JPWO2017222042A1 - Ophthalmic pharmaceutical composition - Google Patents

Abstract

The present application provides an ophthalmic pharmaceutical composition comprising a compound of formula (I) contained in a high density lipoprotein, or an ester, oxide, prodrug, pharmaceutically acceptable salt or solvate thereof. . In formula (I), Ra is halo, hydroxy, alkyl, halo substituted alkyl, aryl, halo or alkyl substituted aryl, alkoxy, hydroxy or carboxy substituted alkoxy, aryloxy, halo or alkyl substituted aryloxy, CHO, C (O) Selected from the group consisting of -alkyl, C (O) -aryl, C (O) -alkyl-carboxyl, C (O) -alkylene-carboxyester and cyano, m is an integer selected from 0-4.

Description

This patent application claims priority with respect to Japanese Patent Application No. 2006-125860, which is hereby incorporated by reference in its entirety.
The present application relates to an ophthalmic pharmaceutical composition characterized in that an active ingredient for the treatment and / or prevention of eye diseases is contained in high-density lipoprotein.

  Certain 4-amino-naphthalene-1-sulfonic acid derivatives having VCP (valosin-containing protein) ATPase inhibitory activity have a cytoprotective effect on various cells through suppression of intracellular ATP decrease and suppression of endoplasmic reticulum stress. -It has a cell death inhibitory effect (Patent Document 1) and is known to be effective for the treatment and / or prevention of eye diseases such as glaucoma, retinitis pigmentosa, age-related macular degeneration, or ischemic eye disease. (Patent Documents 2 to 4, Non-Patent Documents 1 and 2). In these diseases, retinal tissues and optic nerves existing in the posterior eye part are damaged, but it is generally difficult to make the drug reach the posterior eye part by eye drops. In fact, it has been reported that 4-amino-naphthalene-1-sulfonic acid derivatives administered systemically by intraperitoneal administration or the like are effective for the treatment and / or prevention of eye diseases, but were administered by eye drops alone. In some cases, it has not been confirmed that it reaches the posterior eye segment in a sufficient amount.

  In recent years, development of eye drop drug delivery using a drug carrier has been attempted for the purpose of improving the efficiency of drug delivery to the posterior segment. As drug carriers currently applied in the ophthalmic field, liposomes for reaching the posterior segment are known (Patent Documents 5 and 6). The smaller the size of the liposome for reaching the posterior eye part, the higher the reachability to the posterior eye part is expected (Non-patent Document 3), and a report suggesting that the size of the eye drop drug carrier is preferably 20 nm or less. (Non-Patent Document 4). However, there are few reports that liposomes with a size of less than 100 nm can generally be prepared.

  A high-density lipoprotein (cHDL) fused with a cytophilic peptide for the purpose of intracellular delivery of an anticancer agent to malignant tumor cells is known (Non-patent Document 5). Lipoproteins with a particle size of 100 nm or less for the purpose of drug delivery as eye drops are not known.

International Publication No. 2012/014994 Pamphlet International Publication No. 2012/043891 Pamphlet International Publication No. 2014/129495 Pamphlet International Publication No. 2015/129809 Pamphlet International Publication No. 2009/107753 Pamphlet JP 2011-246464 A

Ikeda Hanako et al., Sci Rep 4, 5970, 2014 Noriko Nakano et al., Heliyon 2, e00096, 2016 Kohei Hatanaka et al., J Controlled Release 2009; 136: 247-53 Kompella, U. B. et al., Molecular Vision 2006; 12: 1185-1198 Tatsuya Murakami et al., Nanomedicine (London, U. K.) 2010; 5: 867-79

  The present application aims to provide an ophthalmic pharmaceutical composition comprising a 4-amino-naphthalene-1-sulfonic acid derivative.

〔式中、
Ｒａはハロ、ヒドロキシ、アルキル、ハロ置換アルキル、アリール、ハロまたはアルキル置換アリール、アルコキシ、ヒドロキシまたはカルボキシ置換アルコキシ、アリールオキシ、ハロまたはアルキル置換アリールオキシ、ＣＨＯ、Ｃ（Ｏ）−アルキル、Ｃ（Ｏ）−アリール、Ｃ（Ｏ）−アルキル−カルボキシル、Ｃ（Ｏ）−アルキレン−カルボキシエステルおよびシアノから成る群から選択され、
ｍは０〜４から選択される整数である〕
の化合物、または、それらのエステル、オキシド、プロドラッグ、薬学的に許容される塩もしくは溶媒和物を含む眼用医薬組成物（以下、本願の医薬組成物と称する）を提供する。The present application relates to the formula (I) contained in high density lipoprotein:

[Where,
Ra is halo, hydroxy, alkyl, halo substituted alkyl, aryl, halo or alkyl substituted aryl, alkoxy, hydroxy or carboxy substituted alkoxy, aryloxy, halo or alkyl substituted aryloxy, CHO, C (O) -alkyl, C (O ) -Aryl, C (O) -alkyl-carboxyl, C (O) -alkylene-carboxyester and cyano,
m is an integer selected from 0 to 4]
Or an ester, oxide, prodrug, pharmaceutically acceptable salt or solvate thereof (hereinafter referred to as the pharmaceutical composition of the present application).

  The present application provides an ophthalmic pharmaceutical composition comprising a 4-amino-naphthalene-1-sulfonic acid derivative.

FIG. 1 shows the results of the cytotoxicity test. The figure shows the results of comparing cHDL and rHDL with HBSS / HEPES solution as a negative control and benzalkonium chloride as a positive control. FIG. 2 is a fluorescence micrograph showing the results of a confirmation test for reaching the posterior eye tissue. This figure shows the result of comparing cHDL and HBSS / HEPES solutions 30 minutes after instillation. FIG. 3 shows the fluorescence intensity in the confirmation test for reaching the posterior eye tissue. The figure shows the results compared to cHDL, rHDL, HBSS / HPES solution as a negative control, and DSPC ssLip, a small size liposome as a positive control. FIG. 4 shows the area of choroidal neovascularization after instillation of pazopanib in a model test (choroidal neovascularization model) of the posterior ocular drug delivery system. This drawing shows the result of comparing pazopanib encapsulated in cHDL with pazopanib not encapsulated. FIG. 5 shows the choroidal neovascular area after instillation of pazopanib-encapsulated cHDL in a model test (choroidal neovascularization model) of the posterior ocular drug delivery system. This drawing shows the result of comparison between cHDL containing pazopanib and cHDL not containing pazopanib. FIG. 6 is a fluorescence micrograph showing the results of a confirmation test of coumarin-6 encapsulated in cHDL using DMPC, DPPC, or DSPC as a phospholipid to reach the posterior ocular tissue. This figure shows the result of each cHDL 30 minutes after instillation. FIG. 7 shows the fluorescence intensity in the arrival confirmation test for the posterior segment tissue shown in FIG. This figure shows the results of comparing cHDL using DMPC, DPPC, or DSPC. FIG. 8 shows inclusion in cHDL bound to TAT peptide, penetratin (PEN) peptide, or polyarginine (R8) as a cell membrane permeable peptide (also referred to herein as CPP), and HDL not bound to CPP. It is a fluorescence microscope photograph which shows the result of the arrival confirmation test to the posterior eye part structure | tissue of let coumarin-6 made. This figure shows the result of each cHDL 30 minutes after instillation. FIG. 9 shows the fluorescence intensity in the arrival confirmation test for the posterior segment tissue shown in FIG. This figure shows the result of comparing cHDL bound with TAT peptide, penetratin (PEN) peptide, or polyarginine (R8) and cHDL not bound with CPP. FIG. 10 is a fluorescence micrograph showing the results of a confirmation test of cHDL produced using various concentrations of coumarin-6 to reach the posterior eye tissue. This drawing shows the result of comparing cHDL solutions with a concentration of coumarin-6 of 0.03 mM, 0.05 mM, 0.1 mM, or 0.2 mM 30 minutes after instillation. FIG. 11 shows the fluorescence intensity in the arrival confirmation test for the posterior segment tissue shown in FIG. This drawing shows the result of comparison of each cHDL when the concentration of coumarin-6 is 0.03 mM, 0.05 mM, 0.1 mM, or 0.2 mM. FIG. 12 shows choroidal neovascular areas after instillation of cHDL, captisol, and ssLip encapsulating pazopanib, respectively, in a model test (angiogenesis model) of the posterior segment drug delivery system. This figure shows the result of comparing cHDL and captisol or cHDL and ssLip. FIG. 13 shows the concentration of Compound 1 reached in the retina and vitreous in rats instilled with Compound 1 solution, DSPC-Compound 1 or DC (10) -Compound 1 (shown as PBS, DSPC_HDL, and DC_HDL, respectively). Indicates.

  As used herein, where a numerical value is accompanied by the term “about,” it is intended to include a range of ± 10% of that value. The numerical range includes all numerical values between the end points and numerical values of the end points. “About” a range applies to the endpoints of the range. Therefore, for example, “about 20-30” includes “20 ± 10% -30 ± 10%”.

  Unless otherwise specified, the terms used herein have meanings as commonly understood by one of ordinary skill in the fields of organic chemistry, medicine, pharmacy, molecular biology, microbiology, and the like. The following are definitions for some of the terms used in this specification, but these definitions take precedence over the general understanding herein.

“Alkyl” means a monovalent saturated aliphatic hydrocarbyl group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms. Alkyl is for example linear and branched hydrocarbyl groups such as methyl (CH ₃ —), ethyl (CH ₃ CH ₂ —), n-propyl (CH ₃ CH ₂ CH ₂ —), isopropyl ((CH ₃ ) ₂ CH -), n-butyl _{(CH 3 CH 2 CH 2 CH} 2 -), isobutyl _{((CH 3) 2 CHCH 2} -), sec- butyl _{((CH 3) (CH 3} CH 2) CH -), t - butyl _{((CH 3) 3 C -} ), n- pentyl _{(CH 3 CH 2 CH 2 CH} 2 CH 2 -) and neopentyl _{((CH 3) 3 CCH 2} -) means, but not limited thereto.

