Suppository for immunoprophylaxis of viral infections
FIELD: microbiology, biotechnology, medicine, in particular production of living vaccines against viral infections.
SUBSTANCE: invention relates to living vaccines for rectal administration against viral infections based on attenuated recombinant Salmonella strains bearing protective viral antigens. Suppository for immunoprophylaxis of viral infections contains (per one 2 g suppository, mass %): cell slurry of attenuated recombinant Salmonella strains transformed with pGEX-2T-TBI, pcDNA-TCI, or pKHBc bearing gene of protective viral antigens, mixed with suppository base in amount of 106-109 living cells 1 %; fatty base 94 %; and emulsifier T2 5 %. As fatty solid cooking oil (94 %); or hydrolyzed cotton oil (94 %); or mixture of cooking oil (60 %), Cocoa fat (24 %), and solid wax (10 %).
EFFECT: agent for inducing of humoral and cell immune response to respective viral antigen.
8 tbl, 11 ex, 3 dwg
The invention relates to Microbiology, biotechnology and medicine, specifically to the creation of methods for genetic engineering of live vaccines against viral infections based on attenuated recombinant Salmonella strains carrying protective viral antigens, in the form of suppositories.
Currently in the world there is a rapid spread of a number of viral infections, developing into an epidemic; it is AIDS caused by human immunodeficiency virus (HIV), hepatitis b, hepatitis C, etc. All of these pathogens are highly contagious, virulent and possess antigenic variability.
As in the Arsenal of medicine today, there is not enough effective ways to treat this kind of viral infectious diseases, the only measure to prevent the development of infections is mass vaccination of the population.
The main drawback of existing vaccines is that they can only induce systemic immunity. It is obvious that effective antiviral vaccine should provide both systemic and local mucosal immunity in the field "input gate" infection.
Among the promising new developments in viral vaccine in recent preference for the creation of mucosal vaccines, i.e. drugs, injected mucous membranes. Such vaccines have several advantages in the EU ETS: an introduction without a syringe, for example oral or intranasal; ensures the induction of not only the system, but local immunity, which is very important for sexually transmitted diseases. The main problems creation of mucosal vaccines based on inactivated antigens related to the fact that individual viral proteins not penetrate into the cells of the mucous membrane or degraded in the stomach or intestines.
One of the most promising vectors for mucosal delivery of vaccines are not pathogenic for humans attenuated strains of Salmonella [1, 2].
Interest in living cells of Salmonella as a carrier immunogenic epitopes of infectious agents due to the fact that attenuated strains of this bacterium is able to envirovet mucous and some time to live in the cells of lymphoid tissue of mammals. Cell-vector as a natural depot and adjuvant for antigen, is it in the most immunogenic form, increases the persistence and contributes to the accumulation of the antigen to immunocompetent cells.
The well-known series of works in which it is shown that recombinant attenuated Salmonella producing HIV antigens, induce high system (humoral and cellular) immunity and secretory immunity. An example is the recombinant attenuated Salmonella strains enteitidis E-23 VS  and Salmonella typhimurium T-10 Navy 160 , the first of which provides a fusion protein gp 120 of HIV in mammalian cells, and the second is another protein immunogen HIV gp 160. Known live HIV vaccine based on attenuated strain of Salmonella typhimurium SL 3261 for oral administration, which as immunogens carries protein reverse transcriptase transactivity protein of HIV . In the described works as antigen used only one or two fragments of proteins gp 120 or gp 160 or pol, which is obviously insufficient to create an effective vaccine, because it is believed that an effective HIV vaccine should include epitopes not only of the env protein, but also gag and pol .
Known recombinant attenuated strain of Salmonella enteritidis E-23/pGEX-2T-TBI, and are able to produce artificial protein TBI containing nine T - and b - cell epitopes of HIV, in mammalian cells . Also known recombinant attenuated strain of Salmonella typhimurium SL 7202 pGEX-TBI, and are able to produce artificial protein TBI .
Known recombinant attenuated strain of Salmonella enteritidis E-23/pcDNA-TCI , capable of producing artificial protein TCI containing multiple (more than eighty) cytotoxic T-cell epitopes of HIV-1 in mammalian cells.
All of these strains with the introduction of animals induce a pronounced specific gumor the capacity and cellular immune response to HIV and can be used to design the living DNA vaccines against HIV.
Actively developing approaches to the creation of a live vaccine against hepatitis b based on attenuated strains of Salmonella. Currently undergoing clinical testing of a vaccine based on recombinant Salmonella producing HBcAg-preS2 . However, immunization of volunteers records recombinant strain of Salmonella, the answer to preS2 was only observed in one out of 13 subjects. One of the reasons for such a weak immune response is the fact that reS2-region is minor antigenic determinant, whereas the main neutralizing epitope contained in HBsAg.
Known candidate vaccine strains of bacteria, specifically designed by introducing plasmids encoding the antigens of hepatitis b virus HbsAg and HbcAg in the form of chimeric proteins in recipient attenuated Salmonella typhimurium strains T-10 and Salmonella typhimurium SL 7207 [11, 12]. After immunization of mice received oral structures and errectile (water suspension cells of recombinant strains in the form of an enema) found specific anti - HbsAg and anti-HbcAg IgG in the serum of animals and specific anti-viral IgA in the mucosa of the intestine, and also shows the formation of T-cell response to antigens of hepatitis C.
It is known that aqueous solutions, introduced into the rectum by enema, medicinal substances are absorbed very quickly. However, a lot of which of them is displayed back together with solution therefore, the total amount that absorb from the drug substance is small. The efficiency of absorption increases significantly when using suppozitornoj form. In addition, using suppozitornyj forms of medications can provide rapid and massive or progressive delivery of medicinal substance in the bloodstream .
The advantage of mucosal vaccines in the form of suppositories is that they can provide painless, ease of administration and to promote whiter stable and long-term induction of local immunity, because multiplying in the host organism, vaccinal strain stimulates long-term antibody production to the development of resistance at the input gate of the infection. In addition, this approach does not require expensive cleaning stage antigen.
Therefore, it seems appropriate to develop approaches to the creation of vaccines against viral infectious agents on the basis of living cells recombinant attenuated Salmonella in the form of suppositories.
