WO2019143145A1 - Composition for treating skin inflammation, comprising resolvin d2 as active ingredient - Google Patents

Abstract

The present invention relates to a composition for treating skin inflammation, comprising resolvin D2 as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition, a quasi-drug composition and a cosmetic composition for preventing or treating skin inflammation or hair loss, all of which comprise resolvin D2 or a salt thereof as an active ingredient. A composition comprising resolvin D2, of the present invention, alleviates skin inflammatory actions, inhibits cell death, prevents and treats hair loss caused by inflammation of a fat layer in the skin, and also promotes hair growth, and thus exhibits an effect of restoring overall skin integrity, thereby being effectively usable as an agent for treating skin inflammatory diseases, an agent for preventing or treating hair loss, or a quasi-drug and a cosmetic composition for alleviating skin inflammation or for promoting hair growth.

Description

Composition for the treatment of skin inflammation containing resorcin D2 as an active ingredient

This application claims priority from Korean Patent Application No. 10-2018-0006148 filed on January 17, 2018, the entire contents of which are incorporated herein by reference.

The present invention relates to a composition for the treatment of skin inflammation containing resorcin D2 as an active ingredient. More particularly, the present invention relates to a composition for the prevention or treatment of skin inflammation or hair loss comprising resolol D2 or a salt thereof as an active ingredient, Compositions, quasi-drug compositions and cosmetic compositions.

Inflammation is a physiological response that protects the body from harmful environmental conditions, such as intrusion of external foreign matter such as bacteria and mechanical damage. This inflammation results in a large number of polynuclear leukocytes and immune substances, and these cells are able to treat and defend by secretion of various kinds of inflammatory cell products, proteases and cytokines . Enzymes such as elastase, hyalurinidase and lipoxygenase are known to be proteins and lipolytic enzymes that are produced during inflammation. On the other hand, these actions can cause harmful damage to adjacent tissue cells and non-tissue cell components, resulting in severe tissue damage of chronic inflammation.

Such excessive inflammation damages cells and connective tissues by proteolytic enzymes, and damage of connective tissues not only causes reduction of skin elasticity to cause wrinkles but also adversely affects cell regeneration and proliferation, Resulting in aging of the skin and localized erythema and swelling in the skin. If the connective tissue is severely damaged and the inflammatory response persists, restoring normal tissue structure and function becomes much more difficult.

Lipid-derived signaling molecules regulate a variety of cellular processes including cell survival, apoptosis, and metabolism. Among the lipid-derived signaling molecules, specialized pro-resolving lipid mediators (SPMs) are known to be endogenous lipid species involved in the natural inflammatory cycle. Resolvins are one of the SPMs produced from the metabolism of omega-3 essential fatty acids by lipoxygenase and other lipid regulating enzymes.

Immunomodulatory disorders and inflammation of the skin are a fundamental pathological mechanism of skin disease and the use of resolvin has been suggested as a treatment method. However, the role of lipoxygenase 15, Alox15, and resolvin D2 in relation to skin homeostasis is unknown.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating skin inflammation or hair loss comprising Resolvin D2 or a pharmaceutically acceptable salt thereof as an active ingredient.

It is still another object of the present invention to provide a quasi-drug composition for preventing or treating skin inflammation or hair loss comprising resolol D2 or a pharmaceutically acceptable salt thereof as an active ingredient.

It is still another object of the present invention to provide a cosmetic composition for preventing or ameliorating skin inflammation or hair loss, which comprises losolvin D2 or a cosmetically acceptable salt thereof as an active ingredient.

It is a further object of the present invention to provide a method for the prevention or treatment of skin inflammation or hair loss comprising administering to a subject a resolvin D2 or an acceptable salt thereof.

It is yet another object of the present invention to provide a method for preventing or treating skin inflammation or hair loss of a composition comprising administering to a subject a resorcin D2 or an acceptable salt thereof.

The present invention provides a pharmaceutical composition for preventing or treating skin inflammation or hair loss comprising resololin D2 (Resolvin D2) or a pharmaceutically acceptable salt thereof as an active ingredient.

According to one aspect of the present invention, the skin inflammation or depilation may be inflammation due to the low expression or deletion of the Alox15 gene.

According to one aspect of the present invention, the skin inflammation may be inflammation occurring in the skin fat layer.

According to one aspect of the present invention, the skin inflammation may be chronic skin inflammation.

