Application of antiviral nucleoside analogue in preparation of medicine for treating feline coronavirus

Abstract

According to the application of the antiviral nucleoside analogue shown in the general formula (I) in preparation of the medicine for treating or preventing the feline coronavirus, the antiviral nucleoside analogue can effectively resist the feline coronavirus and has the advantages of being short in treatment cycle, high in treatment index and high in safety.

Description

Use of antiviral nucleoside analogs in the preparation of drugs for treating feline coronavirus Technical Field

[0001] The invention belongs to the field of antiviral drugs, and in particular relates to use of an antiviral nucleoside analog in preparing a drug for treating feline coronavirus. Background Art

[0002] Feline coronavirus (FCoV) belongs to the Coronaviridae family and is a group of enveloped, positive-strand RNA viruses commonly found in felines. In nature, FCoV exists as two distinct biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FIPV is generally considered a mutated form of FECV.

[0003] FECV infection is widespread in cats, with estimates worldwide exceeding 50% of cats carrying and transmitting the virus, and up to 80% of cats in multi-cat settings. FECV chronically infects the gastrointestinal epithelium of cats and is typically transmitted via the fecal-oral route. While most cats infected with FECV are asymptomatic, some may experience diarrhea, vomiting, loss of appetite, and fever.

[0004] In FIPV, more than two-thirds of the 3c gene contains mutations, including deletions and point mutations. These mutations prevent the 3c gene from expressing the complete 3c protein. It is generally believed that mutations in the 3c gene do not affect FIPV virulence, but it cannot be ruled out that such mutations lead to changes in the cell tropism of FECV, thereby increasing the fitness of monocytes and macrophages and leading to the development of FIPV. The coronavirus S protein plays a key role in receptor binding for viral invasion, and the conversion from FECV to FIPV involves mutations in the S protein gene, resulting in changes in target cell tropism.

[0005] Feline infectious peritonitis (FIP) is a chronic, progressive, and fatal infectious disease caused by the feline infectious peritonitis virus. It is characterized by peritonitis, the accumulation of large amounts of ascites, and a high mortality rate. Epidemiological characteristics: FIP primarily infects cats aged 0.5 to 5 years, with increased susceptibility in cats under 12 months of age. Purebred cats, those housed in mixed groups, and stray cats are particularly susceptible. The course of the disease can be sudden (often occurring in kittens) or chronic, lasting several weeks. Initial symptoms are subtle, with possible loss of appetite, poor spirits, weight loss, and persistent, recurring fever. Later, symptoms become distinctly divided into dry and wet forms, or even a combination. With the wet form, cats often die within two months of onset. High-protein exudate is present in the chest and abdominal cavities. Depending on the amount of pleural effusion, symptoms range from no symptoms to wheezing or difficulty breathing. Gradual, painless abdominal enlargement occurs, and male cats may also experience scrotal enlargement. Affected cats may also experience vomiting or diarrhea, and moderate to severe anemia. Clinical examination will reveal progressive muscle wasting and emaciation along the spine, and a progressive enlargement of the abdomen. Dry type: The dry type primarily causes pus-granulomatous lesions in organs, with cats exhibiting symptoms such as progressive weight loss, cloudy eyes, pus in the anterior chamber, miosis, and visual impairment. A minority are accompanied by multiple progressive neurological symptoms, including hind limb paralysis, spasms and tremors, nystagmus, and personality changes. Nodular lesions may develop in the liver, kidneys, spleen, lungs, omentum, and lymph nodes; mesenteric lymph nodes may be palpated on abdominal palpation. A certain percentage of cases also present with anemia and jaundice.

[0006] Traditional treatment for FIP focuses on palliative care, using antibiotics, anti-inflammatory drugs, and interferon to alleviate the inflammatory symptoms and infection caused by FIP. However, traditional therapies are ineffective in controlling the disease, as the virus cannot be controlled or eliminated from the body, ultimately leading to the cat's death. Therefore, FIP is considered a "terminal illness" in cats.

[0007] Currently, there is no approved effective vaccine or safer, more effective antiviral therapy for FIP. No medication is approved specifically for the treatment of this disease. Due to its unique pathogenesis, there is no effective vaccine to prevent it. FIP is a leading cause of death in cats under two years of age, with an estimated mortality rate of 0.3% to 1% worldwide.

