Preparation method and application of an anti-osteoporosis drug

Abstract

The present invention provides a preparation method and application of an anti-osteoporosis drug. The present invention for the first time proposes to construct a bone-targeted nano-therapy system based on phosphorylated dihydromyricetin (p-DMY). By chemical means, the phenolic hydroxyl groups on DMY are transformed into phosphoric acid functional groups to form amphiphilic phosphorylated DMY, which can self-assemble into water-soluble nanoparticles in aqueous solution, thereby increasing its circulation half-life and improving the bioavailability of the drug. In addition, there are a large number of phosphoric acid clusters on the surface of the phosphorylated DMY assembly, which can endow it with bone-targeting characteristics by binding to calcium on the bone surface. Moreover, since the problems of poor water solubility and lack of targeting of DMY are solved, there is no need to repeatedly administer large doses of drugs, effectively alleviating the problem of potential toxic and side effects of DMY.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a preparation method and application of an anti-osteoporosis drug. Background Art

[0002] Osteoporosis is a common systemic metabolic orthopedic disease, characterized by reduced bone mass and destruction of the bone microstructure, leading to bone pain and even fragility fractures. Currently, the commonly used drugs for treating osteoporosis clinically are mainly bisphosphonates, calcitonin and estrogen drugs. However, most anti-osteoporosis drugs are mainly anti-resorptive and anabolic drugs, which only inhibit bone resorption and do not promote bone formation. In addition, it is reported that these drugs will produce serious side effects after long-term use, such as fever, bone pain, nausea, hypocalcemia, hypercalcemia, endometrial examination and breast cancer, as well as cardiovascular diseases. For example, denosumab is a RANKL monoclonal antibody with a definite anti-resorptive effect, but it is found to have a potential risk of inducing malignant tumors in clinical trials, and the drug safety needs to be further demonstrated. Parathyroid hormone, as the first and only anabolic anti-osteoporosis drug with osteogenic effect approved by the FDA, has better therapeutic effects than bisphosphonates, but due to disadvantages such as poor patient compliance and high price, it is not widely used at present. Therefore, it is of great significance to develop new anti-osteoporosis drugs.

[0003] Natural compounds have attracted the attention of many scholars due to their advantages such as low toxicity, easy access and wide range of actions. Among them, dihydromyricetin (DMY) is considered an ideal natural anti-osteoporosis drug because of its good anti-osteoporosis effect, rich sources and low price. However, DMY has poor water solubility and lack of targeting, so large doses of repeated medication are required to achieve better anti-osteoporosis efficacy. However, with the prolongation of the medication time, the accumulation of DMY in other tissues and organs will also produce toxic and side effects, which greatly limits its clinical application. Therefore, how to solve the problems of poor water solubility, lack of targeting and potential toxic and side effects of DMY is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0004] Therefore, the purpose of the present invention is to provide a preparation method and application of an anti-osteoporosis drug to solve the problems of poor water solubility, lack of targeting and potential toxic and side effects of DMY.

[0005] One aspect of the present invention provides a preparation method of an anti-osteoporosis drug, including the following steps:

[0006] Step 1, weigh dihydromyricetin, then add it to anhydrous pyridine, and then place it in a water bath for ultrasonic treatment to fully dissolve dihydromyricetin;

[0007] Step 2: Pour the fully dissolved dihydromyricetin solution into a reaction flask, then add an excessive amount of phosphorus oxychloride, and stir and react the mixture under a nitrogen atmosphere for 4 hours;

[0008] Step 3: After the reaction is completed, transfer the obtained product to a dialysis bag with a molecular weight cut-off of 300, and dialyze alternately in ethanol and ultrapure water;

[0009] Step 4: Finally, transfer the product obtained by dialysis to a rotary evaporation flask, concentrate the product, and then lyophilize it with a freeze dryer to obtain the anti-osteoporosis drug.

[0010] Another aspect of the present invention provides an application of an anti-osteoporosis drug, wherein the anti-osteoporosis drug is prepared by the above method, and the anti-osteoporosis drug is used for the treatment of osteoporosis.

