WO2024078628A1 - 治疗骨质疏松的方法和药物组合物 - Google Patents

治疗骨质疏松的方法和药物组合物 Download PDF

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WO2024078628A1
WO2024078628A1 PCT/CN2023/124591 CN2023124591W WO2024078628A1 WO 2024078628 A1 WO2024078628 A1 WO 2024078628A1 CN 2023124591 W CN2023124591 W CN 2023124591W WO 2024078628 A1 WO2024078628 A1 WO 2024078628A1
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bhg712
nvp
pharmaceutically acceptable
acceptable salt
bone
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PCT/CN2023/124591
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English (en)
French (fr)
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赵跃然
陈子江
宁云娜
马金龙
曹永芝
路钢
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山东大学
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    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/12—Drugs for genital or sexual disorders; Contraceptives for climacteric disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis

Definitions

  • the present invention relates to the field of disease treatment and medicine, and in particular to a method for treating osteoporosis and a related pharmaceutical composition.
  • Osteoporosis is a systemic metabolic disease caused by an imbalance in bone remodeling due to bone absorption exceeding bone formation. Osteoporosis can be divided into three categories according to the cause: primary osteoporosis, secondary osteoporosis and idiopathic osteoporosis.
  • Postmenopausal osteoporosis belongs to primary osteoporosis and occurs 5 to 10 years after menopause.
  • the maintenance of human bone homeostasis is mainly completed by osteoclasts and osteoblasts. Osteoclasts are responsible for bone absorption, and osteoblasts are responsible for bone formation.
  • Bone marrow mononuclear cells (BMMs) are a type of hematopoietic stem cell that can differentiate into osteoclasts. When bone homeostasis is unbalanced and the number of osteoclasts formed by BMMs increases, bone metabolic diseases such as osteoporosis will occur, which seriously affect the patient's living standards.
  • osteoporosis may be caused by a variety of reasons, drug intervention needs to be implemented purposefully, but currently there is no drug that can effectively prevent and treat osteoporosis caused by different reasons.
  • NVP-BHG712 is a specific EphB4 inhibitor that inhibits VEGFR2 and dose-dependently inhibits RTK autophosphorylation in A375 stable melanoma cells.
  • EphB4 is one of the important members of the EPH family in the tyrosine protein kinase receptor family. EphB4 plays an important regulatory role in the occurrence and development of tumors, promotes the metastasis of various tumors, and has an important regulatory effect on tumor prognosis; EphB4 also regulates the biological behavior of endothelial cells and plays a key role in the formation of embryonic blood vessels; EphB4 gene knockout mice show obvious vascular development disorders. There are no reports on the effect of NVP-BHG712 on osteoclasts. Whether it has an inhibitory effect on osteoclast formation and function and whether NVP-BHG712 has a therapeutic effect on bone metabolic diseases such as postmenopausal osteoporosis remains unknown.
  • AMG-900 is a highly selective pan-Aurora kinase inhibitor targeting Aurora A/B/C.
  • AMG-900 is a novel ATP-competitive aminophthalazine small molecule aurora kinase inhibitor.
  • AMG-900 inhibits aurora-A and -B autophosphorylation and inhibits phosphorylation of histone H3 on Ser, a proximal substrate of aurora-B.
  • ONT-380 also known as Tucatinib, Irbinitinib, ARRY-380, is an orally active, reversible, ATP-competitive ErbB2 small molecule inhibitor. ONT-380 can inhibit tumor growth in a variety of HER2-dependent tumor xenograft models and has potential anti-tumor activity.
  • KD025 is an orally active, selective ROCK2 inhibitor.
  • KD025 200 mg/kg, p.o.
  • KD025 200 mg/kg, i.p.
  • KD025 200 mg/kg, i.p.
  • KD025 150 mg/kg, i.p. or p.o.
  • KD025 effectively alleviates chronic graft-versus-host disease.
  • This field also needs further research on the physiological mechanisms and biochemical pathways that affect osteoporosis, as well as the discovery of new drugs for use in the treatment of osteoporosis.
  • the present invention first discovered that NVP-BHG712, AMG-900, ONT-380, and KD025 can effectively inhibit the action of osteoclasts, not only inhibiting the formation of osteoclasts, but also inhibiting the bone resorption function of osteoclasts, and can inhibit the formation of osteoporosis in mammals.
  • the present invention thus provides a new drug for treating osteoporosis.
  • a method for treating osteoporosis comprising administering to a patient in need of such treatment a therapeutically effective amount of one of the following compounds: NVP-BHG712, AMG-900, ONT-380, KD025, or a pharmaceutically acceptable salt thereof.
  • a method for treating osteoporosis comprising administering one of the following compounds: NVP-BHG712, AMG-900, ONT-380, KD025, or a pharmaceutically acceptable salt thereof, the method being an in vitro non-therapeutic method.
  • the method can be used to treat isolated tissues or cells in culture medium for studying osteoporosis.
  • the compound administered is NVP-BHG712 or a pharmaceutically acceptable salt thereof, wherein NVP-BHG712 is a compound having the following formula:
  • NVP-BHG712 or a pharmaceutically acceptable salt thereof is administered at a dose of between 0.1 mg and 50 mg. In one embodiment of the present 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.
  • the compound administered is AMG-900 or a pharmaceutically acceptable salt thereof, wherein AMG-900 is a compound having the following formula:
  • AMG-900 or a pharmaceutically acceptable salt thereof is administered at a dose between 0.1 mg and 50 mg. In another embodiment of the present invention, AMG-900 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, AMG-900 or a pharmaceutically acceptable salt thereof is administered at a dose of 50, 20, 10 or 5 mg/kg body weight.
  • the compound administered is ONT-380 or a pharmaceutically acceptable salt thereof, wherein ONT-380 is a compound having the following formula:
  • ONT-380 or a pharmaceutically acceptable salt thereof is administered at a dose between 10 mg and 1000 mg.
  • ONT-380 or a pharmaceutically acceptable salt thereof is administered at a dose of 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 mg/kg body weight.
  • ONT-380 or a pharmaceutically acceptable salt thereof is administered at a dose of 300, 250, 200 or 100 mg/kg body weight.
  • the compound administered is KD025 or a pharmaceutically acceptable salt thereof, wherein KD025 is a compound having the following formula:
  • KD025 or a pharmaceutically acceptable salt thereof is administered at a dose between 1 mg and 1000 mg.
  • KD025 or a pharmaceutically acceptable salt thereof is administered at a dose of 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 mg/kg body weight.
  • KD025 or a pharmaceutically acceptable salt thereof is administered at a dose of 300, 200, 100 or 50 mg/kg body weight.
  • the compound or a pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier, excipient or diluent.
  • the compound or a pharmaceutically acceptable salt thereof is administered every day or every 2-3 days.
  • the medicament comprises a pharmaceutically acceptable carrier, excipient or diluent.
  • the dosage of the compound or a pharmaceutically acceptable salt thereof is between 0.01 mg and 1000 mg.
  • a method for inhibiting osteoclast differentiation and/or activity comprising administering to a patient in need of such treatment a therapeutically effective amount of one of the following compounds: NVP-BHG712, AMG-900, ONT-380, KD025, or a pharmaceutically acceptable salt thereof.
  • a method for inhibiting osteoclast differentiation and/or activity comprising administering one of the following compounds: NVP-BHG712, AMG-900, ONT-380, KD025, or a pharmaceutically acceptable salt thereof, the method being an in vitro non-therapeutic method.
  • the method can be used to treat isolated tissues or cells in a culture medium.
  • the compound administered is NVP-BHG712 or a pharmaceutically acceptable salt thereof.
  • NVP-BHG712 or a pharmaceutically acceptable salt thereof is administered at a dose between 0.1 mg and 50 mg.
  • 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.
  • the compound administered is AMG-900 or a pharmaceutically acceptable salt thereof.
  • AMG-900 or a pharmaceutically acceptable salt thereof is administered at a dose between 0.1 mg and 50 mg.
  • AMG-900 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, AMG-900 or a pharmaceutically acceptable salt thereof is administered at a dose of 50, 20, 10 or 5 mg/kg body weight.
  • the compound administered is ONT-380 or a pharmaceutically acceptable salt thereof.
  • the compound administered is administered at a dosage between 10 mg ONT-380 or a pharmaceutically acceptable salt thereof is administered at a dose of between 1000 mg and 1000 mg.
  • ONT-380 or a pharmaceutically acceptable salt thereof is administered at a dose of 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 mg/kg body weight. More preferably, ONT-380 or a pharmaceutically acceptable salt thereof is administered at a dose of 300, 250, 200 or 100 mg/kg body weight.
  • the compound administered is KD025 or a pharmaceutically acceptable salt thereof.
  • KD025 or a pharmaceutically acceptable salt thereof is administered at a dose between 1 mg and 1000 mg.
  • KD025 or a pharmaceutically acceptable salt thereof is administered at a dose of 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 mg/kg body weight. More preferably, KD025 or a pharmaceutically acceptable salt thereof is administered at a dose of 300, 200, 100 or 50 mg/kg body weight.
  • the compound or a pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier, excipient or diluent.
  • the compound or a pharmaceutically acceptable salt thereof is administered every day or every 2-3 days.
  • the medicament comprises a pharmaceutically acceptable carrier, excipient or diluent.
  • the dosage of the compound or a pharmaceutically acceptable salt thereof is between 0.01 mg and 1000 mg.
  • the subject in need of the methods and drugs (pharmaceutical compositions) described herein can be a mammal, including a human or a non-human primate such as a monkey.
  • the mammal can also be other animals, such as rats, mice, rabbits, pigs, dogs, etc.
  • the mammal can be a domestic animal, such as a cat or a dog.
  • Bones are constantly undergoing a process called 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 the bones, and when the body needs it, bone cells called osteoclasts attach to the bone surface and break it down, leaving cavities in the bone. Bone-forming cells called osteoblasts then fill the cavities with an organic matrix called osteoid. The osteoid then spontaneously mineralizes with calcium phosphate to reform hard bone.
  • 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).
  • WHO defines bone mineral density within one standard deviation of normal values or a corresponding T-score of less than ⁇ 1 as acceptable bone mineral density.
  • WHO defines bone mineral density within 1 to 2.5 standard deviations of normal values or a corresponding T-score of ⁇ 1 to ⁇ 2.5 as osteopenia.
  • WHO describes the characteristics of osteoporosis as a more severe form of osteopenia and defines osteoporosis as bone mineral density with a standard deviation of less than 2.5 from normal values or a corresponding T-score of less than ⁇ 2.5.
  • osteoporosis includes postmenopausal osteoporosis (PMPO), which is osteoporosis caused by postmenopausal ovarian decline in women and subsequent estrogen deficiency or emergency withdrawal, which seriously affects the physical and mental health of middle-aged and elderly people.
  • PMPO postmenopausal osteoporosis
  • treat refers to methods involving individuals afflicted with or otherwise at risk of developing osteoporosis, and to any method that affects bone mineral density or structure in an individual, including increasing bone mineral density, slowing the rate or onset of loss of bone mineral density, maintaining normal bone mineral density, or reversing some or all loss or deficiency of bone mineral density in an individual afflicted with osteoporosis.
  • the active ingredients in the pharmaceutical compositions of the present invention can be administered in the form of raw compounds.
  • the active ingredients, optionally in the form of pharmaceutically acceptable salts can be introduced into the pharmaceutical compositions together with one or more adjuvants, excipients, carriers, buffers, diluents and/or other conventional pharmaceutical excipients.
