KR101600032B1 - A novel peptide with inhibiting BMP activity and use of the same - Google Patents

Abstract

The present invention relates to the preparation and activity of Noggin-derived peptides. The noggin-derived peptides of the present invention have the same or similar functions as natural noggin and have better thermal stability, better stability in aqueous solution, and superior skin permeability. That is, the present invention effectively inhibits bone morphogenetic protein (BMP), which is a cause of hair loss, and shows an excellent effect in the treatment, prevention or improvement of hair loss. In addition, the present invention solves the problem that a lot of time and cost are consumed in the mass purification of dimeric natural ginseng as an E. coli transformant. Therefore, the excellent activity, stability, and economy of the Noggin-derived peptides can be effectively used for medicines, quasi-drugs, and cosmetics as a hair loss therapeutic agent.

Description

A novel peptide having a BMP activity inhibiting function and a use thereof,

The present invention relates to Noggin-derived peptides using the amino acid sequence of Noggin protein. More particularly, it relates to peptides comprising a Noggin amino acid sequence that specifically binds to BMP (bone morphogenetic protein) to inhibit the activity of BMP.

Noggin is a protein expressed in the Nog gene (Brown, DJ et al., Am J Hu Genet , 71 (3): 618-624 (2002)), University of California-Berkeley, Harland and Smith Richard M. Harland and William C. Smith) first discovered Noggin in a secondary axis formation study of frog embryos (William, CS, and Richard, MH, Cell , 70: 829-840 (1992)). Noggin exists as a dimer in the human body and is 46 kDa in size. In addition, the main role of the currently known Noggin is to inhibit BMP and to bind BMP to the receptor at the site where BMP binds to the BMP receptor (Jay, G., et al., Nature 420: 636 -642 (2002)).

BMP is one of the growth factors that regulate cell growth and differentiation (Reddi, H. Cytokine & Growth Factor Reviews 8: 11-20 (1997)). There are 11 known types of BMPs (Xiao, YT, et al. Biochemical and Biophysical Research Communication 362: 550-553 (2007)) and BMP2 to BMP7 belong to the transforming growth factor-beta (Eskander, JM, and Kang, J. Am Acad Ortho Surgeons 20: 547-552). BMP is generally known to promote bone and cartilage production (Wozney, JM SPINE 27: S2-S8 (2002)) and plays a role in regulating body tissues to maintain correct morphology. BMPs regulate the formation of bone cells and tissues. When problems arise in signal transduction through BMPs, diseases such as osteoporosis, colon cancer, and esophagitis can develop (Kodach, LL, et al. Gastroenterology 134: 1332- 1341 (2008); Milano, F., et al., Gastroeneterology 132: 2412-2421 (2007)). However, recent studies have shown that BMP affects the cycle of hair and causes hair loss (Huelsken, J., et al. Cell Press 105: 533-545 (2001)).

BMP affects the hair follicle cycle and causes hair loss. The period from hair follicle production to excretion is called the cycle of hair. The most important factor affecting the cycle of hair is the presence or absence of beta-catenin. When beta-catenin is present, the stem cells in the bulge of the hair follicle migrate to the bulb in the lower part of the hair follicle, creating hair. However, when beta-catenin is absent, the stem cells in the bulge form a layer of the epidermis that thickens the scalp and consequently has a bad influence on hair loss.

The cycle of the hair follicle consists of five steps. The hair comes out of the hair follicle at the anagen stage. That is, the longer the growth phase is continued, the more the hair loss can be prevented. The cycle of the hair goes from the growing stage to the catagen stage. At the degenerative stage, the growth of the hair ends and the condition of the deformity persists for 2 to 3 weeks. The regenerator phase eventually enters the telogen stage. The dormant phase is also called the dead phase and is the stage where the hair is ready to go out. That is, the longer the hair follicle remains in the resting phase, the worse the hair loss will be. At this stage, if beta-catenin is present, the stem cells in the hair follicle will go down again to prepare for the formation of new hair, which is called the anagen onset stage. Upon entering the exogen stage, the newly created hair exits the existing dead hair and begins to grow again (Cotsarelis, G. The Journal of Clinical Investigation 116: 19-21 (2006)). When BMP binds to cell surface receptors, beta-catenin is degraded by the proteasome, leaving the hair follicle cycle in a resting state and adversely affecting hair loss. However, if the BMP is inhibited by Noggin, beta-catenin promotes hair growth by making the hair follicle cycle grow (Kawano, Y., and Kypta, R. Cell Science 116: 2627-2634 (2003)).

