Pharmaceutical drug on the basis of interleukin-2 (options)


C12N15/26 - Interleukin-2

 

(57) Abstract:

The invention relates to the field of medicine and pharmacology, relates to a pharmaceutical preparation based on interleukin-2 and can be used for the treatment of severe conditions in mammals, caused by an infection, such as sepsis various etiologies, as well as oncological diseases and immunodeficiencies. Pharmaceutical drug (options) on the basis of interleukin-2 as an active substance containing as a stabilizer a substance selected from the group consisting of mannitol, human serum albumin or a mixture, as a solubilizer, a substance selected from the group consisting of guanidine hydrochloride, sodium dodecyl sulphate, as an antioxidant agent is a substance selected from the group consisting of glutathione in the reduced form, 2-mercaptoethanol, dithiothreitol, as a buffer - pharmaceutically acceptable buffer, a stabilizer, a solubilizer, an antioxidant agent, and a buffer at a certain content of the ingredients, provides in solid and liquid forms of high biological activity of interleukin-2, has fewer side effects, technology in manufacturing the pharmaceutical product on the basis of interleukin-2 and can be used for the treatment of severe conditions in mammals, caused by infection, such as sepsis various etiologies, as well as oncological diseases and immunodeficiencies.

Known drug containing interleukin-2, obtained from blood (catalog number 03-020-050, produced by Advanced Biotechnology Inc., USA). This product contains 10% fetal bovine serum, prepared in culture medium for growth of the cell culture RPMI-1640. However, this product contains components of human blood, is potentially biologically hazardous drugs and requires treatment in accordance with the rules of "Level 2 biohazard", according to the manufacturer's recommendations.

Also known recombinant drug interleukin-2 produced by intestinal E. coli genetically engineered, manufactured by Sigma (catalog number E). However, this drug is in lyophilized form contains a buffer PBS (sodium phosphate buffer, pH 7,4) and 0.1% bovine serum albumin as a carrier. This product does not contain a stabilizer or antioxidant agent, whereupon, according to the manufacturer, shows low biological activity, has a limited time hranu otherwise it loses its biological activity.

Also known pharmaceutical drug "Proleukin" in the form of freeze-dried forms of production Chiron, USA. The drug is a recombinant analogue of the interleukin-2 produced by cells of E. coli. It contains as active substance mutant interleukin-2 (1 mg), as a solubilizer is sodium dodecyl sulphate (of 0.13 - 0.23 mg), as a carrier - mannitol (50 mg) and NaH2PO4H2O (0.17 mg) and Na2HPO4(1.2 mg). This product does not contain an antioxidant agent. However, it should be noted that "Proleukin is muteena, i.e., the amino acid sequence of interleukin-2 is amended by removing the cysteine residue at position 125 and its substitution by serine or alanine (U. S. Patent No. 4, 518, 584). This change of the native structure of the protein was carried out in order to obtain the oxidized form of the drug interleukin-2, moreover, the formation of a disulfide bridge at this mutein occurs with only two remaining cysteine residues. However, it should be noted that this drug has many side effects (1). Part of these effects is due to the fact that it mutein, part - bacterial origin is high biological activity significantly surpass those of the natural protein and can stimulate the synthesis of proinflammatory cytokines, causing serious systemic side effects (2). It is known that E. coli is an opportunistic microorganism and even the use of high-tech cleaning is not fully provided for the elimination of E. coli endotoxin. In addition, known methods of oxidation are ethnologica: significantly increased technological scheme and difficult branch of interleukin-2 from impurity proteins. In addition, known methods of oxidation require a large dilution of rastvorov, protein concentrations in these solutions up to 1 /ml (3). Existing technological methods of concentration of large volumes of solutions do not allow to preserve the biological activity of interleukin-2, thus, upon receipt of the oxidized form of interleukin-2 there is some loss of bioactivity.

The present invention is the creation of a pharmaceutical product on the basis of interleukin-2, in such forms and with such a composition, which could produce a biologically active drug with fewer side effects, with the technological scheme of production and composition, allowing to preserve the biological activity of interleukin-2.

This problem can be solved in f the stabilizer, the solubilizer, an antioxidant agent and a buffer. The preparation contains as a stabilizer substance selected from the group consisting of mannitol, human serum albumin or a mixture, as a solubilizer, a substance selected from the group consisting of guanidine hydrochloride, sodium dodecyl sulphate, as an antioxidant agent is a substance selected from the group consisting of glutathione in the reduced form, 2-mercaptoethanol, dithiothreitol, as a buffer - pharmaceutically acceptable buffer, with the following content of components, mg:

Interleukin-2 - 0,1 - 7,0

Stabilizer - 5,0 - 350,0

The solubilizer - 1,0 - 70,0

The antioxidant agent is 0.1 to 3.5

Buffer, mm, pH 7,4 - 8,0 - 5,0 - 10,0

A special case of the implementation of a pharmaceutical preparation in solid form on the basis of interleukin-2 of the present invention are pharmaceutical preparations containing as interleukin-2 or recombinant interleukin-2, or non-recombinant interleukin-2.

