WO2022089417A1

WO2022089417A1 - Drug for treating spondylopathies and osteoarthropathy and relieving pain caused thereby, and application thereof

Info

Publication number: WO2022089417A1
Application number: PCT/CN2021/126366
Authority: WO
Inventors: 韦国荣; 韦建; 郭子湖; 王永华
Original assignee: 陕西秦岭七药协同创新中心有限公司; 北流韦国荣诊所
Priority date: 2020-10-28
Filing date: 2021-10-26
Publication date: 2022-05-05
Also published as: CN112274644B; CN112274644A

Abstract

An application of cephalosporins and a combination of the cephalosporins and a steroidal antiinflammatory drug in preparing a drug for treating spondylopathies and osteoarthropathy and relieving pain caused thereby. Both the cephalosporins and the steroidal antiinflammatory drug are in injection dosage forms, and by intravenous or local lesion administration, can treat aseptic lesions, such as inflammation, edema, and exudation, of spine and bone joints and surrounding muscle tissues, and relieve pain, numbness, muscle adhesions, spasm, and discomfort caused by the lesions. A drug for treating spondylopathies and osteoarthropathy and relieving pain caused thereby is composed of the cephalosporins and the steroidal antiinflammatory drug.

Description

A kind of medicine for treating spine and bone joint disease and alleviating its pain and its application

technical field

The invention relates to the technical field of medicine, and more particularly to a medicine for treating spinal cord and bone joint disease and alleviating its pain and its application.

Background technique

Aseptic lesions of the spine and bones and joints are extremely common painful diseases in clinic. According to medical data, almost everyone will encounter such lesions in their lifetime, and it is difficult to cure. The causes are roughly divided into three types: 1. Muscle overwork 2. Autoimmune disease 3. Hyperuricemia. The common pathological features are inflammation, edema, and exudation in the spine and bone joints, and often involving the surrounding muscle tissue. The main symptoms are swelling, redness, heat, pain, and even limited activity at the lesion site. Most of these lesions are chronic, and the course of the disease is prolonged and repeated, which brings huge economic, physical and mental pressure to the patients. Therefore, the disease has always been a problem that needs to be solved urgently in clinical and basic research.

SUMMARY OF THE INVENTION

In view of this, the present invention provides a medicament with low price, small dosage, and small toxic and side effects, which can effectively treat the pathological changes of the spine and bone and joints and relieve the pain symptoms thereof.

In order to achieve the above purpose, the present invention provides the following technical scheme, which is composed of the following components: a cephalosporin antibiotic and a combination thereof with a steroidal anti-inflammatory drug; the cephalosporin and steroidal anti-inflammatory drug are both injected In dosage form, it is used for spinal and osteoarticular lesions.

Preferably, in the above-mentioned medicament for treating spinal and bone and joint diseases and alleviating their pain, the cephalosporin antibiotics are all in the form of injection: after being dissolved in physiological saline, they can be administered by intravenous drip, intravenous injection, Local lesion injection administration.

Preferably, in the above-mentioned medicines for treating spinal and bone and joint diseases and alleviating their pain, the steroid anti-inflammatory drugs are all in the form of injection: after being dissolved in normal saline, they can be administered by intravenous drip, intravenous injection , Local lesion injection.

Preferably, in the above-mentioned medicament for treating vertebral and bone and joint diseases and alleviating their pain, the cephalosporin antibiotic is selected from any one of the following or a pharmaceutically acceptable salt thereof: cefotaxime, cefradine, Cefalexin, Cefotaxime, Cefacetonitrile, Cefpirin, Ceftiam, Cefmetazole, Cefoxitin, Cefizoxime, Cefmenoxime, Cefpiramide, Cefotetan, Laoxacefa, Cefminox, Cefazolam, cefathiamidine, cefazolin, cefuroxime, cefamandole, ceftizole, cefotaxime, ceftazidime, cefoperazone, ceftriaxone, cefepime, cefpirome, cefidiazine, Cefonicid.

Preferably, in the above-mentioned medicine for the treatment of spinal and bone and joint diseases and alleviating pain, the steroid anti-inflammatory drug is selected from dexamethasone, methylprednisolone, betamethasone, hydrocortisone, Any one of triamcinolone acetonide or a pharmaceutically acceptable salt thereof.

Preferably, in the above-mentioned medicament for the treatment of spinal and bone and joint diseases and alleviating their pain, the spinal and bone and joint diseases are aseptic lesions.

Preferably, in the above-mentioned medicament for treating spine and bone and joint pathology and alleviating its pain, the aseptic pathology is characterized in that it includes lumbar disc herniation, cervical spondylosis, thoracic spondylosis, and uncinate vertebral arthritis. , posterior lumbar arthritis, ankylosing spondylitis, myofasciitis, osteoporosis, carpal tunnel syndrome, flexor tenosynovitis, lateral epicondylitis, medial humeral epicondylitis, shoulder periarthritis, hip joint slip Meningitis, aseptic necrosis of the femoral head, knee arthritis, ankle injury, heel inflammation, rheumatoid arthritis, gouty arthritis.

Preferably, in the above-mentioned medicament for treating spine and bone and joint diseases and alleviating their pain, the steroid anti-inflammatory drug belongs to glucocorticoids, which itself has a strong anti-inflammatory effect and has a rapid onset of action. Combined use with cephalosporin antibiotics can significantly relieve the above-mentioned lesions of the spine and bone and joints, and relieve the symptoms of pain, numbness and discomfort.

