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WO2024167321A1 - Composition pour le traitement de l'alopécie comprenant de la kératine recombinante - Google Patents

Composition pour le traitement de l'alopécie comprenant de la kératine recombinante Download PDF

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Publication number
WO2024167321A1
WO2024167321A1 PCT/KR2024/001856 KR2024001856W WO2024167321A1 WO 2024167321 A1 WO2024167321 A1 WO 2024167321A1 KR 2024001856 W KR2024001856 W KR 2024001856W WO 2024167321 A1 WO2024167321 A1 WO 2024167321A1
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Prior art keywords
keratin
recombinant
recombinant keratin
hair
amino acid
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English (en)
Korean (ko)
Inventor
한송욱
안성영
김우경
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Keramedix Inc
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Keramedix Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1748—Keratin; Cytokeratin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4741—Keratin; Cytokeratin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients

Definitions

  • the present invention relates to a composition for treating hair loss comprising deglycosylated recombinant keratin.
  • 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.
  • composition for treating hair loss 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
  • 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%.
  • 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%.
  • 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).
  • 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
  • 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.
  • growth factor BMP Bis 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.
  • the amino acid sequence of the recombinant keratin 34 may be a sequence of SEQ ID NO: 10.
  • 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.
  • 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.
  • 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.
  • the recombinant keratin may be expressed from a transformed prokaryotic cell.
  • the recombinant keratin may not include recombinant keratin 34.
  • 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.
  • CDS protein coding sequence
  • 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 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 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.
  • 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.
  • the recombinant keratin may include a form of monomer keratin in which a disulfide bond is cleaved by a chemical treatment.
  • 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.
  • the chemical treatment may include treatment with TCEP (Tris-2-Carboxyethylphosphine Hydrochloride), Dithiothreitol, or beta-mercaptoethanol.
  • TCEP Tris-2-Carboxyethylphosphine Hydrochloride
  • Dithiothreitol Dithiothreitol
  • beta-mercaptoethanol 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 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.
  • 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.
  • the average molecular weight of the recombinant keratin may be 20 to 100 kDa.
  • 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
  • the effective concentration of the recombinant keratin may be 10 times lower than the effective concentration of the human hair-derived keratin.
  • 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.
  • the recombinant keratin exhibited relatively higher efficacy than the human hair-derived keratin at the same concentration.
  • 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.
  • 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.
  • 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.
  • 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.
  • the recombinant keratin selected from
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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, ⁇ -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 ⁇ -catenin, ALPase, and CD133.
  • Figure 9C shows the results confirming that rhKRT32 manufactured according to one embodiment increased the expression of ⁇ -catenin, a hair growth-related marker of mammary papilla cells.
  • Figure 10 is a graph showing the mRNA expression of human hair-derived ⁇ -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.
  • ORS outer root sheath cells
  • 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.
  • ORS outer root sheath cells
  • Figure 12A is an image confirming the expression of ⁇ -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 ⁇ -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, ⁇ -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 forming 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
  • 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.
  • 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 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
  • the coding sequence of KRT38 is SEQ ID NO: 9.
  • pKRT31, pKRT32, pKRT33A, pKRT33B, pKRT34, pKRT35, pKRT36, pKRT37, pKRT38 recombinant vectors containing the protein coding region of each KRT.
  • pKRT31, pKRT32, pKRT33A, pKRT33B, pKRT34, pKRT35, pKRT36, pKRT37, pKRT38 may be referred to as pKRT.
  • the recombinant vector 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- ⁇ -D-thiogalactopyranoside (IPTG) through the lac operator.
  • IPTG Isopropyl- ⁇ -D-thiogalactopyranoside
  • the gene sequence of a histidine affinity ligand for affinity chromatography, a protein separation and purification process is present in the N-terminal portion.
  • 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.
  • BL21/pKRT was cultured with LB liquid medium. Specifically, 10 ml of LB liquid medium (containing 500 ⁇ g/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.
  • 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.
  • 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.
  • an inclusion body washing solution (2 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 2% Triton X-100, pH 8.0
  • 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 ⁇ m filter to obtain the inclusion body dissolution solution. The inclusion body dissolution solution was used for the purification of rhKRT.
  • rhKRT was purified by affinity chromatography. 2 ml of HisPurTM 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.
  • Spectra/Por ® Spectrum Lab. Inc. USA
  • the dialyzed rhKRT aqueous solution was filtered through a 0.22 ⁇ m membrane (Millipore SteriflipTM 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.
  • 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%.
  • imageJ image analysis program
