KR20160017313A - Skin permeability enhanced peptide and fusion protein comprising the same - Google Patents
Skin permeability enhanced peptide and fusion protein comprising the same Download PDFInfo
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- KR20160017313A KR20160017313A KR1020140099901A KR20140099901A KR20160017313A KR 20160017313 A KR20160017313 A KR 20160017313A KR 1020140099901 A KR1020140099901 A KR 1020140099901A KR 20140099901 A KR20140099901 A KR 20140099901A KR 20160017313 A KR20160017313 A KR 20160017313A
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- South Korea
- Prior art keywords
- skin
- peptide
- derivative
- fusion protein
- present
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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
- A61K38/18—Growth factors; Growth regulators
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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
-
- 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
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Zoology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Immunology (AREA)
- Pharmacology & Pharmacy (AREA)
- Toxicology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Birds (AREA)
- Engineering & Computer Science (AREA)
- Peptides Or Proteins (AREA)
- Cosmetics (AREA)
Abstract
Description
본 발명은 피부투과성 펩타이드 및 이를 포함하는 융합단백질에 관한 것으로서, 보다 구체적으로 피부를 투과하여 약물을 전달할 수 있는 피부투과성 펩타이드, 이의 유도체 또는 이를 포함하는 융합단백질을 비롯하여, 상기 피부투과성 펩타이드, 이의 유도체 또는 이를 포함하는 융합단백질을 코딩하는 폴리뉴클레오티드, 상기 피부투과성 펩타이드, 이의 유도체 또는 이를 포함하는 융합단백질을 포함하는 피부외용제용 약학적 조성물 및 화장료 조성물에 관한 것이다.
TECHNICAL FIELD The present invention relates to a skin permeable peptide and a fusion protein containing the same, and more particularly, to a skin permeable peptide, a derivative thereof, or a fusion protein comprising the same, which can transmit a drug through the skin, Or a polynucleotide encoding a fusion protein comprising the same, a skin permeable peptide, a derivative thereof, or a fusion protein comprising the same, and a pharmaceutical composition and a cosmetic composition for external use for skin.
피부를 통과하는 약물은 편리한 사용에 근거하여 진통패치, 금연패치, 임신 조절제 등에서 사용되고 있고, 피부를 통하여 대순환계(systemic circulation)에 전달하고자 하는 목적을 가진다. 뿐만 아니라, 아토피 치료제, 미백, 주름 개선용 화장품 등 피부자체에 전달하려는 목적을 가지는 경우도 있다. 이러한 편리성과 기능성에도 불구하고, 피부의 구조상 약물의 전달의 어려움을 갖는다. 피부의 외부에는 약 10 내지 15층의 사세포(corneocyte)가 존재하고, 이는 약 10 ㎛ 내지 45 ㎛ 정도의 두께를 이루고 있다. 이는 모르타르(mortar)와 브릭(brick) 구조라 불리는 형태로 이루어져 있다. 브릭(brick)의 구조는 케라틴이 풍부한 사세포로 이루어져 있고, 그 사이를 세라미드(ceramide), 지방산, 왁스 등의 구조가 모르타르(mortar)를 이루고 있다. 이를 통해서 내부의 수분의 손실을 막고 외부로부터의 침입을 막을 수 있는 구조를 가진다. 따라서 피부투과성이 낮고, 500Da 이하의 저분자 구조 성분들만이 확산(Diffusion) 방식에 의해서 피부 내로 전달된다(Exp. Dermatol., 2000, 9, 165-9.). 이는 모르타르(mortar) 구조의 세포내 구조의 지질층이나, 지질층 사이의 수용성 구조를 통해서 이루어지고, 약물의 성질에 따라서도 크게 좌우된다(Current Drug Delivery, 2005, 2, 23-3). 또한 피부표면을 직접 통과하는 방식 이외에 피부의 땀샘, 모공, 피지선 등을 통하여 전달되기도 한다.
Drugs that pass through the skin are used in analgesic patches, anti-smoking patches, pregnancy regulators, etc., based on convenient use, and have the purpose of delivering them to the systemic circulation through the skin. In addition, there are cases where it has the purpose of delivering to the skin itself, such as atopic treatments, whitening, and cosmetics for improving wrinkles. Despite its convenience and functionality, it has difficulty delivering drugs due to the structure of the skin. Outside the skin there are about 10-15 layers of corneocytes, which are about 10 to 45 microns thick. It consists of mortar and brick structures. The structure of the brick is composed of keratin-rich cells, and ceramides, fatty acids and waxes form mortar. It has a structure that prevents the loss of moisture inside and prevents intrusion from outside. Therefore, only low-molecular-weight components having a low skin permeability and a molecular weight of 500 Da or less are delivered into the skin by a diffusion method (Exp. Dermatol., 2000, 9, 165-9). This is done through the lipid layer in the intracellular structure of the mortar structure or through the water-soluble structure between the lipid layers and is also highly dependent on the nature of the drug (Current Drug Delivery, 2005, 2, 23-3). In addition to passing directly through the surface of the skin, it can also be transmitted through the skin's sweat glands, pores, sebaceous glands, and the like.
이러한 이유로, 약물 분자의 크기나 성질에 관계없이 피부를 투과할 수 있고, 피부의 전체에 고르게 전달할 수 있는 방법을 개발하려는 연구가 활발히 진행되었다.For this reason, research has been conducted actively to develop a method capable of permeating the skin regardless of the size or nature of the drug molecule and delivering it uniformly throughout the skin.
예를 들어, 한국등록특허 제1054519호에는 천연 인간 성장호르몬들과 비교하여 안정성 및 피부 투과도가 우수한 인간 성장호르몬-유래 펩타이드 및 이를 포함하는 조성물이 개시되어 있고, 한국등록특허 제1104223호에는 인간 IL-10의 기능과 동일한 기능을 하며 천연 IL-10과 비교하여 안정성 및 피부투과도가 매우 우수한 IL-10-유래 펩타이드 및 이를 포함하는 조성물이 개시되어 있으나, 이들 펩타이드는 그 자체로 기능성을 나타낼 뿐, 다른 약물의 전달용 담체로서 사용할 수 없다는 단점이 있었다. 또한, 이러한 단점을 극복하기 위하여 펩타이드로서 미국등록특허 제7,659,252호에는 피부질환의 치료 및 약학적 활성제제의 피부투과 촉진에 사용될 수 있는 피부투과성 펩타이드가 개시되어 있다. 상기 펩타이드는 우수한 피부투과도를 나타낼 뿐만 아니라 다른 약물의 경피전달용 담체로서도 사용될 수 있다는 장점이 있으나, 피부를 투과한 다음에는 체내의 순환시스템을 통해 소모되기 때문에, 피부를 표적으로 하는 약물의 경우에는 별다른 효과를 발휘하지 못한다는 단점이 있었다.
For example, Korean Patent Registration No. 1054519 discloses a human growth hormone-derived peptide and a composition containing the human growth hormone-derived peptide, which are superior in stability and skin permeability to natural human growth hormone. In Korean Patent No. 1104223, 10-derived peptides having the same functions as those of natural IL-10 and having excellent stability and skin permeability as compared with natural IL-10, and compositions comprising the same. However, these peptides are merely functional, It can not be used as a delivery carrier for other drugs. In order to overcome this disadvantage, U.S. Patent No. 7,659,252 as a peptide discloses a skin permeable peptide which can be used for treatment of skin diseases and for promoting skin permeation of a pharmacologically active agent. The peptide not only exhibits excellent skin permeability but also can be used as a carrier for transdermal delivery of other drugs. However, since the peptide is consumed through the circulatory system in the body after permeating through the skin, in the case of a drug targeting the skin It has a disadvantage that it can not exert any significant effect.
이러한 배경 하에서, 본 발명자들은 약물의 경피전달용 담체로서 사용될 수 있으면서도 피부에 잔류할 수 있는 특성을 가지는 피부투과성 펩타이드를 개발하기 위하여 예의 연구노력한 결과, 대한민국 등록특허 제10-1329411호를 통하여 피부투과도가 우수한 펩타이드를 등록받은 바 있으며, 추가적으로 연구를 계속한 끝에 본 발명의 신규한 서열의 피부투과성 펩타이드 역시 약물의 경피전달용 담체로서 사용될 수 있으면서도, 생리활성 단백질을 결합하여 제조한 융합단백질 또한 피부투과성이 높고, 피부에 오랫동안 잔류할 수 있음을 확인하고 본 발명을 완성하였다.
Under these circumstances, the inventors of the present invention have made extensive efforts to develop a skin permeable peptide having properties that can be used as a carrier for transdermal delivery of a drug, while remaining in the skin. As a result, Korean Patent No. 10-1329411 The peptide having a novel sequence of the present invention can be used as a carrier for transdermal delivery of a drug, and a fusion protein prepared by binding a physiologically active protein can also be used as a skin permeable peptide Is high and can remain on the skin for a long time, thus completing the present invention.
본 발명의 하나의 목적은 피부투과성 펩타이드 또는 이의 유도체를 제공하는 것이다.It is an object of the present invention to provide skin permeable peptides or derivatives thereof.
본 발명의 다른 목적은 상기 펩타이드 또는 이의 유도체를 포함하는 융합단백질을 제공하는 것이다.It is another object of the present invention to provide a fusion protein comprising the peptide or derivative thereof.
본 발명의 또 다른 목적은 상기 펩타이드, 이의 유도체 또는 상기 융합단백질을 코딩하는 폴리뉴클레오티드를 제공하는 것이다.It is still another object of the present invention to provide a peptide, a derivative thereof, or a polynucleotide encoding the fusion protein.
본 발명의 또 다른 목적은 상기 펩타이드 또는 이의 유도체를 포함하는 피부외용제용 담체를 제공하는 것이다.Still another object of the present invention is to provide a carrier for external preparation for skin comprising the peptide or a derivative thereof.
본 발명의 또 다른 목적은 상기 펩타이드, 이의 유도체 또는 상기 융합단백질을 포함하는 피부외용제용 약학적 조성물을 제공하는 것이다.Still another object of the present invention is to provide a pharmaceutical composition for the external preparation for skin comprising the peptide, a derivative thereof or the fusion protein.
본 발명의 또 다른 목적은 상기 펩타이드, 이의 유도체 또는 상기 융합단백질을 포함하는 화장료 조성물을 제공하는 것이다.
Still another object of the present invention is to provide a cosmetic composition comprising the peptide, a derivative thereof or the fusion protein.
상기 목적을 달성하기 위한 일 실시양태로서, 본 발명은 서열번호 1의 아미노산 서열로 구성되는 피부투과성 펩타이드 또는 이의 유도체를 제공한다.
In one embodiment, the present invention provides a skin permeable peptide or derivative thereof comprising the amino acid sequence of SEQ ID NO: 1.
피부투과성 펩타이드: LQGQSYLSISFP (서열번호 1)
Skin permeable peptide: LQGQSYLSISFP (SEQ ID NO: 1)
본 발명에서 "피부투과성"이란, 펩타이드가 피부를 투과하여 피부 내부로 침투할 수 있는 능력 또는 성질을 의미하며, 본 발명의 펩타이드는 다른 펩타이드에 비하여 현저히 우수한 피부투과성을 나타낸다.
The term "skin permeability" in the present invention means the ability or property of the peptide to penetrate into the skin through the skin, and the peptide of the present invention shows significantly superior skin permeability than other peptides.
상기 피부투과성 펩타이드 또는 이의 유도체는 우수한 피부투과성뿐만 아니라, 우수한 피부잔류성을 나타낼 수 있다.
The skin permeable peptide or derivative thereof may exhibit excellent skin permeability as well as good skin permeability.
본 발명에서 "피부잔류성"이란, 피부를 투과한 펩타이드가 피부조직을 통과하여 순환계로 전달되지 않고, 피부 내의 조직에 결합되어 피부 내에서 잔류하는 능력을 의미한다. 피부조직을 표적조직으로 하는 약학적 제제 또는 화장료의 경우에는, 상기 펩타이드와 결합된 성분이 피부조직 또는 피부세포에 장기간 작용할 수 있도록 피부조직에 잔류하는 특성이 우수한 담체를 사용함이 바람직하다. 본 발명의 펩타이드는 피부투과성뿐만 아니라 피부잔류성이 현저히 우수하므로, 약학적 제제 또는 화장료의 담체로서 사용될 수 있다.
In the present invention, "skin persistence" means the ability of peptides permeating through the skin to pass through the skin tissue and not to be transferred to the circulatory system, but to remain in the skin after being bound to the tissue in the skin. In the case of pharmaceutical preparations or cosmetics containing the skin tissue as a target tissue, it is preferable to use a carrier having excellent properties to remain in the skin tissue so that the component bound to the peptide can act on the skin tissue or skin cells for a long time. The peptides of the present invention can be used as a carrier for pharmaceutical preparations or cosmetics since they have remarkably excellent skin permeability as well as skin retention.
