Use of ip6k2 inhibitors in the preparation of a medicament for the treatment of osteoporosis

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a use of an IP6K2 inhibitor in preparation of a drug for treating osteoporosis. The application finds that IP6K2 regulates osteoclast activity through a series of experiments, and targeting IP6K2 can be used as a treatment method for treating OP. The application mainly finds that IP6K2 is a key target point for regulating osteoclast activity, and inhibiting IP6K2 in osteoclasts (in mature osteoclasts and osteoclast precursors) or the whole body can significantly improve bone mass.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of IP6K2 inhibitors in the preparation of drugs for the prevention, relief or treatment of osteoporosis. Background Technology

[0002] Bone mass maintenance in the human body depends on the dynamic balance between osteoblast and osteoclast activity. The Rank1-Rank signaling axis plays a central role in osteoclast regulation. As key cells maintaining skeletal homeostasis, osteoclasts play a crucial role in regulating bone mass. Therefore, targeting osteoclasts has been proven to be an important strategy for treating osteoporosis (OP).

[0003] The current clinical strategy involves injecting Rank1 neutralizing antibodies (such as denosumab) into the body to effectively reduce Rank1 levels and function, thereby inhibiting osteoclast activity and increasing bone mass. However, this type of therapy has significant limitations, including side effects such as hypocalcemia, drug-related osteonecrosis of the jaw (ONJ), nausea, headache, and gastrointestinal reactions. Of particular note is the potential for rapid bone loss ("rebound effect") after discontinuation of the drug.

[0004] IP6K is a molecule involved in many intracellular signaling pathways through the production of inositol pyrophosphate. Inositol pyrophosphate is a low-energy molecule, and its production is known to be mediated by two enzymes: IP6K with 5-kinase activity and inositol diphosphate pentaphosphate kinase (PPIP5K) with 1-kinase activity. Mammals possess three IP6K subtypes: IP6K1, IP6K2, and IP6K3. Recent studies using knockout mice have explored the physiological roles of each molecule and its potential as a drug discovery target. IP6K1 knockout mice showed increased Akt signaling in the liver, adipose tissue, and muscle, and demonstrated improved glucose tolerance, enhanced insulin sensitivity, and increased muscle mass under high-fat dietary load conditions. IP6K1 protects bone mineral density by promoting osteogenic differentiation and inhibiting bone marrow steatosis, particularly in models of metabolic diseases. IP6K3 is a molecule highly expressed in muscle. IP7, produced by IP6K2, plays a major mediating role in cancer cell migration and tumor metastasis. It has been confirmed that IP6K2 deficiency in cancer cells inhibits cell invasion and metastasis. It is mainly involved in systemic metabolism and phosphate homeostasis, but there are no reports of its correlation with bone metabolism regulation. Given the limitations and safety risks of existing Rank1-targeted therapies, developing novel and safer osteoclast inhibition strategies is of significant scientific value and clinical necessity for the effective and safe treatment of osteoporosis. Summary of the Invention

[0005] This invention aims to provide the use of IP6K2 inhibitors in the preparation of drugs for the prevention, mitigation, or treatment of osteoporosis. This invention experimentally demonstrates that IP6K2 expression increases with osteoclast activation; in vitro and in vivo experiments demonstrate that inhibiting IP6K2 can suppress osteoclast differentiation and increase bone mass; using IP6K2 inhibitors, it is further confirmed that inhibiting IP6K2 can effectively suppress osteoclast activity in vivo and increase bone mass. The combined results indicate that IP6K2 can serve as a novel therapeutic target molecule for osteoporosis.

[0006] In one aspect, the use of an IP6K2 inhibitor in the preparation of a medicament for the prevention, mitigation, diagnosis or treatment of osteoporosis is provided.

[0007] In some embodiments, the IP6K2 inhibitor is a drug used to inhibit osteoclast formation or activity.

[0008] In some embodiments, the IP6K2 inhibitor is a drug used to inhibit bone resorption.

[0009] In some embodiments, the IP6K2 inhibitor is a drug for improving bone mass.

[0010] In some embodiments, the IP6K2 inhibitor is selected from siRNA or UNC7467 that target IP6K2.

