KR101825090B1 - Pharmaceutical Composition Comprising Ribavirin Or Derivatives Thereof For Treating Foot-And-Mouth Disease - Google Patents

Abstract

The present invention relates to a composition for preventing or treating foot-and-mouth disease and a feed composition for alleviating foot-and-mouth disease. The composition of the present invention effectively inhibits proliferation and activity of virus by being bound to enzymes associated with proliferation of foot-and-mouth disease virus with high affinity and can treat foot-and-mouth disease without having to distinguish what type of virus patients are infected from the beginning of disease, thereby being usefully used as an antiviral composition for alleviating symptoms of infected animals and preventing the spread of virus.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for treating foot and mouth disease including ribavirin or a derivative thereof,

The present invention relates to ribavirin and derivatives thereof useful as antiviral agents, and more particularly to the use of ribavirin and derivatives thereof or compositions comprising them for treating viral diseases caused by viruses in mammals.

Foot-and-mouth disease (FMD) is a virus-borne disease that infects animals with split hoofs and is characterized by rapid replication and rapid spreading. The foot-and-mouth disease pathogen is a single stranded bipolar RNA virus that belongs to the genus Picornaviridae and Aphthovirus and has seven different serotypes (A, O, C, Asia 1, SAT 1, SAT 2 and SAT 3) Being classified.

Foot-and-mouth disease viruses can spread air to distant areas within a short time. When foot-and-mouth disease develops, the productivity of animals (pigs, cattle, sheep, goats, etc.) decreases and the disposal of infected animals, Income is becoming a serious problem because of the decrease in the income, and the rate of disposal does not follow the virus propagation rate.

The use of vaccine as a foot and mouth defense method, which is currently being used in addition to live disposal. However, there is a problem that it takes a minimum of 7 days to one month of immune formation period until the antibody is formed by the vaccine and a vaccine effective against the serotypes and subtypes of the vaccine should be applied, which makes it difficult to respond promptly.

Therefore, there is an urgent need for an antiviral agent to be used as an adjuvant and an alternative agent for an emergency vaccine when foot-and-mouth disease has occurred. Of course, research on ribavirin, 5-Fluorouracil, 5-Azacytidine, 2'-C-Methylcytidine and T-1105 compounds for RNA virus defense is under way and T-1105 (Korean Patent Publication No. 10-1388061), there is a report that Ribavirin can help suppress the proliferation and defense of foot-and-mouth disease virus in cells and mice. The recombinant adenovirus was used to express interferon alpha (IFN-α), an antiviral agent, to protect against the foot-and-mouth disease virus in swine. High levels of IFN- . IFN-α, IFN-γ and recombinant adenovirus were used in combination to induce suppression of foot-and-mouth disease virus in pigs and delayed clinical symptoms. Although vaccination with vaccine and adenovirus is possible to protect against foot-and mouth disease virus, high virus titers of 10 ⁹ TCID ₅₀ in suckling pigs and 10 ⁶ to 10 ⁷ TCID ₅₀ in suckling mice are necessary for commercialization. do. In addition, IFN inducers such as polyinosinic: poly (C) and CpG have been used to identify FMDV defenses in adult mice, and the interferon combination is known to protect against foot-and-mouth disease viruses in pigs.

However, commercialization of anti-viral agents for the prevention of foot-and-mouth disease by using these compounds is still insignificant. Actually, there is no example used in the field for treatment or prevention. When foot-and- Suppression has its limits. In addition, since vaccine and antiviral agent can be used as a means for preventing virus transmission in the case of foot-and-mouth disease, a synergistic effect can be expected in combination use, but effective application composition and application method have not been specifically studied. Thus, a viral inhibitor and a method of applying the composition, which exhibit immediate effects immediately upon inoculation, can be highly effective in preventing disease transmission.

Because the foot-and-mouth disease involves the risk of a highly pathogenic virus, studies are only allowed in some licensed BL3 laboratories, and research on foot-and-mouth disease antiviral drugs is actively conducted around the world due to limited research investment such as economic feasibility of livestock medicines. It is not being carried out. Therefore, there is a growing demand for the development of more efficient and non-adverse anti-viral agents for the prevention of foot-and-mouth disease and prevention of the spread of them, and their commercialization.

Korean Patent Registration No. 10-1388061

The present inventors have made extensive efforts to develop an effective therapeutic composition for foot-and-mouth disease virus. As a result, it was found that ribavirin derivatives synthesized from ribavirin have low toxicity and high antiviral activity against foot-and-mouth disease virus, thus completing the present invention.

Accordingly, an object of the present invention is to provide a composition for preventing or treating a foot-and-mouth disease infectious disease.

Another object of the present invention is to provide a method and an amount of the compound of the present invention for the purpose of being used as an initial antiretroviral agent in combination with an emergency vaccine when a foot-and-mouth disease occurs, or as a post-treatment agent for preventing the spread of virus after infection.

Yet another object of the present invention is to provide a feed composition for improving or alleviating a foot-and-mouth disease infectious disease.

Other objects and advantages of the present invention will become more apparent from the following description, claims and drawings.

The present inventors have made extensive efforts to develop an effective therapeutic composition for foot-and-mouth disease virus. As a result, it was found that ribavirin derivatives synthesized from ribavirin have low toxicity and high antiviral activity against foot-and-mouth disease virus, thus completing the present invention.

Accordingly, an object of the present invention is to provide a composition for preventing or treating a foot-and-mouth disease infectious disease.

In order to accomplish the above object, the present invention provides a composition for antiviral against foot-and-mouth disease virus comprising, as an active ingredient, a ribavirin derivative compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

In the above formula (1), R is a ketone group having -COR 'and R' is a linear C ₁ to C ₅ alkyl.

The present invention also provides a composition for the prevention or treatment of foot-and-mouth disease infectious diseases including ribavirin or a ribavirin derivative represented by the following formula (1), foot-and-mouth disease vaccine and adjuvant.

[Chemical Formula 1]

In the above formula (1), R is a ketone group having -COR 'and R' is a linear C ₁ to C ₅ alkyl.

