A method of obtaining a lyophilized preparation on the basis of recombinant-2-interferon against viral diseases of carnivores

 

(57) Abstract:

The invention relates to the field of Virology and biotechnology and can be used for treatment and prevention of immunobiological drugs with antiviral action. The invention consists in obtaining a lyophilized form-based-2-interferon, which is injected serum component containing antibodies to one or more pathogens of viral diseases. As serum components can be used immune serum or immunoglobulins at a final concentration on total protein 0.5 to 10.0%. The technical result of the invention is to improve the antiviral activity and stability during storage. 1 C.p. f-crystals, 3 tab., 1 Il.

The invention relates to the field of Virology and biotechnology and can be used for treatment and prevention of immunobiological drugs against viral diseases of carnivores.

For the treatment and prevention of viral diseases currently used drugs both specific and nonspecific actions. Among the drugs with nonspecific antiviral is bringing a composition of interferon with additional substances, which can have different functional purpose and can be used for stabilization activity during storage, enhance antiviral properties, increasing specificity against a particular infectious agents.

Known methods for producing liquid compositions, and products based on the combination of interferon with substances, stabilizing its activity or enhance the antiviral effect. Thus, the method of obtaining the drug on the basis of recombinant-interferon, which additionally contains as a stabilizing additive glycerin and sodium dodecyl sulphate [1].

Another example of the composition of interferon with substances possessing additional antiviral effect, can serve as a way to obtain complexes of interferon with bactericidal and anti-toxic substances, based on obtaining sera from animals donors who previously, in strictly defined terms, injection injected substance - interferon inducer and the antigen (or toxin), inducing the formation of specific antibodies [2]. The resulting preparation is a healing serum with addition of non-specific, due to the presence of interferon, the specific used at the stage of immunization.

The main disadvantage of these methods [1, 2] preparation due to their liquid form, which is not conducive to long-term preservation of biological activity of interferon, which is especially important for interferon obtained by recombinant technology.

The problem of stability during storage is directly related to drugs based on recombinant-2-interferon. To improve the stability of high-purity from ballast protein substrate cultivation-2-interferon is widely used freeze drying in the presence of the filler, ensuring the preservation of biological activity during the shelf-life of up to 2 years or more. The filler used substances of protein nature, biopolymers or composition of permitted substances for use in immunobiological preparations. Without filler, providing at the stage of lyophilization conditions necessary for obtaining three-dimensional pills and serves as a stabilizer, purified interferon unstable during storage loses its biological activity. At present there are examples of preparation of dry dosage forms in the form of song-2-the AMI.

Known method of preparing a dry, lyophilized drug, including the introduction of poliglyukina in the solution of purified-2-interferon, which serves as a filler and stabilizer subsequent drying [3] (preparation Realigion" manufactured in, Vilnius).

The disadvantage of this method is that the inclusion of this component in the drug does not lead to the enhancement of its antiviral action.

For the treatment of viral diseases, cancer and diabetes, the proposed composition of the two drugs, the first of which consists of a powder that is a-interferon activity 2107IU, dried in the presence of filler, including (in mg/ml): 2,27 NaHPO4, 0,55 NaH2PO420 glycine and 1 albumin, and the second is a solution containing (in mg/ml): 4 (AcO)2Zn, 2.5 preteenlolita NaOH and 0.6. Before applying the powder of the first drug is dissolved in the second solution, and the obtained product is used by injection [4].

This method allows you to obtain a composition with increased antiviral activity and high stability of the component on the basis of interferon, however, has a number of the lyst consisting of glycine and albumin, has no additional antiviral, or therapeutic properties. Uncomfortable and is preparing for the use of the drug by combining components, dissolving one into the other to obtain the final injectable form.

The closest technical solution, selected as a prototype, is a method of producing drug "IFN", Russia [5], which represents a lyophilized composition of purified recombinant-interferon-2 activity 106IU with filler, which is used as human serum albumin (CSA) in a concentration of 0.5%. The drug is used in the complex treatment of infectious diseases of dogs. In the treatment of adenoviroz, distemper and viral enteritis, as well as mixed infections use injections reaferona in the first two or three days of illness dog 1 time a day at a dose of 1 million IU in combination with antibiotics and vitamins.

The difference and advantage of the prototype from analogues is used as a stabilizer of a single component, whey protein, CSA, which provides increased resistance to interferon inactivating factors wysu the soba receiving drug - high resistance of CSA, which is obtained from blood donors, as well as the absence of additional therapeutic or prophylactic antiviral action, due to its introduction in the drug composition in the above-mentioned concentration.

Object of the present invention is to provide such a method of producing drug-based-2-interferon, which would provide the drug higher antiviral activity and stability during storage.

