Application of 5-(tetradecoxy)-2-furfuric acid in the preparation of drugs that inhibit osteoclast differentiation and function

Abstract

This invention relates to the application of 5-(tetradecoxy)-2-furoic acid (TOFA) in the preparation of drugs that inhibit osteoclast differentiation and function. Using a RANKL-induced mouse bone marrow macrophage osteoclast differentiation model, experiments demonstrated that TOFA can effectively inhibit osteoclast differentiation and function by inhibiting ROS and downregulating the expression of key osteoclast differentiation and function proteins such as Nfatc1, Src, and Ctsk. Furthermore, TOFA at concentrations of 40 μM and below showed no significant cytotoxicity, proving that TOFA can serve as a candidate compound for developing drugs that inhibit osteoclast differentiation and function, and for treating diseases caused by excessive activation of osteoclasts, thus possessing significant clinical application value. Additionally, by constructing a mouse ovariectomized osteoporosis model, the preventive and therapeutic effects of TOFA on estrogen-deficiency-induced osteoporosis were demonstrated.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, and in particular relates to the application of 5-(tetradecoxy)-2-furfuric acid in the preparation of drugs that inhibit osteoclast differentiation and function. Background Technology

[0002] Osteolytic diseases are a group of skeletal disorders caused by excessive formation and / or activation of osteoclasts due to pathological factors such as estrogen deficiency, infection, tumors, or inflammation, leading to abnormal bone loss. The most common type is osteoporosis caused by estrogen deficiency. Osteoclasts differentiate and mature from osteoclast precursor cells (such as bone marrow monocytes / macrophages) derived from hematopoietic stem cells under the influence of factors such as M-CSF and RANKL. Mature osteoclasts secrete acidic substances and proteases to dissolve and digest mineralized bone matrix. Given the crucial driving role of osteoclasts in the pathogenesis of osteoporosis, bisphosphonates, denosumab, calcitonin, and raloxifene are commonly used clinically to treat osteoporosis. However, these drugs have certain toxic side effects or are unsuitable for long-term use. Therefore, the development of safe and effective novel drugs that inhibit osteoclast differentiation and function is urgently needed.

[0003] Osteoclast differentiation, maturation, and function are regulated by multiple factors, with the transcription factor NFATc1 playing a particularly crucial role. Simultaneously, intracellular reactive oxygen species (ROS) are indispensable for osteoclast formation. Furthermore, cathepsin K (Ctsk), as an important enzyme for digesting bone matrix components, is essential for osteoclast bone resorption. Therefore, simultaneously inhibiting ROS production and the expression of genes such as NFATc1 and Ctsk can suppress osteoclast formation and bone resorption, potentially becoming an effective means of preventing and treating osteolytic diseases such as osteoporosis.

[0004] TOFA has shown positive therapeutic effects on multiple myeloma, breast cancer, prostate cancer cells, asthma, chikungunya fever, and non-alcoholic fatty liver disease. However, whether TOFA can be used as an inhibitor of osteoclast formation and function for the prevention and treatment of osteolytic diseases remains unclear. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of 5-(tetradecoxy)-2-furfuric acid in the preparation of drugs that inhibit osteoclast differentiation and function.

[0006] This paper provides the application of 5-(tetradecyloxy)-2-furfural acid in the preparation of drugs that inhibit osteoclast differentiation. The molecular formula of 5-(tetradecyloxy)-2-furfural acid is C2. 19 H 32 O4, structural formula is

[0007]

[0008] The application of this 5-(tetradecoxy)-2-furoic acid in the preparation of drugs for the prevention or treatment of osteolytic diseases.

[0009] The application of this 5-(tetradecoxy)-2-furoic acid in the preparation of drugs for the prevention or treatment of osteoporosis.

[0010] The application of this 5-(tetradecoxy)-2-furfuric acid in the preparation of drugs that inhibit NFATc1 gene expression.

[0011] The application of this 5-(tetradecoxy)-2-furfuric acid in the preparation of drugs that inhibit the expression of the Ctsk gene or the Src gene.

[0012] The application of this 5-(tetradecoxy)-2-furfuric acid in the preparation of drugs that inhibit intracellular reactive oxygen species.

[0013] The application of this 5-(tetradecoxy)-2-furfuric acid in the preparation of drugs for the prevention or treatment of bone destruction caused by tumor metastases.

[0014] The use of this 5-(tetradecoxy)-2-furoic acid in the preparation of drugs for the prevention or treatment of rheumatoid arthritis.