[0011] In some embodiments, the IP6K2 inhibitor is administered daily at a dose of 0.001 mg / kg to 15 mg / kg, preferably 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, or 15 mg / kg.

[0012] In some embodiments, the osteoporosis is postmenopausal osteoporosis, osteoporosis caused by estrogen deficiency, or osteoporosis due to aging.

[0013] Secondly, the invention provides the use of a reagent for detecting the expression level of the IP6K2 gene in the preparation of products for diagnosing osteoporosis.

[0014] In some embodiments, the product includes: a product for diagnosing osteoporosis by detecting IP6K2 gene expression using RT-PCR, real-time quantitative PCR, immunoassay, in situ hybridization, chip or high-throughput sequencing platforms.

[0015] On the other hand, the present invention provides a pharmaceutical composition for the prevention, mitigation, diagnosis or treatment of osteoporosis, comprising: a preventive or therapeutically effective amount of an IP6K2 inhibitor and a pharmaceutically acceptable excipient or carrier.

[0016] In some embodiments, the IP6K2 inhibitor includes UNC7467.

[0017] In some embodiments, the pharmaceutical composition may further comprise a suitable carrier, excipient, or diluent typically used in the preparation of the pharmaceutical composition.

[0018] In particular, drugs can be formulated into oral dosage forms, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external dosage forms, suppositories, or sterile injectable solutions, according to conventional methods.

[0019] In this invention, the carrier, excipient, and diluent comprising the composition may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and minerals. These formulations can be prepared using common diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc.

[0020] Examples of solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc. These solid dosage forms are prepared by mixing an extract or a portion thereof with at least one excipient (e.g., starch, calcium carbonate, sucrose, lactose, gelatin, etc.). In addition to simple excipients, lubricants such as magnesium stearate or talc may also be used.

[0021] Examples of liquid formulations for oral administration include suspensions, oral solutions, emulsions, syrups, etc. Besides liquid paraffin or water, liquid formulations may also contain commonly used simple diluents and various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc.

[0022] Preparations intended for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories.

[0023] Non-aqueous solvents and suspensions can be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, or injectable esters such as ethyl oleate. The matrix of suppository formulations can be Witepsol, macrogol, Tween 61, cocoa butter, lauryl acetate, glycerin gel, etc.

[0024] The content of the IP6K2 inhibitor in the pharmaceutical composition of the present invention may be, but is not particularly limited to, for example, 0.0001% to 10% by weight, or 0.01% to 3% by weight.

[0025] The pharmaceutical compositions of the present invention can be administered in pharmaceutically effective amounts. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat or prevent disease with a reasonable benefit / risk ratio suitable for any medical treatment or prevention, and the effective dose level can be determined based on factors including disease severity, drug activity, patient age, weight, health and sex, sensitivity to the drug, time of administration, route of administration and excretion rate of the composition of the present invention, duration of treatment, drugs used concurrently or in combination with the composition of the present invention, and other factors known in the medical field.

[0026] The pharmaceutical compositions of the present invention can be administered alone or in combination with other known treatments for osteoporosis. In view of all the foregoing factors, it is important to administer the composition in the minimum amount necessary to achieve maximum efficacy without causing side effects.

[0027] Those skilled in the art can determine the dosage of the pharmaceutical composition of the present invention by taking into account the intended use, the severity of the patient's disease, age, weight, sex and medical history, and the various components used as the active ingredient. For example, the pharmaceutical composition of the present invention can be administered at a rate of about 0.1 ng / kg / adult to about 100 mg / kg / adult, preferably about 1 ng / kg / adult to about 10 mg / kg / adult, and there is no particular limitation on the frequency of administration of the composition of the present invention, but the composition of the present invention can be administered once daily or in fractional doses. The dosage does not in any way constitute a limitation on the scope of the present invention.

[0028] The pharmaceutical composition of the present invention for treating osteoporosis can be administered via any commonly used route, as long as it can reach the desired tissue. Depending on the intended purpose, the pharmaceutical composition of the present invention can be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonaryly, or rectally, but the invention is not particularly limited thereto. However, since IP6K2 inhibitors may be denatured by gastric acid upon oral administration, the active ingredient of the composition for oral administration should be coated or formulated to prevent degradation in the stomach. Additionally, the composition can be administered using a device capable of delivering the active ingredient to target cells.

