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    • 专利摘要:
    This invention relates to a biological substance extracted from the Mycelium of a Fusarium. This substance has a peptidolic structure with specific amino-acids into. This invention also relates to a process for producing the afore-said biological substance by culturing the strain of Fusarium and extracting the mycelium. The biological substance has a therapeutical use.
    公开号:SU944491A3
    申请号:SU782692749
    申请日:1978-11-30
    公开日:1982-07-15
    发明作者:Симон-Лавуан Николь；Форжо Марсель
    申请人:Сьянс Юньон Э Ко, Сосьете Франсез Де Решерш Медикаль (Фирма)；
    IPC主号:
    专利说明:

    valine, N-methylisoleucine and N-methyl-alloisoleucine.
    The percentages of the various components of the product, designated A, B, C and D, are determined after separation by liquid chromatography at a high pressure in the inverted phase and are measured using an integrator. The percentages of the various components are determined with respect to the total peak area of the four components, depending on their residual absorption in the UV region at 25 nm. The percentage of each of the peptide components is in the range: A 2-5; B 10-16 C 32-39; D.
    Component A, upon partial hydrolysis in an alkaline medium, produces three molecules of lactone, which are formed when the ether bonds are broken and the subsequent cyclization into the lactone of o-hydroxy isovaleryl-N-methylvaline. When fully hydrolyzed in an acidic medium, both ether and amine bonds break. The result of such hydrolysis is the production of three molecules of N-methylvaline and three molecules of o-hydroxyisovaleric acid. Component B with partial hydroli- -j water t
    In an alkaline environment, it produces two lactone molecules formed during cyclization of o-hydroxy isovale l-N-methylvaline and one molecule of lactone formed during cyclization with cii-hydroxy isovaleryl-M-methi / 1-isoleucine or alisoleicle. With complete hydrolysis in the acidic environment of component B, three molecules of hydroxyisovaleric acid, two molecules of N-methylvaline and one molecule of hydroxyisovaleryl-M-methylisoleucine or N-methyl-alloisoleucine are obtained.
    Component C under alkaline hydrolysis produces one molecule of lactone formed during cyclization of 0 1-hydroxy from 5 -valeryl-N-methylvaline and two molecules of lactone formed during cyclization of 0-hydroxy-isovaleryl-N-methylisoleucine or N-methyl-aloisoleucine. When fully hydrolyzed in an acidic medium, three molecules of α-hydroxyisovaleric acid, one molecule of N-methylvaline, and two molecules of N-methylisoleucine or N-methyl-alloisoleucine, are obtained.
    Component D, upon partial hydrolysis in an alkaline medium, produces three molecules of lactone, which are formed during cyclization of o-hydroxyisovaleryl-M-methylisoleucine or N-methyl-alloisoleucine. When fully hydrolyzed in an acidic environment, component D produces three molecules of cc-hydroxyisovaleric acid and three molecules of N-methylisoleucine or N-methyl-alloisoleucine.
    In the hydrolysis of the whole biologically active product according to the proposed method, α-hydroxyisovaleric acid, N-methylvaline, N-methylisoleucine (in erythro form) and N-methyl-alloisoleucine (in trio-form) are obtained, while N-methyl-alloisoleucine cannot be related to no specific component.
    The method is carried out as follows.
    Aerobic fermentation of Fusarium eguiseti strain No. 213107 was carried out in deep vessels at 29 ± 5 ° C and pH within the limits with stirring, the duration of each stage was 36-iS h, the mycelium was collected at the end of the fermentation, its chlorine-containing solution was extracted. trichlorethylene, the organic phase is separated, the solvent is distilled off to dryness, the hydrocarbon {pentane or hexane) is added to the residue, countercurrent alcohol: the aqueous solution of alcohol is a mixture of methanol and water, after which the alcohol phase is separated, the mixture is concentrated , the peptide product is precipitated and purified using chromatography on an alumina column.
    P and me R 1. Identification of the strain.
    Fermentation medium No. 1, g: sucrose 25; glucose 25; ammonium nitrate 10; magnesium sulfate 2.5 monohydrate potassium phosphate 5- pH after sterilization 5- The compact mycelium has a fluffy threadlike structure and an orange color. When turning the Petri dish, the culture takes on a yellow-orange color (peach color), turning into an ocher orange color from a more developed culture.
    Grain Wednesday.
    Mycelium gives a smooth, fluffy culture, arranged in the form of characteristic rings. Pigmentation is weak, the reverse side, when a culture develops, acquires a dark yellow color.