  • Adjuvants include preservatives, wetting agents, suspending agents, sweeteners, flavoring agents, aromatics, emulsifiers and dispersants.
  • protection against the action of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc.
  • isotonic agents such as sugars, sodium chloride, etc.
  • Prolonged absorption of injectable drug forms can be caused by the use of agents that delay absorption (e.g., aluminum monostearate and gelatin).
  • the pharmaceutical composition of the compound may also contain a small amount of auxiliary substances, such as wetting agents or emulsifiers, pH buffering agents, antioxidants, etc., for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylated hydroxytoluene, etc.
  • auxiliary substances such as wetting agents or emulsifiers, pH buffering agents, antioxidants, etc., for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylated hydroxytoluene, etc.
  • a "pharmaceutically acceptable salt” of a compound refers to a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound.
  • Pharmaceutically acceptable salts are non-toxic.
  • Examples of pharmaceutically acceptable acid addition salts include salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; 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, propylene acid, Diacid, 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-naphthalenesul
  • the pharmaceutical composition of the present invention can be administered by any convenient route suitable for the desired therapy.
  • Preferred routes of administration include oral administration, particularly in the form of tablets, capsules, lozenges, powders and liquids; and parenteral administration, particularly cutaneous, subcutaneous, intramuscular and intravenous injections.
  • the pharmaceutical composition 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, a composition suitable for sustained release of the active ingredient can be used.
  • a pharmaceutically acceptable carrier can be a solid or a liquid.
  • Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • a solid carrier can be one or more substances that can also serve as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, binder, preservative, tablet disintegrating agent, or encapsulating material.
  • compositions adapted to give sustained release of the active ingredient may be employed.
  • the pharmaceutical formulation is preferably in unit dosage form.
  • the formulation is subdivided into unit doses containing appropriate quantities of the active ingredient.
  • the unit dosage form can be a packaged formulation containing discrete quantities of the formulation, such as packaged tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or can be a packaged form of the appropriate number of any of these dosage forms.
  • a “therapeutically effective amount” is an amount of a compound of the invention that, when administered to a patient, improves the symptoms of the disease.
  • a therapeutically effective amount is intended to include an amount of a single compound or a combination of compounds and other active ingredients that is effective in treating, ameliorating or reducing the severity of osteoporosis.
  • the compounds of the present disclosure or their pharmaceutically acceptable salts are administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the specific compound used, the metabolic stability and duration of action of the compound, age, body weight, general health, sex, diet, mode and time of administration, excretion rate, drug combination, severity of a particular disease state, and the subject being treated.
  • the compounds of the present invention may be administered to patients at a dosage level ranging from about 0.1 to about 1,000 mg per day. For a normal adult weighing about 70 kg, a dosage ranging from about 0.01 to about 100 mg per kg of body weight per day is an example.
  • the specific dosage used may vary.
  • the dosage may depend on a variety of factors.
  • the dosage of the compound to be used may be determined based on factors including the patient's requirements, the severity of the condition being treated, and the pharmacological activity of the compound being used. Determining the optimal dosage for a particular patient is well within the skill of the art.
  • the dosage form of the compound of the 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.
  • the pharmaceutical dosage form provided by the invention for treating osteoporosis contains between 0.01 mg and 1000 mg of the active ingredient.
  • the dosage of the drug for treating osteoporosis provided by the present invention is between 0.01 mg and 1000 mg/kg body weight.
  • Figure 1 shows that NVP-BHG712 is not cytotoxic to bone marrow mononuclear cells (BMM).
  • Figure 2 shows the effect of NVP-BHG712 on osteoclast differentiation of BMM.
  • A is a photo of osteoclasts detected by TRAP staining;
  • B is a statistical chart of the number of osteoclasts.
  • Figure 3A shows that BMMs cells were treated with 0, 0.1, 0.2, and 0.4 ⁇ M NVP-BHG712 for 6 days, and the changes in bone resorption area were analyzed.
  • Figure 3B shows that BMMs cells were treated with 0.4 ⁇ M NVP-BHG712 for 0, 1-3 days, 3-5 days, 5-6 days, and 1-6 days, and osteoclast formation was observed by TRAP staining.
  • Figure 4 shows that NVP-BHG712 inhibits the expression of CTSK protein and mRNA in osteoclasts.
  • BMMs cells were induced by RANKL and cultured with 0.4 ⁇ M NVP-BHG712 for 1, 2, 3, and 4 days, and Western Blot detection of CTSK protein expression and statistical graph.
  • Figure 5A shows the Western Blot detection of MMP9, CTR, and TRAP protein expression in BMMs cells induced by RANKL and cultured for 4 days with the addition of 0.4 ⁇ M NVP-BHG712.
  • Figure 5B shows the mRNA expression of MMP9, CTR, and TRAP detected by qPCR in BMMs cells induced by RANKL and cultured for 4 days with the addition of 0.4 ⁇ M NVP-BHG712.
  • Figure 6 shows that NVP-BHG712 alleviates bone loss in osteoporosis model mice: A is a two-dimensional structural diagram of femoral trabeculae in osteoporosis model mice treated with NVP-BHG712, and B is a statistical diagram of bone tissue metrology parameters in mice.
  • Figure 7 shows that NVP-BHG712 inhibits bone resorption in ovariectomized mice.
  • (a) Effects of 5, 10, 20, and 40 mg/kg NVP-BHG712 gavage for 5 weeks on serum TRACP-5b in ovariectomized mice.
  • FIG8 shows that AMG-900 is not cytotoxic to bone marrow mononuclear cells (BMM).
  • Figure 9 shows the effect of AMG-900 on osteoclast differentiation of BMM.
  • Figure 9A is a photo of osteoclasts detected by TRAP staining;
  • Figure 9B is a statistical chart of the number of osteoclasts.
  • Figure 10 shows that AMG-900 alleviates bone loss in osteoporosis model mice: A is a two-dimensional structural diagram of femoral trabeculae in osteoporosis model mice treated with NVP-BHG712, and B is a statistical diagram of mouse bone tissue metrology parameters.
  • FIG. 11 shows that ONT-380 is not cytotoxic to bone marrow mononuclear cells (BMM).
  • Figure 12 shows the effect of ONT-380 on osteoclast differentiation of BMM.
  • Figure 12A is a photo of osteoclasts detected by TRAP staining;
  • Figure 12B is a statistical chart of the number of osteoclasts.
  • FIG13 shows that ONT-380 alleviates bone loss in osteoporosis model mice:
  • A is a two-dimensional structural diagram of femoral trabeculae in osteoporosis model mice treated with NVP-BHG712, and B is a statistical diagram of bone tissue metrology parameters in mice.
  • FIG. 14 shows that KD025 is not cytotoxic to bone marrow mononuclear cells (BMM).
  • Figure 15 shows the effect of KD025 on osteoclast differentiation of BMM.
  • Figure 15A is a photo of osteoclasts detected by TRAP staining;
  • Figure 15B is a statistical chart of the number of osteoclasts.
  • FIG16 shows that KD025 alleviates bone loss in osteoporosis model mice:
  • A is a two-dimensional structural diagram of femoral trabeculae in osteoporosis model mice treated with NVP-BHG712, and B is a statistical diagram of bone tissue metrology parameters in mice.
  • BMMs bone marrow mononuclear cells
  • mice M-CSF Resuspend the cells in ⁇ -MEM complete medium, add mouse M-CSF to a final concentration of 5 ng/ml, and the cell concentration is 1-5 ⁇ 10 7 /ml. Inoculate the cells in a culture dish and culture them overnight in a cell culture incubator at 37°C and 5% CO 2 to obtain mouse BMMs.
  • mice Eight-week-old C57BL/6J mice were fasted for 12 hours and anesthetized with sodium pentobarbital injected intraperitoneally before surgery. The hair was removed 3 cm below the rib margin on both sides of the dorsal spine. Two 1 cm incisions were made 1 cm below the rib margin on both sides of the dorsal spine. The ovaries were found, the connection between the ovaries and the fallopian tubes was ligated, the ovaries were removed, the incisions were sutured, and 30,000 units/mouse of penicillin were injected intraperitoneally to prevent infection. The sham operation group used the same method to find the ovaries and remove the fat corresponding to the size of the ovaries. After surgery, the mice were placed on an electric blanket for 1 to 2 hours to rewarm, and then returned to the cage after rewarming.
  • the bone resorption pits on the bone slices are the direct result of bone resorption and are also the most reliable indicator for identifying bone resorption function in vitro.
  • the bone resorption function of osteoclasts can be evaluated by detecting the area of bone pits on the bone slices to reflect the area of bone resorption.
  • the bone absorption function is reflected by analyzing the area of bone pit formation in the bone plate.
  • TRAP staining kit was purchased from Wuhan Saiweier Biotechnology Co., Ltd., China.
  • Alkaline phosphatase assay kit was purchased from Nanjing Jiancheng Bioengineering Institute Co., Ltd., China.
  • NVP-BHG712 (purity 99.53%) was purchased from Shanghai selleckchem Co., Ltd. 503.48 ⁇ g was weighed and dissolved in 1000 ⁇ l dimethyl sulfoxide to prepare NVP-BHG712 mother solution with a final concentration of 1 mM, which was then diluted with culture medium to working solutions of different concentrations.
  • Resuspend BMMs in ⁇ -MEM complete medium containing 50 ng/ml M-CSF to adjust the cell concentration to 5 ⁇ 10 4 /ml take 100 ⁇ l and seed it in a 96-well plate, and incubate it in a cell culture incubator at 37°C and 5% CO 2 for 3 h. Incubate overnight.
  • the tartrate-resistant acid phosphatase (TRAP) staining experiment was used to verify the effect of NVP-BHG712 in inhibiting osteoclast differentiation of BMMs.
  • NVP-BHG712 was added to the final concentrations of 0, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 ⁇ M, respectively, and cultured statically in a cell culture incubator at 37°C and 5% CO 2. Change the medium every 48h, continue to culture for 5-6 days, discard the medium, and add PBS to wash twice.
  • TRAP staining is a standard staining method for osteoclast activity staining. As shown in Figures 2A and 2B, TRAP staining verified that 0.4-1.6 ⁇ M NVP-BHG712 could reduce the osteoclast differentiation ability of BMMs:
  • Figure 2A is a photo of osteoclasts detected by TRAP staining 6 days after induction. The larger the multinuclear giant cells, the more numerous they are, and the more obvious the osteoclasts are;
  • Figure 2B is a statistical chart of the number of osteoclasts.
  • the bone resorption function of osteoclasts can be evaluated by detecting the area of bone pits on the bone plate, which reflects the bone resorption area.
  • NVP-BHG712 In order to observe the effect of NVP-BHG712 on the bone resorption function of osteoclasts, different concentration gradients (0, 0.1, 0.2, 0.4 ⁇ M) of NVP-BHG712 were set to intervene in RANKL-induced osteoclasts and cultured for 6 days. Through the bone plate absorption experiment, it was found that only the M-CSF group had no obvious bone resorption pits, while under the induction of RANKL, BMMs cells differentiated into mature osteoclasts with bone resorption function and produced irregular bone resorption pits on the bone plate, with different sizes and clear boundaries.
  • the area of the absorption pit was about 5.66 mm 2 ; with the addition of NVP-BHG712, the area of the bone absorption pit was significantly reduced.
