Composition comprising sodium polynucleotides for injection into articular cavity of dogs or cats

Abstract

The present invention relates to a composition for injection into the articular cavity of a dog or cat for treating or ameliorating arthritis by injection into the articular cavity of a dog or cat, or for reducing mechanical friction in the articular region via body repair. The invention also relates to a method for administering a composition for injection into the articular cavity of a dog or cat for treating or ameliorating arthritis by injection into the articular cavity of a dog or cat, or for reducing mechanical friction in the articular region by body repair. When the composition of the invention is administered, the dosage of the polynucleotide sodium is 2 to 3 mg per kilogram of body weight of dogs or cats, preferably 2.5 to 3 mg.

Description

Technical Field

[0001] The present invention relates to a composition for injection into the joint cavity of a dog or cat, which comprises sodium polynucleotide. The present invention also relates to a method for administering a composition for injection into the joint cavity of a dog or cat, the composition comprising sodium polynucleotide. The compositions and methods of the present invention can be used to treat or improve arthritis in dogs or cats and can also be used to reduce mechanical friction in the joint area via body repair. Background Art

[0002] Among arthritis diseases, degenerative arthritis is the most common, also known as degenerative joint disease or osteoarthritis. This degenerative arthritis is a disease in which the cartilage protecting the joint is gradually damaged or degeneratively changed, resulting in damage to the bones and ligaments forming the joint, causing inflammation in the synovium around the joint, thereby causing pain and deformation. Degenerative arthritis progresses through a process in which the cartilage tissue in joints (such as knees, ankles, fingers, elbows, and hips) gradually degenerates and is damaged due to aging, obesity, joint trauma, hip dysplasia, a history of arthritis, genetic factors, etc.

[0003] This degenerative arthritis is also gradually increasing in companion dogs or cats. It is one of the age-related diseases that mainly occur in older dogs or cats. The symptoms vary, but they mainly manifest as reluctance to move, showing abnormal gait, and lifting their legs when walking or exercising or lying prone in severe cases.

[0004] General drug treatment helps to improve arthritis in companion dogs or cats. For example, non-steroidal anti-inflammatory drugs have recently been used in various ways to relieve pain and prevent and treat inflammation. However, these non-steroidal anti-inflammatory drugs can cause bloody vomiting, bloody stools, anemia, etc. due to peptic ulcers, and there is also a risk of renal failure.

[0005] Therefore, there is a need for drugs with fewer side effects and effective in improving arthritis when administered to companion dogs or cats. Summary of the Invention

[0006] Technical Problem

[0007] Therefore, the problem that the present invention seeks to solve is to provide a preparation with fewer side effects and effective in improving arthritis when administered to dogs or cats, especially dogs.

[0008] Another problem that the present invention seeks to solve is to provide a method for administering sodium polynucleotide with fewer side effects and effective in improving arthritis when administered to dogs or cats, especially dogs.

[0009] Technical Solution

[0010] To solve the above problems, an embodiment of the present invention provides a composition for injecting sodium polynucleotide into the joint cavity of a dog or a cat, preferably a dog, which is used for treating or improving arthritis in a dog or a cat, preferably a dog, or reducing mechanical friction in the joint area through body repair, wherein the sodium polynucleotide is administered at a dose of 2 to 3 mg / kg of the body weight of the dog or cat.

[0011] Another embodiment of the present invention provides a method for administering a composition for treating or improving arthritis in a dog or a cat, preferably a dog, or reducing mechanical friction in the joint area through body repair by injecting sodium polynucleotide into the joint cavity of the dog or the cat, preferably the dog, wherein the sodium polynucleotide is administered at a dose of 2 to 3 mg / kg of the body weight of the dog or cat.

[0012] In a preferred embodiment of the present invention, the composition and the method of administration of the present invention are such that 2.5 to 3 mg of sodium polynucleotide is administered per kg of the body weight of the dog or cat. In a more preferred embodiment of the present invention, the composition and the method of administration of the present invention are such that 2.5 mg of sodium polynucleotide is administered per kg of the body weight of the dog or cat.