본 발명에서 "유도체"란, 상기 피부투과성 펩타이드의 N-말단, C-말단 등이 화학적으로 수식되거나 또는 아미노산이 추가/치환/결실되어 변형된 펩타이드를 포함한다. 본 발명의 목적상, 상기 유도체는 우수한 피부투과성 및/또는 피부잔류성을 나타내는 한, 특별히 제한되지 않는다. 예를 들면, 본 발명의 피부투과성 펩타이드 유도체는 단실(dansyl)기가 결합된 유도체가 될 수 있다.
In the present invention, the "derivative" includes peptides in which the N-terminal, C-terminal, etc. of the skin permeable peptide are chemically modified or modified by addition / substitution / deletion of amino acid. For the purpose of the present invention, the derivative is not particularly limited so long as it exhibits excellent skin permeability and / or skin persistence. For example, the skin permeable peptide derivative of the present invention may be a derivative to which a dansyl group is bonded.
본 발명의 일 실시예에 의하면, 파지 디스플레이 방법을 이용하여 서열번호 1의 아미노산 서열로 구성된 피부투과성 펩타이드를 선발하였고, 선발된 피부투과성 펩타이드를 포함하는 파지의 우수한 피부투과성을 확인하였다(실시예 2, 표 1). 또한, 상기 펩타이드의 C-말단에 단실(dansyl)기가 결합된 형태의 유도체를 각각 합성하였고, 상기 유도체 역시 우수한 피부투과성을 나타냄을 확인하였다(실시예 3, 표 2). 아울러, 상기 피부투과성 펩타이드 또는 이의 C-말단에 단실기가 결합된 유도체를 포함하는 파지의 우수한 피부잔류성을 확인하였다(실시예 4 및 5, 표 3 및 4).
According to one embodiment of the present invention, a skin permeable peptide composed of the amino acid sequence of SEQ ID NO: 1 was selected using the phage display method and excellent skin permeability of the phage containing the selected skin permeable peptide was confirmed (Example 2 , Table 1). In addition, derivatives in which a dansyl group was bonded to the C-terminal of the peptide were synthesized, respectively, and it was confirmed that the derivative also exhibited excellent skin permeability (Example 3, Table 2). In addition, excellent skin retention of the phage containing the skin permeable peptide or a derivative having a monosaccharide at its C-terminal was confirmed (Examples 4 and 5, Tables 3 and 4).
상기 목적을 달성하기 위한 다른 실시양태로서, 본 발명은 상기 피부투과성 펩타이드 또는 이의 유도체를 포함하는 융합단백질을 제공한다.
As another embodiment for achieving the above object, the present invention provides a fusion protein comprising the skin permeable peptide or a derivative thereof.
본 발명에서 "융합단백질"이란, 상기 피부투과성 펩타이드가 다른 단백질 또는 펩타이드에 결합되도록 인위적으로 합성된 단백질로서, 바람직하게는 상기 피부투과성 펩타이드 및 생리활성 단백질을 포함할 수 있다.In the present invention, the term "fusion protein" refers to a protein artificially synthesized so that the skin permeable peptide binds to another protein or peptide, preferably, the skin permeable peptide and the physiologically active protein.
본 발명에서 “생리활성 단백질”은 치료학적 효과를 위해 사용되는 모든 단백질을 포함한다. 본 발명에서 생리활성 단백질은, 생체의 기능(생리)을 조절하는 단백질을 총칭하며, 생리활성 폴리펩타이드라고도 한다. 본 발명의 생리활성 단백질은 피부에 처리할 수 있는 단백질이면 제한됨이 없고, 상기 생리활성 단백질의 천연형과 실질적으로 동등하거나 증가된 기능, 구조, 활성 또는 안정성을 갖는 한, 임의의 유도체도 본 발명의 생리활성 단백질의 범위에 포함된다. 상기 생리활성 단백질은 호르몬, 사이토카인, 경단백질, 효소, 항체, 성장인자 등을 모두 포괄하는 개념이며, 구체적인 예로서, 인간 성장 호르몬, 성장 호르몬 방출 호르몬, 성장 호르몬 방출 펩타이드, 인터페론류, 인터페론 수용체류, 콜로니 자극인자류, 글루카콘-유사 펩타이드류, 엑센딘-4 펩타이드류, ANP, BNP, CNP, DNP, 지프로테인 관련 수용체 (Gprotein-coupled receptor), 인터루킨류, 인터루킨 수용체류, 효소류, 인터루킨 결합 단백질류, 사이토카인 결합 단백질류, 마크로파지 활성인자, 마크로파지 펩타이드, B 세포인자, T 세포인자, 단백질 A, 알러지 억제인자, 세포 괴사 당단백질, 면역독소, 림포독소, 종양 괴사인자, 종양 억제인자, 전이 성장인자, 알파-1 안티트립신, 알부민, α-락트알부민, 아포리포단백질-E, 적혈구 생성인자, 고 당쇄화 적혈구 생성인자, 안지오포에이틴류, 헤모글로빈, 트롬빈, 트롬빈 수용체 활성펩타이드, 트롬보모듈린, 혈액인자 Ⅶ, Ⅶa, Ⅷ, Ⅸ, 및 XIII, 플라즈미노젠 활성인자, 피브린-결합 펩타이드, 유로키나제, 스트렙토키나제, 히루딘, 단백질 C, C-반응성 단백질, 레닌 억제제, 콜라게나제 억제제, 수퍼옥사이드 디스뮤타제, 렙틴, 혈소판 유래 성장인자, 상피세포 성장인자, 표피세포 성장인자, 안지오스타틴, 안지오텐신, 골 형성 성장인자, 골 형성 촉진 단백질, 칼시토닌, 인슐린, 소마토스타틴, 옥트레오타이드(소마토스타틴 아고니스트), 아트리오펩틴, 연골 유도인자, 엘카토닌, 결합조직 활성인자, 조직인자 경로 억제제, 여포 자극 호르몬, 황체 형성 호르몬, 황체 형성 호르몬 방출 호르몬, 신경 성장인자류, 부갑상선 호르몬, 릴랙신, 씨크레틴, 소마토메딘, 인슐린 유사 성장인자, 부신피질 호르몬, 글루카곤, 콜레시스토키닌, 췌장 폴리펩타이드, 가스트린 방출 펩타이드, 코티코트로핀 방출인자, 갑상선 자극호르몬, 오토탁신, 락토페린, 미오스타틴, 수용체류, 수용체 길항물질, 세포표면항원, 바이러스 유래 백신 항원, 단일클론 항체, 다중클론 항체, 항체 단편류 등을 들 수 있으나, 이에 제한되지 않는다.In the present invention, " physiologically active protein " includes all proteins used for therapeutic effect. In the present invention, the physiologically active protein is collectively referred to as a protein that regulates the function (physiology) of a living body, and is also referred to as a physiologically active polypeptide. The physiologically active protein of the present invention is not limited as long as it is a protein that can be treated on the skin, and any derivative may be used as long as it has substantially the same function, structure, activity or stability as the natural form of the physiologically active protein Of the physiologically active protein. The physiologically active protein is a concept that includes all of hormones, cytokines, light proteins, enzymes, antibodies, growth factors, etc. Specific examples include human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferon, interferon Such as a glucocorticoid-like peptide, an exendin-4 peptide, ANP, BNP, CNP, DNP, a gprotein-coupled receptor, interleukins, interleukin receptors, enzymes , A cytokine binding protein, a macrophage activator, a macrophage peptide, a B cell factor, a T cell factor, a protein A, an allergic inhibitor, a cytotoxic glycoprotein, an immunotoxin, a lymphotoxin, a tumor necrosis factor, a tumor Inhibitors, metastasis growth factors, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein-E, erythropoietic factor, hyperglycosylated erythropoietin , Angiopoietin, hemoglobin, thrombin, thrombin receptor activating peptide, thrombomodulin, blood factor VII, VIIa, VIII, IX, and XIII, plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, An angiostatin, an angiogenic growth factor, an angiogenesis factor, an angiogenesis factor, an angiogenesis factor, an angiogenesis factor, an angiogenesis factor, an angiogenesis factor, a rheumatoid factor, a rheumatoid factor, a rheumatoid factor, An osteogenic stimulating protein, calcitonin, insulin, somatostatin, octreotide (somatostatin agonist), atripeptin, cartilage inducer, elkatonin, connective tissue activator, tissue factor pathway inhibitor, follicle stimulating hormone, luteinizing hormone, Luteinizing hormone-releasing hormone, nerve growth factor, parathyroid hormone, lilacsin, cicletin, somatomedin, insulin-like growth Antagonist, receptor antagonist, cell surface antigens, viral-derived vaccine, antiviral agent, antiviral agent, antiviral agent, antiviral agent, antagonist, antagonist, antagonist, antagonist, corticosteroid, glucagon, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone Antigens, monoclonal antibodies, polyclonal antibodies, antibody fragments, and the like.
본 발명의 융합단백질은 이에 포함된 상기 피부투과성 펩타이드에 의하여 피부투과성 및 피부잔류성이 높아 피부에 사용될 수 있는 의약품 또는 화장품에 효율적으로 적용될 수 있으며, 그에 따라 생리활성 단백질에 특별한 부작용이 없는 한 알려진 모든 생리활성 단백질을 사용할 수 있다.The fusion protein of the present invention has high skin permeability and skin retention due to the skin permeable peptide contained therein and thus can be efficiently applied to medicines or cosmetics which can be used for skin, Physiologically active proteins can be used.
본 발명의 일 실시예에서는 대표적인 생리활성 단백질로서, 상피세포 성장인자(epidermal growth factor, EGF)와 상기 피부투과성 펩타이드를 포함하는 융합단백질을 제조하였으며(실시예 6), 상기 융합단백질의 피부투과성 및 피부잔류성이 우수함을 확인함으로써(표 5, 표 6), 상기 피부투과성 펩타이드에 생리활성 단백질이 융합되더라도 동일한 활성을 유지할 수 있음을 확인하였다.
In one embodiment of the present invention, a typical physiologically active protein, a fusion protein comprising an epidermal growth factor (EGF) and the skin permeable peptide was prepared (Example 6) (Table 5, Table 6), it was confirmed that the same activity can be maintained even if the physiologically active protein is fused to the skin permeable peptide.
본 발명에 있어서, 상기 융합단백질은 상기 피부투과성 펩타이드가 상기 생리활성 단백질의 N-말단에 직접적으로 연결될 수도 있고, 링커를 통해 연결될 수도 있다. 상기 링커는 상기 융합단백질의 활성을 향상시키는 효과를 나타내게 하는 한 특별히 이에 제한되지 않으나, 바람직하게는 글라이신, 알라닌, 루이신, 이소루이신, 프롤린, 세린, 트레오닌, 아스파라긴, 아스파르트산, 시스테인, 글루타민, 글루탐산, 리신, 아르기닌산 등의 아미노산을 사용하여 연결시킬 수 있고, 보다 바람직하게는 발린, 루이신, 아스파르트산, 글라이신, 알라닌, 프롤린 등을 여러개 사용하여 연결시킬 수 있으며, 가장 바람직하게는 유전자 조작의 용이성을 고려하여 글라이신, 발린, 루이신, 아스파르트산 등의 아미노산을 1개 내지 5개씩 연결하여 사용할 수 있다. 예를 들어, 본 발명에서는 피부투과성 펩타이드의 C-말단과 생리활성 단백질의 N-말단을 2개의 글라이신(GG)으로 구성된 링커를 통해 연결시켜서 융합단백질을 제조하였다. In the present invention, the fusion protein may be directly linked to the N-terminal of the physiologically active protein or through the linker. Preferably, the linker is selected from the group consisting of glycine, alanine, leucine, isoleucine, proline, serine, threonine, asparagine, aspartic acid, cysteine, glutamine , Glutamic acid, lysine, arginic acid, and the like, and more preferably, it can be linked using valine, leucine, aspartic acid, glycine, alanine, proline, Considering the ease of manipulation, one to five amino acids such as glycine, valine, leucine, and aspartic acid may be linked and used. For example, in the present invention, a fusion protein was prepared by linking the C-terminal of a skin permeable peptide and the N-terminus of a biologically active protein through a linker composed of two glycines (GG).
