eye drops

Abstract

To provide low-stimulation eyedrops.SOLUTION: The eyedrops contain a hyaluronic acid derivative in which a steryl group is introduced. The hyaluronic acid derivative in which a steryl group is introduced may have a repeating unit represented by general formula (I). The R may be a cholesteryl group. The eyedrops may further contain pharmacologically active ingredients. The eyedrops may further contain pharmaceutically acceptable additives.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an eye drop. [Background technology]

[0002] Dry eye is a disease that begins with the discomfort of dry, gritty eyes, but as it worsens, it can cause significant disruption to daily life. The number of dry eye patients is increasing year by year due to the aging population and the increased use of computers, smartphones, etc.

[0003] Currently, eye drops are the most common treatment for dry eye, and eye drops containing sodium hyaluronate are widely used to treat dry eye.

[0004] For example, Patent Document 1 discloses an eye drop solution in which benzalkonium chloride is added as a preservative to sodium hyaluronate. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2013-28599 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention has been made in view of the above circumstances, and provides an eye drop preparation with low irritation. [Means for solving the problem]

[0007] That is, the present invention includes the following aspects. (1) An eye drop containing a hyaluronic acid derivative having a steryl group introduced therein. (2) The eye drop preparation according to (1), wherein the hyaluronic acid derivative having a steryl group introduced therein has a repeating unit represented by the following general formula (I):

[0008] [ka]

[0009] (In the formula, R 1 , R 2 , R 3 , and R 4 are each independently 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 to 30; X 1 is the following formula: -NR b -R, -NR b -COO-R, -NR b -CO-R, -NR b -CO-NR c -R, -COO-R, -O-COO-R, -SR, -CO-Y a -SR, -O-CO-Y b -SR, -NR b -CO-Y b -SR, and -SSR, is a group selected from the group consisting of groups represented by R a , R b and R c are each independently a hydrogen atom, C 1-20 Alkyl, Amino C 2-20 Alkyl and Hydroxy C 2-20 alkyl, where the alkyl portion of the group is selected from the group consisting of -O- and -NR f - optionally inserted with a group selected from the group consisting of: R f is a hydrogen atom, C 1-12Alkyl, Amino C 2-12 Alkyl and Hydroxy C 2-12 alkyl, the alkyl portion of which may be interrupted by a group selected from the group consisting of -O- and -NH-; R is a steryl group; Y is C 2-30 Alkylene, or -(CH2CH2O) m -CH2CH2-, where the alkylene is -O-, -NR g optionally containing an inserted group selected from the group consisting of - and -SS-; R g is a hydrogen atom, C 1-20 Alkyl, Amino C 2-20 Alkyl and Hydroxy C 2-20 alkyl, the alkyl portion of which may be interrupted by a group selected from the group consisting of -O- and -NH-; Y a is C 1-5 is alkylene; Y b is C 2-8 Alkylene or C 2-8 alkenylene; m is an integer between 1 and 100.

[0010] (3) The eye drop preparation according to (2), wherein R is a cholesteryl group. (4) The eye drop preparation according to any one of (1) to (3), further comprising a pharmacologically active ingredient. (5) The eye drop preparation according to any one of (1) to (4), further comprising a pharmaceutically acceptable additive. [Effects of the Invention]

[0011] According to the eye drops of the above aspect, it is possible to provide an eye drop with low irritation. DETAILED DESCRIPTION OF THE INVENTION

[0012] Below, an embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described in detail, but the present invention is not limited to this and various modifications are possible within the scope of the gist thereof.

[0013] The terms used in this specification will be explained below.

[0014] As used herein, "C 1-20 The term "alkyl" means a straight or branched chain alkyl group having from 1 to 20 carbon atoms, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, etc. 1-4 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, and the like. 1-20 Alkyl has 1 to 12 carbon atoms. 1-12 Alkyl, C with 1 to 6 carbon atoms 1-6 Alkyl groups are also included.

[0015] As used herein, "C 1-6 The term "alkylcarbonyl" refers to the alkyl moiety of the already mentioned C 1-6 It means an alkylcarbonyl group, for example, acetyl, propionyl, n-propylcarbonyl, isopropylcarbonyl, n-butylcarbonyl, sec-butylcarbonyl, isobutylcarbonyl, tert-butylcarbonyl, etc. 1-4 alkylcarbonyl".

[0016] As used herein, "amino C 2-20 The term "alkyl" means a linear or branched alkyl having from 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. 2-20Alkyl includes amino C with 2 to 12 carbon atoms. 2-12 Alkyl is also included.

[0017] As used herein, "hydroxy C 2-20 The term "alkyl" means a linear or branched alkyl group having from 2 to 20 carbon atoms and having a hydroxy group as a substituent, for example, the hydroxy group may be located on the terminal carbon atom of the alkyl group. 2-20 Alkyl has 2 to 12 carbon atoms and hydroxy C 2-12 Alkyl is also included.

[0018] As used herein, "C 2-30 The term "alkylene" means a linear or branched divalent saturated hydrocarbon group having from 2 to 30 carbon atoms, and includes, for example, ethylene, propylene, etc., and has from 2 to 20 carbon atoms. 2-20 Alkylene, C with 2 to 8 carbon atoms 2-8 Alkylene, group -(CH2) n -" (where n is 2 or more and 30 or less, preferably 2 or more and 20 or less, and more preferably 2 or more and 15 or less).

[0019] As used herein, "C 1-5 The term "alkylene" means a straight or branched chain saturated divalent hydrocarbon radical having from 1 to 5 carbon atoms and includes, for example, methylene, ethylene, propylene, and the like.

[0020] The term "C" referred to herein 2-8 The term "alkenylene" refers to a linear or branched divalent saturated hydrocarbon group having from 2 to 8 carbon atoms and containing one or more double bonds, and includes, for example, -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. When geometric isomers exist, each isomer and a mixture thereof are also included.

[0021] <Eye drops> The eye drops of the present embodiment contain a hyaluronic acid derivative into which a steryl group has been introduced (hereinafter, may be simply referred to as "hyaluronic acid derivative").

[0022] In the eye drops of this embodiment, the hyaluronic acid derivative has improved adhesion to cells due to the steryl group introduced into the side chain, and the retention on the eyeball is improved.Therefore, the eye drops of this embodiment can be made into an eye drop with reduced irritation by containing the hyaluronic acid derivative.That is, in one embodiment, the present invention can be said to be a method for improving the retention of eye drops on the eyeball, comprising adding the hyaluronic acid derivative with steryl group introduced into the eye drop. Alternatively, in one embodiment, the present invention can be said to be a method for suppressing eye irritation caused by eye drops, which comprises adding a hyaluronic acid derivative having a steryl group introduced therein to the eye drops. In this specification, whether or not the eye irritation of an eye drop is suppressed can be determined by the method described in the Examples below.

[0023] Next, the components contained in the eye drops of the present embodiment will be described in detail below.

