Application of Remdesivir and its nucleoside combination preparation in anti-porcine epidemic diarrhea virus

Abstract

The present invention provides the use of a combined preparation of remdesivir and its nucleoside in the treatment of porcine epidemic diarrhea virus. Specifically, the present invention provides the use of compound GS-441524 and / or compound GS-5734 in the treatment of porcine epidemic diarrhea virus (PEDV), particularly as (a) an inhibitor of porcine epidemic diarrhea virus (PEDV) replication; and / or (b) a veterinary drug for treating and / or preventing and alleviating related diseases caused by porcine epidemic diarrhea virus (PEDV) infection. GS-441524 and / or compound GS-5734 of the present invention can treat and / or prevent and alleviate related diseases such as porcine epidemic diarrhea caused by porcine epidemic diarrhea virus (PEDV) infection.

Description

Technical Field

[0001] The present invention relates to the field of veterinary drugs, and specifically to the application of a combined preparation of remdesivir and its nucleoside in anti-porcine epidemic diarrhea virus (PDEV). Background Art

[0002] Coronaviruses are a relatively common type of virus at present and can infect a variety of mammals, including bats, pigs, dogs, cats, mice, cattle, horses, camels, etc. Most of these viruses belong to genera α and β. Porcine epidemic diarrhea virus (PEDV) is a coronavirus that can cause acute intestinal infectious diseases in pigs. Pigs of all ages can be infected and develop the disease. Among them, suckling pigs and neonatal piglets are most severely affected, and the breeding industry often suffers heavy losses due to the outbreak of PEDV.

[0003] Porcine epidemic diarrhea (PED) is an acute and highly contagious intestinal infectious disease of pigs caused by porcine epidemic diarrhea virus (PEDV). It spreads rapidly, widely, and has a high lethality rate in piglets, posing a huge threat to the pig-raising industry in China and the world. PEDV is an RNA virus belonging to the genus Alphacoronavirus of the family Nidovirales. It was first discovered abroad in the 1970s. In the 1980s in China, the prevalence of PEDV in the country was determined. Since then, porcine epidemic diarrhea (PED) has become a common disease of viral diarrhea in pig farms. In 2010, a large-scale outbreak of PED caused by a mutant strain of PEDV brought heavy losses to the pig-raising industry.

[0004] PEDV can infect pigs of different age groups, but the virus is most harmful to suckling piglets. After piglets within 7 days of age are infected, they mainly show watery diarrhea, vomiting, and dehydration, with a mortality rate as high as 100%. For this disease, there is currently no specific drug, and clinically, it is mainly controlled by inoculating vaccines.

[0005] In summary, there is an urgent need in this field to develop inhibitors that can inhibit the replication of porcine epidemic diarrhea virus (PDEV) for the treatment of related diseases caused by porcine epidemic diarrhea virus. Summary of the Invention

[0006] The object of the present invention is to provide an active ingredient that can effectively inhibit the replication of porcine epidemic diarrhea virus (PDEV) and its new use in related diseases caused by porcine epidemic diarrhea virus (PDEV) infection.

[0007] In the first aspect of the present invention, there is provided the use of an active ingredient or a preparation containing the active ingredient, wherein the active ingredient is compound GS-441524 (remdesivir nucleoside) or GS-5734 (remdesivir), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof, or a prodrug thereof:

[0008]

[0009] Moreover, the active ingredient or the preparation containing the active ingredient is used for preparing (a) an inhibitor for inhibiting the replication of porcine epidemic diarrhea virus (PEDV); and / or (b) a medicament for treating and / or preventing and alleviating related diseases caused by porcine epidemic diarrhea virus (PEDV) infection.

[0010] In another preferred example, the related disease caused by the virus is porcine epidemic diarrhea.

[0011] In another preferred example, the preparation further contains other antiviral drugs selected from the following group:

[0012] Favipiravir, Galidesivir, NHC (EIDD-1931), EIDD-2801, GC-376, Lopinavir, Ritonavir, Nelfinavir; Chloroquine, Hydroxychloroquine, Cyclosporine, Carrimycin, Baicalin, Baicalein, Forsythoside, Chlorogenic acid, Emodin, Mycophenolic acid, Mycophenolate mofetil, Naphthoquine, Ciclesonide, Ribavirin, Penciclovir, Leflunomide, Teriflunomide, Nafamostat, Nitazoxanide, Darunavir, Arbidol, Camostat, Niclosamide, Baricitinib, Ruxolitinib, Dasatinib, Saquinavir, Beclabuvir, Simeprevir, Palivizumab, Motavizumab, RSV-IGIV MEDI-557, A-60444 (RSV-604), MDT-637, BMS-433771, or a pharmaceutically acceptable salt thereof, or a combination thereof.

[0013] In another preferred embodiment, the formulation is a pharmaceutical composition.

[0014] In another preferred embodiment, the formulation (or pharmaceutical composition) includes: oral formulations and non-oral formulations.

