Methods and pharmaceutical compositions for treating osteoporosis

Abstract

This invention provides a method for treating osteoporosis. This invention also provides a medicine for treating osteoporosis.

Description

[0001] This application claims priority to Chinese Patent Application No. 202211251275.3, filed on October 13, 2022, entitled “Method and Pharmaceutical Composition for Treating Osteoporosis”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the fields of disease treatment and pharmaceuticals. Specifically, this invention relates to treatment methods for osteoporosis, and related pharmaceutical compositions. Background Technology

[0003] Osteoporosis is a systemic metabolic disease caused by an imbalance in bone remodeling, where bone resorption exceeds bone formation. Osteoporosis can be classified into three categories based on its cause: primary osteoporosis, secondary osteoporosis, and idiopathic osteoporosis. Postmenopausal osteoporosis belongs to primary osteoporosis and occurs 5-10 years after menopause. The maintenance of bone homeostasis in the human body is mainly accomplished by osteoclasts and osteoblasts; osteoclasts are responsible for bone resorption, and osteoblasts are responsible for bone formation. Bone marrow mononuclear cells (BMMs) are hematopoietic stem cells that can differentiate into osteoclasts. When bone homeostasis is imbalanced, and the number of osteoclasts formed from BMMs increases, osteoporosis and other bone metabolic diseases occur, severely impacting the patient's quality of life.

[0004] Currently, osteoporosis is generally treated with medication. Because osteoporosis can be caused by a variety of factors, medication intervention needs to be targeted, and there is currently no single drug that can effectively prevent or treat osteoporosis caused by all different factors.

[0005] NVP-BHG712 is a specific EphB4 inhibitor that inhibits VEGFR2 and, in a dose-dependent manner, inhibits RTK autophosphorylation in A375-stabilized melanoma cells. EphB4 is an important member of the EPH family of tyrosine protein kinase receptors. EphB4 plays a crucial regulatory role in tumorigenesis and development, promoting metastasis in various tumors and significantly modulating tumor prognosis. EphB4 also regulates endothelial cell biological behavior and plays a critical role in embryonic angiogenesis; EphB4 knockout mice exhibit significant vascular development disorders. The effects of NVP-BHG712 on osteoclasts have not been reported; whether it inhibits osteoclast formation and function, and whether it has a therapeutic effect on postmenopausal osteoporosis and other bone metabolic diseases, remains unknown.

[0006] Further research is needed in this field on the physiological mechanisms and biochemical pathways affecting osteoporosis, as well as the discovery of new drugs for the treatment of osteoporosis. Summary of the Invention

[0007] This invention is the first to discover that NVP-BHG712 can effectively inhibit osteoclast activity, not only inhibiting osteoclast formation but also inhibiting osteoclast bone resorption, and can also inhibit the formation of osteoporosis in mammals. This invention thus provides a new drug for the treatment of osteoporosis.

[0008] In one aspect of the invention, a method for treating osteoporosis is provided, comprising administering a therapeutically effective amount of NVP-BHG712 or a pharmaceutically acceptable salt thereof to a patient requiring such treatment.

[0009] In one aspect of the invention, a method for treating osteoporosis is provided, the method comprising administering NVP-BHG712 or a pharmaceutically acceptable salt thereof, the method being an in vitro, non-therapeutic method. For example, the method can be used to treat isolated tissues or cells for the study of osteoporosis in a culture medium.

[0010] NVP-BHG712 is a compound with the following formula:

[0011] .

[0012] In another embodiment of the invention, NVP-BHG712 or a pharmaceutically acceptable salt thereof is administered at a dose between 0.1 mg and 50 mg. In another embodiment of the invention, NVP-BHG712 or a pharmaceutically acceptable salt thereof is administered at doses of 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, or 0.01 mg / kg body weight. More preferably, NVP-BHG712 or a pharmaceutically acceptable salt thereof is administered at doses of 40, 20, 10, 5, or 1 mg / kg body weight.

[0013] In one embodiment of the invention, the compound or its pharmaceutically acceptable salt is administered in the form of a pharmaceutical composition, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

[0014] In one embodiment of the invention, the compound or a pharmaceutically acceptable salt thereof is administered daily or every 2-3 days.

[0015] In one aspect of the invention, the use of NVP-BHG712 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating osteoporosis is provided.

[0016] In one embodiment of the invention, the drug comprises a pharmaceutically acceptable carrier, excipient, or diluent.

[0017] In one embodiment of the invention, the dosage of the compound or a pharmaceutically acceptable salt thereof is between 0.01 mg and 1000 mg. In another embodiment of the invention, the dosage of the compound or a pharmaceutically acceptable salt thereof is between 0.1 mg and 50 mg / kg body weight, for example, 50, 40, 20, 10, 5 or 1 mg / kg body weight.

[0018] In one aspect of the invention, a method for inhibiting osteoclast differentiation and / or activity is provided, the method comprising administering a therapeutically effective amount of NVP-BHG712 or a pharmaceutically acceptable salt thereof to a patient requiring such treatment.

