RU2301071C1 - Hepatoprotective agent and method for production thereof - Google Patents

Abstract

FIELD: biologically active substances, medicine.

SUBSTANCE: claimed agent for parantheral administration represents peptide complex containing 70-90 % of low molecular fraction comprising peptide components with molecular mass 70-184 Da, and peptide concentration of 2.5-2.9 mg/ml. Said agent is obtained from liver of calf not older than 12 months or hog by extraction with acetic acid in presence of zinc chloride. Calf or hog liver are frozen at not less -40°C, conditioned at -20-22°C for at least two months, and ground. Then 3 % acetic acid solution in volume ratio of 1:5 is added at 20±5°C. Extraction is carried out under continuous stirring to produce homogenous slurry. Then 1 % zinc chloride solution is added into slurry in volume ratio of 50:1; mixture is cooled under continuous stirring to 7-16°C; stirred during 1 h after each 4 h defecation for 48 hours. Extract is separated from ballast substances; acetone is added to extract in volume ratio of 1:5, followed by conditioning at 3-5°C for 4 hours. Obtained homogenized deposition is deposited again with acetone two time or more. Further active substance containing precipitate is washed on gravity filter with two-fold volumes of acetone cooled to 7-16°C to produce light-gray precipitate. Precipitate is passed through metal sieve, dried, dissolved in distilled water at room temperature and continuous stirring to produce polypeptide concentration of 2.5-2.9 mg/ml. Solution is centrifuged, filtered, subjected to ultrafiltration purification under back pressure of 1.0 kgf/cm² or less trough materials with retentiveness of 15000 Da. Glycocol is added into ultrafiltrate up to finish concentration of 10-20 mg/mg at pH 5.6-6.6. Solution is subjected to sterilizing filtration under pressure of 2.0 kgf/cm² or less, poured in 2 ml ampoules, and autoclaved for 8 min, at 120°C and atmospheric pressure of 1.1 kgf/cm².

EFFECT: method of increased yield, non-toxic and apirogenic agent of improved purity.

2 cl, 5 ex, 4 tbl, 1 dwg

Description

The group of inventions relates to a medicinal product and a method for its preparation from animal organs, in particular to the isolation of a biologically active substance from the liver and the preparation of a parenteral dosage form that can be used in medicine as a hepatoprotective agent.

Known methods for producing biologically active substances and medicines from animal raw materials (RF patent No. 944191, 1994; A.S. SU No. 1112606, 1996; A.S. SU No. 1218521, 1994; A.S. SU No. 1227198, 1986; RF patent 1298879, 1993; RF patent No. 1448443, 1994; RF patent No. 2075944, 1997; RF patent No. 2104702, 1998; RF patent No. 2161501, 2001; RF patent No. 2163129, 2000; US patent 4341765, patent GB 1161896, 1969 ; patent FR 2583982, 1987).

Known substance with hepatoprotective activity, and a method for its preparation from the liver of slaughter animals (RF patent No. 1522486, 1993), which is the closest analogue prototype for the proposed hepatoprotective agent and method for its preparation.

According to the method described in RF patent No. 1522486, polypeptides are extracted from the liver homogenate of slaughter animals with acetic acid at pH 3.5 for 12 ÷ 24 hours at 1 ÷ 5 ° C with the addition of 1 ÷ 1.5 g / l of zinc chloride; polypeptide precipitation is carried out with acetone in an amount of from 6 volumes of acetone per 1 volume of extract to 10 volumes of acetone per 1 volume of extract at a temperature of minus 10 ÷ minus 15 ° C, the precipitate is separated by filtration, dried, and then redissolved in a solution of acetic acid at pH 3, 5 ÷ 4.0; then filtered, and the filtrate lyophilized.

The yield of active substance (i.e., a substance having hepatoprotective activity) is 28 g per 1 kg of feedstock.

The disadvantages of this method include the extraction during the extraction process from the liver tissue of a large amount (more than 70%) of non-peptide substances, which, being impurities, do not determine the biological activity of the isolated active substance, as well as its low yield.

