4-[9-(6-aminopurinyl)]-2(S)-hydroxybutyric acid methyl ester for pharmaceutical use

Abstract

The present application relates to the pharmaceutical use of 4-[9-(6-aminopurine)]-2(S)-hydroxybutyric acid methyl ester, in particular to its use in the preparation of a medicament for preventing and / or treating diseases associated with abnormal osteoclast activity. It has been found that 4-[9-(6-aminopurine)]-2(S)-hydroxybutyric acid methyl ester has a significant inhibitory effect on osteoclast differentiation, and intra-articular injection of 4-[9-(6-aminopurine)]-2(S)-hydroxybutyric acid methyl ester has a therapeutic effect on MIA-induced osteoarthritis. Therefore, 4-[9-(6-aminopurine)]-2(S)-hydroxybutyric acid methyl ester has the prospect of being developed into a therapeutic drug for diseases associated with abnormal osteoclast activity.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to a new use of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate, specifically the use of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate in the preparation of medicaments for the prevention and / or treatment of diseases related to abnormal osteoclast activity. Background Technology

[0002] Osteoarthritis (OA) is a joint disease with a rising global prevalence that severely impacts daily life, commonly affecting high-use, high-load joints such as the fingers and knees. Clinical manifestations include joint swelling, stiffness, and pain, with a high risk of deformity and disability. Contributing factors include age, sports-related wear and tear, and obesity. The pathogenesis involves synovitis and abnormal subchondral bone remodeling. Osteoclasts (OCs) are crucial for bone resorption; they are the only cells with bone-resorbing activity, releasing acidic substances and lysosomes to break down and destroy bone tissue, exacerbating the disease progression. Osteoclasts are currently a hot research area in understanding the pathogenesis of osteoarthritis. Commonly used medications for treating osteoarthritis include antipyretics and analgesics, corticosteroids, and lubricating agents such as sodium hyaluronate. These medications are administered orally, via intra-articular injection, or as joint patches. However, these medications have limitations; they do not effectively target the underlying mechanisms of osteoarthritis, have significant side effects, and cannot truly inhibit the progression of the disease. Therefore, there is a broad clinical need to develop highly effective and low-toxicity drugs for the treatment of osteoarthritis, targeting osteoclast abnormalities.

[0003] 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate methyl ester (as shown below) is a reversible (type III) inhibitor targeting S-adenosyl-L-homocysteine ​​hydrolase (SAHH). Currently, there are no reports or applications of 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate methyl ester for the treatment of osteoclast-related diseases.

[0004] Summary of the Invention

[0005] Based on the above, the inventors of this application have discovered that methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate has a significant inhibitory effect on osteoclast differentiation, and for the first time have found that intra-articular injection of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate has a therapeutic effect on MIA-induced osteoarthritis.

[0006] According to one aspect of the invention, the use of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, hydrate or solvate thereof in the preparation of a medicament for the prevention and / or treatment of diseases associated with abnormal osteoclast activity is provided.

[0007] According to the present invention, preferably, the diseases associated with abnormal osteoclast activity include osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, periodontitis, tooth loss, osteoporosis, rickets, myeloma bone disease, giant cell tumor of bone, and bone destruction caused by cancer bone metastasis.

[0008] According to the present invention, preferably, in the above-described uses, the drug can be an injection, capsule, tablet, granule, oral or enema liquid preparation, for example, it can be various dosage forms for local administration to the lesion site related to the occurrence of abnormal osteoclast activity, such as intra-articular administration, including intra-articular injections, as well as dosage forms involving other methods of administration such as: capsules, tablets, granules, oral or enema liquid preparations, or intravenous or intramuscular injections.

[0009] According to the present invention, the medicament may comprise a therapeutically effective amount of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, hydrate or solvate thereof, optional other active agents, and optional one or more pharmaceutically acceptable conventional excipients.

[0010] According to the present invention, other active agents may be therapeutic agents for diseases associated with abnormal osteoclast activity that are well known in the art.