[0019] In one aspect of the invention, a method for inhibiting osteoclast differentiation and / or activity is provided, the method comprising administering NVP-BHG712 or a pharmaceutically acceptable salt thereof, the method being an in vitro, non-therapeutic method. For example, the method can be used to treat isolated tissues or cells in a culture medium.

[0020] In another embodiment of the invention, NVP-BHG712 or a pharmaceutically acceptable salt thereof is administered at a dose between 0.1 mg and 50 mg. In another embodiment of the invention, NVP-BHG712 or a pharmaceutically acceptable salt thereof is administered at a dose of 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, or 0.01 mg / kg body weight. More preferably, NVP-BHG712 or a pharmaceutically acceptable salt thereof is administered at a dose of 50, 40, 20, 10, 5, or 1 mg / kg body weight.

[0021] In one embodiment of the invention, the compound or its pharmaceutically acceptable salt is administered in the form of a pharmaceutical composition, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

[0022] In one embodiment of the invention, the compound or a pharmaceutically acceptable salt thereof is administered daily or every 2-3 days.

[0023] In one aspect of the invention, the use of NVP-BHG712 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting osteoclast differentiation and / or activity is provided.

[0024] In one embodiment of the invention, the drug comprises a pharmaceutically acceptable carrier, excipient, or diluent.

[0025] In one embodiment of the invention, the dosage of the compound or a pharmaceutically acceptable salt thereof is between 0.01 mg and 1000 mg. In another embodiment of the invention, the dosage of the compound or a pharmaceutically acceptable salt thereof is between 0.1 mg and 50 mg / kg body weight, for example, 40, 20, 10, 5 or 1 mg / kg body weight.

[0026] The objects for which the methods and drugs (pharmaceutical compositions) described herein are needed can be mammals, including humans or non-human primates such as monkeys. Mammals can also be other animals, such as rats, mice, rabbits, pigs, dogs, etc. The mammals can be domesticated animals, such as cats or dogs.

[0027] Bones are constantly undergoing a process known as remodeling. Bone loss occurs in osteoporosis because the normal remodeling process, or bone turnover, removes more bone than it replaces. Bone remodeling involves two distinct phases: bone resorption (breakdown) and bone formation. Calcium is stored in bones, and when the body needs it, osteocytes, or bone cells, attach to the bone surface and break it down, leaving cavities in the bone. Bone morphogenetic cells, called osteoblasts, then fill these cavities with an organic matrix called osteoid. Osteoids then spontaneously mineralize with calcium phosphate to reform hard bone.

[0028] Unless otherwise stated, as used herein, the term "osteoporosis" refers to any decrease or reduction in an individual's bone mineral density, including osteoporosis and osteopenia as defined by the World Health Organization (WHO). The WHO defines acceptable bone mineral density as bone mineral density within one standard deviation of the normal value or with a corresponding T score less than -1. The WHO defines osteopenia as bone mineral density within 1 to 2.5 standard deviations of the normal value or with a corresponding T score of -1 to -2.5. The WHO describes osteoporosis as a more severe form of osteopenia and defines osteoporosis as bone mineral density with a standard deviation from the normal value less than 2.5 or a corresponding T score less than -2.5. In one aspect of the invention, "osteoporosis" includes postmenopausal osteoporosis (PMPO), a serious condition affecting the physical and mental health of middle-aged and elderly women due to ovarian decline and subsequent estrogen deficiency or emergency withdrawal after menopause.

[0029] Unless otherwise stated, the terms “treatment” and “treatment and prevention” may be used interchangeably herein to refer to methods relating to an individual suffering from or otherwise at risk of developing osteoporosis, and to any method that affects an individual’s bone mineral density or structure, including increasing bone mineral density in an individual suffering from osteoporosis, slowing the rate or occurrence of bone mineral density loss, maintaining normal bone mineral density, or reversing some or all of a decrease or deficiency in bone mineral density.

[0030] The active ingredient in the pharmaceutical composition of the present invention can be administered in the form of a raw material compound, or alternatively, the active ingredient can be introduced into the pharmaceutical composition, optionally in the form of a pharmaceutically acceptable salt, together with one or more adjuvants, excipients, carriers, buffers, diluents and / or other conventional pharmaceutical excipients.

[0031] Adjuvants include preservatives, wetting agents, suspending agents, sweeteners, flavoring agents, flavoring agents, emulsifiers, and dispersants. For example, various antibacterial and antifungal agents can ensure protection against microbial activity, such as parabens, chlorobutanol, phenol, and sorbic acid. Isotonic agents, such as sugars and sodium chloride, are also preferable. The absorption of injectable drug forms can be prolonged by using agents that delay absorption (e.g., aluminum monostearate and gelatin).

[0032] If desired, the pharmaceutical composition of the compound may also contain small amounts of excipients, such as wetting agents or emulsifiers, pH buffers, antioxidants, etc., for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylated hydroxytoluene, etc.

[0033] A "pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound. Pharmaceutically acceptable salts are non-toxic.

