WO2021193602A1 - Crosslinked polymer, shiga toxin inhibitor, and pharmaceutical composition - Google Patents

Abstract

A crosslinked polymer obtained by crosslinking a random copolymer having a structural unit (a1) containing a Shiga toxin-binding oligosaccharide and at least one different structural unit. A Shiga toxin inhibitor containing the crosslinked polymer and a pharmaceutical composition for treating or preventing infection with Shiga toxin-producing bacteria.

Description

Crosslinked polymers, Shiga toxin inhibitors, and pharmaceutical compositions

The present invention relates to crosslinked polymers, Shiga toxin inhibitors, and pharmaceutical compositions.
The present application claims priority based on Japanese Patent Application No. 2020-051743 filed in Japan on March 23, 2020, the contents of which are incorporated herein by reference.

In recent years, outbreaks of enterohemorrhagic Escherichia coli (EHEC) via foods and the like have become a problem. Cattle are the main carrier and source of infection for EHEC. Since EHEC is highly infectious and has a relatively long incubation period, secondary human-to-human transmission is also frequent.

When infected with EHEC, 20 to 50% of infected people develop hemorrhagic colitis 3 to 4 days after infection. Hemorrhagic colitis has symptoms such as diarrhea, fresh blood stools, strong abdominal pain, and mild fever. Four to seven days after infection, 2 to 20% of infected individuals develop hemolytic-uremic syndrome (HUS). In HUS, symptoms of hemolytic anemia, thrombocytopenia, and acute renal failure are mainly observed, serious complications such as acute encephalopathy develop, and when HUS becomes severe, death may occur.

Currently, no effective treatment method has been established for EHEC infection. In Japan, about 3,000 people are infected per year, and seriously ill and dead people occur every year. Therefore, the establishment of an effective treatment method is required.

There are two types of Shiga toxins produced by EHEC, Stx1 and Stx2. Both Stx and Stx2 are composed of one molecule of A subunit and five molecules of B subunit. The A subunit is a catalytic subunit having RNA-N-glucosidase activity. The A subunit inactivates the 60S ribosome by the above activity, resulting in inhibition of cellular protein synthesis. The B subunit has a binding activity to Gb3 (globotriaosylceramide), which is a glycolipid on the cell surface, and is involved in the invasion into the target cell.
The homology of the amino acid sequences of Stx1 and Stx2 is about 50%. It is known that the amino acid sequence of Stx1 is almost the same as the amino acid sequence of Stx produced by Shigella dysentis.

Since Shiga toxin has a binding property to Gb3 globotrisaccharide, there is an attempt to develop a Shiga toxin inhibitor using globotrisaccharide. For example, Patent Document 1 reports that a polymer containing a globe trisaccharide is used as a therapeutic agent for EHEC infection.

Japanese Unexamined Patent Publication No. 2005-289907

Since no effective treatment method has been established for EHEC infection, the development of an effective therapeutic drug is required. However, it cannot be said that the inhibitory effect of Shiga toxin is sufficiently high among the EHEC therapeutic drug candidates reported so far.

Therefore, an object of the present invention is to provide a crosslinked polymer having high inhibitory activity against Shiga toxin, and a Shiga toxin inhibitor and a pharmaceutical composition containing the crosslinked polymer.

The present invention includes the following aspects.
[1] A crosslinked polymer obtained by cross-linking a random copolymer, wherein the random copolymer has a structural unit (a1) containing a Shiga toxin-binding oligosaccharide and at least one other structural unit.
[2] The at least one other structural unit contains a hydrophobic group having 4 to 20 carbon atoms (a2), a cationic group (a3), and an anionic group. (A4) and 1- (3-sulfopropyl) -2-vinylpyridinium hydroxide Selected from the group consisting of the structural unit (a5) derived from the molecular salt and the structural unit (a6) derived from the cross-linking agent. The crosslinked polymer according to [1].
[3] The crosslinked polymer according to [2], wherein the random copolymer has the structural unit (a2).
[4] The crosslinked polymer according to [2] or [3], wherein the random copolymer has the structural unit (a3) or the structural unit (a4).
[5] The crosslinked polymer according to any one of [2] to [4], wherein the random copolymer has the structural unit (a5).
[6] The crosslinked polymer according to any one of [1] to [5], wherein the Shiga toxin-binding oligosaccharide is a Globotrisaccharide.
[7] A Shiga toxin inhibitor containing the crosslinked polymer according to any one of [1] to [6].
[8] A pharmaceutical composition for treating or preventing a Shiga toxin-producing bacterial infection, which comprises the crosslinked polymer according to any one of [1] to [6] and a pharmaceutically acceptable carrier. thing.

According to the present invention, a crosslinked polymer having high inhibitory activity against Shiga toxin, and a Shiga toxin inhibitor and a pharmaceutical composition containing the crosslinked polymer are provided.

The NMR chart of the monomer (M1-1) synthesized in the Example is shown. The results of evaluating the inhibitory effect of the crosslinked polymer of Example 1 on the cytotoxicity of Stx1 are shown. The results of evaluating the inhibitory effect of the crosslinked polymer of Example 1 on the cytotoxicity of Stx2 are shown. The results of evaluating the IC50 of the crosslinked polymers of Examples 2 to 10 for the cytotoxicity of Stx1 are shown. The results of evaluating the IC50 of the crosslinked polymers of Examples 2 to 10 for the cytotoxicity of Stx2 are shown. The results of evaluating the IC50 of the crosslinked polymers of Examples 7 to 18 for the cytotoxicity of Stx1 are shown. The results of evaluating the IC50 of the crosslinked polymers of Examples 7 to 18 for the cytotoxicity of Stx2 are shown. The test schedule of the crosslinked polymer administration test of Example 7 using O157-infected mice is shown. The survival curve of the O157-infected mouse to which the crosslinked polymer of Example 7 was administered is shown. The results of stool culture of stool collected from O157-infected mice to which the crosslinked polymer of Example 7 was administered are shown.

"Constituent unit" means a monomer unit (monomer unit) constituting a polymer.
When it is described that "may have a substituent", the hydrogen atom (-H) is replaced with a monovalent group, and the methylene group (-CH _2- ) is replaced with a divalent group. Including both.
"Aromatic hydrocarbon group" means a hydrocarbon group having at least one aromatic ring. The aromatic ring is not particularly limited as long as it is a cyclic conjugated system having 4n + 2 π electrons, and may be a monocyclic type or a polycyclic type. The aromatic ring is an aromatic hydrocarbon ring such as benzene, naphthalene, anthracene, phenanthrene; and a part of carbon atoms constituting the aromatic hydrocarbon ring is replaced with a hetero atom (oxygen atom, sulfur atom, nitrogen atom, etc.). Includes the aromatic heterocycles that have been used.
"Alphatic" is a concept relative to aromatics and means groups, compounds, etc. that do not have aromaticity.
The "alkyl group" includes linear, branched and cyclic monovalent saturated hydrocarbon groups unless otherwise specified. The same applies to the alkyl group in the alkoxy group.
The "alkylene group" includes linear, branched and cyclic divalent saturated hydrocarbon groups unless otherwise specified.

The “constituent unit derived from an acrylamide derivative” means a structural unit formed by cleavage of an ethylenic double bond of an acrylamide derivative. An "acrylamide derivative" is a compound in which one or both hydrogen atoms of the amino group of _{acrylamide (CH 2} = CH-CONH _{2) are substituted with an organic group.} In the acrylamide derivative, the hydrogen atom bonded to the carbon atom at the α-position may be substituted with a substituent. Unless otherwise specified, the carbon atom at the α-position of acrylamide is the carbon atom to which the carbonyl group of acrylamide is bonded. Examples of the substituent that replaces the hydrogen atom bonded to the α-position carbon atom of acrylamide include an alkyl group having 1 to 5 carbon atoms (methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, isobutyl group). , Tart-butyl group, pentyl group, isopentyl group, neopentyl group) and the like.

The “constituent unit derived from the acrylic acid ester” means a structural unit formed by cleaving the ethylenic double bond of the acrylic acid ester. The "acrylic acid ester" is a compound in which the hydrogen atom at the terminal of the carboxy group of _{acrylic acid (CH 2 = CH-COOH) is replaced with an organic group.} In the acrylic acid ester, the hydrogen atom bonded to the carbon atom at the α-position may be substituted with a substituent. Unless otherwise specified, the carbon atom at the α-position of the acrylic acid ester is the carbon atom to which the carbonyl group of acrylic acid is bonded. Examples of the substituent that replaces the hydrogen atom bonded to the carbon atom at the α-position of the acrylic acid ester include those similar to those mentioned in the above-mentioned structural unit derived from acrylamide.

