Application of TTP22 in the preparation of drugs for preventing and / or treating osteoporosis

Abstract

The present invention discloses the use of TTP22 in the preparation of a drug for preventing and / or treating osteoporosis. The present invention proposes that TTP22 inhibits the expression of TRAF6 and P-NIP30 proteins in the body, thereby affecting the downstream NF-κB and MAPK signaling pathways, thereby inhibiting osteoclast differentiation. Furthermore, the present invention discloses that TTP22 can significantly improve osteopenia and bone loss in OVX mice. Therefore, the present invention proposes that TTP22 provides a new direction for the research and development of drugs for the prevention, treatment, and prevention of osteoporosis.

Description

Technical Field

[0001] The present invention relates to the field of pharmacotherapeutics, and in particular to a new use of TTP22 in treating osteoporosis. Background Art

[0002] Osteoporosis is a systemic bone disease characterized by low bone mass and damaged bone microstructure, leading to increased bone brittleness and susceptibility to fractures. Based on the cause, it can be clinically divided into primary and secondary types. Primary osteoporosis includes postmenopausal osteoporosis (type I), senile osteoporosis (type II), and idiopathic osteoporosis (juvenile type). Secondary osteoporosis refers to osteoporosis caused by diseases or drugs that affect bone metabolism, or other clear causes.

[0003] A national osteoporosis epidemiological survey shows that the prevalence of osteoporosis in people over 50 years old is 19.2%, including 32.1% in women and 6.9% in men. The prevalence of osteoporosis in people over 65 years old is 32.0%, with 51.6% in women. With the aging population, the prevalence is expected to continue to rise. Osteoporosis leads to a decline in quality of life, increased disability and mortality rates, and a significant economic burden on families and society, making it a serious public health issue.

[0004] Osteoporosis is primarily caused by a disruption in the metabolic balance between osteoblasts and osteoclasts, leading to increased osteoclast activity. Current treatments for osteoporosis primarily focus on inhibiting osteoclast production, with the primary medications used being bisphosphonates, denosumab, teriparatide, and abaloparatide. However, bisphosphonates are poorly absorbed and can damage the digestive tract, while hormone therapy can increase the incidence of other diseases such as cancer and cardiovascular disease. Consequently, the development of safe and effective treatments has garnered widespread attention.

[0005] TTP22 (3-{[5-(4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl]thio}propanoic acid) is an ATP-competitive casein kinase 2 (CK2) inhibitor with enhanced selectivity for CK2 (Golub AG, et al. Synthesis and biological evaluation of substituted (thieno[2,3-d]pyrimidin-4-ylthio)carboxylic acids as inhibitors of human protein kinase CK2. Eur J Med Chem. 2011 Mar; 46(3): 870-6.). CK2 is a highly conserved serine / threonine protein kinase present in all eukaryotes. It is associated with many malignancies such as Alzheimer's disease, Parkinson's disease, Huntington's disease, diabetes, and lung cancer and breast cancer (Roffey SE, Litchfield DW. CK2 Regulation: Perspectives in 2021. Biomedicines. 2021 Sep 30; 9 (10): 1361.). Several studies have shown that CK2 is an upstream kinase of nuclear protein 30 (NIP30) and can promote NIP30 phosphorylation. NIP30 is an important regulatory factor of REGγ (PA28γ, PAME3, Ki antigen) in cells. Previous studies of our research group have shown that REGγ can regulate bone mass, but whether the CK2 / NIP30 / REGγ pathway can affect osteoporosis has not been reported. It is still unclear what the effect and mechanism of TTP22 on osteoclast differentiation and osteoporosis and other diseases are. Summary of the Invention

[0006] In order to overcome the defects of the existing technology, the present invention proposes that CK2 / NIP30 / REGγ is involved in the regulation of osteoporosis, and further proposes that TTP22 can be used to treat osteoporosis by regulating CK2 / NIP30 / REGγ.

[0007] TTP22 (3-{[5-(4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl]thio}propanoic acid) is an ATP-competitive casein kinase 2 (CK2) inhibitor. The specific structure is shown below:

[0008]

[0009] The present invention proposes the use of TTP22 in preparing a medicine for preventing and / or treating osteoporosis.

[0010] Furthermore, the TTP22 inhibits the differentiation of osteoclasts in vitro.

