Injection for treating cat stomatitis through oral soft tissue injection

Abstract

The invention provides an injection for treating cat stomatitis through oral soft tissue injection. The active ingredients of the injection comprise glucocorticoid and B vitamins, and also comprise mesenchymal stem cells at the same time. According to the invention, the mesenchymal stem cells, the glucocorticoid and the vitamin B are combined as the active ingredient of the injection for biological medicine preparation for the first time, so that the long-term effect and stability of the inflammation inhibition and tissue repair functions of the mesenchymal stem cells are fully exerted, and meanwhile, the characteristic of quick inflammation inhibition effect of the glucocorticoid is utilized; the defects that glucocorticoid is large in side effect after being used for a long time and the condition is repeated after medicine withdrawal are overcome, so that the treatment effect on stomatitis is remarkably improved, and the treatment period is greatly shortened; furthermore, the active ingredients are creatively injected into soft tissues of the oral cavity of an affected cat in a multi-point micro-injection manner in an oral soft tissue injection manner, and a better treatment effect is realized by utilizing low-dose and short-term mesenchymal stem cells and glucocorticoid, so that the technical difficulty in treating the cat stomatitis is overcome.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical manufacturing, and relates to an injection for the injection treatment of feline stomatitis in oral soft tissues. Background Art

[0002] Feline stomatitis is an oral inflammatory disease caused by abnormal immune system, characterized by severe inflammation of the gums, buccal mucosa and caudal oral mucosa. It is estimated that about 0.7% to 10% of cats globally are suffering from this disease. Due to its intractability, recurrence and serious impact on the quality of life of cats, it is often called the "incurable terminal illness".

[0003] The cause of feline stomatitis is still unclear. In essence, some people believe that microbial infection and changes in innate immune responses are highly likely to cause the disease. From a histological perspective, the damage characteristics of the cat's oral cavity are lymphocyte inflammation. This inflammation will cause lesions in the oral mucosa, thus significantly reducing the quality of life. The clinical symptoms of feline stomatitis vary, including gum swelling, oral ulcers, and more severe manifestations such as oral mucosal inflammation, loss of appetite, difficulty in eating, decline in mental state, and weight loss.

[0004] So far, there are not many methods for treating feline stomatitis, and there is no 100% curative therapy, which may lead to the euthanasia of some affected cats. After most cats get sick, doctors will perform total or partial tooth extraction on them, which is relatively effective. However, there are still a small number of cats that do not respond to tooth extraction and need to receive antibiotics, glucocorticoids and other painkillers for life to control inflammation. Long-term use of antibiotics or glucocorticoid anti-inflammatory drugs has side effects and is likely to damage other organs of cats. With social progress, the group of cat owners is increasing day by day. As a highly prevalent and painful disease, feline stomatitis has attracted more and more attention. Finding a safer and more effective treatment method has become an urgent problem to be solved clinically and a major challenge in the veterinary field.

[0005] Several studies have shown that mesenchymal stem cells derived from autologous or allogeneic sources also have a therapeutic effect on feline stomatitis. These studies investigated the safety and efficacy of mesenchymal stem cells in the treatment of feline stomatitis and obtained very useful results (1. See "Therapeutic Efficacy of Fresh, Allogeneic Mesenchymal Stem Cells for Severe Refractory Feline Chronic Gingivostomatitis", STEM CELLS TRANSLATIONAL MEDICINE, 2017; 6: 1710–1722; 2. See the Chinese patent of a Belgian company with application number 2022800561511, which discloses mesenchymal stem cells for the treatment of chronic gingivostomatitis). However, existing research and technologies all use the method of intravenous injection. Since the blood passes through the lungs first after intravenous injection, most of the stem cells will be intercepted by the pulmonary capillaries. After the stem cells pass through the lungs and enter the arteries, they will be shunted to various tissues and organs, and finally the number of stem cells that can reach the oral lesion area is limited. Therefore, intravenous injection often requires several times the amount of cells required for the target treatment area and multiple injections to achieve the therapeutic effect, and the treatment cycle is long, the effect is slow, and the cost is high.