"Shiga toxin" means toxin produced by enterohemorrhagic Escherichia coli (EHCE) and Shigella dysentis, and is a concept including Stx1 family, Stx2 family, and Stx.

The "polymerizable monomer mixture" means a mixture of polymerizable monomers to be subjected to a polymerization reaction for obtaining a crosslinked polymer. The "polymerizable monomer" means a monomer containing at least one polymerizable group. The "polymerizable group" means a functional group subjected to a polymerization reaction.

In the present specification and claims, depending on the structure represented by the chemical formula, asymmetric carbon may be present, and an enantiomer or a diastereomer may be present. In that case, one chemical formula is used to represent those isomers. These isomers may be used alone or as a mixture.

[Crosslinked polymer]
In one embodiment, the present invention provides a crosslinked polymer in which a random copolymer is crosslinked. The random copolymer has a structural unit (a1) containing a Shiga toxin-binding oligosaccharide and at least one other structural unit.

<Random copolymer>
The crosslinked polymer of this embodiment is a polymer crosslinked by a random copolymer. The random copolymer has a structural unit (a1) containing a Shiga toxin-binding oligosaccharide and at least one other structural unit. By using a copolymer having a structural unit (a1) and another structural unit, it is possible to combine Shiga toxin with a functional group capable of interacting with various modes. Moreover, by using a random copolymer, it is possible to form a diverse structure. Therefore, there is a high probability that a binding site having high binding to Shiga toxin will be formed. Further, unlike the block copolymer, the random copolymer does not need to carry out the polymerization reaction for each block, so that the production time and the production cost can be suppressed.

The random copolymer contained in the crosslinked polymer may be one kind or two or more kinds. Cross-linking of the random copolymer can be carried out by using a polymerizable monomer mixture to which a cross-linking agent containing two or more polymerizable groups is added in the copolymerization reaction for obtaining the random copolymer. Alternatively, after obtaining the random copolymer, the cross-linking reaction of the random copolymer may be carried out using a cross-linking agent having two or more groups that react with the functional groups in the random copolymer. Since the synthesis and cross-linking of the random copolymer can be performed in one step, a method of copolymerizing using a polymerizable monomer mixture containing a cross-linking agent is preferable.

(Constituent unit (a1)
The random copolymer contained in the crosslinked polymer of the present embodiment has a structural unit (a1) containing a Shiga toxin-binding oligosaccharide. The crosslinked polymer of the present embodiment has a high binding property to Shiga toxin by having the structural unit (a1).
The Shiga toxin-binding oligosaccharide contained in the structural unit (a1) is not particularly limited as long as it has Shiga toxin-binding property. The Shiga toxin-binding oligosaccharide is preferably a Globotrisaccharide or a derivative thereof. Globotrisaccharide is a trisaccharide having a structure of Galα (1-4) -Galβ (1-4) -Glc-. In the following formula, * is a bond.

Examples of the structural unit (a1) include a structural unit represented by the following general formula (a1-1).

[In the formula, R represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. Y ¹ represents a divalent linking group. Gb represents a Shiga toxin-binding oligosaccharide. ]

In the general formula (a1-1), R represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. The alkyl group preferably has 1 to 3 carbon atoms, more preferably a methyl group or an ethyl group, and even more preferably a methyl group. R is particularly preferably a hydrogen atom.

In the general formula (a1-1), Y ¹ represents a divalent linking group. Examples of the divalent linking group include a divalent hydrocarbon group which may have a substituent. The divalent hydrocarbon group may be an aliphatic hydrocarbon group or an aromatic hydrocarbon group.

The aliphatic hydrocarbon group for Y ¹ may be a saturated, or unsaturated. Examples of the aliphatic hydrocarbon group include a linear or branched aliphatic hydrocarbon group and an aliphatic hydrocarbon group having a ring in the structure.

The linear aliphatic hydrocarbon group preferably has 1 to 15 carbon atoms, more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6 carbon atoms. As the linear aliphatic hydrocarbon group, a linear alkylene group is preferable.
The branched aliphatic hydrocarbon group preferably has 2 to 15 carbon atoms, more preferably 2 to 10 carbon atoms, and even more preferably 3 to 6 carbon atoms. As the branched-chain aliphatic hydrocarbon group, a branched-chain alkylene group is preferable.

As the aliphatic hydrocarbon group containing a ring in the structure, a cyclic aliphatic hydrocarbon group which may contain a substituent containing a hetero atom in the ring structure (a group obtained by removing two hydrogen atoms from the aliphatic hydrocarbon ring). ), A group in which the cyclic aliphatic hydrocarbon group is bonded to the terminal of a linear or branched aliphatic hydrocarbon group, and the cyclic aliphatic hydrocarbon group is a linear or branched aliphatic. Examples thereof include a group intervening in the middle of the hydrocarbon group. Examples of the linear or branched aliphatic hydrocarbon group include the same groups as described above. The cyclic aliphatic hydrocarbon group preferably has 3 to 20 carbon atoms, and more preferably 3 to 12 carbon atoms. The cyclic aliphatic hydrocarbon group may be a polycyclic group or a monocyclic group. The cyclic aliphatic hydrocarbon group may be substituted with a substituent containing a hetero atom (oxygen atom, nitrogen atom, sulfur atom, etc.) as a part of the carbon atom constituting the ring structure.

When Y ¹ is an aromatic hydrocarbon group, the aromatic ring contained in the aromatic hydrocarbon group is more preferably 6 to 15 carbon atoms, and particularly preferably 6 to 12 carbon atoms. Examples of the aromatic ring include aromatic hydrocarbon rings such as benzene, naphthalene, anthracene and phenanthrene; and aromatic heterocycles such as triazole ring, pyridine ring and thiophene ring.
Specifically, the aromatic hydrocarbon group is a group obtained by removing two hydrogen atoms from the aromatic hydrocarbon ring or aromatic heterocycle (arylene group or heteroarylene group); an aromatic compound containing two or more aromatic rings. A group obtained by removing two hydrogen atoms from (for example, biphenyl, fluorene, etc.); one of the hydrogen atoms of the group (aryl group or heteroaryl group) obtained by removing one hydrogen atom from the aromatic hydrocarbon ring or aromatic heterocyclic ring. Examples thereof include a group in which one is substituted with an alkylene group (a group obtained by removing one hydrogen atom from an aryl group or a heteroaryl group) and the like. The alkylene group that replaces the hydrogen atom preferably has 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, and even more preferably 1 to 4 carbon atoms.

Hydrocarbon group which may have a substituent in Y ¹ are methylene groups constituting the carbon chain - be those part of has been replaced with a divalent linking group containing a hetero atom (-CH ₂₎ You may. Examples of the divalent linking group containing a heteroatom substituting the methylene group include —O—, —C (= O) —O—, —OC (= O) −, —C (= O). -, -OC (= O) -O-, -C (= O) -NH-, -NH-, -NH-C (= NH)-, -C (= O) -NH-C (= O) - (H alkyl group may be substituted with a substituent such as an acyl group), -. S -, - S (= O) 2 -, and -S _{(= O)} 2 -O-, etc. Can be mentioned.

The structural unit (a1) is preferably a structural unit derived from an acrylamide derivative or a structural unit derived from an acrylic acid ester. Table ^{(Y 11} can also be divalent substituted hydrocarbon group) at - Therefore, preferable examples of ^{Y 1 are, -CO-NH-Y 11 -} , or ^-CO-O-Y 11 include groups, ^{-CO-NH-Y 11} - is a group represented by the more preferable.

Examples of the divalent hydrocarbon group that may have a substituent in Y ¹¹ include those similar to those mentioned in ^{Y 1.} Among them, Y ¹¹ is preferably an alkylene group which may have a substituent, or a group in which one of the hydrogen atoms of an aryl group or a heteroaryl group is substituted with an alkylene group.

The alkylene group which may have the substituent may be linear or branched, but is preferably linear. The linear alkylene group preferably has 1 to 15 carbon atoms, more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6 carbon atoms. The branched alkylene group preferably has 2 to 15 carbon atoms, more preferably 2 to 10 carbon atoms, and even more preferably 2 to 6 carbon atoms.
When Y ¹¹ is an alkylene group which may have a substituent, a part of the methylene group constituting the carbon chain of the alkylene group may be substituted with a divalent linking group containing a heteroatom. Examples of the divalent linking group containing the hetero atom include the same ones as mentioned in Y ^1.