Currently, hair loss is caused by various factors such as genetic factors or aging or mental stress. Especially in males, hair follicles become smaller due to susceptibility to androgens and hair loss occurs. For women, hair loss can be caused by stress or aging, and hair loss can lead to mental stress, social and sexual self-confidence. However, there is no effective hair loss remedy that can cure these problems due to price or efficacy problems. Currently, minoxidil, pinasteride and the like are generally used as therapeutic agents, but they have only an effect of delaying hair loss and have no effect of promoting hair growth.

In the case of Noggin, it has the potential as a good hair loss treatment to promote hair growth. However, there is a limit to producing natural gene of dimer as Escherichia coli transformant, which is costly and time consuming for mass production. In addition, it is important to develop natural analogs that are low in skin permeability and water solubility, sensitive to temperature, and therefore capable of replacing them.

[Related Patent Literature]

Korean Patent Registration No. 10-0844515

Disclosure of the Invention The present invention solves the above problems and is accomplished by the above-mentioned need. It is an object of the present invention to prepare and screen the nongin-derived peptides having excellent thermal stability and stability in an aqueous solution.

It is another object of the present invention to prepare and screen small size-derived peptides to solve the problem of relatively low skin permeability of natural nongen proteins.

It is still another object of the present invention to prepare and screen relatively costly sogngin-derived peptides in order to solve the problem of a large amount of time and cost consumed in mass purification of natural nogin as an E. coli transformant.

The ultimate purpose of the present invention is to provide peptides that exhibit the same or similar activity as the BMP inhibitory protein.

In order to solve the above-mentioned object, the present invention provides a peptide of the following general formula (4) shown in SEQ ID NO: 4, which shows the activity of Noggin protein.

Formula 4

Ac-His-Tyr-Leu- His-Ile-Arg-Pro-Ala-Pro-Ser-Asp-NH 2

In one embodiment of the present invention, the peptide is preferably a peptide of the following formula 5 according to SEQ ID NO: 5 in which the first His is substituted with Arg and the sixth Arg is substituted with Met,

Formula 5

Ac-Arg-Tyr-Leu- His-Ile-Met-Pro-Ala-Pro-Ser-Asp-NH 2

In another embodiment of the present invention, the peptide is preferably a peptide of the following general formula 3 as shown in SEQ ID NO: 3 further comprising Asn-Leu-Pro-Leu at the Asp terminal,

Formula 3

Ac-His-Tyr-Leu- His-Ile-Arg-Pro-Ala-Pro-Ser-Asp-Asn-Leu-Pro-Leu-NH 2

In another embodiment of the present invention, the peptide is preferably a peptide of the following general formula (2) shown in SEQ ID NO: 2, further comprising Val-Asp-Leu at the 15th Leu end,

Formula 2

As-Leu-Val-Asp-Leu-NH2 < / RTI _>

In another embodiment of the present invention, the peptide is preferably a peptide of the following general formula (1) shown in SEQ ID NO: 1, which comprises Gln at the first His-site and further comprises Ile-Glu-His at the 18th Leu But not limited to this.

1

Ac-Gln-His-Tyr- Leu-His-Ile-Arg-Pro-Ala-Pro-Ser-Asp-Asn-Leu-Pro-Leu-Val-Asp-Leu-Ile-Glu-His-NH 2

In one embodiment of the present invention, the peptide binds to a bone forming protein to inhibit the activity of a bone forming protein, but is not limited thereto.

The present invention also provides a pharmaceutical composition for preventing or treating hair loss comprising the peptide of the present invention as an active ingredient.

The present invention also provides a cosmetic composition for preventing or treating hair loss comprising the peptide of the present invention as an active ingredient.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be prepared in a unit dosage form by formulating it with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs Or into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets, capsules or gels (e.g., hydrogels), and may additionally contain dispersing or stabilizing agents .

According to an embodiment of the present invention, the present invention provides a cosmetic composition comprising (a) a cosmetically effective amount of the peptide of the present invention as described above; And (b) an cosmetically acceptable carrier.

The term " cosmetically effective amount " as used herein means an amount sufficient to achieve the hair loss improving effect of the composition of the present invention described above.

The cosmetic composition of the present invention may be prepared in any form conventionally produced in the art and may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant- , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be prepared in the form of a shampoo, a flexible lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

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 .

In the case where 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. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / 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 composition, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether Alkylamido betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester.

The ingredients contained in the cosmetic composition of the present invention include, in addition to peptides and carrier components as an active ingredient, ingredients commonly used in cosmetic compositions and include conventional ingredients such as antioxidants, stabilizers, solubilizers, vitamins, Phosphorous assistant.

In the case of the hair growth promoting and / or hair loss preventing composition of the present invention, when the content of the effective ingredient in the composition is less than 0.00001%, hair growth enhancement and hair loss prevention effect can not be obtained effectively. In a preferred embodiment of the present invention, the hair growth enhancing and depilatory preventing composition of the present invention contains 0.00001 to 50% (ww), preferably 0.0001 to 5% (w / w), more preferably 0.0002 to 5% 1% (w / w).