[0015] This drug is available in tablet, pill, powder, capsule, injection, oral liquid, ointment or cream form; and is administered orally, by injection or external application.

[0016] In this drug, the dosage of 5-(tetradecoxy)-2-furoic acid is 0.5–1 mg / kg.

[0017] The beneficial effects of this invention are:

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

[0019] 2) This invention also demonstrates the preventive and therapeutic effects of TOFA on osteoporosis caused by estrogen deficiency by constructing a mouse ovariectomized osteoporosis model. The experiment showed that TOFA can significantly reverse the downregulation effect of OVX on the relative volume and number of trabeculae and the upregulation effect of OVX on trabeculae separation. Attached Figure Description

[0020] Figure 1 The results of the CCK-8 assay for detecting the cytotoxicity of TOFA on bone marrow monocytes / macrophages (BMMs);

[0021] Figure 2 The staining results for assessing the inhibitory effect of TOFA on osteoclast differentiation using tartrate-resistant acid phosphatase (TRAP) staining.

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

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

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

[0025] Figure 6 The results are a quantitative analysis of the proportion of ROS-positive cells;

[0026] Figure 7 Figure showing the results of Western blot analysis of the effects of TOFA on osteoclast differentiation and the expression of function-related proteins.

[0027] Figure 8 Three-dimensional reconstructed images for evaluating the preventive and therapeutic effects of TOFA on ovariectomy-induced osteoporosis using microcomputed tomography.

[0028] Figure 9 The results of Micro-CT quantitative analysis of femoral cancellous bone-related parameters. Detailed Implementation

[0029] The present invention will be further described below with reference to embodiments. The description of the embodiments below is only for the purpose of helping to understand the present invention. It should be noted that those skilled in the art can make several modifications to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0030] Example 1

[0031] As an example, this first example proposes that 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA) is an allosteric inhibitor of acetyl-CoA carboxylase-α, with the molecular formula C0. 19 H 32 O4, CAS# is 54857-86-2, structural formula is:

[0032]

[0033] The drug comprises the active ingredient 5-(tetradecoxy)-2-furoic acid (TOFA) and medically acceptable pharmaceutical excipients, including diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc., commonly used in the pharmaceutical field; the 5-(tetradecoxy)-2-furoic acid can also be used in combination with other drugs to prepare compound drug formulations.

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

[0035] First, mouse bone marrow mononuclear / macrophage (BMM) cells were obtained:

[0036] Step 1: In a clean bench, sterilize scissors, forceps, and several culture dishes containing DPBS with UV light. Then, under sterile conditions, take the femur and tibia of 6-10 week old male C57BL / 6 mice, remove the muscles and connective tissue from the bones, cut off the distal ends of the joints, and place the cut ends of the bones face down in a 0.6mL sterile centrifuge tube with a pre-perforated bottom.

[0037] Step 2: Insert a 0.6 mL centrifuge tube into a 1.5 mL centrifuge tube, and stop the centrifugation at 10,000 rpm to separate the bone marrow.

[0038] Step 3: After resuspending the bone marrow cells in α-MEM complete medium containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin, transfer them to a 10cm cell culture dish and incubate them in a 37℃, 5% CO2 cell culture incubator.

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

[0040] Step 5: On the third day, after trypsin digestion, centrifuge at 1000 rpm for 4 min at room temperature, discard the supernatant, resuspend the cells in an appropriate amount of BMM medium, and count them using a cell counting chamber. The resulting cells are considered bone marrow monocytes / macrophages (BMMs). Use 4 × 10⁶ cells / wells in 6-well plates from Bio-Tek (USA). 5 / hole, 24-hole plate 1×10 5 Cell density plating was performed in 5000 wells of 96-well and 96-well plates.

[0041] Then, the cytotoxicity of TOFA to BMMs was detected using CCK-8 assay:

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

[0043] Step 2: On the second day, the cells were divided into 7 groups, with 6 replicates in each group. BMM 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 performed in 3 replicates, and incubated for 24, 48 and 72 hours (h) respectively.

[0044] Step 3: After incubation, replace each well with 100 μL of culture medium containing 10% CCK-8 reagent. In this example, CCK-8 reagent from Beyotime Biotech was used. The culture was then placed in a 37°C cell culture incubator and incubated in the dark for another 2 hours.