When Y ¹¹ is a group in which one of the hydrogen atoms of the aryl group or the heteroaryl group is substituted with an alkylene group, the alkylene group substituting the hydrogen atom may be linear or branched. Although it may be used, it is preferably linear. The linear alkylene group preferably has 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, and even more preferably 1 to 3 carbon atoms. The branched alkylene group preferably has 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and even more preferably 2 to 4 carbon atoms. Examples of the aromatic ring contained in the aryl group include a benzene ring. Examples of the heterocyclic ring contained in the heteroaryl group include a triazole ring, an imidazole ring, a pyridine ring, and a thiophene ring.
Of these, Y ¹¹ is preferably a group in which one of the hydrogen atoms of the triazole group is substituted with an alkylene group.

In the general formula (a1-1), Gb represents a Shiga toxin-binding oligosaccharide. Gb is preferably Globotrisaccharide.

A preferable example of the structural unit (a1) is a structural unit represented by the following general formula (a1-1-1).

[In the formula, R represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. L ¹ represents -CO-O- or -CO-NH-. R ¹ represents a linear or branched alkylene group having 1 to 10 carbon atoms. Gb represents a Shiga toxin-binding oligosaccharide. ]

In the general formula (a1-1-1), R and Gb are the same as R and Gb in the general formula (a1-1), respectively.
In the general formula (a1-1-1), L ¹ represents -CO-O- or -CO-NH-. L ¹ is preferably -CO-NH-.
In the general formula (a1-1-1), R ¹ represents a linear or branched alkylene group having 1 to 10 carbon atoms. The alkylene group preferably has 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms, further preferably 1 to 3 carbon atoms, and particularly preferably a methylene group or an ethylene group.

Specific examples of the structural unit (a1) are given below, but the present invention is not limited to this. In the formula, R ^α represents a hydrogen atom or a methyl group.

The structural unit (a1) may be one type or two or more types.
The ratio of the structural unit (a1) in the total random copolymer contained in the crosslinked polymer of the present embodiment is preferably 3 mol% or more with respect to the total (100 mol%) of all the structural units constituting the total random copolymer. It is preferably mol% or more, more preferably 8 mol% or more, and particularly preferably 10 mol% or more. When the ratio of the constituent unit (a1) is equal to or higher than the preferable lower limit value, the Shiga toxin inhibitory effect is further improved. The upper limit of the ratio of the constituent unit (a1) in the all random copolymer is not particularly limited. When the constituent unit (a1) contains a Globotrisaccharide as a Shiga toxin-binding oligosaccharide, the proportion of the constituent unit (a1) is preferably, for example, 40 mol% or less, more preferably 30 mol% or less, from the viewpoint of production cost. Preferably, 20 mol% or less is more preferable, and 15 mol% or less is particularly preferable. The ratio of the structural unit (a1) is preferably 3 to 40 mol%, more preferably 5 to 30 mol%, further preferably 8 to 20 mol%, and particularly preferably 10 to 15 mol%.

(Other building blocks)
The random copolymer has at least one other structural unit in addition to the above structural unit (a1). Examples of the other structural units include a structural unit containing a hydrophobic group having 4 to 20 carbon atoms (a2), a structural unit containing a cationic group (a3), a structural unit containing an anionic group (a4), and 1-. (3-sulfopropyl) -2-vinylpyridinium hydroxide The structural unit (a5) derived from the intramolecular salt (hereinafter, also referred to as “PPS”), the structural unit (a6) derived from the cross-linking agent, and the like can be mentioned. Be done.

≪Constructive unit (a2) ≫
The structural unit (a2) is a structural unit containing a hydrophobic group having 4 to 20 carbon atoms. The random copolymer preferably has a structural unit (a2). When the random copolymer has the constituent unit (a2), the Shiga toxin inhibitory effect is further improved.

Examples of the hydrophobic group having 4 to 20 carbon atoms contained in the structural unit (a2) include a hydrocarbon group having 4 to 20 carbon atoms. The hydrocarbon group may be an aliphatic hydrocarbon group or an aromatic hydrocarbon group.

Examples of the aliphatic hydrocarbon group include a linear or branched aliphatic hydrocarbon group, an aliphatic hydrocarbon group containing a ring in the structure, and the like. The aliphatic hydrocarbon group preferably has 4 to 15 carbon atoms, more preferably 4 to 10 carbon atoms, and even more preferably 4 to 6 carbon atoms. The aliphatic hydrocarbon group may be saturated or unsaturated, but is preferably saturated.
Examples of the linear aliphatic hydrocarbon group include linear alkyl groups such as n-butyl group, n-pentyl group, n-hexyl group, n-heptyl group and n-octyl group. Examples of the branched aliphatic hydrocarbon group include an isobutyl group, a tert-butyl group, an isopentyl group, a neopentyl group, a 1,1-dimethylpropyl group, a 1,1-diethylpropyl group and a 2,2-dimethylbutyl group. Branched chain alkyl groups can be mentioned.
The aliphatic hydrocarbon group containing a ring in the structure may be a monocyclic group or a polycyclic group. Examples of the monocyclic group include cycloalkyl groups such as cyclobutyl group, cyclopentyl group, cyclohexyl group, methylcyclohexyl group, dimethylcyclohexyl group, cycloheptyl group, cyclooctyl group, cyclononyl group and cyclodecyl group. Examples of the polycyclic group include a decahydronaphthyl group, an adamantyl group, a 2-alkyladamantan-2-yl group, a 1- (adamantan-1-yl) alkane-1-yl group, a norbornyl group, and a methylnorbornyl group. Examples thereof include an isobornyl group.

The aromatic hydrocarbon group preferably has 6 to 20 carbon atoms, more preferably 6 to 15 carbon atoms, and even more preferably 6 to 10 carbon atoms. The aromatic hydrocarbon group may be a monocyclic group or a polycyclic group. Examples of the monocyclic aromatic hydrocarbon group include a phenyl group; and a p-methylphenyl group, a p-tert-butylphenyl group, a tolyl group, a xsilyl group, a cumenyl group, a mesityl group, a 2,6-diethylphenyl group, and 2 Examples thereof include a group in which a part of hydrogen atoms of a phenyl group such as a -methyl-6-ethylphenyl group and a p-adamantylphenyl group is substituted with an alkyl group or a cycloalkyl group. Examples of the polycyclic aromatic hydrocarbon group include a biphenyl group, a phenanthryl group, a naphthyl group, an anthryl group and the like.

As the hydrophobic group, a branched chain alkyl group or a monocyclic aromatic hydrocarbon group is preferable, and a tert-butyl group or a phenyl group is more preferable.

Examples of the structural unit (a2) include those represented by the following general formula (a2-1).

[In the formula, R represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. Y ² represents a single bond or a divalent linking group. R ² represents a hydrophobic group having 4 to 20 carbon atoms. ]

In the general formula (a2-1), R is the same as R in the above formula (a1-1).

In the general formula (a2-1), Y ² represents a single bond or a divalent linking group. Examples of the divalent linking group in ^{Y 2} include those similar to those listed as the divalent linking group in ^{Y 1 in the general formula (a1-1).} The structural unit (a2) is preferably a structural unit derived from an acrylamide derivative or a structural unit derived from an acrylic acid ester. Therefore, preferred examples of ^{Y 2 are, -CO-NH-Y 21 -} , or ^{-CO-O-Y 21 - (} Y 21 is 2 may have a single bond or a substituent monovalent hydrocarbon group ) the group represented by may be mentioned, ^{-CO-NH-Y 21} - group represented by are preferred. When Y ²¹ is a divalent hydrocarbon group which may have a substituent, the divalent hydrocarbon group which may have the substituent is Y in the general formula (a1-1). ^The same as those mentioned in 1 can be mentioned. Y ²¹ is preferably a single bond or an aliphatic hydrocarbon group having 1 to 10 carbon atoms, and more preferably a single bond or a linear or branched alkylene group having 1 to 10 carbon atoms. The linear or branched alkylene group preferably has 1 to 6 carbon atoms, more preferably 1 to 3 carbon atoms, and even more preferably 1 or 2 carbon atoms.
In the general formula (a2-1), R ² represents a hydrophobic group having 4 to 20 carbon atoms. Hydrophobic groups include those similar to those listed above.

As the structural unit (a2), a structural unit represented by the following general formula (a2-1-1) is preferable.

