Application of DKK1 monoclonal antibody in the preparation of a pharmaceutical composition for treating osteoporosis

Abstract

The present invention discloses the use of DKK1 antibody in treating male osteoporosis. The present invention provides the use of an agent for activating the WNT signaling pathway in preparing a pharmaceutical composition for treating osteoporosis, a method for promoting osteoblast differentiation or maturation in vitro, a method for screening candidate drugs for treating osteoporosis, and a pharmaceutical composition for treating osteoporosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to the application of DKK1 antibody in treating male osteoporosis. Background Art

[0002] Osteoporosis is a systemic bone disease associated with aging, characterized by decreased bone strength and increased risk of fractures. With the aggravation of the aging of my country's population, the prevalence of osteoporosis in my country has risen significantly, among which the male patient population is large. The prevalence of osteoporosis in men over 50 years old in my country is 6.0%, and the prevalence of osteopenia in the early stage of the disease, which requires prevention and treatment, is as high as 46.9%. Osteoporosis in men can cause pain, bone deformities, and decreased mobility. The most serious consequence is osteoporotic fractures, which trigger acute cardiovascular and cerebrovascular events and seriously threaten the patient's life. The disability and mortality rates after fractures are much higher than those in women. However, my country's prevention and treatment of osteoporosis is mainly anchored in women. The awareness, diagnosis, and treatment rates of osteoporosis in men are low, and the level of disease diagnosis and treatment is backward.

[0003] In addition, there is a lack of research on the treatment of male osteoporosis in my country. The pathogenesis of osteoporosis is mainly due to the imbalance of bone remodeling. However, the only drugs approved for the treatment of male osteoporosis in my country are calcium, vitamin D, bisphosphonates, and denosumab. The latter two drugs are anchored in inhibiting bone resorption, and the treatment strategy is single, which is difficult to meet the major needs of disease prevention and treatment. Therefore, it is of great significance to construct new biological agents and establish new targeted treatment strategies for the precise treatment of male osteoporosis to improve the level of disease treatment.

[0004] Therapeutic studies on postmenopausal women have found that long-term sequential drug treatment for osteoporosis can effectively increase bone density and continuously reduce the risk of fractures, with significant pharmacoeconomic value. Currently, there is a lack of sequential treatment options for male patients. Actively exploring new sequential treatment strategies suitable for male osteoporosis patients will help further improve the level of diagnosis and treatment. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide the use of DKK1 antibody in the treatment of male osteoporosis.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides the use of an agent for activating the WNT signaling pathway in the preparation of a pharmaceutical composition for treating osteoporosis.

[0008] In some embodiments, the WNT signaling pathway includes the canonical WNT signaling pathway and the non-canonical WNT signaling pathway, the canonical WNT signaling pathway is involved in the regulation of gene expression, the non-canonical planar cell polarization pathway regulates the cytoskeleton to control cell shape, and the non-canonical WNT / calcium ion pathway regulates the concentration of intracellular calcium ions; in the present invention, agents that activate the WNT signaling pathway include but are not limited to activators that activate the pathway or inhibitors that antagonize the pathway.

[0009] In some embodiments, the inhibitor that antagonizes the WNT signaling pathway can use specific antibodies to neutralize secreted WNT signaling inhibitors, or can use small molecules to inactivate intracellular enzymatic bodies that inhibit β-catenin activity.

[0010] Furthermore, the agent includes an agent that inhibits the expression or activity of DKK1.

[0011] Furthermore, the agent for inhibiting the expression of DKK1 includes an agent for inhibiting the expression of DKK1 gene mRNA or the expression of DKK1 protein.

