KR20230173058A - Composition for treating alopecia comprising deglycosylated recombinant keratin - Google Patents

Abstract

A composition containing non-glycosylated recombinant keratin as an active ingredient has better hair growth promotion and hair regeneration effects than glycosylated human hair-derived keratin and can be used to prevent and treat hair loss (alopecia).

Description

Composition for treating alopecia comprising deglycosylated recombinant keratin}

The present invention claims the benefit of the filing date of Korean Patent Application No. 10-2022-0016105 filed with the Korea Intellectual Property Office on February 8, 2022, the entire contents of which are included in the present invention.

The present invention relates to a composition for treating hair loss comprising deglycosylated recombinant keratin.

It has been disclosed that keratin has a hair growth promoting effect and can exert a better hair growth promoting effect when injected transdermally or subcutaneously than when applied to the scalp.

Keratin contains various subclass proteins expressed from various genes, such as KRT1 to KRT86. However, it has not been disclosed what the hair growth promoting effect of each subclass keratin is and whether glycosylation of keratin affects the hair growth promoting effect.

Republic of Korea Publication No. 10-1859421 (2018.05.14)

According to one embodiment, a composition for treating hair loss comprising deglycosylated recombinant keratin as an active ingredient is provided.

One aspect includes recombinant keratin 34 (rhKRT34), a recombinant keratin having at least 60% amino acid sequence identity with said recombinant keratin 34, and a recombinant keratin having at least 75% amino acid sequence similarity (positives) with said recombinant keratin 34. Provided is a pharmaceutical composition for preventing or treating hair loss (alopecia), comprising as an active ingredient at least one recombinant keratin selected from the group, wherein the recombinant keratin is not glycosylated. Specifically, the recombinant keratin may be recombinant keratin 34.

The present inventors demonstrated that non-glycosylated recombinant keratin 34 has a better hair growth inducing effect than glycosylated human hair-derived keratin (hair keratin or human hair keratin). Glycosylation is a process performed by post-translatal modification that occurs in eukaryotic cells, and the presence or absence of glycosylation is determined by the folding structure of the protein; Physical properties such as water solubility; Physiological activities such as enzyme activity and antigenicity can be significantly changed. For example, in the case of BMP (Bone morphogenetic proteins), which are growth factors, BMPs that are not glycosylated lose their original physiological activity, so they must be produced in mammalian cell lines, not E. coli. Considering these points, the improved hair growth effect of non-glycosylated recombinant keratin 34 is difficult to predict.

The hair loss refers to a condition in which the hair on the scalp abnormally decreases or becomes thinner. The hair loss prevention or improvement includes not only promoting new hair generation but also allowing existing hair to grow healthily. The hair loss is, for example, male-pattern alopecia, female-pattern alopecia, alopecia areata, telogen alopecia, androgenetic alopecia, pressure alopecia ( It may be Pressure Alopecia, or Alopecia Seborrhecia, but is not limited thereto.

According to one embodiment, the amino acid sequence of the recombinant keratin 34 may consist of the sequence of SEQ ID NO: 10.

According to one embodiment, the recombinant keratin may be expressed from prokaryotic cells. According to one embodiment, the recombinant keratin may be recombinant keratin 34, and the recombinant keratin 34 may be produced by bacteria transformed by introducing a vector cloning the protein coding region of the keratin 34 gene. The prokaryotic cell may be, for example, a strain of the genus Escherichia, a strain of the genus Bacillus, or a strain of the genus Corynebacterium, and according to one embodiment, it may be E. coli. When a recombinant protein is expressed in a eukaryotic cell such as a mammalian cell, glycosylation occurs, but when the recombinant protein is expressed in a prokaryotic cell, glycosylation does not occur. Specifically, the recombinant keratin 34 may be expressed from transformed prokaryotic cells.

The keratin 34 may be expressed from human KRT34, and the recombinant keratin 34 may be expressed from the protein coding sequence (CDS) of the human KRT34 gene. The keratin34 gene information and coding sequence can be obtained from known databases, for example, the National Center for Biological Information (NCBI). The keratin 34 coding sequence (CDS) may be, for example, the nucleotide sequence of SEQ ID NO: 1.

Cloning of the keratin 34, production of host cells expressing recombinant keratin 34, culture, and protein purification can be performed by methods known to those skilled in the art and are not particularly limited.

According to one embodiment, the recombinant keratin may include a form of monomer keratin whose disulfide bonds have been decomposed by chemical treatment. For example, the recombinant keratin may be recombinant keratin 34, and the recombinant keratin 34 may be made of monomer keratin, dimer keratin in which monomer keratin is formed again with a disulfide bond, or a combination thereof. The chemical treatment may include cleavage of disulfide bonds between cysteines contained in keratin. For example, the chemical treatment may include treatment with Tris-2-Carboxyethylphosphine Hydrochloride (TCEP), Dithiothreitol, or beta-mercaptoethanol. According to particle size analysis through Dynamic Light Scattering Analysis, recombinant keratin 34, a monomer, may be particles with an average size of 45 nm or less. Some of the recombinant keratin 34, which is a monomer, may form dimers by spontaneous disulfide bonds, and the recombinant keratin 34, which is a dimer, may contain aggregated keratin particles with a particle size of 100 nm or more. The pharmaceutical composition for treating hair loss according to the above aspect may include an aqueous solution containing recombinant keratin 34, and the average particle size of recombinant keratin 34 in the aqueous solution is 100 nm or less, thereby preventing hair loss without inducing an immune response in the body. It can provide preventive or therapeutic effects. On the other hand, human hair-derived keratin is composed of a keratin composite material containing proteins bound to sugar structures. In an aqueous solution, aggregation between the proteins forms aggregates with an average size of 1000 nm or more, and the aggregates induce various immune responses in the body. can cause

