KR20190009174A - Composition for preventing or treating inflammatory diseases or spinal cord injury comprising ursodeoxycholic acid - Google Patents
Composition for preventing or treating inflammatory diseases or spinal cord injury comprising ursodeoxycholic acid Download PDFInfo
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- KR20190009174A KR20190009174A KR1020170091065A KR20170091065A KR20190009174A KR 20190009174 A KR20190009174 A KR 20190009174A KR 1020170091065 A KR1020170091065 A KR 1020170091065A KR 20170091065 A KR20170091065 A KR 20170091065A KR 20190009174 A KR20190009174 A KR 20190009174A
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- South Korea
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- ursodeoxycholic acid
- spinal cord
- acid
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- udca
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Classifications
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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
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
Abstract
Description
우르소데옥시콜산을 함유하는 염증성 질환 또는 척수 손상 예방 또는 치료용 조성물에 관한 것이다. And a composition for preventing or treating inflammatory diseases or spinal cord injury containing ursodeoxycholic acid.
염증 반응은 여러가지 원인에 의해 생화학적으로 일어나며, 과도한 염증 반응으로 인해 세포나 조직이 손상을 받거나 괴사하게 될 때, 그 피해를 최소화하거나 괴사하는 부위를 원래대로 회복시키기 위해 국소적으로 일어나는 면역반응이다. 이런 염증 반응은, 정상적인 경우 발병 요인을 감소시키거나 손상된 부위를 재생시켜 정상적인 구조로 회복시키지만, 일반적인 범위를 넘은 과도한 염증 반응은 만성 염증을 비롯한 각종 질병을 유발한다. 산화질소 (nitric oxide, NO)는 해당 염증 반응이 진행될 시 주요한 매개체로 활동하는데, 이 산화질소가 TNF-α, 인터루킨 (IL)-1α, IL-1β, IL-6 등의 여러 염증성 사이토카인에 의해서 유도된다. 과도한 염증 반응을 유발하는 여러가지 병인 중, 척수 손상에 의한 염증 반응은 심각한 수준이라 할 수 있으며, 급성 및 만성 염증을 유발한다.Inflammation reactions occur biochemically due to various causes, and when the cells or tissues become damaged or become necrotic due to excessive inflammation, the immune response occurs locally to minimize the damage or restore the necrotic area . These inflammatory reactions usually cause the disease to be reduced or regenerate the injured area to restore normal structure, but excessive inflammation reaction beyond the general range causes various diseases including chronic inflammation. Nitric oxide (NO) acts as a major mediator in the progression of the inflammatory response. This nitric oxide plays a role in inflammatory cytokines such as TNF-α, interleukin (IL) -1α, IL-1β and IL-6 . Among the various pathologies that cause excessive inflammatory response, the inflammatory response due to spinal cord injury is a serious level and causes acute and chronic inflammation.
척수 손상 환자는 매년 50만 명 이상씩 새로 생기고 있고, 그 질환자가 특히 상대적으로 젊은 환자에게서 외상의 형태로 많이 발생하기 때문에 환자의 남은 생애를 감안해 보아도, 개인과 사회에 미치는 손실의 정도는 매우 심각하다 할 수 있다(인생의 직접적 및 간접적인 비용은 3백만 달러에 이른다는 보고가 있음). 매년 미국내에서만 해도 사회 비용이 100 억 달러를 훨씬 상회할 정도라고 하니 이는 이미 전 세계적으로 치료제 개발을 위한 시장이 형성되어 있음을 의미한다.The number of patients with spinal cord injuries is more than 500,000 per year, and the number of patients suffering from trauma is relatively high, especially in relatively young patients. Therefore, considering the patients' remaining lives, (The direct and indirect costs of life are reported to be in the region of $ 3 million). Every year in the US alone, the social cost is well over $ 10 billion, which means that there is already a market for drug development worldwide.
척수 손상 시 조직의 괴사, 캐비테이션(cavitation) 등의 이차 손상이 일어나는데, 이런 이차 손상이 과도한 염증 반응으로 인해 빠르게 촉진된다. 이런 과도한 염증 반응을 억제하기 위한 연구가 이루어지고 있긴 하나, 임상적으로 입증이 된 치료제는 거의 전무한 실정이다. 그나마 고 용량의 스테로이드제를 사용하여 과도한 염증반응을 억제하고 있으나, 그 부작용이 상당히 심각한 것으로 보고되고 있다. In spinal cord injury, secondary damage such as tissue necrosis and cavitation occurs, which is rapidly promoted by excessive inflammatory responses. Although studies have been conducted to suppress this excessive inflammatory response, there have been almost no clinically proven therapeutic agents. Although high doses of steroids are used to inhibit excessive inflammatory responses, the side effects are reported to be quite severe.
따라서, 부작용을 갖지 않으면서 강력한 항염증 활성을 가져, 척수 손상을 치료할 수 있는 치료제에 대한 요구가 존재한다. Thus, there is a need for therapeutic agents that have potent anti-inflammatory activity without side effects and that can treat spinal cord injury.
일 양상은 우르소데옥시콜산(Ursodeoxycholic acid) 또는 그의 약학적으로 허용가능한 염을 유효성분으로 함유하는 척수 손상 예방 또는 치료용 약학적 조성물을 제공하는 것이다. An aspect of the present invention is to provide a pharmaceutical composition for preventing or treating spinal cord injury containing Ursodeoxycholic acid or a pharmaceutically acceptable salt thereof as an active ingredient.
다른 양상은 우르소데옥시콜산(Ursodeoxycholic acid)을 유효성분으로 함유하는 척수 손상 예방 또는 개선용 건강기능식품을 제공하는 것이다. Another aspect is to provide a health functional food for preventing or ameliorating spinal cord injury containing Ursodeoxycholic acid as an active ingredient.
일 양상은 담즙산 또는 담즙산염, 예를 들면, 우르소데옥시콜산(Ursodeoxycholic acid) 또는 그의 약학적으로 허용가능한 염을 유효성분으로 함유하는 염증성 질환 또는 척수 손상 예방 또는 치료용 약학적 조성물을 제공한다. One aspect provides a pharmaceutical composition for preventing or treating inflammatory diseases or spinal cord injury containing bile acid or bile salt, for example, Ursodeoxycholic acid or a pharmaceutically acceptable salt thereof as an active ingredient.
본 명세서에서 용어 "담즙산 또는 담즙산염"은 담즙의 주요 구성 성분으로 콜레스테롤로부터 합성될 수 있다. 담즙산은 간에서 합성되는 l차 담즙산과 장내 세균에 의해 1차 담즙산이 히드록실화(hydroxylation) 되어 형성되는 2차 담즙산을 포함할 수 있다. 예를 들면, 상기 담즙산은 우르소데옥시콜산(Ursodeoxycholic acid, UDCA)일 수 있다. 상기 우르소데옥시콜산은 생물학적, 또는 화학적으로 합성될 수 있으며, 시중에서 판매가능한 것일 수 있다. As used herein, the term " bile acid or bile salt " may be synthesized from cholesterol as a major constituent of bile. The bile acid may include a primary bile acid synthesized in the liver and a secondary bile acid formed by hydroxylation of the primary bile acid by intestinal bacteria. For example, the bile acid may be Ursodeoxycholic acid (UDCA). The above-mentioned ursodeoxycholic acid may be synthesized biologically or chemically, and may be commercially available.
상기 우르소데옥시콜산은 하기 화학식 1로 표시되는 화합물일 수 있다. The above-mentioned ursodeoxycholic acid may be a compound represented by the following general formula (1).
