Use of benzenesulfonamide compounds in the preparation of medicaments for preventing and / or treating osteoarthritis

Abstract

The present invention discloses the use of a benzenesulfonamide compound in the preparation of a medicament for preventing and / or treating osteoarthritis. This invention proposes for the first time the use of a benzenesulfonamide compound in a medicament for preventing and / or treating osteoarthritis. Experiments have shown that administration of a benzenesulfonamide compound can significantly improve joint swelling and wear in osteoarthritis mice; at the same time, the benzenesulfonamide compound can significantly reduce cartilage damage and inhibit the progression of joint fibrosis in osteoarthritis mice; in addition, the benzenesulfonamide compound significantly inhibits the expression of inflammation-related markers in the serum of osteoarthritis model mice. The above experiments demonstrate that benzenesulfonamide compounds can improve osteoarthritis and are of great significance for the future development of drugs and the prevention and treatment of such diseases.

Description

Technical Field

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

[0002] Osteoarthritis (OA) is a chronic, pan-articular disease affecting the articular cartilage, synovium, and subchondral bone. Articular cartilage degeneration is the primary pathological hallmark of OA. Currently, the number of people suffering from OA worldwide exceeds 500 million, with conservative estimates of over 100 million in my country, representing a total prevalence of 15%. With the accelerating aging of the global population, the incidence and disability rates of OA are increasing annually, making it a major global public health issue.

[0003] At present, the treatments for osteoarthritis mainly include drug therapy, physical therapy, and surgical treatment. In terms of drug therapy, although commonly used non-steroidal anti-inflammatory drugs can relieve pain symptoms to a certain extent, long-term use carries the risk of adverse gastrointestinal reactions, liver and kidney damage, etc.; the efficacy of chondroprotective agents is relatively slow, and the effect is poor for patients in the middle and late stages. Physical therapy such as hot compresses, massage, acupuncture, etc. can only temporarily relieve symptoms and cannot fundamentally prevent the progression of the disease. Surgical treatment includes joint replacement, etc., which can significantly improve joint function, but the surgery is traumatic and expensive, and there is a risk of complications such as infection and thrombosis. It also requires a high physical condition of the patient and is not suitable for all patients. There are still obvious deficiencies in the understanding of the pathogenesis of OA, and the existing treatments for osteoarthritis also have many limitations. There is an urgent need for a safer, more effective, and more convenient treatment method or technology to improve this situation. 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 osteoarthritis, so as to overcome the deficiencies in the prior art.

[0005] To achieve the aforementioned object of the invention, the technical solutions adopted by the present invention include:

[0006] The present invention provides a method for preparing a medicament for preventing and / or treating osteoarthritis using a benzenesulfonamide compound. The benzenesulfonamide compound has a structure as shown in formula (I):

[0007]

[0008] The present invention also provides a pharmaceutical composition for preventing and / or treating osteoarthritis, 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 the preparation of a medicament for preventing and / or treating osteoarthritis.

[0012] Compared with existing technologies, the present invention offers the following advantages: It is the first to propose the use of benzenesulfonamide compounds or pharmaceutically acceptable derivatives thereof in pharmaceuticals for the prevention and / or treatment of osteoarthritis. Experiments have shown that administration of benzenesulfonamide compounds significantly reduces joint swelling and cartilage wear in mice with osteoarthritis. Furthermore, benzenesulfonamide compounds significantly increase the number of chondrocytes, counteracting osteoarticular fibrosis. Furthermore, benzenesulfonamide compounds significantly suppress the expression of inflammatory markers in the serum of osteoarthritis model mice. These experiments demonstrate that benzenesulfonamide compounds can improve osteoarthritis and are of great significance for the future development of drugs and the prevention and treatment of this disease. 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 following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any creative work.