[0034] Pharmaceutically acceptable examples of acid addition salts include those formed with the following acids: inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; and organic acids, such as acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, malic acid, citric acid, benzoic acid, cinnamic acid, 3-(4-hydroxybenzoyl)benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheponic acid, 4,4′-methylenebis-(3-hydroxy-2-en-1-carboxylic acid). 3-Phenylacetic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthenic acid, salicylic acid, stearic acid, mucoconic acid, p-toluenesulfonic acid, and salicylic acid, etc.

[0035] The pharmaceutical compositions of the present invention can be administered via any convenient route suitable for the desired therapy. Preferred routes of administration include oral administration, particularly in tablet, capsule, lozenge, powder, and liquid form; and parenteral administration, particularly by subcutaneous, subcutaneous, intramuscular, and intravenous injection. The pharmaceutical compositions of the present invention can be prepared by those skilled in the art using standard methods and conventional techniques suitable for the desired formulation. If desired, compositions suitable for sustained release of the active ingredient can be used.

[0036] For preparing a pharmaceutical composition from the active ingredient in the pharmaceutical composition of the present invention, a pharmaceutically acceptable carrier may be a solid or a liquid. Solid forms of formulations include powders, tablets, pills, capsules, pouches, suppositories, and dispersible granules. The solid carrier may be one or more substances that can also be used as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, binder, preservative, tablet disintegrant, or encapsulation material.

[0037] When needed, a composition suitable for providing sustained release of active ingredients can be applied.

[0038] Pharmaceutical formulations are preferably unit dosage forms. In this type of form, the formulation is subdivided into unit doses containing appropriate amounts of the active ingredient. A unit dosage form can be a packaged formulation containing discrete amounts of the formulation, such as packaged tablets, capsules, or powder in vials or ampoules. Furthermore, a unit dosage form can be the capsule, tablet, sachets, or lozenges themselves, or can be a packaged form of any of these dosage forms in suitable quantities.

[0039] "Therapeutic effective amount" is the amount of the compound of the present invention that improves the symptoms of the disease when administered to a patient. Therapeutic effective amount is intended to include the amount of a single compound or a combination of a compound and other active ingredients that can effectively treat, improve or reduce the severity of osteoporosis.

[0040] The compounds of this disclosure or their pharmaceutically acceptable salts are administered in therapeutically effective amounts, which will vary depending on a number of factors, including the activity of the specific compound used, the metabolic stability and duration of action of the compound, age, weight, general health condition, sex, diet, administration pattern and time, excretion rate, drug combination, severity of the specific disease state, and the subject receiving treatment. The compounds of this invention can be administered to patients at dose levels ranging from about 0.1 to about 1,000 mg per day. For a normal adult weighing about 70 kg, a dose ranging from about 0.01 to about 100 mg per kg of body weight per day is an example. However, the specific dose used may vary. For example, the dose may depend on a number of factors, including the patient's request, the severity of the condition being treated, and the pharmacological activity of the compound used. Determining the optimal dose for a particular patient is well known to those skilled in the art.

[0041] In one embodiment, the dosage form of the compound of the present invention may contain 1000, 500, 400, 300, 250, 200, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01 mg or less of the compound. In one embodiment, the pharmaceutical dosage form for treating osteoporosis provided by the present invention contains an active substance in the range of 0.01 mg to 1000 mg.

[0042] In one embodiment, the dosage of the medicament for treating osteoporosis provided by the present invention is between 0.01 mg and 1000 mg / kg body weight. Attached Figure Description

[0043] Figure 1 The results showed that NVP-BHG712 had no cytotoxicity against bone marrow mononuclear cells (BMM).

[0044] Figure 2 shows the effect of NVP-BHG712 on osteoclast differentiation of BMM. A is an image of osteoclasts detected by TRAP staining; B is a statistical graph of osteoclast count results.

[0045] Figure 3A The study showed that BMMs cells were treated with 0, 0.1, 0.2, and 0.4 μM NVP-BHG712 for 6 days, and the changes in bone resorption area were analyzed.

[0046] Figure 3B The images show BMMs cells treated with 0.4 μM NVP-BHG712 for 0, 1-3, 3-5, 5-6, and 1-6 days, with TRAP staining used to observe osteoclast formation.

[0047] Figure 4The results showed that NVP-BHG712 inhibited the expression of CTSK protein and mRNA in osteoclasts. (a) Western blot analysis of CTSK protein expression levels and statistical graphs in BMMs cells induced by RANKL and cultured with NVP-BHG712 for 4 days. (b) Western blot analysis of CTSK protein expression and statistical graphs in BMMs cells induced by RANKL and cultured with 0.1, 0.2, and 0.4 μM NVP-BHG712 for 4 days. (c) Western blot analysis of CTSK protein expression and statistical graphs in BMMs cells induced by RANKL and cultured with 0.4 μM NVP-BHG712 for 1, 2, 3, and 4 days. (Group 1: BMMs cells without induction; Group 2: BMMs cells induced by RANKL; Group 3: BMMs cells induced by RANKL + cultured with 0.4 μM NVP-BHG712; data are the mean ± SD of three independent experiments. Compared with Group 2, P<0.05, P<0.01, P<0.001; n=3).

[0048] Figure 5A Western blotting showed that BMMs cells induced by RANKL and cultured for 4 days with 0.4 μM NVP-BHG712 expressed MMP9, CTR, and TRAP proteins.