Particular cases of the implementation of a pharmaceutical preparation in solid form on the basis of interleukin-2 in this invention are tablets, capsules, powders, granules, suppositories, liposomal forms, forms that are suitable for n, is received by dissolving a solid form in a pharmaceutically acceptable solvent, the obtained standard technological ways(4, 5, 6, 7).

This task is also solved by a drug in liquid form on the basis of interleukin-2 as the active substance-containing stabilizer, a solubilizer, an antioxidant agent and a buffer. The preparation contains as a stabilizer substance selected from the group consisting of mannitol, human serum albumin or a mixture, as a solubilizer, a substance selected from the group consisting of guanidine hydrochloride, sodium dodecyl sulphate, as an antioxidant agent is a substance selected from the group consisting of glutathione in the reduced form, 2-mercaptoethanol, dithiothreitol, as a buffer - pharmaceutically acceptable buffer, with the following content of components, mg:

Interleukin-2 - 0,1 - 5,0

Stabilizer - 5,0 - 250,0

The solubilizer - 1,0 - 50,0

The antioxidant agent is 0.1 to 2.5

Buffer, mm, pH 7,4 - 8,0 - 5,0 - 10,0

A special case of the implementation of a pharmaceutical preparation in liquid form on the basis of interleukin-2 of the present invention are pharmaceutical preparations containing as interleukin-2 or recombinases preparations based liquid forms of this invention are emulsions, syrups, elixirs, drops, liposomal forms, forms that are suitable for external use, for example, ointments, gels, wipes, received standard technological means (5, 7).

Further, the invention is illustrated by the following examples.

Example 1. Obtaining a pharmaceutical product on the basis of interleukin-2 in solid form (options).

Got a solution containing interleukin-2 person, by purification by standard methods of protein chemistry (8) extract derived from cells of strain 1-GRF18(MSIL) Saccharomyces cerevisiae deposited in the all-Union collection of industrial microorganisms under the number ACIM-U (9).

Measured concentration of interleukin-2 in the eluate. The eluate obtained gelfiltration the above solution on a column of sephacryl S-300, contains interleukin-2 in a concentration of, on average, 1 to 2 mg per 1 ml of ammonium bicarbonate buffer containing 0.1% sodium dodecyl sulphate (LTOs), 1 mm etilendiamintetrauksusnoy acid (EDTA), 5 mm 2-mercaptoethanol (2-me) and 0.01% NaN3.

To 100 ml of the above eluate containing interleukin-2 in a concentration of 1.5 mg/ml, was added 400 ml) cooled to -18oC acetone and left for 15 m is oC. the Acetone was decanted, dried sludge containing interleukin-2, and dissolving it in 150 ml of a buffer of the following composition (mg):

Mannitol - 7500

Sodium dodecyl sulphate - 1500

Dithiothreitol - 48

Ammonium bicarbonate - 120

or in 150 ml of a buffer of the following composition (mg):

Mannitol - 7500

Sodium dodecyl sulphate - 1500

Dithiothreitol - 48

Ammonium bicarbonate - 120

Human serum albumin - 7500

Thus, the concentration of interleukin-2 in this setting the buffer is equal to 1 mg/ml. a Measure of the hydrogen ion concentration of the resulting solution measured in a standard way on the pH meter Beckman, USA amounted to 7.7. The resulting solution was sterilized by cold sterilization using standard methods (8), poured into ampoules of 1 ml and freeze-dried. Thus, one ampoule of a pharmaceutical preparation in solid form on the basis of interleukin-2 contains (mg):

Interleukin-2 - 1,0

Mannitol - 50,0

Sodium dodecyl sulphate - 10,0

Dithiothreitol - 0,32

Ammonium bicarbonate - 0,8

or

Interleukin-2 - 1,0

Mannitol - 50,0

Sodium dodecyl sulphate - 10,0

Dithiothreitol - 0,32

Ammonium bicarbonate - 0,8

Human serum albumin - 50,0

Anal is at-2.

Example 2. Obtaining a pharmaceutical product on the basis of interleukin-2 in liquid form (options).

Received the eluate containing interleukin-2, in the manner described in Example 1.

To 100 ml of the above eluate containing interleukin-2 in a concentration of 1.0 mg/ml, was added 400 ml) cooled to -18oC acetone and left for 15 minutes at -18oC. was Centrifuged for 15 minutes at a speed of 2500 rpm at a temperature of -18oC. the Acetone was decanted, dried sludge containing interleukin-2, and dissolve it in 100 ml of a buffer of the following composition (mg):

Mannitol - 5000

Sodium dodecyl sulphate - 1000

Dithiothreitol - 32

Sodium phosphate disubstituted - 124

Sodium phosphate one-deputizing - 20

or

Mannitol - 5000

Sodium dodecyl sulphate - 1000

Dithiothreitol - 32

Sodium phosphate disubstituted - 124

Sodium phosphate one-deputizing - 20

Human serum albumin - 5000

Thus, the concentration of interleukin-2 in this setting the buffer is equal to 1 mg/ml. a Measure of the hydrogen ion concentration of the resulting solution measured in a standard way on the pH meter Beckman, USA amounted to 7.6. Poluchenii in a stream of sterile air. Thus, one ampoule of a pharmaceutical preparation in liquid form on the basis of interleukin-2 contains (mg):

Interleukin-2 - 1,0

Mannitol - 50,0

Sodium dodecyl sulphate - 10,0

Dithiothreitol - 0,32

Sodium phosphate disubstituted - 1,24

Sodium phosphate one-deputizing - 0,20

or

Interleukin-2 - 1,0

Mannitol - 50,0

Sodium dodecyl sulphate - 10,0

Dithiothreitol - 0,32

Sodium phosphate disubstituted - 1,24

Sodium phosphate one-deputizing - 0,20

Human serum albumin - 50.0

Example 3. Study of biological activity and stability of pharmaceutical preparations on the basis of interleukin-2, made in solid and liquid form (options).