[0012] In some embodiments, the agents for inhibiting the expression of DKK1 of the present invention include, but are not limited to, agents that inhibit the expression of the gene encoding the DKK1 protein or reduce the expression level of the gene, and agents that reduce the activity of the DKK1 protein, for example, agents that inhibit the expression of the DKK1 gene or reduce its expression level, and these agents include, but are not limited to, agents that inhibit the transcriptional activity of the DKK1 gene, agents that inhibit the transcriptional level of the DKK1 mRNA, agents that promote the degradation of the DKK1 mRNA, SiRNA for the DKK1 gene, shRNA for the DKK1 gene, agents that inhibit the translation of the DKK1 mRNA, agents that specifically recognize the guide nucleic acid of the DKK1 gene and cut it to reduce its expression level, dsRNA for the DKK1 gene, microRNA for the DKK1 gene, and antisense nucleic acid for the DKK1 gene. In other embodiments, the whole DKK1 gene can be knocked out by administering a targeting vector, thereby achieving the inhibition or reduction of the expression of the DKK1 gene.

[0013] In other embodiments, the agent that reduces the activity of DKK1 protein can be, for example, a specific antibody targeting DKK1 or a small molecule compound that inhibits the activity of DKK1 protein. In a specific embodiment of the present invention, the agent that inhibits the activity of DKK1 is a monoclonal antibody targeting DKK1.

[0014] In some embodiments, the activity of the DKK1 protein can also be reduced by introducing mutations into the DKK1 protein. In other embodiments, a mutation is introduced into the functional domain of the DKK1 protein that causes the corresponding activity to be weakened or lost. The mutation can be the insertion, deletion or substitution of one or several or even more (e.g., more than 10, more than 20, more than 30) amino acids. By administering an agent that acts on the DKK1 gene, a mutation that causes the relevant biological activity to be weakened or lost can be present in the functional domain of the DKK1 protein encoded by it. Such agents can change the sequence of the DKK1 gene, resulting in the presence of corresponding mutations in the DKK1 protein encoded by it, thereby having weakened activity or loss of activity. For example, the wild-type DKK1 gene can be replaced by a mutant DKK1 gene by homologous recombination technology, resulting in the expression of a weakly active or inactive DKK1 protein.

[0015] Furthermore, the agent for inhibiting the activity of DKK1 includes a DKK1 monoclonal antibody.

[0016] In the present invention, the term "monoclonal antibody" refers to an antibody that obtains a substantially homogeneous antibody population, i.e., the population comprising individual antibodies is identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic determinant or epitope. In addition, "monoclonal antibody" refers to these antibodies prepared by a variety of methods, including but not limited to hybridomas, phage selection, recombinant expression, and transgenic animals.

[0017] Furthermore, the pharmaceutical composition includes a substance that inhibits bone resorption, a substance that promotes bone formation and / or any combination thereof.

[0018] Further, when the pharmaceutical composition described in the present invention is used in combination with other drugs, the other therapeutic compounds can be administered simultaneously with the main active ingredient, or even in the same composition. The other therapeutic compounds can also be administered separately in a separate composition or in a dosage form different from the main active ingredient. Partial doses of the main ingredient can be administered simultaneously with other therapeutic compounds, while other doses can be administered separately. During the treatment process, the dosage of the pharmaceutical composition of the present invention can be adjusted according to the severity of the symptoms, the frequency of recurrence, and the physiological response of the treatment regimen.

[0019] In some embodiments, the suitable dosage of the pharmaceutical composition of the present invention can be prescribed in a variety of ways depending on factors such as the formulation method, administration method, patient's age, weight, gender, morbidity, diet, administration time, administration route, excretion rate and reaction sensitivity. Generally, a skilled physician can easily determine the prescription and the dosage that is effective for the desired treatment.

[0020] In some embodiments, the pharmaceutical composition of the present invention can be prepared into a variety of clinical pharmaceutical dosage forms as needed to serve as a drug for the treatment of male osteoporosis, and the pharmaceutical dosage forms include but are not limited to: parenteral dosage forms or oral preparations, the parenteral dosage forms include injections, aerosols, suppositories or subcutaneous dosage forms; the oral preparations include tablets, capsules, pills, granules, microcapsule tablets, suspensions, pellets, oral liquid preparations. In a specific embodiment of the present invention, the pharmaceutical dosage form is preferably a parenteral dosage form.

