KR20110040768A - Use of a human prolactin - Google Patents

Abstract

PURPOSE: A composition containing human prolactin for improving skin condition is provided to heal wound and to improve skin condition with safety. CONSTITUTION: A composition for improving skin condition contains human prolactin as an active ingredient. A composition for wound healing contains the huma prolactin as an active ingredient. The human prolactin is encapsulated by nano liposomes. The composition is a pharmaceutical composition or functional cosmetic composition. The composition is topically applied on the skin.

Description

Use of human prolactin {USE OF A HUMAN PROLACTIN}

The present invention relates to a composition for improving skin conditions, a method for improving skin condition using the same, a composition for treating a wound or ulcer, and a method for treating a wound or ulcer using the same.

Prolactin, an anterior pituitary hormone, is known to be involved in growth and development, hair growth, reproduction, water and electrolyte balance, metabolism, behavior, and immune function (Bole-Feysot C, et al., Prolactin (PRL) and its receptor: Actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice.Endocrine Reviews 19: 225-268 (1998); Goffin V, et al., Prolactin: the new biology of an old hormone.Annual Review of Physiology 64 : 47-67 (2002)). For example, prolactin is known to regulate hair growth in a variety of species (Lincoln GA, Significance of seasonal cycles in prolactin secretion in male mammals. Perspectives in Andrology 53: 299-306 (1989)).

Recently, prolactin receptors have been found in hair follicles (Choy VJ, et al., Distribution of prolactin receptor immunoreactivity in ovine skin and during the wool follicle growth cycle. Journal of Endocrinology 155: 265-275 (1997)).

Prolactin, a peptide hormone, contains about 200 amino acids and has a molecular weight of 23,000 kDa (Freeman ME, et al., Prolactin: structure, function, and regulation of secretion. Physiological Reviews 80: 1523-1631 (2000)). Prolactin circulating are synthesized and released from certain pituitary cells are named Lactobacillus trough's (lactotrophs) subject to adjustment of the case hypothalamic factor (Freeman ME, et al, Prolactin :.. Structure, function, and regulation of secretion Physiological Reviews 80: 1523-1631 (2000)). In addition, prolactin is synthesized in extra-pituitary tissue, which causes local self-secreting and lateral secretory effects (Craven AJ, et al., Prolactin signaling influences the timing mechanism of the hair follicle: analysis of hair growth cycles in prolactin). . receptor knockout mice Endocrinology 142: 2533-2539 (2001)).

Throughout this specification, numerous citations and patent documents are referenced and their citations are indicated. The disclosures of cited documents and patents are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

Detailed description of the invention

The present inventors have completed the present invention by finding that the topical application of human prolactin to the skin can greatly improve skin condition and wounds / ulcers.

Accordingly, an object of the present invention is to provide a composition for improving skin conditions.

Another object of the present invention to provide a composition for treating wounds or ulcers.

Another object of the present invention is to provide a method for improving skin condition.

Another object of the present invention is to provide a method of treating a wound or ulcer.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a composition for improving skin conditions comprising human prolactin as an active ingredient.

According to another aspect of the present invention, the present invention provides a composition for treating wounds or ulcers comprising human prolactin as an active ingredient.

According to another aspect of the invention, the invention provides a method for improving skin conditions comprising administering to a human skin an effective amount of human prolactin.

According to another aspect of the invention, the invention provides a method of treating a wound or ulcer comprising administering to a human skin an effective amount of human prolactin.

As a result of our research efforts to find new uses of human prolactin, we found that topical application of human prolactin to the skin can greatly improve skin condition and wounds / ulcers.

Human prolactin (hPRL) used as an active ingredient in the present invention can be used any polypeptide that exhibits human prolactin activity, for example, mature hPRL, Met-hPRL, hPRL variants, modified-hPRL, hPRL fragment or Any one of the hPRL analogues can be used. Preferably, it is mature hPRL or Met-hPRL. mature hPRL refers to human prolactin with the amino acid sequence of major human prolactin present in human blood, Met-hPRL refers to human prolactin added to methionine at the N-terminus of mature hPRL, and hPRL variants are present in the human body Refers to human prolactin having an amino acid sequence of human prolactin other than the main human prolactin, and modified hPRL refers to the attachment of an additive including pegylation or glycation to one or more amino acid residues of human prolactin, or chemical modification including deamidation or phosphorylation. The human prolactin or the N-terminal, C-terminal, or other amino acid sequence of the amino acid sequence of hPRL is transformed into a fusion protein using genetic or biochemical methods, and the hPRL fragment is composed of human prolactin. Part of the amino acid sequence Point to the human prolactin was removed by a chemical method, hPRL analogue refers to a human prolactin variant having an amino acid sequence of human prolactin to another amino acid sequence by genetic engineering methods. Human prolactin of the invention can be specified by one of two methods. One can be specified by binding to human prolactin receptors and the other can be specified by methods of measuring the biological activity of human prolactin.

According to a preferred embodiment of the present invention, the composition of the present invention has a liposome composition. That is, human prolactin as an active ingredient is preferably surrounded by liposomes and applied to the skin. According to a more preferred embodiment of the present invention, the composition of the present invention has a composition of nanoliposomes. As used herein, the term "nanoliposomes" refers to liposomes having the form of conventional liposomes with an average particle diameter of less than 20-1000 nm. According to a preferred embodiment of the invention, the average particle diameter of the nanoliposomes is 50-500 nm, more preferably 50-350 nm and most preferably 50-250 nm.

Liposomes are defined as spherical phospholipid vesicles of colloidal particles that associate themselves. Liposomes composed of amphiphilic molecules with water-soluble heads (hydrophilic groups) and insoluble tails (hydrophobic groups) spontaneously bind and form an ordered structure by their interaction. It is classified into Small Unilamellar Vesicle (SUV), Large Unilamellar Vesicle (LUV) and Multi Lamellar Vesicle (MLV) according to its size and lamellarity. Such liposomes exhibiting various laminations have a double membrane structure similar to that of cell membranes.

According to a preferred embodiment of the invention, the nanoliposomes surrounding the prolactin have a small unilamellar vesicle structure.

According to a preferred embodiment of the present invention, the human prolactin surrounded by the nanoliposomes has 90-100% of the activity of the entrapped human prolactin.

The (nano) liposomes in the present invention can be prepared using phospholipids, polyols, surfactants, fatty acids or / and water.

Phospholipids, which are components used in the preparation of the (nano) liposomes of the present invention, are used as amphoteric lipids, including natural phospholipids (eg, egg yolk lecithin or soy lecithin, sphingomyelin) and synthetic phospholipids (eg, dipalmitoyl). Phosphatidylcholine or hydrogenated lecithin), preferably lecithin. More preferably, the lecithin is naturally occurring unsaturated lecithin or saturated lecithin extracted from soybean or egg yolk.

The polyols that can be used in the (nano) liposomes of the present invention are not particularly limited, preferably propylene glycol, dipropylene glycol, 1,3-butylene glycol, glycerin, methylpropanediol, isoprene glycol, pentylene glycol, Erythritol, xyitol and sorbitol.

Surfactants that can be used in the preparation of the (nano) liposomes of the present invention can be any known in the art, for example, anionic surfactants such as alkylacylglutamate, alkylphosphates, alkyllactylates, Dialkylphosphates and trialkylphosphates), cationic surfactants, amphoteric surfactants and nonionic surfactants (e.g. alkoxylatedalkylethers, alkoxylatedalkylesters, alkylpolyglycosides, polyglyceryl esters and sugar esters) Can be used.

Fatty acids that can be used to prepare the (nano) liposomes of the present invention are higher fatty acids, preferably saturated or unsaturated fatty acids of C _12-22 alkyl chains, for example, lauric acid, myristic acid, palmitic acid, stearic acid. Oleic acid and linoleic acid.

The water used to prepare the (nano) liposomes of the present invention is generally deionized distilled water.

According to a preferred embodiment of the present invention, the (nano) liposomes of the present invention are prepared only with phospholipids and water, specific examples of which are described in the following examples.

According to a preferred embodiment of the invention, the hPRL-containing nanoliposomes of the invention are prepared via a process comprising the following steps: (a) phospholipids capable of forming liposomes (preferably egg yolk lecithin or soy lecithin) ) Is dissolved in an aqueous solution containing human prolactin; And (b) repeatedly passing the aqueous solution containing human prolactin and phospholipid into a high pressure homogenizer, and gradually increasing the content of phospholipid and the pressure of the high pressure homogenizer according to the number of passages to prepare nanoliposomes containing human prolactin. .

