KR20230120594A - Composition for treating alopecia comprising deglycosylated recombinant keratin - Google Patents
Composition for treating alopecia comprising deglycosylated recombinant keratin Download PDFInfo
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- KR20230120594A KR20230120594A KR1020230016344A KR20230016344A KR20230120594A KR 20230120594 A KR20230120594 A KR 20230120594A KR 1020230016344 A KR1020230016344 A KR 1020230016344A KR 20230016344 A KR20230016344 A KR 20230016344A KR 20230120594 A KR20230120594 A KR 20230120594A
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- recombinant keratin
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Classifications
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- A—HUMAN NECESSITIES
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- 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
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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
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- 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
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- 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
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- 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
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- 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
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- 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
Abstract
글리코실화되지 않은 재조합 케라틴을 유효성분으로 포함하는 조성물은 글리코실화된 인모유래 케라틴보다 발모 촉진 및 모발 재생 효과가 우수하며 탈모(alopecia) 예방 및 치료 용도로 사용될 수 있다.A composition containing non-glycosylated recombinant keratin as an active ingredient has better hair growth promoting and hair regenerating effects than glycosylated human hair-derived keratin, and can be used for preventing and treating alopecia.
Description
본 발명은 2022년 2월 8일에 한국특허청에 제출된 한국 특허출원 제10-2022-0016105호의 출원일의 이익을 주장하며, 그 내용 전부는 본 발명에 포함된다.The present invention claims the benefit of the filing date of Korean Patent Application No. 10-2022-0016105 filed with the Korean Intellectual Property Office on February 8, 2022, all of which are included in the present invention.
본 발명은 탈당화 재조합 케라틴을 포함하는 탈모 치료용 조성물에 관한 것이다.The present invention relates to a composition for treating hair loss comprising deglycosylated recombinant keratin.
케라틴은 발모 촉진 효과가 있으며, 두피에 도포하는 것보다 경피 또는 피하에 주사하는 경우 더 우수한 발모 촉진 효과를 발휘할 수 있음이 개시되어 있다. It is disclosed that keratin has a hair growth promoting effect, and can exert a better hair growth promoting effect when injected percutaneously or subcutaneously than when applied to the scalp.
케라틴은 KRT1 내지 KRT86 등 다양한 유전자로부터 발현된 다양한 subclass 단백질들을 포함하고 있는데, 각 subclass 케라틴의 발모 촉진 효과는 어떠한지, 그리고 케라틴의 글리코실화 여부가 발모 촉진 효과에 영향을 미치는지 여부는 개시된 바가 없다.Keratin includes various subclass proteins expressed from various genes, such as KRT1 to KRT86. However, it has not been disclosed what the hair growth promoting effect of each subclass keratin is and whether glycosylation of keratin affects the hair growth promoting effect.
일 구체예에 따르면, 글리코실화 되지 않은 재조합 케라틴(deglycosylated recombinant keratin)을 유효성분으로 포함하는 탈모 치료용 조성물을 제공한다.According to one embodiment, a composition for treating hair loss containing deglycosylated recombinant keratin as an active ingredient is provided.
일 양상은 재조합 케라틴34(rhKRT34), 상기 재조합 케라틴34와 60% 이상의 아미노산 서열 일치성(identities)을 갖는 재조합 케라틴 및 상기 재조합 케라틴34와 75% 이상의 아미노산 서열 유사성(positives)을 갖는 재조합 케라틴을 포함하는 군으로부터 선택된 하나 이상의 재조합 케라틴을 유효성분으로 포함하고, 상기 재조합 케라틴은 글리코실화되지 않은 것인 탈모(alopecia) 예방 또는 치료용 약학적 조성물을 제공한다. 구체적으로, 상기 재조합 케라틴은 재조합 케라틴34일 수 있다.One aspect includes recombinant keratin 34 (rhKRT34), recombinant keratin having at least 60% amino acid sequence identities with the recombinant keratin 34 and recombinant keratin having at least 75% amino acid sequence identities with the recombinant keratin 34. It provides a pharmaceutical composition for preventing or treating alopecia, comprising at least one recombinant keratin selected from the group as an active ingredient, wherein the recombinant keratin is not glycosylated. Specifically, the recombinant keratin may be recombinant keratin 34.
본 발명자는 글리코실화되지 않은 재조합 케라틴34가 글리코실화된 인모유래 케라틴(hair keratin; 인모추출 케라틴 또는 인모 케라틴)보다 발모 유도 효과가 우수함을 입증하였다. 글리코실화는 진핵세포에서 일어나는 번역후 변형(post-translatal modification)에 의해 수행되는 과정으로, 글리코실화 유무는 단백질의 폴딩구조; 수용해도 등 물리적 특성; 효소활성, 항원성 등의 생리활성을 크게 변화될 수 있다. 예를 들면 성장인자인 BMP(Bone morphogenetic proteins)의 경우 글리코실화되지 않은 BMP는 본래의 생리활성을 상실하므로 E.coli가 아닌 포유류 세포주(mammalian cell line)에서 생산해야 한다. 이러한 점들을 고려하면, 글리코실화 되지 않은 재조합 케라틴34의 개선된 발모 효과는 예측하기 어려운 것이다. The present inventors have demonstrated that non-glycosylated recombinant keratin 34 is superior to glycosylated human hair-derived keratin (human hair-extracted keratin or human hair keratin) in its hair growth inducing effect. Glycosylation is a process performed by post-translatal modification that occurs in eukaryotic cells. physical properties such as water solubility; Physiological activities such as enzyme activity and antigenicity can be greatly changed. For example, in the case of bone morphogenetic proteins (BMPs), which are growth factors, non-glycosylated BMPs lose their original physiological activity, so they must be produced in mammalian cell lines, not E.coli. Considering these points, the improved hair growth effect of non-glycosylated recombinant Keratin 34 is difficult to predict.
상기 탈모는 두피의 모발이 비정상적으로 감소하거나 가늘어지는 상태를 의미한다. 상기 탈모 예방 또는 개선은 새로운 모발 생성 촉진뿐만 아니라 기존 모발이 건강하게 자라도록 하는 것을 포함한다. 상기 탈모는 예를 들면 남성형 탈모(male-pattern alopecia), 여성형 탈모(female-pattern alopecia), 원형 탈모(alopecia areata), 휴지기성 탈모(telogen alopecia), 안드로겐 탈모(Androgenetic Alopecia), 압박성 탈모(Pressure Alopecia), 또는 지루 탈모(Alopecia Seborrhecia)일 수 있으나 이에 한정되는 것은 아니다. The hair loss refers to a state in which hair on the scalp is abnormally reduced or thinned. The prevention or improvement of hair loss includes promotion of new hair growth as well as healthy growth of existing hair. The hair loss is, for example, male-pattern alopecia, female-pattern alopecia, alopecia areata, telogen alopecia, androgenetic alopecia, and pressure alopecia ( Pressure Alopecia), or seborrheic hair loss (Alopecia Seborrhecia), but is not limited thereto.
일 구체예에 따르면, 상기 재조합 케라틴 34의 아미노산 서열은 서열번호 10의 서열로 이루어진 것일 수 있다. According to one embodiment, the amino acid sequence of the recombinant keratin 34 may consist of the sequence of SEQ ID NO: 10.
일 구체예에 따르면, 상기 재조합 케라틴은 원핵세포로부터 발현된 것일 수 있다. 일 실시예에 따르면, 상기 재조합 케라틴은 재조합 케라틴34일 수 있으며, 상기 재조합 케라틴34는 케라틴34 유전자의 protein coding region을 클로닝한 벡터를 도입하여 형질전환시킨 박테리아로 생산된 것일 수 있다. 상기 원핵세포는 예를 들면 Escherichia 속 균주, Bacillus 속 균주, 또는 Corynebacterium속 균주일 수 있으며, 일 실시예에 따르면 E.coli일 수 있다. 포유동물 세포(mammalian cell)와 같은 진핵세포(Eukaryotic cell)에서 재조합 단백질을 발현시키면 글리코실화(glycosylation)되지만, 원핵세포(prokaryotic cell)로부터 재조합 단백질을 발현시키면 글리코실화가 일어나지 않는다. 구체적으로, 상기 재조합 케라틴34는 형질전환된 원핵세포로부터 발현된 것일 수 있다.According to one embodiment, the recombinant keratin may be expressed from prokaryotic cells. According to one embodiment, the recombinant keratin may be recombinant keratin 34, and the recombinant keratin 34 may be produced by transforming bacteria by introducing a vector in which the protein coding region of the keratin 34 gene is cloned. The prokaryotic cell may be, for example, a strain of the genus Escherichia, a strain of the genus Bacillus, or a strain of the genus Corynebacterium, and according to one embodiment, it may be E.coli. When recombinant proteins are expressed in eukaryotic cells such as mammalian cells, glycosylation occurs, but when recombinant proteins are expressed in prokaryotic cells, glycosylation does not occur. Specifically, the recombinant Keratin 34 may be expressed from a transformed prokaryotic cell.
상기 케라틴34는 인간 KRT34로부터 발현된 것일 수 있고, 재조합 케라틴 34는 인간 KRT34 유전자의 단백질 코딩 서열(Coding Sequence, CDS)으로부터 발현된 것일 수 있다. 상기 케라틴34 유전자 정보 및 코딩 서열은 공지된 데이터베이스로부터 얻을 수 있으며, 예를 들면 미국국립생물정보센터(NCBI)에서 얻을 수 있다. 상기 케라틴34 코딩 서열(CDS)은 예를 들면 서열번호 1의 염기 서열일 수 있다. The keratin 34 may be expressed from human KRT34, and the recombinant keratin 34 may be expressed from a protein coding sequence (CDS) of the human KRT34 gene. The keratin34 gene information and coding sequence can be obtained from a known database, for example, from the US National Center for Biological Information (NCBI). The keratin 34 coding sequence (CDS) may be, for example, the nucleotide sequence of SEQ ID NO: 1.
상기 케라틴 34의 클로닝, 재조합 케라틴 34를 발현하는 숙주세포의 제작, 배양, 및 단백질 정제는 통상기술자에게 알려진 방법으로 실시할 수 있으며 특별히 한정되는 것은 아니다. The cloning of keratin 34, preparation of host cells expressing recombinant keratin 34, culturing, and protein purification can be performed by methods known to those skilled in the art, and are not particularly limited.