[0021] In some embodiments, the administration route of the pharmaceutical composition of the present invention is not limited, as long as it can exert the desired therapeutic effect, and the administration route of the pharmaceutical composition includes but is not limited to: topical, through the skin, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, inhalation, through the pleura, through the mucosa, skin, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, ​​intrahepatic. In some cases, it can be administered systemically, and in some cases, it can be administered locally.

[0022] In some embodiments, the dosage of the pharmaceutical composition of the present invention is not limited as long as the desired therapeutic effect can be obtained, and can be appropriately determined according to the subject's symptoms, gender, age, etc. The dosage of the pharmaceutical composition of the present invention can be determined in detail using, for example, the therapeutic effect on the disease as an indicator.

[0023] Furthermore, the pharmaceutical composition includes alendronate sodium and other bone resorption inhibitors.

[0024] The alendronate sodium described in the present invention is a third-generation aminobisphosphonate bone metabolism regulator, which has a strong affinity with hydroxyapatite in the bone and can enter the hydroxyapatite crystals in the bone matrix. When osteoclasts dissolve the crystals, the drug is released, which can inhibit the activity of osteoclasts and indirectly inhibit bone resorption through osteoblasts.

[0025] Furthermore, the treatment includes promoting osteoblast differentiation or maturation.

[0026] In the present invention, the treatment refers to slowing down, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of existing symptoms, symptoms, conditions or diseases (e.g., osteoporosis). The desired therapeutic effects include, but are not limited to: preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.

[0027] The osteoblasts described in the present invention are cells that play an important role in bone formation and growth and development, and are mainly differentiated from mesenchymal progenitor cells in the periosteum and bone marrow. Osteoblasts can specifically secrete a variety of bioactive substances to regulate and affect the formation and reconstruction process of bones. Osteoblasts originate from multipotent mesenchymal cells of the bone marrow matrix. At different maturation stages, osteoblasts show four different forms in the body: preosteoblasts, osteoblasts, osteocytes and team cells. Preosteoblasts are precursors of osteoblasts and develop along the osteoblast lineage; mature osteoblasts are monolayer cells located on the surface of bones and bear the important function of synthesizing bone matrix. Osteoblasts are the main functional cells of bone formation and are responsible for the synthesis, secretion and mineralization of bone matrix. During bone formation, osteoblasts undergo four stages: proliferation, extracellular matrix maturation, extracellular matrix mineralization and apoptosis. Osteoblasts can secrete a variety of functional proteins, such as type I collagen, osteocalcin, osteopontin, etc., as well as non-collagenous proteins. Osteoblasts are also rich in alkaline phosphatase (ALP) and are involved in the regulation of multiple hormones and growth factors.

[0028] Furthermore, the patient being treated is a male.

[0029] The second aspect of the present invention provides a method for promoting osteoblast differentiation and maturation for non-therapeutic purposes in vitro, the method comprising the following steps: treating osteoblasts with an effective amount of an agent capable of activating the WNT signaling pathway or a pharmaceutical composition comprising an effective amount of an agent capable of activating the WNT signaling pathway.

[0030] Furthermore, methods for detecting osteoblast differentiation and maturation include, but are not limited to: detecting osteoblast alkaline phosphatase activity, detecting osteoblast differentiation by Alizarin red staining, detecting osteoblast-related marker mRNA expression levels by Real-time PCR, detecting osteoblast marker protein expression levels by Western blot, and the like.

[0031] In the present invention, the term "effective" means sufficient to achieve a desired, expected or intended result.

[0032] Furthermore, the osteoblasts are derived from male animals or male subjects.

[0033] Furthermore, the pharmaceutical composition comprises alendronate sodium.

[0034] Furthermore, the agent includes an agent that inhibits the expression or activity of DKK1.

[0035] Furthermore, the agent for inhibiting the expression of DKK1 includes an agent for inhibiting the expression of DKK1 gene mRNA or the expression of DKK1 protein.

[0036] Furthermore, the agent for inhibiting the activity of DKK1 includes a DKK1 monoclonal antibody.

