Peptide showing melanogenesis promoting activity and use thereof
11518785 · 2022-12-06
Assignee
Inventors
Cpc classification
A61K8/64
HUMAN NECESSITIES
International classification
A61K8/64
HUMAN NECESSITIES
Abstract
The present invention provides a peptide exhibiting melanogenesis promoting activity. A peptide according to the present invention increases the activity and expression of tyrosinase and the expression of factors involved in melanogenesis, thereby exhibiting an outstanding effect on melanogenesis. The peptide of the present invention can be used for the prevention, alleviation, and treatment of hypomelanosis. The outstanding activity and stability stated above allow the peptide of the present invention to be very favorably applied to medicines, quasi-medicines, and cosmetics.
Claims
1. A peptide that has an activity against hypomelanosis and consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3, wherein optionally the peptide includes a modification at the N-terminal and/or C-terminal end.
2. The peptide of claim 1, where the peptide includes said N-terminal and/or C-terminal modification.
3. The peptide of claim 2, wherein the peptide includes an N-terminal modification, which is a protecting group.
4. The peptide of claim 3, wherein the protecting group is selected from the group consisting of an acetyl group, a fluorenyl methoxy carbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG).
5. The peptide of claim 2, wherein the peptide includes a C-terminal modification selected from the group consisting of an amino group (—NH.sub.2) and an azide group (—NHNH.sub.2).
6. A pharmaceutical composition for the treatment of hypomelanosis, comprising, as an active ingredient, at least one peptide consisting of a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3, wherein optionally the peptide includes a modification at the N-terminal and/or C-terminal end.
7. The pharmaceutical composition of claim 6, wherein the hypomelanosis is selected from the group consisting of vitiligo, albinism, nevus depigmentosus, pityriasis alba, pityriasis versicolor, post-inflammatory depigmentation, morphea, piebaldism, idiopathic guttate hypomelanosis, and leucoderma punctatum.
8. The pharmaceutical composition of claim 6, where the peptide includes said N-terminal and/or C-terminal modification.
9. The pharmaceutical composition of claim 8, wherein the peptide includes an N-terminal modification, which is a protecting group.
10. The pharmaceutical composition of claim 9, wherein the protecting group is selected from the group consisting of an acetyl group, a fluorenyl methoxy carbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG).
11. The pharmaceutical composition of claim 8, wherein the peptide includes a C-terminal modification selected from the group consisting of an amino group (—NH.sub.2) and an azide group (—NHNH.sub.2).
12. A cosmetic composition for the alleviation of hypomelanosis, comprising, as an active ingredient, at least one peptide consisting of a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3, wherein optionally the peptide includes a modification at the N-terminal and/or C-terminal end.
13. The cosmetic composition of claim 12, wherein the hypomelanosis is selected from the group consisting of vitiligo, albinism, nevus depigmentosus, pityriasis alba, pityriasis versicolor, post-inflammatory depigmentation, morphea, piebaldism, idiopathic guttate hypomelanosis, and leucoderma punctatum.
14. The cosmetic composition of claim 12, where the peptide includes said N-terminal and/or C-terminal modification.
15. The cosmetic composition of claim 14, wherein the peptide includes an N-terminal modification, which is a protecting group.
16. The cosmetic composition of claim 15, wherein the protecting group is selected from the group consisting of an acetyl group, a fluorenyl methoxy carbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG).
17. The cosmetic composition of claim 14, wherein the peptide includes a C-terminal modification selected from the group consisting of an amino group (—NH.sub.2) and an azide group (—NHNH.sub.2).
18. A method for the treatment or alleviation of hypomelanosis in a subject in need thereof, the method comprising administering the peptide of claim 1 to the subject.
19. The method of claim 18, wherein the peptide is comprised within a pharmaceutical composition or a cosmetic composition.