[0011] For example, the intra-articular injection may include a therapeutically effective amount of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate and one or more pharmaceutically acceptable conventional excipients.

[0012] According to the present invention, the pharmaceutically acceptable conventional excipient may be one or more selected from excipients, fillers and diluents.

[0013] As used herein, the term "therapeutic effective amount" means a quantity that has a therapeutic effect and can be used to prevent and / or treat the specific disease, condition, or symptom described herein. For example, "therapeutic effective amount" may refer to the quantity required to provide a therapeutic or desired effect on an individual receiving treatment. As those skilled in the art will appreciate, therapeutic effective amounts can vary depending on the route of administration, the use of excipients, and the possibility of co-administration with other therapies.

[0014] According to another aspect of the invention, the use of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, hydrate or solvate thereof is provided in the preparation of formulations for inhibiting the differentiation of BMM cells into osteoclasts, inhibiting the formation of osteoclast actin rings, inhibiting the bone resorption capacity of osteoclasts, improving the pain response of osteoarthritis, improving cartilage damage or wear, and / or alleviating subchondral bone structure destruction.

[0015] This invention utilizes a rat osteoarthritis model induced by sodium iodoacetate (MIA) to investigate and find that an intra-articular injection formulation containing methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate can alleviate pain response in the MIA-induced rat osteoarthritis model, restore the weight-bearing capacity of the affected limb, protect cartilage structure, reduce the expression of cartilage-degrading enzymes, reverse the decreasing trend of subchondral bone trabeculae in number, thickness, and density, and inhibit the activity of subchondral osteoclasts.

[0016] This invention utilizes an experimental system to induce the differentiation of mouse bone marrow-derived macrophages (BMMs) into osteoclasts (OCs) using a receptor activator of nuclear factor-κB ligand (RANKL / Macrophage-stimulating factor, M-CSF). The study revealed that methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate, in vitro, can reduce the number and area of ​​mature osteoclasts, decrease the formation of actin rings, reduce the area of ​​bone resorption pits, and downregulate the gene transcription and protein expression levels of osteoclast-related factors. Methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate shows promising clinical application potential in diseases related to abnormal osteoclast activity. Attached Figure Description

[0017] Figure 1 The statistical results of the effect of co-incubating BMM cells with methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate (400 μM, 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, or 6.25 μM) for 5 days in Example 1 on the survival of BMM cells are shown.

[0018] Figure 2The images show representative TRAP staining images of each group after intervention with methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate (100 μM and 10 μM) for a 5-day osteoclast differentiation experiment using M-CSF (30 ng / mL) and RANKL (50 ng / mL) in Example 1.

[0019] Figure 3 This shows an osteoclast differentiation experiment conducted in Example 2 using M-CSF (30 ng / mL) and RANKL (50 ng / mL) for 7 days. Methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate (100 μM and 10 μM) was used as the endpoint after intervention. Representative images of each group of cells after actin ring formation and immunofluorescence staining with phalloidin were taken by a fluorescence inverted microscope.

[0020] Figure 4 The image shown is an illustration of the effect of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate in reducing the formation of bone resorption pits in Example 3, taken by a fluorescence inverted microscope.

[0021] Figure 5 This section shows the improvement results of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate on disease indicators in a rat OA model induced by MIA in Example 4. (A) shows the paw withdrawal pain threshold data of rats, analyzed for significance using the GEE model algorithm; (B) shows the difference in weight-bearing between the two paws of rats; (C) shows the degree of joint swelling in rats. The high-dose group was the 50 μg group of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate, and the low-dose group was the 25 μg group of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate. *p<0.05,**p<0.01,***p<0.001 vs. the model group. Data are expressed as Mean ± SEM.

[0022] Figure 6 The image shows the effect of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate in improving MIA-induced cartilage damage in rats in Example 4.

