WO2014208868A1 - Composition for preventing or treating psoriasis, containing bmp6, and dietary supplement for preventing or alleviating psoriasis, containing bmp6 - Google Patents

Abstract

The present invention relates to a composition for preventing or treating psoriasis, containing BMP6 as an active ingredient, and a dietary supplement for preventing or alleviating psoriasis, containing BMP6. If using the present invention, it is possible to remarkably increase the psoriasis treatment efficiency by targeting a keratin cell.

Description

Composition for preventing or treating psoriasis containing BMP6 and dietary supplement for preventing or improving psoriasis containing BMP6

The present invention relates to a composition for preventing or treating psoriasis containing BMP6 (Bone Morphogenetic Protein 6; bone forming protein 6) and a health functional food for preventing or improving psoriasis containing BMP6.

Psoriasis is a chronic recurrent skin inflammatory disease with erythematous papules covered with silvery white scales. About 3% of the world's population (125 million people) are psoriasis patients and 2% of the population in Korea. Psoriasis is characterized by abnormal histological differentiation of the stratum corneum and is one of the representative chronic inflammatory skin diseases that is still difficult to treat. Therefore, the demand for psoriasis treatments is continuously increasing.

Steroids, which are widely used as a treatment for traditional skin diseases, cause side effects such as diabetes and hypertension, and have been a social problem due to their misuse. Recently developed immunomodulators act safely on specific targets to safely immunize. Antibody preparations that were expected to cause inhibitory actions have been reported to cause unexpected and serious side effects such as immunological disorders, infections, cancers, and the like. Thus, a clearer mechanism of psoriasis development and the selection of new targets were required.

HIF-1, which is reported to have increased expression in psoriasis skin tissue, is a transcription factor that maintains intracellular homeostasis by inducing glycolysis, angiogenesis, etc. in order to respond appropriately to changes in external oxygen concentrations in hypoxia. to be. Among them, HIF-1α is a transcription factor of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) structure, which is a skin produced by hypoxic state or genetic binding. Increasing HIF-1α expression and activity in keratinocytes is known to promote skin diseases such as psoriasis by activating dependent signaling systems, inducing proliferation of keratinocytes and promoting blood vessel formation around the epidermis (C. Rosenberger). et al, Journal of Investigative Dermatology , 127: 2445-52, 2007). In addition, the expression of HIF-1α in the tissues of psoriasis patients was confirmed by immunohistochemical staining and reported to be increased compared to normal tissues (M. Ioannou et al, Journal of Cutaneous Pathology , 36 (12): 1255-). 61, 2009). However, the role of overexpression of HIF-1α in the development of psoriasis is not known at all.

In addition, there are about 20 subtypes of bone morphogenetic proteins (BMPs), and they exhibit numerous biological responses in various cellular tissues of developmental stages and adults. BMPs are known to promote the differentiation and proliferation of keratinocytes in normal skin, to control melanoma forming activity, and to inhibit the onset of hair follicle growth.In lesional skin, BMPs are chronic as well as newly formed keratinocytes and skin fibroblasts after skin damage. It is known that the expression of BMP6 is increased in wound tissues.

In addition, it was reported that psoriasis-like skin was observed in transgenic mice overexpressing BMP6 using the K10 promoter. In this animal model, unlike the present invention, overexpression of BMP6 suppresses cell proliferation of the epidermal layer, and reports that the expression of BMP6 is weak and sparse, and the proliferation of basal keratinocytes is increased (Blessing M. et al. , The Journal of Cell Biology , 135 (1): 227-39, 1996). Accordingly, it is known in the art that BMP6 is overexpressed in the case of psoriasis, and on the contrary, treatment of BMP6 could not lead to the prevention or treatment of psoriasis.

Accordingly, the present inventors have made intensive efforts to solve the above problems, and as a result, BMP6 reduced the proliferation of keratinocytes increased upon overexpression of HIF-1 and completed the present invention.

Summary of the Invention

An object of the present invention is to provide a composition for preventing or treating psoriasis containing BMP6 and health functional food for preventing or improving psoriasis containing BMP6.

In order to achieve the above object, the present invention provides a psoriasis prevention or treatment composition containing BMP6 as an active ingredient and a health functional food for psoriasis prevention or improvement containing BMP6.

FIG. 1A shows the number of living cells with CCK-8 after overexpressing HIF-1 in keratinocytes, and FIG. 1B shows the number of living cells confirmed by fluorescence microscopy through calcein-AM staining. Image.

