Anti-enteroviral and immunostimulating agent

FIELD: medicine.

SUBSTANCE: invention represents an oral anti-enteroviral and immunostimulant agent in the form of capsules containing interferon and additives, differing by the fact that a therapeutic substance is human recombinant interferon alpha-2b immobilised on polyethylene glycol, having a molecular weight of 1.5 kD by a physical method of binding by an accelerated electron flow in a dose of 1.5 Mrad. The ingredients in the agent are taken in a certain ratio.

EFFECT: extending the range of anti-enteroviral agents possessing immunostimulant properties.

6 ex, 10 tbl

 

The invention relates to medicine, specifically to pharmacology, and concerns of suppressing the replication of enteroviruses and stimulating the immune system.

In recent years, the world has been a clear trend towards intensification of enterovirus infection, as evidenced by continuously registered in different countries epidemiological rises in the incidence and outbreaks. Enterovirus infection remains malacostracans in the practice of healthcare and is one of the leading infectious diseases that occur with CNS. One of the main features of these infections is a virus, constantly causing sporadic forms and mass diseases, which, like disease, is observed not only among younger and older age, but also among adults.

As specific therapy of enterovirus infection used: leukocyte interferon, recombinant interferon (Viferon, Reaferon, Roferon), interferonogennoy (cycloferon, neovir), immunoglobulin for intravenous administration (sandoglobulin, Pentaglobin).

Along with the high clinical efficacy of interferon (IFN) in their application frequently observed side effects in the form of flu-like syndrome, arthralgia, depression, diarrhea, hallucinations,�boards rash, allergies, diseases of the hematopoietic system, hair loss [1, 2]. In 30% of patients with long-term use of high doses of recombinant interferons were formed, neutralizing the antiviral activity of the drug antibody, which was accompanied by development of resistance to recombinant interferon [3].

Therefore, the urgent task is to develop new approaches to the treatment of viral, and, in particular, enterovirus infections to reduce side effects. One such approach is the immobilization of interferon on the substance-the matrix that allows to reduce the dose and frequency of drug administration, protects tissues from their irritant action. Became known method of attaching molecules of polyethylene glycol (PEG) to interferon by chemical means (tahilramani). At the present time all over the world, including in Russia, we use two types of pegylated interferon:

1. Pegylated interferon Alfa-2b (PegIntron, Schering-Plough, USA).

2. Pegylated interferon Alfa-2A (Pegasys, Roche, Switzerland).

Both pegylated interferon (patent No. 5951974 from USA 14.09.1999 "Interferon polymer conjugates - drug" PegIntron" and patent number EP 0809996 B1 dated 02.04.2003 "Interferon-alpha polypeptides and conjugates - drug" Pegasys") are used for the treatment of chronic hepatitis C. However, the use of pagalilauan�x interferons has several disadvantages: parenteral route of administration of drugs; side effects in form of fever, weakness, headaches, arthralgia, and others; the inability to accurately control the concentration of interferon in the blood because the drug is administered once a week. That fact is important that the cost of medication is very high. In addition, the production of pegylated multi-stage drugs that are used in some stages of toxic substances, total removal of which entails a decrease in the activity of the drug, resulting in increased dosage.

However, the known technology of immobilization of proteins by ionizing radiation, the so-called physical method (patent RU №2409669, "Method of immobilization of biologically active substances (BAS) on the carrier (variants) and conjugate BAS-carrier, the received data methods", publ. 27.02.2010). According to this method, as biologically active substances used substance selected from the group: insulin, proinsulin, G-CSF, hyaluronidase, tyrosyl-2-alanyl-glycyl-phenylalanyl-leucyl-arginine diacetate, somatotropin.

Physical binding of recombinant interferon with substance-matrix will protect the protein molecule interferon from the action of proteolytic enzymes, thus opening the possibility of oral administration without loss of pharmacological activity and therapeutic efficacy.

Task PR�Gagaemauga of the invention is the development of a drug interferon for the treatment of enteroviral infections of the oral method of application. The use of the drug will allow to: 1) address to deliver immobilizovannyi interferon directly to virusinfection cells of the intestinal mucosa; 2) to influence the cells of lymphoid tissue (macrophages, neutrophils) associated with the intestinal mucosa; 3) reduce the dosage of interferon in a single of its introduction; 4) to eliminate the side effects typical for parenteral administration of the interferons, which will provide the possibility of using interferon not only in adults but also in pediatric and obstetric practice.

The task is achieved by the use of immobilized interferon Alfa-2b as protivoavariynogo and immunostimulating agent, obtained by mixing irradiated by a stream of accelerated electrons of an aqueous solution of polyethylene glycol (PEG), molecular weight of 1.5 kDa and a solution of interferon alpha-2b to a final concentration of 10 mg in 1 ml. the Mixture is stirred to obtain opalescense solution.

The use of polyethylene glycol as the base helps to protect the protein molecule interferon from the action of proteolytic enzymes and thereby provides the possibility of oral administration. The product yield is 98%, the activity of the drug is not less than 1×106international single�C (IU)/ml, molecular weight of not less than 19,5±0,5 kDa. Dosage form immobilized interferon Alfa-2b, a hard gelatin capsule with a lid, size "00".

Composition of 1 capsule:

Active substance:

interferon Alfa-2b human recombinant immobilizovannyi - 1000000 IU/caps.

