Composition including glucosaminoglycan derivative and chemokine receptor activity regulator

Abstract

To provide a composition and a pharmaceutical composition each of which exhibits, while suppressing rapid degeneration of tissues caused by administration thereof, excellent regulatory activity for chemokine receptors and excellent medicinal effects on a posterior eye disease and the like.SOLUTION: Provided are a composition comprising a GAG derivative and a chemokine receptor activity regulator, and a pharmaceutical composition comprising the composition.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition and a pharmaceutical composition comprising a glycosaminoglycan derivative and a chemokine receptor activity modulator. [Background technology]

[0002] Age-related macular degeneration (AMD), a posterior segment disease involving pathological neovascularization, is broadly divided into wet and atrophic types. Wet AMD is a disease caused by choroidal neovascularization (CNV) and its proliferation changes, which occur due to changes in the retinal pigment epithelium, Bruch's membrane, and choroid in the macula. It progresses quickly and causes permanent severe vision loss.

[0003] It is known that CCR3, one of the CC chemokine receptors (hereinafter abbreviated as "CCR"), is specifically expressed in vascular endothelial cells of CNV extracted from patients with wet AMD (see, for example, Nature, 2009, 460: 225-230). In a laser-induced mouse CNV model, which is an AMD pathological model, intravitreal administration of a CCR3 inhibitor suppresses CNV and is useful for treating wet AMD (see, for example, U.S. Patent No. 8,592,482).

[0004] On the other hand, intravitreal administration of heparan sulfate or heparin, which are sulfated glycosaminoglycans (hereinafter, glycosaminoglycans are abbreviated as "GAG"), to a laser-induced mouse CNV model has been shown to suppress CNV and to be useful in the treatment of wet AMD (see, for example, International Publication No. 2011 / 122321). In addition, Heebeom Koo et al. administered a compound in which 5β-cholanic acid was introduced into hyaluronic acid (hereinafter, abbreviated as "HA") into the vitreous and evaluated the intraocular distribution using pathological tissue specimens (see, for example, Biomaterials, 2012, 33: 3485-3493). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Pat. No. 8,592,482 [Patent Document 2] International Publication No. 2011 / 122321 [Non-patent literature]

[0006] [Non-Patent Document 1] Nature, 2009, 460: 225-230. [Non-Patent Document 2] Biomaterials, 2012, 33: 3485-3493. Summary of the Invention [Problem to be solved by the invention]

[0007] However, the solution described in U.S. Patent No. 8,592,482, which contains a solubilizing agent such as DMSO in a predetermined amount or more to prepare a CCR3 inhibitor at a concentration having a pharmacological effect, may cause rapid degeneration of tissues (such as the crystalline lens) adjacent to the vitreous body immediately after intravitreal administration. Furthermore, Biomaterials, 2012, 33: 3485-3493 and WO 2011 / 122321 do not disclose or suggest a chemokine receptor activity modulator.

[0008] An object of the present invention is to provide a composition and a pharmaceutical composition which suppress rapid tissue degeneration associated with administration, and which exhibit excellent chemokine receptor activity regulating effects and excellent medicinal effects against posterior ocular diseases and the like. [Means for solving the problem]

[0009] Specific means for solving the above problems are as follows, and the present invention includes the following aspects. <1> A composition comprising a GAG derivative and a chemokine receptor activity modulator. <2> The GAG ​​derivative is a hydrophobic group-introduced GAG. <1> The composition described in <3> The GAG ​​derivative is a cross-linked GAG <1> or <2> The composition described in <4> Contains covalent bonds between GAG derivatives and chemokine receptor activity regulators <1> ~ <3> 2. The composition according to claim 1 . <5> The GAG ​​derivative is a derivative of HA or chondroitin sulfate (hereinafter abbreviated as "CS"). <1> ~ <4> 2. The composition according to claim 1 . <6> The chemokine receptor activity modulator is a chemokine receptor antagonist. <1> ~ <5> 2. The composition according to claim 1 . <7> <1> ~ <6> A pharmaceutical composition comprising the composition according to any one of the preceding claims. <8> A treatment for posterior segment diseases <7> The pharmaceutical composition described in <9> <1> ~ <6> 2. Use of the composition according to any one of claims 1 to 11 as an agent for treating a posterior segment disease. <10> <1> ~ <6> A method for treating a posterior segment disease, comprising administering into the vitreous the composition described in any one of claims 1 to 5. Effect of the Invention

[0010] According to the present invention, it is possible to provide a composition and a pharmaceutical composition which suppress rapid tissue degeneration associated with administration, and which exhibit excellent chemokine receptor modulating activity and excellent medicinal efficacy against posterior ocular diseases and the like. [Brief description of the drawings]

[0011] [Figure 1] 1 is a graph showing the CNV suppression effect when the Ki19003-introduced CS derivative according to this embodiment is administered intravitreally to an animal model. [Diagram 2] 1 is a graph showing the CNV suppression effect when the Ki19003-introduced CS derivative according to this embodiment was administered intravitreally to an animal model. [Diagram 3] 1 is a graph showing the CNV suppression effect when the Ki19003-introduced CS derivative according to this embodiment was administered intravitreally to an animal model. [Figure 4]1 is a graph showing the inhibitory effect of CNV when a composition containing SB328437 and a CS derivative according to this embodiment was intravitreally administered to an animal model. [Diagram 5] 1 is a graph showing the inhibitory effect of CNV when a composition containing SB225002 and an HA derivative according to the present embodiment was administered intravitreally to an animal model. [Figure 6] 1 is a graph showing the inhibitory effect of CNV when a composition containing GW766994 and an HA derivative according to the present embodiment was administered intravitreally to an animal model. [Figure 7] 1 is a graph showing the inhibitory effect of CNV when a composition containing Ki19003 and an HA derivative according to the present embodiment was administered intravitreally to an animal model. [Figure 8] 1 is a graph showing the inhibitory effect of CNV when a composition containing AZD3778 and an HA derivative according to this embodiment is administered intravitreally to an animal model. [Figure 9] 1 is a graph showing the inhibitory effect of CNV when a composition containing SB328437 and an HA derivative according to this embodiment was administered intravitreally to an animal model. [Figure 10] 1 is a graph showing the inhibitory effect of CNV when a composition containing GW766994 and a GAG derivative according to this embodiment is administered intravitreally to an animal model. [Figure 11] 1 is a graph showing the inhibitory effect of CNV when a composition containing Ki19003 and a GAG derivative according to this embodiment was administered intravitreally to an animal model. [Figure 12] 1 is a graph showing the inhibitory effect of CNV when a composition containing GW766994 and a CS derivative according to this embodiment is administered intravitreally to an animal model. [Figure 13] 1 is a graph showing the inhibitory effect of CNV when a composition containing RS504393 and an HA derivative according to the present embodiment was administered intravitreally to an animal model. [Figure 14] 1 is a graph showing the inhibitory effect of CNV when a composition containing PS372424 and an HA derivative according to this embodiment was administered intravitreally to an animal model. [Figure 15]1 is a graph showing the inhibitory effect of CNV when a composition containing GW766994 and a CS derivative according to this embodiment is administered intravitreally to an animal model. [Figure 16] This is a graph confirming the inhibitory effect of CNV when CS was administered intravitreally to an animal model. [Figure 17] 1 is a graph confirming the inhibitory effect of CNV when CS or a CS derivative was administered intravitreally to an animal model. [Figure 18A] 1 shows images showing the intraocular condition after intravitreal administration of Ki19003 solution to animals. [Figure 18B] 1 shows images showing the intraocular condition after intravitreal administration of SB328437 solution to animals. [Figure 18C] 1 shows images of the intraocular condition after intravitreal administration of GW766994 solution to animals. [Figure 18D] 1 shows images showing the intraocular condition after a composition containing Ki19003 and a CS derivative according to this embodiment was intravitreally administered to an animal. [Figure 18E] 1 is an image showing the intraocular condition after a composition containing GW766994 and a CS derivative according to this embodiment was intravitreally administered to an animal. [Figure 18F] 1 shows images showing the intraocular condition after a composition containing Ki19003 and a CS derivative according to this embodiment was intravitreally administered to an animal. [Figure 18G] 1 shows images showing the intraocular condition after a composition containing Ki19003 and a CS derivative according to this embodiment was intravitreally administered to an animal. [Figure 18H] 1 shows images showing the intraocular condition after a composition containing SB328437 and an HA derivative according to this embodiment was intravitreally administered to an animal. [Figure 19A] 1 shows images showing the intraocular condition after intravitreal administration of RS504393 solution to animals. [Figure 19B] 1 shows images showing the intraocular condition after intravitreal administration of PS372424 solution to animals. [Figure 19C]1 shows images showing the intraocular condition after a composition comprising RS504393 and an HA derivative according to the present embodiment was administered intravitreally to an animal. [Figure 19D] 1 shows images showing the intraocular condition of an animal after a composition containing PS372424 and an HA derivative according to this embodiment was administered intravitreally. [Figure 19E] 1 is an image showing the intraocular condition after a composition containing GW766994 and a CS derivative according to this embodiment was intravitreally administered to an animal. [Figure 19F] 1 is an image showing the intraocular condition after a composition containing GW766994 and a CS derivative according to this embodiment was intravitreally administered to an animal. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In this specification, the term "step" includes not only an independent step, but also a step that cannot be clearly distinguished from other steps but achieves the intended purpose of the step. Furthermore, when a component has a content of a component in a composition, the content of the component means the total amount of the components in the composition unless otherwise specified, in cases where the component has a content of a plurality of substances in the composition. The present invention will be described in detail below with reference to embodiments thereof.

[0013] (1) Composition The composition of the present invention contains a GAG derivative and a chemokine receptor activity modulator. The composition of the present invention may contain the GAG ​​derivative and the chemokine receptor activity modulator as independent compounds, or may contain the GAG ​​derivative and the chemokine receptor activity modulator as a complex formed by physical interaction or chemical bond.

[0014] Examples of this "GAG derivative" include hydrophobic group-introduced GAG in which a group derived from GAG and a hydrophobic group are covalently bonded, optionally via a spacer group; GAG crosslinked bodies in which GAGs are crosslinked intramolecularly or intermolecularly; and compounds containing a group derived from GAG and a spacer group.

[0015] Examples of "hydrophobic group-introduced GAG" include compounds in which a hydrophobic group is covalently bonded, optionally via a spacer group, by an amide bond or ester bond with a carboxy group of GAG, or an ether bond or ester bond with a hydroxy group of GAG. Examples of "GAG crosslinked body" include compounds in which a carboxy group or a hydroxy group of GAG is covalently bonded to each other via a crosslinkable group within or between GAG molecules, and compounds in which a carboxy group and a hydroxy group of GAG are crosslinked within or between GAG molecules without a crosslinkable group.

[0016] The GAG ​​that constitutes the GAG ​​derivative is an acidic polysaccharide having a repeating structure of a disaccharide consisting of an amino sugar and uronic acid (or galactose). Examples of such GAG include HA, chondroitin, CS, dermatan sulfate, and keratan sulfate, of which HA and CS are preferred. In addition, the acidic functional group such as a carboxyl group of the GAG ​​may be in a free state without forming a salt, or in a state of forming a pharmaceutical acceptable salt.