[0014] Figure 1 This is a comparative diagram of bone anatomy of mice in a control group and mice treated with a benzenesulfonamide compound under an osteoarthritis model in a typical embodiment of the present invention;

[0015] Figure 2 This is a comparative diagram of H&E staining of a control mouse and a benzenesulfonamide-treated mouse in an osteoarthritis model in a typical embodiment of the present invention;

[0016] Figure 3 This is a comparison of picrosirius red staining of the bones of mice treated with a benzenesulfonamide compound and a control mouse in an osteoarthritis model in a typical embodiment of the present invention;

[0017] Figure 4 This is a comparison of safranin fast green staining of the bones of mice in a control group and mice treated with a benzenesulfonamide compound under an osteoarthritis model in a typical embodiment of the present invention;

[0018] Figure 5-Figure 7 This is a graph showing the levels of serum inflammatory indicators in mice in a control group and mice treated with a benzenesulfonamide compound under an osteoarthritis model in a typical embodiment of the present invention. DETAILED DESCRIPTION

[0019] In view of the shortcomings of the prior art, the inventors of this case, after long-term research and extensive practice, have proposed the technical solution of the present invention. To facilitate understanding of this application, this application will be described in more detail below. 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 facilitate a more thorough and comprehensive understanding of the disclosure of the present invention.

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

[0021]

[0022] The benzenesulfonamide compounds of the present invention synergistically treat osteoarthritis through the following multiple pathways:

[0023] (1) Fight against joint swelling (bone and joint anatomy diagram, such as Figure 1 ) and reduced cartilage wear (H&E staining of bone joints, as shown Figure 2 shown);

[0024] (2) Protect cartilage: maintain collagen fiber arrangement (sirius red staining, such as Figure 3 ), reduced proteoglycan loss and cartilage damage (Safranin fast green staining, as shown Figure 4 shown);

[0025] (3) Inhibit inflammation: reduce serum MMP13, IL-1β and TNF-α levels (such as Figure 5-Figure 7 shown).

[0026] In some embodiments, the osteoarthritis is mechanical anisotropy-induced bone and joint damage.

[0027] Furthermore, the drug can alleviate the degree of joint swelling and cartilage wear caused by bone and joint injuries induced by uneven mechanical forces.

[0028] Furthermore, the drug can reduce the levels of serum inflammatory factors in bone and joint injuries induced by uneven mechanical forces.

[0029] In some embodiments, when the drug is applied to an osteoarthritis mouse model, the drug can at least inhibit joint swelling in the mouse.

[0030] In some embodiments, when the drug is applied to a mouse model of osteoarthritis, the drug can at least reduce joint wear in the mouse. In some embodiments, when the drug is applied to a mouse model of osteoarthritis, the drug can at least reduce the level of serum inflammatory factors in the mouse.

[0031] In some embodiments, when the drug acts on an osteoarthritis mouse model, the drug can at least reduce the expression of osteoarthritis-related inflammatory markers in the mouse by more than 30%.

[0032] Furthermore, the osteoarthritis-related inflammatory markers include any one or more combinations of MMP13, IL-1β, and TNF-α, but are not limited thereto.

[0033] In some embodiments, when the drug acts on an osteoarthritis mouse model, the drug can at least promote the stabilization of the cartilage structure of the mouse.

[0034] In some embodiments, when the drug acts on an osteoarthritis mouse model, the drug can at least promote and inhibit cartilage fibrosis in the mouse.

[0035] The drug of the present invention improves osteoarthritis through at least one of the following mechanisms: (a) inhibiting joint swelling and reducing articular cartilage wear; (b) protecting the structural integrity of cartilage and inhibiting fibrosis; and (c) reducing the level of serum inflammatory factors and combating the progression of osteoarthritis.

[0036] The drug of the present invention can significantly improve the joint pathology indicators in the bone and joint injury model induced by uneven mechanical force.

[0037] As another aspect of the technical solution of the present invention, it relates to a pharmaceutical composition for preventing and / or treating osteoarthritis, which comprises: a benzenesulfonamide compound represented by formula (I) or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable carrier and / or excipient;

[0038]

[0039] Wherein, the pharmaceutically acceptable derivative is selected from at least one of pharmaceutically acceptable salts, polymorphs, co-crystals, radiolabeled forms and combinations thereof.

[0040] In some embodiments, when the pharmaceutical composition acts on an osteoarthritis mouse model, the pharmaceutical composition can at least inhibit joint swelling and reduce cartilage wear in the mouse.

[0041] In some embodiments, when the pharmaceutical composition acts on an osteoarthritis mouse model, the pharmaceutical composition can at least promote stabilization of the cartilage structure of the mouse.

[0042] In some embodiments, when the pharmaceutical composition acts on an osteoarthritis mouse model, the pharmaceutical composition can at least promote and inhibit cartilage fibrosis in the mouse.

