Composition and using composition for preparing drug having hepatoprotective activity. RU patent 2485970.

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, and concerns drugs having hepatoprotective activity. What is presented is a composition possessing hepatoprotective activity consisting of a mixture of the peptides H-Lys-Asp-Glu-OH, H-Asp-Glu-Pro-OH and H-Asp-Glu-Leu-OH taken on a weight basis (1-8):(1-8):(1-8). What is also presented is using the above composition for preparing the drug possessing hepatoprotective activity.

EFFECT: invention extends the range of low-toxic medical products possessing hepatoprotective activity having no side effects.

3 cl, 5 ex, 4 tbl

The invention relates to chemical industry to obtain the means possessing hepatoprotective activity.

According to the world health organization the number of patients with different types of hepatobiliary pathology on the planet is more than 2 billion people (World health statistics, 2010). More than 50% of Russians suffer from disorders of the liver, and every fourth obesity has the authority, and in 25% of cases of chronic liver diseases guilty of alcohol abuse (data of the all-Russian epidemiological study).

As is known, pharmacotherapy is the basic method of treatment of diseases of the liver, and with different pathology requires prolonged administration of drugs-hepatoprotectors (.. et al. 2003. Federal guidelines for the use of medicines, 2010). Effective hepatoprotector - drug, acting on the basic links of the pathogenesis of many diseases of the liver, is able to maximally restore damaged hepatocytes (structure and function), accelerate the growth of healthy cells, prevent the development of fibrosis and protect hepatocytes in the future from exposure to damaging factors. As a result of normalizing the numerous functions of the liver (improves lipid and protein metabolism and others).

Today hepatoprotectors successfully used for pathogenetic therapy of acute, chronic (persistent, active hepatitis, cirrhosis of the liver and fatty hepatosis toxic, drug and alcohol etiology (.. et al., 2005. Federal guidelines for the use of medicines, 2010).

Therefore, development of new effective methods of drug treatment and prevention of diseases of liver - an actual problem of modern pharmacology and clinical medicine.

Known hepatoprotector ademetionine (Heptral), which , and antioxidant action, donator music outfits groups, providing redox mechanism for cellular detoxification. It is widely used in treatment of various diseases of the liver (toxic to liver, alcoholic steatosis, chronic hepatitis, cirrhosis and other). However, the drug has a pretty wide range of side effects, which limits its use (Federal guidelines for the use of medicines, 2010).

Proceeding from the above occurs need to find new tools, including peptide.

Object of the present invention is to obtain funds peptide, which possesses hepatoprotective activity.

The technical result of the invention is an expansion of the medicines, which have hepatoprotective activity with low toxicity and do not have side effects.

This problem is solved in the following way.

Proposed composition, hepatoprotective activity, which consists of a mixture of peptides H-Lys Asp Glu-OH, N-Asp Glu-Pro-OH and H-Asp Glu Leu-OH taken in the weight ratio (1-8):(1-8):(1-8).

Also invited to the application of the above composition for obtaining of medicinal product possesses hepatoprotective activity, the tool can be in the form of pills, tablets can be sublingual prolonged action and covered; or in the form of capsules; or as a nasal spray or drops and nasal; or as an aerosol or oral; or in powder form; or in the form of solution for intravenous and intramuscular injection; or in the form of for preparation of a solution; or in the form of drops for reception inside.

The invention is illustrated by the following examples.

Example 1. Peptide synthesis

Getting lysyl--glutamic acid (N-Lys Asp Glu-OH).

Z-Lys (Z)-Asp(OBzl)-Glu(OBzl) 2 : 19 g (30 mmol) Boc-Asp(OBzl)-Glu(OBzl) 2 was dissolved in 200 ml of TFA in an hour evaporated, the residue was dissolved in 500 ml of dry DMF and improving portions Na 2 CO 3 to termination of allocation of CO 2 . Residue was filtered, washed with 100 ml of DMF added 3.33 ml (30 mmol) N- and 15.36 g (30 mmol) Z-Lys(Z)-ONSu and left at room temperature for 15 hours. The reaction mixture was evaporated, resulting the residue was dissolved in 300 ml of ethyl washed consistently: 2% sulfuric acid, water until neutral 5% solution of soda and water again. Ethyl acetate was evaporated and the residue was added to 500 ml of isopropyl alcohol, kept for 2 hours at +4 C, precipitate was filtered, washed with isopropyl alcohol, ether, dried. Output 21.6 g (90%) of the product.

H-Lys Asp Glu-OH: 21.6 g (27 mmol) connections Z-Lys(Z)-Asp(OBzl)-Glu(OBzl) 2 obtained as described above, dissolve when heated in 0.5 l glacial acetic acid, add 50 ml of water and in the presence of 5 grams of palladium catalyst. Catalyst filtered and the solvents are evaporated. Crystallized from a mixture of acetic acid-ethanol. If necessary product clean the ion-exchange chromatography on a column with SP-Sephadex, put in the water, wash 0.04 M pyridine- buffer (pH 5.4). Fractions containing the desired connection, evaporated. the residue is dissolved in 300 ml of water, evaporated up to half the amount treated with activated carbon and . Get 8.95 g (85%). The purity of the product according to HPLC more than 95%.

