NOVEL PHARMACEUTICAL COMPOSITION FOR TREATING DRYOPHTHALA

Abstract

The present invention provides a novel pharmaceutical composition for treating xerophthalmia, which significantly improves corneal damage and corneal opacity without side effects, thereby restoring the damaged cornea and increasing tear secretion, thereby effectively treating ophthalmic diseases associated with xerophthalmia.

Description

[Technical field]

[0001] The present invention relates to a novel pharmaceutical composition for treating xerophthalmia, and more particularly, to a novel pharmaceutical composition for treating xerophthalmia that can treat ophthalmic diseases associated with xerophthalmia without side effects. [Background technology]

[0002] Dry eye syndrome is a syndrome in which symptoms such as a foreign body sensation, burning sensation, or eye irritation occur due to a decrease in tear production or an increase in tear film loss due to tear evaporation. Existing treatments for dry eye syndrome focus on maintaining a certain amount of tears by using artificial tears to replenish tears that are insufficient depending on the symptoms, or by using conservative methods such as temporarily or permanently blocking the tear duct. However, such symptomatic treatments are not effective enough and cannot directly intervene in improving the cause of the disease. Therefore, the recent trend in treating dry eye syndrome is to prefer direct and ultimate treatments that reduce inflammation in the lacrimal gland and ocular surface to improve dry eye, rather than passive treatments aimed at alleviating symptoms. In addition, for severe cases of dry eye syndrome that are difficult to control with artificial tears, anti-inflammatory treatments such as cyclosporine, steroids, and autologous serum eye drops are used as drugs to control inflammation, rather than simply prescribing artificial tears. However, since the effect of artificial tears is temporary, they must be used several times a day, and they have the disadvantage of not having a protective effect against corneal damage. Steroid preparations may cause fatal side effects such as glaucoma if used for a long period of time, and cyclosporine, a broad-spectrum immunosuppressant, also has problems such as eye pain, burning sensation, foreign body sensation, and congestion, as well as some systemic side effects. In addition, therapeutic contact lenses are inconvenient to use and may cause infection, and punctal occlusion treatment has the disadvantages of being difficult to recover from if side effects occur, as well as a sense of rejection due to the surgery. Therefore, there is an urgent need to develop a preparation that can effectively prevent or treat xerophthalmia without side effects. In this regard, Korean Patent Publication No. 2014-0099526 discloses the use of a cell-permeable peptide inhibitor of the JNK signaling pathway for the treatment of xerophthalmia. Summary of the Invention [Problem to be solved by the invention]

[0003] However, the prior art has a high possibility of inducing local irritation to the ocular mucosa, and is difficult to administer in a fixed dose, which may significantly reduce the efficacy of treatment.

[0004] The present invention is intended to solve various problems including the above problems, and aims to provide a novel pharmaceutical composition for treating xerophthalmia that effectively treats xerophthalmia by significantly suppressing the decrease in tear secretion and the change in corneal morphology caused by xerophthalmia without side effects. However, these problems are merely examples and are not intended to limit the scope of the present invention. [Means for solving the problem]

[0005] According to one aspect of the present invention, there is provided a pharmaceutical composition for treating and preventing dry eye syndrome, comprising, as an active ingredient, a peptide having antibacterial activity and consisting of 7 amino acids, the peptide including an amino acid sequence selected from the group consisting of the following: BOBWRYm (wherein B is a basic amino acid selected from the group consisting of lysine (K) or arginine (R), respectively, and O is an aromatic amino acid selected from the group consisting of phenylalanine (F) or tryptophan (W), respectively).

[0006] According to another aspect of the present invention, there is provided a pharmaceutical composition for treating keratoconjunctivitis, comprising as an active ingredient a peptide having antibacterial activity and consisting of 7 amino acids including an amino acid sequence selected from the group consisting of the following: BOBWRYm (wherein B is a basic amino acid selected from the group consisting of lysine (K) or arginine (R), respectively, and O is an aromatic amino acid selected from the group consisting of phenylalanine (F) or tryptophan (W), respectively).

[0007] According to another aspect of the present invention, there is provided a pharmaceutical composition for improving corneal damage and opacity, comprising as an active ingredient a peptide having antibacterial activity and consisting of 7 amino acids including an amino acid sequence selected from the group consisting of the following: BOBWRYm (wherein B is a basic amino acid selected from the group consisting of lysine (K) or arginine (R), respectively, and O is an aromatic amino acid selected from the group consisting of phenylalanine (F) or tryptophan (W), respectively).

