WO2013109016A1 - Novel use for melatonin - Google Patents

Novel use for melatonin Download PDF

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Publication number
WO2013109016A1
WO2013109016A1 PCT/KR2013/000171 KR2013000171W WO2013109016A1 WO 2013109016 A1 WO2013109016 A1 WO 2013109016A1 KR 2013000171 W KR2013000171 W KR 2013000171W WO 2013109016 A1 WO2013109016 A1 WO 2013109016A1
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WO
WIPO (PCT)
Prior art keywords
disease
melatonin
prion
present
composition
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PCT/KR2013/000171
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French (fr)
Korean (ko)
Inventor
박상열
정재교
이유진
설재원
김재훈
박양규
이주희
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전북대학교산학협력단
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Publication of WO2013109016A1 publication Critical patent/WO2013109016A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates to a composition for preventing and treating prion diseases. More specifically, the present invention relates to a pharmaceutical composition and a food composition for preventing and treating prion diseases, including melatonin as an active ingredient.
  • Prion is a disease infector that is completely different from the existing pathogens such as bacteria, viruses, bears, and parasites. It is much smaller than ordinary viruses and causes infectious diseases that exclude genetic material, nucleic acid. Has When prions infect animals, including humans, sponge-like fears form in the brain, causing neuronal death, leading to degenerative neuropathy.
  • ⁇ 3> Prion disease is not known to date, and many scientists estimate that the occurrence of prion disease may be related to normal prion protein ( ⁇ ).
  • ⁇ 1 ⁇ is a glycoprotein present in the cell membrane, and most of it is expressed in the brain, and little is known about its in vivo function, but it is known to have cell growth and partial signaling functions.
  • ⁇ ! ⁇ has an abnormal form, called modified prion protein (PrP). Unlike ⁇ , they tend to coagulate with each other, and it is estimated that these cohorts cause infectious sponiform encephalopathy (TSE).
  • ⁇ consists of three helices (a helix) and two screens ( ⁇ sheets), which are converted to ⁇ ! ⁇ through a mechanism unknown to date.
  • ⁇ ! ⁇ Has more folding structure than PrP C, and it is condensed with each other to become insoluble, protease
  • Representative prion diseases include human CJD Creutzfeldt-Jakob disease,
  • Bovine spongiform encephalopathy BSE
  • scrapie of sheep CWD
  • CWD chronic wasting disease
  • TEE transmissible mink encephalopathy
  • prion disease spreads homogeneously but causes greater problems when it has spread to heterogeneity.
  • the most representative example of this is human vCJD, which has spread from bovine BSE.
  • the inventors of the present application conducted research to develop a drug capable of effectively preventing and treating prion diseases, and as a result, confirmed that melatonin selectively inhibits prion-induced neurotoxin, and completed the present invention.
  • an object of the present invention is to provide a pharmaceutical composition for preventing and treating prion diseases.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of pre-silver disease containing melatonin as an active ingredient.
  • the prion disease is preferably human Creutzfeldt-Jakob disease
  • the present invention provides a food composition for preventing or improving prion disease containing melatonin.
  • melatonin does not affect cell viability, and selectively inhibits prion-induced neurotoxins
  • the composition comprising the same may be used as an effective medicine and food for preventing or treating prion diseases. Can be.
  • FIG. 1 shows the results of pretreatment with melatonin (12 hours) in proportion to concentration of SH-SY5Y neurons and exposure to 50 ⁇ PrP (106-126) for 24 hours.
  • Cell viability was measured by annexin V assay.
  • Ml represents the distribution of Annexin V positive cells.
  • FIG. 2 is a bar graph showing the average of Annexin V positive cells. The significant difference between the control and the experimental group was * P ⁇ 0.05, ** P ⁇ 0.001, and the significant difference between PrP (106-126) and experimental groups was #p ⁇ 0.01.
  • FIG. 3 shows 50 ⁇ of PrP (106-126), with and without melatonin (12 hours).
  • the present invention provides a pharmaceutical composition for preventing or treating prion diseases containing melatonin as an active ingredient.
  • the inventors of the present invention exposed a neuroblastoma showing PrP-induced apoptosis to melatonin, and performed an annexin V assay.
  • Annexin V as a marker of early apoptosis is a widely used protein in flow cytometry because it has a strong binding affinity for PS.
  • PS is an important phosphorus lipid in the cell membrane that is exposed to the outside of the cell membrane surface early in the apoptosis process. PS is a well known "eat me" signal during apoptosis. Regardless of the type of cell and apoptosis inducer, PS is expressed on the outer cell membrane very early after apoptosis begins.
  • Annexin V has an anion when there is calcium ion It is a protein that binds to the phospholipid surface [14].
  • Annex in V consists of 320 amino acids and has a molecular weight of 36 kDa.
  • annexin V can be modified and stabilized to serve as an appropriate biological marker for assessing cell death.
  • Various fluorescently labeled annexin Vs have been successfully used for the measurement of apoptosis. As a result, it was confirmed that melatonin exhibited the effect of selectively inhibiting the neurotoxin induced by prion in neuroblastoma.
  • Melatonin in the present invention is a hormone secreted by the pineal gland of the brain, regulates sleep and daily rhythm, and devours free radicals derived from oxygen, It has antioxidant properties by stimulating antioxidant enzymes such as peroxidase, glutathione peroxide enzyme, glutathione reductase, and glucose ⁇ 6—phosphate dehydrogenase.
  • Prion diseases in the present invention may include all neurological diseases related to prion-mediated apoptosis, and specifically, human Creutzfeldt-Jakob disease and Kuru disease. ), Gerstmann-Strsyndrome, fatal familial insomnia; bovine spongiform encephalopathy (bovine BSE); scrapie of sheep; chronic wasting of deer disease) or mink's TME (transmissible mink encephalopathy) and the like.
  • composition of the present invention may contain one or more known active ingredients having a prophylactic or therapeutic effect in conjunction with melatonin.
  • the melatonin is 0.001 to 50% by weight, preferably in the total pharmaceutical composition
  • the melatonin may be adjusted in a content so that the appropriate amount per 1kg of the individual to which the pharmaceutical composition is administered in the entire pharmaceutical composition can be administered.
  • the melatonin is included in the pharmaceutical composition to be administered in an amount of 50 mg / kg to 150 mg / kg, preferably 80 mg / kg to 120 mg / kg. Can be controlled.
  • the melatonin is contained in the pharmaceutical composition to be administered in an amount of 0.0001 to 50 (g / kg, preferably 0.001 to 500 mg / kg, more preferably 0.001 to 300 mg / kg The amount included can be adjusted.
