RU2105565C1 - Method of prophylaxis of viral aerogenic infections - Google Patents

Abstract

FIELD: medicine, veterinary science, virology. SUBSTANCE: method involves intramuscular or intranasal administration of reaferon and/or ridostin, and/or polyribonate before or at period of possible infection. EFFECT: enhanced effectiveness of viral aerogenic infections prophylaxis. 4 tbl

Description

 The invention relates to medicine, namely to methods for non-specific immunoprophylaxis of viral infections.

 Venezuelan encephalomyelitis viruses of horses (VEL), Marburg and Ebola are classified as pathogens that cause especially dangerous (generalized) infections.

 In domestic and foreign scientific and technical documentation there is a small amount of information on the assessment of the therapeutic and prophylactic effect of some immunomodulators in infections of Marburg [5, 8] Ebola [6, 7] and VEL [1, 2, 4] All of them relate to diseases caused by infection animals and humans through the skin. At the same time, this kind of data is practically not presented for susceptible macroorganisms infected by the aerogenous route, although the way the virus penetrates through the respiratory tract can be one of the main ones in accidents that can occur in the laboratory when working with viral pathogens.

 The implementation of preventive measures in this case using specific immune serum preparations may turn out to be less effective compared to their effect on a macroorganism infected through the skin [3]. Therefore, the problem of finding effective non-specific drugs for these infections remains urgent.

 Known methods for the prevention and treatment of viral infections using an interferon inducer: double-stranded ribonucleic acid [9] and reaferon (α-2 interferon obtained by genetic engineering) [10] introduced into the body intramuscularly.

 A known method for the prevention of Ebola infection in monkeys infected by intramuscular injection, which includes the introduction of human interferon (intramuscularly) 2 days before infection or 1 hour after infection, followed by a continuation of the course of prophylactic treatment [14] As a result of these experiments, there were no differences in the clinic and the outcome of the disease between the experimental and control groups of monkeys.

 A disadvantage of the known methods is their low prophylactic effectiveness, because In known schemes for introducing the indicated interferon inducers into the body, the features (paths) of infection of the body are not taken into account, which is very important when it is aerogenously infected with the VEL, Marburg and Ebola viruses.

 The closest technical solution (prototype) is a method for the prevention of infection caused by the VEL virus, which includes introducing into the body preparations containing an interferon inducer and / or immunomodulator, before or during the period of possible infection in the body [11] Poly is used as an interferon inducer -IC stabilized by poly-L-lysine and carboxymethyl cellulose (poly-ICLC). Poly-ICLC induces the synthesis of interferon in high concentrations in rodents, primates and humans. The drug is administered intravenously and intramuscularly at a dose of 0.3 and 3.0 mg / kg body weight 8 hours before inoculation of the VEL virus (strain TS-83).

 The disadvantages of the prototype method are the high toxicity of this immunomodulator (hyperthermia, damage to the hematopoietic organs, coagulopathy, secondary hemorrhagic lesions, shock), accumulation (cumulation) when introduced into animals and humans [12] and its extremely high cost [13] All these circumstances extremely complicate the possibility of widespread use of the drug in medicine. Moreover, permissible non-toxic doses of the drug when administered parenterally do not guarantee the prophylactic effect in aerogenic variants of infection of the body with VEL, Marburg and Ebola viruses.

 The objective of the proposed technical solution is to create such a method that would allow for an increase in the preventive and emergency preventive effect in case of possible aerogenic infections caused by VEL, Marburg and Ebola viruses.

This problem is solved in that in a method for the prevention of aerogenic infections caused by VEL, Marburg and Ebola viruses, comprising parenteral administration to the body of preparations containing an interferon inducer and / or immunomodulator, before or during a possible infection by the aerogenous route, according to the invention simultaneously with the intramuscular injection of drugs into the body, these drugs are additionally and intranasally administered at the same doses to ensure their distribution throughout the respiratory tract, and in ETS prophylactic drugs used reaferon or ridostin or reaferon with ridostin or reaferon with poliribonatom or reaferon with ridostin poliribonatom and which is introduced into the body is not more than twice a day in the total doses: reaferon (1,0 100,0) • 10 ⁶ IU / kg; ridostin 1.0 8.0 mg / kg; polyribonate 10.0 100.0 mg / kg.

