WO2016122169A1 - Pharmaceutical composition for treating wounds or dry eye syndrome, containing neuregulin - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating wounds or dry dye syndrome, containing neuregulin. The pharmaceutical composition is effective in promoting the proliferation of corneal epithelial cells, skin epithelial cells and oral epithelial cells, and in facilitating the recovery of a damaged cornea. Therefore, the composition of the present invention can be useful for treating wounds or treating dry dye syndrome and for improving the skin.

Description

 【Specification】

 [Name of invention]

 Pharmaceutical composition for treatment of wound or dry eye including nuregulin [Technical field]

 ^ The present invention relates to a pharmaceutical composition for treating a wound or dry eye, including neurlinol, and a method for treating a wound or dry eye using the composition.

Background Art

 In our body, the skin is responsible for many functions, the most important of which is a barrier to protect our body from the outside. A wound is a state in which the normal structure of the skin that acts as a barrier is destroyed. If you are injured, the wound may be exposed to the invasion of bacteria, so you need to treat it quickly. The cornea, on the other hand, is a transparent, bloodless tissue on the anterior surface of the eye, often called a black spot, to protect the eye from the outside. In addition, it is a transparent tissue that passes and refracts light, and plays a major role in the refraction and transmission of light. The nerve is well developed, so it is sensitive to external foreign matter. The cornea consists of five layers: corneal epithelium, Bowman's membrane, corneal parenchyma, posterior border plate (desmelar membrane) and corneal endothelium.

The corneal surface is directly exposed to the outside, making it vulnerable to trauma and scratches. In particular, the lack of tears, corneal damage occurs frequently in patients suffering from diseases such as dry eye mania due to a significant decrease in eye protection function due to tears. Cornea damage If irritation occurs, irritation symptoms such as irritation, foreign body or dryness, and if the damage is severe may develop keratitis. Therefore, in order to maintain eye health and preserve vision, it is essential to prevent corneal damage, to prevent minor corneal damage, or to treat damaged corneas. Neureglin (NR) is a glycoprotein belonging to the epidermal growth factor (EGF) family, and four subtype proteins are known. Neureglin plays an important role in the development of the nervous system and in embryonic development of vertebrates. In addition, neuroregulin is known to bind to and activate the erbB family of tyrosine kinase receptors.

[Detailed Description of the Invention]

 [Technical problem]

 It is an object of the present invention to provide a pharmaceutical composition for treating wounds comprising nuregulin, and a method of treating a wound using the composition.

 Another object is to provide a cosmetic composition for improving virtues containing nuregulin, and a method for improving skin using the composition.

 Another object is to provide a pharmaceutical composition for preventing or treating dry eye syndrome including nuregulin, and a method for preventing or treating dry eye syndrome using the composition.

Technical Solution In order to achieve the above object, the present invention provides a pharmaceutical composition for wound treatment comprising nuregulin as an active ingredient.

 The present invention also provides a method of treating a wound comprising administering a composition of the present invention to a subject.

 The present invention also provides the use of a composition of the invention for use in the manufacture of a medicament for the treatment of wounds.

 In addition, the present invention provides a cosmetic composition for improving skin, comprising nuregulin as an active ingredient.

 The present invention also provides a method for improving skin comprising applying the cosmetic composition of the present invention to an individual.

 The present invention also provides the use of the compositions of the invention for use in the preparation of cosmetics for skin improvement.

 In addition, the present invention provides a pharmaceutical composition for preventing or treating dry eye syndrome comprising nuregulin as an active ingredient.

 The present invention also provides a method of treating dry eye syndrome comprising administering a composition of the present invention to a subject.

 In addition, the present invention provides an ophthalmic preparation comprising the nuregulin as an active ingredient. 【Effects of the Invention】

The pharmaceutical composition comprising the neregulin of the present invention is corneal epithelial cell, skin epithelial cell And promotes proliferation of oral epithelial cells and is effective in repairing damaged corneas. Therefore, the composition of the present invention can be usefully used not only for the treatment of wounds or dry eye, but also for skin improvement. 【Brief Description of Drawings】

 1 is a graph showing that neuregulin (NR) promotes proliferation of corneal epithelial cell lines.