[0043] In some embodiments, when the pharmaceutical composition acts on an osteoarthritis mouse model, the pharmaceutical composition can at least reduce the serum inflammatory factor content of the mouse.

[0044] In some embodiments, when the pharmaceutical composition acts on an osteoarthritis mouse model, the pharmaceutical composition can at least reduce the expression of osteoarthritis-related inflammatory markers in the mouse by more than 30%.

[0045] Furthermore, the osteoarthritis-related inflammatory markers include any one or more combinations of MMP13, IL-1β, and TNF-α, but are not limited thereto.

[0046] 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 medicament for preventing and / or treating osteoarthritis.

[0047] The present invention is further illustrated by the following examples. The present invention can be better understood according to the following examples. However, it will be readily understood by those skilled in the art that the specific material ratios, process conditions, and results described in the examples are merely illustrative of the present invention and should not, and do not, limit the present invention as described in detail in the claims.

[0048] 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.

[0049] 1. Experimental steps

[0050] Experimental animals:

[0051] Male C57BL / 6 mice used in this example were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., license number SCXK (Beijing) 2007-0001. Mice were housed under standard conditions of 50 ± 10% humidity and 23 ± 2°C for 12 hours per day and night for acclimatization. Mice had free access to water and food. All animal care and handling protocols were approved by the Animal Ethics Committee of Xuzhou Medical University. All experiments were conducted in accordance with the guidelines for the Care and Use of Animals.

[0052] Example 1 Construction of osteoarthritis model

[0053] 1. Animals: C57BL / 6 mice (male, 12 weeks old) were used and randomly divided into a control group (n=8) and a drug-treated group (n=8).

[0054] 2. Surgical induction of OA: A mechanical force unevenness model was established by anterior cruciate ligament transection (ACLT), and the OA phenotype was confirmed 8 weeks after surgery.

[0055] Example 2 H&E staining

[0056] Specific experimental methods include:

[0057] 1. Preparation of paraffin sections

[0058] (1) Bone tissue fixation: bone tissues of mice from each group in Example 1 were collected, soft tissues removed, and fixed in 4% paraformaldehyde at room temperature for 24 hours, wrapped with gauze, marked, and rinsed with running water overnight;

[0059] (2) Dehydration and clearing: Place the dehydration box in a dehydrator and dehydrate in a gradient of alcohols: 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, benzene alcohol for 5-10 minutes, xylene I for 5-10 minutes, and xylene II for 5-10 minutes.

[0060] (3) Wax immersion 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-immersed tissue in an embedding machine. First, place the melted wax in the embedding frame. Before the wax solidifies, remove the tissue from the dehydration box and place it in the embedding frame according to the requirements of the embedding surface and affix the corresponding label. Cool in a -20° freezer. After the wax solidifies, remove the wax block from the embedding frame and trim the wax block;

[0061] (4) Sectioning and spreading: Cut slices into 5 μm thick slices using a microtome, spread the slices in a 50°C water bath, remove the slices and mount them on clean glass slides, and bake them in a 60°C oven overnight. After sectioning, mark the slices and store them for future use.

[0062] 2. Perform H&E staining

[0063] (1) Dewaxing and rehydration: Sections were dewaxed twice in xylene (15 min each time), dehydrated in 100%, 95%, 90%, 80%, 70%, and 50% alcohol for 5 min each, and finally rehydrated in distilled water for 3 min.

[0064] (2) Hematoxylin staining: The sections were placed in hematoxylin staining solution for 15 minutes, rinsed with tap water for 3 minutes, and separated with hydrochloric acid alcohol (70% alcohol 99 ml + concentrated hydrochloric acid 1 ml) for 10 seconds;

[0065] (3) Bluing and dehydration: Rinse with tap water for 10 minutes to turn the slices blue. Dehydrate the slices in 50%, 70%, 80%, and 90% alcohol for 5 minutes each.

[0066] (4) Eosin counterstaining: stain with 1% eosin solution for 2 minutes, dehydrate in 95% alcohol and 100% alcohol for 3 minutes respectively and color until the boundaries are clear;

[0067] (5) Transparent and sealing: After transparentizing with xylene for 3 minutes, seal the slides with neutral gum;

[0068] (6) After sealing, place the slides in a 50°C oven for drying and observe the changes in the tissue structure under a light microscope.

