KR20220135802A - Composition for treating steatohepatitis comprising A-54556A as an active ingredient - Google Patents
Composition for treating steatohepatitis comprising A-54556A as an active ingredient Download PDFInfo
- Publication number
- KR20220135802A KR20220135802A KR1020210041956A KR20210041956A KR20220135802A KR 20220135802 A KR20220135802 A KR 20220135802A KR 1020210041956 A KR1020210041956 A KR 1020210041956A KR 20210041956 A KR20210041956 A KR 20210041956A KR 20220135802 A KR20220135802 A KR 20220135802A
- Authority
- KR
- South Korea
- Prior art keywords
- liver
- steatohepatitis
- diet
- fat
- tissue
- Prior art date
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Images
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
- A61K31/33—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
- A61K31/407—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 other heterocyclic ring systems, e.g. ketorolac, physostigmine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Abstract
Description
본 발명은 A-54556A를 유효성분으로서 포함하는 지방간염 치료용 조성물에 관한 것이다.The present invention relates to a composition for treating steatohepatitis comprising A-54556A as an active ingredient.
전 세계적으로 과체중 및 비만으로 인한 대사질환 환자는 지속적으로 증가하고 있다. Cohen et al.(2011)에 따르면, 현재 미국의 성인 비만인구의 약 34%가 비알콜성 지방간(steatosis) 질환을 가지고 있으며, 결국 보편적인 만성질환인 간이상(liver failure), 간경화(cirohosis), 간암(hepato cellular carcinoma)과 같은 만성간질환으로 발전할 가능성이 높은 것으로 보고되었다. 또한 Stefan et al.(2008) 및 Cohen et al.(2011)에 따르면, 비알콜성 지방간을 지닌 성인의 80%는 인슐린 저항성, 당뇨병 및 심장질환 등 대사질환질병으로 발전할 수 있는 것으로 보고되었다. Globally, the number of patients with metabolic diseases due to overweight and obesity is continuously increasing. According to Cohen et al. (2011), about 34% of adult obese adults in the United States currently have nonalcoholic fatty liver disease, which eventually leads to common chronic diseases such as liver failure and cirrhosis. , has been reported to be highly likely to develop into chronic liver diseases such as hepato cellular carcinoma. Also, according to Stefan et al. (2008) and Cohen et al. (2011), it was reported that 80% of adults with nonalcoholic fatty liver can develop metabolic diseases such as insulin resistance, diabetes and heart disease.
비알콜성 지방간 질환은 알코올과는 독립적으로 간에 지방이 축적된 단순 지방간질환 및 비알코올성 지방간염(non-alcoholic steatohepatitis, NASH) 질환을 포함한다. 비알코올성 지방간염(NASH)은 간에 지방이 정상범위를 넘어서서 축적되는 단순지방간의 단계를 거쳐서 염증반응, 세포섬유화 및 세포 손상을 유발하므로 간경화 또는 간암으로 발전할 가능성이 상당히 높다.Non-alcoholic fatty liver disease includes simple fatty liver disease and non-alcoholic steatohepatitis (NASH) disease in which fat is accumulated in the liver independently of alcohol. Nonalcoholic steatohepatitis (NASH) is highly likely to develop into cirrhosis or liver cancer because it induces inflammatory reactions, cellular fibrosis and cell damage through the simple fatty liver stage in which fat accumulates beyond the normal range.
미토콘드리아 기능 장애는 비알코올성 지방간염(NASH)의 발병에 중요한 인자로 간주된다. 먼저 유전적, 환경적 요인에 의해 비만, 인슐린 저항성이 유발되고 지방산 처리 능력이 부족하여 간조직에 지방이 축적되는 지방간 현상을 나타내며, 추후 독성을 나타내는 지질 유도체(diacylglycerols, ceramides, lysophosphatidic acid) 축적 및 과도한 산화 스트레스, endotoxin, 미토콘드리아 기능이상, 다양한 염증성 인자(TNF-α 및 IL-6)들이 생성되고, 간성상세포의 활성 등 다양한 이차독성을 유발하여 간세포 손상이 일으킴으로써 비알코올성 지방간염(NASH)으로 진행하게 된다(Mota et al, 2016, Alisi A et al, 2011, Jung et al; 2014).Mitochondrial dysfunction is considered an important factor in the pathogenesis of nonalcoholic steatohepatitis (NASH). First, obesity and insulin resistance are induced by genetic and environmental factors, and the fatty acid processing ability is insufficient, indicating a fatty liver phenomenon in which fat is accumulated in the liver tissue. Nonalcoholic steatohepatitis (NASH) due to excessive oxidative stress, endotoxin, mitochondrial dysfunction, the production of various inflammatory factors (TNF-α and IL-6), and various secondary toxicity such as hepatocyte activation (Mota et al, 2016, Alisi A et al, 2011, Jung et al; 2014).
미토콘드리아의 샤페론과 프로테아제는 미토콘드리아에서 단백질 항상성을 유지하는 데 중요한 역할을 한다고 보고되었다(Jovaisaite, V). 특히 Voos et al.에 의하면 protease Lon과 caseinolytic protease complex (ClpXP), matrix protease (iAAA와 mAAA) 등의 ATP 의존성 미토콘드리아 단백질 분해 효소는 미토콘드리아에서 단백질의 이상을 찾아서 감시하는 역할을 수행하고 이상이 있는 단백질은 분해하여 단백질 항상성을 유지하는 데 중요한 역할을 한다고 보고되었다. 이렇게 단백질 항상성을 조절하는 프로테아제의 기능 이상은 미토콘드리아에 잘못 접힌 단백질(misfolded proteins)들의 축적을 유발하여 미토콘드리아 기능에 손상을 유도하고 신경병증을 유발한다고 추측된다. 미토콘드리아 기질의 ClpP (caseinolytic protease P)의 부족으로 인해 발생한 부적절한 미토콘드리아 단백질 품질 관리는 잠재적으로 신경 기능 장애를 유발할 수 있다. 그러나 아직까지 Clp protease의 이상과 지방간, 지방간염과의 관련성은 보고된 바가 없다. It has been reported that mitochondrial chaperones and proteases play an important role in maintaining protein homeostasis in mitochondria (Jovaisaite, V). In particular, according to Voos et al., ATP-dependent mitochondrial proteolytic enzymes, such as protease Lon, caseinolytic protease complex (ClpXP), and matrix protease (iAAA and mAAA), play a role in finding and monitoring protein abnormalities in the mitochondria. has been reported to play an important role in maintaining protein homeostasis by degradation. This malfunction of the protease that regulates protein homeostasis causes the accumulation of misfolded proteins in the mitochondria, leading to damage to the mitochondrial function and neuropathy. Inadequate mitochondrial protein quality control resulting from the lack of caseinolytic protease P (ClpP) in the mitochondrial matrix can potentially lead to neuronal dysfunction. However, the association between abnormal Clp protease and fatty liver or steatohepatitis has not been reported.
