KR20170061084A - Pharmaceutical composition for wound healing containing Humanin or analogue thereof as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the treatment of wounds comprising humanin or an analogue thereof as an active ingredient. Specifically, humanein or an analogue thereof of the present invention can be used not only in a general environment but also in a hyperglycemic environment Skin ulcer). In particular, it exhibits remarkable wound healing effect than EGF, which is a currently available wound healing agent. Therefore, the humanein or its analogue of the present invention can be used as a composition for treating wound, or for treatment of diabetic ulcer It can be usefully used as an active ingredient of the composition.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for wound healing containing human drug or analogue thereof,

The present invention relates to a composition for the treatment of general wounds or hyperglycemia-induced wounds comprising humanin or an analogue thereof as an active ingredient.

Skin is an organ that has the function of protecting the human body and exposed to the dangerous situation when the skin is damaged. External injuries caused by external forces, skin damage due to skin diseases, skin defects such as burns are formed, and efforts are made to minimize skin tissue regeneration and traces of regenerated tissues. Therefore, prompt treatment is required to restore damaged skin tissue without side effects, and wound treatment with an appropriate wound treatment agent is essential. The development of many medicines is actively researching wound healing agents such as wound healing. However, although many chemical synthesis materials have been developed for the purpose of tissue repair and wound healing due to oxidative damage, development of therapeutic agents capable of replacing existing chemical synthetic materials is under way, since it is not possible to apply them for a long time due to side effects caused by tolerance.

In addition, recent diabetic patients are suffering from chronic skin ulcers due to reduced wound healing rates, which is the major cause of limb amputation. Therefore, despite the active interest in chronic skin wound healing in pathological conditions such as diabetes, various therapies have been actively developed. However, therapeutic agents for new wound healing applicable to hyperglycemic patients such as diabetics are still lacking It is a situation.

The global market for wound healing drugs is on the rise and is expected to reach $ 18.3 billion in 2019. The reason why the market for wound healing drugs grows so much is that it is due to an increase in awareness of new concept of wound healing drugs and an increase in the number of patients with diabetes and aging out of the conventional anti-inflammatory and antibiotic drugs. The domestic wound healing drug market is currently showing a large annual growth rate of 100 billion won, attracting interest from many pharmaceutical companies.

Humanin is encoded in the mitochondrial genome and is produced in similar time periods in each of three laboratories that study Alzheimer's, apoptosis, and IGF-1 signaling. Leu-Leu-Leu-Leu-Ser-Glu-Ile-Asp-Leu-Pro-Val-Lys- Arg-Arg-Ala) and has biological activity, thus exhibiting neuroprotective effects in Alzheimer's, Huntington's, and stroke models. In particular, it is known that mitochondrial cytoplasm binds to BAX to inhibit apoptosis. However, the therapeutic effect of hymenin is not known at all.

Therefore, the present inventors have made efforts to develop a wound treatment agent for a living body which can replace a conventional chemical synthetic substance. As a result, the present inventors have found that humanein, which is naturally synthesized in vivo and known as a cell death inhibitor, The inventors of the present invention have found that the hymenin of the present invention exhibits a significant wound healing effect in the environment (diabetic skin ulcer) and shows a wound treatment effect more remarkably than EGF, which is a commercially available wound treatment agent, The present invention can be used as an effective ingredient of a composition for treating ulcers.

It is an object of the present invention to provide a pharmaceutical composition for treating wound or diabetic ulcer comprising humanin or an analogue thereof as an active ingredient, and a skin external preparation.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the treatment of wounds comprising humanin or an analogue thereof as an active ingredient.

The present invention also provides an external preparation for skin wound healing or improvement comprising humanein or an analogue thereof as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating diabetic ulcer comprising humanein or an analogue thereof as an active ingredient.

In addition, the present invention provides an external preparation for skin for preventing or ameliorating diabetic ulcer comprising humanein or an analogue thereof as an active ingredient.

Humanin or an analogue thereof of the present invention exhibits a significant wound healing effect not only in a general environment but also in a hyperglycemic environment (diabetic skin ulcer), and in particular, a wound treatment effect It is possible to effectively use the active ingredient of the composition for treating wound, or the composition for treating diabetic ulcer, which can replace the existing synthetic compound.

