Peptide composition possessing immunoregulatory property

FIELD: medicine, chemistry of peptides, amino acids.

SUBSTANCE: invention relates to novel biologically active substances. Invention proposes the novel composition comprising peptides of the formula: H-Arg-Gly-Asp-OH and H-Tyr-X-Y-Glu-OH wherein X means Gln and/or Glu; Y means Cys(acm) and/or Cys. The composition shows ability to inhibit proliferative activity of mononuclear cells, to induce suppressive activity and their ability for secretion of cytokines TNF-1β (tumor necrosis factor-1β) and IL-10 (interleukin-10 ).

EFFECT: simplified method for preparing composition, valuable medicinal properties of composition.

4 cl, 16 tbl, 9 ex

 

The invention relates to medicine, in particular to new biologically active substances (BAS), with immunoregulatory properties, and can be used in practical medicine, veterinary medicine, as well as in experimental biochemistry.

Known copolymer-1 (COP-1), manufactured by TEVA under the name "Copaxone" (see the Monograph authors E.I. Gusev, TL Demina and A.N. Boyko "Multiple sclerosis", Moscow, 1997, page 317).

This drug has a wide functionality, however it is significant complexity due to the high molecular weight, which increases its production.

Renowned pharmaceutical composition used for treatment of multiple sclerosis by neutralizing antimelanoma antibodies (see RF patent №2121850, class a 61 L 38/10 from 22.10.91 year).

However, the known arrangement does not possess immunoregulatory properties.

The technical result is the development of a new synthetic composition having immunoregulatory capabilities and ease of access.

To solve this technical problem is proposed composition with immunoregulatory properties, comprising at least two peptide of formula H-Arg-Gly-Asp-OH and H-Tyr-X-Y-Glu-OH, where X is Glu and/or Gln, Y - Cys and/or Cys(acm), and the peptides can be taken in equal shares or in shares, zootoxin the e which is 1:4, respectively, in addition, peptides modified by replacing each of the groups H and HE at least one Oligopeptide or a polysaccharide, and also has the ability to induce suppressor activity of mononuclear cells and secretion of cytokines by them TGF-β1 and IL-10.

The invention consists in that the use of compositions with the above formula allows us to provide extended functionality effects on mononuclear cells by the ability to induce high-level secretion of immunosuppressive cytokines.

In addition, low molecular weight components of the proposed composition determines the ease of its manufacture.

The comparison of the proposed composition with the closest analogue suggests the criterion of "novelty", and the lack of analogues distinctive signs under the criterion of "inventive step".

Preliminary tests suggest about the possibility of wide industrial production and use in medicine.

Obtaining the claimed composition can be carried out by known methods of peptide synthesis.

Peptides comprising the inventive composition according to the present invention, can be obtained in accordance with conventional and well known methods of synthesis of polypeptid the RC. For the synthesis of the used activated amino acids with protected functional groups (company: Sigma, Merk).

Synthesis of peptides was carried out by generally accepted methods of solid-phase synthesis: in particular, Fmoc-scheme of formation of the peptide bond, which involves the use of temporary masking of alpha-amino groups fluorenyl-methyloxycarbonyl (Fmoc) group, is stable in an acidic environment, but tsepliaeva bases. The first peptide RGD - arginyl-glycyl-aspartic acid - (H-Arg-Gly-Asp-OH) was synthesized in the form of the free acid in RAS polymer company MilliGen/Biosearch (USA) attached to it starting C-terminal amino acid residue Fmoc-aspartic acid. The condensation reaction was carried out using 1-oxibendazole or pentafluorophenyl esters, consistently using the corresponding esters of glycine and arginine. Completeness of passage of the reaction was monitored using the ninhydrin test. In the case of incomplete passing the reaction they were re-condensation. For cleavage of the peptide from the polymer used a mixture triperoxonane acid (TN) and phenol (95/5). Purification of the peptide was performed by gelfiltration on Sephadex G-10 and characterized data quantitative amino acid analysis, analytical reversed-phase high-performance liquid chromatography and tone what sloinoi chromatography.

The synthesis was carried out on solid phase using polystyrene polymer crosslinked with divinylbenzene. The degree of crosslinking of 1%. For the synthesis of derivatives used L-amino acid of the firm "Bachem": Boc-Glu(Bzl)OH, Boc-Cys(Acm) - OH and BOC2TyrOH.

The synthesis was performed by the sequential build-up of amino acids starting from the C-end using dicyclohexylcarbodiimide with the addition of N-hydroxybenzotriazole. The protective group was removed with 50% triperoxonane acid in chloroform. The peptide was tsalala from the resin using triftoratsetata. The crude product was subjected to purification by HPLC. And after lyophilization received the desired peptide.