There is information on the development of vaccines in the form of suppositories on the basis of inactivated microorganisms. This kind of vaccine against urogenital infections, consisting of inactivated bacteria that originate from cultures of strains uropathogenic BA is clear species Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Proteus morganii and Streptococcus faecalis, and polietilenglikoli basics suppository, described in the patent of the Russian Federation No. 2224542 issued by the American firm Protein Express . This suppozitornyj form of the vaccine promotes uniform distribution of cells of microorganisms in the pharmaceutical form and ensures the stability of the active substance for a long time.
In medical practice are created and used suppositories containing live microorganisms. It suppositories containing microorganisms normoflora person, which are used in the treatment of dysbacteriosis, inflammatory processes in the distal intestine and vagina [15, 16, 17]. Preparations suppositories contain microbial mass of live bifidobacteria or lactobacilli in the amount of 103-108cells per dose  or dry liofilizovannye biomass of microorganisms  and pharmaceutically acceptable additives. In Russia produced candles Azilect, Baydin, Lactobacterin (OJSC "Biochimmash"), which represent the microbial mass of live microorganisms, dried in an environment of cultivation with the addition of Saharsa-gelatin-dairy environment and formed the medical suppositories.
However, not described in literature preparations suppositories containing living cells of recombinant MIC the organisms, bearing protective viral antigens intended for vaccination of viral infections.
An object of the invention is to develop an effective drug for prophylaxis of viral infections (virus vaccines) based on live recombinant Salmonella strains in the form of a suppository.
The problem is solved by the development of production and composition of suppositories for rectal administration, containing live attenuated cell culture, recombinant Salmonella strains carrying genes protective viral antigens incorporated into suppozitornyj basis.
Rectal route opens the possibility of introducing into the body of drugs, collapsing in the stomach or intestines. And for attenuated Salmonella physiological environment with a pH of 6.8 to 7.2, so the environment of the rectum to the minimum extent will affect the manifestation of the biological activity of the vaccine strain.
To solve the task dynamics of the accumulation of biomass of Salmonella carrying the protective antigens of HIV and hepatitis b - Salmonella enteritidis E-23/ pGEX-2T-TBI, Salmonella enteritidis E-23/pcDNA-TCI, Salmonella typhimurium T-10/pKHBc in the fermentation process. It is shown that active cell division continues until OP=2,0. When this plasmid is stably retained in the strains. After culturing the titer of cells is of lionell in the culture fluids should not be below 10 6.
In the manufacturing process of DNA vaccines in the form of suppositories containing living cells with recombinant Salmonella strains, we studied the influence of substrate and temperature the introduction of native biomass in suppozitornyj based on cell viability. It is shown that the type of basics and nature of excipients has a significant impact on cell viability suppositories. From the data presented in Table 1, it is seen that the best cell viability provides the use of a hydrophobic (fat) basis. Based hydrophilic type no viable cells. Basics # 2 and # 6 have poor fluidity. Introduction to the framework as an excipient of Tween-80 acts detrimental to cell viability of Salmonella in suppositories. In the manufacture of suppositories there is a lack of viable cells with the introduction of cells in suppozitornyj base at a temperature of 50°C. Remains the same viability when making cells based at temperatures up to 45°C.
One dose (one suppositories weighing 2 g) contains 1·106-1·109living cells of Salmonella enteritidis E-23/pGEX-2T-TBI or Salmonella enteritidis E-23/rwma-TC1 or Salmonella typhimurium T-10/HBc.
According to studies suppositories are prepared on the basis of fats, nab the emer using cooking oil "Frying" or hydrolyzed cottonseed oil (GHM), or a mixture of solid cooking fat, cocoa butter with addition of solid paraffin oil and an emulsifier T-2 by pouring in suppozitornoj form a molten mass containing cell culture of Salmonella. The optimal temperature for introduction of biomass in suppozitornyj the basis of the temperature is 39 to 45°C.
Suppositories for prophylaxis of viral infections contain components in the following ratio to one candle by weight of 2 g, wt.%:
cell suspension of recombinant attenuated Salmonella strain carrying genes protective viral antigen, in an amount of 106-109living cells - 1%; fat basis - 94% and the emulsifier T-2 - 5%. At the same time as fat basis can be solid cooking fat (94%), or gidrolizovannogo cottonseed oil (94%), or a mixture of cooking oil "Frying" (60%), cocoa butter (24%) and solid paraffin oil (10%).
In the manufacturing process of the vaccine in the form of suppositories containing the recombinant cells of Salmonella, there is the problem of storing for a long time, standardization and stability of biomass. A necessary condition is that the cells of recombinant Salmonella must be in the active state of division, i.e. in the phase of logarithmic growth. The solution to this problem is to lyophilization biomass.
The viability of m is of croorganisms in the process of freeze-drying has a significant influence the composition of the protective environment. In the experiments we used the following protective environment: Saharsa gelatin (SG), milk-glucose (MG), sacharose gelatin with thiourea (GTL). The biomass samples of recombinant strains of Salmonella, liofilizovannye with different security environments, characterized by vitality and stability. The data are shown in Table 2.
|The viability and stability of biomass recombinant strains of Salmonella, liofilizovannyh with different security environments|
|The name of the biomass. Protective environment.||1 month||3 months||6 months|
|Sal.ent. E23 DNA-TCI Environment SG||4,0·109||100%||2,1·109||100%||2,0·108||100%|
|Sal.ent. E23 DNA-TCI Environment MG||4,0·109||100%||3,3·108||100%||2,7·108||100%|
|Sal.ent. E23 DNA-TCI Media is GTL||1,8·109||100%||6,0·108||100%||5,8·108||100%|
|Sal.ent. E23 rcns Environment SG||4,3·109||100%||4,7·109||100%||4,0·109||100%|
|Sal.ent. E23 rcns Environment MG||5,3·109||100%||4,8·109||100%||3,5·109||100%|
|Sal.ent. E23 rcns Environment GTL||4,0·109||100%||3,5·109||100%||7,8·108||100%|
In Table 2 data show that liofilizovannye biomass recombinant Salmonella is stored without loss of cell viability and stability for 6 months, however, the process of freeze-drying is preferable to use environment SG or MG.
