EA031772B1 - Peptides inducing intensified interferon gamma and il-18 formation, and immunomodulatory, antiviral pharmaceutical composition - Google Patents

Abstract

The invention is related to the field of biotechnology, in particular, to synthetic peptides having immuno-stimulating properties, and can be used in medicine. A series of peptides has been synthesized that differ in their chemical structure from prior art biologically active Alloferon and Allostatin peptides. The distinction is that position 6 of the structure His-Gly-Val-Ser-Gly-X-Gly-Gln-His-Gly-Val-His-Gly, instead of histidine or tryptophan characteristic of those peptides, is occupied by other amino acids of aliphatic and aromatic series or by their chemical modifications. The peptides obtained are used as a part of an immunomodulatory and antiviral pharmaceutical composition. The invention makes it possible to obtain peptides that induce efficiently production of interleukin-18 and interferon gamma.

Description

The invention relates to the field of biotechnology, specifically to synthetic peptides with immunostimulating properties, and can be used in medicine. A number of peptides have been synthesized that differ in chemical structure from the known biologically active peptides of Alloferon and Allostatin. The difference is that in position 6 of the His-Gly-ValSer-Gly-X-Gly-Gln-His-Gly-Val-His-Gly structure, instead of the histidine or tryptophan characteristic of these peptides, there are other aliphatic and aromatic amino acids , as well as their chemical modifications. The resulting peptides are used in the immunomodulating and antiviral pharmaceutical compositions. The invention allows to obtain peptides that effectively induce the production of interleukin-18 and interferon gamma.

The invention relates to proteins and biologically active peptides with immunomodulating and antiviral activity.

The biological activity of low molecular weight peptides is known. A number of peptides have been described that have a direct antiviral or antitumor effect [Akiyama N., Hijikata M., Kobayashi A., Yamori T., Tsuruo T., Natori S. Anticancer effect of Y-Z-alanyl-b-B-glutathionyl dioxiphenylalanine ( 5-S-GAD), a new antibacterial substance derived from insects. Anticancer Research, 2000, v. 20, pp. 357-362].

Amphibians and insects peptides occupy a special place in this series [Bulet P., Khetru K., Diamark J., Hoffman D. Antimicrobial peptides in insects: structure and function. Razv. Comp. Immunol., 1999, v. 23, pp. 329-344, Chinchar V.G., Wang J., Murthy G., Carey K., Rolling-Smith L. Inactivation of 3 frogs virus and flow catfish virus escululentin-2P and ranaturin-2P, two antimicrobial peptides isolated from frog skin. Virology, 2001, v. 288, pp. 351-357].

A known group of antiviral peptides isolated from insects with the common name Alloferons, described by the general formula: X ₁ -His-Gly-X ₂ -His-Gly-Val-X ₃ or their pharmaceutically acceptable salts, where X1 is absent or contains at least 1 amino acid, X2 contains at least 1 amino acid or is a peptide bond; X _{3 is} absent or contains at least 1 amino acid, and these amino acids are selected from the groups: aliphatic, aromatic or heterocyclic. (RF patent No. 2172322, IPC C07K 7/06, C07K 7/08, A61K 38/08, A61K 38/10, A61P 37/02, published on 08/20/2001)

Based on the Alloferon-1 peptide, the drug Allokinalfa lyophilisate for subcutaneous injection, developed for the treatment of herpes, acute hepatitis B and papillomavirus infections, has been developed and is being produced.

Close to Alloferons in structure and properties is a group of peptides under the general name Allostatins with the general formula: X ₁ -Trp-Gly-Gln-X ₂ (RF Patent No. 2267496, IPC C07K 7/06, A61K 38/08, A61K 38/10 , published on January 10, 2006)

Allostatin-1, which differs from Alloferon-1 by the presence of two tryptophan (Trp) residues, replacing histidine (His) and valine (Val), has not only a high antiviral activity, but also an antitumor effect.

This example shows that the replacement of amino acid residues in the molecules of these peptides can significantly change their biological properties.

Alloferons are the closest analogues of the invention in terms of chemical structure and mechanism of action.

In addition, previously, by computer-aided modeling of the spatial structure of the Alloferon-1 molecule, the most important role of histidine residues in the conformation of the peptide molecule was shown. Moreover, the most important contribution is made by histidine residues located in positions 6 and 9 (RF Patent No. 2470031, IPC C07K 7/06, A61K 38/08, A61K 38/10, published on December 20, 2012).

The presence of other amino acids in these positions significantly changes the conformation of the molecules and, accordingly, their biological activity.