[0011] Furthermore, the TTP22 inhibits bone loss and bone loss, and increases the number of trabeculae and bone density.

[0012] Furthermore, the TTP22 downregulates the expression of TRAF6 in osteoclasts in vivo, inhibits NIP30 phosphorylation, and blocks signal transduction, thereby affecting its activation of downstream NF-κB and MAPK signaling pathways, thereby inhibiting osteoclast differentiation and improving bone loss.

[0013] The present invention also provides a medicine / pharmaceutical composition comprising the TTP22 as described above and a pharmaceutically acceptable carrier.

[0014] The drug / drug composition can be used alone or in combination with other drugs.

[0015] The medicament / pharmaceutical composition further comprises an excipient, a diluent, an adjuvant, a vehicle or a combination thereof.

[0016] The drug / pharmaceutical composition is formulated as an injectable fluid, aerosol, cream, gel, pill, capsule, syrup, transdermal patch or excipient.

[0017] The present invention also proposes the use of the medicine / drug composition in preparing a medicine for preventing and / or treating osteoporosis.

[0018] Furthermore, the drug / drug composition inhibits the differentiation of osteoclasts in vitro.

[0019] Furthermore, the drug / drug composition inhibits bone loss and bone loss, and increases the number of trabeculae and bone density.

[0020] Furthermore, the drug / drug composition downregulates the expression of TRAF6 in osteoclasts in vivo, inhibits NIP30 phosphorylation, and blocks signal transduction, thereby affecting its activation of downstream NF-κB and MAPK signaling pathways, thereby inhibiting osteoclast differentiation and improving bone loss.

[0021] The present invention has the following beneficial effects: It innovatively proposes the use of TTP22 in the preparation of a drug for the prevention and / or treatment of osteoporosis. TTP22 inhibits the expression of TRAF6 and P-NIP30 proteins in the body, thereby affecting the downstream NF-κB and MAPK signaling pathways, thereby inhibiting osteoclast differentiation. Furthermore, the present invention demonstrates that TTP22 can significantly improve osteopenia and bone loss in OVX mice. Therefore, the present invention provides a new direction for the research and development of drugs for the prevention, treatment, and prevention of osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Figure 2 shows the results of TTP22 inhibiting RANKL-induced osteoclast differentiation. Figure A shows the TRAP staining results of osteoclasts induced by BMMs under different concentrations of TTP22 treatment. Figure B shows the statistical results of TRAP staining of osteoclasts induced by BMMs under different concentrations of TTP22 treatment.

[0023] Figure 2 This is the result of the effect of TTP22 on the body weight of ovariectomized mice;

[0024] Figure 3 TTP22 improves bone loss in ovariectomized mice. Figure A shows representative Micro-CT images of 5-month-old mice in each group. Figure B shows the statistical analysis of relative bone volume and bone density in each group.

[0025] Figure 4 Figure 3: TTP22 inhibits the expression of phosphorylated TRAF6 and NIP30 proteins in osteoclasts in vivo. Figure A is a Western blot analysis of the expression of TRAF6, NIP30, P-NIP30, NF-κB, and MAPK signaling pathway proteins in the tissues of each group. Figure B is a quantitative analysis of the expression of TRAF6, NIP30, P-NIP30, NF-κB, and MAPK signaling pathway proteins in each group. DETAILED DESCRIPTION

[0026] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all of them. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention, its application, or use. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0028] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0029] Example 1 TTP22 has an inhibitory effect on osteoclast differentiation in vitro

[0030] Experimental Materials:

[0031] Ten specific pathogen-free experimental animals (SPF animals) were male mice of the C57BL / 6 strain, 6-8 weeks old, weighing (20 ± 5) g. They were purchased from the Minhang Laboratory Animal Center of East China Normal University and maintained under SPF animal husbandry conditions.

[0032] Experimental methods:

[0033] 1. Isolation, culture, and differentiation of mouse bone marrow-derived macrophages (BMMs) into OCs

[0034] 1) Male wild-type mice aged 6-8 weeks were sacrificed by cervical dislocation;

[0035] 2) The intact hind limbs of the mouse were peeled off and placed in sterile PBS buffer;

[0036] 3) Remove the muscles and tissues from the legs in a clean bench;

[0037] 4) Use a 1 mL syringe to draw PBS buffer to flush the cells in the bone into a centrifuge tube until the bone marrow cavity turns white;

[0038] 5) Centrifuge, resuspend the collected cell pellet in complete culture medium, transfer to a culture dish, and culture in a 37°C, 5% CO2 incubator;

[0039] 6) After 12 hours, collect the supernatant into a 15 mL centrifuge tube and centrifuge to discard the supernatant;

[0040] 7) Add 10 ng / mL M-CSF to the whole culture medium, resuspend the pellet, transfer to a new culture dish and culture for 24 hours. The adherent cells are BMMs.