  A group prefix “substituted” means that one or more hydrogen atoms of the group are replaced by the same or different designated substituents.

  “Alkylene” means a divalent saturated aliphatic hydrocarbyl group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms. Alkylene groups include branched and straight chain hydrocarbyl groups.

  “Alkoxy” means a group of —O-alkyl wherein alkyl is defined herein. Alkoxy includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy and n-pentoxy.

“Aryl” means a monovalent aromatic carbocyclic group of from 6 to 14 carbon atoms having one ring (eg, phenyl) or multiple condensed rings (eg, naphthyl or anthryl). Aryl groups typically include phenyl and naphthyl.
“Aryloxy” means a group of —O-aryl where aryl is defined herein and includes, for example, phenoxy and naphthoxy.

“Cyano” refers to the group —CN.
“Carboxyl” or “carboxy” means —COOH or a salt thereof.
“Carboxyester” means a group of —C (O) O-alkyl, where alkyl is defined herein.
“Halo” means halogens, including fluoro, chloro, bromo and iodo.
“Hydroxy” means the radical —OH.

  Unless otherwise specified, substituent nomenclature not explicitly defined herein is performed by naming the terminal portion of the functional group and then the adjacent functional group toward the point of attachment. For example, the substituent “arylalkyloxycarbonyl” means (aryl)-(alkyl) -O—C (O) —.

  It is understood that the above definitions are not intended to include unacceptable substitution patterns (eg, methyl substituted with 5 fluoro groups). Such unacceptable substitution patterns are well known to those skilled in the art.

  “A compound of formula (I)” as used herein means a compound included in formula (I) as described herein and a specific compound of formula (I). The term further includes stereoisomers and tautomers of the compound. In the present specification, the compounds of formula (I), and their esters, oxides, prodrugs, pharmaceutically acceptable salts and solvates may be collectively referred to as “active ingredients of the present application”. is there. In certain embodiments, a compound of Formula (I) or an ester, oxide, pharmaceutically acceptable salt or solvate thereof is used. In certain embodiments, a compound of Formula (I) or a pharmaceutically acceptable salt or solvate thereof is used.

  As used herein, the term “stereoisomer” means compounds that differ in the chirality of one or more stereocenters. Stereoisomers include enantiomers and diastereomers. The compounds of formula (I) may contain asymmetrically substituted carbon atoms. Such asymmetrically substituted carbon atoms can result in compounds that exist in other stereoisomeric forms that can be defined as absolute stereochemistry, such as enantiomers, diastereomers, and (R)-or (S) -forms. As a result, all such possible isomers of the compounds, the individual stereoisomers in their optically pure form, mixtures thereof, racemic mixtures (or racemates), mixtures of diastereomers as well as one diastereoisomer. Stereomers are intended. The terms “S” and “R” configurations, as used herein, are as defined by IUPAC 1974 RECOMMENDATIONS FOR SECTION E, FUNDAMENTAL STEREOCHEMISTRY, Pure Appl. Chem. 45:13 30 (1976).

  As used herein, the term “tautomer” refers to another form of a compound that differs in the position of the proton, such as enolketo and imine enamine tautomers, or a ring —NH— group and a ring═N— group. By tautomeric forms of heteroaryl groups containing ring atoms attached to both.

  As used herein, the term “ester” means an ester that is hydrolyzed in vivo and includes those that are readily degraded in the human body to release the parent compound or a salt thereof. Suitable ester groups are, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, in particular alkanoic acids, alkenoic acids, cycloalkanoic acids and alkanedioic acids, where each alkyl or alkenyl group is preferably 6 Having no more than carbon atoms). Specific examples of esters include formate, acetate, propionate, butyrate, acrylic acid and ethyl succinate.

  As used herein, the term “oxide” means that the nitrogen ring atom of a heteroaryl group is oxidized to form an N-oxide.

  The term “prodrug” as used herein is within the scope of reasonable medical judgment and is used in contact with human or animal tissue without undue toxicity, irritation, allergic response, etc. Means a prodrug of a compound suitable for use at a reasonable benefit / risk ratio and effective for its intended use, as well as the zwitterionic form of the compound, if possible. Prodrugs are compounds that are rapidly transformed in vivo to yield the parent compound of the above formula, for example by hydrolysis in blood. General explanations can be found in T. Higuchi and V. Stella, Pro drugs as Novel Delivery Systems, Vol. 14 of the ACS Symposium Series and Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press , 1987 (both are hereby incorporated by reference).

  "Pharmaceutically acceptable salt" means pharmaceutically acceptable salts derived from a variety of organic and inorganic counterions well known in the art, such as sodium, potassium, calcium, magnesium, ammonium and tetraalkyl. Salts with ammonium, as well as salts with organic or inorganic acids such as hydrochloric acid, hydrobromic acid, tartaric acid, mesylic acid, acetic acid, maleic acid and oxalic acid. A pharmaceutically acceptable salt of a compound means a pharmaceutically acceptable salt, including a salt of an oxide, ester or prodrug of a compound of formula (I).

  As used herein, the term “pharmaceutically acceptable salt” includes non-toxic acid or alkaline earth metal salts of the compounds of formula (I). These salts can be prepared in situ during the final isolation and purification of the compound of formula (I) or by reacting the base or acid functionality separately with a suitable organic or inorganic acid or base, respectively. Typical salts are: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, Digluconate, cyclopentanepropionate, dodecyl sulfate, ethane sulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrogen bromide Acid salt, hydroiodide salt, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinic acid Salt, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, sulfate, tartrate, thiocyanate, p- Containing toluene sulfonate and undecanoate but not limited thereto. Basic nitrogen-containing groups also include alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfate, decyl, lauryl and myristyl. And quaternized with reactants such as long chain halides such as stearyl chloride, bromide and iodide, aralkyl halides such as benzyl and phenethyl chloride and others. Thereby a product is obtained which dissolves or disperses in water or oil.

  Examples of acids that can be used to form pharmaceutically acceptable acid addition salts are inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid and oxalic acid, maleic acid, methanesulfonic acid, succinic acid and citric acid. Includes organic acids such as acids. Base addition salts may be used in situ during the final isolation and purification of the compound of formula (I), or with a carboxylic acid group and a suitable base such as a pharmaceutically acceptable metal cation hydroxide, carbonate or bicarbonate or ammonia, Alternatively, it can be produced by separately reacting with an organic primary, secondary or tertiary amine. Pharmaceutically acceptable salts include alkali and alkaline earth metal cations, such as sodium, lithium, potassium, calcium, magnesium, aluminum salts, and the like, as well as non-toxic ammonium, quaternary ammonium and amine cations, such as limited Not including, but limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Other representative organic amines useful for the formation of base addition salts include diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

  As used herein, the term “solvate” means a compound as defined above combined with a stoichiometric or non-stoichiometric amount of solvent. Solvates include solvates of pharmaceutically acceptable salts of the compounds of formula (I). The solvent is volatile, non-toxic and / or acceptable for administration to humans in trace amounts. The preferred solvate is a hydrate.

  It will be appreciated that a compound of formula (I) or any ester, oxide, pharmaceutically acceptable salt or solvate thereof may be processed in vivo by metabolism in the human or animal body or cell to yield a metabolite. It will be clear to the contractor. The term “metabolite” as used herein means a derivative of any formula that is produced in a subject after administration of the parent compound. Derivatives may be produced from the parent compound by biochemical transformations such as oxidation, reduction, hydrolysis or conjugation in a variety of subjects, including, for example, oxide and demethylated derivatives. Metabolites of compounds of formula (I) can be identified using routine techniques known in the art. For example, Bertolini, G. et al., J. Med. Chem. 40: 2011 2016 (1997); Shan, D. et al., J. Pharm. Sci. 86 (7): 765 767; Bagshawe K., Drug Dev. Res. 34: 220 230 (1995); Bodor, N., Advances in Drug Res. 13: 224 331 (1984); Bundgaard, H., Design of Prodrugs (Elsevier Press 1985); and Larsen, IK, Design and Application of Prodrugs, Drug Design and Development (Krogsgaard Larsen et al., eds., Harwood Academic Publishers, 1991). It should be understood that individual compounds that are metabolites of compounds of formula (I) or any ester, oxide, pharmaceutically acceptable salt or solvate thereof are included in the active ingredients of the present application. is there.

In certain embodiments, in Formula (I), each Ra is independently selected from the group consisting of halo, hydroxy, alkyl, halo-substituted alkyl and alkoxy.
In certain embodiments, in Formula (I), each Ra is independently selected from the group consisting of halo and alkyl.
In one embodiment, in Formula (I), there are two Ra, one is halo and the other is alkyl.

