RU2712194C1 - ANTICOAGULANT DRUG, WHICH IS A SYNTHETIC DIPEPTIDE Ac-Trp-Arg-Pip·HCl, A PHARMACEUTICAL COMPOSITION COMPRISING SAID ANTICOAGULANT DRUG - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: group of inventions refers to medicine and concerns an anticoagulant drug, which is a synthetic dipeptide Ac-Trp-Arg-Pip⋅HCl or pharmaceutically acceptable salts thereof. Group of inventions also refers to a pharmaceutical composition containing the above anticoagulant medicinal agent and auxiliary substances – microcrystalline cellulose and stearic acid and / or its pharmaceutically acceptable salt as an active substance.EFFECT: group of inventions provides reducing the risk of uncontrolled thrombosis.8 cl, 3 tbl, 5 ex

Description

The invention relates to medicine, namely to the creation of a medicinal product, a direct anticoagulant based on a tripeptide that simultaneously inhibits thrombin and factor Xa (FHA) and is intended for the preventive treatment of ischemic stroke and systemic embolism - cardiovascular pathologies caused by intravascular thrombosis.

The main cause of thrombosis is a malfunction of the blood coagulation system. Any violation of the circulatory system leads to a number of serious diseases. The blood coagulation system is part of the hemostasis system, the function of which is to stop bleeding when a blood vessel is damaged. However, for this process to take place in the right place and at the right time, most of the components in the blood are in the form of inactive precursors, the activation of which is of a cascading nature, most effectively ensuring the formation of a blood clot. However, for each stage of the cascade there is a regulation system in the form of cofactors and components of the fibrinolytic system. [1]]

To prevent thrombosis and normalize the functioning of the hemostatic system, it is possible to influence its various links: use antiplatelet drugs that reduce platelet activation and aggregation (antiplatelet agents), use anticoagulants - vitamin K antagonists that reduce the synthesis of precursors of procoagulant factors, or blood proteinase inhibitors of the coagulation system.

Anticoagulants are divided into direct ones that act directly on thrombin, or factor Xa (FXa), which is directly involved in the formation of active thrombin, and indirect ones that disrupt the formation of coagulation factors in the liver.

The most widely used indirect anticoagulant is warfarin, which is one of the ten most commonly prescribed cardiovascular drugs. It blocks the conversion of vitamin K into its active form, which is involved in biochemical reactions at the final stages of the synthesis of blood coagulation factors II (thrombin), VII, IX, X. Since the anticoagulant effect of the use of warfarin does not occur immediately, this remedy is intended for long or life anticoagulant therapy. [2].

The disadvantages of warfarin are both a narrow therapeutic range that requires careful selection of the dose, and constant monitoring of blood coagulation (MHO) in order to maintain an effective concentration of the drug and the prevention of hemorrhagic complications. In addition, there is a huge list of drug interactions, foods containing vitamin K, as well as the presence of gene polymorphism, which determines the different individual sensitivity to warfarin. [3].

Direct anticoagulant agents are presented:

Firstly, a group of heparins - sulfated glycosaminoglycans of various molecular weights, secreted from the mucous membrane of the intestines of pigs and from the lungs of cattle, as well as synthetic analogues of heparin. Heparin inhibits thrombin and factor Xa only in combination with antithrombin III (AT) .. In domestic practice, the following low molecular weight heparins are used: enoxaparin sodium (Clexane), calcium nadroparin (Fraxiparin), dalteparin sodium (Fragmin), sulodexide (Wessel-duet F )

The main and disadvantages of the use of heparins are hemorrhagic manifestations due to increased vascular permeability, and AT depletion during prolonged use in large doses is what can cause a state of hypercoagulation and cause thrombosis. [4].

Secondly, the protein physiological anticoagulant antithrombin III, which is isolated from the blood plasma of donors, therefore, when administered, the risk of diseases caused by the transfer of infectious pathogens, including of unknown origin, cannot be completely excluded. In addition, as with the introduction of other plasma components, various undesirable phenomena and anaphylactoid or anaphylactic reactions can occur. Moreover, antithrombin alone has a weak anticoagulant effect, inhibiting almost all coagulation proteases (with the exception of factor VII), plasmin, trypsin, and also the Cls complement component.

