Eye drop composition for preventing or treating ocular neovascular diseases, comprising collagen type i and pigment epithelium-derived factor peptide as active ingredients

Abstract

The present invention relates to an eye drop composition for preventing or treating eye neovascular diseases, comprising collagen type I and a pigment epithelium-derived factor peptide as active ingredients. The present invention provides an eye drop composition capable of most stably storing for a long period of time a pigment epithelium-derived factor peptide, which is an active ingredient exhibiting an anti-neovascular effect on ocular neovascular diseases, and as it is confirmed that the eye drop composition, according to the present invention, exhibits a therapeutic or ameliorating effect on ocular neovascular diseases even when the eye drop composition contains a low concentration of a pigment epithelium-derived factor (PEDF) peptide represented by SEQ ID NO: 1, which is an active ingredient, the present invention provides the eye drop composition exhibiting a therapeutic or ameliorating effect on ocular neovascular diseases and having excellent storage stability.

Description

Eye drop composition for the prevention or treatment of ocular neovascular disease comprising collagen type I and pigment epithelial factor peptide as active ingredients

[0001] The present invention relates to an eye drop composition for preventing or treating ocular neovascular disease, comprising collagen type I and pigment epithelium-derived factor peptide as active ingredients.

[0002] The macula is the neural tissue at the center of the retina. It is where most photoreceptor cells are concentrated and where images are formed, responsible for central vision. Macular degeneration, an eye disease that often progresses with age, causes degeneration of the macula, leading to vision impairment. Initially, the condition causes blurred vision and distorted near-vision, eventually leading to blindness.

[0003] Age-related macular degeneration (AMD) is a chronic, progressive degenerative disease of the macula, resulting in loss of central vision. AMD is currently the most common cause of blindness in people aged 65 and older, and the third leading cause of blindness worldwide. Approximately 30 million people worldwide suffer from the disease, and 500,000 patients lose their vision due to the disease each year. In Korea, 16.5% of people aged 65 and older are found to have early-stage macular degeneration, and the age of onset of macular degeneration is gradually decreasing from the 60s to the 40s.

[0004] Age-related macular degeneration (AMD) is broadly classified into two types: atrophic and exudative. Atrophic AMD accounts for 90% of cases and is a disease characterized by abnormalities in the retinal pigment epithelium (RPE) of the macula, leading to a gradual decline in central vision. Currently, there are no treatments to restore vision beyond smoking cessation, UV protection, and antioxidant use. Exudative AMD accounts for approximately 10% of cases and causes faster and more severe visual impairment than atrophic AMD. Exudative AMD is characterized by fundus findings such as choroidal neovascularization, retinal pigment epithelial detachment, sensory retinal detachment, and retinal pigment epithelial rupture. Exudative AMD is responsible for 70-90% of blindness due to AMD. Choroidal neovascularization (CNV) is a disease in which new blood vessels increase in the choroidal layer of the eye and is associated with AMD.

[0005] Pigment epithelium-derived factor (PEDF) is a protein with anti-angiogenic effects and is considered a treatment for various diseases caused by angiogenesis. Currently, research is underway to develop PEDF as a treatment for macular degeneration. However, its short half-life requires repeated injections, and its narrow concentration range for disease treatment has been pointed out as a problem. Therefore, research is needed to address these issues with PEDF and maximize its effectiveness as a treatment for macular degeneration.

[0006] [Prior Art Literature]

[0007] [Patent Document]

[0008] Korean Patent Publication No. 10-2012-0130752 (published on December 3, 2012)

[0009] Korean Patent Publication No. 10-2018-0034518 (Published on April 4, 2018)

[0010] The purpose of the present invention is to confirm that an eye drop composition comprising collagen type I and pigment epithelium-derived factor (PEDF) peptide has an antiangiogenic effect on ocular neovascular disease, to suggest suitable eye drop conditions for most safely storing pigment epithelium-derived factor peptide as an active ingredient in the eye drop, and to provide the most suitable content range of collagen type I and pigment epithelium-derived factor peptide that exhibits a therapeutic or improving effect on ocular neovascular disease when applied to the eye.

[0011] The present invention provides an eye drop composition for preventing or treating ocular neovascular disease, comprising collagen type I and pigment epithelium-derived factor (PEDF) peptide.

[0012] According to the present invention, an eye drop composition is provided that can most safely store pigmented epithelium-derived factor (PEDF) peptide, which is an active ingredient showing an antiangiogenic effect on ocular neovascular disease, for a long period of time, and by confirming that the eye drop composition according to the present invention shows a treatment or improvement effect on ocular neovascular disease even when it contains a low concentration of the pigmented epithelium-derived factor peptide represented by SEQ ID NO: 1, which is an active ingredient, it is possible to provide an eye drop composition that shows a treatment or improvement effect on ocular neovascular disease and has excellent storage stability.

[0013] Figure 1 shows the results of evaluating the therapeutic or improvement effect on choroidal neovascularization of an eye drop composition under 1x A5S buffer conditions manufactured according to Manufacturing Example 1 of the present invention.

[0014] Figure 2 shows the results of evaluating the therapeutic or improvement effect on choroidal neovascularization of an eye drop composition under 1x A55S buffer conditions manufactured according to Manufacturing Example 2 of the present invention.

[0015] Figure 3 shows the results of evaluating the treatment or improvement effect on choroidal neovascularization of an eye drop composition prepared in 0.1x PBS buffer condition according to Manufacturing Example 3 of the present invention.

[0016] FIG. 4a shows the results of evaluating the content range of pigment epithelium-derived factor (PEDF) peptides that exhibit a therapeutic or improving effect on choroidal neovascularization in an eye drop composition prepared according to Manufacturing Example 2 of the present invention under 1x A55S buffer conditions.

[0017] FIG. 4b shows the results of evaluating the maximum content range of pigment epithelium-derived factor (PEDF) peptides that exhibit a therapeutic or improving effect on choroidal neovascularization in an eye drop composition prepared according to Manufacturing Example 2 of the present invention under 1x A55S buffer conditions.

[0018] FIG. 5 shows the results of evaluating the therapeutic or improvement effect on choroidal neovascularization (CNV) using three types of collagen type I (TheraCol (Porcine), Pepsin Soluble Collagen (Bovine), VitroCol (Human)) from different manufacturers and biological sources in an eye drop composition under 1x A55S buffer conditions manufactured according to Manufacturing Example 4 of the present invention.

[0019] Figure 6 shows the results of two types of collagen type I (Matrixen) from different manufacturers and biological sources in an eye drop composition under 1x AS buffer conditions of pH 5.0, 5.5, and 6.0 manufactured according to Manufacturing Example 5 of the present invention. TM-These are the results of evaluating the treatment or improvement effect on choroidal neovascularization (CNV) using PSP (Porcine), Pepsin Soluble Collagen (Bovine)).

[0020] FIG. 7 is a result of evaluating the treatment or improvement effect on choroidal neovascularization (CNV) according to pH in an eye drop composition prepared according to Manufacturing Example 6 of the present invention under 1x AS buffer conditions of pH 4.0, 4.2, 4.5, 4.7, 5.0, or 5.3.

[0021] FIG. 8 is a result of evaluating the therapeutic or improvement effect on choroidal neovascularization (CNV) by adding 0.005% or 0.01% of BAK (Benzalkonium chloride) or 0.001% of PQ1 (polyquaternium-1) to an eye drop composition containing a preservative added to a 1x AS buffer condition manufactured according to Manufacturing Example 7 of the present invention.