Auxiliary substances:

Maltodextrin75-90%0.3 to 0.36 g
Dextran8,98-22,45%0,03592-0,0898 g
Polyethylene glycol1-2,5%0.004-0.01 g
EDTA0,02% to -0.05%0,08-0,2 mg

New in the present invention is that immobilizovannyi on PEG interferon Alfa-2b was first proposed for use as protivoavariynogo and immunostimulating agent for oral administration.

From domestic the interferons used inside the only the drug is "Reaferon-EU-lient" (prototype) (JSC "Vector-Medica", Novosibirsk) is a liposomal preparation of interferon alpha-2b (patent RU №2123328, "Liposomal antiviral drug� remedy for oral use", publ. 20.12.1998). Specified liposomal drug has insufficient shelf life (activity of interferon Alfa-2 is almost at a constant level is maintained up to 12 months) and its application there are a number of side effects: flu-like condition; nausea, vomiting, diarrhea; violation of the blood picture and liver function; dizziness, drowsiness; decreased or increased blood pressure, arrhythmia.

Similar action has drug "Viferon" (LLC "Feron", Moscow) - suppositories, which in addition to recombinant interferon Alfa-2b include vitamins E and C (patent RU №2024253, "Rectal candles and a device for introducing rectal suppository into the cavity of the body" publ. 15.12.1994). Rectal use "Viferon" contributes to a longer circulation of interferon in the blood than by intravenous or intramuscular injection of recombinant interferons. However, the application of rectal suppository uncomfortable, unhygienic, interferon in candles Viferon" contains in small dosages, the use of candles Viferon" may cause local irritation when using the drug in young children.

The use of immobilized on PEG interferon became possible with the discovery of his ability to suppress the replication of enteroviruses different strain�in vitro. Revealed the presence of immunotropic properties, namely the stimulation of humoral immune response and phagocytic reactions when administered orally to laboratory animals, as well as increased spontaneous and stimulated production of interleukin 2 (IL-2) and IFN-γ without additional stimulation by mitogen adding immobilized interferon in vitro. The use of immobilized on PEG interferon for oral administration as protivoavariynogo and immunostimulating agent is described in the literature and are not explicitly derived from the prior art for the specialist. Immobilizovannyi interferon can be used for patients with enterovirus infection, including those associated with immune deficiency.

Thus, this solution meets the criteria of the invention: "novelty", "inventive step", "industrial applicability".

Antiviral properties immobilized on PEG interferon Alfa-2b were found through experimental studies. Conducted a study of the antiviral activity of the drug on the basis of immobilized interferon Alfa-2b in respect of Coxsackievirus A7 (strain JEV-8) and Coxsackievirus B6 (strain JEV-15) on the culture of RD cells (human rhabdomyosarcoma). Cell culture D is an adequate model to study antiviral activity of interferon [9]. To study antiviral activity was implemented with preincubate of RD cells with various concentrations of recombinant interferon Alfa-2b and study medication based immobilized interferon Alfa-2b (both manufactured by JSC "Siberian center for pharmacology and biotechnology, Novosibirsk, Russia). Dilutions of interferon in the culture medium Needle-DMEM was added to cells cultured in standard 96-well culture microplates in the 6 hours prior to infection [9].

Results confirming antiviral properties immobilized on the PEG of recombinant interferon Alfa-2b.

Example 1. Adding recombinant interferon Alfa-2b and study medication based immobilized interferon Alfa-2b in vitro investigational drugs did not exert toxic effects on the cell culture RD across the range of concentrations used (dilution 1:30-1:93750). Infection was carried out by a dose of 100 TCD50the Coxsackie virus A7. The results are presented in table 1 shows the percentage of viable cells after 30 hours after infection).

In the virus controls (to the cells instead of the test drug was added an equal volume of culture medium) by this date n�has been a 100% virusinduced destruction of the cell monolayer. As can be seen from table 1, the addition in vitro as recombinant human IFN alpha-2b and medicines on the basis of immobilized IFN Alfa-2b at a concentration of 2000 IU/ml and above in the culture of RD cells infected with Coxsackievirus A7 (strain JEV-8), resulted in 100% protection from virus-induced death.

Example 2. Infection of RD cells was performed by dose of 100 TCD50of Coxsackievirus B6 (strain JEV-15). To the culture of the RD cells were added various concentrations of recombinant interferon Alfa-2b and study medication based immobilized interferon Alfa-2b for 6 h before infection. The results of the study are presented in table 2 shows the percentage of viable cells after 30 hours after infection). In the virus controls (to the cells instead of the test drug was added an equal volume of culture medium) by this date was observed 100% virusinduced destruction of the cell monolayer.

As can be seen from table 2, the addition of in vitro as recombinant human IFN alpha-2b and medicines on the basis of immobilized IFN Alfa-2b at a concentration of 2000 IU/ml and above in the culture of RD cells infected with Coxsackievirus B6 (strain JEV-15), resulted in 100% protection from virus-induced death. Significant differences of protivovirusny�th activity of the drug on the basis of immobilized interferon alpha-2b against two used enterovirus strains are not noted.

The effectiveness of suppressing the replication of Coxsackievirus A7 (strain JEV-8) of the investigated drug on the basis of immobilized interferon Alfa-2b in infected RD cells was also investigated by quantitative analysis using polymerase chain reaction with reverse transcription real-time (RT-PCR). The monolayer of RD cells with different concentrations of the drug on the basis of immobilized interferon Alfa-2b were incubated for 6 hours, after which cells were infected with a dose of 100 TCD50of Coxsackievirus A7, 30 hours after infection in cell lysates was determined by the number of viral RNA. Quantitative RT-PCR analysis was performed according to the methodology, J. Lu, L. Yi, J. Zhao et al. (2012). The amount of viral RNA in cells, preincubating with interferon, normalized relative to the control virus (KB, the virus-infected RD cells without preincubation with interferon). The results are presented in table 3.