In the spectrum of TMR

Lys - 3.76 (alpha-CH); 1.72 (?- CH 2 ); 1.36, 1.52 (gamma - CH 2 ); 2.74 (e CH 2 ); 8.11 (NH 2 )

Asp - 4.63 (alpha-CH); 2.71, 2.54 (?- CH 2 ); 8.68 (NH)

Glu - 4.19(alpha-CH); 1.98, 1.78 (?-CH 2 ); 2.28 (gamma-CH 2 ); 8.31 (NH)

Getting aspartyl-glutamyl-Proline (H-Asp Glu-Pro-OH)

Z-Asp(OBu t )-GIu(OBu t )-OH · DCHA: 25.3 g (100 mmol) H-Glu(OBu t )-OH dissolved (suspended) in methanol, added to 50 ml of 40% Triton's in methanol and after the complete dissolution of evaporated in a vacuum. The residue was dissolved in 300 ml of dimethylformamide, evaporated on quarter and rose to 42.0 g (100 mmol) Z-Asp(OBu t )-ONSu. After 12 hours (control - TLC in system chloroform: methanol: acetic acid - 9:1:0.5 B) the reaction mixture was evaporated and the residue was dissolved in ethyl acetate, washed consistently 2% sulfuric acid, water, dried over Na 2 SO 4 , evaporated. The residue was dissolved in 150 ml of ether was added 20 ml (100 mmol) and left at room temperature up to complete sedimentation. Residue was filtered, washed with ether, dried on air. Output of 60 g (87%) chromatographically homogeneous matter.

H-Asp Glu-Pro-OH. 28 g (40 mmol) Z-Asp(OBu t )-GlU(OBu t )-OH · DCHA were added to 500 ml of ethyl, washed 2% sulfuric acid, water, dried over Na 2 SO 4 , drier was filtered and the filtrate was evaporated and the residue was dissolved in 150 ml of dimethylformamide, cooled to -25, chilled solution added 4.4 ml (40 mmol) N- and 5.2 ml (40 mmol) ISO-, kept at -20 degrees for 15 minutes and put a cooled to -25 solution 5.6 g (44 mmole) N- 25 degrees in 50 ml of dimethylformamide. After 30 minutes, the solution was added 10.6 g (44 mmole) H-Pro-OBzl · HCl and 5 ml of N- in 50 ml of dimethylformamide and left at room temperature overnight. The reaction mixture was evaporated and the residue was dissolved in ethyl acetate and washed it in the usual way. Ethyl acetate was evaporated and the remainder was evaporated with toluene. The formed oil dissolved in TFA, was kept for an hour, was evaporated and the remainder were treated with a mixture of ether - hexane 1:1, the formed sediment was filtered, washed filter the mixture of ether - hexane 1:1 hexane, dried in a desiccator over alkali.

The resulting product was dissolved in a mixture of ethanol - water - acetic acid (600:100:100) and over 5% Pd/S. Catalyst was filtered and the filtrate was evaporated. Balance from acetic acid isopropyl alcohol, filtered, washed on the filter isopropyl alcohol, ether, dried. Output 12.2 g (85%). Purity according to HPLC more than 95%.

In the spectrum of TMR

Asp - 4.13 (alpha-CH); 2.79, 2.66 (?- CH 2 ); 8.14 (NH 2 )

Glu - 4.59 (alpha-CH); 1.93, 1.72 (?- CH 2 ); 2.36 (a;- CH 2 ); 8.67 (NH).

Pro - 4.23 (alpha-CH); 2.14, 1.84 (?-CH 2 ); 1.90 (gamma-CH 2 ); 3.63 (b CH 2 ).

Getting aspartyl-glutamyl-leucine (H-Asp Glu Leu-OH)

Boc-Glu(Bzl)-Leu-OBzl: To a solution 26.6 g (78 mmol) Boc-Glu(Bzl)-OH 200 ml of dimethylformamide and 8.65 ml of N-, chilled to -15 C, rose 10.4 ml (80 mmol) mixture was stirred at this temperature for 20 minutes and added cooled to minus 15 degrees Celsius solution 30.5 g (78 mmol) H-Leu-OBzl*Tos, pH which previously was reduced to 8.5-9.0. After an hour, the solvents are evaporated, the residue was dissolved in 500 ml of ethyl washed consistently 400 ml of 2% solution of sulfuric acid, water, 5% solution of soda and water. The organic layer is separated and ethyl acetate evaporated. Balance of a mixture of ether-hexane (1:1). Got 37.8 g (90%) chromatographically homogeneous matter.

Boc-Asp(Bzl)-Glu(Bzl)-Leu-OBzl: 27 g (50 mmol) Boc-Glu(Bzl)-Leu-OBzl was dissolved in 100 ml of chloroform and to this solution was added to 100 l acid. The mixture was kept at room temperature for 2 hours and solvents are evaporated. The residue was dissolved in 200 ml of dimethylformamide and portions were added to soda before the end of the allocation of CO 2 . Residue was filtered, washed with 50 ml of dimethylformamide, in the filtrate taken to 8.5 pH and cooled up to -20 degrees (solution 1).

Simultaneously to a solution of 16.5 g (50 mmol) Boc-Asp(Bzl)-OH in 100 ml of dimethylformamide added 5.6 ml of N-, the mixture was cooled to -20 degrees and would flow 6.5 ml (50 mmol) . The mixture was kept at this temperature for 30 minutes (solution 2).