일 구체예에 따르면, 상기 재조합 케라틴은 화학적 처리에 의해 이황화결합이 분해된 monomer 케라틴의 형태를 포함할 수 있다. 예를 들어, 상기 재조합 케라틴은 재조합 케라틴34일 수 있으며, 상기 재조합 케라틴34는 monomer 케라틴, monomer 케라틴이 다시 이황화결합을 형성한 dimer 케라틴 또는 이들의 조합으로 이루어질 수 있다. 상기 화학적 처리는 케라틴에 포함된 시스테인 간의 이황화결합의 절단을 포함할 수 있다. 예를 들어, 상기 화학적 처리는 TCEP(Tris-2-Carboxyethylphosphine Hydrochloride), Dithiothreitol 또는 beta-mercaptoethanol로 처리하는 것을 포함할 수 있다. Dynamic Light Scattering Analysis를 통한 입자크기 분석에 따르면, monomer인 재조합 케라틴34는 평균 크기 45nm 이하인 입자일 수 있다. monomer인 재조합 케라틴34 중 일부는 자발적인 이황화결합에 의해 dimer를 형성할 수 있으며, dimer인 재조합 케라틴34는 입자 크기가 100nm 이상인 응집된 케라틴 입자를 포함할 수 있다. 상기 일 양상에 따른 탈모 치료용 약학적 조성물은 재조합 케라틴34를 포함하는 수용액을 포함할 수 있으며, 상기 수용액 중에서 재조합 케라틴34의 평균 입자 크기는 100nm 이하로서, 체내에서 면역반응을 유발하지 않으면서 탈모의 예방 또는 치료 효과를 제공할 수 있다. 반면, 인모유래 케라틴은 당구조가 결합된 단백질을 포함하는 케라틴 복합물질로 이루어진 것으로, 수용액 중에서 상기 단백질 사이의 응집에 의해 평균 크기가 1000nm 이상인 응집체를 형성하게 되며, 상기 응집체는 체내에서 각종 면역반응을 유발할 수 있다. According to one embodiment, the recombinant keratin may include a form of monomer keratin in which disulfide bonds are broken down by chemical treatment. For example, the recombinant keratin may be recombinant keratin 34, and the recombinant keratin 34 may be composed of monomer keratin, dimer keratin in which monomer keratin forms a disulfide bond again, or a combination thereof. The chemical treatment may include cleavage of disulfide bonds between cysteines included in keratin. For example, the chemical treatment may include treatment with Tris-2-Carboxyethylphosphine Hydrochloride (TCEP), Dithiothreitol, or beta-mercaptoethanol. According to particle size analysis through Dynamic Light Scattering Analysis, recombinant keratin 34 as a monomer may be particles with an average size of 45 nm or less. Some of the recombinant keratin 34, which is a monomer, may form a dimer by spontaneous disulfide bonds, and the recombinant keratin 34, which is a dimer, may 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 may include an aqueous solution containing recombinant keratin 34, and the average particle size of recombinant keratin 34 in the aqueous solution is 100 nm or less, and hair loss does not induce an immune response in the body. of can provide a preventive or therapeutic effect. On the other hand, human hair-derived keratin is composed of a keratin complex material containing proteins to which sugar structures are bound, and aggregates having an average size of 1000 nm or more are formed by aggregation between the proteins in an aqueous solution, and the aggregates respond to various immune reactions in the body. can cause
일 구체예에 따르면, 상기 재조합 케라틴의 평균 분자량은 20 내지 100kDa일 수 있다. 구체적으로 상기 재조합 케라틴은 재조합 케라틴34일 수 있으며, 평균 분자량은 30 내지 100kDa, 40 내지 100kDa, 50 내지 100kDa, 20 내지 90kDa, 30 내지 90kDa, 40 내지 90kDa, 50 내지 90kDa, 20 내지 80kDa, 30 내지 80kDa, 40 내지 80kDa, 50 내지 80kDa, 20 내지 70kDa, 30 내지 70kDa, 40 내지 70kDa, 50 내지 70kDa, 20 내지 60kDa, 30 내지 60kDa, 40 내지 60kDa, 50 내지 60kDa, 또는 55kDa일 수 있다. 바람직하게는, 상기 재조합 케라틴34의 평균 분자량은 40 내지 70kDa일 수 있다.According to one embodiment, the average molecular weight of the recombinant keratin may be 20 to 100 kDa. Specifically, the recombinant keratin may be recombinant keratin 34, and the average molecular weight is 30 to 100 kDa, 40 to 100 kDa, 50 to 100 kDa, 20 to 90 kDa, 30 to 90 kDa, 40 to 90 kDa, 50 to 90 kDa, 20 to 80 kDa, and 30 to 80 kDa. 80 kDa, 40 to 80 kDa, 50 to 80 kDa, 20 to 70 kDa, 30 to 70 kDa, 40 to 70 kDa, 50 to 70 kDa, 20 to 60 kDa, 30 to 60 kDa, 40 to 60 kDa, 50 to 60 kDa, or 55 k may be Da. Preferably, the average molecular weight of the recombinant keratin 34 may be 40 to 70 kDa.
일 구체예에 따르면, 상기 재조합 케라틴의 유효농도는 인모유래 케라틴의 유효농도보다 10배 낮은 것일 수 있다. 구체적으로, 상기 재조합 케라틴은 재조합 케라틴34일 수 있으며, 상기 재조합 케라틴34의 유효농도는 상기 인모유래 케라틴의 유효농도보다 2배, 3배, 4배, 5배, 6배, 7배, 8배 또는 9배 낮은 것일 수 있다. 일 실시예에 따르면 상기 재조합 케라틴34는 동일한 농도에서 인모유래 케라틴보다 상대적으로 높은 효력을 나타냈다. According to one embodiment, the effective concentration of the recombinant keratin may be 10 times lower than the effective concentration of human hair-derived keratin. Specifically, the recombinant keratin may be recombinant keratin 34, and the effective concentration of the recombinant keratin 34 is 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, or 8 times the effective concentration of the human hair-derived keratin. or 9 times lower. According to one embodiment, the recombinant keratin 34 exhibited a relatively higher potency than human hair-derived keratin at the same concentration.
일 구체예에 따르면, 상기 재조합 케라틴은 N-말단에 태그가 결합되지 않은 것일 수 있으며, 예를 들면 GST 태그를 포함하지 않는 것일 수 있다. According to one embodiment, the recombinant keratin may not have a tag attached to its N-terminus, for example, it may not contain a GST tag.
상기 재조합 케라틴은 N-말단에 결합된 His 태그를 포함하고, C-말단에는 태그가 결합하지 않는 것일 수 있다. The recombinant keratin may include a His tag bound to the N-terminus, and the tag may not bind to the C-terminus.
일 구체예에 따르면, 상기 약학적 조성물은 국소투여용으로 제형화된 것일 수 있다. 상기 국소투여는 두피에 국소 투여되는 것일 수 있다. 상기 국소투여용 제형은 예를 들면 경피(transdermal) 투여용 제형, 진피(intradermal) 투여용 제형, 또는 피하(subcutaneous) 투여용 제형일 수 있다. According to one embodiment, the pharmaceutical composition may be formulated for topical administration. The topical administration may be topical administration to the scalp. The formulation for topical administration may be, for example, a formulation for transdermal administration, a formulation for intradermal administration, or a formulation for subcutaneous administration.
상기 경피투여용 제형은 주사제, 점적제, 연고, 로션, 겔, 크림, 스프레이, 현탁제, 유제, 패치일 수 있다. 상기 경피투여용 제형은 예를 들면 크림, 겔, 연고, 피부 유화제, 피부 현탁액, 경피전달성 패치, 약물 함유 붕대, 로션, 또는 이들의 조합일 수 있다. 상기 경피투여용 제형은 수성성분, 유성성분, 알코올류, 보습제, 증점제, 미백제, 방부제, 산화방지제, 계면활성제, 항료, 피부 영양제 등을 포함할 수 있다. The formulation for transdermal administration may be an injection, drop, ointment, lotion, gel, cream, spray, suspension, emulsion, or patch. The formulation for transdermal administration may be, for example, a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or a combination thereof. The formulation for transdermal administration may include an aqueous component, an oil component, alcohol, a moisturizer, a thickener, a whitening agent, a preservative, an antioxidant, a surfactant, a perfume, a skin nutrient, and the like.
상기 약학적 조성물은 주사제로 제형화될 수 있으며, 상기 주사제는 한크액(Hank's solution), 링거액(Ringer's solution), DPBS(Dubelcos Phosphate Buffered Solution), 생리 식염 완충액과 같은 생리학적으로 적합한 완충액을 포함할 수 있다.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, DPBS (Dubelcos Phosphate Buffered Solution), and physiological saline buffer. can
상기 약학적 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물 제형, 투여 경로 및 기간에 따라 달라질 수 있으며, 통상기술자에 의해 적절히 선택될 수 있다. A preferred dosage of the pharmaceutical composition may vary depending on the condition and weight of the patient, the severity of the disease, the drug formulation, the route and period of administration, and may be appropriately selected by a person skilled in the art.
다른 양상은, 재조합 케라틴34, 상기 재조합 케라틴34와 60% 이상의 아미노산 서열 일치성을 갖는 재조합 케라틴 및 상기 재조합 케라틴34와 75% 이상의 아미노산 서열 유사성을 갖는 재조합 케라틴을 포함하는 군으로부터 선택된 하나 이상의 재조합 케라틴을 유효성분으로 포함하고, 상기 재조합 케라틴은 글리코실화되지 않은 것인 탈모(alopecia) 개선용 화장료 조성물을 제공한다. 구체적으로 상기 재조합 케라틴은 상기 재조합 케라틴34일 수 있다.In another aspect, at least one recombinant keratin selected from the group comprising recombinant keratin 34, recombinant keratin having 60% or more amino acid sequence identity with the recombinant keratin 34, and recombinant keratin having 75% or more amino acid sequence similarity with the recombinant keratin 34. It contains as an active ingredient, and the recombinant keratin provides a cosmetic composition for improving hair loss (alopecia) that is not glycosylated. Specifically, the recombinant keratin may be the recombinant keratin 34.
상기 화장료 조성물은 상기 유효성분 이외에 기능성 첨가물 및 일반적인 화장료 조성물에 포함되는 성분을 추가로 포함할 수 있다. 상기 화장료 조성물은 항산화제, 안정화제, 용해화제, 비타민, 안료, 향료 등과 같은 통상적인 보조제 및 담체를 더 포함할 수 있다. 예를 들어, 상기 화장료 조성물에는 글리세린, 부틸렌글리콜, 폴리옥시에칠렌 경화피마자유, 토코페릴 아세테이트, 시트릭산, 스쿠알란, 소듐 시트레이트, 알란토인 등의 보조성분이 추가로 더 포함될 수 있고, 용제로는 헥산디올, 정제수 등을 포함할 수 있다. 또한 상기 기능성 첨가물은 수용성 비타민, 유용성 비타민, 고분자 펩티드, 고분자 다당, 및 스핑고 지질로 이루어진 군에서 선택된 성분을 포함할 수 있다. 이외에 포함되는 배합될 수 있는 성분으로서는 유지 성분, 보습제, 에몰리엔트제, 계면 활성제, 유기 및 무기 안료, 유기 분체, 자외선 흡수제, 방부제, 살균제, 산화 방지제, 식물 추출물, pH 조정제, 알콜, 색소, 향료, 혈행 촉진제, 냉감제, 제한(制汗)제, 정제수 등을 들 수 있다.The cosmetic composition may further include functional additives and ingredients included in general cosmetic compositions in addition to the active ingredient. The cosmetic composition may further include conventional adjuvants and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances, and the like. For example, the cosmetic composition may further include auxiliary ingredients such as glycerin, butylene glycol, polyoxyethylene hydrogenated castor oil, tocopheryl acetate, citric acid, squalane, sodium citrate, and allantoin, and the solvent is Hexanediol, purified water, and the like may be included. 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. Ingredients that can be blended include oil and fat components, humectants, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, Spices, blood circulation accelerators, cooling agents, antiperspirants, purified water and the like can be mentioned.
