Application of loperamide in preparation of medicine for treating osteoporosis

Abstract

The invention discloses an application of loperamide in preparation of a medicine for treating osteoporosis, and belongs to the field of development of new application of medicines. Loperamide has the effects of inhibiting aging, increasing osteogenic differentiation of bone marrow mesenchymal stem cells and preventing osteoporosis of aged and postmenopausal mice; meanwhile, at the cellular level, loperamide can reduce expression of senescence factors in mouse mesenchymal stem cells, promote senescence cell apoptosis and inhibit cell senescence; and a new choice is provided for the treatment of senescence-related osteoporosis drugs.

Description

Technical Field

[0001] The invention belongs to the field of development of new drug uses, and specifically relates to the use of loperamide in the preparation of drugs for treating osteoporosis. Background Art

[0002] Aging can cause a variety of chronic diseases and geriatric syndromes, leading to a decline in physical function and increased mortality. Cellular senescence was first discovered by Hayflick and Moorehead in 1961. It is an irreversible state of cell cycle arrest. Signals related to tissue or cell damage and cancer development can promote cell senescence. These stimulation signals can activate multiple pro-aging transcription factor cascades, aggregate the activation of cyclin-dependent kinase inhibitors (CDKI) such as p16, p21 and p27 through multiple intracellular pathways such as ATR, ATM and p53, and lead to changes in the expression of hundreds of genes and epigenetic changes in DNA. Senolytics were originally discovered based on the gene expression profile of senescent cells through bioinformatics methods. At present, it is defined as a large class of cells that can temporarily disable SCAP (anti-apoptotic pathway) by interfering with the SCs signaling pathway, causing selective apoptosis of tissue-destructive senescent cells, while other cells can still survive. When exploring effective drugs, it is necessary to establish cell and animal models that specifically induce senescence and accurately identify senescent cells as key targets of senolytics.

[0003] Loperamide, also known as chlorpyramide hydrochloride and Loperamide, is a white or off-white powder chemical. Its chemical name is 4-p-chlorobenzene-4-hydroxy-N, N-dimethyl-α, α-diphenyl-1-piperidinylbutyramide, and its molecular formula is C 29 H 33 ClN2O2, molecular weight is 477.03800. Loperamide is a long-acting antidiarrheal drug, which is mainly used to treat acute and chronic diarrhea caused by various causes in clinical practice, and is particularly suitable for the long-term treatment of chronic diarrhea.

[0004] Previous studies have not shown that loperamide has a therapeutic effect on osteoporosis. Our research group's experiments have shown that a certain dose of loperamide can promote the osteogenic differentiation of bone marrow mesenchymal stem cells, increase the cortical bone thickness of wild-type aged mice, and alleviate osteoporosis caused by postmenopause. At the same time, it can also serve as a potential anti-aging drug "senolytics" to alleviate osteoporosis caused by aging.

[0005] Osteoporosis is one of the common chronic diseases among the elderly in my country. The China Osteoporosis Survey released for the first time by the National Health Commission in 2018 showed that the prevalence of osteoporosis in people over 50 years old was 19.2%, and that in people over 65 years old was 32.0%. Bone homeostasis is mainly balanced by the bone formation of osteoblasts and the bone resorption of osteoclasts. Osteoporosis is caused by the activity of osteoclasts exceeding that of osteoblasts. Osteoarthritis is a degenerative osteoarthritis caused by a variety of risk factors, including genetics and epigenetics, race, age, gender, obesity and inflammation. At present, most of the drugs used clinically for osteoporosis are drugs that inhibit bone resorption, which is difficult to solve the essential problem of osteoporosis in the elderly. A few drugs that promote bone formation, such as parathyroid hormone, have disadvantages such as joint pain, decreased bone density after discontinuation of the drug, and may cause hypercalcemia. Therefore, finding a drug delivery site that can enhance osteoblast function and increase bone formation is an important aspect of research. Summary of the invention

[0006] The first aspect of the present invention provides a use of loperamide as a pharmaceutical ingredient in the preparation of an anti-osteoporosis drug for treatment, wherein the drug also includes a pharmaceutically acceptable carrier.

[0007] Furthermore, the anti-osteoporosis drug comprises a mixture of loperamide or its homologues / metabolites;

[0008] Further, the properties are powder, granules, tablets, capsules, pills, solutions, suspensions or injections;

[0009] Furthermore, the anti-osteoporosis drug is administered orally, by drip or injection;

[0010] Furthermore, the subject of administration is a mammal, and the mammal includes a human. Preferably, the animal or human is a postmenopausal animal or human;

[0011] Furthermore, the dosage is 1-2 mg / kg / day, preferably 1.5 mg / kg / day.