The invention also provides the application of the above-mentioned cephalosporin antibiotic and the combination of the cephalosporin antibiotic and the steroidal anti-inflammatory drug in the preparation of a medicament for treating spinal cord and bone and joint diseases and alleviating their pain. The cephalosporin antibiotics and the steroid anti-inflammatory drugs are both in the form of injections, and are used for treating the lesions of the spine and the bone joints; the lesions of the spine and the bone and joints are aseptic lesions.

Preferably, the cephalosporin antibiotics are all in the form of injection: after being dissolved in physiological saline, they can be administered by intravenous drip, intravenous injection, or local lesion injection.

Preferably, the steroidal anti-inflammatory drugs are all in the form of injection: after being dissolved in physiological saline, they can be administered by intravenous drip, intravenous injection, or local lesion injection.

Preferably, the cephalosporin antibiotics are selected from any one of the following or a pharmaceutically acceptable salt thereof: cefotaxime, cefradine, cephalexin, cefotaxime, cefaacetonitrile, cefapirin, cefotiam, cefamet azole, cefoxitin, cefazolin, cefmenoxime, cefpiramide, cefotetan, laoxef, cefminox, cefazolam, cefathiamidine, cefazolin, cefuroxime, cefamandole, Ceftezole, cefotaxime, ceftazidime, cefoperazone, ceftriaxone, cefepime, cefpirome, cefodizime, cefonicil.

Preferably, the steroid anti-inflammatory drug is selected from any one of dexamethasone, methylprednisolone, betamethasone, hydrocortisone, triamcinolone acetonide or a pharmaceutically acceptable salt thereof.

Preferably, the aseptic lesions include lumbar disc herniation, cervical spondylosis, thoracic spondylosis, uncinate arthritis, posterior lumbar arthritis, ankylosing spondylitis, myofasculitis, osteoporosis, carpal tunnel syndrome , flexor tenosynovitis, lateral epicondylitis, medial epicondylitis, shoulder periarthritis, hip synovitis, aseptic femoral head necrosis, knee arthritis, ankle injury, heel inflammation, rheumatoid Arthritis, gouty arthritis.

Compared with the current therapy, the present invention has at least the following advantages: low price, less drug dosage, less toxic and side effects, good anti-inflammatory effect, fast onset of effect, and lasting effect, which can meet the needs of the vast majority of patients of this type, and relieve them. symptoms.

Antibiotics are a class of drugs used to inhibit the growth of bacteria or kill bacteria, and are clinically used to prevent or treat lesions caused by bacterial infections. Cephalosporins are semi-synthetic antibiotics containing cefane in the molecule, which belong to β - Derivatives of 7-aminocephalosporanic acid (7-ACA) among lactam antibiotics, however, we observed in experiments that they also have strong anti-inflammatory and analgesic effects on aseptic lesions.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only It is an embodiment of the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to the provided drawings without creative work.

Figure 1 is a schematic diagram showing the comparison of the degree of redness and swelling of the ankle joints of mice under different treatment conditions of the present invention.

Figure 2 is a schematic diagram of joint scores of mice in different treatment groups of the present invention.

Figure 3 is a schematic diagram of the 50% mechanical paw withdrawal threshold of mice in different treatment groups of the present invention.

Figure 4 is a schematic diagram showing the detection of tissue damage and inflammatory cell infiltration by H&E staining of mouse ankle joints of the present invention.

Figure 5 is a schematic diagram of the present invention using ELISA to detect three symptom indicators in mouse serum.

Figure 6 is a schematic diagram showing the comparison of the degree of redness and swelling of the ankle joints of rats under different treatment conditions of the present invention.

Figure 7 is a schematic diagram showing the comparison of toe volumes of rats in different treatment groups of the present invention.

Figure 8 is a schematic diagram of the present invention using mechanical stimulation to detect pain thresholds of rats in different treatment groups.

The accompanying drawing of FIG. 9 is a schematic diagram of three symptom indexes of rat serum detected by ELISA according to the present invention.

Fig. 10 is a schematic diagram showing the detection of tissue damage and inflammatory cell infiltration by H&E staining of rat ankle joints of the present invention.

Figure 11 is a schematic diagram showing the comparison of the degree of redness and swelling of the ankle joints of mice under different treatment conditions of the present invention.

Figure 12 is a schematic diagram showing the comparison of toe volumes of mice in different treatment groups of the present invention.

Figure 13 is a schematic diagram showing the comparison of toe volumes of mice in different treatment groups of the present invention.

Fig. 14 is a schematic diagram showing the detection of tissue damage and inflammatory cell infiltration by H&E staining of mouse ankle joints of the present invention.

Figure 15 is a schematic diagram of the present invention using ELISA to detect three symptom indicators in mouse serum.

Figure 16 is a schematic diagram showing the comparison of the sensitivity of mice to mechanical stimulation under different treatments of the present invention.

Figure 17 is a schematic diagram showing the comparison of inflammatory indexes of mice in different treatment groups of the present invention.

Figure 18 is a schematic diagram showing the comparison of the sensitivity of mice to mechanical stimulation under different treatments of the present invention.

Figure 19 is a schematic diagram showing the comparison of inflammatory indexes of mice in different treatment groups of the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Please refer to accompanying drawings 1-19, which is a medicine for treating spinal and bone joint diseases and alleviating their pain disclosed in the present invention.

Example 1: Evaluation of the therapeutic and pain relief effects of cefazolin sodium for injection on mice with rheumatoid arthritis

1. Experimental materials and methods

1.1. Experimental animals and groups

Experimental animals: Wild-type SPF male BALB/c mice (6-8 weeks old) were purchased from the Experimental Animal Center of Yangzhou University, and were raised in the experimental center of Northwestern University under standard rearing conditions. The ambient temperature was (22±3) °C, the humidity was (55±5)%, light: 12h/12h day-night alternation, mice can freely eat and drink.