상기 화장료 조성물은 예를 들면 헤어토닉, 헤어컨디셔너, 헤어에센스, 헤어로션, 헤어영양로션, 헤어샴푸, 헤어린스, 헤어트리트먼트, 헤어크림, 헤어영양크림, 헤어모이스처크림, 헤어맛사지크림, 헤어왁스, 헤어 에어로졸, 헤어팩, 헤어영양팩, 헤어비누, 헤어클렌징폼, 머릿기름, 모발건조제, 모발보존처리제, 모발염색제, 모발용 웨이브제, 모발탈색제, 헤어겔, 헤어글레이즈, 헤어드레싱어, 헤어래커, 헤어모이스처라이저, 헤어무스 또는 헤어스프레이의 제형을 포함할 수 있으며, 구체적으로 샴푸, 클렌져, 컨디셔너, 트리트먼트, 헤어팩, 린스, 두피팩, 토닉, 에센스, 로션, 크림, 오일, 미스트, 스프레이, 또는 헤어젤 제형일 수 있다. The cosmetic composition is, for example, hair tonic, hair conditioner, hair essence, hair lotion, hair nutrition lotion, hair shampoo, hair rinse, hair treatment, hair cream, hair nutrition cream, hair moisture cream, hair massage cream, hair wax , hair aerosol, hair pack, hair nutrition pack, hair soap, hair cleansing foam, hair oil, hair drying agent, hair preservative, hair dye, hair waving agent, hair bleach, hair gel, hair glaze, hair dressing, hair lacquer, hair It may include a formulation of moisturizer, hair mousse or hair spray, specifically shampoo, cleanser, conditioner, treatment, hair pack, rinse, scalp pack, tonic, essence, lotion, cream, oil, mist, spray, or hair It may be in a gel formulation.
상기 화장료 조성물은 스킨로션, 스킨소프너, 스킨토너, 아스트린젠트, 로션, 밀크로션, 모이스쳐 로션, 영양로션, 맛사지크림, 영양크림, 모이스처크림, 핸드크림, 파운데이션, 에센스, 영양에센스, 팩, 비누, 클렌징폼, 클렌징로션, 클렌징크림, 바디로션 및 바디클린저로 이루어진 군으로부터 선택된 어느 하나 이상의 제형으로 제조될 수 있다. The cosmetic composition is skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrient lotion, massage cream, nutrient cream, moisture cream, hand cream, foundation, essence, nutrient essence, pack, soap, cleansing It may be prepared in any one or more formulations selected from the group consisting of foam, cleansing lotion, cleansing cream, body lotion and body cleanser.
상기 화장료 조성물의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물섬유, 식물섬유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 또는 산화아연을 포함할 수 있다. When the formulation of the cosmetic composition is a paste, cream or gel, animal fiber, vegetable fiber, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, or zinc oxide may be included as a carrier component. there is.
또 다른 양상은, 글리코실화되지 않은 재조합 케라틴을 유효량만큼 투여하는 단계를 포함하는 탈모 예방, 치료 또는 개선방법을 제공한다. 구체적으로 상기 재조합 케라틴은 재조합 케라틴34일 수 있다.Another aspect provides a method for preventing, treating, or improving hair loss comprising administering an effective amount of non-glycosylated recombinant keratin. Specifically, the recombinant keratin may be recombinant keratin 34.
일 구체예에 따른 조성물은 글리코실화되지 않은 재조합 케라틴을 포함함으로써 인모유래 케라틴을 포함하는 조성물보다 발모 촉진 및 모발 재생 효과가 현저히 향상되어 우수한 탈모 예방, 치료, 또는 개선 효과를 나타낼 수 있다.The composition according to one embodiment, by including non-glycosylated recombinant keratin, has significantly improved hair growth promoting and hair regenerating effects compared to a composition containing human hair-derived keratin, thereby exhibiting excellent hair loss prevention, treatment, or improvement effects.
도 1은 pBT7-N-His의 벡터맵을 나타낸 것이다.
도 2는 일 실시예에 따라 제조된 재조합 인간 케라틴34(rhKRT34)의 분자량을 확인한 결과이다.
도 3은 일 실시예에 따라 제조된 재조합 인간 케라틴34의 구조를 나타낸 모식도이다.
도 4는 일 실시예에 따라 제조된 재조합 인간 케라틴34의 Identity를 MALDI-TOF로 분석한 결과이다.
도 5a는 인모유래 케라틴에 대한 동적 광 산란 분석을 통해 입자의 크기 분포와 응집도 차이를 확인한 결과이다.
도 5b는 재조합 케라틴34에 대한 동적 광 산란 분석을 통해 입자 크기의 분포 및 응집도 차이를 확인한 결과이다.
도 6는 인모유래 케라틴으로서 글리코실화된 것과 재조합 인간 케라틴34의 개념도와 일 실시예에 따라 제조된 재조합 인간 케라틴34가 글리코실화되지 않았음을 확인한 웨스턴 블롯 분석 결과이다.
도 7a는 일 실시예에 따라 제조된 재조합 인간 케라틴34와 인모케라틴의 표면전하 (Surface Charge) 분석 결과이다.
도 7b는 일 실시예에 따라 제조된 재조합 인간 케라틴34와 인모케라틴의 시스테인의 함량 분석 결과이다.
도 8a는 일 실시예에 따라 제조된 재조합 인간 케라틴34와 인모유래 케라틴의 처리에 따른 모유두세포 응집 유도를 확인한 결과이다(Control은 미처리군이며, 인모유래 케라틴은 0.05%, rhKRT34는 0.05%, 0.025%, 0.01% 및 0.005%로 처리하여 비교하였다).
도 8b는 상기 도 8a의 결과로부터 응집물의 크기를 면적으로 계산하여 비교한 그래프이다(각 막대 그래프에 표시된 선은 표준 편차를 나타낸다).
도 9a는 대조군(Control)으로서 미처리군에서 모유두세포의 발모 관련 마커인 β-카테닌, ALPase, CD133의 발현을 확인한 결과이다.
도 9b는 일 실시예에 따라 제조된 재조합 인간 케라틴34이 모유두세포의 발모 관련 마커인 β-카테닌, ALPase, CD133의 발현을 증가시켰음을 확인한 결과이다.
도 10은 일 실시예에 따라 제조된 재조합 인간 케라틴34와 인모유래 케라틴의 처리 농도 및 시간 경과에 따른인모유래 β-카테닌, ALPase, CD133, shh의 mRNA 발현을 확인한 그래프이다(대조군(Control)으로는 미처리군을 사용하였다).
도 11은 일 실시예에 따라 제조된 재조합 인간 케라틴34와 인모유래 케라틴의 처리 농도에 따른 외모근초세포(ORS)의 분화 결과를 확인한 이미지이다.
도 12a는 일 실시예에 따라 제조된 재조합 인간 케라틴34와 인모유래 케라틴의 처리에 따른 외모근초세포의 β-카테닌의 발현을 확인한 이미지이다(대조군(Control)으로는 미처리군을 사용하였다).
도 12b는 일 실시예에 따라 제조된 재조합 인간 케라틴34와 인모유래 케라틴의 처리에 따른 외모근초세포의 Lgr5의 발현을 확인한 이미지이다(대조군(Control)으로는 미처리군을 사용하였다).
도 13a는 일 실시예에 따라 제조된 재조합 인간 케라틴34와 인모유래 케라틴의 처리에 따른 외모근초세포의 CD 34 발현 억제를 확인한 이미지이다(대조군(Control)으로는 미처리군을 사용하였다).
도 13b는 일 실시예에 따라 제조된 재조합 인간 케라틴34와 인모유래 케라틴의 처리에 따른 외모근초세포의 P-cadherin의 발현을 증가를 확인한 이미지이다(대조군(Control)으로는 미처리군을 사용하였다).
도 14는 일 실시예에 따라 제조된 재조합 인간 케라틴34와 인모유래 케라틴의 처리 농도 및 시간 경과에 따른 외모근초세포의 발모 관련 마커인 β-카테닌, ITGA6, Sox9, FOXN1의 mRNA 발현을 확인한 그래프이다(대조군(Control)으로는 미처리군을 사용하였다).
도 15a는 일 실시예에 따라 제조된 재조합 인간 케라틴34와 인모유래 케라틴의 처리 후 시간 경과(28일)에 따른 동물 실험 결과와 조직에 대한 H&E 염색 결과이다.
도 15b는 재조합 인간 케라틴34와 인모유래 케라틴의 처리 28일 후 확인된 모낭의 수를 인간 모발의 성장 주기별로 나타낸 그래프이다.
도 15c는 재조합 인간 케라틴34와 인모유래 케라틴의 처리 28일 후 확인된 모낭의 성장 주기별 비율을 나타낸 그래프이다.
도 15d는 재조합 인간 케라틴34와 인모유래 케라틴의 처리 28일 후 모낭의 크기별 비율을 나타낸 그래프이다.
도 16은 케라틴34와 머리카락을 구성하는 다른 산성케라틴과의 아미노산 배열의 유사성을 상대비교 분석한 결과이다. 도 16a는 KRT34와 KRT31의 비교 결과이며, 도 16b는 KRT34와 KRT32의 비교 결과이며, 도 16c는 KRT34와 KRT33a의 비교 결과이며, 도 16d는 KRT34와 KRT33b의 비교 결과이며, 도 16e는 KRT34와 KRT35의 비교 결과이며, 도 16f는 KRT34와 KRT36의 비교 결과이며, 도 16g는 KRT34와 KRT37의 비교 결과이며, 도 16h는 KRT34와 KRT38의 비교 결과이다.1 shows a vector map of pBT7-N-His.
2 is a result of confirming the molecular weight of recombinant human keratin 34 (rhKRT34) prepared according to one embodiment.
3 is a schematic diagram showing the structure of recombinant human keratin 34 prepared according to one embodiment.
4 is a result of analyzing the identity of recombinant human keratin 34 prepared according to one embodiment by MALDI-TOF.
5a is a result of confirming the difference in particle size distribution and aggregation degree through dynamic light scattering analysis for human hair-derived keratin.
5B is a result of confirming the difference in particle size distribution and aggregation degree through dynamic light scattering analysis for recombinant Keratin 34.
6 is a conceptual diagram of glycosylated and recombinant human keratin 34 as human hair-derived keratin, and Western blot analysis results confirming that the recombinant human keratin 34 prepared according to one embodiment was not glycosylated.
7A is a surface charge analysis result of recombinant human keratin 34 and human keratin prepared according to one embodiment.
7B is an analysis result of cysteine contents of recombinant human keratin 34 and human keratin prepared according to one embodiment.
Figure 8a is a result confirming the induction of dermal papilla cell aggregation according to the treatment of recombinant human keratin 34 prepared according to one embodiment and human hair-derived keratin (Control is an untreated group, human hair-derived keratin is 0.05%, rhKRT34 is 0.05%, 0.025 %, 0.01% and 0.005% were compared).
FIG. 8B is a graph in which the size of aggregates is calculated by area and compared from the results of FIG. 8A (lines in each bar graph represent standard deviations).
Figure 9a shows the results of confirming the expression of β-catenin, ALPase, and CD133, which are markers related to hair growth of dermal papilla cells, in the untreated group as a control group.
Figure 9b is a result confirming that the recombinant human keratin 34 prepared according to one embodiment increased the expression of β-catenin, ALPase, and CD133, which are markers related to hair growth in dermal papilla cells.
10 is a graph confirming the mRNA expression of human hair-derived β-catenin, ALPase, CD133, and shh according to the treatment concentration and time course of recombinant human keratin 34 and human hair-derived keratin prepared according to one embodiment (control group). used the untreated group).
11 is an image confirming the differentiation results of outer root sheath cells (ORS) according to the treatment concentrations of recombinant human keratin 34 and human hair-derived keratin prepared according to one embodiment.
Figure 12a is an image confirming the expression of β-catenin in outer root sheath cells according to the treatment of recombinant human keratin 34 and human hair-derived keratin prepared according to one embodiment (untreated group was used as a control group).
Figure 12b is an image confirming the expression of Lgr5 in outer root sheath cells according to the treatment of recombinant human keratin 34 and human hair-derived keratin prepared according to one embodiment (untreated group was used as a control group).
Figure 13a is an image confirming the inhibition of CD 34 expression in outer root sheath cells according to the treatment of recombinant human keratin 34 prepared according to one embodiment and human hair-derived keratin (untreated group was used as a control group).