According to an embodiment of the present invention, the composition may inhibit the enzymatic activity of 3C protease or 3D polymerase, which is a non-structural protein synthesized by the virus, Lt; / RTI >

According to one embodiment of the present invention, the ribavirin may be administered in an amount of 0.3 to 1.0 mg per 1 g body weight of the subject once a day, by muscle or abdominal inoculation.

According to one embodiment of the present invention, the ribavirin derivative may be administered in an amount of 0.8 to 7.5 mg per 1 g of body weight of the subject animal once a day by inoculation with muscle or abdomen.

According to one embodiment of the present invention, the adjuvant may be aluminum hydroxide or Montanide ISA 201.

According to one embodiment of the present invention, the mixed solution of ribavirin and adjuvant may be administered at a concentration of 18 to 50 mg per pig.

According to one embodiment of the present invention, the mixed solution of ribavirin and adjuvant and foot-and-mouth disease vaccine can be administered simultaneously, separately or sequentially during the same treatment.

According to one embodiment of the present invention, the composition is administered in a muscle or abdominal inoculation, wherein the effective amount of ribavirin is 0.18 mg per day per gram of body weight of the subject animal and the vaccine composition is 0.18 mg / ≪ / RTI >

According to an embodiment of the present invention, the composition may be a composition for adding animal feed for improving or treating disease caused by foot-and-mouth disease virus.

According to another embodiment of the present invention, the present invention provides a method for treating or preventing a foot-and-mouth disease virus infection in an animal, comprising administering to the animal other than human a composition comprising ribavirin or a ribavirin analogue represented by the following formula . ≪ / RTI >

[Chemical Formula 1]

In the above formula (1), R is a ketone group having -COR 'and R' is a linear C ₁ to C ₅ alkyl.

The ribavirin derivative of the present invention or a pharmaceutically acceptable salt thereof can be usefully used for treating or preventing a disease caused by a virus infection or the like.

The present invention provides excellent anti-viral activity against foot-and-mouth disease virus with little toxicity and minimal side effects by providing the combination composition of ribavirin or the ribavirin derivative, foot-and-mouth vaccine and adjuvant, administration method, and single dose.

Further, the present invention has an excellent effect on early defense before inducing immunity effect of vaccine by preventing the virus from propagating immediately in the foot and mouth disease, thereby alleviating the symptom of the infected animal and reducing the amount of virus emission and preventing propagation to other animals.

FIG. 1 shows the antiviral effect of Ribavirin, T-1105, Azauridine and Adeno virus in foot-and-mouth disease mice (A: ribavirin or 6-azauridine at 3 mg / 1105 3 mg / dose was administered to the abdominal cavity at 0, 8, 13, 24, 30 hours and 5 × 10 ⁸ of siRNA or Ad-IFN-α, γ was inoculated into the muscle 1 day before the inoculation with foot- ribavirin 1.5 mg / dose was inoculated into the abdominal cavity over time).
Figure 2 shows the safety of Ribavirin and Ribavirin-TA in mice.
FIG. 3 shows the antiviral effect (survival rate and weight loss) after administration of ribavirin and ribavirin-TA through various routes in experimental animals (mice) (foot-and-mouth disease virus Asia1 / Shamir) 50 LD ₅₀ , Ribavirin is shown in Figures A to F, and ribavirin-TA is shown in Figures GJ).
FIG. 3A shows the survival rate for 10 days after administration of Ribavirin 10 mg / day by intraperitoneal or intramuscular injection for 0 to 3 days.
FIG. 3B shows changes in body weight for 10 days after administration of Ribavirin 10 mg / day by intraperitoneal or intramuscular injection for 0 to 3 days.
FIG. 3C shows the survival rate change for 10 days after oral administration of 5 to 15 mg / day of Ribavirin from 0 to 6 days.
FIG. 3D shows the change in body weight for 10 days after oral administration of 5 to 15 mg / day of Ribavirin through Day 0 to day 6.
FIG. 3E shows the survival rate change for 10 days after oral administration of 5 to 15 mg / day of Ribavirin mixed with feed for 0 to 6 days.
FIG. 3F shows changes in body weight for 10 days after oral administration of 5 to 15 mg / day of Ribavirin mixed with feed for 0 to 6 days.
Figure 3G shows the survival rate change for 10 days after oral administration of 5 to 15 mg / day of Ribavirin-TA from 0 to 6 days.
FIG. 3H shows the change in body weight for 10 days after oral administration of 5 to 15 mg / day of Ribavirin-TA through day 0 to day 6.
FIG. 3I shows changes in survival rate for 10 days after oral administration of 5 to 15 mg / day of Ribavirin-TA to feed for 0 to 6 days.
Fig. 3J shows changes in body weight for 10 days after oral administration of 5 to 15 mg / day of Ribavirin-TA to feed for 0 to 6 days.
4 shows the antiviral effect (A: change in survival rate, B: change in body weight, C: amount of virus in blood) by administering a combination of foot-and-mouth disease vaccine and antiviral agent in an experimental animal (mouse) , 6 mg or 10 mg of the antiviral agent was orally administered daily for 0-6 days, and the footpad virus Asia1 / Shamir 50 LD ₅₀ was inoculated at 6 hours and 10 days. FIG. 3A shows the survival rate change, FIG. 3B shows the body weight change, 3C shows the amount of virus in the blood, the arrows indicate the time of attack, and the negative control group died by 3 days).
FIG. 5 shows antiviral effects (survival rate, body weight change, blood virus concentration) by co-administration of foot-and-mouth disease vaccine, adjuvant and antiviral agent to experimental animals (mice) by muscle inoculation (negative control group, And the arrow indicates the time of attack.
FIG. 5A shows the survival rate change for 10 days after inoculation with foot-and-mouth disease virus Asia1 / Shamir 50 LD ₅₀ after 6 hours of administration of Ribavirin in combination with adjuvant and vaccine.
FIG. 5B shows the change in body weight for 10 days after inoculation with foot-and-mouth disease virus Asia1 / Shamir 50 LD ₅₀ at 6 hours after administration of Ribavirin in combination with adjuvant and vaccine.
FIG. 5C shows the survival rate change for 10 days after inoculation with the foot-and-mouth disease virus Asia1 / Shamir 50 LD ₅₀ after 6 hours of administration of Ribavirin-TA in combination with adjuvant and vaccine.
FIG. 5D shows the change in body weight for 10 days after inoculation with foot-and-mouth disease virus Asia1 / Shamir 50 LD ₅₀ at 6 hours after coadministered with adjuvant, vaccine and intramuscular injection of Ribavirin-TA.
FIG. 5E shows the survival rate change for 10 days after inoculation of the foot-and-mouth disease virus Asia1 / Shamir 50 LD ₅₀ at 6 hours and 3 days with Ribavirin in combination with adjuvant and vaccine.
FIG. 5F shows the change in body weight for 10 days after inoculation with the foot-and-mouth disease virus Asia1 / Shamir 50 LD ₅₀ at 6 hours and 3 days after administration of Ribavirin in combination with adjuvant and vaccine.
FIG. 5G shows the survival rate change for 10 days after inoculation with the foot-and-mouth disease virus Asia1 / Shamir 50 LD ₅₀ at 6 hours and 3 days after administration of Ribavirin-TA in combination with adjuvant and vaccine.
FIG. 5H shows the change in body weight for 10 days after inoculation of the foot-and-mouth disease virus Asia1 / Shamir 50 LD ₅₀ at 6 hours and 3 days with Ribavirin-TA in combination with adjuvant and vaccine.
FIG. 5I shows the result of measuring the amount of foot-and-mouth disease virus in mouse serum by administering a combination of Ribavirin and adjuvant in combination, alone or in combination with a vaccine.
FIG. 5J shows the result of measuring the amount of foot-and-mouth disease virus in mouse serum by administering a combination of Ribavirin-TA and adjuvant in combination, alone or in combination with a vaccine.
Figure 6 shows the clinical symptoms, viremia and excretion of the pigs by the combination of Ribavirin and adjuvant alone or in combination with the vaccine immediately after inoculation with the foot-and-mouth disease virus Asia1 / Shamir 50 LD ₅₀ (A: Ribavirine Adjuvant administered group, B: Ribavirine, adjuvant and vaccine combination administration group, C: untreated control group).
FIG. 7 shows clinical symptoms after intramuscular administration of 2 g of Ribavirin to two pigs and immediate inoculation with foot-and-mouth disease virus Asia1 / Shamir 50 LD ₅₀ .

