Composition for preventing hair loss or promoting hair growth comprising recombinant polypeptide

Abstract

The present invention relates to a composition for preventing hair loss or promoting hair growth, the composition comprising a recombinant polypeptide. In the present invention, the expression of 5ɑ-reductase, which is a cause of hair loss, can be inhibited by using a recombinant polypeptide, and the growth of outer root sheath cells of human hair follicles can be increased. In addition, the composition comprising the recombinant polypeptide can exhibit the effects of improving dandruff and alleviating itching of the scalp, and preventing hair loss while preventing transdermal moisture loss and increasing the moisture content of the scalp. Thus, the present invention can be effectively used in a cosmetic or pharmaceutical composition exhibiting the effect of alleviating, preventing, or treating hair loss.

Description

Composition for preventing hair loss or promoting hair growth comprising a recombinant polypeptide

[0001] The present invention relates to a composition for preventing hair loss or promoting hair growth comprising a recombinant polypeptide, and more specifically, to a cosmetic composition having an excellent effect for preventing hair loss or promoting hair growth and a pharmaceutical composition for preventing or treating hair loss, which comprises a recombinant polypeptide as an active ingredient.

[0002] The human body has approximately 100,000 to 150,000 hairs, each with its own distinct cycle, progressing through the growth (anagen), regression (catagen), and resting (telogen) phases before falling out. Normally, an average of 50 to 100 hairs fall out daily. Alopecia refers to a condition in which the proportion of hairs in the growth phase decreases, while the proportion of hairs in the catagen or resting phase increases, resulting in an abnormally high number of hairs falling out.

[0003] Causes of hair loss have been discussed, including excessive androgen production, seborrheic dermatitis, stress, obesity, peroxides, and bacterial deterioration of the scalp. However, the exact cause remains unknown. Recently, the number of people suffering from hair loss has been increasing due to factors such as changes in diet and increased stress caused by the social environment. The average age of those affected is also decreasing, and the number of women experiencing hair loss is also increasing. Accordingly, active research is being conducted on functional ingredients with hair loss prevention properties.

[0004] For example, Korean Patent Publication No. 10-2023-0090394 discloses a technology for preventing hair loss and promoting hair growth by proliferating hair papilla cells using Sophora chinensis extract, Phellodendron amurense extract, Scutellaria baicalensis extract, Ophiopogon japonicus extract, and Trichophyton rhizome extract, and Korean Patent Publication No. 10-2023-0130405 describes a composition that includes Angelica dentata extract to prevent hair loss and promote hair growth by inducing or promoting the growth of human hair papilla cells. However, these natural products have problems such as lower activity compared to existing pharmaceuticals or difficulty in long-term stable storage, and thus need improvement.

[0005] Meanwhile, recent research has highlighted dihydrotestosterone (DHT) as a major factor in hair loss. Testosterone, a male hormone, is converted to DHT by the action of 5α-reductase. It is argued that the hair follicles of patients with hair loss have high levels of 5α-reductase activity, which contributes to hair loss. Finasteride is currently marketed as a drug that inhibits 5α-reductase activity and prevents hair loss. However, side effects such as depression, sexual dysfunction, and suicidal impulses have been reported, and it is known to increase the risk of birth defects.

[0006] As such, many conventional hair loss treatments have limited use due to low therapeutic efficacy or toxicity and side effects. Therefore, there is a need for the development of functional raw materials that have excellent hair loss prevention and hair growth promotion effects without causing toxicity or skin irritation.

[0007] The purpose of the present invention is to provide a cosmetic composition that exhibits an effect of preventing hair loss or promoting hair growth by using a highly functional recombinant polypeptide that does not cause irritation to the skin.

[0008] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hair loss comprising the recombinant polypeptide.

[0009] To achieve the above purpose, the present invention provides a cosmetic composition for preventing hair loss or promoting hair growth, comprising a recombinant polypeptide in which a polypeptide consisting of an amino acid sequence of SEQ ID NO: 2 is linked to a polypeptide consisting of an amino acid sequence of SEQ ID NO: 1.

[0010] In the present invention, the polypeptide of sequence number 1 and the polypeptide of sequence number 2 may be linked via a linker consisting of 1 to 10 amino acids.

[0011] In the present invention, the recombinant polypeptide may have an amino acid sequence of SEQ ID NO: 3.

[0012] In the present invention, the concentration of the recombinant polypeptide is preferably 0.1 to 10 μM.

[0013] In the present invention, the cosmetic composition may have at least one function selected from the group consisting of hair tonic, hair cream, hair lotion, hair shampoo, hair rinse, hair conditioner, hair spray, hair aerosol, pomade, powder gel, hair pack, hair treatment, eyebrow hair growth agent, eyelash hair growth agent, and eyelash nutrient.

[0014] In the present invention, the cosmetic composition may exhibit activity that inhibits the expression of 5α-reductase or activity that promotes the growth of outer root sheath cells.

