen groups of atoms, x is between 0.005 and 0.7, preferably between 0.05 and 0.7, and preferentially between 0.2 and 0.6, y is between 0.01 and 0.7, preferably between 0.05 and 0.2, the ratio y / x is greater than or equal to 0.05, preferably greater than or equal to 0.15, and the sum x+y is greater than or equal to 0.30, preferably greater than or equal to 0.35. The invention also relates to the use of said polysaccharide in a dialysis method for capturing at least one metal, in an MRI imaging method, in a brachytherapy method, or in a method for marking food to prevent counterfeiting.

Description

Technical Field

[0001] The present disclosure belongs to the field of compounds having a chelate group. In particular, the present invention relates to polysaccharides that are chelated or capable of chelating one or more metals, and their use in various techniques such as dialysis for homeostasis, MRI imaging, brachytherapy, or food marking for anti-counterfeiting.

Background Art

[0002] Polysaccharides are polymers derived from the biomass of plants, fungi, animals, or bacteria. These polymers have very diverse physicochemical properties and can be formed for a wide range of biological applications. Chemical modification of polysaccharides makes it possible to adjust their properties, particularly their solubility in aqueous media at pH values close to neutral. Their function using highly specific chelating agents also enables applications in the biomedical field. In fact, after grafting these entities onto the polysaccharide structure, the polymer can be used as an antidote for decontaminating the living body with respect to pathogenic metals, such as in relation to maintaining homeostasis.

[0003] Maintaining the internal environment of the living body, i.e., the homeostasis of all biological fluids and the body fluids of the living body, is necessary for the proper functioning of the living body. In many pathological conditions, a systemic or local dysregulation of the homeostasis of metals and / or peptides, or proteins, has been demonstrated.

[0004] Regarding metals, chelation therapy aimed at reducing the concentration of metal ions has been used for many years in cases of acute poisoning. Accordingly, a certain number of chelating agents have already been accepted by humans, each associated with a specific group of metals (G. Crisponi et al., Coordination Chemistry Reviews, 2015).

[0005] Increasing scientific research is highlighting the potential for metals, particularly not only iron but also copper, zinc, manganese, and even aluminum and lead, to play important roles in many neurological disorders (E.J. McAllum et al., J. Mol. Neurosci., 2016). This is especially true for rare disorders associated with genetic abnormalities related to iron accumulation in specific regions of the brain, cases of neurodegeneration with iron overload that currently only have palliative treatments (S. Wiethoff et al., Handb. Clin. Neurol., 2017). Furthermore, many studies have shown that iron tends to accumulate in the brain with age (J. Acosta-Cabronero et al., Journal of Neuroscience, 2016). Wilson's disease is also a genetic disorder that causes copper accumulation in the body, leading to various problems, particularly liver and / or neurological problems (Anna Czlonkowskal et al., Nature Rev., 2018).

[0006] Some neurological disorders such as Alzheimer's disease, Parkinson's disease, and Huntington's disease are also associated with increased iron levels in specific regions, causing cell damage and oxidative stress (A.A. Belaidi et al., Journal of Neurochemistry, 2016). For example, Huntington's disease is a neurodegenerative disorder that causes movement disorders, cognitive decline, and psychiatric problems. In this pathological condition, numerous oxidative stress markers are observed in the brain and may be related to dysregulation of iron homeostasis (S.J.A. van den Bogaard et al., International Review of Neurobiology, 2013). Thus, increased iron levels in several regions of the brain (the putamen, caudate nucleus, and globus pallidus) have been verified by several MRI studies, including those by Bartzorkis (G. Bartzorkis et al., Archives of Neurology, 1999).

[0007] In particular, in Alzheimer's disease, an interaction has been demonstrated between specific metal ions, particularly ions derived from metals such as zinc, iron, or copper, and the Aβ peptide, which can lead to an increase in protein aggregation (Tougu et al., Metallomics, 2010).

[0008] Thus, in many proteinopathies, it has been recognized that metal cations play an important role in the formation of abnormal conformations of specific proteins. In particular, some promote the formation of aggregates, fibrils, or other solid deposits. Therefore, in proteinopathies, there appears to be a locally homeostatic double dysregulation, namely, dysregulation of the homeostasis of specific metals and dysregulation of the homeostasis of the target molecules of the protein types that cause aggregation and other solid deposits.

[0009] Patent application WO2019 / 122790 discloses a medical device that can be introduced into the body to maintain metal homeostasis for therapeutic purposes, and the device contains a chelating agent for extracting metals.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Summary of the Invention

Problems to be Solved by the Invention

[0012] While there are satisfactory medical devices in this way, it is still beneficial to improve the final results obtained so far. Furthermore, a device containing a chelating agent that can be used to capture metals in vivo may be introduced into the body in a pre-chelated form. In the latter configuration, a very diverse range of other applications are envisaged, such as magnetic resonance imaging (MRI), brachytherapy, or food marking to prevent counterfeiting.

[0013] Therefore, an object of the present invention is to provide a polysaccharide containing a chelating agent that can be introduced into the body in a chelated or non-chelated form for use in various applications.

Means for Solving the Problems

[0014] The present invention relates to a statistical polysaccharide of the following formula I having a mass average molecular weight of 100 kDa to 1000 kDa:

[0015] [Chemical formula]

[0016] [In the formula, each Rc independently represents a group containing a chelating agent, each Z independently represents a single bond or a linker which may be a hydrocarbon-based chain containing 1 to 12 carbon atoms, said chain may be linear or branched, may contain one or more unsaturations, and preferably may contain one or more heteroatoms selected from the atoms of the nitrogen, oxygen, sulfur, and halogen groups, x is from 0.005 to 0.7, preferably from 0.05 to 0.7, and preferentially from 0.2 to 0.6, y is from 0.01 to 0.7, preferably from 0.05 to 0.2, the y / x ratio is 0.05 or more, preferably 0.15 or more, and the sum of x + y is 0.30 or more, preferably 0.35 or more].