[0017] Examples of pharma- ceutically acceptable salts include salts with alkali metal ions such as sodium salts and potassium salts, and salts with alkaline earth metal ions such as magnesium salts and calcium salts. Among these, from the viewpoint of compatibility and affinity with the living body, salts with pharma- ceutically acceptable alkali metal ions are preferred, and sodium salts are more preferred. The weight-average molecular weight of GAG is not particularly limited and can be appropriately selected depending on the purpose. The weight-average molecular weight of GAG can be exemplified as 500 Da to 10 million Da or 40,000 Da to 5 million Da. The weight-average molecular weight of GAG can be measured by a light scattering method.

[0018] GAG constituting the GAG ​​derivative can be produced by known methods according to its type, such as extraction and purification from animal-derived raw materials, culture and purification from GAG-producing bacteria, glycosylation, and glycosyl synthesis.

[0019] The "group derived from GAG" in the hydrophobic group-introduced GAG is a group formed by removing a hydroxy group from a carboxy group in GAG, a group formed by removing a hydrogen atom from a hydroxy group, etc. in GAG, etc. The "hydrophobic group" in the hydrophobic group-introduced GAG is not particularly limited, and examples include groups derived from alicyclic compounds such as cholic acid, lithocholic acid, and deoxycholic acid, which are bile acids, and cholanic acid, which is the basic skeleton of these acids, and groups derived from fatty acids such as stearic acid and oleic acid. At least one selected from these is preferred, and groups derived from alicyclic compounds are more preferred, with cholanic acid, and particularly 5β-cholanic acid, being even more preferred. Here, the "group derived from an alicyclic compound" refers to a group formed by removing a hydrogen atom or a hydroxyl group from an alicyclic compound, and the "group derived from a fatty acid" refers to a group formed by removing a hydroxyl group from a fatty acid. Furthermore, the introduction rate of the hydrophobic group into the GAG ​​is not particularly limited and can be appropriately selected depending on the purpose, etc., and can be, for example, 0.1 to 80 mol %.

[0020] In the hydrophobic group-introduced GAG, a group derived from the GAG ​​may be covalently bonded to the hydrophobic group via a spacer group. The term "spacer group" refers to a divalent group capable of covalently linking two groups, specifically, -NH-(CH2) m -NH-, -C(=O)-(CH2) m -NH-, -C(=O)-(CH2) m -C(=O)-, -NH-(CH2) m -O-, -NH-CH2-(OCH2) m -NH-, -NH-CH2-(OCH2) m -O-, -C(=O)-CH2-(OCH2) m -NH-, -C(=O)-CH2-(OCH2) m -(C=O)-, -C(=O)-(CH2) m -O-, -C(=O)-CH2-(OCH2)m Examples thereof include —O—, and at least one selected from these is preferable, and the following formula: -NH-(CH2) m -NH- (I) -C(=O)-(CH2) m -NH- (II) or -NH-(CH2) m -O- (III) In this case, m's each independently represent an integer of 2 to 12, preferably 2 to 5, and particularly preferably 2.

[0021] The spacer group is not limited to being linear, and the methylene groups contained in the spacer group may have a substituent such as an alkyl group, an aryl group, a hydroxyl group, a halogen atom, etc. Furthermore, at least a portion of the methylene groups may be replaced with an oxygen atom, an aryl group, etc.

[0022] The hydrophobic group-introduced GAG can be produced by a conventional method in which a GAG, a compound that forms a hydrophobic group, and a compound that forms a spacer group, which is used optionally, are bonded together using a condensation agent or the like.

[0023] The GAG ​​crosslinked body can be formed by, for example, using a crosslinking compound having two crosslinking groups, covalently bonding at least one of the carboxyl group and hydroxyl group of GAG via the crosslinking group. Examples of the crosslinking compound include compounds having a photoreactive group, a polymerizable functional group, an amino group, or a thiol group, and preferably compounds having a photoreactive group or a polymerizable functional group. The method of crosslinking GAG can be, for example, the following four categories, but the method is not limited thereto. (a) Crosslinking with aldehyde crosslinkers such as formaldehyde and glutaraldehyde. (b) Self-crosslinking between a carboxy group of a GAG and a hydroxy group of a GAG without using a crosslinking group. (c) Crosslinking with homobifunctional crosslinking agents such as diepoxide compounds, divinyl sulfones, diamine compounds, and dihydrazide compounds, and heterobifunctional crosslinking agents such as epihalohydrins. (d) Crosslinking by reacting a GAG having a functional group such as a photoreactive group, a polymerizable functional group, an amino group, a thiol group, or a halogen atom introduced therein with a GAG having a reactive group complementary to the above functional group introduced therein or with a crosslinking agent having two of such reactive groups.

[0024] Among these, GAG crosslinked bodies obtained by crosslinking (d) are preferred, and GAG derivatives obtained by photocrosslinking a GAG having a photoreactive group introduced therein, and GAG crosslinked bodies obtained by crosslinking a GAG having a polymerizable functional group introduced therein with a crosslinking agent having two thiol groups are more preferred.

[0025] Examples of the photoreactive group include a group derived from cinnamic acid. Such a photoreactive group may be bonded to a group derived from GAG via a spacer group, and it is preferable that the group derived from cinnamic acid is covalently bonded to GAG via a spacer group represented by the above formula (I), (II) or (III). Examples of the polymerizable functional group include a group derived from (meth)acrylic acid, and these polymerizable functional groups may be bonded to a group derived from GAG via a spacer group, and it is preferable that the group derived from (meth)acrylic acid is covalently bonded to GAG via a spacer group represented by the above formula (I), (II) or (III). Examples of reactive groups complementary to the polymerizable functional group include a thiol group, and examples of crosslinking agents having two thiol groups include thiol-PEG-thiol.

[0026] The content of crosslinkable groups in the crosslinked GAG is not particularly limited and may be appropriately selected depending on the purpose, etc.

[0027] A "compound containing a group derived from GAG and a spacer group" is a compound in which one end of the spacer group is covalently bonded to a group derived from GAG. When a composition contains the compound, it is preferable that a group derived from a chemokine receptor activity modulator is covalently bonded to the other end of the spacer group. The group derived from GAG and the spacer group are as described above, and the group derived from a chemokine receptor activity modulator is a group formed by removing a hydrogen atom or a hydroxy group from a chemokine receptor activity modulator described below. The amount of the spacer group in the compound containing a GAG-derived group and a spacer group is not particularly limited and may be appropriately selected depending on the purpose, etc.

[0028] A "chemokine receptor activity modulator" refers to a compound (drug) that acts on a chemokine receptor or a substance that binds to a chemokine receptor and modulates (suppresses or enhances) signal transduction mediated by the chemokine receptor, and examples of such compounds include chemokine receptor inhibitors, anti-chemokine antibodies, and chemokine receptor agonists. Drugs that suppress signal transduction mediated by the chemokine receptor include, for example, chemokine receptor antagonists that competitively or non-competitively inhibit chemokine receptors, and among these, CCR3 antagonists, CXC receptor (CXCR)2 antagonists, CCR2 antagonists, and the like are preferred. Drugs that enhance signal transduction mediated by the chemokine receptor include, for example, chemokine receptor agonists that act on chemokine receptors and enhance their action, and the like are preferred, for example, CXCR3 agonists, and the like.

[0029] Specifically, examples of CCR3 antagonists include Ki19003 (see, for example, International Publication No. WO 02 / 059081), SB328437 (see, for example, Journal of Biological Chemistry, 2000, 275(47), 36626-31), GW766994 (see, for example, International Publication No. WO 03 / 082292), AZD3778 (see, for example, International Publication No. WO 03 / 004487), and the like. Examples of CXCR2 antagonists include SB225002 (see, for example, Bioorganic & Medicinal Chemistry, 2009, 17(23), 8102-8112). Examples of CCR2 antagonists include RS504393 (see, for example, J. Biol. Chem., 2000, 275(33)). An example of a CXCR3 agonist is PS372424 (see, for example, Biochem. Biophys. Res. Commun., 2006, 349(1), 221-8). Of these, it is particularly preferable to use a CCR3 antagonist. Specific examples of chemokine receptor activity modulators are shown below by their structural formulas, but the chemokine receptor activity modulators used in the present invention are not limited to these compounds.

[0030] [ka]

[0031] Such chemokine receptor activity modulators may form salts (e.g., pharma- ceutically acceptable salts), such as hydrochloride, hydrobromide, hydroiodide, hydrogen sulfate, hydrogen phosphate, methanesulfonate, nitrate, hydrogen maleate, acetate, hydrogen citrate, hydrogen fumarate, hydrogen tartrate, hydrogen oxalate, hydrogen succinate, benzoate, and p-toluenesulfonate.

[0032] The chemokine receptor activity modulator may be contained in the composition as a complex formed by physical interaction with a GAG derivative. A complex containing a GAG derivative, such as a hydrophobic group-introduced GAG or a GAG crosslinker, and a chemokine receptor activity modulator can suppress rapid tissue degeneration associated with administration, and also exhibits excellent chemokine receptor activity modulatory effects and excellent medicinal effects against posterior segment diseases such as AMD.

[0033] The chemokine receptor activity modulator and the GAG ​​derivative may form a complex by chemical bonding, and among these, a covalent bond in which a group derived from GAG is covalently bonded to a group derived from the chemokine receptor activity modulator via a spacer group is preferred. Among these, a covalent bond in which a carboxy group of GAG is covalently bonded to a carboxy group of the chemokine receptor activity modulator via a spacer group represented by the following formula (III) is preferred, and a covalent bond in which the carboxy group of GAG forms an amide bond with the spacer group of formula (III) is particularly preferred. -NH-(CH2) m -O- (III) In the formula, m represents an integer of 2 to 12, preferably 2 to 5, and more preferably 2.

[0034] The form of the covalent bond between the group derived from the chemokine receptor activity modulator and the spacer group in the covalent bond is not particularly limited, and an example of the covalent bond is an ester bond. There are no particular limitations on the method for preparing the covalent bond, the introduction rate of the chemokine receptor activity modulator, and the like, and these can be appropriately selected depending on the purpose, etc. With the composition of the present invention, chemokine receptor activity modulators, which previously had to be dissolved in a solubilizing agent for intravitreal administration even at concentrations so low that they had no pharmacological effect when used alone, can also be administered intravitreally without the use of a solubilizing agent, or if a solubilizing agent is used, the amount is kept to a very small amount. The "solubilizing agent" referred to in the present specification is not particularly limited as long as it can dissolve the poorly water-soluble chemokine receptor activity modulator, and examples thereof include organic solvents such as DMSO, polysorbate 80, macrogol 400, cyclodextrin, etc. The composition of the present invention preferably contains no more than 10% by weight of such a solubilizer (for example, an organic solvent), and more preferably contains substantially no solubilizer. Furthermore, it is preferable that the composition of the present invention does not cause aggravation of intraocular bleeding when administered into the eye. The presence or absence of aggravation of intraocular bleeding can be confirmed by the method described in the Examples below.

[0035] (2) Method for producing the composition Examples of the embodiment of the composition of the present invention include a mixture of a GAG derivative and a chemokine receptor activity modulator, and a covalent bond formed by covalently bonding a GAG derivative and a chemokine receptor activity modulator. In this case, the mixing method and the covalent bonding method are not particularly limited. For example, a mixing method can be a method of stirring and mixing a PBS solution of a GAG derivative and a chemokine receptor activity modulator dissolved in a solvent such as ethanol. In addition, for example, a covalent bonding method can be a method of bonding a chemokine receptor activity modulator to a functional group of a GAG derivative (preferably a GAG derivative containing a spacer group) via an amide bond, ester bond, or ether bond. Thereafter, dialysis, precipitation, lyophilization, concentration to dryness, etc. may be performed as necessary.