[In the formula, R represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. L ² represents a single bond, -CO-O- or -CO-NH-. Y ²² represents a single bond or a linear or branched alkylene group having 1 to 10 carbon atoms. R ² represents a hydrophobic group having 4 to 20 carbon atoms. ]

In the general formula (a2-1-1), R is the same as R in the general formula (a2-1).
In the general formula (a2-1-1), L ² represents a single bond, -CO-O-, or -CO-NH-. L ² is preferably -CO-NH-.
In the general formula (a2-1-1), Y ²² represents a single bond or a linear or branched alkylene group having 1 to 10 carbon atoms. The alkylene group preferably has 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms, further preferably 1 to 3 carbon atoms, and particularly preferably a methylene group or an ethylene group. Y ²² is preferably a single bond or an alkylene group having 1 to 3 carbon atoms, more preferably a single bond or an alkylene group having 1 to 2 carbon atoms, further preferably a single bond or a methylene group, and particularly preferably a single bond.
In the general formula (a2-1-1), R ² represents a hydrophobic group having 4 to 20 carbon atoms. R ² is the same as ^{R 2} in the general formula (a2-1).

Specific examples of the structural unit (a2) are given below, but the present invention is not limited thereto. In the formula, R ^α represents a hydrogen atom or a methyl group.

The structural unit (a2) may be one type or two or more types.
When the random copolymer has a structural unit (a2), the proportion of the structural unit (a2) in the total random copolymer contained in the crosslinked polymer is relative to the total (100 mol%) of all the structural units constituting the total random copolymer. 10 to 80 mol% is preferable, 20 to 80 mol% is preferable, 30 to 75 mol% is more preferable, and 40 to 70 mol% is particularly preferable. By setting the ratio of the constituent unit (a2) within the above preferable range, the Shiga toxin inhibitory effect is further improved.

≪Constructive unit (a3) ≫
The structural unit (a3) is a structural unit containing a cationic group. The random copolymer preferably has a structural unit (a3). When the random copolymer has the constituent unit (a3), the Shiga toxin inhibitory effect is further improved.

A cationic group means a positively charged atomic group. The cationic group is in the form of a salt formed by forming a salt with fluoride ion, chloride ion, bromide ion, iodide ion, hydrochloric acid ion, acetate ion, sulfate ion, hydrofluoride ion, carbonate ion and the like. There may be. Cationic group structural unit (a3) contains is not particularly limited, primary amino group _(-NH 2), 2 amino groups (-NHR ^3), 3 amino groups ^(-NR ₃ 2), 4 grade Examples thereof include, but are not limited to, an amino group (-NR ³ ₃ ⁺ ) and a heterocyclic group containing a cationic nitrogen atom.

^{R 3} in the secondary amino group, tertiary amino group, and quaternary amino group represents an organic group. Tertiary amino group, or quaternary amino group, two or more of R ³ may be each the same or different. Examples of the organic group include a hydrocarbon group which may have a substituent. Examples of the hydrocarbon group which may have a substituent include those similar to those mentioned in ^{R 1 in the general formula (a1-1-1).} The R ^3, an alkyl group, an aryl group, or an aralkyl group is preferable. The alkyl group preferably has 1 to 6 carbon atoms, more preferably 1 to 3 carbon atoms, and even more preferably 1 or 2 carbon atoms. The aryl group or aralkyl group preferably has 6 to 12 carbon atoms, and more preferably 6 to 10 carbon atoms. Specific examples of R ³ include, but are not limited to, a methyl group, an ethyl group, an n-propyl group, a cyclopentyl group, a cyclohexyl group, a phenyl group, a benzyl group and the like.

The heterocyclic group containing a cationic nitrogen atom may be an aliphatic heterocyclic group or an aromatic heterocyclic group. The aliphatic heterocyclic group preferably has 3 to 15 carbon atoms, and more preferably 5 to 10 carbon atoms. Examples of the aliphatic heterocyclic group include, but are not limited to, a piperidinium group, a 1-pyrrolidinium group, a 1-methylpyrrolidinium group and the like. Examples of the aromatic heterocyclic group include an imidazolium group, a 1-methylimidazolium group, a 1-ethylimidazolium group, a benzimidazolium group, a pyrollium group, a 1-methylpyrrolium group, an oxazolium group and a benzoxazolium group. , Benzisooxazolium group, pyrazolium group, isooxazolium group, pyridinium group, 2,6-dimethylpyridinium group, pyrazinium group, pyrimidinium group, pyridadinium group, triazinium group, but are not limited thereto.

As the cationic group, a tertiary amino group, a quaternary amino group, or a heterocyclic group containing a nitrogen atom is preferable, and a dimethylamino group, a trimethylamino group, or an imidazolium group is more preferable.

Examples of the structural unit (a3) include those represented by the following general formula (a3-1).

[In the formula, R represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. Y ³ represents a single bond or a divalent linking group. Rc ³ represents a cationic group. ]

In the general formula (a3-1), R is the same as R in the above formula (a1-1).

In the general formula (a3-1), Y ³ represents a single bond or a divalent linking group. Examples of the divalent linking group in ^{Y 3} include those similar to those listed as the divalent linking group in ^{Y 1 in the general formula (a1-1).} The structural unit (a3) is preferably a structural unit derived from an acrylamide derivative or a structural unit derived from an acrylic acid ester. Therefore, preferred examples of ^{Y 3 is, -CO-NH-Y 31 -} , or ^{-CO-O-Y 31 - (} Y 31 is 2 may have a single bond or a substituent monovalent hydrocarbon group ) the group represented by may be mentioned, ^{-CO-NH-Y 31} - more preferably a group represented by. When Y ³¹ is a divalent hydrocarbon group which may have a substituent, the divalent hydrocarbon group which may have the substituent is Y in the general formula (a1-1). ^The same as those mentioned in 1 can be mentioned. Y ³¹ is preferably an aliphatic hydrocarbon group having 1 to 10 carbon atoms, and more preferably a linear or branched alkylene group having 1 to 10 carbon atoms. The linear or branched alkylene group preferably has 1 to 6 carbon atoms, and more preferably 1 to 3 carbon atoms.
In the general formula (a3-1), Rc ³ represents a cationic group. Examples of the cationic group include those similar to those listed above.

As the structural unit (a3), a structural unit represented by the following general formula (a3-1-1) is preferable.

[In the formula, R represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. L ³ represents a single bond, -CO-O-, or -CO-NH-. Y ³² represents a single bond or a linear or branched alkylene group having 1 to 10 carbon atoms. Rc ³ represents a cationic group. ]

In the general formula (a3-1-1), R is the same as R in the general formula (a3-1).
In the general formula (a3-1-1), L ³ represents a single bond, -CO-O-, or -CO-NH-. L ³ is preferably single bond or -CO-NH-.
In the general formula (a3-1-1), Y ³² represents a single bond or a linear or branched alkylene group having 1 to 10 carbon atoms. The alkylene group preferably has 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms, and even more preferably 1 to 3 carbon atoms. Y ³² is preferably a single bond or an alkylene group having 1 to 3 carbon atoms, more preferably a single bond or an alkylene group having 1 to 2 carbon atoms, further preferably a single bond or a methylene group, and particularly preferably a single bond. Specific examples of Y ³² include a single bond, an n-propylene group, an ethylene group, a methylene group and the like.
In the general formula (a3-1-1), Rc ³ represents a cationic group. Rc ³ is the same as Rc ³ in the general formula (a3-1).

Specific examples of the structural unit (a3) are given below, but the present invention is not limited thereto. In the formula, R ^α represents a hydrogen atom or a methyl group.

The structural unit (a3) may be one type or two or more types.
When the random copolymer has a structural unit (a3), the proportion of the structural unit (a3) in the total random copolymer contained in the crosslinked polymer is relative to the total (100 mol%) of all the structural units constituting the total random copolymer. 1 to 20 mol% is preferable, 1 to 15 mol% is preferable, and 1 to 10 mol% is more preferable. By setting the ratio of the constituent unit (a3) within the above preferable range, the Shiga toxin inhibitory effect is further improved.

≪Constructive unit (a4) ≫
The structural unit (a4) is a structural unit containing an anionic group. The random copolymer may have a structural unit (a4).

Anionic group means a negatively charged atomic group. The anionic group may be in the form of a salt formed by forming a salt with an alkali metal ion such as sodium ion or potassium ion; and an alkaline earth metal ion such as calcium ion. The anionic group contained in the structural unit (a4) is not particularly limited, but includes a hydroxy group, a carboxy group, a sulfo group, a sulfonyl group, a phosphoric acid group, a phosphonic acid group, a phosphinic acid group, a thiol group, a boronic acid group and the like. These include, but are not limited to. The anionic group is preferably a hydroxy group, a carboxy group, a sulfo group, or a sulfonyl group, and more preferably a carboxy group.

Examples of the structural unit (a4) include those represented by the following general formula (a4-1).