[0049] Figure 5B The results showed that qPCR was used to detect the mRNA expression of MMP9, CTR, and TRAP in BMMs cells induced by RANKL and cultured for 4 days with 0.4 μM NVP-BHG712.

[0050] Figure 6 The following diagram illustrates how NVP-BHG712 alleviates bone loss in osteoporosis model mice: A is a two-dimensional structural diagram of the femoral trabeculae in osteoporosis model mice treated with NVP-BHG712, and B is a statistical graph of bone histometry parameters in mice.

[0051] Figure 7 The study showed that NVP-BHG712 inhibited bone resorption in ovariectomized mice. (a) Effect of NVP-BHG712 administered by gavage at doses of 5, 10, 20, and 40 mg / kg for 5 weeks on serum TRACP-5b in ovariectomized mice. (b) Effect of NVP-BHG712 administered by gavage at doses of 5, 10, 20, and 40 mg / kg for 5 weeks on serum ALP activity in ovariectomized mice (compared to the OVX group). P<0.05, P<0.01, P<0.001; n=5). Detailed Implementation

[0052] The following will further illustrate the essence and beneficial effects of the present invention with reference to embodiments. These embodiments are only used to illustrate the present invention and are not intended to limit the present invention.

[0053] Example 1: Methods and Materials

[0054] Isolation and purification of bone marrow mononuclear cells (BMMs)

[0055] (1) Eight-week-old C57BL / 6J mice were sacrificed and soaked in 75% ethanol for 5 minutes. The femurs and tibias were removed from both sides.

[0056] (2) Cut off the epiphyseal ends on both sides and use a 1ml syringe to flush out the bone marrow into a culture dish.

[0057] (3) Use a 1ml pipette to repeatedly aspirate to obtain a single-cell suspension. Filter the suspension through a 300-mesh nylon membrane into a 15ml centrifuge tube, centrifuge at 1200rpm for 5min, and discard the supernatant.

[0058] (4) Add 3 ml of red blood cell lysis buffer, mix well, and incubate at room temperature for 3 min. Add 6 ml of complete culture medium to neutralize the lysis buffer, centrifuge at 1200 rpm for 5 min, and discard the supernatant.

[0059] (5) Add 5 ml of MEM complete culture medium to suspend the cells, centrifuge at 1200 rpm for 5 min, and discard the supernatant. Repeat the washing once.

[0060] (6) Resuspend cells in α-MEM complete medium, add mouse M-CSF to a final concentration of 5 ng / ml, and the cell concentration is 1~5×10⁻⁶. 7 / ml. Cells were seeded in culture dishes and incubated overnight at 37°C in a 5% CO2 cell incubator. Mouse BMMs were obtained.

[0061] Preparation of a mouse model of osteoporosis after oophorectomy

[0062] Eight-week-old C57BL / 6J mice were fasted for 12 hours and anesthetized preoperatively with sodium pentobarbital via intraperitoneal injection. Hair was removed 3 cm below the costal margin on both sides of the spine. Two 1 cm incisions were made 1 cm below the costal margin on each side of the spine. The ovary was located, the connection between the ovary and fallopian tube was ligated, the ovary was removed, and the incisions were sutured. 30,000 units of penicillin per mouse were injected intraperitoneally to prevent infection. In the sham-operated group, the ovary was located using the same method, and fat of corresponding size to the ovary was removed. Postoperatively, mice were placed on an electric blanket for rewarming for 1-2 hours, after which they were returned to their cages.

[0063] Bone resorption test

[0064] Bone resorption lacunae on bone fragments are a direct result of bone resorption and the most reliable indicator of bone resorption function in vitro. Osteoclast bone resorption function can be evaluated by measuring the area of ​​these lacunae, which reflects the area of ​​bone resorption.

[0065] (1) Resuspend BMMs in α-MEM complete medium and adjust the cell concentration to 5×10⁻⁶. 4 / ml. 400 μl of the solution was seeded into bone plates, and mouse M-CSF and RANKL were added to a final concentration of 50 ng / ml and 50 ng / ml, respectively, to establish an in vitro osteoclast differentiation and maturation culture system. Different concentrations of the test compound, such as NVP-BHG712, were added to this osteoclast differentiation and maturation system, and the cells were incubated statically at 37°C in a 5% CO2 cell culture incubator.

[0066] (2) Change the medium every 48 hours and continue culturing for 6 days.

[0067] (3) When the cells are cultured for 7 days, the culture medium is aspirated and the cells are washed 3 times with PBS.

[0068] (4) Add PBS containing 10% hypochlorous acid to the bone plate and let it stand for 10 minutes.

[0069] (5) Discard the PBS containing 10% hypochlorous acid and wash 3 times with PBS.

[0070] (6) Air dry the bone plate.

[0071] (7) Observe the area of ​​the bone lacunae under a light microscope and take pictures.

[0072] (8) The bone resorption function can be reflected by analyzing the area of ​​the bone lacunae formed by the bone plates.