  • the statistical results showed that the area of bone absorption in the group with 0.1 ⁇ M NVP-BHG712 was 5.07 mm 2 , which was about 10% lower than that in the normal induction group; and as the concentration of NVP-BHG712 increased to 0.2 ⁇ M, the bone absorption area was 6.03 mm 2 .
  • the concentration of NVP-BHG712 was 0.4 ⁇ M, the bone absorption area was only 0.80 mm 2 , which was only 14.13% of that in the normal induction group.
  • the results are shown in Figure 3A, indicating that NVP-BHG712 can significantly inhibit the bone absorption function of osteoclasts differentiated from BMMs.
  • NVP-BHG712 was added to RANKL-stimulated BMMs at different stages of osteoclast differentiation (early, middle and late stages), and the results were observed by TRAP staining experiment and F-actin fluorescence experiment.
  • the specific groups were as follows: control group, only M-CSF+RANKL was added; early group, RANKL was added, and 0.4 ⁇ M NVP-BHG712 was added on days 1 to 3; middle group, RANKL was added, and 0.4 ⁇ M NVP-BHG712 was added on days 3 to 5; late group, RANKL was added, and 0.4 ⁇ M NVP-BHG712 was added on days 5 to 6; early, middle and late groups, RANKL was added, and 0.4 ⁇ M NVP-BHG712 was added on days 1 to 6.
  • TRAP staining results showed that in the early, middle and early, middle and late groups, the addition of NVP-BHG712 had a significant inhibitory effect on osteoclasts (P ⁇ 0.001), and the number of TRAP-positive multinucleated giant cells was significantly reduced compared with the control group, and the cells were single and rarely fused; in the late stage, although NVP-BHG712 had a certain inhibitory effect on osteoclasts, it was not as significant as the early intervention effect.
  • the results are shown in Figure 3B, indicating that NVP-BHG712 mainly acts on the early stage of osteoclast differentiation.
  • NVP-BHG712 inhibits osteoclast function, mainly acting at the early stage of osteoclast differentiation.
  • NVP-BHG712 inhibits the expression of CTSK protein in osteoclasts.
  • Example 5 NVP-BHG712 inhibits the expression of osteoclast differentiation-related genes
  • BMMs cells were induced by RANKL and NVP-BHG712 was added at a final concentration of 0.4 ⁇ M. The cells were cultured for 4 days, and the cells were collected and proteins were extracted. The Western Blot method was used to detect the expression of MMP9, CTR, and TRAP proteins, and the qPCR method was used to detect the expression of MMP9, CTR, and TRAP mRNA.
  • NVP-BHG712 had no significant effect on the protein expression and mRNA expression of TRAP, indicating that NVP-BHG712 can inhibit the increase of MMP9 and CTR protein and mRNA expression induced by RANKL.
  • NVP-BHG712 was added to the RANKL-induced BMMs cell culture system, cultured for 4 days, cells were collected, RNA was extracted, and qPCR was used to detect IP3R1, IP3R2, IP3R3, NFATc1, OC-STAMP, DC-STAMP, Atp6v1c1 and ⁇ v-integrin mRNA.
  • qPCR results showed that after 4 days of RANKL induction, osteoclast function-related genes NFATc1, OC-STAMP, DC-STAMP, Atp6v1c1 were upregulated, calcium oscillation-related genes IP3R1, IP3R2, IP3R3 were upregulated, and bone resorption function-related gene ⁇ v-integrin was upregulated.
  • NVP-BHG712 inhibited irregular calcium oscillations and transient calcium oscillations, but had no obvious effect on long-term continuous calcium oscillations.
  • NVP-BHG712 was found to inhibit the upregulation of osteoclast function-related gene OC-STAMP caused by RANKL.
  • NVP-BHG712 Preparation of NVP-BHG712: Weigh 2 mg NVP-BHG712 and dilute it with 1 ml 0.5% CMC-Na to a 20 mg/kg NVP-BHG712 solution.
  • the ovariectomized OVX osteoporosis mouse model was constructed according to the method described in Example 1.
  • NVP-BHG712 was administered orally once every 3 days, 200 ⁇ l, for 5 weeks.
  • femurs of mice were collected for micro-computed tomography ( ⁇ CT) analysis.
  • ⁇ CT micro-computed tomography
  • Figure 6A is a two-dimensional structure of femoral trabeculae in osteoporosis model mice treated with NVP-BHG712, showing that the Sham group had a large number of trabeculae and high density, while the OVX group had a reduced number of trabeculae and low density; compared with the OVX group, the trabecular density increased after NVP-BHG712 administration.
  • Figure 6B is a statistical diagram of mouse bone tissue metrology parameters.
  • mouse bone tissue morphological dosimetry parameters such as bone density (BMD), bone surface area/bone volume ratio (BS/BV), bone surface area/tissue volume ratio (BS/TV), bone surface/volume ratio (BV/TV), trabecular number (Tb.N), trabecular pattern factor (Tb.Pf), trabecular separation (Tb.Sp) and trabecular thickness (Tb.Th)
  • BMD bone density
  • BS/BV bone surface area/bone volume ratio
  • BS/TV bone surface area/tissue volume ratio
  • BV/TV bone surface/volume ratio
  • Tb.N trabecular pattern factor
  • Tb.Sp trabecular separation
  • Tb.Th trabecular thickness
  • NVP-BHG712 inhibits the destruction of trabecular microstructure after ovariectomy.
  • the above results indicate that NVP-BHG712 has a protective effect on bone loss in ovariectomized mice.
  • mice were fixed with 4% paraformaldehyde, decalcified with decalcified solution, and paraffin-sectioned for H&E staining.
  • the results showed that compared with the Sham group, the trabecular area of the OVX group was significantly reduced (P ⁇ 0.05); compared with the OVX group, the trabecular area of the NVP-BHG712-treated group increased (P ⁇ 0.05). This suggests that NVP-BHG712 slows down the reduction of trabecular bone in mice caused by ovariectomy.
  • TRACP-5b and alkaline phosphatase (ALP).
  • ALP alkaline phosphatase
  • the ELISA method was used to detect the content of serum TRACP-5b in mice after ovariectomy, and the microplate reader method was used to detect the activity of ALP.
  • Group A received 5 mg/kg NVP-BHG712 gavage for 5 weeks after ovariectomy
  • Group B received 10 mg/kg NVP-BHG712 gavage for 5 weeks after ovariectomy
  • Group C received 20 mg/kg NVP-BHG712 gavage for 5 weeks after ovariectomy
  • Group D received 40 mg/kg NVP-BHG712 gavage for 5 weeks after ovariectomy.
  • ovariectomy for 6 weeks in female mice can successfully establish an osteoporosis model, simulating postmenopausal osteoporosis; NVP-BHG712 can effectively weaken the bone resorption function of ovariectomized mice and slow down the high-turnover bone loss in mice after ovariectomy.
  • Example 8 NVP-BHG712 has no obvious toxicity to ovariectomized mice
  • mice were treated with 5, 10, 20, and 40 mg/kg NVP-BHG712 by oral gavage for 5 weeks.
  • NVP-BHG712 had no obvious toxicity to ovariectomized mice at doses of 5, 10, 20, and 40 mg/kg.
  • Example 9 CCK-8 method to detect the toxicity of AMG-900 drugs:
  • AMG-900 (purity 99.32%), 503.58 ⁇ g was weighed and dissolved in 1000 ⁇ l of dimethyl sulfoxide to prepare AMG-900 stock solution with a final concentration of 1 mM, which was then diluted with culture medium to working solutions of different concentrations.
  • Tartrate-resistant acid phosphatase (TRAP) staining experiment verifies the effect of AMG-900 on inhibiting osteoclast differentiation of BMMs: BMMs were resuspended in ⁇ -MEM complete medium and the cell concentration was adjusted to 5 ⁇ 10 4 /ml. 100 ⁇ l was seeded in a 96-well plate, and mouse M-CSF and RANKL were added to the final concentrations of 50 ng/ml and 50 ng/ml, respectively, to establish an in vitro osteoclast differentiation and maturation culture system.
  • TRIP Tartrate-resistant acid phosphatase
  • AMG-900 was added to the final concentrations of 0, 0.25, 0.5, 1, 2, and 4 ⁇ M, respectively, and cultured statically in a cell culture incubator at 37°C and 5% CO 2. The medium was changed every 48 h, and the culture was continued for 5 to 6 days. The medium was discarded and PBS was added to wash twice. TRAP staining was performed and osteoclasts were counted. 100 ⁇ l of fixative was added to each well and fixed at room temperature for 20 min. PBS was added to wash 3 times. 100 ⁇ l of permeabilization solution was added to each well and incubated for 5 min. After permeabilization, PBS was added to wash 3 times. Add 100 ⁇ l of colorimetric substrate to each well.
  • Figure 9A is a photo of osteoclasts detected by TRAP staining 6 days after induction. The larger the multinuclear giant cells, the greater their number, and the more obvious the osteoclasts; Figure 9B is a statistical chart of the number of osteoclasts.
  • AMG-900 Preparation of AMG-900: Weigh 0.3 mg AMG-900 and dissolve it in 10 ⁇ l dimethyl sulfoxide to prepare an AMG-900 stock solution with a final concentration of 300 mg/kg, and then dilute it with 0.5% CMC-Na to a working solution of 3 mg/kg.
  • An osteoporosis mouse model was constructed according to the method described in Example 1.
  • AMG-900 was administered intragastrically one week after surgery, once every 3 days, with a dosage of 200 ⁇ l for 5 weeks.
  • the femurs of the mice were collected for micro-computed tomography ( ⁇ CT) analyze.
  • ⁇ CT micro-computed tomography
  • AMG-900 can alleviate bone loss in osteoporosis model mice:
  • A is the two-dimensional structure diagram of femoral trabeculae in osteoporosis model mice treated with AMG-900, and B is the statistical diagram of mouse bone tissue metrology parameters.
  • Example 12 CCK-8 method to detect ONT-380 drug toxicity:
  • ONT-380 (purity 99.30%) was purchased from Shanghai selleckchem Co., Ltd. 480.52 ⁇ g was weighed and dissolved in 1000 ⁇ l dimethyl sulfoxide to prepare an ONT-380 mother solution with a final concentration of 1 mM, which was then diluted with culture medium to different concentrations of working solution.
  • Tartrate-resistant acid phosphatase (TRAP) staining experiment verifies the effect of ONT-380 on inhibiting osteoclast differentiation of BMMs: BMMs were resuspended in ⁇ -MEM complete medium and the cell concentration was adjusted to 5 ⁇ 10 4 /ml. 100 ⁇ l was seeded in a 96-well plate, and mouse M-CSF and RANKL were added to the final concentrations of 50ng/ml and 50ng/ml, respectively, to establish an in vitro osteoclast differentiation and maturation culture system.
  • TRIP Tartrate-resistant acid phosphatase
  • ONT-380 was added to the final concentrations of 0, 0.25, 0.5, 1, 2, and 4 ⁇ M, respectively, and cultured statically in a cell culture incubator at 37°C and 5% CO 2. The medium was changed every 48h, and the culture was continued for 5-6 days. The medium was discarded and PBS was added to wash twice. TRAP staining was performed and osteoclasts were counted. 100 ⁇ l of fixative was added to each well and fixed at room temperature for 20min. PBS was added to wash 3 times. 100 ⁇ l of permeabilization solution was added to each well and incubated for 5min. After permeabilization, PBS was added to wash 3 times. Add 100 ⁇ l of colorimetric substrate to each well.