[0006] Therefore, there is an urgent need to develop a drug for feline stomatitis with a small amount of cells used and a quick effect. Summary of the Invention

[0007] The object of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide an injection for oral soft tissue injection for the treatment of feline stomatitis. The present invention first combines mesenchymal stem cells, glucocorticoids and vitamin B complex as the active ingredients of the injection for the manufacture of biopharmaceuticals, and creatively injects the active ingredients into the soft tissues of the affected cat's oral cavity by means of oral soft tissue injection, multiple-point and micro-injection. On the one hand, it enables the mesenchymal stem cells to be concentratedly administered to the tissues in need of treatment and directly and quickly reach the cell dose required for treatment in the treated tissues; on the other hand, it is combined with low-dose glucocorticoids and vitamin B complex to establish and maintain a high-level treatment condition in the local tissues, inhibit the inflammation to a certain extent, and create a more favorable tissue environment for the continuous treatment after a certain stage of stem cell proliferation.

[0008] The research of the present invention has found that traditional oral administration, intravenous injection, and subcutaneous injection will affect the therapeutic effect of the active ingredients. The reason is that the active ingredients contain mesenchymal stem cells, and these stem cells will not survive after being digested by gastric juice, so oral administration is not feasible. At the same time, intravenous injection will cause the active ingredients to reach the oral tissue only through blood circulation, and ultimately the amount of cells and drug dose available in the oral tissue will be very low, failing to achieve the therapeutic effect. In addition, subcutaneous injection will make it more difficult for mesenchymal stem cells to reach the oral tissue, and at the same time, glucocorticoids may have an impact on skin health. Therefore, the present invention is designed for local oral administration, can control and adjust the cell supply in the target treatment area as needed, can more accurately grasp the therapeutic effect, only a small amount of mesenchymal stem cells can meet the stem cell dose required for treatment in the target area, and the treatment cost will also be greatly reduced.

[0009] The object of the present invention can be achieved by the following solutions:

[0010] In a first aspect, the present invention provides an injection solution for the treatment of feline stomatitis by injecting into oral soft tissues, and its active ingredients include glucocorticoids and B vitamins.

[0011] As an embodiment of the present invention, the active ingredients further include mesenchymal stem cells.

[0012] As an embodiment of the present invention, the mesenchymal stem cells are cells derived from the umbilical cord, fat, or bone marrow of cats and cultured to the 4th to 6th generation.

[0013] Further, the mesenchymal stem cells are prepared by the following steps: after culturing the mesenchymal stem cells to a confluence of 40 - 50%, replace the fresh culture medium, and add 10 - 100 μM glucocorticoid to the culture medium and continue to culture until the cell confluence reaches 80%, and collect the cells for treatment.

[0014] As an embodiment of the present invention, the content of the mesenchymal stem cells is 1 - 5×10 6 cells / ml.

[0015] As an embodiment of the present invention, the glucocorticoids include one or more of hydrocortisone, prednisolone, and dexamethasone.

[0016] Further, the hydrocortisone includes any one of hydrocortisone acetate, hydrocortisone sodium phosphate, and hydrocortisone sodium succinate.

[0017] Further, the prednisolone includes any one of prednisolone acetate, prednisolone sodium phosphate, and prednisolone sodium succinate.

[0018] Further, the dexamethasone includes any one of dexamethasone acetate, dexamethasone sodium phosphate, and dexamethasone sodium succinate.

[0019] As an embodiment of the present invention, the concentration of the glucocorticoid in the injection is 10-100 μM.

[0020] As an embodiment of the present invention, the B vitamins include one or more of vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B9 (folic acid), and vitamin B12 (cobalamin).

[0021] As an embodiment of the present invention, the concentration of the B vitamins is 0.1-20 μg / ml; among them, the concentration of vitamin B2 (riboflavin) is 0.1-5 μg / ml, the concentration of vitamin B3 (niacin) is 1-20 μg / ml, the concentration of vitamin B5 (pantothenic acid) is 1-20 μg / ml, the concentration of vitamin B6 (pyridoxine) is 1-20 μg / ml, the concentration of vitamin B9 (folic acid) is 1-20 μg / ml, and the concentration of vitamin B12 (cobalamin) is 1-20 μg / ml.