또한 최근 논문 (Implications of Mitochondrial Unfolded Protein Response and Mitokines: A Perspective on Fatty Liver Diseases)에 따르면, ClpP와 지방간염과의 연관성을 확인하기 위해 ClpP knockout 마우스를 제작하였으나, 오히려 NASH 발병이 감소하는 것으로 보고되었다. 따라서 ClpP와 비알코올성 지방간염의 연관성은 아직 명확히 입증되지 못한 상태이다.In addition, according to a recent paper (Implications of Mitochondrial Unfolded Protein Response and Mitokines: A Perspective on Fatty Liver Diseases), ClpP knockout mice were produced to confirm the association between ClpP and steatohepatitis, but rather, it was reported that the incidence of NASH decreased. . Therefore, the association between ClpP and nonalcoholic steatohepatitis has not yet been clearly established.
일 구체예에 따르면 A-54556A를 유효성분으로 포함함으로써 지방간염의 치료 또는 예방용 목적으로 사용될 수 있는 약학적 조성물을 제공한다. According to one embodiment, there is provided a pharmaceutical composition that can be used for the treatment or prevention of steatohepatitis by including A-54556A as an active ingredient.
일 양상은 A-54556A 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 지방간염 치료 또는 예방용 약학적 조성물을 제공한다. One aspect provides a pharmaceutical composition for treating or preventing steatohepatitis comprising A-54556A or a pharmaceutically acceptable salt thereof as an active ingredient.
일 실시예에 따르면, 고지방/고과당 식이를 공급한 마우스의 간조직은 ClpP 발현 및 활성이 정상대조군보다 감소하지만, A-54556A를 투여한 실험군은 고열량 식이 공급에도 불구하고 ClpP 발현량 및 활성이 회복되었다. 나아가 A-54556A는 지방간, 간의 염증, 간손상, 및 간 섬유화를 감소시켜 비알코올성 지방간질환의 예방 및 치료 효과를 나타낼 수 있음을 확인하였다. According to one embodiment, the liver tissue of mice fed a high-fat/high-fructose diet decreased ClpP expression and activity than the normal control group, but the experimental group administered with A-54556A showed ClpP expression and activity despite the high-calorie diet. recovered Furthermore, it was confirmed that A-54556A can exhibit the preventive and therapeutic effect of non-alcoholic fatty liver disease by reducing fatty liver, liver inflammation, liver damage, and liver fibrosis.
상기 A-54556A가 최초 공개된 미국 특허공개공보 US2016/0046674에 따르면 A-54556A의 항생제 용도가 알려져 있으나 지방간염, 비알코올성 지방간염, 간섬유화에 대한 치료 또는 개선 효과에 대해서는 알려진 바가 없다. According to US Patent Publication No. US2016/0046674, in which A-54556A was first published, antibiotic use of A-54556A is known, but there is no known information about the therapeutic or improving effect on steatohepatitis, nonalcoholic steatohepatitis, and liver fibrosis.
용어 "치료"는 질환 또는 그의 하나 이상의 증상의 경감, 진행 억제 또는 예방을 의미할 수 있다. The term “treatment” may mean alleviation, inhibition of progression, or prevention of a disease or one or more symptoms thereof.
상기 A-54556A는 하기 화학식 1로 표시되는 화합물일 수 있다.A-54556A may be a compound represented by the following formula (1).
[화학식 1][Formula 1]
상기 용어 "약학적으로 허용 가능한 염"이란, 일 양상에 따른 특정 화합물과 비교적 무독성인 산 또는 염기를 이용해서 조제되는 염을 의미한다. 약학적으로 허용 가능한 염기 부가염은 나트륨, 칼륨, 칼슘, 암모늄, 유기 아민, 혹은 마그네슘의 염 또는 유사한 염이 포함될 수 있다. 약학적으로 허용 가능한 산 부가염은 염산, 브롬화 수소산, 질산, 탄산, 탄산 수소 이온, 인산, 인산 1수소 이온, 인산 2수소 이온, 황산, 황산 수소 이온, 요오드화 수소산 또는 아인산 등의 무기산의 염, 그리고 아세트산, 프로피온산, 이소부티르산, 말레산, 말론산, 안식향산, 숙신산, 수베르산, 푸마르산, 락트산, 만델산, 프탈산, 벤젠술폰산, p-톨릴술폰산, 구연산, 주석산, 메탄술폰산 등의 유기산의 염을 들 수 있고, 나아가 아미노산(예를 들면 아르기닌 등)의 염 및 글루쿠론산 등의 유기산의 염도 포함될 수 있다. The term “pharmaceutically acceptable salt” refers to a salt prepared using a specific compound according to an aspect and a relatively non-toxic acid or base. Pharmaceutically acceptable base addition salts may include salts of sodium, potassium, calcium, ammonium, organic amines, or magnesium or similar salts. Pharmaceutically acceptable acid addition salts include salts of inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate ion, phosphoric acid, phosphate monohydrogen ion, phosphate dihydrogen ion, sulfuric acid, hydrogen sulfate ion, hydroiodic acid or phosphorous acid; and salts of organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, and methanesulfonic acid and salts of amino acids (eg, arginine, etc.) and salts of organic acids such as glucuronic acid may also be included.
상기 지방간염은 비알콜성 지방간(NASH)일 수 있다. The steatohepatitis may be non-alcoholic fatty liver (NASH).