[0023] Figure 7 CT images of the rat knee joints after treatment in each group in Example 4 are shown, demonstrating the protection of cartilage structure in a MIA-induced rat OA model by methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate. Detailed Implementation

[0024] The present invention will be further illustrated below with specific embodiments. The embodiments provided herein are for illustrative or explanatory purposes only and are not intended to limit the scope of protection of the invention.

[0025] Unless otherwise specified, the raw materials, reagents, methods, and equipment used in the implementation of this application are all conventional raw materials, reagents, methods, and equipment in the field, and can be obtained by those skilled in the art from conventional channels based on their knowledge.

[0026] 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate methyl ester can be prepared using synthetic methods known in the art, such as those described in this application with reference to CN101456860A.

[0027] Example 1.4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate methyl ester inhibits BMM cell differentiation into osteoclasts

[0028] 1. Main experimental materials and their sources

[0029] (1) Animals: SPF-grade 6-8 week old BALB / c mice were purchased from the Shanghai Laboratory Animal Center of Chinese Academy of Sciences.

[0030] (2) Main experimental drugs and reagents

[0031] α-MEM was purchased from Gibco BRL; MTT and TRAP staining kits were purchased from Sigma-Aldrich; fetal bovine serum (FBS) was purchased from Hyclone; mouse recombinant RANKL was purchased from R&D; M-CSF was purchased from Peprotech; and dimethyl sulfoxide (DMSO) was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0032] 2. Main Experimental Methods

[0033] (1) Extraction and differentiation of mouse bone marrow-derived macrophages (BMMs)

[0034] Female BALB / c mice aged 6–8 weeks were euthanized. The mice were disinfected by immersing them in 75% alcohol. In a biosafety cabinet, the mice were held in place with their limbs fixed to a foam board wrapped in aluminum foil, abdomen facing the experimenter. The abdominal fur was grasped with forceps, and the abdomen and legs were cut open with surgical scissors. Smaller scissors and forceps were used to remove the flesh, femur, and tibia. The femoral bone marrow was flushed into a regular culture dish containing α-MEM culture medium using a 2.5 mL syringe. The tibia bone marrow was flushed down using a 1 mL syringe. All bone marrow was pipetted using a 1 mL pipette and filtered through a 100 μm filter into a centrifuge tube. The tube was centrifuged at 4°C, 1500 rpm for 6 min. The supernatant was then discarded, the cells were dispersed, and sterile erythrocyte lysis buffer was added (2 mL / 5 mice). The mixture was inverted and stirred until a white cell pellet appeared. 20 seconds later, the tube was filled with α-MEM to stop the cytokine lysis. The tube was centrifuged at 4°C, 1500 rpm for 6 min. Then discard the supernatant, disperse the contents, add the prepared α-MEM complete medium containing 10% FBS, resuspend, filter through a 70μm filter membrane into a new centrifuge tube, and then seed into a cell culture dish and incubate in a cell culture incubator at 37℃ and 5% CO2.

[0035] After 24 hours, collect the suspended cells (not adherent cells) into centrifuge tubes using a pipette. Centrifuge at 1500 rpm for 5 minutes at room temperature. Prepare BMM medium (α-MEM complete medium containing 10 ng / mL M-CSF). After centrifugation, discard the supernatant, disperse the cells, resuspend them, and adjust the cell concentration to 1 × 10⁻⁶ cells / mL. 8 Seed each cell / dish, seeded into a new culture dish, and returned to the cell culture incubator. After 3 days, once the BMM cells have adhered and extended pseudopodia, they can be detached using PBS, centrifuged, counted, and their concentration adjusted for subsequent experiments.

[0036] (2) MTT assay for detecting the cytotoxicity of compounds

[0037] Succinate dehydrogenase in the mitochondria of living cells can reduce tetramethylazothiazolyl blue (MTT) to water-insoluble blue-violet crystals called formazan, while dead cells lack this function. The absorbance of the formazan dissolved in DMSO at 570 nm can indirectly reflect the number of living cells. Within a certain cell number range, the amount of MTT crystals formed is directly proportional to the cell number. This can be used for cytotoxicity analysis.

