Immunostimulating composition for animals

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to chemical-pharmaceutical industry and veterinary science, and represents an immunomodulatory composition for animals which contains lactoferrin milk whey protein as active substance, and distilled water as a solvent. In addition to lactoferrin, the active substance contains lactalbumin and lactoglobulin milk whey proteins, wherein the active substance represents an aqueous solution of mixed milk whey proteins of the total protein concentration of 10 g/l, containing 10-20% of lactoferrin, 20-30% of lactalbumin, 60-70% of lactoglobulin; moreover, the active substance further comprises selenium nanoparticles in the following proportions, wt %: aqueous solution of mixed milk whey proteins of the total protein concentration of 10 g/l containing 10-20% of lactoferrin, 20-30% of lactalbumin, 60-70% of lactoglobulin, 0.1-100 selenium nanoparticles 00001-10, distilled water up to 100.

EFFECT: invention provides a higher immunostimulating effects on the cell and humoral immunity.

3 ex, 3 tbl

 

The invention relates to veterinary medicine, namely to pharmacology, and can be used as an immunostimulatory agent.

Currently in treatment and prevention of diseases, both infectious and non-infectious etiologies include a number of immunostimulatory drugs to increase the overall resistance of the organism or its non-specific immunity, as well as the influence of specific immune reactions. There are several groups of immunostimulatory drugs. The first group of Immunostimulants are synthetic Immunostimulants. It contains: levamisole (decaris), lagadin, diazepan etc. the Second group presents the endogenous Immunostimulants and their synthetic analogues. It includes preparations of thymus, bone marrow, spleen and their synthetic analogues (thymalin, timogen, taktivin (T-activin), timoptic, tinactin, thymostimulin, Imunofan, mielopid, splenin), immunoglobulins (human polyvalent immunoglobulin (Intraglobin)), interferons (human immune interferon-gamma, recombinant interferon-gamma (hammeron, imukin)), interleukins (recombinant interleukin-2 (aldesleukin, Proleukin, Roncoleukin), a recombinant interleukin 1-beta (betalain) etc). The third group includes the products of microbial origin and the x synthetic analogues (pyrogenes, prodigiozan, ribomunyl, Imudon, Likopid, etc.). The fourth group is represented by drugs of different pharmacological classes with immunostimulatory activity. It contains adaptogens and herbal drugs (Dibazol, bemythyl, preparations of Echinacea, Eleutherococcus, ginseng, Rhodiola rosea, tonsillar N and so on), and vitamins (ascorbic acid (vitamin C), tocopherol acetate (vitamin E), retinol acetate (vitamin a)).

Closest to the claimed invention is an injectable therapeutic veterinary drug "poliferrin"containing the active substance lactoferrin secreted from colostrum animals http://www.narvac.com/art_poliferrin.htm, http://www.webvet.ru/equipment.asp?e_id=1954; http://www.veterinarka.ru/content/view/1142). Lactoferrin is a natural mammalian glycoprotein, it belongs to the iron-containing proteins that are responsible for the primary protection of the organism as immunostimulating substance (http://www.narvac.com/art_laktoferrin3.htm: Lactoferrin as a modulator of immune and inflammatory processes. D.Legrand, E.Elass, M.Carpentier and J.Mazurier). However poliferrin not actively stimulates cell-mediated immunity in animals, in some cases, causes allergic reactions.

The technical challenge is to improve immunostimulating effects on cellular and humoral immunity.

The technical result to the th can be obtained from the use of the claimed composition, is an increase of functional activity of the drug, including enhancing the stimulating effect not only on the cell, but also on humoral immunity by direct presentation of the active components of the drug into the cells of the reticuloendothelial system.

