Use of a benzene sulfonamide compound for the manufacture of a medicament for the prevention and / or treatment of osteoporosis

Abstract

The application discloses a use of a benzene sulfonamide compound in preparation of a medicine for preventing and / or treating osteoporosis. The application first proposes the use of the benzene sulfonamide compound in the medicine for preventing and / or treating osteoporosis. Experiments show that the benzene sulfonamide compound can significantly improve bone remodeling of mice, inhibit osteoclasts and promote osteoblasts, and maintain bone metabolism balance; meanwhile, the benzene sulfonamide compound can significantly improve bone volume fraction, trabecular bone surface area, trabecular bone thickness, trabecular bone density, trabecular bone number and bone mineral content of old mice, and significantly reduce the trabecular bone gap of the old mice, which indicates that the benzene sulfonamide compound can improve osteoporosis, and has very important significance for drug development and prevention and treatment of the disease in the future.

Description

TECHNICAL FIELD

[0001] The present application relates to the use of a benzene sulfonamide compound in the preparation of a drug for preventing and / or treating osteoporosis, and belongs to the technical field of biological medicine. BACKGROUND

[0002] According to statistics, the risk of death of the elderly within 3 months after the initial hip fracture increases by 5-8 times, 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 fracture is a serious harm, and is one of the main causes of disability and death in elderly patients. In addition, osteoporosis is easily overlooked because there are no obvious symptoms in the early stage, and is also known as the invisible killer. At present, the exact pathogenesis of osteoporosis is not clear, which hinders the development of effective treatment methods.

[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 means have significant limitations in long-term drug safety, efficacy maintenance stability, and individual difference adaptability. In clinical practice, new methods with the advantages of efficient bone formation promotion, precise risk intervention, and sustainable treatment are urgently needed to fill the gap in the dynamic regulation and systematic treatment needs that cannot be met by existing technology. SUMMARY

[0004] The main purpose of the present application is to provide a use of a benzene sulfonamide compound in the preparation of a drug for preventing and / or treating osteoporosis, in order to overcome the deficiencies in the prior art.

[0005] To achieve the foregoing application purposes, the technical solutions adopted by the present application include:

[0006] The present application provides a use of a benzene sulfonamide compound in the preparation of a drug for preventing and / or treating osteoporosis, and the structure of the benzene sulfonamide compound is shown in formula (I):

[0007]

[0008] The present application also provides a pharmaceutical composition for preventing and / or treating osteoporosis, which comprises a benzene sulfonamide compound shown in formula (I) or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable carrier and / or excipient.

[0009]

[0010] The pharmaceutically acceptable derivative is selected from at least one of a pharmaceutically acceptable salt, a polymorph, a co-crystal, a radio-labeled form, and a combination thereof.

[0011] The application also provides use of the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating osteoporosis.

[0012] Compared with the prior art, the application has the beneficial effects that the application first proposes the use of benzene sulfonamide compounds in the preparation of a drug for preventing and / or treating osteoporosis; experiments show that the administration of benzene sulfonamide compounds can significantly improve bone remodeling of mice, inhibit osteoclasts and promote osteoblasts, and maintain bone metabolism balance; at the same time, the benzene sulfonamide compounds significantly improve the bone volume fraction, trabecular bone surface area, trabecular bone thickness, trabecular bone density, trabecular bone number and bone mineral content of old mice, and significantly reduce the trabecular bone gap of old mice, which indicates that the benzene sulfonamide compounds can improve osteoporosis, and have very important significance for the development and prevention and treatment of drugs for such diseases in the future. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments described in the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0014] Figure 1 A comparison chart of bone micor-CT of old mice and old mice administered with benzene sulfonamide compounds in a typical embodiment of the application;

[0015] Figure 2 A quantitative statistical result chart of bone micor-CT of old mice and old mice administered with benzene sulfonamide compounds in a typical embodiment of the application;

[0016] Figure 3 A comparison chart of bone toluidine blue staining of old mice and old mice administered with benzene sulfonamide compounds in a typical embodiment of the application;

[0017] Figure 4 A comparison chart of bone toluidine blue staining of old mice and old mice administered with benzene sulfonamide compounds in a typical embodiment of the application;

[0018] Figure 5 A comparison chart of bone toluidine blue staining of old mice and old mice administered with benzene sulfonamide compounds in a typical embodiment of the application; DETAILED DESCRIPTION

[0019] In view of the defects of the prior art, the present inventors have long studied and practiced to propose the technical solutions of the present application. 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 application can be realized 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 disclosure of the present application more thorough and comprehensive.

