JP2005239695A - TGF-beta PRODUCTION-INHIBITING PEPTIDE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a peptide having an activity of inhibiting TGF-β production from cells having a TGF-β production capacity. <P>SOLUTION: This peptide having a specific sequence, and a medicinal composition, hair-growing composition, nephritis-treating agent and hepatic cirrhosis-treating agent containing the peptide as the TGF-β production inhibitor are provided. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a peptide that suppresses TGF-β production from cells capable of producing TGF-β.

  TGF-β (transforming growth factor-β) is a cytokine having many activities such as cell growth promoting action or inhibiting action, promoting action or inhibiting action of various cell functions, production promoting action of extracellular matrix, etc. It is known to be produced in various tissues and cells such as cells, epithelial cells such as hepatocytes, and dermal papilla cells. Since TGF-β exhibits an extracellular matrix production promoting action in various cells, glomerulonephritis, nephrosclerosis, liver fibrosis, cirrhosis, pulmonary fibrosis, myelofibrosis, rheumatoid arthritis, proliferative vitreoretinopathy It is considered that the treatment of these diseases becomes possible by suppressing the production of TGF-β. Examples of the inhibitory effects of TGF-β produced by human-derived cells so far include interferons in retinal pigment epithelial cells (see Patent Document 1), macrolide compounds in renal mesangial cells (see Patent Document 2), Various compounds such as camostat mesylate in liver or lung-derived cells (see Patent Document 3, particularly the compound of formula (I)) are known. TGF-β is also deeply involved in hair loss since it suppresses the proliferation and function of keratinocytes and induces apoptosis, and a hair-growth effect by suppressing the production of TGF-β can be expected.

The number of people who suffer from thinning hair, hair loss and hair loss has been increasing in recent years due to stress and changes in dietary habits due to changes in the social environment. Moreover, since this tendency is spreading from the middle-aged group to the young group, it is required to clarify the cause and mechanism of thinning hair, hair loss, and hair loss. Recently, research has been conducted on the mechanism of hair growth, characterized by the inhibitory activity of testosterone to dehydrotestosterone converting enzyme 5α-reductase (see Patent Document 4), and by suppressing the action of TGF-β. What to do (see Patent Document 5) has been developed as a material having hair growth activity. However, until now, no substance showing a hair-growth effect by inhibiting TGF-β production has been known.
JP-A-10-114676 Japanese Patent Application Laid-Open No. 2003-137791 JP-A-8-333249 JP 2003-238435 A JP 2000-342296 A

  An object of the present invention is to provide a peptide characterized by suppressing TGF-β production from a cell having TGF-β production ability.

  As a result of intensive research, the present inventors have cultured TGF-β-producing cells in the presence of a test substance and measured the amount of TGF-β produced to suppress the production of TGF-β. We found a peptide to do. Further, cells having TGF-β production ability and cells having TGF-β signal acceptability are cultured in the presence of a test substance (hereinafter referred to as co-culture) to evaluate the TGF-β production inhibitory effect and TGF- By evaluating the growth promoting action of cells having β signal acceptability, a peptide showing TGF-β production inhibitory effect and at the same time showing growth promoting action of cells having TGF-β signal acceptability was found, and the present invention was completed. It came to do.

  That is, the present invention relates to a peptide characterized by suppressing TGF-β production from cells having TGF-β production ability, and a pharmaceutical composition comprising the peptide as an active ingredient, specifically, Relates to a hair restorer composition, a nephritis therapeutic agent and a cirrhosis therapeutic agent.

  According to the present invention, it becomes possible to suppress TGF-β production from cells capable of producing TGF-β, and it is possible to provide a hair restorer composition, nephritis therapeutic agent and cirrhosis therapeutic agent containing the same. became.

  The present invention will be described in detail below. The TGF-β production inhibitory peptide of the present invention can be produced by a commonly used peptide synthesis method such as a solid phase method or a liquid phase method. The obtained peptide can be analyzed by high performance liquid chromatography (HPLC) using a reverse phase silica gel column or the like, column chromatography such as distribution, adsorption resin, silica gel, alumina, ion exchange resin, gel filtration or thin layer chromatography (TLC), etc. Can be purified. The amino acid sequence of the peptide is analyzed by a protein sequencer, and a standard with a final purity of 90% or more is obtained by this method.