[화학식 1] [Chemical Formula 1]
본 명세서에서 용어, "염증성 질환"은 염증을 주병변으로 하는 질병을 총칭하는 의미로서, 이에 제한되지는 않으나 구체적으로 천식, 만성폐쇄성 폐질환, 알레르기, 전신성 홍반성 루푸스, 경피증, 궤양성 대장염, 크론병, 아토피성 피부염, 건선, 아나필락시스, 피부염, 당뇨병성 망막증, 망막염, 황반 변성, 포도막염, 결막염, 관절염, 류마티스성 관절염, 강직성 척추염, 골관절염, 골다공증, 당뇨, 당뇨성 신장병, 신염, 신장염, 쇼그렌 증후군, 크론씨 병, 자가 면역 췌장염, 치주질환, 이식편 대 숙주 질환, 만성 골반 염증 질환, 자궁내막염, 비염, 편도염, 중이염, 인후염, 방광염 및 만성 전립선염로 이루어진 군으로부터 선택되는 어느 하나에 해당할 수 있다. As used herein, the term " inflammatory disease " means a disease in which inflammation is the main lesion, and includes, but is not limited to, asthma, chronic obstructive pulmonary disease, allergy, systemic lupus erythematosus, scleroderma, ulcerative colitis, Diabetic retinopathy, retinitis, macular degeneration, uveitis, conjunctivitis, arthritis, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, osteoporosis, diabetes, diabetic nephropathy, nephritis, nephritis, Sjogren's syndrome Wherein the disease is selected from the group consisting of chronic obstructive pulmonary disease, autoimmune pancreatitis, periodontal disease, graft versus host disease, chronic pelvic inflammatory disease, endometritis, rhinitis, tonsillitis, otitis media, sore throat, cystitis and chronic prostatitis. have.
상기 우르소데옥시콜산은 그의 약제학적으로 허용 가능한 염으로 조성물에 포함될 수 있다. 상기 염은 산부가염 또는 염기부가염일 수 있다. 상기 산부가염은 무기산 또는 유기산과의 염을 포함한다. 상기 무기산염으로는, 예를 들면, 염산, 브롬화수소산, 인산 또는 황산과의 염이 있으며, 상기 유기산염으로는 아세트산, 트리플루오로아세트산, 프로피온산, 말레산, 푸마르산, 말산, 시트르산, 타르타르산, 락트산, 벤조산, 메탄술폰산, 에탄술폰산, 벤젠술폰산, 톨루엔술폰산 또는 나프탈렌디술폰산 등이 있으며, 이에 한정되는 것은 아니다. 상기 염기부가염으로는 알칼리금속염 (예, 나트륨 또는 칼륨 염), 알칼리토금속염 (예, 칼슘 또는 마그네슘 염), 또는 암모니아염 또는 유기 아민염, 예를 들면, 디에틸아민, 트리에틸아민, 에틸디이소프로필아민, 프로카인, 디벤질아민, N-메틸모르폴린, 또는 디히드로아비에틸아민과의 염 등이 있다. The ursodeoxycholic acid may be included in the composition as a pharmaceutically acceptable salt thereof. The salt may be an acid addition salt or a base addition salt. The acid addition salt includes a salt with an inorganic acid or an organic acid. Examples of the inorganic acid salt include salts with hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid. Examples of the organic acid salt include acetic acid, trifluoroacetic acid, propionic acid, maleic acid, fumaric acid, malic acid, citric acid, , Benzoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid or naphthalenedisulfonic acid, but is not limited thereto. The base addition salts include, for example, alkali metal salts such as sodium or potassium salts, alkaline earth metal salts such as calcium or magnesium salts, or ammonia salts or organic amine salts such as diethylamine, triethylamine, ethyl And salts with diisopropylamine, procaine, dibenzylamine, N-methylmorpholine, or dihydroabiethylamine.
본 명세서에서 우르소데옥시콜산 또는 약제학적으로 허용 가능한 그의 염은 이의 임의의 결정형 및 무정형, 그리고 이의 수화물, 용매화물, 및 공결정으로 구성된 군에서 선택된 임의의 형태로 존재할 수 있는 것으로 해석된다. Ursodeoxycholic acid or a pharmaceutically acceptable salt thereof is herein interpreted to be present in any form selected from the group consisting of any of its crystalline forms and amorphous forms, and hydrates, solvates, and co-crystals thereof.
약학적 조성물은 경구 또는 비경구 투여 제형일 수 있다. 약제학적 제제는 정제, 경질 또는 연질 캅셀제, 액제, 현탁제 등과 같은 경구투여용 제제가 있으며 이들 약제학적 제제는 약제학적으로 허용 가능한 통상의 담체, 예를 들어 경구투여용 제제의 경우에는 부형제, 결합제, 붕해제, 활택제, 가용화제, 현탁화제, 보존제 또는 증량제 등을 사용하여 조제할 수 있다. 또한 약학적 조성물은 크림, 젤, 패취, 분무제, 연고제, 경고제, 로션제 등과 같은 비경구 투여용 제제로 사용될 수 있다. The pharmaceutical composition may be an oral or parenteral dosage form. The pharmaceutical preparations may be formulated for oral administration, such as tablets, hard or soft capsules, liquids, suspensions, etc. These pharmaceutical preparations may contain conventional pharmaceutically acceptable carriers, for example, excipients, binders , A disintegrant, a lubricant, a solubilizing agent, a suspending agent, a preservative or an extender, and the like. In addition, the pharmaceutical composition may be used as a preparation for parenteral administration such as cream, gel, patch, spray, ointment, warning agent, lotion and the like.
상기 약학적 조성물은 통상적으로 사용되는 부형제, 붕해제, 감미제, 활택제, 향미제 등을 추가로 포함할 수 있으며, 통상적인 방법에 의해 정제, 캅셀제, 산제, 과립제, 현탁제, 유제, 시럽제, 기타 액제로 제형화될 수 있다.The pharmaceutical composition may further contain commonly used excipients, disintegrants, sweeteners, lubricants, flavors and the like, and may be formulated into tablets, capsules, powders, granules, suspensions, emulsions, syrups, Other liquid formulations may be formulated.
정제 및 캡슐 등의 제형으로 제제하기 위해 락토오스, 사카로오스, 솔비톨, 만니톨, 전분, 아밀로펙틴, 셀롤로오스 또는 젤라틴과 같은 결합제, 디칼슘 포스페이트와 같은 부형제, 옥수수 전분 또는 고구마 전분과 같은 붕해제, 스테아르산 마그네슘, 스테아르산 칼슘, 스테아릴푸마르산 나트륨 또는 폴리에틸렌글리콜 왁스와 같은 윤활유가 함유된다. 캡슐제형의 경우는 상기에서 언급한 물질 이외에도 지방유와 같은 액체 담체를 함유한다.Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, celluloses or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, and disintegrants such as stearic acid Magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax. In the case of a capsule formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil is contained.
또한, 본 발명의 약학적 조성물은 경구 또는 비경구 투여할 수 있으며, 비경구 투여시 피하주사, 정맥주사, 근육내 주사 또는 흉부내 주사 주입방식을 선택할 수 있다. 비경구 투여용 제형으로 제제화하기 위해서는 본 발명의 담즙산 또는 이들의 염을 안정제 또는 완충제와 함께 물에서 혼합하여 현탁액으로 제조하고 이를 앰플 또는 바이알의 단위 투여형으로 제제한다.In addition, the pharmaceutical composition of the present invention can be administered orally or parenterally. For parenteral administration, subcutaneous injection, intravenous injection, intramuscular injection, or intramuscular injection can be selected. For formulation into parenteral administration formulations, the bile acid or the salt thereof of the present invention is mixed with water or a stabilizer or buffer to prepare a suspension, which is then formulated into a unit dosage form of ampoule or vial.
본 명세서에서 용어, "투여"는 어떠한 적절한 방법으로 염증성 질환, 또는 척수 손상의 의심 개체에게 본 발명의 약학적 조성물을 도입하는 것을 의미하며, 투여 경로는 목적 조직에 도달할 수 있는 한 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있다.As used herein, the term " administering " means introducing the pharmaceutical composition of the present invention into an inflammatory disease or a suspect member of spinal cord injury by any appropriate method, and the administration route may be either oral or parenteral May be administered via various routes of administration.
