WO2018016901A1 - Composition pharmaceutique destinée à la prévention ou au traitement de maladies osseuses - Google Patents

Composition pharmaceutique destinée à la prévention ou au traitement de maladies osseuses Download PDF

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WO2018016901A1
WO2018016901A1 PCT/KR2017/007852 KR2017007852W WO2018016901A1 WO 2018016901 A1 WO2018016901 A1 WO 2018016901A1 KR 2017007852 W KR2017007852 W KR 2017007852W WO 2018016901 A1 WO2018016901 A1 WO 2018016901A1
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bone
disease
formula
pharmaceutical composition
osteoclasts
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PCT/KR2017/007852
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English (en)
Korean (ko)
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정우진
서은경
이지애
홍성은
이혜인
조유진
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이화여자대학교 산학협력단
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Priority claimed from KR1020160091899A external-priority patent/KR101832351B1/ko
Priority claimed from KR1020160091900A external-priority patent/KR101765141B1/ko
Priority claimed from KR1020160091897A external-priority patent/KR101747775B1/ko
Application filed by 이화여자대학교 산학협력단 filed Critical 이화여자대학교 산학협력단
Publication of WO2018016901A1 publication Critical patent/WO2018016901A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/439—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom the ring forming part of a bridged ring system, e.g. quinuclidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings

Definitions

  • the present invention relates to a pharmaceutical composition for the prevention or treatment of bone diseases.
  • Osteoporosis a representative metabolic bone disease, is a disease in which bone mineral density is reduced and bone density becomes thinner, resulting in widening of the bone marrow cavity. It is easy to fracture even in shock.
  • Osteoporosis is characterized by a decrease in bone mass and abnormal microstructures. As aging progresses, the balance between the absorption of old bone and the formation of new bone is compromised, resulting in poor bone remodeling, which leads to bone failure. As a result, the risk of breakage or fracture increases. This bone mass is influenced by several factors, including genetic factors, nutrition, hormonal changes, differences in exercise and lifestyle, and age, lack of exercise, low weight, smoking, low calcium diet, menopause and ovarian ablation. It is a major cause of bone loss.
  • bone mass is usually the highest at 14-18 years of age, and decreases by about 1% per year in old age.
  • women after 30 years of bone reduction continues to progress, and by the hormonal changes, bone reduction rapidly progresses.
  • estrogen concentration rapidly decreases at the end of menopause, in which a large amount of B-lymphocytes are produced, as in the case of IL-7 (interleukin-7), and the B cell precursor (pre-B) is added to the bone marrow. cells) accumulate, thereby increasing the amount of IL-6, increasing the activity of osteoclasts, and eventually reducing bone mass.
  • IL-7 interleukin-7
  • osteoporosis is an unavoidable symptom for elderly people, especially postmenopausal women, although the degree of osteoporosis is increasing, and as the population ages in developed countries, interest in osteoporosis and its therapeutics is gradually increasing.
  • $ 130 billion is being created around the world in the treatment of bone disease, and because it is expected to grow further, research institutions and pharmaceutical companies around the world are investing heavily in developing bone disease treatments.
  • osteoporosis is more common than diabetes or cardiovascular disease, and osteoporosis is a very important health problem when estimating the pain or cost of treating patients who suffer from fractures.
  • Osteoclasts are large multinucleated cells that destroy or absorb bone tissue that has become unnecessary during the growth of bones of vertebrates and are differentiated from osteoclast precursors. Osteoclast progenitor cells differentiate into osteoclasts in the presence of M-CSF and RANKL, and form multinucleated osteoclasts through fusion. Osteoclasts bind to bone through ⁇ v ⁇ 3 integrin and create an acidic environment while secreting various collagenases and proteases, resulting in bone resorption. Can be an effective way of treating bone disease.
  • Euphorbia Factor L1 (hereinafter referred to as EFL1) is a diterpenoid obtained from seeds of Euphorbia lathyris linne (responder / poor), antiseptic, anticancer and apoptosis sensitization. ) Is known (Zang JY. Et al., Molecules, 2013, 18, 12793-12808).
  • Republic of Korea Patent Publication No. 2001-0028373 discloses the use of EFL1 as a drug or cosmetic material as a keratin exfoliation promoter, the osteoclast differentiation control effect of EFL1 of the present invention and the purpose of treating bone disease is not known to date.
  • the scullcapflavone derivative is a flavonoid derived from Scutellaria baicalensis (gold), which is known to have anti-inflammatory and anticancer effects by folk remedies.
  • gold Scutellaria baicalensis
  • the effect of inhibiting allergic asthma is known (Yun-Choi. Et al., Archives of pharmacal research, v. 16 no. 4, 1993, 283-288).
  • Republic of Korea Patent Publication 2013-0120849 discloses the use of the skull cap flavone derivatives as a pharmaceutical material as a composition for the prevention or treatment of asthma, but the osteoclast differentiation control effect of the skull cap flavone derivatives of the present invention and the purpose of treating bone diseases None known until now.
  • dehydrocostus lactone DHCL
  • DHCL dehydrocostus lactone
  • cinchonine is an alkaloid derived from Cinchona bark, but the effects of anti-obesity and anti-diabetic are known (Korean Patent Publication No. 0946641), the effect of controlling the differentiation of osteoclast differentiation and bone diseases of the present invention Is unknown to date.
  • the present inventors while searching for a substance that inhibits the activity of osteoclasts in search of a new substance for treating bone disease, found that EFL1 inhibited the bone resorption by osteoclasts. It has been confirmed that the composition can be usefully used.
  • the skull capflavone derivatives inhibit bone uptake by osteoclasts, thereby confirming that the skull capflavone derivatives may be usefully used as pharmaceutical compositions for treating bone diseases.
  • dihydrocostus lactone inhibits bone resorption by osteoclasts, and it was confirmed that dihydrocostus lactone may be usefully used as a pharmaceutical composition for treating bone diseases.
  • the present invention was completed by confirming that synconin inhibits bone resorption by osteoclasts, and confirms that synconin may be usefully used as a pharmaceutical composition for treating bone diseases.
  • An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of bone diseases.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of bone diseases containing Eupovia factor L1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a dietary supplement for the improvement of bone diseases containing Euphorbia factor or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention also provides a pharmaceutical composition for the prevention or treatment of bone diseases containing a scalcap flavone derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a health functional food for the improvement of bone disease containing a skull cap flavone derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention also provides a pharmaceutical composition for the prevention or treatment of bone diseases containing dihydrocostus lactone or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a dietary supplement for the improvement of bone diseases containing dihydrocostus lactone or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of bone diseases, which contains as a active ingredient cinconin or a pharmaceutically acceptable salt thereof.
  • the present invention provides a dietary supplement for the improvement of bone diseases, which contains as a active ingredient Cynconin or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition for preventing or treating bone diseases containing Euphorbia Factor of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient inhibits important factors related to osteoclast differentiation, thereby differentiating osteoclasts. There is an effect of inhibiting bone absorption by inhibiting.
  • the pharmaceutical composition for the prevention or treatment of bone diseases containing the skull capflavone derivative or a pharmaceutically acceptable salt thereof of the present invention to inhibit bone absorption by inhibiting important factors associated with osteoclast differentiation It works.
  • the pharmaceutical composition for the prevention or treatment of bone diseases containing the dihydrocostus lactone of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient, by inhibiting important factors associated with osteoclast differentiation, differentiation of osteoclasts There is an effect of inhibiting bone absorption by inhibiting.
