KR20010011926A - Pharmaceutical composition for prevention and remedy of osteoporosis - Google Patents

Pharmaceutical composition for prevention and remedy of osteoporosis Download PDF

Info

Publication number
KR20010011926A
KR20010011926A KR1019990031532A KR19990031532A KR20010011926A KR 20010011926 A KR20010011926 A KR 20010011926A KR 1019990031532 A KR1019990031532 A KR 1019990031532A KR 19990031532 A KR19990031532 A KR 19990031532A KR 20010011926 A KR20010011926 A KR 20010011926A
Authority
KR
South Korea
Prior art keywords
osteoporosis
destruxin
osteoclasts
derivatives
prevention
Prior art date
Application number
KR1019990031532A
Other languages
Korean (ko)
Other versions
KR100309888B1 (en
Inventor
윤성준
김종우
한재진
우제태
케이지하수미
나가이카즈오
Original Assignee
황규언
동화약품공업 주식회사
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 황규언, 동화약품공업 주식회사 filed Critical 황규언
Priority to KR1019990031532A priority Critical patent/KR100309888B1/en
Publication of KR20010011926A publication Critical patent/KR20010011926A/en
Application granted granted Critical
Publication of KR100309888B1 publication Critical patent/KR100309888B1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/15—Depsipeptides; Derivatives thereof

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

PURPOSE: A composition containing destruxin derivatives for prevention and treatment of osteoporosis is provided, inwhich cyclodepsipeptide derived from fungi shows excellent osteoclast activity-inhibition effect regardless of presence of PTH(parathyroid hormone) in osteoclast, therefore being used for prevention and treatment of osteoporosis. CONSTITUTION: The various destruxin derivatives can be prepared by a well known method, which is cyclodepsipeptides separated from various fungi(e.g., Metarhizium anisopliae). The composition containing destruxin derivatives for prevention and treatment of osteoporosis can be prepared in the form of tablets, capsules, troches, solution, suspension, or injections, dried powder for injection, a spray, suppositories, or a patch preparation by mixing with pharmaceutically acceptable carriers, and which can be administrated by oral path and parenteral path(e.g., in vein, under subcutaneous, abdominal cavity, or nasal cavity). The composition containing destruxin derivatives can be also administrated in the dosage of 0.1μg-100mg, desirably 100μg-1mg per day.

Description

골다공증 예방 및 치료제 조성물{Pharmaceutical composition for prevention and remedy of osteoporosis}Pharmaceutical composition for prevention and remedy of osteoporosis

본 발명은 골다공증 예방 및 치료제 조성물에 관한 것으로, 더욱 상세하게는, 본 발명은 데스트룩신(destruxin) 유도체를 유효성분으로 함유하는 골다공증 의 예방 및 치료제로 유용한 약제조성물에 관한 것이다.The present invention relates to a composition for preventing and treating osteoporosis, and more particularly, to a pharmaceutical composition useful as an agent for preventing and treating osteoporosis, which contains a destruxin derivative as an active ingredient.

골다공증은 노령화되면서 골밀도가 감소되어 생기는 질환으로 대사성 골질환중 가장 흔한 질병이다. 이같은 골다공증은 선진국에서 이미 큰 사회문제로 대두되어 그 치료를 위해서 많은 연구가 진행되고 있으며, 최근 우리나라에서도 노년사회로 접어들면서 중요한 노인질환의 하나로 대두되고 있다. 미국에서는 약 1,000만명이 골다공증 환자이며 약 1,800만명이 낮은 골밀도를 가지고 있어 골다공증의 예방 및 치료가 시급한 사회적과제로 알려져 있다. 미국의 경우 일생동안 여성 2명중 1명 그리고 백인 남성 8명중 1명이 골다공증과 관련된 골절을 경험할 것으로 예상하고 있으며 이미 2백만 명이상의 미국 남성들이 골다공증으로 고생하고 있으며 매년 9만명의 미국 남성이 골반골절로 고생하고 그중 1/3이 1년내에 사망하고있다. 이미 노령화 사회가된 유럽의 선진국에서도 골다공증치료제의 수요가 최근 매년 약 10%의 급격한 증가세를 보이고 있다.Osteoporosis is a disease caused by the decrease of bone density with aging and is the most common metabolic bone disease. Osteoporosis has become a major social problem in developed countries, and many studies are being conducted for its treatment. Recently, as osteoporosis enters old age society, it has emerged as one of the important elderly diseases. In the United States, about 10 million people with osteoporosis and about 18 million people with low bone density are known as urgent social tasks to prevent and treat osteoporosis. In the United States, one in two women and one in eight white men are expected to experience osteoporosis-related fractures in their lifetimes, with more than 2 million American men suffering from osteoporosis, and 90,000 American men annually Hardly a third of them die within a year. The demand for osteoporosis treatments has increased rapidly in recent years, about 10% each year, even in advanced European countries, which are already aging.

골조직은 골아세포에 의한 형성과 파골세포에 의한 파괴 흡수가 끊임없이 반복되는 동적인 조직이다. 이러한 골흡수와 골형성의 기전에는 불명확한 점이 많으나 골다공증은 골흡수와 골형성의 발란스가 무너져 발생하는 것으로 골흡수가 골형성보다 항진되는데 기인한 질환으로, 현재 파골세포에 대해 강력한 저해활성을 갖는 골흡수억제제의 개발이 활발히 행해지고 있다.Bone tissue is a dynamic tissue in which the formation by osteoblasts and the destruction and absorption by osteoclasts are constantly repeated. Although the mechanism of bone resorption and bone formation is unclear, osteoporosis is a disease caused by the collapse of the balance of bone resorption and bone formation, which is caused by the fact that bone resorption is more advanced than bone formation. The development of a bone resorption inhibitor is being actively performed.

한편, 데스트룩신 유도체는 곤충에 기생하는 곰팡이인 메타르히지움 아니소플리에(Metarhizium anisopliae)를 비롯한 여러 곰팡이들에서 분리된 사이클로뎁시펩타이드(cyclodepsipeptides)로서 살충활성을 갖는 것으로 잘 알려져 있는 물질이며, 최근에는 항바이러스 활성, 류케미아 세포에 대한 세포독성도 있다는 것이 공지되었으며(F. Cavelier et al., J. Peptide Res., 50, 1997, 94-101), 강심제 또는 에리트로포이에틴의 유도물질 등으로 사용할 수 있음도 알려졌다. 그러나, 지금까지 데스트룩신 유도체가 골다공증 예방 및 치료에 유효하다는데 대하여는 전혀 알려진 바는 없으며 또한, 이 물질의 세포 생리활성 작용 기전은 데스트룩신 유도체가 곤충의 Na+-ATPase의 기능을 저해함으로써 살충 효과를 나타내는 것으로 알려져 있다.Deluxuxine derivatives are well known to have insecticidal activity as cyclodepsipeptides isolated from various fungi, including insect parasitic fungi, Metarhizium anisopliae, Recently, it has been known that there is also antiviral activity, cytotoxicity against leuchemia cells (F. Cavelier et al., J. Peptide Res., 50, 1997, 94-101), cardiac agents or inducers of erythropoietin, etc. It can also be used as. However, until now, there is no known fact that deluxux derivatives are effective for the prevention and treatment of osteoporosis, and the mechanism of cellular physiological activity of the substance is that the dexlux derivatives inhibit the function of Na + -ATPase in insects and have an insecticidal effect. It is known to represent.

