KR102414285B1 - Pharmaceutical composition for prevention or treatment of bone disease containing Flunarizine or pharmaceutically acceptable salts thereof as an active ingredient - Google Patents
Pharmaceutical composition for prevention or treatment of bone disease containing Flunarizine or pharmaceutically acceptable salts thereof as an active ingredient Download PDFInfo
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- KR102414285B1 KR102414285B1 KR1020200066630A KR20200066630A KR102414285B1 KR 102414285 B1 KR102414285 B1 KR 102414285B1 KR 1020200066630 A KR1020200066630 A KR 1020200066630A KR 20200066630 A KR20200066630 A KR 20200066630A KR 102414285 B1 KR102414285 B1 KR 102414285B1
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- bone
- flunarizine
- prevention
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- disease
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P19/00—Drugs for skeletal disorders
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention
Abstract
본 발명은 플루나리진 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 골질환의 예방 또는 치료용 약학적 조성물에 관한 것으로, 상기 조성물은 파골세포 분화와 관련된 중요한 인자들을 억제함으로써, 파골세포의 분화를 억제하여 골 흡수를 저해하는 효과가 있으며 전임상 및 임상 연구에 유용하게 활용될 수 있다. 또한, 파골세포의 불균형에 의하여 유발되는 골 관련 질환을 치료할 수 있으므로, 골 관련 질환의 예방 또는 치료제 개발에 널리 활용될 수 있다.The present invention relates to a pharmaceutical composition for the prevention or treatment of bone disease containing flunarizine or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the composition inhibits important factors related to osteoclast differentiation, It has the effect of inhibiting bone resorption by inhibiting the differentiation of In addition, since it can treat bone-related diseases caused by imbalance of osteoclasts, it can be widely used for preventing or developing therapeutic agents for bone-related diseases.
Description
본 발명은 플루나리진 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 골질환의 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating bone disease containing flunarizine or a pharmaceutically acceptable salt thereof as an active ingredient.
대표적인 대사성 골 질환인 골다공증(osteoporosis)은 골 조직의 석회가 감소되어 뼈의 치밀질이 엷어지고 그로 인해 골수강(骨髓腔)이 넓어지게 되는 질환으로, 증세가 진전됨에 따라 뼈가 약해지기 때문에 작은 충격에도 골절되기가 쉽다. Osteoporosis, a representative metabolic bone disease, is a disease in which the bone marrow becomes thinner due to a decrease in calcification of the bone tissue, and as a result, the bone marrow cavity is widened. It is easy to fracture even on impact.
골다공증은 골량의 감소와 미세구조의 이상을 특징으로 갖는데, 노화가 진행되면서 낡은 뼈의 흡수와 새로운 뼈의 형성 사이에 균형이 무너져 새로운 뼈의 대체(bone remodeling)가 원활히 이루어지지 않아 뼈가 엉성해지고, 부러지거나 부서질 위험성이 커지게 된다. 이러한 골량은 유전적 요인, 영양 섭취, 호르몬의 변화, 운동 및 생활 습관의 차이 등 여러 가지 요인들에 의해 영향을 받으며, 노령, 운동 부족, 저체중, 흡연, 저칼슘 식이, 폐경, 난소 절제 등이 골량 감소의 주요한 원인이다. Osteoporosis is characterized by a decrease in bone mass and abnormalities in the microstructure. As aging progresses, the balance between resorption of old bone and the formation of new bone is broken, and bone remodeling is not performed smoothly, resulting in sloppy bones. , increasing the risk of breaking or breaking. Such bone mass is affected by several factors, such as genetic factors, nutritional intake, hormonal changes, and differences in exercise and lifestyle. It is a major cause of bone loss.
한편, 개인차는 있지만 대개 골량은 14~18세에 가장 높고 노후에는 1년에 약 1%씩 감소한다. 특히, 여성의 경우 30세 이후부터 골 감소가 지속적으로 진행되며, 폐경기에 이르면 호르몬 변화에 의해 골 감소가 급격히 진행된다. 즉, 폐경기에 이르면 에스트로겐 농도가 급속히 감소하는데, 이때, IL-7(interleukin-7)에 의한 것처럼 B-임파구(B-lymphocyte)가 다량 생성되어 골수(bone marrow)에 B 세포 전구체(pre-B cell)가 축적되고, 이로 인해 IL-6의 양이 증가하여 파골세포의 활성을 증가시키므로 결국 골량이 감소하게 된다.On the other hand, although there are individual differences, in general, bone mass is the highest at the age of 14 to 18, and decreases by about 1% per year in old age. In particular, in the case of women, bone loss continuously progresses after the age of 30, and when they reach menopause, bone loss progresses rapidly due to hormonal changes. That is, the estrogen concentration rapidly decreases upon reaching menopause. At this time, as by IL-7 (interleukin-7), a large amount of B-lymphocytes are produced and B-cell precursors (pre-B) in the bone marrow (bone marrow). cells) accumulate, and this increases the amount of IL-6, which increases the activity of osteoclasts, resulting in a decrease in bone mass.
이와 같이, 골다공증은 정도에 차이는 있으나 노년층, 특히 폐경기 이후의 여성에게 있어서는 피할 수 없는 증상으로, 선진국에서는 인구가 노령화됨에 따라 골다공증 및 그 치료제에 대한 관심이 점차 증가하고 있다. 또한, 전 세계적으로 골질환 치료와 관련되어 약 1,300억 달러의 시장이 형성되어 있으며, 앞으로 더 증가할 것으로 예상되기 때문에 세계적인 각 연구 기관과 제약회사에서는 골질환 치료제 개발에 많은 투자를 하고 있다.As such, osteoporosis is an unavoidable symptom for the elderly, particularly postmenopausal women, although there is a difference in degree, and interest in osteoporosis and its therapeutic agents is gradually increasing in developed countries as the population ages. In addition, around the world, a market of about 130 billion dollars is formed related to the treatment of bone diseases and is expected to increase further in the future.
국내에서도 근래에 평균수명이 80세에 육박하면서 골다공증 유병률이 급격하게 증가하고 있는데, 최근 지역 주민을 대상으로 실시된 연구에 의하면 전국 인구로 표준화하였을 경우 남성의 4.5%, 여성의 19.8%가 골다공증을 갖고 있다고 보고되었다. 이는 골다공증이 당뇨병이나 심혈관계 질환보다 더 흔한 질환이며, 골절로 인해 받는 환자들의 고통이나 치료를 위해 들어가는 비용을 추정할 때 골다공증은 매우 중요한 보건 문제임을 시사한다.In Korea, the prevalence of osteoporosis is rapidly increasing as life expectancy approaches 80 years of age. reported to have This suggests that osteoporosis is a more common disease than diabetes or cardiovascular disease, and that osteoporosis is a very important health problem when estimating the pain and cost of treatment for patients suffering from fractures.
지금까지 여러 물질이 골다공증 치료제로 개발되었다. 그 중 골다공증 치료제로 가장 많이 사용되는 에스트로겐은 그 실제적인 효능이 아직 검증되지 않은 상태이며 생애 동안 계속 복용해야 하는 단점이 있으며, 장기간 투여하는 경우 유방암이나 자궁암이 증가하는 부작용이 있다. 알렌드로네이트(alrendronate)도 그 효능이 명확하지 않고 소화관에서의 흡수가 더디며 위장과 식도점막에 염증을 유발하는 문제가 있다. 칼슘제제는 부작용이 적으면서도 효과가 우수한 것으로 알려져 있지만 치료제라기보다는 예방제에 해당한다. 그 외에 칼시토닌과 같은 비타민 D 제제가 알려져 있으나 아직 효능 및 부작용에 대한 연구가 충분히 되어있지 않은 상태이다. 이에, 부작용이 적고 효과가 우수한 새로운 대사성 골 질환 치료제가 요구되고 있는 실정이다. So far, several substances have been developed for the treatment of osteoporosis. Among them, estrogen, which is most often used as a treatment for osteoporosis, has a disadvantage in that its practical efficacy has not yet been verified, and it has to be taken continuously for life. Alendronate also has problems in that its efficacy is not clear, absorption in the digestive tract is slow, and it causes inflammation in the stomach and esophageal mucosa. Calcium preparations are known to be effective with few side effects, but they are a preventive agent rather than a therapeutic agent. In addition, vitamin D preparations such as calcitonin are known, but their efficacy and side effects have not been sufficiently studied. Accordingly, there is a need for a new therapeutic agent for metabolic bone disease with fewer side effects and excellent effects.
파골세포는 척추동물의 뼈가 성장하는 과정에서 불필요하게 된 뼈조직을 파괴 또는 흡수하는 대형의 다핵세포로써, 파골세포 전구체(osteoclast precursor)로부터 분화되는 세포이다. 파골세포 전구세포들은 M-CSF 및 RANKL(Receptor activator of nuclear factor kappa-Β ligand) 존재 하에서 파골세포로 분화되며, 융합을 통해 다핵 파골세포(multinucleated osteoclast)를 형성한다. 파골세포는 αvβ3 인테그린(integrin) 등을 통해 골에 결합하며 산성 환경을 조성하는 한편 각종 콜라게네이즈(collagenase) 및 프로테아제(protease)를 분비하여 골 흡수(bone resorption)를 일으키는데, 이러한 파골세포의 억제는 골질환 치료의 효과적인 방법이 될 수 있다.Osteoclasts are large multinucleated cells that destroy or absorb bone tissue that becomes unnecessary in the process of bone growth in vertebrates, and are cells differentiated from osteoclast precursors. Osteoclast progenitor cells are differentiated into osteoclasts in the presence of M-CSF and receptor activator of nuclear factor kappa-Β ligand (RANKL), and through fusion, they form multinucleated osteoclasts. Osteoclasts bind to bone through αvβ3 integrin and create an acidic environment, while secreting various collagenases and proteases to cause bone resorption. Inhibition of these osteoclasts can be an effective method for the treatment of bone disease.
한편, 플루나리진(Flunarizine)은 T형 칼슘 차단제로, 칼슘통로를 막는 칼슘 채널 길항제로 알려져 있으며, 미국 FDA의 승인을 받아 주로 편두통 치료제로 쓰이고 있는 물질이다. 또한 여러 연구에 따르면 플루나리진은 L형 칼슘 채널, N형 칼슘 채널 및 이노시톨 1,4,5-트리포스페이트(IP3) 수용체를 포함한 다른 종류의 칼슘 채널을 억제한다고 보고되었다. 그러나, 본 발명의 파골세포 분화 조절 효과 및 골질환 치료용도는 현재까지 알려진 바가 없다. On the other hand, flunarizine is a T-type calcium blocker, known as a calcium channel blocker that blocks calcium channels, and is a substance that has been approved by the US FDA and is mainly used as a treatment for migraine. Several studies have also reported that flunarizine inhibits other types of calcium channels, including L-type calcium channels, N-type calcium channels, and
이에 본 발명자들은, 골질환을 치료하기 위한 새로운 물질을 찾고자 파골세포의 활성을 억제하는 물질을 탐색하던 중, 플루나리진이 파골세포에 의한 골 흡수를 저해하고 파골세포 형성을 억제함을 규명하여, 플루나리진이 골질환 치료용 약학적 조성물로 유용하게 사용될 수 있음을 확인함으로써, 본 발명을 완성하였다.Accordingly, the present inventors identified that flunarizine inhibits bone resorption by osteoclasts and inhibits osteoclast formation while searching for substances that inhibit osteoclast activity to find new substances for treating bone diseases, By confirming that flunarizine can be usefully used as a pharmaceutical composition for treating bone disease, the present invention has been completed.
본 발명의 목적은 골다공증, 류마티스 관절염 등과 같은 과도한 파골세포 수나 활동에 의해 유발되는 골질환을 예방 또는 치료할 수 있는 신규 약물을 제공하는 것이다.It is an object of the present invention to provide a novel drug capable of preventing or treating bone diseases caused by excessive number or activity of osteoclasts, such as osteoporosis and rheumatoid arthritis.
