JP2024028857A - Hyaluronic acid derivative, and method for producing hyaluronic acid derivative - Google Patents

Abstract

To provide a proinflammatory cytokine production inhibitor that exhibits sufficient antiinflammatory action.SOLUTION: The present invention relates to a hyaluronic acid derivative having a repeating unit represented by formula (1), or a pharmaceutically acceptable salt thereof (where, R1 is a group represented by formula (1-1) or a group with any one terminal amino group removed from a polyethyleneimine with a molecular weight of 100 to 1200).SELECTED DRAWING: Figure 1

Description

The present invention relates to hyaluronic acid derivatives and methods for producing hyaluronic acid derivatives.

In Japan, which has entered a super-aging society, the development of treatments for acute and chronic inflammatory diseases is even more desirable. While the inflammatory response is a biological defense system against external factors such as infection, it is known to be associated with diseases such as arthritis, arteriosclerosis, pressure ulcers, and cancer.

Steroids, nonsteroidal drugs, and antibody drugs are used as drugs to suppress inflammation, but side effects such as adrenal insufficiency, infection, osteoporosis, and autoimmune diseases are a problem. In addition, there were problems with drug stability, development costs, and manufacturing costs.

Therefore, research is underway into drugs that are safer and have anti-inflammatory properties. For example, hydrogels containing steroids (Non-Patent Document 1), polysaccharides that exhibit anti-inflammatory properties (Non-Patent Document 2), and high molecular weight hyaluronic acid that exhibits anti-inflammatory effects (Non-Patent Document 3) have been proposed. ing.

Uri Soiberman, Siva P. Kambhampati, Tony Wu, Manoj K. Mishra, Yumin Oh, Rishi Sharma, Jiangxia Wang, Abdul Elah Al Towerki, Samuel Yiu, Walter J. Stark, Rangaramanujam M. Kannan, Subconjunctive injectable dendrimer-dexamethasone gel for the treatment of corneal inflammation. Biomaterials, 125, 38-53 (2017). Hong Chen, JiAN SUN, JUN LIU, YARUN GOU, XIN ZHANG, XIAONAN WU, RUI SUN, SIXUE TANG, JUAN KAN, CHUNLU QIAN, CHUNLU QIANG, C HANGHAI JIN, STRUCTURAL CHARACTERIZATION And and INFLAMMATY ACTIVITY OF ALKALI -SOLUBLE POLYSACCHARIDES FROM PURPLE PURPLE sweet potato. International Journal of Biological Macromolecules, 131, 484-494 (2019) Jamie E. Rayahin, Jason S. Buhrman, Yu Zhang, Timothy J. Koh, Richard A. Gemeinhart, High and low molecular weight hyaluronic acid differentially influence macrophage activation. ACS Biomaterials Science and Engineering, 1, 481-493 (2015).

In the steroid-encapsulating hydrogel described in Non-Patent Document 1, it is difficult to control the sustained release rate of the steroid, and side effects originating from the steroid are a problem.
Although some polysaccharides isolated from natural products such as those described in Non-Patent Document 2 exhibit anti-inflammatory properties, their effects are weak and it is difficult to mass produce them.
Although the high molecular weight hyaluronic acid described in Non-Patent Document 3 exhibits anti-inflammatory properties compared to low molecular weight hyaluronic acid, its effect is weak and insufficient.

Therefore, an object of the present invention is to provide an inflammatory cytokine production suppressing material that exhibits a sufficient anti-inflammatory effect without containing a steroid compound, in other words, has excellent safety and anti-inflammatory properties. shall be. Another object of the present invention is to provide a hyaluronic acid derivative and a method for producing the hyaluronic acid derivative.

As a result of intensive studies to achieve the above-mentioned problems, the present inventors have found that the above-mentioned problems can be achieved by the following configuration.

[1] An inflammatory cytokine production suppressing material containing a hyaluronic acid derivative and a compound represented by formula A1 described below, or a specific hyaluronic acid derivative having a group represented by formula A2.
[2] The specific hyaluronic acid derivative according to [1], wherein the specific hyaluronic acid derivative has a repeating unit represented by formula 1 described below, or a pharmaceutically acceptable salt, ester, or glucoside thereof. Inflammatory cytokine production inhibitor.
[3] The inflammatory cytokine production inhibitor according to [2], wherein R ² is an amino group.
[4] The inflammatory cytokine production inhibitor according to [3], wherein the content of the amino group is 0.10 mmol/g or more.
[5] The inflammatory cytokine production inhibitor according to [3], wherein the content of the amino group is 0.40 mmol/g or more.
[6] The inflammatory cytokine production inhibitor according to [3], wherein the content of the amino group is 0.50 mmol/g or more.
[7] The inflammatory cytokine production suppressing material according to any one of [2] to [6], wherein the content of the amino group is 1.10 mmol/g or less.
[8] The inflammatory cytokine production inhibitor according to any one of [1] to [7], which acts on macrophages to suppress the production of inflammatory cytokines.
[9] The inflammatory cytokine production suppressing material according to any one of [1] to [8], which is a hydrogel containing water.
[10] A hyaluronic acid derivative having a repeating unit represented by formula 1 described below, or a pharmaceutically acceptable salt, ester, or glucoside thereof.
[11] The hyaluronic acid derivative according to [10], wherein R ² is an amino group.
[12] The hyaluronic acid derivative according to [11], wherein the amino group content is 0.10 mmol/g or more.
[13] The hyaluronic acid derivative according to [11], wherein the content of the amino group is 0.40 mmol/g or more.
[14] The hyaluronic acid derivative according to [11], wherein the content of the amino group is 0.50 mmol/g or more.
[15] The hyaluronic acid derivative according to any one of [11] to [14], wherein the content of the amino group is 1.10 mmol/g or less.
[16] In an aqueous solution containing alcohol, in the presence of a carboxy group activator, hyaluronic acid or a salt thereof and a compound represented by formula A1 described below are bonded by an amidation reaction, [ A method for producing a hyaluronic acid derivative, for producing the hyaluronic acid derivative according to any one of [11] to [15].

According to the present invention, an inflammatory cytokine production suppressing material having excellent safety and anti-inflammatory properties can be provided. The present invention can also provide hyaluronic acid derivatives and methods for producing hyaluronic acid derivatives.

These are the results of measuring the structure of a hyaluronic acid derivative into which ethylenediamine has been introduced by ¹ H nuclear magnetic resonance spectroscopy ( ¹ H NMR). These are the results of measuring the structure of a hyaluronic acid derivative into which ethylenediamine has been introduced using Fourier transform infrared spectroscopy (FT-IR). These are the results of measuring the viscosity of an aqueous hyaluronic acid derivative solution using a viscoelasticity measuring device. These are the measurement results of viscoelastic properties. These are the measurement results of viscoelastic properties. These are the results of measuring changes in shear elastic modulus with respect to changes in strain of a 10% by mass HA3 aqueous solution using a viscoelasticity measuring device. These are the results of cell viability measured using a cell number counting kit regarding the influence of hyaluronic acid derivatives on cell viability and cell proliferation. Regarding the influence of hyaluronic acid derivatives on cell survival rate and cell proliferation, these are the results of the proliferation rate relative to the number of seeded cells measured using a cell number counting kit. These are the results of measuring cell viability when polyethyleneimine was used instead of ethylenediamine. This is a phase-contrast microscopic image for investigating the effect of hyaluronic acid derivatives on cell morphology. This is the adhesion area calculated from the fluorescence microscope image of the same sample as in FIG. 10. These are the results of evaluating the production of inflammatory cytokines. These are the results of evaluating the production of inflammatory cytokines. These are the results of evaluating the production of inflammatory cytokines. These are the results of measuring the effects of hyaluronic acid derivative production conditions on anti-inflammatory properties. These are the results of measuring the effects of hyaluronic acid derivative production conditions on anti-inflammatory properties. FIG. 3 is a diagram showing the relationship between the content of polyethyleneimine in the reaction solution and the inhibitory effect on inflammatory cytokine production when polyethyleneimine is used as a specific amine. FIG. 3 is a diagram showing the relationship between the content of EDC in the reaction solution and the inhibitory effect on inflammatory cytokine production. These are the results of evaluating the anti-inflammatory effect of modified ethanolamine instead of EDA. These are the results of evaluating the production of inflammatory cytokines when polyethyleneimine and polyethyleneimine-modified hyaluronic acid were added. These are the results of investigating the effects of polyethyleneimine and polyethyleneimine-modified hyaluronic acid on cell survival rate. These are the results of investigating the effects of polyethyleneimine-modified hyaluronic acid and a mixture of polyethyleneimine and hyaluronic acid on cell viability. These are fluorescence microscopy images (upper row) and their evaluation results for evaluating phagocytosis. FIG. 2 is a diagram showing the influence of hyaluronic acid derivatives on cell survival rate and cell proliferation. These are the viscoelasticity measurement results of a hydrogel produced by crosslinking.