[0029] Compared with the prior art, one of the above technical solutions has the following advantages or beneficial effects: This invention, through a series of experiments, confirms that IP6K2 regulates osteoclast activity, and that targeting IP6K2 can be used as a therapeutic approach for osteoporosis (OP). The main finding of this invention is that IP6K2 is a key target for regulating osteoclast activity; inhibiting IP6K2 in osteoclasts (both mature and precursors) or systemically can significantly improve bone mass. Therefore, gene editing technologies (Cre-Loxp or CRSPR / Cas, CasRx), gene knockdown technologies (siRNA or shRNA, etc.), and inhibitors (UNC) can all be used to treat OP. Attached Figure Description

[0030] Figure 1 This image shows the expression level of IP6K2 in osteoclasts after Rank1 stimulation. A is the cell fluorescence result; B is the quantitative statistical graph of relative fluorescence intensity.

[0031] Figure 2 This graph shows the decrease in osteoclast activity after IP6K2 osteoclast siRNA inhibition. A is a Western blotting result; B is a quantitative statistical graph.

[0032] Figure 3 The images show the results of IP6K2 knockout in osteoclast precursors. A is a flowchart of obtaining knockout mice; B is a bone tissue micrograph; C is a bone mineral density map; D is a bone volume fraction map; and E is a Trap staining map.

[0033] Figure 4 This is a graph showing the expression level of IP6K2 in bone marrow from a mouse single-cell sequencing database.

[0034] Figure 5 The diagram shows the effect of IP6K2 on 5-IP7 expression levels. A shows that there was no difference in intracellular 5-IP7 levels after IP6K2 knockdown. B shows that IP6K1 and IP6K3 compensatorily increased 5-IP7 levels after IP6K2 knockdown.

[0035] Figure 6 The figure shows the results of protecting mice from estrogen deficiency-induced bone loss after IP6K2 knockout, a precursor to osteoclasts. A is a morphological view of bone microstructure; B is a bone mineral density map of trabeculae; C is a trabecular bone volume fraction map; D is a histological section with TRAP staining (labeling osteoclasts); Figure E is a quantitative analysis of the proportion of osteoclasts on the bone surface (OC.S / BS).

[0036] Figure 7 The diagram shows the results of IP6K2 knockout in mature osteoclasts increasing bone mass in mice. A is a flowchart of obtaining knockout mice; B is a graph of mouse weight changes; C is a bone tissue micrograph; D is a bone mineral density graph; E is a bone volume fraction graph; F is a Trap staining graph; G is the number of osteoclasts.

[0037] Figure 8 The graph shows the results of using the IP6K2 inhibitor UNC for the treatment of osteoporosis. A is the Western Blot result; B is the quantitative statistical graph; C is the experimental flowchart; D is the body weight graph; E is the bone tissue micrograph; F is the bone mineral density graph; G is the bone volume fraction graph; H is the trap staining graph; I is the osteoclast count. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0039] All reagents used in this invention can be purchased commercially or prepared by the methods described in this invention.

[0040] Example 1: Knocking out IP6K2 in osteoclast precursors increases bone mass in mice (1) Purchased IP6K2 flox mice were mated with Lyz2-Cre mice, and IP6K2 mice were obtained through genotyping (PCR detection of LoxP site and Cre gene). flox / flox Lyz2-Cre conditional knockout (cKO) mice and their control mice (IP6K2) flox / flox Bone marrow cells from the femur and tibia of 6-8 week old mice were obtained. After removing red blood cells with ACK lysis buffer, they were induced and cultured for 3 days in α-MEM medium (containing 10% FBS and 1% penicillin / streptomycin) with M-CSF (10 ng / mL) and RANKL (50 ng / mL) to obtain bone marrow-derived macrophages (BMM).

