The method of prevention and treatment of gallstones


The invention relates to medicine and for the treatment and prevention of gallstones. How is that sick gallstone disease administered orally 50-100 ml aqueous acidic glycoprotein selected isoelectric focusing of bile mammals, at a concentration of 10-14-10-22mol/L. Introduction exercise 3-4 times a day for 5-40 minutes before eating for 10-30 days. The method provides a reduction of the crystallization of bile and formation of the "germ" of gallstones. 1 Il.

The invention relates to medicine and veterinary medicine and for the prevention and treatment of gallstones.

In the basis of the etiology of cholelithiasis (ICB) are: cholestasis (blockage of the bile ducts), inflammatory processes, violation of the chemical composition of bile (a decrease in bile of bile acids glycocholic and human beings need it delivered in the intestinal tract liver through the bile duct, which, being effective detergents, emuleret fats and facilitate the action of a lipase).

On the background of gallstones develop acute cholecystitis, cholecystoenteric, gallstone ileus.

Treatment of gallstone boleznei. Patients JCB often incapacitated, they are often forced to seek medical help while on outpatient, inpatient and sanatorium treatment.

A significant increase in the incidence JCB observed after 40-ies of our century in all European countries and North America. The prevalence of gallstone disease is highest in the age group of 50-60 years and 14% for men and 20.2% for women (Glambek J., Kvna Le G., Amesye Century , Soreide // Scand. J. Gastroenterology, 1987, vol. 22, p. 1089-1094; Gracie W. A, Ransohff D. F. // Gallstone, 1985, p. 27-48). In recent years, the incidence of JCB worldwide has increased dramatically (Gorchakov L. A. et al. // Clinically. med., 1989, 11, 65-67 C.; Loginov, A. S., Ilchenko, A. A., Shibaeva Leningrad region // Russian gastroenterology journal, 1997, 4, S. 29-33).

In recent years there has been a tendency to increase the frequency JCB and in childhood, mainly students. High frequency JCB in girls is associated with hormonal features of their development, including increased estrogenicity (Zaprudnik A. M., L. Kharitonov A.// In the book: Perspective problems in gastroenterology. So 1. - Moscow. 1994. S. 192-193).

At the same time, many remain controversial medical aspects of prevention, diagnosis and treatment is cnyh stones or changes in the gallbladder wall.

Current methods of treatment and prevention are as follows.

I. Prevention (Radbill O. C. Problems of gastroenterology, 1991, 3, 4(5), C. 10-14): 1) diet with calorie restriction, a diet of foods rich in fiber, with the addition of vegetable fats (Mansurov H. H. Prevention of cholelithiasis. Clinical medicine, 1985, 1, 10-16 C.); 2) regular physical activity; 3) pharmacological stimulation of synthesis and secretion of bile acids, as well as suppression of the synthesis or secretion of cholesterol (these requirements more other drugs correspond chenodeoxycholic (CDCA) and ursodeoxycholic (UDCA) acid) (Mansurov H. H., F. Mansurov Agricultural Experience prolonged use chenodesoxycholic acid in patients with gallstone disease // Ter. archive, 1984, 1, S. 43-47); 4) treatment and prevention of opportunistic diseases associated with GCB, such as: - diseases of the stomach - duodenum - pancreas - diabetes - kidney stones
pathology of the biliary system and other pathologies contributing to the stagnation of bile (Menshutkin O. N. etc. // ROS. Zhur. Gastroenterol., gepatol. , croprect. , 1996, T. 6, 4, S. 260; Menshutkin O. N., Maslovsky of Propriety most commonly used surgical treatment - cholecystectomy (laproscopy and laprotomy) (Small invasive medicine. Ed. Bronstein A. S. corresponding member cor. MAI and Professor Rivkin. C. L. - M: Varnish, 1998, S. 16-26; Small medical encyclopedia. - so 2, - M.: Soviet encyclopedia, 1991, S. 179-182).

Currently used in clinical practice solvents bile concrements can achieve a positive effect in only 50-60% of patients. This circumstance is due to insufficient capacity of the used solvents, no generic drugs, able to lyse rocks of different composition. Each of used solvents is valid only for separate patterns included in the biliary calculus. Also a negative point of application litolitic drugs is the duration of their use (at least 1.5-2 years).

Thus, pharmacotherapy JCB currently is not a settled issue. It is obvious that a great prospect has a preventive focus, which requires less pharmaceutical costs, and reduces the possibility of side effects of drug therapy and are able to provide greater social and economic effect.