[In the formula, R represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. Y ⁴ represents a single bond or a divalent linking group. Ra ⁴ represents an anionic group. ]

In the general formula (a4-1), R is the same as R in the formula (a1-1).

In the general formula (a4-1), Y ⁴ represents a single bond or a divalent linking group. Examples of the divalent linking group in ^{Y 4} include those similar to those listed as the divalent linking group in ^{Y 1 in the general formula (a1-1).} The structural unit (a4) is preferably a structural unit derived from an acrylamide derivative or a structural unit derived from an acrylic acid ester. Therefore, preferred examples of ^{Y 4 is, -CO-NH-Y 41 -} , or ^{-CO-O-Y 41 - (} Y 41 is 2 may have a single bond or a substituent monovalent hydrocarbon group ) is a group represented by the mentioned, ^{-CO-NH-Y 41} - group is more preferably represented by. When Y ⁴¹ is a divalent hydrocarbon group which may have a substituent, the divalent hydrocarbon group which may have the substituent is Y in the general formula (a1-1). ^The same as those mentioned in 1 can be mentioned. Y ⁴¹ is preferably an aliphatic hydrocarbon group having 1 to 10 carbon atoms, and more preferably a linear or branched alkylene group having 1 to 10 carbon atoms. The linear or branched alkylene group preferably has 1 to 6 carbon atoms, and more preferably 1 to 3 carbon atoms.
In the general formula (a4-1), Ra ⁴ represents an anionic group. Examples of the anionic group include those similar to those listed above.

As the structural unit (a4), a structural unit represented by the following general formula (a4-1-1) is preferable.

[In the formula, R represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. L ⁴ represents a single bond, -CO-O-, or -CO-NH-. Y ⁴² represents a single bond or a linear or branched alkylene group having 1 to 10 carbon atoms. Ra ⁴ represents an anionic group. ]

In the general formula (a4-1-1), R is the same as R in the general formula (a4-1).
In the general formula (a4-1-1), L ⁴ represents a single bond, -CO-O-, or -CO-NH-. L ⁴ is preferably single bond or -CO-NH-.
In the general formula (a4-1-1), Y ⁴² represents a single bond or a linear or branched alkylene group having 1 to 10 carbon atoms. The alkylene group preferably has 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms, and even more preferably 1 to 3 carbon atoms. Y ⁴² is preferably a single bond or an alkylene group having 1 to 3 carbon atoms, more preferably a single bond or an alkylene group having 1 to 2 carbon atoms, further preferably a single bond or a methylene group, and particularly preferably a single bond.
In the general formula (a4-1-1), Ra ⁴ represents an anionic group. Ra ⁴ is the same as Ra ⁴ in the general formula (a4-1).

Specific examples of the structural unit (a4) are given below, but the present invention is not limited to this. In the formula, R ^α represents a hydrogen atom or a methyl group.

The structural unit (a4) may be one type or two or more types.
When the random copolymer has a structural unit (a4), the proportion of the structural unit (a4) in the total random copolymer contained in the crosslinked polymer is relative to the total (100 mol%) of all the structural units constituting the total random copolymer. 1 to 20 mol% is preferable, 1 to 15 mol% is preferable, and 1 to 10 mol% is more preferable. By setting the ratio of the constituent unit (a4) within the above preferable range, the Shiga toxin inhibitory effect is further improved.

≪Constructive unit (a5) ≫
The structural unit (a5) is a structural unit derived from the 1- (3-sulfopropyl) -2-vinylpyridinium hydroxide intramolecular salt. The random copolymer preferably has a structural unit (a5). When the random copolymer has the constituent unit (a5), the binding property to Shiga toxin is improved, and the Shiga toxin inhibitory effect is further improved.

The structural unit (a5) is a structural unit represented by the following formula (a5-1).

When the random copolymer has a structural unit (a5), the proportion of the structural unit (a5) in the total random copolymer contained in the crosslinked polymer is relative to the total (100 mol%) of all the structural units constituting the total random copolymer. 1 to 20 mol% is preferable, 1 to 15 mol% is preferable, 1 to 10 mol% is more preferable, and 2 to 8 mol% is particularly preferable. By setting the ratio of the constituent unit (a5) within the above preferable range, the Shiga toxin inhibitory effect is further improved.

≪Constructive unit (a6) ≫
The structural unit (a6) is a structural unit derived from the cross-linking agent. The random copolymer preferably has a structural unit (a6). Since the random copolymer having the structural unit (a6) can carry out the copolymerization reaction and the cross-linking reaction in one step, the production cost can be reduced.

Examples of the cross-linking agent include compounds containing two or more polymerizable groups. As the polymerizable group, a group containing an ethylenic double bond is preferable. Examples of the group containing an ethylenic double bond include a vinyl group and a (meth) acryloyl group. The (meth) acryloyl group means either a methacryloyl group or an acryloyl group. The structural unit (a6) is a structural unit formed by cleaving the polymerizable group of the cross-linking agent.

Examples of the cross-linking agent include a bifunctional or higher functional (meth) acrylamide compound or a bifunctional or higher functional (meth) acrylate compound. The (meth) acrylamide compound means either a methacrylamide compound or an acrylamide compound. The (meth) acrylate compound means either a methacrylate compound or an acrylate compound.
Examples of the bifunctional or higher functional (meth) acrylamide compound include N, N'-methylenebisacrylamide, N, N'-methylenebismethacrylamide, N, N'-ethylenebisacrylamide, ethylenediaminedimethylacrylamide, ethylenediaminediacrylamide and the like. However, it is not limited to these.
Examples of the bifunctional or higher functional (meth) acrylate compound include tetraethylene glycol diacrylate, triethylene glycol diacrylate, ethylene glycol diacrylate, diethylene glycol diacrylate, tetraethylene glycol dimethacrylate, triethylene glycol dimethacrylate, and ethylene glycol dimethacrylate. Examples thereof include, but are not limited to, diethylene glycol dimethacrylate, trimethylopropane trimethacrylate, pentaerythritol tetramethacrylate, bisphenol A dimethacrylate, and glycerol dimethacrylate.

Specific examples of the structural unit (a6) are given below, but the present invention is not limited thereto. In the formula, R ^α independently represents a hydrogen atom or a methyl group. n represents an integer of 0 to 2, and 1 is preferable.

The structural unit (a6) may be one type or two or more types.
When the random copolymer has a structural unit (a6), the proportion of the structural unit (a6) in the total random copolymer contained in the crosslinked polymer is relative to the total (100 mol%) of all the structural units constituting the total random copolymer. 5 to 20 mol% is preferable, 5 to 15 mol% is preferable, and 8 to 12 mol% is more preferable. By setting the ratio of the structural unit (a6) within the above preferable range, the particle size of the crosslinked polymer can be controlled within an appropriate range.

≪Other: Structural unit (a7) ≫
The random copolymer may have a structural unit other than the above (a1) to (a6) (hereinafter, also referred to as “constituent unit (a7)”) as long as the effect of the present invention is not impaired. The structural unit (a7) is not particularly limited. Examples of the structural unit (a7) include a structural unit derived from an acrylamide derivative such as N-isopropylacrylamide, N-ethylacrylamide, and N-methylacrylamide.

The structural unit (a7) may be one type or two or more types.
When the random copolymer has a structural unit (a7), the proportion of the structural unit (a7) in the total random copolymer contained in the crosslinked polymer is relative to the total (100 mol%) of all the structural units constituting the total random copolymer. It is preferably 10 to 80 mol%, preferably 10 to 60 mol%, and even more preferably 10 to 50 mol%.

The random copolymer contained in the crosslinked polymer may be one kind or two or more kinds. The combination of structural units contained in the random copolymer includes a combination of structural units (a1), structural units (a2), and structural units (6); structural units (a1), structural units (a2), structural units (a3), and Combination of constituent units (6); Combination of constituent units (a1), constituent units (a2), constituent units (a3), constituent units (6), and constituent units (a7); constituent units (a1), constituent units ( Combination of a2), constituent unit (a3), constituent unit (a5), constituent unit (6), and constituent unit (a7); constituent unit (a1), constituent unit (a3), constituent unit (a6), and configuration Combination of units (a7); Combination of constituent units (a1), constituent units (a6), and constituent units (a7); constituent units (a1), constituent units (a4), constituent units (a6), and constituent units (a6) The combination of a7); and the like can be mentioned.
Preferred combinations include a configuration unit (a1), a configuration unit (a2), a configuration unit (a3), and a configuration unit (a6); a configuration unit (a1), a configuration unit (a2), a configuration unit (a3), Combination of constituent unit (a5) and constituent unit (a6); constituent unit (a1), constituent unit (a2), constituent unit (a3), constituent unit (a5), constituent unit (a6), and constituent unit (a7) ); Combinations of the constituent unit (a1), the constituent unit (a2), the constituent unit (a3), the constituent unit (a6), and the constituent unit (a7); and the like.
Among them, the combination of the structural unit (a1), the structural unit (a2), the structural unit (a3), the structural unit (a5), the structural unit (a6), and the structural unit (a7) is particularly preferable.

