RU2140283C1 - Interleukin-2-base pharmaceutical preparation (variants) - Google Patents

Abstract

FIELD: medicine, pharmacology, pharmacy. SUBSTANCE: invention relates to a pharmaceutical preparation based on interleukin-2 as an active substance has a stabilizing agent taken among the group including mannitol, human serum albumin or their mixture, a dissolving agent taken among the group including guanidine hydrochloride, sodium dodecyl sulfate, an antioxidant taken among the group including reduced glutathione, 2-mercaptoethanol or dithiothreitol. The preparation has a pharmaceutically acceptable buffer, stabilizing agent, dissolving agent, antioxidant and buffer taken at the definite ratio of components. The preparation can be prepared in the solid and liquid form and used for treatment of mammals with severe states caused by infection, for example, sepsis of different etiology and oncological illnesses and immunodeficient states also. EFFECT: retention of high biological activity of interleukin-2, decreased adverse effects, improved technology of the preparation producing. 20 cl, 3 tbl, 4 ex

Description

 The invention relates to medicine and pharmacology, relates to a pharmaceutical preparation based on interleukin-2 and can be used to treat severe conditions in mammals caused by infection, for example, sepsis of various etiologies, as well as oncological diseases and immunodeficiency states.

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

Also known is the recombinant preparation of human interleukin-2, produced by intestinal E. coli, obtained by genetic engineering, manufactured by Sigma (catalog number E3267). However, this freeze-dried preparation contains PBS buffer (sodium phosphate buffer, pH 7.4) and 0.1% bovine serum albumin as a carrier. This preparation does not contain a stabilizer or antioxidant agent, due to which, according to the manufacturer, it exhibits low biological activity, has a limited storage time and should be used immediately after dissolution. In addition, the drug must be stored at -70 ^o C, otherwise it loses its biological activity.

Also known is the pharmaceutical preparation "Proleukin" in the form of a lyophilized form manufactured by Chiron, USA. The drug is a recombinant human interleukin-2 analogue produced by E. coli E. coli cells. It contains mutated interleukin-2 (1 mg) as an active substance, sodium dodecyl sulfate (0.13 - 0.23 mg) as a solubilizer, mannitol (50 mg) as well as NaH ₂ PO ₄ as a carrier. • H ₂ O (0.17 mg) and Na ₂ HPO ₄ (1.2 mg). This medication does not contain an antioxidant agent. However, it should be noted that Proleukin is a mutein, i.e. the amino acid sequence of interleukin-2 is altered by removing the cysteine residue at position 125 and replacing it with serine or alanine (US Patent No. 4, 518, 584). This change in the native structure of the protein was carried out in order to obtain the oxidized form of the preparation of interleukin-2, and the formation of a disulfide bridge in such a mutein occurs only with the participation of the two remaining cysteine residues. However, it should be noted that this drug has a large number of side effects (1). Part of these effects is due to the fact that it is mutein, part to the bacterial origin of this recombinant interleukin-2, and part to the fact that the high biological activity artificially given to oxidized interleukin-2 significantly exceeds that of a natural protein and can stimulate the synthesis of pro-inflammatory cytokines, provoking serious systemic effects (2). It is known that E. coli is a conditionally pathogenic microorganism, and even the use of high-tech cleaning does not fully ensure the removal of E. coli endotoxins. In addition, the known methods of oxidation are not technologically advanced: the technological scheme is significantly extended and the separation of interleukin-2 from impurity proteins is difficult. In addition, the known methods of oxidation require a large dilution of solutions; the concentration of proteins in such solutions is up to 1 γ / ml (3). Existing technological methods for the concentration of large volumes of solutions do not allow preserving the biological activity of interleukin-2, thus, when obtaining the oxidized form of interleukin-2, some loss of bioactivity occurs.

 The objective of the present invention is to provide a pharmaceutical preparation based on interleukin-2, in such forms and with such a composition that would make it possible to obtain a biologically active drug with fewer side effects, with a production flow chart and composition allowing to preserve the biological activity of interleukin-2.