  • the pharmaceutical composition for the prevention or treatment of bone diseases including the present mykonin or a pharmaceutically acceptable salt thereof as an active ingredient inhibits the differentiation of osteoclasts by inhibiting important factors related to osteoclast differentiation
  • the compositions can be usefully used for preclinical and clinical studies, and can treat bone-related diseases caused by the imbalance of osteoclasts, and thus are widely used for the prevention or treatment of bone-related diseases. Can be utilized.
  • EFL1 is a diagram showing the concentration and time-dependent effects of the composition of the present invention EFL1 on the differentiation of RANKL treated osteoclast progenitor cells (BMMs) according to the concentration of EFL1:
  • FIG. 2 is a diagram showing the effect of EFL1, a composition of the present invention, on the expression of NFATc1 and target genes in RANKL treated osteoclast progenitor cells (BMMs):
  • FIG. 3 is a diagram showing the effect of EFL1, the composition of the present invention on NF- ⁇ B activity and c-FOS expression in RANKL treated osteoclast progenitor cells (BMMs):
  • Figure 4 is a diagram showing the effect of EFL1 composition of the present invention on the phosphorylation of MAPKs and AKT in RANKL-treated osteoclast progenitor cells (BMMs).
  • FIG. 5 is a diagram showing the effect of EFL1, the composition of the present invention on the activity of CREB-PGC-1 ⁇ in RANKL treated osteoclast progenitor cells (BMMs):
  • FIG. 6 is a diagram showing the effect of EFL1, a composition of the present invention, on the activity and production of free radicals of Nrf2 and its target genes in RANKL treated osteoclast progenitor cells (BMMs):
  • FIG. 7 is a diagram showing the effects of exposure to EFL1, the composition of the present invention, on osteoclast differentiation and expression of NFATc1 and PGC-1 ⁇ , and their target genes:
  • FIG. 8 is a diagram showing the effects of the exposure time point of EFL1, the composition of the present invention, on actining formation of RANKL-treated osteoclast progenitor cells (BMMs) and bone resorption on dentine discs:
  • FIG. 9 is a diagram showing the effect of EFL1, the composition of the present invention on the death of osteoclasts:
  • FIG. 10 is a diagram showing the inhibitory effect of the composition of the present invention EFL1 against bone damage caused by LPS-induced inflammation:
  • FIG. 11 is a diagram showing the concentration and time-dependent effect of SFII, the composition of the present invention on the differentiation of RANKL-treated osteoclast progenitor cells (BMMs):
  • FIG. 12 is a diagram showing the effect of SFII, the composition of the present invention on the expression of NFATc1 and target genes in RANKL treated osteoclast progenitor cells (BMMs):
  • FIG. 13 is a diagram showing the effect of SFII, the composition of the present invention on NF- ⁇ B activity and c-FOS expression in RANKL treated osteoclast progenitor cells (BMMs):
  • Figure 14 shows the effect of SFII, the composition of the present invention on phosphorylation of MAPKs and Src-AKT and FOXO1 in RANKL treated osteoclast progenitor cells (BMMs):
  • FIG. 15 is a diagram showing the effect of SFII, the composition of the present invention on the activity of CREB-PGC-1 ⁇ in RANKL treated osteoclast progenitor cells (BMMs):
  • FIG. 16 is a diagram showing the effect of SFII, a composition of the present invention, on the activity of Nrf2 and its target genes, caveolin-1 and production of free radicals in RANKL treated osteoclast progenitor cells (BMMs):
  • FIG. 17 is a diagram showing the effects of exposure time of SFII, a composition of the present invention, on osteoclast differentiation and expression of NFATc1 and its target genes:
  • FIG. 18 is a diagram showing the effects of SFII, the composition of the present invention, on the actining formation of RANKL-treated osteoclast progenitor cells (BMMs) and bone resorption on dentine discs:
  • FIG. 19 is a diagram showing the effect of SFII, a composition of the present invention, on bone damage caused by LPS-induced inflammation:
  • DHCL dihydrocostus lactone
  • FIG. 21 is a diagram showing the effect of dehydrocostus lactone (DHCL), a composition of the present invention, on the expression of NFATc1 and target genes in RANKL treated osteoclast progenitor cells (BMMs):
  • DHCL dehydrocostus lactone
  • FIG. 22 is a diagram showing the effect of dihydrocostus lactone (DHCL), a composition of the present invention, on NF- ⁇ B activity and c-FOS expression in RANKL treated osteoclast progenitor cells (BMMs):
  • DHCL dihydrocostus lactone
  • FIG. 23 is a diagram showing the effect of dihydrocostus lactone (DHCL), a composition of the present invention, on phosphorylation of MAPKs and Src-AKT and FOXO1 in RANKL treated osteoclast progenitor cells (BMMs):
  • DHCL dihydrocostus lactone
  • FIG. 24 is a diagram showing the effect of DHCL, a composition of the present invention, on the activity of Nrf2 and its target genes, caveolin-1 and production of free radicals in RANKL treated osteoclast progenitor cells (BMMs):
  • FIG. 25 is a diagram showing the effects of DHCL at the time of exposure of the composition of the present invention on differentiation of RANKL treated osteoclast progenitor cells (BMMs) and expression of NFATc1 and its target genes and osteoclast fusion related genes:
  • FIG. 26 is a diagram showing the effects of DHCL at the time of exposure of the composition of the present invention on actining formation of RANKL-treated osteoclast progenitor cells (BMMs) and bone resorption on dentine discs:
  • FIG. 27 is a diagram showing the inhibitory effect of DHCL, a composition of the present invention, on bone damage caused by LPS-induced inflammation:
  • CN cinconin
  • BMMs progenitor cells
  • CN cinconin
  • BMMs RANKL treated osteoclasts
  • FIG. 30 is a diagram showing the effect of cinconin (CN), a composition of the present invention, on NF- ⁇ B activity and c-FOS expression in progenitor cells (BMMs) of RANKL treated osteoclasts:
  • Figure 31 shows the effect of cinconin (CN), a composition of the present invention, on phosphorylation of MAPKs and Src-AKT and FOXO1 in RANKL treated osteoclasts (BMMs):
  • FIG. 32 is a diagram showing the effect of cinconin (CN), the composition of the present invention on the activity of CREB-PGC-1 ⁇ in RANKL treated osteoclasts (BMMs):
  • Figure 33 shows the effect of the exposure time of the composition of the present invention, Shinkonin (CN) on osteoclast differentiation and expression of NFATc1 and PGC-1 ⁇ and their target genes, and RANKL-treated osteoclast progenitor cells (BMMs)
  • Fig. 1 shows the effects of the composition of the present invention, cinconin (CN) on actining formation and bone resorption in dentine discs.
  • CN cinconin
  • Euphorbia Factor L1 which is an active ingredient of the present invention, is a diterpenoid obtained from seeds of Euphorbia lathyris linne (Successor / Platinum). Has:
  • the present invention provides a pharmaceutical composition for preventing or treating bone diseases containing the compound represented by Formula 1, or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the Euphorbia factor L1 of this invention is 0.1-20 micrometers concentration.
  • Euphorbia factor L1 composition having a concentration of less than 0.1 ⁇ M has a problem in that the treatment efficiency of bone disease is lowered, and if 20 ⁇ M or more, there is a problem that toxicity may occur in cells.
  • the concentration of the pharmaceutical composition containing Euphoria factor L1 as an active ingredient may be preferably 1 to 10 ⁇ M, more preferably 6 ⁇ M, but is not limited thereto.