[0024] <Hyaluronic acid derivatives> In the hyaluronic acid derivative, the steryl group may be directly bound to the hyaluronic acid or may be bound via a linker. The "linker" referred to here can be any peptide linker or synthetic compound linker that can be introduced by genetic engineering, but in hyaluronic acid derivatives, peptide linker is preferred.The length of peptide linker is not particularly limited, and those skilled in the art can appropriately select it according to the purpose, but the preferred length is 2 amino acids or more (the upper limit is not particularly limited, but usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids.The peptide linkers contained in hyaluronic acid derivatives may all be peptide linkers of the same length, or peptide linkers of different lengths may be used.

[0025] [Steryl group] The term "steryl group" used herein is not particularly limited as long as it is a group having a steroid skeleton. Specific examples of steroids include cholesterol, cholestanol, campestanol, ergostanol, stigmastanol, coprostanol, stigmasterol, sitosterol, lanosterol, ergosterol, simiarenolol, bile acids, testosterone, estradiol, progesterone, cortisol, cortisone, aldosterone, corticosterone, and deoxycortisterone. Examples of steryl groups include cholesteryl, stigmasteryl, lanosteryl, and ergosteryl groups, with cholesteryl groups (particularly cholest-5-en-3β-yl groups) being preferred.

[0026] [Steryl group introduction rate] The introduction rate of steryl groups into the hyaluronic acid derivative (hereinafter sometimes simply referred to as "steryl group introduction rate") is preferably 0.1% or more and less than 60%, more preferably 5% or more and 50% or less, and even more preferably 6% or more and 45% or less. When the steryl group introduction rate is within the above range, the hyaluronic acid derivative has the property of forming nanoparticles well in water and dispersing. When the steryl group introduction rate is within the above range, the hyaluronic acid derivative-drug conjugate composition in which the hyaluronic acid derivative in the eye drop is complexed with a drug can become a preparation that dissolves in water and can be sterilized and filtered. In addition, hyaluronic acid derivative has steryl group that has high affinity with cells, so it has higher affinity and retention on eyeball than hyaluronic acid.Furthermore, by controlling the introduction rate of steryl group, hyaluronic acid derivative can be precipitated under physiological salt concentration conditions, and can further improve retention on eyeball.Therefore, the pharmacologically active ingredient contained in eye drops can be retained on eyeball for a long time.In this way, when hyaluronic acid derivative is precipitated when instilled into the eye, from the viewpoint of retention on eyeball, the introduction rate of steryl group is preferably 0.1% or more and less than 35%, more preferably 5% or more and 33% or less, more preferably 6% or more and 22% or less, and particularly preferably 6% or more and 20% or less.

[0027] The steryl group introduction rate is 1 It can be measured by H-NMR measurement. 1 It can be calculated based on the following formula using the integral value of the peak derived from the steryl group of the hyaluronic acid derivative in the H-NMR spectrum and the integral value of the peak derived from the acetyl group of N-acetyl-D-glucosamine contained in the hyaluronic acid derivative (COCH3, 1.6 ppm to 2.0 ppm, 3H). H represents the number of hydrogen atoms corresponding to the peak. Specifically, the measurement can be carried out, for example, according to the method described in the Examples below.

[0028] [Steryl group introduction rate] (%) = [(steryl group-derived peak integral value × 3 / n H ) / (peak integral value derived from the acetyl group of N-acetyl-D-glucosamine)] × 100

[0029] [Molecular weight of hyaluronic acid derivatives] The molecular weight of the hyaluronic acid derivative is not particularly limited, but from the viewpoint of increasing the number of steryl groups introduced per molecule of the hyaluronic acid derivative, forming a drug conjugate, and enhancing molecular entanglement and retention on the eyeball, a hyaluronic acid derivative with a relatively large molecular weight is preferred.The molecular weight of such a hyaluronic acid derivative is preferably 1,000 (1k) or more and 1,000,000 (1,000k) or less, more preferably 5k or more and 500k or less, and even more preferably 10k or more and 300k or less.When the molecular weight of the hyaluronic acid derivative is above the lower limit, the molecular entanglement can be further enhanced, and the retention on the eyeball of the eyedrops of this embodiment can be further improved.On the other hand, when the molecular weight of the hyaluronic acid derivative is below the upper limit, the increase in viscosity can be suppressed, and a higher concentration of the hyaluronic acid derivative can be dissolved in the eyedrops.The molecular weight of the hyaluronic acid derivative can generally be adjusted by using a raw material with a corresponding molecular weight.

[0030] Here, "molecular weight of hyaluronic acid derivative" refers to the weight-average molecular weight determined by size exclusion chromatography multi-angle light scattering detector (SEC-MALS).Specifically, it can be measured according to the method described in the examples below.

[0031] Specific examples of preferred hyaluronic acid derivatives include hyaluronic acid derivatives having one or more repeating units represented by the following general formula (I) (hereinafter, sometimes referred to as "repeating unit (I)").

[0032] [ka]

[0033] (In the formula, R 1 , R 2 , R 3 , and R 4 are each independently 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 to 30; X 1 is the following formula: -NR b -R, -NR b -COO-R, -NR b -CO-R, -NR b -CO-NR c -R, -COO-R, -O-COO-R, -SR, -CO-Y a -SR, -O-CO-Y b -SR, -NR b -CO-Y b -SR, and -SSR, is a group selected from the group consisting of groups represented by R a , R b and R c are each independently a hydrogen atom, C 1-20 Alkyl, Amino C 2-20 Alkyl and Hydroxy C 2-20 alkyl, where the alkyl portion of the group is selected from the group consisting of -O- and -NR f - optionally inserted with a group selected from the group consisting of: R f is a hydrogen atom, C 1-12 Alkyl, Amino C 2-12 Alkyl and Hydroxy C 2-12 alkyl, the alkyl portion of which may be interrupted by a group selected from the group consisting of -O- and -NH-; R is a steryl group; Y is C 2-30 Alkylene, or -(CH2CH2O) m -CH2CH2-, where the alkylene is -O-, -NR goptionally containing an inserted group selected from the group consisting of - and -SS-; R g is a hydrogen atom, C 1-20 Alkyl, Amino C 2-20 Alkyl and Hydroxy C 2-20 alkyl, the alkyl portion of which may be interrupted by a group selected from the group consisting of -O- and -NH-; Y a is C 1-5 is alkylene; Y b is C 2-8 Alkylene or C 2-8 alkenylene; m is an integer between 1 and 100.

[0034] 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)").

[0035] [ka]

[0036] (In the formula, R 1 , R 2 , R 3 , and R 4 are each independently a hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 alkylcarbonyl; X is -NR a -Y-NR b -COO-R is a hydrophobic group; R a and R b are each independently a hydrogen atom and C 1-6 selected from the group consisting of alkyl; R is a steryl group; Y is C 2-30 Alkylene, or -(CH2CH2O) m -CH2CH2-, m is an integer between 1 and 100.