Figure 13b is an image confirming the increase in the expression of P-cadherin in outer root sheath cells according to the treatment of recombinant human keratin 34 and human hair-derived keratin prepared according to one embodiment (untreated group was used as a control group) .
14 is a graph confirming the mRNA expression of β-catenin, ITGA6, Sox9, and FOXN1, which are markers related to hair growth, of outer root sheath cells according to the treatment concentrations and time lapse of recombinant human keratin 34 and human hair-derived keratin prepared according to one embodiment. (The untreated group was used as a control group).
15A shows the results of animal experiments and H&E staining results of tissues according to the time elapsed (28 days) after treatment of recombinant human keratin 34 and human hair-derived keratin prepared according to one embodiment.
15B is a graph showing the number of hair follicles identified 28 days after treatment with recombinant human keratin 34 and human hair-derived keratin for each human hair growth cycle.
FIG. 15C is a graph showing the ratio of hair follicles identified by growth cycle after 28 days of treatment of recombinant human keratin 34 and human hair-derived keratin.
15D is a graph showing the ratio of hair follicle sizes after 28 days of treatment of recombinant human keratin 34 and human hair-derived keratin.
16 is a result of comparative analysis of the similarity of the amino acid sequence between keratin 34 and other acid keratin constituting hair. 16a is a comparison result of KRT34 and KRT31, FIG. 16b is a comparison result of KRT34 and KRT32, FIG. 16c is a comparison result of KRT34 and KRT33a, FIG. 16d is a comparison result of KRT34 and KRT33b, and FIG. 16e is a comparison result of KRT34 and KRT35 16f is a comparison result of KRT34 and KRT36, FIG. 16g is a comparison result of KRT34 and KRT37, and FIG. 16h is a comparison result of KRT34 and KRT38.
이하 하나 이상의 구체예를 실시예를 통해 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.
실시예 1: 재조합 케라틴(KRT) 34 제조 Example 1: Preparation of Recombinant Keratin (KRT) 34
1-1. 재조합 벡터 제작1-1. Construction of recombinant vectors
인간 케라틴 31 (Homo sapiens keratin 31, KRT31), 인간 케라틴 33A (Homo sapiens keratin 33A, KRT33A), 인간 케라틴 33B (Homo sapiens keratin 33B, KRT33B), 인간 케라틴 34 (Homo sapiens keratin 34, KRT34), 인간 케라틴 37 (Homo sapiens keratin 37, KRT37)의 유전자 정보를 미국 국립생물정보센터 (www.ncbi.nlm.nih.gov)에서 얻었다. Human keratin 31 (Homo sapiens keratin 31, KRT31), human keratin 33A (Homo sapiens keratin 33A, KRT33A), human keratin 33B (Homo sapiens keratin 33B, KRT33B), human keratin 34 (Homo sapiens keratin 34, KRT34), human keratin 37 (Homo sapiens keratin 37, KRT37) was obtained from the National Center for Biological Information (www.ncbi.nlm.nih.gov).
KRT34, KRT31, KRT33B, KRT37의 mRNA 서열에서 단백질 코딩 서열(Coding Sequence, CDS)을 발췌하였다. KRT34의 코딩 서열은 서열번호 1이고, KRT31의 코딩 서열은 서열번호 2이고, KRT33B 코딩 서열은 서열번호 5이고, KRT37 코딩 서열은 서열번호 8이다. Protein coding sequences (CDS) were extracted from the mRNA sequences of KRT34, KRT31, KRT33B, and KRT37. The coding sequence of KRT34 is SEQ ID NO: 1, the coding sequence of KRT31 is SEQ ID NO: 2, the coding sequence of KRT33B is SEQ ID NO: 5, and the coding sequence of KRT37 is SEQ ID NO: 8.
바이오니아사(www.bioneer.co.kr)에 의뢰하여 각각의 KRT의 단백질 코딩영역이 포함된 재조합 벡터(pKRT31, pKRT33B, pKRT34, pKRT37)를 제작하였다. (이하 pKRT31, pKRT33B, pKRT34, pKRT37는 pKRT로 지칭할 수 있다) Bioneer (www.bioneer.co.kr) was commissioned to construct recombinant vectors (pKRT31, pKRT33B, pKRT34, pKRT37) containing the protein coding regions of each KRT. (hereinafter, pKRT31, pKRT33B, pKRT34, and pKRT37 may be referred to as pKRT)
도 1에 따르면, 상기 재조합 벡터(pKRT)는 단백질 발현에 적합한 pBT7-N-His 벡터에 KRT 유전자를 도입한 것으로, T7 프로모터를 포함하고 있어 대량의 mRNA를 전사할 수 있으며, lac 오퍼레이터를 통해 Isopropyl-β-D-thiogalactopyranoside (IPTG)로 발현을 유도할 수 있다. 또한 단백질 분리정제 과정인 친화크로마토그래피 (affinity chromatography)를 위한 히스티딘 (histidine) 친화성 리간드 (affinity ligand)의 유전자 서열이 N-terminal 부분에 존재한다. 상기 재조합 벡터를 이용하여 형질전환 미생물 제조 및 배양, 재조합 케라틴 발현 및 정제를 진행하였다. According to FIG. 1, the recombinant vector (pKRT) is a KRT gene introduced into the pBT7-N-His vector suitable for protein expression, and contains a T7 promoter to transcribe a large amount of mRNA, and isopropyl through the lac operator. Expression can be induced with -β-D-thiogalactopyranoside (IPTG). In addition, the gene sequence of histidine affinity ligand for affinity chromatography, a protein separation and purification process, is present in the N-terminal part. Using the recombinant vector, preparation and cultivation of transformed microorganisms, expression and purification of recombinant keratin were performed.
1-2. 재조합 케라틴 발현 형질전환 미생물 제작1-2. Production of recombinant keratin-expressing transgenic microorganisms
E. coli BL21(DE3)를 재조합 케라틴 발현용 대장균으로 사용하였다. 대장균에 CaCl2 버퍼를 처리하여 컴피턴트(competent) 세포를 제작하였다. E. coli competent cell에 상기 재조합 벡터(pKRT31, pKRT33B, pKRT34, pKRT37)를 각각 첨가하고 얼음에서 10분간 방치한 후에 42℃로 90초간 열충격을 가하였다. 앰피실린(ampicillin, Sigma)이 도포된 LB 고체배지에 재조합 벡터가 도입된 E. coli를 도말하고 18시간 배양한 후 BL21/pKRT의 콜로니를 수득하였다. (이하 pKRT31, pKRT33B, pKRT34, pKRT37 각각의 재조합 벡터가 도입된 형질전환 E.coli를 BL21/pKRT로 지칭함)E. coli BL21 (DE3) was used as E. coli for recombinant keratin expression. E. coli was treated with CaCl 2 buffer to prepare competent cells. Each of the recombinant vectors (pKRT31, pKRT33B, pKRT34, and pKRT37) was added to E. coli competent cells, and after standing on ice for 10 minutes, heat shock was applied at 42° C. for 90 seconds. E. coli introduced with the recombinant vector was plated on LB solid medium coated with ampicillin (Sigma) and cultured for 18 hours to obtain BL21/pKRT colonies. (hereinafter referred to as BL21/pKRT)
1-3. 형질전환 미생물 배양 및 발현 1-3. Transgenic microorganism culture and expression
LB 액체배지로 BL21/pKRT를 배양하였다. 구체적으로, 50ml 튜브에 10ml의 LB 액체배지(500㎍/ml ampicillin 포함)를 넣고 BL21/pKRT 콜로니 2 ~ 3개를 접종하여 18시간 배양하였다. 미생물 배양액을 2,000ml의 LB 배지가 들어있는 5,000ml 플라스크에 접종하였다. 배양액의 흡광도 (OD600)값이 0.5~0.8이 되었을 때, 유도물질인 IPTG를 0.5mM이 되도록 첨가하여 재조합 단백질인 rhKRT(recombinant human Keratin)의 발현을 유도하였다. 추가로 16시간 동안 25℃에서 교반하며 배양하고, 배양액을 3,000rpm에서 20분간 원심분리하여 상등액을 제거하고 세포를 회수하였다. 회수된 세포를 -80℃에서 보관하고 실험에 사용하였다.BL21/pKRT was cultured in LB broth. Specifically, 10 ml of LB liquid medium (including 500 μg/ml ampicillin) was placed in a 50 ml tube, and 2 to 3 BL21/pKRT colonies were inoculated and cultured for 18 hours. The microbial culture was inoculated into a 5,000 ml flask containing 2,000 ml of LB medium. When the absorbance (OD600) value of the culture medium reached 0.5 to 0.8, IPTG, an inducer, was added to a concentration of 0.5 mM to induce the expression of rhKRT (recombinant human Keratin), a recombinant protein. The culture was further cultured at 25° C. for 16 hours with agitation, and the culture medium was centrifuged at 3,000 rpm for 20 minutes to remove the supernatant and recover the cells. The recovered cells were stored at -80 °C and used in experiments.
1-4. 봉입체(Inclusion body) 분리 및 용해1-4. Inclusion body separation and dissolution
rhKRT는 불용성 단백질로서 E. coli 내부에서 응집되어 봉입체(Inclusion body)를 형성한다. E. coli에서 rhKRT가 응집된 봉입체를 분리하였다. 구체적으로, -80℃에서 보관된 대장균 세포에 파쇄용액 (20mM Tris-HCl, pH8.0)을 넣고 상온에서 재현탁시키고, 초음파 분쇄기를 이용하여 5초 ~ 15초 pulse on-off로 90초간 파쇄하였다. 파쇄된 대장균 시료를 2,000xg에서 20분간 원심분리하여 상등액을 제거하였다. As an insoluble protein, rhKRT aggregates inside E. coli to form an inclusion body. RhKRT-aggregated inclusion bodies were isolated from E. coli. Specifically, disruption solution (20mM Tris-HCl, pH 8.0) was added to E. coli cells stored at -80 ° C, resuspended at room temperature, and disrupted for 90 seconds with a pulse on-off of 5 to 15 seconds using a sonicator. did The disrupted E. coli sample was centrifuged at 2,000xg for 20 minutes to remove the supernatant.
봉입체 세척을 위하여 펠렛에 봉입체 세척용액 (2M urea, 20mM Tris-HCl, 0.5M NaCl, 2% Triton X-100, pH 8.0)을 넣고 교반한 후 초음파 파쇄기를 이용하여 90초간 파쇄하였다. 시료를 2,000xg에서 10분간 원심분리하여 상등액을 제거하였다. 상기 세척버퍼를 이용한 봉입체 세척 과정을 4회 반복하였다. 세척이 완료된 봉입체는 -20℃에서 보관하여 필요시에 사용하였다. To clean the inclusion body, an inclusion body cleaning solution (2M urea, 20mM Tris-HCl, 0.5M NaCl, 2% Triton X-100, pH 8.0) was added to the pellet, stirred, and then disrupted using an ultrasonic disruptor for 90 seconds. The supernatant was removed by centrifuging the sample at 2,000xg for 10 minutes. The process of washing the inclusion body using the washing buffer was repeated 4 times. The washed inclusion body was stored at -20 ° C and used when necessary.
봉입체에 봉입체 용해용액(5M Urea, 2.5M Thiourea, 25mM Tris-HCl, pH 8.0)을 넣고 95℃에서 10분간 가열한다. 시료를 12,000xg에서 20분간 원심분리하고 상등액을 0.22㎛ 필터로 여과하여 봉입체 용해액을 수득한다. 봉입체 용해액은 rhKRT의 정제에 사용하였다. An inclusion body dissolution solution (5M Urea, 2.5M Thiourea, 25mM Tris-HCl, pH 8.0) was added to the inclusion body and heated at 95°C for 10 minutes. The sample is centrifuged at 12,000xg for 20 minutes and the supernatant is filtered through a 0.22 μm filter to obtain an inclusion body lysate. The inclusion body lysate was used for the purification of rhKRT.
