Method for experimental therapy of hepatitis

FIELD: medicine.

SUBSTANCE: viral hepatitis in simulated in laboratory animals (rats) with D-galactosamine. Hepatitis is treated by single oral administration of immobilised hyaluronidase 25 unit/kg. Administration either precedes simulation, or is combined with the beginning of stimulation.

EFFECT: method enables reducing a severity of metabolic and morphological hepatic disorders effectively in stimulating hepatitis identical to viral hepatitis in a human with underlying reduced xenobiotic load on the body.

1 ex, 3 tbl


The invention relates to medicine, specifically to pharmacology and hepato-biology and concerns a method for the treatment of hepatitis.

For the treatment of diseases of the liver using a large number of pharmacological and non-pharmacological methods [1, 2].

The known method the experimental therapy of chronic hepatitis caused by the introduction of tetrachlorophenol using double or course introduction immobilizovannoi hyaluronidase [3]. This method is the closest to the claimed and selected as a prototype.

The disadvantage of the prototype is its lack of efficiency, relatively high xenobiotica load on the body and limited by a chronic course of the pathological process and its toxic etiology scope.

The problem solved by this invention is to improve the efficiency and safety of the method, and the expansion of indications for its use.

This object is achieved by a technical solution, consisting of a single oral administration to laboratory animals (rats) immobilizovannoi hyaluronidase at a dose of 25 IU/kg for modeling viral hepatitis by using the D-galactosamine.

New in the present invention is a single use immobilizovannoi hyaluronidase at a dose of 25 IU/kg is the simulation of hepatitis.

Diseases of the liver and biliary tract are very common worldwide and occupy one of leading places in the structure of morbidity and mortality in the population of our country. Danger to the health and social importance of this pathology of the liver to determine the need for constant development of new effective pathogenetic methods of pharmacological therapy and prophylaxis of these diseases [1, 2]. When this has been actively conducting research aimed at developing tools for the treatment of various diseases, including liver diseases, by pharmacological stimulation of endogenous stem cells (SC) body [4, 5]. On the model of chronic hepatitis caused by the course introduction tetrachlorophenol shown the presence of hepatoprotective properties immobilizovannoi hyaluronidase [3], defined by its stimulating effect on stem cells and liver tissue depot. The best technical result is achieved only in the course of introduction of this tool in doses of 25-50 IU/kg, of course, accompanied by considerable xenobiotics load on the body [6], and only after modeling liver pathology. In addition, to date there are no data on the presence of hepatoprotective effects in immobilizovannoi g is laronidase in other models of liver disease, including diseases of viral etiology.

The fact that a single application immobilizovannoi hyaluronidase before modeling or simultaneously with the beginning of the simulation viral hepatitis by using the D-galactosamine to achieve the expressed therapeutic effects for professionals is obvious. The experiment showed unexpected results.

The essential features of the claimed showed together new properties that are not derived explicitly from the prior art in this field. New features allow for effective therapy of acute experimental liver lesions simulating disease of viral etiology, and the present invention can be used in medicine. Identical set of features not found in the study of the prior art in the patent and scientific and medical literature.

Based on the above, you should consider the claimed solution meets the criteria of "Novelty", "Inventive step", "Industrial applicability".

The method is as follows:

Laboratory animal (rat) before modeling or simultaneously with the beginning of the simulation viral hepatitis by using the D-galactosamine once orally administered mobilisierung hyaluronidase at a dose of 25 IU/kg

PR is lagueny method was studied in experiments on outbred rats in the amount of 46 pieces, weighing 250-300 Animals, obtained from the nursery of the Department of experimental biomedical modeling NII pharmacology" SB RAMS (certificate available). For modeling of acute hepatitis have used D-galactosamine in aqueous solution (Galactosamine hydrochloride, Applichem, cat no.A). which causes acute hepatitis, identical in morphological and biochemical changes in the liver viral hepatitis human. For this purpose, rats were injected D-galactosamine at a dose of 300 mg/kg intraperitoneally for 3 days [7].