  • the pharmaceutical composition of the present invention does not increase the efficacy, but It may further include ingredients that are commonly used to improve the smell, taste, vision and the like.
  • the composition may be inorganic or organic, such as vitamins B1, B2, B6, C, E, niacin, carnitine, betaine, folate pantothenic acid, biotin, zinc, iron, calcium, cream, magnesium, and combinations thereof. Additives may further be included.
  • the composition may include a substance that is used alone or has a prophylactic or therapeutic activity against a previously used prion disease.
  • the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier and may be formulated for human or veterinary use for oral or parenteral use.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants can be used.
  • Solid form preparations for oral administration include sperm, pills, powders, granules and capsules. Such solid form preparations may comprise at least one excipient such as starch, calcium carbonate in a composition comprising a compound of the present invention. ), Sucrose or lactose, and 3 ⁇ 4 Latin.
  • lubricants such as magnesium, styrate and talc may be used.
  • Oral liquid preparations include suspensions, solvents, emulsions and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are commonly used simple limes.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and suppositories.
  • non-aqueous and suspending solvent vegetable oils such as propylene glycol, polyethylene glycol and olive oil, injectable esters such as ethyl oleate and the like can be used.
  • the pharmaceutical composition of the present invention may be administered to a subject to prevent or treat free silver disease.
  • the term "individual” has a disease caused by prion disease or its direct or indirect cause, including humans having a disease that can be improved by administering the pharmaceutical composition of the present invention. Mammals such as cattle, sheep deer, and mink.
  • the term "administration" means introducing the pharmaceutical composition of the present invention to a subject in any suitable manner.
  • the route of administration may be oral or parenteral via any general route so long as it can reach the desired tissue.
  • the pharmaceutical composition of the present invention may be administered by any device that allows migration to target cells.
  • composition of the present invention is administered in a pharmaceutically effective amount.
  • “Pharmaceutically effective amount” for a given dose means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the sex, age, severity, drug activity or drug of the patient. Sensitivity to, time of administration, route of administration and rate of administration, duration of treatment, factors including concomitant drug use, and other factors well known in the medical arts.
  • the compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may be single or multiple doses.
  • the administration method of the composition containing the compound prepared according to the preparation method of the present invention is preferably oral administration or intravenous administration. Dosage levels for a particular patient may vary depending on gender, age, health diet, time of administration, method of administration, drug combination and severity of disease.
  • the present invention provides a food composition for preventing and improving prion diseases, including melatonin.
  • the food composition of the present invention may be added to a health food for the purpose of suppressing prion disease.
  • the composition of the present invention When the composition of the present invention is used as a food additive, the composition may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method.
  • the amount of active ingredient can be determined appropriately depending on the purpose of use (prevention, health or therapeutic treatment).
  • the compositions of the invention are added in an amount of up to 15% by weight, preferably up to 10% by weight relative to the raw materials.
  • the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.
  • Examples of foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, 3 ⁇ 4, ice cream, various soups, drinks, tea, Drink preparations, alcoholic beverages and vitamin complexes, including all health foods in the conventional sense.
  • the natural carbohydrates mentioned above are the parent Disaccharides such as nosaccharide, maltose and sucrose, and natural sweeteners such as dextrin and cyclotex trine, and synthetic sweeteners such as saccharin and aspartame.
  • the ratio of the natural carbohydrate is generally about 0.001 to 0.4g, preferably about 0.002 to 0.03g per 100 food compositions of the present invention.
  • the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, Glycerin, alcohol, carbonation agent used in carbonated drinks, and the like.
  • the food composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0.001 to 0.1 parts by weight per 100 parts by weight of the food composition of the present invention.
  • Cells Obtained from Culture collection (ATCC, Rockville, MD, USA). Cells are minimal essential medium (Hyclone Laboratories, Logan) containing 10% fetal bovine serum (FBS; Invitrogen-Gibco, Grand Island, NY, USA) and gentamycin (0.1 mg / ml) , UT, USA) and were maintained in a humidified incubator at 37 ° C and 5% CO 2 conditions.
  • FBS fetal bovine serum
  • gentamycin 0.1 mg / ml
  • Peptron (Seoul, Korea). Peptides were dissolved in sterile dimethylsulfoxide at a concentration of 12.5 mM and stored at -80 ° C.
  • Apoptosis was determined by performing Annexin V assay on detached cells (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the protocol provided by the producer.
  • the measurement of Annexin V is by fluorescence measurements at an excitation wavelength of 488 nm and an emission wavelength of 525-530 nm, which is equivalent to the Guava EasyCyte HT apparatus (Millipore, Billerica, Mass., USA). Use.
  • TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling
  • TUNEL analysis was performed using an in situ ApoBrdU DNA fragmentation assay kit (BioVision, San Francisco, CA, USA) according to the protocol provided by the producer, which was performed to measure the degree of apoptosis.
  • Cells were washed with phosphate buffer saline (PBS) and fixed for 15 minutes with paraformaldehyde.
  • PBS phosphate buffer saline
  • the average of the annexin V-positive cells is represented by a bar graph as shown in FIG. 2.
  • the present invention relates to a composition for the prevention and treatment of prionone disease, and is applicable to the industry for the treatment and prevention of prion disease.

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Abstract

The present invention relates to a pharmaceutical composition and a food composition for preventing and treating prion diseases which comprises melatonin as an active ingredient. According to the present invention, a composition comprising melatonin can be used advantageously as a medicinal product and food that is effective in preventing or treating a prion disease since melatonin does not have any effect on the cell survival rate while selectively suppressing prion-induced cytotoxicity.

Description

【명세서】  【Specification】
【발명의 명칭】  [Name of invention]
멜라토닌의 새로운 용도  New Uses of Melatonin
【기술분야】  Technical Field
<1> 본 발명은 프리온 질병 예방 및 치료용 조성물에 관한 것이다. 보다 구체적 으로 본 발명은 멜라토닌을 유효성분으로 포함하는 프리온 질병 예방 및 치료용 약 학적 조성물 및 식품 조성물에 관한 것이다.  <1> The present invention relates to a composition for preventing and treating prion diseases. More specifically, the present invention relates to a pharmaceutical composition and a food composition for preventing and treating prion diseases, including melatonin as an active ingredient.
