KR20200037536A - Composition for diagnosing or treating inflammatory diseases tageting PI3KC2γ - Google Patents
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- KR20200037536A KR20200037536A KR1020180116871A KR20180116871A KR20200037536A KR 20200037536 A KR20200037536 A KR 20200037536A KR 1020180116871 A KR1020180116871 A KR 1020180116871A KR 20180116871 A KR20180116871 A KR 20180116871A KR 20200037536 A KR20200037536 A KR 20200037536A
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Abstract
Description
본 발명은 포스포이노시티드 3-키나제-C2γ(Phosphoinositide 3-Kinase-C2γ; PI3KC2γ)를 표적으로 하는 염증 질환 진단 및 치료용 조성물에 관한 것이다. The present invention relates to a composition for diagnosing and treating inflammatory diseases targeting phosphoinositide 3-kinase-C2γ (Phosphoinositide 3-Kinase-C2γ; PI3KC2γ).
류마티스 관절염은 만성 염증성 자가면역 질환으로 윤활막염과 이의 결과로 발생하는 뼈와 연골의 파괴를 특징으로 한다. 정확한 병인은 밝혀지지 않았으나 유전적 소인과 환경적인 자극이 자가면역 반응을 촉발하여 자가항체 생산을 유발하고 윤활막염이 발생하며, 이 과정에 많은 면역 세포와 시토카인이 서로 유기적 영향 관계를 가지면서 염증 반응을 악화시키며 결과적으로 인접한 연골과 뼈를 파괴하게 된다. 이러한 병인에 착안하여 개발된 종양괴사인자(tumor necrosis factor, TNF) 저해제, 인터루킨(IL)-6 차단제 그리고 다양한 면역 세포 저해제와 같은 생물학적 제제의 도입으로 근 20년간 류마티스 관절염에 대한 치료는 획기적인 변화를 맞게 되었다. 기존의 항류마티스 제제에 불충분한 반응을 보이는 환자에게 우수한 임상적 효과를 보였고, 관절 파괴에 대한 뼈와 연골의 보호 효과를 나타내었다. 그러나 이러한 생물학적 제제에도 반응하지 않는 환자가 적지 않게 존재한다는 점, 감염이나 종양 위험성 등의 부작용, 주사를 이용해야 하는 투여 방법의 침습성과 불편성, 높은 약제 가격 등의 문제점을 안고 있다. 이에 비해 소분자 억제제(small molecular inhibitor)는 대부분 세포내 신호 전달 과정이나 케모카인과 수용체의 결합 작용을 억제하는 약물로, 대표적으로 Janus kinase (JAK) 저해제와 spleen tyrosine kinase (Syk) 저해제가 임상 사용의 승인을 받았거나 임상 연구가 거의 종료 단계에 있다. 이 약물들은 메토트렉세이트의 병합 투여 없이도 생물학적 제제에 버금가는 관절염 조절 효과를 보일 뿐 아니라, 경구 투여가 가능해 투여가 편리하고, 비교적 저렴한 비용으로 이러한 화합물의 생산이 가능하다는 장점을 가지고 있다. 현재 S1P lyase 저해제인 LX3305와 케모카인 수용체 저해제인 CCX354-C 등의 소분자 억제제가 임상 연구 중에 있으며, 병인에 기초한 연구를 통해 지속적으로 새로운 약물들이 개발되고 있다. 또한, 항염증제로 이용하고 있는 물질로는 비스테로이드계에 이부프로펜, 인도메타신 등이 있고 스테로이드계에는 덱사메타손 등이 있으나, 이들의 부작용으로 인해 그 사용이 제한되고 있어서 새로운 항염증 제제의 개발이 요구되는 실정이다.Rheumatoid arthritis is a chronic inflammatory autoimmune disease characterized by synoviitis and the destruction of bone and cartilage as a result. The exact etiology has not been identified, but genetic predisposition and environmental stimulation trigger an autoimmune reaction, leading to autoantibody production, lubritis, and many immune cells and cytokines in this process have an organic relationship to each other, resulting in an inflammatory reaction. And exacerbate adjacent cartilage and bones. With the introduction of biological agents such as tumor necrosis factor (TNF) inhibitors, interleukin (IL) -6 blockers, and various immune cell inhibitors developed in view of this etiology, treatment for rheumatoid arthritis has changed dramatically. It was right. It showed excellent clinical effect in patients with insufficient response to the existing anti-rheumatic agents, and showed the protective effect of bone and cartilage against joint destruction. However, there are a small number of patients who do not respond to such biological agents, side effects such as infection or tumor risk, invasiveness and discomfort of the administration method requiring injection, and high drug prices. In contrast, small molecule inhibitors are mostly drugs that inhibit the intracellular signal transduction process or the binding action of chemokines and receptors. Representatively, Janus kinase (JAK) inhibitors and spleen tyrosine kinase (Syk) inhibitors are approved for clinical use. Or clinical studies are nearing completion. These drugs have the advantage of controlling arthritis, which is comparable to that of biologics, without the combined administration of methotrexate, and they can be administered orally, making it convenient to administer and producing these compounds at a relatively low cost. Currently, small molecule inhibitors such as the S1P lyase inhibitor LX3305 and the chemokine receptor inhibitor CCX354-C are under clinical studies, and new drugs are constantly being developed through pathogenesis-based studies. In addition, materials used as anti-inflammatory agents include ibuprofen and indomethacin in nonsteroidal systems, and dexamethasone in steroid systems, but their use is limited due to their side effects, which requires the development of new anti-inflammatory agents. This is true.
본 발명은 PI3KC2γ를 유효성분으로 포함하는 염증 질환 진단용 바이오마커 조성물, PI3KC2γ의 발현수준을 측정할 수 있는 제제를 유효성분으로 포함하는 염증 질환 진단용 조성물, PI3KC2γ 단백질 발현 또는 활성 억제제를 유효성분으로 포함하는 염증 질환 예방 또는 치료용 약학조성물, PI3KC2γ 단백질 발현 또는 활성 억제제를 유효성분으로 포함하는 염증 질환 예방 또는 개선용 건강기능식품 조성물 및 PI3KC2γ 단백질 발현 수준 측정을 통한 염증 질환 치료제 스크리닝 방법을 제공하고자 한다. The present invention is a biomarker composition for diagnosing inflammatory diseases comprising PI3KC2γ as an active ingredient, a composition for diagnosing an inflammatory disease comprising an agent capable of measuring the expression level of PI3KC2γ as an active ingredient, a PI3KC2γ protein expression or an activity inhibitor as an active ingredient It is intended to provide a pharmaceutical composition for preventing or treating inflammatory diseases, a health functional food composition for preventing or improving inflammatory diseases comprising PI3KC2γ protein expression or activity inhibitor as an active ingredient, and a screening method for treating inflammatory diseases through measurement of PI3KC2γ protein expression level.
상기 과제의 해결을 위해, 본 발명은 PI3KC2γ를 유효성분으로 포함하는 염증 질환 진단용 바이오마커 조성물을 제공한다. To solve the above problems, the present invention provides a biomarker composition for diagnosing inflammatory diseases comprising PI3KC2γ as an active ingredient.
또한, 본 발명은 PI3KC2γ의 발현수준을 측정할 수 있는 제제를 유효성분으로 포함하는 염증 질환 진단용 조성물을 제공한다.In addition, the present invention provides a composition for diagnosing an inflammatory disease comprising an agent capable of measuring the expression level of PI3KC2γ as an active ingredient.
또한, 본 발명은 PI3KC2γ의 발현 수준 측정을 통한 염증 질환 진단에 필요한 정보를 제공하는 방법을 제공한다. In addition, the present invention provides a method for providing information necessary for diagnosing inflammatory diseases through measuring the expression level of PI3KC2γ.
또한, 본 발명은 PI3KC2γ 단백질 발현 또는 활성 억제제를 유효성분으로 포함하는 염증 질환 예방 또는 치료용 약학조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising PI3KC2γ protein expression or activity inhibitor as an active ingredient.
또한, 본 발명은 PI3KC2γ 단백질의 발현 수준 측정을 통한 염증 질환 치료제 스크리닝 방법을 제공한다.In addition, the present invention provides a method for screening a therapeutic agent for an inflammatory disease by measuring the expression level of PI3KC2γ protein.
또한, 본 발명은 PI3KC2γ 단백질 발현 또는 활성 억제제를 유효성분으로 포함하는 염증 질환 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving an inflammatory disease comprising PI3KC2γ protein expression or activity inhibitor as an active ingredient.
본 발명은 PI3KC2γ를 표적으로 하는 염증 질환 진단 및 치료용 조성물에 관한 것으로서, 본 발명에 따른 PI3KC2γ 저해 활성을 갖는 PBT-6는 염증 유발성 사이토카인 등과 같은 다양한 염증 매개 물질들의 발현 및 활성을 억제시켜 우수한 항염증 효과를 나타냈을 뿐만 아니라, 이에 따른 유효한 치료효과를 콜라겐 유도 류마티스 관절염 동물모델에서 확인할 수 있었는 바, 기존의 염증성 질환 치료제를 대체할 수 있는 유효 물질로 활용될 수 있을 것이다.The present invention relates to a composition for diagnosing and treating inflammatory diseases targeting PI3KC2γ, and PBT-6 having PI3KC2γ inhibitory activity according to the present invention inhibits the expression and activity of various inflammatory mediators such as inflammatory cytokines Not only did it show an excellent anti-inflammatory effect, it was confirmed that an effective therapeutic effect according to this can be found in an animal model of collagen-induced rheumatoid arthritis, and could be used as an effective substance to replace the existing treatment for inflammatory diseases.
도 1은 골관절염 환자와 류마티스 관절염 환자의 활막액에서 염증 유발성 사이토 카인 (IL-6, TNF-α, IL-1β )및 PI3KC2γ의 발현량을 확인한 결과이다.
도 2는 골관절염 환자와 류마티스 관절염 환자의 활막 조직에서 H&E 염색과 PI3KC2γ를 IHC로 염색하여 발현량을 확인한 결과이다.
도 3은 건강한 사람과 류마티스 관절염 환자의 PBMC에서 PI3KC2γ의 단백질 발현량을 확인한 결과이다.
도 4는 PBT-6의 구조를 나타낸다.
도 5는 PBT-6의 PI3KC2γ 결합 억제 결과이다.
도 6은 PBT-6의 PI3 키나제 결합 분석 결과이다.
도 7은 류마티스 관절염 세포에서 PBT-6의 세포 사멸을 확인한 결과이다.
도 8은 류마티스 관절염 세포에서 PBT-6에 따른 IL-6의 분비량 변화를 확인한 결과이다.
도 9는 류마티스 관절염 세포에서 PBT-6에 따른 신호 전달물질(PI3KC2γ, p-AKT2, p-mTOR)의 발현량 변화를 확인한 결과이다.