Thus, the antiviral activity of the drug on the basis of immobilized interferon Alfa-2b have shown that this drug has comparable specific activity source neemalirovannym recombinant human interferon Alfa-2b(production of JSC "SCFB"). Thus, the method of quantitative RT-PCR, it was shown that drug-based immobilized interferon Alfa-2b is approximately 5 times less active in comparison with neemalirovannym interferon Alfa-2b. These data are in good agreement with the results for pegylated proteins (cytokines) in in vitro models[10, 11, 12].

Immunotropic properties immobilized on PEG interferon were found through experimental studies.

To establish the immunotropic activity of immobilized recombinant interferon Alfa-2b (Imin-α2b) was studied its influence on the phagocytosis of peritoneal macrophages and neutrophils, the humoral immune response of laboratory animals after oral method of use of an investigational drug. Evaluation of immunotropic activity of the studied drug were also carried out in vitro on the culture of peripheral blood mononuclear cells of healthy donors. It was investigated the influence of Imin-α2b on the synthesis of IL-1β, IL-2, IL-4 and IFN-γ by peripheral blood mononuclear cells of healthy donors spontaneously and stimulate polysaccharide of enterobacteria and phytohemagglutinin.

As the comparison drug used Reaferon-EU-lient (JSC "Vector-Medica p. Koltsov, Novosibirsk reg., reg. beats PN000821/01). �the drug is recombinant interferon Alfa-2 for oral use, enclosed in liposomes that interferon protects from destruction in the digestive tract. As a comparison drug in vitro, when added to a culture of mononuclear cells of healthy donors was used instead reaferona-EU-Lipina its analogue for injecting Reaferon-EU (JSC "Vector-Medica p. Koltsov, Novosibirsk region, per. beats PN000642/01).

Study of the immunotropic activity immobilized on PEG interferon Alfa-2b (Imin-α2b) and reaferona-EU-lient (CPAR-EU-l) was carried out according to Methodical recommendations on the preclinical study of the immunotropic activity of drugs" [13].

The study used 160 mice-males line CBA/CaLac 6-8 weeks of age with body weight of 18-22 g. conventional Mouse 1st category (certificate health of laboratory animals, issued by the "NBMT" RAMS from 17.10.12). Content: in accordance with rules adopted by the European Convention for the protection of vertebrate animals (Strasbourg, 1986). Mice were according to the norms of Seating animals in plastic cages firms VELAZ on the bed of small wood chips. The temperature in the vivarium 18-24°C, humidity 50±20%, the volume of air (exhaust/inflow) - 8:10, light regime (day/night) - 1:1. Mice had continuous access to water and food. Food - complete extruded feed �ecept PC-120 (content) for laboratory animals (mice, rats, hamsters), balanced amino acid composition, mineral substances and vitamins, production of LLC "Laboratorium", Moscow. As drinking water distilled water was used. Work with animals was conducted in accordance with the regulations on the keeping of animals: "Laboratory animals". - M., 2003 [14]. The content of animals comply with good laboratory practices (GLP) and the Order of the MOH of the Russian Federation No. 708 dated 23.08.2010 "On approval of rules of laboratory practice".

In experiments in vitro as the source material used was the peripheral blood of 10 healthy donors ranging in age from 22 to 36 years.

Mice immobilizovannyi interferon Alfa-2b and IFN-EU-lient administered orally the course within 5 days once a day therapeutic dose of 1.8×105IU/kg), which was recalculated based on the average therapeutic dose of interferon Alfa-2b (1000000 ME), in a volume of 0.2 ml of phosphate-buffered saline (PBS) (MP Biomedicals, USA), control animals received the same volume of solvent. In experiments in vitro immobilizovannyi interferon Alfa-2b and IFN-EU was added to the cell culture at doses of 15 IU/ml, 150 IU/ml, 1500 IU/ml. the effect of drugs on the phagocytic activity of peritoneal macrophages and neutrophils in the background required�whether intact animals of the corresponding sex and age. When studying the effect of drugs on the humoral immune response as the background used mice that received the antigen, but of course without the solvent. Scored animals by decapitation after SB2camera either by an overdose of chloroform.

Phagocytic activity of peritoneal macrophages and neutrophils was assessed after 24 h after 5-day course introducing immobilized interferon Alfa-2b and reaferona-EU-lient on the ability of phagocytes to engulf the daily culture of Staph. aureus, strain 209. On smears the contents of the peritoneal cavity was taken into account the percentage of macrophages or neutrophils engulfing microbes (phagocytosis index - FI) and the average number of staphylococci absorbed by one cell (phagocytic number - FC) [13].

The influence of immobilized interferon Alfa-2b and reaferona-EU-lient on humoral immune response was assessed by determining the number of antibody-forming cells (ASC) in the spleen and antibody titers in serum after immunization of mice with sheep red blood cells (ZAO Ecolab, Russia). Immunization of the animals was performed after a 5-day course introducing immobilized interferon Alfa-2b and reaferona-EU-lient minimum dose of sheep erythrocytes to 5×106the mice intraperitoneally. The definition of an ASC was carried out on 4th and 7th day after immuni�ation by the method of local hemolysis [15] with a preliminary estimate of the total number of splenocytes (ACS), and the level of specific antibodies (IgM and IgG haemagglutinins) contained in the serum of immunized animals was determined using the reaction of haemagglutination (DSA) [16]. The number of antibody-forming cells were expressed in relative (%) and absolute (106/organ) units, and the titer of antibody - value of log2T.