Hereinafter, the present invention will be described.

In order to solve the above-mentioned problems, the present invention provides a crystal structure as shown in FIG. 1, wherein the active site of the Noggin that directly inhibits BMP is set, and the peptides including the sequence of the active site are prepared for each size.

The noggin-derived peptides according to the present invention showed the same activity as natural noggin and showed high safety. Therefore, through the production of products using Noggin-derived peptides, it is possible to produce high quality hair loss treatment products superior to the existing natural Noggin products during distribution and storage.

The nongin-derived peptides of the present invention have the same or similar activity as the native nongen. In addition, it is excellent in heat stability and safety in an aqueous solution state, and it is possible to produce the nitrogen-derived peptides with a relatively high skin permeability and relatively low cost as compared with the natural type.

1 shows a crystal structure in which a dimer nodine and a dimeric BMP are combined. The squares in the left figure are the parts where Nogin combines with the BMP, and the figure on the right is an enlarged view of the parts where the Nogin combines with the BMP. The blue loop in the figure on the right is a symbol and the yellow and red parts represent a dimeric BMP. The ring portion of the proline residue 37 of Noggin enters the pocket of the dimeric BMP and binds to the BMP by hydrophobic action (Groppe, J., and et al., Nature 420: 636-642 (2002)).
Fig. 2 shows the results of experiments on the activity of the native nongin and nongin-derived peptides against BMP inhibition under the conditions shown in Table 2.
FIG. 3 shows the result of measurement of circular dichroism at near-UV and far-UV conditions at 20 ° C and 95 ° C for N22 peptide.
FIG. 4 shows results of measurement of circular dichroism at near-UV and far-UV conditions at 20 ° C and 95 ° C for N18 peptide.
FIG. 5 shows the result of measurement of circular dichroism at near-UV and far-UV conditions at 20 ° C and 95 ° C for N15 peptide.
FIG. 6 shows the result of measurement of circular dichroism at near-UV and far-UV conditions at 20 ° C and 95 ° C for N11 peptide.
FIG. 7 shows results of measurement of circular dichroism at near-UV and far-UV conditions at 20 ° C and 95 ° C for N11 (A) peptide.

Examples and experimental examples are provided to facilitate understanding of the present invention. The following examples and experimental examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the examples and experimental examples.

≪ Example 1: Preparation of Noggin-derived peptide >

Synthesis Example 1: Synthesis of Ac-QHYLHIRPAPSDNLPLVDLIEH-NH ₂ (SEQ ID NO: 1)

10 mmole of Fmoc-Rink amide resin (GL Biochem Cat No. 49001) was placed in a reaction vessel, and 50 ml of methylene chloride (MC) was added thereto, followed by stirring for 20 minutes to prepare a peptide having the above sequence Then, the solvent was removed again. 60 ml of the deprotection solution (20% piperidine / DMF) was added to the reaction vessel and stirred for 30 minutes at room temperature. The solution was then removed and washed three times with DMF and once with MC. To the new reactor, 50 ml of DMF solution was added, followed by 15 mmole of Nsc-His-OH, 15 mmole of HoBt and 15 mmole of HBTU, followed by stirring and dissolution. The dissolved amino acid mixture solution was placed in a reaction vessel with the deprotected resin, and 20 mmole of DIPEA (N, N'-Diisopropyl ethylamine) was added thereto, followed by stirring at room temperature for 2 hours.

The reaction solution was removed, and the mixture was stirred with DMF solution three times for 5 minutes to remove unreacted residues. A small amount of the reaction resin was taken and the degree of reaction was checked using a Ninhydrine test. The deprotection solution was subjected to the same deprotection reaction twice as described above to prepare His-Rink amide resin. Washed with DMF and MC, once again subjected to the Kaiser test, and then subjected to the same experiment as described above. OH, Nsc-Leu-OH, Nsc-Pro-OH, Nsc-Leu-OH, Nsc-Asp-OH, Nsc- OH, Nsc-Pro-OH, Nsc-Arg-OH, Nsc-Asp-OH, Nsc-Pro-OH, Nsc- After chain reaction in the order of Ile-OH, Nsc-His-OH, Nsc-Leu-OH, Nsc-Tyr-OH, Nsc- Dean / DMF) was added to the reaction vessel and stirred for 30 minutes at room temperature. The solution was then removed and washed three times with DMF and once with MC. Acetylation reaction was carried out with stirring at room temperature for 30 minutes. DMF (20% -Acetic anhydride) was added and the solution was removed. The solution was removed, washed three times with DMF, three times with MC and dried with N 2 air to obtain Ac-Gln-His Asp-Leu-Val-Asp-Leu-Ile-Glu-His-Rink amidoresin.