[0015] The present invention also provides a method for preventing hair loss, comprising the step of applying a cosmetic composition comprising the recombinant polypeptide.

[0016] The present invention also provides a method for promoting hair growth, comprising the step of applying a cosmetic composition comprising the recombinant polypeptide.

[0017]

[0018] The present invention also provides a pharmaceutical composition for preventing or treating hair loss, comprising a recombinant polypeptide in which a polypeptide comprising an amino acid sequence of SEQ ID NO: 2 is linked to a polypeptide comprising an amino acid sequence of SEQ ID NO: 1.

[0019] In the present invention, the pharmaceutical composition may be a topical skin preparation.

[0020] The present invention also provides a method for preventing or treating hair loss, comprising administering a pharmaceutical composition comprising the recombinant polypeptide.

[0021]

[0022] The present invention utilizes a recombinant polypeptide to provide excellent hair loss prevention or hair growth promotion effects without cytotoxicity or skin irritation. Specifically, the present invention utilizes a recombinant polypeptide to reduce the expression of 5α-reductase, a cause of hair loss, and to promote the growth of outer root sheath cells of human hair follicles. Furthermore, a composition comprising the recombinant polypeptide can improve scalp dandruff, relieve itching, prevent transdermal moisture loss, increase scalp moisture content, and prevent hair loss. Therefore, the present invention can be effectively utilized in cosmetics or pharmaceutical compositions that exhibit hair loss improvement, prevention, or treatment effects.

[0023]

[0024] Figure 1 shows the results of a cell viability test of a recombinant polypeptide according to one embodiment of the present invention.

[0025] Figures 2a and 2b show the results of a 5α-reductase RT-qPCR test of a recombinant polypeptide according to one embodiment of the present invention (a) and the results of comparison with a control polypeptide (b), respectively.

[0026] Figure 3 shows the results of an analysis of the growth of the outer root of a recombinant polypeptide according to one embodiment of the present invention.

[0027] Figures 4a and 4b show the results of dandruff analysis (a) and experimental photographs (b) according to whether a product containing a recombinant polypeptide was used in one embodiment of the present invention, respectively.

[0028] Figure 5 shows the results of an itching analysis according to whether a product containing a recombinant polypeptide is used in one embodiment of the present invention.

[0029] Figure 6 shows the results of analysis of transepidermal water loss according to whether a product containing a recombinant polypeptide is used in one embodiment of the present invention.

[0030] Figure 7 shows the results of measuring scalp moisture content according to whether or not a product containing a recombinant polypeptide is used in one embodiment of the present invention.

[0031] Figure 8 shows the results of measuring the number of hair loss depending on whether a product containing a recombinant polypeptide is used in one embodiment of the present invention.

[0032] Hereinafter, specific implementations of the present invention will be described in more detail. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0033]

[0034] The present invention relates to a composition for preventing hair loss or promoting hair growth comprising a recombinant polypeptide.

[0035] In describing the present invention, “hair loss” means the absence of hair in an area where hair should normally exist, and includes a phenomenon in which the number of hairs decreases compared to the normal state.

[0036] In the present invention, a recombinant polypeptide designed to have a specific amino acid sequence can be used to provide an excellent hair loss prevention or hair growth promotion effect without being cytotoxic or causing skin irritation.

[0037] The recombinant polypeptide used in the present invention is characterized in that a polypeptide consisting of an amino acid sequence of SEQ ID NO: 2 is linked to a polypeptide consisting of an amino acid sequence of SEQ ID NO: 1 below.

[0038]

[0039] [Sequence number 1]

[0040] TNRPDYNDDEEDDYKPPVYKPSPSKYRPVNPCLKKPCKYNGVCKPRGGSYKCFCKGGYYGYNCLKNACKPNQKNKSRCVPVGKTFKCVCRNGNFGRLCEKNVCSPNPCKNNGKCSPLGKTGYKCTCSGGYTGPRCEVHACKNPCKNKGRCFPDGKTGYKCRCVDGYSGPTC

[0041]

[0042] [Sequence number 2]

[0043] AKRHHGYKRKFH

[0044]

[0045] In the present invention, the polypeptide of sequence number 1 has an amino acid sequence derived from a mussel adhesive protein. While the mussel adhesive protein has been reported to exhibit adhesive functions such as natural adhesives and bioadhesive materials, it has not been reported that it can be used to design a recombinant polypeptide that improves the scalp environment and prevents hair loss.

[0046] The present invention was completed by discovering that a hair loss prevention effect is exhibited when a specific amino acid sequence is conjugated to the polypeptide of the above sequence number 1. In the present invention, the amino acid sequence of the above sequence number 1 can exhibit hair loss prevention and hair growth promotion functionality by being combined with the amino acid sequence of the above sequence number 2.