[0038] Mature BMMs were divided into 1×10 4Cells were seeded at 100 μL / well in 96-well plates. After overnight cell adhesion, a Control group (blank control group) and various concentrations of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate (400 μM, 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, or 6.25 μM; 100 μL / well) were set up. Cells were incubated for 5 days, with the medium changed every 2 days. Two hours before the incubation endpoint, 20 μL / well of MTT (5 mg / ml) was added. When a certain number of formazan crystals appeared at the bottom of the plate, the supernatant was aspirated with a pipette, and 150 μL / well of DMSO was added. At this point, the formazan crystals turned purple. The plate was shaken well, and the OD value was read using a microplate reader at 570 nm. Cell viability % = (OD of each concentration group / OD of the Control group) × 100%

[0039] Figure 1 Compared with the Control group, methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate showed no significant effect on BMM cell survival in any of the in vitro administration groups, even at the highest in vitro concentration of 400 μM, demonstrating high safety.

[0040] (3) Osteoclast differentiation experiment and TRAP staining

[0041] Mature BMMs were divided into 1×10 4 Cells were seeded at 100 μL / well in 96-well plates, with Blank group (blank control), Control group (stimulation control / model control), and various drug concentration groups (methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate 100 μM, 10 μM). After the cells adhered firmly overnight, stimulants and different concentrations of compounds were added to begin the experiment. The stimulants were M-CSF (final concentration 30 ng / mL) and RANKL (final concentration 50 ng / mL). The total volume was 200 μL. The cells were returned to the incubator, and the medium was changed every 2 days. Differentiation was observed for 3 and 5 days according to the experimental design. TRAP staining was performed at the differentiation endpoint.

[0042] Before the experimental endpoint, osteoclast differentiation was observed under a microscope. After the Control group developed multinucleated giant cells, i.e., osteoclast differentiation, the TRAP staining kit was used according to the instructions, as briefly described below: Prepare ddH2O in advance and preheat it to 37°C for the preparation of the following reagents; allow the fixative to reach room temperature and equilibrate; prepare the required reagents in 100 μl / well of a 96-well plate, using a 1:1 volume ratio, adding 0.5 mL of Fast Garnet GBC Base solution and 0.5 mL of Sodium nitrate to a centrifuge tube simultaneously, inverting for 30 seconds to mix thoroughly, and label this as Reagent X; take two centrifuge tubes, labeling them A and B respectively. Add 45 mL of preheated ddH2O, 1 mL of Reagent X, 0.5 mL of Naphthol AS-BI, and 2 mL of Acetate to tube A; add 45 mL of preheated ddH2O, 1 mL of Reagent X, and Naphthol AS-BI to tube B. Mix 0.5 mL of acetate, 2 mL of tartaric acid, and 1 mL of water to form TRAP staining solution. Protect from light and incubate at 37°C in an incubator or water bath. First, centrifuge and discard the cell supernatant. Immerse the cells in fixative using a multi-channel pipette (100 μL / well) for 30 seconds, then discard. Wash thoroughly with 200 μL / well of ddH₂O at 37°C, repeating twice. Next, add 100 μL / well of TRAP staining solution and incubate at 37°C for 1 hour, protected from light. After the reaction, wash three times with ddH₂O. Then, counterstain the cell nuclei with hematoxylin (50 μL / well) for 2 minutes, then discard. Finally, wash away excess hematoxylin with 1M NaOH (100 μL / well) for 5 minutes, then discard. This process can be repeated 1-2 times to ensure thorough washing. After the residual liquid was air-dried, images of cell morphology were captured using a bright-field inverted microscope.

[0043] Figure 2 The images show representative TRAP staining images of each group at the endpoint after intervention with methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate (100 μM and 10 μM) in a 5-day osteoclast differentiation experiment using M-CSF (30 ng / mL) and RANKL (50 ng / mL).

