AIMP1 protein fragment and hair growth-promoting composition containing same as active ingredient

Abstract

The present invention relates to novel fragments of AIMP1 protein and a composition for improving alopecia and promoting hair growth comprising the same, more specifically it relates to a polypeptide consisting of 4 to 21 consecutive amino acids from the amino acid sequence of SEQ ID NO: 1, wherein the polypeptide comprises the 28.sup.th to 31.sup.st amino acid residue (KEKA) of amino acid sequence of SEQ ID NO: 1, or a polypeptide consisting of an amino acid sequence having 70% or more sequence homology with the polypeptide; a polynucleotide encoding the polypeptide; a pharmaceutical, cosmetic and food composition for improving alopecia and promoting hair growth comprising the polypeptide.

Claims

1. A composition comprising as an active ingredient one or more polypeptides consisting of one of the amino acid sequences selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 wherein the polypeptide promotes hair growth, promotes proliferation of hair follicle stem cells, and/or treats alopecia and wherein the composition further comprises a compound or pharmaceutically acceptable salt thereof selected from the group consisting of minoxidil, cromakalim, pinacidil, naminidil, diphenylcyclopropenone, cyproterone acetate, danazol, and 5-alpha reductase inhibitors selected from the group consisting of finasteride, turosteride, LY-191704, and dutasteride.

2. The composition of claim 1, wherein the composition is a pharmaceutical composition, a cosmetic composition, or a food composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In FIG. 1, the upper part of the figure shows an outline of a mouse experiment for confirming the in vivo hair growth promoting effect of the polypeptide of the present invention, and the lower part of the figure shows a state in which hair of a dorsal skin of the mouse is removed before the beginning of the experiment (Day 0, left panel), and about two weeks later in which hair of the dorsal skin of the mouse grew (Day 13, right panel) [Control (Con): 20% Glycerol/PBS; 3% Minoxidil (MNX, 140 mM); Neo-Pep polypeptide (polypeptide of SEQ ID NO: 1, 100 nM); FGF7 (100 nM)].

(2) In FIG. 2, in the left part of the figure, the cross-sectional and longitudinal section images of the skin tissue and the hair follicle region observed through a phase contrast microscope are shown in order to compare the hair growth in the control group and the Neo-Pep polypeptide (polypeptide of SEQ ID NO: 1) treated group at Day 13 after depilation, and the right part of the figure shows a graph comparing the number of hair follicle cell in anagen phase and the thickness of the dermis quantitatively.

(3) FIG. 3 shows the cross-sectional images of the skin tissue and hair follicle region observed through a phase contrast microscope in order to compare the hair growth in the control group and the Neo-Pep polypeptide (polypeptide of SEQ ID NO: 1) treated group at Day 6 after depilation. As can be seen from the results of the proliferation of BrdU (cell proliferation marker), β-catenin (signaling marker of hair follicle cell) and Cytokeratin 15 (hair follicle stem cell marker), it was confirmed that the anagen phase starts earlier in Neo-Pep polypeptide (polypeptide of SEQ ID NO: 1) treated group than the control group [Control (Con): 20% Glycerol/PBS; Neo-Pep polypeptide (polypeptide of SEQ ID NO: 1, 100 nM)].

(4) FIG. 4 shows in vitro assay results showing that the Neo-Pep polypeptide (polypeptide of SEQ ID NO: 1) induces the proliferation of CD34+ hair follicle stem cells in a concentration dependent manner (1 nM, 10 nM, 100 nM, 1000 nM).

(5) FIG. 5 is a graph showing in vitro assay results that the Neo-Pep polypeptide (polypeptide of SEQ ID NO: 1, 100 nM) induces the proliferation of CD34+ hair follicle stem cells through the β-catenin pathway, and it was confirmed that the mRNA expression of AXIN, CD44 and TCF which are target gene of β-catenin was increased by treatment of polypeptide of the present invention[Control (Con): 20% Glycerol/PBS; Neo-Pep polypeptide (polypeptide of SEQ ID NO: 1, 100 nM)].