[0008] Sofosbuvir is a drug for treating hepatitis C caused by the hepatitis C virus. Its chemical name is (S)-isopropyl-2-((S)-((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-(phenoxy)phosphoamino)propionate. The hepatitis C virus belongs to the Flaviviridae family, and the mechanism of action of sofosbuvir is to specifically bind to the NS5B protein of the hepatitis C virus to prevent the replication of the virus. In the treatment of hepatitis C, sofosbuvir has an extremely high cure rate and is extremely safe, but it has not yet been successfully applied to other types of antiviral treatments. Due to the different types of viruses and different pathogenic mechanisms, sofosbuvir cannot theoretically be successfully used to treat different types of viruses.

[0009] Therefore, there is a great need to develop a safe and effective drug for the treatment of FIP, which is of practical significance for solving the problem of no drugs available in this field. Summary of the Invention

[0010] In order to overcome the deficiencies of the prior art, the present invention provides an antiviral nucleoside analogue represented by general formula (I) or a pharmaceutically acceptable salt thereof for use in preparing a medicament for treating or preventing feline coronavirus.

[0011] Wherein, R1 is hydrogen, deuterium, n-alkyl, branched alkyl, cycloalkyl, or phenyl; as one embodiment, one or more H in the phenyl group may be optionally substituted by at least one of the following substituents: alkyl, alkenyl, alkynyl, alkoxy, F, Cl, Br, I, nitro, cyano, haloalkyl, N(R1′)2, C 1-6 Acylamino, -NHSO2, C 1-6 Alkyl, or -SO2; said R1' is independently hydrogen or alkyl;

[0012] R2 is hydrogen, deuterium, or alkyl;

[0013] R3 and R4 are hydrogen, deuterium, n-alkyl, branched alkyl, haloalkyl, halogen, alkenyl, alkynyl, cycloalkyl, cyano, alkoxy, aryl, or heterocyclic group;

[0014] The R5 is hydrogen, deuterium, alkyl, halogen, amino, nitro, hydroxy, cyano, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

[0015] In the present invention, as one of the embodiments, the antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof is used in the preparation of a drug for treating or preventing feline enteric coronavirus.

[0016] In the present invention, as one embodiment, the antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof is used in the preparation of a drug for treating or preventing feline infectious peritonitis virus.

[0017] In the present invention, as one of the embodiments, the antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof is used in the preparation of a drug for treating or preventing feline infectious peritonitis.

[0018] In the present invention, as one embodiment, the antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof includes its isotopes, stereoisomers, tautomers, esters or amides.

[0019] In the present invention, as one embodiment, the antiviral nucleoside analog represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the compound represented by formula (Ia) (also known as sofosbuvir):

[0020] It should also be understood that modifications of the pharmacodynamic structure or basic pharmacodynamic structure of sofosbuvir (such as Formula I) are also within the scope of the present invention.

[0021] In the present invention, as one of the embodiments, the disease caused by the feline coronavirus infection of the present invention is feline infectious peritonitis caused by feline infectious peritonitis virus.

[0022] In the present invention, as one embodiment, the effective dose of the antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof is 0.1-500 mg / kg;

[0023] In the present invention, as one embodiment, the pharmacologically effective dose of the compound of formula (Ia) is 0.1-500 mg / kg.

[0024] In the present invention, as one embodiment, the antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof is used in combination with "other antiviral drugs".

[0025] The "other antiviral drugs" mentioned in the present invention refer to antiviral drugs other than the antiviral nucleoside analogs represented by general formula (I) or pharmaceutically acceptable salts thereof, and the other antiviral drugs include but are not limited to remdesivir, elbavir, ravidasvir, monolavir, tenofovir, entecavir, parovird, olfosbuvir, grazoprevir, voxilaprevir, mindevir, azithromycin, favipiravir, senoxan, leretovir, benoforvir, ensetvir, clopavir, danoprevir or imipenem, or a combination of two or more thereof.

[0026] In the present invention, as one embodiment, it is preferred that the antiviral nucleoside analog represented by general formula (Ia) or a pharmaceutically acceptable salt thereof is used in combination with "other antiviral drugs".