1-5. rhKRT의 정제 및 투석 1-5. Purification and dialysis of rhKRT
친화크로마토그래피법(affinity chromatography)으로 rhKRT를 정제하였다. 크로마토그래프관에 HisPur™ Cobalt Superflow Agarose (Thermo Scientific) 2ml를 넣고 평형용액 (20mM sodium phosphate, 300mM NaCl, 8M Urea, 5mM imidazole, pH 8.0) 4ml를 흘려보내며 수지를 평형화(equilibration)하였다. rhKRT를 포함하는 봉입체 용해액을 레진이 충전된 크로마토그래프관에 적용하여 단백질과 결합하도록 하고, 4ml의 세척용액 (20mM sodium phosphate, 300mM NaCl, 8M Urea, 15mM imidazole, pH 8.0)을 이용하여 수지를 세 번 세척하였다. 용출용액 (20mM sodium phosphate, 300mM NaCl, 8M Urea, 150mM imidazole, pH 8.0) 4ml을 세 번 흘려보냄으로써 rhKRT를 포함하는 용출버퍼를 수득하였다. rhKRT was purified by affinity chromatography. 2 ml of HisPur™ Cobalt Superflow Agarose (Thermo Scientific) was put in a chromatograph tube, and the resin was equilibrated by flowing 4 ml of an equilibration solution (20 mM sodium phosphate, 300 mM NaCl, 8 M Urea, 5 mM imidazole, pH 8.0). The inclusion body solution containing rhKRT was applied to a chromatograph tube filled with resin to bind to the protein, and the resin was washed using 4 ml of a washing solution (20 mM sodium phosphate, 300 mM NaCl, 8 M Urea, 15 mM imidazole, pH 8.0). washed three times. An elution buffer containing rhKRT was obtained by flowing 4 ml of an elution solution (20 mM sodium phosphate, 300 mM NaCl, 8 M Urea, 150 mM imidazole, pH 8.0) three times.
rhKRT를 포함하는 용출버퍼 부피의 3배만큼의 증류수를 천천히 추가하며 희석하였다. 이후, 50kDa MWCO의 투석용 막(Spectra/Por®;Spectrum Lab. Inc. USA)에 희석된 rhKRT 포함 수용액(50ml)을 넣고, Spectra/Por RC 투석 튜빙 클로져 (최대 폭 55mm, Orange, Spectrum)를 양쪽 튜브에 패킹하였다. 5L 플라스틱 비커에 증류수를 채워 실온에서 투석을 실시하고, 4시간마다 새로운 5L의 증류수로 교체하여 불순물을 제거하였다. Distilled water three times the volume of the elution buffer containing rhKRT was slowly added and diluted. Then, the diluted rhKRT-containing aqueous solution (50ml) was added to the 50kDa MWCO dialysis membrane (Spectra/Por ® ; Spectrum Lab. Inc. USA), and the Spectra/Por RC dialysis tubing closure (maximum width 55mm, Orange, Spectrum) was used. Packed in both tubes. Distilled water was filled in a 5L plastic beaker and dialysis was performed at room temperature, and impurities were removed by replacing with new 5L distilled water every 4 hours.
1-6. rhKRT 농축1-6. rhKRT enrichment
투석한 rhKRT 수용액을 0.22㎛ 크기의 여과막(Millipore Steriflip™ Sterile Disposable Vacuum Filter)으로 여과하고, 30kDa의 분자량 컷 오프 (cut off) 필터 튜브(Amicon® Ultra-15 Centrifugal Filter Unit, 30kDa MWCO)를 이용하여 3,500rpm, 25℃ 조건으로 원심분리하여 1/50로 농축하였다. 이후, 초저온 냉장고에서 -80℃로 24시간 동안 완전히 동결시켰다. 동결된 rhKRT를 동결건조기(IlShin Lab Freeze Dryer)에 넣고 -85℃의 진공상태에서 완전히 파우더 형태가 되도록 3일 동안 건조하였다. 마지막으로 SDS-PAGE를 실시하여, 분자량을 확인하였다.The dialyzed rhKRT aqueous solution was filtered through a 0.22 μm filtration membrane (Millipore Steriflip™ Sterile Disposable Vacuum Filter), and a 30 kDa molecular weight cut off filter tube (Amicon® Ultra-15 Centrifugal Filter Unit, 30 kDa MWCO) was used to It was centrifuged at 3,500 rpm and 25°C and concentrated to 1/50. Then, it was completely frozen for 24 hours at -80 ° C in an ultra-low temperature refrigerator. The frozen rhKRT was placed in a freeze dryer (IlShin Lab Freeze Dryer) and dried for 3 days in a vacuum at -85°C to completely form a powder. Finally, SDS-PAGE was performed to confirm the molecular weight.
도 2에 따르면 rhKRT34의 분자량은 약 55kDa이었다. 이는 발현시키고자 하는 단백질의 예상 아미노산 서열과 동일한 분자량이었다. 또한 분자량이 약 100kDa인 단백질이 검출되었으며, MALDI-TOF 질량분석법을 이용하여 rhKRT34의 이합체 (Dimer)임을 확인하였다. 이는 하기 설프하이드릴기 차단 과정에서 단량체 (Monomer)로 분리된다. 이미지 분석 프로그램 (ImageJ, https://imagej.nih.gov/ij/)을 이용하여 밴드의 밀도를 측정하여 순도를 확인하였으며, 95% 이상의 순도를 나타냈다. According to Figure 2, the molecular weight of rhKRT34 was about 55 kDa. This was the same molecular weight as the expected amino acid sequence of the protein to be expressed. In addition, a protein having a molecular weight of about 100 kDa was detected, and it was confirmed to be a dimer of rhKRT34 using MALDI-TOF mass spectrometry. This is separated into monomers in the process of blocking the sulfhydryl group below. The purity was confirmed by measuring the density of the band using an image analysis program (ImageJ, https://imagej.nih.gov/ij/), and the purity was 95% or higher.
1-7. 설프하이드릴기 차단 rhKRT 제조1-7. Preparation of rhKRT with sulfhydryl group blocking
동결건조된 rhKRT(100 mg)를 100 mM DTT를 포함하는 8M 우레아(100 ml)에 용해시켜 0.1%(w/v) 농도의 rhKRT 수용액을 제조하고, 50℃에서 18시간 동안 방치하여 이황화결합 제거 반응을 하였다. 이후 L-시스테인염산염 50 mg을 가하여 상온에서 2시간 동안 교반하며 결합 반응을 하였다. 반응이 완료된 수용액(100ml)을 상기 1-5 및 1-6과 동일한 방법으로 투석하고 동결건조하여 분말화하였다. Lyophilized rhKRT (100 mg) was dissolved in 8M urea (100 ml) containing 100 mM DTT to prepare a 0.1% (w/v) aqueous solution of rhKRT, and left at 50°C for 18 hours to remove disulfide bonds. reacted. Thereafter, 50 mg of L-cysteine hydrochloride was added and the mixture was stirred at room temperature for 2 hours to perform a bonding reaction. The reaction-completed aqueous solution (100ml) was dialyzed in the same manner as 1-5 and 1-6, and lyophilized to powder.
실시예 2: rhKRT34 특성 분석(characterization)Example 2: rhKRT34 Characterization
상기 실시예 1에서 제조한 rhKRT34의 물성을 확인하였다. The physical properties of rhKRT34 prepared in Example 1 were confirmed.
2-1. rhKRT34의 아미노산 서열, 구조, 입자크기 분석2-1. Amino acid sequence, structure and particle size analysis of rhKRT34
rhKRT34의 아미노산 서열은 서열번호 10의 서열이고, rhKRT34의 분자량은 약 55kDa이다. The amino acid sequence of rhKRT34 is the sequence of SEQ ID NO: 10, and the molecular weight of rhKRT34 is about 55 kDa.
도 3에 따르면, rhKRT34의 폴딩구조는 크게 Head, Coil, linker, Tail로 나눌 수 있으며, 각 구조에 해당되는 아미노산 잔기는 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 Tail이다. According to FIG. 3, the folding structure of rhKRT34 can be largely divided into Head, Coil, linker, and Tail, and amino acid residues corresponding to each structure are 1-56 Head; 57 - 91 Coil 1A; 92 - 102 Linker 1; 103 - 203 Coil 1B; 204 - 219 Linker 2; 220 - 363 Coil 2; 364 - 394 Tail.
MALDI-TOF/TOF는 프로테인웍스사(http://www.proteinworks.co.kr/)에서 수행하였으며, 펩타이드의 질량은 MALDI-TOF/TOF 질량 분석기(Model: 4700 MALDI-TOF/TOF, Applied Biosystems)를 사용하여 측정하였다. 펩타이드 질량은 Mascot 검색 프로그램을 사용하여 NCBI 데이터베이스의 모든 단백질의 이론적인 펩타이드를 기록하였으며, 그 결과를 도 4에 나타내었다.MALDI-TOF/TOF was performed by Protein Works (http://www.proteinworks.co.kr/), and the mass of the peptide was measured using a MALDI-TOF/TOF mass spectrometer (Model: 4700 MALDI-TOF/TOF, Applied Biosystems ) was measured using. The theoretical peptides of all proteins in the NCBI database were recorded for the peptide mass using the Mascot search program, and the results are shown in FIG. 4 .
인모유래 케라틴과 재조합 케라틴34(rhKRT34)를 둘베코 인산완충 식염수 (DPBS)에 0.1%(w/v) 농도로 용해시키고 37℃에서 인큐베이션을 한 후, Dynamic Light Scattering(DLS) Analysis 방법으로 인모케라틴과 rhKRT34의 입자크기를 상대분석하여 도 5a 및 도 5b에 나타내었다. 도 5에 따르면, 재조합케라틴 (rhKRT34)는 monomer 입자와 dimer 입자가 혼재되어 있으며, 평균 입자 크기는 100nm이하인 것으로 확인되었다. 37℃에서 인큐베이션 시에도 100nm를 초과하는 크기의 응집체는 관찰되지 않았다. 이에 비해 인모유래 케라틴은 37℃에서 인큐베이션시 케라틴 분자끼리의 agglomeration에 의해 크기가 1000nm 이상인 응집체를 형성하는 것으로 나타나, 재조합 케라틴34(rhKRT34)이 인모유래 케라틴 대비 수용액 중에서 상대적으로 안정적으로 존재함이 확인되었다. 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, followed by Dynamic Light Scattering (DLS) Analysis. Relative analysis of the particle size of rhKRT34 and rhKRT34 is shown in FIGS. 5a and 5b. According to FIG. 5, it was confirmed that the recombinant keratin (rhKRT34) had a mixture of monomer particles and dimer particles, and had an average particle size of 100 nm or less. Aggregates larger than 100 nm were not observed even when incubated at 37°C. In contrast, human hair-derived keratin was found to form aggregates larger than 1000 nm in size by agglomeration of keratin molecules when incubated at 37°C, confirming that recombinant keratin 34 (rhKRT34) exists relatively stably in an aqueous solution compared to human hair-derived keratin. It became.
2-2. rhKRT34의 탈당화 확인 2-2. Confirmation of deglycosylation of rhKRT34
인모유래 케라틴과 rhKRT34에 대해 anti-O-GLCNAc antibody(Cell signaling Technology, Cat#9875)를 이용한 웨스턴 블롯 분석을 실시하여 글리코실화 차이를 분석하였다. Differences in glycosylation of human hair-derived keratin and rhKRT34 were analyzed by Western blot analysis using an anti-O-GLCNAc antibody (Cell signaling Technology, Cat# 9875).
