Peptides having immunostimulatory activity, the retrieval method, the drug based on splendid and its application

 

(57) Abstract:

The invention relates to medicine, namely to preparative biochemistry, pharmaceutical industry and biotechnology and relates to peptides having immunostimulatory activity, the method of their derivation, drug Splendida created on the basis of these peptides and its application. The invention consists in obtaining peptides mol. m, from 400 to 50000 Yes fabric spleen by mitogenic stimulation of cells in the tissue of the spleen in the process of washing, homogenizing body by dispersing in distilled water and the destruction of the cells by freeze-thawing and ultrasonic treatment, separation of the extract and its ultrafiltration through a filter with pore size with boundary 50000 Yes. Adding to selected peptides of the filler of the drug is produced in dried form, is stable during storage for two years. The invention provides a high efficiency of the drug in the treatment of severe forms of septic and autoimmune diseases. 4 C. and 3 h.p. f-crystals, 5 PL.

The invention relates to medicine, namely to preparative biochemistry and pharmaceutical is the only drug Splendid, created on their basis, and its applications.

Currently shown clinical efficacy in the treatment of infectious diseases, septic and hematologic diseases cytokines are soluble factors of the immune cells and presents peptides. Cytokines obtain in pure form from natural sources raw: thymus and bone marrow of mammals, as well as from cells stimulated by mitogens. It is now widely known cytokines obtained by the chemical and recombinant synthesis. To improve the efficiency of their use are being established pharmaceutical compositions containing an extensive set of synthetic cytokines.

As for spleen mammals, it is known that the perfusion solution of the spleen of a pig has a pronounced immunostimulating activity, caused by a complex of native cytokines: interleukin-1, interleukin-2, interferon-gamma, tumor necrosis factor-alpha, granulocyte-macrofouling colony-stimulating factor [1]. However, attempts to obtain pure biologically active peptides from the spleen of the relatively few. Thus, a method of obtaining immunostimulant peptide washed with water or acetone, crushed, frozen at -5oC, extracted with 3% acetic acid at pH 3.0 to 4.0 in the presence of zinc chloride, centrifuged, mixed with acetone to obtain a residue which is dissolved in water in the presence of acetic acid followed by filtration and lyophilization. However, the resulting preparation contains a set of peptides with indefinite molecular weight, does not have a high stimulating activity, and also contains large amounts of impurities.

Known peptidemia fraction isolated from the spleen of a pig or cattle, with a molecular weight of from 10,000 to 140,000, Dalton [3] . Specified peptidemia fraction contains not only pure proteins, but also about 20-25 amino acids. As for how it is received, it is analytic because the allocation fraction with the specified molecular weight necessary for division peptidase mixture using chromatography high pressure. The method consists of washing the spleen, grinding, freezing, extraction of phosphate buffer, the separation of the precipitate by centrifugation, ultrafiltration of the supernatant liquid, adsorption on halfspace calcium and allocate the specified f is a method of obtaining and studied some properties of immunomodulator peptide from the spleen of a pig with a molecular mass of peptides from 700 to 15,000 daltons, followed by lyophilization of the product [4]. The resulting modulator has a high stimulating activity, but also contains impurities. As for the pure peptides isolated from spleen, known peptide hil-2, isolated from the supernatant of spleen cells stimulated by concanavalin And that is the kind of interleukin-2 and consists of 140 amino acids, featuring many of the properties of interleukin-2 [5], as well as splenopathy, which pentapeptides, immunological properties, such splenin [6]. These peptides can be used in a neutral or salt form, may include non-protein part, and various additives, which make them suitable for use in injection form, but their use is very limited. At the same time, the literature describes the use of perfusate xenoculture for the treatment of patients with extensive purulent-destructive diseases of lungs and pleura with complicated course [7] , and selenoprotein for the treatment of such autoimmune diseases as systemic lupus erythematosus, rheumatoid arthritis and bronchial asthma [8], while therapeutic effect is due to the properties of this body to produce large amounts of immunomodulatory substances peptide.