상기 A-54556A 또는 이의 약학적으로 허용가능한 염은 임상 투여시에 경구 및 비경구의 여러가지 제형으로 투여될 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등 이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용된다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테로 등이 사용될 수 있다.The A-54556A or a pharmaceutically acceptable salt thereof may be administered in various oral and parenteral formulations during clinical administration. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose or lactose ( lactose), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, and emulsions. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
상기 A-54556A 또는 이의 약학적으로 허용가능한 염을 유효 성분으로 하는 약학적 조성물은 비경구 투여할 수 있으며, 비경구 투여는 피하주사, 정맥주사, 근육 내 주사 또는 흉부 내 주사를 주입하는 방법에 의할 수 있다. 이때, 비경구 투여용 제형으로 제제화하기 위하여 상기 A-54556A 또는 이의 약학적으로 허용가능한 염을 안정제 또는 완충제와 함께 물에 혼합하여 용액 또는 현탁액으로 제조하고, 이를 앰플 또는 바이알단위 투여형으로 제조할 수 있다. 상기 조성물은 멸균되고/되거나 방부제, 안정화제, 수화제 또는 유화 촉진제, 삼투압 조절을 위한 염 및/또는 완충제 등의 보조제, 및 기타 치료적으로 유용한 물질을 함유할 수 있으며, 통상적인 방법인 혼합, 과립화 또는 코팅 방법에 따라 제제화할 수 있다. 경구 투여용 제형으로는 예를 들면 정제, 환제, 경/연질 캅셀제, 액제, 현탁제, 유화제, 시럽제, 과립제, 엘릭시르제, 트로키제 등이 있는데, 이들 제형은 유효성분 이외에 희석제(예: 락토즈, 덱스트로즈, 수크로즈, 만니톨, 솔비톨, 셀룰로즈 및/또는 글리신), 활택제(예: 실리카, 탈크, 스테아르산 및 그의 마그네슘 또는 칼슘염 및/또는 폴리에틸렌 글리콜)를 함유할 수 있다. 정제는 마그네슘 알루미늄 실리케이트, 전분 페이스트, 젤라틴, 메틸셀룰로즈, 나트륨 카복시메틸셀룰로즈 및/또는 폴리비닐피롤리딘 등과 같은 결합제를 함유할 수 있으며, 경우에 따라 전분, 한천, 알긴산 또는 그의 나트륨 염 등과 같은 붕해제 또는 비등 혼합물 및/또는 흡수제, 착색제, 향미제, 및 감미제를 함유할 수 있다.The pharmaceutical composition comprising A-54556A or a pharmaceutically acceptable salt thereof as an active ingredient may be administered parenterally, and parenteral administration is subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. can depend At this time, in order to formulate a formulation for parenteral administration, the A-54556A or a pharmaceutically acceptable salt thereof is mixed with water together with a stabilizer or buffer to prepare a solution or suspension, which may be prepared in an ampoule or vial unit dosage form. can The composition may be sterilized and/or contain adjuvants such as preservatives, stabilizers, wetting agents or emulsification accelerators, salts and/or buffers for regulating osmotic pressure, and other therapeutically useful substances, and mixing, granulation, in a conventional manner. It can be formulated according to the method of formulation or coating. Formulations for oral administration include, for example, tablets, pills, hard/soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, troches, and the like. , dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine), lubricants (such as silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). Tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine and the like, and optionally boron such as starch, agar, alginic acid or its sodium salt, etc. It may contain releasing or boiling mixtures and/or absorbents, colorants, flavoring agents, and sweetening agents.
다른 양상은 A-54556A 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 간섬유화 치료 또는 예방용 약학적 조성물을 제공한다. Another aspect provides a pharmaceutical composition for treating or preventing liver fibrosis comprising A-54556A or a pharmaceutically acceptable salt thereof as an active ingredient.
상기 A-54556A 또는 이의 약학적으로 허용 가능한 염에 대한 설명은 상술한 바와 동일하다. The description of A-54556A or a pharmaceutically acceptable salt thereof is the same as described above.
또 다른 양상은 A-54556A 또는 이의 약학적으로 허용 가능한 염을 개체에 투여하는 단계를 포함하는 지방간염을 치료 또는 예방하는 방법을 제공한다. Another aspect provides a method for treating or preventing steatohepatitis, comprising administering to a subject A-54556A or a pharmaceutically acceptable salt thereof.
A-54556A 또는 이의 약학적으로 허용 가능한 염에 대한 설명은 상술한 바와 동일하다. Description of A-54556A or a pharmaceutically acceptable salt thereof is the same as described above.
상기 용어 "투여"란 적절한 방법으로 개체에게 소정의 물질을 도입하는 것을 의미하며, "개체"란 지방간염 또는 비알코올성 지방간염을 보유할 수 있는 인간을 포함한 쥐, 생쥐, 가축 등의 모든 생물을 의미한다. 구체적으로 인간을 포함한 포유동물일 수 있다. 투여 경로는 구강, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장이 포함될 수 있으며, 특별히 한정되는 것은 아니다. The term "administration" means introducing a predetermined substance to an individual by an appropriate method, and "individual" refers to all living organisms, including humans, rats, mice, livestock, etc. that may have steatohepatitis or nonalcoholic steatohepatitis. it means. Specifically, it may be a mammal including a human. The route of administration may include, but is not particularly limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. .
상기 A-54556A 또는 이의 약학적으로 허용 가능한 염은 약학적으로 유효한 양으로 투여한다. 상기 용어, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 환자의 질환의 종류, 중증도, 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. 구체적으로, 상기 약학적 조성물은 0.01 내지 1000 mg/kg/day로, 보다 구체적으로 0.1 내지 500 ㎎/kg/day로 투여될 수 있다. 상기 투여는 하루에 한 번 투여되는 것일 수도 있고, 수 회 나누어 투여되는 것일 수도 있다. 구체적으로, 상기 약학적 조성물의 유효량은 환자의 연령, 성별, 상태, 체중, 체내에 활성 성분의 흡수도, 불활성율, 배설 속도, 질병 종류, 병용되는 약물에 따라 달라질 수 있으며, 투여 경로, 비만의 중증도, 성별, 체중, 연령 등에 따라 증감될 수 있다.The A-54556A or a pharmaceutically acceptable salt thereof is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level depends on the type, severity, activity of the drug, and the drug. Sensitivity to, administration time, administration route and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. Specifically, the pharmaceutical composition may be administered at 0.01 to 1000 mg/kg/day, more specifically, at 0.1 to 500 mg/kg/day. The administration may be administered once a day or may be administered in several divided doses. Specifically, the effective amount of the pharmaceutical composition may vary depending on the patient's age, sex, condition, body weight, absorption of the active ingredient into the body, inactivation rate, excretion rate, disease type, drug used in combination, administration route, obesity It can be increased or decreased according to the severity of the disease, sex, weight, age, etc.
ClpP 활성제인 A-54556A는 지방간염 동물모델에 투여시 지방간 감소, 염증반응 억제, 간 독성 억제, 및 간조직 섬유화 억제 효과가 확인되었으므로 지방간염 또는 비알콜성 지방간염의 치료 목적으로 사용될 수 있다.ClpP activator A-54556A has been confirmed to reduce fatty liver, inhibit inflammatory response, inhibit liver toxicity, and inhibit liver tissue fibrosis when administered to an animal model of steatohepatitis, so it can be used for the treatment of steatohepatitis or nonalcoholic steatohepatitis.
도 1은 본 발명의 실시예에 사용된 고열량 식이 마우스의 준비과정을 나타낸 것이다.
도 2는 고열량 식이에 의해 감소한 ClpP 활성이 A-54556A 투여에 의해 회복됨을 확인한 결과이다.
도 3은 고열량 식이에 의해 감소한 ClpP 발현이 A-54556A 투여에 의해 회복됨을 확인한 결과이다.
도 4는 고열량 식이에 의한 지방간 증가가 A-54556A에 의해 억제됨을 확인한 결과이다.
도 5는 고열량 식이에 의한 간의 염증성 사이토카인 발현 증가가 A-54556A에 의해 억제됨을 확인한 결과이다.
도 6은 고열량 식이에 의한 간조직의 JNK와 p65의 인산화 증가가 A-54556A에 의해 억제됨을 확인한 결과이다.
도 7은 고열량 식이에 의한 혈중 AST, ALT 수준 및 NAS 스코어 증가가 A-54556A에 의해 억제됨을 확인한 결과이다.
도 8은 고열량 식이에 의해 증가한 Sirius red 염색 및 고열량 식이에 의해 증가한 콜라겐 및 TIMP 발현 증가가 A-54556A에 의해 감소함을 확인한 결과이다.1 shows the preparation process of the high-calorie diet mouse used in the Examples of the present invention.