The technical result is achieved by the fact that the immunomodulating composition for animals contains as active substance protein whey milk lactoferrin, the solvent distilled water, according to the invention the active substance includes, in addition to lactoferrin whey proteins of milk lacto albumin and lactoglobulin, while actively active substance is an aqueous solution of a mixture of whey proteins of milk with a concentration of total protein 10 g/l, containing 10-20% lactoferrin, 20-30% milk albumin, 60-70% lactoglobulin, in addition, the specified active substance further comprises nanoparticles of selenium in the following ratio of components, wt.%:

an aqueous solution of a mixture of whey proteins milk
with the concentration of total protein 10 g/l, containing
10-20% lactoferrin, 20-30% lactoalbumin is a,
60-70% lactoglobulin0,1-10,0
the selenium nanoparticlesof 0.0001 to 1.0
distilled waterup to 100.

What is new is that the joint application active substance 3 whey proteins of milk lactoferrin, milk albumin and lactoglobulin, and the introduction of active substance selenium nanoparticles was first proposed for use as an immunostimulating drug. These three protein in combination with each other increases the stimulatory action of active substances. The selenium nanoparticles included in the composition of active substances, which give the songs an extra antioxidant effect.

Immunostimulirutuyu composition for animals prepared as follows. From whole cow's milk get sour whey, consisting of lactoglobulin, milk albumin, immunoglobulins, lactoferrin and other proteins by yearning citric acid. Proteins from the serum is extracted by precipitation of their ammonium sulfate. The precipitate is dissolved with distilled water to obtain a protein suspension, from which the separated mixture lactate is Rina, milk albumin, lactoglobulin: protein suspension is subjected to dialysis using a dialysis membrane with a pore diameter of 12000-14000 Da to remove proteins with a molecular mass of less than 12000 Yes. At the end of dialysis receive an aqueous solution of a mixture of three proteins, which contains 10-20% lactoferrin (molecular weight 75000-80000 Yes), 20-30% milk albumin (molecular weight 18000 Yes), 60-70% lactoglobulin (molecular weight 67000 Yes). With this method of obtaining a mixture of three of these proteins and their percentages are constant, which is confirmed by the inventors through research of an aqueous solution of a mixture of three proteins by the method of native polyacrylamide gel electrophoresis using standard (http://molbiol.ru/protocol/17_01.html). These studies treated statistically using t-test. This way electrophoresis confirmed the above indicators of molecular weight of proteins lactoferrin, milk albumin, lactoglobulin.

In the obtained aqueous solution of a mixture of three proteins (lactoferrin, lacto albumin, lactoglobulin) determine the concentration of total protein. If the concentration of total protein greater than 10 g/l, the solution was diluted with distilled water to a concentration of 10 g/L. Then the composition is administered selenium nanoparticles trail is accordingly. In an aqueous solution of a mixture of three proteins add a 1M solution of hydrazine hydrochloric acid and 1M solution of sodium Selenite NaSeO4then the volume of the mixture was adjusted with distilled water to 100 wt.%. Stop the reaction by bringing the pH of the solution to 7.2 1M sodium hydroxide solution and freed from low molecular weight compounds by dialysis against distilled water. In the end you get immunostimulirutuyu composition for animals, containing:

an aqueous solution of a mixture of whey proteins milk
with the concentration of total protein 10 g/l, containing
10-20% lactoferrin, 20-30% milk albumin,
60-70% lactoglobulin0,1-10,0
the selenium nanoparticlesof 0.0001 to 1.0
distilled water100

Selenium in immunostimulatory composition for animals is in the nano state: particle size colloidal selenium in the product ranges from 60 to 140 nm, which is confirmed by electronic microscope electronic microscope LIBRA 120 (Carl eiss, Germany).