[0011] Beneficial effects:

[0012] The present invention firstly proposes to construct a bone-targeted nano-therapeutic system based on phosphorylated dihydromyricetin (p-DMY). By chemical means, the phenolic hydroxyl groups on DMY are transformed into phosphoric acid functional groups to form amphiphilic phosphorylated DMY, which can self-assemble into water-soluble nanoparticles in aqueous solution, thereby increasing its circulation half-life and improving the bioavailability of the drug. In addition, there are a large number of phosphoric acid clusters on the surface of the phosphorylated DMY assembly, which can endow it with bone-targeting properties by binding to calcium on the bone surface. Moreover, since the problems of poor water solubility and lack of targeting of DMY are solved, there is no need to repeatedly administer large doses of the drug, effectively alleviating the problem of potential toxic and side effects of DMY. In summary, the present invention phosphorylates dihydromyricetin by using its own phenolic hydroxyl group structure, improves its water solubility and endows it with bone-targeting anti-osteoporosis effects, and solves the problems of poor water solubility, lack of targeting, and potential toxic and side effects of DMY in the prior art. Description of the drawings

[0013] Figure 1 It is the synthetic route diagram of the present invention;

[0014] Figure 2 It is the nuclear magnetic resonance phosphorus spectrum diagram of p-DMY;

[0015] Figure 3 It is the Fourier transform infrared spectrum diagram of p-DMY;

[0016] Figure 4 It is the particle size diagram of p-DMY;

[0017] Figure 5 It is the ultraviolet-visible absorption spectrum diagram of p-DMY;

[0018] Figure 6 It is the transmission electron microscope diagram of p-DMY;

[0019] Figure 7 Element Mapping diagram of p-DMY;

[0020] Figure 8 Result diagram of the viability of BMMs cells after treatment with different concentrations of p-DMY for 48 hours determined by the CCK-8 method;

[0021] Figure 9 Result diagram of the viability of BMMs cells after treatment with different concentrations of p-DMY for 96 hours determined by the CCK-8 method;

[0022] Figure 10 Result diagram of TRAP staining of osteoclasts cultured with osteoclast induction medium for 7 days in the presence of different concentrations of p-DMY;

[0023] Figure 11 Number of mature osteoclasts in each group and proportion of osteoclast area to well area counted after osteoclasts were cultured with osteoclast induction medium for 7 days in the presence of different concentrations of p-DMY;

[0024] Figure 12 Process diagram showing that p-DMY inhibits osteoclast formation during a specific period by TRAP staining;

[0025] Figure 13 Process diagram showing that p-DMY inhibits osteoclast formation during a specific period by TRAP staining, and number of mature osteoclasts in each group and proportion of osteoclast area to well area counted;

[0026] Figure 14 Effect diagram of p-DMY on c-Fos gene, Nfatc1 gene, Mmp9 gene, DC-STAMP gene, CTSK gene, TRAP gene expression;

[0027] Figure 15 Western blot diagram of the effect of p-DMY on osteoclast-related proteins;

[0028] Figure 16 Quantitative evaluation of Nfatc1 Ratio data diagram of gray value relative to GAPDH band;

[0029] Figure 17 Quantitative evaluation of c-Fos Ratio data diagram of gray value relative to GAPDH band;

[0030] Figure 18 Quantitative evaluation of CTSK Ratio data diagram of gray value relative to GAPDH band. Detailed implementation manners

[0031] For the convenience of understanding the present invention, the present invention will be described more comprehensively below with reference to each embodiment. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the disclosure of the present invention more thorough and comprehensive.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0033] The present invention provides a preparation method of an anti-osteoporosis drug. For the synthetic route, please refer to Figure 1 , and the preparation method includes the following steps:

[0034] Step 1: Weigh dihydromyricetin, then add it to anhydrous pyridine, and then place it in a water bath for ultrasonic treatment to fully dissolve dihydromyricetin.

[0035] Step 2: Pour the fully dissolved dihydromyricetin solution into a reaction flask, then add an excessive amount of phosphorus oxychloride, and stir and react the mixture under a nitrogen atmosphere for 4 hours.

[0036] Step 3: After the reaction is completed, transfer the obtained product to a dialysis bag with a molecular weight cut-off of 300, and dialyze it alternately in ethanol and ultrapure water.

[0037] Step 4: Finally, transfer the product obtained by dialysis to a rotary evaporation flask, concentrate the product, and then freeze-dry it with a freeze dryer to obtain the anti-osteoporosis drug.

[0038] Among them, in Step 1, the molar concentration of dihydromyricetin in anhydrous pyridine is 0.005 mol / L.

[0039] Among them, in Step 1, the ultrasonic treatment time is 5 minutes.

[0040] Among them, in Step 2, at room temperature, stir and react the mixture under a nitrogen atmosphere for 4 hours.

[0041] Among them, in Step 3, transfer the obtained product to a dialysis bag with a molecular weight cut-off of 300, and dialyze it alternately in ethanol and ultrapure water for 3 days.