[0008] According to another aspect of the invention there is provided a method for treating xerophthalmia in an individual suffering from xerophthalmia comprising administering to said individual the composition described above.

[0009] According to another aspect of the present invention, there is provided a use of a peptide having antibacterial activity and consisting of 7 amino acids, the peptide including an amino acid sequence selected from the group consisting of the following, in the manufacture of a medicament for treating dry eye syndrome: BOBWRYm (wherein B is a basic amino acid selected from the group consisting of lysine (K) or arginine (R), respectively, and O is an aromatic amino acid selected from the group consisting of phenylalanine (F) or tryptophan (W), respectively). Effect of the Invention

[0010] The novel pharmaceutical composition for treating xerophthalmia of the present invention as described above is excellent in the effects of significantly suppressing the decrease in tear secretion, keratoconjunctivitis, and changes in corneal morphology caused by xerophthalmia, restoring the damaged cornea, and increasing tear secretion, and can be used to develop a safe and effective therapeutic agent for xerophthalmia. Of course, the scope of the present invention is not limited by these effects. [Brief description of the drawings]

[0011] [Figure 1] 1 shows images of the extent of corneal damage in a mouse model treated with the peptide of the present invention, photographed by fluorescent staining and a microscope. [Diagram 2] 1 is a graph showing the quantitative analysis of the degree of corneal damage caused by treatment with the peptide of the present invention. [Diagram 3]1 shows an image of opacity of the eyeball taken through a microscope after treating a mouse model with the peptide of the present invention. [Figure 4] 1 is a graph showing the quantitative analysis of ocular opacity following treatment with the peptide of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The term "antimicrobial peptide" as used herein generally refers to a cationic peptide compound having a relatively simple structure, which has a broad antimicrobial spectrum against gram-positive bacteria, gram-negative bacteria, fungi, viruses, etc., and although the mechanism of action is not fully understood, it is generally known to exert antimicrobial activity through the mechanism of action of destroying the cell membrane of microorganisms.

[0013] The term "dry eye syndrome" as used in the present invention refers to an eye disease in which the ocular surface is damaged due to a lack of tears, excessive evaporation of tears, or an imbalance in the components of tears, causing irritating symptoms such as eye stinging, irritation, foreign body sensation, and dryness. It is not simply a lack of tears, but inflammation of the tears and ocular surface (cornea and conjunctiva) causes eye discomfort, decreased vision, and instability of the tear layer, which damages the ocular surface and increases the risk of developing pain, irregular corneal surface, blurred and fluctuating vision, and corneal ulcers.

[0014] The term "corneal opacity" used in the present invention refers to a state in which the cornea, which is normally a transparent tissue, becomes opaque in some areas due to surface dryness, injury, inflammation, etc., or the cornea as a whole becomes opaque.

[0015] Detailed Description of the Invention According to one aspect of the present invention, there is provided a pharmaceutical composition for treating and preventing dry eye syndrome, comprising, as an active ingredient, a peptide having antibacterial activity and consisting of 7 amino acids, the peptide including an amino acid sequence selected from the group consisting of the following: BOBWRYm (wherein B is a basic amino acid selected from the group consisting of lysine (K) or arginine (R), respectively, and O is an aromatic amino acid selected from the group consisting of phenylalanine (F) or tryptophan (W), respectively).

[0016] In the pharmaceutical composition, the dry eye syndrome is aqueous tear deficiency or evaporative dry eye syndrome. In this regard, the aqueous tear deficiency dry eye syndrome is caused by insufficient tear secretion from the lacrimal gland, and the evaporative dry eye syndrome is caused by excessive water loss from the exposed ocular surface in the presence of normal lacrimal gland secretion function. In addition, an octanoyl group may be added to the N-terminus of the peptide, or an amine group may be added to the C-terminus.

[0017] According to another aspect of the present invention, there is provided a pharmaceutical composition for treating keratoconjunctivitis, comprising as an active ingredient a peptide having antibacterial activity and consisting of 7 amino acids including an amino acid sequence selected from the group consisting of the following: BOBWRYm (wherein B is a basic amino acid selected from the group consisting of lysine (K) or arginine (R), respectively, and O is an aromatic amino acid selected from the group consisting of phenylalanine (F) or tryptophan (W), respectively).

[0018] According to another aspect of the present invention, there is provided a pharmaceutical composition for improving corneal damage and opacity, comprising as an active ingredient a peptide having antibacterial activity and consisting of 7 amino acids including an amino acid sequence selected from the group consisting of the following: BOBWRYm (wherein B is a basic amino acid selected from the group consisting of lysine (K) or arginine (R), respectively, and O is an aromatic amino acid selected from the group consisting of phenylalanine (F) or tryptophan (W), respectively).