[0027] In the present invention, as one of the embodiments, the antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof, or the antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof is combined with other antiviral drugs in the form of a pharmaceutical composition, and the pharmaceutical composition comprises: an effective dose of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a combination of an antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof and other antiviral drugs, and pharmaceutically acceptable excipients.

[0028] In the present invention, as one of the embodiments, the present invention provides a pharmaceutical composition for treating or preventing diseases caused by feline coronavirus infection, wherein the pharmaceutical composition comprises sofosbuvir.

[0029] In the present invention, as one embodiment, the pharmaceutical composition of the present invention generally contains 0.1 to 100% by weight of the antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof; as one embodiment, the antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof in a unit dosage form generally contains 0.1 to 500 mg.

[0030] In the present invention, as one embodiment, the pharmaceutical composition of the present invention generally contains 0.1 to 100% by weight of sofosbuvir; as one embodiment, the content of the compound of the present invention in a unit dosage form is generally 0.1 to 500 mg.

[0031] The pharmaceutical composition of the compounds of this invention can be prepared according to methods well known in the art. When used for this purpose, if necessary, the compounds of this invention can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants to make suitable application forms or dosage forms for veterinary use.

[0032] The compound of the present invention or the pharmaceutical composition containing the same can be administered in a unit dosage form. The main routes of administration are oral administration and injection. If necessary, other routes can be used, such as intestinal administration, peritoneal administration, mucosal administration, vaginal administration or rectal administration.

[0033] The dosage form can be a liquid, semisolid, or solid dosage form. For example, liquid dosage forms can be true solutions, colloids, microparticles, emulsions, or suspensions. Other dosage forms include tablets, capsules, pellets, pills, powders, solutions, emulsions, granules, and suppositories.

[0034] The compound of the present invention can be prepared into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle delivery systems.

[0035] For example, during treatment, the dosage form can be prepared as an injection, and various solvents and additives known in the art can be widely used to prepare solutions, suspensions, emulsions, and freeze-dried powder injections. Such preparations can be aqueous or non-aqueous and can contain one or more pharmacologically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants, dispersants, osmotic pressure regulators, solubilizers, and pH regulators. For example, the diluent can be water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitol esters, fatty acid esters, or a combination of two or more thereof. The osmotic pressure regulator can be sodium chloride, mannitol, glycerol, glucose, phosphate, acetate, etc., or a combination of two or more thereof; the solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl β-cyclodextrin, etc., or a combination of two or more thereof; the pH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc., or a combination of two or more thereof. If a lyophilized powder injection is prepared, mannitol, glucose, etc., or a combination of the two can also be added as a support. In addition, if necessary, colorants, preservatives, spices, flavoring agents, sweeteners, or spices can also be added to the pharmaceutical preparation. These excipients are commonly used in the art.

[0036] For example, in order to prepare a unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc., or a combination of two or more thereof; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc., or a combination of two or more thereof; disintegrants, such as dry starch, Alginate, agar powder, brown seaweed starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, or combinations of two or more thereof; disintegration inhibitors such as sucrose, tristearin, cocoa butter, hydrogenated oil, or combinations of two or more thereof; absorption enhancers such as quaternary ammonium salts and sodium lauryl sulfate; lubricants such as talc, silicon dioxide, corn starch, stearates, boric acid, liquid paraffin, polyethylene glycol, or combinations of two or more thereof. Tablets can also be further prepared as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets.

[0037] For example, to prepare the dosing unit into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, glyceryl monostearate, kaolin, talc, or combinations of two or more thereof; binders such as gum arabic, tragacanth gum, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste, or combinations of two or more thereof; and disintegrants such as agar powder, dry powder, alginate, sodium lauryl sulfate, methylcellulose, ethylcellulose, or combinations of two or more thereof.

[0038] For example, to prepare a dosing unit in the form of a capsule, the active ingredient compound of the present invention, such as sofosbuvir, is mixed with the various carriers described above, and the resulting mixture is placed in a hard gelatin capsule or soft capsule. Alternatively, the active ingredient compound of the present invention may be formulated into microcapsules, suspended in an aqueous medium to form a suspension, which may be encapsulated in a hard capsule or formulated as an injection.