[0037] A third aspect of the present invention provides a system for promoting differentiation and maturation of osteoblasts, the system comprising a treatment unit for treating osteoblasts with a therapeutically effective amount of an agent capable of activating the WNT signaling pathway.

[0038] The term "effective amount" refers to the therapeutic amount required to alleviate at least one or more symptoms of a disease or condition, and relates to a sufficient amount of a drug that provides the desired effect. Therefore, the term "therapeutically effective amount" refers to a therapeutic amount sufficient to cause a specific effect when applied to a typical subject. In various contexts, an effective amount as used herein also includes an amount sufficient to delay the development of a disease condition, change the course of the disease (for example, but not limited to, slowing the progression of disease symptoms), or reverse the disease condition. It should be understood that there are many ways known in the art to determine the effective amount for a given application. For example, pharmacological methods for dose determination can be used in the treatment context. In the context of therapeutic or preventive applications, the amount of the composition applied to the subject will depend on the type and severity of the disease and the characteristics of the individual, such as overall health, age, sex, weight and tolerance to the drug. It also depends on the degree, severity and type of the disease. Those skilled in the art will be able to determine the appropriate dose based on these and other factors. For example, the therapeutically effective amount of Daidzin can be determined by referring to its current safe use amount for the treatment of arterial thrombotic disease patients for the treatment of arterial thrombotic disease, and by clinical investigation. The appropriate effective dosage also needs to take into account therapeutic factors such as the dosage form of the drug, the constitution, weight, age, disease progression, and administration site of the individual being administered.

[0039] Further, the agent includes an agent that inhibits the expression or activity of DKK1;

[0040] Preferably, the agent for inhibiting the expression of DKK1 includes an agent for inhibiting the expression of DKK1 gene mRNA or inhibiting the expression of DKK1 protein;

[0041] Preferably, the agent that inhibits the activity of DKK1 comprises a DKK1 monoclonal antibody.

[0042] Furthermore, the system also includes a determination unit, which is used to determine the amount of DKK1 monoclonal antibody used by osteoblasts.

[0043] In certain specific embodiments, the system provided by the present invention includes a computer-readable storage medium for storing computer-readable programs or instructions. When the programs or instructions are executed by the processor, the functions of the treatment unit or the judgment unit in the system of the present invention can be realized.

[0044] A fourth aspect of the present invention provides a method for screening candidate drugs for treating osteoporosis, the method comprising the following steps:

[0045] Treating a system expressing or containing DKK1 with a test substance;

[0046] Detecting the expression or activity of DKK1 in the system;

[0047] Test substances that can inhibit the expression or activity of DKK1 are selected as candidate drugs.

[0048] Furthermore, the system is selected from: a cell system (such as MG-63, ROS17 / 2, MC3T3-E1, hFOB, etc.), a subcellular system, a solution system, a tissue system, an organ system or an animal system.

[0049] Furthermore, the test substances include but are not limited to: nucleic acid inhibitors and small molecule compounds designed for DKK1;

[0050] Preferably, the nucleic acid inhibitor is selected from: dsRNA, antisense nucleic acid, small interfering RNA, micro RNA; or a construct capable of expressing or forming the dsRNA, antisense nucleic acid, small interfering RNA, micro RNA;

[0051] Preferably, the source of the small molecule compound is selected from: newly synthesized or existing databases; wherein the existing databases include but are not limited to general natural product databases (COCONUT, SuperNatural II, NPASS), plant natural product databases (KNApSaCK, CMAUP, TriForC, Alkamid, NPACT DB, BioPhytMol), Chinese medicine natural product databases (TCM@Taiwan, CEMTDD, CHDD, ETCM, TM-MC, TCMID, YaTCM), microbial natural product databases (StreptomeDB, NPAltas, ProCarDB, PAMDB, Lichen Database), marine natural product databases (MNPD, SWMD), natural product databases of different countries and regions (IMPPAT, NeMedPlant, MedPServer, TlPdb, AfroDB, ANPDB, BIOFACQUIM, NUBBEDB), food natural product databases (FooDB, BitterDB, Phenol-Explorer, PhytoHub, SuperSweet database), toxic natural product databases (Exposome-Explorer, T3DB, Snake Neurotoxin Database, TPPT), natural product industry catalogs (Greenpharma, AnalytiConDiscovery, InterBioScreen, Indofine Chemical Company, Pi Chemicals Systems\Specs, TargetMol), databases for deduplication using MS data (MoNA, MassBank, METLIN, HMDB, YMDB, ReSpect, GNPS), databases for deduplication using NMR data (NMRShiftDB, NAPROC-13), etc.