According to one aspect of the present invention, the present invention relates to an antiviral composition comprising a ribavirin derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Chemical Formula 1]

Wherein R is a ketone group which is -COR 'and R' may be a straight chain C ₁ to C ₅ alkyl, and R is preferably -COCH ₃ , -COC ₃ H ₇ , -COC ₅ H ₁₁ It is, and may more preferably -COCH ₃ days.

According to a specific embodiment of the present invention, the compound of the formula (1) wherein R = -COCH ₃ is ribavirin-2 ', 3', 5'- triacetate, 5'-Tri-O-acetyl-1 -? - D-ribofuranosyl-1,2,4-triazole-3-carbozamide. The term ribavirin-TA is used herein to refer to the compound.

The present inventors have made extensive efforts to develop an effective therapeutic composition for foot-and-mouth disease virus, which is a representative causative virus of foot-and-mouth disease. As a result, it has been found that ribavirin derivatives synthesized from ribavirin significantly improve the survival rate of foot-and-mouth disease virus-infected cells and exhibit low cytotoxicity, thus being usefully used as a safe and effective anti-virus composition.

According to one aspect of the present invention, there is provided a composition for preventing or treating a foot-and-mouth disease infectious disease comprising a compound selected from the group consisting of ribavirin or a ribavirin derivative represented by the following formula (1), foot-and- .

[Chemical Formula 1]

In the above Formula 1, R is a ketone group having -COR 'and R' can be a straight chain C ₁ to C ₅ alkyl, and R is preferably -COCH ₃ , -COC ₃ H ₇ , -COC ₅ H can be _11, can -COCH ₃ days and more preferably.

The ribavirin and ribavirin-TA used in the present invention all exhibit significant anti-viral activity against foot-and-mouth disease virus. According to the present invention, among these, ribavirin showed the best antiviral activity, but the stability of the body due to the use of a high dose was superior to ribavirin-TA.

The ribavirin or ribavirin-TA exhibits anti-viral activity through the action of inhibiting the enzymatic activity of 3C protease or 3D polymerase, which is a non-structural protein synthesized by foot-and-mouth disease virus.

According to the present invention, ribavirin or ribavirin-TA has a three-dimensional structure capable of specifically binding with 3C protease and 3D polymerase, effectively inhibiting the production of structural proteins of foot-and-mouth disease virus, Have excellent anti-viral activity.

The ribavirin or ribavirin-TA may be chemically synthesized or purified in nature if it has enzymatic inhibitory activity of 3C protease or 3D polymerase.

As used herein, the term " anti-viral activity "refers to a period of viral infection, specifically virus penetration into host cells, viral replication, virus assembly and virus release Quot; means an activity that directly or indirectly interferes or inhibits one or more steps of the virus infection cycle. This includes any effects that either nonspecifically inhibit the virus titer increase in the viral infected subject, or reduce the virus titer level to nonspecifically.

As used herein, the term " foot-and-mouth viral infectious disease "means any disease or pathologic condition that occurs, progresses or worsens due to a foot-and-mouth disease virus infection either directly or indirectly.

As used herein, the term "treatment" includes (a) inhibiting the development of a disease, disorder or condition; (b) relief of the disease, disorder or condition; Or (c) eliminating the disease, disease or condition. The composition of the present invention enhances the survival rate of cells infected with foot-and-mouth disease virus and inhibits the effect of virus infection, thereby suppressing, eliminating or alleviating the development of a disease caused by a virus infection or symptoms thereof. Accordingly, the composition of the present invention may itself be a therapeutic composition for a foot-and-mouth disease infectious disease, or may be applied as a therapeutic adjunct to inhibit viral activity by being administered together with other anti-viral compositions.