상기 융합단백질은 이에 포함되는 각 도메인의 야생형의 아미노산 서열과 하나 이상의 아미노산 잔기가 상이한 서열을 가지는 폴리펩타이드를 포함할 수 있다. 분자의 활성을 전체적으로 변경시키지 않는 단백질 및 폴리펩타이드에서의 아미노산 교환은 당해 분야에 공지되어 있다. 가장 통상적으로 일어나는 교환은 아미노산 잔기 Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly 간의 교환이다. 또한, 아미노산 서열상의 변이 또는 수식에 의해서 단백질의 열, pH등에 대한 구조적 안정성이 증가하거나 단백질 활성이 증가한 단백질을 포함할 수 있다.The fusion protein may include a polypeptide having a sequence having a different amino acid sequence from the wild-type amino acid sequence of each domain contained in the fusion protein. Amino acid exchange in proteins and polypeptides that do not globally alter the activity of the molecule is known in the art. The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. In addition, it may include a protein having increased structural stability or increased protein activity due to mutation or modification of the amino acid sequence, for example, heat, pH, etc. of the protein.
상기 융합단백질 또는 상기 융합단백질을 구성하는 폴리펩타이드는 당해 분야에 공지된 화학적 펩타이드 합성방법으로 제조하거나, 상기 융합단백질을 코딩하는 유전자를 PCR(polymerase chain reaction)에 의해 증폭하거나 공지된 방법으로 합성한 후 발현벡터에 클로닝하여 발현시켜서 제조할 수 있다.
The fusion protein or the polypeptide constituting the fusion protein may be prepared by a chemical peptide synthesis method known in the art, or may be prepared by amplifying a gene encoding the fusion protein by PCR (polymerase chain reaction) or by a known method Followed by expression in a post-expression vector.
본 발명은 서열번호 1의 아미노산 서열로 구성되는 피부투과성 펩타이드가, 서열번호 4의 아미노산 서열의 N-말단에 결합되어, 서열번호 5의 아미노산 서열을 포함하는 융합단백질을 제공할 수 있다.The present invention can provide a fusion protein comprising the amino acid sequence of SEQ ID NO: 1, wherein the skin permeable peptide comprising the amino acid sequence of SEQ ID NO: 1 is bound to the N-terminus of the amino acid sequence of SEQ ID NO:
본 발명의 일 실시예에 의하면, 파지라이브러리와 경피제제의 용출 실험 방법을 조합한, 파지 디스플레이 방법을 수행하여 서열번호 1의 아미노산 서열을 포함하는 신규한 피부투과성 펩타이드를 발굴하였다. 상기 발굴된 피부투과성 펩타이드에 서열번호 4의 아미노산 서열을 포함하는 생리활성 단백질(EGF)을 결합하여, 서열번호 5의 아미노산 서열을 포함하는 융합단백질(T4-EGF)을 제조하였다. 상기 제조된 융합단백질의 활성을 피부투과성 펩타이드와 결합하지 아니한 생리활성 단백질의 활성과 비교한 결과, 피부투과성 및 피부잔류성이 현저히 향상되었음을 확인하였다(표 5 및 표 6). According to one embodiment of the present invention, a novel skin permeable peptide containing the amino acid sequence of SEQ ID NO: 1 was discovered by performing a phage display method combining a phage library and a dissolution test method of a transdermal preparation. A fusion protein (T4-EGF) comprising the amino acid sequence of SEQ ID NO: 5 was prepared by binding a physiologically active protein (EGF) comprising the amino acid sequence of SEQ ID NO: 4 to the excised skin permeable peptide. As a result of comparing the activity of the prepared fusion protein with the activity of a physiologically active protein not binding to a skin permeable peptide, it was confirmed that skin permeability and skin retention were remarkably improved (Table 5 and Table 6).
따라서, 본 발명에서 제공하는 융합단백질은 생리활성 단백질에 피부투과성 펩타이드가 결합되어, 생리활성 단백질 자체의 효능을 유지하면서도, 피부투과성과 피부잔류성을 현저하게 향상시킬 수 있으므로, 기능성 화장료 조성물 및 피부외용제용 약학적 조성물의 유효성분으로서 유용하게 사용될 수 있다.Therefore, the fusion protein provided by the present invention can bind skin-permeable peptides to physiologically active proteins and significantly improve skin permeability and skin retention while maintaining the efficacy of the physiologically active protein itself, And can be usefully used as an active ingredient of a pharmaceutical composition.
또 하나의 양태로서, 본 발명은 상기 펩타이드, 이의 유도체 또는 상기 융합단백질을 코딩하는 폴리뉴클레오티드를 제공한다.
In another embodiment, the present invention provides a peptide, a derivative thereof, or a polynucleotide encoding the fusion protein.
상기 폴리뉴클레오티드는 하나 이상의 염기가 치환, 결실, 삽입 또는 이들의 조합에 의해 변이될 수 있다. 뉴클레오티드 서열을 화학적으로 합성하여 제조하는 경우, 당업계에 널리 공지된 합성법, 예를 들어 문헌(Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988)에 기술된 방법을 이용할 수 있으며, 트리에스테르, 포스파이트, 포스포르아미다이트 및 H-포스페이트 방법, PCR 및 기타 오토프라이머 방법, 고체 지지체상의 올리고뉴클레오타이드 합성법 등을 이용하여 합성할 수 있다.The polynucleotide may be mutated by substitution, deletion, insertion, or a combination thereof, of one or more bases. When the nucleotide sequence is prepared by chemically synthesizing, it is possible to use a method well known in the art, for example, a method described in Engels and Uhlmann, Angew Chem IntEd Engl., 37: 73-127, 1988 , Triesters, phosphites, phosphoramidites and H-phosphate methods, PCR and other auto primer methods, and oligonucleotide synthesis on solid supports.
예를 들어, 본 발명의 피부투과성 펩타이드 또는 이의 유도체를 코딩하는 폴리뉴클레오티드는 하기 서열번호 2의 염기서열로 구성된 것일 수 있으나, 이에 제한되는 것은 아니다.
For example, the polynucleotide encoding the skin permeable peptide or derivative thereof of the present invention may be composed of the nucleotide sequence shown in SEQ ID NO: 2 below, but is not limited thereto.
피부투과성 펩타이드를 코딩하는 폴리뉴클레오티드: Polynucleotides encoding skin permeable peptides:
TTG CAG GGT CAG TCT TAT CTG TCG ATT TCT TTT CCG (서열번호 2)
TTG CAG GGT CAG TCT TAT CTG TCG ATT TCT TTT CCG (SEQ ID NO: 2)
또 하나의 양태로서, 본 발명은 상기 폴리뉴클레오티드를 포함하는 발현벡터, 상기 발현벡터를 포함하는 형질전환체 및 상기 형질전환체를 이용하여 상기 피부투과성 펩타이드, 이의 유도체 또는 융합단백질을 생산하는 방법을 제공한다.
In another embodiment, the present invention provides a method for producing the skin permeable peptide, a derivative or a fusion protein thereof using an expression vector comprising the polynucleotide, a transformant containing the expression vector, and the transformant to provide.
본 발명에서 "발현벡터"란, 목적하는 숙주세포에서 목적 펩타이드를 발현할 수 있는 재조합 벡터로서, 유전자 삽입물이 발현되도록 작동가능하게 연결된 필수적인 조절 요소를 포함하는 유전자 제작물을 의미한다. 상기 발현벡터는 개시코돈, 종결코돈, 프로모터, 오퍼레이터 등의 발현조절 요소들을 포함하는데, 상기 개시코돈 및 종결코돈은 일반적으로 폴리펩타이드를 암호화하는 뉴클레오티드 서열의 일부로 간주되고, 유전자 제작물이 투여되었을 때 개체에서 반드시 작용을 나타내어야 하며 코딩 서열과 인프레임(in frame)에 있어야 한다. 벡터의 프로모터는 구성적 또는 유도성일 수 있다. As used herein, the term "expression vector" refers to a recombinant vector capable of expressing a target peptide in a desired host cell, and a gene construct containing an essential regulatory element operatively linked to the expression of the gene insert. The expression vector includes expression control elements such as an initiation codon, a termination codon, a promoter, an operator, etc. The initiation codon and the termination codon are generally regarded as part of the nucleotide sequence encoding the polypeptide, and when the gene product is administered, And must be in coding sequence and in frame. The promoter of the vector may be constitutive or inducible.
상기 "작동가능하게 연결(operably linked)"이란, 일반적 기능을 수행하도록 핵산 발현조절 서열과 목적하는 단백질 또는 RNA를 코딩하는 핵산 서열이 기능적으로 연결(functional linkage)되어 있는 상태를 의미한다. 예를 들어 프로모터와 단백질 또는 RNA를 코딩하는 핵산 서열이 작동가능하게 연결되어 코딩서열의 발현에 영향을 미칠 수 있다. 발현 벡터와의 작동적 연결은 당해 기술분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용할 수 있다.The term " operably linked "means a state in which a nucleic acid sequence encoding a desired protein or RNA and a nucleic acid expression control sequence are functionally linked to perform a general function. For example, a nucleic acid sequence encoding a promoter and a protein or RNA may be operably linked to affect the expression of the coding sequence. The operative linkage with an expression vector can be produced using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be performed using enzymes generally known in the art.
또한, 상기 발현벡터는 세포 배양액으로부터 단백질의 분리를 촉진하기 위하여 폴리펩타이드의 배출을 위한 시그널 서열을 포함할 수 있다. 특이적인 개시 시그널은 또한 삽입된 핵산 서열의 효율적인 번역에 필요할 수도 있다. 이들 시그널은 ATG 개시코돈 및 인접한 서열들을 포함한다. 어떤 경우에는, ATG 개시코돈을 포함할 수 있는 외인성 번역 조절 시그널이 제공되어야 한다. 이들 외인성 번역 조절 시그널 및 개시코돈은 다양한 천연 및 합성 공급원일 수 있다. 발현 효율은 적당한 전사 또는 번역 강화 인자의 도입에 의하여 증가될 수 있다.In addition, the expression vector may comprise a signal sequence for the release of the polypeptide to facilitate the separation of the protein from the cell culture fluid. A specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG start codon and adjacent sequences. In some cases, an exogenous translational control signal, which may include the ATG start codon, should be provided. These exogenous translational control signals and initiation codons can be of various natural and synthetic sources. Expression efficiency can be increased by the introduction of suitable transcription or translation enhancers.
아울러, 상기 발현벡터는 피부투과성 펩타이드, 이의 유도체 또는 이를 포함하는 융합단백질의 검출이 용이하도록 하기 위하여, 임의로 엔도펩티아이제를 사용하여 제거할 수 있는 단백질 태그를 추가로 포함할 수 있다. In addition, the expression vector may further include a protein tag that can be optionally removed using endopeptidase, in order to facilitate detection of the skin permeable peptide, derivative thereof, or a fusion protein comprising the peptide.
상기 "태그(tag)"란, 정량가능한 활성 또는 특성을 나타내는 분자를 의미하며, 플로오레세인과 같은 화학적 형광물질(fluoracer), 형광 단백질(GFP) 또는 관련 단백질과 같은 폴리펩타이드 형광물질을 포함한 형광분자일 수도 있고; Myc 태그, 플래그(Flag) 태그, 히스티딘 태그, 루신 태그, IgG 태그, 스트랩타비딘 태그 등의 에피톱 태그일 수도 있다. 특히, 에피톱 태그를 사용할 경우, 바람직하게는 6개 이상의 아미노산 잔기로 구성되고, 보다 바람직하게는 8개 내지 50개의 아미노산 잔기로 구성된 펩타이드 태그를 사용할 수 있다.The term "tag" refers to a molecule that exhibits quantifiable activity or characteristics and includes a fluorophore such as fluorescein such as fluorescein, a fluorescent protein (GFP), or a fluorescent substance containing a polypeptide fluorescent substance such as a related protein Lt; / RTI > Myc tag, a Flag tag, a histidine tag, a Lucin tag, an IgG tag, or a strap tagidine tag. Particularly, in the case of using an epitope tag, a peptide tag composed of preferably 6 or more amino acid residues, more preferably 8 to 50 amino acid residues can be used.
