GRIGORIEV, Evgeny Iosifovich (Zagrebsky bulvar, 5/1-105St.Petersburg, 4, 19228, RU)
MALININ, Vladimir Victorovich (Serebristy bulvar, 16-1-92St.Petersburg, 7, 19722, RU)
RYZHAK, Galina Anatolievna (ul. Partizana Germana 8/2-91, St.Petersburg, 5, 19820, RU)
KHAVINSON, Vladimir Khatskelevich (Bolshaya Raznochinnaya 3-21, St.Petersburg, 19711, RU)
GRIGORIEV, Evgeny Iosifovich (Zagrebsky bulvar, 5/1-105St.Petersburg, 4, 19228, RU)
MALININ, Vladimir Victorovich (Serebristy bulvar, 16-1-92St.Petersburg, 7, 19722, RU)
RYZHAK, Galina Anatolievna (ul. Partizana Germana 8/2-91, St.Petersburg, 5, 19820, RU)
CLAIMS
1. Peptide glutamyl-aspartyl-leucine with general formula H-GIu- Asp- Leu-OH sequence 1 [SEQ ID NOrI].
2. Peptide glutamyl-aspartyl-leucine with general formula H-GIu- Asp- Leu-OH sequence 1 [SEQ ID NO:1] capable of stimulating the regeneration of liver tissue.
3. Pharmaceutical composition stimulating the regeneration of liver tissue, containing active base and pharmaceutically acceptable carrier, characterized by the fact, that it contains an effective amount of peptide glutamyl-aspartyl-leucine of the general formula H-GIu- Asp- Leu-OH sequence 1 [SEQ ID NO:1] as its active base.
4. Pharmaceutical composition according to Claim 3, characterized by the fact that it exists in the form intended for parenteral administration.
5. Method of stimulation of liver tissue regeneration, which consists in the administration to the patient of the pharmaceutical composition containing peptide glutamyl-aspartyl-leucine with general formula H- Glu-Asp-Leu-OH sequence 1 [SEQ ID NO:1] as its active base in the dose of 0,01 - 100 μg/kg of body weight at least once a day during a period necessary for attaining the therapeutic effect.
6. Method according to Claim 5 characterized by the fact that such administration is performed parenterally. |
Peptide substance stimulating liver tissue regeneration, pharmaceutical composition on its base and the method of its application
The invention is related to medicinal means of treatment for liver diseases and may be used as a means of stimulating liver tissue regeneration.
Presently there are known preparations improving metabolic processes in the liver, including natural antioxidants, in particular B group vitamins (B 1 , B 6 , B 12 ), ascorbic acid, vitamin E 5 vitamin A; herbal preparations Liv.52 (The Comprehensive Russian Encyclopaedia of Medicinal Means, Moscow, Remedium publishing house, V.2, 2002 (rus.), p. 435).
There are known medicinal means of improving metabolism in liver cells, among which Silibinin is noteworthy (The Comprehensive Russian Encyclopaedia of Medicinal Means, Moscow, Remedium publishing house, V.2, 2002 (rus.), p. 719); as well as Sirepar (The Comprehensive Russian Encyclopaedia of Medicinal Means, Moscow, Remedium publishing house, V.2, 2002 (rus.), p. 722); Essentiale (FR Patent No. 2556050); and synthetic peptide preparations (RU Patent No. 2166957, 2000).
The known medications reveal only one-way effect, i.e. they improve metabolic processes in the liver and enhance its resistance against pathogenic impacts.
It is necessary to note, that no medications aimed at hepatocytes regeneration were found in the prior art.
All this points out the importance of designing new groups of medications, including biologically active peptide compounds, revealing the effect of liver tissue regeneration.
The claimed peptide substance does not have structural analogues in the prior art.
The claimed invention has set and resolved the task of obtaining the new peptide, possessing biological activity, which manifests itself in the stimulation of liver tissue regeneration.