[0044] 3. Experimental Results

[0045] Over-differentiation of osteoclasts is a major cause of increased bone resorption, exacerbating subchondral bone destruction. Osteoclastia ossificans (OA) is a multifactorial joint destructive disease, and its effects are positively correlated with subchondral bone destruction and osteophyte formation. Therefore, targeting osteoclasts (OC) is a crucial entry point for discovering drugs that can inhibit or alleviate OA. Figure 2As can be seen, methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate inhibits the differentiation of BMM cells into osteoclasts in a concentration-dependent manner.

[0046] Example 2.4: Methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate inhibits the formation of osteoclast actin rings.

[0047] 1. Main experimental materials and their sources

[0048] (1) Animals: SPF-grade 6-8 week old BALB / c mice were purchased from the Shanghai Laboratory Animal Center of Chinese Academy of Sciences.

[0049] (2) Main experimental drugs and reagents

[0050] α-MEM was purchased from GibcoBRL; TRAP staining kit was purchased from Sigma-Aldrich; fetal bovine serum (FBS) was purchased from Hyclone; mouse recombinant RANKL was purchased from R&D; M-CSF was purchased from Peprotech; FITC-labeled phalloidin was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; and DAPI was purchased from Abcam.

[0051] 2. Main Experimental Methods

[0052] (1) Extraction and differentiation of mouse BMM cells were the same as in Example 1.

[0053] (2) Inoculate BMM into 24-well plates containing the prepared slides at a density of 1×10⁻⁶. 5 Cells / well were prepared as follows: Blank group (blank control group), Control group (stimulation control / model control group), and various concentrations of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate 100 μM, 10 μM. After the cells adhered firmly, different concentrations of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate were added for pre-incubation for 2 h. Then, the cells were replaced with osteoclast stimulants, namely M-CSF (30 ng / ml) and RANKL (50 ng / ml), and incubated for 7 days. After the osteoclasts in the Control group differentiated and matured, staining was performed according to the FITC Phalloidin product instructions. After DAPI coagulation, the cells could be photographed using an Olympus IX73 fluorescence inverted microscope.

[0054] Figure 3This image shows representative images of osteoclast differentiation experiments conducted over 7 days using M-CSF (30 ng / mL) and RANKL (50 ng / mL), with methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate (100 μM and 10 μM) as the endpoint after intervention. The images also show the results of immunofluorescence staining with phalloidin after the formation of actin rings in each group of cells.

[0055] 3. Experimental Results

[0056] The cytoskeleton is a fibrous network of proteins in the cytoplasm of cells. It is in a state of motion, undergoing processes of destruction, reconstruction, and renewal. Cellular movement and physiological activities depend on the movement of the cytoskeleton. F-actin, a microfilamentous structure, is an important component of the cytoskeleton, providing mechanical support, facilitating the extension and contraction of the skeleton, and playing a role in transporting substances. The F-actin ring is a characteristic structure. Osteoblasts (OCs) attach to the surface of the bone matrix and play a role in bone resorption; this role is closely related to the structure of the actin ring. Therefore, the size of the actin ring can not only visually represent the degree of OC differentiation but also the level of OC bone resorption capacity. To quantify this structure, phalloidin staining was used. Phalloidin is a cyclic peptide extracted from Amanita phalloides that specifically binds to the F-actin structure and effectively prevents F-actin depolymerization. After coupling with a fluorescent group, immunofluorescence detection vividly displays the morphology of the cytoskeleton.

[0057] like Figure 3 As shown, methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate inhibited the formation of osteoclast actin rings in vitro in a concentration gradient-dependent manner.

[0058] Example 3.4: Methyl 9-[9-(6-aminopurine)]-2(S)-hydroxybutyrate reduces the formation of bone resorption pits.

[0059] 1. Main experimental materials and their sources

[0060] (1) Animals: SPF-grade 6-8 week old BALB / c mice were purchased from the Shanghai Laboratory Animal Center of Chinese Academy of Sciences.