[0019] In the pharmaceutical composition, the corneal damage and opacity is due to xerophthalmia or keratoconjunctivitis, and may have a dosage form selected from the group consisting of ointments, topical skin preparations, aerosols, sprays, eye drops, oral preparations, and injections.

[0020] According to another aspect of the invention there is provided a method for treating xerophthalmia in an individual suffering from xerophthalmia comprising administering to said individual the composition described above.

[0021] According to another aspect of the present invention, there is provided a use of a peptide having antibacterial activity and consisting of 7 amino acids, the peptide including an amino acid sequence selected from the group consisting of the following, in the manufacture of a medicament for treating dry eye syndrome: BOBWRYm (wherein B is a basic amino acid selected from the group consisting of lysine (K) or arginine (R), respectively, and O is an aromatic amino acid selected from the group consisting of phenylalanine (F) or tryptophan (W), respectively).

[0022] The pharmaceutical composition according to the present invention may further include a pharma- ceutically acceptable carrier or additive. The term "pharmaceutical acceptable" means that it is physiologically acceptable and does not cause typical gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions when administered to humans. Examples of the additives include excipients, disintegrants, binders, lubricants, wetting agents, dispersants, stabilizers, etc. Examples of the excipients include lactose, mannitol, isomalt, microcrystalline cellulose, silicified microcrystalline cellulose, powdered cellulose, etc. Examples of the disintegrants include low-substituted hydroxypropyl cellulose, crospovidone, sodium starch glycolate, croscarmellose sodium, starch, etc. Examples of the binders include hydroxypropyl cellulose, hypromellose, povidone, copovidone, fully gelled starch, etc. Examples of the lubricants include stearic acid, magnesium stearate, sodium stearyl fumarate, etc. Wetting agents include polyoxyethylene sorbitan fatty acid ester derivatives, poloxamer, polyoxyethylene castor oil derivatives, etc. Dispersing agents include hypromellose, hydroxypropyl cellulose, povidone, copovidone, sodium carboxymethylcellulose, methylcellulose, etc. Stabilizing agents include citric acid, fumaric acid, succinic acid, etc. The pharmaceutical composition of the present invention may further include an anti-agglomerating agent, a flavoring, an emulsifier, a preservative, etc.

[0023] Furthermore, the pharmaceutical composition of the present invention can be formulated using methods known to those skilled in the art so as to provide quick, sustained or delayed release of the active ingredient after administration to a mammal, such as powder, granules, tablets, suspensions, emulsions, syrups, aerosols, or soft or hard gelatin capsules.

[0024] The term "treatment" as used herein refers to any action in which the symptoms of dry eye are ameliorated or beneficially altered by administration of the compositions of the present invention.

[0025] The pharmaceutical composition may be administered orally, intravenously, intramuscularly, intraarterially, transdermally, subcutaneously, intraperitoneally, intranasally, intestinal, topically, sublingually or rectally, for example, by topical application. The parenteral route includes subcutaneous, intradermal, intravenous, intramuscular and intralesional injection or infusion techniques.

[0026] The pharmaceutical composition of the present invention can be administered in a pharma- ceutical effective amount. In the present invention, the term "pharma-ceutical effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined by factors including the type and severity of the individual, age, sex, type of disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, co-used drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. It can be administered singly or multiple times. It is important to administer an amount that provides maximum effect at a minimum amount without side effects, taking into consideration all of the above factors, and this can be easily determined by one skilled in the art.

[0027] The dosage of the pharmaceutical composition varies depending on various factors including the age, body weight, general health, sex, administration time, administration route, excretion rate, drug formulation, and the severity of a specific disease. The pharmaceutical composition of the present invention can be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using biological response modifiers. The pharmaceutical composition is administered at a dosage of 0.001 to 200 mg / kg for adults, and when the pharmaceutical composition is an external preparation, it is recommended to apply 1.0 to 3.0 ml for adults 1 to 5 times a day for one month or more, but the dosage does not limit the scope of the present invention.