도 10은 대식세포에서 PBT-6의 세포 사멸을 확인한 결과이다.
도 11은 대식세포에서 PBT-6에 따른 IL-6 및 TNF-α의 분비량 변화를 확인한 결과이다.
도 12는 대식세포에서 PBT-6에 따른 신호 전달물질 (PI3KC2γ, p-AKT2, p-mTOR)의 발현량 변화를 확인한 결과이다.
도 13은 대식세포에서 PBT-6에 따른 파골세포 분화 억제를 확인한 결과이다.
도 14는 대식세포에서 PBT-6에 따른 파골세포 관련 유전자 (NFATc1, RANKL)의 발현량 변화를 확인한 결과이다.
도 15는 대식세포와 류마티스 관절염 세포에서의 세포이동 실험을 보여주는 그림이다.
도 16은 대식세포에서 PBT-6에 따른 세포이동의 변화를 확인한 결과이다.
도 17은 콜라겐 유도 류마티스 관절염 동물모델에서 PBT-6의 처리에 따른 발바닥의 부종 변화를 보여주는 사진이다.
도 18은 콜라겐 유도 류마티스 관절염 동물모델에서 PBT-6의 처리에 따른 관절염 진행지수의 변화를 확인한 결과이다.
도 19는 콜라겐 유도 류마티스 관절염 동물모델에서 PBT-6의 처리에 따른 발바닥 두께의 변화를 확인한 결과이다.
도 20은 콜라겐 유도 류마티스 관절염 동물모델에서 PBT-6의 처리에 따른 IL-6 및 TNF-α의 분비량 변화를 확인한 결과이다.
도 21은 콜라겐 유도 류마티스 관절염 동물모델에서 PBT-6의 처리에 따른 H&E staining과 PI3KC2γ, p-AKT2를 IHC 로 염색하여 발현량을 확인한 결과이다.
도 22는 콜라겐 유도 류마티스 관절염 동물모델에서 PBT-6의 처리에 따른 병리학적 요인들의 변화를 확인한 결과이다.
도 23은 콜라겐 유도 류마티스 관절염 동물모델에서 PBT-6의 처리 후 발목과 손가락 관절을 micro-CT로 관찰한 결과이다.
도 24는 콜라겐 유도 류마티스 관절염 동물모델에서 PBT-6의 처리 후 발목과 손가락 관절의 병적 요인을 확인한 결과이다.1 is a result of confirming the expression levels of inflammation-causing cytokines (IL-6, TNF-α, IL-1β) and PI3KC2γ in synovial fluid in patients with osteoarthritis and rheumatoid arthritis.
2 is a result of confirming the expression level by staining H & E and PI3KC2γ with IHC in synovial tissue of osteoarthritis patients and rheumatoid arthritis patients.
3 is a result of confirming the protein expression level of PI3KC2γ in PBMC of healthy people and rheumatoid arthritis patients.
4 shows the structure of PBT-6.
5 is a result of inhibiting PI3KC2γ binding of PBT-6.
Figure 6 is the result of PBT-6 PI3 kinase binding analysis.
7 is a result of confirming the cell death of PBT-6 in rheumatoid arthritis cells.
8 is a result of confirming the change in the amount of IL-6 secretion according to PBT-6 in rheumatoid arthritis cells.
9 is a result of confirming a change in expression level of a signal transducer (PI3KC2γ, p-AKT2, p-mTOR) according to PBT-6 in rheumatoid arthritis cells.
10 is a result of confirming the cell death of PBT-6 in macrophages.
11 is a result of confirming the change in the secretion amount of IL-6 and TNF-α according to PBT-6 in macrophages.
12 is a result of confirming a change in expression level of a signal transducer (PI3KC2γ, p-AKT2, p-mTOR) according to PBT-6 in macrophages.
13 is a result confirming the inhibition of osteoclast differentiation according to PBT-6 in macrophages.
14 is a result of confirming the change in expression level of osteoclast-related genes (NFATc1, RANKL) according to PBT-6 in macrophages.
15 is a diagram showing a cell migration experiment in macrophages and rheumatoid arthritis cells.
16 is a result of confirming the change in cell migration according to PBT-6 in macrophages.
17 is a photograph showing the change in the swelling of the sole according to the treatment of PBT-6 in an animal model of collagen induced rheumatoid arthritis.
18 is a result of confirming a change in the arthritis progression index according to the treatment of PBT-6 in an animal model of collagen induced rheumatoid arthritis.
19 is a result of confirming the change in the thickness of the sole according to the treatment of PBT-6 in an animal model of collagen induced rheumatoid arthritis.
20 is a result of confirming the change in the secretion amount of IL-6 and TNF-α according to the treatment of PBT-6 in an animal model of collagen induced rheumatoid arthritis.
21 is a result of confirming the expression level by staining H & E staining and PI3KC2γ, p-AKT2 according to the treatment of PBT-6 and collagen-induced rheumatoid arthritis with IHC.
22 is a result of confirming the change of pathological factors according to the treatment of PBT-6 in an animal model of collagen induced rheumatoid arthritis.
23 is a result of observing the ankle and finger joints by micro-CT after treatment of PBT-6 in an animal model of collagen induced rheumatoid arthritis.
24 is a result of confirming the pathological factors of the ankle and finger joints after treatment of PBT-6 in an animal model of collagen induced rheumatoid arthritis.
이에, 본 발명자들은 PI3K classⅡγ가 류마티스 관절염을 가진 환자의 활막액과 활막 조직에서 많이 발현되는 것을 확인하였고, 선택적인 PI3K classⅡγ 억제제인 PBT-6가 류마티스 관절염 세포인 MH7A cell과 대식세포인 Raw 264.7 cell에서 세포사멸 효과를 가져다 주는 것을 확인하였다. 또한 PBT-6에 의해서 파골세포의 분화가 억제되는 것을 확인하였고, 세포이동을 현저히 감소시키는 것을 보여주었다. 또한 콜라겐 유도 류마티스 관절염 동물모델 (CIA mouse)에서 발목의 붓기나 부종, 관절의 뒤틀림 정도가 PBT-6에 의해 현저히 감소 되는 것을 확인하고, 본 발명을 완성하였다.Thus, the present inventors confirmed that PI3K classIIγ is highly expressed in synovial fluid and synovial tissue of patients with rheumatoid arthritis, and the selective PI3K classIIγ inhibitor, PBT-6, is a rheumatoid arthritis cell MH7A cell and a macrophage raw 264.7 cell. It was confirmed that it brings apoptosis effect. In addition, it was confirmed that differentiation of osteoclasts was inhibited by PBT-6, and it was shown that cell migration was significantly reduced. In addition, in the animal model of collagen-induced rheumatoid arthritis (CIA mouse), it was confirmed that the degree of swelling, swelling of the ankle, and joint distortion was significantly reduced by PBT-6, and the present invention was completed.
본 발명은 PI3KC2γ를 유효성분으로 포함하는 염증 질환 진단용 바이오마커 조성물을 제공한다. The present invention provides a biomarker composition for diagnosing inflammatory diseases comprising PI3KC2γ as an active ingredient.
본 명세서에서 용어 “진단”은 특정 질병 또는 질환에 대한 한 객체의 감수성(susceptibility)을 판정하는 것, 한 객체가 특정 질병 또는 질환을 현재 가지고 있는지 여부를 판정하는 것, 특정 질병 또는 질환에 걸린 한 객체의 예후(prognosis)를 판정하는 것, 또는 테라메트릭스(therametrics)(예컨대, 치료 효능에 대한 정보를 제공하기 위하여 객체의 상태를 모니터링하는 것)을 포함한다.The term “diagnosis” herein refers to determining the susceptibility of an object to a particular disease or condition, determining whether an object currently has a specific disease or condition, as long as the disease or condition is affected Determining the prognosis of the object, or terrametrics (eg, monitoring the condition of the object to provide information about treatment efficacy).
또한, 본 발명은 PI3KC2γ의 발현수준을 측정할 수 있는 제제를 유효성분으로 포함하는 염증 질환 진단용 조성물을 제공한다. In addition, the present invention provides a composition for diagnosing an inflammatory disease comprising an agent capable of measuring the expression level of PI3KC2γ as an active ingredient.
상세하게는, 상기 PI3KC2γ의 발현수준을 측정할 수 있는 제제는 상기 PI3KC2γ 유전자에 특이적으로 결합하는 프라이머 또는 프로브, 상기 PI3KC2γ 단백질에 특이적으로 결합하는 항체, 펩타이드, 앱타머 또는 화합물일 수 있으나, 이에 한정되는 것은 아니다. Specifically, the agent capable of measuring the expression level of PI3KC2γ may be a primer or probe specifically binding to the PI3KC2γ gene, an antibody, peptide, aptamer or compound specifically binding to the PI3KC2γ protein, It is not limited to this.
상세하게는, 상기 염증 질환은 류마티스 관절염, 염증성 장질환 또는 췌장염일 수 있으나, 이에 한정되는 것은 아니다.Specifically, the inflammatory disease may be rheumatoid arthritis, inflammatory bowel disease or pancreatitis, but is not limited thereto.
또한, 본 발명은 상기 조성물을 포함하는 염증 질환 진단용 키트를 제공한다.In addition, the present invention provides a kit for diagnosing inflammatory diseases comprising the composition.
본 명세서에서 용어 "프라이머"는 짧은 자유 3-말단 수산화기(free 3' hydroxyl group)를 가지는 핵산 서열로 상보적인 템플레이트(template)와 염기쌍을 형성할 수 있고 템플레이트 가닥 복사를 위한 시작 지점으로서 작용하는 짧은 핵산 서열을 말한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응을 위한 시약(즉, DNA 폴리머라제 또는 역전사효소) 및 상이한 4 가지의 뉴클레오사이드 트리포스페이트의 존재하에서 DNA 합성을 개시할 수 있다. PCR 조건, 센스 및 안티센스 프라이머의 길이는 당업계에 공지된 기술에 따라 적절히 선택될 수 있다.The term "primer" herein is a short nucleic acid sequence having a short free 3-terminal hydroxyl group, capable of forming complementary templates and base pairs and acting as a starting point for template strand copying. Refers to the nucleic acid sequence. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. PCR conditions, sense and antisense primer length can be appropriately selected according to techniques known in the art.
