New application of amiodarone or amiodarone salt

Abstract

The invention discloses an application of amiodarone or amiodarone salt in preparation of an intra-articular injection medicine for treating knee osteoarthritis pain, and also discloses a pharmaceutical composition containing amiodarone or amiodarone salt. The amiodarone articular cavity injection is developed, and the curative effect on pain symptoms caused by knee osteoarthritis is definite; the cost is low, the tolerance is good, and the medicine is accepted by patients. The invention develops a new application of the traditional arrhythmia medicine amiodarone, is safe under an effective dose, and has no obvious side effect on articular cartilage.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a new use of amiodarone or a salt of amiodarone, and also to a pharmaceutical composition containing amiodarone or a salt of amiodarone. Background Art

[0002] Osteoarthritis (OA) is one of the major health problems facing society today. It is estimated that around 240 million people suffer from OA worldwide. OA not only causes pain in patients, but also leads to functional loss and decreased quality of life as the disease progresses. OA mainly occurs in the hip joints, knee joints, and small joints of the hands. The prevalence of knee osteoarthritis (KOA) with painful symptoms in people over 45 years old in my country is 8.1%, which is the most common OA clinically. KOA seriously affects the health and quality of life of patients, causing huge economic and social burdens.

[0003] Intra-articular injection has many advantages over systemic administration. Compared with systemic administration, intra-articular administration has advantages including increased local bioavailability, reduced systemic exposure, reduced adverse events and reduced costs. In addition, the entire joint is involved in the course of KOA, and cartilage, synovium, muscles and ligaments all play an important role. Therefore, intra-articular administration, that is, concentrating the therapeutic dose in the joint capsule, may be the ideal mode of administration for the treatment of KOA. At present, the overall goal of clinical drug treatment of KOA is to relieve patient pain and protect articular cartilage. However, the clinical selection of analgesics for intra-articular injection is very limited. In addition, existing clinical drugs still have problems with safety and efficacy. For example, studies have shown that local anesthetics such as lidocaine and bupivacaine have certain chondrotoxicity to articular chondrocytes.

[0004] Amiodarone is currently one of the most effective drugs for the treatment of life-threatening arrhythmias. Amiodarone is classified as a Vaughan-Williams class III antiarrhythmic drug, but it is a multichannel blocker with properties covering all four classes of drugs. Previous studies have shown that amiodarone exhibits lidocaine-like inhibitory activity on voltage-gated sodium channels. At the same time, the protein binding rate of amiodarone is close to or higher than that of bupivacaine (96%), and its distribution coefficient value is very high (>100,000). However, there have been no reports that amiodarone can be used to relieve the pain symptoms caused by KOA. Summary of the invention

[0005] The purpose of the present invention is to address the deficiencies in the prior art, provide the use of amiodarone or a salt of amiodarone in the preparation of an intra-articular injection drug for treating knee osteoarthritis pain, and also provide a pharmaceutical composition containing amiodarone or a salt of amiodarone.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect is to provide the use of amiodarone or a salt of amiodarone in the preparation of an intra-articular injection drug for treating knee osteoarthritis pain.

[0008] The second aspect is to provide a pharmaceutical composition, which contains amiodarone or a salt of amiodarone.

[0009] Furthermore, the salt of amiodarone is amiodarone hydrochloride.

[0010] Furthermore, the pharmaceutical composition also contains molecules for enhancing the extracellular matrix, and the weight ratio of the amiodarone or the salt of amiodarone to the molecules for enhancing the extracellular matrix is ​​1:1 to 1:5.

[0011] Furthermore, the molecules that enhance the extracellular matrix are collagen or glycosaminoglycan.

[0012] Furthermore, the pharmaceutical composition also contains an active substance, and the weight ratio of the amiodarone or the salt of amiodarone to the active substance is 1:1 to 1:5.

[0013] Furthermore, the active substance is a growth factor or a hormone.

[0014] Furthermore, the pharmaceutical composition also contains an excipient, and the weight ratio of the amiodarone or the salt of amiodarone to the excipient is 1:50 to 1:200.

[0015] Furthermore, the dosage form of the pharmaceutical composition is an injection.

[0016] The present invention will be further described below:

[0017] The amiodarone or a pharmaceutically acceptable salt thereof of the present invention is formulated into a dosage form suitable for intra-articular injection and is used for relieving the pain symptoms of knee osteoarthritis. It can effectively relieve the pain symptoms caused by knee osteoarthritis and has less side effects. Amiodarone or a pharmaceutically acceptable salt thereof and one or more other pharmaceutically acceptable carriers / excipients are used to prepare a pharmaceutical composition for treating knee osteoarthritis pain, preferably in a form suitable for intra-articular injection such as a solution and a gel.