【배경기술】  Background Art
<2> 프리온 (Prion)은 박테리아, 바이러스， 곰광이 , 기생충과 같은 기존의 병원체 와는 전혀 다른 종류의 질병 감염인자로서， 보통의 바이러스보다 훨씬 작고， 유전 물질인 핵산이 배제된 감염성 질환을 일으키는 특징을 가진다. 프리온이 사람을 포 함하여 동물에 감염되면 뇌에서 스펀지 형태의 공포가 형성되어 신경세포사를 일으 켜 퇴행성 신경질환을 유발한다.  <2> Prion is a disease infector that is completely different from the existing pathogens such as bacteria, viruses, bears, and parasites. It is much smaller than ordinary viruses and causes infectious diseases that exclude genetic material, nucleic acid. Has When prions infect animals, including humans, sponge-like fears form in the brain, causing neuronal death, leading to degenerative neuropathy.
<3> 프리온 질병은 현재까지도 정확한 발생기전이 밝혀져 있지 않으며， 많은 과 학자들은 프리온 질병의 발생이 정상적인 프리온 단백질 ( ^)과 연관이 있을 것으 로 추정하고 있다. ^1^는 세포막에 존재하는 당단백질로서 주로 대부분이 뇌에서 발현되며， 생체 내 기능에 대해서는 많이 밝혀져 있지 않으나 세포성장 및 부분적 인 신호전달 기능을 갖는 것으로 알려져 있다. 특정 상황에서 ^！^는 비정상적인 형 태를 취하는데， 이를 변형 프리온 단백질 (PrP )이라고 한다. ！^는 ^^와 달리 서로 웅집하는 경향이 있고， 이러한 ！^！^의 웅집체가 전염성 해면상 뇌증 (tansmissible spongiform encephalopathy; TSE)의 전염성을 일으킨다고 추정하고 있다. ^^는 3개의 나선구조 (a helix)와 2개의 병풍구조 (β sheet)로 이루어져 있 으며， 현재까지 잘 알려지지 않은 기전을 통해 ^！^로 전환된다. ^！^는 PrPC보다 병풍구조가 더 많으며， 서로 웅집하여 수불용성 (insoluble)이 되고， 단백분해효소<3> Prion disease is not known to date, and many scientists estimate that the occurrence of prion disease may be related to normal prion protein (^). ^ 1 ^ is a glycoprotein present in the cell membrane, and most of it is expressed in the brain, and little is known about its in vivo function, but it is known to have cell growth and partial signaling functions. In certain situations, ^！ ^ has an abnormal form, called modified prion protein (PrP). Unlike ^^, they tend to coagulate with each other, and it is estimated that these cohorts cause infectious sponiform encephalopathy (TSE). ^^ consists of three helices (a helix) and two screens (β sheets), which are converted to ^！ ^ through a mechanism unknown to date. ^！ ^ Has more folding structure than PrP C, and it is condensed with each other to become insoluble, protease
KCproteinase K)에 의해 완전히 분해되지 않는다. 또한, ！^는 ^！^와 입체구조가 전혀 다름에도 불구하고， 생체의 면역시스템은 PrPSc를 외부물질로 인식하지 못하고 있다. It is not completely degraded by KCproteinase K). In addition, although！ ^ is completely different from ^！ ^, the immune system of the living body does not recognize PrP Sc as an external substance.
<4> 모든 전염성 해면상 뇌증 질환에 있어서 발병 기전은 신경세포사를 유도함으 로써 뇌에 스폰지 형태의 공동과 같은 병변을 일으켜 뇌의 손상을 야기하는 것으로 알려져 있다. 그러나， 현재까지도 이러한 병변들이 PrP 의 단독 작용에 의한 것인 지 ^！^도 일부 관여를 하는지는 밝혀져 있지 않다. ? 는 다양한 포유류에서 진 행성 신경질환을 일으키며， 그 치사율은 100%에 이른다. <4> In all infectious spongiform encephalopathy diseases, the pathogenesis mechanism causes neuronal cell death and causes brain-like lesions in the brain by causing sponge-like cavities. Known. However, it is still unknown whether these lesions are caused by PrP alone or ^! ^. ? Causes severe planetary neuropathy in various mammals, with a mortality rate of 100%.
<5> 대표적인 프리온 질병으로는 인간의 CJD Creutzfeldt -Jakob disease), 소의 Representative prion diseases include human CJD Creutzfeldt-Jakob disease,
BSE (bovine spongiform encephalopathy) , 양의 스크래피 (scrapie)， 사슴의 CWD (chronic wasting disease) , 밍크의 TME(transmissible mink encephalopathy) 등이 알려져 있다. 일반적으로， 프리온 질병은 동종 내에서 전파되지만 이종으로 전이되 었을 때 더 큰 문제를 야기한다. 이의 가장 대표적인 예로는， 소의 BSE로부터 전이 되어 발병한 인간의 vCJD를 들 수 있다. Bovine spongiform encephalopathy (BSE), scrapie of sheep, chronic wasting disease (CWD) of deer, and transmissible mink encephalopathy (TME) of mink are known. In general, prion disease spreads homogeneously but causes greater problems when it has spread to heterogeneity. The most representative example of this is human vCJD, which has spread from bovine BSE.
<6> 현재 알려져 있는 모든 프리온 질병들에 대한 예방 또는 치료제는 연구 및 개발 단계에 있어서 아직 실용화되어 있지 않다. 항프리은 효과를 갖는 물질로는 퀴나크린， 퀴놀린， 丽 앱타머 및 항 PrP 항체 등이 알려져 있으나， 이들 물질들은 세포 및 동물 실험단계에 머물러 있을 뿐 만족할 만한 효과를 얻지 못하여， 아직까 지 이에 대한 연구 결과는 미미한 실정이다.  Preventive or therapeutic agents for all currently known prion diseases are not yet in practical use in the research and development stages. Quinacrine, quinoline, lia aptamer, and anti-PrP antibodies are known as antifreezing agents, but these substances remain in the experimental stages of cells and animals, but have not been satisfactory. The results are insignificant.
【발명의 상세한 설명】  [Detailed Description of the Invention]
【기술적 과제】  [Technical problem]
<7> 본 출원의 발명자들은 효과적인 프리온 질병 예방 및 치료가 가능한 약물을 개발하기 위해 연구를 수행한 결과， 멜라토닌이 프리온으로 유발된 신경독소를 선 택적으로 억제한다는 것을 확인하고 본 발명을 완성하였다.  The inventors of the present application conducted research to develop a drug capable of effectively preventing and treating prion diseases, and as a result, confirmed that melatonin selectively inhibits prion-induced neurotoxin, and completed the present invention.
<8> 이에 따라， 본 발명의 목적은 프리온 질병 예방 및 치료용 약학적 조성물을 제공하는 것이다.  Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing and treating prion diseases.
