KR20210048208A - Composition for preventing or treating TNF-mediated diseases comprising tetrahydropapaverine and method for inhibiting TNF-activity with the same - Google Patents
Composition for preventing or treating TNF-mediated diseases comprising tetrahydropapaverine and method for inhibiting TNF-activity with the same Download PDFInfo
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Abstract
Description
본 발명은 테트라하이드로파파베린을 유효성분으로 포함하는 TNF-관련 질환 예방 또는 치료용 조성물 및 이를 이용한 TNF 활성 억제 방법에 관한 것이다.The present invention relates to a composition for preventing or treating TNF-related diseases comprising tetrahydropaberine as an active ingredient, and a method for inhibiting TNF activity using the same.
류마티스 관절염(rheumatoid arthritis; RA)은 주로 관절 조직과 뼈에 영향을 미치는 만성 염증성 질환이다. 류마티스 관절염 환자의 관절에서 활막세포의 활성화는 IL-6, TNF 및 IL-1β를 포함하는 염증성 사이토카인의 생성을 증가시켜 연골과 뼈의 파괴를 유발한다. 상기 사이토카인 중 TNF는 주요한 전염증성 사이토카인으로 활막세포에서 IL-6, IL-8, GM-CSF 및 심지어 그 자체의 생성을 강력하게 유도한다.Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects joint tissues and bones. Activation of synovial cells in the joints of patients with rheumatoid arthritis increases the production of inflammatory cytokines including IL-6, TNF, and IL-1β, causing cartilage and bone destruction. Among these cytokines, TNF is a major pro-inflammatory cytokine and strongly induces the production of IL-6, IL-8, GM-CSF and even itself in synovial cells.
현재, IL-17을 생성하는 Th17이 류마티스 관절염의 발병에 관여한다는 연구 결과가 보고되고 있다. 관절에서 IL-17의 억제 또는 과발현은 각각 관절염을 억제시키거나 악화시킬 수 있다. 건강한 대조군 및 류마티스 관절염 환자로부터 활성화된 단핵구는 시험관 내에서 IL-1β/TNF-α에 의존적으로 Th17 반응을 유도하고 류마티스 관절염의 염증 부위로부터 활성화된 단핵구는 세포 접촉에 의존적으로 증가된 Th17 반응을 유도하는 것으로 보고되었다. 마우스 콜라겐 유도 관절염 모델의 초기 단계에서 IL-17은 TNF-α에 의존하는 반면, 후기 단계에서 IL-17은 주도적이며, TNF-α 독립적으로 작용한다. 비록 류마티스 관절염의 기전이 명확하게 밝혀지지는 않았으나, Th17 반응과 IL-17 생산의 상향 조절이 중요한 역할을 하는 것으로 보고되고 있다.Currently, research results have been reported that Th17 producing IL-17 is involved in the development of rheumatoid arthritis. Inhibition or overexpression of IL-17 in the joint can inhibit or worsen arthritis, respectively. Activated monocytes from healthy controls and rheumatoid arthritis patients induce a Th17 response dependent on IL-1β/TNF-α in vitro, and monocytes activated from the inflammatory site of rheumatoid arthritis induce an increased Th17 response dependent on cell contact. It has been reported to be. In the early stages of the mouse collagen-induced arthritis model, IL-17 is dependent on TNF-α, whereas in the later stages, IL-17 is dominant and acts independently of TNF-α. Although the mechanism of rheumatoid arthritis has not been clearly elucidated, it has been reported that Th17 response and upregulation of IL-17 production play an important role.
TNF-α는 NF-κB 경로의 활성화를 통해 광범위한 전염증 특성을 나타내며, 류마티스 활막염에 결정적으로 관여하는 다면성 사이토카인이다. 예를 들어, TNF-α는 전염증성 사이토카인(IL-1, IL-6 등)과 케모카인(IL-8, MCP-1, MIP-1, RANTES 등)의 합성을 유도하여 대식세포를 활성화시켜 자가면역 병리학에서 전염증성 사이토카인 환경을 지속시킨다. 또한, TNF-α 수용체(TNFR1 및 TNFR2)를 통한 NF-κB의 활성화는 항-세포사멸 단백질을 상향 조절하여 염증 세포의 연장된 생존 및 지속적인 염증을 초래한다.TNF-α exhibits a wide range of proinflammatory properties through activation of the NF-κB pathway, and is a pleiotropic cytokine crucially involved in rheumatoid synovitis. For example, TNF-α activates macrophages by inducing the synthesis of pro-inflammatory cytokines (IL-1, IL-6, etc.) and chemokines (IL-8, MCP-1, MIP-1, RANTES, etc.). Sustaining the pro-inflammatory cytokine environment in autoimmune pathology. In addition, activation of NF-κB through TNF-α receptors (TNFR1 and TNFR2) upregulates anti-apoptotic proteins resulting in prolonged survival and persistent inflammation of inflammatory cells.
1988년에 활성화된 대식세포 또는 림프구에 의해 분비된 사이토카인인 TNF를 표적으로 하는 생물학적 제제가 개발되었다. 항-TNF 바이오 신약에는 etanercept(Enbrel), infliximab(Remicade), adalimumab(Humira) 등이 있고, 상이한 기전을 갖는 항염증성 바이오 신약 중 tocilizumab(Actemra)은 신호 전달을 차단하기 위해 IL-6 수용체에 결합하는 모노클로날 항체이며, Anakinra(Kineret)는 IL-1 수용체에 결합하는 길항제이다. 상기 바이오 신약은 단기간에 효과를 나타내지만 반복 주사 치료로 인한 잦은 병원 방문, 높은 저항력, 내성 면역 부작용, 고비용, 저온 저장 등의 단점을 가진다. 따라서, TNF에 직접 결합하여 류마티스 관절염과 같은 자가면역질환을 치료할 수 있는 소분자 화합물의 개발이 필요한 실정이다.In 1988, a biological agent targeting TNF, a cytokine secreted by activated macrophages or lymphocytes, was developed. New anti-TNF biologic drugs include etanercept (Enbrel), infliximab (Remicade), and adalimumab (Humira), and among anti-inflammatory biologic drugs with different mechanisms, tocilizumab (Actemra) binds to the IL-6 receptor to block signal transduction. Is a monoclonal antibody, and Anakinra (Kineret) is an antagonist that binds to the IL-1 receptor. The new bio-drug is effective in a short period of time, but has disadvantages such as frequent hospital visits due to repeated injection treatment, high resistance, immune side effects, high cost, and low temperature storage. Therefore, there is a need to develop a small molecule compound capable of directly binding to TNF to treat autoimmune diseases such as rheumatoid arthritis.
테트라하이드로파파베린(tetrahydropapaverine; THP)은 테트라하이드로이소 퀴놀린 중 하나로, 살소리롤(salsolinol) 및 테트라하이드로파파베롤린의 유사체이며, 도파민 뉴런에 신경 독성을 나타내는 것으로 알려져 있다. TNF 억제 효과 또는 자가면역질환 치료에 있어서 테트라하이드로파파베린의 효과는 아직까지 밝혀진 바가 없다.Tetrahydropapaverine (THP) is one of the tetrahydroisoquinolines, an analogue of salsorol and tetrahydropaberolin, and is known to exhibit neurotoxicity to dopamine neurons. The TNF inhibitory effect or the effect of tetrahydropaberine in the treatment of autoimmune diseases has not yet been revealed.