One of the ways non-surgical treatment of gallstones are the two who esteem.

These methods of treatment JCB include, for example, a method of treating JCB by oral administration a pharmaceutical-based compositions containing N-hydroxymethylcytosine (Nikodin"), starch, talc, stearic acid (RU 2155041, 27.08.2000), or based on vegetable raw materials - based grass astrogal (EN 2155056, 27.08.2000), based on vegetable raw materials rhizomes and elecampane root, Sophora, Burnet, elderberry, hawthorn fruit (EN 2159123, 20.11.2000), based on grass subcode ordinary (EN 2160598, 20.12.2000). These funds have increased choleretic and anti-inflammatory activity.

Another way of treatment JCB are ways of removing gallstones from the body of the patient:
a) direct contact dissolution of gallstones using solvent funds supplied through the drains and catheters (Alien M. J., Barody T. J, Thistle, J. L. In vitro dissolution of cholesterol gallstones. A study of factors influencing rate and comparison of solvents // Gastroenterology, 1985, v. 89, 5, p. 1097-1103.);
b) extracorporeal lithotripsy (Korolev P. A., Pikovsky D. L. Extracorporeal surgery of the biliary tract. - M.: Medicine, 1990, S. 240; Ivannikov Acting, Ulyanov Century. And., Bogachev Century B. Efficiency lithotripsy combined with cholelitiasis therapy in patients with gallstone boleskine the destruction of the stones using physical methods (laser, BONDS and other force) (Zak, Century, Zorin, A. N., Montyan S. A. , Pererva A. N. A new method of prevention of cholelithiasis // Materials I Grew up. gastroenterological week. November 27 - December 2, 1995, St. Petersburg., S. 78; Vorobiev, L. P., Salova L. C., Bags C. M., Chubarov Century, the treatment of diskenesia biliary tract with the aid of laser radiation // Clinically. medicine, 1990, 5, 110-115 C.);
g) mechanical instrumental fragmentation of gallstones (Lygidakis N. J.// Heterogastroenterology, 1989, v. 36, 3, R. 212-222.)
Known, for example, a method of treating JCB by removing gallstones from the body of the patient by using as sequestro (substances that lower level of reabsorption of bile acids) alkylated Poperechnaya polymer (EN 2160742, 20.12.2000).

However, the non-surgical methods of treatment JCB give up to 50%: relapse (Yakovlev, L. M., Ivanov L. N. // In the book: New in gastroenterology. - Moscow. so 2, 1996, S. 158-159).

The closest in technical essence to the present invention is a method of prevention and treatment of gallstone disease by oral administration a pharmaceutical drugs chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) (Mansurov H. H., Mansurov, 1984, 1, S. 43-47).

These drugs stimulate the synthesis and secretion of bile acids, as well as inhibit the synthesis and secretion of cholesterol, promote dissolution of stones.

The technical purpose of this invention is to provide a method for preventing the formation of gallstones and their further growth.

The goal of the project is achieved by a method for the treatment and prevention of gallstone disease by oral administration to the patient (patient) pharmacological tools. As pharmacological tools use acidic aqueous solution of a protein, glycoprotein, a selected isoelectric focusing of bile mammals possess biological activity at concentrations of 10-14-10-22mol/l, the input (received) portions of 50-100 ml 3-4 times a day for 5-40 minutes before eating for 10-30 days.

Used in the method according to the invention acidic protein - glycoprotein from the bile of mammals exhale, using in particular the following steps:
a) salting out of proteins bile;
b) actual allocation (separation and purification) acid glycoprotein (protein) by the method of isoelectric focusing;
C) identification and definition BIOLOGIChESKIKh blood of animals and humans // Journal of General biology, 1984, T. 45, 3, S. 373-382).

The method of isoelectric focusing distinguish the different fractions of proteins, while acidic glycoproteins migrate to the anode. The method of isoelectric focusing is one of the traditional methods of separation and purification of proteins. The method is widely used in physico-chemical and analytical chemistry.

Isoelectric forcing is based on the creation under the action of an external electric field of a stable pH gradient, and the pH value increases from the anode to the cathode. In such a system, each protein is moved in one direction or another in accordance with the sign of their charge until then, until you reach the area in which the pH value coincides with its isoelectric point (PI). In this area further its movement under the action of the electric field is stopped, because the charge of the protein is equal to 0.