[0042] The present invention also provides an application of an anti-osteoporosis drug. The anti-osteoporosis drug is prepared by the above method, and the anti-osteoporosis drug is used for the treatment of osteoporosis.

[0043] The present invention will be described below in conjunction with specific embodiments. The embodiments of the present invention are not limited to the following specific embodiments. Within the scope of the main rights unchanged, appropriate changes can be made for implementation.

[0044] An embodiment of the present invention provides a preparation method of an anti-osteoporosis drug, comprising the following steps:

[0045] Step 1: Accurately weigh 36.8 mg (0.1 mmol) of dihydromyricetin with an electronic balance, and then add it to 20 mL of anhydrous pyridine (Py), and place it in a water bath ultrasonic bath for ultrasonic treatment for 5 minutes to fully dissolve the dihydromyricetin;

[0046] Step 2: Pour the fully dissolved dihydromyricetin solution into a reaction flask, and then add an excessive amount of phosphorus oxychloride. At room temperature, stir and react the mixture under a nitrogen atmosphere for 4 hours;

[0047] Step 3: After the reaction is completed, transfer the obtained product to a dialysis bag with a molecular weight of 300, and dialyze it alternately in ethanol and ultrapure water for 3 days;

[0048] Step 4: Finally, transfer the product obtained by dialysis to a rotary evaporation flask, concentrate the product, and then freeze-dry it with a freeze dryer to obtain the anti-osteoporosis drug, that is, phosphorylated dihydromyricetin (p-DMY), and store it in a dry place.

[0049] The following is an experimental test

[0050] 1. Material characterization of p-DMY

[0051] Perform nuclear magnetic resonance phosphorus spectrum ( 31 P NMR), ultraviolet-visible absorption spectrum (UV-Vis), and Fourier transform infrared spectrum (FTIR) detection on p-DMY, and then further characterize p-DMY with X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), and transmission electron microscopy (TEM); and measure its bone targeting ability through in vitro evaluation of bone mineral affinity.

[0052] The test results are as Figures 2 to 7 .

[0053] According to Figure 2 the nuclear magnetic resonance phosphorus spectrum of p-DMY in, it can be seen that dihydromyricetin is successfully phosphorylated.

[0054] According to Figure 3 the Fourier transform infrared spectrum of p-DMY in, it can be seen that the phenolic hydroxyl peak (3348 -1 ) of p-DMY disappears and is replaced by the characteristic peaks of phosphate groups (931 -1 , 1054 -1 , 678 -1 ), indicating that the phenolic hydroxyl group is replaced by a phosphate group.

[0055] According to Figure 4 the particle size diagram of p-DMY in , it can be seen that the hydrodynamic diameter of p-DMY is 181 ± 20.18 nm, and the polydispersity index (PDI) is 0.124.

[0056] According to Figure 5 the ultraviolet-visible absorption spectrum diagram of p-DMY in , it can be seen that the maximum ultraviolet absorption peak of p-DMY is located at 255 nm. Before adsorbing hydroxyapatite (HAp), the absorbance value at the maximum absorption wavelength is about 1.69. After being adsorbed by hydroxyapatite (HAp), the absorbance value at the same wavelength drops to about 0.67. The significant decrease in absorbance indicates that the binding ability of p-DMY to HAp is very strong. It is calculated that the binding rate of HAp to p-DMY is 60.5%, which is equivalent to the effect of alendronic acid (ALN). The above results show that p-DMY can potentially target bone tissue.

[0057] Please combine Figure 6 and Figure 7 to obtain that elements C, O, and P appear in p-DMY.

[0058] 2. Evaluation of the cytotoxicity of p-DMY and its effect on osteoclast differentiation

[0059] 2.1 Toxicity evaluation of tissue cells

[0060] Experimental procedure: For the CCK-8 assay to detect cell proliferation, add 100 μL of 2000 primary mouse bone marrow cells (BMMs) to each well. After the cells grow and adhere, conduct the experiment. Perform toxicity detection on p-DMY. Incubate with drugs at concentrations of 0, 5, 10, 20, 40, 80, 160, and 320 μg / mL in sequence, and detect the drug toxicity at 48 h and 96 h respectively. Repeat the experiment in 5 wells for each group. During the experiment, aspirate the culture medium in the well plate, then add 90 μL of complete culture medium, and then add 10 μL of CCK-8 reagent to each well. Place it in the incubator for 1 - 4 hours. Then measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader to detect the proliferation rate of cells or the toxicity of the intervention.

