JP5610257B2 - Pharmaceutical composition and use thereof for the treatment and prevention of feline infectious peritonitis - Google Patents

Description

  The present invention relates to a pharmaceutical composition comprising cyclosporine as an active ingredient for treating and preventing feline infectious peritonitis. The present invention also relates to a method for treating and preventing FIPV by administering a cyclosporine preparation.

  Feline Infectious Peritonitis (hereinafter referred to as FIP) is a disease caused by a feline infectious peritonitis virus (hereinafter referred to as FIPV) in which the coronavirus has been mutated in the cat body. The rate is a very terrible disease, almost 100%.

  FIPV belongs to a coronavirus (also a SARS virus) and has a single-stranded plus-strand RNA as a viral genome.

  FIPV has a gene homology of 90% or more with feline intestinal coronavirus (hereinafter referred to as FCoV) and almost the same antigenicity. For this reason, it is impossible to distinguish between both viruses using a commercially available serodiagnosis kit. At present, FIPV is considered to be a variant of FCoV because it cannot be genetically distinguished from each other, and the two are only distinguished by differences in their pathogenicity.

  Clinical symptoms of FIP include infectious serositis, omental inflammation, uveitis, and granulomatous lesions of the parenchymal organs such as the kidney, mesenteric lymph nodes, liver, spleen, and central nervous system. There are two types of clinical features of FIP: exudative type (wet type) in which inflammatory exudation is observed and non-exudable type (dry type). The “wet type”, which has many cases, causes severe vasculitis in the abdomen and chest, as well as fever, diarrhea, and anemia. Ascites and pleural effusion accumulate, the abdomen swells, and dyspnea is observed. The “dry type” has a large lump like lymphoma in the abdomen. In addition, this type may cause inflammation in the brain, causing neurological symptoms such as paralysis and convulsions.

  At present, there is no radical cure for FIP, and once FIP develops, there is only symptomatic treatment to relieve symptoms by administering interferon or steroids, and in most cases it does not help. Moreover, since there is no effective vaccine, it is difficult to obtain a sufficient therapeutic effect (Non-patent Document 1).

  Macrophages are known to be FIPV reservoirs. FIPV has a virus propagation efficiency 100 times or more higher than that of FCoV in macrophages (Non-patent Document 2). However, it is not clear by what mechanism FIPV enhances replication efficiency in macrophages.

  Cyclosporine is one of the cyclic polypeptide antibiotics produced by fungi and has a function of suppressing the production and release of interleukins by T lymphocytes. Today, it is generally used as an immunosuppressant in the suppression of rejection by organ transplantation and the treatment of autoimmune diseases. The mechanism of action is not due to direct cytotoxicity, but is known to act specifically and reversibly on lymphocytes and exhibit a strong immunosuppressive effect. It is also sold as an animal drug and is used for immune related diseases such as atopic dermatitis, allergies, and dry keratoconjunctivitis. In the field of viruses, cyclosporine has been reported to exhibit growth-inhibiting effects against human immunodeficiency virus (HIV-1), cytomegalovirus (CMV), and hepatitis C virus (HCV) (non-patented). Document 3, Non-Patent Document 4, Non-Patent Document 5). However, the effect of cyclosporine on FIPV has not been clarified.

Niels C. Pedersen, Feline Infectious Disease, American Veterinary Publications, Inc. Dewerchin HL et al., Replication of feline coronaviruses in peripheral blood monocytes. Arch Virol. 2005 Dec; 150 (12): 2483-500. Epub 2005 Aug 1. Wainberg MA et al., The effect of cyclosporine A on infection of susceptible cells by human immunodeficiency virus type 1. Blood. 1988 Dec; 72 (6): 1904-10. Watashi K et al., Cyclosporin A suppresses replication of hepatitis C virus genome in cultured hepatocytes. Hepatology. 2003 Nov; 38 (5): 1282-8. Kawasaki H et al., Cyclosporine inhibits mouse cytomegalovirus infection via a cyclophilin-dependent pathway specifically in neural stem / progenitor cells.J Virol. 2007 Sep; 81 (17): 9013-23.Epub 2007 Jun 6.

  In the conventional method, since FIP could not be sufficiently prevented and / or treated, a new method capable of effectively preventing and / or treating FIP has been desired.

  Therefore, the present invention provides a pharmaceutical composition capable of efficiently inhibiting the growth of FIPV and a method for treating FIP using the pharmaceutical composition.

