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

Abstract

The invention discloses an application of TTP22 in preparation of a medicine for preventing and / or treating osteoporosis. According to the application disclosed by the invention, TTP22 influences downstream NF-kappa B and MAPK signal channels by inhibiting expression of TRAF6 and P-NIP30 proteins in vivo, so that osteoclast differentiation is inhibited. In addition, the invention also discloses TTP22 which can obviously improve bone mass reduction and bone loss of OVX mice. Therefore, the TTP22 provided by the invention provides a new direction for research and development of medicines for preventing and treating osteoporosis.

Description

Technical Field

[0001] The present invention relates to the field of pharmacological therapy, 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 damage to bone tissue microstructure, which leads to increased bone brittleness and susceptibility to fractures. Based on the cause, it can be clinically divided into two categories: primary and secondary. 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] The national epidemiological survey on osteoporosis 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%, including 51.6% in women. As the population ages, the prevalence will continue to rise. Osteoporosis has led to a decline in quality of life, an increase in disability and mortality, and a huge economic burden on families and society, and has become a serious public health issue.

[0004] Osteoporosis is mainly caused by the disruption of the metabolic balance between osteoblasts and osteoclasts, which leads to the enhancement of osteoclast activity. At present, the treatment of osteoporosis mainly focuses on inhibiting the production of osteoclasts, and the main drugs used are bisphosphonates, denosumab, teriparatide and abaloparatide. However, bisphosphonates are not easily absorbed by the human body and are harmful to the digestive tract, and hormone therapy is likely to increase the incidence of other diseases such as cancer and cardiovascular disease. Therefore, the development of safe and effective drugs has attracted 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 stronger 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 that exists in all eukaryotes. It is associated with many malignant tumors such as Alzheimer's disease, Parkinson's disease and Huntington's disease, diabetes, lung cancer, breast cancer, etc. (Roffey SE, Litchfield DW. CK2 Regulation: Perspectives in 2021. Biomedicines. 2021 Sep 30; 9 (10): 1361.). Many studies have shown that CK2 is an upstream kinase of nuclear protein 30 (NIP30) and can promote NIP30 phosphorylation. NIP30 is an important regulator 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 prior art, 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, which comprises 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 into 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 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 beneficial effect of the present invention is that the present invention innovatively proposes the use of TTP22 in the preparation of drugs for preventing and / or treating osteoporosis. TTP22 affects the downstream NF-κB and MAPK signaling pathways by inhibiting the expression of TRAF6 and P-NIP30 proteins in the body, thereby inhibiting osteoclast differentiation. In addition, the present invention also proposes that TTP22 can significantly improve the bone loss and bone loss of OVX mice. Therefore, the present invention provides a new direction for the research and development of drugs for preventing, treating and treating osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The results of TTP22 inhibiting the differentiation of osteoclasts induced by RANKL; Figure A shows the TRAP staining results of osteoclasts induced by BMMs under the conditions of different concentrations of TTP22 treatment; Figure B shows the statistical results of TRAP staining of osteoclasts induced by BMMs under the conditions of different concentrations of TTP22 treatment;

[0023] Figure 2 The results show the effect of TTP22 on the body weight of ovariectomized mice;

[0024] Figure 3 The results of TTP22 improving bone loss in ovariectomized mice; Figure A is a representative Micro-CT image of 5-month-old mice in each group; Figure B is a statistical analysis of the relative bone volume and bone density of mice in each group;

[0025] Figure 4 The results of TTP22 inhibiting the expression of phosphorylated TRAF6 and NIP30 proteins in osteoclasts in vivo; Figure A is the 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 the quantitative analysis 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 in the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are 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 as 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] Specific pathogen-free experimental animals (SPF animals) 10 male mice, C57BL / 6 strain, 6-8 weeks old, weight (20±5) g, purchased from Minhang Experimental Animal Center of East China Normal University, and raised according to SPF animal breeding 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 killed by cervical dislocation;

[0035] 2) The complete 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 with complete culture medium, transfer to a culture dish, and culture in a 37°C, 5% CO2 incubator;

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

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

[0041] 8) The BMMs cells were digested with trypsin, centrifuged, resuspended in complete 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 and the culture was continued 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 min at room temperature and washed with PBS buffer;

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

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

[0047] 4) Perform TRAP staining on the incubated cells using a TRAP staining kit (Sigma) in dark conditions, incubate in a 37°C incubator in dark for 60 min, and wash 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, California, USA). Data were expressed as mean ± standard deviation (x-±s), and the means of two samples were compared using independent sample t-test. The difference was 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 induces BMMs with RANKL and treats them with 5nM and 10nM TTP22 respectively. Figure 1TRAP staining results showed that with the increase of TTP22 concentration, the number and area of ​​osteoclasts showed a significant downward trend. This indicates that TTP22 has an inhibitory effect on the differentiation of osteoclasts in vitro, and this effect is dose-dependent. ***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 breeding conditions.