상기 목적을 달성하기 위하여,In order to achieve the above object,
본 발명은 플루나리진 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 골질환의 예방 또는 치료용 약학적 조성물을 제공한다,The present invention provides a pharmaceutical composition for preventing or treating bone disease containing flunarizine or a pharmaceutically acceptable salt thereof as an active ingredient,
아울러, 본 발명은 플루나리진 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 골질환의 예방 또는 개선용 건강기능식품을 제공한다.In addition, the present invention provides a health functional food for preventing or improving bone disease containing flunarizine or a pharmaceutically acceptable salt thereof as an active ingredient.
본 발명의 플루나리진 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 골질환의 예방 또는 치료용 약학적 조성물은 파골세포 분화와 관련된 중요한 인자들을 억제함으로써, 파골세포의 분화를 억제하여 골 흡수를 저해하는 효과가 있으며 전임상 및 임상 연구에 유용하게 활용될 수 있다. 또한, 파골세포의 불균형에 의하여 유발되는 골 관련 질환을 치료할 수 있으므로, 골 관련 질환의 예방 또는 치료제 개발에 널리 활용될 수 있다.The pharmaceutical composition for the prevention or treatment of bone diseases containing flunarizine or a pharmaceutically acceptable salt thereof of the present invention as an active ingredient suppresses important factors related to osteoclast differentiation, thereby inhibiting the differentiation of osteoclasts. It has an effect of inhibiting absorption and can be usefully used in preclinical and clinical studies. In addition, since it can treat bone-related diseases caused by imbalance of osteoclasts, it can be widely used for preventing or developing therapeutic agents for bone-related diseases.
도 1은 플루나리진(Flunarizine)의 농도에 따른 RANKL이 처리된 파골세포 전구세포(BMMs)의 분화에 대한 본 발명의 조성물인 플루나리진(Flunarizine)의 농도 및 시간 별 효과를 나타내는 도이다.
도 1a는 다양한 농도의 플루나리진 존재 하에서, 3일 또는 4일 동안 RANKL(50ng/ml) 및 M-CSF(40ng/ml)와 함께 배양된 파골세포의 TRAP 염색 사진이다(스케일바 100 μm).
도 1b는 TRAP 염색된 다핵의 파골세포 수의 정량분석 그래프이다(3개 초과의 핵을 함유하는 TRAP-양성 다핵 세포(MNC)를 계수).
도 1c는 플루나리진의 농도별 세포독성 분석(M: M-CSF/ M+R: M-CSF 및 RANKL) 그래프이다.
도 2는 RANKL이 처리된 파골세포 전구세포(BMMs)의 NFATc1 및 타겟 유전자들의 발현에 대한 본 발명 플루나리진의 효과를 나타내는 도이다.
도 2a는 NFATc1의 발현에 대한 플루나리진의 농도별 효과를 확인한 면역블롯팅(immunoblotting) 결과를 나타내는 도이다.
도 2b는 NFATc1의 발현에 대한 플루나리진의 시간 별 효과를 확인한 면역블롯팅(immunoblotting) 결과를 나타내는 도이다.
도 2c는 RT-PCR을 통해 정량화한 플루나리진 처리에 따른 NFATc1 및 그의 타겟 유전자들의 mRNA 발현 변화를 나타낸 정량 분석 그래프이다.
도 3은 플루나리진이 파골세포의 칼슘 진동에 영향을 미치는지 확인하기 위해 fura-2 형광을 이용한 도이다.
도 3a의 왼쪽 도면은 BMM 세포의 fura-2 이미지를 나타내고, 오른쪽 그래프는 각 실험의 단일 세포에서 fura-2 형광 비율의 변화의 흔적을 나타내는 도이다.
도 3b는 칼슘 진동을 나타내는 세포를 정량화한 그래프이다.
도 4는 플루나리진에 의한 Ca+ 관련 신호 전달 체계 조절을 확인한 도이다.
도 4a는 세포용해물에 p-CaMKIV, CaMKIV, p-CREB 및 CREB에 대한 항체를 사용한 면역블롯팅(immunoblotting) 결과를 나타내는 도이다.
도 4b는 세포를 6μM 플루나리진의 존재 하에서 12시간 동안 RANKL과 함께 인큐베이션하여 c-fos의 표적 유전자의 mRNA 수준을 실시간 PCR에 의해 정량화한 도이다.
도 4c는 세포용해물에 c-fos에 대한 항체를 사용한 면역블롯팅(immunoblotting) 결과를 나타내는 도이다.
도 4d는 RAW264.7 세포를 0.45mg의 pAP-1-Luc(AP-1 reporter plasmid) 및 0.15mg의 pRL-SV40(대조군)으로 24시간 동안 형질감염시켜 상기 세포의 용해물을 이중 루시퍼라제 분석 시스템을 이용하여 측정한 도이다.
도 4e는 세포를 6μM 플루나리진의 존재 하에서 72시간 동안 RANKL과 함께 인큐베이션하여 PGC1β의 표적 유전자의 mRNA 수준을 실시간 PCR에 의해 정량화한 도이다.
도 4f는 세포용해물에 PGC1β에 대한 항체를 사용한 면역블롯팅(immunoblotting) 결과를 나타내는 도이다.
도 5는 플루나리진이 칼슘 신호 전달을 차단하여 파골세포 분화를 억제하는지 여부를 확인하기 위해, 세포 내 칼슘 상승 화합물인 이오노마이신(ionomycin)을 처리한 도이다.
도 5a는 세포를 고정시키고 TRAP 염색을 실시한 뒤 광학 현미경으로 검사한 도이다(스케일바 200μM).
도 5b는 도 5a에서 10개 이상의 핵을 함유하는 TRAP 양성 MNC를 계수하여 정량화한 그래프이다.
도 6은 플루나리진 처리한 분화된 파골세포에서의 액틴링 및 흡수 기능을 측정한 도이다.
도 6a는 BMM 세포를 3일 동안 RANKL로 처리한 후 6μM 플루나리진을 24시간 동안 분화된 파골세포에 첨가하고 시간에 따라 광학 현미경으로 관찰한 도이다(스케일바, 100μm).
도 6b는 상기 세포를 고정시키고 TRAP 염색한 후 광학 현미경으로 관찰한 도이다(스케일바, 400μm).
도 6c는 상기 도 6b에서의 TRAP 양성 다핵세포(MNC)를 정량화한 그래프이다.
도 6d는 BMM 세포를 RANKL의 존재 하에 유리에서 3일 동안 배양한 후 12시간 동안 6μM의 플루나리진에 노출시킨 후 형광 현미경으로 관찰한 도이다(스케일바, 200μm).
도 6e는 BMM 세포를 RANKL의 존재 하에 dentine disc에서 3일 동안 배양한 후 0일 또는 4일에 6μM의 플루나리진에 노출시킨 후 resorption pits를 hematoxylin으로 염색화하여 시각화한 도이다.
도 6f는 상기 도 6e에서의 Pit area를 수치화한 그래프이다.
도 7은 플루나리진에 의한 파골세포 성숙 및 기능에 필요한 신호의 하향 조절을 확인한 도이다.
도 7a는 BMM 세포를 3일 동안 RANKL과 함께 배양한 후 분화된 파골세포를 24시간 동안 6μM의 플루나리진과 배양한 후 세포용해물을 인테그린 β3, p130Cas 및 c-Src에 특이적인 항체를 사용하여 면역블롯팅(immunoblotting)을 분석한 도이다.
도 7b는 파골세포 성숙 및 기능에 관여하는 유전자의 mRNA 수준을 real-time PCR에 의해 정량화한 도이다.
도 8은 플루나리진에 의한 LPS 또는 OVX 유도성 골 파괴 억제효과를 확인한 도이다.
도 8a는 TRAP 및 hematoxylin으로 염색된 두개골의 사진 이미지를 나타낸 도이다.
도 8b는 TRAP으로 염색된 두개골의 절단면 사진 이미지를 나타낸 도이다.
도 8c는 bone cavity 및 TRAP으로 염색된 파골세포를 정량화한 그래프이다.
도 8d는 모의 수술 또는 난소 절제술(OVX)을 받은 마우스의 대퇴골 micro-CT 이미지를 나타낸 도이다.
도 8e는 상기 도 8d의 대퇴골을 조직학적으로 분석한 그래프이다(BMD, 골 무기질 밀도; BV/TV, 총 골량의 일부인 골량; Tb.N, trabecular 수; Tb.Th, trabecular 두께; Tb.Sp, 섬유 분리; BS/BV, 체적비로서의 뼈 표면; BS/TV, 골 표면 밀도; Tb.pf, trabecular 패턴 계수).1 is a diagram showing the concentration and time-dependent effects of flunarizine, a composition of the present invention, on the differentiation of RANKL-treated osteoclast progenitor cells (BMMs) according to the concentration of flunarizine.
1A is a photograph of TRAP staining of osteoclasts cultured with RANKL (50 ng/ml) and M-CSF (40 ng/ml) for 3 or 4 days in the presence of various concentrations of flunarizine (
1B is a graph of quantitative analysis of the number of TRAP-stained multinucleated osteoclasts (counting TRAP-positive multinucleated cells (MNCs) containing more than 3 nuclei).
Figure 1c is a graph showing the cytotoxicity analysis (M: M-CSF / M + R: M-CSF and RANKL) by concentration of flunarizine.
2 is a diagram showing the effect of flunarizine of the present invention on the expression of NFATc1 and target genes in RANKL-treated osteoclast progenitor cells (BMMs).
Figure 2a is a diagram showing the results of immunoblotting (immunoblotting) confirming the effect of each concentration of flunarizine on the expression of NFATc1.
Figure 2b is a diagram showing the results of immunoblotting (immunoblotting) confirming the time-dependent effect of flunarizine on the expression of NFATc1.
Figure 2c is a quantitative analysis graph showing the mRNA expression change of NFATc1 and its target genes according to flunarizine treatment quantified through RT-PCR.
3 is a diagram using fura-2 fluorescence to determine whether flunarizine affects the calcium vibration of osteoclasts.
The left figure of FIG. 3A shows a fura-2 image of BMM cells, and the right graph shows traces of changes in the fura-2 fluorescence ratio in single cells of each experiment.
3B is a graph quantifying cells exhibiting calcium oscillations.
4 is a diagram confirming the regulation of Ca + -related signal transduction system by flunarizine.
4A is a diagram showing the results of immunoblotting using antibodies against p-CaMKIV, CaMKIV, p-CREB and CREB in cell lysates.
Figure 4b is a diagram illustrating the quantification of the mRNA level of the target gene of c-fos by real-time PCR by incubating cells with RANKL in the presence of 6 μM flunarizine for 12 hours.
Figure 4c is a diagram showing the results of immunoblotting (immunoblotting) using an antibody against c-fos in the cell lysate.
Figure 4d shows that the lysates of RAW264.7 cells were transfected with 0.45 mg of pAP-1-Luc (AP-1 reporter plasmid) and 0.15 mg of pRL-SV40 (control) for 24 hours for double luciferase analysis. It is a figure measured using the system.
Figure 4e is a diagram illustrating the quantification of the mRNA level of the target gene of PGC1β by real-time PCR by incubating cells with RANKL in the presence of 6 μM flunarizine for 72 hours.
Figure 4f is a diagram showing the results of immunoblotting (immunoblotting) using the antibody against PGC1β in the cell lysate.
5 is a diagram illustrating treatment with ionomycin, an intracellular calcium-increasing compound, in order to determine whether flunarizine inhibits osteoclast differentiation by blocking calcium signal transduction.
