EA010156B1 - Peptide stimulating regeneration of lever tissue, pharmaceutical composition based thereon and use thereof - Google Patents

Abstract

The invention relates to medicaments for use in the treatment of lever diseases and can be applied as a means stimulating regeneration of lever tissue.A pharmaceutical composition is proposed stimulating regeneration of lever tissue comprising an effective amount of glutamyl-aspartyl-leucine peptide as an active compound of general formula: H-Glu-Asp-Leu-OH sequence 1 [SEQ ID NO:1] and pharmaceutically-acceptable carrier, wherein the pharmaceutical composition being in a form suitable for parenteral administration.The glutamyl-aspartyl-leucine peptide of general formula: H-Glu-Asp-Leu-OH sequence 1 [SEQ ID NO:1] is proposed being able to stimulate regeneration of lever tissue and use of said peptide for manufacturing a medicament able to stimulate regeneration of lever tissue.A method is also proposed for stimulating regeneration of lever tissue comprising administration a patient a pharmaceutical composition comprising glutamyl-aspartyl-leucine peptide as an active compound of general formula: H-Glu-Asp-Leu-OH sequence 1 [SEQ ID NO:1] in dose of 0.01-100 mkg/kg to the body weight, at least once a day during a period required to reach a therapeutic effect, the medicament is administered parenterally.

Description

The invention relates to medicines for the treatment of liver diseases and can be used as a means of stimulating the regeneration of liver cells.

Currently known drugs that enhance the metabolic processes in the liver, which include natural antioxidants, in particular vitamins of group B (B _s B _6, B _12), ascorbic acid, vitamin E, vitamin A; preparations from medicinal plants - Liv-52 (Big Russian Encyclopedia of Medicines, M .: Remediium Publishing House, t.2, 2002, p.435). Known drugs that improve metabolism in liver cells, among which should be noted silibinin (Big Russian Encyclopedia of medicines, M .: Publishing house Remedium, t.2, 2002, p.719); Sirepar (Big Russian Encyclopedia of Medicines, M .: Publishing House Remedium, t.2, 2002, p. 722); Essential (French Patent No. 2556050); as well as synthetic peptide preparations (RF patent No. 2166957, 2000).

Known drugs have only a unidirectional effect, namely they improve metabolic processes in the liver and increase its resistance to pathogenic effects.

It should be noted that drugs aimed at the regeneration of hepatocytes are not found in the prior art.

In this regard, the development of new groups of drugs, including biologically active peptide compounds with the ability to regenerate liver tissue, is relevant.

The structural analogues represented in the prior art, the proposed peptide does not have.

The present invention has set and solved the problem of obtaining a peptide that stimulates the regeneration of liver cells and a pharmaceutical composition containing this peptide as an active principle, as well as a method for its use.

The technical result of the invention is to create a peptide with biological activity, which is manifested in stimulating the regeneration of liver cells, as well as a pharmaceutical composition containing this peptide as an active principle, the use of which stimulates the regeneration of liver cells by restoring the synthesis of tissue-specific proteins and normalizing the functions of liver cells.

To solve the problem and achieve the technical result, a group of inventions is proposed, united by a common inventive concept.

One of the objects of the proposed group of inventions is a pharmaceutical composition that stimulates the regeneration of liver cells, containing as an active principle an effective amount of a glutamyl-aspartyl-leucine peptide of the general formula H-C1i-A5p-Bsi-OH of the sequence 1 | 8EO Ш N0: 1] and pharmaceutically acceptable carrier.

In this case, the pharmaceutical composition is in a form suitable for parenteral administration.

Another aspect of the present invention relates to a glutamyl-aspartyl-leucine peptide of general formula H-C1i-A5p-Bsi-OH of the sequence 1 | 8EO 1E N0: 1], which has the ability to stimulate the regeneration of liver cells. The use of the glutamyl-aspartyl-leucine peptide of the general formula H-C1i-A5p-Bsi-OH of the sequence 1 | 8EO W N0: 1] for the preparation of a medicament stimulating the regeneration of liver cells is proposed.

A further aspect of the present invention relates to a method for promoting liver cell regeneration, which comprises administering to a patient a pharmaceutical composition comprising, as an active principle, a glutamyl-aspartyl-leucine peptide of general formula H-C1i-A5p-Bsi-0H of sequence 1 | 8E0 W N0: 1] in a dose of 0.01-100 μg / kg body weight at least 1 time per day for the period necessary to achieve a therapeutic effect. In this case, administration is carried out parenterally.