  • Figures 12A and 12B The results are shown in Figures 12A and 12B.
  • TRAP staining verified that 2-4 ⁇ M ONT-380 could reduce the osteoclast differentiation ability of BMMs.
  • Figure 12A is a photo of osteoclasts detected by TRAP staining after 6 days of induction. The larger the multinuclear giant cells, the more numerous they are, and the more obvious the osteoclasts are.
  • Figure 12B is a statistical chart of the number of osteoclasts.
  • ONT-380 Weigh 2.5 mg of ONT-380 and dilute it with 1 ml of 0.5% CMC-Na to make a 25 mg/kg ONT-380 solution.
  • ONT-380 was administered orally one week after surgery, once every 3 days, with a dosage of 200 ⁇ l, for 5 weeks.
  • femurs of the mice were collected for micro-computed tomography ( ⁇ CT) analysis.
  • A is a two-dimensional structure diagram of femoral trabeculae in osteoporosis model mice treated with ONT-380;
  • B is a statistical diagram of mouse bone tissue metrology parameters.
  • KD025 (purity 99.59%) was purchased from Shanghai selleckchem Co., Ltd. 452.51 ⁇ g was weighed and dissolved in 1000 ⁇ l dimethyl sulfoxide to prepare a KD025 mother solution with a final concentration of 1 mM, which was then diluted with culture medium to working solutions of different concentrations.
  • Tartrate-resistant acid phosphatase (TRAP) staining experiment verified the effect of KD025 in inhibiting osteoclast differentiation of BMMs: BMMs were resuspended in ⁇ -MEM complete medium and the cell concentration was adjusted to 5 ⁇ 10 4 /ml. 100 ⁇ l was seeded in a 96-well plate, and mouse M-CSF and RANKL were added to the final concentrations of 50ng/ml and 50ng/ml, respectively, to establish an in vitro osteoclast differentiation and maturation culture system.
  • TRIP Tartrate-resistant acid phosphatase
  • KD025 was added to the final concentrations of 0, 0.25, 0.5, 1, 2, and 4 ⁇ M, respectively, and cultured statically in a cell culture incubator at 37°C and 5% CO2. The medium was changed every 48h, and the culture was continued for 5 to 6 days, and the culture medium was discarded. Add PBS to the culture medium and wash twice. Perform TRAP staining and count osteoclasts. Add 100 ⁇ l of fixative to each well and fix at room temperature for 20 minutes. Add PBS to wash three times. Add 100 ⁇ l of permeabilization solution to each well and incubate for 5 minutes. After permeabilization, add PBS to wash three times. Add 100 ⁇ l of color substrate to each well.
  • Figure 15A is a photo of osteoclasts detected by TRAP staining after 6 days of induction. The larger the multinuclear giant cells, the more numerous they are, and the more obvious the osteoclasts are;
  • Figure 15B is a statistical chart of the number of osteoclasts.
  • KD025 Weigh 6 mg KD025 and dilute it with 1 ml 0.5% CMC-Na to make 60 mg/kg KD025 solution.
  • KD025 improves osteoporosis: An OVX osteoporosis mouse model was constructed according to the method described in Example 1. KD025 was gavaged one week after surgery and administered once every 3 days, 200 ⁇ l, for 5 weeks. The femurs of the mice were collected for microcomputed tomography ( ⁇ CT) analysis in the 6th week.
  • ⁇ CT microcomputed tomography
  • A is a two-dimensional structural diagram of femoral trabeculae in osteoporosis model mice treated with KD025, and B is a statistical diagram of bone tissue metrology parameters in mice.
  • the temperature unit "degree” appearing in this document refers to Celsius, that is, °C.

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Abstract

提供了治疗骨质疏松的方法，还提供了治疗骨质疏松的药物，包括治疗有效量的ONT‐380或其药学上可接受的盐，或ONT‐380或其药学上可接受的盐与以下化合物中的一种的组合：NVP‐BHG712、AMG‐900、KD025或其药学上可接受的盐。

Description

治疗骨质疏松的方法和药物组合物
本申请要求2022年10月13日提交的、申请号为202211251275.3、发明名称为“治疗骨质疏松的方法和药物组合物”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及疾病治疗和药物领域。具体的，本发明涉及骨质疏松的治疗方法，以及相关的药物组合物。
背景技术
骨质疏松症是骨吸收大于骨形成所致的骨重建失衡引起的全身性代谢性疾病。骨质疏松症按照原因分类可分为原发性骨质疏松症、继发性骨质疏松症和特发性骨质疏松症三类。绝经后骨质疏松症属于原发性骨质疏松症，发生于绝经后5～10年。人体骨稳态的维持主要由破骨细胞和成骨细胞共同完成，破骨细胞负责骨吸收，成骨细胞负责骨形成。骨髓单核细胞(BMMs)是一种造血干细胞，可以分化为破骨细胞。当骨稳态失衡，BMMs形成破骨细胞增多时，引起骨质疏松症等骨代谢性疾病，严重影响患者的生活水平。
目前一般都是采用药物对骨质疏松进行干预治疗。由于骨质疏松可能由多种原因引起，导致在进行药物干预时需要有目的地进行实施，而目前没有一种药物可以针对不同原因引起的骨质疏松进行有效的防治。
NVP‐BHG712是一种特异性的EphB4抑制剂，可抑制VEGFR2，呈剂量依赖性抑制A375稳转黑色素瘤细RTK自磷酸化。EphB4是酪氨酸蛋白激酶受体家族中EPH家族的重要成员之一。EphB4在肿瘤的发生发展中起着重要的调节作用，促进多种肿瘤的转移，对肿瘤预后有重要的调节；EphB4也调节内皮细胞生物学行为，对胚胎血管形成过程中发挥及其关键的作用；EphB4基因敲除小鼠表现出明显的血管发育障碍。NVP‐BHG712对破骨细胞作用尚无报导，对破骨细胞形成和功能是否有抑制作用以及NVP‐BHG712对绝经后骨质疏松症等骨代谢性疾病是否有治疗作用仍然未知。
AMG‐900是一种高选择性的pan‐Aurora kinases抑制剂，作用于Aurora A/B/C。AMG‐900是一种新型ATP竞争性氨基酞嗪小分子aurora激酶抑制剂。在HeLa细胞中，AMG‐900抑制aurora‐A和‐B自身磷酸化，并抑制一种aurora‐B的近端底物，Ser上组蛋白H3的磷酸化。
ONT‐380别名为Tucatinib、Irbinitinib,ARRY‐380等，是一种具有口服活性、可逆的、ATP竞争性的ErbB2小分子抑制剂。ONT‐380能够在多种HER2依赖性的肿瘤异种移植模型中抑制肿瘤的生长，具有潜在的抗肿瘤活性。
KD025是一种具有口服活性、选择性的ROCK2抑制剂。在小鼠中，KD025(200mg/kg,p.o.)降低脑和心脏中ROCK的活性；小鼠模型中，KD025(200mg/kg,i.p.)通过作用于Th17介导的信号通路，降低关节炎的发展；在闭塞性支气管炎综合征以及硬皮症小鼠模型中，KD025(150mg/kg,i.p.or p.o.)有效地缓解慢性移植物抗宿主病。
