阅读说明：本技术 伊快霉素在制备破骨细胞分化抑制剂中的应用 (Application of ixomycin in preparation of osteoclast differentiation inhibitor ) 是由 谭艳辉 罗小卫 李智超 谭小辉 刘永宏 于 2020-12-01 设计创作，主要内容包括：本发明公开了伊快霉素在制备破骨细胞分化抑制剂中的应用。本发明中公开了伊快霉素能有效破骨细胞生成及分化,且仅在0.1μM时即可产生抑制效果,且在4μM或以下浓度时不会对细胞生存带来影响,不产生细胞毒性,因此,能有效替代现有的双磷酸盐药物及地诺昔单抗,降低破骨细胞在骨片上形成的骨凹陷面积,抑制破骨细胞的骨吸收活性,同时下调NF-κB和NFATc1的表达,具有极高的应用价值。(The invention discloses application of ixomycin in preparing an osteoclast differentiation inhibitor. The invention discloses that the ixomycin can effectively inhibit the generation and differentiation of osteoclast, can generate an inhibiting effect only at 0.1 mu M, does not influence the survival of cells at a concentration of 4 mu M or below, and does not generate cytotoxicity, so that the iximab can effectively replace the existing diphosphate drugs and dinoteximab, reduce the bone pit area formed on bone chips by the osteoclast, inhibit the bone resorption activity of the osteoclast, and simultaneously reduce the expression of NF-kB and NFATc1, thereby having extremely high application value.)

1. The use of ixomycin in the preparation of an osteoclast differentiation inhibitor.

2. The use of ixomycin in the preparation of a nuclear factor expression inhibitor.

3. The use of claim 2, wherein said nuclear factor comprises NF-kb and NFATc 1.

4. The use of claim 3, wherein said NF- κ B comprises NF- κ B p65 and NF- κ B p 50.

5. The use of ixomycin in the preparation of a therapeutic preparation for a condition associated with abnormal osteoclast differentiation or nuclear factor expression;

wherein, the diseases related to abnormal nuclear factor expression comprise osteoporosis, Paget's disease, multiple myeloma, osteolytic bone metastasis of malignant tumor and osteogenesis imperfecta.

6. The use according to claim 1, the use according to claim 2 and the use according to claim 5, wherein the concentration of ixomycin is greater than or equal to 0.1 μ M; preferably, the concentration of the ixomycin is 0.1-4 mu M.

7. A drug for inhibiting osteoclast differentiation, which comprises ivermectin, wherein the concentration of the ivermectin in the drug is more than or equal to 0.1 mu M.

8. The drug according to claim 7, wherein the concentration of the ixomycin in the drug is 0.1-4 μ M.

9. Pharmaceutical according to claim 7 or 8, characterized in that it further comprises other pharmaceutically acceptable adjuvants.

10. The medicament of claim 9, wherein the dosage form of the medicament comprises a gastrointestinal administration dosage form and a parenteral administration dosage form.

Technical Field

The invention belongs to the field of pharmaceutical preparations, and particularly relates to application of ixomycin in preparation of an osteoclast differentiation inhibitor.

Background

Osteoclasts (OCs) are the only cells in the human body that have a bone resorption function. Osteoclasts originate from bone marrow mononuclear-macrophage lineage cells and are large multinucleated cells formed from mononuclear precursor cells fused in a variety of ways. Excessive osteoclast activation is not only commonly seen in menopausal osteoporosis, but also in bone destructive diseases such as bone loss caused by inflammatory diseases (e.g., bacterial infection), rheumatoid arthritis, tumor bone metastasis, and the like. Therefore, drugs that target osteoclasts, inhibit osteoclastogenesis or function, and may only be important means for preventing and treating the associated bone destructive diseases.

Currently, the clinically relevant inhibitors targeting osteoclast differentiation are denoximab (RANKL (Receptor activator of nuclear factor kappa B ligand)) and bisphosphonates capable of inhibiting osteoclast bone resorption. Both of these drugs, while inhibiting osteoclast differentiation or function, have serious complications and intolerable side effects. If the bisphosphonate medicines are taken for a long time, the natural renewal of bone tissues can be inhibited, so that the fracture is easily caused; and the denoxib needs subcutaneous injection and is expensive, and most importantly, serious complications such as mandibular necrosis and the like exist in the process of long-term use of the denoxib.

