Compositions containing peptides and uses thereof

Abstract

The present invention provides a composition comprising a novel peptide, and a use thereof (for example, the treatment of a macular hole).

Description

[Technical Field]

[0001] The present invention relates to a composition containing a novel peptide and its use. [Background technology]

[0002] The macula is the part of the retina that is most closely related to vision. A macular hole is a condition in which a hole develops in the macula, causing difficulty in seeing. This macular hole is believed to be caused by aging of the eye, particularly age-related changes in the vitreous. Because the retina and vitreous cortex are strongly attached, it is thought that age-related atrophy of vitreous collagen fibers exerts tension on the retina, resulting in forward traction that causes cracks in the retina and leads to macular holes. This disease is common among the elderly, and with the rapid increase in the elderly population in recent years, the number of patients is steadily increasing.

[0003] The only treatment for macular holes is vitrectomy, which removes the vitreous that is causing the hole and closes it. When symptoms are mild and the hole is small, the closure rate is high; however, when symptoms progress and the hole becomes large, the closure rate drops and vision recovery is unlikely. Vitrectomy has a 70% vision recovery rate and requires a hospital stay of 3 to 7 days. Furthermore, while macular hole treatments can close the hole, removing the basement membrane (internal vitreous membrane) leaves no scaffold for macular cell reconstruction after surgery, which means the quality of healing cannot be guaranteed. Thus, there is a need for the development of effective treatments for macular holes. Summary of the Invention [Problem to be solved by the invention]

[0004] Under these circumstances, an object of the present invention is to provide a novel therapeutic agent for macular holes, with the aim of recovering visual function QOV and improving postoperative QOL after vitreous surgery for macular holes. [Means for solving the problem]

[0005] The present inventors have conducted extensive research into artificial collagen-like polypeptide membranes, and as a result have discovered that a composition containing a special peptide can provide the scaffold necessary for retinal cell regeneration, and that simply applying the membrane can close holes, thereby restoring and improving visual function and reducing the burden on patients. Further research led to the completion of this invention.