Moreover, it is shown that the suppositories manufactured using liofilizovannyh biomass can be stored for 6 months at 4-8 t With°, and the cells do not lose their viability and stability of the strain (see Table 3).
|A study of one hundred is innosti and viability liofilizovannyh cells of recombinant strains of Salmonella in suppositories|
|The name of the biomass. Protective environment.||1 month||3 months||6 months|
|Sal.ent. E23 DNA-TCI Environment MG||8,0·106||100%||1,0·106||100%||1,2·106||100%|
|Sal.ent. E23 rcns Environment MG||3,5·108||100%||2,7·107||100%||1,5·107||100%|
During preclinical studies in mice and Guinea pigs shows the formation of immunity against HIV infection and hepatitis b through education in animals of specific antibodies and the formation of a clone of memory cells. The maximum increase in antibody titer is achieved at 28 days and lasts for weeks after a single immunization. Two immunization held within 28 days after the first, the maximum antibody titer is logged on 42-56 days. In the pre-clinical testing performed in accordance with the Rules for state tests is s and the registration of new medical immunobiological preparations (SP 188.8.131.521-96), shows the lack of significant deviations in the condition of the vital organs, hematological and morphological indicators, the absence of allergenic activity that demonstrates the safety and tolerability of the vaccine.
The invention is illustrated in the following graphic.
Figure 1. Analysis of the systemic antibody response to purified HIV-1 sera of mice immunized with strain Salmonella enteritidis E-23/pcDNA-TCI according to ELISA.
Figure 2. Analysis of the systemic antibody response to purified protein TCI sera of mice immunized with strain Salmonella enteritidis E-23/pcDNA-TCI according to ELISA.
S.enteritidis E-23/ pcDNA-TCI
Figure 3 Enzyme-linked immunosorbent assay to HBcAg sera of mice immunized with suppositories containing Salmonella typhimurium T10 rcns.
For a better understanding of the invention the following specific examples of its implementation.
Example 1. The cultivation of recombinant strains of Salmonella. The cultivation of recombinant strains of Salmonella enteritidis E-23/pGEX-2T-TBI or Salmonella enteritidis E-23/pcDNA-TCI or Salmonella typhimurium T-10/pKHBc spend on the medium S with ampicillin. Before cultivation spend the transformation of cells of Salmonella by which electroporation. For this "night" culture of Salmonella enteritidis E-23 or Salmonella typhimurium T-10 diluted in the ratio 1: 100 L-broth and pokasivaut in LB medium without ampicillin on the rocking chair at a temperature of (37±2)°C to an optical density of D550=0,2. The cell suspension is cooled in ice for 20 min and centrifuged 2 min at 6000 rpm After washing with water the precipitate cells resuspended in 40 μl of 10%glycerin solution, make a 1 µl plasmid DNA pGEX-2T-TBI or pcDNA-TCI or rcns, incubated in ice for 2 min and hold electroporation with the use of device "Ecobio LTD. The pulse time is 4 seconds, the voltage 2500 Century After electroporate in the same cuvette add 300 μl of L-broth and incubated 1 h at a temperature of (37±2)°C. the Obtained transformants plated on solid medium L - agar with ampicillin and grown overnight at a temperature of (37±2)°C. After checking for the presence of plasmids carried out clones producer strain cultivated on LB medium with ampicillin at temperature-controlled shaker at a temperature of (37±2)°With a rotation speed of 200 rpm, the Cultivation is continued for 3-4 h until the optical density D550=2,0. After culturing in the culture fluid determine the titer of cells, which should not be below 106. By centrifugation at 5000 rpm for 5 min to separate the biomass cells recombinant Salmonella that COI is lsout for the preparation of suppositories.
Example 2. Preparation of suppository Sal-HIV "B", containing living cells attenuated recombinant strain of Salmonella enteritidis E-23/pGEX-2T-TBI.
Main components of the suppository in the form of hanging (cooking oil "Frying" - 300,0±1.0 g, emulsifier T2 - 25,0±0.1 g cocoa butter - 120,0±0.1 g and paraffin - 50,0±0.1 g) quantitatively transferred into a beaker with a capacity of 1 l, which is placed in a water bath heated to 75°S, and incubated until complete melting of the mixture. Glass with molten base is placed in a mixer-homogenizer and stirred at 200 rpm until a homogeneous mass. The basis is cooled with constant stirring to a temperature of (39±1)°C. the Biomass of cells of Salmonella enteritidis E-23/pGEX-2T-TBI combined with 5 ml of sterile purified water and resuspended to obtain a homogeneous suspension which is injected into a chilled suppozitornyj basis, mixing thoroughly to obtain a homogeneous mass. Suppozitornyj was poured into the prepared forms and is cooled at a temperature of minus 15±2)°C for 60 minutes
Example 3. Preparation of suppository Sal-HIV "D"containing living cells attenuated recombinant strain of Salmonella enteritidis E-23/pcDNA-TCI.
Main components of the suppository in the form of sub-samples (solid cooking fat - 470,0±1.0 g emulsifier T2 - 25,0±0.1 g) quantitatively transferred into a glass in which estimatio 1 l, which is placed in a water bath heated to 55°S, and incubated until complete melting of the mixture. Glass with molten base is placed in a mixer-homogenizer and stirred at 200 rpm until a homogeneous mass. The basis is cooled with constant stirring to a temperature of (39±1)°C. the Biomass of cells of Salmonella enteritidis E-23/pcDNA-TCI combined with 5 ml of sterile purified water and resuspended to obtain a homogeneous suspension which is injected into a chilled suppozitornyj basis, mixing thoroughly to obtain a homogeneous mass. Suppozitornyj was poured into the prepared forms and is cooled at a temperature of minus 15±2)°C for 60 minutes
Example 4. Determination of viable cells in rectal suppositories.
One suppository weighing 2.0 g containing cells attenuated strain of Salmonella enteritidis E-23 with integrated plasmid pcDNA-TCI or pGEX-2T-TB, soften at 37°in phosphate buffer until smooth and cook 10 fold dilution of the suspension. From solutions of the 6th, 7th and 8th breeding do the sowing 100 ál of Petri-dish with L-agar containing 100 μg/ml ampicillin, and thermostatic day at 37°C. Upon expiration of the time visually counting the number of colonies on plates. The number of viable colonies of recombinant Salmonella in suppositories (X) is found by the formula:
where: X is the number of viable colonies;
N - number of colonies;
10pnumber of breeding;
Vp- volume sowing cultivation, ál; (100 μl).
The number of viable colonies in suppositories after the counting is from 106up to 109.
Example 5. Determination of the toxicity of drugs suppositories.