[0039] In addition, if necessary, coloring agents, preservatives, spices, flavoring agents, sweeteners or spices can also be added to the pharmaceutical preparations. These excipients are commonly used in the art.

[0040] The sterile media used in the present invention can be prepared by standard techniques well known to those skilled in the art. They can be sterilized, for example, by filtering through a bacterial filter, by adding a sterilizing agent to the composition, by irradiating the composition, or by heating the composition. They can also be prepared into sterile injectable media immediately prior to use.

[0041] To achieve the intended use and enhance the therapeutic effect, the medicaments or pharmaceutical compositions of the present invention can be administered using any known method of administration. The optimal route of administration for administering the compounds of the present invention will, of course, depend on the disease and the site of treatment. Because the pharmacokinetic and pharmacodynamic characteristics of the compounds of the present invention vary to some extent, the most preferred method for achieving therapeutic concentrations in tissues is to gradually increase the dosage and monitor the clinical effects. For such gradually increasing therapeutic doses, the initial dose will depend on the route of administration.

[0042] The dosage of the pharmaceutical compositions of the compounds of the present invention administered to any particular patient depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, personality and individual response of the patient or animal, the route of administration, the number of doses, and the purpose of the treatment. Therefore, the therapeutic dosage of the present invention may vary widely. Depending on the condition of the patient being treated, some variation in dosage may be necessary, and in all cases, the physician will determine the appropriate dosage for an individual patient.

[0043] The dosage refers to the weight of the compound excluding the weight of the carrier (when a carrier is used). Generally speaking, the dosages of the pharmaceutical ingredients of the present invention are well known to those skilled in the art. The actual amount of drug contained in the final formulation of the compound composition of the present invention can be appropriately adjusted to achieve the therapeutically effective amount required to achieve the preventive or therapeutic purpose of the present invention. Administration can be in the form of a single dose or divided into several doses, such as two, three, or four doses; this is limited by the clinical experience of the administering physician and the dosing regimen including the use of other treatment methods.

[0044] The compound or composition of the present invention can be taken alone, or used in combination with other therapeutic drugs or symptomatic drugs and the dosage can be adjusted.

[0045] In the present invention, we surprisingly discovered for the first time that sofosbuvir, as an anti-hepatitis C virus compound, can effectively achieve the effect of resisting feline infectious peritonitis virus, and can treat and improve various symptoms of feline infectious peritonitis caused by feline infectious peritonitis virus. In the examples, all cases were cured after receiving treatment for no more than 14 days, and no recurrence or treatment-related adverse reactions were found in the follow-up after treatment. It has the advantages of short treatment cycle, high therapeutic index, and high safety. Beneficial effects

[0046] Many compounds in the prior art use cat kidney cells as an in vitro model for anti-feline infectious peritonitis virus. We were surprised to find for the first time that in vitro cat kidney cells and cat lung cells cannot be used as a good in vitro model for sofosbuvir to screen related antiviral drugs. For drug metabolic transformation, inconsistent expression of cell-related metabolic enzymes in different tissues can lead to different drug metabolic transformations, and inappropriate in vitro models cannot actually explain the in vivo drug metabolic transformation and pharmacological effects. In the present invention, we were surprised to find for the first time that sofosbuvir, as an anti-hepatitis C virus compound, can effectively treat feline infectious peritonitis cases, eliminate feline coronavirus in the case, and can treat and improve various symptoms of feline infectious peritonitis caused by feline infectious peritonitis virus, and improve physiological and biochemical indicators. In the examples, all cases were cured after receiving treatment for no more than 21 days, and no recurrence or treatment-related adverse reactions were found in the follow-up after treatment. It has the advantages of short treatment cycle, high therapeutic index, and high safety.

[0047] In the present invention, the treatment of cases suffering from feline infectious peritonitis shows that sofosbuvir can safely and effectively treat various symptoms caused by feline infectious peritonitis, eliminate feline infectious peritonitis virus in the body, and restore body functions (for example, body temperature returns to normal, weight gradually increases, and white-globulin ratio gradually increases to above 0.6). It has the advantages of short treatment cycle, high therapeutic index, and high safety. No drug-related adverse reactions occur during treatment, and no recurrence or related adverse reactions are found after long-term follow-up.