According to one aspect of the present invention, the hair loss may be caused by inflammation of the skin fat layer.

According to one aspect of the present invention, the composition reduces the expression level of RIP3 (receptor-interaction serine / threonine-protein kinase 3), p-RIP3 (phosphorylated- RIP3) or ZBP1 Inflammation or hair loss.

In addition, the present invention provides a quasi-drug composition for preventing or treating skin inflammation or hair loss comprising resolol D2 or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a cosmetic composition for preventing or ameliorating skin inflammation or hair loss comprising, as an active ingredient, resololin D2 or a cosmetically acceptable salt thereof.

The present invention also provides a method of preventing or treating skin inflammation or hair loss comprising administering to a subject lesolin D2 or an acceptable salt thereof.

The present invention also provides a method of preventing or treating skin irritation or depilation of a composition comprising administering to a subject lesolin D2 or an acceptable salt thereof.

The composition comprising the resorcin D2 of the present invention alleviates skin inflammation, suppresses cell death, promotes hair growth as well as prevents and treats hair loss caused by inflammation of the skin fat layer, and can be usefully used as a therapeutic agent for skin inflammation disease, prevention or treatment of hair loss, quasi-drugs for improving skin irritation or promoting hair growth, and cosmetic composition.

FIG. 1 shows the results of (a, WT; b, Alox 15 KO) expression of Alox15 through H / E staining and immunofluorescence staining in skin such as mouse.

Figure 2 shows quantitative analysis (b) of immunoblot analysis (a) and expression of Alox15 in brown adipose tissue (BAT), skin white adipose tissue (dWAT) and dorsal skin of WT and Alox15 KO mice (mean ± SEM; n = 4, *** p <0.001).

FIG. 3 shows the result of checking the hair loss phenomenon in the back skin of WT and Alox 15 knockout mice: FIG. 3a is a photograph showing the hair loss phenomenon after 16 weeks, and FIG. 3b is a graph quantitatively showing the cumulative hair loss occurrence rate until the 18th week Hair loss is defined as hair loss if more than 1 cm ^{2 is} seen).

FIG. 4 shows H / E staining and immunofluorescence staining of PLIN1, keratin 14 and keratin 15 in paraffin sections of skin such as mice (staining with DAPI, Bars = 20 μm).

FIG. 5 shows results of an increase in inflammation and an increase in the expression of fibrosis markers in the Alox15 knockout mouse. FIG. 5A shows the result of immunofluorescence staining of F4 / 80, FIG. 5B shows the result of double staining of Plin1 and Ly6C, 5D is a result of double staining of SMA and tdTomato, FIG. 5F shows the expression of immunological marker and fibrosis marker by qPCR < RTI ID = 0.0 &gt; (Mean ± SEM; n = 4, * p <0.05, ** p <0.01).

FIG. 6 shows the results of immunohistochemical analysis of HMGB1 (nuclei are stained with DAPI, Bars = 20 μm). FIG. 6B shows the results of immunohistochemical analysis of p-RIP3, RIP3, Figure 6C is a quantitative graph of the immunoblot analysis (mean ± SEM; n = 4, * p < 0.05, ** p < 0.001) of ZBP1 expression.

7A is a PCA analysis of eicosanoid and derivatives thereof in the dorsal skin of mice.

Figure 7b shows the heat maps of 24 lipid species that are differentially regulated in Alox15 KO mice and the control group.

FIG. 7C shows the results of confirming the expression of skin RvD1, RvD3, 9-HODE and 13-OxoHODE in mice.

Fig. 8 shows the result of treating RvD2 with Alox15 KO mice. Fig. 8a shows improvement of hair loss after RvD2 treatment, Fig. 8b-c shows H / E staining of CD11b and immunofluorescence FIG. 8D shows the result of immunoblot analysis of the expression of cell death markers, and FIG. 8E shows quantitative analysis of immunoblot analysis (mean ± SEM; n = 4, * p <0.05, ** p <0.01 ).

Hereinafter, the present invention will be described in detail.