[0018] For preparing liquid forms suitable for injection, water is the preferred solvent.

[0019] If desired, it is possible to combine amiodarone or a pharmaceutically acceptable salt thereof with other molecules useful for strengthening the extracellular matrix, such as collagen or glycosaminoglycans, or with other pharmacologically and / or biologically active substances, such as growth factors, hormones.

[0020] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0021] 1) The present invention develops an intra-articular injection formulation of amiodarone, which has a definite therapeutic effect on the pain symptoms caused by knee osteoarthritis; it is low in cost, well tolerated, and has been accepted by patients.

[0022] 2) The present invention explores a new use of the traditional arrhythmia drug amiodarone, which is safe at an effective dose and has no obvious side effects on articular cartilage. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the effect of intra-articular injection of normal saline, intra-articular injection of amiodarone and intra-articular injection of bupivacaine on the mechanical withdrawal reflex threshold of the affected side of OA rats 7 days after modeling in Example 1.

[0024] Figure 2 This is the effect of intra-articular injection of normal saline, intra-articular injection of amiodarone and intra-articular injection of bupivacaine on the mechanical withdrawal reflex threshold of the affected side of OA rats 21 days after modeling in Example 1.

[0025] Figure 3 This is the effect of intra-articular injection of normal saline, intra-articular injection of amiodarone and intra-articular injection of bupivacaine on the total intensity percentage of the affected side paw print of OA rats 7 days after modeling in Example 1.

[0026] Figure 4 This is the effect of intra-articular injection of normal saline, intra-articular injection of amiodarone and intra-articular injection of bupivacaine on the total intensity percentage of the affected side paw print of OA rats 21 days after modeling in Example 1.

[0027] Figure 5 This is the effect of intra-articular injection of normal saline, intra-articular injection of amiodarone and intra-articular injection of bupivacaine on the affected side Knee-Bend score of OA rats 7 days after modeling in Example 1.

[0028] Figure 6 This is the effect of intra-articular injection of normal saline, intra-articular injection of amiodarone and intra-articular injection of bupivacaine on the affected side Knee-Bend score of OA rats 21 days after modeling in Example 1.

[0029] Figure 7 This is the effect of 0.9% saline, 0.5% amiodarone treatment solution and 0.5% bupivacaine treatment solution on chondrocyte activity in Example 2. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0031] Drugs and reagents: Amiodarone was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., monosodium iodoacetate (MIA) was purchased from Sigma-Aldrich, and bupivacaine was purchased from Sigma-Aldrich. Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0032] Example 1 Animal behavior experiment verifies that amiodarone effectively relieves the pain symptoms of knee osteoarthritis

[0033] 1. Materials and Methods

[0034] 1.1 Experimental animals

[0035] Twenty-four 6-week-old male Sprague-Dawley rats were selected for the experiment. All rats were housed in the experimental animal room of Shanghai Sixth People's Hospital, fed with SPF-grade standard feed, and had free access to food and water.

[0036] 1.2 Experimental drugs

[0037] Amiodarone was dissolved in 80°C water and cooled to 20°C, then diluted to 0.5%. Bupivacaine was dissolved in saline and diluted to 0.5%. The drugs were prepared as needed.

[0038] 1.3 Experimental methods

[0039] 1.3.1 Animal modeling

[0040] Rats were anesthetized with 1-2% isoflurane, the left leg was shaved, and the skin was disinfected with iodine. A single intra-articular injection of 5 mg sodium iodoacetate in a total volume of 30 μl saline solution was given through the patellar ligament of the left knee using a 1 cc syringe with a 25G needle.

[0041] 1.3.2 Grouping and intervention methods

[0042] The rats were randomly divided into the following three groups.

[0043] 1) Left knee intra-articular injection of normal saline group: 7 days after the establishment of the MIA injection rat model, normal saline (20 μl) was injected into the left knee intra-articular cavity once a week for 3 consecutive weeks;

[0044] 2) Amiodarone injection group: 7 days after the establishment of the MIA injection model, 0.5% amiodarone (20 μl) was injected into the left knee joint cavity once a week for 3 consecutive weeks;

[0045] 3) Left knee intra-articular injection of bupivacaine group: 7 days after the establishment of the MIA injection rat model, 0.5% bupivacaine (20 μl) was injected into the joint cavity once a week for 3 consecutive weeks;

[0046] 1.3.3 Evaluation methods

[0047] 1.3.3.1 Pain assessment in rats

[0048] The rats in each group were evaluated for pain on the 7th and 21st day after injection. The von Frey analgesia was used to evaluate the mechanical allodynia on the affected side of the rats before and 30min, 60min, 120min, 180min, and 240min after drug injection. The gait parameters and Knee-Bend test were used to evaluate the pain sensation induced by movement on the affected side at 40min.

