Pharmaceutical composition

Abstract

The present invention provides a pharmaceutical composition capable of accelerating the regeneration rate of damaged areas in nerves. [Solution] A pharmaceutical composition for promoting nerve regeneration, comprising (A) a hyaluronic acid derivative into which a cholesteryl group has been introduced, (B) one or more factors selected from the group consisting of secretory sialic acid-binding immunoglobulin-like lectin-9 and monocyte chemotactic factor-1, and (C) a cell adhesion material derived from collagen; the average molecular weight of the hyaluronic acid derivative into which the cholesteryl group has been introduced is 5,000 or more and 50,000 or less; and a method for promoting nerve regeneration, comprising contacting any of the pharmaceutical compositions with a damaged site in nerve tissue.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for promoting nerve regeneration, which uses one or more regenerative factors selected from the group consisting of secreted Siglec (sialic acid-binding immunoglobulin-like lectin)-9 and monocyte chemotactic factor-1 (MCP-1), and uses hyaluronic acid derivatives and collagen-derived materials as retaining materials for the regenerative factors and as scaffold materials. [Background technology]

[0002] Regenerative medicine using stem cells is attracting attention as a versatile alternative technology for diseases that are difficult to treat with conventional medicine. There are many diseases to which regenerative medicine is applicable or expected to be effective, and numerous studies are being conducted toward clinical application. Neurological diseases, particularly intractable neurological diseases such as spinal cord injury, are among those for which treatment with regenerative medicine is expected.

[0003] While human fetal or ES cell-derived neural stem cells are considered as resources for regenerative medicine, they present significant ethical and safety concerns. Therefore, attempts are being made to use bone marrow and adipose-derived stem cells as in vivo stem cells. Dental pulp stem cells collected from deciduous or permanent teeth are also a useful resource for regenerative medicine (Patent Document 1).

[0004] In regenerative medicine for damaged tissue, in addition to directly using stem cells, it has been reported that regeneration is more likely to progress by inducing proliferation and differentiation or suppressing inflammation in damaged tissue and surrounding cells. For example, it has been reported that secreted Siglec-9 has the function of suppressing the differentiation of bone marrow cells and macrophages into osteoclasts, increasing bone density, and suppressing inflammation in fibroblasts, and that administration of secreted Siglec-9 can be applied to the treatment of fractures (Patent Document 2). Furthermore, it has been reported that a combination of three components, secreted Siglec-9, MCP-1, and chondroitin sulfate or chondroitin sulfate proteoglycan, contributes to the repair of damaged tissue where an inflammatory response is occurring (Patent Document 3).

[0005] On the other hand, a method is known in which hyaluronic acid derivatives are used as carriers in the administration of various drugs. Hyaluronic acid derivatives spontaneously associate in aqueous solutions and can efficiently encapsulate poorly water-soluble drugs while maintaining their biological activity (Patent Document 4). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2011-219432 [Patent Document 2] International Publication No. 2023 / 033130 [Patent Document 3] International Publication No. 2014 / 098249 [Patent Document 4] International Publication No. 2010 / 053140 [Overview of the project] [Problems that the invention aims to solve]

[0007] Conventional tissue regeneration methods have faced challenges in terms of regeneration speed. Therefore, the present invention aims to provide a pharmaceutical composition that can accelerate the regeneration speed of nerve damage sites by using one or more regenerative factors selected from the group consisting of secreted Siglec-9 and MCP-1, and by using hyaluronic acid derivatives and collagen-derived materials as retaining materials for the regenerative factors and as scaffold materials. [Means for solving the problem]

[0008] In other words, the present invention includes the following embodiments. [1] A pharmaceutical composition for promoting nerve regeneration, (A) Hyaluronic acid derivatives into which a cholesteryl group has been introduced, (B) One or more factors selected from the group consisting of secreted sialic acid-binding immunoglobulin-like lectin-9 and monocyte chemotactic factor-1, and (C) Collagen-derived cell adhesion material A pharmaceutical composition containing the hyaluronic acid derivative having the (A) cholesteryl group introduced therein and having one or more repeating units represented by the following general formula (I).

[0009]

Chemical formula

[0010] (In the formula, R 1 , R 2 , R 3 , and R 4 are each independently a group selected from the group consisting of a hydrogen atom, C 1-6 alkyl, formyl, and C 1-6 alkylcarbonyl. Z represents a direct bond or a peptide linker consisting of two or more and 30 or less arbitrary amino acid residues. X 1 is a group selected from the group consisting of -NR b -R, -NR b -COO-R, -NR b -CO-R, -NR b -CO-NR c -R, -COO-R, -O-COO-R, -S-R, -CO-Y a -S-R, -O-CO-Y b -S-R, -NR b -CO-Y b -S-R, and -S-S-R. R a , R b and R c are each independently a group selected from the group consisting of a hydrogen atom, C 1-20 alkyl, amino C 2-20 alkyl, and hydroxy C 2-20 alkyl. The alkyl moieties of R a , R b and R c may have a group selected from the group consisting of -O- and -NR f - inserted therein. R f is a hydrogen atom, C 1-12 alkyl, amino C 2-12 alkyl, and hydroxy C2-12 It is a group selected from the group consisting of alkyl groups. f The alkyl portion may have a group selected from the group consisting of -O- and -NH- inserted into it. R is a cholesteryl group. Y is C 2-30 Alkylene, or -(CH2CH2O) m It is -CH2CH2-. Here, the alkylenes of Y are -O- and -NR g A group selected from the group consisting of - and -SS- may be inserted. R g C is a hydrogen atom. 1-20 Alkyl, amino C 2-20 Alkyl and hydroxy C 2-20 It is a group selected from the group consisting of alkyl groups. g The alkyl portion may have a group selected from the group consisting of -O- and -NH- inserted into it. Y a C 1-5 It is alkylene. Y b C 2-8 Alkylene or C 2-8 It is alkenylene. m is an integer between 1 and 100 (inclusive). [2] The pharmaceutical composition according to [1], wherein the average molecular weight of the hyaluronic acid derivative to which the (A) cholesteryl group is introduced is 5,000 or more and 50,000 or less. [3] The pharmaceutical composition according to [1] or [2], wherein the cholesteryl group introduction rate of the hyaluronic acid derivative into which the cholesteryl group of (A) has been introduced is 8% or more and 36% or less. [4] Any of the pharmaceutical compositions of [1] to [3], wherein the (C) cell adhesion material is a porous material. [5] A method for promoting nerve regeneration, comprising bringing one of the pharmaceutical compositions described in [1] to [4] into contact with a site of injury in nerve tissue. [Effects of the Invention]

[0011] According to the present invention, it is possible to provide a pharmaceutical composition that can accelerate the regeneration rate of nerve damage sites. [Brief explanation of the drawing]

[0012] [Figure 1] This figure shows the changes in behavioral evaluation scores over time for facial nerve injury model mice in Example 1, specifically for the CS group (group with collagen sponge only), the CS+CHHA group (group administered collagen sponge and CHHA), the CS+M / S group (group administered collagen sponge, secretory Siglec-9, and MCP-1), and the CS+CHHA+M / S group (group administered collagen sponge, CHHA, secretory Siglec-9, and MCP-1). [Figure 2] The images in Example 1 show photographs of the facial nerve injury site in each group of mice 8 weeks after treatment. [Modes for carrying out the invention]

[0013] The following describes in detail an embodiment of the present invention (hereinafter referred to as "this embodiment"), but the present invention is not limited thereto, and various modifications are possible without departing from its essence.

[0014] The following is a definition of terms used in this specification.

[0015] The term "C" used in this specification 1-20 The term "alkyl" refers to linear or branched alkyl groups having 1 to 20 carbon atoms, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, etc. 1-4 It contains alkyl, and further includes n-pentyl, 3-methylbutyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, n-hexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-ethylbutyl, 2-ethylbutyl, etc. 1-20 Alkyl compounds have 1 to 12 carbon atoms. 1-12 Alkyl, C with 1 to 6 carbon atoms 1-6 Alkyl groups are also included.

[0016] The term "C" used in this specification 1-6 The term "alkylcarbonyl" refers to the alkyl part which has already been mentioned as C. 1-6 This refers to alkylcarbonyl groups, such as acetyl, propionyl, n-propylcarbonyl, iso-propylcarbonyl, n-butylcarbonyl, sec-butylcarbonyl, iso-butylcarbonyl, tert-butylcarbonyl, etc. 1-4 It contains "alkylcarbonyl".

[0017] The term "amino C" used in this specification 2-20 The term "alkyl" refers to a linear or branched alkyl group having 2 to 20 carbon atoms and having an amino group as a substituent. For example, the amino group may be located on the terminal carbon atom of the alkyl group. 2-20 Alkyl compounds include amino acids with 2 to 12 carbon atoms. 2-12 Alkyl compounds are also included.

[0018] The term "hydroxy C" used in this specification 2-20 The term "alkyl" refers to a linear or branched alkyl group having 2 to 20 carbon atoms and having a hydroxyl group as a substituent. For example, the hydroxyl group may be located on the terminal carbon atom of the alkyl group. 2-20 Alkyl compounds include hydroxyl atoms with 2 to 12 carbon atoms. 2-12 Alkyl compounds are also included.

[0019] The term "C" used in this specification 2-30 The term "alkylene" refers to a linear or branched divalent saturated hydrocarbon group having 2 to 30 carbon atoms, including, for example, ethylene and propylene, and having 2 to 20 carbon atoms. 2-20 Alkylene, C with 2 to 8 carbon atoms 2-8 Alkylene, base "-(CH2)" n - (where n is between 2 and 30, preferably between 2 and 20, and more preferably between 2 and 15).