Figure 5a is a diagram examined by an optical microscope after fixing the cells and performing TRAP staining (
FIG. 5B is a graph quantified by counting TRAP-positive MNCs containing 10 or more nuclei in FIG. 5A .
6 is a diagram illustrating the measurement of actinling and absorption functions in flunarizine-treated differentiated osteoclasts.
FIG. 6a is a diagram illustrating BMM cells treated with RANKL for 3 days, followed by addition of 6 μM flunarizine to differentiated osteoclasts for 24 hours and observed with an optical microscope over time (scale bar, 100 μm).
Figure 6b is a view observed under an optical microscope after fixing the cells and staining with TRAP (scale bar, 400 μm).
FIG. 6c is a graph showing quantification of TRAP-positive multinucleated cells (MNCs) in FIG. 6b.
FIG. 6d is a diagram illustrating BMM cells observed under a fluorescence microscope after culturing on glass in the presence of RANKL for 3 days and then exposing them to 6 μM of flunarizine for 12 hours (scale bar, 200 μm).
FIG. 6e is a visualization of resorption pits stained with hematoxylin after BMM cells were cultured on a dentine disc in the presence of RANKL for 3 days and then exposed to 6 μM flunarizine on
6F is a graph in which the pit area in FIG. 6E is digitized.
7 is a diagram confirming the down-regulation of signals required for osteoclast maturation and function by flunarizine.
Figure 7a shows that BMM cells were cultured with RANKL for 3 days, then differentiated osteoclasts were cultured with 6 μM flunarizine for 24 hours, and then cell lysates were prepared using antibodies specific for integrin β3, p130Cas and c-Src. It is a diagram analyzing immunoblotting.
7B is a diagram illustrating quantification of mRNA levels of genes involved in osteoclast maturation and function by real-time PCR.
8 is a view confirming the inhibitory effect of LPS or OVX-induced bone destruction by flunarizine.
Figure 8a is a diagram showing a photographic image of the skull stained with TRAP and hematoxylin.
Figure 8b is a diagram showing a cross-sectional photographic image of the skull stained with TRAP.
8c is a graph quantifying osteoclasts stained with bone cavity and TRAP.
8D is a diagram showing a micro-CT image of a femur of a mouse that has undergone simulated surgery or ovariectomy (OVX).
FIG. 8E is a histologically analyzed graph of the femur of FIG. 8D (BMD, bone mineral density; BV/TV, bone mass as a fraction of total bone mass; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp; , fiber separation; BS/BV, bone surface as a volume ratio; BS/TV, bone surface density; Tb.pf, trabecular pattern coefficient).
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명이 유효성분으로 함유하는 플루나리진(Flunarizine)은 하기 화학식 1로 기재되는 구조를 갖는다:Flunarizine, which the present invention contains as an active ingredient, has a structure represented by the following Chemical Formula 1:
본 발명은 상기 화학식 1로 표시되는 화합물, 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 것을 특징으로 하는, 골질환의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for the prevention or treatment of bone diseases, characterized in that it contains the compound represented by
또한, 본 발명의 플루나리진은 0.1 내지 10 μM 농도인 것을 특징으로 한다. 농도 0.1 μM 미만의 농도를 갖는 플루나리진 조성물은 골질환의 치료 효율이 저하되는 문제점이 있고, 10 μM 이상이면 세포에 독성이 생길 수 있는 문제점이 있다. 이러한 측면에서 플루나리진을 유효성분으로 함유하는 약학적 조성물의 농도는 2 내지 8 μM 인 것이 바람직할 수 있으며, 더욱 바람직하게는 6μM 일 수 있다.In addition, flunarizine of the present invention is characterized in that the concentration of 0.1 to 10 μM. The flunarizine composition having a concentration of less than 0.1 μM has a problem in that the treatment efficiency of bone disease is lowered, and when it is 10 μM or more, there is a problem that toxicity may occur to cells. In this aspect, the concentration of the pharmaceutical composition containing flunarizine as an active ingredient may be preferably 2 to 8 μM, more preferably 6 μM.
또한, 상기 조성물은 NFATc1, Acp5, Mmp9, Ctsk, CalcrC, Dcstamp, c-FOS, p-CaMKIV, p-CREB, PGC1β, Dock5, Src, Atp6v0d2 및 Itgb3로 이루어진 군으로부터 선택되는 어느 하나 이상의 발현 또는 활성을 감소시킬 수 있다.In addition, the composition is NFATc1, Acp5, Mmp9, Ctsk, CalcrC, Dcstamp, c-FOS, p-CaMKIV, p-CREB, PGC1β, Dock5, Src, Atp6v0d2 and Itgb3 any one or more expression or activity selected from the group consisting of can reduce
상기 조성물에서 Acp5, Mmp9, Ctsk, CalcrC 및 Dcstamp은 NFATc1의 표적 유전자로, 플루나리진은 NFATc1의 유전자 발현을 억제할 수 있고, Acp5, Mmp9, Ctsk, CalcrC 및 Dcstamp의 유전자 발현도 억제할 수 있다In the composition, Acp5, Mmp9, Ctsk, CalcrC and Dcstamp are NFATc1 target genes, and flunarizine may inhibit NFATc1 gene expression, and Acp5, Mmp9, Ctsk, CalcrC and Dcstamp gene expression may also be inhibited.
또한, 상기 조성물은 염증 또는 난소 절제술에 의한 골 손상을 억제할 수 있다. 상기 염증은 염증유도물질인 마우스 모델에 LPS(Lipopolysaccharide)를 주입하여 유도할 수 있다.In addition, the composition may inhibit bone damage caused by inflammation or oophorectomy. The inflammation can be induced by injecting LPS (Lipopolysaccharide) into a mouse model, which is an inflammation-inducing substance.
또한, 상기 조성물은 파골세포의 칼슘 진동(calcium oscillation)을 억제할 수 있다.In addition, the composition may suppress calcium oscillation of osteoclasts.
상기 칼슘 진동은 플루나리진에 의해 억제될 수 있고, 이는 p-CaMKIV, p-CREB 및 c-Fos의 하향 조절로 이어질 수 있고, PGC1β의 단백질 발현 및 PGC1β 표적 유전자의 mRNA 수준도 하향 조절될 수 있다.The calcium oscillation can be inhibited by flunarizine, which can lead to down-regulation of p-CaMKIV, p-CREB and c-Fos, and the protein expression of PGC1β and mRNA levels of PGC1β target genes can also be down-regulated. have.
아울러, 본 발명의 조성물이 적용될 수 있는 골질환으로는 성장기 발육부진, 골절, 과도한 파골세포의 골 흡수에 의한 골다공증(osteoprosis), 류마티스성 관절염(rheumatoid arthritis), 치주질환(periodontal disease), 파제트병(Paget disease) 및 전이성 골암(metastatic bone cancers)으로 구성된 군으로부터 선택되는 어느 하나 이상인 것이 바람직하나, 이에 한정되지는 않는다.In addition, bone diseases to which the composition of the present invention can be applied include growth retardation, fracture, osteoporosis due to excessive bone resorption of osteoclasts, rheumatoid arthritis, periodontal disease, Paget It is preferable that at least one selected from the group consisting of disease (Paget disease) and metastatic bone cancers, but is not limited thereto.
본 발명의 구체적인 실험예에서, 상기 화학식 1로 기재되는 플루나리진(Flunarizine)이 RANKL에 의한 파골세포 전구세포(BMMs)의 분화를 효과적으로 억제함을 확인하였다(도 1 참조). 또한 플루나리진을 처리하였을 때, 파골세포 분화에서 가장 중요하게 알려져 있는 transcription factor인 NFATc1 단백질과 그 타겟 유전자들인 Acp5, Mmp9, Ctsk, CalcrC 및 Dcstamp 유전자의 mRNA 발현이 억제되었다(도 2 참조).In a specific experimental example of the present invention, it was confirmed that flunarizine represented by
또한, 플루나리진 존재 하에서 파골세포의 칼슘 진동이 유의적으로 억제되는 것을 확인하였고(도 3 참조), NFATc1의 upstream 조절자로 알려진 c-Fos 단백질과 mRNA의 발현이 유의적으로 억제되는 것을 확인하였으며(도 4 참조), CaMKIV, CREB 등의 인산화가 억제되는 것을 확인하였다(도 4 참조). In addition, it was confirmed that calcium oscillation of osteoclasts was significantly suppressed in the presence of flunarizine (see FIG. 3), and it was confirmed that the expression of c-Fos protein and mRNA, known as upstream regulators of NFATc1, was significantly suppressed. (See FIG. 4), it was confirmed that phosphorylation of CaMKIV, CREB, etc. is inhibited (see FIG. 4).
또한, 플루나리진이 액틴링의 형성과 dentine disc에서 파골세포에 의한 골 흡수를 저해하며(도 6 참조), 파골세포 생성(osteoclastogenesis)의 말기를 억제하며(도 7 참조), 파골세포 성숙 및 기능에서 중요한 역할을 담당하는 Dcstamp, Dock5, MMP9, Ctsk, Src, Atp6v0d2 및 Itgb3 유전자의 mRNA 수준을 감소시키는 것을 확인하였고(도 7 참조), LPS 염증 또는 난소 절제술에 의한 골의 손상을 억제함을 확인하였다(도 8 참조).In addition, flunarizine inhibits the formation of actin rings and bone resorption by osteoclasts in the dentine disc (see FIG. 6), suppresses the end of osteoclastogenesis (see FIG. 7), and osteoclast maturation and function It was confirmed that the mRNA levels of Dcstamp, Dock5, MMP9, Ctsk, Src, Atp6v0d2 and Itgb3 genes, which play an important role in the (see FIG. 8).
따라서, 본 발명의 플루나리진을 유효성분으로 함유하는 조성물은 골질환의 예방 또는 치료를 위해 유용하게 사용될 수 있다.Therefore, the composition containing flunarizine of the present invention as an active ingredient can be usefully used for the prevention or treatment of bone diseases.
또한, 본 발명은 상기 화학식 1로 표시되는 화합물뿐만 아니라, 이의 약학적으로 허용되는 염, 이로부터 제조될 수 있는 가능한 용매화물, 수화물, 라세이체 또는 입체이성질체를 모두 포함한다.In addition, the present invention includes not only the compound represented by
본 발명은 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용되는 염의 형태로 사용할 수 있으며, 염으로는 약학적으로 허용 가능한 유리산(free acid)에 의해 형성된 산 부가염이 유용하다. 산 부가염은 염산, 질산, 인산, 황산, 브롬화수소산, 요오드화수소산, 아질산 또는 아인산과 같은 무기산류와 지방족 모노 및 디카르복실레이트, 페닐-치환된 알카노에이트, 하이드록시 알카노에이트 및 알칸이도에이트, 방향족 산류, 지방족 및 방향족 설폰산류와 같은 무독성 유기산으로부터 얻는다. 이러한 약학적으로 무독한 염류로는 설페이트, 피로설페이트, 바이설페이트, 설파이트, 바이설파이트,니트레이트, 포스페이트, 모노하이드로겐 포스페이트, 디하이드로겐 포스페이트, 메타포스페이트, 피로포스페이트 클로라이드, 브로마이드, 아이오다이드, 플루오라이드, 아세테이트, 프로피오네이트, 데카노에이트, 카프릴레이트, 아크릴레이트, 포메이트, 이소부티레이트, 카프레이트, 헵타노에이트, 프로피올레이트, 옥살레이트, 말로네이트, 숙시네이트, 수베레이트, 세바케이트, 푸마레이트, 말리에이트, 부틴-1,4-디오에이트, 헥산-1,6-디오에이트, 벤조에이트, 클로로벤조에이트, 메틸벤조에이트, 디니트로 벤조에이트, 하이드록시벤조에이트, 메톡시벤조에이트, 프탈레이트, 테레프탈레이트, 벤젠설포네이트, 톨루엔설포네이트, 클로로벤젠설포네이트, 크실렌설포네이트, 페닐아세테이트, 페닐프로피오네이트, 페닐부티레이트, 시트레이트, 락테이트, 하이드록시부티레이트, 글리콜레이트, 말레이트, 타트레이트, 메탄설포네이트, 프로판설포네이트, 나프탈렌-1-설포네이트, 나프탈렌-2-설포네이트 또는 만델레이트를 포함한다.The present invention can be used in the form of the compound represented by
본 발명에 따른 산 부가염은 통상의 방법, 예를 들면 상기 화학식 1로 표시되는 화합물을 과량의 산 수용액 중에 용해시키고, 이 염을 수혼화성 유기 용매, 예를 들면 메탄올, 에탄올, 아세톤 또는 아세토니트릴을 사용하여 침전시켜서 제조할 수 있다. 또한, 동량의 상기 화학식 1로 표시되는 화합물, 및 산 수용액 또는 알코올을 가열하고, 이어서 이 혼합물을 증발시켜서 건조하거나 또는 석출된 염을 흡입 여과시켜 제조할 수도 있다.The acid addition salt according to the present invention is prepared by a conventional method, for example, by dissolving the compound represented by
또한, 염기를 사용하여 약학적으로 허용가능 한 금속염을 만들 수 있다. 알칼리 금속 또는 알칼리 토금속 염은, 예를 들면 화합물을 과량의 알칼리 금속 수산화물 또는 알칼리 토금속 수산화물 용액 중에 용해하고, 비용해 화합물 염을 여과하고, 여액을 증발, 건조 시켜 얻는다. 이때, 금속염으로는 나트륨, 칼륨 또는 칼슘염을 제조하는 것이 제약상 적합하다. 또한, 이에 대응하는 은 염은 알칼리 금속 또는 알칼리 토금속 염을 적당한 음염(예, 질산은)과 반응시켜 얻는다.In addition, a pharmaceutically acceptable metal salt can be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg silver nitrate).