According to one embodiment, the average molecular weight of the recombinant keratin may be 20 to 100 kDa. Specifically, the recombinant keratin may be recombinant keratin 34, and the average molecular weight is 30 to 100 kDa, 40 to 100 kDa, 50 to 100 kDa, 20 to 90 kDa, 30 to 90 kDa, 40 to 90 kDa, 50 to 90 kDa, 20 to 80 kDa, 30 to 90 kDa. It may be 80 kDa, 40 to 80 kDa, 50 to 80 kDa, 20 to 70 kDa, 30 to 70 kDa, 40 to 70 kDa, 50 to 70 kDa, 20 to 60 kDa, 30 to 60 kDa, 40 to 60 kDa, 50 to 60 kDa, or 55 kDa. Preferably, the average molecular weight of the recombinant keratin 34 may be 40 to 70 kDa.

According to one embodiment, the effective concentration of the recombinant keratin may be 10 times lower than the effective concentration of human hair-derived keratin. Specifically, the recombinant keratin may be recombinant keratin 34, and the effective concentration of the recombinant keratin 34 is 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, and 8 times that of the human hair-derived keratin. Or it could be 9 times lower. According to one example, the recombinant keratin 34 showed relatively higher efficacy than human hair-derived keratin at the same concentration.

According to one embodiment, the recombinant keratin may not have a tag attached to the N-terminus, for example, may not contain a GST tag.

The recombinant keratin may include a His tag bound to the N-terminus, but no tag is bound to the C-terminus.

According to one embodiment, the pharmaceutical composition may be formulated for topical administration. The topical administration may be topical administration to the scalp. The formulation for local administration may be, for example, a formulation for transdermal administration, a formulation for intradermal administration, or a formulation for subcutaneous administration.

The formulation for transdermal administration may be an injection, drop, ointment, lotion, gel, cream, spray, suspension, emulsion, or patch. The formulation for transdermal administration may be, for example, a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or a combination thereof. The formulation for transdermal administration may include aqueous ingredients, oily ingredients, alcohols, moisturizers, thickeners, whitening agents, preservatives, antioxidants, surfactants, fragrances, skin nutrients, etc.

The pharmaceutical composition may be formulated as an injection, and the injection may contain a physiologically appropriate buffer such as Hank's solution, Ringer's solution, DPBS (Dubelcos Phosphate Buffered Solution), and physiological saline buffer. You can.

The preferred dosage of the pharmaceutical composition may vary depending on the patient's condition and weight, degree of disease, drug formulation, administration route and period, and may be appropriately selected by a person skilled in the art.

In another aspect, at least one recombinant keratin selected from the group comprising recombinant keratin 34, a recombinant keratin having at least 60% amino acid sequence identity with the recombinant keratin 34, and a recombinant keratin having at least 75% amino acid sequence similarity with the recombinant keratin 34. It provides a cosmetic composition for improving hair loss (alopecia), which includes as an active ingredient, and wherein the recombinant keratin is not glycosylated. Specifically, the recombinant keratin may be the recombinant keratin 34.

In addition to the active ingredients, the cosmetic composition may further include functional additives and components included in general cosmetic compositions. The cosmetic composition may further include conventional auxiliaries and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances, etc. For example, the cosmetic composition may further include auxiliary ingredients such as glycerin, butylene glycol, polyoxyethylene hydrogenated castor oil, tocopheryl acetate, citric acid, squalane, sodium citrate, and allantoin, and the solvent may include It may contain hexanediol, purified water, etc. Additionally, the functional additive may include ingredients selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high molecular weight peptides, high molecular weight polysaccharides, and sphingolipids. Other ingredients that can be mixed include fat ingredients, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, disinfectants, antioxidants, plant extracts, pH adjusters, alcohol, pigments, Fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, etc. are included.

The cosmetic composition includes, for example, hair tonic, hair conditioner, hair essence, hair lotion, hair nutrition lotion, hair shampoo, hair rinse, hair treatment, hair cream, hair nutrition cream, hair moisture cream, hair massage cream, and hair wax. , hair aerosol, hair pack, hair nutrition pack, hair soap, hair cleansing foam, hair oil, hair dryer, hair preservation treatment, hair dye, hair waving agent, hair bleach, hair gel, hair glaze, hair dresser, hair lacquer, hair It may include formulations of moisturizer, hair mousse, or hair spray, specifically shampoo, cleanser, conditioner, treatment, hair pack, rinse, scalp pack, tonic, essence, lotion, cream, oil, mist, spray, or hair treatment. It may be a gel formulation.