본 발명에 있어서, 상기 발현벡터는 상술한 본 발명의 피부투과성 펩타이드, 이의 유도체 또는 이를 포함하는 융합단백질을 코딩하는 뉴클레오티드 서열을 포함할 수 있는데, 이때 사용되는 벡터는 본 발명의 피부투과성 펩타이드, 이의 유도체 또는 이를 포함하는 융합단백질을 생산할 수 있는 한, 특별히 이에 제한되지 않으나, 바람직하게는 플라스미드 DNA, 파아지 DNA 등이 될 수 있고, 보다 바람직하게는 상업적으로 개발된 플라스미드(pUC18, pBAD, pIDTSAMRT-AMP 등), 대장균 유래 플라스미드(pYG601BR322, pBR325, pUC118, pUC119 등), 바실러스 서브틸리스 유래 플라스미드(pUB110, pTP5 등), 효모-유래 플라스미드(YEp13, YEp24, YCp50 등), 파아지 DNA(Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP 등), 동물 바이러스 벡터(레트로바이러스(retrovirus), 아데노바이러스(adenovirus), 백시니아 바이러스(vaccinia virus) 등), 곤충 바이러스 벡터(배큘로바이러스(baculovirus) 등)가 될 수 있다. 상기 발현벡터는 숙주세포에 따라서 단백질의 발현량과 수식 등이 다르게 나타나므로, 목적에 가장 적합한 숙주세포를 선택하여 사용함이 바람직하다.
In the present invention, the expression vector may include a nucleotide sequence encoding the skin permeable peptide of the present invention, a derivative thereof, or a fusion protein comprising the same, wherein the vector used herein is a skin permeable peptide of the present invention (PUC18, pBAD, pIDTSAMRT-AMP, or the like), as long as it is capable of producing a fusion protein comprising the same or a derivative thereof or a fusion protein comprising the same, but may be preferably plasmid DNA, phage DNA, Derived plasmids (pEG601BR322, pBR325, pUC118 and pUC119), Bacillus subtilis plasmids (pUB110 and pTP5), yeast-derived plasmids (YEp13, YEp24 and YCp50), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, lambda gt10, lambda gt11, lambda ZAP), animal virus vectors (retrovirus, adenovirus, There may be a virus (vaccinia virus), etc.), insect viral vectors (viruses (baculovirus as baculovirus), etc.). Since the amount of expression of the protein and the expression of the expression vector are different depending on the host cell, it is preferable to select and use the host cell most suitable for the purpose.
본 발명에서 제공하는 형질전환체는 상기 본 발명에서 제공하는 발현벡터를 숙주에 도입하여 형질전환시킴으로써 제작될 수 있고, 상기 발현벡터에 포함된 폴리뉴클레오티드를 발현시켜서, 본 발명의 피부투과성 펩타이드, 이의 유도체 또는 이를 포함하는 융합단백질을 생산하는데 사용될 수 있다. 상기 형질전환은 다양한 방법에 의하여 수행될 수 있는데, 본 발명의 피부투과성 펩타이드, 이의 유도체 또는 이를 포함하는 융합단백질을 생산할 수 있는 한, 특별히 이에 제한되지 않으나, CaCl2침전법, CaCl2침전법에 DMSO(dimethyl sulfoxide)라는 환원물질을 사용함으로써 효율을 높인 Hanahan 방법, 전기천공법(electroporation), 인산칼슘 침전법, 원형질 융합법, 실리콘 카바이드 섬유를 이용한 교반법, 아그로박테리아 매개된 형질전환법, PEG를 이용한 형질전환법, 덱스트란 설페이트, 리포펙타민 및 건조/억제 매개된 형질전환 방법 등이 사용될 수 있다. 또한, 상기 형질전환제의 제작에 사용되는 숙주 역시 본 발명의 피부투과성 펩타이드, 이의 유도체 또는 이를 포함하는 융합단백질을 생산할 수 있는 한, 특별히 이에 제한되지 않으나, 대장균(E. coli), 스트렙토마이세스, 살모넬라 티피뮤리움 등의 박테리아 세포; 사카로마이세스 세레비지애, 스키조사카로마이세스 폼베 등의 효모 세포; 피치아 파스토리스 등의 균류 세포; 드로조필라, 스포도프테라 Sf9 세포 등의 곤충 세포; CHO, COS, NSO, 293, 보우 멜라노마 세포 등의 동물 세포; 또는 식물 세포가 될 수 있다.
The transformant provided in the present invention can be produced by introducing the expression vector provided in the present invention into a host and transforming the polynucleotide contained in the expression vector to obtain a skin permeable peptide of the present invention Derivative or a fusion protein comprising the same. The transformation can be carried out by various methods. As long as it is capable of producing the skin permeable peptide of the present invention, a derivative thereof or a fusion protein comprising the same, the CaCl 2 precipitation method and the CaCl 2 precipitation method The method of Hanahan method, which employs a reducing material called DMSO (dimethyl sulfoxide), electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fibers, agrobacterium mediated transformation, PEG , Dextran sulfate, lipofectamine, and dry / inhibition-mediated transformation methods. In addition, the host used in the production of the transformant is not particularly limited as long as it can produce the skin permeable peptide of the present invention, a derivative thereof, or a fusion protein comprising the same. However, the host may be E. coli, , Bacterial cells such as Salmonella typhimurium; Yeast cells such as Saccharomyces cerevisiae, and ski-inspected caromyces pombe; Fungal cells such as Pichia pastoris; Insect cells such as Drosophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NSO, 293, Bowmanella cells; Or plant cells.
상기 형질전환체는 또한 본 발명의 피부투과성 펩타이드, 이의 유도체 또는 융합단백질을 생산하는 방법에 사용될 수 있다. 구체적으로, 본 발명의 피부투과성 펩타이드, 이의 유도체 또는 융합단백질을 생산하는 방법은 (a) 상기 형질전환체를 배양하여 배양물을 수득하는 단계; 및 (b) 상기 배양물로부터 본 발명의 본 발명의 피부투과성 펩타이드, 이의 유도체 또는 융합단백질을 회수하는 단계를 포함할 수 있다.
The transformants may also be used in methods of producing the skin permeable peptides, derivatives or fusion proteins of the invention. Specifically, the method for producing the skin permeable peptide, derivative or fusion protein of the present invention comprises the steps of: (a) culturing the transformant to obtain a culture; And (b) recovering the skin permeable peptide, derivative or fusion protein of the present invention of the present invention from the culture.
본 발명에서 "배양"이란, 미생물을 적당히 인공적으로 조절한 환경조건에서 생육시키는 방법을 의미한다. 본 발명에 있어서, 상기 형질전환체를 배양하는 방법은 당업계에 널리 알려져 있는 방법을 이용하여 수행할 수 있다. 구체적으로 상기 배양은 본 발명의 피부투과성 펩타이드, 이의 유도체 또는 융합단백질을 발현시켜서 생산할 수 있는 한 특별히 이에 제한되지 않으나, 배치 공정 또는 주입 배치 또는 반복 주입 배치 공정(fed batch or repeated fed batch process)에서 연속식으로 배양할 수 있다.In the present invention, the term "cultivation" means a method of growing the microorganism under an appropriately artificially controlled environmental condition. In the present invention, the method for culturing the transformant may be carried out by a method well known in the art. Specifically, the culturing may be performed in a batch process or an injection batch or a repeated fed batch process, as long as it can produce the skin permeable peptide of the present invention, a derivative thereof or a fusion protein thereof, It can be cultured continuously.
배양에 사용되는 배지는 적당한 탄소원, 질소원, 아미노산, 비타민 등을 함유한 통상의 배지 내에서 호기성 조건 하에서 온도, pH 등을 조절하면서 적절한 방식으로 특정 균주의 요건을 충족해야 한다. 사용될 수 있는 탄소원으로는 글루코즈 및 자일로즈의 혼합당을 주 탄소원으로 사용하며 이외에 수크로즈, 락토즈, 프락토즈, 말토즈, 전분, 셀룰로즈와 같은 당 및 탄수화물, 대두유, 해바라기유, 피마자유, 코코넛유 등과 같은 오일 및 지방, 팔미트산, 스테아린산, 리놀레산과 같은 지방산, 글리세롤, 에탄올과 같은 알코올, 아세트산과 같은 유기산이 포함된다. 이들 물질은 개별적으로 또는 혼합물로서 사용될 수 있다. 사용될 수 있는 질소원으로는 암모니아, 황산암모늄, 염화암모늄, 초산암모늄, 인산암모늄, 탄산안모늄, 및 질산암모늄과 같은 무기질소원; 글루탐산, 메티오닌, 글루타민과 같은 아미노산 및 펩톤, NZ-아민, 육류 추출물, 효모 추출물, 맥아 추출물, 옥수수 침지액, 카세인 가수분해물, 어류 또는 그의 분해생성물, 탈지 대두 케이크 또는 그의 분해생성물 등 유기질소원이 사용될 수 있다. 이들 질소원은 단독 또는 조합되어 사용될 수 있다. 상기 배지에는 인원으로서 인산 제1칼륨, 인산 제2칼륨 및 대응되는 소듐-함유 염이 포함될 수 있다. 사용될 수 있는 인원으로는 인산이수소칼륨 또는 인산수소이칼륨 또는 상응하는 나트륨-함유 염이 포함된다. 또한, 무기화합물로는 염화나트륨, 염화칼슘, 염화철, 황산마그네슘, 황산철, 황산망간 및 탄산칼슘 등이 사용될 수 있다. 마지막으로, 상기 물질에 더하여 아미노산 및 비타민과 같은 필수 성장 물질이 사용될 수 있다. The medium used for the culture should meet the requirements of the specific strain in a suitable manner while controlling the temperature, pH and the like under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, amino acid, vitamin, and the like. The carbon sources that can be used include glucose and xylose mixed sugar as main carbon sources, and sugar and carbohydrates such as sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, Oils and fats such as oils and the like, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Nitrogen sources that may be used include inorganic sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine and glutamine, and organic substances such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or their decomposition products, defatted soybean cake or decomposition products thereof . These nitrogen sources may be used alone or in combination. The medium may include potassium phosphate, potassium phosphate and the corresponding sodium-containing salts as a source. Potassium which may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to these materials, essential growth materials such as amino acids and vitamins can be used.
또한, 배양 배지에 적절한 전구체들이 사용될 수 있다. 상기된 원료들은 배양과정에서 배양물에 적절한 방식에 의해 회분식, 유가식 또는 연속식으로 첨가될 수 있으나, 특별히 이에 제한되지는 않는다. 수산화나트륨, 수산화칼륨, 암모니아와 같은 기초 화합물 또는 인산 또는 황산과 같은 산 화합물을 적절한 방식으로 사용하여 배양물의 pH를 조절할 수 있다.In addition, suitable precursors may be used in the culture medium. The above-mentioned raw materials can be added to the culture in the culture process in a batch manner, in an oil-feeding manner or in a continuous manner by an appropriate method, but it is not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to adjust the pH of the culture.
또한, 지방산 폴리글리콜 에스테르와 같은 소포제를 사용하여 기포 생성을 억제할 수 있다. 호기 상태를 유지하기 위해 배양물 내로 산소 또는 산소-함유 기체(예, 공기)를 주입한다. 배양물의 온도는 보통 27℃ 내지 37℃, 바람직하게는 30℃ 내지 35℃이다. 배양은 상기 본 발명의 피부투과성 펩타이드, 이의 유도체 또는 이를 포함하는 융합단백질의 생성량이 최대로 얻어질 때까지 계속한다. 이러한 목적으로 보통 10 내지 100 시간에서 달성된다.In addition, bubble formation can be suppressed by using a defoaming agent such as a fatty acid polyglycol ester. An oxygen or oxygen-containing gas (e.g., air) is injected into the culture to maintain aerobic conditions. The temperature of the culture is usually 27 ° C to 37 ° C, preferably 30 ° C to 35 ° C. The culture is continued until the amount of the skin permeable peptide of the present invention, its derivative, or the fusion protein containing the same is maximized. Usually for 10 to 100 hours for this purpose.
아울러, 배양물로부터 상기 본 발명의 피부투과성 펩타이드, 이의 유도체 또는 이를 포함하는 융합단백질을 회수하는 단계는 당업계에 공지된 방법에 의해 수행될 수 있다. 구체적으로, 상기 회수 방법은 생산된 본 발명의 피부투과성 펩타이드, 이의 유도체 또는 이를 포함하는 융합단백질을 회수할 수 있는 한, 특별히 이에 제한되지 않으나, 바람직하게는 원심분리, 여과, 추출, 분무, 건조, 증방, 침전, 결정화, 전기영동, 분별용해(예를 들면 암모늄 설페이트 침전), 크로마토그래피(예를 들면 이온 교환, 친화성, 소수성 및 크기배제) 등의 방법을 사용할 수 있다.
In addition, the step of recovering the skin permeable peptide of the present invention, a derivative thereof, or a fusion protein comprising the same may be carried out by a method known in the art. Specifically, the recovering method is not particularly limited as long as it is capable of recovering the skin permeable peptide of the present invention, a derivative thereof, or a fusion protein containing the same, but is preferably subjected to centrifugation, filtration, extraction, spraying, drying (For example, ammonium sulfate precipitation), chromatography (for example, ion exchange, affinity, hydrophobicity and size exclusion), and the like can be used.