[0036] (2-1) Method for producing a composition containing a hydrophobic group-introduced GAG and a chemokine receptor activity modulator (2-1-1) Method for producing hydrophobic group-introduced GAG Hydrophobic group-introduced GAG can be obtained, for example, by covalently bonding a functional group (e.g., carboxy group) of a GAG molecule to a functional group (carboxy group) of a hydrophobic group via a spacer group. Hereinafter, as an example of a method for producing hydrophobic group-introduced GAG, a case where the compound forming the hydrophobic group is 5β-cholanic acid will be described, but the present invention is not limited thereto. Hydrophobic group-introduced GAG, which is obtained by covalently bonding a group derived from GAG and a group derived from 5β-cholanic acid via a spacer, can be produced, for example, by a method including the following steps.

[0037] (A) condensing the carboxyl group of 5β-cholanic acid with the amino group of the spacer-forming molecule to form a covalent bond (amide bond); (B) condensing a carboxy group of the GAG ​​with an amino group of the spacer-forming molecule to form a covalent bond (amide bond).

[0038] In step (A), the carboxyl group of 5β-cholanic acid is condensed with the amino group of the spacer-forming molecule to form a covalent bond. The amino group of the spacer-forming molecule that is to react with GAG may be protected by a commonly used method as necessary. In step (B), the carboxy group of the GAG ​​and the amino group of the spacer-forming molecule are condensed to form a covalent bond. The method for producing a GAG derivative is only required to include steps (A) and (B), and the order in which the steps are carried out is not limited.

[0039] As the spacer-forming molecule, a compound represented by the following formula can be mentioned. H2N-(CH2) m -NH2 In the formula, m represents an integer of 1 to 12, preferably 2 to 5, and more preferably 2.

[0040] The condensation (esterification, amidation) method used in the production of hydrophobic group-introduced GAG may be appropriately selected from commonly used methods. For example, methods using condensation agents such as water-soluble carbodiimide (e.g., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (WSC)), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride hydrate (DMT-MM), dicyclohexylcarbodiimide, symmetric acid anhydride method, mixed acid anhydride method, active ester method, etc. Condensation reaction conditions are appropriately selected depending on the condensation reaction to be applied. Examples of the solvent used in the condensation reaction include water, DMSO, methanol, ethanol, propanol, butanol, acetonitrile, DMF, THF, formamide, and mixed solvents thereof, and preferred are mixed solvents of ethanol and water and mixed solvents of DMF and formamide.

[0041] (2-1-2) Method for mixing hydrophobic group-introduced GAG and chemokine receptor activity modulator The hydrophobic group-introduced GAG in PBS solution is mixed with a chemokine receptor activity modulator dissolved in ethanol, followed by dialysis and freeze-drying to obtain the product. The mixing method, dialysis method, and freeze-drying method are not limited and may be appropriately selected from commonly used methods.

[0042] (2-2) Method 1 for producing a composition containing a GAG crosslinker and a chemokine receptor activity modulator (2-2-1) Method for producing GAG crosslinked body As an example of the GAG ​​crosslinked product, for example, a photocrosslinkable GAG ​​can be prepared by the method described in JP 2002-249501 A. Specifically, a photoreactive group (e.g., a cinnamic acid derivative) can be introduced into the carboxy group of GAG by a condensation reaction to obtain a photoreactive crosslinkable GAG. The obtained photocrosslinkable GAG ​​can be subjected to a photoreaction to produce a GAG crosslinked product.

[0043] (2-2-2) Method for mixing GAG crosslinker and chemokine receptor activity modulator The desired composition can be prepared by mixing the chemokine receptor activity modulator with the aqueous solution of the GAG ​​crosslinked product obtained above by stirring.

[0044] (2-3) Method 2 for producing a composition containing a GAG crosslinker and a chemokine receptor activity modulator (2-3-1) Method for producing GAG crosslinked precursor For example, a GAG crosslinked precursor can be prepared by introducing a methacryl group, optionally via a spacer group, to the carboxy group of GAG by the method described in the Examples below. The resulting GAG crosslinked precursor can be reacted with, for example, thiol-PEG-thiol to produce a crosslinked product, but is not limited to this compound.

[0045] (2-3-2) Method for producing the composition The desired composition can be obtained by mixing the GAG ​​crosslinked precursor obtained above with a chemokine receptor activity modulator, and then carrying out a crosslinking reaction with a crosslinking agent such as thiol-PEG-thiol.

[0046] (2-4) Method 3 for producing a covalent bond between a GAG derivative and a chemokine receptor activity modulator A GAG derivative having a chemokine receptor activity modulator covalently bonded thereto can be obtained by covalently bonding a functional group (e.g., a carboxy group) possessed by the GAG ​​to a functional group (e.g., a carboxy group) possessed by the chemokine receptor activity modulator via a spacer group.

[0047] (2-4-1) Method for producing GAG covalently bound to a chemokine receptor activity modulator A GAG derivative in which a group derived from GAG and a group derived from a chemokine receptor activity modulator are covalently bonded via a spacer can be produced, for example, by a method including the following steps. (A) condensing a carboxy group of the chemokine receptor activity modulator with a hydroxy group of the spacer-forming molecule to form a covalent bond (ester bond); (B) condensing a carboxy group of the GAG ​​with an amino group of the spacer-forming molecule to form a covalent bond (amide bond).

[0048] In step (A), the carboxy group of the chemokine receptor activity modulator is covalently bonded to the hydroxy group of the spacer-forming molecule by condensation. At this time, the amino group of the spacer-forming molecule that is to react with GAG may be protected by a commonly used method, if necessary. In step (B), the carboxy group of the GAG ​​and the amino group of the spacer-forming molecule are condensed to form a covalent bond. The method for producing a GAG derivative is only required to include steps (A) and (B), and the order in which the steps are carried out is not limited.

[0049] The spacer-forming molecule may be a compound represented by the following formula: HO-(CH2) m -NH2 In the formula, m represents an integer of 1 to 12, preferably 2 to 5, and more preferably 2. The condensation (esterification, amidation) method may be appropriately selected from commonly used methods.

[0050] GAG derivatives can have various structures. Specific examples of the structure of GAG derivatives include at least one structural unit represented by the following chemical formula (IVa) in the case of CS derivatives, and at least one structural unit represented by the following chemical formula (Va) in the case of HA derivatives.

[0051] [ka]

[0052] R 12 or R 13 The group represented by the formula is covalently bonded to the carbonyl group derived from GAG at the position * in the formula.

[0053] Specific examples of the structure of a covalent bond between a GAG derivative and a chemokine receptor activity modulator include a structure containing at least one structural unit represented by the following chemical formula (IVb) in the case of a CS derivative, and at least one structural unit represented by the following chemical formula (Vb) in the case of an HA derivative.

[0054] [ka]

[0055] R 22 or R 23 The group represented by the formula is covalently bonded to the carbonyl group derived from GAG at the position * in the formula.

[0056] (3) Pharmaceutical composition The pharmaceutical composition of the present invention contains the above-mentioned composition, and may further contain other components such as pharma- ceutically acceptable excipients, etc., as necessary. Examples of other components include pharma- ceutically acceptable excipients, surfactants, stabilizers, liquid media, etc. The use of the pharmaceutical composition is not particularly limited, but it is preferably used, for example, for the treatment of posterior segment diseases.

[0057] (4) Treatment of posterior ocular diseases The agent for treating a posterior ocular disease is a pharmaceutical composition containing the composition, and is used for treating a posterior ocular disease. As used herein, the term "treatment" refers to any treatment administered to treat a disease, and examples thereof include cure, amelioration, and inhibition of progression (prevention of aggravation) of a disease.

[0058] The form of the agent for treating posterior ocular diseases is not particularly limited as long as it is a formulation or pharmaceutical form that can be administered to human eyes. The form when administered is preferably liquid, such as a solution or suspension. The solution or suspension can also be administered by dissolving the powder of the composition when used.

[0059] The dosage of the agent for treating posterior ocular diseases, in the case of a liquid, can be, for example, about 10 to 1000 μL for intravitreal administration. In addition, when the agent for treating a posterior ocular disease is in the form of a liquid, the concentration of the chemokine receptor activity regulator is, for example, 0.001 to 5% by weight, and the concentration of the GAG ​​derivative is, for example, 0.01 to 10% by weight.

[0060] Examples of the method of administration of the agent for treating a posterior ocular disease include intravitreal administration, subconjunctival administration, conjunctival sac administration, sub-Tenon administration, eye drop administration, and administration to a device placed in the eye, with intravitreal administration being preferred, and among the intravitreal administrations, intravitreal injection being the most preferred. The frequency of administration of the agent for treating posterior ocular diseases may be appropriately determined depending on the pathology, drug concentration, etc. Alternatively, the agent may be administered as necessary when the pathology worsens, but is not limited thereto.

[0061] The posterior ocular disease treatment agent is intended to be applied to the treatment of posterior ocular diseases. Here, "posterior ocular disease" refers to a disease or other abnormality that occurs in the posterior ocular segment that progresses to a pathological condition caused by neovascularization or exhibits neovascularization, and includes, for example, diabetic retinopathy, diabetic macular edema, retinal artery occlusion, branch retinal vein occlusion (BRVO), central retinal vein occlusion (CRVO), retinopathy of prematurity, central serous chorioretinopathy, central exudative chorioretinopathy, neovascular maculopathy, AMD (exudative AMD with CNV lesions and atrophic AMD with risk of developing CNV and / or progressing to exudative AMD), etc. Among these, the more preferred applicable diseases of the posterior ocular disease treatment agent of the present invention are AMD, diabetic retinopathy, and diabetic macular edema, and the particularly preferred applicable disease is exudative AMD.

[0062] Examples of the agent for treating posterior ocular diseases include a liquid preparation that does not cause aggravation of intraocular bleeding and contains 10% by weight or less of a solubilizing agent; a liquid preparation that does not cause aggravation of intraocular bleeding and contains a crosslinked GAG and a chemokine receptor activity modulator; a liquid preparation that does not cause aggravation of intraocular bleeding and contains 10% by weight or less of a solubilizing agent and contains a crosslinked GAG and a chemokine receptor activity modulator; a liquid preparation that does not cause aggravation of intraocular bleeding and contains photocrosslinked HA and a chemokine receptor antagonist; a liquid preparation that does not cause aggravation of intraocular bleeding and contains 10% by weight or less of a solubilizing agent and contains photocrosslinked HA and a chemokine receptor antagonist; a liquid preparation that does not cause aggravation of intraocular bleeding and contains a hydrophobic group-introduced GAG and a chemokine receptor activity modulator. A liquid preparation comprising a hydrophobic group-introduced GAG and a chemokine receptor activity regulator, which does not cause aggravation of intraocular bleeding and has a solubilizer content of 10% by weight or less; A liquid preparation comprising a hydrophobic group-introduced CS and a chemokine receptor antagonist, which does not cause aggravation of intraocular bleeding and has a solubilizer content of 10% by weight or less and a hydrophobic group-introduced CS and a chemokine receptor antagonist; A liquid preparation comprising a CS and a CCR3 inhibitor into which a group derived from cholanic acid has been introduced, which does not cause aggravation of intraocular bleeding; A liquid preparation comprising a CS and a CCR3 inhibitor into which a group derived from cholanic acid has been introduced, which has a solubilizer content of 10% by weight or less and a CS and a CCR3 inhibitor into which a group derived from cholanic acid has been introduced; and the like. These embodiments of the posterior ocular disease treatment agent can be used as an agent for suppressing the progression of exudative AMD, which is characterized by being administered by injection into the vitreous body of a human eye.