[0047] In this regard, in an embodiment of the present invention, it was confirmed that when another functional peptide was linked to the polypeptide of sequence number 1, it had little effect on the expression of 5α-reductase, whereas when the polypeptide of sequence number 2 was linked to form a recombinant peptide, the expression level of 5α-reductase was greatly reduced. From this, it can be confirmed that the recombinant polypeptide of the present invention exhibits an excellent hair loss prevention effect through a combination of unique amino acid sequences.

[0048] In the present invention, the polypeptide of SEQ ID NO: 2 can be linked to the C-terminus, N-terminus, or both ends of the polypeptide of SEQ ID NO: 1.

[0049] Additionally, the polypeptide of SEQ ID NO: 1 and the polypeptide of SEQ ID NO: 2 may be directly linked, or may be linked via an amino acid linker. In this case, the amino acid linker may be composed of 1 to 10 amino acids, and preferably 2 to 6 amino acids. For example, the linker may be in the form of a KL dipeptide.

[0050] In a preferred embodiment of the present invention, the recombinant polypeptide may be comprised of a polypeptide comprising an amino acid sequence of SEQ ID NO: 1; a KL linker; and a polypeptide comprising an amino acid sequence of SEQ ID NO: 2. The amino acid sequence of the recombinant polypeptide according to the embodiment may be represented by SEQ ID NO: 3 below.

[0051]

[0052] [Sequence number 3]

[0053] TNRPDYNDDEEDDYKPPVYKPSPSKYRPVNPCLKKPCKYNGVCKPRGGSYKCFCKGGYYGYNCLKNACKPNQKNKSRCVPVGKTFKCVCRNGNFGRLCEKNVCSPNPCKNNGKCSPLGKTGYKCTCSGGYTGPRCEVHACKNPCKNKGRCFPDGKTGYKCRCVDGYSGPTCKLAKRHHGYKRKFH

[0054]

[0055] In the present invention, the recombinant polypeptide can be produced using a method known in the field of biotechnology, and for example, can be produced by transforming a host cell with a vector containing a gene encoding the polypeptide.

[0056] In describing the present invention, the term "gene" is interpreted to mean a nucleic acid, such as a DNA sequence, containing a coding sequence necessary for producing a polypeptide. The gene may be encoded by the entire region or a portion of the coding sequence.

[0057] In the present invention, the vector may be a recombinant vector, i.e., an expression vector, capable of expressing a desired peptide in a host cell. The expression vector may include expression control elements such as a promoter, operator, and initiation codon, and may include a signal sequence for the release of the fusion polypeptide.

[0058] Plasmid DNA, phage DNA, etc. can be used as the above expression vector. Specific examples of plasmid DNA include pUC18, pIDTSAMRT-AMP, etc., and in the present invention, plasmids derived from Escherichia coli (pET-22b(+), pYG601BR322, pBR325, pUC118, and pUC119), plasmids derived from Bacillus subtilis (pUB110 and pTP5), and plasmids derived from yeast (YEp13, YEp24, and YCp50) can be used. The above expression vector can be appropriately selected and used depending on the type of host cell.

[0059] In the present invention, the expression vector can be transformed into a host cell such as Escherichia coli (E. coli) to produce a transformant, which can then be cultured to obtain a recombinant polypeptide. The recombinant polypeptide can be used in a composition after additional isolation and purification processes.

[0060] In the present invention, the recombinant polypeptide may be used in the composition at a concentration of 0.1 to 10 μM. When the polypeptide of the present invention is included in the above concentration range, it can alleviate dandruff and itching symptoms without causing skin irritation or toxicity, and improve hair loss symptoms while preventing moisture loss from the scalp.

[0061] In this aspect, the recombinant polypeptide may be included in an amount of 0.0001 to 0.1 wt%, for example, 0.001 to 0.01 wt%, based on the total weight of the composition.

[0062] The composition of the present invention can provide an effect of preventing hair loss and / or promoting hair growth by including the recombinant polypeptide. Specifically, the composition of the present invention can promote the formation and / or proliferation of human dermal papilla (HDP) cells; the migration and / or proliferation of keratinocytes; the development, regeneration, and / or elongation of hair follicles; and hair growth.

[0063] In particular, the recombinant polypeptide of the present invention can promote the growth of outer root sheath (ORS) cells of human hair follicles. The ORS cells are a type of keratinocyte located at the outermost layer of the hair follicle and play a very important role in the growth of the hair follicle. Since they are located outside the dermal papilla cells, they are cells that are easily attacked by inflammatory factors. In this regard, in the examples of the present invention, it was confirmed that the cell growth of the ORS cells of human hair follicles increased when the recombinant polypeptide was treated, and through this, the effect of improving hair loss can be confirmed.