2 is a result confirming that ClpP activity decreased by a high-calorie diet is restored by A-54556A administration.
3 is a result confirming that ClpP expression decreased by a high-calorie diet is restored by A-54556A administration.
4 is a result confirming that the increase in fatty liver caused by a high-calorie diet is inhibited by A-54556A.
5 is a result confirming that the increase in the expression of inflammatory cytokines in the liver by a high-calorie diet is suppressed by A-54556A.
6 is a result confirming that the increase in phosphorylation of JNK and p65 in liver tissue by a high-calorie diet is inhibited by A-54556A.
7 is a result confirming that the increase in blood AST, ALT level and NAS score by a high-calorie diet is inhibited by A-54556A.
8 is a result confirming that Sirius red staining increased by a high-calorie diet and collagen and TIMP expression increased by a high-calorie diet are decreased by A-54556A.
이하 하나 이상의 구체예를 실시예를 통해 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. Hereinafter, one or more specific embodiments will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.
실시예1: 고열량 식이 마우스 준비Example 1: Preparation of high-calorie diet mice
고지방식/고과당 식이 마우스의 준비과정을 도 1에 기초하여 설명한다. 6주령의 수컷 C57BL/6J mice를 일본의 SLC에서 입수하였다. 이 동물을 아주대학교 의과대학 동물 실험실에서 케이지마다 3-4 마리씩 분리하여 온도 22±0.5℃, 습도 55±5%에서 12시간 밤낮의 주기로 사육하였다. The preparation process of the high-fat/high-fructose-fed mice will be described based on FIG. 1 . Six-week-old male C57BL/6J mice were obtained from SLC in Japan. In the animal laboratory of Ajou University College of Medicine, 3-4 animals per cage were separated and bred at a temperature of 22±0.5° C. and a humidity of 55±5% for 12 hours day and night cycle.
8주령의 C57BL/6J mice를 3개의 군으로 나누었다. 1개의 군은 정상대조군으로써 6주간 정상식이를 공급하고 6주는 10% fat을 함유하는 정상지방식이(Research diet, D12450)를 공급하였다.(해당 실험군은 정상식이를 공급한 실험군으로 볼 수 있다) 다른 2개의 군은 고지방(HF) 및 고과당(HFr) 식이를 6주간 공급하였다. 고지방 및 고과당 식이는 60% fat 식이(Research diet, D12492) 및 30% 과당을 포함하였다. 상기 고지방 및 고과당 식이를 6주간 공급한 2개의 군에 대해서, 하나의 군은 고지방 및 고과당 식이를 6주 더 공급하면서 DMSO를 복강투여하고, 다른 군은 고지방 및 고과당 식이를 6주 더 공급하면서 A-54556A 20 mg/kg을 DMSO에 녹여서 복강 투여하였다. (도 1 참고) 이후 마우스를 희생하고 간조직을 분리하여 실험에 사용하였다.8-week-old C57BL/6J mice were divided into 3 groups. As a normal control group, one group was fed a normal diet for 6 weeks and a normal fat diet (Research diet, D12450) containing 10% fat for 6 weeks. The other two groups were fed a high fat (HF) and high fructose (HFr) diet for 6 weeks. The high fat and high fructose diet included a 60% fat diet (Research diet, D12492) and 30% fructose. For the two groups supplied with the high-fat and high-fructose diet for 6 weeks, one group was intraperitoneally administered with DMSO while supplying the high-fat and high-fructose diet for 6 weeks, and the other group received a high-fat and high-fructose diet for 6 more weeks. While supplying, 20 mg/kg of A-54556A was dissolved in DMSO and administered intraperitoneally. (See FIG. 1) Afterwards, the mouse was sacrificed and liver tissue was isolated and used for the experiment.
실시예 2: 고지방/고과당 식이 마우스의 ClpP 단백질 발현량 확인Example 2: Confirmation of ClpP protein expression level in high fat / high fructose diet mice
2-1. 간조직에서 ClpP 활성 측정2-1. Measurement of ClpP activity in liver tissue
마우스를 희생하여 간조직을 분리한 후 간조직 내 ClpP 활성을 측정하였다. 간조직 100 mg으로부터 미토콘드리아 분리 kit를(Thermo Fisher Scientific, Mitochondria Isolation Kit for Tissue, 89801) 이용하여 미토콘드리아를 분리하고 Protease Fluorescent Detection Kit (Sigma-Aldrich)을 이용하여 ClpP 활성을 조사하였다. 각각의 군에서 분리한 미토콘드리아 단백질과 FITC-casein을 넣고 0.5시간 RT 배양한 후 절단된 fluorescein isothiocyanate (FITC)의 흡광도를 ELISA leader로 SpectraMax®iD3 Multi-Mode Microplate Reader (Molecular Devices) 측정하였다 (excitation 485 /emission wavelengths 535). After sacrificing a mouse to isolate liver tissue, ClpP activity in the liver tissue was measured. Mitochondria were isolated from 100 mg of liver tissue using a mitochondria isolation kit (Thermo Fisher Scientific, Mitochondria Isolation Kit for Tissue, 89801), and ClpP activity was investigated using Protease Fluorescent Detection Kit (Sigma-Aldrich). After adding the mitochondrial protein and FITC-casein isolated from each group, and incubating for 0.5 hours at RT, the absorbance of the cut fluorescein isothiocyanate (FITC) was measured as an ELISA leader with a SpectraMax®iD3 Multi-Mode Microplate Reader (Molecular Devices) (excitation 485) /emission wavelengths 535).
2-2. 간조직에서 ClpP 단백질 발현량 측정2-2. Measurement of ClpP protein expression in liver tissue
간조직 및 분리된 미토콘드리아 100 mg에 RIPA buffer (1% triton X-100m 1% sodium deoxcolate, 50 mM NaCl2, 50 mM tris-HCl, 1 mM sodium vanadate, 2 mM PMSF, protease inhibitor cocktail (Roche Applied Science))를 이용하여 단백질을 분리하였다. 정량 후 sample buffer (50 mmol/l Tris-HCl at pH 6.8, 2% SDS, 100 mmol/l DL-dithiothreitol, 10% glycerol)에 단백질을 희석하여 5분간 가열하고, 8 내지 12%의 SDS-polyacrylamid gel에 로딩하여 전기영동하고, PVDF membrane으로 이동시킨 후 5% skim milk 로 blocking 하였다. 하기 표 1의 1차 항체를 반응시킨 후 조건에 맞는 2차 항체를 반응시키고 enhanced chemiluminescence system(pierce, rockford, IL, USA)을 사용해 밴드를 확인하였다. 밴드는 One-Dimensional Quantity One® 1D Image Analysis 프로그램을 이용하여 정량화하였다 (Bio-Rad, Hercules, CA).RIPA buffer (1
2-3. 실험결과 2-3. Experiment result
도 2 및 도 3에 따르면, 고지방/고과당 식이를 공급한 마우스의 간조직은 ClpP 단백질 발현 및 활성이 모두 감소하였다. 그리고 ClpP에 의해 분해되는 것으로 알려진 Gfm1과 Atp5a의 발현량이 증가하였다. According to FIGS. 2 and 3 , both ClpP protein expression and activity were decreased in liver tissue of mice fed a high fat/high fructose diet. And the expression levels of Gfm1 and Atp5a, which are known to be degraded by ClpP, increased.