  Thus, as a result of intensive studies to solve the above-mentioned problems, the present inventors have found that cyclosporine can efficiently suppress the intracellular proliferation of FIPV and have completed the present invention.

That is, the present invention is as follows.
[1] A pharmaceutical composition for treating and / or preventing FIP, comprising cyclosporine as an active ingredient.
[2] The pharmaceutical composition according to [1], which suppresses the growth of FIPV.
[3] The pharmaceutical composition of [1] or [2], wherein the cyclosporine is cyclosporin A.
[4] A method for treating and / or preventing FIP, comprising administering the pharmaceutical composition of any one of [1] to [3] to a cat suffering from or likely to suffer from FIP .
[5] A method for treating and / or preventing FIP comprising administering a cyclosporine preparation to a cat suffering from or likely to suffer from FIP.

  Intracellular growth of FIPV can be significantly suppressed using a pharmaceutical composition containing cyclosporine as an active ingredient. Moreover, FIP can be efficiently prevented or treated by administering a cyclosporine preparation.

FIG. 1 is a graph showing the number of FIPV copies in cells treated with various concentrations of cyclosporin A prior to FIPV infection. FIG. 2 is a photograph of cells treated with various concentrations of cyclosporin A prior to FIPV infection. (A) Mock cells, (B) Control (dimethylsulfoxide administration treatment) cells, (C) Cyclosporin A 0.025 mg / ml treated cells, (D) Cyclosporin A 0.0125 mg / ml treated cells, (E) Cyclosporin A 0.00625 mg / ml treated cells. FIG. 3 is a graph showing the number of FIPV copies in cells treated with various concentrations of cyclosporin A after FIPV infection. FIG. 4 is a photograph of cells treated with various concentrations of cyclosporin A after FIPV infection. (A) Mock cells, (B) Control (dimethylsulfoxide administration treatment) cells, (C) Cyclosporin A 0.025 mg / ml treated cells, (D) Cyclosporin A 0.0125 mg / ml treated cells, (E) Cyclosporin A 0.00625 mg / ml treated cells. FIG. 5 is a graph showing the number of FIPV copies in cells treated with FK506 at various concentrations prior to FIPV infection. FIG. 6 is a photograph of cells treated with various concentrations of FK506 prior to FIPV infection. (A) Mock cells, (B) Control (dimethylsulfoxide administration treated) cells, (C) FK506 1 μM treated cells, (D) FK506 0.5 μM treated cells, (E) FK506 0.25 μM treated cells.

  Cyclosporine contained as an active ingredient in the pharmaceutical composition of the present invention is a known compound, and currently A to Z types such as cyclosporin A, cyclosporin B, cyclosporin C, cyclosporin D, cyclosporin H and the like are known. In the present invention, any type of cyclosporine can be used, but cyclosporin A is particularly preferable.

  Further, in the present invention, “cyclosporine” is not limited to the A to Z type cyclosporin, and includes structural analogs, derivatives, salts, and the like of these cyclosporines.

  Cyclosporine can be obtained by various methods known to those skilled in the art, and examples thereof include fermentation, biotransformation, derivatization, and partial or total synthesis. Cyclosporine can be purchased from a chemical manufacturer such as Novartis.

  The pharmaceutical composition of the present invention is administered orally or parenterally (for example, intravenous administration, intraarterial administration, local administration by injection, administration to the abdominal cavity or thoracic cavity, subcutaneous administration, intramuscular administration, sublingual administration, transdermal For example, by absorption or rectal administration).

  The pharmaceutical composition of the present invention can be made into a suitable dosage form depending on the administration route. Specifically, injections, suspensions, emulsifiers, ointments, cream tablets, capsules, granules, powders, pills, fine granules, troches, rectal administration, oily suppositories, water-soluble suppositories It can be prepared in various pharmaceutical forms such as an agent.

  These various preparations are commonly used excipients, binders, disintegrants, lubricants, diluents, solubilizers, suspending agents, isotonic agents, pH adjusting agents, buffering agents, stable agents. It can manufacture by a conventional method using an agent, a coloring agent, a corrigent, a corrigent, etc.