Brief Description of the Drawings Fig. 1 is a view showing the wound healing effect of humanin of the present invention. Fig.
T0: Cell image of skin wound time;
T14: Cell image after 14 hours after wounding the skin;
HN: an image of a cell treated with humenin of the present invention after 14 hours after wounding the skin; And
HN + PD98059: An image of a cell treated with Humanein and PD98059 of the present invention after 14 hours after wounding the skin.
FIG. 2 is a view showing the effect time of the signal of humanein through Western blot.
3 shows an Erk path.
Figure 4 shows the mechanism of action of wound healing in humans:
HN: Humenin treated group; And
HCL: Negative control vehicle (HCL) treated group.
FIG. 5 is a graph showing the results of cell migration assay in fibroblasts of humanein.
FIG. 6 is a chart for confirming the therapeutic effect of wound heminenin in a hyperglycemic environment.
FIG. 7 is a chart for confirming the effect of wound treatment of humanein in a mouse induced by type 1 diabetes.
Fig. 8 shows the effect of wound healing of the humane analogs of the present invention:
T0: Cell image of skin wound time;
T21: Cell image after 21 hours after wounding the skin;
EGF: EGF treated group, a wound healing agent, as a positive control group after 21 hours after skin injury;
HN: an image of a cell treated with humenin of the present invention after 21 hours after wounding the skin;
HNG1: Cell image treated with S14G-HN, a hermannin analog of the present invention, after 21 hours, after wounding the skin;
HNG2: Cell image treated with hymenin analogue HN (? A) of the present invention after 21 hours after wounding the skin; And
HNG3: An image of a cell treated with a hymenin analogue HN (GABA) of the present invention after 21 hours after skin injury.
FIG. 9 is a chart for confirming the wound healing effect of humane analogs in a hyperglycemic environment.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the treatment of wounds comprising humanin or an analogue thereof as an active ingredient.

The hymenin is a peptide which is encoded by the mitochondrial genome and has the amino acid sequence of SEQ ID NO: 1.

The humanein may be either isolated in vivo or synthesized based on the amino acid sequence of humanein.

The analogue preferably has an amino acid sequence having 80% or more homology with humansin having the amino acid sequence of SEQ ID NO: 1, more preferably 90% or more homology, ≪ / RTI > amino acid sequence. For example, in the amino acid sequence of SEQ ID NO: 1, the 14th amino acid is glycine, alanine, gamma-butyric acid, valine, leucine, methionine ) And isoleucine may be used as the amino acid sequence of SEQ ID NO:

name SEQ ID NO: order humanin One MAPRGFSCLLLLTSEIDLPVKRRA S14G-HN (HNG1) 2 MAPRGFSCLLLLTGEIDLPVKRRA
C8A-HN (HNA) 3 MAPRGFSALLLLTSEIDLPVKRRA
D-Ser14-HN 4 MAPRGFSCLLLLTsEIDLPVKRRA
AGA-HNG 5 MAPAGASCLLLTGEIDLPVKRRA
AGA- (D-Ser14) -HN 6 MAPAGASCLLLLTsEIDLPVKRRA
AGA- (D-Ser14) -HN17 7 PAGASCLLLLTsEIDLP
AGA- (C8R) -HNG17 8 PAGASRLLLLTGEIIDLP
EF-HN 9 EFLIVIKSMAPRGFSCLLLLTSEIDLPVKRRA
EF-HNA 10 EFLIVIKSMAPRGFSALLLLTSEIDLPVKRRA
EF-HNG 11 EFLIVIKSMAPRGFSCLLLLTGEIDLPVKRRA
EF-AGA-HNG 12 EFLIVIKSMAPAGASCLLLLTGEIDLPVKRRA
Colivelin 13 SALLRSIPAPAGASRLLLLTGEIDLP
P3R-HN 14 MARRGFSCLLLSTTATDLPVKRRT
F6A-HN 15 MAPRGASCLLLLTSEIDLPVKRRA
HNGF6A 16 MAPRGASCLLLLTGEIDLPVKRRA
F6AK21A 17 MAPRGASCLLLLTGEIDLPVARRA
Z-HN 18 MAKRGLNCLPHQVSEIDLSVQKRI
HN (? A) 19 MAPRGFSCLLLLT (? A) EIDLPVKRRA
HN (GABA) 20 MAPRGFSCLLLLT (GABA) EIDLPVKRRA
HNA (? A) 21 MAPRGFSALLLLT (? A) EIDLPVKRRA
AGA-HN (? A) 22 MAPAGASCLLLLT (? A) EIDLPVKRRA
HNA (GABA) 23 MAPRGFSALLLLT (GABA) EIDLPVKRRA
AGA-HN (GABA) 24 MAPAGASCLLLLT (GABA) EIDLPVKRRA

In addition, in the humane analog of Table 1, HNG1 represented by SEQ ID NO: 2 is described as S14G-HN or HNG1 in the present specification in which S, the 14th amino acid of humanein, is substituted by G, and SEQ ID NO: 19 HN ([beta] A) described is HN ([beta] A) or HNG2 in this specification in which S, the 14th amino acid of humenin, is replaced by beta-alanine, HN (GABA) Is substituted with GABA (gamma-aminobutyric acid), which is referred to herein as HN (GABA) or HNG3.