The second peptide and recrystallized liofilizirovanny. The presence and sequence of amino acids, as well as the purity of the peptide was checked by thin layer chromatography, using as a single peptide, and the products of its partial or complete hydrolysis. Received second peptide 1 type corresponded to the formula: H-Tyr-Gln-Cys(acm)-Glu-OH, where acm - atsetamidometil, the protective group for the sulfur of cysteine, which was not removed because it spontaneously is easily removed in the conditions of the body and in cell cultures, resulting in the effective agent is a peptide with the formula H-Tyr-Gln-Cys(asm)-Glu-OH.

N - the beginning of a peptide; Tyr - amino acid residue tyrosine; Gln - amino acid residue glutamine; Cys is the amino acid is hydrated cysteine residue, Glu - glutamic acid (glutamate) or its remainder, IT is the end of the peptide.

For synthesis of the second peptide of the 2nd type with the formula H-Tyr-Glu-Cys(acm)-Glu-OH used the same principle with the replacement at the corresponding step of adding Boc-Glu(Bzl)OH hydroxysuccinimide ether on Boc-Glu-OH. All reagents and activated amino acids with protected groups were brand (Sigma, Merk).

Treating the peptide with the above formula in the presence of mercury ions, remove acetamidomethyl group, freeing the SH group of cysteine. Thus obtain peptides with formula without (asm).

Upon receipt of a composition according to claim 2 of the formula mix the first peptide with a second peptide of each type separately in the proportion of 1:4, respectively.

Upon receipt of a composition according to claim 3 of the formula mix the first peptide with a second peptide of each type separately in equal shares.

Upon receipt of a composition according to claim 4 of the formula peptides modified by replacing each of the groups H and HE at least one Oligopeptide or a polysaccharide.

The result can be obtained compositions of four different types. In addition, to obtain a composition of other types used instead of X at the second peptide is simultaneously a mixture of Gln and Glu. It should be noted that carried out the preparation of the composition of the two peptides in various unequal shares. However, the benefits perceived by the Oia composition before composition with peptides in fractions, the ratio which is 1:4, was not observed.

The biological activity of the inventive compositions of each of the four types was determined as follows.

Example 1.

1. Studied the effect of a composition consisting of two peptides of formulas H-Arg-Gly-Asp-OH and H-Tyr-X-Y-Glu-OH, where X is Glu, Y - Cys(acm) on the proliferative activity of mononuclear peripheral blood cells isolated by the method of Boyum 1969

The range of used doses amounted to 0.03 μg/ml to 3.00 mg/ml

The research results are reflected in table 1.

Dose in mcg/ml0,020,030,080,150,30,61,22,43,03,6
Index Inhib %05172433453624120

The results showed that the composition inhibits the proliferative activity of mononuclear peripheral blood cells caused by phytohemagglutinin (PHA).

2. Investigated the influence of the composition on the ability to induce suppressor activity of mononuclear cells in peripheral blood.

At the first stage receives the suspension OLS method sedimentation of the cells is to a single-stage gradient ficoll-protracta (the method of Boyum).

Peripheral blood is collected from the donor by venipuncture at the same time and placed in a test tube with a solution of heparin per 1 ml of blood 20-30 units of heparin. Next, the blood is diluted with Hanks solution without CA++and Mg++in the ratio of 1:2 and layer on the gradient ficoll-eurotrust (density 1,078).

This was followed by centrifugation mode 400 g for 30 minutes Suspended OOC out of interphase transferred into a centrifuge tube, add the Hanks solution without CA++and Mg++and produce 3 consecutive centrifugation for 10 min to launder cells from solution ficoll-eurotrust. After the 3rd centrifugation the precipitate OLS resuspension in 1 ml of medium 199 and count the number of mononuclear cells with the camera Goryaeva.

In the second stage OLS divided into two equal parts, the first of which is cultivated without activator suppressor, the second - with activator suppressor, which is used as the analyzed composition consisting of 2 peptides.

MNCs were cultured in penicillin vials closed with rubber corks No. 14,5 at a temperature of 37°C. the Environment of the cultivation of RPM1-1640 with the addition of 20% serum IV AB blood group and glutamine.

In each vial cultivate 5×106cells in 2.0 ml of complete medium. To culture for induction of suppressor type composition of the 2 peptides at doses of 0.03 to 3.0 is kg/ml

The cell cultivation carried out 48 hours After that MNCs were washed off the environment of the cultivation and are blocking proliferation by treatment with mitomycin C - 40 µg/ml for 30 min at 37°C. Then washed with medium 199 with 5% serum IV AB (chilled) three times. Sediment cells resuspension, count the number of nucleated cells, determine the percent viability of the cells with 0.1%Trypanosoma blue solution and diluted to the required concentration. In this case, all operations are done separately with the control cells and stimulated with the song, and to launder cells use silikonizirovannaya the dishes.

