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

Abstract

The invention discloses application of a benzenesulfonamide compound in preparation of a medicine for preventing and / or treating osteoporosis. The invention provides the application of the benzenesulfonamide compound in the medicine for preventing and / or treating osteoporosis for the first time. Experiments show that the administration of the benzenesulfonamide compound can significantly improve bone remodeling of mice, inhibit osteoclast, promote osteoblasts and maintain bone metabolism balance; meanwhile, the benzenesulfonamide compound remarkably improves the bone volume fraction, the bone trabecula surface area, the bone trabecula thickness, the bone trabecula bone density, the bone trabecula number and the bone mineral content of the old mice and remarkably reduces the bone trabecula gaps of the old mice, and experiments show that the benzenesulfonamide compound can achieve the purpose of improving osteoporosis; and the compound has very important significance on drug development and prevention and treatment of the diseases in the future.

Description

Technical Field

[0001] The invention relates to use of a benzenesulfonamide compound in preparing a medicine for preventing and / or treating osteoporosis, and belongs to the technical field of biomedicine. Background Art

[0002] According to statistics, the risk of death in the elderly increases 5-8 times within 3 months after the first hip fracture, and the proportion of death from various complications within 1 year is as high as 20%, and about half of the survivors cannot take care of themselves. Osteoporotic fractures are serious hazards. Osteoporotic fractures are extremely harmful and are one of the main causes of disability and death in elderly patients. In addition, since there are no obvious symptoms in the early stage of osteoporosis, it is easily ignored by people, so it is also called an invisible killer. At present, the exact pathogenesis of osteoporosis is still unclear, which hinders the development of effective treatments.

[0003] At present, the treatment of osteoporosis mainly includes drug and non-drug treatment, such as anti-bone resorption drugs, bone formation-promoting drugs, and some lifestyle adjustments. However, these methods have significant limitations in terms of long-term medication safety, stability of efficacy maintenance, and adaptability to individual differences. In clinical practice, new methods that have the advantages of efficient bone formation promotion, precise risk intervention, and sustainable treatment are urgently needed to fill the dynamic regulation and systematic treatment needs that cannot be met by existing technologies. Summary of the invention

[0004] The main purpose of the present invention is to provide a use of a benzenesulfonamide compound in the preparation of a medicament for preventing and / or treating osteoporosis, so as to overcome the deficiencies in the prior art.

[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0006] The present invention provides a use of a benzenesulfonamide compound in preparing a drug for preventing and / or treating osteoporosis. The structure of the benzenesulfonamide compound is shown in formula (I):

[0007]

[0008] The embodiment of the present invention also provides a pharmaceutical composition for preventing and / or treating osteoporosis, comprising: a benzenesulfonamide compound represented by formula (I) or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable carrier and / or excipient;

[0009]

[0010] Wherein, the pharmaceutically acceptable derivative is selected from at least one of pharmaceutically acceptable salts, polymorphs, co-crystals, radiolabeled forms and combinations thereof.

[0011] The embodiments of the present invention also provide use of the aforementioned pharmaceutical composition in preparing a drug for preventing and / or treating osteoporosis.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention proposes for the first time the use of benzenesulfonamide compounds in drugs for preventing and / or treating osteoporosis; experiments show that administration of benzenesulfonamide compounds can significantly improve bone reconstruction in mice, inhibit osteoclasts and promote osteoblasts, and maintain bone metabolism balance; at the same time, benzenesulfonamide compounds significantly improve the bone volume fraction, trabecular surface area, trabecular thickness, trabecular bone density, trabecular number, and bone mineral content of elderly mice, and significantly reduce the trabecular gaps in elderly mice. The above experiments show that benzenesulfonamide compounds can improve osteoporosis, which is of great significance to the future drug development and prevention and treatment of such diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 This is a bone micor-CT comparison diagram of aged mice and aged mice given benzenesulfonamide compounds in a typical embodiment of the present invention;

[0015] Figure 2 A graph showing the quantitative statistical results of bone micor-CT in aged mice and aged mice given benzenesulfonamide compounds in a typical embodiment of the present invention;

[0016] Figure 3 This is a comparison of bone toluidine blue staining of aged mice and aged mice given benzenesulfonamide compounds in a typical embodiment of the present invention;