Technical result of the invention consists in the creation of a new peptide, stimulating liver tissue regeneration, as well as a pharmaceutical composition, containing the new peptide as an active base, being used for stimulating liver tissue regeneration by means of restoring the synthesis of tissue specific proteins and normalization of liver cells functions.
This invention is related to the peptide glutamyl-aspartyl-leucine with general formula H-Glu-Asp-Leu-OH sequence 1 [SEQ ID NO: I].
Peptide glutamyl-aspartyl-leucine with general formula H-Glu-Asp-Leu-OH sequence 1 [SEQ ID NO:1], possesses the ability to stimulate the regeneration of liver tissue.
The other aspect of this invention is related to the pharmaceutical composition stimulating liver tissue regeneration, which contains an effective amount of peptide glutamyl-aspartyl-leucine with general formula H-GIu- Asp-Leu-
OH sequence 1 [SEQ ID NO:1] as its active base and a pharmaceutically acceptable carrier.
In this case the pharmaceutical composition exists in the form suitable for parenteral administration.
The next aspect of this invention is related to the method of stimulating liver tissue regeneration, consisting in the administration to the patient of the pharmaceutical composition containing an effective amount of peptide glutamyl- aspartyl-leucine with general formula H-Glu-Asp-Leu-OH sequence 1 [SEQ ID NO:1] in the dose of 0,01-100 μg/kg of body weight as its active base at least once a day during the period necessary for attaining the therapeutic effect.
In this case the administration is parenteral. Peptide glutamyl-aspartyl-leucine with general formula H-Glu-Asp-Leu-OH is obtained using the classical method of peptide synthesis in the solution.
The possibility of objective achievement of the technical result by the use of this invention is confirmed by reliable data displayed in examples showing the results of experimental studies performed using the methods common for this field. The stimulating effect of peptide H-Glu-Asp-Leu-OH on the regeneration of liver tissue has been revealed in the course of its experimental study.
The study of biological activity was performed on liver explants as well as in experimental models of partial hepatectomy and liver cirrhosis.
The notion "pharmaceutical composition" implies such different medicinal forms containing the new peptide, which may find therapeutic application as a means of stimulating liver tissue regeneration.
To obtain pharmaceutical compositions according to this invention, the effective amount of peptide H-Glu-Asp-Leu-OH as the active base (active substance) is mixed with a pharmaceutically acceptable carrier according to methods of compounding, which are traditional for pharmaceutics.
The notion "effective amount" implies the use of such amount of active base, which must be effective in this medicinal form according to the quantitative indices of its activity and toxicity, as well as basing on the knowledge of a specialist in this field.
The carrier may have different forms, depending on the medicinal form of the preparation desirable for the administration into the organism.
For parenteral administration the carrier usually includes physiological saline solution or sterile water, although other ingredients promoting stability or preserving sterility may also be included.
The subject matter of the invention is explained by a Figure and tables.
Table 1 shows the effect of peptide H-GIu- Asp-Leu-OH on the quantity of dividing cells in regenerating rat tissue in 32 and 96 hours after partial hepatectomy (% of total quantity of liver cells).
Table 2 displays the effect of peptide H-Glu-Asp-Leu-OH on blood biochemical indices in rats with experimental liver cirrhosis.
Table 3 displays the effect of peptide H-GIu- Asp-Leu-OH on total glycogen content (conv. units) in the liver of rats with experimental liver cirrhosis. Table 4 displays the effect of peptide H-GIu- Asp-Leu-OH on morphological and biochemical indices of guinea pig peripheric blood in the study of toxicity.
Table 5 displays the effect of peptide H-Glu-Asp-Leu-OH on biochemical indices of peripheric blood of patients with chronic hepatitis.
The Figure displays the effect of peptide H-Glu-Asp-Leu-OH on the development of liver explants.
The invention is illustrated by an example of synthesis of peptide glutamyl- aspartyl-leucine with general formula (H-GIu- Asp-Leu-OH) (Example 1), examples of peptide biological activity studies (Examples 2, 3, 4), example of toxicity study (Example 5), as well as by an example of peptide clinical
administration results, demonstrating its pharmacological properties and confirming the possibility of attaining the therapeutic effect (Example 6).