[0020] The term "C" used in this specification 1-5 The term "alkylene" refers to a linear or branched divalent saturated hydrocarbon group having 1 to 5 carbon atoms, including, for example, methylene, ethylene, propylene, butylene, pentylene, etc. The term "C" as used in this specification refers to a linear or branched divalent saturated hydrocarbon group having 1 to 5 carbon atoms. 1-6 The term "alkylene" refers to a linear or branched divalent saturated hydrocarbon group having 1 to 6 carbon atoms, and includes, for example, methylene, ethylene, propylene, butylene, pentylene, hexylene, etc.

[0021] The term "C" used in this specification 2-8 The term "alkenylene" refers to a divalent saturated hydrocarbon group having 2 to 8 carbon atoms, either linear or branched, containing one or more double bonds. Examples include -CH=CH-, -C(CH3)=CH-, 2-butene-1,4-diyl, hepta-2,4-diene-1,6-diyl, octa-2,4,6-triene-1,8-diyl, etc. If geometric isomerism exists, each isomer and mixtures thereof are also included.

[0022] <Pharmaceutical composition> The pharmaceutical composition according to this embodiment is a pharmaceutical composition for promoting nerve regeneration, and comprises (A) a hyaluronic acid derivative into which a cholesteryl group has been introduced, (B) one or more factors selected from the group consisting of secretory Siglec-9 and MCP-1, and (C) a cell adhesion material derived from collagen. In the pharmaceutical composition according to this embodiment, the hyaluronic acid derivative into which a cholesteryl group has been introduced is used as a scaffold material when administering factor (B) to the target nerve tissue to be regenerated. By retaining factor (B) in the hyaluronic acid derivative into which a cholesteryl group has been introduced, the sustained release and stability in vivo are improved, so that nerve regeneration by the factor is further promoted and a higher nerve regeneration effect can be obtained.

[0023] In the pharmaceutical composition of this embodiment, (A) the hyaluronic acid derivative forms a complex with factor (B) (hereinafter sometimes referred to as the "factor-hyaluronic acid derivative complex"). Specifically, the cholesteryl group in the hyaluronic acid derivative and the hydrophobic portion of factor (B) form a complex through hydrophobic interaction. The form of the complex is not particularly limited and is appropriately determined by the size of the hyaluronic acid derivative, the type and size of factor (B), the content ratio of the hyaluronic acid derivative to factor (B), etc. For example, the complex may exhibit a cylindrical structure in which factor (B) and the cholesteryl group are located in the center and hydrophilic portions such as the hyaluronic acid-derived portion in the hyaluronic acid derivative are located on the outer edge, that is, the complex may exhibit a structure in which factor (B) is encapsulated or contained within the hyaluronic acid derivative. Alternatively, the hyaluronic acid derivative and the active ingredient may be intertwined with each other, and factor (B) may be amorphous (non-crystalline structure).

[0024] In the pharmaceutical composition of this embodiment, the average particle size of the structure containing the factor-hyaluronic acid derivative complex is not particularly limited, but can be 10 nm or more, preferably 20 nm or more, and more preferably 30 nm or more. In the pharmaceutical composition of this embodiment, the average particle size of the structure can be 500 nm or less, preferably 400 nm or less, more preferably 300 nm or less, even more preferably 220 nm or less, and can be 150 nm or less, and can be 100 nm or less. When the average particle size is within the above numerical range, sterile filtration is possible, the factor-hyaluronic acid derivative complex can exist stably in the body, and the effect of the hyaluronic acid derivative as a drug base or scaffold material can be more strongly exhibited.

[0025] The average particle size of a structure containing a factor-hyaluronic acid derivative complex can be measured by methods such as DLS (Dynamic Light Scattering), nanotracking particle analyzers, size exclusion chromatography, high-performance liquid chromatography, and electron microscopy. More specifically, for example, the hyaluronic acid derivative is diluted in a 10 mM phosphate buffer containing 10 w / v% sucrose to a concentration of 1 mg / mL using a DLS instrument, and the 50% cumulative value of the resulting particle size distribution (cumulative distribution curve) is determined as the average particle size.

[0026] Next, the components of this embodiment will be described in detail below.

[0027] ≪Hyaluronic acid derivatives with added cholesteryl groups≫ The pharmaceutical composition of this embodiment includes (A) a hyaluronic acid derivative into which a cholesteryl group has been introduced. Hereafter, "(A) a hyaluronic acid derivative into which a cholesteryl group has been introduced" may be referred to as "(A) a hyaluronic acid derivative".

[0028] The cholesteryl group may be directly bonded to hyaluronic acid or bonded via a linker. Here, "linker" refers to a group in which a group that reacts with the carboxyl group and a group that reacts with the cholesteryl group are linked by a linear group (spacer). Examples of groups that react with the carboxyl group and the cholesteryl group include amino groups and hydroxyl groups. As the linear group, for example, a linear hydrocarbon group, a polyethylene glycol (PEG) chain, any peptide linker that can be introduced by genetic engineering, or a synthetic compound linker can be used. The length of the linker is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose. Examples of linear hydrocarbon groups include C 1-30 Alkylene is preferred, C 1-10 Alkylene is more preferred, C 1-6Alkylenes are more preferred. The PEG group is preferably one with 1 to 15 linked ethylene glycol groups, more preferably one to 10, and even more preferably one to 5. The peptide linker is 2 or more amino acids (the upper limit is not particularly limited, but is usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. In the hyaluronic acid derivative of this embodiment, it is preferable that at least a portion of the carboxyl groups in the glucuronic acid portion are linked to cholesteryl groups by a linker having a chain-like hydrocarbon group.

[0029] [Cholesteryl group introduction rate] (A) The rate of introduction of cholesteryl groups into the hyaluronic acid derivative (hereinafter sometimes simply referred to as "cholesteryl group introduction rate") is preferably 5% to 36%. When the cholesteryl group introduction rate is within the above range, (A) the hyaluronic acid derivative can strongly interact with the hydrophobic portion of poorly water-soluble drugs.

[0030] The rate of introduction of cholesteryl groups is 1 It can be measured by 1H-NMR measurement. That is, the pharmaceutical composition 1 The integral value of the peak originating from the cholesteryl group of (A) hyaluronic acid derivative in the 1H-NMR spectrum and the integral value of the peak originating from the acetyl group of N-acetyl-D-glucosamine contained in (A) hyaluronic acid derivative (COCH3, 1.6 ppm to 2.0 ppm, 3H) can be used to calculate the following based on the formula below. Note that in the formula, n H This represents the number of hydrogen atoms corresponding to the peak. Specifically, it can be measured according to the method described in the examples below.

[0031] [Cholesteryl group introduction rate] (%) =[(Peak integral value derived from cholesteryl group × 3 / n H ) / (Peak integral value derived from the acetyl group of N-acetyl-D-glucosamine)] × 100

[0032] [Cholesteryl group] (A) When the hyaluronic acid derivative contains a cholesteryl group, the cholesteryl group in the hyaluronic acid derivative self-associates in water, and single or multiple molecules associate to form a nano-sized hydrogel.

[0033] (A) The cholesteryl group introduction rate of the hyaluronic acid derivative is preferably 5% to 36%, more preferably 8% to 36%, and (B) even more preferably 12% to 36% from the viewpoint that the sustained release due to the delayed diffusion of factor (B) is better.

[0034] (A) The average molecular weight of the hyaluronic acid derivative is not particularly limited and can be adjusted as appropriate depending on the dosage form. For example, from the viewpoint of improving the sustained-release function derived from the delayed diffusion of factor (B), a hyaluronic acid derivative (A) with a relatively large molecular weight is preferred, preferably between 5,000 (5k) and 50,000 (50k). In addition, if the final dosage form is a solution formulation, a hyaluronic acid derivative (A) with a relatively small molecular weight is preferred from the viewpoint of syringeability, for example, it can be between 5,000 (5k) and 40,000 (40k).

[0035] The "average molecular weight of hyaluronic acid derivatives" referred to here is the weight-average molecular weight determined by size exclusion chromatography multi-angle light scattering detector (SEC-MALS). The weight-average molecular weight of hyaluronic acid derivatives can generally be adjusted by using raw materials with corresponding molecular weights.

[0036] Preferred (A) hyaluronic acid derivatives include, for example, hyaluronic acid derivatives having one or more repeating units represented by the following general formula (I) (hereinafter sometimes referred to as "repeating unit (I)").

[0037] [ka]

[0038] (In the formula, R 1, R 2 , R 3 , and R 4 is, independently of each other, a group selected from the group consisting of a hydrogen atom, C 1-6 alkyl, formyl and C 1-6 alkylcarbonyl. Z represents a direct bond or a peptide linker consisting of any amino acid residues of 2 or more and 30 or less. X 1 is -NR b -R, -NR b -COO-R, -NR b -CO-R, -NR b -CO-NR c -R, -COO-R, -O-COO-R, -S-R, -CO-Y a -S-R, -O-CO-Y b -S-R, -NR b -CO-Y b -S-R, and -S-S-R, and is a group selected from the group consisting of the represented groups. R a , R b and R c are, independently of each other, a group selected from the group consisting of a hydrogen atom, C 1-20 alkyl, amino C 2-20 alkyl and hydroxy C 2-20 alkyl. Here, the alkyl moiety of R a , R b and R c may have a group selected from the group consisting of -O- and -NR f - inserted therein. R f is a group selected from the group consisting of a hydrogen atom, C 1-12 alkyl, amino C 2-12 alkyl and hydroxy C 2-12 alkyl. The alkyl moiety of R f may have a group selected from the group consisting of -O- and -NH- inserted therein. R is a cholesteryl group. Y is C 2-30 alkylene, or -(CH2CH2O) mIt is -CH2CH2-. Here, the alkylenes of Y are -O- and -NR g A group selected from the group consisting of - and -SS- may be inserted. R g C is a hydrogen atom. 1-20 Alkyl, amino C 2-20 Alkyl and hydroxy C 2-20 It is a group selected from the group consisting of alkyl groups. g The alkyl portion may have a group selected from the group consisting of -O- and -NH- inserted into it. Y a C 1-5 It is alkylene. Y b C 2-8 Alkylene or C 2-8 It is alkenylene. m is an integer between 1 and 100 (inclusive).

