Composition for intra-articular injection in dogs or cats containing sodium polynucleotide

Abstract

The present invention relates to a composition for intra-articular injection in dogs or cats, which is used to treat or ameliorate arthritis or reduce mechanical friction at joint sites by physical repair when injected into the joint cavity of dogs or cats. The present invention also relates to a method for administering a composition for intra-articular injection in dogs or cats, which is used to treat or ameliorate arthritis or reduce mechanical friction at joint sites by physical repair when injected into the joint cavity of dogs or cats. The composition of the present invention is administered so that the dosage of polynucleotide sodium is 2 to 3 mg, preferably 2.5 to 3 mg, per kg of body weight of the dog or cat.

Description

[Technical Field]

[0001] The present invention relates to a composition for intra-articular injection in dogs or cats, comprising sodium polynucleotide. The present invention also relates to a method for administering a composition for intra-articular injection in dogs or cats, comprising sodium polynucleotide. The compositions and methods of the present invention are useful for treating or ameliorating arthritis in dogs or cats, and are also useful for reducing mechanical friction at joint sites through physical repair. [Background technology]

[0002] Osteoarthritis, the most common joint disease, is also known as degenerative joint disease or osteoarthritis. It is a disease caused by gradual damage and degenerative changes to the cartilage that protects the joint, resulting in damage to the bones and ligaments that make up the joint, and inflammation of the synovial membrane surrounding the joint, resulting in pain and deformation. Osteoarthritis progresses through a process in which cartilage tissue in joints such as the knee, ankle, finger, elbow, and hip joint gradually degenerates and is destroyed due to factors such as aging, obesity, joint trauma, hip dysplasia, a history of arthritis, and genetic factors.

[0003] Osteoarthritis is increasingly affecting pet dogs and cats. It is an age-related disease that is common in older dogs and cats. Symptoms vary, but primarily include a reluctance to move, abnormal gait, raising of the legs during walks or exercise, and in severe cases, the animal may even sit down.

[0004] Conventional drug therapies are effective in improving arthritis in pet dogs and cats. For example, nonsteroidal anti-inflammatory drugs (NSAIDs) have recently been widely used to relieve pain and prevent or treat inflammation. However, these NSAIDs can cause bloody vomiting, bloody stools, anemia, and other symptoms due to gastrointestinal ulcers, and also pose a risk of renal failure.

[0005] Therefore, there is a need for a formulation that is effective in improving arthritis and has few side effects when administered to pet dogs and cats. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, the problem to be solved by the present invention is to provide a preparation that has few side effects when administered to pet dogs and cats, particularly pet dogs, and is effective in improving arthritis.

[0007] Another problem to be solved by the present invention is to provide a method for administering polynucleotide sodium to pet dogs and cats, particularly pet dogs, which has few side effects when administered and is effective in improving arthritis. [Means for solving the problem]

[0008] In order to solve the above-mentioned problems, one aspect of the present invention provides a composition for treating or ameliorating arthritis in dogs or cats, preferably dogs, or reducing mechanical friction at joint sites through physical repair, by injecting polynucleotide sodium into the joint cavity of the dog or cat, characterized in that the polynucleotide sodium is administered at a dose of 2 to 3 mg per kg of body weight of the dog or cat.

[0009] Another aspect of the present invention provides an administration method for a composition for treating or ameliorating arthritis in a dog or cat, preferably a dog, or reducing mechanical friction at a joint site through physical repair, by injecting polynucleotide sodium into the joint cavity of the dog or cat, preferably a dog, characterized in that the polynucleotide sodium is administered at a dose of 2 to 3 mg per kg of the dog or cat's body weight.

[0010] In a preferred embodiment of the present invention, the composition and administration method of the present invention administers 2.5 to 3 mg of polynucleotide sodium per kg of dog or cat body weight. In a more preferred embodiment of the present invention, the composition and administration method of the present invention administers 2.5 mg of polynucleotide sodium per 10,000 kg of dog or cat body weight.

[0011] In a preferred embodiment of the present invention, the composition of the present invention is a liquid composition containing 20 mg / ml of polynucleotide sodium. In another preferred embodiment of the present invention, the administration method of the present invention administers a liquid composition containing 20 mg / ml of polynucleotide sodium.