[0013] In a preferred embodiment of the present invention, the composition of the present invention is a liquid composition having a sodium polynucleotide content of 20 mg / ml. In another preferred embodiment of the present invention, the method of administration of the present invention administers a liquid composition having a sodium polynucleotide content of 20 mg / ml.

[0014] In a preferred embodiment of the present invention, the liquid composition having a sodium polynucleotide content of 20 mg / ml in the composition and the method of administration is administered at a dose of 0.1 to 0.15 ml / kg of the body weight of the dog or cat, more preferably at a dose of 0.125 to 0.15 ml. In a more preferred embodiment of the present invention, the liquid composition is administered at a dose of 0.125 ml / kg of the body weight of the dog or cat.

[0015] The composition of the present invention is administered into the joint cavity of a dog or a cat, and is preferably administered at the above dose of 2 to 3 mg of sodium polynucleotide / kg of body weight (more preferably 2.5 to 3 mg / kg of body weight), and in particular, considering the administration volume, it is preferably injected into the joint cavity as a 20 mg / ml liquid composition to achieve these doses.

[0016] The sodium polynucleotide according to the present invention is a viscous transparent liquid substance, which is a DNA fragment extracted from the testes of salmonid fish. For example, such a sodium polynucleotide can be prepared by the method described in Korean Patent No. 10-0986603. In an embodiment of the present invention, the average molecular formula of the deoxyribonucleotides in the DNA fragment mixture constituting the sodium polynucleotide is C 9.83 H12.33 N 3.72 O 6.01 P·Na. In one embodiment of the present invention, the molecular weight of the sodium polynucleotide shows a maximum distribution of the sodium polynucleotide at 250 to 1,000 base pairs (bp) when tested by electrophoresis, shows a maximum absorption at 260 ± 2 nm when measuring absorbance, and the ratio of the absorbance at 260 nm to that at 280 nm is 1.6 to 2.0. In addition, the pH of a 1 wt% aqueous solution of the sodium polynucleotide according to the present invention is 6.5 to 7.5.

[0017] In one embodiment of the present invention, the content of the sodium polynucleotide contained in the composition of the present invention is 20 mg / ml, and the viscosity of the composition of the present invention is 100 to 300 Pas when measured according to the viscosity measurement method of the general test method of the Korean Pharmacopoeia, and the elasticity is 50 to 250 Pa when measured under the conditions of geometry: PU20, temperature: 25 °C, and frequency: 0.1 Hz.

[0018] In one embodiment of the present invention, the liquid composition Conjuran with a sodium polynucleotide content of 20 mg / ml TM can be used as the composition of the present invention.

[0019] In another embodiment of the present invention, the sodium polynucleotide composition can be prepared as described in Korean Patent Publication No. 10-2017-0100236, and the content described in Korean Patent Publication No. 10-2017-0100236 is incorporated herein by reference in its entirety.

[0020] In one embodiment of the present invention, the composition of the present invention is administered to the joint cavity at intervals of 2 to 4 weeks for a total of 2 to 4 times. In a preferred embodiment of the present invention, the composition of the present invention is administered to the joint cavity at intervals of 2 weeks for a total of 2 times.

[0021] Advantageous Effects

[0022] The present invention provides a composition for injection into the joint cavity of a dog or a cat, preferably a dog, which is used for treating or improving arthritis by injecting into the joint cavity, or reducing mechanical friction in the joint area through body repair. In particular, the composition of the present invention is administered such that the dose of the sodium polynucleotide is 2 to 3 mg / kg of the dog or cat body weight, preferably 2.5 to 3 mg, more preferably 2.5 mg. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a photograph of administering a 20 mg / ml sodium polynucleotide preparation into the joint cavity of a companion dog showing arthritis symptoms.

[0024] Figure 2 and Figure 3 are the results of the behavioral evaluation and the results of the behavioral index and the standing index, respectively. Values of the behavioral index and the standing index for the knees of the affected dogs in the saline and sodium polynucleotide administration groups during the 30-day study period.