The present invention will be explained in detail below.
Although the description of the constituent elements described below may be made based on typical embodiments of the present invention, the present invention is not limited to such embodiments.
In this specification, a numerical range expressed using "~" means a range that includes the numerical values written before and after "~" as the lower limit and upper limit.

In the description of groups (atomic groups) in this specification, descriptions that do not indicate substituted or unsubstituted include those without a substituent as well as those with a substituent to the extent that the effects of the present invention are not impaired. It is something to do. For example, the term "alkyl group" includes not only an alkyl group having no substituent (unsubstituted alkyl group) but also an alkyl group having a substituent (substituted alkyl group). This also applies to each compound.
Moreover, in this specification, "(meth)acrylate" represents both or either of acrylate and methacrylate, and "(meth)acrylic" represents both or either of acrylic and methacrylic. Moreover, "(meth)acryloyl" represents both or either acryloyl and methacryloyl.

[Inflammatory cytokine production inhibitor]
The inflammatory cytokine production suppressing material (hereinafter also simply referred to as "the present suppressing material") according to an embodiment of the present invention contains a hyaluronic acid derivative as an active ingredient and a compound represented by formula A1 (hereinafter referred to as " or a specific hyaluronic acid derivative having a group represented by formula A2 described below. As described in the Examples below, this inhibitor has the effect of acting on macrophages to suppress the production of inflammatory cytokines.
In addition, since the present inhibitory material contains a hyaluronic acid derivative and/or a specific hyaluronic acid derivative, it can form a hydrogel and can be injected into the site where administration is required using a device such as a syringe (injectable). ).

Furthermore, in addition to the effect of suppressing the production of inflammatory cytokines, it surprisingly also has the effect of lower toxicity to cells, as shown in the Examples below. Below, each component contained in the present inhibitory material will be explained in detail.

<Specific mixture>
A first embodiment of the present inhibitory material is a specific mixture containing a hyaluronic acid derivative and a specific amine. Below, the hyaluronic acid derivative and the specific amine contained in the specific mixture will be explained in detail.

(Specific amine)
The specific mixture contains a specific amine represented by the following formula A1. The content of the specific amine in the specific mixture is not particularly limited, but in general, the content of the specific amine is 1 to 100% based on the total mass of the specific mixture, since the inflammatory cytokine production suppressing material having better effects of the present invention can be obtained. 50% by mass is preferred. Note that the specific mixture may contain one type of specific amine alone, or may contain two or more types of specific amines. When the specific mixture contains two or more types of specific amines, it is preferable that the total content is within the above numerical range.

In formula A1, L ¹ , L ² , and L ³ each independently represent a single bond or a divalent group, and R ² is a hydroxy group, a methyl group, an amino group, a carboxy group, a sulfonic acid group, and represents at least one specific substituent selected from the group consisting of mercapto groups, X represents a hydrogen atom, a halogen atom, or a monovalent substituent different from the above specific substituent, and ^M Represents a bond or a p+q+1-valent group; when ^M1 is a single bond, p is 1 and q is 0; when ^M1 is a p+q+1-valent group, p is an integer of 1 or more, and q is an integer of 0 or more. Each of the plurality of L ² , R ² , L ³ and X representing an integer may be the same or different.

The divalent groups of L ¹ , L ² , and L ³ are not particularly limited, but include -O-, -NR- (R represents a hydrogen atom or a substituent W described later), -C (= O)-, -S-, a linear, branched, or cyclic hydrocarbon group that may have a hetero atom, and combinations thereof.

Hydrocarbon groups include methylene group, ethylene group, 1,2-propylene group, 1,3-propylene group, 1,2-butylene group, 1,3-butylene group, 1,4-butylene group, 1,5 - Divalent saturated hydrocarbon groups having 1 to 20 carbon atoms such as pentylene group, 1,6-hexylene group, 1,9-nonylene group, and 1,12-dodecylene group; ethenylene group, propenylene group , alkenylene groups such as 3-butenylene group, 2-butenylene group, 2-pentenylene group, 2-hexenylene group, 2-nonenylene group, 2-dodecenylene group, and ethynylene group having 2 to 20 carbon atoms. Divalent unsaturated hydrocarbon group; 3 to 20 carbon atoms such as cyclopropylene group, cyclobutylene group, cyclopentylene group, cyclohexylene group, cyclononylene group, cyclododecylene group, norbornylene group, and adamantylene group divalent cyclic saturated hydrocarbon groups; 1,3-phenylene group, 1,4-phenylene group, 1,4-naphthylene group, 1,5-naphthylene group, 2,6-naphthylene group, and biphenyl- Examples include arylene groups having 6 to 20 carbon atoms such as 4,4'-diyl groups, divalent saturated hydrocarbon groups are preferred, and alkylene groups which may have substituents are more preferred.
Among these, the number of carbon atoms in the divalent saturated hydrocarbon group is preferably 10 or less, more preferably 8 or less, from the viewpoint of obtaining an inflammatory cytokine production suppressing material that has a better inflammatory cytokine production suppressing effect. , more preferably 5 or less, particularly preferably 4 or less, and most preferably 3 or less. The lower limit is not particularly limited, but is preferably one or more, more preferably two or more.

Note that some of the hydrogen atoms possessed by each of the above hydrocarbon groups may be substituted with a substituent W described later.

Examples of the heteroatom include an oxygen atom, a nitrogen atom, a sulfur atom, and a phosphorus atom.

The divalent group of M ¹ is not particularly limited, but includes the same groups as L ¹ already explained, and the preferred forms are also the same.

The trivalent or higher-valent group for M ¹ is not particularly limited, but includes, for example, groups represented by the following formulas (3BRC) to (6BRC). In addition, "*" in the following formula represents a bonding position.

In formula 3BRC, L ₃ represents a trivalent group. T ₃ represents a single bond or a divalent group, and three T ₃ may be the same or different from each other.
L ₃ is a trivalent hydrocarbon group (preferably having 1 to 10 carbon atoms. The hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group), or a trivalent heterocarbon group. Cyclic groups (preferably 5- to 7-membered heterocyclic groups, such as triazine rings and isocyanurate rings), and hydrocarbon groups include heteroatoms (for example, -O-). You can leave it there. Specific examples of _L3 include glycerin residue, trimethylolpropane residue, phloroglucinol residue, and cyclohexanetriol residue.
Furthermore, examples of L ₃ include groups represented by the following formulas.

In the above formula, Lm ₁ to Lm ₃ are each independently a single bond or a divalent group. The divalent group is not particularly limited, but includes saturated or unsaturated, linear, branched, or cyclic hydrocarbon groups that may have a heteroatom. Moreover, * represents a bonding position.

In formula 4BRC, L ₄ represents a tetravalent group. T ₄ represents a single bond or a divalent group, and the four T _4s may be the same or different from each other.
Preferred forms of L ₄ include a tetravalent hydrocarbon group (preferably having 1 to 10 carbon atoms; the hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group); The hydrocarbon group may include a valent heterocyclic group (preferably a 5- to 7-membered heterocyclic group), and the hydrocarbon group may contain a heteroatom (for example, -O-). Specific examples of _L4 include pentaerythritol residues, ditrimethylolpropane residues, and the like.