Keeping cooling, two solutions are mixed and left to get mixed up reaching room temperature (approximately 2 hours). Solvents are evaporated, the residue was dissolved in 300 ml of ethyl then washed with 2% solution of sulfuric acid, water, 5% solution of soda, water and solvent was evaporated. After crystallization of ether received 32.4 g (87%) chromatographically homogeneous matter.

14.9 g (20 mmol) of the protected Boc-Asp (Bzl)-Glu(Bzl)-Leu(Bzl) was dissolved in 50 ml and to this solution was added to 50 ml acid. The mixture was kept for 1 hour at room temperature and solvents are evaporated. The residue was dissolved in 300 ml 80% acetic acid and over 5% Pd/C at room temperature. After the reaction catalyst , washed on the filter, acetic acid, and solvents are evaporated. To the residue would flow 200 ml of isopropyl alcohol. The formed sediment was filtered, dried in a vacuum above the CON. Received 7 g (93%) of product purity according to HPLC more than 95%.

In the spectrum of TMR

Asp - 4.13 (alpha-CH); 2.81, 2.65 (?- CH 2 ); 8.15 (NH 2 )

Glu - 4.34 (alpha-CH); 1.94, 1.80 (?- CH 2 ); 2.30 (gamma-CH 2 ); 8.58 (NH)

Leu - 4.19 (alpha-CH); 1.52 (?- CH 2 ); 1.53, 1.62 (gamma-CH 2 ); 0.83 (d-CH 3 ); 0.89 (d-CH3); 8.22 (NH).

Example 2. A composition

In the reactor, supplied with an anchor mixer, pour 8 l of water for injection. Then open the valve to feed the reactor sterile nitrogen and water bubbled within 5 minutes. After that, the reactor consistently download 30 grams of the substance of each of the peptides included in composition, with constant stirring and solution nitrogen. Mixing lead to dissolve (if necessary, the solution can be added excipients). The received solution is brought to volume of 10 l water for injection and passed through a sterilizing filter. In aseptic conditions carry out the filling of vessels and mortar . Thus receive the substance composition, ready to receive the medicinal forms.

Example 3. Obtaining of medicinal forms songs

5 g of substance dissolve at hashing and nitrogen in 1 l of water for injection. Spend filtering of the obtained solution and in aseptic conditions is realized under nitrogen its filling of ampoules 2 ml 1 ml in ampoules. Filled vials sealed under nitrogen.

In the other variant of the cassette with the ampoules are placed in a sublimation camera where it is frozen to a temperature of minus 40 C with a speed of freezing 5-10 Deg/h, and then subjected to drying in a vacuum. Ampoules with the obtained dry freeze-dried form of the medication sealed under nitrogen.

Example 4. Studying the toxicity of peptide songs

General toxic effect of peptide composition tested in accordance with the requirements of the Manual on experimental (pre-clinical) study of new pharmacological substances» (2005)) acute toxicity after acute administration of the drug, and subacute toxicity.

Studies of acute toxicity was rats-males weighing 210-260, The drug was administered to the animals once in doses of 0.6 mg/kg, 3 mg/kg and 6 mg/kg in a sterile physiological solution. Animals of the control group were injected with saline to the same extent.

Study subacute toxicity was rats-males weighing 210-260, Once daily to the animals of the experimental group were injected the preparation in doses of 600 mcg/kg and 3 mg/kg of Animals of the control group were injected with the same amount of saline solution. Before drug administration and 30, 60 and 90 day after the start of the experiment, the animals studied blood parameters. Determined the quantity of erythrocytes, hemoglobin, platelet count, reticulocyte, leukocytes and deposition rate of erythrocytes. In addition, determine the total protein content by the method of Lowry and composition of electrolytes.

When studying acute toxicity shown that a single injection of the studied composition at a dose of 6 mg/kg (exceeding the intended therapeutic dose of 10 times) does not cause toxic reactions.

The study of subacute toxicity also did not reveal side effects with long-term use of the drug.

Example 5. Study of biological activity of new composition

Studies performed on the white nonlinear rats-males weighing 210-260 g according to the procedures described in the «Manual on the experimental (pre-clinical) study of new pharmacological substances» (2005).

In rats was induced acute toxic hepatitis subcutaneous introduction of carbon tetrachloride (CCl 4 , carbon tetrachloride; 4 ml/kg of 50% oil solution) within 4 days. As is known, CCl 4 - classical poison, which is widely used in the experiment for the modelling of acute toxic hepatitis; in particular, it causes intensive peroxide (lipid) oxidation of lipids (Pol) bilayer of cell membranes of hepatocytes (.., .., 1995; .. et al., 2005).

In the experimental groups of animals were injected intraperitoneally (I.P) a new mixture of peptides ( composition; in doses of 100 and 600 mcg/kg), as well as the preparation of comparison, Heptral (ademetionine, lyophilizate for solution for intravenous and intramuscular injection; Hospira S.p.A. Italy) for 1 hour before the introduction of the CCl 4 1 once a day for 7 days.

In the control group rats were treated in b/W only isotonic solution of sodium chloride (NaCl) in quantity.