本领域还需要对影响骨质疏松的生理机制和生化途径进一步的研究，以及从中发掘新的药物在治疗骨质疏松中的应用。
发明内容
本发明首次发现NVP‐BHG712、AMG‐900、ONT‐380、KD025能有效抑制破骨细胞作用，不仅抑制破骨细胞形成，也抑制破骨细胞骨吸收功能，并且能够抑制哺乳动物的骨质疏松症的形成。本发明由此提供了治疗骨质疏松症的新药物。
在本发明的其中一个方面，提供了一种治疗骨质疏松症的方法，包括对需要这种治疗的患者施用治疗有效量的以下化合物中的一种：NVP‐BHG712、AMG‐900、ONT‐380、KD025，或其药学上可接受的盐。
在本发明的其中一个方面，提供了一种处理骨质疏松症的方法，所述方法包括施用以下化合物中的一种：NVP‐BHG712、AMG‐900、ONT‐380、KD025，或其药学上可接受的盐，所述方法为体外非治疗性的方法。例如，所述方法可用于在培养液中处理用于研究骨质疏松症的分离的组织或细胞。
在本发明的其中一种实施方式中，其中施用的化合物为NVP‐BHG712或其药学上可接受的盐，其中NVP‐BHG712为具有下式的化合物：
在本发明的其中又一种实施方式中，以介于0.1mg与50mg之间的剂量施用NVP‐BHG712或其药学上可接受的盐。在本发明的其中一种实施方式中，以50、45、40、35、30、25、20、15、10、5、4、3、2、1、0.5、0.1、0.05或0.01mg/kg体重的剂量施用NVP‐BHG712或其药学上可接受的盐。更优选的，以50、40、20、10、5或1mg/kg体重的剂量施用NVP‐BHG712或其药学上可接受的盐。
在本发明的其中一种实施方式中，其中施用的化合物为AMG‐900或其药学上可接受的盐，其中AMG‐900为具有下式的化合物：
在本发明的其中又一种实施方式中，以介于0.1mg与50mg之间的剂量施用AMG‐900或其药学上可接受的盐。在本发明的其中又一种实施方式中，以50、45、40、35、30、25、20、15、10、5、4、3、2、1、0.5、0.1、0.05或0.01mg/kg体重的剂量施用AMG‐900或其药学上可接受的盐。更优选的，以50、20、10或5mg/kg体重的剂量施用AMG‐900或其药学上可接受的盐。
在本发明的其中一种实施方式中，其中施用的化合物为ONT‐380或其药学上可接受的盐，其中ONT‐380为具有下式的化合物：
在本发明的其中又一种实施方式中，以介于10mg与1000mg之间的剂量施用ONT‐380或其药学上可接受的盐。优选的，以1000、500、400、300、250、200、100、95、90、85、80、75、70、65、60、55、50、45、40、35、30、25、20、15、10mg/kg体重的剂量施用ONT‐380或其药学上可接受的盐。更优选的，以300、250、200或100mg/kg体重的剂量施用ONT‐380或其药学上可接受的盐。
在本发明的其中一种实施方式中，其中施用的化合物为KD025或其药学上可接受的盐，其中KD025为具有下式的化合物：
在本发明的其中又一种实施方式中，以介于1mg与1000mg之间的剂量施用KD025或其药学上可接受的盐。优选的，以1000、500、400、300、250、200、100、95、90、85、80、75、70、65、60、55、50、45、40、35、30、25、20、15、10mg/kg体重的剂量施用KD025或其药学上可接受的盐。更优选的，以300、200、100或50mg/kg体重的剂量施用KD025或其药学上可接受的盐。
在本发明的其中一种实施方式中，以药物组合物的形式施用所述化合物或或其药学上可接受的盐，所述药物组合物另外包含药学上可接受的载体、赋形剂或稀释剂。
在本发明的其中一种实施方式中，所述化合物或其药学上可接受的盐以每天或每隔2‐3天给药。
在本发明的其中一个方面，提供了以下化合物中的一种：NVP‐BHG712、AMG‐900、ONT‐380、KD025，或其药学上可接受的盐在用于制备治疗骨质疏松症的药物中的用途。
在本发明的其中一种实施方式中，所述药物包含药学上可接受的载体、赋形剂或稀释剂。
在本发明的其中一种实施方式中，所述化合物或其药学上可接受的盐的剂量为介于0.01mg与1000mg之间。
在本发明的其中一个方面，提供了一种抑制破骨细胞分化和/或活性的方法，所述方法包括对需要这种治疗的患者施用治疗有效量的以下化合物中的一种：NVP‐BHG712、AMG‐900、ONT‐380、KD025，或其药学上可接受的盐。
在本发明的其中一个方面，提供了一种抑制破骨细胞分化和/或活性的方法，所述方法包括施用以下化合物中的一种：NVP‐BHG712、AMG‐900、ONT‐380、KD025，或其药学上可接受的盐，所述方法为体外非治疗性的方法。例如，所述方法可用于在培养液中处理分离的组织或细胞。
在本发明的其中一种实施方式中，其中施用的化合物为NVP‐BHG712或其药学上可接受的盐。在本发明的其中又一种实施方式中，以介于0.1mg与50mg之间的剂量施用NVP‐BHG712或其药学上可接受的盐。在本发明的其中一种实施方式中，以50、45、40、35、30、25、20、15、10、5、4、3、2、1、0.5、0.1、0.05或0.01mg/kg体重的剂量施用NVP‐BHG712或其药学上可接受的盐。更优选的，以50、40、20、10、5或1mg/kg体重的剂量施用NVP‐BHG712或其药学上可接受的盐。
在本发明的其中一种实施方式中，其中施用的化合物为AMG‐900或其药学上可接受的盐。在本发明的其中又一种实施方式中，以介于0.1mg与50mg之间的剂量施用AMG‐900或其药学上可接受的盐。在本发明的其中又一种实施方式中，以50、45、40、35、30、25、20、15、10、5、4、3、2、1、0.5、0.1、0.05或0.01mg/kg体重的剂量施用AMG‐900或其药学上可接受的盐。更优选的，以50、20、10或5mg/kg体重的剂量施用AMG‐900或其药学上可接受的盐。
在本发明的其中一种实施方式中，其中施用的化合物为ONT‐380或其药学上可接受的盐。在本发明的其中又一种实施方式中，以介于10mg 与1000mg之间的剂量施用ONT‐380或其药学上可接受的盐。优选的，以1000、500、400、300、250、200、100、95、90、85、80、75、70、65、60、55、50、45、40、35、30、25、20、15、10mg/kg体重的剂量施用ONT‐380或其药学上可接受的盐。更优选的，以300、250、200或100mg/kg体重的剂量施用ONT‐380或其药学上可接受的盐。
在本发明的其中一种实施方式中，其中施用的化合物为KD025或其药学上可接受的盐。在本发明的其中又一种实施方式中，以介于1mg与1000mg之间的剂量施用KD025或其药学上可接受的盐。优选的，以1000、500、400、300、250、200、100、95、90、85、80、75、70、65、60、55、50、45、40、35、30、25、20、15、10mg/kg体重的剂量施用KD025或其药学上可接受的盐。更优选的，以300、200、100或50mg/kg体重的剂量施用KD025或其药学上可接受的盐。
在本发明的其中一种实施方式中，以药物组合物的形式施用所述化合物或或其药学上可接受的盐，所述药物组合物另外包含药学上可接受的载体、赋形剂或稀释剂。
在本发明的其中一种实施方式中，所述化合物或其药学上可接受的盐以每天或每隔2‐3天给药。
在本发明的其中一个方面，提供了以下化合物中的一种：NVP‐BHG712、AMG‐900、ONT‐380、KD025，或其药学上可接受的盐在用于制备抑制破骨细胞分化和/或活性的药物中的用途。
在本发明的其中一种实施方式中，所述药物包含药学上可接受的载体、赋形剂或稀释剂。
在本发明的其中一种实施方式中，所述化合物或其药学上可接受的盐的剂量为介于0.01mg与1000mg之间。
需要本文所述的方法和药物(药物组合物)的对象可以是哺乳动物，包括人或者非人灵长类如猴。哺乳动物还可以是其它动物，例如大鼠、小鼠、兔、猪、狗等。所述哺乳动物可以是家养动物，例如猫或者狗。
骨骼不断地经历所谓的重建过程。在骨质疏松症中发生骨质流失，因为正常的重建过程或骨转换移除的骨骼比其取代的多。骨重建涉及两个不同的阶段：骨再吸收(分解)和骨形成。钙储存于骨骼中，当身体需要它的时候，被称作破骨细胞的骨细胞附着于骨表面并将其分解，在骨中留下空腔。然后被称作成骨细胞的骨形成细胞用被称作类骨质的有机基质填充空腔。然后类骨质自发地与磷酸钙矿化以重新形成硬骨骼。
除非另作说明，如本文所用术语“骨质疏松症”指个体骨矿物质密度的任何降低或减少，包括世界卫生组织(WHO)所定义的骨质疏松症和骨质减少。WHO将在正常值一个标准偏差内或相应的T分数小于‐1的骨矿物质密度定义为可接受的骨矿物质密度。WHO将骨矿物质密度在正常值的1至2.5个标准偏差内或相应的T分数为‐1至‐2.5定义为骨质减少，WHO将骨质疏松症的特征描述为更为严重形式的骨质减少，并将骨矿物质密度与正常值的标准偏差小于2.5或相应的T分数小于‐2.5定义为骨质疏松症。在本发明的其中一个方面，“骨质疏松症”包括绝经后骨质疏松症(Postmenopausal Osteoporosis,PMPO)，是由于女性绝经后卵巢衰退进而雌激素缺乏或紧急撤退引起的严重影响中老年人身心健康的骨质疏松症。
除非另作说明，术语“治疗”和“治疗和预防”在本文可相互使用，指涉及受骨质疏松症折磨或以其他方式有发生骨质疏松症危险的个体的方法，并且指任何影响个体骨矿物质密度或结构的方法，包括使受骨质疏松症折磨的个体增加骨矿物质密度，延缓骨矿物质密度减少的速率或发生，保持正常的骨矿物质密度，或逆转某些或全部的骨矿物质密度减少或不足。
本发明的药物组合物中的活性成分可以以原料化合物的形式给药，另外也可将活性成分，任选地以药学上可接受的盐的形式，与一种或多种佐剂、赋形剂、载体、缓冲剂、稀释剂和/或其他常规的药物辅料一起引入药物组合物。
佐剂包括防腐剂、润湿剂、助悬剂、甜味剂、矫味剂、芳香剂、乳化剂和分散剂。例如可通过各种抗细菌剂和抗真菌剂来确保防止微生物的作用，例如对羟基苯甲酸酯类、氯丁醇、苯酚、山梨酸等。还可取的是包含等渗剂，例如糖、氯化钠等。可通过使用延迟吸收的试剂(例如，单硬脂酸铝和明胶)引起可注射的药物形式的吸收延长。
如果需要的话，所述化合物的药物组合物还可含有少量的辅助性物质，如润湿剂或乳化剂、pH缓冲剂、抗氧化剂等，例如像柠檬酸、失水山梨醇单月桂酸酯、油酸三乙醇胺、丁基化羟基甲苯等。
化合物的“药学上可接受的盐”表示药学上可接受的并且具有母体化合物的所需药理活性的盐。药学上可接受的盐是无毒的。
药学上可接受的酸加成盐的例子包括与以下酸形成的盐：无机酸，如盐酸、氢溴酸、硫酸、硝酸、磷酸等；以及有机酸，如乙酸、三氟乙酸、丙酸、己酸、环戊烷丙酸、乙醇酸、丙酮酸、乳酸、草酸、马来酸、丙 二酸、琥珀酸、富马酸、酒石酸、苹果酸、柠檬酸、苯甲酸、肉桂酸、3‐(4‐羟基苯甲酰基)苯甲酸、扁桃酸、甲磺酸、乙磺酸、1，2‐乙二磺酸、2‐羟基乙磺酸、苯磺酸、4‐氯苯磺酸、2‐萘磺酸、4‐甲苯磺酸、樟脑磺酸、葡庚糖酸、4，4′‐亚甲基双‐(3‐羟基‐2‐烯‐1‐羧酸)、3‐苯基丙酸、三甲基乙酸、叔丁基乙酸、月桂基硫酸、葡糖酸、谷氨酸、羟基萘甲酸、水杨酸、硬脂酸、粘康酸、对甲苯磺酸和水杨酸等。
