RU2645630C1 - Method of prevention of bleeding caused by the application of antiagregants, experiment stage - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, in particular to experimental surgery, and can be used to prevent bleeding when applying double antiplatelet therapy before surgery. To this end, a solution containing fibrin monomer at a concentration of 11 mg/ml and urea at a concentration of 150 mg/ml is given to a rabbit-male chinchilla one hour before the operation. In this case, the dose of fibrin monomer is 0.25 mg/kg.

EFFECT: method is highly effective, since it not only reduces the amount of blood loss, but also is safe when applied.

1 cl, 2 dwg

Description

The invention relates to medicine, namely to experimental surgery, and can be used to prevent bleeding while using antiplatelet agents.

The use of a combination of acetylsalicylic acid and clopidogrel in a large number of cardiac patients allows for higher efficacy in the prevention of serious cardiovascular events. These advantages are especially pronounced in patients with acute coronary syndrome, as well as in patients who undergo percutaneous coronary intervention, especially after coronary stenting. However, in low-risk patients (for example, with a stable course of cardiovascular disease), such a combination is not justified because the potential benefits overlap with the risk of hemorrhagic complications (M. Koval. Double antiplatelet therapy - from emergency therapy to long-term maintenance treatment / M. Koval // Medicine Review. - 2008. - No. 3 (03). - S. 46-50).

The risk of severe blood loss in patients taking dual antiplatelet therapy increases sharply with injuries or emergency surgical interventions performed for health reasons. This is due to the fact that after discontinuation, the disaggregant effect of aspirin and clopidogrel persists for about 5 days. Therefore, it is currently relevant to create effective methods for the prevention of bleeding caused by the use of antiplatelet agents, which allows quickly, with a minimum number of adverse reactions, to reduce the amount of blood loss.

E, Filipescu D, Hunt BJ, Komadina R, Nardi G, Neugebauer EA, Ozier Y, Riddez L, Schultz A, Vincent JL, Spahn DR. // Crit Care. - 2016 Apr, p. 35-41), которая содержит ресуспендированный осадок тромбоцитов в небольшом объеме плазмы. Стандартную дозу получают из 450 мл цельной крови с содержанием тромбоцитов не менее чем 55×10⁹ клеток. Этого для одной трансфузии недостаточно. Для эффективной терапии требуется 50-70×10⁹ клеток крови на каждые 10 кг массы тела пациента. Необходимое для переливания тромбомассы количество клеток получают от 6-10 донорских доз крови.A known method for the prevention and treatment of bleeding caused by the use of antiplatelet agents by transfusion of platelet mass (Rossaint R. The European guideline on management of major bleeding and coagulopathy following trauma: fourthedition. / Rossaint R. Bouillon B, Cerny V, Coats TJ, Duranteau J,

E, Filipescu D, Hunt BJ, Komadina R, Nardi G, Neugebauer EA, Ozier Y, Riddez L, Schultz A, Vincent JL, Spahn DR. // Crit Care. - 2016 Apr, p. 35-41), which contains a resuspended platelet pellet in a small plasma volume. A standard dose is obtained from 450 ml of whole blood with a platelet content of at least 55 × 10 ⁹ cells. This is not enough for a single transfusion. Effective therapy requires 50-70 × 10 ⁹ blood cells for every 10 kg of patient body weight. The number of cells required for transfusion of thrombomass is obtained from 6-10 donor doses of blood.

However, the use of platelet mass is associated with risk, because there is a chance of infection with both viral hepatitis and human immunodeficiency virus (HIV). In addition, side effects are observed, such as febrile and anaphylactic reactions, which are caused by sensitization by plasma proteins, which contain platelets (Akwaa, F. Treatment of bleeding complications when using oral anticoagulants for prevention of strokes. / F. Akwaa, AC Spyropoulos // Curr Treat Options Cardiovasc - 2013 .-- Vol. 15, No. 3. - P. 288-298).

A known method of treating bleeding caused by the use of antiplatelet agents, by the use of sodium ethamylate. Ethamsylate sodium is administered intravenously in a dose of 125-250 mg, a multiplicity of 3-4 times a day. The well-known drug inhibits the effects of prostacyclin, in connection with which platelet aggregation is enhanced, it stimulates platelet enzyme reactions, their formation from megakaryocytes and exit from the depot, enhances the platelet release reaction. In addition, sodium etamzilate moderately accelerates the formation of tissue factor (V. Cherniy. Principles of intensive therapy of disorders in the hemostatic system in critical conditions / V. Cherniy // “Medicine of emergency conditions”, Journal. - 2009, No. 5 - p. 24).

