Application of 5-(tetradecyloxy)-2-furoic acid in preparation of medicine for inhibiting osteoclast differentiation and function

Abstract

The invention relates to an application of 5-(tetradecyloxy)-2-furoic acid in preparation of a medicine for inhibiting osteoclast differentiation and function, an RANKL-induced mouse bone marrow macrophage osteoclast differentiation model is adopted, and experiments prove that TOFA can inhibit osteoclast differentiation and function of osteoclast by inhibiting ROS and down-regulating expression of osteoclast differentiation and function key proteins such as Nfatc1, Src and Ctsk. The application has the advantages that the osteoclast differentiation and functions can be effectively inhibited, the TOFA with the concentration of 40 mu M or below has no obvious cytotoxicity, and the TOFA can be used as a candidate compound for researching and developing the medicines for inhibiting the osteoclast differentiation and the functions, can be used for treating diseases caused by overexpression of the osteoclast, and has important clinical application value; a mouse ovariectomized osteoporosis model is constructed, and the prevention and treatment effect of TOFA on osteoporosis caused by estrogen deficiency is proved.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to the application of 5-(tetradecyloxy)-2-furoic acid in the preparation of a drug for inhibiting osteoclast differentiation and function. Background Art

[0002] Osteolytic diseases are a type of bone disease caused by excessive formation and / or activation of osteoclasts due to pathological factors such as estrogen deficiency, infection, tumors or inflammation, which leads to abnormal bone loss in the body. The most common one is osteoporosis caused by estrogen deficiency. Osteoclasts are differentiated and matured from osteoclast precursor cells (such as bone marrow monocytes / macrophages) derived from hematopoietic stem cells under the action of factors such as M-CSF and RANKL. Mature osteoclasts secrete acidic substances and proteases to dissolve and digest mineralized bone matrix. In view of the key driving role of osteoclasts in the pathogenesis of osteoporosis, bone resorption inhibitors such as bisphosphonates, Denosumab, calcitonin, and raloxifene are currently used clinically to treat osteoporosis. However, the above drugs have certain toxic side effects or are not suitable for long-term use. Therefore, it is urgent to develop safe and effective new osteoclast differentiation and function inhibitory drugs.

[0003] The differentiation, maturation and function of osteoclasts are regulated by multiple factors, among which the role of transcription factor NFATc1 is particularly critical. At the same time, intracellular reactive oxygen species (ROS) are also indispensable for osteoclast formation. In addition, cathepsin K (Ctsk), as an important enzyme for digesting the organic matrix components of bone, is essential for the bone resorption function of osteoclasts. Therefore, inhibiting the production of ROS and the expression of genes such as NFATc1 and Ctsk at the same time can inhibit osteoclast formation and its bone resorption function, which is expected to become an effective means of preventing and treating osteolytic diseases such as osteoporosis.

[0004] TOFA has positive therapeutic effects on multiple myeloma, breast cancer, prostate cancer cells, asthma, chikungunya fever and non-alcoholic fatty liver disease. However, it is still unclear whether TOFA can be used as an inhibitor of osteoclast formation and function to prevent and treat osteolytic diseases. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide an application of 5-(tetradecyloxy)-2-furoic acid in the preparation of a drug for inhibiting osteoclast differentiation and function.

[0006] Provided is the use of 5-(tetradecyloxy)-2-furoic acid in the preparation of a drug for inhibiting osteoclast differentiation, wherein the molecular formula of 5-(tetradecyloxy)-2-furoic acid is C 19 H 32 O4, the structural formula is

[0007]

[0008] The invention relates to an application of the 5-(tetradecyloxy)-2-furoic acid in preparing medicine for preventing or treating osteolytic diseases.

[0009] The invention relates to an application of the 5-(tetradecyloxy)-2-furoic acid in preparing a medicine for preventing or treating osteoporosis.

[0010] The invention relates to an application of the 5-(tetradecyloxy)-2-furoic acid in preparing a drug for inhibiting the expression of NFATc1 gene.