【기술적 해결방법】  Technical Solution
<9> 상기와 같은 목적을 달성하기 위하여， 본 발명은 멜라토닌을 유효성분으로 함유하는 프리은 질병의 예방 또는 치료용 약학 조성물을 제공한다.  In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of pre-silver disease containing melatonin as an active ingredient.
<10> 상기 프리온 질병은 바람직하게는 인간의 크로이츠펠트-야코프병 <10> The prion disease is preferably human Creutzfeldt-Jakob disease
(Creutzfeldt— Jakob disease), 쿠루병 (Kuru disease), 게르스트만 -슈트로이슬러-샤 인커병 (Gerstmann-Strsyndrome), 치명적 가족성 불면증 (fatal familial insomnia); 소의 BSE (bovine spongiform encephalopathy)； 양의 스크래피 (scrapie)； 사슴의 CWD (chronic wasting disease)； 또는 밍크의 TME(transmissible mink encephalopathy)일 수 있다.  (Creutzfeldt— Jakob disease), Kuru disease, Gerstmann-Strsyndrome, fatal familial insomnia; Bovine spongiform encephalopathy (BSE); scrapie of sheep; chronic wasting disease (CWD) of deer; or transmissible mink encephalopathy (TME) of mink.
<ιι> 또한， 본 발명은 멜라토닌을 함유하는 프리온 질병 예방 또는 개선용 식품 조성물을 제공한다. 【유리한 효과】 In addition, the present invention provides a food composition for preventing or improving prion disease containing melatonin. Advantageous Effects
<12> 본 발명에 따르면 멜라토닌은 세포의 생존율에는 영향을 미치지 아니하며， 프리온으로 유발된 신경독소를 선택적으로 억제하는 바， 이를 포함하는 조성물은 프리온 질병 예방 또는 치료에 효과적인 의약품과 식품으로 유용하게 이용할 수 있 다.  According to the present invention, melatonin does not affect cell viability, and selectively inhibits prion-induced neurotoxins, and the composition comprising the same may be used as an effective medicine and food for preventing or treating prion diseases. Can be.
【도면의 간단한 설명】  [Brief Description of Drawings]
<13> 도 1은 SH-SY5Y 신경세포에 농도 비례적으로 멜라토닌 (12시간)을 선처리하 고 24시간 동안 50 μΜ의 PrP (106-126)에 노출한 결과이다. 세포생존력은 아넥신 V 에세이 (annexin V assay)로 측정하였다.. Ml은 아넥신 V 양성 세포의 분포를 나 타낸다.  FIG. 1 shows the results of pretreatment with melatonin (12 hours) in proportion to concentration of SH-SY5Y neurons and exposure to 50 μΜ PrP (106-126) for 24 hours. Cell viability was measured by annexin V assay. Ml represents the distribution of Annexin V positive cells.
<|4> 도 2는 아넥신 V 양성 세포의 평균을 나타내는 막대그래프이다. 대조구와 실 험구 간의 유의적 차이는 *P < 0.05, **P < 0.001, PrP (106-126) 처리구와 실험 구와의 유의적 차이는 #p < 0.01 이다.  <| 4> FIG. 2 is a bar graph showing the average of Annexin V positive cells. The significant difference between the control and the experimental group was * P <0.05, ** P <0.001, and the significant difference between PrP (106-126) and experimental groups was #p <0.01.
<15> 도 3은 멜라토닌의 존재 (12시간) 및 비존재하에서， 50 μΜ의 PrP (106-126)  FIG. 3 shows 50 μΜ of PrP (106-126), with and without melatonin (12 hours).
에 노출된 TUNELᅳ양성 (초록색) SH-SY5Y세포의 면역형광 이미지이다. 세포는 세포 의 핵을 나타내기 위하여 PI (빨간색)로 대비염색되었다. 확대율 400X , 스케일 바 (scale bar) = 100 y m.  Immunofluorescence image of TUNEL® positive (green) SH-SY5Y cells exposed to. Cells were counterstained with PI (red) to indicate cell nuclei. Magnification 400X, scale bar = 100 y m.
<!6>  <! 6>
<I7> <I7>
【발명의 실시를 위한 형태】  [Form for implementation of invention]
<18> 본 발명은 멜라토닌을 유효성분으로 함유하는 프리온 질병의 예방 또는 치료 용 약학 조성물을 제공한다.  The present invention provides a pharmaceutical composition for preventing or treating prion diseases containing melatonin as an active ingredient.
<19> 이하， 본 발명에 대해 상세히 설명한다.  Hereinafter, the present invention will be described in detail.
<20> 본 발명의 발명자들은 PrP로 유발된 세포자멸을 나타내는 신경아세포종을 멜 라토닌에 노출하였으며， 이를 대상으로 아넥신 V 에세이를 실시하였다. 초기 세포 자멸의 표지자 (marker)로서의 annexin V는 PS에 대하여 강한 결합 친화력을 가지고 있기 때문에 유세포 분석에서 널리 사용되는 단백질이다. PS는 세포막의 중요한 인 지질로서 세포자멸 과정 초기에 세포막 표면의 바깥쪽으로 노출된다. PS는 세포자 멸 과정에서 잘 알려진 "eat me" 신호이다. 세포와 세포자멸 유발제의 종류에 관 계없이 세포자멸이 시작된 후 매우 초기에 PS가 외부 세포막에 발현된다. PS 수용 체에 세포자멸에 의해 발현된 PS가 결합하는 것은 대식세포의 음세포 작용 (pinocytosis)을 활성화시킨다. Annexin V는 칼슘 이온이 있을 시에 음이온을 가진 인지질 표면과 결합하는 단백질이다 [14]. Annex in V는 320 개의 아미노산으로 구성 되어 있으며 분자량은 36 kDa이다. PS의 위치 이동 (PStranslocat ion)을 이용한 세 포자멸의 검출에 annexin V를 사용한 여러 가지 연구들이 수행되어 왔다. 또한 annex in V를 개질하고 안정화시켜 세포의 죽음을 평가하는데 있어서 적절한 생물학 적 표지로 사용할 수 있다는 것이 증명되어 왔다. 여러 가지 형광 물질이 표지된 annexin V가 세포자멸의 측정에 성공적으로 사용되어 왔다. 그 결과， 멜라토닌이 신경아세포종에서 프리온이 유발하는 신경독소를 선택적으로 억제하는 효과를 나타 냄을 확인하였다. The inventors of the present invention exposed a neuroblastoma showing PrP-induced apoptosis to melatonin, and performed an annexin V assay. Annexin V as a marker of early apoptosis is a widely used protein in flow cytometry because it has a strong binding affinity for PS. PS is an important phosphorus lipid in the cell membrane that is exposed to the outside of the cell membrane surface early in the apoptosis process. PS is a well known "eat me" signal during apoptosis. Regardless of the type of cell and apoptosis inducer, PS is expressed on the outer cell membrane very early after apoptosis begins. The binding of PS expressed by apoptosis to the PS receptor activates the pinocytosis of macrophages. Annexin V has an anion when there is calcium ion It is a protein that binds to the phospholipid surface [14]. Annex in V consists of 320 amino acids and has a molecular weight of 36 kDa. Several studies have been performed using annexin V to detect cell death by PStranslocat ion. It has also been demonstrated that annex in V can be modified and stabilized to serve as an appropriate biological marker for assessing cell death. Various fluorescently labeled annexin Vs have been successfully used for the measurement of apoptosis. As a result, it was confirmed that melatonin exhibited the effect of selectively inhibiting the neurotoxin induced by prion in neuroblastoma.
