RU2527688C2 - Method of treating infectious diseases caused by influenza virus with type h1n1 surface antigen and therapeutic agent for treating infectious diseases caused by influenza virus with type h1n1 surface antigen - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present group of inventions refers to medicine, namely to therapy and virology, and concerns treating and preventing infectious diseases caused by an influenza virus with type H1N1 surface antigen. That is ensured by administering a therapeutic agent containing an active substance in the form of a homoeopathically activated potentiated form prepared by multiple serial dilution of a stock solution of affine purified antibodies to human gamma-interferon in a concentration of 0.3÷5.0 mg/ml.

EFFECT: administering this therapeutic agent provides effective treatment and prevention of the above infectious diseases.

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Description

The invention relates to medicine and can be used for the effective treatment and prevention of infectious diseases caused by influenza virus with the type of surface H1N1 antigen in children and adults.

The prior art method for the treatment of influenza by using the drug "Interferon tablets 4000 IU" (RU 2005132848 A, A61K 39/395, 2008). However, this method is ineffective in the treatment of infectious diseases caused by influenza virus with the type of surface antigen H1N1.

The antiviral drug Tamiflu is also known (international non-proprietary name for the active ingredient: oseltamivir phosphate), manufacturer: F.HOFFMANN-LA ROCHE Ltd., recommended by the World Health Organization for the treatment of influenza A with type H1N1 surface antigen (see Clinical management of human infection with new influenza A (H1N1) virus: initial guidance. 21 May 2009.

http://www.emro.who.int/csr/h1n1/pdf/clinical_management_21_5_2009.pdf). The disadvantages of this drug include the presence of side effects.

Also known is an antiviral drug in the form of drops of a potentized aqueous solution of monoclonal antibodies to gamma interferon C12, which can be used to treat acute respiratory viral infections (RU 2181297 C2, A91K 39/395, 2002), but its effectiveness in treating influenza with the H1N1 type of surface antigen like a person.

The closest analogue to the claimed invention is a method for the treatment of type H1N1 flu in humans by introducing an interferon inducer, anaferon, which is an activated potentiated form of antibodies to human gamma-interferon, obtained by serial dilution of a matrix solution in a neutral solvent with multiple shakes of each dilution obtained (V.K. Verevshchikov et al., “Evaluation of the effectiveness and tolerability of immunotropic therapy of acute respiratory diseases in ial with a history of cardiac history ", 2008). However, the effectiveness of treatment for influenza type H1N1 is determined by the choice of the concentration of the matrix solution, which does not follow from the prior art.

The invention is aimed at creating a universal, effective and safe method for the treatment of infectious diseases caused by influenza A virus with the type of surface antigen H1N1, and a drug with antiviral and immunomodulating activity, which provides effective treatment and prevention of infectious diseases caused by influenza A virus with the type of surface antigen H1N1, as in children and adults.

The solution to this problem is provided by the fact that in the method for the treatment and prevention of infectious diseases caused by influenza virus with the type of surface H1N1 antigen in humans, according to the invention, the active substance is used in the form of an activated potentiated form obtained by serial dilution and intermediate multiple shaking of each dilution obtained from matrix solution of affinity purified antibodies to human gamma interferon in a neutral solvent with a concentration of 0.3 ÷ 5.0 mg / ml.

Moreover, in the treatment method, an activated potentiated form of the active substance is used, which is obtained from a matrix solution of affinity-purified antibodies to human gamma-interferon with a concentration of 0.3 ÷ 5.0 mg / ml.

The claimed choice of the concentration range of the matrix (primary) solution is due to the fact that, at a concentration of less than 0.3 mg / ml, antibodies to human gamma-interferon sharply reduce their specific activity (effectiveness), stability and shelf life, and the use of antibodies with a concentration above 5, 0 mg / ml is technologically difficult.

In this case, the active substance comprises a mixture of various homeopathic dilutions, preferably, centigrade C 12, C 30, C 50 or C 12, C 30, C 200.

In addition, the task is also achieved by the fact that the drug for the treatment and prevention of infectious diseases caused by influenza virus with the type of surface antigen H1N1, according to the invention, includes an active substance in the form of a homeopathically activated potentiated form of affinity-purified human anti-gamma interferon antibodies.

In this case, a homeopathically activated potentiated form of the active substance of the drug is obtained from a matrix solution of affinity-purified antibodies to human gamma-interferon with a concentration of 0.3 ÷ 5.0 mg / ml.