[0022] As an embodiment of the present invention, the injection further includes pharmaceutically acceptable excipients.

[0023] Further, the excipient includes physiological saline.

[0024] As an embodiment of the present invention, the injection is a preparation for injection into oral soft tissues.

[0025] Further, the oral soft tissues include the pharynx, jaw, periodontium, and gingiva.

[0026] Further, the injection method is to select injection points at intervals of 2-10 mm and perform multi-point micro-injection. The injection points should be evenly distributed and comprehensively cover the inflamed tissue.

[0027] In a second aspect, the present invention provides a method for preparing the injection for treating feline stomatitis by injecting into oral soft tissues, including: mixing the active ingredients with excipients to prepare a solution, which is the injection. In the present invention, the active ingredient solution is injected into the inflamed tissue at the oral cavity of the diseased cat with a syringe.

[0028] In a third aspect, the present invention provides the use of the injection in the preparation of a drug for preventing and / or treating feline stomatitis. The present invention uses mesenchymal stem cells, glucocorticoids, and B vitamins as the main active ingredients of the injection for the manufacture of biological drugs for feline stomatitis.

[0029] The traditional administration method of glucocorticoids is oral administration, and the traditional administration method of mesenchymal stem cells is intravenous injection. It is well known in the art that arbitrarily changing the administration method will lead to serious adverse reactions. For the first time, the present invention combines mesenchymal stem cells, glucocorticoids and vitamin B group as the active ingredients of the injection for the manufacture of biological drugs, and creatively injects the active ingredients into the soft tissues of the affected cat's oral cavity by multi-point and micro-injection through the oral soft tissue injection method. At the same time, it is verified that the injection of the present invention does not show adverse reactions, and can significantly improve the treatment effect of stomatitis, greatly shorten the treatment cycle, and overcome the defects of the latter with large side effects during long-term use and recurrence of the disease after drug withdrawal.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The present invention designs the active ingredients of the injection of mesenchymal stem cells, glucocorticoids and vitamin B group for the manufacture of biological drugs, which can effectively treat various feline stomatitis by injecting into the soft tissues of the oral cavity, including eliminating inflammation, repairing ulcerated and damaged tissues, improving immunity, and preventing the recurrence of the disease.

[0032] 2. For the first time, the present invention combines mesenchymal stem cells and glucocorticoid drugs, giving full play to the long-term and stable anti-inflammatory and tissue repair functions of the former, while utilizing the fast anti-inflammatory effect of the latter, and overcoming the defects of the latter with large side effects during long-term use and recurrence of the disease after drug withdrawal, significantly improving the treatment effect of stomatitis and greatly shortening the treatment cycle.

[0033] 3. The present invention creatively injects the active ingredients into the soft tissues of the affected cat's oral cavity by multi-point and micro-injection through the oral soft tissue injection method, solving the limitations of the traditional administration method such as large dosage of stem cells, long treatment cycle, unstable curative effect and high treatment cost, and overcoming the technical difficulties in the treatment of feline stomatitis. Description of the Drawings

[0034] By reading the following detailed description of the non-limiting embodiments with reference to the accompanying drawings, other features, objects and advantages of the present invention will become more obvious:

[0035] Figure 1 Schematic diagram of dot injection of oral soft tissue in Example 1;

[0036] Figure 2 Grade A state of intraoral inflammation of caudal stomatitis in Example 1;

[0037] Figure 3 Grade B state in the oral cavity after obvious improvement in Example 1;

[0038] Figure 4 Grade C state in the oral cavity close to recovery in Example 1;

[0039] Figure 5 The D - level state in the oral cavity after cure in Example 1;

[0040] Figure 6 The comparison of the treatment effects of Groups 1 - 6 in Example 1;

[0041] Figure 7 The treatment effect of the stomatitis cases cured in Group 6 in Example 1; among them, the left picture is before treatment and the right picture is after treatment. Detailed implementation manners

[0042] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The following examples are implemented on the premise of the technical solution of the present invention, providing detailed implementation manners and specific operation processes, which will help those skilled in the art to further understand the present invention. It should be noted that the protection scope of the present invention is not limited to the following embodiments. Several adjustments and improvements made on the premise of the concept of the present invention all belong to the protection scope of the present invention.