[0015] In another preferred embodiment, the formulation includes: powders, granules, capsules, injections, inhalants, tinctures, oral liquids, tablets, lozenges, or dripping pills.

[0016] In a second aspect of the present invention, there is provided a veterinary pharmaceutical composition, the veterinary pharmaceutical composition containing:

[0017] (a1) A first active ingredient, the first active ingredient being the compound GS-441524 or GS-5734, or a pharmaceutically acceptable salt thereof or a hydrate thereof or a solvate thereof or a prodrug thereof:

[0018]

[0019] and (b) a pharmaceutically acceptable carrier (or a veterinarily pharmaceutically acceptable carrier).

[0020] In another preferred embodiment, the composition further contains (a2) a second active ingredient;

[0021] Among them, the second active ingredient is an antiviral drug, which is selected from the following group: interferon, RNA-dependent RNA polymerase inhibitors (such as favipiravir, Galidesivir, NHC (EIDD-1931), EIDD-2801), 3CL protease inhibitors (such as GC-376), Lopinavir, Ritonavir, Nelfinavir; Chloroquine, hydroxychloroquine, cyclosporine, Carrimycin, baicalin, baicalein, forsythoside, chlorogenic acid, emodin, mycophenolic acid, Mycophenolate mofetil, Naphthoquine, Ciclesonide, Ribavirin, Penciclovir, Leflunomide, Teriflunomide, nafamostat, nitazoxanide, Darunavir, Arbidol, Camostat, Niclosamide, baricitinib, Ruxolitinib, Dasatinib, Saquinavir, Beclabuvir, Simeprevir, Palivizumab, Motavizumab, RSV-IGIV MEDI-557, A-60444 (RSV-604), MDT-637, BMS-433771, or a pharmaceutically acceptable salt thereof, or a combination thereof;

[0022] and / or the second active ingredient is selected from the group consisting of: Zinc, Fingolimod, Vitamin C, Olmesartan Medoxomil, valsartan, Losartan, Thalidomide, glycyrrhizic acid, Artemisinin, dihydroartemisinin, Artesunate, Artemisone, Azithromycin, Escin, Naproxen, or a combination thereof.

[0023] In another preferred embodiment, the composition further contains a feed additive and / or a disinfectant.

[0024] In another preferred embodiment, the second active ingredient is selected from the group consisting of: (Y1) RNA replicase inhibitors (such as favipiravir, Galidesivir, NHC, EIDD-2801); (Y2) Lopinavir; (Y3) Ritonavir; (Y4) Chloroquine, hydroxychloroquine, or a pharmaceutically acceptable salt thereof (such as chloroquine phosphate), (Y5) Nelfinavir; (Y6) any combination of the above Y1 to Y5.

[0025] In another preferred embodiment, the pharmaceutical composition is used to inhibit the replication of porcine epidemic diarrhea virus (PEDV).

[0026] In a third aspect of the present invention, there is provided a use of the veterinary pharmaceutical composition described in the second aspect of the present invention for preparing (a) an inhibitor for inhibiting porcine epidemic diarrhea virus (PEDV); and / or (b) a drug for treating and / or preventing and alleviating related diseases caused by porcine epidemic diarrhea virus (PEDV) infection.

[0027] In a fourth aspect of the present invention, there is provided a method for inhibiting the replication of porcine epidemic diarrhea virus, comprising the steps of:

[0028] contacting the first active ingredient or a preparation containing the first active ingredient with porcine epidemic diarrhea virus (PEDV) so as to inhibit the replication of the virus;

[0029] Among them, the first active ingredient is compound GS-441524 or GS-5734, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof, or a prodrug thereof:

[0030]

[0031] In a fifth aspect of the present invention, there is provided a method for (a) inhibiting the replication of porcine epidemic diarrhea virus (PEDV) and / or (b) treating and / or preventing and alleviating related diseases caused by porcine epidemic diarrhea virus (PEDV) infection, comprising the step of: administering a first active ingredient to the environment to be treated or to a subject in need; wherein the first active ingredient is compound GS-441524 or GS-5734, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof, or a prodrug thereof:

[0032]

[0033] In another preferred example, the disease is porcine epidemic diarrhea caused by porcine epidemic diarrhea virus (PEDV).

[0034] In another preferred example, the subject is a pig.

[0035] In another preferred example, the method is an in vitro method.

[0036] In another preferred example, the method is non-therapeutic and non-diagnostic.

[0037] In another preferred example, the environment to be treated is a pig breeding farm.

[0038] In another preferred example, the method is a treatment method.

[0039] In another preferred example, the dosage is an effective amount for inhibiting virus replication.

[0040] In another preferred example, the method further comprises the step of: administering a disinfectant or a second active ingredient to the environment to be treated, or administering a second active ingredient to a subject in need; wherein the second active ingredient is selected from the following group:

[0041] Favipiravir, NHC (EIDD-1931), EIDD-2801, Baicalin, Baicalein, Forsythoside, Chlorogenic Acid, Emodin, Glycyrrhizic Acid, Chloroquine, Hydroxychloroquine, Nelfinavir.