Example 1

An aqueous solution of a mixture of whey proteins milk
with the concentration of total protein 10 g/l, containing
10-20% lactoferrin, 20-30% milk albumin,
60-70% lactoglobulin0,1
the selenium nanoparticles0,0001
distilled water100

Example 2

An aqueous solution of a mixture of whey proteins milk
with the concentration of total protein 10 g/l, containing
10-20% lactoferrin, 20-30% milk albumin,
60-70% lactoglobulin5,0
the selenium nanoparticles0,01
distilled water100

Example 3

An aqueous solution of a mixture of whey proteins milk
with the concentration of total protein 10 g/l, containing
10-20% lactoferrin, 20-30% milk albumin,
60-70% lactoglobulin10,0
the selenium nanoparticles1,0
distilled water100

To establish enhancing immunostimulatory properties of the claimed composition in comparison with analogues were studied:

the immunostimulating effect of the composition on the cellular immune response (studies on the impact of immunostimulatory composition on the survival of Staphylococcus aureus in the presence of peritoneal cells of animals);

the immunostimulating effect of the composition on the humoral immune response (studies on the impact of immunostimulatory composition on the effectiveness of immunization of animals);

the immunostimulating effect of the composition on nonspecific resistance of the organism (studies on the impact of immunostimulatory composition on the redox activity of the cells).

p> When conducting research on the impact of immunostimulatory composition on the survival of Staphylococcus aureus in the presence of peritoneal cells of animals received peritoneal cells from mice (PKM) by a standard method at a concentration of 3 million cells/ml In experiment used a cell culture of microorganisms Staphylococcus aureus 209-P in logarithmic growth phase at mesopatamia broth to obtain a concentration of Staphylococcus 200 million CFU/ml and 20 million CFU/ml

In the 1st version of the research (table 1) was mixed:

I. peritoneal cells from mice at a concentration of 3 million cells/ml, cell culture of these microorganisms at a concentration of 200 million CFU/ml and immunostimulirutuyu composition.

II. peritoneal cells from mice at a concentration of 3 million cells/ml, cell culture of these microorganisms at a concentration of 20 million CFU/ml and immunostimulirutuyu composition.

In the 2nd version of the research (table 1) was mixed:

I. peritoneal cells from mice at a concentration of 3 million cells/ml, cell culture of these microorganisms at a concentration of 200 million CFU/ml

II. peritoneal cells from mice at a concentration of 3 million cells/ml, cell culture of these microorganisms at a concentration of 20 million CFU/ml

In the 3rd version of the research (table 1) was mixed:

I. cell culture of these microorganisms at a concentration of 200 million CFU/ml and immunity olyroos composition.

II. cell culture of these microorganisms at a concentration of 20 million CFU/ml and immunostimulirutuyu composition.

In the 4th version of the research (table 1) were studied:

I. cell culture of these microorganisms at a concentration of 200 million CFU/ml

II. cell culture of these microorganisms at a concentration of 20 million CFU/ml

In all variants of the obtained cultures were incubated 40 min at 37°C, determine the number of CFU of Staphylococcus planting of dilutions of the original samples for salt mastopathy agar and counting the number of grown colonies. Expected activity of phagocytosis PKM by the formula:

AF=100×(a-b)/a,

where a is the concentration of Staphylococcus aureus in the 4th embodiment, b is the concentration of Staphylococcus aureus in the 1st option or 2nd option, or the 3rd version of the study.

The research results are reflected in columns 7 and 8 table 1.

When evaluating the results of the studies (table 1):

- compared the concentration indices of the cells of Staphylococcus aureus after incubation of the 4th variant of studies with similar indices 1, 2 and 3rd options: in the 1st case, the survival of Staphylococcus decreases significantly in the 2nd version, a slight inhibition of growth of Staphylococcus and 3-m variant found that immunostimulirutuyu composition does not possess bacteriostatic effect against the taphylococcus, indicating the absence of the negative effects of immunostimulatory composition to the cells of a living organism;

- analyzed the activity of phagocytosis PKM: 1-m option in the feedback immunostimulatory composition PKM activates cellular immunity, resulting in stunted growth of Staphylococcus by increasing phagocytic activity PKM 2-8 times in comparison with the 2nd option.