[0017] The present invention also relates to the use of said polysaccharide in a dialysis method for capturing at least one metal.

[0018] The present invention also relates to the use of said polysaccharide in an MRI imaging method.

[0019] The present invention is also directed to the use of said polysaccharide in a brachytherapy method.

[0020] The present invention also relates to the use of said polysaccharide in a food marking method for preventing forgery.

[0021] Other features, details, and advantages will become apparent by reading the following detailed description and analyzing the accompanying drawings.

Brief Description of the Drawings

[0022]

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Figure 9

Mode for Carrying Out the Invention

[0023] As described above, the present invention relates to a statistical polysaccharide of the following formula I having a weight average molecular weight of 100 kDa to 1000 kDa:

[0024] [Chemical formula]

[0025] [In the formula, each Rc independently represents a group containing a chelating agent, each Z independently represents a single bond or a linker that can be a hydrocarbon-based chain containing 1 to 12 carbon atoms, said chain may be linear or branched, may contain one or more unsaturations, and may contain one or more heteroatoms, preferably one or more heteroatoms selected from the group consisting of nitrogen, oxygen, sulfur, and halogen atoms, x is 0.005 to 0.7, preferably 0.05 to 0.7, preferentially 0.2 to 0.6, and more preferentially 0.25 to 0.4, y is 0.01 to 0.7, preferably 0.05 to 0.2, the y / x ratio is 0.05 or more, preferably 0.15 or more, and the sum of x + y is 0.30 or more, preferably 0.35 or more].

[0026] In the above formula I, it is understood that some Rc groups may be present in the polysaccharide. These Rc groups may be the same or different from each other. They are all independently selected from groups having a chelating agent. The same is true for the linker Z. There may be several linkers Z, and they may be the same or different from each other.

[0027] According to one embodiment, in formula I, x is 0.005 to 0.6, y is 0.1 to 0.9, the y / x ratio is greater than 0.16, and the sum of x + y is greater than 0.4.

[0028] Preferably, the polysaccharide according to the present invention has a complex formation constant of at least 10 15 for transition element d or f.

[0029] According to one embodiment, the polysaccharide of formula I is a polysaccharide of formula II below:

[0030]

Chemical formula

[0031] [wherein, Rc1 and Rc2 are different and are groups containing a chelating agent, Z1 and Z2 may be the same or different and are linkers which may be a single bond or a hydrocarbon-based chain containing 1 to 12 carbon atoms, said chain may be linear or branched, and may contain one or more unsaturations and may contain one or more heteroatoms, preferably one or more heteroatoms selected from the group consisting of nitrogen, oxygen, sulfur and halogen atoms, x is from 0.005 to 0.7, preferably from 0.05 to 0.7, preferentially from 0.2 to 0.6, and more preferentially from 0.25 to 0.4, y is from 0.01 to 0.7, preferably from 0.05 to 0.2, the y / x ratio is 0.05 or more, preferably 0.15 or more, the sum of x + y is 0.30 or more, preferably 0.35 or more, and z is from 0.5 to 1].

[0032] In this particular embodiment, the polysaccharide of formula II is - when z = 1, a single type of group containing the chelating agent Rc1, or - when 0.5 ≤ z < 1, two types of groups containing the chelating agents Rc1 and Rc2 may be included.

[0033] According to one embodiment, z is from 0.8 to 0.99, and thus the Rc1 group is mainly dominant.

[0034] According to another embodiment, in formula II, x is from 0.005 to 0.6, y is from 0.1 to 0.9, The y / x ratio is greater than 0.16, the sum of x + y is greater than 0.4, and z is between 0.5 and 1.

[0035] <The Rc-type groups (Rc, Rc1, and Rc2)> The term "Rc-type group" is intended to mean the Rc group in the polysaccharide of formula I and, when the Rc2 group is present, the Rc1 and Rc2 groups in the polysaccharide of formula II.

[0036] According to the present invention, the Rc, Rc1, and Rc2 groups are chelating agents. In other words, the Rc, Rc1, and Rc2 groups enable the chelation of one or more metals when forming a complex.

[0037] Each of the Rc, Rc1, and Rc2 groups can contain one or more coordination sites. Preferably, the coordination site is a nitrogen or oxygen atom. Advantageously, each of the Rc, Rc1, and Rc2 groups contains from 4 to 8 coordination sites, more advantageously from 6 to 8 coordination sites, and even more advantageously, each of the Rc, Rc1, and Rc2 groups contains 8 coordination sites.

[0038] The term "coordination site" is intended to mean a single function capable of chelating a metal. For example, an amine functional group represents a site of coordination by the formation of a donor bond between a nitrogen atom and a metal, and a hydroxamic acid functional group also represents a site of coordination through the formation of a donor bond between the oxygen of the carbonyl moiety and the oxygen of the N-oxide moiety, thus forming a 5-membered ring.

[0039] In one embodiment of the present invention, for the polysaccharide of formula I, each Rc group is independently selected from the group consisting of 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-glutaric acid-4,7-diacetic acid), DOTAGA (2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid), DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), NOTAM (1,4,7-tetrakis(carbamoylmethyl)-1,4,7-triazacyclononane), DOTP (1,4,7,10-tetraazacyclododecane 1,4,7,10-tetrakis(methylenephosphonate)), NOTP (1,4,7-tetrakis(methylenephosphonate)-1,4,7-triazacyclononane), TETA (1,4,8,11-tetraazacyclotetradecane-N,N’,N’’,N’’’-tetraacetic acid), TETAM (1,4,8,11-tetraazacyclotetradecane-N,N’,N’’,N’’’-tetrakis(carbamoylmethyl)), DTPA (diethylenetriaminepentaacetic acid) and DFO (deferoxamine), and preferably independently selected from the group consisting of DOTAGA, DFO, DOTAM, and DTPA, and more preferably, the Rc group is DOTAGA.