상기 조성물을 제제화할 경우, 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 제조된다.When formulating the composition, it is usually prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant.
경구 투여를 위한 고형제에는 정제, 환제, 산제, 과립제, 캡슐제, 트로키제 등이 포함되며, 이러한 고형 제제는 하나 이상의 본 발명의 상기 화학식 1로 표시되는 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로스(sucrose) 또는 락토오스(lactose) 또는 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제 또는 시럽제 등이 해당되는데, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc., and these solid preparations include at least one excipient in one or more compounds represented by
비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁용제, 유제, 동결건조제제, 좌제 등이 포함된다.Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspension solutions, emulsions, lyophilized formulations, suppositories, and the like.
비수성용제, 현탁용제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 81, 카카오지, 타우린지, 글리세롤, 젤라틴 등이 사용될 수 있다.Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 81, cacao butter, taurine paper, glycerol, gelatin, etc. may be used.
본 발명에 따른 조성물은 약제학적으로 유효한 양으로 투여한다. 본 발명에 있어서, "약제학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , can be determined according to factors including sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.
구체적으로, 본 발명에 따른 화합물의 유효량은 환자의 나이, 성별, 체중에 따라 달라질 수 있으며, 일반적으로는 체중 1 kg 당 0.1 mg 내지 100 mg, 바람직하게는 0.5 mg 내지 10 mg을 매일 또는 격일 투여하거나, 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나 투여 경로, 질환의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.Specifically, the effective amount of the compound according to the present invention may vary depending on the age, sex, and weight of the patient, and in general, 0.1 mg to 100 mg per kg body weight, preferably 0.5 mg to 10 mg per kg body weight, is administered daily or every other day Or, it can be administered in divided
본 발명의 조성물은 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용될 수 있다.The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.
또한, 본 발명은 상기 화학식 1로 표시되는 플루나리진 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 골질환의 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving bone disease containing flunarizine represented by
또한, 본 발명의 플루나리진은 0.1 내지 10 μM 농도인 것을 특징으로 한다. 농도 0.1 μM 미만의 농도를 갖는 플루나리진 조성물은 골질환의 치료 효율이 저하되는 문제점이 있고, 10 μM 이상이면 세포에 독성이 생길 수 있는 문제점이 있다. 이러한 측면에서 플루나리진을 유효성분으로 함유하는 건강기능식품 조성물의 농도는 2 내지 8 μM 인 것이 바람직할 수 있으며, 더욱 바람직하게는 6μM 일 수 있다.In addition, flunarizine of the present invention is characterized in that the concentration of 0.1 to 10 μM. The flunarizine composition having a concentration of less than 0.1 μM has a problem in that the treatment efficiency of bone disease is lowered, and when it is 10 μM or more, there is a problem that toxicity may occur to cells. In this aspect, the concentration of the health functional food composition containing flunarizine as an active ingredient may be preferably 2 to 8 μM, more preferably 6 μM.
또한, 상기 조성물은 NFATc1, Acp5, Mmp9, Ctsk, CalcrC, Dcstamp, c-FOS, p-CaMKIV, p-CREB, PGC1β, Dock5, Src, Atp6v0d2 및 Itgb3로 이루어진 군으로부터 선택되는 어느 하나 이상의 발현 또는 활성을 감소시킬 수 있다.In addition, the composition is NFATc1, Acp5, Mmp9, Ctsk, CalcrC, Dcstamp, c-FOS, p-CaMKIV, p-CREB, PGC1β, Dock5, Src, Atp6v0d2 and Itgb3 any one or more expression or activity selected from the group consisting of can reduce
상기 조성물에서 Acp5, Mmp9, Ctsk, CalcrC 및 Dcstamp은 NFATc1의 표적 유전자로, 플루나리진은 NFATc1의 유전자 발현을 억제할 수 있고, Acp5, Mmp9, Ctsk, CalcrC 및 Dcstamp의 유전자 발현도 억제할 수 있다In the composition, Acp5, Mmp9, Ctsk, CalcrC and Dcstamp are NFATc1 target genes, and flunarizine may inhibit NFATc1 gene expression, and Acp5, Mmp9, Ctsk, CalcrC and Dcstamp gene expression may also be inhibited.
또한, 상기 조성물은 염증 또는 난소 절제술에 의한 골 손상을 억제할 수 있다.In addition, the composition may inhibit bone damage caused by inflammation or oophorectomy.
상기 염증은 염증유도물질인 마우스 모델에 LPS(Lipopolysaccharide)를 주입하여 유도할 수 있고, 상기 난소 절제술에 의한 골 손상은 폐경기 골다공증의 OVX-유도 모델을 이용할 수 있다.The inflammation can be induced by injecting LPS (Lipopolysaccharide) into a mouse model, which is an inflammation-inducing substance, and bone damage by ovariectomy can be performed using an OVX-induced model of postmenopausal osteoporosis.
또한, 상기 조성물은 파골세포의 칼슘 진동(calcium oscillation)을 억제할 수 있다.In addition, the composition may suppress calcium oscillation of osteoclasts.
상기 칼슘 진동은 플루나리진에 의해 억제될 수 있고, 이는 p-CaMKIV, p-CREB 및 c-Fos의 하향 조절로 이어질 수 있고, PGC1β의 단백질 발현 및 PGC1β 표적 유전자의 mRNA 수준도 하향 조절될 수 있다.The calcium oscillation can be inhibited by flunarizine, which can lead to down-regulation of p-CaMKIV, p-CREB and c-Fos, and the protein expression of PGC1β and mRNA levels of PGC1β target genes can also be down-regulated. have.
아울러, 본 발명의 조성물이 적용될 수 있는 골질환으로는 성장기 발육부진, 골절, 과도한 파골세포의 골 흡수에 의한 골다공증(osteoprosis), 류마티스성 관절염(rheumatoid arthritis), 치주질환(periodontal disease), 파제트병(Paget disease) 및 전이성 골암(metastatic bone cancers)으로 구성된 군으로부터 선택되는 어느 하나 이상인 것이 바람직하나, 이에 한정되지는 않는다.In addition, bone diseases to which the composition of the present invention can be applied include growth retardation, fracture, osteoporosis due to excessive bone resorption of osteoclasts, rheumatoid arthritis, periodontal disease, Paget It is preferable that at least one selected from the group consisting of disease (Paget disease) and metastatic bone cancers, but is not limited thereto.
본 명세서의 "건강기능식품"이란 일상 식사에서 결핍되기 쉬운 영양소나 인체에 유용한 기능을 가진 원료나 성분 (기능성 원료)을 사용하여 제조한 것으로, 인체의 정상적인 기능을 유지하거나 생리기능 활성화를 통하여 건강을 유지하고 개선하는 식품으로 식품의약품안전처장이 정한 것을 의미하나, 이에 한정되지 않으며 통상적인 의미의 건강식품을 배제하는 의미로 사용된 것이 아니다."Health functional food" as used herein refers to a nutrient that is easily deficient in daily meals or manufactured using raw materials or ingredients (functional raw materials) having useful functions in the human body, and maintains normal functions of the human body or promotes health through physiological function activation. It refers to food that maintains and improves food safety, but is not limited thereto and is not used in the meaning of excluding health food in the ordinary sense.
본 발명의 조성물은 식품에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합량은 그의 사용 목적(예방 또는 개선용)에 따라 적합하게 결정될 수 있다. 일반적으로, 건강기능식품 중의 상기 화합물의 양은 전체 식품 중량의 0.01 내지 90 중량부로 가할 수 있다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취 시에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.The composition of the present invention may be added to food as it is or used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of its use (for prevention or improvement). In general, the amount of the compound in the health functional food may be added in an amount of 0.01 to 90 parts by weight based on the total weight of the food. However, during long-term ingestion for health and hygiene or health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than or equal to the above range.
본 발명의 건강 기능성 음료 조성물은 지시된 비율로 필수 성분으로서 상기 조성물을 함유하는 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 단당류, 예를 들어, 포도당, 과당 등; 이당류, 예를 들어 말토스, 수크로스 등; 및 다당류, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제{타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등)} 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 g 당 일반적으로 약 1 내지 20 g, 바람직하게는 약 5 내지 12 g이다.The health functional beverage composition of the present invention is not particularly limited in other ingredients except for containing the composition as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides, for example, conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents {thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)} and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used have. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 g of the composition of the present invention.
상기 외에 본 발명의 조성물은 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 본 발명의 조성물은 천연 과일 쥬스 및 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다.In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages.
이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0.1 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.
이하, 본 발명을 실험예에 의해서 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of experimental examples.
단, 하기 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실험예에 의해서 한정되는 것은 아니다. However, the following experimental examples only illustrate the present invention, and the content of the present invention is not limited by the following experimental examples.
<실시예 1> 실험 준비<Example 1> Experimental preparation
1-1. 시약 및 항체 준비1-1. Reagent and Antibody Preparation
가용성 마우스 RANKL을 암호화하는 DNA 단편(아미노산 157 내지 316)은 pET-28b(Novagen)의 salI 및 NotI 부위에 클로닝하였다. 성숙한(mature) 인간 M-CSF(아미노산 33 내지 190)를 암호화하는 DNA 단편은 pET-11a(Novagen)의 NdeI 및 BamHI 부위에 클로닝하였다.A DNA fragment encoding soluble mouse RANKL (amino acids 157 to 316) was cloned into the salI and NotI sites of pET-28b (Novagen). A DNA fragment encoding mature human M-CSF (amino acids 33 to 190) was cloned into the NdeI and BamHI sites of pET-11a (Novagen).
