KR20240124223A - Composition for treating alopecia comprising recombinant keratin - Google Patents

Abstract

The present invention relates to a composition for treating alopecia comprising deglycosylated recombinant keratin as an active ingredient. Provided is a pharmaceutical composition for preventing or treating alopecia comprising, as an active ingredient, one or more recombinant keratin selected from the group consisting of recombinant keratin, recombinant keratin having 90% or more amino acid sequence identities with the recombinant keratin, and recombinant keratin having 95% or more amino acid sequence positives with the recombinant keratin. The composition according to one embodiment includes deglycosylated recombinant keratin, thereby significantly improving hair growth promotion and hair regeneration effects compared to a composition including human hair-derived keratin, and thus can exhibit excellent effects of preventing, treating, or alleviating hair loss.

Description

{Composition for treating alopecia comprising recombinant keratin}

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 promotion effect, and can exhibit a better hair growth promotion effect when injected percutaneously or subcutaneously than when applied to the scalp.

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

Republic of Korea Public Notice No. 10-1859421 (2018.05.14)

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

One aspect provides a pharmaceutical composition for preventing or treating alopecia, comprising as an active ingredient at least one recombinant keratin selected from the group consisting of recombinant keratin, recombinant keratin having 90% or more amino acid sequence identity (identities) with the recombinant keratin, and recombinant keratin having 95% or more amino acid sequence similarity (positives) with the recombinant keratin, wherein the recombinant keratin is any one selected from the group consisting of recombinant keratin 31, recombinant keratin 32, recombinant keratin 33A, recombinant keratin 33B, recombinant keratin 35, recombinant keratin 36, recombinant keratin 37, and recombinant keratin 38, and wherein the recombinant keratin is non-glycosylated.

Specifically, the sequence identity may be greater than or equal to 60%, and may be greater than or equal to 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%. Specifically, the sequence similarity may be greater than or equal to 75%, and may be greater than or equal to 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%.

The present inventors demonstrated that non-glycosylated recombinant keratin 34 has a superior hair growth induction effect than glycosylated hair-derived keratin (human hair-extracted keratin or human hair keratin). In addition, the present inventors demonstrated that non-glycosylated recombinant keratin derived from 8 other classes of keratins constituting human hair (KRT31, KRT32, KRT33A, KRT33B, KRT35, KRT36, KRT 37, KRT38) other than keratin 34 has a superior hair growth induction effect than human hair-derived keratin.

The sequence of the recombinant keratin 34 may be an amino acid sequence of SEQ ID NO: 10, the sequence of the recombinant keratin 31 may be an amino acid sequence of SEQ ID NO: 11, the sequence of the recombinant keratin 32 may be an amino acid sequence of SEQ ID NO: 12, the sequence of the recombinant keratin 33A may be an amino acid sequence of SEQ ID NO: 13, the sequence of the recombinant keratin 33B may be an amino acid sequence of SEQ ID NO: 14, the sequence of the recombinant keratin 35 may be an amino acid sequence of SEQ ID NO: 15, the sequence of the recombinant keratin 36 may be an amino acid sequence of SEQ ID NO: 16, the sequence of the recombinant keratin 37 may be SEQ ID NO: 17, and the sequence of the recombinant keratin 38 may be SEQ ID NO: 18.

Glycosylation is a process performed by post-translational modification that occurs in eukaryotic cells, and the presence or absence of glycosylation can significantly change the folding structure of a protein; physical properties such as water solubility; and physiological activities such as enzyme activity and antigenicity. For example, in the case of growth factor BMP (Bone morphogenetic proteins), non-glycosylated BMP loses its original physiological activity, so it must be produced in a mammalian cell line, not E. coli. Considering these points, it is difficult to predict the improved hair growth effect of non-glycosylated recombinant keratin 34.

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

According to one specific example, the amino acid sequence of the recombinant keratin 34 may be a sequence of SEQ ID NO: 10. According to one embodiment, the recombinant keratin may be any one selected from the group consisting of recombinant keratin 31, recombinant keratin 32, recombinant keratin 33A, recombinant keratin 33B, recombinant keratin 35, recombinant keratin 36, recombinant keratin 37 and recombinant keratin 38, and the recombinant keratin may be produced by transforming a bacterium by introducing a vector in which the protein coding region of the keratin 31, 32, 33A, 33B, 34, 35, 36, 37 or 38 gene is cloned. According to one specific example, the recombinant keratin may be expressed from a prokaryotic cell. The prokaryotic cell may be, for example, an Escherichia genus strain, a Bacillus genus strain, or a Corynebacterium genus strain, and according to one embodiment, may be E. coli. When the recombinant protein is expressed in a eukaryotic cell, such as a mammalian cell, glycosylation occurs, but when the recombinant protein is expressed from a prokaryotic cell, glycosylation does not occur. Specifically, the recombinant keratin may be expressed from a transformed prokaryotic cell. According to one specific example, the recombinant keratin may not include recombinant keratin 34. According to one specific example, the recombinant keratin may be a recombinant keratin excluding recombinant keratin 34.

The above recombinant keratin may be expressed from human KRT31, 32, 33A, 33B, 34, 35, 36, 37 or 38, and the above recombinant keratin may be expressed from a protein coding sequence (CDS) of human KRT31, 32, 33A, 33B, 34, 35, 36, 37 or 38 gene. The KRT31, 32, 33A, 33B, 34, 35, 36, 37 or 38 gene information and coding sequence can be obtained from a known database, for example, can be obtained from the National Center for Biotechnology Information (NCBI).

The keratin 34 coding sequence (CDS) may be, for example, a base sequence of SEQ ID NO: 1. The keratin 31 coding sequence (CDS) may be, for example, a base sequence of SEQ ID NO: 2. The keratin 32 coding sequence (CDS) may be, for example, a base sequence of SEQ ID NO: 3. The keratin 33A coding sequence (CDS) may be, for example, a base sequence of SEQ ID NO: 4. The keratin 33B coding sequence (CDS) may be, for example, a base sequence of SEQ ID NO: 5. The keratin 35 coding sequence (CDS) may be, for example, a base sequence of SEQ ID NO: 6. The keratin 36 coding sequence (CDS) may be, for example, a base sequence of SEQ ID NO: 7. The keratin 37 coding sequence (CDS) may be, for example, a base sequence of SEQ ID NO: 8. The keratin 38 coding sequence (CDS) may be, for example, a base sequence of SEQ ID NO: 9.