Aqueous solutions containing human prolactin are preferably buffered at pH 6-8, preferably about pH 7, such as sodium phosphate buffer. If sodium phosphate buffer is used, the concentration is 5-100 mM, more preferably 5-60 mM, even more preferably 10-30 mM, most preferably about 20 mM.

In the method of the present invention, the most specific aspect is to pass the mixture of phospholipid and hPRL-containing aqueous solution several times through a high pressure homogenizer, in which the amount of phospholipid and the pressure of the homogenizer are sequentially increased as the number of passage increases. According to a preferred embodiment of the invention, the pressure of the homogenizer is sequentially increased to 0-1000 bar, preferably 0-800 bar. The output can be increased by 50 bar or 100 bar, preferably by 100 bar. According to a preferred embodiment of the invention, the amount of phospholipids is sequentially increased to 5-40 w / v (%), preferably 5-30 w / v (%).

Through this high pressure homogeneous process with sequential increase in phospholipid content and pressure, hPRL-containing nanoliposomes are prepared, preferably liquid hPRL-containing nanoliposomes.

The compositions of the present invention can be used to improve various skin conditions. Preferably, the composition of the present invention is effective in improving wrinkles, removing blemishes or blemishes (blemishes or freckles) produced or promoted by ultraviolet rays, treating acne, whitening, improving skin elasticity, preventing skin aging, and improving skin moisturizing. It is preferably effective for improving wrinkles, removing blemishes (including blemishes and acne), whitening and improving skin elasticity.

The compositions of the present invention can be used for the healing of various wounds or ulcers. Preferably, the compositions of the present invention can be used to treat wounds and ulcers in elderly or diabetic patients. Ulcers that can be cured by the compositions of the present invention include chronic ulcers and acute ulcers. The chronic ulcers include aneurysm ulcers, varicose ulcers, varicose vein ulcers, diabetic ulcers, diabetic foot ulcers, compression ulcers, systemic lupus ulcer ulcers and pressure sores, wherein the acute ulcers are thermal injury), trauma, surgery, ablation of skin cancer, fungal or bacterial infections, vasculitis, scleroderma or flares, and burns.

The composition of the present invention may be prepared as a pharmaceutical composition, a cosmetic cosmetic composition or a cosmetic composition.

Ingredients included in the cosmetic or functional cosmetic composition of the present invention include ingredients commonly used in cosmetic compositions in addition to human prolactin or hPRL-containing liposomes as active ingredients, and include, for example, stabilizers, solubilizers, vitamins, pigments and flavorings. Conventional adjuvants, and carriers.

The cosmetic composition for protecting the skin of the present invention may be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactants- Containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, sprays and the like, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, in particular in the case of spray, additionally nitrogen, chlorofluorohydrocarbons. Propellant such as propane / butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or aerosolizing agent is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

When the composition of the present invention is prepared as a pharmaceutical composition, it may include a pharmaceutically acceptable carrier, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate Gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and / or mineral oils can do. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

Preferably, the pharmaceutical composition of the present invention is developed for topical topical administration.

Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. Meanwhile, the topical dose effective amount of the pharmaceutical composition of the present invention is preferably 0.001-100 ng / cm ² (skin surface area) per day, more preferably 0.01-10 ng / cm ² , most preferably 0.1- 2 ng / cm ² . As used herein, the term "effective amount" means a dose sufficient to achieve the pharmaceutical effect described above.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation is most preferably in the form of a solution containing nanoliposomes.

Overall, the composition of the present invention greatly inhibits skin aging, serves to improve wrinkles and blemishes, treats acne, removes blotch, and shows whitening efficacy. Moreover, the composition of this invention can make wound or ulcer healing high efficiency. In addition, the composition of the present invention is not only very safe for the human body, but also excellent in stability when prepared with nanoliposomes.

The features and advantages of the present invention are summarized as follows:

(a) The composition for improving skin conditions, treating wounds or ulcers of the present invention contains human prolactin as an active ingredient.

(b) The composition of the present invention can successfully exert skin condition improvement, wound or ulcer treatment efficacy even when applied topically to the skin surface.

(c) The composition of the present invention is very safe for human body.

[표 2]

실시예 3: His-tagging 된 narK 프로모터와 시그널 서열이 제거된 Met-프로락틴이 결합된 발현벡터 제조
narK 프로모터 뒤에 6 개의 히스티딘(histidine)을 암호화하는 유전자 서열이 들어간 벡터에 Met-프로락틴 단백질을 암호화하는 유전자를 연결시키는 PCR 을 실행하였는데, 여기서 연결부위의 18 개 염기들이 서로 염기쌍을 이루어 연결되도록 하였다. 1 차 PCR 방법은 다음과 같다. pNKmut 플라스미드(-10 돌연변이된 narK 프로모터를 포함, (주)리제론, 참조: 대한민국 특허출원공개 제 2006-0089086 호)를 주형으로 OY-17 와 His-narK D 프라이머 쌍을 이용하여 PCR(30 싸이클; 98℃ 10 초, 55℃ 5 초, 72℃ 25 초)을 수행하여 368 bp 의 6 개의 histidine 을 암호화하는 유전자 서열을 포함하는 narK 프로모터를 얻었다. 실시예 2 에서 기술한 pUC-narK-Met-hPRL 플라즈미드를 주형으로 hPL 11U 와 hPL 8D 프라이머 쌍을 이용하여 PCR(30 싸이클; 98℃ 10 초, 55℃ 5 초, 72℃ 55 초)을 수행하여 626 bp 의 Met-프로락틴 유전자를 얻었다.
2 차 PCR 반응은 1 차 PCR 반응에서 획득한 368 bp(6 개의 히스티딘을 암호화하는 유전자 서열을 포함하는 narK 프로모터)와 626 bp(Met-hPRL 유전자)를 주형으로 EcoRI 절단부위(GAATTC)가 포함된 OY-17 과 KpnI 절단부위(GGTACC)가 포함된 hPL 8D 프라이머 쌍을 넣고 PCR(30 싸이클; 98℃ 10 초, 55℃ 5 분, 72℃ 1 분)을 수행하여 976 bp 의 절편(narK 프로모터-6 개의 histidine-Met-프로락틴 유전자 절편)을 얻었다. 다음에 EcoRI 과 KpnI 으로 double-digestion 된 976 bp 의 절편(narK 프로모터-6 개의 histidine-Met-프로락틴 유전자 절편)과 pUC-rrnB(pUC18 플라스미드에 rrnB 터미네이터 삽입, (주)리제론) 벡터를 T4 DNA 리가제로 연결시킨 후 Top10F' 균주에 형질전환시켰다. 이후 37℃에서 15 시간동안 배양한 후, 3 개의 콜로니들을 임의 선정하여 배양하고, 알칼리 라이시스 방법으로 플라스미드들을 얻은 후, 1％ 아가로스 겔에서 전기영동하여 크기를 확인 하고 염기서열을 분석함으로써 원하는 플라스미드를 선택하였다(pUC-narK-his-Met-hPRL).
[표 3]