[0028] Based on the peptides discovered through conventional basic screening, the present inventors synthesized modified peptides by attaching various lipid moieties such as palmitoyl group and octanoyl group to the N-terminus of the peptides in order to further increase antibacterial activity and penetrance, and developed a novel antibacterial and immunomodulatory peptide (hereinafter, abbreviated as "Peptide I") that is effective in treating immune diseases such as atopic dermatitis and diseases caused by pathogenic bacteria infections (Korea Patent Registration No. 1855170). However, when the peptide was applied to an animal model induced with xerophthalmia, a significant improvement effect on corneal damage and corneal opacity was confirmed compared to the control group, and the present invention was completed.

[0029] The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to the following examples, and may be embodied in various different forms. The following examples are provided to fully disclose the present invention and to fully convey the scope of the invention to those skilled in the art.

[0030] Example 1: Production of immunomodulatory and antimicrobial peptides The present inventors have developed a peptide (KFKWRYm) having immunomodulatory and antibacterial activity through conventional basic screening and have secured a patent for it (Korean Patent No. 10-1855170). In order to develop an improved peptide with better immunomodulatory and antibacterial abilities than the above peptide, various variants of the amino acid sequence of the above patented peptide were devised and synthesized using a conventional amino acid synthesis method (Umbarger, HE, Ann. Rev. Biochem., 47:533-606, 1978), and used as immunomodulatory and antibacterial candidate peptides. In addition, the present inventors added / added an octanoyl group or an acetyl group to the N-terminus of the peptide synthesized from the above, or modified the form by substituting an amine group instead of the carboxyl group at the C-terminus.

[0031] Example 2: Experiments on FPR2 activation and antibacterial activity of novel peptides The present inventors observed changes in calcium ion permeability to confirm whether the immunoregulatory and antimicrobial peptide candidates of the present invention activate the immune regulatory function of the living body. Specifically, the intracellular calcium ion concentration was measured to confirm whether the peptides activate FPR2. For this purpose, RBL cells not expressing FPR2, RBL cells overexpressing FPR1 (FPR1-RBL), and RBL cells overexpressing FPR2 (FPR2-RBL) were used, and Fura-2 / AM, a dye with strong calcium binding ability, was used as a method for sensitively measuring the release of calcium ions in the cells. That is, the cells were cultured in RPMI medium containing 10% fetal bovine serum, and when they were in the log phase (mid-log phase, 1-3×10 7 The cells were harvested by centrifugation, washed several times with RPMI medium without fetal bovine serum, and then diluted to 1 × 10 7 The cells were resuspended in RPMI medium to give a final concentration of 3 μM Fura-2 / AM and incubated for 45 minutes in a 37°C, 5% CO2 incubator with constant agitation. After an appropriate time had elapsed, the cells were harvested and washed several times with RPMI medium, and then suspended in an appropriate amount of RPMI medium containing 250 μM sulfinpyrazone to prevent Fura-2 that had entered the cells from being lost to the outside of the cells. Approximately 2×10 6 Each time, 10 cells were harvested by rapid centrifugation, resuspended in 1 ml of Locke's solution containing EGTA but no calcium ions, and the absorbance ratio at two wavelengths, 340 nm and 380 nm, was monitored using a spectrophotometer. After treatment with different concentrations of a peptide according to one embodiment of the present invention (1 μM, 0.1 μM, and 0.01 μM) at intervals of about 1 minute, the absorbance difference at the two wavelengths was investigated and later converted into the concentration of calcium ions released into the cells by the method of Grynkiewicz.

[0032] In addition, to measure the antibacterial activity of the peptide, gram-positive bacteria Staphylococcus aureus and gram-negative bacteria Pseudomonas aeruginosa were prepared, smeared on an agar plate medium, and cultured overnight in an incubator at 36°C. The next day, the colonies of the strains formed on the agar plate medium were inoculated into 3 ml of nutrient medium and cultured overnight in an agitator at 36°C and 220 rpm. The next day, the bacteria were diluted and the absorbance was measured at 600 nm, adjusted to 0.5, and diluted in a nutrient medium at a ratio of 1:100. Then, the peptides prepared in the above examples were sequentially diluted in nutrient medium at concentrations of 0, 1.25, 2.5, 5, 10, 20, and 40 μM to prepare 1 ml each, and 1 ml of the diluted bacteria was inoculated. Subsequently, the mixture was stirred at 220 rpm at 36° C. for 18 hours, after which the absorbance at 600 nm was measured.

[0033] As a result, most of the peptides showed FPR2 activation efficacy and showed high antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus (Table 1). The results of FPR2 activation and antibacterial activity of the peptides are summarized in Table 1 below.