The glutamyl-aspartyl-leucine peptide of the general formula H-C1i-A5p-Bsi-0H is obtained by the classical method of peptide synthesis in solution.

The possibility of an objective manifestation of a technical result when using the invention is confirmed by reliable data given in the examples, which contain experimental information obtained in the process of conducting research using methods adopted in this field.

The stimulating effect of the peptide H-C1i-A5p-Bsi-0H on the regeneration of liver cells was revealed during its experimental study.

The study of biological activity was carried out on liver explants, in experimental models of partial hepatectomy and cirrhosis.

The term pharmaceutical composition refers to various dosage forms containing the peptide H-C1i-A5p-Bei-0H. which can find therapeutic use in medicine as a means of stimulating the regeneration of liver tissue.

To obtain pharmaceutical compositions that meet the invention, an effective amount of the peptide H-C1i-A5p-Bay-0H. as an active principle, it is mixed with a pharmaceutically acceptable carrier according to pharmaceutical compounding methods.

The term effective amount implies the use of such an amount

- 1 010156 beginning, which in accordance with its quantitative indicators of activity and toxicity, as well as on the basis of specialist knowledge should be effective in this dosage form.

The carrier may take various forms, which depend on the dosage form of the drug desired for administration to the body.

For parenteral administration, the carrier usually includes saline or sterile water, although other ingredients that promote stability or to maintain sterility may be included.

The invention is illustrated by the figure and tables.

The figure shows the effect of the peptide H-C1i-A8p-Ley-OH on the development of liver explants.

In the table. Figure 1 shows the effect of the peptide H-C1i-L8p-Ley-OH on the number of dividing cells in the regenerating rat liver 32 and 96 hours after partial hepatectomy (% of the total number of liver cells).

In the table. Figure 2 shows the effect of the peptide Н-С1и-Л8р-Лей-OH on blood biochemical parameters in rats with experimental liver cirrhosis.

In the table. Figure 3 shows the effect of the peptide Н-С1и-Л8р-Леи-ОН on the content of total glycogen (conventional units) in the liver of rats with experimental cirrhosis.

In the table. Figure 4 shows the effect of the peptide Н-С1и-Л8р-Лей-OH on the morphological and biochemical parameters of the peripheral blood of guinea pigs in the study of toxicity.

In the table. Figure 5 shows the effect of the peptide Н-С1и-Л8р-Лей-OH on the biochemical parameters of the peripheral blood of patients with chronic hepatitis.

The invention is illustrated by an example of the synthesis of the glutamyl-aspartyl-leucine peptide of the general formula H-C1i-L8p-Lye-OH (example 1), examples of testing the biological activity of the peptide (examples 2, 3, 4), an example of the study of toxicity (example 5), and an example of the results of the clinical use of the peptide, demonstrating its pharmacological properties and confirming the possibility of achieving a therapeutic effect (example 6).

Example 1. The synthesis of the peptide H-S1-L8p-Ley-OH.

1. Name of compound: glutamyl-aspartyl-leucine.

2. Structural formula: H-S1i-L8r-Ley-OH.

3. Gross formula without counterion: C | ₅ H ₂ ^ ₃ O-.

4. Molecular weight without counterion: 375.37.

5. Counterion: acetate.

6. Appearance: odorless white amorphous powder.

7. Method of synthesis: the peptide obtained by the classical method of synthesis in solution according to the scheme

В0С-61и (0Вг1) -0Н

HO5i

OSS

BOC-C1i (OVg1) -O8i

ΒΟ € -61υ (ΟΒζ1) -Α3ρ (ΟΒζ1) -ΟΗ

I N-lei-ΟΒζΙ ψ OSS

BOC-01i (OVg1) -A5r (OVD) -Ei-OV21

1) H ₂ / P1

2) TRA

N-SI-Azr-Ley-OH

BOC - tert-butyloxycarbonyl group, OZi - Ν-oxysuccinimide ether,

OSS - Ν, Ν'-dicyclohexylcarbodiimide,

OV / 1 - benzyl ether,

TPA - trifluoroacetic acid.