The present invention posed and solved the problem of developing a method for producing a hepatoprotective agent in the form of a parenteral dosage form isolated from the liver of calves no older than 12 months of age or pigs, the technical result of which is the optimal technology for the isolation of the peptide complex with a low molecular weight fraction of 70 to 90% with a molecular weight of its peptide components ranging from 70 to 184 Yes and obtaining an aqueous solution of the extract with a concentration of polypeptides 2 , 5 ÷ 2.9 mg / ml, which allows not only to clean the resulting product from impurities, but also to increase its yield.

Experimental verification showed that the proposed method provides pharmaceutical stability, maximum biological value and therapeutic efficacy of a hepatoprotective agent made in the form of a parenteral dosage form, which is confirmed by the experimental results given in the examples illustrating the invention.

Another aspect of the invention is a hepatoprotective agent in the form of a parenteral dosage form, which is a peptide complex with a low molecular weight fraction from 70 to 90% with a molecular weight of its peptide components ranging from 70 to 184 Da, with a polypeptide concentration of 2.5 ÷ 2.9 mg / ml, the technical result of which is that the isolated substance differs from known substances obtained previously from animal raw materials in molecular weight (from 500 to 1500 Da) of the peptides included in it ide components (RF patents Nos. 1298879, 2104702, 2163129), as well as its non-toxicity and pyrogen-free nature due to complete purification from impurities.

In the course of studies in animal experiments, it was found that the obtained aqueous solution of the agent with a concentration of polypeptides of 2.5 ÷ 2.9 mg / ml is a therapeutically effective dose, which allows us to consider the use of the agent for various types of this pathology. This is confirmed by the following examples.

In the process of conducting research, the importance of the following stages of the method for producing a hepatoprotective agent made in the form of a dosage form for parenteral administration (hereinafter referred to as the drug) was established:

- repeated precipitation of the resulting homogenized precipitate with double volumes of acetone at least two times and subsequent washing on the suction filter with double volumes of acetone until a light gray precipitate is obtained, which indicates complete purification of the precipitate from impurities such as lipid, protein, phospholipid and others;

- obtaining an aqueous solution of the extract in a concentration of polypeptides of 2.5 ÷ 2.9 mg / ml; its centrifugation, filtration, followed by ultrafiltration cleaning at the installation with a back pressure of not more than 1.0 kgf / cm ² through materials with a retention capacity of 15,000 Da and the addition of glycocol to the ultrafiltrate to a final concentration of 10 ÷ 20 mg / ml at pH 5.6 ÷ 6, 6;

- sterilizing filtration, ampouling and autoclaving for 8 minutes at a temperature of 120 ° C and atmospheric pressure of 1.1 kgf / cm ² , which ensures the stability of the drug while maintaining therapeutic efficacy.

The mode and time of autoclaving were established experimentally.

The specified technical result is achieved in that the hepatoprotective agent is in the form of a parenteral dosage form and is a peptide complex with a low molecular weight fraction from 70 to 90% with a molecular weight of the peptide components included in it ranging from 70 to 184 Da, with a concentration of polypeptides 2.5 ÷ 2.9 mg / ml and obtained from the liver of calves not older than 12 months of age or pigs by extraction with acetic acid in the presence of zinc chloride.