[0006] That is, the present invention relates to the following items. (1) Formula (I) -(P1,P2,P3)- (I) (In formula (I), P1, P2, and P3 may be the same or different, and each independently represent a peptide chain represented by the following formula (II): P1, P2, and P3 form a trimer with a triple helix structure, and are polymerized by oxidative cross-linking through disulfide bonds via cysteine ​​(Cys) residues contained in each peptide chain. Formula (II) R 1 -(Pro-Hyp-Gly)mX-(Pro-Hyp-Gly)nR 2 (II) (In formula (II), R 1 and R 2 are peptide groups each having an amino terminus and a carboxy terminus, and each consisting of any 2 to 10 amino acid residues containing at least two Cys (cysteine) residues, independently of one another; Pro is a proline residue, Hyp is a hydroxyproline residue, Gly is a glycine residue, m and n are independently integers of 4 or greater; X is a peptide group represented by 1 to 4 repetitions of -Xaa-Yaa-Gly-, and Xaa and Yaa each independently represent an amino acid residue. (2) The composition described in (1), wherein P1, P2 and P3 are the same peptide chain. (3) The composition according to (1), wherein X is a peptide group consisting of amino acid residues selected from -Phe-Hyp-Gly-Glu-Arg-Gly-, -Pro-Arg-Gly-Gln-Hyp-Gly-Val-Met-Gly-Phe-Hyp-Gly-, and -Pro-Lys-Gly-His-Arg-Gly-Phe-Ser-Gly-Ler-Hyp-Gly-. (4) The composition according to (1), wherein X is a peptide group consisting of an amino acid residue selected from -Pro-Arg-Gly- or -Phe-Hyp-Gly-Glu-Arg-Gly-. (5) R 1 and R 2 The composition according to (1), wherein each of the amino acid residues is independently a peptide group consisting of two or more Cys (cysteine) residues. (6) The peptide chain represented by formula (II) is R 1 -(Pro-Hyp-Gly)m-Pro-Arg-Gly-(Pro-Hyp-Gly)nR 2 (i) and R 1 -(Pro-Hyp-Gly)m-Phe-Hyp-Gly-Glu-Arg-Gly-(Pro-Hyp-Gly)nR 2 (ii) The composition according to (1), selected from the group consisting of: (7) The peptide chain represented by formula (II) is represented by the following sequence number: H-Cys-Cys-Cys-(Pro-Hyp-Gly)5-Pro-Arg-Gly-(Pro-Hyp-Gly)4-Cys-Cys-Cys-OH SEQ ID NO: 1 H-Cys-Cys-(Pro-Hyp-Gly)5-Pro-Arg-Gly-(Pro-Hyp-Gly)4-Cys-Cys-OH SEQ ID NO: 2 H-Cys-Cys-Cys-(Pro-Hyp-Gly)4-Phe-Hyp-Gly-Glu-Arg-Gly-(Pro-Hyp-Gly)4-Cys-Cys-Cys-OH SEQ ID NO: 3 The composition described in (1). (8) The composition according to any one of (1) to (7), further comprising a water-soluble filler. (9) The composition according to (8), wherein the water-soluble filler is a sugar. (10) The composition according to (9), wherein the sugar is one or more of glucose, lactose, sucrose, mannitol, trehalose, and sorbitol. (11) The composition according to (10), wherein the sugar is glucose. (12) The composition according to any one of (8) to (11), wherein the water-soluble filler is present in an amount of 10 to 50% based on the total weight of the composition. (13) The composition according to any one of (8) to (11), wherein the polymerized peptide is present in an amount of 50 to 90% based on the total weight of the composition. (14) The composition according to any one of (1) to (13), which is in the form of a gel. (15) The composition according to any one of (1) to (13), which is in a transparent form. (16) A pharmaceutical product comprising the composition according to any one of (1) to (15). (17) A device comprising the composition according to any one of (1) to (15). (18) A therapeutic agent for eye diseases, comprising the composition according to any one of (1) to (15). (19) The therapeutic agent for an eye disease according to (18), wherein the eye disease is a macular hole or retinal detachment. (20) A method for treating an eye disease, comprising the step of administering a therapeutically effective amount of the composition according to any one of (1) to (15) to a subject. (21) The method for treatment according to (20), wherein the eye disease is macular hole or retinal detachment. (22) A method for producing the composition according to any one of (1) to (15), which comprises preparing a homogeneous physical mixture of the polymeric peptide represented by formula (I) and a water-soluble filler. (23) Use of the composition according to any one of (1) to (15) in the manufacture of a pharmaceutical for treating an eye disease. [Effects of the Invention]

[0007] The peptide film made of the composition of the present invention has high transparency and is ideal for treating macular holes.

[0008] According to the present invention, it is possible to process a peptide membrane into a sheet of an optimum thickness while maintaining its strength.

[0009] According to the present invention, it is possible to impart specific physiological functions and adjust the combination of their strengths.

[0010] According to the present invention, it is possible to control cell adhesion, proliferation, differentiation, etc. by changing the hardness of the scaffold.

[0011] According to the present invention, it is possible to control the decomposition speed of the peptide membrane.

[0012] According to the present invention, it is possible to industrially produce peptide membranes. [Brief explanation of the drawings]

[0013] [Figure 1] 1 shows an application test of the peptide membrane of the present invention to defective areas of MIO-M1 cells. [Figure 2] 1 shows that MIO-M1 cells adhere to and grow on the peptide film of the present invention. [Figure 3] Using a peptide membrane without added water-soluble filler, we will demonstrate efficacy and safety (in vivo) in a white rabbit model. [Figure 4] Using a peptide membrane containing a water-soluble filler, we will demonstrate its efficacy and safety (in vivo) in a white rabbit model. [Figure 5] Efficacy and safety in a cynomolgus monkey model (in vivo) are demonstrated. DETAILED DESCRIPTION OF THE INVENTION

[0014] The composition of the present invention contains two or more types of polymerized peptides.