The technical problem to be solved by the claimed invention is directed is the creation of new peptides with increased biological activity, which can be used to create drugs for the treatment and prevention of viral and microbial infections.

The stated technical problem is solved by the fact that biologically active peptides are synthesized: (Peptide I) (Peptide II) (Peptide III) (Peptide IV) (Peptide V)

His-Gly-Val-Ser-Gly-Tyr-Gly-Gln-His-Gly-Val-His-Gly

His-Gly-Val-Ser-Gly-Ala-Gly-Gln-His-Gly-Val-His-Gly His-Gly-Val-Ser-Gly-Arg-Gly-Gln-His-Gly-Val-His-Gly

His-Gly-Val-Ser-Gly-Lys-Gly-Gln-His-Gly-Val-His-Gly

His-Gly-Val-Ser-Gly-Phe -Gly-Gln-His-Gly-Val-His-Gly

His-Gly-Val-Ser-Gly-Phe (p-OMet) -Gly-Gln-His-Gly-Val-His-Gly (neimifl VI)

His-Gly-Val-Ser-Gly-Phe (p-Cl) -Gly-Gln-His-Gly-Val-His-Gly (Peptide VII) having a high immunomodulatory and antiviral effect.

The synthesized peptides are distinguished by the presence of various amino acids of the aliphatic and aromatic series at position 6. For these peptides, the biological activity of the synthesized peptides was determined.

The technical result consists in the fact that the obtained peptides I-VII have high immunomodulating and antiviral activity, which will be confirmed by examples.

It should be noted that the similarity of the chemical structure of the obtained peptides I-VII with Alloferon 1 and Allostatin-1 is purely external. They do not fit into the formulas of these groups of peptides.

The synthesis of peptides was carried out according to the standard Fmoc procedure by the solid-phase method. Delete

- 1,031,772 protection was carried out with a 20% solution of piperidine in dimethylformamide. The coupling reaction was carried out using HBTU in the presence of HOBt and NMM for 2 hours at room temperature. The final separation of the peptides was carried out using TFA, EDT and water (95: 2.5: 2.5) for 2 hours at room temperature.

The crude peptides were washed with cold diethyl ether, dissolved in water and lyophilized. Peptides were purified by HPLC using TOSOH Bioscience C18 columns (21.5 mm x 300 mm) (Tosoh, Japan) with a 210/240 nm UV detector. The process was carried out in a gradient of water acetonitrile containing 0.1% TFA at a flow rate of 7 ml / min. The purified lyophilized peptides had a purity of more than 95%.

The final step in the preparation of peptides is lyophilization from a solution in 50% acetic acid.

The chemical identification of the peptides was confirmed by mass spectrometry using a Microflex LT MALDI-TOF type mass spectrometer (Bruker Daltonics GmbH).

The ability to achieve the objectives of the invention is confirmed by the following examples.

Example 1. The study of the effect of biologically active peptides I-VII on the induction of IL-18

Induction was carried out by administering subcutaneously Alloferon-1 and biologically active peptides I-VII once at a dose of 2 mg per kg of weight of laboratory animals. For the experiment, BALB / C mice were used, females weighing 16-22 g, age 14 weeks, obtained from the laboratory animal nursery and passed the necessary quarantine. The ambient temperature was 22 ± 2 ° C, relative humidity 60 ± 5%, the ammonia content was 10 rpm, carbon dioxide did not exceed 0.15% of the air volume, the air exchange rate was 10-15 times / h at an air velocity of 0.3 m / s. Air filtration was provided using coarse filters. The mice were fed with granular feed made in accordance with the standards for the preparation of feed for laboratory animals. The food was pre-sterilized in an autoclave using vacuum at 121 ° C and 1.2 atm for 20 minutes. Feeding was carried out once a day at the rate of 8.0 g per mouse after cleaning the cells. Drinking water was poured into drinking bottles with a capacity of 200 ml and sterilized at 121 ° C and 1.2 atm for 45 minutes, the corks were autoclaved separately. Drinking bowls were replaced daily with new ones. The level of interleukin-18 (IL-18) in the blood serum was determined by enzyme-linked immunosorbent assay using specific antibodies. The amount of IL-18 in serum was determined at 1, 2, 3, 4, 5, 6, 8, 12, 16, 24, 32, 40, 48, 56 hours after drug administration.

In parallel, the background amount of IL-18 present in the blood serum of the studied animals was studied. For this purpose, 14 mice were used as a control group, which received a subcutaneous injection of saline. In the experimental group, 336 animals were used. In the case of the control group, the confidence interval was calculated from the dilution measurements of the same sample. In the experimental group, the confidence interval was calculated relative to three samples from three animals for each measured point.