The cosmetic composition includes skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, moisture cream, hand cream, foundation, essence, nutritional essence, pack, soap, cleansing product. It can be manufactured in one or more formulations selected from the group consisting of foam, cleansing lotion, cleansing cream, body lotion, and body cleanser.

When the formulation of the cosmetic composition is a paste, cream or gel, the carrier component may include animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, or zinc oxide. there is.

Another aspect provides a method for preventing, treating, or improving hair loss comprising administering an effective amount of non-glycosylated recombinant keratin. Specifically, the recombinant keratin may be recombinant keratin 34.

The composition according to one embodiment includes non-glycosylated recombinant keratin, thereby significantly improving hair growth promotion and hair regeneration effects compared to a composition containing human hair-derived keratin, and may exhibit excellent hair loss prevention, treatment, or improvement effects.

Figure 1 shows the vector map of pBT7-N-His.
Figure 2 shows the results of confirming the molecular weight of recombinant human keratin 34 (rhKRT34) prepared according to an example.
Figure 3 is a schematic diagram showing the structure of recombinant human keratin 34 prepared according to an example.
Figure 4 shows the results of MALDI-TOF analysis of the identity of recombinant human keratin 34 prepared according to an example.
Figure 5a shows the results of confirming differences in particle size distribution and cohesion through dynamic light scattering analysis of human hair-derived keratin.
Figure 5b shows the results of confirming differences in particle size distribution and cohesion through dynamic light scattering analysis of recombinant keratin34.
Figure 6 is a conceptual diagram of human hair-derived keratin that is glycosylated and recombinant human keratin 34, and the results of Western blot analysis confirming that recombinant human keratin 34 prepared according to an example is not glycosylated.
Figure 7a shows the results of surface charge analysis of recombinant human keratin 34 and human keratin prepared according to an example.
Figure 7b shows the results of cysteine content analysis of recombinant human keratin 34 and human keratin prepared according to an example.
Figure 8a is a result confirming the induction of dermal papilla cell aggregation according to the treatment of recombinant human keratin 34 and human hair-derived keratin prepared according to an example (Control is the untreated group, human hair-derived keratin is 0.05%, rhKRT34 is 0.05%, 0.025 %, 0.01% and 0.005% were treated and compared).
Figure 8b is a graph comparing the size of the aggregates calculated by area from the results of Figure 8a (the line in each bar graph represents the standard deviation).
Figure 9a shows the results of confirming the expression of hair growth-related markers β-catenin, ALPase, and CD133 in dermal papilla cells in the untreated group as the control group.
Figure 9b shows the results confirming that recombinant human keratin 34 prepared according to an example increased the expression of hair growth-related markers β-catenin, ALPase, and CD133 in dermal papilla cells.
Figure 10 is a graph confirming the mRNA expression of human hair-derived β-catenin, ALPase, CD133, and shh according to the treatment concentration and time course of recombinant human keratin 34 and human hair-derived keratin prepared according to an example (control) used the untreated group).
Figure 11 is an image confirming the differentiation results of outer root sheath cells (ORS) according to the treatment concentration of recombinant human keratin 34 and human hair-derived keratin prepared according to an example.
Figure 12a is an image confirming the expression of β-catenin in outer root sheath cells following treatment with recombinant human keratin 34 and human hair-derived keratin prepared according to an example (an untreated group was used as a control).
Figure 12b is an image confirming the expression of Lgr5 in outer root sheath cells following treatment with recombinant human keratin 34 and human hair-derived keratin prepared according to an example (an untreated group was used as a control).
Figure 13a is an image confirming the inhibition of CD 34 expression in outer root sheath cells by treatment with recombinant human keratin 34 and human hair-derived keratin prepared according to an example (an untreated group was used as a control).
Figure 13b is an image confirming the increase in the expression of P-cadherin in outer root sheath cells following treatment with recombinant human keratin 34 and human hair-derived keratin prepared according to an example (an untreated group was used as a control) .
Figure 14 is a graph confirming the mRNA expression of β-catenin, ITGA6, Sox9, and FOXN1, hair growth-related markers in outer root sheath cells, according to the treatment concentration and time course of recombinant human keratin 34 and human hair-derived keratin prepared according to an example. (An untreated group was used as the control).
Figure 15a shows the results of animal experiments and H&E staining of tissues over time (28 days) after treatment of recombinant human keratin 34 and human hair-derived keratin prepared according to an example.
Figure 15b is a graph showing the number of hair follicles identified after 28 days of treatment with recombinant human keratin 34 and human hair-derived keratin by growth cycle of human hair.
Figure 15c is a graph showing the ratio of hair follicles identified by growth cycle after 28 days of treatment with recombinant human keratin 34 and human hair-derived keratin.
Figure 15d is a graph showing the ratio of hair follicles by size after 28 days of treatment with recombinant human keratin 34 and human hair-derived keratin.
Figure 16 shows the results of a relative comparison analysis of the similarity of the amino acid sequence between keratin 34 and other acid keratins that make up hair. Figure 16a is the result of comparison between KRT34 and KRT31, Figure 16b is the result of comparison between KRT34 and KRT32, Figure 16c is the result of comparison between KRT34 and KRT33a, Figure 16d is the result of comparison between KRT34 and KRT33b, and Figure 16e is the result of comparison between KRT34 and KRT35. Figure 16f is the comparison result of KRT34 and KRT36, Figure 16g is the comparison result of KRT34 and KRT37, and Figure 16h is the comparison result of KRT34 and KRT38.