[0015] Polymerized peptide (first component) The polymerized peptide of the present invention is Formula (I) -(P1,P2,P3)- (I) (In formula (I), P 1、 P2 and P3 may be the same or different and each independently represent a peptide chain represented by the following formula (II): P1, P2 and P3 form a trimer with a triple helix structure, and are polymerized through oxidative cross-linking via disulfide bonds via cysteine ​​(Cys) residues contained in each peptide chain. Formula (II) R 1 -(Pro-Hyp-Gly)mX-(Pro-Hyp-Gly)nR 2 (II) (In formula (II), R 1 and R 2 are peptide groups each having an amino terminus and a carboxy terminus, and each consisting of any 2 to 10 amino acid residues containing at least two Cys residues, independently of one another; m and n are independently integers of 4 or greater; X is a peptide group represented by 1 to 4 repetitions of -Xaa-Yaa-Gly-.

[0016] The polymeric peptide of the present invention is a polymeric peptide characterized by having a trimeric peptide structural unit formed from three peptide chains represented by the following formula (I) and being oxidatively crosslinked. -(P1,P2,P3)- (I) [In formula (I), P1, P2, and P3 may be the same or different and each independently represent a peptide chain represented by the following formula (II), and P1, P2, and P3 form a trimer having a triple helix structure and may be cross-linked by disulfide bonds via cysteine ​​(Cys) residues contained in each peptide chain, and the trimer is polymerized by oxidative cross-linking at the disulfide bonds of the Cys. R 1-(Pro-Hyp-Gly)mX-(Pro-Hyp-Gly)nR 2 (II) (In formula (II), R 1 and R 2 are the amino and carboxy termini, respectively, and are independently a peptide group consisting of any 2 to 10 amino acid residues containing at least two Cys residues. X is a peptide group represented by 1 to 4 repeats of -(Xaa-Yaa-Gly)- as a basic unit. m and n are independently an integer of 4 or more.)

[0017] The Xaa and Yaa each independently represent an amino acid residue, such as a proline (Pro or P) residue, a hydroxyproline (Hyp or O) residue, an arginine (Arg or R) residue, a lysine (Lys or K) residue, a valine (Val or V) residue, a leucine (Leu or L) residue, an isoleucine (Ile or I) residue, a serine (Ser or S) residue, a threonine (Thr or T) residue, an alanine (Ala or A) residue, a glycine (Gly or G) residue, a phenylalanine (Phe or F) residue, methionine (Met or M) residue, glutamic acid (Glu or E) residue, aspartic acid (Asp or D) residue, asparagine (Asn or N) residue, glutamine (Gln or Q) residue, histidine (His or H) residue, tryptophan (Trp or W) residue, or tyrosine (Tyr or Y) residue, wherein the proline residue may be modified with an amino group or a fluorine atom, and N-isobutyl glycine residues may be used at positions Xaa and Yaa.

[0018] The number of Cys residues contained within 10 residues from the N-terminus and C-terminus of the peptide chain may be independently the same or different, and may be 2 or more, 3 or more, 4 or more, or 5 or more.

[0019] In the constitution of the above-mentioned polymerized peptide, the peptide group from each of the amino terminal and the carboxy terminal containing at least two cysteine ​​(Cys) residues may be 10 residues or less, or may be 9 residues or less, 8 residues or less, 7 residues or less, 6 residues or less, 5 residues or less, 4 residues or less, or 3 residues or less.

[0020] As used herein, the term "polymerized peptide" refers to a peptide polymerized via disulfide bonds formed by oxidative crosslinking between cysteine ​​residues contained in the peptide.

[0021] The degree of polymerization of the polymerized peptide of the present invention is 2 or more and is not particularly limited as long as it is a degree of polymerization that allows the formation of a gel, preferably a hydrogel, containing the polymerized peptide of the present invention, but the average degree of polymerization may be less than 100, 100 to 500, 500 to 1000, 1000 to 5000, 5000 to 10000, or 10000 or more.