Direct thrombin inhibitors include hirudin, a protein first found in the salivary glands of Hirudo medicinalis medical leeches. Selectively binding to thrombin, hirudin irreversibly inhibits not only free, but also associated with a thrombus. In the Russian Federation, the Registration Certificate for Angioks is reissued, the active ingredient in which is bivalirudin - a 20-membered hirudin polypeptide.

All the anticoagulants discussed above are injectable drugs, which significantly complicates their use outside of clinical conditions, in addition, most of them are imported.

In recent years, a number of new generation anticoagulants for oral use have been developed - new oral anticoagulants (PLA). These are low molecular weight synthetic compounds that effectively inhibit thrombin and PCA. All of them are exclusively imported.

The drug Pradax (Germany) registered in the Russian Federation is a prodrug, based on the thrombin inhibitor dabigatran. The drug is widely used in clinical practice for the prevention of stroke, especially in patients with non-valve atrial fibrillation [5], although its use is quite high risk of MI. The main side effect associated with the use of dabigatran is gastrointestinal bleeding.

Rivaroxaban - developed and manufactured by Bayer AG in cooperation with Johnson & Johnson Pharmaceutical Research & Development, is registered in the Russian Federation under the name Xarelto. Rivaroxaban competitively inhibits coagulation factor Xa. The officially approved indications of treatment with rivaroxaban are the prevention of stroke and systemic embolism in non-valve atrial fibrillation, the treatment of deep vein thrombosis and pulmonary embolism, as well as the prevention of venous thromboembolism.

The company Pfizer Inc. (USA) produces the drug Elikvis, the active substance in which is apixaban - a factor Xa inhibitor. The main indications for the appointment of the drug Elikvis completely repeat those for rivaroxaban.

A common side effect for these drugs is an increased risk of developing latent or overt bleeding, and, as a result, the development of posthemorrhagic anemia. The risk of bleeding during therapy with oral anticoagulants is exacerbated by the absence of antidotes to them. If the administration of vitamin K can correct the situation during warfarin therapy, then there are no antidotes to PLA. In addition, the absence of the need for regular anticoagulation monitoring can lead to late detection of bleeding or thrombosis. [6].

In addition, it should be noted that dabigatran (Pradaxa) is eliminated from the body by the kidneys unchanged, therefore, for patients with renal failure, its use is limited. Rivaroxaban (Xarelto) and apixaban (Elikvis), on the contrary, are mainly metabolized in the liver, therefore their use is limited for patients with renal failure.

Thus, all currently available drugs have limitations in use and side effects, which limits their use.

For the treatment and prevention of a number of diseases, standards and clinical recommendations have been developed, guided by which the doctor prescribes drugs of one or another pharmacological group, taking into account the patient’s diseases, drug interactions and possible side processes [7-9]. Xarelto, Elikvis and Pradaxa are included in the list of vital and important medicines (Vital and Essential Drugs), but are not included in all treatment standards. To expand the range of indications, additional clinical studies are necessary.

Considering all the above, the creation of domestic effective and safe low molecular weight synthetic inhibitors of thrombosis for the preventive treatment of ischemic stroke and systemic embolism of various origins is a very urgent task of modern pharmaceuticals and pharmacology.

Very promising from this point of view are low molecular weight peptide compounds that specifically act only on target enzymes and have low toxicity, since they are nature-like substances.

It is known that in the active center of thrombin, peptides containing the arginine residue are effectively bound. Applicants have studied the inhibitory properties of a number of arginine-containing peptides and patented the most effective reversible thrombin inhibitor, Fta-Gly-Arg-Pip (RU 2377246). However, in the preparation of this compound, phthalic anhydride having a general toxic effect is used. In addition, with the decomposition of this peptide, quite toxic phthalic acid is formed in the body, which has a negative effect on the endocrine system.

An object of the present invention is to expand the arsenal of anticoagulant drugs.