In certain embodiments, the pharmaceutical composition comprises the following compound included in formula (I) or an ester, oxide, prodrug, pharmaceutically acceptable salt or solvate thereof:
4-amino-3- (6-phenylpyridin-3-ylazo) naphthalene-1-sulfonic acid;
4-Amino-3- (6-p-toluylpyridin-3-ylazo) naphthalene-1-sulfonic acid;
4-Amino-3- (6-m-toluylpyridin-3-ylazo) naphthalene-1-sulfonic acid;
4-amino-3- (6-o-toluylpyridin-3-ylazo) naphthalene-1-sulfonic acid;
4-Amino-3- (6-biphenyl-2-ylpyridin-3-ylazo) naphthalene-1-sulfonic acid;
3- [6- (2-acetylphenyl) pyridin-3-ylazo] -4-aminonaphthalene-1-sulfonic acid;
3- [6- (3-acetylphenyl) pyridin-3-ylazo] -4-aminonaphthalene-1-sulfonic acid;
3- [6- (4-acetylphenyl) pyridin-3-ylazo] -4-aminonaphthalenesulfonic acid;
4-Amino-3- [6- (2,4-dichlorophenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-trifluoromethylphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4-trifluoromethylphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-chlorophenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (3-chlorophenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4-chlorophenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-methoxyphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4-methoxyphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-isopropoxyphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4-isopropoxyphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-phenoxyphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (3-methoxyphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2,3-dimethylphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2,5-dimethylphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (3,5-dimethylphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (3-trifluoromethylphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4- {4- [5- (1-amino-4-sulfonaphthalen-2-ylazo) pyridin-2-yl] phenyl} -4-oxobutyric acid methyl ester;
4-Amino-3- (6-biphenyl-3-ylpyridin-3-ylazo) naphthalene-1-sulfonic acid;
4-Amino-3- [6- (3-cyanophenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4-cyanophenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (3,5-bistrifluoromethylphenyl) pyridin-3-ylazo] naphthalenesulfonic acid;
4-Amino-3- [6- (4-benzoylphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-propoxyphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4-fluoro-2-methylphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (5-fluoro-2-propoxyphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-fluoro-6-propoxyphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4-fluoro-2-propoxyphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (5-fluoro-2-methylphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-fluoro-5-methylphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-butoxyphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-hexyloxyphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4-butylphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-hydroxyphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-amino-3- {6- [2- (6-hydroxyhexyloxy) phenyl] pyridin-3-ylazo} naphthalene-1-sulfonic acid;
4- {2- [5- (1-amino-4-sulfonaphthalen-2-ylazo) pyridin-2-yl] phenoxy} butyric acid;
4-amino-3- {6- [2- (3-hydroxypropoxy) phenyl] pyridin-3-ylazo} naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-isobutoxyphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (5-chloro-2-hydroxyphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4-methylbiphenyl-2-yl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4′-chloro-4-methylbiphenyl-2-yl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4,3 ′, 5′-trimethylbiphenyl-2-yl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (3′-chloro-4-methylbiphenyl-2-yl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2,6-dimethylphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-amino-3- [6- (3-formyl-2-isopropoxy-5-methylphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (3-formyl-2-butoxy-5-methylphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid.

で表される４−アミノ−３−［６−（４−フルオロ−２−メチルフェニル）ピリジン−３−イルアゾ］ナフタレン−１−スルホン酸ナトリウム塩、またはその遊離形、または、そのエステル、オキシド、プロドラッグ、薬学的に許容される塩もしくは溶媒和物である。In certain embodiments, the active ingredient has the formula

4-amino-3- [6- (4-fluoro-2-methylphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid sodium salt represented by the following formula: Prodrug, pharmaceutically acceptable salt or solvate.

  The properties of compounds of formula (I), in particular the above compounds and the synthesis methods are described in WO 2012/014994, WO 2012/043891, WO 2014/129495, WO 2015/129809. No. pamphlet (Patent Documents 1 to 4). All of these documents are hereby incorporated by reference.

  In the context of this application, “high density lipoprotein (HDL)” means a lipoprotein including apolipoprotein AI (apoA-I), which is chemically synthesized from naturally occurring HDL or apolipoprotein and phospholipid. Any of reconstructed (ie, artificial) HDL manufactured by a technique or a genetic engineering technique may be used. HDL contains apolipoprotein and phospholipid as main components. HDL is obtained as a cytophilic peptide-binding HDL (also referred to as cHDL herein) by binding a cytophilic peptide (also referred to as CP herein) as a functional peptide. be able to. HDL may contain a fluorescent labeling substance as an optional component.

  For the present application, the density of HDL is in the range of about 1.063 to 1.210 g / mL for naturally derived HDL (see Antonio M. Gotto, Jr. et al. Methods Enzymol. 1986; 128: 3-41). is there. In the case of reconstituted HDL, it typically follows the size of naturally occurring HDL, but can be adjusted to the desired density at the time of manufacture, for example, the density of natural low density lipoprotein (LDL) of about 1 A range of 0.019 to about 1.063 g / mL (see Antonio M. Gotto, Jr. et al., Ibid.) May also be included.

  The particle size of HDL is in the range of about 5-12 nm for naturally derived HDL (see the same document by Antonio M. Gotto, Jr. et al.). In the case of reconstituted HDL, the size is typically in accordance with the size of naturally occurring HDL, but can be adjusted to a desired particle size at the time of manufacture. For example, the diameter of natural LDL is about 18 to about The range of 25 nm (see Antonio M. Gotto, Jr. et al., Ibid.) Can also be included and can be adjusted to a particle size of less than about 1000 nm, less than about 200 nm, or less than about 100 nm. Regardless of the presence or absence of cytophilic peptide binding, HDL is expected to increase reachability to the posterior segment as the size is small, and therefore the particle size is typically less than about 100 nm in diameter, preferably Is about 10 to about 50 nm, more preferably about 10 to about 20 nm. In the present application, “particle size” means a volume average diameter measured by a dynamic light scattering method, and is an ordinary measuring device such as a commercially available Nanotrac UPA-EX250 particle size analyzer (Nikkiso Co., Ltd .; Tokyo, Japan).

  “Apolipoprotein” as a constituent component means a protein portion other than lipids constituting lipoprotein. As used herein, the term “apolipoprotein” includes apolipoproteins generally known to be contained in natural lipoproteins and variants thereof. The apolipoprotein is, for example, a protein belonging to the group of apolipoproteins A to E or a variant thereof, and preferably apolipoprotein AI (apoA-I), apolipoprotein A-II (apoA-II), apolipoprotein C (ApoC), apolipoprotein E (apoE), or a variant thereof. The HDL in the present application includes apoA-I or a variant thereof, and may further include another apolipoprotein. In the present specification, a variant of an apolipoprotein means a variant or analog having a function equivalent to that of an apolipoprotein (for example, a lipid binding function) (that is, a functional equivalent). Examples of apolipoprotein variants include partial fragments of apolipoproteins and combinations thereof. Variants of apoA-I include an N-terminal globular domain deficient apoA-I, such as an N-terminal 43 amino acid deficient apoA-I, lacking an N-terminal globular domain (eg, N-terminal 1-43 amino acids), or N-terminal 44 amino acid deletion apoA-I is mentioned.

  “Phospholipid” as a constituent means a lipid having one or more phosphate ester moieties. The phospholipid in the present application includes phospholipids generally known to be contained in natural lipoproteins. In the present application, the phospholipid is preferably a phospholipid known to be contained in natural HDL, but is not limited thereto. Examples of the phospholipid include sphingophospholipid having a sphingosine skeleton and glycerophospholipid having a glycerin skeleton.

  Examples of sphingophospholipids include sphingomyelin, sphingosine-1-phosphate, and ceramide. Examples of the glycerophospholipid include phosphatidylglycerol, phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine, and phosphatidylcholine is preferable. The glycerophospholipid is preferably a glycerophospholipid having a long-chain alkyl group. For example, the acyl group in the acyl group has about 9 to about 23 carbon atoms (that is, a fatty acid group having about 10 to about 24 carbon atoms), preferably about 11 Preferred are glycerophospholipids of ˜about 17 (ie, fatty acid groups of about 12 to about 18 carbon atoms), more preferably about 13 to about 17 (ie, fatty acid groups of about 14 to about 18 carbon atoms). The fatty acid group may contain one or more carbon-carbon unsaturated double bonds, but preferably does not contain it. Typical examples of phosphatidylcholines include dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), egg phosphatidylcholine (PC), and 1-palmitoyl-2- Examples include, but are not limited to oleoyl phosphatidylcholine (POPC). Phospholipids can be used alone or in combination of two or more.

HDL may contain additional lipids in addition to phospholipids. Further lipids include, for example, cholesterol, 1,2-dioleoyl-3-trimethylammoniopropane (DOTAP), N- (2,3-dioleoyloxypropan-1-yl) -N, N, N-trimethyl chloride. Ammonium (DOTMA), 1,2-dioleyl-3-dimethylammoniumpropane (DODAP), N, N-dimethyl- (2,3-dioleyloxy) propylamine (DODMA), 2,3-dioleyloxy-N -[2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetic acid (DOSPA), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE) 1,2-dioleoyloxypropyl-3-diethylhydroxyethyl bromide Ni (DORIE), 3β- [N- (N′N′-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol), cholesteryl hemisuccinate (CHEMS), 1,2-dioleoylphosphatidylethanolamine ( DOPE), general formula: R ₁ R ₂ NX (XH ₂ ) ₂ , R ₁ R ₂ NX (X (XH ₂ ) ₂ ) ₂ or R ₁ R ₂ NX (X (X (XH ₂ ) ₂ ) ₂ ) ₂ Wherein X is —CH ₂ CH ₂ CONHCH ₂ CH ₂ N <; R ₁ and R ₂ are the same or different from each other and are saturated or unsaturated C ₆ -C ₂₀ aliphatic group And the like (for example, dendron lipids D1, D2, D3, D12, D22, D32 (Funakoshi)). The ratio of additional lipids to total lipids in HDL can be, for example, about 0-80 mol%, about 0.001-80 mol%, about 1-50%, or about 3-25 mol%. Suitable phospholipid and additional lipid combinations and their molar ratios are appropriately selected based on the properties of the phospholipid and the additional lipid (eg, degree of unsaturation, charge, molecular weight, hydrophobicity, solubility, etc.). For example, if the phospholipid is DSPC and the additional lipid is DC-Chol, the ratio of DC-Chol to total lipid in HDL is about 3 to 28 mol%, about 5 to 25 mol%, or about 7 to 15 It can be mol%, for example about 10 mol%.