<2i> 본 발명에서의 멜라토닌 (melatonin, 5-methoxy-N— acetyl-trypamine)은 뇌의 송과선에서 분비되는 호르몬으로， 수면과 일일 주기성 리듬을 조절하고, 산소에서 유래한 자유라디칼을 탐식하고， 과산화 환원효소， 글루타치온 퍼옥사이드 효소, 글 루타치온 환원효소， 글루코스ᅳ 6—인산 탈수소 효소와 같은 항산화 효소를 자극하여 항산화 특성을 가지고 있다.  <2i> Melatonin (melatonin, 5-methoxy-N—acetyl-trypamine) in the present invention is a hormone secreted by the pineal gland of the brain, regulates sleep and daily rhythm, and devours free radicals derived from oxygen, It has antioxidant properties by stimulating antioxidant enzymes such as peroxidase, glutathione peroxide enzyme, glutathione reductase, and glucose ᅳ 6—phosphate dehydrogenase.
<22> 본 발명에서의 프리온 질병은 프리온 매개 세포자멸 (apoptosis) 와 관련된 신경질환을 모두 포함할 수 있으며， 구체적으로는 인간의 크로이츠펠트ᅳ야코프병 (Creutzfeldt-Jakob disease), 쿠루병 (Kuru disease), 게르스트만 -슈트로이슬러-샤 인커병 (Gerstmann-Strsyndrome), 치명적 가족성 불면증 (fatal familial insomnia)； 소의 BSE (bovine spongiform encephalopathy)； 양의 스크래피 (scrapie)； 사슴의 CWD( chronic wasting disease)； 또는 밍크의 TME(transmissible mink encephalopathy) 등을 포함하나 이에 한정되지 않는다.  Prion diseases in the present invention may include all neurological diseases related to prion-mediated apoptosis, and specifically, human Creutzfeldt-Jakob disease and Kuru disease. ), Gerstmann-Strsyndrome, fatal familial insomnia; bovine spongiform encephalopathy (bovine BSE); scrapie of sheep; chronic wasting of deer disease) or mink's TME (transmissible mink encephalopathy) and the like.
<23> 본 발명의 조성물은 멜라토닌과 함께 프리온 질병의 예방 또는 치료 효과를 갖는 공지의 유효성분을 1종 이상 함유할 수 있다.  The composition of the present invention may contain one or more known active ingredients having a prophylactic or therapeutic effect in conjunction with melatonin.
<24> 상기 멜라토닌은 전체 약학적 조성물에서 0.001 내지 50중량 %, 바람직하게는  The melatonin is 0.001 to 50% by weight, preferably in the total pharmaceutical composition
0.01 내지 20중량 % 포함될 수 있다. 또한， 상기 멜라토닌은 전체 약학적 조성물에 서 상기 약학적 조성물이 투여되는 개체의 1kg당 적정량이 투여될 수 있도록 함량 을 조절할 수 있다. 예를 들어， 상기 투여되는 개체가 래트 (Rat)인 경우 상기 멜 라토닌은 50mg/kg 내지 150mg/kg, 바람직하게는 80mg/kg 내지 120mg/kg의 함량으로 투여될 수 있도록 약학적 조성물에 포함되는 함량이 조절될 수 있다. 또한， 상기 투여되는 개체가 인간인 경우 상기 멜라토닌은 0.0001 내지 50( g/kg, 바람직하게 는 0.001 내지 500mg/kg, 보다 바람직하게는 0.001 내지 300mg/kg의 함량으로 투여 될 수 있도록 약학적 조성물에 포함되는 함량이 조절될 수 있다.  0.01 to 20% by weight may be included. In addition, the melatonin may be adjusted in a content so that the appropriate amount per 1kg of the individual to which the pharmaceutical composition is administered in the entire pharmaceutical composition can be administered. For example, when the subject to be administered is a rat, the melatonin is included in the pharmaceutical composition to be administered in an amount of 50 mg / kg to 150 mg / kg, preferably 80 mg / kg to 120 mg / kg. Can be controlled. In addition, when the subject is a human, the melatonin is contained in the pharmaceutical composition to be administered in an amount of 0.0001 to 50 (g / kg, preferably 0.001 to 500 mg / kg, more preferably 0.001 to 300 mg / kg The amount included can be adjusted.
<25> 본 발명의 약학적 조성물은 약효를 증가시키지는 않으나， 약학적 조성물에 통상 사용되어 냄새， 맛， 시각 등을 향상시킬 수 있는 성분을 추가로 포함할 수 있 다. 또한， 상기 조성물은 비타민 Bl, B2, B6, C, E, 니아신， 카르니친， 베타인， 엽 산 판토텐산， 비오틴， 아연， 철， 칼슘， 크름， 마그네슘 및 이들의 흔합물 등의 무 기 또는 유기 첨가물들을 추가로 포함할 수 있다. 또한， 상기 조성물은 단독 사용 하거나 기존에 사용된 프리온 질병에 대한 예방 또는 치료 활성을 가지는 물질을 포함할 수 있다. <25> The pharmaceutical composition of the present invention does not increase the efficacy, but It may further include ingredients that are commonly used to improve the smell, taste, vision and the like. In addition, the composition may be inorganic or organic, such as vitamins B1, B2, B6, C, E, niacin, carnitine, betaine, folate pantothenic acid, biotin, zinc, iron, calcium, cream, magnesium, and combinations thereof. Additives may further be included. In addition, the composition may include a substance that is used alone or has a prophylactic or therapeutic activity against a previously used prion disease.