[0011] The invention relates to an application of the 5-(tetradecyloxy)-2-furoic acid in preparing a drug for inhibiting the expression of Ctsk gene or Src gene.

[0012] The invention relates to an application of the 5-(tetradecyloxy)-2-furoic acid in preparing a medicine for inhibiting active oxygen in cells.

[0013] The invention relates to an application of the 5-(tetradecyloxy)-2-furoic acid in preparing medicine for preventing or treating bone destruction of tumor metastasis.

[0014] The invention relates to the application of the 5-(tetradecyloxy)-2-furoic acid in preparing medicine for preventing or treating rheumatoid arthritis.

[0015] This medicine is in the form of tablets, pills, powders, capsules, injections, oral solutions, ointments or creams; it is administered orally, by injection or external application.

[0016] The drug, 5-(tetradecyloxy)-2-furoic acid, is in a dosage of 0.5 to 1 mg / kg.

[0017] The beneficial effects of the present invention are:

[0018] 1) The present invention adopts a RANKL-induced mouse bone marrow macrophage osteoclast differentiation model. Experiments have shown that TOFA can effectively inhibit osteoclast differentiation and its function by inhibiting ROS and down-regulating the expression of key osteoclast differentiation and function proteins such as Nfatc1, Src, and Ctsk. TOFA at a concentration of 40 μM and below has no obvious cytotoxicity, which proves that TOFA can be used as a candidate compound for the development of drugs that inhibit osteoclast differentiation and function, and treat diseases caused by excessive osteoclast activation, and has important clinical application value.

[0019] 2) The present invention also constructs an ovariectomized mouse osteoporosis model, and experimentally obtains that TOFA can significantly reverse the down-regulation effect of OVX on the relative volume and number of trabeculae and the up-regulation effect of OVX on the separation of trabeculae, thus proving the preventive and therapeutic effect of TOFA on osteoporosis caused by estrogen deficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of the experiment using CCK-8 method to detect the cytotoxicity of TOFA on bone marrow mononuclear / macrophages (BMMs);

[0021] Figure 2 This is the staining result of tartrate-resistant acid phosphatase (TRAP) staining to evaluate the inhibitory effect of TOFA on osteoclast differentiation;

[0022] Figure 3 The results of TRAP staining quantitative analysis of osteoclast numbers;

[0023] Figure 4 TRAP staining quantitative analysis results of the relative size of osteoclasts;

[0024] Figure 5 Fluorescence image of the effect of TOFA on RANKL-induced reactive oxygen species (ROS) in BMM cells detected by DCFH-DA probe;

[0025] Figure 6 This is the quantitative analysis result of the proportion of ROS-positive cells;

[0026] Figure 7 The figure shows the results of Western blotting to detect the effect of TOFA on osteoclast differentiation and expression of function-related proteins;

[0027] Figure 8 Three-dimensional reconstruction images of micro-computed tomography to evaluate the protective effect of TOFA on ovariectomy-induced osteoporosis;

[0028] Fig. 9 The results of Micro-CT quantitative analysis of femoral cancellous bone related parameters. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with embodiments. The description of the following embodiments is only used to help understand the present invention. It should be noted that for ordinary persons in the art, without departing from the principle of the present invention, the present invention can also be modified in some ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0030] Embodiment 1

[0031] As an example, this example 1 proposes that 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA for short) is an allosteric inhibitor of acetyl-CoA carboxylase-α, and its molecular formula is C 19 H 32 O4, CAS# is 54857-86-2, structural formula is:

[0032]

[0033] The drug comprises an active ingredient 5-(tetradecyloxy)-2-furoic acid (TOFA) and medically acceptable pharmaceutical excipients, wherein the excipients include conventional diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field; the 5-(tetradecyloxy)-2-furoic acid can also be used in combination with other drugs to prepare a compound pharmaceutical preparation.