뼈대사에 있어서, 석회화된 뼈성분은 골흡수(bone resorotion)에 관여하며 이때 파골세포는 뼈표면에 존재하면서 골흡수를 담당하며 이 과정에서 형성된 세포 찌꺼기를 제거한다( Ross 등, 1995). 또, 골흡수를 담당하는 상기 파골세포는 여러가지 호르몬 작용을 받게되는데 이때 부갑상선 호르몬(parathyroid hormone;PTH)은 뼈기질의 흡수를 촉진시키는 파골세포의 수를 증가시키고 활성화시켜 칼슘을 유리시킨다.In bone metabolism, calcified bone components are involved in bone resorotion, where osteoclasts are present on the bone surface, responsible for bone resorption and remove cell debris formed during this process (Ross et al., 1995). In addition, the osteoclasts responsible for bone resorption are subjected to various hormonal actions, wherein parathyroid hormone (PTH) increases calcium by activating and increasing the number of osteoclasts that promote the absorption of bone matrix.

이처럼 골다공증 유발에 중요한 역할을 하는 파골세포는 그 특성상 뼈를 분해하기 위하여 강한 산을 분비하는데 이것을 위하여 많은 H+-ATPase를 사용하고 있다. 본 발명자들은 Na+-ATPase와 H+-ATPase는 유전자수준에서 상이한 부분도 있지만 같은 공포성 에티피아제 계열(vacuolar ATPase family)로서 공통적인 부분도 갖고 있음에 착안하여 데스트룩신 유도체에 대한 연구를 계속한 결과 데스트룩신 유도체가 파골세포 증가 및 활성화의 저해능을 가지고 있을 뿐만 아니라 파골세포 저해능이 매우 우수함을 확인하게 되었고 이에 의하여 본 발명을 완성하게 되었다.As such, osteoclasts, which play an important role in inducing osteoporosis, secrete strong acids to break down bones, and many H + -ATPases are used for this purpose. The present inventors have focused on the deluxuxine derivatives, considering that Na + -ATPase and H + -ATPase have different parts at the gene level, but also have common parts as the same vacuolar ATPase family. As a result, it was confirmed that the destruxin derivative not only has the ability to inhibit osteoclast growth and activation, but also very excellent ability to inhibit osteoclasts, thereby completing the present invention.

따라서, 본 발명의 목적은 파골세포의 저해능이 있는 덱스트룩스 유도체를 함유하는 골다공증 예방 및 치료용 약제조성물을 제공함에 있다.Accordingly, an object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of osteoporosis containing a dextrox derivative having an inhibitory ability of osteoclasts.

상기 본 발명의 목적은 골다공증 유발 억제 활성을 갖는 데스트룩신 유도체를 조사하고 이와 같은 유도체를 유효성분으로 함유하고 있는 약제 조성물을 조제한 다음 이들 유도체가 골다공증 예방 및 치료제에 유용한지 어떤지 확인하기 위하여 파골세포를 준비하여 골흡수능을 검정하고 칼슘유리 저해능과 파골세포 저해능을 각각 조사하므로써 달성하였다.The object of the present invention is to investigate the destructin derivatives having osteoporosis-induced inhibitory activity and to prepare a pharmaceutical composition containing such derivatives as an active ingredient and then to determine whether the osteoclasts are useful for the prevention and treatment of osteoporosis. This was accomplished by assaying bone resorption capacity and investigating calcium free and osteoclast inhibition.

도 1은 TRAP 염색한 성숙한 파골세포를 나타낸다.1 shows mature osteoclasts stained with TRAP.

도 2의 (A)는 정상적인 파골세포 모양을 나타내고, (B)는 골표면에서 파골세포의 골흡수작용으로 형성된 피트를 나타내며 (C)는 데스트룩신 E로 처리한 후 파골세포가 변형되어 위축된 모습을 나타낸다. 또 (D)는 데스트룩신 E 처리한 후 감소된 피트를 나타낸다.Figure 2 (A) shows the normal osteoclast shape, (B) shows the pits formed by the bone resorption of the osteoclasts on the bone surface (C) shows that the osteoclasts are deformed and contracted after treatment with Destinxine E Show the appearance. (D) shows reduced pits after treatment with Destinlux E.

도 3의 (A)는 데스트룩신 E가 포함된 배지의 농도에 따라서 이 배지에서 성장한 파골세포가 생성한 피트의 수가 크게 감소하고 있음을 보여주는 그래프를 나타내고 (B)는 데스트룩신 E로 처리한 파골세포 액틴링의 정상여부를 정상적인 파골세포와 상대적으로 비교한 그래프를 나타낸다.FIG. 3 (A) shows a graph showing that the number of pits generated by osteoclasts grown in this medium is greatly reduced according to the concentration of the medium containing dexluxin E, and (B) is a osteoclast treated with dexluxin E Shows a graph comparing the normal activity of the cell actining with normal osteoclasts.

도 4는 골흡수작용을 유발하는 부갑상선호르몬(PTH)를 첨가하였으나 데스트룩신 E에 의해 골흡수작용이 다시 감소되었음을 나타내는 그래프이다.Figure 4 is a graph showing that the addition of parathyroid hormone (PTH) to induce bone resorption action, but the bone resorption action was reduced again by Deluxux E.

본 발명은 하기 화학식(I)로 표시되는 데스트룩신(destruxin) 유도체를 유효성분으로 함유하는 약제조성물을 그 특징으로 한다.The present invention is characterized by a pharmaceutical composition containing a destruxin derivative represented by the following formula (I) as an active ingredient.

단, 상기 식에서 R1는 CH3,CH2-CH=CH2, CH2CH(CH3)2, CH2CH(CH3)CH2OH,, CH2CH(CH3)COOH, CH2CH(OH)CH2Cl, CH2-C≡CH 또는 CH2CH(OCOCH3)CH2Cl이고, R2, R4및 R6는 H 또는 CH3이고, R3와 R5는 CH(CH3)CH2CH3또는 CH(CH3)2이며, n는 2 또는 3이다.In which R 1 is CH 3, CH 2 -CH = CH 2 , CH 2 CH (CH 3 ) 2 , CH 2 CH (CH 3 ) CH 2 OH, , CH 2 CH (CH 3 ) COOH, CH 2 CH (OH) CH 2 Cl, CH 2 -C≡CH or CH 2 CH (OCOCH 3 ) CH 2 Cl, R 2 , R 4 and R 6 are H or CH 3 , R 3 and R 5 are CH (CH 3 ) CH 2 CH 3 or CH (CH 3 ) 2 , and n is 2 or 3.