The peptide compound exhibiting a TGF-β production inhibitory effect may be any peptide consisting of 17 amino acids and having a common sequence described in SEQ ID NO: 1, and specific examples include the peptides described in SEQ ID NO: 2-7. it can. In particular, the peptide compound described in SEQ ID NO: 2 or 3 is preferable.
Cys-Asn-Xaa-Gly-Xaa-Ser-Xaa-Asn-Ser-Xaa-Xaa-Ser-Xaa-Xaa-Ser-Arg
(SEQ ID NO: 1: common sequence; Xaa represents any amino acid)
Ser-Cys-Asn-Ser-Gly-Thr-Ser-Tyr-Asn-Ser-Ile-Ser-Ser-Val-Val-Ser-Arg (SEQ ID NO: 2 native17aa)
Ser-Cys-Asn-Ser-Gly-Leu-Ser-Tyr-Asn-Ser-Ile-Ser-Ser-Val-Val-Ser-Arg (SEQ ID NO: 3 T6L)
Ser-Cys-Asn-Ser-Gly-Thr-Ser-Phe-Asn-Ser-Ile-Ser-Ser-Val-Val-Ser-Arg (SEQ ID NO: 4 Y8F)
Ser-Cys-Asn-Ser-Gly-Leu-Ser-Phe-Asn-Ser-Ala-Ala-Ser-Ala-Ala-Ser-Arg (SEQ ID NO: 5: 6mut)
Ala-Cys-Asn-Ser-Gly-Thr-Ser-Tyr-Asn-Ser-Ile-Ser-Ser-Val-Val-Ser-Arg (SEQ ID NO: 6: S1A)
Ser-Cys-Asn-Ala-Gly-Thr-Ser-Tyr-Asn-Ser-Ile-Ser-Ser-Val-Val-Ser-Arg (SEQ ID NO: 7: S4A)
These peptides described in SEQ ID NOs: 2 to 7 have a TGF-β production inhibitory effect and also have a cell growth promoting effect under co-culture.

On the other hand, the peptide in which the 10th Ser from the N-terminus of the peptide described in SEQ ID NO: 2 is replaced with Ala (SEQ ID NO: 8) or the 13th Ser in the peptide replaced with Ala (SEQ ID NO: 9) is TGF-β produced. Since the inhibitory effect is not recognized, it can be seen that the 10th and 13th Ser residues from the N-terminus are important for the TGF-β production inhibitory effect. These peptides of SEQ ID NOs: 8 and 9 not only have no TGF-β production inhibitory effect, but also have no cell growth promoting effect under co-culture.
Ser-Cys-Asn-Ser-Gly-Thr-Ser-Tyr-Asn-Ala-Ile-Ser-Ser-Val-Val-Ser-Arg (SEQ ID NO: 8: S10A)
Ser-Cys-Asn-Ser-Gly-Thr-Ser-Tyr-Asn-Ser-Ile-Ser-Ala-Val-Val-Ser-Arg (SEQ ID NO: 9: S13A)
Furthermore, a peptide in which 6 amino acids have been deleted from the N-terminus of the peptide described in SEQ ID NO: 2 (SEQ ID NO: 10), a peptide in which 9 amino acids have been deleted from the N-terminal of the peptide in SEQ ID NO: 2 (SEQ ID NO: 11), and SEQ ID NO: 2 described Since the peptide (SEQ ID NO: 12) in which 7 amino acids were deleted from the C-terminal of the peptide of No. 2 has no effect of inhibiting TGF-β production, the 10th and 13th Ser residues from the N-terminal of the peptide shown in SEQ ID NO: 2 In addition, it is clear that the amino acid sequence on the N-terminal side is important. These peptides of SEQ ID NOs: 10 to 12 show not only a TGF-β production inhibitory effect but also a cell growth promoting effect under co-culture.
Ser-Tyr-Asn-Ser-Ile-Ser-Ser-Val-Val-Ser-Arg
(SEQ ID NO: 10: dN6)
Ser-Ile-Ser-Ser-Val-Val-Ser-Arg
(SEQ ID NO: 11: dN9)
Ser-Cys-Asn-Ser-Gly-Thr-Ser-Tyr-Asn-Ser
(SEQ ID NO: 12: dC7)
A peptide having an inhibitory effect on TGF-β production on TGF-β-producing cells according to the present invention may be used together with a pharmaceutically acceptable carrier, a diluent, an excipient for formulation, an adjuvant and the like as necessary. It can be provided as a pharmaceutical composition. The dosage forms include solid pharmaceutical compositions such as granules and powders, liquid pharmaceutical compositions such as emulsions, injections and liquid injections, and semisolid preparations such as patches, can do. Moreover, the peptide as an active ingredient of the pharmaceutical composition according to the present invention has an action of controlling TGF-β production in TGF-β producing cells, and the compound having a function of suppressing TGF-β production is a hair restorer, Since it is known to be useful as a nephritis therapeutic agent and a cirrhosis therapeutic agent, it can be used as an active ingredient of these pharmaceuticals.