20. The method of claim 18, wherein the hypomelanosis is selected from the group consisting of vitiligo, albinism, nevus depigmentosus, pityriasis alba, pityriasis versicolor, post-inflammatory depigmentation, morphea, piebaldism, idiopathic guttate hypomelanosis, and leucoderma punctatum.
21. The method of claim 18, where the peptide includes said N-terminal and/or C-terminal modification.
22. The method of claim 21, wherein the peptide includes an N-terminal modification, which is a protecting group.
23. The method of claim 22, wherein the protecting group is selected from the group consisting of an acetyl group, a fluorenyl methoxy carbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG).
24. The method of claim 21, wherein the peptide includes a C-terminal modification selected from the group consisting of an amino group (—NH.sub.2) and an azide group (—NHNH.sub.2).
Description
BRIEF DESCRIPTION OF DRAWINGS
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BEST MODE
(16) Provided is a peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, or 3.
MODE OF DISCLOSURE
(17) Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are only for illustrative purposes, and the scope of the present invention is not limited by these examples.
Synthetic Example 1: Peptide Synthesis
(18) 70 g of chlorotrityl chloride resin (CTC resin, Nova Biochem Cat No. 01-64-0021) was added into a reaction container, and 490 ml of methylene chloride (MC) was added followed by stirring for 3 minutes. After the solution was removed, 490 ml of dimethyl formamide (DMF) was added, followed by stirring for 3 minutes, and then the solvent was again removed. 700 ml of a dichloromethane solution was added to a reaction container, and 200 mmole of Fmoc-Leu-OH (Bachem, Swiss) and 400 mmole of diisopropyl ethylamine (DIEA) were added. Thereafter, the mixture was well dissolved with stirring, and then the reaction was conducted with stirring for 1 hour. After the reaction, washing was conducted, and then methanol and DIEA (2:1) were dissolved in dichloromethane (DCM), followed by reaction for 10 minutes, and then the resultant was washed with excess DCM/DMF (1:1). After the solution was removed, 490 ml of dimethyl formamide (DMF) was added, followed by stirring for 3 minutes, and then the solvent was again removed. 700 ml of a deprotection solution (20% piperidine/DMF) was added to a reaction container, followed by stirring at room temperature for 10 minutes, and then the solution was removed. An equal amount of a deprotection solution was added, and then the reaction was again maintained for 10 minutes, and thereafter, the solution was removed, followed by washing twice with DMF, once with MC, and once with DMF, for 3 minutes each, thereby preparing Leu-CTC resin.
(19) 700 ml of a DMF solution was added to a new reaction container, and 200 mmol Fmoc-Ser(tBu)-OH (Bachem, Swiss), 200 mmol HoBt, and 200 mmole of HBTu were added, and the mixture was well dissolved with stirring. 400 mmole DIEA was added to the reaction container in two divided portions, and then stirring was conducted for at least 5 minutes until all solids were dissolved. The dissolved amino acid mixed solution was added to the reaction container containing the deprotected resin, and the reaction was conducted with stirring at room temperature for 1 hour. After the reaction solution was removed, the stirring was conducted using a DMF solution three times for 5 minutes each, followed by removal. A small amount of the reaction resin was taken to check the extent of reaction using the Kaiser test (Ninhydrin test). The deprotection reaction was twice conducted using a deprotection solution in the same manner as described above, thereby preparing Ser(tBu)-Leu-CTC resin. After sufficient washing with DMF and MC, the Kaiser test was again conducted, and then the following amino acid attachment test was conducted in the same manner as described above. A chain reaction was conducted in the order of Fmoc-Trp-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH on the basis of the selected amino acid sequence. The Fmoc-protecting group was removed by reaction twice with the deprotection solution for 10 minutes for each and then favorable washing. The peptidyl resin was washed with DMF, MC, and methanol three times for each, dried under the flow of nitrogen gas, and completely dried by decompression under vacuum in P.sub.2O.sub.5. Thereafter, 1,900 ml of a leaving solution [81.5% of trifluoroacetic acid (TFA), 5.0% of distilled water, 5.0% of thioanisole, 5.0% of phenol, 2.5% of ethanedithiol (EDT), and 1.0% of triisopropylsilane (TIS)] was added, and the reaction was maintained for 2 hours while stirring the mixture at room temperature. The resin was obtained through filtration, washed with a small amount of TFA solution, and then mixed with the stock solution. Cold ether was added to 2,090 ml of the resultant with the stock solution to induce precipitation, and the precipitates were collected by centrifugation, followed by washing twice with cold ether. The stock solution was removed, followed by sufficient drying under nitrogen atmosphere, and thus 129.8 g of a peptide consisting of the amino acid sequence of SEQ ID NO: 1 (yield: 92.8%) was synthesized, before purification. The molecular weight was determined as 1398.6 (theoretical value: 1398.6) by using a molecular weight analysis system.