- 2 010156

8. Characteristics of the finished product.

The content of the main substance

TLC

Moisture content pH 0.001% solution

Specific optical rotation

98.15% (by HPLC, 220 nm)

Individual, I (= 0.65 (acetonitrile-water 1: 3) 6%

4.57 [a] _s ²² : -30 ° (c = 1, H ₂ O), Po1ata1 A, Cag1 Ζοίκκ 1epa

Synthesis example.

1) BOC-C1i (OV / 1) -O5> and. I-tert-butyloxycarbonyl- (y-benzyl) glutamic acid окс-oxysuccinimide ester (I).

No. Tert. Butyloxycarbonyl- (y-benzyl) glutamic acid BOC-C1P (OBx1) -OH (33.7 g, 0.1 mol) was dissolved in 50 ml of Ν, Ν'-dimethylformamide, cooled to a temperature of -10 ° C, under stirring, cooled (4-6 ° C) solutions of Ν, Ν'-dicyclohexylcarbodiimide (23.0 g, 0.11 mol) in 30 ml of Ν, Ν'-dimethylformamide and Ν-hydroxysuccinimide (13.0 g, 0, 11 mol) in 20 ml of Ν, Ν'-dimethylformamide. The reaction mixture was stirred for 12 hours while cooling with ice and then for 1 day at room temperature. The precipitated Ν, Ν'-dicyclohexylurea was filtered off and the resulting activated ether solution was used without isolation in the next step.

2) BOC-SCHCHOV / O-Akr / OV / O-OH, No. tert.butyloxycarbonyl- (y-benzyl) glutamyl- (b-benzyl) aspartate (II).

((b-Benzyl) aspartic acid H-Acre (OBx1) -OH (28.0 g, 0.12 mol) and 36 ml (0.12 mol) of triethylamine were suspended in 50 ml of Ν, Ν'-dimethylformamide and stirred in within 1 h. Then, a solution of the activated BOC'-C1i (OB / 1) -O8i (I) ester obtained in the previous step was added in portions. The reaction mixture was stirred at room temperature for 2 days. Then it was acidified with 0.5N sulfuric acid to pH 2-3, and extracted with ethyl acetate 4x50 ml. Extractions were combined and washed successively with 0.5N. H ₂ 8o ₄ 3x50 ml, water 2x50 ml, 5% solution of ₃ №1NS'O 2x50 ml, water 2x50 ml, nasy . ennym №1S1 2x50 ml solution The organic layer was dried over № ₂ 8o _4, the solvent was evaporated in vacuo, the residue is crystallized under hexane to give 50 g of product (92%) R (= 0.34 (benzene-acetone 2:.. 1).

3) BOS-SbKOV / O-Akr / OV / O-Vei-OVhk benzyl ester No. tert.butyloxycarbonyl- (y-benzyl) glutamyl- (b-benzyl) aspartylleucine (III).

The BOC-C1i (OB / 1) -A8p (OBx1) -OH (II) dipeptide (1.20 g, 2.2 mmol) and Ν-hydroxybenzotriazole (0.4 g, 3 mmol) were dissolved in 5 ml of tetrahydrofuran and cooled to a temperature of -10 ° C. To a suspension of To8H-Bei-OV / 1 (1.38 g, 3.5 mmol) in 5 ml of tetrahydrofuran was added 0.5 ml (3.5 mmol) of triethylamine and also cooled. Ν, Ν'-Dicyclohexylcarbodiimide (0.62 g, 3.0 mmol) was dissolved in 5 ml of tetrahydrofuran and cooled. The cooled solutions were combined and stirred for 1 day while cooling with ice. The precipitated Ν, Ν-dicyclohexylurea was filtered off, and the solvent was evaporated in vacuo. The residue was dissolved in 50 ml of ethyl acetate and washed successively with 0.5N. H ₂ 8O ₄ 2x520 ml, water 2x20 ml, 5% NaHCO3 solution 2x20 ml, water 2x20 ml, saturated solution No. 1C1 2x20 ml. The organic layer was dried over No. ₂ 8O ₄ , the solvent was evaporated in vacuo, and the residue was crystallized under hexane. Recrystallization from isopropyl alcohol. Received 0.97 g of the product (68%). I (= 0.88 (benzene-acetone 1: 1).

4) H-SY-Acre-Ley-OH, glutamyl-aspartyl-leucine.