In the context of this application, “cytophilic peptide (CP)” means a peptide exhibiting cell affinity capable of binding to the cell membrane and then translocating into the cell. “Cell affinity” includes, but is not limited to, cell membrane permeability, cell adhesion, vascular endothelial cell accumulation, endosome escape, cell nucleus accumulation, mitochondrial accumulation, and the like. In certain embodiments, the cytophilic peptide is a cell membrane permeable peptide (also referred to herein as CPP). Cytophilic peptides include peptides derived from natural proteins and artificially produced peptides (eg, chimeric peptides). Cytophilic peptides include basic cytophilic peptides, amphiphilic cytophilic peptides, and hydrophobic cytophilic peptides due to the chemical properties of their amino acid sequences. Basic cell affinity peptides are preferred, and basic cell membrane permeable peptides are more preferred. For example, a basic cell affinity peptide (e.g., TAT protein (T rans- a ctivator of t ranscription protein) derived TAT peptide of HIV-1 virus, derived from Drosophila penetratin (PEN), polyarginine (e.g., arginine 8 Mer (R8)), polyhistidine (for example, histidine 16-mer (H16)), LL-37 peptide derived from antibacterial peptide, amphiphilic cytophilic peptide (for example, transportan which is a chimeric peptide) And Pep-1 peptide), and a hydrophobic cell affinity peptide (for example, mitochondrial targeting signal (MTS) which is a peptide derived from a signal peptide of a membrane protein). Non-limiting embodiments Is at least one selected from the group consisting of TAT peptide, penetratin, polyarginine (R8), LL-37, transportan, Pep-1 and MTS. The affinity peptide is at least one selected from the group consisting of TAT peptide, penetratin and polyarginine (R8) In a further embodiment, the cytophilic peptide is from the group consisting of penetratin and polyarginine (R8). In a further embodiment, the cytophilic peptide is penetratin.

  Cytophilic peptides bind to HDL to form cytophilic peptide-bound HDL (cHDL). The binding technique may be a chemical synthesis technique (eg, a coupling method) or a biological technique (eg, a genetic engineering method). The functional group on HDL that binds the cytophilic peptide and the position thereof can be appropriately modified. The cytophilic peptide is preferably bound on the apolipoprotein, more preferably at the C-terminus of the apolipoprotein. The apolipoprotein to which the cell affinity peptide is bound may be a fusion protein of the cell affinity peptide and the apolipoprotein. A fusion protein can be obtained by binding and expressing a gene encoding a cytophilic peptide and a gene encoding an apolipoprotein by a genetic engineering method. The fusion protein of a cell affinity peptide and an apolipoprotein can be a fusion protein in which one cell affinity peptide and one apolipoprotein are fused.

  Examples of genetic engineering techniques include DNA editing using methylase and DNA polymerase I / DNA ligase, and polymerase chain reaction (PCR). Specifically, a cell affinity peptide gene having a restriction enzyme recognition sequence added at both ends is inserted into a restriction enzyme recognition site of an E. coli expression vector using DNA ligase. A vector having a cytophilic peptide gene inserted in the forward direction is selected by DNA sequence analysis. Next, a gene in which a restriction enzyme recognition sequence is added to both ends of the apolipoprotein gene is prepared by PCR, and inserted into a restriction enzyme recognition site of an E. coli expression vector using DNA ligase. By the above operation, a gene for a polypeptide in which an apolipoprotein is fused with a cytophilic peptide can be prepared. For example, a cell affinity peptide gene in which a restriction enzyme Pst I or Xba I recognition site of an E. coli expression vector pCOLD I is added with a Pst I at the 5 ′ end and an Xba I recognition sequence at the 3 ′ end, for example, penetratin gene is DNA ligase. insert. Next, the apoA-I gene, for example, a gene having a restriction enzyme Kpn I recognition sequence added to the 5 ′ end side and a restriction enzyme Pst I recognition sequence added to the 3 ′ end side of the N-terminal globular domain-deficient apoA-I gene And inserted using DNA ligase between the Kpn I and Pst I recognition sites of pCOLD I described above. By the above operation, a gene for a fusion protein in which a cytophilic peptide is fused to the C-terminal side of apoA-I can be prepared.

HDL can contain one or more active ingredients of the present application per molecule. In some embodiments, HDL may contain, for example, about 3 to about 15 moles, or about 4 to about 12 moles of active ingredient of the present invention per mole of apolipoprotein.
In some embodiments, the HDL can include, for example, about 50 to about 500 moles, about 70 to about 300 moles, or about 80 moles to about 180 moles of phospholipid per mole of apolipoprotein. Also, in embodiments where the HDL includes phospholipids and additional lipids, the HDL contains phospholipids, for example, from 50 to about 500 moles, from about 70 to about 300 moles, from about 80 moles to about 180 moles per mole of apolipoprotein. Or about 100 moles to about 130 moles.

  In some embodiments, the HDL is, for example, about -30 to about 30 mV, about -20 to about 20 mV, about -15 to about 15 mV, about -10 to about 10 mV, about -5 to about 5 mV, about -2 to about 2 mV. Or an electrokinetic potential (zeta potential) of about -1.5 to 1.5 mV. In further embodiments, the HDL has a positive zeta potential, eg, a zeta potential of 0 to about 30 mV, 0 to about 15 mV, 0 to about 5 mV, 0 to about 2 mV, or 0 to about 1.5 mV. The zeta potential can be measured by an ordinary measuring apparatus such as a commercially available Zetasizer Nano Z (Malvern Instruments, Malvern, U. K.) by light scattering electrophoresis or the like.

  In certain aspects, the present application provides HDL that can be used as a carrier for posterior ocular drug delivery, comprising HDL in addition to apolipoprotein and phospholipid. The “rear eye part drug delivery carrier” means a carrier for delivering a drug to the rear eye part. A posterior segment drug delivery system can be constructed by combining the posterior segment drug delivery carrier with a drug effective for diagnosis, prevention or treatment of posterior segment disease.

  In the context of this application, “ophthalmic pharmaceutical composition” means a pharmaceutical composition for topical administration to the eye. The ophthalmic pharmaceutical composition is preferably an eye drop administered non-invasively. Depending on the solubility of the active ingredient of the present application in water and the stability in an aqueous solution, for example, aqueous eye drops, in-use eye drops, suspension eye drops, or oily eye drops / It can be an eye ointment.

  The pharmaceutical composition of the present application may contain pharmaceutically acceptable additives for ophthalmic use, such as buffering agents, tonicity agents, pH adjusting agents, surfactants, stabilizers, preservatives and the like. The additive is not particularly limited as long as it does not interfere with the effect of the pharmaceutical composition of the present application, and can be appropriately blended within a concentration range that does not cause problems such as eye irritation according to various uses. Examples of the buffering agent include phosphate (for example, sodium phosphate), acetate (for example, sodium acetate) and the like. Examples of the isotonic agent include inorganic salts such as sodium chloride and glycerin such as concentrated glycerin. Examples of the pH adjuster include organic acids such as boric acid. Surfactants include nonionic surfactants (eg, polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monooleate), amphoteric surfactants (eg, glycine type such as alkyldiaminoethylglycine, lauryl dimethyl). Betaine acetate forms such as aminoacetic acid betaine), anionic surfactants (eg, sulfonates such as sodium tetradecene sulfonate, alkyl sulfates such as sodium lauryl sulfate), and cationic surfactants (eg, Alkyl quaternary ammonium salts such as benzalkonium chloride), and nonionic surfactants are preferred. Examples of the stabilizer include organic acid salts (for example, sodium citrate, mannitol) and the like. Examples of the preservative include benzalkonium chloride and paraben. The pharmaceutical composition of the present application may contain additives such as a thickener (for example, sodium alginate), a chelating agent (for example, sodium edetate), and a fragrance (for example, menthol). The pH of the pharmaceutical composition of the present application is usually preferably in the range of 4 to 8 as long as it is within the range acceptable for ophthalmic preparations.

  It is also possible to combine the pharmaceutical composition of the present application with further active ingredients. Further active ingredients include, for example, drugs for the treatment of glaucoma or ocular hypertension (e.g. pilocarpine, distigmine, carteolol, timolol, betaxolol, nipradilol, levobunolol, bunazosin, dipivefrin, brimonidine, isopropylunoprostone, latanoprost, travoprost , Tafluprost, bimatoprost, lipazil, dorzolamide, brinzolamide, etc.), vascular regression drugs (eg, ranibizumab, pegaptanib, aflibercept, etc.), but are not limited thereto.

  Further active ingredients and their therapeutically effective amounts can be determined within the skill and judgment of the ordinary clinician. Additional active ingredients may be present in the pharmaceutical composition of the present application and may be provided separately from the pharmaceutical composition of the present application.

  In the context of this application, “combining” or “in combination with” a pharmaceutical composition of the present application and an additional active ingredient includes not only the use of dosage forms containing all ingredients and the use of a combination of dosage forms containing each ingredient separately. As long as they are used for the treatment and / or prophylaxis of eye diseases, it also means that each component is administered simultaneously or sequentially, or any component is delayed. Two or more additional active ingredients can be used in combination with the pharmaceutical composition of the present application.

  Treatment of ocular diseases may be performed before, after, or at the same time as administration of the pharmaceutical composition of the present application, including but not limited to laser surgery, surgery, photodynamic therapy, and the like.

