Peptide with xerophthalmia relieving activity and application thereof

Abstract

The peptide of the present invention has ability to bind to pituitary adenylate cyclase 1 receptor PAC1R, promotes activation of a PAC1R signaling pathway, and induces nerve cell differentiation, and thus has tear secretion promoting activity. In addition, the peptide of the present invention promotes the expression and secretion of mucoprotein of a tear component in conjunctival goblet cells, and has an activity of inhibiting inflammatory responses and cellular damage in human corneal epithelial cells. The peptide of the present invention can be used as an active substance for treating, preventing and ameliorating xerophthalmia.

Description

Cross-reference to related applications

[0001] This application claims the benefit of priority to Korean Patent Application No. 10-2023-0104783, filed with the Korean Intellectual Property Office on August 10, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to novel peptides with activity in improving dry eye syndrome and their uses. Background Technology

[0003] Dry eye syndrome is a condition or disease characterized by dryness of the cornea or conjunctiva, even under normal tear production conditions, due to reduced tear secretion or changes in tear composition. The causes of dry eye syndrome are diverse, including age-related reductions in tear secretion or changes in tear composition; diseases such as rheumatoid arthritis, Sjogren's syndrome, lupus, scleroderma, diabetes, and vitamin A deficiency leading to reduced tear production; chronic conjunctivitis causing reduced mucus secretion; hyperthyroidism leading to excessive tear evaporation; decreased female hormones causing reduced tear production; medications such as antibiotics and antihistamines causing tear evaporation; eye irritation caused by dry environments, smoke, dust, sunlight, wind, etc.; and decreased blinking frequency.

[0004] Dry eye disease begins with desiccating stress on the cornea and conjunctiva, and the expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 increases due to this stress. Consequently, antigen-presenting cells (APCs) around the cornea and conjunctiva are activated, then migrate to the lymph nodes and induce the differentiation of Th1 and Th17 cells. The differentiated Th1 and Th7 cells then migrate back to the lesion area and promote the secretion of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-17, and IFN-γ, thereby further exacerbating inflammation and impairing the production of matrix metalloproteinases (MMPs) that are increased due to stimulation (Mucosal Immunol. 2009 Jul; 2(4): 375-6. Role of Th17 cells in the immunopathogenesis of dry eye disease).

[0005] Pituitary adenylate cyclase-activating polypeptide (PACAP) is a peptide hormone in vivo, and its known receptors are VPAC1, VPAC2, and PAC1 receptors. Recently, it has been reported that activating the PACAP / PAC1R mechanism in animal models of dry eye can promote tear secretion in the lacrimal glands, thus confirming the effect of improving dry eye disease (Int J Mol Sci. 2022 Jan 8; 23(2): 664., Potential Therapeutic Role of Pituitary Adenylate Cyclase-Activating Polypeptide for Dry Eye Disease). Furthermore, results have been reported demonstrating that synthetic peptides derived from PACAP, which possess PAC1R-specific agonist activity, have the activity of healing corneal injuries and increasing lacrimal secretion (InvestOphthalmol Vis Sci. 2015 Jul; 56(8): 4336-49., A New Recombinant PACAP-Derived Peptide Efficiently Promotes Corneal Wound Repairing and Lacrimal Secretion). To date, PACAP has been shown to participate in improving the symptoms of dry eye through anti-inflammatory activity, corneal injury inhibition activity, and tear secretion induction activity.

[0006] [Documents in related fields] [Non-patent documents] Mucosal Immunol. 2009 Jul;2(4):375-6. Int J Mol Sci. 2022 Jan 8;23(2):664. Invest Ophthalmol Vis Sci. 2015 Jul;56(8):4336-49. Summary of the Invention Technical issues The inventors conducted research and efforts to develop stable and effective active materials with activity in improving dry eye syndrome. As a result, experiments confirmed that the novel peptide synthesized by the inventors possesses excellent activity in improving dry eye syndrome by promoting tear secretion, promoting the secretion of mucin, a component of the tear film, and preventing damage to corneal epithelial cells, thus completing this invention.

[0007] Therefore, one aspect of the present invention provides a novel peptide with activity in improving dry eye syndrome.

[0008] Another aspect of the present invention provides a pharmaceutical composition for treating or preventing dry eye syndrome, comprising a peptide as an active ingredient.

[0009] Technical solution To achieve the objectives of this invention, this disclosure provides a peptide comprising the amino acid sequence disclosed in SEQ ID NO: 1.

[0010] This disclosure provides a pharmaceutical composition for treating or preventing dry eye syndrome, comprising a peptide as an active ingredient.

[0011] This disclosure will be described in detail below.

[0012] Peptides and their activities According to one aspect of the invention, a peptide comprising the amino acid sequence disclosed in SEQ ID NO: 1 is provided.