Fence liver for morphological (light microscopy) research and blood biochemical analysis produced both before and on the 5th, 7th and 10th day of the study.

Fence liver for morphological (light microscopy) research and blood biochemical analysis produced both before and on the 8th and the 10th day of the study.

In plasma of rats was determined (taking into account the recommendations .. /editor/, 1987):

- activity aspartate aminotransferase and alanine aminotransferase optimized UV method of International Federation of clinical chemistry and laboratory medicine (IFCC);

- activity of alkaline phosphatase - kinetic optimized standard method of the German society for clinical chemistry (DGKC);

- activity αamylase - by enzymatic kinetic test (substrate EPS-G7);

- concentration of total protein was measured biuret method;

- concentration of albumin photometric test with green;

- urea - urease kinetic UV method;

- concentration of creatinine - kinetic test Jaffe without ;

- content of cholesterol and triglycerides - methods.

The concentration of total and indirect (free) bilirubin was measured by the method of -Grof sets of reagents company "Eco-service" (Russia).

Measurements were carried out on the biochemical analyzer "Targa 3000 W" (Biotecnica Instruments, Italy).

Determination of the concentration of lipid peroxidation products (diene conjugates and Schiff bases) and antioxidant vitamins (retinol and a-tocopherol) was performed by UV absorption and methanol extracts (.., .., 1985). Measurements were carried out on "Hitachi 650-60" (Japan) and spectrophotometer "Beckman DU-7 (USA).

Collected the pieces of the liver for morphological study, which was conducted by standard methods of light microscopy. For light microscopy study the pieces recorded 10% neutral formalin, then cooked histological sections were stained with hematoxylin and eosin and spent the microscopy.

Investigation of hepatoprotective action of peptide compositions on the model of acute toxic hepatitis caused by (CCl 4 , carbon tetrachloride)

It was found that in the control group CCl 4 (subcutaneous administration of 4 ml/kg over 4 days) caused the death of 22% (7 rats from 32) animals (table 1). Peptide composition in the dose of 100 mg/kg, did not significantly reduce mortality of rats, a dose of 600 mcg/kg significantly (p<0.005) reduced it to 0%.

Drug comparison hepatoprotector Heptral in a dose of 100 mg/kg reduced mortality of animals up to 15%, but insignificant (p>0.005 percent).

On severity of actions in relation to mortality composition (600 mcg/kg) significantly exceeded Heptral (100 mg/kg) 15% (p<0.05).

Table 1

Changing mortality of rats with toxic CCl 4-hepatitis under the influence of peptide composition and drug comparison hepatoprotector due to treatment with Heptral

Substance

Number rats

Rate, %

Control

32

22

PEPTIDE COMPOSITION (100 mcg/kg)

20

20

PEPTIDE COMPOSITION (600 mcg/kg)

29

0 *#

Heptral (100 mg/kg)

27

15

Note. This difference was statistically significant compared with the control and respectively (exact method Fischer): * and # p<0.05.

It is established that animals of the control group CCL 4 caused by 5-10 days of research development of syndrome of endogenous intoxication, including . So, for example, on the 5th day activity-amylase activity in plasma significantly (p<0.001) increased 1.3 times compared with a group of intact rats and on the 10th day exceeded the initial level (here and below refers to the level of intact animals) 1.1 times (p<0.001) (table 2).

Peptide composition in the dose of 100 mg/kg, did not significantly change the activity-amylase activity in the plasma of blood and in a dose of 600 mcg/kg, starting with the 5-day study, significantly (p<0.05) reduced this figure is 1.1-1.2 times.

Drug comparison Heptral in a dose of 100 mg/kg on days 5-7, did not significantly affect the activity of-amylase activity significantly (p<0.05) reducing its only on the 10th day, 1.2 times. On severity of actions in respect of-amylase activity blood plasma peptide composition (600 mcg/kg) significantly exceeded Heptral (100 mg/kg) 7-10 days 1.1 times (p<0.05).

It is revealed that the animals of the control group CCl 4 caused by 5-10 days of research development of cytolysis syndrome. So, for example, on the 5th day activity in the blood plasma aminotransferase - aspartate (ACT) and alanine aminotransferase (ALT) - increased 5.0 and 4.2 times (p<0.001), respectively, compared with the group of intact rats, and on the 10th day she exceeded the initial level 2,3 and 1.5 times, respectively (p<0.001) (table 2).

Peptide composition in the dose of 100 mg/kg was not significantly changed the activity of the ACT and ALT levels in plasma and in a dose of 600 mcg/kg and 5-day study significantly (p<0.001) reduced this figure 1.3-2.3 and 1.5-1.8 times, respectively, reducing it to the 10 th day prior to the initial level.

Drug comparison Heptral in a dose of 100 mg/kg 5-day study significantly (p<0.001) reduced activity ACT and ALT 1.2 to 2.3 and 1.3-1.9 times, respectively, reducing it to the 10 th day almost to the initial level.

On severity of actions in respect to the activity of the ACT and ALT blood plasma peptide composition (600 mcg/kg) significantly exceeded Heptral (100 mg/kg) on the 5th day in 1.1 and 1.2 times respectively (p<0.05).