Herein, the term " treatment "or" therapeutic agent "includes the meaning of" therapeutic aid "

As used herein, the term "prophylactic " means inhibiting the development of a disease or disease in a subject who has never been diagnosed as having a disease or disease, but is likely to suffer from such disease or disease.

The term " administering "or" administering "as used herein refers to the administration of a therapeutically effective amount of a composition of the present invention directly to a subject so that the same amount or a corresponding amount is formed in the body of the subject.

A "therapeutically effective amount " of a composition refers to the amount of a composition comprising ribavirin, a derivative thereof, or a composition thereof, sufficient to provide a therapeutic or prophylactic effect to the individual to whom the composition is to be administered, It means.

The term "animal" used in the present invention may include any kind of animals that suffer from foot-and-mouth disease or belonging to a risk group in which foot-and-mouth disease can occur. For example, artiodactyla such as cattle, .

According to a specific embodiment of the present invention, the foot and mouth disease virus is Asia-1.

According to a specific embodiment of the present invention, the foot-and-mouth disease virus may be O, A, C, SAT-1, SAT-2 or SAT-3 viruses.

In the present invention, ribavirin or a derivative thereof may be in the form of a pharmaceutically acceptable salt, a solvate, a polymorph, a prodrug, a codon, a co-crystal, a tautomer, a racemate, an enantiomer or a diastereomer, May be in the form of a mixture.

When the compounds of the present invention are used in the form of a pharmaceutically acceptable salt, acid addition salts formed by an acceptable free acid are useful. As the free acid, inorganic acid and organic acid can be used.

Preferably, the pharmaceutically acceptable salts of the compounds of the present invention include the hydrochloride, bromate, sulfate, phosphate, citrate, acetate, trifluoroacetate, lactate, tartrate, maleate, fumarate, But are not limited to, sulfonic acid salts, glycolic acid salts, succinic acid salts, 4-toluenesulfonic acid salts, glucuronic acid salts, embronic acid salts, glutamic acid salts or aspartic acid salts, And salts formed using various inorganic acids and organic acids.

The compounds of the present invention may also exist in the form of solvates (e.g., hydrates).

The composition of the present invention may be prepared from a pharmaceutical composition containing a pharmaceutically effective amount of ribavirin or a derivative thereof of the present invention.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to achieve the preventive, palliative or therapeutic efficacy or activity of the foot-and-mouth disease infectious disease of the present invention described above.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. But is not limited thereto. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, topical administration, transdermal administration, nasal administration and the like. Preferably peritoneal or muscular.

A suitable dosage of the pharmaceutical composition of the present invention can be variously prescribed by such factors as the formulation method, the age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate and responsiveness of the animal .

On the other hand, when the pharmaceutical composition of the present invention is used as a composition for muscle (or abdominal cavity) inoculation, the preferred unit dose of ribavirin is 15 to 20 mg per mouse having an average weight of 18 g. A unit dosage of less than 15 mg is less effective in achieving the desired effect, and a unit dosage of more than 20 mg may result in diarrhea, anorexia, hemolytic anemia, fetal malformations and insomnia due to overuse of the ribavirin reagent. It can cause side effects .

The preferred single dose of ribavirin-TA is 15 to 70 mg per mouse with an average body weight of 18 g, and a more preferred unit dose is 15 to 60 mg. When the unit dose is less than 15 mg, the amount of the composition is small and it is difficult to obtain a desired effect. A unit dose of more than 60 mg has a problem of side effects due to overuse of the ribavirin-TA reagent. Weight change was observed in the mice administered with 70 mg even when the change in body weight was observed for the stability study (FIG. 2D).

In the present invention, ribavirin and its derivatives used as an antiviral agent can be used in combination with a known foot-and-mouth disease vaccine.

In the present invention, ribavirin or a derivative thereof used as an antiviral agent can be used in combination with a known adjuvant, and the adjuvant can be aluminum hydroxide or Montanide ISA 201.

The mixed solution of the antiviral agent (ribavirin or its derivative) and the adjuvant may be administered in a dose of 1.6 to 2.0 g, though the dosage may be 1.5 to 5.0 g per day per pig having an average weight of 10 kg. Unit doses less than 1.6 grams are difficult to defend against foot-and mouth viruses, and unit doses above 2.0 grams can cause side effects (diarrhea, anorexia, hemolytic anemia, weight loss, etc.) due to overuse of the ribavirin reagent. When two pigs (10 weeks old, 10kg) were inoculated with 2g each of ribavirin and then infected with foot-and-mouth disease virus, one of the two mice died at 5dpi, but the other clinical index was almost absent. Of ribavirin was found to be helpful in defense against foot-and mouth disease but could cause mortality, thus requiring dose reduction (Fig. 7).

Treatment of foot-and-mouth disease virus infections in swine by administering a combination of ribavirin and its derivatives, foot-and-mouth vaccines and adjuvants has not been disclosed in the prior art at all.

In the present invention, the administration of the antiviral agent (ribavirin or its derivative), adjuvant and foot-and-mouth vaccine may be simultaneous (with a single agent), sequential or separated over time.

When administered simultaneously, the preferred ratio of ribavirin: adjuvant may be 1: 1. If the mixing ratio is exceeded, the mixing of ribavirin and adjuvant becomes inadequate and the continuous diffusion of ribavirin may be inhibited or the adjuvant may remain in the muscles.

The combination of antiviral and known foot-and-mouth vaccines has been observed to show higher antiviral response (synergistic effect) in both in vitro and in vivo than in the case of using antiviral alone.

 In another aspect, the present invention provides a feed composition for preventing or ameliorating a virus-related disease, comprising a composition for an antiviral agent.

The antiviral composition refers to a compound containing ribavirin or a derivative thereof.