The protected BOC-C1i (OB / 1) -A8p (OB / 1) -Vey-OBx1 (III) tripeptide (0.90 g) was dissolved in methyl alcohol-water (4: 1) and hydrogenated over a Pb / C catalyst ( 5%) for 4 hours. The catalyst was filtered off, the solvent was evaporated in vacuo, and the residue was dried in vacuo over KOH and P ₂ O ₅ . Next, the product was dissolved in 2 ml of a mixture of methylene chloride-trifluoroacetic acid (5: 1) and kept at room temperature for 2 hours. The completion of the release reaction was monitored by TLC in an acetonitrile-water system (1: 3). The solvent was evaporated in vacuo, the residue was dried in vacuo over KOH.

For purification, 300 mg of the preparation was dissolved in 4 ml of 0.01% trifluoroacetic acid and subjected to high-performance liquid chromatography on a reverse phase column of 50x250 mm I1a5Ot-130C16T, 7kt. Chromatograph Vesktap 8uk1et 6o1b, 126 8o1uep1 Mobiе, 168 June Aggau Sе1еs1og Mobiе. Chromatography conditions: A: 0.1% TPA; B: MeC # 0.1% TPA; Gradient B 0 ^ 50% in 100 min. Sample volume: 5 ml, detection: at 215 nm, scanning: 190-600 nm, flow rate: 10 ml / min. The fraction 47.0-54.0 min was taken. The solvent was evaporated in vacuo at a temperature not exceeding 40 ° С, evaporation was repeated (5 times) with 10 ml of 10% acetic acid solution. Finally, the residue was dissolved in 20 ml of deionized water and lyophilized.

Received 196 mg of a purified preparation in the form of an odorless amorphous white powder.

5) Analysis of the finished product.

The content of the main substance was determined by HPLC on a Riepotepech C 18 ΒυΝΛ 4.6 x 150 mm column. A: 0.1% TPA; In: MeCI; Gradient B 0 ^ 100% in 10 min. Flow rate: 1 ml / min. Detection at 220 nm, scanning: 190-600 nm, sample: 20 tsl. The content of the main substance: 98.15%.

- 3 010156

TLC: individual,,, = 0.65 (acetonitrile-water 1: 3, PTCX-P-B-UV 8gb1 plates, CTX-1BE silica gel 8-12 μm, manifestation of chlorine / benzidine).

Moisture content: 6% (gravimetrically by weight loss during drying 20 mg at 100 ° C). pH 0.001% solution: 4.57 (potentiometric).

Specific optical rotation: | α | _υ ²² : -30 ° (с = 1, Н ₂ О), Ро1ата1 А, Саг1 Ζοίδδ 1ея.

Example 2. The effect of the peptide H-Cl-A8p-Ley-OH on the development of liver explants.

The experiments were carried out on 27 fragments of the liver of rats of the UMag rat with a body weight of 150-200 g. Nutrient medium for explant cultivation consisted of 35% Eagle's solution, 25% fetal calf serum, 35% Hanks' solution, 5% chicken embryonic extract; glucose (0.6%), insulin (0.5 units / ml), penicillin (100 units / ml), glutamine (2 mm). Liver fragments were placed in this medium and cultured in Petri dishes in an incubator at a temperature of 36.7 ° C for 2 days. The peptide Н-С1и-А8р-беи-ОН at concentrations of 1.10 and 100 ng / ml was added to the experimental medium. The criterion for biological activity was the area index (PI) - the ratio of the area of the entire explant along with the growth zone to the outgoing area of the liver fragment. The IP values were expressed as a percentage, the control IP value was taken as 100%.

The figure shows the effect of the peptide H-C1i-A8p-Ley-OH on the development of liver explants.

It was found that after 1 day of cultivation, the explants spread on a collagen substrate and the proliferation of proliferating and migrating cells began on the periphery of the explant. On the 3rd day of cultivation, at a concentration of the peptide Н-СШ-Akr-Ley-ОН 10 ng / ml, a significant increase in the explant PI by 28% was observed compared with the control PI values. When studying liver explants for longer periods of cultivation (7 days), a similar stimulating effect of the peptide H-NW-Acr-Ley-OH in the same concentration was revealed.

Thus, with respect to liver tissue, the peptide Н-СЫ-Akr-Lei-OH has a tissue-specific effect, which manifests itself in stimulating the growth of explants.