[0041] 8) BMMs cells were trypsinized, centrifuged, resuspended in complete culture medium supplemented with M-CSF (10 ng / mL), plated, and divided into three groups;

[0042] 9) Different groups were treated with RANKL (50 ng / mL), RANKL+TTP22 (5 nM), and RANKL+TTP22 (10 nM), respectively. The culture medium was replaced every other day for 5 days. Obvious osteoclasts (OC) were obtained after about 6 days of induction.

[0043] 2. Tartrate-resistant acid phosphatase (TRAP) staining of osteoclasts

[0044] 1) The mature osteoclasts obtained above were fixed with 4% paraformaldehyde solution for 15 minutes at room temperature and then washed with PBS buffer;

[0045] 2) Permeabilize with 0.1% TritonX-100 at room temperature for 15 min, and then wash with PBS buffer;

[0046] 3) Incubate in PBST at room temperature for 20 min, then wash with PBS buffer;

[0047] 4) TRAP staining was performed on the incubated cells using a TRAP staining kit (Sigma) in the dark. The cells were incubated in a 37°C incubator in the dark for 60 min and then washed with PBS buffer.

[0048] 5) Add new PBS buffer to infiltrate the cells for observation and photography.

[0049] 3. Statistical methods

[0050] Data were processed using Prism software 8.0 (Graph Pad Software, San Diego, CA, USA). Data are presented as mean ± standard deviation (x-±s). Two sample means were compared using the independent sample t-test, and differences were considered statistically significant when P < 0.05.

[0051] Experimental results:

[0052] 1. TTP22 inhibits osteoclast differentiation

[0053] After being induced by RANKL, mouse bone marrow macrophages (BMMs) will form a large number of TRAP-positive osteoclasts. In order to study the effect of TTP22 on osteoclast differentiation, the present invention induced BMMs with RANKL and treated them with 5nM and 10nM TTP22, respectively. Figure 1TRAP staining results showed that the number and area of ​​osteoclasts decreased significantly with increasing TTP22 concentration. This suggests that TTP22 inhibits osteoclast differentiation in vitro in a dose-dependent manner. ***P < 0.001.

[0054] Example 2 TTP22 improves bone loss in ovariectomized mice

[0055] Experimental Materials:

[0056] Twenty-four specific pathogen-free experimental animals (SPF animals) female mice, C57 strain, 3 months old, weighing (25±2) g, were purchased from the Minhang Experimental Animal Center of East China Normal University and raised according to SPF animal husbandry conditions.

[0057] Experimental methods:

[0058] 1. Modeling and Grouping and Dosing

[0059] The above mice were randomly divided into four groups, namely Sham group, OVX group, TTP2215 mg / kg group, and TTP2230 mg / kg group, with 6 mice in each group. The Sham group was a sham operation group. The other three groups of mice had their ovaries removed surgically to simulate osteoporosis. One week after the wounds of the mice healed, they began to receive medication. The TTP22 group was given TTP2215 mg / kg and TTP2230 mg / kg intraperitoneal injections, respectively, once every other day for eight weeks. The body weights of the four groups of mice were measured and recorded before each medication.

[0060] 2. Micro-CT Scanning of Mouse Tibia

[0061] The mouse tibiae were dissected and fixed with 4% paraformaldehyde. Micro-CT scanning was then performed to measure bone mineral density (BMD), bone volume fraction (BV / TV) and other related indicators. The three-dimensional structure of the bones was reconstructed using NRcon and CTVOX software.

[0062] 3. Statistical methods

[0063] The statistical method is the same as that of “Example 1 of the present invention. TTP22 has an inhibitory effect on osteoclast differentiation in vitro”.

[0064] Experimental results:

[0065] 1. TTP22 has no significant effect on the body weight of ovariectomized mice.