    Wednesday dream, g: carboxymethylcellulose 15; ammonium nitrate 1; odnozamemekstraktsii hydrocarbon phase puchenny potassium phosphate 1; magnesium sulfate 0.5; yeast extract 1; distilled water 1000. This medium can be supplemented by adding to it dry fragments of the Phragmites communis culture. Mycelium rare very even, imperceptible, not having coloring. The effect of light on fungal sporulation. When exposed to white light, which is given by white fluorescent light sources, with an intensity of 1000 l for 12 hours daily, macroconidia is observed in a medium based on CMC and in an environment supplemented with Phragmites culture. When using a Wood source of light (the emission wavelength of BdO, Zb5nm), located at a distance of 33 cm from the test cultures, macroconidia are formed in large quantities after -6 days. The color of the culture media cultivated under the influence of the source of the Wood source of light is somewhat weakened by comparison with the color of the media obtained under cultivation under the influence of white light. Culture Fusariurn eguiseti No. 21310 can be obtained on classical media used to obtain cultures of the species Fusariurn. Nasred containing tO g / l oat flour, medium based on potatoes (50 g potato pulp, 10 g glucose and 20 g agar per 1 l water) ,. Malt-agar medium (2) agarized at 2, conidiogenesis is rarely observed with the formation of abnormal conidia, with the mixing of unripe macroconidia With microconidia. The appearance of color in the form of concentric zones is observed to a greater extent when cultivated under the influence of light than for cultures obtained in the dark (on a potato-based medium). The culture on oat flour produces compact, even, heterogeneous, slightly colored mycelium. Conidiogenesis is not observed in this case. Morphological characteristics of maroconidium. Macroconidia have an elongated serous shape, the final cells have the shape of an open beak, the intermediate cells are somewhat enlarged, the main cell has the shape of a foot. The dimensions vary considerably and depend on the cultivation conditions: length 25–60 µm, width – µm. Painted conidia have a glassy appearance. The number of partitions in young cultures of A. Microconidiophores P. P. POCHO are distinguishable and occur rarely, give a branch in the form of a brush. Characteristics of chlamydospores. Intermediate chlamydospores, single or in the form of chains, have the form of globules with more dense walls with a diameter of microns. They are formed in more mature cultures when cultivated for more than 20 days. Preparation of substrates. Oblique agar, 2 ml of sterilized water is extracted from the contents of the ampoule with lyophilized mycelium. The resulting suspension is used for sowing the culture in four tubes containing 10 ml each of beveled agar having the following composition, g: oat flakes 30; glucose 10; agar 20; distilled water 1000; sterilization conditions of 20 min at 120 ° C; initial pH 6.6; pH after sterilization 5.8. . The medium is cultured for 6-8 days in a thermostat at 28 ° C, after which it is irradiated with sunlight for a week in the laboratory at room temperature. Cup Ru. From aerial mycelium obtained on oblique agar, extract 10 ml of sterilized water. The resulting suspension is used for sowing in 5 cups of Ru containing 200 ml of an agar composition identical in composition. The medium is cultured for 68 days in a thermostat at 28 ° C, after which it is irradiated with sunlight for a week under laboratory conditions. Getting a vaccination material. The first stage. A suspension of the mycelium in 100 ml of sterilized water is obtained from the culture obtained in the Ru plate by removing the culture from the entire surface. The resulting suspension is used for sowing a culture in a flask containing a Q l medium of the following composition, g / l: sucrose 25.0; glucose 25; ammonium nitrate 10.0; mono potassium phosphate magnesium sulfate 2.5; tap water 1.0 liters. Sterilization conditions: 30 min., Initial pH 5.5, pH after sterilization A, 75-Aerobic cultivation was carried out at 28 ° C. in the device, which provides sparging sterilized air with stirring for k8 h (average value 72 h). The second stage. Crops of culture () are made from one liter flask of the preparation obtained in the first stage and 75 liters of the medium of the following composition, g / l sucrose kO; celerosis 5; ammonium nitrate or 10; mono potassium phosphate 5; magnesium sulfate 2.5; tap water 1.0 liters. Sterilization conditions are 30 minutes at 120 ° C, the pH after sterilization is 5.6. Fermentation conditions: aeration 3.5 m / h, mixing 70 times per minute, temperature duration 36 hours. During fermentation, sterility monitoring, pH measurements, microscopic studies and quantitative analysis of residual sugars are carried out under sterile conditions. Third stage. The inoculum obtained in the second stage is used for sowing in 1200 l of nutrient medium having a composition identical to that described (sowing 6.25) - Fermentation conditions: aeration 70 m / h, stirring 50 times per minute, temperature + 29-1 ° C, the duration of the hour. During the fermentation, the same countertruck studies were carried out as in the previous stage. Production. The 1200 l of inoculum