RU2647455C1 - Composition having immunostimulating action for sublingual administration - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to pharmaceutical industry and medicine, namely to immunology, and is a composition having immunostimulating effect for sublingual use consisting of a double-stranded RNA bacteriophage F6 in amount of 0.5 ± 0.1 mg, a single-chain yeast RNA of 4.5 ± 0.5 mg, pectin in amount of 21 ± 3 mg, calcium lactate in amount of 15 ± 3 mg, dextran in the amount of 100 ± 10 mg, sodium chloride in amount of 9 ± 1 mg and water purified to 1 ml.

EFFECT: invention provides immunostimulating action, as well as the possibility of obtaining a stable composition.

1 cl, 2 tbl, 3 ex

Description

Technical field

The invention relates to biologically active additives for the prevention of human viral diseases and is intended for sublingual use during the period of threat of epidemics of viral infections and for diseases that lead to a weakening of immunity.

State of the art

A promising pharmacological protection of the human body consists in the use of such drugs, when under their influence any cell of the body performs its function assigned to it by nature. The individual characteristics of any organism are that the immune system perceives exogenous ribonucleic acid (RNA) as foreign, as an encroachment on the sovereignty (status) of the body's systems. As a result of this, the introduction of any form of RNA stimulates its protective factors and mobilizes forces to meet the enemy pathogen.

RNA-based drugs are increasingly used in the development of drugs for the prevention and treatment of topical infectious diseases. The presence of RNA in the pharmacological agent allows you to activate a whole range of cell defense mechanisms and expands the possibilities of immunoprophylaxis, especially in the case of high variability of pathogens: influenza viruses, human papillomas, HIV. Such drugs are interferon inducers based on double-stranded RNA of microbiological origin - ridostin, larifan, rifastin [1-4].

Ridostin is a high molecular weight inducer of interferon of natural origin, its active principle is double-stranded ribonucleic acid (dsRNA) of killer strains of Saccharomyces cerevisiae yeast.

Larifan, a high molecular weight inducer of interferon of natural origin, is a ds-RNA of phage f2 [4].

Rifastin, an inducer of interferon of natural origin, is a double-stranded RNA of bacteriophage ϕ6.

Preparations based on the interferon inducer, double-stranded RNA, are effective in the treatment of not only viral, but also other infectious diseases, as can be seen from table 1 [5].

Since the active principle has a physicochemical structure close to the structure of the genetic material of pathogenic viruses and bacteria, the introduction of a double-stranded RNA causes a powerful immune response in the body, including the induction of interferons, stimulation of the phagocytic activity of macrophages and peripheral blood neutrophils and other processes that counteract infections.

The objective of the invention was to develop the composition of a biologically active additive composition based on RNA, an immunity stimulant, for sublingual use.

Analogs and their disadvantages

The manufactured drug Ridostin [1-3], which is a mixture of sodium salts of double-stranded and single-stranded ribonucleic acid, is produced by the Russian pharmaceutical company Dia-Pharm LLC on the basis of a license agreement for the transfer of rights under RF Patent No. 2083221, IPC A61K 38/20, publ. . 07/10/1997 with the Federal State Institution Scientific Center of the World Bank "Vector" of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare

It is known that this drug is recommended for the prevention of influenza and acute respiratory infections, injection at a dose of 8 mg per person 1 time: before the disease or on the day the patient goes to the doctor, and in the treatment of influenza and acute respiratory infections, the drug is prescribed 8 mg per person 2 times: per day the patient’s visit to the doctor and after 2 days.

The disadvantages of the method: for the prevention and treatment of diseases caused by strains of the H5N1 subtype of avian influenza virus, this drug scheme does not give a reliable effect, especially in the treatment, and requires the development of a different scheme for the use of this interferon inducer in combination with other antiviral drugs.