[0034] In this example, CCK-8 was used to detect the cytotoxicity of TOFA to BMMs:

[0035] First, mouse bone marrow monocytes / macrophages (BMMs) were obtained:

[0036] Step 1: Use ultraviolet disinfection on a clean bench to sterilize scissors, tweezers, and several culture dishes containing DPBS. Then, under sterile conditions, take the femur and tibia of a 6-10 week old C57BL / 6 male mouse, remove the muscles and connective tissue on the bones, cut off the distal ends of each joint, and place the cut end of the bone downward in a 0.6 mL sterile centrifuge tube with a pre-perforated bottom.

[0037] Step 2: Insert the 0.6mL centrifuge tube into the 1.5mL centrifuge tube, increase the speed to 10,000 rpm and then stop to achieve instant centrifugation to separate the bone marrow.

[0038] Step 3: Resuspend the bone marrow cells with α-MEM complete medium containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin, transfer to a 10 cm cell culture dish, and culture in a 37° C., 5% CO 2 cell culture incubator.

[0039] Step 4. On the next day, collect the supernatant, centrifuge at 1000 rpm at room temperature for 4 min, discard the supernatant, resuspend the cells in BMM culture medium containing α-MEM complete medium containing 15 ng / mL M-CSF, and spread them in a 10 cm culture dish for further culture.

[0040] Step 5. On the third day, after trypsin digestion, centrifuge at 1000 rpm at room temperature for 4 min, discard the supernatant, resuspend the cells in an appropriate amount of BMM medium, and count them using a cell counting plate. The obtained cells are considered bone marrow mononuclear / macrophages (BMMs). Use 4×10 5 / well, 24-well plate 1×10 5 The cells were plated at a density of 5000 / well and 96-well plates at a density of 5000 / well.

[0041] CCK-8 was then used to detect the cytotoxicity of TOFA on BMMs:

[0042] Step 1: Press 5×10 3 BMMs were inoculated into 96-well plates, 100 μL of BMM culture medium was added to each well, and the plates were cultured overnight in a cell culture incubator.

[0043] Step 2. On the next day, the cells were divided into 7 groups, each with 6 replicate wells, and BMM culture medium containing 0 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM or 40 μM TOFA was added for cell culture; the above treatment was repeated for a total of 3 plates, and incubated for 24, 48, and 72 hours (h), respectively.

[0044] Step 3: After the incubation, each well was replaced with 100 μL of culture medium containing 10% CCK-8 reagent. In this embodiment, the CCK-8 reagent of Bio-Time was used, and the cells were placed in a 37° C. cell culture incubator and incubated for 2 h in the dark.

[0045] Step 4: Use a multifunctional microplate reader to measure the OD value at a wavelength of 450nm 450 , the results are as follows Figure 1 As shown:

[0046] After 24 h, 48 h or 72 h of treatment, the OD of cells in the 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM or 40 μM TOFA treatment groups was 450, OD compared with the group without TOFA addition 450 There was no significant difference in comparison, indicating that TOFA at concentrations of 40 μM and below had no cytotoxicity to BMMs.

[0047] Embodiment 2

[0048] As another example, based on Example 1, Example 2 proposes that TOFA has a direct inhibitory effect on osteoclast differentiation, and TRAP staining is used for detection:

[0049] In this example, the same method as in Example 1 was used to obtain mouse bone marrow mononuclear / macrophages (BMMs), and then TRAP staining was used to detect the effect of TOFA on osteoclast formation:

[0050] Step 1: According to 1×10 5 BMMs were plated in 24-well plates at 100 μM / well and the cells were divided into 6 groups, including 0, 0.625, 1.25, 2.5, 5 and 10 μM TOFA treatment groups, with 3 replicate wells in each group, and cultured with BMM medium for 1 day.