Moreover, the drug may include a mixture of various homeopathic dilutions, preferably hundreds of C 12, C 30, C 50 or C 12, C 30, C 200.

The therapeutic efficacy of the claimed solution for influenza viruses with the H1N1 type of surface antigen for treating humans is due to the immunomodulating and antiviral effects of the homeopathically activated potentiated form of affinity-purified human anti-gamma interferon antibodies. At the same time, the drug of the claimed composition reduces the concentration of the virus in the affected tissues, affects the system of endogenous interferons and their associated cytokines, and induces the formation of endogenous “early” interferons (IFN α / β) and gamma interferons (IFN γ). In addition, the drug stimulates the humoral and cellular immune response, increases the production of antibodies (including secretory IgA), activates the functions of T-effectors, T-helpers (Tx), normalizes their ratio, increases the functional reserve of Tx and other cells involved in the immune response, It is an inducer of a mixed Tx1 and Tx2-type immune response: it normalizes the production of Tx1 cytokines (IFN γ, IL-2) and Tx2 (IL-4, IL-10), normalizes (modulates) the balance of Tx1 / Tx2 activities, increases the activity of phagocytes and natural killer cells (EC cells) that obus infects the antiviral activity of the drug against influenza viruses with the type of surface antigen H1N1.

Moreover, in contrast to the action of therapeutic doses of known drugs, including those based on interferon, the use of ultra-low doses of dilutions of the activated potentiated form of antibodies to human gamma-interferon does not cause side effects, resistance to the claimed drug does not develop, which makes it possible to use it for long-term safe and effective antiviral prophylaxis. In addition, the claimed drug is characterized by the absence of side effects while maintaining the therapeutic effect, environmental cleanliness and low cost and expands the arsenal of drugs intended for the treatment of influenza with the type of surface H1N1 antigen in humans.

The drug can be prepared on the basis of polyclonal or monoclonal antibodies to human gamma-interferon obtained by known methods.

The method of obtaining polyclonal and monoclonal antibodies is described, for example, in the book: Immunological methods / ed. G. Frimel, M., Medicine, 1987, pp. 9-33, or, for example, in the article Laffly E., Sodoyer R. Hum. Antibodies. Monoclonal and recombinant antibodies, 30 years after. - 2005 - Vol.14. - N 1-2. P.33-55.

The method of obtaining recombinant antibodies is described, for example, in the article Laffly E., Sodoyer R. Hum. Antibodies. Monoclonal and recombinant antibodies, 30 years after. - 2005 - Vol.14. - N 1-2. P.33-55.

The method for producing antibodies to human gamma-interferon is described, for example, in the article Monoclonal antibodies to human-γ: production, affinity, purification and radioimmunoassay. - The EMBO Journal, Vol. 2, No. 9, pp. 1527-1530, 1983.

To obtain polyclonal immune antibodies, as well as in hybridoma technology to obtain recombinant monoclonal and polyclonal antibodies, human gamma interferon, including recombinant, or a fragment or mixture of various fragments of human gamma interferon can be used as an immunogen for immunization of laboratory animals.

Antibodies to human gamma interferon obtained by known methods are purified mainly by affinity chromatography.

When implementing the claimed solution, for example, for research purposes, ready-made antibodies to human gamma-interferon produced by various companies can also be used, for example:

- Anti-Interferon-gamma antibody produced in goat; species reactivity: human (see the catalog "Products for research in the field of natural sciences" (SIGMA) for 2008-2009, p. 2153);

- Rabbit polyclonal antibodies to Interferon gamma (Immunogen: Highly pure (> 98%) recombinant human Interferon-gamma (see http://www.abcam.com/Interferon-gamma-antibody-ab9657.html);

Mouse monoclonal antibodies to Interferon gamma (Immunogen: Recombinant full length protein (Human)) (see http://www.abcam.com/Interferon-gamma-antibody-ab9658.html);

Chicken Anti-Human Interferon gamma Polyclonal Antibody, Unconjugated (see http://www.biocompare.com/ProductDetails/1269192/Chicken-Anti-Human-Interferon-gamma-Polyclonal-Antibody,-Unconjugated.html);

Rabbit Anti-Human Interferon gamma Monoclonal Antibody, Unconjugated, Clone EP1109Y (see http://www.biocompare.com/ProductDetails/880680/Rabbit-Anti-Human-Interferon-gamma-Monoclonal-Antibody,-Unconjugated,-Clone EP1109Y.html) and others.