[0025] Figure 4 and Figure 5 are the results of the pain relief evaluation and the evaluation results of "degree of pain discomfort during palpation and movement" and "weight-bearing degree of the affected hind limb", respectively. Values of the behavioral index and the standing index for the knees of the affected dogs in the saline and sodium polynucleotide administration groups during the 30-day study period.

[0026] Figure 6 Shows the results of evaluating the effect of the test substance on the IL-1β concentration in the serum of dogs with osteoarthritis 48 weeks after the first intra-articular injection.

[0027] In Figures 2 to 6 , the bar graphs represent G1(NC) (no bar), G2(OA), G3(0.5), G4(1.5), G5(2.5), and G6(3.0) from left to right in sequence. In Figures 2 to 6 , * and ** represent data showing statistically significant differences (p < 0.05 and p < 0.01, respectively) compared to the OA group at specific time points, and the meaning of each abbreviation is as follows:

[0028] G1(NC): normal group, G2(OA): osteoarthritis group induced by anterior cruciate ligament transection and medial meniscectomy (ACLT / MMx), G3(0.5): ACLT / MMx surgery and treated with sodium polynucleotide 0.5 mg / kg, G4(1.5): ACLT / MMx surgery and treated with sodium polynucleotide 1.5 mg / kg, G5(2.5): ACLT / MMx surgery and treated with sodium polynucleotide 2.5 mg / kg, G6(3.0): ACLT / MMx surgery and treated with sodium polynucleotide 3.0 mg / kg, WILL: willing to allow the clinician to lift the limb contralateral to the affected limb. Detailed Description of the Invention

[0029] Hereinafter, in order to assist in understanding the present invention, examples and the like will be described in detail. However, the embodiments of the present invention can be modified in many different forms, and the scope of the present invention should not be construed as being limited to the following embodiments. The embodiments of the present invention are provided to more completely illustrate the present invention to those of ordinary skill in the art to which the present invention pertains.

[0030] In preliminary experiments, when doses lower than 2.5 mg / kg body weight were administered, especially when doses lower than 2.0 mg / kg body weight were administered, no satisfactory effects were observed. Additionally, considering the intra-articular volume of companion dogs, a 20 mg / ml preparation of sodium polynucleotide was most suitable, i.e., approximately 0.125 ml / kg body weight was administered, such that the dose was 2.5 mg / kg.

[0031] Therefore, in the following experiments, a 20 mg / ml preparation of sodium polynucleotide (Conjuran TM ) was intra-articularly injected into the joints of 10 companion dogs of different species (body weight: 3 kg to 10 kg) showing arthritis symptoms, such that 2.5 mg of sodium polynucleotide / kg body weight (approximately 0.125 ml / kg body weight) was administered (see Figure 1 ).

[0032] Experimental Example 1. Evaluation of pain discomfort during palpation and movement

[0033] Pain relief was evaluated by the degree of pain discomfort during palpation and movement. The pain level was scored according to the classification in the grading scale specified for each evaluation index in Table 1 below. Pain evaluations were performed before and after administration.

[0034]

Table 1

[0035]

[0036] The evaluation results, including the degree of pain discomfort during palpation and movement, are summarized in Table 2 below.

[0037]

Table 2

[0038]

[0039]

[0040] The test substance has physical properties that make it easy to inject into the joint cavity, and individuals (dogs) 1 to 10 administered showed a reduction in pain.

[0041] Experimental Example 2. Standing evaluation

[0042] The standing grade was evaluated according to the classification in the rating scale specified in Table 3 below. In other words, the behavioral state was scored. During the behavioral evaluation, the animals were allowed to walk freely while allowing an adaptation time of 5 to 10 minutes. The corresponding standing index was assigned a score while walking.

[0043]

Table 3

[0044]

[0045] The evaluation results are summarized in Table 4 below.

[0046] [Table 4]

[0047]

[0048]

[0049] As shown in Table 4 above, when the test substance was injected into the joint cavity, the overall foot strength increased and the standing test showed improvement.