데스트룩신 유도체는 공지된 방법(Phytochemistry, Vol. 20, pp. 715-723, 1981 ; J. Chem. Soc. Perkin. Trans. Ｉ, 2347-2357, 1989 ; J. Peptide Res. 50, 1997, 94-101 ; J. Antibiotics, Vol. 50, 1007- 1013, 1997 ; J. Nat. Prod., 61, 290-293, 1998 등)에 기술된 방법 1에 따라서 제조될 수 있다. 이에 의하면 많이 알려져 있는 데스트룩신 유도체인 데스트룩신 A, B, C, D, E, A1,A2,B1,B2,C2,D1,D2,E1및 E2등 뿐만 아니라 로제오톡신 B, 로제오카르딘, 데스메틸데스트룩신 B 그리고 R1이 클로로하이드린 또는 아세틸화클로로하이드린 등으로 치환된 각종 데스트룩신 유도체들의 제조방법, 분리방법과 그의 용도 등이 공지되어 있다. 본 발명의 데스트룩신 유도체는 약제학적으로 허용되는 염으로 사용될 수도 있는데 통상의 무기산 또는 유기산과의 염일 수 있다.Destruxine derivatives are known methods (Phytochemistry, Vol. 20, pp. 715-723, 1981; J. Chem. Soc. Perkin. Trans. I, 2347-2357, 1989; J. Peptide Res. 50, 1997, 94 -101; J. Antibiotics, Vol. 50, 1007-1013, 1997; J. Nat. Prod., 61, 290-293, 1998, etc.). According to this, as well as the destructin derivatives A, B, C, D, E, A 1, A 2, B 1, B 2, C 2, D 1, D 2, E 1 and E 2 which are well known There are known methods for the preparation, separation and use of various dexruxin derivatives in which rototoxin B, rozeocardine, desmethyldestrexine B and R 1 are substituted with chlorohydrin or acetylated chlorohydrin and the like. . The destruxin derivatives of the present invention may also be used as pharmaceutically acceptable salts, which may be salts with conventional inorganic or organic acids.

본 발명에 따라 사용될 수 있는 데스트룩신 유도체는 다음 표 1과 같다Destruxin derivatives that can be used according to the present invention are shown in Table 1 below.

본 발명에 따른 데스트룩신 유도체Destruxin derivatives according to the invention 종류Kinds R1 R 1 R2 R 2 R3 R 3 R4 R 4 R5 R 5 R6 R 6 nn AA CH2-CH=CH2 CH 2 -CH = CH 2 HH CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 22 BB CH2-CH(CH3)2 CH 2 -CH (CH 3 ) 2 HH CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 22 CC CH2CH(CH3)CH2OHCH 2 CH (CH 3 ) CH 2 OH HH CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 22 DD CH2CH(CH3)COOHCH 2 CH (CH 3 ) COOH HH CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 22 EE HH CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 22 A1A1 CH2CH=CH2 CH 2 CH = CH 2 HH CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 33 A2A2 CH2CH=CH2 CH 2 CH = CH 2 HH CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 22 B1B1 CH2CH(CH3)2 CH 2 CH (CH 3 ) 2 HH CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 33 B2B2 CH2CH(CH3)2 CH 2 CH (CH 3 ) 2 HH CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 22 C2C2 CH2CH(CH3)CH2OHCH 2 CH (CH 3 ) CH 2 OH HH CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 22 D1D1 CH2CH(CH3)COOHCH 2 CH (CH 3 ) COOH HH CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 33 D2D2 CH2CH(CH3)COOHCH 2 CH (CH 3 ) COOH HH CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 22 E1E1 HH CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 33 E2E2 HH CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 22 로제오톡신 BRoseotoxin B CH2CH=CH2 CH 2 CH = CH 2 CH3 CH 3 CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 22 로제오카르딘Roseocardine CH2CH(CH3)2 CH 2 CH (CH 3 ) 2 CH3 CH 3 CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 22 데스메틸데스트룩신BDesmethyldeluxexin B CH2CH(CH3)2 CH 2 CH (CH 3 ) 2 HH CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 HH CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 22 클로로하이드린Chlorohydrin CH2CH(OH)CH2ClCH 2 CH (OH) CH 2 Cl HH CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 22 아세틸화클로로하이드린Acetylated Chlorohydrin CH2CH(OCOCH3)CH2ClCH 2 CH (OCOCH 3 ) CH 2 Cl HH CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 22 A4 클로로하이드린A4 Chlorohydrin CH2CH(OH)CH2ClCH 2 CH (OH) CH 2 Cl HH CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 22 A4A4 CH2CH=CH2 CH 2 CH = CH 2 HH CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 22 호모데스트룩신 BHomo estluxine B CH2CH(CH3)2 CH 2 CH (CH 3 ) 2 HH CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 22 Lac-6덱스트룩신 ELac-6dextrinsine E CH3 CH 3 HH CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 22 2-하이드록시-4-펜티노익애시드2-hydroxy-4-pentinoic acid CH2-C≡CHCH 2 -C≡CH HH CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 CH3 CH 3 CH(CH3)2 CH (CH 3 ) 2 CH3 CH 3 22 프로토데스트룩신Protoestruxin CH2CH(CH3)2 CH 2 CH (CH 3 ) 2 HH CH(CH3)CH2CH3 CH (CH 3 ) CH 2 CH 3 HH CH(CH3)2 CH (CH 3 ) 2 HH 22

본 발명에 따른 상기 화학식(I)로 표시되는 데스트룩신 유도체는 파골세포에 대한 억제효과가 매우 강력하므로 임상적으로 유용한 골다공증 예방 및 치료제로서 사용될 수 있다. 따라서, 본 발명은 상기 화학식(I)로 표시되는 데스트룩신 유도체와 그의 약제학적으로 허용 가능한 염을 활성성분으로 함유하는 약제조성물에 관한 것이다.The destruxin derivative represented by Formula (I) according to the present invention has a very strong inhibitory effect on osteoclasts, and thus can be used as a clinically useful prevention and treatment of osteoporosis. Therefore, the present invention relates to a pharmaceutical composition containing as an active ingredient a destruxin derivative represented by Formula (I) and a pharmaceutically acceptable salt thereof.

본 발명의 약제조성물을 임상적으로 이용할 때에는 약제학적 분야에서 통상적인 담체와 함께 배합하여 통상적인 제제, 예컨대 정제, 캅셀제, 트로키제, 액제, 현탁제 등의 경구투여용 제제 그리고 주사용 용액 또는 현탁액, 또는 주사시에 주사용 증류수로 제조하여 사용할 수 있는 즉시 사용형 주사용 건조분말 등의 형태인 주사용 제제, 기타 스프레이로 사용할수 있는 액제 더 나아가 좌제 또는 패취제 등의 다양한 제제로 제형화 할 수 있다.When clinically using the pharmaceutical composition of the present invention, it is combined with a conventional carrier in the pharmaceutical field, and a conventional preparation such as tablets, capsules, troches, oral administration preparations such as solutions, suspensions, and injectable solutions or suspensions Or injectable preparations in the form of ready-to-use injectable dry powders that can be prepared and used as injectable distilled water at the time of injection. have.