[0064] In addition, the recombinant polypeptide of the present invention can exert an effect of reducing the expression level of 5α-reductase. The 5α-reductase is an enzyme that reduces androgenetic hormone testosterone and converts it into dihydrotestosterone (DHT), and is known to play an important role in the cause of hair loss. In this regard, in the examples of the present invention, it was confirmed that the recombinant polypeptide has an effect of reducing the expression level of 5α-reductase, and through this, hair loss improvement activity can be confirmed.

[0065] In addition, in the examples of the present invention, it was confirmed that the recombinant polypeptide plays a role in reducing the amount of dandruff occurring on the scalp, improving itchiness, suppressing moisture loss from the scalp, and increasing moisture content, and when applied to humans, it was confirmed that the number of hair loss was significantly reduced compared to the control group.

[0066] Therefore, the present invention can be effectively used in a cosmetic or pharmaceutical composition that exhibits an effect of improving, preventing, or treating hair loss, or an effect of promoting hair growth.

[0067] In describing the present invention, the term "improvement" refers to any action that improves or beneficially alters the symptoms of a disease. In addition, the term "prevention" refers to reducing the risk of contracting a disease, and refers to any action that inhibits or delays the onset of a disease by preventing the progression of one or more clinical symptoms of the disease in a subject who is exposed to or susceptible to the disease but has not yet contracted the disease or shown symptoms of the disease. In addition, the term "treatment" refers to alleviating a disease, and refers to any action that improves or beneficially alters the symptoms of the disease by inhibiting or reducing the progression of the disease or one or more clinical symptoms thereof.

[0068]

[0069] Specifically, the composition for preventing hair loss or promoting hair growth comprising the recombinant polypeptide of the present invention may be a cosmetic composition.

[0070] Accordingly, the present invention also provides a method for preventing hair loss, comprising the step of applying a cosmetic composition comprising the recombinant polypeptide.

[0071] The present invention also provides a method for promoting hair growth, comprising the step of applying a cosmetic composition comprising the recombinant polypeptide.

[0072] Specifically, the cosmetic composition of the present invention can be applied topically to the skin, preferably to the scalp or eyelash skin.

[0073] The subject of application of the above cosmetic composition may be a subject, specifically a subject in need of hair loss prevention or hair growth promotion, and the subject may be an animal, and typically a mammal, for example, a human.

[0074] In the present invention, the formulation of the cosmetic composition can be prepared in any formulation commonly manufactured in the art, and for example, can be a formulation selected from the group consisting of a solution, emulsion, suspension, paste, gel, cream, lotion, powder, soap, cleansing, oil, powder or spray.

[0075] In the present invention, when the formulation of the cosmetic composition is a solution or emulsion, a solvent, a solvating agent, or an emulsifying agent may be used as the carrier component. For example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or sorbitan fatty acid ester may be used.

[0076] In the present invention, when the formulation of the cosmetic composition is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, etc. can be used as a carrier component.

[0077] In the present invention, when the formulation of the cosmetic composition is a paste, gel, cream or lotion, animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0078] In the present invention, when the formulation of the cosmetic composition is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether may be additionally included.

[0079] In the present invention, when the formulation of the cosmetic composition is cleansing, a carrier component may be used, such as an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyl taurate, a sarcosinate, a fatty acid amide ether sulfate, an alkyl amidobetaine, a fatty alcohol, a fatty acid glyceride, a fatty acid diethanolamide, a vegetable oil, a linolenic derivative, or an ethoxylated glycerol fatty acid ester.

[0080] The cosmetic formulation of the present invention can also be easily selected and blended with other ingredients, etc., which can preferably provide a synergistic effect to the main effect, within a range that does not impair the main effect aimed at by the present invention, depending on the formulation or purpose of use of the cosmetic.

[0081] In the present invention, the formulation and use of the cosmetic composition are not particularly limited and may be appropriately selected depending on the purpose. For example, the cosmetic composition of the present invention may have one or more formulations and uses including a solution, suspension, emulsion, gel, lotion, essence, cream, powder, soap, shampoo, rinse, and pack mask.

[0082] In particular, the cosmetic composition of the present invention may have at least one function selected from the group consisting of hair tonic, hair cream, hair lotion, hair shampoo, hair rinse, hair conditioner, hair spray, hair aerosol, pomade, powder gel, hair pack, hair treatment, eyebrow hair growth agent, eyelash hair growth agent, and eyelash nutrient.

[0083] In addition to the recombinant polypeptide, the cosmetic composition of the present invention may further include components commonly used in cosmetic compositions, such as conventional auxiliary agents such as antioxidants, stabilizers, moisturizers, solubilizers, vitamins, ultraviolet absorbers, pigments, and fragrances, and may further include preservatives, sterilizers, and the like.

[0084]

[0085] In addition, the present invention can provide a pharmaceutical composition for preventing or treating hair loss comprising the recombinant polypeptide.