<26> 본 발명의 상기 약학적 조성물은 약학적으로 허용 가능한 담체를 포함하고 경구 또는 비경구용의 인체 또는 수의용으로 제형화될 수 있다. 본 발명의 조성물 을 제제화하는 경우 충진제, 증량제， 결합제, 습윤제， 붕해제 및 계면활성제 등의 희석제 또는 부형제를 사용할 수 있다. 경구투여를 위한 고형제제에는 정게, 환제， 산제， 과립제 및 캡슐제 등이 포함되몌 이러한 고형제제는 본 발명의 화합물을 포 함하는 조성물에 적어도 하나 이상의 부형제 예를 들면， 전분， 칼슘카보네이트 (Calcium carbonate), 수크로스 (Sucrose) 또는 락토오스 (Lactose) 및 ¾라틴 등 을 섞어 조제한다. 또한 단순한 부형제 이외에 마그네슘， 스티레이트， 탈크 같은 윤활제를 사용할 수 있다. 경구를 위한 액상제제로는 현탁제， 내용액제， 유제 및 시럽제 등이 해당되는데 흔히 사용되는 단순회석제인 물 및 리퀴드 파라핀 이외에 여러 가지 부형제， 예를 들면 습윤제， 감미제， 방향제 및 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제， 유제， 동 결건조제제 및 좌제가 포함된다. 비수성용제 및 현탁용제로는 프로필렌글리콜 (Propylene glycol), 폴리에틸렌 글리콜 및 올리브 오일과 같은 식물성 기름， 에틸 올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다.  The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier and may be formulated for human or veterinary use for oral or parenteral use. When formulating the composition of the present invention, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants can be used. Solid form preparations for oral administration include sperm, pills, powders, granules and capsules. Such solid form preparations may comprise at least one excipient such as starch, calcium carbonate in a composition comprising a compound of the present invention. ), Sucrose or lactose, and ¾ Latin. In addition to simple excipients, lubricants such as magnesium, styrate and talc may be used. Oral liquid preparations include suspensions, solvents, emulsions and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are commonly used simple limes. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and suppositories. As the non-aqueous and suspending solvent, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, injectable esters such as ethyl oleate and the like can be used.
<27> 본 발명의 상기 약학적 조성물은 개체에 투여하여 프리은 질병을 예방 또는 치료할 수 있다. 본 발명에서 사용된 용어， "개체" 는 프리온 질병 또는 이의 직， 간접적 원인에 의해 유발된 질환을 가지고 있으며， 본 발명의 상기 약학적 조성물 을 투여하여 증상이 호전될 수 있는 질환을 가진 인간을 포함한 소， 양 사슴， 밍크 등의 포유동물을 의미한다 .  The pharmaceutical composition of the present invention may be administered to a subject to prevent or treat free silver disease. As used herein, the term "individual" has a disease caused by prion disease or its direct or indirect cause, including humans having a disease that can be improved by administering the pharmaceutical composition of the present invention. Mammals such as cattle, sheep deer, and mink.
<28> 본 발명에서 사용된 용어， "투여" 는 어떠한 적절한 방법으로 개체에 본 발 명의 약학적 조성물을 도입하는 것을 의미한다. 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 경구 또는 비경구 투여될 수 있다. 또 한， 본 발명의 약학적 조성물이 표적 세포로 이동할 수 있도록 하는 임의의 장치에 의해 투여될 수 있다.  As used herein, the term "administration" means introducing the pharmaceutical composition of the present invention to a subject in any suitable manner. The route of administration may be oral or parenteral via any general route so long as it can reach the desired tissue. In addition, the pharmaceutical composition of the present invention may be administered by any device that allows migration to target cells.
<29> 본 발명의 조성물은 약학적으로 유효한 양으로 투여한다 . 본 발명에서 사용 된 용에 "약제학적으로 유효한 양" 은 의학적 치료에 적용 가능한 합리적인 수혜 /위험 비율로 질환을 치료하기에 충분한 양을 의미하며 , 유효 용량 수준은 환자의 성별, 연령， 중증도， 약물의 활성， 약물에 대한 민감도， 투여 시간， 투여 경로 및 배출 비율， 치료 기간， 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거 나 다른 치료제와 병용하여 투여될 수 있고， 종래의 치료제와는 순차적 또는 동시 에 투여될 수 있다. 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요 하며， 당업자에 의해 용이하게 결정될 수 있다. 본 발명의 제조 방법에 따라 제조 된 화합물을 포함하는 조성물의 투여방법은 경구투여 또는 정맥투여가 바람직하다 . 특정 환자에 대한 투여용량 수준은 성별， 연령， 건강상태 식이， 투여시간， 투여 방법， 약제흔합 및 질환의 중증도에 따라 변화될 수 있다. The composition of the present invention is administered in a pharmaceutically effective amount. Use in the present invention "Pharmaceutically effective amount" for a given dose means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the sex, age, severity, drug activity or drug of the patient. Sensitivity to, time of administration, route of administration and rate of administration, duration of treatment, factors including concomitant drug use, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may be single or multiple doses. In consideration of all the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art. The administration method of the composition containing the compound prepared according to the preparation method of the present invention is preferably oral administration or intravenous administration. Dosage levels for a particular patient may vary depending on gender, age, health diet, time of administration, method of administration, drug combination and severity of disease.
<3()> <3 ( ) >
<31> 다른 하나의 양태로서， 본 발명은 멜라토닌을 포함하는 프리온 질병 예방 및 개선용 식품 조성물을 제공한다.  As another aspect, the present invention provides a food composition for preventing and improving prion diseases, including melatonin.
<32> 본 발명의 식품 조성물은 프리온 질병 억제를 목적으로 건강식품에 첨가될 수 있다. 본 발명의 조성물을 식품 첨가물로 사용할 경우， 상기 조성물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고， 통상적인 방법에 따 라 적절하게 사용될 수 있다. 유효성분의 흔합양은 사용 목적 (예방， 건강 또는 치 료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로， 식품 또는 음료의 제조 시에 본 발명의 조성물은 원료에 대하여 15 중량 % 이하， 바람직하게는 10 중량 % 이 하의 양으로 첨가된다. 그러나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며， 안전성 면 에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.  The food composition of the present invention may be added to a health food for the purpose of suppressing prion disease. When the composition of the present invention is used as a food additive, the composition may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The amount of active ingredient can be determined appropriately depending on the purpose of use (prevention, health or therapeutic treatment). In general, in the manufacture of food or beverages the compositions of the invention are added in an amount of up to 15% by weight, preferably up to 10% by weight relative to the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, it may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.