[0061] (2) Main experimental drugs and reagents

[0062] α-MEM was purchased from Gibco BRL; toluidine blue and TRAP staining kits were purchased from Sigma-Aldrich; fetal bovine serum (FBS) was purchased from Hyclone; mouse recombinant RANKL was purchased from R&D; M-CSF was purchased from Peprotech; and DAPI was purchased from Abcam.

[0063] 2. Main Experimental Methods

[0064] (1) Extraction and differentiation of mouse BMM cells were the same as in Example 1.

[0065] (2) Using tweezers, place the bovine bone slices into the wells of the 96-well plate corresponding to the group number, 1 slice / well, BMM according to 1×10 4 Cells were seeded at 100 μl / well onto bovine bone slices, with the following groups: Blank group (blank control), Control group (stimulation control / model control), and various concentrations of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate (100 μM, 10 μM). After cell adhesion, stimulants M-CSF (final concentration 30 ng / ml) and RANKL (final concentration 50 ng / ml), along with different concentrations of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate (100 μM, 10 μM), were added, for a total volume of 200 μl. Cells were incubated at 37°C in a 5% CO2 incubator, with the medium changed every 2 days. Seven days later, the cell supernatant was aspirated, the cells were washed twice with PBS, and 2.5% glutaraldehyde was added. The plate was then placed at 4°C for 7 minutes to fix the cells. Next, the bovine bone slices were removed and placed in 0.25M ammonium hydroxide aqueous solution, sonicated for 15 minutes to remove cells, and then washed three times with ddH2O. The bovine bone slices were then blotted dry on absorbent paper and stained with 0.5% toluidine blue aqueous solution for 3 minutes. After staining, the bovine bone slices were washed again with ddH2O and air-dried. Images could then be taken using an Olympus IX73 inverted microscope.

[0066] Figure 4 Images show the effect of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate on the formation of bone resorption pits.

[0067] 3. Experimental Results

[0068] BMM was inoculated onto bovine bone slices for osteoclast differentiation experiments. Mature osteoclasts differentiated into bone resorption cells, resulting in pitted formation on the cell attachment surface of the bovine bone slices. At the endpoint, toluidine blue staining visualized the morphology of these pits, allowing for comparison of pit formation in different groups and evaluating the ability of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate to inhibit pit formation. Figure 4 It can be seen that methyl 4-[9-(6-aminopurinyl)]-2(S)-hydroxybutyrate can dose-dependently reduce the number and relative area of bone resorption pits on bovine bone slices, which further provides evidence that methyl 4-[9-(6-aminopurinyl)]-2(S)-hydroxybutyrate can inhibit the bone resorption ability of OC.

[0069] Example 4. Methyl 4-[9-(6-aminopurinyl)]-2(S)-hydroxybutyrate on a sodium iodoacetate (MIA)-induced osteoarthritis model of SD rats

[0070] 1. Main experimental materials and sources

[0071] (1) Animals: Sprague Dawley (SD) rats weighing about 200 g, male (license: SCXK (Beijing) 2019-0008), purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0072] (2) Main experimental drugs and reagents

[0073] Dexamethasone sodium phosphate injection (Chenxin Pharmaceutical) was purchased from Shanghai Xuhui Central Hospital; sodium iodoacetate was purchased from Shanghai Macklin Biochemical Co., Ltd.; formaldehyde aqueous solution, normal saline, chloroform, isopropanol, and 75% ethanol were all purchased from Sinopharm Chemical Reagent Co., Ltd.

[0074] 2. Main experimental methods

[0075] (1) Establishment of a sodium iodoacetate (MIA)-induced rat osteoarthritis model

[0076] SD rats were modeled by intra-articular injection of MIA (2 mg / 20 μL). Three days later, they were grouped and administered according to pain measurement.