[0045] Step 4: Measure the OD value at a wavelength of 450nm using a multi-functional microplate reader. 450 The result is as follows Figure 1 As shown:

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

[0047] Example 2

[0048] As another embodiment, this embodiment two, based on embodiment one, proposes that TOFA has a direct inhibitory effect on osteoclast differentiation, and this is detected using TRAP staining:

[0049] In this embodiment, mouse bone marrow mononuclear / macrophages (BMMs) were first obtained using the same method as in Example 1, 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 seeded into 24-well plates and divided into 6 groups, including 0, 0.625, 1.25, 2.5, 5 and 10 μM TOFA treatment groups, with 3 replicates per group, and cultured in BMM medium for 1 day.

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

[0052] Step 3: After culturing for 5 consecutive days, tartrate-resistant acid phosphatase (TRAP) staining was performed on each group of cells. TRAP-positive multinucleated cells with more than 3 nuclei were identified as osteoclasts.

[0053] TRAP staining results are as follows Figure 2 As shown, TOFA dose-dependently inhibits the RANKL-induced osteoclast formation, such as... Figure 2 As shown.

[0054] The number and size of osteoclasts in each group were counted using ImageJ software, and the results of TRAP staining quantitative analysis are as follows: Figure 3 and Figure 4 As shown, Figure 3 and Figure 4 Further, it was shown that TOFA reduced the number and size of osteoclasts in a dose-dependent manner. These 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 as or similar to those in Embodiment 1 can be referred to each other, and will not be repeated in this application.

[0056] Example 3

[0057] As another embodiment, this third embodiment, based on the first embodiment, proposes that TOFA can inhibit the induction of reactive oxygen species (ROS) production in BMM cells by RANKL, and this can be detected using the DCFH-DA probe:

[0058] In this embodiment, mouse bone marrow mononuclear / macrophages (BMMs) were first obtained using the same method as in Example 1, and then the effect of TOFA on osteoclast formation was detected using the DCFH-DA probe:

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

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

[0061] Step 3: Add BMM osteoclast induction medium containing the corresponding concentration 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 the TOFA treatment group to the OM group; add α-MEM complete medium containing 30ng / mL M-CSF and the same dose of DMSO as the TOFA treatment group to the NM group; after two days of treatment, the reactive oxygen species level was detected using a reactive oxygen species assay kit.

[0062] The simplified steps are as follows: First, DCFH-DA was diluted 1:1000 with serum-free culture medium. After mixing, 300 μL of the diluted DCFH-DA probe was added to each well. The cells were incubated at 37°C for 20 minutes and then washed three times with serum-free culture medium to remove excess DCFH-DA. Subsequently, fluorescence and bright-field images were acquired under an inverted fluorescence microscope, and the proportion of ROS-positive cells was quantified using ImageJ software to observe the effect of TOFA on reactive oxygen species.

[0063] ROS fluorescence images such as Figure 5 As shown in the figure, the green signal represents the DCFH-DA label, and the corresponding ROS quantitative analysis results are as follows. Figure 6 As shown, the data are expressed as mean ± standard deviation. In the figure, *: p < 0.05, **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. Figure 5 and Figure 6As shown, the reactive oxygen species (ROS) level in the OM group was significantly higher than that in the NM control group, while 5-(tetradecyloxy)-2-furonic acid (TOFA) administration dose-dependently reduced the intracellular ROS level. These results indicate that 5-(tetradecyloxy)-2-furonic acid (TOFA) can inhibit the RANKL-induced ROS production in BMM cells.

[0064] It should be noted that the parts in this embodiment that are the same as or similar to those in Embodiment 1 can be referred to each other, and will not be repeated in this application.

[0065] Example 4

[0066] As another embodiment, this fourth embodiment, based on the first embodiment, proposes that TOFA can inhibit osteoclast differentiation and the expression of function-related proteins, and this can be detected using Western blot:

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

[0068] Step 1: According to 4×10 per hole 5 BMMs were seeded into 6-well plates and divided into a control group and a drug treatment group, with 3 replicates in each group; the cells were cultured in BMM medium for 1 day and then the medium was changed.

[0069] Step 2: The drug treatment group and the control group were respectively added to BMM osteoclastosis induction medium containing 10 μM TOFA and an equal volume of DMSO; the medium was changed every other day, and cell proteins were extracted with RIPA lysis buffer containing phosphatase and protease inhibitor after 0, 1, 3 and 5 days of treatment.

[0070] Step 3: Determine the concentration of the above protein samples using the BCA kit, and adjust the protein concentration of each group to a uniform level 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 samples are separated, electroporate them (90V, 2h) onto an NC membrane.