Therefore, there is a need to find a safe, effective, convenient and economical drug targeting osteoclasts to replace the existing therapeutic drugs with obvious defects, which is not only related to the treatment of diseases related to osteoclast differentiation, but also can solve the huge use requirement of the osteoclast differentiation inhibitor in the market.

Disclosure of Invention

The invention aims to provide the application of the ixomycin in preparing an osteoclast differentiation inhibitor;

the invention also aims to provide the application of the ixomycin in preparing the nuclear factor expression inhibitor;

the invention also aims to provide the application of the ixomycin in preparing a preparation for treating diseases related to abnormal osteoclast differentiation or nuclear factor expression;

another object of the present invention is to provide a medicament for inhibiting osteoclast differentiation.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided:

the use of ixomycin in the preparation of an osteoclast differentiation inhibitor.

The ixomycin (Equisetin, YINHUICHUANG, FE1) is a metabolite secreted by Fusarium equiseti, and has antibacterial effect.

The chemical structural formula of the ixomycin is shown as a formula I:

the aforementioned ixomycin has a remarkable inhibitory effect on osteoclastic activity, and the bone resorption activity of osteoclasts can be inhibited by reducing the area of bone pits formed on the bone fragments by osteoclasts only by including the aforementioned ixomycin at a concentration of 0.1. mu.M.

Further, the content of the aforementioned ixomycin is 0.1. mu.M or more.

In a second aspect of the present invention, there is provided:

the use of ixomycin in the preparation of a nuclear factor expression inhibitor.

Further, the above nuclear factors include NF-. kappa.B and NFATc 1.

The ligand (RANKL) for activating nuclear factor NF-kB receptor is the most direct factor for inducing the generation and differentiation of osteoclast in vivo, and the RANKL can be combined with RANK receptor (nuclear factor NF-kB receptor) on the membrane of osteoclast precursor cell to recruit TRAF6 and c-Src complex, so as to activate the expression of nuclear factor NF-K B, AP-1 and NFATc1, induce the OCs-related genes (such as alkali phosphatase against Tartrate (TRAP), matrix metalloproteinase 9(MMP-9), integrin beta 3(integrin beta 3) and cathepsin K, and the like) and promote the differentiation of OCs.

Moreover, the NF-kB signal channel is also involved in the regulation of various physiological functions such as inflammation, apoptosis, tumor, immunity and the like, and plays an important role in the differentiation process of the osteoclast induced by the RANKL. Under the induction of RANKL, the arrestin I kappa Ba combined with NF-kappa B p65 and p50 dimer is rapidly phosphorylated and degraded, and the combined dimer of p65 and p50 is released, so that the dimer of p65 and p50 can enter the nucleus from cytoplasm, and the transcription of OCs related genes is promoted.

Activated NF-. kappa.B also binds to the promoter region Of NFATc1(Recombinant Nuclear Factor Of Activated T-Cells, Cytoplasmic 1, Activated T-Nuclear Factor 1) to activate NFATc 1. NFATc1 is not only an important regulator of immune response, but is also an important transcription factor for osteoclastogenesis. The activated NFATc1 is transferred from cytoplasm to nucleus to start the expression of OCs related genes, and finally leads to the differentiation and maturation of osteoclasts, increase in the number of osteoclasts and generation of hyperfunction of bone resorption.

Further, the NF-. kappa.B mentioned above includes NF-. kappa. B p65 and NF-. kappa. B p 50.

Further, the content of the aforementioned ixomycin is 0.1. mu.M or more.

In a third aspect of the present invention, there is provided:

the use of ixomycin in the preparation of a therapeutic preparation for diseases related to osteoclast differentiation or nuclear factor expression abnormality.