[0017] Figure 4 A comparison of safranin-stained osteoblasts in aged mice and aged mice given benzenesulfonamide compounds in a typical embodiment of the present invention;

[0018] Figure 5 This is a graph showing the bone PCR results of aged mice and aged mice administered benzenesulfonamide compounds in a typical embodiment of the present invention. DETAILED DESCRIPTION

[0019] In view of the defects of the prior art, the inventor of this case has proposed the technical solution of the present invention after long-term research and extensive practice. In order to facilitate the understanding of the present application, the present application will be described in more detail as follows. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0020] Specifically, as one aspect of the technical solution of the present invention, it involves the use of a benzenesulfonamide compound in the preparation of a medicament for preventing and / or treating osteoporosis, and the structure of the benzenesulfonamide compound is shown in formula (I):

[0021]

[0022] The benzenesulfonamide compound of the present invention synergistically treats osteoporosis through the following multiple pathways:

[0023] (1) Improve the spatial morphology of trabecular bone and increase bone density and bone mineral content (such as Figure 1 , Figure 2 );

[0024] (2) Regulate bone metabolism: reduce the number of osteoclasts and promote osteoblast activity (such as Figure 5 );

[0025] (3) Protect cartilage: Maintain the arrangement of collagen fibers (toluidine blue staining, such as Figure 3 ), reduce the loss of proteoglycans (safranin fast green staining, such as Figure 4 ).

[0026] Furthermore, the drug of the present invention can improve the disorder of the spatial morphology of trabeculae and the decrease of bone density and bone mineral content caused by primary osteoporosis.

[0027] Furthermore, the drug of the present invention can significantly inhibit the increase of osteoclasts caused by primary osteoporosis, and promote the number of osteoblasts to maintain bone balance.

[0028] In some preferred embodiments, the osteoporosis is senile osteoporosis.

[0029] In some preferred embodiments, when the drug acts on a mouse model, it can at least improve the morphological structure of trabecular bone in the mouse in the mouse model.

[0030] In some preferred embodiments, when the drug acts on a mouse model, it can at least increase the bone density of mice in the mouse model.

[0031] In some preferred embodiments, when the drug acts on a mouse model, it can at least increase the bone mineral content of mice in the mouse model.

[0032] In some preferred embodiments, the drug can at least protect the integrity of the mouse cartilage structure when acting on a mouse model.

[0033] In some preferred embodiments, when the drug acts on a mouse model, it can at least reduce the number of mouse osteoclasts in the mouse model.

[0034] In some preferred embodiments, when the drug acts on a mouse model, it can at least increase the osteoblast activity of the mouse.

[0035] The drug of the present invention improves osteoporosis through at least one of the following mechanisms:

[0036] (1) Reduce the number of osteoclasts and increase the activity of osteoblasts to maintain the balance of bone metabolism;

[0037] (2) Improve the spatial morphology of trabecular bone and increase bone density and bone mineral content;

[0038] (3) Protect the structural integrity of cartilage.

[0039] As another aspect of the technical solution of the present invention, it relates to a pharmaceutical composition for preventing and / or treating osteoporosis, which comprises: a benzenesulfonamide compound represented by formula (I) or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable carrier and / or excipient;

[0040]

[0041] Wherein, the pharmaceutically acceptable derivative is selected from at least one of pharmaceutically acceptable salts, polymorphs, co-crystals, radiolabeled forms and combinations thereof.

[0042] In some preferred embodiments, when the pharmaceutical composition acts on a mouse model, it can at least improve the morphological structure of trabecular bone in the mice in the mouse model.

[0043] In some preferred embodiments, when the pharmaceutical composition acts on a mouse model, it can at least increase the bone density of mice in the mouse model.

[0044] In some preferred embodiments, when the pharmaceutical composition acts on a mouse model, it can at least increase the bone mineral content of mice in the mouse model.

[0045] In some preferred embodiments, when the pharmaceutical composition acts on a mouse model, it can at least reduce the number of mouse osteoclasts in the mouse model.

[0046] In some preferred embodiments, when the pharmaceutical composition acts on a mouse model, it can at least increase the osteoblast activity of the mouse.

