CN110960524A - Application of pseudo guaianolide sesquiterpene compound in preparation of osteoclast differentiation inhibitor - Google Patents
Application of pseudo guaianolide sesquiterpene compound in preparation of osteoclast differentiation inhibitor Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
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Abstract
The invention discloses an application of a pseudo guaianolide sesquiterpene compound separated from Euphorbia lathyris (Euphorbia thymifolia L.) in preparing an osteoclast differentiation inhibitor. The applicant proves through experiments that the compound shown in the formula (I) has the characteristics of direct and remarkable inhibitory action on osteoclastogenesis and osteoclastic activity, so that the compound can be used as an osteoclast differentiation inhibitor drug and is used for treating diseases treated by the osteoclast differentiation inhibitor.
Description
Technical Field
The invention relates to the field of medicines, in particular to application of a pseudo guaianolide sesquiterpene compound separated from Chimonanthus praecox in preparing an osteoclast differentiation inhibitor.
Background
Osteoclasts are the only cells with bone resorption function in the body, and excessive activation of osteoclasts can lead to the development of various osteolytic diseases, including osteoporosis, tumor bone metastasis, and inflammatory diseases (such as sepsis, rheumatoid arthritis RA, and psoriatic arthritis), among others. Among these diseases, osteoporosis and rheumatoid arthritis alone occur in hundreds of millions of patients all over the world, and lack of estrogen leads to increase of ligands (RANKL) activating nuclear factor NF- κ B receptors, overactivation of osteoclasts, and increase of bone resorption, which leads to the occurrence of osteoporosis; osteoclast positively stained by TRAP exists in joint synovium of rheumatoid arthritis patients, inflammatory factors TNF-a, IL-1 and the like in the joint synovium can promote the generation of osteoclast and accelerate the generation of bone destruction; in addition, bone metastasis is very common in advanced tumors, and patients with advanced breast cancer, prostate cancer, lung cancer and the like of about 3/4 suffer from tumor bone metastasis, activate osteoclasts to generate bone destruction, and cause severe pain to the patients. These diseases seriously affect the quality of life of patients, and in the last 20 years, the treatment of bone destruction related diseases has been much advanced, but the treatment regimen is still far from ideal.
In recent years, drugs targeting osteoclasts, inhibiting osteoclastogenesis or function, have become important targets for preventing and treating bone destruction, fracture and pain in related osteolytic diseases, and are hot spots of current research. Menopausal osteoporosis is a disease which is firstly approved and applied by targeting osteoclast drugs, inhibits osteoclast from increasing bone density of patients and reduces fracture risk. In 2012, denoximab, a RANKL antibody directed to osteoclast differentiation, has become one of the 50 top-off-the-shelf drugs in the united states. Although the clinical application range of the osteoclast differentiation inhibitor is gradually expanded in recent years, currently, related marketed drugs are not many, and representative drugs for clinically targeting osteoclasts are mainly RANKL antibody denoximab (denosumab), bisphosphonate, zoledronic acid and the like. Although the medicines can inhibit the differentiation or the function of osteoclast, the medicines have serious complications and intolerable side effects, and the bisphosphonate can inhibit the natural renewal of bone tissues to cause fracture after being taken for a long time; denosumab not only requires subcutaneous injection and is expensive, but also has serious complications such as mandibular necrosis. Therefore, finding a safe, effective, convenient to take, and economical drug targeting osteoclasts is not only relevant to the development of bone destructive diseases related to osteoclasts, but also has a great practical need.