본 발명의 목적은 TNF-관련 질환 예방 또는 치료용 약학 조성물을 제공하는 데에 있다.An object of the present invention is to provide a pharmaceutical composition for preventing or treating TNF-related diseases.
본 발명의 다른 목적은 TNF-관련 질환 예방 또는 개선용 건강식품 조성물을 제공하는 데에 있다.Another object of the present invention is to provide a health food composition for preventing or improving TNF-related diseases.
본 발명의 또 다른 목적은 TNF-관련 질환을 치료하는 방법을 제공하는 데에 있다.Another object of the present invention is to provide a method for treating TNF-related diseases.
본 발명의 또 다른 목적은 인 비트로(in vitro)에서 TNF 활성을 억제하는 시약 조성물을 제공하는 데에 있다.Another object of the present invention is to provide a reagent composition that inhibits TNF activity in vitro.
본 발명의 또 다른 목적은 TNF 활성 억제 방법을 제공하는 데에 있다.Another object of the present invention is to provide a method for inhibiting TNF activity.
상기 목적을 달성하기 위하여, 본 발명은 테트라하이드로파파베린(tetrahydropapaverine)을 유효성분으로 포함하는 TNF-관련 질환 예방 또는 치료용 약학 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating TNF-related diseases comprising tetrahydropapaverine as an active ingredient.
또한, 본 발명은 테트라하이드로파파베린(tetrahydropapaverine)을 유효성분으로 포함하는 TNF-관련 질환 예방 또는 개선용 건강식품 조성물을 제공한다.In addition, the present invention provides a health food composition for preventing or improving TNF-related diseases comprising tetrahydropapaverine as an active ingredient.
또한, 본 발명은 테트라하이드로파파베린(tetrahydropapaverine)을 약학적으로 유효한 양으로 처리하는 단계를 포함하는, TNF-관련 질환을 치료하는 방법을 제공한다.In addition, the present invention provides a method for treating TNF-related diseases, comprising the step of treating tetrahydropapaverine in a pharmaceutically effective amount.
또한, 본 발명은 테트라하이드로파파베린(tetrahydropapaverine)을 유효성분으로 포함하며, 인 비트로(in vitro)에서 TNF 활성을 억제하는 시약 조성물을 제공한다.In addition, the present invention provides a reagent composition comprising tetrahydropapaverine as an active ingredient and inhibiting TNF activity in vitro.
또한, 본 발명은 사람을 제외한 동물에 테트라하이드로파파베린(tetrahydropapaverine)을 처리하는 단계를 포함하는, TNF 활성 억제 방법을 제공한다.In addition, the present invention provides a method for inhibiting TNF activity, comprising the step of treating tetrahydropapaverine to animals other than humans.
본 발명에서는 천연 화합물 라이브러리를 스크리닝하여 TNF의 직접 결합제 및 억제제로 작용하는 테트라하이드로파파베린을 동정하였다. 상기 테트라하이드로파파베린은 TNF 활성을 억제하고, 콜라겐 유도 관절염 모델에서 뼈 및 연골을 보호하며, 염증성 사이토카인을 감소시킬 뿐만 아니라 Th17 세포의 분화 및 섬유아세포 유사 활막세포의 증식을 억제하는 효과를 나타내는 바, 상기 테트라하이드로파파베린은 류마티스 관절염을 포함하는 TNF-관련 질환의 예방 또는 치료용 약학 조성물, TNF-관련 질환의 예방 또는 개선용 건강식품 조성물 등으로 유용하게 활용될 수 있다. In the present invention, a natural compound library was screened to identify tetrahydropaberine acting as a direct binding agent and inhibitor of TNF. The tetrahydropaberine inhibits TNF activity, protects bone and cartilage in a collagen-induced arthritis model, reduces inflammatory cytokines, and inhibits the differentiation of Th17 cells and proliferation of fibroblast-like synovial cells. Bar, the tetrahydropaberine may be usefully used as a pharmaceutical composition for preventing or treating TNF-related diseases including rheumatoid arthritis, a health food composition for preventing or improving TNF-related diseases, and the like.
도 1은 TNF-TNF 수용체의 결합을 억제하는 소분자 화합물 검출에 관한 것으로, (A)는 TNF-TNF 수용체 결합 ELISA를 이용한 천연 단일 화합물의 스크리닝, (B)는 테트라하이드로파파베린의 화학 구조, (C)는 TNF에 대한 THP의 직접 결합 활성, (D)는 LM 세포에서 THP의 TNF 특이적 중화 활성을 나타낸 결과이다.
도 2는 콜라겐 유도 관절염(CIA) 모델에서 THP의 치료 효과에 관한 것으로, (A)는 관절염 점수, (B)는 혈청 사이토카인 수준, (C)는 조직학적 이미지를 나타낸 결과이다.
도 3은 마우스 비장세포를 이용한 Th17 세포의 분화 및 기능에서 THP의 억제 효과에 관한 것으로, (A)는 유세포 분석을 이용한 IL-17A의 발현, (B)는 ELISA 및 qRT-PCR을 이용한 IL-17A의 발현, (C)는 qRT-PCR을 이용한 RORγt의 발현을 나타낸 결과이다.
도 4는 인간 PBMC를 이용한 Th17 세포의 분화 및 기능에서 THP의 억제 효과에 관한 것으로, (A)는 유세포 분석을 이용한 IL-17A의 발현, (B)는 ELISA 및 qRT-PCR을 이용한 IL-17A의 발현, (C)는 qRT-PCR을 이용한 RORγt의 발현을 나타낸 결과이다.
도 5는 MH7A 세포에서 THP에 의한 세포 증식 억제 활성을 나타낸 결과이다.Figure 1 relates to the detection of a small molecule compound that inhibits the binding of TNF-TNF receptor, (A) is a screening of a natural single compound using a TNF-TNF receptor binding ELISA, (B) is the chemical structure of tetrahydropaberine, ( C) is the direct binding activity of THP to TNF, (D) is the result showing the TNF-specific neutralizing activity of THP in LM cells.
FIG. 2 shows the therapeutic effect of THP in a collagen-induced arthritis (CIA) model, where (A) is an arthritis score, (B) is a serum cytokine level, and (C) is a histological image.
Figure 3 relates to the inhibitory effect of THP on the differentiation and function of Th17 cells using mouse splenocytes, (A) is the expression of IL-17A using flow cytometry, (B) is IL- using ELISA and qRT-PCR. Expression of 17A, (C) is a result showing the expression of RORγt using qRT-PCR.
Figure 4 relates to the inhibitory effect of THP on the differentiation and function of Th17 cells using human PBMC, (A) is the expression of IL-17A using flow cytometry, (B) is IL-17A using ELISA and qRT-PCR. Expression of, (C) is a result showing the expression of RORγt using qRT-PCR.
5 is a result showing the cell proliferation inhibitory activity by THP in MH7A cells.