  An eye disease can be treated by administering a pharmaceutical composition of the present application to a patient suffering from an eye disease, particularly a posterior eye disease. In the context of the present application, “treating” or “treatment” refers to reducing or eliminating the cause of a disease, delaying or stopping the progression of the disease, and / or symptoms of the disease in a subject suffering from the disease. Means mitigation, mitigation, improvement or elimination.

  The eye disease can be prevented by administering the pharmaceutical composition of the present application to a patient at risk of suffering from an eye disease, particularly a posterior eye disease. In the context of the present application, “prevent” or “prevention” means preventing the onset of the disease in a subject who is not afflicted with the disease but who is at risk of being afflicted. Subjects to be prevented include subjects with any abnormality in the eye tissue (eg, high intraocular pressure, drusen accumulation under the retinal pigment, myopia), genetic risk of eye disease (eg, causative genes such as retinitis pigmentosa and glaucoma, Subjects with a genetic polymorphism disease genotype that has been shown to have a high risk of disease, other diseases that increase the prevalence of eye diseases (eg, metabolic diseases such as diabetes, dyslipidemia, Subjects suffering from thrombosis, autoimmune diseases, renal dysfunction, hypertension), subjects using drugs that induce eye diseases (eg, steroids, etc.).

  Examples of ocular diseases that can be treated and / or prevented by the pharmaceutical composition of the present application include glaucoma, retinitis pigmentosa, age-related macular degeneration, ischemic ocular diseases and myopia, etc. Glaucoma includes normal-tension glaucoma or glaucoma that is refractory to intraocular pressure-lowering therapy. Retinitis pigmentosa includes hereditary retinitis pigmentosa and sporadic retinitis pigmentosa. Age-related macular degeneration includes wet age-related macular degeneration and atrophic age-related macular degeneration. Ischemic eye diseases include all eye diseases resulting from or associated with ischemia of the eye, such as central retinal artery occlusion, branch retinal artery occlusion, ischemic optic neuropathy, central retinal vein occlusion, Includes retinal vein branch occlusion, diabetic retinopathy and ocular hypertension or optic neuropathy or optic neuropathy. Ischemic eye disease can be acute and can involve ischemia-reperfusion injury.

  In one aspect, a method for the treatment and / or prophylaxis of ophthalmic diseases is provided that comprises administering to a subject a pharmaceutical composition of the present application. In another aspect, there is provided the use of the pharmaceutical composition of the present application in the treatment and / or prevention of eye diseases.

HDL contained in the pharmaceutical composition of the present application is, for example,
i) a step of producing crude HDL by mixing an apolipoprotein solution and a liposome containing phospholipid (and further lipid if necessary) (Spontaneous interaction method); and ii) removing crude HDL by ultracentrifugation. Purification to obtain HDL,
In which the active ingredient of the present application is contained in an apolipoprotein solution, in a liposome, or mixed with the crude HDL obtained in step i) prior to step ii). .

Alternatively, HDL contained in the pharmaceutical composition of the present application is, for example,
i) micellized by mixing phospholipids (and optionally additional lipids) and cholic acid (eg, at the phase transition temperature of the phospholipids), mixing the resulting micelle solution with the apolipoprotein solution, Ii) to produce crude HDL (cholate-dialysis method); and ii) purification of crude HDL by ultracentrifugation to obtain HDL,
In which the active ingredient of the present application is micellized with a phospholipid or the like, included in an apolipoprotein solution, or mixed with the crude HDL obtained in step i) prior to step ii). be able to.

  When producing HDL, typically, for example, several to several thousand times mole, several tens to several hundred times mole, such as about 5 times to about 2000 times mole, about 1 mole of apolipoprotein, 20-fold to about 500-fold moles, about 30-fold to about 200-fold moles of phospholipid are used as starting materials.

  In producing HDL, typically, for example, about 0.01 to about 15 g, about 0.05 to about 10 g, about 0.07 to about 7 g, or about 0.1 to 1 g of apolipoprotein. ~ 3.5 g of the active ingredient of the present application is used as starting material. In some embodiments, about 0.3 to about 3.5 g of the active ingredient of the present application can be used as a starting material.

  The obtained HDL can be adjusted to fine particles using a gel filtration chromatography method, an ultracentrifugation method, an ultrasonic irradiation method, a freeze-thaw method, a homogenization method, or the like, but is not limited to these methods.

In one embodiment, the present application provides a cytophilic peptide-binding high-density lipoprotein (cHDL) {which is apolipoprotein, phospholipid, and cytophilic peptide (wherein the phospholipid is a fatty acid having 12 to 18 carbon atoms). The glycerophospholipid of the group, the cytophilic peptide is a basic cell membrane permeable peptide) and the particle size is less than about 100 nm in diameter (eg, about 10-20 nm in diameter)}
Provided is a pharmaceutical composition for the treatment and / or prophylaxis of eye diseases comprising the active ingredient of the present application contained therein.

In certain embodiments, the present application provides a cytophilic peptide-bound high density lipoprotein (cHDL) {which comprises an N-terminal globular domain-deficient apoA-I, DSPC, DC-Chol and penetratin and has a particle size of about 10-20 nm in diameter. Is}
A pharmaceutical composition for treatment and / or prevention of at least one disease selected from the group consisting of glaucoma, retinitis pigmentosa, age-related macular degeneration and ischemic eye disease, comprising the active ingredient of the present application contained therein I will provide a.

〔式中、
Ｒａはハロ、ヒドロキシ、アルキル、ハロ置換アルキル、アリール、ハロまたはアルキル置換アリール、アルコキシ、ヒドロキシまたはカルボキシ置換アルコキシ、アリールオキシ、ハロまたはアルキル置換アリールオキシ、ＣＨＯ、Ｃ（Ｏ）−アルキル、Ｃ（Ｏ）−アリール、Ｃ（Ｏ）−アルキル−カルボキシル、Ｃ（Ｏ）−アルキレン−カルボキシエステルおよびシアノから成る群から選択され、
ｍは０〜４から選択される整数である〕
の化合物またはそのエステル、オキシド、薬学的に許容される塩もしくは溶媒和物を含む眼用医薬組成物。
［２］高密度リポタンパク質が細胞親和性ペプチドと結合している、［１］に記載の医薬組成物。
［３］細胞親和性ペプチドが細胞膜透過性ペプチドである、［２］に記載の医薬組成物。
［４］細胞親和性ペプチドが、ＴＡＴペプチド、ペネトラチン、ポリアルギニン（Ｒ８）、ＬＬ−３７、トランスポータン、Ｐｅｐ−１およびＭＴＳからなる群から選択される少なくとも１種である、［２］または［３］に記載の医薬組成物。
［５］細胞親和性ペプチドが、ＴＡＴペプチド、ペネトラチンおよびポリアルギニン（Ｒ８）からなる群から選択される少なくとも１種である、［２］ないし［４］のいずれかに記載の医薬組成物。
［６］細胞親和性ペプチドがペネトラチンである、［２］ないし［５］のいずれかに記載の医薬組成物。
［７］高密度リポタンパク質が、ジミリストイルホスファチジルコリン（ＤＭＰＣ）、ジパルミトイルホスファチジルコリン（ＤＰＰＣ）およびジステアロイルホスファチジルコリン（ＤＳＰＣ）からなる群から選択される少なくとも１種のリン脂質を含む、［１］ないし［６］のいずれかに記載の医薬組成物。
［８］高密度リポタンパク質がジステアロイルホスファチジルコリン（ＤＳＰＣ）を含む、［１］ないし［７］のいずれかに記載の医薬組成物。
［９］高密度リポタンパク質がさらなる脂質を含む、［７］または［８］に記載の医薬組成物。
［１０］さらなる脂質が３β−［Ｎ−（Ｎ'Ｎ'−ジメチルアミノエチル）カルバモイル］コレステロール（ＤＣ−Ｃｈｏｌ）である、［９］に記載の医薬組成物。
［１１］高密度リポタンパク質がジステアロイルホスファチジルコリン（ＤＳＰＣ）および３β−［Ｎ−（Ｎ'Ｎ'−ジメチルアミノエチル）カルバモイル］コレステロール（ＤＣ−Ｃｈｏｌ）を含み、ＤＳＰＣとＤＣ−Ｃｈｏｌの総量に対するＤＣ−Ｃｈｏｌの割合が約７〜１５モル％である、［１］ないし［１０］のいずれかに記載の医薬組成物。
［１２］高密度リポタンパク質が細胞親和性ペプチドとアポリポタンパク質との融合タンパク質を含む、［１］ないし［１１］のいずれかに記載の医薬組成物。
［１３］高密度リポタンパク質が、Ｎ末端球状ドメイン欠損アポリポタンパク質Ａ−Ｉを含む、［１］ないし［１２］のいずれかに記載の医薬組成物。
［１４］Ｎ末端球状ドメイン欠損アポリポタンパク質Ａ−Ｉが、Ｎ末端４３アミノ酸欠損アポリポタンパク質Ａ−ＩまたはＮ末端４４アミノ酸欠損アポリポタンパク質Ａ−Ｉである、［１３］に記載の医薬組成物。
［１５］高密度リポタンパク質が、ペネトラチンとＮ末端球状ドメイン欠損アポリポタンパク質Ａ−Ｉとの融合タンパク質、ジステアロイルホスファチジルコリン（ＤＳＰＣ）およびＤＣ−コレステロールを含む、［１］ないし［１４］のいずれかに記載の医薬組成物。
［１６］Ｎ末端球状ドメイン欠損アポリポタンパク質Ａ−Ｉが、Ｎ末端４４アミノ酸欠損アポリポタンパク質Ａ−Ｉである、［１４］に記載の医薬組成物。
［１７］高密度リポタンパク質の粒径が直径約１０〜２０ｎｍである、［１］ないし［１６］のいずれかに記載の医薬組成物。
［１８］式（Ｉ）中、Ｒａが、それぞれ独立して、ハロ、ヒドロキシ、アルキル、ハロ置換アルキルおよびアルコキシから成る群から選択される、［１］ないし［１７］のいずれかに記載の医薬組成物。
［１９］式（Ｉ）中、Ｒａが、それぞれ独立して、ハロおよびアルキルから成る群から選択される、［１］ないし［１８］のいずれかに記載の医薬組成物。
［２０］式（Ｉ）中、Ｒａが２個存在し、一方がハロであり、他方がアルキルである、［１］ないし［１９］のいずれかに記載の医薬組成物。
［２１］式（Ｉ）の化合物が、式