[0022] FIG. 9 is a result of evaluating the maximum content range of pigment epithelium-derived factor (PEDF) peptides that exhibit a therapeutic or improving effect on choroidal neovascularization (CNV) in a composition containing 0.01% BAK (Benzalkonium chloride) added to 1x A5S buffer conditions manufactured according to Manufacturing Example 8 of the present invention.

[0023] FIG. 10 shows the results of evaluating the therapeutic or ameliorating effect on choroidal neovascularization (CNV) using four different pigment epithelium-derived factor (PEDF) peptides (manufactured by PolyPeptide Laboratories, manufacturing numbers FSC04053, FSC04084, AW19111, or 23-22-0147-01) in a composition containing 0.01% BAK in 1x A5S buffer conditions manufactured according to Manufacturing Example 9 of the present invention.

[0024] FIG. 11a shows the results of evaluating the maximum content range of collagen type I (collagen type I, Pepsin Soluble Collagen, Bovine) that exhibits a therapeutic or improving effect on choroidal neovascularization (CNV) in an eye drop composition under 1x A5S buffer conditions manufactured according to Manufacturing Example 10 of the present invention.

[0025] FIG. 11b shows the results of evaluating the minimum content range of collagen type I (collagen type I Pepsin Soluble Collagen, Bovine) that exhibits a therapeutic or improving effect on choroidal neovascularization (CNV) in a composition containing 0.01% BAK (Benzalkonium chloride) added to 1x A5S buffer conditions manufactured according to Manufacturing Example 11 of the present invention.

[0026] Figure 12 shows the results of analyzing the final eye drop compositions manufactured according to Manufacturing Example 12 of the present invention, which were stored for 4 weeks under refrigerated conditions (5±3°C) or room temperature conditions (25±2°C), weekly visual inspections for color, transparency, and formation of precipitates, pH measurement, and the content of preservatives BAK (Benzalkonium chloride) and pigment epithelium-derived factor (PEDF) peptides contained in the eye drop compositions using RP-UPLC (reverse phase ultra-high performance liquid chromatography). Figure 12a shows the results of analyzing the transparency, BAK, and PEDF content at week 0 of storage of the final eye drop compositions of the present invention manufactured according to Manufacturing Example 12. FIG. 12b shows the results of evaluating the color, transparency, and the formation of sediment through weekly visual inspection after storing the final eye drop composition of the present invention manufactured according to Manufacturing Example 12 under refrigerated conditions (5±3°C) for 4 weeks. FIG. 12c shows the results of evaluating the change in BAK and PEDF contents after storing the final eye drop composition of the present invention manufactured according to Manufacturing Example 12 under refrigerated conditions (5±3°C) for 4 weeks. FIG. 12d shows the results of evaluating the color, transparency, and the formation of sediment through weekly visual inspection after storing the final eye drop composition of the present invention manufactured according to Manufacturing Example 12 under room temperature conditions (25±2°C) for 4 weeks. FIG. 12e shows the results of evaluating the change in BAK and PEDF contents after storing the final eye drop composition of the present invention manufactured according to Manufacturing Example 12 under room temperature conditions (25±2°C) for 4 weeks.FIG. 12f is a table showing the results of analyzing changes in the content of the preservative BAK (Benzalkonium chloride) and pigment epithelium-derived factor (PEDF) peptide contained in the eye drop composition, as well as the color, transparency, and precipitate formation through weekly visual inspection of the final eye drop compositions manufactured according to Manufacturing Example 12 of the present invention after storing them under refrigerated conditions (5±3°C) or room temperature conditions (25±2°C) for 4 weeks.

[0027] The terms used in this specification have been selected from widely used, current terms, taking into account the functions of the present invention. However, these terms may vary depending on the intentions of those skilled in the art, precedents, the emergence of new technologies, etc. Furthermore, in certain cases, terms may be arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should not be defined simply as names, but rather based on their inherent meanings and the overall content of the present invention.

[0028] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0029] Numerical ranges are inclusive of the values ​​defined in the ranges above. Any maximum numerical limitation given throughout this specification includes any lower numerical limitation, as if that lower numerical limitation were explicitly stated. Any minimum numerical limitation given throughout this specification includes any higher numerical limitation, as if that higher numerical limitation were explicitly stated. Any numerical limitation given throughout this specification will include any better numerical range within the broader numerical range, as if that narrower numerical limitation were explicitly stated.

[0030] Hereinafter, the present invention will be described in more detail.

[0031] The present invention provides an eye drop composition for preventing or treating ocular neovascular disease, comprising collagen type I and pigment epithelium-derived factor (PEDF) peptide.

[0032] The above pigment epithelial factor (PEDF) peptide is composed of a 34 amino acid sequence represented by sequence number 1 (DPFFK VPVNK LAAAV SNFGY DLYRV RSSTS PTTN) and can be included in an eye drop composition in a therapeutically effective amount that exhibits an effect of inhibiting intraocular neovascularization.

[0033] The therapeutically effective amount may vary depending on the subject, and if the subject is a human, it may vary depending on factors such as age, weight, height, sex, general medical condition, and previous medical history. The subject may be a mammal including a human, and mammals may include humans, mice, rats, dogs, and cats.

[0034] In a specific embodiment of the present invention, when the subject is a mouse, the eye drop composition of the present invention may include the pigment epithelium-derived factor (PEDF) peptide in an amount of 0.188 ng / 10 μL (0.005 μM) to 37.6 μg / 10 μL (1000 μM), and specifically, the concentration exhibiting choroidal neovascularization (CNV) efficacy may be included in an amount of 7.52 ng / 10 μL to 37.6 μg / 10 μL. The eye drop composition of the present invention can be used by instilling the pigment epithelium-derived factor (PEDF) peptide into the eye once or several times a day at a dosage of 10 μL in a range of 7.52 ng to 37.6 μg, and preferably, the effective amount of the pigment epithelium-derived factor (PEDF) peptide can be used by instilling the eye locally in a dosage range of 0.376 μg / kg / day (7.52 ng / eye / day) to 1.88 mg / kg / day (37.6 μg / eye / day).

[0035] In addition, in another specific embodiment of the present invention, when the subject is a human, the eye drop composition of the present invention may contain the pigment epithelium-derived factor (PEDF) peptide in an amount of 0.94 ng / 50 μL to 94 mg / 50 μL, and specifically, the concentration exhibiting choroidal neovascularization (CNV) efficacy may be contained in an amount of 37.6 ng / 50 μL to 94 mg / 50 μL. The eye drop composition of the present invention can be used by instilling the pigment epithelium-derived factor (PEDF) peptide into the eye once a day at a dosage of 50 μL to 100 μL in a range of 37.6 ng to 94 mg, and preferably, the pigment epithelium-derived factor (PEDF) peptide can be used by instilling the eye locally at a dosage range of 0.63 ng / kg / day (37.6 ng / eye / day) to 1.57 mg / kg / day (94 mg / eye / day).

[0036] The above eye drop composition may include a buffer that is harmless to the human body so as to safely store the active ingredient, and the buffer may include sodium acetate, sorbitol, and polysorbate 80, and may include BAK (Benzalkonium chloride) or PQ-1 (polyquaternium-1) as a preservative. More specifically, the eye drop composition may include a buffer containing 9.9 mM to 10.1 mM sodium acetate, 4.9 wt% to 5.1 wt% sorbitol, and 0.009 wt% to 0.011 wt% polysorbate 80 to safely store the active ingredient under various temperature conditions for a long period of time, and may include 0.005% or 0.01% BAK (Benzalkonium chloride) or 0.001% PQ-1 (polyquaternium-1) as a preservative. The pH of the buffer may be adjusted by adding acetic acid, and the A5S buffer may form a pH of 4.9 to 5.1, and the A55S buffer may form a pH of 5.4 to 5.6. Since the above eye drop composition contains the buffer, it can be stored for 4 weeks at a temperature ranging from 1°C to 30°C.