The specified technical result is also achieved by the fact that the proposed method for producing a hepatoprotective agent is characterized in that the liver of calves not older than 12 months of age or pigs are frozen at a temperature of at least minus 40 ° C, kept at a temperature of minus 20 ÷ 22 ° C for at least two months, then crushed, add a 3% solution of acetic acid in a volume ratio of 1: 5 at a temperature of 20 ± 5 ° C, the extraction is carried out with constant stirring, after obtaining a homogeneous suspension, add a 1% solution of chlorine of zinc in a volume ratio of 50: 1, cooled with constant stirring to a temperature of 7 ÷ 16 ° C, then stirred for 1 hour after every 4 hours of settling for 48 hours, the extract is separated from the ballast substances by separation, acetone is added to the extract in a volume ratio 1: 5, kept at a temperature of 3 ÷ 5 ° C for 4 h, the resulting homogenized precipitate is re-precipitated with acetone at least 2 times, then the precipitate containing the active substance is washed on a suction filter with twice volumes cooled to a temperature of 7 ÷ 16 ° C ac until a light gray precipitate is obtained, it is wiped through a metal sieve, dried, dissolved in distilled water at room temperature and constant stirring to a concentration of polypeptides of 2.5–2.9 mg / ml, the solution is centrifuged, filtered, and subjected to ultrafiltration purification on a plant with a backpressure of not more than 1.0 kgf / cm ² through materials with a retention capacity of 15,000 Yes, glycol is added to the ultrafiltrate to its final concentration of 10 ÷ 20 mg / ml at pH 5.6 ÷ 6.6, the solution is subjected to sterilizing filtration according to d pressure not more than 2.0 kgf / cm ² , poured into ampoules of 2 ml and autoclaved for 8 minutes at a temperature of 120 ° C and atmospheric pressure of 1.1 kgf / cm ² .

The invention is illustrated by tables and drawing.

The drawing shows the effect of the drug on the development of liver explants.

Table 1 shows the effect of the drug on the morphological and biochemical parameters of the peripheral blood of guinea pigs in the study of toxicity.

Table 2 shows the effect of the drug on biochemical blood parameters in rats with experimental cirrhosis.

Table 3 shows the effect of the drug on the total glycogen content (conventional units) in the liver of rats with experimental cirrhosis.

Table 4 shows the effect of the drug on the biochemical parameters of the peripheral blood of patients with chronic hepatitis.

The invention is illustrated by the following examples: example 1 - a method of obtaining a preparation; example 2 - the study of the toxicity of the drug; examples 3 and 4, confirming the hepatoprotective activity of the drug; example 5, confirming the therapeutic effectiveness of the drug, made in the form of a dosage form for parenteral administration.

Example 1. The method of obtaining the drug

As raw materials, the liver of calves (not older than 12 months of age) or pigs is used, which is frozen at a temperature of at least minus 40 ° C and kept at a temperature of minus 20 ÷ 22 ° C for at least two months.

300 l of a 3% solution of acetic acid are pumped into the extraction reactor and cooled to a temperature of 20 ± 5 ° C, then 60 kg of crushed material are loaded into the reactor with constant stirring until a homogeneous mass of raw material is obtained. The extraction is carried out with constant stirring, after obtaining a homogeneous suspension, a 1% solution of zinc chloride in a volume ratio of 50: 1 is added to it, cooled with constant stirring to a temperature of plus 7 ÷ 16 ° C, then it is stirred for 1 hour after every 4 hours of settling for 48 h

The extract is separated from the ballast substances by separation at 5000 ± 500 rpm for 1 hour. Acetone in a volume ratio of 1: 5 is added to the extract. It is kept at a temperature of plus 3-5 ° C for 4 hours. The precipitate formed is homogenized and re-precipitated with acetone at least two times. Then the precipitate containing the active substance is washed on a suction filter with twice volumes of acetone cooled to a temperature of 7-16 ° C until a light gray precipitate is obtained. The washed precipitate is wiped through a metal sieve, spread in a thin layer into enameled cuvettes, covered with a double layer of cotton cloth and dried with periodic stirring in a fume hood until the smell of acetone is completely removed.

The output of the active substance (powder of a biologically active peptide complex isolated from the liver) is 70 g per 1 kg of feedstock.

The resulting powder is dissolved in distilled water with constant stirring at room temperature for 40 min to a concentration of peptides 2.5 ÷ 2.9 mg / ml The resulting solution was centrifuged at 3000 ± 200 rpm for 20 ± 5 minutes. The centrifuge is filtered through an AP-15 filter or similar.

The filtrate is subjected to ultrafiltration purification in an ultrafiltration unit with a backpressure of not more than 1.0 kgf / cm ² through materials with a retention capacity of 15,000 Da.