[0057] Experimental methods:

[0058] 1. Modeling and grouping and drug administration

[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 underwent bilateral ovarian removal surgery to simulate osteoporosis. One week after the wounds of the mice healed, medication was started. The TTP22 group was given intraperitoneal injections of TTP2215 mg / kg and TTP2230 mg / kg, 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 stripped and fixed with 4% paraformaldehyde. Micro-CT scanning was performed to detect bone mineral density (BMD), bone volume fraction (BV / TV) and other related indicators. The 3D 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 osteoporotic 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, which indicated that TTP22 administration did 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 the TTP22 group were significantly higher than those of the OVX group ( Figure 3 B), which indicates that TTP22 can improve bone loss after ovariectomy in mice and increase bone mass in ovariectomized mice, and the effect increases with the increase of TTP22 concentration, in a dose-dependent manner. ***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 breeding conditions.

[0072] Experimental methods:

[0073] 1. Modeling and grouping and drug administration

[0074] The mice were randomly divided into two groups, OVX group and TTP22 group, with 5 mice in each group. The ovaries of both groups of mice were surgically removed to simulate osteoporosis. One week later, the mice were given medication after their wounds healed. The TTP22 group was given an intraperitoneal injection of 30 mg / kg, once every other day for eight weeks.

[0075] 2. Western Blot Detection of Protein Expression

[0076] 1) Kill the mice by cervical dislocation;

[0077] 2) stripping the complete hind limbs of mice;

[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 place on ice for 30 minutes;

[0080] 5) Centrifuge at 12000 rpm, 4°C for 10 min;

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

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

[0083] 3. The specific steps of Western Blot 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 of protein marker (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 sample in the concentrated gel part. When a clear marker band can be seen on the separation gel, adjust the voltage to 120V. When the indicator bromophenol blue runs to the bottom, terminate the electrophoresis.

[0086] 3) Prepare 1× transfer buffer, put the transfer clip into the transfer buffer, and put the sponge, 3 layers of filter paper, protein glue, NC membrane, 3 layers of filter paper, and sponge on the transfer clip from the black side to the white side; put the clip into the transfer tank, with the white side close to the positive electrode and the black side close to the negative electrode. The transfer time is 70 minutes, and the constant current is 200mA;

[0087] 4) After the 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, and then rinse with PBS buffer for 3 × 5 minutes;

[0089] 6) Cut the membrane according to the molecular weight of the target protein, put the cut membrane into a dark box, 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 diluted antibodies) 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, California, USA). ImageJ software was used to analyze the intensity of Western blot. Data were expressed as mean ± standard deviation (x-±s), and the means of two samples were compared using independent sample t-test. The difference was 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 , set up OVX group and TTP22 treatment group. The results showed that NIP30 did not change significantly in the TTP22 treatment group, while P-NIP30 was significantly inhibited. This indicates that TTP22 can inhibit osteoclast differentiation through the CK2 / NIP30 / REGγ axis. ***P<0.001; NS: no 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 performed 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 an OVX group and a TTP22 group. The results are as follows Figure 4The results showed that after TTP22 treatment, the level of TRAF6 protein decreased significantly. The corresponding downstream P-p38, P-JNK, P-ERK, and P-p65 were significantly inhibited, and the background protein expression did not change significantly, indicating that TTP22 inhibited the activation of NF-κB and MAPK signaling pathways by downregulating TRAF6. ***P<0.001; NS: no significant difference.

[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 contents.

[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 such modifications are protected by patent law as long as they are within the scope of the claims of the present application.

Claims

1. Use of TTP22 in the preparation of a drug for preventing and / or treating osteoporosis, It is characterized in that The structure of the TTP22 is as follows:

2. The use according to claim 1, It is characterized in that The TTP22 inhibits the differentiation of osteoclasts in vitro.

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

4. The use according to claim 1, It is 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 the differentiation of osteoclasts and improving bone loss.

5. A drug / drug composition, It is characterized in that It contains TTP22, the structure of which is shown below:

6. The drug / pharmaceutical composition according to claim 5, It is characterized in that The drug / drug composition can be used alone or in combination with other drugs.

7. Use of the medicine / pharmaceutical composition according to claim 5 in the preparation of a medicine for preventing and / or treating osteoporosis.

8. The use according to claim 7, It is characterized in that The medicament / pharmaceutical composition inhibits the differentiation of osteoclasts in vitro.

9. The use according to claim 7, It is characterized in that The drug / drug composition inhibits bone loss and increases the number of trabeculae and bone density.

10. The use according to claim 7, It is 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 the activation of downstream NF-κB and MAPK signaling pathways, thereby inhibiting the differentiation of osteoclasts and improving bone loss.

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