All animals were acclimated in this environment for one week before starting the experiment.

According to the experimental needs, the experimental animals were divided into 6 groups: blank group, arthritis group, dexamethasone group, cefazolin low-dose group, cefazolin medium-dose group, and cefazolin high-dose group.

1.2. Modeling method of rheumatoid arthritis

Use sterile acetic acid with a concentration of 0.01 mol/L to prepare bovine type II collagen (Chondrex) to a final concentration of 2 g/L, and place it at 4°C in the dark, and rotate overnight to complete the dissolution. Take an equal amount of complete Freund's adjuvant (Chondrex) or incomplete Freund's adjuvant (Chondrex) on ice, and fully emulsify with bovine type II collagen to prepare a collagen emulsion for later use.

Except for the blank group, rheumatoid arthritis models were established in other groups. On day 0, a collagen emulsion made of 100 μL of bovine type II collagen and Freund's complete adjuvant was injected intradermally at the base of the mouse tail. In addition, on the 21st day, 100 μL of collagen emulsion made of bovine type II collagen and incomplete Freund's adjuvant was injected to enhance the immune effect. The blank group was injected with an equal volume of 0.9% sodium chloride solution.

1.3. Method of administration

After the rheumatoid arthritis model was successfully established (ie, after immune boosting), the dexamethasone group was injected with 1 mg/kg dexamethasone sodium phosphate injection through the tail vein, once every three days, for a total of 7 injections . The low-dose cefazolin group, the cefazolin medium-dose group, and the cefazolin high-dose group were injected with cefazolin sodium (Original Bio) 200 mg/kg, 400 mg/kg and 800 mg/kg, respectively. It was administered once every three days for a total of 7 injections. Cefazolin sodium for injection uses 0.9% sodium chloride solution as a dissolving agent. The blank group and model group were injected with equal volume of normal saline.

1.4. Animal Evaluation of Rheumatoid Arthritis

From the day of booster immunization, the sub-joint index scores of mice in each group were performed every 3 days. The degree of redness and swelling of the ankle joint of the mice, the activity level of the mice and the joint index of the mice in different groups were observed and recorded. The joint index was scored as follows: normal joints and no obvious redness and swelling (0 points); redness and swelling of one toe joint in mice (1 point); redness and swelling of two or more toe joints in mice (2 points); Paws red and swollen (3 points); mice were severely red and swollen throughout the paws, and the ankle or wrist joint was deformed or rigid (4 points). The joints of the limbs of the mice were scored separately and accumulated, and the total score was 0-16 points.

1.5. Detection of pain threshold by mechanical stimulation method

From the first administration, the pain thresholds of mechanical stimulation in each group were measured 3 hours after each administration. First, the mice were placed in the pain measuring cage of the mechanical pain measuring instrument for 20 minutes, and the Von-Frey mechanical stimulation needle was used to stimulate the middle of the mouse's sole vertically, and then the reaction of the mouse was recorded. If the mouse appeared quickly within the stimulation time Activity such as foot bounce, foot retraction or foot licking was recorded as a positive reaction. If the mouse did not show such response, it was recorded as negative; the 50% paw withdrawal threshold was determined and analyzed using the up-down method.

1.6. Histopathological examination

Mice were sacrificed after anesthesia, and joint tissues were rinsed with normal saline, then fixed in 10% paraformaldehyde solution, embedded in paraffin, and cut into 4-micron sections for H&E staining. The stained sections were observed using an Olympus BX51 fluorescence microscope to observe inflammatory cell infiltration and tissue damage.

1.7. ELISA to detect the level of inflammatory factors

The mice were sacrificed after anesthesia, and blood was collected from the eyeballs of the mice. After standing at room temperature for 2 hours, centrifugation was performed at a speed of 3000 r/min for 10 minutes, and then the serum was recovered. Then, the levels of TNF-α, IL-6 and IL-1β in the serum of mice in different groups were determined according to the official instructions of the ELISA kit.

1.8. Experimental results

Figure 1 shows the comparison of the degree of redness and swelling of the ankle joints of mice under different treatment conditions

A: Blank group B: Arthritis group C: Dexamethasone group D: Cefazolin low-dose group E: Cefazolin medium-dose group F: Cefazolin high-dose group;

Figure 2 shows the joint scores of mice under different treatment conditions. Different lowercase letters indicate that the significance between the two groups is p<0.01, and the same letter indicates that there is no significance between the two groups;

Figure 3 shows the sensitivity of mice to mechanical stimulation (50% mechanical paw withdrawal threshold) under different treatments. Different lowercase letters indicate that the significance between the two groups is p<0.01, and the same letter indicates that there is no significance between the two groups;

Figure 4 is a schematic diagram of inflammatory cell infiltration and tissue damage in mouse ankle joint tissue under different treatment conditions. The results of H&E staining of rat ankle joints to detect tissue damage and inflammatory cell infiltration showed that A: blank group B: arthritis group C: dexamethasone group D: cefazolin low-dose group E: cefazolin medium-dose group F: cephalosporin Zolin high-dose group

Figure 5 is a schematic diagram of the comparison of inflammatory indexes in mice under different treatment conditions. The contents of three arthritis inflammatory indexes (A) TNF-α (B) IL-1β and (C) IL-6 in the serum of mice were detected by ELISA. Different lowercase letters indicate that the significance between the two groups is p<0.01, and the same letter indicates that there is no significance between the two groups;

As can be seen from Figure 1 and Figure 2, the ankle joints of the mice in the model group were obviously red and swollen, and the joint index score was significantly higher than that of the other groups. Both dexamethasone and different doses of cefazolin reduced ankle redness and reduced joint scores. The moderate and high doses of cefazolin had the best effect, better than the dexamethasone group.