[0040] In one embodiment of the present invention, for the polysaccharide 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, and DFO, preferably from the group consisting of DOTAGA, DFO, DOTAM, and DTPA.

[0041] According to one embodiment, for the polysaccharide of formula II, the Rc1 group is DOTAGA, and preferably z = 1.

[0042] According to one embodiment, for the polysaccharide of formula II, the Rc1 group is DOTAGA and the Rc2 group is DFO.

[0043] <Z-type linker (Z, Z1, and Z2)> The term "Z-type linker" is intended to mean linker Z in the polysaccharide of formula I and, when linker Z2 is present, linkers Z1 and Z2 in the polysaccharide of formula II.

[0044] The selection of linkers Z, Z1, and Z2 in formulas I and II is essentially determined by the Rc, Rc1, and Rc2 groups, as well as the metal to be chelated. This is because, particularly for steric reasons, the Rc, Rc1, and Rc2 groups may be more or less proximate to the nitrogen 6-membered ring of the glucosamine unit.

[0045] Preferably, in formula I, each Z is independently a single bond or a hydrocarbon-based chain containing 1 to 12 carbon atoms, said chain may be linear or branched, may contain one or more unsaturations, and may contain one or more heteroatoms, preferably one or more heteroatoms selected from the group consisting of nitrogen, oxygen, sulfur, and atoms of the halogen group.

[0046] According to one embodiment, in formula I, each Z is independently selected from the group consisting of a bond, a linear or branched alkyl chain containing 1 to 12 carbon atoms, and a linear or branched alkenyl chain containing 2 to 12 carbon atoms, said alkyl chain and alkenyl chain may be interrupted by one or more C6-C 10 aryl group, 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)-, -NR'-C(O)-NR'-, -NR'-C(O)-O-, -O-C(O)NR', -C(S)NR'-, -NR'-C(S)-, -NR'-C(S)-NR'. The alkyl chain and alkenyl chain may be substituted with one or more groups selected from the group consisting of halogen, -OR', -COOR', -SR', -NR'2, each R' is independently H or C1-C6 alkyl.

[0047] Advantageously, in formula I, each Z is independently selected from the group consisting of a bond and a straight or branched alkyl chain containing 1 to 12 carbon atoms, the alkyl chain is one or more C6-C 10 aryl group, and / or interrupted by 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'-C(S)-NR'. each R' is independently H or C1-C6 alkyl.

[0048] In one particular embodiment, each Z is an alkyl chain containing 1 to 12 carbon atoms.

[0049] In another particular embodiment, each Z is polyethylene glycol (PEG).

[0050] Preferably, in formula II, Z1 and Z2 are independently a single bond or a hydrocarbon-based chain containing 1 to 12 carbon atoms, the chain may be straight or branched, and may contain one or more unsaturations, and preferably contains one or more heteroatoms selected from the group consisting of nitrogen, oxygen, sulfur, and atoms of the halogen group.

[0051] According to one embodiment, in formula II, Z1 and Z2 are independently selected from the group consisting of a bond, a straight or branched alkyl chain containing 1 to 12 carbon atoms, and a straight or branched alkenyl chain containing 2 to 12 carbon atoms, the alkyl chain and alkenyl chain are one or more C6-C 10The aryl group and / or may be interrupted by one or more heteroatoms or groups selected from the group consisting of -O-, -S-, -C(O)-, -NR'-, -C(O)NR'-, -NR'-C(O)-, -NR'-C(O)-NR'-, -NR'-C(O)-O-, -O-C(O)NR', -C(S)NR'-, -NR'-C(S)-, -NR'-C(S)-NR', The alkyl chain and alkenyl chain may be substituted with one or more groups selected from the group consisting of halogen, -OR', -COOR', -SR' and -NR'2, Each R' is independently H or C1-C6 alkyl.

[0052] Advantageously, in formula II, Z1 and Z2 are independently selected from the group consisting of a bond and a straight-chain or branched alkyl chain containing 1 to 12 carbon atoms, The alkyl chain is one or more C6-C 10 The aryl group and / or may be interrupted by 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'-C(S)-NR', Each R' is independently H or C1-C6 alkyl.

[0053] In one particular embodiment, Z1 and / or Z2 is an alkyl chain containing 1 to 12 carbon atoms.

[0054] In another particular embodiment, Z1 and / or Z2 is polyethylene glycol (PEG).

[0055] <Monomer unit of the polysaccharide according to the present invention> The polysaccharide according to the present invention is composed of three monomer units, namely, a unit A of the N-acetylglucosamine type, a unit B of the glucosamine type, and a unit C of the glucosamine type functionalized with a chelating agent (type Rc) bonded to the nitrogen of glucosamine by a linker (type Z).

[0056] The polysaccharide according to the present invention is a random polymer. In other words, the linkage of various monomer units of type A, type B, and type C is random.

[0057] In Formulas I and II, x represents the ratio of unit A, and x can be from 0.05 to 0.7, preferably x is from 0.2 to 0.6, and more preferably x is from 0.25 to 0.4.

[0058] In Formulas I and II, y represents the proportion of type C units, and y can be from 0.01 to 0.7.

[0059] The remainder of the monomer units in Formulas I and II is unit B. Therefore, in Formulas I and II, the proportion of unit B is equal to 1 - x - y.

[0060] According to the present invention, in Formulas I and II, the y / x ratio can be 0.05 or more, preferably 0.15 or more. In fact, the effectiveness of the product depends on the number of chelating sites, which is directly related to the number of metals required for detection by, for example, imaging.