본 발명의 발명자들은 천연 단일 화합물 라이브러리에서 TNF 결합 스크리닝을 통해 테트라하이드로파파베린을 동정하였으며, 상기 테트라하이드로파파베린이 TNF에 결합하여 TNF 활성을 억제하고, 염증성 사이토카인의 분비, Th17 세포의 분화, Th17 세포와 관련된 분자의 발현, FLS 세포의 증식을 억제하여 류마티스 관절염을 개선시키는 것을 확인하며 본 발명을 완성하였다.The inventors of the present invention identified tetrahydropaberine through TNF binding screening in a natural single compound library, and the tetrahydropaberin binds to TNF and inhibits TNF activity, secretion of inflammatory cytokines, differentiation of Th17 cells, The present invention was completed by confirming the improvement of rheumatoid arthritis by inhibiting the expression of molecules related to Th17 cells and proliferation of FLS cells.
이에, 본 발명은 테트라하이드로파파베린(tetrahydropapaverine)을 유효성분으로 포함하는 TNF-관련 질환 예방 또는 치료용 약학 조성물을 제공한다.Accordingly, the present invention provides a pharmaceutical composition for preventing or treating TNF-related diseases comprising tetrahydropapaverine as an active ingredient.
상기 테트라하이드로파파베린은 종양괴사인자(TNF)에 직접 결합하여 TNF 활성을 억제할 수 있다.The tetrahydropaberine can inhibit TNF activity by directly binding to tumor necrosis factor (TNF).
상기 TNF-관련 질환은 자가 면역 질환, 염증성 질환, 심혈관 질환, 대사성 질환, 면역 장애, 신경 질환, 안과 질환, 피부 질환, 정신 질환, 감염 질환 및 암으로 이루어진 군에서 선택되는 어느 하나일 수 있으나, 이에 제한되는 것은 아님을 명시한다.The TNF-related disease may be any one selected from the group consisting of autoimmune diseases, inflammatory diseases, cardiovascular diseases, metabolic diseases, immune disorders, neurological diseases, eye diseases, skin diseases, mental diseases, infectious diseases and cancer, It should be noted that it is not limited thereto.
상세하게는, 상기 TNF-관련 질환은 류마티스 관절염, 소아류마티스관절염, 염증성 장질환, 크론병, 궤양성 대장염, 건선, 판상건선, 소아판상건선, 건선성관절염, 다관절형소아특발성관절염, 베체트장염, 강직성척추염, 축성척추관절염, 소아골부착부위염관련관절염, 류마티스다발근통, 다발성경화증, 갑상선염, 지연과민증, 알레르기, 접촉성피부염, 아토피성피부염, 전신성홍반루푸스, 전신경화증, 성인형스틸병, 천식, 자가면역성갑상선장애, 쇼그렌증후군, 가와사키병, 췌장염, 신장염, 간염, 폐렴, 만성폐쇄성폐질환, 중이염, 맥관증식신염, 골수형성이상증후군, 골관절염, 유육종증, 고리육아종, 베게너육아종증, 낭창, 용혈성요독증후군, 동맥경화증, 혈관염, 심부전, 뇌졸중, 심근경색, 심근허혈-재관류손상, 성기능장애, 비만, 고혈압, 당뇨병 및 당뇨합병증, 고지혈증, 자간전증, 신장병, 간장병, 신장 손상, 간손상, 뱀교상, 동종이식거부, 장기이식, 이식편대숙주병, 치매, 알츠하이머병, 파킨슨병, 근위축성측색경화증, 통증, 중추신경계질환, 포도막염, 베체트병, 당뇨황반부종, 황반변성, 안와병증, 녹내장, 화농성한선염, 다중심성망상조직구증식증, 모공성홍색비강진, 호산구성근막염, 지방층염, 당뇨병성유지방성괴사생성, 흉터유사천포창, 괴저성농피증, 스위트증후군, 각질하농포성피부증, 피부경화증, 호중구성피부염, 독성표피괴사융해, 농포성피부염, 피부근염, 다발근육염, 물집피부병, 결절홍반, 탈모증, 우울증, 양극성장애, 불안장애, 결핵, 바이러스감염, 박테리아감염, 진균감염, 원충감염, 뇌말라리아, 패혈증, 패혈성 쇼크, 전립선암, 피부암, 대장암, 신장암, 췌장암, 난소암, 유방암, 방광암, 전립선암, 림프종, 교종, 골육종, 백혈병, 다발성골수종 및 악액질로 이루어진 군에서 선택될 수 있으나, 이에 제한되는 것은 아님을 명시한다.Specifically, the TNF-related diseases are rheumatoid arthritis, juvenile rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriasis, plaque psoriasis, juvenile plaque psoriasis, psoriatic arthritis, polyarticular pediatric idiopathic arthritis, Behcet's enteritis. , Ankylosing spondylitis, axial spondyloarthritis, pediatric bone-attached gastritis-related arthritis, rheumatoid polymyalgia, multiple sclerosis, thyroiditis, delayed hypersensitivity, allergies, contact dermatitis, atopic dermatitis, systemic lupus erythematosus, systemic sclerosis, adult steel disease, asthma , Autoimmune thyroid disorder, Sjogren's syndrome, Kawasaki's disease, pancreatitis, nephritis, hepatitis, pneumonia, chronic obstructive pulmonary disease, otitis media, vascular proliferative nephritis, myelodysplastic syndrome, osteoarthritis, sarcoidosis, ring granuloma, Wegener's granulomatosis, lupus, hemolytic Uremic syndrome, arteriosclerosis, vasculitis, heart failure, stroke, myocardial infarction, myocardial ischemia-reperfusion injury, sexual dysfunction, obesity, hypertension, diabetes and diabetes complications, hyperlipidemia, preeclampsia, kidney disease, liver disease, kidney injury, liver injury, snake bite, Allograft rejection, organ transplantation, graft versus host disease, dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, pain, central nervous system disease, uveitis, Behcet's disease, diabetic macular edema, macular degeneration, orbitopathy, glaucoma, purulent Adenitis, multicentric reticulocytosis, follicular erythema, eosinophilic fasciitis, steatitis, diabetic milk fat necrosis, scar-like pemphigus, gangrene pyoderma, sweet syndrome, subkeratinous pustular dermatosis, scleroderma, neutrophilic dermatitis , Toxic epidermal necrosis fusion, pustular dermatitis, dermatitis, polymyositis, blister dermatitis, nodular erythema, alopecia, depression, bipolar disorder, anxiety disorder, tuberculosis, viral infection, bacterial infection, fungal infection, protozoal infection, cerebral malaria, sepsis , Septic shock, prostate cancer, skin cancer, colon cancer, kidney cancer, pancreatic cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, lymphoma, glioma, osteosarcoma, leukemia, multiple myeloma, and cachexia, but It should be noted that it is not limited.
상기 조성물은 TNF, IL-6 및 IL-1β의 발현을 감소시킬 수 있고, Th17 세포의 분화 및 섬유아세포 유사 활막세포의 증식을 억제시킬 수 있다.The composition can reduce the expression of TNF, IL-6 and IL-1β, and can inhibit the differentiation of Th17 cells and the proliferation of fibroblast-like synovial cells.
