EP1239923A2 - Stabilized veterinary compositions comprising more than one antiviral agent - Google Patents

Stabilized veterinary compositions comprising more than one antiviral agent

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Publication number
EP1239923A2
EP1239923A2 EP00983353A EP00983353A EP1239923A2 EP 1239923 A2 EP1239923 A2 EP 1239923A2 EP 00983353 A EP00983353 A EP 00983353A EP 00983353 A EP00983353 A EP 00983353A EP 1239923 A2 EP1239923 A2 EP 1239923A2
Authority
EP
European Patent Office
Prior art keywords
composition according
volume
weight
ribavirin
amount
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00983353A
Other languages
German (de)
French (fr)
Inventor
Abdul Rahman Shoa'a
El-Banna Hossny
Da'as Kefah
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
NEW PHARMA RESEARCH SWEDEN AB
Original Assignee
New Pharma Research Sweden AB
Newpharmaresearch Sweden AB
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Filing date
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Priority to EP00983353A priority Critical patent/EP1239923A2/en
Publication of EP1239923A2 publication Critical patent/EP1239923A2/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals

Definitions

  • the present invention concerns compositions comprising ribavirin and at least another antiviral agent as essential active ingredients, and a stabilizing agent, the compositions being intended to be used preferably in veterinary medicine.
  • RNA and DNA viruses like the Newcastle disease, infectious bursal disease (Gumboro disease) , infectious bronchitis, influenza disease or Marek' s viral disease, are known to cause heavy losses in the poultry industry due to high mortality and the rapid spreading of the disease to healthy flocks.
  • Newcastle disease is an acute, highly contagious rapidly spreading viral disease caused by a group of viruses which form the avian paramyxovirus of type 1.
  • Previous studies made on chickens have shown that the incubation period of the virus is generally of between 2 and 15 days.
  • the virus is generally transmitted through the upper respiratory system, from which it will penetrate into the blood. There will then follow a damage of the blood vessels and a spreading of the virus to other organs (spleen, liver, bone marrow) . After generally three days, all organs are infected, including the nervous system.
  • the clinical signs of the disease are greenish diarrhea (due to haemorrhagic lesions present in the intestinal tract), respiratory symptoms (difficult respiration, coughing, sneezing, rales due to inflammation, oedema and mucoid exudate present in the trachea and pharynx) and nervous signs (stiff neck, dropped wings, absence of coordination, paralysis) , which leads generally to the death of the animal.
  • a decrease of egg production in layers or even a cessation of laying have also been observed.
  • the virus can also infect humans and cause severe conjunctivitis.
  • the infectious bursal disease (or Gumboro disease) is an important viral disease of poultry caused by a group of viruses called birnaviruses . It affects young chickens and is characterized by a sudden onset and a short course with massive destruction of lymphocytes, particularly in the bursa .
  • Infectious bronchitis is a highly infectious and contagious respiratory disease of chickens. In some cases and with some virus strains, the infection may also affect the oviduct or the kidneys. A significant decrease in egg production and egg quality in layers and a decrease of production in broilers is observed in infected flocks.
  • influenza disease is caused by a viral infection originated by viruses belonging to the orthomyxoviruses family. Diseases specific to some animal species have been described, like for example the avian influenza, turkey influenza, swine influenza or equine influenza. The clinical signs of the disease are broncho-intestinal pneumonia and lung lesions. Many different subtypes of viruses have been described and isolated. These subtypes may be introduced from one species to another species, like for example from birds to mammals, and may then cause major pandemics in the unprotected population.
  • Marek' s viral disease is a widespread disease affecting domestic chickens in all sections of the world. It is characterized by lesions affecting the nervous system (demyelination of peripheral nerves leading to progressive paralysis of the wings and legs), organs and other tissues. Young chickens under sixteen weeks of age are most susceptible. Once the birds are infected, there is no known treatment to stop or reduce the infection. In its acute form, the disease leads to the death of the animal. The only actual treatment available is by vaccination.
  • vaccination against the viral agents causing the disease has been shown to be helpful, but however insufficient.
  • the vaccination is made either by intraocular or intra-nasal instillation, by inhalation (spray or aerosol) , by adding the vaccine to the feed or by injection.
  • agents like amantadine, methisazone or cytarabine have been described. However, these agents have only a limited spectrum antiviral activity. These agents also have the drawback of being toxic to the animal when administered at an effective therapeutic or prophylactic dosage, which is not desirable, especially if they are intended to be used for prophylaxis.
  • an antiviral agent developed against viral human diseases, ribavirin or tribavirin (sold under the name
  • Ribavirin is a synthetic non-interferon inducing broad- spectrum antiviral agent, which is used against many viral human diseases including type A hepatitis, measles and influenza B. Ribavirin has been shown to have a significant effect against respiratory syncytial virus (RSV) , parainfluenza and influenza B viruses, and a lesser activity against herpes, varicella, Lassa fever, infectious hepatitis, dengue fever, measles and AIDS viruses.
  • RSV respiratory syncytial virus
  • ribavirin has been shown to be active against influenza virus types A and F infections in mice.
  • ribavirin is 1- -D-Ribofuranosyl-lH-1, 2, 4- triazole-3-carboxamide, a synthetic nucleoside. Due to its analogy with the nucleosides used for the replication of the virus in the target infected cell, this molecule intervenes as an inhibitor of the 5' capping of viral mRNA, so that ultimately viral protein synthesis of both DNA and RNA viruses are affected.
  • Ribavirin is sold as a powder. At 25 °C, the maximum aqueous solubility of ribavirin is 142 mg/ml and the drug is slightly soluble in alcohol.
  • the drug ribavirin is approved for use only in the medical treatment of severe upper respiratory infections caused by RSV in infants and children.
  • Ribavirin has also demonstrated significant teratogenic and/or embryocidal potential in all animal species in which adequate studies have been conducted (rodents and rabbits) . Studies in which the drug has been administered systemically demonstrate that ribavirin is concentrated in the red blood cells and persists for the life of the erythrocyte .
  • the composition becomes turbid or a precipitate forms, which is not desirable in certain forms of administration or for storage, as will be explained hereafter.
  • the compound is administered in a specific form, generally selected from injection, topical application, inhalation, ingestion or spraying.
  • a specific form generally selected from injection, topical application, inhalation, ingestion or spraying.
  • the spraying or inhalation form by way of an aerosol will be most preferable in the case of viral respiratory diseases where the broncho-pulmonary epithelium is primarily affected, since the administration of the active substance through the respiratory tract in the form of micronized particles (the size of the particles is generally comprised between 0.3 and 5 micrometers for an aerosol and 5 to 100 micrometers for a spray) will be most effective. Therefore, in the case of aerosol administration, the stability of the active compound in the composition is of upmost importance, since precipitates from aerosols can interfere with the optimal operation of respirators or spraying apparatus.
  • antiviral agents whose activity is based on an inhibition of the viral DNA polymerase enzyme of the virus have been developed by the pharmaceutical industry, like
  • acyclovir 9- [2-hydroxyethoxymethyl) guanine] .
  • This chemical agent is known for its activity against a limited number of viruses, the herpes simplex virus type 1 and type 2, the varicella zoster virus and the Epstein-Barr virus.
  • Acyclovir is sold in its sodium salt form as a white, crystalline powder.
  • the solubility of acyclovir sodium in water exceeds 100 mg/ml and shows a pH of approximately 11. However, at physiologic pH, acyclovir exists as the non- ionized form and has only a maximum solubility of 2.5 mg/ml .
  • Blough also mentions in the same patent that it is preferred to administer the compounds separately to the patient, i.e. not in the same composition.