2A and 2B show the results of overexpressing HIF-1 in keratinocytes and analyzing the cell cycle using a flow cytometer after PI staining, and FIG. 2C shows Western blots of Examples 1-2. Expression of each cell cycle regulator identified as shown in Figure 2, D, E, F and G of Figure 2 shows the expression of differentiation markers confirmed by the real-time PCR method of Example 1-2, respectively K5, K10, FLG and LOR is shown.

3 shows a decrease in BMP6 expression due to overexpression of HIF-1, where A is a change in BMP6 mRNA and B is a change in BMP6 protein.

4A is a schematic diagram of the BMP6 promoter, and FIG. 4B shows the activity of the BMP6 promoter after overexpression of HIF-1.

5A shows the number of living cells with CCK-8 after BMP6 treatment to keratinocytes, and FIG. 5B is an image of living cells confirmed by fluorescence microscopy through calcein-AM staining on the cells. .

6A and 6B show the results of cell cycle analysis using a flow cytometer after PI staining after BMP6 treatment to keratinocytes, and FIG. Expression of cell cycle regulators is shown, D, E, F and G of Figure 6 shows the expression of the differentiation markers confirmed by the real-time PCR method of Example 2-2, respectively showing K5, K10, FLG and LOR will be.

FIG. 7A shows the number of living cells with CCK-8 after BMP6 treatment to HIF-1 overexpressed keratinocytes, and FIG. 7B shows fluorescence microscopy through calcein-AM staining. It is an image of confirmed living cells, C and D of Figure 7 shows the results of analyzing the cell cycle using a flow cytometer after PI staining at all times the cells.

Figure 8 shows the decrease in proliferation of keratinocytes and increased filaggrin expression by treatment with BMP6 in the mouse psoriasis model of Example 3.

Detailed Description of the Invention and Preferred Embodiments

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, the increase of keratinocytes and S-phase keratinocyte proliferation following HIF-1 overexpression, the decrease of BMP6 expression by HIF-1, and the reduction of keratinocytes and S-phase keratinocyte proliferation by BMP6 treatment were analyzed. As a result, it was confirmed that BMP6 reduces the proliferation of keratinocytes increased upon overexpression of HIF-1.

That is, in one embodiment of the present invention it was confirmed that BMP6 reduces the proliferation of keratinocytes increased when HIF-1 overexpression.

Therefore, in one aspect, the present invention relates to a composition for preventing or treating psoriasis containing BMP6 as an active ingredient.

In the present invention, the BMP6 reduces the proliferation of keratinocytes increased when HIF-1 overexpression.

In addition, in the present invention, the composition is characterized by containing a pharmaceutically acceptable carrier, the carrier is ion exchange resin, alumina, aluminum stearate, lecithin, serum protein, buffer material, water, salt, electrolyte , At least one selected from the group consisting of colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol and wool.

In the present invention, the composition is formulated for intravenous, intraperitoneal, intramuscular, intraarterial, oral, intracardiac, intramedullary, intradural, transdermal, intestinal, subcutaneous, sublingual or topical administration. The composition further comprises any one or more auxiliaries selected from the group consisting of buffers, antimicrobial preservatives, surfactants, antioxidants, tonicity modifiers, preservatives, thickeners and viscosity modifiers, wherein the composition comprises a solution, It is characterized by having a formulation selected from the group consisting of suspensions, emulsions, gels and powders.

In another aspect, the present invention relates to a psoriasis prevention or improvement health functional food containing the composition as an active ingredient.

As used herein, the term “composition” is considered to include not only products comprising specific components, but also any products made directly or indirectly by the combination of specific components.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intraperitoneal injection, intravenous injection, subcutaneous injection, intramuscular injection, or the like.

Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as the severity of the symptoms, the weight, age, sex, mode of administration, and time of administration of the patient, and the skilled practitioner is usually effective to administer the desired treatment or prevention. The amount can be easily determined.

In the present invention, the composition is characterized in that it contains 0.0001 to 100 mg per kg body weight of the treatment subject, preferably 0.01 to 50 mg BMP6 per kg body weight of the treatment subject.

The pharmaceutical composition of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier or excipient according to a method which can be easily carried out by those skilled in the art. It can be prepared by incorporation into a dose container. Such carriers are commonly used in the preparation, and include lactose, dextrose, sucrose, sorbitol, acacia rubber, calcium phosphate, gelatin, cellulose, water, syrup, methylhydroxybenzoate, talc, mineral oil, etc. It is not limited to this. The composition may include preservatives such as methyl paraoxybenzoate and potassium sorbate, natural flavors such as stabilizers, plum and lemon flavors, natural pigments such as chlorophyllin, sweeteners such as fructose, honey, sugar, dyes, anti-oxidants, and the like. It may further contain.