Selected acidic glycoproteins used in this method according to the invention show a biological activity in midget doses, corresponding to concentrations of 10-14-10-22mol/l, and have a biological effect from 135 to 170%, and possibly higher (drawing).

The drawing shows the dependence of the viscoelastic properties of the plasma membrane GE is s short multiple organ cultivation in vitr ([S] - the concentration of the studied glycoprotein mol/l, To control the experience).

Biological effects caused by these acidic glycoproteins, is determined by a formula below. Selected acidic glycoproteins from the bile of mammals soluble in a saturated (100%) solution of ammonium sulfate, retain their activity during repeated freeze-thawing, and after heating at 100oC for 10 minutes. The apparent molecular weight glycoproteins of this invention is from 8 to 100 kDa; degree of glycosylation of from 0.1 to 75%; isoelectric point (PI) of selected acidic glycoproteins below 3.0.

Below are examples of allocation used in the method of the glycoprotein from the bile of mammals and illustrating the method according to the invention.

Example 1. Selection acidic protein from the bile of cattle.

1.1. Salting out of proteins bile.

In bile canned health (registration number 70/529/5; JSC "Samson", St.-Petersburg) with vigorous stirring gradually add dry ammonium sulphate to the formation of saturated salt solution (4oC, pH 8.0 to 8.5). The precipitate of impurity proteins precipitated centrify, using cellulose film domestic production (GOST 7730-89). During dialysis distilled water repeatedly replace with fresh. After complete removal of ammonium ions in the supernatant concentrated to a volume of 100 ml using evaporation on a rotary evaporator under vacuum at a temperature of 35-40oWith, and repeat the procedure vysalivaniya in the same conditions. Similarly, the received second supernatant also cialiswhat against distilled water until complete removal of ammonium ions and then divide by the method of isoelectrofocusing (IEF).

1.2. Isoelectrofocusing supernatant of tissue extract.

Isoelectrofocusing carried out in a sucrose gradient, using a column LKB-440 (LKB, Sweden) and ampholytes (Serva, Germany) pH range of 3.5 to 10.0, at a temperature of 4oWith over 100 hours and at a voltage of 500-1500 C. the Supernatant in the form of a solution injected into the heavy gradient solution in an amount of not more than 100 mg of total protein. Detection factions carried out spectrophotometrically at 280 nm. Dialysis of the fractions after IEF is carried out in bags of cellulose film against distilled water. Collect the fraction of acidic glycoproteins that migrate to the positively charged electrode.

Identified gli-10-22mol/l (and possibly also below) aqueous solution of glycoprotein.

1.3. Study of biological activity.

Study of biological activity conducted on mice CBAxC57BL (males weighing 18-20 g) using adhesiometres method, based on experimental estimation of the parameter characterizing the viscoelastic properties of tissue by standard deformation forces exerted on the piece of cloth (Yamskov B. N., N. Tumanova.B. "Izv. Acad. Sciences, vol. Biol., 1994, 5, S. 732-737).

Study of biological activity carried out by multiple organ culturing fragments of liver tissue weighing 1.0-2.0 mg in 1 ml of culture medium Needle containing 0.1 ml of bovine serum, which contribute a certain amount of the investigated protein preparation - acid glycoprotein bile, and incubated for 2 h at 37oC.

The change in the viscoelastic properties of the tissue is assessed through adhesiometres method, the essence of which is as follows: each piece of fabric after cultivation dried and subjected to dispersion in 0.1 ml of culture medium containing 1% solution Trypanosoma blue, in a special cage fabric with SASO is examined microscopically in the camera Goryaeva.

For the study of biological activity in the concentration range from 10-6mol/l to 10-22mol/l of the drug serially diluted in culture medium Needle. In the control experience of the study drug did not add. In each experiment for each experimental point estimate 5 pieces of fabric. Biological effects are calculated according to the formula, taking the control as 100% (Yamskov I. A., Yamskov B. N., Danilenko A. N., Klimenkova the legislative Assembly, Antipov, B., and other Experimental evidence for the role of physico-chemical factors in the mechanism of biological action of ultra-low doses // ROS. chem. W. (J. ROS. chem. of the society to them. D. I. Mendeleev), 1999, T. 43, 5, S. 34-39):

where Nonthe number of cell nuclei released by deformation of the standard exposure of 1-2 mg tissue - organ culture of the liver in the experience;
Nkthe number of cell nuclei released by standard deformation the effect of organ culture of the liver in the control.