[0051] Step 2: After the cells adhered, change the medium. Add BMM osteoclast induction medium containing an equal volume of DMSO to the cells in the 0μM TOFA treatment group. The BMM osteoclast induction medium is α-MEM complete medium containing 30ng / mL M-CSF and 50ng / mL RANKL, and DMSO is the solvent of TOFA; add BMM osteoclast induction medium containing corresponding concentrations of TOFA to the cells in the 0.625, 1.25, 2.5, 5 and 10μM TOFA treatment groups respectively; change the medium every other day.

[0052] Step 3: After 5 days of continuous culture, cells in each group were stained with tartrate-resistant acid phosphatase (TRAP). TRAP-positive multinucleated cells with >3 nuclei were identified as osteoclasts.

[0053] TRAP staining results Figure 2 As shown, TOFA dose-dependently inhibited the induction of osteoclast formation by RANKL. Figure 2 shown.

[0054] ImageJ software was used to count the number and size of osteoclasts in each group. The results of TRAP staining quantitative analysis were as follows: Figure 3 and Figure 4 As shown, Figure 3 and Figure 4 It was further shown that TOFA dose-dependently reduced the number and size of osteoclasts. The above results indicate that 5-(tetradecyloxy)-2-furoic acid (TOFA) has a direct inhibitory effect on osteoclast differentiation.

[0055] It should be noted that the parts in this embodiment that are the same or similar to those in the first embodiment can be referenced to each other and will not be described in detail in this application.

[0056] Embodiment 3

[0057] As another example, based on Example 1, Example 3 proposes that TOFA can inhibit the induction of RANKL on the production of reactive oxygen species (ROS) in BMM cells, and uses a DCFH-DA probe for detection:

[0058] In this example, the same method as in Example 1 was used to obtain mouse bone marrow mononuclear / macrophages (BMMs), and then the DCFH-DA probe was used to detect the effect of TOFA on osteoclast formation:

[0059] Step 1: BMMs are divided into 1×10 5 The cells were inoculated in 24-well plates at a concentration of 100 μM / well, and the cells were divided into 7 groups, including NM (negative control group), RANKL-induced group (OM group), and different concentrations of TOFA treatment groups (including OM+0.625μM TOFA, OM+1.25μM TOFA, OM+2.5μM TOFA, OM+5μM TOFA, OM+10μM TOFA groups), with 3 replicates in each group. The cells were cultured in BMM medium for 1 day.

[0060] Step 2: Add 500 μL of BMM culture medium and incubate overnight in a 37°C, 5% CO2 incubator. Change the medium the next day.

[0061] Step 3. Add BMM osteoclast induction medium containing corresponding concentrations of TOFA to the cells in the OM+0.625μM TOFA, OM+1.25μM TOFA, OM+2.5μM TOFA, OM+5μM TOFA, and OM+10μM TOFA groups, respectively; add BMM osteoclast induction medium containing the same dose of DMSO as that in the TOFA-treated group to the OM group; add α-MEM complete medium containing 30ng / mL M-CSF and the same dose of DMSO as that in the TOFA-treated group to the NM group; after two days of treatment, use the reactive oxygen species kit to detect the level of reactive oxygen species.

[0062] The brief steps are as follows: first, dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000, mix well, add 300 μL of diluted DCFH-DA probe to each well, incubate in a 37°C cell culture incubator for 20 minutes, and then wash the cells three times with serum-free culture medium to fully remove excess DCFH-DA; then, collect fluorescence and bright field images under an inverted fluorescence microscope, and use ImageJ software to quantify the proportion of ROS-positive cells to observe the effect of TOFA on reactive oxygen species.

[0063] ROS fluorescence images Figure 5 As shown in the figure, the green signal is DCFH-DA marker, and the corresponding ROS quantitative analysis results are shown in Figure 6 The data are shown as mean ± standard deviation, *: p < 0.05, **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. Figure 5 and Figure 6As shown in the figure, the level of reactive oxygen species in the OM group was significantly higher than that in the NM control group, while the administration of 5-(tetradecyloxy)-2-furoic acid (TOFA) reduced the level of reactive oxygen species in the cells in a dose-dependent manner. The above results indicate that 5-(tetradecyloxy)-2-furoic acid (TOFA) can inhibit the induction of RANKL on the production of reactive oxygen species (ROS) in BMM cells.

