WO2025123371A1 - Drug for preventing or treating steroid-induced osteonecrosis of femoral head, and use thereof - Google Patents

Abstract

A drug for preventing or treating steroid-induced osteonecrosis of the femoral head, comprising an active ingredient cycloastragenol. The drug can remarkably improve intraosseous blood supply in the steroid-induced osteonecrosis of the femoral head, reduce formation of necrotic areas and empty lacunae, decrease excessive bone resorption and reduce CTSK protein expression, and provide a potential prevention and treatment scheme for treatment of steroid-induced osteonecrosis of the femoral head.

Description

A medicine for preventing or treating hormone-induced femoral head necrosis and its application Technical Field

The invention belongs to the technical field of biomedicine, and specifically relates to a medicine for preventing or treating hormone-induced femoral head necrosis, and also relates to the application of the medicine in preparing a medicine for preventing or treating hormone-induced femoral head necrosis.

Background Art

Osteonecrosis of the femoral head (ONFH) is an orthopedic disease that seriously endangers people's health. Patients experience hip pain and limited mobility. In the past, the femoral head may collapse and develop hip arthritis in the late stage, which may lead to hip replacement. There are currently 10 million patients with ONFH in my country, and the main patients are young and middle-aged people, which brings a heavy burden to individuals and society. Trauma, drinking, and hormone use are the main causes of ONFH. Among them, hormones are an important cause of ONFH in my country. The use of hormones increased significantly during the COVID-2019 epidemic, increasing the risk of steroid-induced ONFH.

There is currently a lack of effective preventive and therapeutic drugs for hormonal femoral head necrosis. Patients need to undergo surgical treatment, such as core decompression, iliac bone grafting and other operations. In addition to the risks of the operation itself, patients also need to face problems such as a long postoperative recovery period, which is undoubtedly a heavy burden. In the late stage, joint replacement is required, and the operation will face the risk of prosthesis repair, prosthesis loosening, and even catastrophic periprosthetic infection. Traditional Chinese medicine has been used for a long time in the treatment of orthopedic diseases. In-depth exploration of effective ingredients will provide potential compounds for the development of new preventive and therapeutic drugs. The present invention provides a drug for preventing or treating hormonal femoral head necrosis, which can prevent and treat hormonal femoral head necrosis, is expected to delay disease progression, postpone hip replacement for patients, and improve the quality of life.

Summary of the invention

The invention provides a medicine for preventing or treating hormone-induced femoral head necrosis, which solves the deficiency that the prior art for treating hormone-induced femoral head necrosis mostly adopts surgical treatment and lacks definite drug treatment means.

To achieve the above object, the present invention adopts the following technical solutions:

A medicine for preventing or treating steroid-induced femoral head necrosis, comprising cycloastragenol as an active ingredient.

Furthermore, the drug for preventing or treating hormone-induced femoral head necrosis is an inhibitor of CTSK protein expression in the femoral head.

A pharmaceutical composition comprises an effective dose of the above-mentioned drug for preventing or treating hormone-induced femoral head necrosis and a pharmaceutically acceptable carrier.

Furthermore, the carrier includes one or more of a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, an excipient, a filler, a coagulant and a regulator, a surfactant, a dispersant or a defoaming agent.

In the present invention, the medicine further comprises a pharmaceutically acceptable diluent.

Furthermore, the diluent is one of distilled water, physiological sodium chloride or phosphate buffered saline, and glucose solution.

Furthermore, the pharmaceutical composition is administered by gastrointestinal administration or parenteral administration.

In the present invention, the dosage form of the pharmaceutical composition is tablets, capsules, granules, pills, One of oral solution or injection.

Furthermore, the drug dosage for preventing or treating hormone-induced femoral head necrosis is 5 mg/kg-15 mg/kg.

A drug for preventing or treating hormone-induced femoral head necrosis is used in the preparation of a drug for preventing or treating hormone-induced femoral head necrosis.

Furthermore, the steroid-induced femoral head necrosis is femoral head necrosis caused by the use of supraphysiological doses of glucocorticoids.