본 발명의 조성물이 약학 조성물인 경우, 투여를 위하여, 상기 기재한 유효성분 이외에 약학적으로 허용 가능한 담체, 부형제 또는 희석제를 포함할 수 있다. 상기 담체, 부형제 및 희석제로는 락토오스, 덱스트로오스, 수크로오스, 소르비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.When the composition of the present invention is a pharmaceutical composition, for administration, it may include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the above-described active ingredients. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.
본 발명의 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용할 수 있다. 상세하게는 제형화할 경우 통상 사용하는 충진제, 중량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형 제제로는 정제, 환제, 산제, 과립제, 캡슐제 등을 포함하나, 이에 한정되는 것은 아니다. 이러한 고형 제제는 상기 유효성분 외에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘 카보네이트, 수크로오스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구를 위한 액상물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등을 첨가하여 조제될 수 있다. 비경구 투여를 위한 제제는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제 및 과제를 포함한다. 비수성 용제 및 현탁제로는 프로필렌글리콜, 폴리에틸렌글리콜, 올리브 오일과 같은 식물성 오일, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로솔, 트윈 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical compositions of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injectable solutions according to a conventional method. . Specifically, when formulated, it may be prepared using diluents or excipients such as fillers, weight agents, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc. in addition to the active ingredient. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. It can be prepared by adding various excipients, such as wetting agents, sweetening agents, fragrances, preservatives, and the like, in addition to oral liquids and liquid paraffin. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and tasks. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like may be used. As a base for suppositories, witepsol, macrosol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.
본 발명의 약학 조성물의 적합한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 시간에 따라 다르지만, 당 업자에 의해 적절하게 선택될 수 있는 바, 상기 조성물의 일일 투여량은 바람직하게는 0.001 mg/kg 내지 50 mg/kg이며, 필요에 따라 일일 1회 내지 수회로 나누어 투여할 수 있다.A suitable dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the patient, the severity of the disease, the form of the drug, and the time, but may be appropriately selected by a person skilled in the art. The daily dosage of the composition is preferably It is 0.001 mg/kg to 50 mg/kg, and it can be administered once to several times a day, if necessary.
또한, 본 발명은 테트라하이드로파파베린(tetrahydropapaverine)을 유효성분으로 포함하는 TNF-관련 질환 예방 또는 개선용 건강식품 조성물을 제공한다.In addition, the present invention provides a health food composition for preventing or improving TNF-related diseases comprising tetrahydropapaverine as an active ingredient.
본 발명의 조성물이 건강식품 조성물인 경우, 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 천연 과일 주스, 합성 과일 주스 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 또한, 건강식품 조성물은 육류, 소세지, 빵, 초콜릿, 캔디류, 스넥류, 과자류, 피자, 라면, 껌류, 아이스크림류, 스프, 음료수, 차, 기능수, 드링크제, 알코올 및 비타민 복합제 중 어느 하나의 형태일 수 있다.When the composition of the present invention is a health food composition, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and salts thereof , Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like. In addition, it may contain pulp for the manufacture of natural fruit juices, synthetic fruit juices and vegetable beverages. These components may be used independently or in combination. In addition, the health food composition may be in the form of any one of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcohol and vitamin complex. I can.
또한, 상기 건강식품 조성물은 식품첨가물을 추가로 포함할 수 있으며, 식품첨가물로서의 적합 여부는 다른 규정이 없는 한 식품의약품안전처에 승인된 식품첨가물공전의 총칙 및 일반 시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다.In addition, the health food composition may additionally contain food additives, and the suitability as a food additive is determined according to the general rules and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified. It is judged according to the standards and standards.
상기 식품첨가물공전에 수재된 품목으로 예를 들어, 케톤류, 글리신, 구연산 칼륨, 니코틴산, 계피산 등의 화학적 합성품, 감색소, 감초추출물, 결정셀룰로오스, 고랭색소, 구아검 등의 천연첨가물, L-글루타민산나트륨 제제, 면류 첨가 알칼리제, 보존료제제, 타르색소 제제 등의 혼합 제제류 등을 들 수 있다.For example, ketones, glycine, potassium citrate, nicotinic acid, cinnamic acid, chemical synthetic products such as reduced pigment, licorice extract, crystalline cellulose, high cooling pigment, natural additives such as guar gum, L-glutamic acid And mixed preparations such as sodium preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations.
이때, 건강식품 조성물을 제조하는 과정에서 식품에 첨가되는 본 발명에 따른 조성물은 필요에 따라 그 함량을 적절히 가감할 수 있다.At this time, the content of the composition according to the present invention added to food in the process of manufacturing the health food composition may be appropriately added or subtracted as needed.
또한, 본 발명은 테트라하이드로파파베린(tetrahydropapaverine)을 약학적으로 유효한 양으로 처리하는 단계를 포함하는, TNF-관련 질환을 치료하는 방법을 제공한다.In addition, the present invention provides a method for treating TNF-related diseases, comprising the step of treating tetrahydropapaverine in a pharmaceutically effective amount.
또한, 본 발명은 테트라하이드로파파베린(tetrahydropapaverine)을 유효성분으로 포함하며, 인 비트로(in vitro)에서 TNF 활성을 억제하는 시약 조성물을 제공한다.In addition, the present invention provides a reagent composition comprising tetrahydropapaverine as an active ingredient and inhibiting TNF activity in vitro.
또한, 본 발명은 사람을 제외한 동물에 테트라하이드로파파베린(tetrahydropapaverine)을 처리하는 단계를 포함하는, TNF 활성 억제 방법을 제공한다.In addition, the present invention It provides a method for inhibiting TNF activity, comprising the step of treating an animal other than humans with tetrahydropapaverine.
이하에서는 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
실시예 1: 마우스Example 1: Mouse
6주령 수컷 DBA/1J 또는 C57BL/6 마우스는 Orientbio(Gyeonggi-do, Korea)에서 구입하였다. 마우스는 12시간 명/암 주기, 온도(22 ± 3℃) 및 습도(40-60%)가 조절된 환경에서 사육하였으며, 식이 및 물은 자유롭게 섭취할 수 있게 하였다. 마우스는 1주일의 순응 기간을 거친 후 실험에 사용하였다. 모든 동물실험은 가톨릭대학교 동물실험윤리위원회(IACUC)가 승인한 프로토콜(허가번호: 2016-018-02)을 준수하였다.6-week-old male DBA/1J or C57BL/6 mice were purchased from Orientbio (Gyeonggi-do, Korea). Mice were bred in an environment in which 12 hours light/dark cycle, temperature (22 ± 3°C) and humidity (40-60%) were controlled, and diet and water were freely ingested. Mice were used in the experiment after an acclimatization period of 1 week. All animal experiments followed the protocol (license number: 2016-018-02) approved by the Catholic University's Animal Experimental Ethics Committee (IACUC).