Found that the animals of the control group CCL 4 caused by 5-10 days of research development of cholestasis syndrome. So, for example, on the 5th day activity of alkaline phosphatase (ALP - the main indicator of cholestasis syndrome) in plasma significantly (p<0.001) increased 2.4 times in comparison with a group of intact rats and on the 10th day exceeded the initial level of 1.3 times (p<0.001) (table 2).

Peptide composition in the dose of 100 mg/kg was not significantly changed the activity of alkaline phosphatase in the plasma of blood and in a dose of 600 mcg/kg and 5-day study significantly (p<0.001) reduced this indicator 1.4-1.6 times, reducing it to the 10 th day prior to the initial level.

Drug comparison Heptral in a dose of 100 mg/kg 5-day study significantly (p<0.001) reduced activity of alkaline phosphatase in 1.3-1.4 times, reducing it to the 10 th day prior to the initial level.

On severity of actions in respect to the activity of alkaline phosphatase blood plasma composition (600 mcg/kg) significantly exceeded Heptral (100 mg/kg) on the days 5-7 1.2 times (p<0.05).

Other indicators cholestasis syndrome in blood plasma also showed the increase of General and indirect (free) bilirubin (table 3). So, for example, on the 5th day, the concentration of total and indirect cium in plasma significantly (p<0.001) increased 2.5 and 3.5 times respectively compared with a group of intact rats and on the 10th day exceeded the initial level of 1.5 and 1.9 times respectively (p<0.001).

Peptide composition in the dose of 100 mg/kg was not significantly changed the content of General and indirect bilirubin in blood plasma and in a dose of 600 mcg/kg and 5-day study significantly (p<0.001) reduced this figure 1.5-1.9 and 1.8-2.6 times, respectively.

Drug comparison Heptral in a dose of 100 mg/kg 5-day study significantly (p<0.001) reduced content of General and indirect bilirubin 1.4-1.6 and 1.7-1.8 times respectively.

On severity of actions in respect of the content of General and indirect bilirubin blood plasma peptide composition (600 mcg/kg) significantly exceeded Heptral (100 mg/kg) for 10 day of 1.3 and 1.6 times respectively (p<0.05).

Table 3

The dynamics of other biochemical indices of blood in rats toxic CCL 4-hepatitis under the influence of peptide composition and drug comparison hepatoprotector due to treatment with Heptral

Indicator

In intact rats

Day study

Series

5-e (n=8)

7-th (n=8)

10-e (n=8)

Total protein, g/l

76,6 ą 1.3 (n=24)

58,0±1,4 ooo

67,1±1,4 ooo

73,9±1,7

Control

60,9 ą1.0 ooo

69,2 ą1.0 o

73,0 about 1.6

PEPTIDE COMPOSITION (100 mcg/kg)

68,7±1,9 ***#

72,8 ą1.5 *#

75,6 ą 1.3

PEPTIDE COMPOSITION (600 mcg/kg)

64,2±0,8 *

68,4±1,4

72,2 about 1.6

Heptral (100 mg/kg)

Albumin (g/l)

42,8±0,8 (n=24)

34,7 about 1.1 ooo

36,9±1,2 ooo

39,7 ą1.0 o

Control

37,6±0,9 ooo

38,3±1,3 o

39,0 about 1.1 o

PEPTIDE COMPOSITION (100 mcg/kg)

39,4±1,2 *

41,4 ą1.0*

41,8±0,6

PEPTIDE COMPOSITION (600 mcg/kg)

38,6±0,7*

39,0±1,2

40,0 about 1.1

Heptral (100 mg/kg)

Total cholesterol (LDL), mm/l

2,21±0,08 (n=26)

1,18±0,11 ooo

1,32±0,12 ooo

2,14±0,11

Control

1,20±0,10 ooo

1,32±0,11 ooo

2,11±0,12

PEPTIDE COMPOSITION (100 mcg/kg)

1,72±0,06 ***#

2,21±0,13***#

2,62±0,24

PEPTIDE COMPOSITION (600 mcg/kg)

1,48±0,08 *

1,85±0,10 *

2,20±0,19

Heptral (100 mg/kg)

Triglycerides (TG), mm/l

1,70 ħ 0.09 (n=26)

0,34±0,03 ooo

0,46±0,04 ooo

1,53±0,10

Control

0,41±0,03 ooo

0,50±0,02 ooo

1,62 ą0,19

PEPTIDE COMPOSITION (100 mcg/kg)

0,60±0,06 *

0,92±0,10 **#

1,97±0,10 *

PEPTIDE COMPOSITION (600 mcg/kg)

0,52±0,03 *

0,68±0,04*

1,68 ħ 0.09

Heptral (100 mg/kg)

Bilirubin total, m/l

6,2±0,1 (n=25)

15,7±0,3 ooo

12,2±0,4 ooo

9,3±0,2 ooo

Control

13,9±0,9 ooo

10,5±0,7 ooo

6,7 approximately 0.5 *

PEPTIDE COMPOSITION (100 mcg/kg)

9,8±0,4 ***

7,9±0,3 ***

4,8±0,2 ***##

PEPTIDE COMPOSITION (600 mcg/kg)

10,1±0,6 ***

8,6 approximately 0.5 ***

6,0±0,3 ***

Heptral (100 mg/kg)