The present inventors have found that hair loss occurs in the knockout mouse model in which lipoxygenase 15 (Alox15) is deleted and that the structure of skin is destroyed. As a result, hair follicle stem cells decrease, , And found that skin adipose tissues undergo abnormal migration to fibroblasts. Deletion of Alox15 increased infiltration of proinflammatory macrophages and increased expression of proinflammatory and skin cell death signaling genes in dorsal skin. In particular, it was confirmed that Resolvin D2 in the back skin of Alox15 knockout mouse was significantly reduced. As a result of administering RvD2 to Alox15 knockout mouse, the present inventors have completed the present invention by confirming that the hair loss is improved, the hair loss is progressed, the expression of skin cell death marker is decreased, and the inflammatory index is improved.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating skin inflammation or hair loss comprising Resolvin D2 or a pharmaceutically acceptable salt thereof as an active ingredient.

The term " resorcin "of the present invention refers to an inflammatory clearing lipid mediator produced by the body from omega-3 fatty acids, eicosapentaenoic acid and docosahexaenoic acid. In particular, "Resolvin D2, RvD2" of the present invention is 7S, 16R, 17S-trihydroxy-DHA (7S, 16R, 17S-trihydroxy- DHA) represented by the following formula: DHA (Docosahexaenoic acid) Lt; / RTI &gt;

[Chemical Formula]

In the present invention, the skin inflammation or alopecia may be inflammation due to low expression or deletion of the Alox 15 gene.

In addition, the skin inflammation according to the present invention may be an inflammation occurring in the skin fat layer due to the low expression or deletion of Alox15.

In the present invention, the skin inflammation may be chronic skin inflammation.

The term "hair loss" of the present invention means a phenomenon in which hair completely escapes from the scalp. A person undergoing hair loss has a short growing period and a long dormant hair cycle, as demonstrated in the following examples, RvD2 converts hair from resting to growing, inhibiting hair growth and wool-related growth factors It was confirmed that hair loss, which increases expression, is prevented, improved or treated.

In the present invention, the hair loss may be caused by inflammation of the skin fat layer.

In the present invention, the terms &quot; pharmaceutically acceptable salt &quot;, &quot; cosmetically acceptable salt &quot;, or &quot; salt thereof &quot; may be an acid addition salt formed by a free acid. Acid addition salts can be prepared in a conventional manner, for example, by dissolving the compound in an excess amount of an aqueous acid solution and precipitating the salt with a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It is also possible to heat an equimolar amount of the compound and an acid or alcohol (e.g., glycol monomethyl ether) in water, and then evaporate the mixture to dryness, or the precipitated salt may be subjected to suction filtration.

RvD2 of the present invention can be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihalogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluene sulfonate, chlorobenzene sulfoxide But are not limited to, naphthalene-1-yne, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, Sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be obtained by a conventional method, for example, by dissolving RvD2 in an excess amount of an aqueous acid solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile . Heating the same amount of RvD2 and an acid or alcohol in water, and then evaporating and drying the mixture, or by suction filtration of the precipitated salt.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

In addition, the RvD2 of the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates which can be prepared by conventional methods.

The addition salt according to the present invention may be prepared by a conventional method. For example, RvD2 may be dissolved in a water-miscible organic solvent such as acetone, methanol, ethanol, acetonitrile, etc. and added with an excess amount of an organic acid, Followed by precipitation or crystallization. Subsequently, in this mixture, a solvent or an excess acid is evaporated and then dried to obtain an additional salt, or the precipitated salt can be produced by suction filtration.

When the composition of the present invention is used as a medicament, the pharmaceutical composition containing RvD2 or a pharmaceutically acceptable salt thereof as an active ingredient may be formulated into various oral or parenteral dosage forms at the time of clinical administration, , But is not limited thereto.

Examples of the formulations for oral administration include tablets, pills, light / soft capsules, liquids, suspensions, emulsions, syrups, granules and elixirs. These formulations may contain, in addition to the active ingredient, a diluent (e.g., lactose, dextrose, (E.g., silica, talc, stearic acid and magnesium or calcium salts thereof and / or polyethylene glycols), such as, for example, water, rosin, sucrose, mannitol, sorbitol, cellulose and / or glycine. The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine and may optionally contain additives such as starch, agar, alginic acid or its sodium salt A disintegrating or boiling mixture and / or an absorbent, a colorant, a flavoring agent, and a sweetening agent.

The pharmaceutical composition containing RvD2 of the present invention or its pharmaceutically acceptable salt as an active ingredient can be administered parenterally, and parenteral administration can be carried out by injecting subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection And. At this time, in order to formulate parenteral administration, RvD2 or a pharmaceutically acceptable salt thereof may be mixed with water or a stabilizer or a buffer to prepare a solution or suspension, which may be prepared into an ampoule or vial unit dosage form . The compositions may contain sterilized and / or preservatives, stabilizers, wettable or emulsifying accelerators, adjuvants such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, Or may be formulated according to the coating method.