[0042] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 are the results of the cytotoxicity test of Compound GS-441524 (A) and Compound GS-5734 (B) on Vero cells.

[0044] Figure 2 are micrographs for evaluating the anti-porcine epidemic diarrhea virus (PEDV) effect of Compound GS-441524 using the CPE method (cytopathic effect).

[0045] Figure 3 are micrographs for evaluating the anti-porcine epidemic diarrhea virus (PEDV) effect of Compound GS-5734 using the CPE method (cytopathic effect).

[0046] Figure 4 are the EC 50 determination curves of the in vitro 72h anti-porcine epidemic diarrhea virus (PEDV) activities of Compound GS-441524 (left) and Compound GS-5734 (right).

[0047] Figure 5 are the determination results of the in vitro 48h anti-porcine epidemic diarrhea virus (PEDV) activities of Compound GS-441524 (A) and Compound GS-5734 (B).

[0048] Figure 6 are the results of the indirect immunofluorescence assay for the anti-porcine epidemic diarrhea virus (PEDV) activity of Compound GS-441524.

[0049] Figure 7 are the results of the indirect immunofluorescence assay for the anti-porcine epidemic diarrhea virus (PEDV) activity of Compound GS-5734. DETAILED DESCRIPTION OF THE INVENTION

[0050] Through extensive and in - depth research and a large number of screenings, the present inventor unexpectedly discovered for the first time that remdesivir and its nucleosides can effectively inhibit the replication of porcine epidemic diarrhea virus. Experiments show that the active ingredients of the present invention can efficiently inhibit the replication and viability of porcine epidemic diarrhea virus, and thus can be used to inhibit the replication of porcine epidemic diarrhea virus and for the preparation of drugs for preventing and treating related diseases caused by porcine epidemic diarrhea virus. Based on this, the present invention was completed.

[0051] Specifically, the present invention discloses the use of compound GS - 441524 (remdesivir nucleoside) or compound GS - 5734 (remdesivir) and their pharmaceutical compositions in the anti - porcine epidemic diarrhea virus (PEDV). Compound GS - 441524 or compound GS - 5734 and their pharmaceutical compositions have a significant inhibitory effect on the replication of porcine epidemic diarrhea virus (PEDV), and have good clinical application prospects.

[0052] The term

[0053] As used herein, the "active compound of the present invention", the "active ingredient of the present invention", and the "active compound for inhibiting porcine epidemic diarrhea virus (PDEV) of the present invention" can be used interchangeably, and refer to compound GS - 441524 (remdesivir nucleoside) and compound GS - 5734 (remdesivir) which have excellent activity in inhibiting the replication of porcine epidemic diarrhea virus (PDEV), or their pharmaceutically acceptable salts, or their hydrates (such as crystalline hydrates), or their solvates, or their prodrugs, or combinations thereof.

[0054]

[0055] As used herein, the "preparation of the present invention" refers to a preparation containing the active compound of the present invention, including veterinary drug compositions, feeds, inhibitor products, etc.

[0056] As used herein, the term "comprising" or its variants such as "including" or "including with" etc. is understood to include the stated elements or components, without excluding other elements or other components.

[0057] Porcine epidemic diarrhea virus (PEDV)

[0058] Porcine epidemic diarrhea virus (PEDV) belongs to the genus Coronavirus of the family Coronaviridae. Porcine epidemic diarrhea is an acute intestinal infectious disease of piglets and fattening pigs caused by PEDV virus.

[0059] After oral and nasal infection with PEDV virus, it directly enters the small intestine. The replication of PEDV virus can occur in the cytoplasm of intestinal and colonic villous epithelial cells. PEDV can cause diarrhea, which belongs to osmotic diarrhea. Severe diarrhea causes dehydration, which is the main cause of death of diseased pigs.

[0060] The active compounds and active ingredients of the present invention

[0061] The compound GS-441524 or GS-5734 of the present invention has the structure shown below:

[0062]

[0063] GS-441524 is a 1'-cyano-substituted nucleoside analog containing unnatural bases. This compound first appeared in the patent WO2009132135 published in 2009 and was developed by Gilead Sciences for the treatment of Flaviviridae virus infections, especially diseases caused by hepatitis C virus infection. The compound GS-5734 is an amino-phosphonate prodrug of GS-441524, that is, remdesivir. Compared with the nucleoside form, the prodrug compound GS-5734 has stronger in vitro antiviral activity. In recent years, successive studies have found that the compounds GS-441524 (remdesivir nucleoside) and GS-5734 (remdesivir) have inhibitory effects on the replication of a variety of viruses, including Ebola virus, Marburg virus, respiratory syncytial virus, Middle East respiratory syndrome coronavirus (MERS-CoV), Severe acute respiratory syndrome coronavirus (SARS-CoV), and a variety of paramyxoviruses, etc. (Sci Rep. 2017, 7, 43395; J. Med. Chem. 2017, 60, 1648-1661; mBio. 2018, 9, e00221-18). After the outbreak of the novel coronavirus (SARS-CoV-2) at the end of 2019, scholars found that GS-5734 could effectively inhibit the proliferation of the virus in an in vitro cell model, and its EC 50 was 0.77 μM (Cell Research, 2020, 0:1–3).