[0073] Antibodies used

[0074] Antibody name company Item number Rabbit anti-Cathepsin K polyclonal antibody Abcam ab19027 Rabbit anti-MMP9 monoclonal antibody Abcam Ab76003 Rabbit anti-CT-R polyclonal antibody Abcam ab11042 Rabbit anti-TRAP1 polyclonal antibody Invitrogen PA5-27596 Mouse anti-β-actin monoclonal antibody PTG 66009-1-Ig HRP-labeled goat anti-rabbit IgG secondary antibody CST #7074 HRP-labeled equine anti-mouse IgG secondary antibody CST #7076 Rabbit anti-CSF-1-R monoclonal antibody Abcam ab254357 Mouse anti-RANK monoclonal antibody Abcam ab13918 Alexa Fluor 594-labeled goat anti-rabbit IgG (H+L) secondary antibody Spark Jade EF0011 Alexa Fluor 594-labeled goat anti-mouse IgG (H+L) secondary antibody Spark Jade EF0010

[0075] Primers or probes used

[0076]

[0077] The TRAP staining kit was purchased from Wuhan Saive Biotechnology Co., Ltd., China.

[0078] The Alkaline phosphatase assay kit was purchased from Nanjing Jiancheng Biotechnology Research Institute Co., Ltd., China.

[0079] Example 2: CCK-8 assay for NVP-BHG712 drug toxicity:

[0080] 1. Preparation of NVP-BHG712: NVP-BHG712 (purity 99.53%) was purchased from Shanghai Selleckchem Co., Ltd. Weigh 503.48 μg and dissolve it in 1000 μl of dimethyl sulfoxide to prepare a stock solution of NVP-BHG712 with a final concentration of 1 mM. Then, it was diluted with culture medium to different concentrations of working solution.

[0081] 2. Cytotoxicity of NVP-BHG712 against bone marrow monocytes (BMMs)

[0082] BMMs were resuspended in α-MEM complete medium containing 50 ng / ml M-CSF, and the cell concentration was adjusted to 5 × 10⁻⁶ cells / ml. 4 / ml, take 100μl and seed it into a 96-well plate, and incubate it statically overnight in a 37°C, 5% CO2 cell culture incubator. Add NVP-BHG712 to the final concentrations of 0, 0.1, 0.2, 0.4, 0.8, and 1.6μM, and incubate for 72h. Replace each well with 90ul of complete culture medium + 10μl of CCK-8 solution, and incubate at 37°C for 1-2h. Measure the absorbance at 450nm using a microplate reader.

[0083] See results Figure 1 The results showed that 0.1-1.6 μM NVP-BHG712 had no cytotoxic effect on BMMs. Furthermore, the experimental results revealed that concentrations of NVP-BHG712 above 3.2 μM affected the normal proliferation of BMMs.

[0084] Example 3: Effect of NVP-BHG712 on osteoclast differentiation of BMMs

[0085] 1. The inhibitory effect of NVP-BHG712 on osteoclast differentiation of BMMs was verified by tartrate-resistant acid phosphatase (TRAP) staining experiment.

[0086] BMMs were resuspended in α-MEM complete medium and the cell concentration was adjusted to 5 × 10⁻⁶. 4 / ml. 100 μl of the culture medium was seeded into 96-well plates, and mouse M-CSF and RANKL were added to a final concentration of 50 ng / ml and 50 ng / ml, respectively, to establish an in vitro osteoclast differentiation and maturation culture system. NVP-BHG712 was added to this osteoclast differentiation and maturation system at final concentrations of 0, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 μM, respectively, and the cells were incubated statically at 37°C in a 5% CO2 cell culture incubator. The medium was changed every 48 h, and the cells were cultured for another 5–6 days. The culture medium was discarded, and the cells were washed twice with PBS. TRAP staining was performed, and osteoclasts were counted. 100 μl of fixative was added to each well, and the cells were fixed at room temperature for 20 min. The cells were washed three times with PBS. 100 μl of permeabilization buffer was added to each well, and the cells were incubated for 5 min. After permeabilization, the cells were washed three times with PBS. 100 μl of chromogenic substrate was added to each well. Incubate the 96-well plate at 37°C in the dark for 60–90 min. Wash with deionized water, discard the supernatant, and repeat 3 times. Add 100 μl of methyl green solution to each well, covering the bottom of the well, and stain the cell nuclei for 5 min. After staining, wash 3 times with deionized water, discard the supernatant, and air dry. Count osteoclasts under a microscope (giant cells with ≥3 nuclei are osteoclasts).

[0087] TRAP staining is a standard staining method for detecting osteoclast viability. For example... Figure 2A and Figure 2B As shown, TRAP staining confirmed that treatment with 0.4-1.6 μM NVP-BHG712 reduced the osteoclast differentiation capacity of BMMs. Figure 2A The image shows an osteoclast detected by TRAP staining 6 days after induction. The larger and more numerous the multinucleated giant cells, the more obvious the osteoclast. Figure 2B This is a statistical chart showing the results of osteoclast count.

[0088] 2. The osteoclast bone resorption function can be evaluated by detecting the area of ​​the bone lacunae in the bone plate, which reflects the bone resorption area.