The biological activity of pharmaceutical preparations in solid form on the basis of interleukin-2 were investigated for their ability to stimulate proliferation, cytotoxic T-lymphocytes mouse line CTLL-2. Specific biological activity of IL-2 is expressed in International Units (IU), 1 mg of IL-2 in restored form must have biological activity in the range from 300 thousand up to 1.5 million IU IU.

Continuous proliferation of the cell line CTLL-2 was supported by the km 2-mercaptoethanol, 100 units/ml of penicillin or ampicillin, 100 μg/ml streptomycin and 50 μg/ml gentamicin, 16 μm/ml sodium bicarbonate, 25 μm/ml HEPES and 10% V / V heat inactivated at +5oC for 1 hour serum fetal fetal cows (SEC) containing IL-2 at a concentration of 35 to 45 IU/ml as the source of IL-2 used the supernatant of cells of rat spleen, stimulated concanavalin A. Spleen of rats Wystar destroyed gentle methods to cellular state. The spleen cells were twice washed with RPMI medium 1640. Received cell suspension at a concentration of about 5 to 106cells/ml) was Added 5% fetal serum and of concanavalin a to a concentration of 5 μg/ml of the Mixture were incubated for 30 h at 37oC in an atmosphere of 5% CO2. The cells were separated by centrifugation. The supernatant was sterilized by filtration through membrane filters with a pore diameter of 0.22 μm. Evaluated the presence and concentration of IL-2 in the resulting solution. Sterile solutions were stored in a frozen state during the year.

The cultivation of the used CO2the incubator, which was maintained at constant levels of humidity, carbon dioxide concentration (5,0 + 1,0)% and temperature (37,0 + 0,2)oC. Cells were cultured in a volume of the I (TU 64-2-56-81) with loosely closed plastic covers. Produced subcultures after the cell suspension has reached a concentration of 1 to 106cells/ml (usually every 2 - 3 days). For laying new cultures used an initial concentration of 5 103live cells/ml in fresh medium. Before determining the activity of the drug for the removal of traces of IL-2 cells thoroughly, at least twice, washed described above environment.

The contents of each ampoule with options 1 - 4 of pharmaceuticals on the basis of IL-2 in solid form was dissolved 1.00 0.01 ml of 0.1% - ordinator. The resulting solution in 0.1% LTOs were diluted 100 times using 0.1% of LTOs series two serial dilutions, and then were diluted 10 times with RPMI medium 1640 without supplements. When it is necessary to determine the biological activity of the oxidized form of pharmaceutical preparations based on interleukin-2 in solid or liquid form, the contents of the vial were diluted in 400 ml of 0.9% isotonic NaCl at room temperature. The resulting solution retireval the holes filled with 100 μl of RPMI medium 1640 without supplements, by a two-fold dilutions by transferring 100 ál from wells in the hole, mixing thoroughly before each subsequent breeding so that not less than five consecutive two variants of cultivation used at least 3 holes.

As a positive control options used the solutions of a standardized drug interleukin-2 (10), rostirolla it in a similar way, using at least 3 holes on each dilution. As a negative control options (control background) used at least 3 holes that were made in 100 μl of medium containing the mixture of excipients included in the composition of the drug mannitol, sodium dodecylsulfate, dithiothreitol, ammonium bicarbonate), at concentrations corresponding to their content in the test samples.

All wells with samples analyzed drug and control options were added 100 μl of the washed medium RPMI 1640 without supplements containing 5% fetal serum fetal cow, cells CTLL-2 (1 of 105living cells/ml) and covered the tablet is gas-permeable film or a leaky cap. Samples were incubated CC2-the incubator in a humid atmosphere containing 5% carbon dioxide at 37oC for 40 to 42 hours. In samples containing interleukin-2, observed cell proliferation CTLL-2.

After incubation, each well was brought to 10 μl of MTT solution (3-[4,5-dimethylthiazol-2-yl] -2,5 of diphenyltetrazolium bromide) (0,005% MTT in RPMI medium 164 is, containing 5% carbon dioxide at 37oC.

In viable cells stimulated by interleukin-2, was formed water-insoluble dark blue crystals formazan. The plates were centrifuged for 5 min at 2000 rpm and the supernatant was removed from the wells by a sharp Strahovanie. In each Lenk added a 100 - 200 ál of dimethyl sulfoxide (DMSO) and shook the tablets on the shaker for 15 minutes to dissolve the formed crystals formazan.

In samples containing interleukin-2 person, watching the deep blue colour of the solution, which was Kosovo dependent on the concentration of interleukin-2 in the culture medium.