[0061] In order to be introduced in a liquid state in vivo and at the same time produce the desired effect, the polysaccharide according to the present invention must be soluble at physiological pH. For this reason, the sum of x + y can be 0.30 or more, preferably 0.35 or more.

[0062] The specific ratio between the number of unit A and the number of type C units, and the combination of the sum of the proportion of unit A and the proportion of type C units enable the achievement of chelation and solubility suitable for using the polysaccharide according to the present invention in various fields such as dialysis for capturing at least one metal, MRI imaging, brachytherapy, and food marking for preventing forgery.

[0063] Advantageously, x is from 0.2 to 0.6, and more preferably x is from 0.25 to 0.4.

[0064] Advantageously, y is from 0.01 to 0.7, preferably from 0.05 to 0.2.

[0065] According to the present invention, z is from 0.5 to 1. In other words, the C-type unit may be only a unit containing Z1 as a linker and Rc1 as a group having a chelating agent.

[0066] The polysaccharide according to the present invention has a weight average molecular weight of 100 kDa to 1000 kDa. Advantageously, the mass average molecular weight of the polysaccharide according to the present invention is from 250 kDa to 750 kDa, more advantageously from 400 kDa to 600 kDa, and even more advantageously from 450 kDa to 550 kDa.

[0067] According to one embodiment, the polysaccharide is selected from the following polysaccharides: - a polysaccharide of formula II where z = 1, Rc1 is DOTAGA, and Z1 is a bond, - a polysaccharide of formula II where z = 1, Rc1 is DTPA, and Z1 is a bond, and - a polysaccharide of formula II where 0.5 ≤ z < 1, Rc1 is DOTAGA, Z1 is a bond, Rc2 is DFO, Z2 is a bond and is selected from the group consisting of a straight-chain or branched alkyl chain containing 1 to 12 carbon atoms, wherein the alkyl chain is interrupted by one or more C6 - C 10 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'-C(S)-NR' may be interrupted, each R' is independently H or C1 - C6 alkyl.

[0068] <Method for obtaining the polysaccharide according to the present invention> The polysaccharide of formula I or II according to the present invention can be obtained from chitosan, which is optionally partially acetylated, in particular by acetylation of some of the amine functional groups and functionalization of at least some of the amine functional groups still present after said acetylation.

[0069] In one particular embodiment, the method for obtaining a polysaccharide according to the invention comprises at least the following three successive steps: · Step 1: solubilization of chitosan in an acid solution at a pH of 4 to 5; · Step 2: partial acetylation of the amine functional groups of the chitosan solubilized in Step 1 (formation of unit A); · Step 3: functionalization of at least some of the amine functional groups still present at the end of Step 2 (formation of type C units).

[0070] If the polysaccharide contains several different groups of the Rc type, Step 3 of the above method can be repeated several times. For example, if the polysaccharide of formula II contains Rc1 groups and Rc2 groups, the method can include two Steps 3 of functionalizing at least some of the amine functional groups, namely, first a step of introducing Rc1 groups and second a step of introducing Rc2 groups.

[0071] Step 3 can be subdivided into several sub-steps, particularly when the linker Z is a hydrocarbon-based chain as defined above (a chain based on hydrocarbons).

[0072] In an embodiment where the Z-type linker is a hydrocarbon-based chain as defined above, Step 3 can include sub-step 3-1, which consists of grafting the hydrocarbon-based chain onto at least some of the amine functional groups still present at the end of Step 2, and then sub-step 3-2, which consists of grafting an Rc-type group onto the hydrocarbon-based chain. Alternatively, Step 3 does not include sub-steps. In this alternative method, before Step 3, the hydrocarbon-based chain is coupled with an Rc-type group, and then Step 3 is carried out using a molecule containing the Rc-type group and the hydrocarbon-based chain.

[0073] Alternatively, the polysaccharide according to the invention can be obtained from chitosan having the desired degree of acetylation, and thus, in this embodiment, unit A already exists and does not need to be formed. In this embodiment, the method for obtaining a polysaccharide according to the invention comprises at least the following two successive steps: · Step 1b: solubilization of partially acetylated chitosan (unit A) in an acid solution at a pH of 4 to 5; · Step 2b: functionalization of at least a part of the amine functional groups of the partially acetylated chitosan solubilized in Step 1b (formation of C-type units).

[0074] Similarly, when there are several different Rc-type groups, Step 2b of the above method can be repeated several times.

[0075] Similar to Step 3 above, Step 2b can be subdivided into several sub-steps, especially when the Z-type linker is a hydrocarbon-based chain as defined above.

[0076] In an embodiment where the Z-type linker is a hydrocarbon-based chain as defined above, Step 2b can include Sub-step 2b-1 which consists of grafting the hydrocarbon-based chain to at least a part of the amine functional groups, and then Sub-step 2b-2 which consists of grafting the Rc-type group to the hydrocarbon-based chain. Alternatively, Step 2b does not include sub-steps. In this alternative method, the hydrocarbon-based chain is coupled with the Rc-type group before Step 2b, and then Step 2b is carried out using the molecule containing the Rc-type group and the hydrocarbon-based chain.

[0077] When the polysaccharide according to the present invention is in a chelated form, the above method can include Step 4 of chelating the polysaccharide with at least one metal.

[0078] <Use of the Polysaccharide According to the Present Invention> The polysaccharides according to the invention can be used for various applications due to their solubility at physiological pH. The Rc-type groups enable chelation of metals, and as a result, the polysaccharides can be introduced into the body in two different forms: (i) a chelated form, i.e., a form in which the polysaccharide chelates at least one metal, or (ii) a free form, i.e., a form in which the polysaccharide does not chelate a metal. When the polysaccharide is in the free form, this means that less than 5% of the Rc-type chelating groups are complexed, in particular, less than 5% of the Rc-type chelating groups chelate the target ions. When the polysaccharide is in the chelated form, this means that at least 80% of the Rc-type chelating groups form a complex with the metal of interest.