 Figure 2 is a cell photo showing that neuregulin is effective in healing wounds of corneal epithelial cell line.

 Figure 3 is a graph showing the quantification of the wound healing effect of the corneal epithelial cell line induced by nereglin.

 Figure 4 shows that neureglin has the effect of healing the damage to the cornea. 5 is a graph showing the quantification of the area of recovery of corneas damaged by neuregulin.

 FIG. 6 is a cell photograph showing that neregulin is effective in healing wounds of skin epithelial cell lines.

 7 is a graph showing the quantification of the wound healing effect of dermal epithelial cell line by neuregulin.

 FIG. 8 is a cell photograph showing that neregulin is effective in healing wounds of oral epithelial cell lines.

9 is to quantify the wound healing effect of oral epithelial cell line induced by neureglin This is a graph.

[Best form for implementation of the invention]

Hereinafter, the present invention will be described in more detail. The present invention provides a pharmaceutical composition for wound treatment containing nuregulin as an active ingredient. ^"As used herein, the term" IGNERAIE gyulrin (neuregul in) "is a multi-cells in combination with the erbB family of tyrosine Chi Nei's receptor means a cell cross signal passed protein that mediates the action. The neuroregulin of the present invention may have at least about 70%, 80%, 90% or 95% homology with the amino acid sequences of known nuregulins. The nuregulin may be a polypeptide having any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4. In one embodiment, the nuregulin may be a polypeptide having the amino acid sequence of SEQ ID NO: 1.

 The concentration of nuregulin included in the composition of the present invention may be 0.002 to 200 ng / m. The concentration of nuregulin may be 0.02 to 20 ng / u, preferably 0.2 to 2 ng / m £.

As used herein, the term "wound" means that the living body is damaged and is also called a wound. The wound may be a corneal wound, a skin wound or an intraoral wound. As used herein, the term "corneal wound" refers to a wound on the cornea, which is the surface of the eye, and may be caused by factors not only by external stratification but also by the internal part of the body. Can be. Such damage can include both physical and chemical damage. Physical damage may include damage caused by lens wear, surgical damage such as LASIK or LASEK. On the other hand, chemical damage may include damage by chemicals such as acids or bases. Such wounds include wounds that may occur in any one of corneal epithelium, Bowman's membrane, corneal parenchyma, posterior border plate or corneal endothelium. The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier.

 The pharmaceutically acceptable carriers are commonly used in the manufacture of medicines, such as lactose, dextrose, sucrose, sorbbi, manny, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, fine Crystalline cellulose, polyvinylpyridone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxy benzoate, talc, stearic acid magnesium, mineral oil, etc./ It is not limited to this.

 In addition, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive selected from the group consisting of lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspensions, preservatives, and combinations thereof.

 The carrier is about 1% by weight to about based on the total weight of the pharmaceutical composition of the present invention

99.99% by weight, preferably from about 90% by weight to about 99.99% by weight, and the pharmaceutically acceptable additive may be included from about 0.01% to about 20% by weight. The invention also provides a method of treating a wound comprising administering a composition of the invention to a subject.

 The pharmaceutical compositions of the present invention may be administered parenterally, and may preferably be administered directly to the skin in a topical manner.

 Formulations of the pharmaceutical compositions of the present invention may be in the form of external skin preparations such as transdermal injections, ointments, solutions, cream crabs, bills, sprays, patches.

 Suitable dosages of the pharmaceutical compositions of the present invention may be determined in view of various related factors such as formulation method, mode of administration, age of patient, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to response. . The present invention also provides the use of a composition containing the nuregulin described above as an active ingredient for use in the manufacture of a medicament for the treatment of wounds. In addition, the present invention provides a cosmetic composition for skin improvement comprising the nuregulin as an active ingredient. The cosmetic composition has the same active ingredient as the pharmaceutical composition as described above.