In the next step, each part of the control and stimulated composition OOC add freshly isolated MNCs stimulated phytohemagglutinin (PHA), which are responsible of the test cells in equal proportions (0,5×106: 0,5×106cells/ml) to obtain the test cultures. The cultivation is carried out within 72 hours After using the N3-thymidine assess the proliferation of the test cultures, and the amount of suppression is judged by the degree of reduction of proliferation in them. Index suppression (IP) is determined by the formula:

The results of studies of the effect of the composition consisting of two peptides of formulas H-Arg-Gly-Asp-OH and H-Tyr-X-Y-GluOH, where X is Glu, Y - Cys(acm), the ability to induce suppressor activity MTL reflected in table 2.

The dose range of 0.02-3.6 µg/ml.

Table 2.
Dose0,020,030,080,150,30,61,22,43,03,6
mg/ml
Index029182543281450
SUPR. %

It is established that the composition in doses of 0.03 µg/ml of 3.00 μg/ml induces suppressor activity of mononuclear cells in peripheral blood.

Example 2.

In the study of the composition of the 2nd type H-Arg-Gly-Asp-OH and H-Tyr-X-Y-Glu-OH, where X is Gln, Y - Cys(acm), mimicked the actions of paragraphs 1 and 2 of example 1.

The research results are reflected in alizah 3 and 4.

Table 3.

The effect of the composition consisting of two peptides of formulas (H-Arg-Gly-Asp-OH and H-Tyr-X-Y-Glu-OH, where X is Gln, Y - Cys(acm)on the proliferative activity of MNCs.
Dose0,020,030,080,150,30,61,22,43,03,6
mg/ml
Index0516212844291870
Inhib %
Table 4.

The influence of the composition consisting of two peptides of formulas (H-Arg-Gly-Asp-OH and H-Tyr-X-Y-Glu-OH, where X is Gln, Y - Cys(acm), the ability to induce suppressor activity of MNCs.
Dose0,020,030,080,150,30,61,22,43,03,6
mg/ml
Index03152432392312 40
SUPR. %

Thus, this arrangement also inhibits the proliferative activity of mononuclear peripheral blood cells caused by the PHA, and induces suppressor activity of mononuclear peripheral blood cells when used in doses of 0.03 µg/ml of 3.00 mg/ml

Example 3.

In the study of the action of the composition of the 3rd and 4th types of the 2 peptides with H-Arg-Gly-Asp-OH and H-Tyr-Gln-Cys-Glu-OH and H-Arg-Gly-Asp-OH and H-Tyr-Glu-Cys(acm)-Glu-OH, taken in equal parts and in the ratio of 1:4, repeated action item 1 and item 2 of example 1.

The results of the action research of the composition of the two peptides with H-Arg-Gly-Asp-OH and H-Tyr-Gln-Cys-Glu-OH on proliferative activity and ability to induce suppressor activity of the OLS regression are shown in tables 5 and 6.

The dose range of 0.02-3.6 µg/ml.

Table 5.
Dose0,020,030,080,150,30,61,2 2,43,03,6
mg/ml
Index04183037433122110
Inhib %

Table 6.
Dose0,020,030,080,150,30,61,22,43,03,6
mg/ml
Index0411182541261130
SUPR. %

The results of the action research of the composition of the two peptides with N-Arg-Gly-Asp-OH and H-Tyr-Glu-Cys(acm)-Glu-OH on proliferative activity and ability to induce suppressor activity of the OLS regression are shown in tables 7 and 8.

The dose range of 0.02-3.6 µg/ml.

Table 7.
Dose0,020,030,080,150,30,61,22,43,03,6
mg/ml
Index06143035 462918120
Inhib %
Table 8.
Dose0,020,030,080,150,30,61,22,43,03,6
mg/ml
Index041017314933 2150
SUPR. %

The result was that these compositions inhibit the proliferative activity of mononuclear peripheral blood cells caused by the PHA, and induce suppressor activity of mononuclear peripheral blood cells when used in doses of 0.03 µg/ml of 3.00 mg/ml

In addition, we investigated other types (variants) of the claimed composition, consisting of two peptides, which are also confirmed their ability to inhibit the proliferative activity and to induce suppressor activity of mononuclear cells in human peripheral blood. It should be noted the considerable ease of access to all variants of the compositions of the two peptides.

Example 4.