[0079] As shown above, the invention also relates to the use of said polysaccharides in a dialysis method for capturing at least one metal, an MRI imaging method, a brachytherapy method, or a food marking method for preventing counterfeiting.

[0080] When the polysaccharide is used in a dialysis method for capturing at least one metal, the polysaccharide is in the free form to enable the capture of at least one metal.

[0081] When the polysaccharide is used in an MRI imaging method, a brachytherapy method, or a food marking method for preventing counterfeiting, the polysaccharide chelates at least one metal.

[0082] The polysaccharides according to the invention can be used as such or incorporated into a composition. When the polysaccharide is incorporated into a composition, the polysaccharide is preferably in the form of a hydrogel.

[0083] <Use in a dialysis method for capturing at least one metal> In one particular embodiment, the dialysis is MARS dialysis or CSF dialysis.

[0084] When a polysaccharide is used in a dialysis method for capturing at least one metal, the metal preferably belongs to the group consisting of copper, iron, lead, zinc, aluminum, gadolinium, and manganese, and more preferably belongs to the group consisting of copper, iron, and lead.

[0085] When a polysaccharide is used in a dialysis method for capturing at least one metal, the Rc type group is preferably selected from the group consisting of DOTAGA, DFO, DOTAM, and DTPA.

[0086] When a polysaccharide is used in a dialysis method for capturing at least one metal, x is preferably 0.25 to 0.4.

[0087] When a polysaccharide is used in a dialysis method for capturing at least one metal, y is preferably 0.05 to 0.2.

[0088] When a polysaccharide is used in a dialysis method for capturing at least one metal, the y / x ratio is preferably 0.15 to 1.5.

[0089] When a polysaccharide is used in a dialysis method for capturing at least one metal, the sum of x + y is preferably 0.35 to 0.8.

[0090] <Use in MRI imaging method> When a polysaccharide is used in an MRI imaging method, the polysaccharide chelates at least one metal. The metal preferably belongs to the group consisting of gadolinium, iron, manganese, and dysprosium, and more preferably the metal is gadolinium.

[0091] When a polysaccharide is used in an MRI imaging method, z is preferably equal to 1 and the Rc1 group is preferably DOTAGA.

[0092] When a polysaccharide is used in an MRI imaging method, x is preferably 0.05 to 0.35.

[0093] When the polysaccharide is used in the MRI imaging method, y is preferably from 0.1 to 0.7.

[0094] <Use in brachytherapy> When the polysaccharide is used in brachytherapy, the polysaccharide chelates at least one metal. This metal is a radioisotope.

[0095] Therefore, when the polysaccharide is used in brachytherapy, the metal is preferably 177 Lu, 166 Ho, 212 Bi, 213 Bi, 90 Y, and 225 belongs to the group of radioisotope elements consisting of At.

[0096] When the polysaccharide is used in brachytherapy, z is preferably equal to 1, and the Rc1 group is preferably selected from the group consisting of DOTAGA.

[0097] <Use in a food marking method for preventing forgery> When the polysaccharide is used in a food marking method for preventing forgery, the polysaccharide chelates at least one metal. The metal preferably belongs to the group consisting of lanthanides and bismuth.

[0098] When the polysaccharide is used in a food marking method for preventing forgery, the Rc-type group is preferably independently selected from the group consisting of DOTA, NOTA, NODAGA, DOTAGA, DOTAM, NOTAM, DOTP, NOTP, TETA, and TETAM. When the polysaccharide according to the present invention is used in a food marking method for preventing forgery, the cyclic chelating agent is advantageous for actually avoiding transmetalation.

Example

[0099] <Materials and methods> Anhydrous acetic acid (>99%) and 1,2-propanediol were supplied by Sigma-Aldrich (France), DOTAGA anhydride, DTPA-dianhydride, and p-NCS-Bz-DFO (N1-hydroxy-N1-(5-(4-(hydroxy(5-(3-(4-isothiocyanatophenyl)thioureido)pentyl)amino)-4-oxobutanamide)pentyl)-N4-(5-(N-hydroxyacetamide)pentyl)succinamide) were supplied by CheMatech (France), DMSO was supplied by Fischer-Chemicals, and ultrapure water (Milli-Q water) was obtained by an Eolia filtration system. Chitosan was from Mahtani, GdCl3 was from Sanofi, KBr and all metal salts (nitrates of Al, Mn, Cu, Pb, and Zn) used in Example 4 were provided by Sigma-Aldrich (France).

[0100] Tangential flow filtration was carried out using a Sartoflow Smart tangential flow filter equipped with a Sartocon Slice200 polyethersulfone membrane with a cut-off threshold of 100 kDa.

[0101] HPLC-UV was carried out on an Agilent 1200 device equipped with a DAD detector. The size exclusion column used was a Polysep-GFC-P-4000 using 0.1 M acetic acid / ammonium acetate buffer as the eluent. Detection was carried out using a UV detector at a wavelength of 295 nm. The substances to be analyzed were injected at a concentration of approximately 10 g / l.

[0102] Studies by UV-visible spectrophotometry were carried out on a Varian Cary50 UV-VIS device.

[0103] Proton NMR spectroscopy was carried out at room temperature on a Brucker Avance III 400 MHz. All samples were dissolved at 8 mg / ml in deuterium oxide (D2O) containing 5 μL of 12 N HCl and placed in 5 mm NMR tubes. TMPSA was used as an internal standard.

[0104] Elemental analysis was carried out at the Institut des Sciences Analytiques UMR5280, Pole Isotopes & Organique, 5 rue de la Doua 69100 Villeurbanne.