도 6에 따르면, 인모유래 케라틴은 글리코실기에 해당되는 positive band가 관찰되었으나, rhKRT34에서는 글리코실기에 대응하는 positive band가 관찰되지 않았다. 따라서 rhKRT34는 탈당화되어 있음을 확인하였다. According to FIG. 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. rhKRT34의 화학적 특성 분석 2-3. Chemical characterization of rhKRT34
케라틴의 표면 전하를 측정하기 위해 rhKRT34와 인모 케라틴의 농도가 각각 0.1(w/v)%가 되도록 정제수에 완전히 녹여 케라틴 용액을 준비하였다. pH 측정장치를 사용하여 상기 케라틴 용액에 수산화나트륨 용액을 첨가하면서 용액의 pH가 11이 되도록 하였다. Dynamic Light Scattering 장비의 zeta potential 측정 기능을 이용하여 단백질 표면 전하를 측정하였다. 이어서 상기 용액에 염산을 첨가하여 pH를 0.5 내지 1.5 씩 낮추어가며 상기 장비로 표면 전하를 pH 3 부근까지 측정하여 도 7a에 나타내었다. To measure the surface charge of keratin, a keratin solution was prepared by completely dissolving rhKRT34 and human hair keratin in purified water to a concentration of 0.1 (w/v)%, respectively. While adding sodium hydroxide solution to the keratin solution using a pH measuring device, the pH of the solution was adjusted to 11. The protein surface charge was measured using the zeta potential measurement function of Dynamic Light Scattering equipment. Subsequently, hydrochloric acid was added to the solution to lower the pH by 0.5 to 1.5, and the surface charge was measured up to around pH 3 with the equipment, as shown in FIG. 7a.
케라틴을 구성하는 주 아미노산인 시스테인(Cysteine)의 설프하이드릴기(free sulfhydryl group)에 대한 분석을 Invitrogen사의 Thiol Assay Kit(M30550)로 수행하였다. rhKRT34 및 인모 케라틴 샘플을 각각 0.2(w/v)% 농도가 되도록 정제수에 녹인 용액으로 분석을 진행하였다. 이후 과정은 상기 키트 제조사의 지침에 따라 수행하였으며, Fluorescence microplate reader에서 ex 494 nm / em 517 nm으로 측정하여 결과를 도 7b에 나타내었다.Analysis of the free sulfhydryl group of cysteine, a major amino acid constituting keratin, was performed using Invitrogen's Thiol Assay Kit (M30550). Analysis was performed using a solution in which rhKRT34 and human hair keratin samples were dissolved in purified water to a concentration of 0.2 (w/v)%, respectively. The subsequent process was performed according to the kit manufacturer's instructions, and the results were measured using a fluorescence microplate reader at ex 494 nm / em 517 nm, and the results are shown in FIG. 7B.
도 7a에 따르면, 인모 케라틴의 경우 표면 전하가 음성을 나타내고 있으며, pH 범위에 걸쳐 음성을 유지하여 PI값을 나타내지 않음을 확인하였으며, rhKRT34의 경우는 pH 4 부근에서 표면 전하값이 0이 되는 PI값을 나타내고 있음을 확인하여, 인모 케라틴과 rhKRT34는 화학적으로 상이함을 확인하였다. 또한, 인모 케라틴의 경우 설프하이드릴기(free sulfhydryl group)이 거의 남아 있지 않았으며, rhKRT34의 경우 설프하이드릴기(free sulfhydryl group)이 많이 남아 있음을 확인하였다.According to FIG. 7a, in the case of human hair keratin, the surface charge was negative, and it was confirmed that the PI value remained negative throughout the pH range, and in the case of rhKRT34, the PI value became 0 at around pH 4. By confirming that the values were shown, it was confirmed that human hair keratin and rhKRT34 were chemically different. In addition, it was confirmed that almost no free sulfhydryl groups remained in the case of human hair keratin, and many free sulfhydryl groups remained in the case of rhKRT34.
실시예 3: rhKRT34의 발모 효과 분석Example 3: Analysis of hair growth effect of rhKRT34
3-1. 모유두세포의 세포응집체 형성 분석3-1. Analysis of cell aggregate formation of dermal papilla cells
실시예 1의 rhKRT34를 모유두세포(Dermal papilla cell; DP cell)에 처리하고 세포응집체(cell aggregate) 형성을 확인하였다. 세포응집체가 증가하면 모발성장 및 모발재생을 유도하는 효과가 있는 것으로 평가할 수 있다. Dermal papilla cells (DP cells) were treated with rhKRT34 of Example 1, and cell aggregate formation was confirmed. An increase in cell aggregates can be evaluated as having an effect of inducing hair growth and hair regeneration.
인간 모유두세포를 24웰 플레이트에 5×104/well로 접종하고, 24시간 후에 배지를 실시예 1의 rhKRT34가 0.05%(w/v), 0.025%(w/v), 0.01%(w/v) 또는 0.005%(w/v)로 포함된 high glucose DMEM 배지로 교체하고 2일간 배양하였다. 대조군(Control)으로 아무것도 처리하지 않은 high glucose DMEM 배지, 그리고 비교군으로 인모유래 케라틴이 0.05%(w/v)로 포함된 high glucose DMEM 배지에서 배양된 모유두세포를 준비하였다. 2일간 배양 후, 각 웰에 형성된 모유두세포 유래 구형 응집체의 개수를 광학현미경으로 확인한 이미지를 도 8a에 나타냈으며, 이를 counting하였다. 상기 이미지로부터 구형 응집물의 크기를 측정하여 도 8b에 나타내었다.Human dermal papilla cells were inoculated in a 24-well plate at 5×10 4 /well, and after 24 hours, the rhKRT34 of Example 1 was added to the medium 0.05% (w/v), 0.025% (w/v), 0.01% (w/v). v) or high glucose DMEM medium containing 0.005% (w/v) and cultured for 2 days. Dermal papilla cells cultured in a high glucose DMEM medium without any treatment as a control group and in a high glucose DMEM medium containing 0.05% (w/v) of human hair-derived keratin as a control group were prepared. After culturing for 2 days, an image confirming the number of dermal papilla cell-derived spherical aggregates formed in each well with an optical microscope is shown in FIG. 8a, and it was counted. The size of the spherical aggregates was measured from the images and shown in FIG. 8B.
도 8에 따르면, 재조합 케라틴34는 0.05%(w/v)의 낮은 농도에서도 모유두세포의 응집을 유도하였으며 같은 농도의 인모 케라틴에 비해 응집유도능이 뛰어났다. 또한, 재조합 케라틴34는 0.005%(w/v)의 농도에서 0.05(w/v) 농도의 인모 케라틴과 유사한 응집유도능을 나타내었는 바, 재조합 케라틴34는 그 유효 농도가 인모유래 케라틴보다 10배 낮은 것으로 확인되었다. According to FIG. 8, recombinant keratin 34 induced aggregation of dermal papilla cells even at a low concentration of 0.05% (w/v), and was superior in aggregation inducing ability compared to human hair keratin at the same concentration. In addition, the recombinant keratin 34 exhibited similar aggregation inducing ability at a concentration of 0.005% (w/v) to that of human hair keratin at a concentration of 0.05 (w/v). found to be low.
3-2. 모유두세포의 발모 관련 마커 발현 분석3-2. Expression analysis of markers related to hair growth in dermal papilla cells
면역화학염색을 통해 배양된 모유두세포의 발모 관련 마커인 베타카테닌, CD133, 및 Alkaline phosphatase(ALPase)의 발현을 확인하였다. The expression of beta-catenin, CD133, and alkaline phosphatase (ALPase), which are markers related to hair growth in cultured dermal papilla cells, was confirmed through immunochemical staining.
실시예 1의 rhKRT34가 0.05%(w/v) 포함된 high glucose DMEM 일반 배지에서 모유두세포를 2일간 배양하였다. 배지를 제거하고 DPBS로 세척하였다. 4% 파라포름알데하이드를 10분간 처리하여 고정시킨 후 다시 DPBS로 세척하였다. 고정 후 0.1% triton X-100를 30분간 처리하여 투과성을 부여하고(permeabilization), 10% (w/v)의 정상염소혈청(normal goat serum, NGS)을 1시간 동안 처리하였다. 그 후, 1:200으로 희석된 토끼 항-인간 베타-카테닌 항체(rabbit anti-human beta-catenin antibody), 토끼 항-인간 CD133항체(rabbit anti-human CD34 antibody), 또는 쥐 항-인간 ALPase 항체(mouse anti-human ALPase antibody)를 처리하고 24시간동안 4℃에서 반응시켰다. The dermal papilla cells were cultured for 2 days in a regular high glucose DMEM medium containing 0.05% (w/v) of rhKRT34 of Example 1. Media was removed and washed with DPBS. After treatment with 4% paraformaldehyde for 10 minutes to fix it, it was washed again with DPBS. After fixation, 0.1% Triton X-100 was treated for 30 minutes to provide permeabilization, and 10% (w/v) normal goat serum (NGS) was treated for 1 hour. Then, rabbit anti-human beta-catenin antibody, rabbit anti-human CD133 antibody, or rat anti-human ALPase antibody at a dilution of 1:200. (mouse anti-human ALPase antibody) and reacted at 4°C for 24 hours.
24시간 반응 후, DPBS로 세 번 세척하고, Counter staining을 위해 PE-conjugated Palloidin antibody를 상온에서 1시간동안 처리하였고 2차항체로는 Alexa Fluor 488 conjugated goat anti-rabbit IgG 또는 Alexa Fluor 594 conjugated goat anti-mouse IgG를 상온에서 1시간동안 처리하였다. 그 후, DPBS로 세 번 세척하였고, 4',6-diamidino-2-phenylindole(DAPI)를 처리하였다. 그 후, 염색된 세포들을 형광현미경(OlympusI ×71)으로 관찰하여 그 이미지를 도 9b에 나타내었다.After 24 hours of reaction, washed three times with DPBS, treated with PE-conjugated Palloidin antibody for 1 hour at room temperature for counter staining, and as a secondary antibody, Alexa Fluor 488 conjugated goat anti-rabbit IgG or Alexa Fluor 594 conjugated goat anti -Mouse IgG was treated at room temperature for 1 hour. Then, it was washed three times with DPBS and treated with 4',6-diamidino-2-phenylindole (DAPI). Thereafter, the stained cells were observed under a fluorescence microscope (OlympusI × 71), and the images are shown in FIG. 9B.
대조군으로는 상기 high glucose DMEM 일반 배지를 사용한 미처리군을 사용하였으며, 상기와 같은 방법으로 준비 및 형광현미경 관찰하여 그 이미지를 도 9a에 나타내었다.As a control group, an untreated group using the high glucose DMEM general medium was used, prepared in the same way and observed under a fluorescence microscope, and the image is shown in FIG. 9a.
도 9에 따르면, rhKRT34 처리군은 미처리군보다 모유두세포의 발모 관련 마커인 베타카테닌, CD133 및 Alkaline phosphatase의 발현이 증가하였다. According to FIG. 9, the expression of beta-catenin, CD133, and alkaline phosphatase, which are markers related to hair growth of dermal papilla cells, increased in the rhKRT34-treated group compared to the untreated group.