Z. // Scandinavian Journal of Clinical and Laboratory Investigation. - 2013. - Vol. 73 - Issue 5).However, the use of sodium ethamylate is associated with a risk of adverse reactions, such as: a marked decrease in blood pressure, anaphylactic shock, asthma attacks, asthenia, thromboembolism (Etamsylate): instructions, use and formula // CAS code 2624-44-4 // Drug Register, Radar // 2016), as well as an increase in serum creatinine concentration (Wiewiorka O. Strong negative interference of ethamsylate (Dicynone®) in serum creatinine quantification via enzymatic assay using Trinder reaction. / Wiewiorka O, Dastych M,

Z. // Scandinavian Journal of Clinical and Laboratory Investigation. - 2013 .-- Vol. 73 - Issue 5).

The authors propose an effective method for the prevention of bleeding caused by the use of antiplatelet agents in the experiment, which allows clinically significant prevention of hemorrhage with the minimum number of adverse reactions through the use of fibrin monomer, which is a protein obtained from sterile human blood plasma. Fibrin monomer is not an antidote of antiplatelet agents and can be used as a systemic hemostatic agent, does not require additional use of platelet mass. The fibrin monomer provides a quick and stable stop of intraoperative parenchymal bleeding, which is confirmed in an animal experiment, as well as in vitro when a patient receiving antiplatelet agents is added to human plasma and leads to the rapid formation of a persistent fibrin clot.

Fibrin monomer is the first systemic hemostatic that can be used to prevent bleeding caused by the use of antiplatelet agents, not being their antidote and not acting directly on them.

The technical result of the claimed invention is the creation of an effective method for the prevention of bleeding caused by the use of antiplatelet agents before surgery.

The technical result is achieved in that male rabbits of the chinchilla breed are injected one hour before surgery with a solution containing fibrin monomer at a concentration of 11 mg / ml and urea at a concentration of 150 mg / ml, while the dose of fibrin monomer is 0.25 mg / kg

The inventive method is illustrated by figures 1 and 2 in the form of tables.

In FIG. 1 shows the selection of an effective and safe dose of fibrin monomer.

In FIG. 2 illustrates indicators of bleeding and hemostasis in animals of the control and experimental groups.

In figures 1 and 2, the data are presented as follows:

, где

- выборочное среднее; m - стандартная ошибка выборочного среднего.one.

where

- sample mean; m is the standard error of the sample mean.

2. The volume of blood loss,% of the bcc - part of the lost blood of its total circulating volume (bcc), expressed as a percentage.

3. The concentration of D-dimers in blood plasma, ng / ml - the content of degradation products of stabilized fibrin in blood plasma, which are markers of the formation of fibrin and its dissolution under the action of plasmin.

4. ADP-induced platelet aggregation,% - platelet aggregation, with the induction of ADP.

5. p - level of statistical significance of the differences of the compared indicators.

6. n is the number of animals in the group.

The method is as follows.

In the experiment, male chinchilla rabbits weighing 2-4 kg are used. For the prevention of bleeding, a fibrinogen derivative from human blood plasma is used - fibrin monomer (LLC Technologiya Standard), which is a freeze-dried soluble white powder with urea, packaged in a penicillin bottle, closed with a rubber stopper and crimped with an aluminum cap, in the amount of 22 mg in a bottle, which is prepared as follows: in a bottle with freeze-dried fibrin monomer with urea, 2 ml of sterile distilled water for injection was added, mix accurately nym shaking without foaming until complete dissolution of the substance during 2 to 3 minutes. The resulting solution contained fibrin monomer at a concentration of 11 mg / ml and urea at a concentration of 150 mg / ml.

A placebo solution — a urea solution at a concentration of 150 mg / ml, which does not contain the active substance — fibrin monomer — was used as a control. 2 ml of sterile distilled water for injection is added to the vial with the lyophilized dried urea solution, mixed with gentle swaying until the substance is completely dissolved within 2 to 3 minutes.

The most effective dosage of fibrin monomer was selected experimentally. In the experiment, 82 male chinchilla rabbits were used. All animals were divided into 7 groups. The dose of fibrin monomer was selected from a dosage range from 0.1 mg / kg to 5.0 mg / kg body weight. Data confirmed by figure 1.