Preparations for parenteral administration of the pharmaceutical composition of the present invention include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like, and propylene glycol, olive oil, and the like for zero non-aqueous solvents or suspensions. The same vegetable oil, injectable esters such as ethyl oleate, and the like can be used, and as a base of suppositories, Utopepsol, macrogol, cacao butter, glycerogelatin and the like can be used, but are not limited thereto.

The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents.

The health functional food of the present invention means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to the Act No. 6722 of the Health Functional Food Act. It means a food consumed for the purpose of obtaining a useful effect for health use such as physiological action.

The dietary supplement of the present invention may be formulated into a formulation of a conventional dietary supplement known in the art, wherein the dietary supplement is a granule, tablet, pill, suspension, emulsion, syrup, gum, tea, jelly, Various beverages, drinks, alcoholic beverages and the like can be prepared, the type of health functional food is not particularly limited.

The nutraceuticals of the present invention may be in any form of herbal suitable for administration to an animal body, including the human body, more specifically in any form customary for oral administration, for example food or feed, additives and supplements of food or feed, fortification. Formulated foods or solid forms such as feeds, tablets, pills, granules, capsules and foam formulations and the like, or liquid forms such as solutions, suspensions, emulsions, beverages, pastes, and the like, nutritional supplements, vitamins, electrolytes, sweeteners, colorants, organic acids, preservatives And the like, and these components may be used independently or in combination.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Example 1 Regulation of BMP6 by HIF-1

1-1: Increased proliferation of keratinocytes following HIF-1 overexpression

In order to confirm the over-proliferation of keratinocytes, HIF-1 was overexpressed in keratinocytes, and the proliferation of keratinocytes was determined by CCK-8 (Ungbi, Korea) over time. The result of increasing the proliferation was confirmed (A of FIG. 1). Subsequently, on the fifth day of the experiment, the cells were easily absorbed into the cell by a hydrophobic substance that was not originally fluoresced, and entered the living cell, changed into calcein by the intracellular esterase, and became green fluorescence. Using a 2uM calcein acetomethyl ester (calcein acetoxymethyl ester; calcein-AM; Sigma, USA) can be confirmed after staining for 10 minutes at 37 ℃ (FIG. 1 B).

1-2: Increase of proliferative S-phase keratinocytes following HIF-1 overexpression

In order to confirm whether the proliferation of keratinocytes is related to cell cycle changes, after overexpressing HIF-1, keratinocytes were identified by flow cytometry analysis after propidium iodide (PI) staining. Specifically, transfection of pcDNA3-no insert and pcDNA-HIF-1α (P402A, P564A) was performed on human keratinocytes (Millipore, USA) using lipofectamine (lipofectamin 2000). After fixed for 1 hour in ethanol and washed, it was reacted for 1 hour in 0.5mg / ml RNase (Sigma, USA) solution at 37 ℃. The cell cycle was then analyzed by flow cytometry (BD, USA) by adding 10 ug / ml PI solution. As a result, it was confirmed that S-phase cells, which are proliferative cells, were increased in keratinocytes overexpressing HIF-1 (A and B of FIG. 2).

In addition, in order to confirm which cell cycle regulators increased the cell cycle, the expression of those involved in the cell cycle was confirmed by Western blot. Specifically, 48 hours after transfection of pcDNA3-no insert and pcDNA-HIF-1α (P402A, P564A) using lipofectamine (lipofectamin 2000) to human keratinocytes (Millipore, USA), RIPA buffer (Elpis) , Korea) and left to stand on ice for 30 minutes. Thereafter, the mixture was centrifuged at 13000 rpm for 30 minutes in a refrigerated centrifuge, and only the supernatant was taken. After quantification of the protein, 50 μg of protein was loaded on the SDS-PAGE gel. The gel loaded in PVDF was then transferred and blocked for 1 hour with 5% BSA, incubated after diluting CKD2, CDK4, CDK6 or Actin antibody (Cell Signaling, USA) at 1: 1000. After HRP-attached secondary antibody reaction and washing process, the band of the protein was confirmed using ECL solution. As a result, it was confirmed that the expression of CDK2, which is known to increase the expression of S phase, increased more than two times when HIF-1 overexpression (FIG. 2C). It can be seen that the differentiation and proliferation of keratinocytes is closely related and regulated.