[0013] [Amino acid sequence of SEQ ID NO: 1] TLKKNKRFSK In this specification, the term "peptide" refers to a linear molecule formed by amino acid residues linked together by peptide bonds.

[0014] The peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention may be used without modification, but amino acid variants or fragments having different sequences by deletion, insertion or substitution of amino acid residues or combinations thereof may also be used to the extent that the natural activity of the peptide (e.g., activity to improve dry eye) is not affected.

[0015] The peptides of the present invention can be modified by phosphorylation, sulfation, acrylate, glycosylation, methylation, farnesylation, etc., without changing their activity.

[0016] The peptides of the present invention include peptides comprising an amino acid sequence substantially identical to that of the peptide comprising the amino acid sequence of SEQ ID NO: 1, and variants or active fragments thereof. A substantially identical amino acid sequence is defined as an amino acid sequence that is 75% or higher, for example 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, or 98% or higher, identical to the amino acid sequence of SEQ ID NO: 1. Furthermore, the peptide may also include amino acid sequences prepared for specific purposes such as increasing targeting sequences, tags, labeled residues, and half-life or peptide stability.

[0017] The peptides of the present invention can be obtained by selecting a portion of the amino acid sequence and inducing N-terminal and / or C-terminal modifications to increase activity. The stability of the peptides of the present invention can be significantly improved by N-terminal and / or C-terminal modifications, and, for example, the half-life of the peptide can be increased when administered in vivo. The term "stability" encompasses not only the meaning of in vivo stability that protects the peptides of the present invention from attack by protein-cleaving enzymes, but also storage stability (e.g., storage stability at room temperature).

[0018] N-terminal modification can be a modification in which a protecting group selected from the group consisting of acetyl, fluorenylmethoxycarbonyl, formyl, palmitoyl, myristyl, stearoyl, and polyethylene glycol (PEG) is attached to the N-terminus of the peptide. C-terminal modification can be a modification in which a hydroxyl (-OH), amino (-NH2), hydrazide (-NHNH2), etc., are attached to the C-terminus of the peptide, but is not limited to these.

[0019] The peptides of the present invention can be prepared by a variety of methods widely known in the art to which this invention pertains. For example, the peptides of the present invention can be prepared using chemical synthesis methods known in the art, especially solid-phase synthesis techniques (Merrifield, J. Amer. Chem. Soc. 85: 2149-54 (1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co.: Rockford, 111 (1984)) or liquid-phase synthesis techniques (US Patent Registration No. 5,516,891).

[0020] The peptides of this invention have activity in improving, treating, or preventing dry eye syndrome.

[0021] In exemplary embodiments, the peptides of the present invention have the following activities related to promoting tear secretion: (i) It has the ability to bind to the pituitary adenylate cyclase type 1 receptor (PAC1R) and promote the activation of the PAC1R signaling pathway. (ii) Promotes the migration of aquaporin 5 (AQP5) protein towards the plasma membrane, and (iii) Inducing neuroblast differentiation.

[0022] In an exemplary embodiment, the peptide of the present invention has the activity of promoting the expression and secretion of mucin, which is a component of tear fluid in conjunctival goblet cells.

[0023] In an exemplary embodiment, the peptide of the present invention has the activity of inhibiting IFN-γ-induced inflammatory response and cell damage in human corneal epithelial cells.

[0024] In an exemplary embodiment, the peptide of the present invention has the activity of inhibiting inflammatory responses and cell damage induced by ultraviolet (UVB) irradiation in human corneal epithelial cells.

[0025] Compositions for the treatment or prevention of dry eye syndrome According to another aspect of the invention, a pharmaceutical composition for treating or preventing dry eye syndrome is provided, comprising a peptide containing the amino acid sequence disclosed in SEQ ID NO: 1 as an active ingredient.

[0026] As used in this manual, the term "dry eye syndrome" refers to an eye disease that causes damage to the ocular surface and symptoms of irritation, such as visual impairment, eye pain, irritation, foreign body sensation, or dryness, due to lack of tears, excessive tear evaporation, tear film instability, or an imbalance in tear composition.

[0027] The term "dry eye syndrome" as used in this specification may include one or more disorders characterized by insufficient or defective tear production, such as, but not limited to, the following disorders or diseases: dry eye, dry eye syndrome, dry eye disease, evaporative dry eye, dehydrated dry eye, dry keratitis (a dry and inflammatory disease of the cornea), keratoconjunctivitis sicca (dryness of both the cornea and conjunctiva), and dysfunctional tear syndrome (insufficient tear quality or quantity).

[0028] The term “treatment” as used in this instruction manual means the prevention, elimination, cure, suppression, and reduction of at least one symptom of a disease, illness, or disorder.

[0029] The pharmaceutical compositions of the present invention may comprise a therapeutically effective amount of a peptide comprising the amino acid sequence disclosed in SEQ ID NO: 1 and a pharmaceutically acceptable carrier.