Experiment design

The first group consisted of 11 animals that were given a 0.5 ml placebo intravenously. One hour after the administration of a placebo solution, blood was drawn from the marginal vein of the ear to assess the concentration of D-dimers in blood plasma. It is described that an increase in the concentration of D-dimers in blood plasma accompanies venous thrombosis and pulmonary thromboembolism (Savelyev VS, Chazov EI, Gusev EI, Kiriyenko AI, etc. Russian clinical recommendations for diagnosis, treatment and prevention of venous thromboembolic complications // Phlebology. - 2010. - T. 4, No. 1. - Issue 2. - P. 2-37). After this, the animals were anesthetized and they underwent median laparotomy along the white line of the abdomen under general anesthesia. The left lobe of the liver was removed into the wound and a standard injury in area and depth was inflicted on its diaphragmatic surface using a special limiting device (a metal plate with a round hole in the center). The cut-off segment in a vertical projection had the form of a circle or an ellipse, its size and shape were constant (Manual on the experimental (preclinical) study of new pharmacological substances / edited by R.Yu. Khabriev. - 2 ed., Revised and ext. - M .: OJSC - Publishing house "Medicine", 2005. - 828 p.). The resulting bleeding wound with smooth edges and uniform curvature had a diameter of about 1.5 cm, a depth of about 0.5 cm. After applying a standard injury, the volume of blood loss was measured. This indicator was determined by blotting previously weighed dry sterile wipes with blood from a liver wound, until the bleeding stopped completely with the formation of a stable clot. Wipes were weighed on an electronic balance immediately after blotting. The weight of all wipes was summarized and the volume of blood lost was taken into account. The volume of blood loss was calculated as a percentage of its total circulating volume.

The second group consisted of 12 animals, which were injected intravenously with a fibrin monomer at a dose of 0.1 mg / kg.

The third group consisted of 15 animals, which were injected intravenously with a fibrin monomer at a dose of 0.25 mg / kg.

The fourth group consisted of 12 animals that were injected with the fibrin monomer at a dose of 0.5 mg / kg.

The fifth group consisted of 10 animals, which were injected intravenously with fibrin monomer at a dose of 1.0 mg / kg

The sixth group consisted of 13 animals, which were injected intravenously with a fibrin monomer at a dose of 2.5 mg / kg.

The seventh group consisted of 9 animals, which were injected intravenously with fibrin monomer at a dose of 5.0 mg / kg

One hour after the injection of the fibrin monomer solution in animals of each group, blood was drawn from the marginal vein of the ear to assess the concentration of D-dimers in blood plasma. After this, the animals were anesthetized and they underwent median laparotomy along the white line of the abdomen under general anesthesia with a liver injury and determination of the volume of blood loss according to the scheme described above.

As can be seen from FIG. 1, the most effective doses of fibrin monomer for a statistically significant reduction in blood loss are 0.25 mg / kg and 2.5 mg / kg (p <0.001 in both cases). At the same time, doses of fibrin monomer above 1.0 mg / kg have significant thrombogenicity, which was assessed by an increase in the concentration of D-dimers in blood plasma, which are a classic marker of fibrin formation and fibrinolysis. Thus, the dose of fibrin monomer 0.25 mg / kg was chosen as the most effective and safe for further experiments.

To study the effectiveness of the proposed method, all animals were divided into two groups: the first group - control, consisting of 9 animals, received Plavix in combination with thrombotic ACC and placebo; the second group - experimental, consisting of 9 animals, received Plavix in combination with thrombotic ACC and fibrin monomer.

Experiment design

1. Two days before the experiment, blood was taken from the marginal ear vein from animals of the control group (Guidelines for the experimental (preclinical) study of new pharmacological substances / edited by R.Yu. Khabriev - 2nd ed., Revised and additional - M. : OJSC - Publishing House "Medicine", 2005. - 828 p.) For the study of initial indicators of the hemostatic system. A specific test of the coagulogram for the action of antiplatelet agents is ADP-induced platelet aggregation (Barkagan Z. S., Momot A. P. Diagnosis and controlled therapy of hemostatic disorders. / M.: Publishing House: "Newdiamed-AO", 2001. - 296 p. ) On the day of the experiment, animals in the control group were enterically administered Plavix (Plavix (Plavix) (Clopidogrel (Clopidogrel)), Sanofi-Winthrop Industrie (France)), registration number: P No. 015542/01) at a dose of 8 mg per kg of body weight and thrombo ACC (thrombo ASS (Thrombo ASS) (Acetylsalicylic acid (Acetylsalicylic acid)), Lannacher Heilmittel GmbH (Austria)), registration number: P No. 013722 / 01-2002) at a dose of 2 mg per kg of body weight. Doses of Plavix and thrombotic ACC were one and a half times the dose for therapeutic use in humans during coronary artery bypass grafting.

An hour after administration of antiplatelet agents, a 0.5 ml placebo was administered intravenously. One hour after the administration of a placebo solution, blood was drawn from the marginal ear vein to evaluate the hemostatic system of animals. After blood sampling, the animals were anesthetized and they underwent median laparotomy along the white line of the abdomen under general anesthesia. The left lobe of the liver was removed into the wound and a standard injury in area and depth was inflicted on its diaphragmatic surface using a special limiting device (a metal plate with a round hole in the center). The cut-off segment in a vertical projection had the form of a circle or an ellipse, its size and shape were constant (Manual on the experimental (preclinical) study of new pharmacological substances. Edited by R.Yu. Khabriev, 2nd ed., Revised and enlarged; M. ; OJSC "Publishing house" Medicine ", 2005. - 828 p.). The resulting bleeding wound with smooth edges and uniform curvature had a diameter of about 1.5 cm, a depth of about 0.5 cm.