Suppositories weighing 100 mg, containing from 106up to 109cells of Salmonella enteritidis E-23/pGEX-2T-TBI or Salmonella enteritidis E-23/pcDNA-TCI enter not less than 5 Guinea pigs (weighing 250-350 g), once in the rectum to a depth of 0.5 to 1 cm and fixed in 2-3 minutes during the first day and for 7 days after the administration of suppositories conduct a daily visual inspection of animals, evaluating their appearance, mobility, measure body weight. During the whole observation period no animal died, visually recorded signs of intoxication and loss of body weight of the animals was not observed. Thus, the proposed product is non-toxic and harmless.
Example 6. Determination of specific activity of suppositories containing Salmonella enteritidis E-23/pcDNA-TCI
The immunization. For immunization using mice of BALB/c mice, weighing 12-15 grams, which were obtained from the vivarium of the SRC VB "Vector". The animals were kept on a standard diet.
Immunization of animals with suppositories p is avodat once, parentale (one suppository contains 108cells of S. enteritidis E-23/pcDNA-TCI). Blood sampling to produce 0, 7, 14, 21, 28, 35 the day after the introduction of Salmonella.
Enzyme-linked immunosorbent assay. The specificity of the resulting antibodies in sera of mice analyzed by ELISA. For the detection of antibodies against HIV-1 using purified inactivated HIV-1 virus and purified protein TCI. In each experiment, take blood samples from three animals. After obtaining serum samples are mixed, and then determine the titre of specific antibodies. Titer is defined as the excess of the titer of the sera of mice immunized with suppositories containing S. enteritidis E-23/pcDNA-TCI above the titer of the sera of mice immunized with the suppositories from the original strain of S.enteritidis E-23. At each point do two repetitions. The results are presented in figures 1, 2. Starting with the third week after immunization of mice with suppositories S. enteritidis E-23/pcDNA-TCI detect products of IgG to HIV-1, and antibodies production remains at a high level throughout the observation period.
The determination of the number of CTL lymphocytes on the basis of the reaction ELISPOT
When setting the ELISPOT response at the first stage are sorption anti-INF-γ MAT with a concentration of 5 μg/ml per well of 96-hole tablet ImmunoSpot M200. After incubation for 12 h at 4°With each of the wells washed twice with a solution of PBS and blocked the Ute medium RPMI 1640, containing 10% fetal bovine serum for 2 hours as cell-effectors using splenocytes of immunized animals at a concentration of 106Jr. For the stimulation of product INFγ suspension cells using protein TCI (1 μg/ml) and two peptide: N15 (DRVIEVVQGAYRAIR), N16 (KQIINMWQEVGKAMYA), to assess nonspecific products make use of the peptide of EHEC (negative control). Cells cultured in the presence of 5% CO2at 37°C for 24 h INFγ-secreting cells render using 0.5 μg/ml biotinylating anti - INF-γ antibodies and 0.25 μg/ml conjugate Avidin-HRP. Staining produced by adding a substrate for peroxidase (4-chloro-1-naphthol, diaminobenzidin phosphate). The reaction is stopped by removal of the reagents, the wells washed 3 times with distilled water. Counting the number of INFγ-producing cells is carried out using a microscope. The results of the experiments are summarized in Table 4.
The results of the evaluation of cytotoxic T-lymphocytes of animals immunized with suppositories containing S. enteritidis E-23/pcDNA-TCI in the ELISPOT
|Antigen||The period from the beginning of immunization (day) / number of INF-gamma producing cells|
Example 7. Evaluation of the neutralizing activity of sera.
Neutralizing activity of sera determined by the accounting method of inhibiting the reproduction of the virus HIV-1 by reducing the infectious titer. This is done using human lymphoblastoid cells of a human MT-4. Cells were cultured at a concentration of 3.0 to 5.0×105cells in 1 ml of RPMI medium 1640 with 10% serum of cow embryos, 100 μg/ml of gentamicin. In the reaction of virusneutralizing use strain of HIV-1899A(type X4, subtype b), obtained from the collection of strains of human immunodeficiency Institute of Virology. DI Ivanovo RAMS. The multiplicity of infection of 100 TCD50. Serum inactivate in a water bath at a temperature of 56°C for 30 min In the wells of a plastic 96-hole panel (Costar, USA) add 50 ál of the following breeding sera: 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, etc. and add 50 μl of the suspension of the virus. The mixture is cuberoot for 1 h at 37° C. After incubation, make a 100 μl suspension of sensitive cells MT-4 in the amount of 90-100×103cells/well.
Panel incubated at 37°C in an atmosphere with 5% CO2and 98% humidity for 5-7 days prior accounting results. As controls use: control of virus - virus with the addition of medium without serum, "control cells" - cells in a nutrient medium, "control of cytotoxicity sera" - cells + serum. Analysis performed 5-7 days after the start of the experiment. Inhibition of virus reproduction judged by the reduction in infectious titer, which is determined by TCD50(50% tissue cytopathic dose). The data for detection of neutralizing antibody titers in the blood serum of immune animals are summarized in Table 5.
The neutralizing activity of sera
|The period from the beginning of immunization (day)|
|groups of animals||Titers of neutralizing antibodies|
|Sera from mice immunized||0||1:20||1:80||1:640|
|the suppositories S. Enteritidis E-231|
|Sera from mice immunized||0||0||0||0|
|the suppositories S. Enteritidis E-23|
The data presented in Table 5, suggest that formed after insertion of suppositories S. enteritidis E-231 pcDNA-TCI antibodies have neutralizing activity.
Thus, suppositories containing Salmonella enteritidis E-23/pcDNA-TCI with the introduction of animals induces a pronounced specific humoral and cellular immune response to HIV-1. Proposed suppositories can be used as a living DNA vaccines against HIV-1.
Example 8. Determination of specific activity of suppositories containing Salmonella typhimurium T10 rcns.
The immunization. For immunization using mice of BALB/c mice, weighing 12-15 grams, obtained from the vivarium of the SRC VB "Vector". The animals were kept on a standard diet.
Immunization of mice conduct the introduction suppositories perfectline, twice with an interval of 3 weeks. The dose of S. typhimurium T10 rcns is 107cells on the suppository. The blood produce the via 14, 21, 28, 36 and 44 days after the first injection of Salmonella.
Enzyme-linked immunosorbent assay. The specificity of the resulting antibodies in sera of mice analyzed by ELISA. For the detection of antibodies against HBcAg using purified recombinant HBcAg. In each experiment, take samples from three animals. These samples are mixed, and then determine the titre of specific antibodies. Titer is defined as the excess of the titer of the sera of mice immunized with suppositories containing Salmonella typhimurium T10 rcns, above the titer of the sera of mice immunized suppositories with the original strain of Salmonella typhimurium T10. At each point do two repetitions. The results are presented in figure 3.