Rats: expected weight of the liver (ratio of body mass to the mass of the animal), the indicator determined for the integrated assessment of the availability of hepatotropic influence of xenobiotics; conducted biochemical studies content in blood serum aspartate and alanine aminotrasferase (AST, Alt), bilirubin, alkaline phosphatase activity (alkaline phosphatase); and morphological study of the liver [7]. The enzyme activity of blood serum were determined by conventional methods using semi-automatic biochemical analyzer company Cormay and standard sets of firms Cormay and Vital Diagnostics" (Saint-Petersburg). Blood for the study was obtained through a catheter implanted in the femoral artery with subsequent ligation of the vessels. On histological preparations of liver stained with hematoxylin and eosin, podschitivala the number of hepatocytes in a state of necrosis and shapes mitoses per 1000 hepatocytes. Using an ocular grid, Avtandilov was determined by the relative area of infiltration of the hepatic parenchyma by macrophages and leukocytes and the relative size of the collagen fibers. For detection of RNA preparations of liver were stained with help of gallotsianin on Einarson. Glycogen was revealed using Schiff-iodine reaction in J. Mc.Manus. Quantitative determination of the content in the cell specified substances was carried out on the scanning cytophotometry UNIVAR ("REICHERT-JUNG, Austria) at a wavelength of 500 nm, the filter No. 42, the probe diameter 2 (RNA) and 3 (glycogen) microns. Each animal was measured content of RNA and glycogen 50 hepatocytes. For the detection of lipids were prepared cryostate liver slices with a thickness of 10 μm. Slices were fixed with 4% calcium-formula and stained with Sudan black B. Degree of fatty degeneration was evaluated in points [7]. Analysis was performed by the method of variation statistics using t-test, t-test and non-parametric U-test, Wilcoxon-Mann-Whitney. Was performed with the program Statistica 6.0.

Example 1.

On the first day of the experiment, labeled "day 1", began to enter D-galactosamine, the total observation period was 4 days. Mobilisierung hyaluronidase (imgd) (OOO "Scientific future management, Novosibirsk), a high-purity testicular bullish hyaluronate-endo-P-N-acetilene is Amiridze, administered orally in three schemes. The first circuit assumed the administration of a medicinal product according to the method prototype: exchange double injection at 1 and 2 days after injection of D-galactosamine at a dose of 35 IU/kg (dose and mode of administration were determined in preliminary experiments as the most effective). The second circuit assumed a single injection immobilizovannoi hyaluronidase after the first injection of D-galactosamine at a dose of 25 U/kg of a Third scheme would impose imgd on zero day before modeling of acute hepatitis. Control animals received the same pattern in the equivalent volume was administered distilled water.

Groups of animals:

1) the Intact animals. Group 1.

2) Control animals, the introduction of D-galactosamine. Group 2.

3) Group course with twice the introduction of drugs 1 and 2 days after injection of D-galactosamine. Group 3.

4) Group with a single administration of a medicinal product in 1 day introduction D-galactosamine. Group 4.

5) the Group with prior administration of a medicinal product on day 0 before vvedenim D-galactosamine. Group 5.

The morphological examination of the liver of rats with acute liver D-galactosamine treated with distilled water, was detected violation of the beam structure. Prevailed hydrobikes the traveler (protein) degeneration of hepatocytes. Met a large number of hepatocytes in the state of apoptosis of varying severity from degradation of DNA to the formation of the "classic" Taurus Councilmen that humans are indirect markers of viral hepatitis [Serov V.V., Lapis K., 1989]. Portal and vnutridolkovom stroma was diffusely infiltrated by lymphocytes and macrophages mixed with neutrophilic leukocytes.

From morphometric parameters in group 3 did not reveal significant differences with a group of acute hepatitis caused by the introduction of D-galactosamine (table 1). At the same time in groups 4 and 5 there was a decrease in the number of apoptotic hepatocytes, which was accompanied by a reduction in the degree of fatty infiltration of the liver in the form of statistically significant trends.

However, in rats 3 days after the beginning of the introduction of D-galactosamine was rising ratio of the mass of the liver. In the serum were statistically significantly increased activity of Alt and AST, the content of bilirubin, and also had a tendency to increase the activity of alkaline phosphatase (table 2). It should also be noted in rats of this group increased time thiopental sleep, probably due to inhibition of enzyme systems of hepatocytes responsible for the antitoxic function of the liver (table 3).