Unconjugated bilirubin, m/l

1,81±of 0.01 (n=25)

6,28±0,34 ooo

5,12±0,31 000

3,41±0,29 ooo

Control

5,71±0,29 ooo

4,41±0,19 ooo

2,68±0,17 ooo

PEPTIDE COMPOSITION (100 mcg/kg)

3,44±0,14 ***

2,72±0,12 ***

1,29±0,03 ***##

PEPTIDE COMPOSITION (600 mcg/kg)

3,51±0,17 ***

2,98±0,11 ***

2,03±0,08 ***

Heptral (100 mg/kg)

Urea, mm/l

8,2±0,1 (n=26)

20,8 ħ 2.6 ooo

14,8±0,6 ooo

9,3 approximately 0.5 o

Control

19,3±2,4 ooo

12,4±0,8 *

8.9 ħ 0,6

PEPTIDE COMPOSITION (100 mcg/kg)

13,2±9 *#

9,5 of - / + 6 ***#

7,4±0,1 *

PEPTIDE COMPOSITION (600 mcg/kg)

16,6 ą1.0

11,9±0,8 *

8,1±0,4

Heptral (100 mg/kg)

Creatinine, m/l

46,8±0,2 (n=26)

100,2±8,2 ooo

71,4±1,7 ooo

60,9 about 1.6 ooo

Control

97,6±8,5 ooo

66,9±1,8 ooo

59,7 ą1.5 ooo

PEPTIDE COMPOSITION (100 mcg/kg)

62,1±1,4 ***

59,5±1,9 ***

46,3 about 1.1 ***#

PEPTIDE COMPOSITION (600 mcg/kg)

65,9±1,8 ***

63,2 ą 1.3 **

50,1 ą1.0 ***

Heptral (100 mg/kg)

It was found that animals of the control group CCl 4 caused by 5-10 days of research development of syndrome failure synthetic processes in hepatocytes. So, for example, on the 5th day, the level of total protein and albumin in the blood plasma was significantly (p<0.001) declined 1.3 and 1.2 times respectively compared with a group of intact rats. On the 10th day, the concentration of total protein was not significantly differ from the original one, and the level of albumin was significantly slightly below the original 1.1 times (p<0.001) (table 3).

Peptide composition in the dose of 100 mg/kg was not significantly changed the content of total protein and albumin in the blood plasma, but in a dose of 600 mcg/kg on days 5-7 research significantly (p<0.05) increased these figures 1.1-1.2 and 1.1 times respectively.

Drug comparison Heptral in a dose of 100 mg/kg also only on the 5th day of the study significantly (p<0.05) increased the levels of total protein and albumin in 1.1 times, and 7-10 days did not significantly affect this indicator.

On severity of actions peptide composition (600 mcg/kg) significantly exceeded Heptral (100 mg/kg) only in respect of raising the level of total protein in blood plasma on the days 5-7 1.1 times (p<0.05).

Also in the blood plasma was observed a decrease in total cholesterol (cholesterol; LDL) and triglycerides (triacylglycerols, TG) (table 3). So, for example, on the 5th day, the level of total cholesterol and triglycerides in the plasma significantly (p<0.001) was decreased in 1.9-5.0 times respectively compared with a group of intact rats. On the 10th day, the concentration of total cholesterol and triglycerides did not differ significantly from baseline (table 3).

Peptide composition in the dose of 100 mg/kg was not significantly changed the content of total cholesterol (cholesterol) and TG (triglycerides) in the blood plasma, and in a dose of 600 mcg/kg on days 5-7 research significantly increased these indicators 1.5-1.7 (p<0.001) and 1.8-2.0 times (p<0.05) respectively, increasing the concentration of total cholesterol to 7-day to its original level, and TG - to 10-m

On severity of actions regarding the content of total cholesterol blood plasma peptide composition (600 mcg/kg) significantly (p<0.05) exceeded Heptral (100 mg/kg) on the days 5-7 1.2 times and in relation to the contents of TG - only on the 7th 1.4 times.

It was found that in plasma was increased content of urea (the main end product of protein metabolism, synthesized in the liver) and creatinine (table 3). So, for example, on the 5th day, the level of urea and creatinine in the plasma significantly (p<0.001) increased by 2.5 and 2.1 times respectively compared with the group intact . 10 day of the concentration of urea and creatinine exceeded the initial level of 1.1 (p<0.05) and 1.3 times (p<0.001), respectively (table 3).

Peptide composition in the dose of 100 mg/kg was not significantly changed the content of creatinine in the blood plasma, and the level of urea significantly (p<0.05) reduced only on the 7th day, 1.2 times at a dose of 600 mcg/kg and 5-day study significantly reduced these indicators 1.3 1.6 (p<0.05) and 1,2-1,6 times (p<0.001), respectively.

Drug comparison Heptral in a dose of 100 mg/kg only on the 7th day of the study significantly (p<0.05) decreased the level of urea in 1,2 times, and the content of creatinine, beginning with 5 days, 1.2-1.5 times (p<0.01).

On severity of actions regarding the content of urea blood plasma peptide composition (600 mcg/kg) significantly (p<0.05) exceeded Heptral (100 mg/kg) for 5-7 days in 1,3 times, and regarding the content of creatinine - only 10th in 1.1 times.