The present invention has the following beneficial effects:

The drug for preventing or treating steroid-induced femoral head necrosis of the present invention can significantly improve the blood supply in the femoral head of steroid-induced femoral head necrosis, reduce the formation of necrotic areas and empty bone pits, reduce excessive bone absorption and reduce CTSK protein expression, providing a potential prevention and treatment plan for the treatment of steroid-induced femoral head necrosis.

BRIEF DESCRIPTION OF THE DRAWINGS

The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods.

FIG1 is a flowchart of the experiment of cycloastragenol intervention on steroid-induced femoral head necrosis in rats;

Figure 2 shows the changes in rat body weight during the experiment;

Figure 3 shows the femoral head angiography in different treatment groups;

Figure 4 shows the Micro-CT scans of the femoral heads of rats in different treatment groups;

FIG5 is a two-dimensional image of the frontal plane of the femoral head of rats in different treatment groups scanned by Micro-CT and two-dimensional and three-dimensional images of ROI1 and ROI2;

Figure 6 shows the bone parameters of femoral head ROI1 in rats of different treatment groups;

Figure 7 shows the bone parameters of femoral head ROI2 in rats in different treatment groups;

Figure 8 shows the expression of bone resorption protein CTSK in bone tissue of rats with steroid-induced femoral head necrosis by cycloastragalool;

FIG9 is HE staining images of femoral head tissue sections of rats in different treatment groups;

Figure 10 shows the statistics of empty bone pit rates in different treatment groups.

DETAILED DESCRIPTION

Cycloastragenol is one of the main components of Astragalus, a triterpenoid saponin compound, and studies have reported that it can resist aging and oxidation, etc. The inventors have found that cycloastragenol can inhibit RANKL-induced osteoclast formation and bone resorption function, and can improve bone loss in osteoporotic mice, but there are no reports on its prevention and treatment of steroid-induced femoral head necrosis. The present invention clarifies through animal experiments that cycloastragenol can inhibit methylprednisolone-induced femoral head necrosis in rats, and the mechanism of action is related to the inhibition of osteoclasts.

As shown in Figure 1, the experimental flow chart of cycloastragenol intervention in rats, with the caption: Gluteus injection: gluteal muscle injection; Intraperitoneal injection. 9-week-old female SD rats were randomly divided into a solvent control group (Control), a model group (MPS), a low-dose cycloastragenol intervention group (MPS+CAG (5 mg/kg)), and a high-dose cycloastragenol intervention group (MPS+CAG (15 mg/kg)), with 6 rats in each group. Each group was marked with a different color, and each dot represented a rat. Modeling method: methylprednisolone gluteal muscle injection (20 mg/kg), on the 1st to 3rd day of each week, for three consecutive weeks, a total of 9 times, and then the intervention was stopped for 3 weeks. At the 6th week after the first modeling, 3 rats were randomly selected from each group for barium sulfate cardiac perfusion angiography. After completion, all rats were killed and samples were collected for Micro-CT scanning to obtain the femoral head vascular conditions and bone structure conditions, and bone tissue protein expression was detected by Western Blot. The specific process is as follows:

1. Acquisition and grouping of SPF-grade SD rats for experiments

Twenty-four 9-week-old SPF female SD rats were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine (Sanyuanli, SCXK [粤] 2013-0034), SPF Animal Certificate No. 44005800008479. The design and ethics of this experiment were approved by the Animal Experiment Ethics Review Committee of Guangzhou University of Chinese Medicine (ethics number: 20190722001). All rats were weighed and numbered in ascending order after purchase. EXCEL was used to establish a function and randomly divided into a solvent control group (weight 222.4±15.84g), a GIONFH model group (weight 222.9±13.28g), a low-dose cycloastragenol intervention group (dose of 5mg/kg) (weight 223.6±14.46g) and a high-dose cycloastragenol intervention group (dose of 15mg/kg) (weight 224.1±12.81g), with 6 rats in each group. After grouping, each group was placed in a standard cage and the model was established after one week of adaptive feeding. All rats were fed with ordinary feed, free access to food and water, and 24-hour light cycle. The animals were checked regularly every day.