可以通过任意便利的适合于期望疗法的途径给予本发明的药物组合物。优选的给药途径包括口服给药，特别是以片剂、胶囊、锭剂、散剂和液体形式；和胃肠外给药，特别是皮肤、皮下、肌内和静脉内注射。本发明的药物组合物可以由本领域技术人员通过使用适合于期望制剂的标准方法和常规技术制备。如果需要，则可以使用适合于使活性成分缓释的组合物。
为从本发明药物组合物中的活性成分制备药物组合物，药学上可接受的载体可以是固体或者液体。固体形式的制剂包括散剂、片剂、丸剂、胶囊、扁囊剂、栓剂以及可分散的颗粒剂。固体载体可以是一种或多种还能用作稀释剂、矫味剂、增溶剂、润滑剂、悬浮剂、粘合剂、防腐剂、片剂崩解剂或包囊材料的物质。
需要时，可以应用适合提供活性成分缓释的组合物。
药物制剂优选为单位剂型。这类形式中，制剂被细分为含有适量活性组分的单位剂量。单位剂型可以是包装的制剂，该包装含有离散量的制剂，如包装的片剂、胶囊，以及小瓶或安瓿中的粉末。此外，单位剂型可以是胶囊、片剂、扁囊剂或锭剂本身，或者可以是适合数量的任何这些剂型的包装形式。
“治疗有效量”是当对患者施用时会改善疾病的症状的本发明化合物的量。治疗有效量旨在包括能有效治疗、改善或减轻骨质疏松症的严重程度的单独的化合物或化合物与其它活性成分组合的量。
以治疗有效量来施用本公开的化合物或它们的药学上可接受的盐，该量将根据多种因素情况而有所不同，所述因素包括所使用的具体化合物的活性、化合物的代谢稳定性和作用时间长短、年龄、体重、一般健康状况、性别、饮食、施用模式和时间、排泄速率、药物组合、特定疾病状态的严重程度以及接受治疗的主体。可以按每天约0.1至约1,000mg范围的剂量水平对患者施用本发明所述的化合物。对于体重约70公斤的正常成年人，每天每公斤体重约0.01至约100mg范围的剂量就是一个例子。然而，所使用的具体剂量可有所不同。例如，剂量可取决于多种因 素，包括患者的要求、被治疗的病症的严重程度以及所使用的化合物的药理活性。为特定的患者确定最佳剂量是本领域普通技术人员所熟知的。
在一个实施方案中，本发明所述化合物的剂型可含有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、0.01mg或更少的所述化合物。在一个实施方案中，本发明提供的治疗骨质疏松症的药物剂型含有的活性物质在0.01mg与1000mg之间。
在一个实施方案中，本发明提供的治疗骨质疏松症的药物的剂量介于0.01mg与1000mg/kg体重之间。
附图说明
图1显示NVP‐BHG712对骨髓单核细胞(BMM)无细胞毒性。
图2显示NVP‐BHG712对BMM破骨分化的影响。A为TRAP染色检测破骨照片图；B为破骨细胞数量结果统计图。
图3A显示0、0.1、0.2、0.4μM NVP‐BHG712处理BMMs细胞6天，分析骨吸收面积变化。
图3B显示0.4μM NVP‐BHG712处理BMMs细胞0、1～3天、3～5天、5～6天、1～6天，TRAP染色观察破骨细胞形成。
图4显示NVP-BHG712抑制破骨细胞CTSK蛋白和mRNA表达。(a)Western Blot检测BMMs细胞经RANKL诱导和加入NVP-BHG712培养4天，CTSK蛋白表达水平及统计图。(b)BMMs细胞经RANKL诱导和加入0.1、0.2、0.4μM NVP-BHG712，培养4天，Western Blot检测CTSK蛋白表达及统计图。(c)BMMs细胞经RANKL诱导和加入0.4μM NVP-BHG712培养1、2、3、4天，Western Blot检测CTSK蛋白表达及统计图。(第1组：BMMs细胞无诱导；第2组BMMs细胞经RANKL诱导；第3组BMMs细胞经RANKL诱导+0.4μM NVP-BHG712培养；数据为三次独立实验结果平均值±SD，与第2组相比，*P<0.05，*P<0.01，***P<0.001；n＝3)。
图5A显示Western Blot检测BMMs细胞经RANKL诱导，加入0.4μM NVP‐BHG712培养4天，MMP9、CTR、TRAP蛋白表达。
图5B显示qPCR检测BMMs细胞经RANKL诱导，加入0.4μM NVP‐BHG712培养4天，MMP9、CTR、TRAP的mRNA表达。
图6显示NVP‐BHG712缓解骨质疏松症模型小鼠的骨量丢失：A为骨质疏松症模型小鼠经NVP‐BHG712治疗的股骨骨小梁二维结构图，B为小鼠骨组织计量学参数统计图。
图7显示NVP‐BHG712抑制卵巢切除小鼠骨吸收功能。(a)5、10、20、40mg/kg NVP‐BHG712灌胃5周，对卵巢切除小鼠血清TRACP‐5b的影响。(b)5、10、20、40mg/kg NVP‐BHG712灌胃5周，对卵巢切除小鼠血清ALP活性的影响(与OVX组相比，*P<0.05，**P<0.01，***P<0.001；n＝5)。
图8显示AMG‐900对骨髓单核细胞(BMM)无细胞毒性。
图9显示AMG‐900对BMM破骨分化的影响。图9A为TRAP染色检测破骨照片图；图9B为破骨细胞数量结果统计图。
图10显示AMG‐900缓解骨质疏松症模型小鼠的骨量丢失：A为骨质疏松症模型小鼠经NVP‐BHG712治疗的股骨骨小梁二维结构图，B为小鼠骨组织计量学参数统计图。
图11显示ONT‐380对骨髓单核细胞(BMM)无细胞毒性。
图12显示ONT‐380对BMM破骨分化的影响。图12A为TRAP染色检测破骨照片图；图12B为破骨细胞数量结果统计图。
图13显示ONT‐380缓解骨质疏松症模型小鼠的骨量丢失：A为骨质疏松症模型小鼠经NVP‐BHG712治疗的股骨骨小梁二维结构图，B为小鼠骨组织计量学参数统计图。
图14显示KD025对骨髓单核细胞(BMM)无细胞毒性。
图15显示KD025对BMM破骨分化的影响。图15A为TRAP染色检测破骨照片图；图15B为破骨细胞数量结果统计图。
图16显示KD025缓解骨质疏松症模型小鼠的骨量丢失：A为骨质疏松症模型小鼠经NVP‐BHG712治疗的股骨骨小梁二维结构图，B为小鼠骨组织计量学参数统计图。
具体实施方式
下面将结合实施例进一步说明本发明的实质内容和有益效果，该实施例仅用于说明本发明而非对本发明的限制。
实施例1方法和材料
骨髓单核细胞(BMMs)的分离与纯化
(1)8周龄C57BL/6J小鼠处死，用75％乙醇浸泡5min。取出双侧股骨和胫骨。
(2)剪断两侧骨骺端，使用1ml注射器冲洗出骨髓至培养皿中。
(3)用1ml移液器反复抽吸获得单细胞悬液。用300目尼龙膜过滤悬液于15ml离心管中，1200rpm离心5min，弃上清。
(4)加入3ml红细胞裂解液，混匀，室温孵育3min。加入6ml完全培养基中和裂解液，1200rpm离心5min，弃上清。
(5)加入5ml‐MEM完全培养基悬浮细胞，1200rpm离心5min，弃上清。重复洗涤一次。
(6)α‐MEM完全培养基重悬细胞，加入小鼠M‐CSF，终浓度为5ng/ml，细胞浓度为1～5×107/ml。细胞接种于培养皿中，于37℃、5％CO2细胞培养箱中静置培养过夜。获得小鼠BMMs。
卵巢切除骨质疏松症小鼠模型制备
8周龄C57BL/6J小鼠禁食12h，术前腹腔注射戊巴比妥钠麻醉。去除背部脊柱两侧肋缘下3cm毛发。在背部脊柱两侧1cm肋缘下1cm处，分别切两个1cm切口。找到卵巢，将卵巢与输卵管连接处结扎，切除卵巢，缝合切口，腹腔注射3万单位/只青霉素预防感染。假手术组用相同方法找到卵巢，切除与卵巢相应大小脂肪。术后小鼠放于电热毯上复温1～2h，复温后小鼠放回鼠笼。
骨吸收实验
骨片上的骨吸收陷窝是骨吸收的直接结果，也是体外鉴别骨吸收功能的最可靠指标。通过检测骨板骨陷窝面积反应骨吸收面积可评价破骨细胞骨吸收功能。
(1)用α‐MEM完全培养基重悬BMMs，调整细胞浓度为5×104/ml。取400μl种于骨板中，加入小鼠M-CSF和RANKL，终浓度分别为50ng/ml和50ng/ml，建立破骨细胞体外分化成熟培养体系。在该破骨细胞分化成熟体系中，加入不同浓度的待测化合物如NVP-BHG712，于37℃、5％CO2细胞培养箱中静置培养。
(2)每48h换液，继续培养6天。
(3)细胞培养至第7天时，吸出培养基，用PBS洗3遍。
(4)将含10％次氯酸的PBS加入骨板，静置10min。
(5)吸弃含10％次氯酸的PBS，用PBS洗3遍。
(6)将骨板风干。
(7)光镜下观察骨陷窝形成面积并拍照。
(8)通过分析骨板骨陷窝形成面积反映骨吸收功能。
使用的抗体

使用的引物或探针
TRAP染色试剂盒购于中国武汉赛维尔生物科技有限公司。
Alkaline phosphatase assay kit购于中国南京建成生物工程研究所有限公司。
实施例2 CCK-8法检测NVP-BHG712药物毒性：
1.NVP-BHG712的配制：由上海selleckchem公司购得NVP-BHG712(纯度99.53％)，称取503.48μg后用1000μl二甲基亚砜溶解，制成终浓度为1mM的NVP-BHG712母液，再用培养基稀释至不同浓度的工作液。
2.NVP-BHG712对骨髓单核细胞(BMMs)的细胞毒性
用含50ng/ml M-CSF的α-MEM完全培养基重悬BMMs，调整细胞浓度为5×104/ml，取100μl种于96孔板，于37℃、5％CO2细胞培养箱中静 置培养过夜。加入NVP-BHG712，终浓度分别为0、0.1、0.2、0.4、0.8、1.6μM，孵育72h。每孔更换为90ul完全培养基+10μl CCK-8溶液，孵箱37℃孵育1～2h。用酶标仪检测450nm处吸光度。
结果见图1。结果表明，0.1-1.6μM NVP-BHG712对BMMs无细胞毒性。另根据实验结果发现，高于3.2μM的NVP-BHG712对BMMs的正常增殖有影响。
实施例3 NVP‐BHG712对BMMs破骨分化的影响
1.通过抗酒石酸酸性磷酸酶(TRAP)染色实验验证NVP‐BHG712抑制BMMs破骨分化的作用。
用α‐MEM完全培养基重悬BMMs，调整细胞浓度为5×104/ml。取100μl种于96孔板中，加入小鼠M‐CSF和RANKL，终浓度分别为50ng/ml和50ng/ml，建立破骨细胞体外分化成熟培养体系。在该破骨细胞分化成熟体系中，加入NVP‐BHG712，终浓度分别为0、0.05、0.1、0.2、0.4、0.8、1.6μM，于37℃、5％CO2细胞培养箱中静置培养。每48h换液，继续培养5～6天，弃去培养基，加入PBS洗2次。行TRAP染色，破骨细胞计数。每孔加入100μl固定液，室温固定20min。加入PBS洗3次。每孔加入100μl透化液孵育5min。透化后，加入PBS洗3次。每孔加入100μl显色底物。将96孔板置于37℃孵箱避光孵育60～90min。加入去离子水洗涤，弃上清，重复3次。每孔加入100μl甲基绿溶液，覆盖孔底，染细胞核5min。染色结束后，加入去离子水洗3次，弃上清。自然晾干，显微镜计数破骨细胞(细胞核≥3个的巨细胞为破骨细胞)。
TRAP染色是作为破骨细胞活性染色的标准染色方法。如图2A和图2B所示，TRAP染色验证0.4‐1.6μM的NVP‐BHG712作用后能够使BMMs的破骨分化能力降低：图2A为诱导6天后TRAP染色检测破骨照片图，多核巨细胞越大，数量越多，破骨越明显；图2B为破骨细胞数量结果统计图。
2.通过检测骨板骨陷窝面积反应骨吸收面积可评价破骨细胞骨吸收功能。