The above diseases related to abnormal osteoclast differentiation or nuclear factor expression include, but are not limited to, bisphosphonates, and clinical indications corresponding to denoximab, such as postmenopausal osteoporosis, rheumatoid arthritis, tumors, and bone destruction caused by tumor metastasis, and also include prevention, treatment and/or prognosis of diseases related to abnormal expression of NF- κ B and NFATc1, including, but not limited to, inflammatory diseases, autoimmune diseases, tumors, and the like.

Further, the above-mentioned disorders associated with abnormal nuclear factor expression specifically include osteoporosis, Paget's disease, multiple myeloma, osteolytic bone metastasis of malignant tumor, and osteogenesis imperfecta.

NFATc1 is an important transcription regulator, participates in the regulation and expression of OC specific genes, and is important for OC differentiation, fusion, adhesion and bone resorption functions. Activation of RANK by osteoclast precursor cells (OCP) induces activation of NFATc1 through multiple pathways, whereas NFATc1 is a target gene for NF- κ B signaling. Osteoporosis, Paget's disease, multiple myeloma, osteolytic bone metastasis from malignant tumors, and osteogenesis imperfecta have been shown to be associated with the expression of NFATc1, and if the expression of NFATc1 is effectively inhibited, the risk, progression, or related conditions of osteoporosis, Paget's disease, multiple myeloma, osteolytic bone metastasis from malignant tumors, and osteogenesis imperfecta can be reduced, alleviated, or treated.

Further, the content of the aforementioned ixomycin is 0.1. mu.M or more.

In a fourth aspect of the present invention, there is provided:

a drug for inhibiting osteoclast differentiation, which contains ivermectin, wherein the concentration of the ivermectin in the drug is more than or equal to 0.1 mu M.

The inventor finds that the medicament in the embodiment of the invention can effectively inhibit the generation and differentiation of osteoclast, and can generate a remarkable inhibiting effect only when the concentration of the ixomycin is 0.1 mu M. The content of the composition in the medicine is increased to 4 mu M, so that the cell survival is still not influenced, and the cytotoxicity is not generated.

Further, the concentration of the ixabepilin in the above-mentioned medicine is 0.1-4. mu.M.

Furthermore, the medicine also contains other pharmaceutically acceptable auxiliary agents.

Further, the dosage forms of the above drugs include gastrointestinal administration dosage forms and parenteral administration dosage forms.

Wherein, the gastrointestinal administration dosage forms comprise common oral dosage forms, such as powder, tablet, granule, capsule, solution, emulsion, suspension, etc.

The parenteral administration forms include injection, respiratory administration, skin administration, mucosal administration and cavity administration.

Of course, the dosage form of the above-mentioned drugs can be reasonably adjusted by those skilled in the art according to the actual use requirements, so as to carry out the administration.

The invention has the beneficial effects that:

1. the inventors found that ixomycin is effective in osteoclastogenesis and differentiation, has an inhibitory effect at 0.1. mu.M, and does not affect cell survival at a concentration of 4. mu.M or less, and does not cause cytotoxicity.

2. The medicament can effectively replace the existing bisphosphate medicament and dinoteximab, can obviously reduce the bone pit area formed by osteoclasts on bone chips and inhibit the bone resorption activity of the osteoclasts on one hand, and can regulate and control the expression abnormality of NF-kB and NFATc1 on the other hand, thereby having extremely high application value and market prospect.

Drawings

FIG. 1 shows the effect of 0.1. mu.M, 1. mu.M, 2. mu.M and 4. mu.M of ixomycin on the activity of osteoclast precursor RAW264.7 cells;

FIG. 2 is a graph of the effect of 0.1. mu.M, 1. mu.M, 2. mu.M and 4. mu.M of ixomycin on the activity of BMMs;

FIG. 3 is a graph showing the effect of ixomycin on the differentiation of osteoclast precursor RAW264.7 cells, wherein A is a graph showing the result of TRAP staining of RAW264.7 cells, and B is the result of the effect of ixomycin on the differentiation of osteoclast precursor RAW264.7 cells;