[0041] like Figure 1 As shown in Figure A, the blue signal represents the cell nucleus, which is present in both groups, indicating a similar cell number; the green signal represents the IP6K2 protein. In the left (Ctrl) group: almost no green fluorescence is visible, indicating very low IP6K2 expression levels in unstimulated osteoclast precursor cells. In the right (rhRANKL) group: abundant bright, clear green fluorescence appears in the cells, indicating that IP6K2 expression is strongly induced and surges under RANKL stimulation.

[0042] Figure 1 B Figure 1The intensity of green fluorescence in column A was precisely quantified. The IP6K2 fluorescence intensity of the treatment group (red column) was several times higher than that of the control group (black column).

[0043] Studies have found that osteoclasts can be activated after Rankl induction, during which the expression level of IP6K2 increases significantly.

[0044] (2) In order to investigate whether IP6K2 can be used as a target for osteoclast activity and thus for OP treatment, siRNA technology was used for verification. In the control group BMM cells extracted above, siRNAs of siNC and siIP6K2 were added to knock down IP6K2, and mouse osteoclast activity marker genes were detected by WB and qPCR.

[0045] Figure 2 In this dataset, Ip6k2 is the protein product of the target gene. Runx2 and NFATc1 are core transcription factors regulating osteoclast differentiation. Ctsk is cathepsin K, a key functional enzyme used by mature osteoclasts to degrade the bone matrix. Gapdh is a housekeeping protein used as an internal control to ensure consistent sample loading and comparability. siNC is the negative control group, using nonsense siRNA to represent the protein expression level under normal conditions. silp6k2-1 and silp6k2-2 are two different IP6K2-specific siRNA treatment groups used as biological replicates.

[0046] Figure 2 A showed that compared with the siNC group, the Ip6k2 protein band was significantly lighter in both silp6k2 groups, proving that the gene knockdown was successful. The downstream Runx2, NFATc1, and Ctsk protein bands also showed a synchronous and significant lightening. The Gapdh band was uniform across all groups, indicating that the change was specific. Figure 2 B quantified the four target proteins (excluding the internal control Gapdh) in Figure A using grayscale values ​​and presented them as bar charts of relative protein levels (with the siNC group as 1). The results showed that osteoclast activity decreased after IP6K2 osteoclast siRNA inhibition. IP6K2 knockdown significantly inhibited osteoclast activity.

[0047] (3) IP6K2 flox mice were purchased and mated with Lyz2-Cre mice. The IP6K2 mice were obtained through genotyping (PCR detection of LoxP site and Cre gene). flox / flox (like Figure 3 As shown in A); Lyz2-Cre conditional knockout (cKO) mice and their control mice (IP6K2). flox / flox This system can knock out IP6K2 in osteoclast precursors. Left femurs were harvested from mice at 3 months of age (e.g.,...). Figure 3(As shown in B) µCT scans (SkyScan 1176, 50 kV, 200 µA, 9 µm resolution) were performed to analyze trabecular bone parameters in the distal femoral metaphysis. MicroCT examination revealed a significant increase in bone mass in mice, specifically a significant increase in bone mineral density (BMD) and bone volume fraction (BV / TV) (e.g., ...). Figure 3 C and Figure 3 (As shown in D). This demonstrates that IP6K2 deficiency alone leads to a high bone mass phenotype in mice. This suggests that IP6K2 could serve as a therapeutic target for osteoporosis. Trap staining analysis revealed a significant downregulation of osteoclast activity in mice (e.g., ...). Figure 3 As shown in E), this indicates that the high bone mass phenotype caused by IP6K2 deficiency alone is due to decreased osteoclast activity.

[0048] (4) Furthermore, it is noteworthy that analysis of IP6K2-expressing cells in mouse bone marrow using the publicly available mouse single-cell sequencing database (Tabula Muris) revealed that IP6K2 was highly expressed in hematopoietic stem cells rather than MSCs (osteoblast precursors), indicating that it is specifically distributed in osteoclast precursor cells rather than osteoblast precursors (e.g., Figure 4 (As shown). After siRNA knockdown, the level of 5-IP7 in the cell supernatant was detected by SDS-PAGE. 5-IP7 is the active product of IP6K1, IP6K2, and IP6K3. Knockdown of IP6K2 alone did not affect the level of 5-IP7, indicating that knockdown of IP6K2 alone does not affect the metabolism of IP7 in cells (e.g., ...). Figure 5 (As shown in A). This differs from its traditionally understood function. For example... Figure 5 The qPCR assay shown in Figure B indicates that knocking down IP6K2 leads to a compensatory increase in IP6K1 and IP6K3, which may explain why the 5-IP7 level remained unchanged. This also suggests that the function of IP6K2 is independent of IP6K1 and IP6K3.