[0030] Therapeutic effective dose (TAD) refers to the amount of an active substance or preparation that produces the desired effect at a reasonable profit / risk ratio. A TAD is the amount required or sufficient to treat dry eye syndrome or its symptoms. TAD can vary depending on factors such as the disease or condition being treated, the specific composition being applied, or the severity of the disease or condition.

[0031] Pharmaceutically acceptable carriers are commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0032] In addition to the above-mentioned components, the pharmaceutical composition of the present invention may also contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, isotonic agents, buffers, surfactants, lubricants, excipients, pH adjusters, etc., but is not limited thereto.

[0033] Suitable pharmaceutically acceptable carriers and formulations are detailed in Remington: The Science and Practice of Pharmacy (19th ed., 1995, Williams & Wilkins).

[0034] The pharmaceutical compositions of the present invention may include solutions, suspensions or gels suitable for topical application to the eyes, comprising the peptides of the present invention and any suitable carriers, such as saline solutions or artificial tears.

[0035] The pharmaceutical compositions of the present invention may also contain one or more components known in the art of preparing compositions for topical application to the eye.

[0036] The pharmaceutical compositions of the present invention can be administered via all suitable routes for the treatment or prevention of dry eye syndrome, and for example, can be administered orally or parenterally, and in the case of parenterally administration, can be administered intravenously, subcutaneously, intramuscularly, intraperitoneally, topically, percutaneously, etc. Since the pharmaceutical compositions of the present invention are for the treatment of dry eye syndrome, topical application to the eye is preferred.

[0037] The dosage of the drug composition may be from 0.0001 μg to 100 mg, 0.001 μg to 100 mg, 0.01 μg to 100 mg, 0.1 μg to 100 mg, or 1.0 μg to 1000 mg per day, but is not limited thereto; and may be prescribed differently according to factors such as preparation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and responsiveness.

[0038] According to methods readily practiced by those skilled in the art, the pharmaceutical compositions of the present invention can be formulated into unit dosage forms using pharmaceutically acceptable carriers and / or excipients, or prepared by injection into multi-volume containers. In this document, formulations may be in the form of solutions, suspensions, or emulsions in oily or aqueous media, or in the form of extracts, powders, granules, tablets, or capsules, and may further contain dispersants or stabilizers.

[0039] In exemplary embodiments, the pharmaceutical compositions of the present invention have the following activities related to tear secretion promotion activity: (i) It has the ability to bind to the pituitary adenylate cyclase type 1 receptor (PAC1R) and promote the activation of the PAC1R signaling pathway. (ii) Promotes the migration of aquaporin 5 (AQP5) protein towards the plasma membrane, and (iii) Inducing neuroblast differentiation.

[0040] In an exemplary embodiment, the pharmaceutical composition of the present invention has the activity of expressing and secreting mucin, which is a component of tear fluid in conjunctival goblet cells.

[0041] In an exemplary embodiment, the pharmaceutical composition of the present invention has the activity of inhibiting IFN-γ-induced inflammatory response and cell damage in human corneal epithelial cells.

[0042] In an exemplary embodiment, the pharmaceutical composition of the present invention has the activity of inhibiting inflammatory responses and cell damage induced by ultraviolet (UVB) irradiation in human corneal epithelial cells.

[0043] Uses of peptides According to another aspect of the invention, the use of a peptide comprising the amino acid sequence disclosed in SEQ ID NO: 1 in the treatment, prevention or improvement of dry eye syndrome is provided.

[0044] According to another aspect of the invention, the use of a peptide comprising the amino acid sequence disclosed in SEQ ID NO: 1 in the preparation of a medicament for treating, preventing or improving dry eye syndrome is provided.

[0045] According to another aspect of the invention, a method for treating, preventing or improving dry eye syndrome is provided, comprising administering to a patient or subject requiring treatment, prevention or improvement of dry eye syndrome a peptide comprising the amino acid sequence disclosed in SEQ ID NO: 1 or a pharmaceutical composition comprising said peptide as an active ingredient.

[0046] The terms “patient” or “subject” as used in this specification refer to human or non-human animals, such as humans, primates, mammals and vertebrates.

[0047] The descriptions of the peptides, their activities, and pharmaceutical compositions thereof of the present invention can be applied in the same manner to the above-described uses and treatment methods, and will not be repeated hereto avoid making the specification too complicated.

[0048] Advantages of the present invention The peptides of this invention have the ability to bind to PAC1R, promote the activation of the PAC1R signaling pathway, and induce neuronal differentiation, thereby having tear secretion-promoting activity.

[0049] Furthermore, the peptides of the present invention promote the expression and secretion of mucin, a component of tear fluid in conjunctival goblet cells, and have the activity of inhibiting inflammatory responses and cell damage in human corneal epithelial cells.