통상적인 담체를 사용하여 제조된 약제학적 제제는 경구로 투여하거나, 비경구로 예를들면 정맥내, 피하, 복강내, 비강 또는 구강 등의 체강 또는 국소로 투여할 수 있다. 또한, 본 발명에 따른 데스트룩신 유도체의 인체에 대한 투여량은 일반적으로 성인에게 1일에 0.1㎍ ~ 100㎎, 바람직하게는 100㎍ ~ 1㎎의 양이 투여되도록 하며 의사 또는 약사의 판단에 따라 일정시간 간격으로 1일 수회, 바람직하기로는 1회 내지는 6회 분할투여할 수 있다.Pharmaceutical formulations prepared using conventional carriers can be administered orally or parenterally, for example intravenously, subcutaneously, intraperitoneally, nasal or orally, into body cavity or topically. In addition, the dosage of the destruxin derivative according to the present invention to the human body is generally such that an amount of 0.1 µg to 100 mg, preferably 100 µg to 1 mg per day is administered to an adult, at the discretion of a doctor or pharmacist. It can be administered several times a day, preferably once or six times at regular time intervals.

본 발명에서는 데스트룩신 유도체가 골흡수를 억제하는 정도를 측정하여 이를 골다공증 억제활성으로 조사하기 위하여 파골세포의 골흡수 억제능 시험법을 확립하여 검색에 사용하였다. 시험법은 뼈절편에 생성된 파골세포의 골흡수 흔적인 피트(pit)의 수와 면적을 측정하는 것이며, 이때 골흡수가 억제된 만큼 피트의 면적이 줄게 됨으로 대조군과 구별될 수 있다. 또한 다른 시험법은 파골세포의 골격을 유지하는 기능을 가지고 있는 액틴링(actin ring)을 관찰하는 것이며, 이때 파골세포가 저해받은 만큼 액틴링이 정상적인 구조를 가지지 못하게 됨으로 대조군과 구별될 수 있다.In the present invention, in order to determine the degree of inhibition of bone resorption by the dexluxine derivative and to investigate it as an osteoporosis inhibitory activity, it was used to establish a test for the inhibition of bone resorption of osteoclasts. The test method is to measure the number and area of pit, the bone resorption traces of osteoclasts generated in the bone fragments, which can be distinguished from the control group because the area of the pit decreases as the bone resorption is suppressed. In addition, another test is to observe the actin ring (actin ring) that has the function to maintain the skeleton of the osteoclasts, which can be distinguished from the control group because the actin ring does not have a normal structure as the osteoclasts are inhibited.

본 발명의 구체적인 구성 및 작용을 다음의 실시예에 의거 더욱 상세히 설명하지만 본 발명의 권리범위가 이들 실시예에만 한정되는 것은 아니며 당업자간에 용이하게 실시할 수 있는 어떠한 변경이 가능하다.Although the specific configuration and operation of the present invention will be described in more detail based on the following examples, the scope of the present invention is not limited only to these examples, and any modifications which can be easily implemented by those skilled in the art can be made.

실시예 1: 파골세포의 준비Example 1 Preparation of Osteoclasts

본 실시예에서는 골아세포와 골수세포의 공존배양에 의하여 파골세포를 다음과 같이 준비하였다.In this example, osteoclasts were prepared by co-culture of osteoblasts and bone marrow cells as follows.

실험예 1: 골아세포의 준비Experimental Example 1: Preparation of Osteoblasts

생후 1일된 마우스 50마리를 70%에탄올에 익사시킨 후 각 마우스로부터 두개골(calvaria)를 얻고 이를 α-MEM 배지(Gibco사 제품)에 디스파아제(dispase, 베링거만하임사 제품)와 콜라게나아제(collagenase, Wako사 제품)를 각각 0.2%, 0.1%가 되도록 용해한 배지 10mL가 담긴 50mL 원심분리용 튜브에 넣는다. 37℃에서 5분 동안 200rpm으로 진탕한 후 배지를 회수하고 배지가 제거된 시험관에 다시 0.2% 디스파아제, 0.1% 콜라게나아제가 포함된 α-MEM배지 10mL을 넣고 37℃에서 10분간 200rpm으로 진탕한 후 또 배지를 회수하였다. 이 조작을 3회 반복 실시하여 골아세포가 포함된 배지 40mL을 회수하였다. 이 액을 1000rpm 으로 5분간 원심분리 한 후 배지는 버리고 침전된 골아세포에 혈청(FCS: Fetal Calf Serum)이 10% 포함된 α-MEM(10%FCS)배지 10mL를 넣어 현탁시키고 다시 α-MEM(10%FCS)배지 15mL를 넣어 25mL로 만들었다. 상기 용액 5mL를 미리 α-MEM(10%FCS)배지 10mL이 포함된 100 x 20mm 배양접시 5개에 각각 넣고 37C, 5% CO2에서 배양하였다. 2일간 배양 후 세포를 관찰하고 회수하였다. 이때 파골세포의 회수는 배양접시의 배지를 제거하고 0.2% 콜라게나아제가 포함된 α-MEM (10%FCS(-))배지를 2mL씩 넣고 약 20분간 37℃, 5% CO2에 방치한 후 표면에 부착된 세포를 떼어내고 떼어낸 배지는 모두 모아서 1000rpm에서 원심분리하여 완료하였다. 상기 골아세포의 세포액을 다시 α-MEM(10%FCS)배지로 현탁하여 준비하고 다음실험에 공시재료로 사용하였다.Fifty days old mice were drowned in 70% ethanol, and then skulls were obtained from each mouse, which were then dissolved in α-MEM medium (manufactured by Gibco) and collagenase (from Boehringer Mannheim). collagenase, manufactured by Wako) were placed in a 50 mL centrifuge tube containing 10 mL of the dissolved medium to 0.2% and 0.1%, respectively. After shaking at 200 ° C. for 5 minutes at 37 ° C., the medium was recovered, and 10 mL of α-MEM medium containing 0.2% dispase and 0.1% collagenase was added to the test tube from which the medium was removed. After shaking, the medium was also recovered. This operation was repeated three times to recover 40 mL of the medium containing osteoblasts. Centrifuge this solution at 1000rpm for 5 minutes, discard the medium, and suspend 10mL of α-MEM (10% FCS) medium containing 10% serum (FCS: Fetal Calf Serum) in precipitated osteoblasts. 15 mL of (10% FCS) medium was added to make 25 mL. 5 mL of the solution was previously added to each of 100 x 20 mm culture plates containing 10 mL of α-MEM (10% FCS) medium and incubated at 37C and 5% CO 2 . After incubation for 2 days, cells were observed and recovered. At this time, the recovery of osteoclasts was carried out by removing the culture plate medium and adding 2 mL of α-MEM (10% FCS (-)) medium containing 0.2% collagenase, and leaving it at 37 ° C. and 5% CO 2 for about 20 minutes. After detaching the cells attached to the surface and remove all the removed medium was completed by centrifugation at 1000rpm. Cell solution of the osteoblasts was prepared by suspending again in α-MEM (10% FCS) medium and used as a test material for the next experiment.