This problem is solved by a pharmaceutical preparation in solid form based on interleukin-2 as an active substance, containing a stabilizer, a solubilizer, an antioxidant agent and a buffer. The preparation contains as a stabilizer a substance selected from the group consisting of mannitol, human serum albumin or a mixture thereof, as a solubilizer, a substance selected from the group consisting of guanidine hydrochloride, sodium dodecyl sulfate, and as an antioxidant agent, a substance selected from the group containing glutathione in reduced form, 2-mercaptoethanol, dithiothreitol, as a buffer - a pharmaceutically acceptable buffer, with the following components, mg:
Interleukin-2 - 0.1 - 7.0
Stabilizer - 5.0 - 350.0
Solubilizer - 1.0 - 70.0
Antioxidant Agent - 0.1 - 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 based on interleukin-2 of the present invention are pharmaceutical preparations containing, as interleukin-2, either recombinant interleukin-2 or non-recombinant interleukin-2.

 Particular cases of the implementation of a pharmaceutical preparation in solid form based on interleukin-2 of the present invention are tablets, capsules, powders, granules, suppositories, liposome forms, forms suitable for external use, for example, ointments, gels, moistened wipes, or liquid preparations based solid forms obtained by dissolving the solid form in a pharmaceutically acceptable solvent, obtained by standard technological methods (4, 5, 6, 7).

This problem is also solved by a preparation in liquid form based on interleukin-2 as an active substance, containing a stabilizer, a solubilizer, an antioxidant agent and a buffer. The preparation contains as a stabilizer a substance selected from the group consisting of mannitol, human serum albumin or a mixture thereof, as a solubilizer, a substance selected from the group consisting of guanidine hydrochloride, sodium dodecyl sulfate, and as an antioxidant agent, a substance selected from the group containing glutathione in reduced form, 2-mercaptoethanol, dithiothreitol, as a buffer - a pharmaceutically acceptable buffer, with the following components, mg:
Interleukin-2 - 0.1 - 5.0
Stabilizer - 5.0 - 250.0
Solubilizer - 1.0 - 50.0
Antioxidant Agent - 0.1 - 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 based on interleukin-2 of the present invention are pharmaceutical preparations containing either recombinant interleukin-2 or non-recombinant interleukin-2 as interleukin-2.

 Emulsion, syrups, elixirs, drops, liposome forms, forms suitable for external use, for example, ointments, gels, moistened wipes obtained by standard technological methods, are also special cases of pharmaceutical preparations based on the liquid form of the present invention (5, 7).

 The invention is further illustrated by the following examples.

 Example 1. Obtaining a pharmaceutical preparation based on interleukin-2 in solid form (options).

 A solution containing human interleukin-2 was obtained by standard methods of protein chemistry (8) purification of the extract obtained from cells of strain 1-GRF18 (MSIL) of Saccharomyces cerevisiae yeast deposited in the All-Union Collection of Industrial Microorganisms under the number VKPM-U791 (9).

The concentration of interleukin-2 in the eluate was measured. The eluate obtained by gel filtration of the above solution on a Sephacryl S-300 column contains interleukin-2 at a concentration of, on average, from 1 to 2 mg per 1 ml of ammonium bicarbonate buffer containing 0.1% sodium dodecyl sulfate (SDS), 1 mM ethylenediaminetetrachloroacetic acid (EDTA), 5 mM 2-mercaptoethanol (2-ME) and 0.01% NaN ₃ .