[0050] The peptides of this invention can be used as active materials for treating, preventing and improving dry eye syndrome.

[0051] However, the effects of the present invention are not limited to those described above, and those skilled in the art can clearly understand from the following description other effects not mentioned herein. Attached Figure Description

[0052] Figures 1A and 1B show the results of SPR analysis, indicating that the peptides of the present invention have the ability to bind to PAC1R.

[0053] Figure 2A shows the results of Western blotting, indicating that the peptides of the present invention activate the PAC1R signaling pathway in the human neuroblastoma cell line SH-SY5H to increase the phosphorylation of PKA and CREB; Figure 2B is a graph showing the band intensity of the Western blotting results of Figure 2A in the form of a relative ratio to the control group.

[0054] Figure 3 The results of Western blotting indicate that the peptides of the present invention promote the migration of AQP5 protein to the cell membrane in the human neuroblastoma cell line SH-SY5Y.

[0055] Figure 4 The results of Western blotting indicate that the peptides of the present invention increase the expression level of the mature neural marker GAP43 in the human neuroblastoma cell line SH-SY5Y in a concentration-dependent manner.

[0056] Figure 5 The experimental results demonstrate that the peptides of the present invention increase the gene expression levels of mucin 2 (MUC2) and mucin 5AC (MUC5AC) in conjunctival goblet cells.

[0057] Figures 6A and 6B show the results of Western blotting and ELISA, indicating that the peptides of the present invention increase the expression levels of MUC2 and MUC5AC proteins in conjunctival goblet cells.

[0058] Figure 7A shows the experimental results demonstrating the peptide of the present invention inhibiting the expression levels of COX-2, IL-1β and IL-6 genes in human corneal epithelial cells increased by IFN-γ. Figure 7B shows the band intensity of Figure 7A in the form of a relative ratio with the control group.

[0059] Figure 7C shows the experimental results demonstrating the peptide of the present invention inhibiting the expression levels of MMP3 and MMP9 genes in human corneal epithelial cells increased by IFN-γ. Figure 7D shows the band intensity of Figure 7C in the form of a relative ratio with the control group.

[0060] Figure 8A shows the experimental results demonstrating that the peptide of the present invention reduces the expression levels of COX-2, IL-1β and IL-6 genes in human corneal epithelial cells that are increased due to UVB irradiation. Figure 8B shows the band intensity of Figure 8A in the form of a relative ratio with the control group.

[0061] Figure 8C shows the experimental results of the peptide of the present invention inhibiting the expression levels of MMP3 and MMP9 genes in human corneal epithelial cells increased by UVB irradiation. Figure 8D shows the band intensity of Figure 8C in the form of a relative ratio with the control group.

[0062] Figure 9A is a schematic diagram of the five regions of the cornea used to score corneal damage according to the National Institute of Eye (NIE) guidelines when assessing clinical corneal injury.

[0063] Figure 9B shows the experimental results demonstrating the tear film retention effect of the peptide of the present invention in a mouse model of dry eye. Figure 9C is a table showing information on the experimental group configuration, application materials, and application dosage related to the experimental results in Figure 9B.

[0064] Figures 10A to 10C show the experimental results demonstrating the effect of the peptides of the present invention on improving corneal damage in a dry eye model mouse, and a table showing information on the experimental group configuration, application materials, and application dosage. Detailed Implementation

[0065] The present invention will be described in detail below through embodiments. However, the following embodiments are only for illustrating the present invention, and the content of the present invention is not limited to the following embodiments.

[0066] Preparation Example 1: Preparation of Peptides Peptides having the amino acid sequence SEQ ID NO: 1 shown in Table 1 were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptides were isolated into purified form using C18 high-performance liquid chromatography (HPLC, Waters Associates, USA). An ACQUITY UPLC BEH300 C18 column (2.1 mm × 100 mm, 1.7 μm, Waters Co, USA) was used as the chromatographic column.

[0067] [Table 1]

[0068] The efficacy of the peptide prepared above, SEQ ID NO: 1, was evaluated through the following experiments.

[0069] Experimental Example 1: Surface Plasmon Resonance (SPR) Analysis SPR analysis was used to assess whether the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 has the ability to bind to pituitary adenylate cyclase type 1 receptor (PAC1R).

[0070] This analysis was performed using a Biacore T200 (Cytiva, USA) instrument. Recombinant human PAC1R protein (LSBio, USA) was immobilized on a CM5 chip (Cytiva, USA) using the amine coupling method. Samples were prepared in which the peptide was diluted with 1X HBS-EP buffer as a running buffer, and then the prepared samples were flown onto the chip surface. The reaction units (RU) showing the binding of the peptide to PAC1R at different concentrations were analyzed using Biacore T200 evaluation software version 3.1 (Cytiva, USA) as a function of increasing concentration.

