RU2219948C2 - Method for treatment of acute experimental pancreatitis - Google Patents

Abstract

FIELD: experimental surgery. SUBSTANCE: method involves using serum raised against true viper venom taken as measured 75 AE/kg. Preparation is administrated under parietal abdomen of anterior abdominal wall in 6-9 min after onset of disease reproduction. Method provides inhibition of activity of pancreatic enzymes that results to the enhancement of effectiveness of treatment. EFFECT: improved treatment method. 4 ex

Description

 The invention relates to experimental medicine, namely to gastroenterology, and can be used to treat acute pancreatitis.

 A known method of treating acute pancreatitis with 5-fluorouracil aimed at inhibiting the synthesis of pancreatic enzymes [1].

 The disadvantage of this method is the toxicity of 5-fluorouracil, the manifestations of which include changes in the protein spectrum of blood serum and potassium ions in red blood cells, inhibition of red blood cells and neutrophilic bone marrow growth, degenerative changes in the small intestine mucosa with subsequent dyspeptic disorders, decreased secretory and acid-forming function of the stomach.

 A known method of treating acute pancreatitis with sandostatin [2].

 Sandostatin in a daily dose of 300-600 μg suppresses exocrine pancreatic function, having an inhibitory cytoprotective effect, inhibits the synthesis of enzymes, vasoactive peptides and amines.

In addition to the existing advantages, the use of sandostatin has the following disadvantages [3]:
- sandostatin increases the tone and contractility of the sphincter of Oddi, resulting in a delay in pancreatic secretion in the duct system, which enhances hypertension syndrome and promotes the release of active enzymes into the pancreatic tissue and enhances autocatalytic processes;
- Sandostatin does not have a significant effect on the indices of the blood inhibitory system: general antitryptic activity and level of alpha-2-macroglobulin. Under these conditions, alpha-2-macroglobulin associated with trypsin retains kininogenase activity, thereby reducing the effect of neutralization of active enzymes, therefore, blood circulation in the microvasculature does not improve, which contributes to the progression of the pathological process;
- Sandostatin is an expensive drug, which makes it inaccessible to the bulk of patients.

 The closest known method is the treatment of acute experimental pancreatitis [4] by triple parenteral (intraperitoneal) administration of the drug in the form of a complex of antioxidant enzymes superoxide dismutase and catalase in a ratio of 1: 2 wt.h. 3, 24, 48 hours after the reproduction of the disease.

 The disadvantages of this method is that enzymatic antioxidants cannot have a direct effect on the intacinar activity of proteolytic enzymes, and therefore completely prevent autocatalytic processes, which are the basis for the development of acute pancreatitis.

 The aim of the invention is to increase the effectiveness of the treatment of acute pancreatitis with a single injection of the drug by inhibiting the activity of enzymes, as well as the possibility of using this method in wide clinical practice.

 This goal is achieved by the fact that animals with acute experimental pancreatitis caused, for example, by a solution of active crystalline trypsin, are administered parenterally against serum viper venom after 6-9 minutes from the start of disease reproduction at a rate of 75 (AE) / kg body weight.

 Serum is commercially available and is a protein fraction of horse serum containing specific immunoglobulins that is hyperimmunized with venom of the common viper, purified and concentrated by peptic digestion and salt fractionation [5].

 The technical result is expressed in the suppression of the enzymatic activity of the pancreas, due to which there is a relief of the pathological process and leads to 100% survival of animals.

 The method is as follows.

Animals under general ether anesthesia produce a median laparotomy, the duodenum and stomach are removed to the wound. A solution of active crystalline trypsin at a dose of 25 mg / kg body weight is injected into the pancreatic tissue through a syringe needle. As a result, acute pancreatitis in the form of a hemorrhagic spot 0.5-0.8 cm in size against the background of serous edema and dilated pancreatic vessels and hemorrhagic effusion (exudate) in the abdominal cavity of 1.2-2 ml with amylase activity from 1300-2000 is simulated EDU
After 6-9 minutes from the beginning of the disease, a specific serum is introduced under the parietal peritoneum of the anterior abdominal wall against the venom of the ordinary viper at the rate of 75 (AE) / kg body weight. For 3 hours, they monitor changes in the pancreas and other organs of the abdominal cavity, after which the abdominal cavity is sutured tightly. After 24 hours, a relaparotomy is performed to study the consequences of the treatment.