본 명세서에서 용어 "프로브"는 mRNA와 특이적으로 결합을 이룰 수 있는 짧게는 수 염기 내지 길게는 수백 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하며 라벨링되어 있어서 특정 mRNA의 존재 유무, 발현양을 확인할 수 있다. 프로브는 올리고뉴클레오타이드(oligonucleotide) 프로브, 단쇄 DNA(single strand DNA) 프로브, 이중쇄DNA(double strand DNA)프로브, RNA 프로브 등의 형태로 제작될 수 있다. 적절한 프로브의 선택 및 혼성화 조건은 당해 기술 분야에 공지된 기술에 따라 적절히 선택할 수 있다.As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to short to several bases to hundreds of bases, which is capable of specifically binding to mRNA, and is labeled to determine whether or not a specific mRNA exists or not. You can check the amount. The probe may be produced in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, or an RNA probe. The appropriate probe selection and hybridization conditions may be appropriately selected according to techniques known in the art.
본 명세서에서 용어 "항체"는 당해 기술분야에 공지된 용어로서 항원성 부위에 대하여 지시되는 특이적인 면역 글로불린을 의미한다. 본 발명에서의 항체는 본 발명의 PI3KC2γ에 대해 특이적으로 결합하는 항체를 의미하며, 당해 기술분야의 통상적인 방법에 따라 항체를 제조할 수 있다. 상기 항체의 형태는 폴리클로날 항체 또는 모노클로날 항체를 포함하며, 모든 면역글로불린 항체가 포함된다. 상기 항체는 2개의 전체 길이의 경쇄 및 2 개의 전체 길이의 중쇄를 갖는 완전한 형태를 의미한다. 또한, 상기 항체는 인간화 항체 등의 특수 항체도 포함된다.The term "antibody" as used herein refers to a specific immunoglobulin directed against an antigenic site as a term known in the art. The antibody in the present invention means an antibody that specifically binds to PI3KC2γ of the present invention, and the antibody can be prepared according to a conventional method in the art. The form of the antibody includes a polyclonal antibody or a monoclonal antibody, and all immunoglobulin antibodies are included. The antibody refers to a complete form with two full-length light chains and two full-length heavy chains. In addition, the antibody also includes special antibodies such as humanized antibodies.
또한 본 발명의 키트는 마커 성분에 특이적으로 결합하는 항체, 기질과의 반응에 의해서 발색하는 표지체가 접합된 2차 항체 접합체(conjugate), 상기 표지체와 발색 반응할 발색 기질 용액, 세척액 및 효소반응 정지용액 등을 포함할 수 있으며, 사용되는 시약 성분을 포함하는 다수의 별도 패키징 또는 컴파트먼트로 제작될 수 있다.In addition, the kit of the present invention is an antibody that specifically binds to a marker component, a secondary antibody conjugate conjugated with a marker to be colored by reaction with a substrate, a chromogenic substrate solution to react with the marker, a washing solution, and an enzyme. It may include a reaction stop solution and the like, and may be manufactured in a number of separate packaging or compartments containing reagent components used.
본 명세서에서 용어 "펩타이드"는 표적 물질에 대한 결합력 높은 장점이 있으며, 열/화학 처리시에도 변성이 일어나지 않는다. 또한 분자 크기가 작기 때문에 다른 단백질에 붙여서 융합 단백질로의 이용이 가능하다. 구체적으로 고분자 단백질 체인에 붙여서 이용이 가능하므로 진단 키트 및 약물전달 물질로 이용될 수 있다. The term "peptide" in this specification has an advantage of high binding to a target material, and does not denature even when subjected to heat / chemical treatment. In addition, because the molecular size is small, it can be used as a fusion protein by attaching it to other proteins. Specifically, it can be used by attaching it to a polymer protein chain, so it can be used as a diagnostic kit and a drug delivery material.
본 명세서에서 용어 "앱타머(aptamer)"란, 그 자체로 안정된 삼차 구조를 가지면서 표적 분자에 높은 친화성과 특이성으로 결합할 수 있는 특징을 가진 특별한 종류의 단일가닥 핵산(DNA, RNA 또는 변형핵산)으로 구성된 폴리뉴클레오티드의 일종을 의미한다. 상술한 바와 같이, 앱타머는 항체와 동일하게 항원성 물질에 특이적으로 결합할 수 있으면서도, 단백질보다 안정성이 높고, 구조가 간단하며, 합성이 용이한 폴리뉴클레오티드로 구성되어 있으므로, 항체를 대체하여 사용될 수 있다.As used herein, the term "aptamer" is a special kind of single-stranded nucleic acid (DNA, RNA or modified nucleic acid) that has a stable tertiary structure in itself and has the characteristics of being capable of binding to a target molecule with high affinity and specificity. ) Is a kind of polynucleotide. As described above, aptamers are capable of specifically binding to antigenic substances in the same manner as antibodies, but are composed of polynucleotides that are more stable than proteins, have simple structures, and are easy to synthesize. You can.
또한, (1) 환자에서 분리된 시료로부터 PI3KC2γ 유전자의 mRNA 발현 수준 또는 PI3KC2γ 단백질의 발현 수준을 측정하는 단계; (2) 상기 PI3KC2γ 유전자의 mRNA 발현 수준 또는 PI3KC2γ 단백질의 발현 수준을 대조군 시료와 비교하는 단계; 및 (3) 상기 PI3KC2γ 유전자의 mRNA 발현 수준 또는 PI3KC2γ 단백질의 발현 수준이 대조군 시료보다 높을 경우 염증 질환으로 판단하는 단계를 포함하는 염증 질환 진단에 필요한 정보를 제공하는 방법을 제공한다. In addition, (1) measuring the mRNA expression level of the PI3KC2γ gene or the expression level of the PI3KC2γ protein from the sample isolated from the patient; (2) comparing the mRNA expression level of the PI3KC2γ gene or the expression level of the PI3KC2γ protein with a control sample; And (3) provides a method for providing information necessary for diagnosing an inflammatory disease, including determining the inflammatory disease when the mRNA expression level of the PI3KC2γ gene or the expression level of the PI3KC2γ protein is higher than the control sample.
상세하게는, 상기 염증 질환은 류마티스 관절염, 염증성 장질환 또는 췌장염일 수 있으나, 이에 한정되는 것은 아니다.Specifically, the inflammatory disease may be rheumatoid arthritis, inflammatory bowel disease or pancreatitis, but is not limited thereto.
상세하게는, 상기 mRNA 발현 수준을 측정하는 방법은 RT-PCR, 경쟁적 RT-PCR(Competitive RT-PCR), 실시간 RT-PCR (Real-time RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블랏팅 (Northern blotting) 및 DNA 칩을 이용하지만, 이에 한정되는 것은 아니다.Specifically, the method for measuring the mRNA expression level is RT-PCR, competitive RT-PCR (RT-PCR), real-time RT-PCR (RT-PCR), RNase protection assay (RPA) ), Northern blotting and DNA chips are used, but are not limited thereto.
상세하게는, 상기 단백질 발현 수준을 측정하는 방법은 웨스턴 블랏, ELISA(enzyme linked immunosorbent asay), 방사선면역분석(Radioimmunoassay; RIA), 방사면역확산법(radioimmunodiffusion), 오우크테로니(Ouchterlony) 면역 확산법, 로케이트(rocket) 면역전기영동, 조직면역염색, 면역침전 분석법(Immunoprecipitation assay), 보체고정분석법 (Complement Fixation Assay), FACS 및 단백질 칩을 이용하지만, 이에 한정되는 것은 아니다.Specifically, the method for measuring the protein expression level is Western blot, ELISA (enzyme linked immunosorbent asay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion method, Rocket (rocket) immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip are used, but are not limited thereto.
본 명세서에서 용어 "환자에서 분리된 시료"란 염증 질환 진단용 바이오 마커인 상기 PI3KC2γ 유전자 또는 PI3KC2γ 단백질의 발현 수준에 있어서 대조군과 차이가 나는 조직, 세포, 전혈, 혈청, 혈장, 타액, 객담, 뇌척수액, 또는 뇨와 같은 시료를 포함하지만, 이에 한정되는 것은 아니다. The term "sample isolated from a patient" herein refers to tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, which are different from the control in the expression level of the PI3KC2γ gene or PI3KC2γ protein, which is a biomarker for diagnosing inflammatory diseases, Or a sample such as urine, but is not limited thereto.
또한, 본 발명은 PI3KC2γ 단백질 발현 또는 활성 억제제를 유효성분으로 포함하는 염증 질환 예방 또는 치료용 약학조성물을 제공한다. In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising PI3KC2γ protein expression or activity inhibitor as an active ingredient.
상세하게는, 상기 PI3KC2γ 단백질 발현 억제제는 PI3KC2γ 유전자의 mRNA에 상보적으로 결합하는 안티센스 뉴클레오타이드, 작은 간섭 RNA(small interfering RNA; siRNA) 또는 짧은 헤어핀 RNA(short hairpin RNA; shRNA)일 수 있고, 상기 PI3KC2γ 단백질 활성 억제제는 PI3KC2γ단백질에 특이적으로 결합하는 화합물, 펩티드, 펩티드 미메틱스, 앱타머, 항체 또는 천연물일 수 있으나, 이에 한정되는 것은 아니다.Specifically, the PI3KC2γ protein expression inhibitor may be an antisense nucleotide that complementarily binds to the mRNA of the PI3KC2γ gene, small interfering RNA (siRNA), or short hairpin RNA (shRNA), and the PI3KC2γ The protein activity inhibitor may be, but is not limited to, a compound, peptide, peptide mimetics, aptamer, antibody or natural product that specifically binds to the PI3KC2γ protein.
보다 상세하게는, 상기 화합물은 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염일 수 있으나, 이에 한정되는 것은 아니다.More specifically, the compound may be a compound represented by
[화학식 1][Formula 1]
상기 화학식 1에서, In
R1 및 R2는 각각 같거나 다를 수 있으며, 수소, (C1~C4)알킬, (C1~C4)알콕시 및 할로겐으로 이루어진 군에서 선택되는 어느 하나임.R 1 and R 2 may each be the same or different, and are any one selected from the group consisting of hydrogen, (C1 to C4) alkyl, (C1 to C4) alkoxy and halogen.
보다 더 상세하게는, 상기 화합물은 하기 화학식 2로 표시되는 화합물일 수 있으나, 이에 한정되는 것은 아니다.In more detail, the compound may be a compound represented by
[화학식 2][Formula 2]
상세하게는, 상기 염증 질환은 류마티스 관절염, 염증성 장질환 또는 췌장염일 수 있으나, 이에 한정되는 것은 아니다.Specifically, the inflammatory disease may be rheumatoid arthritis, inflammatory bowel disease or pancreatitis, but is not limited thereto.