[0037] When the hyaluronic acid derivative contains two or more repeating units (I) or two or more repeating units (Ia), the repeating units may be the same or different.

[0038] The hyaluronic acid derivative may be modified at a position other than the repeating unit (I) or the repeating unit (Ia), for example, the hydroxy group may be modified by -O(C 1-6 alkyl), -O(formyl), -O(C 1-6 The carboxy group may be converted to an amide or ester, or may form a salt.

[0039] Repeating Unit The group "-ZN(R a )YX 1 " is the formula: -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 (where mz is an integer between 2 and 30, and R 8 is a hydrogen atom or a methyl group, and R and m are as defined above in this specification. The group includes a group selected from the group consisting of groups represented by the formula: Examples of the group include: -NH-(CH2) mz -NH-COO-R; -NH-(CH2CH2O) m -CH2CH2-NH-COO-R; and -NH-(CH2) mz -SSR (wherein mz, ​​R, and m are as previously defined herein.) A group selected from the group consisting of: is preferred.

[0040] (Z) In general formula (I), Z is preferably a direct bond. In another embodiment, when Z is a peptide linker, X 1 Ha-NR b Preferably, Z is -COO-R. In another embodiment, Z is -NH-[CH(-Z a )-CONH] n-1 -CH(-Z a )-CO-, where n is an integer of 2 or more and 30 or less, and Z a are each independently H2N-CH(-Z aThe peptide linker is attached to the carboxy group of the glucuronic acid moiety at the N-terminus and to the group -N(-R)-COOH at the C-terminus. a )-YX 1 Examples of amino acids that can be used as amino acid residues of the peptide linker include α-amino acids, such as natural (L-form) 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 D-forms thereof, and all α-amino acids, including synthetic amino acids, can be used. That is, Z a Examples of such linkers include -CH3, H2NC(NH)NH(CH2)3-, H2NCOCH2-, and the like. Furthermore, n Zs may be the same or different. n is an integer of 2 to 30, preferably 2 to 10, and more preferably 2 to 4. Preferred examples of peptide linkers include -Gly-Phe-Leu-Gly-, -Asn-Phe-Phe-, -Phe-Phe-, Phe-Gly-, and the like.

[0041] (Y) In general formula (I), Y is —(CH2) n1 -and-(CH2CH2O) m1 A group selected from the group consisting of -CH2CH2- (where n1 is an integer of 2 or more and 20 or less, preferably an integer of 2 or more and 15 or less, more preferably an integer of 2 or more and 12 or less, and even more preferably an integer of 2 or more and 6 or less; m1 is an integer of 1 or more and 4 or less) is preferred. Specifically, -(CH2)2-, -(CH2)6-, -(CH2)8-, -(CH2) 12 -, or -(CH2CH2O)2-CH2CH2- is preferred. From the viewpoint of realizing high solubility in pure water or at low salt concentrations and exhibiting high precipitate-forming ability at physiological salt concentrations, Y is preferably -(CH2)2-, -(CH2)6-, -(CH2)8-, or -(CH2) 12A group selected from the group consisting of - is preferred, with -(CH2)6- being more preferred.

[0042] Y may 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-, -(CH2CH2O)2-CH2CH2-SS-(CH2CH2O)2-CH2CH2-, and the like.

[0043] (Y a ) Y a is preferably -CH2- or -CH2-CH2-.

[0044] (Y b ) Y b As the alkyl group, -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 is preferred, and -CH2-CH2- or -CH(CH3)CH2- is more preferred.

[0045] Group "-ZN(R a )YX 1" is exemplified by -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-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-N(-(CH2)3-NH2)-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(C -NH-(CH)-NH-(CH)-COO-cholesteryl, -NH-(CH)-NH-(CH)-N(-(CH)-NH-(CH)-NH)-COO-cholesteryl, -NH-(CH)-NH-(CH)-N(-(CH)-NH)-CO-NH-cholesteryl, -NH-(CH)-NH-(CH)-N(-(CH)-NH)-CO-cholesteryl, -NH-(CH)-NH-(CH)-N(-(CH)-NH)-cholesteryl, and the like are preferred. a )YX 1 " as R a , R b and R c 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 alkylene, or Y b However, linear C 2-8 Alkylene or linear C 2-8 It is alkenylene.

[0046] Repeating unit (Ia) In the 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.

[0047] The hyaluronic acid derivative may further contain, in addition to the repeating unit (I), a repeating unit represented by general formula (II) (hereinafter, sometimes referred to as "repeating unit (II)").

[0048] [ka]

[0049] (In the formula, R 1a , R 2a , R 3a , and R 4a are each independently a hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 alkylcarbonyl; X a is hydroxy and -OQ + wherein Q is selected from the group consisting of + is the countercation.)

[0050] When the hyaluronic acid derivative contains two or more repeating units (II), the repeating units may be the same or different. In another embodiment, the hyaluronic acid derivative may be a hyaluronic acid derivative consisting essentially of repeating units (I), (Ia) and (II).

[0051] [Repeating unit (II)] In general formula (II), Q +is not particularly limited as long as it is a counter cation that forms a salt with a carboxy group in water, and if it is divalent or higher, it forms a salt with multiple carboxy groups depending on the valence. Examples of counter cations include metal ions such as lithium ion, sodium ion, rubidium ion, cesium ion, magnesium ion, and calcium ion; + R j R k R l R m (In the formula, R j , R k , R l and R m are each independently a hydrogen atom and C 1-6 Among them, ammonium ions represented by Q + is preferably a sodium ion, a potassium ion, or a tetraalkylammonium ion (e.g., a tetra-n-butylammonium ion). j , R k , R l and R m is C 1-6 Preferably, they are identical groups selected from the group consisting of alkyl, and preferably n-butyl groups.

[0052] R 1 , R 2 , R 3 , and R 4 , and R 1a , R 2a , R 3a , and R 4a are preferably all hydrogen atoms. a and R b are preferably all hydrogen atoms.

[0053] Among them, the hyaluronic acid derivative is preferably substantially composed of repeating unit (I) and repeating unit (II).In the hyaluronic acid derivative, among the repeating units of the disaccharide consisting of D-glucuronic acid and N-acetyl-D-glucosamine contained in the derivative, for example, 80% or more, preferably 90% or more, more preferably 95% or more are repeating unit (I) and repeating unit (II).The hyaluronic acid derivative may be composed only of repeating unit (I) and repeating unit (II).

[0054] The content of the hyaluronic acid derivative in the eye drops of the present embodiment is not particularly limited and can be designed as appropriate.