In-vitro experiments studied the effect of immobilized interferon Alfa-2b and reaferona-EU when they are added to the culture of monocular peripheral blood of healthy donors on the production of IL-1β, IL-2, IL-4 and IFN-γ. To do this, the quality of the material used the supernatants of cultures of mononuclear cells obtained on the first day of their cultivation in vitro at 37°C in a CO2incubator containing 5% CO2. For the isolation of mononuclear cells blood was diluted 1:2. medium 199 (FSIS SRC VB "Vector", Russia), then layered on a gradient of ficoll-Pak ("Pharmacia", Sweden) with a density of 1,077 and centrifuged at 1500 rpm for 40 min. White ring formed at the section of phases and containing mononuclear cells, gently aspirated by pipette and 2 times washed with medium 199 with centrifugation for 10 min at 1000 Rev/min the Precipitate was resuspended in complete growth medium (PRS), consisting of medium RPMI-1640 ("HyClone", USA), 10% fetal calf serum ("HyClone", USA), 10 mm HEPES (Sigma, USA), 2 mm L-glutamine ("Sigma", USA) and 40 µg/ml gentam�Qing (JSC "dalkhimfarm, Russia), and mononuclear cells was adjusted to a concentration of 2 million/ml and were cultured day. Received 4 types of supernatant: 1) from cells stimulated with lipopolysaccharide (LPS, 10 μg/ml) (Sigma, USA) for induction of production of IL-1β, or phytohemagglutinin (PHA, 10 μg/ml) (Sigma, USA) for induction of production of IL-2, IL-4 and IFN-γ in the presence of the test preparations at a final concentration of 15 IU/ml, 150 IU/ml, 1500 IU/ml; 2) from cells stimulated with LPS or PHA; 3) from cells cultivated in the presence of the test preparations at a final concentration of 15 IU/ml, 150 IU/ml, 1500 IU/ml; 4) from cells cultivated only in DRC. Cytokines (IL-1β, IL-2, IL-4 and IFN-γ) in the supernatants of cultures of mononuclear cells was determined using enzyme immunoassay method using the corresponding sets of production of CJSC "Vector-best" (Novosibirsk) and LLC "Protein contour" (St. Petersburg). Evaluated the ability of investigational drugs to enhance (mononuclear cells stimulated with LPS or PHA + drug) and to induce (mononuclear cells stimulated only with medication) production of cytokines.

Statistical processing of results was carried out using statistical software package Statistica for Windows (version 5.0) with a preliminary estimate of the normal distribution and the t-student criterion.

Results confirming the immunotropic properties�and immobilized on the PEG of recombinant interferon Alfa-2b, the oral method of application, illustrated by the following examples.

Example 3. The study of the phagocytic activity of peritoneal macrophages and neutrophils was assessed after 24 h after 5-day course introducing immobilized interferon Alfa-2b (group 4), reaferona-EU-lient (group 3) or solvent (group 2) by the ability of phagocytes to engulf the daily culture of Staph. aureus, strain 209. Course introduction CPAR-EU-l did not lead to significant changes in the phagocytic index and phagocytic number of peritoneal macrophages of experimental animals compared with the control group, only relative to the background was observed a statistically significant increase in the phagocytic activity of macrophages (FC) (PL. 4).

Course introduction Imin-α2b did not lead to significant changes in the phagocytic index of peritoneal macrophages of experimental animals as compared with control group and with the background, but statistically significantly increased the average number of absorbed single bacteria cell (FC). In the mice that received Imin-α2b, showed a significant increase of phagocytic number of peritoneal macrophages compared with the group of animals with the use of CPAR-EU-L.

Course introduction CPAR-EU-l did not lead to a significant change�m phagocytic index and phagocytic number of neutrophils in experimental animals compared with the control group, only relative to the background was observed a statistically significant increase in the number of absorbed bacteria (FC) (PL. 5). Course introduction Imin-α2b resulted in a significant relative increase in the number of phagocytic neutrophils in experimental animals as compared with control group and background. The number of absorbed bacteria (FC) were not significantly altered. In the mice that received Imin-α2b, there was a significant relative increase in the number of phagocytic neutrophils in comparison with the group of animals with the use of CPAR-EU-l in the same dose.

Example 4. The influence of immobilized IFN Alfa-2b and reaferona-EU-lient on humoral immune response was assessed by determining the number of antibody-forming cells (ASC) in the spleen and antibody titers in serum after immunization of mice with sheep erythrocytes (tab. 6). Course introduction CPAR-EU-l resulted in a significant increase in the number of splenocytes, relative and absolute number of ASC in the spleen, the titer of IgM in serum for 4 days after immunization, but only relative to background values, as compared with the control group there was a statistically significant decrease in the relative numbers of ASC and IgG titers, although the titer of IgM was significantly higher.

Course introduction Imin-α2b resulted in a significant increase in the number of splenocytes, relative and absolute number of ASC in the spleen of experimental animals at 4 days after immunization as compared with the control group, and with a background, but the activity of the ASC, judging by the titles of specific hemagglutinin in the serum, increased slightly. In the mice that received Imin-α2b, showed a statistically significant increase in absolute and relative numbers of antibody-forming cells, titers of IgG and total antibodies in comparison with the group of animals with the use of CPAR-EU-L.