  Hereinafter, the present invention will be described more specifically with reference to Examples and Comparative Examples, but the scope of the present invention is not limited to these Examples.

Example 1 (Establishment of immortalized human dermal papilla cell line with high TGF-β production)
Papilla cells are isolated from male-type hair loss scalp obtained as a by-product of cosmetic surgery, so that fetal bovine serum (jrh Biociences) is 10% in Dulbecco's modified Eagle medium (DMEM; Nissui Pharmaceutical) And further cultured at 37 ° C. in the presence of 5% CO ₂ using a medium supplemented with penicillin G having a final concentration of 50 U / mL and streptomycin sulfate at 50 μg / mL (hereinafter referred to as a growth medium) (J Investig. Dermatol. Symp. Proc., Vo. 8, 69-71, (2003)). The hair papilla cells were seeded on a 6-well plate (Costar) at 5 × 10 ⁵ cells / well, and plasmid SV-ori-8-16 (JCRB) lacking the ori of SV40 was introduced into the hair papilla cells. Cell introduction conditions were as follows: after replacement with 1 mL serum-free DMEM medium according to the attached manual using FuGene6 (Roche), 1 μg / well SV-ori-8-16 and 3 μL / well FuGene6 were added. After time treatment, the medium was returned to the growth medium. About 20 days after the introduction, 9 proliferating colonies consisting of slightly smaller and faster-growing cells appeared from the cells into which the SV40 plasmid had been introduced, and cloning was performed using glass rings. Finally, DP2-SV40-No.1, DP2-SV40-No.2, DP2-SV40-No.5, DP2-SV40-No.6, DP2-SV40-No.7, DP2-SV40-No. 8. Seven immortalized cells of DP2-SV40-No.9 were established. The dermal papilla cells into which SV-ori-8-16 had not been introduced stopped growing after about the 10th passage, but the immortalized dermal papilla cells continued to grow after the 10th generation. The doubling time of dermal papilla cells before immortalization was about 2 weeks, but the doubling time of immortalized dermal papilla cells was 1.5 to 3 days, and the growth rate was fast.

In order to examine the TGF-β1 concentration in the culture supernatant when the immortalized hair papilla cells were cultured alone, the immortalized hair papilla cells were seeded on a 6-well plate at 5 × 10 ⁵ cells / well in a 3.5 mL growth medium. After 4 days of culture, 3.5 mL of serum-free-DMEM medium was replaced, and after 2 days, the culture supernatant was collected. The TGF-β1 concentration of the culture supernatant was measured using an ELISA kit (R & D Systems) according to the method of the attached manual. As a result, DP2-SV40-No.1, DP2-SV40-No.2, DP2-SV40-No. .5, DP2-SV40-No.6, DP2-SV40-No.7, and DP2-SV40-No.8 cell culture supernatants were 700 pg / mL-1100 pg / mL compared to the dermal papilla cells before immortalization. High TGF-β1 production was observed.