Example 1. Synthesis of peptide H-Glu-Asp-Leu-OH
1. Compound name: glutamy-aspartyl-leucine.
2. Structural formula: H-GIu-Asp-Leu-OH
3. Molecular formula without ion pair: C 15 H 25 N 3 O 7 .
4. Molecular weight without ion pair: 375,37.
5. Ion pair: acetate.
6. Appearance: white amorphous powder without smell.
7. Method of synthesis: the peptide is obtained using a classical method of synthesis in the solution by the following scheme:
H-Asp(OBzl)-OH
BOC-Glu(OBzl)-Asp(OBzl)-OH
H-Leu-OBzl DCC
BOC-Glu(OBzl)-Asp(OBzl)-Leu-OBzl
1) H 2 /Pt
2) TFA
H-Glu-Asp-Leu-OH
BOC - tert. butyloxycarbonyl group,
OSu - N-oxysuccinimide ester, DCC - NjN'-dicyclohexylcarbodiimide, OBzI - benzyl ester, TFA - trifluoracetic acid. Characteristics of the product:
• base substance content: 98,15 % (no B3)KX, 220 HM),
• TLC - individual, R f =0,65 (acetonitrile-water 1 :3),
• moisture content: 6 %, • pH of 0,001% solution: 4,57,
• Specific rotary power: [α] D 22 : -30° (c=l, H 2 O), "Polamat A", Carl Zeiss Jena.
Example of synthesis:
1) BOC-GIU(OBZI)-OSU, N-oxysuccinimide ester of N- tert.butyloxycarbonyl-(γ-benzyl)glutamic acid (I).
N-tert.butyloxycarbonyl-(γ-benzyl)glutamic acid BOC-GIU(OBZI)-OH (33,7 g, 0,1 mole) is dissolved in 50 ml of N,N'-dimethylformamide, cooled up to -1O 0 C; while stirring, cooled (4-6 0 C) solutions of N^'-dicyclohexylcarbodiimide (23,0 g, 0,11 mole) in 30 ml of N,N'-dimethylformamide and N-hydroxysuccinimide (13,0 g, 0,11 mole) in 20 ml of N,N'-dimethylformamide are added. The reactive solution is stirred for 12 hours, being cooled with ice, and then for 24 hours more at a room temperature. The fallout N,N'-dicyclohexylurea is filtered out, and the obtained solution of activated ester without extraction is used during the following stage. 2) BOC-GIu(OBzl)-Asp(OBzl)-OH, N-tert.butyloxycarbonyl.-(γ- benzyl)glutamyl-(β-benzyl)aspartate (II).
(β-benzyl)aspartic acid H-Asp(OBzl)-OH (28,0 g, 0,12 mole) and 36 ml (0,12 mole) of triethylamine is suspended in 50 ml of N,N'-dimethylformamide and stirred for 1 hour. Then activated ester BOC-GIU(OBZI)-OSU solution (I), obtained during the previous stage, is added. Reactive mixture is stirred at room temperature for 48 hours. Then it is acidified by 0,5 N sulfur acid up to pH 2-3 and extracted with ethyl acetate 4x50 ml. The extracts are united and subsequently washed with 0,5 N H 2 SO 4 3x50 ml, water 2x50 ml, 5% NaHCO 3 solution 2x50 ml,
water 2x50 ml, saturated NaCl solution 2x50 ml. Organic layer is dried over Na 2 SO 4 , solvent is removed in vacuo, and the residue is crystallized over hexane. 5O g of product (92%) are obtained. R f = 0,34 (benzene-acetone 2:1).
3) BOC-Glu(OBzI)-Asp(OBzI)-Leu-OBzl, benzyl ester of N- tert.butyloxycarbonyl-(γ-benzyl)glutamyl-(β-benzyl)asparty lleucine
(in).