Investigated the influence of the composition consisting of two peptides of formulas (H-Arg-Gly-Asp-OH and H-Tyr-X-Y-Glu-OH, where X is Gln and/or Glu, Y - Cys(acm) and/or Cys, on the ability to induce the production of cytokines, transforming growth factor beta 1 (TGF-β1) and interleukin-10 (IL-10) mononuclear the mi cells (MNCs) in the peripheral blood.

In the first phase have received a suspension OLS method sedimentation of cells in a single-stage gradient ficoll-ultratrust (the method of Boyum).

MNCs were divided into 2 equal parts, the first of which were cultivated without composition, consisting of two peptides, one with a composition consisting of two peptides.

MNCs were cultured in penicillin vials closed with rubber corks No. 14,5 at a temperature of 37°C. the cultural Medium RPMI-1640 with the addition of 20% serum IV AB blood group and glutamine.

In each vial were cultured 5×106MNCs in 1 ml of complete medium. The culture of MNCs to induce the production of cytokines TGF-β1 and IL-10 were added to a composition consisting of two peptides.

The cultivation was carried out for 24 hours. Then the culture medium was separated from cells by centrifugation (1000 g 10 minutes) and it took 500 ál needed to perform the analysis.

When determining the amount of TGF-β1 in the culture medium was originally produced by extraction of the samples. This stage of analysis allows the release of TGF-β1 package, making it available for analysis.

To do this in a polypropylene tube was made of 0.25 ml (250 μl) of culture medium and 0.05 (50 ál) of the extracting solution. Mixed contents of the vortex. Incubated for 30 minutes at 4°C. was Added in a test tube 250 ál of raboteg the buffer solution for dilution of standards. At this stage, the dilution of culture medium 2.2 times.

Then took required for analysis, the number 8 hole strips of collapsible microplate coated with antibodies to TGF-β1.

To improve the reliability of the results of the analysis and control samples were placed in duplicate, using for each sample two holes. Contributed by 200 µl of each standard and extracted sample or cotrona into the appropriate wells. Added 500 μl of biotinylated antibodies to TGF-β1 all wells were mixed by tapping on the tablet. Covered the plate with foil and incubated for 30 minutes at room temperature. Completely delete the contents of the wells. Washed all the wells 4 times with working buffer solution, adding it each time with 400 ml to all wells. Each time was completely filled and removed from the wells strips solution. After washing away excess moisture released tapping the inverted stripe on filter paper. Added 100 ál of working strength conjugate streptavidin-peroxidase to each well, except chromogenic Blanca. Covered the plate with foil and incubated for 30 minutes at room temperature. Completely delete the contents of the wells. Washed the wells 4 times with working buffer solution. Next was introduced into each well, 100 ál of Romodanovo solution of tetramethylbenzidine (TMB). Incubated for 20-30 minutes at room temperature in the dark until the blue color in the wells with the calibration breakdown with a maximum content of TGF-β1.

Contributed to each well 100 ál of stop solution (solution 1 N. sulfuric acid) to stop the enzymatic reaction. They mixed the reagents by gently tapping the strip holder. The color of the solution changed from blue to yellow.

The registration results were photometrically on the photometer for assay at a wavelength of 450 nm immediately after stopping the enzymatic reaction.

To determine the concentration of TGF-β1 in the samples were building a calibration curve in the coordinates: the x - axis the concentration of TGF-β1 in the calibration samples (WG/ml); y-axis is the corresponding value of optical density.

By means of a calibration curve based on the obtained values of optical density was determined the concentration of TGF-β1 in the samples. If the samples were pre-diluted, obtained values of the concentrations were multiplied by the dilution factor (10, 100, 1000 and so on).

To determine the amount of IL-10 in culture medium were taken required for analysis, the number 8 hole strips of collapsible microplate coated with antibodies to IL-10.

To improve the reliability of the results and the aleesa study and control samples were placed in duplicate, using for each sample two holes.

Made of 50 µl of each standard, test sample or control sample into the appropriate wells. Added 50 μl of incubation buffer to the wells containing standards, and 50 µl of the dilution of the working solution in the wells containing culture medium.

Closed the tablet film and incubated 2 hours at room temperature.

Completely delete the contents of the wells.

Washed all the wells 4 times with working buffer solution, adding it each time in 400 ál to all wells. Each time was completely filled and removed from the wells strips solution. Added 100 μl of biotinylated antibodies to IL-10 in all wells were mixed by tapping on the tablet.

Closed the tablet film and incubated 2 hours at room temperature. Completely delete the contents of the wells. Washed all the wells 4 times with working buffer solution. Added 100 ál of working strength conjugate streptavidin-peroxidase to each well except chromogenic form.

Closed the tablet film and incubated for 30 minutes at room temperature. Completely delete the contents of the wells. Washed the wells 4 times with working buffer solution.