The experimental results in Figure 3 show that the mice in the model group are significantly less sensitive to mechanical stimulation than other groups. The use of dexamethasone and different doses of cephalosporin can improve the pain sensitivity threshold, and the middle-dose and high-dose treatment groups have the best improvement, and there is no statistical difference with the blank group.

The H&E histopathological staining results in Figure 4 showed that there was no tissue damage and inflammatory cell infiltration in the ankle joints of the mice in the normal group. The ankle joint tissue of the mice in the model group was severely damaged, and there was obvious inflammatory cell infiltration. The dexamethasone group showed decreased inflammatory cell infiltration. Inflammatory cells in the low, medium and high dose groups of cefazolin were less than those in the model group, and the infiltration of inflammatory cells in the high dose group was the least. And in cefazolin, the inflammatory cell infiltration in the high-dose group was less than that in the dexamethasone group.

The results in Figure 5 show that the three inflammatory indicators of inflammation in the rheumatoid model group were significantly increased compared with the normal group. The dexamethasone group, as well as the cefazolin low, medium, and high groups, reduced these inflammatory markers. And in cefazolin, the inflammatory indexes in the high-dose group were lower than those in the dexamethasone group.

Example 2: Evaluation of the treatment and pain relief effects of cefuroxime sodium for injection on gouty arthritis rats

2. Experimental animals and methods

2.1 Animals and groups

Experimental animals: Wild-type SPF healthy male SD rats (180±20g) were purchased from the Experimental Animal Center of Yangzhou University, and were raised in the experimental center of Northwestern University under standard rearing conditions. 5)%, light: 12h/12h day-night alternation, all rats can eat and drink freely.

According to the experimental needs, the experimental animals were divided into 6 groups: blank group, gout group, positive drug group (dexamethasone sodium phosphate injection), cefuroxime low-dose group, cefuroxime medium-dose group, cefuroxime high-dose group Group.

2.2 Modeling method of gouty arthritis

Acute gout model was established in rats except for the blank group by using sodium urate to induce gout in the toes of rats. First, sodium urate was formulated into a 25 mg/ml sodium urate suspension with normal saline and Tween 80 (9:1 by volume). After the rats were anesthetized, 0.2 mL of sodium urate suspension was injected into the bone joint cavity of the ankle joint of the rat to be modeled. The rats in the control group were injected with the same volume of normal saline at the same site. 1-2 hours after modeling, observe whether there is obvious swelling of the toes of the rats.

2.3 Method of administration

The administration of gouty arthritis was started 24 hours after the completion of the modeling. For the dexamethasone group, 0.5 mg/kg dexamethasone sodium phosphate injection was injected through the tail vein, once a day, for a total of 3 injections. For cefuroxime low dose group 100mg/kg, cefuroxime medium dose 200mg/kg, cefuroxime high dose group 400mg/kg. Administered once a day, a total of 3 injections. Cefuroxime sodium for injection uses 0.9% sodium chloride solution as a dissolving agent. The blank group and model group were injected with equal volume of normal saline.

2.4 Evaluation of gouty arthritis animals

At 0h, 2h, 4h, 8h, 16h, 24h, 36h, 48h, 60h and 72h, the volume value of the same part of the ankle joint of each group of rats was measured with a toe volume meter, and the swelling of the ankle joint of the rats was recorded. Spend.

2.5 Detection of pain threshold by mechanical stimulation method

From the first day of administration, the pain thresholds of mechanical stimulation of rats in each group were measured at 0h, 2h, 4h, 8h, 16h, 24h, 36h, 48h, 60h and 72h, respectively. First, the rats were placed in the pain measuring cage of the mechanical pain meter to adapt for 5 minutes, and the Von-Frey mechanical stimulation needle was used to stimulate the middle of the rat's sole vertically, and then the reaction of the rat was recorded. If the experimental animal appeared rapidly within the stimulation time Activity such as foot bounce, foot retraction or foot licking was recorded as a positive reaction. If no such reaction occurs, it is negative; the 50% paw withdrawal threshold is determined and analyzed according to the up-down method.

2.6 Histopathological examination

After the last administration and detection, the rats were anesthetized and sacrificed, the ankle joint capsule was incised, and the synovial tissue of the ankle joint was rinsed with normal saline, then fixed in 10% paraformaldehyde solution and embedded in paraffin. Tissues were cut into 4 micron sections and stained by the H&E method. The stained sections were observed using an Olympus BX51 fluorescence microscope to observe inflammatory cell infiltration and tissue damage.

2.7 ELISA detection

After the last administration and detection, blood was collected from the eyeballs of the rats, and after standing at room temperature for 2 hours, centrifuged at 3000 r/min for 10 min with a low-temperature high-speed centrifuge to recover serum. Follow the official instructions of the ELISA kit, draw a standard curve, and measure the levels of IL-1β, IL-8, and TNF-α in the supernatant.

