KR102581090B1 - Pharmaceutical composition for the treatment of porcine epidemic diarrhea virus comprising statin compounds - Google Patents

Abstract

The present invention relates to a pharmaceutical composition, food composition, feed composition, and health functional food for preventing, treating or improving porcine infectious diarrhea virus containing a statin compound or a pharmaceutically acceptable salt thereof as an active ingredient.

Description

Pharmaceutical composition for the treatment of porcine epidemic diarrhea virus comprising statin compounds}

The present invention relates to a pharmaceutical composition, food composition, and feed composition for treating porcine epidemic diarrhea virus (PEDV) containing a statin compound.

Porcine infectious diarrhea virus is a member of the Alphacoronavirus genus of the Coronaviridae family and causes intestinal symptoms in pigs of all ages. When suckling piglets are infected with the porcine infectious diarrhea virus, they die from watery diarrhea and rapid dehydration, with a high mortality rate of 50 to 95%. Therefore, an uncontrolled outbreak of the virus can cause enormous economic losses to farms. bring about

A commercially available vaccine against porcine infectious diarrhea virus has been developed, but with the emergence of new mutant strains, virus infection is increasing even in pigs that have received the existing vaccine, and the development of an effective treatment to treat porcine infectious diarrhea virus is insufficient. This is the situation.

Statins are inhibitors of 3-methylgludaryl-CoA (HMG-CoA) reductase, which is involved in cholesterol biosynthesis. Statins are clinically approved for the prevention of primary and secondary cardiovascular disease. Meanwhile, Martiez-Gutierrez et al. reported that statins were effective against dengue virus (Statins Reduce Dengue Virus Production via Decreased Virion Assembly. Intervirology 54 , 202-216 (2011)), and Andrus et al. reported that statins were effective against hepatitis C virus. has reported its effectiveness (Use of statins in patients with chronic Hepatitis C. Southern Medical Journal 103 , 1018-1024 (2010). In addition, Shrivastava-Ranjan et al. applied statins to Ebola virus, and Mehrbod et al. applied statins to Ebola virus. There are examples of its application to treat influenza viruses, etc. (a: Suppress Ebola Virus Infectivity by Interfering with Glycoprotein Processing. mBio 9 , e00660-00618 (2018); b: Mechanisms of Action and Efficacy of Statins against Influenza. BioMed Research International 2014 , 872370 (2014). However, there has been no report yet on the inhibitory effect of statins on porcine infectious diarrhea virus.

The problem to be solved by the present invention relates to a pharmaceutical composition for preventing or treating porcine infectious diarrhea virus containing a statin compound as an active ingredient.

Another problem to be solved by the present invention is to provide a food composition, feed composition, and health functional food for preventing or improving porcine infectious diarrhea virus containing a statin compound.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating porcine infectious diarrhea virus containing a statin compound or a pharmaceutically acceptable salt thereof as an active ingredient.

According to the present invention, the statin compound is fluvastatin, mevastatin, simvastatin, lovastatin, somatostatin, nystatin, pravastatin, and cilastatin. It may be any one or more selected from the group consisting of cilastatin, atorvastatin, cervastatin, ulinastatin, and rosuvastatin.

According to the present invention, the statin compound may preferably be selected from the group consisting of fluvastatin, mevastatin, and simvastatin.

According to the present invention, the fluvastatin is a compound represented by the following formula (1).

[Formula 1]

According to the present invention, the mevastatin is a compound represented by the following formula (2).

[Formula 2]

According to the present invention, the simvastatin is a compound represented by the following formula (3).

[Formula 3]

According to the present invention, a statin compound can prevent or treat porcine infectious diarrhea virus by inhibiting cell death or cell transformation caused by the porcine infectious diarrhea virus.

According to the present invention, the statin compound can prevent or treat porcine infectious diarrhea virus by directly killing the porcine infectious diarrhea virus or inhibiting virus proliferation.

