JP7680046B2 - Crosslinked polymers, shiga toxin inhibitors, and pharmaceutical compositions - Google Patents

Description

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

In recent years, outbreaks of Enterohemorrhagic Escherichia coli (EHEC) infections via food and other sources have become a problem. Cattle are the main carriers and source of infection for EHEC. As EHEC is highly infectious and has a relatively long incubation period, secondary person-to-person infections are also frequent.

When infected with EHEC, 20-50% of infected individuals develop hemorrhagic colitis within 3-4 days of infection. Symptoms of hemorrhagic colitis include diarrhea, fresh blood in the stool, severe abdominal pain, and mild fever. Within 4-7 days of infection, 2-20% of infected individuals develop hemolytic-uremic syndrome (HUS). Symptoms of HUS include hemolytic anemia, thrombocytopenia, and acute renal failure, and can lead to severe complications such as acute encephalopathy, and if HUS becomes severe, it can be fatal.

Currently, there is no established effective treatment for EHEC infection. In Japan, approximately 3,000 people are infected each year, with serious cases and deaths occurring every year. Therefore, there is a need to establish an effective treatment.

There are two types of Shiga toxins produced by EHEC: Stx1 and Stx2. Both Stx and Stx2 are composed of one A subunit molecule and five B subunit molecules. The A subunit is a catalytic subunit with RNA-N-glucosidase activity. The A subunit inactivates 60S ribosomes through said activity, resulting in inhibition of cellular protein synthesis. The B subunit has binding activity to Gb3 (globotriaosylceramide), a glycolipid on the cell surface, and is involved in invading target cells.
The amino acid sequence of Stx1 and Stx2 share approximately 50% homology. The amino acid sequence of Stx1 is known to be nearly identical to the amino acid sequence of Stx produced by Shigella dysenteriae.

Because Shiga toxin has a binding affinity to the globotrisaccharide of Gb3, there have been attempts to develop Shiga toxin inhibitors using the globotrisaccharide. For example, Patent Document 1 reports the use of a polymer containing the globotrisaccharide as a therapeutic agent for EHEC infection.

JP 2005-289907 A

As there is no established effective treatment for EHEC infection, there is a need to develop an effective drug to treat it. However, the EHEC treatment candidates reported so far have not been found to have a sufficiently high inhibitory effect on Shiga toxin.

Therefore, the present invention aims to provide a cross-linked polymer having high inhibitory activity against Shiga toxin, as well as a Shiga toxin inhibitor and a pharmaceutical composition containing the cross-linked polymer.

The present invention includes the following aspects.
[1] A crosslinked polymer in which a random copolymer is crosslinked, the random copolymer having a structural unit (a1) containing a Shiga toxin-binding oligosaccharide and at least one other structural unit.
[2] The crosslinked polymer according to [1], wherein the at least one other structural unit is selected from the group consisting of structural units (a2) containing a hydrophobic group having 4 to 20 carbon atoms, structural units (a3) containing a cationic group, structural units (a4) containing an anionic group, and structural units (a5) derived from a 1-(3-sulfopropyl)-2-vinylpyridinium hydroxide inner salt, and structural units (a6) derived from a crosslinking agent.
[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 described in any one of [1] to [5], wherein the Shiga toxin-binding oligosaccharide is a globotrisaccharide.
[7] A Shiga toxin inhibitor comprising 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, comprising the crosslinked polymer according to any one of [1] to [6] and a pharma- ceutical acceptable carrier.

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

1 shows an NMR chart of a monomer (M1-1) synthesized in an example. 1 shows the results of evaluating the inhibitory effect of the crosslinked polymer of Example 1 against the cytotoxicity of Stx1. 1 shows the results of evaluating the inhibitory effect of the crosslinked polymer of Example 1 against the cytotoxicity of Stx2. The results of evaluating the IC50 of the crosslinked polymers of Examples 2 to 10 against the cytotoxicity of Stx1 are shown. The results of evaluating the IC50 of the crosslinked polymers of Examples 2 to 10 against the cytotoxicity of Stx2 are shown. The results of evaluating the IC50 of the crosslinked polymers of Examples 7 to 18 against the cytotoxicity of Stx1 are shown. The results of evaluating the IC50 of the crosslinked polymers of Examples 7 to 18 against the cytotoxicity of Stx2 are shown. 1 shows the test schedule for the crosslinked polymer administration test of Example 7 using O157-infected mice. 1 shows the survival curve of O157-infected mice administered the crosslinked polymer of Example 7. 1 shows the results of fecal culture of feces collected from O157-infected mice administered the crosslinked polymer of Example 7.

The term "structural unit" refers to a monomer unit that constitutes a polymer.
The phrase "may have a substituent" includes both the case where a hydrogen atom (--H) is replaced with a monovalent group and the case where a methylene group (--CH ₂ -) is replaced with a divalent group.
The term "aromatic hydrocarbon group" refers to 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 monocyclic or polycyclic. The aromatic ring includes aromatic hydrocarbon rings such as benzene, naphthalene, anthracene, and phenanthrene; and aromatic heterocycles in which a part of the carbon atoms constituting the aromatic hydrocarbon ring is replaced with a heteroatom (oxygen atom, sulfur atom, nitrogen atom, etc.).
The term "aliphatic" is a relative concept to aromatic, and refers to groups, compounds, etc. that do not have aromaticity.
Unless otherwise specified, the term "alkyl group" includes linear, branched and cyclic monovalent saturated hydrocarbon groups. The same applies to the alkyl group in an alkoxy group.
Unless otherwise specified, the term "alkylene group" includes linear, branched and cyclic divalent saturated hydrocarbon groups.

The term "structural unit derived from an acrylamide derivative" refers to a structural unit formed by cleavage of the ethylenic double bond of an acrylamide derivative. The term "acrylamide derivative" refers to a compound in which one or both of the 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. The carbon atom at the α-position of acrylamide refers to the carbon atom to which the carbonyl group of acrylamide is bonded, unless otherwise specified. Examples of the substituent substituting the hydrogen atom bonded to the carbon atom at the α-position of acrylamide include alkyl groups having 1 to 5 carbon atoms (methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, and neopentyl).