본 명세서의 약학적 조성물은 약학적으로 유효한 양으로 투여할 수 있다.The pharmaceutical compositions herein may be administered in a pharmaceutically effective amount.
본 명세서에서 용어, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 질병의 종류, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다. 본 발명의 약학적 조성물의 투여 용량은, 환자의 상태, 연령, 성별 및 합병증 등의 다양한 요인에 따라 전문가에 의해 결정될 수 있지만 일반적으로는 성인 1kg 당 0.1㎎ 내지 10g, 또는, 10 mg 내지 5g의 용량으로 투여될 수 있다. 또한, 단위 제형당 상기 약학적 조성물의 1일 용량 또는 이의 1/2, 1/3 또는 1/4의 용량이 함유되도록 하며, 하루 1 내지 6 회 투여될 수 있다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로도 사용될 수 있다. As used herein, the term " pharmaceutically effective amount " means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and the effective dose level will vary depending on the species and severity, age, sex, The type of drug, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art. The dosage of the pharmaceutical composition of the present invention may be determined by a specialist depending on various factors such as the condition of the patient, age, sex, and complications, but is generally from 0.1 mg to 10 g per kg of the adult, or from 10 mg to 5 g Dose. ≪ / RTI > Also, the daily dosage of the pharmaceutical composition per unit dosage form, or a half, 1/3, or 1/4 dose thereof, may be contained, and may be administered 1 to 6 times per day. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and the active ingredient may be used in an amount of more than the above range since there is no problem in terms of safety.
일 구체예에 있어서, 상기 우르소데옥시콜산은 대식세포에서 NO 분비량을 감소시키거나, 염증성 사이토카인 유전자 및 단백질의 발현을 억제시키는 것일 수 있다. 상기 염증성 사이토카인은 TNF-α, IL-1α, IL-1β, 및 IL-6로 이루어진 군으로부터 선택된 어느 하나 이상인 것일 수 있다. In one embodiment, the ursodeoxycholic acid may reduce NO secretion in macrophages or inhibit the expression of inflammatory cytokine genes and proteins. The inflammatory cytokine may be at least one selected from the group consisting of TNF-a, IL-1 alpha, IL-1 beta, and IL-6.
다른 구체예에 있어서, 우르소데옥시콜산은 MAPKs 경로 또는 NF-κB 경로에서의 인산화를 억제하는 것일 수 있다. 상세하게는 MAPKs 경로의 ERK, JNK, 또는 p38의 인산화를 억제하는 것일 수 있고, NF-κB 경로의 IκBα의 인산화를 억제하는 것일 수 있다. In another embodiment, the ursodeoxycholic acid may be one that inhibits phosphorylation in the MAPKs pathway or the NF-KB pathway. Specifically, it may inhibit ERK, JNK, or p38 phosphorylation of the MAPKs pathway, and may inhibit the phosphorylation of IκBα in the NF-κB pathway.
또 다른 구체예에 있어서, 우르소데옥시콜산은 척수 손상된 개체의 운동기능을 향상시커나, 손상된 척수의 조직의 복구를 촉진하는 것일 수 있다. In yet another embodiment, the ursodeoxycholic acid may be one that enhances the motor function of spinal cord injured individuals, or promotes repair of injured spinal cord tissue.
따라서, 일 구체예에 따른 우르소데옥시콜산은 염증성 질환 또는 척수 손상의 예방, 개선, 또는 치료용 조성물에 유용하게 사용될 수 있다. Thus, the ursodeoxycholic acid according to one embodiment may be usefully used in compositions for the prevention, amelioration, or treatment of inflammatory diseases or spinal cord injury.
다른 양상은 우르소데옥시콜산(Ursodeoxycholic acid)을 유효성분으로 함유하는 염증성 질환, 또는 척수 손상 예방 또는 개선용 건강기능식품을 제공한다. Another aspect provides an inflammatory disease containing Ursodeoxycholic acid as an active ingredient, or a health functional food for preventing or ameliorating spinal cord injury.
식품은 건강보조식품, 건강기능식품, 기능성 식품 등이 될 수 있으나 이에 제한되는 것은 아니며, 천연식품, 가공식품, 환자식품, 일반적인 식자재 등에 본 발명의 화합물을 첨가하는 것도 포함된다. The food may be, but is not limited to, a health supplement food, a health functional food, a functional food, and the like, and includes the addition of the compound of the present invention to natural foods, processed foods, patient foods, and general food ingredients.
본 발명의 식품 조성물은, 상기 본 발명의 담즙산 또는 이들의 염을 그대로 첨가하거나 다른 식품 또는 식품 조성물과 함께 사용될 수 있으며, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합양은 그의 사용 목적(예방, 개선 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 본 발명의 담즘산 또는 이들의 염은, 식품 또는 음료의 제조 시에 식품 또는 음료의 원료 100 중량부에 대하여 0.001 내지 99.99 중량부, 바람직하게는 0.001 내지 30 중량부 첨가될 수 있다. 상기 본 발명의 담즘산 또는 이들의 염의 유효용량은 상기 약학적 조성물의 유효용량에 준해서 사용할 수 있으나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 상기 성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로도 사용될 수 있다.The food composition of the present invention can be used as it is or in combination with other foods or food compositions, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, improvement, or therapeutic treatment). In general, the chalcometric acid or salt thereof of the present invention may be added in an amount of 0.001 to 99.99 parts by weight, preferably 0.001 to 30 parts by weight, based on 100 parts by weight of the raw material for food or beverage in the production of food or beverage. The effective dose of the chimic acid or salt thereof of the present invention can be used in accordance with the effective dose of the pharmaceutical composition. However, in the case of long-term intake for the purpose of health and hygiene or health control, And the above components can be used in an amount in excess of the above range because there is no problem in terms of safety.
상기 식품의 종류에는 특별한 제한은 없다. 상기 식품 조성물은 정제, 경질 또는 연질 캅셀제, 액제, 현탁제 등과 같은 경구투여용 제제의 형태로 이용될 수 있으며, 이들 제제는 허용 가능한 통상의 담체, 예를 들어 경구투여용 제제의 경우에는 부형제, 결합제, 붕해제, 활택제, 가용화제, 현탁화제, 보존제 또는 증량제 등을 사용하여 조제할 수 있다.There is no particular limitation on the kind of the food. The food composition may be used in the form of tablets, hard or soft capsules, liquids, suspensions, and the like, which may contain conventional excipients, such as excipients in the case of oral preparations, Binders, disintegrators, lubricants, solubilizers, suspending agents, preservatives or extenders.
또한, 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제, 기타 영양제 등을 들 수 있으나 이들 종류의 식품으로 제한되는 것은 아니다.Examples of the food include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen and other noodles, gums and ice cream, various soups, drinks, tea, drinks, alcoholic beverages and vitamins , And other nutrients, but are not limited to these kinds of foods.
일 양상에 따른 우르소데옥시콜산은 염증성 사이토카인의 분비를 억제하고, MAPKs 경로의 ERK, JNK, 또는 p38의 인산화를 억제하며, NF-κB 경로의 IκBα의 인산화를 억제할 뿐만 아니라, 척수 손상된 개체의 운동기능을 향상시커나, 손상된 척수의 조직의 복구를 촉진하는 효과가 있어, 염증성 질환 또는 척수 손상의 예방, 개선, 또는 치료용 조성물에 유용하게 사용될 수 있다. Ursodeoxycholic acid according to one aspect inhibits the secretion of inflammatory cytokines, inhibits the phosphorylation of ERK, JNK, or p38 in the MAPKs pathway, inhibits the phosphorylation of IκBα in the NF-κB pathway, Improving the motor function of the injured spinal cord, or repairing damaged spinal cord tissues, and thus can be usefully used in compositions for preventing, ameliorating, or treating inflammatory diseases or spinal cord injuries.
도 1a의 다양한 농도에서의 우르소데옥시콜산의 세포 독성을 정량적으로 분석한 결과를 나타낸 그래프이다.
도 1b는 다양한 농도에서의 우르소데옥시콜산의 세포 독성을 정성적으로 분석한 결과를 나타낸 그래프이다.