According to the present invention, the porcine infectious diarrhea virus that can be prevented or treated with the statin compound of the present invention is one or more selected from the group G1a, G1b, G2a, and G2b, or the G1a, G1b, G2a, and G2b, depending on the genotype of the spike gene. Porcine infectious diarrhea viruses belong to the group and have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%. %, or may have sequence homology of at least 99%. The “% sequence homology” for a polynucleotide is determined by comparing a comparison region with two optimally aligned sequences, where a portion of the polynucleotide sequence in the comparison region is a reference sequence (additions or deletions) for the optimal alignment of the two sequences. may contain additions or deletions (i.e. gaps) compared to those that do not contain .

In the present invention, pharmaceutically acceptable salts refer to salts commonly used in the pharmaceutical industry, for example, inorganic ionic salts made of calcium, potassium, sodium and magnesium, hydrochloric acid, nitric acid, phosphoric acid, bromic acid, and iodine. Inorganic acid salts made from acids, perchloric acid, tartaric acid and sulfuric acid, acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid. , organic acid salts made of galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p -There are sulfonates made from toluenesulfonic acid and naphthalenesulfonic acid, amino acid salts made from glycine, arginine, lysine, etc., and amine salts made from trimethylamine, triethylamine, ammonia, pyridine, picoline, etc. The types of salts meant in the present invention are not limited by these listed salts.

In the present invention, the term "treatment" refers to the act of alleviating symptoms caused by porcine infectious diarrhea virus by administering the composition, and refers to the disease, symptoms of the disease, secondary diseases of the disease or disease, or related thereto. To subjects (humans or animals) with a disease, symptoms of a disease, secondary conditions of a disease or disease, or a predisposition therefor, with the purpose of treating, alleviating, alleviating, remedying, or improving the predisposition. It is defined as the application or administration of the composition.

As used herein, “subject” and “patient” refer to humans or non-humans, such as primates, mammals, and vertebrates. In certain embodiments, the subject is a pig.

As used herein, “therapeutic benefit” or “therapeutically effective” refers to anything that promotes or increases the health of a subject in connection with medical treatment of such condition. This includes, but is not limited to, a reduction in the frequency or severity of signs or symptoms of the disease.

The pharmaceutical composition of the present invention may contain 0.01 to 95% by weight, preferably 1 to 80% by weight, of a statin compound or a pharmaceutically acceptable salt thereof, based on the total weight of the composition.

According to one embodiment of the present invention, the statin compound or a pharmaceutically acceptable salt thereof is mixed with a conventional carrier, adjuvant or diluent and formulated by a conventional formulation method to prepare a form suitable for oral or parenteral administration. It can be.

The carriers, adjuvants or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, poly Examples include vinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

In addition, when formulated, conventional fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. may be further included, and lubricants such as magnesium styrate and talc may also be added.

For oral administration, it can be manufactured in the form of tablets, capsules, solutions, syrups, suspensions, etc., and for parenteral administration, it can be injected into veins, intrauterine dura, abdominal cavity, subcutaneous, muscle, transdermal, and cerebrovascular injection. It can be manufactured in the form of an injection.

The pharmaceutical composition of the present invention is an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal, or human as considered by a researcher, veterinarian, physician, or other clinician, i.e., the symptom of the disease or disorder being treated. It can be administered in a therapeutically effective amount that induces remission. It is obvious to those skilled in the art that the therapeutically effective dosage and frequency of administration of the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by a person skilled in the art, depending on the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of dosage form, the patient's age, weight, and general health condition. , gender and diet, administration time, administration route and secretion rate of the composition, treatment period, and various factors including concurrently used drugs. The pharmaceutical composition of the present invention can be administered to an individual through various routes. For example, it may be administered intravenously, intraperitoneally, intramuscularly, intraarterially, bucally, intracardiacally, intramedullary, intrathecally, transdermally, enterally, subcutaneously, sublingually, or topically, but is not limited thereto.

The pharmaceutical composition of the present invention can be administered in an amount of 1 to 10,000 mg/kg/day, preferably 1 to 200 mg/kg/day, and may be administered once a day or divided into several times. .

In addition, another problem to be solved by the present invention is to provide a food composition for preventing or improving porcine infectious diarrhea virus containing a statin compound or a pharmaceutically acceptable salt thereof.