[0039] (A) The hyaluronic acid derivative preferably includes a hyaluronic acid derivative having one or more repeating units represented by the following general formula (Ia) (hereinafter sometimes referred to as "repeating unit (Ia)").

[0040] [ka]

[0041] (In the formula, R 1 , R 2 , R 3 , and R 4 These are, independently, hydrogen atoms and C 1-6 Alkyl, formyl and C 1-6 It is a group selected from the group consisting of alkylcarbonyl groups. X is -NR a -Y-NR b R is a hydrophobic group represented by -COO-R. a and R b These are, independently, hydrogen atoms and C 1-6 It is a group selected from the group consisting of alkyl groups. R is a cholesteryl group. Y is C 2-30 Alkylene, or -(CH2CH2O)m The equation is -CH2CH2-, where m is an integer between 1 and 100 (inclusive).

[0042] Here, if the hyaluronic acid derivative contains two or more repeating units (I) or repeating units (Ia), these repeating units may be the same or different.

[0043] Hyaluronic acid derivatives may be modified at positions other than the repeating unit (I) or repeating unit (Ia), for example, the hydroxyl group may be -O(C 1-6 Alkyl), -O (formyl), -O (C 1-6 The carboxyl group may be converted to an alkylcarbonyl group, and the carboxyl group may be converted to an amide or ester, and a salt may be formed.

[0044] [Repeating Unit (I)] The base "-Zn(R)" in general formula (I) a )YX 1 The expression is as follows: -NH-(CH2) mz -NH-R; -NH-(CH2) mz -NH-COO-R; -NH-(CH2CH2O) m -CH2CH2-NH-COO-R; -NH-(CH2) mz -COO-R; -NH-(CH2CH2O) m -CH2CH2-COO-R, -NH-(CH2) mz -O-COO-R; -NH-(CH2CH2O) m -CH2CH2-O-COO-R, -NH-(CH2) mz -SR; -NH-(CH2CH2O) m -CH2CH2-SR; -NH-(CH2) mz -O-CO-CH(R 8 )-CH2-SR; -NH-(CH2) mz -NHCO-CH(R 8 )-CH2-SR; -NH-(CH2CH2O) m -CH2CH2-NHCO-CH(R 8 )-CH2-SR; -NH-(CH2CH2O) m -CH2CH2-O-CO-CH(R 8 )-CH2-SR; -NH-(CH2) mz -SSR; and -Z-NR a -Y-NR b -COO-R (Here, mz is an integer between 2 and 30, and R 8 (where R is a hydrogen atom or a methyl group, and R and m are as already defined in this specification.) It includes a group selected from the group consisting of groups represented by . The group in question is, -NH-(CH2) mz -NH-COO-R; -NH-(CH2CH2O) m -CH2CH2-NH-COO-R; and -NH-(CH2) mz -SSR (Here, mz, R, and m are as already defined in this specification.) A group selected from the group consisting of is preferred.

[0045] (Z) In general formula (I), Z is preferably a direct bond. In another embodiment, when Z is a peptide linker, X 1 -NR b It is preferable that -COO-R. Furthermore, in another embodiment, Z is -NH-[CH(-Z a )-CONH] n-1 -CH(-Z a It may also be a peptide linker represented by )-CO-, where n is an integer between 2 and 30, and Z aEach of these is independently H2N-CH(-Z a This represents a substituent in an α-amino acid, represented as )-COOH. The peptide linker is bonded to the carboxyl group of the glucuronic acid moiety at the N-terminus and to the group -N(-R) at the C-terminus. a )-YX 1 It binds to. Examples of amino acids that can be used as amino acid residues of the peptide linker include α-amino acids, such as alanine, arginine, asparagine (Asn), aspartic acid, cysteine, glutamine, glutamic acid, glycine (Gly), histidine, isoleucine, leucine (Leu), lysine, methionine, phenylalanine (Phe), proline, serine, threonine, tryptophan, tyrosine, and valine, as well as their D forms, and all α-amino acids, including synthesized amino acids, can be used. That is, Z a Examples of these include -CH3, H2NC(NH)NH(CH2)3-, H2NCOCH2-, etc. Also, the n Zs may be the same or different. n is an integer between 2 and 30, but is preferably between 2 and 10, and more preferably between 2 and 4. Preferred examples of peptide linkers include, for example, -Gly-Phe-Leu-Gly-, -Asn-Phe-Phe-, -Phe-Phe-, Phe-Gly-, etc.

[0046] (Y) In general formula (I), Y is -(CH2) n1 -and-(CH2CH2O) m1 A base selected from the group consisting of -CH2CH2- (where n1 is an integer between 2 and 20, preferably between 2 and 15, more preferably between 2 and 12, and even more preferably between 2 and 6; m1 is an integer between 1 and 4) is preferred. Specifically, -(CH2)2-, -(CH2)6-, -(CH2)8-, -(CH2) 12-, or -(CH2CH2O)2-CH2CH2- is preferred. Furthermore, from the viewpoint of achieving high solubility in pure water or low salt concentrations while exhibiting high precipitation ability under physiological salt concentrations, Y is preferably -(CH2)2-, -(CH2)6-, -(CH2)8- and -(CH2) 12 A group selected from the group consisting of - is preferred, and -(CH2)6- is more preferred.

[0047] Y can be, for example, -CH2CH2O-CH2CH2-SS-CH2CH2O-CH2CH2-, -(CH2CH2O)2-CH2CH2-SS-CH2CH2O-CH2CH2-, -CH2CH2O-CH2CH2-SS-(CH2CH2O)2-CH2CH2-, -(CH2CH2O)2-CH2CH2-SS-(CH2CH2O)2-CH2CH2-, etc.

[0048] (Y a ) Y a -CH2- or -CH2-CH2- is preferred.

[0049] (Y b ) Y b The preferred members are -CH2-CH2-, -CH(CH3)CH2-, 2-butene-1,4-diyl, hepta-2,4-diene-1,6-diyl, or octa-2,4,6-triene-1,8-diyl, with -CH2-CH2- or -CH(CH3)CH2- being more preferred.

[0050] Base "-ZN(R a )YX 1Specific examples of "" include -NH-(CH2)2-NH-CO-cholesteryl, -NH-(CH2)4-NH-(CH2)3-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-N(-(CH2)3-NH2)-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(C Examples include H2)3-NH2)-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH-(CH2)3-NH2)-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH2)-CO-NH-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH2)-CO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(CH2)3-NH2)-cholesteryl, etc. Preferred group "-Zn(R a )YX 1 " is R a , R b and R c However, it is a hydrogen atom, and Y is a linear C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2- and Y a However, linear C 1-5 It is alkylene, or Y b However, linear C 2-8 Alkylene or linear carbon 2-8 It is alkenylene.

[0051] [Repeating Unit (Ia)] In general formula (Ia), X is -NH-(CH2)2-NH-COO-cholesteryl, -NH-(CH2)6-NH-COO-cholesteryl, -NH-(CH2) 12-NH-COO-cholesteryl or -NH-(CH2CH2O)2-CH2CH2-NH-COO-cholesteryl is preferred, and -NH-(CH2)2-NH-COO-cholesteryl, -NH-(CH2)6-NH-COO-cholesteryl, or -NH-(CH2CH2O)2-CH2CH2-NH-COO-cholesteryl is more preferred.

[0052] The content of (A) hyaluronic acid derivative relative to the total amount of the pharmaceutical composition of this embodiment is preferably 0.001 wt% or more and less than 10 wt%, more preferably 0.01 wt% or more and 2 wt%, and even more preferably 0.1 wt% or more and 2 wt%.

[0053] (Method for producing hyaluronic acid derivatives) The (A) hyaluronic acid derivative to be contained in the pharmaceutical composition of this embodiment can be obtained, for example, by converting the carboxyl group of glucuronic acid to an amide and introducing a cholesteryl group directly or via a linker to at least a portion of it. The rate of cholesteryl group introduction can be controlled by adjusting the amount of the compound having a cholesteryl group to be reacted with the raw material hyaluronic acid or its derivative. The method for introducing a cholesteryl group into the raw material hyaluronic acid can be carried out by appropriately modifying the methods described in, for example, Japanese Patent Publication No. 2021-123597, Japanese Patent Publication No. 2022-013861, Japanese Patent Publication No. 2022-044579, etc.

[0054] A specific method for converting the carboxyl group of glucuronic acid to an amide and introducing a cholesteryl group is, for example, to ion-exchange a starting material hyaluronic acid or a derivative thereof with a tetraalkylammonium salt (e.g., tetrabutylammonium (TBA) salt), and then react the hyaluronic acid salt with an amine to which a cholesteryl group has been introduced in a solvent in the presence of a suitable condensing agent.