The dose of the compound of the present invention to the human body may vary depending on the patient's age, weight, sex, dosage form, health condition, and disease severity. In general, when referring to an adult patient weighing 60 kg, And may be 0.01 to 500 mg / day, preferably 0.01 to 500 mg / day, and may be administered once or several times a day at regular intervals according to the judgment of a doctor or pharmacist.

The present invention also provides a quasi-drug composition for preventing or treating skin inflammation or hair loss comprising RvD2 or a pharmaceutically acceptable salt thereof as an active ingredient. The specific contents of RvD2 are as described above.

The term "quasi-drug product" used in the present invention means products that are less active than drugs, among the products used for diagnosing, treating, improving, alleviating, treating or preventing diseases of human or animal. For example, Quasi-drugs are products that are used for the purpose of treating or preventing diseases of humans or animals, products that are mild or have no direct action on the human body.

When RvD2 of the present invention is used as an active ingredient of a quasi-drug product composition, RvD2 which exhibits skin inflammation prevention or treatment, prevention of hair loss, and hair growth promoting effect can be directly added or used together with other quasi-drugs or quasi drug components, And the like. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

The quasi-drug composition of the present invention is not particularly limited in its formulation and may be variously formulated in the form of quasi-drugs known in the art. The quasi-drug composition of the present invention is used for the purpose of preventing or treating skin inflammation or hair loss, and is not particularly limited in its formulation. Examples of the quasi-drug composition include softening longevity, nutritional lotion, massage cream, , A mask sheet, a gel or a skin adhesive type cosmetic composition, and may be a transdermal dosage form such as lotion, ointment, gel, cream, patch or spray. In addition, the formulated quasi-drugs can be used in various forms such as scalp tonic, scalp lotion, scalp cream, scalp serum, scalp essence, scalp ampoule, scalp treatment, scalp conditioner, scalp shampoo, scalp pack, hair tonic, , Hair mousse, hair gel, hair conditioner, hair shampoo, hair conditioner, hair pack, hair treatment, eyebrow hair extender, eyelash hair extender, eyelash hair conditioner, eyelash nourishing agent, pet shampoo, pet rinse, hand cleaner, detergent soap, A wetting agent, a mask, an ointment agent, a patch or a filter filler, and includes quasi-drugs in a conventional sense.

In addition, the quasi-drug composition in each formulation can be blended by arbitrarily selecting other components according to the formulation of the other quasi-drugs, the purpose of use, and the like. The mixing amount of the active ingredient can be appropriately determined according to the intended use (inhibition or relaxation). For example, conventional adjuvants such as thickeners, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers and the like.

The content of the composition is preferably 0.0001 to 10% by weight based on the total weight of the composition. If it is more than 10% by weight, the color and stability of the composition are poor. When the composition is less than 0.0001% by weight, . The quasi-drug composition containing RvD2 of the present invention as an active ingredient has little toxicity and side effects to cells as confirmed by the treatment index, and thus can be useful as a quasi-drug material.

The present invention also provides a cosmetic composition for preventing or ameliorating skin inflammation or hair loss comprising RvD2 or a cosmetically acceptable salt thereof as an active ingredient.

The components included in the cosmetic composition of the present invention include components commonly used in cosmetic compositions in addition to RvD2 or its pharmaceutically acceptable salts as an active ingredient, and examples thereof include antioxidants, stabilizers, solubilizers, vitamins, , And a carrier.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters.

The present invention also provides a method of preventing or treating skin inflammation or hair loss comprising administering to a subject lesolin D2 or an acceptable salt thereof.

The present invention also provides a method of preventing or treating skin irritation or depilation of a composition comprising administering to a subject lesolin D2 or an acceptable salt thereof.

The pharmaceutical composition or cosmetic composition comprising RvD2 or an acceptable salt thereof according to the present invention as an active ingredient is useful as an agent for inhibiting the expression or deletion of the Alox15 gene and for alopecia caused by the Rox3 receptor-interactiong serine / threonine-protein kinase 3), p-RIP3 (phosphorylated-RIP3) or ZBP1 (Z-DNA binding protein 1) .