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

According to one specific example, the folding structure of the recombinant keratin may be divided into a head, a coil, a linker, and a tail. The folding structure of the recombinant keratin may be arranged in the order of head-first coil-first linker-second coil-second tail-third coil-tail. The coil may mean an alpha helix structure. The folding structure of the recombinant keratin may include two or more regions of alpha helix structures. The folding structure of the recombinant keratin may include three or more regions of alpha helix structures.

According to one specific example, the recombinant keratin may include a form of monomer keratin in which a disulfide bond is cleaved by a chemical treatment. For example, the recombinant keratin may be recombinant keratin 34, and the recombinant keratin 34 may be composed of a monomer keratin, a dimer keratin in which a monomer keratin forms a disulfide bond again, or a combination thereof. The chemical treatment may include cleavage of a disulfide bond between cysteines included in the keratin. For example, the chemical treatment may include treatment with TCEP (Tris-2-Carboxyethylphosphine Hydrochloride), Dithiothreitol, or beta-mercaptoethanol. According to particle size analysis through Dynamic Light Scattering Analysis, the monomeric recombinant keratin may be a particle having an average size of 45 nm or less. Some of the monomeric recombinant keratins can form dimers by spontaneous disulfide bonds, and the recombinant keratins as dimers can include aggregated keratin particles having a particle size of 100 nm or more. The pharmaceutical composition for treating hair loss according to the above aspect can include an aqueous solution containing the recombinant keratin, and the average particle size of the recombinant keratin in the aqueous solution is 100 nm or less, so that it can provide a preventive or therapeutic effect on hair loss without inducing an immune response in the body. On the other hand, human hair-derived keratin is composed of a keratin complex containing proteins to which a sugar structure is bound, and forms aggregates having an average size of 1000 nm or more by aggregation between the proteins in an aqueous solution, and the aggregates can induce various immune responses in the body.

According to one specific example, the average molecular weight of the recombinant keratin may be 20 to 100 kDa. Specifically, the recombinant keratin may be any one selected from the group consisting of recombinant keratin 34, recombinant keratin 31, recombinant keratin 32, recombinant keratin 33A, recombinant keratin 33B, recombinant keratin 35, recombinant keratin 36, recombinant keratin 37 and recombinant keratin 38, and has an average molecular weight of 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 80 kDa, 40 to 80 kDa, 50 to 80 kDa, 20 to 70 kDa, 30 to 70 kDa, 40 The average molecular weight of the recombinant keratin may be from 30 to 70 kDa, from 50 to 70 kDa, from 20 to 60 kDa, from 30 to 60 kDa, from 40 to 60 kDa, from 50 to 60 kDa, or from 55 kDa. Preferably, the average molecular weight of the recombinant keratin may be from 30 to 70 kDa.

In one specific example, the effective concentration of the recombinant keratin may be 10 times lower than the effective concentration of the human hair-derived keratin. Specifically, the effective concentration of the recombinant keratin may be 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times or 9 times lower than the effective concentration of the human hair-derived keratin. In one embodiment, the recombinant keratin exhibited relatively higher efficacy than the human hair-derived keratin at the same concentration.

In one specific example, the recombinant keratin may not have a tag attached to its N-terminus, for example, may not comprise a GST tag.

The above recombinant keratin may comprise a His tag linked to the N-terminus and may not have a tag linked to the C-terminus.

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

The above transdermal administration formulation may be an injection, a drop, an ointment, a lotion, a gel, a cream, a spray, a suspension, an emulsion, or a patch. The above transdermal administration formulation may be, for example, a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal delivery patch, a drug-containing bandage, a lotion, or a combination thereof. The above transdermal administration formulation may include an aqueous component, an oily component, an alcohol, a moisturizer, a thickener, a whitening agent, a preservative, an antioxidant, a surfactant, a fragrance, a skin nutrient, and the like.

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

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

Another aspect provides a cosmetic composition for improving alopecia, comprising as an active ingredient at least one recombinant keratin selected from the group consisting of recombinant keratin, recombinant keratin having 90% or more amino acid sequence identity to the recombinant keratin, and recombinant keratin having 95% or more amino acid sequence similarity to the recombinant keratin, wherein the recombinant keratin is any one selected from the group consisting of recombinant keratin 31, recombinant keratin 32, recombinant keratin 33A, recombinant keratin 33B, recombinant keratin 35, recombinant keratin 36, recombinant keratin 37, and recombinant keratin 38, and wherein the recombinant keratin is non-glycosylated. Specifically, the recombinant keratin may be recombinant keratin 34.

The cosmetic composition may further include functional additives and components included in general cosmetic compositions in addition to the effective ingredient. The cosmetic composition may further include conventional auxiliary agents 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, allantoin, etc., and the solvent may include hexanediol, purified water, etc. In addition, the functional additive may include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high molecular peptides, high molecular polysaccharides, and sphingolipids. In addition, other ingredients that can be mixed include maintenance ingredients, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohol, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, etc.

The above cosmetic composition may include, for example, a formulation of a hair tonic, a hair conditioner, a hair essence, a hair lotion, a hair nutrition lotion, a hair shampoo, a hair rinse, a hair treatment, a hair cream, a hair nutrition cream, a hair moisture cream, a hair massage cream, a hair wax, a hair aerosol, a hair pack, a hair nutrition pack, a hair soap, a hair cleansing foam, a hair oil, a hair drying agent, a hair preservative, a hair dye, a hair waving agent, a hair bleaching agent, a hair gel, a hair glaze, a hair dressing agent, a hair lacquer, a hair moisturizer, a hair mousse or a hair spray, and may specifically be a formulation of a shampoo, a cleanser, a conditioner, a treatment, a hair pack, a rinse, a scalp pack, a tonic, an essence, a lotion, a cream, an oil, a mist, a spray or a hair gel.

The above cosmetic composition can be manufactured into at least one formulation selected from the group consisting of skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, foundation, essence, nutrition essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, and body cleanser.