The reaction besttransport. The reaction besttransport of splenocytes to carry through the 81 day after the first injection of Salmonella according to previously described methods . As a specific antigen using HBcAg, as a non - specific antigen-virus enteritis of mink. The hole in the tablet make 100 ál of each component. The antigen concentration of equal to 2 μg/ml as a mitogen use of concanavalin a (manufactured by Sigma, USA) at a concentration of 5 µg/ml Cells stimulated by antigen, incubated for 78 hours For 18 h before the end of the incubation period make3H-thymidine (St. Petersburg) in the dose of 2 µci per well. Cellular response estimate by pokazatelyakh stimulation, which characterizes the ratio of the proliferation of splenocytes induced by specific antigens to spontaneous proliferation of splenocytes (table 6). The formation of a clone of splenocytes sensitized to HBcAg, is registered in the group of animals immunized with the suppositories with S. typhimurium T10/pKHBc. The stimulation index in the group of S. typhimurium T10/pKHBc significantly exceeding that in the control group S. typhimurium T10, and stimulation of splenocytes heterologous antigen mink enteritis virus. The ability of splenocytes to respond to the stimulation of HBcAg in the 81st day of the start of immunization S. typhimurium T10/pKHBc indicates the formation of a specific memory cell.
|The proliferative activity of splenocytes of mice immunized with the suppositories, carriers of recombinant Salmonella (stimulation index) on day 81 after the beginning of immunization|
|The group of animals||HbcAg||Virus enteritis of mink||Of concanavalin|
|S. typhimurium T-10||1,0||0,9||7,4|
|S. typhimurium T-10/rcns||3,3||1,0||7,5|
The development of HIV-specific humoral immune response in mice immunized with cells of recombinant Salmonella producing TBI
|Group||Week 2 title||Week 6 : the titer of anti-TBI||12-week titer anti-TBI|
|S. enteriditis E23/ pGEX-2T-TBI rectal||1:300||1:100||1:1450||1:600||1:800||1:400|
|S. enteriditis E23 rectal (contr)||1:100||1:50||1:50||1:50||1:50||1:50|
The reaction besttransport. The reaction besttransport of splenocytes spend 6 weeks after the introduction of Salmonella according to previously described methods . As a specific antigen using gst-TBI and the lysate of HIV-1 as a non - specific antigen-virus enteritis of mink. The hole in the tablet make 10 µl of each component. The antigen concentration of equal to 2 μg/ml as a mitogen use of concanavalin a (manufactured by Sigma, USA) at a concentration of 5 µg/ml Cells stimulated by antigen, incubated for 78 hours For 18 h before the end of the incubation period make3H-thymidine (St. Petersburg) in the dose of 2 µci per well. The cellular response is evaluated in terms of stimulation index, which characterizes the ratio of the proliferation of splenocytes induced by specific antigens, to spontaneous proliferation of splenocytes (table 8). The formation of a clone of splenocytes sensitized to gst-TBI, is registered in the group of animals immunized with the suppositories with S. enteriditis E23/pGEX-2T-TBI. The stimulation index in the group of S. enteriditis E23/pGEX-2T-TBI significantly exceeding that in the control group S. typhimurium 7207 and stimulation of splenocytes heterologous antigen mink enteritis virus. The ability of splenocytes to respond to the stimulation gst-TBI 6 week from the beginning of immunization with S. enteriditis E23/pGEX-2T-TBI indicates the formation of a specific memory cell.
|Induction of T-cell response. The stimulation index|
|The group of animals immunized with recombinant Salmonella||The lysate of HIV-1||gst-TBI||antigen mink enteritis virus||ConA|
|S. enteriditis E23/pGEX-2T - TBI rectal||3,4||3,5||1,1||7,9|
|S. enteriditis E23 rectal(contr)||1,1||1,2||1,2||7,9|
Note.In each experiment took cells from 3 animals. The error in the determination of the stimulation index of not more than 0.1.
The data suggests that suppositories containing with S. enteriditis E23/pGEX-2T-TBI, with the introduction of animals induce distinct HIV-1 specific humoral and cellular immune response.
Example 11. Preparation of suppository Sal-HIV "D"containing living cells attenuated recombinant strain of Salmonella enteritidis E-23/pcDNA-TCI.
Main components of the suppository in the form of hanging (gidrolizovannogo cottonseed oil (GHM) - 425,0±1.0 g emulsifier T2 - 25,0±0.1 g) quantitatively transferred into a beaker with a capacity of 1 l, which is placed in a water bath heated to 55°S, and incubated until complete melting of the mixture. Glass with molten base is placed in a mixer-homogenizer and stirred at 200 rpm until a homogeneous mass. The basis is cooled with constant stirring to a temperature of (39±1)°C. the Biomass of cells of Salmonella enteritidis E-23/pcDNA-TCI enter into a chilled suppozitornyj basis then what s under stirring, suspended until a homogeneous suspension. Suppozitornyj was poured into the prepared forms and is cooled at a temperature of minus 15±2)°C for 60 minutes, the Number of viable colonies in suppositories after the counting is from 1.1×106up to 4×108.
Thus, for the first time received suppositories for prophylaxis of viral infections, containing living attenboroughii recombinant Salmonella strains carrying protective viral antigens, inducing when introducing animals expressed specific humoral and cellular immune response to the corresponding viral antigen that can be used as living antiviral vaccines.
1. International application WO No. 98/48026, CL 12N 15/74, publ. 1998
2. International application WO No. 0014240, CL 12N 15/31, publ. 2000
3. RF patent №2192277, CL AC 39/12, publ. BI No. 31, 2002
4. RF patent №2192886, CL AC 39/19, publ. BI No. 32 of 2002
5. RF patent №2223784, CL AC 39/385, publ. BI # 4, 2004
6. Eroshkin A.M., Karginova E.A., Gileva I.P. et al. Design of four-helical protein as a candidate for HIV vaccine. // Protein engineering-1995 - 8 - p.167-173.
7. Nekrasov N.A., Ignatyev, G.M., Agafonov A.P., Il A.A. Karpenko LI Study of systemic and mucosal immune response after immunization of mice with candidate HIV-1 vaccine. // Siberian medical journal of the L. - 2004. - T. - P.60-62.