(6) FIG. 6 is images showing the effect of promoting hair growth using C3H/HeJ mice having a skin graft cell-induced alopecia areata [(Control treated mice: 100 μl of 20% glycerol/PBS); Neo-Pep polypeptide-treated group (Peptide-treated mice, 100 μl of 100 nM Neo-Pep polypeptide (SEQ ID NO: 1) in 20% glycerol/PBS).

(7) FIG. 7 is images showing the state of hair follicles and presence or absence of inflammation in the skin tissue sections of C3H/HeJ mice having a skin graft cell-induced alopecia areata [(Control treated mice: 100 μl of 20% glycerol/PBS); Neo-Pep polypeptide-treated group (Peptide-treated mice, 100 μl of 100 nM Neo-Pep polypeptide (SEQ ID NO: 1) in 20% glycerol/PBS).

(8) FIG. 8 is a graph showing in vitro assay results confirming the effect on promoting the proliferation of CD34+ hair follicle stem cells with the Neo-Pep polypeptide of the present invention (SEQ ID NO: 1) and its fragment having 19 or 20 amino acids (N1: SEQ ID NO: 2, N2: SEQ ID NO: 3, N3: SEQ ID NO: 4, N4: SEQ ID NO: 5, N5: SEQ ID NO: 6, N6: SEQ ID NO: 7, N7: SEQ ID NO: 8 and N8: SEQ ID NO: 9)(each peptide was treated at the concentration of 100 nM).

(9) FIG. 9 is images confirming the in vivo hair growth promoting effect of fragments of Neo-Pep polypeptide of the present invention (SEQ ID NO: 1), wherein the fragments consist of 15 amino acids (N9: SEQ ID NO: 10, N10: SEQ ID NO: 11, N11: SEQ ID NO: 12, N12: SEQ ID NO: 13, N13: SEQ ID NO: 14 and N14: SEQ ID NO: 15) [Control (con): 20% Glyceol/PBS; 3% Minoxidil (MNX, 140 mM); N9 to N14 polypeptide fragments (SEQ ID NO: 10 to 15, 100 nM)]

MODE FOR CARRYING OUT THE INVENTION

(10) Hereinafter, the present invention will be described in detail.

(11) However, the following examples are merely for illustrating the present invention and are not intended to limit the scope of the present invention.

(12) Method

(13) 1. In Vivo Study

(14) The hair of the dorsal skin of mice (7 weeks old male C57BL6, purchased from Orient Bio Inc.) was cut out with a clipper, and hair removal cream (Niclean, Ildong Pharmaceutical) was applied. The depilated area was about 2×2.5 cm. Mice were treated with 20% glycerol/PBS (control group), 3% Minoxidil (MNX, 140 mM), FGF7 (100 nM) or Neo-Pep of the present invention (polypeptide of SEQ ID NO: 1, 100 nM), respectively, once a day in 80 μl using brush, for 13 days (n=6 per group).

(15) 2. Hematoxylin-eosin (H&E) Staining

(16) Skin samples of 7 weeks old male C57BL6 mice (purchased from Orient Bio Inc.) were collected and placed in a cryomold using an OCT compound (Tissue-Tek) and stored at −80° C. in a freezer. After tissue sections were prepared with Microm HM 525 (Thermo Scientific), the tissue sections prepared in the slide glass were fixed with 4% paraformaldehyde for 10 minutes, washed twice with PBS for 5 minutes, and then stained with 50 μl of hematoxylin solution (Sigma Aldrich) for 6 minutes and washed with water and PBS for 5 minutes. Then, stained with eosin (Sigma Aldrich), briefly washed with water and washed with PBS for 5 min, then briefly twice with 95% ethanol and 100% ethanol, respectively. After treatment with xylene (Duksan) twice for 2 min, the stained tissue sections were covered with cover slides and fixed.