obtained in the third stage is transferred for sowing in 7 m of nutrient pulp having the following composition, g / l: sucrose 50; celerosis 5.0; ammonium nitrate 10.0; potassium phosphate monobasic 3.0; magnesium sulfate 2.5; zinc sulfate 0, calcium carbonate 2.0 tap water 1.0 L; The conditions of sterilization were 30 minutes at +20 pH after sterilization of 6.5. Conditions of fermentation: aeration 250, stirring 20 times per minute, temperature + 29 41 ° C, duration 0 hours. During the fermentation, the same control studies, as in the previous stages, the pH value close to 3 per 2A h, is slowly increased and stabilized at a value of 5.3-5.5 by the end of the fermentation. The targets are separated from the fermentation bed by pressure filtration and dried in a ventilated oven. In this way, after grinding, an average of 120 kg of dry mycelium is obtained. Extraction. Extraction of the crude product. Per 100 kg of mycelium. 400 l of trichlorethylene is extracted from the crushed mycelium, stirred for 2 hours and dried. The residue obtained is washed with 100 liters of trichlorethylene and secondary extraction is carried out with 300 liters of trichlorethylene with stirring for 1 hour, after which it is dried again. The residue is washed with 100 liters of trichlorethylene, the extracts are combined, and they are concentrated under reduced pressure to a thick mass. 10 kg of the resulting mass. The mass is dissolved in 90 l of hexane, clarified by filtration, and extracted from a hexane solution with an aqueous solution of methanol in countercurrent (cor becoming an aqueous solution of water: 80:20 alcohol by volume); the first extraction was 50 liters of the mixture, the second 25 liters, the third 12.5 liters and the fourth 10 liters. The methanol extracts are combined and concentrated to dryness under reduced pressure. Chromatography on acidic alumina. Getting alumina. 1 kg of alumina is suspended in 5 liters of distilled water and, with gentle stirring, concentrated hydrochloric acid is added in an amount necessary to maintain a pH of 0 for 2 ° C. The suspension is filtered through a nylon filter and washed extensively with distilled water, after which washed with acetone to remove residual water; dried alumina in a drying oven and a cupboard overnight. Chromatography 20 kg of methanol extract. The methanol extract is dissolved in 80 l of methylene chloride, the solution is clarified by filtration, and subjected to chromatographic separation in methylene chloride on a 0.8 m high glass column filled with 0 kg of alumina so that the solution consumption in methylene chloride is 15 / h The aluminum oxide is washed with 80 l of methylene chloride at the same hourly flow rate. Isolation of the active substance. The methylene chloride washed substance is concentrated under reduced pressure to obtain a thick mass, which is then dissolved in 200 l of ethanol and clarified by filtration. The solution in ethyl alcohol is cooled to-C and added with slow stirring for 8 hours while maintaining the temperature and under stirring for 20 hours, after which the separated crystals are separated by filtration. The resulting residue is washed with a mixture of methanol: water 1: 2, pre-cooled, then 50 l of distilled-15 liquefied water. The crystalline septic peptide is dried in a drying cabinet under reduced pressure and 40 ° C to a weight. Depsipeptide get in the form of a crystalline powder of white or light yellow color, odorless, with a bitter taste. It is very poorly soluble in water and readily soluble in chloroform, methyl alcohol and ethyl alcohol at 95 ° C. The melting point of the anhydrous product, determined by the Kofler method, is 121i5 ° C, specific rotation C °, -80 ° ± 5 ° C 5, methanol. The total content of azbt 6,2 ± 0,3%. Identification of components by thin layer chromatography. Cooking media. Based on 5 chromatographic plates of 20–20 cm, a homogeneous mixture of 4 g of cellulose Mp 300.8 g of cellulose Ip 300 G and b5 ml of water is prepared, then the resulting suspension is applied to carefully defatted plates. as a layer thickness of 0.25 mm. The chromatographic plates treated in this way are dried in air at 110 ° C for 2 h, cooled and immersed in a vertical position in the formamide: acetone 1 mixture: to a depth of about 2 cm from the edge. They are then dried for 1 minute in the open air to remove acetone. Chromatography solvent. Heptane saturated with formamide (supersaturated solution) is used. Prepare a solution in methanol based on 10 mg of substance per liter. (About 1 ml of the solution is placed at a distance of 1 cm from the edge of the plate, treated with formalin. After the solution has been spread for a distance of about 10 cm, the plate is dried for 1 hour and sprayed with 10% 0.5% solution of iodine in chloroform. four yellow spots are formed, two of which, the most intense, correspond to Rf about 0.55 and 0.5, one less intense, Rf about 0.35, and one very weak Rf about 0.3. Separation of the dip-peptide into components by the liquid method High Pressure Chromatography (HPLC): Relative Content, Component A , B, C and D are determined for different industrial designs after their separation by HPLC and peak measurement using an integrator. The relative percentages of the components are calculated