When conducting research on the impact of immunostimulatory composition on the humoral immune response carried out the study of the influence of immunostimulatory composition on the effectiveness of immunization. The experiment used a 3 mice groups of 3 animals each. Mice of the first group (control) throughout the immunization was administered intraperitoneally biomass field isolate Salmonella thyphimurium, killed by heating. The mice of the second group were injected intraperitoneally mixture of biomass Salmonella and immunostimulatory composition, the dose of which was 0.4 ml per 20 g body weight. Mice of the third group received intraperitoneally immunostimulirutuyu composition in the above dose and after a day or biomass Salmonella. Dose of wet biomass of Salmonella in all groups was 0.2 mg per 20 g body weight. Immunization was performed with an interval of 2 weeks - 4 injection. Before slaughter conducted beads the licensing biomass of Salmonella. The titer of agglutinating antibodies in the serum of experimental animals was determined using the method of radial immunodiffusion 1.5% agar gel. Table 2 presents the results of determining the titer of agglutinating AT in the sera of immunized mice.

8
Table 2
The agglutinating titers AT against Salmonella in sera of mice immunized with biomass Salmonella in conjunction or in parallel with immunostimulatory composition
№ p/pThe degree of dilution serumArea precipitation
group 1group 2group 3
1156,256452,5625
224956,2549
34364936
430,2542,2530,25
51620,253627,5625
63220,2530,2520,25
76412,2520,2512,25
8128012,250
9256090
10512000

As can be seen from table 2, the highest titer AT detected in the serum of mice immunized with a mixture of Salmonella and immunostimulatory composition. In mice immunized with only biomass, and mice immunized with biomass through day after injection immunostimuliruyushchie song titles AT similar and significantly lower than alive is the shaft, receiving immunostimulirutuyu composition and antigen simultaneously. Immunization biomass of Salmonella together with immunostimulatory composition is more effective, while preliminary, the day before immunization, injection of immunostimulatory composition does not affect the level AT in serum compared with the control. Conclusion: immunostimulirutuyu composition for immunization stimulates phagocytosis corpuscular antigen, thereby facilitating more complete and effective presentation to the immune system of the host.

The effect of immunostimulatory composition on nonspecific resistance of organism conducted a study of the influence of immunostimulatory composition on the redox activity of the cells. In one embodiment of the study drug on the prototype was made in monoclonal culture cells in a dose of 0.5 ml per 10 ml of medium, in the second embodiment of the study drug on the proposed composition was made in monoclonal culture cells in a dose of 0.5 ml per 10 ml of medium. Studies were performed on cell lines SPEV-2. Cultivation was carried out for 48 hours, after which the cells were removed by trypsinization and they determined the intensity of respiration in the MTT test. Table 3 outlines the comparative indicators of changes in respiratory activity in the cell population when it is litwinowii them in the presence of the drug on the prototype and the proposed immunostimulatory composition (P< =0,05).

Table 3
Comparative indicators of changes in respiratory activity in the cell population during culturing them in the presence of the drug on the prototype and the proposed immunostimulatory composition (P<=0,05)
IndexMonoclona culture cell lines SPEV-2 (Control)The preparation of the prototype + monoclona culture cell lines SPEV-2Immunostimulirutuyu composition + monoclona culture cell lines SPEV-2
The concentration of formazan in 1 cage, Ng/ml0,015217±0,02545±0,071245±
0,0033810,0056530,012305

As can be seen from table 3, when culturing cells in the presence of immunostimulatory composition increases respiratory activity 4-fold, compared with control, as well as more than 2 times in comparison with the preparation of the prototype that proves the ability of the proposed immunostimulatory composition for animals to activate acyclical the but-reduction processes of cellular metabolism.

Immunomodulating composition for animals comprising as activitiesthese substances whey protein milk lactoferrin, the solvent distilled water, characterized in that activetestsuite substance includes, in addition to lactoferrin whey proteins of milk lacto albumin and lactoglobulin, while activetestsuite substance is an aqueous solution of a mixture of whey proteins of milk with a concentration of total protein 10 g/l, containing 10-20% lactoferrin, 20-30% milk albumin, 60-70% of lactoglobulin, in addition, specified activetestsuite substance further comprises nanoparticles of selenium in the following ratio, wt.%:
an aqueous solution of a mixture of whey proteins of milk with a concentration of total protein 10 g/l, containing 10-20% lactoferrin, 20-30% milk albumin, 60-70% of lactoglobulin 0.1 to 100, selenium nanoparticles of 0.0001 to 1.0, distilled water to 100.