[0037] The above eye drop composition can exhibit a pharmacological effect by instilling the pigment epithelium-derived factor (PEDF) peptide into the eye once a day at a dosage ranging from 37.6 ng to 94 mg, and the dosage can be from 50 μL to 100 μL.

[0038] The above ocular neovascular disease may be at least one disease selected from corneal neovascularization, diabetic retinopathy, choroidal neovascularization, and age-related macular degeneration.

[0039] The composition of the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains.

[0040] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0041] In the present invention, the content of the additive included in the composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0042] The composition of the present invention may further comprise pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starches, sugars, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinyl pyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbates, cetyl alcohol, and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents, and / or dispersion media.

[0043] As used herein, the term "prevention" refers to any action that suppresses or delays the onset of a disease by administering a composition according to the present invention. The term "treatment" refers to any action that improves or beneficially alters the symptoms of a disease by administering a composition according to the present invention. As used herein, "improvement" refers to any action that improves the worsening condition of a disease by administering or ingesting a composition according to the present invention to a subject.

[0044]

[0045] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0046]

[0047] <Manufacturing Example> Manufacturing of eye drop composition

[0048] The eye drop composition of the present invention comprises collagen type I and a pigment epithelium-derived factor (PEDF) peptide represented by sequence number 1, and was manufactured under 12 conditions as follows.

[0049] Collagen type I was prepared using four different collagens from different manufacturers and biological sources. Manufacturing examples 1, 2, 3, or 5 were prepared using Matrixen as collagen type I. TM -PSP (HYUNDAI BIOLAND Co., Ltd., Porcine) is used, Manufacturing Example 3 or 4 uses TheraCol (Sewon Cellontech Co., Ltd., Porcine), Manufacturing Example 4 uses VitroCol (ADVANCED BIOMETRIX, Human), and Manufacturing Examples 6, 7, 8, 9, 10, 11, or 12 uses Pepsin Soluble Collagen (COLLAGEN SOLUTIONS, Bovine).

[0050] Pigment epithelium-derived factor (PEDF) peptides represented by sequence number 1 were prepared using four different lot numbers FSC04053, FSC04084, AW19111, or 23-22-0147-01 manufactured by PolyPeptide Laboratories. Preparation example 1, 2, 3, 4, 5, 6, 7, 8, or 10 uses lot number FSC04053, Preparation example 9 uses lot number FSC04053, FSC04084, AW19111, or 23-22-0147-01, and Preparation example 11 or 12 uses lot number 23-22-0147-01.

[0051]

[0052] Manufacturing Example 1. Eye drop composition under 1x A5S buffer conditions

[0053] Collagen type I (Matrixen) in 2x A5S buffer containing 20 mM sodium acetate, 10% sorbitol, and 0.02% polysorbate80, adjusted to pH 5.0±0.1 with acetic acid. TM An eye drop composition was prepared under 1x A5S buffer conditions so that the final concentration of -PSP (Porcine) was 0.1% and the final eye drop concentration of pigment epithelium-derived factor (PEDF) peptide (manufacturing number FSC04053) represented by sequence number 1 was 1 μM, 5 μM, or 25 μM, and 10 μL was used for eye drop.

[0054] The doses administered to evaluate the efficacy of the above-mentioned manufactured eye drop composition were 1.88 μg / kg / day (37.6 ng / eye / day), 9.4 μg / kg / day (188 ng / eye / day), or 47.0 μg / kg / day (940 ng / eye / day) to mice. When the doses set for mice are converted to humans, they correspond to a range of 3.13 ng / kg / day (188 ng / eye / day) to 40 μg / kg / day (2.35 mg / eye / day). The pigment epithelial factor (PEDF) peptide represented by sequence number 1 is 3.7632 kDa, and when 1 μM is administered in 10 μL eye drops, it is 37.632 ng / eye / day.

[0055]

[0056] Manufacturing Example 2. Eye drop composition under 1x A55S buffer conditions

[0057] Porcine collagen type I (Matrixen) from different manufacturers was prepared using 2x A55S buffer containing 20 mM sodium acetate, 10% sorbitol, and 0.02% polysorbate80, and pH adjusted to 5.5±0.1 with acetic acid. TM -PSP or TheraCol) to a final concentration of 0.1%, pigmented epithelium-derived factor (PEDF) peptide represented by sequence number 1 (manufactured number FSC04053) was prepared in 1x A55S buffer conditions to a final concentration of 0.2 μM, 1 μM, 5 μM, 25 μM, 50 μM / 10 μL, 100 μM, 200 μM, or 400 μM, and 10 μL was used for eye drop.

[0058] The administration doses for evaluating the efficacy of the above-mentioned manufactured eye drop composition were 0.376 μg / kg / day (7.52 ng / eye / day), 1.88 μg / kg / day (37.6 ng / eye / day), 9.4 μg / kg / day (188 ng / eye / day), 47 μg / kg / day (940 ng / eye / day), 94 μg / kg / day (1.88 μg / eye / day), 188 μg / kg / day (3.76 μg / eye / day), 376 μg / kg / day (7.52 μg / eye / day), or 752 μg / kg / day (15 μg / eye / day). When the dose set for the above mice is converted to a human dose, it is equivalent to a range of 0.63 ng / kg / day (37.6 ng / eye / day) to 0.63 mg / kg / day (37.6 mg / eye / day).

[0059]

[0060] Manufacturing Example 3. Eye drop composition under 0.1x PBS buffer conditions

[0061] Collagen type I (Matrixen) was prepared by diluting 1x PBS (phosphate buffer saline) with distilled water to a concentration of 0.1% in 0.1x PBS buffer. TM -PSP, Porcine) was mixed to a final concentration of 0.1%, and the pigment epithelium-derived factor (PEDF) peptide (manufacturing number FSC04053) represented by sequence number 1 was mixed to a final concentration of 1 μM, 5 μM, or 25 μM to prepare an eye drop composition, and 10 μL was used for eye drop.

[0062] The administration doses for evaluating the efficacy of the above-mentioned manufactured eye drop composition were 1.88 μg / kg / day (37.6 ng / eye / day), 9.4 μg / kg / day (188 ng / eye / day), or 47 μg / kg / day (940 ng / eye / day). When the administration doses set for the mice are converted to humans, they correspond to a range of 3.13 ng / kg / day (188 ng / eye / day) to 40 μg / kg / day (2.35 mg / eye / day).

[0063]

[0064] Manufacturing Example 4. Eye drop composition manufactured with collagen type I from different manufacturers and biological sources.

[0065] An eye drop composition was prepared by mixing 2x A55S buffer to a final concentration of 1x so that the pH of the final eye drop composition was 5.5±0.3, containing 20 mM sodium acetate, 10% sorbitol, and 0.02% polysorbate80, and a pigmented epithelium-derived factor (PEDF) peptide represented by SEQ ID NO: 1 (manufacturing number FSC04053) to a final concentration of 5 μM, and using TheraCol (Porcine), Pepsin Soluble Collagen (Bovine), or VitroCol (Human) as collagen type I to a final concentration of 0.1%, and using 10 μL for eye drop application.