The invention consists in obtaining peptides from the cells of the tissue of the spleen with a molecular mass of 400-50000 daltons obtained by mitogen stimulation in the process of washing the tissue of the spleen, destruction of cells dispersed in water, pre-digested tissue and ultrafiltration separated extract through a filter with pore size with boundary 50000 daltons.

The method of producing peptides having immunostimulatory activity, involves the stimulation of the cells in the tissue of the spleen tissue, dispersed in distilled water after pre-homogenization body with subsequent ultrasonic treatment, the separation of the extract and its ultrafiltration through capillary filters with pore size with boundary 50000 daltons.

Drug Splendid having immunostimulatory activity, represents the peptides from the cells of the tissue of the spleen with a molecular mass of 400-50000 daltons obtained by mitogen stimulation in the process of washing the tissue of the spleen, destruction of cells pre-digested tissue, dispersed in distilled water, and ultrafiltration to separate the extract through a filter with pore size with boundary 50000 daltons, taken in an amount of 10-15 mg protein dose, and physiologically suitable filler, which can be used relational with gentamicin. The drug is produced in dried form by freeze drying of peptides with filler, including the freezing of freeze-dried at -35oC and drying for 48 hours in the freeze-drying chamber at -30oand the vacuum of 0.1 Torr.

The method of treatment of purulent-septic diseases includes comprehensive background thereto within one day in amounts of 5-20 mg protein within 3-10 days.

The method of treatment of autoimmune diseases includes additional intramuscular patient drug drug Splendid daily, once a day in amounts of 5-20 mg protein within 3-10 days.

The invention is implemented as follows.

Sterile taken spleen pigs or cattle washed with saline containing mitogenic stimulator phytohemagglutinin or concanavalin And mitogenic stimulation of splenocytes. The spleen is placed in the transport container at a temperature of 4-6oC and delivered in a sterile box, spleen shredded with scissors, and then in the homogenizer, the homogenate add distilled water and the destruction of the cells are repeated freeze-thawing followed by sonication. Then the mass is centrifuged or filtered through a coarse filter and the resulting extract is subjected to ultrafiltration through a capillary filters with boundary 50000 daltons. Ultrafiltrate represents the fraction containing peptides, polypeptides and low molecular weight proteins with a molecular weight of 400 to 50,000 daltons. To obtain stable when stored is hydrated relational and the solution of gentamicin, poured into 5 ml vials and subjected to freeze drying at the temperature of freezing -35oC, the temperature in the freeze-drying chamber is -30oC, the vacuum level -0,1 tor, time sublimation - 48 hours. The dried preparation is tightly closed with a rubber cap, sealcoat aluminum cap and label. To test the biological activity of the contents of the vial are dissolved in distilled water and check the proliferative activity of lymphocytes, as well as the influence of the drug on the course of autoimmune process in experimental animals and induced experimental sepsis and experimental mielodepressiu. Next, perform a test of the drug Splendid in the clinic for the treatment of severe forms of autoimmune (systemic lupus erythematosus, rheumatoid arthritis, and so on) and purulent-septic diseases.

In the treatment of severe forms of septic and autoimmune diseases the patient on the traditional treatment intramuscularly drug Splendid in the amount of 5-20 mg of protein in the daily dose, once a day for 3-10 days.

Examples of the method.