[0048] The present invention can fill the gap in feline infectious peritonitis drugs in the veterinary drug market and has broad market application value prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1: Chest and abdominal X-rays before and after Lanbei treatment. A and B are before treatment, and C and D are after treatment.

[0050] Figure 2: Chest and abdominal X-rays before and after treatment with Laifu. A and B are before treatment, and C and D are after treatment.

[0051] Figure 3: Chest and abdominal X-rays before and after Tangtang treatment. A and B are before treatment, and C and D are after treatment. DETAILED DESCRIPTION

[0052] The following examples are used to further illustrate the present invention, but are not intended to limit the effective scope of the present invention in any way.

[0053] Example 1: Determination of the antiviral effect of sofosbuvir using different cell lines in vitro

[0054] Feline kidney cells (CRFK, Crandell Reese Feline Kidney), feline lung cells (AK-D) and feline infectious peritonitis virus (FIPV-79-1146) were purchased from the American Type Culture Collection (ATCC). After cell recovery, the cells were placed in a 5% CO2, 37°C incubator in DMEM (Dulbecco's Modified Eagle's Medium) culture medium containing 10% FBS. When the cells grew to 80%-90% density, 0.25% trypsin was used to digest the cells. After digestion, the cells were pipetted into the culture medium, centrifuged to obtain the cells, and the cells were counted. 8,000 cells were plated in each well of a 96-well plate and cultured for 12 hours. After 12 hours, sofosbuvir solutions with different concentration gradients and equal amounts of virus were added to the relevant groups, with 5 replicates per group. The virus control group was added with equal amounts of solutions that did not contain sofosbuvir but contained equal amounts of virus. The blank control group was added with equal amounts of solutions that contained neither sofosbuvir nor virus. Each well contained 0.1% DMSO. The cells were incubated for 48 hours. After 48 hours, the viral inhibition effect was determined using the CCK8 assay.

[0055] Results: As shown in Tables 1 and 2, sofosbuvir failed to achieve a significant antiviral effect on CRFK and AK-D. Sofosbuvir requires metabolization to triphosphate nucleoside analogs to exert its pharmacological activity. Different tissues and cells in the body express different metabolic enzymes. Therefore, this example demonstrates that CRFK and AK-D are unable to convert sofosbuvir into an effective antiviral active structure.

[0056] Table 1 CRFK virus inhibition rate of FIPV infection at different drug concentrations of sofosbuvir

[0057] Table 2 Inhibition rate of AK-D virus against FIPV infection at different drug concentrations of sofosbuvir

[0058] Example 2: Intravenous administration of sofosbuvir to treat cases of feline infectious peritonitis

[0059] The cases in this embodiment were recruited from animals that were seen daily at a formally qualified pet hospital. They were diagnosed with feline infectious peritonitis by clinical diagnosis during the visit. Sofosbuvir intravenous injection was prepared by pharmaceutically feasible means, and the preparation method was as follows: 5g of pharmaceutical grade sofosbuvir compound was weighed, placed on a magnetic stirrer, water for injection was added, a pH regulator was added to stabilize the pH at 6.5-7.0, and the solution was diluted to 1L. The final concentration of sofosbuvir was 5mg / mL. After sufficient dissolution, the sofosbuvir intravenous injection was filtered using a 0.22μm filter membrane in a biological safety cabinet and packaged into a sterile, pyrogen-free container for use. 5-40mg / kg was preferably administered for treatment during clinical treatment. Preferably, according to body weight, after calculating the dosage, each case was divided into two injections per day for treatment. During the treatment, the symptoms and physiological and biochemical indicators of the cats were observed. Other manifestations included loss of appetite, poor spirits, weight loss, and continued recurrent fever.