Below, The present invention will be described in more detail by way of examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

<Experimental Method>

1. Experimental animals

All animal tests were carried out according to regulations set by the Korea Food and Drug Administration and Yonsei University. During the experiment, the mice were fed a regular solid chow with water and maintained in a 22 ° C, 12 hour night and day cycle environment.

Alox15 KO mice (JAX Mice Stock # 002778, Bar Harbor, ME, USA) were purchased and reared. Male C57BL / 6 mice 5-6 weeks old were purchased from Orient Biotech and used as a normal phenotype control. AdipoqCreER mice (JAX Mice Stock # 024671: B6.129-Tg (Adipoqcre / Esr1 *) 1Evdr / J) and tdTomato mice (JAX MICE Stock # 007914: B6.Cg- Gt (ROSA) 26Sortm9 (CAG-tdTomato) Hze / J) were purchased, Alox15 ^{- / -} KO mice crossed with Alox15 ^- was prepared adipoqCreER_tdTomato triple transgenic mice ^{- /.} For Cre transformation, sunflower seed oil (Sigma, 50 mg / kg body weight) in which tamoxifen was dissolved in 4-week old mice was taken daily for 5 days. Alox15 + / + (WT) / adipoqCreER_tdTomato mice were used as WT controls. For RvD2 (Cayman) treatment, 8 wk old WT mice and Alox15 KO mice were injected intraperitoneally with RvD2 at 25 ng / kg for 2 weeks once every other day.

2. Gene Expression Analysis

RNA was extracted using TRIzol® reagent (Invitrogen, Carlsbad, Calif., USA), and 1 μg of RNA reverse transcription was performed using a cDNA synthesis kit (High-capacity cDNA Reverse Transcription Kit; Applied Biosystems, Foster City, Respectively. (IQ SYBR Green Supermix; Bio-Rad, Hercules, Calif., USA) with 100 nM cDNA in a reaction volume of 20 μL with 100 nM of primer, SYBR Green dye and CFX Connect Real- (Kwon HJ et al., Cell Death & Disease 2016, 7 (6)) were used as a housekeeping gene in the mRNA expression analysis, : e2285).

3. Western blot analysis

10 μg of sample was electrophoresed and blotted onto PVDF membrane (Bio-Rad, Hercules, Calif., USA). Western blot was used to detect the presence of 15-lipoxygenase-1 (Mouse, Abcam, Cambridge, UK), PLIN1 (Rabbit, Cell Signaling, Danvers, MA, USA), Keratin 14 (Rabbit, Cell Signaling), phospho-RIP3 The primary antibody and the secondary anti-mouse (RIB3) to RIP3 (Rabbit, Cell signaling), ZBP1 (Rabbit, Cell Signaling), Tubulin (Cell Signaling), and β-actin (Mouse, Santa Cruz Biotechnology, Dallas, And anti-rabbit horse-radish peroxidase (HRP) antibody (Cell signaling). The results were visualized using SuperSignal West Dura Substrate (ThermoFisher Scientific).

4. Flow cytometry

For flow cytometry, a total of 500,000 collagenase degraded cell surface markers were incubated with AlexaFluor 488 or anti-CD4b conjugated with FITC (fluorescein isothiocyanate) conjugated with APC, and Alexa Fluor 488 or APC (Rat, 1: 200, Biolegend, San Diego, Calif., USA). 30,000 samples were analyzed in 500,000 cells / 500 μl PBS. Flow cytometry was performed using BD FACSAria III (BD Biosciences, San Jose, Calif., USA) and raw data were processed using FlowJo software (Tree Star, Ashland, OR, USA).

5. Histological analysis

The back skin of the mice was cut into histological sections and H / E staining or immunohistological analysis was performed with a 5-μm thick paraffin section. Antibodies used for immunochemical detection were anti-15-lipoxygenase-1 antibody (Abcam), Collagen I (Abcam), Perilipin A (Cell Signaling), Keratin 14 (Cell Signaling), Keratin 15 (Cell Signaling), PDGFRA systems, Smooth muscle actin-FICT (Sigma), HMGB1 (Cell Signaling), tdTomato (Clonetech), Ly6C (Biolegend) and F4 / 80 antibody (AbD Serotec). Alexa Fluor 488, donkey anti-goat-Alexa Fluor 594, and donkey anti-Alexa Fluor 594 (ThermoFisher Scientific, Molecular Probes ) Was used.