[0055] <Pharmacologically active ingredients> The eye drops of the present embodiment may further contain a pharmacologically active ingredient. The pharmacologically active ingredient is not particularly limited as long as it is an active ingredient commonly used in eye drops. Examples of such pharmacologically active ingredients include antibiotics such as tetracycline, chlortetracycline, bacitracin, neomycin, polymyxin, gramicidin, cephalexin, oxytetracycline, chloramphenicol, kanamycin, rifampicin, tobramycin, gentamicin, ciprofloxacin, aminoglycosides, erythromycin, penicillin, quinolones, ceftazidime, vancomycin, and imipenem; antifungal agents such as amphotericin B and miconazole; sulfones, etc. Antibacterial agents such as amide, sulfadiazine, sulfacetamide, sulfamethizole, sulfisoxazole, nitrofurazone, sodium propionate, etc.; antiviral agents such as idoxuridine, trifluorothymidine, acyclovir, ganciclovir, cidofovir, interferon, etc.; antibacterial substances such as nitrofurazone, sodium propionate, etc.; iodine preparations, triclosan, chlorhexidine, etc., non-antibiotic, infection-preventing antibacterial or antibacterial agents; sodium cromoglycate, antazoline, methapyrilin, chlorohexidine, etc. Antiallergic agents such as thalidomide, cetirizine, pyrilamine, and profenpyridamine; antiproliferative agents such as thalidomide; synthetic glucocorticoids and mineralocorticoids; hormonal derivatives derived from cholesterol metabolism such as androgenic hormones such as progesterone, estrogen, testosterone, DHEA, and their derivatives; hydrocortisone, hydrocortisone acetate, dexamethasone, dexamethasone 21-phosphate, fluorouracil, medrysone, prednisolone acetate, fluorouracil, triamcinolone, anti-inflammatory agents such as acetaminophen, triamcinolone, and their derivatives; non-steroidal anti-inflammatory agents such as salicylates, indomethacin, ibuprofen, diclofenac, flurbiprofen, and piroxicam; COX2 inhibitors such as rofecoxib, diclofenac, nimesulide, and nepafenac; antitumor drugs such as carmustine, cisplatin, mitomycin, and fluorouracil; immunological drugs such as vaccines and immune enhancers; insulin, calcitonin, parathyroid hormone, peptides, and vasopressin-releasing hypothalamic factor;Timolol maleate, levobunol hydrochloride, betaxolol hydrochloride, timolol base, betaxolol, atenolol, dipivalyl, oxonorol; beta-adrenergic blockers such as acetazolamide base and methazolamide; interleukins; growth factors such as epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, transforming growth factor beta, ciliary nerve growth factor, glial-derived neurotrophic factor, NGF, EPO, and PlGF; antibodies or antibody fragments; oligoaptamers, aptamers; oligonucleotides, plasmids, ribozymes, and short interfering RNA. , nucleic acid fragments, peptides, gene fragments such as antisense sequences; immunosuppressants such as cyclosporine, sirolimus, tacrolimus; immunomodulators such as endoxan and tamoxifen; antithrombin agents, rtPA; vasodilators such as urokinase, plasmin, nitric oxide donors; antioxidants such as lutein and vitamins, and their derivatives and optically acceptable salts thereof; epinephrine, epinephrine hydrochloride, ephedrine hydrochloride, tetrahydrozoline hydrochloride, naphazoline hydrochloride, naphazoline nitrate, phenylephrine hydrochloride, dl-methylephedrine hydrochloride, neostigmine methylsulfate, epsilon-aminated protic acid, allantoin, berberine chloride, berberine sulfate, sodium azulene sulfonate, dipotassium glycyrrhizinate, zinc sulfate, zinc lactate, lysozyme chloride, diphenylhydrochloride Antihistamines, chlorpheniramine maleate, flavin adenine dinucleotide sodium, cyanocobalamin, retinol acetate, retinol palmitate, pyridoxine hydrochloride, panthenol, calcium pantothenate, sodium pantothenate, tocopheryl acetate, potassium L-aspartate, magnesium L-aspartate, magnesium and potassium L-aspartate (equal mixture), aminoethylsulfonic acid, sodium chondroitin sulfate, sulfatomethoxazole, sulfatomethoxazole sodium, sulfisomidine sodium, cyclosporine A, diquafosol sodium, carteolol hydrochloride, isopropyl unoprostone, dorzolamide hydrochloride, latanoprost, travoprost, tafluprost, bimatoprost, etc.; Since the hyaluronic acid derivative has the function of improving the solubility of poorly soluble drugs, it is possible to dissolve poorly soluble drugs such as cyclosporine A at high concentrations in the eye drops of this embodiment.

[0056] <Other additives> The eye drops of the present embodiment may further contain a pharmaceutically acceptable additive. Examples of pharmaceutically acceptable additives include carriers (aqueous solvents, aqueous or oily bases, etc.) commonly used in the preparation of eye drops, solubilizers, stabilizers, isotonicity agents, buffers, pH adjusters, preservatives, bactericides or antibacterial agents, thickeners, etc.

[0057] Examples of the carrier include aqueous solvents such as water and aqueous ethanol. Examples of solubilizers include polyoxyethylene hydrogenated castor oil, polyoxyl 40 stearate, povidone, polysorbate 80, and the like. Examples of stabilizers include sodium edetate hydrate, polyvinylpyrrolidone (povidone), polysorbate 80, and the like. Examples of isotonic agents include potassium chloride, calcium chloride, sodium chloride, concentrated glycerin, glucose, D-mannitol, and the like. Examples of buffering agents include sodium citrate hydrate, sodium acetate hydrate, sodium bicarbonate, dry sodium carbonate, sodium carbonate, magnesium sulfate, trometamol, boric acid, borax, sodium hydrogen phosphate hydrate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate. Examples of pH adjusters include hydrochloric acid and sodium hydroxide. Examples of the antiseptic, disinfectant or antibacterial agent include benzalkonium chloride, methyl parahydroxybenzoate, propyl parahydroxybenzoate, chlorobutanol, sorbic acid, alkylpolyaminoethylglycine and the like. Examples of thickening agents include carboxyvinyl polymer, povidone, polyvinyl alcohol (partially saponified), hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hypromellose, methyl cellulose, and glycerin.

[0058] <Manufacturing method of eye drops> The eye drops of the present embodiment can be produced by producing a hyaluronic acid derivative and then adding the above-mentioned pharmacologically active ingredient and additives to the hyaluronic acid derivative.

[0059] First, as the method for producing hyaluronic acid derivative, for example, the carboxyl group of glucuronic acid is converted into amide, and steryl group is introduced to obtain hyaluronic acid derivative.In addition, by adjusting the compound amount of the compound that has steryl group that reacts with raw material hyaluronic acid or its derivative, the steryl group introduction rate can be made to be 1% or more and less than 60%.

[0060] Specifically, a method for converting the carboxy group of glucuronic acid into an amide and introducing a steryl group is, for example, to ion-exchange the starting material hyaluronic acid or a derivative thereof, preferably hyaluronic acid or a derivative thereof composed only of the repeating unit (II), with a tetraalkylammonium salt (e.g., tetrabutylammonium (TBA) salt), and then reacting the hyaluronic acid salt with a compound of the formula: "HNR a -Y-NR b -R, NHR a -Y-NR b -COO-R, HNR a -Y-NR b -COO-R, HNR a -Y-NR b -CO-R, HNR a -Y-NR b -CO-NR c -R, HNR a -Y-COO-R, HNR a -YO-COO-R, HNR a -YSR, HNR a -Y-CO-Ya -SR, HNR a -YO-CO-Y b -SR, HNR a -Y-NR b -CO-Y b -SR, HNR a -YSSR or -Z-NR a -Y-NR b -COO-R(wherein, R a , R b , R c , Y, Y a , Y b , Z and R are as defined above) with an amine having a steryl group (particularly, a cholesteryl group) introduced therein.