For the quantitative determination of biological activity used 96-well plates, suitable for photometry (for example, Titertek production Flow Laboratoris, England). We measured the optical density of the solution in all holes spectrophotometrically at a wavelength of 540 and 690 nm using a computerized spectrophotometer reader (Uniplan" firm bioservice, Moscow). All wells, except for one or more "zero", made in 100 μl of RPMI medium 1640 containing interleukin-2.

He worked with the values obtained as rasati 0,2.

Build new, reflecting the dependence of the measured values of the difference of optical densities in the wells from the number listed in the standard sample of interleukin-2 (in semi-logarithmic scale). On the same graph built a similar curve for the dependency of the measured values of optical density in the wells from the dilution were added the analyzed drug. Both curves were sigmoidal (S-arbusow) form and located in parallel.

For standard IL-2 sloping segment of the curve must be in the range of 0.2 to 5 IU IU.

To evaluate the activity of the analyzed drug used sheet probabilities (probitas) paper, where on the x-axis (arithmetic scale) postponed the degree of dilution, expressed in logarithms to base 2 (log2), and on the ordinate axis (probability scale), color grade, which is expressed in fractions (%) of the maximum level of color made in the positive control version with standard drug interleukin-2.

The data obtained is displayed at the specified scale as points with the corresponding coordinate values.

Opcina-2, which corresponds to a 50% degree of coloration, and K2 are equal to the value of the test drug.

To calculate the activity And the test drug in the international activity units (IU) by the formula: A = 2K2/2K1.

Statistical analysis was performed by standard methods.

Below are the results of the study of biological activity and stability of pharmaceutical preparations on the basis of interleukin-2, made in solid form (options).

Option 1 mg

interleukin-2 - 1,0

mannitol - 50,0

sodium dodecyl sulphate - 10,0

dithiothreitol - 0,32

ammonium bicarbonate - 0,8

the pH of 7.7

Option 2 mg

interleukin-2 - 1,0

mannitol - 50,0

sodium dodecyl sulphate - 10,0

ammonium bicarbonate - 0,8

the pH of 7.7

Option 3 mg

interleukin-2 - 1,0

mannitol - 50,0

human serum albumin - 50,0

sodium dodecyl sulphate - 10,0

dithiothreitol - 0,32

ammonium bicarbonate - 0,8

the pH of 7.7

Option 4 mg

interleukin-2 - 1,0

human serum albumin - 50,0

sodium dodecyl sulphate - 10,0

dithiothreitol - 0,32

ammonium bicarbonate - 0,8

the pH of 7.7 is, containing the recombinant protein in a reduced state are the same convenient for clinical use, as a similar product containing interleukin-2 in the oxidized state, but, unlike the latter, has virtually no side effects and has higher biological stability. It is known that the reduced form of the interleukin-2 is rapidly oxidized under physiological conditions, for example, saline solution, plasma, blood.

To stabilize and prevent the loss of biological activity of interleukin-2 in the form of pharmaceutical preparations of the present invention introduced human serum albumin (CSA). This also makes for more convenient use of the drug for parenteral administration, because there is no need of adding albumin in physiological solutions, which are used interleukin-2 system. In those cases, when a pharmaceutical preparation based on interleukin-2 is administered topically, use of the pharmaceutical preparations of the present invention that does not contain human serum albumin.

From the presented data in table 1 (paragraph 1) shows that the biological activity of the drug of option 1 (threitol increases the biological activity of drugs in solid form on the basis of interleukin-2 (paragraph 1), and also contributes to the conservation of biological stability during two years of storage (paragraphs 2 and 3 of Table 1).

The biological activity of the drug of option 3 (containing together CSA and mannitol) is slightly superior to that of option 4 (containing CSA, not containing mannitol) and option 1 (containing mannitol, not containing CSA) therefore, the joint use of mannitol, CSA promote more effective conservation of the biological activity of drugs in solid form on the basis of interleukin-2.

Stable aforementioned substances interleukin-2 may be stored at a temperature of from -20oC up to +4oC within two years, as demonstrated by the experiments on the study of the biological activity of various pharmaceutical preparations of the invention on the basis of interleukin-2 (paragraphs 2 and 3 of Table 1).

The biological activity of variants of drugs in solid form on the basis of interleukin-2 during the first year is reduced by 12.5% of drugs in options 1 and 4, 5.5%, the drug of option 3, by the end of 2 years of storage is reduced by 20.0% of drugs in options 1 and 4, 14.0% of the drug of option 3, therefore, the most stabil is.

The biological activity of variants of solid drug forms of the present invention, the oxidized state, comparable to that of natural interleukin-2 (item 4, Table 1). Demonstrated enhancement of biological activity associated with the oxidation is natural for biologically active molecules. From the presented data shows that the oxidized state of the biological activity of the drug of option 3 (containing together mannitol, CSA) surpass those of option 1 (containing mannitol), but inferior to that of option 4 (containing CSA), therefore, CSA better stabilizes the oxidized Il-2 in the form of pharmaceutical preparations in solid form. It should be noted that the activity of the same drug, "Proleukin" produced in oxidized form, ranges from 11 to 24 million IU per 1 mg protein.