The wound treatment refers to the inhibition of development of skin damage, the alleviation of skin damage, or the removal of skin damage, and the wound may be burned, burned, traumatic, post-surgical, Or damage due to dermatitis.

Preferably, the image is an image caused by sun, chemical, radiation, or heat, and the ulcer is preferably a diabetic ulcer, and the chronic wound is preferably a pressure ulcer or a pressure ulcer. The dermatitis is impetigo ), Intertrigo, folliculitis, and eczema. However, the present invention is not limited thereto.

In a specific example of the present invention, it was confirmed that the wound healing effect of humanein was confirmed, indicating that humanein of the present invention activates Erk and thus exhibits significant wound healing effects (see FIG. 1).

In addition, the present inventors confirmed by Western blot that the appropriate effect time of the signal of humanein is transmitted, and as a result, Humenin activates Erk and JNK in a shorter time than EGF (Epithelial Growth factor) Indicating significant wound healing effects (see FIG. 2).

In addition, the present inventors confirmed that the humenin of the present invention promotes cell migration through the phosphorylation of JNK or phosphorylation of Erk, P90RSK phosphorylation, and the phosphorylation pathway of integrin [beta] 4 3 and 4).

In addition, cell migration assay was performed to examine the wound healing effect of humemannin in fibroblasts, another cell layer of the skin. As a result, the group treated with humenin of the present invention showed a time point T0), and vehicle treated group (see FIG. 5).

In addition, as a result of confirming the effect of wound healing in hyperglycemic conditions, it was confirmed that the wound treatment effect was slowed in the hyperglycemic environment, and the treatment with hymenin significantly increased the wound treatment speed even in the hyperglycemic environment (See FIG. 6).

Also, check the check result, the first type added to the wound in a mouse leads to diabetes After a daily treatment an amount of Hugh maenin represents a powerful wound healing, such as in vitro results for wound healing Hugh maenin in in vivo models (See FIG. 7).

The HUMANIN analogues HNG1 (S14G-HN), HNG2 (HN (βA)) and HNG3 (HN (GABA)) showed the same wound healing effect as Humenin (See FIG. 8).

In addition, it was confirmed that the treatment effect of wound healing in hyperglycemic conditions was slowed in hyperglycemic environment, and the treatment of hymenin analogue significantly increased wound treatment speed even in hyperglycemic environment (See Fig. 9).

Accordingly, the humanein or analog thereof of the present invention exhibits a significant wound treatment effect in a hyperglycemic environment (diabetic skin ulcer) as well as a general environment, and exhibits a wound treatment effect remarkably more remarkable than EGF, which is a commercially available wound treatment agent, The humanein of the present invention or an analog thereof can be usefully used as an active ingredient of a composition for treating or preventing wound healing.

When the composition is formulated, it is prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used.

Solid formulations for oral administration include tablets, patients, powders, granules, capsules, troches and the like, which may contain one or more excipients such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like are included in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically 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 will depend on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. 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, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of humanein according to the present invention may vary depending on the age, sex, and body weight of the patient. In general, 0.1 mg to 100 mg, preferably 0.5 mg to 10 mg, Or one to three divided doses per day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

The present invention also provides an external preparation for skin wound healing or improvement comprising humanein or an analogue thereof as an active ingredient.

The humanein of the present invention or its analogue exhibits a significant wound treatment effect not only in a general environment but also in a hyperglycemic environment (diabetic skin ulcer), and in particular, exhibits remarkable wound healing effect than EGF, which is a currently available wound healing agent, There is no side effect, and it can be usefully used as an external preparation for skin for safe wound treatment or improvement.

The external preparation for skin of the present invention may be formulated for topical application to skin containing a cosmetically or dermatologically acceptable medium or base. These are all formulations suitable for topical application, for example as a solution, a gel, a solid, a paste anhydrous product, an emulsion obtained by dispersing an oil phase in water, a suspension, a microemulsion, a microcapsule, a microgranule or an ionic form (liposome) In the form of ionic fibrin dispersions, or in the form of creams, skins, lotions, powders, ointments, sprays or conical sticks. It can also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant. These compositions may be prepared according to conventional methods in the art.

In addition, the external preparation for skin of the present invention can be used in the form of a skin preparation, a skin lotion, a thickening agent, a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, The active ingredient may be combined with any other ingredients commonly used in cosmetics, such as ionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, And may contain adjuvants commonly used in the same cosmetic or dermatological fields. Such adjuvants are introduced in amounts commonly used in the cosmetics or dermatological fields.

The present invention also provides a pharmaceutical composition for preventing or treating diabetic ulcer comprising humanein or an analogue thereof as an active ingredient.