[0061] The condensing agent that can be used in the above reaction is not particularly limited, and examples thereof include 4-(4,6-dimethoxy-1,3,5-triazine)-4-methylmorpholinium (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).

[0062] In particular, although not limited to, DMT-MM is preferred because the reaction proceeds efficiently even in a mixed solvent of water and an organic solvent.In addition, by using DMT-MM as a condensing agent, in a system where a large number of hydroxyl groups coexist, it is possible to suppress the formation of ester bonds and highly selectively form amide bonds between amino groups and carboxyl groups.The use of this condensing agent can, for example, prevent the reaction of the solvent alcohol with the carboxyl group of the hyaluronic acid moiety, or prevent the intramolecular or intermolecular bonding of the carboxyl group and the hydroxyl group simultaneously present in the hyaluronic acid moiety, resulting in the formation of undesired crosslinks.

[0063] Examples of solvents used in the steryl 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.

[0064] Alternatively, the starting hyaluronic acid or its derivative can be ion-exchanged with a tetraalkylammonium salt (e.g., tetrabutylammonium (TBA) salt), and then react this hyaluronic acid salt with a spacer moiety in a solvent in the presence of a suitable condensing agent (at this time, protection and deprotection reactions can be carried out as necessary), converting the carboxyl group (-COOH) of the starting hyaluronic acid or its derivative, and then reacting it with a suitable reagent. Examples of the combination of groups derived from carboxyl groups and reaction reagents are shown below. -CONR a -Y-NR b H + Hal-R; -CONR a -Y-NRb 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 a -Y-OCOOH + Hal-R; -CONR a -Y-OCO-Hal + HO-R; -CONR a -Y-SH + Hal-R; -CONR a -Y-Hal + HS-R; -CONR a -Y-CO-Y a -Hal + HS-R; -CONR a -Y-CO-Y a -SH + Hal-R; -CONR a -Y-O-CO-CH=CH2+ HS-R; -CONR a -Y-NRb -CO-CH(CH3)=CH2+ HS-R; -CONR a -Y-SH + HS-R; -COZ-OH + HNR a -Y-NR b -COO-R; -COZ-NR a -Y-NR b H + Hal-COO-R (In the formula, R a , R b , R c , Y, Y a , Y b and Z are as previously defined herein, and Hal represents a halogen atom selected from the group consisting of a fluorine atom, a chlorine atom, a bromine atom and an iodine atom).

[0065] Examples of reaction modes include dehydrohalogenation 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. When the conversion product or reaction product has a carboxy group, it may be converted into an N-hydroxysuccinimide (hereinafter also referred to as "NHS") ester and then reacted.

[0066] Another method is to react the carboxyl group of the raw material hyaluronic acid or its derivative with 2-aminoethyl 2-pyridyl disulfide to prepare a hyaluronic acid derivative having a spacer with a mercapto group modified with a leaving group at the end, and then subject this to a nucleophilic substitution reaction with thiocholesterol to form a disulfide bond.

[0067] Furthermore, the method of preparing the carboxyl group of hyaluronic acid or its derivatives that introduces a part of spacer, and the method of introducing a part of spacer into steryl group, and then reacting them.Some specific examples have been mentioned above, but when -SS- is inserted into Y, the carboxyl group of hyaluronic acid can also be prepared by introducing the spacer that has mercapto group at the end of the hyaluronic acid derivative, and the steryl group that has mercapto group at the end of the spacer, and then oxidatively react them to form disulfide bond.At this time, one mercapto group can be reacted with 2-mercaptopyridine to form disulfide, and then be replaced with the other mercapto group.

[0068] After preparing the hyaluronic acid derivative, other substituents may be introduced. For example, 0.1% to 99.5%, preferably 40% to 65%, of the carboxyl groups in the hyaluronic acid derivative essentially consisting of the repeating unit (I) and the repeating unit (II) may be replaced with -CO-X z , where X z is 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 (where R 17 is a hydrogen atom or C 1-6 p1 is an alkyl group, p2 is an integer of 2 or more and 10 or less, q is an integer of 1 or more and 200 or less, and r is an integer of 1 or more and 3 or less. By converting the compound into the above, cross-linking can be effected within a molecule or between molecules including other molecules to form a gel.

[0069] The conditions for the step of gelling the hyaluronic acid derivative by chemical crosslinking may be appropriately selected, including the crosslinking method, polymer concentration, crosslinking agent concentration, solvent, solvent pH, salt concentration, temperature, time, etc.

[0070] In the process of gelling a hyaluronic acid derivative, the crosslinking density of the resulting gel can be increased by increasing the reaction conditions for crosslinking, for example, by increasing the polymer concentration during chemical crosslinking and the introduction rate of groups capable of crosslinking.

[0071] When using a crosslinking agent having a group capable of forming crosslinks at both ends in the process of gelling hyaluronic acid derivative, it is preferable to add it at a concentration that allows the group to participate in the crosslinking reaction quickly and without excess or deficiency.For example, when a polymer having methacryloyl groups (MA groups) introduced therein is crosslinked by Michael addition reaction using DTT, the ratio of MA groups to SH groups is preferably 3:1 to 1:3, and particularly preferably 2:1 to 1:2.

[0072] The solvent used in the gelation step of the hyaluronic acid derivative is preferably one that can sufficiently dissolve the polymer and the crosslinking agent. It is not particularly limited, but it is preferable to use water, dimethyl sulfoxide (DMSO), dimethylacetamide (DMAc), dimethylformamide (DMF), N-methylpyrrolidone (NMP), and mixed solvents selected from these. It is also possible to use a mixture of these solvents with a miscible organic solvent. Although not particularly limited, examples of miscible organic solvents include methanol, ethanol, propanol, isopropanol, butanol, polyhydric alcohols, acetone, acetonitrile, etc. 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, 1,6-hexanediol, etc. Examples of trihydric alcohols include glycerin, trimethylolpropane, etc.

[0073] Hyaluronic acid derivative forms nanoparticles in aqueous solution, so can form nano-sized particle gel by crosslinking in solution.The concentration of solution is preferably more than 0.1mg / mL, more preferably more than 1mg / mL.On the other hand, the concentration of solution is preferably less than 100mg / mL, more preferably more than 50mg / mL, and even more preferably more than 20mg / mL. That is, the solution concentration is preferably 0.1 mg / mL or more and 100 mg / mL or less, more preferably 1 mg / mL or more and 50 mg / mL or more, and even more preferably 1 mg / mL or more and 20 mg / mL or more. When the solution concentration is equal to or higher than the lower limit, a dilute solution can be obtained, and the microparticle gels do not aggregate with each other, allowing them to remain as small particles. On the other hand, when the solution concentration is equal to or lower than the upper limit, the microparticle gels aggregate with each other, thereby improving the retention of the eyedrops on the eyeball.

