RU2161483C2 - Agent for inhibition of platelet activity - Google Patents

Abstract

FIELD: medicine, hematology. SUBSTANCE: invention proposes to use N-chloro-2- -aminoethanesulfonic or N,N-dichloro-2-aminoethanesulfonic acid for inhibition of functional activity of platelets. Agent inhibits platelet aggregation and secretion of granules content. EFFECT: enhanced effectiveness of agent. 2 cl, 4 tbl, 6 ex

Description

 The invention relates to medicine, to substances that act on the state of aggregation of the blood, namely, substances that inhibit platelet activity.

In medicine, there is a need for substances that suppress intravascular thrombogenesis associated with platelets. Known antiplatelet activity of acetylsalicylic acid - aspirin. This substance is used to prevent the formation of blood clots, depending on the increased activity of platelets. Acetylsalicylic acid inhibits platelets by chemical modification of prostaglandin H ₂ synthetase. The antiplatelet effect of acetylsalicylic acid is manifested if platelets are activated with the participation of the specified enzyme.

 Known anti-platelet substance, which is an aqueous solution of a chloramine derivative of amino acids or a chlorimine derivative of amino acids. This substance is described in USSR patent N 1834659. Its activity is due to chemical modification of the plasma membrane of platelets under the action of the active chloramine or chlorimine group. This substance is selected as a prototype. Its disadvantage is the short shelf life of several hours, the inability to obtain the active agent in dry form. The prototype substance is not obtained in the solid state, decomposes upon drying.

 The objective of the invention is to develop a substance that effectively inhibits platelet activity, regardless of the nature of activation, existing in a dissolved and solid state, characterized by an increased shelf life.

N,N-дихлор-2-аминоэтансульфоновая кислота, или N,N-дихлортаурин

В отличие от прототипа в заявленных соединениях отсутствует карбоксильная группа. Заявленные соединения в растворенном состоянии сохраняются до нескольких дней против нескольких часов в случае прототипа.The objective of the invention in one case is solved by the fact that as an antithrombotic agent, chloramine derivatives of 2-aminoethanesulfonic acid, which is a product of the oxidation of the amino acid cysteine, are used:
N-chloro-2-aminoethanesulfonic acid, or N-chlorothaurine

N, N-dichloro-2-aminoethanesulfonic acid, or N, N-dichlorotaurin

Unlike the prototype, the claimed compounds lack a carboxyl group. The claimed compounds in dissolved state last up to several days against several hours in the case of the prototype.

 The objective of the invention is solved in another case by the fact that N, N-dichloro-2-aminoethanesulfonic acid is obtained in the solid state.

The invention is illustrated by the following drawings:
FIG. 1 - absorption spectra of a solution of N-chloro-2-aminoethanesulfonic acid before and after storage;
FIG. 2 - absorption spectra of a solution of N, N-dichloro-2-aminoethanesulfonic acid before and after storage;
FIG. 3 - absorption spectra of N, N-dichloro-2-aminoethanesulfonic acid after dissolution of its solid sample.

 The creation of the claimed substance to inhibit platelet activity is confirmed by the following examples.

Example 1
Received N, N-dichloro-2-aminoethanesulfonic acid and N-chloro-2-aminoethanesulfonic acid by mixing a solution of 2-aminoethanesulfonic acid and sodium hypochlorite solution in accordance with the known method (Thomas EL, Jefferson MM, Grisham MB Myeloperoxidase-catalyzed incorporation of amines into proteins: Role of hypochlorous acid and dichloramines. - Biochemistry. 1982, vol. 21, p. 6299-6308).

Human or rabbit blood stabilized with a 3.8% sodium citrate solution (9: 1 by volume) was centrifuged at 210 g for 20 minutes. The supernatant, which is a platelet-rich plasma, was used to study platelet aggregation. For this, a Chrono-Log turbidimetric aggregometer or a turbidimetric aggregometer assembled on the basis of the KFK-2MP photoelectrocolorimeter was used. In the latter case, the thickness of the cuvette for the platelet sample was 1 cm; the instrument records the kinetic curve of the transmittance at 670 nm. The quantitative index of platelet aggregation ability was the change in light transmission of the sample, measured 5-10 min after the administration of adenosine diphosphoric acid in a final concentration of 10 μmol per 1 l; this compound served as an agonist (platelet activator). The anti-aggregation effect of the claimed substances was characterized by the ratio ΔT / ΔT _k , where ΔT _k and ΔT are the change in the transmittance of the sample without substance (control) and the sample with the addition of the studied substance.