3-4 h after administration, all peptides show a small significant peak in the increase in IL-18 concentration. After 24 hours, there is a second peak of a significant increase in the serum IL-18 content in the experimental group of animals, which lasts about 20 hours. The presence of the first peak for 3-4 hours is probably due to nonspecific induction. A significant increase in IL-18 on the 2-3rd day of the experiment indicates the specific ability of the drugs to induce the studied cytokine.

The test results of the drug are given in table. one.

Table 1. The dynamics of the concentration of IL-18 (PG / ml) in the blood of mice after the introduction of physical. solution, Alloferon-1 and peptides I-VII

The interval between observations, hours The control Alloferon-1 Peptide I Peptide II Peptide III Peptide IV Peptide V Peptide VI Peptide VII one 216 ± 15 218 ± 12 231 ± 15 225 ± 19 218 ± 26 222 ± 12 222 ± 14 218 ± 25 230 ± 29 2 210 ± 18 216 ± 18 236 ± 17 227 ± 18 218 ± 19 225 ± 17 221 ± 16 218 ± 24 216 ± 21 3 208 ± 20 245 ± 15 238 ± 20 241 ± 23 222 ± 30 231 ± 21 238 ± 21 225 ± 17 243 ± 17 four 213 ± 17 250 ± 16 238 ± 14 245 ± 14 228 ± 15 230 ± 13 245 ± 17 238 ± 14 243 ± 25 5 223 ± 12 220 ± 25 232 ± 22 235 ± 17 225 ± 17 225 ± 14 240 ± 23 225 ± 16 236 ± 19 6 203 ± 29 212 ± 30 230 ± 28 227 ± 12 218 ± 19 218 ± 15 221 ± 24 227 ± 20 226 ± 14 8 211 ± 21 213 ± 17 230 ± 19 223 ± 28 216 ± 23 216 ± 19 218 ± 25 218 ± 14 221 ± 15 12 213 ± 17 224 ± 12 231 ± 20 228 ± 20 220 ± 17 220 ± 21 228 ± 15 221 ± 19 225 ± 18 16 218 ± 12 229 ± 11 235 ± 25 235 ± 25 222 ± 22 225 ± 18 235 ± 19 226 ± 23 228 ± 19 24 212 ± 22 240 ± 15 300 ± 14 340 ± 14 295 ± 26 256 ± 23 256 ± 21 249 ± 26 275 ± 24 32 220 ± 15 362 ± 22 410 ± 19 387 ± 27 375 ± 30 405 ± 11 370 ± 25 372 ± 18 387 ± 28 40 200 ± 30 354 ± 14 328 ± 23 315 ± 19 328 ± 29 365 ± 22 365 ± 21 375 ± 28 394 ± 30 48 204 ± 28 242 ± 18 241 ± 27 235 ± 21 240 ± 14 241 ± 26 240 ± 18 245 ± 14 256 ± 29 56 213 ± 17 291 ± 19 257 ± 21 257 ± 19 257 ± 15 271 ± 16 280 ± 30 285 ± 15 287 ± 16

The introduction of both Alloferon-1 and peptides I-VII caused the induction of IL-18, which lasted up to 3 days. The maximum concentration of IL-18 is achieved 30 hours after a single injection of peptides. The nature of induction - the intensity and time range - is the same for peptides.

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For all studied peptides, the induction of interleukin-18 was recorded, the level of which exceeds the value achieved by Alloferon-1. The maximum level of interleukin-18 after 32 hours (410 pg / ml) was recorded for peptide I. The average background level in the group of control animals was 200 pg per ml.

Example 2. The study of interferonogenic activity of the studied peptides I-VII

Activity was determined by administering subcutaneously studied peptides once at a dose of 1 mg per kg of weight of laboratory animals. For the experiment we used mice of the BALB / C line, females, weighing 16-22 g, age 18 weeks, contained in accordance with the protocol described above in the example

1. The level of interferon gamma in the blood serum was determined by enzyme-linked immunosorbent assay using specific antibodies. The amount of interferon in the serum was determined at 3, 6, 12, 16, 24, 32, 40 and 48 hours after administration of the drug.

At the same time, the background amount of interferon gamma present in the serum of the studied animals was studied. For this purpose, 8 mice were used as a control group, which received a subcutaneous injection of saline. The average value in the control group at a 5% significance level was 29 ± 8 pg / ml of the studied serum. In each experimental group, 8 mice were also used, which received a subcutaneous injection of drugs at a dose of 25 μg with the studied peptides I-VII, as well as Alloferon-1. The test results of the drugs are presented in table. 2.