  Examples of excipients include lactose, sucrose, sodium chloride, glucose, maltose, mannitol, erythritol, xylitol, maltitol, inositol, dextran, sorbitol, albumin, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silica Examples include acid, methylcellulose, glycerin, sodium alginate, gum arabic, and mixtures thereof. Examples of the lubricant include purified talc, stearate, borax, polyethylene glycol, and a mixture thereof. Examples of the binder include simple syrup, glucose solution, starch solution, gelatin solution, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, carboxymethyl cellulose, shellac, methyl cellulose, ethyl cellulose, water, ethanol, potassium phosphate, and a mixture thereof. Can be mentioned. Examples of the disintegrant include dry starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose and mixtures thereof. Is mentioned. Examples of the diluent include water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, and mixtures thereof. Examples of the stabilizer include sodium pyrosulfite, ethylenediaminetetraacetic acid, thioglycolic acid, thiolactic acid, and a mixture thereof. Examples of the isotonic agent include sodium chloride, boric acid, glucose, glycerin and a mixture thereof. Examples of the pH adjuster and buffer include sodium citrate, citric acid, sodium acetate, sodium phosphate, and mixtures thereof.

  The present invention relates to a method for treating and preventing FIP by administering cyclosporine.

  In this method, the cyclosporin may be a pharmaceutical composition containing the cyclosporin as an active ingredient, or a cyclosporin preparation known for humans and animals is administered instead of and / or in addition to the pharmaceutical composition. May be. Known cyclosporine formulations that can be used in the present invention include, but are not limited to, the following:

  The dose of cyclosporine can vary depending on factors such as the age, weight and severity of disease of the subject of administration, but 1 mg to 100 mg / kg, preferably 2 mg to 50 mg / kg, more preferably 5 to 5 per day. It can be appropriately selected from the range of 20 mg / kg. In some cases, less than this may be sufficient, and vice versa. Moreover, it can also be divided and administered to 2 to 3 times a day.

  Cyclosporine can be administered prophylactically to cats that are susceptible to FIP or who are infected with FIPV and who do not develop FIP, or who are not infected with FIPV, or who have FIP. It can also be administered therapeutically to cats. “Prophylactic” refers to preventing the recurrence, onset or onset of symptoms of FIP in a subject administered due to administration of cyclosporine. “Therapeutic” refers to complete or amelioration of symptoms of FIP in a subject administered due to administration of cyclosporine.

  By administering cyclosporine prophylactically, FIPV proliferation after FIPV infection can be significantly suppressed in the administration subject. In addition, by administering cyclosporine therapeutically, the growth of FIPV after administration can be significantly suppressed.

  Cyclosporine may be administered in combination with other prophylactic and / or therapeutic agents. “In combination” means a combination with one or more prophylactic and / or therapeutic agents. The use of the term “in combination” does not limit the order in which cyclosporine and prophylactic and / or therapeutic agents are administered to a subject. The first drug is administered to the subject before the second drug is administered (for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with administration, or after administration (eg, 5 minutes, 15 Minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks later).

  Other prophylactic and / or therapeutic agents that can be administered in combination include agents known to be used for the treatment and prevention of FIP, such as steroids, antibiotics, interferons, vitamins, nutrients, etc. Although it is mentioned, it is not limited to these. Furthermore, other prophylactic and / or therapeutic agents include RNAi molecules that target mRNA of specific FIPV genes (for example, matrix gene and 3CLpro gene) (Japanese Patent Application Nos. 2008-1382 and 2008). -1419).

  The effects of cyclosporine administration can be evaluated using as an indicator that the symptoms of FIP (severity, range, etc. of the affected area) are mitigated compared to individuals who have not been administered cyclosporine or before administration. is there. The effect of cyclosporine administration is that the number of FIPV copies in the subject of administration (FIPV total mRNA indicating the number of FIPV copies or the expression level of mRNA or protein of a specific gene (marker gene)) is not administered to cyclosporine. Alternatively, it is possible to evaluate using an index that is lower than that before administration. When the measurement target is mRNA, it can be measured by Northern hybridization, RT-PCR, in situ hybridization, and the like. When the measurement target is a protein, it can be measured by Western blotting, ELISA, measurement using a protein chip to which an antibody is bound, measurement of protein activity, or the like.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

(Example 1) Virus growth inhibitory effect of cyclosporin A administered before FIP virus infection
A fetal fetal cell line fcwf-4 (ATCC CRL-2787) capable of infecting and replicating FIPV was cultured in D-MEM containing 10% fetal bovine serum in the presence of 5% CO ₂ . Specifically, the cells were seeded in a 24-well plate (2 × 10 ⁵ cells / well), and the next day, cyclosporin A (Sigma-Aldlich Japan) was added at a final concentration of 0.025 mg / ml, 0.0125 mg / ml or 0.00625 mg / ml. Diluted with dimethyl sulfoxide to a concentration and added to each well 2 hours prior to FIPV infection. Only dimethyl sulfoxide was added to the control group wells. Thereafter, the cells in each well were infected with FIPV (final concentration 10 ⁴ copies / ml). 24 hours after addition of cyclosporin A, images of cells were taken with Axiovert 200M / ApoTome (Carl Zeiss), then cells were collected from each well, and total RNA was extracted using Isogen (Nippon Gene) according to the manufacturer's recommended protocol. did.