Hereinafter, one or more embodiments will be described in more detail through examples. However, these examples are intended to illustrate one or more embodiments and the scope of the present invention is not limited to these examples.

Example 1: Preparation of recombinant keratin (KRT) 34

1-1. Recombinant vector construction

Human Keratin 31 (Homo sapiens keratin 31, KRT31), Human Keratin 33A (Homo sapiens keratin 33A, KRT33A), Human Keratin 33B (Homo sapiens keratin 33B, KRT33B), Human Keratin 34 (Homo sapiens keratin 34, KRT34), Human Keratin Genetic information of 37 (Homo sapiens keratin 37, KRT37) was obtained from the U.S. National Center for Biological Information (www.ncbi.nlm.nih.gov).

Protein coding sequences (CDS) were extracted from the mRNA sequences of KRT34, KRT31, KRT33B, and KRT37. The coding sequence of KRT34 is SEQ ID NO: 1, the coding sequence of KRT31 is SEQ ID NO: 2, the coding sequence of KRT33B is SEQ ID NO: 5, and the coding sequence of KRT37 is SEQ ID NO: 8.

Bioneer (www.bioneer.co.kr) was commissioned to produce recombinant vectors (pKRT31, pKRT33B, pKRT34, pKRT37) containing the protein coding region of each KRT. (hereinafter pKRT31, pKRT33B, pKRT34, and pKRT37 may be referred to as pKRT)

According to Figure 1, the recombinant vector (pKRT) is a KRT gene introduced into the pBT7-N-His vector suitable for protein expression. It contains a T7 promoter and can transcribe a large amount of mRNA, and isopropyl Expression can be induced with -β-D-thiogalactopyranoside (IPTG). In addition, the gene sequence of histidine affinity ligand for affinity chromatography, a protein separation and purification process, is present in the N-terminal portion. Using the above recombinant vector, transformed microorganisms were prepared and cultured, and recombinant keratin was expressed and purified.

1-2. Production of recombinant keratin-expressing transformed microorganisms

E. coli BL21(DE3) was used as E. coli for recombinant keratin expression. Competent cells were produced by treating E. coli with CaCl ₂ buffer. The above recombinant vectors (pKRT31, pKRT33B, pKRT34, pKRT37) were added to E. coli competent cells, left on ice for 10 minutes, and then heat shocked at 42°C for 90 seconds. E. coli into which the recombinant vector was introduced was plated on LB solid medium coated with ampicillin (Sigma) and cultured for 18 hours to obtain BL21/pKRT colonies. (Hereinafter, transformed E. coli into which recombinant vectors of pKRT31, pKRT33B, pKRT34, and pKRT37 were introduced are referred to as BL21/pKRT)

1-3. Transformed microorganism culture and expression

BL21/pKRT was cultured in LB liquid medium. Specifically, 10 ml of LB liquid medium (containing 500 μg/ml ampicillin) was added to a 50 ml tube, and 2 to 3 BL21/pKRT colonies were inoculated and cultured for 18 hours. The microbial culture was inoculated into a 5,000 ml flask containing 2,000 ml of LB medium. When the absorbance (OD600) value of the culture medium reached 0.5-0.8, IPTG, an inducer, was added to 0.5mM to induce the expression of rhKRT (recombinant human Keratin), a recombinant protein. The culture was cultured with stirring at 25°C for an additional 16 hours, and the culture was centrifuged at 3,000 rpm for 20 minutes to remove the supernatant and recover the cells. The recovered cells were stored at -80°C and used in experiments.

1-4. Separation and dissolution of inclusion bodies

rhKRT is an insoluble protein that aggregates inside E. coli to form inclusion bodies. Inclusion bodies containing aggregated rhKRT were isolated from E. coli. Specifically, E. coli cells stored at -80°C were added with a disruption solution (20mM Tris-HCl, pH 8.0), resuspended at room temperature, and disrupted for 90 seconds using an ultrasonicator with pulses on and off for 5 to 15 seconds. did. The crushed E. coli sample was centrifuged at 2,000xg for 20 minutes and the supernatant was removed.

To wash the inclusion bodies, an inclusion body washing solution (2M urea, 20mM Tris-HCl, 0.5M NaCl, 2% Triton The sample was centrifuged at 2,000xg for 10 minutes and the supernatant was removed. The inclusion body washing process using the washing buffer was repeated four times. After washing, the inclusion body was stored at -20°C and used when necessary.

Add inclusion body dissolution solution (5M Urea, 2.5M Thiourea, 25mM Tris-HCl, pH 8.0) to the inclusion body and heat at 95°C for 10 minutes. The sample was centrifuged at 12,000xg for 20 minutes and the supernatant was filtered through a 0.22㎛ filter to obtain an inclusion body solution. The inclusion body solution was used for purification of rhKRT.