[0077] (2) von Frey filament mechanical behavior test

[0078] Von Frey fibers are commonly used to assess tactile sensitivity in animals. In animal models of osteoarthritis (OA), especially MIA-induced OA models, they are frequently used to observe the animals' pain responses during drug administration trials. Successful behavioral testing requires sufficient acclimatization time for the animals. Before starting, rats are placed in a transparent acrylic test chamber with a wire mesh support provided with the fibers. Up to eight rats can be tested consecutively at a time. After approximately 15 minutes of acclimatization, the rats will become calm and composed in the chamber, and mechanical pain testing can begin. Fibers of 4g, 6g, 8g, 10g, 15g, and 26g were selected sequentially. Using an "up and down" method, pressure was applied to the sole of the rat's left paw until the fiber formed a C-shape, held for 5 seconds. If the rat exhibited a positive response such as lifting its paw, withdrawing its paw, backing away, licking its paw, or screaming, the pressure was stopped, and the rat was allowed to calm down for 1-2 minutes. The procedure was repeated. If a positive response occurred in 3 out of 5 attempts, an "X" was recorded under that value on the recording paper; otherwise, an "O" was recorded. The next higher fiber level was used for testing until 4 positive responses were recorded, or if the rat did not respond to the stimulation of the 26g fiber, an "O" was recorded. The final value was calculated using the formula 50% PWT(g) = 10^(Xf + kδ), and the differences between groups were compared.

[0079] (3) Bipedal balance test

[0080] The bipedal balance test involves having rats stand cooperatively on a metal detector. In rats with MIA (muscular atrophy) modeling, pain on the affected side (left side) causes uneven weight-bearing on both hind limbs. The sensitive metal detector displays the pressure felt by each hind limb on an electronic screen. During the test, the rats should be guided to face forward with their heads and bodies. Rats may resist by twisting their bodies in the induction box, leading to significant testing errors. Each rat is tested three times. The differences in values ​​between the left and right sides allow for comparison of pain levels in different groups and the effectiveness of medication in relieving pain associated with OA.

[0081] (4) Grouping of experimental animals and administration time

[0082] Based on the pain test results, SD rats were divided into a normal group, a model group (administered with methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate solution, i.e., physiological saline containing 30% 0.2% HPMC, 25 μl / rat; intra-articular injection; once a week), a positive control group (dexamethasone sodium phosphate injection, 40 μg; intra-articular injection; once a week), and a methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate treatment group (50 μg (high dose group), 25 μg (low dose group); intra-articular injection; once a week).

[0083] (5) Micro-CT

[0084] Twenty-eight days after modeling, rats were euthanized by CO2 asphyxiation. Half of the rats in each group had their left knee joints examined. These joints were then immersed in 4% formaldehyde at room temperature and fixed for 48 hours. Micro-CT scans were performed at a resolution of 8.5 μm, a current of 500 mA, and a voltage of 80 kV. Longitudinal images of the tibial subchondral bone of the knee joint were used to construct a 3D histological analysis.

[0085] (6) Knee joint structural pathological analysis

[0086] The complete rat knee joint tissues of the above groups were obtained by "fixation-decalcification-dehydration-paraffin embedding-rehydration-sectioning" to obtain pathological sections, and hematoxylin-eosin (HE) staining was performed to observe the pathological condition of each group under the corresponding indicators.

[0087] 3. Experimental Results

[0088] (1) Intra-articular injection of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate significantly improved pain response in a rat OA model induced by MIA.

[0089] Intra-articular injection of sodium iodoacetate (MIA) to induce osteoarthritis is a classic pain model. The principle behind this model is that MIA is a selective inhibitor of glyceraldehyde-3-phosphate dehydrogenase, which can induce articular cartilage apoptosis, leading to cartilage damage and a sharp increase in the concentration of inflammatory factors within the joint cavity, causing destruction of other tissues within the joint cavity. This can simulate the symptoms of late-stage osteoarthritis in humans. One of the typical symptoms of osteoarthritis is joint pain, and severe osteoarthritis (KOA) can also significantly affect a patient's gait and posture.