  • composition is characterized by reducing the expression or activity of NFATc1, NF- ⁇ B, c-FOS and PGC-1 ⁇ , characterized by increasing the expression or activity of Nrf2, inhibits the production of free radicals in the cell It promotes the death of osteoclasts and is characterized by inhibiting bone damage caused by inflammation.
  • bone diseases to which the composition of the present invention can be applied include growth stage of development, fracture, osteoporosis due to excessive absorption of osteoclasts (osteoporosis), rheumatoid arthritis, periodontal disease (periodontal disease), Paget It is preferably at least one selected from the group consisting of Paget disease and metastatic bone cancers. It is not limited to this.
  • EFL1 Euphorbia factor L1
  • Chemical Formula 1 effectively inhibits osteoclast differentiation by RANKL
  • NFATc1 protein which is the most important transcription factor in osteoclast differentiation
  • target genes CK, MMP9 and TRAP genes
  • EFL1 inhibited the mRNA expression of PGC-1 ⁇ and its target genes, ND4, COX1, and COX3 (see FIG. 5), and mRNA expression of Nrf2 and NQO1 and Srx, which are representative target genes, increased together with EFL1. It confirmed that it became. At the same time, the expression of Nrf2 target proteins Srx, Prxl, PrxV, PrxVI, Trx1 and TrxR1 antioxidant enzymes, including Nrf2 and antioxidant enzymes, also increased. In addition, it was confirmed that the generation of free radicals is reduced by EFL1 (see Fig. 6).
  • EFL1 inhibited maintenance of osteoclasts differentiated, actin ring formation, and bone resorption in the dentine disc (see FIGS. 7 and 8), and promoted the death of osteoclasts (see FIG. 9). It was confirmed that the bone damage by inhibiting (see Fig. 10).
  • composition containing the euphoria factor L1 of the present invention as an active ingredient can be usefully used for the prevention or treatment of bone diseases.
  • the scullcapflavone derivative containing the present invention as an active ingredient is a flavonoid derived from Scutellaria baicalensis (golden), and has a structure represented by the following formula (2):
  • R 1 and R 2 are H or OCH 3 .
  • the present invention provides a pharmaceutical composition for preventing or treating bone diseases containing the compound represented by Formula 2, or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the skull cap flavone derivatives of the present invention is characterized in that the skull cap flavone I or skull cap flavone II, wherein the skull cap flavone derivatives R 1 and R 2 are each independently hydrogen (H) or methoxy (OCH 3 ) group.
  • R 1 and R 2 is hydrogen (H).
  • Scapcapflavone II, R 1 and R 2 in the formula (4) is a methoxy (OCH 3 ) group.
  • the skullcapflavone derivative of the present invention is preferably in a concentration of 0.1 to 10 ⁇ M.
  • Skullcapflavone derivative composition having a concentration of less than 0.1 ⁇ M has a problem that the treatment efficiency of bone disease is lowered, there is a problem that can cause toxicity to cells if more than 10 ⁇ M.
  • the concentration of the pharmaceutical composition containing the scullcapflavone derivative as an active ingredient may be preferably 1 to 5 ⁇ M, more preferably 2 ⁇ M, but is not limited thereto.
  • composition is characterized by reducing the expression or activity of NFATc1, c-FOS, MAPKS, Src, AKT, CREB, PGC-1 ⁇ and caveolin-1, and increases the expression or activity of Nrf2 and FOXO1 and catalase Characterized in that it inhibits the production of free radicals in the cell and inhibits bone damage caused by inflammation.
  • bone diseases to which the composition of the present invention can be applied include growth stage of development, fracture, osteoporosis due to excessive absorption of osteoclasts (osteoporosis), rheumatoid arthritis, periodontal disease (periodontal disease), Paget It is preferably one or more selected from the group consisting of paget disease and metastatic bone cancers, but not limited thereto.
  • the skull capflavone II (hereinafter SFII) of the present invention
  • the skull cap flavone derivative described in Formula 2 effectively inhibits the differentiation of osteoclast progenitor cells (BMMs) by RANKL (See FIG. 11).
  • mRNA treatment of CR and DC-STAMP genes was inhibited with SFII, along with NFATc1 protein, the most important transcription factor in osteoclast differentiation, and its target genes CK, MMP9, and TRAP (see FIG. 12). ).
  • SFII was found to significantly inhibit the mRNA expression of CREB and PGC-1 ⁇ and its target genes ND4, COX1, COX3 and Cyt b (see FIG. 15).
  • SFII and NQO1 and Srx are representative target genes by SFII. MRNA expression was increased together.
  • Nrf2 and Nrf2 target proteins including antioxidant enzymes, NQO1, Srx, PrxI, PrxII, PrxIII, PrxIV, PrxV, PrxVI, Trx1, Trx2, TrxR1 and TrxR2, were also found to increase expression.
  • the generation of reactive oxygen by SFII was reduced, and the expression of caveolin-1, which is known to inhibit Nrf2 activity and promote the differentiation of osteoclasts, was also inhibited by SFII (see FIG. 16).
  • SFII inhibits the formation of actin ring and bone resorption by osteoclasts in dentine disc (see FIGS. 17 and 18) and inhibits bone damage due to LPS inflammation (see FIG. 19).
  • composition containing the skull cap flavone derivative of the present invention as an active ingredient can be usefully used for the prevention or treatment of bone diseases.
  • DHCL dihydrocostus lactone
  • the present invention provides a pharmaceutical composition for preventing or treating bone diseases, comprising the compound represented by Formula 5, or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the dihydrocostus lactone of the present invention is characterized in that the concentration of 0.1 to 10 ⁇ M.
  • Dihydrocostus lactone composition having a concentration of less than 0.1 ⁇ M has a problem that the treatment efficiency of bone disease is lowered, there is a problem that can cause toxicity to cells if more than 10 ⁇ M.
  • the concentration of the pharmaceutical composition containing dihydrocostus lactone as an active ingredient may be preferably 1 to 5 ⁇ M, more preferably 1.5 ⁇ M.
  • composition is characterized by reducing the expression or activity of NFATc1, c-FOS, JNK, ERK, Src, AKT and caveolin-1, increases the phosphorylation of CREB and the expression of PGC-1 ⁇ , Nrf2 and FOXO1 And increasing expression or activity of catalase, and inhibiting the production of free radicals in cells and inhibiting bone damage caused by inflammation.
  • bone diseases to which the composition of the present invention can be applied include growth stage of development, fracture, osteoporosis due to excessive absorption of osteoclasts (osteoporosis), rheumatoid arthritis, periodontal disease (periodontal disease), Paget It is characterized in that any one or more selected from the group consisting of (Paget disease) and metastatic bone cancer (metastatic bone cancers).
  • DHCL dehydrocostus lactone
  • Nrf2 and NQO1 and Srx which are representative target genes, was increased by DHCL, and Nrf2 target proteins including Nrf2 and antioxidant enzymes were also increased.
  • DHCL DHCL
  • Nrf2 target proteins including Nrf2 and antioxidant enzymes were also increased.
  • the generation of free radicals is reduced by DHCL
  • the expression of caveolin-1 which inhibits the activity of Nrf2 and is known as a differentiation promoter of osteoclasts, is also inhibited by DHCL (see FIG. 24).
  • DHCL inhibits the formation of actin ring and bone uptake by osteoclasts in dentine disc (see FIGS. 25 and 26) and inhibits bone damage due to LPS inflammation (see FIG. 27).