[0060] Results: See Table 3. Among the cases that came for treatment, a typical moderate case, case 3, had its fever under control and its appetite gradually recovered after three days of continuous treatment. In order to completely cure it, after 21 days of supportive treatment, the case completely recovered to normal and its weight gradually increased. Case 2 and case 3 were more serious when they were enrolled, with some abdominal effusion, but after seven days of medication, their body temperature returned to normal, their mental state changed significantly, their appetite returned, their weight gradually increased, and the abdominal effusion disappeared. No adverse reactions occurred during the treatment of the cases in this example, and no recurrence was observed during the 90-day follow-up observation after the end of treatment.

[0061] Table 3 Statistics of confirmed types and recovery status of cases treated with intravenous injection

[0062] Example 3: Subcutaneous and oral administration of sofosbuvir in the treatment of feline infectious peritonitis

[0063] The cases in this example were recruited in the same manner as in Example 2. Sofosbuvir subcutaneous injection and sofosbuvir oral tablets were prepared by pharmaceutically feasible means, and 5-60 mg / kg was preferably administered for treatment during clinical treatment. Preparation of Sofosbuvir subcutaneous injection: Use pharmaceutical grade PEG 400 for injection and water for injection to prepare a 20% (v / v) PEG400 solution as a solvent. Weigh 10 g of pharmaceutical grade sofosbuvir compound, add solvent to dissolve thoroughly, add a pH regulator to stabilize the pH at 6.5-7.0, add 20% PEG solution to the solution and make the volume to 250 mL, so that the final concentration of sofosbuvir is 40 mg / mL. After sufficient dissolution, filter the sofosbuvir subcutaneous injection using a 0.22 μm filter membrane in a biological safety cabinet and package it into a sterile, pyrogen-free container for use. Preparation of sofosbuvir oral tablets: Weigh 15g of pharmaceutical grade sofosbuvir compound, 13.62g of pharmaceutical grade mannitol, 11.38g of microcrystalline cellulose, 1.12g of cross-linked carboxymethyl cellulose sodium, 0.22g of colloidal silicon dioxide, and 0.34g of magnesium stearate and place them in a mixer. Stir thoroughly and evenly, place the mixed powder in a tablet press, control the tablet weight to 0.208g, and compress 200 sofosbuvir oral tablets. The oral tablets are placed in a pharmaceutical packaging bottle for use. Preferably, based on body weight, after calculating the dosage, each case is divided into two subcutaneous injections per day for treatment on days 0-14. Oral tablets are given to support stable improvement of case symptoms on days 15-21. During the treatment, the symptoms and physiological and biochemical indicators of the cats are observed. Other manifestations include loss of appetite, poor spirits, weight loss, and persistent and recurrent fever.

[0064] Results: See Table 4. Among the cases that came for treatment, case 2 had severe ascites, repeated fever, and loss of appetite. After 14 days of continuous subcutaneous injection treatment, the ascites was absorbed, the fever was controlled, and the appetite gradually recovered. In order to completely cure the disease, oral supportive treatment was subsequently used for seven days of medication. The case completely recovered to normal and the weight gradually increased. Among them, case 1 and case 3 were more serious, with a certain amount of abdominal effusion. Case 1 was determined to be a mixed case of dry type with concurrent wet type. However, after seven days of medication, the body temperature of these two more serious cases returned to normal, the mental state obviously changed to normal, the appetite recovered, the weight gradually increased, and the abdominal effusion disappeared. The dry confirmed cases had obvious symptoms subsided, the body temperature recovered, the eating returned to normal, and the mental state was good. No adverse reactions occurred during the treatment of the cases in this embodiment, and no recurrence occurred during the 90-day follow-up observation after drug discontinuation.

[0065] Table 4 Statistics of confirmed cases and recovery status of subcutaneous injection and oral administration cases

[0066] Example 4: Subcutaneous injection of sofosbuvir clears the virus from the body and cures a case of feline infectious peritonitis

[0067] To further demonstrate that sofosbuvir is not applicable to in vitro cell models but can achieve the actual therapeutic effect of clearing the virus in vivo, this example recruited 8 cases of feline infectious peritonitis. In this recruitment, wet feline infectious peritonitis required ascites to be tested positive for feline infectious peritonitis virus by qPCR before inclusion in treatment, and dry feline infectious peritonitis required fine needle aspiration of the mesenteric lymph node and then tested positive for feline infectious peritonitis virus by qPCR before inclusion in treatment. During treatment, various physiological and biochemical indicators and changes in serum amyloid protein (SAA) were tested, changes in viral load during treatment were monitored, and the clinical manifestations of the cats were observed. Changes in the chest and abdomen were observed by X-ray imaging before and after treatment.