추가적으로, 인모유래 케라틴과 rhKRT34의 농도별 처리에 따른 모유두세포의 발모 관련 마커로서 베타카테닌, Alkaline phosphatase, CD133 및 shh의 발현을 RT-Qpcr 분석으로 확인하였다. RT-qPCR 분석은 Corbett사로부터의 RG-6000 RT-qPCR 기계를 이용하여 삼중 샘플에서 수행하였다. RT-qPCR을 1 μL의 cDNA 주형, 1 μL 센스 및 안티센스 프라이머, Bioneer사로부터의 10 μL qPCR Master Mix, 그리고 20 μL의 최종 부피의 DEPC-water를 이용하여 수행하였다. 상기 목적에 사용된 프라이머는 GAPDH (정방향 5'-ACTTTGGTATCGTGGAAGGAC-3' 및 역방향 5'-GCAGGGATGATGTTCTGGAG-3'), CD133 (정방향 5'-GTGAAACCTGCAACAGCATC-3' 및 역방향 5'-TGTCAAGTTCTGCATCCACG-3'), β-Catenin (정방향 5'-TGTAGAAGCTGGTGGAATGC-3' 및 역방향 5'-GCTGAACAAGAGTCCCAAGG-3'), ALPase (정방향 5'-CTCGTTGACACCTGGAAGAG-3' 및 역방향 5'-GGCTCGAAGAGACCCAATAG-3'), 및 Shh (정방향 5'-GATGTCTGCTGCTAGTCCTCG-3' 및 역방향 5'- CACCTCTGAGTCATCAGCCTG-3')이었다. 샘플을 하기 프로토콜로 처리하였다: 95℃에서 10분 동안의 가열, 및 이후 10초 동안 95℃ 20초 동안 60 ~ 63℃의 45 사이클 후, 및 융해 곡선을 위한 가열 1회 사이클, 후 12℃에서의 냉각. 데이터를 GAPDH 수준에 대해 표준화시키고, 도 10에 나타내었다.Additionally, the expression of beta-catenin, alkaline phosphatase, CD133, and shh as markers related to hair growth in dermal papilla cells according to concentrations of human hair-derived keratin and rhKRT34 was confirmed by RT-Qpcr analysis. RT-qPCR analysis was performed on triplicate samples using an RG-6000 RT-qPCR machine from Corbett. RT-qPCR was performed using 1 μL of cDNA template, 1 μL sense and antisense primers, 10 μL qPCR Master Mix from Bioneer, and DEPC-water in a final volume of 20 μL. Primers used for this purpose were GAPDH (forward 5'-ACTTTGGTATCGTGGAAGGAC-3' and reverse 5'-GCAGGGATGATGTTCTGGAG-3'), CD133 (forward 5'-GTGAAACCTGCAACAGCATC-3' and reverse 5'-TGTCAAGTTCTGCATCCACG-3'), β -Catenin (forward 5'-TGTAGAAGCTGGTGGAATGC-3' and reverse 5'-GCTGAACAAGAGTCCCAAGG-3'), ALPase (forward 5'-CTCGTTGACACCTGGAAGAG-3' and reverse 5'-GGCTCGAAGAGACCCAATAG-3'), and Shh (forward 5'- GATGTCTGCTGCTAGTCCTCG-3' and reverse 5'-CACCTCTGAGTCATCAGCCTG-3'). Samples were subjected to 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 a melting curve, followed by 12°C. of cooling. Data were normalized to GAPDH levels and are shown in FIG. 10 .
도 10에 따르면, rhKRT34 처리군은 미처리군보다 모유두세포의 발모 관련 마커인 베타카테닌, CD133, Alkaline phosphatase 및 shh의 mRNA 발현이 증가하였다. According to FIG. 10, in the rhKRT34-treated group, the mRNA expression of beta-catenin, CD133, alkaline phosphatase, and shh, which are markers related to hair growth in dermal papilla cells, increased compared to the untreated group.
3-3. 외모근초세포의 군집형성능 및 분화유도능 평가3-3. Evaluation of cluster formation ability and differentiation inducing ability of outer root sheath cells
외모근초세포(outer root sheath cell, ORS Cell)는 2차모발배아세포(Secondary hair germ cell, SHG)로 분화하는 세포로서 모근형성에 중요한 역할을 담당한다. 외모근초세포의 군집유도능 및 분화유도능이 모근 형성과 관련이 높다고 알려져 있다. 특히 CD34 마커를 발현하는 외모근초세포는 2차모발배아세포의 형성에 직접적으로 관여하는 CD34-/Lgr5+ 세포군으로 분화되는 것으로 알려져 있다.The outer root sheath cell (ORS Cell) is a cell that differentiates into a secondary hair germ cell (SHG) and plays an important role in hair root formation. It is known that the ability to induce clustering and differentiation of outer root sheath cells is highly related to hair root formation. In particular, it is known that hair follicle cells expressing the CD34 marker are differentiated into a CD34 - /Lgr5 + cell group directly involved in the formation of secondary hair embryonic cells.
실시예 1의 rhKRT34를 0.05%(w/v), 0.025%(w/v) 또는 0.01%(w/v)의 농도로 포함하는 high glucose DMEM 배지에서 외모근초세포를 2일간 배양하고 군집형성 및 분화유도를 관찰하였다. 대조군(Control)으로는 미처리군을 사용하였으며, 인모유래 케라틴을 0.1%(w/v) 또는 0.01%(w/v)의 농도로 포함하는 high glucose DMEM 배지에서 외모근초세포를 2일간 배양하고 군집형성 및 분화유도를 광학 현미경(OLYMPUS, IX71, ×100)으로 관찰하여 그 결과를 도 11에 나타내었다.In high glucose DMEM medium containing the rhKRT34 of Example 1 at a concentration of 0.05% (w/v), 0.025% (w/v) or 0.01% (w/v), the outer root sheath cells were cultured for 2 days, and colony formation and Differentiation induction was observed. An untreated group was used as a control group, and the outer root sheath cells were cultured for 2 days in a high glucose DMEM medium containing human hair-derived keratin at a concentration of 0.1% (w/v) or 0.01% (w/v), and then colonized. Formation and induction of differentiation were observed under an optical microscope (OLYMPUS, IX71, ×100), and the results are shown in FIG. 11 .
도 11에 따르면, 인모유래 케라틴 처리군은 0.1%(w/v) 이상의 높은 농도에서 외모근초세포의 군집유도 및 연장된(elongated) 형태로의 분화능을 나타냈으나, 이보다 낮은 0.01%(w/v)의 농도에서는 이러한 효과를 나타내지 않았다. 그러나, 실시예 1의 rhKRT34 처리군은 0.01%(w/v)의 낮은 농도에서도 외모근초세포의 군집유도 및 연장된(elongated) 형태로의 분화능을 나타내었다. According to FIG. 11, the human hair-derived keratin-treated group showed the ability to induce colonization of outer root sheath cells and differentiate into an elongated form at a high concentration of 0.1% (w/v) or more, but at a lower concentration of 0.01% (w/v). The concentration of v) did not show this effect. However, the rhKRT34-treated group of Example 1 showed the ability to induce colonization of outer root sheath cells and differentiate into an elongated form even at a low concentration of 0.01% (w/v).
추가적으로, 상기 rhKRT34 처리군과 인모유래 케라틴 처리군으로부터 β-카테닌, Lgr5, CD34 및 P-cadherin의 발현 변화를 확인하였다. 도 12a는 β-카테닌의 발현 변화를 확인한 이미지이며, 도 12b는 Lgr5의 발현 변화를 확인한 이미지이며, 도 13a는 CD34의 발현 변화를 확인한 이미지이며, 도 13b는 P-cadherin의 발현 변화를 확인한 이미지이다.Additionally, changes in the expression of β-catenin, Lgr5, CD34, and P-cadherin were confirmed in the rhKRT34-treated group and the human hair-derived keratin-treated group. Figure 12a is an image confirming the expression change of β-catenin, Figure 12b is an image confirming the expression change of Lgr5, Figure 13a is an image confirming the expression change of CD34, Figure 13b is an image confirming the expression change of P-cadherin am.
도 12에 의하면, 인모유래 케라틴은 0.1%(w/v) 이상의 높은 농도를 처리한 외모근초세포에서 β-카테닌 및 Lgr5의 발현이 증가하였으나, 0.01%(w/v)의 농도에서는 이러한 효과를 나타내지 않았다. 그러나, 실시예 1의 rhKRT34은 0.05%(w/v), 0.025%(w/v) 및 0.01%(w/v)의 낮은 농도에서도 β-카테닌 및 Lgr5의 발현이 증가하였다.According to FIG. 12, human hair-derived keratin increased the expression of β-catenin and Lgr5 in outer root sheath cells treated with a high concentration of 0.1% (w/v) or more, but at a concentration of 0.01% (w/v), these effects were suppressed. did not show 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).
도 13에 의하면, 인모유래 케라틴은 0.1%(w/v) 처리시 CD34의 발현이 나타나지 않았고, 0.01%(w/v) 처리시에는 CD34 발현이 나타났으나, 실시예 1의 rhKRT34는 0.01%(w/v)의 낮은 농도에서도 CD34의 발현이 억제되었다. 또한, rhKRT34는 hair germ형성시 발현되는 마커인 P-cadherin 발현을 증가시킴을 확인하였다.According to FIG. 13, human hair-derived keratin did not show CD34 expression when treated with 0.1% (w/v), and CD34 expression appeared when treated with 0.01% (w/v), but rhKRT34 of Example 1 showed 0.01% CD34 expression was suppressed even at a low concentration of (w/v). In addition, it was confirmed that rhKRT34 increased the expression of P-cadherin, a marker expressed during hair germ formation.
상기 결과에 따르면, rhKRT34는 인모케라틴보다 낮은 농도에서도 외모근초세포의 군집유도, 연장된(elongated) 형태로의 분화가 일어나며 CD34-/Lgr5+/P-cadherin+ 세포로의 분화가 유도될 수 있음을 확인하였다. According to the above results, even at a lower concentration than human keratin, rhKRT34 induces colonization of outer root sheath cells and differentiation into an elongated form, and differentiation into CD34 - /Lgr5 + /P-cadherin + cells can be induced. confirmed.
인모유래 케라틴과 rhKRT34의 농도별 처리에 따른 외모근초세포의 발모 관련 마커로서 베타카테닌, ITGA6, Sox9 및 FOXN1의 발현을 RT-Qpcr 분석으로 확인하였다. RT-qPCR 분석은 앞선 실시예와 동일한 조건으로 수행하였다. 상기 목적에 사용된 프라이머는 GAPDH (정방향 5'-ACTTTGGTATCGTGGAAGGAC-3' 및 역방향 5'-GCAGGGATGATGTTCTGGAG-3'), Sox9 (정방향 5'-GGACCACCCGGATTACAAGT-3' 및 역방향 5'-AAGATGGCGTTGGGGGAGAT-3'), β-Catenin (정방향 5'-TGTAGAAGCTGGTGGAATGC-3' 및 역방향 5'-GCTGAACAAGAGTCCCAAGG-3'), ITGA6 (정방향 5'-GAAGGTGGCTGCGGTAGCA-3' 및 역방향 5'-ATCACGTTGTCCTCCCGAGT-3'), 및 FOXN1 (정방향 5'-AGTCCTGGGTTCAGAGGTCA-3' 및 역방향 5'-CTGGCGAGTACTGGTGGAAG-3')이었다. RT-Qpcr 결과를 도 14에 나타내었다.Expressions of beta-catenin, ITGA6, Sox9, and FOXN1 as markers related to hair growth in outer root sheath cells according to concentrations of human hair-derived keratin and rhKRT34 were confirmed by RT-Qpcr analysis. RT-qPCR analysis was performed under the same conditions as in the previous example. The primers used for this purpose were GAPDH (forward 5'-ACTTTGGTATCGTGGAAGGAC-3' and reverse 5'-GCAGGGATGATGTTCTGGAG-3'), Sox9 (forward 5'-GGACCACCCGGATTACAAGT-3' and reverse 5'-AAGATGGCGTTGGGGGAGAT-3'), β -Catenin (forward 5′-TGTAGAAGCTGGTGGAATGC-3′ and reverse 5′-GCTGAACAAGAGTCCCAAGG-3′), ITGA6 (forward 5′-GAAGGTGGCTGCGGTAGCA-3′ and reverse 5′-ATCACGTTGTCCTCCCGAGT-3′), and FOXN1 (forward 5′- AGTCCTGGGTTCAGAGGTCA-3' and reverse 5'-CTGGCGAGTACTGGTGGAAG-3'). The RT-Qpcr results are shown in FIG. 14 .