In formula 5BRC, L ₅ represents a pentavalent group. T ₅ represents a single bond or a divalent group, and the five T ₅ may be the same or different from each other.
A preferred form of L ₅ is a pentavalent hydrocarbon group (preferably having 2 to 10 carbon atoms. The hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group), or , a pentavalent heterocyclic group (preferably a 5- to 7-membered heterocyclic group), and the hydrocarbon group may contain a heteroatom (for example, -O-). Specific examples of L ₅ include arabinitol residue, phloroglucidol residue, and cyclohexanepentaol residue.

In formula 6BRC, L ₆ represents a hexavalent group. T ₆ represents a single bond or a divalent group, and the six T ₆ may be the same or different from each other.
A preferred form of L ₆ is a hexavalent hydrocarbon group (preferably having 2 to 10 carbon atoms. The hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group), or , a hexavalent heterocyclic group (preferably a 6- to 7-membered heterocyclic group), and the hydrocarbon group may contain a heteroatom (for example, -O-). Specific examples of L ₆ include mannitol residue, sorbitol residue, dipentaerythritol residue, hexahydroxybenzene, and hexahydroxycyclohexane residue.

In formulas 3BRC to 6BRC, specific examples and preferred forms of the divalent groups represented by T ₃ to T ₆ may be the same as the divalent group of M ¹ described above.
Furthermore, when M ¹ is a group having a valence of 7 or more, a group that is a combination of groups represented by formulas 3BRC to 6BRC can be used.

The specific substituent for R ² is at least one selected from the group consisting of a hydroxy group, a methyl group, an amino group, a carboxy group, a sulfonic acid group, and a mercapto group; A group is preferable, and an amino group is more preferable.

The monovalent substituent for X that is different from the specific substituent is not particularly limited, and includes those exemplified as the substituent W described later.

As A1, a compound represented by A1' below is more preferable in that a material for suppressing inflammatory cytokine production having more excellent effects of the present invention can be obtained.

In formula A1', L ¹² and L ²² are each independently a single bond or a divalent group, and examples of the divalent group include the group explained as L ¹ in formula 2, and preferred forms are also included. The same is true.
In formula A1', p' is an integer of 1 or more, and ^M12 is a single bond or a p'+1-valent group. R ²² represents a specific substituent, and the preferred form is the same as R ² described above. Examples of M ¹² include the same groups as M ¹ described above, and preferred forms are also the same.
Although the specific amine is not particularly limited, for example, a compound represented by the following formula can be used.

Moreover, the following compounds can also be used as the specific amine.

Further, as the specific amine, polyethyleneimine is preferable. The molecular weight of polyethyleneimine is not particularly limited, but is generally preferably from 100 to 1,200, more preferably from 200 to 1,000.

(hyaluronic acid derivative)
The specific mixture contains a hyaluronic acid derivative. The content of the hyaluronic acid derivative in the specific mixture is not particularly limited, but it is generally 0.00000000000000000000000000000000000000000. 01 to 10% by mass is preferred. Note that the specific mixture may contain one type of hyaluronic acid derivative alone, or may contain two or more types of hyaluronic acid derivatives. When the composition contains two or more types of hyaluronic acid derivatives, it is preferable that the total content is within the above numerical range.

As used herein, hyaluronic acid derivatives include hyaluronic acid and derivatives thereof. As the hyaluronic acid, hyaluronic acid (HA) or a salt thereof can be used. Examples of hyaluronates include alkali metal salts such as sodium salts, potassium salts, and lithium salts, and tetraalkylammonium salts (e.g., tetrabutylammonium (TBA) salts), which are frequently used as pharmaceuticals. The sodium salt can be converted into a tetraalkylammonium salt such as tetrabutylammonium (TBA) salt and used. HA or a pharmaceutically acceptable salt can be produced using various known methods such as extraction from biological sources such as chicken comb and pig skin, and biological fermentation. Alternatively, commercially available products can be purchased and used (for example, from Denki Kagaku Kogyo Co., Ltd., Shiseido Co., Ltd., Seikagaku Kogyo Co., Ltd., R&D System Co., Ltd., etc.).
The hyaluronic acid derivative is not particularly limited, but specific hyaluronic acid derivatives described below are preferred.

The molecular weight of HA is not particularly limited, but the number average molecular weight is preferably 100,000 to 3,000,000, more preferably 600,000 to 1,200,000.

<Specific hyaluronic acid derivative>
The inflammatory cytokine production suppressing material according to the second embodiment of the present invention contains (as an active ingredient) a hyaluronic acid derivative having a group represented by the following formula A2.

In formula A2, L ¹ , L ² , and L ³ each independently represent a single bond or a divalent group, and R ² is a hydroxy group, a methyl group, an amino group, a carboxy group, a sulfonic acid group, and represents at least one specific substituent selected from the group consisting of mercapto groups, X represents a hydrogen atom, a halogen atom, or a monovalent substituent different from the above specific substituent, and ^M Represents a bond or a p+q+1-valent group; when ^M1 is a single bond, p is 1 and q is 0; when ^M1 is a p+q+1-valent group, p is an integer of 1 or more, and q is an integer of 0 or more. Each of the plurality of L ² , R ² , L ³ and X representing an integer may be the same or different.
Note that L ¹ , L ² , L ³ , R ² , M ¹ , p, and q in formula A2 have the same meanings as each symbol in formula A1, and preferred forms are also the same.

In order to have better effects of the present invention, it is more preferable that the specific hyaluronic acid derivative has a group represented by A2' below.

In formula A2', L ¹² and L ²² are each independently a single bond or a divalent group, and examples of the divalent group include the group explained as L ¹ in formula 2, and preferred forms are also included. The same is true.
In formula A2', p' is an integer of 1 or more, and ^M12 is a single bond or a p'+1-valent group. R ²² represents a specific substituent, and the preferred form is the same as R ² described above. Examples of M ¹² include the same groups as M ¹ described above, and preferred forms are also the same. Further, in formula A2', * represents a bonding position.

The group represented by A2 above is not particularly limited, but includes groups represented by the following formulas. In addition, * in the formula represents a bonding position.

Further, as the group represented by formula A2, a group obtained by removing one arbitrary terminal amino group of polyethyleneimine is also preferable. The molecular weight of the polyethyleneimine is not particularly limited, but is generally preferably from 100 to 1,200, more preferably from 200 to 1,000.

- Preferred forms of specific hyaluronic acid derivatives Among these, specific hyaluronic acid derivatives are hyaluronic acid having a repeating unit represented by the following formula 1 from the viewpoint of obtaining an inflammatory cytokine production suppressing material having more excellent effects of the present invention. Preferably, it is a derivative, or a pharmaceutically acceptable salt, ester, or glucoside thereof.

In Formula 1, R ¹ is the group represented by A2 as described above.
The specific hyaluronic acid derivative has a group represented by formula A2 introduced into the 6-position carbon of D-glucuronic acid in the disaccharide repeating unit (hereinafter also referred to as "unit") represented by formula 1 above. Has a structure.
The content of the unit represented by formula 1 in the specific hyaluronic acid derivative is not particularly limited, but when the total repeating units of the present hyaluronic acid derivative is 100 mol%, it is preferably 0.1 to 99 mol%, and 1 to 99 mol%. 90 mol% is more preferable.

The molecular weight of the specific hyaluronic acid derivative is not particularly limited, but is typically preferably from 100,000 to 3,000,000, more preferably from 600,000 to 1,200,000. In addition, in this specification, the molecular weight of a hyaluronic acid derivative is a number average molecular weight measured by gel permeation chromatography (GPC) method.

The content of the specific substituent in the specific hyaluronic acid derivative is not particularly limited, but from the standpoint of having more excellent effects of the present invention, it is preferably 0.10 mmol/g or more, more preferably 0.40 mmol/g or more, and 0. More preferably, it is .50 mmol/g or more. The upper limit is not particularly limited, but is generally preferably 2.00 mmol/g or less, more preferably 1.10 mmol/g or less.
As a method for measuring the content of a specific substituent, for example, in the case of an amino group, it can be determined by a colorimetric test using 2,4,6-trinitrobenzenesulfonic acid.