[0020] Specifically, as one aspect of the technical solutions of the present application, the use of the benzene sulfonamide compound in the preparation of a drug for preventing and / or treating osteoporosis, the structure of the benzene sulfonamide compound is shown in formula (I):

[0021]

[0022] The benzene sulfonamide compound in the present application can treat osteoporosis through the following multiple pathways:

[0023] (1) improving the trabecular bone space morphology, increasing bone density and bone mineral content (such as Figure 1 , Figure 2 ) ;

[0024] (2) regulating bone metabolism: reducing the number of osteoclasts and promoting the activity of osteoblasts (such as Figure 5 ) ;

[0025] (3) protecting cartilage: maintaining collagen fiber arrangement (toluidine blue staining, such as Figure 3 ), reducing the loss of proteoglycans (picro-sirius red staining, such as Figure 4 ).

[0026] Further, the drug in the present application can improve the trabecular bone space morphology disorder, the decrease of bone density and bone mineral content caused by primary osteoporosis.

[0027] Further, the drug in the present application 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, the drug can at least improve the trabecular bone morphology of mice in a mouse model when the drug acts on the mouse model.

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

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

[0032] In some preferred embodiments, the drug is capable of at least protecting the integrity of cartilage structure in mice in a mouse model when the drug acts on the mouse model.

[0033] In some preferred embodiments, the drug is capable of at least reducing the number of osteoclasts in mice in a mouse model when the drug acts on the mouse model.

[0034] In some preferred embodiments, the drug is capable of at least increasing osteoblast activity in mice in a mouse model when the drug acts on the mouse model.

[0035] The drug in the present application improves osteoporosis by at least one of the following mechanisms:

[0036] (1) reducing the number of osteoclasts and increasing osteoblast activity to maintain bone metabolism balance;

[0037] (2) improving the trabecular bone space structure, increasing bone density and bone mineral content;

[0038] (3) protecting the integrity of cartilage structure.

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

[0040]

[0041] 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, the pharmaceutical composition is capable of at least improving the trabecular bone space structure in mice in a mouse model when the pharmaceutical composition acts on the mouse model.

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

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

[0045] In some preferred embodiments, the pharmaceutical composition is capable of at least reducing the number of osteoclasts in mice in a mouse model when the pharmaceutical composition acts on the mouse model.

[0046] In some preferred embodiments, the pharmaceutical composition is capable of at least increasing osteoblast activity in a mouse model when the pharmaceutical composition is administered to the mouse model.

[0047] In some preferred embodiments, the pharmaceutical composition is capable of at least protecting the integrity of cartilage structure in a mouse model when the pharmaceutical composition is administered to the mouse model.

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

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

[0050] The present application is further illustrated by the following examples, which serve to better illustrate the present application. However, those skilled in the art will readily understand that the specific materials, conditions and procedures described in the examples are merely illustrative of the present application and are not intended to limit the scope of the application as described in the claims.

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

[0052] I. Experimental procedures

[0053] Experimental animals:

[0054] The male C57BL / 6 mice used in this example were purchased from Jiangsu Jizhuangkang Biotechnology Co., Ltd., license number SCXK (Beijing) 2007-0001. The mice were placed in standard conditions with a humidity of 50±10% and a temperature of 23±2℃, with 12h of light and 12h of darkness each day for acclimation. The mice were allowed to drink water and eat freely. 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 Guide for the Care and Use of Laboratory Animals.

[0055] Example 1 Construction of a primary osteoporosis mouse model

[0056] As the mice age, they will spontaneously develop osteoporosis. C57 mice naturally grown to 2 years old were selected as experimental subjects, half of which were injected intraperitoneally with normal saline and half of which were injected intraperitoneally with a benzenesulfonamide compound.

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

[0058] The specific experimental methods include:

[0059] 1. Paraffin section preparation

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

[0061] (2) Dehydration and transparency: The dehydration box was placed in the dehydration machine and dehydrated with gradient alcohol in sequence, 75% alcohol for 4h, 85% alcohol for 2h, 90% alcohol for 2h, 95% alcohol for 1h, anhydrous ethanol I for 30min, anhydrous ethanol II for 30min, alcohol-benzene for 5-10min, dimethylbenzene I for 5-10min, dimethylbenzene II for 5-10min;

[0062] (3) Wax immersion and embedding: 65°C melting paraffin I for 1h, 65°C melting paraffin II for 1h, 65°C melting paraffin III for 1h. The wax- immersed tissues were embedded in the embedding machine. The melted wax was first placed in the embedding frame, and before the wax solidified, the tissues were taken out from the dehydration box and placed in the embedding frame according to the requirements of the embedding surface and labeled accordingly. The-20°C freezing table was cooled, and after the wax solidified, the wax block was taken out of the embedding frame and trimmed;

[0063] (4) Sectioning and mounting: The sectioning machine was used to section at a thickness of 5μm, and the sections were mounted on clean glass slides in a 50°C water bath, and then baked in a 60°C oven overnight. After sectioning, the sections were labeled and stored for use.