1-5. Purification and dialysis of rhKRT

rhKRT was purified by affinity chromatography. 2ml of HisPur™ Cobalt Superflow Agarose (Thermo Scientific) was added to the chromatographic tube, and 4ml of equilibration solution (20mM sodium phosphate, 300mM NaCl, 8M Urea, 5mM imidazole, pH 8.0) was added to equilibrate the resin. The inclusion body solution containing rhKRT was applied to the resin-filled chromatographic tube to bind to the protein, and the resin was washed using 4ml of washing solution (20mM sodium phosphate, 300mM NaCl, 8M Urea, 15mM imidazole, pH 8.0). Washed three times. An elution buffer containing rhKRT was obtained by flowing 4 ml of the elution solution (20mM sodium phosphate, 300mM NaCl, 8M Urea, 150mM imidazole, pH 8.0) three times.

Dilution was performed by slowly adding distilled water 3 times the volume of the elution buffer containing rhKRT. Afterwards, the diluted aqueous solution (50 ml) containing rhKRT was added to a 50 kDa MWCO dialysis membrane (Spectra/Por ^® ; Spectrum Lab. Inc. USA), and the Spectra/Por RC dialysis tubing closure (maximum width 55 mm, Orange, Spectrum) was placed. Both tubes were packed. A 5L plastic beaker was filled with distilled water and dialysis was performed at room temperature, and impurities were removed by replacing it with new 5L of distilled water every 4 hours.

1-6. rhKRT enrichment

The dialyzed rhKRT aqueous solution was filtered through a 0.22㎛ filtration membrane (Millipore Steriflip™ Sterile Disposable Vacuum Filter) and filtered using a 30kDa molecular weight cut off filter tube (Amicon® Ultra-15 Centrifugal Filter Unit, 30kDa MWCO). It was concentrated to 1/50 by centrifugation at 3,500 rpm and 25°C. Afterwards, it was completely frozen at -80°C for 24 hours in an ultra-low temperature refrigerator. The frozen rhKRT was placed in a freeze dryer (IlShin Lab Freeze Dryer) and dried in a vacuum at -85°C for 3 days to completely form a powder. Finally, SDS-PAGE was performed to confirm the molecular weight.

According to Figure 2, the molecular weight of rhKRT34 was about 55 kDa. This was the same molecular weight as the expected amino acid sequence of the protein to be expressed. Additionally, a protein with a molecular weight of approximately 100 kDa was detected, and it was confirmed to be a dimer of rhKRT34 using MALDI-TOF mass spectrometry. It is separated into monomers in the sulfhydryl group blocking process below. Purity was confirmed by measuring the density of the band using an image analysis program (ImageJ, https://imagej.nih.gov/ij/), and the purity was over 95%.

1-7. Manufacture of rhKRT blocking sulfhydryl group

Freeze-dried rhKRT (100 mg) was dissolved in 8M urea (100 ml) containing 100 mM DTT to prepare a 0.1% (w/v) aqueous solution of rhKRT, and left at 50°C for 18 hours to remove disulfide bonds. I reacted. Afterwards, 50 mg of L-cysteine hydrochloride was added and stirred at room temperature for 2 hours to perform a binding reaction. The reaction-completed aqueous solution (100 ml) was dialyzed in the same manner as 1-5 and 1-6 above, lyophilized, and powdered.

Example 2: rhKRT34 characterization

The physical properties of rhKRT34 prepared in Example 1 were confirmed.

2-1. Amino acid sequence, structure, and particle size analysis of rhKRT34

The amino acid sequence of rhKRT34 is SEQ ID NO: 10, and the molecular weight of rhKRT34 is about 55 kDa.

According to Figure 3, the folding structure of rhKRT34 can be largely divided into Head, Coil, linker, and Tail, and the amino acid residues corresponding to each structure are 1-56 Head; 57 - 91 Coil 1A; 92 - 102 Linker 1; 103 - 203 Coil 1B; 204 - 219 Linker 2; 220 - 363 Coil 2; 364 - 394 Tail.

MALDI-TOF/TOF was performed by Proteinworks (http://www.proteinworks.co.kr/), and the mass of the peptide was measured using a MALDI-TOF/TOF mass spectrometer (Model: 4700 MALDI-TOF/TOF, Applied Biosystems). ) was measured using . The peptide mass was recorded from the theoretical peptides of all proteins in the NCBI database using the Mascot search program, and the results are shown in Figure 4.

Human hair-derived keratin and recombinant keratin 34 (rhKRT34) were dissolved in Dulbecco's phosphate-buffered saline (DPBS) at a concentration of 0.1% (w/v), incubated at 37°C, and human hair keratin was analyzed using Dynamic Light Scattering (DLS) Analysis. The particle sizes of rhKRT34 and rhKRT34 were compared and shown in Figures 5a and 5b. According to Figure 5, the recombinant keratin (rhKRT34) is a mixture of monomer particles and dimer particles, and the average particle size was confirmed to be less than 100 nm. Even during incubation at 37°C, no aggregates larger than 100 nm were observed. In comparison, human hair-derived keratin was found to form aggregates larger than 1000 nm in size due to agglomeration of keratin molecules when incubated at 37°C, confirming that recombinant keratin 34 (rhKRT34) exists relatively stably in aqueous solution compared to human hair-derived keratin. It has been done.

2-2. Confirmation of deglycosylation of rhKRT34

Western blot analysis using anti-O-GLCNAc antibody (Cell signaling Technology, Cat#9875) was performed on human hair-derived keratin and rhKRT34 to analyze differences in glycosylation.