A calculated sample of glycocol is added to the ultrafiltrate, to its final concentration of 10–20 mg / ml, mixed until the glycol is completely dissolved while maintaining a pH of 5.6–6.6.

The solution is subjected to sterilizing filtration, which is carried out under pressure not exceeding 2.0 kgf / cm ² .

The resulting solution is poured into 2.0 ml ampoules and autoclaved, and the ampoules with the preparation are sterilized for 8 minutes at a temperature of 120 ° C and atmospheric pressure of not more than 1.1 kgf / cm ² .

The preparation is a colorless, transparent solution and contains a peptide complex with a concentration of polypeptides of 2.5 ÷ 2.9 mg / ml.

Testing for the absence of high molecular weight protein components is carried out by adding to the contents of 1 ampoule of the drug 1 ml of a 10% solution of trichloroacetic acid. The transparency of the solution indicates the absence of high molecular weight protein components.

To determine peptide bonds in a preparation, a biuret reagent is added to its solution. Staining the solution with violet color indicates the peptide bonds present in the preparation.

In order to determine the polypeptides and their fractions in the preparation, the methods of ultraviolet spectrophotometry, gel chromatography, mass spectrometry, high performance liquid chromatography and polyacrylamide gel electrophoresis are used.

The ultraviolet spectrum of the drug solution is recorded in the region of wavelengths from 250 to 350 nm: the absorption maximum is observed at a wavelength of 270 ± 5 nm.

The molecular weight of the polypeptides that make up the drug is determined by the following methods:

by gel chromatography on Sephadex G-25 and G-50 (Pharmacia, Sweden). Peptide Molecuar Weight Kit MS III (Serva, Germany) was used to calibrate a 1.6 x 60 cm column;

mass spectrometry method. Spectra are obtained on a Voyager DE Biospectrometry time-of-flight mass spectrometer with laser desorption and ionization using a matrix (MALDI-TOF) at a relative intensity of a nitrogen laser of 2300 ÷ 2400, an accelerating voltage of 25000 V, a delay time of 90 ns, and a pressure in the vacuum chamber of 2.6 × 10 ⁶ torr;

by electrophoresis in a 15% polyacrylamide gel in comparison with a standard set of marker proteins.

Using the methods listed above, it was found that the composition of the drug includes polypeptides with a molecular weight of from 70 to 184 Da.

Using reverse-phase high-performance liquid chromatography in an acetonitrile gradient (Lichrosorb C18 sorbent, column 2 × 62 mm), it was found that the preparation contains mainly low molecular weight fractions - from 70 to 90%, and high molecular weight components are absent in the preparation.

Pyrogenicity of the drug is determined in rabbits by the generally accepted method (GF XI, issue 2, p. 183) with a test dose of 0.25 mg per 1 kg of animal weight in 1.0 ml of isotonic 0.9% sodium chloride solution for injection. It is shown that the drug is pyrogen-free.

Thus, in the above method, a preparation was obtained - a parenteral dosage form, which is a peptide complex with a low molecular weight fraction from 70 to 90%, with a molecular weight of its peptide components ranging from 70 to 184 Da, with a concentration of 2.5 polypeptides ÷ 2.9 mg / ml.

Example 2. The study of the toxicity of the drug

The general toxic effect of the drug was investigated 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 drug.

A study of acute toxicity was conducted on 72 white mongrel male mice with a body weight of 20–22 g. Animals were randomly divided into 6 equal groups. The drug was administered to animals once intramuscularly at doses of 35, 50, 100, 150, 200 mg / kg in 0.25 ml of sterile 0.9% NaCl solution. Animals of the control group were injected with the same volume of 0.9% NaCl solution.