Cosmetic composition according to the present invention is a group consisting of suppressing the occurrence of wrinkles, skin presence ^ ᅵ, improving skin elasticity, skin regeneration, wound or wound healing (wound healing), corneal regeneration, blood, irritation relief and combinations thereof It can be used to protect the skin from functional degradation or loss of skin cells or to improve skin condition. The cosmetic composition of the present invention can be applied directly to the skin for the purpose of improving the skin condition.

 The cosmetic composition may be formulated into a cosmetic formulation commonly prepared in the art. The cosmetic composition may include, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, sprays, and the like. It may be formulated as, but the present invention is not limited thereto. More specifically, it may be formulated in the form of a flexible lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing product, a cleansing water, a pack, a spray or a powder.

 When the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica ᅳ talc, zinc oxide and It may include a carrier component selected from the group consisting of a mixture thereof.

 When the formulation of the cosmetic composition of the present invention is a powder or a spray, it may include a carrier component selected from the group consisting of lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, and mixtures thereof. In the case of a spray in particular, it may further include chlorofluorohydrocarbon, propane / butane or dimethyl ether.

The formulation of the cosmetic composition of the present invention may include a carrier component selected from the group consisting of a solvent, a solvating agent, an emulsifying agent and a combination thereof which is a solution or an emulsion. Examples thereof include water, ethanol isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol, sorbitan fatty acid ester, and mixtures thereof. Etc. can be mentioned.

 When the formulation of the cosmetic composition of the present invention is a suspension, suspensions such as water, liquid diluents such as ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbide esters and polyoxyethylene sorbitan esters First, it may include a carrier component selected from the group consisting of microcrystalline cellulose, aluminum metahydroxy, bentonite, agar, tragacanth and mixtures thereof.

 When the formulation of the cosmetic composition of the present invention is a surfactant-containing cleansing agent, the aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate imidazolinium derivative methyl taurate, sarcosinate, fatty acid amide ether And a carrier component selected from the group consisting of sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, ethoxylated glycerol fatty acid esters, and combinations thereof. The "carrier component" in the cosmetic composition of the present invention is a compound or composition that is already known and used that can be included in cosmetic preparations, and is a component that has no toxicity, instability or irritation that is more than acceptable to the human body upon contact with the skin.

In addition to the carrier component, the cosmetic composition of the present invention may further include an adjuvant selected from the group consisting of antioxidants, stabilizers, solubilizers, humectants, pigments, flavors, sunscreens, colorants, surfactants, and combinations thereof. The adjuvant is a cosmetic There is no limit to the use of the adjuvant conventionally used in the preparation of the composition. Therefore, the present invention provides a method for improving the skin condition using the cosmetic composition.

 The method for improving the skin condition may include applying to the skin of the subject in need thereof. The subject may be a mammal, specifically a human.

 Applying to the skin may include applying directly to the skin or spraying, according to the form of the cosmetic composition according to the invention. At this time, the application amount of the cosmetic composition and the number of times of use may be appropriately set according to the age, sex, use, degree of symptoms, etc. of the user, for example, an appropriate amount of the cosmetic composition 1 to 6 times a day Can be applied to the skin. The present invention also provides the use of the compositions of the invention for use in the preparation of cosmetics for skin improvement. The present invention provides a pharmaceutical composition for preventing or treating dry eye syndrome comprising nuregulin as an active ingredient. The pharmaceutical composition according to the present invention is the same as the active ingredient as described above.