[0086] Accordingly, the present invention also provides a method for preventing or treating hair loss, comprising administering a pharmaceutical composition comprising the recombinant polypeptide. In this case, the subject of administration of the pharmaceutical composition may be a subject, specifically a subject in need of hair loss prevention or treatment, and the subject may be an animal, and typically a mammal, such as a human.

[0087] In the present invention, hair loss to be prevented or treated is a concept that includes both non-scarring hair loss and scarring hair loss depending on whether hair follicles are destroyed, and includes, for example, hereditary androgenetic alopecia, alopecia areata, tinea capitis caused by fungal infection, telogen effluvium, trichotillomania, hair growth disorder disease, hair loss caused by lupus, folliculitis pilaris, lichen planus, hair loss caused by burns and / or trauma, etc.

[0088] In addition to the recombinant polypeptide, the pharmaceutical composition may further comprise an appropriate pharmaceutically acceptable carrier, excipient or diluent according to a conventional method. The pharmaceutically acceptable carrier is one commonly used in formulation, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0089] The pharmaceutical composition of the present invention may further include, in addition to the above ingredients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. With regard to suitable pharmaceutically acceptable carriers and formulations, each ingredient can be preferably formulated according to the method disclosed in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0090] In a preferred embodiment of the present invention, the pharmaceutical composition may be a topical skin preparation. A topical skin preparation is a preparation that can be applied to the outside of the skin. When the composition of the present invention is used as a topical skin preparation, it can be applied topically to the skin, for example, to the scalp or eyelashes, and specifically, to the scalp where hair loss has occurred or to the scalp where hair growth is desired to be promoted.

[0091] The above skin external preparation may be a cream, gel, ointment, emulsifier, suspension, transdermal delivery patch, drug-containing bandage, lotion or a combination thereof, and may be appropriately mixed with ingredients commonly used in skin external preparations, such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients or combinations thereof, as needed.

[0092] The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, factors including concurrently used drugs, and other factors well known in the medical field. The composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take all of the above factors into consideration and administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by a skilled artisan.

[0093]

[0094] Example

[0095]

[0096] The present invention is described in more detail through the following examples. However, these examples are intended to illustrate some experimental methods and configurations of the present invention, and the scope of the present invention is not limited to these examples.

[0097]

[0098] Manufacturing example: Production of recombinant polypeptide

[0099]

[0100] The recombinant MGFP-2 gene was cloned based on the amino acid sequence of sequence number 3. After synthesizing template DNA, the gene was amplified by PCR using primers and inserted into the pET22b(+) vector (Cat. No. 69744-3, Novagen).

[0101]

[0102] [Sequence number 3]

[0103] TNRPDYNDDEEDDYKPPVYKPSPSKYRPVNPCLKKPCKYNGVCKPRGGSYKCFCKGGYYGYNCLKNACKPNQKNKSRCVPVGKTFKCVCRNGNFGRLCEKNVCSPNPCKNNGKCSPLGKTGYKCTCSGGYTGPRCEVHACKNPCKNKGRCFPDGKTGYKCRCVDGYSGPTCKLAKRHHGYKRKFH

[0104]

[0105] Escherichia coli (E. coli) DH5α cells for cloning and E. coli BL21(DE3) cells for protein expression were made competent using CaCl2 buffer, and then transformed with the pET22b(+) Mgfp2 / histatin vector using a heat shock method in which the cells were left at 42°C for 1 minute. Transformed colonies were selected using ampicillin (Gold bio) to obtain E. coli DH5α pET22b(+) MGFP-2 / histatin and E. coli BL21(DE3) mgfp2 / histatin transformants.

[0106] Escherichia coli BL21 / pET22b(+) was cultured in LB medium (5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl), and when the absorbance of the culture medium reached approximately 0.7 at 600 nm, IPTG was added to induce the expression of an antimicrobial recombinant polypeptide conjugated with a recombinant MGFP-2 polypeptide and an antimicrobial peptide. The expressed antimicrobial recombinant polypeptide was then isolated and purified to obtain a final high-purity protein.

[0107]

[0108] Experimental Example 1: Cell Viability Test using MTT Assay

[0109]

[0110] The recombinant polypeptide of the manufacturing example was treated on HFDPC (human follicular dermal palilla cells, PromoCell, Germany), and cell viability was tested using the MTT (Thiazolyl Blue Tetrazolium Bromide) assay.

[0111] 100mm 2 1 Х 10 HFDPC in a culture dish 6 After inoculation into cells / dish, Human Dermal Papilla Growth Medium containing penicillin (100 IU / ml), streptomycin (100 μg / mL) and supplements was added and cultured in an incubator (Sanyo, Japan) containing 5% carbon dioxide at 37°C.

[0112] To test cell viability, subcultured HFDPC cells were seeded in 96-well plates at 1 Х 10 4 Cells were seeded per well and cultured for 24 hours in an incubator containing 5% carbon dioxide at 37°C. Subsequently, the polypeptide of the manufacturing example was treated to HFDPC cells at final treatment concentrations of 0.125, 0.25, 0.5, and 1 μM, respectively, and cultured under cell culture conditions for 24 hours.