[0022] In this specification and in the sequence listing attached hereto, peptide structures are described using three-letter or one-letter amino acid codes commonly used by those skilled in the art. The amino acids herein are in the L-form. The amino acids herein include the 20 L-amino acids known to be commonly used in protein translation in molecular biology, as well as modified amino acid residues well known in the art, such as 4-hydroxy-L-proline, 4-fluoro-L-proline, and N-isobutylglycine. Hydroxyproline, as used herein, is 3-hydroxyproline or 4-hydroxy-L-proline, and is represented by the three-letter code "Hyp" or the one-letter code "O."

[0023] The polymeric peptides of the present invention can be produced by known methods for chemically synthesizing peptides, including but not limited to, using commercially available amino acids (Japanese Patent No. 6455862). Alternatively, a nucleic acid sequence encoding a desired amino acid sequence can be prepared and incorporated into an expression vector to create a recombinant expression vector by known methods. This vector can then be introduced into an appropriate host, such as Escherichia coli or another microorganism, to produce a transformant. The resulting transformant can be cultured in an appropriate medium to produce a recombinant peptide chain. The recombinant peptide chain produced from the culture can then be recovered to prepare the recombinant peptide chain used in the present invention.

[0024] Such peptide chains can be obtained by separation and purification using a separation means such as high performance liquid chromatography, and can be used to produce triple-chain peptides.

[0025] In the present invention, a polymerized peptide is produced in which multiple cysteine ​​residues are incorporated into the peptide chain, and then oxidatively cross-linked using an oxidizing agent such as, but not limited to, dimethyl sulfoxide (DMSO) or air oxidation, thereby providing a polymerized peptide in which the peptides are cross-linked by disulfide bonds.

[0026] In addition to the above-mentioned DMSO, examples of oxidizing agents used in producing the polymerized peptide of the present invention include, but are not limited to, oxygen, iodine, hydrogen peroxide, sodium bromate (sodium bromide), potassium bromate, sodium perborate, and potassium perborate.

[0027] The formation of crosslinks by disulfide bonds in the present invention can be confirmed, for example, by quantifying the remaining thiol groups after the oxidative crosslinking reaction using Ellman's reagent.

[0028] Furthermore, by drying the hydrogel made of polymerized peptides produced in an aqueous solvent according to the present invention, a polymerized peptide thin film with improved strength can be provided, as described below.

[0029] The composition of the present invention contains two or more different polymerized peptides. Different polymerized peptides can be selected based on the physiological activity and physical strength of the polymerized peptide thin film. The ratio of different polymerized peptides in the composition can be adjusted. For example, the following three peptides can be used to prepare films at ratios of 5:5:1, 5:4:1, and 5:3:1. C2s:CCPO(GPO)4GPR(GPO)4GCC H-Cys-Cys-(Pro-Hyp-Gly)5-Pro-Arg-Gly-(Pro-Hyp-Gly)4-Cys-Cys-OH SEQ ID NO: 2 C3s:CCCPO(GPO)4GPR(GPO)4GCCC H-Cys-Cys-Cys-(Pro-Hyp-Gly)5-Pro-Arg-Gly-(Pro-Hyp-Gly)4-Cys-Cys-Cys-OH SEQ ID NO: 1 C3s-GFOGER:CCCPO(GPO)3GFOGER(GPO)4GCCC H-Cys-Cys-Cys-(Pro-Hyp-Gly)4-Phe-Hyp-Gly-Glu-Arg-Gly-(Pro-Hyp-Gly)4-Cys-Cys-Cys-OH SEQ ID NO: 3 Here, a high number of cysteines results in a hard membrane, while a low number of cysteines results in a soft membrane. Therefore, the physical strength is determined by the number of cysteines. On the other hand, GFOGER has an integrin-binding sequence, which allows cells to adhere. Therefore, the bioactivity of C3s-GFOGER is increased by combining it with other C3s-GFOGER molecules.