The results of the experimental data is shown in the drawing.

As can be seen from the drawing, an acidic glycoprotein isolated from the bile of cattle, exhibits biological activity with Valier 2. The influence of acidic glycoprotein of bile on the size of the particles in human bile in vitro.

The study in this way is performed on bile at least 8 patients JCB.

Patient M. , 53 years history 700). Was treated in the PDB 71 17.01. on 28.01.00). Was admitted to the surgical Department with complaints of constant aching pain in the right hypochondrium, nausea after ingestion of fatty foods, bitter taste in the mouth, yellow sclera and skin. Gallstone disease is suffering for 4 years.

Objective examination of the sclera and skin, jaundice, tongue moist, coated thick root yellow tinge, there are imprints of teeth. Palpation of the abdomen is painful, in right hypochondrium positive symptoms Kera-Hausman, Ortner. Cardiovascular, respiratory systems without features.

Operated on for acute calculous cholecystitis, mechanical jaundice. Produced cholecystectomy, drainage choledochus by Halstead. In the postoperative period were studied ductal bile obtained by drainage, and gallbladder bile obtained intraoperatively.

To 10 ml of bile was added to 1 ml aqueous acidic glycoprotein bile (KHZ) at a concentration of 10-14M. the resulting suspension straw D. C., Gulmetova Century. And. // international agricultural journal, 1993, 3, S. 52-54; Luchnikov D. S. and others // Methodical instructions for practical lessons on discipline "physical and chemical methods of research". M: CEA them. G. C. Plekhanov. 2000, S. 63).

When the action KHZ average particle diameter was decreased from 56,9 to 28.9 nm, whereas in the control dimension (bile with distilled water) particle radii remained practically unchanged, the average radius of the particles ranged from 63.3 to 45.5 nm. For the control and the experimental study carried out at least 8 measurements for each sample of bile with a measurement interval of 20-40 minutes. In all cases, with CGI was a marked decrease in the radius of the particles.

Example 3. The influence of aqueous solution KHZ at a concentration of 10-14M npu oral on the particle size of human bile.

The study was conducted on volunteers (a total of 8 people).

Volunteer L., male, 60 years.

No complains. Ultrasonography (ultrasound) hepatobiliary zone detected moderate compaction of the gallbladder, bile homogeneous. Signs of fatty liver.

Took aqueous solution KHZ at a concentration of 10-14mol/l for 21 days 100 ml 3 times a day 30 minutes before meals. Bile Adam examination of patients with diseases of the gastrointestinal tract, liver, biliary tract. Ed. by L. P. Vorobiev. - M.: Moscow Museum of modern art, 1989. S. 59-63).

We investigated the portion "B" - before taking the drug, through 1, 3, 6, 10, 16, 21 day (the interval between the fences bile taken empirically). Bile was investigated microdiffusion method.

The average particle size was decreased from 52 nm (reference dose without drug) to 17.0 nm on the 16th day of receiving KHZ and remained approximately at the same level in the 5th sample (on day 21). While receiving drug diet food is not changed.

Volunteer K., woman, 23 years.

No complains. Ultrasound of the hepatobiliary zone revealed no pathology. Bile homogeneous.

Took aqueous solution CGI prepared in a similar way (see the previous example) 100 ml 3 times a day 30 minutes before meals (diet did not change). Also studied was the bile portions, obtained by duodenal intubation:
1) prior to taking the drug;
2) after 1 day after administration of the drug;
3) after 4 days;
4) after 8 days;
5) after 14 days;
6) after 20 days.

The study of bile was conducted microdiffusion method. The average radius of the particles before the start of the experiment corresponded to 38.4 nm. The observed decrease of particle size on the 8th day after admission preparationnaires.

Thus, the radius of the particles decreased from 38.4 nm in the control sample to 14.5 nm on the 20th day after taking the drug.

The above examples show that the method according to the invention allows to improve the condition of patients with gallstone disease and can be used for the treatment and prevention of gallstones.


Method for the prevention and treatment of gallstone disease by oral administration of pharmacological means, characterized in that as pharmacological tools use acidic aqueous solution of a protein, glycoprotein, a selected isoelectric focusing of bile mammals possessing biological activity, at concentrations of 10-14-10-22mol/l, which receive portions of 50-100 ml 3-4 times a day for 5-40 minutes before eating for 10-30 days.


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