또 하나의 양태로서, 본 발명은 상기 펩타이드 또는 이의 유도체를 포함하는 피부외용제용 담체 또는 피부외용제용 조성물을 제공한다. 또한 본 발명은 상기 융합단백질을 포함하는 피부외용제용 조성물을 제공한다.
In another aspect, the present invention provides a carrier for external preparation for skin or a composition for external preparation for skin comprising the peptide or a derivative thereof. The present invention also provides a composition for external application for skin comprising the fusion protein.
본 발명에서 "피부외용제"란, 유지류, 바셀린, 라놀린, 글리세롤 등의 다양한 기제에 약품을 혼합하여 쉽게 피부에 바를 수 있도록 한 고형, 반고형 또는 액상의 외용약을 의미한다. 외용 제형은 특별히 한정되지 않으나, 파우더, 젤, 연고, 크림, 액체 또는 에어로졸 제형이 바람직하다. The term "external preparation for skin " in the present invention means a solid, semi-solid or liquid external preparation which can be easily applied to skin by mixing drugs with various bases such as oils, petrolatum, lanolin and glycerol. Formulations for external use are not particularly limited, but powders, gels, ointments, creams, liquids or aerosol formulations are preferred.
본 발명의 목적상 상기 피부외용제는 본 발명의 펩타이드 또는 이의 유도체를 포함하고, 적절한 피부외용제용 담체인 기제를 포함하는 제제로 해석될 수 있으나, 특별히 이에 제한되지는 않는다.
For the purpose of the present invention, the external preparation for skin may be interpreted as a preparation containing the peptide of the present invention or a derivative thereof and a base which is a carrier for a suitable external preparation for skin, but is not particularly limited thereto.
한편, 본 발명의 피부외용제용 조성물은 약학적 조성물일 수 있다. Meanwhile, the composition for external application for skin of the present invention may be a pharmaceutical composition.
본 발명의 피부투과성 펩타이드, 이의 유도체 또는 이를 포함하는 융합단백질을 포함하고 추가적으로 피부를 통과하여 체내에 도달하려는 약물 또는 피부 자체의 기관에 전달하려는 약물이 포함될 수 있다. A skin permeable peptide of the present invention, a derivative thereof, or a fusion protein containing the fusion protein, and additionally a drug to reach the body through the skin or a drug to be delivered to an organ of the skin itself.
본 발명의 약학적 조성물은, 약학 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형체 또는 희석제를 추가로 포함할 수 있다. 이때, 상기 조성물에 포함되는 피부투과성 펩타이드, 이의 유도체 또는 이를 포함하는 융합단백질의 함량은 특별히 이에 제한되지 않으나, 조성물 총 중량에 대하여 0.01 중량% 내지 50.0 중량%로, 바람직하게는 5 중량% 내지 20 중량%로 포함될 수 있다. The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions. In this case, the content of the skin permeable peptide, its derivative, or the fusion protein comprising the skin permeable peptide contained in the composition is not particularly limited, but may be 0.01 to 50.0% by weight, preferably 5 to 20% % ≪ / RTI > by weight.
상기 약학적 조성물은 정제, 환제, 산제, 과립제, 캡슐제, 현탁제, 내용액제, 유제, 시럽제, 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결 건조제 및 좌제으로 이루어진 군으로부터 선택되는 어느 하나의 제형을 가질 수 있으며, 경구 또는 비경구의 여러 가지 제형일 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용된다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical composition may be any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, nonaqueous solvents, suspensions, emulsions, And may be oral or parenteral formulations of various forms. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.
본 발명의 조성물은 약학적으로 유효한 양으로 투여할 수 있다.The composition of the present invention may be administered in a pharmaceutically effective amount.
본 발명에서 용어, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 질병의 종류, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다. 본 발명의 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물 형태, 투여경로 및 기간에 따라 다르며, 적합한 총 1일 사용량은 올바른 의학적 판단범위 내에서 처치의에 의해 결정될 수 있으나, 일반적으로 0.001 내지 1000 mg/kg의 양, 바람직하게는 0.05 내지 200 mg/kg, 보다 바람직하게는 0.1 내지 100 mg/kg의 양을 일일 1회 내지 수회로 나누어 투여할 수 있다. 상기 조성물의 투여 대상개체는 특별히 한정되지 않는다. 예를 들면, 원숭이, 개, 고양이, 토끼, 모르모트, 랫트, 마우스, 소, 양, 돼지, 염소 등과 같은 비인간동물 및 인간 등 어느 개체에나 적용할 수 있으며, 투여의 방식은 당업계의 통상적인 방법이라면 제한없이 포함한다. 예를 들어, 국소도포 등을 통한 경피투여 방식을 사용할 수 있으나, 이에 제한되는 것은 아니다.
The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will vary depending on the species and severity, age, sex, The type of drug, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art. The preferred dosage of the composition of the present invention will depend on the condition and the weight of the patient, the severity of the disease, the type of drug, the route of administration and the period of time, and the appropriate total daily dose may be determined by treatment within the proper medical judgment, Generally, an amount of 0.001 to 1000 mg / kg, preferably 0.05 to 200 mg / kg, more preferably 0.1 to 100 mg / kg, can be administered in a single dose, divided into several times a day. The subject to which the composition is to be administered is not particularly limited. For example, it can be applied to any individual such as a monkey, a dog, a cat, a rabbit, a guinea pig, a rat, a mouse, a cattle, a pig, a goat, Including without limitation. For example, transdermal administration through topical application may be used, but is not limited thereto.
또 하나의 양태로서, 본 발명은 피부투과성 펩타이드, 이의 유도체 또는 상기 융합단백질을 포함하는 화장료 조성물 및 상기 조성물을 경피투여하는 단계를 포함하는, 피부 상태의 개선 방법을 제공한다.In another aspect, the present invention provides a skin condition improving method comprising the step of administering a skin permeable peptide, a derivative thereof, or a cosmetic composition comprising the fusion protein and the composition.
본 발명의 피부투과성 펩타이드, 이의 유도체 또는 융합단백질은 피부투과성 및 피부잔류성이 현저히 우수하므로, 피부를 개선시킬 수 있는 화장료 조성물에 포함될 수 있다.
The skin permeable peptide, derivative or fusion protein of the present invention is remarkably excellent in skin permeability and skin retention, and thus can be included in a cosmetic composition capable of improving skin.
본 발명에서 "피부개선"이란, 피부의 내재적 요인 또는 외인적인 요인에 의하여 유발되는 피부의 손상을 치료, 경감, 완화시키는 과정 또는 그의 효과 등을 포괄적으로 의미한다. 구체적인 예로는 주름개선, 피부 보습, 피부 미백, 피부 탄력 유지 및/또는 증진, 상처회복, 노화 억제, 피부염 완화 또는 개선 등의 효과를 의미하는 것으로 해석될 수 있다.In the present invention, "skin improvement" means a process of treating, alleviating, or alleviating damage to the skin caused by intrinsic or extrinsic factors of the skin or its effect. Specific examples can be interpreted to mean the effects of wrinkle improvement, skin moisturization, skin whitening, skin elasticity maintenance and / or enhancement, wound restoration, aging inhibition, dermatitis relief or improvement.
상기 피부투과성 펩타이드, 이의 유도체 또는 이를 포함하는 융합단백질은 전체 화장료 조성물의 중량 대비 0.0001 내지 50중량%로 포함되는 것이 바람직하다. 상기 피부투과성 펩타이드, 이의 유도체 또는 이를 포함하는 융합단백질의 함량이 전체 화장료 조성물의 중량 대비 0.0001중량% 미만일 경우에는 실질적인 피부개선 효과를 기대하기 어렵고, 50중량% 이상일 경우에는 제형이 불안정해지는 등의 문제가 발생할 수 있다.Preferably, the skin permeable peptide, derivative thereof, or fusion protein comprising the same is contained in an amount of 0.0001 to 50% by weight relative to the total weight of the cosmetic composition. If the content of the skin permeable peptide, derivative thereof or a fusion protein thereof is less than 0.0001% by weight of the total cosmetic composition, it is difficult to expect substantial skin improvement effect. If the content is more than 50% by weight, the formulation becomes unstable May occur.
본 발명의 화장료 조성물은 화장 분야에서 통상적인 보조제 예를 들어, 지방 물질, 유기용매, 용해제, 농축제, 겔화제, 연화제, 항산화제, 현탁화제, 안정화제, 발포제(foaming agent), 방향제, 계면활성제, 물, 이온형 또는 비이온형 유화제, 충전제, 금속이온봉쇄제, 킬레이트화제, 보존제, 비타민, 차단제, 습윤화제, 필수 오일, 염료, 안료, 친수성 또는 친유성 활성제, 지질 소낭 뿐만 아니라 화장품에 통상적으로 사용되는 임의의 다른 성분과 같은 화장품학 또는 피부과학 분야에서 통상적으로 사용되는 보조제와 함께 일반 화장품 또는 기능성 화장품의 구성요소로서 사용될 수 있다.
The cosmetic composition of the present invention can be used as an adjuvant commonly used in the cosmetics field, for example, a lipid material, an organic solvent, a solubilizing agent, a thickening agent, a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, It is also possible to use the active agent, water, ionic or nonionic emulsifier, filler, sequestering agent, chelating agent, preservative, vitamin, blocking agent, wetting agent, essential oil, dye, pigment, hydrophilic or lipophilic active agent, And may be used as a component of general cosmetics or functional cosmetics together with adjuvants commonly used in cosmetics or dermatology, such as any other ingredient commonly used.
본 발명은 상기 화장료 조성물을 포함하는 기능성 화장품을 제공한다.
The present invention provides a functional cosmetic comprising the cosmetic composition described above.
상기 "기능성 화장품(cosmedical, cosmeceutical)"이란 화장품에 의약품의 전문적인 치료기능이 도입되어, 생리활성적인 효능, 효과가 강조된 전문적인 기능성을 갖는 제품으로서, 피부의 미백에 도움을 주는 제품, 피부 주름개선에 도움을 주는 제품, 피부를 곱게 태우거나 자외선으로부터 피부를 보호하는데 도움을 주는 화장품 등을 포함한다.
The above-mentioned "cosmedical, cosmeceutical" refers to a product that has professional functions that emphasize physiologically active effects and effects by introducing professional therapeutic functions of medicines into cosmetics, products that help to whiten skin, Products that help improve the appearance of the skin, cosmetics that help to protect the skin from ultraviolet light.
본 발명의 기능성 화장품은 콜라겐, 엘라스틴, 라미닌, HAS2(hyaluronan synthase 2) 등의 기능성 물질의 합성 촉진효과 등을 통하여 피부개선 효과를 나타낼 수 있다. 그의 제형은 특별히 제한되는 것은 아니나, 예를 들면, 용액, 유탁액, 현탁액, 페이스트, 크림, 로션, 겔, 파우더, 스프레이, 계면활성제-함유 클린징, 오일, 비누, 액체 세정료, 입욕제, 파운데이션, 메이크업베이스, 에센스, 화장수, 폼, 팩, 유연수, 선 스크린 크림, 선오일 등의 제형으로 제조될 수 있고, 바람직하게는 피부외용연고, 유연화장수, 영양화장수, 영양크림, 마사지크림, 에센스, 팩, 에멀젼 또는 오일젤의 제형으로 제조될 수 있는데, 이때, 사용되는 담체는 화장품의 제형에 따라 선택적으로 사용될 수 있다.
The functional cosmetic composition of the present invention can exhibit the skin improving effect by promoting synthesis of functional materials such as collagen, elastin, laminin, and hyaluronan synthase 2 (HAS2). The formulation may be, for example, but not limited to, solutions, emulsions, suspensions, pastes, creams, lotions, gels, powders, sprays, surfactant-containing cleansing oils, soaps, liquid cleansing agents, The composition may be formulated as a make-up base, an essence, a lotion, a foam, a pack, a soft water, a sunscreen cream, a line oil and the like, preferably an external skin ointment, a softening longevity, , Emulsions or oil gels, wherein the carrier used can be optionally used according to the formulation of the cosmetic.
또 하나의 양태로서, 본 발명은 상기 펩타이드 또는 이의 유도체를 이용하여 약물의 피부투과를 촉진시키는 방법을 제공한다.
In another embodiment, the present invention provides a method for promoting skin permeation of a drug using the peptide or a derivative thereof.