To 100 ml of the above-mentioned eluate containing interleukin-2 at a concentration of 1.5 mg / ml, 400 ml of acetone cooled to -18 ^° C was added and left for 15 minutes at -18 ^° C. Centrifuged for 15 minutes at a speed of 2500 rpm minute at a temperature of -18 ^o C. the Acetone was discharged, the precipitate containing interleukin-2 was dried, and dissolved in 150 ml of a buffer of the following composition (mg):
Mannitol - 7500
Sodium dodecyl sulfate - 1500
Dithiothreitol - 48
Ammonium Bicarbonate - 120
or in 150 ml of the buffer of the following composition (mg):
Mannitol - 7500
Sodium dodecyl sulfate - 1500
Dithiothreitol - 48
Ammonium Bicarbonate - 120
Human Serum Albumin - 7500
Thus, the concentration of interleukin-2 in this formulation buffer is 1 mg / ml. The concentration of hydrogen ions in the resulting solution, measured by a standard method on a Beckman pH meter, USA, was 7.7. The resulting solution was sterilized by cold sterilization in a standard way (8), poured into 1 ml ampoules and freeze-dried. Thus, one ampoule of a pharmaceutical preparation in solid form based on interleukin-2 contains (mg):
Interleukin-2 - 1.0
Mannitol - 50.0
Sodium dodecyl sulfate - 10.0
Dithiothreitol - 0.32
Ammonium bicarbonate - 0.8
or
Interleukin-2 - 1.0
Mannitol - 50.0
Sodium dodecyl sulfate - 10.0
Dithiothreitol - 0.32
Ammonium bicarbonate - 0.8
Human Serum Albumin - 50.0
In a similar way, a pharmaceutical preparation is obtained in solid form based on non-recombinant interleukin-2.

 Example 2. Obtaining a pharmaceutical preparation based on interleukin-2 in liquid form (options).

 An eluate containing interleukin-2 was obtained by the method described in Example 1.

To 100 ml of the above eluate containing interleukin-2 at a concentration of 1.0 mg / ml, 400 ml of acetone cooled to -18 ^° C was added and left for 15 minutes at -18 ^° C. Centrifuged for 15 minutes at a speed of 2500 rpm minute at a temperature of -18 ^o C. the Acetone was discharged, the precipitate containing interleukin-2 was dried, and dissolved in 100 ml of a buffer of the following composition (mg):
Mannitol - 5000
Sodium dodecyl sulfate - 1000
Dithiothreitol - 32
Sodium Phosphate Disubstituted - 124
Monosubstituted sodium phosphate - 20
or
Mannitol - 5000
Sodium dodecyl sulfate - 1000
Dithiothreitol - 32
Sodium Phosphate Disubstituted - 124
Monosubstituted sodium phosphate - 20
Human Serum Albumin 5000
Thus, the concentration of interleukin-2 in this formulation buffer is 1 mg / ml. The concentration of hydrogen ions in the resulting solution, measured by the standard method on a Beckman pH meter, USA, was 7.6. The resulting solution was sterilized by cold sterilization in a standard way (8), poured into 1 ml ampoules and sealed in a stream of sterile air. Thus, one ampoule of a pharmaceutical preparation in liquid form based on interleukin-2 contains (mg):
Interleukin-2 - 1.0
Mannitol - 50.0
Sodium dodecyl sulfate - 10.0
Dithiothreitol - 0.32
Disubstituted sodium phosphate - 1.24
Monosubstituted sodium phosphate - 0.20
or
Interleukin-2 - 1.0
Mannitol - 50.0
Sodium dodecyl sulfate - 10.0
Dithiothreitol - 0.32
Disubstituted sodium phosphate - 1.24
Monosubstituted sodium phosphate - 0.20
Human Serum Albumin - 50.0
Example 3. The study of the biological activity and stability of pharmaceutical preparations based on interleukin-2, made in solid and liquid form (options).

 The biological activity of pharmaceutical preparations in solid form based on interleukin-2 was studied by their ability to stimulate the proliferation of mouse cytotoxic T-lymphocytes of the CTLL-2 line. The specific biological activity of IL-2 is expressed in International Units (IU), 1 mg of IL-2 in reduced form should have biological activity ranging from 300 thousand IU to 1.5 million IU.

Continuous proliferation of CTLL-2 cells was maintained in complete culture medium RPMI 1640 with the necessary additives (2 mM L-glutamine, 2 mM pyruvic acid, 50 μM 2-mercaptoethanol, 100 units / ml benzylpenicillin or ampicillin, 100 μg / ml streptomycin or 50 μg / ml gentamicin, 16 μM / ml sodium bicarbonate, 25 μM / ml HEPES and 10% heat inactivated at +5 ^o C for 1 hour of fetal fetal cow serum (SEC) containing IL-2 at a concentration of 35 - 45 IU / ml as a source of IL-2 used the supernatant of rat spleen cells, stimulus nested with concanavalin A. The spleen of Wystar rats was destroyed by mild methods to a cellular state. Spleen cells were washed twice with RPMI 1640 medium. A suspension of cells was obtained at a concentration of about 5 • 10 ⁶ cells / ml. 5% fetal serum and concanavalin A were added to a concentration of 5 μg / ml, the Mixture was incubated for 30 h at 37 ^o C in an atmosphere of 5% CO _2. Cells were separated by centrifugation. The supernatant was sterilized by filtration through membrane filters with a pore diameter of 0.22 μm. The presence and concentration of IL-2 in the resulting solution were evaluated. Sterile solutions were stored frozen for a year.