[0071] SPR analysis confirmed that the peptide of SEQ ID NO:1 showed an RU value that increased in a concentration-dependent manner and that the peptide had the ability to bind to PAC1R (Figure 1A and Figure 1B).

[0072] Experimental Example 2: Activation Analysis of the PAC1R Signaling Pathway The Western blotting method was used to assess whether the peptide prepared in Preparation Example 1, SEQ ID NO: 1, activated the PAC1R signaling pathway.

[0073] The human neuroblastoma cell line SH-SY5Y was injected with 1.5 × 10⁻⁶ cells. 6Cells were seeded at a density of 10 nM / well in 6-well plates and cultured for 24 hours. Cells were pretreated with 10 nM PA8 (PAC1R antagonist) for 1 hour. Subsequently, cells were treated with peptides at concentrations (10 μM, 50 μM, 100 μM) for 15 minutes, washed twice with PBS, and lysed by adding cell lysis buffer. Cells were treated with 5X sample buffer and then subjected to SDS-PAGE using a 10% SDS-PAGE gel. The proteins separated by SDS-PAGE were transferred to a PVDF membrane. Blocking was performed with 5% skim milk at room temperature for 1 hour. Subsequently, the primary antibody for p-PKA or p-CREB as described in Table 2 was diluted 1:1000 in 5% skim milk and reacted with the membrane for 2 hours. The membrane was washed three times with 0.1% PBS-T (0.1% Tween-20 in PBS) for 10 minutes each time. Subsequently, the corresponding secondary antibodies described in Table 2 were diluted 1:3000 in 5% skim milk and reacted with the membrane for 1 hour. The membrane was then treated with ECL solution (GE Healthcare, RPN2232, USA), and the expression levels of p-PKA and p-CREB were analyzed using an ImageQuant 800 (Cytiva, 29-3994-81, USA).

[0074] As described above, Western blot analysis confirmed that treatment with the peptide of SEQ ID NO: 1 activated the PAC1R signaling pathway, leading to an increase in phosphorylated protein kinase A (PKA) and cAMP response element binding protein (CREB). Pretreatment with PA8, a PAC1R-specific antagonist, reduced the increase in PKA and CREB phosphorylation (Figures 2A and 2B). These results confirm that the peptide of SEQ ID NO: 1 acts as an agonist for PAC1R.

[0075] Experimental Example 3: Analysis of plasma membrane migration of aquaporin 5 (AQP5) The Western blotting method was used to assess whether the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 promoted the migration of aquaporin 5 (AQP5) to the plasma membrane after activation of the PAC1R signaling pathway.

[0076] The human neuroblastoma cell line SH-SY5Y was injected with 1.5 × 10⁻⁶ cells. 6Cells were seeded at a density of 10 cells / well in 6-well plates and cultured for 24 hours. Cells were treated with peptides at concentrations (10 μM, 50 μM, 100 μM) for 1 hour, washed twice with PBS, and then treated with a plasma membrane protein extraction kit (Abcam, catalog number: ab65400, USA). After treatment with 5X sample buffer, SDS-PAGE was performed using a 10% SDS-PAGE gel. The proteins separated by SDS-PAGE were transferred to a PVDF membrane. Blocking was performed with 5% skim milk at room temperature for 1 hour. The primary antibody for AQP5 (Table 2) was diluted 1:1000 in 5% skim milk and reacted with the membrane for 2 hours. The membrane was washed three times with 0.1% PBS-T (0.1% Tween-20 in PBS) for 10 minutes each time, and the secondary antibody (Table 2) was diluted 1:3000 in 5% skim milk and reacted with the membrane for 1 hour. The AQP5 protein was processed with ECL solution (GE Healthcare, catalog number: RPN2232, USA) and then the expression level was analyzed using ImageQuant 800 (Cytiva, catalog number: 29-3994-81, USA).

[0077] It is known that when the PAC1R signaling pathway is activated, tear secretion occurs as AQP5 migrates towards the plasma membrane. Based on the results of the above experiments, the concentration-dependent increase of AQP5 protein in the plasma membrane after treatment with the peptide of SEQ ID NO:1 was confirmed by measuring the levels of AQP5 protein found in the plasma membrane (see [link to experiment]). Figure 3 ).

[0078] Experiment Example 4: Analysis of Neuron Differentiation The peptide of SEQ ID NO: 1 prepared in Preparation Example 1 was evaluated to assess whether it induces neuroblast differentiation by measuring the expression level of growth-associated protein 43 (GAP43), a marker of mature neurons.

