RU2497520C1 - Method for correction of disturbed functional activity of platelets - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present invention refers to medicine, particularly to pharmacology, and describes a method for correction of disturbed functional activity of platelets, consisting in using Melaxen as a corrector for the disaggregation and hyperaggregation state of the platelets to be orally administered into white non-linear mature rats in a dose of 1 mg/kg once a day within the 7-day therapeutic course.

EFFECT: invention aims at extending the range of preparations having an ability to control the aggregation properties of platelets depending on the nature of the haemostatic disorders.

4 tbl, 4 ex

Description

The invention relates to medicine, in particular to pharmacology, and can be used to prevent thrombohemorrhagic disorders in the hemostatic system.

One of the key mechanisms of hemostasis is the balanced functional activity of platelets, where an increase in their aggregation ability can provoke an increase in thrombosis, and a decrease in the development of hemorrhagic syndrome in organs (Barkagan Z.S., Momont A.P. Diagnosis and controlled therapy of hemostatic disorders. Edition 2 supplemented. M: Newdiamed, 2001, 296 p.). Numerous experimental and clinical studies have shown that increased platelet aggregation ability accompanies a number of severe pathological conditions: myocardial infarction, cerebrovascular accident, diabetes mellitus, hypertension, etc. It is known that with the progression of the above diseases, blood hypercoagulation and depression of fibrinolysis increase. In the treatment and prevention of diseases and conditions accompanied by platelet hyperaggregation, antiplatelet agents are currently widely used: pentoxifylline (trental); ticlopedin (ticlide), acetylsalicylic acid, etc., the disadvantage of which are hemorrhagic complications, thrombopenia, leukopenia, agranulocytosis, severe allergic reactions, as well as the inability to use these drugs due to their contraindications for severe sclerosis of coronary vessels and cerebral vessels, with exacerbation diseases of the stomach and duodenum, liver diseases, pregnancy and breastfeeding (Belousov Yu.B., Leonova MV, Belousov D.Yu. et al. Fundamentals of clinical pharmacology and rational rmakoterapii / Hand-in for doctors // Edited by Belousov YB, Leonova MV - M .: Bionics, 2002. - 368 c).. With their prolonged use, tolerance to drugs with activation of thrombus formation processes is also possible (Chen W., Lee P., Ng W. et al. Aspirin resistance is associated with a high incidence ofmyonecrosis after non-urgent percutaneous coronary intervention despite clopidogrel pretreatment // J. Am. Coll. Cardiol. - 2004. - Vol. 43. - P.1122-1126) (Dalen JE Aspirin Resistance: Is it Real? Is it Clinically Significant? // Am. J. Med. - 2007. - Vol.120. - No. 1. - P.1-4).

Reduced platelet aggregation ability, which is often formed during the use of drugs with antiaggregation activity, is the cause of hemorrhagic complications. In practical medicine, there are ways to eliminate them, which indirectly have a hemostatic effect. For example, the use of thromboconcentrate is aimed at replenishing platelets in the bloodstream, and drugs such as adroxon and dicinone have antihyaluronidase activity and help stimulate the formation of thromboplastin (I.B.Mikhailov. Doctor’s handbook on clinical pharmacology. - A guide for doctors. - St. Petersburg: Publishing House "Foliant", 2001. - 736 p.). However, the use of these methods is justified in the presence of clinical symptoms of hemorrhagic syndrome, and not for the prevention of its occurrence.

In view of the foregoing, it becomes apparent that many drugs affecting the functional activity of platelets have a unidirectional effect. In this regard, this invention is aimed at expanding the arsenal of drugs with the ability to regulate the aggregation properties of platelets depending on the direction of the disorders in the hemostatic system, which is the objective of the proposed method.

The specified technical result is achieved by the fact that melaxen is used as a corrector for the disaggregation and hyperaggregation state of platelets, which is administered orally to white non-linear adult rats at a dose of 1 mg / kg once a day for 7 days.

A distinctive feature of the proposed method is that the authors for the first time to correct the disaggregation and hyperaggregation state of platelets arising under the conditions of an “aspirin” model of hypoaggregation, as well as enhance their functional activity by calcium chloride (hyperaggregation model), use melaxen in the appropriate dosage range, determining the degree of effectiveness each of the doses in the experiment.

The proposed method allows to exclude a complete blockade of the platelet aggregation process, which is physiological in the body and necessary to maintain balance in all links of hemostasis. In addition, taking into account the features of the selected model - “aspirin” hypoaggregation, it is possible to predict the success of the prevention of hemorrhagic complications with the use of acetylsalicylic acid.