Next was introduced into each well, 100 μl of chromogenic solution of tetramethylbenzidine (TMB).

Incubated for 20-30 minutes at room is temperature in the dark until the blue color in the wells with the calibration breakdown with the highest levels of IL-10.

Contributed to each well 100 ál of stop solution (solution 1 N. sulfuric acid) to stop the enzymatic reaction. They mixed the reagents by gently tapping the strip holder. The color of the solution changed from blue to yellow.

Check results also carried out photometrically on the photometer for assay at a wavelength of 450 nm immediately after stopping the enzymatic reaction.

To determine the concentration of IL-10 in the samples were building a calibration curve in the coordinates: the x - axis the concentration of IL-10 in the calibration samples (WG/ml); y-axis is the corresponding value of optical density.

By means of a calibration curve based on the obtained values of optical density was determined by the concentration of IL-10 in the samples. If the samples were pre-diluted, obtained values of the concentrations must be multiplied by the dilution factor (10, 100, 1000 and so on).

As a result of the research it was found that a composition consisting of two peptides has the ability to induce the production of two cytokines, TGF-β1 and IL, mononuclear cells of peripheral blood.

Example 5-1.

The effect of the composition consisting of two peptides of formulas H-Arg-Gly-Asp-OH and H-Tyr-X-Y-Glu-OH, where X is Glu, Y - Cys(acm), proliferative asset is ity MNCs when the ratio of the first peptide with a second 4:1.

The range of the used dose was 0.02 μg/ml to 3.6 mcg/ml

The research results are reflected in table 9.

Table 9.
Dose0,020,030,080,150,30,61,22,43,03,6
mg/ml
Index0412212841271140
Inhib %

Example 5-2.

The results of studies of the effect of the composition consisting of two peptides of formulas H-Arg-Gly-Asp-OH and H-Tyr-X-Y-Glu-OH, where X is Glu, Y - Cys(acm), the ability to induce suppressor activity MNF when the ratio of the first peptide with a second peptide 4:1 is presented in table 10.

Diapasons of 0.02-3.6 µg/ml.

Table 10.
Dose0,020,030,080,150,30,61,22,43,03,6
mg/ml
Index037152639251140
SUPR. %

Example 6-1 and 6-2.

The results of the action research of the composition consisting of two peptides of formulas H-Arg-Gly-Asp-OH and H-Tyr-X-Y-Glu-OH, where X is Gln, Y - Cys(acm)on proliferative activity and ability to induce suppressor activity of MNCs with a ratio of the first peptide with a second peptide 10:1 are shown in tables 11 and 12.

The dose range of 0.02-3.6 µg/ml.

Table 11.
Dose0,020,030,080,150,30,61,22,43,03,6
mg/ml
Index0712172229201050
Inhib %
Table 12.
Dose0,020,030,080,150,30,61,22,43,03,6
mg/ml
Index0612182332201150
SUPR. %

Example 7-1 and 7-2.

The results of the action research of the composition of the two peptides with H-Arg-Gly-Asp-OH and H-Tyr-Gln-Cys-Glu-OH on proliferative activity and ability to induce suppressor activity of MNCs with a ratio of the first peptide with a second peptide 1:4 are shown in tables 13 and 14.

The dose range of 0.02-3.6 µg/ml.

Table 13.
Dose0,020,030,080,150,30,61,22,4 3,03,6
mg/ml
Index0716222839251960
Inhib %
Table 14.
Dose in mcg/ml0,020,030,080,150,30,61,22,43,03,6
Index PMPs. %03 9192638241240

Example 8-1 and 8-2.

The results of the action research of the composition of the two peptides with N-Arg-Gly-Asp-OH and H-Tyr-Glu-Cys(acm)-Glu-OH on proliferative activity and ability to induce suppressor activity of MNCs with a ratio of the first peptide with a second peptide of 1:10 are shown in tables 15 and 16.

The dose range of 0.02-3.6 µg/ml.

Table 15.
Dose0,020,030,080,150,30,61,22,43,03,6
mg/ml
Index0 510182431211260
Inhib %
Table 16.
Dose0,020,030,080,150,30,61,22,43,03,6
mg/ml
Index0 37122229231660
SUPR. %

Thus, examples 5-1, 5-2, 6-1, 6-2, 7-1, 7-2, 8-1, 8-2, in which the peptides were mixed in the ratio 4:1, 10:1, 1:4 and 1:10, indicate the absence of benefits of getting these songs before composition with peptides in fractions, the ratio of which is 1:1.

Example 9.