For cultivation, a CO ₂ incubator was used, in which humidity, carbon dioxide concentration (5.0 + 1.0)% and temperature (37.0 + 0.2) ^o C were maintained at constant levels. Cells were cultured in a volume of 1.0 - 1.5 ml of the above medium with additives in 24-well plates for single-use cell cultures (TU 64-2-56-81) with loose-fitting plastic lids. Reseeds were performed after the cell suspension reached a concentration of 1 • 10 ⁶ cells / ml (usually every 2 to 3 days). For laying new cultures used an initial concentration of 5 • 10 ³ living cells / ml in fresh medium. Before determining the activity of the drug to remove traces of IL-2, the cells were washed thoroughly, at least two times, with the medium described above.

 The contents of each ampoule with options 1-4 of pharmaceutical preparations based on IL-2 in solid form were dissolved in 1.00 ± 0.01 ml of 0.1% SDS. The resulting solution in 0.1% SDS was diluted 100 times with 0.1% SDS in series of two successive dilutions, after which it was diluted 10 times with RPMI 1640 medium without additives. In the case 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 ampoule were diluted in 400 ml of a 0.9% isotonic NaCl solution at room temperature. The resulting solution was triturated in wells filled with 100 μl of RPMI 1640 medium without additives by double dilutions, transferring 100 μl from well to well, and mix thoroughly before each subsequent dilution so that at least five consecutive double dilutions of the analyzed sample contained rIL-2 in concentrations from about 50 IU / ml to 0.1 IU / ml. At least 3 wells were used for each dilution option.

 Solutions of a standardized human interleukin-2 preparation (10) were used as positive control options, triturated in the same way, using at least 3 wells for each dilution. At least 3 wells were used as negative control options (background control), in which 100 μl of medium containing a mixture of excipients included in the preparation (mannitol, sodium dodecyl sulfate, dithiothreitol, ammonium bicarbonate) was added at concentrations corresponding to their content in test samples.

100 μl of washed RPMI 1640 medium without additives containing 5% fetal bovine fetal serum, CTLL-2 cells (1 × 10 ⁵ live cells / ml) was added to all wells with samples of the analyzed preparation and with control variants and the plate was closed with a gas-permeable film or loose cover. Samples were incubated in a CC ₂ incubator in a humid atmosphere containing 5% carbon dioxide at 37 ^° C for 40 to 42 hours. In samples containing human interleukin-2, proliferation of CTLL-2 cells was observed.

After incubation, 10 μl of MTT solution (3- [4,5-dimethylthiazol-2-yl] -2.5 diphenyltetrazoline bromide) (0.005% MTT in RPMI 1640 medium without additives or standard PBS buffer) was added to each well and incubated even 4 - 6 hours in a CO ₂ incubator in a humid atmosphere containing 5% carbon dioxide at 37 ^o C.

 In viable cells stimulated by interleukin-2, water-insoluble dark blue formazan crystals formed. The plates were centrifuged for 5 min at 2000 rpm and the supernatant was removed from the wells by sharp shaking. 100-200 μl of dimethyl sulfoxide (DMSO) was added to each ribbon and the plates were shaken on a shaker for 15 minutes until the formed formazan crystals were completely dissolved.

 In samples containing human interleukin-2, a dark blue staining of the solution was observed, which was dose-dependent on the concentration of interleukin-2 in the culture medium.

