RU2063760C1 - Method for treating hepatic insufficiency in the cases of chronic liver diseases - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves introducing per os suspended isolated hepatocytes in Hencks solution into the gastrointestinal tract at a dose of 2 ml per 100 g mass, cell concentration 2•10⁷ cells per ml in 2 days before stable remittance takes place. The preparation is administered to a patient 30-40 min before taking food. The patient has to drink half a glass of mineral water before and after giving the preparation. EFFECT: simplified and low cost treatment.

Description

 The invention relates to medicine, namely to hepatology and can be used in the treatment of liver failure in chronic liver diseases, and can also be used in surgical departments for preoperative preparation of patients, infectious, intensive care units and intensive care.

 Currently, liver failure in chronic liver diseases requires considerable effort and modern medicines for its treatment. Medicines are often ineffective and mortality in this case reaches high numbers. With liver damage, a failure of many organ functions develops simultaneously. Modern therapy does not have the ability to simultaneously replace several liver functions. So if detoxification of the body is carried out (by the method of perfusion of detoxification solutions, dialysis or sorption), then it is not possible to simultaneously synthesize vitamins or correct the protein spectrum of the plasma by introducing certain components during intravenous perfusion of solutions.

 Solving the problem by donor liver transplantation has little effect due to the extremely high sensitivity of the transplanted liver to hypoxia and, as a consequence, its very rapid death, in addition, technical difficulties in performing this operation, and lack of donor organs.

 More promising are currently methods that combine the detoxifying effect by passing blood, lymph, plasma, biological fluid through sorbents, AIP, peritoneal dialysis. A more significant effect in the detoxification function of the liver was obtained when using cell suspension of isolated hepatocytes planted on sorbents (Margulis M.S. Erufimov A.S. et al. 1985), as well as in cell hemodialysis (Bruslik V.G. et al. 1975) and cell peritoneal dialysis (Ostroverkhov G.E. Bruslik V.G. Berelavichus V.Yu. Shimanko I.I. et al. 1979), which made it possible to significantly reduce the percentage of adverse results. However, these methods have their drawbacks: they need special equipment, specially trained personnel, they are difficult to perform surgical procedures to isolate and catheterize blood vessels and ensure extracorporeal blood flow.

 There is also a method of treating liver failure by introducing cells into the subcutaneous tissue by cellular subcutaneous dialysis (Bruslik V.G. et al. Auth. Certificate N 1202593, class A 61M 1/14, 1985), which prevents complications. This method does not require special equipment to perform, technically simple, but still requires surgical procedures.

 The aim of the invention is to simplify the method of treatment by approximating living, functioning liver cells to the site of metabolic processes, i.e. to the depot of toxic substances in the gut.

The goal is achieved by the fact that a suspension of isolated hepatocytes is administered orally into the gastrointestinal tract at a dose of 2 ml per 100 g / mass at a concentration of 2.10 ⁷ cells per ml. after 2 days on the third until the onset of stable remission.

 To implement the method, experimental studies were carried out, which amounted to 3 experimental groups: 1 group (120 healthy rats) search for the optimal dose of cell suspension; Group II (160) evaluation of the results of treatment of animals with experimental liver damage; Group III (30) studying the fate of orally administered isolated cells.

 So in the first group healthy animals through a probe installed in the stomach, were administered various doses of cell suspension (0.5; 1.0; 2.0; 3.0; 4.0; 8.0; 10.0; 12.0 ml per 100 g / animal mass), which did not cause the development of any complications and changes in their condition. Animals remained active, ate food. Repeated injections were carried out every two days on the third. The permissible dose in healthy animals varies widely.