According to Figure 6, a positive band corresponding to the glycosyl group was observed in human hair-derived keratin, but a positive band corresponding to the glycosyl group was not observed in rhKRT34. Therefore, it was confirmed that rhKRT34 was deglycosylated.

2-3. Chemical characterization of rhKRT34

To measure the surface charge of keratin, a keratin solution was prepared by completely dissolving rhKRT34 and human hair keratin in purified water so that the concentration of each was 0.1 (w/v)%. Using a pH measuring device, sodium hydroxide solution was added to the keratin solution so that the pH of the solution was 11. Protein surface charge was measured using the zeta potential measurement function of the Dynamic Light Scattering equipment. Next, hydrochloric acid was added to the solution to lower the pH by 0.5 to 1.5, and the surface charge was measured up to pH around 3 using the above equipment, as shown in Figure 7a.

Analysis of the free sulfhydryl group of cysteine, the main amino acid constituting keratin, was performed using Invitrogen's Thiol Assay Kit (M30550). Analysis was conducted with rhKRT34 and human hair keratin samples each dissolved in purified water to a concentration of 0.2 (w/v)%. The subsequent process was performed according to the kit manufacturer's instructions, and measured at ex 494 nm / em 517 nm in a fluorescence microplate reader, and the results are shown in Figure 7b.

According to Figure 7a, in the case of human hair keratin, the surface charge is negative, and it is confirmed that it remains negative throughout the pH range and does not show a PI value. In the case of rhKRT34, the surface charge value becomes 0 around pH 4, a PI By confirming that the values are shown, it was confirmed that human hair keratin and rhKRT34 are chemically different. In addition, it was confirmed that in the case of human hair keratin, almost no free sulfhydryl groups remained, and in the case of rhKRT34, many free sulfhydryl groups remained.

Example 3: Analysis of hair growth effect of rhKRT34

3-1. Analysis of cell aggregate formation in dermal papilla cells

Dermal papilla cells (DP cells) were treated with rhKRT34 of Example 1, and cell aggregate formation was confirmed. It can be evaluated that an increase in cell aggregates has the effect of inducing hair growth and hair regeneration.

Human dermal papilla cells were inoculated in a 24-well plate at 5 × 10 ⁴ /well, and 24 hours later, the medium was mixed with rhKRT34 of Example 1 at 0.05% (w/v), 0.025% (w/v), and 0.01% (w/). v) or replaced with high glucose DMEM medium containing 0.005% (w/v) and cultured for 2 days. Dermal papilla cells cultured in high glucose DMEM medium without any treatment were prepared as a control group, and in high glucose DMEM medium containing 0.05% (w/v) of human hair-derived keratin as a comparison group. After culturing for 2 days, an image confirming the number of spherical aggregates derived from dermal papilla cells formed in each well using an optical microscope is shown in Figure 8a, and these were counted. The size of the spherical aggregates was measured from the image and is shown in Figure 8b.

According to Figure 8, recombinant keratin 34 induced aggregation of dermal papilla cells even at a low concentration of 0.05% (w/v) and had superior aggregation inducing ability compared to human hair keratin at the same concentration. In addition, recombinant keratin 34 showed an aggregation-inducing ability similar to that of human hair keratin at a concentration of 0.05% (w/v) at a concentration of 0.005% (w/v), and the effective concentration of recombinant keratin 34 was 10 times that of human hair-derived keratin. It was confirmed to be low.

3-2. Analysis of hair growth-related marker expression in dermal papilla cells

Expression of hair growth-related markers beta-catenin, CD133, and alkaline phosphatase (ALPase) in cultured dermal papilla cells was confirmed through immunochemical staining.

Dermal papilla cells were cultured in high glucose DMEM general medium containing 0.05% (w/v) of rhKRT34 of Example 1 for 2 days. The medium was removed and washed with DPBS. It was fixed with 4% paraformaldehyde for 10 minutes and then washed again with DPBS. After fixation, permeabilization was achieved by treating with 0.1% Triton Then, rabbit anti-human beta-catenin antibody, rabbit anti-human CD34 antibody, or rat anti-human ALPase antibody diluted 1:200. (mouse anti-human ALPase antibody) and reacted at 4°C for 24 hours.

After 24 hours of reaction, the cells were washed three times with DPBS and treated with PE-conjugated Palloidin antibody for 1 hour at room temperature for counter staining. The secondary antibodies used were Alexa Fluor 488 conjugated goat anti-rabbit IgG or Alexa Fluor 594 conjugated goat anti. -mouse IgG was treated at room temperature for 1 hour. Afterwards, it was washed three times with DPBS and treated with 4',6-diamidino-2-phenylindole (DAPI). Afterwards, the stained cells were observed under a fluorescence microscope (OlympusI ×71), and the image is shown in Figure 9b.

As a control group, an untreated group using the high glucose DMEM general medium was used, prepared in the same manner as above and observed under a fluorescence microscope, and the image is shown in Figure 9a.

According to Figure 9, the rhKRT34-treated group had increased expression of beta-catenin, CD133, and alkaline phosphatase, which are hair growth-related markers in dermal papilla cells, than the untreated group.