This objective is achieved in that in a method of producing a lyophilized antiviral drug based on recombinant-2-interferon, comprising the purification of the recombinant-2-interferon, blending it with filler-stabilizer and drying, according to the invention as a filler-stabilizer use of immune serum or immunoglobulins containing antibodies to one or more viral agents.

Mixing-2-interferon immune serum (immunoglobulins) are in a ratio that provides the number-2-interferon in a single dose equal to 1106- 1107IU, and the final concentration of serum immunoglobulins) in General parvovirus enteritis, and/or infectious hepatitis mixing serum or immunoglobulin C-2-interferon conduct of the calculation to obtain the final concentration of specific antibodies in the product, expressed in the title: not less than 1:100 against distemper in the neutralization; not less than 1:128 against virus enteritis in response inhibition of haemagglutination; not less than 1:4 against the virus of infectious hepatitis in the reaction diffuse precipitation.

New, distinctive features of the prototype are the following characteristics of the method of obtaining the drug. As an additional matter, ensuring stabilization-2-interferon and at the same time possessing antiviral activity, use of immune serum or immunoglobulins containing antibodies to one or more viral agents. The final concentration of the serum component of the total protein is 0.5 to 10.0% and is derived from the need to achieve the required quantity of specific antibodies in a single dose.

A comparison of the proposed method of producing an antiviral drug with known shows that the distinctive features of the prototype signs are new, previously unknown its the th activity, moreover, the Association-2-interferon serum protein component containing antibodies to one or more viruses, does not affect the biological activity of each of them; 2) use as a component of immunoglobulin from the serum of humans and animals or antiviral immune sera of humans and animals allows to avoid additional drug substances for stabilization-2-interferon, since their introduction in preparation to final concentrations of 0.5 to 10.0% provides stabilization effect, equal to or exceeding that of other fillers on the basis of serum albumin and polymers.

The decrease in the content of the component in serum of nature to a concentration of less than 0.5% is impractical because below this concentration, firstly, it is difficult to achieve the level of specific antibodies, providing curative and preventive action, and secondly, there is a decrease in stabilizing properties with respect to-2-interferon in the drying of the drug. The excess concentration of protein above 10% also impractical because this value corresponds to the optimal upper limit for serum immunobiological preparations intended for in the way of it is produced by mixing the individual, pre-computed components: purified recombinant-2-interferon and immune serum (immunoglobulins). The method allows to obtain the product with specified rates on the content of specific antibodies and a-interferon-2, which is almost impossible to achieve, using the technique of immunization of animals, due to their existing individual differences in the nature of the immune response to the introduction of antigens and inducers of interferon.

In connection with the above technical solution meets the criterion of "inventive step".

The invention is illustrated charts, where the drawing (a, b, C) shows the results of the test storage-interferon for 1 year at a temperature of 42oC, lyophilized in the presence of fillers differing in protein concentration (a-2-interferon, dried in the presence of CSA; b - the same when used as a filler immunoglobulin against parvovirus enteritis; b - same, when used as filler serum against parvovirus enteritis).

The ordinate axis on the left - the contents of interferon in a single dose in IU, right - titer of specific antibodies in the initial dilution of the drug in reacts the mu protein.

The graphs show the following legend:

- 0 - - level source content interferon;

- *- the content of interferon after 2 years of storage;

- x - - titers of antiviral antibodies;

- . - - the same after a year of storage.

The method of obtaining the drug is as follows.

Example 1. Obtaining a drug for the prevention and treatment of plague carnivorous animals of the family dog.

For preparation of drug use cake mix-2-recombinant interferon on VFS 42-WS-89, representing a sterile solution with a pH of 7.0 and 7.6, with a protein concentration of 0.1-0.2 mg/ml and specific activity (1-2) 107IU/ml of This mix cake mix 1:5-1:9 serum against distemper on THE 10-07-03-92 or specific antibodies against canine distemper beyond 10-07-04-92, bring the pH to values of 7.2 to 7.4. The resulting mixture was poured into a 3.0 or 5.0 ml ampoule (inflaton), frozen in a refrigerator at a temperature not higher than -30oC, then lyophilizer. The drying time on the installation type TG-50 is 48 hours at a residual pressure in the chamber lyophilizate 10-50 m RT.article and the temperature of the heated shelves not exceeding 20oC.