[0079] The human neuroblastoma cell line SH-SY5Y was used at 5×10 6Cells were seeded at a density of 10 cells / well in 6-well plates and cultured for 24 hours. Cells were treated with peptides at concentrations (10 μM, 50 μM, 100 μM) for 24 hours, washed twice with PBS, and lysed by adding cell lysis buffer. Cells were treated with 5X sample buffer and then subjected to SDS-PAGE using a 10% SDS-PAGE gel. The proteins separated by SDS-PAGE were transferred to a PVDF membrane. Blocking was performed with 5% skim milk at room temperature for 1 hour. Subsequently, the primary antibody for GAP43 (Table 2) was diluted 1:1000 in 5% skim milk and reacted with the membrane for 2 hours. Cells were washed three times with 0.1% PBS-T (0.1% Tween-20 in PBS) for 10 minutes each time. Subsequently, the secondary antibody (Table 2) was diluted 1:3000 in 5% skim milk and reacted with the membrane for 1 hour. The protein was treated with ECL solution (GE Healthcare, catalog number: RPN2232, USA) and then the expression level of GAP43 protein was analyzed using ImageQuant 800 (Cytiva, catalog number: 29-3994-81, USA).

[0080] Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide and a peptide hormone in vivo. PAC1, VPAC2, and PAC1 receptors are known to act as receptors for this peptide hormone. It is known that neuroblasts differentiate via PACAP to increase synapse formation, thereby increasing the secretion of tear film component factors (Int J Mol Sci. 2022 Jan 8;23(2):664.). Experimental results confirmed that when nerve cells were treated with the peptide of SEQ ID NO: 1, the expression level of GAP43, a marker of mature neurons, increased in a concentration-dependent manner (see [link to SEQ ID NO: 1]). Figure 4 ).

[0081] Experiment Example 5: Analysis of Mucin Gene Expression Levels To assess whether the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 increases the expression level of mucin genes in conjunctival goblet cells.

[0082] The human colon adenocarcinoma cell line (well-differentiated human colon goblet cell line) LS174T was injected with 2×10 6Cells were seeded at a density of 10 cells / well in 6-well plates and cultured for 24 hours. After culturing in serum-free medium for 24 hours, cells were treated with peptides at concentrations (10 μM, 50 μM, 100 μM) for 4 hours. Cells were washed twice with PBS and RNA was isolated using Easy Blue (iNtRON, catalog number: 17061, Korea). The isolated RNA was quantified, and cDNA was then synthesized using an RT kit (enzynomics, catalog number: RT200, Korea). Subsequently, PCR was performed using a PCR kit (enzynomics, catalog number: P581T, Korea). Table 3 below shows the primer sequences used in the PCR.

[0083] Mucin secreted by conjunctival goblet cells is another component of the tear film. Experimental results confirmed that treatment of conjunctival goblet cells with the peptide of SEQ ID NO: 1 increased the expression levels of mucin 2 (MUC2) and mucin 5AC (MUC5AC) (see [link to SEQ ID NO: 1]). Figure 5 ).

[0084] Experiment Example 6: Analysis of Mucin Protein Expression Levels To assess whether the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 increases the expression level of mucin protein in conjunctival goblet cells.

[0085] The LS174T cells from the human colon adenocarcinoma cell line (a well-differentiated human colon goblet cell line) were injected at a dose of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / well in 6-well plates and cultured for 24 hours. After culturing in serum-free medium for 24 hours, cells were treated with peptides at concentrations (10 μM, 50 μM, 100 μM) for 24 hours. Cells were washed twice with PBS and lysed by adding cell lysis buffer.

[0086] First, Western blotting of the MUC2 protein was performed as follows. Cell lysates were treated with 5X sample buffer and then subjected to SDS-PAGE using a 10% SDS-PAGE gel. The protein separated by SDS-PAGE was transferred to a PVDF membrane. Blocking was performed with 5% skim milk at room temperature for 1 hour. Subsequently, the primary antibody for MUC2 (Table 2) was diluted 1:1000 in 5% skim milk and reacted with the membrane for 2 hours. Washing was performed three times with 0.1% PBS-T (0.1% Tween-20 in PBS) for 10 minutes each time. Subsequently, the secondary antibody (Table 2) was diluted 1:3000 in 5% skim milk and reacted with the membrane for 1 hour. The membrane was treated with ECL solution (GE Healthcare, catalog number: RPN2232, USA) and then the expression level of the MUC2 protein was analyzed using an ImageQuant 800 (Cytiva, catalog number: 29-3994-81, USA).

[0087] In addition, the expression level of MUC5AC protein in cell lysates was measured using the MUC5AC ELISA kit (LSBIO, catalog number: LS-F38060, USA).

[0088] As a result of the experiment, it was confirmed that treatment of goblet cells with the peptide of SEQ ID NO: 1 increased the expression levels of mucin 2 (MUC2) and mucin 5AC (MUC5AC) proteins (Figures 6A and 6B).

[0089] Experiment Example 7: Inhibition of IFN-γ-induced inflammation and cell damage in human corneal epithelial cells To assess whether the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 has the activity of inhibiting IFN-γ-induced cell damage in human corneal epithelial cells.