[0050] Experimental Example 3. Evaluation of the effect according to the dose

[0051] As follows, osteoarthritis was induced in beagle dogs (18 males, 2.5 to 5 years old, 12 to 16 kg) for about 3 months by anterior cruciate ligament resection and medial meniscectomy, and the effects according to the dose of the composition of the present invention were compared and evaluated.

[0052] The beagle dog species used was Canis familiaris (Woojungbio., Co., Ltd.). The beagle dogs selected for this study can be used as a surgically induced osteoarthritis model, and it is an animal species that is easy to evaluate the analgesic and behavioral improvement effects of the test substance, and because there are accumulated previous research data, the test results can be easily interpreted and evaluated. After about 2 weeks of adaptation and drug withdrawal before the test, healthy animals suitable for the experiment were used. On the day of surgery and the day of administration of the test substance, the individuals were anesthetized under 16-hour fasting, and after recovery from anesthesia, about 300 g / day / head of solid food was supplied to the feeder and they were allowed to eat freely. In addition, 300 g / day / head was fed once a day in the afternoon at some time during the test period and they were allowed to eat freely.

[0053] Thereafter, the composition of the present invention was administered according to Table 5 below. Intra-articular administration was performed for all administration groups. Before administration of the test substance, the animals were anesthetized under 16-hour fasting and placed in the supine position. The administration composition was administered using a 3 cc syringe (connected to a 23G needle). With the right knee bent, the injection was performed into the knee joint cavity through the patellar tendon according to the administration volume of each group. After induction of arthritis, the test substance was administered a total of two times at 2-week intervals (day 0, day 14), and the groups were separated.

[0054] [Table 5]

[0055]

[0056]

[0057] OA: Osteoarthritis, ACLT: Anterior cruciate ligament transection, MMx: Medial meniscectomy, I.A: Intra-articular

[0058] The test group consisted of a total of 6 groups (n = 3), the negative control group was group G1, the induction group was group G2, and the test groups were groups G3 to G6. In groups G2 to G6, osteoarthritis was induced by anterior cruciate ligament transection and medial meniscectomy through a right knee joint incision. On the day of test substance administration, group G1 did not receive any treatment alone, while group G2 received the same amount of 0.9% saline into the joint cavity. The first day of test substance administration was designated as day 0.

[0059] Osteoarthritis was induced as follows. Before the surgical procedure under 16-hour fasting, fifteen animals to be induced with osteoarthritis were injected subcutaneously with atropine sulfate (0.03 mg / kg) and prednisolone as an oral medicine. Cefazolin (20 mg / kg) and meloxicam (0.2 mg / kg) were injected intramuscularly and subcutaneously, respectively, as an antibiotic and an anti-inflammatory analgesic before the surgery. Anesthesia was maintained by intravenous injection of Zoletil 50 (10 mg / kg) and xylazine (0.2 mg / kg). The hair on the right knee joint and the thigh around the knee joint of each animal was removed using a hair remover, and disinfected with betadine. To ensure smooth hemostasis and infiltration anesthesia during the surgery, 2 mL of 2% lidocaine (1:10,000 epinephrine) was injected as a local anesthetic around the knee joint, and then a longitudinal incision of about 5 cm centered on the tibial tubercle was made from the medial parapatellar approach. After the joint cavity was exposed by the incision, the patella was positioned laterally, and the knee was flexed to expose the anterior cruciate ligament and the meniscus under the surgical field. The middle part of the anterior cruciate ligament was completely cut with a surgical blade (No. 15), and the medial meniscus was clamped with forceps and pulled, and the meniscus ligament was cut with surgical scissors from its surrounding attachments to completely remove the medial meniscus. After that, the loosening of the ligament's support for the tibialis muscle was confirmed by the anterior drawer test, and the joint cavity and subcutaneous tissue were sutured with absorbable sutures respectively, and the skin was sutured with non-absorbable sutures. To control postoperative pain, meloxicam (0.2 mg / kg) was injected subcutaneously for 5 days. At the same time, the surgical site was disinfected with betadine for about 7 days to prevent infection, and cefazolin was injected intramuscularly at a dose of 20 mg / kg twice a day for 7 days. All animals were allowed to move freely in separate cages for about 12 weeks to aid recovery without external fixation. All animals underwent a stabilization period of about 4 weeks to stabilize the skin at the surgical incision site. After that, to induce osteoarthritis, the animals were positioned supine on a table with a corrector for about 3 to 5 minutes once a day for 8 weeks, while another experimenter repeated flexion and relaxation exercises on the surgically treated right knee joint. Then the animals were returned to their cages and allowed to move freely without separate external fixation.