실험예 2: 골수세포의 준비Experimental Example 2: Preparation of Bone Marrow Cells

ddY 마우스(6-9주령 수컷)를 척추탈구로 희생시킨 후 70%에탄올로 소독한 후[이하, 조작은 크린벤치(Clean Bench)에서 수행하였다] 대퇴골 및 경골부분의 피층을 절개하고 부착근육을 제거하였다. 경골원심부를 절단하고 무릎관절을 탈구시켜 경골 및 대퇴골을 적출하였다. 적출된 골 끝을 절단 하고 25게이지(Gage)의 주사기를 이용하여 대퇴골 및 경골의 원심 말단부분에 α-MEM(10%FCS) 배지 1-2mL을주입하여 골수세포를 모았으며 2-3마리씩 각각 이 조작을 수행하였다. 1000rpm에서 원심분리하여 세포를 모은 후 배지를 제거하고 침전된 세포중 적혈구를 제거하기 위해 0.83% NH4Cl가 포함된 10mM Tris HCl 완충액(pH7.5) 3mL을 첨가하였다. 다시, 1000rpm으로 원심분리한 후 상층용액을 제거하였다. 이렇게 하여 경골 한 개당 약 1 × 107개의 골수세포를 얻었으며 이 골수세포는 하기 실험예 3의 공존배양에 바로 사용되었다.ddY mice (6-9 week old males) were sacrificed by spinal dislocation and then disinfected with 70% ethanol (hereinafter, the manipulation was performed on a clean bench). Removed. The tibia center was cut and the knee joint was dislocated to remove the tibia and the femur. Bone marrow cells were collected by injecting 1-2 mL of α-MEM (10% FCS) medium into the distal ends of the femur and tibia using a 25-gauge syringe. This operation was performed. Cells were collected by centrifugation at 1000 rpm, medium was removed, and 3 mL of 10 mM Tris HCl buffer (pH 7.5) containing 0.83% NH 4 Cl was added to remove red blood cells from precipitated cells. Again, the supernatant was removed after centrifugation at 1000rpm. In this way, about 1 × 10 7 bone marrow cells were obtained per tibia, and these bone marrow cells were immediately used in the co-culture of Experimental Example 3 below.

실험예 3: 파골세포의 준비Experimental Example 3: Preparation of Osteoclasts

파골세포의 형성과 분리는 콜라겐 겔용액이 코팅된 배지 위에서 이루어졌으며 콜라겐 겔용액 코팅배지의 제조는 다음과 같이 하였다. 콜라겐 겔, 5배농도의 α-MEM배지(NaHCO3를 포함함), 2.2% NaHCO3또는 200mM HEPES를 포함한 0.05M NaOH 완충액(pH7.4)을 각각 7 : 2 : 1의 비율로 저온에서 혼합한 후 약 4-5mL를 100mL 배양접시에 붓고 콜라겐 용액이 배양접시 전체면에 고루 도포 되도록 잘 흔든 후 37℃에서 5분간 겔화 시키고 4℃에 보관하였다. 도포된 콜라겐 겔용액 배지에 상기 실험예 1과 2에서 준비한 골아세포( 약 1x105세포) 와 골수세포(약 1x107세포)를 활성비타민 D3(10-8M)이 포함된 α-MEM(10% FCS)배지에서 2일마다 배지를 교환하면서 6∼8일간 공존배양을 수행하였다. 배양액을 제거하고 디스파아제와 콜라게나아제가 포함된 α-MEM배지(10% FCS가 포함되지 않음)를 3mL 첨가한 후 37℃에서 약 300rpm으로 진탕시켜 콜라겐 겔이 용해되게 하였다.The formation and separation of osteoclasts was done on the collagen gel-coated medium. The collagen gel solution-coated medium was prepared as follows. Collagen gel, 5-fold α-MEM medium (containing NaHCO 3 ), 0.05M NaOH buffer (pH7.4) containing 2.2% NaHCO 3 or 200 mM HEPES, respectively, at a low ratio of 7: 2: 1 Then, about 4-5mL was poured into a 100mL culture dish, shaken well so that the collagen solution was evenly applied to the whole surface of the culture dish, and then gelled at 37 ° C for 5 minutes and stored at 4 ° C. In the applied collagen gel solution medium, the osteoblasts (about 1x10 5 cells) and bone marrow cells (about 1x10 7 cells) prepared in Experimental Examples 1 and 2 contained α-MEM containing active vitamin D 3 (10 -8 M). Co-cultures were performed for 6-8 days with medium change every 10 days in 10% FCS) medium. The culture solution was removed, and 3 mL of α-MEM medium (not containing 10% FCS) containing dispase and collagenase was added, followed by shaking at 37 ° C. at about 300 rpm to dissolve the collagen gel.

입구가 넓은 폴리프로필렌 피펫으로 세포를 혼탁시킨 후 50mL 원심관으로 옮기고 200rpm으로 5분간 원심분리 한 후 α-MEM배지(10% FCS 포함)로 현탁하여 파골세포 용액을 준비하였다.The cells were turbid with a polypropylene pipette with a wide mouth, then transferred to a 50 mL centrifuge tube, centrifuged at 200 rpm for 5 minutes, and suspended in α-MEM medium (including 10% FCS) to prepare osteoclast solutions.

파골세포의 전형적인 특징은 TRAP(tartrate-resistant acid phospatase)활성, 다량의 칼시토닌 수용체 및 골흡수능을 가진다는 점이다. 따라서, 본 발명에서는 파골세포를 다른 세포와 구분하기 위하여 TRAP 염색을 수행함으로써 쉽게 확인하였다.A typical feature of osteoclasts is that they have tartrate-resistant acid phospatase (TRAP) activity, abundant calcitonin receptors, and bone resorption capacity. Therefore, in the present invention, it was easily confirmed by performing TRAP staining to distinguish osteoclasts from other cells.

