Composition for treating viral diseases in animals

FIELD: medicine.

SUBSTANCE: invention refers to veterinary science, and aims at treating viral diseases in animals. What is declared is a composition for treating viral diseases in animals, containing two subtypes of vertebrate recombinant interferon mixed in equal molar proportions.

EFFECT: use of the declared composition enables ensuring the substantial increase of the therapeutic effect of recombinant interferons and minimising the potential adverse effects associated with their use.

4 ex

 

The technical FIELD TO WHICH the INVENTION RELATES.

The invention relates to veterinary medicine, specifically to compositions and methods for enhancing therapeutic effects of recombinant interferons, and can be used in the preparation of recombinant interferon for treatment of various viral diseases of animals.

The LEVEL of TECHNOLOGY

Interferons are a group of biologically active proteins, synthesized by lymphocytes during defensive reaction to foreign agents - viral infection, bacteria, parasites - antigenic or mitogenic effects. Interferons are secreted in the extracellular fluid and through specific receptors act on other cells, which is initiated by a complex sequential intracellular reactions leading to the induction of several enzymes and proteins such as proteinase R, oligoadenylates and others), which increase the resistance of cells to alien agents. As a result of action of interferon suppresses viral replication in infected cells, increased phagocytic activity of macrophages and increases the specific cytotoxicity of lymphocytes for target cells. In certain conditions, the interferon is also able to inhibit the development of malignant tumors.

Interferons can be divided into two chapters, the s-type. The type I interferons one common receptor IFN-alpha (IFNAR), consisting of an alpha subunit (IFNAR1) and short or long beta-subunit (IFNAR2) (M.S.Kunzi and P.P.Rowe (2001) in J.J.Oppenheim et al. (eds) Cytokine Reference, v.1, pp.627-639). In mammals are four types of interferons - alpha, beta and omega and Tau. Interferon type II are associated with receptor IFNGR and presents only one view - interferon-gamma.

All so far studied mammals have genes encoding interferons alpha, beta and gamma. Interferon omega and Tau are presented only in some animal species. Many types of mammalian cells, such as fibroblasts, epithelial cells and endothelial, lymphoid cells and astrocytes, are capable of producing beta-interferon. Whereas alpha-interferon is synthesized mainly by cells in response to viral infection (M.S.Kunzi and P.P.Rowe (2001) in J.J.Oppenheim et al. (eds) Cytokine Reference, v.1, pp.627-639). Gamma interferon is known as immune interferon, is produced mainly by activated T-cells and NK-cells (G.R. Stark (1998) Annu. Rev. Biochem. 67, 227-264).

In all mammals the most commonly presents interferons type alpha, and some groups of animals - also omega and Tau (R.M.Roberts et. al. (1998) J. Interferon Cytok. Res. 19(4), 427). These interferons are encoded by genes with no introns, and form extensive collection. For example, in cats was identified 14 genes inter is Aronov alpha and 13 genes interferon omega (see GenBank). The dog was found 8 genes interferon alpha (E.g Osamu et al. (2005) J. Vet. Med. Sci. 67(10), 1059-1062). Within families of proteins are very similar (the percentage of homology is 90%). Between proteins belonging to different families, homology below and is approximately 70%. Interferons differ mainly by the presence of insertions in several amino acid residues at the C-end interferon omega (R.M.Roberts, L.Liu, A.Alexenko (1997) Nucl. Acids Res. Mol. Biol. 56, 287-325). In rodents and humans functional beta-interferon encoded by only one gene.

The presence of multiple subtypes of interferon alpha and omega raises the question of their functional differences. Indeed, in a number of papers was found the difference in activity of interferon against various kinds of viruses (W.-S.Yeow et al. (1998) J. Immunol. 160, 2932-2939; G.R.Foster et al. (1996) J. Interferon Cytok. Res. 16, 1027-1033; R.Wonderling et al. (2002) Veterin. Immun. Immunopath. 89, 13-27).

Interferons can be characterized by inhibition cytopathological action of the virus on the cells (Rubinstein. Familletti, and Pestka, J. Virol., 37: 755 (1981); Armstrong. "Cytopathic Effect Inhibition Assay for Interferon: Microculture Plate Assay," in Methods in Enzymology, 78: 381-387 (1981); Familletti, Rubinstein, and Pestka, "A Convenient and Rapid Cytopathic Effect Inhibition Assay for Interferon," in Methods in Enzymology, 78: 387-394 (1981)). One unit of activity of interferon is defined as the amount of interferon that reduce virus-induced cytopathologically effect by 50%, and is calibrated by mezhdunarodnaia-standard international units.