<33> 상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식 품의 예로는 육류， 소세지， 빵， 쵸코렛， 캔디류， 스넥류， 과자류， 피자, 라면， 기 타 면류， ¾류， 아이스크림류를 포함한 낙농제품， 각종 스프， 음료수， 차, 드링크 제， 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모 두 포함한다.  There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, ¾, ice cream, various soups, drinks, tea, Drink preparations, alcoholic beverages and vitamin complexes, including all health foods in the conventional sense.
<34> 음료의 경우 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모 노사카라이드， 말토스， 슈크로스와 같은 디사카라이드， 및 덱스트린， 사이클로텍스 트린과 같은 천연 감미제나， 사카린， 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 식품 조성물 100 당 일반적으 로 약 0.001 내지 0.4g, 바람직하게는 약 0.002 내지 0.03g 이다. In the case of beverages, various flavors, natural carbohydrates, and the like may be contained as additional ingredients, as in general beverages. The natural carbohydrates mentioned above are the parent Disaccharides such as nosaccharide, maltose and sucrose, and natural sweeteners such as dextrin and cyclotex trine, and synthetic sweeteners such as saccharin and aspartame. The ratio of the natural carbohydrate is generally about 0.001 to 0.4g, preferably about 0.002 to 0.03g per 100 food compositions of the present invention.
<35> 상기 외에 본 발명의 식품 조성물은 여러 가지 영양제， 비타민， 전해질， 풍 미제， 착색제， 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산， 보호성 콜로이드 증점제， pH 조절제， 안정화제， 방부제， 글리세린, 알콜， 탄산 음료에 사용되는 탄 산화제 등을 함유할 수 있다. 그 밖에 본 발명의 식품 조성물은 천연 과일쥬스， 과 일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 식품 조성물 100 중량부 당 0.001 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다. In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, Glycerin, alcohol, carbonation agent used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0.001 to 0.1 parts by weight per 100 parts by weight of the food composition of the present invention.
<36>  <36>
<37> 이하， 본 발명을 실시예에 의거하여 보다 구체적으로 설명한다. 실시예는 오 로지 본 발명을 보다 구체적으로 설명하기 위한 것으로， 본 발명의 요지에 따라 발 명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식 을 가진 자에 있어서 자명할 것이다.  Hereinafter, the present invention will be described in more detail with reference to Examples. The examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.
<38>  <38>
<3 > <3>
<40> *시료와 방법  <40> * Samples and methods
<41> <41>
<42> 세포 배양  <42> cell culture
<43> 인간 신경아세포종 (neuroblastoma) 세포주인 SHᅳ SY5Y는 American Type  <43> SH 세포 SY5Y, a human neuroblastoma cell line, is an American type
Culture collection (ATCC, Rockville, MD, USA)로부터 얻었다. 세포는 10%의 소태 아혈청 (fetal bovine serum) (FBS; Invitrogen-Gibco, Grand Island, NY, USA)과 겐타마이신 (gentamycin) (0.1 mg/ml)을 포함하는 Minimum Essential Medium (Hyclone Laboratories, Logan, UT, USA)에 배양되었으며， 가습 배양기 (humidified incubator)에 37° C에서 5% C02조건에서 유지되었다. Obtained from Culture collection (ATCC, Rockville, MD, USA). Cells are minimal essential medium (Hyclone Laboratories, Logan) containing 10% fetal bovine serum (FBS; Invitrogen-Gibco, Grand Island, NY, USA) and gentamycin (0.1 mg / ml) , UT, USA) and were maintained in a humidified incubator at 37 ° C and 5% CO 2 conditions.
<44>  <44>
<45> PrP (106-126) 처리  <45> PrP (106-126) processing
<46> 합성 PrP (106-126) (sequence, Lys-Thr-Asn-Met -Lys-H i s-Met -A 1 a-G 1 y-A 1 a- Synthetic PrP (106-126) (sequence, Lys-Thr-Asn-Met -Lys-H i s-Met -A 1 a-G 1 y-A 1 a-
A 1 a-A 1 a-A 1 a-G 1 y-A 1 a-Va 1 -Va 1 -G 1 y-G 1 y-Leu-G 1 y , 서열번호 1)은 Peptron (Seoul , Korea)에 의하여 합성되었다. 펩타이드를 살균 다이메틸설폭시화물 (sterile dimethylsulfoxide)에 12.5mM의 농도로 용해하였으며， —80° C에서 보관하였다.A 1 aA 1 aA 1 aG 1 y A 1 a-Va 1 -Va 1 -G 1 yG 1 y-Leu-G 1 y, SEQ ID NO: 1) Peptron (Seoul, Korea). Peptides were dissolved in sterile dimethylsulfoxide at a concentration of 12.5 mM and stored at -80 ° C.
<47> <47>
<48> 아넥신 V에세이 (Annexin V assay)  <48> Annexin V assay
<49> 세포자멸은 생산자가 제공하는 프로토콜에 따라 분리된 세포 (detached cells) (Santa Cruz Biotechnology, Santa Cruz, CA, USA)에 대하여 아넥신 V 에세 이를 수행하여 결정하였다. 아넥신 V의 측량은 488 nm 의 여기파장 (excitation wavelength) 및 525 -530 nm 의 방출파장 (emission wavelength)에서의 형광측정에 의하며, 이는 Guava EasyCyte HT apparatus (Millipore, Billerica, MA, USA)를 이 용한다.  Apoptosis was determined by performing Annexin V assay on detached cells (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the protocol provided by the producer. The measurement of Annexin V is by fluorescence measurements at an excitation wavelength of 488 nm and an emission wavelength of 525-530 nm, which is equivalent to the Guava EasyCyte HT apparatus (Millipore, Billerica, Mass., USA). Use.
<50>  <50>
<5i> Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) 에 세이  <5i> Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
<52> TUNEL 분석은 생산자가 제공하는 프로토콜에 따라서， in situ ApoBrdU DNA fragmentation assay kit (BioVision, San Francisco, CA, USA)를 이용하여 수행되 었으며， 이는 세포자멸 정도를 측정하기 위하여 수행되었다. 세포는 인산완충식염 수 (phosphate buffer saline, PBS)으로 워시되었으며 파라포름알데하이드 (paraformaldehyde)로 15분동안 고정되었다.  TUNEL analysis was performed using an in situ ApoBrdU DNA fragmentation assay kit (BioVision, San Francisco, CA, USA) according to the protocol provided by the producer, which was performed to measure the degree of apoptosis. Cells were washed with phosphate buffer saline (PBS) and fixed for 15 minutes with paraformaldehyde.