그러나 A-54556A를 투여한 고지방/고과당식 공급 마우스의 간조직은 ClpP 발현 및 활성이 증가하였으며, Gfm1과 Atp5a의 발현량도 정상 수준으로 감소하였다. However, ClpP expression and activity were increased in liver tissues of high fat/high fructose fed mice administered with A-54556A, and the expression levels of Gfm1 and Atp5a were also decreased to normal levels.
실시예 3: A-54556A의 간 지방 감소효과 확인 Example 3: Confirmation of liver fat reduction effect of A-54556A
3-1. 간의 지방(liver TG) 측정3-1. Measurement of liver TG
간 조직 내 지방 축적량을 측정하기 위해 마우스로부터 분리한 간 조직 100 mg을 1 ml의 5% NP-40 DW에 homogenize 하였다. 샘플을 water bath에서 2-3 분 동안 80-100℃까지 천천히 가열하여 Triglyceride(TG)를 추출하였다. Buffer가 추출된 TG에 의해 혼탁해지면 상온에서 자연냉각시키고 다시 추출하는 과정을 2 내지 3회 반복하였다. 샘플을 원심분리하여 불용성 조직을 침전시키고 TG가 포함된 상등액을 얻었다. 상등액을 Triglyceride Quantification kit (K-622-100/ Biovision)를 이용하여 간조직의 TG 농도를 측정하였다. To measure the amount of fat accumulation in the liver tissue, 100 mg of liver tissue isolated from the mouse was homogenized in 1 ml of 5% NP-40 DW. Triglyceride (TG) was extracted by slowly heating the sample to 80-100°C in a water bath for 2-3 minutes. When the buffer becomes turbid by the extracted TG, the process of naturally cooling at room temperature and extracting again is repeated 2-3 times. The sample was centrifuged to precipitate insoluble tissue and a supernatant containing TG was obtained. The supernatant was measured for TG concentration in liver tissue using a Triglyceride Quantification kit (K-622-100/Biovision).
3-2. Hematoxylin-Eosin (H&E) 염색3-2. Hematoxylin-Eosin (H&E) staining
마우스로부터 얻은 간조직을 4℃에서 4% paraformaldehyde로 24 시간 고정한 후 흐르는 물에 수세한 다음 파라핀으로 포매하여(Paraffin embedding) 조직을 10 μm 두께로 박절(microtome cutting)하였다. 박절한 조직을 코팅 슬라이드 위에 붙인 후 xylen으로 파라핀을 제거하고 100% 에탄올, 95% 에탄올, 90% 에탄올, 80% 에탄올, 70% 에탄올 순서로 함수 과정을 거친 후 면역 조직학적 염색을 시행하였다. 박절한 조직을 Harry's hematoxylin 염색액으로 5 분간 염색 후 흐르는 물에 5 분간 수세하고 1% HCl alcohol과 ammonia로 탈염색하였다. 이후 흐르는 물에 10 분간 수세한 후 다시 eosin 염색액으로 1 분간 대조염색하고 상기 함수 과정의 역방향으로 탈수 과정을 거친 후 봉입하여 현미경(Olympus, Tokyo, Japan)으로 관찰하였다. The liver tissue obtained from the mouse was fixed with 4% paraformaldehyde at 4°C for 24 hours, washed with running water, and then embedded with paraffin (Paraffin embedding), and the tissue was microtome cut to a thickness of 10 μm. After attaching the sliced tissue to the coated slide, paraffin was removed with xylen, and after hydrolysis in the order of 100% ethanol, 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol, immunohistological staining was performed. The sliced tissue was stained with Harry's hematoxylin staining solution for 5 minutes, washed with running water for 5 minutes, and destained with 1% HCl alcohol and ammonia. After washing with running water for 10 minutes, counterstaining with eosin dye solution for 1 minute was performed, dehydration was performed in the reverse direction of the water-containing process, and then sealed and observed under a microscope (Olympus, Tokyo, Japan).
3-3. Oil-red O 염색3-3. Oil-red O dyeing
분리한 조직은 수세한 후 물기를 제거하고 동결조직 포매제인 Optical cutting temperature (O.C.T) compound (SAKURA, U.S.A)에 고정시켜 동결조직 블럭을 만들었으며 -70℃ 냉동고에 보관하였다. 간조직은 10 ㎛ 두께로 microtome으로 잘라 간조직 절편을 슬라이드에 신속히 붙인 후 실온에서 1 시간 가량 건조 후 -70℃ deep freezer에서 슬라이드를 보관하였다. 3 g/L의 Oil-red-O (Sigma, U.S.A) powder를 99％ Isopropanol 용액에 넣고 실온에서 진탕하여 포화용액을 만든 후, 60℃ oven에 보관하며 염색 직전에 포화용액과 증류수를 6:4의 비율로 섞은 용액을 Whatman Filter Paper #2 (Whatman, England)로 여과하여 사용하였다. -70℃ deep freezer에 보관하고 있던 슬라이드는 실험 직전 꺼내어 실온에서 1 내지 2 시간 가량 건조 후, 슬라이드를 물에 수세하고 70％ isopropanol에 10 분간 수세한다. 여과한 Oil-red-O에 슬라이드를 넣고 15 분간 염색 후 70％ isopropanol에 넣고 3 분간 탈색한다. 다시 슬라이드를 흐르는 물에 5분간 수세하고 hematoxylin solution (Sigma,U.S.A)에 5 분간 염색하고 다시 흐르는 물에 5 분간 수세하였다. 염색 후 Faramount Aqueous Mounting Medium (Dako. cat. S3025, Japan)로 봉합하고 현미경으로 관찰하였다.The separated tissue was washed with water, dried, fixed in Optical cutting temperature (O.C.T) compound (SAKURA, U.S.A), a frozen tissue embedding agent, to make a frozen tissue block, and stored in a -70°C freezer. The liver tissue was cut with a microtome to a thickness of 10 μm, and the liver tissue section was quickly attached to the slide, dried at room temperature for about 1 hour, and then stored in a -70°C deep freezer. Add 3 g/L of Oil-red-O (Sigma, U.S.A) powder to 99% Isopropanol solution and shake at room temperature to make a saturated solution, store in a 60℃ oven, and mix saturated solution and distilled water at 6:4 The mixed solution was used by filtration with Whatman Filter Paper #2 (Whatman, England). Slides stored in -70°C deep freezer are taken out immediately before the experiment, dried at room temperature for 1 to 2 hours, then washed with water and then washed with 70% isopropanol for 10 minutes. Put the slide in the filtered Oil-red-O, dye for 15 minutes, put it in 70% isopropanol, and decolorize for 3 minutes. The slides were again washed with running water for 5 minutes, stained with hematoxylin solution (Sigma, U.S.A) for 5 minutes, and washed again with running water for 5 minutes. After staining, it was sutured with Faramount Aqueous Mounting Medium (Dako. cat. S3025, Japan) and observed under a microscope.
