A means for oppression of activity of platelets


(57) Abstract:

The invention relates to medicine. It is proposed to use N-chloro-2-aminoethanesulfonic or N, N-dichloro-2-aminoethanesulfonic acid for inhibition of the functional activity of platelets. Means inhibits platelet aggregation and secretion of the contents of the granules. 2 C. p. F.-ly, 4 tab., 3 Il.

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

In medicine there is a need for substances that suppress intravascular thrombogenesis associated with platelets. Known protivotromboznoe activity of acetylsalicylic acid - aspirin. This substance is used to prevent blood clots that depend on increased activity of platelets. Acetylsalicylic acid inhibits platelets by chemical modification of prostaglandin synthetase H2. Protivotromboznoe action of acetylsalicylic acid appears if the platelets are activated with the participation of the specified enzyme.

Known protivotromboznoe substance, which is an aqueous solution chloraminated proizvodnog what you want to make due to the chemical modification of the plasma membrane of platelets under the action of the active chloramines or chlorimines group. This substance is selected prototype. Its disadvantage is a small shelf life, amounting to a few hours, the inability to obtain an active agent in dry form. Substance-prototype is not obtained in the solid state, dissolves when drying.

The objective of the invention is to develop substances, effectively inhibiting platelet activity irrespective of the nature of the activation, existing in solution and solid state, characterized by an increased shelf life.

The invention in one case is solved by the fact that as protivotromboznoe funds are used chloramine derivatives of 2-aminoethanesulfonic acid, which is a product of the oxidation of the amino acids cysteine:

N-chloro-2-aminoetansulfonovaya acid, or N-chlorotaurine

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N,N-dichloro-2-aminoetansulfonovaya acid, or N,N-dichlorotaurine

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Unlike the prototype in the claimed compounds lacking the carboxyl group. The claimed compounds in the dissolved state are preserved up to a few days versus a few hours in the case of the prototype.

The objective of the invention is solved in another case, the fact that N,N-dichloro-2-aminoethanesulfonic acid on the s absorption 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 the firm sample.

The creation of the stated substances for inhibition of platelet activity, confirmed by the following examples.

Example 1

Received N, N-dichloro-2-aminoethanesulfonic acid and N-chloro-2-aminoethanesulfonic acid by mixing solution of 2-aminoethanesulfonic acid and sodium hypochlorite solution in accordance with known methods (Thomas E. L. , Jefferson M. M., Grisham, M. B. Myeloperoxidase-catalyzed incorporation of amines into proteins: Role of hypochlorous acid and dichloramines. - Biochemistry. 1982, vol. 21, p. 6299-6308).

Human blood or rabbit, stable and 3.8% solution of sodium citrate (9:1 by volume), was centrifuged at 210 g for 20 minutes the Supernatant, representing the platelet-rich plasma, was used to study the aggregation of platelets. This used the turbidimetric aggregometer Chrono-Log or turbidimetric aggregometry collected on the basis of fotoelektrokalorimetry CPK-2MP. In the latter case, the thickness of the cell for the sample of platelets was 1 cm, the instrument records kinetics acetow served as a change of transmittance of the sample, measured in 5-10 min after injection by acid at a final concentration of 10 μmol per 1 liter; this compound was an agonist (activator of platelets). Protivogrippoznoe action stated substances characterized by the ratio T/Tkwhere Tkand T is the change of the transmittance, respectively, of the sample without substance (control) and sample with the addition of the analyte.

N, N-Dichloro-2-aminoetansulfonovaya acid at a final concentration of 0.25-1.0 mmol/l inhibited the platelet aggregation comprising platelet-rich human plasma and rabbit (PL. 1).

Introduced in the blood of human or rabbit N,N-dichloro-2-aminoethanesulfonic acid. After 10 minutes of this blood sample received platelet-rich plasma, measured aggregation activity of platelets caused by acid at a final concentration of 10 µmol/l (table. 2). N, N-Dichloro-2-aminoetansulfonovaya acid at its introduction in whole blood showed a pronounced protivogrippoznoe effect on platelets (PL. 2).

Example 2

Received as described in example 1, method N-chloro-2-aminoethanesulfonic acid and N,N-dichloro-2-aminoethanesulfonic acid is the N,N-dichloro-2-aminoethanesulfonic acid compared to the activity in control. Aggregation caused by collagen at a final concentration of 5 μg/ml or adrenaline at a final concentration of 10 µmol/L.

Got isolated rabbit platelets from platelet-rich plasma by centrifugation. To analyze the aggregation activity of platelets used their suspension in a medium containing NaCl 134 mm, KCl 5 mm MgSO41 mm, Na2HPO40.5 mm, HEPES 10 mm, glucose 5 mm (pH = 7,4). Aggregation caused 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-aminoetansulfonovaya acid inhibited aggregation activity of platelets when they are activated by collagen or epinephrine (PL. 3). N-Chloro-2-aminoetansulfonovaya acid inhibited the aggregation of platelets activated by thrombin or epinephrine (PL. 3).

Thus, the claimed substances are able to inhibit aggregation activity of platelets irrespective of the nature of the activation.