Known patent 2398596, which describes a method for the prevention and treatment of diseases caused by strains of the H5N1 subtype of avian influenza virus using an interferon inducer and a neuraminidase inhibitor, comprising administering to the body an interferon inducer preparation or together an interferon inducer and an oseltamivir neuraminidase inhibitor according to the invention as an inducer interferon use the drug Ridostin, which is injected into the body during the prevention of diseases in the dose range from 0.05 to 0.5 mg / kg once a day and then 1 and 3 days after the first injection of Ridostin. In the treatment of diseases, Ridostin is injected into the body in a dose range from 0.05 to 0.5 mg / kg once a day per day of infection and then 1 and 3 days after infection and the neuraminidase inhibitor oseltamivir orally in a dose range from 0.2 up to 2 mg / kg 2 times a day for 5-7 days after infection with the H5N1 influenza virus.

The disadvantages of the method: the drug Ridostin must be injected several times, under the supervision of a medical professional, combining with the use of the drug oseltamivir (Tamiflu). This method significantly stimulates the immune system and is justified in case of illness or the threat of infection with especially dangerous infections. With the likelihood of contracting ARI or seasonal flu, such stimulation of the immune system is excessive.

Also known patent of the Russian Federation 2191594. A tool and method for increasing resistance to infection. IPC A61K 38/21, publ. October 27, 2002. The essence of the invention is that the preparation of interferon (IFN) is developed, for which sublingual granules containing low doses of the drug reaferon or realdiron or an IFN inhibitor (ridostin, larifan, half-dan, amixin, etc.) are used. The content of interferon inducers in sublingual granules is 0.01-5 mg in a granule without a clear dosage recommendation for each drug from the claimed group: amixin, cycloferon, neovir, ridostin, poludan, larifan, kagocel, as well as from the group: dibazole, papaverine, caffeine , chimes. Sublingual granules weighing 0.05 g are prepared on the basis of sucrose or lactose. For diabetics, the use of drops is possible.

This tool has the following disadvantages: sublingual granules contain an interferon inducer in small doses, for this reason, as indicated in the patent, even for the prevention of the disease, it is necessary to use up to 5 granules simultaneously and to achieve the effect, take them 1-3 times a day. There are no data on the effectiveness of sublingual granules containing an interferon inducer when used to treat a disease. In this case, the drug exhibits the claimed effect when used together with the drug alpha-2 interferon. In addition, there is no information about the therapeutic effect of the drug in relation to diseases caused by strains of the H5N1 subtype of avian influenza virus.

The disadvantages of the method: the method involves the use of recombinant human interferon α2β obtained by microbiological synthesis and special technology for its purification. It is known that with prolonged use of recombinant cytokines, the body begins to produce antibodies to them that neutralize the activity of the introduced proteins, and the doses of the latter have to be increased.

In addition, these drugs are drugs and require medical prescription in appropriate therapeutic doses, which does not allow their wide use for preventive and health purposes as dietary supplements

Promising as a prophylactic agent is the use of a sublingual form of RNA.

Disclosure of invention

The objective of the invention was to develop the composition of a biologically active supplement based on RNA, an immunity stimulator, for sublingual use, which, along with the use of the beneficial properties of RNA, would have a wider spectrum of effective action on the body, which would allow replenishing the range of nutritional supplements for general strengthening purposes.

The problem is solved by incorporating RNA into microcapsules formed from calcium lactate and pectin, on the basis of which a sublingual form of a biologically active additive is created to stimulate immunity.

Content of components: double-stranded RNA 0.5 ± 0.1 mg, single-stranded RNA 4.5 ± 0.5 mg, pectin and calcium lactate are used in amounts necessary for the formation of microcapsules. To dissolve pectin and calcium lactate, a solution of dextran with a molecular weight of 40,000 Da is used, which is used in medicine as a blood substitute (Reopoliglyukin).

Case Studies

1. The choice of the ratio of components for the formation of microcapsules

The ratios of the components: pectin and calcium lactate were selected so that uniform capsules formed. A number of ratios of the main components are presented in table 2.

The most homogeneous microcapsules were observed at a ratio of components, mg / g: pectin - 20, calcium lactate - 15, which were chosen for further studies. The drug was called Ricalcin.