の化合物である、［１］ないし［２０］のいずれかに記載の医薬組成物。
［２２］高密度リポタンパク質が、アポリポタンパク質１モル当たり、約３〜約１５モルの式（Ｉ）の化合物またはそのエステル、オキシド、薬学的に許容される塩もしくは溶媒和物を含む、［１］ないし［２１］のいずれかに記載の医薬組成物。
［２３］眼疾患の処置および／または予防のための、［１］ないし［２２］のいずれかに記載の医薬組成物。
［２４］眼疾患が、緑内障、網膜色素変性症、加齢黄斑変性または虚血性眼疾患である、［２３］に記載の医薬組成物。
［２５］点眼剤である、［１］ないし［２４］のいずれかに記載の医薬組成物。
［２６］［１］ないし［２５］のいずれかに記載の医薬組成物を対象に投与することを含む、眼疾患の処置および／または予防方法。
［２７］眼疾患の処置および／または予防における［１］ないし［２５］のいずれかに記載の医薬組成物の使用。For example, the present application provides the following embodiments.
[1] Formula (I) contained in high-density lipoprotein:

[Where,
Ra is halo, hydroxy, alkyl, halo substituted alkyl, aryl, halo or alkyl substituted aryl, alkoxy, hydroxy or carboxy substituted alkoxy, aryloxy, halo or alkyl substituted aryloxy, CHO, C (O) -alkyl, C (O ) -Aryl, C (O) -alkyl-carboxyl, C (O) -alkylene-carboxyester and cyano,
m is an integer selected from 0 to 4]
Or an ester, oxide, pharmaceutically acceptable salt or solvate thereof.
[2] The pharmaceutical composition according to [1], wherein the high-density lipoprotein is bound to a cytophilic peptide.
[3] The pharmaceutical composition according to [2], wherein the cell affinity peptide is a cell membrane permeable peptide.
[4] The cytophilic peptide is at least one selected from the group consisting of TAT peptide, penetratin, polyarginine (R8), LL-37, transportan, Pep-1 and MTS, [2] or [ [3] The pharmaceutical composition according to [3].
[5] The pharmaceutical composition according to any one of [2] to [4], wherein the cytophilic peptide is at least one selected from the group consisting of a TAT peptide, penetratin and polyarginine (R8).
[6] The pharmaceutical composition according to any one of [2] to [5], wherein the cytophilic peptide is penetratin.
[7] The high-density lipoprotein comprises at least one phospholipid selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC). 6] The pharmaceutical composition according to any one of the above.
[8] The pharmaceutical composition according to any one of [1] to [7], wherein the high-density lipoprotein comprises distearoylphosphatidylcholine (DSPC).
[9] The pharmaceutical composition according to [7] or [8], wherein the high-density lipoprotein comprises an additional lipid.
[10] The pharmaceutical composition according to [9], wherein the further lipid is 3β- [N- (N′N′-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol).
[11] The high-density lipoprotein comprises distearoylphosphatidylcholine (DSPC) and 3β- [N- (N′N′-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol), and DC relative to the total amount of DSPC and DC-Chol -Pharmaceutical composition in any one of [1] thru | or [10] whose ratio of Chol is about 7-15 mol%.
[12] The pharmaceutical composition according to any one of [1] to [11], wherein the high-density lipoprotein comprises a fusion protein of a cytophilic peptide and an apolipoprotein.
[13] The pharmaceutical composition according to any one of [1] to [12], wherein the high-density lipoprotein comprises N-terminal globular domain-deficient apolipoprotein AI.
[14] The pharmaceutical composition according to [13], wherein the N-terminal globular domain-deficient apolipoprotein AI is N-terminal 43 amino acid-deficient apolipoprotein AI or N-terminal 44 amino acid-deficient apolipoprotein AI.
[15] Any one of [1] to [14], wherein the high-density lipoprotein comprises a fusion protein of penetratin and N-terminal globular domain-deficient apolipoprotein AI, distearoylphosphatidylcholine (DSPC) and DC-cholesterol The pharmaceutical composition as described.
[16] The pharmaceutical composition according to [14], wherein the N-terminal globular domain-deficient apolipoprotein AI is N-terminal 44 amino acid-deficient apolipoprotein AI.
[17] The pharmaceutical composition according to any one of [1] to [16], wherein the high-density lipoprotein has a diameter of about 10 to 20 nm.
[18] The medicament according to any one of [1] to [17], wherein in formula (I), each Ra is independently selected from the group consisting of halo, hydroxy, alkyl, halo-substituted alkyl and alkoxy. Composition.
[19] The pharmaceutical composition according to any one of [1] to [18], wherein in formula (I), each Ra is independently selected from the group consisting of halo and alkyl.
[20] The pharmaceutical composition according to any one of [1] to [19], wherein in formula (I), two Ra are present, one is halo and the other is alkyl.
[21] The compound of formula (I) is of the formula

The pharmaceutical composition according to any one of [1] to [20], which is a compound of
[22] The high density lipoprotein comprises from about 3 to about 15 moles of the compound of formula (I) or an ester, oxide, pharmaceutically acceptable salt or solvate thereof per mole of apolipoprotein [1] ] Thru | or the pharmaceutical composition in any one of [21].
[23] The pharmaceutical composition according to any one of [1] to [22], for the treatment and / or prevention of eye diseases.
[24] The pharmaceutical composition according to [23], wherein the eye disease is glaucoma, retinitis pigmentosa, age-related macular degeneration or ischemic eye disease.
[25] The pharmaceutical composition according to any one of [1] to [24], which is an eye drop.
[26] A method for treating and / or preventing an eye disease, comprising administering to the subject the pharmaceutical composition according to any one of [1] to [25].
[27] Use of the pharmaceutical composition according to any one of [1] to [25] in the treatment and / or prevention of eye diseases.

  The above description is all non-limiting and can be modified without departing from the scope of the invention as defined in the appended claims. Furthermore, the following examples are all non-limiting examples and serve only to illustrate the invention.

(Experiment 1)
Production of high density lipoprotein (HDL) (method)
A fusion protein was produced in which a TAT peptide having a cell-affinity action was linked to the C-terminus of apoA-I lacking the N-terminal 43 amino acids lacking the N-terminal globular domain (1-43 amino acids). CHDL was prepared by mixing liposomes composed of dimythryl phosphatidylcholine (DMPC) and coumarin-6 which is a fluorescent labeling substance with a particle size of about 100 nm into this fused apoA-I (Spontaneous interaction method). In order to remove unreacted liposomes, phospholipid micelles, and apolipoproteins not bound to phospholipids or fluorescent substances, cHDL was purified by ultracentrifugation. In the same manner, reconstituted HDL (hereinafter referred to as rHDL) containing no cytophilic peptide was prepared. As a comparative control, submicron sized small uni-lamella vesicle (ssLip) of distearoylphosphatidylcholine (DSPC) was prepared according to the method described in International Publication No. 2009/107753 pamphlet.

The obtained cHDL, rHDL, and liposomes were examined for protein, phospholipid and coumarin-6 content, particle size, and surface potential. The protein concentration was determined by the Lowry method, the phospholipid concentration was determined by the C test (C test Wako ^{(registered trademark)} ), and the coumarin-6 concentration was determined by fluorescence spectroscopy using a fluorescence spectrophotometer FluoroMax. The particle size and surface charge state were determined as volume average diameter (MV) and zeta potential using dynamic light scattering (DLS), respectively. As the measurement apparatus, Nanotrac UPA-EX250 particle size analyzer (Nikkiso Co., Ltd .; Tokyo, Japan) and Zetasizer Nano Z (Malvern Instruments, Malvern, UK) were used. When measuring the particle size, the sample was diluted with a 100% PBS solution, and when measuring the zeta potential, the sample was diluted 10-fold with ultrapure water.

(result)
By the spontaneous interaction method and the ultracentrifugation method, HDL having a particle diameter of 10 to 20 nm could be obtained. Table 1 below shows the size of each HDL and the concentrations of the proteins, phospholipids and coumarin-6, and the zeta potential.

(Experiment 2)
Toxicity assessment (method) of HDL
In order to examine the corneal toxicity of each HDL obtained in Experiment 1, cytotoxicity tests were performed using cultured human cornea cells. Enzymatic activity of cells treated with cHDL, rHDL, and control benzalkonium chloride was measured by a colorimetric method. CCK-8 (Cell Counting Kit-8) was used as a detection reagent.

(result)
Both cHDL and rHDL showed cell survival rates equivalent to or better than the negative control HBSS / HEPES solution, and were significantly more cells than the positive control benzalkonium chloride (preservative contained in commercial eye drops). Toxicity was low (Figure 1).

(Experiment 3)
HDL Reaching Efficiency (Method)
3 μL each of cHDL, rHDL, and 100 nm diameter DSPC liposomes obtained in Experiment 1 were applied to the C57 / B6 strain of mice on the ocular surface. After 30 minutes, the eyeballs were excised and frozen sections were prepared. The fluorescence intensity of the inner retina was observed with a confocal microscope, and the results were compared.
(result)
By confocal microscopic observation, in the cHDL eye drop group, fluorescence that was not seen in the eye drop group of the HBSS / HEPES solution without coumarin-6 was observed in the inner layer of the retina (FIG. 2).