Reaferon (VFS-42-227 BC-89) is a protein with a molecular weight of 18 KD, synthesized as a result of incorporation into the genetic apparatus of pseudomonas using plasmid G3 of the human leukocyte interferon gene α-2. As a stabilizer of biological activity, it contains human albumin and mannitol in a final concentration 5 mg / cell. The specific activity of 4 • 10 ⁸ IU / mg protein.

 Ridostin (TU 9291-008-00479979-94) VFS 42-245-7-94 dosage form dS-RNA of the killer strain of Sac. cerevisiae, produced in NIKTI BAS SSC WB "Vector", Novosibirsk. Obtained by enzymatic and mechanical destruction of the yeast cell walls and extraction of dS-RNA by fractionation with a solution of lithium chloride.

 Polyribonate (TU 9291-007-00479979-94) VFS 42-264-3-95 dosage form of yeast RNA, produced by NIKTI BAS SSC WB "Vector". The RNA content in terms of dry matter reaches 93% and the protein is not more than 0.7% VFS - temporary pharmacopoeial articles were developed at the State Research Center of the World Bank "Vector" and approved by the State Institute of Standardization. Tarasevich (Moscow).

 With the introduction into the body of doses of drugs lower than the doses of drugs indicated in the proposed technical solution, there is no prophylactic effect. With the introduction into the body of doses of drugs larger than the doses of drugs specified in the present invention, there is a significant increase in their toxicity.

 Example 1. The method of infection of laboratory animals in the study of the preventive action of drugs.

 We use the VEL virus (Trinidad strain), the Marburg virus (Porr strain) and the Ebola virus (Zaire strain variant 8 M.S.).

 The experiments are carried out on guinea pigs weighing 180,200 g.

Aerosol infection of guinea pigs is carried out in a small dynamic chamber at a relative humidity of 50 to 70% and a temperature of 20 - 24 ^o C.

 When infected with the VEL virus, a virus-containing suspension obtained by culturing the VEL pathogen in a Vero cell culture using a roller method is used. Doses of infection range from 3 to 100 respiratory LD50 for experimental animals.

 When infected with Marburg and Ebola viruses, liver homogenates of guinea pigs containing these viruses are used. Doses of infection are 5 to 20 respiratory doses of LD50 for guinea pigs.

 The biological concentration of the Marburg and Ebola viruses in microcyclone samples is determined by titration on guinea pigs, which are injected intraperitoneally with 0.5 ml of material, and by the plaque method on VERO cell culture, respectively.

 The camera allows you to simultaneously infect 10 guinea pigs. The aerosol flow rate in the exposure channel is 10 cm / s, and the passage time by the aerosol flow of the entire chamber is 5 7 s.

 Spraying of infectious material is carried out using a biological aerosol generator (BG-2). The median-mass particle diameter of the aerosol is 1.1 ± 0.6 μm.

 Dilution of the aerosol is carried out using a metering aerosol attachment, changing the diaphragm of the outlet from 0.5 to 6 mm. Aerosol samples are taken into microcyclones, into which 10 ml of a sorbing liquid are preliminarily filled, consisting of Hanks solution containing 2% (by volume) inactivated serum of cattle, 100 units / ml of penicillin and 100 μg / ml of streptomycin. The aerosol volumetric sampling rate is 10.0 ± 0.5 l / min.

 The biological concentration of the virus in samples of microcyclones is determined by titration on a culture of chicken embryo fibroblast cells by the plaque method.

 Observation of the animals is carried out for 11 days in case of infection with the VEL virus and for 21 days in case of infection with the Marburg and Ebola viruses. The specificity of the death of guinea pigs is assessed by the presence of plaques on the chicken embryo fibroblast cell culture when 0.2 ml of a 10% brain suspension of a fallen animal is introduced onto the cell monolayer.

 Statistical processing of the results is carried out according to the student method and according to Genes tables.

 Before starting the experimental work on the selection of optimal dosage regimens, additional studies are carried out related to the study of the pathogenetic picture of the disease in guinea pigs, aerogenically infected with VEL, Marburg and Ebola viruses. Animals are infected in an aerosol chamber with a dose of 10 respiratory LD50. At certain intervals, the infected animals are opened (after preliminary intravital washing of organs and tissues from the blood) and various biological samples are taken for virological studies. The concentration of the virus in all selected samples is determined by titration according to the method of plaques on a monolayer of fibroblast cells of a chicken embryo.