  • composition containing the dihydrocostus lactone (DHCL) of the present invention as an active ingredient can be usefully used for the prevention or treatment of bone diseases.
  • cinchonine (CN) which is contained as an active ingredient of the present invention, is an alkaloid derived from Cinchona bark, and has a structure represented by Formula 6 below:
  • the present invention provides a pharmaceutical composition for preventing or treating bone diseases, which contains the compound represented by Chemical Formula 6, or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the cinconin of the present invention is characterized in that the concentration of 0.1 to 50 ⁇ M.
  • Synconin composition having a concentration of less than 0.1 ⁇ M has a problem in that the treatment efficiency of bone disease is lowered, there is a problem that can cause toxicity to cells if more than 50 ⁇ M.
  • the concentration of the pharmaceutical composition containing cinnaconin as an active ingredient is 1 to 30 ⁇ M, more preferably 20 ⁇ M.
  • composition is characterized by reducing the expression or activity of NFATc1, NF- ⁇ B, ERK, Src, AKT, FOXO1, c-FOS and PGC-1 ⁇ , it is characterized by inhibiting bone damage caused by inflammation.
  • bone diseases to which the composition of the present invention can be applied include growth stage of development, fracture, osteoporosis due to excessive absorption of osteoclasts (osteoporosis), rheumatoid arthritis, periodontal disease (periodontal disease), Paget It is preferably one or more selected from the group consisting of paget disease and metastatic bone cancers, but not limited thereto.
  • composition containing the present mykonin (CN) as an active ingredient can be usefully used for the prevention or treatment of bone diseases.
  • the present invention includes not only the compound represented by Chemical Formula 1 to Chemical Formula 6, but also a pharmaceutically acceptable salt thereof, and possible solvates, hydrates, racemates or stereoisomers which can be prepared therefrom.
  • the present invention can be used in the form of a compound represented by Formula 1 to Formula 6 or a pharmaceutically acceptable salt thereof, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful as the salt.
  • Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. Obtained from non-toxic organic acids such as doates, aromatic acids, aliphatic and aromatic sulfonic acids.
  • Such pharmaceutically toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide and iodide Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suverate , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, meth Oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfon
  • Acid addition salts according to the present invention are dissolved in conventional methods, for example, the compounds represented by the above formulas (1) to (6) in an excess of aqueous acid solution, and the salts are water-miscible organic solvents such as methanol, ethanol, acetone. Or by precipitation with acetonitrile.
  • the same amount of the compound represented by the formula (1) to formula (6), and the acid aqueous solution or alcohol may be heated, and then the mixture is evaporated to dryness or the precipitated salt may be produced by suction filtration.
  • Bases can also be used to make pharmaceutically acceptable metal salts.
  • Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt.
  • Corresponding silver salts are also obtained by reacting alkali or alkaline earth metal salts with a suitable negative salt (eg, silver nitrate).
  • composition When formulating the composition, it is prepared using commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants.
  • Solids for oral administration include tablets, pills, powders, granules, capsules, troches and the like, and such solid preparations include at least one excipient example in the compound represented by Formula 1 to Formula 6 of the present invention.
  • it is prepared by mixing starch, calcium carbonate, sucrose or lactose or gelatin.
  • lubricants such as magnesium styrate talc are also used.
  • Liquid preparations for oral administration include suspensions, solvents, emulsions, or syrups, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like.
  • non-aqueous solvent and the suspension solvent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.
  • injectable ester such as ethyl oleate, and the like
  • base of the suppository witepsol, macrogol, tween 81, cacao butter, taurine, glycerol, gelatin and the like can be used.
  • composition according to the invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level means the type, severity, and activity of the patient's disease. , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts.
  • the compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
  • 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, preferably 0.5 mg to 10 mg per 1 kg of body weight is administered daily or every other day. Alternatively, the administration may be divided into 1 to 3 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.
  • compositions of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.
  • the present invention provides a dietary supplement for the improvement of bone disease containing Euphorbia factor L1 represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a health functional food for the improvement of bone disease containing the skull cap flavone derivative represented by the formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the skull cap flavone derivative is characterized in that the skull cap flavone I or skull cap flavone II.
  • the present invention provides a dietary supplement for improving bone diseases containing dihydrocostus lactone represented by the formula (5) or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a health functional food for improving bone diseases containing the compound represented by the formula (6), or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the bone diseases include growth stage development, fracture, osteoporosis (osteoporosis) due to excessive bone resorption of osteoclasts (rheumatoid arthritis), periodontal disease (periodontal disease), Paget disease (metastatic) and metastatic bone cancer (metastatic) It is preferably at least one selected from the group consisting of bone cancers), but is not limited thereto.
  • the "health functional food” of the present specification is manufactured by using nutrients or ingredients (functional raw materials) having useful functions to the human body, which are easily deficient in a daily meal, and maintaining health through physiological functions or maintaining normal functions of the human body.
  • the food is to maintain and improve the food as defined by the Commissioner of Food and Drug Safety, but is not limited to this and is not used to exclude the health food in the normal sense.
  • the composition of the present invention may be added as it is to food or used with other food or food ingredients, and may be appropriately used according to conventional methods.
  • the mixing amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement).
  • the amount of the compound in the dietary supplement may be added at 0.01 to 90 parts by weight of the total food weight.
  • the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.
  • the health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the composition as an essential ingredient in the indicated ratios, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages.
  • natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • natural flavoring agents tautin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) ⁇ and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
  • the proportion of said natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 g of the composition of the present invention.
  • the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like.
  • the composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages.
  • additives can be used independently or in combination.
  • the proportion of such additives is not so 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.
  • the present invention also provides a method for preventing or treating bone diseases, comprising administering Euphorbia Factor L1 or a pharmaceutically acceptable salt thereof to a mammal, which is represented by Formula 1 below:
  • the present invention also provides the use of Euphorbia Factor L1 or a pharmaceutically acceptable salt thereof, as described in Formula 1, for use in the manufacture of a medicament for the prevention or treatment of bone diseases.
  • the present invention also provides a method for preventing or treating bone diseases, comprising administering to a mammal a skullcapflavone derivative represented by Formula 2 or a pharmaceutically acceptable salt thereof:
  • R 1 and R 2 are H or OCH 3 .
  • the present invention also provides the use of a scapcapflavone derivative according to Formula 2 or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for the prevention or treatment of bone diseases.
  • the present invention provides a method for preventing or treating bone diseases, comprising administering to a mammal a dihydrocostus lactone represented by the following formula (5) or a pharmaceutically acceptable salt thereof:
  • the present invention also provides the use of a dihydrocostus lactone or a pharmaceutically acceptable salt thereof according to Formula 5, for use in the manufacture of a medicament for the prevention or treatment of a bone disease:
  • the present invention provides a method for preventing or treating bone diseases, comprising administering to a mammal, a neoconsin or a pharmaceutically acceptable salt thereof represented by the following formula:
  • the present invention also provides the use of cinconin or a pharmaceutically acceptable salt thereof as described in Formula 6 for use in the manufacture of a medicament for the prevention or treatment of bone diseases.
  • Euphorbia factor L1 contained in the present invention as an active ingredient was used in an experiment using CFN92883 / 76376-43-7 manufactured by ChemFaces.