The problem is solved

- the creation of an anticoagulant drug, which is a synthetic dipeptide Ac-Trp-Arg-Pip⋅HCl or its pharmaceutically acceptable salts.

- the development of a pharmaceutical composition comprising, as an active ingredient, the synthetic dipeptide Ac-Trp-Arg-Pip⋅HCl or its pharmaceutically acceptable salts and excipients:

microcrystalline cellulose and stearic acid and / or its pharmaceutically acceptable salt, in the following ratio, wt. %:

The inventive synthetic dipeptide Ac-Trp-Arg-Pip HCl is a low toxic drug and acts simultaneously on two main enzymes of the blood coagulation system: thrombin and factor Xa, effectively reducing the concentration of active thrombin in the blood and preventing thrombosis caused by a malfunction in the functioning of the blood coagulation system .

The invention is confirmed by the following examples:

Example 1. Obtaining the dipeptide Ac-Trp-Arg-Pip⋅HCl

The peptide (Ac-acetyl, Pip-piperidin-4-yl) is obtained by sequentially building up the peptide chain, starting with the substituted arginine amide.

The substituted arginine amide 2HCl⋅H-Arg-Pip is obtained by reaction

Boc-Arg-OH (27.5 g) with N, N'-dicyclohexylcarbodiimide (20.7 g) and N-hydroxysuccinimide (6.3 g) and piperidine (10 ml) in dimethylformamide [10]. The Boc-protecting group with Boc-Arg-Pip was removed by the standard method by treatment with a 4 M solution of hydrogen chloride in dioxane [11]. Yield HCl⋅H-Arg-Pip 25.0 g (90%)

Activated Boc-Trp-OPƒp ester is obtained by the standard method by reaction of Boc-Trp-ОН with N, N'-dicyclohexylcarbodiimide in tetrahydrofuran, followed by crystallization of products from a mixture of diethyl ether and hexane [11].

Preparation of 2HCl * H-Trp-Arg-Pip Dipeptide To a solution of Boc-Trp-OPfp (25.9 g) in 120 ml cooled to 0 ° C in DMF, a solution of 17.5 g of 2HCl * H-Arg-Pip in 150 ml of DMF and 10 ml of DIPEA was added and the reaction mixture was stirred in ice-cold bath for 2 hours, then left to mix overnight at room temperature. The reaction mixture is evaporated on a rotary evaporator, the obtained residue is dissolved in a mixture of water (350 ml), methanol (40 ml) and diethyl ether (350 ml), the aqueous layer is separated from the ether in a separatory funnel, acidified with 10% HCl to pH = 3-3 5 with vigorous stirring and washed with diethyl ether, 5 M NaOH was added dropwise to pH = 9.5-10, and washed again with dtetyl ether. Dry NaCl (75 g) and n-butanol (300 ml) are added to the aqueous solution, the two-phase solution is stirred until the salts are completely dissolved, transferred to a separatory funnel, the aqueous layer is washed with butanol (150 ml), the combined butanol extracts are dried over anhydrous Na ₂ SO ₄ for 10 minutes. The solution was filtered using a glass filter and evaporated on a rotary vacuum evaporator to a viscous oil. The residue was dissolved in acetone (500 ml), the resulting cloudy solution was filtered with celite and evaporated. The Boc-protecting group from the resulting HCl * Boc-Trp-Arg- oil was removed by the standard method by treatment with a 4 M solution of hydrogen chloride in dioxane [11]. Yield 2HCl * H-Arg-Pip 15.6 g, 62%.