Course introduction CPAR-EU-l resulted in a significant increase in the number of splenocytes and the absolute number of ASC in the spleen as compared to control and group with background (PL. 7), and on this group increased percentage content of antibody productive cells and the titer of IgG in the serum at 7 days after immunization.

Course introduction Imin-α2b resulted in a significant increase in the number of splenocytes, the absolute number of ASC in the spleen of experimental animals and the titer of specific hemagglutinin in the serum at 7 days after immunization as compared to control group and background, and on this g�uppy increased titer of IgG. The studied parameters in the group of mice that received Imin-α2b, did not differ significantly compared to the group of animals with the use of CPAR-EU-L.

Example 5. The influence of immobilized interferon Alfa-2b and reaferona-EU when they are added to the culture of peripheral blood mononuclear cells of healthy donors on the production of IL-1β, IL-2, IL-4 and IFN-γ. To do this, the quality of the material used the supernatants of cultures of mononuclear cells obtained on the first day of their cultivation in vitro.

When you add in the culture of mononuclear cells of Imin-α2b and CPAR-EU in various concentrations (15 IU/ml, 150 IU/ml, 1500 IU/ml) spontaneous and induced production of IL-1β and IL-4 did not change significantly compared with the control group (without addition of drug) (table. 8, 9). It was not observed statistically significant differences in the development of these cytokines when comparing appropriate doses for insertion of drugs groups.

By the addition in culture of mononuclear cells of Imin-α2b and CPAR-EU increased production of IL-2 in all studied doses (15 IU/ml, 150 IU/ml, 1500 IU/ml) compared with the control. When you make Imin-α2b at a dose of 150 IU/ml was observed a significant enhancement of spontaneous production of IFN-γ. Add CPAR-EU in the culture of mononuclear cells in doses of 15 IU/ml and 150 IU/ml without mitogen statistically Zn�chimo increased production of the studied cytokine as compared to the control, and at a dose of 15 IU/ml group with the introduction of Imin-α2b at the same concentration.

When you add in the culture of mononuclear cells of Imin-α2b in conjunction with PHA increased production of IL-2 in all studied doses of the drug (15 IU/ml, 150 IU/ml, 1500 IU/ml) compared to control (table. 9). Maximum production of the studied cytokine was observed in making them-IFN-α2b at a concentration of 15 IU/ml. adding CPAR EU-statistically significant increased production of IL-2 was observed only at the dose of insertion of the drug 150 IU/ml When comparing appropriate doses of the drugs were observed statistically significant differences in the development of the investigated cytokine.

When you add in the culture of mononuclear cells of Imin-α2b and CPAR-EU together with PHA increased production of IFN-γ was observed at the insertion-dose 15 IU/ml and 1500 IU/ml, but statistically insignificant.

Thus, using the proposed method physical immobilization of recombinant interferon Alfa-2b on the polyethylene glycol, the weight of 1.5 kDa, allows to obtain a preparation for oral administration, which manifests protivoavariynoy activity in vitro and a significant immunostimulating activity in vivo, resulting in increased phagocytic activity of peritoneal macrophages and neutrophils and humoral and�Moonen answer comparable or exceeding those under the influence of reaferona-EU-lient, and in vitro, resulting in increased spontaneous and stimulated production of IL-2, is more effective than the reference drug CPAR-EU and IFN-γ without additional stimulation with mitogen.

Example 6. Production of encapsulated drugs. Preparation of the composition for freeze drying:

The composition for freeze drying are shown in table 10.

Table 10
The composition of the components of the composition for freeze dryers
The name of the componentNumber
1. Interferon Alfa-2b human recombinant immobilizovannyi 2×107IU/ml60,0 ml
2. Dextran-4050,00 g
3. Phosphate buffered saline (10 mM NaH2PO4, pH 7.4±0,2)450,0 ml
3. EDTA1.00 g
4. PEG-15002.00 g

A suspension of dextran-40 50,00±0.01 g was dissolved in 450.0 ml of standard phosphate buffered saline� (10 mM NaH 2PO4, pH 7,4±0,2). On an electronic balance "Ohaus Adventurer RV 313" in a beaker of 1000 ml was weighed 449,0±0.1 g of a solution of dextran-40, 10% in phosphate-buffered saline and dissolved in the sample substances are 1.00±0.01 g of EDTA and 2.00±0.01 g of PEG-1500. To the resulting solution is added to 60.0 ml of a solution of the substance immobilized interferon alpha-2b with the activity of 2×107IU/ml and stirred. The resulting solution was filtered through a 0.2 μm filter using vacuum sterilization system "Stericup" Millipore to reduce contamination of the product was poured into sterile trays for drying and subjected to a deep freeze at -70°C in hibernator "UTLF-80" during the day.

Freeze drying the composition to dry:

Freeze drying the frozen composition for drying was performed on laboratory freeze dryer "MLW LGA 05" at a temperature of -50°C and a pressure in the drying chamber is not more than 0.4 mm Hg.PT. during the day. Dry the extract is poured into a plastic bag.

Preparation of medicinal mixtures for filling capsules:

Sample lyophilizate compositions for drying (45,0±0.1 g) and maltodextrin (of 405, 0±0.1 g) was transferred into the mixing vessel and the resulting mixture was stirred on a combine for 30 minutes to obtain a homogeneous, evenly mixed and crumbly mixture.