[0074] The gelation process of the hyaluronic acid derivative may be carried out in bulk or in a discontinuous phase such as an emulsion or spray droplets. For example, when carrying out the process in a W / O emulsion, the aqueous phase containing the polymer, crosslinker, etc. is emulsified in a water-immiscible solvent, and the gelation reaction is carried out. Examples of the water-immiscible solvent include, but are not limited to, hexane, chloroform, dichloromethane, ethyl acetate, medium-chain triglyceride (MCT), liquid paraffin, and soybean oil. A surfactant may be added to stabilize the emulsion. Alternatively, the process may be carried out in a solvent capable of desolvation, such as supercritical carbon dioxide or PEG. In this case, the aqueous or organic solvent phase containing the polymer, crosslinker, etc. is emulsified and dispersed in the aforementioned solvent, resulting in the concentration of the polymer due to desolvation (solvent diffusion), thereby enabling the production of a gel with a higher crosslink density.

[0075] The process of gelling the hyaluronic acid derivative and subsequent processes may include terminating the crosslinking reaction and deactivating or washing the remaining crosslinkable functional groups. It is preferable to remove crosslinkable functional groups not involved in the reaction, groups bonded to only one end of the crosslinking agent, and remaining crosslinking agents from the viewpoints of safety, stability during storage, and side reactions with the encapsulated drug. While not particularly limited, for example, if unreacted crosslinking agents remain, they may be removed by washing with excess water or the like. Furthermore, for example, if methacryloyl groups substituted on the polymer remain, they may be removed by adding excess mercaptoethanol or the like to deactivate the methacryloyl groups, followed by washing the excess mercaptoethanol with excess water or the like. Furthermore, for example, if mercapto groups remain, they may be removed by adding excess 3-maleimidopropionic acid, iodoacetic acid, or the like to deactivate the mercapto groups, followed by washing the excess 3-maleimidopropionic acid and iodoacetic acid with excess water or the like.

[0076] After the process of gelling hyaluronic acid derivative, pulverization process can be carried out.Pulverization method can be pulverization using pestle and mortar or pulverization using mill, but pulverization using mill is preferred.As mill pulverization device, can be pulverization device of rotating disk type such as centrifugal pulverizer (Nihon Seiki Seisakusho) and impact mill (Dalton Co., Ltd.), pulverization device of screen mill such as atomizer (Tokyo Atomizer Manufacturing Co., Ltd.), sample mill (Tokyo Atomizer Manufacturing Co., Ltd.), bantam mill (Tokyo Atomizer Manufacturing Co., Ltd.) and SK mill (Tokken), jet pulverization device such as ultra-micro laboratory jet mill (AO jet mill, Seishin Enterprise), and can pulverize at ultra-low temperature Rinrex mill (Liquid Gas Co., Ltd.), but SK mill and Rinrex mill are preferred.

[0077] After the process of gelling hyaluronic acid derivative, drying process can be carried out.Drying method can be for example ventilation drying, drying in thermostatic oven, drying under reduced pressure, hot air circulation drying etc.Air velocity, drying time, temperature, pressure etc. are suitably selected within the range that the gel of hyaluronic acid derivative does not decompose or change in quality.

[0078] <Physical properties of eye drops> The pH of the eye drops of the present embodiment is not particularly limited as long as it is within a medicamentarily, pharmacologically (pharmaceutical), or physiologically acceptable range. The pH of the eye drops can be, for example, 4.0 to 9.5, 5.0 to 9.0, or 5.5 to 8.5.

[0079] The osmotic pressure of the eye drops of this embodiment is not particularly limited as long as it is within a range acceptable to the body. The osmotic pressure ratio of the eye drops can be, for example, 0.5 to 5.0, 0.6 to 3.0, or 0.7 to 2.0. The osmotic pressure can be adjusted by methods known in the art using inorganic salts, polyhydric alcohols, sugar alcohols, sugars, or the like. The osmotic pressure ratio is defined as the ratio of the osmotic pressure of the sample to 286 mOsm (the osmotic pressure of a 0.9 w / v% sodium chloride aqueous solution) in accordance with the 16th Edition of the Japanese Pharmacopoeia, and the osmotic pressure is measured using the osmotic pressure measurement method (freezing point depression method) described in the Japanese Pharmacopoeia. The standard solution for measuring osmolality ratios (0.9 w / v% sodium chloride aqueous solution) can be prepared by drying sodium chloride (Japanese Pharmacopoeia standard reagent) at a temperature of 500°C to 650°C for 40 to 50 minutes, allowing it to cool in a desiccator (silica gel), accurately measuring 0.900 g of the dried solution, and dissolving it in purified water to make exactly 100 mL. Alternatively, a commercially available standard solution for measuring osmolality ratios (0.9 w / v% sodium chloride aqueous solution) can be used.

[0080] The viscosity of the eye drops of this embodiment can be, for example, 1 mPa or more and 9000 mPa or less. Viscosity measurements are performed in accordance with the viscosity measurement method described in the General Test Methods of the 16th Edition of the Japanese Pharmacopoeia, using a single-cylinder rotational viscometer (Brookfield viscometer) if the viscosity at 25°C is 100 mPa·s or greater, and a cone-plate rotational viscometer (cone-plate viscometer) if the viscosity at 25°C is less than 100 mPa·s. The rotor, rotation speed, and other conditions for viscosity measurement should be selected in accordance with the device's instruction manual, and the viscosity is measured at 25°C.

[0081] <One drop of eye drops> The eye drops of this embodiment are designed so that the amount of each drop administered is, for example, 5 μL to 25 μL. By designing the amount of each drop administered of this embodiment to be within the above range, the eye drops can exhibit even more pronounced effects of retention on the eyeball and low irritation.

[0082] <Container for filling eye drops> The eye drop of the present embodiment can be filled in any container. The internal volume, shape, material, etc. of the container can be appropriately set and are not generally specified.

[0083] <Uses of eye drops> As described above, the eye drops of the present embodiment have improved retention on the eyeball, and are therefore suitable for use in the treatment or prevention of various ophthalmic diseases, including, but not limited to, corneal and conjunctival epithelial disorders associated with endogenous diseases such as dry eye (xerophthalmia), Sjögren's syndrome, and Stevens-Johnson syndrome; and corneal and conjunctival epithelial disorders associated with exogenous diseases such as postoperative, drug-induced, traumatic, and contact lens wear disorders.

[0084] In this specification, the term "treatment" means alleviating or mitigating the symptoms of various ophthalmic diseases or slowing down the rate of progression.