2.8 Experimental results

Figure 6 is a comparison of the degree of swelling of the ankle joints of rats under different treatment conditions. A: Blank group B: Arthritis group C: Dexamethasone group D: Cefuroxime low dose group E: Cefuroxime medium dose group F: Cefuroxime high dose group;

Figure 7 shows the comparison of toe volumes of rats under different treatment conditions. Different lowercase letters indicate that the significance between the two groups is p<0.01, and the same letter indicates that there is no significance between the two groups;

Figure 8 is the detection of pain thresholds of rats in different treatment groups using mechanical stimulation method. Different lowercase letters indicate that the significance between the two groups is p<0.01, and the same letter indicates that there is no significance between the two groups;

Figure 9 shows the content of (A) IL-1β (B) TNF-α and (C) IL-8 in the serum of three commonly used inflammatory indicators of gouty arthritis in rats using ELISA respectively. Different lowercase letters represent the significance between the two groups at p<0.01, and the same letters represent no significance between the two groups.

Figure 10 shows tissue damage and inflammatory cell infiltration detected by H&E staining of rat ankle joints. A: Blank group B: Arthritis group C: Dexamethasone group D: Cefuroxime low dose group E: Cefuroxime medium dose group F: Cefuroxime high dose group

The results shown in Figures 6 and 7 show that the right ankle swelling of the gouty arthritis model group is significantly higher than that of the blank group, and both dexamethasone and different doses of cefuroxime can relieve the swelling of the ankle joint of the rats. The cefuroxime medium and high dose groups were better than the dexamethasone group, and there was no statistical difference with the blank group.

The experimental results shown in Figure 8 show that the sensitivity of the rats in the model group to mechanical stimulation is significantly lower than that in other groups. The use of different doses of cefuroxime can improve the pain sensitivity threshold, the middle-dose and high-dose treatment groups have the best improvement, and there is no statistical difference with the blank group

The results in Figure 9 show that the three inflammatory indexes of inflammation in the gout model group were significantly increased compared with the normal group. The dexamethasone group as well as the cefuroxime low, medium and high groups were able to reduce these inflammatory markers. And in cefuroxime, high-dose group, the inflammatory indexes were lower than dexamethasone group.

The H&E histopathological staining results of the rat ankle joints in Figure 10 showed that there was no tissue damage and inflammatory cell infiltration in the ankle joints of the rats in the normal group. In the gout model group, the ankle joint tissue of the rats was severely damaged, and there was obvious inflammatory cell infiltration. The dexamethasone group showed decreased inflammatory cell infiltration. Inflammatory cells in the low, medium and high dose groups of cefuroxime were less than those in the model group, and the infiltration of inflammatory cells in the high dose group was the least. And in cefuroxime, the inflammatory cells in the high-dose group were less than those in the dexamethasone group.

Example 3: Evaluation of cefotaxime sodium for injection and its combination with dexamethasone sodium phosphate injection on the treatment and pain relief in mice with chronic inflammatory pain

3. Materials and methods

3.1 Experimental animals and groups

According to the experimental needs, the experimental animals were divided into 7 groups: blank group, model group, dexamethasone group, cefotaxime low-dose group, cefotaxime high-dose group, cefotaxime low + dexamethasone group, cefotaxime High + dexamethasone group.

3.2 Chronic inflammatory pain modeling method

In this example, a mouse model of chronic inflammatory pain was established by injecting Freund's complete adjuvant. After anesthetizing the mice to be modeled, 20 μL of Freund's complete adjuvant solution was injected into the subcutaneous tissue of the left ankle joint to create a chronic inflammatory pain model. After 48 hours of modeling, the ankle joints were observed to have obvious redness and swelling, and the mice moved slowly, indicating that the modeling was successful.

3.3 Method of administration

After the chronic inflammatory pain model was successfully established, the dexamethasone group was injected with 1 mg/kg dexamethasone sodium phosphate injection through the tail vein, once a day, for a total of 7 injections. To the cefotaxime low-dose group and the cefotaxime high-dose group, cefotaxime sodium (Origin Leaf Bio) 200mg/kg and 600mg/kg were injected respectively. Administered once a day, a total of 7 injections. The cefotaxime low + dexamethasone group and the cefotaxime high + dexamethasone group received the same dose as the single drug injection. Cefotaxime sodium for injection uses 0.9% sodium chloride solution as a dissolving agent. The blank group and model group were injected with equal volume of normal saline.

3.4 Animal evaluation of chronic inflammatory pain model

Before modeling and 2 hours after daily administration, the volume value of the same part of the foot of each group of mice was measured with a toe volume meter, and the swelling degree of the ankle joint of the mice was recorded.

3.5 Detection of pain threshold by mechanical stimulation method

From the first day of administration, the pain thresholds of mechanical stimulation in each group were measured 3 hours after each administration. First, the mice were placed in the pain-measuring cage of the mechanical pain meter to adapt for 5 minutes, and the Von-Frey mechanical stimulation needle was used to stimulate the middle of the mouse's sole vertically, and then the reaction of the mouse was recorded. If the mouse appeared rapidly within the stimulation time Activity such as foot bounce, foot retraction or foot licking was recorded as a positive reaction. If the mice did not have such reactions, they were recorded as negative; the mechanical pain threshold of mice was determined and analyzed according to the up-down method.

3.6 Histopathological examination

The mice were sacrificed after anesthesia, and the mouse ankle joint tissues were obtained by dissection, rinsed with normal saline, and subsequently fixed in 10% paraformaldehyde solution. After embedding in paraffin, the tissues were cut into 4-micron sections and stained by H&E method. . The stained sections were observed using an Olympus BX51 fluorescence microscope to observe inflammatory cell infiltration and tissue damage.

3.7 ELISA to detect the level of inflammatory factors

The mice were sacrificed after anesthesia, and blood was collected from the eyeballs of the mice. After standing at room temperature for 2 hours, centrifugation was performed at a speed of 3000 r/min for 10 minutes, and then the serum was recovered. Then, according to the official instructions of the ELISA kit, a standard curve was drawn to measure the levels of TNF-α, IL-6 and IL-1β respectively.