[0055] The coupling agent that can be used in the above reaction is not particularly limited and includes, for example, 4-(4,6-dimethoxy-1,3,5-triazine)-4-methylmorphorium (DMT-MM), N,N'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 2-benzotriazole-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HODhbt), benzotriazole-1-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), and benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium Examples include hexafluorophosphate (BOP), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS).

[0056] In particular, although not limited to, DMT-MM is preferred because the reaction proceeds with high efficiency even in a mixed solvent of water and an organic solvent. Furthermore, by using DMT-MM as a condensing agent, it is possible to selectively form amide bonds between amino groups and carboxyl groups while suppressing ester bond formation in systems where many hydroxyl groups coexist. By using this condensing agent, it is possible to prevent, for example, the reaction of the solvent alcohol with the carboxyl groups of the hyaluronic acid portion, or the formation of unwanted crosslinks by intramolecular or intermolecular bonding between carboxyl groups and hydroxyl groups simultaneously present in the hyaluronic acid portion.

[0057] Solvents used in the cholesteryl group introduction reaction include water, DMSO, methanol, ethanol, propanol, butanol, isopropanol, polyhydric alcohols, acetonitrile, DMF, THF, dichloromethane, chloroform, hexane, diethyl ether, ethyl acetate, and mixtures thereof. The polyhydric alcohol may be a dihydric alcohol or a trihydric alcohol. Examples of dihydric alcohols include ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, neopentyl glycol, 1,4-butanediol, and 1,6-hexanediol. Examples of trihydric alcohols include glycerin and trimethylolpropane.

[0058] Alternatively, the raw material hyaluronic acid or its derivative may be ion-exchanged with a tetraalkylammonium salt (e.g., tetrabutylammonium (TBA) salt), and the hyaluronic acid salt and the spacer portion may be reacted in a solvent in the presence of a suitable condensing agent (protection and deprotection reactions may be performed as needed) to convert the carboxyl group (-COOH) of the raw material hyaluronic acid or its derivative, and then reacted with a suitable reagent. Examples of combinations of groups derived from the carboxyl group and reaction reagents are shown below. -CONR a -Y-NR b H + Hal-R; -CONR a -Y-NR b H + Hal-COOR; -CONR a -Y-NR b H + HOCO-R; -CONR a -Y-NR b H + Hal-CO-R; -CONR a -Y-NR b -COOH + HNR c -R; -CONR a -Y-NR b -CO-NR c H + Hal-R; -CONR a -Y-NR b H + HOCO-NR c -R; -CONR a -Y-NR b H + Hal-CO-NR c -R; -CONR a -Y-COOH + HO-R; -CONR a -Y-OH + Hal-COO-R; -CONR a -Y-OCOOH + HO-R; -CONR​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​(wherein Z is as already defined in this specification, and Hal represents a halogen atom selected from the group consisting of fluorine, chlorine, bromine, and iodine).

[0059] Reaction modes include dehalogenation reactions, condensation reactions, dehydration reactions, nucleophilic addition reactions such as Michael addition, and oxidative disulfide formation reactions. These are well-known reactions and can be appropriately selected by those skilled in the art, and carried out under preferred reaction conditions. If the converted product or reactant has a carboxyl group, it may be reacted with an N-hydroxysuccinimide (hereinafter also referred to as "NHS") ester.

[0060] Another method involves reacting the carboxyl group of hyaluronic acid or its derivative with 2-aminoethyl 2-pyridyl disulfide to prepare a hyaluronic acid derivative into which a spacer having a mercapto group modified with a leaving group at its terminal end is introduced, and then forming a disulfide bond by nucleophilic substitution of thiocholesterol with this derivative.

[0061] Furthermore, a method can be described in which a portion of the spacer is introduced into the carboxyl group of hyaluronic acid or its derivative, and a portion of the spacer is introduced into the cholesteryl group, and these are reacted. Some specific examples have been described above, but further, if -SS- is inserted into Y, a method can be described in which a hyaluronic acid derivative in which a spacer having a mercapto group at the terminal is introduced into the carboxyl group of hyaluronic acid, and a cholesteryl group in which a spacer having a mercapto group at the terminal is introduced, and these are reacted oxidatively to form a disulfide bond. In this case, one mercapto group can be reacted with 2-mercaptopyridine to form a disulfide, and then substituted with the other mercapto group.

[0062] Furthermore, other substituents may be introduced after the preparation of the hyaluronic acid derivative. For example, 0.1% to 95.0%, preferably 10% to 60%, of the carboxyl groups in the hyaluronic acid derivative may be -CO-X z [Here, X zis the following group: -NH-(CH2) p1 -O-CO-C(R 17 )=CH2; -NH-(CH2) p1 -O-CO-CH(R 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH; -NH-(CH2) p1 -SH; -NH-(CH2) p1 -NH-CO-C(R 17 )=CH2; -NH-(CH2) p1 -NH-C(=NH)-(CH2)3-SH; -NH-(CH2) p1 -NH-CO-(CH2) r -SH; -NH-(CH2) p1 -NH-CO-CH(R 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH; -NH-(CH2) p1 -NH-CO-CH(NH2)-CH2-SH; -NH-(CH2) p1 -NH-CO-CH(NH2)-(CH2)2-SH; -NH-NH-CO-(CH2)4-CO-NH-NH-C(=NH)-(CH2)3-SH; -NH-(CH2-CH2-O) q -CH2-CH2-O-CO-C(R 17 )=CH2; -NH-(CH2-CH2-O) q -CH2-CH2-O-CO-CH(R 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH; -NH-(CH2-CH2-O) q -CH2-CH2-SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-C(R 17 )=CH2; -NH-(CH2-CH2-O) q-CH2-CH2-NH-C(=NH)-(CH2)3-SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-(CH2) r -SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-CH(R 17 )-CH2-S-CH2-CH(OH)-CH(OH)-CH2-SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-CH(NH2)-CH2-SH; -NH-(CH2-CH2-O) q -CH2-CH2-NH-CO-CH(NH2)-(CH2)2-SH; -NH-CH(CO2H)-(CH2)-SH; -NH-CH(CO2H)-(CH2)2-SH; and -NH-CH(CO2H)-(CH2)2-CONH-CH(CONH-CH2-CO2H)-CH2-SH (Here, R 17 is a hydrogen atom or C 1-6 [Selected from the group consisting of alkyl groups, where p1 is an integer between 2 and 10, q is an integer between 1 and 200, and r is an integer between 1 and 3] It can also be converted to [this format].

[0063] ≪Factor≫ The pharmaceutical composition of this embodiment contains (A) a hyaluronic acid derivative, as well as (B) one or more factors selected from the group consisting of secretory Siglec-9 and MCP-1. Both secretory Siglec-9 and MCP-1 are factors (nerve regeneration factors) involved in the regeneration of nerve damage sites (Patent Documents 2 and 3). Because the pharmaceutical composition of this embodiment contains factor (B), it is effective for nerve regeneration and is particularly effective for the treatment of nerve damage.

[0064] MCP-1, also known as CCL2, is a member of the CC chemokine family, and homologs are known in various animals, including humans. (B) The MCP-1 used as factor may be natural MCP-1 derived from animals such as humans (natural type MCP-1), recovered from natural raw materials, or obtained through genetic engineering or chemical means. The amino acid sequences of natural type MCP-1 from various animals can be obtained from protein amino acid sequence databases such as NCBI. For example, the amino acid sequence of human MCP-1 (SEQ ID NO: 1) is registered under NCBI accession number: NP02973.1.

[0065] (B) The MCP-1 used as factor can be any protein that possesses MCP-1 activity. That is, it can be any known native MCP-1, or any protein that has been newly confirmed to possess MCP-1 activity. It can also be a modified version of native MCP-1 that possesses MCP-1 activity. Note that MCP-1 activity is, for example, the activity that enhances chemotaxis against monocytes.

[0066] Examples of modified MCP-1 include proteins (mutant MCP-1) that have MCP-1 activity, consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence of natural MCP-1. Examples of amino acid substitutions include conservative substitutions, specifically substitutions within the following groups: (glycine, alanine) (valine, isoleucine, leucine) (aspartic acid, glutamic acid) (asparagine, glutamine) (serine, threonine) (lysine, arginine) (phenylalanine, tyrosine).

[0067] Examples of mutant MCP-1 include proteins that have an amino acid sequence with 60% or more identity to the amino acid sequence of natural MCP-1 and that possess MCP-1 activity. The identity is more preferably 65% ​​or more, even more preferably 70% or more, even more preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and most preferably 99% or more. Furthermore, the identity of amino acid sequences can be determined using publicly available programs such as the BLAST (Basic Local Alignment Search Tool) program by Altschul et al. (e.g., Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410 (1990), Altschyl SF, Madden TL, Schaffer AA, Zhang J, Miller W, Lipman DJ., Nucleic Acids Res. 25: p3389-3402 (1997)).

[0068] (B) The MCP-1 used as factor may be any other CC chemokine family protein that has MCP-1 activity. Examples of such proteins include CCL13, CCL7, CCL8, and CCL11, with human CCL13 (NCBI accession number: NP_005399.1), human CCL7 (NCBI accession number: NP_6264.2), human CCL8 (NCBI accession number: NP_005614.2), and human CCL11 (NCBI accession number: NP_002977.1) being preferred.

[0069] (B) The MCP-1 used as factor (B) is preferably human MCP-1, a partial protein of human MCP-1 that has MCP-1 activity, a mutant protein of human MCP-1 that has MCP-1 activity, a mutant protein of a partial protein of human MCP-1 that has MCP-1 activity, or a chimeric protein of any of these proteins and another protein that has MCP-1 activity. (B) A commercially available recombinant protein of human MCP-1 is preferably used as factor (B).