[0069] Example 3 Observation of cartilage tissue fibrosis by picrosirius red staining

[0070] Specific experimental methods include:

[0071] 1. Preparation of paraffin sections

[0072] (1) Fixation of tissue specimens: bone tissues from each group of mice in Example 1 were fixed in 4% paraformaldehyde at room temperature for 24 hours, wrapped with gauze, marked, and rinsed with running water overnight;

[0073] (2) Dehydration and transparency: Place the dehydration box in the dehydrator and dehydrate with graded alcohols in sequence: 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, alcohol benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes.

[0074] (3) Wax immersion 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-immersed tissue in an embedding machine. First, place the melted wax in the embedding frame. Before the wax solidifies, remove the tissue from the dehydration box and place it in the embedding frame according to the requirements of the embedding surface and affix the corresponding label. Cool in a -20° freezer. After the wax solidifies, remove the wax block from the embedding frame and trim the wax block;

[0075] (4) Sectioning and spreading: Cut slices into 5 μm thick slices using a microtome, spread the slices in a 50°C water bath, remove the slices and place them on clean glass slides, and bake them in a 60°C oven overnight. After sectioning, mark the slices and store them for future use.

[0076] 2. Sirius red staining

[0077] (1) Dewaxing and rehydration: Sections were dewaxed twice in xylene (15 min each time), dehydrated in 100%, 95%, 90%, 80%, 70%, and 50% alcohol for 5 min each, and finally rehydrated in distilled water for 3 min.

[0078] (2) Staining: Immerse in picric acid red stain (1% in saturated picric acid solution) for 60 minutes.

[0079] (3) Differentiation and washing: Rapid acid ethanol differentiation (1% HCl-ethanol, 1 to 3 seconds), and rinse with running water for 5 minutes.

[0080] (4) Dehydration and sealing: Dehydrate with gradient ethanol, make transparent with xylene, and seal with neutral resin.

[0081] Example 4 Observation of bone tissue by Safranin Fast Green staining

[0082] 1. Preparation of paraffin sections

[0083] Same as above-mentioned embodiment 2.

[0084] 2. Safranin Fast Green Dyeing

[0085] (1) Dewaxing and rehydration: Sections were dewaxed twice in xylene (15 min each time), dehydrated in 100%, 95%, 90%, 80%, 70%, and 50% alcohol for 5 min each, and finally rehydrated in distilled water for 3 min.

[0086] (2) Safranin staining: Add safranin stain solution, incubate at room temperature for 2 hours, and rinse with running water;

[0087] (3) Fast green staining: Fast green staining for about 1 minute;

[0088] (4) Dehydration, transparency, and mounting: Dehydrate the sections in 50%, 70%, 80%, 90%, 95%, and 100% alcohol for 5 minutes each. Transparent the sections in xylene for 3 minutes, and then mount the sections in neutral gum.

[0089] (5) After sealing, place the slides in a 50°C oven for drying and observe the changes in the tissue structure under a light microscope.

[0090] Example 5 Detection of serum inflammation-related indicators using mouse ELISA detection kit

[0091] (1) Remove the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips in a ziplock bag and return them to 4°C.

[0092] (2) Set up standard wells and sample wells, and add 50 μL of standard of different concentrations to each standard well;

[0093] (3) First add 10 μL of the sample to be tested to the sample well, and then add 40 μL of the sample diluent; do not add anything to the blank well.

[0094] (4) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard and sample wells, seal the reaction wells with a sealing film, and incubate in a 37°C water bath or incubator for 60 min.

[0095] (5) Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 5 times.

[0096] (6) Add 50 μL of substrate A and substrate B to each well and incubate at 37°C in the dark for 15 min.

[0097] (7) Add 50 μL of stop solution to each well. Within 15 min, measure the OD value of each well at a wavelength of 450 nm and calculate relevant indicators according to the instructions.

[0098] 2. Experimental Results

[0099] 1. Benzenesulfonamide compounds significantly improve joint swelling in osteoarthritis model mice

[0100] Mice of appropriate age were selected and anterior cruciate ligament transection (ACLT) surgery was performed to establish an osteoarthritis model. The mice after surgery were randomly divided into two groups, one group was given normal saline, and the other group was given benzenesulfonamide compounds. Samples were collected and analyzed 8 weeks after administration. The results showed that the joints of mice in the normal saline group were significantly swollen, showing typical arthritis symptoms. In contrast, the joint swelling of mice in the drug group was significantly improved, such as Figure 1 As shown, it suggests that benzenesulfonamide compounds may play a protective role against joint damage in osteoarthritis mice.