[0064] In the present invention, the inventors first discovered that the compound GS-441524 or GS-5734 can effectively inhibit the replication of porcine epidemic diarrhea virus, thereby effectively preventing and / or treating diseases caused by porcine epidemic diarrhea virus, such as porcine epidemic diarrhea, vomiting, dehydration and other diseases.

[0065] In the present invention, the active ingredient is the compound GS-441524 (remdesivir nucleoside) or GS-5734 (remdesivir), or a pharmaceutically acceptable salt thereof, or a prodrug thereof, or a combination thereof, or a crystal thereof, or a solvate thereof.

[0066] The preferred structural formulas of the compound GS-441524 (remdesivir nucleoside) and the compound GS-5734 (remdesivir) are the compounds prepared in the examples or a pharmaceutically acceptable salt thereof, or a prodrug thereof, or a combination thereof, or a crystal thereof, or a solvate thereof.

[0067] Tests have shown that the active ingredient of the present invention can effectively inhibit the replication of porcine epidemic diarrhea virus (PEDV), thereby preventing, treating and / or alleviating related diseases caused by porcine epidemic diarrhea virus (PEDV).

[0068] As used herein, "the active compound of the present invention" and "the active compound of the present invention that inhibits virus replication" are used interchangeably and refer to compounds having excellent activity in inhibiting virus replication, including compound GS-441524 (remdesivir nucleoside), compound GS-5734 (remdesivir), or a pharmaceutically acceptable salt thereof, or a crystal thereof, or a solvate thereof.

[0069] It should be understood that the active ingredient of the present invention includes remdesivir nucleoside and remdesivir, or a pharmaceutically acceptable salt, enantiomer, diastereomer or racemate thereof, or a prodrug thereof. It should be understood that the active ingredient of the present invention also includes forms such as crystal forms of the active compound of the present invention and amorphous compounds.

[0070] The "pharmaceutically acceptable salt" is a conventional non-toxic salt formed by the reaction of the active compound of the present invention with an inorganic acid or an organic acid. For example, the conventional non-toxic salt can be prepared by reacting the active compound of the present invention with an inorganic acid or an organic acid. The inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid, phosphoric acid, etc., and the organic acids include citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, isethionic acid, etc.; or the sodium salt, zinc salt, potassium salt, calcium salt, aluminum salt or ammonium salt formed by reacting the active compound of the present invention with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid or glutamic acid to form an ester and then reacting with an inorganic base; or the corresponding inorganic acid salts formed by reacting the active compound of the present invention with lysine, arginine, ornithine to form an ester and then reacting with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid or the corresponding organic acid salts formed by reacting with formic acid, acetic acid, picric acid, methanesulfonic acid or ethanesulfonic acid.

[0071] In addition, the active ingredient of the present invention is also particularly suitable for combination with other antiviral drugs. Representative other antiviral drugs include (but are not limited to): interferons, RNA-dependent RNA polymerase inhibitors (such as favipiravir, Galidesivir, NHC, EIDD-2801); 3CL protease inhibitors (such as GC-376), Lopinavir, Ritonavir, Nelfinavir; Chloroquine (Sigma-C6628), Hydroxychloroquine, Cyclosporine, Carrimycin, Baicalin, Baicalein, Forsythoside, Chlorogenic acid, Emodin, Mycophenolic acid, Mycophenolate mofetil, Naphthoquine, Ciclesonide, Ribavirin, Penciclovir, Leflunomide, Teriflunomide, Nafamostat, Nitazoxanide, Darunavir, Arbidol, Camostat, Niclosamide, Baricitinib, Ruxolitinib, Dasatinib, Saquinavir, Beclabuvir, Simeprevir, Palivizumab, Motavizumab, RSV-IGIV MEDI-557, A-60444 (RSV-604), MDT-637, BMS-433771 or a pharmaceutically acceptable salt thereof, or a combination thereof. The interferons include one or more of interferon α-2a, interferon α-2b, interferon α-n1, interferon α-n3, interferon β-1a, and interferon β-1b.