Osteoclasts originate from cells of the myeloid monocyte-macrophage lineage and must be induced in vivo by some trigger to differentiate into multinucleated mature osteoclasts and eventually become activated. The endogenous molecule currently believed to most directly activate osteoclasts is the ligand (RANKL) secreted by osteoblasts or bone marrow stromal cells that activates the nuclear factor NF- κ B receptor. RANKL binds to an activation nuclear factor NF-kB Receptor (RANK) on an osteoclast membrane, recruits a tumor necrosis factor-related receptor 6 (associated receptor 6, TRAF6) and a c-Src tyrosine kinase (c-Src kinase, c-Src) complex, and activates downstream signaling pathways (NF-k B, MAPK and Ca)2+Pathways) activate nuclear transcription factor κ B (NF- κ B), activin 1 (AP-1) and activated T cell nuclear factor c1(nuclear factor of activated tcellscytoplasmic micl, NFATc1) through intracellular signaling pathways, activate osteoclast-associated gene expression, such as tartrate-resistant alkaline phosphatase (TRAP), TRAP, matrix metalloproteinase 9 (matrixmetallotensinase 9, MMP-9), integrin β 3(integrin β 3) and cathepsin k (cathepsin k), promote osteoclast differentiation, enhance bone resorption activity, inhibit apoptosis, ultimately leading to increased osteoclast number and hyperfunction.
The pseudo-guaianolide sesquiterpene compound 6-O-angeloylplenolin is a compound isolated from Euphorbia lathyris L, and has a structural formula shown in the following formula (I) (also referred to as 6-OAP in the present application):
at present, no report related to the application of the pseudo guaianolide sesquiterpene compound in the preparation of an osteoclast differentiation inhibitor is found.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an application of a pseudo-guaianolide sesquiterpene compound separated from Euphorbia lathyris L in preparing an osteoclast differentiation inhibitor.
The technical scheme of the invention is as follows: the application of the compound shown in the formula (I) or the pharmaceutically acceptable salt thereof in preparing an osteoclast differentiation inhibitor;
the compounds of formula (I) are isolated from the plant Euphorbiae Lathyridis (Europodia Thymelioa) and are available in the literature (Liu J.L. et al (2019) Sesquiterpinenes and dicentenes from Europodia Thymeliota. Fitotepia 139: 104408).
In the technical scheme of the invention, the osteoclast differentiation inhibitor is a medicine for correspondingly treating the clinical indications of diphosphate and denoxizumab. Furthermore, the osteoclast differentiation inhibitor is an anti-postmenopausal osteoporosis drug and/or an anti-rheumatoid arthritis drug and/or an anti-tumor metastasis bone destruction drug.
The invention also provides a pharmaceutical composition which contains a therapeutically effective dose of a compound shown in the formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable auxiliary material;
the pharmaceutical composition can be a post-menopausal osteoporosis resistant drug and/or a rheumatoid arthritis resistant drug and/or a tumor metastasis bone destruction resistant drug.
The dosage form of the pharmaceutical composition can be any pharmaceutically acceptable dosage form, such as an internal preparation, an injection or an external preparation. Specifically, the oral preparation can be conventional preparations such as granules, tablets, pills or capsules, and the external preparation can be conventional preparations such as plasters.
The applicant proves through experiments that the compound shown in the formula (I) has the characteristics of direct and remarkable inhibitory action on osteoclastogenesis and osteoclastic activity, so that the compound can be used as an osteoclast differentiation inhibitor drug and used for treating diseases treated by the osteoclast differentiation inhibitor.
Drawings
FIG. 1 shows the results of the experiment of the cell survival effect of 6-OAP on osteoclast precursor RAW264.7 cells in Experimental example 1 of the present invention (n ═ 3);
FIG. 2 shows the results of the cell survival test of mouse Bone Marrow Macrophages (BMMs) with 6-OAP in Experimental example 2 of the present invention (n-3);
FIG. 3 is an experimental result of the effect of 6-OAP on differentiation of RAW264.7 cells into osteoclasts in Experimental example 3 of the present invention, wherein (a) is a photomicrograph of the inhibitory effect of 6-OAP at various concentrations on RANKL-induced osteoclasts, and (b) is a bar graph after counting osteoclasts in each well; RANKL is a ligand for activating nuclear factor NF-. kappa.B receptors (note: P <0.001 in the blank control group; P <0.01 in the RANKL group, n 3);
FIG. 4 is an experimental result of the effect of 6-OAP on the differentiation of BMMs into osteoclasts in Experimental example 4 of the present invention, in which (a) is a photomicrograph of the inhibitory effect of 6-OAP at various concentrations on RANKL-induced osteoclasts and (b) is a bar graph after counting osteoclasts in each well; RANKL is a ligand for activating nuclear factor NF-kB receptor, M-CSF is macrophage colony stimulating factor (note: compared with blank control group, # # P < 0.001; compared with RANKL group, # P <0.01, # P <0.001, n ═ 3);
FIG. 5 is an experimental result of the effect of 6-OAP on osteoclast activity in Experimental example 5 of the present invention, wherein (a) is a photomicrograph showing the inhibitory effect of 6-OAP at different concentrations on the bone resorption function of RANKL-induced osteoclasts, and (b) is a bar graph showing the bone resorption area formed on bone chips in each well; RANKL is a ligand for activating nuclear factor NF-. kappa.B receptors, and M-CSF is macrophage colony stimulating factor (note: P <0.001 # compared to blank control group; P <0.01 # P < 0.001; n ═ 3) compared to RANKL group.