도 2는 다양한 농도에서의 우르소데옥시콜산이 LPS 유도된 세포의 NO 분비에 미치는 영향을 나타낸 그래프이다.
도 3a는 우르소데옥시콜산이 LPS 유도된 세포의 TNF-α mRNA 발현에 미치는 영향을 나타낸 그래프이다.
도 3b는 우르소데옥시콜산이 LPS 유도된 세포의 IL-1α mRNA 발현에 미치는 영향을 나타낸 그래프이다.
도 3c는 우르소데옥시콜산이 LPS 유도된 세포의 IL-1β mRNA 발현에 미치는 영향을 나타낸 그래프이다.
도 3d는 우르소데옥시콜산이 LPS 유도된 세포의 IL-6 mRNA 발현에 미치는 영향을 나타낸 그래프이다.
도 3e는 우르소데옥시콜산이 LPS 유도된 세포의 IL-10 mRNA 발현에 미치는 영향을 나타낸 그래프이다.
도 4a는 우르소데옥시콜산이 LPS 유도된 세포의 TNF-α 단백질 발현에 미치는 영향을 나타낸 그래프이다.
도 4b는 우르소데옥시콜산이 LPS 유도된 세포의 IL-1α 단백질 발현에 미치는 영향을 나타낸 그래프이다.
도 4c는 우르소데옥시콜산이 LPS 유도된 세포의 IL-1β 단백질 발현에 미치는 영향을 나타낸 그래프이다.
도 4d는 우르소데옥시콜산이 LPS 유도된 세포의 IL-6 단백질 발현에 미치는 영향을 나타낸 그래프이다.
도 4e는 우르소데옥시콜산이 LPS 유도된 세포의 IL-10 단백질 발현에 미치는 영향을 나타낸 그래프이다.
도 5a는 우르소데옥시콜산이 LPS 유도된 세포의 ERK의 인산화에 미치는 영향을 나타낸 그래프이다.
도 5b는 우르소데옥시콜산이 LPS 유도된 세포의 JNK의 인산화에 미치는 영향을 나타낸 그래프이다.
도 5c는 우르소데옥시콜산이 LPS 유도된 세포의 p-38의 인산화에 미치는 영향을 나타낸 그래프이다.
도 5d는 우르소데옥시콜산이 LPS 유도된 세포의 IκBα의 인산화에 미치는 영향을 나타낸 그래프이다.
도 6은 우르소데옥시콜산이 척수 손상 동물모델에서 운동기능의 회복에 미치는 영향을 평가한 그래프이다.
도 7a는 우르소데옥시콜산 처리 1일 후, 우르소데옥시콜산이 척수 손상 동물모델에서 조직의 회복에 미치는 영향을 나타낸 사진이다.
도 7b는 우르소데옥시콜산 처리 3일 후, 우르소데옥시콜산이 척수 손상 동물모델에서 조직의 회복에 미치는 영향을 나타낸 사진이다.
도 7c는 우르소데옥시콜산 처리 7일 후, 우르소데옥시콜산이 척수 손상 동물모델에서 조직의 회복에 미치는 영향을 나타낸 사진이다.
도 8은 우르소데옥시콜산이 척수 손상 동물모델에서 염증 반응의 억제 활성을 나타낸 그래프이다. FIG. 1A is a graph showing the results of quantitative analysis of cytotoxicity of ursodeoxycholic acid at various concentrations. FIG.
FIG. 1B is a graph showing the qualitative analysis of cytotoxicity of ursodeoxycholic acid at various concentrations. FIG.
Figure 2 is a graph showing the effect of ursodeoxycholic acid at various concentrations on NO secretion in LPS-induced cells.
3A is a graph showing the effect of ursodeoxycholic acid on the expression of TNF-α mRNA in LPS-induced cells.
3b is a graph showing the effect of ursodeoxycholic acid on IL-1? MRNA expression of LPS-induced cells.
3c is a graph showing the effect of ursodeoxycholic acid on the expression of IL-1? MRNA in LPS-induced cells.
FIG. 3D is a graph showing the effect of ursodeoxycholic acid on IL-6 mRNA expression of LPS-induced cells.
3E is a graph showing the effect of ursodeoxycholic acid on IL-10 mRNA expression of LPS-induced cells.
4A is a graph showing the effect of ursodeoxycholic acid on the expression of TNF-a protein in LPS-induced cells.
4b is a graph showing the effect of ursodeoxycholic acid on IL-1 alpha protein expression of LPS-induced cells.
4c is a graph showing the effect of ursodeoxycholic acid on the expression of IL-1 beta protein in LPS-induced cells.
4d is a graph showing the effect of ursodeoxycholic acid on the expression of IL-6 protein in LPS-induced cells.
4E is a graph showing the effect of ursodeoxycholic acid on IL-10 protein expression of LPS-induced cells.
5A is a graph showing the effect of ursodeoxycholic acid on the phosphorylation of ERK in LPS-induced cells.
5B is a graph showing the effect of ursodeoxycholic acid on the phosphorylation of JNK in LPS-induced cells.
5c is a graph showing the effect of ursodeoxycholic acid on the phosphorylation of p-38 of LPS-induced cells.
FIG. 5d is a graph showing the effect of ursodeoxycholic acid on the phosphorylation of IκBα in LPS-induced cells.
6 is a graph showing the effect of ursodeoxycholic acid on recovery of motor function in an animal model of spinal cord injury.
FIG. 7A is a photograph showing the effect of ursodeoxycholic acid on the recovery of tissue in an animal model injured in spinal cord after 1 day of ursodeoxycholic acid treatment.
Fig. 7b is a photograph showing the effect of ursodeoxycholic acid on recovery of tissue in an animal model injured in spinal cord after 3 days of treatment with ursodeoxycholic acid.
7C is a photograph showing the effect of ursodeoxycholic acid on the recovery of tissue in an animal model injured in spinal cord after 7 days of treatment with ursodeoxycholic acid.
Figure 8 is a graph showing the inhibitory activity of the inflammatory response in an animal model of spinal cord injured ursodeoxycholic acid.
이하 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
실험예Experimental Example 1. One. UDCA의UDCA's 세포독성 측정 Cytotoxicity measurement
세포수 측정 키트(cell counting kit) (CCK-8, Dojindo Molecular Technologies Inc., Japan) 및 생존/사멸 염색 키트(live/dead staining kit)(Invitrogen, USA) 를 사용하여, 다양한 농도의 우르소데옥시콜산(UDCA)의 세포독성을 측정하였다. Using a cell counting kit (CCK-8, Dojindo Molecular Technologies Inc., Japan) and a live / dead staining kit (Invitrogen, USA), various concentrations of ursodeoxy The cytotoxicity of cholic acid (UDCA) was measured.
구체적으로, 우르소데옥시콜산(Tokyo Chemical Industry Co., LTD, Japan) 을 99.9% 울트라 퓨어 등급(ultra-pure grade)의 에탄올에 매우 높은 농도로 녹인 다음, 10% 소 태아 혈청(fetal bovine serum) (FBS, Grand Island, NY, GIBCO) 및 1% 페니실린-스트렙토마이신 (PS, GIBCO)이 함유된 둘베코 변형 이글 배지(Dulbecco's modified eagle medium) (DMEM, GIBCO) 에 필요한 농도 만큼 희석 후 사용하였다. Specifically, ursodeoxycholic acid (Tokyo Chemical Industry Co., Ltd., Japan) was dissolved in 99.9% ultra-pure grade ethanol at a very high concentration, and then 10% fetal bovine serum (DMEM, GIBCO) containing 1% penicillin-streptomycin (FBS, Grand Island, NY, GIBCO) and 1% penicillin-streptomycin (PS, GIBCO).