한편, 본 명세서에서는 상기 화학식 2로 표시되는 화합물을 6-(4-피리디닐)-2-벤조티아졸라민(6-(4-pyridinyl)-2-benzothiazolamine; PBT-6)으로도 명명하였다.Meanwhile, in the present specification, the compound represented by
본 발명의 약학 조성물은 화학물질, 뉴클레오타이드, 안티센스, siRNA 올리고뉴클레오타이드 및 천연물 추출물을 유효성분으로 포함할 수 있다. 본 발명의 약학 조성물 또는 복합 제제는 유효 성분 이외에 약제학적으로 적합하고 생리학적으로 허용되는 보조제를 사용하여 제조될 수 있으며, 상기 보조제로는 부형제, 붕해제, 감미제, 결합제, 피복제, 팽창제, 윤활제, 활택제 또는 향미제 등의 가용화제를 사용할 수 있다. 본 발명의 약학 조성물은 투여를 위해서 유효 성분 이외에 추가로 약제학적으로 허용 가능한 담체를 1 종 이상 포함하여 약학 조성물로 바람직하게 제제화할 수 있다. 액상 용액으로 제제화되는 조성물에 있어서 허용 가능한 약제학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. The pharmaceutical composition of the present invention may include chemicals, nucleotides, antisense, siRNA oligonucleotides, and natural product extracts as active ingredients. The pharmaceutical composition or combination preparation of the present invention may be prepared using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the active ingredients, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, expanders, lubricants , Solubilizing agents such as lubricants or flavoring agents. The pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. Acceptable pharmaceutical carriers in compositions formulated as liquid solutions include, as sterile and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets.
본 발명의 약학 조성물의 약제 제제 형태는 과립제, 산제, 피복정, 정제, 캡슐제, 좌제, 시럽, 즙, 현탁제, 유제, 점적제 또는 주사 가능한 액제 및 활성 화합물의 서방출형 제제 등이 될 수 있다. 본 발명의 약학 조성물은 정맥내, 동맥내, 복강내, 근육내, 동맥내, 복강내, 흉골내, 경피, 비측내, 흡입, 국소, 직장, 경구, 안구내 또는 피내 경로를 통해 통상적인 방식으로 투여할 수 있다. 본 발명의 약학 조성물의 유효성분의 유효량은 질환의 예방 또는 치료 요구되는 양을 의미한다. 따라서, 질환의 종류, 질환의 중증도, 조성물에 함유된 유효 성분 및 다른 성분의 종류 및 함량, 제형의 종류 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료 기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다. 이에 제한되는 것은 아니나, 예컨대, 성인의 경우, 1일 1회 내지 수회 투여시, 본 발명의 조성물은 1일 1회 내지 수회 투여시, 화합물일 경우 0.1ng/kg~10g/kg, 폴리펩타이드, 단백질 또는 항체일 경우 0.1ng/kg~10g/kg, 안티센스 뉴클레오타이드, siRNA, shRNAi, miRNA일 경우 0.01ng/kg~10g/kg의 용량으로 투여할 수 있다. Pharmaceutical formulation forms of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained release formulations of active compounds, etc. You can. The pharmaceutical composition of the present invention is a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. Can be administered. The effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for prevention or treatment of diseases. Thus, the type of disease, the severity of the disease, the type and content of active and other ingredients contained in the composition, the type of formulation and the patient's age, weight, general health status, gender and diet, time of administration, route of administration and composition It can be adjusted according to various factors, including the rate of secretion, the duration of treatment, and drugs used simultaneously. Without being limited thereto, for example, in the case of an adult, once to several times a day, the composition of the present invention is administered once to several times a day, when the compound is 0.1ng / kg to 10g / kg, a polypeptide, In the case of protein or antibody, 0.1ng / kg ~ 10g / kg, antisense nucleotide, siRNA, shRNAi, miRNA may be administered at a dose of 0.01ng / kg ~ 10g / kg.
또한, 본 발명은 (1) 염증 질환 동물 모델에 시험물질을 접촉시키는 단계; (2) 상기 시험물질을 접촉한 염증 질환 동물 모델에서 PI3KC2γ 단백질의 발현 또는 활성 정도를 측정하는 단계; 및 (3) 대조군 시료와 비교하여 상기 PI3KC2γ 단백질의 발현 또는 활성 정도가 감소한 시험물질을 선별하는 단계를 포함하는 염증 질환 치료제 스크리닝 방법을 제공한다. In addition, the present invention (1) contacting the test substance to the inflammatory disease animal model; (2) measuring the expression or activity level of PI3KC2γ protein in an animal model of inflammatory disease in contact with the test substance; And (3) provides a screening method for the treatment of inflammatory diseases comprising the step of selecting a test substance having a reduced expression or activity level of the PI3KC2γ protein compared to the control sample.
상세하게는, 상기 염증 질환은 류마티스 관절염, 염증성 장질환 또는 췌장염일 수 있으나, 이에 한정되는 것은 아니다.Specifically, the inflammatory disease may be rheumatoid arthritis, inflammatory bowel disease or pancreatitis, but is not limited thereto.
본 발명의 스크리닝 방법을 언급하면서 사용되는 용어 "시험물질"은 유전자의 발현량에 영향을 미치거나, 단백질의 발현 또는 활성에 영향을 미치는지 여부를 검사하기 위하여 스크리닝에서 이용되는 미지의 후보 물질을 의미한다. 상기 시료는 화학물질, 뉴클레오타이드, 안티센스-RNA, siRNA(small interference RNA) 및 천연물 추출물을 포함하나, 이에 제한되는 것은 아니다. The term "test substance" used while referring to the screening method of the present invention means an unknown candidate substance used in screening to test whether it affects the expression level of a gene or the expression or activity of a protein. do. The sample includes, but is not limited to, chemicals, nucleotides, antisense-RNA, small interference RNA (siRNA), and natural product extracts.
또한, 본 발명은 PI3KC2γ 단백질 발현 또는 활성 억제제를 유효성분으로 포함하는 염증 질환 예방 또는 개선용 건강기능식품 조성물을 제공한다. In addition, the present invention provides a health functional food composition for preventing or improving an inflammatory disease comprising PI3KC2γ protein expression or activity inhibitor as an active ingredient.
상세하게는, 상기 PI3KC2γ 단백질 발현 또는 활성 억제제는 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염일 수 있으나, 이에 한정되는 것은 아니다.Specifically, the PI3KC2γ protein expression or activity inhibitor may be a compound represented by
[화학식 1][Formula 1]
상기 화학식 1에서, In
R1 및 R2는 각각 같거나 다를 수 있으며, 수소, (C1~C4)알킬, (C1~C4)알콕시 및 할로겐으로 이루어진 군에서 선택되는 어느 하나임.R 1 and R 2 may each be the same or different, and are any one selected from the group consisting of hydrogen, (C1 to C4) alkyl, (C1 to C4) alkoxy and halogen.
보다 더 상세하게는, 상기 화합물은 하기 화학식 2로 표시되는 화합물일 수 있으나, 이에 한정되는 것은 아니다.In more detail, the compound may be a compound represented by
[화학식 2][Formula 2]
상세하게는, 상기 염증 질환은 류마티스 관절염, 염증성 장질환 또는 췌장염일 수 있으나, 이에 한정되는 것은 아니다.Specifically, the inflammatory disease may be rheumatoid arthritis, inflammatory bowel disease or pancreatitis, but is not limited thereto.
본 발명의 조성물이 건강기능식품 조성물인 경우, 건강기능식품 조성물은 분말, 과립, 정제, 캡슐, 시럽 또는 음료의 형태로 제공될 수 있으며, 상기 건강기능식품 조성물은 유효성분 이외에 다른 식품 또는 식품 첨가물과 함께 사용되고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 그의 사용 목적 예를 들어 예방, 건강 또는 치료적 처치에 따라 적합하게 결정될 수 있다.When the composition of the present invention is a health functional food composition, the health functional food composition may be provided in the form of a powder, granule, tablet, capsule, syrup or beverage, and the health functional food composition is other food or food additives other than the active ingredient It can be used in conjunction with, and can be suitably used according to conventional methods. The mixing amount of the active ingredient may be appropriately determined according to its purpose of use, for example, prophylactic, health or therapeutic treatment.
상기 건강기능식품 조성물에 함유된 유효성분의 유효용량은 상기 약학조성물의 유효용량에 준해서 사용할 수 있으나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로도 사용될 수 있음은 확실하다.The effective dose of the active ingredient contained in the dietary supplement composition can be used in accordance with the effective dose of the pharmaceutical composition, but in the case of long-term intake for health and hygiene purposes or for health control purposes, the effective dose is below the above range. It can be, it is clear that the active ingredient can be used in an amount above the range because there is no problem in terms of safety.
상기 건강식품의 종류에는 특별한 제한이 없고, 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등을 들 수 있다.There are no particular restrictions on the type of the health food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinks, tea, Drinks, alcoholic beverages, and vitamin complexes.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be described in detail to help understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.
<< 실험예Experimental example >>
하기의 실험예들은 본 발명에 따른 각각의 실시예에 공통적으로 적용되는 실험예를 제공하기 위한 것이다. The following experimental examples are intended to provide an experimental example commonly applied to each embodiment according to the present invention.
1. 시약 및 항체1. Reagents and antibodies
PI3KC2γ에 대한 1차 항체는 Thermo Fisher Scientific (Rockford, IL, USA)으로부터, p-AKT2 (S474), p-mTOR 및 β-actin은 Cell Signaling Technology (Danvers, MA, USA)로부터, RANKL은 Abcam (Cambridge, MA, UK)으로부터, NFATc1는 Santa Cruz Biotechnology (Dallas, CA, USA)로부터 구입하였다. RANKL 및 M-CSF는 R&D systems (Minneapolis, MN, USA)으로부터 얻었다. 6-(4-피리디닐)-2-벤조티아졸라민(6-(4-pyridinyl)-2-benzothiazolamine; PBT-6)은 성균관대학교 약학대학에서 합성하였다. 1차 단계는 온화한 조건하에서 피리딘(pyridine) 및 아세트산 무수물(acetic anhydride)로 NH2를 아세틸화시켰다. 2차 단계는 포스핀 리간드, 염기 및 Pd 촉매를 이용하여, Suzuki 반응을 통해 브로모 벤조티아졸(bromo benzothiaol) 및 보로닉산(boronic acid)을 커플링시켰다. 마지막 단계는 이를 NaOH 용액에서 가수분해시켰다. 6-(pyridin-4-yl) benzo[d]thiazol-2-amine: 1H NMR (400 MHz, DMSO): 8.58 (d, J = 6, 2H-Ar), 8.18 (d, J = 1.6, 1H-Ar), 7.70-7.66 (m, 5H), 7.42 (d, J = 8, 1H-Ar).Primary antibodies against PI3KC2γ from Thermo Fisher Scientific (Rockford, IL, USA), p-AKT2 (S474), p-mTOR and β-actin from Cell Signaling Technology (Danvers, MA, USA), RANKL from Abcam ( Cambridge, MA, UK), NFATc1 was purchased from Santa Cruz Biotechnology (Dallas, CA, USA). RANKL and M-CSF were obtained from R & D systems (Minneapolis, MN, USA). 6- (4-pyridinyl) -2-benzothiazolamine (6- (4-pyridinyl) -2-benzothiazolamine; PBT-6) was synthesized by Sungkyunkwan University College of Pharmacy. The first step was acetylation of NH 2 with pyridine and acetic anhydride under mild conditions. In the second step, bromo benzothiaol and boronic acid were coupled through a Suzuki reaction using a phosphine ligand, a base, and a Pd catalyst. The last step was to hydrolyze it in NaOH solution. 6- (pyridin-4-yl) benzo [d] thiazol-2-amine: 1H NMR (400 MHz, DMSO): 8.58 (d, J = 6, 2H-Ar), 8.18 (d, J = 1.6, 1H -Ar), 7.70-7.66 (m, 5H), 7.42 (d, J = 8, 1H-Ar).