It is important to separate preparations of interferon on the way they are received, which causes the difference in their composition, and therefore, the effects that they will cause in the body. Leukocyte interferon produced by stimulation of non-pathogenic viruses, double-stranded RNA, etc. of donor leukocytes. In fact, the thus obtained mixture of all subtypes of alpha-interferon, the content of which in the product varies from a few percent up to 90%, depending on the methods of purification and concentration. When applying gentle cleaning methods possible to get a comprehensive preparations enriched mediators of cell interactions, cytokines. The main drawback of this method of producing interferon is the possibility of contamination of the final product of viruses, such as hepatitis, HIV and other currently more promising recognized as the method of producing interferon microbiological synthesis. Recombinant interferons produced by expression of the gene encoding interferon, comprising the plasmid prokaryotic cells. Drugs are very homogeneous in composition and contain protein, corresponding to a specific subtype of interferon (Rafalski CENTURIES Clinical application of interferons).

For the treatment of viral diseases in dogs (distemper, bolezn Odeski, parvovirus enteritis, viral hepatitis) is widely used genetically engineered interferon for the veterinary mixoterin, representing a mixture of alpha, beta and gamma interferons person. However, treatment with exogenous gennoinzhenernym interferon (myxopyronin) is not effective enough: being a foreign protein drug, mixoterin can cause the phenomenon anaphylaxie and immunosuppression. In addition, given the different sensitivity groups and strains of the virus to interferon, it is not always possible to enter from the outside in the number of interferon, sufficient to suppress the persistence of the virus and does not cause anaphylactic shock.

Attempts were repeatedly made use of interferons in the form of mixtures (for example, patent number WO 0039280 (A2), CN 1935256 (A), RO 118842 (), CN 1449740 (A), RU 0002327486 27.06.2008 S1). However, it is surprising that in the descriptions of the inventions often no indication of the source of origin of interferons, which makes it impossible to interpret the results of experiments presented in the examples. As already mentioned, drugs leukocyte interferons contain many other serum proteins, including cytokines. It is possible that these proteins (not interferons), by themselves or in combination with other proteins (including interferon), can cause biological efficiency is s, described by the authors. In contrast, recombinant interferon homogeneous in composition, and therefore, the effects derived from their combination (mixing), strictly reproducible and can be attributed to interferon within the composition.

One of the factors limiting the widespread use of the drugs interferon in veterinary medicine, is that they have side effects depending on the dose and duration of therapy.

It is known that interferon beta and gamma are species-specific. However, interferons alpha, omega and Tau detect antiviral activity on heterologous animal cells (M.Kubes, N.Fuchsberger, R.V.H.Pollock (1994) J. Interferon Res. 14, 57-59; L.Liu et al. (1996) Biochim. Biophys. Acta 1294, 55-62). However, the effectiveness of long-term treatment of exogenous interferon in virus-living animals is problematic because it is already in the few weeks they have acquired neutralizing antibodies, making further treatment interferon impossible (N.S.Zeidner et al. (1990) Antimicrob. Agents Chemother. 34(9), 1749-1756).

Description of compositions for the treatment of viral diseases in animals, containing a mixture of species-specific recombinant interferon animals in the patent database is missing.

The INVENTION

The objective of the invention is to increase therapeutic efficacy of recombinantly interferon and reduction on the basis of therapeutic doses of interferons.

The invention is a method for enhancing therapeutic effect of interferon animal impose endogenous interferon in an amount of from 1 to 1×107ME on the day where the interferon is a mixture of recombinant interferons one subspecies alpha or omega, or a mixture of recombinant interferon different subtypes alpha and omega.

Due to increased therapeutic effect of interferon therapeutic effect can be achieved with a smaller amount of each recombinant interferon than normal therapeutic doses. Therefore, using the present invention, it becomes possible to reduce the cost of treatment and to minimize the potential side effects associated with large therapeutic doses of recombinant interferon, and, nevertheless, to achieve therapeutic effect.

DETAILED description of the INVENTION

The proposed composition for the treatment of viral diseases of animals contains:

two subspecies of recombinant interferon Alfa, or

two subspecies of recombinant interferon omega, or

two subspecies of recombinant interferon alpha and omega, mixed in equal molar proportions.

In the method of enhancing therapeutic effect of interferon mixture of recombinant interferons can be used in various dosage is karstenii forms, including aqueous solutions and powders. Pharmaceutical ingredients that can be used in the composition of the dosage forms may include buffers, salts and stabilizers.

EXAMPLES

The following examples illustrate the invention.

Example 1. The introduction of the mixture of recombinant interferon-alpha enhances their antiviral effects in cell lines cat (Fells catus).