Example 1. In the Department of biological preparations of meat osushestvliayut 1 l of 0.9% sodium chloride solution with the addition of 30-40 mg of phytohemagglutinin. The spleen in a sterile plastic bag and put the transport container (temperature +4-6oC) transferred to a sterile Boxing lab. The drug is produced by rough grinding the tissue of the spleen scissors with subsequent fine grinding homogenizer, add 1600 ml of distilled water. The subsequent destruction of the cell mass exercise 3x freeze-thawing, followed by ultrasonic destruction (22 kHz 60 sec). Next, the mass centrifuged at a temperature of +4-6oC for 30 minutes at 3000 Rev/min the Supernatant is collected in tanks for subsequent filtration through capillary polysulfone filters with boundary 50000 daltons. Get a 1 l ultrafiltrate with a molecular mass of 400-50000 daltons. As filler and stabilizer before sublimation 1000 ml ultrafiltrate add 1663,0 ml officinal of Gelatines and 1,20,1 ml of 4% officinal solution of gentamicin. The mixture is poured into vials of 10 ml (from neutral glass) 5 ml 5 ml contains 8 to 10 ml total protein determination by Lowry). Then hold the freeze drying of ultrafiltrate on the machine LZ-45 (Czech Republic). Mode lyophilization has the Amer - 30oC, the level of vacuum in the freeze-drying chamber is 0.1 Torr. Time sublimation 48 hours. The dried preparation is tightly closed with the lid and sealcoat aluminum cap and label. The drug is porous mass of pale yellow, soluble in water and in 0.9% sodium chloride solution. The humidity of the drug is 5%, the amount of protein 8-10 mg. At sowing 2 vials of the drug is a sterile, non-toxic, aerogenes, stable when stored at +4-6oC for 2 years, the pH of the preparation when dissolved in water is 7.0-8.5 in.

The product had a specific immunostimulirute action, identified by increased proliferative activity of lymphocytes.

Specific and non-specific impurities. Exercise control on the absence of peptides with molecular weight more than 50,000 daltons. For this purpose the contents of one vial was dissolved in 5 ml of 10% aqueous solution of formic acid in water and 0.4 ml of solution is injected in a glass chromatographic column h filled with Sephadex G-50 (Fine Pharmacia, Sweden). Sample chromatographic at a flow rate of 0.15 ml/min Registration produced by UV absorption at a wavelength of 280 nm. Pre-determine the chromatograph exit area off the Ana and 0.2 ml of 0.01% aqueous solution ciankobalamin in 10% formic acid. The exit area on the chromatogram blue dextran corresponds to the free volume of the column.

The chromatogram of the sample of the drug should not be peaks of UV-absorbing material in the exit area of the free volume.

Example 2. In a similar way secrete biologically active fraction of peptides from tissue spleen of cattle, except that the laundering of the fabric is done by adding concavalin And intact and ruined cells are removed by filtration through a coarse cloth filters. And 1 kg spleen receive 1 liter of ultrafiltrate.

Determination of proliferative activity of lymphocytes. Getting lymphocytic suspension: blood taken from the ulnar vein, transferred into sterile tubes with 2.7% R-rum Na-EDTA (pH of 7.4) at a rate of 1 ml per 10 ml of blood. Thoroughly mixed with an anticoagulant to prevent clotting.

- Obtained blood bred HEPES-buffer (without ions Ca and Mg) in the ratio 1:3.

In the centrifuge tube pour 3 ml of R-RA ficol-urografin (with a density of 1.077 g/cm). Using a Pasteur pipette 8 ml of diluted blood gently layer on the wall of the tube on the solution ficol-urografin, close spoke centrifugation of whitish rings, formed on the border of the mixture ficol-urografin and diluted blood, the Pasteur pipette select a suspension of mononuclear cells (lymphocytes and monocytes) and count the number of (P) by the formula:

< / BR>
where K is the number of shared blood, M is the number of leukocytes in 1 ml of blood, F is the fraction of lymphocytes, V is the volume of the suspension of lymphocytes after separation of blood in ml, N is the concentration of lymphocytes in 1 ml of suspension.

The output of lymphocytes, of not less than 70 - 90% is considered satisfactory. The concentration of the selected cells in sterile conditions lead up to 510 6 degree cells/ml of medium RPMJ 1640 containing 10% human serum IV(AB) group or veal; 12 mm HEPES, 300 μg/ml glutamine, 100 μg/ml of each antibiotic and drug Splendid concentrations from 0,00012 to 0.12 mg protein/ml and covered with a sterile tubes.