Studies on the study of subacute toxicity were carried out on 65 white outbred male rats weighing 150 ÷ 180 g. Daily, once a day, animals of the experimental groups were injected intramuscularly with doses of 1, 10, 100 mg / kg in 0.5 ml of sterile 0 9% NaCl solution. The animals of the control group were injected in the same volume with a sterile 0.9% NaCl solution. 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 prescription of the drug in 84 male guinea pigs with a body weight of 250–280 g. Animals of the experimental groups received intramuscular preparation once daily for 6 months at doses of 1 mg / kg, 10, 100 in 0.5 ml of a sterile 0.9% NaCl solution. In the control group, animals were injected in a similar fashion with a sterile 0.9% NaCl solution in the same volume. In animals, peripheral blood was determined by conventional methods: the number of red blood cells, hemoglobin, reticulocytes, platelets, white blood cells, white blood cell count, erythrocyte sedimentation rate (ESR), and erythrocyte resistance. 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 was completed, 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, thymus, spleen, lymph nodes, bone marrow.

When studying acute toxicity, it was found that a single administration of the studied drug to animals in 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 drug indicates the absence of side effects with prolonged use of the drug in doses exceeding the therapeutic 300-3000 times. When studying the effect of the drug on the morphological composition and biochemical parameters of the peripheral blood of guinea pigs after 3 and 6 months after the start of its administration, no significant change in the indicators was revealed (Table 1).

When assessing the general condition of animals, morphological and biochemical parameters of peripheral blood, the morphological state of internal organs, the state of the cardiovascular and respiratory systems, and liver and kidney function, pathological changes in the body were not detected.

Therefore, the drug obtained by the proposed method, with long-term administration to animals does not have toxic properties that prevent its further use as a medicine, made in the form of a dosage form for parenteral administration.

Example 3. The effect of the drug on the development of liver explants

The experiments were performed on 27 fragments of the liver of Wistar rats weighing 150–200 g. The culture medium for explant cultivation consisted of 35% Eagle's solution, 25% fetal calf serum, 35% Hanks' solution, 5% chicken embryonic extract, on Wednesday glucose (0.6%), insulin (0.5 units / ml), penicillin (100 units / ml), glutamine (2 mM) were added. Liver fragments were placed in this medium and cultured on a collagen substrate in Petri dishes in a thermostat at a temperature of 36.7 ° C for 2 days. The preparation was added to the experimental medium at concentrations of 1, 10, and 100 ng / ml.

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%.

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 drug concentration of 10 ng / ml, a significant increase in the explant PI by 28% was observed compared with the control PI values.

The drawing shows the effect of the drug on the development of liver explants.

When studying liver explants for longer periods of cultivation (7 days), a similar stimulating effect of the drug at the same concentration was revealed.

Thus, in relation to liver tissue, the drug had a tissue-specific effect, manifested in stimulating the growth of explants.

Example 4. The effect of the drug on the functional activity of hepatocytes in experimental liver 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 (CCl ₄ ) for 6 months.

24 animals comprised the experimental group: half of the animals were injected intraperitoneally with a preparation isolated from the liver of the claimed method, 1.0 mg each, and the other half, 5.0 mg per injection per month for 5 consecutive days in order to select a therapeutically effective dosage.

Animals of the control group (n = 21) received injections of saline according to a similar scheme.

The liver condition in animals was evaluated according to biochemical analysis, determining the concentration of protein, bilirubin, cholesterol, alanine aminotransferase (ALT) and aspartate aminotransferase (ACT).

The content of total glycogen in hepatocytes was determined cytofluorimetrically in smear preparations after the fluorescence PAS 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 CCl 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 1).

With the simultaneous administration of CCl ₄ and the study drug, biochemical parameters were restored to normal values and indicated a less pronounced liver destruction (Table 1).

The data presented indicate that the studied drug in both dosages 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 2). It was found that after the use of the drug in both dosages, the increased glycogen content decreases almost to the norm.

Thus, the obtained data showed a positive effect of the action of the drug isolated from the liver of the claimed method in two doses on the glycogen-forming function of hepatocytes of a cirrhotically altered liver, indicating the restoration of the metabolic viability of organ tissue and the hepatoprotective activity of the drug.

Example 5. The effectiveness of the drug in patients with chronic hepatitis

The study was conducted with the participation of 35 patients aged 32 ÷ 53 years. The duration of the disease ranged from 10 to 20 years. 79% of patients had a history of viral hepatitis A, 11% had viral hepatitis B.