In Fig. Figure 1 shows the appearance of microcapsules during microscopy (magnification 100, 10 × 10).

2. Obtaining biologically active additives based on RNA

2.1. Obtaining a biologically active additive based on yeast RNA

The tool is obtained in two stages: the preparation of solutions (A and B) and the formation of microcapsules when they are mixed.

To prepare solution A, a sample of ridostin (lyophilized porous mass according to FS 42-3862-99 [6, 7], containing a mixture of sodium salts of nucleic acids: 10% double-stranded RNA and 90% single-stranded RNA) with a mass of 5 mg is dissolved in 0.7 ml of solution reopoliglyukin (dextran with a molecular weight of 30000-40000 Yes in a 0.9% NaCl solution according to FSP 42-0088-7009-05,2006 [8]), then 20-22 mg of pectin are added and dissolved.

Solution B is prepared by dissolving a portion of calcium lactate weighing 15-17 mg in 0.3 ml of a solution of reopoliglyukin.

Solution B is quickly added to solution A with vigorous stirring and left for 10-15 minutes to form three-dimensional microcapsules.

2.2. Obtaining biologically active additives based on double-stranded bacteriophage RNA and single-stranded yeast RNA

To prepare solution A, a sample of rifastin containing dsRNA of bacteriophage ϕ6 (production of IMBT SSC WB Vector), weighing 0.5 mg, is dissolved in 0.7 ml of reopoliglukin solution (dextran with a molecular weight of 30000-40000 Da in a 0.9% NaCl solution according to FSP 42-0088-7009-05,2006 [8]), a portion of polyribonate is added and dissolved (preparation of high-polymer RNA from baker's yeast according to TU 9381-018-00479979-01 [9]) and then 20-22 mg are added and dissolved pectin.

Solution B is prepared by dissolving a portion of calcium lactate weighing 15-17 mg in 0.3 ml of a solution of reopoliglyukin.

Solution B is quickly added to solution A with vigorous stirring and left for 10-15 minutes to form three-dimensional microcapsules.

3. The study of the kinetics of RNA release from microcapsules in saline and hydrochloric acid at 37 ° C by measuring the optical density at 260 nm. When studying the kinetics of RNA release, the drug plates with the dimensions: 1.5 × 1 × 0.25 cm were placed in boxes, 5 ml of physiological saline (or 0.5% hydrochloric acid) was added. At certain intervals, the optical density was measured at a wavelength of 260 nm of the solution (supernatant fraction). Based on the measurement results, we plotted the dependence of optical density in optical units on time in minutes.

The maximum concentration of RNA during diffusion into saline was recorded after 1.5-2.5 hours, it was 3.8 -4 mg of RNA (75-80% of the initial content), remaining at this level for 12 hours. At the same time, the appearance of the microcapsules during microscopy did not significantly change.

During diffusion into a solution of 0.5% hydrochloric acid, the maximum concentration of RNA was recorded after 2 hours, the concentration value was equal to 1.5-1.8 mg, then the RNA content gradually decreases to 0 over the next 3 hours. The microcapsules were deformed.

4. Assessment of drug stability

The stability of the preparations was evaluated by electrophoresis in a 1% agarose gel with ethidium bromide staining.

An analysis by agarose electrophoresis in a 1% gel showed that the mobility of RNA in the preparation was reduced compared to the mobility of the original (RNA substance), but its structure was preserved (Fig. 1). A similar conservation of RNA was established after storage of the drug for 90 days in a refrigerator at 6 ° C.

In Fig. Figure 2 shows the Electrophoregram (1% agarose gel, staining with ethidium bromide) of RNA samples diffused into the solution after 2, 4 and 8 hours from microcapsules. And - the original (RNA substance).

On the left is a yeast-based RNA preparation, on the right is a double-stranded bacteriophage RNA.