Example 3. The effect of the peptide H-SSh-Akr-Ley-OH on compensatory regeneration of the liver after partial hepatectomy in rats.

The study was conducted on 18 white outbred male rats weighing 150-200 g.

Animals were divided into 3 groups: group 1 — healthy animals, group 2 — control (rats that underwent partial hepatectomy with removal of 2/3 of the liver), group 3 — operated animals, which were then injected subcutaneously 2 and 24 hours after surgery 0.1 μg of the HC1i-A8p-Ley-OH peptide per rat in 0.5 ml of sterile 0.9% physiological solution of IaCl. At the same time, animals of the 1st and 2nd groups were injected with sterile saline in the same volume.

The operated rats were sacrificed with ether 32 and 96 hours after surgery. At the same time, the rats of the control group were killed. Rat liver was fixed in formalin. After staining the preparations with hematoxylin-eosin, the mitotic index in the liver cells was determined, as well as the number of polyploid cells in the 8th phase of the cell cycle (the number of dividing cells).

A study of the mitotic activity of cells of the regenerating liver 32 hours after partial hepatectomy showed that the number of mitoses and cells in the 8th phase of the cell cycle becomes 2 times greater than in the liver of healthy animals. These differences are unreliable in the case of the introduction of physiological saline, whereas after injections of the peptide НСЫ-Akr-Lei-OH, the increase in the number of mitoses, DNA synthesizing cells, and the total amount of dividing cells becomes significant (Table 1).

When studying liver preparations 96 hours after hepatectomy, it turned out that both rats treated with saline and rats treated with the H-SSh-Akr-Ley-OH peptide showed a significant increase in the mitotic activity of hepatocytes. When comparing the data of the experimental (third) and control (second) groups, it was found that in rats that were injected with the peptide H-Cl-A8p-Ley-OH, the number of mitoses was observed, 2 times greater than in rats treated with saline. The number of cells in the 8-phase of the mitotic cycle in rats of the experimental group did not differ significantly from the number of hepatocytes in the 8-phase in the control group, although, in general, the number of dividing cells 96 hours after hepatectomy in the rat regenerating liver with the introduction of the HC1 peptide -A8p-Ley-OH was 75% more than in rats after administration of physiological saline (Table 1).

Thus, it was found that when rats were injected with the peptide H-SSh-Akr-Ley-OH, 96 hours after partial hepatectomy, an increase in the mitotic activity of hepatocytes was observed, indicating an acceleration of reparative processes in the liver.

Example 4. The effectiveness of the use of the peptide HCH-Acr-Ley-OH in rats with experimental cirrhosis.

We used 45 white outbred male rats, the mass of which at the beginning of the experiment was 120-180 g. The animals were inhaled with carbon tetrachloride (CC14) for 6 months. Half of the animals were injected intramuscularly with the peptide H-SSh-Akr-Ley-OH at 0.5 μg per rat in 0.5 ml of sterile physiological 0.9% solution No. C1 monthly with courses of 5 consecutive days. Animals of the control group received injections of sterile saline according to a similar scheme.

The liver condition in animals was evaluated according to biochemical analysis, determining the concentration

- 010156 tion of protein, bilirubin, cholesterol, alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

The content of total glycogen in hepatocytes was determined cytofluorimetrically in smear preparations after conducting a fluorescent PA8 reaction.

Glycogen content is one of the most important indicators of the functional activity of hepatocytes. It is known that cirrhosis of the liver is characterized by significant violations of the glycogen-forming function of hepatocytes.

As a result of the studies, it was found that animals after exposure to CC1 ₄ developed severe disorders in the liver. A decrease in total protein, bilirubin and cholesterol levels was observed. The activity of aminotransferases sharply increased (Table 2).

With the simultaneous administration of CC1 ₄ and the peptide H-OSh-Akr-Lei-OH, biochemical parameters were restored to normal values and indicated less pronounced liver destruction than in animals of the control group (Table 2).

The data presented indicate that the peptide H-C1i-A8p-Ley-OH limits the development of the pathological process in the liver. It reduces fermentemia, limits the signs of hepatocellular insufficiency and thrombohemorrhagic syndrome, increasing foci of regeneration in the liver.

Against the background of exposure to hepatotropic poison, the content of total glycogen in hepatocytes increases, on average, 2.5 times (Table 3).