[0060] All experimental results obtained in the experiment were expressed as mean ± standard deviation and tested using SPSS (version 20, IBM SPSS Statistics, USA). In the evaluation results, the comparison between the normal group (Group G1) and the induced group (Group G2), and the comparison between the induced group (Group G2) and the test group were tested using the student T-test method at the p < 0.05 level (significance levels: two-sided 5%, 1%, and 0.1%).

[0061] Behavior evaluation (evaluation of behavior index and standing index)

[0062] The behavioral evaluation consists of a behavior index and a standing index, and the behavior index and standing index of test groups G1 to G6 are evaluated according to Table 6 below or Table 3 above, respectively. When evaluating the behavior, the animal to be evaluated is placed in an empty space of about 8 tsubo without people and allowed to freely walk for an adaptation time of about 5 to 10 minutes. The observer gives the corresponding score for each behavior evaluation index through an open window that allows the room to be seen while walking. A total of 4 behavioral evaluations are conducted, and the evaluations are carried out 2 days after the end of the test substance administration on the first (day 0) and second (day 14) administration days, and 2 weeks after the second administration. The results are shown in Figure 2 and Figure 3 respectively.

[0063]

Table 6

[0064]

[0065]

[0066] As a result of the behavior index evaluation, the G5(2.5) group in the test group showed the best result of 1.00 2 days after the first administration. When evaluated on day 16 (2 days after the second administration), the G5(2.5) group and the G6(3.0) group showed average values of 1.00 and 1.00, respectively, and when evaluated on day 30 (16 days after the second administration), the G5(2.5) group and the G6(3.0) group showed average values of 1.00 and 0.67, respectively. When compared with the OA group, which showed 1.67, 1.83, and 1.67 at each evaluation time point, a significant decrease was confirmed in these high-dose (G5(2.5) group, G6(3.0) group) test groups (p < 0.05, Figure 2 ).

[0067] In the evaluations on day 2 and day 16 after the first administration, no test group showed a significant difference in the standing index evaluation result compared with the OA group. In the final evaluation on day 30, the average values of the G4(1.5) group, the G5(2.5) group, and the G6(3.0) group were 1.33, 1.17, and 1.17, respectively, and the G4(1.5) group and the G5(2.5) group showed a significant decrease compared with the OA group, which showed 2.17 (p < 0.05, Figure 3 ).

[0068] Pain relief evaluation

[0069] The pain relief evaluation consists of the degree of pain discomfort during palpation and movement, and the weight-bearing degree of the affected hind limb. The pain degree is scored according to the classification table specified for each evaluation index in Table 1 above and Table 7 below. A total of 4 pain evaluations are conducted, and the evaluation times are on the day after the first (day 0) and second (day 14) administrations, and 2 weeks after the second administration. All pain evaluations are conducted on the day after the behavioral evaluation. The results are shown in Figure 4 and Figure 5 shown below.

[0070]

Table 7

[0071]

[0072]

[0073] The evaluation results of pain during palpation / movement showed that at all evaluation time points (days 3, 17, and 31), the mean values of groups G4 (1.5) and G5 (2.5) in the test group were 1.00 and 0.83 respectively, showing a significant decrease compared with the OA group, which showed the same value of 2.50 at each evaluation time point (p < 0.05, Figure 4 ).

[0074] As a result of evaluating the willingness to allow the clinician to lift the contralateral limb (WILL) of the affected limb, the high-dose group G5 (2.5) in the test group showed 1.67, 1.50, and 1.33 at all evaluation time points (days 3, 17, and 31), which was significantly lower compared with the OA group, which showed 2.67 on day 31. In the highest-dose group, the mean value was jointly confirmed as 1.33, which was significantly lower compared with the OA group, which showed 2.67 at all days 3, 17, and 31 (p < 0.05, Figure 5 ).