Table 2. The study of the dynamics of the induction of interferon gamma biologically active derivatives of Alloferon-1 (peptides I-VII)

The interval between observations, hours The control Alloferon one Peptide I Peptide II Peptide III Peptide IV Peptide V Peptide VI Peptide VII 3 30 ± 7 91 ± 16 101 ± 17 89 ± 15 95 ± 14 90 ± 16 95 ± 13 100 ± 15 97 ± 13 6 28 ± 5 88 ± 12 99 ± 13 90 ± 12 87 ± 14 89 ± 12 90 ± 12 98 ± 15 89 ± 12 12 29 ± 8 62 ± 5 63 ± 6 71 ± 7 64 ± 12 70 ± 12 64 ± 7 64 ± 7 71 ± 7 16 31 ± 6 46 ± 7 40 ± 12 42 ± 7 42 ± 14 46 ± 12 46 ± 7 42 ± 6 46 ± 7 24 28 ± 9 38 ± 6 36 ± 9 29 ± 9 21 ± 6 40 ± 12 36 ± 9 40 ± 6 34 ± 11 32 28 ± 12 28 ± 7 34 ± 14 23 ± 8 22 ± 8 35 ± 7 30 ± 6 36 ± 9 28 ± 7 40 30 ± 6 22 ± 7 22 ± 10 21 ± 7 26 ± 5 25 ± 7 26 ± 5 26 ± 5 25 ± 8 48 29 ± 13 29 ± 12 25 ± 8 29 ± 9 29 ± 8 27 ± 6 25 ± 8 25 ± 8 21 ± 7

The data obtained indicate that the studied peptides I-VII cause the induction of interferon gamma, exceeding the level of induction of Alloferon-1.

Example 3. The study of the effect of biologically active peptides I-VII on the resistance of mice to influenza A virus

The antiviral effect of biologically active peptides I-VII was studied in a model of lethal viral infection of mice with influenza A. The virus suspension was administered intranasally at a dose corresponding to 10 LD50. Alloferon-1 and peptides I-VII in 0.5 ml of a 0.9% sodium chloride solution were administered intraperitoneally one day before the virus inoculation, then 1, 2, 4, 6, and 8 days after the inoculation. The drugs were tested at a dose of 25 mcg. In control, mice were injected with an equal volume of solvent. The criterion of effectiveness was the survival of animals 10 days after infection with the virus. For the experiment, sexually mature white mice, males weighing 20.0-22.0 g, obtained from the laboratory animal nursery and passed the necessary quarantine, were selected. The mice were maintained according to the protocol described above in Example 1. 180 mice were used in the experiment.

Administration of Alloferon-1 and biologically active peptides I-VII to infected animals caused a dose-dependent protective effect (Table 3). Drugs at a dose of 25 μg caused a significant increase in the number of survivors during the observation period of animals. The effect of this dose is highly reliable.

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Table 3. The study of the effect of biologically active peptides I-VII on the resistance of mice to influenza A virus

A drug Dose, mcg Qty ANIMALS Mortality 10 days after the introduction of the virus N % The control - twenty fourteen 70 Alloferon-1 25 twenty 5 25 ** Peptide I 25 twenty 0 Peptide II 25 twenty 3 fifteen** Peptide III 25 twenty four twenty** Peptide IV 25 twenty four twenty** Peptide V 25 twenty 2 10** Peptide VI 25 twenty one ^ *** Peptide VII 25 twenty 3 fifteen**

** P <0.01 *** P <0.001

Thus, the results of example 3 indicate that biologically active peptides I-VII at a dose of 25 μg have a pronounced antiviral effect on mice infected with influenza A virus, exceeding the antiviral effect of Alloferon-1.

Example 4. The study of the effect of biologically active peptides I-VII on the resistance of mice to influenza B virus

A study of the effect of biologically active peptides I-VII of the resistance of mice to influenza B virus was carried out on 200 mice. For the experiment, sexually mature white mice, males weighing 20.0-22.0 g, obtained from the laboratory animal nursery and passed the necessary quarantine, were selected. The mice were maintained according to the protocol described in Example 1 above. The mice were infected with a strain of LEE 1/40 of influenza B virus taken in an infectious dose corresponding to 30 LD ₅₀ . Specific antiviral drug Ribavirin was used as a positive control.

Alloferon-1 and biologically active peptides I-VII were administered intraperitoneally at a dose of 25 μg 1 day before infection with the virus, then after 1, 2, 4, 6 and 8 days.

