A 3cl protease inhibitor composition for treating porcine epidemic diarrhea

Abstract

The present application relates to the technical field of 3CL protease inhibitor group, and discloses a 3CL protease inhibitor group for treating porcine epidemic diarrhea, which comprises the following ingredients in parts by mass: 10-15 parts of composite 3CL protease inhibitor, 5-10 parts of glutamine, 5-10 parts of glycyrrhizic acid, 2-3 parts of sodium caprate, 12-15 parts of starch and 8-12 parts of lactose.The composite 3CL protease inhibitor, glutamine, glycyrrhizic acid, sodium caprate, soluble starch and lactose are uniformly mixed, granulated and dried to obtain the 3CL protease inhibitor group, which has the effect of efficiently treating porcine epidemic diarrhea and has good targeted therapy effect, can directionally inhibit the 3CL protease of PEDV, in addition, the 3CL protease inhibitor group has small irritation, low toxicity, no residue, is not easy to produce drug resistance and has high safety.

Description

Technical Field

[0001] This invention relates to the field of 3CL protease inhibitor compounds, specifically a 3CL protease inhibitor compound for treating porcine epidemic diarrhea. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) is a porcine alpha coronavirus, an intestinal infectious disease caused by porcine epidemic diarrhea virus. It belongs to the family Coronaviridae and is an RNA virus. It mainly causes symptoms of vomiting, diarrhea, and dehydration, and pigs of all ages are susceptible. 3CL protease can effectively inhibit the activity of 3CL protease in various coronaviruses, thereby exerting an antiviral effect. 3CL protease is a cysteine ​​protease whose main function is to cleave coronavirus polyproteins to produce various functional proteins, including RNA-dependent RNA polymerase, helicase, single-stranded RNA-binding protein, exonuclease, endonuclease, etc. These functional proteins all play an important role in viral replication, leading to the occurrence of porcine epidemic diarrhea.

[0003] 3CL protease inhibitors can block the cleavage of polyproteins by the 3CL protease of PEDV, inhibit viral replication, and at the same time alleviate intestinal inflammation in piglets and repair intestinal mucosal damage, thereby treating porcine epidemic diarrhea. However, the inhibitory activity of a single 3CL protease inhibitor is limited, and the oral bioavailability of 3CL protease inhibitors is low. In addition, 3CL protease inhibitor drugs are randomly distributed throughout the body, which can easily lead to intestinal inflammation and damage. Summary of the Invention

[0004] This invention provides a 3CL protease inhibitor composition for treating porcine epidemic diarrhea, which solves the problems of limited inhibitory activity of 3CL protease inhibitors, low oral bioavailability of 3CL protease inhibitors, and the random distribution of 3CL protease inhibitor drugs throughout the body, which can easily lead to intestinal inflammation and damage.

[0005] The technical solution of the present invention: A 3CL protease inhibitor compound for treating porcine epidemic diarrhea comprises the following raw materials in parts by weight: 10-15 parts of compound 3CL protease inhibitor, 5-10 parts of glutamine, 5-10 parts of glycyrrhizic acid, 2-3 parts of sodium decanoate, 12-15 parts of starch, and 8-12 parts of lactose. The composite 3CL protease inhibitor is formed by coating 3CL protease inhibitor with lipid nanoparticles to form lipid nanoparticles loaded with 3CL protease inhibitor; it is obtained by mixing phospholipid-polyethylene glycol-N-hydroxysuccinimide ester, lactobacillus, montmorillonite and lipid nanoparticles loaded with 3CL protease inhibitor, and then mixing with ethyl caffeate.

[0006] A method for preparing a 3CL protease inhibitor compound for treating porcine epidemic diarrhea includes the following steps: mixing a compound 3CL protease inhibitor, glutamine, glycyrrhizic acid, sodium decanoate, starch, and lactose, stirring evenly, granulating, and drying to obtain the 3CL protease inhibitor compound.

[0007] Furthermore, the lactose is β-lactose.

[0008] Furthermore, the starch is corn starch or sweet potato starch.

[0009] Furthermore, the composite 3CL protease inhibitor is specifically prepared by the following steps: A1. 3CL protease inhibitor, stearic acid and soybean lecithin were added to ethanol and stirred to obtain an oil phase. Poloxamer 188 was added to deionized water and stirred to obtain an aqueous phase. The oil phase and aqueous phase were mixed and stirred. After stirring, the mixture was stirred at 1-3℃ for 3-5 hours to form granules. After centrifugation, washing and freeze-drying, lipid nanoparticles loaded with 3CL protease inhibitor were obtained. A2. Mix phospholipid-polyethylene glycol-N-hydroxysuccinimide ester and anhydrous ethanol, stir until completely dissolved, add lactobacillus and montmorillonite, stir evenly, then add lipid nanoparticles loaded with 3CL protease inhibitor, stir at 40-60℃ for 2-3 hours, remove organic solvent by rotary evaporation to obtain modified lipid nanoparticles. A3. Add ethyl caffeate to ethanol, stir well, add modified lipid nanoparticles, stir at 60-70℃ and 100-120r / min for 20-30min, and dry at room temperature overnight to obtain the composite 3CL protease inhibitor.