As used herein, the term “dry eye” refers to a condition in which the amount of tears is reduced, deficient, or excessively evaporated, causing discomfort to the eye. This dry eye syndrome It may cause damage to the oculo ar surf ace between the upper and lower eyelids (interpalpebral). Symptoms of dry eye syndrome may include eye stiffness, foreign body sensation, stinging, or related conjunctival epithelial injuries due to lack of tears in patients with suppressed tear production. The composition may be a formulation that can be used for ophthalmic use. Specifically, the formulation may be in the form of a solution, lotion, plaster, gel, cream, paste, spray, suspension, dispersion, hydrogel, ointment, oil or blowing agent. The compositions of the present invention may be formulated by mixing with pharmaceutically acceptable, particularly ophthalmic, acceptable nontoxic excipients or carriers. In addition, the formulation can be used to adjust to the appropriate pH.

 Preferred carriers which can be used according to the invention include water, cellulose derivatives such as methyl cellulose, alkali metal salts of carboxymethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, methyl hydroxypropyl cell, and the like. Ross and hydroxypropylcelrose, neutral carbopol, or combinations thereof.

 Stabilizers that can be used in the ophthalmic composition of the present invention are, for example, tyloxapol, fatty acid glycerol poly-lower alkylene glycol esters, fatty acid poly-lower alkylene glycol esters, polyethylene glycols, glycerol ethers or these Mixtures of compounds. These stabilizers can be used in amounts sufficient to dissolve the active ingredient.

Complete agents that can be used in the compositions of the present invention include borate, hydrogen carbonate / carbonate, gluconate, phosphate, propionate and TRIS (tromethamine) buffers. It may be any one selected from the group consisting of. Preferably tromethamine and borohydride agents can be used. Added buffer substrates are necessary to ensure and maintain a physiologically acceptable pH range.

 Preservatives that can be used in the compositions of the present invention include quaternary ammonium salts, alkyl-mercury salts of thiosalicylic acid, parabens, alcohols, guanidine derivatives and the like. Preferred preservatives are cestlimide, benzalkonium chloride, benzoxium chloride and parabens. The preservative may be added in an appropriate amount to prevent secondary contamination during use caused by bacteria and fungi.

 Tonics can be used to control physiological tonicity (eg 0.9% saline). For example, sodium chloride, potassium chloride, calcium chloride, dextrose and / or manny can be added to the composition of the present invention. The amount of tonic agent varies depending on the specific agent to be added. In general, certain inventive compositions may have an osmotic permissible for ophthalmic use of 150-450 mOsm, preferably 250-350 mOsm.

 In addition, monomeric polyols, polymeric polyols, cellulose derivatives (cells of the cellulose family), dextran, water soluble proteins, vinyl polymers, other polys are also used to maintain the appropriate viscosity in ophthalmic formulations. , Carbomers, polysaccharides / glycosamino glycans, and the like. One or more viscosity enhancers may be added to the compositions of the present invention to increase the viscosity of the carrier (vehicle).

The amount and type of excipient added may vary depending on the particular requirements, but can generally be used in the range from about 0.0001 to about 90% by weight, and can be used within the range normally used by those skilled in ophthalmology. In addition, the pH range of ophthalmic formulations is 3.5 to 9, preferably 4.5 to 8 and most preferably pH 5.5 to 7.8, and may be pH 7.0.

The present invention also provides a method for treating dry eye syndrome comprising the step of administering to the subject a composition of the present invention as described above.

 The administration can be direct administration, which includes direct application of a solution, lotion, plaster, gel, cream, paste, spray, suspension, hydrogel, ointment, oil or blowing agent to the tissue.

 In addition, the composition of the present invention may be administered alone or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with known therapeutic agents. The administration may be administered in a total of 0.08 to 2.0 daily, divided once or several times a day. Specifically, it may be administered dividedly twice to five times a day. However, the administration of the composition according to the present invention takes into account all factors that can affect the effect of the composition, such as the age, sex, degree of disease, drug activity, drug sensitivity, duration of treatment, etc. Can be easily determined.

 Examples of routes of administration of the pharmaceutical compositions of the present invention are preferably in the form of transdermal (such as topical) dosage forms, including but not limited to parenteral, intranasal (such as inhalation), administration via mucous membranes.