실시예 4: Nanolipo-hPRL 과 Nanolipo-his-hPRL 의 제조
인간 프로락틴과 his-인간 프로락틴을 나노리포좀 제형화하였다. 이용된 인지질은 대두 레시틴(soybean lecithin; (주)신동방, South Korea), Metarin P(Degussa Texturant Systems Deutschland GmbH ＆ Co. KG), Nutripur S(Degussa Texturant Systems Deutschland GmbH ＆ Co. KG) 또는 Emultop(Degussa Texturant Systems Deutschland GmbH ＆ Co. KG)이다.
기기 출구(outlet)의 온도가 30℃가 넘지 않게 열교환기를 장치한 고압균질기(max. output 5 L/hr, 최대압력 1200 bar, Model HS-1002, (주)화성기계 (Korea))의 열교환기를 얼음물속에 장치한 후 증류수로 기기 내부를 세척하여 작동준비한 후, 완충용액(20 mM NaH₂PO₄ pH 6.5-7.5, 1 mM EDTA)에 용해되어 있는 2 mg/㎖ 농도의 인간 프로락틴 혹은 his-인간 프로락틴 용액에 인지질을 10 w/v％ 비율로 첨가하여 충분히 수화시켜 교반하고, 이를 상온에서 균질기를 이용하여 저압 0 bar 에서 3 회 이상 균질기를 통과시켰다. 이어, 상기 균질기 과정을 거친 용액에 인지질을 14 w/v％ 비율이 되게 첨가하여 충분히 수화시켜 교반하고, 100 bar 에서 3 회 이상 균질기를 통과시켰다. 그런 다음, 상기 균질기의 과정을 거친 용액에 인지질을 18 w/v％ 비율이 되게 첨가하여 충분히 수화시켜 교반하고, 200 bar 에서 3 회 이상 균질기를 통과시켰다. 그리고 나서, 상기 균질기의 과정을 거친 용액에 인지질을 20 w/v％ 비율이 되게 첨가하여 충분히 수화시켜 교반하고, 300 bar 에서 3 회 이상 균질기를 통과시켰다. 이어, 상기 균질기의 과정을 거친 용액에 인지질을 22 w/v％ 비율이 되게 첨가하여 충분히 수화시켜 교반하고, 400 bar 에서 3 회 이상 균질기를 통과시켰다. 그런 다음, 상기 균질기의 과정을 거친 용액에 인지질을 24 w/v％ 비율이 되게 첨가하여 충분히 수화시켜 교반하고, 500 bar 에서 3 회 이상 균질기를 통과시켰다. 그리고 나서, 상기 균질기의 과정을 거친 용액에 인지질을 26 w/v％ 비율이 되게 첨가하여 충분히 수화시켜 교반하고, 600 bar 에서 3 회 이상 균질기를 통과시켰다. 이어, 상기 균질기의 과정을 거친 용액에 인지질을 28 w/v％ 비율이 되게 첨가하여 충분히 수화시켜 교반하고, 700 bar 에서 3 회 이상 균질기를 통과시켰다. 그런 다음, 상기 균질기의 과정을 거친 용액을 800 bar 에서 3 회 이상 균질기를 통과한 후 균질기로부터 용액을 배출하고 15,000 x g 에서 30 분간 고속원심분리하여 상등액을 분리하였다. 이때 리포좀 내에 들어가지 않은 프로락틴과 his-프로락틴은 젤 투과 크로마토그래피(GE Healthcare, USA)로 제거하고 액상의 리포좀을 수득하였다.
실시예 5: 무모 마우스(Hairless nude mouse)를 이용한 주름/피부탄력/수분손실/표피두께 평가
1. 실험동물의 준비
실험동물은 4 주령의 암컷 SKH-1 무모 마우스를 두열 바이오텍에서 구입하였다(Sungnam, Korea). 실험동물은 온도 24± 2℃, 상대습도 50± 10％, 명암순환이 12 시간 단위로 조절되는 조건에서 사육하였으며 2 주간 순치시킨 후 임의로 11개의 군으로 나누어 이후 실험을 진행하였다.
UV 조사기기는 VLX-3W stimulator(Vilber Lourmat, Marne la Vallee, France)를 사용하였다. UVB 조사는 주당 3 회씩 총 11 주간 조사하였다. 조사량은 1-2 주는 30 mJ/cm², 3-4 주는 40 mJ/cm², 5-9 주는 50 mJ/cm², 10-11 주는 60 mJ/cm² 로 조사하였다. 단백질 군 및 vehicle 군의 처리는 총 11 주 동안 매주 5 회 각각 300 ㎕씩 도포했다. EtOH(Merck)군과 레티노산(retinoic acid, Sigma) 군은 100 ㎕씩 처리하였다. UV 를 처리하게 되는 날의 경우 UV 조사 한 시간 전에 샘플을 도포하여 주었다. 도포는 미리 준비된 샘플을 미술용 3 호 붓(Hwasung, Chuncheon, Korea)을 이용하며 등 전체에 골고루 펴 발라 주었다. 각각의 단백질 처리 농도는 다음 과 같다.
[표 4]

2. 무모 마우스를 이용한 Nanolipo-hPRL 의 주름개선 효능 분석
장기간에 걸쳐 지속적인 UV 가 조사될 경우 SKH-1 무모 마우스에서 굵고 깊은 주름이 생기는 것은 널리 알려져 있다. 본 실험의 육안적 피부 주름의 평가의 기준 또한 UV 조사에 의해 형성되는 깊은 주름의 생성을 기준으로 하여 11 주 동안의 UV 와 샘플 처리 종료 후 다음과 같은 기준으로 평가했다.
- : 관찰되지 않음(Not observed)
* : 경도 두께 및 깊이의 주름(Mild thick and deep winkle)
** : 경도 초과 중등도 미만 두께 및 깊이의 주름(Mild to Morderate thick and deep winkle)
*** : 중등도 두께 및 깊이의 주름(Morderate thick and deep winkle)
**** : 중등도 초과 중증 미만 두께 및 깊이의 주름(Morderate to severe thick and deep winkle)
***** : 중증 두께 및 깊이의 주름(Severely thick and deep winkle)
실험결과, 그룹 2 내지 3 및 그룹 5 에서 중등도 이상의 주름이 발견되었으나, 그룹 4 및 6 내지 7에서는 경도 또는 중등도 미만의 두께 및 깊이의 주름을 관찰할 수 있었다(도 1).
3. 무모 마우스를 이용한 Nanolipo-hPRL 의 피부탄력도 및 수분손실도 효능 분석
피부 탄력도 및 수분손실량 측정은 11 주 동안의 UV 와 샘플 처리 종료 후 항온 항습실(온도 22± 2℃, 습도 50± 10％)에서 피실험동물을 20 분간 안정화 시킨 다음 측정을 수행하였다. 피부 효능 기기 평가에 널리 사용되는 Multi Probe Adaptor systems, MPA580(Courage+Khazaka, Germany)를 이용하여 등(dorsal) 부위에 수분손실량을 Texameter, TM300 probe(Courage+Khazaka, Germany)를 사용하여 측정하고 탄력도는 Cutometer SEM 575 probe(Courage+Khazaka, Germany)로 각각 3 회 측정한 수치의 평균을 결과로 하였다(Pressure : 500 mbar, Probe aperture: 2 mm, On-time: 1 seconds, Off-time: 1 seconds, Repetitions: 5).
실험결과, Nanolipo-hPRL 의 피부탄력도에 대한 영향은 미미하였으나 다른 실험(수분손실도, 주름개선, H＆E 염색, 인공피부실험) 결과들과 비교 유추할 때 무모 마우스 마리 수를 늘리거나 장기적으로 적용하면 피부탄력도 증가시키리라 생각된다(도 2a). 하지만, 수분손실도에서 EtOH군(그룹 3), UV(+)군(그룹 2) 및 리포좀 군(그룹 5)과 비교하여 Nanolipo-hPRL 군(그룹 6) 및 Nanolipo-his-hPRL(그룹 7) 군은 명확한 수분 손실 방지 효과를 나타내었다(도 2b).
4. 무모 마우스를 이용한 Nanolipo-hPRL 의 피부손상 억제 효능 분석
조직 분석을 통한 피부 표피 두께 측정은 11 주 동안 UV 와 각각의 도포제를 처리한 암컷 SKH-1 무모 마우스의 등 피부조직을 적출하여 4％ 파라포름알데히드가 첨가된 0.1M 인산염 고정액에 담가 4℃에 O/N 처리하였고, 조직의 탈수과정을 거친 후 파라핀으로 포매하여 블록을 제작하였다. 다음에 Leica microtome 를 이용하여 조직을 3 ㎛의 두께의 절편으로 자른 후, 절편슬라이드를 제작하여 탈파라핀과정과 함수과정을 거친 후, Herris' s 헤마톡실린(Hematoxylin, Sigma, U.S.A)과 에오신(eosin, Alpha Chem, Inc, U.K.)으로 염색하였다. 그리고나서 Zeiss Axiostar Plus 현미경으로 관찰 후, AxioCam MR 카메라를 이용하여 촬영하였고, Axiovision 소프트웨어를 이용하여 표피두께를 측정하였다.
UV 조사에 의해 주름이 유발된 실험 동물에서 주름 개선에 효과가 있다고 알려진 많은 물질들이 UV 조사에 의한 피부 표피(epidermis) 비후화를 예방하는 것으로 보고되고 있다. 도 3 에 나타난 바와 같이, UV 조사에 의해 유발된 피부 주름이 Nanolipo-hPRL 또는 Nanolipo-his-hPRL 처리로 현저하게 개선됐고 또한 조직학적으로 피부 표피 비후화가 감소되었다. 아마도 UV 조사에 의해 기저세포의 복제, 분화, 탈피되는 일련의 과정이 비정상적으로 이루어져 나타나는 피부 표피 비후화가 프로락틴에 의해 피부 표피 기저세포의 항상성(homeostasis)이 정상적으로 이루어져 표피 비후화를 예방하는 것으로 사료된다.
실시예 6: Nanolipo-hPRL 의 세포내 멜라닌 생성 억제 여부 측정
B16F1 흑색종(melanoma) 세포를 6 웰 플레이트에 깔고(6 × 10⁴ 세포/웰) 배양기에서 24 시간 배양한(5％ CO₂, 37℃) 후 α-MSH(sigma)가 함유된 DMEM 배지로 교체하고 arbutin(sigma)과 Nanolipo-hPRL 을 농도별로 처리하여 72 시간 배양한다. 세포 펠렛을 회수하여 원심 분리하고 상등액이 제거된 펠렛을 건조시킨 후 세포내의 멜라닌을 1N NaOH/10％ DMSO 로 용해 시켜서 SPECTRA MAX 190(Molecular Devices)으로 405 nm에서 흡광도를 측정하고 melanin standard(Sigma) curve 를 이용하여 멜라닌 생성 저해율(％)을 계산하였다. 도 4 에 나타난 바와 같이, 본 발명의 Nanolipo-hPRL 은 농도에 따라 유의미한 멜라닌 생성 억제율을 나타내었다.
실시예 7: 인공피부를 이용한 Nanolipo-hPRL 의 표피층 두께 변화 측정
4.5cm² 면적의 인공피부(Tegoscience., South Korea)에 리포좀(1％), lipo-hPRL(2 ㎍/㎖), 10％ EtOH, 레티노산(0.01％ in 10％ EtOH)을 300 ㎕ 씩 4 일 동안 처리하였다. 마지막 처리 후 24 시간이 지나 마우스를 희생시켜 이로부터 인공피부를 얻은 다음, 4％ 포르말린(Yakuri, Japan)으로 상온에서 5 시간 동안 고정시키고, 파라핀(McCormick, U.S.A.) 블록을 준비하였다. 이 파라핀 블록을 5 ㎛ 크기로 절단하여 헤마톡실린(Hematoxylin, Sigma, U.S.A.)과 에오신(eosin, Alpha Chem, Inc, U.K.)으로 염색하여 광학현미경 400 배로 관찰하였다.
도 5 에서 나타난 바와 같이, Nanolipo-hPRL 처리군에서 인공피부의 분화되는 세포층의 두께가 두꺼워진 것을 통해 볼 때, 본 발명의 Nanolipo-hPRL 은 피부의 상태를 건강하게 하는데 효과가 있는 것으로 보인다.
실시예 8: Nanolipo-hPRL 의 주름 개선 효능 분석
Nanolipo-hPRL 의 주름 개선 효능을 실제 사용 테스트를 통하여 평가하였다. 제형예 3 의 영양 크림을 사용하였다. 20 명의 40-50 세 여성을 무작위로 2 개군으로 나누어 제형예 3 의 크림을 매일 아침/저녁 2 회씩 세안 후 크림 적당량을 눈가를 중심으로 2 개월간 연속적으로 바르게 하였다. 각 피검자의 주름 개선 효과를 육안 관찰을 통하여 평가하였다. 실험 결과는 하기의 표 5 에 기재되어 있다.
[표 5]