[0010] Furthermore, in the A1 reaction process described above, 3CL protease inhibitor, stearic acid, and soybean lecithin are mixed at 60°C to form an oil phase, while poloxamer 188 is dissolved in deionized water as the aqueous phase. The oil phase is added to the aqueous phase, and the hydrophobic segments of poloxamer 188 extend into the oil phase, while the hydrophilic segments of poloxamer 188 extend into the aqueous phase, thereby forming lipid nanoparticles coated with stearic acid and soybean lecithin to encapsulate the 3CL protease inhibitor, thus obtaining lipid nanoparticles loaded with the 3CL protease inhibitor.

[0011] Furthermore, in the above A2 reaction process, phospholipid-polyethylene glycol-N-hydroxysuccinimide ester and anhydrous ethanol are mixed and stirred until completely dissolved. Lactobacillus and montmorillonite are added and mixed evenly. The lipid nanoparticles loaded with 3CL protease inhibitors have a large number of hydrophilic phosphate groups on their surface, which allows the hydrophilic phosphate groups in phospholipid-polyethylene glycol-N-hydroxysuccinimide ester to bind with the hydrophilic phosphate groups on the surface of the lipid nanoparticles loaded with 3CL protease inhibitors. This results in phospholipid-polyethylene glycol-N-hydroxysuccinimide ester, lactobacillus, and montmorillonite being evenly distributed on the surface of the lipid nanoparticles, thus obtaining modified lipid nanoparticles.

[0012] Furthermore, during the A3 reaction described above, the hydrophilic phosphate groups on the surface of the modified lipid nanoparticles can bind with the hydroxyl groups carried by ethyl caffeate through hydrogen bonds, allowing ethyl caffeate to be adsorbed onto the surface of the modified lipid nanoparticles, thus obtaining a composite 3CL protease inhibitor.

[0013] Further, in step A1, the mass ratio of the 3CL protease inhibitor, stearic acid, soybean lecithin, and ethanol is (1-1.2):(5-5.5):(1.6-2):(45-55).

[0014] Further, in step A1, the mass ratio of poloxamer 188 to deionized water is 1:(45-55).

[0015] Further, in step A2, the mass ratio of the phospholipid-polyethylene glycol-N-hydroxysuccinimide ester, anhydrous ethanol, lactobacillus, montmorillonite, and lipid nanoparticles loaded with 3CL protease inhibitor is (0.5-1):(40-50):(0.1-0.3):(0.1-0.3):(2.2-2.5).

[0016] Further, in step A3, the mass ratio of ethyl caffeate, ethanol and modified lipid nanoparticles is (1-1.2):(10-12):(2-2.3).

[0017] Furthermore, in step A3, the mass ratio of the oil phase to the water phase is 1:(4-4.5).

[0018] The present invention has the following beneficial effects: (1) In the technical solution of the present invention, nimatelavir, as a 3CL protease inhibitor, can block the 3CL protease of PEDV from cleaving polyproteins, effectively inhibit the activity of 3CL protease in various coronaviruses, inhibit viral replication, and at the same time relieve intestinal inflammation in piglets and repair intestinal mucosal damage, thereby treating porcine epidemic diarrhea; montmorillonite relies on its layered porous structure to physically adsorb porcine epidemic diarrhea virus, pathogens and inflammatory toxins in the intestine, and at the same time forms a protective film on the surface of the damaged intestinal epithelium, adsorbs excess water in the intestine, quickly relieves watery diarrhea, and avoids dehydration in livestock; lactobacillus can regulate the homeostasis of intestinal flora, promote the proliferation of beneficial bacteria, inhibit the reproduction of harmful bacteria, reduce the risk of secondary bacterial infection, and stimulate intestinal mucosal repair, thereby reducing intestinal inflammatory damage; lactobacillus and montmorillonite combined produce synergistic antidiarrheal and intestinal repair effects, and lactobacillus and montmorillonite assist nimatelavir in treating porcine epidemic diarrhea from multiple dimensions of antiviral, mucosal protection and flora regulation.

[0019] (2) In the technical solution of the present invention, 3CL protease inhibitor, stearic acid and soybean lecithin are mixed to form an oil phase, and poloxamer 188 is used as an aqueous phase. The oil phase and aqueous phase are mixed and reacted to form lipid nanoparticles loaded with 3CL protease inhibitor. On the one hand, the lipid nanoparticles formed by stearic acid and soybean lecithin have good gastric acid resistance, which can improve the acid resistance of 3CL protease inhibitor and avoid the 3CL protease inhibitor being easily affected by gastric acid and degraded during the process of entering the intestine through gastric acid, resulting in insufficient utilization of 3CL protease inhibitor and poor treatment effect of porcine epidemic diarrhea. On the other hand, the formed lipid nanoparticles can bind to the intestinal epithelial cell membrane, so that 3CL protease inhibitor can better act on the intestinal mucosa. Moreover, the lipid nanoparticles can penetrate the viscous mucus layer of the pig intestine, increase the probability of intestinal 3CL protease inhibitor contact with the pig intestinal epithelium, and have a highly effective treatment effect of porcine epidemic diarrhea.