[0049] Example 2: IP6K2 knockout protects against OVX-induced osteoporosis in mice Since estrogen deficiency can lead to bone loss, this study investigated whether estrogen deficiency-induced bone loss can be counteracted by IP6K2 deficiency.

[0050] A mouse ovariectomy (OVX) model was used to simulate human osteoporosis caused by estrogen deficiency. Two-month-old mice underwent bilateral ovariectomy via abdominal surgery, while a control group underwent sham surgery (abdominal surgery only, without ovariectomy). Two months post-OVX surgery, bone mass in the mice was assessed using microCT.

[0051] Figure 6Image A shows a microscopic image of bone microstructure in OVX mice lacking the IP6K2 osteoclast precursor after simulating osteoporosis. This image displays a micro-CT 3D reconstruction of the distal femoral metaphysis (top row shows 3D morphology, bottom row shows cross-sections). Visual comparison shows: sham-operated group (Sham): control group (Ctrl) vs. IP6K2 knockout group (Ip6k2...). LKO No significant differences were observed in bone mass and trabecular bone structure in the Ovarian Removal Group (OVX): OVX control group (Ctrl) mice showed significant bone loss, with sparse and broken trabeculae. In contrast, the OVX and IP6K2 knockout group (Ip6k2...) showed... LKO In mice, the trabecular bone structure was significantly preserved, bone density was higher, and the trabecular bone network was more continuous. This directly demonstrates that IP6K2 deficiency can effectively reduce bone destruction caused by OVX surgery.

[0052] Figure 6 B is a statistical plot of trabecular bone mineral density (Tb.BMD) in OVX-induced osteoporosis-simulated mice with IP6K2 osteoclast precursor deficiency, analyzing the mineral density of trabecular bone. Under sham surgery conditions, IP6K2 knockout had little effect on baseline bone mineral density. After OVX induction, bone mineral density decreased in both groups of mice. Although the IP6K2 knockout group (OVX's Ip6k2...) showed a lower bone mineral density... LKO The mean bone mineral density of the OVX group was higher than that of the OVX control group, but the difference was not statistically significant (marked as "ns").

[0053] Figure 6 C represents the statistical plot of trabecular bone volume fraction (Tb.BV / TV) in IP6K2 osteoclast precursor-deficient mice after OVX-induced osteoporosis, analyzing the percentage of trabecular bone volume to total tissue volume, a key indicator of bone mass. Under sham surgery conditions, there was no significant difference between the two groups. After OVX induction, the bone volume fraction in the control group decreased sharply. In stark contrast, the bone volume fraction in IP6K2 knockout mice was significantly protected, with values ​​far exceeding those in the OVX control group, reaching a very high level of statistical significance. This indicates that IP6K2 deficiency can extremely effectively prevent trabecular bone loss.

[0054] Figure 6 D shows the trap staining image of bone fragments from mice lacking IP6K2 osteoclast precursors after osteoporosis was simulated using OVX mice. The OVX-Ctrl group shows a large number of purplish-red osteoclasts, while the OVX-Ip6k2 group shows... LKO The number of osteoclasts in the group was significantly reduced.

[0055] Figure 6E shows the percentage of osteoclasts on the bone surface of mice lacking IP6K2 precursor osteoclasts after osteoporosis was simulated using OVX mice. OVX surgery significantly increased the number of osteoclasts in Ctrl mice, while IP6K2 deficiency significantly inhibited this OVX-induced osteoclast overactivation.