 The subject may be a mammal, specifically a human.

In addition, the present invention is intended for use in the manufacture of a medicament for treating dry eye syndrome Provided are uses of the compositions of the invention.

[Form for implementation of invention]

 Hereinafter, the present invention will be described in more detail based on the following examples. The following examples are only for illustrating the present invention, but the scope of the present invention is not limited thereto. Experimental Example 1: Comparison of Cell Proliferation Rate

First, the corneal epithelial cell line (HCE) (ATCC, USA) was added to 15% fetal bovine serum (FBS), 5 zg / insulin, and 10 ng / m £ human epidermal growth factor ( hEGF) was cultured in DMEM / F12 culture medium containing 37 ^° C, 5% C0 ₂ conditions. Cultured cells were treated with 0.05% (w / v) trypsin to obtain only cells. Thereafter, it was aliquoted into 96-well plates to be 3xl0 ³ per well and incubated for 12 hours. The culture medium of the cultured cells was replaced with a medium not containing FBS, and then cultured for another 12 hours. On the other hand, nereglin (SEQ ID NO: 1XR & D system, USA) was added to the medium without FBS at a concentration of 0.02, 0.2, 2 or 20 ng /. The medium containing the nuregulin was added to the cultured cells, and only the culture medium containing no serum was added to the control group. After incubation for 12, 24 or 48 hours, respectively, 10 ^ CCK-8 reaction solution (Doj indo) was added to each well. The reaction was stirred for 1 hour at 37 ^° C., followed by microplate reader (Bio-rad, CA,

USA) and absorbance at 450 nm. 1 shows the results of comparing the absorbance values of each well based on the absorbance values of the cells grown in the culture medium without the addition of neurgline. The addition of nuregulin increased the proliferation rate of the corneal epithelial cell line compared to the control group (see FIG. 1). Experimental Example 2: Confirmation of wound healing effect

Corneal epithelial cell lines prepared as described in Experimental Example 1 were dispensed into 24-well plates to be 2xl0 ^{4 per} well and incubated for 12 hours. The culture medium of the cultured cells was replaced with a medium not containing FBS, and then cultured for another 12 hours. A gap was created in the layer of cells and damaged at intervals of 2 mm 3. On the other hand, nuregulin was added to the medium without FBS at a concentration of 0.002, 0.02, 0.2, 2, 20, 50, 100 or 200 ng / me. The media containing the neuroregulin was added to the cultured cells, and only the culture medium without serum was added to the control group. After culturing for 24 hours, it was photographed using an inverted microscope (Olympus). From the images taken, the distance of damage was assessed using T-Scratch software (Computat ional Science & Engineering Laboratory, Switzerland) and compared with the control. As a result, the corneal epithelial cell line grew when the neregulin treatment resulted in a narrowing of the damaged area (see FIGS. 2 and 3). Experimental Example 3: Confirmation of the repair effect of the damaged cornea

Six-week-old BABL / C mice (30 g) females were divided into two groups to be 6 per group. 2 wet paper disk of aim with 20% (w / v) ethanol The cornea of the prepared mouse was covered for 60 seconds to induce corneal damage. Thereafter, the cornea was wounded with a 2 mm blade punch.

 Tobramycin Ahn Ointment (Taejun Pharmaceutical Co., Ltd., Korea) containing 2 ng / ^ nuregulin was applied to the cornea once daily. At this time, only tobramycin ophthalmic ointment was applied to the control group. The wound healing effect of the corneal epithelium was confirmed by corneal fluorescein staining 16, 24 or 48 hours after applying the ophthalmic ointment. Specifically, fluorescein (f luoresce in X Sigma-Aldr i ch, USA) was applied to the corneal epithelium, and after 1 minute, it was washed with sterile phosphate physiological saline. The stained cornea was observed under a blue light under a microscope. The cured area was photographed and measured with the Adobe Acrobat 9.5 program.