The data on dynamics of indicators peroxide (peroxide) oxidation of lipids (Pol) and reflecting the state of the system endogenous antioxidant protection for the study of antioxidants such as vitamins a-tocopherol vitamin E and retinol /vitamin-A/) represented in table 4. It showed that, in the blood plasma of rats control group had (due to the impact of CCl 4 ) a significant increase in the content of diene conjugates /DK/appearing at the initial stages FLOOR (table 4). So, on the 5th day the concentration of DK in the plasma significantly (p<0.001) increased 4.2 times compared with a group of intact rats, and on the 10th day returned to the original level (table 4).

Peptide composition in the dose of 100 mg/kg, did not significantly affect the DK. Composition in a dose of 600 mcg/kg and 5-day study significantly (p<0.001) reduced the content DK 1.5-3.2 times.

Drug comparison Heptral in a dose of 100 mg/kg also significantly (p<0.001) reduced DK 1.5-3.2 times.

On severity of actions in respect of DC plasma blood peptide composition (600 mcg/kg) significantly exceeded Heptral (100 mg/kg) on the days 5-7 1.2-1.7 times (p<0.05).

Also in the blood plasma was observed a significant increase of the contents of Schiff bases - secondary products of interaction of N-terminal residues of proteins, amino acids and amino groups of phospholipids with aldehydes, arising in the course of the reactions FLOOR (table 4). So, for example, on the 5th day the concentration of Schiff bases in plasma significantly (p<0.001) increased 6.5 times compared with a group of intact rats and on the 10th day exceeded the initial level in 1.2 times (p<0.001) (table 4).

Peptide composition in the dose of 100 mg/kg for 5 days did not significantly affect the level of Schiff bases, and on the 7th day significantly (p<0.05) reduced this figure 1.2 times. Composition in a dose of 600 mcg/kg and 5-day study significantly (p<0.001) reduced the content of Schiff bases 1.4-2.8 times.

Drug comparison Heptral in a dose of 100 mg/kg significantly (p<0.01) reduced level of Schiff bases 1.3-2.0 times.

On severity of actions regarding the content of Schiff bases blood plasma peptide composition (600 mcg/kg) significantly exceeded Heptral (100 mg/kg) on the days 5-7 1.1-1.4 times (p<0.001).

Table 4

Dynamics of some indicators of peroxide (peroxide) oxidation of lipids (Pol) in the blood rats with toxic CCl 4-hepatitis under the influence of peptide composition and drug comparison hepatoprotector due to treatment with Heptral

Indicator

In intact rats

Day study

Series

5-e (n=8)

7-th (n=8)

10-e (n=8)

Content of diene conjugates (DC), Rel. units

0,51±of 0.01 (n=22)

2,15±0,08 ooo

1,08±0,06 ooo

0,55±0,02

Control

1,95±0,10 ooo

0,97±0,09 ooo

0,44±0,05

PEPTIDE COMPOSITION (100 mcg/kg)

1,45±0,07 ***#

0,42±0,05 ***###

0,17±0,01 ***

PEPTIDE COMPOSITION (600 mcg/kg)

1,75±0,08 **

0,73±0,04 ***

0,17±0,02 ***

Heptral (100 mg/kg)

Schiff bases, Rel. units

20,2 ą 0.3 (n=22)

131,8±4,2 ooo

122,8 ħ 2.6 ooo

24,6 ą1.0 ooo

Control

118,5±5,8 ooo

106,2 ħ 4.9 *

22,8±1,7

PEPTIDE COMPOSITION (100 mcg/kg)

96,3±3,9 ***###

43,6±1,4 ***###

15,2±0,7 ***

PEPTIDE COMPOSITION (600 mcg/kg)

104,1±5,7 **

60,1±2,2 ***

15,9±0,8 ***

Heptral (100 mg/kg)

So, the animals of the control group dynamics of DC and Schiff bases indicates expressed destructive of free-radical oxidation of phospholipids membranes of hepatocytes. It should be emphasized that the peptide composition (600 mcg/kg) could substantially lessen the severity of these processes.

Thus, CCl 4 in rats causes of acute hepatitis with inflammatory destruction of the liver (confirmed by the data of morphological studies), as evidenced by zitoliz hepatocytes and violation of basic liver functions (synthetic and others), also noted cholestasis (increase activity of alkaline phosphatase, the content of free faction bilirubin, total bilirubin).

Peptide composition (600 mcg/kg) and Heptral (100 mg/kg), with a hepatoprotective action, normalizes the most biochemical indicators reflecting various liver function (ACT, ALT, alkaline phosphatase, total protein, albumin, total cholesterol, triglycerides), the degree of enzymatic toxemia (alpha-amylase), the level of the FLOOR (the content of DK and Schiff bases) for 7-10 days. The efficiency peptide composition (600 mcg/kg) exceeds or is not inferior drug comparison .

Morphological confirmation

The results of the above biochemical studies are supported by the data of morphological studies of the liver in rats.

In 5-10 days in all animals of the control group was observed characteristic picture: first celebrated stage fatty degeneration of hepatocytes, accurately delimited tumour localised and covering a considerable part of the cell, and then pathological changes in hepatocytes are amplified, which is expressed in the fatty degeneration of cells and their hydropic dystrophy. Is growth of connective frame. Reactive inflammation enters the phase of proliferation, as reflected in the absence of infiltration and thickened connective tissue around the blood vessels. In hepatocytes - hydropic degeneration and decay. Kernel many hepatocytes that testifies to the irreversibility of pathological changes and starting the cell death.