Preferred for the preparation of the claimed drug is the use of polyclonal antibodies to human gamma interferon.

The antibodies obtained after purification by affinity chromatography in a phosphate-saline buffer solution with a concentration of 0.5 ÷ 5.0 mg / ml (preferably 1.0 ÷ 3.0 mg / ml) are used as a matrix solution for the preparation of the active substance in the form of homeopathic activated potentiated form, which is obtained by repeated serial dilution of a matrix solution of affinity-purified antibodies to human gamma-interferon and intermediate external exposure, preferably by homeopathic technology (see, For example, homeopathic medicines. Guidance on the description and production. B. Schwabe. Moscow 1967, s.12-38).

In this case, a uniform decrease in concentration is made by sequentially diluting 1 volume part of a matrix solution of affinity purified antibodies to human gamma-interferon in 9 volume parts (for decimal dilution D) or in 99 volume parts (for hundred percent dilution C) or in 999 volume parts (for thousandth dilution M) of a neutral solvent with multiple vertical shaking of each dilution obtained and using predominantly separate containers for each subsequent dilution until obtained the required ultra-low dose (potency). Moreover, the first homeopathic dilution is carried out in a neutral solvent: preferably in 10 ÷ 15% aqueous solution of ethyl alcohol or distilled water, the following dilutions in 20 ÷ 30% aqueous solution of ethyl alcohol and the final (last two to three) dilutions are carried out in 35 ÷ 75% aqueous ethyl alcohol solution or distilled water.

When using a mixture of various, mainly hundred, dilutions (potencies) as an active biologically active potentized substance, each component of the composition (for example, C 12, C 30, C 50 or C 200) is prepared separately according to the above technology until they are last but one diluted (respectively , to obtain C11, C29, C49 or C199) and then added in accordance with the composition of the mixture in one container in one part and mixed with the required amount of solvent (respectively, with 97 parts for a hundredth dilution).

External influence in the process of concentration reduction can be carried out both in the form of multiple vertical shaking, and, for example, ultrasound, electromagnetic or other similar physical action.

The claimed range of the concentration of the matrix solution is optimal for obtaining a drug with increased antiviral activity and is due to the fact that when the concentration of the matrix solution is less than 0.5 mg / ml, antibodies to human gamma-interferon during their further processing-potentiation sharply reduce their specific activity (effectiveness ), stability and shelf life, and the use of antibodies with a concentration above 5.0 mg / ml is technologically difficult and expensive.

Thus prepared active substance in the form of a homeopathically activated potentiated form can be used in conventional dosage forms, both in the form of water-alcohol solutions and for the preparation of solid dosage forms.

Example 1

A study of the effectiveness of ultra-low doses of antibodies to human gamma-interferon under the conditions of influenza infection in female mice of the Balb / c strain (n = 80) was carried out on the basis of the State Research Institute of Influenza RAMS (St. Petersburg). The infection process was modeled by intranasal administration of the influenza virus A / California / 07 / 2009swl (H1N1) at a dose of 10 LD50 (lethal infection). Ultra-low doses (a mixture of homeopathic dilutions C12, C30, C50) of rabbit polyclonal antibodies to human gamma interferon, prepared from a matrix solution with a concentration of 0.5 mg / ml and a final dilution in distilled water, were administered to 20 mice intragastrally at 0.2 ml / mouse (daily dose of 0.4 ml / mouse) for 5 days before infection and for 21 days after infection in combination with the addition of the drug to drinkers for animals. Comparison drug oseltamivir was administered intragastrically to 20 mice 25 and 1 hour before infection at a dose of 10 mg / kg, then within 3 days after infection - 2 times a day at 10 mg / kg (in a volume of 0.2 ml / mouse); starting from 4 days after infection, instead of oseltamivir, animals were injected with distilled water 2 times a day at 0.2 ml / mouse. In the control group, 20 mice were intragastrically injected with distilled water 2 times a day at 0.2 ml / mouse. 20 mice were not infected to assess the condition of animals throughout the experiment (intact control). The survival rate of animals was evaluated.