relative to the sum of the peak areas of all four components under the assumption that the residual absorption in the UV region at 25 nm remains the same for all components. Separation conditions: the Bonsapak 18 phase carrier (Waters Associates) length 30 cm, internal diameter 3.9 mm. Eluent methanol: water 8: 2 by volume. A flow rate of 1.5 ml / min. Working solutions: 10 mg. substances per 1 liter of methanol, dosing of 10 ml. Detection: UV region, wavelength 25 nm. Sensitivity D 0.02 of the entire scale. Area measurement using the ICAP 5 integrator. The following results correspond to the average composition for different samples of the depsipeptide product,%: A 3.9; At 15.3; C 38.1; D k2.7. The presence of the product is also confirmed by IR spectra. Example 2. Depsipeptide-based pharmaceutical compositions. Curd solution in isopropyl myristate, g: depsipeptide extract Fusarlum eguiseti 0,050; Essenti Neroli 0,050; eucalyptol 0,050; saccharin O, 001 25; isopropyl myristate 2.5 ml; Flugen 12-7.5 ml. Aqueous suspension, g: extract of depsipeptide Fusarium eguiseti 0,050; polyoxyethylene sorbitan monooleate tween 80 0.010; sodium chloride GJ80; Essenti Neroli 0,050 .; Evcamptol 0.05; sodium saccharin 0,010, distilled water 20. In both cases, the product is distributed using a dosing syringe with a capacity of 0.080 g per dose, which corresponds to 200 mg of the active drug per dose.
    1 fermented solution, g: extract of depsipeptide Fusarium eguiseti 0,015; flavoring agent 0,0075; isopropyl myristate 0.75 ml, freon 122, 25 ml. The vessel is equipped with a 25 µl valve and contains 3 ml of solution. Each time the valve is triggered, a dose of depsipeptide of 125 µg is obtained.
    Curd solution, g: extract of depsipeptide Fusarium ego i set 0,030 flavoring agent 0.015; ISO-Pilmyristate 0.750 El, Freon 122, 23 ML- This recipe makes it possible to have 250 μg of depsipeptide in each dose with a total number of doses of 120. Oily solution, g: extract of depsipeptide Fusarium eguiseti 0.05; saccharin 0.0015; flavoring agent 0.05; isopropyl myristate 6 ml; the vessel is equipped with a dispenser that allows a dose of 30 µl to be obtained with each press. Aqueous suspension, mg: depsipeptide extract Fusarium eguiseti 50; polyoxyethylene sorbitan monooleate 15; sodium chloride 120; disubstituted sodium phosphate 17.25; dibasic potassium phosphate 3; calcium chloride 1,5; magnesium chloride 1.5; mouse cohesive mercaptide sodium 0.0375; water 15- The suspension is placed in a 15 ml vessel equipped with a metering unit, providing a dose of 75 μl at each injection. Each injection contains 250 μg of active substance. Aqueous solution for injection: depsipeptide Fusarium eguiseti 100 mg, Cremophor EL 0.10 g, sodium chloride 0.8 g, water 100 ml. Example 3 - Pharmacological study of depsipeptide extract. The study of humoral immunostimulation in mice in relation to sheep erythrocytes (GRM). Immunization. Swiss mice with 5 mice each weighing 5 gs, a threshold threshold of 10 million sheep erythrocytes was injected intravenously. The blood of the ram is pretreated with a solution of Elsevier. The erythrocytes are washed three times with physiological serum, and then transferred to suspension in the same medium for carrying out vaccinations. Each group of immunized mice consists of 8 individuals. Handled the animals. In each experiment, have one group of control animals and two
    groups of animals that have undergone special treatment. Mice treated, after a different time after immunization in various ways: intravenously or intraperitoneally. Culture pseudo-diphtheria sticks Soupé bacterium Parvum is prepared as a suspension in physiological serum. Depsipeptide is dissolved in propylene glycol, based on a concentration of mg / ml, after which the concentrated solution is diluted with physiological serum in a ratio of 1:20 for mice treated with a dose of 50 µg depsipeptide, and in a ratio of 1: 100 for mice treated with 10 µg of depsipeptide. Sampling. On certain days, mice are sampled for blood by retroorbital puncture. Serums are stored to prevent decomposition. For each given group of animals studied, the characteristics of the individual sera as well as the characteristics of the pooled serum are studied. Antibody analysis. Determination of the degree of hemagglutination. The method of microhemagglutination on the plates. To 0.75 ml of a suspension of sheep erythrocytes deposited on a Mayer swab with a titer of ml (0, them 0.700) was added 0.025 ml of serum diluted in a ratio of 2: 2 on the same tampon. The plates are left for 2 hours at room temperature, then overnight at C, Hemagglutination is observed with a magnifying mirror. The degree of hemagglutination is determined by the highest serum dilution at which free hemagglutination is observed. The degree is determined as a logarithm at base 2 of the dilution degree, so when diluted 1, the degree of hemagglutination is 6. Free hemagglutination can be counted only if the degree value is greater than or equal to 4, with smaller values of this index, the score becomes too difficult. Definition of hemolysis. Microhemolysis technique on plates. To 0.025 ml of a sheep erythrocyte suspension on a Mayer swab (10 per ml) was added 0.025 ml of the elixin of a Mericux guinea