The amide resin prepared was placed in a round bottom flask. 100 ml of a previously prepared solution (TFA 81.5%, distilled water 5%, thioanisole 5%, phenol 5%, EDT 2.5% and TIS 1%) was added and the reaction was kept for 2 hours with shaking at room temperature. After filtration of the resin by filtration, the resin was washed with a small amount of TFA solution and then combined with the mother liquor. The precipitate was distilled using a vacuum to distill the entire volume to about half, and 300 ml of cold ether was added to induce precipitation. The precipitate was collected by centrifugation and washed twice with cold ether. The mother liquor was removed and dried under nitrogen to obtain an acetyl-docosapeptide (Ac-Gln-His-Tyr-Leu-His-Ile- Arg-Pro-Ala-Pro-Ser-Asp-Asn- the Val-Asp-Leu-Ile- Glu-His-NH 2) was obtained. The pre-tablet acetyl-docosapeptide was purified via ID 5 cm preparative LC and lyophilized to give 2.81 g in powder form. As a result of HPLC analysis of the obtained powder, the acetyl-docosapeptide (Ac-Gln-His-Tyr-Leu-His-Ile- Arg-Pro-Ala-Pro- Ser-Asp-Asn- Val-Asp-Leu-Ile- Glu-His-NH 2) of a purity of 86.68% were, 2621.6 (theory is as measured by using a molecular weight measuring molecular weight values: was 2619.00).

Synthesis Example 2: Synthesis of Ac-HYLHIRPAPSDNLPLVDL-NH ₂ (SEQ ID NO: 2)

10 mmole of Fmoc-Rink amide resin (GL Biochem Cat No. 49001) was added to the reaction vessel, and 50 ml of MC was added thereto, followed by stirring for 20 minutes. Subsequently, Respectively. 60 ml of the deprotection solution (20% piperidine / DMF) was added to the reaction vessel and stirred for 30 minutes at room temperature. The solution was then removed and washed three times with DMF and once with MC. To the new reactor, 50 ml of DMF solution was added, followed by 15 mmole of Nsc-Leu-OH, 15 mmole of HoBt and 15 mmole of HBTU, followed by stirring and dissolution. The dissolved amino acid mixture solution was placed in a reaction vessel with deprotected resin, and 20 mmole of DIPEA was added thereto, followed by stirring at room temperature for 2 hours.

The reaction solution was removed, and the mixture was stirred with DMF solution three times for 5 minutes to remove unreacted residues. A small amount of the reaction resin was taken and the degree of reaction was checked using a Ninhydrine test. The reaction solution was subjected to the same deprotection reaction twice as described above to prepare a Leu-Rink amide resin. Washed with DMF and MC, once again subjected to the Kaiser test, and then subjected to the same experiment as described above. Asp-OH, Nsc-Leu-OH, Nsc-Pro-OH, Nsc-Leu-OH, Nsc-Asn-OH, Nsc- OH, Nsc-Pro-OH, Nsc-Pro-OH, Nsc-Pro-OH, Nsc-Arg-OH, Nsc- Tyr-OH and Nsc-His-OH in that order. 60 ml of deprotection solution (20% piperidine / DMF) was added to the reaction vessel and stirred for 30 minutes at room temperature. And washed once with MC. Acetylation reaction was carried out at room temperature for 30 minutes with DMF 60 ml (20% -Acetic anhydride), and the solution was removed. The solution was washed three times with DMF, three times with MC, and dried with N 2 air to obtain Ac-His-Tyr -Leu-His-Ile-Arg-Pro-Ala-Pro-Ser-Asp-Asn-Leu-Pro-Leu-Val-Asp-Leu-Rink amide resin.

The amide resin prepared was placed in a round bottom flask. 100 ml of a previously prepared solution (TFA 81.5%, distilled water 5%, thioanisole 5%, phenol 5%, EDT 2.5% and TIS 1%) was added and the reaction was kept for 2 hours with shaking at room temperature. After filtration of the resin by filtration, the resin was washed with a small amount of TFA solution and then combined with the mother liquor. The precipitate was distilled using a vacuum to distill the entire volume to about half, and 300 ml of cold ether was added to induce precipitation. The precipitate was collected by centrifugation and washed twice with cold ether. After removing the mother liquor and drying under nitrogen, the acetyl-octadecapeptide (Ac-His-Tyr-Leu-His-Ile-Arg-Pro-Ala-Pro-Ser-Asp-Asn- the Asp-Leu-NH ₂₎ was obtained. The pre-tablet acetyl-octadecapeptide was purified via ID 5 cm preparative LC and lyophilized to give 2.78 g in powder form. As a result of HPLC analysis of the powder obtained after purification, the acetyl-octadecapeptide (Ac-His-Tyr-Leu-His-Ile-Arg-Pro-Ala-Pro-Ser-Asp-Asn- Asp-Leu-NH ₂ ) was 86.68%, and the molecular weight was 2113.92 (theoretical value: 2111.45) when measured using a molecular weight meter.