[0052] Furthermore, the method for detecting DKK1 mRNA expression includes, but is not limited to, reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, ribonuclease protection assay (RPA), Northern blotting and DNA chip.

[0053] Preferably, the reagent for detecting the expression level of DKK1 mRNA comprises a primer, a probe or an antisense nucleotide that specifically binds to the mRNA encoding the DKK1 gene. Information about the DKK1 protein can be obtained through NCBI, and those skilled in the art can design primers, probes or antisense nucleotides that specifically bind to the mRNA of the gene encoding the protein based on the information.

[0054] The term "primer" is a chain of a short nucleic acid sequence that recognizes a target gene sequence, which includes a pair of forward and reverse primers. Specifically, the "primer" includes a pair of primers that provide specific and sensitive analysis results. Primers are believed to provide a high degree of specificity when used to amplify a target gene sequence, but they do not cause amplification of non-target sequences that are inconsistent with or complementary to the target gene sequence.

[0055] The term "probe" refers to a substance that specifically binds to a target to be detected in a sample. Through the binding, the probe can determine the presence of the target in the sample. As long as it is commonly used in the art, any probe can be used in the present disclosure. In particular, the probe can be PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA, most preferably PNA. Specifically, the probe is a biological material that can come from an organism or can be synthesized in vitro or is a mimetic thereof. For example, the probe can be an enzyme, protein, antibody, microorganism, animal or plant cell or organ, neuron, DNA or RNA. DNA can include cDNA, genomic DNA and oligonucleotides. Similarly, genomic RNA, mRNA and oligonucleotides can fall within the scope of RNA. Examples of proteins include antibodies, antigens, enzymes and peptides.

[0056] The term "antisense" refers to an oligomer having a nucleotide base sequence and a subunit, subunit backbone that allows the antisense oligomer to hybridize to a target sequence in RNA by Watson-Crick base pairing to form an RNA:oligomer heteroduplex nucleic acid molecule in the target sequence.

[0057] Further, the method for detecting the expression level of DKK1 protein includes but is not limited to: protein chip assay, immunoassay, ligand binding test, MALDI-TOF (matrix-assisted laser desorption / ionization time-of-flight mass spectrometry), SELDI-TOF (surface-enhanced laser desorption / ionization time-of-flight mass spectrometry), radioimmunoassay, radial immunodiffusion, bidirectional immunodiffusion (Ouchterlony immunodiffusion), rocket immunoelectrophoresis, immunohistochemical staining, complement fixation test, 2-D electrophoresis, liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), immunoblotting, and ELISA (enzyme-linked immunosorbent assay).

[0058] Preferably, the reagent for detecting the expression level of DKK1 protein may comprise an antibody, an oligopeptide, a ligand, a PNA (peptide nucleic acid) or an aptamer that can specifically bind to the protein DKK1.

[0059] The term "antibody" refers to a substance that specifically binds to an antigen to induce an antigen-antibody reaction. For the purpose of the present disclosure, the term "antibody" means an antibody that specifically binds to a DKK1 protein. Within the scope of antibodies of the present disclosure are polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. These antibodies can be easily prepared using techniques well known in the art. In addition, the antibodies that can be used in the present disclosure can be complete antibodies consisting of two full-length light chains and two full-length heavy chains, or functional fragments of complete antibody molecules. The term "functional fragment" of an antibody molecule means a fragment that retains the antibody binding function.