The crosslinkable polymer preferably has a theoretical molecular weight of 100 to 500, more preferably 120 to 300, and even more preferably 150 to 200. The "theoretical molecular weight" of the crosslinkable polymer is a theoretical value calculated from the charged molar ratio of each polymerizable monomer at the time of synthesizing the crosslinkable polymer and the molecular weight of each polymerizable monomer. In the present specification, the "theoretical molecular weight" is a value calculated as follows.
The molar ratio of each polymerizable monomer when the total amount of all the polymerizable monomers in the polymerizable monomer mixture is 1 ( _RA : the value in [] in Table 1 divided by 100) is calculated. The molar ratio R _A, calculates the molecular weight obtained by multiplying the value of the polymerizable monomer (M _A). Value which is the sum of M _A calculated for each monomer of the polymerizable monomer mixture is a molecular weight of theory.

The crosslinked polymer of the present embodiment preferably has a molecular weight of 1000 to 5000, more preferably 1200 to 3000, and even more preferably 1500 to 2000. The "molecular weight per sugar unit" of the crosslinkable polymer is a value obtained by dividing the theoretical molecular weight of the crosslinkable polymer by the molar ratio ( _{RA) of the polymerizable monomer that induces the structural unit (a1).}

The crosslinked polymer of the present embodiment preferably has a Z average particle size of about 30 to 2000 at 35 ° C. measured by a dynamic scattering method. Further, the crosslinked polymer of the present embodiment preferably has a Z average particle size of about 30 to 2000 at 40 ° C. measured by a dynamic scattering method.

<Manufacturing method of crosslinked polymer>
The crosslinked polymer of the present embodiment is produced by radically polymerizing a polymerizable monomer mixture containing a polymerizable monomer for inducing each structural unit of a random copolymer and a crosslinking agent in the presence of a polymerization initiator. Can be done. Alternatively, after radical polymerization of the polymerizable monomer that induces each structural unit to obtain a random copolymer, a cross-linking reaction of the random copolymer is carried out using a cross-linking agent having two or more groups that react with functional groups in the random copolymer. You may go. Since the synthesis and cross-linking of the random copolymer can be performed in one step, a method of copolymerizing using a polymerizable monomer mixture containing a cross-linking agent is preferable. Examples of the cross-linking agent include those listed in the above-mentioned structural unit (a6).

As the polymerization initiator, one generally used for radical polymerization may be used, for example, V-501 (4,4'-Azobis (4-cyanovalic acid)) or AAPD (2,2'-Azobis (2-2'-Azobis)). (Methlypolymeridine) Dihydrochloride) and the like can be mentioned. Further, as the reaction solvent for radical polymerization, an aqueous medium containing a surfactant can be used. As the surfactant, for example, sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide (CTAB), or the like can be used.

The crosslinked polymer of the present embodiment exhibits high inhibitory activity against Shiga toxin by having a structure in which a random copolymer having a structural unit (a1) and at least one other structural unit is crosslinked. be able to. It is considered that this is because the cross-linking of the random copolymer forms various structures and can bind to Shiga toxin at multiple sites.
Since the crosslinked polymer of the present embodiment has high inhibitory activity against Shiga toxin, it can be used as a Shiga toxin inhibitor described later and a pharmaceutical composition for treating or preventing a Shiga toxin-producing bacterial infection.

[Shiga toxin inhibitor]
In one embodiment, the present invention provides a Shiga toxin inhibitor containing the crosslinked polymer of the embodiment.

Since the crosslinked polymer of the above embodiment has high Shiga toxin inhibitory activity, it can be used as a Shiga toxin inhibitor. The crosslinked polymer of the above embodiment contained in the Shiga toxin inhibitor may be one kind or two or more kinds.
Further, the crosslinked polymer may be a pharmaceutically acceptable salt of the crosslinked polymer), a pharmaceutically acceptable solvate of the crosslinked polymer, or a solvate of a pharmaceutically acceptable salt of the crosslinked polymer. good.

The Shiga toxin inhibitor may contain other components in addition to the crosslinked polymer of the above embodiment. The other ingredients are not particularly limited, and those commonly used in the pharmaceutical field can be used without particular limitation. Examples of other components include pharmaceutically acceptable carriers described later. The Shiga toxin inhibitor can be formulated by a known method by mixing the crosslinked polymer of the above embodiment with other components as appropriate.

The route of administration of the Shiga toxin inhibitor is not particularly limited as long as it can reach the intestinal tract, but oral administration is preferable. The Shiga toxin inhibitor can be an orally administered preparation.

Targets for administration of the Shiga toxin inhibitor include humans and mammals other than humans. Mammals other than humans are not particularly limited, but are primates (monkeys, chimpanzees, gorillas, etc.), rodents (mouses, hamsters, rats, etc.), rabbits, dogs, cats, cows, pigs, goats, sheep, horses, etc. Can be mentioned.

The Shiga toxin inhibitor of this embodiment can be used in vitro or in vivo to neutralize the toxicity of Shiga toxin. Alternatively, it can be administered to animals such as humans as a pharmaceutical composition described later.

[Pharmaceutical composition]
In one embodiment, the present invention provides a pharmaceutical composition comprising the crosslinked polymer of the embodiment and a pharmaceutically acceptable carrier for treating or preventing a Shiga toxin-producing bacterial infection.

Shiga toxin-producing bacterial infection means a disease caused by the production of Shiga toxin in the intestinal tract by bacteria that produce Shiga toxin. Examples of Shiga toxin-producing bacteria include EHEC. Examples of EHEC include Escherichia coli in which the O antigen is O157, O111, O26, O103, O104, O118, O121, O145, O165 and the like.
Examples of the application target of the pharmaceutical composition of the present embodiment include the same targets as the above-mentioned Shiga toxin inhibitor.

The crosslinked polymer of the above embodiment contained in the pharmaceutical composition of the present embodiment may be one kind or two or more kinds. The pharmaceutical composition of the present embodiment may contain at least one pharmaceutically acceptable carrier in addition to the crosslinked polymer. The "pharmaceutically acceptable carrier" means a carrier that does not inhibit the physiological activity of the active ingredient and does not exhibit substantial toxicity to the administration subject. By "not showing substantial toxicity" is meant that the ingredient is not toxic to the subject at commonly used doses. In the pharmaceutical composition of the present embodiment, the pharmaceutically acceptable carrier is a carrier that does not inhibit the Shiga toxin inhibitory activity of the crosslinked polymer and does not exhibit substantial toxicity to the administration subject thereof. The pharmaceutically acceptable carrier includes any known pharmaceutically acceptable ingredient, which is typically considered an inactive ingredient. Pharmaceutically acceptable carriers are not particularly limited, but include, for example, solvents, diluents, vehicles, excipients, flow promoters, binders, granulators, dispersants, suspending agents, wetting agents, etc. Lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, fillers, preservatives (eg antioxidants), chelating agents, flavoring agents, sweeteners, thickeners, buffers, colorants, etc. Can be mentioned. As the pharmaceutically acceptable carrier, one type may be used alone, or two or more types may be used in combination.

The pharmaceutical composition of the present embodiment may contain other components other than the crosslinked polymer and the pharmaceutically acceptable carrier. The other ingredients are not particularly limited, and those commonly used in the pharmaceutical field can be used without particular limitation. In addition, the pharmaceutical composition of the present embodiment may contain an active ingredient other than the crosslinked polymer. Examples of the active ingredient include, but are not limited to, antibiotics, antidiarrheal agents, antidiarrheal agents, antipyretics, analgesics and the like. As for the other components, one type may be used alone, or two or more types may be used in combination.

The dosage form of the pharmaceutical composition of the present embodiment is not particularly limited, and can be a dosage form generally used as a pharmaceutical preparation. The pharmaceutical composition of the present embodiment may be an oral preparation or a parenteral preparation. Examples of the oral preparation include tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, liquids, drop loves, emulsions and the like. Examples of parenteral preparations include suppositories, nasal drops, enteral preparations, inhalants and the like. The pharmaceutical compositions of these dosage forms can be formulated according to a conventional method (for example, the method described in the Japanese Pharmacopoeia). The pharmaceutical composition of the present embodiment is preferably an oral preparation.