[0030] The content (%) of the "polymerized peptide" in the entire composition of the present invention (a composition containing a polymerized peptide and a water-soluble filler) is preferably 50 to 90% (throughout this specification, "%" refers to percent by weight unless otherwise specified).

[0031] Water-soluble filler (second component) The composition of the present invention may contain a water-soluble filler in addition to the polymeric peptide. Examples of the water-soluble filler include potassium carbonate, sodium carbonate, ammonium carbonate, calcium lactate, mannitol, urea, inositol, magnesium succinate, sorbitol, and carbohydrates (e.g., sugars such as glucose, lactose, sucrose, mannitol, trehalose, and sorbitol) or a combination thereof, with glucose being preferred.

[0032] The content (%) of the "water-soluble filler" in the entire composition according to the present invention (a composition containing a polymerized peptide and a water-soluble filler) is preferably 10 to 50%.

[0033] The composition of the present invention may contain an excipient in addition to the polymeric peptide and the water-soluble filler.

[0034] Examples of excipients include mannitol, croscarmellose sodium, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyethylene glycol, polyvinylpyrrolidone, crystalline cellulose, lactose, sucrose, starch, corn starch, titanium oxide (TiO2), light anhydrous silicic acid, etc. These excipients may be used alone or in combination of two or more.

[0035] How to make a peptide membrane Different polymerized peptides can be dissolved in a solvent and then gelled to produce a hydrogel of a peptide film. Furthermore, different polymerized peptides can be dissolved in a solvent, added to a water-soluble filler, and gelled to produce a hydrogel of a peptide membrane.

[0036] Furthermore, the hydrogel can be dried to produce a polymerized peptide thin film in the form of a sheet, which can be used, for example, as a material for treating macular holes.

[0037] The polymerized peptide thin film of the present invention can be stored for a long period of time at room temperature by drying, compared to a hydrogel composed of polymerized peptides before drying, and the interactions between the peptide molecules change, resulting in greater strength even after rehydration compared to the original hydrogel. Thus, the polymerized peptide thin film of the present invention has improved strength and can be used as a heat-resistant medical material that can be heat sterilized.

[0038] As used herein, "gel stiffness" refers to the resistance of a gel to degradation by phagocytes such as macrophages in vivo. The stiffness of a gel is affected by the degree of cross-linking polymerization of the gel and the branching structure of the cross-linked peptide chains. For example, a gel containing a large number of disulfide bond cross-links forms a stiff gel.

[0039] The compositions of the present invention may be used directly as a therapeutic agent for macular hole or retinal detachment, or may be formulated into various dosage forms using pharmaceutically acceptable carriers or excipients by methods known to those skilled in the art. The carriers or excipients used are known to those skilled in the art and can be selected appropriately. The pharmaceutical agents of the present invention can be manufactured using means and methods known to those skilled in the art. For example, when preparing injections or infusions, pharmaceutically acceptable carriers such as saline and phosphate-buffered saline can be used. When preparing the pharmaceutical agents of the present invention, pharmaceutically acceptable additives such as thickeners, absorption enhancers, pH adjusters, preservatives, dispersants, wetting agents, stabilizers, antiseptics, suspending agents, and surfactants may be used.

[0040] The dosage form of the drug of the present invention is not particularly limited and can be appropriately selected depending on the site, size, and type of macular hole or retinal detachment to be treated, the patient's condition, etc. The drug of the present invention may be liquid, semi-solid, or solid. Examples of dosage forms of the drug of the present invention include, but are not limited to, thin films. Alternatively, the drug of the present invention may be in the form of a lyophilized product that is suspended in a pharmaceutically acceptable carrier such as saline or phosphate-buffered saline at the time of administration.