The feed composition may include a feed additive. The feed additive of the present invention corresponds to an auxiliary feed in the feed control method.

The term "feed" as used herein in the context of the present invention may mean any natural or artificial diet, single meal, or the like ingredients for feeding, ingesting, digesting or suitable for the animal.

The kind of the feed is not particularly limited, and feeds conventionally used in the art can be used. Non-limiting examples of such feeds include vegetable feeds such as cereals, muscle roots, food processing busines logistics, algae, fibers, pharmaceutical buses, oils, fats, pastes or grain by-products; Animal feeds such as proteins, inorganic substances, fats, oils, fats, oils, monocellular proteins, animal plankton, or food. These may be used alone or in combination of two or more.

In addition, the feed additive may additionally contain a carrier that is acceptable to the unit animal. In the present invention, the feed additive may be added as it is or a known carrier, stabilizer and the like may be added. If necessary, various nutrients such as vitamins, amino acids and minerals, antioxidants and other additives may be added. Powders, granules, pellets, suspensions, and the like. When the feed additive of the present invention is supplied, it can be supplied to the unit animal singly or mixed with the feed.

Foot-and-mouth disease is transmitted by direct contact through infected animal fluids, saliva, milk, semen, air at the time of breathing, and feces. Also indirect infections are possible through work clothes, gloves, shoes, vehicles, feeds, etc. of farmers, farm workers, veterinarians, artificial fertilizers, feed suppliers, manure disposal companies and slaughterhouse shipment vehicle drivers. Not only that, but it can also be transmitted by air.

The feed composition of the present invention is excellent in antiviral efficacy, thereby enhancing resistance and defense against virus-related diseases, thereby preventing mortality and reduced productivity due to virus-related diseases, thereby preventing human infection.

In another aspect, the present invention provides a method for treating or preventing a foot-and-mouth disease virus infection in an animal, comprising administering to the animal, excluding human, a composition comprising ribavirin or a ribavirin analogue represented by the following formula

[Chemical Formula 1]

Wherein R is a ketone group which is -COR 'and R' may be a straight chain C ₁ to C ₅ alkyl, and R is preferably -COCH ₃ , -COC ₃ H ₇ , -COC ₅ H ₁₁ It is, and may more preferably -COCH ₃ days.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following examples.

Experimental material

(Sigma Aldrich A1882, USA), T-1105 (Alfa Aesar, A Johnson Matthey Company, United Kingdom) and Ribavirin-TA (GyeongGi Bio-Center) (3.25-400 μM) medium is replaced.

The new antiviral agent, 2 ', 3', 5'-Tri-O-acetyl-1-β-D-ribofuranosyl-1,2,4-triazole-3-carbozamide (ribavirin-TA) In order to increase the uptake rate of the drug in the gastrointestinal tract, the hydrophilic substituent of ribavirin was treated at 60 ° C for 6 hours to convert the secondary alcohol to a hydrophobic substituent such as acetonide to obtain 2 ', 3', 5'-Tri-O- acetyl-1-β-D-ribofuranosyl-1,2,4-triazole-3-carbozamide (ribavirin-TA).

Test Example 1. Antiviral activity and cytotoxicity of Ribavirin and Ribavirin-TA compared to conventional drugs for foot-and-mouth disease virus

The cells were cultured in pig kidney cells (IBRS-2, Swine kidney cells) for 1 day at 37 ° C in a 5% CO ₂ incubator (Minimum Essential Medium, 10% fetal bovine serum ) And 1% antibiotics) were used for the experiments. IBRS-2 cells cultured for 1 day were infected with virus type O / SKR / 2002 (100 TCID ₅₀ ) for 1 hour. After 1 hour, the test substances listed in the following Table 1 were serially diluted (3.25-400 μM) and the medium was changed. After 48 hours from the addition of the test substance, the surviving cells were stained with MTS (CellTiter 96 Aqueous One Solution Proliferation Assay (Promega, USA)) for 3 hours and then incubated with a microplate reader (Molecular device, USA) And the antiviral and cytotoxic effects were compared. Specifically, 50% effective concentration (EC ₅₀ ) and 50% cytotoxic concentration (CC ₅₀ ) are calculated using GraphPad 5.0 software. The EC ₅₀ (50% effective concentration) is the concentration of the drug that can induce 50% survival rate of cells in the virus-infected well. The smaller the value, the better the antiviral efficacy. When the drug does not show any toxicity, it will show 100% survival rate like control cells with no added drug and virus, and 0% survival rate when all cells are killed due to strong toxicity. In addition, the higher the concentration of the drug, CC ₅₀ (50% cytotoxic concentration), which can kill 50% of the cells, means less toxicity. Table 1 below shows the cytotoxic and antiviral effects of Ribavirin, T-1105, Azauridine and Ribavirin-TA in pig kidney cell line IBRS-2 of foot-and-mouth disease virus.

Antiviral agent ^a CC ₅₀ (uM) ^b EC ₅₀ (μM) ^c Selectivity Index (CC ₅₀ / EC ₅₀ ) Ribavirin 1898.50 ^d 28.09 67.59 T-1105 341.05 68.65 4.97 Azauridine 1893.00 343.70 5.51 Ribavirin-TA 18190.00 1730.50 10.51

^a IBRS-2 cells were inoculated and inoculated with FMDV, O / SKR / 2002 100 TCID ₅₀ for 1 hour, and the inoculation supernatant was removed and the antiviral agent was administered.

^b 50% cytotoxicity compared to negative control.

^c Effective concentration of 50% Effect concentration showing cytopathic effect.

^d This data is mean ± standard deviation (SD)

Referring to Table 1, ribavirin selectivity index (SI) showed the highest value. In the case of cytotoxicity (CC ₅₀ ), ribavirin, azauridine and ribavirin-TA showed similar values, but T-1105 showed the highest toxicity to the cells. In the case of foot-and-mouth disease virus resistance (EC ₅₀ ), ribavirin was found to be antiviral even at the lowest concentration. SI had lower ribavirin-TA than ribavirin.