为观察NVP-BHG712对破骨细胞骨吸收功能的影响，设置了不同浓度梯度(0、0.1、0.2、0.4μM)的NVP-BHG712干预RANKL诱导的破骨细胞，培养6天。通过骨板吸收实验，发现仅加入M-CSF组，未见明显骨吸收陷窝，而在RANKL诱导下，BMMs细胞分化为具有骨吸收功能的成熟破骨细胞，并在骨板上产生不规则形骨吸收陷窝，大小不一，边界清晰，此时骨板的骨 吸收陷窝面积约为5.66mm2；随着NVP-BHG712的加入，骨吸收陷窝面积明显减少，统计结果显示，加入0.1μM的NVP-BHG712组骨吸收的面积为5.07mm2，骨吸收面积较正常诱导组而言，下降了约10％；而随着NVP-BHG712的浓度升高至0.2μM，骨吸收面积为6.03mm2。当加入NVP-BHG712浓度为0.4μM时，骨吸收面积仅为0.80mm2，仅为正常诱导组的14.13％。结果如图3A所示，表明NVP-BHG712能显著抑制BMMs分化而来的破骨细胞的骨吸收功能。
为进一步研究NVP-BHG712作用于破骨分化的阶段，在破骨细胞分化的不同时段(早中晚阶段)添加0.4μM NVP-BHG712于RANKL刺激的BMMs中，经TRAP染色实验和F-actin荧光实验观察结果，具体分组如下：对照组，仅加入M-CSF+RANKL；早期组，加入RANKL，1～3天加入0.4μM NVP-BHG712；中期组，加入RANKL，3～5天加入0.4μM NVP-BHG712；晚期组，加入RANKL，5～6天加入0.4μM NVP-BHG712；早中晚期组，加入RANKL，1～6天加入0.4μM NVP-BHG712。TRAP染色结果发现，在早期、中期和早中晚期组，加入NVP-BHG712对破骨细胞抑制作用显著(P<0.001)，TRAP阳性多核巨细胞数量与对照组相比明显减少，细胞单个，少有融合；在晚期加入NVP-BHG712，虽然有一定破骨细胞抑制作用，但不如早期的干预效果显著。结果如图3B所示，表明NVP-BHG712主要作用于破骨分化的早期阶段。
F-actin实验发现，BMMs细胞在RANKL的诱导下，能分化为带有F-actin环的多核巨细胞，F-actin数量与破骨细胞数的比值为7.83％；但当早期阶段和早中晚阶段加入0.4μM的NVP-BHG712，F-actin环形成数量较正常破骨诱导组明显减少(P<0.05)，F-actin数量与破骨细胞数的比值为1.48％和0.41％。当中期阶段和晚期阶段加入NVP-BHG712，F-actin数量较正常诱导组减少不明显，其与破骨细胞数的比值为2.76％和5.88％，结果表明，NVP-BHG712抑制RANKL诱导的破骨F-actin形成以早期为主。
综上所述，NVP-BHG712抑制破骨细胞功能，主要作用于破骨分化的早期阶段。
实施例4 NVP-BHG712抑制破骨细胞CTSK蛋白表达
采用Western Blot技术检测BMMs细胞中的CTSK表达。为了进一步检测NVP-BHG712浓度及处理时间对CTSK蛋白的影响，在RANKL诱导的条件下加入0、0.1、0.2、0.4μM浓度的NVP-BHG712，处理细胞4天，收集细胞，提取蛋白，Western Blot检测CTSK蛋白表达；在RANKL诱导的 条件下加入NVP-BHG712，终浓度为0.4μM，分别培养1、2、3、4天，收集细胞，提取蛋白，Western Blot检测CTSK蛋白表达。
结果如图4所示，发现BMMs细胞加入0.4μM NVP-BHG712，培养4天，RANKL诱导的CTSK蛋白表达显著降低(P<0.05)(图4a)；0.1、0.2、0.4μM NVP-BHG712加入BMMs培养体系，CTSK蛋白表达显著下降(P<0.05)(图4b)；BMMs细胞加入0.4μM NVP-BHG712，培养1天和2天CTSK蛋白表达没有明显改变，培养3天和4天CTSK蛋白表达显著下降(P<0.05)(图4c)。
综上所述，NVP-BHG712抑制破骨细胞CTSK蛋白表达。
实施例5 NVP-BHG712抑制破骨细胞分化相关基因表达
为了研究NVP-BHG712对破骨细胞分化相关基因的影响，使用BMMs细胞，经RANKL诱导，加入NVP-BHG712，终浓度为0.4μM，培养4天，收集细胞，提取蛋白，采用Western Blot方法检测MMP9、CTR、TRAP蛋白表达，采用qPCR方法检测MMP9、CTR和TRAP mRNA表达。
结果发现BMMs经RANKL诱导后，MMP9、CTR和TRAP蛋白表达升高，mRNA表达升高，加入0.4μM NVP-BHG712培养后，MMP9和CTR蛋白表达显著下降(P<0.01)(图5A)，MMP9和CTR mRNA表达显著下降(P<0.05)(图5B)，而NVP-BHG712对TRAP的蛋白表达和mRNA表达没有明显作用，提示NVP-BHG712能抑制RANKL诱导的MMP9和CTR蛋白和mRNA表达升高。
类似的，在RANKL诱导的BMMs细胞培养体系中加入0.4μM NVP-BHG712，培养4天，收集细胞，提取RNA，采用qPCR方法检测IP3R1、IP3R2、IP3R3、NFATc1、OC-STAMP、DC-STAMP、Atp6v1c1和αv-integrin mRNA。qPCR结果发现，加入RANKL诱导4天后，破骨细胞功能相关基因NFATc1、OC-STAMP、DC-STAMP、Atp6v1c1上调，钙震荡相关基因IP3R1、IP3R2、IP3R3上调，骨吸收功能相关基因αv-integrin上调。加入NVP-BHG712培养4天后，NVP-BHG712抑制不规律性钙震荡和瞬时钙震荡，而对长时间持续性钙震荡没有明显作用。另外发现，NVP-BHG712抑制RANKL引起的破骨细胞功能相关基因OC-STAMP的上调。
实施例6 NVP‐BHG712减缓卵巢切除小鼠骨量的丢失
NVP‐BHG712的配制：称取2mg NVP‐BHG712，用1ml 0.5％CMC‐Na稀释为20mg/kg的NVP‐BHG712溶液。
根据实施例1描述的方法构建卵巢切除OVX骨质疏松症小鼠模型，术 后一周灌胃NVP‐BHG712，每3天给药一次，给药200μl，持续5周，第6周收小鼠股骨进行微计算机断层扫描(μCT)分析。统计学处理：组间均值比较采用方差分析。
骨微观结构退化、骨小梁变细及变稀和骨量减少是骨质疏松症的特征之一。通过μCT扫描从二维角度观察卵巢切除后骨小梁密度和数量的变化。图6结果表明，NVP‐BHG712能够缓解骨质疏松症模型小鼠的骨量丢失。图6A为骨质疏松症模型小鼠经NVP‐BHG712治疗的股骨骨小梁二维结构图，显示Sham组骨小梁数量多、密度高，OVX组骨小梁数量减少、密度低；与OVX组相比，NVP‐BHG712给药后，骨小梁密度增多。
图6B为小鼠骨组织计量学参数统计图。通过对小鼠骨组织形态剂量学参数骨密度(BMD)、骨表面积/骨体积比(BS/BV)、骨表面积/组织体积比(BS/TV)、骨表面/体积比(BV/TV)、骨小梁数量(Tb.N)、骨小梁模式因子(Tb.Pf)、骨小梁分离度(Tb.Sp)和骨小梁厚度(Tb.Th)的统计，可以看出，与Sham组相比较，双侧卵巢切除后，小鼠骨密度(P<0.05)、骨表面积/组织体积比(P<0.05)、骨表面/体积比(P<0.05)、骨小梁数量(P<0.05)、骨小梁厚度(P<0.05)显著降低，骨表面积/骨体积比(P<0.05)、骨小梁模式因子(P<0.05)、骨小梁分离度显著升高(P<0.05)(图10c)；与OVX相比较，NVP‐BHG712组骨密度(P<0.05)、骨表面积/组织体积比(P<0.05)、骨表面/体积比(P<0.05)、骨小梁数量(P<0.05)、骨小梁厚度(P<0.05)升高和骨表面积/骨体积比(P<0.05)、骨小梁模式因子(P<0.05)、骨小梁分离度显著升高(P<0.05)降低有统计学意义。提示NVP‐BHG712抑制卵巢切除后骨小梁微结构的破坏。以上结果说明，NVP‐BHG712对卵巢切除小鼠的骨量丢失有保护作用。
为进一步验证μCT的结果，将小鼠股骨4％多聚甲醛固定、脱钙液脱钙、石蜡切片，进行H&E染色实验。结果显示，与Sham组相比，OVX组的骨小梁面积明显减少(P<0.05)；与OVX组相比，NVP-BHG712治疗组骨小梁面积增加(P<0.05)。提示NVP-BHG712减缓卵巢切除引起的小鼠骨小梁减少。
实施例7 NVP‐BHG712减弱卵巢切除小鼠骨吸收功能
为了检测体内NVP-BHG712是否对骨代谢活动有作用，选取两个血清指标：TRACP-5b和碱性磷酸酶(Alkaline phosphatase,ALP)。采用ELISA方法检测卵巢切除后小鼠血清TRACP-5b的含量，采用微量酶标仪法检测ALP活性。每组小鼠术后恢复一周，随机分组，各分组如下：Sham组为假手术后溶剂CMC-Na灌胃5周，OVX组为卵巢切除后溶剂CMC-Na灌胃5周， A组为切除卵巢后5mg/kg NVP-BHG712灌胃5周，B组为切除卵巢后10mg/kg NVP-BHG712灌胃5周，C组为切除卵巢后20mg/kg NVP-BHG712灌胃5周，D组为切除卵巢后40mg/kg NVP-BHG712灌胃5周。
ELISA结果如图7所示，发现与Sham组相比，OVX组小鼠血清TRACP-5b含量明显升高(P<0.001)(图7a)；微量酶标法结果显示，与Sham组相比，OVX组小鼠血清ALP活性显著降低(P<0.01)(图7b)，提示造模成功；当给予5、10、20、40mg/kg NVP-BHG712灌胃5周后，与OVX组相比，TRACP-5b含量明显下降(P<0.01)，ALP活性明显升高(P<0.01)，提示NVP-BHG712抑制小鼠卵巢切除后血清TRACP-5b水平升高，促进血清ALP活性。
综上所述，雌性小鼠卵巢切除6周可成功构建骨质疏松症模型，模拟绝经后骨质疏松症；NVP-BHG712能有效减弱卵巢切除小鼠骨吸收功能，减缓切除卵巢后小鼠体内高转换型骨丢失。
实施例8 NVP‐BHG712对卵巢切除小鼠无明显毒性
为研究长期给予NVP‐BHG712治疗过程中，是否产生除骨之外的影响，检测卵巢切除小鼠经5、10、20、40mg/kg NVP‐BHG712灌胃处理5周，小鼠体重变化、心脏指数(心脏重量/体重)、脾脏指数(脾脏重量/体重)、肾脏指数(肾脏重量/体重)、抓力指数(前肢抓力/体重)、心脏肥大(心脏重量/胫骨长度)指标。
结果发现，与OVX组相比，5、10、20、40mg/kg NVP‐BHG712剂量组，体重增长没有明显差异，提示在该剂量下NVP‐BHG712不影响卵巢切除小鼠体重增长；与OVX组相比，5、10、20、40mg/kg NVP‐BHG712剂量组，小鼠心脏指数、脾脏指数、肾脏指数没有明显差异，提示在该剂量下NVP‐BHG712不影响卵巢切除小鼠脏器指数；与OVX组相比，5、10、20、40mg/kg NVP‐BHG712剂量组，小鼠前肢肌肉力量没有明显差异，提示在该剂量下NVP‐BHG712不影响卵巢切除小鼠抓力指数；与OVX组相比，5、10、20、40mg/kg NVP‐BHG712剂量组，小鼠心脏肥大没有明显差异，提示在该剂量下NVP‐BHG712不影响卵巢切除小鼠心脏肥大。
综上所述，NVP‐BHG712在5、10、20、40mg/kg剂量下，对卵巢切除小鼠无明显毒性。
实施例9 CCK‐8法检测AMG‐900药物毒性：
1.AMG‐900的配制：由上海selleckchem公司购得AMG‐900(纯度 99.32％)，称取503.58μg后用1000μl二甲基亚砜溶解，制成终浓度为1mM的AMG‐900母液，再用培养基稀释至不同浓度的工作液。
2.AMG‐900对BMMs的细胞毒性