[0034] [Table 1]

[0035] Example 3: Creation of an animal model The present inventors have synthesized the peptide of the present invention, Peptide I (SEQ ID NO: 2In order to confirm the efficacy of Peptide I in improving dry eye and inflammation, a mouse animal model was prepared. Specifically, 12-week-old male mice of C57BL / 6 strain were used as experimental animals, and 0.2% benzalkonium chloride was administered twice a day for 15 days to induce dry eye and keratoconjunctivitis, except for a control group that did not induce dry eye. After 3 days, the degree of corneal damage of the mice was evaluated through corneal staining, and mice with sufficient corneal damage were selected and classified into experimental groups through a randomized block design. Subsequently, from the 4th day, Peptide I of the present invention (0.005% and 0.01%) was administered twice a day at intervals, one hour after administration of 0.2% benzalkonium chloride. The Peptide I was dissolved in saline according to the concentration of the administration solution and administered. The positive control group was administered with commercially available Restasis (0.05% cyclosporin) from Allergan, and the negative control group was administered with saline.

[0036] Example 4: Improvement of corneal damage The present inventors investigated the therapeutic effect of dry eye and keratoconjunctivitis by administration of Peptide I of the present invention. Specifically, on the 3rd, 7th, 11th, and 15th days after dry eye induction (0th, 4th, 8th, and 12th days after drug administration), the mice were anesthetized by inhalation with isoflurane, and fluorescein, a fluorescent dye, was instilled into the eyes to stain the cornea. After that, the residual fluorescein was washed off with saline, and the eyeball was photographed under a fluorescent microscope to obtain an image. The fluorescently stained area was calculated and quantified using an image analysis program (Image J) to evaluate the degree of corneal damage caused by dry eye and keratitis.

[0037] As a result, it was shown that administration of Peptide I of the present invention significantly improved corneal damage at a lower concentration compared to the control group (FIGS. 1 and 2).

[0038] Example 5: Improvement effect of corneal opacity The present inventors investigated the effect of administration of Peptide I of the present invention on improving opacity caused by corneal injury. Specifically, on the 3rd, 7th, 11th, and 15th days after dry eye induction (0th, 4th, 8th, and 12th days after drug administration), the mice were anesthetized by inhalation with isoflurane. Images were obtained by photographing the eyeballs under a microscope. The degree of corneal opacity was quantified and evaluated by giving a score from 0 to 4 points, with 0 point being given if the iris was clearly visible without opacity, 1 point being given if partial mild opacity was observed, 2 points being given if partial opacity or mild opacity was observed overall, 3 points being given if opacity was observed throughout the cornea, and 4 points being given if severe opacity and vascular development were observed. As a result, it was shown that administration of Peptide I of the present invention significantly improved corneal opacity at a lower concentration compared to the control group (FIGS. 3 and 4).

[0039] In conclusion, when the novel pharmaceutical composition for treating xerophthalmia of the present invention was administered to a mouse animal model, corneal damage and corneal opacity were significantly improved without side effects compared to the control group, and the composition can be used as a material to effectively treat xerophthalmia by recovering damaged cornea and increasing tear secretion.

[0040] The present invention has been described with reference to the above-mentioned embodiment, but it is to be understood that the embodiment is merely illustrative and that various modifications and equivalent embodiments are possible. Therefore, the true technical scope of the present invention should be determined by the technical spirit of the claims.

Claims

1. A pharmaceutical composition for treating and preventing dry eye syndrome, comprising as an active ingredient a peptide having antibacterial activity comprising the amino acid sequence of SEQ ID NO:

2.

2. 2. The pharmaceutical composition of claim 1, wherein the dry eye syndrome is aqueous tear deficiency or evaporative dry eye syndrome.

3. The pharmaceutical composition of claim 1 , wherein the peptide has an octanoyl group attached to the N-terminus or an amine group attached to the C-terminus.

4. A pharmaceutical composition for treating keratoconjunctivitis induced by dry eye, comprising as an active ingredient a peptide having antibacterial activity consisting of the amino acid sequence of SEQ ID NO:

2.

5. A pharmaceutical composition for improving corneal damage and opacity induced by dry eye, comprising as an active ingredient a peptide having antibacterial activity consisting of the amino acid sequence of SEQ ID NO:

2.

6. The pharmaceutical composition according to any one of claims 1 to 5, having a dosage form selected from the group consisting of ointments, topical skin preparations, aerosols, sprays, eye drops, oral preparations and injections.

7. 13. A method for treating xerophthalmia in a non-human mammalian individual suffering from xerophthalmia, comprising administering to said individual a pharmaceutical composition according to claim 1.

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