The food composition of the present invention can be used as a health functional food. The term “health functional food” refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with the Health Functional Food Act, and “functional” refers to food that is related to the structure and function of the human body. It means ingestion for the purpose of controlling nutrients or obtaining useful health effects such as physiological effects.

The food composition of the present invention may contain common food additives, and its suitability as a “food additive” is determined in accordance with the general provisions and general test methods of the food additive code approved by the Ministry of Food and Drug Safety, unless otherwise specified. The decision is made based on the specifications and standards for the item.

Items listed in the "Food Additives Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as subchromic pigment, licorice extract, crystalline cellulose, high-liquid pigment, and guar gum; Examples include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations.

The food composition of the present invention may contain 0.01 to 95% by weight, preferably 1 to 80% by weight, of a statin compound or a pharmaceutically acceptable salt thereof, based on the total weight of the composition.

Additionally, the food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc.

For example, the health functional food in the form of tablets is made by granulating a mixture of statins, excipients, binders, disintegrants, and other additives in a conventional manner, and then adding a lubricant and compression molding, or the mixture is directly manufactured. Compression molding is possible. In addition, the health functional food in the form of tablets may contain a flavoring agent, etc., if necessary, and may be peeled with a suitable peeling agent, if necessary.

Among health functional foods in the form of capsules, hard capsules can be manufactured by filling a regular hard capsule with a mixture of additives such as statins and excipients, or their granules or peeled granules, while soft capsules contain statins and excipients. It can be manufactured by filling a mixture with additives such as gelatin into a capsule base. The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary.

Health functional foods in the form of pills can be prepared by molding a mixture of statins, excipients, binders, disintegrants, etc. in an appropriate manner. If necessary, the pills can be coated with white sugar or other suitable coating agents, or the pills can be coated with starch, talc, or other suitable substances. You can also wear it.

Health functional foods in granular form can be manufactured into granules from a mixture of statins, excipients, binders, disintegrants, etc., using an appropriate method, and may contain flavoring agents, flavoring agents, etc. as needed. For health functional foods in granular form, use No. 12 (1680 μm), No. 14 (1410 μm), and No. 45 (350 μm) sieves. When the next particle size test is performed, the entire amount passes through the No. 12 sieve and what remains on the No. 14 sieve is the total amount. Less than 5.0% and passing through No. 45 sieve may be less than 15.0% of the total amount.

Definitions of terms such as excipients, binders, disintegrants, lubricants, corrigants, flavoring agents, etc. are described in literature known in the art and include those with the same or similar functions (Korean Pharmacopoeia Commentary, Munseongsa, Korea) Association of Colleges of Pharmacy, 5th edition, p33-48, 1989).

There are no particular restrictions on the type of food. Examples of foods to which the statin compound of the present invention can be added include beverages, gums, vitamin complexes, drinks, etc., and include all health functional foods in the conventional sense.

The food composition may include food supplements, and foods include, for example, beverages, gum, tea, vitamin complexes, health supplements, capsules, tablets, powders, granules, liquids, pills, flakes, and pastes. It can be used in the form of tablets, syrups, gels, beverages, jellies or vines.

The content of statin may be 0.01 to 15% by weight based on the total weight of the health functional food, and in the case of a health drink composition, it may be contained at 0.02 to 10 g, preferably 0.3 to 1 g, based on a total of 100 g. You can.

The food supplements include food additives common in the art, such as flavoring agents, flavoring agents, colorants, fillers, stabilizers, etc. In addition, the health drink composition is an essential ingredient in the indicated ratio, and there is no particular limitation on ingredients added other than the statin, and it may contain various flavoring agents or natural carbohydrates as additional ingredients like a regular drink. Examples of the natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; polysaccharides such as dextrins, cyclodextrins; These are common sugars such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g. rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. there is.

In addition to the above, food compositions, diet supplements, or health functional foods containing statin compounds or pharmaceutically acceptable salts thereof include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, and coloring agents. and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated drinks, etc. can do.

Additionally, the present invention provides a feed composition for preventing or improving porcine infectious diarrhea virus containing a statin compound or a pharmaceutically acceptable salt thereof.