  • BMM Bone Marrow-Derived Macrophages
  • BMM (Bone Marrow-Derived Macrophages) cells were treated with 50 ng / ml RANKL for 3 days in the presence of various concentrations (0, 4, 6, 8 ⁇ M) of EFL1 to differentiate into osteoclasts and 4% paraformaldehyde after PBS washing. After fixation with TRAP staining using leukocyte acid phosphatase cytochemistry kit (Sigma Aldrich), the number of osteoclasts with three or more multinucleated stained TRAP was analyzed under a microscope.
  • cytotoxicity was analyzed by EASY Cytox (WST-1) assay kit in order to exclude that the osteoclast differentiation inhibitory effect of EFL1 is due to cytotoxicity.
  • BMM cells were treated with 50 ng / ml of RANKL for 2 days in the presence of various concentrations (0, 4, 6, 8 ⁇ M) of EFL1 to differentiate into osteoclasts, and osteoclasts.
  • various concentrations (0, 4, 6, 8 ⁇ M) of EFL1 to differentiate into osteoclasts, and osteoclasts.
  • immunoblot using an antibody against NFATc1 was performed.
  • Proteins were separated using 10% polyacrylamide gel, and the separated proteins were transferred to nitrocellulose membranes. All nonspecific 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 secondary antibodies were treated at room temperature for 1 hour and analyzed by West save Up (Ab Frontier).
  • NFATc1 protein was significantly inhibited from 4 ⁇ M EFL1 concentration (FIG. 2A), and it was confirmed that the inhibitory effect was continued up to 5 days (FIG. 2B).
  • CK, MMP9 and TRAP genes which are target genes of NFATc1 were confirmed using real time-PCR.
  • Trizol reagent Invitrogen
  • ABI 7300 real at a final 20 ⁇ l volume containing 10 ⁇ l of 2 ⁇ SYBR Green PCR Master Mix (M Biotech) and 1 ⁇ l of 10 ⁇ M of each gene-specific primer (Genotech). Amplification was carried out using a time PCR system (Applied Biosystems), and the amplification was performed by performing 40 cycles for 2 minutes at 50 ° C, 2 minutes at 95 ° C, then 15 seconds at 95 ° C, and 1 minute at 60 ° C. The sequence of Primer used in the experiment is shown in Table 1 below.
  • EFL1 modulates NF- ⁇ B activity and c-Fos expression, known as upstream regulators of NFATc1
  • immunoblot using I ⁇ B ⁇ and p-I ⁇ B ⁇ (rabbit polyclonal antibodies, Cell Signaling Technology) was used to determine the degree of phosphorylation of I ⁇ B ⁇ .
  • SOD2 and c-FOS protein expression was also confirmed by immunoblot using antibodies (SOD2 / rabbit polyclonal antibody, Upstate; c-FOS / rabbit polyclonal antibody, Santa Cruz Biotechnology, Inc), and SOD2 and c-Fos mRNA expression was confirmed. It was analyzed by real-time PCR by the method described in Experimental Example 2 (see Table 1 primer sequence).
  • EFL1 (6 ⁇ M) significantly inhibited the phosphorylation of I ⁇ B ⁇ in the presence of RANKL (50 ng / ml) (FIG. 3A) and significantly inhibited protein and mRNA expression of SOD2, an NF- ⁇ B target gene. (B and D of FIG. 3). In addition, EFL1 significantly inhibited c-Fos protein and mRNA expression (Fig. 3 C and D).
  • PGC-1 ⁇ is a transcription factor well known to be activated by RANKL and involved in osteoclast differentiation.
  • RANKL a transcription factor well known to be activated by RANKL and involved in osteoclast differentiation.
  • mRNA expression of PGC-1 ⁇ and its target genes ND4, COX1 and COX3 was confirmed by real-time PCR (see Table 1 primer sequence).
  • EFL1 (6 ⁇ M) significantly inhibited PGC-1 ⁇ , ND4, COX1 and COX3 mRNA expression by RANKL (50 ng / ml) (FIG. 5A).
  • EFL1 also inhibited the expression of PGC-1 ⁇ (rabbit polyclonal antibody, Abcam) protein, but CREB (phospho-CREB rabbit monoclonal antibody, Cell Signaling Technology; CREB mouse monoclonal antibody, Santa Cruz Biotechnology) , Inc) did not affect the phosphorylation (B and C of Figure 5).
  • Nrf2 Redox-sensitive transcription factor Nrf2 is known to be involved in osteoclast differentiation by regulating the expression of GSH synthase and antioxidant enzymes in vivo, and to investigate the inhibitory effect of osteoclast differentiation by regulating Nrf2 activity of EFL1.
  • Nrf2 NQO1 and Srx gene expression was confirmed by real-time PCR (see Table 1 primer sequence), Nrf2 target proteins NQO1, Srx, Prxl, PrxV, PrxVI, Trx1 and TrxR1 (rabbit polyclonal antibodies, Young In Frontier) protein expression was confirmed by immunoblot.
  • Nrf2 and its target genes NQO1 and Srx were increased by EFL1 (6 ⁇ M) treatment in the presence of RANKL (50 ng / ml) (FIG. 6A).
  • Nrf2 and its target proteins NQO1, Srx, Prxl, PrxV, PrxVI, Trx1 and TrxR1 antioxidant enzymes were also confirmed to increase the expression (Fig. 6B).
  • ROS was measured by oxidant sensitive fluorescence detection using CM-H2DCFDA. Since CM-H2DCFDA reacts with ROS and fluoresces, cells are treated with RANKL, washed with HBSS, and then treated with 5 ⁇ M of CM-H2DCFDA for 10 minutes in the dark, using a FACS Calibur flow cytometer (BD Biosciences). Analyzed.
  • EFL1 (6 ⁇ M) was pretreated for 30 minutes and RANKL (A in FIG. 6, bottom) or H 2 O 2 (FIG. A, lower right) was treated for 15 minutes, and then active oxygen was measured using CM-H2DCFDA as described above.
  • E0, RANKL treatment; E1, RANKL 1 day treatment; E2, RANKL 2 days) during RANKL treatment to differentiate BMM cells into osteoclasts Post-treatment was exposed to EFL1 and analyzed for the number of TRAP stained multinucleated osteoclasts.
  • BMM cells were treated with RANKL for 3 days to differentiate into osteoclasts, and then treated with EFL1, and EFL1
  • the cells treated with RANKL were fixed as follows and stained with actining and nuclei or analyzed for bone resorption on dentine discs.
  • the cells were fixed in PBS with 3.7% formaldehyde and treated with 0.1% Triton X-100, followed by Alexa Fluor 488-phalloidin (Invitrogen) for 20 minutes, followed by DAPI (4 ', 6-diamidino-2-phenylindole). , Roche) and observed through a fluorescence microscope.
  • the degree of bone resorption was treated with BMM cells cultured in dentine disc (Immunodiagnostic Systems Ltd) under the same conditions as above, followed by removal of the cells using a cotton tip, staining the resorption pit with hematoxylin, and then using a microscope 100x magnification. The photographs taken were analyzed with Image-Pro Plus 4.5 software (Media Cybernetics).
  • the osteoclasts were treated with EFL1 (6 ⁇ M) and the shape change of cells with time (0, 6, 12, 18h) was observed through a microscope.
  • the osteoclasts were fixed as follows, followed by DAPI staining and TUNEL assay: The osteoclasts were fixed with 4% formaldehyde for 60 minutes at room temperature and washed with PBS. After staining the cells in which apoptosis occurred using In Situ Cell Death Detection Kit TMR red (Roche), the cells were stained with DAPI for 3 minutes at room temperature, and observed with a fluorescence microscope. As a result, it was confirmed that EFL1 induces the death of differentiated osteoclasts (C and D of FIG. 9).