[0090] Von Frey fibers are currently a commonly used tool for scientifically assessing mechanical pain responses in small animals. The rat's foot has a rich area of ​​nerve fiber sensory regions, which can be used to measure the rat's mechanical pain threshold, thereby comparing the pain threshold exhibited by the model control group after drug administration. Figure 5 Figure A shows the paw withdrawal pain threshold of each group of rats, and the significance was analyzed by the GEE model algorithm. The pain threshold decreased as osteoarthritis symptoms worsened. The bipedal balance test can flexibly measure and compare the changes and effects of unilateral intra-articular injection of MIA on the weight-bearing of both hind limbs of rats. The results are expressed as the average difference between the weight-bearing readings of the left and right hind limbs in three measurements for each rat. Figure 5 Figure B shows the difference in weight-bearing capacity of the rat's two feet. Figure 5 The value in C shows the degree of joint swelling in the rat, obtained by measuring the knee joint diameter with calipers. Figure 5 As shown, methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate improved disease indicators in a rat OA model induced by MIA, and methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate significantly improved pain response in a rat OA model induced by MIA.

[0091] (2) Intra-articular injection of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate significantly alleviated cartilage damage in a rat model of MIA-induced osteoarthritis.

[0092] In osteoarthritis, cartilage lesions are the most visually apparent within the joint cavity. MIA is a chemical inducer targeting chondrocytes. At the endpoint, the joint cavity was dissected. Taking the femoral cartilage as an example, macroscopic photographs were taken from the proximal to the distal end of the rat. The degree of cartilage damage was observed. In the normal group, the cartilage surface was smooth, bright in color, and without vascular proliferation; the cartilage angles were gentle. In the model group, the cartilage damage was severe, exhibiting a moth-eaten lesion. Significant vascular proliferation and invasion were observed in the central depression, with multiple punctate or patchy purplish-red areas. The cartilage structure was severely damaged; the cartilage angles became swollen and white, and multiple defects appeared (e.g., Figure 6 The arrow indicates that weekly intra-articular injections of the positive control drug (40 μg) and methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate (50 μg) provided some relief. While the pathological features of cartilage swelling and whitening were not improved, no significant cartilage defects were observed, and vascular invasion at the depressions was inhibited, without the appearance of moth-eaten lesions. 25 μg of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate did not improve vascular invasion, but the cartilage structure remained more intact than in the model group. In conclusion, methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate can, in a dose-dependent manner, reverse the cartilage pathological changes in a MIA-induced osteoarthritis model and improve MIA-induced cartilage damage in rats (see...). Figure 6 ).

[0093] (3) Intra-articular injection of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate significantly alleviated subchondral bone structure destruction in a rat OA model induced by MIA.

[0094] Micro-computed tomography (Micro-CT) can reconstruct the internal microstructure of a sample without damaging it. CT images of the rat knee joint were captured at a specified resolution, and trabecular bone analysis was performed. In 3D imaging, compared to the normal group, the cartilage in the model group showed severe damage: the cartilage surface was uneven, especially at the femoral condyle, with multiple defects, indicating significant cartilage wear and loss. The cartilage in the positive control group was relatively smooth, with no obvious cartilage defects at the femoral condyle. The overall cartilage structure of both doses of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate injected into the joint cavity was relatively intact (e.g., ...). Figure 7 (As shown).

Claims

1. Use of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention and / or treatment of osteoarthritis.

2. Use according to claim 1, wherein, The drug is an injection, capsule, tablet, granule, oral or enema liquid preparation.

3. Use according to claim 2, wherein, The injectable is an intra-articular injection, an intravenous injection, or an intramuscular injection.

4. The use according to claim 1, wherein, The drug comprises a therapeutically effective amount of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate or a pharmaceutically acceptable salt thereof, optional other active agents, and optional one or more pharmaceutically acceptable conventional excipients.

5. Use of methyl 4-[9-(6-aminopurine)]-2(S)-hydroxybutyrate or a pharmaceutically acceptable salt thereof in the preparation of formulations for improving pain response in osteoarthritis, improving cartilage damage or wear, and / or alleviating subchondral bone destruction.

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