Similar patterns observed in experiments with variants 5, 6, 7 liquid form of pharmaceutical preparations based on interleukin-2.

Option 5 mg

Interleukin-2 - 1,0

Mannitol - 50,0

Sodium dodecyl sulphate - 10,0

Dithiothreitol - 0,32

Sodium phosphate disubstituted - 1,24

Sodium phosphate one-deputizing - 0,20
the in - 50,0

Sodium dodecyl sulphate - 10,0

Dithiothreitol - 0,32

Sodium phosphate disubstituted - 1,24

Sodium phosphate one-deputizing - 0,20

pH 7,6

Option 7 - mg

Interleukin-2 - 1,0

Human serum albumin - 50,0

Sodium dodecyl sulphate - 10,0

Dithiothreitol - 0,32

Sodium phosphate disubstituted - 1,24

Sodium phosphate one-deputizing - 0,20

pH 7,6

The biological activity of these drugs were investigated for their ability to stimulate the proliferation of cytotoxic T-cell line mouse CTLL-2 by the method described above in this example. The difference lay in the fact that the contents of the capsules already in the liquid state, was diluted 100 times with 0.1% LTOs and then retireval according to the described method. Further, as a negative control options (control background) used a mixture of excipients included in a liquid form (mannitol, sodium dodecylsulfate, dithiothreitol, sodium phosphate and one-deputizing sodium phosphate disubstituted), at concentrations corresponding to their content in the test samples. Specific biological activity of IL-2 was also expressed in International Units (IU). Re the local CSA and mannitol) is slightly superior to that of option 7 (containing CSA, does not contain mannitol) and option 5 (containing mannitol and not containing CSA) therefore, the joint use of mannitol, CSA promote more effective conservation of the biological activity of drugs in liquid form on the basis of interleukin-2.

The biological activity of variants of drugs in liquid form on the basis of interleukin-2 by the end of 2 years of storage at 25% of drugs in options 5 and 7, 18.0% of the drug of option 6, therefore, the most stable is version 6 of drugs in liquid form, containing together mannitol, CSA.

The biological activity of variants liquid form preparations of the present invention, the oxidized state, comparable to that of natural interleukin-2 and several surpass those of the variants of the drug in solid form in the oxidized state. It should be noted that the biological activity of oxidized variant 7 (containing CSA) surpass those of the oxidized version 6 (containing together mannitol, CSA), and activity of oxidized variants 6 and 7 above activity of oxidized option 5.

Therefore, under physiological conditions, the biological activity is higher for drugs in solid and liquid form at the core is the one with CSA in solid form.

The biological stability of the above medicaments on the basis of interleukin-2 in solid form than that of the drugs in liquid form, most - of the drug in solid form, containing together mannitol, CSA.

Example 4. Research on the effects of pharmaceutical medicine on the basis of interleukin-2 in solid form (option 1).

Study of General toxic effect of interleukin-2 were performed on rodents (50 randombred white mice, 230 white rats and 12 rabbits of the Chinchilla breed). The study included a Toxicological study of a single exposure of the drug in a dose of 5 mg/kg, exceeding therapeutic dose 50 times, and chronic Toxicological experiment.

Toxicological study of a single exposure of the drug to the maximum-tolerated dose of 5 mg/kg intravenous testifies to the stability of these types of mammalian interleukin-2. In this experiment, the drug caused a short-term hyperthermia, had no effect on cellular elements of the peripheral blood (erythrocytes, leukocytes) were slightly changed behavioral responses. Sex differences in mice to the toxic effects of the drug with an introduction not seen the signs of anxiety. Analysis of ECG 24 hours after drug administration to rabbits did not reveal any cardioprotective actions.

Histological examination of organs and tissues of rats after a single injection of the drug at a dose of 5 mg/kg did not find any structural changes in the brain, pituitary gland, spleen, lymph nodes, testes. The thymus gland of Wistar rats did not differ from the structure of the thymus of control animals.

Thus, detected no significant toxic effects in experiment on acute toxicity after a single dose maximum tolerated dose.

Chronic Toxicological experiment lasted for three months at 230 randombred white rats of both sexes. Males had a body weight of 300 to 315 g, females - 310 - 320, Animals were kept in plastic cages area of 2150 cm28 individuals were fed piketirovany and natural foods. The drug was administered intraperitoneally at a standard experimental technique for chronic toxicity in the dose calculated for rats Y. R. Rybolovlev, R. C. Rybolovlev (1979), corresponding to 0.1 mg/kg At the end of the experiment part of the experimental animals was decapotable, cracked, op is neither fixed in neutral formalin, histological specimens were prepared by the conventional method of filling in paraffin and staining hematoxylin-eosin. All materials experiments were processed by the conventional method by the Student.

Revealed mainly functional and behavioral changes. Table 3 presents the results of the comparison side effects of the drug of option 1 with those of the drug Proleukin in the experiment for the study of chronic toxicity on white rats.

From Table 3 it is evident that the drug of option 1 is missing most of the side effects, exhibited similar drug Proleukin.

Next, the effects of option 1 compared with control animals not receiving the drug.

Within three months of chronic experiment in laboratory animals was not observed signs of severe intoxication and significant changes in behavior.