Additionally, the expression of beta-catenin, alkaline phosphatase, CD133, and shh as hair growth-related markers in dermal papilla cells according to treatment with human hair-derived keratin and rhKRT34 at different concentrations was confirmed by RT-Qpcr analysis. RT-qPCR analysis was performed on triplicate samples using an RG-6000 RT-qPCR machine from Corbett. RT-qPCR was performed using 1 μL of cDNA template, 1 μL sense and antisense primers, 10 μL qPCR Master Mix from Bioneer, and DEPC-water in a final volume of 20 μL. The primers used for this purpose were GAPDH (forward 5'-ACTTTGGTATCGTGGAAGGAC-3' and reverse 5'-GCAGGGATGATGTTCTGGAG-3'), CD133 (forward 5'-GTGAAACCTGCAACAGCATC-3' and reverse 5'-TGTCAAGTTCTGCATCCACG-3'), β -Catenin (forward 5'-TGTAGAAGCTGGTGGAATGC-3' and reverse 5'-GCTGAACAAGAGTCCCAAGG-3'), ALPase (forward 5'-CTCGTTGACACCTGGAAGAG-3' and reverse 5'-GGCTCGAAGAGACCCAATAG-3'), and Shh (forward 5'- GATGTCTGCTGCTAGTCCTCG-3' and reverse 5'- CACCTCTGAGGTCATCAGCCTG-3'). Samples were treated with the following protocol: heating at 95°C for 10 minutes, then 45 cycles of 95°C for 20 seconds, 60-63°C for 10 seconds, and one cycle of heating for melting curves, then at 12°C. cooling. Data were normalized to GAPDH levels and shown in Figure 10.

According to Figure 10, the rhKRT34 treated group had increased mRNA expression of beta-catenin, CD133, Alkaline phosphatase, and shh, hair growth-related markers of dermal papilla cells, than the untreated group.

3-3. Evaluation of colony forming ability and differentiation inducing ability of outer root sheath cells

Outer root sheath cells (ORS Cells) are cells that differentiate into secondary hair germ cells (SHG) and play an important role in hair root formation. It is known that the colony-inducing and differentiation-inducing abilities of outer root sheath cells are highly related to hair root formation. In particular, outer root sheath cells expressing the CD34 marker are known to be differentiated into the CD34 ^- /Lgr5 ⁺ cell group, which is directly involved in the formation of secondary hair germ cells.

The outer root sheath cells were cultured for 2 days in high glucose DMEM medium containing rhKRT34 of Example 1 at a concentration of 0.05% (w/v), 0.025% (w/v), or 0.01% (w/v), and colony formation and Induction of differentiation was observed. An untreated group was used as the control, and outer root sheath cells were cultured for 2 days in high glucose DMEM medium containing human hair-derived keratin at a concentration of 0.1% (w/v) or 0.01% (w/v), and clustered. Formation and differentiation induction were observed under an optical microscope (OLYMPUS, IX71, ×100), and the results are shown in Figure 11.

According to Figure 11, the human hair-derived keratin treated group showed the ability to induce clusters of outer root sheath cells and differentiate into an elongated form at a high concentration of 0.1% (w/v) or higher, but at a lower concentration of 0.01% (w/v). The concentration of v) did not show this effect. However, the rhKRT34-treated group of Example 1 showed the ability to induce clusters of outer root sheath cells and differentiate into an elongated form even at a concentration as low as 0.01% (w/v).

Additionally, changes in the expression of β-catenin, Lgr5, CD34, and P-cadherin were confirmed in the rhKRT34-treated group and the human hair-derived keratin-treated group. Figure 12a is an image confirming the expression change of β-catenin, Figure 12b is an image confirming the expression change of Lgr5, Figure 13a is an image confirming the expression change of CD34, and Figure 13b is an image confirming the expression change of P-cadherin. am.

According to Figure 12, the expression of β-catenin and Lgr5 increased in outer root sheath cells treated with human hair-derived keratin at a high concentration of 0.1% (w/v) or higher, but this effect was not achieved at a concentration of 0.01% (w/v). did not indicate However, rhKRT34 of Example 1 increased the expression of β-catenin and Lgr5 even at low concentrations of 0.05% (w/v), 0.025% (w/v), and 0.01% (w/v).

According to Figure 13, human hair-derived keratin did not show expression of CD34 when treated with 0.1% (w/v), and CD34 expression appeared when treated with 0.01% (w/v), but rhKRT34 of Example 1 showed 0.01% Even at low concentrations of (w/v), the expression of CD34 was suppressed. In addition, rhKRT34 was confirmed to increase the expression of P-cadherin, a marker expressed during hair germ formation.

According to the above results, rhKRT34 induces clusters of outer root sheath cells and differentiates them into an elongated form even at a lower concentration than human keratin, and can induce differentiation into CD34 ^- /Lgr5 ⁺ /P-cadherin ⁺ cells. was confirmed.