[0072] Additional therapeutic agents, such as other anti-inflammatory agents that act through anti-inflammatory cascade mechanisms, can also be used in combination with compound GS-441524 or compound GS-5734 to treat viral infections. For example, the application of "anti-inflammatory signal transduction modulators" (referred to herein as AISTMs) such as phosphodiesterase inhibitors (e.g., specific for PDE-4, PDE-5, or PDE-7), transcription factor inhibitors (e.g., blocking NFκB through IKK inhibition), or kinase inhibitors (e.g., blocking P38MAP, JNK, PI3K, EGFR, or Syk) is a logical way to cut off inflammation because these small molecules target a limited number of common intracellular pathways - those signal transduction pathways that are key points of anti-inflammatory therapeutic intervention (see review by P.J. Barnes, 2006). These non-limiting additional therapeutic agents include: 5-(2,4-difluoro-phenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (2-dimethylamino-ethyl)-amide (P38Map kinase inhibitor ARRY-797); 3-cyclopropylmethoxy-N-(3,5-dichloro-pyridin-4-yl)-4-difluoromethoxy-benzamide (PDE-4 inhibitor roflumilast); 4-[2-(3-cyclopentyloxy-4-methoxyphenyl)-2-phenyl-ethyl]-pyridine (PDE-4 inhibitor CDP-840); N-(3,5-dichloro-4-pyridinyl)-4-(difluoromethoxy)-8-[(methylsulfonyl)amino]-1-dibenzofuran-carboxamide (PDE-4 inhibitor omalizumab); N-(3,5-dichloro-pyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-hydroxy-1H-indol-3-yl]-2-oxo-acetamide (PDE-4 inhibitor AWD12-281); 8-methoxy-2-trifluoromethyl-quinoline-5-carboxylic acid (3,5-dichloro-1-oxo-pyridin-4-yl)-amide (PDE-4 inhibitor Sch351591); 4-[5-(4-fluorophenyl)-2-(4-methylsulfinyl-phenyl)-1H-imidazol-4-yl]-pyridine (P38 inhibitor SB-203850); 4-[4-(4-fluoro-phenyl)-1-(3-phenyl-propyl)-5-pyridin-4-yl-1H-imidazol-2-yl]-but-3-yn-1-ol (P38 inhibitor RWJ-67657); 4-cyano-4-(3-cyclopentyloxy-4-methoxy-phenyl)-cyclohexanecarboxylic acid 2-diethylamino-ethyl ester (prodrug of cilomilast's 2-diethyl-ethyl ester, PDE-4 inhibitor); (3-chloro-4-fluorophenyl)-[7-methoxy-6-(3-morpholin-4-yl-propoxy)-quinazolin-4-yl]-amine (gefitinib, EGFR inhibitor); and 4-(4-methyl-piperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamide (imatinib, EGFR inhibitor).

[0073] The active ingredient of the present invention can inhibit the infectivity of porcine epidemic diarrhea virus. Therefore, when the active ingredient of the present invention is administered or given therapeutically, the infection of porcine epidemic diarrhea virus can be inhibited, thereby achieving an antiviral effect.

[0074] Any compound of the present invention can also be combined with one or more other active therapeutic agents in a unit dosage form for simultaneous or sequential administration to a patient. The combination therapy can be administered as a simultaneous or sequential regimen. When administered sequentially, the combination can be administered in two or more administrations.

[0075] The co-administration of the compound of the present invention with one or more other active therapeutic agents generally means the simultaneous or sequential administration of the compound of the present invention and one or more other active therapeutic agents such that a therapeutically effective amount of the compound of the present invention and one or more other active therapeutic agents are both present in the patient's body.

[0076] Co-administration includes administering a unit dose of the compound of the present invention before or after administering a unit dose of one or more other active therapeutic agents. For example, the compound of the present invention is administered within seconds, minutes or hours of administering one or more other active therapeutic agents. For example, a unit dose of the compound of the present invention can be administered first, followed by a unit dose of one or more other active therapeutic agents within seconds or minutes. Or, a unit dose of one or more other therapeutic agents can be administered first, followed by a unit dose of the compound of the present invention within seconds or minutes. In some cases, it may be necessary to administer a unit dose of the compound of the present invention first and then, after a period of hours (e.g., 1 - 12 hours), administer a unit dose of one or more other active therapeutic agents. In other cases, it may be necessary to administer a unit dose of one or more other active therapeutic agents first and then, after a period of hours (e.g., 1 - 12 hours), administer a unit dose of the compound of the present invention.

[0077] Combination therapies can provide "synergistic effects" and "synergism", i.e., the effect obtained when the active ingredients are used together is greater than the sum of the effects obtained when the compounds are used separately. Synergism can be obtained when the active ingredients: (1) are co-formulated and administered or delivered simultaneously in the form of a combination preparation; (2) are administered alternately or delivered in parallel as separate preparations; or (3) are delivered by some other administration regimen. When delivered by alternate therapy, synergism can be obtained when the compounds are administered or delivered sequentially, e.g., as separate tablets, pills or capsules, or by different injections from separate syringes. Generally, during alternate therapy, the effective dose of each active ingredient is administered sequentially, i.e., continuously, while in combination therapy, the effective doses of two or more active ingredients are administered together. A synergistic antiviral effect means an antiviral effect greater than the predicted purely additive effect of the individual compounds in the combination.

[0078] Pharmaceutical composition and use

[0079] The present invention also provides the use of a mixture of one or more of the active compounds that inhibit viral replication of the present invention, or pharmaceutically acceptable salts thereof, or prodrugs thereof, as an active ingredient in the preparation of a medicament (or veterinary drug) for treating and / or preventing and alleviating porcine epidemic diarrhea caused by porcine epidemic diarrhea virus (PEDV) infection.