The term "structural unit derived from an acrylic acid ester" refers to a structural unit formed by cleavage of the ethylenic double bond of an acrylic acid ester. An "acrylic acid ester" is a compound in which the hydrogen atom at the carboxyl end of acrylic acid (CH ₂ ═CH-COOH) is substituted with an organic group. In an 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 an acrylic acid ester refers to the carbon atom to which the carbonyl group of acrylic acid is bonded. Examples of the substituent substituting the hydrogen atom bonded to the carbon atom at the α-position of an acrylic acid ester include the same ones as those listed for the structural unit derived from acrylamide.

"Shiga toxin" refers to toxins produced by enterohemorrhagic Escherichia coli (EHCE) and Shigella dysenteriae, and is a concept that encompasses the Stx1 family, Stx2 family, and Stx.

"Polymerizable monomer mixture" means a mixture of polymerizable monomers that undergoes a polymerization reaction to obtain a crosslinked polymer. "Polymerizable monomer" means a monomer that contains at least one polymerizable group. "Polymerizable group" means a functional group that undergoes a polymerization reaction.

In this specification and claims, some structures represented by chemical formulas may have asymmetric carbons, and enantiomers or diastereoisomers may exist. In such cases, a single chemical formula represents all of the 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 having a constitutional unit (a1) containing a Shiga toxin-binding oligosaccharide and at least one other constitutional unit.

<Random copolymer>
The crosslinked polymer of this embodiment is a 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. By using a copolymer having the structural unit (a1) and other structural units, it is possible to combine functional groups capable of interacting with Shiga toxin in various modes. In addition, by using a random copolymer, it is possible to form a diverse structure. Therefore, the probability of forming a binding site having high binding affinity with Shiga toxin is increased. Furthermore, unlike a block copolymer, a random copolymer does not require a polymerization reaction for each block, so that the production time and production costs can be reduced.

The crosslinked polymer may contain one type of random copolymer or two or more types. Crosslinking of the random copolymer can be carried out by using a polymerizable monomer mixture to which a crosslinking agent containing two or more polymerizable groups has been added during the copolymerization reaction to obtain the random copolymer. Alternatively, after obtaining the random copolymer, a crosslinking reaction of the random copolymer can be carried out using a crosslinking agent having two or more groups that react with the functional groups in the random copolymer. Since the synthesis and crosslinking of the random copolymer can be carried out in one step, a method of copolymerization using a polymerizable monomer mixture containing a crosslinking agent is preferred.

(Structural unit (a1)
The random copolymer contained in the crosslinked polymer of this embodiment has a structural unit (a1) containing a Shiga toxin-binding oligosaccharide. The crosslinked polymer of this embodiment has a high binding affinity to Shiga toxin due to the presence of 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 ability. The Shiga toxin-binding oligosaccharide is preferably globotrisaccharide or a derivative thereof. Globotrisaccharide is a trisaccharide having the structure Galα(1-4)-Galβ(1-4)-Glc-. In the following formula, * represents a bond.

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

［式中、Ｒは、水素原子、又は炭素原子数１～５のアルキル基を表す。Ｙ^１は、２価の連結基を表す。Ｇｂは、志賀毒素結合性オリゴ糖を表す。］

[In the formula, R represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. ^Y1 represents a divalent linking group. Gb represents a Shiga toxin-binding oligosaccharide.]

In 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 general formula (a1-1), ^Y1 represents a divalent linking group. Examples of the divalent linking group include divalent hydrocarbon groups which may have a substituent. The divalent hydrocarbon group may be an aliphatic hydrocarbon group or an aromatic hydrocarbon group.

The aliphatic hydrocarbon group in Y ¹ may be saturated or unsaturated. Examples of the aliphatic hydrocarbon group include linear or branched aliphatic hydrocarbon groups and aliphatic hydrocarbon groups containing 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 chain aliphatic hydrocarbon group preferably has 2 to 15 carbon atoms, more preferably has 2 to 10 carbon atoms, and even more preferably has 3 to 6 carbon atoms. As the branched chain aliphatic hydrocarbon group, a branched chain alkylene group is preferable.

Examples of the aliphatic hydrocarbon group containing a ring in its structure include a cyclic aliphatic hydrocarbon group (a group in which two hydrogen atoms have been removed from an aliphatic hydrocarbon ring) which may contain a substituent containing a heteroatom in the ring structure, a group in which the cyclic aliphatic hydrocarbon group is bonded to the end of a linear or branched aliphatic hydrocarbon group, and a group in which the cyclic aliphatic hydrocarbon group is interposed in the middle of a linear or branched aliphatic hydrocarbon group. Examples of the linear or branched aliphatic hydrocarbon group include the same as those described above. The cyclic aliphatic hydrocarbon group preferably has 3 to 20 carbon atoms, more preferably 3 to 12 carbon atoms. The cyclic aliphatic hydrocarbon group may be a polycyclic group or a monocyclic group. A part of the carbon atoms constituting the ring structure of the cyclic aliphatic hydrocarbon group may be substituted with a substituent containing a heteroatom (oxygen atom, nitrogen atom, sulfur atom, etc.).

When ^Y1 is an aromatic hydrocarbon group, the aromatic ring contained in the aromatic hydrocarbon group more preferably has 6 to 15 carbon atoms, 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 a triazole ring, a pyridine ring, and a thiophene ring.
Specific examples of the aromatic hydrocarbon group include a group in which two hydrogen atoms have been removed from the aromatic hydrocarbon ring or aromatic heterocycle (arylene group or heteroarylene group); a group in which two hydrogen atoms have been removed from an aromatic compound containing two or more aromatic rings (e.g., biphenyl, fluorene, etc.); a group in which one hydrogen atom of a group in which one hydrogen atom has been removed from the aromatic hydrocarbon ring or aromatic heterocycle (aryl group or heteroaryl group) has been substituted with an alkylene group (a group in which one further hydrogen atom has been removed from an aryl group or heteroaryl group), etc. The alkylene group substituting the hydrogen atom preferably has 1 to 10 carbon atoms, more preferably has 1 to 6 carbon atoms, and even more preferably has 1 to 4 carbon atoms.