  • LPS Lipopolysaccharide, 12.5 mg / kg body weight
  • an inflammatory inducer was injected into the mouse skull twice daily at intervals.
  • vehicle 50% DMAC + 10% Tween 80 + 50% distilled water
  • EFL1 10 mg / kg
  • decalcification was performed with 0.5 M ethylenediaminetetraacetic acid for 7 days, paraffin block was made and then cut and stained with TRAP and Hematoxylin.
  • Skullcapflavone II (hereinafter referred to as SFII) containing the present invention as an active ingredient was used in an experiment using CFN92216 / 55084-08-7 manufactured by ChemFaces.
  • BMM Bone Marrow-Derived Macrophages
  • Bone Marrow-Derived Macrophages (BMM) cells were treated with 25 ng / ml M-CSF (macrophage colony stimulating factor) and 50 ng / ml RANKL in the presence of SFII at various concentrations (0, 1, 2, 3 ⁇ M). After treatment for 1 day, differentiated into osteoclasts, fixed with 4% paraformaldehyde after PBS wash, followed by TRAP staining using leukocyte acid phosphatase cytochemistry kit (SigmaAldrich), followed by TRAP staining. Analyzed.
  • cytotoxicity was analyzed by EASY Cytox (WST-1) assay kit in order to exclude that the osteoclast differentiation inhibitory effect of SFII is due to cytotoxicity.
  • BMM cells were prepared using 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium mono-sodium salt (WST-1) reagent (Roche Applied Science), respectively. ⁇ l was added and incubated at 37 ° C. for 2-4 hours, and the absorbance at 450 nm was measured.
  • BMM cells were treated with 50 ng / ml of RANKL for 2 days in the presence of SFII at various concentrations (0, 1, 2, 3 ⁇ M), and differentiated into osteoclasts.
  • SFII serum-derived factor
  • Proteins were separated using 10% polyacrylamide gel, and the separated proteins were transferred to nitrocellulose membranes. All nonspecific 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 secondary antibodies were treated at room temperature for 1 hour and analyzed by West save Up (Ab Frontier).
  • DC-STAMP genes associated with CK, MMP9 and TRAP genes which are target genes of NFATc1, and CR and osteoclasts, which are calcitonin receptor genes, were confirmed by real time-PCR.
  • Trizol reagent Invitrogen
  • ABI 7300 real at the final 20 ⁇ l volume containing 0.8 ⁇ l of the diluted cDNA template (1: 2.5) containing 10 ⁇ l of 2X SYBR Green PCR Master Mix (M Biotech) and 1 ⁇ l of 10 ⁇ M of each gene-specific primer (Genotech).
  • Amplification was carried out using a time PCR system (Applied Biosystems), and the amplification was performed by performing 40 cycles for 2 minutes at 50 ° C, 2 minutes at 95 ° C, then 15 seconds at 95 ° C, and 1 minute at 60 ° C.
  • the sequence of Primer used in the experiment is shown in Table 2 below.
  • PGC-1 ⁇ is a transcription factor well known to be activated by RANKL and involved in osteoclast differentiation.
  • RANKL a transcription factor well known to be activated by RANKL and involved in osteoclast differentiation.
  • mRNA expression of PGC-1 ⁇ and its target genes ND4, COX1, COX3 and Cyt b was confirmed by real-time PCR (see Table 2 primer sequence).
  • Nrf2 The redox-sensitive transcription factor Nrf2 is known to be involved in the differentiation of osteoclasts by regulating the expression of GSH synthase and antioxidant enzymes in vivo, and to investigate the inhibitory effect of osteoclast differentiation by regulating Nrf2 activity of SFII.
  • Nrf2, NQO1 and Srx gene expression was confirmed by real-time PCR (see Table 2 primer sequence), Nrf2 and other target proteins of Nrf2 including Nrf2 and antioxidant enzymes NQO1, Srx, PrxI, PrxII, PrxIII, PrxIV, PrxV, Expression of PrxVI, Trx1, Trx2, TrxR1 and TrxR2 (rabbit polyclonal antibodies, Young In Frontier) proteins was confirmed by immunoblot and real-time PCR.
  • Nrf2 and its target genes NQO1 and Srx were increased by SFII (2 ⁇ M) treatment in the presence of RANKL (50 ng / ml) (FIG. 16A).
  • Nrf2 and antioxidant enzymes including NQO1, Srx, PrxI, PrxII, PrxIII, PrxIV, PrxV, PrxVI, Trx1, Trx2, TrxR1 and TrxR2 were also found to increase the expression (Fig. 16B).
  • ROS was measured by oxidant sensitive fluorescence detection using CM-H2DCFDA. Since CM-H2DCFDA reacts with ROS and fluoresces, cells are treated with RANKL, washed with HBSS, and then treated with 5 ⁇ M of CM-H2DCFDA for 10 minutes in the dark, using a FACS Calibur flow cytometer (BD Biosciences). Analyzed.
  • SFII (2 ⁇ M) was pretreated for 30 minutes and RANKL (C, M in Figure 16) or H 2 O 2 (C in Figure 16) to determine the possibility of SFII to remove the active oxygen directly as an antioxidant. , Right) was treated for 15 minutes and the active oxygen was measured using CM-H2DCFDA as described above.
  • osteoclast formation was significantly inhibited when SFII (2 ⁇ M) was treated with RANKL (50 ng / ml) (E0), and osteoclasts when exposed to SFII after RANKL 1 day treatment (E1). Formation was significantly inhibited.
  • E2 when exposed after 2 days (E2), there was a significant difference in the number of osteoclasts of 10 or more nuclei but little effect on the number of osteoclasts of 3 or more nuclei (FIG. 17A). ).
  • BMM cells were treated with RANKL for 3 days to differentiate into osteoclasts, and then treated with SFII and SFII.
  • the cells treated with RANKL were fixed as follows and stained with actining and nuclei or analyzed for bone resorption on dentine discs.
  • the cells were fixed in PBS added with 3.7% formaldehyde and treated with 0.1% Triton X-100, followed by 20 minutes treatment with Alexa Fluor 488-phalloidin (Invitrogen), followed by DAPI (4 ', 6-diamidino-2-phenylindole). , Roche) and observed through a fluorescence microscope.
  • the degree of bone resorption was obtained by removing the cells cultured on dentine disc (Immunodiagnostic Systems Ltd) using cotton tip, and staining the resorption pit with hematoxylin. (Media Cybernetics).
  • the mouse skull was injected twice a day with an inflammatory inducer, LPS (Lipopolysaccharide, 12.5 mg / kg body weight).
  • LPS Lipopolysaccharide, 12.5 mg / kg body weight
  • vehicle 10% DMAC + 10% Tween 80 + 80% distilled water
  • SFII 2 mg / kg
  • decalcification was performed with 0.5 M ethylenediaminetetraacetic acid for 7 days, paraffin blocks were made and then cut and stained with TRAP and Hematoxilin.
  • DHCL Dehydrocostus lactone
  • BMM Breast-Derived Macrophages
  • BMM cells are precursor cells capable of differentiating into osteoclasts.
  • cells extracted from femur and tibia bone marrow of 8-week-old C57BL / 6 male mice were used.