The method was carried out as follows: for the study used white non-linear rats of both sexes aged 5-7 months. In each series of animals, they were divided into the following groups (n = 8): the 1st group received an equivalent volume of 0.9% sodium chloride solution as a “placebo” (control 1); The 2nd group is represented by animals with a model of “aspirin” hypoaggregation induced by oral administration of acetylsalicylic acid at a dose of 20 mg / kg (control 2); 3rd and 4th groups - animals received melaxen in doses of 1 mg / kg and 10 mg / kg, respectively (control 3 and 4); 5th — formed from animals with a model of hyperaggregation induced by intraperitoneal administration of 0.1 ml of 10% calcium chloride solution per 1 ml of 0.9% sodium chloride solution (control 5); 6th and 7th - animals received melaxen in doses of 1 mg / kg and 10 mg / kg, respectively, under the conditions of an “aspirin” hypoaggregation (experiment).

The functional activity of platelets was evaluated on the basis of the aggregation activity of one platelet (AAT) at the maximum (-2) and minimum (-6) dose of hemolysate as an inducer of ADP platelet aggregation and platelet activation index (IAT) obtained during the hemolysate aggregation test ( GAT) according to the method of L.Z. Barkagan et al. (Barkagan L.Z., Arkhipova B.F., Kutskogo V.M. Medical Laboratory Technologies and Diagnostics: Reference. Medical Laboratory Technologies / Edited by A.I. Karpishchenko. - St. Petersburg: Intermedika, 1999. - P. 273-275).

Studies in all series were carried out in the autumn-winter period, all animals were kept under standard vivarium conditions with alternating natural and artificial lighting to block the synthesis of endogenous melatonin and its effect on hemostasis and were synchronized in nutrition with free access to water. At the end of the experiments, the animals were killed by decapitation under chloroform anesthesia.

All manipulations with animals were performed in accordance with the International GLP Rules. All data obtained were statistically processed using Student's t-test with Bonferroni correction for multiple comparisons. The proposed method was tested on animals at the Department of Pharmacognosy, Pharmaceutical Technology and Biotechnology SBEI HPE "Astrakhan State Medical Academy". Below are the results of testing

Example 1

We studied the ability of melaxen at a dose of 1 mg / kg to maintain the aggregation properties of platelets under the conditions of taking acetylsalicylic acid, which is capable of irreversibly blocking the cyclooxygenase route of thromboxane synthesis, thereby causing persistent platelet disaggregation.

The experiment was carried out according to the previously described scheme. The animals of the experimental group while taking acetylsalicylic acid orally received melaxen 1 time per day at a dose of 1 mg / kg for 7 days. The results are shown in table 1.

As shown in table 1, the administration of acetylsalicylic acid was not accompanied by a significant decrease in the number of platelets in control group 2, but their aggregation activity was completely “blocked”, which was manifested by a decrease in indicators reflecting the aggregation activity of one platelet (AAT) at the maximum (-2 ) and a minimum (-6) dose of hemolysate as an inducer of ADP-platelet aggregation, as well as a platelet activation index. In control group 3, there was a significant decrease in platelet count relative to control 1, but the level of active platelets (IAT) did not change. In the experimental group, the platelet count significantly increased only relative to the third and second groups. Under the influence of melaxen at a dose of 1 mg / kg, the disaggregation effect of acetylsalicylic acid was reliably leveled, since platelet aggregation occurred under the influence of both high and low doses of ADP, and the level of active platelets significantly increased relative to all control groups.

Example 2

We studied the ability of melaxen at a dose of 10 mg / kg to maintain the aggregation properties of platelets under the conditions of taking acetylsalicylic acid, which is capable of irreversibly blocking the cyclooxygenase pathway for the synthesis of thromboxane, thereby causing persistent platelet disaggregation.

The experiment was carried out according to the previously described scheme. The animals of the experimental group while taking acetylsalicylic acid orally received melaxen once a day at a dose of 10 mg / kg for 7 days. The results are shown in table 2.

As shown in table 2, the use of melaxen in a dose of 10 mg / kg was not accompanied by significant changes in the number of platelets, but at the same time their aggregation ability under the influence of acetylsalicylic acid remained. This follows from the fact that the indicators of functional activity in the experimental group reliably approached the control values of the first and fourth groups and the level of active platelets increased almost 1.5 times.

Example 3

We studied the ability of melaxen at a dose of 1 mg / kg to suppress the functional activity of platelets under the conditions of platelet hyperaggregation under the influence of calcium chloride.