[0066] To determine whether TTP22 treatment would affect the body weight of OVX-induced osteoporosis mice, the body weights of mice in each group were statistically analyzed. Figure 2The results showed that after four weeks of treatment, the weight of mice in the two TTP22 groups was slightly lower than that of mice in the OVX group, but there was no significant difference. This indicates that TTP22 treatment does not have a significant effect on the weight of mice. NS: No significant difference.

[0067] 2.TTP22 improved bone loss in ovariectomized mice.

[0068] Micro-CT detects various indicators and reconstructs the three-dimensional structure of bones. The results are as follows Figure 3 , Figure 3 A is the three-dimensional image of the bones of mice in each group. Figure 3 B is the statistical analysis of bone mineral density (BMD) and bone volume fraction (BV / TV). Compared with the Sham group, the number of trabeculae in the OVX group decreased significantly, showing bone loss, while the number of trabeculae in the TTP22 group increased compared with the OVX group, and with the increase of TTP22 concentration, the number of trabeculae increased ( Figure 3 A). Further statistical analysis of bone mineral density (BMD) and bone volume fraction (BV / TV) showed that the BMD and BV / TV of TTP22 group mice were significantly higher than those of OVX group ( Figure 3 B) This indicates that TTP22 can alleviate bone loss and increase bone mass in ovariectomized mice, with the effect increasing in a dose-dependent manner with increasing TTP22 concentration. ***P < 0.001.

[0069] Example 3 TTP22 inhibits the expression of TRAF6 and P-NIP30 proteins in vivo.

[0070] Experimental Materials:

[0071] Ten specific pathogen-free experimental animals (SPF animals) female mice, C57 strain, 3 months old, weighing (25±2) g, were purchased from the Minhang Experimental Animal Center of East China Normal University and raised according to SPF animal husbandry conditions.

[0072] Experimental methods:

[0073] 1. Modeling and Grouping and Dosing

[0074] The mice were randomly divided into two groups, the OVX group and the TTP22 group, with 5 mice in each group. Both groups of mice underwent bilateral ovarian removal surgery to simulate osteoporosis. One week after the wounds healed, the mice were given medication again. The TTP22 group was given an intraperitoneal injection of 30 mg / kg every other day for eight weeks.

[0075] 2. Western Blot Detection of Protein Expression

[0076] 1) Kill mice by cervical dislocation;

[0077] 2) stripping the intact hind limb of the mouse;

[0078] 3) Remove the muscles and tissues from the legs, place them in a 1.5 mL centrifuge tube, and place on ice;

[0079] 4) Add 1 mL of RIPA lysis buffer and magnetic beads, grind, and lyse on ice for 30 minutes;

[0080] 5) Centrifuge at 12,000 rpm, 4°C for 10 minutes;

[0081] 6) Transfer the supernatant to a new pre-chilled EP tube and determine the protein concentration using the BCA assay;

[0082] 7) Adjust the protein to an appropriate concentration with 6× loading buffer, incubate in a metal bath for 15 minutes, and store at -80°C.

[0083] 3. Western Blot specific steps are:

[0084] 1) Place the protein gel in the tank, add 1× electrophoresis buffer to the tank, load 10 μL of protein sample, add 1 μL and 3 μL protein markers (Thermo Scientific) to the wells on both sides respectively. TM , Cat No.: 26616) to distinguish left and right and fill to 10 μL with 1× loading buffer;

[0085] 2) Use 80V constant voltage for the stacking gel portion of the sample. When a clear marker band can be seen on the separating gel, adjust the voltage to 120V. When the indicator bromophenol blue reaches the bottom, terminate the electrophoresis.

[0086] 3) Prepare 1× transfer buffer. Place the transfer cassette into the transfer buffer. Place a sponge, three layers of filter paper, protein gel, NC membrane, three layers of filter paper, and a sponge on the cassette, starting from the black side to the white side. Place the cassette in the transfer tank, with the white side facing the positive electrode and the black side facing the negative electrode. Transfer for 70 minutes at a constant current of 200 mA.