[0080] The pharmaceutical composition (or veterinary pharmaceutical composition) provided by the present invention preferably contains 0.001-99 wt% of the active ingredient, and the preferred ratio is that the active compound of the present invention as the active ingredient accounts for 0.1 wt% to 90 wt% or 1 wt% to 50 wt% of the total weight, and the remaining part is a pharmaceutically acceptable carrier (or a veterinarily pharmaceutically acceptable carrier), diluent or solution or salt solution.

[0081] When needed, one or more pharmaceutically acceptable carriers can also be added to the medicament of the present invention. The carriers include conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0082] The compounds and pharmaceutical compositions provided by the present invention can be in various forms, such as tablets, capsules, powders, syrups, solutions, suspensions and aerosols, etc., and can be present in a suitable solid or liquid carrier or diluent and in a suitable sterilized apparatus for injection or infusion.

[0083] The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to the conventional preparation methods in the pharmaceutical field. The unit dosage of its formulation usually contains 0.05-1000 mg of the active compound of the present invention, and preferably, the unit dosage of the formulation contains 1 mg-500 mg of the active compound of the present invention.

[0084] The compounds and pharmaceutical compositions of the present invention can be clinically used in mammals (such as pigs), and can be administered through routes such as oral, nasal, skin, lung, or gastrointestinal tract. The most preferred route is oral administration. The most preferred daily dose is 0.01 - 400 mg / kg body weight, taken once, or 0.01 - 200 mg / kg body weight taken in divided doses. Regardless of the administration method, the optimal dose should be determined according to the specific treatment. Usually, it starts with a small dose and gradually increases until the most suitable dose is found.

[0085] The drugs or inhibitors of the present invention can be administered in various different ways. For example, they can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, and mucosal tissues by injection, spraying, nasal dropping, eye dropping, penetration, absorption, physical or chemical mediated methods; or be mixed or encapsulated with other substances and then introduced into the body.

[0086] Typically, the active ingredient of the present invention or the pharmaceutical composition containing it can be administered in unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eye, lung and respiratory tract, skin, rectum, etc.

[0087] The dosage forms can be liquid dosage forms, solid dosage forms or semi - solid dosage forms. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including O / W type, W / O type and multiple emulsions), suspensions, injections (including aqueous injections, powder injections and infusions), eye drops, nasal drops, lotions and liniments, etc.; solid dosage forms can be tablets (including ordinary tablets, enteric - coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric - coated capsules), granules, powders, pellets, dripping pills, suppositories, membranes, patches, aerosols (powder aerosols), sprays, etc.; semi - solid dosage forms can be ointments, gels, pastes, etc.

[0088] The active ingredient of the present invention can be made into conventional preparations, as well as sustained - release preparations, controlled - release preparations, targeted preparations and various particulate drug delivery systems.

[0089] In order to prepare the active ingredient of the present invention into tablets, various excipients well-known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and glidants. The diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agents can be water, ethanol, isopropanol, etc.; the binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfate, etc.; the lubricants and glidants can be talc powder, silicon dioxide, stearates, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0090] The tablets can be further prepared into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets.

[0091] In order to prepare the dosage unit into capsules, the active ingredient of the present invention as the effective ingredient can be mixed with diluents and glidants, and the mixture can be directly placed into hard capsules or soft capsules. The effective ingredient can also be first made into granules or pellets with diluents, binders, and disintegrants, and then placed into hard capsules or soft capsules. The varieties of diluents, binders, wetting agents, disintegrants, and glidants used for preparing the tablets of the present invention can also be used for preparing the capsules of the present invention.

[0092] In order to prepare the active ingredient of the present invention into injections, water, ethanol, isopropanol, propylene glycol or their mixtures can be used as solvents and appropriate solubilizers, cosolvents, pH adjusters, and osmotic pressure regulators commonly used in the art are added. The solubilizers or cosolvents can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjusters can be phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulators can be sodium chloride, mannitol, glucose, phosphates, acetates, etc. When preparing freeze-dried powder injections, mannitol, glucose, etc. can also be added as supporting agents.

[0093] In addition, if necessary, coloring agents, preservatives, flavors, flavoring agents or other additives can also be added to the pharmaceutical preparations.

[0094] The active ingredient or composition of the present invention can be administered alone or in combination with other therapeutic drugs or symptomatic drugs.

[0095] When there is a synergistic effect between the active ingredient of the present invention and other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0096] The main advantages of the present invention include:

[0097] (a) The active compounds of the present invention can efficiently inhibit the replication of porcine epidemic diarrhea virus (PEDV). The half-maximal effective concentration (EC 50 ) of compound GS-441524 and compound GS-5734 against PEDV are 0.31 μM and 0.74 μM respectively, and the therapeutic indices (SI) are greater than 1600 and 600 respectively.