- Automatic pipette the cell suspension is poured into 20 μl to the wells of the 96-well plate to immunological reactions. In the wells of the control cultures for evaluation of spontaneous proliferative activity of lymphocytes add 20 µl of RPMI medium 1640, in the wells of the control cultures for the evaluation of mitogen-induced proliferative activity of lymphocytes in addition to 20 µl of RPMI medium 1640 concentrations of the drug. Each culture takes on 3 holes.

Culture for 72 hours placed in a thermostat at 37oC in a humid atmosphere containing 5% CO. 4 hours before the end of cultivation in each well is made of 20 ál R-RA H-thymidine with beats. activity 10 µci/ml without sterility.

After completion of cultivation every Wednesday automatic pipette (preferably using automatic collection of cell Harvester) carefully resuspended and transferred to glass fiber filters, dried, and each section of the filter with a breakdown of individual wells is transferred into a vial with 2 ml of scintillation fluid (1 kg of toluene, 4 PPO and 061 g - POPOP).

The count enable H-thymidine produce scintillation counter (counts per minute), calculates the average value is included in 3 of the control and without mitogen (spontaneous proliferation - SP) and 3 with mitogen (induced proliferation - PI), and proliferation when exposed to different concentrations of the drug cultures of lymphocytes; Proliferative activity is expressed by the "stimulation index" is calculated by the formula:

< / BR>
Normal IP - 50-60%.

In low doses, the drug should stimulate lymphocyte proliferative activity, in high doses - the influence of the drug Splendid study on inbred mice-female NZB 10 months of age with an active experimental lupus erythematosus, with severe lupus-nephritis. Experimental mice injected the drug in the dose of 0.2 mg per animal. The drug is administered 2 times a week for 2 months from 10 months of age (a total of 16 injections). Control animals injected fiziologicheskii solution in the same volume. Blood for the study of anti-DNA antibodies and the CEC taken from the retro-bulbar plexus prior to the introduction of the drug, at the age of 11 months (8 injections) and 13 months (1 month after completion of the course of injection). The content of antibodies to native DNA is determined using radioisotope techniques and standard kits Kit company Amersham (UK). The CEC identified by the method of 3% PEG-precipitation on a laser turbidity meter company "Haechat-Bechring" (Germany) and print on the computer Hewlett-Packard-85 (USA). Deposition of immune complexes in the kidneys examined in histological sections fluorescing serum against immunoglobulins of mice, labeled with isothiocyanates of flourescein (research Institute of epidemiology and Microbiology. N. F. Gamaleya RAMS). The area occupied by deposits of immune complexes (IC) in vascular glomerulus of the kidney assess on a 5-point system. They control the survival rate of mice and determine the protein content in daily Issledovanii presented in table 1.

From this table it is seen that under the influence of drugs is a significant reduction of anti-DNA antibodies and the CEC in serum, decreased content of immune complexes in the glomeruli of the kidneys, as well as the concentration of protein in the daily portions of urine. It was also established that the average lifespan of mice and the number of surviving animals to 13-months of age was more than 3 times higher in the experimental group.

Thus, the drug Splendid NZB mice with a pronounced lupus process is able to reduce the activity of the autoimmune process.

Example 4. The effect of the drug on the course of experimental sepsis. Impact on septic process of the preparation of the tissue of the spleen is studied in experiments on outbred mice. Experimental sepsis cause intravenous animals day living culture Staph. aureus strain Wood-46" in a dose of 0.2 ml with a concentration of 1 billion cells in 1 ml of Sepsis developed over 3 days with LD-50. After infection, animals were divided into 2 groups (30 animals in each). In group I at 3, 4, 5 days after infection intravenously injected in 0.2 ml of saline. In group II during the same period were injected with 0.2 ml of the drug Splendid.

Rasula, while in the control survival rate of mice was 38%.

To explore mechanisms for increasing the resistance of mice to septic process was studied by changing physiological characteristics of cells (phagocytosis, luminal-dependent chemiluminescence, NST-test) under the influence of the drug spleen. It is established that under the influence of the drug increase the absolute phagocytic index of leukocytes by approximately 100% (experience - 906925231; control - 459202145, p<0,05); capture of microbes by leukocytes (Wright) 92% (control 8,50,05, experience - 16,31,2, p<0.05) and the percentage of digesting microbes by 55%.