[0087] 4) After transfer, remove the NC membrane and rinse with PBS buffer for 3 × 5 min;

[0088] 5) Incubate with 7% milk solution at room temperature for 1 hour, then rinse with PBS buffer for 3 × 5 minutes;

[0089] 6) Cut the membrane according to the molecular weight of the target protein, place the cut membrane in a dark box, and add the corresponding antibodies (β-Actin, NIP30, P-NIP30, TRAF6, p65, P-p65, p38, P-p38, JNK, P-JNK, ERK, P-ERK; 3% BSA 1:1000 / 2000 dilution) and incubate at 4°C overnight;

[0090] 7) Recover the corresponding primary antibody and wash with PBST buffer for 3 × 5 min;

[0091] 8) Fluorescent secondary antibody (M680 / R800; 3% BSA 1:10000 dilution), incubate at 4°C in the dark for 1 hour, recover the secondary antibody, and rinse with PBST for 3 × 5 minutes;

[0092] 9) Add clean PBS buffer, scan the membrane using an Odessy machine, and process the data.

[0093] 4. Statistical methods:

[0094] Data were processed using Prism software 8.0 (Graph Pad Software, San Diego, CA, USA). ImageJ software was used to analyze Western blot intensities. Data are presented as mean ± standard deviation (x-±s). Two sample means were compared using an independent t-test. Differences were considered statistically significant when P < 0.05.

[0095] Experimental results:

[0096] 1. TTP22 downregulates the expression level of P-NIP30 in osteoclasts.

[0097] CK2 / NIP30 / REGγ may play an important role in osteoporosis. To further explore the molecular mechanism of TTP22 inhibiting osteoclast differentiation, the present invention used Western Blot to detect the expression changes of NIP30 and P-NIP30 protein levels. Figure 4 OVX and TTP22-treated groups were established. Results showed no significant change in NIP30 in the TTP22-treated group, while P-NIP30 was significantly suppressed. This suggests that TTP22 may inhibit osteoclast differentiation through the CK2 / NIP30 / REGγ axis. ***P < 0.001; NS: not significant difference.

[0098] 2. TTP22 inhibited the activation of NF-κB and MAPK signaling pathways caused by TRAF6.

[0099] In order to further explore the potential mechanism of TTP22 inhibiting osteoclast differentiation, the present invention conducted Western Blot analysis on the classic signaling pathway of osteoclast differentiation. TRAF6, as an important adaptor protein, can cause the activation of downstream NF-κB and MAPK signaling pathways. The present invention set up OVX group and TTP22 group, and the results are as follows Figure 4Results showed that TTP22 treatment significantly decreased TRAF6 protein levels. Corresponding downstream proteins, P-p38, P-JNK, P-ERK, and P-p65, were significantly suppressed, while baseline protein expression remained unchanged. This suggests that TTP22 inhibits activation of the NF-κB and MAPK signaling pathways by downregulating TRAF6. ***P < 0.001; NS: not significant.

[0100] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0101] As used in the present invention, the term "comprising" is an open expression, that is, including the contents specified in the present invention, but not excluding other aspects.

[0102] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0103] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. Use of TTP22 in the preparation of a medicament for preventing and / or treating osteoporosis, characterized in that: The structure of the TTP22 is as follows: TTP22 inhibits osteoclast differentiation in vitro through the CK2 / NIP30 / REGγ axis.

2. The use according to claim 1, characterized in that The TTP22 inhibits bone loss and increases the number of trabeculae and bone density.

3. The use according to claim 1, characterized in that The TTP22 downregulates the expression of TRAF6 in osteoclasts in vivo, inhibits NIP30 phosphorylation, and blocks signal transduction, thereby affecting its activation of downstream NF-κB and MAPK signaling pathways, thereby inhibiting osteoclast differentiation and improving bone loss.

4. Use of a drug / drug composition in the preparation of a drug for preventing and / or treating osteoporosis, characterized in that: The drug / pharmaceutical composition comprises TTP22, the structure of which is shown below: TTP22 inhibits osteoclast differentiation in vitro through the CK2 / NIP30 / REGγ axis.

5. The use according to claim 4, characterized in that The drug / drug composition is used alone or in combination with other drugs.

6. The use according to claim 4, characterized in that The drug / drug composition inhibits bone loss and increases the number of trabeculae and bone density.

7. The use according to claim 4, characterized in that The drug / drug composition downregulates the expression of TRAF6 in osteoclasts in vivo, inhibits NIP30 phosphorylation, and blocks signal transduction, thereby affecting its activation of downstream NF-κB and MAPK signaling pathways, thereby inhibiting osteoclast differentiation and improving bone loss.

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