[0060] The term "PNA (peptide nucleic acid)" refers to an artificially synthesized polymer similar to DNA or RNA, first introduced in 1991 by Professors Nielsen, Egholm, Berg and Buchardt (University of Copenhagen, Denmark). DNA has a phosphate-ribose backbone, while the backbone of PNA consists of repeated N-(2-aminoethyl)-glycine units connected by peptide bonds. Due to this structure, PNA significantly enhances the affinity and stability of DNA or RNA and is therefore effectively used in molecular biology research, diagnosis, and antisense therapy.

[0061] The term "aptamer" is an oligonucleotide or peptide molecule that binds to a specific target molecule.

[0062] Furthermore, the step includes: detecting changes in the expression or activity level of DKK1 in the system, and comparing with a control group, wherein the control group is a system containing DKK1 without adding the candidate substance; if the candidate substance statistically inhibits (preferably significantly inhibits, such as being lower by 20% or more, preferably lower by 50% or more; more preferably lower by 80% or more) the expression or activity level of DKK1, it indicates that the candidate drug is a potential substance for treating osteoporosis.

[0063] A fifth aspect of the present invention provides a pharmaceutical composition for treating osteoporosis.

[0064] Furthermore, the pharmaceutical composition comprises an agent for activating the WNT signaling pathway and alendronate sodium.

[0065] Furthermore, the agent includes an agent that inhibits the expression or activity of DKK1.

[0066] Furthermore, the agent for inhibiting the expression of DKK1 includes an agent for inhibiting the expression of DKK1 gene mRNA or the expression of DKK1 protein.

[0067] Furthermore, the agent for inhibiting the activity of DKK1 includes a DKK1 monoclonal antibody.

[0068] Furthermore, the dosage form of the pharmaceutical composition described in the present invention is a dosage form that is prepared by conventional methods and is convenient for administration, including but not limited to: aqueous solution injection, powder injection, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder sprays, sustained-release agents and controlled-release agents, etc.

[0069] In the present invention, unless otherwise specified, the terms "comprise", "include" and "contain" are open expressions, meaning that in addition to the listed elements, components and steps, other unspecified elements, components and steps may also be included.

[0070] In the present invention, unless the context clearly indicates otherwise, the term "or" is used herein to mean the term "and / or" and can be used interchangeably with the terms.

[0071] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0072] The present invention proposes for the first time a new use of DKK1 antibody in the treatment of male osteoporosis, and finds that DKK1 antibody followed by sodium alendronate has a better therapeutic effect on the orchiectomized osteoporosis mouse model. The present invention provides a theoretical basis and a new treatment idea for the treatment of male osteoporosis, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 : is the SDS-PAGE electrophoretogram of the DKK1 antibody used in the present invention, wherein A is a typical electrophoretogram of non-reducing SDS-PAGE, and B is a typical electrophoretogram of reducing SDS-PAGE;

[0074] Figure 2 This is a diagram to verify the results of establishing an osteoporosis model in testicular mice;

[0075] Figure 3 This is a graph showing the effects of different treatment groups on bone density in orchiectomized mice. DETAILED DESCRIPTION

[0076] The present invention demonstrates the application of DKK1 monoclonal antibody in treating male osteoporosis by constructing an orchiectomized mouse osteoporosis model (ORX), and conducts exploratory treatment on the orchiectomized mouse osteoporosis model by sequential administration of DKK1 monoclonal antibody and alendronate sodium, thereby exploring the potential application value of DKK1 monoclonal antibody and sequential administration of alendronate sodium in treating male osteoporosis.

[0077] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Simple improvements to the present invention made according to the essence of the present invention all fall within the scope of protection claimed in the present invention.

[0078] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present invention. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0079] Example 1 Construction of orchiectomized osteoporosis model

[0080] 1. Experimental methods

[0081] (1) A total of 40 12-week-old C57BL6 mice were used in the present invention, of which 32 mice underwent orchiectomy and 8 mice underwent sham surgery.