Under the influence of the drug spleen leukocyte chemiluminescence in vitro was increased 2.5 times (experience 3722253+856712; control 1507456+242052, and NST is the reaction of cells in 2 times (experience 386+11,2; control 194+9,5). Thus the introduction of anti-infective drug increases protection in animals with experimental sepsis.

Example 5. The drug Splendid on experimental myelodepression.

The study was conducted on 2 groups of outbred rats-males with ciclofosfamida mielodepressiey:

group 1 - control with the introduction of cyclophosphamide;

group 2 - the experience with the introduction of cyclophosphamide and so the AZ daily for 2 days. The white blood cell count fell by more than 5 times, lymphocytes about 6 times, neutrophils 1/3 from baseline (table 2). Preparation of spleen injected intraperitoneally at a dose of 1.6 mg protein/kg once daily for 3 days. Blood for research is taken from the tail vein. The number of leukocytes, erythrocytes and platelets determined by standard methods. In the experimental rats after drug administration (table 2) there was an increase in leukocytes from 1.8+0.6 V control to 4.4+0,4 10/L. Stab leukocytes with 0,86+0.03 in control until 1,29+0,04 10/l lymphocytes from 1.2+0.04 in the control to 3.1+0,03 10/l and monocytes from 0.03+0.001 in the control to 0.11+0,001 10/l For 5-th day the favorable effect of the drug on the studied parameters was more pronounced; to 14 days in the experimental group did not differ from the outcome, whereas in the control group, they were still below normal (table 2).

Thus, the experiments indicate that the drug Splendid has a strong immunocorrective action of various pathological conditions associated with dysfunction of the immune system. It is established that the drug Splendid received by our technology, stimulates metabolic and functional activity is Noah lupus that clearly associated with increased activity faguoqitirute cells. It is likely that these mechanisms play an important role in the implementation of therapeutic actions of the drug in experimentally induced pathological conditions, sepsis, systemic lupus erythematosus and pharmacological mielodepressii.

Examples of clinical application.

Example No. 1. Patient P., aged 27. Transferred to the surgical Department of the city clinical hospital N 67 21.04.89, the Diagnosis of Peritoneal sepsis, catabolic phase septicopyemia. Sick with 20.03.89, 5.04.89, the operation was performed for acute appendicitis. After 11 days developed purulent peritonitis. The patient is listless, dinamico, pale skin, cyanosis of the lips, acrocyanosis tachypnea to 34 per minute. Heart tones are deaf, tachycardia of 120 / min, BP 115/80 mm RT.art., ECG signs of myocardial hypoxia, liver 2.5 cm stands from under the costal arch, the edge of the liver is painful. The abdominal wall is tense, symptom Shchetkina-Blouberg positive. Urine protein 0,99 g/l, leukocytes 2-3 in eyeshot. Blood leukocytosis 14,5109/l with neutrophilic shift to the left, p/I-neutrophils - 25%/I - 55%, eosinophils - 1%, lymphocytes - 12%, monocytes - 7%. The hematocrit 33%, ESR - 3 mm/h, LII, and 7.1%. The reaction HCT leukocyte - 115 unit density. Ferociter>oC (night). The patient had previously undergone intensive treatment: a massive dose of antibiotics; blood and plasma; detoxification therapy. Despite treatment, septicopyemia progressed. In this regard, on the background of intensive therapy were treated with the drug Splendid. The drug was administered intramuscularly at a dose of 20 ml, 1 times a day, for 4 days. On the background of the overall poor condition of the positive dynamics appeared on the 2nd day after the first injection of Splendid. The patient actively been in contact, improved General condition, appetite, mucous covers became more pink, decreased cyanosis of the lips, body temperature decreased to values: 36,5oC (in the morning) to 37.5oC (night). The pulse rate of 75 minutes, HELL - 120/70 mm RT. Art., heart sounds are clear. Symptom Blomberg became negative. The liver decreased to 1.5 see the Amount of protein in the urine decreased - 0.33 g/L. the Number of leukocytes - 11,4109/l, p/I - 15%, with/I - 55%, eosinophils - 1%, lymphocytes 20%, monocytes - 9%, LII - 4,2%, ESR 25 mm/h, Hct - 45%, HCT - 159. Phagocytic index 72, phagocytic number is 6.4 percentage digestion - 46.