RANKL 및 M-CSF 단백질은 isopropyl-β-D-thiogalactopyranoside 유도에 의해 Escherichia coli BL21(DE3)에서 발현되었다. His-태그된 RANKL은 음이온 교환 및 Ni-chelating column chromatography를 사용하여 정제하고, 불용성 단백질의 리 폴딩으로부터 음이온 교환 및 hydrophobic column chromatography를 통해 정제하여 M-CSF를 수득하였다. 플루나리진(Flunarizine, FN) 이염산염은 AK sci로부터 구입 하였다. 플루나리진은 DMSO 중 28.64mg/ml(60mM) 스톡으로 준비되었고 -80℃에 저장되었다. 이오노마이신(ionomycin)은 Agscientific에서, Fura-2 AM은 Molecular probe에서, Pluronic F-127은 Invitrogen에서, 리포폴리사카라이드(LPS)는 시그마(Sigma)에서 구입하였다.RANKL and M-CSF proteins were expressed in Escherichia coli BL21(DE3) by isopropyl-β-D-thiogalactopyranoside induction. His-tagged RANKL was purified using anion exchange and Ni-chelating column chromatography, and M-CSF was obtained by anion exchange and hydrophobic column chromatography from refolding of insoluble protein. Flunarizine (FN) dihydrochloride was purchased from AK sci. Flunarizine was prepared as a 28.64 mg/ml (60 mM) stock in DMSO and stored at -80°C. Ionomycin was purchased from Agscientific, Fura-2 AM from Molecular probe, Pluronic F-127 from Invitrogen, and lipopolysaccharide (LPS) from Sigma.
β-액틴, PGC1β 및 phospho-CaMKIV에 대한 토끼 다클론성 항체는 Abcam으로부터 구입하였다.Rabbit polyclonal antibodies to β-actin, PGC1β and phospho-CaMKIV were purchased from Abcam.
phospho-JNK, phospho-ERK, phospho-p38 및 phospho-CREB에 대한 토끼 다클론성 항체는 Cell Signaling Technology에서 구입하였다. JNK1, p38 및 c-Fos에 대한 토끼 다클론성 항체, 및 ERK2, NFATc1, 인테그린 β3, 5 CaMKIV 및 CREB에 대한 마우스 다클론성 항체는 Santa Cruz Biotechnology, Inc.에서 구입하였다.Rabbit polyclonal antibodies against phospho-JNK, phospho-ERK, phospho-p38 and phospho-CREB were purchased from Cell Signaling Technology. Rabbit polyclonal antibodies to JNK1, p38 and c-Fos, and mouse polyclonal antibodies to ERK2, NFATc1, integrins β3, 5 CaMKIV and CREB were purchased from Santa Cruz Biotechnology, Inc.
1-2. 파골세포 전구세포(bone marrow-derived macrophage) 준비1-2. Preparation of osteoclast progenitor cells (bone marrow-derived macrophage)
파골세포 전구세포(bone marrow-derived macrophage, BMM)는 6 내지 8주령 C57BL/6 수컷 마우스의 대퇴골 및 경골로부터 제조하였다.Bone marrow-derived macrophage (BMM) was prepared from the femur and tibia of 6-8 week old C57BL/6 male mice.
구체적으로, 골수세포는 10% 태아 소 혈청(fetal bovine serum, FBS), 100U/ml 페니실린 및 100g/ml 스트렙토마이신을 함유하는 α-최소 필수 배지(α-MEM)를 사용하여 골수강(bone-marrow cavity)으로부터 플러싱하였고, 실온에서 800 rpm에서 원심 분리를 통해 수확하였다. 이어서 세포 펠릿을 동일한 배지에 재현탁시켰다. 37℃에서 1일 동안 배양한 후, 비부착성 세포를 수확하고 Gey's solution에서 10분 동안 배양하여 적혈구를 제거하였다. 원심 분리를 통한 정화 후, 세포를 10% FBS, 100U/ml 페니실린 및 100g/ml 스트렙토마이신 및 40ng/ml 재조합 인간 M-CSF를 함유하는 α-MEM에서 배양하였다. 3일 후, 부착 세포를 파골세포 생성을 위한 파골세포 전구세포로 사용하였다.Specifically, bone marrow cells were isolated from bone marrow using α-minimum essential medium (α-MEM) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 g/ml streptomycin. marrow cavity) and harvested by centrifugation at 800 rpm at room temperature. The cell pellet was then resuspended in the same medium. After incubation at 37°C for 1 day, non-adherent cells were harvested and incubated in Gey's solution for 10 minutes to remove red blood cells. After clarification by centrifugation, cells were cultured in α-MEM containing 10% FBS, 100 U/ml penicillin and 100 g/ml streptomycin and 40 ng/ml recombinant human M-CSF. After 3 days, adherent cells were used as osteoclast progenitors for osteoclast generation.
<실험예 1> 플루나리진의 파골세포 분화 억제<Experimental Example 1> Inhibition of osteoclast differentiation of flunarizine
본 발명자들은 플루나리진의 파골세포 분화 억제 효과를 확인하기 위하여, BMM 세포를 대상으로 하여 실험을 수행하였다. The present inventors performed an experiment on BMM cells in order to confirm the inhibitory effect of flunarizine on osteoclast differentiation.
구체적으로, 다양한 농도(0, 2, 4, 6 및 8μM)의 플루나리진 존재 하에서 BMM 세포에 40ng/ml의 M-CSF(macrophage-colony stimulating factor)와 50ng/ml의 RANKL(Receptor activator of nuclear factor kappa-Β ligand)을 3일 또는 4일 동안 처리하여 파골세포로 분화시키고 PBS 워싱처리 후 4% 파라포름알데하이드(paraformaldehyde)로 고정한 뒤 leukocyte acid phosphatase cytochemistry kit (SigmaAldrich)를 사용하여 TRAP 염색한 후, TRAP 염색된 3개 이상의 다핵을 가진 파골세포의 수를 현미경으로 분석하였다. Specifically, 40 ng/ml of macrophage-colony stimulating factor (M-CSF) and 50 ng/ml of RANKL (Receptor activator of nuclear) in BMM cells in the presence of various concentrations (0, 2, 4, 6 and 8 μM) of flunarizine factor kappa-Β ligand) for 3 or 4 days to differentiate into osteoclasts, and after PBS washing treatment, fixation with 4% paraformaldehyde, leukocyte acid phosphatase cytochemistry kit (SigmaAldrich), followed by TRAP staining , The number of TRAP-stained osteoclasts with 3 or more multinuclear cells was analyzed under a microscope.
그 결과, RANKL을 3일 동안 처리하여 파골세포로 분화시켰을 때 4μM의 플루나리진에서부터 파골세포 분화가 유의적으로 억제되었으며(도 1a), 4일 동안 처리하였을 때는 6μM의 플루나리진에서부터 파골세포 분화가 유의적으로 억제되었다(도 1b).As a result, osteoclast differentiation was significantly inhibited from 4 μM flunarizine when treated with RANKL for 3 days to differentiate into osteoclasts (FIG. 1a), and osteoclasts from 6 μM flunarizine when treated for 4 days Differentiation was significantly inhibited (Fig. 1b).
또한, 플루나리진의 파골세포 분화 억제효과가 세포독성에 의한 것임을 배제하기 위하여, EASY Cytox (WST-1) assay kit로 세포독성을 분석하였다. BMM 세포를 각각 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) reagent (Roche Applied Science)를 10μl씩 넣어주고 37 ℃에서 2-4시간 배양한 뒤, 450nm에서의 흡광도를 측정하였다.In addition, in order to exclude that the osteoclast differentiation inhibitory effect of flunarizine is due to cytotoxicity, cytotoxicity was analyzed with the EASY Cytox (WST-1) assay kit. Add 10 μl of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) reagent (Roche Applied Science) to each BMM cell. After incubation at 37 °C for 2-4 hours, absorbance at 450 nm was measured.
그 결과, 다양한 농도의 플루나리진(0, 2, 4, 6 및 8μM)을 3일 동안 처리하였을 때 8μM 농도까지 플루나리진에 의한 세포독성이 나타나지 않는 것을 확인하였다(도 1c). 이는 플루나리진의 파골세포 분화 억제효과가 세포사멸에 의한 것이 아님을 보여준다.As a result, it was confirmed that when flunarizine at various concentrations (0, 2, 4, 6 and 8 μM) was treated for 3 days, cytotoxicity by flunarizine did not appear up to a concentration of 8 μM ( FIG. 1c ). This shows that the inhibitory effect on osteoclast differentiation of flunarizine is not due to apoptosis.
<실험예 2> 플루나리진에 의한 NFATc1 발현 억제<Experimental Example 2> Inhibition of NFATc1 expression by flunarizine
플루나리진의 파골세포 분화 억제 효과를 확인하기 위하여, 다양한 농도(0, 2, 4, 6 및 8μM)의 플루나리진 존재 하에서 BMM 세포에 2일 동안 50ng/ml의 RANKL을 처리하여 파골세포로 분화시키고, 파골세포의 분화에서 가장 중요하게 알려져 있는 전사 인자(transcription factor)인 NFATc1 단백질의 발현을 확인하기 위해 NFATc1에 대한 항체를 사용한 면역 블롯(immunoblot)을 수행하였다. To confirm the osteoclast differentiation inhibitory effect of flunarizine, BMM cells were treated with 50ng/ml of RANKL for 2 days in the presence of flunarizine at various concentrations (0, 2, 4, 6 and 8 μM) to differentiate them into osteoclasts. In order to confirm the expression of NFATc1 protein, which is the most important transcription factor known in the differentiation of osteoclasts, an immunoblot using an antibody against NFATc1 was performed.
구체적으로, 10% 폴리아크릴아미드 겔(polyacrylamide gel)을 이용하여 단백질을 분리하고, 분리된 단백질을 니트로 셀룰로오스 막(nitrocellulose membranes)에 옮겨주었다. TBST(0.05% Tween-20 in Tris-buffered saline, pH 7.4)에 녹인 5% 탈지유를 이용하여 막에 있는 비특이적 결합부위를 모두 블로킹하였다. 일차항체(mouse monoclonal antibodies, Santa Cruz Biotechnology Inc)를 14시간 동안 4℃에서 처리한 후 이차항체를 1시간 동안 상온에서 처리하여 West save Up(Ab Frontier)으로 분석하였다.Specifically, the protein was separated using a 10% polyacrylamide gel, and the separated protein was transferred to nitrocellulose membranes. All non-specific binding sites on the membrane were blocked using 5% skim milk dissolved in TBST (0.05% Tween-20 in Tris-buffered saline, pH 7.4). Primary antibodies (mouse monoclonal antibodies, Santa Cruz Biotechnology Inc) were treated at 4°C for 14 hours, and then secondary antibodies were treated at room temperature for 1 hour and analyzed by West save Up (Ab Frontier).
그 결과, NFATc1 단백질은 6μM의 플루나리진 농도에서부터 상당히 억제되었으며(도 2a), 4일까지 억제효과가 지속되는 것을 확인하였다(도 2b). As a result, it was confirmed that the NFATc1 protein was significantly inhibited from the flunarizine concentration of 6 μM ( FIG. 2A ), and the inhibitory effect continued until 4 days ( FIG. 2B ).
또한, NFATc1 및 그의 타겟 유전자인 Acp5, Mmp9, Ctsk, CalcrC 및 Dcstamp 유전자의 mRNA 발현 변화를 real time-PCR로 확인하였다. Also, mRNA expression changes of NFATc1 and its target genes Acp5, Mmp9, Ctsk, CalcrC and Dcstamp genes were confirmed by real time-PCR.