*총 피검자 중에서 주름 개선 효과가 있는 것으로 판정된 피검자의 수의 비율
**프로락틴이 없는 Nanoliposome 제형
상기 표 5 에서 알 수 있듯이, 프로락틴은 명확한 주름 개선 효과를 나타내고 있다.
실시예 9: Nanolipo-hPRL 의 여드름 치료 효능 분석
본 발명의 Nanolipo-hPRL 의 여드름 치료 효능을 다음과 같이 조사하였다: 15-40 세의 여성 중 얼굴 피부에 여드름을 갖는 60 명을 20 명씩 무작위로 3 그룹으로 나눈 다음, Nanolipo-hPRL 또는 리포좀만 있는 비교용액(Nanolipo)을 각각 3 주간 아침 저녁으로 하루 2 번씩 세안 후 가장 먼저 사용하게 하였다. 그 외에는 평소에 사용하는 화장품에 대한 특별한 제한은 두지 않았다. 그런 다음, 사용자의 의견에 따라 여드름에 대한 개선 정도를 하기의 평가기준에 따라 판정 하였다. 시험 결과는 표 6 에 나타나 있다. 평가 기준:
+++ : 매우 양호한 개선효과가 있음
++ : 상당한 개선효과가 있음
+ : 약간의 개선효과가 있음
± : 개선효과는 없으나 악화되지도 않음
- : 악화됨.
[표 6]

표 6 에서 보는 바와 같이, 본 발명의 Nanolipo-hPRL 은 여드름에 대한 개선 정도가 매우 우수함을 알 수 있으며, 여드름 개선 효과는 적용 후 약 2 주부터 명백하게 나타나기 시작하였다. 더욱이, 본 발명의 Nanolipo-hPRL 은 피부에 대한 자극, 예컨대, 홍반 또는 가려움증을 거의 유발하지 않았다.
실시예 10: Nanolipo-hPRL 의 검버섯 제거 효능 분석
본 발명의 Nanolipo-hPRL 의 검버섯 제거 효능을 다음과 같이 조사하였다: 40-60 세의 여성 중 얼굴 피부에 검버섯을 갖는 60 명을 20 명씩 무작위로 3 그룹으로 나눈 다음, Nanolipo-hPRL 또는 리포좀만 있는 비교용액을 각각 8 주간 아침 저녁으로 하루 2 번씩 세안 후 가장 먼저 사용하게 하였다. 그 외에는 평소에 사용하는 화장품에 대한 특별한 제한은 두지 않았다. 그런 다음, 사용자의 의견에 따라 검버섯에 대한 개선 정도를 하기의 평가기준에 따라 판정 하였다. 시험 결과는 표 7 에 나타나 있다. 평가 기준:
+++ : 매우 양호한 개선효과가 있음
++ : 상당한 개선효과가 있음
+ : 약간의 개선효과가 있음
± : 개선효과는 없으나 악화되지도 않음
- : 악화됨.
[표 7]

표 7 에서 보는 바와 같이, 본 발명의 Nanolipo-hPRL 은 검버섯에 대한 개선 정도가 상당히 우수함을 알 수 있으며, 검버섯 개선 효과는 적용 후 약 5 주부터 명백하게 나타나기 시작하였다.
실시예 11: Nanolipo-hPRL 의 미백 효능 분석
본 발명의 Nanolipo-hPRL 의 미백 효능을 다음과 같이 조사하였다: 20-60 세의 여성 중 얼굴 피부에 기미 또는 주근깨가 있거나 자신의 피부가 칙칙하다고 생각하는 60 명을 20 명씩 무작위로 3 그룹으로 나눈 다음, Nanolipo-hPRL 또는 리포좀만 있는 비교용액을 각각 8 주간 아침 저녁으로 하루 2 번씩 세안 후 가장 먼저 사용하게 하였다. 그 외에는 평소에 사용하는 화장품에 대한 특별한 제한은 두지 않았다. 그런 다음, 사용자의 의견에 따라 기미, 주근깨, 또는 피부의 칙칙함에 대한 개선(휘광(Radiance)효과) 정도를 하기의 평가기준에 따라 판정 하였다. 시험 결과는 표 8 에 나타나 있다. 평가 기준:
+++ : 기미.주근깨를 없애고 피부 휘광 회복에 매우 양호한 개선효과가 있음
++ : 상당한 개선효과가 있음
+ : 약간의 개선효과가 있음
± : 개선효과는 없으나 악화되지도 않음
- : 악화됨.
[표 8]