[0075] Analysis of cytokines related to inflammation in blood

[0076] According to previous studies, osteoarthritis is a non-inflammatory arthritis, but as cartilage destruction progresses, inflammation-related changes occur, and the joint capsule shows a mild to moderate inflammatory response, which is partly due to the inflammatory response caused by damaged cartilage fragments in the synovial fluid. Once an inflammatory response occurs in the synovium, synoviocytes produce cartilage-destructive enzymes such as matrix metalloproteinases, interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), which stimulate chondrocytes to produce more destructive enzymes. In addition, effective inflammatory mediators are released, and IL-1, which is the most effective in damaging cartilage, mediates the inflammatory response by causing synoviocytes to produce prostaglandin E2, downregulating the synthesis of the extracellular matrix, and upregulating matrix metalloproteinase (MMP) through the production of nitric oxide (NO) in chondrocytes. Therefore, we aimed to evaluate the degree of treatment of canine arthritis by the blood level of IL-1β.

[0077] After 2 weeks of the second administration of the test substance under 16-hour fasting, blood collection was performed on all subjects. 10 ml of blood was collected from each subject and divided into two SST (BD Vacutainer SST tubes, 5 ml) tubes. The blood was centrifuged at 4,000 rpm for 10 minutes at 4 °C. The separated supernatant was divided into 5 groups, 1 ml per group in 1.5 ml EP tubes, and stored in a -80 °C freezer until analysis.

[0078] The representative inflammatory cytokine IL-1β in serum was measured using an ELISA kit (CUSABIO, CSB-E13836c), and the analysis was performed using an ELISA reader (SpectraMax M2, Molecular Devices, USA). The results are shown in Figure 6 shown.

[0079] As Figure 6As shown, the average serum levels (pg / ml) of the quantified IL-1β results were as follows: NC group (G1, 9.27), OA group (G2, 29.56), G3 (0.5) group (23.96), G4 (1.5) group (7.63), G5 (2.5) group (5.22), and G6 (3.0) group (6.74). In the serum IL-1β index results, all test groups showed lower levels than OA, and among them, the G4 (1.5), G5 (2.5), and G6 (3.0) groups showed lower levels than the NC group. In other words, it was confirmed that the composition of the present invention showed a result of generally reducing the elevated level of the inflammatory cytokine (IL-1β), thus showing a positive effect on pain and behavior improvement.

[0080] Conclusion

[0081] In summary, in a surgically induced beagle osteoarthritis model under test conditions, after intra-articular administration, compared with the induction group, the 20 mg / ml preparation of sodium polynucleotide as an injection, which was the test substance, showed significantly lower scores in terms of the behavioral assessment indices (behavioral index and standing index) and pain assessment indices (palpation of the affected hind limb, pain level during movement, and weight-bearing level), and it was confirmed that the mechanism was to inhibit the increase in the level of the inflammatory cytokine (IL-1β), and showed an effect of behavior improvement and analgesia. In particular, it was confirmed that this effect was most effective at the same level as the doses of the 20 mg / ml preparations of sodium polynucleotide at 2.5 mg / kg and 3.0 mg / kg.

Claims

1. A composition for treating or improving arthritis in dogs or cats or reducing mechanical friction in the joint area via body repair by intra-articular injection of sodium polynucleotide, wherein the composition is characterized in that the sodium polynucleotide is administered at a dose of 2 to 3 mg / kg of the body weight of the dog or cat.

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

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

4. The composition according to claim 1, wherein the composition is a liquid composition with a sodium polynucleotide content of 20 mg / ml.

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

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

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

8. The composition according to any one of claims 1 to 7, wherein the composition is for intra-articular administration to dogs or cats.

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

10. The composition according to any one of claims 1 to 7, wherein the sodium polynucleotide contained in the composition is a DNA fragment extracted from the testes of salmonid fish.

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