[0085] Alternatively, the eye drops of the present embodiment are suitably used as artificial tears, or as eye drops or contact lens wetting agents that can be applied while wearing contact lenses. The eye drops or contact lens wetting agent that can be applied while wearing contact lenses can be applied to any contact lenses, including hard and soft contact lenses. Soft contact lenses include both ionic and non-ionic contact lenses, and also include both silicone hydrogel contact lenses (hereinafter sometimes abbreviated as SHCL) and non-silicone hydrogel contact lenses (soft contact lenses that are not silicone hydrogel lenses).

[0086] The subject to which the eye drops of the present embodiment are applied is not particularly limited, and examples thereof include mammals including humans, with humans being preferred.

[0087] <How to apply eye drops> The method of instilling the eye drops of the present embodiment is not particularly limited, and the eye drops can be instilled from any angle depending on the shape of the eye dropper container and nozzle, the purpose of use, etc. From the viewpoint of suppressing variation in the amount of drops dispensed, it is preferable to dispense the drops with the instillation port facing straight down. [Example]

[0088] The present invention will be described in detail below with reference to examples, but it is not intended that the scope of the present invention be limited to these examples.

[0089] <Methods for measuring physical properties> (Molecular weight of hyaluronic acid derivative) The molecular weight of the hyaluronic acid derivative is the weight average molecular weight determined by size exclusion chromatography multi-angle light scattering detector (SEC-MALS). Specifically, the molecular weight of the hyaluronic acid derivative was measured by dissolving 20 mg of the hyaluronic acid derivative in 10 mL of ultrapure water and stirring at room temperature for 12 hours or more to obtain a 2 mg / mL aqueous solution of the hyaluronic acid derivative. 750 μL of 300 mM hydroxypropyl-β-cyclodextrin (HP-β-CD) aqueous solution was added to this 750 μL aqueous solution of the hyaluronic acid derivative, mixing for 10 seconds using a shaker, and incubating at 37°C for 1 hour. The resulting sample was then subjected to SEC-MALS analysis to determine the weight-average molecular weight. The SEC-MALS measurement conditions are shown below.

[0090] (Measurement conditions) Column: TSKgel GMPWXL (Tosoh Corporation) x 2 Column temperature: 30℃ Eluent: 10 mM HP-β-CD in phosphate buffered saline (pH 7.4) Flow rate: 1mL / min Injection volume: 200μL

[0091] <Production of hyaluronic acid derivatives> [Manufacturing Example 1] (Production of hyaluronic acid derivative HA-a1) The hyaluronic acid derivative was prepared according to the following steps 1 to 3.

[0092] 1.Process 1 (Synthesis of cholesteryl 6-aminohexylcarbamate hydrochloride) Cholesteryl 6-aminohexylcarbamate hydrochloride (Chol hydrochloride) was synthesized according to the following steps 1-1 and then 1-2.

[0093] (1) 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-(tert-butoxycarbonyl)amino-1-aminohexane (1.12 mL, 5 mmol) was added dropwise, and the mixture was stirred for 30 minutes under ice-cooling. After warming 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 evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate:n-hexane = 1:4). The fractions containing the target compound were combined, and the solvent was evaporated under reduced pressure.

[0094] (2) Process 1-2 The resulting residue was dissolved in ethyl acetate (40 mL), and a 4N hydrochloric acid / ethyl acetate solution (40 mL) was added and stirred overnight at room temperature. The resulting precipitate was collected by centrifugation. The resulting solid was washed four times with ethyl acetate and then dried under reduced pressure to obtain 1.2 g of cholesteryl 6-aminohexylcarbamate hydrochloride (Chol hydrochloride).

[0095] 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 2-2.

[0096] (1) Process 2-1 DOWEX® 50WX-8-400 (manufactured by Aldrich) was suspended in ultrapure water, and the resin was washed with ultrapure water approximately three times by decantation. Approximately 1.5 molar equivalents of 40 wt% tetrabutylammonium hydroxide (TBA-OH) (manufactured by Aldrich) based on the cation exchange capacity of the resin was added and stirred for 30 minutes. After removing excess TBA-OH solution by decantation, the resin was further washed with excess ultrapure water to obtain a TBA salt-modified cation exchange resin.

[0097] (2) Process 2-2 Hyaluronic acid sodium salt (HA-Na) with a molecular weight of 53,000 (53 kDa) was dissolved in ultrapure water at a concentration of 15 mg / mL. The suspension of the cation exchange resin converted to TBA salt in "(1) Step 2-1" was added in an amount equivalent to 5 times the molar ion exchange capacity of the resin relative to the number of moles of HA units (unit molecular weight 401.3). After stirring for 15 minutes, the solution was filtered through a 0.45 μm filter, and the filtrate was freeze-dried to obtain the TBA salt of hyaluronic acid (HA-TBA) as a white solid.

[0098] 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 to the HA-TBA synthesized in "1. Step 1" so that the molar ratio relative to the disaccharide repeating units (HA units) present was 16 / 100. Next, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) was added so that the molar ratio relative to the HA units was 21 / 100, and the mixture was stirred overnight at room temperature. The reaction solution was dialyzed (Spectrapore 7, molecular weight cutoff (MWCO): 2,000) against 0.3 M ammonium acetate / DMSO solution, 0.15 M NaCl aqueous solution, and ultrapure water, in that order. The resulting dialyzed solution was lyophilized to obtain the target product (HA-C6-Chol) as a white solid. The product 1In the H-NMR spectrum, a peak derived from the acetyl group of N-acetyl-D-glucosamine (COCH3, 1.6 ppm to 2.0 ppm, 3H) and a peak derived from the methyl group in the cholesteryl group (CH3, 0.7 ppm, 3H) were confirmed, and the cholesterol introduction rate was 15%.

[0099] [Manufacturing Example 2] (Production of hyaluronic acid derivative HA-a2) Hyaluronic acid with a molecular weight of 10,000 (10 kDa) was used, and the amount of Chol hydrochloride added to the disaccharide repeating unit (HA unit) present in HA-TBA was 9 / 100 in molar ratio, and further, the amount of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) added to the HA unit was 11 / 100 in molar ratio, except that the hyaluronic acid derivative HA-a2 was produced using the same method as in Production Example 1. The cholesterol introduction rate was 8%.

[0100] [Manufacturing Example 3] (Production of hyaluronic acid derivative HA-a3) Hyaluronic acid derivative HA-a3 was produced using hyaluronic acid with a molecular weight of 10,000 (10 kDa), with the addition of cholesterol hydrochloride in a molar ratio of 42 / 100 relative to the disaccharide repeating unit (HA unit) present in HA-TBA, and with the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) in a molar ratio of 54 / 100 relative to the HA unit, using the same method as in Production Example 1. The cholesterol introduction rate was 40%.

[0101] [Examples 1 to 3] (Production of eye drops D-1 to D-3) Each of the hyaluronic acid derivatives obtained in Production Examples 1 to 3 was dissolved in water to a concentration of 15 mg / mL to obtain eye drops D-1 to D-3.