The administration route of the pharmaceutical composition of the present embodiment is not particularly limited and can be administered by an oral or parenteral route, but oral administration is preferable.

The pharmaceutical composition of the present embodiment can administer a therapeutically effective amount of the crosslinked polymer. "Therapeutically effective amount" means the amount of a drug effective for the treatment or prevention of a target disease. For example, the therapeutically effective amount of the crosslinked polymer may be an amount capable of alleviating, suppressing, or inhibiting symptoms such as diarrhea, bloody stool, abdominal pain, fever, lytic anemia, thrombocytopenia, acute renal failure, and encephalopathy caused by Shiga toxin. .. The therapeutically effective amount may be appropriately determined depending on the patient's symptoms, body weight, age, gender, etc., the dosage form of the pharmaceutical composition, the administration method, and the like. For example, the pharmaceutical composition of the present embodiment can have a single dose of the crosslinked polymer of 0.01 to 1000 mg per 1 kg of body weight of the subject to be administered. The dose may be 0.05 to 500 mg / kg, 0.1 to 300 mg / kg, 0.2 to 200 mg / kg, or 0.3 to 100 mg / kg. It may be kg.

The pharmaceutical composition of this embodiment may contain a therapeutically effective amount of the crosslinked polymer per unit dosage form. For example, the content of the crosslinked polymer in the pharmaceutical composition of the present embodiment may be 0.01 to 90% by mass, 0.05 to 80% by mass, or 0.1 to 60% by mass. May be%.

The administration interval of the pharmaceutical composition of the present embodiment may be appropriately determined depending on the patient's symptoms, body weight, age, gender, etc., the dosage form of the pharmaceutical composition, the administration method, and the like. The administration interval can be, for example, every few hours, 2 to 3 times a day, once a day, once every 2 to 3 days, once a week, or the like.

Since the pharmaceutical composition of the present embodiment contains the crosslinked polymer of the above-described embodiment having high inhibitory activity against Shiga toxin, it is possible to effectively treat or prevent Shiga toxin-producing bacterial infection. Further, in the pharmaceutical composition of the present embodiment, the content of Shiga toxin-binding oligosaccharide such as Globotrisaccharide can be reduced by using the crosslinkable polymer, so that the production cost can be suppressed. ..

[Other aspects]
In one embodiment, the present invention provides a method for treating or preventing a Shiga toxin-producing bacterial infection, which comprises administering the crosslinked polymer of the embodiment to a subject.
In one embodiment, the present invention provides the use of the crosslinked polymer of the embodiment in the production of a Shiga toxin inhibitor.
In one embodiment, the present invention provides the use of the crosslinked polymer of the embodiment in the manufacture of a pharmaceutical composition for treating or preventing a Shiga toxin-producing bacterial infection.
In one embodiment, the present invention provides the crosslinked polymer of said embodiment for inhibiting Shiga toxin.
In one embodiment, the present invention provides the crosslinked polymer of the embodiment for treating or preventing a Shiga toxin-producing bacterial infection.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to the following Examples.

<Synthesis of Monomer (M1-1) Containing Globotrisaccharide>
The synthesis of the monomer (M1-1) containing the globotrisaccharide was carried out according to the reaction formula shown below.
Gb _3- β-MP (product code: M1767, Tokyo Chemical Industry Co., Ltd.) 617.9 mg, _{16 mL of acetic anhydride (Ac 2} O) and 32 mL of pyridine were mixed and reacted overnight at room temperature. The reaction mixture was concentrated under reduced pressure and then extracted with ethyl acetate. The cells were washed successively with 1N hydrochloric acid, saturated aqueous sodium hydrogen carbonate solution, water and saturated brine. The organic layer was dried over magnesium sulfate. The filtrate was concentrated under reduced pressure and then vacuum dried to give compound (I).
Then, 1664.4 mg of ammonium hexanitratocerium (IV) and 20 mL of a mixed solvent of acetonitrile (MeCN) / water (MeCN: H ₂ O = 4: 1 (volume ratio)) were added, and the mixture was added at 0 ° C. It was allowed to react for 3 hours. The reaction mixture was extracted with ethyl acetate and washed successively with water, saturated aqueous sodium hydrogen carbonate solution and saturated brine. The organic layer was dried over magnesium sulfate. After concentrating the filtrate under reduced pressure, the residue is subjected to flash chromatography (SiO ₂ , toluene / acetone, gradient of 9/1 to 7/3 (volume ratio); flash automatic purification device: Isolera One, column: SNAP Ultra 25 g. Purified by Biotage). After concentration under reduced pressure, the reaction product was recovered by vacuum drying to obtain 846.8 mg of compound (II) (yield 90.5%).
Next, 15 mg of sodium methoxide (NaOMe) and 10 mL of methanol (super-dehydrated) were added to 808.7 mg of compound (II), and the mixture was reacted at room temperature for 2 hours. After neutralization with Amberlist, the filtrate was concentrated under reduced pressure, and the reaction product was recovered by lyophilization to obtain 406.9 mg of compound (III) (yield 92.3%).
Next, the compound of 389.3mg (III), sodium azide _{(NaN 3) 503.8mg, N,} N- diisopropylethylamine (DIPEA) 1.21 mL, and it was added deuterated water _(D 2 O) _3.1mL , Cooled to 0 ° C. This solution was added to 391.5 mg of 2-chloro-1,3-dimethylimidazolinium chloride (DMC) and reacted at 0 ° C. for 4 hours. The reaction mixture was concentrated under reduced pressure, N, N-dimethylformamide (DMF) was added, and the solid was removed by filtration. The filtrate was concentrated under reduced pressure, extracted with water, and washed with dichloromethane. After passing through a cation exchange resin column activated with 1N aqueous sodium hydroxide solution, flash chromatography (reverse phase (C18), water / methanol, 10/0 to 0/10 (volume ratio) gradient; flash automatic Purification apparatus: Isolera One, column: SNAP Ultra C18 30 g; manufactured by Biotage). After concentration under reduced pressure, the reaction product was recovered by freeze-drying to obtain 331.5 mg of compound (IV) (yield 81.1%).
Next, in 293.9 mg of compound (IV), 75.2 mg of 3-butynylacrylamide, _{8.9 mg of copper (II) sulfate (CuSO 4} ), and tris [(1-benzyl-1H-1,2,3-) triazol-4-yl) methyl] amine (TBTA) 29.5 mg, L-ascorbic sodium acid (L-Asc-Na) 22.0mg and water / methanol mixed _{solvent, (H 2 O: MeOH =} 1: 1 ( Volume ratio)) 6 mL was added, and the mixture was reacted overnight at 30 ° C. under a nitrogen atmosphere. After concentrating the reaction solution under reduced pressure, the residue was subjected to flash chromatography (reverse phase (C18), water / methanol, gradient from 10/0 to 0/10 (volume ratio); flash automatic purification device: Isolera One, column: SNAP Ultra C18. Purified with 30 g; manufactured by Biotage). Then the metal scavenger ^(SiliaMetS TM Imidazole, 300mg) was stirred at room temperature overnight. The metal scavenger was removed by filtration, concentrated under reduced pressure, and then the reaction product was recovered by freeze-drying to obtain 315.7 mg of monomer (M1-1) (yield 87.2%).
The obtained monomer (M1-1) was subjected to NMR measurement (JNM-ECZ400; manufactured by JEOL Ltd.), and its structure was confirmed from the following analysis results. FIG. 1 shows an NMR chart of the monomer (M1-1).
1H-NMR (400 MHz, D ₂ O) δ 8.09 (s, 1H, triazole), 6.14-6.28 (m, 2H, vinyl), 5.75-5.79 (m, 2H, vinyl and H-1), 5.00 (d , J = 3.7 Hz, 1H, H-1'), 4.60 (d, J = 7.8 Hz, 1H, H-1 ”), 4.40 (t, J = 6.4 Hz, 1H, sugar-H), 3.60-4.10 (m, 19H, sugar-H and -CH ₂ -NH-), 3.03 (t, J = 6.6 Hz, 2H, -CH ₂ -CH ₂ -NH-)

<Synthesis of crosslinked polymer>
(Examples 1 to 16)
The polymerizable monomer mixture of each example was prepared at the molar ratio shown in Table 1 and subjected to a radical polymerization reaction at 70 ° C. for 3 hours in a nitrogen atmosphere to produce a crosslinked polymer of each example. In the preparation of the polymerizable monomer mixture, the monomer (M2-1) was dissolved in methanol and added. When the polymerizable monomer mixture contained an anionic monomer, water containing 6.21 mM sodium dodecyl sulfate (SDS) was used as the reaction solvent. When the polymerizable monomer mixture contained a cationic monomer, water containing 2.07 mM hexadecyltrimethylammonium bromide (CTAB) was used as a reaction solvent. As the polymerization initiator, 0.69 mM V-501 (4,4'-Azobis (4-cyanovalic acid)) or AAPD (2,2'-Azobis (2-methylpropionamide) Dihydrochloride) was used. V-501 was dissolved in dimethyl sulfoxide (DMSO) and added to the radical polymerization reaction solution. AAPD was dissolved in water and added to the radical polymerization reaction solution. After the radical polymerization reaction, the reaction solution was dialyzed against a dialysis membrane (Fisherland) of MWCO 12,000-14,000, and the crosslinked polymer was recovered by freeze-drying.