[0066] For TheraCol, the pH of the final eye drop composition was 5.31 using 2x AS buffer adjusted to pH 5.5 with acetic acid, for Pepsin Soluble Collagen, the pH of the final eye drop composition was 5.29 using 2x AS buffer adjusted to pH 6.91 with sodium hydroxide, and for VitroCol, the pH of the final eye drop composition was 5.30 using 2x AS buffer adjusted to pH 11.59 with sodium hydroxide.

[0067] The dosage for evaluating the efficacy of the above-mentioned manufactured eye drop composition was 9.4 μg / kg / day (188 ng / eye / day). When the dosage set for mice is converted to a human dosage, it corresponds to a range of 15.7 ng / kg / day (940 ng / eye / day) to 7.83 μg / kg / day (470 μg / eye / day).

[0068]

[0069] Manufacturing Example 5. Eye drop compositions containing two types of collagen I from different manufacturers and biological sources and three pH conditions.

[0070] 2x AS buffer containing 20 mM sodium acetate, 10% sorbitol, and 0.02% polysorbate80, adjusted to pH 5.0, 5.5, or 6.0 with acetic acid to a final concentration of 1x, and Pepsin Soluble Collagen (Bovine) or Matrixen as collagen type I. TM - An eye drop composition was prepared by mixing PSP (Porcine) to a final concentration of 0.1% and the pigmented epithelium-derived factor (PEDF) peptide (manufacturing number FSC04053) represented by sequence number 1 to a final concentration of 5 μM, and 10 μL was used for eye drop. Matrixen TM -The final pH of the eye drop composition manufactured with PSP is 4.90, 5.31, or 5.60, and the final pH of the eye drop composition manufactured with Pepsin Soluble Collagen is 4.68, 4.96, or 5.10.

[0071] The dosage for evaluating the efficacy of the above-mentioned manufactured eye drop composition was 9.4 μg / kg / day (188 ng / eye / day). When the dosage set for mice is converted to a human dosage, it corresponds to a range of 15.7 ng / kg / day (940 ng / eye / day) to 7.83 μg / kg / day (470 μg / eye / day).

[0072]

[0073] Manufacturing Example 6. Eye drop compositions prepared with Pepsin Soluble Collagen under various pH conditions

[0074] An eye drop composition was prepared by mixing 2x AS buffer containing 20 mM sodium acetate, 10% sorbitol, and 0.02% polysorbate80, and adjusted to pH 4.0, 4.2, 4.5, 4.7, 5.0, or 5.3 with acetic acid to a final concentration of 1x, collagen type I (Pepsin Soluble Collagen, Bovine) to a final concentration of 0.1%, and pigmented epithelium-derived factor (PEDF) peptide represented by SEQ ID NO: 1 (manufacturing number FSC04053) to a final concentration of 5 μM. 10 μL is used for eye drop. The final pH of the eye drop composition is 3.89, 4.04, 4.29, 4.48, 4.65, or 4.85.

[0075] The dosage for evaluating the efficacy of the above-mentioned manufactured eye drop composition was 9.4 μg / kg / day (188 ng / eye / day). When the dosage set for mice is converted to a human dosage, it corresponds to a range of 15.7 ng / kg / day (940 ng / eye / day) to 7.83 μg / kg / day (470 μg / eye / day).

[0076]

[0077] Manufacturing Example 7. Eye drop composition with added preservative BAK or PQ1

[0078] An eye drop composition is prepared by mixing 2x AS buffer containing 20 mM sodium acetate, 10% sorbitol, 0.02% polysorbate80, 0.01% or 0.02% BAK (Benzalkonium chloride) as a preservative, or 0.002% PQ-1 (polyquaternium-1) and adjusted to pH 5.3±0.2 with acetic acid to a final concentration of 1x, using collagen type I (Pepsin Soluble Collagen, Bovine) to a final concentration of 0.1%, and mixing the pigmented epithelium-derived factor (PEDF) peptide represented by sequence number 1 (manufacturing number FSC04053) to a final concentration of 5 μM or 200 μM. It was manufactured and 10 μL is used for eye drops.

[0079] The administration dose for evaluating the efficacy of the above-mentioned manufactured eye drop composition was 9.4 μg / kg / day (188 ng / eye / day) or 376 μg / kg / day (7.52 μg / eye / day). When the administration dose set for the mouse is converted to a human dose, it is equivalent to a range of 15.7 ng / kg / day (940 ng / eye / day) to 0.31 mg / kg / day (18.8 mg / eye / day).

[0080]

[0081] Manufacturing Example 8. Eye drop compositions with various peptide concentrations with added preservative BAK

[0082] 2x A5S buffer containing 20 mM sodium acetate, 10% sorbitol, 0.02% polysorbate80, 0.02% BAK (Benzalkonium chloride) as a preservative, pH adjusted to 5.0 with acetic acid to a final concentration of 1x, collagen type I (Pepsin Soluble Collagen, Bovine) to a final concentration of 0.1%, and pigmented epithelium-derived factor (PEDF) peptide represented by SEQ ID NO: 1 (manufacturing number FSC04053) to a final concentration of 0.005 μM, 0.05 μM, 0.5 μM, 5 μM, 50 μM, 200 μM, 500 μM or An eye drop composition was prepared by mixing to make 1000 μM, and 10 μL was used for eye drop.

[0083] The administration doses for evaluating the efficacy of the above-mentioned manufactured eye drop composition were 9.4 ng / kg / day (0.188 ng / eye / day), 94 ng / kg / day (1.88 ng / eye / day), 940 ng / kg / day (18.8 ng / eye / day), 9.4 μg / kg / day (188 ng / eye / day), 94 μg / kg / day (1.88 μg / eye / day), 376 μg / kg / day (7.52 μg / eye / day), 940 μg / kg / day (18.8 μg / eye / day), or 1.88 mg / kg / day (37.6 μg / eye / day). When the dose set for the above mice is converted to a human dose, it is equivalent to a range of 15.7 pg / kg / day (0.94 ng / eye / day) to 1.57 mg / kg / day (94 mg / eye / day).

[0084]

[0085] Manufacturing Example 9. Eye drop composition manufactured with peptides of four manufacturing numbers

[0086] An eye drop composition was prepared by mixing 2x A5S buffer containing 20 mM sodium acetate, 10% sorbitol, 0.02% polysorbate80, 0.02% BAK (Benzalkonium chloride) as a preservative and adjusted to pH 5.0 with acetic acid to a final concentration of 1x, collagen type I (Pepsin Soluble Collagen, Bovine) to a final concentration of 0.1%, and pigmented epithelium-derived factor (PEDF) peptide represented by sequence number 1 to a final concentration of 5 μM. 10 μL is used for eye drop application. The pigment epithelial factor (PEDF) peptides used in the manufacture of the eye drop composition were manufactured under four different manufacturing numbers (manufacturing numbers FSC04053, FSC04084, AW19111, 23-22-0147-01).

[0087] The dosage administered to evaluate the efficacy of the above-mentioned manufactured eye drop composition was 9.4 μg / kg / day (188 ng / eye / day). When the dosage set for mice is converted to a human dosage, it corresponds to a range of 15.7 ng / kg / day (940 ng / eye / day) to 7.83 μg / kg / day (470 μg / eye / day).