[0043] Example 1

[0044] 1. Experimental animals and grouping

[0045] Select cats with non - curable caudal stomatitis after tooth extraction as the treatment objects, regardless of gender, with a body weight of 3 - 5 kg. Typical symptoms include: the color of the pharyngeal mucosa is dark red ( Figure 2 ) or accompanied by ulcers, salivation, pain when touching the chin, and difficulty in eating. Among them, the color of the pharyngeal mucosa is the most core inspection index. The treatment plan was agreed by the cat owners. Before treatment, the physical condition of the cats was checked to ensure that there was no interference from other diseases. The injection was prepared by the laboratory of Magic Hair Knight Biotechnology Co., Ltd. The stomatitis cats were divided into 6 groups, namely Group 1: oral glucocorticoid control group, Group 2: stem cell intravenous injection group, Group 3: injection intravenous injection group, Group 4: stem cell oral injection group, Group 5: oral control group, Group 6: injection oral injection group, with 5 experimental cats in each group.

[0046] 2. Treatment methods:

[0047] (1) Group 1: oral glucocorticoid control group. The experimental cats were continuously orally administered prednisolone acetate at a dose of 1 mg / kg body weight for 30 days, and the drug was stopped after 30 days. Observed continuously for 2 months, during which the oral cavity was rinsed with normal saline every day.

[0048] (2) Group 2: stem cell intravenous injection group. Mesenchymal stem cells were intravenously injected into the experimental cats. According to 5x10 6 stem cells / kg body weight, injected once a month for two months. Observed continuously for 2 months, during which the oral cavity was rinsed with normal saline every day.

[0049] (3) Group 3: Intravenous injection group of injection. Intravenous injection of 1 ml of mesenchymal stem cell injection into the experimental cats. The injection contains: normal saline, 1×10 6 mesenchymal stem cells, 20 μM of prednisolone sodium phosphate, 2 μg / ml of vitamin B2 (riboflavin), 10 μg / ml of vitamin B3 (niacin), 10 μg / ml of vitamin B5 (pantothenic acid), 10 μg / ml of vitamin B6 (pyridoxine), 10 μg / ml of vitamin B9 (folic acid), 10 μg / ml of vitamin B12 (cobalamin). Inject once a month for 2 consecutive months, and rinse the oral cavity with normal saline every day during the period.

[0050] (4) Group 4: Oral injection group of stem cells: Inject 1 ml of mesenchymal stem cell solution into the oral soft tissue of the experimental cats. The solution is prepared by suspending 1×10 6 mesenchymal stem cells in 1 ml of normal saline. Inject only once and observe continuously for 2 months, and rinse the oral cavity with normal saline every day during the period.

[0051] (5) Group 5: Oral injection control group: Inject 1 ml of glucocorticoid and B vitamin solution into the oral soft tissue of the experimental cats. The solution contains: normal saline, 20 μM of prednisolone sodium phosphate, 2 μg / ml of vitamin B2 (riboflavin), 10 μg / ml of vitamin B3 (niacin), 10 μg / ml of vitamin B5 (pantothenic acid), 10 μg / ml of vitamin B6 (pyridoxine), 10 μg / ml of vitamin B9 (folic acid), 10 μg / ml of vitamin B12 (cobalamin). Inject only once and observe continuously for 2 months, and rinse the oral cavity with normal saline every day during the period.