  • Pancheva et al . (Acta Microbiologica Bulgaria, 1993, Vol. 29, pp. 61-64) disclosed that a combined treatment with acyclovir and ribavirin is more effective for the treatment of herpes simplex keratoconjunctivitis in rabbits than the treatment with the individual drugs.
  • this reference does not address the problem of combining antiviral agents in a single composition and the problem of the stability upon storage of a composition which would comprise the active agents or the problems related to the way of administration of such a composition.
  • antibiotics are chemical compounds which are active against bacteria, but have generally no activity against viruses. Therefore, viral and bacterial diseases are generally treated separately and with distinctive agents, and the compositions which are intended to be used to treat viral diseases do not contain antibiotics, as well as antibiotic compositions do generally not contain antiviral agents.
  • a composition comprising essentially as active ingredients ribavirin and at least another antiviral agent like acyclovir is effective as treatment or prophylaxis of the Newcastle disease, infectious bursal disease (Gumboro disease) , infectious bronchitis, influenza disease or Marek' s viral disease in poultry.
  • compositions are effective against other animal viral diseases and are therefore broad- spectrum antiviral compositions.
  • compositions comprising the active analogue chemical derivatives of ribavirin or of the at least other antiviral agent are also effective as broad-spectrum antiviral compositions .
  • the inventors of the present invention have also found that when combined with a vaccination treatment the efficacy of the treatment with the compositions in accordance with the present invention is improved and a synergistic effect is observed.
  • the inventors of the present invention have also found that when adding other agents like quinolone-type antibiotics in the compositions, the efficacy of the composition as broad- spectrum antiviral is similar or even improved.
  • the inventors of the present invention have also found that the stability of the composition comprising ribavirin and at least another antiviral agent like acyclovir as essential active ingredients is achieved when a stabilizing agent is added to the composition, the stabilizing agent being preferably benzyl alcohol, N- methylpyrrolidone, glacial acetic acid, 2-pyrrolidone , dimethylacetamate (DMA) or dimethylformamide (DMF) or a combination thereof.
  • a stabilizing agent being preferably benzyl alcohol, N- methylpyrrolidone, glacial acetic acid, 2-pyrrolidone , dimethylacetamate (DMA) or dimethylformamide (DMF) or a combination thereof.
  • the main object of the present invention is to provide new compositions which are effective as antiviral agents, and preferably as broad-spectrum antiviral agents effective as treatment or prophylaxis of viral diseases in animals in general, and specifically in poultry, like the Newcastle, infectious bursal (Gumboro) , infectious bronchitis, influenza or Marek' s viral diseases.
  • Another object of the present invention is to provide these compositions as compositions which are stable upon storage.
  • a further object of the present invention is to provide compositions effective as treatment or prophylaxis of viral diseases in animals which do not interact negatively with other treatments, like for example vaccination, and which can be used in an effective manner without being toxic to the treated animal.
  • One of the advantages of the present invention is therefore that the composition can be stored in a stable manner in the form in which it will be administered.
  • composition is efficient against the viral diseases at doses which are not toxic to the treated animal.
  • therapeutic index of the composition is increased.
  • composition can be used for the treatment or prophylaxis of viral diseases in animal even if a treatment by vaccination or with antibiotics is already applied on the animal. Both treatments can coexist and generally will show an improved efficacy.
  • the present invention provides a solution to the aforementioned problems.
  • the aforementioned object is achieved by a composition having the features defined in claim 1 and comprising as essential active ingredients ribavirin and at least another antiviral agent like acyclovir, and a stabilizing agent, the stabilizing agent being preferably benzyl alcohol, N- methylpyrrolidone, glacial acetic acid, 2-pyrrolidone or a combination thereof.
  • the aforementioned object is also achieved by the use of the compositions for the treatment of viral diseases, and preferably animal viral diseases, and most preferably those affecting poultry.
  • Preferred embodiments of the invention including the use of chemical derivatives of the essential compounds of the composition, i.e. ribavirin and the other antiviral agents, the addition of optional ingredients in the composition like for example quinolone-type antibiotics, preservatives, vitamins or further stabilizing agents, or the use of the composition for the treatment of specific viral diseases in animal are defined in the dependent claims.
  • compositions are defined as weight per volume.
  • Viruses are classified according to the type of nucleic acid they contain, i.e. RNA or DNA. Further subdivision is made into groups based upon physical and biochemical properties.
  • RNA viruses are subdivided into the families paramixoviridae, orthomyxoviridae, coronaviridae, arenaviridae, retroviridae, reoviridae, birnarividae, picornaviridae, toroviridae, caliciviridae, togaviridae, bunyaviridae, filoviridae and rhabdoviridae .
  • the DNA viruses are subdivided into poxviridae, herpesviridae, papovaviridae, adenoviridae, hepadoviridae, parvoviridae and circoviridae .
  • Adenoviruses are responsible of diseases such as the infectious canine hepatitis and infectious canine tracheobronchitis .
  • Herpesviruses are subdivided into alpha-, beta- and gamma- herpesviruses.
  • Alpha-herpesviruses are associated with numerous diseases affecting different animal species such as infectious bovine rhinotracheitis, infectious pustular vulvovaginitis, haemorrhagic disease of pups, feline viral rhinotracheitis, infectious laryngotracheitis (due to avian herpesvirus 1), Marek' s disease (due to avian herpesvirus 2) and duck plague (due to duck herpesvirus 1).
  • Orthomyxoviruses are responsible of influenza diseases such as equine influenza, swine influenza and avian influenza.
  • Paramyxoviruses are associated with the Newcastle disease.
  • Birnaviruses are associated with the infectious chicken bursal disease.
  • the viral diseases which can be treated by the compositions according to the present invention are any viral diseases already known which affect humans or animals, and more specifically those which affect poultry.
  • the diseases caused by the following viruses are aimed at being treated: Newcastle disease virus (NDV) , Marek' s disease virus, infectious bursal disease (IBD; synonym Gumboro disease) virus, infectious bronchitis virus (IBV), avian pneumovirus (APV) , equine herpes virus type 2 (EHV-2), transmissible gastroenteritis (TGE) virus, porcine respiratory coronavirus (PRCV) , bovine parainfluenza-3 virus, lymphocytic choriomeningitis (LCM) virus, bovine viral diarrhoea virus (BVDV) , equine arteritis virus (EAV) , porcine parvovirus (PPV), classical swine fever virus (CSFV; synonym: hog cholera virus, HCV) , border disease virus (B
  • EHV-1 and EHV-4 feline coronavirus
  • FCV feline coronavirus
  • rotaviruses rotaviruses
  • poxviruses like fowl poxvirus, turkey poxvirus, pigeon poxvirus, quail poxvirus, sparrow poxvirus
  • avian influenza A viruses turkey influenza virus, chicken anaemia virus, duck enteritis virus and Aleutian disease virus .
  • compositions according to the present invention comprise as essential active ingredients ribavirin or an active chemical derivative thereof and at least another antiviral agent or an active chemical derivative thereof and at least one stabilizing agent.
  • the amount of ribavirin or its active chemical derivative in the composition is preferably between 2.5 and 20% weight/volume. Most preferably, the composition contains 10% weight/volume of ribavirin.
  • the other antiviral agent or its active chemical derivative is present in the composition in amounts of preferably between 0.1 and 20% weight/volume. Most preferably, the composition contains between 1 and 10% weight/volume of the other antiviral agent or of its active chemical derivative. If more than one antiviral agent or active chemical derivative thereof is added, the above proportions apply individually to each antiviral agent or active chemical derivative thereof added.