3-4. 실험결과3-4. Experiment result
도 4A에 따르면, 고지방/고과당 식이 마우스의 지방간은 정상 대조군보다 약 6배 증가하였다. 그러나 A-54556A를 투여한 마우스는 고지방/고과당 식이에도 불구하고 지방간 형성이 억제되었다. According to FIG. 4A, the fatty liver of the high-fat/high-fructose-fed mice increased about 6-fold compared to the normal control group. However, in mice administered with A-54556A, fatty liver formation was suppressed despite the high-fat/high-fructose diet.
A-54556A의 지방간 감소 효과를 더 자세히 확인하기 위하여 간 조직을 Hematoxylin-Eosin (H&E)와 Oil Red O로 염색하고 실험군과 대조군을 비교하였다. 도 4B에 따르면, A-54556A를 투여한 마우스의 간 조직은 고과당/고지방 식이만 공급한 실험군에 비해서 지방간이 감소하였다.To further confirm the fatty liver reduction effect of A-54556A, liver tissue was stained with Hematoxylin-Eosin (H&E) and Oil Red O, and the experimental group and the control group were compared. According to FIG. 4B , the liver tissue of mice administered with A-54556A had reduced fatty liver compared to the experimental group fed only high-fructose/high-fat diet.
실시예 4: A-54556A의 지방간염 억제 효과Example 4: The inhibitory effect of A-54556A on steatohepatitis
4-1. 대식세포 조직염색4-1. macrophage tissue staining
간조직의 형태 계측을 위해 마우스로부터 얻은 간조직을 4℃에서 4% paraformaldehyde로 24 시간 고정 후 흐르는 물에 수세한 다음 파라핀으로 포매하여 조직을 5 ㎛ 두께로 박절하였다. 박절한 조직을 코팅 슬라이드 위에 붙인 후 xylen으로 파라핀을 제거하고 100% 에탄올, 95% 에탄올, 90% 에탄올, 80% 에탄올, 70% 에탄올 순서로 함수 과정을 거친 후 F4/80 항체로 면역 조직학적 염색을 시행 후 대조염색하였다. 상기 함수 과정의 역방향으로 탈수 과정을 거친 후 봉입하여 현미경 (Olympus, Tokyo, Japan)으로 관찰하였다. For morphometry of the liver tissue, the liver tissue obtained from the mouse was fixed with 4% paraformaldehyde at 4°C for 24 hours, washed with running water, and then embedded with paraffin, and the tissue was sliced to a thickness of 5 μm. After attaching the sliced tissue onto the coated slide, removing paraffin with xylen, performing a hydrolysis process in the order of 100% ethanol, 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol, and then immunohistochemical staining with F4/80 antibody. After performing the counterstaining. After the dehydration process in the reverse direction of the hydrous process, it was sealed and observed with a microscope (Olympus, Tokyo, Japan).
4-2. qRT-PCR4-2. qRT-PCR
간조직의 형태 계측을 위해 마우스를 희생하여 간을 적출한 후, RNA TRI Zol을 이용하여 RNA를 추출하여 정량하였다. 2 ㎍의 RNA를 1000U AMV 역전사 효소 0.5 ㎕, 2.5 mM dNTP 4 ㎕, Random 9 mer 1 ㎕, RNase inhibitor 0.5 ㎕, MgCl2 4 ㎕, 10X RT buffer 2 ㎕와 혼합하여 30℃에서 10 분, 42℃에서 30 분, 99℃에서 5 분 동안 역전사 반응을 시켜 cDNA를 합성하였다. 이렇게 얻어진 cDNA로 하기 표 2의 primer들과 다카라 qPCR kit (cybergreen)를 이용하여 qPCR을 수행하였다.For morphometry of liver tissues, mice were sacrificed and livers were removed, and RNA was extracted and quantified using RNA TRI Zol. 2 μg of RNA was mixed with 1000U AMV reverse transcriptase 0.5 μl, 2.5
4-3. 염증 기전 조사 (p-JNK, p-p65)4-3. Investigation of inflammatory mechanisms (p-JNK, p-p65)
간조직 100 mg을 RIPA buffer (1% triton X-100m 1% sodium deoxcolate, 50mM NaCl2, 50mM tris-HCl, 1mM sodium vanadate, 2mM PMSF, protease inhibitor cocktail(Roche Applied Science))에 넣고 파쇄 후 단백질을 분리하였다. 정량 후 sample buffer(50 mmol/l Tris-HCl at pH 6.8, 2% SDS, 100 mmol/l DL-dithiothreitol, 10% glycerol)에 단백질을 희석하여 5분간 열탕처리한 후 8 내지 12%의 SDS-polyacrylamid gel에서 전기영동하여 단백질을 PVDF membrane으로 이동시킨 후 5% skim milk 로 blocking 하였다. 각각의 1차 항체를 반응시킨 후 조건에 맞는 2차 항체를 반응시켜 enhanced chemiluminescence system(pierce, rockford, IL, USA)을 사용해 밴드를 확인하였다. 밴드는 One-Dimensional Quantity One® 1D Image Analysis 프로그램을 이용하여 정량화하였다(Bio-Rad, Hercules, CA). 실험에 사용된 항체는 하기 표 3에 기재되어 있다.100 mg of liver tissue was placed in RIPA buffer (1
NASH는 지방간에서 지방간염으로의 진행이 되며, 단순 지방간과는 다르게 염증세포들이 간 조직으로 침투하여 염증을 유발하고 간세포의 기능을 교란시킨다.NASH progresses from fatty liver to steatohepatitis, and unlike simple fatty liver, inflammatory cells penetrate into the liver tissue, causing inflammation and disrupting the function of hepatocytes.
고지방 및 고과당 식이를 공급한 C57BL/6J mice와, 고지방 및 고과당 식이와 함께 A-54556A를 투여한 실험군의 염증성 사이토카인 및 면역세포 침윤 상태를 조사한 결과 그 차이가 명확하였다. 도 5에 따르면, 고지방 및 고과당 식이를 한 마우스의 간 조직은 IL-1beta, MCP-1등의 염증성 사이토카인의 mRNA가 유의적으로 증가하였다. 그러나 A-54556A를 투여한 군의 간조직은 염증성 사이토카인(IL-1beta, MCP-1)의 mRNA가 유의하게 감소하였다. As a result of investigating the inflammatory cytokine and immune cell infiltration status of C57BL/6J mice fed a high-fat and high-fructose diet and the experimental group administered with A-54556A along with a high-fat and high-fructose diet, the difference was clear. According to FIG. 5 , mRNA of inflammatory cytokines such as IL-1beta and MCP-1 was significantly increased in liver tissue of mice fed a high fat and high fructose diet. However, the mRNA of inflammatory cytokines (IL-1beta, MCP-1) was significantly decreased in the liver tissue of the group administered with A-54556A.