pig, diluted with a 1/100 ratio on a Mayer tampon, and 0.025 ml of serum, 13S diluted 2: 2. The plates are placed for 1 hour in an oven at ZSU, then for 2 hours at C, and the calculation is carried out using optical devices. The hemolytic index is determined for the strongest dilution of the serum, at which the erythrocyte sediment of the ram's red blood is still completely dissolved. This indicator is determined in the same way as the hemagglutination index. Processing results The average values of individual parameters (± standard deviation), as well as the combined serum values for each group, are calculated depending on the time of sampling. Functional changes for a group that has not been specifically treated are determined from an estimate of the difference between the averages and the difference of the indicators by groups. In the calculations, the Student’s bilateral t distribution or method x is used in cases where the Student method cannot be used. The study of threshold phenomena. A large number of different doses of sheep erythrocyte blood was studied to find the threshold value of the humoral response in mice of the Swiss breed and to detect a superthreshold effect. 10 erythrocytes provide an opportunity to study the kinetics in the existing conditions of the experiment. Kinetic response studies in control animals. Several groups of animals immunized with 10 red blood cells were studied. Hemagglutination indices. The appearance of hemagglutinant antibodies is observed in some groups of animals on the s-day after immunization, e In some cases, only for a day, and the results, which have a strong scatter on the s-th day, tend to approach each other on the 11th day. Indicators do not exceed k.
    Hemolysis rates.
    The appearance of hemolytic antibodies is practically not observed until the results show a tendency toward convergence in a later stage of the experiment.
    The study of comparative immunostimulant.
    introduction of labeled prior substance.
    Reference conditions.
    The determination of the optimal dose of mitogen is made from the curves, depending on the effect / dose bridges to initiate the onset of plateau activity on these curves. The concentrations of the mitogen, at which the greatest stimulating effect is achieved under the conditions of the test, are as follows, µg / ml: phytohemaglutinin in a solution of 100 µg / ml physiological serum (PHA) 1; LipopoI The introduction of a Corynebacterium parvum culture on the 3-6th day of immunization promotes an immune effect. The study of the preliminary phase of the superthreshold effect (4th day) makes it possible to detect the formation of hemolytic and hemagglutinant antibodies in animals exposed to the stimulator. Observations at a later stage (day) confirm the results obtained. The scatter of results is explained by the heterogeneity of the Swiss strain used in the experiments. Study of the activity of the proposed depsipeptide. Depsipeptide, administered for 3 days, from the 3rd to the 6th day before immunization, causes an increase in the number of hemagglutinating and hemolytic antibodies, as well as the appearance of hemolytic antibodies on the C-th day of the immune test, with doses of 10 and 50 µg / day per mouse give similar results. The stimulation obtained by using the depsipeptide under the proposed conditions is similar to the stimulation achieved by using the G. parvum culture intraperitoneally in doses of 500 µg / day per mouse from the point of view of the kinetics of the process. At the same time, a comparable effect is obtained when using depsipeptide in active doses that are 10-50 times smaller. The study of lymphoblasttransformation. In the case of TTL dosing of a dipsepeptide, conditions of 2j are reproduced with respect to the optimal concentration of methogen, while the test product remains in contact during the entire conversion time, the most complete and reproducible introduction of the preceding substance occurs, and the biological effect of the product can be represented through inhibition of Elysaccharide cultures of Escherichia Co Ii 026 b solution at a concentration of 1 µg / ml in physiological serum (LPS) 5- Lymphocytes transformed by PHA are only T cells, and LPS concentrate Only in cells B are used. Kinetic studies allow to determine the onset of the maximum stimulating activity of PHA at the 66th hour, and LPS at the 8th At the indicated dosage, PHA represents the best mitogen, the introduction in this case is twice as large as in the case of LPS, and the results obtained with its use are more reproducible. From the obtained results, it follows that the Fusarium eguiseti depsipetide extract is not a mitogenic agent and inhibits the introduction of tritium-substituted thymidine caused by mitogenic agents. The study of the immune effect of depsipeptide. The stimulating or inhibiting effect of this drug has been studied in relation to the humoral and cellular effect. The humoral effect is studied with the introduction of LPS, which causes an increase in cells in the spinal substance. The cellular effect is studied with the administration of oxazolone. The effect caused by this product is concentrated mainly in the cells. The study of cells producing antiterla. The number of cells producing antibodies in