[0020] (3) In the technical solution of the present invention, phospholipid-polyethylene glycol-N-hydroxysuccinimide ester, lactobacillus, and montmorillonite are uniformly distributed on the surface of lipid nanoparticles to obtain modified lipid nanoparticles. Ethyl caffeate is then adsorbed onto the surface of the modified lipid nanoparticles. On the one hand, the long chain of phospholipid-polyethylene glycol-N-hydroxysuccinimide ester forms a hydration layer on the surface of lipid nanoparticles, inhibiting the aggregation of lipid nanoparticles, avoiding flocculation and precipitation in intestinal fluid, prolonging intestinal retention time, and improving the effect of treating porcine epidemic diarrhea. On the other hand, it can avoid non-specific adsorption of lipid nanoparticles with intestinal mucus proteins, reduce ineffective drug retention in the intestinal lumen, and enable lipid nanoparticles to reach the PEDV infection site in the small intestine to treat porcine epidemic diarrhea. In addition to treating porcine epidemic diarrhea (PEDD), phospholipid-polyethylene glycol-N-hydroxysuccinimide ester provides a covalent binding site to firmly couple ethyl caffeate, making the ethyl caffeate-modified layer less likely to detach in the digestive tract environment. Furthermore, ethyl caffeate directly targets the 3CL protease of PEDV and inhibits its enzyme activity, thereby inhibiting PEDV replication in vitro, improving the survival rate of PEDV-infected suckling piglets, and inhibiting viral replication in vivo. It exhibits high anti-PEDV activity and exerts a highly effective therapeutic effect on porcine epidemic diarrhea. In addition, lactobacillus and montmorillonite, through the long-chain action of phospholipid-polyethylene glycol-N-hydroxysuccinimide ester, are tightly bound to the surface of lipid nanoparticles, exerting a therapeutic effect on porcine epidemic diarrhea.

[0021] (4) In the technical solution of the present invention, the compound 3CL protease inhibitor, glutamine, glycyrrhizic acid, sodium decanoate, soluble starch and lactose are mixed evenly, granulated and dried to obtain the 3CL protease inhibitor composition, which has a high efficacy in treating porcine epidemic diarrhea and has a good targeted therapeutic effect, which specifically inhibits the 3CL protease of PEDV. In addition, the 3CL protease inhibitor composition has low irritation, low toxicity and no residue, is not easy to produce drug resistance, and has high safety. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] The raw materials used in the embodiments of this invention are shown below, and all reagents used are analytical grade.

[0024] Among them, lactose is β-lactose, catalog number L812331, containing 70% β-lactose and 30% α-lactose; glutamine catalog number D807113, with a content of 98%; glycyrrhizic acid catalog number G886327, ≥80% (HPLC); sodium decanoate catalog number S833402, with a content of 98%; and ethyl caffeate catalog number E842655, with a content of 98%. All of these were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0025] The starch is corn starch or sweet potato starch; the 3CL protease inhibitor is nimatetravir.

[0026] Example 1 A 3CL protease inhibitor compound for treating porcine epidemic diarrhea comprises the following raw materials in parts by weight: 10 parts of compound 3CL protease inhibitor, 5 parts of glutamine, 5 parts of glycyrrhizic acid, 2 parts of sodium decanoate, 12 parts of corn starch, and 8 parts of lactose. A method for preparing a 3CL protease inhibitor compound for treating porcine epidemic diarrhea includes the following preparation steps: mixing a compound 3CL protease inhibitor, glutamine, glycyrrhizic acid, sodium decanoate, corn starch and β-lactose, stirring evenly, granulating by stirring, and drying at 45°C for 30 min to obtain the 3CL protease inhibitor compound.

[0027] The complex 3CL protease inhibitor is prepared by the following steps: A1. 3CL protease inhibitor, stearic acid, and soybean lecithin were added to ethanol and stirred at 60°C for 25 min to obtain an oil phase. Poloxamer 188 was added to deionized water and stirred at 60°C for 25 min to obtain an aqueous phase. The oil phase was added to the aqueous phase, and the mixture was stirred at 60°C and 2000 r / min for 15 min, then stirred at 1°C for 3 h to form granules. The granules were collected by centrifugation at 17000 r / min, washed twice with deionized water, and freeze-dried at -20°C for 12 h to obtain lipid nanoparticles loaded with 3CL protease inhibitor. The mass ratio of 3CL protease inhibitor, stearic acid, soybean lecithin, and ethanol was 1:5:1.6:45; the mass ratio of poloxamer 188 and deionized water was 1:45. A2. Phospholipid-polyethylene glycol-N-hydroxysuccinimide ester and anhydrous ethanol were mixed and stirred until completely dissolved. Lactobacillus and montmorillonite were added and stirred evenly. Then, lipid nanoparticles loaded with 3CL protease inhibitors were added. The mixture was stirred at 40°C for 2 hours. The organic solvent, anhydrous ethanol, was removed by rotary evaporation at 37°C and 100 r / min to obtain modified lipid nanoparticles. The mass ratio of phospholipid-polyethylene glycol-N-hydroxysuccinimide ester, anhydrous ethanol, lactobacillus, montmorillonite, and lipid nanoparticles loaded with 3CL protease inhibitors was 0.5:40:0.1:0.1:2.2. A3. Add ethyl caffeate to ethanol, stir well, add modified lipid nanoparticles, stir at 60℃ and 100r / min for 20min, and dry at room temperature overnight to obtain a composite 3CL protease inhibitor; the mass ratio of ethyl caffeate, ethanol and modified lipid nanoparticles is 1:10:2.

[0028] Example 2 A 3CL protease inhibitor compound for treating porcine epidemic diarrhea comprises the following raw materials in parts by weight: 13 parts of compound 3CL protease inhibitor, 8 parts of glutamine, 8 parts of glycyrrhizic acid, 2.5 parts of sodium decanoate, 13 parts of corn starch, and 10 parts of lactose. A method for preparing a 3CL protease inhibitor compound for treating porcine epidemic diarrhea includes the following preparation steps: mixing a compound 3CL protease inhibitor, glutamine, glycyrrhizic acid, sodium decanoate, corn starch and β-lactose, stirring evenly, granulating by stirring, and drying at 45°C for 30 min to obtain the 3CL protease inhibitor compound.