2. Rat GIONFH Modeling and Cycloastragenol Intervention

All rats were weighed again after 1 week of adaptive feeding, and the required amount of methylprednisolone (20 mg/kg) for intramuscular injection and different doses of cycloastragenol (5 mg/kg and 15 mg/kg) for intraperitoneal injection was calculated according to the body weight, and sterile saline (0.5% DMSO) was used as the solvent to dissolve methylprednisolone and cycloastragenol. During modeling, methylprednisolone was injected into the gluteal muscle of the model group, and attention was paid to alternating the gluteal muscles on both sides. In the intervention groups with different doses of cycloastragenol, the cycloastragenol solution was injected intraperitoneally after the intramuscular injection of methylprednisolone, while the solvent control group was given an equal volume of sterile saline (0.5% DMSO) for intraperitoneal injection. Modeling was performed on the 1st to 3rd day of each week, 3 times in a row, with an interval of 24 hours each time, for 3 consecutive weeks, with a total of 9 interventions. During this period, a new syringe was replaced for each injection. No antibiotics were used, and the intervention was stopped for 3 weeks. During the experiment, the rats were observed every day, recorded and processed in time, and the weight changes of the rats were recorded once a week, and the drug dosage was adjusted accordingly. From the 6th week after the first modeling, the three rats with the lowest, middle and highest weights were selected in each group (the two rats ranked 3rd and 4th in weight in the 6 rats in the high-dose cycloastragenol intervention group were randomly selected by establishing a function in EXCEL). After anesthetizing the above three rats in each group, barium sulfate cardiac perfusion angiography was performed, and the other rats were killed after overdose of anesthesia. After that, bone tissue samples were taken from all rats, and the bilateral femurs were preserved in 4% PFA, and the bilateral tibias were washed with ultrapure water and stored in a -80℃ refrigerator.

As shown in Figure 2, there were no significant differences in the baseline weight levels of the rats in each group (week 0 and week 1), see Figure 2A and Figure 2B. The weight changes of the rats during the entire cycloastragenol intervention process (once a week) are shown in Figure 2C.

3. Barium Sulfate Suspension Cardiac Perfusion Angiography

The amount of anesthesia required was calculated based on the weight of the rats at the end of the sixth week after modeling. Before anesthesia, a barium sulfate suspension (15 g barium sulfate: 50 ml gel) with a mass volume concentration of 30% was prepared in advance. The specific method was to first put the sterilized gel into a 50 ml centrifuge tube and heat it in a 40°C water bath until it was completely dissolved in water. Sub-nanometer barium sulfate (diameter < 0.8 μm) was gradually added in small amounts and multiple times, and continuous shaking during the period promoted the continuous dissolution of the two, and finally formed a thick milk-like barium sulfate suspension, which was continuously heated and shaken at 40°C for standby use.

After the anesthesia takes effect, the rat is fixed in the beach position on a rack modified from a mouse cage cover. The bilateral forelimbs are fixed with a long tail clip. The surgical scissors make a longitudinal incision upward from the junction of the chest and abdomen in the midline of the abdomen, gradually exposing the thoracic diaphragm, and the diaphragm is completely cut from the middle to both sides along the inner wall of the thorax. During this period, the rat has pneumothorax and shakes. When the rat calms down, the surgical scissors cut the ribs upward along both sides of the sternum (because there are accompanying thoracic arteries and veins close to both sides of the sternum, which may affect the perfusion effect after injury) until the root of the heart and lungs are completely exposed. Flip the sternum backward and fix it to keep the heart surgical field clear. Use tissue forceps to open the thymus and see the white translucent aortic arch above the heart. At this time, the operator holds the heart with the thumb and index finger of one hand, and holds a homemade perfusion needle with a diameter of 1.2mm (the tip of a 20ml syringe needle is smoothed and the tail is tied with silk thread) in the other hand. Pierce the heart from the apex, and continue to slide slowly toward the aortic arch after feeling the sense of emptiness until the needle tip is seen through the aortic root. Fix the perfusion needle to the chest with the silk thread at the tail to prevent it from slipping out during the operation. After completing the fixation of the perfusion needle, the operator carefully lifts the right atrial appendage with micro forceps and cuts it with micro scissors to allow venous blood to flow out. Then hold a 50ml syringe and continue to slowly perfuse the heart with 50IU/ml heparin sodium (25000IU: 500ml saline) until the mesentery becomes completely transparent and clear heparin sodium solution flows out of the right atrial appendage (about 150ml/mouse). After heparinization, the operator wears a protective mask, replaces the syringe and slowly perfuses the heart with 4% PFA, and maintains the perfusion until the right atrial appendage flows out of clear PFA (about 50 ml/rat). After PFA fixation, replace a new 50 ml syringe to extract the barium sulfate solution, maintain a certain pressure and slowly push it at a uniform speed. It can be seen that the mesenteric microvessels are gradually filled with barium sulfate. Continue to perfuse until it can no longer be pushed. Finally, it is best if the right atrial appendage can flow out of the barium sulfate solution. After the rat is cleaned with ultrapure water, it is transferred to a 4° refrigerator overnight. The next day, the femurs on both sides are sampled and fixed in 4% PFA at room temperature.