8. Karpenko LI, Ignatyev, G.M., Agafonov A.P., Poryvaev V.A., Lebedev LR, Veremail T.A., N.A. Nekrasov, Klimov N.A., Kozlov A.P., Il A.A. Design and study of the antigenic properties of the recombinant strain of Salmonella, producing protein TBI. // Virology - 2002 - N.2 - C-28.
9. Application for patent of the RF No. 2003111095/13 from 17.04.2003, "Recombinant plasmid DNA pcDNA-TCI, ensuring the expression of the synthetic gene TCI in eukaryotic cells, and recombinant attenuated strain of Salmonella enteritidis E-23/pcDNA-TCI as a candidate for constructing living DNA vaccines against HIV."
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11. RF patent №2216590, CL 12N 1/21, publ. BI No. 32 for 2003
12. Karpenko LI, Ignatyev, G.M., Simon E.M. and other Receipt and study of recombinant strains of Salmonella typhimurium SL 7207, producing HbcAg HbcAg-HBs. Questions Virology - 2000 - No. 2 - s.43-46.
13. Gregg M.R., Jack D.B., Smith S.R., Kendall, M.J. The pharmacokinetics ofoxprenolol following oral and rectal dosing-a comparison of delivery systems and routes of administration. // J Clin Pharm Ther. 1987 Apr; 12 (2): 91-9.
14. RF patent №2224542, CL AC 39/116, publ. BI No. 6, 2004
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1. Suppositories for prophylaxis of viral infections, containing live attenuated cell culture, recombinant Salmonella strains, transformed with pGEX-2T-TBI, pcDNA-TCI or rcns bearing genes protective viral antigens, hydrophobic suppozitornyj basis and emulsifier T2 in the following ratio of components on one candle by weight of 2 g, wt.%:
|Suspension of living cells in quantities of 1·106-1·109||1|
|Hydrophobic suppozitornyj base||94|
2. Suppositories according to claim 1, characterized in that as a live attenuated cell culture recombinant Salmonella strains contain liofilizovannye biomass recombinant microorganisms.
3. Suppositories according to claim 1, characterized in that the hydrophobic suppozitornoj bases contain solid cooking fat (TKJ) or gidrolizovannogo cottonseed oil (GHM) in the following ratio, wt.%:
|Suspension of living cells in quantities of 1·106-1·191||1|
|TKJ or GHM||94|
4. Suppositories according to claim 1, characterized in that the hydrophobic bases contain a mixture of cooking oil "Frying", cocoa butter and paraffin wax in the following ratio, wt.%:
|Suspension of living cells in quantities of 1·106-1·10||1|
|Cooking fat "Frying"||60|
FIELD: biotechnology, gene engineering, medicine, pharmaceutical industry.
SUBSTANCE: method production of preparative amounts of plasmid DNA from recombinant microorganism cells includes cultivation of recombinant cells in gas-vortex bioreactor, plasmid DNA isolation and centrifugal purification.
EFFECT: increased yield of target product.
2 cl, 3 ex, 3 tbl, 2 dwg
FIELD: microbiological and medicine industry, gene and protein engineering.
SUBSTANCE: disclosed is recombinant plasmide DNA pU8HBc-preS1. Said plasmide encodes chimerical protein gene of hepatitis B virus core antigen bearing pre-S1 region (27-37 a.b.) epitope of hepatitis B virus surface protein. Also disclosed is bacterium strain Escherichia coli DH5αF'/pU8HBc-preS1, bearing of such plasmide and producing hybrid protein HBc-preS1.
EFFECT: invention useful in medicine.
2 cl, 8 dwg, 3 ex
SUBSTANCE: method for production of purine nucleosides and purine nucleotides, in particular 5'-inosinic acid is disclosed. Claimed method includes using of bacterium belonging to genus Esherichia or genus Bacillus. Production of purine nucleosides and purine nucleotides by said bacterium is increased due to increased activity of YdhL pritein. Also disclosed is amino acid sequence of YdhL pritein from Bacillus amyloliquefaciens and gene encoding thereof.
EFFECT: method for industry scale production of nucleosides and nucleotides.
25 cl, 2 dwg, 6 tbl, 9 ex
FIELD: biotechnology, food processing industry.
SUBSTANCE: invention relates to transformed lactic-acid bacteria which express polypeptide including tolerogenic peptide and L.bulgaris protease fragment. Food composition containing said microorganisms and supernatant thereof also are disclosed.
EFFECT: decreased individual susceptibility to allergic reactions.
9 cl, 6 ex, 1 tbl, 5 dwg
FIELD: biotechnology, microbiology, antibiotics, genetic engineering, molecular biology.
SUBSTANCE: invention relates to a polynucleotide comprising nucleotide sequences encoding product of gene aveC molecules of that can be used for changing the ratio of amount of avermectins of class 2:1 produced in fermentation cultures of S. avermitilis. This nucleotide sequence is similar essentially with aveC allele sequence from S. avermitilis, plasmid pSE186 (ATCC 209604) sequence encoding product of gene aceC from S. avermitilis, nucleotide sequence of aveC open reading frame but this nucleotide sequence comprises mutations encoding combination of amino acids owing to the strain S. avermitilis ATCC 53692 in cells of them allele aveC of wild type is inactivated is able to produce avermectins of class 2:1 in the ratio 0.35:1 or less (when avermectins represent cyclohexyl B2 and cyclohexyl B1, respectively). Also, invention relates to vectors, host-cells and mutant strains of S. avermitilis wherein aveC gene is inactivated or mutated with the possibility for changing the ratio of amount of avermectins of class 2:1. Also, invention relates to a method for preparing the mutated strain (combination of amino acid changes are given in the invention claim) and a method for preparing avermectins of class 2:1 in the reduced state and to a composition prepared by a method given above. Using the proposed invention provides preparing the avermectins composition of class 2:1 with the reduced amount of avermectin B2 in the mixture. Invention can be used in producing avermectin antibiotics.
EFFECT: improved preparing method of avermectins.
17 cl, 21 dwg, 4 tbl, 8 ex
FIELD: molecular biology, veterinary.
SUBSTANCE: invention proposes isolated DNA sequence (variants) encoding Ehrlichia canis protein of size 30 kDa. Also, invention proposes vector comprising such sequence, recombinant Ehrlichia canis 28 kDa protein encoded by this sequence, a cell-host comprising this sequence, a method for preparing the protein, immunoreactive antibody specific to this protein and a method for inhibition of Ehrlichia canis infection in subject. Recombinant protein of size 28 kDa from Ehrlichia canis shows immune reactivity with respect to serum against Ehrlichia canis. Proposed group of inventions can be used in development of vaccines and serodiagnosticum that shows high effectiveness for prophylaxis of diseases and for carrying out the serodiagnosis.