구체적으로, 본 발명의 약물의 피부투과를 촉진시키는 방법은 (ⅰ) 목적하는 약물 또는 물질을 상기 펩타이드 또는 이의 유도체에 결합시켜 결합체를 형성하는 단계; 및 (ⅱ) 상기 결합체를 포함하는 약학적 조성물을 피부에 도포하는 단계를 포함할 수 있다.Specifically, a method for promoting skin permeation of a drug of the present invention comprises the steps of (i) binding a desired drug or substance to the peptide or a derivative thereof to form a conjugate; And (ii) applying the pharmaceutical composition comprising the conjugate to the skin.
이때, 상기 약물은 상기 펩타이드 또는 이의 유도체와 직접적으로 또는 링커 등을 이용하여 간접적으로 결합할 수 있으면서도, 그의 원천적인 활성이 저하되지 않는 모든 약물을 사용할 수 있고, 상기 물질은 생리활성 단백질과 같은 물질일 수 있으나, 이에 제한되지 않는다. 상기 결합체는 상기에서 설명한 융합단백질을 포함할 수 있다.
At this time, the drug can be indirectly bound to the peptide or its derivative directly or by using a linker, and all the drugs that do not deteriorate its original activity can be used, and the substance can be used as a substance such as a physiologically active protein But is not limited thereto. The conjugate may comprise a fusion protein as described above.
본 발명의 피부투과성 펩타이드 또는 이의 유도체는 우수한 피부 침투활성뿐만 아니라 우수한 피부 잔류 효과를 나타내고, 여기에 생리활성 단백질을 결합하여 제조한 융합단백질 또한 생리활성 효과를 나타내는 물질의 합성능이 유지 또는 향상되면서도, 피부투과성과 피부잔류성을 현저하게 향상시키므로, 상기 피부투과성 펩타이드, 이의 유도체 또는 이를 포함하는 융합단백질은 피부조직을 표적조직으로 하는 기능성 화장료 조성물 및 피부외용제용 약학적 조성물의 유효성분으로 널리 활용될 수 있다.
The skin permeable peptide or derivative thereof of the present invention exhibits excellent skin penetration activity as well as excellent skin retention effect and the fusion protein prepared by binding a physiologically active protein thereto also maintains or improves the ability to synthesize a substance exhibiting a physiological activity effect, The skin permeability peptide and the derivative thereof or the fusion protein comprising the peptide can be widely used as an effective ingredient of a functional cosmetic composition having a skin tissue as a target tissue and a pharmaceutical composition for external application for skin have.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
실시예 1: 피부투과성 펩타이드의 선발Example 1: Screening of skin permeable peptides
피부투과성 펩타이드를 선별하기 위해서 경피제제의 용출 실험 방법을 응용하였다. 이를 위해 Franz glass cell(Standard 직경 9mm, Receiver 5㎖, Permgear)을 이용하였다. Glass cell의 상단과 하단 사이에 돼지 피부(0.7mm 두께, Medikinetics)를 장착하고 상단에 파지를 처리한 뒤, 돼지피부를 투과하여 하단의 수용부(Receiver)에 잔류하는 파지를 증폭하였다. 이 과정을 1회 진행하는 것을 1 라운드로 선별한 것으로 정의하였다. 1 라운드에서 증폭된 파지를 가지고 다시 2 라운드를 진행하는 방식으로 피부 투과력이 좋은 파지를 경쟁하는 방식으로 선별하였고 총 3 라운드를 수행하였다. To elucidate skin permeable peptides, the dissolution test method of transdermal preparation was applied. For this purpose, a Franz glass cell (Standard diameter 9 mm, Receiver 5 ml, Permgear) was used. The pig skin (0.7 mm in thickness, Medikinetics) was placed between the top and bottom of the glass cell, and the upper part was treated with phage, and the pig skin was permeated to amplify the remaining phage in the lower receiver. This process was defined as one round of selection. In the second round with the phage amplified in the first round, the phages with good permeability of the skin were selected in such a manner as to compete with each other, and a total of three rounds were performed.
피부와 파지의 코팅(coating) 단백질 사이에 생길 수 있는 비특이적인 결합을 줄이기 위하여, 상단과 하단에는 각각 500㎕와 5㎖의 TBS(50mM Tris pH 7.5, 150mM NaCl)용액에 1% BSA(Sigma)를 용해시켜서 사용하였다.In order to reduce the non-specific binding between the skin and the coating of the phage, 500 μl and 5 ml of TBS (50 mM Tris pH 7.5, 150 mM NaCl) Was used.
무작위적인 펩타이드 서열을 가지는 파지를 가지고 시작하기 위해서, Ph.D-12 phage library kit(New England Biolab)를 사용하였다. 500㎕의 TBS(1% BSA)에 109개의 파지를 처리하였다. 이후 16시간동안 배양하였고, 상기 수용부의 TBS 5㎖를 모두 사용하여 파지를 증폭하였다. To start with a phage having a random peptide sequence, a Ph.D-12 phage library kit (New England Biolab) was used. 10 < 9 > phages were treated with 500 [mu] l of TBS (1% BSA). Thereafter, the cells were cultured for 16 hours, and the phage was amplified using 5 ml of TBS in the above-mentioned receiving portion.
파지를 증폭하기 위한 숙주세포로서 E.coli ER2738(New England Biolab)를 이용하였다. 25㎖의 LB배지에 진탕 배양된 ER2738를 접종하고 O.D.(550nm) 0.5에서 투과한 파지가 있는 TBS 5㎖를 모두 처리하고 4시간 배양하였다. 이후 8000G로 원심 분리하여 E.coli와 파지를 분리하였다. 파지가 있는 상층액에 6㎖의 침전액(20% PEG6000, 2.5M NaCl)을 처리하여 파지를 침전시켰다. 8000G로 원심 분리후 침전된 파지에 TBS용액을 처리하여 파지를 분리하여 보관하였다. E. coli ER2738 (New England Biolab) was used as a host cell to amplify the phage. 25 mL of LB medium was inoculated with shake cultured ER2738, and 5 mL of phage-containing TBS permeated at OD (550 nm) 0.5 was treated and cultured for 4 hours. After centrifugation at 8000G, E. coli and phage were separated. The phage-containing supernatant was treated with 6 ml of sediment (20% PEG6000, 2.5M NaCl) to precipitate the phage. After centrifugation at 8000G, the precipitated phages were treated with TBS solution to separate the phage.
최종적으로, 피부투과성 파지가 가지는 펩타이드를 확인하기 위해서 파지를 단일 콜로니에서 증폭시켰다. 파지를 가지는 ER2738 E.coli는 LB/X-gal/IPTG 플레이트에서 파란색을 띄는 성질을 이용하였다. ER2738 배양액에 300㎕에 파지를 TBS로 적정량 희석하고 2㎕를 처리한 뒤, 이를 TOP agar 4㎖에 섞은 후 LB/X-gal/IPTG plate 상단에 처리하고 16시간 추가 배양한 뒤 청색의 콜로니를 선별하였다. 각각의 콜로니를 5㎖의 ER2738 배양액에 6시간동안 추가로 배양하고, Ph.D-13 phage spin kit(Qiagen)을 이용하여 DNA를 분리한 다음, 이들의 염기서열을 분석함으로써, 1종의 피부투과성 펩타이드(서열번호 1)를 선별하였다.
Finally, the phage were amplified in a single colony to identify peptides with a skin permeable phage. ER2738 E. coli with phage exploited the blue character in LB / X-gal / IPTG plates. To the ER2738 culture medium, 300 μl of the phage was diluted with TBS to an appropriate volume, treated with 2 μl, mixed with 4 ml of TOP agar, treated on the top of LB / X-gal / IPTG plate and further cultured for 16 hours. Respectively. Each of the colonies was further cultured in 5 ml of ER2738 culture medium for 6 hours, DNA was isolated using a Ph.D-13 phage spin kit (Qiagen), and the nucleotide sequences of these colonies were analyzed, The permeable peptide (SEQ ID NO: 1) was selected.
실시예 2: 피부투과성 펩타이드를 포함하는 파지의 피부투과성 확인Example 2: Confirmation of skin permeability of phage containing skin permeable peptide
상기 실시예 1에서 선발된 서열번호 1의 아미노산 서열을 가지는 각각의 펩타이드를 발현시킬 수 있는 파지들의 피부투과성을 확인하였다.
The skin permeability of the phages capable of expressing each of the peptides having the amino acid sequence of SEQ ID NO: 1 selected in Example 1 was confirmed.
구체적으로, Franz glass cell의 상단과 하단 사이에 돼지 피부를 장착하고, 피부와 파지의 코팅(coating) 단백질 사이에 생길 수 있는 비특이적인 결합을 줄이기 위하여, 상단과 하단에는 각각 500㎕와 5㎖의 TBS용액에 1% BSA를 용해시켜서 사용하였다. 처리하는 파지의 수를 1010 개로 일정하게 맞추어서 상기 Franz glass cell의 상단에 처리하였다. 이때, 피부를 투과하지 않고 Franz cell 상단에 잔류하는 phage를 무작위 추출하여 그 펩타이스 서열을 대조군으로 사용하였는데, 본 발명에서의 대조군으로는 "TDMNKTEIRFVR" (서열번호 3) 펩타이드 서열을 발현시킬 수 있는 파지를 이용하였다. 6시간동안 반응시킨 후에 투과하여 수용부에 있는 파지의 수를 측정하였다. 이를 위해 ER2738 배양액 300㎕ 및 파지를 포함하는 수용부의 TBS 10㎕를 혼합하고, 이를 TOP agar 4㎖에 섞은 후, LB/X-gal/IPTG plate 상단에 처리하고, 16시간 동안 추가로 배양한 다음, 청색 콜로니의 수를 계수하였다(표 1). Specifically, pig skin was placed between the upper and lower ends of the Franz glass cell, and 500 μl and 5 ml of the supernatant were added at the top and bottom, respectively, to reduce nonspecific binding between the skin and the coating protein of the phage 1% BSA was dissolved in TBS solution and used. The number of treated phages was fixed to 10 10 and the top of the Franz glass cell was treated. At this time, the phage remaining on the upper part of the Franz cell without permeation of the skin was randomly extracted and used as a control. As a control group in the present invention, a peptide sequence "TDMNKTEIRFVR" (SEQ ID NO: 3) Phage were used. After 6 hours of reaction, permeation was measured to determine the number of phage in the receiving portion. For this, 300 μl of the ER 2738 culture medium and 10 μl of TBS in the receiver containing the phage were mixed and mixed with 4 ml of TOP agar, treated on the top of LB / X-gal / IPTG plate and further cultured for 16 hours , And blue colonies were counted (Table 1).
서열번호 1 발현가능한 파지Control phage
SEQ ID NO: 1 Expressible fingers
1760011
17600
상기 표 1에서 보듯이, 상기 선발된 1종의 펩타이드는 모두 대조군보다도 우수한 피부투과성을 나타냄을 확인할 수 있었다.
As shown in Table 1, it was confirmed that all of the selected peptides showed better skin permeability than the control group.
실시예 3: 피부투과성 펩타이드 유도체의 피부투과성 확인Example 3: Determination of skin permeability of skin permeable peptide derivative
상기 실시예 1에서 선발된 서열번호 1의 아미노산 서열을 가지는 각각의 펩타이드의 C 말단에 단실(Dansyl)기가 결합된 형태의 유도체를 각각 합성하고, 이들 유도체의 피부투과성을 확인하고자 하였다.
Dansyl group-binding derivatives were synthesized at the C-terminus of each of the peptides having the amino acid sequence of SEQ ID NO: 1 selected in Example 1, and the skin permeability of these derivatives was examined.
구체적으로, 상기 각각의 펩타이드들을 화학적으로 합성하고, C-말단에 Dansyl기를 추가한 유도체를 각각 합성하였으며, 대조군으로는 C-말단에 Dansyl기를 추가한 "TDMNKTEIRFVR(서열번호 3)"의 아미노산 서열로 구성된 펩타이드를 사용하였다. 각각 펩타이드의 합성은 펩트론에 의뢰하여 제작하였다. 1회 실험에 약 40㎍의 펩타이드를 사용하였는데 대조군과 실험군 펩타이드의 형광세기를 측정하여 동일한 형광세기를 나타내는 펩타이드를 처리하였다. Specifically, each of the peptides was synthesized chemically, and a derivative to which a Dansyl group was added at the C-terminus was synthesized. As a control group, an amino acid sequence of "TDMNKTEIRFVR (SEQ ID NO: 3)" in which a Dansyl group was added at the C- The constructed peptides were used. The synthesis of each peptide was made with reference to a peptide. In one experiment, about 40 μg of the peptide was used. The fluorescence intensities of the control and experimental peptides were measured, and the peptides exhibiting the same fluorescence intensity were treated.