표 8 에서 보는 바와 같이, 본 발명의 Nanolipo-hPRL 은 기미나 주근깨 개선 또는 칙칙한 피부에 대한 휘광 효과가 상당히 우수함을 알 수 있으며, 이러한 미백 효과는 적용 후 약 4 주부터 명백하게 나타나기 시작하였다.
실시예 12: Nanolipo-hPRL 의 피부탄력 개선 능력 분석
Nanolipo-hPRL 의 피부탄력 개선 효능을 실제 사용 테스트를 통하여 평가하였다. 제형예 3 의 영양 크림을 사용하였다. 20 명의 40-50 세 여성을 무작위로 2 개군으로 나누어 제형예 3 의 크림을 매일 아침/저녁 2 회씩 세안 후 크림 적당량을 얼굴 및 목에 2 개월간 연속적으로 바르게 하였다. 각 피검자의 피부탄력 개선 효과를 설문조사를 통하여 아래와 같이 평가하였다.
평가 기준: +++ (매우 양호한 개선효과가 있음), ++ (상당한 개선효과가 있음), + (약간의 개선효과가 있음), ± (개선효과는 없으나 악화되지도 않음), - (악화됨).
설문조사 결과는 하기의 표 9 에 기재되어 있다.
[표 9]

상기 표 9 에서 알 수 있듯이, 프로락틴은 명확한 피부탄력 개선 효과를 나타내고 있다.
실시예 13: Nanolipo-hPRL 의 창상 치유 능력 분석
본 발명의 Nanolipo-hPRL 의 창상 또는 궤양 치유 능력을 다음과 같이 조사하였다.
본 발명의 Nanolipo-hPRL 을 창상 또는 궤양의 대표적인 예, 욕창, 당뇨병성 족부궤양 및 전신성 홍반성낭창 궤양 환자에게 적용하였다. 상기 창상을 갖는 환자 60 명을 20 명씩 무작위로 3 그룹으로 나눈 다음, Nanolipo-hPRL 또는 리포좀만 있는 비교 용액을 각각 8 주간 아침/저녁으로 하루 2 번씩 창상 부위에 도포하였다. 그런 다음, 사용자의 의견에 따라 창상에 대한 개선 정도를 하기의 평가기준에 따라 판정하였다. 시험 결과는 표 10a-10c 에 나타나 있다. 평가 기준: +++ (매우 양호한 개선효과가 있음), ++ (상당한 개선효과가 있음), + (약간의 개선효과가 있음), ± (개선효과는 없으나 악화되지도 않음), - (악화됨).
[표 10a]

[표 10b]

[표 10c]

표 10a-10c 에서 보는 바와 같이, 본 발명의 Nanolipo-hPRL 은 창상 또는 궤양의 대표적인 예, 즉 욕창, 당뇨병성 족부궤양 및 전신성 홍반성낭창 궤양에 대한 개선 효과를 나타내었다. 이러한 창상 개선 효과는 적용 후 약 3-4 주부터 명백하게 나타나기 시작 하였다.
실시예 14: Nanolipo-hPRL 의 국소요법이 부분층식피술공요부의 창상치유에 미치는 영향 분석
본 발명의 Nanolipo-hPRL 의 창상 또는 궤양 치유 능력을 다음과 같이 조사하였다.
실험기간은 3 주(21 일)간 실시 하였으며, micropig (생후 4 개월, 수컷, 9.5 kg, 살색, 단모(short hair))/1 마리를 이용하였다. 마취는 전마취와 심마취를 하였으며, 전마취의 경우 ketamine: 1 ml/10 kg(유한양행), xylazine: 1 ml/10 kg(인터벳코리아), 심마취의 경우 isoforane : 산소 = 2.5 : 2.5 (중외제약)로 마리당 10 개의 창상을 만들기 위해 심마취를 실시 : 24 시간 전부터 금식- 마취후 12 시간 절식시켰다.
창상의 유도는 먼저 심마취를 시킨 후, cliper 를 사용하여 피부 채취할 부위의 털을 제거해주고, 등부위에 2×2cm(4cm²)의 크기, 4cm 간격으로 펜으로 적출부위를 표시하였다. 베타딘으로 적출할 등부위를 소독한 후 피부채취전동기를 사용하여 부분층식피편을 0.6mm 두께로 2×2cm(4cm²)넓이의 피부를 등부위에서 제거하여 창상을 만들어준 후 피부를 채취하였다.
머리쪽으로부터 우측 1 번째, 좌측 2 변째, 우측 3 번째, 좌측 4 번째 및 우측 5 번째 부위를 대조군(C), 머리쪽으로부터 좌측 1 번째, 우측 2 번째, 좌측 3 번째, 우측 4 번째 및 좌측 5 번째 부위를 실험군(P)로 하였다.
도포방식은 표 11 과 같다.
[표 11]

드레싱은 대조군과 실험군 모두에 필름드레싱(Opsite)을 사용하여 폐쇄드레싱을 하고 mephix(tape)로 고정한 후 창상보호를 위해 붕대(EB)로 몸통 전체를 감싸준 후 stockinet(4 번째 사진에서 보이는 탄력성이 있는 큰 천)으로 마무리 하였다. 상기의 과정을 나타낸 도면이 도 6 이다.
창상처치(드레싱)는 2 일 마다 시행, 다음을 병행하였다. 마취는 간단한 드레싱과 피부채취이므로 흡입마취만으로 가능하였다. 1) 창상면적측정- 일주일 간격(7 일, 14 일, 21 일)으로 OHP 필름을 이용하여 창상부위 모양을 본뜬 후 planimeter 면적측정프로그램을 이용하여 면적을 측정한 다음, 최초 상처면적크기에 대한 상처크기를 비율로 환산한다. 2) 2 일마다 시료를 창상부위에 1ml 양의 시료를 도포 한다. 3) 각각의 시료가 상처에서 떨어지지 않도록 4 cm x 4 cm 크기의 밀착포를 첩부한다. 4) 밀착포 주위를 micropore(3M 사)로 처리한다. 5) stokinet 을 사용하여 몸 전체를 감싸 주고, 상처 부위를 피하여 접착테이프로 고정한다
실험결과 상처크기의 변화는 표 12 와 같다(도 7):
[표 12]