정량적인 세포 독성 평가를 위해 48 웰 세포 배양 플레이트(Falcon Becton Dickinson, Lincoln Park, NJ)에 웰 당 2 x 104개의 RAW 266.7 대식세포를 분주한 후, 1시간 동안 안정화 시켰다. 이후에, 0(대조군), 0.5 mM, 1 mM, 2 mM 또는 5 mM 농도의 UDCA(각 그룹당 n =3)를 처리한 후 24시간 동안 세포를 배양하였다. 24시간 후 둘베코 인산-버퍼 염수(Dulbecco's phosphate-buffered saline) (DPBS, Invitrogen Life Sciences, Carlsbad, CA, USA)로 2번 세척하고, 500 μL (0.1 mL/mL) 의 CCK-8 용액을 처리하고 2 시간 더 배양하였다. 2 시간 후, 마이크로플레이트 리더(Bio-Rad, Hercules, CA, USA)를 이용하여 450 nm 파장에서 각 그룹의 흡광도를 측정하였다. 대조군 그룹의 흡광도 값을 100%일 때의 상대적인 값을 계산하여 수치화하였고, 그 결과를 도 1a에 나타내었다. For quantitative cytotoxicity evaluation, 2 x 10 4 RAW 266.7 macrophages per well were plated on 48 well cell culture plates (Falcon Becton Dickinson, Lincoln Park, NJ) and stabilized for 1 hour. Cells were then cultured for 24 hours after treatment with 0 (control), 0.5 mM, 1 mM, 2 mM or 5 mM UDCA (n = 3 per group). After 24 hours, the cells were washed twice with Dulbecco's phosphate-buffered saline (DPBS, Invitrogen Life Sciences, Carlsbad, Calif., USA) and treated with 500 μL (0.1 mL / mL) of CCK- And cultured for another 2 hours. After 2 hours, the absorbance of each group was measured at a wavelength of 450 nm using a microplate reader (Bio-Rad, Hercules, Calif., USA). The relative value of the absorbance value of the control group at 100% was calculated and quantified. The results are shown in FIG. 1A.
또한, 정성적인 세포독성 평가를 위해, 동일한 조건에서 생존/사멸 염색 키트(Invitrogen)를 사용해 제조사의 지시에 따라 세포를 염색하였다. 15분 동안 세포를 염색한 후, 형광 현미경(Olympus IX71, Japan)을 사용하여 40배율로 세포를 관찰하였고, 그 결과를 도 1b에 나타내었다. In addition, for qualitative cytotoxicity evaluation, cells were stained according to the manufacturer's instructions using a survival / killing dye kit (Invitrogen) under the same conditions. After staining the cells for 15 minutes, the cells were observed with a fluorescence microscope (Olympus IX71, Japan) at 40 magnification, and the results are shown in FIG.
도 1a의 다양한 농도에서의 우르소데옥시콜산의 세포 독성을 정량적으로 분석한 결과를 나타낸 그래프이다. FIG. 1A is a graph showing the results of quantitative analysis of cytotoxicity of ursodeoxycholic acid at various concentrations. FIG.
도 1b는 다양한 농도에서의 우르소데옥시콜산의 세포 독성을 정성적으로 분석한 결과를 나타낸 그래프이다. FIG. 1B is a graph showing the qualitative analysis of cytotoxicity of ursodeoxycholic acid at various concentrations. FIG.
도 1a 및 도 1b에 나타낸 바와 같이, 일 구체예에 따른 우르소데옥시콜산은 높은 농도에서도 유의적인 세포독성이 나타나지 않음을 확인하였고, 이들 농도의 우르소데옥시콜산을 아래의 실험에 사용하였다. As shown in Figs. 1A and 1B, it was confirmed that ursodeoxycholic acid according to one embodiment did not show significant cytotoxicity even at a high concentration, and these concentrations of ursodeoxycholic acid were used in the following experiments.
실험예Experimental Example 2. 2. UDCA의UDCA's 항염증 활성 분석 Analysis of anti-inflammatory activity
UDCA의 항염증 활성을 확인하기 위해, RAW 264.7 대식세포를 사용하였다. To confirm the anti-inflammatory activity of UDCA, RAW 264.7 macrophages were used.
구체적으로, 리포폴리사카라이드(LPS, Sigma, St. Louis, MO) 를 3차 증류수에 100 μg/ml 농도로 녹인 다음, DMEM 에 1 μg/ml 농도로 희석하여 RAW 264.7 대식세포(한국세포주은행)에 처리하였다. 이때, 실험에 사용된 모든 세포는 충분한 계대 배양을 통한 안정화 기간을 거친 후, 이후의 실험에 사용하였다. 실험은 총 4 종류의 그룹으로 분류하였다. 아무것도 처리하지 않은 대조군 그룹, UDCA 만 단독으로 처리한 UDCA 그룹, LPS 만 단독으로 처리한 LPS 그룹, UDCA 와 LPS 를 함께 처리한 UDCA + LPS 그룹 이렇게 총 4 그룹으로 구분하였다. 이 때, UDCA 단독 그룹과 LPS 와 UDCA 를 함께 처리한 그룹은 미리 지정된 시작 시간 이전에, 한 시간씩 같은 농도의 UDCA 를 세포에 전처리 하였다. Specifically, lipopolysaccharide (LPS, Sigma, St. Louis, MO) was dissolved in distilled water at a concentration of 100 μg / ml and then diluted with DMEM to a concentration of 1 μg / ml to prepare RAW 264.7 macrophages ). At this time, all the cells used in the experiment were used for the subsequent experiments after a stabilization period through sufficient subculture. The experiment was divided into four groups. A UDCA group treated only with UDCA alone, a LPS group treated with LPS alone, and a UDCA plus LPS group treated with UDCA and LPS were classified into four groups. At this time, the group treated with the UDCA alone group, the LPS and the UDCA group were pretreated with the same concentration of UDCA for one hour before the predetermined start time.
2.1. NO 유도 활성2.1. NO induction activity
UDCA 가 RAW 264.7 대식세포에서 NO(Nitric Oxide) 를 얼마나 유도하는지 측정하기 위해 그리스 시약 키트(Griess Reagent kit) (Promega, Madison, WI) 를 사용하였다. A Griess Reagent kit (Promega, Madison, Wis.) Was used to determine how UDCA induced nitric oxide (NO) in RAW 264.7 macrophages.
구체적으로, 96-웰 배양 플레이트(Falcon) 에 웰 당 1 x 105의 대식세포를 분주하고, (n=3 per group) 1 시간 동안 안정화 시켰다. LPS 에 의해 촉진된 NO를 UDCA 가 얼마나 억제할 수 있는지 확인하기 위해 LPS 만 단독 처리한 그룹에서의 NO 와 UDCA + LPS 그룹에서의 NO 발현 량을 같은 세포의 조건에서 비교, 분석하였다. 이 때, LPS 단독 그룹은 제외하고, UDCA + LPS 그룹만 같은 농도의 UDCA 를 1 시간 동안 전처리 하였다. UDCA는 0.5, 또는 1 mM 의 용량으로 처리하였고, 그 결과를 도 2b에 나타내었다. Specifically, 1 × 10 5 macrophages per well were dispensed into 96-well culture plates (Falcon) and stabilized (n = 3 per group) for 1 hour. To determine how much UDCA could inhibit NO stimulated by LPS, NO expression levels in NO and UDCA + LPS groups in the LPS alone treatment group were compared and analyzed under the same cell conditions. At this time, except for the LPS alone group, only the UDCA + LPS group was pretreated with the same concentration of UDCA for 1 hour. UDCA was treated at a dose of 0.5, or 1 mM, and the results are shown in Figure 2b.
도 2는 다양한 농도에서의 우르소데옥시콜산이 LPS 유도된 세포의 NO 분비에 미치는 영향을 나타낸 그래프이다. Figure 2 is a graph showing the effect of ursodeoxycholic acid at various concentrations on NO secretion in LPS-induced cells.
도 2에 나타낸 바와 같이, 우르소데옥시콜산은 염증 반응이 유도된 대식세포에서 NO의 분비량을 현저히 억제하여 항염증 활성이 있음을 알 수 있다. As shown in Fig. 2, ursodeoxycholic acid significantly inhibited the secretion amount of NO in inflammatory response-induced macrophages, indicating anti-inflammatory activity.