[0102] [Test Example 1] (Eye irritation confirmation test) The test was conducted in accordance with PECD GUIDELINE FOR TESTING OF CHEMICALS Test No. 492 and EYE IRRITATION TEST METHOD: LIQUID IN VITRO PREDICTION ASSAY FOR ACUTE OCCULAR IRRITATION OF LIQUID CHEMICALS - Standard Operation Procedure (SOP). The principle of eye irritation evaluation was based on the cell viability (MTT viability (%)) of a corneal epithelial regeneration model using the MTT (3-(4,5-Dimethyl-2-thiozolyl)-2,5-diphenyl-2H-tetrazolium bromide) method.

[0103] Specifically, first, SkinEthic TM HCE (corneal epithelial regeneration model) was preincubated overnight in maintenance medium and then exposed to 30 μL of each eye drop solution from the epithelial side. PBS(-) was used as a negative control, and methylene acetate was used as an irritant control. After 30 minutes of exposure to each eye drop solution from the epithelial side, the corneal epithelial regeneration model was washed and removed. The corneal epithelial regeneration model was transferred to maintenance medium containing 1.0 mg / mL MTT and cultured for 3 hours. The corneal epithelial regeneration model was then immersed in isopropanol for at least 4 hours to extract blue formazan, and the absorbance at 570 nm was measured using a microplate reader. The MTT viability (%) was expressed as a percentage of the absorbance of the corneal epithelial regeneration model exposed to the negative control. The criteria for test success and the criteria for test sample irritation are shown in Tables 1 and 2, respectively.

[0104] [Table 1]

[0105] [Table 2]

[0106] The results of the negative control and irritant control met all of the criteria for determining whether the test was successful, as shown in Table 1, and therefore it was determined that the test was conducted properly. Table 3 shows the cell viability and absorbance after exposure to each eye drop, as well as the results of the eye irritation assessment for each eye drop. In Table 3, "NC" indicates the negative control (PBS(-)) and "PC" indicates the irritant control (methyl acetate). Each group was tested with n=2.

[0107] [Table 3]

[0108] As shown in Table 3, each eye drop was determined to be non-irritating.

[0109] [Manufacturing Example 4] (Production of hyaluronic acid derivative HA-a4) Hyaluronic acid derivative HA-a4 was produced using hyaluronic acid with a molecular weight of 35,000 (35 kDa), with the addition of cholesterol hydrochloride in a molar ratio of 21 / 100 relative to the disaccharide repeating units (HA units) present in HA-TBA, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) in a molar ratio of 24 / 100 relative to the HA units, using the same method as in Production Example 1. The cholesterol introduction rate was 19%.

[0110] [Example 4] (Manufacturing of eye drops D-4) 208 mg of the hyaluronic acid derivative HA-a4 obtained in Preparation Example 4 was weighed and dissolved in 41.6 g of ultrapure water. 0.627 g of a 41 mg / mL cyclosporine A-containing methanol solution was added dropwise to the mixture while stirring to form a complex. The mixture was then left to stand at 4°C for 16 hours, and then freeze-dried to obtain a complex powder (powdered hyaluronic acid derivative HA-a4 / cyclosporine A (10% by mass) complex). 5.5 mg of the complex powder was dissolved in 1 mL of 10% by mass sucrose solution to prepare eye drops D-4 consisting of a 10% by mass sucrose isotonic solution of the hyaluronic acid derivative HA-a4 / cyclosporine A (10% by mass) complex.

[0111] [Example 5] (Manufacturing of eye drops D-5) A composite powder (powdered hyaluronic acid derivative HA-a3 / cyclosporine A (15% by mass) composite) was obtained using the same method as in Example 4, except that the hyaluronic acid derivative HA-a3 prepared in Preparation Example 3 was used instead of the hyaluronic acid derivative HA-a4, and the amount of 41 mg / mL cyclosporine A-containing methanol solution added was 0.941 g. 57.5 mg of the powdered hyaluronic acid derivative HA-a3 / cyclosporine A (15% by mass) composite was dissolved in 1 mL of 10% by mass sucrose solution to prepare eye drops D-5 consisting of a 10% by mass sucrose isotonic solution of the hyaluronic acid derivative HA-a3 / cyclosporine A (15% by mass) composite. As described above, by conjugating the hyaluronic acid derivative, an eye drop solution was obtained in which the poorly soluble cyclosporine A was dissolved at a high concentration of 7.5 mg / mL. [Industrial Applicability]

[0112] According to the eye drops of this embodiment, it is possible to provide an eye drop with low irritation.

Claims

1. An eye drop for treating or preventing an ophthalmic disease, comprising a hyaluronic acid derivative having a steryl group introduced therein, The hyaluronic acid derivative having a steryl group introduced therein has a repeating unit represented by the following general formula (I) and a repeating unit represented by the following general formula (II), The eye drops comprise 80% or more of repeating units of a disaccharide consisting of D-glucuronic acid and N-acetyl-D-glucosamine contained in the hyaluronic acid derivative, which are repeating units represented by the following general formula (I) and repeating units represented by the following general formula (II): 【Chemical 1】 wherein R 1 , R 2 , R 3 , and R 4 are each independently 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 to 30; X 1 is a group represented by the following formula: -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 -SR, -O-CO-Y b -SR, —NR b —CO—Y b —S—R, and -S-S-R, is a group selected from the group consisting of groups represented by R a , R b and R c are each independently selected from the group consisting of a hydrogen atom, C 1-20 alkyl, aminoC 2-20 alkyl and hydroxyC 2-20 alkyl, wherein the alkyl portion of the group may be interrupted by a group selected from the group consisting of —O— and —NR f —; R f is selected from the group consisting of a hydrogen atom, C 1-12 alkyl, aminoC 2-12 alkyl and hydroxyC 2-12 alkyl, the alkyl portion of which may be inserted with a group selected from the group consisting of —O— and —NH—; R is a steryl group; Y is C 2-30 alkylene or —(CH 2 CH 2 O) m —CH 2 CH 2 —, wherein the alkylene may be inserted with a group selected from the group consisting of —O—, —NR g — and —S—S—; R g is 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 portion of which may be inserted with a group selected from the group consisting of —O— and —NH—; Y a is C 1-5 alkylene; Y b is C 2-8 alkylene or C 2-8 alkenylene; m is an integer of 1 or more and 100 or less. 【Chemistry 2】 (In the formula, R １ａ , R ２ａ , R ３ａ , and R ４ａ are each independently a hydrogen atom, C １－６ Alkyl, formyl and C １－６ alkylcarbonyl; X ａ is hydroxy and -O-Q ＋ wherein Q is selected from the group consisting of ＋ is the countercation.)

2. The eye drop according to claim 1, wherein R is a cholesteryl group.

3. The eye drop preparation according to claim 1 or 2, further comprising a pharmacologically active ingredient.

4. The eye drop preparation according to any one of claims 1 to 3, further comprising a pharmaceutically acceptable additive.

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