도 14에 따르면, rhKRT34 처리군은 미처리군보다 외모근초세포의 발모 관련 마커인 베타카테닌, ITGA6, Sox9 및 FOXN1의 mRNA 발현이 증가한 것으로 확인되었다. According to FIG. 14, it was confirmed that the mRNA expression of beta-catenin, ITGA6, Sox9, and FOXN1, which are hair growth-related markers of outer root sheath cells, increased in the rhKRT34-treated group than in the untreated group.
3-4. 동물실험을 통한 발모효과 검증3-4. Verification of hair growth effect through animal experiments
YoungBio(Samtako, 1404957265)에서 구입한 수컷 C57BL/6 마우스를 사용하여 동물실험을 통한 발모효과를 확인하였다.The hair growth effect was confirmed through animal experiments using male C57BL/6 mice purchased from YoungBio (Samtako, 1404957265).
상기 마우스를 23 ± 2 ℃의 온도, 50 ± 5 %의 습도, 12시간의 명암 주기로 조절된 조건 하에 사육하였다. 마우스 등의 털은 모낭주기를 동기화를 하기 위해 전기 이발기를 사용하여 반복적으로 면도하였다. 케라틴의 발모 촉진 효과를 알아보기 위해 둘베코인산완충식염수, 0.05(w/v)% 인모 케라틴, 0.05(w/v)% 재조합 케라틴, 0.025(w/v)% 재조합 케라틴 및 0.01(w/v)% 재조합 케라틴 주사액을 사용하였으며, 10주령 마우스를 시술 전 시판되는 제모 크림 Veet®(Reckitt Benckiser, 62200809951)을 사용하여 등쪽 털을 완전히 제거하고 무작위로 할당하여 주사했다. 28일간 관찰 및 기록(도 15a 참조)을 하였으며, 마우스를 희생하여 피부 조직을 10% 중성 완충 포르말린(BBC Biochemical, 0141)으로 고정하였다. 조직을 파라핀에 포매하고 4 μm 두께로 절단한 후 조직학적 분석을 위해 헤마톡실린과 에오신으로 염색(H&E staining)하였다. 각주기의 모낭 수와 비율 및 성장기 모낭의 직경은 Х 100 배율에서 광학 현미경으로 관찰 및 기록하여 도 15b 내지 도 15d에 나타내었다.The mice were reared under conditions controlled by a temperature of 23±2° C., humidity of 50±5%, and a light/dark cycle of 12 hours. The fur of the mouse's back was shaved repeatedly 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 was used, and 10-week-old mice were completely depilated using commercially available hair removal cream Veet ® (Reckitt Benckiser, 62200809951) before the procedure, and randomly assigned and injected. Observation and recording were performed for 28 days (see FIG. 15a), and mice were sacrificed and skin tissues were fixed with 10% neutral buffered formalin (BBC Biochemical, 0141). Tissues were embedded in paraffin, cut to a thickness of 4 μm, 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 anagen phase were observed and recorded under an optical microscope at Х 100 magnification, and are shown in FIGS. 15B to 15D.
도 15에 따르면, 동일 농도인 0.05%(w/v) 인모케라틴과 rhKRT34를 주사시, rhKRT34를 주사한 마우스에서 높은 발모효과를 확인하였다. rhKRT34 주사시 모발의 성장 주기 전반에 걸쳐 모낭의 수가 상대적으로 크게 증가하였고, 성장기 모낭의 비율이 증가하였으며, 모낭의 직경 또한 rhKRT34의 주사시 상대적으로 크게 증가함을 확인하였다.According to FIG. 15, when rhKRT34 and 0.05% (w/v) human hair keratin at the same concentration were injected, high hair growth effect was confirmed in the mice injected with rhKRT34. Upon injection of rhKRT34, it was confirmed that the number of hair follicles increased relatively greatly throughout the hair growth cycle, the ratio of hair follicles in the anagen phase increased, and the diameter of hair follicles also increased relatively greatly when rhKRT34 was injected.
3-5. rhKRT34와 인모를 구성하는 산성케라틴(acidic keratin)의 유사성 분석3-5. Similarity analysis between rhKRT34 and acidic keratin, which constitutes human hair
탈당화 재조합 케라틴34와 머리카락을 구성하는 다른 산성케라틴과의 아미노산 배열의 유사성을 상대비교하기 위하여, NCBI의 Basic Local Alignment Search Tool(BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi)에서 단백질과 단백질을 비교할 때 사용하는 blastp 알고리즘을 이용하여 케라틴34 서열과 인모를 구성하는 다른 class 8개(KRT31, KRT32, KRT33A, KRT33B, KRT35, KRT36, KRT37, KRT38)의 산성케라틴을 비교하였다. 유사한 서열의 경우 blastp를 통한 유사도를 기준으로 관련 정보(유사도 점수 (score, 서열들간의 유사도를 알고리즘을 통해 점수화), 일치성 (Identities, 두 서열간 동일한 아미노산으로 일치하는 비율), 유사성 (Positives, 두 서열간 동일한 아미노산은 아니지만 유사한 성질의 아미노산으로 일치하는 비율)) 및 각각의 쿼리(query)와 케라틴34 서열간의 얼라인먼트(alignment) 결과를 확인할 수 있다.In order to compare the similarity of the amino acid sequence between deglycosylated recombinant keratin 34 and other acidic keratins constituting hair, NCBI's Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast .cgi), acid keratins of 8 different classes (KRT31, KRT32, KRT33A, KRT33B, KRT35, KRT36, KRT37, KRT38) constituting keratin 34 sequence and human hair were analyzed using the blastp algorithm used to compare proteins. compared. In the case of similar sequences, related information (similarity score (score, scoring similarity between sequences through an algorithm), identity (ratio of identical amino acids between two sequences), similarity (Positives, It is not the same amino acid between the two sequences, but the ratio of matching amino acids of similar nature)) and alignment results between each query and the Keratin 34 sequence can be confirmed.
도 16에 따르면, 케라틴34와 머리카락을 구성하는 다른 산성케라틴과의 아미노산 배열의 일치성(identities) 및 유사성(positives)을 상대비교 분석한 결과, 상기 재조합 케라틴34과 60% 이상의 일치성을 갖는 아미노산 배열 또는 75% 이상의 유사성을 갖는 아미노산 배열을 가지고 있음을 확인하였으며, 인모를 구성하는 케라틴들은 그 아미노산 배열의 유사성이 매우 높음을 알 수 있다.According to FIG. 16, as a result of comparative analysis of the identities and positives of the amino acid sequence of keratin 34 and other acidic keratins constituting hair, amino acids having 60% or more identity with the recombinant keratin 34 It was confirmed that they had sequences or amino acid sequences having a similarity of 75% or more, and keratins constituting human hair had very high amino acid sequence similarities.
두 단백질간의 아미노산 배열 일치성이 60% 미만이면 단백질의 과도한 구조적 변형을 의미하여 동일 또는 유사한 기능을 확보할 수 없는 것으로 알려져있다. 구체적으로, 상기 산성케라틴이 상기 재조합 케라틴34 대비 아미노산 배열 일치성이 60% 미만이거나 아미노산 배열의 유사성이 75% 미만이면, 케라틴의 기능을 상실하거나 감소하여 상기 재조합 케라틴34에서와 같은 탈모 예방 또는 치료 효과를 확보할 수 없다. 상기 8종의 산성케라틴은 상기 재조합 케라틴34의 아미노산 서열과 일치성 60% 이상 또는 유사성 75% 이상이거나, 일치성 60% 이상 및 유사성 75% 이상일 것을 만족하여, 상기 재조합 케라틴34와 동일 또는 유사한 탈모 예방 또는 치료 효과를 나타낼 수 있다.It is known that if the amino acid sequence identity between the two proteins is less than 60%, it means excessive structural modification of the protein, so that the same or similar function cannot be secured. Specifically, if the acidic keratin has an amino acid sequence identity of less than 60% or an amino acid sequence similarity of less than 75% compared to the recombinant keratin 34, the function of keratin is lost or reduced to prevent or treat hair loss as in the recombinant keratin 34. effect cannot be obtained. The 8 types of acidic keratins are identical or similar to those of the recombinant keratin 34, as they satisfy the requirement that they have at least 60% identity or at least 75% similarity with the amino acid sequence of the recombinant keratin 34, or at least 60% identity and 75% similarity. May have preventive or therapeutic effects.
Claims (8)
상기 재조합 케라틴은 글리코실화되지 않은 것인,
탈모(alopecia) 예방 또는 치료용 약학적 조성물. At least one selected from the group consisting of recombinant keratin 34, recombinant keratin having 60% or more amino acid sequence identity with the recombinant keratin 34, and recombinant keratin having 75% or more amino acid sequence positives with the recombinant keratin 34. Contains recombinant keratin as an active ingredient,
The recombinant keratin is not glycosylated,
A pharmaceutical composition for preventing or treating alopecia.
상기 재조합 케라틴 34의 서열은 서열번호 10의 아미노산 서열로 이루어진 것인,
탈모 치료용 약학적 조성물. According to claim 1,
The sequence of the recombinant keratin 34 consists of the amino acid sequence of SEQ ID NO: 10,
A pharmaceutical composition for treating hair loss.
상기 재조합 케라틴은 형질전환 원핵세포로부터 발현된 것인,
탈모 치료용 약학적 조성물. According to claim 1,
The recombinant keratin is expressed from a transformed prokaryotic cell,
A pharmaceutical composition for treating hair loss.
상기 재조합 케라틴은 이황화결합이 분해된 것으로,
평균 크기가 100 nm 이하의 입자인,
탈모 치료용 약학적 조성물.According to claim 1,
The recombinant keratin has disulfide bonds broken down,
Particles with an average size of 100 nm or less,
A pharmaceutical composition for treating hair loss.
상기 재조합 케라틴의 평균 분자량은 40 내지 70 kDa인,
탈모 치료용 약학적 조성물. According to claim 1,
The average molecular weight of the recombinant keratin is 40 to 70 kDa,
A pharmaceutical composition for treating hair loss.
상기 재조합 케라틴의 유효농도는 인모유래 케라틴의 유효농도보다 10배 낮은 농도인,
탈모 치료용 약학적 조성물. According to claim 1,
The effective concentration of the recombinant keratin is 10 times lower than the effective concentration of human hair-derived keratin.
A pharmaceutical composition for treating hair loss.
상기 조성물은 국소투여용으로 제형화된 것인,
탈모 치료용 약학적 조성물. According to claim 1,
The composition is formulated for topical administration,
A pharmaceutical composition for treating hair loss.
상기 재조합 케라틴은 글리코실화되지 않은 것인,
탈모(alopecia) 개선용 화장료 조성물.At least one recombinant keratin selected from the group comprising recombinant keratin 34, recombinant keratin having 60% or more amino acid sequence identity with the recombinant keratin 34, and recombinant keratin having 75% or more amino acid sequence similarity with the recombinant keratin 34 as an active ingredient. include,
The recombinant keratin is not glycosylated,
A cosmetic composition for improving alopecia.
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