Investigated the influence of the composition consisting of two peptides of formulas H-Arg-Gly-Asp-OH and H-Tyr-X-Y-Glu-OH, where X is Glu and/or Gln, Y - Cys and/or Cys(acm) - modified by replacing each group of H or HE Oligopeptide or polysaccharide, proliferative activity, the ability to induce suppressor activity of MNCs and the production of cytokines, TGF-β1 and IL-10.

Example replacement of the N groups on the polysaccharide dextran.

To 10 ml of 1% solution of dextran molecular weight of 10000 (firm Sigma), distilleria the Oh water was added 100 ml of periodate sodium (firm Sigma) and left on a magnetic stirrer for 3 hours, then the solution were dialyzed and added composition with equal content of peptides in quantities of 1 mg After hours incubation were added 2 g of sodium borohydride (firm Sigma) and continued incubation for another hour. Then the solution were dialyzed, and subjected to sterilization by passing through membrane filters and used in immunological methods. That has been the accession of the polysaccharide groups.

To study the security of the claimed composition of the two peptides was performed to study its acute toxicity. Study of acute toxicity was carried out in accordance with the Methodological recommendations of the Pharmacological Committee of the Russian Federation "Requirements for preclinical study obmotochnogo action of new pharmacological substances", Moscow, 2001, the results of the study showed that intraperitoneal injection of 1000-fold dose of the composition there were no acute toxic effect, and at these doses has not been possible to reach his LD50. Thus, the proposed composition consisting of two peptides, is not toxic.

HE is known for a number of molecules which have immunoregulatory properties: alpha-fetoprotein, fibronectin and some cell adhesion molecules. Therefore, modification of the peptide H-Arg-Gly-Asp-OH by replacing H and/or HE at least one Oligopeptide the om or polysaccharide, increasing the size of the molecule, it does not change the specificity of action and immunoregulatory capabilities inherent in the peptide H-Arg-Gly-Asp-OH. Similarly, the possible modification of the second peptide by replacing it with H and Oh groups of a polysaccharide or peptide groups.

To study the security of the claimed composition of the two peptides was performed to study its acute toxicity. Study of acute toxicity was carried out in accordance with the Methodological recommendations of the Pharmacological Committee of the Russian Federation "Requirements for preclinical study obmotochnogo action of new pharmacological substances", Moscow, 2001, the results of the study showed that intraperitoneal injection of 1000-fold dose of the composition there were no acute toxic effect, and at these doses has not been possible to reach his LD50. Thus, the proposed composition consisting of two peptides, is not toxic.

Although the above invention has been described in detail to illustrate, for the average person skilled in the art will be quite clear, in light of the description of this invention that can be made certain changes and modifications of the invention without departing from the idea or the scope of the proposed claims.

1. The composition having immunoregulatory property, on the expectation of at least two peptide of formula H-Arg-Gly-Asp-OH and H-Tyr-X-Y-Glu-OH, where X is Gln and/or Glu, Y-Cys(acm) and/or Cys, which may be modified by replacing each of the groups H and HE at least one Oligopeptide or a polysaccharide.

2. The composition according to claim 1, characterized in that the peptides are taken in fractions, the ratio of which is 1:4, respectively.

3. The composition according to claim 1, characterized in that the peptides are taken in equal parts.

4. The composition according to claim 1, characterized in that it has the ability to inhibit the proliferative activity of mononuclear cells to induce their suppressor activity and the secretion of cytokines by them TGF-β1 and IL-10.



 

Same patents:

FIELD: medicine, immunology, peptides.

SUBSTANCE: invention relates to a new composition of biologically active substances. Invention proposes the composition comprising of peptides of the formula: Arg-Gly-Asp and H-Tyr-X-Y-Glu-OH wherein X means Gln and/or Glu; Y means Cys(acm) and/or Cys that elicits ability to inhibit the proliferative response for phytohemagglutinin, to induce the suppressive activity of mononuclear cells and ability of peptides to induce secretion of immunosuppressive cytokines of grouth-transforming factor-β1 and interleukin-10 (IL-10). The composition can be prepared by a simple procedure.

EFFECT: valuable biological properties of composition.

3 cl, 16 tbl, 9 ex

The invention relates to novel soluble synthetic polimersvarka the anthracyclines, exhibiting antitumor activity, to a method of receiving and containing pharmaceutical compositions

FIELD: medicine, immunology, peptides.

SUBSTANCE: invention relates to a new composition of biologically active substances. Invention proposes the composition comprising of peptides of the formula: Arg-Gly-Asp and H-Tyr-X-Y-Glu-OH wherein X means Gln and/or Glu; Y means Cys(acm) and/or Cys that elicits ability to inhibit the proliferative response for phytohemagglutinin, to induce the suppressive activity of mononuclear cells and ability of peptides to induce secretion of immunosuppressive cytokines of grouth-transforming factor-β1 and interleukin-10 (IL-10). The composition can be prepared by a simple procedure.