[0105] Time-resolved luminescence studies were performed using an Agilent Cary Eclipse instrument.

[0106] The relaxation rate at 1.5 T was measured using a Minispec mq-60 instrument provided by Brucker (Karlsruhe, Germany).

[0107] <Example 1: Preparation of a polysaccharide according to the invention starting from chitosan with an average mass of 250 kDa, having a degree of acetylation of 40% (x = 0.4) and a degree of grafting with DOTAGA of 10% (y = 0.1)> In step 1, 60 g of chitosan, 4 L of ultrapure water, and 45 ml of glacial acetic acid are introduced into a 10 L reactor and stirred at pH 4.5 ± 0.5 for 16 h. A pale yellow solution is obtained.

[0108] In step 2, 1.2 L of 1,2-propanediol is added to the pale yellow solution obtained in step 1 and stirring is maintained for 1 h. Next, a solution consisting of 14 ml of acetic anhydride dissolved in 600 ml of 1,2-propanediol is slowly added over 10 min in order to obtain a uniform acetylation along the polymer chains, and the reaction medium is stirred for a further 4 h.

[0109] The degree of acetylation can be determined by elemental analysis. The molar mass of the non-acetylated units (unit B) of the polysaccharide is 161.2 g·mol -1 (C6NO4H 11 ) whereas the molar mass of the acetylated units (unit A) is 203.2 g·mol -1 (C8NO5H 13) It is as follows. The elemental analysis of the polysaccharide obtained at the end of acetylation step 2 is as follows. C: 39.22%, H: 7.55%, and N: 6.77%, which corresponds to a degree of acetylation of 40% (x = 0.4) of the acetylated unit (unit A).

[0110] In step 3, 2 L of the solution obtained in acetylation step 2 is put into the reactor while stirring. Then, 120 g of DOTAGA anhydride is added and stirring is maintained for 16 hours. At the end of this reaction, the solution is diluted 10-fold with ultrapure water and purified by tangential flow filtration using a 100 kDa membrane. After the first step of re-concentrating to 16 L, the solution is filtered using 480 L of a 0.1 M acetic acid solution of a constant volume (16 L), followed by another step of using 320 L of ultrapure water and re-concentrating to 8 L. By HPLC-UV, it can be confirmed that DOTAGA has actually been removed (Figure 1). The peak at about 7 minutes corresponds to the polymer, and the peak at about 11 minutes corresponds to non-grafted DOTAGA. A solution with a polysaccharide concentration of 10 g / L is filtered through a nylon membrane (0.4 μm) before lyophilization.

[0111] If the degree of acetylation x is known, the degree of functionalization y of the polysaccharide with DOTAGA can be determined by proton NMR. Non-grafted and non-acetylated units (unit B) are composed of 7 protons covalently bonded to a carbon atom and having a chemical shift of 2.9 ppm to 4.3 ppm. The acetylated unit (unit A) has these same 7 protons and 3 protons covalently bonded to the carbon atom present in the acetyl, and is characterized by a chemical shift of 2 ppm to 2.2 ppm. Finally, the unit grafted with DOTAGA (unit C) contains 34 protons covalently bonded to a carbon atom, 32 of which are integrated at 2.9 ppm to 4.3 ppm, and 2 of which are integrated at 2 ppm to 2.2 ppm. The NMR spectrum shown in Figure 2 enables the value of y to be determined by the integration of various peaks according to the following formula.

[0112]

Equation

[0113] The degree of the grafted unit (unit C) is approximately 0.1.

[0114] Therefore, the obtained polysaccharide has approximately 0.4 degree (x = 0.4) of unit A, approximately 0.5 degree (1 - x - y = 0.5) of unit B, and approximately 0.1 degree (y = 0.1) of unit C.

[0115] The degree of the grafted unit (unit C) can also be determined by the fluorescence by europium. In fact, europium mainly emits light centered around approximately 590 nm ( 5 D0 → 7 F1) and 615 nm ( 5 D0 → 7 F2). This emission disappears when the europium ions are coordinated only to water molecules. The principle of the method for determining the degree (extent) of the grafted unit is to add an increasing amount of europium to chelate the europium, and then the emission increases and reaches a plateau (steady state) when all the chelation sites are filled. In fact, the polysaccharide obtained at the end of step 3 was put into an acetate buffer at pH 5, and then europium chloride salt dissolved in the acetate buffer was added. Next, a quantitative analysis curve was traced by exciting at 396 nm and recording the emission at 590 nm (Figure 3). This quantitative analysis makes it possible to return to an amount of 0.4 μmol of chelate per 1 mg of polymer, that is, approximately 10% (y = 0.1) of the degree (extent).

[0116] <Example 2: Preparation of a polysaccharide according to the invention starting from chitosan with an average mass of 600 kDa and a degree of acetylation of 0.5%, having a degree of acetylation of 0.5% (x = 0.005) and a degree of grafting of DOTAGA of 60% (y = 0.6)> In step 1b, 3 g of chitosan with an average mass of 600 kDa and a degree of acetylation of 0.5%, 150 ml of ultrapure water, and 2.1 ml of glacial acetic acid were introduced into a 1 L reactor and stirred at a pH of 4.5 ± 0.5 for 16 hours. A light yellow solution was obtained.

[0117] Following the solubilization step 1b, 150 ml of 1,2-propanediol is added to the previous solution and stirring is maintained for 1 hour. Next, 11 g of DOTAGA anhydride is added and stirring is maintained for 16 hours. At the end of this reaction, the light brown solution is diluted 10-fold with ultrapure water and purified by tangential flow filtration using a 100 kDa membrane. Following this first purification cycle, a second purification cycle with 50-fold purification using ultrapure water is continued. Subsequent filtration is carried out in 0.01 M HCl for a final purification factor of 1250.