<5 > 세포는 DNA-라벨링 용액 (10 의 TdT reaction buffer, 0.75 yL의 TdT enzyme, 8 의 Br-dUTP)과 함께 1시간동안 37°C에서 선배양된 뒤， 5 μί 항 -BrdU_ 풀루오레세인 (fluorescein) 이소티오시안산 ( isothiocynate) 헝체와 0.5시간동안 상온 (20OC)에서 배양되었다. 최종적으로 세포는 DakoCytomation 형광 배지에 고정 되었으며, 형광현미경을 이용하여 관찰되었다. 세포의 핵은 propodium iodide (PI) 로 대조염색되었다. <5> Cells were preincubated at 37 ° C for 1 hour with DNA-labeling solution (10 TdT reaction buffer, 0.75 yL TdT enzyme, 8 Br-dUTP), followed by 5 μί anti-BrdU_ pullulose is (fluorescein) was cultured in iso thiocyanate (isothiocynate) at room temperature (20 O C) for heongche and 0.5 hours. Finally, the cells were fixed in DakoCytomation fluorescent medium and observed using a fluorescence microscope. Cell nuclei were counterstained with propodium iodide (PI).
<54>  <54>
<55> 결과  <55> results
<56> 멜라토닌의 프리은 매개 신경독소 억제의 확인  <56> Confirmation of Free Silver-mediated Neurotoxin Inhibition of Melatonin
<57> 아넥신 V 에세이 (annexin V assay)를 통하여 SH-SY5Y 세포에서의 PrP (106ᅳ  PrP (106 에서 의) in SH-SY5Y cells via annexin V assay
126)—매개 세포독성에 대한 멜라토닌의 영향을 실험하였다. SH-SY5Y 세포는 PrP(106-126)가 존재하거나 존재하지 않는 조건에서 멜라토닌에 노출되었다. 세포 는 PrP (106-126) 처리에 즉각 반웅하였으며 (annexin V 양성 세포가 56.3« 로 증가)， 멜라토닌 자체는 세포의 생존력에는 아무런 영향을 미치지 못하였다. 그러 나， 멜라토닌과 PrP (106-126)가 동시에 처리된 경우 annex in V 양성 세포가 감소 하였으며, 이러한 효과는 멜라토닌의 농도 비례적이었다 (도 1). 126) —The effect of melatonin on mediated cytotoxicity was tested. SH-SY5Y cells were exposed to melatonin in the presence or absence of PrP (106-126). Cells immediately responded to PrP (106-126) treatment (annexin V positive cells increased to 56.3 «) and melatonin itself had no effect on cell viability. Like that In addition, when melatonin and PrP (106-126) were treated simultaneously, annex in V positive cells were decreased, and this effect was proportional to the concentration of melatonin (FIG. 1).
<58> 상기 실험결과를 바탕으로， 아넥신 V 양성 세포의 평균을 막대그래프로 나타 낸 것은 도 2와 같다. Based on the above experimental results, the average of the annexin V-positive cells is represented by a bar graph as shown in FIG. 2.
<5 >  <5>
<60> <60>
<6i> *TUNEL 에세이 결과  <6i> * TUNEL Essay Results
<62> 멜라토닌의 존재 (12시간) 및 비존재하에서, 50 μΜ의 PrP (106-126)에 노출 된 TUNEL-양성 (초록색) SHᅳ SY5Y세포의 면역형광 이미지는 도 3과 같다.  Immunfluorescence images of TUNEL-positive (green) SHVIII SY5Y cells exposed to 50 μΜ of PrP (106-126) in the presence (12 hours) and no melatonin are shown in FIG. 3.
<63> PrP(106-126)을 세포에 처리하는 경우， DNA분절이 유발되어 비오틴화 디옥 시우디린 표지 DNA (초록색)의 양이 증가된 것을 확인할 수 있었다. 여기에 멜라토 닌을 처리하는 경우 PrP(106-126)을 처리해도 PI만 염색 (붉은색)되어 DNA의 분절이 일어나지 않음을 확인할 수 있었다.  When the cells were treated with PrP (106-126), it was confirmed that DNA fragmentation was induced to increase the amount of biotinylated dioxidirin-labeled DNA (green). In the case of melatonin treatment, even if PrP (106-126) treatment, only PI was stained (red), so that DNA fragmentation did not occur.
【산업상 이용가능성】  Industrial Applicability
<64> 본 발명은 프리오논 질병의 예방 및 치료에 관한 조성물에 관한 것으로， 프 리온 질병의 치료 및 예방에 관한 산업에 이용가능하다.  The present invention relates to a composition for the prevention and treatment of prionone disease, and is applicable to the industry for the treatment and prevention of prion disease.

Claims

【청구의 범위】 [Range of request]
【청구항 1】  [Claim 1]
멜라토닌을 유효성분으로 함유하는 프리온 질병의 예방또는 치료용 약학 조 성물.  A pharmaceutical composition for preventing or treating prion diseases containing melatonin as an active ingredient.
【청구항 2]  [Claim 2]
제 1항에 있어서, 상기 프리온 질병은 인간의 크로이츠펠트-야코프병 (Creutzfeldt- Jakob disease), 쿠루병 (Kuru disease), 게르스트만 -슈트로이슬러-샤인커병 (Gerstmann-Str syndrome) , 치명적 가족성 불면증 (fatal familial insomnia)； 소의 BSE (bovine spongiform encephalopathy)； 양의 스크래피 (scrapie)； 사슴의 CWD( chronic wasting disease)； 및 밍크의 TME(transmissible mink encephalopathy)로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는 프 리온 질병의 예방 또는 치료용 약학 조성물. The method of claim 1, wherein the prion disease is human Creutzfeldt-Jakob disease (Kreutzfeldt- Jakob disease), Kuru disease (Kuru disease), Gerstmann-Stroisler-Shinker disease (Gerstmann-Str syndrome), fatal family Fatal familial insomnia; bovine spongiform encephalopathy (bovine BSE); sheep scrapie; chronic wasting disease (CWD) of deer; and transmissible mink encephalopathy (TME) in mink Pharmaceutical composition for the prevention or treatment of prion disease.
【청구항 3】  [Claim 3]
멜라토닌을 함유하는 프리온 질병 예방 또는 개선용 식품 조성물.  Food composition for the prevention or improvement of prion diseases containing melatonin.
PCT/KR2013/000171 2012-01-16 2013-01-09 Novel use for melatonin WO2013109016A1 (en)

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CN117159539B (en) * 2023-10-31 2024-01-23 南京农业大学三亚研究院 Application of melatonin in resisting porcine rotavirus

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