 To quantify the biological activity, 96-well plates suitable for photometry were used (e.g., Titertek manufactured by Flow Laboratoris, England). The optical density of the solution was measured in all wells spectrophotometrically at a wavelength of 540 and 690 nm using a computerized photometer reader (Uniplan firm Bioservice, Moscow). 100 μl of RPMI 1640 medium containing no interleukin-2 was added to all wells, with the exception of one or more “null” ones.

 Then we worked with the values obtained as the difference of these two measurements. The optical density values in the wells with negative control should not exceed 0.2.

 A new one was constructed, reflecting the dependence of the measured values of the difference in optical densities in the wells on the amount of a standard human interleukin-2 sample introduced into them (on a semi-logarithmic scale). A similar curve was constructed on the same graph for the dependence of the measured values of optical density in the wells on the dilution degree of the analyzed preparation introduced into them. Both curves had a sigmoid (S-orbase) shape and were located in parallel.

 For the standard sample IL-2, the inclined portion of the curve should range from 0.2 IU to 5 IU.

To evaluate the activity of the analyzed drug, a sheet of probabilities of the (probit) paper was used, where the degree of dilution of the drug, expressed in logarithms on base 2 (log ₂ ), was plotted on the x-axis (on an arithmetic scale), and the degree of coloring was plotted on the ordinates (on a probability scale) , which is expressed in fractions (%) of the maximum color level achieved in the positive control variant with the standard preparation of human interleukin-2.

 The obtained data were displayed in the indicated scale in the form of points with corresponding coordinate values.

 The degree indicators K1 and K2 were determined. K1 is equal to the value of the degree of dilution of the standard sample of interleukin-2, which corresponds to a 50% degree of color, and K2 is equal to the same value for the test drug.

The activity A of the test drug in international units of activity (ME) was calculated by the formula: A = 2 ^k2 / 2 ^k1 .

 Statistical processing of the results was carried out by standard methods.

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

Option 1 - mg
interleukin-2 - 1.0
mannitol - 50.0
sodium dodecyl sulfate - 10.0
dithiothreitol - 0.32
ammonium bicarbonate - 0.8
pH - 7.7
Option 2 - mg
interleukin-2 - 1.0
mannitol - 50.0
sodium dodecyl sulfate - 10.0
ammonium bicarbonate - 0.8
pH - 7.7
Option 3 - mg
interleukin-2 - 1.0
mannitol - 50.0
human serum albumin - 50.0
sodium dodecyl sulfate - 10.0
dithiothreitol - 0.32
ammonium bicarbonate - 0.8
pH - 7.7
Option 4 - mg
interleukin-2 - 1.0
human serum albumin - 50.0
sodium dodecyl sulfate - 10.0
dithiothreitol - 0.32
ammonium bicarbonate - 0.8
pH - 7.7
The pharmaceutical preparations based on interleukin-2 in solid or liquid form of the present invention containing recombinant protein in the reduced state are as convenient for clinical use as a similar preparation containing interleukin-2 in the oxidized state, but, in contrast to the latter, practically It has no side effects and exhibits higher biological stability. It is known that the reduced form of interleukin-2 is rapidly oxidized under physiological conditions, for example, in physiological saline, plasma, and blood.

 To stabilize and prevent the loss of biological activity of interleukin-2, human serum albumin (HSA) has been introduced into the pharmaceutical preparations of this invention. It also makes it more convenient to use the drug for parenteral administration, as there is no need to add albumin to physiological solutions, in the composition of which interleukin-2 is used systemically. In cases where a pharmaceutical preparation based on interleukin-2 is administered topically, the pharmaceutical preparations of the present invention that do not contain human serum albumin are used.

 From the data presented in table 1 (paragraph 1), it is clear that the biological activity of the preparation of option 1 (containing dithiothreitol) exceeds that of option 2 (not containing dithiothreitol), therefore, dithiothreitol increases the biological activity of the preparations in solid form based on interleukin-2 (paragraph 1 ), and also contributes to the preservation of biological stability during two years of storage (paragraphs 2, 3 of Table 1).

 The biological activity of the drug of option 3 (containing both HSA and mannitol) is slightly higher than that of option 4 (containing HSA, not containing mannitol) and option 1 (containing mannitol, not containing HSA), therefore, the combined use of mannitol and HSA contributes to more effective conservation of biological activity solid preparations based on interleukin-2.