Missing primary antibody or common rabbit, rat, goat or mouse IgG control (Santa Cruz Biotechnology) was used as a negative control. DAPI (Sigma) was used for contrast dyeing of the nuclei and the cells were visualized by Zeiss confocal laser-scanning microscope (LSM 710 META, Zeiss, Jena, Germany).

6. Lipidomics analysis

Tissue samples were ground (3 X 10 sec) by probe sonication in ice. The cell lysates were analyzed by LC-MS-based lipid analysis to determine the lipid content of fatty acids (Lee YH et al., American Journal of Physiology, 2016; 310 (1): R55-65). Lipid extraction and LC-MS / MS analysis were performed at the Pathology Department of Wayne State University. Resolvin D2 ELISA Kit (Cayman Chemical) was used to measure the level of RvD2 expression in the skin.

7. Statistical Analysis

Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA). Data values were expressed as means ± SD (mean ± S.E.M), and statistical significance between the two groups was determined by unpaired t-test. When comparing multiple groups, two-way ANOVA was used with Bonferroni post hoc tests to determine the associated p-value. Principal component analysis (PCA) was performed using XLStat software (Addinsoft, New York, NY) and Heatmap was generated using the PermutMatrix program.

Example 1. Local expression of Alox15 in dorsal skin

To determine the role of Alox15 in skin phenotype, we examined the expression level of Alox15 in the dorsal skin of mice. Specifically, the expression of Alox15 in epithelial / hair follicle and dermal adipose tissues was confirmed by histological analysis (H / E staining and immunofluorescence staining). As a result, Alox15 was not observed in all dermal layers of Alox15 knockout mouse (Fig. 1).

Immunoblot analysis of Alox15 was also performed in the adipose tissue layer of dermis and dermis and brown adipose tissue. Perilipin (PLIN1) and keratin 14 (keratin 14) were used as adipocytes and skin-specific markers, respectively. As a result, it was found that Alox15 was highly expressed in the dorsal skin and dermal fat layer as compared with brown adipose tissue (Fig. 2).

Example 2. Confirmation of hair loss in Alox15 knockout mouse

Observation of the Alox15 knockout mouse over 16 weeks revealed that hair loss (> 1 cm &lt; ² &gt;) was noticeable compared to the wild type model (Fig. 3).

Immunostaining of follicular stem cell markers revealed that keratin 15 + follicular stem cells were reduced in Alox15 KO mice, whereas keratin 14+ cells were increased (Fig. 4). The present inventors also observed histological analysis of thickening of the epidermal layer or increase of the myeloid cells in the dermal fat tissue layer. From these results, the integrity of the skin layer was destroyed by the decrease of Alox15 expression and the hair follicle growth cycle And it became abnormal.

Example 3: Increased inflammation in Alox15 knockout mice

It is known that inflammation is a major mechanism underlying skin disease, and Alox15 is involved in the production of anti-inflammatory and inflammatory lipid mediators (pro-resolving lipid mediators). Immunohistochemical analysis revealed that F4 / 80 + macrophages and Lys6C + proinflammatory monocytes were increased in the dorsal skin of the Alox15 knockout model (Fig. 5a, b). Flow cytometry analysis also confirmed that the infiltration of CD11b + and Ly6C + myeloid cells, which are important in chronic inflammatory skin diseases such as psoriasis, is increased (Fig. 5c, d).

The AdipoqCreER / Rosa26-loxP-stop-loxP-tdTomato system was used to track adipose cells in the dorsal skin of Alox15 knockout mice. Adipocyte- expressing adipocytes in this system were labeled with the red fluorescent protein tdTomato after treatment with tamoxifen. TdTomato expression was observed only in dermal adipocytes and not in myofibroblasts in the wild type control group. However, tdTomato + cells were observed in the skin of Alox15 KO mouse, which indicates adipocytes that are differentiated into myoblasts (Fig. 5e).

These double-positive fibroblasts are mainly surrounded by hair follicles in the dermal fat layer. The expression profile of the proinflammatory gene markers (Tnfa, IL1 [beta], Emr1) and fibrogenic markers (Fibronectin Tgfb1, Tgfb3) was increased through the dorsal skin expression profile of Alox15 mice (Fig. 5f).