  The fluorescence intensity results were quantified for cHDL, rHDL, HBSS / HPES solution as negative control, and DSPC ssLip as positive control (FIG. 3). Compared with the HBSS / HEPES ophthalmic group, the fluorescence intensity of the inner layer of the retina was improved in all of the cHDL, rHDL, and DSPC liposome ophthalmic groups. In addition, the fluorescence intensity was higher in the cHDL eye drop group than in either the rHDL or DSPC liposome eye drop group. This indicates that HDL allows coumarin-6 to reach the inner layer of the retina, and in particular, cHDL bound with a cytophilic peptide can reach coumarin-6 to the retina to a very high extent.

(Experiment 4)
Model test of posterior ocular drug delivery system (choroidal neovascularization model)
(Method)
A choroidal neovascularization model was tested as a model test for the posterior ocular drug delivery system.
The following ophthalmic solutions were prepared: 1) 1 mL of cHDL solution having 1 mg / mL apolipoprotein concentration and 1 mL of cHDL-free buffer solution, each containing 1 mg of pazopanib, 2) equimolar ratio with apolipoprotein A cHDL solution containing pazopanib and a cHDL solution not containing pazopanib (both apolipoprotein concentration: 1.5 mg / mL). Here, the cHDL solution containing pazopanib was prepared using pazopanib instead of coumarin-6 in Experiment 1.

  The retina of 6-8 week old C57BL6 mice was irradiated with argon laser to produce choroidal neovascularization. One week immediately after laser irradiation, 3 μL of ophthalmic solution was instilled three times a day, and the eyeball was removed one week later. The choroidal neovascularization was stained by immunostaining, and the area of the choroidal neovascularization was measured under a confocal microscope by the flat mount method.

(result)
The comparison results of 1) and 2) are shown in FIGS. 4 and 5, respectively. In either case, the area of choroidal neovascularization was reduced by instillation of cHDL solution encapsulating pazopanib, suggesting that pazopanib having an angiogenesis-inhibiting action has reached the disease site through encapsulation in cHDL. This suggests that pazopanib reaches the posterior eye and suppresses angiogenesis.

(Experiment 5)
CHDL using various phospholipids
1) Preparation (method) of cHDL
Using different phospholipids (specifically, three types of DMPC, DPPC, and DSPC), cHDL was prepared by the following method. Carbon numbers of fatty acid groups of DMPC, DPPC, and DSPC are 14, 16, and 18, respectively.
Specifically, a micelle composed of coumarin-6, phospholipid, and cholic acid was prepared in advance, and cHDL was prepared by mixing the micelle with the same fusion type apoA-I as in Experiment 1 at the phase transition temperature of phospholipid. (Cholic acid dialysis method). Here, the phase transition temperature of each phospholipid is as follows: DMPC is 24 ° C, DPPC is 40-41 ° C, and DSPC is 55 ° C. The concentration of coumarin-6 was unified to 0.05 μmol / ml. The obtained cHDL was purified by ultracentrifugation according to the method of Experiment 1. According to the method of Experiment 1, the contents of cHDL protein, phospholipid and coumarin-6 obtained, and the particle size were measured.

(result)
By cholic acid dialysis and ultracentrifugation, cHDL having a particle diameter of 10 to 20 nm could be obtained. The particle size of each cHDL and each concentration of protein, phospholipid and coumarin-6 constituting the cHDL are shown in Table 2 below.

2) Efficiency of cHDL reaching the retina (method)
The arrival efficiency of the various cHDL produced above to the retina was examined. Specifically, the fluorescence intensity of the inner layer of the retina was observed with a confocal microscope in the same manner as in Experiment 3, and the results were compared.

(result)
The fluorescence intensity of the inner retina was compared by confocal microscopy. The results are shown in FIG. The fluorescence intensity was higher in the order of DSPC>DPPC> DMPC. In addition, these fluorescence intensity results were quantified according to the method of Experiment 3 (FIG. 7). This result shows that the above-mentioned cHDL allows coumarin-6 to reach the inner layer of the retina, and suggests that the higher the carbon number of the alkyl group in the acyl group of glycerophospholipid, the higher the efficiency of cHDL reaching the retina. It was.

(Experiment 6)
CHDL using various cell membrane permeable peptides
1) Preparation (method) of cHDL
Different CPPs (specifically, three types of TAT peptide, penetratin (PEN) peptide, and polyarginine (R8)) are converted into N-terminal 43 amino acid-deficient apoA-I (TAT peptide) or N-terminal 44 by genetic engineering techniques. A fusion protein fused to the C-terminus of amino acid deficient apoA-I (PEN and R8) was produced. Next, cHDL was prepared using DSPC and coumarin-6 in the same manner as in Experiment 5. The concentration of coumarin-6 was used uniformly at 0.03 μmol / ml. The obtained cHDL was purified by ultracentrifugation according to the method of Experiment 1. According to the method of Experiment 1, the content, particle size, and surface potential of the obtained cHDL protein, phospholipid, and coumarin-6 were measured.

(result)
By cholic acid dialysis and ultracentrifugation, cHDL having a particle diameter of 10 to 20 nm could be obtained. Table 3 below shows the particle size of each cHDL, each concentration of protein, phospholipid and coumarin-6, and zeta potential of each cHDL.

2) Efficiency of cHDL reaching the retina (method)
The arrival efficiency of the various cHDL produced above to the retina was examined. Specifically, the fluorescence intensity of the inner layer of the retina was observed with a confocal microscope in the same manner as in Experiment 3, and the results were compared.

(result)
The fluorescence intensity of the inner retina was compared by confocal microscopy. The results are shown in FIG. The fluorescence intensity increased in the order of PEN> R8 ≧ TAT> CPP. In addition, the fluorescence intensity results were quantified according to the method of Experiment 3 (FIG. 9). This result shows that the above-mentioned HDL allows coumarin-6 to reach the inner layer of the retina. In particular, cHDL bound with a cytophilic peptide can reach coumarin-6 to the retina to a very high degree. . It was also suggested that PEN improves the retina arrival efficiency most.

(Experiment 7)
Concentration dependence on drug delivery to the retina The concentration dependence on drug delivery to the retina was verified using cHDL containing PEN peptide, DSPC and coumarin-6.
(Method)
A cHDL solution prepared by adjusting the concentrations of coumarin-6 to 0.03 μmol / ml, 0.05 μmol / ml, 0.1 μmol / ml, and 0.2 μmol / ml, respectively, was prepared using the same method as in Experiment 5 or 6 above. did. Subsequently, the fluorescence intensity of the inner layer of the retina of the mouse administered with each solution was compared in the same manner as in Experiment 3.

(result)
The results are shown in FIG. A tendency was found that the higher the concentration of coumarin-6, the higher the fluorescence intensity. In addition, these fluorescence intensity results were quantified according to the method of Experiment 3 (FIG. 11). This result suggests that if the concentration of the drug contained in the cHDL is high, a high concentration of the drug can reach the retina. Therefore, the amount of the drug reaching the retina is contained in the applied cHDL. It turned out to be dependent on the amount of drug being used.

(Experiment 8)
Comparison experiment (method) between cHDL and conventional carrier
Pazopanib was selected as a drug, and captisol generally used in clinical trials and the like as a positive control carrier, and a DSPC liposome (DSPC ssLip) having a particle size of 100 nm, which was compared in Experiment 3, were selected. CHDL prepared by the same method as in Experiment 4 2) was used. As a comparison object, a comparative captisol solution was prepared by dissolving pazopanib in a 7% captisol solution, similarly to the method described in Yafai et al. Eur J Pharmacol. 2011; 666: 12-8 and JP 2013-525501 A Was made. Further, pazopanib dissolved in DMSO was mixed with ssLip prepared in the same manner as in Experiment 1, and then purified by removing unencapsulated pazopanib using a gel filtration column to prepare a comparative ssLip solution.

  In this experiment, in order to unify the concentration of pazopanib, ultra high performance liquid chromatography (UPLC) according to the method described in International Publication No. 2011-069053 pamphlet and Escudero-Ortiz et al. Ther Drug Monit. 2015; 37: 172-9. ) To measure the pazopanib concentration of each sample. A calibration curve was prepared by confirming the retention time of chromatography using a pazopanib standard substance solution with a known concentration and measuring the area of the peak located at the retention time. In each sample, the peak area located at the same retention time was weighed, the pazopanib concentration was quantified from the calibration curve, and diluted appropriately so that each sample had the same concentration.

  Each of these prepared solutions was instilled into an angiogenic model mouse according to the method described in Experiment 4 except that the number of instillations on mice was changed to twice a day, and choroidal neovascularization (CNV) The effect of reducing the area was investigated.

(result)
The results are shown in FIG. When cHDL was used as a carrier, the area of choroidal neovascularization was significantly reduced as compared with the case of using either captisol or ssLip as a comparison target (FIG. 12). From this, it was suggested that the drug having an angiogenic action effectively reaches the disease site through the inclusion in cHDL.

を有する化合物（化合物１：４−アミノ−３−［６−（４−フルオロ−２−メチルフェニル）ピリジン−３−イルアゾ］ナフタレン−１−スルホン酸ナトリウム塩）を内包するｃＨＤＬを作製した。(Experiment 9)
Production of cHDL (DSPC-compound 1) containing compound 1 The following structure

CHDL encapsulating a compound having a compound (compound 1: 4-amino-3- [6- (4-fluoro-2-methylphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid sodium salt) was prepared.