Table 2 below shows antibody reactivity in mice treated with Ribavirin or Ribavirin-TA. Antibody reactivity in the mouse was also measured and the antibody formation formed by the infection was not observed until day 16 in the vaccinated and inoculated Ribavirin compared to the other inoculated groups. The vaccine was effective against Ribavirin.

Antiviral agents (doses) Positivity to structural protein antibody 0 DPI ^a 10 DPI 16 DPI Ribavirin (15 mg) - ^b - + ^c Ribavirin-TA (15 mg) - + + Ribavirin (10 mg) - - + Ribavirin with vaccine (6 mg) - - - Ribavirin-TA with vaccine (10 mg) - NA ^d N.A. Ribavirin-TA with vaccine (6 mg) - N.A. N.A.

^a Date of infection DPI, Days post infection

^b (FMDV type Asia 1 ELISA), PI < 50%

^c positive (FMDV type Asia 1 ELISA), PI > 50%

^d We can not check the results with our mouse. N. A

The detection of FMDV non-structural protein (NSP) antibodies in pig and goat serum was performed by ELISA using PrioCHECK FMDV NSP (Prionics AG, Schliern-Zurich, Switzerland). As a result, the antiviral ability of ribavirin and ribavirin-TA was confirmed by antibody test in the group administered with 15 mg / day ribavirn-TA orally at 10 days, and 15 mg / day ribavirin was orally administered to the mice. In addition, it was confirmed that the antibody was formed on the 16th day of inoculation in the group to which 10 mg / day of ribavirin was orally administered while the vaccine was inoculated.

Test Example 2. Comparison of proliferative defense and therapeutic effect of foot-and-mouth disease with conventional drugs and Ribavirin and Ribavirin-TA

C57 / BL6 mice (7 weeks old, 17-19 g) required for the experiment were purchased from Orient Co., Ltd. Purchased from Republic of Korea and inoculated intraperitoneally with the Asian1 Shamir virus, which is known to be pathogenic to C57 / BL6 mice. The determination of the amount of foot-and-mouth disease virus to be vaccinated was performed in bovine kidney cells (LF-BK), and the existing compound (ribavirin, 6-Azauridine, T-1105, recombinant adenovirus) Were injected into mice to compare the proliferation and treatment effects of the virus. After 3 days, 50% tissue culture infective dose (TCID) ₅₀ was calculated by the Reed and Muench method.

Specifically, 3 mg of ribavirin, 6-azauridine, and T-1105 were intraperitoneally administered at intervals of 12 hours from day 0 to day 3. Six hours after the first drug was administered, 100 LD ₅₀ of Asia 1 / Shamir was intraperitoneally inoculated (IP). The recombinant adenovirus Ad-3 siRNA, Ad-IFN-α, γ of ₅ × ^{10 8} TCID ₅₀ was inoculated intramuscularly (IM) one day before challenge with mouse and inoculated with Asia 1 / Shamir at 50 LD ₅₀ on Day 0. Survival of all mice was monitored for 10 days.

<Results>

As shown in FIG. 1A, Azauridine and T-1105 showed similar tendency to the negative control group, and there was no anti-foot-and-mouth effect in mouse models compared to other drugs of the same dose. The survival rate was 40% and Ad-3 siRNA was 80% when Ad-IFN-α, γ was inoculated among adult adenoviruses in adult mice. However, when ribavirin was administered at 3 mg every day from day 0 to day 3, all mice survived (FIG. 1A).

Example 1 Inhibition of proliferation of foot-and-mouth disease virus according to administration time of Ribavirin in mouse and its therapeutic effect

5 groups, and 1.5 mg of ribavirin was intraperitoneally inoculated into mature mice at different administration times (Fig. 1B). The first group is 0, 24 hours, the second group is 0, 24, 48 hours, the third group is 0, 8, 24, 30 hours, the fourth group is 0, 24, The fifth group was inoculated intraperitoneally to mature mice at 0, 8, 24, 30, 48, and 56 hours. All groups, including the negative control, were inoculated with 100 LD ₅₀ of Asia 1 / Shamir intraperitoneally 6 hours after the first ribavirin treatment. Survival of mice was monitored for 10 days.

<Results>

According to FIG. 1B, the survival rate of mice in the group (0, 24 h / 0, 24, 48 h / 0, 24, 48, 72 h) once inoculated once a day rapidly decreased from 1 day to 4 days All died on the 5th ~ 6th day. The final mouse survival rate of 80% was observed in the groups that received ribavirin twice a day (0, 8, 24, 30 h / 0, 8, 24, 30, 48, 56 h). Inoculation of mature mice with different doses of Ribavirin resulted in a survival rate of 80% or more after inoculation of 2 or more days, twice a day.

Example 2. Safety of Ribavirin and Ribavirin-TA in Mice

For safety studies, 20-70 mg of Ribavirin and Ribavirin-TA were inoculated intramuscularly into mice and monitored for survival and weight changes for 7 days.

<Results>

Referring to FIG. 2, 30 mg of ribavirin did not affect the survival of mature mice, but decreased in weight and recovered, while mice over 40 mg ribavirin died. However, ribavirin-TA survived in mature mice at all test concentrations and showed little change in weight up to 60 mg ribavirin-TA. In the case of safety, ribavirin-TA does not affect the survival of mature mice at a concentration 2 times higher than that of ribavirin. Therefore, it is considered that the safety of ribavirin-TA is higher than that of ribavirin. In addition, when ribavirin and TA were compared with ribavirin-TA at the same dose, ribavirin increased the survival rate of mature mouse. However, ribavirin-TA showed less weight change in mouse than 15 mg, The antiviral effect is judged to be.

Example 3: Determination of administration method of an anti-hypersensitizer in an experimental animal

When different doses of ribavirin were administered, 10 mg of ribavirin per day was injected intraperitoneally or intramuscularly until day 0 to 3, Six hours after the first dose of Ribavirin, all groups received 50 LD ₅₀ of Asia 1 / Shamir foot-and-mouth disease virus peritoneally, and the weight change and survival rate of the mice were monitored for 10 days (FIGS. 3A, 3B).