구체적으로, real time-PCR을 위해, Trizol reagent(Invitrogen)을 사용하여 총 RNA를 추출하여 260nm의 흡광도에서 정량 분석하였으며, 총 RNA(2㎍)를 0.5㎍의 oligo(dT) primers를 포함, 총 15㎕가 되게 하여 70℃에서 5분간 인큐베이션한 후, 얼음에서 바로 식혀주었다. cDNA 처음 가닥은 M-MLV reverse transcriptase (Promega) 200 units, ribonuclease inhibitor 24 units 및 각각의 dNTP 0.25mM를 포함한 최종볼륨 25㎕에서 42℃에서 1시간, 70℃에서 10분 간 합성하였다. 희석 cDNA template의 0.8㎕씩(1:2.5)을 2X SYBR Green PCR Master Mix(M Biotech) 10㎕, 각 gene-specific primer(제노텍) 10μM 의 1㎕씩을 포함한 최종 20㎕ 볼륨에서 ABI 7300 real time PCR System(Applied Biosystems)을 사용하여 증폭시켰으며, 증폭은 50℃에서 2분간, 95℃에서 2분간, 다음으로 95℃에서 15초간, 60℃에서 1분간 40 사이클을 수행하여 실험하였다. 실험에서 사용한 Primer의 서열은 하기 표 1과 같다.Specifically, for real-time-PCR, total RNA was extracted using Trizol reagent (Invitrogen) and quantitatively analyzed at absorbance at 260 nm. Total RNA (2㎍) containing 0.5㎍ of oligo(dT) primers After incubation at 70° C. for 5 minutes to make 15 μl, it was immediately cooled on ice. The first strand of cDNA was synthesized in a final volume of 25 μl containing 200 units of M-MLV reverse transcriptase (Promega), 24 units of ribonuclease inhibitor and 0.25 mM each dNTP, at 42°C for 1 hour and at 70°C for 10 minutes. ABI 7300 real time in the final 20 μl volume including 0.8 μl of diluted cDNA template (1:2.5), 10 μl of 2X SYBR Green PCR Master Mix (M Biotech) and 1 μl of each gene-specific primer (Genotech) 10 μM It was amplified using a PCR System (Applied Biosystems), and amplification was tested by performing 40 cycles at 50° C. for 2 minutes, 95° C. for 2 minutes, then 95° C. for 15 seconds, and 60° C. for 1 minute. The sequences of the primers used in the experiment are shown in Table 1 below.
size (bp)Amplicon
size (bp)
5′-CCAATGAACAGCTGTAGCG-3′5'-CTTCAGCTGGAGGACACC-3'
5'-CCAATGAACAGCTGTAGCG-3'
서열번호 2SEQ ID NO: 1
SEQ ID NO: 2
5′-CGTGGCGTTATACATACAAC-3′5'-ACCACTGCCTTCCAATACG-3'
5'-CGTGGGCGTTATACATACAAC-3'
서열번호 4SEQ ID NO: 3
SEQ ID NO: 4
5′-TGCTGTCGGCTGTGGTTC-3′5'-AGACGACATAGACGGCATC-3'
5'-TGCTGTCGGCTGTGGTTC-3'
서열번호 6SEQ ID NO: 5
SEQ ID NO: 6
5′-AGAGACGTTGCCAAGGTGAT-3′5'-CCAGCGACAAGAGGTTCC-3'
5'-AGAGACGTTGCCAAGGTGAT-3'
서열번호 8SEQ ID NO: 7
SEQ ID NO: 8
5′-ACTGGATCAATCTGTAGGAG-3′5'-TGATGACTCTCAGGACAATG-3'
5'-ACTGGATCAATTCTGTAGGAG-3'
서열번호 10SEQ ID NO: 9
SEQ ID NO: 10
5′-ACAGAAGAGAGCAGGGCAACG-3′ 5'-TTATGTGTTTCCACGAAGCCCTA-3'
5'-ACAGAAGAGAGCAGGGCAACG-3'
서열번호 12SEQ ID NO: 11
SEQ ID NO: 12
5′-CACCCCAACTATGCACGTGG-35'-TGGTGACACAGGGACAGTGG-3'
5'-CACCCCAACTATGCACGTGG-3
서열번호 14SEQ ID NO: 13
SEQ ID NO: 14
5′-GAGAAACGGAGAATCCGAAG-3′5'-GAGAAACGGAGAATCCGAAG-3'
5'-GAGAAACGGAGAATCCGAAG-3'
서열번호 16SEQ ID NO: 15
SEQ ID NO: 16
5′-TGTCCCCCACCATTGAACTT-35'-ATTAACGCGCAGATCATGCA-3'
5′-TGTCCCCCACCATTGAACTT-3
서열번호 18SEQ ID NO: 17
SEQ ID NO: 18
5′-GTCTGTCCAGAGTTTCACC-3′5'-GATCATAAGCGAGGACCTG-3'
5'-GTCTGTCCAGAGTTTCACC-3'
서열번호 20SEQ ID NO: 19
SEQ ID NO: 20
5′-TCTGACCATCTTCCCTGTCC-3′ 5'-GGAGTGGCTGATCCAGATGT-3'
5′-TCTGACCATCTTCCCTGTCC-3′
서열번호 22SEQ ID NO: 21
SEQ ID NO: 22
5′-TGCCAAATGAGTTCAG-3′5'-CAGAGATGGAAGCTGT-3'
5'-TGCCAAATGAGTTCAG-3'
서열번호 24SEQ ID NO: 23
SEQ ID NO: 24
5′-GGGCCAGAAGTTCCCTTAGG-3′5'-CTCCAGGCAGGTTCAACCC-3'
5'-GGGCCAGAAGTTCCCTTAGG-3'
서열번호 26SEQ ID NO: 25
SEQ ID NO: 26
5′-TTGCTAGGGCCGCGATAAT-35'-GCCACCTTGACCCGATTCT-3'
5'-TTGCTAGGGCCGCGATAAT-3
서열번호 28SEQ ID NO: 27
SEQ ID NO: 28
5′-TCCTCGGGCCATGATTATAGTAC-3′ 5'-CATCACTCCTATTCTGCCTAGCAA-3'
5'-TCCTCGGGCCATGATTATAGTAC-3'
서열번호 30SEQ ID NO: 29
SEQ ID NO: 30
5′-GAAGAATGTTATGTTTACTCCTACGAATATG-3′ 5'-TTTTCAGGCTTCACCCTAGATGA-3'
5'-GAAGAATGTTATGTTTACTCCTACGAATATG-3'
서열번호 32SEQ ID NO: 31
SEQ ID NO: 32
5′-CAGCAGCCTCCTAGATCATGTG-3′ 5'-CGGAAGTATTTTTCTTTGCAGGAT-3'
5'-CAGCAGCCTCCTAGATCATGTG-3'
서열번호 34SEQ ID NO: 33
SEQ ID NO: 34
5′-GCCTGGATGGCTACGTAC-3′5'-ACCCTAAGGCCAACCGTG-3'
5'-GCCTGGATGGCTACGTAC-3'
서열번호 36SEQ ID NO: 35
SEQ ID NO: 36
그 결과, 6μM 플루나리진 존재 하에서 2일 동안 50ng/ml RANKL을 처리하였을 때 NFATc1 및 그의 타겟 유전자인 Acp5, Mmp9, Ctsk, CalcrC 및 Dcstamp 유전자의 mRNA도 발현이 현저히 억제되는 것을 확인하였다(도 2c).As a result, it was confirmed that the mRNA expression of NFATc1 and its target genes Acp5, Mmp9, Ctsk, CalcrC and Dcstamp genes was significantly suppressed when 50 ng/ml RANKL was treated in the presence of 6 μM flunarizine for 2 days (Fig. 2c). ).
<실험예 3> 플루나리진에 의한 파골세포 전구세포에서의 RANFL-자극된 Ca+ 신호 전달 및 하류 경로의 감쇠<Experimental Example 3> Attenuation of RANFL-stimulated Ca+ signal transduction and downstream pathways in osteoclast progenitor cells by flunarizine
RANKL에 의해 유도된 칼슘 진동(calcium oscillation)은 파골세포의 분화에서 중요한 역할을 한다. 따라서 플루나리진이 파골세포의 칼슘 진동에 영향을 미치는지 여부를 조사하였다.Calcium oscillation induced by RANKL plays an important role in osteoclast differentiation. Therefore, we investigated whether flunarizine affects calcium oscillations in osteoclasts.
구체적으로 BMM 세포를 12-well plate의 바닥에서 커버 슬립 유리 상에 도말하고 40ng/ml M-CSF 및 50ng/ml RANKL과 함께 48시간 동안 배양하였고, 상기 세포를 6μM 플루나리진을 첨가하거나 첨가하지 않고 48시간 동안 처리하였다. 이어서, 세포에 5mM Fura-2 AM 및 5μM Pluronic F12를 실온에서 30분 동안 RT에서 규칙적인 완충액: 10mM HEPES, 140mM NaCl, 10mM 포도당, 5mM KCl, 1mM CaCl 및 1mM MgCl2, pH 7.4로 로딩하였다. 세포의 세포질 칼슘 진동은 340 및 380 nm의 여기 파장 및 510 nm의 방출 파장에서 형광 현미경(Axio Observer A1; Zeiss)을 사용하여 측정되었다. 변동-양성 세포는 기초 조건 하에서 600초 관찰 기간 동안 적어도 3 개의 자발적 사건(> 0.05 in Fura-2 ratio)을 나타내는 세포로서 정의되었다.Specifically, BMM cells were plated on a cover slip glass at the bottom of a 12-well plate and incubated with 40 ng/ml M-CSF and 50 ng/ml RANKL for 48 hours, and the cells were incubated with or without 6 μM flunarizine. and treated for 48 hours. Cells were then loaded with 5 mM Fura-2 AM and 5 μM Pluronic F12 at room temperature for 30 min at RT with regular buffers: 10 mM HEPES, 140 mM NaCl, 10 mM glucose, 5 mM KCl, 1 mM CaCl and 1 mM MgCl 2 , pH 7.4. Cytoplasmic calcium oscillations of cells were measured using a fluorescence microscope (Axio Observer A1; Zeiss) at excitation wavelengths of 340 and 380 nm and emission wavelengths of 510 nm. Fluctuation-positive cells were defined as cells exhibiting at least 3 spontaneous events (>0.05 in Fura-2 ratio) during a 600 sec observation period under basal conditions.
도 3에서 왼쪽은 상기 세포의 fura-2 이미지를 나타내고, 오른쪽 그래프는 각 실험의 단일 세포에서 fura-2 형광 비율의 변화의 흔적을 나타낸다(도 3a). 칼슘 진동은 플루나리진이 없는 대조군과 비교하여 6μM 플루나리진에 의해 유의하게 억제되었다 (도 3b). 플루나리진에 의한 손상된 칼슘 진동은 파골세포 분화에 중요한 역할을 하는 p-CaMKIV, p-CREB 및 c-Fos의 하향 조절로 이어진다 (도 4a 내지 4d). 또한, 세포가 플루나리진과 함께 배양될 때 PGC1β의 단백질 발현 및 PGC1β의 표적 유전자의 mRNA 수준도 하향 조절되었다(도 4e 및 4f).In FIG. 3, the left side shows the fura-2 image of the cells, and the right graph shows the traces of changes in the fura-2 fluorescence ratio in a single cell of each experiment (FIG. 3a). Calcium oscillations were significantly inhibited by 6 μM flunarizine compared to controls without flunarizine ( FIG. 3b ). Impaired calcium oscillations by flunarizine lead to downregulation of p-CaMKIV, p-CREB and c-Fos, which play important roles in osteoclast differentiation ( FIGS. 4a to 4d ). In addition, protein expression of PGC1β and mRNA levels of target genes of PGC1β were also down-regulated when cells were incubated with flunarizine ( FIGS. 4E and 4F ).
플루나리진이 칼슘 신호 전달을 차단하여 파골세포 분화를 억제하는지 여부를 확인하기 위해, 세포 내 칼슘 상승 화합물인 이오노마이신(ionomycin)을 처리하여 파골세포에 대한 플루나리진의 효과를 구제(rescue)하였다(도 5). 이러한 결과는 플루나리진이 칼슘 진동을 약화시킴으로써 파골세포의 분화를 억제하고 방해된 칼슘 신호 전달은 파골세포에서 관련 신호의 하향 조절을 야기한다는 것을 보여준다.In order to determine whether flunarizine inhibits osteoclast differentiation by blocking calcium signal transduction, the effect of flunarizine on osteoclasts was rescued by treatment with an intracellular calcium-increasing compound, ionomycin. (Fig. 5). These results show that flunarizine inhibits the differentiation of osteoclasts by attenuating calcium oscillations, and that disrupted calcium signaling causes downregulation of related signals in osteoclasts.