The second group of animals with a model of toxic hepatitis, which was created by oral seeding of animals CCl ₄ in vegetable oil, after a day or two received an oral cell suspension of hepatocytes, as in the 1st group. In animals with a disease model (group 2), the minimum dose of cell suspension at which a stable therapeutic effect was obtained (reduction in the percentage of mortality and life expectancy of experimental animals) was 2 ml of cell suspension per 100 g of animal weight at a concentration of 20 million / ml. We obtained similar results when using other doses of 3, 4, 5, 6, 7 ml of cell suspension per 100 g of animal mass. When analyzing the materials, we came to the conclusion that with an increase in the dose, the results of treatment of animals did not change, and the protein load increased, which can affect the course of the disease and allergization of the body. Therefore, a dose of 2 ml per 100 g of mass can be taken as the minimum therapeutic. With an increase in dose above 7 ml (8.0, 9, 10, 11, 12, 14 ml) mm, a decrease in the therapeutic effect was noted, which was the basis for choosing the minimum dose. As a rule, 73.3 80% of the animals died in the control group when CCl _{4 was} seed and the average life expectancy was 6.3 8.1 days. When using isolated hepatocytes for treating animals with experimental liver damage, the percentage of death of animals decreases to 33.3% and their average life expectancy increased to 17.1 19.6 days. Thus, it was found that the used isolated cells have a therapeutic effect in experimental liver damage, which led to the conclusion about the advisability of oral administration of isolated hepatocytes for the treatment of liver failure.

 An equally important issue for us was the fate of orally isolated hepatocytes. 2 p / groups of experiments were conducted on healthy animals and animals with experimental liver damage. The animals were controlled and the animals were slaughtered at certain time intervals. The estimated dose of the cell mass was stained with a 0.2% solution of methylene blue and introduced into the stomach through a probe, then the first rat was slaughtered an hour later. Up to 1.5 2 hours, the cells were in the stomach, and then passed into the 12 duodenum, skinny, ileum, etc. By the end of 2 days, the cells were in the rectum. In animals with experimental liver damage, the movement of the cell mass was faster after 1.5 days, the cells were already in the rectum. We were not able to calculate the number of hepatocytes in the gastrointestinal tract-chyme and therefore it is not currently possible to draw a conclusion about the degree of destruction of cells in various parts of the gastrointestinal tract. However, the calculation of dark and light cells in the intestinal chyme and their ratio indicates that as they move along the gastrointestinal tract, the number of dark cells increases in relation to light cells, and therefore their destruction occurs and the degree of destruction of hepatocytes also increases.

 Thus, the conducted experimental studies on animals in the third group allow us to conclude that hepatocytes pass the gastric barrier and fall into the lower lying sections of the intestinal tube, the timing of advancement varies within 1.5–2 days, as the cell mass moves along the gastrointestinal tract they are destroyed in the intestinal tract, but what mass of hepatocytes reaches the rectum is currently difficult to say, but light cells are in the smears from the rectum, in the mass of the microbial flora. Therefore, a certain percentage of them reaches the rectum.

 Thus, cells that pass through the gastrointestinal tract can exhibit their detoxification properties, as evidenced by the therapeutic effect in animals with experimental liver damage. In addition, the duration of their advancement along the gastrointestinal tract indicates the timing of reintroduction of the cell mass for treatment. All this was an experimental justification for the use of this method in clinical practice.

The method is as follows. A suspension of hepatocytes after isolation was placed in vials with 199 medium, in each ml of which there are 20 million cells (2.10 ⁷ ), which is 20 25 ml of cell sediment in the vial. The initial dose for each patient is calculated and, after sedimentation (centrifugation), the supernatant is poured off and a sterile Hanks solution is poured into the cell vial and the pellet is well mixed. The cell suspension settles and the supernatant is again drained. This manipulation is repeated 3 to 4 times in order to remove the destroyed cells, cell debris, enterobacteria present in the cell suspension. Cellular sediment of 20 25 ml is diluted with Hanks solution to 100 ml of volume and it is recommended that the patient drink it in 30-40 minutes. before eating, while before and after taking cell suspension, the patient should drink 0.5 cups of mineral water. This manipulation is repeated every 2 days on the 3rd day until a stable remission is obtained.

 Clinical example. Patient U.Z.A. 52 years old. entered the Central Research Institute of Gastroenterology 05/05/1988 (and / b 2832) with a diagnosis of Cirrhosis of the viral etiology, active with moderate cholestasis. Peptic ulcer with localization of the process in the duodenal bulb in the phase of remission. Chronic gastritis.

 At admission, the patient complained of pain in the epigastric region and right hypochondrium, weakness, itching, poor sleep, nosebleeds, weight loss. About 5 years ago, redness of the palms began to be noted, and after a while, the appearance of telangiectasias. During the examination, chronic active hepatitis was diagnosed. On this occasion, she was treated at the Central Research Institute of Scientific Research in 1986.