[0012] In a preferred embodiment of the present invention, the liquid composition containing 20 mg / ml of polynucleotide sodium is administered to a dog or cat at a dose of 0.1 to 0.15 ml, preferably 0.125 to 0.15 ml, per kg of body weight. In a more preferred embodiment of the present invention, the liquid composition is administered at a dose of 0.125 ml per kg of body weight.

[0013] The composition of the present invention is administered into the joint cavity of a dog or cat, preferably at the aforementioned dose of 2 to 3 mg of polynucleotide sodium per kg of body weight (more preferably 2.5 to 3 mg per kg of body weight). Taking into consideration the administration volume in particular, it is preferable to inject a liquid composition containing 20 mg / ml of polynucleotide sodium into the joint cavity to achieve this dose.

[0014] The sodium polynucleotide according to the present invention is a DNA fragment extracted from the testes of salmonid fish, and is a transparent liquid substance having a dotted shape. For example, such sodium polynucleotide can be prepared by the method described in Korean Patent Registration No. 10-0986603. In one embodiment of the present invention, the average molecular formula of the deoxyribonucleotides constituting the DNA fragment mixture of the sodium polynucleotide is C 9.83 H 12.33 N 3.72 O 6.01P·Na. In one embodiment of the present invention, the molecular weight of the sodium polynucleotide exhibits a maximum distribution of 250 to 1,000 base pairs (bp) when tested using electrophoresis, and when absorbance is measured, it exhibits an absorption maximum at 260±2 nm and an absorbance ratio of 1.6 to 2.0 at 260 nm and 280 nm. Furthermore, the pH of a 1 wt % aqueous solution of the sodium polynucleotide according to the present invention is 6.5 to 7.5.

[0015] In one embodiment of the present invention, the content of polynucleotide sodium contained in the composition of the present invention is 20 mg / ml, and the composition of the present invention has a viscosity of 100 to 300 Pas when measured by the viscosity measurement method of the general test method in the Korean Pharmacopoeia, and an elasticity of 50 to 250 Pa when measured under the conditions of geometry: PU20, temperature: 25°C, and frequency: 0.1 Hz.

[0016] In one embodiment of the present invention, Conjuran (trademark), a liquid composition containing 20 mg / ml of sodium polynucleotide, may be used as the composition of the present invention.

[0017] In another aspect of the present invention, the polynucleotide sodium composition can be prepared by the method described in Korean Patent Publication No. 10-2017-0100236, the entire contents of which are incorporated herein by reference.

[0018] In one embodiment of the present invention, the composition of the present invention is administered into the articular cavity a total of 2 to 4 times at intervals of 2 to 4 weeks. In a preferred embodiment of the present invention, the composition of the present invention is administered into the articular cavity a total of 2 times at intervals of 2 weeks. [Effects of the Invention]

[0019] The present invention provides a composition for intra-articular injection in dogs or cats, preferably dogs, which is used to treat or improve arthritis or reduce mechanical friction at joint sites by injecting it into the joint cavity. In particular, the composition of the present invention is administered so that the dose of polynucleotide sodium is 2 to 3 mg, preferably 2.5 to 3 mg, more preferably 2.5 mg per kg of body weight of the dog or cat. [Brief explanation of the drawings]