Interleukin-2 stabilized by the aforementioned substances can be stored at temperatures from -20 ^o C to +4 ^o C for two years, as demonstrated by experiments to study the conservation of biological activity of various pharmaceutical preparations of the invention based on interleukin-2 (paragraphs 2, 3 of Table 1) .

 The biological activity of the variants of the preparations in solid form based on interleukin-2 during the first year decreases by 12.5% in the preparations of options 1 and 4, by 5.5% - in the preparation of option 3, by the end of 2 years of storage it decreases by 20.0 % in the preparations of variants 1 and 4, by 14.0% - in the preparation of variant 3, therefore, the most stable is the variant of the preparation based on interleukin-2 in solid form, containing both mannitol and HSA.

 The biological activity of the solid form variants of the preparations of the present invention in an oxidized state is comparable to that of natural interleukin-2 (paragraph 4 of Table 1). The demonstrated increase in biological activity associated with oxidation is natural for biologically active molecules. It can be seen from the presented data that in the oxidized state the biological activity of the preparation of option 3 (containing mannitol and HSA together) is superior to that of option 1 (containing mannitol), but inferior to that of option 4 (containing HSA), therefore, HSA better stabilizes oxidized interleukin-2 in solid pharmaceutical formulations. It should be noted that the activity of the similar drug "Proleikin", produced in oxidized form, ranges from 11 to 24 million IU per 1 mg of protein.

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

Option 5 - mg
Interleukin-2 - 1.0
Mannitol - 50.0
Sodium dodecyl sulfate - 10.0
Dithiothreitol - 0.32
Disubstituted sodium phosphate - 1.24
Monosubstituted sodium phosphate - 0.20
pH - 7.6
Option 6 - mg
Interleukin-2 - 1.0
Mannitol - 50.0
Human Serum Albumin - 50.0
Sodium dodecyl sulfate - 10.0
Dithiothreitol - 0.32
Disubstituted sodium phosphate - 1.24
Monosubstituted sodium phosphate - 0.20
pH - 7.6
Option 7 - mg
Interleukin-2 - 1.0
Human Serum Albumin - 50.0
Sodium dodecyl sulfate - 10.0
Dithiothreitol - 0.32
Disubstituted sodium phosphate - 1.24
Monosubstituted sodium phosphate - 0.20
pH - 7.6
The biological activity of these drugs was investigated by their ability to stimulate the proliferation of cytotoxic T-lymphocytes of the mouse CTLL-2 line by the method described above in this example. The difference was that the contents of the ampoule, already in a liquid state, were diluted 100 times with 0.1% SDS and then titrated according to the described procedure. Further, as a negative control option (background control), we used a mixture of auxiliary substances that are part of the liquid form (mannitol, sodium dodecyl sulfate, dithiothreitol, monosubstituted sodium phosphate and disubstituted sodium phosphate), in concentrations corresponding to their content in the test samples. The specific biological activity of IL-2 was also expressed in International Units (IU). The results are presented in Table 2.

 The biological activity of the drug of option 6 (containing both HSA and mannitol) is slightly higher than that of option 7 (containing HSA, not containing mannitol) and option 5 (containing mannitol and not containing HSA), therefore, the combined use of mannitol and CSA contributes to more effective conservation of biological activity preparations in liquid form based on interleukin-2.

 The biological activity of the variants of preparations in liquid form based on interleukin-2 by the end of 2 years of storage by 25% in preparations of variants 5 and 7, by 18.0% - in the preparation of option 6, therefore, the most stable is option 6 of liquid preparations containing co-mannitol and HSA.

 The biological activity of the variants of liquid forms of the preparations of the present invention in the oxidized state is comparable to that of natural interleukin-2 and slightly exceeds that of the variants of the drug in solid form in the oxidized state. It should be noted that the biological activity of oxidized version 7 (containing HSA) exceeds that of oxidized version 6 (containing mannitol and HSA), and the activity of oxidized variants 6 and 7 is higher than the activity of oxidized version 5.