Proteron in accordance with THE 10-07-04-92, it should be at least 2,3 lg. Antiviral activity-2-interferon determine the cell culture L-68 vesicular stomatitis virus (air force). Air force pre-passedout on chicken embryos beyond 42-14-99-77, spend at least 3 passages when infecting dose of 100-1000 TCD50/0,1 ml Contagious material should be 1106-1107TCD50/ml. Monolayer cells L-68 is grown on the mattress with a capacity of 1000 ml in the presence of growth medium (medium Needle MEME with a double set of ingredients - 90% FS 42-WS-90, serum fruits cows on FS 42-WS-90 - 10%) with antibiotics (kanamycin 100000 U/ml, gentamicin 16 U/ml), after which cells are removed from the glass, suspended in 40 ml of growth medium and count the number in the counting chamber Goryaeva. Then the cell suspension is diluted growth medium based to 1.0 ml contained 200 thousand/ml In the wells of the microplate contribute to 0.1 ml of cell suspension and after 2-3 days incubation at 37oC get monoclonal culture. The content of the active-2-interferon determine in the finished, dry the product for which it is dissolved using water for injection, up to the original volume. Then prepare two dilutions (above and below the intended TII fruits cows and antibiotics (penicillin, streptomycin). For each dilution using not less than 4 wells with cell culture. From the wells to remove the environment and contribute to 0.1 ml of each dilution of interferon, 16 holes left to control the infective dose and 4 holes for the control cell culture. Incubation is carried out in one day at 37oC in an atmosphere with 5,00,5% CO2then in each well, including the control, make the dose of the air force, equal to 100 TCD500.1 ml. After making indicator of the virus in cell culture is incubated for 2-3 days. The accounting is done, if there are no signs of degeneration in the control culture. The titer of interferon take the reciprocal dilution of the drug, wherein the cell culture 50% of the holes were completely protected from the cytopathic effect of the virus. Conversion activity in IU perform in the formula

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This method of cooking produces a drug, one dose of which contains a therapeutic dose of serum, or of antibodies with neutralizing activity of at least 1:100 and interferon in an amount not less than 1106IU.

Example 2. Receiving the drug for the prevention and treatment of parvovirus enteritis in animals of the family dog.

Cake mix-2-the th protein 0.1-0.2 mg/ml and specific activity (1-2)107IU/ml, mixed 1:5-1:9 serum against parvovirus enteritis carnivores in THE 10-07-01-92 or specific antibodies against parvovirus enteritis carnivores in THE 10-07-02-92, bring the pH to values of 7.2 to 7.4. The resulting mixture was poured into a 3.0 or 5.0 ml ampoule (inflaton), frozen in a refrigerator at a temperature not higher than -30oC, then lyophilizer define the contents-2-interferon in the dry preparation analogously to example 1. Determination of antiviral activity of serum component in the finished drug, after its dissolution in the original volume of saline solution (solution for injection) by the statement of response inhibition of haemagglutination (rtga) in accordance with THE 10-07-92-02. Title speficically antibodies must be at least 1: 128. The content of interferon in a single dose of the drug should not be less than 1106IU.

Example 3. Receiving the drug for the prevention and treatment of viral diseases of carnivores (distemper, parvovirus enteritis, infectious hepatitis).

The drug is prepared by mixing 1:1 to 1:9 HF-2-recombinant interferon activity 106- 107IU/ml bulk product specific polyvalent immunoglobulin immunoglobulin in the product before drying should be at least 0.5%, pH of 7.2 to 7.4. Immunoglobulins before mixing with interferon can be diluted with saline solution taking into account the maximum of the minimum concentration of protein (0.5%) and by maintaining a sufficient level of specific activity in accordance with THE 10-07-008-92. The resulting mixture was poured into ampoules (inflaton) for 2, 3 or 5 ml and lyophilizers with subsequent determination of interferon in the same dose as in example 1. One dose of the drug must contain-2-inteferon not less than 106IU.

To determine the specific activity of the drug against the virus enteritis use rtha, while the titre of specific antibodies must be at least 1: 128. The activity of antibodies to the causative agent of infectious hepatitis determine the reaction diffuse precipitation (RDP), it should be observed in the separation of at least 1:4. The activity of specific antibodies to distemper define in the source solution of immunoglobulins, before mixing with interferon, in the neutralization. It should be at least 2.3 to lg.

Example 4. A study of the effectiveness of the drug, manufactured by the proposed method.

The effectiveness of the preparations prepared according to the joint application for the prevention of canine distemper and parvovirus enteritis drugs "Kieron" and "IFN" with antibodies against canine distemper and parvovirus enteritis, respectively, according to the instructions on the application of these therapeutic agents. The drugs were administered for preventive purposes healthy dogs, and it was noted the absence of side effects beyond specified for each product separately.