In the present invention, the animal may be one or more selected from mammals and birds. Preferably, the animal has purified wild habits as defined in Article 2, Paragraph 1 of the Livestock Act and Article 2 of the Enforcement Rules of the same Act and is suitable for breeding. It can be a livestock that can contribute to increasing the income of farmers. The livestock may include cattle, horses, mules, donkeys, goats, goats, sheep, deer, pigs, rabbits, poultry, etc., and the poultry may include chickens, turkeys, ducks, ostriches, geese, quails, etc., preferably chickens. It is not limited to this as long as it is suitable for breeding and obtaining livestock products. The above “livestock products” are defined by Article 2, Paragraph 3 of the Livestock Act, as defined by the Ordinance of the Ministry of Agriculture, Food and Rural Affairs, as meat, milk, eggs, honey and their processed products, raw skins (including raw fur), raw wool, and other livestock products produced from livestock. means that Additionally, it may be a dog, cat, etc., including companion animals, but is not limited thereto.

The term "feed" of the invention means any natural or artificial diet, meal, etc., or a component of the meal, for or suitable for eating, ingestion, and digestion by animals. In one aspect, the feed composition containing the indenone derivative compound of the present invention or a pharmaceutically acceptable salt thereof may include concentrate feed, roughage feed, and/or special feed.

Concentrated feed includes seeds and fruits, including grains such as wheat, oats, and corn; bran, which includes rice bran, bran, and barley bran, which are by-products obtained from refining grains; soybeans; oil, sesame seeds, linseed, and coco oil; Fish soluble, meat powder, which is a concentrated product of fresh liquid obtained from fish meal, fish waste, fish meal, fish waste, etc., such as residual starch, which is the main component of the starch residue that is left after removing the starch from the by-products such as seed jelly, sweet potatoes, potatoes, etc. , animal feeds such as blood meal, feather meal, skim milk powder, dried whey, which is the residue from milk to cheese, and casein from skim milk, yeast, chlorella, seaweed, etc.

Forage includes raw grass feed such as wild grasses, grasses, and green cuttings, turnips for feed, beets for feed, root vegetables such as rutterbearger, a type of turnip, raw grass, green cuttings, and grains, etc., in silos. There are silage, a stored feed that is stuffed and fermented with lactic acid, field grass, hay made by cutting and drying grass, straw from crops for breeding livestock, and leaves from legumes. Special feeds include mineral feeds such as oyster shells and rock salt, urea feeds such as urea and its derivative diureide isobutane, and mixed feeds to supplement ingredients that are likely to be lacking when mixing only natural feed raw materials, or to increase the storability of the feed. There are feed additives, which are substances added in trace amounts.

The statin compound according to the present invention can increase the cell survival rate against porcine epidemic diarrhea virus, lower the cell deformation rate, and protect, prevent, and treat an individual against infection by porcine epidemic diarrhea virus, and therefore can prevent, treat, or treat porcine epidemic diarrhea virus. It is useful as a pharmaceutical composition, food composition or feed composition for improvement.

Figure 1 shows the cell survival rate according to the administered dose of statins (FLU, MEV, SIM) in PEDV-infected Vero cells.
Figure 2a is a relative inhibitory dose-response curve for the dose of statin in PEDV-infected Vero cells, and Figure 2b is a cytotoxic dose-response curve in uninfected Vero cells.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, these examples are for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited thereto.

Example

cells and viruses

African green monkey (Vero; ATCC® CCL-81TM) cells were grown in growth medium consisting of Minimal Eagle's Medium (MEM, Gibco) supplemented with 10% fetal calf serum, antifungal antibiotics (Gibco), and L-glutamine (Gibco). I ordered it. Porcine epidemic diarrhea virus (PEDV) infection medium consisted of Minimal Eagle's Medium supplemented with 0.3% bovine serum albumin, antifungal antibiotics, and L-glutamine.

reagent

Fluvastatin sodium hydrate (FLU) and simvastatin (SIM) were purchased from Sigma-Aldrich. Mevastatin sodium salt (MEV) was purchased from Calbiochem. Statin compounds were reconstituted at 20 mM in dimethyl sulfoxide. All statin compounds were stored at -20°C in aliquots and diluted to working concentrations in infection medium on the day of use.