3.8 Experimental results

Figure 11 shows the degree of redness and swelling of the ankle joints of mice in different treatment groups. A: Blank group B: Arthritis group C: Dexamethasone group D: Cefotaxime low dose group E: Cefotaxime medium dose group F: Cefotaxime low + dexamethasone group G: Cefotaxime high + dexamethasone dexamethasone;

Figure 12 shows the volume detection of mouse toes under different treatments. Different lowercase letters indicate that the significance between the two groups is p<0.01, and the same letter indicates that there is no significance between the two groups;

Figure 13 is the detection of pain threshold of mice in different treatment groups using mechanical stimulation method. Different lowercase letters indicate that the significance between the two groups is p<0.01, and the same letter indicates that there is no significance between the two groups;

Figure 14 shows the detection of tissue damage and inflammatory cell infiltration by H&E staining of mouse ankle joints. A: Blank group B: Arthritis group C: Dexamethasone group D: Cefotaxime low dose group E: Cefotaxime medium dose group F: Cefotaxime low + dexamethasone group G: Cefotaxime high + dexamethasone dexamethasone;

Figure 15. The content of three inflammatory markers (A) IL-1β (B) IL-6 and (C) TNF-α in mouse serum were detected by ELISA. Different lowercase letters represent the significance between the two groups at p<0.01, and the same letters represent no significance between the two groups.

It can be seen from Figures 11 and 12 that the feet of the mice in the model group were obviously red and swollen, and the toe volume was significantly higher than that of the other groups. The use of dexamethasone and different doses of cefotaxime can relieve the swelling of the ankle joint and reduce the volume of the toes of the mice. The therapeutic effect of high-dose cefotaxime was better than that of dexamethasone group, and the combined effect of different doses of cefotaxime and dexamethasone was better than that of single use.

The experimental results shown in Figure 13 show that the mice in the model group are significantly less sensitive to mechanical stimulation than other groups. The use of different doses of cefotaxime can improve the pain sensitivity threshold, and the improvement effect is better than that of the dexamethasone group. The combined effect of different doses of cefotaxime and dexamethasone is better than that of single use.

As shown in Figure 14, the H&E histopathological staining results of mouse ankle joints showed that there was no tissue damage and inflammatory cell infiltration in the ankle joints of mice in the normal group. The ankle joint tissue of the mice in the model group was severely damaged, and there was obvious inflammatory cell infiltration. The dexamethasone group showed decreased inflammatory cell infiltration. There were fewer inflammatory cells in the low-dose and high-dose cefotaxime groups than in the model group, and there was less inflammatory cell infiltration in the high-dose group. The combination of different doses of cefotaxime and dexamethasone has better effect on reducing inflammatory cell infiltration than single use.

As shown in the results in Figure 15, the three inflammatory indicators of inflammation in the chronic inflammatory pain model group were significantly increased compared with the normal group. Both the dexamethasone group and the cephalosporin low and high groups significantly reduced these inflammatory markers. In particular, the levels of three inflammatory markers in the high-dose cefotaxime group were lower than those in the dexamethasone group. In addition, the combination of different doses of cefotaxime and dexamethasone has a better effect on reducing inflammatory factor indexes than single use. There was no statistically significant difference between the three inflammatory indexes in the combination of high-dose cefotaxime and dexamethasone and the blank group.

Example 4: Evaluation of the therapeutic and pain relief effects of cefoperazone sodium for injection on mice with sciatica

4 Experimental Materials and Methods

4.1 Experimental animals and groups

Wild-type SPF male BALB/c mice (6-8 weeks old) were purchased from the Experimental Animal Center of Yangzhou University, and were raised in the Experimental Center of Northwestern University under standard rearing conditions, with an ambient temperature of (22±3) °C and a humidity of (55 ±5)%, light: 12h/12h day-night alternation, mice can freely eat and drink.

According to the experimental needs, the experimental animals were divided into 6 groups: blank group, arthritis group, dexamethasone group, cefoperazone low-dose group, cefoperazone medium-dose group, and cefoperazone high-dose group.

4.2 Sciatica modeling method

Anesthetize the mice that need to be modeled, place their backs on the operating table, prepare the skin on the left thigh, apply alcohol for disinfection, cut the skin, and use catgut to ligate the sciatic nerve trunk. Mild muscle tremor response. The ligated sciatic nerve was repositioned, the skin was sutured, and penicillin sodium was injected into the wound of the model mice to prevent infection. After modeling, the mice were reared for 7 days. For the sham operation group, the wound was sutured only after incision of the skin, and no nerve ligation was performed.

4.3 Method of administration

Seven days after modeling, the dexamethasone group was injected with 1 mg/kg dexamethasone sodium phosphate injection through the tail vein, once a day, for a total of 7 injections. Cefoperazone sodium low-dose group, cefoperazone medium-dose group, and cefoperazone high-dose group were injected with cefoperazone sodium (source leaf) 100 mg/kg, 200 mg/kg and 600 mg/kg, respectively. Administered once a day, a total of 7 injections. Cefoperazone sodium for injection uses 0.9% sodium chloride solution as a dissolving agent. The blank group and model group were injected with equal volume of normal saline.