Dipeptide BOC-Glu(OBzl)-Asp(OBzl)-OH (II) (1,20 g, 2,2 mmole) and N- oxybenzotriazol (0,4 g, 3 mmole) are dissolved in 5 ml of tetrahydrofuran and cooled up to -1O 0 C. Suspension Tosη-Leu-OBzl (1,38 g, 3,5 mmole) in 5 ml of tetrahydrofuran is added with 0,5 ml (3,5 mmole) of triethylamine and cooled again. N,N'-dicyclohexylcarbodiimide (0,62 g, 3,0 mmole) is dissolved in 5 ml of tetrahydrofuran and cooled. Cooled solutions are united and stirred for 24 hours while being cooled with ice. The fallout N,N-dicyclohexylurea is filtered out, the solvent is removed in vacuo. The residue is dissolved in 50 ml of ethyl acetate and subsequently washed with 0,5 N H 2 SO 4 2x20 ml, water 2x20 ml, 5% NaHCO 3 solution 2x20 ml, water 2x20 ml, saturated NaCl solution 2x20 ml. Organic layer is dried over Na 2 SO 4 , solvent is removed in vacuo, residue is crystallized over hexane. Re-crystallization from isopropyl spirit. 0,97 g of product (68%) are obtained. R f = 0,88 (benzene-acetone 1:1).
4) H-Glu-Asp-Leu-OH, glutamyl-aspartly-Ieucine.
The protected peptide BOC-Glu(OBzl)-Asp(OBzl)-Leu-OBzl (III) (0,90 g) is dissolved in the mixture of methyl spirit and water (4:1) and hydrated over catalyst
Pd/C (5%) for 4 hours. Catalyst is filtered out, solvent is removed in vacuo, residue is dried in vacuo over KOH and P 2 O 5 . Then the product is dissolved in 2 ml of mixture of chlorous methylene - trifluoracetic acid (5:1) and kept at room temperature for 2 hours. The fullness of deblocking reaction is controlled by TLC in acetonitrile-water (1 :3) system. The solvent is removed in vacuo, residue is dried in vacuo over KOH.
For purification 300 mg of the substance is dissolved in 4 ml of 0,01% trifluoracetic acid and subjected to highly productive liquid chromatography on reverse phase column 50x250 mm Diasorb-130-C16T, 7 μm. Chromatograph
Beckman System Gold, 126 Solvent Module, 168 Diode Array Detector Module.
Conditions of chromatography: A: 0,1% TFA; B: MeCN/0,1% TFA 5 gradient B 0 -> 50% in 100 minutes. Sample volume 5 ml, detection at 215 nm, scanning 190- 600 nm, flow rate 10 ml/min. Fraction 47,0-54,0 min was selected. Solvent is removed in vacuo at the temperature not higher than 40°C, removal is multiply (5 times) repeated with 10 ml of 10% acetic acid solution. In the end the residue is dissolved in 20 ml of deionized water and lyophilized.
196 mg of rectified substance is obtained in the form of amorphous white powder without smell. 5) Analysis of the ready substance.
• Base substance content was identified using the method of HPLC on the column Phenomenex C 18 LUNA 4,6x150 mm. A: 0,1% TFA, B: MeCN; grad.B 0-100% in 10 min. Flow rate 1 ml/min. Detection at 220 nm, scanning 190-600 nm, sample 20 μl. Base substance content 98,15%.
• TLC: individual, R f = 0,65 (acetonitrile-water 1:3, Sorbfil plates, silicagel 8-12 μm, developing with chlorine/benzidine).
• Moisture content: 6% (gravimetrically, judging by weight loss by drying 20 mg at 100 0 C). • pH of 0,001% solution: 4,57 (potentiometrically).
• Specific rotary power: [α] D 22 : -30° (c=l, H 2 O), "Polamat A", Carl Zeiss Jena.