EFFECT: improved preparing method, valuable medicinal and veterinary properties of protein.
19 cl, 17 dwg, 8 ex
FIELD: biotechnology, microbiology, genetic engineering, molecular biology.
SUBSTANCE: invention relates to a method for preparing anthrax protective antigenic protein (PA). Protective antigenic protein is prepared by using the modified E. coli culture in its culturing with feeding. E. coli cells are transformed with the recombinant constitutive expression plasmid DH5α comprising PA gene to obtain E. coli DH5α cells expressing the protein PA. After culturing high density cells are selected, solubilized with 6-8 M urea and separated by centrifugation. Urea-denaturated PA is isolated from supernatant, purified and eluted. Also, method describes anthrax PA as 6X-histidine-fused protein prepared by this method. In culturing method involves using the primary additive chosen from the group consisting of polythiol, carbohydrate and organic acid, and LB medium (Luria broth) is used for feeding beginning from middle log-phase. As a plasmid for transformation of E. coli cells method involves vector of series pQE comprising phage promoter recognized by E. coli cells. Using the invention provides enhancing the level for producing anthrax protective antigen wherein antigen is obtained in form of protein fused with 6X-histidine.
EFFECT: improved preparing method, high level of antigen.
13 cl, 3 dwg, 1 tbl
FIELD: biotechnology, amino acids, microbiology.
SUBSTANCE: invention relates to a method for preparing L-glutamic acid by fermentation including culturing microorganisms possessing the ability for producing L-glutamic acid in medium with pH 5.0 or less wherein the total content of organic acid that inhibits the microorganism growth at this pH corresponds to the amount wherein the growth of microorganisms is not in inhibited. Also, L-glutamic acid is prepared by fermentation that includes culturing microorganism possessing the ability for producing L-glutamic acid at the first pH value wherein organic acid in medium doesn't inhibit the growth of microorganism followed by culturing microorganism at the second pH value suitable for production of L-glutamic acid by microorganism and at value lower as compared with the first value of pH. Microorganism can metabolize the carbon source in liquid medium containing L-glutamic acid at the saturation concentration and carbon source at pH 5 or less and able to accumulate L-glutamic acid in the amount exceeding the saturation concentration of L-glutamic acid. The claimed invention provides preparing L-glutamic acid with the high effectiveness degree.
EFFECT: improved preparing method.
10 cl, 13 dwg, 2 tbl, 1 ex
FIELD: biotechnology, biochemistry, enzymes, amino acids, microbiology.
SUBSTANCE: invention relates to a method for producing and preparing L-amino acids, such as L-tryptophan, L-phenylalanine, L-tyrosine and L-histidine that are prepared by culturing the modified microorganism of genus Escherichia with enhanced activity of 6-phosphogluconolactonase. The claimed invention provides preparing indicated amino acids with the high degree of effectiveness.
EFFECT: enhanced yield of amino acids.
15 cl, 9 dwg, 5 tbl, 14 ex
FIELD: biotechnology, biochemistry, amino acids, genetic engineering.
SUBSTANCE: invention relates to a method for producing L-threonine. Method involves partial inactivation of gene encoding enzyme threonine hydratase (tdc) in genome DNA of microorganism by using recombinant technology procedure. Strain of microorganism Escherichia coli pGm TN-PPC12 (KSMM-10236) is transformed with DNA fragment Δtdc::1oxpKan and the strain E. coli TRN212 (KSSM-1053) is prepared. This strain is cultured for preparing L-threonine. The claimed invention provides significant increasing the yield of L-threonine.
EFFECT: valuable properties of strain.
4 cl, 3 dwg, 4 tbl, 5 ex
FIELD: organic chemistry, medicine, pharmacy.
SUBSTANCE: invention describes a compound of the formula (I): wherein X means alkylene group; Y means -CO-, -CS- or -SO2-group; Z represents a simple bond or -NR5-group; R1 represents unsubstituted phenyl or phenyl substituted with halogen atom. (C1-C20)-alkyl group; R2 is chosen from -alkyl, -alkyl-O-alkyl; R3 and R4 represent alkyl; R5 represents hydrogen atom or (C1-C10)-alkyl group; Also, invention describes intermediate compounds - derivatives of imidazopyridine-4-amine, 2-phenoxypyridine and 4-phenoxypyridine. Proposed compounds and pharmaceutical compositions are able to stimulate biosynthesis of different cytokines and can be used in treatment of viral and tumor diseases.
EFFECT: valuable medicinal properties of compounds and pharmaceutical compositions.
32 cl, 1 tbl, 9 ex
FIELD: veterinary virology, biotechnology.
SUBSTANCE: the suggested vaccine contains avirulent and purified antigenic material out of strain A (Georgia) being homologous to infection agent 1999/N1721-DEP obtained in passaged cell culture VNK-21 being the suspension that contains, predominantly, 146S and 75S immunogenic components of foot-and-mouth disease virus, adjuvants aluminum hydroxide with saponin and maintenance medium in efficient ratios. The vaccine is of high immunogenicity and is capable to provide efficient protection against homologous infection agent circulating in Transcaucasian countries and those of Central Asia, Near and Middle East.
EFFECT: higher efficiency.
10 cl, 1 dwg, 4 ex, 10 tbl
FIELD: veterinary virology, biotechnology.
SUBSTANCE: the suggested vaccine contains avirulent and purified antigenic material out of strain A (Georgia) being homologous to infection agent 1999/N1721 obtained in passaged cell culture VNK-21 being the suspension that contains, predominantly, 146S and 75S immunogenic components of foot-and-mouth disease virus. Moreover, the vaccine contains maintenance medium and butyric adjuvant in efficient ratios. The strain has been deposited in collection of FGU VGNKI under registration number - industrial culture strain of foot-and-mouth disease virus A (Georgia) 1999/N1721-DEP of serotype A. As maintenance medium it is necessary to apply serum-free Earle's solution at addition of FGMS, GBCS and antibiotics at pH being 7.4-7.6. Out of butyric adjuvants the vaccine contains butyric adjuvant of the All-Russia Research Institute of Animal Protection (VNIIZZH) or butyric adjuvant of Montanide ISA-70 or Montanide ISA-260 marks by "Seppic" (France). The vaccine provides efficient protection against homologous infection agent circulating in Transcaucasian countries and those of Central Asia, Near and Middle East.