2. 세포 배양2. Cell culture
MH7A 세포는 Riken cell bank (Tsukuba, Japan)로부터 구입하였고, 10% 열-불활화된 우태아혈청(fetal bovine serum; FBS) 및 1% 페니실린/스트렙토마이신(penicillin/streptomycin)이 첨가된 RPMI1640 배지에서 배양하였다. Raw 264.7 세포는 Korean Cell Line Bank로부터 구입하였고, 10% 열-불활화된 우태아혈청(fetal bovine serum; FBS) 및 1% 페니실린/스트렙토마이신(penicillin/streptomycin)이 첨가된 Dulbecco's Modified Eagle's Medium (DMEM) 배지에서 배양하였다. 세포 배양을 위한 FBS 및 모든 다른 시약은 Invitrogen (Carlsbad, CA, USA)으로부터 구입하였다. 95% air 및 5% CO2 조성으로 습도가 조절되는 배양기에서 세포 배양을 37℃로 유지하였다.MH7A cells were purchased from Riken cell bank (Tsukuba, Japan) and in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin / streptomycin. Cultured. Raw 264.7 cells were purchased from Korean Cell Line Bank, and Dulbecco's Modified Eagle's Medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin / streptomycin added ) Cultured in the medium. FBS and all other reagents for cell culture were purchased from Invitrogen (Carlsbad, CA, USA). The cell culture was maintained at 37 ° C. in an incubator with humidity control of 95% air and 5% CO 2 composition.
3. 세포 3. Cell 생존능Viability 측정 Measure
세포 생존능은 MTT 분석을 사용하여 측정하였다. 간단히 설명하면, 세포는 96-웰 플레이트에서 8×103 cells/well의 밀도로 접종한 후, 밤새도록 배양하였다. 다음 날, 배지를 제거하였고 세포들을 유도체인 TNF-α (50 ng/mL) 또는 LPS (1 μg/mL) 중 하나와, 다양한 농도의 PBT-6 (0.1-100 μM)로 처리하였다. 72시간 배양 후, 10% MTT 용액 (2 mg/mL)을 각 웰에 첨가하였고, 세포들을 37℃에서 4시간 동안 배양하였다. 얻어낸 포르마잔 결정은 DMSO (100 μL/well)에서 5분 동안 일정하게 흔들어 녹였다. 그 후, 플레이트의 흡광도를 540 nm에서 microplate reader로 측정하였다. 각 분석에 대해서 3번 반복하여 측정하였다.Cell viability was measured using MTT assay. Briefly, cells were inoculated at a density of 8 × 10 3 cells / well in a 96-well plate, and then cultured overnight. The next day, the medium was removed and cells were treated with either the derivative TNF-α (50 ng / mL) or LPS (1 μg / mL) and various concentrations of PBT-6 (0.1-100 μM). After incubation for 72 hours, 10% MTT solution (2 mg / mL) was added to each well, and the cells were incubated at 37 ° C for 4 hours. The obtained formazan crystals were dissolved in DMSO (100 μL / well) with constant shaking for 5 minutes. Thereafter, the absorbance of the plate was measured at 540 nm with a microplate reader. Each analysis was repeated three times.
4. 4. 웨스턴Western 블랏팅Blotting (Western blotting)(Western blotting)
프로테아제 및 포스파타아제 억제제가 포함된 용해 완충액에서 용해시키기 전, 세포들을 DPBS로 씻어냈다. 8, 12% 소듐 도데실 설페이트(sodium dodecyl sulfate; SDS)-폴리아크릴아미드 젤 전기영동을 사용하여, 동등한 양의 단백질을 분리하였고, 폴리비닐리덴 플루오라이드 멤브레인 상으로 옮겼다. Ponceau S staining solution (Sigma Aldrich, St. Louis, MO, USA)을 사용하여, 단백질 이동을 확인하였다. 그 후, 블랏들을 적절한 1차 항체(1:1000)로 면역염색한 후, 겨자무 퍼록시다아제(horseradish peroxidase)가 접합된 적절한 2차 항체(1:5000)로 반응시켰다. 관심 단백질에 특이적인 1차 항체가 사용되었고, enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, UK)를 통해, X-Ray 필름을 사용하여 검출하였다. Cells were washed with DPBS prior to lysis in lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein were separated using 8, 12% sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. Protein migration was confirmed using Ponceau S staining solution (Sigma Aldrich, St. Louis, MO, USA). Thereafter, the blots were immunostained with an appropriate primary antibody (1: 1000), and then reacted with an appropriate secondary antibody (1: 5000) conjugated with horseradish peroxidase. Primary antibodies specific to the protein of interest were used and were detected using X-Ray film via enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, UK).
5. 효소-결합 면역흡착 분석(Enzyme-linked immunosorbent assay)5. Enzyme-linked immunosorbent assay
모든 환자로부터 동의서를 받았고, 실험 프로토콜은 인하대병원 윤리위원회로부터 승인 받았다(INHAUH IRB 2015-09-016-002). 건강한 지원자(n = 8), 골관절염 환자(n = 9) 및 류마티스 관절염 환자(n = 9)로부터 수집한 말초 혈액 및 윤활액(synovial fluid)을 기록하였고, ELISA 키트는 R&D systems (Minneapolis, MN, USA)으로부터 구입하였으며, 사이토카인 수준은 혈청 또는 윤활액으로부터 제조사의 지시에 따라 측정하였다. 간단히 설명하면, 각 웰은 0.1 g의 TNF-α, IL-1β 및 IL-6로 24시간 동안 코팅하였고, 2% BSA가 포함된 PBS로 1시간 동안 상온에서 차단시켰다. 혈청 또는 윤활액을 각 웰에 첨가하였고, 4℃에서 24시간 동안 반응시켰다. 0.1% Tween 20이 포함된 PBS로 플레이트를 씻어낸 후, 비오틴화된 항체를 각 웰에 첨가하였고, 스트렙타비딘(streptavidin)-HRP로 반응시켰다. 세척 완충액으로 플레이트를 씻어낸 후, 3, 3', 5, 5' - 테트라메틸벤지딘(3, 3', 5, 5' - tetramethylbenzidine) 용액을 20분 동안 첨가하였다. 1 N 황산(sulfuric acid)을 첨가함으로써 반응을 중단시켰고, 450 nm에서 신호를 측정하였다. 시험관 내(in vitro) 사이토카인 분비 분석을 위해, MH7A cells (5×104) 또는 Raw 264.7 cells (5×106)를 12-웰 플레이트에 접종하였고, 1, 10 μM의 PBT-6 화합물로 6시간 반응시킨 후, LPS 또는 TNF-α를 첨가하여 24시간 동안 반응시켰다. 배양 배지를 수확하였고, 사이토카인 측정을 위해 사용하였다.Consent was obtained from all patients, and the experimental protocol was approved by the Inha University Hospital Ethics Committee (INHAUH IRB 2015-09-016-002). Peripheral blood and synovial fluid collected from healthy volunteers (n = 8), osteoarthritis patients (n = 9) and rheumatoid arthritis patients (n = 9) were recorded, and ELISA kits were used for R & D systems (Minneapolis, MN, USA). ), And cytokine levels were measured according to the manufacturer's instructions from serum or lubricant. Briefly, each well was coated with 0.1 g of TNF-α, IL-1β and IL-6 for 24 hours, and blocked with PBS containing 2% BSA for 1 hour at room temperature. Serum or lubricant was added to each well and reacted at 4 ° C. for 24 hours. After washing the plate with PBS containing 0.1
6. 생체 내(6. In vivo ( in in vivovivo ) 콜라겐-유도 관절염 모델) Collagen-induced arthritis model
모든 동물 실험은 인하대학교 의과대학의 INHA 실험동물운영위원회(INHA Institutional Animal Care and Use Committee; INHA IACUC)의 가이드라인에 따라 수행되었다(approval ID: INHA 170718-501). 5주령의 비병원성 DBA/1 mice (수컷)을 Orient Bio (Orient Bio, Seoul, Korea)로부터 구입하였다. 면역화를 위해, 200㎍ 미변성 bovine type II collagen (Chondrex Inc, Redmond, WA, USA)을 동등 부피의 complete Freund's adjuvant (Chondrex)와 상온에서 15분 동안 섞어 완전히 혼합하였다. 6주령 수컷 DBA/1 mice를 100 ㎍의 상기 혼합물로 꼬리 기저부에서 피부 내 면역화시켰다. 21일 째, 마우스를 200 ㎍ incomplete Freund's adjuvant (Chondrex)에 녹인 200 ㎍ bovine type II collagen으로 추가 접종하였다. 추가 접종 14일 후에, CIA가 완전히 확립되었다. 관절염 점수가 4보다 높은 마우스를 선별하여 그룹화하였다. 15마리의 실험 마우스를 3 그룹으로 고르게 나누었다(group 1, 정상 대조군; group 2, vehicle 처리; group 3, 10 mg/kg PBT-6 처리). 정상 대조군을 제외한 모든 마우스는 PBT-6를 하루에 한 번 경구 투여하였다. 1차 면역화 후, 마우스를 40일 동안 일주일에 한 번 관측하였다. 버니어 캘리퍼스(vernier caliper)로 발 두께를 측정하였다. 관절염은 점수화하였고, 발 두께는 2명의 독립된 측정자에 의해 측정되었다. 마우스의 4개 다리 모두 측정하였고, 다음의 등급에 따라 0 내지 4로 점수화하였다. 0, 관절염 증상 없음; 1, 발 또는 발가락 하나에 발적(redness); 2, 발가락 각각에 부기(swelling)를 동반하는 발목 또는 손목의 약한 부기; 3, 발목 또는 손목의 중간 정도의 부기; 4, 발가락을 포함한 전체 발의 심각한 부기. AI = 사지 관절 부기 등급 점수의 총합 (1 등급은 1포인트를 나타내고, 전체는 16포인트).All animal experiments were performed according to the guidelines of the INHA Institutional Animal Care and Use Committee (INHA IACUC) of the Inha University School of Medicine (approval ID: INHA 170718-501). Non-pathogenic DBA / 1 mice (male) at 5 weeks of age were purchased from Orient Bio (Orient Bio, Seoul, Korea). For immunization, 200 μg unmodified bovine type II collagen (Chondrex Inc, Redmond, WA, USA) was mixed with an equal volume of complete Freund's adjuvant (Chondrex) for 15 minutes at room temperature and mixed thoroughly. Male DBA / 1 mice, 6 weeks old, were immunized in the skin at the base of the tail with 100 μg of this mixture. On the 21st day, mice were additionally inoculated with 200 μg bovine type II collagen dissolved in 200 μg incomplete Freund's adjuvant (Chondrex). 14 days after booster inoculation, the CIA was fully established. Mice with arthritis scores higher than 4 were selected and grouped. The 15 experimental mice were evenly divided into 3 groups (
7. 마이크로-CT 이미지화7. Micro-CT imaging
마우스를 케타민으로 마취시켰고, 희생시켰다. 그 후, 이들의 다리를 잘라내어 4% 포르말린에서 2일 동안 고정시켰다. 실험 마우스로부터 얻어낸 발[발가락 끝에서 끝마디뼈(distal phalanx) 끝까지]을 스캔하였고, 이미지는 13.38 mm의 복셀 크기(13.38 mm)를 가진 micro-CT (Sky Scan 1172; Bruker)로 3차원 구조로 재구성하였다. 0.5 mm 두께의 알루미늄 필터를 가진 X ray 튜브 전압은 59 kV이었고, 전류는 167 mA이었다. 노출 시간은 1160ms이었다. X ray 투사이미지는 180°각도 회전으로 스캔하여 0.45°간격에서 얻었다.Mice were anesthetized with ketamine and sacrificed. Thereafter, their legs were cut off and fixed in 4% formalin for 2 days. The foot (from the tip of the toe to the tip of the distal phalanx) obtained from the experimental mouse was scanned, and the image was reconstructed into a three-dimensional structure by micro-CT (Sky Scan 1172; Bruker) with a voxel size of 13.38 mm (13.38 mm). Did. The X ray tube voltage with a 0.5 mm thick aluminum filter was 59 kV and the current was 167 mA. The exposure time was 1160 ms. X-ray projection images were scanned at 180 ° rotation and were obtained at 0.45 ° intervals.