FIG. 4 is a schematic representation of the effect of iximacin on the differentiation of BMMs, wherein A is the result of TRAP staining of BMMs, and B is the result of the effect of iximacin on the differentiation of BMMs;

FIG. 5 is a graph of the effect of ixomycin on osteoclast activity, wherein A is a graph of the staining results of cellular TRAP staining and B is the area of bone depression formed by the ixomycin osteoclasts on the bone plate at concentrations of 0.1. mu.M, 0.5. mu.M and 1. mu.M;

FIG. 6 shows the effect of ixabepilin on NF- κ B and NFATc1 induced by RANKL, wherein a is the effect of ixabepilin on the activity of NF- κ B of osteoclast precursor cells, B is the effect of ixabepilin on the activity of NFATc1 of osteoclast precursor cells, c is the fluorescence spectrum of ixabepilin on NF- κ B induced by RANKL, d is the band intensity of ixabepilin on NF- κ B induced by RANKL, e is the fluorescence spectrum of ixabepilin on NFATc1 induced by RANKL, and d is the band intensity of NFATc1 induced by ixabepilin on RANKL.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.

Cytotoxicity assays for ixomycin

In this example, MTT method and CCK-8 method were used to detect the cytotoxicity of ixomycin, respectively, to ensure the accuracy and stringency of the assay.

(1) MTT method for detecting survival influence of ixomycin on osteoclast precursor RAW264.7 cells

RAW264.7 cells (1X 10) in good growth status were collected³One/well) were inoculated in a 96-well plate, DMEM medium (containing 10% fetal bovine serum, 100IU/mL penicillin and 100IU/mL streptomycin) was added to 200. mu.L per well, and 5% CO was added at 37 ℃₂Incubate overnight at ambient. After the cells are adherent and stable, different concentrations of the ixomycin are respectively added to ensure that the final concentration of the ixomycin in the holes is 0.1 mu M, 1 mu M, 2 mu M and 4 mu M, each group is provided with 3 multiple holes, and the incubation is carried out for 4 days. After completion of incubation, the supernatant was discarded, and 100. mu.L of MTT (0.5 mg/mL) was added to each well at 37 ℃ with 5% CO₂Incubation was continued for 4 h. After the incubation is finished, the supernatant is discarded, 150 mu L of DMSO solution is added into each hole, the mixture is shaken for 15min on a micro-oscillator, the optical density value (OD value) at the wavelength of 570nm is measured by a TECANGENIOsPro multifunctional microplate reader, and the survival rate of each group of cells is calculated.

The results are shown in FIG. 1.

As shown in FIG. 1, after the addition of 0.1. mu.M, 1. mu.M, 2. mu.M and 4. mu.M of ixomycin, there was no significant difference in the viability of the osteoclast precursor RAW264.7 cells, indicating that the concentration of 4. mu.M or less of ixomycin was not cytotoxic to RAW264.7 cells in the in vitro assay.

(2) CCK-8 method for detecting survival influence of ixomycin on mouse Bone Marrow Macrophages (BMMs)

a. Preparation of mouse Bone Marrow Macrophages (BMMs):

under aseptic conditions, the femurs of 8-12 week old C57BL/6 female mice were harvested, the joint sites at both ends of the femurs were cut, and the femurs were repeatedly flushed with phenol red-free alpha-MEM medium (containing 10% fetal bovine serum, 100IU/mL penicillin and 100IU/mL streptomycin) until the femoral bone cavities became white.

Placing the washed femoral medullary cavity cells at 37 deg.C and 5% CO₂And (3) incubating the cell culture box for 2h, sucking the supernatant, cracking the red blood cells by using a lysate, centrifuging, and re-suspending to obtain the BMMs.