(20) In addition, the peptide composed of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 was synthesized in the same manner as described above.
(21) TABLE-US-00001 TABLE 1 Analysis value SEQ (Mass spectrometer) ID Sequence listing Analytical Theoretical NO: (5′->3′) value value 1 FCLGPCPYIWSL 1398.6 1398.6 2 KVTAMRCFLL 1181.5 1181.5 3 RVTAMRCFLL 1209.5 1209.5
Example 1: Melanogenesis Assay
(22) After seeding melanocytes (B16F10 cell line) in 6-well plates at the density of 5×10.sup.4 cells/well, the melanocytes were cultured in an incubator at a temperature of 37° C. for 24 hours, and the medium of each plate was removed and replaced with 2% serum-containing media, followed by treatment with the present peptide at different concentrations and then incubation of 72 hours.
(23) Then, the culture medium was removed, and the cells were taken off and then transferred into 1.5-ml tubes, followed by centrifugation at 13,000 rpm for 3 minutes to remove the supernatant. Next, cell pellets were collected to observe melanin. 150 μl of 1 N NaOH was added to the cell pellets to lyse intracellular melanin at a temperature of 60° C. for 30 minutes. Thereafter, 100 μl of the supernatant obtained from the lysis was added into each well of 96-well plates, and the absorbance was measured at 450 nm, and the results are shown in
(24) TABLE-US-00002 TABLE 2 Control SEQ ID NO: 1 SEQ ID NO: 1 a-MSH group (10 μM) (50 μM) (200 nM) Melanin 100 125 264 276 content (%)
(25) TABLE-US-00003 TABLE 3 Control SEQ ID NO: 2 SEQ ID NO: 2 a-MSH group (10 μM) (50 μM) (200 nM) Melanin 100 109 155 263 content (%)
(26) TABLE-US-00004 TABLE 4 Control SEQ ID NO: 3 SEQ ID NO: 3 a-MSH group (10 μM) (50 μM) (200 nM) Melanin 100 106 164 263 content (%)
(27) As it may be confirmed in
Example 2: Tyrosinase Activity Assay
(28) Melanoma cell line (B16F10) cells were cultured in 6-well culture plates for 24 hours, and treated with the peptide with different concentrations, followed by culture for 72 hours. The 6-well culture plates were loaded on ice and washed with cool PBS, and then 300 μl of 0.1 M sodium phosphate buffer (pH 6.8, lysis buffer) containing 1% Triton X-100 was added. The cells were collected in 1.5-ml tubes, and then cell membranes were disrupted by repeating five times rapid-freezing at −270° C. and thawing. After centrifugation at 15,000 rpm for 10 minutes, the supernatant was collected in other 1.5-mL tubes, and the protein of the samples was quantified. The samples were diluted to have the same protein concentration and then dispensed in every three wells in 96-well culture plates, and then 20 μl of 10 mM L-DOPA was added, followed by incubation at 37° C. for 1 hour The blank and positive control were as shown in Table 5.