 N, N-Dichloro-2-aminoethanesulfonic acid at a final concentration of 0.25-1.0 mmol / L suppressed the ability of platelets to aggregate in platelet-rich human and rabbit plasma (Table 1).

 N, N-dichloro-2-aminoethanesulfonic acid was introduced into the blood of a person or rabbit. After 10 min, platelet-rich plasma was obtained from such a blood sample; platelet aggregation activity induced by adenosine diphosphoric acid at a final concentration of 10 μmol / L was measured (Table 2). N, N-Dichloro-2-aminoethanesulfonic acid, when introduced into whole blood, showed a pronounced anti-aggregation effect on platelets (Table 2).

Example 2
N-chloro-2-aminoethanesulfonic acid and N, N-dichloro-2-aminoethanesulfonic acid were obtained by the method described in Example 1. A platelet-rich rabbit blood plasma was obtained and platelet aggregation activity was measured under the action of N, N-dichloro-2-aminoethanesulfonic acid in comparison with the activity in the control. Aggregation was caused by collagen at a final concentration of 5 μg / ml or adrenaline at a final concentration of 10 μmol / L.

Isolated rabbit platelets were obtained from platelet-rich plasma by centrifugation. To analyze the aggregation activity of platelets, their suspension was used in a medium containing NaCl 134 mM, KCl 5 mM, MgSO ₄ 1 mM, Na ₂ HPO ₄ 0.5 mM, HEPES 10 mM, glucose 5 mM (pH = 7.4). Aggregation was induced by thrombin at a final concentration of 0.1 U / ml or adrenaline at a final concentration of 10 μmol / L.

 N, N-Dichloro-2-aminoethanesulfonic acid inhibited the aggregation activity of platelets upon their activation by collagen or adrenaline (Table 3). N-Chloro-2-aminoethanesulfonic acid inhibited platelet aggregation activated by thrombin or adrenaline (Table 3).

 Thus, the claimed substances are able to inhibit platelet aggregation activity, regardless of the nature of activation.

Example 3
A platelet-rich rabbit blood plasma was obtained, acridine orange was introduced into this sample at a final concentration of 10 μmol / L and the mixture was incubated for 5 min at room temperature to accumulate the dye probe in intracellular granules. Then the cells were separated from the plasma by centrifugation, suspended in the medium described in Example 2. Calcium chloride was introduced into this platelet suspension at a final concentration of 1 mmol / L and then thrombin at a final concentration of 0.1 U / ml. The time dependence of the fluorescence intensity of a platelet sample passing through a ZhS-17 filter and excited by light at 450 nm was measured. The intensity of this fluorescence initially increased with time as a result of the secretion process and the release of acridine orange into the external environment and, upon completion of this process, reached a plateau. The maximum change in fluorescence intensity was a quantitative indicator of the efficiency of secretion. The thrombin N, N-dichloro-2-aminoethanesulfonic acid was injected into the suspension of isolated platelets before the thrombin was administered at a final concentration of 0.14 and 0.25 mmol / L. The secretion efficiency was 43 and 29%, respectively, of the efficiency in the initial platelet sample. Thus, the claimed substance also inhibits another important type of functional activity of platelets - the secretion of the contents of the granules.

Example 4
N, N-dichloro-2-aminoethanesulfonic acid dissolved in physiological saline was injected into the bloodstream of the rabbit through the marginal vein of the ear at the rate of about 0.4 mmol per 1 liter of blood. Blood was taken from a rabbit before administration of the claimed substance and 1 and 24 hours after its administration. Platelet-rich plasma was obtained from all blood samples and the aggregation activity of platelets activated by adenosine diphosphoric acid, adrenaline at a final concentration of 10 μmol / L or collagen at a final concentration of 5 μg / ml was measured (Table 4). The claimed substance, introduced into the bloodstream, strongly suppressed platelet aggregation activity after 1 hour. The inhibition effect persisted for 24 hours (Table 4).