[0029] The complex 3CL protease inhibitor is prepared by the following steps: A1. 3CL protease inhibitor, stearic acid, and soybean lecithin were added to ethanol and stirred at 60°C for 25 min to obtain an oil phase. Poloxamer 188 was added to deionized water and stirred at 60°C for 25 min to obtain an aqueous phase. The oil phase was added to the aqueous phase, and the mixture was stirred at 60°C and 2000 r / min for 15 min, then at 2°C for 4 h to form granules. The granules were collected by centrifugation at 17000 r / min, washed twice with deionized water, and freeze-dried at -20°C for 12 h to obtain lipid nanoparticles loaded with 3CL protease inhibitor. The mass ratio of 3CL protease inhibitor, stearic acid, soybean lecithin, and ethanol was 1.1:5.3:1.8:50; the mass ratio of poloxamer 188 and deionized water was 1:50. A2. Phospholipid-polyethylene glycol-N-hydroxysuccinimide ester and anhydrous ethanol were mixed and stirred until completely dissolved. Lactobacillus and montmorillonite were added and stirred evenly. Then, lipid nanoparticles loaded with 3CL protease inhibitors were added. The mixture was stirred at 50°C for 2.5 h. The organic solvent anhydrous ethanol was removed by rotary evaporation at 37°C and 100 r / min to obtain modified lipid nanoparticles. The mass ratio of phospholipid-polyethylene glycol-N-hydroxysuccinimide ester, anhydrous ethanol, lactobacillus, montmorillonite and lipid nanoparticles loaded with 3CL protease inhibitors was 0.8:45:0.2:0.2:2.3. A3. Add ethyl caffeate to ethanol, stir well, add modified lipid nanoparticles, stir at 65℃ and 110r / min for 25min, and dry at room temperature overnight to obtain a composite 3CL protease inhibitor; the mass ratio of ethyl caffeate, ethanol and modified lipid nanoparticles is 1.1:11:2.2.

[0030] Example 3 A 3CL protease inhibitor compound for treating porcine epidemic diarrhea comprises the following raw materials in parts by weight: 15 parts of compound 3CL protease inhibitor, 10 parts of glutamine, 10 parts of glycyrrhizic acid, 3 parts of sodium decanoate, 15 parts of corn starch, and 12 parts of lactose. A method for preparing a 3CL protease inhibitor compound for treating porcine epidemic diarrhea includes the following preparation steps: mixing a compound 3CL protease inhibitor, glutamine, glycyrrhizic acid, sodium decanoate, corn starch and β-lactose, stirring evenly, granulating by stirring, and drying at 45°C for 30 min to obtain the 3CL protease inhibitor compound.

[0031] The complex 3CL protease inhibitor is prepared by the following steps: A1. 3CL protease inhibitor, stearic acid, and soybean lecithin were added to ethanol and stirred at 60°C for 25 min to obtain an oil phase. Poloxamer 188 was added to deionized water and stirred at 60°C for 25 min to obtain an aqueous phase. The oil phase was added to the aqueous phase, and the mixture was stirred at 60°C and 2000 r / min for 15 min, then at 3°C ​​for 5 h to form granules. The granules were collected by centrifugation at 17000 r / min, washed twice with deionized water, and freeze-dried at -20°C for 12 h to obtain lipid nanoparticles loaded with 3CL protease inhibitor. The mass ratio of 3CL protease inhibitor, stearic acid, soybean lecithin, and ethanol was 1.2:5.5:2:55; the mass ratio of poloxamer 188 and deionized water was 1:55. A2. Phospholipid-polyethylene glycol-N-hydroxysuccinimide ester and anhydrous ethanol were mixed and stirred until completely dissolved. Lactobacillus and montmorillonite were added and stirred evenly. Then, lipid nanoparticles loaded with 3CL protease inhibitors were added. The mixture was stirred at 60°C for 3 hours. The organic solvent, anhydrous ethanol, was removed by rotary evaporation at 37°C and 100 r / min to obtain modified lipid nanoparticles. The mass ratio of phospholipid-polyethylene glycol-N-hydroxysuccinimide ester, anhydrous ethanol, lactobacillus, montmorillonite, and lipid nanoparticles loaded with 3CL protease inhibitors was 1:50:0.3:0.3:2.5. A3. Add ethyl caffeate to ethanol, stir well, add modified lipid nanoparticles, stir at 70℃ and 120r / min for 20-30min, and dry at room temperature overnight to obtain a composite 3CL protease inhibitor; the mass ratio of ethyl caffeate, ethanol and modified lipid nanoparticles is 1.2:12:2.3.