[0063] (5) Methods for treating posterior segment diseases The method for treating a posterior ocular disease includes a step of intraocularly administering a therapeutic agent for a posterior ocular disease. The method for treating a posterior ocular disease may further include other steps as necessary. The method for treating a posterior ocular disease can be carried out in the same manner as described in (4) Medication for treating a posterior ocular disease above, and the preferred conditions, administration frequency, etc. of the therapeutic agent are the same as those described above. EXAMPLES

[0064] The following examples and test examples of the present invention will be described in more detail. However, the technical scope of the present invention is not limited by these. The content of GAG substituents and chemokine receptor activity modulators in the composition was measured by the methods described below. The molecular weight of GAG is the weight average molecular weight.

[0065] (1) Preparation of GAG Derivatives and Compositions Example 1: Synthesis of aminoethyl 5β-cholanoamide Methanol (5 ml) and concentrated hydrochloric acid (0.18 ml) were added to 5β-cholanic acid (1 g, Aldrich), and the mixture was stirred at 60°C for 6 hours, after which the reaction solution was cooled to room temperature and the precipitated solid was collected by filtration. Ethylenediamine (5 ml, Wako Pure Chemical Industries, Ltd.) was added to the obtained compound and stirred at 130°C for 5 hours. After confirming the target product by LCMS, the mixture was returned to room temperature and the obtained solid was collected by filtration, washed with distilled water and dried to obtain compound 1 (895 mg). ESI-MS; Calcd for C267H3746N2O [M + H] + , 404; found 404

[0066] (Example 2) Synthesis of 5β-cholanic acid-introduced HA Formamide (40 ml) was added to HA (average molecular weight approximately 210,000, Lifecore Biomedical, 500 mg) and dissolved by heating and stirring at 40°C for 2 hours, and WSC (Wako Pure Chemical Industries, 205 mg) and N-hydroxysuccinimide (Watanabe Chemical Industry, 123 mg) were added and stirred. Compound 1 (96 mg) in DMF solution (10 ml) was added, and DMF (30 ml) was further added and stirred at room temperature for 24 hours. The reaction solution was placed in a dialysis membrane (Spectra / Pore RC Biotech Membrane MWCO 8-10 kDa, purchased from Funakoshi) and dialyzed for three days against methanol:distilled water (3:1), methanol:distilled water (1:1), and distilled water in that order. The dialysate was collected and cation exchange resin (DOWEX TM50Wx8 50-100, 2 g, manufactured by Wako Chemical Co., Ltd.) was added and stirred for 30 minutes. The reaction solution was filtered and freeze-dried to obtain compound 2 (590 mg). The introduction rate of 5β-cholanic acid was 4.7 mol%.

[0067] (Example 3) Synthesis of 5β-cholanic acid-introduced CS Using CS (average molecular weight about 40,000, manufactured by Seikagaku Corporation, 500 mg), a reaction was carried out according to the method of Example 2 to obtain compound 3 (490 mg). The introduction rate of 5β-cholanic acid was 10.0 mol%. The drug solution for administration to animals was prepared by dissolving the lyophilized product in PBS to 10 mg / ml and filtering through a 0.22 μm filter.

[0068] Example 4 Preparation of Ki19003-containing HA derivative Ki19003 (synthesized according to WO 02 / 059081A2, 35 mg) was dissolved in ethanol (2 ml), and a solution of compound 2 (40 mg) synthesized in Example 2 in PBS (8 ml, pH 7.4) was added and dissolved, then the mixture was mixed and stirred, and dialyzed against distilled water for 7 hours (Spectra / Pore RC Biotech Membrane MWCO 3.5-5 kDa, Spectrum Laboratories). The dialysate was collected, filtered through a 0.45 μm filter, and freeze-dried to obtain composition 4 (35 mg). The Ki19003 content was 14.0 wt%. The obtained composition 4 was dissolved in PBS to a concentration of 3 mg / ml, and filtered through a 0.22 μm filter to prepare a sample for administration to animals.

[0069] Example 5 Preparation of Ki19003-containing CS derivative Using a solution of compound 3 (35 mg) synthesized in Example 3 in PBS (8 ml, pH 7.4), a reaction was carried out in accordance with the method of Example 4 to obtain composition 5 (33 mg). The Ki19003 content was 22.0 wt%. The obtained composition 5 was dissolved in PBS to a concentration of 3 mg / ml, and filtered through a 0.22 μm filter to prepare a sample for administration to animals.

[0070] Example 6 Preparation of HA derivatives containing GW766994 GW766994 (synthesized according to WO 03 / 082292A1, 30 mg) was dissolved in ethanol (2 ml), and a solution of compound 2 (40 mg) synthesized in Example 2 in PBS (8 ml, pH 7.4) was added and dispersed. The resulting dispersion was dialyzed (Spectra / Pore RC Biotech membrane, MWCO 3.5-5 kDa) against distilled water for 7 hours. The dialysate was collected, filtered through a 0.45 μm filter, and freeze-dried to obtain composition 6 (35 mg). The GW766994 content was 23.5 wt%. The resulting composition 6 was dissolved in PBS to a concentration of 3 mg / ml, and filtered through a 0.22 μm filter to prepare a sample for administration to animals.

[0071] (Example 7) Preparation of GW766994-containing CS derivative (1) Using a solution of compound 3 (35 mg) synthesized in Example 3 in PBS (8 ml, pH 7.4), a reaction was carried out in accordance with the method of Example 6 to obtain composition 7 (31 mg). The GW766994 content was 27.7% by weight. The obtained composition 7 was dissolved in PBS to a concentration of 3 mg / ml, and filtered through a 0.22 μm filter to prepare a sample for administration to animals.

[0072] (Example 8) Preparation of GW766994-containing CS derivative (2) GW766994 (12 mg) was dissolved in ethanol (2 ml), and a solution of compound 3 (60 mg) synthesized in Example 3 in PBS (8 ml, pH 7.4) was added and mixed and stirred. The mixture was dialyzed (Slide-A-Lyzer, MWCO 3.5 kDa, Thermo Fisher Scientific) against distilled water for 7 hours. The dialysate was collected, filtered through a 0.22 μm filter, and freeze-dried to obtain composition 8 (40 mg). The GW766994 content was 9.4% by weight. The obtained composition 8 was dissolved in PBS to a concentration of 10 mg / ml, and prepared as a sample for administration to animals.

[0073] (Example 9) Synthesis of 2-aminoethyl esterified Ki19003 Ki19003 (190 mg) was dissolved in methylene chloride (2 ml), dimethylaminopyridine (Wako Pure Chemical Industries, Ltd., 13 mg), WSC (Wako Pure Chemical Industries, Ltd., 206 mg), and N-Boc-ethanolamine (Wako Pure Chemical Industries, Ltd., 174 mg) were added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 3 hours. After confirming the completion of the reaction by LCMS, the reaction solution was washed with saturated saline, dehydrated with anhydrous magnesium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography (hexane: ethyl acetate = 1:2) to obtain a white solid (81 mg). 4M HCl / AcOEt (10 ml, Kokusan Chemical Industries, Ltd.) was added to this, and the mixture was stirred at room temperature for 1 hour. After confirming the target product by LCMS, the reaction solution was concentrated to obtain compound 9 (71 mg). ESI-MS; Calcd for C27H37Cl3N4O3 [M + H] + , 573; found 573

[0074] (Example 10) Synthesis of Ki19003-introduced CS Distilled water (4 ml) was added to CS (average molecular weight about 40,000, manufactured by Seikagaku Corporation, 270 mg), and the mixture was stirred for 30 minutes to dissolve. An ethanol solution (4 ml) of compound 9 (64 mg) was added, and DMT-MM (manufactured by Wako Pure Chemical Industries, 47 mg) was further added and stirred at room temperature overnight. Salt (270 mg) was added to the reaction solution, and the mixture was dropped into ethanol (40 ml). The precipitated white solid was filtered, washed three times with 90% ethanol water, and dried overnight with a vacuum pump to obtain compound 10 (281 mg, introduction rate 18.0 wt%). The obtained compound 10 was dissolved in PBS to 3 mg / ml, and filtered again with a 0.22 μm filter to obtain composition 10. This was used as a sample for administration to animals.

[0075] (Example 11) Synthesis of Ki19003-introduced CS 2 CS (average molecular weight about 150,000, manufactured by Seikagaku Corporation, 150 mg) was reacted according to the method described in Example 10. Sodium chloride (150 mg) was added to the reaction solution, and the mixture was dropped into ethanol (40 ml). The precipitated white solid was collected by filtration, washed three times with 90% ethanol water, and dried overnight with a vacuum pump to obtain a white solid. The obtained solid was dissolved in distilled water, filtered through a 0.22 μm filter, and lyophilized to obtain compound 11 (91 mg, introduction rate 5.3 wt%). The obtained compound 11 was dissolved in PBS to a concentration of 10 mg / ml to obtain composition 11. This was used as a sample for administration to animals.

[0076] (Example 12) Synthesis of 2-aminoethyl methacrylate hydrochloride Methacryloyl chloride (10.5mL, Tokyo Chemical Industry Co., Ltd.) was added dropwise over 3 minutes to a solution of N-Boc-ethanolamine (15.5g, Tokyo Chemical Industry Co., Ltd.) and diisopropylethylamine (25.2mL, Tokyo Chemical Industry Co., Ltd.) in methylene chloride (180mL) under an argon atmosphere at -78°C, and the mixture was stirred for 1 hour. The mixture was then stirred at room temperature for 14 hours. The reaction solution was washed with water, extracted with methylene chloride, and dried over sodium sulfate. The solid was removed by filtration, and the oily matter obtained by concentration was purified by silica gel chromatography (hexane:ethyl acetate=4:1). The yellow solid obtained was washed with diethyl ether and hexane to obtain the N-Boc-modified ester (12.3g) as a white solid. 1 H-NMR (CDCl3) δ6.71 (1H, br-s), 5.75 (1H, s), 5.33-5.33 (1H, m), 4.96 )1H, br-s), 3.40-3.43 (2H, m), 3.32-3.35 (2H, m), 1.96-1.96 (3H, m), 1.44 (9H, s) To this solid, 4M HCl / Dioxane (120 mL, manufactured by Kokusan Chemical Industry Co., Ltd.) was added and stirred at room temperature for 3 hours. The reaction solution was concentrated, and the precipitated solid was washed with hexane. It was dried under reduced pressure at room temperature to obtain compound 12 (8.83 g) as a white solid. 1H-NMR (D2O) δ5.73 (1H, s), 5.47 (1H, s), 3.15 (2H, t, J=6.0 Hz), 2.54 (2H, t, J=6.0 Hz), 1.90 (3H, s)

[0077] (Example 13) Synthesis of CS with methacryl group CS (average molecular weight approximately 40,000, manufactured by Seikagaku Corporation, 4.03 g) was dissolved in deionized water (120 mL), and compound 12 (0.23 g) and DMT-MM (0.36 g) were added successively at room temperature and stirred for 18 hours. Sodium bicarbonate (3.0 g) was added to the reaction solution and stirred for 30 minutes, then neutralized to pH 7.0 with acetic acid. After stirring for 30 minutes, salt (12.0 g) was added and stirred for 30 minutes. 90% ethanol (240 mL) was added and stirred for 30 minutes, and the supernatant was discarded. 90% ethanol (240 mL) was added again and stirred for 30 minutes, and the supernatant was discarded. After performing this operation two more times, the solid was dialyzed overnight against distilled water (cellulose tube 36 / 32, MWCO 10 kDa, EIDIA Co., Ltd.). Compound 13 (4.34 g) was obtained as a white solid by lyophilization. The incorporation rate was 9.5 mol%.