Obtaining the claimed dipeptide Ac-Trp-Arg-Pip HCl. 15.42 g of 2HCl⋅H-Trp-Arg-Pip was dissolved in DMF (100 ml), acetic anhydride (5.6 g;) was added and the solution was cooled in an ice bath to 0 ° C. Then, 10 ml of DIPEA is added dropwise to the reaction mixture over 10 minutes, the reaction mixture is stirred for 3 hours at room temperature, and the solution is evaporated on a rotary evaporator. The resulting residue was triturated in diethyl ether (300 ml) for 4 hours, then left overnight in the refrigerator. The precipitated crystals are separated on a glass filter, washed with ether (3 × 80 ml) and hexane (100 ml) and dried in vacuum. The resulting product was dissolved in ethanol (150 ml) and activated carbon (10 g) was added, the mixture was stirred for 30 minutes, the carbon was filtered off, washed with ethanol (3 × 100 ml), the combined fractions of the ethanol solution were evaporated, the oily residue was dissolved in isopropanol (120 ml), the solution is filtered and evaporated to the state of hardened foam. Acetone (150 ml) was added to the residue and the residue was triturated in this acetone for 4 hours, then left in the refrigerator overnight. The precipitated crystals are filtered, washed with cold acetone (2 × 50 ml) and hexane (50 ml) and dried in vacuum. Yield Ac-Trp-Arg-Pip HCl 6.78 g, 43.2%,

Example 2. Obtaining the dosage form of the inventive drug tablets

Obtaining the mass for tabletting is carried out by mixing pre-sieved 294.00 g of microcrystalline cellulose, 300.00 g of dipeptide and 6.00 g of magnesium stearate. The resulting mixture was stirred at a stirrer speed of 300 rpm for 15 minutes. The tablet mass was pressed into tablets of 0.300 g in a standard manner [12]. After dedusting the finished tablets, they are covered with a film coating of the specified composition in a standard manner [13]

Example 3. The study of the inhibitory effect of the claimed drug on the amidase activity of serine proteases of the hemostatic system and digestive enzymes

When choosing the structure of a peptide inhibitor of blood coagulation enzymes, the conformational requirements of the active centers of thrombin and PXa were taken into account, however, the study of the effect of various concentrations of Ac-Trp-Arg-Pip HCl on the own amidase activities of enzymes of the plasmin fibrinolytic system and urokinase is taken into account. as well as digestive enzymes that enter the blood from the pancreas, were carried out in order to confirm the selectivity of the inhibitor.

The influence of the Ac-Trp-Arg-Pip HCl_ dipeptide on the activity of enzymes is assessed by the standard method, by determining kinetic constants by measuring the initial hydrolysis rates of the corresponding chromogenic substrates [14]. The hydrolysis rates of various substrate concentrations by enzymes in the presence of an inhibitor are determined from the initial sections of the kinetic curves A ₄₀₅ - t on a UV-1202 spectrophotometer (Shimadzu, Germany). Each experiment is duplicated. Initial speeds are determined by the dependence of the change in A ₄₀₅ over time. The kinetic parameters of the inhibition of substrate hydrolysis IC50 and the inhibition constant K _{1 were} calculated on the basis of the Linuiver-Burke dependences. All experiments were performed in triplicate.

The absence of inhibition of plasmin, urokinase, trypsin and chymotrypsin indicates the absence of the binding of the dipeptide to these enzymes, and, therefore, does not change its effective concentration in the body.

Example 4. Evaluation of the anticoagulant effect of the claimed drug in vivo with intravenous administration

Evaluation of the antithrombotic activity of the drug was carried out on a model of venous stasis in male rats of the Wistar strain, according to the Guide to Preclinical Drug Research edited by A. Mironov.

Anesthetized animals (Nembutal, 40 mg / kg, intraperitoneally) injected the Ac-Trp-Arg-Pip HCl dipeptide in a dose of 30 mg / kg in a volume of 1 ml into the left jugular vein. Control animals were injected with 0.9% NaCl solution in an equivalent volume. In order to suppress the protective reaction of the anticoagulant system, a solution of atropine sulfate at a dose of 5 mg / kg was introduced into the same vein. To simulate thrombosis, 15 minutes after the administration of the Ac-Trp-Arg-Pip HCl dipeptide or 0.9% NaCl solution in the control, the rat blood coagulation system is activated with human serum. Then, a section of a vein (0.7 cm) is bandaged with a thread, which is not used for the administration of substances (right jugular vein).

The effectiveness of antithrombotic activity is evaluated:

1) in the form of a blood clot extracted from a ligated section of a vein,

2) by weight of a wet thrombus on an analytical balance.