2.2. 염증 관련 유전자 발현 분석2.2. Analysis of inflammatory gene expression
UDCA의 항염증 활성을 확인하기 위하여, UDCA의 처리에 따른, 항염증 관련 인자인 TNF-α, IL-1α, IL-1β, IL-6, 또는 IL-10의 mRNA 발현을 qRT-PCR로 측정하였다. In order to confirm the anti-inflammatory activity of UDCA, mRNA expression of anti-inflammatory factors TNF-α, IL-1α, IL-1β, IL-6 or IL-10 following treatment with UDCA was measured by qRT-PCR Respectively.
구체적으로, 세포배양 플레이트(Falcon) 에 2 x 105 개의 세포를 분주한 후 1 시간 동안 안정화 시켰다. 그 다음, UDCA, LPS, 또는 UDCA + LPS 를 처리하여 1, 6, 또는 24 시간 동안 배양하였다. 이 때, UDCA 그룹과 UDCA + LPS 그룹은 1 mM 농도의 UDCA 를 1시간 동안 전처리 하였다. 세포 처리 1시간, 6시간, 및 24시간 후에, 세포로부터 트리졸 시약(Invitrogen)을 이용하여 제조사의 지시에 따라 RNA를 추출하였다. 그룹당 1 μg 의 RNA 를 정량하여, cDNA 합성 kit (TAKARA, Shiga, Japan) 를 사용해 cDNA 로 합성 후, qRT-PCR 하였다. PCR 기계는 ABI Step One Real-time PCR System (Applied Biosystems, Warrington, UK) 제품을 사용하였고, 하우스키핑 유전자인 GAPDH(glyceraldehyde 3-phosphate dehydrogenase) 같이 측정하여 결과 값을 보정하였으며, 이 때, 2-△△CT 방법(SCHMITTGEN, TD, and KJ LIVAK. "ANALYZING REAL-TIME PCR DATA BY THE COMPARATIVE C (T) METHOD." (2008): 1101-1108)을 사용하였다.Specifically, the cells after the division 2 x 10 5 cells on a culture plate (Falcon) were stabilized for 1 hour. UDCA, LPS, or UDCA + LPS were then treated and incubated for 1, 6, or 24 hours. At this time, UDCA group and UDCA + LPS group were pretreated with 1 mM UDCA for 1 hour. After 1, 6, and 24 hours of cell treatment, RNA was extracted from the cells according to the manufacturer's instructions using a trizol reagent (Invitrogen). 1 μg of RNA per group was quantified and synthesized with cDNA using a cDNA synthesis kit (TAKARA, Shiga, Japan), followed by qRT-PCR. PCR was performed using ABI Step One Real-time PCR System (Applied Biosystems, Warrington, UK) and corrected with GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as a housekeeping gene. (T) method (SCHMITTGEN, TD, and KJ LIVAK. "ANALYZING REAL-TIME PCR DATA BY THE COMPARATIVE C (T) METHOD." (2008): 1101-1108) was used.
도 3a는 우르소데옥시콜산이 LPS 유도된 세포의 TNF-α mRNA 발현에 미치는 영향을 나타낸 그래프이다. 3A is a graph showing the effect of ursodeoxycholic acid on the expression of TNF-α mRNA in LPS-induced cells.
도 3b는 우르소데옥시콜산이 LPS 유도된 세포의 IL-1α mRNA 발현에 미치는 영향을 나타낸 그래프이다. 3b is a graph showing the effect of ursodeoxycholic acid on IL-1? MRNA expression of LPS-induced cells.
도 3c는 우르소데옥시콜산이 LPS 유도된 세포의 IL-1β mRNA 발현에 미치는 영향을 나타낸 그래프이다. 3c is a graph showing the effect of ursodeoxycholic acid on the expression of IL-1? MRNA in LPS-induced cells.
도 3d는 우르소데옥시콜산이 LPS 유도된 세포의 IL-6 mRNA 발현에 미치는 영향을 나타낸 그래프이다. FIG. 3D is a graph showing the effect of ursodeoxycholic acid on IL-6 mRNA expression of LPS-induced cells.
도 3e는 우르소데옥시콜산이 LPS 유도된 세포의 IL-10 mRNA 발현에 미치는 영향을 나타낸 그래프이다. 3E is a graph showing the effect of ursodeoxycholic acid on IL-10 mRNA expression of LPS-induced cells.
도 3a 내지 3e에 나타낸 바와 같이, 우르소데옥시콜산은 염증성 사이토카인인 TNF-α, IL-1α, IL-1β, 및 IL-6의 발현은 억제하면서, 염증 반응을 억제시키는 인자인 IL-10의 발현은 증가시킴을 확인할 수 있었다. As shown in Figs. 3A to 3E, ursodeoxycholic acid inhibited the expression of IL-10, which inhibits the inflammatory response, while suppressing the expression of inflammatory cytokines TNF-a, IL-la, IL- And the expression of the gene was increased.
따라서, 일 구체예에 따른 우르소데옥시콜산은 현저한 항염증 활성을 가짐을 확인할 수 있었다. Thus, it was confirmed that the ursodeoxycholic acid according to one embodiment has a remarkable anti-inflammatory activity.
2.3. 염증 관련 단백질 발현 분석2.3. Analysis of inflammation-related protein expression
상기 실시예 2.2.와 동일한 염증관련 인자들에 대해 단백질 발현을 ELISA로 분석하였다. Protein expression was analyzed by ELISA for the same inflammatory-related factors as in Example 2.2.
구체적으로, 96-웰 세포 배양 프레이트(Falcon)에 1 x 105개의 세포를 분주한 후 1 시간 동안 안정화 시켰다. 그 다음, UDCA, LPS, 또는 UDCA + LPS 를 처리하고 배양하였다. 이 때, UDCA 그룹과 UDCA + LPS 그룹은 1 mM 농도의 UDCA 를 1시간 동안 전처리 하였다. 1, 6, 또는 24 시간 후에 세포 상층액을 걷어 ELISA 전용 플레이트 (Koma Biotech, Seoul, Korea) 제조사의 지시에 따라 처리하였다. 전용 플레이트 표면에 흡착된 각각의 사이토카인의 양을 분석하였고, 그 결과를 도 4에 나타내었다. Specifically, 96-well cell division after the 1 x 10 5 cells in culture freight (Falcon) were stabilized for 1 hour. UDCA, LPS, or UDCA + LPS were then treated and cultured. At this time, UDCA group and UDCA + LPS group were pretreated with 1 mM UDCA for 1 hour. After 1, 6, or 24 hours, the cell supernatants were plated and processed according to the manufacturer's instructions for ELISA plates (Koma Biotech, Seoul, Korea). The amount of each cytokine adsorbed on the surface of the dedicated plate was analyzed, and the results are shown in FIG.
도 4a는 우르소데옥시콜산이 LPS 유도된 세포의 TNF-α 단백질 발현에 미치는 영향을 나타낸 그래프이다. 4A is a graph showing the effect of ursodeoxycholic acid on the expression of TNF-a protein in LPS-induced cells.
도 4b는 우르소데옥시콜산이 LPS 유도된 세포의 IL-1α 단백질 발현에 미치는 영향을 나타낸 그래프이다. 4b is a graph showing the effect of ursodeoxycholic acid on IL-1 alpha protein expression of LPS-induced cells.
도 4c는 우르소데옥시콜산이 LPS 유도된 세포의 IL-1β 단백질 발현에 미치는 영향을 나타낸 그래프이다. 4c is a graph showing the effect of ursodeoxycholic acid on the expression of IL-1 beta protein in LPS-induced cells.
도 4d는 우르소데옥시콜산이 LPS 유도된 세포의 IL-6 단백질 발현에 미치는 영향을 나타낸 그래프이다. 4d is a graph showing the effect of ursodeoxycholic acid on the expression of IL-6 protein in LPS-induced cells.