In addition, the present invention provides an external preparation for skin for preventing or ameliorating diabetic ulcer comprising humanein or an analogue thereof as an active ingredient.

Because the diabetic ulcer is a disease that occurs in diabetic patients, chronic diabetic patients develop chronic skin ulcers due to a slower wound healing rate compared with the general population. Therefore, a substance that increases the rate of wound treatment in a diabetic state (hyperglycemic state) Can be treated.

In a specific example of the present invention, it has been confirmed that the wound treatment effect is slowed in a hyperglycemic environment, wherein the treatment of humemannin or its analogue increases the rate of wound healing even in a hyperglycemic environment, (See FIGS. 6 and 9).

Accordingly, the humenin or analog thereof of the present invention exhibits a significant wound healing effect on ulcers caused by hyperglycemia including diabetes, so that the humemann or its analogue of the present invention can be used as a pharmaceutical composition for the treatment of diabetic ulcer, . ≪ / RTI >

The present invention also provides a cosmetic composition for improving skin condition containing humanein or an analogue thereof.

It is preferable that the skin condition improvement is any one selected from the group consisting of wrinkle improvement, skin elasticity improvement, skin regeneration and skin aging prevention.

The humenin or analog thereof of the present invention has wound healing effect and thus can be effectively used as a cosmetic composition for skin condition improvement including skin remnants.

Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Preparation Examples.

However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Experimental Examples and Production Examples.

< Example  1> Cell culture

Human keratinocyte cells, HaCaT cells and mouse embryonic fibroblasts were cultured in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% FBS (heat-inactivated fetal bovine serum) at 37 ° C and 5% CO ₂ incubator and cultured.

< Experimental Example  1> Humanein ( humanin ) Of wound treatment

In order to confirm the wound healing effect of humanein, a wound healing assay was performed on human keratinocyte cells, and the titration time for the signal of humanein was confirmed by Western blot Respectively.

Specifically, in the wound healing assay, human keratinocytes were dispensed into 6-well plates, and cells were confluently cultured at 37 ° C, and then the cell surface was constantly drawn using a P100 pipet tip. Then, the cells were washed and cultured in 1 ml growth medium containing 20 [mu] M humanein. In contrast, the control group was cultured in 1 ml growth medium containing vehicle (DMSO; dimethyl sulfoxide) containing 50 μM of the Erk inhibitor PD98059. Images were measured at × 10 magnification using an AxioObserver FL microscope (Advanced Microscopy Group, Bothell, Wash. WA), and the intervals of the wounds were measured at three or more sites. The percentage of wound healing was measured using ImageJ software (http: //imagej.nih. gov / ij / index.html). In addition, the experiment was repeated three times.

 Western blot was prepared by treating human keratin cells with the same huminin and the same amount of EGF (Epithelial Growth Factor), washing the cells with 1X PBS, adding RIPA buffer containing protease and phosphatase inhibitor (150 mM NaCl, 50 nM Tris, 1% Triton-X-100, 0.5% sodium deoxycholate and 0.1% SDS). The protein concentration was measured using Bio-Rad Protein Assay reagent (Bio-Rad Laboratories, Hercules, Calif., USA) after centrifugation at 12,000 g for 1 minute. The same amount of protein was loaded, electrophoresed using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and then transferred to nitrocellulose membranes. The membranes were blocked for 1 hour with Tris-buffered saline containing 5% skim milk and 0.1% Tween 20 and incubated with primary antibodies (pErk (Cell Signaling, Danvers, MA), pJNK (Cell Signaling), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, Calif.) Overnight at 4 ° C and then reacted with HRP (horeseradish peroxidase) -conjugated secondary antibody for 1 hour at room temperature, followed by a Chemiluminescence Western Blot Detection System 600 Imaging System CA USA).

As a result, as shown in Fig. 1, it was confirmed that a wide wound area appeared in human keratinocytes after 14 hours (T14) after wounding and 14 hours after injury, and after 14 hours of injury, The group treated with Humenin significantly reduced the injured area and confirmed that the wound treatment effect was remarkable. In addition, through simultaneous treatment of Erk inhibitor PD98059 and humanein, it was confirmed that the humemann of the present invention activates Erk and JNK and shows wound healing effect (FIG. 1).

In addition, as shown in FIG. 2, the optimal effect time of the signal of humanein was confirmed by Western blot. As a result, Humenin showed Erk in a shorter time than EGF (Epithelial Growth factor) (Fig. 2).