실시예 2: 콜라겐 유도 관절염 동물모델 제작 및 테트라하이드로파파베린(THP) 주사Example 2: Collagen-induced arthritis animal model production and tetrahydropaberine (THP) injection
콜라겐 유도 관절염(collagen-induced arthritis; CIA)은 하기와 같이 유도되었다. 간략하게, 소 타입 II 콜라겐(CII)(Chondrex, ES) 및 Complete Freund’s adjuvant(Sigma, US) 50 μg를 꼬리 기저에 피내 주사하였다. 2주 후, CII 및 Incomplete Freund’s adjuvant(Sigma, US) 50 μg를 피내 주사하여 부스팅하였다. THP(5 mg/kg), 또는 비히클 대조군(10% DMSO가 포함된 증류수)은 첫 면역 후 CIA 마우스(N = 10)에 주 6회 경구 투여하였다.Collagen-induced arthritis (CIA) was induced as follows. Briefly, 50 μg of bovine type II collagen (CII) (Chondrex, ES) and Complete Freund's adjuvant (Sigma, US) were injected intradermally at the base of the tail. After 2 weeks, 50 μg of CII and Incomplete Freund's adjuvant (Sigma, US) was boosted by intradermal injection. THP (5 mg/kg) or vehicle control (distilled water containing 10% DMSO) was orally administered 6 times a week to CIA mice (N = 10) after the first immunization.
실시예 3: CIA의 임상 관절염 점수 평가Example 3: Evaluation of CIA's Clinical Arthritis Score
마우스의 4개 발에서 관절염의 중증도는 하기의 방법을 이용하여 3명의 맹검 관찰자에 의해 평가되었다. 앞발 및 뒷발의 관절염의 중증도는 첫 면역 후 0 내지 56일 사이에 주 2회 평가하였다: 0 = 정상 발, 1 = 1개의 발에 염증이 있고 부어오름, 2 = 1~3개의 발에 염증이 있고 부어오름, 3 = 발 전체에 염증이 있고 부어오름, 4 = 염증이 심하고 부어오르거나 유착된 발. 각 마우스의 관절염 점수는 4개 발의 관절염 중증도의 합으로 나타내었다. 가장 높은 점수는 16점이다.The severity of arthritis in the four paws of mice was evaluated by three blind observers using the following method. The severity of arthritis of the forefoot and hind paw was assessed twice a week between 0 and 56 days after the first immunization: 0 = normal foot, 1 = 1 foot is inflamed and swollen, 2 = 1 to 3 feet are inflamed. And swollen, 3 = inflamed and swollen all over the foot, 4 = inflamed, swollen or adherent foot. The arthritis score of each mouse was expressed as the sum of the arthritis severity of the four paws. The highest score is 16 points.
실시예 4: 조직학적 분석Example 4: Histological analysis
뒷발을 절개하고 10% 포름산으로 21일 동안 탈석회 과정을 수행하였다. 탈석회 후, 표본을 파라핀에 포매시킨 후 조직 절편을 제작하였다. 조직 절편은 탈파라핀 후 재수화시킨 다음, 헤마톡실린 & 에오신, 또는 사프라닌 O로 염색하였다.The hind paw was dissected and the deliming process was performed for 21 days with 10% formic acid. After deliming, the specimen was embedded in paraffin, and a tissue section was prepared. Tissue sections were rehydrated after deparaffining, and then stained with hematoxylin & eosin, or safranin O.
실시예 5: 생체 외 마우스 Th17 세포 분화Example 5: In vitro mouse Th17 cell differentiation
naive 마우스를 희생시킨 후, 비장을 적출하여 단일세포로 분리하였다. 이후 세포를 얼음 위에 놓고 3분 동안 RBC 용해 완충액(Thermo, US)과 반응시켰다. PBS로 세척한 후, 마우스 CD4 MACS 마이크로비드(MiltenyiBiotec, Germany)를 사용하여 naive CD4+ T 세포를 분리하였다.After the naive mouse was sacrificed, the spleen was excised and separated into single cells. Thereafter, the cells were placed on ice and reacted with RBC lysis buffer (Thermo, US) for 3 minutes. After washing with PBS, naive CD4 + T cells were isolated using mouse CD4 MACS microbeads (MiltenyiBiotec, Germany).
마우스 Th17 분화 키트(R&D system, US)를 사용하여 마우스 비장세포에 대한 생체 외 실험을 수행하였다. naive CD4+ T 세포(1 X 106 세포/ml)를 마우스 Th17 분화 배지에 현탁시켰다. 부유 세포(1 X 105 세포/웰)를 96 웰 플레이트(Gibco, US)에 접종하고 미리 코팅된 햄스터 항-CD3 마우스 항체로 자극시켰다. 이들 세포를 37℃, 5% CO2 배양기에서 3일 동안 배양하였다. 3일째에 동일한 부피의 새로운 마우스 Th17 분화 배지를 첨가하여 Th17 분화 배지를 교체하고, 2일 동안 추가 배양하였다. 이후, 세포 및 배양 상층액을 회수하고, 수집된 세포는 유세포 분석을 이용하여 Th17 세포 분화 여부를 측정하였다. 또한, 배양 상층액은 IL-17A ELISA 키트(Biolegend, US)를 사용하여 마우스 IL-17A 분비를 측정하였다.In vitro experiments were performed on mouse splenocytes using a mouse Th17 differentiation kit (R&D system, US). naive CD4 + T cells (1
실시예 6: 인간 말초혈액 단핵세포(PBMC)에서 CD4Example 6: CD4 in human peripheral blood mononuclear cells (PBMC) ++ T 세포 분리 및 Th17 세포 분화 T cell isolation and Th17 cell differentiation
건강한 기증자 3명의 시료는 적십자로부터 제공받았으며, TrimaAccel 자동 혈액 수집 시스템을 사용하여 혈액을 수집하였다. 키트에 부착된 LRS(Leukoreduction system) 챔버는 상용화 제품을 구입하여 사용하였다. LRS 챔버에 연결된 input 및 output 튜브는 알코올로 멸균하고 절단하여 사용하였다. 세포를 50 ml 튜브에 넣고, 동량의 2% 우태아혈청(FBS, Corning, US)이 포함된 인산완충식염수(PBS, Welgene)를 첨가하였다. 이어서 SepMateTM-50 튜브(STEMCELL technology, Canada)에 Ficoll-PoqueTM PLUS ratio 1.077(GE Healthcare, US) 15 ml을 첨가하고, 그 위에 LRS 챔버로 분리된 15 ml의 세포를 포개어 넣었다. 브레이크 오프 상태에서 1200 g로 20분 동안 원심분리한 후, 말초혈액 단핵세포를 새로운 50 ml 튜브로 옮겼으며, 2% FBS가 포함된 PBS로 2회 세척하였다.Samples from three healthy donors were provided by the Red Cross, and blood was collected using the TrimaAccel automated blood collection system. The LRS (Leukoreduction system) chamber attached to the kit was used by purchasing a commercial product. The input and output tubes connected to the LRS chamber were sterilized with alcohol and cut. The cells were placed in a 50 ml tube, and phosphate buffered saline (PBS, Welgene) containing an equal amount of 2% fetal calf serum (FBS, Corning, US) was added. Subsequently, 15 ml of Ficoll-Poque™ PLUS ratio 1.077 (GE Healthcare, US) was added to a SepMate™-50 tube (STEMCELL technology, Canada), and 15 ml of cells separated by an LRS chamber were superimposed thereon. After centrifugation at 1200 g for 20 minutes in the break off state, the peripheral blood mononuclear cells were transferred to a new 50 ml tube, and washed twice with PBS containing 2% FBS.