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

Another aspect provides a method for preventing, treating or improving hair loss, comprising administering an effective amount of a non-glycosylated recombinant keratin. Specifically, the recombinant keratin may be any one selected from the group consisting of recombinant keratin 31, recombinant keratin 32, recombinant keratin 33A, recombinant keratin 33B, recombinant keratin 35, recombinant keratin 36, recombinant keratin 37 and recombinant keratin 38.

A composition according to one specific example can exhibit excellent hair loss prevention, treatment, or improvement effects by significantly improving hair growth promotion and hair regeneration effects compared to a composition containing human hair-derived keratin by including non-glycosylated recombinant keratin.

Figure 1 shows a vector map of pBT7-N-His.
Figure 2A shows the results of confirming the molecular weight of recombinant human keratin 34 (rhKRT34) manufactured according to one embodiment.
Figure 2B shows the SDS-PAGE analysis results for rhKRT31, rhKRT32, rhKRT33A, rhKRT33B, rhKRT34, rhKRT35, rhKRT36, rhKRT37, and rhKRT38 manufactured according to one embodiment.
FIG. 3A is a schematic diagram showing the structure of recombinant human keratin 34 manufactured according to one embodiment.
Figure 3B shows the structures of rhKRT31, rhKRT32, rhKRT33A, rhKRT33B, rhKRT34, rhKRT35, rhKRT36, rhKRT37, and rhKRT38 using AlphaFold, a protein structure database.
Figure 3C is a schematic diagram of the mechanism that induces hair growth.
Figure 4 shows the results of analyzing the identity of recombinant human keratin 34 manufactured according to one embodiment using MALDI-TOF.
Figure 5A shows the results of confirming the size distribution and cohesion differences of particles through dynamic light scattering analysis of human hair-derived keratin.
Figure 5B shows the results of dynamic light scattering analysis for recombinant keratin 34 to confirm the distribution of particle size and differences in aggregation.
Figure 6 is a conceptual diagram of human hair-derived keratin as glycosylated and recombinant human keratin 34, and the results of a Western blot analysis confirming that recombinant human keratin 34 produced according to one embodiment is not glycosylated.
Figure 7A shows the results of surface charge analysis of recombinant human keratin 34 and human hair keratin manufactured according to one embodiment.
Figure 7B shows the results of analyzing the cysteine content of recombinant human keratin 34 and human hair keratin manufactured according to one embodiment.
Figure 8A shows the results of confirming the induction of hair papilla cell aggregation according to treatment with recombinant human keratin 34 and human hair-derived keratin manufactured according to one embodiment (Control is an untreated group, human hair-derived keratin was treated at 0.05%, and rhKRT34 was treated at 0.05%, 0.025%, 0.01%, and 0.005% for comparison).
Figure 8B is a graph comparing the size of the aggregates calculated as area from the results of Figure 8A (the line indicated on each bar graph represents the standard deviation).
Figure 8C shows that aggregation of mammary papilla cells was induced when treated with 0.05% (w/v) of rhKRT32.
Figure 8D shows that aggregation of mammary papilla cells was induced when treated with 0.05% (w/v) of rhKRT33A, rhKRT35, and rhKRT37.
Figure 8E shows that aggregation of mammary papilla cells was induced when treated with 0.05% (w/v) of rhKRT33A, rhKRT35, and rhKRT37, as confirmed by phalloidin staining.
Figure 8F shows that aggregation of mammary papilla cells was induced when treated with 0.05% (w/v) of rhKRT31, rhKRT33B, rhKRT36, and rhKRT38.
Figure 9A shows the results of confirming the expression of hair growth-related markers of mammary papilla cells, β-catenin, ALPase, and CD133, in the untreated group as a control group.
Figure 9B shows the results confirming that recombinant human keratin 34 manufactured according to one embodiment increased the expression of hair growth-related markers of dermal papilla cells, such as β-catenin, ALPase, and CD133.
Figure 9C shows the results confirming that rhKRT32 manufactured according to one embodiment increased the expression of β-catenin, a hair growth-related marker of mammary papilla cells.
Figure 10 is a graph showing the mRNA expression of human hair-derived β-catenin, ALPase, CD133, and shh according to the treatment concentration and time of recombinant human keratin 34 and human hair-derived keratin manufactured according to one embodiment (an untreated group was used as a control group).
Figure 11A 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 manufactured according to one embodiment.
Figure 11B is an image confirming the differentiation results of outer root sheath cells (ORS) according to the treatment concentration of recombinant human keratin 32 and human hair-derived keratin manufactured according to one embodiment.
Figure 12A is an image confirming the expression of β-catenin in outer root sheath cells according to treatment with recombinant human keratin 34 and human hair-derived keratin manufactured according to one embodiment (an untreated group was used as a control group).
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 manufactured according to one embodiment (an untreated group was used as a control group).
Figure 12C is an image confirming the expression of β-catenin in outer root sheath cells according to treatment with recombinant human keratin 32 and human hair-derived keratin manufactured according to one embodiment (an untreated group was used as a control group).
Figure 13A is an image confirming the inhibition of CD 34 expression of outer root sheath cells by treatment with recombinant human keratin 34 and human hair-derived keratin manufactured according to one embodiment (an untreated group was used as a control group).
Figure 13B is an image confirming an increase in the expression of P-cadherin in outer root sheath cells following treatment with recombinant human keratin 34 and human hair-derived keratin manufactured according to one embodiment (an untreated group was used as a control group).
Figure 13C is an image confirming an increase in the expression of P-cadherin in outer root sheath cells following treatment with recombinant human keratin 32 and human hair-derived keratin manufactured according to one embodiment (an untreated group was used as a control group).
Figure 13D is an image confirming an increase in the expression of P-cadherin in outer root sheath cells according to treatment with recombinant human keratin 32 and human hair-derived keratin manufactured according to one embodiment, and shows the result of using phalloidin staining together (an untreated group was used as a control group).
Figure 13E is an image confirming an increase in the expression of P-cadherin in outer root sheath cells according to treatment with recombinant human keratin 33, recombinant human keratin 35, recombinant human keratin 37, and human hair-derived keratin manufactured according to one embodiment (an untreated group was used as a control group).
Figure 13F is an image confirming an increase in the expression of P-cadherin in outer root sheath cells according to treatment with recombinant human keratin 31, recombinant human keratin 33B, recombinant human keratin 36, recombinant human keratin 38 and human hair-derived keratin manufactured according to one embodiment (an untreated group was used as a control group).
Figure 14 is a graph showing the mRNA expression of hair growth-related markers of outer root sheath cells, β-catenin, ITGA6, Sox9, and FOXN1, according to the treatment concentration and time of recombinant human keratin 34 and human hair-derived keratin manufactured according to one embodiment (an untreated group was used as a control group).
Figure 15A shows the results of animal experiments and H&E staining of tissues over time (28 days) after treatment with recombinant human keratin 34 and human hair-derived keratin manufactured according to one embodiment.
Figure 15B is a graph showing the number of hair follicles confirmed 28 days after treatment with recombinant human keratin 34 and human hair-derived keratin according to the human hair growth cycle.
Figure 15C is a graph showing the growth cycle ratio of hair follicles confirmed 28 days after treatment with recombinant human keratin 34 and human hair-derived keratin.
Figure 15D is a graph showing the size ratio of hair follicles 28 days after treatment with recombinant human keratin 34 and human hair-derived keratin.
Fig. 16 shows the results of a comparative analysis of the amino acid sequence similarity between keratin 34 and other acidic keratins constituting hair. Fig. 16A shows the results of comparison between KRT34 and KRT31, Fig. 16B shows the results of comparison between KRT34 and KRT32, Fig. 16C shows the results of comparison between KRT34 and KRT33a, Fig. 16D shows the results of comparison between KRT34 and KRT33b, Fig. 16E shows the results of comparison between KRT34 and KRT35, Fig. 16F shows the results of comparison between KRT34 and KRT36, Fig. 16G shows the results of comparison between KRT34 and KRT37, and Fig. 16H shows the results of comparison between KRT34 and KRT38.
Figure 17 shows the results of a phylogenetic analysis of human keratin, which shows that it can be divided into type I keratin and type II keratin.