[0101] 2. Benzenesulfonamide compounds significantly reduce bone tissue wear in osteoarthritis model mice

[0102] Subsequently, the fixed bone tissue was further subjected to paraffin sectioning and H&E staining. The staining revealed that the bone joints in the saline group were significantly swollen and the articular cartilage was severely worn, showing typical arthritis symptoms, such as Figure 2 In contrast, after administration of benzenesulfonamide compounds, the swelling and wear of the bone joints of mice were significantly reduced, as shown in Figure 2. Figure 2 As shown, Figure 1 These results again confirm that benzenesulfonamide compounds have a significant protective effect on bone damage caused by osteoarthritis.

[0103] 3. Benzenesulfonamide compounds significantly reduced the degree of fibrosis in osteoarthritis model mice

[0104] Healthy articular cartilage has a smooth extracellular matrix, but once the cartilage is damaged, chondrocytes proliferate abnormally. Eventually, the proliferating chondrocytes transform into a fibroblast-like phenotype, synthesizing type I collagen and inducing the degradation and dedifferentiation of nearby hyaline cartilage to form a thick layer of fibrocartilage-like tissue. These changes cause the cartilage to become harder and less durable, and ultimately change the phenotype of the articular cartilage, accelerating the progression of OA. Subsequently, picrosirius red staining found that the degree of fibrosis in arthritic mice after administration of benzenesulfonamide compounds was significantly reduced compared to the saline control group mice, such as Figure 3 This suggests that benzenesulfonamide compounds may counteract the progression of fibrosis during arthritis.

[0105] 4. Benzenesulfonamide compounds can reduce cartilage damage in arthritic mice

[0106] The typical characteristic of osteoarthritis is cartilage damage. Subsequently, the effect of benzenesulfonamide compounds on chondrocytes in arthritis mice was tested. By staining with safranin fast green, it was found that the positive staining of chondrocytes in the drug-treated group was significantly higher than that in the control group. Figure 4 As shown, it suggests that benzenesulfonamide compounds play a counteracting role in the damage of chondrocytes during arthritis.

[0107] 5. Benzenesulfonamide compounds can inhibit the inflammatory process in arthritic mice

[0108] The process of osteoarthritis is often accompanied by the release of inflammatory factors. These pro-inflammatory factors promote the production of proteolytic enzymes, which degrade the extracellular matrix and further damage joint tissue. ELISA tests found that the levels of MMP13, IL-1β and TNF-α, markers of inflammation, in the serum of mice were significantly reduced after administration of benzenesulfonamide compounds. Figure 5-Figure 7 As shown, this suggests that the drug has an inhibitory effect on the inflammatory process of osteoarthritis.

[0109] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments using other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0110] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention as claimed.

Claims

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

2. The use according to claim 1, characterized in that: When the drug acts on an osteoarthritis mouse model, the drug can at least inhibit the joint swelling of the mouse.

3. The use according to claim 1, characterized in that: When the drug acts on an osteoarthritis mouse model, the drug can at least reduce joint wear of the mouse.

4. The use according to claim 1, characterized in that: When the drug acts on an osteoarthritis mouse model, the drug can at least reduce the serum inflammatory factor content of the mouse.

5. The use according to claim 1, characterized in that: When the drug acts on an osteoarthritis mouse model, the drug can at least reduce the expression of osteoarthritis-related inflammatory markers in the mouse by more than 30%; the osteoarthritis-related inflammatory markers include any one or more combinations of MMP13, IL-1β, and TNF-α.

6. The use according to claim 1, characterized in that: When the drug acts on an osteoarthritis mouse model, the drug can at least promote the stabilization of the cartilage structure of the mouse.

7. The use according to claim 1, characterized in that: When the drug acts on an osteoarthritis mouse model, the drug can at least promote and inhibit cartilage fibrosis in the mouse.

8. Use of a pharmaceutical composition for preparing a medicament for preventing and / or treating osteoarthritis; the pharmaceutical composition comprising: A benzenesulfonamide compound represented by formula (I), and a pharmaceutically acceptable carrier and / or excipient; Formula (I).

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