[0078] (Example 14) Preparation of CS derivative containing SB328437 1 Compound 13 (50.0 mg) and SB328437 (2.0 mg, synthesized according to Journal of Biological Chemistry, 2000, 275 (47), 36626-31.) were added with 65 μL of a saline solution (0.2 mg / mL) of thiol-PEG-thiol (molecular weight 3400, manufactured by Laysan Bio), and then 135 μL of saline was added. After stirring for 1 minute, the mixture was left to stand for 48 hours to obtain a gel-like compound. The gel-like compound was added to saline (3 mL) and then left to stand for 24 hours. Only the gel-like compound was transferred to a sterile syringe with two connected ends, and the plunger was inserted into the syringe barrel. After removing the air inside the syringe, the gel-like compound was transferred to one of the syringes by pressing the plunger alternately 30 times, and composition 14 was obtained. This was used as a sample for administration to animals.

[0079] (Example 15) Preparation of CS derivative containing SB328437 2 The reaction was carried out in the same manner as in Example 14 except that the amount of the thiol-PEG-thiol saline solution (0.2 mg / mL) added was 100 μL, and then the amount of saline added was 100 μL, to obtain Composition 15. This was used as a sample for administration to animals.

[0080] (Example 16) Preparation of CS derivative containing SB328437 3 The reaction was carried out in the same manner as in Example 14, except that the amount of thiol-PEG-thiol in saline solution (0.2 mg / mL) added was 200 μL, and composition 16 was obtained. This was used as a sample for administration to animals.

[0081] (Example 17) Synthesis of aminoethyl oleic acid amide Oleic acid (500mg, Tokyo Chemical Industry Co., Ltd.) was dissolved in dimethylformamide (8ml, Wako Pure Chemical Industries Co., Ltd.), triethylamine (Wako Pure Chemical Industries Co., Ltd., 0.49ml), WSC (Wako Pure Chemical Industries Co., Ltd., 408mg), HOBT (Kokusan Chemical Industries Co., Ltd., 406mg), and N-Boc-diethylamine (Wako Pure Chemical Industries Co., Ltd., 312mg) were added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 3 hours. After confirming the completion of the reaction by LCMS, ethyl acetate was added to the reaction solution, and the organic layer was washed with saturated sodium bicarbonate water, saturated ammonium chloride water, and saturated saline in sequence, dehydrated with anhydrous magnesium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography (hexane:ethyl acetate = 1:1) to obtain a white solid (360mg). Methanol (2ml, Wako Pure Chemical Industries Co., Ltd.) and 4M HCl / AcOEt (10ml, Kokusan Chemical Industries Co., Ltd.) were added to the mixture, and the mixture was stirred at room temperature overnight. After confirming the target product by LCMS, the reaction mixture was concentrated to obtain compound 17 (250 mg). ESI-MS; Calcd for C 20 H 40 N2O [M+H]+, 325; found 325

[0082] (Example 18) Synthesis of oleic acid-introduced CS Distilled water (20 ml) was added to CS (average molecular weight approximately 40,000, manufactured by Seikagaku Corporation, 500 mg) and stirred for 30 minutes to dissolve. An ethanol solution (20 ml) of compound 17 (36 mg) was added, and DMT-MM (manufactured by Tokuyama Corporation, 55 mg) was further added and stirred at room temperature overnight. 1N aqueous sodium hydroxide solution (2.5 mL) was added to the reaction solution and stirred for 30 minutes. The entire reaction solution was placed in a dialysis membrane (Slide-A-Lyzer G2, MWCO 10K, 30 mL, purchased from Funakoshi Co., Ltd.) and dialyzed against ethanol:distilled water (1:1) and distilled water in that order for two days. The dialysate was collected and washed with a cation exchange resin (DOWEX TM 50Wx8 50-100, 2 g, manufactured by Wako Chemical Co., Ltd.) was added and stirred for 30 minutes. The reaction solution was filtered and freeze-dried to obtain compound 18 (277 mg). The introduction rate of 5β-oleic acid was 10.3 mol%.

[0083] (Example 19) Synthesis of CS containing GW766994 and oleic acid GW766994 (15 mg) was dissolved in ethanol (2 ml), and a solution of compound 18 (90 mg) synthesized in Example 18 in PBS (9 ml, pH 7.4) was added and mixed, and the resulting solution was dialyzed (Slide-A-Lyzer G2, MWCO 3.5K, 15 mL, purchased from Funakoshi) against distilled water for 7 hours. The dialysate was collected, filtered through a 0.22 μm filter, and freeze-dried to obtain composition 19 (75 mg). The GW766994 content was 8.62 wt%. The obtained composition 19 was dissolved in PBS to a concentration of 10 mg / ml, and filtered through a 0.22 μm filter to prepare a sample for administration to animals.

[0084] (Example 20) Synthesis of aminoethyl lithocholic acid amide Lithocholic acid (500mg, Tokyo Chemical Industry Co., Ltd.) was dissolved in dimethylformamide (8ml, Wako Pure Chemical Industries Co., Ltd.), WSC (Wako Pure Chemical Industries Co., Ltd., 305mg), HOBT (Kokusan Chemical Industries Co., Ltd., 304mg), and N-Boc-diethylamine (Wako Pure Chemical Industries Co., Ltd., 254mg) were added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 3 hours. After confirming the completion of the reaction by LCMS, ethyl acetate was added to the reaction solution, and the organic layer was washed with saturated sodium bicarbonate water, saturated ammonium chloride water, and saturated saline, dehydrated with anhydrous magnesium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography (hexane:ethyl acetate = 1:1) to obtain a white solid (310mg). This was added with THF (2ml, Wako Pure Chemical Industries Co., Ltd.) and 4M HCl / AcOEt (10ml, Kokusan Chemical Industries Co., Ltd.), and stirred at 45 degrees for 1 hour. After confirming the target product by LCMS, the reaction mixture was concentrated to obtain compound 20 (305 mg). ESI-MS; Calcd for C 26 H 46 N2O2[M+H]+, 419; found 419

[0085] (Example 21) Synthesis of lithocholic acid-introduced CS Distilled water (18 ml) was added to CS (average molecular weight approximately 40,000, manufactured by Seikagaku Corporation, 400 mg) and stirred for 30 minutes to dissolve. An ethanol solution (18 ml) of compound 20 (36 mg) was added, and DMT-MM (manufactured by Tokuyama Corporation, 55 mg) was further added and stirred at room temperature overnight. 1N aqueous sodium hydroxide solution (2.0 ml) was added to the reaction solution and stirred for 30 minutes. The entire reaction solution was placed in a dialysis membrane (Slide-A-Lyzer G2, MWCO 10K, 30 ml, purchased from Funakoshi Co., Ltd.) and dialyzed against ethanol:distilled water (1:1) and distilled water in that order for two days. The dialysate was collected and washed with a cation exchange resin (DOWEX TM 50Wx8 50-100, 2 g, manufactured by Wako Chemical Co., Ltd.) was added and stirred for 30 minutes. The reaction solution was filtered and lyophilized to obtain compound 21 (250 mg). The introduction rate of 5β-lithocholic acid was 10.0 mol%.

[0086] (Example 22) Synthesis of lithocholic acid-introduced CS containing GW766994 GW766994 (11 mg) was dissolved in ethanol (2 ml), and a solution of compound 21 (66 mg) synthesized in Example 21 in PBS (6 ml, pH 7.4) was added and mixed, and the obtained solution was dialyzed (Slide-A-Lyzer G2, MWCO 3.5K, 15 mL, purchased from Funakoshi) against distilled water for 7 hours. The dialysate was collected, filtered through a 0.22 μm filter, and freeze-dried to obtain composition 22 (75 mg). The GW766994 content was 10.3 wt%. The obtained composition 22 was dissolved in PBS to a concentration of 10 mg / ml, and filtered through a 0.22 μm filter to prepare a sample for administration to animals.

[0087] (Method for measuring the content of GAG substituents and chemokine receptor activity modulators) (A) 5β-cholanic acid introduction rate measurement method The introduction rate of 5β-cholanic acid in Examples 1 and 2 was measured as follows. In a mixed solvent of deuterium oxide and deuterium oxide (1:1) 1 H-NMR was measured and calculated from the following formula. 5β-cholanic acid introduction rate (mol%) = (Integral value derived from the 21st methyl group of 5β-cholanic acid) / (Integral value derived from the N-acetyl group of GAG)

[0088] (B) Methacrylic group introduction rate measurement method The methacryl group introduction rate in Example 13 was measured as follows. In heavy water 1 H-NMR was measured and calculated according to the following formula. Methacrylic group introduction rate (mol%) = (Integral value derived from methacryl groups) / (Integral value derived from N-acetyl groups of GAG)

[0089] (C) Method for measuring the content of chemokine receptor activity modulator Method for measuring Ki19003 content in Examples 4 and 5 A calibration curve was prepared using a spectrophotometer (Shimadzu Corporation, UV SPECTROPHOTOMETER, measurement wavelength 220 nm), and measurements were performed. The drug content was calculated using the following formula. Content rate (wt%)= (Weight of Ki19003 in the composition) / (Weight of the composition)×100

[0090] Method for measuring GW766994 content in Examples 6, 7 and 8 A calibration curve was prepared using HPLC under the following conditions, and then measurements were performed. Column: ODS-3, 4.6x150mm, 5um (GL Science) Flow: 0.8ml / min Detector: 225 nm Eluent: acetonitrile: 20 mM ammonium acetate aqueous solution = 5:5 (isocratic)

[0091] The drug content was calculated according to the following formula. Content rate (wt%)= (Weight of GW766994 in the composition) / (Weight of the composition) x 100

[0092] Method for calculating Ki19003 content in Examples 10 and 11 Weighed amounts of Compounds 10 and 11 were hydrolyzed by heating in a 2M aqueous sodium hydroxide solution at 37° C. for 2 hours, and the liberated Ki19003 was measured after preparing a calibration curve under the following HPLC conditions. Column: ODS-3, 4.6x150mm, 5um (GL Science) Flow: 0.8ml / min Detector: 225 nm Eluent: acetonitrile: 50 mM formic acid aqueous solution = 3:7 (isocratic) The content was calculated according to the following formula. Content rate (wt%)= (Weight of Ki19003 released from the compound) / (Weight of the compound)×100

[0093] (2) Evaluation method (Test Example 1) Verification of CNV suppression effect using Composition 10 A laser-induced CNV model was created in rats, and the antiangiogenic effect of intravitreal administration of Composition 10 or Ki19003 was examined.

[0094] <Test substance> Ki19003 was dissolved in DMSO to obtain a solution of Ki19003 (0.6 mg / mL). The following were used as test substances: 1) Composition 10 (containing 0.54 mg / mL Ki19003) 2) Ki19003 (0.6 mg / mL) 3)DMSO (Composition 10 was prepared using the sample for administration to animals prepared in Example 10)

[0095] <Method> (1) Preparation of laser-induced CNV model and administration of test substances BN / CrlCrlj rats (male, Charles River Japan, Inc.) were used as the animal model. Under general anesthesia by intraperitoneal administration of an anesthetic mixture (saline:somnopentyl = 9:1) (approximately 2mL / body), both eyes were dilated by instillation of Mydrin P eye drops. The retina around the optic disc was irradiated with a laser to induce CNV. For laser irradiation, Scopisol ophthalmic solution, Fundus 5.4mm laser lens, laser photocoagulation device, and slit lamp irradiation system were used. Immediately after laser irradiation, 5μL / eye of the test substance was administered once into the vitreous of both eyes. Immediately after administration, one drop of an antibiotic (Vegamox eye drops 0.5%) was instilled into the eyes.