[0056] Figure 6 A, Figure 6 B and Figure 6 C showed that the deletion of the IP6K2 gene in osteoclast precursor cells significantly improved OVX-induced osteoporosis. Although the increase in bone mineral density was not statistically significant, IP6K2 deficiency significantly maintained the volume and microstructural integrity of bone trabeculae, thus playing a protective role for the bone as a whole. Figure 6 D- Figure 6 E revealed the cellular mechanism by which IP6K2 exerts its protective effect—namely, by inhibiting osteoclast production and / or activity, thereby reducing bone resorption and ultimately preserving bone mass.

[0057] Figure 6 The results showed that knocking out the osteoclast precursor IP6K2 during OVX surgery in mice significantly protected against OVX-induced bone loss, as evidenced by a significant increase in BMD and BV / TV in the knockout group. These results indicate that targeting IP6K2 can protect against estrogen deficiency-induced osteoporosis. This suggests that IP6K2 is a potential therapeutic target for preventing and treating postmenopausal bone loss.

[0058] Example 3: Knocking out IP6K2 in mature osteoclasts increases bone mass in mice Osteoclast differentiation occurs in two stages: osteoclast precursors and mature osteoclasts. To further validate the role of targeting IP6K2 in the treatment of osteoporosis (OP), mature osteoclasts (Cre) were used to investigate IP6K2 deficiency.

[0059] like Figure 7 As shown in Figure A, purchased IP6K2 flox mice were mated with CTSK-Cre mice, and IP6K2 mice were obtained through genotyping (PCR detection of the LoxP site and Cre gene). flox / flox CTSK-Cre conditional knockout (cKO) mice and their control mice (IP6K2) flox / flox Bone mass analysis was performed on mice at 3 months of age.

[0060] Figure 7 The following figures (A~7E) show the changes in body weight and bone mass in mice after IP6K2 knockout in mature osteoclasts: [Example data would be inserted here] Figure 7As shown in Figure B, there was no significant difference in body weight between the knockout group and the control group. The observed skeletal phenotype was not due to differences in systemic growth, development, or metabolism in the mice, but rather to specific changes in the skeletal system itself. Figure 7 As shown in C, the knockout group (Ip6k2) CKO The trabeculae in the CT group were denser, thicker, and more connected than those in the control group (Ctrl). Figure 7 Both bone mineral density (D) and bone volume fraction (7E) showed a significant increase. Bone mineral density reflects the mineral content of bone, while bone volume fraction reflects the amount of trabecular bone. The simultaneous increase in both clearly confirms that IP6K2 knockout directly leads to net bone mass increase. Specific knockout of IP6K2 in mature osteoclasts does not affect the overall physiological state of mice (such as body weight), but it significantly and specifically enhances bone and prevents bone loss, manifested as an increase in bone mineral density and bone mass.

[0061] Figure 7 F and Figure 7 G shows that knocking out IP6K2 in mature osteoclasts reduces the number of osteoclasts in mice. Figure 7 F is a TRAP staining image. TRAP is a marker enzyme of mature osteoclasts and appears dark after staining. Figure 7 F indicates: Knockout group (Ip6k2) CKO The number of stained osteoclasts on the bone sections was significantly less than that in the control group (Ctrl). Figure 7 The G assay showed a significant decrease in osteoclast numbers, statistically confirming a reduction in the number of mature osteoclasts. IP6K2 knockout resulted in reduced formation or impaired survival of functional osteoclasts on the bone surface. Since osteoclasts are the only cells in the body responsible for degrading (absorbing) bone tissue, a decrease in their number signifies weakened bone resorption activity.

[0062] Figure 7 The results showed that microCT scans revealed a significant increase in bone mass in mice. Trap staining analysis revealed a significant downregulation of osteoclast activity in mice. Knockout of IP6K2 in mature osteoclasts inhibited bone resorption by reducing the number of functional osteoclasts, resulting in increased bone mass in mice. This demonstrates that IP6K2 is a key regulator of osteoclast function in degrading bone tissue and plays a crucial role in bone metabolism.

[0063] Example 4: IP6K2 inhibitors for the treatment of osteoporosis (1) Based on the preliminary results of Examples 1-3, the treatment with IP6K2 inhibitors was validated in vitro. The results showed that the IP6K2 inhibitor UNC (UNC7467, CAS No.: 2922283-43-8) was effective. It can significantly inhibit key indicators of osteoclast differentiation.