The expression of beta-catenin, ITGA6, Sox9, and FOXN1 as hair growth-related markers in outer root sheath cells following treatment with human hair-derived keratin and rhKRT34 at different concentrations was confirmed by RT-Qpcr analysis. RT-qPCR analysis was performed under the same conditions as the previous example. The primers used for this purpose were GAPDH (forward 5'-ACTTTGGTATCGTGGAAGGAC-3' and reverse 5'-GCAGGGATGATGTTCTGGAG-3'), Sox9 (forward 5'-GGACCACCCGGATTACAAGT-3' and reverse 5'-AAGATGGCGTTGGGGGAGAT-3'), β -Catenin (forward 5'-TGTAGAAGCTGGTGGAATGC-3' and reverse 5'-GCTGAACAAGAGTCCCAAGG-3'), ITGA6 (forward 5'-GAAGGTGGCTGCGGTAGCA-3' and reverse 5'-ATCACGTTGTCCTCCCGAGT-3'), and FOXN1 (forward 5'- AGTCCTGGGTTCAGAGGTCA-3' and reverse 5'-CTGGCGAGTACTGGTGGAAG-3'). The RT-Qpcr results are shown in Figure 14.

According to Figure 14, the rhKRT34 treated group was confirmed to have increased mRNA expression of beta-catenin, ITGA6, Sox9, and FOXN1, hair growth-related markers of outer root sheath cells, compared to the untreated group.

3-4. Verification of hair growth effect through animal testing

The hair growth effect was confirmed through animal testing using male C57BL/6 mice purchased from YoungBio (Samtako, 1404957265).

The mice were raised under controlled conditions with a temperature of 23 ± 2 °C, humidity of 50 ± 5%, and a 12-hour light/dark cycle. The hair on the back of the mouse was shaved repeatedly using electric clippers to synchronize the hair follicle cycle. To determine the hair growth promoting effect of keratin, Dulbeccoic acid buffered saline solution, 0.05(w/v)% human hair keratin, 0.05(w/v)% recombinant keratin, 0.025(w/v)% recombinant keratin, and 0.01(w/v) )% recombinant keratin injection solution was used, and 10-week-old mice had their dorsal hair completely removed using commercial hair removal cream Veet ^® (Reckitt Benckiser, 62200809951) before the procedure, and were randomly assigned and injected. Observation and recording were made for 28 days (see Figure 15a), and the mice were sacrificed and skin tissues were fixed with 10% neutral buffered formalin (BBC Biochemical, 0141). The tissue was embedded in paraffin, cut into 4 μm thick sections, and stained with hematoxylin and eosin (H&E staining) for histological analysis. The number and ratio of hair follicles in each cycle and the diameter of hair follicles in the growth phase were observed and recorded under an optical microscope at Х 100 magnification and shown in Figures 15b to 15d.

According to Figure 15, when 0.05% (w/v) human hair keratin and rhKRT34 were injected at the same concentration, a high hair growth effect was confirmed in mice injected with rhKRT34. It was confirmed that the number of hair follicles increased relatively significantly throughout the hair growth cycle upon injection of rhKRT34, the proportion of hair follicles in the growth phase increased, and the diameter of hair follicles also increased relatively significantly upon injection of rhKRT34.

3-5. Similarity analysis between rhKRT34 and acidic keratin that makes up human hair

To compare the similarity of the amino acid sequence between deglycosylated recombinant keratin 34 and other acid keratins that make up hair, NCBI's Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast Using the blastp algorithm, which is used to compare proteins in .cgi), the keratin 34 sequence and the acid keratins of 8 other classes (KRT31, KRT32, KRT33A, KRT33B, KRT35, KRT36, KRT37, KRT38) that make up human hair were compared. compared. In the case of similar sequences, related information (similarity score (score, scoring the similarity between sequences through an algorithm), identity (percentage of identical amino acid matches between two sequences), similarity (Positives) is based on the similarity through blastp. Although it is not the same amino acid between the two sequences, it is possible to check the ratio of matching amino acids with similar properties) and the alignment results between each query and the Keratin 34 sequence.

According to Figure 16, as a result of relative comparative analysis of the identities and similarities (positives) of the amino acid sequence between keratin 34 and other acid keratins constituting hair, amino acids having more than 60% identity with the recombinant keratin 34 It was confirmed that the keratins that make up human hair have a very high amino acid sequence similarity.

It is known that if the amino acid sequence identity between two proteins is less than 60%, it means excessive structural modification of the protein, making it impossible to secure the same or similar function. Specifically, if the acid keratin has an amino acid sequence identity of less than 60% or an amino acid sequence similarity of less than 75% compared to the recombinant keratin 34, the function of the keratin is lost or reduced, thereby preventing or treating hair loss as in the recombinant keratin 34. The effect cannot be secured. The eight types of acid keratins satisfy the requirement that they have 60% or more identity or 75% or more similarity with the amino acid sequence of the recombinant keratin 34, or 60% or more identity and 75% or more similarity, and are identical or similar to the recombinant keratin 34 for hair loss. It may have a preventive or therapeutic effect.

Claims (1)

At least one selected from the group consisting of recombinant keratin 34, recombinant keratin having 60% or more amino acid sequence identity with the recombinant keratin 34, and recombinant keratin with 75% or more amino acid sequence similarity (positives) with the recombinant keratin 34. Contains recombinant keratin as an active ingredient,
The recombinant keratin is not glycosylated,
Pharmaceutical composition for preventing or treating alopecia.