(17) 3. Separation of CD34+ Cells for Proliferation Assay

(18) The hair of the dorsal skin of 7 weeks old male C57BL6 mice (purchased from Orient Bio Inc.) were cut with a clipper and skin tissue samples of the depilated dorsal region of the mice were collected. The collected skin tissue samples were immersed in medium using collagenase, DNase I, and hyaluronidase, and cells were isolated using Gentle MACSTM Dissociator (Miltenyi Biotec). After the cells were separated, the separated cells were incubated with a mouse CD34-Biotin antibody (Miltenyi Biotec). Cells and antibody solutions were incubated with streptavidin microbeads (Miltenyi Biotec) and then separated using LS column (Miltenyi Biotec). The separated cells were cultured in DMEM medium containing 10% FBS, 1% penicillin/streptomycin solution in humidified air of 95% O.sub.2 and 5% CO.sub.2 at 37° C.

(19) 4. BrdU Proliferation Assay

(20) Cell proliferation can be confirmed using the BrdU cell proliferation assay kit (Cell Signaling). Proliferation assays were performed using protocols presented by Miltenyi Biotec. Briefly, the cells were incubated with BrdU for 2 h. The primary BrdU detection antibody was incubated for 1 hour and the secondary anti-mouse IgG, HRP-conjugate antibody was incubated for 30 minutes.

(21) 5. In Vitro Hair Growth Assay and Observation of mRNA Expression Level

(22) A hair growth in vitro assay was performed using the Cyquant Assay (Thermo Fisher). The assay conditions were as follows: Cell—CD34+ hair follicle stem cells; Culture plate—96 well plate; Cell number—3×10.sup.3 cells/well; Concentration of final polypeptide—100 nM; incubation time 24 hours.

(23) In order to confirm the induction of CD34+ hair follicle stem cell proliferation through β-catenin pathway, mRNA expression levels of β-catenin target genes AXIN, CD44 and TCF7 were confirmed by the following method. Specifically, each test substance was treated and CD34+ cells were cultured for 8 hours. Then, the cultured cells were collected, RNA was isolated using GeneJET RNA purification kit (Thermo Fisher, K0731), and cDNA was synthesized with the Maxima first strand cDNA synthesis kit (Thermo Fisher, K1642). qRT-PCR was performed using each of the gene-specific primers (TCF7: 5′-ATCCTTGATGCTGGGATTCTG-3′ (SEQ ID NO: 36) and 5′-CTTCTCTTGCCTTGGGTTCTG-3′ (SEQ ID NO: 37), AXIN2: 5′-CTCCTTGGAGGCAAGAGC-3′ (SEQ ID NO: 38) and 5′-GGCCACGCAGCACCGCTG-3′ (SEQ ID NO: 39), CD44: 5′-CCACAGCCTCCTTTCAATAACC-3′ (SEQ ID NO: 40) and 5′-GGAGTCTTCGCTTGGGGTA-3′ (SEQ ID NO: 41)), Maxima SYBR green/ROX qPCR master mix (Thermo Fisher, K0222) and 7500 Real-time PCR system (Applied Biosystems, 2720; condition: 40 cycles, 2 step (95° C. for 15 sec, and 54° C. for 60 sec). The expression level of each gene was calculated by the ΔΔC.sub.T method.

(24) 6. Histological Analysis

(25) A skin sample of 7 weeks old male C57BL6 mice (purchased from Orient Bio Inc.) was collected and placed in a paraffin block. IHC (Immunohistochemistry) was performed using Cytokeratin 15 antibody (Abcam), BrdU antibody (Novusbio), and β-catenin antibody.

(26) For reference, β-catenin is a hair follicle signaling marker, Cytokeratin 15 is a hair follicle stem cell marker, and BrdU is a cell proliferation marker.

Example 1

Hair Growth Promoting Effect of the Polypeptide of the Present Invention

(27) The effects of the polypeptide according to the present invention in promoting hair growth and preventing or treating alopecia in vivo were confirmed using mice. The hair was removed from the dorsal skin of the mice, and the hair regrowth pattern after treatment with the polypeptide according to the present invention was compared with the control group (see FIG. 1).