The CCK-8 method is used for detecting the survival condition of the cells:

taking the BMMs (1X 10) prepared in the step a⁵One/well) in a 96-well plate, phenol red-free alpha-MEM medium was added to 200. mu.L (containing 10% fetal bovine serum, 100IU/mL penicillin and 100IU/mL streptomycin) per well, while macrophage colony stimulating factor (M-CSF, final concentration of 50ng/mL) was added to each well, and then the 96-well plate was placed at 37 ℃ in a 5% CO system₂The cell culture chamber of (a) was incubated overnight. After the cells are attached to the wall stably, different concentrations of the ixomycin are respectively added to ensure that the final concentration of the ixomycin in the holes is 0.1 mu M, 1 mu M, 2 mu M and 4 mu M, each group is provided with 3 multiple holes, and the incubation is carried out for 4 days. After completion of incubation, the supernatant (100. mu.L) was discarded, 5. mu.L of CCK-8 reagent (Cell Counting Kit-8 Cell Counting reagent) was added to each well, shaken, and placed at 37 ℃ in 5% CO₂And (3) continuously incubating for 3h in the environment, measuring the optical density value (OD value) at the wavelength of 450nm by using a TECANGENIOsPro multifunctional enzyme-linked immunosorbent assay, and calculating the survival rate of each group of cells.

The results are shown in FIG. 2.

As shown in FIG. 2, after the addition of 0.1. mu.M, 1. mu.M, 2. mu.M and 4. mu.M of ixomycin, no significant difference occurred in the survival rate of BMMs, indicating that the concentration of 4. mu.M or less of ixomycin was not cytotoxic to BMMs in the in vitro assay.

Effect of Ixabemycin on cellular differentiation into osteoclasts

This example uses osteoclast precursor RAW264.7 cells and BMMs as test subjects to examine the effect of ixomycin on differentiation of cells into osteoclasts.

(1) Effect of Iflunomycin on differentiation of RAW264.7 cells into osteoclasts

RAW264.7 cells (1X 10) in good growth status were collected³One/well) in 96-well plates at 37 ℃ with 5% CO₂Incubate overnight at ambient. After the cells are attached to the wall stably, different concentrations of the iximazam are respectively added to ensure that the final concentration of the iximazam in the holes is 0.1 mu M, 1 mu M, 2 mu M and 4 mu M, each group is provided with 3 multiple holes, and the incubation is carried out for 4 hours. Adding RANKL (final concentration of 100ng/mL), changing the solution every two days, and culturing for 4-5 d.

TRAP staining of the incubated cells, photographing under an inverted microscope and counting, wherein TRAP positive cells with a nucleus larger than 3 are osteoclasts.

The results are shown in FIG. 3.

As shown in FIG. 3, it is understood that the inhibition effect of ixomycin is exhibited at an effective concentration of 0.1. mu.M, and the induction of RAW264.7 osteoclast precursor cells into osteoclasts by RANKL is significantly inhibited at an effective concentration of 1. mu.M.

(2) Effect of Ixabemycin on the differentiation of BMMs into osteoclasts

Taking the BMMs (2X 10) prepared in the step a⁴One/well) in a 96-well plate, phenol red-free alpha-MEM medium was added to 200. mu.L (containing 10% fetal bovine serum, 100IU/mL penicillin and 100IU/mL streptomycin) per well, while macrophage colony stimulating factor (M-CSF, final concentration of 50ng/mL) was added to each well, and then the 96-well plate was placed at 37 ℃ in a 5% CO system₂The cell culture chamber of (a) was incubated overnight.

After the cells are attached to the wall stably, the iximazac with different concentrations is added respectively, so that the final concentration of the iximazac in the holes is 0.1 mu M, 1 mu M, 2 mu M and 4 mu M, each group is provided with 3 compound holes, and the incubation is carried out for 4 hours. After the incubation was completed, RANKL (final concentration of 100ng/mL) was added and cultured for 3-4 d.

TRAP staining of the incubated cells, photographing under an inverted microscope and counting, wherein TRAP positive cells with a nucleus of more than 5 are osteoclasts.

The results are shown in FIG. 4.

As can be seen from fig. 4, ixomycin significantly inhibits RANKL from inducing BMMs to generate osteoclasts.

In conclusion, by combining the cytotoxicity detection results of the ixabepilin, the skilled person can completely select the ixabepilin with the concentrations of 0.1 μ M, 0.5 μ M and 1 μ M to intervene the generation of osteoclasts, and when the effective concentration of the ixabepilin reaches 0.5 μ M, the in vitro RANKL can be remarkably inhibited from inducing RAW264.7 cells and BMMs cells to form osteoclasts.