Detailed Description
The present invention will be better understood from the following detailed description of specific examples, which should not be construed as limiting the scope of the present invention.
Example 1: preparation of 6-OAP
1) Taking 10kg of aerial parts of the thousand-rooted salvia, crushing, carrying out reflux extraction for 3 times by using 95 v/v% ethanol (100L) for 3 hours each time, combining extracting solutions, and carrying out reduced pressure concentration to obtain 2.1kg of extract;
2) suspending the obtained extract with water, extracting with ethyl acetate, and concentrating to obtain 726.9g of ethyl acetate extract;
3) subjecting the ethyl acetate extract to silica gel column chromatography, gradient eluting with a first eluent, and detecting the combined fractions by thin layer chromatography to obtain 8 fractions A-H respectively; wherein the first eluent is prepared from dichloromethane and methanol according to the ratio of 100: 1-6: 1 in a volume ratio;
4) subjecting the D fractions (215.3g) to MCI column chromatography, performing gradient elution with a second eluent, and detecting the combined fractions by thin layer chromatography to obtain 8 fractions D1-D8; the second eluent is a mixed solvent composed of methanol and water according to the volume ratio of 20:80-100: 0;
5) subjecting D8 fractions (10.9g) to RP-C18 reverse phase chromatography, gradient eluting with a third eluent, and identifying the combined fractions by thin layer chromatography to obtain 20 fractions D8a-D8t respectively; the third eluent is a mixed solvent composed of methanol and water according to the volume ratio of 50:50-100: 0;
6) subjecting D8i (282.5mg) to Sephadex column chromatography, eluting with methanol, collecting eluate, subjecting to preparative high performance liquid chromatography (innovative constant LC3000 HPLC, YMC-pack ODS-A column (250X 20mm, 5 μm)), and separating with mixed solvent composed of acetonitrile and water at volume ratio of 43:57 as mobile phase (flow rate of 8mL/min) to obtain white amorphous powder (17.5mg, tR ═ 55.4 min);
the white amorphous powder obtained was characterized by the following specific spectral data:
(+)HR-ESIMS m/z 369.1675[M+Na]+calculating the value C20H26O5Na,369.1672)。
1H NMR(methanol-d4,400MHz)δ7.89(1H,dd,J＝6.4,2.0Hz,H-2),6.09(1H,m,H-18),6.06(1H,dd,J＝6.4,2.8Hz,H-3),5.49(1H,s,H-6),4.87(1H,m,H-8),3.28(1H,m,H-11),3.13(1H,m,H-1),2.97(1H,m,H-7),2.47(1H,m,H-9a),2.19(1H,m,H-10),1.88(3H,d,J＝7.2Hz,H-19),1.73(3H,s,H-20),1.73(1H,m,H-9b),1.48(3H,d,J＝7.2Hz,H-13),1.26(3H,d,J＝6.8Hz,H-14),1.03(3H,s,H-15)。
13C NMR(methanol-d4,100MHz)δ212.4(C-4),181.7(C-12),167.7(C-16),165.2(C-2),139.5(C-18),130.0(C-3),128.8(C-17),81.5(C-8),73.1(C-6),56.2(C-5),56.0(C-1),50.2(C-7),41.9(C-9),41.5(C-11),27.2(C-10),20.6(C-20),20.0(C-15),18.1(C-19),15.9(C-14),11.1(C-13)。
Thus, the obtained white amorphous powder was determined to be the target compound 6-OAP.