The antiviral effect of a mixture of interferon was evaluated by inhibition cytopathological effect of the following viruses: virus, vesicular stomatitis virus, kalitsiviroza cats, virus infectious peritonitis in cats and herpes virus cats on cell lines cat CRFK and FCWF-4 (Armstrong, Methods in Enzymol., v.78 (PtA), pp.381-387 (1981)). Subjects compositions were prepared either from powdered liofilizatow, or of an aqueous solution of interferon alpha No. 2 (GenBank: AAM78029) and No. 7 (GenBank: BAC75983), mixed in various molar proportions 1:5, 1:4, 1:3, 1:2, 5:1, 4:1, 3:1, 2:1 and 1:1 in acetate buffer containing salt NaCl and stabilizers acid and sorbitol. A mixture of interferon were made in cell culture, which was kept during the day in CO2-incubator with 5% carbon dioxide, 95% humidity at 37,5°C. Environment decantation, and the cells were treated with virus and incubated for 72 hours prior development cytopathological effect under the same conditions as before. The ability of the mixture of interfero the s to inhibit virus-induced cytopathologically effect was evaluated in terms of the final titers of interferon. For the final titer of interferon took the reciprocal of the dilution that provided 50% protection of the cells in each of three independent experiments. The findings suggest that the introduction of a mixture of interferon alpha was greatly enhanced the antiviral effect of interferon. The greatest effect of cell protection was achieved with an equal ratio of interferon Alfa 2 and 7 in the mixture. While the current total concentration of the mixture of interferon was 1.45 times less than in control, where each of the subtypes of interferon were used separately.

Example 2. The introduction of the mixture of recombinant interferon omega enhances their antiviral effects in cell lines cat (Felis catus).

The antiviral effect of a mixture of interferon was evaluated as described in example 1. As previously, the introduction of a mixture of interferon omega was greatly enhanced the antiviral effect of interferon. The greatest effect of cell protection was achieved with an equal ratio of interferon omega No. 1 (GenBank: ABD78704) and omega No. 2 (GenBank: ABD78705) in the mixture. While the current total concentration of the mixture of interferon was 1.6 times less than in the control, where each of the subtypes of interferon were used separately.

Example 3. The introduction of the mixture of recombinant interferon alpha and omega enhances their antiviral effects in cell lines is x cat (Felis catus).

The antiviral effect of a mixture of interferon was evaluated as described in example 1. The introduction of a mixture of interferon omega was greatly enhanced the antiviral effect of interferon. The greatest effect of cell protection was achieved with an equal ratio of interferon alpha No. 2 (GenBank: AAM78029) and omega No. 1 (GenBank: ABD78704) in the mixture. Thus the effective concentration of a mixture of interferon was 1.7 times less than in the control, where each of the subtypes of interferon were used separately.

Example 4. The introduction of the mixture of recombinant interferon-alpha enhances their antiviral effects in cell lines dogs (Canisfamiliaris).

The antiviral effect of a mixture of interferon was evaluated by inhibition cytopathological effect of vesicular stomatitis virus and herpes virus dogs on cell lines dog MDCK, A72 and Cf2Th (Armstrong, Methods in EnzymoL, v.78 (PtA), pp.381-387 (1981)). Subjects compositions were prepared either from powdered liofilizatow, or of an aqueous solution of interferon alpha No. 1 (NCBI: NP_001006655.1) and alpha No. 8 (NCBI: NP_001007131.1), mixed in various molar proportions 1:5, 1:4, 1:3, 1:2, 5:1, 4:1, 3:1, 2:1 and 1:1 in acetate buffer containing salt NaCl and stabilizers acid and sorbitol. A mixture of interferon were made in cell culture, which was kept during the day in CO2-incubator with 5% carbon dioxide, 95% humidity at 37,5°C. Environment DECA is sabotaged, cells were treated with virus and incubated for 72 hours prior development cytopathological effect under the same conditions as before. The ability of a mixture of interferon-alpha to inhibit virus-induced cytopathologically effect was evaluated in terms of the final titers of interferon. For the final titer of interferon took the reciprocal of the dilution that provided 50% protection of the cells in each of three independent experiments. The findings suggest that the introduction of a mixture of interferon alpha was greatly enhanced the antiviral effect of interferon. The greatest effect of cell protection was achieved with an equal ratio of interferon alpha No. 1 (NCBI: NP_001006655.1) and 8 (NCBI: NP_001007131.1) in the mixture. While the current total concentration of the mixture of interferon was 1.8 times lower than in the control, where each of the subtypes of interferon were used separately.

The above data confirm the ability of the proposed method with the achievement of a technical result.

1. Composition for the treatment of viral diseases in animals, containing two subspecies of recombinant interferon vertebrates, mixed in equal molar proportions.

2. The composition according to claim 1, characterized in that the subspecies of recombinant interferon in the mixture are alpha interferon, omega interferon is, or a mixture of alpha and omega interferons vertebrates.

3. The composition according to claim 1, characterized in that it can contain pharmaceutical ingredients, which can be used in the composition of the dosage forms, and represent the buffers, salts and stabilizers.

4. The composition according to claims 1 to 3, characterized in that it presents in a variety of dosage forms, including aqueous solutions and powders.



 

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