The hydrocarbon group which may have a substituent in ^Y1 may be one in which a part of the methylene groups (-CH ₂ -) constituting the carbon chain is substituted with a divalent linking group containing a heteroatom. Examples of the divalent linking group containing a heteroatom substituting the methylene group include -O-, -C(=O)-O-, -O-C(=O)-, -C(=O)-, -O-C(=O)-O-, -C(=O)-NH-, -NH-, -NH-C(=NH)-, -C(=O)-NH-C(=O)- (H may be substituted with a substituent such as an alkyl group or an acyl group), -S-, -S(=O) ₂ -, and -S(=O) ₂ -O-.

The structural unit (a1) is preferably a structural unit derived from an acrylamide derivative or an acrylic acid ester, and therefore preferred examples of ^Y1 include groups represented by -CO-NH- ^Y11- or -CO-O- ^Y11- ( ^Y11 is a divalent hydrocarbon group which may have a substituent), with -CO-NH- ^Y11- being more preferred.

Examples of the divalent hydrocarbon group for ^Y11 which may have a substituent include the same as those exemplified for ^Y1 . Among them, ^Y11 is preferably an alkylene group which may have a substituent, or an aryl group or heteroaryl group in which one of the hydrogen atoms is substituted with an alkylene group.

The alkylene group which may have a 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 ^Y11 is an alkylene group which may have a substituent, a part of the methylene groups 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 a heteroatom include the same as those exemplified for ^Y1 .

When Y ¹¹ is 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 substituting the hydrogen atom may be linear or branched, but 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. An example of the aromatic ring possessed by the aryl group is a benzene ring. An example of the heterocyclic ring possessed by the heteroaryl group is a triazole ring, an imidazole ring, a pyridine ring, and a thiophene ring.
Among these, Y ¹¹ is preferably a group in which one hydrogen atom of a triazole group has been substituted with an alkylene group.

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

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

［式中、Ｒは、水素原子、又は炭素原子数１～５のアルキル基を表す。Ｌ^１は、－ＣＯ－Ｏ－又は－ＣＯ－ＮＨ－を表す。Ｒ^１は、炭素原子数１～１０の直鎖状若しくは分岐鎖状のアルキレン基を表す。Ｇｂは、志賀毒素結合性オリゴ糖を表す。］

[In the formula, R represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. ^L1 represents -CO-O- or -CO-NH-. ^R1 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 formula (a1-1-1), L ¹ represents -CO-O- or -CO-NH-. ^{L 1} is preferably -CO-NH-.
In 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 has 1 to 4 carbon atoms, and even more preferably has 1 to 3 carbon atoms, and is particularly preferably a methylene group or an ethylene group.

Specific examples of the structural unit (a1) include, but are not limited to, the following: In the formula, R ^α represents a hydrogen atom or a methyl group.

The structural unit (a1) may be of one type, or of two or more types.
The proportion of the structural unit (a1) in the entire random copolymer contained in the crosslinked polymer of this embodiment is preferably 3 mol% or more, more preferably 5 mol% or more, more preferably 8 mol% or more, and particularly preferably 10 mol% or more, based on the total (100 mol%) of all structural units constituting the entire random copolymer. When the proportion of the structural unit (a1) is equal to or more than the above-mentioned preferable lower limit, the Shiga toxin inhibitory effect is further improved. The upper limit of the proportion of the structural unit (a1) in the entire random copolymer is not particularly limited. When the structural unit (a1) contains globotrisaccharide as a Shiga toxin-binding oligosaccharide, from the viewpoint of production costs, the proportion of the structural unit (a1) is, for example, preferably 40 mol% or less, more preferably 30 mol% or less, even more preferably 20 mol% or less, and particularly preferably 15 mol% or less. The proportion of the structural unit (a1) is preferably 3 to 40 mol%, more preferably 5 to 30 mol%, even more 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 structural unit (a1). Examples of the other structural units include a structural unit (a2) containing a hydrophobic group having 4 to 20 carbon atoms, a structural unit (a3) containing a cationic group, a structural unit (a4) containing an anionic group, a structural unit (a5) derived from 1-(3-sulfopropyl)-2-vinylpyridinium hydroxide inner salt (hereinafter also referred to as "PPS"), and a structural unit (a6) derived from a crosslinking agent.

<Structural unit (a2)>
The structural unit (a2) is a structural unit containing a hydrophobic group having 4 to 20 carbon atoms. The random copolymer preferably contains the structural unit (a2). When the random copolymer contains the structural unit (a2), the Shiga toxin inhibitory effect is further improved.

The hydrophobic group having 4 to 20 carbon atoms contained in the structural unit (a2) may be 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 linear or branched aliphatic hydrocarbon groups and aliphatic hydrocarbon groups containing a ring in the structure. The aliphatic hydrocarbon group preferably has 4 to 15 carbon atoms, more preferably has 4 to 10 carbon atoms, and further preferably has 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, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of the branched aliphatic hydrocarbon group include branched alkyl groups such as isobutyl, tert-butyl, isopentyl, neopentyl, 1,1-dimethylpropyl, 1,1-diethylpropyl, and 2,2-dimethylbutyl groups.
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 a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a methylcyclohexyl group, a dimethylcyclohexyl group, a cycloheptyl group, a cyclooctyl group, a cyclononyl group, and a 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, a methylnorbornyl group, and 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 monocyclic aromatic hydrocarbon groups include a phenyl group; and groups in which some of the hydrogen atoms of a phenyl group are substituted with an alkyl group or a cycloalkyl group, such as a p-methylphenyl group, a p-tert-butylphenyl group, a tolyl group, a xylyl group, a cumenyl group, a mesityl group, a 2,6-diethylphenyl group, a 2-methyl-6-ethylphenyl group, and a p-adamantylphenyl group. Examples of polycyclic aromatic hydrocarbon groups include a biphenyl group, a phenanthryl group, a naphthyl group, and an anthryl group.

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

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

［式中、Ｒは、水素原子、又は炭素原子数１～５のアルキル基を表す。Ｙ^２は、単結合又は２価の連結基を表す。Ｒ^２は、炭素原子数４～２０の疎水性基を表す。］

[In the formula, R represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. ^Y2 represents a single bond or a divalent linking group. ^R2 represents a hydrophobic group having 4 to 20 carbon atoms.]