[0089] To observe the effect of NVP-BHG712 on osteoclast bone resorption function, different concentration gradients (0, 0.1, 0.2, 0.4 μM) of NVP-BHG712 were used to treat RANKL-induced osteoclasts for 6 days. Bone plate resorption assays showed that only the M-CSF group showed no obvious bone resorption lacunae, while under RANKL induction, BMMs differentiated into mature osteoclasts with bone resorption function, producing irregularly shaped bone resorption lacunae of varying sizes with clear boundaries on the bone plate. At this stage, the area of ​​the bone resorption lacunae on the bone plate was approximately 5.66 mm². 2 With the addition of NVP-BHG712, the area of ​​bone resorption depressions was significantly reduced. Statistical results showed that the area of ​​bone resorption in the group with 0.1 μM NVP-BHG712 was 5.07 mm². 2Compared to the normal induction group, the bone resorption area decreased by approximately 10%; however, as the concentration of NVP-BHG712 increased to 0.2 μM, the bone resorption area was 6.03 mm². 2 When NVP-BHG712 was added at a concentration of 0.4 μM, the bone resorption area was only 0.80 mm². 2 It was only 14.13% of the normal induction group. The results are as follows... Figure 3A As shown, this indicates that NVP-BHG712 can significantly inhibit the bone resorption function of osteoclasts differentiated from BMMs.

[0090] To further investigate the effects of NVP-BHG712 on the stages of osteoclast differentiation, 0.4 μM NVP-BHG712 was added to RANKL-stimulated bone marrow mesenchymal stem cells (BMMs) at different stages of osteoclast differentiation (early, middle, and late stages). The results were observed by TRAP staining and F-actin fluorescence assays. The specific groups were as follows: control group, only M-CSF + RANKL were added; early stage group, RANKL was added, and 0.4 μM NVP-BHG712 was added 1-3 days later; middle stage group, RANKL was added, and 0.4 μM NVP-BHG712 was added 3-5 days later; late stage group, RANKL was added, and 0.4 μM NVP-BHG712 was added 5-6 days later; early-middle-late stage group, RANKL was added, and 0.4 μM NVP-BHG712 was added 1-6 days later. TRAP staining results showed that in the early, middle, and early-middle-late stages, the addition of NVP-BHG712 significantly inhibited osteoclast activity (P<0.001). The number of TRAP-positive multinucleated giant cells was significantly reduced compared to the control group, with cells being singular and rarely fused. While the addition of NVP-BHG712 in the late stage showed some osteoclast inhibition, the effect was not as significant as that of early intervention. The results are as follows... Figure 3B As shown, this indicates that NVP-BHG712 mainly acts on the early stages of osteoclast differentiation.

[0091] F-actin assays revealed that BMMs, induced by RANKL, differentiated into multinucleated giant cells with F-actin loops, with a F-actin-to-osteoclast ratio of 7.83%. However, when 0.4 μM NVP-BHG712 was added in the early and early-mid-late stages, the number of F-actin loops was significantly reduced compared to the normal osteoclast-induced group (P<0.05), with F-actin-to-osteoclast ratios of 1.48% and 0.41%, respectively. When NVP-BHG712 was added in the mid- and late-stage stages, the reduction in F-actin levels was not significant compared to the normal induction group, with F-actin-to-osteoclast ratios of 2.76% and 5.88%, respectively. These results indicate that NVP-BHG712 primarily inhibits RANKL-induced osteoclast-induced F-actin formation in the early stage.

[0092] In summary, NVP-BHG712 inhibits osteoclast function, primarily acting on the early stages of osteoclast differentiation.

[0093] Example 4: NVP-BHG712 inhibits CTSK protein expression in osteoclasts.

[0094] CTSK expression in BMM cells was detected using Western blotting. To further investigate the effects of NVP-BHG712 concentration and treatment time on CTSK protein, cells were treated with 0, 0.1, 0.2, and 0.4 μM NVP-BHG712 for 4 days under RANKL-induced conditions. Cells were then collected, proteins were extracted, and CTSK protein expression was detected by Western blotting. Additionally, cells were cultured for 1, 2, 3, and 4 days under RANKL-induced conditions with a final concentration of 0.4 μM NVP-BHG712. Cells were then collected, proteins were extracted, and CTSK protein expression was detected by Western blotting.

[0095] The results are as follows Figure 4 As shown, the addition of 0.4 μM NVP-BHG712 to BMMs cells and culturing for 4 days significantly reduced RANKL-induced CTSK protein expression (P<0.05). Figure 4 a); Adding 0.1, 0.2, and 0.4 μM NVP-BHG712 to the BMMs culture system significantly decreased CTSK protein expression (P<0.05). Figure 4 b); After adding 0.4 μM NVP-BHG712 to BMMs cells, there was no significant change in CTSK protein expression after 1 and 2 days of culture, but CTSK protein expression decreased significantly after 3 and 4 days of culture (P<0.05). Figure 4 c).

[0096] In summary, NVP-BHG712 inhibits the expression of CTSK protein in osteoclasts.

[0097] Example 5: NVP-BHG712 inhibits the expression of osteoclast differentiation-related genes.