The introduced inventive dipeptide gives a pronounced antithrombotic effect, namely, in the field of view of several microscopic threads; in control - there is no effect - in the field of view is one large blood clot.

From the results presented in table 2, it follows that the introduced inventive dipeptide gives a pronounced effect - the weight of the formed thrombus in the presence of the dipeptide is almost 4 times less than in the control.

Example 5. Evaluation of the anticoagulant effect of the claimed drug in vivo with intragastric administration

The drug based on the synthetic dipeptide Ac-Trp-Arg-Pip HCl is part of the tablet. To administer this agent to rats, a model of venous stasis was developed in male Wistar rats by intragastric administration of the anticoagulant Ac-Trp-Arg-Pip HCl dipeptide in an adequate dosage form in the form of tablet mass.

The tablet mass prepared as in Example 2 is poorly soluble in water; therefore, a tablet mass suspension is prepared in a 1% starch solution. The resulting suspension is transferred into sterile tubes with screw caps.

Before administration, the mixture is shaken and, using a special probe for intragastric administration with a club-shaped tip, rats are administered in doses of 1 ETD, 2ETD and 3 ETD (ETD is an effective therapeutic dose) of 1 ml. Control animals were intragastrically administered with a 1% starch solution in an appropriate volume.

Animals were anesthetized with 1.5% isoflurane anesthesia. Rats breathed on their own, without intubation. To simulate thrombosis, 30 minutes after intragastric administration of the suspension, the blood coagulation system of rats with human serum is activated. Then, a section of a vein (0.5-0.7 cm) is ligated with a thread (right jugular vein).

The effectiveness of antithrombotic activity is evaluated by:

форме тромба, извлеченного из перевязанного участка вены (баллы);

the form of a blood clot extracted from a ligated section of a vein (points);

весу влажного тромба на аналитических весах (г).

the weight of the wet thrombus on an analytical balance (g).

Assessing the shape of a blood clot extracted from a ligated section of a vein

- 0 and 1 point - pronounced antithrombotic effect, in the field of view of the clot or not, or several microscopic threads;

- 2 points - moderate antithrombotic effect, several small thrombi in the field of view;

- 3 and 4 points - the effect is absent or insignificant, in the field of view one large or 2-3 thrombi smaller).

Recalculation of the scoring system by the percent prevention of thrombosis was carried out according to the following formula: [1- (Sa / 4n)] × l00,

where a is the antithrombotic effect in points, n is the number of experiments;

The results of a study of the antithrombotic activity of the claimed dipeptide, evaluated in accordance with the "Guidelines for preclinical drug research" edited by A. Mironov, are presented in table 3. Doses are given for the active substance.

The effectiveness of the antithrombotic action of the claimed dipeptide in experimental groups with a single intragastric administration at doses of 30-90 mg / kg was statistically significantly different and amounted to 75%, 82.5% and 87.5%, respectively.

The number of points in the experimental groups of rats reflecting the form of a blood clot is significantly lower than in the control group of animals with intragastric administration of a 1% starch solution. In the field of view, either a clot was not observed, or there was 1 small thrombus in the form of a microscopic thread. In the control group of rats, large blood clots are visible in the field of view of the jugular vein after its isolation.

The wet thrombus mass in the experimental groups was significantly less than in the control group of animals.

Thus, an anticoagulant drug was obtained, which is a synthetic dipeptide Ac-Trp-Arg-Pip HCl, which, in contrast to the closest analogue, inhibits two enzymes of the blood coagulation system and does not contain a toxic phthalyl group. The effectiveness of the anticoagulant action of the claimed drug and pharmaceutical compositions based on it is confirmed by in vivo experiments with intravenous and intragastric administration to experimental animals.