도 4e는 우르소데옥시콜산이 LPS 유도된 세포의 IL-10 단백질 발현에 미치는 영향을 나타낸 그래프이다. 4E is a graph showing the effect of ursodeoxycholic acid on IL-10 protein expression of LPS-induced cells.
도 4a 내지 4e에 나타낸 바와 같이, 우르소데옥시콜산은 상기 실시예 2.2.와 동일하게 염증성 사이토카인인 TNF-α, IL-1α, IL-1β, 및 IL-6의 발현은 억제하면서, 염증 반응을 억제시키는 인자인 IL-10의 발현은 증가시킴을 확인할 수 있었다. 특히 LPS 에 의해 발현 되었어야 할 염증성 사이토카인의 값들이 거의 우르소데옥시콜산에 의해 거의 0 에 가깝게 억제되어 발현되지 못했음을 확인할 수 있었다. 이는 우르소데옥시콜산이 매우 강하게 염증성 사이토카인을 억제함을 의미한다.As shown in Figs. 4A to 4E, ursodeoxycholic acid inhibited the expression of inflammatory cytokines TNF-α, IL-1α, IL-1β and IL-6 in the same manner as in Example 2.2, IL-10 < / RTI > In particular, it was confirmed that the values of the inflammatory cytokines to be expressed by LPS were almost suppressed to almost 0 by ursodeoxycholic acid and could not be expressed. This means that ursodeoxycholic acid inhibits inflammatory cytokines very strongly.
또한, 추가적으로 우르소데옥시콜산이 염증 관련 인자인 ERK(Extracellular signal-regulated kinase), JNK(c-Jun N-terminal kinase), p38, IκBα(nuclear factor kappa-light polypeptide gene enhancer in B-cells inhibitor, alpha)의 인산화에 미치는 영향을 웨스턴 블랏팅으로 분석하였다. In addition, ursodeoxycholic acid is also known to be involved in inflammatory factors such as ERK (Extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), p38, IκBα (nuclear factor kappa-light polypeptide enhancer in B- alpha) phosphorylation was analyzed by Western blotting.
구체적으로, 100-mm 배양 플레이트(Falcon)에 2 x 106 개의 세포를 분주한 후, 1 시간 동안 안정화 시켰다. 그 다음, UDCA, LPS, 또는 UDCA + LPS 를 처리하여 24 시간 동안 배양하였다. 이 때, UDCA 그룹과 UDCA + LPS 그룹은 1 mM 농도의 UDCA를 1시간 동안 전처리 하였다. 24 시간 후, 세포 스크래퍼(cell scraper) (SPL, Seoul, Korea)를 사용하여 세포를 걷고 튜브에 수집하였다. 수집된 세포들을 차가운 RIPA 라이시스 버퍼(2.5% 디옥시콜산, 0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 10% NP-40, 10 mM EDTA, 프로테아제 억제제 (Roche Applied Science, Indianapolis, IN), 포스파타아제 억제제 (Sigma)함유)를 이용하여 용해시킨 후, 제조사의 지시에 따라 단백질을 추출 후 정량하였다. 각 그룹의 시료를 각각 40 μg으로 만든 다음, 10%의 소듐 도데실 설페이트 폴리아크릴아미드 젤에 전기영동 한 후, 젤에 처리된 단백질들에 항체를 붙이기 위해 니트로셀룰로오스 트랜스퍼 멤브레인(nitrocellulose transfer membranes) (Protran, Whatman, Germany) 으로 옮겼다. 1차 항체를 붙이기 전에 5% 의 탈지 우유를 1 시간 처리하여 비 특이적인 단백질들의 결합을 차단하였으며, 먼저 인산화 형태의 단백질을 확인한 후, 같은 멤브레인에 흡착된 단백질을 스트립 용액을 이용해 다 떼어내고, 그 다음 인산화 전 형태의 단백질을 처리하는 방법으로 웨스턴 블랏을 수행하였다. 밴드의 값은 ChemiDoc XRS (Bio-Rad)를 이용하여 측정하였다. 처리된 1차 항체(Cell Signaling Technology, Danvers, MA, USA)의 농도는 모두 1:1000 이었다. 대조군으로 사용된 β-액틴(actin) 의 1차 항체(Abcam (Cambridge, UK) 농도는 1:2000이었다. 대조군의 (인산화/인산화 전) 비율을 1로 하고, 그에 상대적인 각 그룹들의 (인산화/인산화 전) 밴드 값을 ChemiDoc 프로그램 (Bio-Rad) 을 이용하여 계산하였고, 그 결과를 도 5에 나타내었다. Specifically, 2 x 10 6 cells were placed in 100-mm culture plates (Falcon) and stabilized for 1 hour. UDCA, LPS, or UDCA + LPS were then treated and cultured for 24 hours. At this time, UDCA group and UDCA + LPS group were pretreated with 1 mM UDCA for 1 hour. After 24 hours, the cells were harvested by using a cell scraper (SPL, Seoul, Korea). The collected cells were washed with cold RIPA lysis buffer (2.5% deoxycholate, 0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 10% NP-40, 10 mM EDTA, protease inhibitor (Roche Applied Science, , Phosphatase inhibitor (Sigma)), and the protein was extracted and quantified according to the manufacturer's instructions. Each sample was made up to 40 μg each, and then electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gel. Nitrocellulose transfer membranes (hereinafter referred to as "nitrocellulose") were used to attach the antibodies to the gel- Protran, Whatman, Germany). Prior to attaching the primary antibody, 5% of skim milk was treated for 1 hour to block the binding of nonspecific proteins. First, the phosphorylated form of the protein was identified, the protein adsorbed on the same membrane was removed using the strip solution, Western blotting was then performed by a method of treating pre-phosphorylated forms of the protein. The values of the bands were measured using ChemiDoc XRS (Bio-Rad). The concentration of the treated primary antibody (Cell Signaling Technology, Danvers, MA, USA) was 1: 1000. The concentration of the primary antibody (Abcam (Cambridge, UK)) of β-actin used as a control was 1: 2000. The ratio of the control group (before phosphorylation / phosphorylation) was set to 1, The pre-phosphorylation band values were calculated using the ChemiDoc program (Bio-Rad) and the results are shown in FIG.
도 5a는 우르소데옥시콜산이 LPS 유도된 세포의 ERK의 인산화에 미치는 영향을 나타낸 그래프이다. 5A is a graph showing the effect of ursodeoxycholic acid on the phosphorylation of ERK in LPS-induced cells.
도 5b는 우르소데옥시콜산이 LPS 유도된 세포의 JNK의 인산화에 미치는 영향을 나타낸 그래프이다. 5B is a graph showing the effect of ursodeoxycholic acid on the phosphorylation of JNK in LPS-induced cells.
도 5c는 우르소데옥시콜산이 LPS 유도된 세포의 p-38의 인산화에 미치는 영향을 나타낸 그래프이다. 5c is a graph showing the effect of ursodeoxycholic acid on the phosphorylation of p-38 of LPS-induced cells.
도 5d는 우르소데옥시콜산이 LPS 유도된 세포의 IκBα의 인산화에 미치는 영향을 나타낸 그래프이다. FIG. 5d is a graph showing the effect of ursodeoxycholic acid on the phosphorylation of IκBα in LPS-induced cells.
도 5a 내지 5d에 나타낸 바와 같이, 우르소데옥시콜산은 MAPKs(ERK, JNK, p38) 및 NF-κB 경로에서의 인산화를 현저하게 억제함을 알 수 있다. As shown in Figs. 5A to 5D, it can be seen that ursodeoxycholic acid significantly inhibited phosphorylation in MAPKs (ERK, JNK, p38) and the NF-kB pathway.