3 days after completion of the course of drug treatment the patient's condition has improved. Biochemical and immunol. , 32 years. He joined the Institute of pulmonology 5.10.89, the Diagnosis of infectious-allergic bronchial asthma, severe form. Asthma attacks are 10-20 times a day. The disease is very short periods of remission (no longer than one month). Treated with broncholytics (Berotec when the kinks) and steroid hormones from 2 to 5 mg per day. After therapy drug BAS noted: first, the significant decrease in the severity and frequency of asthma attacks, and then complete their disappearance. The quantity of broncholytics decreased significantly: the patient prophylactically used 1-2 doses per day. Steroid hormones gradually ceased altogether. The content of the CEC in blood plasma significantly decreased 1.8 times after the first injection of the drug Splendid and remained at this level until discharge of the patient. The concentration of IgE was reduced to 10 days after application of the drug in 2.2 times compared to the initial value. Phagocytosis was increased in 2 times. Research prior to treatment the patient BAS T-cell immunity using monoclonal antibodies showed a decrease in the total number of T-lymphocytes and the imbalance of subpopulations of T-lymphocytes. After treatment BAWS (3 intramuscular through de 16,3% to 20.3%).

Within 1.5 years after the drug treatment of asthma, the patient was not observed. Harbingers of the coming attack was easily stopped 1 dose birotica.

Example No. 3. Patient S., 38 years. He entered the clinic of the Institute of Rheumatology 6.12.89, Diagnosis: Rheumatoid arthritis (RA), activity III. Complaints of pain in the joints - knee, right hip, ankle, small joints of the hands. Severe morning stiffness of the joints. Reducing the force of compression in the brushes. Swelling of the joints. Treatment of corticosteroid therapy, the effectiveness of the treatment gameable was not satisfactory, and therefore underwent a course of treatment by intramuscular injection of 1 time per day and for 10 days and 15 ml of the drug Splendid.

After the injection there was a significant positive trend (table. 3). The earliest positive symptom was a significant decrease in pain in the joints, which was observed already at day 2 after the introduction of BAS. A week after the second treatment, morning stiffness decreased by 37%, the remaining indicators articular syndrome has changed insignificantly. Significant positive dynamics of articular syndrome with reliable changes pokazatelei, the number of inflamed and painful joints, there was a significant reduction in joint pain and articular index Richie. The compression force of the brushes and the circumference of the proximal interphalangeal joints has not changed. During the year, monitoring the progression of articular syndrome was not observed.

Of interest is the effect of the drug on extra-articular manifestations of the disease. One month after treatment with BAS in 3 patients disappeared rheumatoid nodules, 2 decreased manifestations of neuropathy and 3 - digital arthritis. Clinical efficacy of BAS was confirmed by the reduction of inflammatory activity according to the results of laboratory parameters (table. 4).

There was a significant decrease in erythrocyte sedimentation rate, fibrinogen, alpha-2 and gamma-globulin. A significant decrease rheumatoid factor, decreased concentration of circulating immune complexes. The disease activity decreased to the I-II degree. In the study of phagocytosis indices (table. 5) there was a sharp increase in HCT-test, the percentage of phagocytosis and phagocytic number immediately after injection, indicating that the stimulating effect of the drug on the phagocytic activity of polymorphonuclear leukocytes in patients with R number of T-lymphocytes (CD1+) primarily due to a subpopulation of suppressor/killer cells (CD8+). Immunoregulatory index was sharply increased by the reduction of a subpopulation of CD8+ unchanged when the CD4+ count. After the drug was increasing the content of suppressor/killer, immunoregulatory index approached normal values. Therefore, the impact of Splendid on T-lymphocytes when they reduced the number can be defined as immunostimulatory.