  • Bone Marrow-Derived Macrophages (BMM) cells were treated with 25 ng / ml M-CSF (macrophage colony stimulatory factor) and 50 ng / ml RANKL in the presence of various concentrations (0, 0.5, 1, 1.5 ⁇ M) of DHCL. After treatment for 1, 5 days to differentiate into osteoclasts, fixed with 4% paraformaldehyde after PBS washing, and then TRAP stained using leukocyte acid phosphatase cytochemistry kit (SigmaAldrich), followed by TRAP staining of osteoclasts with three or more multinucleated cells. The numbers were analyzed under a microscope.
  • M-CSF macrophage colony stimulatory factor
  • osteoclast differentiation was significantly inhibited from 0.5 ⁇ M of DHCL (A and B in FIG. 20), and 0.5 ⁇ M of DHCL was also treated when treated for 5 days. Osteoclast differentiation was significantly inhibited (D and E in FIG. 20).
  • cytotoxicity was analyzed by EASY Cytox (WST-1) assay kit to exclude that the osteoclast differentiation inhibitory effect of DHCL is due to cytotoxicity.
  • BMM cells were treated with 50 ng / ml RANKL for 2 days in the presence of various concentrations (0, 0.5, 1, 1.5 ⁇ M) of DHCL to differentiate into osteoclasts, and osteoclasts.
  • various concentrations (0, 0.5, 1, 1.5 ⁇ M) of DHCL to differentiate into osteoclasts, and osteoclasts.
  • immunoblot using an antibody against NFATc1 was performed.
  • Proteins were separated using 10% polyacrylamide gel, and the separated proteins were transferred to nitrocellulose membranes. All nonspecific 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 secondary antibodies were treated at room temperature for 1 hour and analyzed by West save Up (Ab Frontier).
  • DC-STAMP genes associated with CK, MMP9 and TRAP genes which are target genes of NFATc1, and CR and osteoclasts, which are calcitonin receptor genes, were confirmed by real time-PCR.
  • Trizol reagent Invitrogen
  • ABI 7300 real at the final 20 ⁇ l volume containing 0.8 ⁇ l of the diluted cDNA template (1: 2.5) containing 10 ⁇ l of 2X SYBR Green PCR Master Mix (M Biotech) and 1 ⁇ l of 10 ⁇ M of each gene-specific primer (Genotech).
  • Amplification was carried out using a time PCR system (Applied Biosystems), and the amplification was performed by performing 40 cycles for 2 minutes at 50 ° C, 2 minutes at 95 ° C, then 15 seconds at 95 ° C, and 1 minute at 60 ° C.
  • the sequence of Primer used in the experiment is shown in Table 3 below.
  • DHCL regulates NF- ⁇ B activity and c-Fos expression known as upstream regulators of NFATc1
  • immunoblot using I ⁇ B ⁇ and p-I ⁇ B ⁇ (rabbit polyclonal antibodies, Cell Signaling Technology) was used to determine the degree of phosphorylation of I ⁇ B ⁇ .
  • SOD2 and c-FOS proteins was also confirmed by immunoblot using these antibodies (SOD2 / rabbit polyclonal antibody, Upstate; c-FOS / rabbit polyclonal antibody, Santa Cruz Biotechnology, Inc), and SOD2 and c-Fos.
  • Expression of mRNA was analyzed by real-time PCR by the method described in Experimental Example 19 (see Table 3 primer sequence).
  • DHCL significantly inhibited the phosphorylation of I ⁇ B ⁇ in the presence of RANKL (50 ng / ml) (FIG. 22A) and significantly inhibited protein and mRNA expression of SOD2, an NF- ⁇ B target gene. (FIGS. 22B and D). In addition, it was confirmed that DHCL also significantly inhibits the expression of c-Fos protein and mRNA (C and D of Figure 22).
  • Nrf2 Redox-sensitive transcription factor Nrf2 is known to be involved in osteoclast differentiation by regulating the expression of GSH synthase and antioxidant enzymes in vivo, and to investigate the inhibitory effect of osteoclast differentiation by regulating Nrf2 activity of DHCL.
  • Nrf2 NQO1 and Srx gene expression was confirmed by real-time PCR (see Table 3 primer sequence), and target proteins of Nrf2 including Nrf2 and antioxidant enzymes NQO1, Srx, Prx, PrxV, PrxVI and Trx1 (rabbit polyclonal antibodies, Young In Frontier) protein expression was confirmed by immunoblot and real-time PCR.
  • Nrf2 and its target genes were increased by DHCL (1.5 ⁇ M) treatment in the presence of RANKL (50 ng / ml) (FIG. 24A).
  • Nrf2 and antioxidant enzymes including its target proteins NQO1, Srx, Prx I, PrxV, PrxVI and Trx1 was also confirmed that the expression is increased (Fig. 24B).
  • ROS was measured by oxidant sensitive fluorescence detection using CM-H2DCFDA. Since CM-H2DCFDA reacts with ROS and fluoresces, the cells are treated with RANKL, washed with HBSS, and then treated with 5 ⁇ M of CM-H2DCFDA for 20 minutes in the dark, using a FACS Calibur flow cytometer (BD Biosciences). Analyzed.
  • DHCL 1.5 ⁇ M was pretreated for 30 minutes and RANKL (C in FIG. 24) or H 2 O 2 (C in FIG. 24) to determine the possibility of DHCL being able to directly remove active oxygen as an antioxidant. After treating for 15 minutes, the right oxygen was measured using CM-H2DCFDA as described above.
  • DHCL is caveolin-1 (rabbit monoclonal) induced by RANKL. It was confirmed that the expression of antibody, Trnasduction Laboratories) is suppressed (FIG. 24D). This suggests that DHCL inhibits the expression of caveolin-1, thereby reducing the binding of Nrf2 to promote Nrf2 activity, resulting in lowering free radicals in cells.
  • DHCL osteoclasts In order to investigate the differentiation inhibitory effect of DHCL osteoclasts, different time points (E0, RANKL treatment; E1, RANKL 1 day treatment; E2, RANKL 2 days) during RANKL treatment to differentiate BMM cells into osteoclasts After treatment; after 3 days of treatment with E3, RANKL), DHCL was exposed and the number of TRAP stained multinucleated osteoclasts was analyzed.
  • NFATc1 its target genes MMP9, TRAP and DC-STAMP genes, CK and CR was also measured by real-time PCR (see Table 3 primer sequence), indicating that NFATc1 and its target genes Expression was also inhibited at the beginning of the differentiation of osteoclasts but hardly inhibited in mature osteoclasts (FIG. 25B).
  • BMM cells were treated with RANKL for 3 days to differentiate into osteoclasts, and then treated with DHCL and DHCL.
  • the cells treated with RANKL were fixed as follows and stained with actining and nuclei or analyzed for bone resorption on dentine discs.
  • the cells were fixed in PBS with 3.7% formaldehyde and treated with 0.1% Triton X-100, followed by treatment with Alexa Fluor 488-phalloidin (Invitrogen) for 30 minutes, followed by DAPI (4 ', 6-diamidino-2-phenylindole). , Roche) and observed through a fluorescence microscope.
  • the degree of bone resorption was obtained by removing the cells cultured on dentine disc (Immunodiagnostic Systems Ltd) using cotton tip, and staining the resorption pit with hematoxylin. (Media Cybernetics).
  • DHCL may inhibit osteoclast fusion and bone resorption.
  • LPS Lipopolysaccharide, 12.5 mg / kg body weight
  • DHCL DHCL (2 mg / kg)
  • vehicle 10% DMAC + 10% Tween 80 + 80% distilled water
  • DHCL 2 mg / kg
  • decalcification was performed with 0.5 M ethylenediaminetetraacetic acid for 7 days, paraffin blocks were made and then cut and stained with TRAP and Hematoxylin.