5. Determination of immunostimulating properties of drugs

To determine the phagocytic activity, two-month-old Syrian (golden) hamsters with a body weight of 90-110 g were used, 5 goals in each group obtained from the nursery. To do this, they were fed 0.2 g of the drug. After 6 and 24 hours, blood was taken for analysis and macrophages were isolated. As an object of phagocytosis, carcass particles were used. Phagocytic activity was determined by the phagocytic index (PI) in a conventional manner [10].

The biological activity of Ricalcin samples was evaluated on hamsters by its ability to increase the phagocytic activity of macrophages, determining the phagocytic index.

In Fig. 3 shows the phagocytic index of macrophages after administration of the drug Ricalcin.

Differences with control (capsules not containing RNA) were significant at P <0.05.

As can be seen from the graph (Fig. 3), an increase in activity was observed 6 hours after drug administration, the stimulation effect intensified after 24 hours. These data are consistent with the results of the authors of [11], who used the introduction of the preparation of virus-like particles from yeast containing RNA.

The result achieved by using the invention consists in the selection of ingredients, which allows, on the one hand, to maintain and stimulate the body's immune system, due to the presence of RNA in the composition. On the other hand, the proposed dietary supplement will provide the body with trace elements necessary for its normal functioning and dietary fiber, which have a regulatory effect on metabolism.

Also, the sublingual method of administration should be attributed to the advantages of dietary supplements: in this case, the components will not be subjected to the degrading effects of gastric juice and digestive enzymes.

The manufacturing process is attractive due to the lack of technically complex techniques and consists of the following simple stages: dissolution, mixing, molding.

The usefulness of dietary supplements and the lack of technical complex solutions in the manufacture, can contribute to the development of a wide industrial release of the drug.

The drug, obtained in the form of plates, is used sublingually as a dietary supplement. Recommended prophylactic dose on one plate daily in case of threat of possible infection.

Information Sources Used

1. Interferonogens: prospects for clinical use. A guide for doctors. Edited by M.G. Romantsova. NTFF "Polisan", Moscow - St. Petersburg, 1998.

2. FS 42-3862-99 was approved on January 20, 2000 “RIDOSTIN - an antiviral drug, immunostimulant, interferon inducer”.

3. FSP 42-0197-06-16-00 approved December 6, 2000 "RIDOSTIN-OINTMENT - antiviral drug, interferon inducer."

4. What is Larifan. URL http://www.larifans.lv/

5. The use of ridostine for the treatment of viral and bacterial infections and the prospects for its use in the treatment of diseases of non-infectious nature. Sat round-table materials scientific. conf. / Ministry of Health of the Russian Federation. Sib. Department of RAMS. SSC WB "Vector". NIKTI BAS SSC WB "Vector". Interregion. Siberian Healthcare Association, - Berdsk, 1998. - 81 p.

6. FS 42-3862-99, approved on January 20, 2000.

7. FSP 42-0197-5044-04 (lyophilisate) / LS-000381 from 08/03/2010.

8. FSP 42-0088-7009-05,2006, Reopoliglyukin.

9. TU 9381-018-00479979-01, Polyribonate.

10. Stenko M.I. Handbook of clinical research methods. - M., 1975 .-- S. 56-57.

11. Shevchenko Z.A., Lebedev L.R., Klimenko V.P. et al. Production methods and properties of virus-like particles of Saccharomyces cerevisiae yeast killer strain / Applied Biochemistry and Microbiology. 2015. T. 51. No. 8. C. 812-817.

Claims (4)

A composition having an immunostimulating effect for sublingual administration, consisting of double-stranded RNA of bacteriophage F6, single-stranded yeast RNA, pectin, calcium lactate, dextran, sodium chloride and purified water, in the following ratio of components: double-stranded RNA bacteriophage F6 0.5 ± 0.1 mg single stranded yeast RNA 4.5 ± 0.5 mg pectin 21 ± 3 mg calcium lactate 15 ± 3 mg dextran 100 ± 10 mg sodium chloride 9 ± 1 mg purified water up to 1 ml

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