[0070] Siglec-9 is a transmembrane protein expressed in monocytes, granulocytes, and macrophages, and homologs are known in various animals, including humans. Siglec-9 possesses an extracellular domain, a transmembrane domain, and a cytoplasmic domain, and the extracellular domain is known to contain an immunoglobulin-like domain. Secreted Siglec-9, used as factor (B), is a protein that possesses the extracellular domain activity of Siglec-9. As factor (B), a partial protein of the extracellular domain portion of natural Siglec-9 derived from animals such as humans can be used. The amino acid sequences of natural Siglec-9 from various animals can be obtained from protein amino acid sequence databases such as NCBI.

[0071] For example, human Siglec-9 (NCBI accession numbers: NP_055256.1, NP_001185487.1) consists of an amino acid sequence of 463 amino acids (SEQ ID NO: 2), of which the amino acid sequence from position 1 to position 17 is a signal peptide. (B) The factor is preferably a protein containing the amino acid sequence from position 18 to position 348 of the amino acid sequence of SEQ ID NO: 2, or it may be a protein consisting of the amino acid sequence from position 18 to position 348 of the amino acid sequence of SEQ ID NO: 2, or it may be a protein consisting of the amino acid sequence from position 18 to position 463 of the amino acid sequence of SEQ ID NO: 2, or it may be a protein consisting of the full sequence of SEQ ID NO: 2.

[0072] The secreted Siglec-9 used as factor (B) can be any protein that possesses the extracellular domain activity of Siglec-9. That is, the secreted Siglec-9 used as factor (B) may be a modified partial protein of the extracellular domain portion of the natural Siglec-9 derived from animals such as humans, and which possesses the extracellular domain activity of Siglec-9. Examples of secreted Siglec-9 modified from the extracellular domain portion of the natural Siglec-9 include proteins that have an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of the extracellular domain portion of the natural Siglec-9, and which possess the extracellular domain activity of Siglec-9. As examples of amino acid substitutions, the conservative substitutions mentioned above are preferred.

[0073] The extracellular domain activity of Siglec-9 is, for example, the activity of promoting the production of anti-inflammatory cytokines. Whether or not a substance has anti-inflammatory cytokine production promoting activity can be evaluated, for example, by supplying the substance to cultured microglia / macrophage cells isolated and cultured from mice, etc., in the presence of MCP-1, and confirming the production of anti-inflammatory cytokines such as IL-10 and TGF-β1.

[0074] (B) Examples of secreted Siglec-9 used as factor include proteins having an amino acid sequence that has 60% or more identity with the amino acid sequence of the extracellular domain portion of natural Siglec-9, and that possess the extracellular domain activity of Siglec-9. The identity is more preferably 65% ​​or more, even more preferably 70% or more, even more preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and most preferably 99% or more.

[0075] (B) The secreted Siglec-9 used as factor may be a partial protein containing the extracellular domain of another Siglec family protein that has the extracellular domain activity of Siglec-9. Examples of such proteins include Siglec-7, Siglec-12, Siglec-8, and CD33, with human Siglec-7 (NCBI accession numbers: NP_00055220.1, NP_057627.2), human Siglec-12 (NCBI accession numbers: NP_2015856.1, NP_443729.1), human Siglec-8 (NCBI accession number: 055257.2), and human CD33 (NCBI accession numbers: NP_001171079.1, NP055257.2, NP_001076087.1).

[0076] (B) The secreted Siglec-9 used as factor (B) is preferably a partial protein of the extracellular domain portion of human Siglec-9, a mutant protein of the partial protein of the extracellular domain portion of human Siglec-9 that has Siglec-9 extracellular domain activity, or a chimeric protein of any of these proteins and another protein that has Siglec-9 extracellular domain activity. (B) The secreted Siglec-9 used as factor (B) is particularly preferably a protein consisting of the amino acid sequence from position 18 to 348 of the amino acid sequence of SEQ ID NO: 2, a protein consisting of the amino acid sequence from position 18 to 463 of the amino acid sequence of SEQ ID NO: 2, a mutant protein of these proteins that has Siglec-9 extracellular domain activity, or a chimeric protein of any of these proteins and another protein that has Siglec-9 extracellular domain activity. (B) Commercially available recombinant proteins of human secreted Siglec-9 are also preferably used as factor (B).

[0077] In the pharmaceutical composition of this embodiment, factor (B) may be included alone or in combination of two types. In order to obtain a higher regeneration-promoting effect and to suppress scar formation, resulting in a better condition than after regeneration, it is particularly preferable that the pharmaceutical composition of this embodiment uses both secretory Siglec-9 and MCP-1 as factor (B).

[0078] The pharmaceutical composition of this embodiment may contain, as factor (B), a cell culture supernatant or a lysate obtained by homogenizing tissue. For example, a culture supernatant obtained by culturing neural stem cells or dental pulp stem cells, which have been reported to have nerve cell inductive and anti-inflammatory effects (Patent Document 1, International Publication No. 2014 / 126176, International Publication No. 2019 / 230859), can be used as factor (B).

[0079] In this embodiment, the content of factor (B) per 100 parts by mass of hyaluronic acid derivative (A) is not particularly limited and is appropriately determined considering the type of factor (B), the molecular weight of the hyaluronic acid derivative (A), the cholesteryl group introduction rate, etc. In this embodiment, the content of factor (B) per 100 parts by mass of hyaluronic acid derivative (A) is preferably 1.0 part by mass or more, and more preferably 2.0 parts by mass or more. In this embodiment, the content of factor (B) per 100 parts by mass of hyaluronic acid derivative (A) can be 10,000 parts by mass or less, preferably 1,000 parts by mass or less, more preferably 100 parts by mass or less, even more preferably 75 parts by mass or less, and even more preferably 50 parts by mass or less.

[0080] ≪Cell adhesive material≫ The pharmaceutical composition of this embodiment further contains (C) a cell adhesion material in addition to (A) a hyaluronic acid derivative and (B) a factor. The inclusion of (C) the cell adhesion material allows the complex of (A) the hyaluronic acid derivative and (B) a factor to adhere more stably to the target tissue, resulting in a high regeneration rate promoting effect.

[0081] (C) As the cell adhesion material, it is preferable that it be a porous material because it can more stably support (A) hyaluronic acid derivatives and (B) factors. The porous material is not particularly limited, and can be appropriately selected from known cell adhesion materials, taking into consideration the type and structure of the target tissue to which the regeneration rate is to be accelerated. As the (C) cell adhesion material, it is preferable to use collagen sponge because it has good adhesion to various tissues. The collagen used is preferably soluble (acid-soluble collagen, alkali-soluble collagen, enzyme-soluble collagen, etc.).

[0082] In this embodiment, the content of (C) cell adhesion material per 100 parts by mass of (A) hyaluronic acid derivative is not particularly limited and is appropriately determined considering the volume, density, and shape of (C) cell adhesion material, the molecular weight and cholesteryl group introduction rate of (A) hyaluronic acid derivative, etc. In this embodiment, the content of (C) cell adhesion material per 100 parts by mass of (A) hyaluronic acid derivative is preferably 10 parts by mass or more, and more preferably 100 parts by mass or more. In this embodiment, the content of (C) cell adhesion material per 100 parts by mass of (A) hyaluronic acid derivative can be 1,000,000 parts by mass or less, preferably 500,000 parts by mass or less, more preferably 100,000 parts by mass or less, and even more preferably 50,000 parts by mass or less. The volume of (C) cell adhesion material is 0.001 mm 3 The above is preferable, 0.1 mm 3 The above is more preferable, 1 mm 3 The above is even more preferable. In this embodiment, (C) the volume of the cell adhesion material is 10,000,000 mm³. 3 It can be as follows: 1,000,000 mm 3 The following is preferable, 100,000 mm 3 The following is more preferable: 10,000 mm 3 The following is even more preferable: 1,000 mm 3The following are even more preferable. In this embodiment, the length of (C) cell adhesion material is preferably 0.01 cm or more, more preferably 0.1 cm or more, and even more preferably 1 cm or more. In this embodiment, the length of (C) cell adhesion material can be 10,000 cm or less, preferably 1,000 cm or less, more preferably 100 cm or less, and even more preferably 10 cm or less. In this embodiment, the thickness of (C) cell adhesion material is preferably 0.001 mm or more, more preferably 0.01 mm or more, and even more preferably 0.1 mm or more. In this embodiment, the thickness of (C) cell adhesion material can be 10,000 mm or less, preferably 1,000 mm or less, more preferably 100 mm or less, and even more preferably 10 mm or less.

[0083] <<Other additives>> The pharmaceutical composition of this embodiment may consist only of (A) a hyaluronic acid derivative, (B) a factor, and (C) a cell adhesion material, or it may contain other additives in addition to these. The other additives are not particularly limited as long as they are pharmacologically acceptable, and are appropriately selected according to the dosage form. Examples of such additives include water or other physiologically acceptable liquids (e.g., physiological saline, phosphate-buffered saline (PBS)), vehicles, buffers, surfactants, pH adjusters, isotonic agents, viscosity modifiers, excipients, binders, disintegrants, preservatives, stabilizers, cryoprotectants, colorants, etc. These additives may be used alone, or two or more additives of the same type or different types may be used in combination.

[0084] Examples of buffering agents include Tris, sodium citrate hydrate, sodium acetate hydrate, sodium bicarbonate, anhydrous sodium carbonate, sodium carbonate, magnesium sulfate, trometamol, boric acid, borax, sodium hydrogen phosphate hydrate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, and histidine.