Most patients complained of pain in the right hypochondrium, general weakness and fatigue, in 45% of patients dyspeptic disorders were noted.

All patients previously received conventional drugs.

All patients by randomization were divided into 2 groups. 23 patients who made up the main group were injected with a dose of 5 mg in 2 ml of saline intramuscularly once daily for 10-15 days, depending on the severity of the disease.

The control group consisted of 12 similar patients who were prescribed conventional treatment.

The complaints of patients were evaluated in dynamics, a general clinical study of blood and urine was performed, a biochemical blood test was performed on a REFLOTRON apparatus (Boehringer Mannheim, Germany), and an immunological study of peripheral blood (determination of immunoglobulins according to Mancini). Ultrasound examination of the liver was carried out on an ultrasound machine (ALOKA, Japan).

Against the background of the treatment with the use of the studied drug, 93% of patients noted the disappearance of weakness, increased appetite and performance. In 51% of patients, the intensity of the pain syndrome significantly decreased.

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

72% of the examined patients had hyperbilirubinemia, an increase in the level of alanine aminotransferase (ALT) and a slight increase in the γ-globulin fraction of blood proteins, mainly due to IgM, which indicates a certain activity of the chronic inflammatory process.

The use of the drug contributed to the normalization of bilirubin levels and ALT activity (table 3).

The results of the study showed that the use of the drug in patients with chronic hepatitis helps to normalize metabolic processes in the liver, reduce the activity of the inflammatory process and prevents the death of hepatocytes.

Thus, the results of a clinical study of the effectiveness of the drug indicate its hepatoprotective properties and the appropriateness of its use in the complex treatment of chronic hepatitis in the phase of exacerbation and remission in a therapeutically effective dose of 5 mg per injection (2.5 mg / ml) intramuscularly once daily for 10 ÷ 15 days.

Table 1 Hepatoprotective agent and method for its preparation Indicator The introduction of the drug (10 mg / kg) 3 months 6 months Control (n = 21) Drug (n = 21) Control (n = 21) Drug (n = 21) Red blood cells, × 10 ¹² / l 5.2 ± 0.5 5.3 ± 0.6 5.4 ± 0.6 5.3 ± 0.4 Hemoglobin, g / l 14.1 ± 2.5 14.3 ± 2.4 14.2 ± 1.9 14.3 ± 2.2 Reticulocytes,% 1.2 ± 0.06 1.2 ± 0.08 1.3 ± 0.03 1.1 ± 0.08 Platelets × 10 ⁹ / L 144.3 ± 6.7 145.2 ± 7.3 143.4 ± 7.1 145.5 ± 6.3 White blood cells × 10 ⁹ / L 9.5 ± 0.1 9.6 ± 0.3 9.5 ± 0.2 9.7 ± 0.1 Band neutrons,% 0.28 ± 0.06 0.32 ± 0.08 0.29 ± 0.05 0.31 ± 0.09 Segmented neutrophils,% 45.7 ± 1.2 46.8 ± 1.6 44.2 ± 1.2 45.3 ± 2.0 Eosinophils,% 0.67 ± 0.07 0.71 ± 0.05 0.70 ± 0.07 0.71 ± 0.05 Basophils,% 0.57 ± 0.05 0.58 ± 0.08 0.62 ± 0.06 0.61 ± 0.04 Monocytes,% 2.4 ± 0.03 2.3 ± 0.02 2.3 ± 0.07 2.5 ± 0.06 Lymphocytes,% 52.6 ± 2.9 53.6 ± 2.5 52.9 ± 2.6 49.9 ± 2.18 ESR, mm / h 1.76 ± 0.01 1.78 ± 0.08 1.83 ± 0.06 1.81 ± 0.08 Erythrocyte resistance,% NaCl - maximum 0.44 ± 0.02 0.43 ± 0.05 0.41 ± 0.05 0.45 ± 0.07 - minimum 0.32 ± 0.04 0.31 ± 0.03 0.32 ± 0.08 0.33 ± 0.06 Total protein in blood serum, g / l 74.7 ± 1.7 75.3 ± 1.6 73.7 ± 1.7 75.3 ± 1.8 Sodium in blood serum, mmol / l 153.1 ± 5.5 154.2 ± 5.1 155.7 ± 5.0 152.6 ± 5.7 Serum potassium, mmol / l 5.2 ± 2.3 5.4 ± 2.1 5.3 ± 1.9 5.5 ± 2.3