As shown in Figure 3, the red color indicates that the blood vessels are filled with contrast agent. Among them, angiography showed that the blood supply in the femoral head was abundant in the solvent control group, and the blood supply in the femoral head was reduced in the model group. The intervention of cycloastragenol improved the blood supply of the femoral head in a dose-dependent manner. Femoral head angiography proved that cycloastragenol improved the reduced blood supply in the femoral head of rats with steroid-induced femoral head necrosis.

4. Bone parameter analysis and vascular image extraction after Micro-CT scanning reconstruction

After the rat bone tissue was fixed for 48 hours, Micro-CT scanning was performed on the part above the lesser trochanter to the femoral head. The scanning parameters were: 80kV, 100uA, 0.5mm AI filter, 1K resolution, 0.6° rotation step, 12μm pixel. After scanning, NRecon was used for 3D reconstruction. The reconstruction parameters were: ring artifact correction = 7, smoothing = 1, beam hardening correction = 33%, and the image conversion threshold was uniformly set to 0-0.0771. The reconstructed 3D image was loaded into CTvox, and after confirming that the femoral head was completely scanned, it was imported into Dataviewer. The posture of each cross-section of the femoral head was manually adjusted to try to make the line passing through the apex of the femoral head and the center of the epiphysis approximately perpendicular to the horizontal plane in the virtual space. All samples were performed in this way, so that the ROI between samples tended to be consistent. After the adjustment, the two-dimensional images of the three sections of the femoral head were exported respectively. The two-dimensional images of the horizontal plane were imported into CTan software, and two ROIs were selected above and below the epiphyseal line respectively [132]. Specifically, the highest point of the center of the growth plate was first determined as the reference, and the upward and downward offsets were set to 0.203/0.799 mm (about 1 mm in total). The cylindrical areas with a height of 0.3 mm above the upper area and a radius of 0.65 mm were selected as ROI1 and ROI2 respectively. The grayscale threshold of the polarization was set to 100-255. The three-dimensional analysis function was used to obtain the bone parameters of the corresponding ROI, including bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). For the femoral head with barium sulfate angiography, the three-dimensional image reconstructed by the above scan was imported into CTvox, and the curve in the channel was manually adjusted to separate the blood vessels filled with barium sulfate from the femoral head.

As shown in Figure 4, the trabecular structure of the femoral head was normal in the solvent control group, and necrotic cavities were formed in the femoral head of the model group (the white arrow in the femoral head indicates the necrotic area). The cycloastragenol intervention improved the formation of necrotic cavities in the femoral head in a dose-dependent manner. Micro-CT reconstruction proved that cycloastragenol improved the formation of necrotic areas in rats with steroid-induced femoral head necrosis.