[0047] In some preferred embodiments, the pharmaceutical composition can at least protect the integrity of the mouse cartilage structure when acting on a mouse model.

[0048] In some preferred embodiments, when the pharmaceutical composition acts on a mouse model, the effective amount of the benzenesulfonamide compound is 0.5 mg / kg.

[0049] As another aspect of the technical solution of the present invention, it also relates to the use of the aforementioned pharmaceutical composition in the preparation of a drug for preventing and / or treating osteoporosis.

[0050] The present invention is further described by the following examples: The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0051] Unless otherwise specified, the various raw materials, reaction equipment, testing equipment and testing methods used in the following examples are all well known in the art.

[0052] 1. Experimental steps

[0053] Experimental Animals:

[0054] The male C57BL / 6 mice used in this example were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., license number SCXK (Beijing) 2007-0001. The mice were placed under standard conditions of 50±10% humidity and 23±2°C for 12 hours per day and night to adapt to survival. The mice were free to drink water and eat. All animal management and treatment protocols were approved by the Animal Ethics Committee of Xuzhou Medical University. All experiments were conducted in accordance with the recommendations of the Code of Ethics for the Management and Use of Animals.

[0055] Example 1 Construction of primary osteoporosis mouse model

[0056] As age increases, mice will spontaneously develop osteoporosis. C57 mice that grew naturally to 2 years old were selected as experimental subjects, half of them were intraperitoneally injected with saline, and the other half were intraperitoneally injected with benzenesulfonamide compounds.

[0057] Example 2 Observation of bone tissue by toluidine blue staining

[0058] Specific experimental methods include:

[0059] 1. Preparation of paraffin sections

[0060] (1) Fixation of tissue specimens: bone tissues of mice in each group in Example 1 were fixed in 4% paraformaldehyde at room temperature for 24 hours, wrapped with gauze, marked, and rinsed with running water overnight;

[0061] (2) Dehydration and transparency: Place the dehydration box in a dehydrator and dehydrate with graded alcohols in sequence: 75% alcohol for 4 h, 85% alcohol for 2 h, 90% alcohol for 2 h, 95% alcohol for 1 h, anhydrous ethanol I for 30 min, anhydrous ethanol II for 30 min, alcohol benzene for 5-10 min, xylene I for 5-10 min, and xylene II for 5-10 min;

[0062] (3) Wax dipping and embedding: melt paraffin I at 65° for 1 hour, melt paraffin II at 65° for 1 hour, and melt paraffin III at 65° for 1 hour. Embed the wax-dipping tissue in an embedding machine. First, put the melted wax into the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and put it into the embedding frame according to the requirements of the embedding surface and attach the corresponding label. Cool in a -20° freezer. After the wax solidifies, take the wax block out of the embedding frame and trim the wax block;

[0063] (4) Sectioning and spreading: Cut slices with a microtome to a thickness of 5 μm, spread the slices in a 50°C water bath, pick up the slices and mount them on a clean glass slide, and bake them in a 60°C oven overnight. After sectioning, mark them and store them for later use.

[0064] 2. Toluidine blue staining

[0065] (1) Dewaxing and rehydration: The sections were dewaxed twice in xylene (15 min / time), dehydrated in 100%, 95%, 90%, 80%, 70%, and 50% alcohol for 5 min each, and finally rehydrated in distilled water for 3 min.

[0066] (2) Toluidine blue staining: Place the sections in toluidine blue staining solution for 25 minutes and then rinse with running water to remove excess staining solution.

[0067] (3) Color separation: Use 95% ethanol for color separation and control the color separation effect under a microscope;

[0068] (4) Transparency and sealing: After xylene is transparent for 3 minutes, the slides are sealed with neutral gum;

[0069] (5) After sealing, the slides were placed in a 50°C oven for drying and the changes in the tissue structures were observed under a light microscope.

[0070] Example 3 Bone CT Observation

[0071] (1) Prepare mouse bone samples. Take the required part of the mouse bone, remove the surrounding materials irrelevant to the study, fix it in 4% paraformaldehyde for 24 hours, and rinse it with running water overnight.

[0072] (2) Decalcification. Place the bone sample in nitric acid decalcification solution with the liquid level about 3 ml higher than the specimen. The decalcification endpoint is determined by whether a pin can be easily inserted when tested.