Synthesis Example 3: Synthesis of Ac-HYLHIRPAPSDNLPL-NH ₂ (Sequence Listing 3)

10 mmole of Fmoc-Rink amide resin (GL Biochem Cat No. 49001) was added to the reaction vessel, and 50 ml of MC was added thereto, followed by stirring for 20 minutes. Subsequently, Respectively. 60 ml of the deprotection solution (20% piperidine / DMF) was added to the reaction vessel and stirred for 30 minutes at room temperature. The solution was then removed and washed three times with DMF and once with MC. To the new reactor, 50 ml of DMF solution was added, followed by 15 mmole of Nsc-Leu-OH, 15 mmole of HoBt and 15 mmole of HBTU, followed by stirring and dissolution. The dissolved amino acid mixture solution was placed in a reaction vessel with deprotected resin, and 20 mmole of DIPEA was added thereto, followed by stirring at room temperature for 2 hours.

The reaction solution was removed, and the mixture was stirred with DMF solution three times for 5 minutes to remove unreacted residues. A small amount of the reaction resin was taken and the degree of reaction was checked using a Ninhydrine test. The reaction solution was subjected to the same deprotection reaction twice as described above to prepare a Leu-Rink amide resin. Washed with DMF and MC, once again subjected to the Kaiser test, and then subjected to the same experiment as described above. Nsc-Pro-OH, Nsc-Pro-OH, Nsc-Asn-OH, Nsc-Asp-OH, Nsc- Nsc-Pro-OH, Nsc-Arg-OH, Nsc-Ile-OH, Nsc-His-OH, Nsc-Leu-OH, Nsc- Solution (20% piperidine / DMF) was added to the reaction vessel and stirred for 30 minutes at room temperature. Then, the solution was removed and washed three times with DMF and once with MC. Acetylation reaction was carried out at room temperature for 30 minutes with DMF 60 ml (20% -Acetic anhydride), and the solution was removed. The solution was washed three times with DMF, three times with MC, and dried with N 2 air to obtain Ac-His-Tyr -Leu-His-Ile-Arg-Pro-Ala-Pro-Ser-Asp-Asn-Leu-Pro-Leu-Rink amide resin.

The amide resin prepared was placed in a round bottom flask. 100 ml of a previously prepared solution (TFA 81.5%, distilled water 5%, thioanisole 5%, phenol 5%, EDT 2.5% and TIS 1%) was added and the reaction was kept for 2 hours with shaking at room temperature. After filtration of the resin by filtration, the resin was washed with a small amount of TFA solution and then combined with the mother liquor. The precipitate was distilled using a vacuum to distill the entire volume to about half, and 300 ml of cold ether was added to induce precipitation. The precipitate was collected by centrifugation and washed twice with cold ether. Purification by removing the mother liquid and dried under a nitrogen pre-acetyl-penta-deca-peptide (Ac-His-Tyr-Leu -His-Ile-Arg-Pro-Ala-Pro-Ser-Asp-Asn-Leu-Pro-Leu-NH 2 ). The pre-tablet acetyl-pentadecapeptide was purified via ID 5 cm preparative LC and lyophilized to give 2.90 g in powder form. The analysis of the powder obtained after purification by HPLC-acetyl-penta-deca-peptide (Ac-His-Tyr-Leu -His-Ile-Arg-Pro-Ala-Pro-Ser-Asp-Asn-Leu-Pro-Leu-NH 2 ) Was 83.8%. The molecular weight when measured using a molecular weight analyzer was 1786.39 (theoretical value: 1784.07).

Synthesis Example 4: Synthesis of Ac-HYLHIRPAPSD-NH ₂ (Sequence Listing 4)

10 mMole of Fmoc-Rink amide resin (GL Biochem Cat No. 49001) was added to the reaction vessel, and 50 ml of MC was added thereto, followed by stirring for 20 minutes. Subsequently, Respectively. 60 ml of the deprotection solution (20% piperidine / DMF) was added to the reaction vessel and stirred for 30 minutes at room temperature. The solution was then removed and washed three times with DMF and once with MC. In a new reactor, 50 ml of DMF solution was added, followed by 15 mmole of Nsc-Asp-OH, 15 mmole of HoBt and 15 mmole of HBTU, followed by stirring and dissolution. The dissolved amino acid mixture solution was placed in a reaction vessel with deprotected resin, and 20 mmole of DIPEA was added thereto, followed by stirring at room temperature for 2 hours.