< Experimental Example  2> Humanein's  Confirm mechanism of wound healing action

JNK is known to directly participate in various cell signal transduction including cell proliferation, differentiation and migration. JNK phosphorylation is essential for cell migration and keratinocyte migration through paxillin, one of the proteins involved in intercellular binding (Huang C et al . Nature 10: 424 (6945): 219-23). In addition, it is known that cell migration increases with Erk activation (Faure E et al . J Cell Sci . 2012; 125: 4264-4277) (FIG. 3) Western blot and immunofluorescence were performed in order to identify the sub-signal pathway of humemannin through the membrane.

Specifically, Western blot was prepared in the same manner as in Experimental Example 1 except that pErk (Cell Signaling, Danvers, MA), pP90RSK (Cell Signaling) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, , And immunofluorescence was performed by immobilizing the cells with 4% paraformaldehyde and washing with PBS. The cells were incubated with primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) Corresponding to integrin β4, washed, and incubated with Alexa Fluor 594 or Alexa Fluor 488 conjugated secondary antibody. were captured and visualized using a confocal microscope (Leica Microsystems, GmbH, Wetzlar, Germany).

As a result, as shown in FIG. 4, it was confirmed that humanein (20 μM) of the present invention promotes cell migration through Erk phosphorylation, P90RSK phosphorylation, and phosphorylation pathway of integrin β4 (FIG. 4).

< Experimental Example  3> In fibroblasts Humanein's  Check wound treatment effect

From the above <Experimental Example 1> and <Experimental Example 2>, humectinin increased cell migration and confirmed wound healing effect. To examine the effect of humemannin on fibroblasts, another cell layer of the skin, Cell migration assay was performed.

Cell migration was assessed by dividing the cell surface with a P100 pipette tip, then washing the cells and culturing in 1 ml growth medium containing Mitomycin C (5 ug / ml of proliferation inhibitor). The images were then measured at × 10 magnification using an AxioObserver FL microscope (Advanced Microscopy Group, Bothell, Wash.) After treatment with 20 μM humane or vehicle (HCL) The distance of cell movement was calculated using ImageJ software (http://imagej.nih.gov/ij/index.html).

As a result, as shown in FIG. 5, it was confirmed that the group treated with Humenin of the present invention significantly increased cell migration compared to the time point of injury (T0) and the group treated with vehicle 5).

< Experimental Example  4> In hyperglycemic conditions Humanein's  Check wound treatment effect

After application of an environment similar to the delayed wound healing seen in diabetic patients (hyperglycemic environment) to the cells, the degree of delayed wound healing and wound healing effect by humenine treatment were confirmed.

Specifically, the surface of HaCaT cells was stained with a P100 pipette tip in the same manner as in Experimental Example 1, and then a low level (5.5 mM; low glucose, LG) and a high level (50 mM; High glucose (HG) glucose was treated, and humenin of the present invention was treated at a concentration of 20 μM, and the wound healing effect was confirmed in the same manner as in Experiment 1.

As a result, as shown in FIG. 6, it was confirmed that the wound treatment effect was slowed in the hyperglycemic environment, and that the treatment with humemannin significantly increased the wound healing rate even in the hyperglycemic environment (FIG. 6).

< Experimental Example  5> Through animal experiments Humanein's  Check wound treatment effect

In order to confirm the wound healing effect of humanein in vivo model, mice with type 1 diabetes induced by injection of general mouse and Streptozotocin were used.

Specifically, huymanin was dissolved in 3 ml of 100% ethanol, and then mixed with 7 ml of polyethylene glycol (PEG) to prepare a huminin treatment drug at a concentration of 50 μM. In addition, 8-week-old BL6 female mice were prepared, and groups of general conditions and streptozotocin 50 mg / kg were injected for 5 days to induce type 1 diabetes. Both groups were subjected to a body weight trace for a period of time to check for changes in blood glucose. Two days before the wound on the mouse model, use a depilator to remove all of the hairs from the back and give it a period of time to stabilize. After 4 weeks of inhalation anesthesia, 4 equal-sized wounds were applied using a 4 mm diameter biopsy punch and then humans and vehicle (PEG) were treated. Each morning and afternoon, 50 μM of humemann drug was applied and the degree of wound healing was observed.

As a result, as shown in FIG. 7, it was confirmed that when a wound was applied and a certain amount of Humenin was treated daily, it showed a strong wound healing effect as in vitro results (FIG. 7).

< Experimental Example  6> Humanein  Identifying wound healing effects of analogs

A wound healing assay was performed on human keratinocyte cells in the same manner as in Experimental Example 1, in order to confirm the wound healing effect of humane analogues.

Specifically, HNG1 (S14G-HN), HNG2 (HN (βA)) and HNG3 (HN (GABA)) were used as the three HNG analogues shown in Table 1, 1000 times lower concentration was used because it is visible.

As a result, as shown in Fig. 8, it was confirmed that the hymenin analogue had a significant wound healing effect as compared with the negative control group, like hymenin (Fig. 8).