Effect of ixomycin on osteoclast Activity

By adopting the technical scheme of the influence of the ixomycin on the differentiation of the BMMs into osteoclasts, the BMMs are inoculated to the bone slices coated with the artificial bone slicesCells were washed out on day 7 post-induction in Osteo Assay 96-well plates, photographed under 40 XNikon inverted light microscope light microscopy and the percentage of bone resorption area per well was calculated by Image-Pro Plus software.

The results are shown in FIG. 5.

The experimental results prove that the ixomycin at the concentration of 0.1 mu M shows the bone resorption inhibiting activity (the bone resorption area is smaller than that of the control group), and the ixomycin at the concentrations of 0.5 mu M and 1 mu M can obviously reduce the bone depression area formed on the bone plate by the osteoclast and has the inhibiting effect on the bone resorption activity of the osteoclast.

Effect of ixomycin on RANKL-induced NF- κ B and NFATc1

Taking experimental cell strain (NF-kB-luc stable RAW264.7 cell strain or NFATc1-luc plasmid DNA transient transfection RAW264.7 cell strain), adding RANKL for stimulation, and simultaneously adding ixomycin (final concentration of 0.1 μ M, 1 μ M, 2 μ M and 4 μ M) and positive control drugSubstance (5. mu.M BAY or 1. mu.M CsA), 5% CO at 37 ℃₂After 8h incubation, luciferase expression was detected, and the effect of the drug on the transcriptional expression of the osteoclast NF-kappa B, NFATc1 was observed, as shown in a in FIG. 6 and b in FIG. 6.

Meanwhile, Western blot detection medicines are used for detecting the expression of nuclear transcription factor NF-kappa B, NFATc1 related protein activated by RNAKL, and the method comprises the following specific steps:

the above test cell line (1X 10) was used⁶Or 2X 10⁵One/well) in 96-well plates, 37 ℃ with 5% CO₂Incubate overnight. Adding ixomycin (final concentration of 0.1. mu.M, 1. mu.M, 2. mu.M and 4. mu.M) and RANKL (final concentration of 100ng/mL), incubating for 30min (detection of NF-kappa B) or 24h (detection of NFATc1), extracting nucleoproteins (Lamin A/C, Lamin A/C; beta-actin, beta-actin), and detecting the expression of NF-kappa B (NF-kappa Bp65) or NFATc1 by the conventional method in the art respectively, wherein the results are shown as C, d, e and f in FIG. 6.

As can be seen from FIG. 6, the luciferase activity of NF-kappa B, NFATc1 induced by Iemomycin and RANKL is in a concentration-dependent inhibition relationship, wherein the inhibition effect of 1 μ M of Iemomycin on NF-kappa B, NFATc1 is stronger than that of a positive control drug with the same concentration, and therefore, the Iemomycin may inhibit the generation and function of osteoclasts by inhibiting the NF-kappa B, NFATc1 induced by RANKL.

Taken together with the above results, at a concentration of 0.1. mu.M, ixomycin can exhibit NF-. kappa. B, NFATc1 expression inhibitory activity, whereas at a concentration of 0.1. mu.M, ixomycin can significantly inhibit in vitro RANKL-induced osteoclast differentiation formation and bone resorption function, and it is possible to inhibit osteoclast production and function by inhibiting NF-. kappa. B, NFATc 1. It is expected that the ixomycin can be used for replacing the bisphosphate drug and the denoxib in the field with the same self action mechanism, so as to treat the prevention, treatment and/or prognosis of the diseases related to the abnormal expression of the bisphosphate drug and the denoxib in NF-kB and NFATc1, wherein the diseases related to the abnormal expression of the NF-kB and NFATc1 include but are not limited to inflammatory diseases, autoimmune diseases, tumors and the like, and particularly include osteoporosis, Paget's disease, multiple myeloma, osteolytic bone metastasis of malignant tumors, osteogenesis imperfecta and other symptoms.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.