8. 8. 주석산염Tartrate -저항 산성 인산분해효소(-Resistance acid phosphatase ( TartrateTartrate -resistant acid -resistant acid phosphatasephosphatase ; TRAP)-양성 염색 분석; TRAP) -positive staining analysis
Raw 264.7 세포를 100 ng/mL RANKL 및 30 ng/mL M-CSF가 포함되고, 다양한 농도의 PBT-6 (1-10 μM)가 포함된 분화 배지에서 6일 동안 배양하였다. TRAP staining kit (Sigma Aldrich, OH)를 사용하여, TRAP 발현을 측정하였다. 광학 현미경을 이용하여, 3개 이상의 핵이 포함된 TRAP (+) 다핵 세포를 파골세포로서 계수하였고, Image Pro Plus를 사용하여 데이터를 정량하였다. Raw 264.7 cells were cultured for 6 days in differentiation medium containing 100 ng / mL RANKL and 30 ng / mL M-CSF and various concentrations of PBT-6 (1-10 μM). TRAP expression was measured using a TRAP staining kit (Sigma Aldrich, OH). Using an optical microscope, TRAP ( + ) multinuclear cells containing three or more nuclei were counted as osteoclasts, and data were quantified using Image Pro Plus.
9. 9. PI3KC2γPI3KC2γ 키나제Kinase 분석 analysis
ADP-Glo kinase assay는 Promega (Madison, WI, USA)로부터 구입하였다. 키나제 반응은 다음과 같이 진행하였다. 1.5 mL 마이크로원심분리 튜브에 25 μM APT, 0.1 mg/mL PI3K 기질, 여러 농도의 PBT-6 (0.1-100 μM)을 잘 혼합하고, 최종적으로 5 ng/μL PI3KC2γ 효소를 첨가하였다. 30℃에서 40분 동안 반응시킨 후, 10 μL ADP-Glo™ 시약을 첨가하였다. 30℃에서 40분 동안 반응시킨 후, 키나제 검출 시약 10 μL를 첨가하였고, 스핀 다운하였다. 최종적으로, 화이트 플레이트로 옮긴 후, 이의 발광도를 측정하였다.ADP-Glo kinase assay was purchased from Promega (Madison, WI, USA). The kinase reaction proceeded as follows. In a 1.5 mL microcentrifuge tube, 25 μM APT, 0.1 mg / mL PI3K substrate, various concentrations of PBT-6 (0.1-100 μM) were mixed well, and finally 5 ng / μL PI3KC2γ enzyme was added. After reacting at 30 ° C. for 40 minutes, 10 μL ADP-Glo ™ reagent was added. After reacting at 30 ° C. for 40 minutes, 10 μL of kinase detection reagent was added and spin down. Finally, after transferring to a white plate, its luminescence was measured.
10. 10. 키나제Kinase 프로파일링 Profiling
10 μM 농도의 ATP를 사용한 1 μM 농도에서, Eurofins Kinase Profiler Selectivity Testing Service에서 제공하는 Eurofins (https://www.eurofinsdiscoveryservices.com/)를 사용하여, PI3 키나제에 대한 PBT-6의 활성을 스크리닝하였다. At 1 μM concentration using ATP at 10 μM concentration, the activity of PBT-6 against PI3 kinase was screened using Eurofins ( https://www.eurofinsdiscoveryservices.com/ ) provided by Eurofins Kinase Profiler Selectivity Testing Service. .
11. 11. 트랜스웰Transwell 이동 분석 Movement analysis
지름 6.5 mm 및 기공 크기 8.0 μm를 갖는 폴리카보네이트 필터(Corning Costar, Cambridge, MA, USA)가 장착된 24-트랜스웰 유닛을 사용하여, 제조사의 지시에 따라 대식세포(macrophages) 이동 분석을 수행하였다. 간단히 설명하면, 배양 배지에 PBT-6를 포함하거나 또는 포함하지 않고, TNF-α-처리된 MH7A 세포를 24-웰 플랫-바텀(flat-bottom) 플레이트에서 배양하였다. 그 후, Raw 264.7 세포를 3시간 동안 굶겼고, 2% FBS DMEM에 녹인 LPS가 포함된 트랜스웰 삽입물의 상단 챔버에 세포를 넣었다. 48시간 후, 이동하지 않은 세포를 제거하기 위해서, 상단 부분에 있는 Raw 264.7 세포를 면봉으로 조심스럽게 제거하였고, 트랜스웰 내 멤브레인의 하단으로 이동한 세포들은 20% 메탄올로 고정시켰고, 0.2% 크리스탈 바이올렛으로 30분 동안 염색시켰다. 각각 독립된 샘플당 5개의 임의적 스팟을 계수함으로써, 이동된 대식세포(macrophages)를 정량하였다. Macrophages migration analysis was performed using a 24-transwell unit equipped with a polycarbonate filter (Corning Costar, Cambridge, MA, USA) having a diameter of 6.5 mm and a pore size of 8.0 μm, according to the manufacturer's instructions. . Briefly, TNF-α-treated MH7A cells with or without PBT-6 in the culture medium were cultured in 24-well flat-bottom plates. Thereafter, Raw 264.7 cells were starved for 3 hours, and the cells were placed in the upper chamber of a transwell insert containing LPS dissolved in 2% FBS DMEM. After 48 hours, in order to remove the cells that did not migrate, the raw 264.7 cells in the upper part were carefully removed with a cotton swab, and the cells moving to the bottom of the membrane in the transwell were fixed with 20% methanol, 0.2% crystal violet Stain for 30 minutes. Moved macrophages were quantified by counting 5 random spots per independent sample.
12. 면역조직화학12. Immunohistochemistry
CIA 마우스로부터 얻은 관절 조직을 탈석회화시켰고, 10% 완충된 포름알데히드에서 4℃로 밤새도록 고정시켰으며, 파라핀에 포매하여 절단하였다. 탈파라핀화 후, 8-mm 관절 샘플 절편 상에서 면역염색을 수행하였다. PBS에 녹인 0.2 mg/mL Proteinase K (Thermo Fisher Scientific, Grand Island, NY, USA)를 상온에서 15분 동안 반응시켜, 항원 복구(Antigen retrieval)를 수행하였다. PBS로 2번 조심스럽게 씻어낸 후, 증류수에 녹인 0.3% H2O2를 사용하여 상온에서 15분 동안 내재성 퍼록시다아제를 차단시켰다. 조직 절편을 PBS로 씻어냇고, 정상 염소 또는 말 혈청(Vector Laboratories, Burlingame, CA, USA)으로 1시간 동안 차단시켰으며, PI3KC2γ 및 p-AKT2 (S474)에 대한 1차 항체의 1:50 희석액으로 4℃에서 밤새도록 반응시켰다. 그 후, 절편들을 비오틴화된 2차 항체(1:100)로 1시간 동안 반응시켰다. ABC kit (Vector Laboratories)를 사용하여 아비딘-비오틴 퍼록시다아제(avidin-biotin peroxidase) 복합체 용액으로 절편들을 가시화시켰고, PBS로 씻어냈으며, 디아미노벤지딘 테트라하이드로클로라이드(diaminobenzidine tetrahydrochloride; DAB) 기질로 5분 동안 반응시킨 후, 헤마톡실린으로 대비 염색하였다. 각각의 절편에 대해 최소한 3개의 임의로 선택된 영역에서 200×배율로 측정 및 분석하였다.The joint tissue obtained from CIA mice was decalcified, fixed overnight at 4 ° C. in 10% buffered formaldehyde, embedded in paraffin and cut. After deparaffinization, immunostaining was performed on 8-mm joint sample sections. Antigen recovery (Antigen retrieval) was performed by reacting 0.2 mg / mL Proteinase K (Thermo Fisher Scientific, Grand Island, NY, USA) dissolved in PBS for 15 minutes at room temperature. After carefully washing twice with PBS, intrinsic peroxidase was blocked for 15 minutes at room temperature using 0.3% H 2 O 2 dissolved in distilled water. Tissue sections were washed with PBS and blocked with normal goat or horse serum (Vector Laboratories, Burlingame, CA, USA) for 1 hour, with a 1:50 dilution of primary antibody against PI3KC2γ and p-AKT2 (S474). The reaction was carried out overnight at 4 ° C. Thereafter, the sections were reacted with a biotinylated secondary antibody (1: 100) for 1 hour. Sections were visualized with avidin-biotin peroxidase complex solution using ABC kit (Vector Laboratories), washed with PBS, and 5 with diaminobenzidine tetrahydrochloride (DAB) substrate. After reacting for a minute, it was counter-stained with hematoxylin. For each section, at least three randomly selected regions were measured and analyzed at 200 × magnification.