[0041] The route of administration of the drug of the present invention is not particularly limited and can be appropriately selected depending on the site, size, and type of macular hole or retinal detachment to be treated, the condition of the patient, etc. Examples of routes of administration of the drug of the present invention include, but are not limited to, injection or placement by incision.

[0042] The dosage of the agent of the present invention can be determined appropriately by a physician depending on the type, location, size, and type of macular hole or retinal detachment to be treated, the condition of the patient, and the like.

[0043] The drug of the present invention can be administered once or multiple times. The number of times can be determined by a physician, taking into consideration the location and severity of the macular hole, the patient's condition, etc.

[0044] In yet another aspect, the present invention provides use of the above composition for producing a medicament for treating macular hole or retinal detachment.

[0045] In yet another aspect, the present invention provides use of the above composition for the treatment of macular holes or retinal detachment.

[0046] In yet another aspect, the present invention provides a method for treating a macular hole, which comprises administering the above-described composition to a patient suffering from a macular hole or retinal detachment.

[0047] The present invention will be explained in more detail and specifically below by showing examples, but the examples should not be construed as limiting the scope of the present invention. [Example]

[0048] The abbreviations used in the examples are listed below. Amino acid residues (all L-form) Arg(R): Arginine Asp(D): Aspartic acid Cys(C): Cysteine Gln(Q): Glutamine Glu(E): glutamic acid Gly(G): Glycine His(H): histidine Hyp(O): 4-hydroxyproline Lys(K): Lysine Pro(P): Proline Tyr(Y): Tyrosine Ile(I): Isoleucine Leu(L): Leucine Met(M): methionine Phe(F): phenylalanine Ser(S): serine Val(V): Valin

[0049] protecting group Fmoc: 9-fluorenylmethoxycarbonyl tBu: tert-butyl Trt: triphenylmethyl (trityl) Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl Boc: tert-butoxycarbonyl

[0050] Resin CTC: 2-chlorotrityl chloride

[0051] reagent BCA: Bicinchoninic acid BSA: bovine serum albumin CHCA: α-cyano-4-hydroxycinnamic acid DCM: dichloromethane DIC: N,N'-diisopropylcarbodiimide DIEA: N,N-diisopropylethylamine DMAP: N,N-dimethylaminopyridine DMF: N,N-dimethylformamide DMSO: dimethyl sulfoxide DTNB: 5,5'-dithiobis-2-nitrobenzoic acid DTT: Dithiothreitol EDT: Ethanedithiol EDTA: Ethylenediaminetetraacetic acid FBS: fetal bovine serum HOBt: 1-hydroxybenzotriazole MeCN: acetonitrile MeOH: Methanol MOPS: 3-morpholinopropanesulfonic acid NEM: N-ethylmaleimide PBS: phosphate buffer solution PMSF: Phenylmethylsulfonyl fluoride SDS: sodium dodecyl sulfate TBS: Tris-buffered saline TFA: Trifluoroacetic acid TMSO: tetramethylene sulfoxide

[0052] device RP-HPLC: reversed-phase high-performance liquid chromatography MALDI-TOF-MS: Matrix-assisted laser desorption / ionization time-of-flight mass spectrometer

[0053] others CD: circular dichroism DDR: discoidin domain receptor ECM: extracellular matrix FAK: focal adhesion kinase HDF: human dermal fibroblasts HRP: Horseradish peroxidase HSPG: Heparan sulfate proteoglycan PEDF: Pigment epithelium-derived factor SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis vWF: von Willebrand factor

[0054] The following examples of the present invention are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. The present invention may be modified, for example, by adding, deleting, or substituting components of the present invention, provided that the modifications do not depart from the spirit of the present invention. [Example]