1) Examination of the weight ratio of cHDL to Compound 1 (method)
Phospholipid (DSPC) 10 mg was dispersed in 706 μl of a 30 mg / ml sodium cholate solution (buffer solution: PBS) to prepare cholate micelles. A fusion protein in which penetratin was connected to the C-terminus of N-terminal 44 amino acid-deficient apoA-I was dissolved in 4M urea (buffer solution: PBS) to a concentration of 1.5 mg / ml. Cholic acid micelle and fusion protein solution were mixed at a molar ratio of DSPC: fusion protein = 200: 1 at 55 ° C. for 30 minutes. The mixture was dialyzed overnight against a PBS solution, and the precipitate was removed by ultracentrifugation according to Experiment 1 (HDL purification). The obtained HDL and Compound 1 were mixed at various weight ratios at 55 ° C. for 30 minutes. The cHDL was purified using a gel filtration column, and the contents of the fusion protein and Compound 1 were measured using the Lowry method and the colorimetric method (472 nm), respectively.

(result)
As shown in Table 4, cHDL (DSPC-compound 1) containing compound 1 was obtained.

化合物１／ＨＤＬ比が表５に示す結果と近似するＤＳＰＣ−化合物１が得られた。ＤＬＳにより測定した粒径は１４.９５ｎｍであり、ＨＤＬの粒径として合理的な範囲にあった。 2) Reproducibility of DSPC-compound 1 production The addition amount of compound 1 was 1500 μg, and DSPC-compound 1 was produced again, and the contents of fusion protein, compound 1, and phospholipid (C test) were measured. The results are shown in Table 5.

A DSPC-compound 1 with a compound 1 / HDL ratio approximating to the results shown in Table 5 was obtained. The particle size measured by DLS was 14.95 nm, which was in a reasonable range as the particle size of HDL.

(Experiment 10)
Examination of lipids in HDL containing compound 1 (method)
In order to confirm the effectiveness of the sample in which the cationic potential (DOTAP, DC-Chol) was added to the phospholipid to change the zeta potential, it was prepared by changing the content ratio of each lipid in the same manner as in Experiment 9. did. The amount of Compound 1 used was prepared by adding both DOTAP-Compound 1 and DC-Compound 1 at a ratio of 2 g to 1 g of the fusion protein.

(result)
Table 6 shows the zeta potential of the obtained DOTAP-compound 1, DC-compound 1 and DSPC-compound 1 and the particle sizes before and after purification. The numbers in parentheses after DOTAP and DC indicate the mole percent of DOTAP or DC-cholesterol relative to the total amount of DSPC and DOTAP or DC-cholesterol.

  DOTAP-Compound 1 had very large particle sizes and varied. DC-Compound 1 has a relatively large particle size, but when purified by ultracentrifugation, particles having a particle size within the HDL theoretical value were obtained.

(Experiment 11)
Preparation of cHDL (DC-compound 1) containing compound 1 (method)
DSPC-DC-chol mixture (DSPC: DC-chol = 9: 1, 8: 2, or 7: 3 (molar ratio)) dissolved in 100% ethanol at a concentration of 5 mg / ml Evaporated and lyophilized. This was dispersed in a 30 mg / ml sodium cholate solution (buffer solution: PBS) at a ratio of 706 μl per 10 mg of DSPC to prepare cholate micelles. A fusion protein in which penetratin was connected to the C-terminus of the N-terminal 44 amino acid-deficient apoA-I was dissolved in 4 M urea (buffer solution: PBS) at a concentration of 1.5 mg / ml, and Compound 1 was dissolved therein (750 μg of fusion). 750 μg of compound 1) per protein. This solution and cholate micelles were mixed at a molar ratio of DSPC-DC-chol: fusion protein = 200: 1 at 55 ° C. for 30 minutes. The mixture was dialyzed overnight against PBS solution and the precipitate was removed by ultracentrifugation (286,000 g, 106 min) (HDL purification). The HDL fraction was collected and diluted and concentrated (Amicon Ultra 100K). PBS was used as a diluent. DSPC-compound 1 was also prepared in the same manner as described above.

(result)
The obtained fusion protein of DSPC-compound 1 and DC (10) -compound 1, content of compound 1 and phospholipid, particle size, and zeta potential were measured according to Experiments 1 and 9. The results are shown in Table 7.

(Experiment 12)
The arrival efficiency of Compound 1 contained in the high-density lipoprotein to the retina and vitreous tissue was examined.
Efficiency of reaching the retina and vitreous tissue of Compound 1 (method)
6-8 week old male SD rats were divided into (1) Compound 1 solution (compound 1 concentration: 4 mM, solvent PBS), (2) DSPC-compound 1 (compound 1 concentration 4.7 mM), (3) DC ( 10)-3 groups instilled with Compound 1 (Compound 1 concentration of 3.9 mM) were divided into 6 eyes, 3 animals each, and were instilled once under awakening (5 μl). DSPC-compound 1 and DC (10) -compound 1 listed in Table 7 were used concentrated until compound 1 was at the above concentration. After 30 minutes in the awake state, the animal was euthanized and the eyeball was removed. Immediately wash the removed eyeball with physiological saline, freeze it to -80 ° C, halve the eyeball in the coronal direction with a scalpel blade, and use the insulator together with the vitreous body that has become a sherbet shape. The retina was detached and collected. In order to avoid touching the cornea and sclera at that time, the insulator touching the eyeball wall and the intraocular tissue was used separately. The collected samples were weighed together for 6 eyes, frozen at −80 ° C., and homogenized for measurement of Compound 1 concentration. In preparing the homogenate, the frozen sample was crushed with a multi-bead shocker, and 5 times the sample weight of pure water was added and mixed. The concentration of Compound 1 in the sample was measured by LC_MSMS.

  The results are shown in FIG. In rats instilled with DSPC-Compound 1 or DC (10) -Compound 1, Compound 1 was detected in the retina and vitreous tissue 30 minutes after instillation. This indicates that Compound 1 contained in both high-density lipoproteins reaches the retina and vitreous tissue, and the delivery efficiency of DC (10) -Compound 1 is high.

  The pharmaceutical composition of the present application can reach the posterior eye segment by topical administration of a compound of formula (I) or an ester, oxide, prodrug, pharmaceutically acceptable salt or solvate thereof. Therefore, the risk of side effects due to systemic administration can be reduced. In addition, the pharmaceutical composition of the present application can be instilled, but instillation is safer and simpler than intravitreal injection, which is also used for local administration to the eye.

Claims (19)

Formula (I) contained in high density lipoprotein:

[Where,
Ra is halo, hydroxy, alkyl, halo substituted alkyl, aryl, halo or alkyl substituted aryl, alkoxy, hydroxy or carboxy substituted alkoxy, aryloxy, halo or alkyl substituted aryloxy, CHO, C (O) -alkyl, C (O ) -Aryl, C (O) -alkyl-carboxyl, C (O) -alkylene-carboxyester and cyano,
m is an integer selected from 0 to 4]
Or an ester, oxide, pharmaceutically acceptable salt or solvate thereof.   The pharmaceutical composition according to claim 1, wherein the high-density lipoprotein is bound to a cytophilic peptide.   The pharmaceutical composition according to claim 2, wherein the cytophilic peptide is at least one selected from the group consisting of TAT peptide, penetratin, polyarginine (R8), LL-37, transportan, Pep-1 and MTS. object.   The pharmaceutical composition according to claim 2 or 3, wherein the cytophilic peptide is at least one selected from the group consisting of a TAT peptide, penetratin and polyarginine (R8).   The pharmaceutical composition according to any one of claims 2 to 4, wherein the cytophilic peptide is penetratin.   The high-density lipoprotein comprises at least one phospholipid selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC). A pharmaceutical composition according to any one of the above.   The pharmaceutical composition according to any one of claims 1 to 6, wherein the high-density lipoprotein comprises distearoylphosphatidylcholine (DSPC).   8. A pharmaceutical composition according to claim 6 or claim 7, wherein the high density lipoprotein comprises further lipids.   9. The pharmaceutical composition according to claim 8, wherein the further lipid is 3 [beta]-[N- (N'N'-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol).   The high density lipoprotein comprises distearoylphosphatidylcholine (DSPC) and 3β- [N- (N′N′-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol), and the amount of DC-Chol relative to the total amount of DSPC and DC-Chol The pharmaceutical composition according to any one of claims 1 to 9, wherein the ratio is about 7 to 15 mol%.   The pharmaceutical composition according to any one of claims 1 to 10, wherein the high-density lipoprotein comprises a fusion protein of a cytophilic peptide and an apolipoprotein.   The pharmaceutical composition according to any one of claims 1 to 11, wherein the high-density lipoprotein comprises N-terminal globular domain-deficient apolipoprotein AI.   The medicament according to any one of claims 1 to 12, wherein the high-density lipoprotein comprises a fusion protein of penetratin and N-terminal globular domain-deficient apolipoprotein AI, distearoylphosphatidylcholine (DSPC) and DC-cholesterol. Composition.   The pharmaceutical composition according to any one of claims 1 to 13, wherein the high-density lipoprotein has a diameter of about 10 to 20 nm. The compound of formula (I) is of formula

The pharmaceutical composition according to any one of claims 1 to 14, which is a compound of   The high density lipoprotein comprises from about 3 to about 15 moles of a compound of formula (I) or an ester, oxide, pharmaceutically acceptable salt or solvate thereof per mole of apolipoprotein. Item 16. A pharmaceutical composition according to any one of Items 15.   The pharmaceutical composition according to any one of claims 1 to 16, for the treatment and / or prevention of an eye disease.   The pharmaceutical composition according to claim 17, wherein the eye disease is glaucoma, retinitis pigmentosa, age-related macular degeneration or ischemic eye disease.   The pharmaceutical composition according to any one of claims 1 to 18, which is an eye drop.

Images