It has been established that after the use of the peptide H-O1i-A8p-Ley-OH, the increased glycogen content decreases almost to the norm.

Thus, the data obtained indicate a positive effect of the peptide H-O1 and Acr-Ley-OH on the glycogen-forming function of hepatocytes of a cirrhotically altered liver, as well as the restoration of the metabolic viability of liver tissue.

Example 5. The study of the toxicity of the peptide H-O1i-A8p-Ley-OH.

The general toxic effect of the peptide H-O1i-A8p-Ley-OH was studied in accordance with the requirements of the Guidelines for the experimental (preclinical) study of new pharmacological substances (2000): acute toxicity with a single administration of the drug, as well as subacute and chronic toxicity with prolonged administration of the peptide.

A study of acute toxicity was conducted on 66 white outbred male mice weighing 20-22 g. Animals were randomly divided into 6 equal groups. The drug was administered to animals once intramuscularly at doses of 1, 2, 3, 4, 5 mg / kg in 0.25 ml of a sterile 0.9% solution of IAl. Animals of the control group were injected with the same volume of 0.9% solution No. 1C1.

Studies on the study of subacute toxicity were carried out on 55 white mongrel rats with a body weight of 140-180 g. Daily, once a day, animals of the experimental groups were injected intramuscularly with doses of 1 μg / kg, 0.1, 1 mg / kg in 0.5 ml of sterile 0.9% solution of IAl. The animals of the control group were injected in the same volume with a sterile 0.9% solution of IAl. Before administration of the drug on the 30, 60 and 90 days after the start of drug administration, the morphological composition and properties of peripheral blood were studied in animals. At the end of the experiment, biochemical and coagulological blood parameters were examined.

Studies of chronic toxicity were carried out for 6 months, based on the duration of the recommended clinical purpose of the drug, in 88 male guinea pigs weighing 260-300 g. Animals of the experimental groups received a daily intramuscularly peptide for 6 months in doses of 1 μg / kg, 0.1, 1 mg / kg in 0.5 ml of a sterile 0.9% solution of IaS1. In the control group, animals were injected according to a similar scheme to a sterile 0.9% solution No. C1 in the same volume. In animals, the number of erythrocytes, hemoglobin, reticulocytes, platelets, leukocytes, leukocyte formula, erythrocyte sedimentation rate (ESR), and erythrocyte resistance were determined by the generally accepted methods in peripheral blood. Along with this, the content of total protein in blood serum was determined by the method of Lowry, potassium and sodium by plasma spectrophotometry. After the experiment, a pathomorphological study of the brain and spinal cord, spinal ganglia, thyroid gland, parathyroid glands, adrenal glands, testes, pituitary gland, heart, lungs, aorta, liver, kidney, bladder, pancreas, stomach, small intestine, colon intestines, thymus, spleen, lymph nodes, bone marrow.

When studying acute toxicity, it was found that a single administration of the studied peptide to animals at a dose exceeding the therapeutic recommended for clinical use by more than 5000 times does not cause toxic reactions, which indicates a large therapeutic breadth of the drug.

The study of subacute and chronic toxicity of the peptide indicates the absence of side effects with prolonged use of the drug in doses exceeding the therapeutic by 100-1000 times. When studying the effect of the peptide on the morphological composition of the blood of guinea pigs, there was no significant change in the morphological and biochemical parameters of the peripheral blood of guinea pigs (Table 4).

When assessing the general condition of animals, morphological and biochemical parameters of peripheral blood, the morphological state of internal organs, the state of cardiovascular and

- 5 010156 respiratory systems, liver and kidney function, pathological changes in the body were not detected. The absence of a general toxic effect makes it possible to recommend a pharmaceutical composition containing the peptide H-NW-Acr-bie-OH as an active principle for conducting clinical trials. Example 6. The effectiveness of the use of the peptide H-O1i-L8p-Ley-OH in patients with chronic hepatitis. The study was conducted on 34 patients with chronic hepatitis aged 30 to 56 years, who were divided into 2 groups by randomization.

patients of the main group were injected with a pharmaceutical composition containing the peptide H-O1i-L8p-Ley-OH at a dose of 10 μg in 1.0 ml of a sterile 0.9% saline solution intramuscularly once daily for 10 days. The control group consisted of 15 similar patients who were prescribed conventional treatment.

Patients' complaints and biochemical blood parameters were evaluated in dynamics.