[0032] Comparative Example 1 The only difference between this comparative example and Example 3 is the preparation of the compound 3CL protease inhibitor, as detailed below: The complex 3CL protease inhibitor is prepared by the following steps: A1. Phospholipid-polyethylene glycol-N-hydroxysuccinimide ester and anhydrous ethanol were mixed and stirred until completely dissolved. Lactobacillus and montmorillonite were added and stirred evenly. Then, 3CL protease inhibitor was added and stirred at 60°C for 3 hours. The organic solvent anhydrous ethanol was removed by rotary evaporation at 37°C and 100 r / min to obtain the complex. The mass ratio of phospholipid-polyethylene glycol-N-hydroxysuccinimide ester, anhydrous ethanol, lactobacillus, montmorillonite and 3CL protease inhibitor was 1:50:0.3:0.3:2.5. A2. Add ethyl caffeate to ethanol, stir well, add the complex, stir at 70℃ and 120r / min for 20-30min, and dry at room temperature overnight to obtain the complex 3CL protease inhibitor; the mass ratio of ethyl caffeate, ethanol and complex is 1.2:12:2.3.

[0033] Comparative Example 2 The only difference between this comparative example and Example 3 is the preparation of the compound 3CL protease inhibitor, as detailed below: The complex 3CL protease inhibitor is prepared by the following steps: A1. 3CL protease inhibitor, stearic acid, and soybean lecithin were added to ethanol and stirred at 60°C for 25 min to obtain an oil phase. Poloxamer 188 was added to deionized water and stirred at 60°C for 25 min to obtain an aqueous phase. The oil phase was added to the aqueous phase, and the mixture was stirred at 60°C and 2000 r / min for 15 min, then at 3°C ​​for 5 h to form granules. The granules were collected by centrifugation at 17000 r / min, washed twice with deionized water, and freeze-dried at -20°C for 12 h to obtain lipid nanoparticles loaded with 3CL protease inhibitor. The mass ratio of 3CL protease inhibitor, stearic acid, soybean lecithin, and ethanol was 1.2:5.5:2:55; the mass ratio of poloxamer 188 and deionized water was 1:55. A2. Phospholipid-polyethylene glycol-N-hydroxysuccinimide ester and anhydrous ethanol were mixed and stirred until completely dissolved. Montmorillonite was added and stirred evenly. Then, lipid nanoparticles loaded with 3CL protease inhibitors were added. The mixture was stirred at 60°C for 3 hours. The organic solvent, anhydrous ethanol, was removed by rotary evaporation at 37°C and 100 r / min to obtain modified lipid nanoparticles. The mass ratio of phospholipid-polyethylene glycol-N-hydroxysuccinimide ester, anhydrous ethanol, montmorillonite, and lipid nanoparticles loaded with 3CL protease inhibitors was 1:50:0.6:2.5. A3. Add ethyl caffeate to ethanol, stir well, add modified lipid nanoparticles, stir at 70℃ and 120r / min for 20-30min, and dry at room temperature overnight to obtain a composite 3CL protease inhibitor; the mass ratio of ethyl caffeate, ethanol and modified lipid nanoparticles is 1.2:12:2.3.

[0034] Comparative Example 3 The only difference between this comparative example and Example 3 is the preparation of the compound 3CL protease inhibitor, as detailed below: The complex 3CL protease inhibitor is prepared by the following steps: A1. 3CL protease inhibitor, stearic acid, and soybean lecithin were added to ethanol and stirred at 60°C for 25 min to obtain an oil phase. Poloxamer 188 was added to deionized water and stirred at 60°C for 25 min to obtain an aqueous phase. The oil phase was added to the aqueous phase, and the mixture was stirred at 60°C and 2000 r / min for 15 min, then at 3°C ​​for 5 h to form granules. The granules were collected by centrifugation at 17000 r / min, washed twice with deionized water, and freeze-dried at -20°C for 12 h to obtain lipid nanoparticles loaded with 3CL protease inhibitor. The mass ratio of 3CL protease inhibitor, stearic acid, soybean lecithin, and ethanol was 1.2:5.5:2:55; the mass ratio of poloxamer 188 and deionized water was 1:55. A2. Phospholipid-polyethylene glycol-N-hydroxysuccinimide ester and anhydrous ethanol were mixed and stirred until completely dissolved. Lactobacillus was added and stirred evenly. Then, lipid nanoparticles loaded with 3CL protease inhibitors were added. The mixture was stirred at 60°C for 3 hours. The organic solvent, anhydrous ethanol, was removed by rotary evaporation at 37°C and 100 r / min to obtain modified lipid nanoparticles. The mass ratio of phospholipid-polyethylene glycol-N-hydroxysuccinimide ester, anhydrous ethanol, lactobacillus, and lipid nanoparticles loaded with 3CL protease inhibitors was 1:50:0.6:2.5. A3. Add ethyl caffeate to ethanol, stir well, add modified lipid nanoparticles, stir at 70℃ and 120r / min for 20-30min, and dry at room temperature overnight to obtain a composite 3CL protease inhibitor; the mass ratio of ethyl caffeate, ethanol and modified lipid nanoparticles is 1.2:12:2.3.