After applying a standard injury, the volume of blood loss was measured. This indicator was determined by blotting previously weighed dry sterile wipes with blood from a liver wound, until the bleeding stopped completely with the formation of a stable clot. Wipes were weighed on an electronic balance immediately after blotting. The weight of all wipes was summarized and the volume of blood lost was taken into account. The volume of blood loss was calculated as a percentage of its total circulating volume.

2. Two days before the experiment, blood was taken from the marginal ear vein from animals of the control group (Guidelines for the experimental (preclinical) study of new pharmacological substances / edited by R.Yu. Khabriev. - 2nd ed., Revised and additional - M .: OJSC - Publishing house "Medicine", 2005. - 828 p.) For the study of the initial indicators of the hemostatic system. A specific test of the coagulogram for the action of antiplatelet agents is ADP-induced platelet aggregation (Barkagan Z.S., Momot A.P. Diagnostics and controlled therapy of hemostatic disorders. / M., Publishing House: "Newdiamed-AO", 2001. - 296 p. ) On the day of the experiment, animals in the control group were enterically administered Plavix (Plavix (Plavix) (Clopidogrel (Clopidogrel)), Sanofi-Winthrop Industrie (France)), registration number: P No. 015542/01) at a dose of 8 mg per kg of body weight and thrombo ACC (thrombo ASS (Thrombo ASS) (Acetylsalicylic acid (Acetylsalicylic acid)), Lannacher Heilmittel GmbH (Austria)), registration number: P No. 013722 / 01-2002) at a dose of 2 mg per kg of body weight. Doses of Plavix and thrombotic ACC were one and a half times the dose for therapeutic use in humans during coronary artery bypass grafting.

An hour after taking antiplatelet agents, a fibrin monomer at a dose of 0.25 mg / kg was intravenously administered. One hour after the administration of the fibrin monomer solution, the animals were anesthetized and they underwent median laparotomy along the white line of the abdomen under general anesthesia. The left lobe of the liver was removed into the wound and a standard injury in area and depth was inflicted on its diaphragmatic surface using a special limiting device (a metal plate with a round hole in the center). The cut-off segment in a vertical projection had the form of a circle or an ellipse, its size and shape were constant (Manual on the experimental (preclinical) study of new pharmacological substances. Edited by R.Yu. Khabriev, 2nd ed., Revised and enlarged; M. ; OJSC "Publishing house" Medicine ", 2005, - 828 p.). The resulting bleeding wound with smooth edges and uniform curvature had a diameter of about 1.5 cm, a depth of about 0.5 cm.

After applying a standard injury, the volume of blood loss was measured. This indicator was determined by blotting previously weighed dry sterile wipes with blood from a liver wound, until the bleeding stopped completely with the formation of a stable clot. Wipes were weighed on an electronic balance immediately after blotting. The weight of all wipes was summarized and the volume of blood lost was taken into account. The volume of blood loss was calculated as a percentage of its total circulating volume.

The effectiveness of the proposed method is illustrated in FIG. 2, which shows comparative data on the volume of blood loss, the concentration of D-dimers and the hemostatic system, which allows us to demonstrate that the volume of blood loss (in% of the volume of circulating blood) in the experimental group was 5.2 times lower than the control group (p <0.001 )

According to the data presented, when comparing the hemostasis system parameters in both groups of animals, the effect of antiplatelet agents in the form of a decrease in ADP-induced platelet aggregation was demonstrated, which corresponded to the previously described changes in the hemostasis of human blood plasma (A.P. Momot Monitoring the use of dabigatran as a means of prevention thromboembolic complications after arthroplasty of the hip joint / A.P. Momot, E.V. Grigoriev, L.P. Tsyvkina // Clinical Laboratory Diagnostics: Scientific and Practical Journal -. 2012. - №5 -. S. 40-42 -. ISSN 0869-2084).

Thus, the claimed method is highly effective, allows not only to reduce the amount of blood loss, but also does not increase the likelihood of thrombosis with a minimum of adverse reactions.

Claims (1)

A method for the prevention of bleeding caused by the use of double antiplatelet therapy in an experiment, which consists in the fact that male rabbits of the chinchilla breed are injected one hour before surgery with a solution containing fibrin monomer at a concentration of 11 mg / ml and urea at a concentration of 150 mg / ml while the dose of fibrin monomer is 0.25 mg / kg

Images