실시예 2: 파골세포의 생성 확인Example 2: Confirmation of production of osteoclasts

실험예 1: TRAP(tartrate-resistant acid phospatase) 반응액 조제Experimental Example 1: Preparation of a TRAP (tartrate-resistant acid phospatase) reaction solution

기질 나프톨(Naphtol) AS-MX 포스페이트(sigma사 제품) 5mg을 N,N-디메틸포름아마이드 0.5mL에 용해한 다음 50mM 타르타르산(sigma사 제품)을 포함한 0.1N NaHCO3완충액(pH 5)을 50mL이 되게 첨가하고 색소( Fast red violet LB salt(sigma사 제품)) 30mg을 넣어 용해시켰다.Dissolve 5 mg of substrate Naphtol AS-MX phosphate (from Sigma) in 0.5 mL of N, N-dimethylformamide and then 50 mL of 0.1 N NaHCO 3 buffer (pH 5) containing 50 mM tartaric acid (from Sigma). 30 mg of the pigment (Fast red violet LB salt, manufactured by sigma) was dissolved.

실험예 2: TRAP 염색Experimental Example 2: TRAP Staining

파골세포가 형성된 플레이트로부터 배지를 제거하고 10%포르말린을 넣어 5-10분 고정한 후 건조하였다. 상기 실험예 1에서 조제한 에탄올/아세톤(1/1)용액에 재차 고정한 후 또다시 건조하였다. TRAP 용액을 넣고 실온에서 10-15분간 방치한 후 반응액을 제거하였으며 이를 수세 건조하고 현미경으로 관찰한 결과 다량의 파골세포가 생성되었음이 도 1가 같이 확인되었다.The medium was removed from the plate on which the osteoclasts were formed, fixed with 10% formalin for 5-10 minutes, and dried. After fixing again to the ethanol / acetone (1/1) solution prepared in Experimental Example 1 and dried again. After the TRAP solution was added and left at room temperature for 10-15 minutes, the reaction solution was removed, and the resultant was washed with water and observed under a microscope. A large amount of osteoclasts were produced as shown in FIG. 1.

실시예 3: 파골세포의 골흡수능 검정Example 3: Bone resorption capacity assay of osteoclasts

실험예 1: 피트 측정법Experimental Example 1: Feet Measurement

파골세포의 가장 중요한 특성은 뼈를 분해하는 것이므로 뼈표면에서 성장하고 있는 파골세포가 뼈표면의 골흡수 흔적(피트, pit)을 측정함으로써 파골세포의 골흡수능의 유무를 조사하였다.Since the most important characteristic of osteoclasts is the breakdown of bones, osteoclasts growing on the bone surface were examined for the bone resorption capacity of osteoclasts by measuring bone resorption traces (pits) on the bone surface.

뼈절편을 1mm두께로 절단하여 메탄올에 넣고 U.V 살균등이 켜진 상기 크린벤치에서 살균시킨 후 준비된 뼈절편을 96well plate에 하나씩 넣고 α-MEM배지(10% FCS 포함) 100ul를 넣었다. 파골세포의 피트 형성 저해활성을 측정할 데스트룩신 E(Destruxin E)를 최고 3ul 넣은 후 준비된 파골세포 용액을 100ul 넣고 잘 흔들어 혼합한 뒤 37℃, 5% CO2하에서 1일간 배양하였다. 배양 후 현미경으로 뼈절편 주변의 파골세포 상태를 관찰하고 4℃에서 보관하였다.The bone fragments were cut to 1 mm thickness, put into methanol, sterilized in the clean bench with UV sterilization lamps, and then prepared bone fragments were placed in 96well plates one by one and 100ul of α-MEM medium (including 10% FCS) was added thereto. After inserting up to 3ul of Destructin E (Destruxin E) to measure the pit formation inhibitory activity of the osteoclasts, 100ul of the prepared osteoclast solution was mixed well, followed by incubation at 37 ° C. and 5% CO 2 for 1 day. After incubation, the osteoclast state around the bone fragments was observed under a microscope and stored at 4 ° C.

뼈절편에 생성된 피트의 수 와 면적을 측정하여 대조군과 비교하면 활성을 측정할 수 있는 것이므로 피트를 현미경으로 관찰하기 위해서 뼈절편을 파골세포가 성장한 부분을 위로 가게 하여 96well plate로부터 꺼내서 종이타올에 놓고 염색액 헤마톡신용액(Sigma)을 6ul 뼈절편 위에 첨가하였다. 3-5분 후에 상기 헤마톡신 용액(sigma)을 제거하고 산성용액에서 염색액을 완전히 제거한 후 뼈절편을 현미경으로 관찰하여 피트의 수를 측정한 결과, 도 2 및 도 3(A)와 같았다. 도 2(A)는 정상적인 파골세포의 모양을 나타내고 (B)는 골표면에서 파골세포의 골흡수작용으로 인하여 형성된 피트의 모습이며 (C)는 데스트룩신 E로 처리한 후 파골세포가 변형되어 위축된 모습을 나타내고, (D)는 데스트룩신 E를 처리한 후 파골세포의 골흡수작용 결과 감소된 피트모양을 나타내고 있다. 도 3(A)는 데스트룩신 E가 처리된 배지에서 성장한 파골세포가 생성한 피트수와 정상배지에서 성장한 파골세포수를 수치화한 그래프인 바 데스트룩신 E의 농도가 증가할 수록 피트수가 감소하고 있음을 알 수 있었다. 또한, 데스트룩신 E는 다음 표 2에서 볼 수 있듯이 10nM에서 50%의 저해활성(IC50)을 갖는 것으로 확인되었다.Since the number and area of pits generated on the bone fragments can be measured and compared to the control group, the activity can be measured compared with the control group. Stain and hematoxylin solution (Sigma) were added onto 6ul bone sections. After 3-5 minutes, the hematoxylin solution (sigma) was removed, the dye solution was completely removed from the acidic solution, and the bone fragments were observed under a microscope to measure the number of pits, as shown in FIGS. 2 and 3 (A). Figure 2 (A) shows the shape of the normal osteoclast (B) is the appearance of the pit formed by the bone resorption of the osteoclast at the bone surface (C) is deformed atrophy of osteoclasts after treatment with Destinxine E (D) shows reduced pit shape as a result of bone resorption of osteoclasts after treatment with Destinlux E. FIG. 3 (A) shows that the number of feet produced by osteoclasts grown in desluxxin E-treated medium and the number of osteoclasts grown in normal medium are decreased as the concentration of bar destranxin E increases. And it was found. In addition, Dexluxin E was found to have a 50% inhibitory activity (IC 50 ) at 10 nM as shown in Table 2.