The reaction solution was removed, and the mixture was stirred with DMF solution three times for 5 minutes to remove unreacted residues. A small amount of the reaction resin was taken and the degree of reaction was checked using a Ninhydrine test. Asp-Rink amide resin was prepared by the same deprotection reaction twice as the above-described deprotection solution. Washed with DMF and MC, once again subjected to the Kaiser test, and then subjected to the same experiment as described above. (Trt) -OH, Nsc-Pro-OH, Nsc-Ala-OH, Nsc-Pro-OH, Nsc-Arg-OH, Nsc- (20% piperidine / DMF) were added to the reaction vessel, and the mixture was reacted for 30 minutes in the presence of N, N, After stirring at room temperature, the solution was removed and washed three times with DMF and once with MC. Acetylation reaction was carried out at room temperature for 30 minutes with DMF 60 ml (20% -Acetic anhydride), and the solution was removed. The solution was washed three times with DMF, three times with MC, and dried with N 2 air to obtain Ac-His-Tyr -Leu-His-Ile-Arg-Pro-Ala-Pro-Ser-Asp-Rink amide resin.

The amide resin prepared was placed in a round bottom flask. 100 ml of a previously prepared solution (TFA 81.5%, distilled water 5%, thioanisole 5%, phenol 5%, EDT 2.5% and TIS 1%) was added and the reaction was kept for 2 hours with shaking at room temperature. After filtration of the resin by filtration, the resin was washed with a small amount of TFA solution and then combined with the mother liquor. The precipitate was distilled using a vacuum to distill the entire volume to about half, and 300 ml of cold ether was added to induce precipitation. The precipitate was collected by centrifugation and washed twice with cold ether. To give a undeca peptide (Ac-His-Tyr-Leu -His-Ile-Arg-Pro-Ala-Pro-Ser-Asp-NH 2) - the mother liquor and removed before drying under nitrogen to give acetyl. The pre-tablet acetyl-undeca peptide was purified via ID 5 cm preparative LC and lyophilized to give 2.92 g in powder form. The purity of the acetyl-undeca peptide (Ac-His-Tyr-Leu-His-Ile-Arg-Pro-Ala-Pro-Ser-Asp-NH ₂ ) was 88.95% The molecular weight when measured using a molecular weight meter was 1347.47 (theoretical value: 1346.52).

Synthesis Example 5: Ac-RYLHIMPAPSD-NH ₂ Synthesis of (Sequence Listing, SEQ ID No. 5)

10 mmole of Fmoc-Rink amide resin (GL Biochem Cat No. 49001) was put in a reaction vessel, and 50 ml of MC was added thereto, followed by stirring for 20 minutes The solvent was removed again. 60 ml of the deprotection solution (20% piperidine / DMF) was added to the reaction vessel and stirred for 30 minutes at room temperature. The solution was then removed and washed three times with DMF and once with MC. In a new reactor, 50 ml of DMF solution was added, followed by 15 mmole of Nsc-Asp-OH, 15 mmole of HoBt and 15 mmole of HBTU, followed by stirring and dissolution. The dissolved amino acid mixture solution was placed in a reaction vessel with deprotected resin, and 20 mmole of DIPEA was added thereto, followed by stirring at room temperature for 2 hours.

The reaction solution was removed, and the mixture was stirred with DMF solution three times for 5 minutes to remove unreacted residues. A small amount of the reaction resin was taken and the degree of reaction was checked using a Ninhydrine test. Asp-Rink amide resin was prepared by the same deprotection reaction twice as the above-described deprotection solution. Washed with DMF and MC, once again subjected to the Kaiser test, and then subjected to the same experiment as described above. (Trt) -OH, Nsc-Pro-OH, Nsc-Ala-OH, Nsc-Pro-OH, Nsc-Met-OH, Nsc- ), 60 ml of a deprotection solution (20% piperidine / DMF) was added to the reaction vessel, and the mixture was reacted for 30 minutes in the presence of NSC-Tyr-OH and Nsc- After stirring at room temperature, the solution was removed and washed three times with DMF and once with MC. Acetylation reaction was carried out at room temperature for 30 minutes with DMF 60 ml (20% -Acetic anhydride), and the solution was removed. The solution was washed three times with DMF, three times with MC and dried with N 2 air to obtain Ac-Arg-Tyr -Leu-His-Ile-Met-Pro-Ala-Pro-Ser-Asp-Rink amide resin.