Encapsulation and packaging of medical CME�and:

Encapsulation of drug mixture by weight 420,0±0.1 g in a gelatin capsule No. 00 yellow (weight of the capsule 0.4 g) was carried out on semi-automatic machine for filling capsules "CGN-208" according to technological instructions. For cleaning capsules from dust used machine for polishing and dust removing capsules "S&P-100". Dust-free capsules (950 PCs) Packed in transparent plastic bags on the machine for counting and packaging of capsules RA" and hermetically sealed.

Sources of literature

1. Patten S. B. What is the best approach to treating interferon-induced depression in people with multiple sclerosis? // J. Neurosci. - 2001. - V. 26. - P. 66.

2. Wills R. J. Clinical pharmacokinetics of interferons // Clin. Pharmacokinet. - 1990. - Vol. 19. - No. 5. - P. 390-399.

3. Prummer via O., Streichan U., Porzsolt F. Treatment-induced interferon (IFN)-α antibodies: Differential neutralisation of the antiviral and the antiproliferative IFN-α activity in vitro // J. Interferon Res. - 1991. - No. 11. - P. 265.

4. US Pat. No. 5951974 A "Interferon polymer conjugates", publ. 14.09.1999.

5. EP Pat. No. 0809996 B1 "Interferon-alpha polypeptides and conjugates", publ. 02.04.2003.

6. The patent (RU) for the invention №2409669 "Method of immobilization of biologically active substances (BAS) on the carrier (variants) and conjugate BAS-carrier, the received data methods", publ. 27.02.2010.

7. The patent (RU) for the invention №2123328 "Liposomal antiviral drug for oral use", publ. 20.12.1998.

8. The patent (RU) No. 2024253 "Rectal candles and a device for introducing rectal �eternal in the cavity of the body" publ. 15.12.1994.

9. Lu J., Yi L., Zhao J., Yu J., Chen Y., Lin M. C., Kung H. F., M. L. Hea Enterovirus 71 Disrupts Interferon Signaling by Reducing the Level of Interferon Receptor 1 // Journal of Virology. - 2012. - V. 86, No. 7. - P. 3767-3776.

10. Pasut G. a PEGylated Interferons: two different strategies to achieve increased effi cacy. In: PEGylated protein drugs: basic science and clinical applications (ed. Veronese FM) // Birkhauser Verlag, Basel, 2009.

11. Pasut G., Vemose F. PEGylation of proteins as tailored chemistry for optimized bioconju-gates // Adv Polym Sci. - 2006. -Vol. 192. - P. 95-134.

12. Veronese F. M. G. Pasut Protein PEGylation In: Long Acting Injections and Implants (Eds: J. C. Wright, D. J. Burgess) // Springer, New York, Dordrecht, Heidelberg, London, in 2012. - P. 295-314.

13. Guidance on the preclinical testing of medicines. Part one. - M. Cole, 2013. - 944 p

14. Laboratory animals (Regulation and management) // under the editorship of Prof. interviewer RAMS I. N. Karkishchenko. - M.: publishing house "military industrial complex". - 2003. - 138 p.

15. Cunningham A. I. A method of increased sensitivity for detecting single antibody-forming cells // Nature. - 1965. - V. 207, No. 5001. - P. 1106-1107.

16. Ling N. R., Cathy D. Hemagglutinate and reactions antibody-dependent hemolysis. Antibodies. Methods / ed. by D. catty. kN. 1. M.: Mir, 1991. Pp. 238-243.

Protivokariosnoe and immunostimulatory agent for oral administration in capsule form containing interferon and excipients, characterized in that the medicinal substance contains interferon Alfa-2b human recombinant, immobilizovannyi on the polyethylene glycol molecular weight of 1.5 kDa by physical binding thread� accelerated electrons at a dose of 1.5 mrad in the following ratio of ingredients per capsule:

interferon Alfa-2b human
recombinant immobilizovannyi1000000 ME
auxiliary substances:
maltodextrin0.3 to 0.36 g
dextran0,03592-0,0898 g
polyethylene glycol0.004-0.01 g
EDTA0,08-0,2 mg



 

Same patents:

FIELD: medicine.

SUBSTANCE: immunomodulatory agent contains 3-O-propionate allobetulenole (19beta,28-epoxy-18alpha-oleanane-3beta-yl and propionate) as an active substance. The immunomodulatory agent stimulates a humoral immune response. The T-cell immune response develops in a delayed-type hypersensitivity test of the immunomodulatory agent of 3-O-propionate allobetulenole (19beta,28-epoxy-18alpha-oleanane-3beta-yl and propionate).

EFFECT: reduced oedema.

1 dwg, 2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: drops possessing antiviral and immunomodulatory effects characterised by the fact that they represent a 95% ethanol infusion of wild strawberry leaves and fruit specified in: red raspberry fruit, mountain ash fruit, bilberry fruit, blood-red hawthorn fruit, cinnamon rose fruit; 15-25 mg of the substance in 1 ml of the infusion.

EFFECT: drops possess pronounced antiviral and immunomodulatory effects.

15 tbl, 5 ex

FIELD: biotechnology.

SUBSTANCE: increase in biocidal and immunobiological action due to the use of distilled water ionised with silver ions and adding into the composition of succinic, ascorbic, citric acid and aethonium.

EFFECT: increase in biocidal immunobiological properties of the antiseptic-stimulant of Dorogov ASD-2F.