[0049] 1) Mechanical withdrawal reflex threshold

[0050] Mechanical pain threshold was measured using the von Frey analgesia instrument:

[0051] a. Place the rats in a clean plexiglass box with a wire mesh bottom and allow them to adapt to the environment for 15-30 minutes (until the rats stop foraging for food, playing around, etc. in the cage);

[0052] b. The stimulating bending force is 0.41 to 15.10 g, from small to large;

[0053] c. Each time lasts for 3 seconds. If the rat lifts its foot, licks its foot, or avoids, it is considered a positive reaction; otherwise, it is considered a negative reaction.

[0054] d. If the mice and rats do not show the above reaction, change to a needle with a higher strength; if a positive reaction occurs, give a needle with a lower strength;

[0055] e. The mechanical withdrawal reflex threshold of the affected side was measured according to the up-and-down method proposed by Dixon et al.

[0056] 2) Gait analysis

[0057] Gait analysis using the Catwalk test:

[0058] a. Place the rat on a glass platform for illumination so that light is reflected downward only at the point where the paw contacts the glass surface, thereby obtaining a clear and bright image of the paw print;

[0059] b. The platform is monitored by a camera equipped with a wide-angle objective lens. The camera is installed under the glass platform and connected to a computer equipped with video acquisition software;

[0060] c. The footprints captured by a high-speed camera placed under the glass floor were analyzed using VisuGait software (Xinruan, Shanghai, China). By analyzing the area and pressure applied by each paw of the rat, the load-bearing of different paws during the movement of the animals can be compared. The results are expressed as the percentage of the total intensity of the paw print of the affected hindlimb (left hindlimb) to the total intensity of both hindlimbs.

[0061] d. Measure three times continuously, with an interval of 3 minutes between each measurement, and take the average value.

[0062] 3) Knee-Bend score

[0063] The Knee-Bend test was used to determine the Knee-Bend score:

[0064] a. Knee-Bend test involves counting the screaming and / or struggling response during five alternating flexion and extension of the knee joint on the affected side of the rat within the physiological limits of flexion / extension;

[0065] b. Scoring criteria are:

[0066] 0-no response to any type of knee movement;

[0067] 0.5—struggle during maximum flexion / extension;

[0068] 1-struggling in moderate flexion / extension or screaming in maximum flexion / extension;

[0069] 2-Screaming during moderate flexion / extension.

[0070] 2. Experimental results

[0071] Results Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6

[0072] 3. Results Analysis

[0073] 3.1 Mechanical withdrawal reflex threshold

[0074] like Figure 1As shown in the data, on Day 7 (early and inflammatory stage of knee osteoarthritis), the withdrawal reflex thresholds of the affected side of the amiodarone group were greater than those of the saline group at 30 min, 60 min, 120 min, 180 min, and 240 min (P < 0.001), the withdrawal reflex thresholds of the affected side of the amiodarone group were greater than those of the bupivacaine group at 60 min (P < 0.01), and the withdrawal reflex thresholds of the affected side of the amiodarone group were greater than those of the bupivacaine group at 120 min, 180 min, and 240 min (P < 0.001). Figure 2 As shown, on Day 21 (knee osteoarthritis was chronic and fully established), the paw withdrawal reflex thresholds of the affected side of the amiodarone intra-articular injection group were greater than those of the saline intra-articular injection group at 30min, 60min, 120min, 180min, and 240min (P < 0.001), and the paw withdrawal reflex thresholds of the affected side of the amiodarone intra-articular injection group were greater than those of the bupivacaine intra-articular injection group at 120min, 180min, and 240min (P < 0.001).

[0075] 3.2 Gait analysis

[0076] like Figure 3 As shown in the figure, on Day 7 (early and inflammatory stage of knee osteoarthritis), there was no significant difference in the total intensity percentage of the affected side paw prints in the amiodarone group before injection, compared with the saline group and the bupivacaine group; 40 minutes after injection, the total intensity percentage of the affected side paw prints in the amiodarone group was greater than that in the saline group (P < 0.001), and the total intensity percentage of the affected side paw prints in the amiodarone group was greater than that in the bupivacaine group (P < 0.05); Figure 4 As shown, on Day 21 (knee osteoarthritis was chronic and fully established), there was no significant difference in the total intensity percentage of the paw print on the affected side between the amiodarone group and the saline group or the bupivacaine group before injection; 40 minutes after injection, the total intensity percentage of the paw print on the affected side between the amiodarone group and the saline group was greater than that between the bupivacaine group (P < 0.001); there was no significant difference in the total intensity percentage of the paw print on the affected side between the amiodarone group and the bupivacaine group.