4.4 Detection of pain threshold by mechanical stimulation method

From the first day of administration, the pain thresholds of mechanical stimulation in each group were measured 3 hours after each administration. First, the mice were placed in the pain-measuring cage of the mechanical pain meter to adapt for 5 minutes, and the Von-Frey mechanical stimulation needle was used to stimulate the middle of the mouse's sole vertically, and then the reaction of the mouse was recorded. If the mouse appeared rapidly within the stimulation time Activity such as foot bounce, foot retraction or foot licking was recorded as a positive reaction. If the mice did not show such response, it was recorded as negative; the pain threshold was determined and analyzed according to the up-down method.

4.5 Detection of inflammatory factor levels by ELISA

After the last administration and detection, blood was collected from the eyeballs of the mice, left standing at room temperature for 2 hours, and then centrifuged at 3000 rpm for 10 minutes to recover serum. Then, according to the official instructions of the ELISA kit, a standard curve was made to measure the levels of IL-1β, IL-6 and TNF-α respectively.

4.6 Experimental results

Figure 16 shows the pain threshold of mice detected by mechanical stimulation under different treatment conditions. Different lowercase letters represent significant p<0.01 between the two groups, and the same letter represents no significance between the two groups

Figure 17. Detecting the content of three inflammatory indexes (A) IL-1β (B) IL-6 and (C) TNF-α in the serum of mice of different groups by ELISA. Different lowercase letters represent the significance between the two groups at p<0.01, and the same letters represent no significance between the two groups.

The experimental results shown in Figure 16 show that the mice in the model group are significantly less sensitive to mechanical stimulation than other groups. The use of different doses of cefoperazone can improve the pain sensitivity threshold, and the middle-dose and high-dose treatment groups have the best improvement, and there is no statistical difference with the blank group.

As shown in Figure 17, the three inflammatory indicators of inflammation in the sciatica model group were significantly increased compared with the normal group. The dexamethasone group as well as the cefoperazone low, medium and high groups all reduced these inflammatory markers. In particular, the cefoperazone high-dose group had the lowest levels of three inflammatory markers. And in the high-dose cefoperazone group, the inflammatory indexes were lower than those in the dexamethasone group.

Example 5: Evaluation of the therapeutic and pain relief effects of ceftazidime for injection in mice with ankylosing spondylitis

5. Materials and methods

5.1 Experimental animals and groups

Wild-type SPF male BALB/c mice (6-8 weeks old) were purchased from the Experimental Animal Center of Yangzhou University, and were raised in Northwestern University under standard rearing conditions. The ambient temperature was (22±3)°C, and the humidity was (55±5) %, light: 12h/12h day-night alternation, the mice can eat and drink freely.

According to the experimental needs, the experimental animals were divided into 6 groups: blank group, arthritis group, dexamethasone group, ceftazidime low-dose group, ceftazidime medium-dose group, and ceftazidime high-dose group.

5.2 Modeling method of ankylosing spondylitis

First, 75 μg of proteoglycan and 150 μL of Freund's complete adjuvant were taken, and the two were emulsified, shaken and mixed. On day 0, mice that need to be modeled were injected intraperitoneally. On the 7th day, 75 μg of proteoglycan and 150 μL of incomplete Freund’s adjuvant were emulsified and shaken to mix, and injected into mice intraperitoneally for boosting immunization. One week after the last intraperitoneal injection, an ankylosing spondylitis mouse model was formed.

5.3 Method of administration

After the ankylosing spondylitis mouse model was established, the dexamethasone group was injected with 1 mg/kg dexamethasone sodium phosphate injection through the tail vein, once a day, for a total of 7 injections. The ceftazidime low-dose group, ceftazidime medium-dose group, and ceftazidime high-dose group were injected with ceftazidime (Original Bio) 100 mg/kg, 200 mg/kg and 600 mg/kg, respectively. Administered once a day, a total of 7 injections. Ceftazidime for injection uses 0.9% sodium chloride solution as a dissolving agent. The blank group and model group were injected with equal volume of normal saline.

5.4 Detection of Pain Threshold by Mechanical Stimulation

From the first day of administration, the pain thresholds of mechanical stimulation in each group were measured 3 hours after each administration. First, the mice were placed in the pain measuring cage of the mechanical pain measuring instrument for 20 minutes, and the Von-Frey mechanical stimulation needle was used to stimulate the middle of the mouse's sole vertically, and then the reaction of the mouse was recorded. If the mouse appeared quickly within the stimulation time Activity such as foot bounce, foot retraction or foot licking was recorded as a positive reaction. If the mouse did not show such a reaction, it was recorded as negative; the method was based on Dixon's up-down method to measure and analyze the paw withdrawal threshold 50%.

5.5 Detection of inflammatory factor levels by ELISA

The mice were sacrificed after anesthesia, and blood was collected from the eyeballs of the mice. After standing at room temperature for 2 hours, centrifugation was performed at a speed of 3000 r/min for 10 minutes, and then the serum was recovered. Then, the levels of IL-1β, IL-6 and TNF-α were measured according to the official instructions of the ELISA kit.

5.6 Experimental results

Figure 18. Detection of pain thresholds in mice of different treatment groups using mechanical stimulation. Different lowercase letters indicate that the significance between the two groups is p<0.01, and the same letter indicates that there is no significance between the two groups;

Figure 19. Using ELISA to detect the content of three inflammatory indexes (A) IL-6 (B) TNF-α and (C) IL-1β in the serum of mice of different groups respectively. Different lowercase letters represent the significance between the two groups at p<0.01, and the same letters represent no significance between the two groups.

The experimental results shown in Figure 18 show that the mice in the model group are significantly less sensitive to mechanical stimulation than other groups. The use of different doses of ceftazidime and dexamethasone can improve the pain sensitivity threshold, and the middle-dose and high-dose ceftazidime treatment groups have the best improvement, and there is no statistical difference with the blank group.