Example 2. Effect of peptide H-Glu-Asp-Leu-OH on the development of liver explants
The experiments were performed on 27 fragments of Wistar rats liver with body weight 150-200 g. Nutritious medium for explants cultivation consisted of 35% Eagle's solution, 25% calf fetal serum, 35% Hanx solution, 5% chicken embryonic extract, with the addition of glucose (0,6%), insulin (0,5 units/ml), penicillin (100 units/ml), glutamine (2 mM). Liver fragments were placed into this medium and cultivated in Petri dishes in a thermostat at the temperature of 36,7 0 C for 48 hours. Peptide H-Glu-Asp-Leu-OH was added in the cultivation medium in concentrations 1, 10 H 100 ng/ml. Area index (AI), i.e. the ratio of total explant area together with the growth zone to the initial area of the explant, was a criterion
of biological activity. AI values were expressed in per cent, the control AI value being 100%.
The Figure shows the effect of peptide H-Glu-Asp-Leu-OH on the development of liver explants. It was found, that after 24 hours of cultivating the explants were spread over the collagen base, and proliferating and migrating cells began to settle down on the periphery of the explant. By the 3 rd day of cultivating in case of peptide H-GIu- Asp-Leu-OH concentration of 10 ng/ml there was observed a reliable increase in the AI of explants by 28% as compared to control AI values. The study of liver explants after longer terms of cultivation (7 days) revealed a similar stimulating effect of peptide H-GIu- Asp-Leu-OH in the same concentration.
Thus, it was shown, that peptide H-Glu-Asp-Leu-OH exerts a tissue specific effect on liver tissue cells, which is manifested in the stimulation of explants growth.
Example 3. Effect of H-Glu-Asp-Leu-OH peptide on liver compensatory regeneration after partial hepatectomy in rats
The study was conducted on 18 white mongrel male rats with body weight of 150-200 g. The animals were divided into 3 groups: healthy rats in the first one, control rats (subjected to partial hepatectomy with removal of 2/3 of the liver) in the second one and operated animals treated with H-Glu-Asp-Leu-OH peptide in the dose of 0,1 μg in 0,5 ml of sterile 0,9% NaCl solution per rat in 2 and 24 hours after the surgery, in the third group. The animals in the first and the second groups were injected with sterile physiological saline solution in the same volume and by the same schedule.
Animals subjected to the surgery were slaughtered with ether in 32 and 96 hours after the surgery. Control animals were also slaughtered. Their liver was fixed in formalin. Histological preparations were stained with haematoxylin-eosin, then assessed mitotic index of the liver cells and the number of polyploid cells in S- phase of the cellular cycle (the number of dividing cells) were assessed.
Studies of the cellular mitotic activity of the regenerating liver in 32 hours after partial hepatectomy revealed that the number of mitoses and cells in S-phase of the cellular cycle becomes 2-fold higher then in the liver of healthy animals.
These differences are not statistically reliable in case of sterile physiological saline solution administration, while after H-Glu-Asp-Leu-OH peptide injections there was registered an increase in the number of mitoses, DNA synthesizing cells and total amount of the dividing cells is reliable (Table 1). Studies of the preparations in 96 hours after hepatectomy showed that both in rats treated with physiological saline solution and animals treated with H-Glu-Asp- Leu-OH peptide, revealed significant intensification of hepatocytes mitotic activity. The comparison of the results in the experimental (third) group and control (second) group showed that rats treated with H-Glu-Asp-Leu-OH peptide the number of mitoses is twice higher than in rats administered with physiological saline solution. The number of cells in the S-phase of the mitotic cycle in rats of the experimental group did not reliably differ from the number of hepatocytes in S- phase in the control, although in general the number of dividing cells in 96 hours after hepatectomy in regenerating liver of the rats treated with H-Glu-Asp-Leu-OH peptide was by 75% higher than that in rats administered with physiological saline solution (Table 1).
Thus, it was revealed that H-Glu-Asp-Leu-OH peptide administration to rats in 96 hours after partial hepatectomy intensified the mitotic activity of hepatocytes, evidencing the intensification of reparative processes in the liver.