[0088]

[0089] Manufacturing Example 10. Eye drop composition containing 0.025% to 0.2% of collagen type I

[0090] An eye drop composition was prepared by mixing 2x A5S buffer containing 20 mM sodium acetate, 10% sorbitol, and 0.02% polysorbate80 and adjusted to pH 5.0±0.1 with acetic acid to a final concentration of 1x, a pigmented epithelium-derived factor (PEDF) peptide represented by SEQ ID NO: 1 (manufacturing number FSC04053) to a final concentration of 5 μM, and collagen type I (Pepsin Soluble Collagen, Bovine) to a final concentration of 0.025%, 0.05%, 0.075%, 0.1%, or 0.2%, and using 10 μL for eye drop. The final eye drop composition pH depending on the concentration of collagen type I (collagen type I, Pepsin Soluble Collagen, Bovine) is 4.89, 4.79, 4.71, 4.63, or 4.35.

[0091] The dosage administered to evaluate the efficacy of the above-mentioned manufactured eye drop composition was 9.4 μg / kg / day (188 ng / eye / day). When the dosage set for mice is converted to a human dosage, it corresponds to a range of 15.7 ng / kg / day (940 ng / eye / day) to 7.83 μg / kg / day (470 μg / eye / day).

[0092]

[0093] Manufacturing Example 11. Eye drop composition containing 0.001% to 0.1% of collagen type I

[0094] An eye drop composition is prepared by mixing 2x A5S buffer containing 20 mM sodium acetate, 10% sorbitol, 0.02% polysorbate80, 0.02% BAK (Benzalkonium chloride) as a preservative and adjusted to pH 5.0 with acetic acid to a final concentration of 1x, a pigmented epithelium-derived factor (PEDF) peptide represented by sequence number 1 (manufacturing number 23-22-0147-01) to a final concentration of 5 μM, and collagen type I (collagen type I, Pepsin Soluble Collagen, Bovine) to a final concentration of 0.001%, 0.005%, 0.01%, 0.05%, or 0.1%. It was manufactured and 10 μL is used for eye drop application. The final eye drop composition pH depending on the concentration of collagen type I (collagen type I, Pepsin Soluble Collagen, Bovine) is 5.17, 5.15, 5.12, 4.99, or 4.85.

[0095] The dosage administered to evaluate the efficacy of the above-mentioned manufactured eye drop composition was 9.4 μg / kg / day (188 ng / eye / day). When the dosage set for mice is converted to a human dosage, it corresponds to a range of 15.7 ng / kg / day (940 ng / eye / day) to 7.83 μg / kg / day (470 μg / eye / day).

[0096]

[0097] Manufacturing Example 12. Eye drop composition for storage stability evaluation

[0098] In order to evaluate the storage stability of the final eye drop composition showing the effect of inhibiting choroidal neovascularization (CNV), the pigmented epithelium-derived factor (PEDF) peptide represented by sequence number 1 (manufacture number 23-22-0147-01) was dissolved in water for injection as preparation A to prepare a final concentration of 800 μM, and as preparation B, 2x A5S buffer containing 20 mM sodium acetate, 10% sorbitol, 0.02% polysorbate 80, and 0.02% BAK (Benzalkonium chloride) as a preservative and adjusted to pH 5.0±0.3 with acetic acid was prepared, and as preparation C, 6 mg / ml collagen type I (collagen type I, Pepsin Soluble Collagen, Bovine) was prepared. Get ready.

[0099] To prepare 3 mL of a 50 μM, 0.1% collagen type I eye drop composition, mix 188 μL of preparation A, 1500 μL of preparation B, 500 μL of preparation C and 812 μL of water for injection.

[0100] To prepare 3 mL of a 200 μM, 0.1% collagen type I eye drop composition, mix 752 μL of preparation A, 1500 μL of preparation B, 500 μL of preparation C and 248 μL of water for injection.

[0101]

[0102] <Example 1> Collagen Type I Matrixen in an animal model of choroidal neovascularization TM -Evaluation of the therapeutic or improvement efficacy of each eye drop composition manufactured with PSP

[0103] In order to identify the eye drop composition having the best treatment or improvement effect on choroidal neovascularization (CNV) among the eye drop compositions of each manufacturing example manufactured according to the present invention, an animal model of choroidal neovascularization (CNV) formation was established, and the improvement effect on neovascular lesions according to administration of each eye drop composition was evaluated. The animal model used in the experiment was a 5-week-old C57BL / 6 mouse, purchased from Nara Biotech, and used in this experiment when the mice were 6 to 8 weeks old after a breeding adaptation period.

[0104] Mice were instilled with 5 mg / mL tropicamide (Santen Pharmaceutical Co. Ltd., Kita-ku, Osaka, Japan) to dilate the pupils, and anesthetized with 100 mg / kg ketamine (Huons, SEONGNAM-SI, GYEONGGI-DO, Korea) and 10 mg / kg xylazine (BAYER, Leverkusen, Germany) intraperitoneally. After anesthesia, the mice were fixed on a mouse fixture, and a slit-lamp microscope laser system was used to irradiate the retina with a 210 mW laser for 0.1 s to create four to five lesions, each 100 μm in size, at the 3, 6, 9, and 12 o'clock positions.

[0105] In the mouse model in which a lesion was created in the retina, each eye drop composition in the above manufacturing example contained pigmented epithelium-derived factor (PEDF) peptide at an administration dose of 1.88 μg / kg / day (37.6 ng / eye / day), 9.4 μg / kg / day (188 ng / eye / day), or 47 μg / kg / day (940 ng / eye / day), and 10 μL was instilled into the right eye once a day for 14 days.

[0106] In the control group, 4 μg / 2 μl of Eylea (Bayer AG) was injected intravitreally (IVT) using a 35 Ultra Micropump and Microcontroller system. Mice receiving each eye drop for 14 days after laser irradiation were anesthetized with ketamine (100 mg / kg) and xylazine (10 mg / kg), and 100 μl of 25 mg / mL FITC-dextran (Sigma-Aldrich, St. Louis, MO, USA) was injected retro-orbitally to stain the retinal blood vessels for 30 minutes. The eyes were then enucleated, fixed in 10% formalin for 1 hour at room temperature, and the muscles, cornea, iris, lens, and vitreous around the fixed eyes were carefully removed under a microscope without damaging the retina. After cutting four parts in the 3, 6, 9, and 12 o'clock directions, a cover slip was placed, and the lesion was photographed using a fluorescence microscope. The area of ​​the lesion was measured and compared with the control group.

[0107] In the case of mice administered with the eye drop composition in the 1x A5S buffer condition prepared according to Manufacturing Example 1, the effect of inhibiting choroidal neovascularization was not significantly observed compared to the control group under all conditions, as shown in Fig. 1. On the other hand, in the case of mice administered the eye drop composition under 1x A55S buffer conditions prepared according to Manufacturing Example 2, as shown in FIG. 2, when the content of pigment epithelial factor (PEDF) peptide in the eye drop composition under 1x A55S buffer conditions was 1.88 μg / kg / day (37.6 ng / eye / day), 9.4 μg / kg / day (188 ng / eye / day), or 47 μg / kg / day (940 ng / eye / day), the area of ​​choroidal neovascularization (CNV) was significantly reduced by 57.1±23.9%, 59.4±29.5%, or 65.1±29.1%. In the case of mice administered the eye drop composition in 0.1x PBS buffer condition prepared according to Manufacturing Example 3, as shown in Fig. 3, when the content of pigment epithelium-derived factor (PEDF) peptide in the eye drop composition in 0.1x PBS buffer condition was 9.4 μg / kg / day (188 ng / eye / day) and 47 μg / kg / day (940 ng / eye / day), the area of ​​choroidal neovascularization was significantly reduced by 64.4±32.9% or 62.9±23.4%, but when the content was 1.88 μg / kg / day (37.6 ng / eye / day), the area of ​​choroidal neovascularization was 86.5±37.0%, showing a low effect of inhibiting choroidal neovascularization (CNV).