Increase in body weight of rats of both sexes did not differ significantly from control animals. Observed short-term reduction in the emotional component of behavior of male rats under the influence of a therapeutic dose of the drug, increasing the number of "washings" and "cleansing" of male rats. The drug is established in the open field during the chronic experiment revealed no significant natural changes.

Within three months of chronic experiment detoxifying, secretory and absorptive functions of the liver of Wistar rats did not differ significantly from the norm. Structure of the rat liver in normal limits. Inflammation was absent.

At autopsy, the animals macroscopically not detected any significant changes. The study of the histological structure of organs and tissues of experimental animals compared to control rats revealed no significant structural changes caused by the influence of the drug. The heart muscle does not have any significant deviations from the norm.

Observed changes in the structure of the spleen of Wistar rats. The lymph follicles of the white pulp of the spleen, in the main, had the centers of reproduction, in which it noted more macrophages, as well as a greater number of mitoses than in the control group. In the red pulp increased number of megakaryocytes and macrophages. These changes are inevitable on the basis of immunotropic nature of the drug on the basis of interleukin-2, causing the proliferation of the above-mentioned cells of the immune system.

After a month of exposure to the drug of option 4 under the influence of a therapeutic dose noble was temporary and was normalized to the end of the experiment rats-males. In female rats after three months of administration of the drug was noted only decrease the speed of blood clotting in the third minute.

In the thymus noted the plethora of capillaries. In the testes was observed plethora of capillaries of the interstitial tissue.

Analysis of the local irritant action of the drug of option 1 was performed by macroscopic and histological examination of tissues at the injection site. The analysis did not reveal any significant macro - and microscopic changes of the peritoneum, blood vessels and surrounding tissue at the injection site.

In General, the drug is characterized as non-lethal, highly portable, does not have specific side effects.

References:

1. Sedlacek, H.-H, Moroy T./Immune reactions. p. 15 - 17. Springer, 1995.

2. Hack, C. E. et al. The vascular leak syndrom induced by interleukin-2: pathogenesis and treatment with C1-elastase inhibitor. In: The immune consequences of trauma, shock and sepsis - mechanisms and therapeutic approaches, vol. 1, p. 72 - 77. 1996. E. Faist et al. (Eds).

3. Weil, M. P. , Sparks J. Purification and renaturation of recombinant human interleukin-2. Biochemical Journal, v. 245, p. 85 - 91. 1987.

4. Ant I. A. Technology of drugs. So 1. M.: Medicine, 1981, S. 391.

5. Ant I. A. Technology of drugs. So 2. M.: Medicine, 1981, S. 703.

6. With the logically active dispersive environments. Thesis of doctor in pharmaceutical Sciences. - Kharkiv; 1992.

7. Cats M. Technique of lipid. M.: 1975, S. 324.

8. Methods of enzymology. V. 182. Guide to protein purification. Ed. Murray P. Deutscher. P. 894. 1993. Academic Press.

9. Patent SU N 1770359. Myasnikov A. N., Smirnov A. N., Avot A. J., Gren, E. J., Romanchikova N. In., Simanis A. Y. Recombinant plasmid DNA pJDB(MSIL), which provides a synthesis of interleukin-2 in cells of the yeast Saccharomyces cervisiae, its preparation and strain of Saccharomyces cerevisiae producing interleukin-2 human".

10. Gearing, A. J., Thorpe R. The international standard for human interleukin-2. Calibration by international study. Journal of Immunological Methods, v. 114, p. 3 - 9. 1988. Elsevier Science Publishers B. V. (Biomedical Division).

1. Pharmaceutical drug on the basis of interleukin-2 as the active substance-containing stabilizer, a solubilizer, an antioxidant agent and a buffer, wherein the solid form of the drug contains as a stabilizer substance selected from the group consisting of mannitol, human serum albumin or a mixture, as a solubilizer, a substance selected from the group consisting of guanidine hydrochloride, sodium dodecyl sulphate, as an antioxidant agent is a substance selected from the group consisting of glutathione in return the ith component, mg:

Interleukin-2 - 0,1 - 7,0

Stabilizer - 5,0 - 350,0

The solubilizer - 1,0 - 70,0

The antioxidant agent is 0.1 to 3.5

Buffer, mm - 5,0 - 10,0

2. Pharmaceutical drug under item 1, characterized in that as interleukin-2 it contains recombinant interleukin-2.

3. Pharmaceutical drug under item 1, characterized in that as interleukin-2, it contains non-recombinant interleukin-2.

4. Pharmaceutical drug on the PP.1 to 3, characterized in that it is made in the form of tablets.

5. Pharmaceutical drug on the PP.1 to 3, characterized in that it is made in the form of capsules.

6. Pharmaceutical drug on the PP.1 to 3, characterized in that it is made in the form of a powder.

7. Pharmaceutical drug on the PP.1 to 3, characterized in that it is made in the form of granules.

8. Pharmaceutical drug on the PP.1 to 3, characterized in that it is made in the form of a suppository.

9. Pharmaceutical drug on the PP.1 to 3, characterized in that it is made in the form of a liposomal form.

10. Pharmaceutical drug on the PP.1 to 3, characterized in that it is made in the form, suitable for outdoor use.

11. Pharmaceutical drug on the PP.1 - reallemon solvent.

12. Pharmaceutical drug on the basis of interleukin-2 as the active substance, optionally containing a stabilizer, a solubilizer, an antioxidant agent and a buffer, wherein the liquid form of the drug contains as a stabilizer substance selected from the group consisting of mannitol, human serum albumin or a mixture, as a solubilizer, a substance selected from the group consisting of guanidine hydrochloride, sodium dodecyl sulphate, as an antioxidant agent is a substance selected from the group consisting of glutathione in the reduced form, 2-mercaptoethanol, dithiothreitol, as a buffer - pharmaceutically acceptable buffer, the next component content, mg:

Interleukin-2 - 0,1 - 5,0

Stabilizer - 5,0 - 250,0

The solubilizer - 1,0 - 50,0

The antioxidant agent is 0.1 to 2.5

Buffer, mm - 5,0 - 10,0

13. Pharmaceutical drug under item 12, characterized in that as interleukin-2 it contains recombinant interleukin-2.

14. Pharmaceutical drug under item 12, characterized in that as interleukin-2, it contains non-recombinant interleukin-2.

15. Pharmaceutical drug on the PP.12 to 14, characterized in that it is the imp is the IDA syrup.

17. Pharmaceutical drug on the PP.12 to 14, characterized in that it is made in the form of an elixir.

18. Pharmaceutical drug on the PP.12 to 14, characterized in that it is made in the form of drops.

19. Pharmaceutical drug on the PP.12 to 14, characterized in that it is made in the form of a liposomal form.

20. Pharmaceutical drug on the PP.12 to 14, characterized in that it is made in the form, suitable for outdoor use.

 

Same patents:

The invention relates to biotechnology, in particular to the protein containing two subunits R40 interleukin-12, which are associated with one another preferably by means of at least one disulfide bond with mol
The invention relates to medicine, namely to neurosurgery and neurology

The invention relates to biotechnology, namely to medicines immunomodulating activity

The invention relates to medicine, namely to drugs and compositions for the treatment of poorly healing and surgical wounds, lesions, erosions, trophic ulcers, burns, bedsores, etc., especially complicated by purulent-inflammatory processes

The invention relates to medicine, in particular to the treatment of severe conditions in humans caused by infection
The invention relates to medicine, namely to a gastroenterologist may be used in the treatment of stomach polyps

FIELD: medicine, phthisiology.

SUBSTANCE: method involves administration of roncoleukin by lymphotropic route: subcutaneously, in region of pretracheal cellular tissue in the equal dose 1/4-1/5 of the average daily therapeutic dose, once in one or two days, 3 injections for a course. Invention provides the local delivery of roncoleukin to the injure zone directly that promotes to making depot of the latter in the injure focus and to the rapid elimination of pathogen from body of patients. Invention can be used for correction of immune insufficiency in patients with pulmonary tuberculosis.

EFFECT: improved method for correction.

2 ex

FIELD: medicine, immunology.

SUBSTANCE: method involves inhalation administration of combination of immunocorrecting agents wherein recombinant interleukin-2 is used or its combination with genetic engineering α2-interferon and with the complex immunoglobulin preparation in the daily doses for 5 days depending on age of patient. Before sanitation with an immunocorrecting agent the method involves assay of carrying type by anti-lysozyme activity of microorganisms (resident or transitory) and repeated examination after sanitation course is carried out. Carriers are subjected for additional sanitation with anti-bacterial preparations in change of carrying type from resident to transitory and with taking into account antibiotic-resistance property of the carrier strain. Method allows carrying out the effective sanitation and immune reablement of germ carriers due to recovery of the adequate natural resistance and complex of nonspecific factor of regional and systemic immunity.

EFFECT: improved and effective method for sanitation.

2 cl, 2 tbl, 5 ex

FIELD: cardiovascular disorders.

SUBSTANCE: invention provides methods for modulating tissue antiogenesis using Raf and/or Ras protein, modified Raf and Ras proteins, nucleic acids encoding them. Antiogenesis is inhibited using inactive Raf or Ras proteins or nucleic acids encoding them, and antiogenesis is potentiated using active Raf or Ras proteins or nucleic acids encoding them. Use of gene transportation system to provide nucleic acids encoding Raf or Ras proteins or modified forms thereof.

EFFECT: enlarged choice of tissue antiogenesis control methods and means.

44 cl, 20 dwg

FIELD: medicine, gastroenterology, pharmacy.

SUBSTANCE: invention relates to agents used in treatment of ulcerous-erosion injures in gastroduodenal region. Method involves diluting 100 mcg of dry lyophilized powder of immunomodulating agent "Superlimf" in 3-5 ml of 0.9% isotonic solution and irrigation of ulcer or erosion with this solution 1 time per a day by the endoscopy method. The treatment course is 3-4 procedures with break for 4-5 days. Method provides alteration of cytokine pattern of tissues, induction of influx of mononuclear phagocytes to the injure focus that results to localization of inflammation and the complete epithelization of ulcers and erosions.

EFFECT: improved and effective method for treatment.

1 ex

FIELD: medicine, oncology.