[0035] Comparative Example 4 The only difference between this comparative example and Example 3 is the preparation of the compound 3CL protease inhibitor, as detailed below: The complex 3CL protease inhibitor is prepared by the following steps: A1. 3CL protease inhibitor, stearic acid, and soybean lecithin were added to ethanol and stirred at 60°C for 25 min to obtain an oil phase. Poloxamer 188 was added to deionized water and stirred at 60°C for 25 min to obtain an aqueous phase. The oil phase was added to the aqueous phase, and the mixture was stirred at 60°C and 2000 r / min for 15 min, then at 3°C ​​for 5 h to form granules. The granules were collected by centrifugation at 17000 r / min, washed twice with deionized water, and freeze-dried at -20°C for 12 h to obtain lipid nanoparticles loaded with 3CL protease inhibitor. The mass ratio of 3CL protease inhibitor, stearic acid, soybean lecithin, and ethanol was 1.2:5.5:2:55; the mass ratio of poloxamer 188 and deionized water was 1:55. A2. Anhydrous ethanol, lactobacillus, and montmorillonite were mixed and stirred evenly. Then, lipid nanoparticles loaded with 3CL protease inhibitors were added. The mixture was stirred at 60°C for 3 hours. The organic solvent, anhydrous ethanol, was removed by rotary evaporation at 37°C and 100 r / min to obtain modified lipid nanoparticles. The mass ratio of anhydrous ethanol, lactobacillus, montmorillonite, and lipid nanoparticles loaded with 3CL protease inhibitors was 50:0.3:1.3:2.5. A3. Add ethyl caffeate to ethanol, stir well, add modified lipid nanoparticles, stir at 70℃ and 120r / min for 20-30min, and dry at room temperature overnight to obtain a composite 3CL protease inhibitor; the mass ratio of ethyl caffeate, ethanol and modified lipid nanoparticles is 1.2:12:2.3.

[0036] Comparative Example 5 The only difference between this comparative example and Example 3 is the preparation of the compound 3CL protease inhibitor, as detailed below: The complex 3CL protease inhibitor is prepared by the following steps: A1. 3CL protease inhibitor, stearic acid, and soybean lecithin were added to ethanol and stirred at 60°C for 25 min to obtain an oil phase. Poloxamer 188 was added to deionized water and stirred at 60°C for 25 min to obtain an aqueous phase. The oil phase was added to the aqueous phase, and the mixture was stirred at 60°C and 2000 r / min for 15 min, then at 3°C ​​for 5 h to form granules. The granules were collected by centrifugation at 17000 r / min, washed twice with deionized water, and freeze-dried at -20°C for 12 h to obtain lipid nanoparticles loaded with 3CL protease inhibitor. The mass ratio of 3CL protease inhibitor, stearic acid, soybean lecithin, and ethanol was 1.2:5.5:2:55; the mass ratio of poloxamer 188 and deionized water was 1:55. A2. Phospholipid-polyethylene glycol-N-hydroxysuccinimide ester and anhydrous ethanol were mixed and stirred until completely dissolved. Lactobacillus and montmorillonite were added and stirred evenly. Then, lipid nanoparticles loaded with 3CL protease inhibitors were added. The mixture was stirred at 60°C for 3 hours. The organic solvent anhydrous ethanol was removed by rotary evaporation at 37°C and 100 r / min to obtain a composite 3CL protease inhibitor. The mass ratio of phospholipid-polyethylene glycol-N-hydroxysuccinimide ester, anhydrous ethanol, lactobacillus, montmorillonite and lipid nanoparticles loaded with 3CL protease inhibitors was 1:50:0.3:0.3:2.5.

[0037] The performance of the 3CL protease inhibitor compositions prepared in Examples 1-3 and Comparative Examples 1-5 was then tested.

[0038] Safety testing: The CCK8 assay was used to detect the toxicity of the drug composition to Vero cells (African green monkey kidney cells); the prepared 3CL protease inhibitor composition was mixed with DMEM medium to form DMEM medium containing the inhibitor composition at concentrations of 5 µg / mL, 10 µg / mL, 20 µg / mL, 40 µg / mL and 80 µg / mL.

[0039] Vero cells were divided at a ratio of 1×10 5Cells were seeded at a density of 100 cells / mL in 96-well plates and cultured at 37°C with 5% CO2 for 24 h. Once the cells had adhered and grown into a monolayer, the original cell culture medium was removed and replaced with DMEM medium containing the drug. The final drug concentration in each well was controlled at 5 µg / mL, 10 µg / mL, 20 µg / mL, 40 µg / mL, and 80 µg / mL. A cell control group (only Vero cells and an equal volume of DMEM medium) and a blank control group (only an equal volume of DMEM medium) were set up. The cells were then cultured at 37°C with 5% CO2 for another 48 h. After the culture period, CCK-8 solution was added to each well, and the cells were cultured for 1 h. The absorbance of each well was measured at 450 nm, and the cell viability was calculated. Cell viability (%) = (OD450 value of experimental group - OD450 value of blank control group) / (OD450 value of cell control group - OD450 value of blank control group) × 100%. The experimental results are shown in Table 1.

[0040] Table 1 ; As shown in Table 1, the 3CL protease inhibitor group prepared above maintained a survival rate of over 99% for Vero cells within a concentration range of 5 μg / mL to 80 μg / mL, with no significant difference compared to the cell control group. This indicates that the 3CL protease inhibitor group prepared above has no cytotoxicity to Vero cells and has good safety, providing a safety guarantee for its subsequent application.

[0041] Validation of the antiviral efficacy of the drug composition: Vero cells were divided into 2×10 5 Cells / mL were seeded into 96-well cell culture plates and cultured at 37°C in a 5% CO2 incubator until a monolayer was formed. PEDV strain (multiple of infection (MOI) of 0.1) was added and incubated at 37°C for 2 hours. After removing the supernatant, uninvaded virus particles were washed with PBS, and then the corresponding drugs were added according to Examples 1-3 and Comparative Examples 1-5, and cultured for another 16 hours. Viral supernatant from each group was collected, and the viral titer (TCID) was tested. 50 ); Uninfected Vero cells were divided into groups of 2 × 10⁻⁶. 5 Cells were seeded at a rate of 1 / mL into 96-well plates. After a confluent monolayer was formed, the collected supernatant was seeded in 10-fold serial dilutions, with 8 replicates for each dilution. Supernatant from PEDV-infected cells without drug treatment was used as a viral control. The seeded 96-well plates were incubated at 37°C in a 5% CO2 incubator. Cytopathic effects were observed daily, and the number of wells showing cytopathic effects was recorded until the number of wells showing cytopathic effects stopped increasing. The number of wells producing CPE was recorded, and the TCID of the virus was calculated. 50 The results are shown in Table 2.