[0061] Experimental results: The results are as shown in Figure 8 and Figure 9 It can be seen from Figure 8 and Figure 9 that p-DMY has no obvious toxicity to bone marrow cells and has no effect on cell proliferation within the concentration range of 0 - 320 μg / mL for 48 and 96 hours.

[0062] 2.2 Effect on osteoclast differentiation

[0063] Experimental procedure:

[0064] a. Mouse bone marrow primary cells (BMMs) were isolated from mouse femurs, and M-CSF was added to stimulate their directional differentiation into monocytes / macrophages;

[0065] b. The above cells were seeded in 96-well plates at a density of 8×10 3 cells / well, incubated overnight in an incubator, and then different concentrations of p-DMY were added. At the same time, M-CSF (30 ng / mL) and RANKL (osteoclast differentiation factor, 50 ng / mL) were added to induce osteoclast differentiation, and the culture medium was changed every other day;

[0066] c. After 5 - 7 days of culture, when mature osteoclasts could be seen under a light microscope in the blank group (i.e., without adding p-DMY), the cells were fixed with 4% paraformaldehyde, and then the osteoclasts in each group were stained with TRAP staining solution. The number of mature osteoclasts and the spreading area of osteoclasts in each group were counted under an optical microscope (an osteoclast with three or more nuclei is called a mature osteoclast).

[0067] Experimental results:

[0068] Figure 10 The figure shows the results of TRAP staining of osteoclasts cultured with osteoclast induction medium for 7 days in the presence of different concentrations of p-DMY (0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL respectively). It can be Figure 10 seen that p-DMY has a significant inhibitory effect on the differentiation of mature osteoclasts.

[0069] Figure 11 The figure shows the number of mature osteoclasts in each group and the proportion of the osteoclast area to the well area counted after osteoclasts were cultured with osteoclast induction medium for 7 days in the presence of different concentrations of p-DMY. It can be Figure 11 seen that p-DMY has a significant inhibitory effect on the differentiation of mature osteoclasts.

[0070] Figure 12 The figure shows the process of p-DMY inhibiting the formation of osteoclasts during a specific period as shown by TRAP staining, Figure 13It shows that TRAP staining reveals that p-DMY inhibits osteoclast formation during a specific period. The number of mature osteoclasts in each group and the proportion of the osteoclast area to the pore area were counted. Among them, CTRL represents the blank group without p-DMY added. 1-3d means that there is 20 μg / mL of p-DMY present on the 1st, 2nd, and 3rd days. 3-5d means that there is 20 μg / mL of p-DMY present on the 3rd, 4th, and 5th days. 1-5d means that there is 20 μg / mL of p-DMY present on the 1st, 2nd, 3rd, 4th, and 5th days.

[0071] Comprehensively Figures 10 to 13 It can be seen that the inhibitory effect of p-DMY on osteoclast differentiation is dose-dependent and mainly occurs during the early differentiation process.

[0072] 2.3 Study the effect of p-DMY on osteoclast formation at the gene level

[0073] Experimental procedure 1:

[0074] a. Inoculate the isolated BMMs cells into a 6-well plate at a cell density of 8×10 4 cells / well, and then add the medium containing M-CSF and place it in an incubator for 24 hours;

[0075] b. After the cells adhere, add p-DMY at different concentrations to stimulate the cells, and then add M-CSF and RANKL to induce osteoclast differentiation. The culture medium is changed every other day and continuously cultured for 5-7 days. After the blank group (i.e., without p-DMY added) shows mature osteoclasts under a light microscope, fix the cells and then extract the total RNA of each group of cells;

[0076] c. Use Real Time PCR technology to detect osteoclast-specific genes, such as: c-Fos gene, Nfatc1 gene, Mmp9 gene, DC-STAMP gene, CTSK gene, TRAP the expression of the gene.

[0077] Experimental procedure 2:

[0078] a. Isolate and culture mouse bone marrow-derived BMMs in vitro, and under the stimulation of M-CSF, direct their differentiation into monocytes / macrophages. Inoculate them into a 6-well plate, with 8×10 4 cells per well, and then add the medium containing M-CSF and place it in an incubator for 24 hours;

[0079] b. After the cells adhered, one group was treated with p-DMY (20 μg / mL), and the other group was added with phosphate buffer solution (PBS) as the negative control group;

[0080] c. Both groups of cells were stimulated with RANKL, and the total cell proteins were extracted on the 0th, 1st, and 3rd days after RANKL stimulation;

[0081] d. Using Western Blot technology, Nfatc1 and c-fos genes closely related to osteoclast formation were detected one by one to study the effect of p-DMY on the above genes.