In general formula (a2-1), R is the same as R in 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 the same as those exemplified 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 include groups represented by -CO-NH-Y ²¹ - or -CO-O-Y ²¹ - (Y ²¹ is a single bond or a divalent hydrocarbon group which may have a substituent), and a group represented by -CO-NH-Y ²¹ - is preferred. When Y ²¹ is a divalent hydrocarbon group which may have a substituent, examples of the divalent hydrocarbon group which may have a substituent include the same as those exemplified for Y ¹ in the general formula (a1-1). Y ²¹ is preferably a single bond or an aliphatic hydrocarbon group having 1 to 10 carbon atoms, 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 has 1 to 3 carbon atoms, and even more preferably has 1 or 2 carbon atoms.
In the general formula (a2-1), ^R2 represents a hydrophobic group having 4 to 20 carbon atoms. Examples of the hydrophobic group include the same groups as those listed above.

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

［式中、Ｒは、水素原子、又は炭素原子数１～５のアルキル基を表す。Ｌ^２は、単結合、－ＣＯ－Ｏ－又は－ＣＯ－ＮＨ－を表す。Ｙ^２２は、単結合又は炭素原子数１～１０の直鎖状若しくは分岐鎖状のアルキレン基を表す。Ｒ^２は、炭素原子数４～２０の疎水性基を表す。］

[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 general formula (a2-1-1), R is the same as R in general formula (a2-1).
In formula (a2-1-1), ^L2 represents a single bond, -CO-O-, or -CO-NH-. ^L2 is preferably -CO-NH-.
In 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 has 1 to 4 carbon atoms, even more preferably has 1 to 3 carbon atoms, and is 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, even more preferably a single bond or a methylene group, and is particularly preferably a single bond.
In 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 general formula (a2-1).

Specific examples of the structural unit (a2) include, but are not limited to, the following: In the formula, R ^α represents a hydrogen atom or a methyl group.

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

<Structural unit (a3)>
The structural unit (a3) is a structural unit containing a cationic group. The random copolymer preferably contains the structural unit (a3). When the random copolymer contains the structural unit (a3), the Shiga toxin inhibitory effect is further improved.

The cationic group refers to an atomic group bearing a positive charge. The cationic group may be in the form of a salt formed with a fluoride ion, a chloride ion, a bromide ion, an iodide ion, a hydrochloride ion, an acetate ion, a sulfate ion, a hydrofluoride ion, a carbonate ion, or the like. The cationic group contained in the structural unit (a3) is not particularly limited, and examples thereof include, but are not limited to, a primary amino group (-NH ₂ ), a secondary amino group (-NHR ³ ), a tertiary amino group (-NR ³ ₂ ), a quaternary 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. In the case of a tertiary amino group or a quaternary amino group, the two or more R ³ may be the same or different. The organic group may be a hydrocarbon group which may have a substituent. Examples of the hydrocarbon group which may have a substituent include those exemplified as R ¹ in the general formula (a1-1-1). R ³ is preferably an alkyl group, an aryl group, or an aralkyl group. The alkyl group preferably has 1 to 6 carbon atoms, more preferably has 1 to 3 carbon atoms, and even more preferably has 1 or 2 carbon atoms. The aryl group or aralkyl group preferably has 6 to 12 carbon atoms, and more preferably has 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, and a benzyl group.

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, 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, and a 1-methylpyrrolidinium group. Examples of the aromatic heterocyclic group include, but are not limited to, an imidazolium group, a 1-methylimidazolium group, a 1-ethylimidazolium group, a benzimidazolium group, a pyrrolium group, a 1-methylpyrrolium group, an oxazolium group, a benzoxazolium group, a benzisoxazolium group, a pyrazolium group, an isoxazolium group, a pyridinium group, a 2,6-dimethylpyridinium group, a pyrazinium group, a pyrimidinium group, a pyridazinium group, and a triazinium group.

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

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

［式中、Ｒは、水素原子、又は炭素原子数１～５のアルキル基を表す。Ｙ^３は、単結合又は２価の連結基を表す。Ｒｃ^３は、カチオン性基を表す。］

[In the formula, R represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. ^Y3 represents a single bond or a divalent linking group. ^Rc3 represents a cationic group.]

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

In 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 the same as those exemplified as the divalent linking group in Y ¹ in 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 include groups represented by -CO-NH-Y ³¹ - or -CO-O-Y ³¹ - (Y ³¹ is a single bond or a divalent hydrocarbon group which may have a substituent), and a group represented by -CO-NH-Y ³¹ - is more preferred. When Y ³¹ is a divalent hydrocarbon group which may have a substituent, examples of the divalent hydrocarbon group which may have a substituent include the same as those exemplified for Y ¹ in general formula (a1-1). 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 has 1 to 3 carbon atoms.
In formula (a3-1), ^Rc3 represents a cationic group. Examples of the cationic group include the same groups as those exemplified above.

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

［式中、Ｒは、水素原子、又は炭素原子数１～５のアルキル基を表す。Ｌ^３は、単結合、－ＣＯ－Ｏ－、又は－ＣＯ－ＮＨ－を表す。Ｙ^３２は、単結合又は炭素原子数１～１０の直鎖状若しくは分岐鎖状のアルキレン基を表す。Ｒｃ^３は、カチオン性基を表す。］

[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 general formula (a3-1-1), R is the same as R in the general formula (a3-1).
In formula (a3-1-1), ^L3 represents a single bond, -CO-O-, or -CO-NH-. ^L3 is preferably a single bond or -CO-NH-.
In 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 has 1 to 4 carbon atoms, and even more preferably has 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, even more 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, and a methylene group.
In formula (a3-1-1), ^Rc3 represents a cationic group. ^Rc3 is the same as ^Rc3 in formula (a3-1).

Specific examples of the structural unit (a3) include, but are not limited to, the following: In the formula, R ^α represents a hydrogen atom or a methyl group.

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

<Structural unit (a4)>
The structural unit (a4) is a structural unit containing an anionic group. The random copolymer may contain the structural unit (a4).

Anionic group means an atomic group carrying a negative charge. The anionic group may be in the form of a salt formed with an alkali metal ion such as a sodium ion or a potassium ion; or an alkaline earth metal ion such as a calcium ion. The anionic group contained in the structural unit (a4) is not particularly limited, and examples thereof include, but are not limited to, 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, and a boronic acid group. 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 structural unit (a4) include those represented by the following general formula (a4-1):

［式中、Ｒは、水素原子、又は炭素原子数１～５のアルキル基を表す。Ｙ^４は、単結合又は２価の連結基を表す。Ｒａ^４は、アニオン性基を表す。］

[In the formula, R represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. ^Y4 represents a single bond or a divalent linking group. ^Ra4 represents an anionic group.]