When the specific substituent is an amino group, the content of the carboxy group in the specific hyaluronic acid derivative is not particularly limited, but is generally preferably 0.1 to 1.2 mmol, and 0.1 to 0.7 mmol/g. More preferably, 0.1 to 0.5 mmol/g is even more preferable.
The amount of carboxyl groups in a specific hyaluronic acid derivative can be determined by neutralization titration using potentiometry using an aqueous sodium hydroxide solution.

As long as the specific hyaluronic acid derivative has the unit represented by Formula 1, it may have other units and partial structures within the scope of achieving the effects of the present invention. Other units include, but are not particularly limited to, hyaluronic acid (a structure in which disaccharide units of N-acetylglucosamine and D-glucuronic acid are linked), and the unit represented by formula 1, which is intermolecular and/or intermolecular. Examples include structures that are cross-linked within.

・Manufacturing method Although there are no particular restrictions on the manufacturing method of the specific hyaluronic acid derivative, in that the specific hyaluronic acid derivative can be produced more easily, hyaluronan is Specified hyaluronic acid, which involves bonding an acid or a salt thereof (hereinafter also referred to as "hyaluronic acid etc.") and a compound represented by formula A1 (hereinafter also referred to as "specific amine") by an amidation reaction. Preferred are methods for producing acid derivatives.
In other words, the above production method involves preparing and reacting a reaction solution containing a carboxy group activator, a specific amine represented by formula A1, hyaluronic acid, etc., alcohol, and water. This is a method for producing a hyaluronic acid derivative.
Below, each component contained in the above reaction solution will be explained in detail.

The reaction solution contains a specific amine represented by formula A1. The specific amine has the function of cross-coupling with hyaluronic acid through an amidation reaction and introducing a group having a specific substituent into the hyaluronic acid molecule. The specific amine is as already explained as the specific amine contained in the specific mixture, and the preferred form is also the same.

The content of the specific amine in the reaction solution is not particularly limited, but generally, when the D-glucuronic acid unit possessed by hyaluronic acid in the reaction solution is 100 mol%, it is preferably 1 to 1000 mol%, and 10 to 200 mol%. Mol% is preferred. In addition, the reaction solution may contain one kind of specific amine independently, and may contain two or more kinds. When the reaction solution contains two or more types of specific amines, it is preferable that the total content is within the above numerical range.

(hyaluronic acid, etc.)
As the hyaluronic acid, hyaluronic acid (HA) or a salt thereof can be used. Examples of hyaluronates include alkali metal salts such as sodium salts, potassium salts, and lithium salts, and tetraalkylammonium salts (e.g., tetrabutylammonium (TBA) salts), which are frequently used as pharmaceuticals. The sodium salt can be converted into a tetraalkylammonium salt such as tetrabutylammonium (TBA) salt and used. HA or a pharmaceutically acceptable salt can be produced using various known methods such as extraction from biological sources such as chicken comb and pig skin, and biological fermentation. Alternatively, commercially available products can be purchased and used (for example, from Denki Kagaku Kogyo Co., Ltd., Shiseido Co., Ltd., Seikagaku Kogyo Co., Ltd., R&D System Co., Ltd., etc.).
The molecular weight of HA is not particularly limited, but the number average molecular weight is preferably 100,000 to 3,000,000, more preferably 600,000 to 1,200,000.

The content of hyaluronic acid, etc. in the reaction solution is not particularly limited, but it is 0.01 to 50 parts by mass when the content of the solvent in the reaction solution is 100 parts by mass, since the reaction tends to proceed more uniformly. part is preferred.
Note that the reaction solution may contain one type of hyaluronic acid or the like alone, or may contain two or more types of hyaluronic acid. When the reaction solution contains two or more types of hyaluronic acid, the total content is preferably within the above numerical range.

(Carboxy group activator)
The carboxy group activator is not particularly limited, but preferably contains a carbodiimide compound. Examples of carbodiimide compounds include EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride) and 1-alkyl-3-(3-dimethylaminopropyl)carb, which has a structure similar to EDC. odiimides, ETC(1-ethyl-3 -(3-(trimethylammonio)propyl)carbodiimide), and CMC (1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide).

The content of the carboxy group activator in the reaction solution is not particularly limited, but since the reaction tends to proceed more uniformly, when the D-glucuronic acid unit such as hyaluronic acid in the reaction solution is 100 mol%. , preferably 1 to 1000 mol%, and preferably 10 to 200 mol%. The reaction solution may contain one type of carboxyl group activator alone or may contain two or more types of carboxyl group activators. When contained, it is preferable that the total content is within the above numerical range.

(alcohol)
It is generally known that when alcohol is added to an aqueous solution in which hyaluronic acid is dispersed, the hyaluronic acid aggregates, typically becoming particulate, and it is thought that the amidation reaction tends to proceed unevenly. It's here. Therefore, from the viewpoint of making the amidation reaction proceed more uniformly, adding alcohol to the reaction solution has not been considered so far.

However, the present inventor's extensive studies have revealed that the amount of specific substituents introduced can be easily controlled by allowing the above amination reaction to proceed in an aqueous solution containing alcohol, and in particular, the amount of specific substituents introduced can be further improved. In other words, it has been found that the reaction efficiency can be further improved.
The above mechanism is not necessarily clear, but it is thought that the presence of alcohol in the reaction solution further inhibits hydrogen bonds between and within molecules of hyaluronic acid, etc., making it easier to introduce specific substituents. It will be done.

The alcohol is not particularly limited, and both monohydric alcohols having one hydroxy group in the molecule and polyhydric alcohols having two or more hydroxy groups can be used, and mixtures thereof can also be used.

Further, the number of carbon atoms in the alcohol is not particularly limited, but from the viewpoint of obtaining a more uniform reaction solution, the number of carbon atoms is preferably 1 to 10, more preferably 1 to 6, and even more preferably 1 to 4.
Examples of alcohols having 1 to 4 carbon atoms include methanol, ethanol, propanol, 2-propanol, 2-methoxyethanol, 2-ethoxyethanol, 2-methoxypropanol, and 2-methoxy-2-propanol. Monohydric alcohol (monol); ethylene glycol, propylene glycol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, Dihydric alcohols (diols) such as 2-methyl-1,2-propanediol, 1,4-butynediol, and diethylene glycol; polyols such as glycerin, 1,2,4-butanetriol, and erythritol. It will be done.

Among these, the alcohol is preferably a monohydric alcohol, and more preferably at least one selected from the group consisting of methanol, ethanol, propanol, and 2-propanol, from the standpoint of more efficiently introducing a specific substituent. At least one selected from the group consisting of , ethanol, propanol, and 2-propanol is more preferred.

The content of alcohol in the reaction solution is not particularly limited, but it is generally 10 to 200 volume% when the water content in the solvent is 100 volume%, since the introduction rate of the specific substituent can be more easily controlled. % is preferred, and 20 to 130 volume % is more preferred.
The reaction solution may contain one type of alcohol alone, or may contain two or more types of alcohol. When the reaction solution contains two or more types of alcohol, it is preferable that the total content is within the above numerical range.

(Other ingredients)
The reaction solution may contain components other than those listed above. Examples of components other than those mentioned above include reaction aids, pH adjusters, and the like.

Preferably, the reaction aid is a substance capable of forming active esters. Examples of reaction aids include NHS (N-Hydroxysuccinimide), HOBt (1-hydroxybenzotriazole), and HOOBt (3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazin). e), HOAt(1 -hydroxy-7-azabenzotriazole) and Sulfo-NHS (N-hydroxysulfosuccinimide). The reaction aids may be used alone or in combination of two or more.

The pH adjuster is not particularly limited, but includes potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium hydroxide, lithium hydroxide, potassium hydroxide, ammonia, diethanolamine, triethanolamine, triisopropanolamine, potassium carbonate, carbonic acid. Sodium, sodium bicarbonate, trishydroxymethylaminomethane (THAM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), morpholinoethanesulfonic acid (MES), carbamoylmethyliminobisacetic acid (ADA) , piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), colamine hydrochloride, N,N-bis(2-hydroxyethyl )-2-aminoethanesulfonic acid (BES), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), acetamidoglycine, tricine, glycinamide, and the like. The pH adjusters may be used alone or in combination of two or more.