(28) Specifically, the hair of the dorsal skin of a mouse (7 weeks old male C57BL6, purchased from Orient Bio Inc.) was cut out with a clipper, and hair removal cream (Niclean, Ildong Pharmaceutical) was applied. The depilated area was about 2×2.5 cm. Mice were treated with 20% glycerol/PBS(control group), 3% Minoxidil (MNX, 140 mM), FGF7 (100 nM) or Neo-Pep of the present invention (polypeptide of SEQ ID NO: 1, 100 nM), respectively, once a day in 80 μl using brush, for 13 days (n=6 per group). For comparative observation, the dorsal skin of the mouse was photographed before and after the beginning of the test.

(29) As can be seen from the images of the skin of the mice at the bottom of FIG. 1, 13 days after depilation of dorsal skin, the dorsal hair of the mice treated with the polypeptide of the present invention “Neo-Pep” (polypeptide of SEQ ID NO: 1) grew much more than that of control and other experimental groups (i.e., MNX and FGF7).

(30) In addition, dorsal skin samples of the mice were collected and placed in a cryomold using an OCT compound (Tissue-Tek) and stored at −80° C. in a freezer. After tissue sections were prepared with Microm HM 525 (Thermo Scientific), the tissue sections prepared in the slide glass were fixed with 4% paraformaldehyde for 10 minutes, washed twice with PBS for 5 minutes, and then stained with 50 μl of hematoxylin solution (Sigma Aldrich) for 6 minutes and washed with water and PBS for 5 minutes. Then, stained with eosin (Sigma Aldrich), briefly washed with water and washed with PBS for 5 min, then briefly twice with 95% ethanol and 100% ethanol, respectively. After treatment with xylene (Duksan) twice for 2 min, the stained tissue sections were covered with cover slides and fixed. The number of hair follicles of anagen phase (growth phase) was counted and compared quantitatively in dorsal skin sections of each group stained with hematoxylin and eosin (H & E).

(31) As shown in FIG. 2, as a result of comparing the number of hair follicles of anagen phase in the mouse skin sections stained with H & E, in the dorsal skin of the mice treated with the polypeptide of the present invention “Neo-Pep” (polypeptide of SEQ ID NO: 1), more than twice as much hair follicles of anagen phase were observed as compared with the control (Con), and the dermis thickness was also much thicker.

(32) Through the test result, it was confirmed that the polypeptide of the present invention “Neo-Pep” (SEQ ID NO: 1) has effect on promoting hair growth, and improving and treating alopecia in vivo.

Example 2

Effect of the Polypeptide of the Present Invention on Inducing Proliferation of Hair Follicle Stem Cell Through β-catenin Signaling

(33) β-catenin is a very important protein for hair growth and a very important factor in follicular stem cell proliferation playing a role as a signaling marker for hair follicle cells. In addition, Cytokeratin 15 acts as a biomarker of hair follicle stem cells, and BrdU is known to be a factor that plays a role as a cell growth marker. FIG. 3 shows the cross-sectional images of the skin tissue and hair follicle region observed through a phase contrast microscope in order to compare the hair growth in the control group and the Neo-Pep polypeptide (polypeptide of SEQ ID NO: 1) treated group at Day 6 after depilation. As can be seen from the results of the BrdU (cell proliferation marker), β-catenin (signaling marker of hair follicle cell) and Cytokeratin 15 (hair follicle stem cell marker), it was confirmed that the anagen phase starts earlier in Neo-Pep polypeptide (polypeptide of SEQ ID NO: 1) treated group than the control group.

(34) In addition, as can be seen from the graph of in vitro assay of hair growth in FIG. 4, the Neo-Pep polypeptide (polypeptide of SEQ ID NO: 1) induced proliferation of CD34+ hair follicle stem cells in a dose-dependent manner (1 nM, 10 nM, 100 nM and 1000 nM). As shown in the in vitro assay result graph of FIG. 5, it was confirmed that the Neo-Pep polypeptide (polypeptide of SEQ ID NO: 1, 100 nM) induces the proliferation of CD34+ hair follicle stem cells through the β-catenin pathway. Specifically, when the Neo-Pep polypeptide (polypeptide of SEQ ID NO: 1, 100 nM) according to the present invention is used, the mRNA expression level of AXIN, CD44 and TCF7, which are the target genes of β-catenin, increased 5-fold compared to the control group.