[0096] (2) Preparation of flat mount Ten days after model preparation, the mice were euthanized by CO2. The eyeballs were removed and immersed in 10% neutral buffered formalin (room temperature, approximately 60 minutes). Eye cups were prepared, washed with PBS, dehydrated with methanol, and then immersed in PBS containing 1% bovine serum albumin and 0.5% Triton X-100 (room temperature, approximately 60 minutes). After removing the retina, 60 μL of 0.5% fluorescein griffonia simplicifolia lectin I, FITC-conjugate was added to the eye cup and left to stand (refrigerated, overnight). This allowed the vascular endothelial cells of CNV to be fluorescently stained with FITC-lectin. Eight radial cuts were made in the eye cup to prepare a flat mount. The flat mount was washed twice with PBS containing 0.1% Triton X-100, and then mounted on a slide glass using approximately 120 μL of Prolong Gold Antifade Reagent.

[0097] (3) CNV imaging and area measurement Fluorescent images of CNV in flat-mounted slides were taken using a fluorescent microscope, and the CNV area was measured using image analysis software (Image pro exp).

[0098] <Statistical analysis> The CNV area in each group was analyzed using unpaired t-test with a two-sided significance level of 5%.

[0099] <Test Results> The results of CNV area measurement are shown in the table below and in FIG. Composition 10 showed a significant CNV suppression effect compared to DMSO and Ki19003.

[0100] [Table 1]

[0101] (Test Example 2) Verification of CNV suppression effect using Composition 11 1 A laser-induced CNV model was created in rats, and the antiangiogenic effect of intravitreal administration of Composition 11 or Ki19003 was examined.

[0102] <Test substance> Ki19003 was dissolved in DMSO to obtain a solution of Ki19003 (0.53 mg / mL). The following were used as test substances: 1) Composition 11 (containing 0.53 mg / mL Ki19003) 2) Ki19003 (0.53 mg / mL) 3)DMSO (Composition 11 was prepared using the sample for administration to animals prepared in Example 11)

[0103] <Method> The test was carried out in the same manner as in Test Example 1.

[0104] <Statistical analysis> The CNV area in each group was analyzed using unpaired t-test with a two-sided significance level of 5%.

[0105] <Test Results> The results of CNV area measurement are shown in the table below and in FIG. Composition 11 showed a significant CNV-suppressing effect compared to DMSO and Ki19003.

[0106] [Table 2]

[0107] <Conclusion> Test Examples 1 and 2 demonstrated that Ki19003-introduced CS can be used as a posterior segment disease treatment agent, particularly an AMD treatment agent, which shows a pharmacological effect superior to that of Ki19003. It was demonstrated that CS can be used as GAG constituting the GAG ​​derivative.

[0108] (Test Example 3) Verification of CNV suppression effect using Composition 11 2 A laser-induced CNV model was created in rats, and the antivascular effects of intravitreal administration of Composition 11 or a combination of CS and Ki19003 were examined.

[0109] <Test substance> CS (19 mg, the same as the CS in Example 11, manufactured by Seikagaku Corporation) and Ki19003 (1 mg) were mixed with PBS (2 mL) and shaken to prepare a combination preparation 1 of CS and Ki19003. The following were used as test substances. 1) Composition 11 (containing 0.53 mg / mL Ki19003) 2) Combination drug 1 (containing 0.5 mg / mL Ki19003) 3)DMSO (Composition 11 was prepared using the sample for administration to animals prepared in Example 11)

[0110] <Method> The test was carried out in the same manner as in Test Example 1.

[0111] <Statistical analysis> The CNV area in each group was analyzed using unpaired t-test with a two-sided significance level of 5%.

[0112] <Test Results> The results of CNV area measurement are shown in the table below and in FIG. Composition 11 showed a significant CNV suppression effect compared to DMSO.

[0113] [Table 3]

[0114] <Conclusion> It was shown that Ki19003-introduced CS can be used as a treatment for posterior segment diseases, especially AMD, with efficacy superior to that of a combination of CS and Ki19003. It was shown that the mere combination of GAG and a chemokine receptor antagonist is not sufficient as a treatment for posterior segment diseases, and the usefulness of using a covalent bond between a GAG derivative and a chemokine receptor antagonist was confirmed.

[0115] (Test Example 4) Verification of CNV suppression effect using Composition 14, Composition 15 and Composition 16 A laser-induced CVN model was created in rats, and the antivascular effects of Composition 14, Composition 15, Composition 16, and SB328437 administered intravitreously were examined.

[0116] <Test substance> SB328437 was dissolved in DMSO to obtain a solution of SB328437 (2 mg / mL). The following were used as test substances. 1) Composition 14 (containing 1.33 mg / mL SB328437) 2) Composition 15 (containing 1.67 mg / mL SB328437) 3) Composition 16 (containing 3.33 mg / mL SB328437) 4) SB328437 (2 mg / mL) (Compositions 14, 15, and 16 were prepared using the animal administration samples prepared in Examples 14, 15, and 16, respectively.)

[0117] <Method> The test was carried out in the same manner as in Test Example 1.

[0118] <Statistical analysis> The CNV area in each group was analyzed using unpaired t-test with a two-sided significance level of 5%.

[0119] <Test Results> The results of CNV area measurement are shown in the table below and in FIG. Compositions 14, 15 and 16 exhibited significant CNV suppression effects compared to SB328437.

[0120] [Table 4]

[0121] <Conclusion> It was shown that a composition containing SB328437 and a CS crosslinker can be used as a posterior segment disease treatment agent, particularly an AMD treatment agent, that shows a pharmacological effect superior to that of SB328437. It was shown that a GAG crosslinker can be used as a GAG derivative, and that CS can be used as a GAG. It was also shown that SB328437 can be used as a chemokine receptor antagonist.

[0122] (Test Example 5) Verification of CNV suppression effect using a composition containing SB225002 and HA crosslinker (Gel-One) A laser-induced CNV model was created in rats, and the antiangiogenic effects of intravitreal administration of a composition containing SB225002 and Gel-One (photocrosslinked HA: manufactured by Seikagaku Corporation) and SB225002 were examined.

[0123] <Test substance> SB225002 (synthesized according to Bioorganic & Medicinal Chemistry, 2009, 17(23), 8102-8112) was dissolved in DMSO to obtain a solution of SB225002 (0.02 mg / mL). SB225002 was dissolved in DMSO to obtain a solution of SB225002 (0.2 mg / mL), and then 0.15 mL of the SB225002 (0.2 mg / mL) solution was mixed with Gel-One (1.5 mL) and stirred to obtain composition 31. SB225002 was dissolved in DMSO to obtain SB225002 (2 mg / mL), and then 2 mg / mL of SB225002 (0.015 mL) was mixed with Gel-One (1.5 mL) and stirred to obtain composition 32. The following test substances were used. 1) Composition 31 (containing 0.018 mg / mL SB225002) 2) Composition 32 (containing 0.02 mg / mL SB225002) 3) SB225002 (0.02 mg / ml) 4)DMSO

[0124] <Method> The test was carried out in the same manner as in Test Example 1.

[0125] <Statistical analysis> The CNV area in each group was analyzed using unpaired t-test with a two-sided significance level of 5%.

[0126] <Test Results> The results of CNV area measurement are shown in the table below and in FIG. Compositions 31 and 32 showed significant CNV suppression effects compared to DMSO and SB225002.

[0127] [Table 5]

[0128] <Conclusion> It was shown that a composition containing SB225002 and Gel-One can be used as a posterior segment disease treatment agent, particularly an AMD treatment agent, that shows a pharmacological effect superior to that of SB225002. It was shown that a GAG crosslinker can be used as a GAG derivative, and that HA can be used as a GAG. It was also shown that SB225002 can be used as a chemokine receptor antagonist.

[0129] (Test Example 6) Verification of CNV suppression effect using a composition containing GW766994 and HA crosslinker (Gel-One) A laser-induced CNV model was created in rats, and the antiangiogenic effects of intravitreal administration of a composition containing GW766994 and Gel-One (photocrosslinked HA: manufactured by Seikagaku Corporation) and GW766994 were examined.

[0130] <Test substance> GW766994 was dissolved in DMSO to obtain a solution of GW766994 (2 mg / mL). GW766994 was dissolved in DMSO to obtain GW766994 (50 mg / mL), and then 50 mg / mL GW766994 (0.0417 mL) was mixed with Gel-One (1.0 mL) and stirred to obtain composition 33. The following was used as the test substance. 1) Composition 33 (containing 2 mg / mL GW766994) 2) GW766994 (2 mg / ml) 3)DMSO

[0131] <Method> The test was carried out in the same manner as in Test Example 1.

[0132] <Statistical analysis> The CNV area in each group was analyzed using unpaired t-test with a two-sided significance level of 5%.

[0133] <Test Results> The results of CNV area measurement are shown in the table below and in FIG. Composition 33 showed a significant CNV inhibitory effect compared to DMSO and GW766994.

[0134] [Table 6]

[0135] <Conclusion> It has been demonstrated that a composition containing GW766994 and Gel-One can be used as a therapeutic agent for posterior segment diseases, particularly AMD, which shows a pharmacological effect superior to that of GW766994. It has been demonstrated that GW766994 can be used as a chemokine receptor antagonist.

[0136] (Test Example 7) Verification of CNV suppression effect using a composition containing Ki19003 and HA crosslinker (Gel-One) A laser-induced CNV model was created in rats, and the antiangiogenic effects of intravitreal administration of a composition containing Ki19003 and Gel-One (photocrosslinked HA: manufactured by Seikagaku Corporation) and Ki19003 were examined.

[0137] <Test substance> Ki19003 was dissolved in DMSO to obtain a solution of Ki19003 (0.5 mg / mL). Ki19003 was dissolved in DMSO to obtain Ki19003 (25 mg / mL), and then 25 mg / mL Ki19003 (0.0204 mL) was mixed with Gel-One (1.0 mL) and stirred to obtain composition 34. The following test substances were used. 1) Composition 34 (containing 0.5 mg / mL Ki19003) 2) Ki19003 (0.5 mg / ml) 3)DMSO

[0138] <Method> The test was carried out in the same manner as in Test Example 1.

[0139] <Statistical analysis> The CNV area in each group was analyzed using unpaired t-test with a two-sided significance level of 5%.

[0140] <Test Results> The results of CNV area measurement are shown in the table below and in FIG. Composition 34 showed a significant CNV suppression effect compared to DMSO.

[0141] [Table 7]

[0142] <Conclusion> It was shown that a composition containing Ki19003 and Gel-One can be used as a posterior segment disease treatment agent, particularly an AMD treatment agent, that shows a pharmacological effect superior to that of Ki19003. In addition, Test Examples 1, 2, 3, and 7 showed that Ki19003 could be combined with multiple GAG ​​derivatives (hydrophobic group-introduced GAG or GAG crosslinked body). This shows that the combination of GAG derivatives and chemokine receptor antagonists is not necessarily a one-to-one correspondence.