[0090] Human corneal epithelial cells were used at a rate of 5 × 10 5 Cells were seeded at a density of 10 cells / well in 6-well plates and cultured for 24 hours. The peptide of SEQ ID NO: 1 was treated with peptide at concentrations (10 μM, 50 μM, 100 μM) for 24 hours. Currently, in the case of the induced group, it was also treated simultaneously with 100 ng / ml IFN-γ. Subsequently, the cells were washed twice with PBS and RNA was isolated using Easy Blue (iNtRON, catalog number: 17061, Korea). The isolated RNA was quantified and then cDNA was synthesized using an RT kit (enzynomics, catalog number: RT200, Korea). PCR was then performed using a PCR kit (enzynomics, catalog number: P581T, Korea). The primers used in the PCR are shown in Table 3.

[0091] It is known that tear film disruption leading to corneal and conjunctival dryness stress triggers Th1 cell activation, and the Th1 cell activation-promoted IFN-γ secretion exacerbates damage to corneal and conjunctival tissues. Experimental results confirmed that IFN-γ treatment of human corneal epithelial cells induced an increase in the expression levels of pro-inflammatory molecules COX-2, IL-1β, and IL-6 genes, and this increase in gene expression levels of pro-inflammatory molecules COX-2, IL-1β, and IL-6 was inhibited by the peptide in SEQ ID NO:1 (Figures 7A and 7B).

[0092] In addition, it was confirmed that when human corneal epithelial cells were treated with IFN-γ, the expression levels of MMP (MMP3 and MMP9) genes increased, and the increased expression levels of MMP (MMP3 and MMP9) genes were inhibited by the peptide of SEQ ID NO: 1 (Fig. 7C and Fig. 7D).

[0093] Experimental Example 8: Inhibition of UVB-induced inflammation and cell damage in human corneal epithelial cells To assess whether the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 has the activity of inhibiting UVB-induced cell damage in human corneal epithelial cells.

[0094] Human corneal epithelial cells were used at a rate of 5 × 10 5 Cells were seeded at a density of 10 cells / well in 6-well plates and cultured for 24 hours. After replacing the medium with serum-free medium, the peptide of SEQ ID NO: 1 was treated with peptide at concentrations (10 μM, 50 μM, 100 μM) for 1 hour. The concentration was 20 mJ / cm². 2 Cells were irradiated with UVB and treated with the peptide of SEQ ID NO: 1 at concentrations (10 μM, 50 μM, 100 μM) in serum-free medium for 24 hours. Cells were washed twice with PBS, and RNA was isolated using Easy Blue (iNtRON, catalog number: 17061, Korea). The isolated RNA was quantified, and cDNA was then synthesized using an RT kit (enzynomics, catalog number: RT200, Korea). PCR was subsequently performed using a PCR kit (enzynomics, catalog number: P581T, Korea). The primers used for PCR are shown in Table 3.

[0095] Ultraviolet (UV) radiation is known to be another stressor that can induce dry eye syndrome. This study evaluated the effect of the peptide in SEQ ID NO:1 on mitigating cell damage after UV irradiation-induced corneal epithelial cell injury. Experimental results confirmed that UVB irradiation of human corneal epithelial cells increased the expression levels of pro-inflammatory molecules COX-2, IL-1β, and IL-6 genes, and these increased expression levels were subsequently reduced by the peptide in SEQ ID NO:1 (Figs. 8A and 8B). Furthermore, UVB irradiation of human corneal epithelial cells was confirmed to increase the expression levels of MMP (MMP3 and MMP9) genes, which were then reduced by the peptide in SEQ ID NO:1 (Figs. 8C and 8D).

[0096] Experiment Example 9: Activity in improving symptoms of a mouse model of dry eye The activity of the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 in improving dry eye symptoms in a mouse model of dry eye was evaluated.

[0097] (1) Induction and maintenance of dry eye syndrome Experimental animals (C57BL / 6 mice, 10 weeks old, female) were housed in a desiccation stress chamber with a relative humidity of 30% or lower. Dry eye syndrome was induced by subcutaneous injection of scopolamine hydrobromide (Sigma-aldrich, catalog number: S0929, USA) at a concentration of 2.5 mg / mL, prepared in saline (0.9% physiological saline, JW Pharmaceutical), three times daily for 10 days (TID, 9:30 am, 13:30 pm, and 17:30 pm). After the start of test material application, scopolamine was injected subcutaneously into the neck once daily (QD, 9:30 am) while the mice were housed under conditions of relative humidity of 30% or lower.

[0098] (2) Preparation of application materials The peptide of SEQ ID NO: 1 was dissolved in saline (0.9% physiological saline, JW Pharmaceutical) at a concentration of 50 μM, filtered through a 0.22 μm filter, and then used in the experimental group during administration (corresponding to CG-T1 in the table of Figure 9C). As an excipient control group, the same saline was filtered through a 0.22 μm filter and administered during administration. As a positive control group, 0.05% Restasis ophthalmic emulsion (Restasis®, Allergan) was administered.