이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.1 is a view showing the wrinkle improvement effect of Nanolipo-hPRL or Nanolipo-his-hPRL using a hairless mouse. In the figure, Lipo-hPRL represents Nanolipo-hPRL and Lipo-his-hPRL represents Nanolipo-his-hPRL.
2 is a view showing the effect on skin elasticity and water loss of Nanolipo-hPRL or Nanolipo-his-hPRL using a hairless mouse. Figure 2a shows the results for the skin elasticity, Figure 2b shows the results for the moisture loss, the average of the values measured three times as a result.
3 is a view showing the effect on the skin epidermal thickening prevention of Nanolipo-hPRL or Nanolipo-his-hPRL using hairless mice.
4 is a diagram showing the effect on the inhibition of melanogenesis of Nanolipo-hPRL using B16F1 melanoma cells.
5 is a diagram showing the effect on skin cell differentiation of Nanolipo-hPRL using Tegoscience Neoderm artificial skin.
6 is a view showing a process of applying the control group and the experimental group to pigs induced wounds.
7 is a view showing a change in wound size when applying Nanolipo-hPRL.
Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.
EXAMPLE
Example 1: Cloning of Prolactin Gene
Genomic DNA was obtained from HEF (Human embryonic fibroblast). This was digested with Bam HI (Takara, Japan; same as the place where the restriction enzyme was used), and then used as a template to perform PCR to obtain five exon DNA fragments encoding prolactin. To connect each exon DAN fragment, primers were designed so that some bases of the linking sites were base paired with each other, and PrimeSTAR ™ HS DNA polymerase (Takara, Japan) was used for all PCR.
The primary PCR method for amplifying exons 1, 2, 3, 4 and 5 of prolactin is as follows. PCR using genomic DNA digested with Bam HI (Takara, Japan) as a template and using hPL 1U primers (Bioneer, Korea; same as where to buy primers used below) and hPL 2D primer pairs containing exon 1 (30 cycles; 98 ° C. 10 seconds, 55 ° C. 5 seconds, 72 ° C. 15 seconds), 222 bp of exon 1,2 was amplified and obtained. In the same way, 131 bp exon 3 using hPL 3U and hPL 4D primer pairs, 202 bp exon 4 using hPL 5U and hPL 6D primer pairs, and 209 bp using hPL 7U and hPL 8D primer pairs. Exon 5 was obtained.
In the secondary PCR, exons 2 (222 bp) and exon 3 (131 bp) containing exon 1 obtained by the above-described method were used as templates, and the hPL 1U and hPL 4D primer pairs were put into PCR (30 cycles; 98 ° C). 10 seconds, 55 ° C 5 seconds, 72 ° C 25 seconds) was performed to amplify and acquire 334 bp (Exon 1 + Exon 2 + Exon 3). Exon 3 (195 bp) and Exon 4 (224 bp) were obtained in the same manner. Using a template, hPL 5U and hPL 8D primer pairs were added, and PCR was carried out in the same manner to obtain amplification of 392 bp (exon 4 + exon 5).
Tertiary The PCR into the secondary PCR product is 334 bp and the 392 bp used as a template to containing the Eco RI cleavage site (GAATTC) hPL 1U and Kpn I cleavage site (GGTACC) containing the hPL 8D primer pair PCR (30 cycles; 98 ° C. 10 seconds, 55 ° C. 5 seconds, 72 ° C. 45 seconds) was performed to obtain 701 bp (exon 1 + exon 2 + exon 3 + exon 4 + exon 5) containing DNA encoding mature prolactin. Amplification was obtained.
DNA and pUC-rrnB (rrnB terminator inserted into pUC18 plasmid, Rigeron Co., Ltd.) encoding the mature prolactin obtained by the above method were digested with Eco RI and Kpn I, ligated with T4 DNA ligase, and then transformed into Top10F 'strain. Switched. After incubation at 37 ° C. for 15 hours, three colonies were randomly selected and cultured, plasmids were obtained by alkali lysis method, and then electrophoresed on a 1% agarose gel to confirm the size and sequence analysis. Plasmids were selected (pUC-hPRL).
Example 2: Preparation of expression vector combining narK promoter and Met-prolactin from which signal sequence was removed
PCR was performed to link the narK promoter with Met-prolactin from which the signal sequence was removed, and the 18 bases at the linking site were linked to each other in base pairs. The first PCR method is as follows. pNKmut plasmid (including 10 mutated narK promoters, Ligeron Co., Ltd., Korean Patent Application Publication No. 2006-0089086) was used as a template, and PCR (using OY-17 and r-narK D primer pairs) was used as a template. 30 cycles; 98 ° C., 10 seconds, 55 ° C., 5 seconds, 72 ° C., 25 seconds) to obtain 350 bp of narK promoter. Using pUC-hPRL as a template, PCR (30 cycles; 98 ° C. 10 sec, 55 ° C. 5 sec, 72 ° C. 55 sec) was performed using hPL 9U and hPL 8D primer pairs to remove 626 bp signal sequence. Met-prolactin was obtained.
In the secondary PCR, OY-17 and KpnI cleavage sites (GGTACC) containing EcoRI cleavage sites (GAATTC) were prepared using narK promoter (350 bp) and Met-prolactin (626 bp) as templates. Insert the included hPL 8D primer pair and perform PCR (30 cycles; 98 ° C 10 seconds, 55 ° C 5 minutes, 72 ° C 1 minute) to include 958 bp fragments (narK promoter and Met-prolactin without signal sequence). Section). 958 bp fragments (narK promoter and fragments containing Met-prolactin without signal sequence) and pUC-rrnB (rrnB terminator inserted into pUC18 plasmid, Rigeron Co., Ltd.) were digested with EcoRI and KpnI and T4 DNA ligase. After zero linkage, the Top10F 'strain was transformed. After incubation at 37 ° C. for 15 hours, three colonies were randomly selected and cultured, plasmids were obtained by alkali lysis method, and then electrophoresed on a 1% agarose gel to confirm the size and sequence analysis. Plasmids were selected (pUC-narK-Met-hPRL).
TABLE 1

TABLE 2

Example 3 Preparation of an Expression Vector Combining His-tagging narK Promoter and Met-Prolactin Removed from Signal Sequence
PCR was performed to connect a gene encoding Met-prolactin protein to a vector containing a gene sequence encoding six histidines after the narK promoter, in which 18 bases of the linking sites were linked in base pairs. The first PCR method is as follows. PCR (30 cycles) using OY-17 and His-narK D primer pairs as a template with a pNKmut plasmid (including 10 mutated narK promoters, Ligeron Co., Ltd., Korean Patent Application Publication No. 2006-0089086) 98 ° C., 10 seconds, 55 ° C., 5 seconds, 72 ° C., 25 seconds) to obtain a narK promoter containing a gene sequence encoding 6 histidines of 368 bp. Using the pUC-narK-Met-hPRL plasmid described in Example 2 as a template, PCR (30 cycles; 98 ° C 10 seconds, 55 ° C 5 seconds, 72 ° C 55 seconds) was carried out using hPL 11U and hPL 8D primer pairs. 626 bp of Met-prolactin gene was obtained.
Secondary PCR reactions included EcoRI cleavage site (GAATTC) with 368 bp (narK promoter containing 6 histidine encoding gene sequences) and 626 bp (Met-hPRL gene) obtained from the first PCR reaction. Insert a pair of hPL 8D primers containing OY-17 and KpnI cleavage site (GGTACC) and perform PCR (30 cycles; 98 ° C 10 seconds, 55 ° C 5 minutes, 72 ° C 1 minute) to obtain a 976 bp fragment (narK promoter- Six histidine-Met-prolactin gene segments) were obtained. Next, 976 bp fragments (narK promoter-6 histidine-Met-prolactin gene segments) double-digestion with EcoRI and KpnI and pUC-rrnB (rrnB terminator insertion into pUC18 plasmid, Ligeron) vector were added to T4 DNA. After linking with ligase, the strain was transformed into Top10F 'strain. After culturing at 37 ° C. for 15 hours, three colonies were randomly selected and cultured, plasmids were obtained by alkaline lysis method, and then electrophoresed on a 1% agarose gel to confirm the size and sequence analysis. Plasmids were selected (pUC-narK-his-Met-hPRL).
TABLE 3

Example 4 Preparation of Nanolipo-hPRL and Nanolipo-his-hPRL
Human prolactin and his-human prolactin were formulated nanoliposomes. Phospholipids used were soybean lecithin (Sindongbang, South Korea), Metarin P (Degussa Texturant Systems Deutschland GmbH & Co. KG), Nutripur S (Degussa Texturant Systems Deutschland GmbH & Co. KG) or Emultop (Degussa). Texturant Systems Deutschland GmbH & Co. KG.
Heat exchanger of high pressure homogenizer (max. Output 5 L / hr, max. Pressure 1200 bar, Model HS-1002, Hwasung Machinery Co., Ltd.) equipped with heat exchanger so that the temperature of equipment outlet does not exceed 30 ℃ The device was placed in ice water, washed with distilled water, and then prepared for operation. Human prolactin or his- at a concentration of 2 mg / mL dissolved in a buffer solution (20 mM NaH ₂ PO ₄ pH 6.5-7.5, 1 mM EDTA) was prepared. Phospholipid was added to the human prolactin solution at a rate of 10 w / v%, sufficiently hydrated and stirred, and passed through the homogenizer three times or more at low pressure 0 bar using a homogenizer at room temperature. Subsequently, phospholipid was added to a solution having undergone the homogenizer at a ratio of 14 w / v%, sufficiently hydrated and stirred, and passed through the homogenizer three times or more at 100 bar. Then, to the solution passed through the homogenizer, phospholipid was added at an 18 w / v% ratio, sufficiently hydrated and stirred, and passed through the homogenizer three times or more at 200 bar. Then, to the solution passed through the homogenizer, phospholipid was added at a ratio of 20 w / v%, sufficiently hydrated and stirred, and passed through the homogenizer three times or more at 300 bar. Subsequently, to the solution passed through the homogenizer, phospholipid was added at a ratio of 22 w / v%, sufficiently hydrated and stirred, and passed through the homogenizer three times or more at 400 bar. Then, to the solution passed through the homogenizer, phospholipid was added to a ratio of 24 w / v%, sufficiently hydrated and stirred, and passed through the homogenizer three times or more at 500 bar. Then, to the solution passed through the homogenizer, phospholipid was added at a ratio of 26 w / v%, sufficiently hydrated and stirred, and passed through the homogenizer three times or more at 600 bar. Subsequently, to the solution passed through the homogenizer, phospholipid was added at a ratio of 28 w / v%, sufficiently hydrated and stirred, and passed through the homogenizer three times or more at 700 bar. Then, the solution passed through the homogenizer was passed through the homogenizer three times or more at 800 bar, the solution was discharged from the homogenizer, and the supernatant was separated by high-speed centrifugation at 15,000 xg for 30 minutes. At this time, prolactin and his-prolactin not contained in liposomes were removed by gel permeation chromatography (GE Healthcare, USA) to obtain liquid liposomes.
Example 5: Evaluation of wrinkles / skin elasticity / moisture loss / epidermal thickness using hairless nude mouse
1. Preparation of experimental animals
The experimental animals were purchased from Dooyeol Biotech, female SKH-1 hairless mice, 4 weeks old (Sungnam, Korea). Experimental animals were raised under conditions of 24 ± 2 ℃, 50 ± 10% relative humidity, and 12 hours of contrast cycle. The animals were incubated for 2 weeks and then divided into 11 groups.
UV irradiator was used VLX-3W stimulator (Vilber Lourmat, Marne la Vallee, France). UVB irradiation was carried out three times a week for a total of 11 weeks. The irradiation dose was 30 mJ / cm ² for 1-2 weeks, 40 mJ / cm ^{2 for} 3-4 weeks, 50 mJ / cm ^{2 for} 5-9 weeks and 60 mJ / cm ² for 10-11 weeks. Treatment of the protein and vehicle groups was applied 300 μl each 5 times a week for a total of 11 weeks. EtOH (Merck) group and retinoic acid (retinoic acid, Sigma) group was treated with 100 μl each. In the case of UV treatment, the sample was applied one hour before UV irradiation. For application, the sample prepared in advance was spread evenly over the entire back using an art No. 3 brush (Hwasung, Chuncheon, Korea). Each protein treatment concentration is as follows.
TABLE 4