13. 통계 분석13. Statistical Analysis
데이터는 평균 ± SD로 나타냈고, ANOVA 및 unpaired Student's t-test로 분석하였다. 0.05 이하의 P-value는 통계적 유의성이 있는 것으로 나타냈다. 결과 비교는 Student's t-test를 사용하여 수행하였다.Data are presented as mean ± SD and analyzed by ANOVA and unpaired Student's t-test. P-values of 0.05 or less were shown to be statistically significant. Results were compared using Student's t-test.
<< 실시예Example 1> RA 환자 유래 윤활액, 1> RA patient-derived lubricant, 윤활막Lubricant film 조직 및 Organization and PBMC에서의At PBMC PI3KC2γPI3KC2γ 발현 증가 Increased expression
우선, 본 발명자들은 윤활액에서의 PI3KC2γ 및 전-염증성 사이토카인의 발현을 측정하였다. ELISA를 통한 윤활액 분석 결과, PI3KC2γ, IL-6, IL-1β 및 TNF-α 발현은 골관절염(OA)에서의 발현 수준에 비해 류마티스 관절염(RA)에서 더 증가하였다(도 1). 또한, 본 발명자들은 RA 및 OA의 윤활막 조직에서 염증 반응 및 PI3KC2γ의 단백질 발현을 측정하였다. RA 환자 윤활막 조직에서, 염증 세포 침습을 동반한 활막 내피 과다 증식(synovial endothelial hyperplasia), 과립 형성, 섬유아세포 및 혈관 증식이 명확하게 관측되었다. 또한, PI3KC2γ은 RA 그룹에서 높게 발현되는 것을 면역조직화학 및 웨스턴 블랏 분석을 통해 확인하였다(도 2). 이외에도, 건강한 대조군의 PBMC에 비해 RA PBMC에서 PI3KC2γ의 단백질 수준이 증가되었다(도 3). 상기 결과는 RA 윤활액, 윤활막 조직 및 PBMC에서, 염증 및 PI3KC2γ가 증가된다는 것을 뒷받침한다. First, we measured the expression of PI3KC2γ and pro-inflammatory cytokines in the lubricant. As a result of lubricating fluid analysis through ELISA, PI3KC2γ, IL-6, IL-1β and TNF-α expression increased more in rheumatoid arthritis (RA) compared to the expression level in osteoarthritis (OA) (FIG. 1). In addition, the present inventors measured the inflammatory response and protein expression of PI3KC2γ in the lubricating tissue of RA and OA. In the synovial tissue of RA patients, synovial endothelial hyperplasia, granulation, fibroblast and vascular proliferation were clearly observed with inflammatory cell invasion. In addition, PI3KC2γ was confirmed to be highly expressed in the RA group through immunohistochemistry and Western blot analysis (FIG. 2). In addition, the protein level of PI3KC2γ was increased in RA PBMC compared to PBMC in the healthy control group (FIG. 3). The results support the increased inflammation and PI3KC2γ in RA lubricant, synovial tissue and PBMC.
<< 실시예Example 2> 2> PI3KC2γ의PI3KC2γ ATP 결합 사이트에 강한 결합 친화도로 결합하는 PBT-6 PBT-6 binds to ATP binding site with strong binding affinity
PI3KC2γ 억제제인 PBT-6의 화학구조는 도 4에 나타냈다. 다음으로, PBT-6의 결합 친화도를 측정하기 위하여, 키나제 분석을 수행하였다. 그 결과, PBT-6의 결합 포화 곡선을 분석하였고, Kd 수치는 826 nM이었다(도 5). PBT-6의 보다 자세한 특성을 확인하기 위해서, 본 발명자들은 Eurofins를 통해 균일 시간 분해 형광(homogeneous time-resolved fluorescence; HTRF) 키나제 분석을 수행하였다. 도 6에 나타낸 바와 같이, PBT-6는 다른 PI3K 이성질체와 다르게 1 μM 농도에서 PI3KC2γ을 완전히 선택적으로 억제시켰다.The chemical structure of PBT-6, a PI3KC2γ inhibitor, is shown in FIG. 4. Next, in order to measure the binding affinity of PBT-6, kinase analysis was performed. As a result, the binding saturation curve of PBT-6 was analyzed, and K d The figure was 826 nM (FIG. 5). To confirm the more detailed properties of PBT-6, the inventors performed homogeneous time-resolved fluorescence (HTRF) kinase analysis through Eurofins. As shown in Fig. 6, PBT-6 completely inhibited PI3KC2γ at a concentration of 1 μM unlike other PI3K isomers.
<< 실시예Example 3> 3> TNFTNF -α 유도 -α induction MH7AMH7A 세포에서 세포 Cell to Cell 생존능Viability , , 전염증성Infectious 사이토카인 및 PI3K/AKT 활성화를 감소시키는 PI3KC2γ 억제 PI3KC2γ inhibition that reduces cytokine and PI3K / AKT activation
세포 생존능에 있어 PBT-6의 효과를 시험하기 위해서, 본 발명자들은 류마티스 관절염 활막세포(MH7A)를 이용하여 MTT 분석을 수행하였다. 그 결과, PBT-6는 농도 의존적 방식으로 세포 생존능을 감소시켰다. 특히, 10 μM 농도로 PBT-6를 처리하면, 세포 성장이 40-50% 감소하였다(도 7). TNF-α 및 IL-6과 같은 전-염증성 사이토카인은 RA의 발병을 촉진시키는 것으로 잘 알려져 있다. PI3KC2γ 및 염증 간의 관련성을 더 입증하기 위해서, 본 발명자들은 세포 상등액을 사용하여 ELISA로 PBT-6의 효과를 확인하였다. 그 결과, TNF-α 유도 MH7A 세포에서 PBT-6에 의해 IL-6는 약간 감소하였다(도 8). PBT-6가 PI3K/AKT 경로의 활성화를 억제하는지 확인하기 위해서, 본 발명자들은 MH7A 세포에서 PI3K/AKT 신호 경로의 억제를 조사하였다. 그 결과, PBT-6는 AKT2 및 mTOR의 인산화를 상당히 억제하였다(도 9).In order to test the effect of PBT-6 on cell viability, the present inventors performed MTT analysis using rheumatoid arthritis synovial cells (MH7A). As a result, PBT-6 reduced cell viability in a concentration dependent manner. In particular, when treated with PBT-6 at a concentration of 10 μM, cell growth was reduced by 40-50% (FIG. 7). It is well known that pro-inflammatory cytokines such as TNF-α and IL-6 promote the development of RA. To further demonstrate the association between PI3KC2γ and inflammation, we confirmed the effect of PBT-6 by ELISA using cell supernatant. As a result, IL-6 was slightly decreased by PBT-6 in TNF-α induced MH7A cells (FIG. 8). To confirm that PBT-6 inhibits the activation of the PI3K / AKT pathway, we investigated the inhibition of the PI3K / AKT signaling pathway in MH7A cells. As a result, PBT-6 significantly inhibited phosphorylation of AKT2 and mTOR (Fig. 9).
<< 실시예Example 4> 4> LPSLPS 유도 Raw 264.7 세포에서 세포 Cells from induced Raw 264.7 cells 생존능Viability , , 전염증성Infectious 사이토카인 및 Cytokines and PI3KPI3K // AKTAKT 활성화를 감소시키는 Reducing activation PI3KC2γPI3KC2γ 억제 control
대식세포에서도 상기와 유사한 결과를 나타내는지 확인하기 위해서, 본 발명자들은 LPS 유도 Raw 264.7 세포에서 PBT-6의 효과를 확인하였다. 예상대로, PBT-6는 농도 의존적 방식으로 세포 생존능을 감소시켰고(도 10), Raw 264.7 세포에서 PBT-6에 의해 TNF-α를 크게 감소시켰다(도 11). 게다가, PBT-6는 AKT2 및 mTOR의 인산화를 상당히 억제하였는데(도 12), 이는 PBT-6가 PI3K/AKT/mTOR 신호 전달 체계를 차단함으로써 염증성 사이토카인 발현을 억제한다는 것을 나타낸다.In order to confirm that macrophages showed similar results to the above, the present inventors confirmed the effect of PBT-6 on LPS-induced Raw 264.7 cells. As expected, PBT-6 reduced cell viability in a concentration dependent manner (Figure 10), and significantly reduced TNF-α by PBT-6 in Raw 264.7 cells (Figure 11). In addition, PBT-6 significantly inhibited phosphorylation of AKT2 and mTOR (FIG. 12), indicating that PBT-6 inhibits inflammatory cytokine expression by blocking the PI3K / AKT / mTOR signaling system.
<< 실시예Example 5> 파골세포형성 및 이동을 감소시키는 5> Reducing osteoclast formation and migration PI3KC2γPI3KC2γ 억제 control
Raw 264.7 세포는 RANKL-발현 세포로서 알려져 있고, 파골세포 형성 및 기능을 연구하기 위한 파골세포 전구 모델로서 널리 사용되고 있다. 파골세포형성에 있어 PI3KC2γ의 역할을 연구하기 위해서, 본 발명자들은 Raw 264.7 세포의 파골세포로의 RANKL-유도 분화에 있어 비-독성 농도 범위에서의 PBT-6 효과를 확인하였다. 파골세포 분화 정도를 측정하기 위해서, 파골세포에서 높게 발현되는 마커 효소인 TRAP 염색을 수행하였다. 3개 이상의 핵을 가진 TRAP-양성 다핵성 세포를 파골세포로 간주하였다. PBT-6는 Raw 264.7 세포의 파골세포 분화를 농도 의존적 방식으로 크게 억제하였다(도 13). NFATc1은 파골세포로의 분화 초기 단계에서 RANKL 촉진에 의해 가장 강력하게 유도되는 전사 인자로 알려져 있는데, 이는 파골세포 분화에 필요한 여러 유전자들의 발현을 조절한다. 도 14에 나타낸 바와 같이, RANKL-촉진 Raw 264.7 세포에서, NFATc1 발현에 상당한 증가가 관측되었으나, 1-10 μM의 PBT-6의 존재 하에서는 NFATc1의 발현이 분명히 억제되었다. 다음으로, 대식세포로부터 활막세포로의 주화성(chemo-attraction)에서의 PBT-6 저항성을 시험하기 위해, 대식세포 이동에 있어서의 PI3KC2γ 효과를 Transwell 챔버 분석을 통해 측정하였다. 그 결과, LPS-처리 Raw 264.7 세포의 이동은 증가하였으나, 플레이트 하단에 PBT-6가 존재시에는 억제되었다(도 15 및 도 16).Raw 264.7 cells are known as RANKL-expressing cells and are widely used as osteoclast progenitor models for studying osteoclast formation and function. In order to study the role of PI3KC2γ in osteoclast formation, the present inventors confirmed the effect of PBT-6 in the non-toxic concentration range in RANKL-induced differentiation of Raw 264.7 cells to osteoclasts. In order to measure the degree of osteoclast differentiation, TRAP staining, a marker enzyme highly expressed in osteoclasts, was performed. TRAP-positive polynuclear cells with three or more nuclei were considered as osteoclasts. PBT-6 significantly inhibited osteoclast differentiation of Raw 264.7 cells in a concentration dependent manner (FIG. 13). NFATc1 is known to be the most potently induced transcription factor by RANKL promotion in the early stage of osteoclast differentiation, which regulates the expression of several genes required for osteoclast differentiation. As shown in FIG. 14, in RANKL-promoted Raw 264.7 cells, a significant increase in NFATc1 expression was observed, but the expression of NFATc1 was clearly inhibited in the presence of 1-10 μM of PBT-6. Next, in order to test the PBT-6 resistance in chemo-attraction from macrophages to synovial cells, the effect of PI3KC2γ on macrophage migration was measured through Transwell chamber analysis. As a result, the migration of LPS-treated Raw 264.7 cells was increased, but was suppressed in the presence of PBT-6 at the bottom of the plate (FIGS. 15 and 16).