Example 4. Efficacy of H-Glu-Asp-Leu-OH peptide application in rats with experimental liver cirrhosis
The study was conducted on 45 white mongrel male rats, with body weight 120-180 g before the experiment onset. The animals were subjected to inhalations of CCl 4 for 6 months. Half of the animals were injected with H-GIu- Asp-Leu-OH peptide in the dose of 0,5 μg per rat in 0,5 ml of sterile NaCl solution every month in courses of 5 consequent days. The animals of the control group received sterile physiological saline solution by the same schedule. The assessment of the liver status was performed using the results of the biochemical analysis: protein concentration, bilirubin, cholesterol, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) indices.
The content of total glycogen in hepatocytes was assessed using cytofluorimetric analysis in smears after the fluorescent PAS-reaction.
Glycogen content is one of the most important indicators of hepatocytes functional activity. It is known that liver cirrhosis is characterized by severely disordered hepatocytes function of glycogen production.
The study revealed that after the CCl 4 inhalations the animals showed significant liver disorders. There was registered a decrease in the total protein content, bilirubin and cholesterol level. The activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was distinctly increased (Table 2).
In case of concurrent administration of CCl 4 and H-GIu- Asp-Leu-OH peptide biochemical indices were restored up to normal ones, which shows that liver destruction in the animals of the main group was less pronounced than that in the control (Table 2).
These data evidence that H-Glu-Asp-Leu-OH peptide constrains the development of the pathologic process in the liver. It reduces fermentemia, limits hepatocellular insufficiency and thrombohemorrhagic syndrome, enhancing regeneration foci in the liver.
Against the background of the hepatotropic poison effect the content of the total glycogen in hepatocytes increases 2-fold in the average (Table 3).
It was revealed that after H-GIu- Asp-Leu-OH peptide administration the high glycogen content decreases almost to the normal level. Thus, the data confirm the positive effect of H-GIu- Asp-Leu-OH peptide on glycogen production by hepatocytes in the cirrhotic liver.
Example 5. Study of peptide H-Glu-Asp-Leu-OH for toxicity
Common toxicity of peptide H-Glu-Asp-Leu-OH was studied according to the requirements stated in the "Manual for experimental (pre-clinical) study of new pharmacological substances" (2000): acute toxicity in case of single administration of the substance and sub-acute and chronic toxicity in case of long-term administration of the peptide.
The study of acute toxicity was performed on 66 white mongrel male mice with body weight of 20-22 g. The animals were randomly subdivided into 6 equal groups. The substance was administered to the animals once, intramuscularly, in the doses of 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg in 0,25 ml of sterile 0,9% NaCl solution. The control animals received 0,9% NaCl solution in the same volume.
The study of sub-acute toxicity was performed on 55 white mongrel male rats with body weight of 140-180 g. Experimental animals received the substance daily, intramuscularly for 90 days in the doses of 1 μg/kg, 0,1 mg/kg, 1 mg/kg in 0,5 ml of sterile 0,9% NaCl solution. Control animals received sterile 0,9% NaCl solution in the same volume. Morphology and properties of the animals' peripheric blood were studied before the administration of the substance, as well as on the 30* , 60* and 90* day after the beginning of the administration. Upon completion of the experiment biochemical and coagulologic indices of the blood were also evaluated.
The studies of chronic toxicity were conducted for 6 months, basing on the term of recommended clinical administration of the substance, on 88 male guinea pigs with body weight of 260-300 g. Experimental animals received the peptide daily, once a day, intramuscularly, for 6 months in the doses of 1 μg/kg, 0,1 mg/kg, 1 mg/kg in 0,5 ml of sterile 0,9% NaCl solution. Control animals received sterile 0,9% NaCl solution in the same volume and by the same schedule. Traditional methods were used for the evaluation of the following indices of the animals' peripheric blood: the quantity of erythrocytes, hemoglobin, reticulocytes, thrombocytes, leukocytes, leukocyte formula, erythrocyte sedimentation rate (ESR), erythrocyte resistance. Alongside with that, the content of total protein in the serum was identified using Lo wry 's method, as well as potassium and sodium content using the method of plasma spectrophotometry. After the completion of the experiment pathomorphologic studies of animal brain and spinal cord, spinal cord ganglia, thyroid gland, parathyroid glands, adrenal glands, testis, pituitary body, heart, lungs, aorta, liver, kidneys, urinary bladder, pancreas, stomach, small intestine, large intestine, thymus, spleen, lymph nodes and bone marrow were performed.