In Table 1, each abbreviation indicates the polymerizable monomer shown in Table 2 and Table 3, respectively. The value in [] is the molar ratio in the polysynthetic monomer mixture.

<Calculation of molecular weight of crosslinked polymer>
(Theoretical molecular weight)
_{The molar ratio of each polymerizable monomer (RA} : the value in [] in Table 1 divided by 100) was calculated when the total amount of all the polymerizable monomers in the polymerizable monomer mixture was 1. The molar ratio R _A, was calculated molecular weight multiplied by the value of the polymerizable monomer (M _A). Summing the M _A calculated for each monomer of the polymerizable monomer mixture, the total value "molecular weight of theory", shown in Table 4.

(Molecular weight per sugar unit)
The value calculated by dividing the "theoretical molecular weight" calculated above by the molar ratio ( _{RA) of the globotrisaccharide monomer (M1-1) was calculated.} This is shown in Table 4 as "molecular weight per sugar unit".

<Analysis of crosslinked polymers by dynamic light scattering (DLS)>
The crosslinked polymer was dissolved in PBS to a concentration of 0.1 mg / mL, and the Z average and PDI of the crosslinked polymer were measured using a DLS device (Zetasizer Nano ZS, Malvern). The Z mean and PDI measurements were taken at 35 ° C and 40 ° C. The results are shown in Table 4.

<Evaluation of Shiga toxin inhibitory activity of crosslinked polymer>
Vero cells (RCB0001, RIKEN BRC) were used to evaluate the inhibitory activity of the crosslinked polymer against Shiga toxin (Stx1, Stx2) prepared from the EHEC O157 Sakai strain. Vero cells were cultured at 37 ° C. in DMEM high-glucose medium (Sigma) supplemented with 2 mM glutamine and 5% fetal bovine serum. Stx1 (final concentration 3.6 pg mL ^-1 ) or Stx2 (final concentration 13.3 pg mL ^-1 ) was added to 100 μL of 1 × 10 ^{5 cells / mL Vero cell culture medium.} Then, a PBS suspension of the crosslinked polymer was added to the culture solution so that the final concentration of the crosslinked polymer was 0 to 50 μg / mL. For negative controls, PBS (without crosslinked polymer) was added to the culture. Then, it was cultured at 37 ° C. for 48 hours. After culturing, the cell viability of Vero cells was measured by CellTiter-Glo 2.0 Assay (Promega). Furthermore, the IC50 of the crosslinked polymer with respect to Stx1 and Stx2 was calculated from the cell viability.

FIG. 2A shows the results of evaluating the cell viability by adding the crosslinked polymer of Example 1 to the Vero cell culture medium to which Stx1 was added. FIG. 2B shows the results of evaluating the cell viability by adding the crosslinked polymer of Example 1 to the Vero cell culture medium to which Stx2 was added. From this result, in the crosslinked polymer of Example 1, the IC50 for Stx1 was calculated to be 4.7E-04 ± 9.9E-05 μg / mL, and the IC50 for Stx2 was calculated to be 31.0 ± 11.0 μg / mL.

FIG. 3A shows IC50s of the crosslinked polymers of Examples 2-10 for Stx1. FIG. 3B shows IC50s of the crosslinked polymers of Examples 2-10 for Stx2. All of the crosslinked polymers of Examples 2 to 10 showed high Shiga toxin inhibitory activity. Among these crosslinked polymers, the crosslinked polymer of Example 7 has a low IC50 with respect to both Stx1 and Stx2, and the molar concentrations of the IC50 are Stx1 (1.52 nM) and Stx2 (181 nM). there were. The molar concentration of IC50 was calculated by dividing the concentration of IC50 (w / v) by the above-mentioned "theoretical molecular weight".

FIG. 4A shows IC50s of the crosslinked polymers of Examples 7, 11-16 for Stx1. FIG. 4B shows IC50s of the crosslinked polymers of Examples 7, 11-16 for Stx2. The crosslinked polymers of Examples 11-16 had an even lower IC50 than the crosslinked polymers of Example 7 for both Stx1 and Stx2.

<Animal test>
Animal tests were performed using the crosslinked polymer of Example 7. FIG. 5 shows the test schedule of animal tests. As experimental animals, 4-week-old BALB / c sterile mice (4 males and 1 female) were used.
One day before the start of administration of the crosslinked polymer, mice were infected with Enterohemorrhagic Escherichia coli O157 strain producing Stx2 (Fig. 5, day (-1)). Infection of the O157 strain was carried out by oral administration of 1E + 7 CFU / mL O157 strain bacterial solution by free water supply. The crosslinked polymer of Example 7 was dissolved in water so as to be 0.05 mg / mL, and administered to O157-infected mice (n = 3) by free water supply. As a negative control, water (without crosslinked polymer) was administered to O157-infected mice (n = 2) by free water supply. Feces were collected at the timing of the white arrow in FIG. 5, and the number of O157 strains in the feces was examined by stool culture.

The survival curve of O157-infected mice is shown in FIG. 6A. The result of stool culture is shown in FIG. 6B. As shown in FIG. 6A, all O157-infected mice to which water was administered died 5 days after the start of administration. On the other hand, all the mice to which the crosslinked polymer of Example 7 was administered were alive during the test period. As shown in FIG. 6B, no change was observed in the number of O157 strains in the mice to which the crosslinked polymer of Example 7 was administered during the test period. From these results, it was confirmed that the crosslinked polymer of Example 7 neutralized the toxicity of Shiga toxin without affecting the O157 strain.

Furthermore, the concentration of the crosslinked polymer of Example 7 to be administered was changed, and an animal test was conducted in the same manner as above. The concentration of the crosslinked polymer of Example 7 in free water supply was adjusted to 0.05 mg / mL, 0.025 mg / mL, 0.01 mg / mL, or 0.005 mg / mL and administered to O157-infected mice. The results are shown in Table 5. Table 5 shows the survival rate 10 days after the start of administration.

As shown in Table 5, the survival rate was 100% even when the concentration of the crosslinked polymer of Example 7 was 0.025 mg / mL. The intake of the crosslinked polymer calculated from the amount of water supplied at this time was 0.275 mg / day per mouse. Moreover, even when the concentration of the crosslinked polymer was 0.005 mg / mL, the survival rate was 30%. From these results, it was confirmed that even a low-dose cross-linked polymer showed a Shiga toxin inhibitory effect.

According to the present invention, a crosslinked polymer having high inhibitory activity against Shiga toxin, and a Shiga toxin inhibitor and a pharmaceutical composition containing the crosslinked polymer are provided.

Claims (8)

1. A crosslinked polymer in which a random copolymer is crosslinked,
   The random copolymer has a structural unit (a1) containing a Shiga toxin-binding oligosaccharide and at least one other structural unit.
   Crosslinked polymer.
2. The at least one other structural unit includes a hydrophobic group having 4 to 20 carbon atoms (a2), a cationic group (a3), and an anionic group (a4). , 1- (3-sulfopropyl) -2-vinylpyridinium hydroxide, selected from the group consisting of a structural unit (a5) derived from an intramolecular salt and a structural unit (a6) derived from a cross-linking agent.
   The crosslinked polymer according to claim 1.
3. The crosslinked polymer according to claim 2, wherein the random copolymer has the structural unit (a2).
4. The crosslinked polymer according to claim 2 or 3, wherein the random copolymer has the structural unit (a3) or the structural unit (a4).
5. The crosslinked polymer according to any one of claims 2 to 4, wherein the random copolymer has the structural unit (a5).
6. The crosslinked polymer according to any one of claims 1 to 5, wherein the Shiga toxin-binding oligosaccharide is a Globotrisaccharide.
7. A Shiga toxin inhibitor containing the crosslinked polymer according to any one of claims 1 to 6.
8. A pharmaceutical composition for treating or preventing a Shiga toxin-producing bacterial infection, which comprises the crosslinked polymer according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

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