 

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9 cl, 12 dwg, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to novel derivatives of imidazo[4,5-c]chinoline of general formula or to its pharmaceutically acceptable salts, where R1 represents straight-chained C1-C6alkyl, possibly substituted with one substituent, selected from C1-C3alkoxy; Z1 represents C2-C6alkylene; X1 represents NR5 or >NCOR5; Y1 represents C1-C6alkylene; R3 represents C1-C6alkyl, possibly substituted with C1-C6alkoxy; R5 represents hydrogen, piperidinyl, possibly substituted by piperidinyl nitrogen with group R10, group C1-C6alkyl, where the last group is possibly substituted with one substituent, independently selected from NR7R8 or R9; or R5 represents C1-C6alkylene, which can be bound with carbon atom in C2-C6alkylene group Z1 with formation of piperidine ring; each of R7 and R8 independently represents tetrahydropyranyl, piperidinyl, possibly substituted by piperidinyl nitrogen atom with group R10a, C1-C6alkyl, where the last group is possibly substituted with one group, independently selected from OR12; or R7 and R8 together with nitrogen atom, to which they are bound, form 4-7-membered saturated heterocyclic ring, selected from asetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, azepanyl, 1,4-oxazepanyl and 1,4-diazepanyl, where heterocyclic ring is possibly substituted with one or two substituents, independently selected from S(O)qR15, OR15, CO2R15, COR15, CONR15R16, NR15CO2R16, pyrimidinyl and C1-C6alkyl, where the last group is possibly substituted with one group, independently selected from OR18 and CO2R18; R9 represents S(O)qR20; R10 and R10a independently represent COR2 or group C1-C6alkyl; each of R12, R15, R16, R18, R20 and R24 independently represents hydrogen or C1-C6alkyl; q equals 2; m and n both equal 0; and A represents phenyl. Invention also relates to method of obtaining formula (I) compound, based on it pharmaceutical composition, and to method of treating said pathological conditions.

EFFECT: obtained are novel derivatives of imidazo[4,5-c]chinoline, useful modulation of TLR7 activity.

17 cl, 18 dwg, 81 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry and concerns an ergogenic formulation possessing adaptogenic, hepatoprotective and immunomodulatory action, and may be used in hepatic diseases, in decreases in immunity and increase in performance efficiency and tolerance. The composition contains burnut extract, zinc aspartate, magnesium aspartate, vitamin B6, water in certain proportions of the ingredients.

EFFECT: composition possess higher pharmacological activity and bioavailability.

3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, namely immunology and may be used for treating rheumatoid arthritis. That is ensured by the use of a pharmaceutical composition containing an active principle presented by a monoclonal antibody which recognises a CD6 human leukocyte differentiation antigen and produced by secreting hybridoma IOR-TIA with deposition No. ECACC 96112640, and an applicable excipient. What is also presented is a method for the use of the pharmaceutical composition in the form of injections, as well as the use of a monoclonal antibody which recognises the CD6 human leukocyte differentiation antigen, for preparing a therapeutic agent.

EFFECT: group of inventions provides a prolonged therapeutic effect in the patients suffering rheumatoid arthritis after a short period of administration.

3 cl, 3 dwg, 3 ex

Hematogen // 2485961

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely to hematogen. Hematogen containing edible black albumin, glucose syrup, condensed milk with sugar added, granulated sugar, vitamin C, a flavouring agent, butter and drinking water with a certain grain size and ratio of the ingredients.

EFFECT: presented formulation of hematogen enables better nutritional properties, caloric and biological content, improved organoleptic properties of hematogen, higher bioavailability of the biologically active ingredients, prepared product with a shelf life prolonged to 12 months.

4 cl, 1 tbl, 3 ex

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