Example 3

Effect on Promoting Hair Growth in Alopecia Areata Mouse Model

(35) Alopecia areata is known to be a cell-mediated autoimmune disease that targets hair follicles of anagen phase in various mammalian species. In humans, alopecia areata is divided into three classes: alopecia areata (patchy hair loss), alopecia totalis (hair loss on the head), and alopecia universalis (total body hair loss).

(36) In order to confirm the effect of the polypeptide of the present invention on the human hair growth cycle, C3H/HeJ mouse (Jackson Laboratory, USA) was used as an animal model reflecting the pathological state of human alopecia areata. The advantage of using the C3H/HeJ mouse is that when full thickness skin graft is applied to an aged mouse having alopecia areata from young age, patchy alopecia develops within 8 to 10 weeks, and after 20 weeks, alopecia extends to the whole body skin and result in a chronic state of alopecia universalis.

(37) In this test, C3H/HeJ female mice having alopecia universalis that was induced 25 weeks after skin transplantation as described above were used. Without any pretreatment such as shaving or depilation, control group (Control-treated mice, 100 μl of 20% glycerol/PBS) and Neo-Pep polypeptide treated group (Peptide-treated mice, 100 μl of 100 nM Neo-Pep polypeptide (SEQ ID NO: 1) in 20% glycerol/PBS) were treated with each test material topically around the alopecia region using brush, once daily, for 14 weeks. Control group and Neo-Pep treated group consisted of 4 mice, respectively.

(38) Photographs were taken every 2 weeks from the beginning of the experiment. FIG. 6 shows the state of hair growth of mice at 1 week and 14 weeks after the beginning of the experiment. In the case of Neo-Pep polypeptide-treated group (peptide-treated mice, 100 nM Neo-Pep polypeptide (SEQ ID NO: 1) in 20% glycerol/PBS), hair began to grow again after 11 weeks of administration, and mild to moderate hair growth was observed in 3 out of 4 mice (#11, #13, and #14 mice) showing consistent hair growth effect. In contrast, the control group showed mild hair growth effect in only 1 mouse (#18 mouse) out of 4 mice, alopecia of 2 mice (#15 and #17 mouse) became more severe, and 1 (#16 mouse) died before the end of the experiment, thus showing no consistent hair growth effect.

(39) Meanwhile, in the images of the skin tissue of the mouse in FIG. 7, the skin tissue at the alopecia area and the area without alopecia around it was collected, fixed in Fekete's acid alcohol formalin and analyzed for H&E staining. And FIG. shows results confirming the presence or absence of deformation or inflammation, and the state of hair follicle.

(40) As can be seen from FIG. 7, especially in the skin tissue of the control group, complex infiltration of inflammatory cells in the hair follicles and around the hair follicles was observed, and especially lymphocyte infiltration was frequently found. These are main characteristics used for diagnosing alopecia areata. And follicular dystrophy is a phenomenon that occurs frequently in alopecia areata, which means that hair follicles are twisted and deformed or have weak or disrupted hair shaft.

(41) As can be seen from FIG. 7, inflammation and deformed hair shaft were developed in the skin of the control group. On the other hand, the development of inflammation and deformed hair shaft was significantly lower in Neo-Pep polypeptide treated group compared to the control group. The overall alopecia state changes depending on the hair shaft breakage near the skin surface or skin surface. And the alopecia state was more clearly observed in the control group where the malformed hair shaft such as hair shaft breakage was relatively more.