The results are presented in table. four.

Table 4. The study of the effect of biologically active peptides I-VII on the resistance of mice to influenza B virus

A drug Dose the virus (LD50) Qty animals Mortality 10 days after the introduction of the virus N % The control thirty twenty 16 80 Ribavirin thirty twenty 12 60 Alloferon-1 thirty twenty 6 thirty Peptide I thirty twenty one Peptide II thirty twenty four twenty** Peptide III thirty twenty 5 25 ** Peptide IV thirty twenty 5 25 ** Peptide V thirty twenty 3 fifteen** Peptide VI thirty twenty 2 10** Peptide VII thirty twenty four twenty**

** P <0.01 *** P <0.001

In control, intranasal administration of the virus caused severe pneumonia with high mortality (80%). Against this background, Ribavirin with a high infectious load (30 LD ₅₀ ) in the conditions of this experiment was ineffective. At the same time, the biologically active peptides I-VII had a pronounced protective effect that exceeded the antiviral effect of Alloferon-1.

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Thus, the results of example 4 indicate that biologically active peptides I-VII at a dose of 25 μg have a pronounced antiviral effect on mice infected with influenza B virus, significantly exceeding the protective effect of Ribavirin, a well-established antiviral agent, as well as Alloferon- 1 and can serve as the basis for the creation of new antiviral drugs.

SEQUENCE LISTING <110> Alloferon Limited Liability Company <120> Peptides inducing increased formation of interferon-gamma and IL18, and immunomodulating, antiviral pharmaceutical composition <130> RU2013141485 <140> RU 2013141485 <141> 2013-09-10 < 160> 7 <170> PatentIn version 3.5 <210> 1 <211> 13 <212> Protein

<213> artificial sequence <220> <223> Peptide 1 <400> one His Gly Val Ser Gly Tyr Gly Gln His Gly Val His Gly one 5 10 <210> 2 <211> 13 <212> Protein <213> artificial sequence <220> <223> Peptide 2 <400> 2 His Gly Val Ser Gly Ala Gly Gln His Gly Val His Gly one 5 10

<210> 3 <211> 13 <212> Protein <213> artificial sequence <220>

<223> Peptide 3 <400> 3

His Gly Val Ser Gly Arg Gly Gln His Gly Val His Gly

- 5 031772 <210> 4 <211> 13 <212> Protein <213> artificial sequence <220>

<223> Peptide 4 <400> 4

His Gly Val Ser Gly Lys Gly Gln His Gly Val His Gly

510 <210> 5 <211> 13 <212> Protein <213> artificial sequence <220>

<223> Peptide 5 <400> 5

His Gly Val Ser Gly Phe Gly Gln His Gly Val His Gly

510 <210> 6 <211> 13 <212> Protein <213> artificial sequence <220>

<223> Peptide 6 <220>

<221> misk_feature <222> (6) .. (6) <223> Xaa may be phenylalanine having a methoxy group in the para position.

<400> 6

His Gly Val Ser Gly Xaa Gly Gln His Gly Val His Gly

510 <210> 7 <211> 13 <212> Protein <213> artificial sequence <220>

<223> Peptide 7 <220>

<221> misk_feature <222> (6) .. (6) <223> Xaa may be phenylalanine having chlorine in the para position <400> 7

- 6 031772

His Gly Val Ser Gly Xaa Gly Gln His Gly Val His Gly

5 10

Claims (2)

CLAIM 1. Biologically active peptide having an amino acid sequence selected from His-Gly-Val-Ser-Gly-Tyr-Gly-Gln-His-Gly-Val-His-Gly, His-Gly-Val-Ser-Gly-Ala- Gly-Gln-His-Gly-Val-His-Gly, His-Gly-Val-Ser-Gly-Arg-Gly-Gln-His-Gly-Val-His-Gly, His-Gly-Val-Ser-Gly- Lys-Gly-Gln-His-Gly-Val-His-Gly, His-Gly-Val-Ser-Gly-Phe-Gly-Gln-His-Gly-Val-His-Gly, His-Gly-Val-Ser- Gly-Phe (p-OMet) -Gly-Gln-His-Gly-Val-His-GlyH His-Gly-Val-Ser-Gly-Phe (p-Cl) -Gly-Gln-His-Gly-Val-His -Gly, and inducing increased formation of gamma-interferon and interleukin-18. 2. Immunomodulatory and antiviral pharmaceutical composition containing one or more peptides according to claim 1 or their pharmaceutically acceptable salts in an effective amount in combination with auxiliary substances.