As a result of the experiment, it was found that ultralow doses of antibodies to human gamma-interferon under the conditions of lethal infection of mice with the influenza A / H1N1 virus increased animal survival by 3.7 times compared with the control group. On the 10th day after infection, 17.5% of the mice survived in the control group, 30% of the mice in the oseltamivir group, and 65% of the mice in the ultra-low dose group of antibodies to human gamma interferon. In the intact control group, all mice were alive until the end of the experiment.

Thus, the claimed drug has high antiviral efficacy in the treatment of influenza A / H1N1 caused by strain A / California / 07 / 2009swl, superior to the reference drug for the treatment of influenza A - oseltamivir.

Example 2

The study of the effectiveness of ultra-low doses of antibodies to human gamma-interferon under the conditions of influenza infection in female mice of the Balb / c line (n = 96) was carried out on the basis of the Federal State Institution Scientific Center of the WB “Vector” (Koltsovo). The infection process was modeled by intranasal administration of influenza virus A / California / 07/2009 (H1N1) v at a dose of 10ID50 (non-lethal infection). Ultra-low doses (a mixture of homeopathic dilutions C12, C30, C50) of rabbit polyclonal antibodies to human gamma-interferon, prepared from a matrix solution with a concentration of 5.0 mg / ml and final dilution in distilled water, were administered to 32 mice intragastrally at 0.2 ml / mouse (daily dose of 0.4 ml / mouse) for 5 days before infection and for 8 days after infection. Comparison drug oseltamivir was administered to 32 mice intragastrally 1 hour before infection at a dose of 20 mg / kg, then within 5 days after infection - 2 times a day at 10 mg / kg (in a volume of 0.2 ml / mouse); starting from 6 days after infection, instead of oseltamivir, animals were injected with distilled water 2 times a day at 0.2 ml / mouse. In the control group, 20 mice were intragastrically injected with distilled water 2 times a day at 0.2 ml / mouse. The titer of influenza virus in the lungs of mice was evaluated (on the 2nd, 4th, 6th, 8th day after infection).

As a result of the experiment, it was found that the use of ultra-low doses of antibodies to gamma-interferon in mice infected with A / California / 07/2009 (H1N1) v was comparable in efficiency with the use of oseltamivir and contributed to a reliable, more than 10-fold compared with the control group, reducing the production of influenza virus in the lungs of mice on the 4th, 6th and 8th day after infection (see Table 1).

Table 1 The dynamics of the production of influenza virus in the lungs of mice receiving ultra-low doses of antibodies to interferon gamma or oseltamivir when infected with 10ID50 strain of influenza virus A / California / 07/2009 (H1N1) v Groups The average titer values (log TCD ₅₀ / ml ± m) of influenza A (H1N1) v virus in lung homogenates in infected animals through: 2 days 4 days 6 days 8 days Ultra-low doses of antibodies to interferon gamma 0.54 ± 0.03, n = 8 0.59 ± 0.03, n = 8, * 1.63 ± 0.19, n = 8, * 0.98 ± 0.17, n = 8, * Oseltamivir 0.52 ± 0.02, n = 8 0.54 ± 0.03, n = 8, * 1.77 ± 0.09, n = 8, * 1.10 ± 0.16, n = 8, * The control 0.56 ± 0.03, n = 8 1.96 ± 0.14, n = 8 2.86 ± 0.11, n = 8 2.65 ± 0.23, n = 8 Note: * - p = <0.05 compared with control

Thus, the claimed drug has high antiviral efficacy in the treatment of influenza A / H1N1 caused by strain A / California / 07/2009 (H1N1) v, not inferior to the standard drug for the treatment of influenza A - oseltamivir.

Example 3

The study of the effectiveness of ultra-low doses of antibodies to human gamma-interferon in influenza infections in female mice of the Balb / c strain (n = 80) was conducted at APcis (Maison-Alfort, France). The infection process was modeled by intranasal administration of the A / New Caledonia / 20/99 (H1N1) influenza virus at a dose of 10 LD50 (lethal infection). Ultra-low doses (a mixture of homeopathic dilutions C12, C30, C50) of rabbit polyclonal antibodies to human gamma-interferon, prepared from a matrix solution with a concentration of 1.5 mg / ml and final dilution in distilled water, were administered intragastrically to 20 mice of 0.2 ml / mouse (daily dose of 0.4 ml / mouse) for 5 days before infection and for 21 days after infection in combination with the addition of the drug to drinkers for animals. The effectiveness of the claimed drug was compared with oseltamivir at a dose of 4 mg / kg / day. In the control group, 20 mice were intragastrically injected with distilled water 2 times a day at 0.2 ml / mouse. 20 mice were not infected to assess the condition of animals throughout the experiment (intact control). The survival rate of animals was evaluated.