Below, one or more specific examples are described in more detail by way of examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Preparation of recombinant keratin (KRT)

1-1. Production of recombinant vector

Genetic information of human keratin 31 (Homo sapiens keratin 31, KRT31), human keratin 32 (Homo sapiens keratin 32, KRT32), 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 35 (Homo sapiens keratin 35, KRT35), human keratin 36 (Homo sapiens keratin 36, KRT36), human keratin 37 (Homo sapiens keratin 37, KRT37), and human keratin 38 (Homo sapiens keratin 38, KRT38) is available from the National Center for Biotechnology Information in the United States. Obtained from (www.ncbi.nlm.nih.gov).

The protein coding sequences (CDS) were extracted from the mRNA sequences of KRT34, KRT31, KRT32, KRT33A, KRT33B, KRT35, KRT36, KRT37, and KRT38. The coding sequence of KRT34 is SEQ ID NO: 1, the coding sequence of KRT31 is SEQ ID NO: 2, the coding sequence of KRT32 is SEQ ID NO: 3, the coding sequence of KRT33A is SEQ ID NO: 4, the coding sequence of KRT33B is SEQ ID NO: 5, the coding sequence of KRT35 is SEQ ID NO: 6, the coding sequence of KRT36 is SEQ ID NO: 7, the coding sequence of KRT37 is SEQ ID NO: 8, and the coding sequence of KRT38 is SEQ ID NO: 9.

We commissioned Bioneer (www.bioneer.co.kr) to produce recombinant vectors (pKRT31, pKRT32, pKRT33A, pKRT33B, pKRT34, pKRT35, pKRT36, pKRT37, pKRT38) containing the protein coding region of each KRT. (Hereinafter, pKRT31, pKRT32, pKRT33A, pKRT33B, pKRT34, pKRT35, pKRT36, pKRT37, pKRT38 may be referred to as pKRT.)

According to Figure 1, the recombinant vector (pKRT) is a pBT7-N-His vector suitable for protein expression, which introduces the KRT gene, contains a T7 promoter so that a large amount of mRNA can be transcribed, and can induce expression with Isopropyl-β-D-thiogalactopyranoside (IPTG) through the lac operator. In addition, the gene sequence of a histidine affinity ligand for affinity chromatography, a protein separation and purification process, is present in the N-terminal portion. Using the recombinant vector, the production and cultivation of transformed microorganisms, and the expression and purification of recombinant keratin were performed.

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 recombinant vectors (pKRT31, pKRT32, pKRT33A, pKRT33B, pKRT34, pKRT35, pKRT36, pKRT37, pKRT38) were each added to E. coli competent cells, left on ice for 10 minutes, and then heat-shocked at 42°C for 90 seconds. E. coli introduced with the recombinant vector were plated on LB solid medium coated with ampicillin (ampicillin, Sigma), and after culturing for 18 hours, colonies of BL21/pKRT were obtained. (Hereinafter, the transformed E. coli into which each of the recombinant vectors pKRT31, pKRT32, pKRT33A, pKRT33B, pKRT34, pKRT35, pKRT36, pKRT37, and pKRT38 has been introduced is referred to as BL21/pKRT)

1-3. Culture and expression of transformed microorganisms

BL21/pKRT was cultured with LB liquid medium. Specifically, 10 ml of LB liquid medium (containing 500 ㎍/ml ampicillin) was added to a 50 ml tube, 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 was 0.5 to 0.8, the inducer IPTG was added to 0.5 mM to induce the expression of the recombinant protein rhKRT (recombinant human Keratin). Additionally, the culture was stirred at 25°C for 16 hours, and the culture was centrifuged at 3,000 rpm for 20 minutes to remove the supernatant and harvest the cells. The harvested cells were stored at -80°C and used in the experiment.

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 resuspended at room temperature with a disruption solution (20 mM Tris-HCl, pH 8.0), and disrupted for 90 seconds using an ultrasonic disruptor with pulse on-off for 5 to 15 seconds. The disrupted E. coli sample was centrifuged at 2,000 × g for 20 minutes, and the supernatant was removed.

To wash the inclusion bodies, the pellet was added with an inclusion body washing solution (2 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 2% Triton X-100, pH 8.0), stirred, and then disrupted using an ultrasonic disrupter for 90 seconds. The sample was centrifuged at 2,000 × g for 10 minutes, and the supernatant was removed. The inclusion body washing process using the washing buffer was repeated 4 times. The washed inclusion bodies were stored at -20°C and used when necessary.