[0118] Grafting with DOTAGA (units C) was shown by thermogravimetric analysis using a TA Instruments TGA-Q500 apparatus, heating from 30 °C to 700 °C at 10 °C / min under an inert atmosphere. Grafting of DOTAGA causes a decrease in the decomposition temperature proportional to the degree of grafting. Thus, by comparing the thermogravimetric analysis (graph (A) in Figure 4) and the derivative curve (graph (B) in Figure 4) of the starting chitosan and the polysaccharide according to the present invention, it can be inferred that the functionalization step was indeed effective.

[0119] The IR spectra were normalized using a band with a maximum absorption (1072 cm -1 ) (Figure 5). The formation of amide bonds was verified and they were quantified by examining the deconvolution of the spectra and the area of the band at 1590 cm -1 . The calculation is carried out by comparing the areas of the spectral bands located around 1590 cm -1 . To determine the coefficient z = 100×(1 - x - y) of the following equation, it is necessary to determine the degree of functionalization of the chitosan / DOTAGA product by other methods. In this example, the IR spectrum of chitosan + DOTAGA (6.0%) was used as the reference spectrum (spectrum (A)).

[0120]

Number

[0121] [wherein, D s is the degree of substitution of the analyzed chitosan, and A’ (1590) is the area of the band at 1590 cm -1 in the IR spectrum of the analyzed chitosan, and A’ T is the total area of the IR spectrum of the chitosan to be analyzed, and A ref(1590) is the area of band IV at 1590 cm -1 in the IR spectrum of the reference chitosan, and A Tref is the total area of the reference chitosan spectrum, and z is the percentage of free amine groups of the chitosan (x = 0.02, y = 0.06) used as a reference (z = 92)]. Thus, a degree of grafting of DOTAGA close to 60% is obtained (spectrum (B)).

[0122] <Example 3: Preparation of a polysaccharide according to the invention starting from chitosan with an average mass of 250 kDa and having a degree of acetylation of 60% (x = 0.6) and a degree of grafting of DTPA close to 5% (y ≒ 0.05)> In step 1, 0.25 g of chitosan, 25 ml of ultrapure water, and 0.18 ml of glacial acetic acid are introduced into a 250 ml reactor and stirred at pH 4.5 ± 0.5 for 16 hours. A pale yellow solution is obtained.

[0123] In step 2, 5 ml of 1,2-propanediol is added to the pale yellow solution obtained in step 1 and stirring is maintained for 1 hour. Next, a solution consisting of 0.045 ml of acetic anhydride dissolved in 5 ml of 1,2-propanediol is gradually added over 10 minutes in order to obtain uniform acetylation along the polymer chain. The reaction medium is continuously stirred for 4 hours.

[0124] In Step 3, a solution composed of 0.346 g of DTPA-dianhydride dissolved in 15 ml of 1,2-propanediol and 16.58 μl of ultrapure water is continuously stirred for 1.5 hours. Then, this solution is added to the solution obtained in Step 2, and stirring is maintained for 16 hours. At the end of this reaction, ultrapure water is added to the solution to dilute it 10-fold, and it is purified by tangential flow filtration using a 100 kDa membrane. The subsequent filtration is carried out in ultrapure water due to a final purification factor of 6250. Then, this solution with a concentration of 5 g / L is lyophilized.

[0125] <Example 4: Use of the Polymer of Example 1 for Capturing Metals> The polysaccharide synthesized in Example 1 was formulated to be incorporated into the hemodialysis fluid for the purpose of demonstrating its ability to perform metal extraction in the context of conventional hemodialysis. As test requirements, a Dialife DIAPH06 high-flux hemodialysis device, a Cole-Parmer Masterflex 7555-05 peristaltic pump, and a Masterflex L / S PharMed BPT tube L / S #16 were used. The metal solution used here was prepared to have a content of approximately 50 ppb of Cu, Zn, Pb, Al, and Mn in 5 L of water. The flow rate was 200 ml / min, and the temperature was 37 °C. A dialysate containing only water was compared with a dialysate containing 0.001 mol / L of DOTAGA corresponding to 1.6 g / L of the polysaccharide synthesized in Example 1. For all metals except aluminum, greater capture is observed for the dialysate containing the functionalized polymer at 25 minutes after the start of dialysis by ICP / MS analysis (Figure 6). After dialysis using the solution of the polysaccharide of Example 1, the amounts of metals present in the dialyzed solution are actually 30 vs. 42 ppb for manganese, 18 vs. 46 ppb for zinc, 7 vs. 46 ppb for copper, and 8 vs. 55 ppb for lead, compared to dialysis performed using an aqueous solution without the polysaccharide according to the present invention.

[0126] <Example 5: Use of the Polymer of Example 1 in a Method for Dialyzing Porcine Blood to Capture Metals> To demonstrate the capture ability in a biological medium, a test similar to that described in Example 4 was carried out using porcine blood instead of the aqueous metal solution described in Example 4. As test requirements, a Dialife 20HP high-flow hemodialysis device, three Cole-Parmer Masterflex peristaltic pumps (7555-05, 7523-80, and 7523-90), Masterflex L / S PharMed BPT tubing L / S#16, and Ismatec SC0328 PharMed BPT tubing were used. 10 l of porcine blood was dialyzed at different flow rates: 200 ml / min (0 - 10 min and 70 - 80 min), 100 ml / min (10 - 30 min), and 50 ml / min (30 - 70 min) respectively. The dialysate was formulated with the polysaccharide synthesized in Example 1 at a concentration of 0.001 mol / L. The research results after ICP / MS analysis are shown in Figure 7. In the dialysate containing the polysaccharide synthesized in Example 1, a significant increase in the contents of Zn, Fe, and Pb was observed compared to dialysis performed without the polysaccharide synthesized in Example 1. Therefore, the amounts of metals present in the dialysis solution are 83 vs 20 ppb for zinc, 257 vs 55 ppb for copper, and 2604 vs 62 ppb for iron.