[0085] Surfactants are also used as association promoters for (A) hyaluronic acid derivatives. Examples of such surfactants include polysorbates, polyoxyethylene fatty acid esters, and cremophor.

[0086] Examples of pH adjusting agents include hydrochloric acid and sodium hydroxide. Examples of isotonic agents include potassium chloride, calcium chloride, sodium chloride, concentrated glycerin, glucose, and D-mannitol. Examples of viscosity-concentrating agents include carboxyvinyl polymer, povidone, polyvinyl alcohol (partially saponified), hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hypromellose, methylcellulose, and glycerin.

[0087] Examples of preservatives include benzalkonium chloride, methyl parahydroxybenzoate, propyl parahydroxybenzoate, chlorobutanol, sorbic acid, and alkyl polyaminoethylglycine. Examples of stabilizers include sodium edetate hydrate and polyvinylpyrrolidone (povidone).

[0088] The cryoprotectant is not particularly limited as long as it is known as a "freeze-protectant" or "freeze-drying protectant," and examples include disaccharides, sorbitol, dextran, polyethylene glycol, propylene glycol, glycerin, glycerol, polyvinylpyrrolidone, dimethyl sulfoxide, and the like.

[0089] The disaccharides are not particularly limited, and examples include sucrose, lactulose, lactose, maltose, trehalose, cellobiose, kojibiose, nigerose, isomaltose, isotrehalose, neotrehalose, sophorose, laminaribiose, genthiobiose, turanose, maltulose, palatinose, genthiobiulose, mannobiose, melibiose, melibiulose, neolactose, galactosucrose, sylabiose, neohesperidose, rutinose, rutinulose, bicyanose, xylobiose, and primevelose. Among these, sucrose, trehalose, maltose, or lactose are preferred because they are widely used as cryoprotective agents. Furthermore, sucrose is more preferred from the viewpoint of its track record as a pharmaceutical additive and its ability to more effectively suppress the increase in particle size of the fine particles formed by the hyaluronic acid derivative during freeze-drying.

[0090] Excipients include, for example, cellulose, starch acrylate, L-aspartic acid, aminoethylsulfonic acid, aminoacetic acid, candy (powder), gum arabic, gum arabic powder, alginic acid, sodium alginate, pregelatinized starch, pumice granules, inositol, ethylcellulose, ethylene vinyl acetate copolymer, sodium chloride, olive oil, kaolin, cocoa butter, casein, fructose, pumice granules, carmellose, carmellose sodium, hydrated silicon dioxide, dried yeast, dried aluminum hydroxide gel, dried sodium sulfate, dried magnesium sulfate, agar, and Gluten powder, citric acid, sodium citrate, disodium citrate, glycerin, calcium glycerophosphate, sodium gluconate, L-glutamine, clay, clay 3, clay granules, croscarmellose sodium, crospovidone, magnesium aluminosilicate, calcium silicate, magnesium silicate, light anhydrous silicic acid, light liquid paraffin, cinnamon powder, aster, synthetic aluminum silicate, synthetic hydrotalcite, sesame oil, wheat flour, wheat germ flour, rice flour, rice starch, potassium acetate, calcium acetate, cellulose phthalate acetate, safflower Oil, bleached beeswax, zinc oxide, titanium dioxide, magnesium oxide, β-cyclodextrin, dihydroxyaluminum aminoacetate, 2,6-di-butyl-4-methylphenol, dimethylpolysiloxane, tartaric acid, potassium bitartrate, calcined gypsum, sucrose fatty acid ester, aluminum magnesium hydroxide, aluminum hydroxide gel, aluminum hydroxide / sodium bicarbonate coprecipitate, magnesium hydroxide, squalane, stearyl alcohol, stearic acid, calcium stearate, polyoxyl stearate, magnesium stearate, dye Hydrogenated soybean oil, refined gelatin, refined shellac, refined sucrose, refined sucrose spherical granules, cetostearyl alcohol, polyethylene glycol 1000 monocetyl ether, gelatin, sorbitan fatty acid ester, tricalcium phosphate, soybean oil, soybean unsaponifiables, soybean lecithin, skim milk powder, talc, ammonium carbonate, calcium carbonate, magnesium carbonate, neutral anhydrous sodium sulfate, low-substituted hydroxypropyl cellulose, dextran, dextrin, natural aluminum silicate, tragacanth powder, silicon dioxide, calcium lactate, perfiller 101, white shellac,White petrolatum, white barley, sucrose, sucrose starch spherical granules, hulless barley green leaf extract powder, hulless barley leaf green juice dried powder, honey, paraffin, potato starch, semi-digested starch, human serum albumin, hydroxypropyl starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose phthalate, phytic acid, glucose, glucose monohydrate, partially pregelatinized starch, pullulan, propylene glycol, powdered reduced maltose syrup, pectin, bentonite, sodium polyacrylate, polyoxyethylene alkyl ether, polyoxyethylene hydrogenated castor oil, polyoxyethylene (105) polyoxypropylene (5) glycol, polyoxyethylene (160) polyoxypropylene (30) glycol, polystyrene Examples include sodium sulfonate, polysorbate 80, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, polyethylene glycol, maltose, corn syrup, isopropyl myristate, anhydrous lactose, anhydrous calcium hydrogen phosphate, anhydrous calcium phosphate granules, magnesium aluminometasilicate, methylcellulose, cottonseed flour, cottonseed oil, Japanese wax, aluminum monostearate, glyceryl monostearate, sorbitan monostearate, medicinal charcoal, peanut oil, aluminum sulfate, calcium sulfate, granular corn starch, liquid paraffin, dl-malic acid, calcium hydrogen phosphate, calcium hydrogen phosphate granules, sodium hydrogen phosphate, potassium dihydrogen phosphate, calcium dihydrogen phosphate, sodium dihydrogen phosphate, etc.

[0091] Examples of binders include sugars such as sucrose, fructose, lactose or lactose monohydrate, fructooligosaccharides, glucose, palatinose, maltose, reduced maltose, powdered sugar, powdered candy, isomerized lactose, and honey sugar; sugar alcohols such as mannitol, xylitol, maltitol, erythritol, sorbitol, and lactitol; water-soluble polysaccharides such as gelatin, pullulan, carrageenan, locust bean gum, agar, glucomannan, xanthan gum, tamarind gum, pectin, sodium alginate, and gum arabic; celluloses such as hydroxypropyl cellulose and methylcellulose; starches such as corn starch, potato starch, rice starch, pregelatinized starch, and starch paste; synthetic polymers such as polyvinylpyrrolidone, carboxyvinyl polymer, and polyvinyl alcohol; and inorganic compounds such as calcium hydrogen phosphate, calcium carbonate, synthetic hydrotalcite, and magnesium aluminosilicate.

[0092] Examples of disintegrants include celluloses such as croscarmellose sodium, carmellose, carmellose calcium, carmellose sodium, and low-substituted hydroxypropyl cellulose; starches such as carboxymethyl starch sodium, hydroxypropyl starch, rice starch, wheat starch, potato starch, pregelatinized starch, and partially pregelatinized starch; and synthetic polymers such as crospovidone and crospovidone copolymer.

[0093] ≪Form≫ The dosage form of the pharmaceutical composition of this embodiment is not particularly limited and can be determined as appropriate according to the route of administration. For example, the pharmaceutical composition of this embodiment may be in solid or semi-solid form. In the case of solids, examples of forms include cubes, tubes, and sheets. In the case of semi-solids, examples include gels and creams. Furthermore, it can also be carried on an implantable medical device or similar.

[0094] In this embodiment, the pharmaceutical composition contains (A) a hyaluronic acid derivative, (B) a factor, and (C) a cell adhesion material, and (C) the cell adhesion material is a porous material, it is preferable that the complex of (A) the hyaluronic acid derivative and (B) the factor is supported on (C) the cell adhesion material. The complex of (A) the hyaluronic acid derivative and (B) the factor is absorbed into the porous structure of (C) the cell adhesion material.

[0095] ≪Manufacturing method≫ The pharmaceutical composition of this embodiment can be produced, for example, by mixing a solution (Solution (A)) in which a hyaluronic acid derivative is dissolved and a solution (Solution (B)) in which a factor (B) is dissolved, thereby forming a complex of the hyaluronic acid derivative (A) and factor (B). Solution (A) is prepared by dissolving the hyaluronic acid derivative (A) in water or PBS, etc. Solution (B) can be prepared by dissolving factor (B) in a solution to which various organic solvents or solubilizers such as vehicles are added to water. Due to the interaction between the cholesteryl group in the hyaluronic acid derivative (A) and the hydrophobic part of factor (B), and the self-assembly of the hyaluronic acid derivatives themselves, a complex of the hyaluronic acid derivative (A) and factor (B) is formed simply by mixing and stirring the two solutions.

[0096] In the pharmaceutical composition of this embodiment, when the amount of hyaluronic acid derivative (A) is relatively large relative to factor (B), the hyaluronic acid derivative (A) readily forms nanoparticles containing factor (B). For example, by setting the amount of hyaluronic acid derivative (A) and factor (B) in the solution at the time of complex formation to 10 parts by mass or more and less than 30 parts by mass, preferably 10 parts by mass or more and 25 parts by mass or less, and more preferably 10 parts by mass or more and 20 parts by mass or less, a complex of hyaluronic acid derivative (A) and factor (B) can be formed as nanoparticles.

[0097] When the amount of hyaluronic acid derivative (A) is relatively small relative to factor (B), aggregation of factor (B) and hyaluronic acid derivative (A) is easily suppressed. For example, by setting the amount of hyaluronic acid derivative (A) and factor (B) in the solution at the time of complex formation to 30 parts by mass or more and less than 100,000 parts by mass, preferably 30 parts by mass or more and 1,000 parts by mass or less, and more preferably 30 parts by mass or more and 100 parts by mass or less, a complex can be formed in which factor (B) is retained as amorphous.