[0064] It should be noted that the parts in this embodiment that are the same or similar to those in the first embodiment can be referenced to each other and will not be described in detail in this application.

[0065] Embodiment 4

[0066] As another example, based on Example 1, Example 4 proposes that TOFA can inhibit osteoclast differentiation and expression of function-related proteins, and Western blot is used for detection:

[0067] In this example, the same method as in Example 1 was used to obtain mouse bone marrow mononuclear / macrophages (BMMs), and then Western blot was used to detect the effect of TOFA on osteoclast formation:

[0068] Step 1: 4×10 per hole 5 The BMMs were plated into 6-well plates and divided into control group and drug treatment group, with 3 replicates in each group; the medium was changed after culturing with BMM medium for 1 day;

[0069] Step 2: Add BMM osteoclast induction medium containing 10 μM TOFA and an equal volume of DMSO to the drug treatment group and the control group, respectively; change the medium every other day, and extract cell proteins with RIPA lysis buffer containing phosphatase and protease inhibitors after 0, 1, 3, and 5 days of treatment, respectively;

[0070] Step 3: Determine the concentration of the above protein samples using a BCA kit, and adjust each group of proteins to a uniform concentration using RIPA lysis buffer; prepare a 10% SDS-PAGE gel, load 15 μg of protein sample into each well, and separate by electrophoresis; after the protein sample is separated, transfer it to the NC membrane by electrophoresis (90V, 2h);

[0071] Step 4: Cut the NC into strips containing each target protein, and then block it with TBST containing 5% skim milk for 1.5 h; then add Ctsk, Src, or Nfatc1 antibodies diluted 1:2000, or use membrane regeneration solution to strip the above antibodies and add β-actin antibodies diluted 1:7500, and incubate overnight at 4°C.

[0072] Step 5. The next day, wash three times with 1×TBST on a shaker, add secondary antibody diluted 1:4000, and incubate at room temperature for 1.5 hours; wash three times with 1×TBST, add ECL luminescent solution for development and collect images.

[0073] The results are as follows Figure 7 As shown in the figure, 10 μM TOFA can significantly inhibit the induction of RANKL on osteoclast differentiation and function-related proteins Ctsk, Src, and Nfatc1. These results further prove that TOFA can inhibit osteoclast differentiation and function.

[0074] It should be noted that the parts in this embodiment that are the same or similar to those in the first embodiment can be referenced to each other and will not be described in detail in this application.

[0075] Embodiment 5

[0076] As another example, based on Example 1, Example 5 proposes that TOFA can prevent and treat osteoporosis caused by ovariectomy in mice, and uses micro-computed tomography (Micro-CT) scanning for detection:

[0077] In this example, the same method as in Example 1 was used to obtain mouse bone marrow mononuclear / macrophages (BMMs), and then Micro-CT scanning was used to detect the effect of TOFA on osteoclast formation:

[0078] Step 1. Mouse grouping and drug preparation: 28 C57BL / 6 mice were randomly divided into 4 groups, namely, sham group, OVX group, TOFA-L group and TOFA-H group, with 7 mice in each group; a solution of 10% DMSO + 90% corn oil was prepared as TOFA solvent.

[0079] Step 2. OVX mouse ovariectomized osteoporosis model: After anesthesia, the sham operation group underwent a longitudinal incision on the back under sterile conditions, and the subcutaneous fascia and muscle were bluntly separated. The muscle was cut at the left and right dorsal kidney projections, and the muscle, fascia, and skin were directly sutured after removing some fat near the ovaries; after successful anesthesia in the OVX group, TOFA-L group, and TOFA-H group, a longitudinal incision on the back was made under sterile conditions, and the subcutaneous fascia and muscle were bluntly separated. The muscle was cut at the left and right dorsal kidney projections, the fallopian tubes and ovaries were clamped, and the ovaries were cut off after ligation below the ovaries, and the muscles, fascia, and skin were sutured.