[0068] In this example, the administration method was subcutaneous injection, and sofosbuvir was administered twice a day. The preparation of sofosbuvir subcutaneous injection was the same as that in Example 3. The time of enrollment was recorded as D0.

[0069] Results: As shown in Table 5, Table 6, Table 7, Table 8, Figure 1, Figure 2, and Figure 3.

[0070] Table 5 shows the information of the enrolled cases. At the time of enrollment, each case was positive for feline infectious peritonitis virus (CT < 30 was positive; 30 < CT < 35 was suspected; CT > 35 was negative). The SAA value was abnormally increased, and the white-globulin ratio was abnormally decreased.

[0071] Table 6 shows the viral load of feline infectious peritonitis patients during treatment. Seven of the eight patients tested negative for the virus after treatment and were discharged. Case Guaiguai's viral load decreased during treatment, but due to his frail condition, he was unable to sustain further treatment after a week and died on Day 8.

[0072] Table 7 shows the results of serum amyloid protein (SAA) treatment. This level can be abnormally elevated in cats with infectious peritonitis or other inflammatory conditions. After sofosbuvir treatment, 6 / 8 cases showed effective improvement and reduction. However, one / 8 cases experienced a broken hind leg upon entry and exit from the group, which the veterinarian determined was the cause of the elevated SAA level at the end of the group.

[0073] Table 8 shows the albumin / globulin (A / G) ratio. In feline infectious peritonitis, albumin levels are low and globulin levels are high, resulting in a decreased A / G ratio. After 1-3 weeks of treatment, 6 / 8 cases showed significant recovery, reaching 0.6 or higher. Hairballs, due to a broken leg, did not experience a significant increase in the A / G ratio.

[0074] Figures 1, 2, and 3 show chest and abdominal radiographs of wet-type feline infectious peritonitis before and after treatment with sofosbuvir. The wet-type feline infectious peritonitis case showed abdominal enlargement due to pleural and abdominal effusions caused by complications of the viral infection (Figures 1A, 1B; Figures 2A, 2B; Figures 3A, 3B). The pleural and abdominal effusions significantly resolved after treatment with sofosbuvir (Figures 1C, 1D; Figures 2C, 2D; Figures 3C, 3D).

[0075] Table 5 Information of each case at the time of enrollment

[0076] Table 6 Virus clearance in patients at each examination time point

[0077] Table 7 Statistics of changes in SAA values ​​at each checkpoint in cases

[0078] Table 8 Statistics of changes in the white-globulin ratio (A / G) at each examination point in the cases

Claims

1. Use of an antiviral nucleoside analogue represented by general formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing feline coronavirus, in, R1 is hydrogen, deuterium, n-alkyl, branched alkyl, cycloalkyl, or phenyl; One or more H in the phenyl group is optionally substituted by one or more of the following substituents: alkyl, alkenyl, alkynyl, alkoxy, F, Cl, Br, I, nitro, cyano, haloalkyl, N(R1')2, C 1-6 Acylamino, -NHSO2, C 1-6 Alkyl, -SO2; Said R1' is independently hydrogen or alkyl; R2 is hydrogen, deuterium, or alkyl; The R3 or R4 are each independently hydrogen, deuterium, n-alkyl, branched alkyl, haloalkyl, halogen, alkenyl, alkynyl, cycloalkyl, cyano, alkoxy, aryl, or heterocyclic group; The R5 is hydrogen, deuterium, alkyl, halogen, amino, nitro, hydroxy, cyano, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

2. The use according to claim 1, characterized in that The use of the antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing feline enteric coronavirus.

3. The use according to claim 1, characterized in that Use of the antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof in preparing a drug for treating or preventing feline infectious peritonitis virus.

4. The use according to claim 1, characterized in that Use of the antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof in preparing a drug for treating or preventing feline infectious peritonitis.

5. The use according to claim 1, characterized in that The antiviral nucleoside analog represented by the general formula (I) or a pharmaceutically acceptable salt thereof includes its isotopes, stereoisomers, tautomers, esters or amides.