<실험예 4> 플루나리진의 파골세포 형성 및 흡수 기능 억제<Experimental Example 4> Inhibition of osteoclast formation and absorption function of flunarizine
플루나리진이 파골세포 형성 및 기능을 조절하는 방법을 발견하기 위해, 플루나리진 처리한 분화된 파골세포에서의 액틴링 감소 및 골흡수 기능을 측정하는 실험을 수행하였다. In order to discover how flunarizine regulates osteoclast formation and function, an experiment was performed to measure actinling reduction and bone resorption function in flunarizine-treated differentiated osteoclasts.
구체적으로, BMM 세포를 RANKL과 함께 3일 동안 인큐베이션함으로써 파골세포로 분화시킨 후, 6μM 플루나리진을 12시간, 24시간 또는 48시간 동안 분화된 파골세포에 첨가하였다. 그 후, 액틴링을 염색하기 위해 BMM 세포를 PBS 중 3.74% 포름알데히드 용액으로 고정시키고, 0.1% Triton X-100으로 투과시키고, 20분 동안 Alexa Fluor 488-phalloidin(Invitrogen)과 함께 배양하였다. PBS로 세척한 후, 세포를 4', 6-diamidino-3-phenylindole(Roche)과 함께 2분간 배양하고 형광 현미경으로 사진을 찍었다. 그리고 골흡수 측정을 위해, BMM 세포를 dentine disc(Immunodiagnostic Systems, Ltd.)에 플레이팅하고 40ng/ml M-CSF 및 50ng/ml RANKL로 처리하였다. 면 팁으로의 마모를 통해 dentine disc로부터 세포를 완전히 제거하고, dentine disc를 hematoxylin으로 염색하였다. resorption pits의 사진을 ×100의 배율에서 광학 현미경으로 촬영하고, 이들의 면적을 Image-Pro Plus 4.5 software(Media Cybernetics)를 통해 측정하였다.Specifically, after the BMM cells were differentiated into osteoclasts by incubation with RANKL for 3 days, 6 μM flunarizine was added to the differentiated osteoclasts for 12 hours, 24 hours or 48 hours. Then, to stain actinling, BMM cells were fixed with a 3.74% formaldehyde solution in PBS, permeabilized with 0.1% Triton X-100, and incubated with Alexa Fluor 488-phalloidin (Invitrogen) for 20 minutes. After washing with PBS, cells were incubated with 4',6-diamidino-3-phenylindole (Roche) for 2 minutes and photographed under a fluorescence microscope. And for the measurement of bone resorption, BMM cells were plated on a dentine disc (Immunodiagnostic Systems, Ltd.) and treated with 40ng/ml M-CSF and 50ng/ml RANKL. Cells were completely removed from the dentine disc through abrasion with a cotton tip, and the dentine disc was stained with hematoxylin. Photographs of the resorption pits were taken with an optical microscope at a magnification of ×100, and their areas were measured through Image-Pro Plus 4.5 software (Media Cybernetics).
그 결과, 플라나리진이 처리된 파골세포의 액틴링은 사라진 반면, 대조 파골세포의 액틴링은 두껍고 커졌다(도 6a). 플라나리진에 의한 사라진 액틴링 및 감소된 파골세포는 TRAP 염색(도 6b), TRAP 양성 세포 계수(도 6c) 및 액틴링 염색(도 6d)에 의해 확인되었다. 플루나리진이 파골세포의 흡수 기능을 억제하는지 여부를 결정하기 위해 플루나리진의 존재 또는 부재 하에 dentine disc 상에 RANKL을 갖는 파골세포로 BMM을 분화시켰고(도 6e), 플루나리진이 파골세포의 흡수 기능을 억제하는 것을 확인하였다(도 6f). As a result, the actin ring of the osteoclasts treated with planarizin disappeared, whereas the actin ring of the control osteoclasts became thicker and larger (FIG. 6a). The disappeared actinling and reduced osteoclasts by planarizin were confirmed by TRAP staining (FIG. 6b), TRAP-positive cell counting (FIG. 6c) and actinling staining (FIG. 6d). To determine whether flunarizine inhibits the uptake function of osteoclasts, BMMs were differentiated into osteoclasts with RANKL on dentine discs in the presence or absence of flunarizine (Figure 6e), and flunarizine inhibits the uptake function of osteoclasts. was confirmed to inhibit (Fig. 6f).
<실험예 5> 플루나리진의 파골세포 생성(osteoclastogenesis) 말기 억제<Experimental Example 5> Late inhibition of osteoclastogenesis of flunarizine
파골세포 생성의 말기에, 파골세포는 세포-세포 융합(cell-cell fusion) 및 이동(migration)에 의해 다핵 세포를 형성하고, 골흡수가 발생하는 밀봉 구역을 형성하기 위해 액틴링을 개발한다. 따라서, 세포 이동 또는 융합에 관여하는 유전자를 억제하거나 액틴링을 형성하는 것은 파골세포 성숙 또는 기능에 상당한 영향을 미친다. 플루나리진의 파골세포 생성 관련 유전자의 발현이 감소되는지 여부를 확인하기 위해, 실험예 2의 실험방법과 동일한 real-time PCR 방법을 이용하여 실험을 수행하였고, 이 때, 표 1의 Primer를 사용하였다.At the end of osteoclastogenesis, osteoclasts form multinucleated cells by cell-cell fusion and migration, and develop actin rings to form a sealing zone where bone resorption occurs. Thus, repressing genes involved in cell migration or fusion or forming actin rings have significant effects on osteoclast maturation or function. In order to determine whether flunarizine reduces the expression of osteoclast-related genes, an experiment was performed using the same real-time PCR method as in Experimental Example 2, and in this case, the Primer of Table 1 was used. .
파골세포는 인테그린 수용체 그리고 src, p130cas를 포함하는 다운스트림(downstream) 경로에 의해 뼈 표면에 부착되며, 이는 플루나리진에 의해 하향 조절되었다(도 7a). 플루나리진은 또한 세포-세포 융합을 촉진시키는 Dcstamp, 이동을 조절하는 Dock5, 골흡수에 관여하는 MMP9 및 Ctsk(Cathepsin K), 그 외에 Src, Atp6v0d2, Itgb3를 포함한 파골세포 성숙 및 기능에서 중요한 역할을 하는 유전자의 mRNA 수준을 감소시켰다(도 7b).Osteoclasts adhere to the bone surface by downstream pathways including integrin receptors and src and p130cas, which are down-regulated by flunarizine (Fig. 7a). Flunarizine also plays an important role in osteoclast maturation and function, including Dcstamp that promotes cell-cell fusion, Dock5 that regulates migration, MMP9 and Ctsk (Cathepsin K) that are involved in bone resorption, as well as Src, Atp6v0d2 and Itgb3. reduced the mRNA level of the gene (Fig. 7b).
<실험예 6> 플루나리진에 의한 염증 및 난소 절제술로 인한 골 손상 억제<Experimental Example 6> Inhibition of bone damage due to inflammation and ovariectomy by flunarizine
플루나리진의 염증에 의해 유도되는 골 손상 억제효능을 동물모델에서 확인하기 위해, 마우스 두개골에 염증유도물질인 LPS(Lipopolysaccharide, 12.5mg/kg body weight)를 하루 간격을 두고 2회 주입하였다. 이 때 Vehicle(10% DMAC + 10% Tween 80 + 80% distilled water) 또는 플루나리진(10mg/kg)이 함께 투여되었고, 첫 주입 5일 후에 두개골을 4% 파라포름알데하이드(paraformaldehyde)로 고정 후, PBS 워싱하여 TRAP 염색 후 관찰하였다. 또한 0.5M ethylenediaminetetraacetic acid로 7일간 석회질을 제거하고, 파라핀 블록을 만든 후 절단하여 TRAP과 Hematoxylin으로 염색한 뒤 관찰하였다.To confirm the inhibitory effect of flunarizine on bone damage induced by inflammation in an animal model, LPS (Lipopolysaccharide, 12.5 mg/kg body weight), an inflammation-inducing substance, was injected twice a day into the mouse skull. At this time, vehicle (10% DMAC + 10
그 결과, LPS만 처리한 두개골에서는 TRAP 염색된 부분이 많았으나, 플루나리진을 함께 처리한 두개골에서는 TRAP 염색된 부분이 현저히 감소된 것을 확인하였다(도 8a 및 8b). 또한 이를 정량적으로 분석한 결과, LPS 처리에 의해 증가된 bone cavity와 파골세포의 수가 플루나리진에 의해 상당히 감소되는 것을 확인하였다(도 8c). 이는, 플루나리진이 염증 유도성 골질환을 예방할 수 있음을 제시한다.As a result, it was confirmed that there were many TRAP-stained parts in the skull treated with LPS only, but the TRAP-stained part was significantly reduced in the skulls treated with flunarizine ( FIGS. 8a and 8b ). In addition, as a result of quantitative analysis, it was confirmed that the number of bone cavities and osteoclasts increased by LPS treatment was significantly reduced by flunarizine (FIG. 8c). This suggests that flunarizine can prevent inflammation-induced bone disease.
또한, 폐경기 골다공증의 OVX-유도 모델을 사용하여 플루나리진의 치료 가능성을 확인하였다. In addition, the therapeutic potential of flunarizine was confirmed using an OVX-induced model of postmenopausal osteoporosis.
구체적으로, 난소 절제술(OVX)과 마이크로 컴퓨터 단층 촬영(μCT)은 전술 한 바와 같이 수행되었다. 암컷 12주령 마우스는 모의 수술 또는 OVX를 받았고, 플루나리진(10mg/kg)을 주당 6회 복강 내 주사하였다. 수술 3주 후 마우스를 안락사시키고, 대퇴골을 μCT를 사용하여 평가하였다.Specifically, ovariectomy (OVX) and microcomputed tomography (μCT) were performed as described above. Female 12-week-old mice underwent sham surgery or OVX, and were injected intraperitoneally with flunarizine (10 mg/kg) 6 times per week. Mice were euthanized 3 weeks after surgery, and femurs were evaluated using µCT.
그 결과, OVX-유도 모델은 골 무기질 밀도, 골 표면, 골 뼈 수 및 골 부피가 감소하였고, 골 패턴 인자 및 골 분리가 OVX에 의해 증가되었다. OVX의 이러한 영향 중 일부는 플루나리진 치료에 의해 상당히 역전되었으며(도 8d 및 8e), 이는 플루나리진은 골다공증과 같은 병리학적 골질환을 치료할 수 있는 치료 가능성이 있음을 나타낸다.As a result, in the OVX-induced model, bone mineral density, bone surface, bone number and bone volume were decreased, and bone pattern factor and bone separation were increased by OVX. Some of these effects of OVX were significantly reversed by flunarizine treatment ( FIGS. 8d and 8e ), indicating that flunarizine has therapeutic potential to treat pathological bone diseases such as osteoporosis.