Example 2 (TGF-β1 production inhibitory effect of peptide described in SEQ ID NO: 2 in immortalized hair papilla cell line)
The obtained immortalized dermal papilla cell line (DP2-SV40-No.8) is seeded on a 6-well plate at 5 × 10 ⁵ cells / well, cultured for 1 day in 3.5 mL growth medium, and SEQ ID NO: 2 at a final concentration of 30 μM. The medium was replaced with 3.5 mL serum-free-DMEM medium containing the described peptides, and further cultured for 5 days, and then the culture supernatant was collected. The TGF-β1 concentration of the culture supernatant was measured using an ELISA kit (R & D Systems) according to the method of the attached manual. The results are shown in FIG. The peptide described in SEQ ID NO: 2 showed a remarkable TGF-β1 production inhibitory effect.

Comparative Example 1
The same procedure as described in Example 2 was performed except that the peptide described in SEQ ID NO: 2 at a final concentration of 30 μM was not included. The results are shown in FIG.

Example 3
The obtained immortalized dermal papilla cell line (DP2-SV40-No.8) was seeded on a 6-well plate at 5 × 10 ⁵ cells / well and cultured in a 3.5 mL growth medium for 1 day. In addition, keratinocyte cells were seeded on a 6-well plate at 5 × 10 ⁴ cells / well and cultured in a 3.5 mL growth medium for 1 day. After culturing, both cells were further co-cultured in 3.5 mL serum-free-DMEM medium containing one selected from peptides of SEQ ID NOs: 2 to 7 having a final concentration of 30 μM, and the culture supernatant was collected. The TGF-β1 concentration of the culture supernatant was measured using an ELISA kit (R & D Systems) according to the method of the attached manual. The TGF-β production inhibitory effect of each peptide described in SEQ ID NOs: 2 to 7 is shown in FIG. Further, the proliferation of keratinocyte cells after co-culture was calculated by counting the number of cells. The results are shown in FIG.

Comparative Example 2
The same procedure as described in Example 3 was performed except that the peptide described in SEQ ID NOs: 2 to 7 at a final concentration of 30 μM was not included. The results are shown in FIG. 2, FIG. 3, FIG. 4 and FIG.

Example 4
It carried out like the method of Example 3 except having carried out the coculture in the serum-free-DMEM culture medium containing 1 type chosen from the peptide of sequence number 8-12 instead of the peptide of sequence number 2-7. The results are shown in FIG. 4 and FIG.

It is a figure which shows the TGF- (beta) 1 production suppression effect of the peptide which has an amino acid sequence of sequence number 2 in an immortalized hair papilla cell. It is a figure which shows the TGF- (beta) 1 production inhibitory effect of the peptide which has an amino acid sequence of sequence number 2-7 under co-culture with an immortalized hair papilla cell and a keratinocyte cell. It is a figure which shows the keratinocyte cell growth promotion effect of the peptide which has an amino acid sequence of sequence number 2-7 in the coculture of an immortalized hair papilla cell and a keratinocyte cell. It is a figure which shows the TGF- (beta) 1 production inhibitory effect of the peptide which has an amino acid sequence of sequence number 2, 8-12 under co-culture with an immortalized hair papilla cell and a keratinocyte cell. It is a figure which shows the keratinocyte cell growth promotion effect of the peptide which has an amino acid sequence of sequence number 2, 8-12 under co-culture with an immortalized hair papilla cell and a keratinocyte cell.

Claims (8)

  A peptide characterized by suppressing TGF-β production from a cell capable of producing TGF-β.   The peptide according to claim 1, wherein the cell having the ability to produce TGF-β is a human dermal papilla cell.   The peptide according to claim 1, which has a growth promoting effect on a cell having TGF-β signal acceptability.   The peptide according to any one of claims 1 to 3, which has at least an amino acid sequence consisting of the sequence of SEQ ID NO: 1.   The peptide according to claim 4, which has an amino acid sequence consisting of the sequence of SEQ ID NO: 2.   A hair restorer composition comprising the peptide according to any one of claims 1 to 5 as an active ingredient.   A therapeutic agent for nephritis comprising the peptide according to any one of claims 1 to 5 as an active ingredient.   A cirrhosis therapeutic agent comprising the peptide according to any one of claims 1 to 5 as an active ingredient.

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