또한 대표적으로 염증성 사이토카인을 분비하는 것으로 알려져 있는 마크로파지 침윤여부를 알아보기 위하여 F4/80으로 간조직을 염색해본 결과 고지방 및 고과당 식이를 공급한 마우스의 간에서는 대식세포의 침윤이 유의적으로 증가하였으나, A-54556A 처리한 마우스는 대식세포의 침윤이 유의적으로 감소하였다. 이 결과에 따르면, A-54556A 투여군은 대조군에 비해서 지방간 염증개선 효능이 있음을 알 수 있었다. In addition, as a result of staining liver tissue with F4/80 to examine whether macrophages, which are known to secrete inflammatory cytokines, were infiltrated, macrophage infiltration was significantly increased in the liver of mice fed a high-fat and high-fructose diet. However, A-54556A-treated mice showed a significant decrease in macrophage infiltration. According to these results, it was found that the A-54556A administration group had an effect on improving inflammation of fatty liver compared to the control group.
염증 억제 기전을 조사하기 위해서 대표적인 스트레스 신호로 알려진 JNK와 p65의 인산화를 조사하였다. 도 6에 따르면, 고지방 및 고과당 식이를 공급한 마우스의 간조직은 JNK와 p65 의 인산화가 증가하였다. 그러나 A-54556A를 투여한 실험군은 JNK 및 p65 의 인산화가 유의적으로 감소하였다. To investigate the mechanism of inhibition of inflammation, phosphorylation of JNK and p65, known as representative stress signals, was investigated. According to FIG. 6 , phosphorylation of JNK and p65 was increased in liver tissues of mice fed a high fat and high fructose diet. However, in the experimental group administered with A-54556A, phosphorylation of JNK and p65 was significantly reduced.
실시예 5: A-54556A의 간세포 손상 억제 효과 Example 5: Inhibitory effect of A-54556A on hepatocellular injury
마우스를 희생하고 혈중 AST, ALT를 측정하였다. 도 7A에 따르면, 고지방 및 고과당 식이를 공급한 마우스의 혈중 AST, ALT의 수준은 대조군에 비해 현저히 증가하였다. 이는 고열량 식이를 공급받은 마우스는 정상 식이를 공급받은 마우스보다 간세포의 손상이 증가함을 알 수 있는 결과이다. 그러나 고열량 식이와 함께 A-54556A를 투여한 실험군의 AST, ALT 수준은 고과당 및 고지방 식이만을 공급받은 동물군에 비해 현저히 감소되었다. Mice were sacrificed and blood AST and ALT were measured. According to FIG. 7A , the levels of AST and ALT in the blood of mice fed a high-fat and high-fructose diet were significantly increased compared to the control group. This is a result that can be seen that the mice fed the high-calorie diet increased liver cell damage than the mice fed the normal diet. However, the AST and ALT levels of the experimental group administered with A-54556A along with the high-calorie diet were significantly reduced compared to the animal group fed only the high-fructose and high-fat diet.
A-54556A의 간세포 보호 효과를 좀 더 자세히 확인하기 위하여 각각의 조직을 Hematoxylin-Eosin (H&E) 염색하고 지방증, Ballooning, 소엽염증 (steatosis, lobular inflammation, ballooning)등 NAS score를 비교하였다. 도 7B에 따르면, A-54556A를 투여한 마우스의 간 조직은 고과당 및 고지방식만을 투여한 실험군에 비해서 NAS score(NAFLD activity score, 비알콜성 지방간질환 스코어)가 감소함을 확인하였다.To confirm the hepatoprotective effect of A-54556A in more detail, each tissue was stained with Hematoxylin-Eosin (H&E) and NAS scores such as steatosis, ballooning, and lobular inflammation (steatosis, lobular inflammation, ballooning) were compared. According to FIG. 7B, it was confirmed that the NAS score (NAFLD activity score, non-alcoholic fatty liver disease score) was decreased in the liver tissue of the mice administered with A-54556A compared to the experimental group administered only with high fructose and high-fat diet.
실시예 6: A54556A의 간섬유화 억제 효과Example 6: The inhibitory effect of A54556A on hepatic fibrosis
Sirius Red 염색을 통해 고과당 및 고지방 식이를 12주 동안 공급한 C57BL/6J mice에서 간 섬유화의 진행되는지 여부를 조사하였다.We investigated whether liver fibrosis progressed in C57BL/6J mice fed a high-fructose and high-fat diet for 12 weeks through Sirius Red staining.
NASH는 단순 지방간과는 다르게 염증세포가 간조직으로 침투하여 염증을 유도하고 간세포의 부피가 정상크기에 비해 커지며, 병증이 더욱 진행되면 간조직의 섬유화를 유발하게 된다. 간조직의 섬유화에 기여하는 가장 중요한 세포는 간세포의 15%를 차지하는 간성상세포(hepetic stellate cells, HSCs)이다. 손상된 간세포나 면역세포는 PDGF 나 TGF-beta1를 분비하여 간성상세포를 활성화시키고, 활성화된 간성상세포는 근섬유모세포와 유사한(myofibroblast-like) 세포로 변질되어 type I 콜라겐 및 type III 콜라겐과 같은 세포외기질 요소들(Extracellular matrix component)을 분비하고, 이들이 축적되어 간섬유화가 진행된다. In NASH, unlike simple fatty liver, inflammatory cells penetrate into the liver tissue to induce inflammation, and the volume of hepatocytes becomes larger than the normal size. The most important cells contributing to hepatic fibrosis are hepetic stellate cells (HSCs), which account for 15% of hepatocytes. Damaged hepatocytes or immune cells secrete PDGF or TGF-beta1 to activate hepatocytes, and the activated hepatocytes are transformed into myofibroblast-like cells, such as type I collagen and type III collagen. Extracellular matrix components are secreted, and they accumulate and liver fibrosis proceeds.