[0064] like Figure 8 As shown in Figure A, when cells were treated with osteoclast differentiation inducers (such as RANKL) and different concentrations of UMC7467 were added, the protein expression levels of key indicators decreased in a dose-dependent manner with increasing UMC7467 concentration. Figure 8 As shown in Figure B, the WB bands in Figure A were quantified by grayscale values, and the resulting bar chart confirmed a significant decrease in the protein expression of the key indicator. Figure 8 Figures A and 8B show that the IP6K2 inhibitor UNC7467 effectively inhibits osteoclast differentiation in vitro. It downregulates the expression of transcription factors and functional proteins essential for osteoclast formation by blocking IP6K2 activity. This demonstrates at the molecular and cellular levels that the mechanism of action of UNC7467 is consistent with previous gene knockout studies, namely, that targeting IP6K2 can block the osteoclast generation pathway.

[0065] (2) In addition to estrogen deficiency, the pathogenic factors of osteoporosis also include aging, among which aging is one of the most important factors. To clarify whether targeting IP6K2 can be used for age-related osteoporosis, such as... Figure 8 As shown in Figure C, 18-month-old mice (equivalent to 60 years old in humans) were treated with UNC7467 via daily intraperitoneal injection at a dose of 5 mg / kg. Bone mass was measured and analyzed after 6 weeks of treatment.

[0066] Figure 8 D shows no significant change in weight. Figure 8 E shows a bone tissue micrograph, indicating that the trabeculae of mice treated with UNC7467 were denser and had better structure than those of the untreated aged control group. Figure 8 F, Figure 8 G and Figure 8 The changes in key indicators, including bone density, bone volume fraction, and osteoclast count, were significantly better than those in the control group, indicating that the bone tissue of aged mice was effectively restored after treatment. UNC7467 treatment can significantly increase bone mass in mice, and the number of osteoclasts on the bone surface of mice in the treatment group was significantly reduced. Figure 8 Trap staining of H cells revealed a large number of purplish-red osteoclasts adhering to the bone surface of the untreated elderly control group, while the number of osteoclasts in the treatment group (UNC) was significantly reduced. These results indicate that targeting IP6K2 can effectively protect against age-related osteoporosis. In an animal model simulating age-related osteoporosis, the IP6K2 inhibitor UNC7467 significantly increased bone mineral density, improved bone microstructure, and effectively reversed or prevented age-related bone loss. This strongly demonstrates that UNC7467 has a clear anti-osteoporosis efficacy at the whole animal level.

[0067] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. Use of an IP6K2 inhibitor in the preparation of a medicament for the prevention, mitigation, diagnosis or treatment of osteoporosis.

2. Use according to claim 1, characterized in that, The IP6K2 inhibitor is a drug used to inhibit osteoclast formation or activity, inhibit bone resorption, or improve bone mass.

3. The use according to claim 1, characterized in that, The IP6K2 inhibitor is selected from siRNA or UNC7467 that target IP6K2.

4. The use according to claim 1, characterized in that, The IP6K2 inhibitor is administered daily at a dose of 0.001 mg / kg to 15 mg / kg.

5. The use according to claim 1, characterized in that, The osteoporosis mentioned refers to postmenopausal osteoporosis, osteoporosis caused by estrogen deficiency, or osteoporosis caused by aging.

6. The use of a reagent for detecting IP6K2 gene expression in the preparation of products for diagnosing osteoporosis.

7. The use according to claim 5, characterized in that, The products include those for diagnosing osteoporosis by detecting IP6K2 gene expression using RT-PCR, real-time quantitative PCR, immunoassay, in situ hybridization, chip or high-throughput sequencing platforms.

8. A pharmaceutical composition for the prevention, relief, diagnosis, or treatment of osteoporosis, comprising: An effective amount of IP6K2 inhibitor for prevention or treatment and a pharmaceutically acceptable excipient or carrier.

9. The pharmaceutical composition according to claim 8, characterized in that, The IP6K2 inhibitor includes UNC7467.

10. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition is formulated into powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external dosage forms, suppositories, or sterile injectable solutions.

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