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

1-5. Purification and dialysis of rhKRT

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

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

1-6. rhKRT concentration

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

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

Figure 2B shows the SDS-PAGE analysis results for rhKRT31, rhKRT32, rhKRT33A, rhKRT33B, rhKRT34, rhKRT35, rhKRT36, rhKRT37, and rhKRT38. In Figure 3, rhKRT34 was detected at the 38 kDa position by the succinylation process. rhKRT31, rhKRT32, rhKRT33A, rhKRT33B, rhKRT35, rhKRT36, rhKRT37, and rhKRT38 all showed molecular weights in the range of 30 kDa to 70 kDa.

1-7. Manufacturing of rhKRT with sulfhydryl group blocking

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

Example 2: Characterization of rhKRT34

The physical properties of rhKRT34 manufactured in Example 1 above were confirmed.

2-1. Analysis of amino acid sequence, structure, and particle size of rhKRT34

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

According to Fig. 3A, 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. According to Fig. 3B, rhKRT31, rhKRT32, rhKRT33A, rhKRT33B, rhKRT35, rhKRT36, rhKRT37, and rhKRT38 have a folding structure similar to that of rhKRT34, and have a head, coil, linker, and tail configuration similar to that of rhKRT34.

Figure 3C is a schematic diagram of the mechanism for inducing hair growth. According to Figure 3, the mechanism for inducing hair growth requires (1) condensation of dermal epithelial cells and (2) production of P-cadherin. Here, rhKRT31, rhKRT32, rhKRT33A, rhKRT33B, rhKRT34, rhKRT35, rhKRT36, rhKRT37, and rhKRT38, which have similar folding structures, were confirmed to activate the above mechanism.

MALDI-TOF/TOF was performed at Proteinworks (http://www.proteinworks.co.kr/), and the peptide mass was measured using a MALDI-TOF/TOF mass spectrometer (Model: 4700 MALDI-TOF/TOF, Applied Biosystems). The peptide mass was recorded using the Mascot search program for theoretical peptides of all proteins in the NCBI database, and the results are shown in Fig. 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) and incubated at 37°C. The particle sizes of human hair keratin and rhKRT34 were relatively analyzed by Dynamic Light Scattering (DLS) Analysis, and the results are shown in Figs. 5A and 5B. According to Fig. 5, recombinant keratin (rhKRT34) was confirmed to be a mixture of monomer and dimer particles, and an average particle size of less than 100 nm. No aggregates exceeding 100 nm in size were observed even when incubated at 37°C. In comparison, human hair-derived keratin was found to form aggregates of 1000 nm or larger in size through 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.

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

2-3. Chemical properties analysis of rhKRT34

To measure the surface charge of keratin, rhKRT34 and human hair keratin were completely dissolved in purified water at a concentration of 0.1 (w/v)% each to prepare a keratin solution. Using a pH measuring device, sodium hydroxide solution was added to the keratin solution so that the pH of the solution became 11. The protein surface charge was measured using the zeta potential measuring function of the Dynamic Light Scattering device. Subsequently, hydrochloric acid was added to the solution to lower the pH by 0.5 to 1.5, and the surface charge was measured using the device up to pH 3, which is shown in Fig. 7A.

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

According to Figure 7A, in the case of human hair keratin, it was confirmed that the surface charge was negative and maintained negative over the pH range, showing no PI value, and in the case of rhKRT34, it was confirmed that the PI value at which the surface charge value became 0 around pH 4, confirming that human hair keratin and rhKRT34 are chemically different. In addition, in the case of human hair keratin, it was confirmed that almost no free sulfhydryl groups remained, and in the case of rhKRT34, it was confirmed that many free sulfhydryl groups remained.

Example 3: Analysis of hair growth effect of rhKRT

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

3-1-1. Analysis of cell aggregate formation of rhKRT34 in mammary papilla cells

In Example 1, rhKRT34 was treated to dermal papilla cells (DP cells) and the formation of cell aggregates was confirmed. If the cell aggregates increase, it can be evaluated as having an effect of inducing hair growth and hair regeneration.

Human papilla cells were seeded at 5 × 10 ⁴ /well in a 24-well plate, and after 24 hours, the medium was replaced with high glucose DMEM medium containing 0.05% (w/v), 0.025% (w/v), 0.01% (w/v), or 0.005% (w/v) of rhKRT34 of Example 1, and cultured for 2 days. As a control, high glucose DMEM medium without any treatment, and as a comparison group, human hair-derived keratin-derived keratin-containing high glucose DMEM medium cultured with 0.05% (w/v) were prepared. After culturing for 2 days, the number of spherical aggregates derived from papilla cells formed in each well was confirmed by an optical microscope. The images are shown in Fig. 8A, and they were counted. The sizes of the spherical aggregates were measured from the images, and are shown in Fig. 8B.

According to Fig. 8, recombinant keratin 34 induced the aggregation of hair papilla cells even at a low concentration of 0.05% (w/v) and had a superior aggregation-inducing ability compared to human hair keratin at the same concentration. In addition, recombinant keratin 34 showed a similar aggregation-inducing ability at a concentration of 0.005% (w/v) to human hair keratin at a concentration of 0.05 (w/v), confirming that the effective concentration of recombinant keratin 34 is 10 times lower than that of human hair-derived keratin.

3-1-2. Analysis of cell aggregate formation of rhKRT31, rhKRT32, rhKRT33A, rhKRT33B, rhKRT35, rhKRT36, rhKRT37, and rhKRT38 in mammary papilla cells

In Example 1, rhKRT31, rhKRT32, rhKRT33A, rhKRT33B, rhKRT35, rhKRT36, rhKRT37, and rhKRT38 were treated to breast papilla cells, and the formation of cell aggregates was confirmed.

Human hair papilla cells were seeded at 5 × 10 ⁴ /well in a 24-well plate, and after 24 hours, the medium was replaced with high glucose DMEM medium containing 0.05% (w/v) or 0.01% (w/v) of rhKRT31, rhKRT32, rhKRT33A, rhKRT33B, rhKRT35, rhKRT36, rhKRT37, and rhKRT38 of Example 1, and cultured for 1 or 2 days. As a control, untreated high glucose DMEM medium, and as a comparison group, hair papilla cells cultured in high glucose DMEM medium containing 0.5% or 0.25% (w/v) human hair-derived keratin were prepared. After culturing for 1 or 2 days, the number of spherical aggregates derived from mammary papilla cells formed in each well was confirmed by an optical microscope, and images are shown in Fig. 8C, Fig. 8D, Fig. 8E, and Fig. 8F.