To determine whether the expression of keratinocyte differentiation factors causes a change, keratin-10 (K10), filaggrin (FLG), which is known to increase expression during normal differentiation of keratinocytes by real time PCR assays ), Loricrin (LOR) and keratin-5 (K-5) expression was increased during keratin differentiation was confirmed. Specifically, 48 hours after transfection of pcDNA3-no insert and pcDNA-HIF-1α (P402A, P564A) using lipofectamine (lipofectamin 2000) to human keratinocytes (Millipore, USA), TRIzol reagent was added. After the fine grinding using a homogenizer (homogenizer) was isolated RNA. The isolated RNA was used to prepare cDNA of each tissue using reverse transcriptase (Super-bio, Korea). Using the prepared cDNA as a template, K5 primer-F (SEQ ID NO: 1) and K5 primer-R (SEQ ID NO: 2), K10 primer-F (SEQ ID NO: 3) and K10 primer-R (SEQ ID NO: 4), FLG primer -F (SEQ ID NO: 5) and FLG Primer-R (SEQ ID NO: 6), LOR Primer-F (SEQ ID NO: 7) and LOR Primer-R (SEQ ID NO: 8), GAPDH Primer-F (SEQ ID NO: 9), and GAPDH Primer PCR was carried out using -R (SEQ ID NO: 10) for 35 cycles of the denaturation step (95 ° C, 30 sec), annealing step (56 ° C, 30 sec) and extension step (72 ° C, 40 sec) as one cycle Amplified. As a result, it was found that the expression of FLG and LOR decreased during HIF-1 overexpression (D, E, F and G in FIG. 2). This result shows that aberrant differentiation of keratinocytes can be induced by HIF-1 overexpression in keratinocytes.

1-3: Targeting of HIF-1 by Microarray

The overexpression of HIF-1 altered not only the proliferation of keratinocytes but also the ability to regulate differentiation because it is assumed that HIF-1 targets differentiation factors. Targets were selected. RNA was extracted from keratinocytes transfected with HIF-1 plasmid and keratinocytes transfected with no insert plasmid, and microarray was performed using a 4x44k array chip (Agilent, Korea). As a result, expression in keratinocytes overexpressing HIF-1 was reduced by about half, BMP6, which has a HIF-1 binding site on the promoter, and is involved in the differentiation of keratinocytes, was selected as a target.

1-4: Confirmation of decreased expression of BMP6 gene by HIF-1

After transfecting the HIF-1 plasmid into keratinocytes, it was confirmed that the expression of BMP6 mRNA and protein was reduced by real-time PCR and Western blotting 24 hours after HIF-1 was overexpressed (FIG. 3). Western blot was the same as in Example 1-2 except that BMP6 (R & D, USA), HIF-1α (BD, USA) or actin antibody (Cell Signaling, USA) were diluted 1: 1000. In addition, for real-time PCR, except for using BMP6 primer-F (SEQ ID NO: 11) and BMP6 primer-R (SEQ ID NO: 12), GAPDH primer-F (SEQ ID NO: 9), and GAPDH primer-R (SEQ ID NO: 10). Is the same as Example 1-2.

1-5: Regulation of BMP6 by HIF-1

To confirm the regulation of BMP6 molecules reduced by HIF-1 overexpression, in bioinformatics, the presence of HIF-1 binding sites in the promoter of BMP6 gene was examined. As a result of analyzing the transcription factor binding to the BMP6 promoter using a matrix-inspector (Genomatix), it was confirmed that the HIF-1 binding site is present in the promoter of the BMP6 gene (FIG. 4A).

In addition, after transfection of keratinocytes with pcDNA3-no insert, pcDNA-HIF-1α (P402A, P564A) plasmid and pGL3-BMP6 promoter plasmid, luciferase (Promega, USA) activity was measured and transfected HIF was detected. As the amount of -1 increased, the promoter activity of BMP6 was found to be inferior (FIG. 4B). That is, it was found that HIF-1 reduces the amount of expression by regulating BMP6 transcription.

Example 2: Psoriasis Treatment by BMP6 Treatment

2-1: BMP6 Treatment Inhibits Cell Proliferation of Keratinocytes

After the BMP6 protein was treated in each concentration of keratinocytes, the degree of proliferation of cells over time was confirmed using CCK-8. As a result, it was found that the proliferation of keratinocytes was reduced depending on the concentration of BMP6 (FIG. 5A). In addition, the cells were confirmed by fluorescence microscopy through calcein-AM staining on the day 5 of the experiment (FIG. 5B).