With the introduction of immobilizovannoi hyaluronidase on the Onet acute D-galactosamine hepatitis the weight of the liver in the third group of animals was superior to the corresponding index of the first group (52,1±2,4 against 44,9±1,9), that was close to the corresponding value groups of animals with hepatitis without pharmacological correction (57,9±1,8). In groups 4 and 5 were studied parameter was not different from the corresponding values in the group of intact animals (group 1). Was also observed normalization of Alt activity in groups of 3, 4 and 5, AST - in groups 4 and 5. Time thiopental sleep was decreased compared with control (hepatitis) in group 4 and 5. In groups of 3, this figure though was decreased compared to control, but did not reach statistical significance (table 3).

Thus, immobilizovannaya hyaluronidase in terms of intoxication D-galactosamine had the most pronounced hepatoprotective effect with its single introduction to the most used low dose (25 U/kg) after simulation of a pathological process or simultaneously with the beginning of the simulation hepatitis.

The proposed method can effectively reduce the severity of metabolic and morphological disorders of the liver, with its acute lesion simulating viral hepatitis, the decline xenobiotics load on the body.

Cited literature

1. Vengerovsky A.I. Effectiveness and mechanism of action of hepatoprotectors in experimental toxic injury of the liver: a Thesis... Dr. med. Sciences. - Tomsk, 1991. - P.4-57.

2. Mashkovsky PPM of Lekarstvo the e tool: in 2 volumes. So 1. - M.: Medicine, 1996. - 624 S.

3. Patent (RU) for the invention №2444569 "Hepatoprotective agent" (publ. 10.03.2012, bull. No. 7). Authors: A.V. Artamonov, Bakharev A.A., Digi A.M., Zhdanov V., Sushkov G.N., Udut CENTURIES

4. Goldberg ED, Digi A.M., Sushkov GN. Hypoxia and blood system. - Tomsk: Izd. University, 2006. - 142 S.

5. Digi A.M., Sushkov GN. Cell therapy: new approaches // Science in Russia - Moscow: Publishing house "Science", 2009. Tom. 169. No. 1. P.4-8.

6. Zborowski A.B., Tyurenkov I.N. Complications of pharmacotherapy. - M.: Medicine, 2003.-543 C.

7. Manual on experimental (preclinical) study of new pharmacological substances / Under the General editorship of the corresponding member. Russian Academy of medical Sciences, Professor RU Khabriev - 2-ed., Rev. and ext. - M, JSC "Publishing house "Medicine", 2005. - 832 S.

Table 1
Influence immobilizovannoi hyaluronidase on morphometric characteristics of the liver of rats with acute liver D-galactosamine, (X±m)
The group of animalsThe degree of fatty infiltration, pointsThe number of apoptotic heparin-tocito, %The relative area of infiltration, %Mitotic index, % RNA srvc. unit optat.Glycogen srvc. unit opt tight
32,44±0,1710,06±0,75*at 8.60±1,080,24±0,070,16±0,010,22±0,01
42,16±0,122,04±0,10#at 8.60±0,510,38±0,080,16±0,010,22±0,01
52,08±0,103,76±0,10#of 8.40±0,750,28±0,040,16±0,010,22±0,01

* - reliability of differences when compared with the group of intact animals (group 1) at p<0,05.

# - reliability of differences when compared with group 2 at p<0,05.

Table 2
Influence immobilizovannoi hyaluronidase on biochemical parameters of blood serum of rats with acute liver D-galactosamine, (X±m)
PAfactor(ákat/l)(ákat/l)De RytisFOSFA-bilirubin
352,1±2,4*0,54±0,030,60±0,03 *0,90±0,04146,5±20,2a 7.62±1,32

* - reliability of differences when compared with the group of intact animals (group 1) at p<0,05.

# - reliability of differences when compared with group 2 at p≤0,05.

Table 3
Influence immobilizovannoi hyaluronidase on time thiopental sleep in rats with acute lesions of pécs is no D-galactosamine
(min, X±W)
Group 1Group 2Group 3Group 4Group 5

* - reliability of differences when compared with the group of intact animals (group 1) at p≤0,05.

# - reliability of differences when compared with group 2 at p≤0,05.

The method of therapy of acute hepatitis induced in the experiment the introduction of D-galactosamine by a single oral administration to an animal immobilizovannoi hyaluronidase at a dose of 25 U/kg before the simulation, either simultaneously with the beginning of the simulation.


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