Specifically, Fig. 8C shows that aggregation of papilla cells was induced when treated with 0.05% (w/v) of rhKRT32. Fig. 8D shows that aggregation of papilla cells was induced when treated with 0.05% (w/v) of rhKRT33A, rhKRT35, and rhKRT37, and Fig. 8E shows that aggregation of papilla cells was induced when treated with 0.05% (w/v) of rhKRT33A, rhKRT35, and rhKRT37, as confirmed by phalloidin staining. Fig. 8F shows that aggregation of papilla cells was induced when treated with 0.05% (w/v) of rhKRT31, 33B, 36, and 38.

According to FIGS. 8C, 8D, 8E, and 8F, rhKRT31, rhKRT32, rhKRT33A, rhKRT33B, rhKRT35, rhKRT36, rhKRT37, and rhKRT38 induced aggregation of hair papilla cells even at a low concentration of 0.05% (w/v) and had a superior aggregation-inducing ability than human hair keratin at the same concentration.

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

3-2-1. Analysis of hair growth-related markers of rhKRT34 in mammary papilla cells

The expression of hair growth-related markers, beta-catenin, CD133, and alkaline phosphatase (ALPase), in cultured mammary papilla cells was confirmed through immunohistochemical staining.

In Example 1, dermal papilla cells were cultured for 2 days in high glucose DMEM general medium containing 0.05% (w/v) rhKRT34. The medium was removed and washed with DPBS. The cells were fixed by treatment with 4% paraformaldehyde for 10 minutes and washed again with DPBS. After fixation, the cells were permeabilized by treatment with 0.1% Triton X-100 for 30 minutes and treated with 10% (w/v) normal goat serum (NGS) for 1 hour. Thereafter, rabbit anti-human beta-catenin antibody, rabbit anti-human CD133 antibody, or mouse anti-human ALPase antibody diluted 1:200 were treated and reacted at 4°C for 24 hours.

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

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

According to Figure 9, the rhKRT34 treatment group showed increased expression of beta-catenin, CD133, and alkaline phosphatase, which are hair growth-related markers of hair papilla cells, compared to the untreated group.

Additionally, the expression of hair growth-related markers, β-catenin, Alkaline phosphatase, CD133, and shh, in human hair papilla cells treated with various concentrations of human hair-derived keratin and rhKRT34 were 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 of sense and antisense primers, 10 μL qPCR Master Mix from Bioneer, and a final volume of 20 μL of DEPC-water. Primers used for the above 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'- CACCTCTGAGTCATCAGCCTG-3'). Samples were treated with the following protocol: heating at 95°C for 10 minutes, followed by 45 cycles of 95°C for 10 seconds, 60-63°C for 20 seconds, and 1 cycle of heating for melting curve, followed by cooling at 12°C. Data were normalized to GAPDH levels and are presented in Figure 10.

According to Figure 10, the rhKRT34-treated group showed increased mRNA expression of beta-catenin, CD133, alkaline phosphatase, and shh, which are hair growth-related markers of hair papilla cells, compared to the untreated group.

3-2-2. Analysis of hair growth-related markers of rhKRT32 in mammary papilla cells

For rhKRT32, the expression of beta-catenin, a hair growth-related marker, in cultured hair papilla cells was confirmed through immunochemical staining.

In Example 1, dermal papilla cells were cultured for 2 days in high glucose DMEM general medium containing 0.01% (w/v) or 0.05% (w/v) of rhKRT32. The medium was removed and washed with DPBS. The cells were fixed by treatment with 4% paraformaldehyde for 10 minutes and washed again with DPBS. After fixation, the cells were permeabilized by treatment with 0.1% Triton X-100 for 30 minutes and treated with 10% (w/v) normal goat serum (NGS) for 1 hour. Thereafter, rabbit anti-human beta-catenin antibody diluted 1:200 was treated and reacted at 4°C for 24 hours.

After 24 hours of reaction, the cells were washed three times with DPBS, and PE-conjugated Palloidin antibody was treated for 1 hour at room temperature for counterstaining, and Alexa Fluor 488 conjugated goat anti-rabbit IgG or Alexa Fluor 594 conjugated goat anti-mouse IgG as secondary antibodies were treated for 1 hour at room temperature. After that, the cells were washed three times with DPBS, and 4',6-diamidino-2-phenylindole (DAPI) was treated. An untreated group using the high glucose DMEM general medium was used as a control group. After that, the stained cells were observed under a fluorescence microscope (OlympusI ×71), and the image is shown in Fig. 9C.

According to Figure 9C, the rhKRT32-treated group showed increased expression of beta-catenin, a hair growth-related marker of hair papilla cells, compared to the untreated group.

3-3. Evaluation of cluster formation ability and differentiation induction ability of outer root cells

3-3-1. Evaluation of cluster formation and differentiation induction ability of rhKRT 34 in 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 ORS cells are closely related to hair root formation. In particular, outer root sheath cells expressing the CD34 marker are known to differentiate into the CD34 ^- /Lgr5 ⁺ cell population, which is directly involved in the formation of secondary hair germ cells.

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

According to Fig. 11A, the human hair-derived keratin treatment group showed cluster induction of outer root sheath cells and differentiation into an elongated form at a high concentration of 0.1% (w/v) or higher, but did not show such effects at a lower concentration of 0.01% (w/v). However, the rhKRT34 treatment group of Example 1 showed cluster induction of outer root sheath cells and differentiation into an elongated form even at a low concentration of 0.01% (w/v).

Additionally, the expression changes of β-catenin, Lgr5, CD34, and P-cadherin were confirmed from the rhKRT34 treatment group and the human hair-derived keratin treatment group. Fig. 12A is an image confirming the expression change of β-catenin, Fig. 12B is an image confirming the expression change of Lgr5, Fig. 13A is an image confirming the expression change of CD34, and Fig. 13B is an image confirming the expression change of P-cadherin.