EFFECT: higher efficiency.
11 cl, 1 dwg, 5 ex, 8 tbl
FIELD: medicine, virology.
SUBSTANCE: invention relates to method for virus controlling using substance based on 2,8-dithioxo-1H-pyrano[2,3-d;6,5-d']dipyrimidine and 10-aza- analogs thereof. Claimed method includes sharing said substances together with integrase inhibitor, reverse transcriptase inhibitor, ore protease inhibitor in various ratio (variants).
EFFECT: method for virus inhibiting of increased effectiveness.
7 cl, 8 tbl, 8 ex
FIELD: medicine, infectious diseases.
SUBSTANCE: method involves administration of drug in the effective dose comprising compound of the formula (A) given in the invention description or acyl derivative of a bioprecursor of the formula (B) given in the invention description 18 h after infection. Invention promotes to prophylaxis of viral etiology in mammals, among them, in humans being even with impaired function of the immune system. Invention can be used in treatment and prophylaxis of infections caused by herpes virus.
EFFECT: valuable medicinal properties of compounds.
4 cl, 3 tbl, 3 ex
FIELD: medicine, virology, biochemistry.
SUBSTANCE: invention relates to a method for inhibition of viruses growth and/or virulicide effect. Method involves biotransformation of pyrazine nucleotide analog of the formula  and pyrazine nucleoside analog of the formula [3z] involving decomposition and phosphorylation resulting to preparing analog of pyrazine nucleotide of the formula [1b] and above said effect. This method can be used as a method for treatment of viral infections. Also, analog of pyrazine carboxamide or its salt can be used as an agent for prophylaxis and treatment of viral infections. Invention provides the development of a novel anti-viral agent and methods for its using.
EFFECT: valuable medicinal properties of analogs, improved method for inhibition.
50 cl, 80 ex
FIELD: medicine, biomolecular pharmacology, pharmacy.
SUBSTANCE: invention proposes a pharmaceutical composition that comprises virion vaccine anti-herpetic preparation containing simple herpes viruses of serotype 1 and 2 inactivated with formalin or γ-radiation, an immunocompetent substance and a pharmaceutically acceptable carrier. The composition comprises polyoxydonium, valine, lysine and isoleucine and also a combination consisting of at least two amino acids, not less, chosen from the following group: phenylalanine, leucine, alanine, threonine, histidine, arginine and methionine. The composition is made as a formulation on a solid carrier as tablet, lozenge, granules, sachet or powder placed into capsule, or on a liquid carrier as solution, gel, emulsion, syrup or liniment, or on a soft carrier as ointment, cream, paste, suppository, implant, chew or pastille. The composition can comprise zinc, chrome, selenium and nickel. The composition can be administrated in body by different ways: topical, oral, sublingual, intranasal, rectal, vaginal, parenteral or subconjunctival route. The composition provides the intense immunomodulating on body as a whole.
EFFECT: valuable medicinal and pharmaceutical properties of composition.
8 cl, 1 tbl, 13 ex
FIELD: organic chemistry, medicine, pharmacy.
SUBSTANCE: invention relates to medicinal agents, namely, to using fumaric acid amides of the formula (I): These compounds are used in preparing a medicinal agent designated for treatment of autoimmune diseases, response reactions "transplant against host", treatment of diseases mediated by NfkappaB, and to fumaric acid amides of the formula (I) and to a medicinal agent comprising fumaric acid amide of the formula (I) taken in the dose corresponding to 1-500 mg of fumaric acid as measured for a single dose and designated for treatment abovementioned diseases. Fumaric acid amides and a medicinal agent comprising thereof are characterized by absence of systemic adverse effect of body and resistance against hydrolysis that allows avoiding their multiply dosing.
EFFECT: valuable medicinal properties of agents.
19 cl, 2 tbl, 3 ex
FIELD: medicine, pharmacy.
SUBSTANCE: invention relates to anhydrous, antifungal, lubricating gel compositions comprising polyhydric alcohol, gelatinizing agent and an antifungal azole compound, and to a method for treatment of a patient with fungal infections comprising administration of indicated composition in a patient. Compositions shows the excellent warming and lubricating effect after its applying on skin and mucosal tissues and provides the effective treatment of fungal infections.
EFFECT: improved and valuable properties of compositions.
21 cl, 2 tbl, 11 dwg, 9 ex
FIELD: veterinary science.
SUBSTANCE: the suggested preparation for preventing and treating respiratory and gastrointestinal infectious diseases of bacterial and viral etiology in farm animals contains tetracycline (oxytetracycline) (8-10%-solution) at the quantity of about 10-15 vol.%, polyethylene glycol (15-20%-solution) - about 10-15 vol.%, polyvinyl pyrrolidone (15-20%-solution) - about 10-20 vol.%, ethylene diamine tetraacetate (0.01-0.02%-Versen's solution) - about 10-15 vol.%, and, also, trypsin (0.01-0.25%-solution) - the rest. As for the method for preventing and treating the above-mentioned diseases, it deals with a single intramuscular injection of the present preparation for farm animals (predominantly, for piglets) at the dosage of about 0.5-1.0 ml/animal. The suggested preparation is of immunostimulating properties and provides efficient therapy of respiratory and gastrointestinal diseases of bacterial and viral etiology.
EFFECT: higher efficiency of prophylaxis and therapy.
2 cl, 6 tbl
FIELD: medicine, gynecology, proctology, pharmacy.
SUBSTANCE: invention relates to medicaments made as vaginal and rectal suppository used in treatment of proctological, non-inflammatory and inflammatory diseases of female pelvis organs, among them complicated with suppurative-inflammatory processes. Invention relates to an agent used in treatment of gynecological and proctological diseases it is made as a suppository and comprises 2-ethyl-6-methyl-3-oxypyridine succinate as an active component and a base chosen from group involving polyethylene oxide-1500, polyethylene oxide-400, cacao butter and hydrogenated fats. Invention provides prolonged release of a curative preparation in mucosa and soft tissues and provides its effect for about 12 h. The procedure is suitable and easy in performance and can be carried out by patients independently under ambulatory conditions.
EFFECT: valuable medicinal and pharmaceutical properties of agent.
3 cl, 2 tbl, 2 ex