 Example 1. For the experiment (control specimen), a white Wistar rat was used, weighing 400 g, medical history 106 dated 10/30/2000. At 12:00 a median laparatomy was performed under general ether anesthesia. The duodenum and stomach were removed to the wound. Active crystalline trypsin at a dose of 25 mg / kg dissolved in 1 ml of distilled water (dose lethal in 100% of cases) was injected into the pancreatic tissue through a syringe needle. Immediately in the pancreatic tissue, a colorless “creeping” infiltrate 1 cm in size formed under the peritoneal leaf. After 3 minutes, uniform pancreatic edema occurred (in the area bounded by the horseshoe duodenum, with a large curvature of the stomach, below the transverse colon). After 6 minutes along the dilated stellate vessels, against the background of mild hyperemia of the peritoneal leaf, hemorrhagic imbibition of pancreatic tissue appeared in the form of punctate and larger hemorrhages with a gradual, within 1-1.5 min, continuous hemorrhagic imbibition in the form of a hematoma up to 1 cm in injection site of trypsin solution. After 8 minutes from the start of the experiment, edema and hemorrhagic impregnation spread to the mesentery of the transverse colon, which is accompanied by paresis and a change in wall color to a bluish-gray color.

 At the same time, similar changes in the cecum are observed. At 12.15 the pancreas acquired a black tint. The duodenum for 5-6 cm is dark red with hemorrhages on the serous membrane, the liver becomes purple-dark in color. From the moment of pancreatic edema and its hemorrhagic impregnation, an effusion appeared in the abdominal cavity of a serous-hemorrhagic nature, which by 12.35 reached a maximum amount of pH 7-8, amylase activity of 1528 units. At 13.00, the pancreas and mesentery of the transverse colon took the form of a black film. By this time, there were changes in the necrotic nature of individual sections of the small and large intestines. Respiration was noted at 13.10, rare breathing at 14.25, at 14.35 respiratory arrest and death.

 A biopsy of the abdominal organs was performed. There is no effusion in the pleural cavity, venous stasis of the vessels of the anterior wall of the heart, spot hemorrhages at the top of the left lung under the pleura.

 Morphology: pancreas - edema, extensive hemorrhage, destruction of the structures of the acini, stasis in the vessels, blood clots. The glandular tissue is saturated with blood, many fields of necrosis. Preserved acini in a state of hypersecretion. A total of 12 experiments (control) were performed to reproduce acute hemorrhagic pancreatitis induced by active crystalline trypsin at a dose of 25 mg / kg of animal body weight. In all cases, there was a similar pattern of changes in the pancreas and other abdominal organs. Mortality in 100% of cases.

 Example 2. For the experiment, we used a white rat of the Wistar strain weighing 350 g, a medical history of 87 from 08/15/2000. Under general ether anesthesia at 11:00 a median laparatomy was performed. The duodenum and stomach were removed to the wound. In the course of the common bile duct, at the place where it enters the duodenum, a solution of active crystalline trypsin (25 mg / kg weight) is introduced into the pancreatic tissue with a syringe. Immediately, a colorless (creeping) infiltrate of 0.8 cm formed in the pancreatic tissue. After 3 minutes from the beginning of the experiment (11.04), a serous uniform edema of the pancreas developed, against which light hyperemia and pinpoint hemorrhages appeared along the dilated stellate vessels. At 11.05 (4 minutes after the disease was reproduced), a specific serum was introduced against the venom of the ordinary viper at the rate of 75 (AE) / kg body weight under the parietal peritoneum of the anterior abdominal wall.