According to Fig. 12, human hair-derived keratin increased the expression of β-catenin and Lgr5 in outer root sheath cells treated at a high concentration of 0.1% (w/v) or higher, but did not show this effect at a concentration of 0.01% (w/v). 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 CD34 expression when treated at 0.1% (w/v), and CD34 expression was shown when treated at 0.01% (w/v), but rhKRT34 of Example 1 suppressed CD34 expression even at a low concentration of 0.01% (w/v). In addition, it was confirmed that rhKRT34 increased the expression of P-cadherin, a marker expressed during hair germ formation.

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

The expression of beta-catenin, ITGA6, Sox9, and FOXN1, markers related to hair growth in outer root sheath cells, was confirmed by RT-qPCR analysis according to the concentration-specific treatment of human hair-derived keratin and rhKRT34. RT-qPCR analysis was performed under the same conditions as in the previous example. Primers used for the above 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 hair growth-related markers of outer root sheath cells, beta-catenin, ITGA6, Sox9, and FOXN1, compared to the untreated group.

3-3-2. Evaluation of cluster formation ability and differentiation induction ability of rhKRT31, rhKRT32, rhKRT33A, rhKRT33B, rhKRT35, rhKRT36, rhKRT37, and rhKRT38 in outer root sheath cells

In Example 1, outer root sheath cells were cultured for 2 days in high glucose DMEM medium containing rhKRT31, rhKRT32, rhKRT33A, rhKRT33B, rhKRT35, rhKRT36, rhKRT37, and rhKRT38 at a concentration of 0.1% (w/v) or 0.05% (w/v), and colony formation and differentiation induction were observed. The untreated group was used as the control group, 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), 0.25% (w/v), or 0.5% (w/v), and colony formation and differentiation induction were observed under an optical microscope (OLYMPUS, IX71, ×100). The results are shown in Fig. 11B.

According to Figure 11B, the human hair-derived keratin treatment group exhibited cluster induction of outer root sheath cells and differentiation into an elongated form at high concentrations of 0.5% (w/v) or higher. On the other hand, the rhKRT32 treatment group of Example 1 exhibited cluster induction of outer root sheath cells and differentiation into an elongated form even at low concentrations of 0.1% (w/v) and 0.05% (w/v).

Additionally, changes in the expression of β-catenin and P-cadherin were confirmed from the rhKRT31, rhKRT32, rhKRT33A, rhKRT33B, rhKRT35, rhKRT36, rhKRT37, rhKRT38 treatment groups and the human hair-derived keratin treatment group. Figure 12C is an image confirming the change in expression of β-catenin according to treatment with rhKRT32, Figure 13C is an image confirming the change in expression of P-cadherin according to treatment with rhKRT32, Figure 13D is an image confirming the change in expression of P-cadherin according to treatment with rhKRT32, Figure 13E is an image confirming the change in expression of P-cadherin according to treatment with rhKRT32 with Phalloidin staining, Figure 13F is an image confirming the change in expression of P-cadherin according to treatment with rhKRT33A, rhKRT35, and rhKRT37, and Figure 13G is an image confirming the change in expression of P-cadherin according to treatment with rhKRT31, rhKRT33B, rhKRT36, and rhKRT38 with Phalloidin staining.

According to Figure 12C, human hair-derived keratin increased the expression of β-catenin in outer root sheath cells treated with a high concentration of 0.5% (w/v) or higher, and rhKRT32 of Example 1 increased the expression of β-catenin even at low concentrations of 0.1% (w/v) and 0.05% (w/v).

According to Figure 13C, human hair-derived keratin increased P-cadherin expression in outer root sheath cells treated at high concentrations of 0.5% (w/v) or higher, and rhKRT32 of Example 1 increased P-cadherin expression even at low concentrations of 0.1% (w/v) and 0.05% (w/v).

According to Figure 13D, human hair-derived keratin increased P-cadherin expression in outer root sheath cells treated with high concentrations of 0.5% (w/v) or higher, and rhKRT32 of Example 1 increased P-cadherin expression even at low concentrations of 0.1% (w/v) and 0.05% (w/v).

According to Figure 13E, human hair-derived keratin showed P-cadherin expression in outer root sheath cells treated with a concentration of 0.1% (w/v) or higher, and rhKRT33A, rhKRT35, and rhKRT37 of Example 1 showed P-cadherin expression even at a low concentration of 0.05% (w/v).

According to Figure 13F, human hair-derived keratin showed P-cadherin expression in outer root sheath cells treated with a concentration of 0.25% (w/v) or higher, and rhKRT31, rhKRT33B, rhKRT36, and rhKRT38 of Example 1 showed P-cadherin expression even at a low concentration of 0.05% (w/v).

According to the above results, rhKRT31, rhKRT32, rhKRT33A, rhKRT33B, rhKRT35, rhKRT36, rhKRT37, and rhKRT38 were confirmed to induce clustering of outer root sheath cells, differentiation into an elongated form, and differentiation into β-catenin ⁺ /P-cadherin ⁺ cells even at concentrations lower than human keratin.

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 above mice were housed under controlled conditions of 23 ± 2 ℃ temperature, 50 ± 5% humidity, and 12-h light/dark cycle. The hair on the back of the mice was repeatedly shaved using an electric clipper to synchronize the hair follicle cycle. To investigate the hair growth promoting effect of keratin, Dulbecco's acid buffered saline, 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 were used. Before the procedure, 10-week-old mice were completely removed from the back hair using commercially available hair removal cream Veet ^® (Reckitt Benckiser, 62200809951), and were randomly assigned to receive the injection. Observations and records were made for 28 days (see Fig. 15A), and the mice were sacrificed and the skin tissues were fixed with 10% neutral buffered formalin (BBC Biochemical, 0141). The tissues were embedded in paraffin, sectioned at 4 μm thickness, 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 Х100 magnification and are shown in Figs. 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. When rhKRT34 was injected, the number of hair follicles increased relatively significantly throughout the hair growth cycle, the proportion of hair follicles in the growth phase increased, and the diameter of the hair follicles also increased relatively significantly when rhKRT34 was injected.