[0098] To investigate the effects of NVP-BHG712 on osteoclast differentiation-related genes, BMMs cells were induced with RANKL, and NVP-BHG712 was added to a final concentration of 0.4 μM. After culturing for 4 days, cells were collected, proteins were extracted, and the expression of MMP9, CTR, and TRAP proteins was detected by Western blotting. The expression of MMP9, CTR, and TRAP mRNA was detected by qPCR.

[0099] The results showed that after RANKL induction, the expression of MMP9, CTR, and TRAP proteins and mRNA in BMMs increased. After culture with 0.4 μM NVP-BHG712, the expression of MMP9 and CTR proteins decreased significantly (P<0.01). Figure 5A MMP9 and CTR mRNA expression were significantly decreased (P<0.05). Figure 5B NVP-BHG712 had no significant effect on the protein and mRNA expression of TRAP, suggesting that NVP-BHG712 can inhibit the RANKL-induced increase in the expression of MMP9 and CTR proteins and mRNA.

[0100] Similarly, 0.4 μM NVP-BHG712 was added to the RANKL-induced BMM cell culture system, and the cells were cultured for 4 days. Cells were then collected, RNA was extracted, and qPCR was used to detect the mRNA levels of IP3R1, IP3R2, IP3R3, NFATC1, OC-STAMP, DC-STAMP, Atp6v1c1, and αv-integrin. qPCR results showed that after 4 days of RANKL induction, osteoclast function-related genes NFATC1, OC-STAMP, DC-STAMP, and Atp6v1c1 were upregulated; calcium oscillation-related genes IP3R1, IP3R2, and IP3R3 were upregulated; and bone resorption-related gene αv-integrin was upregulated. After 4 days of culture with NVP-BHG712, NVP-BHG712 inhibited irregular and transient calcium oscillations, but had no significant effect on prolonged, continuous calcium oscillations. In addition, it was found that NVP-BHG712 inhibited the upregulation of OC-STAMP, a gene related to osteoclast function induced by RANKL.

[0101] Example 6: NVP-BHG712 slows down bone loss in ovariectomized mice.

[0102] Preparation of NVP-BHG712: Weigh 2 mg of NVP-BHG712 and dilute it with 1 ml of 0.5% CMC-Na to make a 20 mg / kg NVP-BHG712 solution.

[0103] A mouse model of osteoporosis (OVX) was established by ovariectomy. Mice were administered NVP-BHG712 by gavage one week post-surgery, 200 μl every 3 days for 5 weeks. At week 6, femurs were harvested for microcomputed tomography (μCT) analysis. Statistical analysis: ANOVA was used to compare means between groups.

[0104] Bone microstructure degeneration, thinning and sparsening of trabeculae, and decreased bone mass are characteristic features of osteoporosis. μCT scanning was used to observe changes in trabecular density and quantity after oophorectomy from a two-dimensional perspective. Figure 6The results showed that NVP-BHG712 could alleviate bone loss in osteoporosis model mice. Figure 6 A shows a two-dimensional structural diagram of femoral trabeculae in osteoporosis model mice treated with NVP-BHG712. The diagram shows that the Sham group had a large number and high density of trabeculae, while the OVX group had a reduced number and low density of trabeculae. Compared with the OVX group, the density of trabeculae increased after administration of NVP-BHG712.

[0105] Figure 6 B represents a statistical graph of biometric parameters of mouse bone tissue. Statistical analysis of the biometric parameters of mouse bone tissue, including bone mineral density (BMD), bone surface area / bone volume ratio (BS / BV), bone surface area / tissue volume ratio (BS / TV), bone surface area / volume ratio (BV / TV), trabecular bone number (Tb.N), trabecular bone pattern factor (Tb.Pf), trabecular bone separation (Tb.Sp), and trabecular bone thickness (Tb.Th), shows that compared with the Sham group, after bilateral ovariectomy, mice exhibited significantly decreased BMD (P<0.05), BMD / tissue volume ratio (P<0.05), BV / volume ratio (P<0.05), trabecular bone number (P<0.05), and trabecular bone thickness (P<0.05), and significantly reduced BMD / BV ratio. Compared with OVX, the NVP-BHG712 group showed significantly increased bone mineral density (P<0.05), bone surface area / tissue volume ratio (P<0.05), bone surface area / volume ratio (P<0.05), trabecular bone number (P<0.05), and trabecular bone thickness (P<0.05), and significantly increased bone surface area / bone volume ratio (P<0.05), trabecular bone pattern factor (P<0.05), and decreased trabecular bone separation (P<0.05). These results suggest that NVP-BHG712 inhibits the destruction of trabecular microstructure after ovariectomy. Therefore, NVP-BHG712 has a protective effect against bone loss in ovariectomized mice.

[0106] To further validate the μCT results, mouse femurs were fixed with 4% paraformaldehyde, decalcified with decalcifying solution, and sectioned in paraffin for H&E staining. The results showed that compared to the Sham group, the OVX group had a significantly reduced trabecular bone area (P<0.05); compared to the OVX group, the NVP-BHG712 treatment group had an increased trabecular bone area (P<0.05). This suggests that NVP-BHG712 alleviates the reduction in trabecular bone in mice induced by ovariectomy.