The method for preparing the reaction solution is not particularly limited, and the above-mentioned components may be mixed. Among these, it is possible to introduce specific substituents more efficiently by dissolving hyaluronic acid, etc. in a solvent containing a pH adjuster and water, and then adding a carboxy group activator and an adjuvant as necessary. A preferred method is to stir and dissolve the alcohol, and then add the alcohol at the end.

The temperature at which the amidation reaction is carried out in the prepared reaction solution is not particularly limited, but it may be stirred at 20 to 30°C for 2 to 6 hours.
Furthermore, the present production method may further include a step of purifying the reaction solution after the reaction. Purification methods include, but are not particularly limited to, dialysis and/or freeze-drying.

<Application>
As shown in the examples, this inhibitory material can act on macrophages to suppress the production of inflammatory cytokines. The present hyaluronic acid derivative can also be used as an inflammation suppressant.
As shown in the Examples, when the present inhibitory material contains water, a hydrogel is formed, and the hydrogel has thixotropic properties and has properties suitable for being injected with a syringe or the like. Injecting the above hydrogel suppresses inflammation in the affected area and promotes healing.

Targeted inflammatory diseases include, but are not particularly limited to, arthritis, arteriosclerosis, myocardial infarction, spinal cord injury, hepatitis, pericarditis, bedsores, and wound healing in the skin and gastrointestinal mucosa.
Furthermore, in the field of regenerative medicine, it can be used as a scaffolding material for cells during cell transplantation. By enclosing transplanted cells in this suppressive material and transplanting them, it is possible to protect them from attack by the host's immune cells (immune rejection).
Furthermore, in the beauty field, by using it as an additive in skin care agents, excessive inflammatory reactions can be suppressed. Furthermore, inflammation can be suppressed by using it as an alternative to hyaluronic acid injections in the field of cosmetic surgery.

The present suppressing material may be used in combination with other components within the range that exhibits the effects of the present invention. Other components include diluents, wetting agents, emulsifiers, dispersants, adjuvants, preservatives, buffers, binders, stabilizers, surfactants, lipids, base materials, and the like. Further, the administration route may be parenteral or oral, but parenteral is preferred.

This inhibitory material can be administered via transmucosal or transdermal administration methods using devices such as sprays, dry powder inhalers, drops, iontophoresis, electroporation, and sonophoresis (ultrasound), microcannulas, microneedles, etc. It can be administered by needle-free injection, transmucosal or transdermal administration methods such as polishing. Oral administration in the form of tablets, vaginal administration, and rectal administration are also possible. Administration using a syringe is also possible. Creams, ointments, and drops are also available.

[Hydrogel]
The hydrogel according to the embodiment of the present invention (hereinafter also referred to as "the present hydrogel") is a hydrogel containing the present inhibitory material and water. Although not particularly limited, it is preferable that the present hydrogel has no fluidity at room temperature and normal pressure.
The present hydrogel contains the present inhibitory material and water, and the water content is not particularly limited and can be adjusted as appropriate depending on the application. Generally, the water content is preferably 1 to 99% by weight, more preferably 80 to 99% by weight, based on the total weight of the hydrogel.

This hydrogel may contain a crosslinking agent. The crosslinking agent is not particularly limited, but it is possible to form a hydrogel by using a compound (may be a polymer compound), particles, fibers, etc. having an aldehyde group, an N-hydroxysuccinimide group, an isocyanate group, etc. It can also be formed. Mechanical strength can be improved by adding a crosslinking agent.

Although the crosslinking agent is not particularly limited, for example, sulfonated cellulose represented by the following formula may be used.

In the above formula, k, m, and n represent mol% of each repeating unit, and are not particularly limited, but k is preferably 25 to 95 mol%, more preferably 40 to 90 mol%. m is preferably 4 to 70 mol%, more preferably 7 to 60 mol%. n is preferably 1 to 70 mol%, more preferably 10 to 30 mol%.

The above-mentioned sulfonated cellulose is a cellulose containing a sulfonated structure in which a ring is opened between the 3rd and 4th positions of a glucose unit shown in the following formula.

The above formula shows a state in which the sulfone group (-SO ₃ H) is dissociated in water or the like, and the counter cation is not limited to Na ⁺ but may be a proton, K ⁺ or the like.

Sulfonated cellulose can be synthesized, for example, by the route shown in the following formula described in Henrikki Liimatainen et al., Cellulose (2013) 20:741-749.

First, raw cellulose is dispersed in water to obtain a dispersion. To the above dispersion, 50 to 200 mol% of sodium periodate (NaIO ₄ ) was added when the amount (k) of glucose units in cellulose was 100 mol%, and the mixture was heated at 40 to 60°C while shielding from light. By stirring for 2 to 6 hours, aldehyde cellulose is produced.

Next, the aqueous solution containing unreacted substances is removed by vacuum filtration, and the procedure of washing with ultrapure water is repeated several times, followed by freeze-drying to obtain a dry powder of aldehyde cellulose. In the above formula, when the initial value of k is 100 mol%, m is 1 to 80 mol%, preferably 10 to 50 mol%.

The amount of aldehyde groups can be determined by neutralization titration using conductivity measurement using an aqueous sodium hydroxide solution, and is 0.5 to 8 mmol/g, preferably 1 to 7 mmol/g, more preferably 1 to 5 mmol/g. mmol/g.

As the raw material cellulose, for example, cellulose obtained from plants, animals, etc. such as softwood pulp, hardwood pulp, and cotton pulp, paper using these, waste paper, etc. can be used.

Next, the aldehyde cellulose was dispersed in ultrapure water, and sodium pyrosulfite (Na2S2O5) was added in an amount of 20 to 200 mol%, preferably 50 to 150 mol%, based on the aldehyde group amount of 100 mol%. Allow to react with stirring for ~24 hours. The product is collected by centrifugation, purified by washing with ultrapure water etc. to remove unreacted substances, and then homogenized for about 10 to 30 minutes using an ultrasonic homogenizer to obtain sulfonated cellulose nanofibers ( (sometimes abbreviated as "sCNF") can be obtained. Yield is approximately 80-90%.

sCNF is biodegradable. In the present invention, "biodegradability" was confirmed by decomposition of 1% by weight or more in 1 day in phosphate buffered saline (PBS) of p7.4 at 37°C. This biodegradability is determined when m+n, that is, ring-opened units, is at least 1 mol%, preferably 10 to 50 mol%, when the initial value of the amount (k) of glucose units in the raw material cellulose is 100 mol%. This is thought to be due to certain reasons. Furthermore, since sCNF has a ring-opening unit, the degree of crystallinity measured by X-ray diffraction is 20 to 70%, which is lower than the raw material cellulose, which has a degree of crystallinity of about 90%.

[Additives, etc.]
The present hydrogel may contain other components, and may be blended with various drugs to be delivered, proteins, etc., and used as a local delivery carrier or sustained release delivery carrier for these.
Examples of drugs include inflammatory drugs, antithrombotic drugs, antibiotics, growth factors such as fibroblast growth factors, vascular endothelial cell growth factors, and hepatocyte growth factors.
Furthermore, it may be used as a vaccine carrier by supporting a virus or cancer antigen protein.