[0082] (2) Surgical procedure:

[0083] ① Before surgery: adjust the flow rate and velocity of the gas anesthesia machine to anesthetize the mouse.

[0084] ②Surgical operation: After the mouse is anesthetized, fix it in a supine position, press the abdomen gently with your fingers to make the testicle enter the scrotum, cut the hair around the surgical site, and disinfect with iodine. Make an incision in the scrotum to cut the scrotal skin, separate the bilateral testicles and epididymis, ligate between the head and tail of the epididymis, and then remove the testicles, and then suture the skin layer by layer.

[0085] ③After surgery: To prevent infection, intramuscular injection of penicillin 20,000 u / d is performed from the day of surgery to 5 days after surgery.

[0086] ④ Operation method for sham-operated mice: make an incision in the scrotum and cut the scrotal skin, separate the bilateral testicles and epididymis, put them back into the scrotum without removal, suture the incision, and the rest of the steps are the same as those for orchiectomized mice.

[0087] ⑤ After the operation, the healing status of the surgical incision and the activity status of the mice were closely observed, and then the osteoporosis induction period of 2 months was entered.

[0088] (3) Model verification method: Two months after surgery, 32 orchidectomized mice were randomly divided into 4 groups and subjected to baseline bone density measurement (small animal dual-energy X-ray absorptiometry, INSIGHT VET DXA, OsteoSys) together with the sham-operated mice to verify whether the osteoporosis model was successfully established.

[0089] 2. Experimental results

[0090] The model validation results are as follows Figure 2 As shown in the figure, the bone density of orchiectomized mice was significantly lower than that of sham-operated mice, indicating that the osteoporosis model was successfully established.

[0091] Example 2 Efficacy and safety of DKK1 monoclonal antibody in the treatment of osteoporotic mice with orchiectomy

[0092] 1. Experimental methods

[0093] (1) Treatment groups and interventions: The treatment was divided into 5 groups. The specific intervention methods, treatment doses and usage are shown in Table 1.

[0094] Table 1 Treatment groups

[0095]

[0096] The orchidectomized mice were divided into 4 groups: the first group was the DKK1 monoclonal antibody treatment group, which received subcutaneous injections of DKK1 monoclonal antibody 25 mg / kg / time twice a week; the second group was the DKK1 monoclonal antibody sequential alendronate sodium treatment group, which received subcutaneous injections of DKK1 monoclonal antibody 25 mg / kg / time twice a week in the first month and subcutaneous injections of alendronate sodium 1 mg / kg / time once a week in the second month; the third group was the alendronate sodium treatment group, which received subcutaneous injections of alendronate sodium 1 mg / kg / time once a week; and the fourth group was the normal saline group, which received subcutaneous injections of normal saline 1 mL / kg / time once a week.

[0097] Sham-operated mice were subcutaneously injected with normal saline (1 mL / kg / time) once a week.

[0098] The amino acid sequence of the DKK1 monoclonal antibody used in the present invention is as follows:

[0099] Light chain variable region:

[0100] DIQMTQSPSSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKLLISYTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYSKLPLTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:1)

[0101] Heavy chain variable region:

[0102] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSSISTGGTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVSEIYWPMDFWGQGTL VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ IDNO:2)

[0103] (2) Determination of bone density during treatment:

[0104] Before treatment, the baseline bone density of each group of mice was measured (small animal dual-energy X-ray absorptiometry, INSIGHTVET DXA, OsteoSys), and the results were recorded;

[0105] After one month of treatment, bone density was measured again to analyze the bone density after one month of treatment.

[0106] After 2 months of treatment, bone density was measured again to analyze the bone density after 2 months of treatment.

[0107] 2. Experimental results

[0108] (1) Construction results of DKK1 antibody

[0109] The SDS-PAGE electrophoresis diagram of the DKK1 monoclonal antibody constructed by the present invention is as follows: Figure 1 As shown, the results showed that the purified DKK1 monoclonal antibody was successfully constructed.