[0127] <Example 6: Addition of Gadolinium to Obtain a Polymer Capable of MRI Imaging> After purification in Example 2, 4.9 g of gadolinium chloride hexahydrate was added to the solution, then the pH was adjusted to 5, and the solution was stirred at 80 °C for 48 hours. The pH was continuously adjusted to 5 with 0.5 M sodium hydroxide until the pH became stable (this indicates that DOTAGA had complexed all Gd 3+ ions).

[0128] <Example 7: Use of the Polysaccharide Complexed with Gadolinium> The polysaccharide according to the invention complexed with gadolinium in Example 6 was used in the MRI imaging method.

[0129] The efficiency of the polymer complex with gadolinium for MRI was 15.2 mM -1 .s by gadolinium at 37 °C in a 1.5 T magnetic field-1 ) and its transverse relaxation rate (r2 = 21.5 mM with gadolinium) -1 .s -1 ) were evaluated by measurement. The r2 / r1 ratio was 1.4, which is typical of positive contrast agents. The longitudinal relaxation is less than 4 times that of commercially available contrast agents (about 4 mM -1 .s -1 ).

[0130] <Example 8: Preparation of a polysaccharide according to the invention starting from chitosan with an average mass of 250 kDa and having a degree of acetylation of 40% (x = 0.4) and a degree of grafting of 11% of DOTAGA and DFO (y = 0.11) in all cases> Chitosan-DOTAGA was pre-synthesized as described in Example 1.

[0131] 720 ml of purified chitosan-DOTAGA with a concentration of 7 g / L is placed in a 2 L round-bottom flask. This solution (pH 6 - 6.5) is made up to a total volume of 900 ml with ultrapure water. In parallel, 143.1 mg of p-NCS-Bz-DFO is weighed and dissolved in 100 ml of DMSO. This solution is then added dropwise to the chitosan-DOTAGA solution. The solution is continuously stirred and heated at 40 °C overnight. Next, the solution is purified with ultrapure water using a Sartoflow Smart purifier equipped with a PES membrane with a cut-off threshold of 100 kDa until a purification degree of 545 is obtained. Then, the solution with a concentration of 5.1 g / L is lyophilized.

[0132] HPLC-UV analysis of the product makes it possible to confirm the grafting of DFO and the removal of residual p-NCS-Bz-DFO. Indeed, the comparison between the HPLC-UV analysis of chitosan-DOTAGA and that of chitosan-DOTAGA-DFO shows an increase in the absorption of the polymer peak (about 7 minutes) when p-NCS-Bz-DFO is grafted (Figure 8).

[0133] The amount of grafted p-NCS-Bz-DFO can be determined by quantitative analysis using ultraviolet-visible spectrophotometry at the maximum absorption wavelength (425 nm) of the DFO-iron complex. Iron(III) at increasing concentrations is added to a 0.1 g / L solution of chitosan-DOTAGA-DFO in acetate buffer (0.1 M ammonium acetate and 0.1 M acetic acid) at pH 4.5. Next, the absorption measured at 425 nm is plotted as a function of iron concentration, and a break in the gradient is observed at 35 μM iron (Figure 9). Since one molecule of DFO can form a complex with one iron atom, 1 gram of the polymer thus contains 35 μmol of DFO. Therefore, the grafted chelating groups contain 90% DOTAGA and 10% DFO (z = 0.9).

Claims

1. A statistical polysaccharide of formula I having a weight average molecular weight of 100 kDa to 1000 kDa: 【Chemical 1】 [wherein, each Rc is DOTAGA, each Z independently represents a single bond or a linker which is a hydrocarbon-based chain containing 1 to 12 carbon atoms, said chain can be linear or branched, and may contain one or more unsaturations, and may contain one or more heteroatoms, x is from 0.005 to 0.7, y is from 0.01 to 0.7, the y / x ratio is 0.05 or more, and the sum of x + y is 0.30 or more].

2. The polysaccharide according to claim 1, wherein x is from 0.05 to 0.

7.

3. The polysaccharide according to claim 1 or 2, wherein x is from 0.2 to 0.

6.

4. The polysaccharide according to claim 1, wherein x is from 0.25 to 0.

4.

5. The polysaccharide according to claim 1, wherein y is from 0.05 to 0.

2.

6. The polysaccharide according to any one of claims 1 to 5, wherein the y / x ratio is 0.15 or more.

7. The polysaccharide according to any one of claims 1 to 6, wherein the sum of x + y is 0.35 or more.

8. The polysaccharide according to any one of claims 1 to 7, characterized in that it is incorporated in a composition in hydrogel form.

9. The polysaccharide according to any one of claims 1 to 8, for use in a dialysis method for capturing at least one metal.

10. The polysaccharide according to any one of claims 1 to 8, wherein said polysaccharide chelates at least one metal.

11. The polysaccharide according to claim 10, for use in MRI imaging methods.

12. The polysaccharide according to claim 10, for its use in brachytherapy.

13. Use of the polysaccharide according to claim 10, in a method of marking food to prevent counterfeiting.

14. At least the following three sequential steps: - Step 1: A step of solubilizing chitosan in an acid solution at a pH of 4 to 5, - Step 2: A step of partially acetylating the amine functional groups of the chitosan solubilized in Step 1 (formation of unit A), - Step 3: A step of functionalizing at least a part of the amine functional groups not acetylated in Step 2 (formation of C-type units) A method for producing the polysaccharide according to any one of claims 1 to 8 from chitosan, which comprises

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