the lymphocyte colony is quantified by studying hemolytic plates in a semi-liquid medium containing labeled red blood cells. The experiment was carried out using direct hemolytic plates, and cells producing IQ.M. A second agar layer (0.6) containing 110 lymphocytes and erythrocytes pooled onto 30 is poured into a Petri dish containing a layer of 3-agar agar. The resulting medium is kept at 37 ° C for 1 hour, after which Aleksin of guinea pigs 1 is added to it. 10 and re-left for an hour at 37 ° C. The study of hemolytic plates is carried out using a binocular loupe and the results are expressed per 10 lymphocytes or per organ. The erythrocytes of ram blood are considered labeled, sensory one16 temporarily in relation to oxazalone and LPS. Immuno-cytochemical study of cell suspensions, rosettes and cells producing antibodies. The study of total lymphocytosis in the lymph node in the spleen. For an autoradiographic study, the cell suspensions were incubated for 30 min at 37 ° C with tritium-substituted thymidine with a radiation dose of 20 microureas / ml, after which they were subjected to treatment to determine the surface immunoglobulin. Cells are applied to glutaraldehyde, treated with diaminobenzidine, according to the method of Grahoma and Karnowski, (1966), to detect peroxidase, then treated with osmic acid and enclosed in plastic (epon). Results. The study of the immune response using oxazalone. The study of the number of cells. On day 5, an increase in the number of cells exceeds 50%, with the most important results being observed with intraperitoneal administration. On day 9, the increase in cell number becomes less noticeable. The number of cells containing tritium-substituted thymidine is comparable in control animals and in animals treated and is in the order of -6 to 100. Study of the number of cells recognizing the antigen. On day 5, there is a very strong increase in the number of cells recognizing the antigen (257 per 100), leading simultaneously to an increase in the number of cells in the lymph node and an increase in the number of cells recognizing the antigen per 10 cells. These changes are not observed on day 9. The study of the number of cells that form hemolytic plates. A marked increase in the number of cells that form hemolytic plates on day 5 leads to an increase in the number of cells, while on day 9, the number of cells that form hemolytic plates on the basis of 10 cells increases. Study of antibody amounts. On day 5, the number of antibodies remains constant, however, on day 9, the number of hemagglutinant and hemolytic antibodies increases, in particular, this concerns hemolytic antibodies. The study of the number of cells per 1 g of the surface. The number of cells per 1 g of the surface is calculated on semi-thin mediums for the initial cell suspension, while the percentage ratio in the control animals and in the treated animals is 35 per 100. Immunocytological examination under an electron microscope. The nature of the cells forming the rosettes and the plates are the same in control animals and in animals that have undergone treatment. We are talking about lymphocyte sockets per 1 g of the surface and T cells, which on the ninth day are in the amount of 0-50 per 100, 8 cases of hemolytic plates are talking about plasma cells. The study of the immune response to LPS. Study the number of cells. Note a marked increase of 9 days in the case of depsipeptide. The study of the number of cells recognizing the antigen. No effect is observed, or W causes a decrease in the case when the results are calculated on 10 cells or organ. This reduction becomes especially noticeable on day 9. The study of the number of cells that form hemolytic plates. The obtained results do not allow to speak about the presence of any action in the direction of decreasing or increasing. Study of the amount of antibodies. Observes a tendency to decrease the number of hemagglutinant antibodies, while the number of hemolytic antibodies has a tendency to a noticeable increase by day 5. Immunocytological examination under an electron microscope. . The cells forming the rosettes and the gels forming the plates, both in the case of control animals and in the case of animals treated, are mainly plasmocytes. In this case, the number of lymphocytes per 1 g of the surface is observed (in the case of rosettes). Among immunocytes, T cells are observed. Depsipeptide works as follows. The response to oxazalon is stimulated. A marked increase in the number of cells in the lymph nodes, reaches a maximum on day 5 (50-80 per 100). The number of cells recognizing the antigen is increased in all cases. An increase in the number of antibodies is always insignificant, especially on day 9. The response to LPS may be more inhibited. An increase in the number of cells in the spleen is not observed, the number of cells recognizing the antigen does not change or rather decreases, the change in the number of cells forming plates is too random, and the number of antibodies either decreases (hemagglutinant antibodies) or remains unchanged (hemolytic antibodies ). The effect of depsipeptide on the immune response. Material. Depsipeptide is converted into a solution at the rate of 100 µg per 50 ml of physiological saline for those experiments where