< Experimental Example  7> In hyperglycemic conditions Humanein  Identifying wound healing effects of analogs

In the same manner as in <Experimental Example 4>, after delaying wound healing in a diabetic patient (hyperglycemic environment), the degree of delay of wound healing and wound healing effect by humemannin analogue treatment were confirmed.

As a result, as shown in FIG. 9, it was confirmed that the rate of wound healing was significantly increased when the hyperglycemic environment treated the humectant analogue with a slow wound treatment (FIG. 9).

The preparation examples for the composition of the present invention are illustrated below.

< Manufacturing example  1> Preparation of pharmaceutical preparations

<1-1> Sanje  Produce

20 mg of the peptide of the present invention

20 mg of lactose

After mixing the above components, the mixture was packed in an airtight container to prepare a powder.

<1-2> Preparation of tablets

10 mg of the peptide of the present invention

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

10 mg of the peptide of the present invention

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium stearate 0.2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

<1-4> Liquid  Produce

20 mg of the peptide of the present invention

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

<1-5> Preparation of Injection

10 [mu] g / ml of the peptide of the present invention

Diluted hydrochloric acid BP until pH 7.6

Sodium chloride up to 1 ml for injection

The peptide of the present invention was dissolved in an appropriate volume of injectable sodium chloride BP. The pH of the resulting solution was adjusted to pH 7.6 using diluted hydrochloric acid BP, and the volume was adjusted using main sodium chloride BP and mixed thoroughly. The solution was filled in a 5 ml type I ampoule made of transparent glass, sealed in an upper lattice of air by dissolving the glass, sterilized by autoclaving at 120 DEG C for 15 minutes or longer, and an injection solution was prepared.

< Manufacturing example  2> Manufacture of cosmetics

<2-1> Flexible longevity skin )

To prepare the softened softening water for antibiosis containing the peptide of the present invention, it may be formulated as described in [Table 2] below and can be produced according to a conventional manufacturing method in the field of cosmetics.

ingredient Content (% by weight) The peptide of the present invention 0.1 to 30% 1,3-butylene glycol 3.0 glycerin 5.0 Polyoxyethylene (60) Hardened castor oil 0.2 ethanol 8.0 Citric acid 0.02 Sodium citrate 0.06 antiseptic a very small amount Spices a very small amount Purified water To 100

<2-2> Nourishing lotion (lotion)

To prepare an antimicrobial nutrition lotion containing the peptide of the present invention, it may be formulated as described in [Table 3] below and may be produced according to a conventional manufacturing method in the field of cosmetics.

ingredient Content (% by weight) The peptide of the present invention 0.1 to 30% 1,3-butylene glycol 8.0 glycerin 5.0 Squalane 10.0 Polyoxyethylene sorbitan monooleate 2.0 Nursery oil 0.1 to 30% 1,3-butylene glycol 3.0 glycerin 5.0 Polyoxyethylene (60) Hardened castor oil 0.2 ethanol 8.0 Citric acid 0.02 Sodium citrate 0.06 antiseptic a very small amount Spices a very small amount Purified water To 100

<2-3> Essence

To prepare an antimicrobial essence containing the peptide of the present invention, it may be formulated as described in [Table 4] below and may be produced according to a conventional production method in the field of cosmetics.

ingredient Content (% by weight) The peptide of the present invention 0.1 to 30% Sitosterol 1.7 The polyglyceryl 2-oleate 1.5 Ceramide 0.7 Setares-4 1.2 cholesterol 1.5 Dicetyl phosphate 0.4 Concentrated glycerin 5.0 Carboxyvinyl polymer 0.2 Xanthan gum 0.2 antiseptic a very small amount Spices a very small amount Purified water To 100

<2-4> Facial wash ( Cleansing Foam )

To prepare an antimicrobial cleanser (cleansing foam) containing the peptide of the present invention, it may be formulated as described in [Table 5] below and may be produced according to a conventional production method in the field of cosmetics.

ingredient Content (% by weight) The peptide of the present invention 0.1 to 30% Sodium N-acylglutamate 20.0 glycerin 10.0 PEG-400 15.0 Propylene glycol 10.0 POE (15) oleyl alcohol ether 3.0 Laurin derivative 2.0 Methyl paraben 0.2 EDTA-4Na 0.03 Spices 0.2 Purified water To 100

<2-5> Nourishing Cream

In order to prepare an antimicrobial nutrition cream containing the peptide of the present invention, the preparation is carried out according to a conventional method in the field of cosmetics as described in the following [Table 6].