  • Synconin (hereinafter referred to as CN) contained in the present invention as an active ingredient was used in an experiment using 27370 / 118-10-5 product of Sigma-Aldrich.
  • BMM Bone Marrow-Derived Macrophages
  • cytotoxicity was analyzed by EASY Cytox (WST-1) assay kit to exclude the fact that the inhibition of osteoclast differentiation of the cytokine is caused by cytotoxicity.
  • BMM cells were prepared using 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium mono-sodium salt (WST-1) reagent (Roche Applied Science), respectively. ⁇ l was added and incubated at 37 ° C. for 2-4 hours, and the absorbance at 450 nm was measured.
  • BMM cells were treated with 50 ng / ml of RANKL for 2 days in the presence of various concentrations (0, 10, 20, 30 ⁇ M) to differentiate into osteoclasts.
  • various concentrations (0, 10, 20, 30 ⁇ M) to differentiate into osteoclasts.
  • immunoblot using an antibody against NFATc1 was performed.
  • Proteins were separated using 10% polyacrylamide gel, and the separated proteins were transferred to nitrocellulose membranes. All nonspecific 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 secondary antibodies were treated at room temperature for 1 hour and analyzed by West save Up (Ab Frontier).
  • CK, MMP9 and TRAP genes which are target genes of NFATc1 were confirmed using real time-PCR.
  • Trizol reagent Invitrogen
  • ABI 7300 real at the final 20 ⁇ l volume containing 0.8 ⁇ l of the diluted cDNA template (1: 2.5) containing 10 ⁇ l of 2X SYBR Green PCR Master Mix (M Biotech) and 1 ⁇ l of 10 ⁇ M of each gene-specific primer (Genotech).
  • Amplification was carried out using a time PCR system (Applied Biosystems), and the amplification was performed by performing 40 cycles for 2 minutes at 50 ° C, 2 minutes at 95 ° C, then 15 seconds at 95 ° C, and 1 minute at 60 ° C.
  • the sequence of Primer used in the experiment is shown in Table 4.
  • PGC-1 ⁇ is a transcription factor well known to be activated by RANKL and involved in osteoclast differentiation.
  • RANKL a transcription factor well known to be activated by RANKL and involved in osteoclast differentiation.
  • mRNA expression of PGC-1 ⁇ and its target genes ND4, Cyt b, COX1 and COX3 was confirmed by real-time PCR (see Table 4 primer sequence).
  • cinconin (20 ⁇ M) significantly inhibited PGC-1 ⁇ , ND4, Cyt b, COX1 and COX3 mRNA expression by RANKL (50 ng / ml) (FIG. 32A).
  • Cinconin inhibited the expression of PGC-1 ⁇ (rabbit polyclonal antibody, Abcam) protein, but CREB (phospho-CREB rabbit monoclonal antibody, Cell Signaling Technology; CREB mouse monoclonal antibody, Santa Cruz, a higher transcription factor of PGC-1 ⁇ ) Biotechnology, Inc) did not affect the phosphorylation (B and C of Figure 32).
  • NFATc1 and its target genes CK, MMP9, TRAP and PGC-1 ⁇ and its target genes ND4, COX1, COX3, and Cyt b genes were also measured by real-time PCR (Table 4). The expression was significantly inhibited when Cynconin was treated with RANKL (E0), but was hardly inhibited by Cynonin exposed after RANKL 2 days treatment (E2) (Fig. 33). B and c).
  • BMM cells were treated with RANKL for 3 days to differentiate into osteoclasts
  • Cells treated with Cinconin with RANKL were fixed as follows and stained for actining and nuclei or analyzed for bone resorption on dentine discs.
  • the cells were fixed in PBS with 3.7% formaldehyde and treated with 0.1% Triton X-100, followed by Alexa Fluor 488-phalloidin (Invitrogen) for 20 minutes, followed by DAPI (4 ', 6-diamidino-2-phenylindole). , Roche) and observed through a fluorescence microscope.
  • the degree of bone resorption was obtained by removing the cells cultured on dentine disc (Immunodiagnostic Systems Ltd) using cotton tip, and staining the resorption pit with hematoxylin. (Media Cybernetics).
  • the mouse skull was infused with LPS (Lipopolysaccharide, 12.5 mg / kg body weight), which is an inflammation-inducing substance, twice daily at intervals.
  • LPS Lipopolysaccharide, 12.5 mg / kg body weight
  • vehicle 20% polyethylene glycol + 20% ethanol + 60% distilled water
  • cinconin 10 mg / kg
  • Decalcification was performed with 0.5 M ethylenediaminetetraacetic acid for 7 days, paraffin blocks were made, cut and stained with TRAP and hematoxilin for observation.

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Abstract

La présente invention concerne une composition pharmaceutique pour prévenir ou à traiter des maladies osseuses et, plus précisément, la composition pharmaceutique comprenant le facteur d'Euphorbia ou un sel de qualité pharmaceutique de ce dernier en tant que principe actif pour prévenir ou à traiter des maladies osseuses de la présente invention inhibe des facteurs importants impliqués dans la différenciation des ostéoclastes, ce qui permet d'inhiber la différenciation des ostéoclastes en vue de supprimer la résorption osseuse. En outre, la composition pharmaceutique de la présente invention comprenant un dérivé de skullcapflavone ou un sel de qualité pharmaceutique de ce dernier en tant que principe actif pour prévenir ou à traiter des maladies osseuses inhibe des facteurs importants impliqués dans la différenciation des ostéoclastes, ce qui permet de supprimer la résorption osseuse. En outre, une composition pharmaceutique de la présente invention comprenant de la déhydrocostus lactone ou un sel de qualité pharmaceutique de cette dernière en tant que principe actif pour prévenir ou à traiter des maladies osseuses inhibe des facteurs importants impliqués dans la différenciation des ostéoclastes, ce qui permet d'inhiber la différenciation des ostéoclastes en vue de supprimer la résorption osseuse. En outre, une composition pharmaceutique de la présente invention comprenant de la cinchonine ou un sel de qualité pharmaceutique de cette dernière en tant que principe actif pour prévenir ou à traiter des maladies osseuses inhibe des facteurs importants impliqués dans la différenciation des ostéoclastes, ce qui permet d'inhiber la différenciation des ostéoclastes en vue de supprimer la résorption osseuse. Par conséquent, les compositions peuvent être utilisées avantageusement dans des études précliniques et cliniques et peuvent traiter des maladies liées aux os provoquées par un déséquilibre des ostéoclastes, et peuvent donc être largement utilisées pour le développement de médicaments pour la prévention ou au traitement de maladies liées aux os.
PCT/KR2017/007852 2016-07-20 2017-07-20 Composition pharmaceutique destinée à la prévention ou au traitement de maladies osseuses WO2018016901A1 (fr)

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KR1020160091899A KR101832351B1 (ko) 2016-07-20 2016-07-20 신코닌 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 골질환의 예방 또는 치료용 약학적 조성물
KR20160091898 2016-07-20
KR10-2016-0091899 2016-07-20
KR10-2016-0091898 2016-07-20
KR1020160091900A KR101765141B1 (ko) 2016-07-20 2016-07-20 스컬캅플라본 유도체 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 골질환의 예방 또는 치료용 약학적 조성물
KR10-2016-0091900 2016-07-20
KR1020160091897A KR101747775B1 (ko) 2016-07-20 2016-07-20 유포비아 인자 l1 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 골질환의 예방 또는 치료용 약학적 조성물
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