[0108] In summary of the above results, the effect of inhibiting choroidal neovascularization (CNV) was found to be the best in the eye drop composition prepared according to Manufacturing Example 2 under the condition of 1x A55S buffer, and the effect of inhibiting choroidal neovascularization (CNV) was also found to be excellent under the condition of the eye drop composition containing a low concentration of pigment epithelial factor (PEDF) peptide of 1.88 μg / kg / day (37.6 ng / eye / day).

[0109]

[0110] <Example 2> Evaluation of therapeutic efficacy concentration range in an animal model of choroidal neovascularization

[0111] Referring to the results of Example 1, the concentration range of pigment epithelium-derived factor (PEDF) peptide showing an effect of inhibiting choroidal neovascularization (CNV) was confirmed using an eye drop composition prepared in 1x A55S buffer condition according to Manufacturing Example 2, which showed the best efficacy. The method of producing a laser-induced choroidal neovascularization (CNV) formation model and analyzing lesions was performed in the same manner as in Example 1. The eye drop compositions prepared according to Manufacturing Example 2 in 1x A55S buffer conditions contained pigment epithelial factor (PEDF) peptide at a dosage of 0.376 μg / kg / day (7.52 ng / eye / day), 1.88 μg / kg / day (37.6 ng / eye / day), or 9.4 μg / kg / day (188 ng / eye / day), and 10 μL was administered to the right eye once a day for 14 days.

[0112] For comparison of efficacy, mice were used as a control group to which the eye drop composition prepared in 0.1x PBS buffer condition according to Manufacturing Example 3 was applied once a day for 14 days. In the eye drop composition of the control group, 10 μL was applied once a day to the right eye for 14 days so that the pigment epithelial factor (PEDF) peptide was included as an administration dose of 9.4 μg / kg / day (188 ng / eye / day).

[0113] Fourteen days after laser irradiation, mice in each experimental group were anesthetized with ketamine (100 mg / kg) and xylazine (10 mg / kg), and 50 μL of 10% Fluorescite (Alcon, Fort Worth, TX, USA) was injected retro-orbitally to stain retinal blood vessels for 5 minutes. The eyes were then enucleated, flat-mounted using the same method as in Example 1, and the lesion area was measured after photographing the lesion and compared with the control group.

[0114] In the case of mice administered an eye drop composition prepared according to Manufacturing Example 2 in a 1x A55S buffer condition, when the pigment epithelial factor (PEDF) peptide content was 0.376 μg / kg / day (7.52 ng / eye / day), 1.88 μg / kg / day (37.6 ng / eye / day), or 9.4 μg / kg / day (188 ng / eye / day), as shown in FIG. 4a, the area of ​​choroidal neovascularization (CNV) was significantly reduced by 72.6±26.6%, 66.9±21.1%, or 64.2±22.3%. Additionally, when the content of pigment epithelial factor (PEDF) peptide was increased to 752 μg / kg / day (15 μg / eye / day), as shown in Fig. 4b, when the eye drop composition prepared according to Manufacturing Example 2 in the 1x A55S buffer condition was administered, the area of ​​choroidal neovascularization (CNV) was 65.62±23% when the content of pigment epithelial factor (PEDF) peptide (manufacturing number FSC04053) was 9.4 μg / kg / day (188 ng / eye / day), 47 μg / kg / day (940 ng / eye / day), 94 μg / kg / day (1.88 μg / eye / day), 188 μg / kg / day (3.76 μg / eye / day), or 376 μg / kg / day (7.52 μg / eye / day). The area of ​​choroidal neovascularization (CNV) was significantly reduced by 72.19±30.49%, 75.36±27.08%, 71.42±30.48%, or 62.28±37.97%. On the other hand, when the pigment epithelial factor (PEDF) peptide content was 752 μg / kg / day (15 μg / eye / day), the inhibitory effect was low at 80.64±34.59%.

[0115] In summary of the above results and the results of Example 1, the content of pigment epithelial factor (PEDF) peptide (manufacturing number FSC04053) in the eye drop composition under 1x A55S buffer conditions manufactured according to Manufacturing Example 4 showed an excellent choroidal neovascularization (CNV) inhibitory effect when the range of the total eye drop composition weight was 0.376 μg / kg / day (7.52 ng / eye / day) to 376 μg / kg / day (7.52 μg / eye / day).

[0116]

[0117] <Example 3> Evaluation of the therapeutic or improvement efficacy range according to the type of collagen type 1 in an animal model of choroidal neovascularization formation

[0118] In order to identify the eye drop composition having the best treatment or improvement effect on choroidal neovascularization (CNV) among the eye drop compositions of each manufacturing example manufactured according to the present invention, the CNV inhibitory effect was identified according to the type of collagen type I using the eye drop composition in 1x A55S buffer condition. The method for producing a laser-induced choroidal neovascularization (CNV) formation model and analyzing lesions was performed in the same manner as in Example 1. According to Manufacturing Example 4 or 5, eye drop compositions prepared with collagen type I from different manufacturers in 1x A55S buffer conditions were administered at a dosage of 9.4 μg / kg / day (188 ng / eye / day) of pigment epithelium-derived factor (PEDF) peptide (manufacturing number FSC04053), and 10 μL was applied to the right eye once a day for 14 days.

[0119] In the case of mice administered an eye drop composition prepared with three types of collagen type I prepared according to Manufacturing Example 4, as shown in FIG. 5, when the collagen type I was TheraCol (Porcine), Pepsin Soluble Collagen (Bovine), or VitroCol (Human), the area of ​​choroidal neovascularization (CNV) was significantly reduced by 68.3±42.4%, 68.1±39.7%, or 69.7±30.3%.

[0120] In the case of mice administered with two types of collagen type I and eye drop compositions with three pH conditions prepared according to Manufacturing Example 5, as shown in Fig. 6, in the eye drop composition prepared using Pepsin Soluble Collagen (Bovine) as collagen type I, the area of ​​choroidal neovascularization (CNV) was significantly reduced by 56.4±30.1%, 53.5±26.2%, or 72.0±35.9% when the pH of the 2x AS buffer was 5.0, 5.5, or 6.0. On the other hand, in the case of collagen type I (collagen type I, Matrixen TM - In the eye drop composition manufactured using PSP, Porcine, when the pH of the 2x AS buffer was 5.0, the effect of inhibiting the area of ​​choroidal neovascularization (CNV) was low at 82.2±38.8%, but when the pH of the 2x AS buffer was 5.5 or 6.0, the effect of significantly reducing the area of ​​choroidal neovascularization (CNV) by 71.9±37.9% or 71.9±40.7% was observed.

[0121] In summary of the above results, it was confirmed that the eye drop compositions manufactured with TheraCol (Porcine), VitroCol (Human), or Pepsin Soluble Collagen (Bovine) showed almost the same inhibitory efficacy on choroidal neovascularization (CNV) at 9.4 μg / kg / day (188 ng / eye / day) of pigment epithelial factor (PEDF) peptide (manufacturing number FSC04053). In addition, when a comparative experiment on the inhibitory efficacy on choroidal neovascularization (CNV) according to pH was conducted, Matrixen TM - In the case of PSP (Porcine), it was confirmed that the inhibitory effect on choroidal neovascularization (CNV) was not observed at pH 5.0 and was only observed at pH 5.5 and 6.0, while in the case of Pepsin Soluble Collagen (Bovine), the inhibitory effect on choroidal neovascularization (CNV) was observed at all pHs of 5.0, 5.5, and 6.0.