Against the background of the treatment, 89% of patients of the main group noted the disappearance of weakness, increased appetite and performance, in 53% of patients the intensity of the pain syndrome significantly decreased.

When examining patients, the most significant attention was paid to assessing the results of biochemical studies characterizing aminotransferase activity, pigment and protein-forming functions of the liver.

81% of the examined patients had hyperbilirubinemia and an increase in the level of alanine aminotransferase (ALT), which indicates a certain activity of the chronic inflammatory process.

The use of a pharmaceutical composition containing the peptide Н-СШ-Akr-Lei-OH contributed to the normalization of the level of bilirubin and ALT activity (Table 5), which indicates the normalization of metabolic processes in the liver, a decrease in the activity of the inflammatory process, and restoration of the synthesis of tissue-specific proteins by liver cells.

Thus, the results obtained indicate the property of a pharmaceutical composition containing the peptide H-OSh-Acr-Ley-OH as an active principle, to stimulate the regeneration of liver tissue. In addition, the research results confirm the feasibility of using a pharmaceutical composition containing H-OSH-Acr-Ley-OH peptide as an active principle in the complex treatment of chronic hepatitis in the phase of exacerbation and remission.

Table 1

Group of animals Study period Mitotic index % of cells in the phase of DNA synthesis Total number of dividing cells Healthy animals + physical. solution - 0.682 ± 0.013 1,752 + 0,463 3.403 ± 0.498 Control (partial hepatectomy + saline solution) 32 h Before 0.431 + 0.019 1,043 ± 0,127 1,474 ± 0,143 After 1,364 ± 0,595 2.063 ± 0.474 3.427 ± 1.066 96 h Before 0.417 ± 0.053 0.924 + 0.091 1.342 ± 0.060 After 2.012 + 0.146 * 3.417 ± 0.295 * 5,429 ± 0,388 * Partial hepatectomy + peptide II-Azr-Azr-Lei IT 32 h Before 0.449 ± 0.065 0.872 ± 0.101 1.321 ± 0.156 After 2.316 ± 0.451 ** 3.885 + 0.838 ** 6.197 ± 1.274 ** 96 h Before 0.296 ± 0.085 0.984 ± 0.139 1.278 ± 0.125 After 4.850 + 0.336 * & 4,667 ± 1,312 ** 9.513 ± 1.608 * #

* P <0.001 compared with indicators before surgery;

** P <0.05 compared with indicators before surgery;

& P <0.001 compared with the animals in the control group;

# P <0.05 compared with indicators in animals of the control group.

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table 2

Index Healthy animals The control Peptide N-SI-Azr-Ley-OH Protein, g / l 85.5 ± 4.2 74.2 + 2.1 * 84.4 + 3.4 ** Total bilirubin, µmol / l 13.4 + 1.3 9.2 + 1.1 * 14.7 + 3.2 ** Cholesterol, mm / l 4,5 + 0,3 3.1 + 0.3 * 4.3 + 0.32 ** ALT, mU / ml 6.1 + 1.1 9.2 + 1.2 * 5.7 + 0.78 ** AST, mU / ml 12.7 + 2.1 18.2 + 1.3 * 12.3 + 1.9 **

* P <0.05 compared with that in healthy animals;

** P <0.05 compared with the indicator in the corresponding control group.

Table 3

Healthy animals Control (exposure to SSC) Exposure to CCK + peptide N-O1i-Akr-1.ei-OH 3.85 + 0.1 8.21 + 0.3 * 3.65 + 0.1 **

* P <0.05 compared with that in healthy animals;