<< 실시예Example 6> 콜라겐 유도 6> collagen induction 류마티스Rheumatism 관절염(CIA) 마우스에서 관절염 Arthritis in arthritis (CIA) mice 심각도를Severity 약화시키는 PBT-6 투여 Weakening PBT-6 administration
시험관 내(in vitro) 실험 결과를 고려하여, RA에서의 활막 염증 및 관절 손상에 대한, PBT-6의 생체 내(in vivo) PI3KC2γ 억제 효과를 CIA 마우스에서 평가하였다. 비히클(vehicle)-처리군(0.5% 메틸 셀룰로오스 경구 투여)과 비교하여, PBT-6-처리군(10 mg/kg PBT-6 경구 투여)은 발바닥 두께 및 평균 관절염 점수를 포함하는 관절염 심각도를 상당히 약화시켰다(도 17 내지 도 19). 또한, 혈청 내 TNF-α 수준은 비히클(vehicle)-처리군에 비해 PBT-6-처리군에서 상당히 낮아졌으나, 혈청 내 IL-6 수준은 약간 감소하였다(도 20). 비히클(vehicle)-처리된 CIA 마우스 유래 발 관절 절편의 조직병리학적 측정 및 면역조직화학 분석 결과, 염증 세포 침습, 석회화된 연골 및 뼈로의 판누스(pannus) 침윤에 따른 활막 과형성이 나타났다. 상기 절편들은 PI3KC2γ 및 p-AKT2에 대한 집중적인 염색이 나타났는데, 이는 관절 주위에 염증 세포가 존재한다는 것을 뒷받침한다(도 21). PBT-6 투여는 염증 세포 침습 및 뼈 손상 범위를 감소시켰을 뿐만 아니라, PI3KC2γ 및 p-AKT2의 발현도 감소시켰다. 일관성 있게, 활막 염증, 연골 손상 및 뼈 손상을 나타내는 조직학적 점수도 CIA 마우스에서 PBT-6에 의해 상당히 감소되었다(도 22). 실험 과정에서 비히클(vehicle)-처리군 및 PBT-6-처리군 사이의 몸무게는 거의 차이가 없었다. 또한, PBT-6-처리군에서 제거된 비장 및 간에서는 비히클(vehicle)-처리군에 비해 유의성 있는 조직병리학적 변화를 확인할 수 없었다. 상기 결과는 CIA 마우스에서의 PBT-6 처리에 대한 안전성을 뒷받침한다. 다음으로, 뒷발에 대한 micro-CT scan의 3차원 재구성 결과, CIA 마우스에서 전형적인 변화를 나타냈는데, 이는 관절 손상, 관절 변위 및 비정상적인 뼈 증식 결과이다. 하지만, PBT-6 처리는 뼈 손상을 완화시켰고, 뼈 부피, 골밀도 및 골주 두께를 상당히 증가시켰다(도 23 및 도 24).Considering the results of in vitro experiments, the in vivo PI3KC2γ inhibitory effect of PBT-6 on synovial inflammation and joint damage in RA was evaluated in CIA mice. Compared to the vehicle-treated group (orally administered 0.5% methyl cellulose), the PBT-6-treated group (orally administered 10 mg / kg PBT-6) significantly affected arthritis severity, including plantar thickness and mean arthritis score. Weakened (Figs. 17-19). In addition, the TNF-α level in the serum was significantly lower in the PBT-6-treated group compared to the vehicle-treated group, but the IL-6 level in the serum was slightly decreased (FIG. 20). Histopathological measurements and immunohistochemical analysis of foot-joint sections from vehicle-treated CIA mice revealed synovial hyperplasia due to inflammatory cell invasion, calcified cartilage and pannus infiltration into bone. The fragments showed intensive staining for PI3KC2γ and p-AKT2, supporting the presence of inflammatory cells around the joint (Figure 21). PBT-6 administration not only reduced the extent of inflammatory cell invasion and bone damage, but also decreased the expression of PI3KC2γ and p-AKT2. Consistently, histological scores indicating synovial inflammation, cartilage damage, and bone damage were also significantly reduced by PBT-6 in CIA mice (FIG. 22). There was little difference in body weight between the vehicle-treated group and the PBT-6-treated group in the experimental process. In addition, in the spleen and liver removed from the PBT-6-treated group, no significant histopathological changes could be confirmed compared to the vehicle-treated group. The results support the safety of PBT-6 treatment in CIA mice. Next, three-dimensional reconstruction of the micro-CT scan of the hind paw revealed typical changes in CIA mice, which are the result of joint damage, joint displacement, and abnormal bone proliferation. However, PBT-6 treatment alleviated bone damage and significantly increased bone volume, bone density and bone thickness (Figures 23 and 24).
이상으로 본 발명을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시예일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다. The present invention has been described in detail above, and it will be apparent to those skilled in the art that this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (18)
(2) 상기 PI3KC2γ 유전자의 mRNA 발현 수준 또는 PI3KC2γ 단백질의 발현 수준을 대조군 시료와 비교하는 단계; 및
(3) 상기 PI3KC2γ 유전자의 mRNA 발현 수준 또는 PI3KC2γ 단백질의 발현 수준이 대조군 시료보다 높을 경우 염증 질환으로 판단하는 단계를 포함하는 염증 질환 진단에 필요한 정보를 제공하는 방법.(1) measuring the mRNA expression level of the PI3KC2γ gene or the expression level of the PI3KC2γ protein from a sample isolated from the patient;
(2) comparing the mRNA expression level of the PI3KC2γ gene or the expression level of the PI3KC2γ protein with a control sample; And
(3) A method for providing information necessary for diagnosing an inflammatory disease, including determining an inflammatory disease when the mRNA expression level of the PI3KC2γ gene or the expression level of the PI3KC2γ protein is higher than the control sample.
[화학식 1]
상기 화학식 1에서,
R1 및 R2는 각각 같거나 다를 수 있으며, 수소, (C1~C4)알킬, (C1~C4)알콕시 및 할로겐으로 이루어진 군에서 선택되는 어느 하나임.The pharmaceutical composition for preventing or treating inflammatory diseases according to claim 10, wherein the compound is a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof.
[Formula 1]
In Chemical Formula 1,
R 1 and R 2 may each be the same or different, and are any one selected from the group consisting of hydrogen, (C1 to C4) alkyl, (C1 to C4) alkoxy and halogen.
[화학식 2]
[Formula 2]
(2) 상기 시험물질을 접촉한 염증 질환 동물 모델에서 PI3KC2γ 단백질의 발현 또는 활성 정도를 측정하는 단계; 및
(3) 대조군 시료와 비교하여 상기 PI3KC2γ 단백질의 발현 또는 활성 정도가 감소한 시험물질을 선별하는 단계를 포함하는 염증 질환 치료제 스크리닝 방법.(1) contacting the test substance to the inflammatory disease animal model;
(2) measuring the expression or activity level of PI3KC2γ protein in an animal model of inflammatory disease in contact with the test substance; And
(3) Screening method for the treatment of inflammatory diseases, comprising the step of selecting a test substance having a reduced expression or activity level of the PI3KC2γ protein compared to a control sample.
[화학식 1]
상기 화학식 1에서,
R1 및 R2는 각각 같거나 다를 수 있으며, 수소, (C1~C4)알킬, (C1~C4)알콕시 및 할로겐으로 이루어진 군에서 선택되는 어느 하나임.The method of claim 16, wherein the PI3KC2γ protein expression or activity inhibitor is a health functional food composition for preventing or improving inflammatory diseases, characterized in that the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
[Formula 1]
In Chemical Formula 1,
R 1 and R 2 may each be the same or different, and are any one selected from the group consisting of hydrogen, (C1 to C4) alkyl, (C1 to C4) alkoxy and halogen.
[화학식 2]
[Formula 2]
Priority Applications (1)
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101538787B1 (en) * | 2013-06-27 | 2015-07-22 | 인하대학교 산학협력단 | Biomarker composition for diagnosing pancreatitis |
| KR20170136281A (en) | 2016-06-01 | 2017-12-11 | 인하대학교 산학협력단 | Composition comprising imidazopyridine derivative with PI3-kinase inhibitory activity for arthritis |
| KR20180100838A (en) * | 2017-03-02 | 2018-09-12 | 인하대학교 산학협력단 | A Composition for Treating or Preventing Inflammatory Diseases Comprising Inhibitors against PI3K and mTOR as an Active Ingredient |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101538787B1 (en) * | 2013-06-27 | 2015-07-22 | 인하대학교 산학협력단 | Biomarker composition for diagnosing pancreatitis |
| KR20170136281A (en) | 2016-06-01 | 2017-12-11 | 인하대학교 산학협력단 | Composition comprising imidazopyridine derivative with PI3-kinase inhibitory activity for arthritis |
| KR20180100838A (en) * | 2017-03-02 | 2018-09-12 | 인하대학교 산학협력단 | A Composition for Treating or Preventing Inflammatory Diseases Comprising Inhibitors against PI3K and mTOR as an Active Ingredient |
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| Emilie Collmann et al, University of Basel (2009.06.23.), pp 1-158. * |
| Montserrat Camps et al, Nature Medicine (2005), vol 11, pp 936-943. * |
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| Thomas Bohnacker et al, Science Signaling (2009), vol 2, issue 74ra27, pp 1-12. * |
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