2. Analysis of wrinkle improvement efficacy of Nanolipo-hPRL using hairless mice
It is well known that thick, deep wrinkles occur in SKH-1 hairless mice when sustained UV irradiation over a long period of time. The criteria for the evaluation of the visual skin wrinkles in this experiment were also evaluated based on the generation of deep wrinkles formed by UV irradiation and the following criteria after 11 weeks of UV and the end of sample treatment.
-: Not observed
*: Mild thick and deep winkle
**: Mild to Morderate thick and deep winkle
***: Morderate thick and deep winkle
****: Order to severe thick and deep winkle
*****: Severely thick and deep winkle
As a result of the experiment, moderate or higher wrinkles were found in groups 2 to 3 and group 5, but groups 4 and 6 to 7 were able to observe wrinkles having a thickness and depth less than moderate or moderate (FIG. 1).
3. Efficacy analysis of skin elasticity and water loss of Nanolipo-hPRL using hairless mice
Skin elasticity and water loss were measured after stabilizing the test animals for 20 minutes in a constant temperature and humidity room (temperature 22 ± 2 ℃, humidity 50 ± 10%) after UV and sample treatment for 11 weeks. Using the Multi Probe Adaptor systems, MPA580 (Courage + Khazaka, Germany), which is widely used for the evaluation of skin efficacy devices, moisture loss was measured using the Texameter, TM300 probe (Courage + Khazaka, Germany) and elasticity. The results are the average of three measurements each with a Cutometer SEM 575 probe (Courage + Khazaka, Germany) (Pressure: 500 mbar, Probe aperture: 2 mm, On-time: 1 seconds, Off-time: 1 seconds , Repetitions: 5).
The experimental results showed that the effect of Nanolipo-hPRL on skin elasticity was minimal, but compared with other experiments (moisture loss, wrinkle improvement, H & E staining, artificial skin test), the number of hairless mice increased or applied in the long term. It is thought to increase skin elasticity (FIG. 2A). However, in terms of water loss, the Nanolipo-hPRL group (Group 6) and Nanolipo-his-hPRL (Group 7) compared to the EtOH group (Group 3), UV (+) group (Group 2) and liposome group (Group 5). The group showed a clear water loss prevention effect (Fig. 2b).
4. Effect of Nanolipo-hPRL on Skin Damage Inhibition using Hairless Mouse
Skin epidermal thickness measurement by tissue analysis was carried out for 11 weeks in the back skin tissue of female SKH-1 hairless mouse treated with UV and each coating agent, soaked in 0.1M phosphate fixative added with 4% paraformaldehyde at 4 ° C. After O / N treatment, the tissue was dehydrated and embedded in paraffin to prepare blocks. Next, the tissue was cut into 3 μm thick sections using a Leica microtome, and then slice slides were prepared to undergo deparaffinization and hydrolysis, followed by Herris's hematoxylin (Hematoxylin, Sigma, USA) and eosin ( eosin, Alpha Chem, Inc, UK). Then, after observation with a Zeiss Axiostar Plus microscope, the image was taken using an AxioCam MR camera and the epidermal thickness was measured using Axiovision software.
Many of the substances known to be effective in improving wrinkles in experimental animals in which wrinkles are induced by UV irradiation have been reported to prevent skin epidermis thickening by UV irradiation. As shown in FIG. 3, skin wrinkles induced by UV irradiation were markedly improved with Nanolipo-hPRL or Nanolipo-his-hPRL treatment and histologically reduced skin epidermal thickening. Perhaps the skin epidermal thickening, which is a result of abnormal processes of replication, differentiation and detachment of basal cells by UV irradiation, is thought to prevent epidermal thickening by prolactin homeostasis of skin epidermal basal cells. .
Example 6 Measurement of Inhibition of Intracellular Melanogenesis by Nanolipo-hPRL
B16F1 melanoma cells were plated in 6 well plates (6 × 10 ⁴ cells / well) and incubated for 24 hours in an incubator (5% CO ₂ , 37 ° C.), followed by DMEM medium containing α-MSH (sigma). Replace and incubate for 72 hours with arbutin (sigma) and Nanolipo-hPRL. Cell pellets were collected and centrifuged, and the supernatant was removed. The pellets were dried, and the melanin in the cells was dissolved in 1N NaOH / 10% DMSO. The absorbance was measured at 405 nm with SPECTRA MAX 190 (Molecular Devices), and the melanin standard (Sigma). Melanin inhibition rate (%) was calculated using the curve. As shown in Figure 4, Nanolipo-hPRL of the present invention showed a significant melanin inhibition rate according to the concentration.
Example 7: Determination of thickness change of epidermal layer of Nanolipo-hPRL using artificial skin
300 μl of liposomes (1%), lipo-hPRL (2 μg / ml), 10% EtOH and retinoic acid (0.01% in 10% EtOH) were placed on 4.5 cm ² artificial skin (Tegoscience., South Korea). Treatment was for days. After 24 hours after the last treatment, the mice were sacrificed to obtain artificial skin, and then fixed with 4% formalin (Yakuri, Japan) at room temperature for 5 hours, and paraffin (McCormick, USA) blocks were prepared. The paraffin block was cut to a size of 5 μm and stained with hematoxylin (Smatma, USA) and eosin (eosin, Alpha Chem, Inc, UK) and observed at 400 times with an optical microscope.
As shown in FIG. 5, the nanolipo-hPRL of the present invention seems to have an effect on health of the skin in view of the thickening of the differentiated cell layer of artificial skin in the nanolipo-hPRL treated group.
Example 8: Analysis of wrinkle improvement efficacy of Nanolipo-hPRL
Wrinkle improvement efficacy of Nanolipo-hPRL was evaluated through a practical test. The nutritional cream of Formulation Example 3 was used. Twenty 40-50 year old women were randomly divided into two groups, and the cream of Formulation Example 3 was washed twice daily each morning / evening, and then an appropriate amount of cream was applied continuously for 2 months around the eyes. Wrinkle improvement effect of each subject was evaluated through visual observation. The experimental results are shown in Table 5 below.
TABLE 5