[0122]

[0123] <Example 4> Evaluation of the therapeutic or improvement efficacy range of eye drops prepared with pigment epithelial factor peptide (manufacturing number FSC04053) in an animal model of choroidal neovascularization

[0124] Referring to the results of Example 3 above, in order to identify the eye drop composition having the best treatment or improvement effect on choroidal neovascularization (CNV) among the eye drop compositions manufactured with collagen type I (collagen type I, Pepsin Soluble Collagen, Bovine), the CNV inhibition effect was evaluated according to the concentration of pigment epithelium-derived factor (PEDF) peptide (manufacturing number FSC04053), the concentration of collagen type I, the pH of AS buffer, or the presence or absence of a preservative.

[0125] In the case of mice administered with eye drop compositions of various pH AS buffer conditions manufactured with Pepsin Soluble Collagen (Bovine) according to Manufacturing Example 6, as shown in Fig. 7, when the pH of the 2x AS buffer was 4.0, 4.2, 4.5, 4.7, 5.0, or 5.3, the area of ​​choroidal neovascularization (CNV) was significantly reduced by 66.3±23.1%, 66.9±31.4%, 66.0±37.2%, 67.4±31.2%, or 65.1±36.1%, but when the pH was 5.3, the area of ​​choroidal neovascularization (CNV) was 82.0±35.6%, showing a low effect of inhibiting choroidal neovascularization (CNV).

[0126] In the case of mice administered an eye drop composition containing a preservative added to a 1x A55S buffer condition according to Manufacturing Example 7, as shown in FIG. 8, when the content of pigment epithelial factor (PEDF) peptide in the eye drop composition containing a 1x A55S buffer condition was 9.4 μg / kg / day (188 ng / eye / day) or 376 μg / kg / day (7.52 μg / eye / day) and 0.005% of the preservative BAK (Benzalkonium chloride) was added, the area of ​​choroidal neovascularization (CNV) was significantly reduced by 71.4±36.6% or 59.9±33.6%, and when 0.01% of the preservative BAK was added, the area of ​​choroidal neovascularization (CNV) was significantly reduced by 66.5±25.1% or 62.1±29.2%. Even when 0.01% of the preservative PQ1 (polyquaternium-1) was added, the area of ​​choroidal neovascularization (CNV) was significantly reduced by 71.2±32.8% or 67.0±37.1%. Referring to the results shown in Fig. 9, the effective concentration range of pigment epithelial factor (PEDF) peptide in an eye drop composition prepared using 0.01% BAK as a preservative was evaluated.

[0127] In the case of mice administered eye drop compositions with various concentrations of pigment epithelial factor (PEDF) peptide to which the preservative BAK was added according to Manufacturing Example 8, as shown in FIG. 9, when the content of pigment epithelial factor (PEDF) peptide in the eye drop composition in 1x A5S buffer condition to which the preservative BAK 0.01% was added was 9.4 ng / kg / day (0.188 ng / eye / day) or 94 ng / kg / day (1.88 ng / eye / day), the area of ​​choroidal neovascularization (CNV) was 87.9±27.5% or 88.8±43.6%, indicating a low effect in inhibiting choroidal neovascularization (CNV). When the content of pigment epithelial factor (PEDF) peptide was 940 ng / kg / day (18.8 ng / eye / day), 9.4 μg / kg / day (188 ng / eye / day), 94 μg / kg / day (1.88 μg / eye / day), 376 μg / kg / day (7.52 μg / eye / day), 940 μg / kg / day (18.8 μg / eye / day), or 1.88 mg / kg / day (37.6 μg / eye / day), the area of ​​choroidal neovascularization (CNV) was significantly reduced by 69.1±32.8%, 66.9±24.4%, 68.4±23.6%, 65.3±34.5%, 71.1±29.4%, or 76.4±25.2%, respectively. When the content of epithelial dermal factor (PEDF) peptide was 940 ng / kg / day (18.8 ng / eye / day) to 1.88 mg / kg / day (37.6 μg / eye / day), choroidal neovascularization (CNV) was inhibited, and in particular, when the content was 940 ng / kg / day (18.8 ng / eye / day) to 376 μg / kg / day (7.52 μg / eye / day), the effect of inhibiting choroidal neovascularization (CNV) was the best.

[0128] In summary of the above results, the effect of inhibiting choroidal neovascularization (CNV) was shown under the conditions of an eye drop composition containing 0.01% BAK as a preservative in 1x A5S buffer according to Manufacturing Example 8, 0.1% collagen type I (collagen type I, Pepsin Soluble Collagen, Bovine), and 940 ng / kg / day (18.8 ng / eye / day) to 1.88 mg / kg / day (37.6 μg / eye / day) of pigment epithelial factor (PEDF) peptide (Manufacturing No. FSC04053).

[0129]

[0130] <Example 5> Evaluation of the therapeutic or improvement efficacy range of eye drops manufactured with pigment epithelial factor (PEDF) peptides with different manufacturing numbers in an animal model of choroidal neovascularization

[0131] In the case of mice administered an eye drop composition prepared with four different batch numbers of pigment epithelium-derived factor (PEDF) peptides manufactured by PolyPeptide Laboratories according to Manufacturing Example 9, as shown in FIG. 10, when the PEDF peptide content was 9.4 μg / kg / day (188 ng / eye / day) and the batch numbers of the peptides were FSC04053, FSC04084, AW19111, or 23-22-0147-01, the area of ​​choroidal neovascularization (CNV) was significantly reduced by 63.5±31.3%, 63.4±28.4%, 61.2±25.5%, or 65.5±24.3%. In summary of the above results, the effect of inhibiting choroidal neovascularization (CNV) was excellent regardless of the manufacturing number when the content of pigment epithelial factor (PEDF) peptide was 9.4 μg / kg / day (188 ng / eye / day).

[0132]

[0133] <Example 6> Evaluation of the effective concentration range of collagen type 1 in an eye drop composition showing therapeutic or improvement efficacy in an animal model of choroidal neovascularization formation

[0134] With reference to the results of Examples 4 and 5 above, an evaluation was conducted to determine the effective concentration range of collagen type I (Pepsin Soluble Collagen, Bovine) in an eye drop composition manufactured using a composition having a pigment epithelium-derived factor (PEDF) peptide content of 9.4 μg / kg / day (188 ng / eye / day) in 1x A5S buffer conditions to show the inhibitory effect on choroidal neovascularization (CNV).

[0135] When an eye drop composition containing 0.025% to 0.2% of collagen type I was administered in a 1x A5S buffer condition manufactured according to Manufacturing Example 10, as shown in FIG. 11a, when the content of pigment epithelial factor (PEDF) peptide (manufacturing number FSC04053) was 9.4 μg / kg / day (188 ng / eye / day) and the content of collagen type I (collagen type I, Pepsin Soluble Collagen, Bovine) was 0.025%, 0.05%, 0.075%, 0.1%, or 0.2%, the area of ​​choroidal neovascularization (CNV) was 67.1±38.7%, 72.5±31.8%, 71.9±36.5%, 68.5±41.4%, or A significant decrease of 62.1±33.4% was observed. To determine the minimum concentration of collagen type I (collagen type I, Pepsin Soluble Collagen, Bovine) that significantly reduces choroidal neovascularization (CNV), additional experiments were conducted including lower concentrations of 0.001%, 0.005%, or 0.01% than 0.025%.