[0143] (Test Example 8) Verification of CNV suppression effect using a composition containing AZD3778 and HA crosslinker (Gel-One) A laser-induced CNV model was created in rats, and the antiangiogenic effect of a composition containing AZD3778 (synthesized in accordance with WO 03 / 004487A1) and Gel-One (photocrosslinked HA: manufactured by Seikagaku Corporation) and AZD3778 administered intravitreously was examined.

[0144] <Test substance> AZD3778 was dissolved in DMSO to obtain a solution of AZD3778 (0.1 mg / mL). AZD3778 was dissolved in DMSO to obtain AZD3778 (5 mg / mL), and then 5 mg / mL AZD3778 (0.0204 mL) was mixed with Gel-One (1.0 mL) and stirred to obtain composition 35. The following test substances were used. 1) Composition 35 (containing 0.1 mg / mL AZD3778) 2) AZD3778 (0.1 mg / ml) 3)DMSO

[0145] <Method> The test was carried out in the same manner as in Test Example 1.

[0146] <Statistical analysis> The CNV area in each group was analyzed using unpaired t-test with a two-sided significance level of 5%.

[0147] <Test Results> The results of CNV area measurement are shown in the table below and in FIG. Composition 35 showed a significant CNV inhibitory effect compared to AZD3778.

[0148] [Table 8]

[0149] <Conclusion> It has been shown that a composition containing AZD3778 and Gel-One can be used as a posterior segment disease treatment agent, particularly an AMD treatment agent, which shows a pharmacological effect superior to that of AZD3778. It has also been shown that AZD3778 can be used as a chemokine receptor antagonist.

[0150] (Test Example 9) Verification of CNV suppression effect using a composition containing SB328437 and HA crosslinker (Gel-One) A laser-induced CNV model was created in rats, and the antiangiogenic effects of intravitreal administration of a composition containing SB328437 and Gel-One (photocrosslinked HA: manufactured by Seikagaku Corporation) and SB328437 were examined.

[0151] <Test substance> SB328437 was dissolved in DMSO to obtain a solution of SB328437 (1 mg / mL). SB328437 was dissolved in DMSO to obtain SB328437 (50 mg / mL), and then 50 mg / mL SB328437 (0.02 mL) was mixed with Gel-One (1.0 mL) and stirred to obtain composition 36. The following was used as the test substance. 1) Composition 36 (containing 1 mg / mL SB328437) 2) SB328437 (1 mg / ml) 3)DMSO

[0152] <Method> The test was carried out in the same manner as in Test Example 1.

[0153] <Statistical analysis> The CNV area in each group was analyzed using unpaired t-test with a two-sided significance level of 5%.

[0154] <Test Results> The results of CNV area measurement are shown in the table below and in FIG. Composition 36 showed a significant CNV inhibitory effect compared to SB328437.

[0155] [Table 9]

[0156] <Conclusion> It was shown that a composition containing SB328437 and Gel-One can be used as a posterior ocular disease treatment agent, particularly an AMD treatment agent, which shows a drug efficacy superior to that of SB328437. It was also shown that SB328437 can be used as a chemokine receptor antagonist. From Test Example 5, Test Example 6, Test Example 7, Test Example 8, and Test Example 9, the GAG ​​crosslinker could be combined with multiple chemokine receptor antagonists (GW766994, Ki19003, AZD3778, SB328437, SB225002). This shows that the combination of GAG derivatives and chemokine receptor antagonists is not necessarily one-to-one correspondence.

[0157] (Test Example 10) Verification of CNV suppression effect using Composition 6 and Composition 7 A laser-induced CNV model was created in rats, and the antiangiogenic effects of Composition 6, Composition 7, and GW766994 administered intravitreously were examined.

[0158] <Test substance> GW766994 was dissolved in DMSO to give a solution of GW766994 (1.5 mg / mL). The following were used as test substances: 1) Composition 6 (containing 0.71 mg / mL GW766994) 2) Composition 7 (containing 0.83 mg / mL GW766994) 3) GW766994 (1.5 mg / mL) 4)DMSO (Compositions 6 and 7 were prepared using the animal administration samples prepared in Examples 6 and 7, respectively.)

[0159] <Method> The test was carried out in the same manner as in Test Example 1.

[0160] <Statistical analysis> The CNV area of ​​the DMSO group and other groups was analyzed by Dunnett's test. The significance level was set to 5% on both sides. The CNV area of ​​the GW766994 group and the composition group was analyzed by Dunnett's test. The significance level was set to 5% on both sides.

[0161] <Test Results> The results of CNV area measurement are shown in the table below and in FIG. Compositions 6 and 7 showed a significant CNV inhibitory effect compared to GW766994.

[0162] [Table 10]

[0163] <Conclusion> It was demonstrated that a composition containing GW766994 and hydrophobic group-introduced HA, and a composition containing GW766994 and hydrophobic group-introduced CS can be used as a posterior segment disease treatment agent, particularly an AMD treatment agent, that shows a medicinal effect superior to that of GW766994. It was demonstrated that HA and CS can be used as GAGs for use in hydrophobic group-introduced GAGs.

[0164] (Test Example 11) Verification of CNV suppression effect using Composition 4 and Composition 5 A laser-induced CNV model was created in rats, and the antiangiogenic effects of Composition 4, Composition 5, and Ki19003 administered intravitreously were examined.

[0165] <Test substance> Ki19003 was dissolved in DMSO to obtain a solution of Ki19003 (0.6 mg / mL). The following were used as test substances: 1) Composition 4 (containing 0.42 mg / mL Ki19003) 2) Composition 5 (containing 0.66 mg / mL Ki19003) 3) Ki19003 (0.6 mg / mL) 4)DMSO (Compositions 4 and 5 were prepared using the samples for administration to animals prepared in Examples 4 and 5, respectively.)

[0166] <Method> The test was carried out in the same manner as in Test Example 1.

[0167] <Statistical analysis> The CNV area in the DMSO group and other groups was analyzed using the Dunnett test with a two-sided significance level of 5%.

[0168] <Test Results> The results of CNV area measurement are shown in the table below and in Figure 11. Compositions 4 and 5 showed a significant CNV inhibitory effect compared to DMSO.

[0169] [Table 11]

[0170] <Conclusion> It was shown that a composition containing Ki19003 and hydrophobic group-introduced HA and a composition containing Ki19003 and hydrophobic group-introduced CS can be used as a treatment for posterior segment diseases, particularly AMD, that exhibits efficacy superior to that of Ki19003. It was shown that HA and CS can be used as GAGs for use in hydrophobic group-introduced GAGs.

[0171] (Test Example 12) Verification of CNV suppression effect and effect on intraocular bleeding using Composition 8 A laser-induced CNV model was created in rats, and the effects of intravitreal administration of Composition 8 or a combination of heparin and GW766994 on the inhibition of neovascularization and intraocular bleeding were examined.

[0172] <Test substance> GW766994 was dissolved in DMSO to obtain a solution of GW766994 (10 mg / mL). Heparin (10 mg, Aldrich) was dissolved in PBS (1 ml) to obtain heparin (10 mg / mL), which was then mixed with 10 mg / mL GW766994 (0.1 mL) to prepare heparin and GW766994 combination agent 2. The following were used as test substances. 1) Composition 8 (containing 0.96 mg / mL GW766994) 2) Combination drug 2 (containing 0.91 mg / mL GW766994) 3) PBS (Composition 8 was prepared using the sample for administration to animals prepared in Example 8)

[0173] <Method> (Angiogenesis inhibitory effect) The test was carried out in the same manner as in Test Example 1.

[0174] (Effects on intraocular bleeding) Immediately after administration of the test substance and 10 days after administration, intraocular bleeding was scored, and the effect on intraocular bleeding was examined based on whether or not the intraocular bleeding worsened.

[0175] (1) Evaluation of intraocular bleeding score immediately after administration of the test substance Intraocular observation was performed using a slit lamp or the like, and intraocular bleeding was scored according to the following intraocular bleeding score criteria. Score 0: No bleeding was observed within the observation area. Score 1: Bleeding was observed in less than 1 / 4 of the observation area. Score 2: Bleeding was observed in 1 / 4 to 1 / 2 of the observation area. Score 3: Bleeding was observed in more than half of the observation area.

[0176] (2) Evaluation of intraocular hemorrhage score on the 10th day after administration Immediately after preparation of the ocular cup, the vitreous humor and retina were observed and scored using the following criteria for scoring vitreous humor and retinal hemorrhage. The average of the vitreous humor and retinal hemorrhage scores was taken as the intraocular hemorrhage score. Score 0: No bleeding was observed within the observation area. Score 1: Bleeding was observed in less than 1 / 4 of the observation area. Score 2: Bleeding was observed in 1 / 4 to 1 / 2 of the observation area. Score 3: Bleeding was observed in more than half of the observation area.

[0177] (3) Calculation of the presence or absence of worsening intraocular hemorrhage The number of eyes with deterioration (eyes with a difference of +) and the number of eyes without deterioration (difference of - or 0) were calculated from the difference in intraocular hemorrhage score immediately after administration of the test substance and 10 days after administration.

[0178] <Statistical analysis> The CNV area in each group was analyzed using unpaired t-test with a two-sided significance level of 5%. The presence or absence of worsening of intraocular hemorrhage in each group was analyzed using a 2 × 2 Fisher's exact probability test with a one-sided significance level of 5%.

[0179] <Test Results> The results of CNV area measurement are shown in Table 12 and Figure 12. Composition 8 showed a significant CNV inhibitory effect compared to PBS. The results of the effect on intraocular bleeding are shown in Table 13. It was shown that the combination of heparin and GW766994 2 caused significantly more exacerbations of intraocular bleeding than PBS and Composition 8.

[0180] [Table 12]

[0181] [Table 13]

[0182] <Conclusion> It was shown that a composition containing GW766994 and hydrophobic group-introduced CS can be used as a posterior segment disease treatment agent, particularly an AMD treatment agent, that shows a pharmacological effect superior to that of combination drug 2 of heparin and GW766994. It was shown that simply combining GAG and a chemokine receptor antagonist is not sufficient as a posterior segment disease treatment agent, and the usefulness of using a composition containing a GAG derivative and a chemokine receptor antagonist was confirmed. Combination drug 2 of heparin and GW766994 worsened intraocular bleeding, confirming that it is not suitable for intravitreal administration.

[0183] (Test Example 13) Verification of tissue degeneration upon intravitreal administration of a chemokine receptor antagonist solution using a solubilizing agent (DMSO) used in Patent Document 1 and the composition according to the present invention The test substance was administered once into the vitreous cavity of rats, and the intraocular condition was observed.

[0184] <Test substance> The following test substances were used: 1) Ki19003 (0.6 mg / mL) 2) SB328437 (1 mg / mL) 3) GW766994 (1.5 mg / mL) 4) Composition 5 (containing 0.66 mg / mL Ki19003) 5) Composition 7 (containing 0.83 mg / mL GW766994) 6) Composition 10 (containing 0.54 mg / mL Ki19003) 7) Composition 11 (containing 0.53 mg / mL Ki19003) 8) Composition 36 (containing 1 mg / mL SB328437) (For Compositions 5, 7, 10, 11, and 36, the animal administration samples prepared in Examples 5, 7, 10, and 11 and Test Example 9 were used, respectively.) Ki19003 was obtained in the same manner as in Test Example 1, SB328437 in the same manner as in Test Example 9, and GW766994 in the same manner as in Test Example 10.