SUBSTANCE: after removing malignant cerebral tumor one should conduct successive course of: cytokinotherapy consisting of 3 intramuscular injections of leukineferon at 48-h-long interval, adaptive immunotherapy with lymphokine-activated killer cells (LAKC) generated in the presence of recombinant interleukin-2 (IL-2). Moreover, LAKC should be introduced into the channel of removed tumor in combination with IL-2 as 2 procedures at 24-h-long interval. Then comes the course of adaptive immunotherapy with cytotoxic lymphocytes (CTL) generated due to cultivating patient's mononuclear blood cells together with dendritic cells loaded with a tumor antigen, in the presence of recombinant IL-2 to be introduced in combination with it into the channel of removed tumor as 2 procedures at 48-h-long interval. For obtaining dendritic cells in patients before operation it is necessary to isolate monocytes to cultivate with granulocytic-macrophageal colony-stimulating factor and interferon-α at maturating dendritic cells in the presence of monocytic conditioned medium and incubation of dendritic cells in the presence of tumor antigenic material for their loading with a tumor antigen. After immunotherapy with CTL on should conduct the course of vaccinotherapy with dendritic cells loaded with a tumor antigen in combination with subcutaneous injections of IL-2. The method enables to induce high specific anti-tumor immune response along with improved quality of life and prolonged duration of relapse-free period.

EFFECT: higher efficiency of immunotherapy.

2 cl, 2 ex

FIELD: medicine.

SUBSTANCE: method involves combining vaccine prepared from 107 cells of autologic tumor lysate with 60 mg of betaleukine and introducing it strictly in intracutaneous paravertebral mode in three points arranged 3 cm far from each other. The introduction takes place every 3 weeks so that the first two vaccinations are combined with 470 mg of betaleukine introduced into anterior abdominal wall. Vaccination treatment is continued on receiving positive retarded type hypersensitivity response to injection after every vaccination with tumor lysate without betaleukine.

EFFECT: enhanced effectiveness in inducing and supporting antitumor immune response.

FIELD: medicine.

SUBSTANCE: method involves applying basic therapy until stroke type is unclear and differentiated therapy after having determined stroke type. Recombinant interleukine-2-roncoleukine as subcutaneous injections at a dose of 500 000 MU into external surface of intact arm at the fourth-fifth day after stroke beginning. Roncoleukine is administered as a course of 1-3 injections given with two-three days pause on the background of traditional stroke treatment.

EFFECT: enhanced effectiveness of treatment.

2 cl, 3 tbl

FIELD: medicine, infectious diseases, psychotherapy.

SUBSTANCE: method involves antiviral therapy, immune correction with thymus hormones and interferon inductors. Since the first day the relapse symptom method involves prescription of antiox+ (1 capsule per a day) and detox+ (1 capsule, 2 times per a day) for 30 days, profluzak (20 mg, 3 times per a day for 5 days) and then in the dose 20 mg, 1 time per a day for 20 days. Derinate is prescribed topically as installation into urethra in the dose 3-5 ml or with tampon into vagina and with simultaneous prescription of microenemas in the dose 10-40 ml for 10 days. Since 10-14 day in exacerbation period in the proliferative stage of an antiherpetic immune response derinate is prescribed by intramuscular injections in the dose 5 ml, 1 time in a day, 10 injections in total number. Then since 6-th day of exacerbation and intake of profluzak psychotherapy seances are carried out. The first seance of rational psychotherapy involves explanation to a patient in available form mechanism of the disease, the necessity of prolonged treatment and motivation for treatment is enhanced by suggestion. The second psychotherapy seance involves neurolinguistic programming wherein a patient colorful and detailed description of desirable function when he imagines achievement of the desire result, and positive emotional and vegetative symptoms are notes and the conditional-reflect association is formed by tactile contact. Under psychotherapist control a patient imagines "part of person" responsible for achievement of the desire result the patient attention is accented for the desire result and arisen physiological responses are fixed by using tactile contact. Also, new behavior methods are proposed to take for a patient that are directed for achievement of the desire result - avoiding sexual contacts during exacerbation of genital herpes in one of partner and during every month hormonal cycles, avoiding stress situations, and in case of each stress situation significant for patient profluzak has to be intake in a single dose 40 mg, using a condom in sexual contact in the exacerbation period. Patient analyzes the proposed new behavior methods that help avoiding relapses, provide good state of health, promotes to recovery process of genitals recovery and selects at least three the most rationally available for him behavior methods. In the case of the positive response that is controlled by physiological symptoms the result is fixed by tactile contact. The third seance involves the suggestive psychotherapy directed for fixing the attained result. The suggestive therapy seance is carried out once per a week for 6 months. Method provides declining the treatment time, to reduce relapse frequency of genital herpes and to recover the emotional state of patient.

EFFECT: improved treatment method.

2 cl, 3 tbl, 1 ex

FIELD: medicine, immunology.

SUBSTANCE: invention relates to compositions and method for immunosuppression achievement. Claimed compositions contain two main agents: namely the first agent targeted to interleukin-15 receptor (IL-15R), and the second agent which inhibits costimulating signal transferred between T-cell and antigen-presenting cell (APC).

EFFECT: diagnosis and therapy of immune deceases, in particular autoimmune deceases with improved effectiveness.

45 cl, 3 dwg

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