  • the antiviral agent can be selected from known antiviral agents, like for example acyclovir ( 9— [2— hydroxyethoxymethyl) guanine] ) , amantadine (tricyclo [3.3.1.1 (3, 7) ] decane-1-amine) , udicil (4-amino-l-
  • the purpose of adding another antiviral agent is to obtain a synergistic and/or additive therapeutic or prophylactic effect.
  • the composition will contain as other antiviral agent 4% weight/volume of acyclovir or a combination of 4% weight/volume of acyclovir and 1% weight/volume of amantadine.
  • the stabilizing agent can be selected from benzyl alcohol, N-methylpyrrolidone, glacial acetic acid or another natural fruit vinegar, 2-pyrrolidone, dimethylacetamate (DMA) or dimethylformamide (DMF) .
  • the amount of stabilizing agent in the composition is preferably between 2.5 and 97.4% weight/volume.
  • the stabilizing agent is benzyl alcohol, N-methylpyrrolidone, glacial acetic acid, 2- pyrrolidone or a combination thereof.
  • the amount of benzyl alcohol in the composition is preferably 4% weight/volume .
  • the amount of N-methylpyrrolidone in the composition is preferably between 5 and 40% weight/volume, but can also reach an amount of up to 70% weight/volume.
  • the amount of glacial acetic acid in the composition is preferably between 2.5 and 90% weight/volume .
  • the composition contains a combination of 4% weight/volume of benzyl alcohol and 70% weight/volume of N- methylpyrrolidone .
  • compositions according to the present invention optionally comprise one or more quinolone-type antibiotics.
  • the antibiotic is selected preferably from enrofloxacin, ciprofloxacin, ofloxacin, flumequine, norfloxacin, nalidixic acid, cinoxacin, clinafloxacin, difloxacin, enoxacin, fleroxacin, miloxacin, nadifloxacin, pefloxacin, pipemidic acid, rosoxacin, rufloxacin, sparfloxacin, temafloxacin, tosufloxacin or trovafloxacin .
  • the amount of antibiotic in the composition is preferably either 5% or
  • enrofloxacin will be used, at a concentration of 5% or 10% weight/volume.
  • the effect obtained by the combination is a higher efficiency in the treatment or prophylaxis of the disease.
  • vaccine i.e. the inactivated virus against which a treatment or prophylaxis is aimed at
  • IBV infectious bronchitis virus
  • NDV Newcastle disease virus
  • compositions may also comprise optionally other agents like vitamins, preservatives, thickeners or any other agent or additive commonly used in veterinary or medical compositions which is for example useful for the stability of the composition or permits to improve the formulation for a specific way of administration without altering the antiviral activity.
  • agents like vitamins, preservatives, thickeners or any other agent or additive commonly used in veterinary or medical compositions which is for example useful for the stability of the composition or permits to improve the formulation for a specific way of administration without altering the antiviral activity.
  • sodium hydroxide or potassium hydroxide may be added in the composition in order to adjust the pH and/or improve the stability of the composition.
  • sodium hydroxide or potassium hydroxide will be added in an amount of 1% weight/volume.
  • composition When necessary, the composition is generally complemented to the required final volume by the addition of deionized water .
  • compositions are administered depends upon the particular virus infection to be treated.
  • the mode of treatment is preferably by spray, for example by aerosol spray, since this mode of administration delivers the agent to the site of infection.
  • injection or oral therapy may be also indicated.
  • Topical application may be used in the case of infections of a topical nature, such as herpesvirus infections.
  • the composition is administered in order that ribavirin is given at a daily dose of about 5 to 10 mg/kg body weight, one time each day and for two days in the case of poultry and between three and ten days in the case of other animals.
  • the amounts of ingredients are given in % of weight/volume The volume is adjusted by addition of deionized water.
  • a composition comprising: Ribavirin 10%
  • a composition comprising:
  • compositions in accordance with, for example, examples 1 or 2 of the present invention were made to evaluate the impact of the compositions in accordance with, for example, examples 1 or 2 of the present invention on the pathogenesis of the disease and the replication of the virus in the case of an infection of chickens with either the Newcastle disease virus or the infectious bronchitis virus.
  • the aim of these studies was to evaluate the protective activity of the compositions according to the present invention against the replication of the Newcastle disease virus or the infectious bronchitis virus in chickens, the effect of the composition on the clinical signs of the disease and the immunostimulating activity of the composition. These effects were evaluated with different routes of administration (spraying and injection) and with or without a combined treatment with a vaccine.
  • compositions in accordance with the present invention have clearly a prophylactic and therapeutic effect on chickens infected by the Newcastle disease virus or the infectious bronchitis virus, and a higher immunostimulating effect on chickens infected by the Newcastle disease virus or the infectious bronchitis virus than the effect which can be obtained by vaccination alone. Furthermore, a synergistic effect is obtained when a combined treatment by vaccination and with a composition according to the invention is used.
  • the method monitors the presence of the active ingredients in the composition in their native active and/or in their degraded inactive form. By varying the conditions of storage of the composition (temperature and time) , the results give a good indication of the stability of the compositions over different temperatures and shorter or longer periods of time.
  • compositions to be assayed are stored in different conditions of temperature (40°C or 60°C) in an incubator. Samples are collected at different times, from 0 days to 52 days, and analysed for their composition by HPLC (high pressure liquid chromatography) . The elution profile of the samples are compared to elution profiles of standard compositions containing the active ingredients either in their native active form or in their degraded inactive form. The results are quantified by measurement of the area of the different peaks of elution in the different samples and comparison of the obtained results.
  • HPLC high pressure liquid chromatography
  • a Shimadzu HPLC chromatograph class-VP equipped with an UV detector, and auto-sampler with computing software class-VP 5.03.
  • Chromatography column stainless steel C-18 silica based Luna column, particle size 5 ⁇ m.
  • Chromatography conditions flow rate 1.2 ml/min, wavelength of measurement 235 nm, temperature 30°C, injection volume 20 ⁇ l, run time 11 minutes, other parameters adjusted to get peak heigh of ca . 80% of scale.
  • Ribavirin standard stock solution 100 mg of ribavirin is dissolved in 50 ml of deionized water; then 1 ml of this solution is diluted again in 50 ml deionized water to get a final concentration of 40 ⁇ g/ml.
  • Acyclovir standard stock solution 40 mg of acyclovir is dissolved in 50 ml of deionized water; then 1 ml of this solution is diluted again in 50 ml deionized water to get a final concentration of 16 ⁇ g/ml.
  • composition to be tested (composition of example 1 in the present case) is stored in an incubator at either 40°C or 60°C.
  • 1 ml of sample is collected from the composition and is diluted in 50 ml of deionized water; then 1 ml of this solution is diluted again in 50 ml deionized water to get a final concentration of ribavirin in the sample of 40 ⁇ g/ml.
  • active ingredient mg/ml (peak area of test sample / peak area of standard sample) x (weight of standard sample in mg / volume of sample in ml) x dilution factor difference between standard sample and test sample
  • active ingredient mg/ml (peak area of test sample / peak area of standard sample) x (weight of standard sample in mg / volume of sample in ml) x dilution factor difference between standard sample and test sample
  • the % of acyclovir is given by active ingredient (mg/ml) /40 mg/ml x 100
  • the % of ribavirin is given by active ingredient (mg/ml) /100 mg/ml x 100.
  • composition according to example 1 of the present invention is stable upon storage at 40°C for up to 52 days with almost no detectable degradation of the active agents acyclovir and ribavirin, and at 60°C for up to 9 days with a degradation of the active agent ribavirin of less than 3% and with no detectable degradation of the active agent acyclovir.