[Substituent W]
The monovalent substituent W is not particularly limited, but includes methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, t-butyl group, pentyl group, hexyl group, nonyl group, dodecyl group, pentadecyl group. , an octadecyl group, and an alkyl group having 1 to 20 carbon atoms such as an eicosyl group; a carbon number such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cyclononyl group, a cyclododecyl group, a norbornyl group, and an adamantyl group. 3 to 20 cycloalkyl groups;
Alkenyl groups having 2 to 20 carbon atoms such as ethenyl group, propenyl group, 3-butenyl group, 2-butenyl group, 2-pentenyl group, 2-hexenyl group, 2-nonenyl group, and 2-dodecenyl group;
Phenyl group, 1-naphthyl group, 2-naphthyl group, 2-methylphenyl group, 3-methylphenyl group, 4-methylphenyl group, 4-ethylphenyl group, 4-propylphenyl group, 4-isopropylphenyl group, 4 -Aryl groups having 6 to 20 carbon atoms such as butylphenyl group, 4-t-butylphenyl group, 4-hexylphenyl group, 4-cyclohexylphenyl group, 4-adamantylphenyl group, and 4-phenylphenyl group;
Phenylmethyl group, 1-phenyleneethyl group, 2-phenylethyl group, 1-phenyl-1-propyl group, 1-phenyl-2-propyl group, 2-phenyl-2-propyl group, 3-phenyl-1-propyl group aralkyl groups having 7 to 20 carbon atoms, such as 4-phenyl-1-butyl groups, 5-phenyl-1-pentyl groups, and 6-phenyl-1-hexyl groups;
A group in which at least some of these hydrogen atoms are substituted with halogen atoms (fluorine, chlorine, and bromine are preferred); and
Alkoxy groups such as methoxy groups, ethoxy groups, and propoxy groups;
Examples include alkoxycarbonyl groups such as methoxycarbonyl group, ethoxycarbonyl group, n-propoxycarbonyl group, isopropoxycarbonyl group, n-butoxycarbonyl group, and t-butoxycarbonyl group.

In addition, the substituent W includes a heterocyclic group (heterocyclic group), a cyano group, a nitro group, an aryloxy group, a silyloxy group, a heterocyclicoxy group, an acyloxy group, a carbamoyloxy group, an alkoxycarbonyloxy group, and an aryloxycarbonyl group. Oxy group, acyl group, aryloxycarbonyl group, alkoxycarbonyl group, aryl or heterocyclic azo group, phosphino group, phosphinyl group, phosphinyloxy group, phosphono group, silyl group, hydrazino group, ureido group, boronic acid group ( -B(OH) ₂ ), phosphato group (-OPO(OH) ₂ ), and the like are also included.

The present invention will be explained in more detail below based on Examples. The materials, usage amounts, proportions, processing details, processing procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the Examples shown below.
Further, all error bars in graphs in the drawings described in the following examples mean standard deviation.

[Preparation of hyaluronic acid derivative]
Hyaluronic acid derivatives HA1-6 shown in Table 1 were synthesized. Unmodified hyaluronic acid (hyaluronic acid before reaction) was designated as HA0.
First, 250 mg of cockscomb-derived hyaluronic acid (molecular weight: 600,000 to 1,200,000) was dissolved in 50 mL of 2-morpholinoethanesulfonic acid buffer while stirring with a stir bar to obtain a dispersion. Next, when the amount (n) of D-glucuronic acid units in hyaluronic acid is 100 mol%, 10 to 200 mol% of ethylenediamine (EDA) and 1-ethyl-3-(3-dimethylamino) are added to the dispersion. Propyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at a final concentration of 10 mM were added and stirred. A hyaluronic acid derivative was synthesized by adding 20% to 130% (volume) of ethanol to the buffer solution and stirring at 20 to 30°C for 2 to 6 hours. Next, unreacted substances were removed by dialysis using a dialysis membrane (molecular weight fraction: 3,500 to 15,000 Da) and freeze-dried to obtain a dry powder of a hyaluronic acid derivative. The amount of amino groups was determined by a colorimetric test using 2,4,6-trinitrobenzenesulfonic acid, and the amount of carboxy groups was determined by neutralization titration using potentiometric measurements.

In Table 1, "HA (g)" is the content of hyaluronic acid (g) in the reaction solution, and "Ethylene diamine (mmol)" is the content of ethylenediamine in the reaction solution. (mmol), "EDC (mmol)" is the content (mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in the reaction solution, and "NHS (mmol)" is the content (mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in the reaction solution. )" is the content of N-hydroxysuccinimide (mmol/L) in the reaction solution, and "MES buffer (mL)" is the content of 2-morpholinoethanesulfonic acid buffer in the reaction solution. "Ethanol (mL)" is the content (mL) of ethanol in the reaction solution, and "Turbidity" indicates the turbidity of the reaction solution. ``Clear'' indicates that the condition was determined to be transparent by visual inspection, and ``Turbid'' indicates that the condition was determined to be turbid by visual inspection. "amino group (mmol/g)" is the amount of (primary) amino groups determined for the obtained hyaluronic acid derivative, and "Redsidual carboxylic group (mmol/g)" is the amount of the obtained hyaluronic acid derivative. The amount of hydroxyl groups quantified for the acid derivative.

In addition, a hyaluronic acid derivative was obtained in the same manner as above except that polyethyleneimine (manufactured by Nippon Shokubai Co., Ltd., trade name "Epomin", average molecular weight 300) was used instead of ethylenediamine. The results are shown in Table 2.

In Table 2, "PEI (mmol)" is the content (mmol) of polyethyleneimine in the reaction solution, and Amine/COOH is the content of amino groups contained in polyethyleneimine and hyaluronic acid. EDC/COOH is the ratio of the number of moles of EDC charged to the number of moles of carboxy groups contained in hyaluronic acid, and the other terms are as defined in Table 1.

<Identification of hyaluronic acid derivatives>
In order to identify the structure of the hyaluronic acid derivative, measurements were performed using 1H nuclear magnetic resonance spectroscopy ( ^1H NMR) and Fourier transform infrared spectroscopy (FT-IR). FIG. 1 shows a ¹ H NMR spectrum of a hyaluronic acid derivative ("PHEIA10" in Table 2) into which polyethyleneimine has been introduced. In addition, in FIG. 1, "HA" represents hyaluronic acid, and "PEIHA" represents the respective spectra of "PEIHA10."

Furthermore, FIG. 2 shows an FT-IR spectrum of a hyaluronic acid derivative into which ethylenediamine has been introduced. In FIG. 2, "HA3" indicates the spectrum of a hyaluronic acid derivative into which ethylenediamine has been introduced, and "HA0" indicates the spectrum of hyaluronic acid.

As shown in FIG. 1, a peak of the methylene moiety of polyethyleneimine was detected in the spectrum of PEIHA. Furthermore, as shown in Figure 2, the C=O stretching peak around 1725 cm ^-1 disappears due to derivatization, which indicates that the carboxy group has reacted with ethylenediamine and that ethylenediamine has been introduced. It could be confirmed.

<Viscosity measurement>
The viscosity of the hyaluronic acid derivative aqueous solution was measured using a viscoelasticity measuring device (“Rheoplus”, Anton Paar). 100 μL of a 1% by mass aqueous solution of HA3 (ultra-pure water, pH=7) was placed on the stage of a viscoelasticity measuring device (rheometer) and sandwiched between 10 mm diameter jigs. During the measurement, the temperature of the stage was 25°C. The results are shown in Figure 3.

From the results shown in Figure 3, when the shear rate was varied and the viscosity was measured, a decrease in viscosity was observed as the shear rate increased, indicating that hyaluronic acid derivatives have shear thinning properties. That's what I found out.
Furthermore, compared to the viscosity of HA0, which is unmodified hyaluronic acid, the viscosity of HA1 and HA2 was increased. This is thought to be due to the increase in molecular weight due to crosslinking of hyaluronic acid. On the other hand, in HA3, the viscosity decreased, and this is considered to be due to a decrease in intramolecular and intermolecular hydrogen bonds due to the introduction of ethylenediamine.

<Viscoelastic properties>
When hyaluronic acid is dissolved in water at high concentrations, it becomes a highly viscous liquid. HA3 was dissolved in ultrapure water at 5, 7.5, and 10% by mass, and 100 μL was placed on the stage of a rheometer and sandwiched between jigs with a diameter of 10 mm. During the measurement, the temperature of the stage was 25°C. The results are shown in FIGS. 4 and 5.