그런 다음, Franz glass cell의 상단과 하단 사이에 돼지 피부를 장착하고, 상기 cell의 상단과 하단에 각각 500㎕와 5㎖의 TBS용액을 가한 다음, 상단에 각각의 유도체를 동일한 농도로 가하면서, 16시간 동안 배양하였다. 배양이 종료된 후, 하단의 수용부에 존재하는 각 유도체의 양을 victor fluorescence meter(340/520)로 측정하여 투과정도를 비교하였다(표 2).Then, pig skin was placed between the upper and lower ends of the Franz glass cell, and 500 μl and 5 ml of TBS solution were added to the upper and lower ends of the cell, respectively. Then, And cultured for 16 hours. After the culture was completed, the amount of each derivative present in the lower receptacle was measured with a victor fluorescence meter (340/520), and the degree of permeation was compared (Table 2).
서열번호 1 유도체 발현가능한 파지Control phage
A phage capable of expressing the SEQ ID NO: 1 derivative
352001280
35200
상기 표 2에서 보듯이, 상기 합성된 펩타이드 유도체는 대조군보다 우수한 피부투과성을 나타냄을 확인할 수 있었다.
As shown in Table 2, it was confirmed that the synthesized peptide derivatives exhibited better skin permeability than the control group.
실시예 4: 피부투과성 펩타이드를 포함하는 파지의 피부잔류성 확인Example 4: Determination of skin persistence of phage containing skin permeable peptide
약물 등이 피부를 투과하게 되는 주요 메커니즘은 피부의 성분에 용해되어서, 최상층에서 하층으로 확산되는 방식으로 진행되는 것이다. 이를 통하여 피부 전체에 유효물질이 효과적으로 전달될 수 있다. 피부의 표면은 각질과 각질간극 물질 등으로 이루어져 있고, 선별된 펩타이드가 피부를 잘 투과하기 위하여서는 피부 표면을 이루는 성분에 대한 용해성이 좋아야 한다. 피부성분에 대한 용해성은, 짧은 시간(10분) 피부와 반응 후 결합된 양을 측정하여 분석할 수 있다. 또한 wash-off 제품에 사용될 경우 피부와의 결합력이 좋아야 한다. 이 경우, 먼저 피부와의 결합 후 내부로 확산되게 된다. 따라서 선별된 파지의 피부 결합성을 측정하여 펩타이드의 우수성을 확인하고자 하였다.
The main mechanism by which drugs and the like are permeated through the skin is dissolving in the constituents of the skin and proceeds in such a way as to diffuse from the uppermost layer to the lower layer. Whereby the effective substance can be effectively delivered to the entire skin. The surface of the skin consists of keratin and keratin materials, and the selected peptides must have good solubility in the ingredients of the skin surface in order to penetrate the skin well. Solubility for skin components can be determined by measuring the combined amount after reaction with the skin for a short time (10 minutes). In addition, when used in wash-off products, it should have good adhesion to the skin. In this case, it first diffuses into the inside after bonding with the skin. Therefore, we tried to confirm the superiority of peptides by measuring the skin binding of the selected phages.
이를 위해, 먼저 돼지 피부를 10mm × 10mm 크기로 절단하고, 상기 실시예 1에서 선별한 각각의 펩타이드를 발현시킬 수 있는 파지를 가한 다음, 상기 돼지 피부에 대한 결합정도를 측정하였다. 이때, 대조군으로는 "TDMNKTEIRFVR(서열번호 3)" 서열을 발현시키는 파지를 이용하였다. To this end, pig skin was cut into a size of 10 mm x 10 mm, and a phage capable of expressing each of the peptides selected in Example 1 was added. Then, the degree of binding to the pig skin was measured. At this time, as a control group, a phage expressing the "TDMNKTEIRFVR (SEQ ID NO: 3)" sequence was used.
상기 돼지 피부에 결합한 파지의 양을 측정하기 위하여, anti-M13 antibody HRP conjugate를 이용하였다. Antibody 자체와 피부와의 결합에 의한 시그널을 제거하기 위해서 파지가 없는 대조군을 음성대조군으로 설정하였다. 절단한 돼지 피부를 1.5㎖ 튜브에 넣고 500㎕의 TBS(1% BSA)를 가한 다음, 상기 음성대조군의 경우 파지를 넣지 않고, 대조군과 측정하고자 하는 파지의 경우, 1010개의 파지를 가하여 10분간 배양하였다. 그런 다음, 1% BSA를 포함하는 TBS용액을 이용하여 12,000 RPM에서 1분간 10회 세척하였다. 이어, anti-M13 antibody HRP(Horseradish peroxidase) conjugate가 포함된 1% BSA를 포함하는 TBS용액 500㎕를 가하고, 5분간 추가로 배양하였다. 상기 배양물에 1% BSA를 포함하는 TBS용액을 가하고, 12,000 RPM에서 1분간 10회 세척한 다음, 잔류하는 완충액을 모두 제거하였으며, TMB(Tetramethylbenzidine) solution(Amersham)을 추가로 가하였다. 이때, 상기 TMB는 항체에 결합된 HRP에 의해서 분해되어 파란색을 발색시킨다. 최종적으로, 0.2N 의 황산을 처리하여 반응을 정지시키고, 파란색을 노란색으로 변화시킨 다음, 흡광도(450nm)를 측정하여 파지-피부간 결합정도를 측정하였다. 각각의 흡광도에서 음성 대조군의 흡광도를 삭제하여 산출한 각각의 활성을 상호 비교하였다(표 3).To measure the amount of phage bound to the porcine skin, anti-M13 antibody HRP conjugate was used. A control group without a phage was set as a negative control group in order to remove the signal due to binding of the antibiotic itself and the skin. The cut pig skin was placed in a 1.5 ml tube and 500 μl of TBS (1% BSA) was added. In the case of the control group and the phage to be measured, 10 10 pieces of phage were added for 10 minutes Lt; / RTI > It was then washed 10 times for 1 minute at 12,000 RPM with TBS solution containing 1% BSA. Then, 500 T of TBS solution containing 1% BSA containing anti-M13 antibody HRP (Horseradish peroxidase) conjugate was added, followed by further incubation for 5 minutes. To the culture, TBS solution containing 1% BSA was added, and the mixture was washed 10 times at 12,000 rpm for 1 minute. Then, the remaining buffer solution was removed, and TMB (Tetramethylbenzidine) solution (Amersham) was further added. At this time, the TMB is degraded by the HRP bound to the antibody to develop blue color. Finally, the reaction was stopped by treating with 0.2 N sulfuric acid, blue was changed to yellow, and absorbance (450 nm) was measured to measure the degree of binding between the phage and the skin. The absorbances of the negative control were subtracted from each absorbance, and the respective activities were compared with each other (Table 3).
대조군 파지
서열번호 1 발현가능한 파지Negative control group
Control phage
SEQ ID NO: 1 Expressible fingers
0.06
0.690.01
0.06
0.69
상기 표 3에서 보듯이, 피부에 잔류하는 각 펩타이드의 수준은 모든 펩타이드가 대조군보다도 현저히 높은 수준을 나타냄을 확인할 수 있었다.
As shown in Table 3, it was confirmed that the level of each peptide remaining on the skin was significantly higher than that of the control group in all the peptides.
실시예 5: 피부투과성 펩타이드 유도체의 피부잔류성 확인Example 5: Skin retention of skin permeable peptide derivatives
상기 실시예 3에서 합성된 각 유도체들의 피부 결합성을 확인하기 위해서, 돼지 피부와 상기 유도체를 반응시키고, 표면에 용해된 펩타이드의 양을 측정하였다. 구체적으로, 잘라낸 돼지 피부를 1.5㎖ 튜브에 넣고 500㎕의 TBS(1% BSA)를 넣었다. 여기에 대조군과 측정하고자 하는 각 유도체를 추가하여 피부와 함께 10분간 배양하였다. 대조군으로는 "TDMNKTEIRFVR(서열번호 3)" 펩타이드를 이용하였다. 각각 펩타이드의 합성은 펩트론에 의뢰하여 제작하였다. 1회 실험에 약 40㎍의 펩타이드를 사용하였는데 대조군과 실험군 펩타이드의 형광세기를 측정하여 동일한 형광세기를 나타내는 펩타이드를 처리 하였다. 배양 이후 TBS(1% BSA)를 이용하여 12,000 RPM에서 1분간 10회 세척하고, 상기 반응이 종료된 돼지 피부를 24웰 플레이트에 옮긴 다음, 표면에 잔류하는 펩타이드의 양을 victor fluorescence meter(340/520)로 측정하여 비교하였다(표 4). In order to confirm skin adhesion of each derivative synthesized in Example 3, pig skin and the derivative were reacted and the amount of peptide dissolved on the surface was measured. Specifically, cut sliced pig skin was placed in a 1.5 ml tube and 500 占 퐇 of TBS (1% BSA) was added. The control and each derivative to be measured were added and incubated with the skin for 10 minutes. As a control group, "TDMNKTEIRFVR (SEQ ID NO: 3)" peptide was used. The synthesis of each peptide was made with reference to a peptide. In one experiment, about 40 μg of the peptide was used. The fluorescence intensities of the control and experimental peptides were measured, and the peptides exhibiting the same fluorescence intensity were treated. After incubation, the cells were washed 10 times with TBS (1% BSA) at 12,000 rpm for 1 minute. The pig skin was transferred to a 24-well plate and the amount of peptide remaining on the surface was measured using a victor fluorescence meter 520) (Table 4).
서열번호 1 유도체 발현가능한 파지Control group
A phage capable of expressing the SEQ ID NO: 1 derivative
3570004000
357000
상기 표 4에서 보듯이, 피부에 잔류하는 각 펩타이드 유도체의 수준은 모든 펩타이드 유도체가 대조군보다도 현저히 높은 수준을 나타냄을 확인할 수 있었다.
As shown in Table 4, it was confirmed that the level of each peptide derivative remaining on the skin was significantly higher than that of the control peptide.
실시예 6: 피부투과 촉진 펩타이드가 상피세포 성장인자(EGF)에 결합된 융합단백질의 제조Example 6: Preparation of fusion protein in which skin permeation-promoting peptide is bound to epithelial growth factor (EGF)
상기 실시예 1에서 수득한 서열번호 1의 아미노산 서열을 가지는 피부투과 촉진용 펩타이드의 C-말단과 서열번호 4의 아미노산 서열을 가지는 상피세포 성장인자(EGF)의 N-말단이 두 개의 아미노산(GG)으로 구성된 링커를 매개로 연결된 형태의 융합단백질인 T4-EGF(서열번호 5)를 생산하였다.
The N-terminal of the epithelial growth factor (EGF) having the amino acid sequence of SEQ ID NO: 4 and the C-terminal of the peptide for promoting skin permeation having the amino acid sequence of SEQ ID NO: 1 obtained in Example 1 was replaced with two amino acids ) To produce T4-EGF (SEQ ID NO: 5), a fusion protein in the form of a linker.
구체적으로, 상기 서열번호 1의 아미노산 서열을 코딩하는 폴리뉴클레오티드와 서열번호 4의 아미노산 서열을 코딩하는 폴리뉴클레오티드를 각각 합성하고, 두 개의 아미노산(GG)을 코딩하는 뉴클레오티드 서열을 중심으로 연결시켜서, 상기 서열번호 5의 아미노산 서열을 코딩하는 폴리뉴클레오티드를 제작하였다. 한편, N-말단의 정확한 가공을 위해서 엔테로키나제(Enterokinase)에 의해 절단될 수 있는 절단부위인 DDDDK(Asp-Asp-Asp-Asp-Lys)를 코딩하는 폴리뉴클레오티드를 합성하고, 이를 상기 제작된 서열번호 5의 아미노산 서열을 코딩하는 폴리뉴클레오티드의 5'-말단에 연결하여, 엔테로키나제 절단부위-피부투과성 펩타이드-EGF로 구성된 융합단백질을 코딩하는 최종 폴리뉴클레오티드를 수득하였으며, 상기 수득한 폴리뉴클레오티드를 GST 발현 벡터에 도입하여 발현벡터를 제작하였다. Specifically, a polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 and a polynucleotide encoding the amino acid sequence of SEQ ID NO: 4 are synthesized, and a nucleotide sequence encoding two amino acids (GG) is linked at the center A polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 was prepared. On the other hand, a polynucleotide encoding DDDDK (Asp-Asp-Asp-Asp-Lys), which is a cleavage site which can be cleaved by enterokinase, is synthesized for accurate processing of the N-terminus, Terminus of the polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 to obtain a final polynucleotide encoding a fusion protein composed of an enterokinase cleavage site-skin permeable peptide-EGF, and the obtained polynucleotide was subjected to GST Into an expression vector to prepare an expression vector.