  • compositions according to the present invention can be used to treat animals infected by the aforementioned viral diseases at an industrial level.
  • compositions of examples 1 or 2 can be used to treat the Newcastle disease, the infectious bursal disease (Gumboro disease), Marek' s disease or the infectious bronchitis, in poultry or in turkeys (administration by spray or orally) , with or without a combined treatment by vaccination.
  • Newcastle disease the infectious bursal disease (Gumboro disease)
  • Marek' s disease the infectious bronchitis
  • poultry or in turkeys administration by spray or orally
  • compositions in accordance with the present invention can also be used for the treatment of viral active diarrhea, calf scours, foot-and-mouth disease and foot diarrhea affecting calves (mainly due to rota- and coronaviruses) , as well as similar diseases affecting foals, sheep, goats, dogs, cats or pigs, equine infectious anaemia, equine herpesvirus, equine encephalomyelitis of horses, rift valley fever affecting most domestic animals such as cattle, sheep and goats, the disease caused by the bovine respiratory syncitial virus and affecting calves, bovine leukosis, blue tongue, caprine arthritis and encephalitis, malignant catarrhal fever, cattle plague, bovine pneumonia, infectious bovine rhinotracheitis, infectious vesicular stomatitis, sheep pox, goat pox, bovine herpesvirus type 2, vulvovaginitis and "peste desproductive ruminants", feline respiratory disease and other viral diseases of dogs or cats due

Abstract

Compositions for the prophylactic and/or therapeutic treatment of viral diseases, preferably in poultry, comprising as essential active ingredients ribavirin and at least another antiviral agent or active chemical derivatives thereof and a stabilizing agent. Optionally, the compositions further comprise one or more antibiotics of the quinolone type, vitamins and/or preservatives. The compositions are stable upon storage in the form in which they are intended to be administered to poultry, i.e. by injection, orally or by spray. A combined therapy with vaccine or a combination with antibiotics is also possible. The invention concerns also the combined use of ribavirin and other antiviral agents for the treatment of animal viral diseases.

Description

Title Veterinary compositions
Field of the invention
The present invention concerns compositions comprising ribavirin and at least another antiviral agent as essential active ingredients, and a stabilizing agent, the compositions being intended to be used preferably in veterinary medicine.
Background of the invention
Diseases caused by RNA and DNA viruses, like the Newcastle disease, infectious bursal disease (Gumboro disease) , infectious bronchitis, influenza disease or Marek' s viral disease, are known to cause heavy losses in the poultry industry due to high mortality and the rapid spreading of the disease to healthy flocks.
The Newcastle disease is an acute, highly contagious rapidly spreading viral disease caused by a group of viruses which form the avian paramyxovirus of type 1. Previous studies made on chickens have shown that the incubation period of the virus is generally of between 2 and 15 days. The virus is generally transmitted through the upper respiratory system, from which it will penetrate into the blood. There will then follow a damage of the blood vessels and a spreading of the virus to other organs (spleen, liver, bone marrow) . After generally three days, all organs are infected, including the nervous system. The clinical signs of the disease are greenish diarrhea (due to haemorrhagic lesions present in the intestinal tract), respiratory symptoms (difficult respiration, coughing, sneezing, rales due to inflammation, oedema and mucoid exudate present in the trachea and pharynx) and nervous signs (stiff neck, dropped wings, absence of coordination, paralysis) , which leads generally to the death of the animal. A decrease of egg production in layers or even a cessation of laying have also been observed. The virus can also infect humans and cause severe conjunctivitis.
The infectious bursal disease (or Gumboro disease) is an important viral disease of poultry caused by a group of viruses called birnaviruses . It affects young chickens and is characterized by a sudden onset and a short course with massive destruction of lymphocytes, particularly in the bursa .
Infectious bronchitis is a highly infectious and contagious respiratory disease of chickens. In some cases and with some virus strains, the infection may also affect the oviduct or the kidneys. A significant decrease in egg production and egg quality in layers and a decrease of production in broilers is observed in infected flocks.
The influenza disease is caused by a viral infection originated by viruses belonging to the orthomyxoviruses family. Diseases specific to some animal species have been described, like for example the avian influenza, turkey influenza, swine influenza or equine influenza. The clinical signs of the disease are broncho-intestinal pneumonia and lung lesions. Many different subtypes of viruses have been described and isolated. These subtypes may be introduced from one species to another species, like for example from birds to mammals, and may then cause major pandemics in the unprotected population.
Marek' s viral disease is a widespread disease affecting domestic chickens in all sections of the world. It is characterized by lesions affecting the nervous system (demyelination of peripheral nerves leading to progressive paralysis of the wings and legs), organs and other tissues. Young chickens under sixteen weeks of age are most susceptible. Once the birds are infected, there is no known treatment to stop or reduce the infection. In its acute form, the disease leads to the death of the animal. The only actual treatment available is by vaccination.
Up to three hundred different immunologic types of virus have been associated with diseases affecting poultry and other farming animals.
In the field of poultry farming or industry, when a vaccine is available, vaccination against the viral agents causing the disease has been shown to be helpful, but however insufficient. The vaccination is made either by intraocular or intra-nasal instillation, by inhalation (spray or aerosol) , by adding the vaccine to the feed or by injection.
Thus, if prophylaxis against some viral diseases can be obtained through immunisation, an efficient immunisation is not possible against some viruses, either because a prolonged immunity is not developed or because cross- immunisation is not possible. In the first case, further vaccinations are necessary, which leads to high costs. In the second case, immunisation will not be efficient against a related virus which is antigenically dissimilar but leads to the same disease (known for example for commonly named cold viruses) .
Due to this lack of universal prophylaxis when using vaccination against viral diseases, studies have been made to find chemotherapeutic agents having an antiviral activity.
In this regard, agents like amantadine, methisazone or cytarabine have been described. However, these agents have only a limited spectrum antiviral activity. These agents also have the drawback of being toxic to the animal when administered at an effective therapeutic or prophylactic dosage, which is not desirable, especially if they are intended to be used for prophylaxis.
Therefore, there is still a need for a chemotherapeutic agent or composition which has a wide spectrum antiviral activity and a limited, or even zero, toxicity.
When looking for agents having a wide spectrum antiviral activity, an antiviral agent developed against viral human diseases, ribavirin or tribavirin (sold under the name
® Virazole by the company ICN Pharmaceuticals Inc., USA, or
® Rebetol by the company Scheπng Corp., USA) was found to be of special interest, since this agent also has an antiviral effect on several viral animal diseases.
Ribavirin is a synthetic non-interferon inducing broad- spectrum antiviral agent, which is used against many viral human diseases including type A hepatitis, measles and influenza B. Ribavirin has been shown to have a significant effect against respiratory syncytial virus (RSV) , parainfluenza and influenza B viruses, and a lesser activity against herpes, varicella, Lassa fever, infectious hepatitis, dengue fever, measles and AIDS viruses.
Moreover, ribavirin has been shown to be active against influenza virus types A and F infections in mice.
Chemically, ribavirin is 1- -D-Ribofuranosyl-lH-1, 2, 4- triazole-3-carboxamide, a synthetic nucleoside. Due to its analogy with the nucleosides used for the replication of the virus in the target infected cell, this molecule intervenes as an inhibitor of the 5' capping of viral mRNA, so that ultimately viral protein synthesis of both DNA and RNA viruses are affected.
Ribavirin is sold as a powder. At 25 °C, the maximum aqueous solubility of ribavirin is 142 mg/ml and the drug is slightly soluble in alcohol.