 Over the next 2 minutes, hemorrhagic imbibition (tissue impregnation) of the pancreas progressively increased, and at 11.10 (9 minutes after the disease was reproduced) a bright red hematoma up to 1.5 cm in size without clear contours formed. Simultaneously with the occurrence of hemorrhagic imbibition (soaking) of pancreatic tissue, hemorrhagic exudate appeared in the abdominal cavity and there was hyperemia and swelling of the wall of the duodenum in the area up to 2.5 cm without clear boundaries at 11.09. The amount of exudate to 1.5 ml with an alkaline reaction (pH 7-8), amylase activity of 220 PIECES. 11 minutes after the disease was reproduced (11.12), hemorrhagic hemorrhagic hemorrhages appeared in the colon mesentery with changes in its color in separate areas in the form of hyperemic spots from 0.5-0.7 cm. Hyperemia in the form of separate spots of the small intestine wall arose at 11.14 (13 min after the disease was reproduced) A change in the color of the liver to a dark cherry hue occurred at 11.15 (14 minutes after the disease was reproduced).

 During a three-hour observation, the condition of the experimental animal remains stable. The amount of fluid (exudate) in the abdominal cavity without a tendency to increase, hemorrhagic impregnation of the pancreas became brighter (red), but of a local nature - up to 0.8 cm in diameter. The hyperemic area of the duodenum increased to 3 cm, but without signs of necrosis. The hyperemic sections of the colon and small remain without acquiring a dark color (without transition to necrosis). The liver has acquired the usual color.

 At 14.00, the abdominal cavity is sutured tightly and the animal is transferred to the cage for further observation in normal mode (water and food intake). After 24 hours, the rat was alive, active, took water and food.

 08.16.2000, operation relaparatomy, biopsy of the abdominal and pleural cavities.

 Under general ether anesthesia, the sutures were removed from the anterior abdominal wall and relaparotomy was performed. There is no effusion in the abdominal cavity. During an audit of the abdominal organs, it was found that in the pancreas at the injection site of trypsin, a disk-shaped infiltrate with the presence of fibrin and single point hemorrhages. The rest of the pancreas is pale gray, slightly infiltrated. Slight swelling of the wall of the duodenum without clear contours. No other abdominal changes were found. A biopsy of the pancreas, liver, and duodenum was performed. There is no fluid in the pleural cavity. In the area of the apex of the left lung under the visceral pleura, single point hemorrhages. Lung biopsy. After euthanasia, deepening of ether anesthesia produced a biopsy of the heart. No changes in the cavities of the heart and muscle were found macroscopically.

 Morphology: the structure of the pancreas is preserved, there are areas of dystrophic changes in the parenchyma of the gland, islet tissue unchanged, the vascular component is absent, islet tissue is unchanged. Liver - swelling of the tissue, signs of fatty degeneration, no necrosis. The duodenum - the structure of the villi is preserved, moderate edema.

 A total of 12 experiments were performed to reproduce acute hemorrhagic pancreatitis induced by active crystalline trypsin at a dose of 25 mg / kg of animal body weight. The treatment was carried out with a specific serum against viper venom ordinary at a dose of 75 (AE) / kg, which was administered within 1-5 minutes from the moment the disease was reproduced.

 With the introduction of specific serum, regardless of the timing of administration, the survival of animals in 100% of cases, while not all organs of the abdominal cavity were involved in the pathological process: liver, duodenum, small and thick.

 Example 3

 For the experiment, we used a white rat of the Wistar line, weighing 420 g, a medical history 105 of 09/14/2000. Under general ether anesthesia, at 13:00 midline laporatomy was performed. The duodenum and stomach were removed into the wound and along the common bile duct at the place where it entered the duodenum, a solution of active crystalline trypsin was introduced into the intestine at a dose of 25 mg / kg of animal body weight dissolved in 1 ml of distilled water.