본 발명에 따른 데스트룩신 E에 의한 파골세포의 피트저해활성Pit inhibitory activity of osteoclasts by destructin E according to the present invention 농도(nM)Concentration (nM) 00 0.50.5 1One 55 1010 5050 100100 피트저해활성(%)Feet inhibition activity (%) 00 22 55 3434 5050 9595 100100

실험예 2: 액틴링 검정법(Actin ring assay)Experimental Example 2: Actin ring assay

또한, 파골세포의 골격을 유지하는 기능을 갖고 있는 액틴링의 관찰을 위해서 상기 실험예 1의 TRAP 염색법 대신 액틴링 검정법을 수행하였다. 배지를 제거하고 포르말린으로 10분간 고정하고 Tween 20으로 1회 세척하여 메탄올에 용해된 300U 로드아민 팔로이딘(Rhodamine phalloidin(sigma사 제품))으로 30분간 빛이 들지 않는 암소에서 처리하고 UV광원 하에서 세포내 액틴링의 분포를 관찰하였다. 실험결과, 표 3에서 보듯이 8nM(IC50)에서 파골세포의 50%가 정상적인 액틴링의 구조를 가지지 못하는 것으로 확인되었다 . 도 3에서 (B)는 데스트룩신 E로 처리된 파골세포의 액틴링 정상유무를 정상적인 파골세포와 상대적으로 비교한 그래프인바, 데스트룩신 E의 농도증가에 따라 정상적인 액틴링의 수가 감소하고 있음을 알 수 있었다.In addition, the actining assay was performed instead of the TRAP staining method of Experimental Example 1 for the observation of actining having a function of maintaining the skeleton of osteoclasts. The medium was removed, fixed in formalin for 10 minutes, washed once with Tween 20, treated with 300 U rhodeamine paloidine (Rhodamine phalloidin (manufactured by Sigma)) dissolved in methanol in a light-free cow for 30 minutes and treated under UV light. The distribution of actining was observed. As a result, as shown in Table 3, it was confirmed that 50% of the osteoclasts at 8 nM (IC 50 ) do not have a normal actin ring structure. 3 (B) is a graph comparing the normal presence or absence of actining of osteoclasts treated with Destinxine E as compared with normal osteoclasts, indicating that the number of normal actining decreases as the concentration of Destinxine E increases. Could.

본 발명에 따른 데스트룩신 E에 의한 파골세포의 액틴링 저해활성Actining Inhibitory Activity of Osteoclasts by Destinoxin E According to the Present Invention 농도(nM)Concentration (nM) 00 0.50.5 1One 55 1010 5050 100100 액틴링저해활성(%)Actining inhibitory activity (%) 00 1One 33 1010 6565 100100 100100

실시예 4: 마우스의 기관배양을 통한 데스트룩신 E의45Ca 유리 저해능 조사Example 4 Investigation of 45 Ca Free Inhibition of Destructin E by Organ Culture of Mice

임신 15-16일의 ddy 마우스에45Ca으로 라벨링된 염화칼슘용액을 피하주사해서 뼈에 라벨링을 한 후 1일 후에 마우스를 에테르(Ether)로 마취시키고 70% 에탄올로 전신을 소독하였다. 다음, 마우스의 배를 자르고 자궁을 잘라 태마우스를 적출하여 소독된 실험접시로 옮겼다. 적출된 태마우스의 앞다리를 잘라내어 뼈를 적출하였는데 이는 실체현미경 아래에서 수행하였으며 피층를 벗긴 후에 요골(radius)와 척골(ulna)에 붙어있는 근육을 떼어내고 각 뼈의 양쪽 말단에 있는 연골부를 잘라내었다. 이때, 뼈가 건조되지 않도록 하며 전배양을 하기 전에 BGJb배지(Gibco사 제품)를 0.5mL씩 24 well 접시에 넣고 스테인레스 스틸 막을 배지에 넣고 그 위에 준비된 뼈를 놓았다. 24시간 배양 후 데스트룩신 E가 포함된 배지로 옮겨서 72시간 배양하였다. 이때, 인체에서 칼슘 방출을 유도하는 부갑상선호르몬(PTH)을 같이 사용하여 그 저해활성을 측정하고자 하였다. 배양종료 후에는 뼈만 건져서 5%의 TCA로 뼈 안에 존재하는45Ca량을 측정하고 동시에 배지 내에 존재하는45Ca양을 측정하였다. 이때 골흡수 활성은 다음과 같이 계산하였다.The bones were labeled subcutaneously with 45 Ca labeled calcium chloride solution at 15-16 days of gestation, and mice were anesthetized with ether and disinfected whole body with 70% ethanol. Next, the mouse's belly was cut and the uterus was cut and the mouse was removed and transferred to a sterile test plate. Bones were extracted by cutting the forelimbs of the extracted mouse, which was carried out under a stereomicroscope, and after removing the cortex, the muscles attached to the radial and ulna were removed and the cartilages at both ends of each bone were cut out. At this time, the bone is not dried and before the pre-culture BGJ b medium (Gibco Co., Ltd.) 0.5mL each 24 well plate and put a stainless steel membrane in the medium to place the prepared bone on it. After incubation for 24 hours, the medium was transferred to a medium containing deluxuxin and incubated for 72 hours. At this time, the parathyroid hormone (PTH) that induces the release of calcium in the human body was used to measure its inhibitory activity. After the end of the culture, only the bones were harvested to measure the amount of 45 Ca present in the bone with 5% TCA and at the same time, the amount of 45 Ca present in the medium was measured. The bone resorption activity was calculated as follows.

골흡수활성= 배양액중의45Ca방출량 / (배양액중의45Ca방출량+뼈중의45Ca량) × 100Bone resorption activity = culture medium 45 Ca (Ca 45 45 Ca + ppyeojung amount of emission in the culture medium) discharge amount / of × 100

실험결과, 도 4에서 알 수 있듯이 본 발명에 따른 데스트룩신 E는 3nM에서 PTH에의한45Ca유리작용을 거의 완전히 저해하고 있음을 확인하였다.As shown in FIG. 4, it was confirmed that the destruxin E according to the present invention almost completely inhibited 45 Ca free action by PTH at 3 nM.

실시예 5: 데스트룩신 B(Destruxcin B)의 파골세포 저해Example 5: Osteoclast Inhibition of Destruxcin B

본 발명에 따른 데스트룩신 B의 파골세포 저해활성 실험을 상기 실시예 4와 동일한 과정을 거쳐 실시하였다. 데스트룩신 B의 경우는 표 4에서 알 수 있듯이 IC50(피트저해활성)와 IC50(액틴링저해활성)가 각각 0.2μM, 0.6μM로서 데스트툭신 E보다 활성은 낮았지만 파골세포 저해활성은 매우 우수한 것으로 판명되었다.The osteoclast inhibitory activity of the destructin B according to the present invention was carried out through the same process as in Example 4. As shown in Table 4, in the case of dexluxin B, IC 50 (pit inhibitory activity) and IC 50 (actinring inhibitory activity) were 0.2 μM and 0.6 μM, respectively, lower in activity than dextuxine E, but excellent in osteoclast inhibitory activity. It turned out to be.