When the strain was varied while being fixed with a disk-shaped jig with a diameter of 10 mm, it exhibited linear viscoelastic properties in the strain range of 0.1% to 100% (Figure 4). In addition, the shear modulus increases depending on the concentration, and the storage modulus (G') exceeds the loss modulus (G'') at 7.5% by weight or more, indicating that gelation occurs in the high concentration region. On the other hand, when frequency dependence was evaluated, G″ exceeded G′ in the low frequency region, indicating that it was a viscous body and exhibited viscoelastic properties similar to those of the vitreous body (Figure 5). .

<Thixotropy>
Thixotropy is a property in which the viscosity decreases in response to shearing, and the viscosity recovers again when the load is stopped, and is a property that is very suitable for injectable materials using syringes and the like.
Using a viscoelasticity measuring device, changes in shear modulus of a 10% by mass HA3 aqueous solution (ultra pure water, pH=7) with respect to changes in strain were measured. During the measurement, the temperature of the stage was 25° C., and the frequency during measurement was 1 Hz. The elastic modulus was measured when the sample was fixed with a disk-shaped jig with a diameter of 10 mm and the strain was changed from 1% to 300% every 2 minutes. The results are shown in FIG.

As shown in Figure 6, when the strain was increased from 1% to 300%, G' decreased significantly, and when the strain was returned to 1%, G' recovered (Figure 6). Even after repeating this operation multiple times, G' recovered to its original value. This result revealed that the hyaluronic acid derivative hydrogel has thixotropic properties.

<Effects of hyaluronic acid derivatives on cell viability and cell proliferation>
The effects of hyaluronic acid derivatives on cytotoxicity and cell proliferation were evaluated using mouse macrophage-like cells (RAW264.7). RAW264.7 cells were cultured in RPMI1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin) in an incubator at 37°C and 5% _CO2 . 1×10 ⁴ RAW264.7 cells were seeded in a 96-well plate and precultured for 24 hours. HA0 and HA3 were dissolved in cell culture medium at 0.3 to 5 mg/mL, 100 μL was added to each well, and cultured for 3 hours for cell viability evaluation and 24 hours for cell proliferation test.
Furthermore, as controls, a sample to which only the medium was added (control) and a sample to which lipopolysaccharide (LPS), a toxin derived from bacteria that causes inflammation, was added at 100 ng/mL were used.

After completion of the culture, the number of cells was quantified using a cell counting kit (WST-8, DOJINDO). The cell survival rate is shown in FIG. 7, and the degree of proliferation (cell proliferation test results) relative to the number of seeded cells is shown in FIG.

As a result, even at the highest concentration of 5 mg/mL, the cell survival rate was high, indicating high cytocompatibility (FIG. 7). Regarding cell proliferation, a slight decrease in the proliferation rate was observed in the high concentration range.

Moreover, FIG. 8 shows the measurement results of cell survival rate when polyethyleneimine was used instead of ethylenediamine. When polyethyleneimine was used, the cell survival rate did not decrease as in the case where ethylenediamine was used. Note that in FIG. 8, PEIHA means "PEIHA10".

<Effect on cell morphology>
1×10 ⁴ RAW264.7 cells were seeded in a 96-well plate and precultured for 24 hours. LPS was added to HA0 and HA3, which were adjusted to 5 mg/mL in a medium, to 100 ng/mL.
100 μL of this solution was added to each well and cultured for 24 hours. As controls, a sample to which only the medium was added (control) and a sample to which LPS was added at 100 ng/mL were used.
When the morphology of the cells after culture was observed using a phase contrast microscope, it was observed that RAW264.7 cells, which were originally round in shape, were elongated by LPS stimulation (FIG. 10). In the sample to which HA0 and LPS were added at the same time, there was no change in the morphology of the cells, while in the sample using HA3, the morphology of the cells was round and close to that of the control. From this, it was found that HA3 has the effect of alleviating the inflammatory state and bringing it closer to an unstimulated state.
FIG. 11 shows the adhesion area calculated from fluorescence microscopic images of similar samples. From the results in FIG. 11, it was found that LPS+HA3 had a narrower adhesion area and was more suppressed in extension due to LPS stimulation than LPS only and LPS+HA0.

<Evaluation of inflammatory cytokine production>
1×10 ⁴ RAW264.7 cells were seeded in a 96-well plate and precultured for 24 hours. LPS was added to HA0 and HA3, which were adjusted to 5 mg/mL in a medium, to 100 ng/mL. 100 μL of this solution was added to each well and cultured for 24 hours. As controls, a sample to which only the medium was added (control) and a sample to which LPS was added at 100 ng/mL were used.

After culturing, the supernatant was collected, and the concentration of tumor necrosis factor (TNF-α), a type of inflammatory cytokine, contained in the supernatant was determined by enzyme-linked immunosorbent assay (ELISA). As a result, when LPS alone was added, 800 pg/mL of TNF-α was produced, whereas by adding HA3 at the same time, the production of TNF-α was suppressed by more than 3.5 times. (Figure 12). On the other hand, unmodified hyaluronic acid H0 did not show any anti-inflammatory effect. When the concentration dependence of HA0 and HA3 was evaluated using the same method, it was found that HA3 decreased the amount of TNF-α produced as the concentration increased, and showed the strongest anti-inflammatory effect at 5 mg/mL. Ta. On the other hand, no anti-inflammatory effect was observed for HA0 at any concentration (FIG. 13).

Further, FIG. 14 shows the results when specific hyaluronic acid derivatives synthesized using polyethyleneimine (average molecular weight 300) and 1,4-diaminobutane were used in place of ethylenediamine.
In FIG. 14, EDA-HA means that ethylenediamine is used, C4 means that 1,4-diaminobutane is used instead of ethylenediamine, and PEIHA means that 1,4-diaminobutane is used instead of ethylenediamine. It uses polyethyleneimine.
From the results shown in FIG. 14, EDA-HA and PEIHA, in which the number of carbon atoms in L ² in formulas A1 and A2 is 3 or less, are superior to C4, in which the number of carbon atoms is 4. It was found to have the effect of suppressing inflammatory cytokine production.
Note that the vertical axis in FIG. 14 represents the production amount of TNF-α in each sample as a ratio when the production amount of TNF-α in LPS is set to 1.

<Effects of hyaluronic acid derivative production conditions on anti-inflammatory properties>
We evaluated the relationship between the amount of ethanol added when producing hyaluronic acid derivatives and the anti-inflammatory effect. Under the conditions for producing HA3, the amount of ethanol was varied from 0 to 65 mL to synthesize a hyaluronic acid derivative. The amount of TNF-α produced was quantified using the same method used to evaluate the production of inflammatory cytokines.
As a result, almost no anti-inflammatory effect was observed when ethanol was not added, and the highest anti-inflammatory effect was observed when 50 mL of ethanol was added (FIG. 15).

Furthermore, the relationship between added EDA and anti-inflammatory effect was similarly evaluated (FIG. 16). Experiments were conducted using HA1 to HA6 in which the amounts of EDA and EDC added during the preparation of hyaluronic acid derivatives were varied. As a result, stronger anti-inflammatory effects were observed in samples with increased amounts of HA1, HA2, HA3, and EDA.

From the results shown in FIG. 16, it was found that HA3 with an amino group content of 0.10 mmol/g or more has excellent anti-inflammatory properties. It was also found that HA3, which has an amino group content of 0.40 mmol/g or more, has better anti-inflammatory properties than HA1. Furthermore, it was found that HA3 with an amino group content of 1.10 mmol/g had better anti-inflammatory properties.

Relationship between the content of polyethyleneimine in the reaction solution and the inhibitory effect on inflammatory cytokine production when polyethyleneimine is used as a specific amine (However, EDC is fixed at 10 mol% with respect to the carboxy group contained in hyaluronic acid) is shown in FIG.
From FIG. 17, it can be seen that specific hyaluronic acid has a better effect of suppressing inflammatory cytokine production when the amount (molar ratio) of the amino group possessed by polyethyleneimine to the carboxy group possessed by hyaluronic acid is 0.5 (50%) or more. It was found that acid derivatives were obtained.

Similarly, the relationship between the content of EDC in the reaction solution and the inhibitory effect on inflammatory cytokine production (however, polyethyleneimine was fixed at 50 mol% relative to the carboxyl group contained in hyaluronic acid) is shown in Figure 18. Indicated.