Compounding ingredient Content (% by weight) The peptide of the present invention 0.1 to 30% vaseline 7.0 Liquid paraffin 10.0 Wax 2.0 Polysorbate 60 2.5 Sorbitan sesquioleate 1.5 Squalane 3.0 Propylene glycol 6.0 glycerin 4.0 Triethanolamine 0.5 Xanthan gum 0.5 Tocophenylacetate 0.1 Incense, preservative a very small amount Purified water To 100

<2-6> Massage Cream

In order to prepare antimicrobial massage cream containing the peptide of the present invention, the preparation is carried out according to a conventional method in the field of cosmetics as described in the following [Table 7].

Compounding ingredient Content (% by weight) The peptide of the present invention 0.1 to 30% Propylene glycol 6.0 glycerin 4.0 Triethanolamine 0.5 Wax 2.0 Tocophenylacetate 0.1 Polysorbate 60 3.0 Sorbitan sesquioleate 2.5 Cetearyl alcohol 2.0 Liquid paraffin 30.0 Xanthan gum 0.5 Incense, preservative a very small amount Purified water To 100

<2-7> Pack

To prepare an antimicrobial pack containing the peptide of the present invention, the preparation is carried out according to the conventional production method in the field of cosmetics as shown in the following [Table 8].

Compounding ingredient Content (% by weight) The peptide of the present invention 0.1 to 30% Propylene glycol 2.0 glycerin 4.0 Polyvinyl alcohol 10.0 ethanol 7.0 Paiji-40 Hydrogenated Castor Oil 0.8 Triethanolamine 0.3 Incense, preservative a very small amount Purified water To 100

<110> Yonsei University Wonju Industry-Academic Cooperation Foundation <120> pharmaceutical composition for wound healing containing Humanin,          analogue thereof or pharmaceutically acceptable salts thereof          an active ingredient <130> P2016-318 <150> KR 10-2015-0165908 <151> 2015-11-25 <160> 24 <170> Kopatentin 1.71 <210> 1 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> humanin <400> 1 Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Ser Glu Ile   1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala              20 <210> 2 <211> 24 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > S14G-HN (HNG) <400> 2 Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Gly Glu Ile   1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala              20 <210> 3 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> C8A-HN (HNA) <400> 3 Met Ala Pro Arg Gly Phe Ser Ala Leu Leu Leu Leu Thr Ser Glu Ile   1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala              20 <210> 4 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> D-Ser14-HN <400> 4 Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Ser Glu Ile   1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala              20 <210> 5 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> AGA-HNG <400> 5 Met Ala Pro Ala Gly Ala Ser Cys Leu Leu Leu Leu Thr Gly Glu Ile   1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala              20 <210> 6 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> AGA- (D-Ser14) -HN <400> 6 Met Ala Pro Ala Gly Ala Ser Cys Leu Leu Leu Leu Thr Ser Glu Ile   1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala              20 <210> 7 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> AGA- (D-Ser14) -HN17 <400> 7 Pro Ala Gly Ala Ser Cys Leu Leu Leu Leu Thr Ser Glu Ile Asp Leu   1 5 10 15 Pro     <210> 8 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> AGA- (C8R) -HNG17 <400> 8 Pro Ala Gly Ala Ser Arg Leu Leu Leu Leu Thr Gly Glu Ile Ile Asp   1 5 10 15 Leu Pro         <210> 9 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> EF-HN <400> 9 Glu Phe Leu Ile Val Ile Lys Ser Met Ala Pro Arg Gly Phe Ser Cys   1 5 10 15 Leu Leu Leu Leu Thr Ser Glu Ile Asp Leu Pro Val Lys Arg Arg Ala              20 25 30 <210> 10 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> EF-HNA <400> 10 Glu Phe Leu Ile Val Ile Lys Ser Met Ala Pro Arg Gly Phe Ser Ala   1 5 10 15 Leu Leu Leu Leu Thr Ser Glu Ile Asp Leu Pro Val Lys Arg Arg Ala              20 25 30 <210> 11 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> EF-HNG <400> 11 Glu Phe Leu Ile Val Ile Lys Ser Met Ala Pro Arg Gly Phe Ser Cys   1 5 10 15 Leu Leu Leu Leu Thr Gly Glu Ile Asp Leu Pro Val Lys Arg Arg Ala              20 25 30 <210> 12 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> EF-AGA-HNG <400> 12 Glu Phe Leu Ile Val Ile Lys Ser Met Ala Pro Ala Gly Ala Ser Cys   1 5 10 15 Leu Leu Leu Leu Thr Gly Glu Ile Asp Leu Pro Val Lys Arg Arg Ala              20 25 30 <210> 13 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Colivelin <400> 13 Ser Ala Leu Leu Arg Ser Ile Pro Ala Pro Ala Gly Ala Ser Arg Leu   1 5 10 15 Leu Leu Leu Thr Gly Glu Ile Asp Leu Pro              20 25 <210> 14 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> P3R-HN <400> 14 Met Ala Arg Arg Gly Phe Ser Cys Leu Leu Leu Ser Thr Thr Ala Thr   1 5 10 15 Asp Leu Pro Val Lys Arg Arg Thr              20 <210> 15 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> F6A-HN <400> 15 Met Ala Pro Arg Gly Ala Ser Cys Leu Leu Leu Leu Thr Ser Glu Ile   1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala              20 <210> 16 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> HNGF6A <400> 16 Met Ala Pro Arg Gly Ala Ser Cys Leu Leu Leu Leu Thr Gly Glu Ile   1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala              20 <210> 17 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> F6AK21A <400> 17 Met Ala Pro Arg Gly Ala Ser Cys Leu Leu Leu Leu Thr Gly Glu Ile   1 5 10 15 Asp Leu Pro Val Ala Arg Arg Ala              20 <210> 18 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Z-HN <400> 18 Met Ala Lys Arg Gly Leu Asn Cys Leu Pro His Gln Val Ser Glu Ile   1 5 10 15 Asp Leu Ser Val Gln Lys Arg Ile              20 <210> 19 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> HA (BETA A) <220> <221> VARIANT <222> (14) <223> x = beta domain <400> 19 Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Xaa Glu Ile   1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala              20 <210> 20 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> HN (GABA) <220> <221> VARIANT <222> (14) <223> X = GABA <400> 20 Met Ala Pro Arg Gly Phe Ser Cys Leu Leu Leu Leu Thr Xaa Glu Ile   1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala              20 <210> 21 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> HNA (BETA A) <220> <221> VARIANT <222> (14) <223> X = beta-alanine <400> 21 Met Ala Pro Arg Gly Phe Ser Ala Leu Leu Leu Leu Thr Xaa Glu Ile   1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala              20 <210> 22 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> AGA-HN (BETAA) <220> <221> VARIANT <222> (14) <223> X = beta-alanine <400> 22 Met Ala Pro Ala Gly Ala Ser Cys Leu Leu Leu Leu Thr Xaa Glu Ile   1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala              20 <210> 23 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> HNA (GABA) <220> <221> VARIANT <222> (14) <223> X = GABA <400> 23 Met Ala Pro Arg Gly Phe Ser Ala Leu Leu Leu Leu Thr Xaa Glu Ile   1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala              20 <210> 24 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> AGA-HN (GABA) <220> <221> VARIANT <222> (14) <223> X = GABA <400> 24 Met Ala Pro Ala Gly Ala Ser Cys Leu Leu Leu Leu Thr Xaa Glu Ile   1 5 10 15 Asp Leu Pro Val Lys Arg Arg Ala              20