In general formula (a4-1), R is the same as R in 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 the same as those exemplified 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 include groups represented by -CO-NH-Y ⁴¹ - or -CO-O-Y ⁴¹ - (Y ⁴¹ is a single bond or a divalent hydrocarbon group which may have a substituent), and a group represented by -CO-NH-Y ⁴¹ - is more preferred. When Y ⁴¹ is a divalent hydrocarbon group which may have a substituent, examples of the divalent hydrocarbon group which may have a substituent include the same as those exemplified for Y ¹ in the general formula (a1-1). 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 has 1 to 3 carbon atoms.
In the general formula (a4-1), ^Ra4 represents an anionic group. Examples of the anionic group include the same groups as those listed above.

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

［式中、Ｒは、水素原子、又は炭素原子数１～５のアルキル基を表す。Ｌ^４は、単結合、－ＣＯ－Ｏ－、又は－ＣＯ－ＮＨ－を表す。Ｙ^４２は、単結合又は炭素原子数１～１０の直鎖状若しくは分岐鎖状のアルキレン基を表す。Ｒａ^４は、アニオン性基を表す。］

[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 general formula (a4-1-1), R is the same as R in general formula (a4-1).
In formula (a4-1-1), ^L4 represents a single bond, -CO-O-, or -CO-NH-. ^L4 is preferably a single bond or -CO-NH-.
In 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 has 1 to 4 carbon atoms, and even more preferably has 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, even more preferably a single bond or a methylene group, and particularly preferably a single bond.
In formula (a4-1-1), ^Ra4 represents an anionic group. ^Ra4 is the same as ^Ra4 in formula (a4-1).

Specific examples of the structural unit (a4) include, but are not limited to, the following: In the formula, R ^α represents a hydrogen atom or a methyl group.

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

<Structural unit (a5)>
The structural unit (a5) is a structural unit derived from 1-(3-sulfopropyl)-2-vinylpyridinium hydroxide inner salt. The random copolymer preferably contains the structural unit (a5). When the random copolymer contains the structural unit (a5), the binding ability 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 the structural unit (a5), the proportion of the structural unit (a5) in the entire random copolymer contained in the crosslinked polymer is preferably 1 to 20 mol%, more preferably 1 to 15 mol%, more preferably 1 to 10 mol%, and particularly preferably 2 to 8 mol%, relative to the sum (100 mol%) of all structural units constituting the entire random copolymer. By setting the proportion of the structural unit (a5) within the above-mentioned preferred range, the Shiga toxin inhibitory effect is further improved.

<Structural unit (a6)>
The structural unit (a6) is a structural unit derived from a crosslinking agent. The random copolymer preferably has the structural unit (a6). The random copolymer having the structural unit (a6) can carry out the copolymerization reaction and the crosslinking reaction in one step, thereby reducing the production cost.

The crosslinking agent may be a compound containing two or more polymerizable groups. The polymerizable group is preferably a group containing an ethylenic double bond. 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 cleavage of the polymerizable group of the crosslinking agent.

Examples of the crosslinking agent include a difunctional or higher functional (meth)acrylamide compound, or a difunctional 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 di- or higher functional (meth)acrylamide compound include, but are not limited to, N,N'-methylenebisacrylamide, N,N'-methylenebismethacrylamide, N,N'-ethylenebisacrylamide, ethylenediaminedimethylacrylamide, and ethylenediaminediacrylamide.
Examples of the difunctional or higher functional (meth)acrylate compound include, but are not limited to, tetraethylene glycol diacrylate, triethylene glycol diacrylate, ethylene glycol diacrylate, diethylene glycol diacrylate, tetraethylene glycol dimethacrylate, triethylene glycol dimethacrylate, ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, trimethylopropane trimethacrylate, pentaerythritol tetramethacrylate, bisphenol A dimethacrylate, and glycerol dimethacrylate.

Specific examples of the structural unit (a6) include, but are not limited to, the following: In the formula, R ^α each independently represents a hydrogen atom or a methyl group, and n represents an integer of 0 to 2, with 1 being preferred.

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

<Others: structural unit (a7)>
The random copolymer may contain a structural unit other than the above (a1) to (a6) (hereinafter also referred to as "structural unit (a7)"), as long as the effects of the present invention are not impaired. The structural unit (a7) is not particularly limited. Examples of the structural unit (a7) include structural units derived from acrylamide derivatives such as N-isopropylacrylamide, N-ethylacrylamide, and N-methylacrylamide.

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

The random copolymer contained in the crosslinked polymer may be one type, or two or more types. The combination of structural units contained in the random copolymer includes the combination of structural unit (a1), structural unit (a2), and structural unit (6); the combination of structural unit (a1), structural unit (a2), structural unit (a3), and structural unit (6); the combination of structural unit (a1), structural unit (a2), structural unit (a3), structural unit (6), and structural unit (a7); the combination of structural unit (a1), structural unit (a2), structural unit (a3), structural unit (a5), structural unit (6), and structural unit (a7); the combination of structural unit (a1), structural unit (a3), structural unit (a6), and structural unit (a7); the combination of structural unit (a1), structural unit (a6), and structural unit (a7); the combination of structural unit (a1), structural unit (a4), structural unit (a6), and structural unit (a7); and the like.
Preferred combinations include a combination of the structural unit (a1), the structural unit (a2), the structural unit (a3), and the structural unit (a6); a combination of the structural unit (a1), the structural unit (a2), the structural unit (a3), the structural unit (a5), and the structural unit (a6); a 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); a combination of the structural unit (a1), the structural unit (a2), the structural unit (a3), the structural unit (a6), and the structural unit (a7); and the like.
Of these, combinations of the structural units (a1), (a2), (a3), (a5), (a6), and (a7) are particularly preferred.

The theoretical molecular weight of the crosslinkable polymer is preferably 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 molar ratio of each polymerizable monomer charged during synthesis of the crosslinkable polymer and the molecular weight of each polymerizable monomer. In this specification, the "theoretical molecular weight" is a value calculated as follows.
The molar ratio of each polymerizable monomer is calculated ( _RA : the value in [ ] in Table 1 divided by 100) when the total amount of all polymerizable monomers in the polymerizable monomer mixture is set to 1. The molar ratio _RA is multiplied by the molecular weight of the polymerizable monomer to calculate a value ( _MA ). The sum of the _MA calculated for each monomer in the polymerizable monomer mixture is the theoretical molecular weight.