In addition, 6, 10, and 15 mg of ribavirin or ribavirin-TA per day were given to mature mice with oral doses ranging from day 0 to day 6 or with reagent-absorbed diets. The first reagent was given and after 6 hours, 50 LD ₅₀ of Asia 1 / Shamir were intraperitoneally inoculated. Weight change and survival rate of mice were monitored for 10 days. In addition, blood samples were collected on days 0, 2, 4, 6, and 8, serum was separated, RNA was extracted, and the amount of virus was quantitatively measured.

<Results>

The difference in the effect of ribavirin on the foot-and-mouth disease virus according to the administration method was observed (Fig. 3). Both intraperitoneal (IP) and intramuscular (IM) methods showed 100% mouse survival (FIG. 3A). However, when the weight change rate of the mice was compared (Fig. 3B), the weight change was significant at 5 to 7 days in the inoculation of the peritoneal cavity, and the largest weight change rate was observed at 4 to 7 days in the muscle inoculation. It has been found to be more effective in defense against foot and mouth disease than muscle inoculation.

After oral administration (PO) method, or when the drug was adsorbed to the feed, oral administration was effective for defense against FMD virus (Figs. 3C-F). The survival rate of mice increased as the dose was increased per day, and the survival rate was 100% for 10 days when 15 mg of ribavirin was orally administered with the feed (Fig. 3C, 3E). The same dose of ribavirin was orally administered to adult mice with diets, and the survival rate was higher than that of oral administration using the Zoner group. In addition, mature mouse weight change was significantly (p <0.05) observed at 1 to 4 days after inoculation with footpad (Fig. 3D). However, no significant change in body weight was observed when the mice were orally administered with feed (Fig. 3F).

Ribavirin-TA also showed a tendency that the overall survival rate of mice increased as the intake dose increased (Fig. 3G, 3I). Survival of 100% was achieved when 15 mg of ribavirin-TA was orally administered with feed per day. In addition, when mature mouse weight changes were compared, when 15 mg of ribavirin-TA was orally administered with feed, there was no significant weight change even when inoculated with foot-and-mouth disease virus (Fig. 3H, 3J). In the case of inoculation, 3mg / day of foot-and-mouth defense effect was shown, but oral administration of 15mg / day or more showed a 100% survival rate in mature mouse. As a result of measuring the amount of FMD RNA in the serum, when the 15 mg / day ribavirin was orally administered, foot-and-mouth disease virus RNA was not detected. When ribavirin-TA was orally administered, less RNA was detected than the negative control group. No RNA was detected in the cells.

As a result, it was found that the use of ribavirin or ribavirin-TA in mature mice was more effective in the treatment of foot and mouth disease than in the case of oral administration of the drug in the feed. When the mice are administered orally by oral route, the absorption of the stress and the high dose of the drug in the mature mouse are reduced and the survival rate is lowered. When the drug was adsorbed to the feed, it was thought that the mouse continuously increased the absorption rate of the drug by oral administration of the drug together with the feed.

Example 4. Confirmation of the therapeutic effect of the combination vaccine with Ribavirin and Ribavirin-TA in mice

Ribavirin or ribavirin-TA was orally administered to mature mice and the vaccine group was inoculated together with a group that did not achieve 100% survival (Fig. 4). Day 0 vaccination and 6 or 10 mg of ribavirin or ribavirin-TA were given to the feed for 0 to 6 days. Six hours after the vaccine and reagent-adsorbed feeds were given, they were inoculated intraperitoneally with 50 LD ₅₀ of Asia 1 / Shamir foot-and-mouth disease virus. After that, we re-challenged with Asia 1 / Shamir of 50 LD ₅₀ on day 10. Mice were monitored for changes in body weight and survival rate for 15 days.

<Results>

When the vaccine was inoculated on the 0th day and 10mg of ribavirin adsorbed to the feed was orally administered from day 0 to day 6, the mature mouse had a survival rate of 100% ( P <0.0001, Fig. 4A) (FIG. 4B) also showed no significant difference with a variation range of less than 4%. Survival rates were low when both 6 mg / day of ribavirin, 10 mg / day of ribavirin-TA, or 6 mg / day of ribavirin-TA were administered orally together with the feed and in the vaccine-inoculated group. When ribavirin or ribavirin-TA was orally administered, the survival rate of mature mouse was also increased as the concentration of the drug was increased, indicating that there was a significant difference from the group vaccinated with foot-and-mouth disease virus only. In addition, when the vaccine was inoculated with 10 mg or 6 mg of ribavirin orally per day, the change in body weight of the adult mice was not significantly different from that before vaccination. On Day 10, secondary challenge to mature mice with the same concentration of foot-and-mouth disease virus did not change survival but tended to increase or decrease with weight change.

Ribavirin or ribavirin-TA orally administered groups had similar or fewer foot-and-mouth viruses on day 2 than the control group (Fig. 4C) and on day 4, virus detection was reduced on day 2 in all groups.

Example 4. Effect of coadministering with an adjuvant

One group received 15 mg of ribavirin or ribavirin-TA alone, the other group received 15 mg of ribavirin or ribavirin-TA and adjuvants, and the other group received 15 mg of ribavirin or ribavirin-TA plus adjuvant and vaccine All adult mice were inoculated with muscle inoculation (IM) on day 0. Adjuvant used Montanide ISA 201 VG (SEPPIC, Paris) or Aluminum Hydroxide (Al (OH) ₃ ). Six hours after the inoculation, 50 LD ₅₀ of Asia 1 / Shamir were intraperitoneally inoculated. Another group of these conditions was set up and inoculated into mature mice with 50 LD ₅₀ of Asia 1 / Shamir at 6 hours and day 3 after inoculation. Mice were monitored for weight change and survival for 10 days. RNA was extracted using the MagNa pure LC 96 system (Roche, Switzerland), and real-time reverse transcriptase polymerase chain reaction (RT-PCR) Respectively.