[0012] The second aspect of the present invention provides the use of loperamide in the preparation of a preparation for promoting osteogenic differentiation of bone marrow mesenchymal stem cells; characterized in that the use is an in vitro application.

[0013] Furthermore, the stem cells are bone marrow mesenchymal stem cells;

[0014] Furthermore, the dosage of loperamide is 5 to 15 μM, preferably 10 μM.

[0015] The third aspect of the present invention provides the use of loperamide in the preparation of a preparation for inhibiting stem cell aging; characterized in that the application is an in vitro application.

[0016] Furthermore, the stem cells are bone marrow mesenchymal stem cells.

[0017] Furthermore, the dosage of loperamide is 5 to 15 μM, preferably 10 μM.

[0018] The fourth aspect of the present invention provides the use of loperamide in the preparation of a preparation for inhibiting stem cell damage; characterized in that the application is an in vitro application.

[0019] Furthermore, the stem cells are bone marrow mesenchymal stem cells.

[0020] Furthermore, the dosage of loperamide is 5 to 15 μM, preferably 10 μM.

[0021] The beneficial effects of the present invention include:

[0022] 1) The present invention has been found through research that, in in vivo experiments on experimental animals, loperamide has the effects of inhibiting aging, increasing osteoblast differentiation and preventing osteoporosis in aged mice;

[0023] 2) The present invention has found through research that after OVX surgery (ovarian removal surgery) osteoporosis modeling in female adult mice, loperamide treatment significantly increased the bone mass / tissue mass and cancellous bone content of the femur, which can prevent osteoporosis in aged postmenopausal mice;

[0024] 3) Loperamide can reduce the expression of senescence factors in mouse bone marrow mesenchymal stem cells, indicating that loperamide promotes apoptosis of senescent cells, thereby inhibiting cell senescence at the overall level;

[0025] 4) Loperamide can promote osteoblast mineralization of bone marrow mesenchymal stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the chemical structural formula of loperamide;

[0027] Figure 2 The changes in cancellous bone thickness in 10-month-old wild-type mice after 8-month treatment with loperamide, where A: 3D schematic diagram of cancellous bone, B: statistics of cancellous bone parameters;

[0028] Figure 3 The changes of aging factor SASPs in bones of 10-month-old wild-type mice after being treated with loperamide for 8 months;

[0029] Figure 4 The changes of cancellous bone in OVX model mice after loperamide treatment, where A: 3D schematic diagram of cancellous bone, B: statistics of cancellous bone parameters;

[0030] Figure 5 The effect of loperamide on mineral nodule staining of primary bone marrow mesenchymal stem cells after differentiation;

[0031] Figure 6 The effect of loperamide on the RNA level of osteogenic differentiation in primary bone marrow mesenchymal stem cells;

[0032] Figure 7 The effect of loperamide on the cell senescence factor p16 after 1ps stimulation of primary bone marrow mesenchymal stem cells;

[0033] Figure 8 The effect of loperamide on cellular senescence factors of primary bone marrow mesenchymal stem cells. DETAILED DESCRIPTION

[0034] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0035] Example 1, Effect of loperamide on bones of wild-type aged mice

[0036] 1. Experimental animals:

[0037] Male 10-month-old C57BL / 6 mice were provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The mice were allowed to drink water and eat freely, and the feed was provided by the Animal Experiment Center of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The mice were intraperitoneally injected with loperamide and solvent every week for 8 months starting from 10 months of age. After 8 months, the mice were euthanized and the femurs were removed for micro-CT scanning to detect bone mass.

[0038] 2. Experimental drugs:

[0039] Loperamide was purchased from Sigma Reagent Company, catalog number L4762. Dimethyl sulfoxide (DMSO) was purchased from Solebol Reagent Company.

[0040] 3. Experimental equipment: weighing scale, scissors, tweezers, syringe.

[0041] 4. Experimental steps:

[0042] 4.1 Animal intraperitoneal injection treatment:

[0043] The 10-month-old mice were treated with loperamide and solvent at 1.5 mg / kg body weight per day, and the control group was injected with dimethyl sulfoxide per day. The body weight was measured and injected per day at 10 a.m. The mice were killed 8 months after the injection and the femurs were collected for scanning and analysis of bone mass and thickness.