EFFECT: valuable biological properties of composition.

3 cl, 16 tbl, 9 ex

The invention relates to new compounds of General formula I

R1-A-B-D-En-R2 (I)

in which R1 represents R12C(O), and R12 is selected from the group consisting of alkenyl, alkenylacyl or alkenylamine; And is a group A1-A2-A3, where A1 represents NH, A2 is a CHR93 in which R93 is 4-amidinophenoxy; A3 represents C(O); is a group B1-B2-B3, where B1 represents NH; B2 is a CHR97 where R97 represents ethyl, which is substituted in position 2 by hydroxycarbonyl or allyloxycarbonyl; B3 represents C(O); D represents a group D1-D2-D3, where D1 represents NH, D2 represents CR81R82 where R81 and R82 are independently selected from the group consisting of hydrogen and unsubstituted or substituted residues of alkyl, aryl, arylalkyl, heteroallyl; D3 represents C(O); Enis a (E1-E2-E3)nin which n is 0 or 1; E1 represents NR70, where R70 is H; E2 represents CR71R72, where R71 and R72 include independently selected from the group consisting of hydrogen and unsubstituted or substituted residues of alkyl, aryl, arylalkyl, heteroallyl; E3 represents C(O); R2 is a NR21R22 where R21 of iillil and geterotsiklicheskikh, moreover, the alkyl contains from 1 to 13 carbon atoms, alkenyl contains from 2 to 13 carbon atoms, aryl and heteroaryl contain from 5 to 13 ring carbon atoms, where in the rest of heteroaryl one or more carbon atoms are replaced by heteroatoms selected from the group consisting of N, O and S; heteroseksualci contains from 3 to 8 ring carbon atoms, of which from one to three carbon atoms are replaced by heteroatoms selected from the group consisting of N, O and S; in any stereoisomeric forms or their mixtures in any ratio, and their pharmaceutically acceptable salts; the method of obtaining compounds of General formula I, including linking protected amino acids; to pharmaceutical compositions which are able to exert an antithrombotic effect by activated factor VII(FVIIa) blood coagulation
The invention relates to a group of new protected linear peptides containing the amino acid sequence, which can be used as starting compounds to obtain RGD-containing cyclopeptides, the General formula

R3-Arg-Gly-Asp(OR1)-OR2,

where R1is benzyl or tert-butyl; R2not equal to R1and is selected from the group of tert-butyl; benzyl; 4-methoxybenzyl; 4-nitrobenzyl; diphenylmethyl; 2,2,2-trichloroethyl; 2,2,2-trichloro-1,1-dimethylethyl; allyl; 9-fluorenylmethyl; carboxamidine; substituted 2-sulfonylated type AND-SO2-CH2-CH2- where a is substituted or unsubstituted phenyl or benzyl; R3is a hydrogen atom or a urethane protective group of the B1O-CO-, where1not equal to R1and can take values tert-butyl, benzyl, 4-methoxybenzyl, 9-fluorenylmethyl, 2-(4-nitrophenyloctyl)ethyl; or is a peptidyl containing from one to three amino acid residues; and the peptides, where R3- peptidyl structure E-Z-Y-X-, in which E is a hydrogen atom or a urethane protective group IN2O-CO-, where2not equal to R1and can take values tert-butyl; benzyl; 4-methoxybenzyl; 9-fluorenylmethyl; 2-(4-nitrophen is IN3O-CO-, in which3= R1attached to the omega-amino group; Z can take values Phe or D-Phe

The invention relates to biotechnology and can be used for Introduzione nucleic acids into cells

The invention relates to a peptide compound of the formula Iwhere R1- cinoxacin-2-alkyl, CORandor CORin, R2Is h or alkyl, R3- C1- C4alkyliden etc., R4- phenylalkyl, R5-alkoxy, alkylamino, hydroxylamino and others, And the group-(CH2)m- (CH2)n

The invention relates to derived-amino acids, which are isostere dipeptide link Gly - Asp

The invention relates to new derivatives of acetic acid, hydrate, solvate and pharmaceutically acceptable salts, which can find application in the pharmaceutical industry

The invention relates to new biologically active peptide, inhibiting the hypersecretion of thyroid-stimulating hormone (TSH) and prolactin (PRL), caused by natural hypothalamic peptide with thyroliberin (tireotropin-releasing hormone, TRH)

FIELD: medicine, phthisiology, pharmacy.