In the group of animals treated with comparator drug hepatoprotector Heptral, in all cases, there is pathology of hepatocytes (fatty infiltration), but its scale is smaller than in the control group. Hydropic degeneration of hepatocytes noted more often than in control, lysis of hepatocytes was not observed. The structure of the liver in all cases does not differ from the intact body except thickening connective tissue. Occurs to a moderate expansion of the connective tissue of the frame, but it is less pronounced than in the control group. One can make a conclusion about the good (or rather, moderate) action due to treatment with Heptral, the calling structure normalization of the liver after its damage CCl 4 .

In the liver the group of animals treated with the peptide composition at the dose of 100 mg/kg, has a similar pathological changes: expressed muscular dystrophy, extending from the the center of the lobules to the periphery, on a scale comparable to the control group more than the group receiving Heptral. Pathological changes involve infiltration cells of connective tissue, less pronounced than in the control group, but more than in the group receiving Heptral. Often added to this is epithelial hyperplasia ducts. Normalization expressed worse than in the group with . Intensive development of the connective tissue of the frame is expressed in the emergence of greater than in control, the number of connective tissue cells and clusters of small cell lung infiltrate, but the number of connective tissue fibers in this group did not increase as compared with the control, though exceeds those in the group with . Therefore, a peptide composition in the dose of 100 mg/kg cause a certain decrease in the severity of pathological processes in hepatocytes, but on a smaller extent than Heptral, i.e. has little (little) hepatoprotective action.

In the group of animals treated with the peptide composition in a dose of 600 mcg/kg, there was a significant decrease in the severity of pathological changes in hepatocytes compared with the control group, on a scale comparable with the group due to treatment with Heptral. Vascular reactions are more pronounced than in the above-mentioned groups, hyperplasia of connective frame expressed somewhat higher than in the group with , but considerably less than in control. Proliferation of the epithelium ducts better expressed than in the comparison groups.

Thus, on the basis of analysis of morphological changes in the liver tissue and directly in hepatocytes as well as the dynamics of the basic biochemical parameters of blood, reflecting the phenomenon syndromes of cytolysis and cholestasis, activation of lipid peroxidation and decrease the activity of antioxidant systems of the body, you can do the conclusion that the composition (600 mcg/kg) has a beneficial effect on the model of acute toxic hepatitis of animals. Effect of peptide composition (600 mcg/kg) is shown, in particular, reduction of , inhibition of the FLOOR, the improvement of the state of antioxidant system in reducing the severity of lesions on the level of hepatocytes, acceleration of the processes of regeneration of cells and activation of metabolism. These data indicate the effectiveness of the composition as a means of hepatoprotective on the model of acute toxic damage of liver. The efficiency composition (600 mcg/kg) exceeds or is not inferior drug comparison (100 mg/kg).

It should be emphasized that the action of the peptide song comes earlier for a 5-7 day, while the comparator drug - Heptral is most effective on the 10th day. This may be important when therapy in cases of acute hepatic lesions.

LITERATURE

3. V.T. Ivashkin, Lapina T.L., Baranskaya E.K. Rational pharmacotherapy of diseases of organs of digestion. - 2003. - M: «». - 1045 C.

4. V.T. Ivashkin, Ulanova I.M. Premature mortality in the Russian Federation and ways of its reduction. // ROS. Journe. . gepatol. koloproktol. - 2006. - T.16., №1. - P.8-14.

5. Kolesov PU, Markin A. Lipid peroxidation and methods of determining products of lipid peroxidation in biological environments. // Laboratory work. 1985. - №1. .540-546.

6. V.V. Menshikov (as amended). Laboratory methods of investigation in the clinic: a Handbook. - M, 1987. - 368 C.

7. World health statistics, 2010. The world health organization, 2010 - 177 C.

8. Saratikov A.S., vengerovsky A.I. New hepatoprotectors of natural origin. // Expert. and the wedge. . - 1995. - .58, №1. - P.8-11.

9. Smolyakova V.I., carpenters MB, Chernysheva, G.A. and others Gemoreologicakie effects lesion of the liver . // Expert. and the wedge. . - 2010. - .73, №8. - P.32-34.

10. Federal guidelines for the use of medicines ( system). Release XI // Under the editorship of .., .Б.Б, .. - M: «Echo», 2010. - 944 C.

1. The composition, which has hepatoprotective activity, consists of a mixture of peptides H-Lys Asp Glu-OH, H-Asp Glu-Pro-OH and H-Asp Glu Leu-HE taken on a weight basis (1-8):(1-8):(1-8).

2. Application of the composition of claim 1, to obtain a medicinal product, which has hepatoprotective activity.

3. Use according to claim 2, where the tool is in the form of pills, tablets can be sublingual prolonged action and covered, or in the form of capsules, or in the form of a spray nasal, or nasal drops, or aerosol or oral, or in powder form or in the form of solution for intravenous and intramuscular injection, or in the form of for preparation of a solution, or in the form of drops for reception inside.