[0052] (6) Group 6: Oral injection group of injection. Inject 1 ml of mesenchymal stem cell injection into the oral soft tissue of the experimental cats. The injection contains: normal saline, 1×10 6 mesenchymal stem cells, 20 μM of prednisolone sodium phosphate, 2 μg / ml of vitamin B2 (riboflavin), 10 μg / ml of vitamin B3 (niacin), 10 μg / ml of vitamin B5 (pantothenic acid), 10 μg / ml of vitamin B6 (pyridoxine), 10 μg / ml of vitamin B9 (folic acid), 10 μg / ml of vitamin B12 (cobalamin). Inject only once (using dot injection in oral soft tissue, as shown in Figure 1 , the injection points should be evenly distributed and fully covered on the inflamed tissue), and observe continuously for 2 months, and rinse the oral cavity with normal saline every day during the period.

[0053] 3. Inspection and evaluation criteria

[0054] On the 1st - 3rd and 7th days after the first treatment, examinations are conducted daily. Then, examinations are carried out once a week. Check the recovery of the inflamed tissues in the oral cavity, as well as the oral pain and feeding situation of the cat. The evaluation is carried out by professional veterinarians according to the following criteria:

[0055] A: No improvement: There has been no improvement or the improvement has reverted to the initial state (see Figure 2 , which is the A - level state of the inflammation in the oral cavity of feline stomatitis). The main symptoms include salivation, difficulty in eating, obvious pain when touching the mouth, and deep red pharyngeal mucosa.

[0056] B: Obvious improvement: No salivation, can eat, the pain response when touching the chin is reduced, and the color of the pharyngeal mucosa changes from deep red to light red (see Figure 3 , which is the B - level state of the inflammation in the oral cavity of feline stomatitis), but it has not completely turned pink.

[0057] C: Near complete recovery: No salivation, smooth eating, can touch the chin without pain, and the color of 80% of the pharyngeal mucosa area changes from deep red to pink (see Figure 4 , which is the C - level state of the inflammation in the oral cavity of feline stomatitis), and the ulcers disappear.

[0058] D: Complete recovery: No salivation, smooth eating, can touch the chin without pain, and the color of the pharyngeal mucosa changes from deep red to pink (see Figure 5 , which is the D - level state of the inflammation in the oral cavity of feline stomatitis), and the ulcers disappear.

[0059] 4. Results

[0060] (1) Group 1: Oral glucocorticoid control group. Oral prednisolone acetate was administered to the experimental cats for 3 days, and a B - level treatment effect could be achieved; after continuing to administer oral prednisolone acetate for 7 days, a C - level treatment effect was achieved; after continuously administering oral prednisolone acetate for 30 days, 3 cats recovered to the D - level complete recovery, and 2 cats recovered to the C - level. After stopping the drug, within one week, the conditions of all the cats relapsed and gradually returned to the A - level state (see Figure 6 ).

[0061] (2) Group 2: Stem cell intravenous injection group. There was no obvious change within one week after injecting stem cells. After two weeks, some treated cats began to show a certain treatment effect. After about one month, only 2 cats recovered to the B - level treatment effect, and the other 3 cats had no obvious improvement. Then, the second injection was given. The condition improved further after about two weeks. By the end of the second month, only 1 cat recovered to the C - level, 2 cats recovered to the B - level, and 2 cats had some improvement but not obvious (see Figure 6 ).

[0062] (3) Group 3: Injection intravenous injection group. During the entire treatment and observation period, the treatment effect was not obvious (see Figure 6 ).

[0063] (4) Group 4: Stem cell oral injection group. Within 3 days after oral injection, the treatment effect on all cats was not obvious; around one week, 3 cats recovered to grade B, and 2 cats had slight changes but not obvious, so they were evaluated as grade A; around two weeks, 3 cats recovered to grade D and were cured, and 1 of them remained cured without recurrence until the end of the two-month treatment and observation period. The other 2 cats had a relapse one month later and returned to grade A at the end of the treatment and observation period. The other 2 cats recovered to grade C, but their conditions started to reverse two weeks later and they returned to grade A at the end of the treatment and observation period (see Figure 6 ).

[0064] (5) Group 5: Oral injection control group: Within 3 days after oral injection, all cats had an obvious treatment effect and reached grade B efficacy. However, the condition quickly reversed and all returned to grade A (see Figure 6 ).