[0113] After incubation, MTT solution was added to each well, incubated for 2 hours in a darkened environment, and the supernatant was removed. The produced formazan was completely dissolved in DMSO, and the absorbance was measured at 540 nm using a microplate reader (Biotek Synergy-HT, USA). Based on the measurement results, the percentage compared to the untreated group was calculated and is shown in Figure 1.

[0114] As a result of the experiment, the polypeptide-treated group showed cell viability of 102.42% at a concentration of 0.125 μM, 104.48% at a concentration of 0.25 μM, 104.16% at a concentration of 0.5 μM, and 100.22% at a concentration of 1 μM, respectively, compared to the untreated group. From the above results, it was confirmed that the polypeptide of the present invention did not affect cytotoxicity for HFDPC cells at all treatment concentrations.

[0115]

[0116] Experimental Example 2: 5α-reductase mRNA expression test

[0117]

[0118] 5α-reductase, an enzyme that converts the androgen testosterone into dihydrotestosterone (DHT), is known to play a key role in hair loss. To quantitatively analyze the mRNA expression of 5α-reductase, a real-time polymerase chain reaction (RT-qPCR) test was performed.

[0119] Subcultured HFDPC cells were plated at 5 Х 10 in 60 mm dishes. 5The cells were seeded into dishes and cultured for 24 hours in an incubator containing 5% carbon dioxide at 37°C. The culture medium was then replaced with serum-free DMEM. Subsequently, 20 nM testosterone was added to the cells to induce 5α-reductase mRNA expression. Simultaneously, the polypeptides of the manufacturing example were diluted to final treatment concentrations of 0.125, 0.25, 0.5, and 1 μM, respectively, and the cells were cultured for 24 hours. After culture, RNA was extracted from the cells, cDNA was synthesized, and RT-qPCR was performed to confirm the level of 5α-reductase mRNA expression.

[0120] Figure 2a shows the results of the RT-qPCR test, and in the polypeptide treatment group according to the present invention, the expression level of 5α-reductase mRNA was measured as 1.28 at a concentration of 0.125 μM, 1.17 at a concentration of 0.25 μM, 1.06 at a concentration of 0.5 μM, and 0.95 at a concentration of 1 μM.

[0121] From the above experimental results, it was confirmed that the polypeptide of the present invention has the effect of lowering the expression level of 5α-reductase mRNA induced by testosterone in HFDPC cells.

[0122] In addition, in order to compare the effect of the polypeptide according to the present invention with a polypeptide having a different amino acid sequence, a control polypeptide having the amino acid sequence of SEQ ID NO: 4 below was produced, and the result was confirmed after 1 μM treatment using the same method as above.

[0123]

[0124] [Sequence number 4]

[0125] TNRPDYNDDEEDDYKPPVYKPSPSKYRPVNPCLKKPCKYNGVCKPRGGSYKCFCKGGYYGYNCLKNACKPNQKNKSRCVPVGKTFKCVCRNGNFGRLCEKNVCSPNPCKNNGKCSPLGKTGYKCTCSGGYTGPRCEVHACKNPCKNKGRCFPDGKTGYKCRCVDGYSGPTCKLLKKLAKLALAF

[0126]

[0127] Figure 2b shows the results of the above comparative experiment, and in the case of the polypeptide treatment group according to the present invention, the expression level of 5α-reductase mRNA was confirmed to be 0.95, while in the polypeptide treatment group of the control group, it was confirmed to be 1.42.

[0128] Accordingly, it was confirmed that the polypeptide of the present invention has the effect of lowering the expression level of 5α-reductase mRNA induced by testosterone in HFDPC cells, whereas the control polypeptide exhibits almost no inhibitory effect despite having an amino acid sequence similar to that of the polypeptide of the present invention.

[0129]

[0130] Experimental Example 3: Expression Test of Outer Root Sheath Cells (ORS) of Human Hair Follicles

[0131]

[0132] The outer root sheath (ORS) cells of human hair follicles are keratinocyte cells located at the outermost layer of the hair follicle and play a crucial role in hair follicle growth. A test was conducted to determine the effect of the polypeptide of the present invention on ORS cell growth.

[0133] First, human ORS cells (1.5 x 10 3Cells) were seeded in 96-well, and the following day, the polypeptides of the manufacturing example were treated at concentrations of 0.125 and 0.25 μM, respectively. Five replicate experimental groups were prepared for each concentration group. The experimental group and the control group (DMSO) were cultured in a cell culture incubator for 3 days, and after 5 days, the culture medium was removed, and 100 μl of 1x EZ-cytox (cell viability, proliferation, cytotoxicity assay kit) diluted in PBS was added each, and the cells were cultured in a cell culture incubator for about 2 hours. After that, the absorbance (OD) was measured at 450 nm using a reader.