 Objectively, when examining the correct physique, satisfactory nutrition, the skin of the upper girdle has an expanded vascular network, spider veins, and hepatic palms. In the lungs and heart without features. The tongue is slightly overweight, the abdomen is slightly swollen, soft, painful in the right hypochondrium. The liver along the edge of the costal arch, compacted. Venous collaterals on the sides of the abdomen. Gastroduodenoscopy. Chronic gastritis. Ultrasound Diffuse liver damage. Spintigraphy of the liver with a colloidal solution of TC-99m signs of diffuse liver disease. Puncture of the liver. Conclusion By indirect signs, it is predominantly micromodular cirrhosis, active against the background of large-drop fatty degeneration.

In biochemical studies, bilirubinemia up to 27.5 μmol / L is observed, where the indirect fraction of bilirubin is 21.5 μmol / L. Increased alkaline phosphatase to 182 μmol / L is also noted, the percentage of albumin is reduced. The patient was treated: essentiale, metacin, almagel, Bourget mixture, B vitamins, romedorm, lipoic acid, auk-phosphate, hemodesis, casein hydrolyzate. However, the patient's condition remained without significant changes and therefore it was decided that the patient should use an oral suspension of isolated hepatocytes. The dose for the patient was determined from a solution of her body weight (62 kg). For each kg of mass, 20 ml of cell suspension in medium 199 is necessary at a concentration of 2 • 10 ⁷ in 1 ml.

 Thus, the patient needs 1240 ml of cell suspension, which is 2.5 bottles with 199 medium, but in this particular case 2 bottles were taken. The supernatant was settled and drained and sterile Hanks solution was poured into the vials with the cells and the sediment in the vials was well mixed. The cell suspension was defended (centrifuged) and the supernatant was again drained. This manipulation was repeated 3-4 times in order to remove destroyed cells, cell debris, enterobacteria, which are in the cell suspension. The cell pellet from two bottles was poured (25 + 25 ml) and diluted with Hanks solution to 100 ml of volume and it was recommended that the patient drink 30-40 minutes before eating. In this case, the patient before and after taking the cell suspension drank 0.5 cup of mineral water. This manipulation was repeated every 2 days on the 3rd day 10 times. The patient's condition stabilized, pain in the right hypochondrium disappeared, the patient became more active, sleep normalized, skin itching, nosebleeds disappeared. For organs unchanged. The tongue is moist, the stomach is soft and painless in the right hypochondrium, the liver is at the edge of the costal arch. During biochemical control, normalization of the concentration of total bilirubin and its indirect fraction, a decrease in alkaline phosphatase and an increase in the percentage of albumin are noted. Thus, the patient had a stable remission of the disease.

Currently, at the Central Research Institute of Gastroenterology, we have used this method in 23 patients with various liver diseases (289 times):
a) in 5 patients with chronic hepatitis, isolated hepatocytes were administered orally 73 times;
b) in 8 patients with primary biliary cirrhosis (one patient repeatedly) used orally isolated hepatocytes 133 times;
c) in patients with alimentary cirrhosis, orally isolated hepatocytes were used 31 times;
d) in 6 patients with post-infectious cirrhosis, 52 times orally isolated hepatocytes were used.

 Compared with the prototype method, the proposed method for oral use of isolated hepatocytes allows you to: significantly reduce the cost and simplify the method of treatment due to the rejection of complex, bulky and expensive devices.

 The method of oral use of isolated hepatocytes in the treatment of liver failure in chronic liver diseases can be included in the arsenal of therapeutic agents both in the hospital and in outpatient settings.

Claims (2)

1. A method of treating liver failure in chronic liver diseases, comprising administering a suspension of isolated functionally active hepatocytes, characterized in that a suspension of hepatocytes in Hanks solution is administered orally at the rate of 2 ml per 100 g of patient’s body weight at a concentration of 2 • 10 ⁷ cells in 1 ml 3O-40 minutes before a meal every 2 days on the 3rd until a stable remission occurs, if necessary, the cycle is repeated after 5-6 months.  2. The method according to claim 1, characterized in that before and after receiving a suspension of hepatocytes, 100 ml of mineral water is administered.