[0055] Method for producing peptide membrane Each peptide powder, C2s, C3s, and C3s-GFOGER, was weighed and dissolved in a degassed 0.05% TFA solution to a concentration of 12.5 mg / mL. After heating at 85°C for 5 minutes, the mixture was left at room temperature for a while, and then left to stand at 4°C for one day. The C2s solution, C3s solution, and C3s-GFOGER solution were mixed in a 5:4:1 ratio, and DMSO was added to a final concentration of 10%. The mixture was poured onto an acrylic mold plate and left to stand at room temperature in a sealed container under humid conditions for three days. When adding a water-soluble filler, heat the mixture at 85°C for 5 minutes, then leave it at room temperature for a while, and then leave it at 4°C for one day. Mix C2s solution, C3s solution, C3s-GFOGER solution with 10% (w / v) glucose solution and DMSO in a ratio of 5:4:1:1:1. Pour the mixture onto an acrylic plate that will serve as a mold and leave it to stand at room temperature in a sealed container under humid conditions for three days. After 3 days, the acrylic plate is immersed in pure water to prevent the gel from breaking down, and left to stand at room temperature for 1 to 2 hours. The pure water is removed, and the acrylic plate of the mold is placed in a dryer and dried overnight. After drying, the peptide membrane is punched out with a 2 mm diameter trephine and subjected to testing. Test Example 1

[0056] Application test of peptide membranes containing water-soluble fillers to MIO-M1 cell defects Test Method: JPEG0007774276000001.jpg71137 Human Müller cell line MIO-M1 is seeded onto a 6-well Transwell plate and cultured in a CO2 incubator. When the cells become confluent, the insert is removed from the plate and transferred to a 60 mm dish. The culture medium inside the insert is aspirated and a hole is punched in the center of the bottom with a 1 mm diameter disposable biopsy trephine to confirm that it has been perforated. The inserts are transferred to a 6-well Transwell plate containing medium, and the medium volume is adjusted to just cover the bottom of the insert where the cells are attached. A peptide membrane is placed over the central perforated area of ​​the insert. Add a small amount of medium to the bottom of the insert, then transfer to a CO2 incubator and culture. Observe under a microscope over time to confirm that MIO-M1 cells are adhering to and growing on the peptide film. result: In the group in which a 1 mm perforation made in a disposable biopsy trephine was covered with a peptide membrane (without GFOGER), the human Müller cell line MIO-M1 was unable to adhere to the peptide membrane, and no cell spreading or proliferation was observed.On the other hand, in the group in which the perforation was covered with a peptide membrane (with GFOGER), the human Müller cell line MIO-M1 adhered to the peptide membrane, and cell spreading and proliferation were observed (Figures 1 and 2). Test Example 2

[0057] Verification of efficacy and safety in a white rabbit model (in vivo) Test Method: The rabbit was anesthetized (ketamine and xylazine) and the implantation test was performed. A topical anesthetic (Benoxil ophthalmic solution 0.4% solution, Santen Pharmaceutical Co., Ltd.) was instilled into the right eye. The rabbit was placed in a supine position, and the right eye was opened with an eye speculum. The eyeball was disinfected and cleaned with isodine solution diluted 40 times with saline or PA / iodine eye drops and eyewash diluted 4 times with saline, followed by a saline rinse. The surgical site was covered with a protective sheet. After vitreous removal by vitrectomy, a circular hole of approximately 500 μm was created. A peptide membrane and a peptide membrane containing a water-soluble filler were then inserted into the eye through the scleral wound and left in place. The test substance was fixed in place using a combination of viscoelastic materials, nitrogen gas replacement, and perfluorocarbon. Optical coherence tomography (OCT) was performed two days and one week after implantation to evaluate adhesion. result: In the cases where a viscoelastic substance was used in combination, adhesion was observed after one week, and no inflammation or other issues were observed (Figure 3: Peptide membrane without added water-soluble filler; Figure 4: Peptide membrane with added water-soluble filler). Test Example 3