  The obtained total RNA (0.8 μg) was synthesized into cDNA using PrimeScript cDNA synthesis kit (Takara Bio) for each sample. cDNA synthesis was performed under conditions of 25 ° C for 10 minutes, 50 ° C for 30 minutes, and 85 ° C for 5 seconds.

  Using the obtained cDNA, real-time PCR by Premix EXTaq (Takara Bio) was performed according to the manufacturer's recommended protocol. Real-time PCR was performed at 95 ° C. for 10 seconds, followed by 40 cycles of 95 ° C. for 5 seconds and 60 ° C. for 36 seconds. The above series of experiments was repeated three times or more.

  The results of real-time PCR are shown in FIG. The graph in FIG. 1 shows FIPV copy number in cells treated with various concentrations of cyclosporin A.

Cyclosporin A 0.025 mg / ml treated cells, 0.0125 mg / ml treated cells and 0.00625 mg / ml treated cells detected 5.05 × 10 ⁷ copies, 3.41 × 10 ⁸ copies and 1.04 × 10 ⁹ copies of FIPV, respectively. In contrast, 2.30 × 10 ⁹ copies of FIPV were detected in the control group not administered with cyclosporin A.

  From the above, it was revealed that FIPV proliferation can be suppressed in a dose-dependent manner by administering cyclosporin A before virus infection.

  Moreover, the image of the cell 24 hours after cyclosporin A addition is shown in FIG. In the infected cells treated with cyclosporin A 0.025 mg / ml, the effect of cytopathic degeneration mainly due to the cell fusion phenomenon was hardly observed, and the cytopathic inhibitory effect was remarkably observed (FIG. 2 (C )). As the concentration of cyclosporin A with 0.0125 mg / ml administration and 0.00625 mg / ml administration decreased, the inhibitory effect was also attenuated (FIGS. 2 (D) and (E)). This result indicates that the cytopathic inhibitory effect is dependent on the concentration of cyclosporin A, and correlates with the result of the real-time PCR showing the amount of FIPV proliferation.

(Example 2) Virus growth inhibitory effect of cyclosporin A administered after infection with FIP virus The above cell line fcwf-4 (2 x 10 ⁵ cells / well) was transferred to a 24-well plate using D-MEM containing 10% fetal bovine serum. The cells were cultured overnight in the presence of scattered 5% CO ₂ . Thereafter, the cells in each well were infected with FIPV (10 ⁴ copies / ml). Two hours after FIPV infection, cyclosporin A was added to each well to a final concentration of 0.025 mg / ml, 0.0125 mg / ml or 0.00625 mg / ml. Only dimethyl sulfoxide was added to the control group wells. 24 hours after adding cyclosporin A, images of the cells were taken with Axiovert 200M / ApoTome (Carl Zeiss), and then the cells were collected from each well, and the total RNA was extracted as described above.

  CDNA synthesis was performed using the obtained total RNA in the same manner as described above, and real-time PCR was performed using the cDNA under the above conditions. The above series of experiments was repeated three times or more.

  The results of real-time PCR are shown in FIG. The graph of FIG. 3 shows FIPV copy number in cells treated with various concentrations of cyclosporin A.

1.98 × 10 ⁷ copies, 1.25 × 10 ⁸ copies and 1.94 × 10 ⁹ copies of FIPV were detected in cyclosporin A 0.025 mg / ml treated cells, 0.0125 mg / ml treated cells and 0.00625 mg / ml treated cells, respectively. In contrast, 2.31 × 10 ⁹ copies of FIPV were detected in the control group not administered with cyclosporin A.

  From the above, it was revealed that FIPV proliferation can be suppressed in a dose-dependent manner by administering cyclosporin A after virus infection.

  Moreover, the image of the cell 24 hours after cyclosporin A addition is shown in FIG. In the infected cells treated with cyclosporin A 0.025 mg / ml, the effect of cytopathic degeneration mainly due to the cell fusion phenomenon was hardly observed, and the cytopathic inhibitory effect was remarkably observed (FIG. 4 (C )). As the concentration of cyclosporin A with 0.0125 mg / ml administration and 0.00625 mg / ml administration decreased, the inhibitory effect was also attenuated (FIGS. 4D and 4E). This result indicates that the cytopathic inhibitory effect is dependent on the concentration of cyclosporin A, and correlates with the result of the real-time PCR showing the amount of FIPV proliferation.