[0110] (2) Therapeutic effect of DKK1 antibody

[0111] The results of bone density measurement before treatment, 1 month after treatment and 2 months after treatment are as follows Figure 3As shown in the figure, after 1 month of treatment, the bone density of mice in the orchidectomy DKK1 monoclonal antibody treatment group was significantly higher than that in the orchidectomy saline group. After 2 months of treatment, the bone density of mice in the orchidectomy DKK1 monoclonal antibody treatment group was higher than that in the orchidectomy saline group, and the bone density of mice in the orchidectomy DKK1 monoclonal antibody sequential alendronate sodium treatment group was significantly higher than that in the orchidectomy saline group.

[0112] Using King's formula expression q = E A+B / (E A +E B -E A ×E B ) To determine whether the combination of the two (DKK1 monoclonal antibody followed by alendronate sodium) has a synergistic effect in the treatment of osteoporosis. A 、E B 、E A+B They represent the cure rates of the two drugs used separately and in combination, respectively. A q value between 0.85 and 1.15 represents an additive effect, a q value > 1.15 represents a synergistic effect, and a q value < 0.85 represents an antagonistic effect.

[0113] The calculation process using King's formula is as follows: normal bone density is 0.0851, and osteoporotic bone density is 0.0788;

[0114] After treatment with DKK1 monoclonal antibody, the bone density became 0.0854, E A =(0.0854-0.0788) /

[0115] (0.0851-0.0788)=1.048;

[0116] After treatment with alendronate, the bone density became 0.0856, E B =(0.0856-0.0788) /

[0117] (0.0851-0.0788)=1.079;

[0118] After sequential treatment with DKK1 monoclonal antibody and alendronate sodium, the bone density became 0.0897, E A+B =(0.0897-0.0788) / (0.0851-0.0788)=1.730;

[0119] q=E A+B / (E A +E B -E A ×E B )=1.737;

[0120] The above results indicate that the sequential administration of DKK1 monoclonal antibody and alendronate sodium has a synergistic effect in the treatment of osteoporosis.

[0121] Example 3 Screening for candidate drugs for treating osteoporosis using the relationship between DKK1 and osteoporosis treatment

[0122] 1. Experimental methods

[0123] (1) Search for small molecule compounds as test substances from existing public databases.

[0124] (2) The test substance is placed in a cell system containing DKK1 protein, and a cell system without the test substance but containing DKK1 protein is used as a control group. RT-PCR is used to detect the DKK1 expression levels in the experimental group and the control group.

[0125] 2. Experimental results

[0126] If the candidate substance statistically inhibits (preferably significantly inhibits, such as by more than 20%, preferably by more than 50%, and more preferably by more than 80%) the expression or activity level of DKK1, it indicates that the candidate drug is a potential substance for treating osteoporosis.

[0127] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of DKK1 monoclonal antibody in the preparation of a pharmaceutical composition for treating osteoporosis; The DKK1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:1; and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

2.

2. Use of DKK1 monoclonal antibody and alendronate sodium in combination for preparing a pharmaceutical composition for treating osteoporosis; The DKK1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:1; and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

2.

3. The use according to claim 2, characterized in that The treatment includes promoting osteoblast differentiation or maturation.

4. The use according to claim 2, characterized in that The patients treated were males.

5. The use according to claim 2, characterized in that The dosage ratio of the DKK1 monoclonal antibody to alendronate sodium is 25 mg / kg:1 mg / kg.

6. The use according to claim 2, characterized in that The DKK1 monoclonal antibody and alendronate sodium are administered sequentially.

7. A pharmaceutical composition for treating osteoporosis, characterized in that: The pharmaceutical composition consists of DKK1 monoclonal antibody and alendronate sodium; The DKK1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:1; and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

2.

8. The pharmaceutical composition according to claim 7, characterized in that The dosage ratio of the DKK1 monoclonal antibody to alendronate sodium is 25 mg / kg:1 mg / kg.

9. The pharmaceutical composition according to claim 7, characterized in that The DKK1 monoclonal antibody and alendronate sodium are administered sequentially.

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