** P <0.05 compared with the indicator in animals of the control group.

Table 4

Index The introduction of the peptide H-O1i-Avr-Ley-OH (1 μg / kg) 3 months 6 months Control (n = 24) Peptide (n = 24) Control (n = 24) Peptide (n = 24) Red blood cells, x10 ^{| 2} / l 5.3 + 0.6 5.4 + 0.1 5.4 + 0.3 5.3 + 0.3 Hemoglobin, g / l 14.2 + 1.4 14.5 + 0.9 14.5 + 1.3 14.1 + 1.5 Reticulocytes,% 1.3 + 0.07 1.1 + 0.05 1.1 + 0.05 1.2 + 0.04 Platelets, xU ’/ l 143.7 + 7.9 144.9 + 4.8 144.5 + 8.6 143.5 + 6.4 White blood cells, x10 ⁹ / l 9.4 + 0.5 9.9 + 0.6 9.6 + 0.5 10.8 + 0.3 Band neutrons,% 0.31 + 0.04 0.29 + 0.06 0.33 + 0.04 0.31 + 0.02 Segmented neutrophils,% 45.8 + 2.1 45.7 + 3.1 46.2 + 3.5 45.4 + 2.6 Eosinophils,% 0.69 + 0.05 0.72 + 0.08 0.72 + 0.04 0.67 + 0.06 Basophils,% 0.61 + 0.04 0.64 + 0.03 0.72 + 0.03 0.67 + 0.04 Monocytes,% 2.5 + 0.02 2.6 + 0.04 2.6 + 0.06 2.4 + 0.07 Lymphocytes,% 48.9 + 2.5 50.2 + 1.9 51.3 + 2.7 49.9 + 1.8 ESR, mm / h 1.69 + 0.05 1.64 + 0.03 2.01 + 0.05 1.87 + 0.07 Erythrocyte resistance,% IaC1 -maximum -minimum 0.41 + 0.02 0.32 + 0.05 0.40 + 0.01 0.29 + 0.05 0.40 + 0.07 0.33 + 0.03 0.43 + 0.05 0.31 + 0.07 Total protein in blood serum, g / l 72.9 + 3.1 72.6 + 3.3 73.1 + 3.4 71.6 + 2.8 Sodium in blood serum, mmol / l 153.9 + 5.7 154.8 + 6.8 155.5 + 6.2 152.8 + 4.9 Serum potassium, mmol / l 5.1 + 2.3 5.3 ± 1.8 5.2 + 2.1 5.2 + 1.9

- 7 010156

Table 5

Indicator (mmol / l) Before treatment After treatment with conventional means After treatment using the peptide H-61i-Azr-Ley-OH Cholesterol 5.1 ± 0.5 5.2 ± 0.2 5.2 ± 0.2 Bilirubin 27.4 ± 1.7 22.3 ± 1.1 * 18.6 ± 1.4 * # AST 40.3 ± 3.2 39.8 ± 2.7 39.5 ± 2.3 ALT 54.5 ± 4.9 46.2 ± 3.8 41.7 ± 2.9 * Triglycerides 2.3 ± 0.4 2.4 ± 0.2 2.4 ± 0.3

* P <0.05 compared with the indicator before treatment;

# P <0.05 compared with indicators after treatment with conventional means.

The list of sequences <110> Limited liability company LED peptides ”<120> peptide that stimulates the regeneration of liver tissue, a pharmaceutical composition based on it and the method of its use <13 0> 5ΙΑ / 04/2006 <160> 1 <170> Ratepp PPG 3.3 <210> 1 <211> 3 <212> RCT <213> Ar11Nac1a1 <22O>

<223> The peptide compound nc! And -A5p-kei-on, which stimulates the regeneration of liver tissue by restoring the synthesis of tissue-specific proteins and normalizing the functions of liver cells.

<400> 1 and Azr

Claims (6)

1. A pharmaceutical composition that stimulates the regeneration of liver cells, characterized in that it contains as an active principle an effective amount of a glutamyl-aspartyl leucine peptide of the general formula H-SinAzr-Bey-OH of the sequence 1 | 8EO ΙΌ N0: 1] and a pharmaceutically acceptable carrier. 2. The pharmaceutical composition according to claim 1, characterized in that it is in a form suitable for parenteral administration. 3. The glutamyl-aspartyl-leucine peptide of the general formula H-01i-Azr-Bsi-0H of the sequence 1 | 8Е0 ΙΌ N0: 1], which has the ability to stimulate the regeneration of liver cells. 4. The use of the peptide according to claim 3 for the preparation of a medicinal product that stimulates the regeneration of liver cells. 5. A method of stimulating the regeneration of liver cells, which consists in administering to the patient a pharmaceutical composition containing, as an active principle, a glutamyl-aspartyl-leucine peptide of the general formula H-C1i-A8p-Bei-OH of the sequence 1 | 8E0 ΙΌ N0: 1] at a dose of 0, 01-100 μg / kg body weight at least 1 time per day for the period necessary to achieve a therapeutic effect. 6. The method according to claim 5, characterized in that the introduction is carried out parenterally.