(Example 3) Examination of FIPV growth inhibitory effect by FK506
A fetal fetal cell line fcwf-4 (ATCC CRL-2787) capable of infecting and replicating FIPV was cultured in D-MEM containing 10% fetal bovine serum in the presence of 5% CO ₂ . Specifically, the cells are seeded in a 24-well plate (2 × 10 ⁵ cells / well), and the next day, FK506 (Sigma-Aldlich Japan), which is generally used as an immunosuppressant like cyclosporin A, has a final concentration of 1 μM and 0.5 μM. Or diluted with dimethyl sulfoxide to 0.25 μM and added to each well 2 hours prior to FIPV infection. Dimethyl sulfoxide was added to the control group wells. Thereafter, the cells in each well were infected with FIPV (final concentration 10 ⁴ copies / ml). 24 hours after addition of FK506, images of cells were taken with Axiovert 200M / ApoTome (Carl Zeiss), then cells were collected from each well, and total RNA was extracted using Isogen (Nippon Gene) according to the manufacturer's recommended protocol. .

  The obtained total RNA (0.8 μg) was synthesized into cDNA using PrimeScript cDNA synthesis kit (Takara Bio) for each sample. cDNA synthesis was performed under conditions of 25 ° C for 10 minutes, 50 ° C for 30 minutes, and 85 ° C for 5 seconds.

  Using the obtained cDNA, real-time PCR by Premix EXTaq (Takara Bio) was performed according to the manufacturer's recommended protocol. Real-time PCR was performed at 95 ° C. for 10 seconds, followed by 40 cycles of 95 ° C. for 5 seconds and 60 ° C. for 36 seconds. The above series of experiments was repeated three times or more.

  The results of real-time PCR are shown in FIG. The graph of FIG. 5 shows the number of FIPV copies in cells treated with each concentration of FK506.

7.55 × 10 ⁸ copies, 1.09 × 10 ⁹ copies and 1.20 × 10 ⁹ copies of FIPV were detected in FK506 1 μM treated cells, 0.5 μM treated cells and 0.25 μM treated cells, respectively. In contrast, 2.36 × 10 ⁸ copies of FIPV were detected in the control group not administered with FK506.

  From the above, as a result, the effect of FK506 on the suppression of viral growth of FIPV was not observed.

  Moreover, the image of the cell 24 hours after FK506 addition is shown in FIG. In the infected cells treated with FK506, no inhibitory effect such as cell degeneration mainly due to cell fusion was observed (FIGS. 6 (C)-(E)). This result correlates with the above real-time PCR result showing the amount of FIPV proliferation.

  From the above results, it was revealed that FIPV proliferation can be significantly suppressed by treating infected cells with cyclosporin A before or after FIPV infection. At this time, no toxic effect of cyclosporin A on the cells was observed, and it was confirmed that cyclosporin A exhibits a virus-specific growth inhibitory effect. In addition, the growth-inhibiting effect is volume-dependent, and it becomes clear that the cell fusion phenomenon, which is a cytopathic effect by FIPV, can be suppressed almost 100% by using cyclosporin A with a final concentration of 0.025 mg / ml. It was. On the other hand, the above effect was not obtained in the infected cells treated with FK506, and it was revealed that this effect is specific to cyclosporin A.

  By administering cyclosporine before or after infection with FIPV, it is possible to significantly suppress the growth of FIPV, and it is expected to contribute to the prevention and / or treatment of FIP.

Claims (6)

  A pharmaceutical composition for treating and / or preventing feline infectious peritonitis, comprising cyclosporine as an active ingredient.   The pharmaceutical composition according to claim 1, which suppresses the growth of feline infectious peritonitis virus.   The pharmaceutical composition according to claim 1 or 2, wherein the cyclosporine is cyclosporin A.   A method for treating and / or preventing feline infectious peritonitis comprising the step of administering the pharmaceutical composition according to any one of claims 1 to 3 to a cat suffering from or at risk of suffering from feline infectious peritonitis. How to do.   A method for treating and / or preventing feline infectious peritonitis comprising administering a cyclosporine formulation to a cat suffering from or at risk of suffering from feline infectious peritonitis. A pharmaceutical composition for suppressing the growth of feline infectious peritonitis virus, comprising cyclosporine as an active ingredient.

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