用含50ng/ml M‐CSF的α‐MEM完全培养基重悬BMMs，调整细胞浓度为5×104/ml，取100μl种于96孔板，于37℃、5％CO2细胞培养箱中静置培养过夜。加入AMG‐900，终浓度分别为0、0.25、0.5、1、2、4μM，孵育72h。每孔更换为90ul完全培养基+10μl CCK‐8溶液，孵箱37℃孵育1～2h。用酶标仪检测450nm处吸光度。
图8结果表明，本发明的0.25‐2μM AMG‐900对BMMs无细胞毒性。
实施例10不同浓度AMG‐900对BMMs破骨分化的影响：
抗酒石酸酸性磷酸酶(TRAP)染色实验验证AMG‐900抑制BMMs破骨分化的作用：用α‐MEM完全培养基重悬BMMs，调整细胞浓度为5×104/ml。取100μl种于96孔板中，加入小鼠M‐CSF和RANKL，终浓度分别为50ng/ml和50ng/ml，建立破骨细胞体外分化成熟培养体系。在该破骨细胞分化成熟体系中，加入AMG‐900，终浓度分别为0、0.25、0.5、1、2、4μM，于37℃、5％CO2细胞培养箱中静置培养。每48h换液，继续培养5～6天，弃去培养基，加入PBS洗2次。行TRAP染色，破骨细胞计数。每孔加入100μl固定液，室温固定20min。加入PBS洗3次。每孔加入100μl透化液孵育5min。透化后，加入PBS洗3次。每孔加入100μl显色底物。将96孔板置于37℃孵箱避光孵育60～90min。加入去离子水洗涤，弃上清，重复3次。自然晾干，显微镜计数破骨细胞(细胞核≥3个的巨细胞为破骨细胞)。
结果如图9A和图9B所示，TRAP染色验证1‐4μM的AMG‐900作用后能够使BMMs的破骨分化能力降低：图9A为诱导6天后TRAP染色检测破骨照片图，多核巨细胞越大，数量越多，破骨越明显；图9B为破骨细胞数量结果统计图。
实施例11 AMG‐900对骨质疏松小鼠骨量的影响：
1.AMG‐900的配制：称取0.3mg AMG‐900后用10μl二甲基亚砜溶解，制成终浓度为300mg/kg的AMG‐900母液，再用0.5％CMC‐Na稀释为3mg/kg的工作液。
2.动物实验验证AMG‐900改善骨质疏松症：根据实施例1描述的方法构建骨质疏松症小鼠模型，术后一周灌胃AMG‐900，每3天给药一次，给药200μl，持续5周，第6周收小鼠股骨进行微计算机断层扫描(μCT) 分析。
统计学处理组间均值比较采用方差分析。
图10结果表明，AMG‐900能够缓解骨质疏松症模型小鼠的骨量丢失：A为骨质疏松症模型小鼠经AMG‐900治疗的股骨骨小梁二维结构图，B为小鼠骨组织计量学参数统计图。
实施例12 CCK‐8法检测ONT‐380药物毒性：
1.ONT‐380的配制：由上海selleckchem公司购得ONT‐380(纯度99.30％)，称取480.52μg后用1000μl二甲基亚砜溶解，制成终浓度为1mM的ONT‐380母液，再用培养基稀释至不同浓度的工作液。
2.ONT‐380对BMMs的细胞毒性
用含50ng/ml M‐CSF的α‐MEM完全培养基重悬BMMs，调整细胞浓度为5×104/ml，取100μl种于96孔板，于37℃、5％CO2细胞培养箱中静置培养过夜。加入ONT‐380，终浓度分别为0、0.25、0.5、1、2、4μM，孵育72h。每孔更换为90ul完全培养基+10μl CCK‐8溶液，孵箱37℃孵育1～2h。用酶标仪检测450nm处吸光度。
结果如图11所示，0.25‐4μM ONT‐380对BMMs无细胞毒性。
实施例13不同浓度ONT‐380对BMMs破骨分化的影响：
抗酒石酸酸性磷酸酶(TRAP)染色实验验证ONT‐380抑制BMMs破骨分化的作用：用α‐MEM完全培养基重悬BMMs，调整细胞浓度为5×104/ml。取100μl种于96孔板中，加入小鼠M‐CSF和RANKL，终浓度分别为50ng/ml和50ng/ml，建立破骨细胞体外分化成熟培养体系。在该破骨细胞分化成熟体系中，加入ONT‐380，终浓度分别为0、0.25、0.5、1、2、4μM，于37℃、5％CO2细胞培养箱中静置培养。每48h换液，继续培养5～6天，弃去培养基，加入PBS洗2次。行TRAP染色，破骨细胞计数。每孔加入100μl固定液，室温固定20min。加入PBS洗3次。每孔加入100μl透化液孵育5min。透化后，加入PBS洗3次。每孔加入100μl显色底物。将96孔板置于37℃孵箱避光孵育60～90min。加入去离子水洗涤，弃上清，重复3次。自然晾干，显微镜计数破骨细胞(细胞核≥3个的巨细胞为破骨细胞)。
结果如图12A和图12B所示，TRAP染色验证2‐4μM的ONT‐380作用后能够使BMMs的破骨分化能力降低。图12A为诱导6天后TRAP染色检测破骨照片图，多核巨细胞越大，数量越多，破骨越明显，图12B为破骨细胞数量结果统计图。
实施例14 ONT‐380对骨质疏松小鼠骨量的影响：
1.ONT‐380的配制：称取2.5mg ONT‐380后用1ml的0.5％CMC‐Na稀释为25mg/kg的ONT‐380溶液。
2.动物实验验证ONT‐380改善骨质疏松症：
根据实施例1描述的方法构建OVX骨质疏松症小鼠模型，术后一周灌胃ONT‐380，每3天给药一次，给药200μl，持续5周，第6周收小鼠股骨进行微计算机断层扫描(μCT)分析。
统计学处理组间均值比较采用方差分析
骨微观结构退化、骨小梁变细及变稀和骨量减少是骨质疏松症的特征之一。通过μCT扫描从二维角度观察卵巢切除后骨小梁密度和数量的变化。图13结果表明，ONT‐380能够缓解骨质疏松症模型小鼠的骨量丢失.A为骨质疏松症模型小鼠经ONT‐380治疗的股骨骨小梁二维结构图；B为小鼠骨组织计量学参数统计图。
实施例15 CCK‐8法检测KD025药物毒性：
1.KD025的配制：由上海selleckchem公司购得KD025(纯度99.59％)，称取452.51μg后用1000μl二甲基亚砜溶解，制成终浓度为1mM的KD025母液，再用培养基稀释至不同浓度的工作液。
2.KD025对BMMs的细胞毒性
用含50ng/ml M‐CSF的α‐MEM完全培养基重悬BMMs，调整细胞浓度为5×104/ml，取100μl种于96孔板，于37℃、5％CO2细胞培养箱中静置培养过夜。加入KD025，终浓度分别为0、0.25、0.5、1、2、4μM，孵育72h。每孔更换为90ul完全培养基+10μl CCK‐8溶液，孵箱37℃孵育1～2h。用酶标仪检测450nm处吸光度。
图14结果表明，0.25‐4μM KD025对BMMs无细胞毒性。
实施例16不同浓度KD025对BMMs破骨分化的影响：
抗酒石酸酸性磷酸酶(TRAP)染色实验验证KD025抑制BMMs破骨分化的作用：用α‐MEM完全培养基重悬BMMs，调整细胞浓度为5×104/ml。取100μl种于96孔板中，加入小鼠M‐CSF和RANKL，终浓度分别为50ng/ml和50ng/ml，建立破骨细胞体外分化成熟培养体系。在该破骨细胞分化成熟体系中，加入KD025，终浓度分别为0、0.25、0.5、1、2、4μM，于37℃、5％CO2细胞培养箱中静置培养。每48h换液，继续培养5～6天，弃去培 养基，加入PBS洗2次。行TRAP染色，破骨细胞计数。每孔加入100μl固定液，室温固定20min。加入PBS洗3次。每孔加入100μl透化液孵育5min。透化后，加入PBS洗3次。每孔加入100μl显色底物。将96孔板置于37℃孵箱避光孵育60～90min。加入去离子水洗涤，弃上清，重复3次。自然晾干，显微镜计数破骨细胞(细胞核≥3个的巨细胞为破骨细胞)。
结果如图15A和图15B所示，TRAP染色验证2‐4μM的KD025作用后能够使BMMs的破骨分化能力降低。图15A为诱导6天后TRAP染色检测破骨照片图，多核巨细胞越大，数量越多，破骨越明显；图15B为破骨细胞数量结果统计图。
实施例17 KD025对骨质疏松小鼠骨量的影响：
1.KD025的配制：称取6mg KD025后用1ml 0.5％CMC‐Na稀释为60mg/kg的KD025溶液。
2.动物实验验证KD025改善骨质疏松症：根据实施例1描述的方法构建OVX骨质疏松症小鼠模型，术后一周灌胃KD025，每3天给药一次，给药200μl，持续5周，第6周收小鼠股骨进行微计算机断层扫描(μCT)分析。
统计学处理组间均值比较采用方差分析
图16结果表明，KD025能够缓解骨质疏松症模型小鼠的骨量丢失，A为骨质疏松症模型小鼠经KD025治疗的股骨骨小梁二维结构图，B为小鼠骨组织计量学参数统计图。
上面是对本发明进行的说明，不能将其看成是对本发明进行的限制。除非另外指出，本发明的实践将使用有机化学、聚合物化学、生物技术等的常规技术，显然除在上述说明和实施例中所特别描述之外，还可以别的方式实现本发明。其它在本发明范围内的方面与改进将对本发明所属领域的技术人员显而易见。根据本发明的教导，许多改变和变化是可行的，因此其在本发明的范围之内。
如无特别表示，本文中出现的温度的单位“度”是指摄氏度，即℃。

Claims (10)

  1. 一种治疗骨质疏松症的方法，包括对需要这种治疗的患者施用治疗有效量的ONT‐380或其药学上可接受的盐，或ONT‐380或其药学上可接受的盐与以下化合物中的一种的组合：NVP‐BHG712、AMG‐900、KD025或其药学上可接受的盐。
  2. 根据权利要求1所述的方法，其中以介于10mg与1000mg之间的剂量施用ONT‐380或其药学上可接受的盐。
  3. 根据权利要求1所述的方法，其中以300、250、200、100或50mg/kg体重的剂量施用ONT‐380或其药学上可接受的盐。
  4. ONT‐380或其药学上可接受的盐，或ONT‐380或其药学上可接受的盐与以下化合物中的一种的组合：NVP‐BHG712、AMG‐900、KD025或其药学上可接受的盐在用于制备治疗骨质疏松症的药物中的用途，其中所述药物包含药学上可接受的载体、赋形剂或稀释剂。
  5. 根据权利要求4所述的用途，其中ONT‐380或其药学上可接受的盐的剂量为介于10mg与1000mg之间，例如为300、250、200、100或50mg。
  6. 一种抑制破骨细胞分化和/或活性的方法，所述方法包括对需要这种治疗的患者施用治疗有效量的ONT‐380或其药学上可接受的盐，或ONT‐380或其药学上可接受的盐与以下化合物中的一种：NVP‐BHG712、AMG‐900、KD025或其药学上可接受的盐的组合。
  7. 根据权利要求6所述的方法，其中以介于10mg与1000mg之间的剂量施用ONT‐380或其药学上可接受的盐。
  8. 根据权利要求6所述的方法，其中以300、250、200、100或50mg/kg体重的剂量施用ONT‐380或其药学上可接受的盐。
  9. ONT‐380或其药学上可接受的盐，或ONT‐380或其药学上可接受的盐与以下化合物中的一种：NVP‐BHG712、AMG‐900、KD025或其药学上可接受的盐的组合在用于制备抑制破骨细胞分化和/或活性的药物中的用途，其中所述药物包含药学上可接受的载体、赋形剂或稀释剂。
  10. 根据权利要求9所述的用途，其中ONT‐380或其药学上可接受的盐的剂量为介于10mg与1000mg之间，例如为300、250、200、100或50mg。
PCT/CN2023/124591 2022-10-13 2023-10-13 治疗骨质疏松的方法和药物组合物 WO2024078628A1 (zh)

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