(29) TABLE-US-00005 TABLE 5 Sample Blank Positive control Sample 90 μl — — Buffer — 90 μl 80 μl Mushroom tyrosinase — — 10 μl (0.1 mg/ml)
(30) Then, the absorbance was measured at 475 nm, and the results are shown in
(31) TABLE-US-00006 TABLE 6 Control SEQ ID NO: 1 SEQ ID NO: 1 a-MSH group (10 μM) (50 μM) (200 nM) Tyrosinase 100 157 201 467 activity (%)
(32) TABLE-US-00007 TABLE 7 Control SEQ ID NO: 2 SEQ ID NO: 2 a-MSH group (10 μM) (50 μM) (200 nM) Tyrosinase 100 144 189 484 activity (%)
(33) TABLE-US-00008 TABLE 8 Control SEQ ID NO: 3 SEQ ID NO: 3 a-MSH group (10 μM) (50 μM) (200 nM) Tyrosinase 100 114 173 484 activity (%)
(34) As it may be confirmed in
Example 3: RT-PCR of Melanogenesis-Related Genes
(35) Melanocytes (B16F10 cell line) were seeded on 6-well culture plates at the density of 5×10.sup.4 cells/well and incubated in an incubator for 24 hours. Then, the medium was replaced with 00, and the cells were treated with the peptides of the present invention with different concentrations and cultured for 72 hours. Next, after RNA extraction of cells and quantification, cDNA was synthesized by using the cDNA synthesis kit (Intron, Korea). Thereafter, as shown in Table 3, PCR was performed using specific primers for MITF, tyrosinase, and TRP1, which are factors involved in melanogenesis. Then, degrees of mRNA expression of the growth factors under each sample treatment conditions were compared by running the resultants on a 5% agarose gel, and the results are shown in
(36) TABLE-US-00009 TABLE 9 SEQ ID NO: Primer name Sequence listing (5′-3′) 4 MITF_F CCAGCCTGGCGATCATGTCAT 5 MITF_R GGTCTGGACAGGAGTTGCTG 6 tyrosinase_F GGCCAGCTTTCAGGCAGAGG 7 tyrosinase_R TGGTGCTTCATGGGCAAAAT 8 TRP1_F TCTGTGAAGGTGTGCAGGAG 9 TRP1_R CCGAAACAGAGTGGAAGGTT
(37) As it may be confirmed in
Example 4: Western Blotting of Melanogenesis-Related Proteins
(38) Melanocytes (B16F10 cell line) were seeded on 6-well culture plates at the density of 5×10.sup.4 cells/well and incubated in an incubator for 24 hours, and the cells were treated with the peptides of the present invention with different concentrations. After 72-hour incubation, the cells were lysed, and the cells were subjected to western blotting using antibodies (antacruz biotechnology, USA) each specific to MITF and tyrosinase, which are the factors involved in melanogenesis. The results are shown in
(39) As it may be confirmed in
Example 5: Melanogenesis-Related Protein Activity Assay
(40) Melanocytes (B16F10 cell line) were seeded on 6-well culture plates at the density of 5×10.sup.4 cells/well and incubated in an incubator for 24 hours, and the cells were treated with the peptides of the present invention with different concentrations. After 48-hour incubation, the cells were lysed, and the cells were subjected to western blotting using specific antibodies (Cell Signaling Technology, USA) to investigate the phosphorylation level of CREB, which is a signaling substance involved in melanogenesis. The results are shown in
(41) As it may be confirmed in
INDUSTRIAL APPLICABILITY
(42) The present invention is related to a peptide showing melanogenesis promoting activity; a pharmaceutical composition for prevention and/or treatment of hypomelanosis including the peptide as an active ingredient; a cosmetic composition for prevention and/or alleviation of hypomelanosis including the peptide as an active ingredient; and the use of the peptide for prevention, alleviation, and/or treatment of hypomelanosis.