Claims (14)

A pharmaceutical composition for the treatment of wounds, comprising humanin or an analogue thereof as an active ingredient.
The pharmaceutical composition for treating wounds according to claim 1, wherein the humansin has the amino acid sequence of SEQ ID NO: 1.
2. The method of claim 1, wherein the analogue is selected from the group consisting of glycine, alanine, gamma-butyric acid, valine, leucine (SEQ ID NO: leucine, methionine, and isoleucine. &lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt;
The pharmaceutical composition for wound healing according to claim 1, wherein the analog is any one selected from the group consisting of the amino acid sequences represented by SEQ ID NOS: 2 to 24.
The pharmaceutical composition for wound healing according to claim 1, wherein the wound is wound, burn, ulcer, trauma, post-surgical, birth, chronic wound or dermatitis Composition.
[Claim 6] The pharmaceutical composition according to claim 5, wherein the ulcer is a diabetic ulcer.
The pharmaceutical composition for wound healing according to claim 5, wherein the chronic wound is a pressure ulcer or pressure ulcer.
The pharmaceutical composition for wound healing according to claim 5, wherein the dermatitis is any one selected from the group consisting of impetigo, intertrigo, folliculitis, and eczema.
An external preparation for wound healing or improvement comprising humanein or an analogue thereof as an active ingredient.
The external preparation for skin treatment or improvement according to claim 9, wherein the external preparation for skin is for topical application to skin.
A pharmaceutical composition for preventing or treating diabetic ulcer comprising humanein or an analogue thereof as an active ingredient.
A dermatological preparation for preventing or ameliorating diabetic ulcer comprising humanein or an analogue thereof as an active ingredient.
A cosmetic composition for improving skin condition containing humanein or an analogue thereof.
The cosmetic composition according to claim 13, wherein the skin condition improvement is any one selected from the group consisting of wrinkle improvement, skin elasticity improvement, skin regeneration, and skin aging.

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