인간 PBMC를 얼음 위에 놓고 5분 동안 RBC 용해 완충액(Thermo, US)과 반응시켰다. PBS로 세척한 후, 인간 CD4 MACS 마이크로비드(MiltenyiBiotec, Germany)를 사용하여 naive CD4+ T 세포를 분리하였다. 인간 Th17 분화 키트(R&D system, US)를 사용하여 PBMC에 대한 생체 외 실험을 수행하였다. 수집된 세포는 유세포 분석을 이용하여 Th17 세포 분화 여부를 측정하였다. 또한, 배양 상층액은 IL-17A ELISA 키트(Biolegend, US)를 사용하여 인간 IL-17A 분비를 측정하였다.Human PBMCs were placed on ice and reacted with RBC lysis buffer (Thermo, US) for 5 minutes. After washing with PBS, naive CD4 + T cells were isolated using human CD4 MACS microbeads (MiltenyiBiotec, Germany). In vitro experiments were performed on PBMCs using the human Th17 differentiation kit (R&D system, US). The collected cells were measured for Th17 cell differentiation using flow cytometry. In addition, the culture supernatant was measured for human IL-17A secretion using an IL-17A ELISA kit (Biolegend, US).
실시예 7: 생체 외 IL-17A 분비 분석Example 7: In vitro IL-17A secretion assay
ELISA 플레이트를 마우스 또는 인간 IL-17A 포획 항체가 포함된 PBS로 4℃에서 밤새 코팅하였다. 코팅 후, 플레이트를 PBST(0.05% 트윈-20이 포함된 PBS)로 세척하고, 1% BSA가 포함된 PBS와 상온에서 진탕하면서 1시간 동안 반응시켜 블로킹하였다. PBST로 세척한 후, PBSA(1% BSA가 포함된 PBS)로 희석된 배양 상층액을 각 웰에 첨가하였다. 플레이트를 4℃에서 진탕하면서 밤새 반응시켰다. 세척 후, 1X 검출 항체를 각 웰에 첨가하고, 플레이트를 상온에서 진탕하면서 1시간 동안 반응시켰다. PBST로 세척한 후, 검출 항체(avidin-HRP)를 첨가하고 플레이트를 상온에서 진탕하면서 1시간 동안 반응시켰다. 세척 후, TMB 용액을 각 웰에 첨가하고 플레이트를 실온에서 진탕하면서 반응시켰다. 이후, 정지 용액(2N HCl)을 각 웰에 첨가하고, 마이크로플레이트 리더기를 이용하여 450 nm에서 흡광도를 측정하였다.ELISA plates were coated overnight at 4° C. with PBS containing mouse or human IL-17A capture antibody. After coating, the plate was washed with PBST (PBS containing 0.05% Tween-20), and blocked by reacting with PBS containing 1% BSA for 1 hour while shaking at room temperature. After washing with PBST, a culture supernatant diluted with PBSA (PBS containing 1% BSA) was added to each well. The plate was reacted overnight while shaking at 4°C. After washing, 1X detection antibody was added to each well, and the plate was reacted for 1 hour while shaking at room temperature. After washing with PBST, a detection antibody (avidin-HRP) was added, and the plate was reacted for 1 hour while shaking at room temperature. After washing, TMB solution was added to each well and the plate was reacted with shaking at room temperature. Thereafter, a stop solution (2N HCl) was added to each well, and absorbance was measured at 450 nm using a microplate reader.
실시예 8: 효소면역분석법Example 8: Enzyme Immunoassay
CIA 마우스에서 TNF, IL-6 및 IL-1β 분비를 확인하기 위해, CIA 실험 56일째에 마우스로부터 혈청을 채취하였다. ELISA 플레이트(Nunc, Denmark)를 1X 마우스 TNF, IL-6 또는 IL-1β 포획 항체가 포함된 PBS로 4℃에서 밤새 코팅하였다. 코팅 후, 플레이트를 PBST(0.05% 트윈-20이 포함된 PBS)로 세척하고, 1% BSA가 포함된 PBS와 상온에서 진탕하면서 1시간 동안 반응시켜 블로킹하였다. PBST로 세척한 후, PBSA(1% BSA가 포함된 PBS)로 희석된 배양 상층액을 각 웰에 첨가하였다. 플레이트를 4℃에서 진탕하면서 밤새 반응시켰다. 세척 후, 1X 검출 항체를 각 웰에 첨가하고, 플레이트를 상온에서 진탕하면서 1시간 동안 반응시켰다. PBST로 세척한 후, 검출 항체(avidin-HRP)를 첨가하고 플레이트를 상온에서 진탕하면서 1시간 동안 반응시켰다. 세척 후, TMB 용액을 각 웰에 첨가하고 플레이트를 실온에서 진탕하면서 반응시켰다. 이후, 정지 용액(2N HCl)을 각 웰에 첨가하고, 마이크로플레이트 리더기를 이용하여 450 nm에서 흡광도를 측정하였다.In order to confirm the secretion of TNF, IL-6 and IL-1β in CIA mice, serum was collected from mice on day 56 of the CIA experiment. ELISA plates (Nunc, Denmark) were coated overnight at 4° C. with PBS containing 1X mouse TNF, IL-6 or IL-1β capture antibody. After coating, the plate was washed with PBST (PBS containing 0.05% Tween-20), and blocked by reacting with PBS containing 1% BSA for 1 hour while shaking at room temperature. After washing with PBST, a culture supernatant diluted with PBSA (PBS containing 1% BSA) was added to each well. The plate was reacted overnight while shaking at 4°C. After washing, 1X detection antibody was added to each well, and the plate was reacted for 1 hour while shaking at room temperature. After washing with PBST, a detection antibody (avidin-HRP) was added, and the plate was reacted for 1 hour while shaking at room temperature. After washing, TMB solution was added to each well and the plate was reacted with shaking at room temperature. Thereafter, a stop solution (2N HCl) was added to each well, and absorbance was measured at 450 nm using a microplate reader.
실시예 9: 유세포 분석Example 9: Flow cytometry
수집된 마우스 또는 인간 Th17 세포를 ep 튜브에 넣고 원심분리한 후 PBS로 세척하였다. 세척 후, 단일세포를 1X stimulation cocktail 및 1X monensin과 37℃에서 4시간 동안 자극시켰다. 자극된 세포를 원심분리하고, 항-마우스 또는 인간 CD4-PEcy7 염색 완충액과 빛 차단 하에 4℃에서 30분 동안 반응시켰다. 세척 후, 시료에 fixation/permeabilization 용액을 첨가하고 빛 차단 하에 4℃에서 18시간 동안 반응시켰다. 세척 후, 세포를 항-마우스 또는 인간 IL-17A-PE가 포함된 1X permeabilization 용액과 빛 차단 하에 4℃에서 30분 동안 반응시켰다. FACS 완충액으로 세척한 후, 세포를 FACS 완충액에 재현탁시키고 유세포 분석기로 세포를 분석하였다.Collected mouse or human Th17 cells were placed in an ep tube, centrifuged, and washed with PBS. After washing, single cells were stimulated with 1X stimulation cocktail and 1X monensin at 37°C for 4 hours. Stimulated cells were centrifuged and reacted with anti-mouse or human CD4-PEcy7 staining buffer at 4° C. for 30 minutes under light blocking. After washing, a fixation/permeabilization solution was added to the sample and reacted at 4° C. for 18 hours under light blocking. After washing, the cells were reacted with 1X permeabilization solution containing anti-mouse or human IL-17A-PE at 4° C. for 30 minutes under light blocking. After washing with FACS buffer, the cells were resuspended in FACS buffer and analyzed by flow cytometry.
