RU2033791C1 - Agent showing antiaggregation activity - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: proposed agent is 5-oxa-6-ketoprostaglandin E₁ shows antiaggregation activity and can be used for bolus administration. It shows high stability at storage and low metabolism in organism. EFFECT: enhanced properties of agent. 6 tbl

Description

 The invention relates to medicine and can be used for stopping conditions accompanied by an increase in the adhesive-aggregation properties of platelets and for preserving platelet-rich plasma.

 The search for platelet aggregation inhibitors that have a pronounced effect upon bolus injection into the body is of great interest for emergency care for patients with increased platelet aggregation ability (myocardial infarction, cerebral ischemia, etc.).

 The purpose of the invention is a tool that effectively reduces the aggregation activity of platelets during bolus administration, which has high storage stability and low ability to biotransformation in the body.

This goal is achieved by using as a means of reducing platelet aggregation, the well-known compound 5-oxa-6-keto-prostaglandin E ₁ . Information on biological activity in the literature is missing.

To detect in vitro antiaggregation activity of freshly prepared solutions of 5-oxa-6-keto-prostaglandin E ₁ , ADPs at concentrations of 10 ^-5 M and 2 ˙ 10 ^-7 M in the final mixture, collagen at a concentration of 14 μg / ml and adrenaline were used as proaggregants at a concentration of 2.5-5 μg / ml. Platelet-rich plasma was obtained by centrifugation of a healthy 3.8% solution of sodium citrate in the blood of healthy donors for 10 min at 1000 rpm. Platelet aggregation was recorded on an aggregometer manufactured by Chronolog Corporation (USA). The degree of aggregation was expressed as a percentage of the decrease in the optical density of platelet-rich plasma after the end of the reaction compared to the initial level. The experimental results showed that when using ADP as a proaggregant at a concentration of 2 × 10 ^-7 M (Table 1) 5-oxa-6-keto-prostaglandin E ₁ already in an amount of 0.001 μg / ml of the final mixture significantly reduced platelet aggregation in vitro. With an increase in the amount of prostanoid added, the anti-aggregation effect increased. Collagen-induced platelet aggregation (Table 2) was significantly reduced if 5-oxa-6-keto-prostaglandin E _{1 was} added to the sample in an amount of 0.01 μg / ml and was almost completely blocked when the concentration of the studied agent was increased to 0.1 mcg / ml. Adrenaline-dependent aggregation (Table 3) decreased by about 20% when a prostanoid was added in a cuvette in an amount of 0.01 μg / ml and doubled with an increase in the concentration of 5-oxa-6-keto-prostaglandin E ₁ to 0.1 μg / ml This substance had approximately the same activity upon induction of ADP aggregation of 10 ^-5 M (Table 4).

Thus, the results of the work showed that 5-oxa-6-keto-prostaglandin E ₁ has a pronounced anti-aggregation effect in vitro.

To detect the anti-aggregation activity of 5-oxa-6-keto-prostaglandin E ₁ ex vivo, a freshly prepared solution of this substance was injected into rabbits in the marginal ear vein in the amount of 1 μg / kg, 2 mg / kg or 5 mg / kg of animal weight. Blood was taken by the method of free fall of drops before administration of the substance, 15, 30, 60, 90, 120, 150 and 180 minutes after administration of the prostanoid and stabilized with a 3.8% sodium citrate solution in a ratio of 9: 1. In platelet rich plasma obtained from these samples, platelet aggregation activity was determined using 10 ^-5 M ADP as an inductor.

The results of the study, presented in table 5, led to the conclusion about the high efficiency of 5-oxa-6-keto-prostaglandin E ₁ as an antiaggregation agent. So, even when administered at a dose of 1 mg / kg of animal weight, this substance significantly reduced rabbit platelet aggregation over 30 minutes. With an increase in the amount of introduced prostanoid to 2 mg / kg of weight, the duration of the effect after a single injection increased to two hours, and the strength of the effect, especially during the first 30 minutes, increased significantly. A further increase in the dose of the administered agent to 5 mg / kg did not increase the duration of the anti-aggregation effect of the drug, but the severity of this effect was such that platelet aggregation ex vivo in the first 30 minutes after administration was almost completely suppressed.

Thus, 5-oxa-6-keto-prostaglandin E ₁ had a pronounced anti-aggregation effect upon intravenous bolus administration.

To assess the stability effect antiagregatsionnogo 5-oxa-6-keto-prostaglandin E ₁ dry samples of the substance stored at 4 ^{° C} in glass or plastic vials. The initial solutions of the prostanoid were prepared by adding 96 ^о ethanol (0.1 ml per 5 mg) to the sample, then an equimolar amount of sodium bicarbonate and physiological saline to obtain a prostanoid solution with a concentration of 1 mg / ml. Stock solutions were stored in glass bottles with ground glass stoppers at 4 ^C. Working solutions were prepared immediately before the experiment is already starting from adding saline in quantities necessary to obtain the desired concentration prostanoid. The anti-aggregation activity of 5-oxa-6-keto-prostaglandin E ₁ was evaluated by its ability to block platelet aggregation of healthy donors in the presence of ADP (10 ^-5 M).

To study the stability of the anti-aggregation properties of 5-oxa-6-keto-prostaglandin E ₁ , preserved in dry form, samples were taken from the same sample of this substance every six months for two years and diluted according to the method described above, after which studied the effect of the obtained solutions on the functional activity of platelets. The results of the experiment showed that the sample of 5-oxa-6-keto-prostaglandin E ₁ did not lose its anti-aggregation activity in dry form over two years of storage.

The stability of samples stored in the form of solutions was evaluated monthly for two years. The data obtained indicate that although the activity of solutions of 5-oxa-6-keto-prostaglandin E ₁ decreased over time, even after 24 months after dilution, this agent significantly inhibited platelet aggregation at a concentration of 1 μg / ml, which indicates its high storage stability.

The anti-aggregation activity of 5-oxa-6-keto-prostaglandin E ₁ can also be used to stabilize platelet-rich plasma, since it is known that many agents that have an anti-aggregation effect and do not damage enzymatic systems that provide functional activity of the cell are able to maintain full platelets in a condition suitable for subsequent transfusion. To confirm this property of a prostanoid, its effect on the functional activity of human platelets during storage was studied. For the experiment, platelet-rich human plasma was placed in equal volumes in two glass tubes, one of the portions being used as a control, and a solution of 5-oxa-6-keto-prostaglandin E _{1 was} added to the other in the amount of 1 μg of substance per 1 ml of rich plasma. Then the test samples were placed in a water bath at 37 ^{° C} for 4 hours, after which they were stored at 4 ^{° C} throughout the experiment. The functional activity of platelets was evaluated by their ability to aggregate in response to the addition of ADP (10 ^-5 M) in combination with adrenaline (5 μg / ml). The results of the study, presented in Table 6, were statistically processed using the method for evaluating dependent choices.

The data obtained show that when storing plasma samples for more than 72 hours, the presence of 5-oxa-6-keto-prostaglandin E ₁ in the mixture significantly increased the functional activity of platelets. This indicates that this substance increased the shelf life of platelets.

Claims (1)

The use of 5-oxa-6-keto-prostaglandin E ₁ as an agent with antiaggregatory activity.

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