The crosslinked polymer of this embodiment preferably has a molecular weight per sugar unit of 1000 to 5000, more preferably 1200 to 3000, and even more preferably 1500 to 2000. The "molecular weight per sugar unit" of a crosslinkable polymer is the value obtained by dividing the theoretical molecular weight of the crosslinkable polymer by the molar ratio (R _A ) of the polymerizable monomer from which the structural unit (a1) is derived.

The crosslinked polymer of this embodiment preferably has a Z-average particle size at 35°C measured by dynamic scattering of about 30 to 2000. The crosslinked polymer of this embodiment preferably has a Z-average particle size at 40°C measured by dynamic scattering of about 30 to 2000.

<Method of producing crosslinked polymer>
The crosslinked polymer of this embodiment can be produced by radically polymerizing a polymerizable monomer mixture containing a polymerizable monomer that derives each structural unit of the random copolymer and a crosslinking agent in the presence of a polymerization initiator. Alternatively, after radically polymerizing the polymerizable monomer that derives each structural unit to obtain a random copolymer, a crosslinking reaction of the random copolymer may be carried out using a crosslinking agent having two or more groups that react with the functional group in the random copolymer. Since the synthesis and crosslinking of the random copolymer can be carried out in one step, a method of copolymerization using a polymerizable monomer mixture containing a crosslinking agent is preferred. Examples of the crosslinking agent include those listed in the structural unit (a6) above.

The polymerization initiator may be one generally used in radical polymerization, such as V-501 (4,4'-Azobis(4-cyanovaleric acid)) or AAPD (2,2'-Azobis(2-methylpropionamidine)Dihydrochloride). In addition, an aqueous medium containing a surfactant can be used as the reaction solvent for radical polymerization. Examples of the surfactant that can be used include sodium dodecyl sulfate (SDS) and hexadecyltrimethylammonium bromide (CTAB).

The crosslinked polymer of the present embodiment has a structure in which a random copolymer having the structural unit (a1) and at least one other structural unit is crosslinked, and thus can exhibit high inhibitory activity against Shiga toxin. This is thought to be because the crosslinking of the random copolymer forms a variety of structures, which enable binding to Shiga toxin at multiple sites.
The crosslinked polymer of this embodiment has high inhibitory activity against Shiga toxin, and can therefore be used as a Shiga toxin inhibitor described below, and as a pharmaceutical composition for treating or preventing infections caused by Shiga toxin-producing bacteria.

[Shiga toxin inhibitors]
In one embodiment, the invention provides a Shiga toxin inhibitor comprising a crosslinked polymer of the above embodiment.

The crosslinked polymer of the embodiment has high Shiga toxin inhibitory activity and can be used as a Shiga toxin inhibitor. The Shiga toxin inhibitor may contain one type of crosslinked polymer of the embodiment, or two or more types of crosslinked polymers.
The crosslinked polymer may also be a pharma- ceutically acceptable salt of the crosslinked polymer, a pharma- ceutically acceptable solvate of the crosslinked polymer, and a solvate of a pharma- ceutically acceptable salt of the crosslinked polymer.

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

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 preferred. The Shiga toxin inhibitor can be prepared as an orally administered formulation.

Shiga toxin inhibitors can be administered to humans or non-human mammals. Non-human mammals include, but are not limited to, primates (monkeys, chimpanzees, gorillas, etc.), rodents (mice, hamsters, rats, etc.), rabbits, dogs, cats, cows, pigs, goats, sheep, horses, etc.

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 below.

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

Shiga toxin-producing bacteria infection refers to a disease caused by bacteria that produce Shiga toxins in the intestinal tract. Examples of Shiga toxin-producing bacteria include EHEC. Examples of EHEC include Escherichia coli with O antigens of O157, O111, O26, O103, O104, O118, O121, O145, O165, etc.
The pharmaceutical composition of this embodiment can be used for the same subjects as the Shiga toxin inhibitors described above.

The crosslinked polymer of the embodiment contained in the pharmaceutical composition of the present embodiment may be one type or two or more types. The pharmaceutical composition of the present embodiment may contain at least one pharma- ceutically acceptable carrier in addition to the crosslinked polymer. The term "pharma-ceutically acceptable carrier" means a carrier that does not inhibit the physiological activity of the active ingredient and does not show substantial toxicity to the subject to which it is administered. The term "not substantially toxic" means that the ingredient does not show toxicity to the subject to which it is administered at a dose normally used. In the pharmaceutical composition of the present embodiment, the pharma-ceutically acceptable carrier is a carrier that does not inhibit the Shiga toxin inhibitory activity of the crosslinked polymer and does not show substantial toxicity to the subject to which it is administered. The pharma-ceutically acceptable carrier includes any known pharma-ceutically acceptable ingredient that is typically considered to be an inactive ingredient. The pharma- ceutically acceptable carrier is not particularly limited, and examples thereof include solvents, diluents, vehicles, excipients, flow promoters, binders, granulating agents, dispersing agents, suspending agents, wetting agents, lubricants, disintegrants, solubilizing agents, stabilizers, emulsifying agents, fillers, preservatives (e.g., antioxidants), chelating agents, flavoring agents, sweetening agents, thickening agents, buffers, coloring agents, etc. One type of pharma- ceutically acceptable carrier may be used alone, or two or more types may be used in combination.

The pharmaceutical composition of this embodiment may contain other ingredients other than the crosslinked polymer and the pharma- ceutically acceptable carrier. The other ingredients are not particularly limited, and those commonly used in the pharmaceutical field can be used without particular limitation. The pharmaceutical composition of this embodiment may also contain an active ingredient other than the crosslinked polymer. Examples of active ingredients include, but are not limited to, antibiotics, intestinal regulators, antidiarrheal agents, antipyretics, analgesics, etc. The other ingredients may be used alone or in combination of two or more.

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

The administration route of the pharmaceutical composition of this embodiment is not particularly limited and can be administered orally or parenterally, with oral administration being preferred.