the maximum concentration is 2 µg / ml. If high concentrations are used, suspensions prepared by stirring for 3 hours at room temperature are used. Animals The humoral immune capacity is male mice C 57 B 6, lymphoblast transformation is CBA mice, males, macrophage activation + FISHER rat / 1 co, females. Antigen. Purified (Kohn fractions) bovine serum albumin Armor Pharmaceutical Company (Chicago). Each mouse was injected with 300 µg of this antigen in the rear plantar pads. Auxiliary means. Difco Comparison Supplements - Complete Supplement for Freund, incomplete Supplement for Freund. Depsipeptide is administered at a rate of 10 µg per mouse for testing humoral immune capacity on day 0 and 5 mg per mouse for inducing immunization. Methods Immunization. Day 0. Immunization of 20 mice at 10 µg / depsipeptide mouse. In addition, each mouse receives 0.2 ml of a mixture consisting of two volumes of phosphate extract of BSA and depsipeptide and three volumes of incomplete reagent to Freund. Ten mice receive the same mixture, but without depsipeptide, and ten mice receive an antigen with complete Freund reagent. Day 21. The halves of mice in each batch are sacrificed to measure the amount of antibodies that act against BSA by passive hemagglutination. I give the second half of the animals an injection to induce the effect of half the initial dose (0.1 ml instead of 0.2 ml). Day 35. The remaining mice die to determine passive haemaglutination and thus determine the number of antibodies specific for BSA. Cell studies. Mitogenic ability of depsipepti is yes. 0, the spleen lymphocytes of CBA mice in 0.1 ml of RPMI are placed on culture for 3 days while the COg content is. with different concentrations of depsipeptide. Groups of mice were injected with PHA at dilution (1/200, Coop A at a dose of 10 µg / ml of ULPA at a dose of 100 µg / ml, 4 hours later after incubation, 1 culture of tritium-substituted thymidine was added to each culture. filtered through a Whatman GF / C filter and washed twice with 10 ml of saline. Third-substituted thymidine fixed to ADN remains on the filter and its radioactivity is studied using an fb-radiation counter. The conversion rate is defined as the ratio of thymidine in the presence of the studied compounds to The iodine entering lymphocytes in the absence of any gene. Macrophage stimulation. Peritoneal macrophages of uninspired Fisher rats taken during abdominal washing and isolated on a cultured mass of NUNCLON were cultured during the night together. with doses of depsipept ranging from 1 pg to 1 ng per 1 ml. Then the protein content is determined with the release of lyso-fi-glucone dase and leucine aminopeptides of the cytoplasm in cells soluble in 0.05 Triton X-100. The LPS culture of Escherichia and muramyldipepside (MDP) are comparative standards. Potentiating effect on non-specific cellular immunity. Lymphocytes: Mitogenic Ability While phytohemagglutinin (PHA) with normal dilution of 1/200 with respect to mouse splenic lymphocytes and concavalin A (ConA at a dose of 10 µg / ml give an indicator of 120 conversions of about 50, and LPS of an Escherichia culture at a dose of 100 µg / ml has an indicator of And, 8, the proposed depsipeptide inhibits the introduction of tritiated thymidine (which corresponds to less than 1.0) at all concentrations tested (from 0.1 pg to 100 mg / ml). Similar inhibition of nuclear metabolism when using very small doses of depsipept a is indicative of a very large activity of the product in the mechanism in which the destroyed cells or even with respect to the metabolism modification and cellular permeability take part. The depsipeptide itself is not a mitogen and this effect stimulating the lymphocytes cannot be expected Based on concepts of classical mitogens. Activation of macrophages. Sn V and Tro. Enzymatic dosing: DPSY-peptide causes a significant increase in macrophage hydrolase at a dose from 1 pg to 10 n. / Ml. The protein yield is significantly increased at a dose of 10-100 mg / r-tn. Starting with a dose of 100 ng / ml, the depsipeptide becomes toxic, as indicated by a drop in the yield of proteins and intracellular ectim of the macrophage observed at a dose equal to or greater than this concentration. On V iVO Depsipeptide noticeably stimulates macrophage metabolism. On vivo, 6 days prior to the washing of the animals, the animals are injected with a dose of 1-100 µg intraperitoneally, as a solution in 1 ml of physiological solution of LPS, MDP, and VSA serve as a standard. Cells selected as a result of peritoneal washing are kept for 2 hours to remove non-adjacent cells, and a population of matched cells consisting of 30% of the macrophage is additionally cultured for an additional hour. At the same time, the cells are dissolved and the yield of proteins and enzymes is determined at the doses given for an in vitro study. Dn vivo enzymatic dosing: at a dose of 1 µg, depsipeptide causes an increase in lysoenzymes (when tested on p-glucuronidase + 35%) and cytoplasmic (so for leucine aminopeptidase +35), as well as an increase in the yield of proteins () that places this product for LPS and VSA on stimulation intensity. The ICA at the same dose causes an increase in hydrolase by 39, and LPS an increase of 42% leucine aminopeptidase and 35 proteins.