The amide resin prepared was placed in a round bottom flask. 100 ml of a previously prepared solution (TFA 81.5%, distilled water 5%, thioanisole 5%, phenol 5%, EDT 2.5% and TIS 1%) was added and the reaction was kept for 2 hours with shaking at room temperature. After filtration of the resin by filtration, the resin was washed with a small amount of TFA solution and then combined with the mother liquor. The precipitate was distilled using a vacuum to distill the entire volume to about half, and 300 ml of cold ether was added to induce precipitation. The precipitate was collected by centrifugation and washed twice with cold ether. To give a undeca peptide (Ac-Arg-Tyr-Leu -His-Ile-Met-Pro-Ala-Pro-Ser-Asp-NH 2) - the mother liquor and removed before drying under nitrogen to give acetyl. The pre-purified acetyl-undeca peptide was purified via ID 5 cm preparative LC and lyophilized to give 3.01 g in powder form. The purity of the acetyl-undeca peptide (Ac-Arg-Tyr-Leu-His-Ile-Met-Pro-Ala-Pro-Ser-Asp-NH ₂ ) was 92.41% The molecular weight when measured using a molecular weight meter was 1339.24 (theoretical value: 1340.58).

SEQ ID NO: Amino acid sequence One Ac-QHYLHIRPAPSDNLPLVDLIEH-NH ₂ 2 Ac-HYLHIRPAPSDNLPLVDL-NH ₂ 3 Ac-HYLHIRPAPSDNLPL-NH ₂ 4 Ac-HYLHIRPAPSD-NH ₂ 5 Ac-RYLHIMPAPSD-NH ₂

≪ Example 2: Confirmation test of BMP2 inhibition of synthetic peptides >

For this experiment, 50 mL FBS (Thermo, Cat No. 21600-010) and 5 mL penicillin-streptomycin preservative (Thermo, Cat No. SV30010) were added to 445 mL of DMEM / High Glucose (Thermo. Cat No. SH30243.01) ) Is added to prepare a cell culture medium. Trypsin-EDTA buffer solution is prepared by adding 5 mL of trypsin 10X stock solution (Thermo, Cat No. SV30037.01) to 45 mL of PBS buffer (Gibco, Cat No. 21600-010). The composition of the ALP disruption buffer was 100 mM glycine (Cat No. 27185-0350), 1 mM MgCl ₂ (Kanto, Cat No. 25009-01), 0.5% Triton X-100 (purified gold, Cat. 8566-4400).

For this experiment, add 10 mL of cell-freezing solution of C ₂ C ₁₂ (ATCC no-CRL1772) cells and mix well. After centrifugation at 800 rpm for 10 minutes, remove the supernatant completely and add 10 mL of the cell culture solution again. Add this to a T175 flask, add 20 mL of the cell culture, and incubate for 48 hours in a carbon dioxide incubator (ASTEC, Cat No. SCI-165D) at 37 ° C. Remove the culture medium from the T175 flask, wash once with 10 mL PBS, and add 5 mL of Trypsin-EDTA. After incubating for 5 minutes in a 37 ° C carbon dioxide incubator, the adherent cells are washed out. To the T175 flask, add 10 mL of PBS, pipet the cells, and transfer the cells to a new 15 mL tube. Centrifuge at 800 rpm for 10 minutes to completely remove the supernatant, add 10 mL of cell culture, and mix well. Here, 20 μL of the sample is taken and the number of cells is confirmed using a hemocytometer. Based on the number of cells identified, the sample is diluted to 1 x 10 ⁵ / mL using cell culture medium. Prepare a 24-well plate, dispense 1 mL per well, and incubate for 12 hours at 37 ° C in a CO2 incubator.

BMP2 (Ostem Implant) and natural type (PnP-Biopharm), which are in a freeze-dried state, and 20 mM acetic acid are added to the above five peptides so that their respective concentrations become 1 mg / ml.

SEQ ID NO: BMP2: Noggin peptide
Reaction condition 1 BMP2: Noggin peptide
Reaction condition 2 BMP2: Noggin peptide
Reaction condition 3 One 100 ng: 100 ng 100 ng: 300 ng 100 ng: 1,000 ng 2 100 ng: 100 ng 100 ng: 300 ng 100 ng: 1,000 ng 3 100 ng: 100 ng 100 ng: 300 ng 100 ng: 1,000 ng 4 100 ng: 100 ng 100 ng: 300 ng 100 ng: 1,000 ng 5 100 ng: 100 ng 100 ng: 300 ng 100 ng: 1,000 ng

BMP2 and NOGIN-derived peptides were reacted for 1 hour in 4 mL of DMEM medium under the three conditions shown in [Table 2]. After removing all of the culture medium from the 24-well plate for 12 hours, the medium in which the BMP2 and the peptides were reacted was added to each well and reacted for 3 days in a carbon dioxide incubator at 37 ° C.