Thus, experimental and clinical studies have shown high efficiency of the drug Splendid and a wide range of its immunostimulating activity, aimed at different parts of humoral and cellular immunity and leading to the successful treatment of pathologies with their violations. The stability of the drug Splendid during long-term storage, as well as a high degree of purification, caused no adverse reactions when used, the simplicity of the method of manufacture, its efficiency and the availability of raw materials creates opportunities for the widespread introduction of the drug in clinical practice.

Literature

1. Nikolaev, S. D. and other Theoretical and experimental background the use of perfusate xenoculture in clinical practice. Experimental and clinical aspects

4. Tsypin, A. B., Onishchenko N.And, Manuilov B. M. and other Development, production and some properties of a new immunomodulator peptide in the tissue of the spleen. J. Immunology, 1995, No. 7, S. 33-36.

5. ERO 21997, 29.04.87.

6. WO 445581, 11.09.91.

7. Methodical recommendations. The use of perfusion solution of xenoculture in the treatment of patients with extensive purulent-destructive diseases of lungs and pleura with complicated course, Novosibirsk, 1992.

8. Tsypin, A. B. , Vedernikov L. A. Selenophene in treatment of severe systemic lupus erythematosus, rheumatoid arthritis and bronchial asthma. In about.: Problems of Transplantology and artificial organs, M., 1994, S. 131-140.

1. Peptides from spleen cells of mammals, having immunostimulatory activity, obtained by extraction of the cells after mitogenic stimulation and separation of the extract, characterized in that they represent peptides with mol. m 400 to 50,000 daltons, obtained by cell stimulation with mitogen in the process of washing the tissue of the spleen, the destruction of the cells dispersed in water, pre-digested tissue and ultrafiltration separated extract through the capillary filters with pore size with border section is stimulating the spleen cells mammalian mitogen, their extraction and separation of the extract, wherein the stimulation of the cells with mitogen realized in the process of washing the tissue of the spleen, the destruction of cells is performed by freeze-thawing tissue, dispersed in distilled water after preliminary homogenization of the body, with subsequent ultrasonic treatment and ultrafiltration separated extract through the capillary filters with pore size with boundary 50000 daltons.

3. Drug, possess immunostimulatory activity, comprising the biologically active substance and a physiologically suitable filler or carrier, characterized in that the biologically active substances are used peptides on p. 1 of 10 - 15 mg per dose.

4. Drug under item 3, characterized in that the filler used relational and the solution of gentamicin.

5. Drug under item 3 or 4, characterized in that it lyophilised freezing ultrafiltrate with filler at -35oC and dried for 48 hours at a temperature in the freeze-drying chamber -30oC and a vacuum of 0.1 Torr.

6. Drug under item 5, characterized in that it is AVI by adjuvant therapy, wherein the patient is additionally administered intramuscularly drug, possess immunostimulatory activity according to any one of paragraphs.3 - 6 daily, once during the day in an amount of 5 to 20 mg of protein within 7 - 10 days.

 

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FIELD: medicine, surgery, transplantology.

SUBSTANCE: embryonic spleen should be sampled, washed in nutritive medium № 199 to be placed into fresh medium № 199 to obtain homogenate in teflon homogenizer followed by centrifuging; then one should isolate the upper, medium and inferior layers, suck off medium layer and the upper part of inferior layer; the cell mixture obtained should be diluted in nutritive medium № 199 to be then introduced by injections into mesentery of small intestine or rectus muscle of abdomen. The present innovation favors the activation of immune system in patients undergone splenectomic operation and in those in case of surgical immunodefficient state due to high functional and regenerating activity of transferred embryonic splenic cells.

EFFECT: higher efficiency of prophylaxis.

6 dwg, 2 tbl

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