[0098] If the pharmaceutical composition of this embodiment contains (A) a hyaluronic acid derivative, (B) a factor, and (C) a cell adhesion material, and (C) the cell adhesion material is a porous material, then, for example, the pharmaceutical composition of this embodiment can be produced by forming a complex of (A) the hyaluronic acid derivative and (B) the factor, and then allowing it to be absorbed into (C) the cell adhesion material.

[0099] Methods to promote nerve regeneration The target of administration of the pharmaceutical composition of this embodiment is not particularly limited to animals, and may be any mammal, reptile, amphibian, bird, fish, etc. Mammals are particularly preferred as the target of administration of the pharmaceutical composition of this embodiment. Examples of mammals to which the pharmaceutical composition of this embodiment may be administered include humans, monkeys, marmosets, mice, rats, cattle, horses, cats, dogs, pigs, sheep, goats, rabbits, etc.

[0100] The tissue whose regeneration is promoted by the pharmaceutical composition of this embodiment is damaged nerve tissue.

[0101] The pharmaceutical composition of this embodiment is intended to treat, for example, neurological diseases of the central nervous system (brain and spinal cord) or the peripheral nervous system. Examples of such neurological diseases include traumatic injuries such as spinal cord injury and peripheral nerve paralysis. Spinal cord injury refers to a condition in which the spinal cord is damaged due to external impact or internal factors such as spinal cord tumors or herniated discs. Depending on the degree of damage, it is divided into complete type (a condition in which the spinal cord is completely severed) and incomplete type (a condition in which the spinal cord is damaged or compressed, but the function of the spinal cord is partially maintained). The cause (for example, primary causes such as trauma or cerebral infarction, or secondary causes such as infection or tumors) is not particularly limited as long as it is a disease or condition in which nerve cells are damaged.

[0102] The administration route of the pharmaceutical composition of this embodiment is not particularly limited and can be used as appropriate in any currently known administration route depending on the intended use and the location of the tissue to be treated. The pharmaceutical composition of this embodiment can also be implanted, for example, by surgical procedure. It is preferable to administer the pharmaceutical composition of this embodiment directly to the site of nerve damage in order to obtain a higher effect in accelerating the nerve regeneration rate.

[0103] In the pharmaceutical composition of this embodiment, the dosage can be appropriately selected considering the type of recipient (including age, sex, etc.). Generally, for example, in a human (assuming a body weight of 60 kg), the daily dosage of factor (B) can be 0.01 μg to 1000 mg, 0.1 μg to 500 mg, or 1 μg to 100 mg.

[0104] The number of administrations may be a single dose of the above-mentioned dosage, or it may be administered two or more times, such as once every 1 day, 2 days, 4 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or every six months. Alternatively, it may be administered to two or more sites in a single administration.

[0105] <<Other Embodiments>> In one embodiment, the present invention provides a method for treating damaged nerves, comprising administering an effective amount of the above-mentioned pharmaceutical composition to a patient or animal. Furthermore, the term "effective dose" here includes the amount that is effective in accelerating the rate of nerve regeneration.

[0106] In one embodiment, the present invention provides the use of the above-mentioned factor-hyaluronic acid derivative complex for producing a pharmaceutical composition for promoting nerve regeneration. [Examples]

[0107] The present invention will be described in detail below with reference to examples, but these are not intended to limit the scope of the present invention to these examples.

[0108] The physical properties of the hyaluronic acid derivatives used in the following examples were investigated by the following method.

[0109] [Physical Properties 1] (Weight-average molecular weight Mw of hyaluronic acid derivatives, and the ratio of weight-average molecular weight to number-average molecular weight Mw / Mn) The weight-average molecular weight (Mw) and number-average molecular weight (Mn) of hyaluronic acid derivatives were measured using size exclusion chromatography with a multi-angle light scattering detector (SEC-MALS). A hyaluronic acid derivative (20 mg) was dissolved in ultrapure water (10 mL) and stirred at room temperature for at least 12 hours to obtain an aqueous solution of the hyaluronic acid derivative (2 mg / mL). To this aqueous solution (750 μL), a 300 mM hydroxypropyl-β-cyclodextrin (HP-β-CD) aqueous solution (750 μL) was added and mixed for 10 seconds using a shaker, then incubated at 37°C for 1 hour. The resulting sample was then subjected to SEC-MALS measurement to determine the weight-average molecular weight (Mw) and number-average molecular weight (Mn). The conditions for SEC-MALS measurement are shown below. Furthermore, the ratio of weight-average molecular weight to number-average molecular weight (Mw / Mn) was calculated from the SEC-MALS measurement results by dividing the weight-average molecular weight (Mw) by the number-average molecular weight (Mn).

[0110] (Measurement conditions) Columns: TSKgel GMPWXL (manufactured by Tosoh Corporation), 2 pieces Column temperature: 30℃ Eluent: 10 mM HP-β-CD-containing phosphate-buffered saline (pH 7.4) Flow rate: 1mL / min Injection volume: 200μL

[0111] [Physical Properties 2] (Cholesteryl group introduction rate) The cholesteryl group introduction rate of hyaluronic acid derivatives is 1 The determination was made by 1H-NMR measurement. First, dimethyl sulfoxide-d6 (99.9 v / v%, containing 0.05 v / v% trimethylsilyl (TMS), manufactured by Fujifilm Wako) and 20% bihydrochloric acid (99.5 v / v%, manufactured by Fujifilm Wako) were mixed in a mass ratio of 99:1 to prepare the measurement solvent. Subsequently, hyaluronic acid derivative (2 mg) was added to this measurement solvent (0.6 mL) and treated in an ultrasonic bath for 30 minutes to completely dissolve it. 1 The samples were subjected to 1H-NMR measurements. 1 ¹H-NMR measurements were performed using a Fourier transform nuclear magnetic resonance spectrometer (FT-NMR spectrometer) (ECS400, JEOL) at a sample temperature of 85°C. The cholesteryl group introduction rate was calculated from the integral value of the peak derived from the acetyl group of N-acetyl-D-glucosamine (COCH3, 1.6 ppm to 2.0 ppm, 3H) and the integral value of the peak derived from the methyl group in the cholesteryl group (CH3, 0.7 ppm, 3H), using the formula shown below. Furthermore, since the peak derived from the acetyl group of N-acetyl-D-glucosamine, in the range of 1.6 ppm to 2.0 ppm, is superimposed with the peak derived from the cholesteryl group (5H), the integral value of the peak derived from the cholesteryl group (0.7 ppm) was calculated by subtracting 5 / 3 times the integral value of the peak derived from the methyl cholesteryl group (0.7 ppm) from the integral value of the peak in the range of 1.6 ppm to 2.0 ppm (i.e., integral value (1.6 ppm to 2.0 ppm) - integral value (0.7 ppm) × 5 / 3) was used as the integral value of the peak derived from the acetyl group of N-acetyl-D-glucosamine and was used in the calculation of the cholesteryl group introduction rate.

[0112] [Cholesteryl group introduction rate (%)] =[(Peak integral value derived from the methyl group in the cholesteryl group) / (Peak integral value derived from the acetyl group of N-acetyl-D-glucosamine)]×100 =[Integral value (0.7 ppm) / {Integral value (1.6 ppm or more and 2.0 ppm or less) - Integral value (0.7 ppm) × 5 / 3}] × 100

[0113] [Example 1] The therapeutic effects of a mixture of a hyaluronic acid derivative with a cholesteryl group introduced, secretory Siglec-9, and MCP-1 were investigated in a mouse model of facial nerve injury.

[0114] <Manufacturing of hyaluronic acid derivative HA-a1> A hyaluronic acid derivative HA-a1, into which a cholesteryl group was introduced, was prepared according to steps 1 to 3 of the following procedure.

[0115] 1.Process 1 (Synthesis of cholesteryl 6-aminohexylcarbamate hydrochloride) Cholesteryl 6-aminohexylcarbamate hydrochloride (Chol hydrochloride) was synthesized according to the following steps 1-1, followed by step 1-2.

[0116] (Process 1-1) To a solution of cholesteryl chloroformate (3.37 g, 7.5 mmol) in anhydrous dichloromethane (20 mL), triethylamine (TEA, 1.05 mL) was added under an argon atmosphere and stirred. Under ice cooling, 6-(t-butoxycarbonyl)amino-1-aminohexane (1.12 mL, 5 mmol) was added dropwise, and the mixture was stirred under ice cooling for 30 minutes. After raising the temperature to room temperature, the mixture was stirred overnight. The reaction mixture was washed with ultrapure water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate:n-hexane = 1:4), and the fractions of the target product were combined and the solvent was removed under reduced pressure.

[0117] (Step 1-2) The obtained residue was dissolved in ethyl acetate (40 mL), and 40 mL of 4N hydrochloric acid / ethyl acetate solution was added and the mixture was stirred overnight at room temperature. The resulting precipitate was collected by centrifugation. The obtained solid was washed four times with ethyl acetate and dried under reduced pressure to obtain 1.2 g of cholesteryl 6-aminohexylcarbamate hydrochloride (Chol hydrochloride).

[0118] 2.Process 2 (Preparation of tetrabutylammonium (TBA) salt of hyaluronic acid) Hyaluronic acid TBA salt (HA-TBA) was prepared according to the following steps 2-1 and then 2-2.