To improve the antiviral activity of ribavirin, studies have been made to chemically modify the molecule. These studies have shown that the antiviral activity of 2', 3', 5' and carboxamide derivatives of ribavirin is in certain cases better than the antiviral activity of the unmodified molecule .
However, some problems with respect to the medical or veterinary use of ribavirin have been addressed in the prior art.
Firstly, the drug ribavirin is approved for use only in the medical treatment of severe upper respiratory infections caused by RSV in infants and children.
Studies have shown that when used orally, it may cause mild gastrointestinal discomfort. At high doses of more than lg per day, erythropoiesis is suppressed, which leads to anaemia and reticulocytosis . Occasionally, hypotension, cardiac arrest or digitalis intoxication occurs.
Other studies have shown that when administered orally or as an aerosol at doses of 30, 36 and 120 mg/kg body weight or greater for 4 weeks or more, ribavirin caused cardiac lesions in rats, mice and monkey. Rash and conjunctivitis can also occur in the case of aerosol application.
Studies to investigate the toxicologic effects of ribavirin in cats have shown that daily administration of ribavirin at 11 to 44 mg/kg induced a dose-related toxic effect on bone marrow, primarily on megakaryocytes and erythroid precursors, and that at the higher dosages it suppressed numbers of circulating leukocytes.
Ribavirin has also demonstrated significant teratogenic and/or embryocidal potential in all animal species in which adequate studies have been conducted (rodents and rabbits) . Studies in which the drug has been administered systemically demonstrate that ribavirin is concentrated in the red blood cells and persists for the life of the erythrocyte .
Furthermore, according to published results concerning studies made with the drug ribavirin on birds having the Newcastle disease, it seems that ribavirin has no curative effect on the advanced stage of the disease, and a toxic effect on developing embryos (Anjum et al., Pakistan Vet. J., Vol. 2, No. 4, pp. 191-192, 1982 and Hussain et al . , Pakistan Vet. J. , Vol. 7, No. 3, pp.92-94, 1987). Some authors have even addressed the problem that ribavirin could interfere with the interferon activities connected with the vaccination of farming animals (Anjum et al., Pakistan Vet. J. , Vol. 2, No. 4, pp. 191-192, 1982), although in other respects the molecule has been used in combination with interferon alfa-2b against hepatitis C viral infection in humans (see Schering Corp.'s Rebetron™ combination therapy product information document issued in 1998) .
Another problem is related to the stability of ribavirin liquid compositions upon storage. Studies concerning solubility and made by the present inventors have shown that when ribavirin is combined in a composition with other compounds like the preservatives methyl- or propyl-paraben
(to avoid the development of microorganisms or fungi in the composition) , the composition becomes turbid or a precipitate forms, which is not desirable in certain forms of administration or for storage, as will be explained hereafter.
Upon the particular virus infection to be treated, the compound is administered in a specific form, generally selected from injection, topical application, inhalation, ingestion or spraying. The spraying or inhalation form by way of an aerosol will be most preferable in the case of viral respiratory diseases where the broncho-pulmonary epithelium is primarily affected, since the administration of the active substance through the respiratory tract in the form of micronized particles (the size of the particles is generally comprised between 0.3 and 5 micrometers for an aerosol and 5 to 100 micrometers for a spray) will be most effective. Therefore, in the case of aerosol administration, the stability of the active compound in the composition is of upmost importance, since precipitates from aerosols can interfere with the optimal operation of respirators or spraying apparatus.
Other antiviral agents whose activity is based on an inhibition of the viral DNA polymerase enzyme of the virus have been developed by the pharmaceutical industry, like
® for example acyclovir (sold under the name Zovirax by
Glaxo-Wellcome, USA) . The chemical name of acyclovir is 9- [2-hydroxyethoxymethyl) guanine] . This chemical agent is known for its activity against a limited number of viruses, the herpes simplex virus type 1 and type 2, the varicella zoster virus and the Epstein-Barr virus.
Acyclovir is sold in its sodium salt form as a white, crystalline powder. The solubility of acyclovir sodium in water exceeds 100 mg/ml and shows a pH of approximately 11. However, at physiologic pH, acyclovir exists as the non- ionized form and has only a maximum solubility of 2.5 mg/ml .
In US patent No. 5,491,135, Blough reports the test of a combination therapy (a treatment whereby several agents are administered separately but simultaneously to the patient) with acyclovir and with a broad spectrum antiviral agent, PALA (N- (phosphonoacetyl) -L-aspartic acid), against the simian varicella virus. The purpose of this study was to see if an additive or synergistic effect could be obtained. The results showed a slight benefit of the combination therapy with acyclovir and with PALA for the treatment of simian varicella virus. In the same patent, Blough reports also the test of a combination therapy with ribavirin and with PALA against the respiratory syncytial virus. However, in this case, no additive or synergistic effect of the combination therapy with ribavirin and with PALA against the virus could be detected.
Blough also mentions in the same patent that it is preferred to administer the compounds separately to the patient, i.e. not in the same composition.
Therefore, no prediction of the result of a combination therapy could be made, even if the agents themselves are individually active with regard to the treatment of the disease .
Pancheva et al . (Acta Microbiologica Bulgaria, 1993, Vol. 29, pp. 61-64) disclosed that a combined treatment with acyclovir and ribavirin is more effective for the treatment of herpes simplex keratoconjunctivitis in rabbits than the treatment with the individual drugs.
However, this reference does not address the problem of combining antiviral agents in a single composition and the problem of the stability upon storage of a composition which would comprise the active agents or the problems related to the way of administration of such a composition.
In other respects, it is also well known that antibiotics are chemical compounds which are active against bacteria, but have generally no activity against viruses. Therefore, viral and bacterial diseases are generally treated separately and with distinctive agents, and the compositions which are intended to be used to treat viral diseases do not contain antibiotics, as well as antibiotic compositions do generally not contain antiviral agents.
Surprisingly, the inventors of the present invention have found that a composition comprising essentially as active ingredients ribavirin and at least another antiviral agent like acyclovir is effective as treatment or prophylaxis of the Newcastle disease, infectious bursal disease (Gumboro disease) , infectious bronchitis, influenza disease or Marek' s viral disease in poultry.
Furthermore, the inventors of the present invention have also found that the compositions are effective against other animal viral diseases and are therefore broad- spectrum antiviral compositions.
The inventors of the present invention have also found that compositions comprising the active analogue chemical derivatives of ribavirin or of the at least other antiviral agent are also effective as broad-spectrum antiviral compositions .
The inventors of the present invention have also found that when combined with a vaccination treatment the efficacy of the treatment with the compositions in accordance with the present invention is improved and a synergistic effect is observed. The inventors of the present invention have also found that when adding other agents like quinolone-type antibiotics in the compositions, the efficacy of the composition as broad- spectrum antiviral is similar or even improved.
The inventors of the present invention have also found that the stability of the composition comprising ribavirin and at least another antiviral agent like acyclovir as essential active ingredients is achieved when a stabilizing agent is added to the composition, the stabilizing agent being preferably benzyl alcohol, N- methylpyrrolidone, glacial acetic acid, 2-pyrrolidone , dimethylacetamate (DMA) or dimethylformamide (DMF) or a combination thereof.
Thus, the main object of the present invention is to provide new compositions which are effective as antiviral agents, and preferably as broad-spectrum antiviral agents effective as treatment or prophylaxis of viral diseases in animals in general, and specifically in poultry, like the Newcastle, infectious bursal (Gumboro) , infectious bronchitis, influenza or Marek' s viral diseases.
Another object of the present invention is to provide these compositions as compositions which are stable upon storage.