[0064] 2. Toluidine blue staining

[0065] (1) De-waxing and rehydration: The sections were de-waxed with xylene twice (15min each time), and then dehydrated in 100%, 95%, 90%, 80%, 70% and 50% alcohol for 5min each time, and finally rehydrated in distilled water for 3min;

[0066] (2) Toluidine blue staining: The sections were stained in toluidine blue staining solution for 25min, and then washed with running water to remove excess staining solution;

[0067] (3) Color separation: Color separation was performed using 95% ethanol under a microscope to control the color separation effect;

[0068] (4) Transparency and mounting: After transparency with xylene for 3min, neutral balsam was used for mounting;

[0069] (5) After mounting, the sections were placed in a 50°C oven for drying, and the changes in the structure of each tissue were observed under a light microscope.

[0070] Example 3. Observation of bone CT

[0071] (1) Preparation of mouse bone samples. The mouse bones at the required sites were taken, and the surrounding materials unrelated to the study were removed, and then fixed in 4% paraformaldehyde for 24 hours and washed with running water overnight.

[0072] (2) Decalcification. Put the bone sample into nitric acid decalcification solution, the liquid level is about 3 ml higher than the sample. The end point of decalcification is determined by the fact that a large needle can be easily inserted.

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

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

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

[0076] 1. Paraffin section preparation

[0077] The same as Example 2.

[0078] 2. Safranin-fast green staining

[0079] (1) De-waxing and rehydrating: de-waxing the section with xylene twice (15 minutes each time), dehydrating in 100%, 95%, 90%, 80%, 70%, and 50% alcohol for 5 minutes each time, and finally rehydrating in distilled water for 3 minutes;

[0080] (2) Safranin staining: add safranin dye, incubate at room temperature for 2 hours, and wash slightly with running water;

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

[0082] (4) Dehydrating, clearing, and mounting: dehydrate the section in 50%, 70%, 80%, 90%, 95%, and 100% alcohol for 5 minutes each time, clear in xylene for 3 minutes, and mount with neutral balsam;

[0083] (5) After mounting, put it in a 50°C oven to dry, and observe the changes in the structure of each tissue under a light microscope.

[0084] Example 5 qRT-PCR

[0085] (1) Put 0.02 g of tissue and 500 μL of Trizol lysis solution into a grinding tube, add non-enzyme grinding beads, use a homogenizer to homogenize until the tissue is completely broken, and let it stand at room temperature for 10 minutes to fully lyse.

[0086] (2) After adding 100 μL chloroform, shake up and down quickly for 15 seconds, and then place the sample in a centrifuge at 12000 rpm for 15 minutes after standing for 10 minutes at room temperature.

[0087] (3) Carefully transfer the upper water phase to a new EP tube, and then add 300 μL isopropanol and stand for 10 minutes at room temperature. Place the sample in a centrifuge at 12000 rpm for 10 minutes.

[0088] (4) After centrifugation, discard the supernatant, add 500 μL of pre-cooled 75% ethanol to the precipitate, and then wash by blowing with a pipette. Place the sample in a centrifuge at 12000 rpm for 10 minutes, and repeat once.

[0089] (5) After centrifugation, discard the supernatant, and then place the EP tube open at room temperature for 10 minutes. Add 80 μL of RNase-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, and the total volume of the reaction system is 10.00 μL.

[0095] Table 2 qRT-PCR reaction system

[0096]

[0097]

[0098] II. Experimental results

[0099] 1. The benzene sulfonamide compound significantly improves the bone structure damage and bone loss of old primary osteoporosis

[0100] Osteoporosis is a systemic metabolic bone disease characterized by reduced bone mass and degenerative bone microstructure, resulting in decreased bone strength, "brittle" bones, and easy fractures. The bone tissue of old mice and old mice administered with benzene sulfonamide compound was detected by micro-CT. The results showed that after administration, the bone tissue morphology of old mice was more complete, the trabecular bone arrangement was more orderly, and the trabecular bone number was significantly more than that of the control group, such as Figure 1The statistical analysis also further showed that the benzene sulfonamide compound could obviously increase the bone volume and weight, improve the bone mineral content and density, and improve the bone loss in the old mice, thereby protecting the osteoporosis symptoms. Figure 2 The statistical analysis also further showed that the benzene sulfonamide compound could obviously increase the bone volume and weight, improve the bone mineral content and density, and improve the bone loss in the old mice, thereby protecting the osteoporosis symptoms.