[0098] (b) The active compounds of the present invention have low toxicity and side effects and good drug-likeness. This indicates that the active compounds of the present invention (compound GS-441524, compound GS-5734) have good application prospects in the field of treating porcine epidemic diarrhea.

[0099] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0100] Example 1, Cytotoxicity Test of Compound GS-441524 and Compound GS-5734

[0101] 1.1 Method

[0102] 1) Digest and passage Vero cells in good growth state, adjust the cell density to 1×10 5 / mL and inoculate into 96-well plates, 100 μL / well, and place in a 37°C, 5% CO2 incubator for 24 h;

[0103] 2) After 24 h, discard the medium in the wells, wash three times with 1×PBS, spin dry, and perform 2-fold serial dilution of the compound with cell maintenance medium to make the final concentration of the test compound 500 μM, 250 μM, 125 μM, 62.5 μM, 31.25 μM. At the same time, set up a cell control;

[0104] 3) After 72 h, perform cell viability detection with CCK-8 reagent. Obtain Figure 1 .

[0105] 1.2 Results

[0106] From Figure 1It can be seen that both compound GS-441524 (Figure A) and compound GS-5734 (Figure B) can inhibit the toxicity of Vero cells, and the Vero cell toxicity decreases with the increase of the compound concentration. However, the compound of the present invention has a relatively small inhibitory effect on the toxicity of Vero cells, and the half cytotoxic concentration (CC 50 ) of both compounds is greater than 500 μM.

[0107] Example 2, in vitro antiviral test (CPE method) of compound GS-441524 and compound GS-5734

[0108] 2.1 Method

[0109] 1) Digest and passage Vero cells in good growth state, adjust the cell density to 1×10 5 / mL and inoculate into 96-well plates, 100 μL / well, and place in a 37°C, 5% CO2 incubator for 24 h;

[0110] 2) After 24 h, discard the medium in the wells, wash three times with 1×PBS, spin dry, and perform 2-fold serial dilution of the test compound with cell maintenance medium. At the same time, add 0.01 MOI of porcine epidemic diarrhea virus (PEDV) to each well. Make the final concentration of the test compound 125 μM, 62.5 μM, 31.25 μM. At the same time, set up cell controls and virus controls;

[0111] 3) When the virus control shows 70% lesions, observe the changes in the cell state after the compound and the virus act together under a microscope and take pictures for preservation.

[0112] 2.2 Results

[0113] The results are shown in Figure 2 (compound GS-441524) and Figure 3 (compound GS-5734). It can be observed from the microscope pictures in the figure that the normal Vero cells grow well, with complete cell morphology and clear cell boundaries; while the cells infected with the virus show large areas of membrane fusion and nuclear aggregation, without complete cell morphology, and the lesions are obvious; however, after treating the cells with compound GS-441524 or compound GS-5734, the degree of cell lesions is significantly reduced. Thus, it can be seen that both compound GS-441524 and compound GS-5734 have obvious inhibitory effects on PEDV at the cell level.

[0114] Example 3, in vitro antiviral activity (EC 50 ) test (72 h) of compound GS-441524 and compound GS-5734

[0115] 3.1 Method

[0116] 1) Digest and passage Vero cells, adjust the cell density to 1×10 5 / mL and inoculate into 96-well plates, 100 μL per well, and place in an incubator at 37°C and 5% CO2 for 24 h;

[0117] 2) After 24 h, dilute PEDV to 0.01 MOI per well with cell maintenance medium, and add test compounds with final concentrations of 125 μM, 62.5 μM, 31.2 μM, 15.6 μM, 7.8 μM, 3.9 μM, 2.0 μM, 1.0 μM, 0.5 μM, 0.2 μM, and 0.1 μM to the dilution solution, and incubate in an incubator at 37°C and 5% CO2 for 1 h;

[0118] 3) After the treatment, take out the 96-well plates, discard the medium in the wells, wash three times with 1×PBS, centrifuge dry, add the virus solutions of each group, and culture in an incubator at 37°C and 5% CO2. At the same time, set up virus controls and cell controls;

[0119] 4) After 72 h, collect cell samples and measure the changes in virus content in different treatment groups.

[0120] 3.2 Results

[0121] The results are shown in Figure 4 . By calculating the inhibition rate of the drug on the virus to reflect the inhibitory effect of the compound on PEDV. It can be seen from the figure that the inhibitory effects of compound GS-441524 and compound GS-5734 on PEDV are both dose-dependent. The half-maximal effective concentration (EC 50 ) of compound GS-441524 and compound GS-5734 are 0.31 μM and 0.74 μM, respectively.