Sources of scientific and technical information

1. M.A. Panteleev, F.I. Ataullakhanov / “Blood coagulation: research methods and regulatory mechanisms.” // Clinical Oncohematology, 2008, t 1, “2,174-1810]

2. Bogachev V.Yu. Warfarin ... Death is delayed // breast cancer. 2013. No.15. S. 797

3 Prevention of thromboembolic complications in atrial fibrillation rat poison or new oral anticoagulants: do we have a choice and should we be afraid to make it? The first clinical experience with the use of rivaroxaban in Nizhny Novgorod ”Koroleva L.Yu., Kolesnichenko IV, Nosov VP, Gritsenko II, Timoshchenko ES. // Rational pharmacotherapy in cardiology, 2014, 10 (3 ), 303-306)

4. A.L. Melkumyan, A.L. Berkovsky, R.S. Chisinau, A.A. Kozlov. “Monitoring the effectiveness of direct anticoagulants” // Hematol. and transfusiol., 2013, v. 58, No. 1, 32-38

5. Nikolaeva I.E., Yanbaeva S.M., Zalaldinova A.M., Ishmanova A.R., Gareeva N.Kh., Khmelevskikh N.A. The experience of using dabigatran etexilate in various clinical situations in patients with atrial fibrillation // breast cancer. 2015. No5. S. 280

Hans-Ruprecht Neuberger,; Stefan H. Hohnloser, Isabelle C. van Gelder,; Gregory Y. H. Lip, Michael

«Novel Anticoagulants for Stroke Prevention in Atrial Fibrillation, Current Clinical Evidence and Future Developments»//J Am Coll Cardiol 2010; Vol. 56, №25: 2067-20766. Stephan N. Schirmer, MD, PHD; Magnus

Hans-Ruprecht Neuberger ,; Stefan H. Hohnloser, Isabelle C. van Gelder ,; Gregory YH Lip, Michael

"Novel Anticoagulants for Stroke Prevention in Atrial Fibrillation, Current Clinical Evidence and Future Developments" // J Am Coll Cardiol 2010; Vol. 56, No.25: 2067-2076

7. The standard of emergency medical care for stroke was approved by Order of the Ministry of Health of the Russian Federation of December 20, 2012 No. 1282 n

8. The standard of emergency medical care for acute cerebrovascular accident, approved by Order of the Ministry of Health of the Russian Federation of July 5, 2016 N466н

9. NATIONAL STANDARD OF THE RUSSIAN FEDERATION GOST R 56377-2015 CLINICAL RECOMMENDATIONS (TREATMENT PROTOCOLS) PREVENTION OF THROMBOEBOLIC SYNDROMES, Bozhkova SA, Bulanov A.Yu., Vavilova TV, Domashenko P. A. A. , Krasnova L.S., Leontiev S.G., Momot A.P., Morozov K.M., Roitman E.V., Susin S.V., Sychev D.A., Yavelov I.S., Yakovlev V.B //. PROBLEMS OF STANDARDIZATION IN HEALTH CARE 2015, No. 7-8, p. 28-68

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Claims (10)

1. An anticoagulant drug, which is a synthetic dipeptide Ac-Trp-Arg-Pip⋅HCl or its pharmaceutically acceptable salts. 2. A pharmaceutical composition comprising, as an active substance, an anticoagulant drug according to claim 1 and excipients: microcrystalline cellulose and stearic acid and / or its pharmaceutically acceptable salt, in the following ratio, wt. %:

3. The pharmaceutical composition according to claim 2, characterized in that the magnesium salt is used predominantly as the stearic acid salt. 4. The pharmaceutical composition according to any one of paragraphs. 2, 3, characterized in that it is made in the form of tablets, capsules, granules, sachets. 5. The pharmaceutical composition according to any one of paragraphs. 2, 3, characterized in that it is made in the form of a tablet coated with an enteric coating. 6. The pharmaceutical composition according to claim 5, characterized in that the average weight of the tablet core is 0.3 g. 7. The pharmaceutical composition according to paragraphs. 5, 6, characterized in that the shell of the tablet is up to 10% by weight of the tablet core. 8. The pharmaceutical composition according to paragraphs. 5-7, characterized in that the tablet shell contains a copolymer of methacrylic acid, silicon dioxide, colloidal anhydrous, sodium bicarbonate, sodium lauryl sulfate, talc, titanium dioxide, triethyl citrate, acceptable dyes or the ready-made mixture for film coating of the brand Acryl-EZE® 93A White ".