[0065] (6) Group 6: Injection oral injection group: Within 3 days after oral injection, all cats reached grade B treatment effect; around one week, 4 cats recovered to grade C and 1 cat remained at grade B, around two weeks, 4 cats recovered to grade D and were cured (treatment cases see Figure 7 , the left picture is before treatment and the right picture is after treatment), and remained cured without recurrence until the end of the two-month treatment and observation period. 1 cat recovered to grade C around two weeks, but had a relapse one month later and returned to grade A at the end of the treatment and observation period (see Figure 6 ).

[0066] In summary, according to groups 1 - 2, it can be known that traditional oral glucocorticoids relapse immediately after drug withdrawal and require long-term medication, and long-term medication will damage the body and have side effects; for traditional stem cell intravenous injection, the number of stem cells that can reach the inflamed oral tissues through blood circulation is limited, the effect is slow, and the treatment effect cannot be achieved. By comparing groups 3 and 6, it can be known that through the intravenous injection method, the amount of cells and drug dose that can be obtained in oral tissues will be very low, and the treatment effect cannot be achieved; while through oral soft tissue injection relaxation, the treatment effect can be significantly improved, thereby reducing the dosage of stem cells, and having the advantages of short treatment cycle, stable curative effect, and low treatment cost. By comparing groups 4, 5, and 6, it can be known that by combining mesenchymal stem cells and glucocorticoid drugs, the treatment effect of stomatitis can be significantly improved, the treatment cycle is greatly shortened, and the defect of the latter having large side effects during long-term use and the condition recurring after drug withdrawal is overcome.

[0067] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which does not affect the essence of the present invention.

Claims

1. An injection solution for the injection treatment of feline stomatitis in oral soft tissues, characterized in that, Its active ingredients include glucocorticoids and B vitamins.

2. The injection according to claim 1, characterized in that The active ingredients further include mesenchymal stem cells; the mesenchymal stem cells are cells derived from the umbilical cord, adipose tissue or bone marrow of a cat and cultured to the 4th to 6th generation.

3. The injection according to claim 2, characterized in that, The content of the mesenchymal stem cells is 1-5×10 6 cells / ml.

4. The injection according to claim 1, wherein The glucocorticoids include one or more of hydrocortisone, prednisolone, and dexamethasone; The hydrocortisone includes any one of hydrocortisone acetate, hydrocortisone sodium phosphate, and hydrocortisone sodium succinate; The prednisolone includes any one of prednisolone acetate, prednisolone sodium phosphate, and prednisolone sodium succinate; The dexamethasone includes any one of dexamethasone acetate, dexamethasone sodium phosphate, and dexamethasone sodium succinate; The concentration of glucocorticoids in the injection is 10 - 100 μM.

5. The injection according to claim 1, wherein The B vitamins include one or more of vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B9, and vitamin B12.

6. The injection according to claim 5, characterized in that, The concentration of the B vitamins is 0.1 - 105 μg / ml; among them, the concentration of vitamin B2 is 0.1 - 5 μg / ml, the concentration of vitamin B3 is 1 - 20 μg / ml, the concentration of vitamin B5 is 1 - 20 μg / ml, the concentration of vitamin B6 is 1 - 20 μg / ml, the concentration of vitamin B9 is 1 - 20 μg / ml, and the concentration of vitamin B12 is 1 - 20 μg / ml.

7. The injection according to claim 1, characterized in that, The injection further includes pharmaceutically acceptable excipients; the excipients include physiological saline.

8. The injection according to claim 1, wherein The injection is a preparation for injection into oral soft tissues; the oral soft tissues include the pharynx, palate, periodontium, and gingiva; the injection method is to select injection points at intervals of 2 - 10 mm and perform multi - point micro - injection.

9. A method for preparing an injection solution for treating feline stomatitis by injecting oral soft tissues as described in any one of claims 1-8, characterized in that, The preparation method includes: mixing the active ingredients with the excipients to prepare a solution, which is the injection.

10. Use of an injection according to any one of claims 1 - 8, or an injection obtained by the preparation method of claim 9, in the preparation of a medicament for preventing and / or treating feline stomatitis.

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