[0020] [Figure 1] 10 is a photograph showing administration of a 20 mg / ml polynucleotide sodium preparation into the joint cavity of a pet dog showing symptoms of arthritis. [Figure 2] 1 shows the behavioral assessment results, which show the behavioral scale scores of the affected knees of dogs in the saline and polynucleotide sodium administration groups during the 30-day study period. [Figure 3] 1 shows the results of behavioral evaluation, including the standing scale evaluation results. These are the standing scale values ​​of the affected knees of dogs in the saline and polynucleotide sodium administration groups during the 30-day study period. [Figure 4] The results of the pain relief evaluation show the evaluation results of "degree of pain complaints on palpation and movement." The behavioral and standing scale scores of the affected knees of dogs in the saline and polynucleotide sodium administration groups during the 30-day study period. [Figure 5] Figure 1 shows the results of the pain relief evaluation, "degree of weight bearing support of the affected hind limb." The standing scale and standing scale values ​​of the affected knee of dogs in the saline and polynucleotide sodium treatment groups during the 30-day study period. [Figure 6]The results show the effects of the test substance on serum IL-1β concentrations in osteoarthritic dogs 48 weeks after the first intra-articular injection. In Figures 2 to 6, the bar graphs show, from left to right, G1 (NC) (no bar), G2 (OA), G3 (0.5), G4 (1.5), G5 (2.5), and G6 (3.0). In Figures 2 to 6, * and ** indicate statistically significant differences (p<0.05, p<0.01, respectively) compared to the OA group at a particular time point. The abbreviations are as follows:G1 (NC): normal group; G2 (OA): anterior cruciate ligament transection with medial meniscectomy (ACLT / MMx)-induced osteoarthritis group; G3 (0.5): ACLT / MMx operated and treated with Sodium Polynucleotide 0.5 mg / kg; G4 (1.5): ACLT / MMx operated and treated with Sodium Polynucleotide 1.5 mg / kg; G5 (2.5): ACLT / MMx operated and treated with Sodium Polynucleotide 1.5 mg / kg; G6(3.0): ACLT / MMx operated and treated with Sodium Polynucleotide 3.0 mg / kg; WILL: Willingness to allow the clinician to lift the limb contralateral to the affected limb. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention will be described in detail below with reference to examples to aid in understanding the present invention. However, the examples according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those having average knowledge in the field to which the present invention pertains.

[0022] In preliminary studies, doses below 2.5 mg / kg body weight, especially below 2.0 mg / kg body weight, did not produce satisfactory results. Furthermore, taking into account the intra-articular volume of pet dogs, it was found that the use of a 20 mg / ml formulation of polynucleotide sodium, i.e., administration of approximately 0.125 ml / kg body weight to achieve a dose of 2.5 mg / kg, was most appropriate.

[0023] Therefore, in the following experiments, Experiments 1 and 2 were conducted by injecting a 20 mg / ml formulation of polynucleotide sodium (Conjuran (trademark)) into the joint cavity of 10 pet dogs (body weight: 3 kg to 10 kg) of various breeds exhibiting arthritis symptoms at a dose of 2.5 mg of polynucleotide sodium per kg of body weight (approximately 0.125 ml per kg of body weight) (see Figure 1).

[0024] Experimental Example 1. Evaluation of the degree of pain during palpation and movement Pain relief was evaluated based on the degree of pain reported during palpation and movement. For each evaluation index, scores were given based on the degree of pain using the rankings shown in Table 1 below. Pain assessment was performed before and after administration.

[0025] [Table 1]

[0026] The evaluation results, including the degree of pain reported during palpation and movement, are summarized in Table 2 below.

[0027] [Table 2]

[0028] The test substance had physical properties suitable for intra-articular injection, and pain relief was observed in dogs 1 to 10 to which the test substance was administered.

[0029] Experimental example 2: Standing position evaluation The standing scale was evaluated using the ranking system shown in Table 3 below. That is, scores were given based on the behavioral state. For behavioral evaluation, the animals were allowed to adapt for 5 to 10 minutes and then allowed to walk freely. During walking, a score corresponding to the standing index was given.

[0030] [Table 3]

[0031] The evaluation results are summarized in Table 4 below.

[0032] [Table 4]

[0033] As shown in Table 4 above, injection of the test substance into the joint cavity resulted in overall leg strength and improvement in the standing scale.

[0034] Experimental Example 3: Effect evaluation by dosage Osteoarthritis was induced in beagle dogs (18 males, 2.5 to 5 years old, weighing 12 to 16 kg) over a period of approximately 3 months by anterior cruciate ligament transection and medial meniscectomy using the following method, and the effects of different doses of the composition of the present invention were compared and evaluated.