A further object of the present invention is to provide compositions effective as treatment or prophylaxis of viral diseases in animals which do not interact negatively with other treatments, like for example vaccination, and which can be used in an effective manner without being toxic to the treated animal. One of the advantages of the present invention is therefore that the composition can be stored in a stable manner in the form in which it will be administered.
Another advantage is that the composition is efficient against the viral diseases at doses which are not toxic to the treated animal. Thus, the therapeutic index of the composition is increased.
Another advantage is that the composition can be used for the treatment or prophylaxis of viral diseases in animal even if a treatment by vaccination or with antibiotics is already applied on the animal. Both treatments can coexist and generally will show an improved efficacy.
Further problems which can be solved by this invention with respect to known prior art compositions will become apparent to the reader of the following description.
Summary of the invention
The present invention provides a solution to the aforementioned problems.
The aforementioned object is achieved by a composition having the features defined in claim 1 and comprising as essential active ingredients ribavirin and at least another antiviral agent like acyclovir, and a stabilizing agent, the stabilizing agent being preferably benzyl alcohol, N- methylpyrrolidone, glacial acetic acid, 2-pyrrolidone or a combination thereof. The aforementioned object is also achieved by the use of the compositions for the treatment of viral diseases, and preferably animal viral diseases, and most preferably those affecting poultry.
Preferred embodiments of the invention, including the use of chemical derivatives of the essential compounds of the composition, i.e. ribavirin and the other antiviral agents, the addition of optional ingredients in the composition like for example quinolone-type antibiotics, preservatives, vitamins or further stabilizing agents, or the use of the composition for the treatment of specific viral diseases in animal are defined in the dependent claims.
Unless otherwise specified, the percentages of ingredients used in the compositions are defined as weight per volume.
Description of preferred embodiments
Viruses are classified according to the type of nucleic acid they contain, i.e. RNA or DNA. Further subdivision is made into groups based upon physical and biochemical properties.
The RNA viruses are subdivided into the families paramixoviridae, orthomyxoviridae, coronaviridae, arenaviridae, retroviridae, reoviridae, birnarividae, picornaviridae, toroviridae, caliciviridae, togaviridae, bunyaviridae, filoviridae and rhabdoviridae . The DNA viruses are subdivided into poxviridae, herpesviridae, papovaviridae, adenoviridae, hepadoviridae, parvoviridae and circoviridae .
Adenoviruses are responsible of diseases such as the infectious canine hepatitis and infectious canine tracheobronchitis .
Herpesviruses are subdivided into alpha-, beta- and gamma- herpesviruses. Alpha-herpesviruses are associated with numerous diseases affecting different animal species such as infectious bovine rhinotracheitis, infectious pustular vulvovaginitis, haemorrhagic disease of pups, feline viral rhinotracheitis, infectious laryngotracheitis (due to avian herpesvirus 1), Marek' s disease (due to avian herpesvirus 2) and duck plague (due to duck herpesvirus 1).
Orthomyxoviruses are responsible of influenza diseases such as equine influenza, swine influenza and avian influenza.
Paramyxoviruses are associated with the Newcastle disease.
Birnaviruses are associated with the infectious chicken bursal disease.
The viral diseases which can be treated by the compositions according to the present invention are any viral diseases already known which affect humans or animals, and more specifically those which affect poultry. Preferably, the diseases caused by the following viruses are aimed at being treated: Newcastle disease virus (NDV) , Marek' s disease virus, infectious bursal disease (IBD; synonym Gumboro disease) virus, infectious bronchitis virus (IBV), avian pneumovirus (APV) , equine herpes virus type 2 (EHV-2), transmissible gastroenteritis (TGE) virus, porcine respiratory coronavirus (PRCV) , bovine parainfluenza-3 virus, lymphocytic choriomeningitis (LCM) virus, bovine viral diarrhoea virus (BVDV) , equine arteritis virus (EAV) , porcine parvovirus (PPV), classical swine fever virus (CSFV; synonym: hog cholera virus, HCV) , border disease virus (BDV) , bovine respiratory syncytial virus (BRSV) , bovine leukaemia virus (BLV) , equine herpesvirus 1 and 4
(EHV-1 and EHV-4), feline coronavirus (FCV), rotaviruses, poxviruses (like fowl poxvirus, turkey poxvirus, pigeon poxvirus, quail poxvirus, sparrow poxvirus), avian influenza A viruses, turkey influenza virus, chicken anaemia virus, duck enteritis virus and Aleutian disease virus .
The compositions according to the present invention comprise as essential active ingredients ribavirin or an active chemical derivative thereof and at least another antiviral agent or an active chemical derivative thereof and at least one stabilizing agent.
The amount of ribavirin or its active chemical derivative in the composition is preferably between 2.5 and 20% weight/volume. Most preferably, the composition contains 10% weight/volume of ribavirin.
The other antiviral agent or its active chemical derivative is present in the composition in amounts of preferably between 0.1 and 20% weight/volume. Most preferably, the composition contains between 1 and 10% weight/volume of the other antiviral agent or of its active chemical derivative. If more than one antiviral agent or active chemical derivative thereof is added, the above proportions apply individually to each antiviral agent or active chemical derivative thereof added.
The antiviral agent can be selected from known antiviral agents, like for example acyclovir ( 9— [2— hydroxyethoxymethyl) guanine] ) , amantadine (tricyclo [3.3.1.1 (3, 7) ] decane-1-amine) , udicil (4-amino-l-
[3, 4-dihydroxy-5- (hydroxymethyl) tetrahydro-2-furanyl] - 2 (1H) -pyrimidinone) , remantadin ( 1- (1-adamantyl) -1- ethanamine) , foscavir (trisodium dioxido (oxo) phosphoranecarboxylate) , ganciclovir (2-amino- 9- ( [2-hydroxy-l- (hydroxymethyl ) ethoxy] methyl ) -1 , 9-dihydro- 6H-purin-6-one) , ziduvudine (l-[4-azido-
5 (hydroxymethyl) tetrahydro-2-furanyl] -5-methyl-2, (1H, 3H) - pyrimidinedione) , tromatidine (N- (1-adamantyl) -2- [2-
(dimethylamino) ethoxy] acetamide) , virudox (1- [4-hydroxy-5- (hydroxymethyl) tetrahydro-2-furanyl] -5-iodo-2, 4 (1H, 3H) - pyrimidinedione) or viroptic (1- [4-hydroxy-5-
(hydroxymethyl) tetrahydro-2-furanyl] -5- (trifluoromethyl) - 2, 4 (1H, 3H) -pyrimidinedione) .
The purpose of adding another antiviral agent is to obtain a synergistic and/or additive therapeutic or prophylactic effect.
Preferably, the composition will contain as other antiviral agent 4% weight/volume of acyclovir or a combination of 4% weight/volume of acyclovir and 1% weight/volume of amantadine. The stabilizing agent can be selected from benzyl alcohol, N-methylpyrrolidone, glacial acetic acid or another natural fruit vinegar, 2-pyrrolidone, dimethylacetamate (DMA) or dimethylformamide (DMF) . The amount of stabilizing agent in the composition is preferably between 2.5 and 97.4% weight/volume. Preferably, the stabilizing agent is benzyl alcohol, N-methylpyrrolidone, glacial acetic acid, 2- pyrrolidone or a combination thereof. The amount of benzyl alcohol in the composition is preferably 4% weight/volume . The amount of N-methylpyrrolidone in the composition is preferably between 5 and 40% weight/volume, but can also reach an amount of up to 70% weight/volume. The amount of glacial acetic acid in the composition is preferably between 2.5 and 90% weight/volume . In one embodiment, the composition contains a combination of 4% weight/volume of benzyl alcohol and 70% weight/volume of N- methylpyrrolidone .