    At a dose of 100 µg, depsipeptide causes the strongest increase
    proteins (+32) and cytoplasmic enzymes (+59) of the four immunostimulators tested. Regarding the increase in hydrolase, depsipeptide is in this line for BCA (+ 37%)
    In tab. i presents the increase in the yield of proteins and enzymes in peritoneal macrophages when stimulated in vivo for 6 days.
    In tab. 2 shows the intra macrophage yield of enzymes and proteins after incubation with various immunostimulants for 16 h. Proteins, µg / 10 cells, glucuronidase nM hydrolyzed substrate (1 (hcl / h)
    23 Note. Degree of significance:
    Increase in the loss of chromium, injected by the larvae of Shistosofna mansoni, with the use of various immunoE + E
    table 2
    no stimuli of the macrophage tori in comparison with the stimulated macrophages. ap 0.05; Lp 0.025; Wed {: 0.001; NS - insignificant. Cytotoxicity: depsipeptide has been studied for its in vivo effect on the cytotoxicity of rat peritoneal macrophages in relation to Shistosoma mansoni larvae. Cells stimulated in vitro or in vivo are placed together with Shistosoma mansoni chromium-5-labeled larvae to detect possible cytotoxic substances.
    Depsipeptide + 0.5 (slightly) LPS
    MDPVSA
    Depsipeptide causes only a slight increase in chromium-51 deposition, which is a radioactive label for schistosomul, to the same extent as MDP, while LPS and BCA produce macrophages that are much more toxic than the larvae than animals. that exist only in saline. Thus, the decipeptide has no in vivo effect on macrophages if the tests are carried out with respect to chromosome-51-labeled schistosomes. 19,67.13,1 I8,21t3,9 2i, 2, k Study of the effect of cyclodepsipeptide on the survival time of mice infected with 55 L1210 leukemia. The cyclodepsipeptide is an immunostimulant in vivo by +15 (slightly) +10 (slightly)
    (p 0.005) +10 (slightly)
    flax) + 30% (p 0.025)
    Pitotoxic ability. Only LPS E. coli, except for depsipeptide, does not cause in vitro a significant increase in the cytotoxicity of macrophages with respect to chromosome-51-labeled schistosomils. Stimulating doses of depsipeptide cause only a small increase in the radioactive label content.
    The total amount of chromium found in schistosomuli incubated for 16 h with normal macrophages stimulated for 2 h and with various doses of depsipeptide is presented in Table. i.
    权利要求:
    Claims (1)
    [1]
    1. For France of France No. 2269965,3. Mate et al. Jmmunotherapie Askl. A 61 K 35/7, 1976.tive des cancers, 1976, ESF - Paris.
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    同族专利:
    公开号 | 公开日
    DK537578A|1979-05-31|
    IT1157737B|1987-02-18|
    FR2410658B1|1980-08-22|
    DE2851629A1|1979-06-07|
    IL56072D0|1979-01-31|
    FI783675A|1979-05-31|
    US4233291A|1980-11-11|
    AT363593B|1981-08-10|
    CA1117882A|1982-02-09|
    GB2010848A|1979-07-04|
    JPS54163893A|1979-12-26|
    OA06095A|1981-06-30|
    NL7811765A|1979-06-01|
    SE7812298L|1979-05-31|
    ZA786716B|1980-02-27|
    IT7852133D0|1978-11-29|
    ES475593A1|1979-04-16|
    DD140046A5|1980-02-06|
    FR2410658A1|1979-06-29|
    ATA858578A|1981-01-15|
    IL56072A|1982-02-28|
    AR220921A1|1980-12-15|
    GB2010848B|1982-04-07|
    NZ189048A|1982-03-23|
    GR68710B|1982-02-03|
    AU4202278A|1979-06-07|
    BE872385A|1979-05-29|
    EG13644A|1982-06-30|
    PT68837A|1978-12-01|
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    法律状态:
    优先权:
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