[0044] 4.2 Detection of animal femur aging factors

[0045] The bone marrow in the femur was flushed out with ice-cold PBS, and then the femur was ground into powder in liquid nitrogen. Then 1 ml of Trizol solution was added, mixed, and placed on ice for 10-15 minutes to allow it to be fully lysed. RNA was then extracted and reverse transcribed. The expression levels of molecular signals such as p16, p53, IL-1β, and CXCL1 were detected using RT-PCR.

[0046] 4.3 Statistical analysis:

[0047] The experimental results were expressed as mean ± SEM, and the significance among different treatment groups was analyzed by two-way ANOVA.

[0048] The results are as follows Figure 2 As shown in the figure, wild-type mice were intraperitoneally injected with loperamide and solvent every day from 10 months of age for 8 M. The loperamide-treated group showed a significant increase in cancellous bone thickness ( Figure 2 A), specifically, increased bone volume fraction (BV / TV), increased trabecular number (Tb.N), increased trabecular thickness (Tb.Th), and decreased trabecular spacing (Tb.Sp) (each group of animals = 6, *p < 0.05) ( Figure 2 B). The above results indicate that loperamide can significantly increase the number of trabeculae and the thickness of cancellous bone in aged wild-type mice.

[0049] Detection of aging factors Figure 3 As shown in Figure 2, the RNA levels of aging secretory factors such as p16, p53, IL-1β, and CXCL1 in the femur of mice treated with loperamide showed a significant downregulation. The above results indicate that loperamide can inhibit bone aging and restore bone mass in the elderly.

[0050] Example 2, Effect of loperamide on the skeleton of wild-type C57BL6J female mice after ovariectomy (OVX)

[0051] 1. Experimental animals:

[0052] C57BL6J mice, female, were provided by Spefor Laboratory Animal Technology Co., Ltd. Mice underwent OVX surgery at 11 weeks of age, and were housed in single cages after surgery with free access to food and water. They were intraperitoneally injected with loperamide and solvent and dimethyl sulfoxide in the control group for 6 months starting at 12 weeks of age, and then the mice were killed, and the femurs were removed for micro-CT scanning to measure bone mass.

[0053] 2. Experimental drugs:

[0054] Loperamide was purchased from Sigma Reagent Company, catalog number L4762. Dimethyl sulfoxide (DMSO) was purchased from Solebol Reagent Company.

[0055] 3. Experimental equipment: weighing scale, scissors, tweezers, and syringe.

[0056] 4. Experimental steps:

[0057] 4.1 OVX surgery:

[0058] After anesthetizing the mice, shave them, leaving no hair around the wound for about 1 cm. Disinfect with iodine, and then disinfect the mouse skin surface with 75% alcohol. Open the abdominal cavity, find the ovaries, and remove the ovaries after ligating the fallopian tubes (the control group opened the abdominal cavity and followed the same steps to find the ovaries, but did not remove them). Suture the peritoneum and skin layers separately, sprinkle antibiotics on the wound surface, and inject the mice with 0.1g / kg body weight of antibiotics and 200μL of 20% glucose.

[0059] 4.2 Animal intraperitoneal injection treatment:

[0060] The mice in the treatment group were intraperitoneally injected with loperamide and solvent at 1.5 mg / kg body weight every day, and the mice in the control group were intraperitoneally injected with dimethyl sulfoxide. The body weight was measured and intraperitoneally injected at 10 a.m. every day. After 6 M intraperitoneal injection, the mice were killed and the femurs were collected for scanning and analysis of bone mass and thickness.

[0061] 4.3 Statistical analysis:

[0062] The experimental results are expressed as mean ± SEM, and the significance between different treatment groups was analyzed by two-way analysis of variance. Figure 4 As shown, the bone volume fraction (BV / TV) increased, the number of trabeculae (Tb.N) increased, the trabecular thickness (Tb.Th) increased, and the trabecular spacing (Tb.Sp) decreased (animals in each group = 6, *p < 0.05), indicating that after weekly intraperitoneal injection of loperamide, loperamide-treated postmenopausal induced osteoporosis model mice showed a significant anti-osteoporosis effect, that is, loperamide can alleviate the osteoporosis phenomenon in mice caused by postmenopause, and the bone mass / tissue mass ratio and the number of cancellous bones in mice have been restored, which are significantly higher than those in the control group.

[0063] Example 3. Effect of loperamide on differentiation of bone marrow mesenchymal stem cells

[0064] 1. Experimental animals:

[0065] 8-12W C57BL / 6 mice were provided by Beijing Sibeifu Laboratory Animal Technology Co., Ltd.