<110> Ewha University - Industry Collaboration Foundation <120> Pharmaceutical composition for prevention or treatment of bone disease containing Flunarizine or pharmaceutically acceptable salts thereof as an active ingredient <130> 2020P-03-017 <160> 36 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Nfatc1 sense primer <400> 1 cttcagctgg aggacacc 18 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Nfatc1 antisense primer <400> 2 ccaatgaaca gctgtagcg 19 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Ctsk sense primer <400> 3 accactgcct tccaatacg 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ctsk antisense primer <400> 4 cgtggcgtta tacatacaac 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Mmp9 sense primer <400> 5 agacgacata gacggcatc 19 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Mmp9 antisense primer <400> 6 tgctgtcggc tgtggttc 18 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Acp5 sense primer <400> 7 ccagcgacaa gaggttcc 18 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Acp5 antisense primer <400> 8 agagacgttg ccaaggtgat 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Calcr sense primer <400> 9 tgatgactct caggacaatg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Calcr antisense primer <400> 10 actggatcaa tctgtaggag 20 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Dcstamp sense primer <400> 11 ttatgtgttt ccacgaagcc cta 23 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Dcstamp antisense primer <400> 12 acagaagaga gcagggcaac g 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Dock5 sense primer <400> 13 tggtgacaca gggacagtgg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Dock5 antisense primer <400> 14 caccccaact atgcacgtgg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> cFos sense primer <400> 15 gagaaacgga gaatccgaag 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> cFos antisense primer <400> 16 gagaaacgga gaatccgaag 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sod2 sense primer <400> 17 attaacgcgc agatcatgca 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sod2 antisense primer <400> 18 tgtcccccac cattgaactt 20 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cox2 sense primer <400> 19 gatcataagc gaggacctg 19 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cox2 antisense primer <400> 20 gtctgtccag agtttcacc 19 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Itgb3 sense primer <400> 21 ggagtggctg atccagatgt 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Itgb3 antisense primer <400> 22 tctgaccatc ttccctgtcc 20 <210> 23 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Atp6v0d2 sense primer <400> 23 cagagatgga agctgt 16 <210> 24 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Atp6v0d2 antisense primer <400> 24 tgccaaatga gttcag 16 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Ppargc1b sense primer <400> 25 ctccaggcag gttcaaccc 19 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ppargc1b antisense primer <400> 26 gggccagaag ttcccttagg 20 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Mt-cyb sense primer <400> 27 gccaccttga cccgattct 19 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Mt-cyb antisense primer <400> 28 ttgctagggc cgcgataat 19 <210> 29 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Mt-nd4 sense primer <400> 29 catcactcct attctgccta gcaa 24 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Mt-nd4 antisense primer <400> 30 tcctcgggcc atgattatag tac 23 <210> 31 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Mt-co1 sense primer <400> 31 ttttcaggct tcaccctaga tga 23 <210> 32 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Mt-co1 antisense primer <400> 32 gaagaatgtt atgtttactc ctacgaatat g 31 <210> 33 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Mt-co3 sense primer <400> 33 cggaagtatt tttctttgca ggat 24 <210> 34 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Mt-co3 antisense primer <400> 34 cagcagcctc ctagatcatg tg 22 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> b-actin sense primer <400> 35 accctaaggc caaccgtg 18 <210> 36 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> b-actin antisense primer <400> 36 gcctggatgg ctacgtac 18 <110> Ewha University - Industry Collaboration Foundation <120> Pharmaceutical composition for prevention or treatment of bone disease containing Flunarizine or pharmaceutically acceptable salts thereof as an active ingredient <130> 2020P-03-017 <160> 36 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Nfatc1 sense primer <400> 1 cttcagctgg aggacacc 18 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Nfatc1 antisense primer <400> 2 ccaatgaaca gctgtagcg 19 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Ctsk sense primer <400> 3 accactgcct tccaatacg 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ctsk antisense primer <400> 4 cgtggcgtta tacatacaac 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Mmp9 sense primer <400> 5 agacgacata gacggcatc 19 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Mmp9 antisense primer <400> 6 tgctgtcggc tgtggttc 18 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Acp5 sense primer <400> 7 ccagcgacaa gaggttcc 18 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Acp5 antisense primer <400> 8 agagacgttg ccaaggtgat 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Calcr sense primer <400> 9 tgatgactct caggacaatg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Calcr antisense primer <400> 10 actggatcaa tctgtaggag 20 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Dcstamp sense primer <400> 11 ttatgtgttt ccacgaagcc cta 23 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Dcstamp antisense primer <400> 12 acagaagaga gcagggcaac g 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Dock5 sense primer <400> 13 tggtgacaca gggacagtgg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Dock5 antisense primer <400> 14 caccccaact atgcacgtgg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> cFos sense primer <400> 15 gagaaacgga gaatccgaag 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> cFos antisense primer <400> 16 gagaaacgga gaatccgaag 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sod2 sense primer <400> 17 attaacgcgc agatcatgca 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sod2 antisense primer <400> 18 tgtccccccac cattgaactt 20 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cox2 sense primer <400> 19 gatcataagc gaggacctg 19 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cox2 antisense primer <400> 20 gtctgtccag agtttcacc 19 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Itgb3 sense primer <400> 21 ggagtggctg atccagatgt 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Itgb3 antisense primer <400> 22 tctgaccatc ttccctgtcc 20 <210> 23 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Atp6v0d2 sense primer <400> 23 cagagatgga agctgt 16 <210> 24 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Atp6v0d2 antisense primer <400> 24 tgccaaatga gttcag 16 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Ppargc1b sense primer <400> 25 ctccaggcag gttcaaccc 19 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ppargc1b antisense primer <400> 26 gggccagaag ttcccttagg 20 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Mt-cyb sense primer <400> 27 gccaccttga cccgattct 19 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Mt-cyb antisense primer <400> 28 ttgctagggc cgcgataat 19 <210> 29 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Mt-nd4 sense primer <400> 29 catcactcct attctgccta gcaa 24 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Mt-nd4 antisense primer <400> 30 tcctcgggcc atgattatag tac 23 <210> 31 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Mt-co1 sense primer <400> 31 ttttcaggct tcaccctaga tga 23 <210> 32 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Mt-co1 antisense primer <400> 32 gaagaatgtt atgtttactc ctacgaatat g 31 <210> 33 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Mt-co3 sense primer <400> 33 cggaagtatt tttctttgca ggat 24 <210> 34 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Mt-co3 antisense primer <400> 34 cagcagcctc ctagatcatg tg 22 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> b-actin sense primer <400> 35 accctaaggc caaccgtg 18 <210> 36 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> b-actin antisense primer <400> 36 gcctggatgg ctacgtac 18
Claims (13)
[화학식 1]
.
A pharmaceutical composition for the prevention or treatment of bone disease, characterized in that it contains flunarizine represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]
.
상기 플루나리진은 0.1 내지 10μM 농도인 것을 특징으로 하는, 골질환의 예방 또는 치료용 약학적 조성물.
The method of claim 1,
The flunarizine is a pharmaceutical composition for preventing or treating bone disease, characterized in that the concentration of 0.1 to 10 μM.
상기 조성물은 NFATc1, Acp5, Mmp9, Ctsk, CalcrC, Dcstamp, c-FOS, p-CaMKIV, p-CREB, PGC1β, Dock5, Src, Atp6v0d2 및 Itgb3로 이루어진 군으로부터 선택되는 적어도 어느 하나 이상의 발현 또는 활성을 감소시키는 것을 특징으로 하는, 골질환의 예방 또는 치료용 약학적 조성물.
According to claim 1,
The composition exhibits at least one expression or activity selected from the group consisting of NFATc1, Acp5, Mmp9, Ctsk, CalcrC, Dcstamp, c-FOS, p-CaMKIV, p-CREB, PGC1β, Dock5, Src, Atp6v0d2 and Itgb3. A pharmaceutical composition for the prevention or treatment of bone disease, characterized in that it reduces.
상기 조성물은 염증에 의한 골 손상을 억제하는 것을 특징으로 하는, 골질환의 예방 또는 치료용 약학적 조성물.
The method of claim 1,
The composition is a pharmaceutical composition for preventing or treating bone disease, characterized in that it suppresses bone damage caused by inflammation.
상기 조성물은 파골세포의 칼슘 진동(calcium oscillation)을 억제하는 것을 특징으로 하는, 골질환의 예방 또는 치료용 약학적 조성물.
According to claim 1,
The composition is characterized in that suppressing calcium oscillation of osteoclasts, a pharmaceutical composition for the prevention or treatment of bone disease.
상기 조성물은 파골세포 생성(osteoclastogenesis)을 억제하는 것을 특징으로 하는, 골질환의 예방 또는 치료용 약학적 조성물.
The method of claim 1,
The composition is characterized in that inhibiting osteoclastogenesis (osteoclastogenesis), a pharmaceutical composition for the prevention or treatment of bone disease.
상기 파골세포 생성은 파골세포 생성 말기(late stage)인 것을 특징으로 하는, 골질환의 예방 또는 치료용 약학적 조성물.
7. The method of claim 6,
The osteoclast generation is characterized in that the osteoclast generation late stage (late stage), a pharmaceutical composition for the prevention or treatment of bone disease.
상기 골질환은 성장기 발육부진, 골절, 과도한 파골세포의 골 흡수에 의한 골다공증(osteoprosis), 류마티스성 관절염(rheumatoid arthritis), 치주질환(periodontal disease), 파제트병(Paget disease) 및 전이성 골암(metastatic bone cancers)으로 구성된 군으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는, 골질환의 예방 또는 치료용 약학적 조성물.
According to claim 1,
The bone diseases include growth retardation, fractures, osteoporosis due to excessive bone resorption of osteoclasts, rheumatoid arthritis, periodontal disease, Paget disease, and metastatic bone cancer (metastatic). Bone cancers), characterized in that any one or more selected from the group consisting of, a pharmaceutical composition for the prevention or treatment of bone diseases.
[화학식 1]
.
A health functional food composition for the prevention or improvement of bone diseases, characterized in that it contains flunarizine or a pharmaceutically acceptable salt thereof represented by the following formula (1) as an active ingredient:
[Formula 1]
.
상기 플루나리진은 0.1 내지 10μM 농도인 것을 특징으로 하는, 골질환의 예방 또는 개선용 건강기능식품 조성물.
10. The method of claim 9,
The flunarizine is a health functional food composition for the prevention or improvement of bone disease, characterized in that the concentration of 0.1 to 10μM.
상기 조성물은 NFATc1, Acp5, Mmp9, Ctsk, CalcrC, Dcstamp, c-FOS, p-CaMKIV, p-CREB, PGC1β, Dock5, Src, Atp6v0d2 및 Itgb3로 이루어진 군으로부터 선택되는 적어도 어느 하나 이상의 발현 또는 활성을 감소시키는 것을 특징으로 하는, 골질환의 예방 또는 개선용 건강기능식품 조성물.
10. The method of claim 9,
The composition exhibits at least one expression or activity selected from the group consisting of NFATc1, Acp5, Mmp9, Ctsk, CalcrC, Dcstamp, c-FOS, p-CaMKIV, p-CREB, PGC1β, Dock5, Src, Atp6v0d2 and Itgb3. A health functional food composition for the prevention or improvement of bone disease, characterized in that it reduces.
상기 조성물은 염증에 의한 골 손상을 억제하는 것을 특징으로 하는, 골질환의 예방 또는 개선용 건강기능식품 조성물.
10. The method of claim 9,
The composition is a health functional food composition for preventing or improving bone disease, characterized in that it suppresses bone damage caused by inflammation.
상기 골질환은 성장기 발육부진, 골절, 과도한 파골세포의 골 흡수에 의한 골다공증(osteoprosis), 류마티스성 관절염(rheumatoid arthritis), 치주질환(periodontal disease), 파제트병(Paget disease) 및 전이성 골암(metastatic bone cancers)으로 구성된 군으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는, 골질환의 예방 또는 개선용 건강기능식품 조성물.10. The method of claim 9,
The bone diseases include growth retardation, fractures, osteoporosis due to excessive bone resorption of osteoclasts, rheumatoid arthritis, periodontal disease, Paget disease, and metastatic bone cancer (metastatic). Bone cancers), characterized in that any one or more selected from the group consisting of, a health functional food composition for the prevention or improvement of bone diseases.
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