The effect of 6-OAP on resistance to osteoblast differentiation was verified by in vitro experiments as follows.
Experimental example 1: MTT method for detecting survival effect of 6-OAP on osteoclast precursor RAW264.7 cells
Taking RAW264.7 cells with good growth state at a ratio of 1 × 103The density of each well was inoculated in a 96-well plate, DMEM medium containing 10% fetal bovine serum and 100IU/ml penicillin and 100IU/ml streptomycin was added to 200. mu.l per well, and then the 96-well plate was placed at 37 ℃ with 5% CO2The cell culture box is used for incubation, after the cells are attached to the wall stably overnight, 6-OAP with different concentrations is respectively added to ensure that the final concentrations are 0.5 mu M, 1 mu M, 2 mu M and 4 mu M, and each group is provided with 3 multiple holes. After 5 days of incubation, the supernatant was discarded and 100. mu.l of MTT was added to each well at 0.5 mg/ml. Placing at 37 ℃ and 5% CO2After the cell culture box is incubated for 4 hours, the supernatant is discarded, 150 μ l of DMSO solution is added into each well, and the mixture is added into a micro-incubatorAfter shaking for 15min on a volume oscillator, measuring the optical density value (OD value) at the wavelength of 570nm by using a TECANGENIOsPro multifunctional microplate reader, and calculating the survival rate of each group of cells. The results are shown in FIG. 1.
As can be seen from FIG. 1, the in vitro concentration of 4. mu.M or less had no significant effect on the survival of RAW264.7 cells.
Experimental example 2: CCK-8 method for detecting survival effect of 6-OAP on mouse Bone Marrow Macrophages (BMMs)
Under aseptic condition, taking femur of 8-12 week old C57BL/6 female mouse, cutting off joint parts at two ends of femur, sucking phenol-free α -MEM culture medium (containing 10% fetal calf serum, 100IU/ml penicillin and 100IU/ml streptomycin) with 1ml syringe, repeatedly washing femur until femoral bone cavity is whitish, placing cells washed out of femoral bone marrow cavity at 37 deg.C and 5% CO2And after the cell culture box is incubated for 2h, sucking the supernatant, cracking red blood cells, centrifuging, and resuspending to obtain the BMMs. Take 1X 105The density of each well was inoculated in a 96-well plate, phenol red-free α -MEM medium containing 10% fetal bovine serum and 100IU/ml penicillin and 100IU/ml streptomycin was added to 200. mu.l per well while M-CSF was added to a final concentration of 50ng/ml per well, and then the 96-well plate was placed at 37 ℃ and 5% CO2The cell culture box is incubated, after the cells are stable overnight, 6-OAP with different concentrations is added respectively to make the final concentration 0.5. mu.M, 1. mu.M, 2. mu.M and 4. mu.M, and each group is provided with 3 multiple wells. After 5 days of incubation, 100. mu.l of supernatant was aspirated, 5. mu.l of CCK-8 was added to each well, shaken, and placed in a cell incubator containing 5% CO2 at 37 ℃ for further incubation, and after 3 hours, the optical density (OD value) at a wavelength of 450nm was measured with a TECANGENiosPro multifunctional microplate reader to calculate the cell survival rate of each group. The results are shown in FIG. 2.
As can be seen from fig. 2, in vitro concentrations <4 μ M had no significant effect on the survival of BMMs.
Experimental example 3: effect of 6-OAP on differentiation of Raw264.7 cells into osteoclasts
Take 1X 103RAW 264.7/well was plated in 96-well plates and after overnight stabilization, 6-OAP was added at different concentrations to give final concentrations of 0.5. mu.M, 1 and 2. mu.M, with 3 duplicate wells per group. After 4h, RANKL was added to each of the other groups except the blank group to give a final concentration of RANKLThe medium was changed every two days for 100ng/ml per well, and after culturing for 4-5 days, TRAP staining was performed. The osteoclasts were photographed and counted under an inverted microscope, wherein TRAP positive cells with nuclei greater than 3 were osteoclasts. The results are shown in FIG. 3.