[0073] (3) Micro-CT scanning. Place the decalcified bone sample on the sample stage of the Micro-CT scanner, fix the position, select the appropriate resolution and scanning parameters, start scanning, observe the image quality during the scanning process, and save the original data after the scanning is completed.

[0074] (4) Image reconstruction and analysis. Use dedicated software to reconstruct the original data to obtain a three-dimensional reconstructed image, perform threshold segmentation on the region of interest, extract two-dimensional and three-dimensional images of cortical bone and cancellous bone, perform quantitative analysis on bone morphology and density and other parameters, and output the analysis results and report.

[0075] Example 4 Observation of bone tissue by safranin fast green staining

[0076] 1. Preparation of paraffin sections

[0077] Same as Example 2.

[0078] 2. Safranin Fast Green Dyeing

[0079] (1) Dewaxing and rehydration: The sections were dewaxed twice in xylene (15 min / time), dehydrated in 100%, 95%, 90%, 80%, 70%, and 50% alcohol for 5 min each, and finally rehydrated in distilled water for 3 min.

[0080] (2) Safranin staining: Add safranin stain solution, incubate at room temperature for 2 hours, and rinse with running water;

[0081] (3) Fast green staining: Fast green staining for about 1 minute;

[0082] (4) Dehydration, transparency, and sealing: Dehydrate the sections in 50%, 70%, 80%, 90%, 95%, and 100% alcohol for 5 minutes each. Transparentize with xylene for 3 minutes, and then seal the sections with neutral gum.

[0083] (5) After sealing, the slides were placed in a 50°C oven for drying and the changes in the tissue structures were observed under a light microscope.

[0084] Example 5 qRT-PCR

[0085] (1) Place 0.02 g of tissue and 500 μL of Trizol lysis buffer into a grinding tube, add nuclease-free grinding beads, and use a homogenizer to homogenize until the tissue is completely broken. Let it stand at room temperature for 10 minutes to allow it to be fully lysed.

[0086] (2) After adding 100 μL of chloroform, the mixture was rapidly shaken up and down for 15 seconds. After standing at room temperature for 10 minutes, the sample was placed in a centrifuge and centrifuged at 12,000 rpm for 15 minutes.

[0087] (3) Carefully transfer the upper aqueous phase to a new EP tube, add 300 μL of isopropanol, and let stand at room temperature for 10 minutes. Place the sample in a centrifuge and centrifuge at 12,000 rpm for 10 minutes.

[0088] (4) After centrifugation, discard the supernatant, add 500 μL of pre-cooled 75% ethanol to the precipitate, wash it with a pipette, place the sample in a centrifuge, and centrifuge it at 12,000 rpm for 10 minutes. Repeat this process.

[0089] (5) After centrifugation, discard the supernatant, leave the EP tube open at room temperature for 10 minutes, and add 80 μL of nuclease-free water to the tube to measure the RNA concentration.

[0090] (6) Prepare the reverse transcription working solution according to Table 1.

[0091] Table 1 RNA reverse transcription reaction system

[0092]

[0093] Reverse transcription conditions: 37°C, 15 min → 85°C, 5 s → 4°C hold;

[0094] (7) Prepare the amplification system according to Table 2, with a total reaction volume of 10.00 μL.

[0095] Table 2 qRT-PCR reaction system

[0096]

[0097]

[0098] 2. Experimental Results

[0099] 1. Benzenesulfonamide compounds significantly improve bone structure damage and bone loss in primary osteoporosis in the elderly

[0100] Osteoporosis is a systemic metabolic bone disease characterized by decreased bone mass and deterioration of bone microstructure, which leads to decreased bone strength, "brittle" bones, and easy fractures. Micro-CT examination of bone tissues of old mice and old mice given benzenesulfonamide compounds was performed. The results showed that after administration, the bone tissue morphology of old mice was more complete, the trabeculae were arranged more neatly, and the number of trabeculae was significantly more than that of the control group. Figure 1At the same time, statistical analysis also further confirmed that benzenesulfonamide compounds can significantly increase bone volume and weight, improve bone mineral content and density, improve the phenomenon of bone loss in elderly mice, and play a good protective role against osteoporosis symptoms, such as Figure 2 shown.