실시예 10: 정량적 실시간 중합효소 연쇄반응(qRT-PCR) 분석Example 10: Quantitative real-time polymerase chain reaction (qRT-PCR) analysis
qRT-PCR은 ABI StepOnePlus Real-Time PCR 시스템(Applied Biosystems, US)에서 SYBR® Green 실시간 PCR 키트(Mbiotech, Korea)를 사용하여 수행하였다. 유전자의 발현 수준은 GAPDH로 정규화하여 분석하였다.qRT-PCR was performed using the SYBR® Green real-time PCR kit (Mbiotech, Korea) in the ABI StepOnePlus Real-Time PCR system (Applied Biosystems, US). The expression level of the gene was analyzed by normalizing to GAPDH.
실시예 11: 세포 배양Example 11: Cell culture
MH7A 인간 류마티스 관절염 활막세포는 Cho YY 교수(Catholic University, Korea)로부터 제공받았다. MH7A 세포는 10% FBS, 페니실린(최종 농도, 100 U/ml), 스트렙토마이신(최종 농도, 0.1 mg/ml) 및 L-글루타민이 포함된 RPMI 1640 배지(Corning)를 이용하여 37℃, 5% C02 배양기에서 배양하였다.MH7A human rheumatoid arthritis synovial cells were provided by Professor Cho YY (Catholic University, Korea). MH7A cells were prepared using RPMI 1640 medium (Corning) containing 10% FBS, penicillin (final concentration, 100 U/ml), streptomycin (final concentration, 0.1 mg/ml) and L-glutamine at 37° C., 5%. Incubated in a CO 2 incubator.
LM 세포는 마우스 섬유아세포(fibroblast)로 Kim TS 교수(Korea university, Korea)로부터 제공받았다. LM 세포는 10% FBS, 페니실린(최종 농도, 100 U/ml), 스트렙토마이신(최종 농도, 0.1 mg/ml)이 포함된 DMEM 배지(Welgene)를 이용하여 37℃, 5% CO2 배양기에서 배양하였다.LM cells were mouse fibroblasts, which were provided by Professor Kim TS (Korea University, Korea). LM cells were cultured in a DMEM medium (Welgene) containing 10% FBS, penicillin (final concentration, 100 U/ml), and streptomycin (final concentration, 0.1 mg/ml) in a 37°C, 5% CO 2 incubator. I did.
실시예 12: 세포 생존율 분석Example 12: Cell viability analysis
세포 증식을 측정하기 위해, 세포(1 X 104 세포/200 μl/웰)를 96 웰 플레이트에 접종하고, 일반 배지를 첨가하여 72시간 동안 배양하였다. 이후, tetrazolim salt WST-1[2-(4-Iodophenyl)-3-(4-nitrophenyl)5-(2,4-disulfophenyl)-2H-tetrazolium]-based EZ-cytox 용액(Daeillab, Korea)을 사용하여 제조사에서 제공한 프로토콜에 따라 450 nm에서 흡광도를 측정하였다. 간략하게, 20 μl의 EZ- cytox 용액을 각 웰에 첨가하고, 37℃, 5% C02 배양기에서 1시간 동안 배양한 후, 450 nm에서 즉시 흡광도를 측정하였다.In order to measure cell proliferation, cells (1
실시예 13: TNF 중화 활성 분석Example 13: TNF neutralizing activity assay
TNF에 대한 독성 중화 활성을 측정하기 위해, 세포(5 X 104세포/100 μl/웰)를 96 웰 플레이트에 접종하고, 일반 배지를 사용하여 Actinomycin-D(AD) 0.5 μg/ml, AD+TNF 20 ng/ml, AD+TNF+THP(200 μM부터 1 nM까지 1/2씩 희석)를 처리한 후, 37℃, 5% CO2 배양기에서 24시간 동안 배양하였다. 이후, thiazolyl blue tetrazolium bromide(MTT, 5 mg/ml, Sigma, US)를 각 웰 당 10 μl씩 첨가하고 4시간 동안 추가 배양하였다. 이후, 상층액을 제거하고 200 μl의 DMSO를 각 웰에 첨가한 후 570 nm에서 흡광도를 측정하였다. To measure the toxic neutralizing activity for TNF, cells (5
실험예 1: 천연 단일 화합물에서 TNF-TNF 수용체의 결합을 억제하는 소분자 화합물 검출Experimental Example 1: Detection of small molecule compounds that inhibit the binding of TNF-TNF receptors in a single natural compound
TNF-TNF 수용체 사이의 결합을 억제하는 소분자 화합물을 검출하기 위해, TNF 경쟁 분석으로 스크리닝을 수행하였다. 플레이트를 TNF로 코팅한 후 TNFR2-Fc(etanercept, Enbrel)를 라이브러리의 각 천연 소분자 화합물과 동시에 첨가하여 TNF에 대한 경쟁적 결합을 유도하고, 대조군과 비교하여 OD 감소를 통해 TNF와 결합된 TNFR2를 검출하였다. TNF 경쟁 분석을 이용하여, 천연 단일 화합물 라이브러리(502 유형)를 스크리닝하였다. 도 1A와 같이, 두 가지 스크리닝 과정을 통해 TNF 표적 소분자 화합물인 THP를 동정하였으며, THP의 화학 구조는 도 1B에 나타내었다.In order to detect small molecule compounds that inhibit the binding between TNF-TNF receptors, screening was performed by TNF competition assay. After coating the plate with TNF, TNFR2-Fc (etanercept, Enbrel) was added simultaneously with each natural small molecule compound in the library to induce competitive binding to TNF, and compared with the control group, TNFR2 bound to TNF was detected through OD reduction. I did. Using the TNF competition assay, a native single compound library (502 type) was screened. As shown in FIG. 1A, THP, which is a TNF target small molecule compound, was identified through two screening processes, and the chemical structure of THP is shown in FIG. 1B.