 Studies show that the primary reproductive organ of the virus in guinea pigs is the lungs. During infection, the virus reaches maximum concentrations in the olfactory bulbs and cerebral hemispheres, spleen and blood.

 It was noted that 4 to 12 hours before the appearance of the virus in the olfactory bulbs of the brain of animals, the pathogen was recorded in the olfactory region of the nasal cavity. This circumstance, together with the data of electron microscopic studies, indicates the possibility of the spread of the virus into the brain of infected animals along the olfactory pathway.

 In connection with the foregoing, in experiments on aerogenically infected guinea pigs, medicinal substances are administered both intramuscularly and intranasally (with the aim of delivering immunomodulators to the organs and cells of the primary and secondary reproduction of the virus).

 Example 2. The study of the effectiveness of the emergency preventive action of drugs on guinea pigs infected with the VEL virus (Strain Trinidad).

 When studying the emergency preventive effect of drugs by airborne infected guinea pigs (10 respiratory LD50), drugs are administered individually and in various combinations as follows: reaferon at a dose of 250 thousand IU / head 2 hours after infection and then 2 times a day for 10 days; ridostin in a dose of 1 mg / head 2 hours, 2 and 4 days after infection; polyribonate at a dose of 5 mg / head 2 hours, 1 and 2 days after infection. All drugs are used, treating animals both intramuscularly and intranasally (under ether anesthesia), dividing the inoculum into 2 equal parts by volume (0.25 ml each). The drug using a microdoser is applied, if possible, to most of the surface of the respiratory tract of animals.

 The results of these studies are presented in table. 1. From the above data it follows that the protective effect of drugs to one degree or another is manifested in each of the tested options for emergency prevention of the disease in guinea pigs. The largest percentage of surviving animals is observed when using ridostin and ridostin with reaferon and is 20% with a probability of an accidental deviation of p 0.05. For the variant using a combination of three drugs (reaferon, ridostin and polyribonate), the percentage of surviving animals is lower, and there is no significant deviation of this value from the control. But the average life expectancy in this case increases by 3.2 days (p 0.02).

 Example 3. The study of the effectiveness of the preventive effect of drugs on guinea pigs infected with the Marburg virus (strain Rohr).

 When conducting studies on the effectiveness of the prophylactic effect of immunomodulators in experiments on animals infected with an aerosol of the Marburg virus, drugs are administered in various combinations as follows: reaferon at a dose of 250 thousand IU / head, ridostine at a dose of 1 mg / head, polyribonate at a dose of 5 mg / head. The introduction of drugs is carried out 48, 24 hours before infection, at the time of infection and 24, 48, 72, 96 hours after infection, treating animals both intramuscularly and intranasally (under ether anesthesia), dividing the inoculum into two equal parts by volume ( 0.25 ml each).

 The results of these studies are presented in table. 2.

 It was noted that all tested drugs with combined use have a significant prophylactic effect (increased survival of animals and their average life expectancy).

Example 4. The study of the emergency preventive effect of drugs on guinea pigs infected with the viruses of Marburg (strain Porr) and Ebola (strain Zaire option 8 MS)
In order to study the emergency preventive effect of immunomodulators in experiments on aerogenically infected (Marburg and Ebola viruses) guinea pigs, drugs are administered individually and in various combinations as follows: reaferon at a dose of 250 thousand IU / head 2 hours after infection and then 2 once a day for 2 weeks; ridostin in a dose of 1 mg / head 2 hours, 2 and 4 days after infection; polyribonate at a dose of 5 mg / head 2 hours, 1 and 2 days after infection.

 All these drugs were used, treating animals both intramuscularly and intranasally (under ether anesthesia), dividing the inoculum into 2 equal parts by volume (0.25 ml each).

 The results of these studies are presented in table. 3 and 4.

 From the analysis of the table. 3 and 4 it is seen that some positive effect of the emergency prevention of Marburg disease in guinea pigs is observed only in the case of ridostine (see table. 3). At the same time, the number of dead experimental animals decreases (p 0.1), and their average life expectancy increases compared to the control group of animals (p 0.15).

 Analyzing the results of the studies presented in table. 4, it should be noted that only the combined administration of ridostin and reaferon to aerosolized Ebola virus in guinea pigs caused a significant positive effect on the increase in the average life expectancy of these animals compared to the control (p 0.04).