As shown in Figure 5, the two-dimensional image of the frontal plane of the rat femoral head and the two-dimensional and three-dimensional images of ROI1 and ROI2, and Figures 6 and 7 are the bone parameters of ROI1 and ROI2 of the rat femoral head, respectively. Notes: COR: frontal plane; TRA: horizontal plane; ROI1: region of interest above the epiphyseal line; ROI2: region of interest below the epiphyseal line; BV/TV: bone volume fraction; Tb.N: number of trabeculae; Tb.Th: trabecular thickness; Tb.Sp: trabecular dispersion. * in the statistical graph represents P < 0.05. The results showed that the trabeculae in different regions of the femoral head of the solvent control group were abundant, the bone resorption in the femoral head of the model group was significantly increased, and the intervention of cycloastragenol improved the bone resorption in the femoral head in a dose-dependent manner.

5. Rat bone tissue protein immunoblotting experiment

The bilateral bone tissues of rats without angiography were taken out from the -80℃ refrigerator and immediately put into a mortar filled with liquid nitrogen for manual grinding. Liquid nitrogen was continuously added during the process to maintain the low temperature. After sufficient grinding, an equal amount of bone powder was weighed for each group, and RIPA protein lysis buffer (containing 10μl PMSF) was added at a ratio of 100mg:1ml. After sufficient shaking and mixing, the samples were lysed on ice for 30min, transferred to a centrifuge at 4℃, centrifuged at 12000r/min for 20min, and the protein supernatant was carefully aspirated with a pipette. 1/4 volume of 5×SDS-PAGE protein loading buffer was added, and the protein was fully denatured in a water bath at 100℃ for 5min. After that, the samples were promptly divided and stored in a -20℃ refrigerator for use. The protein samples were boiled again and centrifuged before electrophoresis. 10% and 12% gels (1.5mm, 15 holes) were prepared according to the instructions, and the primary and secondary antibodies were diluted with 1% skim milk (1×TBST) in advance. The protein loading volume was 15 μl, electrophoresis was performed at room temperature (110 kV, 90 min), membrane transfer was performed on ice (200 mA, 2 h), 5% milk blocking was performed (room temperature, 1 h), primary antibody incubation was performed (4 °C, overnight), membrane washing was performed (room temperature, 3 × 5 min), secondary antibody incubation was performed (room temperature, 2 h), membrane washing was performed (room temperature, 3 × 5 min), the membrane was placed in the imaging system after soaking in ECL developer, automatic exposure was set, and the exported image was quantitatively analyzed for grayscale using Image J. For proteins with close positions, membrane regeneration solution was added for elution, and other proteins were detected according to the above method.

As shown in Figure 8, representative WB images (Figure 8A) and CTSK protein expression (Figure 8B), * in the statistical graph represents P < 0.05. The expression of CTSK in the femoral head of the solvent control group was low, and the expression of CTSK in the femoral head of the model group was significantly increased. The intervention of cycloastragenol improved the expression of CTSK in a dose-dependent manner. It was proved that cycloastragenol improved the expression of bone resorption proteins in the bone tissue of rats with steroid-induced femoral head necrosis.

6. HE staining experiment of rat femoral head tissue sections

After Micro-CT scanning, the same number of rat femoral heads (n=3) were selected from each group for pathological observation. First, the femoral heads were immersed in 14% ethylenediaminetetraacetic acid (EDTA) (NeoFroxx, Germany) at room temperature for 2 weeks for decalcification, and the solution was changed once a day. After that, ethanol was used for routine dehydration and paraffin embedding. The RM 2155 Biocut Microtome paraffin slicer (Leica, Germany) was used to make tissue sections with a thickness of 5 μm and loaded onto slides for HE staining. The Pannoramic MIDI slice automatic scanner (3DHistech, Hungary) was used to complete the panoramic scanning of the slices, and the (empty) bone lacuna in the femoral head was quantitatively analyzed using the SlideViewer software (3DHistech, Hungary) that came with the scanner.

As shown in Figure 9, the HE staining images of rat femoral head tissue sections and the statistics of empty bone lacuna rates are shown in Figure 10. Notes: CAG, cycloastragenol; MPS, methylprednisolone; HE, hematoxylin and eosin staining; GIONFH, glucocorticoid-induced femoral head necrosis. * in the statistical graph represents P < 0.05. The results showed that there were fewer empty bone lacunas in the femoral head in the solvent control group, and more empty bone lacunas in the femoral head in the model group. Cycloastragenol intervention reduced the formation of empty bone lacunas in a dose-dependent manner. The empty bone lacunas (necrotic-like areas) and bone lacunas (normal areas) above the epiphysis are marked with black arrows and green arrows, respectively. The empty bone lacuna rate is the ratio of the number of empty bone lacunas to the total number of (empty) bone lacunas.