The pharmaceutical composition of this embodiment can be administered in a therapeutically effective amount of the crosslinked polymer. The term "therapeutically effective amount" means an amount of a drug effective for treating or preventing a target disease. For example, the therapeutically effective amount of the crosslinked polymer can be an amount that can alleviate, suppress, or inhibit symptoms such as diarrhea, bloody stool, abdominal pain, fever, lytic anemia, thrombocytopenia, acute renal failure, and encephalopathy caused by Shiga toxins. The therapeutically effective amount may be appropriately determined depending on the symptoms, weight, age, and sex of the patient, the dosage form of the pharmaceutical composition, and the administration method. For example, the pharmaceutical composition of this embodiment can be administered in a single dose of the crosslinked polymer in a range of 0.01 to 1000 mg per kg of the body weight of the subject. The dosage 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.

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 this embodiment may be 0.01 to 90% by mass, 0.05 to 80% by mass, or 0.1 to 60% by mass.

The administration interval of the pharmaceutical composition of this embodiment may be appropriately determined depending on the symptoms, body weight, age, sex, etc. of the patient, as well as the dosage form of the pharmaceutical composition and the administration method, etc. The administration interval may be, for example, every few hours, 2 to 3 times a day, once a day, once every 2 to 3 days, once a week, etc.

The pharmaceutical composition of this embodiment contains the crosslinked polymer of the above embodiment that has high inhibitory activity against Shiga toxin, and therefore can effectively treat or prevent Shiga toxin-producing bacterial infections. In addition, the pharmaceutical composition of this embodiment can reduce the content of Shiga toxin-binding oligosaccharides such as globotrisaccharide by using the crosslinked polymer, thereby reducing production costs.

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

The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

<Synthesis of globotrisaccharide-containing monomer (M1-1)>
The synthesis of a monomer (M1-1) containing globotrisaccharide was carried out according to the reaction scheme shown below.
617.9 mg of Gb ₃ -β-MP (product code: M1767, Tokyo Chemical Industry Co., Ltd.), 16 mL of acetic anhydride (Ac ₂ O) and 32 mL of pyridine were mixed and reacted at room temperature overnight. The reaction solution was concentrated under reduced pressure and then extracted with ethyl acetate. It was washed successively with 1N hydrochloric acid, a saturated aqueous solution of sodium bicarbonate, water and saturated saline. The organic layer was dried over magnesium sulfate. The filtrate was concentrated under reduced pressure and then vacuum dried to obtain compound (I).
Then, 1664.4 mg of ammonium hexanitratocerate (IV) (CAN) 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 reacted at 0° C. for 3 hours. The reaction solution was extracted with ethyl acetate, and washed successively with water, a saturated aqueous sodium bicarbonate solution, and saturated saline. The organic layer was dried over magnesium sulfate. The filtrate was concentrated under reduced pressure, and the residue was purified by flash chromatography (SiO ₂ , toluene/acetone, gradient from 9/1 to 7/3 (volume ratio); flash automatic purification apparatus: Isolera One, column: 2 connected SNAP Ultra 25 g; Biotage). After concentration under reduced pressure, the reaction product was collected by vacuum drying, and 846.8 mg of compound (II) was obtained (yield 90.5%).
Next, 15 mg of sodium methoxide (NaOMe) and 10 mL of methanol (ultra-dehydrated) were added to 808.7 mg of compound (II) and reacted at room temperature for 2 hours. The mixture was neutralized with Amberlyst, and the filtrate was concentrated under reduced pressure. The reaction product was then recovered by freeze-drying to obtain 406.9 mg of compound (III) (yield 92.3%).
Next, 503.8 mg of sodium azide (NaN ₃ ), 1.21 mL of N,N-diisopropylethylamine (DIPEA), and 3.1 mL of heavy water (D ₂ O) were added to 389.3 mg of compound (III), and the mixture was cooled to 0° C. This solution was added to 391.5 mg of 2-chloro-1,3-dimethylimidazolinium chloride (DMC), and the mixture was reacted at 0° C. for 4 hours. The reaction solution was concentrated under reduced pressure, and then 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. The mixture was passed through a cation exchange resin column activated with 1N aqueous sodium hydroxide solution, and then purified by flash chromatography (reverse phase (C18), water/methanol, gradient from 10/0 to 0/10 (volume ratio); flash automatic purification apparatus: Isolera One, column: SNAP Ultra C18 30 g; Biotage). After concentration under reduced pressure, the reaction product was recovered by lyophilization to obtain 331.5 mg of compound (IV) (yield 81.1%).
Next, 75.2 mg of 3-butynyl acrylamide, 8.9 mg of copper(II) sulfide (CuSO ₄ ), 29.5 mg of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), 22.0 mg of sodium L-ascorbate (L-Asc-Na), and 6 mL of a mixed solvent of water/methanol (H ₂ O:MeOH=1:1 (volume ratio)) were added to 293.9 mg of compound (IV), and the mixture was reacted overnight under a nitrogen atmosphere at 30° C. The reaction solution was concentrated under reduced pressure, and the residue was purified by flash chromatography (reverse phase (C18), water/methanol, gradient from 10/0 to 0/10 (volume ratio); flash automatic purification apparatus: Isolera One, column: SNAP Ultra C18 30 g; manufactured by Biotage). Subsequently, a metal scavenger (SiliaMetS ^™ Imidazole, 300 mg) was added and the mixture was stirred at room temperature overnight. The metal scavenger was removed by filtration, and the mixture was concentrated under reduced pressure, after which the reaction product was recovered by lyophilization to obtain 315.7 mg of a monomer (M1-1) (yield 87.2%).
The resulting monomer (M1-1) was subjected to NMR measurement (JNM-ECZ400; manufactured by JEOL Ltd.), and its structure was confirmed from the following analytical results: The NMR chart of the monomer (M1-1) is shown in FIG.
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 in the molar ratio shown in Table 1, and a radical polymerization reaction was carried out at 70° C. for 3 hours under a nitrogen atmosphere to produce a crosslinked polymer of each example. When preparing 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 the reaction solvent. As a polymerization initiator, 0.69 mM V-501 (4,4'-Azobis(4-cyanovaleric acid)) or AAPD (2,2'-Azobis(2-methylpropionamidine)Dihydrochloride) was used. V-501 was dissolved in dimethylsulfoxide (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 with a dialysis membrane (Fisher brand) with MWCO 12,000-14,000, and the crosslinked polymer was recovered by freeze-drying.