SUBSTANCE: invention proposes the inhalation preparation in treatment of tuberculosis comprising an antibacterial medicinal agent an aqueous solution. The preparation comprises also a medicinal preparation with the immunomodulating effect as 5-amino-2,3-dihydro-1,4-phthalazinedione potassium salt or 5-amino-2,3-dihydro-1,4-phthalazinedione sodium salt, or 5-amino-2,3-dihydro-1,4-phthalazinedione potassium and sodium salts taken in the equal ratio in the following ratio of components, wt.-%: antibacterial medicinal agent, 1.0-10.0; 5-amino-2,3-dihydro-1,4-phthalazinedione potassium salt or 5-amino-2,3-dihydro-1,4-phthalazinedione sodium salt, or 5-amino-2,3-dihydro-1,4-phthalazinedione potassium and sodium salts taken in the equal ratio, 1.0-10.0; water, the balance. The preparation inhibits activity of macrophages reducing the level of tumor necrosis factor (TNF) and acute-phase proteins that results to weakening intoxication symptoms, activates superoxide-forming function and phagocyte activity of neutrophile granulocytes, enhances the microbiocide system of cells that, in turn, results to arresting the inflammation process. Invention can be used as the inhalation agent in treatment of tuberculosis.

EFFECT: valuable medicinal properties of preparation.

2 cl, 2 ex

FIELD: medicine, rheumatology, pharmacy.

SUBSTANCE: invention proposes ointment as a curative agent that comprises the following components: 5-amino-2,3-dihydro-1,4-phthalazinedione alkaline salt of mixture of alkaline salts, medicinal vaseline, vaseline oil as an ointment base and distilled water taken in the following ratio of components, wt.-%: 5-amino-2,3-dihydro-1,4-phthalazinedione alkaline salt or 5-amino-2,3-dihydro-1,4-phthalazinedione mixture of alkaline salts, 1.0-50.0; medicinal vaseline, 10.0-20.0; vaseline oil, 5.0-10.0, and water, the balance, wherein 5-amino-2,3-dihydro-1,4-phthalazinedione sodium or potassium salt is used as an alkaline salt, and 5-amino-2,3-dihydro-1,4-phthalazinedione sodium and potassium salts mixture is used as mixture of alkaline salts composed of the equal ratio of components, i. e. 5-amino-2,3-dihydro-1,4-phthalazinedione sodium and potassium salts mixture. Indicated ointment normalizes membrane cell to the normal state damaged as result of disease, regulated intra- and intercellular metabolic processes that, in turn, promotes to normalization of metabolism and arresting inflammation for the shortest times. Invention can be used in treatment of autoimmune disease.

EFFECT: improved and valuable medicinal properties of ointment.

3 ex

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to new 6-alkyl-5-(2-isonicotinoylsulfohydrazoyl)uracil hydrochlorides of the general formula (I) wherein R means alkyl comprising 1-4 carbon atom. Compounds possess an anti-mycobacterial and an immunotropic activity and can be used as an immunomodulating agent and an anti-mycobacterial agents. Also, invention relates to a pharmaceutical composition based on thereof.

EFFECT: valuable medicinal properties of compounds.

4 cl, 10 tbl, 2 ex

FIELD: veterinary science.

SUBSTANCE: the present innovation deals with cooling selected fertilized uninfected eggs at 2-4 C up to 7 d followed by homogenization, diluting with physiological solution of egg mass, bactericidal treatment, conservation with phenol solution up to its concentration being 0.3% at subsequent filtration of target product. Moreover, egg mass should be diluted with physiological solution at the ratio of 1:5, then comes thermal treatment upon water bath at 70-80 C for 1 h and before conservation one should cool the target product in bactericidal chamber under the lamp with ultraviolet irradiation for 30-40 min. The method provides increased total and specific body resistance, it enables to obtain cheaper and more acceptable biostimulant and simplified way for preparing.

EFFECT: higher efficiency.

4 ex, 3 tbl

FIELD: medicine, biopharmacology, immunology.

SUBSTANCE: invention relates to manufacturing curative-prophylactic preparations based on leukocyte interferon and natural cytokines. The preparation based on complex of natural cytokines is prepared by induction of human leukocytes with Newcastle disease virus of strain "H". Indicated complex of natural cytokines is prepared by at least two treatments of leukocytes before induction stage with virus with the same natural complex of cytokines prepared in previous stage of induction. Complex is purified from foreign proteins of inductor and comprises 104 IU of antiviral activity of human interferon-alpha in one ampoule, not less, and cellular cytokines, phosphate buffer for maintaining pH 6.9-7.5, sodium chloride and mannitol. The end product is lyophilized for preparing the injection preparation. Invention provides increasing activity of the preparation and to retain the natural complex of cytokines.

EFFECT: improved preparing method, valuable medicinal properties of preparation.

3 cl, 2 ex

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

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