The experiment was carried out according to the previously described scheme. The animals of the experimental group against the background of intraperitoneal administration of 0.1 ml of 10% calcium chloride solution per 1 ml of 0.9% sodium chloride solution were orally given melaxen 1 time per day at a dose of 1 mg / kg for 7 days. The results are shown in table 3.

As shown in table 3, under the influence of calcium chloride, platelet aggregation activity (AAT) significantly increased regardless of the inducer dose, which is probably due to an increase in the influx of Ca ++ ions into platelets, while the number of their active forms did not change (IAT). In animals of the experimental group, under the influence of melaxen, against the background of a significant increase in the number of platelets and an increase in the active forms, their aggregation ability significantly decreased to control values of the first group under the influence of high doses of ADP.

Example 4

We studied the ability of melaxen at a dose of 10 mg / kg to suppress the functional activity of platelets under conditions of hyperaggregation under the influence of calcium chloride.

The experiment was carried out according to the previously described scheme. Animals of the experimental group against the background of intraperitoneal administration of 0.1 ml of 10% calcium chloride solution per 1 ml of 0.9% sodium chloride solution received melaxen once a day at a dose of 10 mg / kg for 7 days. The results are shown in table 4.

As shown in table 4, the number of platelets in the experimental group did not change, but their aggregation activity under the influence of minimum doses of ADP relative to control 1 and 5 (p <0.05), and under the influence of maximum only in comparison with control 1 (p <0.01) significantly increased. The platelet activation index in all groups did not change.

An analysis of the results reflected in the above examples shows that melaxen at a dose of 1 mg / kg body weight has a corrective ability with respect to the functional activity of platelets, depending on the direction of change. So, when this drug is administered under conditions of “aspirin” hypoaggregation, the disaggregation effect of acetylsalicylic acid is leveled almost to background values, while in conditions of hyperaggregation, melaxen also reduces platelet aggregation activity to control values.

The proposed method achieves the correctional effectiveness of functional disorders of platelet aggregation ability that occur against the background of the development of disaggregation caused by the use of acetylsalicylic acid and hyperaggregation under the influence of calcium chloride.

The high efficiency of the proposed method lies in the fact that melaxen is used as a corrector for the disaggregation and hyperaggregation state of platelets and when it is administered at a dose of 1 mg / kg simultaneously with a substance that can cause a sharp decrease in platelet aggregation activity as a cause of hemorrhagic bleeding, it retains the physiological potential platelets to the processes of aggregation, while in conditions of hyperaggregation-reduces their functional activity.

Table 1 The effect of melaxen in a dose of 1 mg / kg on the functional activity of platelets in conditions of "aspirin" hypoaggregation GAT indices of animals Platelet Count (M ± m, x 10 ¹² / L) AAT (-2) (M ± m,%) AAT (-6) (M ± m,%) IAT (M ± m) Control 1: saline (n = 8) 852.0 ± 53.1 150.6 ± 18.2 308.0 ± 22.7 2.6 ± 0.31 Control 2: Acetylsalicylic. acid (20 mg / kg) (n = 8) 766.0 ± 49.5 0 *** 0 *** 0 *** Control 3: Melaxen (1 mg / kg) (n = 8) 720 ± 21.8 * 171.0 ± 28.1 294.5 ± 30.5 2.3 ± 0.31 Experience: acetylsalicylic acid (20 mg / kg) + Melaxen (1 mg / kg) (n = 8) 1007.2 ± 7.1 ^{◇◇◇ #} 72.0 ± 12.0 ^### 275.9 ± 36.7 ^### 4.6 × 0.6 ^{** ### ◇◇} Note: * - p <0.05 in comparison with control 1; ** - p <0.01; *** - p <0.001; # - p <0.05 in comparison with control 2; ## - p <0.01; ### - p <0.001; ◇ - p <0.05 in comparison with control 3; ◇◇ - p <0.01; ◇◇◇ - p <0.001; GAT - hemolysate-aggregation test; AAT (-2) - platelet aggregation activity at the maximum dose of ADP; AAT (-6) - platelet aggregation activity at a minimum dose of ADP; IAT is the platelet activation index.