 As a result, the image of the fluorescently stained mouse cornea is shown in FIG. 4, and the cured area is shown in FIG. In the group to which tobramycin ophthalmic ointment containing neregulin was repaired, the damaged cornea was recovered at a faster rate compared to the control group (see FIGS. 4 and 5). Experimental Example 4: Confirmation of the wound healing effect of the skin

Dermal epithelial ^' cell lines (human adul t ow cal cium high temperature, HaCaT) (ATCC, USA) were incubated with DMEM containing io% FBS. Cultivation was carried out at 37 ^° C, 5% C0 ₂ conditions. Cultured cell lines were aliquoted into 24-well plates to 6xl0 ⁴ cells per well and incubated for 12 hours. The culture medium of the cultured cells was replaced with a medium not containing FBS, and then cultured for another 12 hours. A gap was created in the layer of cells and damaged at intervals of 2 mm 3. Meanwhile, neurlinol 0.002 ， 0.02, 0.2 ， 2 Or to medium without FBS to a concentration of 20 ng / m. The medium containing the neregulin was added to the cultured cells, and only the culture medium containing no serum was added to the control group. This was incubated for 9 hours, and the subsequent process was performed as described in Experimental Example 1.

 As a result, the value of the measured distance of the damaged um was shown in comparison with the control. It was confirmed by neuregulin that regeneration of damaged skin epithelial cell lines occurs rapidly (see FIGS. 6 and 7). Experimental Example 5 Confirming the Wound Treatment Effect of Oral Epithelium

 Per well of human gingival f ibrobl ast-1 (HGF-1) (ATCC, USA)

It was performed in the same manner as Experimental Example 4, except that the aliquots were dispensed into 24-well plates to 2.5xl0 ⁴ and treated with nereglin and incubated for 24 hours.

 As a result, the effect of neuregline wound healing on oral epithelium was confirmed. Compared with the control group, it was confirmed that neurligin had an excellent effect on the healing of oral epithelium wounds (see FIGS. 8 and 9).

Claims

[Range of request]  [Claim 1]  Pharmaceutical composition for wound treatment containing nuregulin as an active ingredient. [Claim 2]  The pharmaceutical composition of claim 1, wherein the neuregline is included at a concentration of 0.002 to 200 ng / m. [Claim 3]  The pharmaceutical composition of claim 2, wherein the neuregline is included at a concentration of 0.02 to 20 ng / ii ^. [Claim 4]  The pharmaceutical composition of claim 1, wherein the neurglin is any one of the polypeptides set forth in SEQ ID NO: 1 to SEQ ID NO: 4. [Claim 5]  The pharmaceutical composition of claim 1, wherein the wound is a corneal wound, a skin wound or an intraoral wound. [Claim 6] The pharmaceutical composition of claim 1, wherein the composition further comprises any one selected from the group consisting of a carrier, a stabilizer, a laxative, a preservative, and a tonic agent. [Claim 7]  A method of treating a wound comprising administering the composition of claim 1 to a subject. [Claim 8]  Use of the composition of claim 1 for use in the manufacture of a medicament for the treatment of wounds. [Claim 9]  Cosmetic composition for skin improvement comprising a neuroregulin as an active ingredient. [Claim 10]  A method of improving skin comprising applying the cosmetic composition of claim 9 to a subject. [Claim 11]  Use of the composition of claim 9 for use in the preparation of cosmetics for skin improvement. [Claim 12] Pharmaceutical composition for the prevention or treatment of dry eye syndrome containing nuregulin as an active ingredient water [Claim 13]  A method of treating dry eye syndrome comprising administering to a subject a composition of claim 12. [Claim 14]  Ophthalmic formulation comprising nuregulin as an active ingredient. [Claim 15]  The ophthalmic preparation according to claim 14, wherein the ophthalmic preparation is an eye drop or an ointment.