[0136] In the case of mice administered an eye drop composition containing 9.4 μg / kg / day (188 ng / eye / day) of pigment epithelial factor (PEDF) peptide (Manufacturing No. 23-22-0147-01) and 0.001% or 0.005% of collagen type I (Pepsin Soluble Collagen, Bovine) in 1x A5S buffer conditions prepared according to Manufacturing Example 11, the area of ​​choroidal neovascularization (CNV) was 93.15±36.6% or 93.84±36.7%, indicating a low effect of inhibiting choroidal neovascularization (CNV), as shown in Fig. 11b. On the other hand, when the content of collagen type I (collagen type I, Pepsin Soluble Collagen, Bovine) was 0.01%, 0.05%, or 0.1%, the area of ​​choroidal neovascularization (CNV) was significantly reduced by 76.53±28.9%, 74.77±24.9%, or 69.91±30.4%.

[0137] In summary, in the eye drop composition prepared according to Manufacturing Example 11 under 1x A5S buffer conditions, when the content of pigment epithelial factor (PEDF) peptide was 9.4 μg / kg / day (188 ng / eye / day) based on the total weight of the eye drop composition, and the content of collagen type I (collagen type I, Pepsin Soluble Collagen, Bovine) was 0.01% to 0.2% based on the total weight of the eye drop composition, an excellent effect of inhibiting choroidal neovascularization (CNV) was observed.

[0138]

[0139] <Example 7> Evaluation of storage stability of the final eye drop composition

[0140] The final eye drop composition of the present invention manufactured according to Manufacturing Example 12 was stored for 4 weeks under refrigerated conditions (5±3℃) or room temperature conditions (25±2℃) depending on the experimental purpose, and its storage stability was evaluated by visual inspection, pH measurement, and RP-UPLC (reverse phase ultra-high performance liquid chromatography) analysis immediately after manufacture, and at the 1st, 2nd, 3rd, and 4th weeks, respectively. The appearance of the final eye drop composition manufactured according to the present invention was analyzed by visual inspection in comparison with a freshly prepared reference solution (ultra-pure water R), and the pH was measured. The content and purity of the preservative BAK and pigment epithelium-derived factor (PEDF) peptide contained in the eye drop composition were evaluated through RP-UPLC analysis using a Shimadzu Nexera UPLC (ultra-high performance liquid chromatography) instrument. For the preservative BAK, the stationary phase of UPLC was Luna 5 μm CN 100 Å, LC column (5 μm / 100 Å 250 X 4.6 mm), and the mobile phase was acetonitrile (ACN) solution containing 0.45% (v / v) 75 mM sodium acetate, pH 5.0 (sodium acetate solution). The mobile phase flow rate was 1 mL / min, the sample injection volume was 20 μL, and the analysis was performed under the conditions of an ultraviolet spectrophotometer wavelength of 254 nm ± 2 nm. The temperature of the column was controlled at 35°C during the analysis, and the analysis time was 35 minutes.

[0141] For pigment epithelium-derived factor (PEDF) peptides, Waters Acquity Peptide CSH C18* (1.7 μm / 130 Å 100 X 2.1 mm) was used as the stationary phase of UPLC, and Solution A, distilled water containing 0.1% (v / v) trifluoroacetic acid (TFA), and Solution B, acetonitrile (ACN) containing 0.1% (v / v) trifluoroacetic acid (TFA) were used as the mobile phase. The mobile phase flow rate was 0.4 mL / min, the sample injection volume was 6 μL, and the analysis was performed under the conditions of an ultraviolet spectrophotometer wavelength of 210 nm ± 4. During analysis, the temperature of the column was controlled at 47°C, and the mixing conditions of the mobile phase solution over time were set such that the mixing volume ratio of solution A and solution B was 72:28 for 50 minutes, 62:38 from 51 to 55 minutes, 0:100 from 56 to 59 minutes, and 72:28 from 60 to 75 minutes.

[0142] The final eye drop compositions of the present invention manufactured according to Manufacturing Example 12 were stored for 4 weeks under refrigerated conditions (5±3℃) and room temperature conditions (25±2℃). As a result, as shown in Fig. 12, there was no change in transparency, particles, degree of color, or pH in the visual inspection and pH measurement results, and it was confirmed that the peaks corresponding to the preservative BAK and the pigment epithelium-derived factor (PEDF) peptide were observed at the same positions in the RP-UPLC analysis results. In summary, the results of analyzing the storage stability according to the storage period of the final eye drop compositions of the present invention manufactured according to the Manufacturing Example under refrigerated conditions (5±3℃) or room temperature conditions (25±2℃) are as shown in Table 1 below. The results of Table 1 below include an error due to sample storage, and the corresponding error range is a sufficient value to evaluate the storage stability of the eye drop according to the storage period. The acceptable standard for storage safety was set to the preservative BAK content in the range of 0.01 ± 0.005%, and the decrease in the content of the active ingredient, peptide, was limited to approximately 10%. If the content of the active ingredient decreases by more than 10%, it means that the product is not stable.

[0143] In summary of the above results, it can be confirmed that the final eye drop compositions of the present invention manufactured according to Manufacturing Example 12 can be stably stored for 4 weeks under refrigerated conditions (5±3°C) and room temperature conditions (25±2°C).

[0144]

[0145] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An eye drop composition for preventing or treating ocular neovascular disease, comprising collagen type I and pigment epithelium-derived factor (PEDF) peptides.

2. An eye drop composition for preventing or treating ocular neovascular disease, characterized in that the pigment epithelium-derived factor (PEDF) peptide in paragraph 1 is composed of an amino acid represented by sequence number 1.

3. An eye drop composition for preventing or treating ocular neovascular disease, characterized in that the eye drop composition comprises the pigment epithelium-derived factor (PEDF) peptide in a concentration range of 37.6 ng / 50 μL to 94 mg / 50 μL in the first paragraph.

4. An eye drop composition for preventing or treating ocular neovascular disease, characterized in that the eye drop composition comprises sodium acetate in a range of 9.9 mM to 10.1 mM in the first paragraph.

5. An eye drop composition for preventing or treating ocular neovascular disease, characterized in that the eye drop composition comprises sorbitol in the range of 4.9 wt% to 5.1 wt%, polysorbate 80 in the range of 0.009 wt% to 0.011 wt%, and BAK (Benzalkonium chloride) in the range of 0.005 wt% to 0.015 wt% in the first paragraph.

6. An eye drop composition for preventing or treating ocular neovascular disease, characterized in that the eye drop composition in paragraph 1 has a pH of 5.0 ± 0.

3.

7. An eye drop composition for preventing or treating ocular neovascular disease, characterized in that the eye drop composition in the first paragraph can be stored for 4 weeks in a range of 1°C to 30°C.

8. In the first paragraph, the eye drop composition is characterized in that the pigment epithelium-derived factor (PEDF) peptide is administered to the eye once a day at a dosage ranging from 0.94 ng / eye / day to 94 mg / eye / day.

9. An eye drop composition for preventing or treating ocular neovascular disease, characterized in that the administration dose in the 8th paragraph is between 50 μL and 100 μL.

10. An eye drop composition for the prevention or treatment of ocular neovascular disease, characterized in that the ocular neovascular disease in paragraph 1 is at least one disease selected from corneal neovascular disease, diabetic retinopathy, choroidal neovascular disease, and age-related macular degeneration.

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