As shown in FIG. 3, 6-OAP inhibits RANKL from inducing RAW264.7 osteoclast precursor cells to generate osteoclasts.
Experimental example 4: effect of 6-OAP on differentiation of BMMs into osteoclasts:
take 1X 105One/well of BMMs were inoculated in a 96-well plate, phenol red-free α -MEM medium containing 10% fetal bovine serum and 100IU/ml penicillin and 100IU/ml streptomycin was added to 200. mu.l per well while M-CSF was added to a final concentration of 50ng/ml per well, and then the 96-well plate was placed at 37 ℃ and 5% CO2After the cells are stabilized overnight, 6-OAP with different concentrations is added to the cells to make the final concentration 0.5. mu.M, 1. mu.M and 2. mu.M, each group is provided with 3 multiple wells. After 4h, RANKL was added to each of the other groups except the blank group to a final concentration of 100ng/ml per well, and TRAP staining was performed after 3-4 days of culture. The osteoclasts were photographed and counted under an inverted microscope, wherein TRAP positive cells with nuclei greater than 5 were osteoclasts. The results are shown in FIG. 4.
As can be seen in FIG. 4, 6-OAP inhibits RANKL from inducing the generation of osteoclasts by BMMs.
Combining the cell survival experiment results of experiment examples 1 and 2, 6-OAP with concentrations of 0.5 mu M, 1 mu M and 2 mu M is selected for intervention, and the 6-OAP is found to be capable of remarkably inhibiting RANKL in vitro from inducing RAW264.7 cells and BMMs cells to form osteoclasts. It is suggested that 6-OAP can inhibit osteoclast generation in vitro.
Experimental example 5: effect of 6-OAP on osteoclast osteoclastic activity:
the same osteoclastogenesis protocol as in Experimental example 4 above, BMMs cells were inoculated onto the coated artificial bone fragments
Osteo Assay 96-well plate culture, washing cells at induction day 7, photographing under 40 XNikon inverted optical microscope light microscope observation, calculating the percentage of bone absorption area in each well by Image-Pro Plus software. The results are shown in FIG. 5.
The experimental results prove that: 6-OAP with concentration of 0.5 μ M, 1 μ M, 2 μ M can significantly reduce the area of bone depression formed on bone plate by osteoclast, and has inhibitory effect on bone resorption activity of osteoclast.
By combining the results, the pseudo-guaianolide sesquiterpene compound 6-OAP shown in the formula (I) can remarkably inhibit in vitro RANKL from inducing osteoclasts to be formed by osteoclasts of RAW264.7 cells and BMMs cells, directly inhibit the differentiation and formation of the osteoclasts and inhibit the bone resorption function of the osteoclasts. Namely, 6-OAP can inhibit the generation, activation and osteoclast activity in vitro.
Claims (6)
1. The application of the compound shown in the formula (I) or the pharmaceutically acceptable salt thereof in preparing an osteoclast differentiation inhibitor;
2. use according to claim 1, characterized in that: the osteoclast differentiation inhibitor is a medicine for treating the clinical indications of diphosphate and dinoteximab correspondingly.
3. Use according to claim 1, characterized in that: the osteoclast differentiation inhibitor is an anti-postmenopausal osteoporosis drug and/or an anti-rheumatoid arthritis drug and/or an anti-tumor metastasis bone destruction drug.
4. A pharmaceutical composition characterized by: contains a therapeutically effective dose of a compound shown in the formula (I) or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable auxiliary materials;
5. the pharmaceutical composition of claim 4, wherein: the medicine is an anti-postmenopausal osteoporosis medicine and/or an anti-rheumatoid arthritis medicine and/or an anti-tumor metastasis bone destruction medicine.
6. The pharmaceutical composition of claim 4, wherein: the medicine is a pharmaceutically acceptable dosage form.
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