6. The use according to any one of claims 1 to 5, characterized in that The antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof is selected from:

7. The use according to claim 1, characterized in that The effective dosage of the antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof administered to a subject is 0.1-500 mg / kg.

8. The use according to claim 6, characterized in that The effective dosage of the antiviral nucleoside analog represented by general formula (Ia) or a pharmaceutically acceptable salt thereof administered to a subject is 0.1-500 mg / kg.

9. The use according to claim 1, characterized in that The antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof is used in combination with "other antiviral drugs", wherein: The “other antiviral drugs” include remdesivir, elbavir, ravidavir, monolavir, tenofovir, entecavir, parovird, olfosbuvir, grazoprevir, voxilaprevir, mindevir, azithromycin, favipiravir, senoxan, levitra, beaufortvir, ensetvir, clopavir, danoprevir or imipenem, or a combination of two or more thereof.

10. The use according to claim 9, characterized in that The antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof is an antiviral nucleoside analog represented by general formula (Ia) or a pharmaceutically acceptable salt thereof.

11. The use according to claim 1 or 9, characterized in that The antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof, or a combination of the antiviral nucleoside analog represented by general formula (I) or a pharmaceutically acceptable salt thereof with other antiviral drugs is in the form of a pharmaceutical composition, and the pharmaceutical composition comprises: An effective dose of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or an effective dose of an antiviral nucleoside analogue of general formula (I) or a pharmaceutically acceptable salt thereof and a combination of other antiviral drugs; and Pharmaceutically acceptable excipients.

12. The use according to claim 11, characterized in that The pharmaceutical composition is a liquid preparation, solid or semisolid preparation, sustained-release preparation, controlled-release preparation, targeted preparation, tablet, capsule, dripping pill, pill, powder, suspension, injection, emulsion, granule, lyophilized powder injection or suppository.

13. The use according to claim 12, characterized in that When the pharmaceutical composition is an injection, solution, suspension, emulsion or lyophilized powder injection, The pharmacologically acceptable excipients include diluents, adhesives, lubricants, preservatives, surfactants, dispersants, osmotic pressure regulators, solubilizers or cosolvents, pH regulators, proppants, or a combination of two or more thereof.

14. The use according to claim 13, characterized in that The diluent includes water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol, fatty acid ester, or a combination of two or more thereof; The osmotic pressure regulator includes sodium chloride, mannitol, glycerol, glucose, phosphate, acetate, or a combination of two or more thereof; The solubilizer or cosolvent includes poloxamer, lecithin, hydroxypropyl β-cyclodextrin, or a combination of two or more thereof; The pH adjuster includes phosphate, acetate, hydrochloric acid, sodium hydroxide, or a combination of two or more thereof; The proppant includes mannitol, glucose or a combination thereof.

15. The use according to claim 12, characterized in that When the pharmaceutical composition is a tablet, The pharmacologically acceptable excipients include diluents, binders, disintegrants, disintegration inhibitors, lubricants, or a combination of two or more thereof.

16. The use according to claim 15, characterized in that The diluent includes starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc., or a combination of two or more thereof; The binder includes water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinyl pyrrolidone, or a combination of two or more thereof; The disintegrant includes dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, or a combination of two or more thereof; The disintegration inhibitor includes sucrose, tristearin, cocoa butter, hydrogenated oil, or a combination of two or more thereof; The absorption enhancer includes quaternary ammonium salt, sodium lauryl sulfate, or a combination of two or more thereof; The lubricant includes talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, or a combination of two or more thereof.

17. The use according to claim 12, characterized in that When the pharmaceutical composition is in the form of a pill, The pharmacologically acceptable excipients include diluents, binders, disintegrants, or a combination of two or more thereof.

18. The use according to claim 17, characterized in that The diluent includes glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, glyceryl monostearate, kaolin, talc or a combination of two or more thereof; The binder includes gum arabic, gum tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste or a combination of two or more thereof; The disintegrant includes agar powder, dry powder, alginate, sodium lauryl sulfate, methyl cellulose, ethyl cellulose or a combination of two or more thereof.

Images