[0101] 2. Benzenesulfonamide compounds can combat cartilage damage caused by osteoporosis in the elderly

[0102] One of the typical characteristics of osteoporosis is cartilage lesions. Subsequently, the cartilage damage of aged osteoporotic mice was evaluated by analyzing the benzenesulfonamide compound by toluidine blue staining. The results showed that after administration, the cartilage boundaries of aged mice were clearer and the positive staining was significantly more than that of the control group aged mice, such as Figure 3 In addition, the results of safranin fast green staining showed that the cartilage structure of the aged mice after administration of benzenesulfonamide compounds was more complete and the subchondral bone damage was reduced, as shown in Figure 4 The above results suggest that benzenesulfonamide compounds play a protective role against cartilage damage in the process of osteoporosis in the elderly.

[0103] 3. Benzenesulfonamide compounds effectively maintain bone metabolism imbalance in elderly mice

[0104] Osteoporosis is mainly caused by an imbalance between bone formation mediated by osteoblasts and bone resorption mediated by osteoclasts. The ratio of bone formation to bone resorption decreases, leading to progressive bone loss. Subsequently, the effects of benzenesulfonamide compounds on the imbalance of bone metabolism in the development of osteoporosis were further analyzed. TRAP staining showed that the number of osteoclasts was significantly reduced after administration. Subsequently, PCR analysis showed that benzenesulfonamide compounds can significantly upregulate the expression of osteoblast marker molecules and downregulate the expression of osteoclast marker molecules, promote bone formation and inhibit bone resorption, thereby maintaining bone metabolism homeostasis, such as Figure 5 The results show that benzenesulfonamide compounds can significantly improve the degree of primary osteoporosis in the elderly and improve bone health.

[0105] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0106] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. Use of a benzenesulfonamide compound in the preparation of a medicament for preventing and / or treating osteoporosis, wherein the structure of the benzenesulfonamide compound is shown in formula (I):

2. The use according to claim 1, characterized in that: The osteoporosis is senile osteoporosis.

3. The use according to claim 1, characterized in that: When the drug acts on a mouse model, it can at least improve the morphological structure of the trabecular bones of the mice in the mouse model; And / or, when the drug acts on a mouse model, it can at least increase the bone density of mice in the mouse model; And / or, when the drug acts on the mouse model, it can at least increase the bone mineral content of the mice in the mouse model.

4. The use according to claim 1, characterized in that: When the drug acts on a mouse model, it can at least protect the integrity of the mouse cartilage structure.

5. The use according to claim 1, characterized in that: When the drug acts on a mouse model, it can at least reduce the number of mouse osteoclasts in the mouse model; And / or, when the drug acts on a mouse model, it can at least increase the osteoblast activity of the mouse.

6. A pharmaceutical composition for preventing and / or treating osteoporosis, characterized in that: include: A benzenesulfonamide compound represented by formula (I) or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable carrier and / or excipient; Wherein, the pharmaceutically acceptable derivative is selected from at least one of pharmaceutically acceptable salts, polymorphs, co-crystals, radiolabeled forms and combinations thereof.

7. The pharmaceutical composition according to claim 6, characterized in that: When the pharmaceutical composition acts on a mouse model, it can at least improve the morphological structure of trabecular bones of mice in the mouse model; And / or, when the pharmaceutical composition acts on a mouse model, it can at least increase the bone density of mice in the mouse model; And / or, when the pharmaceutical composition acts on a mouse model, it can at least increase the bone mineral content of mice in the mouse model; And / or, when the pharmaceutical composition acts on a mouse model, it can at least reduce the number of mouse osteoclasts in the mouse model; And / or, when the pharmaceutical composition acts on a mouse model, it can at least increase the osteoblast activity of the mouse.

8. The pharmaceutical composition according to claim 6, characterized in that: When the pharmaceutical composition acts on a mouse model, it can at least protect the integrity of the mouse cartilage structure.

9. The pharmaceutical composition according to claim 6, characterized in that: When the pharmaceutical composition acts on a mouse model, the effective dosage of the benzenesulfonamide compound is 0.5 mg / kg.

10. Use of the pharmaceutical composition according to any one of claims 6 to 9 in the preparation of a medicament for preventing and / or treating osteoporosis.

Images