After removing the culture medium from the 24-well plate treated for 3 days, add 300 μL of ALP disruption buffer to each well and incubate at room temperature for 30 minutes. After centrifugation at 13,000 rpm for 10 minutes, 100 μL of the supernatant is mixed with 100 μL of pNPP liquid substrate (Sigma, Cat No. P7998-100ML) and transferred to a 96-well plate. After reacting for 2 hours in a 37 ° C carbon dioxide incubator, the absorbance is measured at 405 nm in a multireader (PerkinElmer, 1420 Multilable counter VICTOR3) (see FIG. 1).

As can be seen from Fig. 2, it was confirmed from the reaction condition 3 of [Table 2] that the Noggin-derived peptides had the most similar activity to the native noggin. In particular, NP18 and NP11 were found to effectively inhibit BMP2.

≪ Example 3: Stability test of Noggin-derived peptide through circular dichroism >

The peptides obtained in the above Synthesis Examples were subjected to a temperature and water stability test using a J-810 spectrometer (JASCO) (see FIGS. 2, 3, 4, 5 and 6).

The circular dichroism was measured at 20 ° C and 95 ° C under near-UV (250 nm to 190 nm) conditions after adjusting to pH 7.0 after dissolving the nongin-derived peptides in water to a concentration of 0.5 mg / ml. The detailed conditions are band width 1 nm, response 0.25 sec, data pitch 0.1 nm, scanning speed 20 nm / min, cell length 0.1 cm, accumulation 8 times.

The circular dichroism was measured at 20 ° C and 95 ° C under Far-UV (350 nm to 250 nm) conditions after adjusting to pH 7.0 after dissolving the nongin-derived peptides in water to a concentration of 1 mg / ml. The detailed conditions are band width 1 nm, response 0.25 sec, data pitch 0.1 nm, scanning speed 20 nm / min, cell length 0.5 cm, accumulation 8 times.

As a result of the measurement, all of the five nongin-derived peptides exhibit the same pattern, as shown in Figs.

In the near-UV condition, peaks were observed at 200 nm at both 20 ° C and 95 ° C, and it was confirmed that the peaks existed in a random loop form.

In the Far-UV condition, we measured whether the temperature changes at 270 nm absorbed by the tryptophan residues present in each peptide. When measured at 20 ° C and 95 ° C, no significant changes were observed at 270 nm, confirming that there was no significant change in the structure of the peptides.

These results show that both of the five Noggin-derived peptides have excellent thermal stability and high water solubility.

&Lt; 110 > PnP Biopharm Co., Ltd. <120> A novel peptide with inhibitory BMP activity and use of the same <160> 5 <170> Kopatentin 1.71 <210> 1 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 1 Gln His Tyr Leu His Ile Arg Pro Ala Pro Ser Asp Asn Leu Pro Leu   1 5 10 15 Val Asp Leu Ile Glu His              20 <210> 2 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 2 His Tyr Leu His Ile Arg Pro Ala Pro Ser Asp Asn Leu Pro Leu Val   1 5 10 15 Asp Leu         <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 3 His Tyr Leu His Ile Arg Pro Ala Pro Ser Asp Asn Leu Pro Leu   1 5 10 15 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 4 His Tyr Leu His Ile Arg Pro Ala Pro Ser Asp   1 5 10 <210> 5 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 5 Arg Tyr Leu His Ile Met Pro Ala Pro Ser Asp   1 5 10

Claims (8)

A peptide of the following general formula (4) as set forth in SEQ ID NO: 4, which shows the activity of the Noggin protein.
Formula 4
Ac-His-Tyr-Leu- His-Ile-Arg-Pro-Ala-Pro-Ser-Asp-NH 2 The peptide of the following general formula (5) as set forth in SEQ ID NO: 5 which shows the activity of the Noggin protein.
Formula 5
Ac-Arg-Tyr-Leu- His-Ile-Met-Pro-Ala-Pro-Ser-Asp-NH 2 delete The peptide of the following general formula (2) as set forth in SEQ ID NO: 2 which shows the activity of the Noggin protein.
Formula 2
As-Leu-Val-Asp-Leu-NH2 &lt; / RTI _&gt; A peptide of the following general formula (1) as set forth in SEQ ID NO: 1, which shows the activity of Noggin protein.
1
Ac-Gln-His-Tyr- Leu-His-Ile-Arg-Pro-Ala-Pro-Ser-Asp-Asn-Leu-Pro-Leu-Val-Asp-Leu-Ile-Glu-His-NH 2 The peptide according to any one of claims 1 to 5, wherein the peptide binds to a bone forming protein to inhibit the activity of a bone forming protein. A pharmaceutical composition for preventing or treating hair loss comprising the peptide of any one of claims 1 to 2 and 4 to 5 as an active ingredient. A cosmetic composition for preventing or treating hair loss comprising the peptide of any one of claims 1 to 2 and 4 to 5 as an active ingredient.

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