Test Example 1. Evaluation of the effect and cytotoxicity of statins

To determine the cytotoxicity of fluvastatin, mevastatin and simvastatin, overnight 96-well plate cultures of Vero cells (1.5 × 10 ⁴ cells/well) in growth medium were incubated for fluvastatin, mevastatin and simvastatin. Ten dose response curves were prepared prior to analysis. Before treatment, cells were washed once with phosphate-buffered saline (PBS). Cells were treated with statins and simultaneously infected with PEDV at a multiplicity of infection (MOI) of 0.001 in infection medium up to a volume of 100 μL/well.

After culturing at 37°C and 5% CO ₂ for up to 66 hours, EZ-CYTOX (DoGenBio, Co., Ltd., South Korea) was added to the culture medium (10 μL/well). After 3 to 4 hours, absorbance was checked at 450 nm. Cell viability (%) was calculated per dose compared to cell control (uninfected, using 0.5% DMSO). Virus control (no statin added) was also treated with 0.5% DMSO. Data were normalized and the relative half-maximal effective concentration ( EC ₅₀ ) was determined. All experiments were performed three times.

As shown in Figure 1, statins dose-dependently inhibited porcine epidemic diarrhea virus (PEDV) infection in Vero cells and improved cell survival. In addition, it was confirmed that administration of statins (FLU, MEV, SIM) reduced cell degeneration of PEDV-infected Vero cells in a dose-dependent manner. By type of statin, the most optimal effect was confirmed when administering 5 μM of fluvastatin, 12 μM of mevastatin, and 7.5 μM of simvastatin, and it was confirmed that the cell survival rate was increased to about 85%.

Figure 2A shows the relative inhibitory dose-response curves for doses of statins in PEDV-infected Vero cells. PEDV-infected Vero cells were treated with various doses of statins (FLU, MEV, SIM), and data were normalized based on the maximum and minimum responses. and the relative EC ₅₀ was calculated. Figure 2b is a cytotoxic dose-response curve in which uninfected Vero cells were treated with various doses of statins (FLU, MEV, SIM), and the relative CC ₅₀ was calculated. (Dose is based on nM). The measured EC ₅₀ and CC ₅₀ values and selectivity index (SI = CC ₅₀ /EC ₅₀ ) are shown in Table 1.

statins EC ₅₀ CC ₅₀ SI* fluvastatin 2.07 μM 4.61 μM 2.2 mevastatin 4.72 μM 15.35 μM 3.25 Simvastatin 3.30 μM 14.52 μM 4.4

The results show that statin compounds such as fluvastatin, mevastatin, and simvastatin can effectively inhibit PEDV infection in a dose-dependent manner.

Claims (11)

A pharmaceutical composition for preventing or treating porcine infectious diarrhea virus comprising a statin compound or a pharmaceutically acceptable salt thereof as an active ingredient,
A pharmaceutical composition, wherein the statin compound is at least one selected from the group consisting of fluvastatin, mevastatin, and simvastatin. delete delete The pharmaceutical composition according to claim 1, wherein the statin compound inhibits cell death or cell transformation caused by porcine infectious diarrhea virus. A food composition for preventing or improving porcine infectious diarrhea virus containing a statin compound or a pharmaceutically acceptable salt thereof,
A food composition wherein the statin compound is at least one selected from the group consisting of fluvastatin, mevastatin, and simvastatin. delete delete A feed composition for preventing or improving porcine infectious diarrhea virus containing a statin compound or a pharmaceutically acceptable salt thereof,
The feed composition wherein the statin compound is at least one selected from the group consisting of fluvastatin, mevastatin, and simvastatin. delete delete A health functional food for preventing or improving porcine infectious diarrhea virus containing a statin compound or a pharmaceutically acceptable salt thereof,
The statin compound is a health functional food that is at least one selected from the group consisting of fluvastatin, mevastatin, and simvastatin.