[0099] (3) Application One drop (5 μL) each of test material, positive control material, and excipient was administered to both eyes via eye drops using a micropipette, twice daily (BID, 9:30 am and 5:30 pm) for 16 days. Seven subjects were included in each administration group, and the final analysis included 12 to 14 eyes.

[0100] (4) Assessment of tear break-up time (TBUT) and assessment of clinical corneal damage (corneal fluorescein score). After inducing dry eye by desiccation stress and administration of scopolamine hydrobromide, mice were induced to be generalized anesthetized by intraperitoneal injection of 10 mg / kg toluidine (Rompun®, BAYER) and 75 mg / kg ketamine (Ketamine®, Yuhan Corporation) at grouping time (day 0) and at 6, 11 and 16 days after administration of the test, and were evaluated.

[0101] Tear film breakup time (TBUT) was assessed using the following method. First, a 0.4 M Fluorescein® (Haag-streit AG, Switzerland) test strip was moistened with one drop of saline and instilled into the lower conjunctival sac of a mouse. The mouse blinked 2 or 3 times to allow the fluorescent dye to fully stain the tear film. The stained eye was gently opened, and the time in seconds for the first crack point or crack line to appear in the stained tear film layer under blue light was measured.

[0102] Clinical corneal injury assessment was performed using the following method. First, mydriatic agent (TROPERIN®, Alcon Korea) was instilled into the eyes to induce mydriasis. Then, 10 μL of 0.2% sodium fluorescein (FLUORESCITE®, Alcon Korea) was instilled into each eye to fluoresce the damaged corneal area. Subsequently, the eyes were gently rinsed with saline, and corneal images were taken under blue light (Micron-IV®, Phoenix). The corneal region was divided into 5 regions according to the National Eye Institute (NIE) guidelines, and each region was scored from 0 to 3. These scores were then summed to calculate the fluorescein score (Figure 9A).

[0103] (5) Experimental Results In the tear film breakup time assay, compared with the untreated group and the excipient group, when test material containing the peptide of SEQ ID NO:1 (CG-T1) at a concentration of 50 μM was administered twice daily to dry eye model mice (see Figure 9C), a significant prolongation of breakup time was observed on days 6, 11, and 16. In particular, the results on days 11 and 16 showed higher significance than the positive control group. These results confirm that the peptide of SEQ ID NO:1 exhibits a tear film retention effect even in dry eye animal models by controlling various mechanisms, an effect already confirmed in cellular-level experiments, thereby preventing excessive tear evaporation (Figures 9B and 9C).

[0104] Furthermore, compared to the untreated group, when dry eye mice were administered test material containing a 50 μM peptide of SEQ ID NO: 1 (CG-T1) twice daily, the corneal fluorescence score, which was increased according to corneal damage, showed a decreasing trend on day 11 and decreased to a significant level on day 16 (see Figure 10B). The experimental results indicate that corneal damage caused by dry eye was significantly improved by the peptide of SEQ ID NO: 1 (Figures 10A to 10C).

[0105] [Table 2]

[0106] [Table 3]

[0107] The foregoing has described representative embodiments of this application by way of exemplary implementation; however, the scope of this application is not limited to the specific embodiments described above, and those skilled in the art can make appropriate modifications within the scope of the claims of this application.

Claims

1. A peptide comprising the amino acid sequence of SEQ ID NO:

1.

2. A pharmaceutical composition for treating or preventing dry eye syndrome, comprising the peptide of claim 1 as an active ingredient.

3. The pharmaceutical composition of claim 2, wherein, The pharmaceutical composition has the activity of promoting tear secretion through one or more of the following activities: (i) It has the ability to bind to the pituitary adenylate cyclase type 1 receptor (PAC1R) and promote the activation of the PAC1R signaling pathway. (ii) The activity of promoting the migration of aquaporin 5 (AQP5) protein to the plasma membrane; and (iii) Activity that induces neuroblast differentiation.

4. The pharmaceutical composition of claim 2, wherein, The pharmaceutical composition has the activity of promoting the expression and secretion of mucin in conjunctival goblet cells.

5. The pharmaceutical composition of claim 2, wherein, The pharmaceutical composition has the activity of inhibiting inflammatory responses and cell damage in human corneal epithelial cells.

6. The pharmaceutical composition of claim 2, wherein, The dry eye syndrome includes one or more diseases, disorders, or symptoms selected from the group consisting of evaporative dry eye, dehydrating dry eye, keratoconjunctivitis sicca, keratoconjunctivitis sicca, and dysfunctional tear syndrome.

7. The pharmaceutical composition of claim 2, wherein, The pharmaceutical composition comprises a solution, suspension, or gel suitable for topical application to the eyes.

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