The study of acute toxicity showed, that a single administration of the studied peptide to animals in the dose exceeding the therapeutic one, which is recommended for clinical administration, by more than 5000 times, does not cause toxic reactions, which points out the width of diapason of possible therapeutic doses of the substance.
The study of sub-acute and chronic toxicity of the peptide showed the absence of any side effects in case of long-term administration of the substance in doses exceeding the therapeutic one by 100-1000 times. The study of peptide effect on guinea pig blood morphology and biochemical indices in 3 and 6 months after
the beginning of the substance administration showed that no statistically significant changes in the studied indices took place (Table 4).
The evaluation of the animals' general state, of morphologic and biochemical indices of peripheric blood, of morphological status of the organs, of the status of cardiovascular and respiratory systems, as well as of liver and kidney functions revealed no pathologic alterations in the organism.
The absence of common toxicity allows to recommend the pharmaceutical composition, containing the peptide H-Glu-Asp-Leu-OH as its active base, for clinical studies.
Example 6. Efficacy of H-Glu-Asp-Leu-OH peptide application in patients with chronic hepatitis
The study was conducted on 34 patients with chronic hepatitis aged from 30 to 56, who were randomly divided into 2 groups. The patients of the main group were subdivided into 3 subgroups according to the stage of the disease. Patients with chronic hepatitis in the acute phase were treated with pharmaceutical composition containing peptide H-GIu- Asp-Leu-OH in case of high severity of the disease in the dose of 5,0 mg in 1,0 ml of sterile 0,9% physiological solution intramuscularly, once a day for 10 days, and in case of medium severity of the disease - in the dose of 10,0 μg in 1,0 ml of sterile 0,9% physiological solution intramuscularly, once a day for 10 days. Chronic hepatitis patients in remission were treated with pharmaceutical composition containing peptide H-GIu- Asp-Leu-OH, in the dose of 1,0 μg in 1,0 ml of sterile 0,9% physiological solution intramuscularly, once a day for 10 days. Control group consisted of 15 patients treated by conventional methods.
The patients' complaints and blood biochemical indices were assessed in dynamics.
During the treatment course 89% of the main group patients reported reduced asthenia, increased appetite and working capacity, and in 53% of the patients the intensity of pain syndrome significantly decreased.
In the course of the patients examination most attention was paid to the assessment of the biochemical studies results, defining aminotransferase activity, pigment and protein producing function of the liver.
81% of the examined patients reported hyperbilirubinemia and increased level of alanine aminotransferase (ALT), which shows that there was a certain chronic inflammatory process.
The administration of the pharmaceutical composition containing H-GIu- Asp-Leu-OH peptide in different doses contributed to the normalization of bilirubin level and ALT activity, which confirms the normalization of metabolic processes in the liver, decreased inflammatory process activity and restored tissue specific proteins synthesis by liver cells.
Thus, the obtained results show that pharmaceutical composition, containing H-GIu- Asp-Leu-OH peptide, stimulates liver tissues regeneration. Moreover, they confirm the expedience of application of the pharmaceutical composition containing H-GIu- Asp-Leu-OH peptide in complex treatment of chronic hepatitis in the acute phase and in remission.
Table 1
* - P < 0,001 as compared to the index before surgery; ** - P < 0,05 as compared to the index before surgery; & - P < 0,001 as compared to the index in the control;
# - P < 0,05 as compared to the index in the control.
Table 2
* - P<0,05 as compared to the index in healthy animals;
** - P<0,05 as compared to the index in the corresponding control group.
Table 3
* - P<0,05 as compared to the index in healthy animals; ** - P<0,05 as compared to the index in the control.
Table 4
Table 5
* - P<0,05 as compared to the index before treatment. # - P<0,05 as compared to the indices before treatment using conventional means and methods.
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