[0107] Example 7: NVP-BHG712 attenuates bone resorption in ovariectomized mice.

[0108] To investigate whether NVP-BHG712 has an effect on bone metabolism, two serum markers were selected: TRACP-5b and alkaline phosphatase (ALP). The serum TRACP-5b level in ovariectomized mice was detected by ELISA, and ALP activity was detected by microplate reader. Mice were randomly assigned to groups one week after surgery: the Sham group received CMC-Na via gavage for 5 weeks after sham surgery; the OVX group received CMC-Na via gavage for 5 weeks after ovariectomy; group A received 5 mg / kg NVP-BHG712 via gavage for 5 weeks after ovariectomy; group B received 10 mg / kg NVP-BHG712 via gavage for 5 weeks after ovariectomy; group C received 20 mg / kg NVP-BHG712 via gavage for 5 weeks after ovariectomy; and group D received 40 mg / kg NVP-BHG712 via gavage for 5 weeks after ovariectomy.

[0109] ELISA results as follows Figure 7 As shown, compared with the Sham group, the serum TRACP-5b level in the OVX group mice was significantly increased (P<0.001). Figure 7 a); Microenzyme labeling results showed that, compared with the Sham group, the serum ALP activity of mice in the OVX group was significantly reduced (P<0.01). Figure 7 (b) This indicates successful model establishment. After 5 weeks of gavage administration of 5, 10, 20, and 40 mg / kg NVP-BHG712, compared with the OVX group, the TRACP-5b content was significantly decreased (P<0.01), and the ALP activity was significantly increased (P<0.01), indicating that NVP-BHG712 inhibited the increase in serum TRACP-5b levels and promoted serum ALP activity after ovariectomy in mice.

[0110] In summary, an osteoporosis model can be successfully established in female mice 6 weeks after ovariectomy, simulating postmenopausal osteoporosis; NVP-BHG712 can effectively reduce bone resorption in ovariectomized mice and slow down the loss of high-turnover bone in mice after ovariectomy.

[0111] Example 8: NVP-BHG712 showed no significant toxicity in ovariectomized mice.

[0112] To investigate whether long-term NVP-BHG712 treatment produces effects other than bone damage, ovariectomized mice were treated with 5, 10, 20, and 40 mg / kg NVP-BHG712 by gavage for 5 weeks. The results included changes in body weight, cardiac index (heart weight / body weight), spleen index (spleen weight / body weight), kidney index (kidney weight / body weight), grip strength index (forelimb grip strength / body weight), and cardiac hypertrophy (heart weight / tibia length).

[0113] The results showed that, compared with the OVX group, there were no significant differences in weight gain among the 5, 10, 20, and 40 mg / kg NVP-BHG712 dose groups, indicating that NVP-BHG712 did not affect weight gain in ovariectomized mice at this dose. Compared with the OVX group, there were no significant differences in cardiac, spleen, and kidney indices among the 5, 10, 20, and 40 mg / kg NVP-BHG712 dose groups, indicating that NVP-BHG712 did not affect organ indices in ovariectomized mice at this dose. Compared with the OVX group, there were no significant differences in forelimb muscle strength among the 5, 10, 20, and 40 mg / kg NVP-BHG712 dose groups, indicating that NVP-BHG712 did not affect grip strength index in ovariectomized mice at this dose. Compared with the OVX group, the 5, 10, 20, and 40 mg / kg NVP-BHG712 dose groups showed no significant differences in forelimb muscle strength, indicating that NVP-BHG712 did not affect grip strength index in ovariectomized mice at this dose. In the NVP-BHG712 dose group, there was no significant difference in cardiac hypertrophy in mice, suggesting that at this dose, NVP-BHG712 does not affect cardiac hypertrophy in ovariectomized mice.

[0114] In conclusion, NVP-BHG712 showed no significant toxicity in ovariectomized mice at doses of 5, 10, 20, and 40 mg / kg.

[0115] The foregoing description of the present invention should not be construed as limiting it. Unless otherwise indicated, the present invention will be practiced using conventional techniques such as organic chemistry, polymer chemistry, and biotechnology, and it is obvious that the invention can be implemented in other ways besides those specifically described in the foregoing description and examples. Other aspects and modifications within the scope of the invention will be apparent to those skilled in the art. Many changes and variations are possible based on the teachings of the present invention, and therefore fall within the scope of the invention.

[0116] Unless otherwise specified, the temperature unit "degree" in this article refers to Celsius, or °C.

Claims

1. Use of NVP-BHG712, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of osteoporosis, wherein the medicament comprises a pharmaceutically acceptable carrier, excipient or diluent, wherein the osteoporosis is postmenopausal osteoporosis.

2. Use according to claim 1, wherein the dose of NVP-BHG712, or a pharmaceutically acceptable salt thereof, is between 0.1 mg and 50 mg.

3. Use according to claim 2, wherein the dose of NVP-BHG712, or a pharmaceutically acceptable salt thereof, is 50, 40, 20, 10, 5 or 1 mg.

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