Example 4. Confirmation of cell necrosis increase in dermal layer of Alox15 KO mouse

Since inflammation without infection results in cell dysfunction and apoptosis, the present inventors confirmed the expression of necroptosis markers. HMGB1 (high-mobility group box 1 protein) is a nucleoprotein that regulates apoptosis. It moves from the nucleus to the cytoplasm and is released from the cell by the cell damage signal.

HMGB1 expression was confirmed, indicating that the expression of HMGB1 was highly expressed in the cytoplasm due to apoptosis of dermal cells of Alox15 KO mice (FIG. 6A). Immunoblot analysis showed that phosphorylation of RIP3 (receptor-interaction serine / threonine-protein kinase 3) and expression of ZBP1 (Z-DNA binding protein 1) were increased in Alox15 KO mice (FIG. These results suggest that cell death is increased in the dorsal skin of Alox15 KO mice.

Example 5. Confirmation of therapeutic effect of RvD2 on skin inflammation

The key function of Alox15 is to produce lipid mediators of arachidonic acid and other fatty acids. The LC-MS / MS lipidome analysis of the skin revealed 90 lipid species and the principal component analysis (PCA) showed the lipid metabolite profile of Alox15 mouse. The lipid profile was separated by the secondary component factor (F2) of the principal component analysis, accounting for 21.47% of the total variance. Cluster analysis clearly showed the skin lipid profiles of WT and Alox15 KO mice (Fig. 7A). RvD2 and 9-HODE were identified as major components in the total data, and it was confirmed that the expression of Alox15 KO mice was remarkably decreased in the dorsal skin (Fig. 7 (b), (c)).

To determine if RvD2 could restore skin damage to Alox15 KO mice, Alox15 KO mice were treated with RvD2. As a result, as shown in Fig. 8, the hair began to come back to the hair loss part by RvD2 (Fig. 8A), and the integrity of the skin was restored (Fig. 8B). In addition, it was confirmed that the infiltration of CD11b + myeloma cells was reduced after treatment with RvD2 (FIG. 8c), and the cell death killing was inhibited (FIG. 8d).

Claims (16)

1. A pharmaceutical composition for preventing or treating skin inflammation or hair loss comprising Resolvin D2 or a pharmaceutically acceptable salt thereof as an active ingredient.
2. The method according to claim 1,

   Wherein said skin inflammation or depilation is due to the low expression or deletion of the &lt; RTI ID = 0.0 &gt; Alox15 &lt; / RTI &gt; gene.
3. The method according to claim 1,

   Wherein the skin inflammation is inflammation occurring in the skin fat layer.
4. The method according to claim 1,

   Wherein the skin inflammation is chronic skin inflammation.
5. The method according to claim 1,

   Wherein said hair loss is caused by inflammation of the skin fat layer.
6. The method according to claim 1,

   Wherein the composition reduces expression levels of RIP3 (receptor-interaction serine / threonine-protein kinase 3), p-RIP3 (phosphorylated-RIP3) or ZBP1 (Z-DNA binding protein 1).
7. A quasi-drug composition for preventing or treating skin inflammation or hair loss comprising Resolvin D2 or a pharmaceutically acceptable salt thereof as an active ingredient.
8. 8. The method of claim 7,

   Wherein said skin inflammation or depilation is due to low expression or deletion of the Alox15 gene.
9. A cosmetic composition for preventing or ameliorating skin inflammation or hair loss comprising Resolvin D2 or a cosmetically acceptable salt thereof as an active ingredient.
10. 8. The method of claim 7,

    Wherein the skin inflammation or depilation is due to the low expression or deletion of the &lt; RTI ID = 0.0 &gt; Alox15 &lt; / RTI &gt; gene.
11. A method for preventing or treating skin inflammation or hair loss comprising administering to a subject an effective amount of Resolvin D2 or an acceptable salt thereof.
12. 12. The method of claim 11,

    Wherein the skin inflammation or alopecia is caused by low expression or deletion of the lipoxygenase 15 (Alox15) gene.
13. 12. The method of claim 11,

    Wherein the hair loss is caused by inflammation of the skin fat layer.
14. A method for preventing or treating skin inflammation or hair loss in a composition comprising administering Resolvin D2 or an acceptable salt thereof to a subject.
15. 15. The method of claim 14,

    Wherein the skin inflammation or depilation is due to the low expression or deletion of the Alox 15 gene.
16. 15. The method of claim 14,

    Wherein said hair loss is caused by inflammation of the skin fat layer.

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