Example 5
A solution of N-chloro-2-aminoethanesulfonic acid was prepared in accordance with Example 1. It was stored for 24 hours at room temperature. Using an SF-46 spectrophotometer, its absorption spectrum was measured as the dependence of the optical density (D) on the wavelength (nm). After storage in the absorption spectrum of the solution, a characteristic band of the monochloramine chemical group appeared in the wavelength range of 230-300 nm with a maximum at 250 nm (Fig. 1, solid curve 1), almost coinciding with the spectrum band of the freshly prepared solution (Fig. 1, dashed curve 2 ) During this storage, the optical density of the solution at a maximum at 250 nm, proportional to the concentration of N-chloro-2-aminoethanesulfonic acid, remained practically unchanged. Thus, N-chloro-2-aminoethanesulfonic acid in an aqueous solution persists for 1 day.

A solution of N, N-dichloro-2-aminoethanesulfonic acid was prepared in accordance with Example 1. It was stored for 5 months in a frozen state at a temperature of from -1 to -3 ^° C. The absorption spectra of the N, N- solution were measured using an SF-46 spectrophotometer. dichloro-2-aminoethanesulfonic acid before (Fig. 2, dashed curve 2) and after such storage (Fig. 2, solid curve 1). In the spectra of both solutions, there was an absorption band characteristic of the dichloramine chemical group in the wavelength range of 270-320 nm with a maximum of about 290 nm, i.e. the solution, stored for 5 months, contained N, N-dichloro-2-aminoethanesulfonic acid. Its amount was 80% of the amount in the initial solution.

Example 6
The receipt of the claimed substance in the solid state, inhibiting the activity of platelets, is confirmed by the following example.

A solution of N, N-dichloro-2-aminoethanesulfonic acid was obtained as described in Example 1. The concentration of N, N-dichloro-2-aminoethanesulfonic acid was 11 mmol / L. 5 ml of this solution was frozen in a glass bottle, using a vacuum hose, the bottle was connected to the Rime 3-2 vacuum unit, a reduced gas pressure of 2 Pa was established over the frozen sample, the sample was held at this pressure for 7 hours, and a solid residue was taken. It was stored for 10 months in a refrigerator at 4 ^° C. This stored solid was mixed with 5 ml of water; it dissolved in a few seconds without a trace. The absorption spectrum of the resulting solution was measured on an SF-46 spectrophotometer. The measured absorption spectrum had a characteristic absorption band (Fig. 3, solid curve 1), which practically coincided with the corresponding band in the absorption spectrum of a freshly prepared solution of N, N-dichloro-2-aminoethanesulfonic acid (Fig. 3, dashed curve 2). A freshly prepared solution was prepared so that the concentration of the active chlorine of the dichloramine group was equal to its concentration in the solution obtained from the solid. Thus, the claimed solid substance after 10 months of storage contained an active dichloramine group.

 An aqueous solution was prepared from a solid residue that had been stored for 10 months. 50 mg of potassium iodide was added to 2 ml of this solution. There was a rapid release of molecular iodine, which was found by its ability to stain soluble starch in blue. This indicates that the claimed solid substance showed a characteristic property of the chloramine chemical group to oxidize the iodide anion with the formation of molecular iodine.

 Iodometric titration using sodium thiosulfate showed that the content of N, N-dichloro-2-aminoethanesulfonic acid in the claimed solid after 10 months of storage was 11.4%. An aqueous solution was prepared from the claimed solid substance containing N, N-dichloro-2-aminoethanesulfonic acid and stored for 14 days. This solution was injected into the bloodstream of the rabbit through the marginal vein of the ear at the rate of about 0.1 mmol per 1 liter of blood. Platelet aggregation activity was measured as described in Example 4. The platelet activity induced by adrenaline 1 and 24 hours after administration of the indicated solution to the rabbit was 33 and 56% of the control, respectively. Thus, the claimed method allows to obtain a solid substance that is stored for a long time and after storage has an antiplatelet effect.

Claims (2)

 1. The use of N-chloro-2-aminoethanesulfonic acid or N, N-dichloro-2-aminoethanesulfonic acid as a means of inhibiting the functional activity of platelets.  2. A tool for inhibiting the functional activity of platelets, characterized in that it is N, N-dichloro-2-aminoethanesulfonic acid in the solid state.

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