[0042] Table 2 Performance testing of 3CL protease inhibitor compositions prepared in Examples 1-3 and Comparative Examples 1-5 ; In vivo experiments were conducted to verify the therapeutic effect of the prepared 3CL protease inhibitor group on porcine epidemic diarrhea (PED): Healthy 7-day-old piglets were selected as experimental animals and randomly divided into six groups each: Examples 1-3, Comparative Examples 1-5, a blank control group, and an infection group. Except for the blank control group, the animals were administered 10... 5 TCID 50 The experimental piglets were given a viral load of 1.0 mL of PEDV orally. 12 hours after challenge, the piglets in Examples 1-3 and Comparative Examples 1-5 were given the corresponding drug composition by gavage at a dose of 10 mg / kg. The blank control group was given physiological saline by gavage. The treatment was given once every 6 hours for 66 hours. After treatment, the clinical symptoms of piglets in each group were systematically observed and quantitatively scored. The scoring system included four core indicators: mental state, feeding ability, degree of diarrhea, and coat condition. The score range for each indicator was set from 1 to 4 points. The higher the total score, the more severe the PEDV symptoms and the worse the condition of the piglets. The lower the total score, the better the treatment effect.

[0043] The specific scoring criteria are as follows: A score of 1 indicates that the indicator is normal and the patient is in a normal physiological state. A score of 2 indicates mild symptoms that do not affect normal physiological functions; A score of 3 indicates severe symptoms that significantly impact the physiological condition of piglets. A score of 4 indicates that if a piglet dies, all its clinical indicators will be scored as 4.

[0044] The total clinical score for a single piglet is the sum of the scores of the four indicators.

[0045] The results are shown in Table 3 below.

[0046] Table 3 Performance testing of 3CL protease inhibitor compositions prepared in Examples 1-3 and Comparative Examples 1-5 ; As can be seen from the data in Tables 1, 2 and 3, the 3CL protease inhibitor compositions prepared in Examples 1-3 have a highly effective therapeutic effect on porcine epidemic diarrhea and have a good targeted therapeutic effect, specifically inhibiting the 3CL protease of PEDV.

[0047] Comparative Example 1 showed that replacing the lipid nanoparticles loaded with 3CL protease inhibitor with a composite 3CL protease inhibitor added to the 3CL protease inhibitor composition resulted in a decrease in its therapeutic performance for porcine epidemic diarrhea (PED). This indicates that the lipid nanoparticles formed by stearic acid and soybean lecithin have better gastric acid resistance, which can improve the acid resistance of the 3CL protease inhibitor and prevent it from being easily degraded by gastric acid during its entry into the intestine, leading to insufficient utilization of the 3CL protease inhibitor and poor therapeutic effect on PED. The formed lipid nanoparticles can bind to the intestinal epithelial cell membrane, allowing the 3CL protease inhibitor to act better on the intestinal mucosa. Furthermore, the lipid nanoparticles can penetrate the viscous mucus layer of the pig intestine, increasing the probability of contact between the intestinal 3CL protease inhibitor and the pig intestinal epithelium, thus exhibiting a highly effective therapeutic effect on PED.

[0048] Comparative Example 2, where lactobacillus was replaced with montmorillonite, and Comparative Example 3, where montmorillonite was replaced with lactobacillus, were used to prepare a composite 3CL protease inhibitor. When this was added to the 3CL protease inhibitor group, its therapeutic performance for porcine epidemic diarrhea (PED) decreased. This indicates that montmorillonite, through its layered porous structure, physically adsorbs PED viruses, pathogens, and inflammatory toxins in the intestines. Simultaneously, it forms a protective film on the damaged intestinal epithelium, adsorbing excess intestinal water, rapidly relieving watery diarrhea, and preventing dehydration in livestock. Lactobacillus can regulate intestinal flora homeostasis, promote the proliferation of beneficial bacteria, inhibit the growth of harmful bacteria, reduce the risk of secondary bacterial infections, and stimulate intestinal mucosal repair, thus alleviating intestinal inflammatory damage. The combination of lactobacillus and montmorillonite produces a synergistic antidiarrheal and intestinal repair effect. Furthermore, lactobacillus and montmorillonite, in conjunction with nimatrazor, treat PED from multiple dimensions: antiviral, mucosal protection, and flora regulation.