[0066] 2. Experimental drugs:

[0067] Loperamide was purchased from Sigma Reagent Company, catalog number L4762. Dimethyl sulfoxide (DMSO) was purchased from Solebol Reagent Company. Culture medium was purchased from Corning Company, and other chemical reagents were purchased from Sigma Reagent Company.

[0068] 3. Experimental instruments:

[0069] Cell culture plates, cell culture dishes, pipettes, scissors, tweezers, centrifuge, 50ml centrifuge tubes.

[0070] 4. Experimental steps:

[0071] 4.1. Isolation of primary bone marrow mesenchymal stem cells:

[0072] Primary bone marrow mesenchymal stem cells (BMSCs) were isolated from the bone marrow of 8-12 week-old mice using the adherent screening method. The adherent growth characteristics of BMSCs were utilized, and the culture medium was replaced to gradually remove the floating hematopoietic system cells to obtain more purified BMSCs.

[0073] 4.2. Cell differentiation and drug treatment:

[0074] After the cells grew to 100% density, they were treated with 10 μM loperamide and differentiated. The cell differentiation medium was α-MEM medium with 10% fetal bovine serum supplemented with 10 μg / ml ascorbic acid, 10 mM β-glycerophosphate and 10 nM dexamethasone. After 14 days of osteogenic differentiation, alizarin red staining was performed to visualize the mineralized nodules in the cells. After staining, the color depth was observed to determine the difference in mineralization degree.

[0075] 4.3. Expression of osteogenic differentiation markers in cells:

[0076] The cells were stimulated with 50 ng / ml lipopolysaccharide (LPS) and treated with 10 μM loperamide throughout the differentiation process. After 0, 7 and 14 days of differentiation, the cells were harvested with Trizol at different time points, RNA was extracted and reverse transcribed, and the expression levels of molecular signals such as Ocn were detected by RT-PCR.

[0077] 4.4. Expression of p16 and other SASPs molecular signals in cells:

[0078] After stimulating the cells with 50 ng / ml LPS and 10 μM loperamide for 48 h, the cells were harvested with Trizol, RNA was extracted and reverse transcribed, and the expression levels of p16 and p53 were detected by RT-PCR.

[0079] The results are as follows Figure 5-8As shown, specifically, Figure 5 It shows that under LPS stimulation, the 10μM loperamide treatment group has a significant repair effect, and the mineralized nodules (red) in the 10μM loperamide treatment group are significantly higher than those in the LPS stimulation treatment group;

[0080] Figure 6 It showed that under LPS stimulation, the 10μM loperamide treatment group had a significant repair effect, and the OCN level in the 10μM loperamide treatment group was significantly higher than that in the LPS stimulation treatment group; Figure 7 It was proved that under LPS stimulation, cell p16 increased significantly, and after treatment with loperamide, cell senescence could be effectively inhibited; Figure 8 The results suggest that loperamide treatment can significantly inhibit the expression of senescence factors (p53, MCP1, p16 and IL6), effectively eliminate senescent cells and inhibit cell senescence. The above results show that loperamide has the effect of repairing the damage and senescence of primary bone marrow mesenchymal stem cells caused by LPS.

[0081] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall all fall within the scope of protection of the present invention.

Claims

1. The use of loperamide in the preparation of a drug for treating osteoporosis, wherein: The medicine also includes a pharmaceutically acceptable carrier.

2. The use according to claim 1, characterized in that: The medicine is in the form of powder, granules, tablets, capsules, pills, solutions, suspensions or injections, and the subjects of administration of the medicine are mammals or humans.

3. The use according to claim 1 or 2, characterized in that: The drug is administered orally, by drip or injection, and the subjects are mammals or humans who have undergone menopause or ovarian removal.

4. The use according to claim 3, characterized in that: The dosage of loperamide, the active ingredient of the medicine, is 1-2 mg / kg / day.

5. Use of loperamide in the preparation of a preparation for promoting osteogenic differentiation of bone marrow mesenchymal stem cells, characterized in that: The application is in vitro application.

6. The use according to claim 5, wherein the dosage of loperamide is 5 to 15 μM.

7. Use of loperamide in the preparation of a preparation for inhibiting aging of bone marrow mesenchymal stem cells, characterized in that: The application is in vitro application.

8. The use according to claim 7, characterized in that: The dosage of loperamide is 5 to 15 μM.

9. Use of loperamide in the preparation of a preparation for inhibiting bone marrow mesenchymal stem cell damage, characterized in that: The application is in vitro application.

10. The use according to claim 9, characterized in that: The dosage of loperamide is 5 to 15 μM.

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