Thus, the proposed method of immunoprophylaxis allows to obtain a positive prophylactic effect using ridostin and ridostin in combination with polyribonate in case of aerogenic VEL infection in animals. The combined use of ridostine with reaferon and reaferon with polyribonate before aerogenic infection with the Marburg virus causes an increase in animal survival with a probability of more than 95%. The implementation of emergency preventive measures gives the following positive effect on aerogenically infected animals:
in case of VEL disease, intramuscular and intranasal administration to guinea pigs of ridostin or ridostin with reaferon significantly increases the survival rate of animals (K _z ) to 0.29; similar administration of ridostine with reaferon and polyribonate significantly increases the average life expectancy (LSS) of animals (p 0.02);
in Marburg’s disease, intramuscular and intranasal administration of ridostine provides protection (K _z 0.2; p 0.10) and with a 85% probability it increases LIV by 2.4 days in guinea pigs;
in case of Ebola disease, intramuscular and intranasal administration of ridostin to guinea pigs with reaferon significantly increases P 0.04 by 10.9%.

 Industrial applicability. The invention can be used in medicine and experimental virology in the immunoprophylaxis of especially dangerous viral infections.

 Sources of information.

 1. Shubladze A.K. Gaidamovich S.Ya. Gavrilov V.I. // Q. virusol. 1959, N 3, p. 305 310.

 2. Gibbs E.P. J. // Equine Veterinar J. 1976, Vol. 8, N 2, p. 66 71.

 3. Ryzhikov A.B, Tkacheva N.V. Sergeev A.N. et. al.// 4th International aerosol Conf. Los Angeles, 29 aug. 2 sept. 1994.Abstr. Ed. Richard C. Flagen. Los Angeles, 1994, Vol. 2, p 723,724.

 4. Wedum A.G. // Publ. Health Rep. 1964, Vol. 79, N 7, p. 619 633.

 5. Anonymous // Br.Med.J. 1977, Vol. 1, p. 64 64.

 6. Bowen E.T.W. Baskerville A. Contell K.et al. // Ebola virus haemorrhagic fever / Ed. S.R. Pattyn / -Elsevier.-North Holland, Amsterdam, New York, 1978, p. 245 251.

 7. Markin V. A. Michailov V.V. Bogatikov G.Y. et al.// Int. Symp "100 Years of virol". St. Petersburg, 21 25 sept. 1992. M. 1992, p. 57 58.

 8. Stewart W. The interferon system. New York, 1979, 143 p.

 9. Application of France N 2433535, cl. C 07 H 21/02, publ. 04/18/80.

 10. Auth. testimonial. N 1680200, cl. A 61 K 37/66, publ. 09/30/91.

 11. Hilmos D.E. Stephen E.Z. Spertrel R.O. Levy H.B. Use of poly (ICLC) for the prophylaxis and treatment of venezuelan equine encephalomyelitis virus infection in nonhuman primates // Amer. Soc. Microbiol, 1978, vol. 1, p. 317 319 (prototype).

 12. Inductors of interferon // Series "Interaction of viruses and cells" / Ed. R.A. Kukayn et al. Riga, ed. Zinatne, 1981, p. 11 13.

 13. Dyakov S.I. Chizhov N.P. Sidorenko S.V. Modern antibiotics and antiviral drugs in experimental chemotherapy of bacterial and viral infections. Mn Belarus, 1988, p. 163,164.

 14. Bowen E.T.W. Baskerville A.//Ebola virus haemorragic fever. Ed. by Pattyn S.R. North Holland, Amsterdam, N-Y. Elsevier, 1978, p. 245 251.

Claims (1)

A method for the prevention of viral aerogenic infections, including intramuscular administration of preparations containing an interferon inducer and / or immunomodulator to the body before or during the possible exposure of the causative agent to the body by the aerogenous route, characterized in that intranasal administration is additionally carried out simultaneously with the intramuscular injection of drugs the same drugs in the same doses, and reaferon or p is used as preparations containing an interferon inducer and / or immunomodulator idostin, or reaferon with ridostin, or reaferon with polyribonate, or reaferon with ridostin and polyribonate, which are administered to the body no more than twice a day in total doses: reaferon (1.0 100.0) • 10 ⁶ IU / kg, ridostin 1.0 8.0 mg / kg, polyribonate 10.0 100.0 mg / kg.

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