6 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: method includes carrying out complex treatment at the background of diet therapy. Intake of antihelmintic of vegetable origin and immunomodulator is carried out daily with washing down each of them with 200 ml of radon water. Intestinal lavage is performed every second day. On days of performing intestinal lavage, patient takes bath with radon water in the morning before lavage, with performing underwater hydrodynamic massage (UHM) on other days. On days, when intestinal lavage is not performed, sessions of sound therapy are carried out after UHM.

EFFECT: method provides correction of biological age of organism as prevention of premature ageing.

3 cl, 4 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: before Mantoux test with 2 TE PDD-L, Ergoferon preparation is used in the infants infected with tuberculosis mycobacteria with the suspected early period of primary tuberculosis infection and active tuberculosis infection, in a dose of 1 tablet 20-30 minutes before or after a meal, once a day for 45 days.

EFFECT: invention provides the complex preparation of frequently and chronically ill children with an allergic pathology, for Mantoux test with 2 TE PDD-L.

3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: before Mantoux test with 2 TE PPD-L, general advice for phthisiologist-controlled preparation of the infants are given. That is added with prescribing the immunomodulatory preparation Anaferon for children aged 1 year and older 1 tablet 20-30 minutes before and after meals once a day for 30 days.

EFFECT: invention provides the complex preparation of frequently and chronically ill children for Mantoux test with 2 TE PDD-L.

3 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, specifically to immunostimulating compounds and may be used in medicine. An immunostimulating peptide of an amino acid sequence XLYDKGYTSKEQKDCVGI, where N-terminal X is N-acetylalanine, and may be covalently linked to fatty acids, selected from C2-C25, to form PDAG (peptidyl-2,3-diacylglycerides). The resulted compound may be included in pharmaceutical formulations for stimulating an immune response.

EFFECT: invention provides efficient stimulation of an immune response in subjects and may enhance the immunogenicity of the antigenic peptide when administered with PDAG.

29 cl, 10 dwg, 9 ex

FIELD: medicine.

SUBSTANCE: first stage involves a cervical uterine repair by staged dissection on the 5-7th menstruation day in a combination with laser destruction of exophytic condylomas. Laser light power is 6-9 Wt, spot diameter is 1.5 mm, and exophytic condylomas penetration is 1-1.5mm. The second stage involves the immunomodulatory therapy.

EFFECT: method enables increasing the clinical effectiveness by recovering the immunological homeostasis of the uterine cervix by potentiating two effects: recovering archtechtonics of the cervical canal and immunomodulatory therapy.

4 tbl, 2 ex

Fingolimod salts // 2543621

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new 2-amino-2-[2-(4-C2-20alkylphenyl)ethyl]propan-1,3-diol salts specified in tartrate, lactate benzoate, succinate, malonate, acetate and propionate in the crystalline form. Each of the above salts is characterised by powder X-ray pattern data. Compounds in the therapeutically effective amount can be used in treating autoimmune diseases.

EFFECT: crystalline salts of the present invention possess higher stability, better solubility, more convenient to store and handle.

11 cl, 7 dwg, 1 tbl, 10 ex

FIELD: pharmacology.

SUBSTANCE: invention mainly relates to a food composition, including punicalagins.

EFFECT: improved action in respect to health improvement.

8 cl, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and concerns using rhCC10 protein for preparing a therapeutic agent for the therapeutic or preventive effect on influenza virus.

EFFECT: invention provides reducing a pulmonary titre of influenza virus.

11 cl, 4 dwg, 1 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmacology, in particular, to medication, possessing antiviral, antimicrobial, immunomodulating and anti-inflammatory action, in form of drops, spray, gel and solution for injection for treatment and prevention of infection diseases: herpes, acute respiratory viral infections, hepatitis, HIV-infections, viral disease. Medication according to invention contains complex of cytokines with Trilonum B, immobilised on biologically compatible polymer as carrier.

EFFECT: obtaining medication, possessing antiviral, antimicrobial, immunomodulating and anti-inflammatory action.

3 cl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to 5,5-condensed heteroarylene compounds IIIB, where U2, V1, V2 and W1 are selected from O, N, NH, S or CR3a; U1, W2, X1 and X2 represent C or N; R1 and R2 represents hydrogen, -C(O)CH(NR1bR1c)R1a, -C(O)CH(N(R1c)C(O)OR1b)R1a or -C(O)OR1a; R3a represents hydrogen or R3; R3 represents halogen or -C(O)OR1a; L1 and L2 are such as given in invention formula, each Z1 and Z2 represents bond or -O-; each Rla, R1b and R1c represents hydrogen, C1-6 alkyl or C6-14 aryl; or Rlb and Rlc together with N atom, which they are bound to, form 5-6-membered heterocyclyl; q, r, s, t and u equal 1. Invention also relates to pharmaceutical compositions, containing 5,5-condensed heteroarylene compounds, and methods of treating or preventing HCV infection.

EFFECT: 5,5-condensed heteroarylene derivatives, possessing inhibiting activity with respect to hepatitis C virus.

43 cl, 42 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a novel compound - N,N-bis(1-adamantyl-1-ethylamine) hydrochloride of the structural formula I and a method for production thereof. Compound has biological activity and can be used as a component of a medicinal agent or as an impurity label of a medicinal agent as a standard sample in analytical chemistry of pharmaceutical preparations. A method of producing a compound of formula I includes a) obtaining 1-adamantyl-1-ethylamine from 1-adamantyl-1-ethylamine hydrochloride, b) obtaining a Schiff base of structural formula III by treating with a compound obtained at step (a) with acetyl adamantane, c) treating the Schiff base of formula III with a reducing agent to obtain bis(1-adamantyl-1-ethylamine) hydrochloride of formula I. The compound is characterised by a boiling point, IR, NMR and a mass spectrum data.