The compositions according to the present invention optionally comprise one or more quinolone-type antibiotics.
The antibiotic is selected preferably from enrofloxacin, ciprofloxacin, ofloxacin, flumequine, norfloxacin, nalidixic acid, cinoxacin, clinafloxacin, difloxacin, enoxacin, fleroxacin, miloxacin, nadifloxacin, pefloxacin, pipemidic acid, rosoxacin, rufloxacin, sparfloxacin, temafloxacin, tosufloxacin or trovafloxacin . The amount of antibiotic in the composition is preferably either 5% or
10% weight/volume. Preferably, enrofloxacin will be used, at a concentration of 5% or 10% weight/volume. The effect obtained by the combination is a higher efficiency in the treatment or prophylaxis of the disease.
For the therapy, vaccine, i.e. the inactivated virus against which a treatment or prophylaxis is aimed at, may be administered to the patient or animal. This may improve the antiviral efficacy of the therapy using the compositions according to the present invention. Hence, a combined therapy of this type was successfully carried out by the present inventors for the treatment of the diseases caused by the infectious bronchitis virus (IBV) and the Newcastle disease virus (NDV) in poultry. From the results, it could be deduced that the combined therapy gives better results and shows a synergistic effect.
The compositions may also comprise optionally other agents like vitamins, preservatives, thickeners or any other agent or additive commonly used in veterinary or medical compositions which is for example useful for the stability of the composition or permits to improve the formulation for a specific way of administration without altering the antiviral activity.
For example, sodium hydroxide or potassium hydroxide may be added in the composition in order to adjust the pH and/or improve the stability of the composition. Preferably, sodium hydroxide or potassium hydroxide will be added in an amount of 1% weight/volume.
When necessary, the composition is generally complemented to the required final volume by the addition of deionized water .
It should be noted that the form in which the compositions are administered depends upon the particular virus infection to be treated. For example, if the infection is caused by the Newcastle influenza or parainfluenza virus, and has manifested itself in the upper respiratory tract of the animal, the mode of treatment is preferably by spray, for example by aerosol spray, since this mode of administration delivers the agent to the site of infection. Depending on the severity of the infection, injection or oral therapy may be also indicated. Topical application may be used in the case of infections of a topical nature, such as herpesvirus infections.
For systemic treatment, the composition is administered in order that ribavirin is given at a daily dose of about 5 to 10 mg/kg body weight, one time each day and for two days in the case of poultry and between three and ten days in the case of other animals.
The invention will now be described in more detail with reference to the following examples.
Examples of compositions
The amounts of ingredients are given in % of weight/volume The volume is adjusted by addition of deionized water.
# Example 1 :
A composition comprising: Ribavirin 10%
Acyclovir 4%
Benzyl alcohol 4%
N-methylpyrrolidone 70%
Sodium hydroxide 1% Water q.s.p. 100% # Example 2 :
A composition comprising:
Ribavirin 10%
Acyclovir 4%
Amantadine 1%
Benzyl alcohol 4%
N-methylpyrrolidone 70%
Sodium hydroxide 1%
Water q.s.p. 100%
Evaluation of the efficacy of the compositions in the treatment of viral diseases in poultry
In vivo studies were made to evaluate the impact of the compositions in accordance with, for example, examples 1 or 2 of the present invention on the pathogenesis of the disease and the replication of the virus in the case of an infection of chickens with either the Newcastle disease virus or the infectious bronchitis virus.
The aim of these studies was to evaluate the protective activity of the compositions according to the present invention against the replication of the Newcastle disease virus or the infectious bronchitis virus in chickens, the effect of the composition on the clinical signs of the disease and the immunostimulating activity of the composition. These effects were evaluated with different routes of administration (spraying and injection) and with or without a combined treatment with a vaccine.
The results have shown that the compositions in accordance with the present invention have clearly a prophylactic and therapeutic effect on chickens infected by the Newcastle disease virus or the infectious bronchitis virus, and a higher immunostimulating effect on chickens infected by the Newcastle disease virus or the infectious bronchitis virus than the effect which can be obtained by vaccination alone. Furthermore, a synergistic effect is obtained when a combined treatment by vaccination and with a composition according to the invention is used.
Stability of the compositions upon storage (and results obtained with composition of example 1)
To assess for the stability of the compositions upon storage, a method has been developed by the inventors.
The method monitors the presence of the active ingredients in the composition in their native active and/or in their degraded inactive form. By varying the conditions of storage of the composition (temperature and time) , the results give a good indication of the stability of the compositions over different temperatures and shorter or longer periods of time.
Hence, it has be shown that a composition which is stable for eight days in an incubator at 60 °C is stable on shelf at room temperature for two years.
The principle of the methodology is as follows.
The compositions to be assayed are stored in different conditions of temperature (40°C or 60°C) in an incubator. Samples are collected at different times, from 0 days to 52 days, and analysed for their composition by HPLC (high pressure liquid chromatography) . The elution profile of the samples are compared to elution profiles of standard compositions containing the active ingredients either in their native active form or in their degraded inactive form. The results are quantified by measurement of the area of the different peaks of elution in the different samples and comparison of the obtained results.
The details of the methodology are as follows.
# Apparatus
A Shimadzu HPLC chromatograph class-VP, equipped with an UV detector, and auto-sampler with computing software class-VP 5.03.
Chromatography column: stainless steel C-18 silica based Luna column, particle size 5 μm.
Chromatography conditions: flow rate 1.2 ml/min, wavelength of measurement 235 nm, temperature 30°C, injection volume 20 μl, run time 11 minutes, other parameters adjusted to get peak heigh of ca . 80% of scale.
Mobile phase for chromatography: water/methanol in the proportion 93/7.
# Preparation of standard solutions (in the present case, an example with ribavirin and acyclovir standard solutions is given, since the method will be used to assess the stability of the composition of example 1)
Ribavirin standard stock solution: 100 mg of ribavirin is dissolved in 50 ml of deionized water; then 1 ml of this solution is diluted again in 50 ml deionized water to get a final concentration of 40 μg/ml. Acyclovir standard stock solution: 40 mg of acyclovir is dissolved in 50 ml of deionized water; then 1 ml of this solution is diluted again in 50 ml deionized water to get a final concentration of 16 μg/ml.
# Preparation of sample solution
The composition to be tested (composition of example 1 in the present case) is stored in an incubator at either 40°C or 60°C.
At the times of monitoring (selected from day 0 to day 35) , 1 ml of sample is collected from the composition and is diluted in 50 ml of deionized water; then 1 ml of this solution is diluted again in 50 ml deionized water to get a final concentration of ribavirin in the sample of 40 μg/ml.
All samples (including the standard samples) are filtered through a 0.45 mm filter prior to injection in the chromatograph. 3 injections are performed for each sample to be assayed.
# Calculations
From the elution graphs obtained, the following calculation is made in order to quantify the amount of native active ingredient in the sample:
active ingredient mg/ml = (peak area of test sample / peak area of standard sample) x (weight of standard sample in mg / volume of sample in ml) x dilution factor difference between standard sample and test sample Thus, in the present case of example 1, the % of acyclovir is given by active ingredient (mg/ml) /40 mg/ml x 100 and the % of ribavirin is given by active ingredient (mg/ml) /100 mg/ml x 100.
The experimental results obtained when assaying the composition of example 1 of the present invention are shown in Table I.
The values given in the table represent the percentage of native active ingredient (acyclovir or ribavirin) present in the sample collected at different times from the composition of example 1 stored at either 40°C or 60°C.