본 발명에 따른 데스트룩신 B의 파골세포 저해활성Osteoclast inhibitory activity of destrancin B according to the present invention 농도(μM)Concentration (μM) 00 0.10.1 0.50.5 1One 55 피트저해활성(%)Feet inhibition activity (%) 00 2525 7171 9494 100100 액틴링저해활성(%)Actining inhibitory activity (%) 00 1212 4141 9292 100100

이상, 실시예와 실험예를 통하여 알 수 있는 바와 같이, 본 발명에 따르면 데스트룩신 유도체의 골다공증과 관련된 질환의 예방 및 치료용 약제 조성물로서의 새로운 용도를 제공하는 효과가 있다. 또한 본 발명에 따르면 화합물 데스트룩신 유도체를 유효성분으로 함유한 골다공증의 예방 및 치료용 약제조성물을 제공하는 뛰어난 효과가 있으므로 화학 및 의약산업상 매우 유용한 발명인 것이다.As can be seen through the examples and experimental examples, according to the present invention, there is an effect of providing a new use as a pharmaceutical composition for the prevention and treatment of diseases related to osteoporosis of the destruxin derivative. In addition, according to the present invention is a very useful invention in the chemical and pharmaceutical industry because it has an excellent effect of providing a pharmaceutical composition for the prevention and treatment of osteoporosis containing the compound destruxin derivative as an active ingredient.

Claims (3)

하기 화학식(I)으로 표시되는 데스트룩신 유도체를 유효성분으로 함유하는 골다공증 예방 및 치료제 조성물.A osteoporosis prophylactic and therapeutic composition comprising the destruxin derivative represented by the following formula (I) as an active ingredient. 단, 상기 식에서 R1는 CH3,CH2-CH=CH2, CH2CH(CH3)2, CH2CH(CH3)CH2OH,, CH2CH(CH3)COOH, CH2CH(OH)CH2Cl, CH2-C≡CH 또는 CH2CH(OCOCH3)CH2Cl이고, R2, R4및 R6는 H 또는 CH3이고, R3와 R5는 CH(CH3)CH2CH3또는 CH(CH3)2이며, n는 2 또는 3이다.In which R 1 is CH 3, CH 2 -CH = CH 2 , CH 2 CH (CH 3 ) 2 , CH 2 CH (CH 3 ) CH 2 OH, , CH 2 CH (CH 3 ) COOH, CH 2 CH (OH) CH 2 Cl, CH 2 -C≡CH or CH 2 CH (OCOCH 3 ) CH 2 Cl, R 2 , R 4 and R 6 are H or CH 3 , R 3 and R 5 are CH (CH 3 ) CH 2 CH 3 or CH (CH 3 ) 2 , and n is 2 or 3. 제 1 항에 있어서, 상기 데스트룩신 유도체가 데스트룩신 E인 골다공증 예방 및 치료제 조성물.2. The osteoporosis prophylactic and therapeutic composition according to claim 1, wherein the destructin derivative is destructin E. 제 1항에 있어서, 데스트룩신 유도체가 데스트록신 B인 골다공증예방 및 치료제 조성물The osteoporosis prophylaxis and therapeutic composition according to claim 1, wherein the destruxin derivative is destroxin B.
KR1019990031532A 1999-07-31 1999-07-31 Pharmaceutical composition for prevention and remedy of osteoporosis KR100309888B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1019990031532A KR100309888B1 (en) 1999-07-31 1999-07-31 Pharmaceutical composition for prevention and remedy of osteoporosis

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1019990031532A KR100309888B1 (en) 1999-07-31 1999-07-31 Pharmaceutical composition for prevention and remedy of osteoporosis

Publications (2)

Publication Number Publication Date
KR20010011926A true KR20010011926A (en) 2001-02-15
KR100309888B1 KR100309888B1 (en) 2001-11-01

Family

ID=19605913

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1019990031532A KR100309888B1 (en) 1999-07-31 1999-07-31 Pharmaceutical composition for prevention and remedy of osteoporosis

Country Status (1)

Country Link
KR (1) KR100309888B1 (en)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69519669T2 (en) * 1994-05-26 2001-04-26 Nisshin Flour Milling Co CYCLODEPSIPEPTIDE
JPH0827183A (en) * 1994-07-08 1996-01-30 Riyoushiyoku Kenkyukai Cyclic depsipeptide
JPH09315996A (en) * 1996-05-27 1997-12-09 Meiji Milk Prod Co Ltd Cardiotonic agent and its production

Also Published As

Publication number Publication date
KR100309888B1 (en) 2001-11-01

Similar Documents

Publication Publication Date Title
US8227496B2 (en) Use of 4-[(4-thiazolyl)phenoxy]alkoxy-benzamidine derivatives for treatment of osteoporosis
Fell et al. The effect of hypervitaminosis A on embryonic limb-bones cultivated in vitro
KR20080094950A (en) External preparation for skin containing flavanone derivative
CZ20013110A3 (en) Preparation for treating arthritis
CN1111403C (en) Secondary cataract inhibitor
KR20170137201A (en) Derivatives used in the treatment of muscle atrophy
KR100309888B1 (en) Pharmaceutical composition for prevention and remedy of osteoporosis
JP2006515276A (en) Furan derivative having preventive and therapeutic effects on osteoporosis and pharmaceutical composition containing the same
US20040102365A1 (en) Pharmaceutical composition for prevention and remedy of osteoporosis
CN111920791A (en) Application of cuminaldehyde in preparation of medicine for treating echinococcosis granulosa
HU202104B (en) Process for producing immunstimulating pharmaceutical compositions containing 1-carboxy-3,4-methylenedioxi-8-methoxy-phenantrene
EP4049724A1 (en) Pharmaceutical composition for protecting cartilage
KR100718030B1 (en) Remedies for arthrosis deformans
AU2002231500B2 (en) Antiangiogenic compounds and an assay for inhibitors of cell invasion
KR100705173B1 (en) Use of N,N-diisopropyl-4-[7-4-aminophenoxy heptoxy]-3-methoxybenzamide for treatment of osteoporosis
CN117883439A (en) Application of indole-3-propionic acid in preparation of medicines for inhibiting osteoclast differentiation
US3991192A (en) Method of preventing metastasis and primary tumor growth of H. EP. No. 3
CN109793747A (en) Application of the Vaccarin in terms of the drug of preparation prophylactic treatment bone dissolubility disease
CN117618424A (en) Application of dihydroberberine in inhibiting osteoclast differentiation
US3988463A (en) Method of preventing metastasis of H. Ep. No. 3
CN116919932A (en) Osteogenic agent and use thereof
US20030004149A1 (en) Antiangiogenic compounds and an assay for inhibitors of cell invasion
Zhang et al. Application of cell engineering of herbal Medicine treating bone resorption of osteoclasts
AU2002231500A1 (en) Antiangiogenic compounds and an assay for inhibitors of cell invasion
KR20030024763A (en) Chemical compound for prevention and treatment of osteoporosis

Legal Events

Date Code Title Description
A201 Request for examination
E701 Decision to grant or registration of patent right
GRNT Written decision to grant
FPAY Annual fee payment

Payment date: 20070704

Year of fee payment: 7

LAPS Lapse due to unpaid annual fee