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

[0072] Step 5: The next day, wash 3 times with 1×TBST on a shaker, add secondary antibody diluted 1:4000, and incubate at room temperature for 1.5h; wash 3 times with 1×TBST, add ECL chemiluminescence solution for development and image acquisition.

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

[0074] It should be noted that the parts in this embodiment that are the same as or similar to those in Embodiment 1 can be referred to each other, and will not be repeated in this application.

[0075] Example 5

[0076] As another embodiment, this fifth embodiment, based on the first embodiment, proposes that TOFA can prevent and treat osteoporosis induced by ovariectomy in mice, and this is detected using micro-computed tomography (Micro-CT) scanning:

[0077] In this embodiment, mouse bone marrow mononuclear / macrophages (BMMs) were first obtained using the same method as in Example 1, 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: sham-operated 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 the TOFA solvent.

[0079] Step 2, OVX mouse model of ovariectomized osteoporosis: In the sham surgery group, after anesthesia, a longitudinal incision was made in the back under sterile conditions, and the subcutaneous fascia and muscles were bluntly dissected. The muscles were cut at the projection of the left and right dorsal kidneys, and some fat near the ovary was removed before the muscles, fascia, and skin were directly sutured. In the OVX, TOFA-L, and TOFA-H groups, after successful anesthesia, a longitudinal incision was made in the back under sterile conditions, and the subcutaneous fascia and muscles were bluntly dissected. The muscles were cut at the projection of the left and right dorsal kidneys, the fallopian tubes and ovaries were clamped, and the ovaries were removed after ligation below the ovaries. The muscles, fascia, and skin were then sutured.

[0080] Step 3, Administration and Sample Collection: Three days after surgery, mice in the TOFA-L and TOFA-H groups were administered 5 mg / kg and 10 mg / kg of TOFA solution by gavage, respectively. The solvent was 10% DMSO + 90% corn oil. Mice in the Sham and OVX groups were administered the same amount of the solvent, i.e., a solution of 10% DMSO + 90% corn oil, by gavage. The gavage was repeated every two days for 21 consecutive times. After this, the mice were euthanized by cervical dislocation, and the femurs were collected and fixed in 10% formalin fixative at room temperature for 48 hours.

[0081] Step 4: Micro-CT Scanning and Analysis: After rinsing with PBS, the femurs in each group were subjected to three-dimensional scanning using the NEMO in vivo integrated micro-CT scanner from Ping Sheng Company. The original scan data were then reconstructed and corrected. Subsequently, Avatar software was used to perform three-dimensional reconstruction and quantitative analysis of the cancellous bone in the region 0.2 mm to 1.20 mm below the distal femoral growth plate, obtaining three-dimensional images of the cancellous bone in this region, such as... Figure 8 As shown, the results of quantitative parameters of cancellous bone are as follows: Figure 9 As shown, the data includes relative trabecular volume (BV / TV), number of trabecular bones (Tb.N), and trabecular separation (Tb.Sp).

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

[0083] It should be noted that the parts in this embodiment that are the same as or similar to those in Embodiment 1 can be referred to each other, and will not be repeated in this application.

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

Claims

The use of 1,5-(tetradecoxy)-2-furoic acid in the preparation of a medicament for treating osteolytic diseases, wherein the medicament inhibits osteoclast differentiation, and the molecular formula of 5-(tetradecoxy)-2-furoic acid is C1. 19 H 32 O4, structural formula is 。 2. The use of 5-(tetradecoxy)-2-furoic acid as described in claim 1 in the preparation of a medicament for treating osteolytic diseases, wherein the osteolytic diseases are osteoporosis, bone destruction due to tumor metastasis, or rheumatoid arthritis.

3. The use of 5-(tetradecoxy)-2-furfuric acid as described in any one of claims 1 to 2 in the preparation of a medicament for treating osteolytic diseases, characterized in that, The dosage forms of the drug are tablets, pills, powders, capsules, injections, oral liquids, ointments, or creams.

4. The use of 5-(tetradecoxy)-2-furfuric acid as described in any one of claims 1 to 2 in the preparation of a medicament for treating osteolytic diseases, characterized in that, The dosage of 5-(tetradecoxy)-2-furoic acid in the drug is 0.5–1 mg / kg.

5. The use of 5-(tetradecoxy)-2-furfuric acid as described in any one of claims 1 to 2 in the preparation of a medicament for treating osteolytic diseases, characterized in that, Medications are administered orally, by injection, or topically.

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