[0080] Step 3, drug administration and sample collection: 3 days after postoperative recovery, the mice in the TOFA-L and TOFA-H groups were gavaged with 5 mg / kg and 10 mg / kg of TOFA solution, respectively, and the solvent was 10% DMSO + 90% corn oil. The mice in the Sham and OVX groups were gavaged with an equal dose of solvent, i.e., a solution of 10% DMSO + 90% corn oil. The mice were killed by cervical dislocation after 21 consecutive gavages every two days, and the femurs were obtained and placed in 10% formalin fixative and fixed at room temperature for 48 hours.

[0081] Step 4, Micro-CT scanning and analysis: After rinsing with PBS, the femurs of each group were scanned in three dimensions using the NEMO in vitro integrated Micro-CT scanner of Pingsheng Company, and the original scan data were reconstructed and corrected. Then, the cancellous bone in the area of ​​0.2mm to 1.20mm below the distal femoral growth plate was reconstructed in three dimensions and quantitatively analyzed using Avatar software to obtain a three-dimensional stereoscopic image of the cancellous bone in this area, as shown in Figure 4. Figure 8 The results of the quantitative parameters of cancellous bone are shown in Fig. 9 As shown, including trabecular relative volume (BV / TV), trabecular number (Tb.N) and trabecular separation (Tb.Sp).

[0082] Micro-CT three-dimensional reconstruction results showed that the femoral cancellous bone mass in the OVX group was significantly less than that in the Sham group; while the femoral cancellous bone mass in the TOFA-L and TOFA-H groups was significantly more than that in the OVX group. Micro-CT quantitative analysis further showed that 5mg / kg and 10mg / kg TOFA could significantly reverse the down-regulation effect of OVX on the relative volume and number of trabeculae and the up-regulation effect of OVX on the separation of trabeculae. These results indicate that TOFA has a significant preventive and therapeutic effect on osteoporosis caused by ovariectomy in mice.

[0083] It should be noted that the parts in this embodiment that are the same or similar to those in the first embodiment can be referenced to each other and will not be described in detail in this application.

[0084] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

Claims

1. Use of 5-(tetradecyloxy)-2-furoic acid in the preparation of a drug for inhibiting osteoclast differentiation, wherein the molecular formula of 5-(tetradecyloxy)-2-furoic acid is C 19 H 32 O4, the structural formula is 2. Use of 5-(tetradecyloxy)-2-furoic acid as claimed in claim 1 in the preparation of a medicament for preventing or treating osteolytic diseases.

3. Use of 5-(tetradecyloxy)-2-furoic acid as claimed in claim 1 in the preparation of a medicament for preventing or treating osteoporosis.

4. Use of 5-(tetradecyloxy)-2-furoic acid as claimed in claim 1 in the preparation of a drug for inhibiting NFATc1 gene expression.

5. Use of 5-(tetradecyloxy)-2-furoic acid as claimed in claim 1 in the preparation of a drug for inhibiting Ctsk gene expression or Src gene expression.

6. Use of 5-(tetradecyloxy)-2-furoic acid as claimed in claim 1 in the preparation of a drug for inhibiting intracellular reactive oxygen species.

7. Use of 5-(tetradecyloxy)-2-furoic acid in the preparation of drugs for preventing or treating bone destruction caused by tumor metastasis.

8. Use of 5-(tetradecyloxy)-2-furoic acid in the preparation of drugs for preventing or treating rheumatoid arthritis.

9. The drug according to any one of claims 1 to 8, characterized in that The drug is in the form of tablets, pills, powders, capsules, injections, oral solutions, ointments or creams; it is administered orally, by injection or external application.

10. The drug according to any one of claims 1 to 8, characterized in that The dosage of 5-(tetradecyloxy)-2-furoic acid in the medicine is 0.5 to 1 mg / kg.

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