WO2013109016A1

WO2013109016A1 - Novel use for melatonin

Info

Publication number: WO2013109016A1
Application number: PCT/KR2013/000171
Authority: WO
Inventors: 박상열; 정재교; 이유진; 설재원; 김재훈; 박양규; 이주희
Original assignee: 전북대학교산학협력단
Priority date: 2012-01-16
Filing date: 2013-01-09
Publication date: 2013-07-25
Also published as: KR20130084047A

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

【Specification】

[Name of invention]

New Uses of Melatonin

Technical Field

<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.

Background Art

<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> 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

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> 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%.

Representative prion diseases include human CJD Creutzfeldt-Jakob disease,

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.

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.

[Detailed Description of the Invention]

[Technical problem]

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.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing and treating prion diseases.

Technical Solution

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> The prion disease is preferably human Creutzfeldt-Jakob disease

(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.

In addition, the present invention provides a food composition for preventing or improving prion disease containing melatonin. Advantageous Effects

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.

[Brief Description of Drawings]

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> 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 μΜ of PrP (106-126), with and without melatonin (12 hours).

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>

<I7>

[Form for implementation of invention]

The present invention provides a pharmaceutical composition for preventing or treating prion diseases containing melatonin as an active ingredient.

Hereinafter, the present invention will be described in detail.

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> 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.

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.

The 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

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> 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.

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.

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.

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.

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 ( ⁾ >

As another aspect, 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. 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.

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.

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.

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>

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>

<3>

<40> * Samples and methods

<41>

<42> cell culture

<43> SH 세포 SY5Y, a human neuroblastoma cell line, is an American type

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>

<45> PrP (106-126) processing

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 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>

<48> Annexin V assay

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>

<5i> Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)

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> 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>

<55> results

<56> Confirmation of Free Silver-mediated Neurotoxin Inhibition of Melatonin

PrP (106 에서 의) in SH-SY5Y cells via annexin V assay

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).

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>

<60>

<6i> * TUNEL Essay Results

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.

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.

Industrial Applicability

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

[Range of request]

[Claim 1]

A pharmaceutical composition for preventing or treating prion diseases containing melatonin as an active ingredient.

[Claim 2]

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.

[Claim 3]

Food composition for the prevention or improvement of prion diseases containing melatonin.