 Immediately, a colorless creeping infiltrate of 0.7 cm in size formed in the pancreatic tissue. After 6 minutes (13.36) hyperemia and hemorrhagic impregnation of the pancreatic tissue appeared in the form of separate hemorrhages with subsequent formation of hemorrhages with subsequent formation of 1 min after 6 minutes (13.36). a hemorrhagic spot 0.6 cm in size. After that, an antivenant serum was introduced at a dose of 75 (AE) / kg body weight of the animal under the parietal peritoneum of the anterior abdominal wall at 13.36 (6 minutes after the experiment was reproduced) .

 A hemorrhagic effusion of up to 2 ml appeared at 13.38 in the abdominal cavity (8 minutes after the experiment was reproduced). In the study, the indicator established an alkaline exudate reaction (pH 7-8), amylase activity of 128 PIECES.

 Along with the appearance of hemorrhagic imbibition in 13.36 of the pancreas, mild hyperemia and edema of the duodenum appeared over 2.5 cm. By 13.30 the liver became darker in color (dark cherry, with a purple hue). By this time, hyperiminated areas of the small intestine and colon appeared.

 When observing the animal for three hours, the effusion in the abdominal cavity did not increase. Edema and hemorrhagic pancreatic gland imbalance was localized to 0.8 cm, remaining a bright red color, not acquiring a dark color (without transition to necrosis). Swelling and hyperemia of the duodenum stabilized within 3-4 cm without signs of necrosis. The liver has acquired a normal color. Areas of hyperemia of the colon and small intestine with no tendency to increase, without signs of necrosis.

 The general condition of the experimental animal remained stable, at 16.30 the abdominal cavity was sutured tightly. The animal is transferred to a cage with the usual regimen of water and food intake.

 After 24 hours, September 15, 2000 at 13.00, the surgery was performed relaparotomy, revision and biopsy of the abdominal and pleural cavities. Under general ether anesthesia, the sutures were removed from the anterior abdominal wall and relaparatomy was performed. There is no effusion in the abdominal cavity. When revising the abdominal organs, it was found that in the pancreas there is a disc-shaped infiltrate in the amount of up to 1 cm, a loose consistency, with single hemorrhages. The duodenum is slightly swollen in a limited area of up to 2 cm. No other changes from the abdominal organs were found. A biopsy of the pancreas, liver, and duodenal wall was performed.

 There is no fluid in the pleural cavity. In the area of the apex of the left lung, under the visceral pleura, point hemorrhages. No other changes found. Euthanasia by deepening ether anesthesia. Heart biopsy.

 Morphology: pancreas - the structure is preserved, there is no necrosis, the vascular component is absent, there are areas of dystrophic changes in the parenchyma, islet tissue is preserved. Liver - moderate edema, no necrosis. The duodenum - the structure of the villi is preserved, edema.

 A total of 12 experiments were performed to reproduce acute hemorrhagic pancreatitis induced by active crystalline trypsin at a dose of 25 mg / kg of animal body weight. The treatment was carried out with a specific serum against viper venom at a dose of 75 (AE) / kg, which was administered in a period of 6-9 minutes. At the same time, a clear picture of hemorrhagic pancreatitis with the involvement of other abdominal organs in the pathological process, but without the development of necrosis, developed. In all cases, after 24.00 the pathological process was stopped, the survival rate of animals was 100%.

 Example 4

 For the experiment, a white rat of the Wistar line was used, body weight 320 g, medical history 88, dated 08/15/2000

 Under general ether anesthesia at 11.15 a median laparotomy was performed. The duodenum and stomach were removed to the wound. In the course of the common bile duct at the place where it enters the duodenum, a solution of active crystalline trypsin (25 mg / kg of animal body weight) was injected into the pancreatic tissue with a syringe. Immediately, a colorless creeping infiltrate with a diameter of 0.8 cm formed in the pancreatic tissue. After 2 min (11 h 17 min) a uniform serous edema of the pancreas occurred. After another 5 min (11 h 22 min), against the background of mild hyperemia of the pancreas and its edema, spot hemorrhages appeared, followed by hemorrhagic imbibition in the form of a hematoma in the area up to 1.5 cm at the injection site of trypsin (at 11 h 23 min). At the same time as the onset of hemorrhagic pancreatic gland imbibition, hyperemia and edema of the pancreas occurred in a limited area up to 2.5 cm with a gradual progressive increase in the distal direction. At the same time, a change in the color of the intestinal wall from bright red to black (necrosis) was observed in the first 20 minutes from the beginning of the experiment (at 11 h 35 min).