EFFECT: improved method.

2 cl, 8 dwg, 1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to N-adamantylbenzotriazole derivatives

and where R1 and R2 are hydrogen or a nitro group. The invention also relates to a method of producing the compound of formulae 1 and 2.

EFFECT: obtaining novel N-adamantylbenzotriazole derivatives which exhibit anti-influenza A virus activity.

3 cl, 5 ex

FIELD: medicine.

SUBSTANCE: drops possessing antiviral and immunomodulatory effects characterised by the fact that they represent a 95% ethanol infusion of wild strawberry leaves and fruit specified in: red raspberry fruit, mountain ash fruit, bilberry fruit, blood-red hawthorn fruit, cinnamon rose fruit; 15-25 mg of the substance in 1 ml of the infusion.

EFFECT: drops possess pronounced antiviral and immunomodulatory effects.

15 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: method includes treating crushed Echinacea purpurea (L.) Moench roots and rhizome with steam, extraction with ethyl alcohol, then steeping, stirring, steeping, draining a portion of the extract which is equal to the amount of the loaded Echinacea purpurea (L.) Moench roots and rhizome, after draining a portion of the extract, adding ethyl alcohol to the treated material, draining the whole extract; extracting crushed Echinacea purpurea (L.) Moench herbs with ethyl alcohol, steeping, then stirring, steeping, draining a portion of the extract which is equal to the amount of the loaded Echinacea purpurea (L.) Moench herbs, after draining a portion of the extract, adding ethyl alcohol to the treated herbs, draining the whole extract; all obtained extracts are mixed, cooled and filtered under certain conditions. An Echinacea purpurea (L.) Moench tincture. Use of the method to obtain an Echinacea purpurea (L.) Moench tincture.

EFFECT: method preserves the biological activity of components of the tincture and medicinal properties.

3 cl

FIELD: medicine.

SUBSTANCE: invention represents an encapsulated liposomal antiviral agent based on human interferon alpha-2b for vaginal application, characterised by the fact that each capsule is made in the form of a hollow coating, which encloses a powder excipient and liposomes distributed in the excipient, and sodium alginate, a water-soluble polymer gel former; the excipient consists of lactose, sodium chloride, 12-aqueous disodium hydrogen phosphate and sodium dihydrogen phosphate, whereas each of the liposomes represents a hollow coating containing lecithin, cholesterol and alpha-tocopherol, and a nucleus inside the coating and containing recombinant human interferon alpha-2; the ingredients of the agents are taken in a certain ratio, mg.

EFFECT: maintaining the storage activity of recombinant human interferon alpha-2 and prolonged action in vaginal application.

2 cl, 3 dwg, 6 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel compounds of general formula (I) or their pharmaceutically acceptable salts, which possess properties of RNA-polymerase inhibitor, in particular HCV inhibitor. Such disease can be hepatitis C. In formula (I)

is selected from the group, including simple carbon-carbon bond and double carbon-carbon bond; R1 represents hydrogen atom; R2 represents hydrogen atom; R3 represents hydrogen atom; R4 is selected from the group, including

and ,

R5 is selected from the group, including hydrogen atom, C1-C6alkyl and C1-C6alkyloxy; R6 is selected from the group, including hydrogen atom, C1-C6alkyl and C1-C6alkyloxy; R7 is selected from the group, including hydrogen atom, phenyl, 5-membered heterocycle, carbocycle with 2 condensed cycles, where 5-membered heterocycle contains at least 1 heteroatom, selected from the group, consisting of N, O and S, and where phenyl, heterocycle and carbocycle with 2 condensed cycles are optionally substituted with at least one of RJ and RK.

EFFECT: compounds can be used for treatment of disease, which can be treated by HCV inhibition.

21 cl, 2 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to organic chemistry and particularly to aminoalkyl esters of 5-methoxyindole-3-carboxylic acid and pharmacologically acceptable salts thereof of general formula (I), where R1 is cyclohexyl, C1-3alkyl; R2 is phenylthio, phenyloxy, wherein the phenyl group can have 1-2 halogen substitutes or a C1-4alkoxy group, or R2 is a 5-6-member heterocycloalkyl containing 1-2 heteroatoms selected from nitrogen and oxygen; n equals 1, 2, 3, 4; each R is independently selected from C1-4alkyl; except compounds indicated in the claim. The invention also relates to a method of producing a compound of formula (I) and use thereof.

EFFECT: obtaining novel 5-methoxyindole-3-carboxylic acid derivatives, having antiviral activity.

4 cl, 2 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: presented egg-shaped vaginal suppositories contains the following mass ratio in 1 capsule (egg-shaped vaginal suppository): sulphadimine 0.05 g; metronidazole 0.01 g, potato starch 0.02 g, glucose 0.04 g, 5% polyvinyl alcohol 0.07 g, 15% oily extract of propolis 1.0 g; gelatine 0.2 g; dimethicone 0.04 g; glycerol 0.4 g; purified water 0.37 g.

EFFECT: using the invention increases the therapeutic effect and bioavailability of the capsules, increases the prolonged action time, provides the control release of the active substances.

3 cl, 1 dwg, 1 tbl, 3 ex

Up!