[0082] Figure 14 Effect of p-DMY on c-Fos gene, Nfatc1 gene, Mmp9 gene, DC-STAMP gene, CTSK gene, TRAP gene expression diagram. In Figure 14 , RANKL represents the osteoclast differentiation factor, "-" indicates that the osteoclast differentiation factor was not added, and "+" indicates that 50 ng / mL of the osteoclast differentiation factor was added. It can be seen from Figure 14 that p-DMY can inhibit c-Fos gene, Nfatc1 gene, Mmp9 gene, DC-STAMP gene, CTSK gene, TRAP gene, and shows concentration dependence.

[0083] Figure 15 Western blot diagram of the effect of p-DMY on osteoclast-related proteins. Figure 16 Quantitative evaluation of Nfatc1 gray value ratio data diagram relative to the GAPDH band. Figure 17 Quantitative evaluation of c-Fos gray value ratio data diagram relative to the GAPDH band. Figure 18 Quantitative evaluation of CTSK gray value ratio data diagram relative to the GAPDH band.

[0084] Figure 15 and Figure 16 and Figure 17 and Figure 18Among them, RANKL 0d means that p-DMY is not added and not exposed to RANKL; RANKL1d means that p-DMY is not added and exposed to RANKL treatment for 1 day; RANKL 3d means that p-DMY is not added and exposed to RANKL treatment for 3 days; RANKL and p-DMY 0d means that 20 μg / mL of p-DMY is added and not exposed to RANKL; RANKL and p-DMY means that 20 μg / mL of p-DMY is added and exposed to RANKL treatment for 1 day; RANKL and p-DMY 3d means that 20 μg / mL of p-DMY is added and exposed to RANKL treatment for 3 days.

[0085] From Figure 15 it can be seen that p-DMY can effectively reduce the expression of proteins related to osteoclasts. From Figure 16 it can be seen that p-DMY can effectively reduce Nfatc1 the expression of proteins. From Figure 17 it can be seen that p-DMY can effectively reduce c- Fos the expression of proteins. From Figure 18 it can be seen that p-DMY can effectively reduce CTSK the expression of proteins.

[0086] In summary, the present invention first proposes to construct a bone-targeted nano-therapy system based on phosphorylated dihydromyricetin (p-DMY). By chemical means, the phenolic hydroxyl groups on DMY are transformed into phosphoric acid functional groups to form amphiphilic phosphorylated DMY, which can self-assemble into water-soluble nanoparticles in aqueous solution, thereby increasing its circulation half-life and improving the bioavailability of the drug. In addition, there are a large number of phosphate clusters on the surface of the phosphorylated DMY assembly, which can endow it with bone-targeting properties by binding to calcium on the bone surface. And because the problems of poor water solubility and lack of targeting of DMY are solved, there is no need to repeatedly administer large doses of drugs, effectively alleviating the problem of potential toxic and side effects of DMY. To sum up, the present invention phosphorylates dihydromyricetin using its own phenolic hydroxyl structure, improves its water solubility and endows it with bone-targeting anti-osteoporosis effects, solving the problems of poor water solubility, lack of targeting, and potential toxic and side effects of DMY in the prior art.

[0087] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. Use of an anti-osteoporosis drug in the preparation of a drug for treating osteoporosis, characterized in that, The anti-osteoporosis drug is prepared by the following method: Step 1: Weigh dihydromyricetin, then add it to anhydrous pyridine, and then place it in a water bath for ultrasonic treatment to fully dissolve dihydromyricetin; Step 2: Pour the fully dissolved dihydromyricetin solution into a reaction flask, then add an excessive amount of phosphorus oxychloride, and stir and react the mixture under a nitrogen atmosphere for 4 hours; Step 3: After the reaction is completed, transfer the obtained product to a dialysis bag with a molecular weight of 300, and dialyze it alternately in ethanol and ultrapure water; Step 4: Finally, transfer the product obtained by dialysis to a rotary evaporation flask, concentrate the product, and then freeze-dry it with a freeze dryer to obtain the anti-osteoporosis drug; In Step 1, the molar concentration of dihydromyricetin in anhydrous pyridine is 0.005 mol / L; In Step 1, the ultrasonic time is 5 minutes; In Step 2, at room temperature, stir and react the mixture under a nitrogen atmosphere for 4 hours; In Step 3, transfer the obtained product to a dialysis bag with a molecular weight of 300, and dialyze it alternately in ethanol and ultrapure water for 3 days.

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