[0035] The beagle dogs used were Canis familiaris (Woojungbio., Co. Ltd.). The beagle dogs selected for this study can be used as a surgically induced osteoarthritis model, and are an animal species with a wealth of previous research data. This makes them convenient for evaluating the analgesic and behavioral improvement effects of test substances, and also facilitates the interpretation and evaluation of test results. Healthy animals suitable for the experiment were used after a two-week acclimatization and withdrawal period prior to the test. On the day of surgery and the day of test substance administration, the animals were anesthetized under a 16-hour fasting state. After recovery from anesthesia, approximately 300g / day / head of solid feed was placed in a feeding container and allowed to eat ad libitum. In addition, during the test period, 300g / day / head of solid feed was given once a day at a fixed time in the afternoon and allowed to eat ad libitum.

[0036] Thereafter, the compositions of the present invention were administered according to Table 5 below. All administration groups were administered intra-articularly. Prior to administration of the test substance, the animals were fasted for 16 hours and anesthetized, with the animals placed in the supine position. The administered composition was administered using a 3 cc syringe (with a 23G needle). With the right knee flexed, the composition was injected into the knee joint cavity via the patellar tendon at a dose appropriate for each group. After arthritis was induced and the animals were separated into groups, the test substance was administered twice (days 0 and 14) at two-week intervals.

[0037] [Table 5]

[0038] OA: osteoarthritis, ACLT: anterior cruciate ligament transection, MMx: medial meniscectomy, IA: intra-articular

[0039] The test consisted of a total of six groups (n=3), with the negative control group being group G1, the induction group being group G2, and the test groups being groups G3 to G6. In groups G2 to G6, the right knee joint was opened, and anterior cruciate ligament resection and medial meniscectomy were performed to induce osteoarthritis. On the day of test substance administration, no additional treatment was performed in group G1, while the same volume of 0.9% saline was administered into the joint capsule in group G2. The first day of test substance administration was designated day 0.

[0040] Osteoarthritis was induced in 15 rats using the following method. After a 16-hour fast, atropine sulfate (0.03 mg / kg) and prednisolone were injected subcutaneously as medical treatment before the surgical procedure. Before surgery, cefazolin (20 mg / kg) and meloxicam (0.2 mg / kg) were injected intramuscularly and subcutaneously as antibiotics and anti-inflammatory analgesics, respectively. Anesthesia was maintained with intravenous injections of Zoletil 50 (10 mg / kg) and xylazine (0.2 mg / kg). The hair on the right knee joint and thigh surrounding the knee joint of each rat was removed with a depilator and disinfected with Betadine. To ensure smooth hemostasis and infiltration anesthesia during surgery, 2 mL of 2% lidocaine (1:10,000 epinephrine), a local anesthetic, was injected around the knee joint. A 5 cm longitudinal incision was then made from the medial parapatellar approach, centered on the tibial tubercle. The exposed joint capsule was then incised, the patella was positioned laterally, and the knee was flexed to expose the anterior cruciate ligament and meniscus to the surgical field. A surgical blade (No. 15) was used to completely cut the middle of the anterior cruciate ligament. While grasping and pulling the medial meniscus with forceps, surgical scissors were used to cut the meniscal ligament from its surrounding attachments, completely removing the medial meniscus. Next, an anterior drawer's test was performed to confirm loosening of the tibial ligament support. The joint capsule and subcutaneous tissue were sutured using absorbable sutures, and the skin was sutured with non-absorbable sutures. After surgery, meloxicam (0.2 mg / kg) was injected subcutaneously for 5 days to suppress pain. At the same time, the surgical site was disinfected with betadine for approximately 7 days to prevent infection, and cefazolin was injected intramuscularly at a dose of 20 mg / kg twice daily for 7 days. All animals were allowed to move freely in individual enclosures for approximately 12 weeks to aid recovery without external fixation. All animals underwent a stabilization period of approximately 4 weeks until the skin around the surgical incision site stabilized.To induce osteoarthritis, the rats were restrained on a table in a supine position by a technician, and another technician repeatedly performed flexion and relaxation exercises on the operated right knee joint for approximately 3-5 minutes once a day for an 8-week period. After the exercise, the rats were returned to their cages and allowed to move freely without any external fixation.