table 2 The effect of melaxen in a dose of 10 mg / kg on the functional activity of platelets in conditions of "aspirin" hypoaggregation GAT indices of animals Platelet Count (M ± m, x 10 ¹² / L) AAT (-2) (M ± m,%) AAT (-6) (M ± m,%) IAT (M ± m) Control 1: saline (n = 8) 852.0 ± 53.1 150.6 ± 18.2 308.0 ± 22.7 2.6 ± 0.31 Control 2: Acetylsalicylic. acid (20 mg / kg) (n = 8) 766.0 ± 49.5 0 *** 0 *** 0 *** Control 4: Melaxen (10 mg / kg) (n = 8) 810.0 ± 45.6 179.4 ± 26.4 298.2 ± 24.9 2.5 ± 0.27 Experience: Acetylsalicylic acid (20 mg / kg) + Melaxen (10 mg / kg) (n = 8) 978.6 ± 61.6 159.5 ± 19.2 ^### 351.0 ± 24.1 ^### 4.0 ± 0.16 ^{** ### ◇◇} Note: * - p <0.05 in comparison with control 1; ** - p <0.01; *** - p <0.001; # - p <0.05 in comparison with control 2; ## - p <0.01; M # - p <0.001; ◇ - p <0.05 in comparison with control 3; ◇◇ - p <0.01; ◇◇◇ - p <0.001; GAT - hemolysate-aggregation test; AAT (-2) - platelet aggregation activity at the maximum dose of ADP; AAT (-6) - platelet aggregation activity at a minimum dose of ADP; IAT is the platelet activation index.

Table 3 The effect of melaxen in a dose of 1 mg / kg on the functional activity of platelets with their increased aggregation ability under the influence of calcium chloride GAT indices of animals Platelet Count (M ± m, x 10 ¹² / L) AAT (-2) (M ± m,%) AAT (-6) (M ± m,%) IAT (M ± m) Control 1: saline (n = 8) 852.0 ± 53.1 150.6 ± 18.2 308.0 ± 22.7 2.6 ± 0.31 Control 5: calcium chloride (0.1 ml of a 10% solution) (n = 8) 904.6 ± 67.3 236.3 ± 24.1 ** 421.6 ± 19.9 ** 2.3 ± 0.12 Control 3: Melaxen (1 mg / kg) (n = 8) 720.0 ± 21.8 171.0 ± 28.1 294.5 ± 30.5 2.3 ± 0.31 Experience: calcium chloride (0.1 ml of a 10% solution) + melaxen (1 mg / kg) (n = 8) 1091.0 ± 13.6 170.1 ± 19.0 ^# 385.7 ± 14.8 3.1 ± 0.17 ^### Note: * - p <0.05 in comparison with control 1; ** - p <0.01; *** - p <0.001; # - p <0.05 in comparison with control 2; ## - p <0.01; M # - p <0.001; ◇ - p <0.05 in comparison with control 3; ◇◇ - p <0.01; ◇◇◇ - p <0.001; GAT - hemolysate-aggregation test; AAT (-2) - platelet aggregation activity at the maximum dose of ADP; AAT (-6) - platelet aggregation activity at a minimum dose of ADP; IAT is the platelet activation index.

Table 4 The effect of melaxen in a dose of 10 mg / kg on the functional activity of platelets with their increased aggregation ability under the influence of calcium chloride GAT indices of animals Platelet Count (M ± m, x 10 ¹² / L) AAT (-2) (M ± m,%) AAT (-6) (M ± m,%) IAT (M ± m) Control 1: saline (n = 8) 852.0 ± 53.1 150.6 ± 18.2 308.0 ± 22.7 2.6 ± 0.31 Control 5: calcium chloride (0.1 ml of a 10% solution) (n = 8) 904.6 ± 67.3 236.3 ± 24.1 ** 421.6 ± 19.9 ** 2.3 ± 0.12 Control 3: Melaxen (10 mg / kg) (n = 8) 810.0 ± 45.6 179.4 ± 26.4 298.2 ± 24.9 2.5 ± 0.27 Experience: Calcium chloride (0.1 ml of a 10% solution) + melaxen (10 mg / kg) (n = 8) 888.0 ± 55.2 238.1 ± 17.3 ** 487.3 ± 16.4 * ^# 2.1 ± 0.19 Note: * - p <0.05 in comparison with control 1; ** - p <0.01; *** - p <0.001; # - p <0.05 in comparison with control 2; ## - p <0.01; M # - p <0.001; ◇ - p <0.05 in comparison with control 3; ◇◇ - p <0.01; ◇◇◇ - p <0.001; GAT - hemolysate-aggregation test; AAT (-2) - platelet aggregation activity at the maximum dose of ADP; AAT (-6) - platelet aggregation activity at a minimum dose of ADP; IAT is the platelet activation index.

Claims (1)

A method for correcting impaired functional activity of platelets, which consists in the use of melaxen as a corrector for the disaggregated and hyperaggregated state of platelets, which is administered orally to white non-linear adult rats at a dose of 1 mg / kg once a day for 7 days.