[0134] Figure 3 shows the results of absorbance measurements of the ORS cells, and the experimental results confirmed that the polypeptide of the manufacturing example increased ORS cell growth by 24.1% and 11.9% at concentrations of 0.125 and 0.25 μM, respectively. Accordingly, it was confirmed that the polypeptide of the present invention can effectively promote the growth of outer root sheath cells of human hair follicles.

[0135]

[0136] Experimental Example 4: Evaluation of scalp dandruff in the forehead area

[0137]

[0138] In order to confirm the effect of the polypeptide of the present invention on dandruff of the scalp, the difference in dandruff amount depending on whether or not a test product manufactured using the polypeptide of the manufacturing example was used was analyzed, and the experimental results were compared with the experimental results of the control product use group. In Experimental Examples 4 to 9, the test product was used in which 0.003 g of the recombinant polypeptide of the manufacturing example, 0.25 g of salicylic acid, 0.2 g of dexpanthenol, and 0.3 g of L-menthol were dissolved in purified water and a cosmetic based on 100 g, and a product with the same other raw materials but excluding only the polypeptide was used as a control product.

[0139] The above dandruff analysis was based on the 'Guidelines for the Evaluation of Dandruff and Itching Relief of Hair Cosmetics (Guide-0331-01)', and the number of test subjects was at least 20 people in each of the test and control groups to enable statistical comparison, and the principle was a controlled comparative study, double-blind, and randomized.

[0140] Visioscan (VC98, CK electronics, Germany) was used as a dandruff measuring device, and dandruff in the captured image was analyzed using the analysis program (VS2000, CK electronics, Germany) provided by the Visioscan device.

[0141] Figures 4a and 4b respectively show the dandruff confirmation results graph (a) and experimental photographs (b). In the case of the test product use group, the improvement rate (%) of scalp dandruff in the forehead area was 48.204% after 2 weeks of product use and 86.964% after 4 weeks of product use, and no adverse skin reactions due to the test product occurred during the evaluation period. On the other hand, in the case of the control product use group, it was confirmed that there was no significant difference in the amount of scalp dandruff in the forehead area compared to before product use (0 week).

[0142] According to the above experimental results, in the group using the test product, the amount of scalp dandruff in the forehead area was significantly reduced after 2 weeks and 4 weeks compared to before using the product (week 0), and it was confirmed that the dandruff reduction effect was much better than that of the group using the control product.

[0143]

[0144] Experimental Example 5: Itching Evaluation

[0145]

[0146] To determine the effect of the polypeptide of the present invention on itching symptoms, the difference in itching analysis values ​​depending on whether or not a test product manufactured using the polypeptide of the manufacturing example was used was analyzed. The itching analysis was performed using a Visual Analogue Scale (VAS, unit: cm).

[0147] Figure 5 shows the graph of the VAS analysis values. In the case of the test product (A), the improvement rate was 41.04% after 4 weeks of product use and 57.98% after 8 weeks of product use compared to before product use (week 0). As such, in the case of the test product (A), the degree of itching was significantly reduced according to the use of the product, and in particular, the degree of itching was reduced from moderate to mild after 8 weeks. On the other hand, the control product (B) showed a minimal improvement in itching compared to the test product (A), and it was confirmed that moderate itching was maintained even after product use.

[0148] From the above experimental results, it can be confirmed that the polypeptide of the present invention can very effectively improve itching.

[0149]

[0150] Experimental Example 6: Measurement of transepidermal water loss (TEWL)

[0151]

[0152] In order to confirm the effect of the polypeptide of the present invention on the moisture content of the skin, the difference in the measured transepidermal water loss (TEWL) value depending on whether or not a test product manufactured using the polypeptide of the manufacturing example was used was analyzed.

[0153] Figure 6 shows the results of the above transepidermal water loss analysis. In the case of the test product (A), it was confirmed that the water loss was reduced compared to before use (0 weeks), and in particular, the water loss was significantly reduced after 8 weeks. On the other hand, the control product (B) showed a minimal effect in reducing water loss, and no significant change in the effect was observed even after the use period.

[0154] From the above experimental results, it was confirmed that the polypeptide of the present invention effectively prevents transdermal moisture loss.

[0155]

[0156] Experimental Example 7: Measurement of scalp moisture content

[0157]

[0158] In order to confirm the effect of the polypeptide of the present invention on the moisture content of the scalp, the difference in scalp moisture content depending on whether or not a test product manufactured using the polypeptide of the manufacturing example was used was analyzed.

[0159] Figure 7 shows the results of the scalp moisture content measurement. Both the test product (A) and the control product (B) showed an increase in the scalp moisture content with use of the product, but it was confirmed that the effect of the test product (A) was far superior.