[0041] All experimental results were expressed as mean ± standard deviation and were analyzed using SPSS (version 20, IBM SPSS Statistics, USA). Comparisons between the normal group (G1 group) and the induced group (G2 group) and between the induced group (G2 group) and the test group were analyzed using Student's T-test at a p<0.05 level (significance levels: two-tailed 5%, 1%, and 0.1%).

[0042] Behavioral assessment (assessment using behavioral and postural scales) Behavioral assessment consisted of a behavioral scale and an orthostatic scale. The behavioral and orthostatic scales were evaluated for the G1 to G6 test groups according to Table 6 below or Table 3 above. For behavioral assessment, the animals were placed in an open, unoccupied space of approximately 26.2 m2 and allowed to walk freely after a 5-10 minute adaptation period. While walking, an observer assessed the animals through an open window, allowing observation of the room, and scored them according to each behavioral assessment index. Behavioral assessment was conducted four times in total. The assessment was conducted on the first (day 0) and second (day 14) dosing days, two days after the completion of test substance administration for each dosing day, and two weeks after the second dosing. The results are shown in Figures 2 and 3, respectively.

[0043] [Table 6]

[0044] Two days after the first dose, the G5(2.5) group demonstrated the best behavior index score of 1.00. At 16 days (two days after the second dose), the G5(2.5) and G6(3.0) groups demonstrated mean scores of 1.00 and 1.00, respectively. At 30 days (16 days after the second dose), the G5(2.5) and G6(3.0) groups demonstrated mean scores of 1.00 and 0.67, respectively. Among the test groups, the high-dose groups (G5(2.5) and G6(3.0)) demonstrated significantly lower behavior index scores than the OA group, which demonstrated scores of 1.67, 1.83, and 1.67 at each evaluation time point (p<0.05, Figure 2). No significant differences were observed between the test and OA groups in the standing index evaluations 2 and 16 days after the first administration. However, in the final evaluation 30 days after administration, the G4 (1.5) group, G5 (2.5) group, and G6 (3.0) group showed mean values ​​of 1.33, 1.17, and 1.17, respectively, confirming that the G4 (1.5) and G5 (2.5) groups showed significant decreases compared with the OA group, which showed a mean value of 2.17 (p<0.05, Figure 3).

[0045] Pain relief assessment Pain relief was assessed based on the severity of pain reported during palpation and movement, as well as the ability of the affected hind limb to support weight bearing. Each evaluation index was scored based on the severity of pain using the ranking system shown in Table 1 above and Table 7 below. Pain assessments were performed four times in total, on the day following the first (day 0) and second (day 14) administrations, and two weeks after the second administration. All pain assessments were performed the day following the completion of behavioral assessments. The results are shown in Figures 4 and 5.

[0046] [Table 7]

[0047] The evaluation results for the degree of pain at palpation and mobilization (pain at palpation / mobilization) showed that the G4 (1.5) and G5 (2.5) groups among the test groups showed mean values ​​of 1.00 and 0.83, respectively, at all evaluation time points (days 3, 17, and 31), which showed a significant decrease compared to the OA group, which showed the same value of 2.50 at each evaluation time point (p<0.05, Figure 4). The willingness to allow the clinician to lift the limb contralateral to the affected limb (WILL) was evaluated at all evaluation time points (days 3, 17, and 31) in the high-dose G5 (2.5) group, which showed scores of 1.67, 1.50, and 1.33, respectively, showing a significant decrease compared with the OA group, which showed a score of 2.67 on day 31. The highest-dose G6 (3.0) group showed a mean score of 1.33, showing a significant decrease compared with the OA group, which showed a score of 2.67 on days 3, 17, and 31 (p<0.05, Figure 5).

[0048] Analysis of inflammation-related cytokines in the blood Previous studies have shown that osteoarthritis is a non-inflammatory form of arthritis, but as cartilage destruction progresses, inflammation-related changes occur, and a mild to moderate inflammatory response appears in the articular capsule. This response is partly due to the inflammatory response caused by destroyed cartilage fragments in the synovial fluid. Once an inflammatory response occurs in the synovial capsule, synovial cells begin to produce cartilage-destroying enzymes, such as matrix metalloproteinases, interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), which stimulates chondrocytes to produce more cartilage-destroying enzymes. Furthermore, among the potent inflammatory mediators released, IL-1, the most potent in destroying cartilage, mediates the inflammatory response by inducing synovial cells to produce prostaglandin E2, downregulating extracellular matrix synthesis, and upregulating matrix metalloproteinases (MMPs) through nitric oxide (NO) production in chondrocytes. Therefore, the degree of treatment of canine arthritis was evaluated based on blood levels of IL-1β.