  • Figure 2B shows the SDS-PAGE analysis results for rhKRT31, rhKRT32, rhKRT33A, rhKRT33B, rhKRT34, rhKRT35, rhKRT36, rhKRT37, and rhKRT38.
  • 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.
  • 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.
  • the amino acid sequence of rhKRT34 is the sequence of SEQ ID NO: 10, and the molecular weight of rhKRT34 is about 55 kDa.
  • 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.
  • 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.
  • Fig. 3A 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
  • 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.
  • the mechanism for inducing hair growth requires (1) condensation of dermal epithelial cells and (2) production of P-cadherin.
  • 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.
  • rhKRT34 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.
  • DPBS Dulbecco's phosphate buffered saline
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • hair growth-related markers beta-catenin, CD133, and alkaline phosphatase (ALPase)
  • ALPase alkaline phosphatase
  • 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.
  • NGS normal goat serum
  • 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.
  • DAPI 4',6-diamidino-2-phenylindole
  • 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.
  • RT-qPCR analysis was performed on triplicate samples using an RG-6000 RT-qPCR machine from Corbett. RT-qPCR was performed using 1 ⁇ L of cDNA template, 1 ⁇ L of sense and antisense primers, 10 ⁇ L qPCR Master Mix from Bioneer, and a final volume of 20 ⁇ L 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'), ⁇ -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').
  • the rhKRT34-treated group showed increased mRNA expression of hair growth-related markers of hair papilla cells, such as beta-catenin, CD133, alkaline phosphatase, and shh, compared to the untreated group.
  • beta-catenin a hair growth-related marker
  • 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.
  • 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
  • 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.
  • DAPI 4',6-diamidino-2-phenylindole
  • the rhKRT32-treated group showed increased expression of beta-catenin, a hair growth-related marker of hair papilla cells, compared to the untreated group.
  • Outer root sheath 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.
  • 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.
  • 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).
  • 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).
  • Fig. 12A is an image confirming the expression change of ⁇ -catenin
  • Fig. 12B is an image confirming the expression change of Lgr5
  • Fig. 13A is an image confirming the expression change of CD34
  • Fig. 13B is an image confirming the expression change of P-cadherin.
  • human hair-derived keratin increased the expression of ⁇ -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).
  • rhKRT34 of Example 1 increased the expression of ⁇ -catenin and Lgr5 even at low concentrations of 0.05% (w/v), 0.025% (w/v), and 0.01% (w/v).
  • 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.
  • beta-catenin, ITGA6, Sox9, and FOXN1 markers related to hair growth in outer root sheath cells.
  • RT-qPCR analysis was performed under the same conditions as in the previous example.
  • 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.
  • 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.
  • 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.
  • 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).
  • Figure 12C is an image confirming the change in expression of ⁇ -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
  • 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.
  • human hair-derived keratin increased the expression of ⁇ -catenin in outer root sheath cells treated with a high concentration of 0.5% (w/v) or higher
  • rhKRT32 of Example 1 increased the expression of ⁇ -catenin even at low concentrations of 0.1% (w/v) and 0.05% (w/v).
  • human hair-derived keratin increased P-cadherin expression in outer root sheath cells treated at high concentrations of 0.5% (w/v) or higher
  • rhKRT32 of Example 1 increased P-cadherin expression even at low concentrations of 0.1% (w/v) and 0.05% (w/v).
  • human hair-derived keratin increased P-cadherin expression in outer root sheath cells treated with high concentrations of 0.5% (w/v) or higher
  • rhKRT32 of Example 1 increased P-cadherin expression even at low concentrations of 0.1% (w/v) and 0.05% (w/v).
  • 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).
  • 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).
  • 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 ⁇ -catenin + /P-cadherin + cells even at concentrations lower than human keratin.
  • the hair growth effect was confirmed through animal testing using male C57BL/6 mice purchased from YoungBio (Samtako, 1404957265).
  • mice were housed under controlled conditions of 23 ⁇ 2 °C 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.
  • 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.
  • 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 ⁇ m 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 ⁇ 100 magnification and are shown in Figs. 15B to 15D.
  • BBC Biochemical neutral buffered formalin
  • 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).
  • 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.
  • 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. 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 similar
  • 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%.
  • 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.
  • 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.
  • 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.
  • highly homologous keratin proteins e.g., K5 and K6 paralogs, K14, K16, and K17
  • 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.

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Abstract

Composition comprenant de la kératine recombinante déglycosylée comme principe actif ayant des effets de promotion de pousse des cheveux et de régénération des cheveux supérieurs à ceux de la kératine dérivée de cheveux humains glycosylée des cheveux humains et pouvant ainsi être utilisée pour prévenir et traiter l'alopécie.
PCT/KR2024/001856 2022-02-08 2024-02-07 Composition pour le traitement de l'alopécie comprenant de la kératine recombinante Ceased WO2024167321A1 (fr)

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KR20220016105 2022-02-08
KR1020230016344A KR20230120594A (ko) 2022-02-08 2023-02-07 탈당화 재조합 케라틴을 포함하는 탈모 치료용 조성물
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