3-5. Analysis of the similarity between rhKRT34 and acidic keratin that constitutes human hair

To compare the amino acid sequence similarity of deglycosylated recombinant keratin 34 with other acid keratins that compose hair, the blastp algorithm, which is used to compare proteins in NCBI's Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi), was used to compare the keratin 34 sequence with eight other classes of acid keratins that compose human hair (KRT31, KRT32, KRT33A, KRT33B, KRT35, KRT36, KRT37, KRT38). For similar sequences, you can check the related information (similarity score (score, algorithmically scores the similarity between sequences), identity (Identities, the percentage of identical amino acids between two sequences), similarity (Positives, the percentage of amino acids that are not identical but have similar properties between two sequences)) and the alignment results between each query and the keratin 34 sequence based on the similarity through blastp.

According to Fig. 16, as a result of comparative analysis of the amino acid sequence identities (identities) and similarities (positives) between keratin 34 and other acidic keratins constituting hair, it was confirmed that the recombinant keratin 34 has an amino acid sequence having 60% or more identity or an amino acid sequence having 75% or more similarity, and it can be seen that the keratins constituting human hair have very high similarity in their amino acid sequences. Specifically, Fig. 16A shows that the amino acid sequence identity of KRT34 and KRT31 is 90%, and the amino acid sequence similarity is 93%. Fig. 16B shows that the amino acid sequence identity of KRT34 and KRT32 is 69%, and the amino acid sequence similarity is 82%. Fig. 16C shows that the amino acid sequence identity of KRT34 and KRT33A is 90%, and the amino acid sequence similarity is 93%. Figure 16D shows that the amino acid sequence identity of KRT34 and KRT33B is 88%, and the amino acid sequence similarity is 92%. Figure 16E shows that the amino acid sequence identity of KRT34 and KRT35 is 72%, and the amino acid sequence similarity is 81%. Figure 16F shows that the amino acid sequence identity of KRT34 and KRT36 is 72%, and the amino acid sequence similarity is 82%. Figure 16G shows that the amino acid sequence identity of KRT34 and KRT37 is 62%, and the amino acid sequence similarity is 78%. Figure 16H shows that the amino acid sequence identity of KRT34 and KRT38 is 62%, and the amino acid sequence similarity is 78%.

It is known that if the amino acid sequence identity between two proteins is less than 60%, it means excessive structural deformation of the protein, and the same or similar function cannot be secured. Specifically, if the amino acid sequence identity of the acid keratin is less than 60% or the amino acid sequence similarity is less than 75% with respect to the recombinant keratin 34, the function of the keratin is lost or reduced, and the same hair loss prevention or treatment effect as in the recombinant keratin 34 cannot be secured. The eight kinds of acid keratins satisfy the amino acid sequence identity of 60% or more or the similarity of 75% or more, or the identity of 60% or more and the similarity of 75% or more, and can exhibit the same or similar hair loss prevention or treatment effect as in the recombinant keratin 34.

Figure 17 shows the results of phylogenetic analysis of human keratins, which can be divided into type I keratins and type II keratins. Figure 17 shows that KRT 31, 32, 33a, 33b, 35, 36, 37, and 38, including KRT 34, are all classified into the same type I subgroup. Specifically, all functional type I keratin genes are clustered on the long arm (q) of human chromosome 17, whereas all functional type II keratin genes are located on the long arm of chromosome 12. As highlighted by the color codes used in frames A and B, individual type I and type II keratin genes that belong to the same subgroup based on the basic structure of their protein products tend to cluster in the genome. Furthermore, highly homologous keratin proteins (e.g., K5 and K6 paralogs, K14, K16, and K17) are often encoded by neighboring genes, indicating a key role of gene duplication in generating keratin diversity.

Attach electronic file of sequence list

Claims (7)

A composition comprising at least one recombinant keratin selected from the group consisting of a recombinant keratin, a recombinant keratin having 90% or more amino acid sequence identity (identities) with the recombinant keratin, and a recombinant keratin having 95% or more amino acid sequence similarity (positives) with the recombinant keratin as an active ingredient,
The above recombinant keratin is any one selected from the group consisting of recombinant keratin 31, recombinant keratin 32, recombinant keratin 33A, recombinant keratin 33B, recombinant keratin 35, recombinant keratin 36, recombinant keratin 37 and recombinant keratin 38.
The above recombinant keratin is non-glycosylated.
A pharmaceutical composition for preventing or treating alopecia. In the first paragraph,
The sequence of the above recombinant keratin 31 is the amino acid sequence of sequence number 11,
The sequence of the above recombinant keratin 32 is the amino acid sequence of sequence number 12,
The sequence of the above recombinant keratin 33A is the amino acid sequence of sequence number 13,
The sequence of the above recombinant keratin 33B is the amino acid sequence of SEQ ID NO. 14,
The sequence of the above recombinant keratin 35 is the amino acid sequence of sequence number 15,
The sequence of the above recombinant keratin 36 is the amino acid sequence of sequence number 16,
The sequence of the above recombinant keratin 37 is the amino acid sequence of sequence number 17,
A pharmaceutical composition for treating hair loss, characterized in that the sequence of the above recombinant keratin 38 is the amino acid sequence of sequence number 18. In the first paragraph,
The above recombinant keratin is expressed from a transformed prokaryotic cell.
A pharmaceutical composition for the treatment of hair loss. In the first paragraph,
The above recombinant keratin is one in which disulfide bonds are broken.
Particles with an average size of less than 100 nm,
A pharmaceutical composition for the treatment of hair loss. In the first paragraph,
The average molecular weight of the above recombinant keratin is 30 to 70 kDa.
A pharmaceutical composition for the treatment of hair loss. In the first paragraph,
The effective concentration of the above recombinant keratin is 10 times lower than the effective concentration of human hair-derived keratin.
A pharmaceutical composition for the treatment of hair loss. Containing as an active ingredient at least one recombinant keratin selected from the group consisting of recombinant keratin, recombinant keratin having 90% or more amino acid sequence identity with said recombinant keratin, and recombinant keratin having 95% or more amino acid sequence similarity with said recombinant keratin,
The above recombinant keratin is any one selected from the group consisting of recombinant keratin 31, recombinant keratin 32, recombinant keratin 33A, recombinant keratin 33B, recombinant keratin 35, recombinant keratin 36, recombinant keratin 37 and recombinant keratin 38.
The above recombinant keratin is non-glycosylated.
A cosmetic composition for improving alopecia.