 Therefore, under physiological conditions, the biological activity is higher for preparations in solid and liquid form based on interleukin-2 containing HSA, and the activity of the drug with HSA in liquid form is slightly higher than that of the drug with HSA in solid form.

 Biological stability is higher for preparations based on interleukin-2 in solid form than that for preparations in liquid form, and the highest is for a preparation in solid form containing mannitol and HSA together.

 Example 4. The study of the side effects of a pharmaceutical preparation based on interleukin-2 in solid form (option 1).

 The study of the general toxic effect of interleukin-2 was carried out on rodents (50 randomly bred white mice, 230 white rats and 12 chinchilla rabbits). The study included a toxicological study of a single exposure to the drug at a dose of 5 mg / kg, which is 50 times the therapeutic dose, as well as a chronic toxicological experiment.

 A toxicological study of a single exposure of the drug at a maximum tolerated dose of 5 mg / kg intravenously indicates the resistance of these mammalian species to interleukin-2. In this experiment, the drug caused short-term hyperthermia, did not affect the cellular elements of peripheral blood (erythrocytes, leukocytes), and behavioral reactions changed somewhat. Sexual differences in mice to the toxic effect of the drug with a single administration were not observed. In male rabbits, after a single intravenous dose of 3 mg / kg, rapid breathing and signs of anxiety were observed. Analysis of the ECG, taken 24 hours after administration of the drug to rabbits, did not reveal any cardiotropic effect.

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

 Thus, the absence of significant toxic effects of the drug was found in the acute toxicity experiment with a single dose of the maximum tolerated dose.

A chronic toxicological experiment was carried out for three months on 230 randomly bred white rats of both sexes. Males had a body weight of 300 - 315 g, females - 310 - 320 g. The animals were kept in plastic cages with an area of 2150 cm ² of 8 animals, fed with briquetted and natural feeds. The drug was administered intraperitoneally according to the standard experimental procedure for chronic toxicity in a dose calculated for rats according to Yu.R. Rybolovlev, R.S. Rybolovlev (1979), corresponding to 0.1 mg / kg. At the end of the experiment, part of the experimental animals were decapitated, opened, the relative mass of organs was determined, and material was taken for pathomorphological studies. Organs and tissues were fixed in neutral formalin, histological preparations were prepared by the conventional method of paraffin embedding and hematoxylin-eosin staining. All experimental materials were processed by the conventional student method.

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

 From Table 3 it is seen that the drug of option 1 does not have most of the side effects exhibited by the similar drug Proleukin.

 Next, the effects of the drug of option 1 were compared with the control group of animals that did not receive this drug.

 During the three months of the chronic experiment, the experimental animals showed no signs of severe intoxication and significant changes in behavior.

 The dynamics of body weight gain in rats of both sexes did not differ significantly from control animals. A short-term decrease in the emotional component of the behavior of male rats was observed under the influence of a therapeutic dose of the drug, an increase in the number of “washing” and “cleansing” in male rats. The drug had no effect on the research behavior of experimental animals. Assessment of the animal’s behavioral reactions “in the open field” during a chronic experiment did not allow revealing regular significant changes.

 During the three months of the chronic experiment, the detoxifying, excretory, and absorption functions of the liver of experimental rats did not differ significantly from the norm. The liver structure of rats is normal. Inflammation was absent.

 When opening animals macroscopically not detected any pronounced changes. The study of the histological structure of organs and tissues of experimental animals in comparison with control rats did not reveal significant structural changes caused by exposure to the drug. The heart muscle did not have significant deviations from the norm.

 Changes in the spleen structure of experimental rats were observed. Lymphatic follicles of the white pulp of the spleen mainly had reproduction centers, in which more macrophages and also more mitoses were noted than in the control group. In the red pulp, the number of megakaryocytes and macrophages is increased. These changes are logical based on the immunotropic nature of the drug based on interleukin-2, which causes proliferation of the aforementioned cells of the immune system.

 After a month of exposure to option 4, an increase in hemoglobin level and a decrease in blood coagulation were observed only in male rats under the influence of a therapeutic dose. The decrease in coagulation was temporary and normalized towards the end of the experiment in male rats. In female rats, after three months of drug administration, only a decrease in the blood coagulation rate in the third minute was noted.