[0101] 2. The benzene sulfonamide compound can resist the cartilage damage caused by the old osteoporosis

[0102] One of the typical characteristics of the osteoporosis is the cartilage lesion. Subsequently, the benzene sulfonamide compound was analyzed for the cartilage damage in the old osteoporosis mice by toluidine blue staining. The results showed that after the administration, the cartilage boundary of the old mice was clearer, and the positive staining was obviously more than that of the control group of the old mice, as shown in Figure 3 In addition, the results of the ponceau solid green staining showed that the cartilage structure of the old mice after the administration of the benzene sulfonamide compound was more complete, and the subchondral bone damage was reduced, as shown in Figure 4 The above results indicated that the benzene sulfonamide compound had a protective effect on the cartilage damage in the old osteoporosis process.

[0103] 3. The benzene sulfonamide compound effectively maintains the bone metabolism imbalance in the old mice

[0104] The osteoporosis is mainly caused by the imbalance between the bone formation mediated by the osteoblasts and the bone absorption mediated by the osteoclasts, the bone formation / bone absorption ratio is reduced, and the progressive bone loss is caused. Subsequently, the benzene sulfonamide compound was further analyzed for the influence on the bone metabolism imbalance in the occurrence and development of the osteoporosis. The results of the TRAP staining showed that the number of the osteoclasts was obviously reduced after the administration. Subsequently, the PCR analysis showed that the benzene sulfonamide compound could obviously up-regulate the expression of the osteoblast marker molecules and down-regulate the expression of the osteoclast marker molecules, promote the bone formation, inhibit the bone absorption, and further maintain the bone metabolism homeostasis, as shown in Figure 5 The results showed that the benzene sulfonamide compound could obviously improve the degree of the old primary osteoporosis and improve the bone health.

[0105] In addition, the inventors of the present case also refer to the foregoing examples, and the other raw materials, process operations, process conditions described in the specification are tested, and the ideal results are obtained.

[0106] The above shows and describes the basic principles, main features and advantages of the present application. It should be understood by those skilled in the art that the present application is not limited to the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application.

Claims

1. Use of a benzenesulfonamide compound in the manufacture of a medicament for preventing and / or treating osteoporosis, the benzenesulfonamide compound having a structure as shown in formula (I): ###0001### Formula (I) wherein the osteoporosis is senile osteoporosis. Formula (I).

2. Use according to claim 1, characterized in that: The medicament is capable of at least improving the trabecular bone morphology of a mouse in a mouse model when the medicament is administered to the mouse model.

3. Use according to claim 1, characterized in that: The medicament is capable of at least increasing the bone density of a mouse in a mouse model when the medicament is administered to the mouse model. The medicament is capable of at least increasing the bone mineral content of a mouse in a mouse model when the medicament is administered to the mouse model. The medicament is capable of at least protecting the cartilage structure integrity of a mouse in a mouse model when the medicament is administered to the mouse model.

4. Use according to claim 1, characterized in that: The medicament is capable of at least reducing the number of osteoclasts of a mouse in a mouse model when the medicament is administered to the mouse model.

5. Use according to claim 1, characterized in that: The medicament is capable of at least increasing the osteoblast activity of a mouse in a mouse model when the medicament is administered to the mouse model. The pharmaceutical composition comprises a benzenesulfonamide compound as shown in formula (I) or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable carrier and / or excipient.

6. Use of a pharmaceutical composition for the manufacture of a medicament for the prevention and / or treatment of osteoporosis, characterized in that, The pharmaceutically acceptable derivative is selected from a pharmaceutically acceptable salt. Formula (I) The pharmaceutical composition is capable of at least improving the trabecular bone morphology of a mouse in a mouse model when the pharmaceutical composition is administered to the mouse model.

7. Use according to claim 6, characterized in that: The pharmaceutical composition is capable of at least increasing the bone density of a mouse in a mouse model when the pharmaceutical composition is administered to the mouse model. The pharmaceutical composition is capable of at least increasing the bone mineral content of a mouse in a mouse model when the pharmaceutical composition is administered to the mouse model. The pharmaceutical composition is capable of at least reducing the number of osteoclasts of a mouse in a mouse model when the pharmaceutical composition is administered to the mouse model. The pharmaceutical composition is capable of at least increasing the osteoblast activity of a mouse in a mouse model when the pharmaceutical composition is administered to the mouse model. The pharmaceutical composition is capable of at least protecting the cartilage structure integrity of a mouse in a mouse model when the pharmaceutical composition is administered to the mouse model.

8. Use according to claim 6, characterized in that: The effective amount of the benzenesulfonamide compound in the pharmaceutical composition is 0.5 mg / kg when the pharmaceutical composition is administered to a mouse model.

9. Use according to claim 6, characterized in that: ​

Images