결합 동역학을 측정하기 위해, biacore T200을 사용하여 SPR 분석을 수행하였다. 도 1C와 같이, THP는 CM5 칩에 고정화된 TNF에 대해 농도 의존적이고 특이적으로 포화 결합된 센서그램을 나타내었고, 결합 친화도(KD) 값은 1.663E-5였다. THP의 TNF 결합이 TNF의 시험관 내 세포독성을 억제할 수 있는지 여부를 평가하였다. 악티노마이신 D, TNF 및 THP를 마우스 섬유아세포인 LM 세포주에 처리하여 세포 독성을 유도하였다. 그 결과, 도 1D와 같이, THP는 농도 의존적으로 TNF의 세포독성을 차단하고, THP의 IC50 값은 0.4 μM임을 확인하였다.To measure binding kinetics, SPR analysis was performed using biacore T200. As shown in FIG. 1C, THP showed a concentration-dependent and specifically saturated-bound sensorgram for TNF immobilized on the CM5 chip, and the binding affinity (KD) value was 1.663E -5 . It was evaluated whether the TNF binding of THP could inhibit the cytotoxicity of TNF in vitro. Cytotoxicity was induced by treatment with actinomycin D, TNF, and THP on the LM cell line, which is a mouse fibroblast. As a result, as shown in FIG. 1D, it was confirmed that THP blocked the cytotoxicity of TNF in a concentration-dependent manner, and the IC50 value of THP was 0.4 μM.
실험예 2: CIA 모델에서 THP에 의한 증상 억제 효과Experimental Example 2: Effect of suppressing symptoms by THP in CIA model
CIA 동물 모델에서 THP의 치료 효과를 평가하기 위해, 2차 면역 후 비히클 또는 THP(5 mg/kg)를 매일 경구 투여하였다. 그 결과, 도 2A와 같이, 비히클이 투여된 군에 비해 THP(5 mg/kg)가 투여된 군에서 관절염 점수가 유의하게 감소하는 것을 확인하였다.To evaluate the therapeutic effect of THP in the CIA animal model, vehicle or THP (5 mg/kg) was administered orally daily after the second immunization. As a result, as shown in FIG. 2A, it was confirmed that the arthritis score significantly decreased in the group administered with THP (5 mg/kg) compared to the group administered with the vehicle.
다음으로, 비히클 또는 THP가 투여된 마우스의 혈청을 이용하여 염증성 사이토카인 분비에 대한 THP의 효능을 평가하였다. 그 결과, 도 2B와 같이, 비히클이 투여된 군에 비해 THP가 투여된 군에서 TNF, IL-6 및 IL-1β의 수준이 유의하게 감소하는 것을 확인하였다. 또한, 뒷다리 관절을 헤마톡실린 및 에오신으로 염색하여 조직학적 분석을 수행한 결과, 도 2C(상부)와 같이, THP가 투여된 군에서 염증, 연골 손상 및 뼈의 침식이 약화되는 것을 확인하였고, 사프라닌-O로 염색한 결과, 도 2C(하부)와 같이, 연골 손실이 방지되는 것을 확인할 수 있었다.Next, the efficacy of THP on the secretion of inflammatory cytokines was evaluated using the vehicle or serum of mice administered THP. As a result, as shown in FIG. 2B, it was confirmed that the levels of TNF, IL-6, and IL-1β were significantly reduced in the THP-administered group compared to the vehicle-administered group. In addition, as a result of performing histological analysis by staining the hind limb joints with hematoxylin and eosin, it was confirmed that inflammation, cartilage damage, and bone erosion were weakened in the THP-administered group, as shown in FIG. 2C (top), As a result of staining with safranin-O, it was confirmed that cartilage loss was prevented, as shown in FIG. 2C (lower part).
실험예 3: THP에 의한 마우스 및 인간 Th17 세포의 분화 및 기능 억제 효과Experimental Example 3: Effect of THP on differentiation and function inhibition of mouse and human Th17 cells
분화/활성화된 Th17 세포는 염증성 사이토카인인 IL-17A를 분비하고, 분비된 IL-17A는 관절 조직의 활막 섬유아세포에 영향을 미쳐 염증 반응을 유발하는 것으로 알려져 있다. Differentiated/activated Th17 cells secrete IL-17A, an inflammatory cytokine, and secreted IL-17A is known to affect synovial fibroblasts in joint tissues and induce an inflammatory response.
이에, 마우스 비장세포 및 인간 PBMC를 사용한 Th17 세포 분화에서 THP의 효과를 평가하였다. 그 결과, 도 3A 및 도 3B와 같이, 마우스 비장세포에서, THP는 농도 의존적으로 Th17 세포의 분화를 억제하고, THP(50 μM)는 Th17 세포에 의해 분비된 IL-17A 단백질 및 mRNA의 발현을 감소시키는 것을 확인하였다. 또한, 도 3C와 같이, Th17 관련 분자인 RORγT의 mRNA 발현을 감소시키는 것을 확인하였다.Thus, the effect of THP on Th17 cell differentiation using mouse splenocytes and human PBMCs was evaluated. As a result, as shown in FIGS. 3A and 3B, in mouse splenocytes, THP inhibits the differentiation of Th17 cells in a concentration-dependent manner, and THP (50 μM) inhibits the expression of IL-17A protein and mRNA secreted by Th17 cells. It was confirmed to decrease. In addition, as shown in Fig. 3C, it was confirmed that the mRNA expression of RORγT, a Th17-related molecule, was reduced.
마찬가지로, 도 4A 및 도 4B와 같이, 인간 PBMC에서, THP(12.5, 25 및 50 μM)는 Th17 세포의 분화를 유의하게 억제하고, 농도 의존적으로 Th17 세포에 의해 분비된 IL-17 단백질 및 mRNA의 발현을 유의하게 감소시키는 것을 확인하였다. 또한, 도 4C와 같이, THP(12.5 μM)는 RORγT의 mRNA 발현을 감소시키는 것을 확인하였다.Similarly, as shown in Figs. 4A and 4B, in human PBMCs, THP (12.5, 25 and 50 μM) significantly inhibits the differentiation of Th17 cells, and concentration-dependently of IL-17 proteins and mRNA secreted by Th17 cells. It was confirmed that the expression was significantly reduced. In addition, as shown in Figure 4C, it was confirmed that THP (12.5 μM) reduced the mRNA expression of RORγT.
실험예 4: 류마티스 관절염의 섬유아세포 유사 활막세포에서 THP에 의한 세포 증식 억제 효과Experimental Example 4: Inhibitory effect of THP on cell proliferation in fibroblast-like synovial cells of rheumatoid arthritis
THP가 류마티스 관절염의 섬유아세포 유사 활막세포(fibroblast like synoviocyte; FLS)의 증식을 억제할 수 있는지 여부를 평가하기 위해, 인간 류마티스 관절염 환자의 FLS 세포주인 MH7A 세포를 사용하여 MTT 분석을 수행하였다. 그 결과, 도 5와 같이, THP가 농도 의존적으로 MH7A 세포의 증식을 유의하게 억제 하는 것을 확인하였다.To evaluate whether THP can inhibit the proliferation of fibroblast-like synoviocytes (FLS) of rheumatoid arthritis, MTT analysis was performed using MH7A cells, which are FLS cell lines of human rheumatoid arthritis patients. As a result, as shown in FIG. 5, it was confirmed that THP significantly inhibited the proliferation of MH7A cells in a concentration-dependent manner.
이상으로 본 발명의 특정한 부분을 상세히 기술한 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, it is clear that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.
본 발명의 범위는 후술하는 특허청구범위에 의하여 나타내어지며, 특허청구범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.The scope of the present invention is indicated by the claims to be described later, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.
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