As shown in Figure 19, the three inflammatory indexes of the mice in the ankylosing spondylitis model group were significantly increased compared with those in the normal group. The dexamethasone group, as well as the ceftazidime low, medium, and high groups, reduced these inflammatory markers. In particular, the content of three inflammatory indexes in the high-dose ceftazidime group was the lowest, and the ceftazidime medium and high-dose groups had lower inflammatory indexes than the dexamethasone group.

The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments, and the same and similar parts between the various embodiments can be referred to each other. As for the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant part can be referred to the description of the method.

The above description of the disclosed embodiments enables any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

Application of cephalosporin antibiotics and their compositions with steroidal anti-inflammatory drugs in the preparation of medicines for treating spinal and osteoarticular lesions and alleviating their pain; said cephalosporin antibiotics and steroidal anti-inflammatory drugs are both The dosage form for injection is used for treating the lesions of the spine and the bone joints; the lesions of the spine and the bone joints are all aseptic lesions.
The application according to claim 1, characterized in that, the cephalosporin antibiotics are all in the form of injection: after being dissolved in physiological saline, they can be administered by intravenous drip, intravenous injection, and local lesion injection.
The application according to claim 1, wherein the steroid anti-inflammatory drugs are all in the form of injection: after being dissolved in physiological saline, they can be administered by intravenous drip, intravenous injection, or local lesion injection.
The application according to claim 2, wherein the cephalosporin antibiotic is selected from any one of the following or a pharmaceutically acceptable salt thereof: cefotaxime, cefradine, cephalexin, cefotaxime, cefaacetonitrile, Cefpirin, Ceftiam, Cefmetazole, Cefoxitin, Cefazoxime, Cefmenoxime, Cefpiramide, Cefotetan, Laoxef, Cefminox, Cefazolam, Cefthiamidine, Cefizole Lin, cefuroxime, cefamandole, ceftizole, cefotaxime, ceftazidime, cefoperazone, ceftriaxone, cefepime, cefpirome, cefodizime, cefonicid.
The application according to claim 3, wherein the steroid anti-inflammatory drug is selected from any one of dexamethasone, methylprednisolone, betamethasone, hydrocortisone, triamcinolone acetonide or its medicine Academically acceptable salt.
The use according to claim 1, wherein the aseptic lesions comprise lumbar disc herniation, cervical spondylosis, thoracic spondylosis, uncinate arthritis, posterior lumbar arthritis, ankylosing spondylitis, and myofasciitis , Osteoporosis, Carpal Tunnel Syndrome, Flexor Tenosynovitis, Lateral Epicondylitis, Medial Epicondylitis, Shoulder Periarthritis, Hip Synovitis, Aseptic Femoral Head Necrosis, Knee Arthritis, Ankle Joint damage, heel inflammation, rheumatoid arthritis, gouty arthritis.
A medicament for treating vertebral and bone and joint lesions and alleviating their pain is characterized in that, it is composed of the following components: cephalosporin antibiotics and combinations thereof with steroidal anti-inflammatory drugs; the cephalosporins and Steroidal anti-inflammatory drugs are all in the form of injection, which are used for spinal and bone and joint diseases.
A kind of medicine for treating spine and bone and joint disease and alleviating its pain according to claim 7, it is characterized in that, described cephalosporin antibiotics are all injection dosage forms: after being dissolved in normal saline, can be dripped intravenously Injection, intravenous injection, local lesion injection administration.
The medicament for treating vertebral column and osteoarthropathy and relieving pain according to claim 7, characterized in that, the steroid anti-inflammatory drugs are all in the form of injection: after being dissolved in physiological saline, they can be administered intravenously Instillation, intravenous injection, local lesion injection administration.
A kind of medicine for treating vertebral column and osteoarthropathy and alleviating its pain according to claim 8, is characterized in that, described cephalosporin antibiotic is selected from following any one or its pharmaceutically acceptable salt: Cefotaxime, Cefradine, Cefalexin, Cefotaxime, Cefacetonitrile, Cefpirin, Ceftiam, Cefmetazole, Cefoxitin, Cefizoxime, Cefmenoxime, Cefpiramide, Cefotetan , cefminox, cefazolam, cefathiamidine, cefazolin, cefuroxime, cefamandole, ceftizole, cefotaxime, ceftazidime, cefoperazone, ceftriaxone, cefepime, cefpirome , cefodizime, cefnicil.
A kind of medicine for treating vertebral column and osteoarthropathy and alleviating its pain according to claim 9, is characterized in that, described steroid anti-inflammatory drug is selected from dexamethasone, methylprednisolone, betamethasone, Any one of hydrocortisone, triamcinolone acetonide or a pharmaceutically acceptable salt thereof.
The medicament for treating spinal and bone joint lesions and alleviating their pain according to claim 7, wherein the spinal and bone joint lesions are aseptic lesions.
The medicament for treating vertebral and osteoarticular pathologies and alleviating their pain according to claim 12, wherein the aseptic pathology comprises lumbar disc herniation, cervical spondylosis, thoracic spondylosis, and uncinate arthritis , posterior lumbar arthritis, ankylosing spondylitis, myofasciitis, osteoporosis, carpal tunnel syndrome, flexor tenosynovitis, lateral epicondylitis, medial humeral epicondylitis, shoulder periarthritis, hip joint slip Meningitis, aseptic necrosis of the femoral head, knee arthritis, ankle injury, heel inflammation, rheumatoid arthritis, gouty arthritis.