[0049] In Comparative Example 4, when a 3CL protease inhibitor prepared by replacing phospholipid-polyethylene glycol-N-hydroxysuccinimide ester with montmorillonite was added to the 3CL protease inhibitor group, its therapeutic performance for porcine epidemic diarrhea (PED) decreased. This indicates that the long chain of phospholipid-polyethylene glycol-N-hydroxysuccinimide ester forms a hydration layer on the surface of lipid nanoparticles, inhibiting lipid nanoparticle aggregation, preventing flocculation and precipitation in intestinal fluid, prolonging intestinal retention time, and improving the therapeutic effect on PED. Furthermore, it can prevent non-specific adsorption of lipid nanoparticles to intestinal mucoproteins, reducing ineffective drug retention in the intestinal lumen, allowing lipid nanoparticles to reach the PEDV infection site in the small intestine and treat PED. Phospholipid-polyethylene glycol-N-hydroxysuccinimide ester provides covalent binding sites, firmly coupling with ethyl caffeate, making the ethyl caffeate modified layer less prone to detachment in the digestive tract environment, thus exerting its therapeutic effect on PED.

[0050] In Comparative Example 5, the composite 3CL protease inhibitor prepared without caffeic acid ethyl ester was added to the 3CL protease inhibitor group, resulting in a decrease in its therapeutic performance for porcine epidemic diarrhea (PED). This indicates that caffeic acid ethyl ester directly targets the 3CL protease of PEDV and inhibits its enzyme activity, thereby inhibiting PEDV replication in vitro, improving the survival rate of PEDV-infected suckling piglets, and inhibiting viral replication in vivo. It exhibits high anti-PEDV activity and exerts a highly effective therapeutic effect on porcine epidemic diarrhea. In addition, lactobacillus and montmorillonite, through the long-chain action of phospholipid-polyethylene glycol-N-hydroxysuccinimide ester, are tightly bound to the surface of lipid nanoparticles, thus exerting a therapeutic effect on porcine epidemic diarrhea.

[0051] In the description of this specification, the references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0052] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. A 3CL protease inhibitor complex for treating porcine epidemic diarrhea, characterized in that, The ingredients include the following parts by weight: 10-15 parts of compound 3CL protease inhibitor, 5-10 parts of glutamine, 5-10 parts of glycyrrhizic acid, 2-3 parts of sodium decanoate, 12-15 parts of starch, and 8-12 parts of lactose. The composite 3CL protease inhibitor is formed by coating 3CL protease inhibitor with lipid nanoparticles to form lipid nanoparticles loaded with 3CL protease inhibitor; it is obtained by mixing phospholipid-polyethylene glycol-N-hydroxysuccinimide ester, lactobacillus, montmorillonite and lipid nanoparticles loaded with 3CL protease inhibitor, and then mixing with ethyl caffeate.

2. The 3CL protease inhibitor compound for treating porcine epidemic diarrhea according to claim 1, characterized in that, The complex 3CL protease inhibitor is prepared by the following steps: A1. 3CL protease inhibitor, stearic acid and soybean lecithin were added to ethanol and stirred to obtain an oil phase. Poloxamer 188 was added to deionized water and stirred to obtain an aqueous phase. The oil phase and aqueous phase were mixed and stirred. After stirring, the mixture was stirred at 1-3℃ for 3-5 hours to form granules. After centrifugation, washing and freeze-drying, lipid nanoparticles loaded with 3CL protease inhibitor were obtained. A2. Mix phospholipid-polyethylene glycol-N-hydroxysuccinimide ester and anhydrous ethanol, stir until completely dissolved, add lactobacillus and montmorillonite, stir evenly, then add lipid nanoparticles loaded with 3CL protease inhibitor, stir at 40-60℃ for 2-3 hours, remove organic solvent by rotary evaporation to obtain modified lipid nanoparticles. A3. Add ethyl caffeate to ethanol, stir well, add modified lipid nanoparticles, stir at 60-70℃ and 100-120r / min for 20-30min, and dry at room temperature overnight to obtain the composite 3CL protease inhibitor.

3. The 3CL protease inhibitor compound for treating porcine epidemic diarrhea according to claim 2, characterized in that, In step A1, the mass ratio of the 3CL protease inhibitor, stearic acid, soybean lecithin, and ethanol is (1-1.2):(5-5.5):(1.6-2):(45-55).

4. The 3CL protease inhibitor compound for treating porcine epidemic diarrhea according to claim 2, characterized in that, In step A1, the mass ratio of poloxamer 188 to deionized water is 1:(45-55).

5. A 3CL protease inhibitor compound for treating porcine epidemic diarrhea according to claim 2, characterized in that, In step A2, the mass ratio of the phospholipid-polyethylene glycol-N-hydroxysuccinimide ester, anhydrous ethanol, lactobacillus, montmorillonite, and lipid nanoparticles loaded with 3CL protease inhibitor is (0.5-1):(40-50):(0.1-0.3):(0.1-0.3):(2.2-2.5).

6. A 3CL protease inhibitor compound for treating porcine epidemic diarrhea according to claim 2, characterized in that, In step A3, the mass ratio of ethyl caffeate, ethanol and modified lipid nanoparticles is (1-1.2):(10-12):(2-2.3).

7. A 3CL protease inhibitor compound for treating porcine epidemic diarrhea according to claim 1, characterized in that, The lactose in question is β-lactose.

8. A 3CL protease inhibitor compound for treating porcine epidemic diarrhea according to claim 1, characterized in that, The starch is corn starch or sweet potato starch.

9. A 3CL protease inhibitor compound for treating porcine epidemic diarrhea according to claim 1, characterized in that, The preparation method of the 3CL protease inhibitor composition includes the following steps: mixing the compound 3CL protease inhibitor, glutamine, glycyrrhizic acid, sodium decanoate, starch and lactose, stirring evenly, granulating, and drying to obtain the 3CL protease inhibitor composition.