5-1. qRT-PCR5-1. qRT-PCR
실험군들의 마우스를 희생하여 간을 적출한 후, RNA TRI Zol을 이용하여 RNA를 추출하고 정량하였다. 2 ㎍의 RNA를 1000U AMV 역전사 효소 0.5 ㎕, 2.5 mM dNTP 4 ㎕, Random 9 mer 1 ㎕, RNase inhibitor 0.5 ㎕, MgCl2 4 ㎕, 10X RT buffer 2 ㎕와 혼합하여 30℃ 10 분, 42℃ 30 분, 99℃ 5 분 동안 역전사 반응을 시켜 cDNA를 합성하였다. 이렇게 얻어진 cDNA로 하기 표 4의 primer 및 다카라 qPCR kit(cybergreen)를 이용하여 qPCR을 수행하였다.After sacrificing the mice of the experimental group and removing the liver, RNA was extracted and quantified using RNA TRI Zol. 2 μg of RNA was mixed with 1000U AMV reverse transcriptase 0.5 μl, 2.5
5-2. Sirius Red 염색5-2. Sirius Red dye
실험군들의 마우스를 희생하여 간을 적출하고 4℃에서 4% paraformaldehyde로 24시간 고정 후 흐르는 물에 수세한 다음 파라핀으로 포매하여 조직을 5 μm 두께로 박절하였다. 박절한 조직을 코팅 슬라이드 위에 붙인 후 xylen으로 파라핀을 제거하고 100% 에탄올, 95% 에탄올, 90% 에탄올, 80% 에탄올, 및 70% 에탄올의 순서로 함수 과정을 거쳤다. picro-sirius red로 1 시간 동안 조직학적 염색 및 대조염색을 실시하였다. 상기 함수 과정의 역방향으로 탈수 과정을 거친 후 봉입하고 현미경(Olympus, Japan)으로 관찰하였다.The mice of the experimental group were sacrificed and livers were removed, fixed with 4% paraformaldehyde at 4°C for 24 hours, washed with running water, and then embedded with paraffin, and the tissue was sliced to a thickness of 5 μm. After attaching the sliced tissue onto the coated slide, paraffin was removed with xylen, and the hydrolysis process was performed in the order of 100% ethanol, 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol. Histological staining and counterstaining were performed with picro-sirius red for 1 hour. After the dehydration process in the reverse direction of the hydrous process, it was sealed and observed with a microscope (Olympus, Japan).
도 8에 따르면, 고과당 및 고지방 식이를 공급한 마우스의 간 조직에서 Sirius Red 염색이 유의적으로 증가하였다. 그리고 고과당 및 고지방 식이를 공급한 마우스의 간 조직은 섬유화에 관련된 type I 콜라겐(col1a2) 및 type III 콜라겐(col3a1)의 발현 정도와 세포질의 분해를 촉진하는 MMPs(Matrix metalloproteinases)를 억제하는 TIMP(Tissue inhibitor of metalloproteinases)의 발현이 증가하였다. 이 결과는 고과당 및 고지방 식이를 공급한 마우스는 간손상 후 반흔과정(scarring process)에 의한 간섬유화(fibrosis) 현상이 증가함을 시사한다. 그러나 고지방 및 고과당 식이와 함께 A-54556A를 투여한 마우스의 간조직은 Timp, ctgf, col1a2, col3a1의 발현이 현저히 감소하였고 Sirius Red 염색이 감소하였다. 따라서 A-54556A는 간조직의 섬유화를 유의적으로 감소시킴을 확인하였다.According to FIG. 8, Sirius Red staining was significantly increased in liver tissue of mice fed a high-fructose and high-fat diet. In addition, the liver tissue of mice fed a high-fructose and high-fat diet showed the expression level of type I collagen (col1a2) and type III collagen (col3a1) involved in fibrosis and TIMP (Matrix metalloproteinases), which inhibits MMPs (Matrix metalloproteinases) that promote cytoplasmic degradation. Tissue inhibitor of metalloproteinases) was increased. These results suggest that mice fed a high-fructose and high-fat diet increased liver fibrosis by the scarring process after liver injury. However, the expression of Timp, ctgf, col1a2, and col3a1 in the liver tissue of mice administered A-54556A along with a high fat and high fructose diet was significantly reduced, and Sirius Red staining was decreased. Therefore, it was confirmed that A-54556A significantly reduced liver tissue fibrosis.
<110> AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Composition for treating steatohepatitis comprising A-54556A as an active ingredient <130> PN210066 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rpl32 F primer <400> 1 aagcgaaact ggcggaaacc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rpl32 R primer <400> 2 cccataaccg atgttgggca 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> f4/80 F primer <400> 3 cccgtgttgt tggtggcact 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> f4/80 R primer <400> 4 gctttggctg gatgtgctgg 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> il-1beta F primer <400> 5 tctcgcagca gcacatcaac a 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> il-1beta R primer <400> 6 cctggaaggt ccacgggaaa 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mcp-1 F primer <400> 7 cagccagatg cagttaacgc 20 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> mcp-1 R primer <400> 8 gcctactcat tgggatcatc ttg 23 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> timp F primer <400> 9 acgccaggga accaagaagc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> timp R primer <400> 10 ccacaagtcc cagaaccgca 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ctgf F primer <400> 11 ctttctggct gcaccagtgt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ctgf R primer <400> 12 ggcagagtgg tggttctgtg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> col1a2 F primer <400> 13 ctgggtgaac ctggtcctct 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> col1a2 R primer <400> 14 accaggggca ccattaactc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> col3a1 F primer <400> 15 gtccaaaggg tgaagtcggt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> col3a1 R primer <400> 16 cagctccacc tctagcacca 20 <110> AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Composition for treating steatohepatitis comprising A-54556A as an active ingredient <130> PN210066 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rpl32 F primer <400> 1 aagcgaaact ggcggaaacc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rpl32 R primer <400> 2 cccataaccg atgttgggca 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> f4/80 F primer <400> 3 cccgtgttgt tggtggcact 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> f4/80 R primer <400> 4 gctttggctg gatgtgctgg 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> il-1beta F primer <400> 5 tctcgcagca gcacatcaac a 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> il-1beta R primer <400> 6 cctggaaggt ccacgggaaa 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mcp-1 F primer <400> 7 cagccagatg cagttaacgc 20 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> mcp-1 R primer <400> 8 gcctactcat tgggatcatc ttg 23 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> timp F primer <400> 9 acgccaggga accaagaagc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> timp R primer <400> 10 ccacaagtcc cagaaccgca 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ctgf F primer <400> 11 ctttctggct gcaccagtgt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ctgf R primer <400> 12 ggcagagtgg tggttctgtg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> col1a2 F primer <400> 13 ctgggtgaac ctggtcctct 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> col1a2 R primer <400> 14 accaggggca ccattaactc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> col3a1 F primer <400> 15 gtccaaaggg tgaagtcggt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> col3a1 R primer <400> 16 cagctccacc tctagcacca 20
Claims (4)
A pharmaceutical composition for treating or preventing steatohepatitis comprising A-54556A or a pharmaceutically acceptable salt thereof as an active ingredient.
상기 A-54556A는 하기 화학식 1로 표시되는 화합물인 조성물.
[화학식 1]
The method of claim 1,
The composition of A-54556A is a compound represented by the following formula (1).
[Formula 1]
상기 지방간염은 비알콜성 지방간(NASH)인 조성물.
The method of claim 1,
The composition wherein the steatohepatitis is non-alcoholic fatty liver (NASH).
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| US20160046674A1 (en) | 2011-03-30 | 2016-02-18 | Brown University | Enopeptins, uses thereof, and methods of snythesis thereof |
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| US20160046674A1 (en) | 2011-03-30 | 2016-02-18 | Brown University | Enopeptins, uses thereof, and methods of snythesis thereof |
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