Example 4

Effect of the Polypeptide of the Present Invention on Proliferation of Hair Follicle Stem Cells

(42) The present inventor prepared N1 (SEQ ID NO: 2), N2 (SEQ ID NO: 3), N3 (SEQ ID NO: 4), N4 (SEQ ID NO: 5), N5 (SEQ ID NO: 6), N6 (SEQ ID NO: 7), N7 (SEQ ID NO: 8) and N8 (SEQ ID NO: 9) in order to identify peptide fragments having the effect on promoting hair growth and improving alopecia in the Neo-Pep polypeptide (SEQ ID NO: 1) in which the hair growth promoting effect was confirmed in the above Examples.

(43) Table 1 below shows the amino acid sequence, PI and Tm of Neo-Pep (SEQ ID NO: 1) and its fragments, N1 (SEQ ID NO: 2), N2 (SEQ ID NO: 3), N3 (SEQ ID NO: 4), N4 (SEQ ID NO: 5), N5 (SEQ ID NO: 6), N6 (SEQ ID NO: 7), N7 (SEQ ID NO: 8) and N8 (SEQ ID NO: 9).

(44) TABLE-US-00001 TABLE 1 Polypeptide (SEQ ID) Amino acid sequence PI Tm Neo-Pep (SEQ ID 6                                      46 9.33 >65 NO: 1) AVLKRLEQKGAEADQIIEYLKQQVSLLKEKAILQATLREEK N1 AVLKRLEQKGAEADQIIEYL 4.64 55~65 (SEQ ID NO: 2) N2   LKRLEQKGAEADQIIEYLKQ 7.05 <55 (SEQ ID NO: 3) N3         KGAEADQIIEYLKQQVSLLK 7.01 55~65 (SEQ ID NO: 4) N4           AEADQIIEYLKQQVSLLKEK 4.64 55~65 (SEQ ID NO: 5) N5                 IEYLKQQVSLLKEKAILQAT 9.53 >65 (SEQ ID NO: 6) N6                   YLKQQVSLLKEKAILQATLR 10.56 >65 (SEQ ID NO: 7) N7                     KQQVSLLKEKAILQATLREE 9.8 >65 (SEQ ID NO: 8) N8                       QVSLLKEKAILQATLREEK 9.8 55~65 (SEQ ID NO: 9)

(45) For reference, the polypeptide Neo-Pep (SEQ ID NO: 1) of the present invention corresponds to the 6.sup.th to 46.sup.th amino acid residue in the AIMP1 protein (SEQ ID NO: 16).

(46) TABLE-US-00002 MANNDAVLKRLEQKGAEADQIIEYLKQQVSLLKEKAILQATLREEKKLRV ENAKLKKEIEELKQELIQAEIQNGVKQIPFPSGTPLHANSMVSENVIQST AVTTVSSGTKEQIKGGTGDEKKAKEKIEKKGEKKEKKQQSIAGSADSKPI DVSRLDLRIGCIITARKHPDADSLYVEEVDVGEIAPRTVVSGLVNHVPLE QMQNRMVILLCNLKPAKMRGVLSQAMVMCASSPEKIEILAPPNGSVPGDR ITFDAFPGEPDKELNPKKKIWEQIQPDLHTNDECVATYKGVPFEVKGKGV CRAQTMSNSGIK

(47) CD34+ hair follicle stem cells in vitro assay and BrdU proliferation assay method described above were used. As can be seen in FIG. 8 in which the relative number of CD34+ hair follicle stem cell was compared, Neo-Pep (SEQ ID NO: 1), and its fragments (N5 (SEQ ID NO: 6), N6 (SEQ ID NO: 7), N7 (SEQ ID NO: 8) and N8 (SEQ ID NO: 9)) comprising 28.sup.th to 31.sup.st amino acid residue (KEKA) of Neo-Pep polypeptide and consisting of 19 or 20 consecutive amino acids induced proliferation of CD34+ hair follicle stem cells.

(48) On the other hand, fragments (N1 (SEQ ID NO: 2), N2 (SEQ ID NO: 3), N3 (SEQ ID NO: 4) and N4 (SEQ ID NO: 5)) of Neo-Pep (SEQ ID NO: 1) consisting of 20 consecutive amino acids without 28.sup.th to 31.sup.st amino acid residue (KEKA) of Neo-Pep polypeptide didn't show any effect on inducing proliferation of CD34+ hair follicle stem cells.