상기 제작된 발현벡터를 대장균에 도입하여 형질전환체를 수득하고, 상기 수득한 형질전환체를 배양한 다음, 배양물을 파쇄하여 세포 파쇄물을 수득하였으며, 상기 수득한 세포 파쇄물을 GST 친화성 컬럼에 적용하여, GST-엔테로키나제 절단부위-피부투과성 펩타이드-EGF로 구성된 융합단백질을 회수하였다. 상기 회수된 융합단백질에 엔테로키나제를 처리하여 GST 부분을 제거한 다음, 상기 반응물을 GPC 컬럼 크로마토그래피에 적용함으로써, 최종적으로 피부투과성 펩타이드-EGF로 구성된 융합단백질인 T4-EGF(서열번호 5)를 제조하였다.
The resulting expression vector was introduced into Escherichia coli to obtain a transformant. The obtained transformant was cultured, and then the culture was disrupted to obtain a cell lysate. The obtained cell lysate was subjected to GST affinity column To obtain a fusion protein composed of GST-enterokinase cleavage site-skin permeable peptide-EGF. The recovered fusion protein was treated with enterokinase to remove the GST moiety and the reaction product was applied to GPC column chromatography to finally prepare T4-EGF (SEQ ID NO: 5), a fusion protein composed of skin permeable peptide-EGF Respectively.
실시예 7: 융합단백질의 피부투과성 검증Example 7: Verification of skin permeability of fusion protein
Franz glass cell(Standard 직경 9mm, Receiver 5ml, Permegear)을 이용하여 융합단백질의 피부투과성을 검증하였다.The skin permeability of the fusion protein was verified using a Franz glass cell (Standard diameter 9 mm, Receiver 5 ml, Permegear).
구체적으로, 상기 Glass cell의 상단과 하단 사이에 돼지 피부(0.7mm 두께, Medikinetics)를 장착하고, 1% BSA와 0.01% Tween 20을 포함하는 TBS(50mM Tris pH 7.5, 150mM NaCl)를 준비한 다음, 상기 Glass cell의 상단(Donor chamber)에는 상기 TBS를 500㎕ 가하고, 상기 Glass cell의 하단(Receiver chamber)에는 상기 TBS를 5㎖ 가하였다. 이어, 2㎍의 EGF 또는 T4-EGF를 상단에 가하고, 16시간 동안 반응시킨 후, 하단에 존재하는 EGF 또는 T4-EGF의 농도를 정량분석하고, EGF의 함량에 대한 T-EGF의 양의 상대적인 함량을 투과량으로 산출하였다(표 5).Specifically, TBS (50 mM Tris pH 7.5, 150 mM NaCl) containing 1% BSA and 0.01% Tween 20 was prepared by placing pig skin (0.7 mm in thickness, Medikinetics) between the upper and lower ends of the glass cell, 500 μl of the above TBS was added to the donor chamber of the glass cell and 5 ml of the TBS was added to the lower chamber of the glass cell. Then, 2 μg of EGF or T4-EGF was added to the top and allowed to react for 16 hours. Then, the concentration of EGF or T4-EGF present at the bottom was quantitatively analyzed, and the relative amount of TGF- The content was calculated as the amount of permeation (Table 5).
T4-EGFEGF
T4-EGF
410±21100 ± 19
410 ± 21
상기 표 5에서 보듯이, T4-EGF를 처리할 경우 EGF보다 피부투과성이 약 4배 이상 증가함을 확인하였다.As shown in Table 5, it was confirmed that when T4-EGF was treated, the skin permeability increased about 4 times more than that of EGF.
따라서, 본 발명의 융합단백질을 사용하면, EGF의 피부투과율이 현저하게 증가함을 알 수 있다.
Therefore, it can be seen that the use of the fusion protein of the present invention significantly increases the skin permeability of EGF.
실시예 8: 융합 단백질의 피부잔류성 검증Example 8 Verification of Skin Residues of Fusion Proteins
Franz glass cell (Standard 직경 9mm, Receiver 5ml, Permegear)을 이용하여 융합단백질의 피부잔류성을 검증하였다.The skin persistence of the fusion protein was verified using a Franz glass cell (Standard diameter 9 mm, Receiver 5 ml, Permegear).
구체적으로, 상기 Glass cell의 상단과 하단 사이에 돼지 피부(0.7mm 두께, Medikinetics)를 장착하고, 상단과 하단에는 각각 500 ㎕와 5 ㎖의 TBS(50 mM Tris pH 7.5, 150 mM NaCl)용액에 1% BSA(Sigma), 0.01% Tween 20을 녹여서 사용하였다. 기존의 EGF와 T4-EGF를 porcine skin을 이용한 Franz cell system의 Donor chamber에 처리하고 porcine skin 내에 존재하는 EGF의 양을 측정하기 위하여 porcine skin 조직을 파쇄 후 elisa kit를 이용하였다(표 6).Specifically, pig skin (0.7 mm in thickness, Medikinetics) was mounted between the upper and lower ends of the glass cell, 500 μl and 5 ml of TBS (50 mM Tris pH 7.5, 150 mM NaCl) 1% BSA (Sigma) and 0.01% Tween 20 were dissolved and used. In order to measure the amount of EGF present in the porcine skin, EGF and EGF were treated with donna chamber of Franz cell system using porcine skin and porcine skin tissue was disrupted and elisa kit was used (Table 6).
T4-EGFEGF
T4-EGF
11900±1200100 ± 22
11900 ± 1200
상기 표 6에서 보듯이, T4-EGF를 처리할 경우 EGF보다 피부투과성이 약 120배 증가함을 확인하였다.As shown in Table 6, it was confirmed that when T4-EGF was treated, skin permeability increased about 120 times as compared with EGF.
따라서, 본 발명의 융합단백질을 사용하면, EGF의 피부잔류율이 현저하게 증가함을 알 수 있다.
Therefore, it can be seen that the skin retention rate of EGF is remarkably increased by using the fusion protein of the present invention.
<110> LG HOUSEHOLD & HEALTH CARE LTD. <120> Skin permeability enhanced peptide and fusion protein comprising the same <130> KPA140723-KR <160> 5 <170> KopatentIn 2.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Transdermal peptide <400> 1 Leu Gln Gly Gln Ser Tyr Leu Ser Ile Ser Phe Pro 1 5 10 <210> 2 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Transdermal peptide-coding polynucleotide <400> 2 ttgcagggtc agtcttatct gtcgatttct tttccg 36 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Control peptide <400> 3 Thr Asp Met Asn Lys Thr Glu Ile Arg Phe Val Arg 1 5 10 <210> 4 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> EGF <400> 4 Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His 1 5 10 15 Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30 Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45 Trp Trp Glu Leu Arg 50 <210> 5 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> T4-EGF <400> 5 Leu Gln Gly Gln Ser Tyr Leu Ser Ile Ser Phe Pro Gly Gly Asn Ser 1 5 10 15 Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly 20 25 30 Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val 35 40 45 Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp 50 55 60 Glu Leu Arg 65 <110> LG HOUSEHOLD & HEALTH CARE LTD. <120> Skin permeability enhanced peptide and fusion protein the same <130> KPA140723-KR <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Transdermal peptide <400> 1 Leu Gln Gly Gln Ser Tyr Leu Ser Ile Ser Phe Pro 1 5 10 <210> 2 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Transdermal peptide-coding polynucleotide <400> 2 ttgcagggtc agtcttatct gtcgatttct tttccg 36 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Control peptide <400> 3 Thr Asp Met Asn Lys Thr Glu Ile Arg Phe Val Arg 1 5 10 <210> 4 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> EGF <400> 4 Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His 1 5 10 15 Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30 Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45 Trp Trp Glu Leu Arg 50 <210> 5 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> T4-EGF <400> 5 Leu Gln Gly Gln Ser Tyr Leu Ser Ile Ser Phe Pro Gly Gly Asn Ser 1 5 10 15 Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly 20 25 30 Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val 35 40 45 Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp 50 55 60 Glu Leu Arg 65
Claims (18)
A skin permeable peptide or derivative thereof comprising the amino acid sequence of SEQ ID NO: 1.
상기 유도체는 상기 펩타이드의 C-말단에 단실(Dansyl)기가 결합된 형태인 것인 펩타이드 또는 이의 유도체.
The method according to claim 1,
Wherein the derivative is a form in which a Dansyl group is bonded to the C-terminal of the peptide or a derivative thereof.
상기 펩타이드 또는 이의 유도체는 추가로 피부잔류성을 나타내는 것인 펩타이드 또는 이의 유도체.
The method according to claim 1,
Wherein said peptide or derivative thereof further exhibits skin retention.
A fusion protein comprising a skin permeable peptide or derivative thereof according to any one of claims 1 to 3.
상기 융합단백질은 상기 피부투과성 펩타이드 및 생리활성 단백질을 포함하는 것인 융합단백질.
5. The method of claim 4,
Wherein the fusion protein comprises the skin permeable peptide and a physiologically active protein.
상기 피부투과성 펩타이드는 생리활성 단백질의 N-말단에 결합된 것인 융합단백질.
5. The method of claim 4,
Wherein said skin permeable peptide is bound to the N-terminus of a physiologically active protein.
상기 피부투과성 펩타이드는 생리활성 단백질의 N-말단에 링커(linker)를 통하여 결합된 것인 융합단백질.
5. The method of claim 4,
Wherein the skin permeable peptide is bound to the N-terminal of the physiologically active protein through a linker.
상기 생리활성 단백질은 상피세포 성장인자(EGF)인 융합단백질.
5. The method of claim 4,
Wherein the physiologically active protein is an epithelial growth factor (EGF).
상기 상피세포 성장인자는 서열번호 4의 아미노산 서열을 포함하는 것인 융합단백질.
9. The method of claim 8,
Wherein the epithelial growth factor comprises the amino acid sequence of SEQ ID NO: 4.
상기 융합단백질은 서열번호 5의 아미노산 서열을 포함하는 것인 융합단백질.
5. The method of claim 4,
Wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 5.
A polynucleotide which encodes a peptide according to any one of claims 1 to 3 or a derivative thereof.
상기 폴리뉴클레오티드는 서열번호 2의 염기서열로 구성되는 것인 폴리뉴클레오티드.
12. The method of claim 11,
Wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2.
10. A polynucleotide encoding the fusion protein of any one of claims 4 to 10.
A carrier for external preparation for skin comprising the peptide according to any one of claims 1 to 3 or a derivative thereof.
A pharmaceutical composition for external application for skin comprising the peptide according to any one of claims 1 to 3 or a derivative thereof.
10. A pharmaceutical composition for external application for skin comprising the fusion protein of any one of claims 4 to 10.
A cosmetic composition comprising the peptide of any one of claims 1 to 3 or a derivative thereof.
11. A cosmetic composition comprising the fusion protein of any one of claims 4 to 10.
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Cited By (1)
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KR20180028748A (en) * | 2016-09-09 | 2018-03-19 | 주식회사 엘지생활건강 | Neuron cells penetrability enhanced peptide regulating neurotransmitter secretion |
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KR20090016890A (en) * | 2007-08-13 | 2009-02-18 | 조선대학교산학협력단 | New cell penetrating peptides and method for delivery of biologically active agents using thereof |
KR20100111899A (en) * | 2009-04-08 | 2010-10-18 | 서울대학교산학협력단 | Novel kgf2-fn10 fusion protein and its use for promoting skin regeneration |
KR20120034927A (en) * | 2010-10-04 | 2012-04-13 | 주식회사 바이오셀트란 | Skin-transducing human epidermal growth factor and a producing method therof |
KR20130135207A (en) * | 2013-09-11 | 2013-12-10 | 주식회사 엘지생활건강 | Novel transdermal peptide |
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Patent Citations (4)
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KR20090016890A (en) * | 2007-08-13 | 2009-02-18 | 조선대학교산학협력단 | New cell penetrating peptides and method for delivery of biologically active agents using thereof |
KR20100111899A (en) * | 2009-04-08 | 2010-10-18 | 서울대학교산학협력단 | Novel kgf2-fn10 fusion protein and its use for promoting skin regeneration |
KR20120034927A (en) * | 2010-10-04 | 2012-04-13 | 주식회사 바이오셀트란 | Skin-transducing human epidermal growth factor and a producing method therof |
KR20130135207A (en) * | 2013-09-11 | 2013-12-10 | 주식회사 엘지생활건강 | Novel transdermal peptide |
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KR20180028748A (en) * | 2016-09-09 | 2018-03-19 | 주식회사 엘지생활건강 | Neuron cells penetrability enhanced peptide regulating neurotransmitter secretion |
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