2-2: Cell Cycle Inhibition of Keratinocytes by BMP6 Treatment

As a result of confirming that the decrease in the proliferation rate of keratinocytes by BMP6 treatment was associated with cell cycle inhibition, it was confirmed that the S-phase cells of the BMP6-treated group decreased as opposed to HIF-1 overexpression (FIG. 6). PI staining, western blot, and real-time P CR were performed in the same manner as in Example 1-2 except that the BMP6 protein was treated for 18 hours.

2-3: inhibition of proliferation by BMP6 treatment to keratinocytes increased by HIF-1

To determine whether the increased proliferation of keratinocytes during HIF-1 overexpression is a result of decreased BMP6 expression, the proliferation of keratinocytes increased during HIF-1 overexpression by treating BIF6 protein with HIF-1 overexpressing keratinocytes. It was confirmed that the decrease. As a result, it can be seen that the degree of proliferation decreases upon treatment with BMP6 (A and B of FIG. 7).

In addition, as a result of confirming that the reduction of proliferation of HIF-1 overexpressing keratinocytes by BMP6 treatment was associated with cell cycle inhibition, it was found that the S phase cells of the BMP6 treated group decreased (FIG. 7C and D).

Example 3: Psoriasis treatment efficacy verification experiment of BMP6 protein

In order to verify the efficacy of psoriasis treatment of BMP6 protein, an animal model in which psoriasis was induced by applying imiquimod (IMQ) to mouse skin was used. 62.5 mg IMQ cream (sigma, USA) per day was applied to the back of 5 week old Balb / c mice for 9 days to induce psoriasis-like skin. Thereafter, 5 ug of BMP6 protein was injected subcutaneously at the IMQ application site for 7 days from the 7th day of IMQ treatment, and PBS was injected subcutaneously at the IMQ application site as a control.

The results were analyzed by immunohistochemical staining. Specifically, the skin tissue was collected at the expense of the mouse, fixed in 4% formaldehyde (sigma, USA) and embedded in paraffin to prepare a slide. After the tissue slides were subjected to deparinization using xylene and dehydration using alcohol, the slides were immersed in citrate buffer, rotated in a microwave for 10 minutes, and cooled at room temperature for 1 hour. After washing with distilled water and reacting with 5% goat serum at room temperature for 1 hour, the filaggrin antibody was diluted 1:50 and incubated. After HRP-attached secondary antibody was reacted and developed using DAB kit to confirm the change in the expression of filaggrin in the tissue.

As a result, the group treated with IMQ and PBS was found to have significantly increased thickness of the stratum corneum compared to the group treated with PBS, resulting in psoriasis. In addition, the groups treated with IMQ and BMP6 reduced the thickness of the stratum corneum by about one-half compared to the groups treated with IMQ and PBS. It was confirmed that the expression of the green is increased (Fig. 8).

Therefore, it was found that treatment of psoriasis tissues with BMP6 suppresses the proliferation of keratinocytes and shows psoriasis treatment efficacy.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

In the case of using the composition containing BMP6 according to the present invention as an active ingredient, by targeting keratinocytes, the treatment efficiency of psoriasis can be improved as compared with conventional therapeutic agents targeting immune cells, which is very useful for treating psoriasis.

Electronic file attached.

Claims (7)

1. A composition for preventing or treating psoriasis containing BMP6 (Bone Morphogenetic Protein 6) as an active ingredient.
2. The composition for preventing or treating psoriasis according to claim 1, wherein the BMP6 reduces proliferation of keratinocytes increased when HIF-1 is overexpressed.
3. The composition for preventing or treating psoriasis according to claim 1, which contains a pharmaceutically acceptable carrier.
4. 4. The carrier of claim 3 wherein the carrier is ion exchange resin, alumina, aluminum stearate, lecithin, serum protein, buffer material, water, salt, electrolyte, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose based substrate , Polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol, and wool, at least one selected from the group consisting of psoriasis prevention or treatment composition for the treatment.
5. The method of claim 1, wherein the formulation is for intravenous, intraperitoneal, intramuscular, intraarterial, oral, intracardiac, intramedullary, intradural, transdermal, intestinal, subcutaneous, sublingual or topical administration. Therapeutic composition.
6. The composition for preventing or treating psoriasis according to claim 1, further comprising any one or more adjuvants selected from the group consisting of buffers, antimicrobial preservatives, surfactants, antioxidants, tonicity modifiers, preservatives, thickeners and viscosity modifiers. .
7. Health functional food for psoriasis prevention or improvement containing BMP6 as an active ingredient.

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