(49) Through the above results, it was confirmed that not only Neo-Pep polypeptide (SEQ ID NO: 1) but also polypeptide fragments which comprise 28.sup.th to 31.sup.st amino acid residue of Neo-Pep polypeptide as an active amino acid residue and consist of consecutive amino acids have effect on promoting hair growth and improving alopecia.

Example 5

Effect of the Polypeptide of the Present Invention on Promoting In Vivo Hair Growth

(50) The present inventor prepared N9 (SEQ ID NO: 10), N10 (SEQ ID NO: 11), N11 (SEQ ID NO: 12), N12 (SEQ ID NO: 13), N13 (SEQ ID NO: 14) and N14 (SEQ ID NO: 15), which are polypeptide fragments consisting of 15 amino acids, in order to further identify peptide fragments having the effect on promoting hair growth and improving alopecia in the Neo-Pep polypeptide (SEQ ID NO: 1) in which the hair growth promoting effect was confirmed in the above Examples.

(51) Table 2 below shows the amino acid sequence, PI and Tm of Neo-Pep (SEQ ID NO: 1) and its fragments, N9 (SEQ ID NO: 10), N10 (SEQ ID NO: 11), N11 (SEQ ID NO: 12), N12 (SEQ ID NO: 13), N13 (SEQ ID NO: 14) and N14 (SEQ ID NO: 15).

(52) TABLE-US-00003 TABLE 2 Polypeptide (SEQ ID) Amino acid sequence PI Tm Neo-Pep 6                                      46 9.33 >65 (SEQ ID AVLKRLEQKGAEADQIIEYLKQQVSLLKEKAILQATLREEK NO: 1) N9 AVLKRLEQKGAEADQ 7.08 >65 (SEQ ID NO: 10) N10            EADQIIEYLKQQVSL 3.66 >65 (SEQ ID NO: 11) N11             ADQIIEYLKQQVSLL 3.99 >65 (SEQ ID NO: 12) N12                 IEYLKQQVSLLKEKA 9.53 >65 (SEQ ID NO: 13) N13                     KQQVSLLKEKAILQA 10.41 >65 (SEQ ID NO: 14) N14                           LKEKAILQATLREEK 9.8 <55 (SEQ ID NO: 15)

(53) As can be seen in FIG. 9 (dorsal skin images of mice at 13 days after depilation using the same method as in Example 1), dorsal hair of the mouse group treated with the polypeptide fragments of the present invention (N12 (SEQ ID NO: 13), N13 (SEQ ID NO: 14) and N14 (SEQ ID NO: 15)) comprising 28.sup.th to 31.sup.st amino acid residue (KEKA) of Neo-Pep polypeptide and consisting of 15 consecutive amino acids grew much more than control group (Con, 20% glycerol/PBS treated group), and grew even similar to or much more than positive control group (MNX, 3% Minoxidil treated group).

(54) On the other hand, fragments (N9 (SEQ ID NO: 10), N10 (SEQ ID NO: 11) and N11 (SEQ ID NO: 12)) of Neo-Pep (SEQ ID NO: 1) consisting of 15 consecutive amino acids without 28.sup.th to 31.sup.st amino acid residue (KEKA) of Neo-Pep polypeptide didn't show such effect.

(55) Through the above results, it was further confirmed that polypeptide fragments such as N12 (SEQ ID NO: 13), N13 (SEQ ID NO: 14) and N14 (SEQ ID NO: 15), which comprise 28.sup.th to 31.sup.st amino acid residue of Neo-Pep polypeptide (SEQ ID NO: 1) as an active amino acid residue and consist of consecutive amino acids, have effect on promoting hair growth and improving alopecia. Thus, it was confirmed that comprising the 28.sup.th to 31.sup.st amino acid residue (KEKA) of the Neo-Pep polypeptide (SEQ ID NO: 1) as an active site is essential for effect on promoting hair growth and improving alopecia.