The second phase of the study of the properties of drugs prepared according to the claimed method is the experiment compared their effectiveness in the treatment of canine distemper with the effectiveness of previously used tools on the background of complex therapy (antibiotics, with Dimedrol analgin, b complex vitamins, heart drugs). The scheme of all antivirals ("Kieron", "IFN"; specific antibodies against canine distemper; the product prepared by the present method) was single, consistent with the instructions and was 3 times the introduction of therapeutic doses with an interval of 12-24 hours in the first 2-3 days of illness. Table 1 shows the comparison results of the above treatment options. Based on these results we can conclude with the higher efficiency of the proposed drug, manifested in the reduction of duration of treatment and reduce the number of relapses.

The same pattern of use of the drug, is of the stages of the disease. Compare its efficiency compared to immunoglobulins. Processing of statistical data on the treatment of 36 animals showed that the integrated drug interferon-based and specific antibodies gave a greater number success rate (17%) during the first three days of treatment compared to immunoglobulins.

The efficiency of the product prepared by the present method based on the-2-interferon and polyvalent immunoglobulins (example 3), was shown in the treatment of diseases presumably viral nature in their initial stage. Treatment was subjected dogs to return 6-12 months, clinical signs of disease which was expressed in a depressed condition, slight fever, vomiting, interethnic phenomena. Data comparing efficacy in the treatment of dogs with unknown final diagnosis are shown in table 2.

The results are shown in table 2, indicate a high efficiency of the proposed drug in the case of its application in the initial stage of the disease presumably viral nature. Whilst there was no evidence of recurrence (re-emergence of symptoms within one month after completed which may be associated with bacterial or helminth nature of the disease, that required the change of treatment using antibiotics and antiparasitic funds.

This method of preparation can be used to obtain antiviral drugs for prophylaxis and treatment of various viral diseases of humans and animals, provided that in each case will be experimentally shown therapeutic effect in the joint introduction-2-interferon with specific antiviral antibodies. Presently, we have experimentally found that the serum component within the specified concentrations and containing specific antibodies, equally has a protective action against interferon in the stages of drying and storage regardless of the type and kind of viral agents that received immune serum. So, for example, there were no differences in heat stability when used as a stabilizer immunoglobulin human normal in FS-42-WS-87, immunoglobulin anti-human FC 42-WS-87, immunoglobulin against hepatitis B FS 42-WS-90, immunoglobulin against tick-borne encephalitis on FS 42-WS-93, as well as measles, protivoraketnoi Cheskie advantages of the way

The economic effect of using the product prepared by the present method, due to the reduction of treatment time, and no additional costs associated with the necessity of making the preparation of fillers to stabilize interferon. Table 3 shows data on the cost of the reagents used for the stabilization of interferon in the composition of currently available drugs. Prices are in us dollars catalog "Sigma" (1994).

Thus, when the use of drugs prepared by the present method and combining-2-interferon with antiviral antibodies or sera, savings, due to the non-use of additional substances-stabilizers-2-interferon, may be per 1000 doses not less than 2 $ for veterinary products and up to 70 $ (in case of non-use of CSA) for medical products.

Industrial applicability. The invention can be used in veterinary and human medicine.

Sources of information

1. The application of EPO N 270799, A 61 K 45/02, 47/00, publ. 15.06.88.

2. A PCT application (WO) N 82/03012, A 61 K 45/02, 37/10, publ., 16.09.82 (analog).

3. Methodological basis for the application of reaferona - chelovechestva, 25-29 January 1993, - Koltsovo, 1993, - S. 15-19, 22-23 (analog).

4. U.S. patent N 4853218, MKI A 61 K 45/02, publ. 1.08.88 (analog).

5. Methodological basis for the application of reaferona - human recombinant interferon-2B protein. // Collection of materials of scientific conference for practitioners, Koltsovo, 25-29 January 1993, - Koltsovo, 1993, - S. 115-117 (prototype).

6. U.S. patent N 471461, MKI A 61 K 45/02, publ. 22.12.87 (analog).

1. A method of obtaining a lyophilized preparation on the basis of recombinant-2-interferon against viral diseases of carnivores, including the purification of the recombinant-2-interferon, blending it with filler-stabilizer and drying, characterized in that the filler-stabilizer use of immune serum or immunoglobulins containing antibodies to one or more viral agents, and mixing-2-interferon with immune serum or immunoglobulin is carried out in a ratio that provides the number-2-interferon in a single dose, equal to 1 x 106- 1 x 107IU, the final concentration of serum or immunoglobulins on total protein, 0.5 to 10.0 %.

2. The method according to p. 1, characterized in that for the treatment and prophylact the mixing serum or immunoglobulin-2B-interferon conduct of the calculation to obtain the final concentration of specific Titel in the product expressed in the title: not less than 1 : 100 against distemper in the neutralization; not less than 1 : 128 against virus enteritis in response inhibition of haemagglutination; not less than 1 : 4 against infectious hepatitis in the reaction diffuse precipitation.

 

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