[0160] From the above experimental results, it was confirmed that the polypeptide of the present invention very effectively increases the moisture content of the scalp.

[0161]

[0162] Experimental Example 8: Measurement of the number of hairs lost

[0163]

[0164] In order to confirm the effect of the polypeptide of the present invention on hair loss symptoms, the number of hair loss was measured according to whether or not a test product manufactured using the polypeptide of the manufacturing example was used, and the difference from the results of using the control product was analyzed.

[0165] Figure 8 shows the results of measuring the number of hair shedding (unit: pieces). It was confirmed that the number of hair shedding decreased with use of both the test product (A) and the control product (B), but it was confirmed that the test product (A) prevented hair loss more effectively than the control product (B). Specifically, in the case of the test product (A), the improvement rate was 35.30% after 4 weeks of product use, 45.95% after 8 weeks of product use, and 55.24% after 12 weeks of product use compared to before product use (week 0).

[0166] According to the above experimental results, it was found that hair loss symptoms can be effectively prevented by using the polypeptide of the present invention.

[0167]

[0168] Experimental Example 9: Measurement of Skin Irritation Index

[0169]

[0170] In order to confirm whether the polypeptide of the present invention causes skin irritation, the intensity of skin irritation was measured according to whether a test product manufactured using the polypeptide of the manufacturing example was used, and the difference in results from the negative control group (DW) and the positive control group (1% SLS) was analyzed.

[0171] Patches containing each product were applied to the skin and removed after 30 minutes, 24 hours, and 48 hours, and the irritation intensity was measured. The average irritation index was calculated according to a formula, and the skin irritation intensity was evaluated according to the judgment criteria. The final suitability was determined based on three criteria: the safety zone for each product, whether it caused irritation at a frequency exceeding 20% ​​of the test subjects, and whether an irritation reaction of +2 or higher was observed in more than 10% of the total test subjects at each reading. The results of the skin irritation intensity test are shown in Table 1 below.

[0172]

[0173] Sum of the skin irritation response intensity of the sample Skin irritation index Irritation judgment grade Safety zone Test subjects Over 20% + 2 or more 10% or more Acceptable or Unacceptable judgment 1st 2nd 3rd test Product 0.000.000.000.00 Non-irritating Suitable Suitable Suitable Suitable Negative control 0.000.000.000.00 Non-irritating Suitable Suitable Suitable Suitable Positive control 6.507.506.007.41 Strong irritation Inappropriate Inappropriate Inappropriate Inappropriate

[0174]

[0175] As a result of the experiment, the test product containing the polypeptide of the present invention showed no irritation at 30 minutes, 24 hours, and 48 hours after removal of the adhesive, and was confirmed to have low skin irritation. Accordingly, it was confirmed that the polypeptide of the present invention can be used to produce a safe product with a low risk of causing irritation.

[0176]

[0177] Although some implementation forms of the present invention have been described above, the present invention is not limited to the implementation forms described above, and can be implemented by modifying and changing them within a scope that does not deviate from the gist of the present invention, and it should be understood that forms with such modifications and changes also fall within the technical spirit of the present invention.

Claims

1. A cosmetic composition for preventing hair loss or promoting hair growth, comprising a recombinant polypeptide in which a polypeptide comprising an amino acid sequence of sequence number 2 is linked to a polypeptide comprising an amino acid sequence of sequence number 1.

2. In paragraph 1, A cosmetic composition for preventing hair loss or promoting hair growth, wherein the polypeptide of sequence number 1 and the polypeptide of sequence number 2 are linked via a linker consisting of 1 to 10 amino acids.

3. In paragraph 1, A cosmetic composition for preventing hair loss or promoting hair growth, wherein the recombinant polypeptide has an amino acid sequence of sequence number 3.

4. In paragraph 1, A cosmetic composition for preventing hair loss or promoting hair growth, wherein the concentration of the recombinant polypeptide is 0.1 to 10 μM.

5. In paragraph 1, A cosmetic composition for preventing hair loss or promoting hair growth, wherein the cosmetic composition has at least one function selected from the group consisting of hair tonic, hair cream, hair lotion, hair shampoo, hair rinse, hair conditioner, hair spray, hair aerosol, pomade, powder gel, hair pack, hair treatment, eyebrow hair tonic, eyelash hair tonic, and eyelash nutrient.

6. In paragraph 1, A cosmetic composition for preventing hair loss or promoting hair growth, wherein the cosmetic composition exhibits activity for inhibiting the expression of 5α-reductase or activity for promoting the growth of outer root sheath cells.

7. A pharmaceutical composition for preventing or treating hair loss, comprising a recombinant polypeptide in which a polypeptide comprising an amino acid sequence of sequence number 2 is linked to a polypeptide comprising an amino acid sequence of sequence number 1.

8. In paragraph 7, A pharmaceutical composition for preventing or treating hair loss, wherein the above pharmaceutical composition is an external skin agent.

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