[0122] Example 4, in vitro antiviral activity test of compound GS-441524 and compound GS-5734 (48 h)

[0123] 4.1 Method

[0124] 1) Digest and passage Vero cells, adjust the cell density to 1×10 5 / mL and inoculate into 96-well plates, 100 μL per well, and place in an incubator at 37°C and 5% CO2 for 24 h;

[0125] 2) After 24 h, dilute PEDV to 0.01 MOI per well with cell maintenance medium, and add test compounds with final concentrations of 125 μM, 62.5 μM, 31.2 μM, 15.6 μM, 7.8 μM, 3.9 μM, 2.0 μM, 1.0 μM, 0.5 μM, 0.2 μM, and 0.1 μM to the dilution solution, and incubate in an incubator at 37°C and 5% CO2 for 1 h;

[0126] 3) After the treatment, take out the 96-well plate, discard the culture medium in the wells, wash three times with 1×PBS, spin dry, add the virus solutions of each group, and culture in an incubator at 37°C and 5% CO2. At the same time, set up virus control and cell control.

[0127] 5) After 48 h, collect the cell samples and measure the changes in virus content in different treatment groups.

[0128] 4.2 Results

[0129] The results are shown in Figure 5 . From Figure 5 it can be seen that as the concentration of the test compound increases, the virus inhibition effect becomes more obvious, showing a dose-dependent relationship. For compound GS-441524 (Figure A), no virus was detected at concentrations of 125 μM, 62.5 μM, 31.3 μM, 15.6 μM, 7.8 μM, and 3.9 μM, and the virus proliferation could be completely inhibited; at 2.0 μM, the virus titer could be reduced by about 2000-fold; at 1.0 μM, it could be reduced by about 100-fold; at 0.5 μM, it could be reduced by about 10-fold. For compound GS-5734 (Figure B), no virus was detected at concentrations of 125 μM, 62.5 μM, 31.3 μM, and 15.6 μM, and the virus proliferation could be completely inhibited; at 7.8 μM, the virus titer could be reduced by about 4000-fold; at 3.9 μM, it could be reduced by about 80-fold; at 2.0 μM, it could be reduced by about 10-fold.

[0130] Example 5 Indirect Immunofluorescence Detection of the Viral Activity of Compound GS-441524 and Compound GS-5734

[0131] 5.1 Method

[0132] 1) Seed cells: Digest and passage Vero cells, adjust the cell density to 1×10 5 / mL and inoculate into a 96-well plate, 100 μL / well, and culture in an incubator at 37°C and 5% CO2 for 24 h;

[0133] 2) Virus infection: After 24 h, when the cells grow to confluence, take out the 96-well plate, wash three times with 1×PBS, dilute PEDV to 0.01 MOI per well with cell maintenance medium, add the test compound with a final concentration of 1 μM to the dilution, and at the same time set up virus control and cell control, and continue to culture in a 37°C and 5% CO2 cell incubator;

[0134] 3) Fix the cells: When 70% of the cells in the virus control group show lesions (membrane fusion can be observed and there is no large-scale detachment), discard the culture medium in the wells, wash twice with 1×PBS, add 100 μL of 4% paraformaldehyde to each well, and fix the cells at room temperature for 30 min; discard the paraformaldehyde, wash three times with 1×PBS, add 100 μL of 1% Triton X-100 to each well, and permeabilize at room temperature for 10 min;

[0135] 4) Blocking: Wash the fixed plate three times with 1×PBS, add 100 μL of 5% BSA to each well for blocking, and block at 37 °C for 30 min (blocking can be carried out overnight at 4 °C);

[0136] 5) Add the primary antibody: Discard the blocking solution, wash three times with 1×PBS, add 100 μL of the primary antibody (N protein monoclonal antibody) diluted 1:1000 to each well, and incubate at 37 °C for 1 h;

[0137] 6) Add the secondary antibody: Discard the primary antibody, wash three times with 1×PBS, add 100 μL of the secondary antibody (FITC-labeled goat anti-mouse IgG) diluted 1:500 to each well, and incubate at 37 °C for 1 h (pay attention to operating in the dark);

[0138] 7) Discard the secondary antibody, wash three times with 1×PBS, observe under a fluorescence microscope and take pictures for preservation.

[0139] 5.2 Results

[0140] The results are shown in Figure 6 and Figure 7 respectively, which are fluorescence microscope photos after adding compound GS-441524 and compound GS-5734 at 1 μM. When compound GS-441524 and compound GS-5734 are at 1 μM, a significant decrease in fluorescence can be observed, indicating that after adding compound GS-441524 and compound GS-5734, the expression of porcine epidemic diarrhea virus is reduced. The compounds of the present invention can effectively inhibit the replication of porcine epidemic diarrhea virus (PDEV), thereby helping to relieve, prevent, and reduce the related diseases caused by porcine epidemic diarrhea virus (PDEV).

[0141] All the documents mentioned in the present invention are cited in this application as references, just as each document is cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of compound GS-441524 or a pharmaceutically acceptable salt thereof in the preparation of an inhibitor for (a) inhibiting the replication of porcine epidemic diarrhea virus; and / or (b) a medicament for treating or preventing related diseases caused by porcine epidemic diarrhea virus infection ; Among them, the related disease caused by the porcine epidemic diarrhea virus is porcine epidemic diarrhea.

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