<Results>

Ribavirin or ribavirin-TA together with ISA 201 or Al (OH) ₃ , which are used as adjuvants, were inoculated together (FIG. 5). There was no significant difference ( P > 0.5) between the vaccine alone and the negative control ( P <0.01). However, the ribavirin or ribavirin-TA reagent and adjuvants or adjuvants and vaccine were significantly more effective than the negative control. Also, survival rates of mature mice were higher when the foot-and-mouth disease virus was inoculated once (day 0) to mature mice (FIGS. 5A and 5C) than when they were inoculated twice (days 0 and 3) Similar trends were observed. The amount of foot-and-mouth disease virus in the mature mouse serum was measured (Figures 5I, 5J). The virus amount of all groups inoculated with the reagent was lower than the negative control or not detected. Detection of foot-and-mouth disease virus tended to be faster than that of the group not receiving adjuvants when the vaccine was inoculated with adjuvant or adjuvant with Ribavirin-TA.

When we observed changes in the amount of foot-and-mouth disease virus in the mature mouse, 15 mg / day of ribavirin and ribavirin-TA inhibited the growth of foot-and-mouth disease virus within about 4 days, All adjuvants were used to reduce the concentration of ribavirin or ribavirin-TA to 6 mg / day, and all adult mice survived. Adjuvant ISA 201 or Al (OH) ₃ , when administered to mature mice with the reagent, showed a higher survival rate, suggesting that the adjuvant delayed the uptake of reagents in the mature mouse body. As a result, it was proved that even one injection of high dose, not the continuous injection of the reagent or the oral administration, has the effect of defending against foot and mouth disease virus.

Assuming that the target animal was re-infected with foot-and-mouth disease virus on the farm, secondary challenge to mature mice resulted in slight weight change but no change in survival rate. Immunity generated through the first pathogenic infection is considered to be defensible even if it is secondarily pathogenic and reinfected. In the structural protein antibody study, which was used to quantitatively measure foot-and-mouth disease virus in mature mouse serum, anti-virus was inhibited by vaccine and ribavirin until day 10, and antibody was not detected. Respectively. When ribavirin is orally administered, the virus is protected by the vaccine, but the antibody formation time is delayed. In the case of Ribavirin-TA, it was thought that virus-induced suppression of ribavirin was lower than that of ribavirin due to the relatively rapid antibody formation due to the virus multiplication in the primary virus antigen stimulation.

Example 5 Effect of Combination with Adjuvant in Pig

(Group 1), ISA206 and Ribavirin (1.8g) and vaccine group (Group 2) were used in two groups of SPF pigs (10 weeks old, average body weight 10kg) and two oil adjuvants ISA206 and Ribavirin ) And a control group (Group 3). At the same time, footpad was inoculated with footpad of Asia1 Shamir (10 ⁵ TCID ₅₀ ), and clinical symptoms were observed for 7 days. Clinical symptoms were indexed. (2 points for 40 ° C, 2 points for 40.5 ° C, 3 points for 41 ° C), feed consumption (1 point for appetite reduction, 2 points for unfeeded food or feed left), walking (1 point for drowning, 2 points for drowsiness) (Tongue 1, gingiva or mouth 1, and mouth 1) were observed, and the sum of the scores was calculated as follows: Clinical index.

In Group 1, which contained 1.8 g of the oil adjuvant ISA206 and 1.8 g of Ribavirin, the clinical indices were up to five points in up to three days and viremia and viral clearance were highest at 3 or 4 days, A relatively large amount of about 10 ⁶ was released and detected (Figure 6A). In the vaccinated group, no clinical symptoms were observed, no viremia was observed, and only a relatively small amount of virus was ^detected in 10 ^2-4 days in 2-3 days. There is a possibility that the radio wave is significantly lowered (Fig. 6B). It was confirmed that a relatively large amount of about 10 ⁸ was excreted in the untreated control group (FIG. 6C).

Therefore, in pigs, the safety of Ribavirin is likely to be a problem. A single dose of 1.8 g does not cause mortality in animals. Ribavirin and the vaccine can be used to supplement the initial immunity. Ribavirin side effects (diarrhea, Anorexia) can be significantly reduced.

Production Example 1. Preparation of a feed composition containing ribavirin or a derivative thereof

Dissolve ribavirin or ribavirin-TA in 1 ml of PBS (Phosphate buffered saline) or 75 mg of distilled water (D.W.). When 15 mg of ribavirin or ribavirin-TA is mixed into the feed of the animal, 200 ul of solution is fully absorbed into the feed. Dry the feed at 2 to 8 ° C, where light is blocked. Store the dried feed in a place protected from light at 2 to 8 ° C.

Claims (15)

An antiviral composition for foot-and-mouth disease virus comprising, as an active ingredient, a ribavirin derivative compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[Chemical Formula 1]

In the above formula (1), R is -COCH ₃ . delete delete Ribavirin, foot and mouth vaccines and adjuvants,
A composition for prevention or treatment of foot-and-mouth disease infectious disease which can be administered once a day, which comprises the above-mentioned ribavirin in an amount of 1.8 g. 5. The method of claim 4,
Wherein said foot-and-mouth disease virus is Asia-1 type. 5. The method of claim 4,
Wherein the composition exhibits antiviral activity through the action of inhibiting the enzymatic activity of 3C protease or 3D polymerase, which is a non-structural protein synthesized by the virus. delete delete 5. The method of claim 4,
Wherein the adjuvant is aluminum hydroxide or Montanide ISA &lt; RTI ID = 0.0 &gt; 201. &lt; / RTI &gt; delete 5. The method of claim 4,
Wherein the mixed solution of ribavirin and adjuvant and foot-and-mouth disease vaccine are administered simultaneously or separately or sequentially during the same treatment. delete delete 5. The method of claim 4,
Wherein the composition is a composition for animal feed addition for the improvement or treatment of diseases caused by foot-and-mouth disease virus. A method of treating or preventing a foot-and-mouth disease virus infection in an animal, comprising administering the composition of claim 1 to an animal other than a human.

Images