Example 3

Received a platelet-rich blood plasma of rabbit, introduced in this sample acridine orange at a final concentration of 10 µmol/l, and incubated the mixture for 5 min at room temperature for accumulation of the dye in the intracellular probe the gat suspension of platelets introduced calcium chloride at a final concentration of 1 mmol/l and then thrombin at a final concentration of 0.1 U/ml Measured time dependence of the fluorescence intensity of the sample of platelets passing through the filter LGL-17 and excited by light at 450 nm. The intensity of this fluorescence was initially increased in time in the process of secretion and release of acridine orange into the environment and upon completion of this process has reached a plateau. The maximum change in fluorescence intensity was measure the efficiency of secretion. Introduced into a suspension of isolated platelets prior to the introduction of thrombin N,N-dichloro-2-aminoethanesulfonic acid at a final concentration of 0.14 and 0.25 mmol/l, the Efficiency of secretion were, respectively, 43 and 29% of the efficiency in the original sample of platelets. Thus, the claimed substance also inhibits another important type of functional activity of platelets - the secretion of the contents of the granules.

Example 4

Through the marginal ear vein was injected into the bloodstream rabbit N,N-dichloro-2-aminoethanesulfonic acid, dissolved in saline solution, at the rate of about 0.4 mmol per 1 liter of blood. Took blood from the rabbit before the introduction of the claimed substances and after 1 and 24 h after its injection. From all blood samples received rich thrombocytopenia at a final concentration of 10 µmol/l or collagen at a final concentration of 5 μg/ml (table. 4). The claimed substance introduced into the bloodstream, strongly inhibited aggregation activity of platelets after 1 h the Effect of oppression persisted 24 h (table. 4).

Example 5

Prepared a solution of N-chloro-2-aminoethanesulfonic acid in accordance with example 1. He was kept 24 h at room temperature. With the help of spectrophotometer SF-46 was measured by its absorption spectrum as the dependence of optical density (D) as a function of wavelength (nm). After storage in the absorption spectrum of the solution showed a characteristic band monochloramine chemical groups in the wavelength range of 230-300 nm with a maximum at 250 nm (Fig. 1, the solid curve 1), almost coinciding with the band spectrum of a freshly prepared solution (Fig. 1, dashed curve 2). During the specified storage optical density of the solution at the maximum at 250 nm, is proportional to the concentration of N-chloro-2-aminoethanesulfonic acid, practically has not changed. Thus, N-chloro-2-aminoetansulfonovaya acid in aqueous solution is maintained for 1 day.

Prepared a solution of N, N-dichloro-2-aminoethanesulfonic acid in accordance with example 1. It was stored for 5 months in a frozen state at a temperature of from -1 to -3oC. using spectropho curve 2) and after storage (Fig. 2, the solid curve 1). In the spectra of both solutions were characteristic dichloramine chemical group absorption band in the wavelength interval 270-320 nm with a maximum at 290 nm, i.e., the solution is kept for 5 months, contained N,N-dichloro-2-aminoethanesulfonic acid. It accounted for 80% of the number in the original solution.

Example 6

Obtaining stated substances in the solid state, the inhibitory activity of platelets, is confirmed by the following example.

Received as described in example 1, a solution of N,N-dichloro-2-aminoethanesulfonic acid. The concentration of N, N-dichloro-2-aminoethanesulfonic acid was 11 mmol/L. Froze 5 ml of this solution in a glass vial, using a vacuum hose attached the bottle to the vacuum unit "Frost 3-2", over the frozen sample set the low gas pressure of 2 PA, withstood the sample at the pressure of 7 hours, took a solid residue. It was stored for 10 months in a refrigerator at a temperature of 4oC. It is kept solid was mixed with 5 ml of water; it disappeared for a few seconds without leaving residue. Measured absorption spectrum of the resulting solution in the spectrophotometer SF-46. The measured absorption spectrum had'hara in the absorption spectrum of freshly prepared solution of N,N-dichloro-2-aminoethanesulfonic acid (Fig. 3, dashed curve 2). Fresh solution was prepared so that the concentration of active chlorine dichloramine group was equal to its concentration in the solution obtained from the solid. Thus, the claimed solid after 10 months of storage contained active dichloramine group.

Prepared aqueous solution from the solid residue, stored for 10 months. Added to 2 ml of this solution, 50 mg of potassium iodide. Saw a rapid release of molecular iodine, which is found in his ability to paint soluble starch blue. This suggests that the claimed solid showed characteristic chloramines 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%. Prepared aqueous solution of the stated solids containing N,N-dichloro-2-aminoethanesulfonic acid and stored for 14 days. Introduced into the bloodstream of the rabbit through the marginal ear vein this solution and the ore 4. The activity of platelets caused by adrenaline, after 1 and 24 h after injection of rabbit specified solution were, respectively, 33 and 56% of control. Thus, the claimed method allows to obtain a solid substance, which is permanently stored and after storage has protivotromboznoe action.

1. The use of N-chloro-2-aminoethanesulfonic acid or N,N-dichloro-2-aminoethanesulfonic acid as a tool for the oppression of the functional activity of platelets.

2. Means for inhibition of the functional activity of platelets, wherein a represents N,N-dichloro-2-aminoethanesulfonic acid in the solid state.


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1 tbl, 8 dwg