As a result of the experiment, it was found that the use of ultra-low doses of antibodies to human gamma-interferon in the case of lethal infection of mice with the A / H1N1 influenza virus ensured a later onset of the disease: on the 5th day after infection, the number of dead mice was 25% (p <0.05) less than the placebo group.

Thus, the claimed drug has high antiviral efficacy in the treatment of influenza A / H1N1 caused by strain A / New Caledonia / 20/99, not inferior to oseltamivir (4 mg / kg / day).

Example 4

The study of the effectiveness and safety of ultra-low doses of antibodies to human gamma interferon in the treatment of influenza (A, B, mixed) and acute respiratory viral infections in children was carried out on the basis of the GU Research Institute of Influenza RAMS (St. Petersburg). The double-blind, placebo-controlled study involved 105 children of both sexes, aged 1 to 10 years old, who were admitted 1 ÷ 2 days after the onset of the disease, with a body temperature of at least 37.5 ° C and did not receive immunomodulating or antiviral therapy before the study.

Influenza monoinfection was laboratory confirmed in 53.4% of children, of which A / H1N1 influenza virus was detected in 21.1% (A / New Caledonia / 20/99, A / Beijing / 269/95). Children received ultra-low doses of antibodies to human gamma interferon in the form of a solid dosage form - a tablet saturated with a mixture of homeopathic dilutions C12, C30, C50, prepared from a matrix solution with a concentration of 3.0 mg / ml (3-8 tablets / day) for resorption in the mouth or placebo (an empty tablet that does not contain a homeopathically activated potentiated form of the active substance) in combination with symptomatic therapy for 7-14 days. In children, the duration of the main clinical symptoms, the frequency and severity of complications were evaluated.

As a result of the experiment, it was found that the ultra-low doses of antibodies to human gamma-interferon significantly reduced, compared with placebo, the total duration of the disease (from 7.5 ± 0.28 to 5.8 ± 0.24 days) and the duration of the main symptoms of the disease : fever from 3.67 ± 0.12 to 2.25 ± 0.13 days; symptoms of intoxication on average for more than 1 day; catarrhal symptoms - for 1.5 days. Adverse events associated with taking the drug are not registered. It is also shown that the claimed drug reduces the duration of virus excretion - 1 ÷ 2 days after the start of treatment, the number of detected viral antigens decreased by 3 times compared with the initial data, in the placebo group - by 2.2 times; on the 4th – 5th day of therapy, unlike the placebo group, no antigens were found in the group of the claimed preparation.

Thus, the claimed drug has high clinical efficacy and safety in the treatment of acute respiratory viral infections and influenza, including those caused by the influenza A / H1N1 virus.

Claims (6)

1. A method for the treatment and prevention of infectious diseases caused by influenza virus with the type of surface antigen H1N1 in humans, characterized in that they use the active substance in the form of an activated potentiated form obtained by serial dilution and intermediate multiple shaking of each dilution obtained from the affinity purified matrix solution antibodies to human gamma-interferon in a neutral solvent with a concentration of 0.3 ÷ 5.0 mg / ml 2. The treatment method according to claim 1, characterized in that the use of a medicinal product, comprising a mixture of various homeopathic dilutions. 3. The treatment method according to claim 1, characterized in that the use of a medicinal product, comprising a mixture of homeopathic dilutions of C 12, C 30, C 50 or C 12, C 30, C 200. 4. A drug for the treatment and prevention of infectious diseases caused by influenza virus with the type of surface antigen H1N1, characterized in that it comprises an active substance in the form of an activated potentiated form obtained by serial dilution and intermediate multiple shaking of each dilution obtained from a matrix solution of affinity-purified antibodies to human gamma interferon in a neutral solvent with a concentration of 0.3 ÷ 5.0 mg / ml 5. The drug according to claim 5, characterized in that it includes a mixture of various homeopathic dilutions. 6. The drug according to claim 5, characterized in that it includes a mixture of homeopathic dilutions C 12, C 30, C 50 or C 12, C 30, C 200.