table 2 Hepatoprotective agent and method for its preparation Indicator Healthy animals The control Animals that were given the drug 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 ** ACT, IU / 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 animals of the control group. Table 3 Indicator Healthy animals Control (exposure CCl ₄ ) Drug (exposure to CCl ₄ ) After 2 weeks After 4 weeks After 2 weeks After 4 weeks Glycogen content 3.85 ± 0.1 6.70 ± 0.2 * 8.21 ± 0.3 * 2.84 ± 0.09 ** 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 Hepatoprotective agent and method for its preparation Indicators Before treatment After treatment using conventional means After treatment with the drug Cholesterol (mmol / L) 4.6 ± 0.3 4.7 ± 0.7 5.0 ± 0.6 Bilirubin, (μmol / L) 32.4 ± 1.3 23.1 ± 1.1 * 19.9 ± 1.4 * # ACT, (mmol / h · l) 37.2 ± 2.3 38.7 ± 2.6 35.7 ± 1.8 ALT, (mmol / h · l) 56.8 ± 2.1 46.8 ± 2.7 * 38.5 ± 1.9 * # γ-GT, (mmol / h · l) 45.1 ± 2.3 46.7 + 2.4 40.3 ± 2.5 Triglycerides (mmol / L) 2.5 ± 0.08 2.5 ± 0.09 2.4 ± 0.1 * - P <0.05 compared with the indicator before treatment;
# - P <0.05 compared with the indicator after treatment using conventional means.

Claims (2)

1. Hepatoprotective agent, characterized in that it is in the form of a parenteral dosage form and is a peptide complex with a low molecular weight fraction of 70 to 90% with a molecular weight of its constituent peptide components ranging from 70 to 184 Da with a concentration of polypeptides 2.5-2.9 mg / ml and obtained from the liver of calves not older than 12 months of age or pigs by extraction with acetic acid in the presence of zinc chloride. 2. A method of obtaining a hepatoprotective agent, characterized in that the liver of calves not older than 12 months of age or pigs is frozen at a temperature of at least minus 40 ° C, kept at a temperature of minus 20-22 ° C for at least two months, then crushed, add a 3% solution of acetic acid in a volume ratio of 1: 5 at a temperature of 20 ± 5 ° C, the extraction is carried out with constant stirring, after obtaining a homogeneous suspension, add a 1% solution of zinc chloride in a volume ratio of 50: 1, cool with constant stirring to a temperature of 7-16 ° C, then mix for 1 hour after every 4 hours of settling for 48 hours, the extract is separated from the ballast substances by separation, acetone is added to the extract in a volume ratio of 1: 5, kept at a temperature of 3-5 ° C within 4 hours, the resulting homogenized precipitate is re-precipitated with acetone at least 2 times, then the precipitate containing the active substance is washed on a suction filter with twice volumes of acetone cooled to a temperature of 7-16 ° C until a light gray precipitate is obtained, wiped through metal sieve, dried, dissolved in distilled water at room temperature and constant stirring to a concentration of polypeptides of 2.5-2.9 mg / ml, the solution is centrifuged, filtered, subjected to ultrafiltration purification on the installation with a backpressure of not more than 1.0 kgf / cm ² through materials with a retention capacity of 15,000 Yes, glycol is added to the ultrafiltrate to its final concentration of 10 ÷ 20 mg / ml at pH 5.6-6.6, the solution is subjected to sterilizing filtration under a pressure of not more than 2.0 kgf / cm ² , poured into 2 ml ampoules and autoclave t for 8 min at a temperature of 120 ° C and atmospheric pressure of 1.1 kgf / cm ² .

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