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

 Analysis of the local irritant effect of the preparation of option 1 was carried out by macroscopic and histological studies of tissues at the injection site. The analysis did not reveal any significant macro- and microscopic changes in the peritoneum, vascular wall and surrounding tissues at the injection site.

 In general, the drug is characterized as low toxic, well tolerated, without specific side effects.

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 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).

Claims (18)

1. A pharmaceutical preparation based on interleukin-2 as an active substance, containing a stabilizer, a solubilizer, an antioxidant agent and a buffer, characterized in that the solid form of the preparation contains as a stabilizer a substance selected from the group consisting of mannitol, human serum albumin, or a mixture thereof , as a solubilizer, a substance selected from the group consisting of guanidine hydrochloride, sodium dodecyl sulfate, as an antioxidant agent, a substance selected from the group containing glutathione in reductive form, 2-mercaptoethanol, dithiothreitol, as a buffer - a pharmaceutically acceptable buffer, with the following components, mg:
Interleukin-2 - 0.1 - 7.0
Stabilizer - 5.0 - 350.0
Solubilizer - 1.0 - 70.0
Antioxidant Agent - 0.1 - 3.5
Buffer, mm - 5.0 - 10.0
2. The pharmaceutical preparation according to claim 1, characterized in that as interleukin-2 it contains recombinant interleukin-2.  3. The pharmaceutical preparation according to claim 1, characterized in that as interleukin-2 it contains non-recombinant interleukin-2.  4. The pharmaceutical preparation according to claims 1 to 3, characterized in that it is made in the form of a tablet.  5. The pharmaceutical preparation according to claims 1 to 3, characterized in that it is made in the form of a capsule.  6. The pharmaceutical preparation according to claims 1 to 3, characterized in that it is made in the form of a powder.  7. The pharmaceutical preparation according to claims 1 to 3, characterized in that it is made in the form of granules.  8. The pharmaceutical preparation according to claims 1 to 3, characterized in that it is made in the form of a suppository.  9. The pharmaceutical preparation according to claims 1 to 3, characterized in that it is made in the form of a liposome form.  10. The pharmaceutical preparation according to claims 1 to 3, characterized in that it is made in a form suitable for external use.  11. The pharmaceutical preparation according to claims 1 to 3, characterized in that it is made in liquid form, obtained by dissolving the solid form in a pharmaceutically acceptable solvent. 12. A pharmaceutical preparation based on interleukin-2 as an active substance (option) containing a stabilizer, a solubilizer, an antioxidant agent and a buffer, characterized in that the liquid form of the preparation contains, as a stabilizer, a substance selected from the group consisting of mannitol, human serum albumin or a mixture thereof, as a solubilizer, a substance selected from the group consisting of guanidine hydrochloride, sodium dodecyl sulfate, as an antioxidant agent, a substance selected from the group containing hl tation in reduced form, 2-mercaptoethanol, dithiothreitol in a buffer - a pharmaceutically acceptable buffer, with the following component, mg:
Interleukin-2 - 0.1 - 5.0
Stabilizer - 5.0 - 250.0
Solubilizer - 1.0 - 50.0
Antioxidant Agent - 0.1 - 2.5
Buffer, mm - 5.0 - 10.0
13. The pharmaceutical preparation according to item 12, characterized in that as interleukin-2 it contains recombinant interleukin-2.  14. The pharmaceutical preparation according to claim 12, characterized in that it contains non-recombinant interleukin-2 as interleukin-2.  15. The pharmaceutical preparation according to claims 12-14, characterized in that it is made in the form of an emulsion.  16. The pharmaceutical preparation according to claims 12 to 14, characterized in that it is made in the form of a syrup.  17. The pharmaceutical preparation according to claims 12-14, characterized in that it is made in the form of an elixir.  18. The pharmaceutical preparation according to claims 12 to 14, characterized in that it is made in the form of drops.  19. The pharmaceutical preparation according to claims 12 to 14, characterized in that it is made in the form of a liposome form.  20. The pharmaceutical preparation according to paragraphs 12 to 14, characterized in that it is made in a form suitable for external use.

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