Table I
x means no sample assayed As is clear from the results shown in Table I, the composition according to example 1 of the present invention is stable upon storage at 40°C for up to 52 days with almost no detectable degradation of the active agents acyclovir and ribavirin, and at 60°C for up to 9 days with a degradation of the active agent ribavirin of less than 3% and with no detectable degradation of the active agent acyclovir.
Therefore, the results are surprisingly very satisfactory and show that the composition of example 1 is very stable, if one considers that a solution stable for 8 days at 60°C means that it will be stable for 2 years at room temperature.
Industrial applicability: treatment of viral diseases
The compositions according to the present invention can be used to treat animals infected by the aforementioned viral diseases at an industrial level.
For example, the compositions of examples 1 or 2 can be used to treat the Newcastle disease, the infectious bursal disease (Gumboro disease), Marek' s disease or the infectious bronchitis, in poultry or in turkeys (administration by spray or orally) , with or without a combined treatment by vaccination.
The compositions in accordance with the present invention can also be used for the treatment of viral active diarrhea, calf scours, foot-and-mouth disease and foot diarrhea affecting calves (mainly due to rota- and coronaviruses) , as well as similar diseases affecting foals, sheep, goats, dogs, cats or pigs, equine infectious anaemia, equine herpesvirus, equine encephalomyelitis of horses, rift valley fever affecting most domestic animals such as cattle, sheep and goats, the disease caused by the bovine respiratory syncitial virus and affecting calves, bovine leukosis, blue tongue, caprine arthritis and encephalitis, malignant catarrhal fever, cattle plague, bovine pneumonia, infectious bovine rhinotracheitis, infectious vesicular stomatitis, sheep pox, goat pox, bovine herpesvirus type 2, vulvovaginitis and "peste des petits ruminants", feline respiratory disease and other viral diseases of dogs or cats due to distemper virus, adenovirus, calicivirus or poxvirus infections, diseases affecting pigs like the porcine epidemic diarrhea, Aujeszky's disease and other viral diseases of pigs due to influenza-, rota- or cytomegaloviruses .
Although the present invention has been described with reference to several examples and embodiments of specific compositions and concentrations of ingredients, this is not to be considered as a limitation of the invention but merely illustrative thereof.
Specifically, other compounds like chemical derivatives of the active compounds cited herein could be used, as soon as the modification does not lead to a substantial loss of the activity of the compound. For example, it is well known that acetylation or phosphorylation of the ribosyl hydroxyl groups of ribavirin or conversion of the carboxamide group of the triazole carboxamide moiety of ribavirin to a carboxamidine moiety does not affect the antiviral activity.

Claims

1. A composition comprising:
(i) ribavirin or an active chemical derivative thereof; (ii) at least one other antiviral agent or an active chemical derivative thereof; (iii) a stabilizing agent.
2. A composition according to claim 1 wherein the stabilizing agent is selected from benzyl alcohol, N- methylpyrrolidone, glacial acetic acid or another natural fruit vinegar, 2-pyrrolidone, dimethylacetamate (DMA) or dimethylformamide (DMF) or a combination thereof.
3. A composition according to claim 1 or 2 wherein the amount of stabilizing agent is between 2.5% and 97.5% weight/volume.
4. A composition according to any one of claims 1 to
3 wherein the stabilizing agent is a combination of 4% weight/volume of benzyl alcohol and 70% weight/volume of N- methylpyrrolidone.
5. A composition according to any one of claims 1 to
4 wherein the amount of ribavirin or of its active chemical derivative is between 2.5% and 20% weight/volume.
6. A composition according to any one of claims 1 to 5 wherein the amount of ribavirin or of its active chemical derivative is 10% weight/volume.
7. A composition according to any one of claims 1 to 6 wherein said at least one other antiviral agent is selected from the group consisting of acyclovir, amantadine, udicil, remantadin, foscavir, ganciclovir, ziduvudine, tromatidine, virudox and viroptic.
8. A composition according to claim 7 wherein said at least one other antiviral agent is selected from the group consisting of acyclovir, amantadine, udicil, and remantadin.
9. A composition according to claim 7 or 8 wherein the amount of antiviral agent or of its active chemical derivative is between 0.1% and 20% weight/volume.
10. A composition according to claim 9 wherein the amount of antiviral agent or of its active chemical derivative is between 1% and 10% weight/volume.
11. A composition according to any one of claims 1 to
10 wherein said at least one other antiviral agent is acyclovir.
12. A composition according to claim 11 wherein the amount of acyclovir is 4% weight/volume.
13. A composition according to any one of claims 1 to 12 which comprises as further antiviral agent amantadine.
14. A composition according to claim 13 wherein the amount of amantadine is 1% weight/volume.
15. A composition according to any one of claims 1 to 14 which further comprises one or more antibiotics of the quinolone-type.
16. A composition according to claim 15 wherein the antibiotic is preferably enrofloxacin.
17. A composition according to claims 15 or 16 wherein the antibiotic is present in an amount of either 5% or 10% weight/volume.
18. A composition according to any one of claims 1 to 17 which further comprises other agents or additives commonly used in veterinary or medical compositions like vitamins, preservatives, pH adjusting agents, thickeners or ingredients necessary to permit an oral administration or an administration by way of spray, injection or topical application.
19. A composition according to claim 18 which comprises sodium hydroxide or potassium hydroxide.
20. A composition according to claim 19 wherein the amount of sodium hydroxide or potassium hydroxide is 1% weight/volume .
21. A composition according to any one of claims 1 to 20 for its use as a medicament.
22. Use of a composition according to any one of claims 1 to 20 for the preparation of a medicament for the prophylactic or therapeutic treatment of viral diseases.
23. The use according to claim 22 wherein the viral disease is selected from Newcastle, infectious bursal, infectious bronchitis, influenza or Marek' s disease.
24. The use according to claim 22 or 23 wherein the treatment further comprises a combined treatment with a vaccine .
EP00983353A 1999-12-20 2000-12-20 Stabilized veterinary compositions comprising more than one antiviral agent Withdrawn EP1239923A2 (en)

Priority Applications (1)

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EP99125414 1999-12-20
EP99125414 1999-12-20
PCT/EP2000/013017 WO2001045727A2 (en) 1999-12-20 2000-12-20 Stabilized veterinary compositions comprising more than one antiviral agent
EP00983353A EP1239923A2 (en) 1999-12-20 2000-12-20 Stabilized veterinary compositions comprising more than one antiviral agent

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EP1782826A1 (en) * 2005-11-08 2007-05-09 GBF Gesellschaft für Biotechnologische Forschung mbH PQS and c-diGMP and its conjugates as adjuvants and their uses in pharmaceutical compositions
US7981930B2 (en) * 2007-03-13 2011-07-19 Adamas Pharmaceuticals, Inc. Compositions and kits for treating influenza
CN105021756B (en) * 2015-07-01 2017-03-01 山东世通检测评价技术服务有限公司 Amantadine, rimantadine, ribavirin, the multi-joint detection method of Moroxydine residual in a kind of fowl egg
WO2018203879A1 (en) * 2017-05-01 2018-11-08 Kiani Iraj E Anti-viral compositions and method
CN109431981B (en) * 2018-11-26 2020-02-14 天津市中升挑战生物科技有限公司 Enrofloxacin injection and preparation method thereof

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IL105090A (en) * 1992-03-18 1998-08-16 Us Bioscience N-(phosphonoacetyl)-l-aspartic acid as broad spectrum antiviral
EP0871489A1 (en) * 1995-10-12 1998-10-21 Gs Development Ab A pharmaceutical composition for administration of an active substance to or through a skin or mucosal surface
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