이상의 결과로, 우르소데옥시콜산은 LPS 유도된 대식 세포에서 염증성 사이토카인의 분비를 억제하고, 염증 관련 경로의 인산화를 억제하여 항염증성 질환의 치료에 유용하게 사용될 수 있음을 알 수 있다. As a result, ursodeoxycholic acid suppresses the secretion of inflammatory cytokines from LPS-induced macrophages and inhibits the phosphorylation of inflammation-related pathways, thus being useful for the treatment of anti-inflammatory diseases.
실험예Experimental Example 3. 3. UDCA의UDCA's 척수 손상 개선 활성 Spinal cord injury improving activity
우르소데옥시콜산이 척수 손상 치료제로서 활용될 수 있는지 확인하기 위해, 척수 손상을 가한 동물 모델에 우르소데옥시콜산을 투여하여 회복 정도를 평가하였다. Ursodeoxycholic acid was administered to animal models of spinal cord injury to assess the degree of recovery in order to confirm that ursodeoxycholic acid could be used as a therapeutic agent for spinal cord injury.
구체적으로, 척수 손상 동물 모델을 제작하기 위해 랫드(Sprague Dawley, 9주령 (200 ~ 220 g), 암컷)에 케타민 (80 mg/kg) 및 자일라진 (10 mg/kg)을 복강 주사하여 마취시킨 후, T9 번의 척추를 제거하였다. 노출된 해당 부위의 척수에 35 g의 추를 중력을 이용한 콤프레션(compression) 방법으로 5분 동안 가하여 척수 손상을 유도하였다. 음성 대조군(SHAM)을 위해, T9번의 척추를 제거하였으나, 척수에 손상을 주지 않은 동물모델도 제조하였다. Specifically, animals were anesthetized by intraperitoneal injection of ketamine (80 mg / kg) and xylazine (10 mg / kg) in rats (Sprague Dawley, After that, the spine of T9 was removed. Spinal cord injury was induced by applying a 35 g weight to the exposed spinal cord spinal cord for 5 minutes using a gravity compression method. For the negative control (SHAM), animal models were also prepared that removed spinal T9 but did not damage the spinal cord.
척수 손상 1시간 후에 우르소데옥시콜산을 24.4 mg/kg/day의 용량으로 매일 1회 총 7일간 복강주사하였고, 대조군으로는 동일양의 염수(normal saline)를 복강 투여하였다(각 그룹당 n=27). 각 그룹당 다시 1일차 그룹, 3일차 그룹, 및 7일차 그룹으로 나누어 Basso, Beattie, Bresnahan (BBB) 스코어(운동기능의 회복 정도를 복합적으로 평가할 수 있는 실험방법으로서, 0부터 21 까지 점수를 나누어 뒷다리를 전혀 사용하지 못하는 경우 0점, 정상인 경우를 21점으로 표시함) 실험에 충분한 지식과 경험이 있는 연구원들이 2분 이상 뒷다리의 움직임 정도를 관찰하여 수치화하였고, 그 결과를 도 6에 나타내었다. Ursodeoxycholic acid was intraperitoneally injected once daily for 7 days at a dose of 24.4 mg / kg / day for 1 hour after spinal cord injury, and the same amount of saline (normal saline) was administered intraperitoneally as a control group (n = 27 ). Basso, Beattie, and Bresnahan (BBB) scores were divided into three groups: 1st, 3rd, and 7th groups. And 0 for normal, and 21 for normal.) The researchers who had sufficient knowledge and experience in the experiments observed the degree of movement of the hind legs for more than 2 minutes, and the results are shown in FIG.
또한, 염색 실험을 위해 마취 후 퍼퓨전(perfusion) 방법으로 분리시킨 랫드의 척수를 4% PFA 에 고정시켰다. 이후에 염색의 준비 단계인 파라핀 섹션을 위해 일련의 방법대로 척수 외 성분을 제거한 뒤 필요한 만큼 섹션한 절편들을 헤마톡실린 및 에오신(Hematoxylin & eosin)으로 염색하였다. H&E 염색을 통해 손상된 조직의 복구 및 염증 반응 정도를 광학 현미경(Olympus IX71, Japan)으로 40 x와 100x에서 비교 분석하였고, 1일째, 3일째, 및 7일째의 결과를 각각 도 7a, 7b, 및 7c에 나타내었다. For the staining experiment, the spinal cord of rats isolated by perfusion after anesthesia was fixed to 4% PFA. Subsequently, for the paraffin section, which is the preparation stage for staining, the extra-spinal components were removed in a series of steps, and sections as needed were stained with hematoxylin and eosin. The degree of repair of injured tissue and the degree of inflammation were analyzed by optical microscope (Olympus IX71, Japan) at 40 x and 100x through H & E staining, and the results on
또한, 안락사시킨 쥐의 척수를 분리하여 손상 부위를 정중앙으로 포함시킨 10 mm 길이의 척수를 그룹마다 똑같은 크기로 잘라내어, 실시예 2.2.와 동일한 방법으로 mRNA 수준에서 그 염증 반응 억제 정도를 비교하였고, 그 결과를 도 8에 나타내었다. In addition, the spinal cord of euthanized rats was separated and the 10-mm-long spinal cord including the damaged area at the center was cut to the same size in each group. The degree of inhibition of the inflammatory reaction was compared at the mRNA level in the same manner as in Example 2.2. The results are shown in Fig.
도 6은 우르소데옥시콜산이 척수 손상 동물모델에서 운동기능의 회복에 미치는 영향을 평가한 그래프이다. 6 is a graph showing the effect of ursodeoxycholic acid on recovery of motor function in an animal model of spinal cord injury.
도 7은 우르소데옥시콜산이 척수 손상 동물모델에서 조직의 회복에 미치는 영향을 나타낸 사진이다. 7 is a photograph showing the effect of ursodeoxycholic acid on the recovery of tissue in an animal model of injured spinal cord.
도 8은 우르소데옥시콜산이 척수 손상 동물모델에서 염증 반응의 억제 활성을 나타낸 그래프이다. Figure 8 is a graph showing the inhibitory activity of the inflammatory response in an animal model of spinal cord injured ursodeoxycholic acid.
도 6에 나타낸 바와 같이, UDCA를 척수 손상 동물 모델에 투여한 결과, 대조군에 비해 BBB 스코어가 약 2배 정도 높아져서, 동물 모델의 운동 능력히 현저하게 개선된 것을 확인할 수 있었다. As shown in FIG. 6, when UDCA was administered to an animal model of spinal cord injury, the BBB score was about two times higher than that of the control group, and it was confirmed that the exercise capacity of the animal model was remarkably improved.
또한, 도 7에 나타낸 바와 같이, 척수 손상 동물 모델의 척수 조직을 관찰한 결과, UDCA의 투여에 의해서 척수 조직이 손상이 회복되고 있음을 확인할 수 있었다. As shown in FIG. 7, the spinal cord tissues of the spinal cord injury animal model were observed. As a result, it was confirmed that the spinal cord tissue was recovered by UDCA administration.
또한, 도 8에 나타낸 바와 같이, 염증 관련 인자를 확인한 결과, UDCA가 척수 손상 동물 모델에서 염증성 사이토카인인 TNF-α, IL-1β, 및 IL-6의 발현은 억제하면서, 염증 반응을 억제시키는 인자인 IL-10의 발현은 증가시킴을 확인할 수 있었다. In addition, as shown in Fig. 8, it was confirmed that UDCA inhibited the inflammatory response while inhibiting the expression of inflammatory cytokines TNF-a, IL-l [beta] and IL-6 in spinal cord injury animal models And the expression of IL-10, which is a factor, is increased.
이상의 결과로, 우르소데옥시콜산은 척수 손상된 개체에의 항염증 활성 뿐만 아니라, 개체의 운동 능력을 현저하게 향상 시키고, 손상된 척수 조직을 회복시켜, 척수 손상 치료제로 유용하게 사용될 수 있음을 알 수 있다. As a result, ursodeoxycholic acid remarkably improves the exercise capacity of the individual as well as anti-inflammatory activity on spinal cord injured individuals, restores injured spinal cord tissues, and is useful as a therapeutic agent for spinal cord injury .
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