[0058] Verification of efficacy and safety in a cynomolgus monkey model (in vivo) Test Method: Male cynomolgus monkeys were anesthetized and implantation tests were performed. The skin around the eyelids was disinfected with cotton soaked in 10% isodine solution (Meiji Seika Pharma Co., Ltd.) or sterile gauze. After drying, the eyelids were disinfected with cotton soaked in PA / iodine eye drops and eyewash (Japan Eye Drop Research Institute Co., Ltd.) diluted 4:1 with saline (Otsuka Pharmaceutical Factory). A topical anesthetic (Benoxil eye drops 0.4% solution, Santen Pharmaceutical Co., Ltd.) was instilled into the right eye. The monkey was held in a supine position, and the right eye was opened with a sterilized eyelid speculum. The eyeball was disinfected and cleaned with isodine solution diluted 40:1 with saline or PA / iodine eye drops and eyewash diluted 4:1 with saline, and then rinsed with saline. The surgical site was covered with a protective sheet. The vitreous body is removed by vitrectomy, and vitreous forceps are inserted to partially peel off the internal limiting membrane at the area corresponding to the macula, approximately 4 mm in diameter. After creating a hole (0.5 to 1 optic disc diameter) in the macula, a peptide membrane containing a water-soluble filler is inserted into the eye and left in place in the hole. Gas replacement is performed intraocularly as needed, and the test substance is pressed onto the retina. After implantation, the model is prepared based on the macula, and the test substance implantation site is recorded. result: In cases where intraoperative or postoperative gas replacement was performed, adhesion was observed after 8 days, and no inflammation was observed (Figure 5).

Claims

1. Formula (I) -(P 1 ,P 2 ,P 3 )- (I) (In formula (I), P 1 , P 2 and P 3 may be the same or different, and each independently H-Cys-Cys-Cys-(Pro-Hyp-Gly)5-Pro-Arg-Gly-(Pro-Hyp-Gly)4-Cys-Cys-Cys-OH SEQ ID NO: 1, H-Cys-Cys-(Pro-Hyp-Gly)5-Pro-Arg-Gly-(Pro-Hyp-Gly)4-Cys-Cys-OH SEQ ID NO: 2, and H-Cys-Cys-Cys-(Pro-Hyp-Gly)4-Phe-Hyp-Gly-Glu-Arg-Gly-(Pro-Hyp-Gly)4-Cys-Cys-OH SEQ ID NO: 3, P 1 , P 2 and P 3 The composition comprises two or more polymerized peptides each having a triple-stranded peptide represented by the formula (I) below, which forms a trimer having a triple helix structure and is polymerized by oxidative cross-linking through disulfide bonds via cysteine ​​(Cys) residues contained in each peptide chain.

2. P 1 , P 2 and P 3 The composition of claim 1 , wherein:

3. The composition according to claim 1 or 2, further comprising a water-soluble filler.

4. The composition of claim 3 wherein the water-soluble filler is a sugar.

5. 5. The composition of claim 4, wherein the sugar is one or more of glucose, lactose, sucrose, mannitol, trehalose, and sorbitol.

6. The composition of claim 5 , wherein the sugar is glucose.

7. 7. The composition according to claim 3, wherein the water-soluble filler is present in an amount of from 10 to 50% relative to the total weight of the composition.

8. The composition according to any one of claims 1 to 7, wherein the polymerized peptide is present in an amount of 50 to 90% based on the total weight of the composition.

9. The composition according to any one of claims 1 to 8, which is in the form of a gel.

10. The composition according to any one of claims 1 to 8, which is in a transparent form.

11. A pharmaceutical comprising the composition according to any one of claims 1 to 10.

12. A device comprising the composition of any one of claims 1 to 10.

13. A therapeutic agent for eye diseases, comprising the composition according to any one of claims 1 to 10.

14. The therapeutic agent for an eye disease according to claim 13, wherein the eye disease is a macular hole or retinal detachment.

15. A method for producing the composition of any one of claims 3 to 10, comprising bringing the polymeric peptide of formula (I) and a water-soluble filler into a homogeneous physical mixture.

16. Use of a composition according to any one of claims 1 to 10 in the manufacture of a medicament for treating an eye disease.

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