Example 1

A drug for preventing or treating steroid-induced femoral head necrosis, comprising cycloastragenol as an active ingredient. In this embodiment, cycloastragenol can improve the reduced blood supply to the femoral head of rats with steroid-induced femoral head necrosis, reduce the formation of necrotic areas and empty bone pits, and reduce excessive bone absorption; and reduce the expression of CTSK protein, and can be used as an inhibitor of CTSK protein expression in the femoral head.

Example 2

A pharmaceutical composition comprises an effective dose of the above-mentioned drug for preventing or treating hormone-induced femoral head necrosis. According to the required preparation, it also comprises a pharmaceutically acceptable, non-toxic carrier or diluent. Such diluents are distilled water, physiological sodium chloride or phosphate buffered saline, glucose solution, etc. Such pharmaceutically acceptable carriers may be buffers, emulsifiers, suspending agents, stabilizers, preservatives, excipients, fillers, coagulants and regulators, surfactants, diffusants or defoamers. In addition, the pharmaceutical composition or preparation may also include other carriers, adjuvants or non-toxic, non-therapeutic stabilizers, etc. The pharmaceutical composition may also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid and copolymers (e.g., latex functionalized agarose (TM), agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers and lipid aggregates.

The dosage form of the pharmaceutical composition of the present invention is tablets, capsules, granules, pills, oral liquids or injections. The medicine of the present invention can be administered to the body in a known manner. The administration method is enteral administration or parenteral administration. Parenteral administration methods are, for example, delivered to the tissue of interest by intravenous systemic delivery or local injection. It can be optionally administered via intravenous, transdermal, intranasal, mucosal or other delivery methods. The effective dose of the drug for preventing or treating steroid-induced femoral head necrosis is 5mg/kg-15mg/kg.

Example 3

The invention discloses a drug for preventing or treating steroid-induced femoral head necrosis in the preparation of a drug for preventing or treating steroid-induced femoral head necrosis. Specifically, steroid-induced femoral head necrosis is femoral head necrosis caused by the application of a super-physiological dose of glucocorticoid.

The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims (10)

A medicine for preventing or treating hormone-induced femoral head necrosis, characterized in that it comprises the active ingredient cycloastragenol. The drug for preventing or treating steroid-induced femoral head necrosis according to claim 1 is characterized in that the drug for preventing or treating steroid-induced femoral head necrosis is an inhibitor of CTSK protein expression in the femoral head. A pharmaceutical composition, characterized in that it comprises an effective dose of the drug for preventing or treating hormone-induced femoral head necrosis according to claim 1 or 2 and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 3 is characterized in that the carrier includes one or more of a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, an excipient, a filler, a coagulant and a regulator, a surfactant, a dispersant or a defoaming agent. The pharmaceutical composition according to claim 3 is characterized in that it also includes a pharmaceutically acceptable diluent, and the diluent is one of distilled water, physiological sodium chloride or phosphate buffered saline, and glucose solution. The pharmaceutical composition according to any one of claims 3 to 5, characterized in that the pharmaceutical composition is administered by gastrointestinal administration or parenteral administration. The pharmaceutical composition according to claim 3 is characterized in that the dosage form of the pharmaceutical composition is one of tablets, capsules, granules, pills, oral liquid or injection. The pharmaceutical composition according to claim 3 is characterized in that the drug dosage for preventing or treating steroid-induced femoral head necrosis is 5 mg/kg-15 mg/kg. A use of the drug for preventing or treating steroid-induced femoral head necrosis as claimed in claim 1 in the preparation of a drug for preventing or treating steroid-induced femoral head necrosis. The use according to claim 9 is characterized in that the steroid-induced femoral head necrosis is femoral head necrosis caused by the use of supraphysiological doses of glucocorticoids.