[0049] Two weeks after the second administration of the test substance, blood samples were collected from all animals under a 16-hour fast. 10 ml of blood was collected from each animal and divided into two SST (BD Vacutainer SST™ tubes, 5 ml) tubes. The collected blood was centrifuged at 4,000 rpm for 10 minutes at 4°C. The supernatant was then dispensed into five 1 ml aliquots of 1.5 ml EP tubes and stored in a -80°C deep freezer until analysis.

[0050] IL-1β, a representative inflammatory cytokine, was measured in serum using an ELISA kit (CUSABIO, CSB-E13836c) and analyzed using an ELISA reader (SpectraMax M2, Molecular Devices, USA). The results are shown in Figure 6.

[0051] As shown in Figure 6, the IL-1β quantification results showed that the mean serum IL-1β levels (pg / mL) were observed in the NC group (G1 group, 9.27), the OA group (G2 group, 29.56), the G3 (0.5) group (23.96), the G4 (1.5) group (7.63), the G5 (2.5) group (5.22), and the G6 (3.0) group (6.74). All test groups showed lower levels of IL-1β than the OA group, and among them, the G4 (1.5), G5 (2.5), and G6 (3.0) groups showed lower levels than the NC group. This indicates that the composition of the present invention generally reduced the increase in the inflammatory cytokine (IL-1β) levels, demonstrating a positive effect on improving pain and behavior.

[0052] conclusion In summary, in a surgically induced beagle osteoarthritis model under these experimental conditions, the test substance, polynucleotide sodium 20 mg / ml, was administered intra-articularly. Significantly lower scores were observed in behavioral assessment indices (behavioral scale and standing scale) and pain assessment indices (pain complaints during palpation and movement, and weight-bearing capacity of the affected hind limb) compared to the induced group. Mechanistically, it was confirmed that the polynucleotide sodium 20 mg / ml formulation exhibited behavioral improvement and analgesic effects by suppressing increases in the levels of inflammatory cytokines (IL-1β). In particular, it was confirmed that the polynucleotide sodium 20 mg / ml formulation had the most effective effect at comparable levels at doses of 2.5 mg / kg and 3.0 mg / kg.

Claims

1. A composition for treating or ameliorating arthritis in dogs or cats, or reducing mechanical friction at joint sites through physical repair, by injecting polynucleotide sodium into the joint cavity of a dog or cat, comprising: The composition, wherein the polynucleotide sodium is administered at a dose of 2-3 mg per kg of body weight of the dog or cat.

2. 2. The composition of claim 1, wherein the sodium polynucleotide is administered at a dose of 2.5 to 3 mg per kg of body weight of the dog or cat.

3. 3. The composition of claim 2, wherein the sodium polynucleotide is administered at a dose of 2.5 mg per kg of body weight of the dog or cat.

4. The composition of claim 1, wherein the composition is in a liquid form and has a polynucleotide sodium content of 20 mg / ml.

5. The composition of claim 4, wherein the liquid composition is administered at a dose of 0.1 to 0.15 ml per kg of body weight of the dog or cat.

6. The composition of claim 5, wherein the liquid composition is administered at a dose of 0.125 to 0.15 ml per kg of body weight of the dog or cat.

7. 7. The composition of claim 6, wherein the liquid composition is administered at a dose of 0.125 ml per kg of body weight of a dog or cat.

8. The composition of claim 1 , wherein the composition is for intra-articular administration to dogs or cats.

9. The composition of claim 8, wherein the composition is for intra-articular administration to dogs.

10. 8. A composition according to any one of claims 1 to 7, for intra-articular administration to dogs or cats, wherein the polynucleotide sodium contained in the composition is a DNA fragment extracted from the testes of salmonid fish.

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