[0119] (1) Process 2-1 DOWEX® 50WX-8-400 (manufactured by Aldrich) was suspended in ultrapure water, and the resin was washed approximately three times with ultrapure water by decantation. To the washed resin, approximately 1.5 times the molar equivalent of the resin's cation exchange capacity was added, and the mixture was stirred for 30 minutes. After removing the excess TBA-OH solution by decantation, the resin was further washed with excess ultrapure water to obtain a TBA-chlorinated cation exchange resin.

[0120] (2) Process 2-2 Sodium hyaluronate salt (HA-Na) with a weight-average molecular weight of 10,000 (10 kDa) was dissolved in ultrapure water at a concentration of 15 mg / mL. To the resulting solution, a suspension of cation exchange resin, TBA-chlorinated in "(1) Step 2-1," was added in an amount equivalent to 5 times the molar amount of HA units (unit molecular weight 401.3) in terms of the resin's ion exchange capacity. After stirring for 15 minutes, the mixture was filtered using a 0.45 μm filter, and the filtrate was freeze-dried to obtain hyaluronic acid TBA salt (HA-TBA) as a white solid.

[0121] 3.Process 3 An anhydrous DMSO solution (10 mg / mL) of HA-TBA prepared in "2.(2) Step 2-2" was prepared. Then, Chol hydrochloride was added in a molar ratio of 15 / 100 relative to the disaccharide repeating units (HA units) present in the HA-TBA synthesized in "1. Step 1". Next, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMT-MM) was added in a molar ratio of 21.6 / 100 relative to the HA units, and the mixture was stirred overnight at room temperature. The reaction solution was dialyzed in the following order: 0.3 M ammonium acetate / DMSO solution, 0.15 M NaCl aqueous solution, and ultrapure water (Spectrapore 4, molecular weight cutoff (MWCO): 12,000 to 14,000). The resulting dialysate was freeze-dried to obtain the target hyaluronic acid derivative HA-a1 (HA-C6-Chol) as a white solid. Physical property evaluation of the product revealed that the weight-average molecular weight of the hyaluronic acid derivative was 10,000 (10 kDa), and the cholesteryl group introduction rate was 33%.

[0122] <Preparation of a mixture of hyaluronic acid derivative, secretory Siglec-9, and MCP-1> For the secreted Siglec-9, we used ED-Siglec-9 (recombinant human Siglec-9Fc chimera, manufactured by R&D Systems), a chimeric protein containing Gln18 to Gly348 of human Siglec-9. For MCP-1, we used MCP-1 recombinant protein (recombinant human MCP-1 / CCL2, manufactured by Peprotech).

[0123] A treatment solution for M / S was prepared by dissolving 2 ng of ED-Siglec-9 and 2 ng of MCP-1 recombinant protein in PBS. A solution for M / S+CHHA treatment was prepared by mixing 2 ng of ED-Siglec-9, 2 ng of MCP-1 recombinant protein, and 40 ng of hyaluronic acid derivative HA-a1 in PBS and incubating at 37°C for 24 hours to promote the complexation of the hyaluronic acid derivative and protein. A solution for CHHA treatment was prepared by mixing 40 ng of hyaluronic acid derivative HA-a1 with PBS and incubating at 37°C for 24 hours.

[0124] <Preparation of a facial nerve injury model mouse> Facial nerve resection was performed on ICR mice (male, 8 weeks old, total of 21 mice). First, an incision was made with a No. 10 scalpel from the left corner of the mouth toward below the ear. For each mouse, the parotid gland was dissected, the two facial nerves were visually identified, the fascia was dissected, and 5 mm of each facial nerve was resected.

[0125] <Treatment> The procedure involved placing a collagen sponge impregnated with a treatment solution at the site of facial nerve resection. The collagen sponge used was "atelocollagen sponge" (CSH-10, manufactured by Koken Co., Ltd.). The collagen sponge was impregnated with the treatment solution immediately before the procedure.

[0126] More specifically, five mice that had undergone resection had collagen sponges impregnated with M / S treatment solution placed at the facial nerve resection sites. Another five mice that had undergone resection had collagen sponges (5mm x 5mm x 2.5mm) impregnated with CHHA treatment solution placed at each of the two facial nerve resection sites. Another six mice that had undergone resection had collagen sponges impregnated with M / S + CHHA treatment solution placed at the facial nerve resection sites. The remaining five mice had collagen sponges impregnated with PBS placed. Subsequently, the parotid glands were repositioned in all mice, and the skin was sutured with 3-0 Vicryl.

[0127] <Behavioral Assessment: Vibressa Movement Score> From the day after surgery (procedure) until 8 weeks post-surgery, the movement of the whiskers in the whisker pad, which is innervated by the facial nerve, was evaluated. The degree of paralysis was semi-quantitatively assessed using the following evaluation criteria, comparing the healthy side (the side that did not undergo surgery) with the injured side.

[0128] Behavioral evaluation criteria 0 points: Completely paralyzed. 1 point: The movement is slight compared to the healthy side. 2. Compared to the healthy side, it exhibits fibrillation. 3. Compared to the healthy side, the movement is more flutter. 4 points: Symmetrical. No difference from the healthy side.

[0129] Figure 1 shows the changes in evaluation results for each group of mice over time. The group that received CHHA solution (CS+CHHA group) showed similar behavioral evaluations to the group that did not receive any solution on the collagen sponge (CS group). In contrast, the group that received M / S solution (CS+M / S group) showed significantly greater whisker movement than the CS group from the fourth week after treatment, confirming the therapeutic effect. In the group that received CHHA+M / S solution (CS+CHHA+M / S group), whisker movement was already greater than the CS group from the first week after treatment, indicating that the regeneration rate was accelerated compared to the CS+M / S group.

[0130] <Checking playback status> Eight weeks after surgery, the site of facial nerve resection was opened and the condition of the facial nerve was observed. For control, mice that had not undergone resection surgery were observed in the same manner. Photographs of the facial nerve resection site in each mouse are shown in Figure 2. In the figure, the white dotted line indicates the facial nerve.

[0131] As shown in Figure 2, in the CS group and the CS+CHHA group, both facial nerves remained severed. In contrast, in the CS+M / S group, one facial nerve remained severed, but the other facial nerve was reconnected, and in the CS+CHHA+M / S group, both facial nerves were reconnected. [Industrial applicability]

[0132] According to the present invention, it is possible to provide a pharmaceutical composition that can accelerate the regeneration rate of damaged areas in nerves. For this reason, the present invention is particularly suitable for use in the field of regenerative medicine.

Claims

1. A pharmaceutical composition for promoting nerve regeneration, (A) Hyaluronic acid derivatives into which cholesteryl groups have been introduced, (B) One or more factors selected from the group consisting of secretory sialic acid-binding immunoglobulin-like lectin-9 and monocyte chemotactic factor-1, and (C) Cell adhesion material derived from collagen A pharmaceutical composition comprising the above (A) hyaluronic acid derivative into which the cholesteryl group has been introduced, having one or more repeating units represented by the following general formula (I). 【Chemistry 1】 (In the formula, R １ , R ２ , R ３ , and R ４ These are, independently, hydrogen atoms and C １－６ Alkyl, formyl and C １－６ It is a group selected from the group consisting of alkylcarbonyl groups. Z represents a direct bond or a peptide linker consisting of any 2 to 30 amino acid residues. X １ is —NR ｂ —R, —NR ｂ —COO—R, —NR ｂ —CO—R, —NR ｂ —CO—NR ｃ —R, —COO—R, —O—COO—R, —S—R, —CO—Y ａ —S—R, —O—CO—Y ｂ —S—R, —NR ｂ —CO—Y ｂ —S—R, and —S—S—R, and is a group selected from the group consisting of groups represented by R ａ , R ｂ and R ｃ These are, independently, hydrogen atoms and C １－２０ Alkyl, amino C ２－２０ Alkyl and hydroxy C ２－２０ It is a group selected from the group consisting of alkyl groups. ａ , R ｂ and R ｃ The alkyl portion consists of -O- and -NR ｆ A group selected from the group consisting of - may be inserted. R ｆ C is a hydrogen atom. １－１２ Alkyl, amino C ２－１２ Alkyl and hydroxy C ２－１２ It is a group selected from the group consisting of alkyl groups. ｆ The alkyl portion may have a group selected from the group consisting of -O- and -NH- inserted into it. R is a cholesteryl group. Y is C ２－３０ Alkylene, or -(CH ２ CH ２ O) ｍ -CH ２ CH ２ -. Here, the alkylenes of Y are -O- and -NR ｇ A group selected from the group consisting of - and -S-S- may be inserted. R ｇ C is a hydrogen atom. １－２０ Alkyl, amino C ２－２０ Alkyl and hydroxy C ２－２０ It is a group selected from the group consisting of alkyl groups. ｇ The alkyl portion may have a group selected from the group consisting of -O- and -NH- inserted into it. Y ａ C １－５ It is alkylene. Y ｂ C ２－８ Alkylene or C ２－８ It is alkenylene. m is an integer between 1 and 100 (inclusive).

2. The pharmaceutical composition according to claim 1, wherein the average molecular weight of the hyaluronic acid derivative into which the cholesteryl group (A) is introduced is 5,000 or more and 50,000 or less.

3. The pharmaceutical composition according to claim 1, wherein the cholesteryl group introduction rate of the hyaluronic acid derivative into which the cholesteryl group has been introduced is 8% or more and 36% or less.

4. The pharmaceutical composition according to claim 1, wherein the (C) cell adhesion material is a porous material.

5. A method for promoting nerve regeneration, comprising bringing a pharmaceutical composition according to any one of claims 1 to 4 into contact with a damaged site in nerve tissue.

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