 From the beginning of the appearance of hemorrhagic pancreatic gland imbibition (11 h 22 min), hemorrhagic exudate with amylase activity of 1800 units appeared. The amount of exudate in the abdominal cavity increased, the total amount was 3 ml.

 After 10 minutes (11 hours 25 minutes), hemorrhages appeared in the mesentery of the colon with changes in its color in the form of separate violet-red spots. At 11.25 (10 minutes after the disease was reproduced), a specific serum was introduced under the parietal peritoneum of the anterior abdominal wall against the venom of the ordinary viper at a dose of 75 (AE) / kg of the animal’s body weight. A liver color change from dark cherry to dark purple occurred in the first 15 minutes from the start of the experiment (11 h 30 min).

 Upon further observation of the experimental animal, it was found that changes in the pancreas, liver and duodenum progressively increased in the form of irreversible necrotic processes. Shortness of breath followed by the development of rare breathing and death occurred at 2.10 p.m.

 Morphology: pancreas - extensive hemorrhage, the tissue of the gland is saturated with blood, many fields of necrosis. In the vessels - stasis, blood clots. Preserved acini in a state of hypersecretion. The liver - edema, in the vessels - stasis, blood clots, areas of parenchyma necrosis. The duodenum - edema, necrosis of the villi. In the pleural cavity, a small amount of fluid, under the visceral pleura, point hemorrhage. Heart - a muscle of the heart is pale gray, with the presence of point hemorrhages.

 A total of 12 experiments were performed to reproduce acute hemorrhagic pancreatitis induced by active crystalline trypsin at a dose of 25 mg / kg of animal body weight. The treatment was carried out with a specific serum against viper venom ordinary at a dose of 75 (AE) / kg, which was administered within 10-15 minutes from the moment the disease was reproduced. In all cases, the same type of picture developed with the involvement of the abdominal organs in the pathological process with a mortality rate of 70-100% of cases.

CONCLUSION
The experiments showed that the use of specific serum against viper venom is a highly effective treatment for acute pancreatitis, leading to 100% survival.

 The optimal time for the introduction of antivenom serum, it is advisable to consider the terms 6-9 minutes from the moment of reproduction of the disease. With the early introduction of specific serum, animal survival in 100% of cases, while not all abdominal organs were involved in the pathological process: liver, duodenum, small and thick, with later administration, irreversible processes are observed that can lead to death.

 A dose of 75 (AE) / kg is considered optimal. At a lower dose, a decrease in the percentage of survival due to the progression of the pathological process is observed. At a dose of more than 75 (AE) / kg, the effect remains the same (100% survival), but due to the high cost of serum, increasing the dose is not advisable.

Sources of information
1. A. B. Prayers and others. Modern principles of conservative treatment of acute pancreatitis. // Surgery. - 1994. - 6. - S. 38-41.

 2. B. C. Savelyev et al. Comprehensive treatment of patients with pancreatic necrosis. // Annals of surgery. - 1999. - 1. - S. 18-22.

 3. A.S. Loginov and others. The experience of using sandostatin in patients with chronic pancreatitis. // Therapeutic archive. - 1995. - 7. - S. 60-62.

 4. A method of treating acute experimental pancreatitis. RF patent 2043359, IPC A 61 K 38/43.

 5. Instructions for use of serum against the venom of a viper ordinary horse, purified concentrated liquid.

Claims (1)

A method of treating acute experimental pancreatitis by parenteral administration of a drug, characterized in that serum against the venom of ordinary viper is injected under the parietal peritoneum of the anterior abdominal wall as a drug after 6-9 minutes from the start of disease reproduction at a rate of 75 AU / kg body weight.

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