[0077] 3.3Knee-Bend score

[0078] like Figure 5As shown in the data, on Day 7 (early and inflammatory stage of knee osteoarthritis), there was no significant difference in the Knee-Bend score of the affected side between the amiodarone group and the saline group or the bupivacaine group before injection; 40 minutes after injection, the Knee-Bend score of the affected side between the amiodarone group and the bupivacaine group was lower than that between the saline group (P < 0.001); there was no significant difference in the Knee-Bend score of the affected side between the amiodarone group and the bupivacaine group; Figure 6 As shown in the data, on Day 21 (when knee osteoarthritis was chronic and fully established), there was no significant difference in the Knee-Bend score of the affected side between the amiodarone group and the saline group or the bupivacaine group before injection; 40 minutes after injection, the Knee-Bend score of the affected side between the amiodarone group and the bupivacaine group was lower than that between the saline group (P < 0.001); there was no significant difference in the Knee-Bend score of the affected side between the amiodarone group and the bupivacaine group.

[0079] Example 2 Cell experiment verifies that the effect of amiodarone on rat articular chondrocyte viability is lower than other local anesthetics

[0080] 1. Materials and Methods

[0081] 1.2 Experimental cells

[0082] This study used 3-week-old rat knee chondrocytes. Rat chondrocytes were obtained from the hip, knee and ankle cartilage of 3-week-old rats, and primary rat chondrocytes were cultured.

[0083] 1.3 Cellular intervention

[0084] The first generation of rat knee chondrocytes were cultured in DMEM-F12 medium containing fetal bovine serum (10%) and double antibiotics (penicillin 50U / ml, streptomycin 50U / ml), and placed in a CO2 incubator (37°C constant temperature, 5% CO2, 95% air, 100% humidity). They were first incubated in DMEM / F12 containing 5% FBS for 24 hours to synchronize the cells in the non-proliferation and inactive phases. When the cells grew to 70%-80%, the medium was removed and intervention was performed according to the following groups:

[0085] a. Chondrocyte routine culture control group: treated with 0.9% saline solution, and then placed the sample back in the CO2 incubator for 1 hour. After 1 hour, the treatment solution was aspirated and 10% FBS DMEM / F12 complete medium was added, and the sample was returned to the incubator. After a 24-hour incubation period, the cell viability was determined by flow cytometry;

[0086] b. 0.5% amiodarone chondrocyte intervention group: 200 μL 0.5% amiodarone treatment solution was given, and then the sample was returned to the CO2 incubator for incubation for 1 hour. After 1 hour, the treatment solution was aspirated and 10% FBS DMEM / F12 complete medium was added, and the sample was returned to the incubator. After a 24-hour incubation period, the cell viability was determined by flow cytometry;

[0087] c. 0.5% bupivacaine chondrocyte intervention group: 200 μL 0.5% bupivacaine treatment solution was given, and then the sample was returned to the CO2 incubator for incubation for 1 hour. After 1 hour, the treatment solution was aspirated and 10% FBS DMEM / F12 complete medium was added, and the sample was returned to the incubator. After a 24-hour incubation period, cell viability was determined using flow cytometry.

[0088] 2. Experimental results

[0089] Results Figure 7 .

[0090] 3. Results Analysis

[0091] 3.1 Chondrocyte apoptosis rate

[0092] The experimental results showed that the 0.5% amiodarone group was relatively lacking in chondrocyte toxicity, while the 0.5% bupivacaine group showed a higher level of chondrocyte death. However, compared with the conventional chondrocyte culture control group, both 0.5% bupivacaine and 0.5% amiodarone showed lower chondrocyte viability.

[0093] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of amiodarone or its salt in the preparation of intra-articular injection drugs for treating knee osteoarthritis pain.

2. A pharmaceutical composition, characterized in that The pharmaceutical composition contains amiodarone or a salt of amiodarone.

3. The pharmaceutical composition according to claim 2, characterized in that The amiodarone salt is amiodarone hydrochloride.

4. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition also contains molecules for enhancing the extracellular matrix, and the weight ratio of the amiodarone or the amiodarone salt to the molecules for enhancing the extracellular matrix is ​​1:1 to 1:

5.

5. The pharmaceutical composition according to claim 3, characterized in that The molecules that enhance the extracellular matrix are collagen or glycosaminoglycan.

6. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition also contains an active substance, and the weight ratio of the amiodarone or the salt of amiodarone to the active substance is 1:1 to 1:

5.

7. The pharmaceutical composition according to claim 6, characterized in that The active substance is a growth factor or a hormone.

8. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition also contains an excipient, and the weight ratio of the amiodarone or the salt of amiodarone to the excipient is 1:50 to 1:

200.

9. The pharmaceutical composition according to claim 2, characterized in that The dosage form of the pharmaceutical composition is injection.

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