[0185] <Method> (1) Administration of test substance The animals used were BN / CrlCrlj rats (male, Charles River Japan, Inc.). Under general anesthesia with intraperitoneal administration (approximately 2mL / body) of an anesthetic mixture (saline:somnopentyl = 9:1), both eyes were dilated with instillation of Mydrin P eye drops. Then, 5μL / eye of the test substance was administered once into the vitreous of both eyes. Immediately after administration, one drop of an antibiotic (Vegamox eye drops 0.5%) was instilled into the eyes.

[0186] (2) Intraocular photography The condition inside the rat's eye was photographed using a digital microscope.

[0187] <Test Results> The results of intraocular photography are shown in Figures 18A to 18H. In the case of chemokine receptor antagonist solutions using a solubilizing agent (Ki19003 (FIG. 18A), SB328437 (FIG. 18B), GW766994 (FIG. 18C)), lens tissue degeneration occurred as indicated by the arrows in the figures. On the other hand, in the case of compositions containing a GAG derivative and a chemokine receptor antagonist, Composition 5 (FIG. 18D), Composition 7 (FIG. 18E), Composition 10 (FIG. 18F), Composition 11 (FIG. 18G), and Composition 36 (FIG. 18H), lens tissue degeneration was not observed.

[0188] <Conclusion> Compared to a chemokine receptor antagonist solution using a solubilizing agent, a composition containing a GAG derivative and a chemokine receptor antagonist was shown to suppress the occurrence of rapid tissue degeneration and to have excellent chemokine receptor antagonistic activity.

[0189] (Test Example 14) Verification of CNV suppression effect using a composition containing RS504393 and HA crosslinker (Gel-One) A laser-induced CNV model was created in rats, and the antiangiogenic effects of intravitreal administration of RS504393 and a composition containing RS504393 and Gel-One (photocrosslinked HA: manufactured by Seikagaku Corporation) were examined.

[0190] <Test substance> RS504393 (10 mg, Abcam) was dissolved in DMSO to obtain a solution of RS504393 (0.01 mg / mL). RS504393 was dissolved in DMSO to obtain RS504393 (1 mg / mL), and then 1 mg / mL RS504393 (0.01 mL) was mixed with Gel-One (1.0 mL) and stirred to obtain composition 37. The following test substances were used. 1) Composition 37 (containing 0.01 mg / mL RS504393) 2) RS504393 (0.01 mg / ml) 3)DMSO

[0191] <Method> The test was carried out in the same manner as in Test Example 1.

[0192] <Statistical analysis> The CNV area in each group was analyzed using unpaired t-test with a two-sided significance level of 5%.

[0193] <Test Results> The results of CNV area measurement are shown in the table below and in FIG. Composition 37 showed a significant CNV inhibitory effect compared to DMSO and RS504393.

[0194] [Table 14]

[0195] <Conclusion> It was shown that a composition containing RS504393 and Gel-One can be used as a posterior segment disease treatment agent, particularly an AMD treatment agent, which shows a pharmacological effect superior to that of RS504393. It was also shown that RS504393 can be used as a chemokine receptor antagonist.

[0196] (Test Example 15) Verification of CNV suppression effect using a composition containing PS372424 and HA crosslinker (Gel-One) A laser-induced CNV model was created in rats, and the antiangiogenic effects of intravitreal administration of a composition containing PS372424 and Gel-One (photocrosslinked HA: manufactured by Seikagaku Corporation) and PS372424 were examined.

[0197] <Test substance> PS372424 (10 mg, Calbiochem) was dissolved in DMSO to obtain a solution of PS372424 (0.1 mg / mL). PS372424 was dissolved in DMSO to obtain PS372424 (1 mg / mL), and then 1 mg / mL PS372424 (0.1 mL) was mixed with Gel-One (1.0 mL) and stirred to obtain composition 38. The following test substances were used: 1) Composition 38 (containing 0.091 mg / mL PS372424) 2) PS372424 (0.1 mg / ml) 3)DMSO

[0198] <Method> The test was carried out in the same manner as in Test Example 1.

[0199] <Statistical analysis> The CNV area in each group was analyzed using unpaired t-test with a two-sided significance level of 5%.

[0200] <Test Results> The results of CNV area measurement are shown in the table below and in FIG. Composition 38 exhibited a significant CNV suppression effect compared to DMSO and PS372424.

[0201] [Table 15]

[0202] <Conclusion> It was demonstrated that a composition containing PS372424 and Gel-One can be used as a therapeutic agent for posterior segment diseases, particularly AMD, which shows a pharmacological effect superior to that of PS372424. It was also demonstrated that PS372424 can be used as a chemokine receptor agonist.

[0203] (Test Example 16) Verification of CNV suppression effect using Composition 22 (lithocholic acid-introduced CS: Example 22) and Composition 19 (oleic acid-introduced CS: Example 19) A laser-induced CNV model was created in rats, and the antiangiogenic effects of Composition 22 and Composition 19 administered intravitreously were examined.

[0204] <Test substance> The following test substances were used: 1) PBS 2) Composition 22 (containing 0.86 mg / mL GW766994) 3) Composition 19 (containing 1.03 mg / mL GW766994) (Compositions 22 and 19 were prepared using the animal administration samples prepared in Examples 22 and 19, respectively.)

[0205] <Method> The test was carried out in the same manner as in Test Example 1.

[0206] <Statistical analysis> The CNV area in the PBS group and other groups was analyzed using unpaired t-tests with a two-sided significance level of 5%.

[0207] <Test Results> The results of CNV area measurement are shown in the table below and in Figure 15. Compositions 22 and 19 showed significant CNV inhibitory effects compared to PBS.

[0208] [Table 16]

[0209] As described in Test Examples 6 and 10, GW766994 dissolved in DMSO (2 mg / mL and 1.5 mg / mL) had no CNV inhibitory effect at either concentration compared to DMSO.

[0210] <Conclusion> It was shown that a composition containing GW766994 and hydrophobic group-introduced CS can be used as a posterior segment disease treatment agent, particularly an AMD treatment agent, that shows a medicinal effect superior to that of GW766994. It was shown that a group derived from an alicyclic compound such as cholanic acid, which is the basic skeleton of lithocholic acid, a bile acid, or a group derived from a fatty acid such as oleic acid can be used as the "hydrophobic group" in the hydrophobic group-introduced GAG.

[0211] (Test Example 17) Verification of tissue degeneration upon intravitreal administration of a chemokine receptor activity modulator solution using a solubilizing agent (DMSO) used in the specification of U.S. Patent No. 8,592,482 and the composition according to the present invention The test substance was administered once into the vitreous cavity of rats, and the intraocular condition was observed.

[0212] <Test substance> The following test substances were used: 1) RS504393 (0.01 mg / mL) 2) PS372424 (0.1 mg / mL) 3) Composition 37 (containing 0.01 mg / mL RS504393) 4) Composition 38 (containing 0.1 mg / mL PS372424) 5) Composition 22 (containing 0.86 mg / mL GW766994) 6) Composition 19 (containing 1.03 mg / mL GW766994) (For composition 37 and RS504393, the samples for administration to animals prepared in Test Example 14, for composition 38 and PS372424, in Test Example 15, for composition 22, in Example 22, and for composition 19, in Example 19 were used.)

[0213] <Method> The test was carried out in the same manner as in Test Example 13.

[0214] <Test Results> The results of intraocular photography are shown in Figures 19A to 19F. In the case of solutions of chemokine receptor activity modulators using a solubilizing agent (RS504393 (FIG. 19A), PS372424 (FIG. 19B)), lens tissue degeneration occurred, as indicated by the arrows in the figures. On the other hand, in the case of compositions containing a GAG derivative and a chemokine receptor activity modulator, Composition 37 (FIG. 19C), Composition 38 (FIG. 19D), Composition 22 (FIG. 19E), and Composition 19 (FIG. 19F), lens tissue degeneration was not observed.

[0215] <Conclusion> Compared to a chemokine receptor activity modulator solution using a solubilizing agent, a composition containing a GAG derivative and a chemokine receptor activity modulator was shown to suppress the occurrence of rapid tissue degeneration and to have excellent chemokine receptor activity modulatory effect.

[0216] (Test Example 18) Verification of CNV suppression effect using CS A laser-induced CNV model was established in rats, and the inhibitory effect of intravitreal administration of CS on angiogenesis was examined.

[0217] <Test substance> CS (average molecular weight: about 140,000, manufactured by Seikagaku Corporation) was dissolved in PBS to obtain CS (20 mg / mL). The following were used as test substances. 1) CS (20 mg / mL) 2) PBS

[0218] <Method> The test was carried out in the same manner as in Test Example 1.

[0219] <Statistical analysis> The CNV area in each group was analyzed using unpaired t-test with a two-sided significance level of 5%.

[0220] <Test Results> The results of CNV area measurement are shown in the table below and in FIG. CS did not show a significant inhibitory effect on CNV compared with PBS.

[0221] [Table 17]

[0222] CS alone did not show significant efficacy as a treatment for age-related macular degeneration.

[0223] (Test Example 19) Verification of CNV suppression effect using compound 3 A laser-induced CNV model was established in rats, and the inhibitory effect of intravitreal administration of compound 3 on angiogenesis was examined.

[0224] <Test substance> CS (average molecular weight: about 40,000, manufactured by Seikagaku Corporation) was dissolved in PBS to obtain CS (10 mg / mL). The following were used as test substances. 1) Compound 3 2) CS (10 mg / mL)

[0225] <Method> The same procedure as in Test Example 1 was repeated, except that the flat mount in the <Method> of Test Example 1 was prepared 4 days after the model was prepared instead of 10 days after the model was prepared.

[0226] <Statistical analysis> The CNV area in each group was analyzed using unpaired t-test with a two-sided significance level of 5%.

[0227] <Test Results> The results of CNV area measurement are shown in the table below and in FIG. Compound 3 did not show significant CNV inhibitory activity compared to CS.

[0228] [Table 18]

[0229] <Conclusion> Cholanic acid-loaded CS alone did not show any significant efficacy as a treatment for age-related macular degeneration, similar to CS alone.

[0230] The disclosure of Japanese Patent Application No. 2015-110784 (filing date: May 29, 2015) is incorporated herein by reference in its entirety. All publications, patent applications, and standards mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or standard was specifically and individually indicated to be incorporated by reference.

Claims

1. A composition comprising a glycosaminoglycan derivative and a chemokine receptor activity modulator.

2. 2. The composition according to claim 1, wherein the glycosaminoglycan derivative is a hydrophobic group-introduced glycosaminoglycan.

3. 3. The composition according to claim 1 or 2, wherein the glycosaminoglycan derivative is a crosslinked glycosaminoglycan.

4. The composition according to any one of claims 1 to 3, comprising a covalent bond between a glycosaminoglycan derivative and a chemokine receptor activity modulator.

5. The composition according to any one of claims 1 to 4, wherein the glycosaminoglycan derivative is a derivative of hyaluronic acid or chondroitin sulfate.

6. The composition according to any one of claims 1 to 5, wherein the chemokine receptor activity modulator is a chemokine receptor antagonist.

7. A pharmaceutical composition comprising the composition according to any one of claims 1 to 6.

8. The pharmaceutical composition according to claim 7, which is an agent for treating a posterior segment eye disease.

9. Use of the composition according to any one of claims 1 to 6 as an agent for treating posterior segment diseases.

10. A method for treating a posterior segment disease, comprising administering the composition according to any one of claims 1 to 6 into the vitreous body.

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