In Table 1, each abbreviation indicates a polymerizable monomer described in Tables 2 and 3. The numbers in [ ] are the molar ratios in the polymerized monomer mixture.

<Calculation of molecular weight of crosslinked polymer>
(Theoretical molecular weight)
The molar ratio of each polymerizable monomer was calculated when the total amount of all polymerizable monomers in the polymerizable monomer mixture was taken as 1 ( _RA : the value in [ ] in Table 1 divided by 100). The molar ratio _RA was multiplied by the molecular weight of the polymerizable monomer to calculate a value ( _MA ). The _MA calculated for each monomer in the polymerizable monomer mixture was totaled, and the total value is shown in Table 4 as the "theoretical molecular weight".

(Molecular weight per sugar unit)
The theoretical molecular weight calculated above was divided by the molar ratio (R _A ) of the globo3 sugar monomer (M1-1) to calculate the molecular weight per sugar unit.

<Dynamic Light Scattering (DLS) Analysis of Crosslinked Polymer>
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-average and PDI measurements were performed at 35° C. and 40° C. The results are shown in Table 4.

<Evaluation of Shiga toxin inhibitory activity of crosslinked polymers>
The inhibitory activity of the cross-linked polymer against Shiga toxins (Stx1, Stx2) prepared from EHEC O157 Sakai strain was evaluated using Vero cells (RCB0001, RIKEN BRC). 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 Vero cell culture solution at ¹ ×10 ⁵ cells/mL. Then, a PBS suspension of the cross-linked polymer was added to the culture solution so that the final concentration of the cross-linked polymer was 0 to 50 μg/mL. As a negative control, PBS (containing no cross-linked polymer) was added to the culture medium. The cells were then cultured at 37° C. for 48 hours. After the culture, the cell viability of Vero cells was measured using CellTiter-Glo 2.0 Assay (Promega). Furthermore, the IC50 of the cross-linked polymer against Stx1 and Stx2 was calculated from the cell viability.

Figure 2A shows the results of evaluating cell viability when the crosslinked polymer of Example 1 was added to Vero cell culture medium containing Stx1. Figure 2B shows the results of evaluating cell viability when the crosslinked polymer of Example 1 was added to Vero cell culture medium containing Stx2. From these results, the IC50 for the crosslinked polymer of Example 1 against 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.

Figure 3A shows the IC50 of the crosslinked polymers of Examples 2 to 10 against Stx1. Figure 3B shows the IC50 of the crosslinked polymers of Examples 2 to 10 against 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 had a low IC50 against both Stx1 and Stx2, with the molar concentrations of IC50 being Stx1 (1.52 nM) and Stx2 (181 nM). The molar concentration of IC50 was calculated by dividing the concentration of IC50 (w/v) by the above-mentioned "theoretical molecular weight."

Figure 4A shows the IC50 of the cross-linked polymers of Examples 7 and 11 to 16 against Stx1. Figure 4B shows the IC50 of the cross-linked polymers of Examples 7 and 11 to 16 against Stx2. The cross-linked polymers of Examples 11 to 16 had an even lower IC50 than the cross-linked polymer of Example 7 against both Stx1 and Stx2.

<Animal testing>
An animal test was carried out using the crosslinked polymer of Example 7. Figure 5 shows the test schedule of the animal test. Four-week-old BALB/c germ-free mice (4 males, 1 female) were used as experimental animals.
One day before the start of administration of the crosslinked polymer, mice were infected with the enterohemorrhagic Escherichia coli O157 strain producing Stx2 (FIG. 5, day (-1)). Infection with the O157 strain was performed by orally administering 1E+7 CFU/mL of the O157 strain liquid by free water supply. The crosslinked polymer of Example 7 was dissolved in water to a concentration of 0.05 mg/mL, and administered to O157-infected mice (n=3) by free water supply. As a negative control, water (not containing the crosslinked polymer) was administered to O157-infected mice (n=2) by free water supply. Feces were collected at the timing indicated by the white arrow in FIG. 5, and the number of O157 strains in the feces was examined by fecal culture.

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

Furthermore, the concentration of the cross-linked polymer of Example 7 administered was changed, and animal tests were conducted in the same manner as above. The cross-linked polymer of Example 7 was administered in free water at concentrations of 0.05 mg/mL, 0.025 mg/mL, 0.01 mg/mL, or 0.005 mg/mL 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, even when the concentration of the cross-linked polymer of Example 7 was 0.025 mg/mL, the survival rate was 100%. The intake amount of the cross-linked polymer calculated from the amount of water supplied at this time was 0.275 mg/day per mouse. Furthermore, even when the concentration of the cross-linked polymer was 0.005 mg/mL, the survival rate was 30%. From these results, it was confirmed that even a low dose of the cross-linked polymer exhibited a Shiga toxin inhibitory effect.

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

Claims (7)

A crosslinked polymer obtained by crosslinking a random copolymer,
The random copolymer has a structural unit (a1) containing a Shiga toxin-binding oligosaccharide and at least one other structural unit,
The at least one other structural unit has a structural unit (a5) derived from a 1-(3-sulfopropyl)-2-vinylpyridinium hydroxide inner salt.
Crosslinked polymer. the at least one other structural unit further comprises a structural unit selected from the group consisting of a structural unit (a2) containing a hydrophobic group having 4 to 20 carbon atoms, a structural unit (a3) containing a cationic group, a structural unit (a4 ) containing an anionic group, and a structural unit (a6) derived from a crosslinking agent;
The crosslinked polymer of claim 1. The crosslinked polymer according to claim 2, wherein the random copolymer has the structural unit (a2). The crosslinked polymer according to claim 2 or 3, wherein the random copolymer has the structural unit (a3) or the structural unit (a4). The crosslinked polymer of any one of claims 1 to 4 , wherein the Shiga toxin-binding oligosaccharide is a globotrisaccharide. A Shiga toxin inhibitor comprising the crosslinked polymer according to any one of claims 1 to 5 . A pharmaceutical composition for treating or preventing infections caused by Shiga toxin-producing bacteria, comprising the crosslinked polymer according to any one of claims 1 to 5 and a pharma- ceutical acceptable carrier.

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