*% Of the total number of subjects determined to have an anti-wrinkle effect
** Nanoliposome Formulation without Prolactin
As can be seen in Table 5, prolactin has a clear wrinkle improvement effect.
Example 9 Analysis of Acne Treatment Efficacy of Nanolipo-hPRL
The acne treatment efficacy of the Nanolipo-hPRL of the present invention was investigated as follows: 60 randomly divided acne on the facial skin among 20 15-40 year old women were divided into 3 groups of 20 randomly, followed by only Nanolipo-hPRL or liposomes. The comparative solution (Nanolipo) was used first after washing the face twice a day for 3 weeks morning and evening. Other than that, there are no special restrictions on the cosmetics that are used. Then, according to the user's opinion, the degree of improvement for acne was determined according to the following evaluation criteria. The test results are shown in Table 6. Evaluation standard:
+++: Very good improvement
++: Significant improvement
+: Slight improvement
±: No improvement but no deterioration
-: Worsened.
TABLE 6

As shown in Table 6, the nanolipo-hPRL of the present invention can be seen that the degree of improvement for acne is very excellent, the acne improvement effect began to appear clearly from about 2 weeks after application. Moreover, Nanolipo-hPRL of the present invention hardly caused irritation to the skin such as erythema or itching.
Example 10 Analysis of Scavenging Effect of Nanolipo-hPRL
The efficacy of the nanolipo-hPRL of the present invention was investigated as follows: Among 40-60 year old females, 60 randomly having blemishes on the facial skin were randomly divided into 3 groups of 20, followed by only Nanolipo-hPRL or liposomes. The comparative solution was first used after washing face twice a day for 8 weeks morning and evening. Other than that, there are no special restrictions on the cosmetics that are used. Then, according to the user's opinion, the degree of improvement for the mushroom was determined according to the following evaluation criteria. The test results are shown in Table 7. Evaluation standard:
+++: Very good improvement
++: Significant improvement
+: Slight improvement
±: No improvement but no deterioration
-: Worsened.
TABLE 7

As shown in Table 7, the nanolipo-hPRL of the present invention can be seen that the degree of improvement for the mushroom was considerably excellent, and the effect of improving the mushroom was apparent from about 5 weeks after application.
Example 11 Analysis of Whitening Efficacy of Nanolipo-hPRL
The whitening efficacy of Nanolipo-hPRL of the present invention was investigated as follows: 60 of 60-60 year old females who had spots or freckles on their facial skin or thought their skin was dull were randomly divided into 3 groups of 20 people. Next, nanolipo-hPRL or liposome-only comparative solution was used first after washing twice a day for 8 weeks each morning and evening. Other than that, there are no special restrictions on the cosmetics that are used. Then, according to the opinion of the user, the degree of improvement (radiance effect) on blemishes, freckles, or skin dullness was determined according to the following evaluation criteria. The test results are shown in Table 8. Evaluation standard:
+++: Removes spots and freckles and has a very good improvement effect on skin brightening
++: Significant improvement
+: Slight improvement
±: No improvement but no deterioration
-: Worsened.
TABLE 8

As shown in Table 8, it can be seen that the Nanolipo-hPRL of the present invention has a very good brightening effect on blemishes and freckles or dull skin, and this whitening effect began to be apparent from about 4 weeks after application.
Example 12 Analysis of Skin Elasticity Improvement Ability of Nanolipo-hPRL
The skin elasticity improvement effect of Nanolipo-hPRL was evaluated through a practical test. The nutritional cream of Formulation Example 3 was used. Twenty 40-50 year old women were randomly divided into two groups, and the cream of Formulation Example 3 was washed twice daily each morning / evening, and then the appropriate amount of cream was applied to the face and neck continuously for two months. The skin elasticity improvement effect of each subject was evaluated through the questionnaire as follows.
Evaluation criteria: +++ (very good improvement), ++ (significant improvement), + (slight improvement), ± (no improvement, but no deterioration),-(deterioration) ).
The survey results are listed in Table 9 below.
TABLE 9

As can be seen in Table 9, prolactin shows a clear skin elasticity improving effect.
Example 13: Analysis of wound healing ability of Nanolipo-hPRL
The wound or ulcer healing ability of the Nanolipo-hPRL of the present invention was investigated as follows.
Nanolipo-hPRL of the present invention has been applied to patients with wounds or ulcers, such as bedsores, diabetic foot ulcers and systemic lupus ulcer ulcers. Sixty patients with these wounds were randomly divided into three groups of 20, and then nanolipo-hPRL or liposome-only comparative solutions were applied to the wound site twice daily for eight weeks each morning / evening. Then, according to the opinion of the user, the degree of improvement for the wound was judged according to the following evaluation criteria. The test results are shown in Tables 10a-10c. Evaluation criteria: +++ (very good improvement), ++ (significant improvement), + (slight improvement), ± (no improvement, but no deterioration),-(deterioration) ).
TABLE 10a

TABLE 10b

TABLE 10c

As shown in Table 10a-10c, Nanolipo-hPRL of the present invention showed an improvement effect on the representative examples of wounds or ulcers, namely pressure sores, diabetic foot ulcers and systemic lupus ulcer ulcers. This wound improvement began to manifest itself about 3-4 weeks after application.
Example 14: Effect of topical therapy of Nanolipo-hPRL on wound healing
The wound or ulcer healing ability of the Nanolipo-hPRL of the present invention was investigated as follows.
The experiment was conducted for 3 weeks (21 days) and micropig (4 months, male, 9.5 kg, flesh color, short hair) / 1 was used. Anesthesia was pre- and deep-anesthesia, ketamine: 1 ml / 10 kg (Yuhan), xylazine: 1 ml / 10 kg (intervet Korea), isoforane: oxygen = 2.5: 2.5 Cardiac anesthesia was performed to make 10 wounds per head (Sino-Pharmaceuticals): 24 hours before fasting-12 hours after anesthesia.
Induction of wounds was performed by anesthesia first, and then the hair was removed using a cliper, and the extraction area was marked with a pen at a size of 2 × 2 cm (4 cm ² ) at intervals of 4 cm on the back. After sterilizing the dorsal area to be extracted with betadine, the skin was collected by using a skin motor to remove the partial layered skin of 0.6mm thickness and removing the skin of 2 × 2cm (4cm ² ) area from the back.
Right 1st, left 2nd edge, right 3rd, left 4th and 5th right from head side control (C), left 1st, right 2nd, left 3rd, right 4th and left 5 from head side The first site was used as the experimental group (P).
Application methods are shown in Table 11.
TABLE 11

The dressing was closed dressing using film dressing (Opsite) in both the control and experimental groups, fixed with mephix (tape), wrapped around the entire body with bandages (EB) to protect the wound, and then the stockinet (the elasticity shown in the fourth photo) Large cloth). 6 illustrates the above process.
Wound treatment (dressing) was performed every two days, and the following were performed simultaneously. Anesthesia was possible only with inhalation anesthesia because of simple dressing and skin extraction. 1) Wound area measurement- Model the wound site using OHP film at weekly intervals (7 days, 14 days, 21 days), measure the area using the planimeter area measurement program, and then cut the wound for the initial wound area size. Convert size to proportions. 2) Apply the sample in 1ml amount to the wound every 2 days. 3) Apply a 4 cm x 4 cm adhesive cloth to prevent each sample from falling off the wound. 4) Treat the adhesive cloth around with micropore (3M). 5) Wrap the whole body with stokinet and fix it with adhesive tape to avoid the wound
Experimental results show that the wound size changes are shown in Table 12 (Figure 7):
TABLE 12

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (10)

Composition for improving skin conditions (human conditions) comprising human prolactin as an active ingredient. A composition for treating wounds or ulcers comprising human prolactin as an active ingredient. The composition of claim 1 or 2, wherein said human prolactin is surrounded by liposomes. The composition of claim 3, wherein the liposomes are nanoliposomes. The composition of claim 1, wherein the improvement of the skin condition is wrinkle improvement, blotch removal, skin blemish removal, whitening, acne treatment, skin elasticity improvement or skin aging prevention. 3. The wound or ulcer of claim 2 wherein the wound or ulcer is an aneurysm ulcer, varicose ulcer, varicose vein ulcer, diabetic ulcer, diabetic foot ulcer, compression ulcer, systemic lupus ulcer ulcer or pressure sore. Ulcerative composition for treatment. The composition of claim 1 or 2, wherein the composition is a pharmaceutical composition, a cosmetic composition or a cosmetic composition. The composition according to claim 1 or 2, wherein the composition is for topical application of skin. A method of improving skin conditions comprising administering to a human skin an effective amount of human prolactin. A method of treating a wound or ulcer comprising administering to a human skin an effective amount of human prolactin.

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