<Effects of molecular modifications other than EDA>
In order to evaluate the effect of functional groups modifying hyaluronic acid, ethanolamine was modified instead of EDA and the anti-inflammatory effect was evaluated. Regarding ethanolamine, HA11 was synthesized by adding 30 mol% ethanolamine. Anti-inflammatory properties were evaluated using these hyaluronic acid derivatives. The results are shown in FIG.
As a result of the above, an anti-inflammatory effect was also obtained for HA11.

<Anti-inflammatory effect on primary macrophage cells>
3 × 10 ⁴ mouse bone marrow-derived macrophage (BMDM) cells were seeded in a 96-well plate and precultured for 24 hours. LPS was added to HA0 and PEIHA10, which were adjusted to 5 mg/mL in a medium, or polyethyleneimine (PEI300), which was adjusted to 0.5 mg/mL, to 100 ng/mL. 100 μL of this solution was added to each well and cultured for 24 hours.
Further, as a control, a sample to which LPS was added at 100 ng/mL was used.

After culturing, the supernatant was collected, and the concentration of TNF-α contained in the supernatant was determined by ELISA. The results are shown in FIG. In addition, in FIG. 20, "LPS" means the above control, "PEI" means the one using only polyethyleneimine (average molecular weight 300), and "HA" means the one using hyaluronic acid. "PEIHA" means hyaluronic acid modified with the above-mentioned polyethyleneimine.

As shown in FIG. 20, it was found that PEIHA suppressed TNF-α production compared to PEI and HA used alone.

<Influence on survival rate of primary macrophage cells>
3 × 10 ⁴ BMDMs were seeded in a 96-well plate and precultured for 24 hours. LPS was added to polyethyleneimine (PEI300), HA0, and PEIHA10 prepared in a medium to give a concentration of 100 ng/mL. Note that the concentrations of PEI300 and PEIHA10 solutions were adjusted so that the amount of primary amino groups was 0.3 to 9 mM. HA0 was made to be the same mass % as PEIHA10. 100 μL of this solution was added to each well and cultured for 24 hours. As a control, a sample to which LPS was added at 100 ng/mL was used. After culturing, cell viability was evaluated using the WST-8 method.

As shown in FIG. 21, it was revealed that PEIHA showed a higher cell survival rate than when PEI was used alone.

<Influence on survival rate of primary macrophage cells>
3 × 10 ⁴ BMDMs were seeded in a 96-well plate and precultured for 24 hours. LPS was added to a mixture of PEIHA10 or PEI300 and HA prepared in a medium to give a concentration of 100 ng/mL. Note that the concentrations of PEI300 and PEIHA10 solutions were adjusted so that the amount of primary amino groups was 0.3 to 9 mM. The concentration of HA was the same mass % as PEIHA10. 100 μL of this solution was added to each well and cultured for 24 hours. As a control, a sample to which LPS was added at 100 ng/mL was used. After culturing, cell viability was evaluated using the WST-8 method.

As shown in FIG. 22, it was revealed that both the mixture of PEI and HA (indicated as "PEI+HA" in the figure) and PEIHA exhibited high cell survival rates.

<phagocytosis>
Phagocytosis refers to phagocytosis by phagocytic cells such as macrophages, which are carried out by macrophages to remove foreign substances. The effect of hyaluronic acid derivatives on phagocytosis was investigated using fluorescent beads (fluorescein immobilized on microparticles, particle size 100 nm). 1×10 ⁴ RAW264.7 cells were seeded in a 96-well plate and precultured for 24 hours.
Fluorescent beads were added to HA0 and HA3 adjusted to 5% by weight in a medium, 100 μL of each well was added, and cultured for 24 hours. As a control, a sample to which only the fluorescent bead suspension was added (control) was used. After culturing, each well was washed with phosphate buffered saline (PBS) and observed under a fluorescence microscope (in the upper row of FIG. 23, the white part on the image is the fluorescently stained part).
Fluorescently stained areas were seen in each cell in the control, but not in the HA3-added sample.

In addition, the cells were lysed by adding 100 μL of 1% sodium dodecyl sulfate/0.1M NaOH, and the fluorescence intensity of each well was calculated using a fluorescence microplate reader (lower row of FIG. 23). As a result, phagocytosis was significantly suppressed in the sample containing HA0 and HA3 compared to the control (lower panel of FIG. 23). These results suggested that hyaluronic acid derivatives exert anti-inflammatory effects by suppressing macrophage function.

<Effects of hyaluronic acid derivatives on cell viability and cell proliferation>
To evaluate cellular uptake of hyaluronic acid derivatives, fluorescently labeled HA3 was produced. In the process of synthesizing HA3, when EDA was added, fluorescein amine (0.02 mmol) was added at the same time. A fluorescent dye can be introduced into hyaluronic acid by the reaction between the amino group of fluorescein amine and the carboxy group of hyaluronic acid. The obtained fluorescent HA3 was dissolved in a medium and added to RAW264.7 (1× ¹⁰ cells seeded and precultured for 24 hours) at a concentration of 0.6 to 5 mg/mL. After culturing, each well was washed with PBS, the cells were lysed by adding 100 μL of 1% sodium dodecyl sulfate/0.1M NaOH, and the fluorescence intensity of each well was calculated using a fluorescence microplate reader. As a result, it was confirmed that the amount of uptake increased as the concentration of fluorescent HA3 increased (FIG. 24).

<Preparation of hydrogel by crosslinking>
Sulfonated nanocellulose (sNC) containing aldehyde groups was used to improve the mechanical strength of the hydrogel. Since the aldehyde group of sNC and the amino group introduced into the hyaluronic acid derivative are intermolecularly bonded by imine formation, it is possible to crosslink the hyaluronic acid derivative with sNC. A hydrogel was prepared by quickly mixing 50 μL of 2 mass % sNC solution (ultrapure water, pH = 8) and 5 mass % HA3 solution (0.1 M boric acid buffer, pH = 9), and its viscoelastic The properties were evaluated using a viscoelasticity measuring device. 100 μL of the pregel solution was placed on the stage of a viscoelasticity measuring device, fixed with a disk-shaped jig with a diameter of 10 mm, and measurement was performed at 25 ^° C., strain of 1%, and frequency of 1 Hz. As a result, it was observed that the storage modulus G' (black circle) of the solution increased over time, confirming that gelation had occurred (FIG. 25). After about 2 hours, G' reached a plateau, indicating that the imine formation reaction was completed. Note that the loss modulus G'' is indicated by a white circle in the figure.

This acid derivative can form an injectable gel that exhibits high safety and anti-inflammatory properties, and is very useful for medical purposes as an anti-inflammatory material.

Claims (7)

It has a repeating unit represented by the following formula 1, has a number average molecular weight of 600,000 to 1,200,000, and has an amino group content of 0.10 to 1.10 mmol/g in R ¹ of formula 1. A certain hyaluronic acid derivative or a pharmaceutically acceptable salt thereof.

(In formula 1, R ¹ is a group represented by formula 1-1 or a group obtained by removing one arbitrary terminal amino group of polyethyleneimine having a molecular weight of 100 to 1200, and * represents the bonding position. .) The hyaluronic acid derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the polyethyleneimine has a molecular weight of 200 to 1000. The hyaluronic acid derivative according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the content of amino groups contained in R ¹ is 0.40 mmol/g or more. The hyaluronic acid derivative according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the content of amino groups contained in R ¹ is 0.50 mmol/g or more. The hyaluronic acid derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein R ¹ is a group represented by formula 1-1. The hyaluronic acid derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein R ¹ is a group obtained by removing one arbitrary terminal amino group of the polyethyleneimine. A method for producing a hyaluronic acid derivative according to any one of claims 1 to 6, comprising:
In an aqueous solution containing alcohol, in the presence of a carboxyl group activator, hyaluronic acid or a salt thereof and at least one amine selected from the group consisting of ethylenediamine and polyethyleneimine are bonded by an amidation reaction. A method for producing a hyaluronic acid derivative, comprising: