RU2709017C1 - Pharmaceutical composition possessing antiaggregant activity - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to a pharmaceutical composition, specifically to a solution for intravenous administration containing as an active substance a Fur-Lys-His-Ala-Asp-Asp heteropeptide, where "Fur" is carboxymethylimidazo[4,5-e]benzo[1,2-c; 3,4-c'] difuroxane, and as auxiliary – citric acid monohydrate, sodium hydroxide to bring pH to 5.35 and water for injections.

EFFECT: invention is proposed for use as an anti-aggregant-inhibitor of GPIIb/IIIa-receptor thrombocytes.

2 cl, 4 dwg, 12 tbl, 8 ex

Description

The invention relates to a pharmaceutical composition, namely, an intravenous solution containing the active substance Fur-Lys-His-Ala-Asp-Asp heteropeptide, where “Fur” is carboxymethylimidazo [4,5-e] benzo [1,2 -c; 3,4-c '] difuroxan, and as auxiliary citric acid monohydrate, sodium hydroxide 1M or 2M solution for adjusting pH to 5.35 and water for injection for use as an antiplatelet inhibitor of GPIIb / IIIa platelet receptors .

Thrombosis and thromboembolism, which play a key role in the development of various cardiovascular complications, continue to be one of the most pressing problems of modern medicine. Thromboembolism in its modern interpretation is a disease characterized by thrombosis on the wall of veins or arteries with a narrowing or “blocking” of their lumen as a result of the growth of a thrombus or the movement of its fragments distal to the bloodstream. The decisive role in the violation of the rheological parameters of the blood is played by the aggregation state of platelets and red blood cells. Currently, drugs are used as prophylaxis of thrombosis that affect different parts of the hemostatic system, both separately and in various combinations. At the same time, antiaggregation therapy is an integral part of the pathogenetic therapy of diseases of the cardiovascular system. Of the disaggregants, the most famous ones used in practical medicine include acetylsalicylic acid, clopidogrel, ticlopidine, dipyridamole (curantyl), butadione, anturan, indomethacin (methindole), papaverine, clofibrate, parmidin, etc. These drugs reduce the functional activity of thrombocytes the turn is their ability to aggregate. The mechanisms of action of antiplatelet agents are due to the ability of drugs to disrupt the metabolism of arachidonic acid, increase the intracellular level of cyclic AMP (cAMP), change the state of the cytoplasmic membrane of platelets and others. The blockade of aggregation can be achieved by inhibiting the effects of thromboxane A2 (cyclooxygenase inhibitor, acetylsalicylic acid inhibitors) ADP receptors - thienopyridines - clopidogrel and ticlopidine) or by neutralization of platelet glycoprotein receptors IIb / IIIa (antagonists IIb / IIIa likoproteinovyh receptors - abciximab, eptifibatide and tirofiban). Thus, the prevention of thrombosis and thromboembolism, which is a key point in the development of cardiovascular pathologies, with the help of antiplatelet drugs is a determining direction in the treatment of patients in therapeutic and surgical practice [2-6].

Abciximab, eptifibatide and tirofiban IIb / IIIa glycoprotein receptor inhibitors uncompetitively block the final aggregation pathway, which consists in cross-linking platelet glycoprotein receptors under the influence of fibrinogen [Alekseev, A.A. Platelet Aggregation Peptide Inhibitors: Mathematical Modeling, Synthesis, and Evaluation of the Specific Activity of New Compounds in vitro / A.A. Alekseev, M.I. Brylev, V.L. Korolev [et al.] // Butlerov Communications. - 2012. - T. 32. - No. 11. - S. 96-100]. Having a moderate effect on the last stage of hemostasis - fibrinolysis, they also prevent the binding of the primary thrombus to this adhesive protein. The speed of the onset of antiplatelet and fibrinolytic effects, as well as the duration of the latter are the main distinguishing features of this pharmacological group [Alekseev, A.A. Peptide Platelet Aggregation Inhibitors / A.A. Alekseev, M.I. Brylev, V.L. Korolev [et al.] // Scientific notes. Electronic scientific journal of Kursk State University. - 2013. - T. 2. - No. 3 (27). - S. 32-37].

The negative side of the use of inhibitors of glycoprotein receptors IIb / IIIa is a high risk of bleeding and prolongation of its time, as well as the risk of thrombocytopenia, in connection with which, when administered, the parameters of the coagulogram and platelet count should be monitored. In addition, the lack of alternative parenteral forms of these drugs limits their scope, narrowing it to the inpatient stage of medical care.

Thus, eptifibatide, abciximab, and tirofiban found their application in unstable angina and ACS (with or without ST segment elevation) both before resolving the need for revascularization interventions and after percutaneous myocardial revascularization (PCI). Due to the fact that surgical removal of atherosclerotic plaque triggers platelet aggregation mechanisms, the main goal of UTI glycoprotein receptor inhibitors is the prevention of intracoronary thrombosis with a high risk of reocclusion of the operated vessel [Alekseev, A.A. Synthesis and study of the biological activity of new antiaggregational pentapeptides / A.A. Alekseev, M.I. Brylev, V.L. Korolev [et al.] // Sat. doc. The First All-Russian Scientific and Practical Conference "Problems of the Development of New Medicines". - Moscow. - 2013. - P.5.].

The range of antiplatelet inhibitors of the GPIIb / IIIa platelet receptor is presented in clinical practice by only four drugs - Abcicimab (ReoPro), Ruciromab (Monafram), Eptifibatide (Integrilin), Tirofiban (Agrastat). All of these drugs are available in parenteral dosage forms.

However, a significant drawback of these drugs is the pronounced side effects, the most significant of which are bleeding, which limits their widespread use [L. Jovanovic, N. Antonijevic, T. Novakovic, N. Savic et al., Semin. Thromb. Hemost., 2017, 43 (1): 14-23); Q. Shi, J. Xu, T. Zhang, B. Zhang, H. Liu. Front Pharmacol., 2017, 8:58]. In addition, there are other negative consequences - such as thrombocytopenia and immediate allergic reactions (urticaria, angioedema or bronchospasm), and in case of an overdose - the possibility of hemorrhage.

Since the drugs of the antiplatelet inhibitors of the GPIIb / IIIa platelet receptor are produced only in solutions for intravenous administration, their use is limited to the inpatient phase of medical care. This is due to unsuccessful attempts at the pharmaceutical development of oral dosage forms - peptidomimetics, inhibitors of GPIIb / IIIa-platelet receptors (Ximelofiban, Orbofiban, Sibrafiban, Lotrafiban) for the long-term prevention of thrombosis. Numerous clinical studies (phase III, more than 40,000 patients) have demonstrated their inefficiency and / or high mortality due to excessive bleeding and adverse cardiovascular events. Presumably, this is due to incomplete blockade of the GPIIb / IIIa platelet receptor by oral GPIIb / IIIa inhibitors.

Accordingly, it is an object of the present invention to expand the range of domestic preparations of antiplatelet inhibitors of GPIIb / IIIa platelet receptors having less toxicity and improved specific activity.

The technical result to which the claimed invention is directed is to create an effective intravenous pharmaceutical composition based on a heteropeptide that has an order of magnitude higher specific activity with appropriate dosage analogs, as well as reduced toxicity and pronounced side effects.

The technical result is achieved through the development of a pharmaceutical composition, which, in addition to a synthetic cyclic substance, consists of two pharmacophore groups (peptide and furoxane): Fur-Lys-His-Ala-Asp-Asp, where “Fur” is carboxymethylimidazo [4,5 ] benzo [1,2-s; 3,4-s'] difuroxane - a heteropeptide as an active substance, excipients - citric acid monohydrate, sodium hydroxide 1M or 2M solution to bring the pH to 5.35 and water for injection, when the following ratio of components, mg / ml:

Heteropeptide 0.5-2.5 Citric Acid Monohydrate 5-6 Sodium hydroxide 1M or 2M solution to bring the pH to 5.35 How much is required to bring the pH to 5.35 Water for injections Up to 1.0 ml

The closest analogue for comparing formulations by the principle of action is Integrilin.

The innovative drug Heteropeptide, a solution for intravenous administration, belongs to the pharmacotherapeutic group of antiplatelet agents. The active substance is a heteropeptide, a synthetic cyclic substance consisting of two pharmacophore groups (peptide and furoxan): Fur-Lys-His-Ala-Asp-Asp, where "Fur" is carboxymethylimidazo [4,5-e] benzo [1,2 -s; 3,4-s'] difuroxan was first described in the dissertation “DEVELOPMENT AND RESEARCH OF A MEDICINAL PRODUCT BASED ON NEW PEPTIDE THROMBOCYTES AGGREGATION INHIBITORS” Alekseev A. A., Moscow 2015. The peptide pharmacophore receptor group III inhibitor is III. The furoxan pharmacophore group is a nitric oxide donor. The presence of two pharmacophores with different mechanisms of action has a synergistic antiplatelet effect. Prevents the binding of fibrinogen, von Willebrand factor and other adhesive ligands to glycoprotein IIb / IIIa platelet receptors, reversibly inhibits platelet aggregation. The drug is a platelet receptor GP IIb / IIIa antagonist and an exogenous nitric oxide donor. The composition of the finished dosage form includes excipients: water for injection as a solvent, citric acid monohydrate as a solution stabilizer, sodium hydroxide as a pH regulator. The main indications for use: antiplatelet therapy in acute coronary syndrome.

The results obtained in experimental studies indicate the promising use of the drug Heteropeptide, a solution for intravenous administration, 0.75 mg / ml and 2 mg / ml to suppress platelet aggregation. It was found that the activity of the studied medicinal product is 25% higher than that of the comparison drug Integrilin.

Peptide drugs have a number of drawbacks related to their structure and pharmacokinetic characteristics: thermolability, metabolic instability (enzymatic degradation and susceptibility to hydrolysis in the acidic environment of the gastrointestinal tract), short half-life, rapid metabolism and high clearance.

The heteropeptide due to the furoxane fragment has a more compact and protected structure than the original linear short-chain polypeptides. However, due to the thermolability of the pharmaceutical substance, its storage is carried out at a temperature of minus 5 to minus 18 ° C.

The correct choice of excipients is important in the technology of dosage forms. Previously, only indifferent excipients were seen in excipients, the significance of which was to give the pharmaceutical substance an appropriate shape and volume for the convenience of its administration, transportation, storage. However, the results of studies of recent decades have led to a review of the biological role of excipients. They can enhance, reduce the therapeutic effectiveness of pharmaceutical substances or change the nature of their action under the influence of various factors. Excipients must meet the basic requirement: to disclose the entire gamut of pharmacological properties of the drug, to ensure its optimal effect. The correct choice of excipients allows you to reduce the dose of the pharmaceutical substance while maintaining (or increasing) its therapeutic effect. From modern positions, there are no indifferent or universal excipients, therefore, the composition of each specific drug should be scientifically substantiated.

Excipients not only have formative and technological functions, but are also active components of the drug composition. It has been established that not a single pharmaceutical factor has such a significant and complex effect on the action of a drug as excipients. The effect of excipients on bioavailability and therapeutic activity is great. It is important to consider the safety of the used excipients, since their unreasonable selection can lead to a weakening of therapeutic activity, as well as to all kinds of adverse reactions.

Pharmaceutical substances of the peptide and protein structure are thermolabile and, in most cases, are not highly stable, hydrolytically unstable, subject to transformation, and easily metabolized in the body [21. Banga, A. K., Therapeutic peptides and proteins: Formulation, Processing, and Delivery Systems. 3rd ed. Atlanta: CRC Press, 2015. - P. 139-167].

A brief description of the drawings.

Figure 1 - The dependence of the degree of platelet aggregation on the concentration of inhibitors of Integrilin and heteropeptide (PRC-007) when adding ADP 8 μm.

Figure 2 - Change in the concentration of the drug over time in the blood plasma of rats after a single intravenous administration.

Figure 3 - Assessment of the linear dependence of AUC (0-t) on the dose.

Figure 4. The change in the concentration of the drug over time in the plasma of rabbits after repeated intravenous administration.

Example 1. The study of the finished dosage form

The negative experience of numerous clinical trials of oral dosage forms of antiplatelet inhibitors of GPIIb / IIIa-platelet receptors determines the feasibility of pharmaceutical development of a heteropeptide in a dosage form for parenteral use. Dosage forms for parenteral use include:

injection and infusion dosage forms; concentrates for the preparation of injection and infusion dosage forms; solid dosage forms for the preparation of injection and infusion dosage forms; dosage forms for implantation.

At the stage of preliminary experimental studies, a solid dosage form of a heteropeptide for parenteral use, a lyophilisate for the preparation of a solution for injection, was proposed. The dosage form of the lyophilisate was not very convenient, taking into account the specifics of the use of the drug in clinical practice, since antiplatelet inhibitors of inhibitors of the GPIIb / IIIa platelet receptor require first bolus injection (loading dose) followed by infusion (maintenance dose). Therefore, the volume of the injected drug solution should be at least 10 ml (in the first case) and at least 100 ml (in the second case). The heteropeptide lyophilisate (8 mg / ml) proposed at the preliminary stage of the study is packaged in 2 ml ampoules into which it is possible to add only 2 ml of solvent. The use of a heteropeptide lyophilisate in small ampoules in clinical practice can provoke the risk of microbial contamination, solution “confusion”, and dosage failure (human factor) during the final preparation of the drug (lyophilisate dissolution before administration). The theoretical replacement of the capacity of the ampoules with the lyophilisate of the heteropeptide from 2 ml to 10 ml does not solve the problem of administering a maintenance dose that requires the administration of large volumes of a drug solution.

Given the specifics of the administration of drugs, inhibitors of the GPIIb / IIIa receptors of platelets (into the intravascular bloodstream, often with percutaneous coronary interventions, the introduction of loading and then maintenance doses per 1 kg of patient’s body weight), the optimal dosage form of the heteropeptide is injection (infusion) solution for intravenous administration.

The intravenous injection route has undoubted advantages: it prevents the destruction of the pharmaceutical substance under the influence of enzymes of the gastrointestinal tract; ensures the dosage accuracy of the pharmaceutical substance, 100% bioavailability of the dosage form (the pharmaceutical substance enters the bloodstream immediately, bypassing the gastrointestinal tract and liver); desired speed and place of administration; the possibility of introducing drip large volumes of liquids; the possibility of immediate termination of the administration process in case of side effects.

Example 2. Research and selection of excipients. The choice of solvent is the first stage in the development of the composition of liquid dosage forms, including solutions. As solvents, individual chemical compounds or mixtures thereof are used, capable of dissolving various medicinal substances (forming homogeneous systems with them - solutions). Requirements for pharmaceutical solvents: high solubility, chemical indifference and biological harmlessness, microbiological purity and availability. In the pharmaceutical technology of liquid dosage forms, the most affordable, almost universal solvent is water. In the case of the solubility of the pharmaceutical substance in water, the stability of the resulting solutions during storage of the dosage form, the preference when choosing a solvent is given primarily to water.

When choosing a solvent for compounds of the peptide structure, the physiological indifference of water and its physicochemical compatibility with the pharmaceutical substance play a special role. The heteropeptide is soluble in water at a ratio of 1: 300 - 1: 320 at a temperature of 8 ° C and at a ratio of 1: 100 - 1: 120 at a temperature of 20 ° C. The developed dosage form of the heteropeptide is a solution for intravenous administration. Given the relatively low dosage of the heteropeptide in the dosage form (0.75 mg / ml and 2 mg / ml), the dissolution of the heteropeptide in the indicated concentrations in water at 8 and at 20 ° C will be complete. Given the peptide structure of a pharmaceutical substance, which determines its thermal lability, the upper limit of the temperature regime of dissolution in the production process should be limited to no more than 22 ° C.

As a result of an experimental study, it was confirmed that the dissolution of the heteropeptide (0.75 mg / ml and 2 mg / ml) in water for injection at temperatures of 8 and 20 ° C occurs completely, the resulting solutions are colorless transparent liquids.

Stabilizer selection

In the pharmaceutical development of protein and peptide drugs, special attention is paid to stabilizing the structure of the pharmaceutical substance, increasing shelf life, protecting against the effects of damaging factors, standardizing technological processes and controlling the quality of finished products in order to produce safe drugs.

The issues of drug stability are currently given great attention. In the process of production and storage, a change in the physicochemical properties of drugs often occurs, proceeding at different rates and degrees of manifestation. Such changes affect the shelf life of drugs. The stability of drugs depends on storage temperature, lighting, the composition of the surrounding atmosphere, production technology, type of packaging and many other factors.

The stability of the parenteral dosage form is composed of chemical, physical and microbiological stability. Chemical stability is determined mainly by the resistance of the pharmaceutical substance to hydrolytic decomposition and oxidation; physical concerns two-phase systems and determines their ability to remain in a finely dispersed state for a certain period of time; microbiological determines the resistance of drugs to the action of microorganisms during storage.

Stabilizers can slow down or accelerate undesirable chemical reactions, create certain pH values of solutions, increase the solubility of pharmaceutical substances, and the choice of stabilizer primarily depends on the nature of the pharmaceutical substance. To stabilize solutions for intravenous administration, buffer solutions are often used to maintain an optimal pH value.

Buffers and buffer solutions are solutions that are able to maintain an almost constant pH when small amounts of acid or alkali are added to them. Most often, phosphate, citrate and citrate-phosphate buffer systems are introduced into the composition of injection solutions.

In the course of the studies, the compositions of antiaggregants (GPIIb / IIIa platelet receptor inhibitors) formulations registered in the Russian Federation for medical use in the form of solutions for intravenous administration were analyzed (table 1). To stabilize the solutions for intravenous administration of Monafram and ReoPro medicines, a phosphate buffer was added to their compositions. Integrilin contains citrate buffer.

Thus, it may be appropriate to introduce a phosphate or citrate buffer into the heteropeptide solution. However, in a published study by Adamo et al. (2016) studied the effect of buffer excipients that are part of the medication of inhibitors of GPIIb / IIIa platelet receptors on the development of thrombocytopenia. Initially, phosphate buffer was used to maintain a slightly acidic pH of the Tirofiban peptidomimetic solution, however, due to insufficient stabilizing properties, it was replaced in the formulation with citrate. The clinical use of Tirofiban phosphate buffered saline formulations has shown a significantly greater number of cases of thrombocytopenia (first phase of clinical trials) than the use of citrated buffered formulations (second phase of clinical trials). Thus, the nature of the buffer solution significantly influenced the degree of thrombocytopenia, and priority was given to the inclusion of the citrate buffer system in the composition of the drug-inhibitors of platelet GPIIb / IIIa receptors.

The composition of dosage forms for parenteral administration allows the introduction of stabilizers, which include citric acid and sodium hydroxide - components of a citrate buffer system.

Accordingly, there is a need to solve the problem for pharmaceutical development - to choose the optimal composition of a citrate buffer system with a target pH range that maximally supports the stability of the heteropeptide in the solution for intravenous administration. To achieve the stability of the heteropeptide, auxiliary substances were used - citric acid monohydrate and sodium hydroxide, based on their functional and physicochemical properties. Citrate buffer systems with different pH values and composition of the buffering agents were investigated.

In the course of the work, samples of the heteropeptide solution were controlled. The behavior of the pharmaceutical substance heteropeptide in solution was studied at pH values characteristic and critical for it in terms of transparency, color and impurity content (table 2). Targetable identifiable limits on the content of impurities are set for any single unidentified impurity - not more than 0.1%; for the sum of all impurities - not more than 1.0%. The analysis of heteropeptide solutions in therapeutic concentrations (0.75 mg / ml and 2.0 mg / ml) at various pH levels indicates the need to maintain the pH level for the dosage form in the range from 5.0 to 5.5 in order to avoid the appearance of degradation impurities and slightly yellow staining solution. The study of the quality indicators of the heteropeptide solution was carried out at 8 ° C without temperature exposure, taking into account the peptide structure of the pharmaceutical substance (thermal lability).

Thus, as a result of experimental studies, analysis of the scientific literature and content analysis of the assortment of antiplatelet drugs, inhibitors of GPIIb / IIIa platelet receptors, scientifically substantiated and proposed optimal solutions of heteropeptide for intravenous administration with antiaggregatory properties:

1) Heteropeptide 0.75 mg / ml; citric acid monohydrate 5.25 mg and sodium hydroxide 1.70-220 mg / ml to pH 5.35, water for injection up to 100 ml;

2) Heteropeptide 2.0 mg / ml; citric acid monohydrate 5.25 mg and sodium hydroxide 1.70-220 mg / ml to pH 5.35, water for injection up to 10 ml.

For the pharmaceutical substance and excipients in the composition of the dosage form and in the production standard load, excesses are not provided.

Example 3. Determining the effectiveness of the investigated composition.

The study of specific activity must be carried out in comparison with the reference drug. Integrilin, which also has a peptide structure, was chosen as the reference drug for the study of the antiplatelet properties of the heteropeptide. Integrilin is a synthetic substance with a peptide structure similar to the RGD sequence (Arg-Gly-Asp).

Drug Information Comparison Eptifibatide (Integrilin); CAS Code: 188627-80-7; Dosage form: solution for intravenous administration. Integrilin is a synthetic cyclic heptapeptide containing 6 amino acids and a mercaptopropionyl residue, desaminocysteinyl. The drug prevents the binding of fibrinogen, von Willebrand factor, and other adhesive ligands to glycoprotein IIb / IIIa platelet receptors. Thus, the mechanisms of action of Integrilin and the heteropeptide are identical.

The activity of the antiplatelet dosage form (heteropeptide) was studied on the model of ADP-induced platelet aggregation on human donor blood (platelet-rich plasma) in comparison with the drug Integrilin, solution for intravenous administration of 0.75 mg / ml, (Glaxo Operations UK (Great Britain)).

Blood sampling technique: Vacutest vacuum systems with 3.8% sodium citrate were used to take a venous blood sample. Platelet-rich plasma was obtained by centrifuging whole blood at 200 g for 20 minutes. To prepare experimental platelets, platelet-rich plasma was acidified to pH 6.5 with 1 M citric acid; the sample was centrifuged at 1500g for 20 min to obtain a precipitate that was resuspended in Hepes Ca2 ⁺ buffer [152 mm NaCl, 2.8 mm KCl, 8.9 mm NaHCO3, 0.8 mm KH2PO4, 0.8 mm MgCl2, 5, 6 mM glucose, 10 μM EGTA, BSA (3.5 mg / ml) and 10 mM Hepes, pH 6.5]. Platelets were washed once with the above buffer and suspended in the same buffer and the pH was adjusted to 7.4. Platelet concentration was standardized to 2.5 × 10 ⁸ cells / ml by dilution with Hepes buffer. Platelet counts were determined using a Coulter hematology analyzer.

Platelet Aggregation Analysis: The anti-aggregation activity of the test samples was studied in platelet-rich plasma using adenosine diphosphate (ADP) as an inducer of platelet aggregation. All procedures were carried out in polystyrene dishes with thromboresistant properties. Throughout the study period, rich and platelet-free plasma was at room temperature, and platelet aggregation was recorded at 37 ° C.

Platelet aggregation was studied using the Born turbidimetric method, based on the change in light transmission (540 nm) through the plasma under study with constant stirring (1000 rpm). Platelet-free plasma was used as a reference sample. Light transmission through platelet-free plasma was taken as 100%, and light transmission through platelet-rich plasma was taken as 0%. The platelet concentration was adjusted to 2.5 × 10 ⁸ cells / ml in platelet-rich plasma by diluting platelet-poor plasma. ADP at a concentration of 8 μM was used as an inductor. The studied compound (in the form of an aqueous solution, if necessary, containing DMSO up to 0.2%) at various concentrations (from 5 to 60 μg / ml) was added to the sample before introducing the aggregation inductor (ADP) in three independent experiments. In the statistical processing of the results, the differences were considered significant at a 95% significance level (p≤0.05). Platelet aggregation inhibition data by Integrilin and heteropeptide (PRC-007) are presented in table 3 and figure 1.

The results show the presence of dose-dependent inhibition of ADP-induced platelet aggregation of healthy blood donors under the action of peptide inhibitors of GP IIb / IIIa - platelet receptors.

As a result of the studies, the IC50 values of Integrilin were ~ 15-20 μg / ml and IC50 of the heteropeptide (PRC-007) ~ 8-12 μg / ml. Thus, it was found that the antiaggregation activity of the heteropeptide (PRC-007) was significantly 25% higher compared to the drug Integrilin. The high antiaggregation activity of the heteropeptide, which exceeds the activity of Integrilin (a GPIIb / IIIa peptide inhibitor) used in clinical practice, can be explained by the synergistic effect of excipients and the active substance.

The dosage of the heteropeptide in the solution for intravenous administration was selected based on its anti-aggregation activity, which is 25% more effective than the anti-aggregation activity of the comparison drug Integrilin.

Example 4. Determination of loading and maintenance doses.

The recommendations on the route of administration and the dose of Integrilin (INN: Eptifibatide) in acute coronary syndrome were investigated. Appendix 8c to the Recommendations of the Society of Specialists in Emergency Cardiology "Diagnosis and treatment of patients with acute coronary syndrome without raising the ST segment of the electrocardiogram" (2015) shows the method of administration and dose of Integrilin: When starting administration before percutaneous coronary intervention: intravenously, with a bolus of 180 mcg / kg , then an infusion of 2 μg / kg / min for 72-96 hours (after percutaneous coronary intervention, continue for 18-24 hours). At the beginning of administration immediately before percutaneous coronary intervention: intravenously; a bolus of 180 mcg / kg followed by infusion of 2 mcg / kg / min, after 10 minutes a second bolus of 180 mcg / kg; start before the procedure, continue during it and for the next 18-24 hours. In patients with glomerular filtration rate <50 ml / min / 1.73 m ^2, reduce the infusion rate to 1 μg / kg / min; not recommended for glomerular filtration rate <30 ml / min / 1.73 m ² . The recovery time of platelet function by 50% after the termination of iv administration is 2-4 hours.

Thus, the loading and maintenance doses of the heteropeptide were determined by analogy with the doses of Integrilin. In the process of anti-aggregation therapy using a heteropeptide, it is recommended to recalculate taking into account the activity of the load and maintenance dose of the drug per 1 kg of body weight.

Given the proposed method of administration, loading and maintenance doses of an antiplatelet drug, we substantiated two effective therapeutic dosages of the heteropeptide in the solution for intravenous administration: 0.75 mg / ml and 2 mg / ml.

Example 5. Obtaining a solution of a heteropeptide.

The main technological process for the production of a heteropeptide solution (0.75 mg / ml and 2 mg / ml) for intravenous administration is carried out under aseptic conditions by dissolving the heteropeptide in water for injection at (20 + 2) ° С, followed by stabilization of the heteropeptide solution with a citrate buffer system to a pH value of 5.35; membrane sterilization using polyethersulfone or cellulose filters; pouring into sterile glass bottles, corking the bottles with sterile rubber stoppers and running in aluminum caps.

Stages of solution preparation and stabilization of a heteropeptide solution

In the process of developing a technological process for the production of a heteropeptide solution, the optimal procedure for introducing ingredients into the solution at the stages of dissolution and stabilization of the solution with a citrate buffer system was determined.

It was found that the order of introduction of buffering agents (citric acid monohydrate and sodium hydroxide) does not affect the stability of the heteropeptide solution.

The preparation of an aqueous solution of the heteropeptide was carried out in a stainless steel container, which would avoid its spill taking into account the likely risk of damage to the glass container. For the complete dissolution of the pharmaceutical substance and components of the citrate buffer system, an overhead portable paddle-type mixer (mixer) was used. The optimal speed of rotation of the mixer is 100-120 rpm. The time of complete dissolution of the heteropeptide is about 20 minutes at a temperature of (20 ± 2) ° C. The limiting temperature regime of dissolution of the heteropeptide is not higher than 22 ° C.

After complete dissolution of the pharmaceutical substance, the resulting heteropeptide solution was stabilized with a citrate buffer system. Then analyzed the pH of the solution, the value of which should be in the range from 5.0 to 5.5. In case of non-compliance with the specified standard, an adjustment was made with 1 M sodium hydroxide solution.

Filtration and sterilization stages

One of the requirements for dosage forms for injection is the lack of mechanical impurities and sterility. Injection solutions should not contain particles visible to the naked eye.

In almost all stages of production, contamination of drugs can occur. Three types of parenteral drug contamination are distinguished: mechanical, chemical (soluble) and microbial. Mechanical and microbial pollution are closely related: often their sources are the same, methods of dealing with them are similar. Mechanical inclusions present in the injection solution can lead to blood clots, granulomas, allergic reactions and other pathological phenomena. Therefore, one of the most important tasks to be solved in the production process of sterile solutions is the complete release from mechanical impurities, for this, filtration is used.

Currently, the simplest, most cost-effective technologies for producing dosage forms are gaining importance. In the case of pharmaceutical development of sterile solutions for intravenous administration, it is rational to use a technology that includes combining the two stages of filtration and solution sterilization in order to minimize the influence of technological pharmaceutical factors on the bioavailability of the drug. The peptide structure of the heteropeptide determines the thermolability of the pharmaceutical substance; as a result, thermal methods of sterilization of the heteropeptide solution are unacceptable. It is advisable to use a gentle membrane filtration method, which combines both the filtration stage and the sterilization stage.

In membrane (or sieve) filtration, particles larger than the pore size of the filter are retained on its surface. Depending on the ratio of pore size and particle size in the membrane filters, deep, screen or mixed retention mechanisms are implemented. Membrane filters have a certain pore size, mediated by the rigidity of the frame and mainly the sieve separation mechanism. The compatibility of the material of the filtration system and the filtered liquid is attached significant importance, since the filter may be in contact with the liquid during the filtration process for a long time (from 8 to 30 hours). Filters based on cellulose derivatives are most often used for sterilization; the working pH range of aqueous solutions for these filters is 1.0-10.0.

For sterilization, membrane filters with a pore size of 0.22 μm or less are used. In the process of developing technology for preparing a heteropeptide solution for intravenous administration, the stability of the heteropeptide solution during sterilization through various types of membranes was investigated. In laboratory conditions, filtration units with hydrophilic polyethersulfone membranes of the PTGC type (Millipore, USA) were used for final sterilization; membranes made of cellulose acetate Corning ^® Vacuum Filters (USA); nylon membranes (Pall Corporation, USA). Cellulose acetate filtration membranes have low adsorption characteristics, polyethersulfone membranes are able to provide high flow rates, and are also applicable over a wide range of pH values. Used glass filter holders for reuse with the ability to replace filters. The filtration process in these plants depends on the volume and viscosity of the true solution and occurs under vacuum.

Evaluation of sterile solutions for intravenous injection when choosing a material for filtration was carried out according to the indicators: color, transparency, lack of mechanical impurities, change in pH of the solution, quantitative content of the heteropeptide. Evaluation was carried out before and after sterilization (table 4).

The results of the studies confirmed the possibility of using both polyethersulfone and cellulosic materials in the process of sterilizing membrane filtration of a heteropeptide solution. All quality indicators of sterilized solutions of the heteropeptide corresponded to the target values. Filtration of the heteropeptide solution through the installation with nylon filters led to a significant (more than 10% of the initial content) decrease in the concentration of the heteropetide, which is probably associated with the adsorption of the heteropeptide on this type of filter.

To remove mechanical impurities from the solution and its sterilization, a sterilizing filtration unit was used, consisting of a peristaltic pump (model SP 311, VELP SCETIFICA, Italy) and a filter module using a membrane filter with a pore diameter of 0.22 μm, which allows obtaining pure solutions . This method is used to sterilize thermolabile solutions. Polyethersulfone membranes of the PTGC type (Millipore, USA) were used as ultrafiltration membranes. Filtration results in sterilization of the solution, separation of proteins and other macromolecular compounds and mechanical impurities.

A study of the specific pharmacological activity of the drug. Antiplatelet activity was studied in platelet-rich plasma using adenosine diphosphate (ADP) as an inducer of platelet aggregation. The obtained results showed the presence of dose-dependent inhibition of ADP-induced platelet aggregation of healthy blood donors under the action of the studied funds. As a result of the analysis of the specific action, it was found that the activity of the studied medicinal product is 25% higher than that of the comparison drug Integrilin. IC ₅₀ for the studied drug ~ 8-12 μg / ml.

Example 5. The study of pharmacokinetics.

The pharmacokinetics research methods were carried out in accordance with standard practices and are known from the prior art to an average specialist.

The study was conducted as open prospective, with the inclusion of animals in various groups to study pharmacokinetics. According to the results of the selection of animals, a sequential set of three groups of rats was made. A total of 220 male rats were used (Table 5). 18-20 hours before the study, the animals were deprived of food with open access to water. Samples of the study drug were administered intravenously once at doses of 260 μg / kg, 1300 μg / kg and 2600 μg / kg to rats.

Before blood sampling, rats were placed in a CO2 chamber until breathing was completely stopped. Blood was taken from the jugular vein 1 ml with EDTA at the following time points: 0, 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 24 hours. There were 5 animals per time point. Also, at a dose of 2600 μg / kg in animals, organs were taken (brain, thymus, heart, lungs, liver, kidneys, spleen) to study the distribution of the drug at the following time points: 0, 0.1, 0.25, 0.5, 0.75, 1, 1.5 , 2, 3, 4, 6, 8, 12, 24, 36, 48 hours. Plasma was obtained from blood in the usual way: centrifugation at 3500 rpm at 4 ° C and preservation at minus 20 ° C in 1 ml aliquots.

During the entire experiment, including its biological part, there were no differences in the behavior, appearance and manifestations of physiological functions in experimental rats before and after they received the drug.

The figure 2 presents the experimental data for determining the concentration of the test drug in rat plasma samples at different times after a single intravenous dose of 260 μg / kg, 1300 μg / kg and 2600 μg / kg.

Individual values of the concentration of the drug in the blood plasma of rats after a single intravenous administration are presented in tables 6-8.

The values of the pharmacokinetic parameters were obtained from experimental data in an automatic mode using the PK Solver Excel module program (table 9).

After a single intravenous administration of the drug to rats in three increasing doses of 260 μg / kg, 1300 μg / kg and 2600 μg / kg, the linear pharmacokinetics of the drug was studied. Based on the data obtained, the hypothesis of linear pharmacokinetics of the drug was put forward. To test this hypothesis, the statistical significance of the deviation from zero of the free member of the linear regression AUC (0-t) was evaluated. The calculation is presented in figure 3. The results showed that the free term is slightly different from zero and the hypothesis should be considered true.

An analysis of the concentrations and distribution of the drug in the organs of animals was also carried out. The main pharmacokinetic parameters of the drug in rat organs after a single intravenous dose of 2600 μg / kg are presented in table 10.

Table 10. The main pharmacokinetic parameters of the drug in rat organs after a single intravenous dose of 2600 mcg / kg

The analysis showed that the greatest retention time (MRT) of the drug was in the following organs: thymus (5.88 h), lungs (3.88 h) and spleen (3.60 h). The half-life (T1 / 2) of the drug was the largest for the following organs: thymus (3 hours), spleen (2.05 hours) and liver (1.92).

Based on the data obtained during the study of the pharmacokinetics of a drug with antiplatelet activity based on a heteromeric peptide with a single intravenous dose of 260 μg / kg, 1300 μg / kg and 2600 μg / kg, the pharmacokinetic parameters of the drug were calculated on SD rats. The dose dependence of the pharmacokinetic parameters of the drug in rats was evaluated. The hypothesis of linear pharmacokinetics is confirmed.

T1 / 2 of the drug at doses of 260 μg / kg, 1300 μg / kg and 2600 μg / kg when administered intravenously was 1.77 hours, 1.90 hours and 1.83 hours, respectively. The average retention time (MRT) of the drug at doses of 260 μg / kg, 1300 μg / kg and 2600 μg / kg when administered intravenously was 2.26 hours, 2.24 hours and 2.23 hours, respectively. The clearance (Cl_obs) of the drug in doses of 260 μg / kg, 1300 μg / kg and 2600 μg / kg when administered intravenously was 0.09 (μg / kg) / (ng / ml) / h for all doses studied.

To study the pharmacokinetics of the drug with repeated administration, 6 male rabbits were used. 18-20 hours before the study, the animals were deprived of food with open access to water. Samples of the study drug were administered intravenously at a dose of 1300 μg / kg to rabbits. Animals received the drug every 24 hours for 6 days (144 hours).

Blood was taken from the central ear vein 1 ml EDTA at the following time points: 0, 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 120.1, 120.25, 120.5, 120.75, 121, 121.5, 122, 123, 124, 126, 128, 132, 144 hours. At one time point there were 6 animals. Plasma was obtained from blood in the usual way: centrifugation at 3500 rpm at 4 ° C and preservation at minus 20 ° C in 1 ml aliquots.

The figure 4 presents the experimental data for determining the concentration of the studied drug in blood plasma samples of rabbits at different times after repeated intravenous administration at a dose of 1300 μg / kg

The individual plasma concentration values of the drug after repeated intravenous administration are presented in Table 11. The pharmacokinetic parameters were obtained from the experimental data automatically using the PK Solver Excel module program (table 12).

A comparison of the pharmacokinetics of the studied medicinal product after repeated administration to rabbits at a dose of 1300 μg / kg with a dosing interval of 24 hours did not show an acceleration of the elimination of the drug from the body after repeated administration. Ground clearance has not changed, mean retention time (MRT) has not changed. The maximum concentration created in plasma immediately after the next injection was 9150 ng / ml and did not significantly differ from the maximum concentration in the first 24 hours of the experiment. Thus, repeated administration of a drug at a dose of 1300 mcg / kg does not affect its main pharmacokinetic parameters. Example 6. The study of toxicity.

Methods for assessing toxicity were carried out in accordance with standard practices and are known in the art to a person skilled in the art.

The acute toxicity of a drug with antiplatelet activity based on a heteromeric peptide was studied in fourteen experimental groups of CD rats and fourteen experimental groups of CD-1 mice, ten females and ten males in each group. The studied drug was administered intravenously in doses: 7.5, 22.5 and 45 mg / kg (HLF 0.75 mg / ml) and 20, 60, 120 mg / kg (HLF 2 mg / ml) to rats; 18.75, 56.25, 112.5 mg / kg (GLF 0.75 mg / ml) and 50, 150, 300 mg / kg (GLF 2 mg / ml) in mice and intraperitoneally in doses: 18.75, 56 25, 112.5 mg / kg (GLF 0.75 mg / ml) and 50, 150, 300 mg / kg (GLF 2 mg / ml) to rats; 37.5, 112.5, 225 mg / kg (GLF 0.75 mg / ml) and 100, 300, 600 mg / kg (GLF 2 mg / ml) in mice.

In all doses, the studied medicinal product did not cause death or worsening of the animals, spontaneous locomotor activity was similar to the control group, the lag in the increase in body weight of animals of the experimental groups compared with the control group was also not registered. Deviations in hematological and biochemical parameters in all studied groups in relation to the control were not observed. There was no significant toxic effect of the studied drug on the mass of rat organs in an acute experiment. Calculation of lethal dose indicators was not possible.

A study of the chronic toxicity of a drug with antiplatelet activity based on a heteromeric peptide in two dosage forms of 0.75 mg / ml and 2 mg / ml was carried out in rats and rabbits (males and females). Samples of the study drug were administered intravenously at doses of 260 μg / kg, 1300 μg / kg and 2600 μg / kg to rats and 100 μg / kg, 500 μg / kg and 1000 μg / kg to rabbits. Which is equivalent to equal doses when recalculated taking into account the species coefficient and amounts to 1, 5 and 10 TD, respectively. Medicines were administered daily for 30 days.

In all doses, the studied medicinal product did not cause deterioration of the condition and death of animals during the entire observation period. Animals retained their normal appearance, coat, the nature of the discharge and behavioral reactions that did not differ from those in animals in the control group. The increase in body weight did not slow down and was similar to the increase in the control group.

Chronic administration of a drug with antiplatelet activity based on a heteromeric peptide in two finished dosage forms: 0.75 mg / ml and 2 mg / ml does not reduce the motor and cognitive activity of rats compared to animals in the control group.

The urine analysis data of animals at a dose of 2600 μg / kg for rats and 1000 μg / kg for rabbits indicate the absence of pathological changes in the kidneys after 30 days of drug administration. In the urine of animals of the experimental group and the comparison group, erythrocytes and protein were not detected, which indicates the integrity of the filtration apparatus of renal nephrons.

The toxic effect of the studied medicinal product in both finished dosage forms on the blood system of rats and rabbits in a chronic experiment at low and medium doses was not detected. Fluctuations of all quantities were within the physiological norm. In rats at a maximum dose of 2600 μg / kg, in both dosage forms of HFL 0.75 mg / ml and HLF 2 mg / ml, thrombocytopenia and a decrease in hemoglobin and red blood cells were observed. The platelet count decreased by 32.9% and 31.9% in both dosage forms of GLF 0.75 mg / ml and GLF 2 mg / ml, respectively. The hemoglobin level decreased by 53.9% and 58.2% in both dosage forms of GLF 0.75 mg / ml and GLF 2 mg / ml, respectively. The level of red blood cells decreased by 55.2% and 76.5% in both dosage forms of GLF 0.75 mg / ml and GLF 2 mg / ml, respectively. In rabbits at a maximum dose of 1000 μg / kg in both dosage forms of HFL of 0.75 mg / ml and HFL of 2 mg / ml, thrombocytopenia and a decrease in the level of hemoglobin and red blood cells were also observed. Platelet count decreased by (31.9–25.4)% (male-female) and (26.0–26.0)% (male-female) in both dosage forms of HLF 0.75 mg / ml and HLF 2 mg / ml, respectively. Hemoglobin level decreased by (29.3-25.0)% (male-female) and (28.0-25.4)% (male-female) in both dosage forms of HLF 0.75 mg / ml and HLF 2 mg / ml, respectively. The level of red blood cells decreased by (31.6-31.6)% (male-female) and (28.1-33.3)% (male-female) in both dosage forms of HLF 0.75 mg / ml and HLF 2 mg / ml, respectively. These changes are the result of the specific activity of the studied medicinal product and are described in the literature for this class of drugs - antiplatelet agents. All changes returned to normal after a recovery period of drug withdrawal.

A biochemical study of the blood serum of animals of experimental groups (rats and rabbits) that received the studied drug with antiplatelet activity based on a heteromeric peptide in two dosage forms of 0.75 mg / ml and 2 mg / ml did not reveal a statistically significant change in enzyme activity compared to the control group. All values of biochemical parameters were within the physiological norm for a given animal species.

Assessment of the level and nature of pathological changes in internal organs (systems of internal organs) of experimental animals did not reveal pathological changes. Histological analysis of the injection site of the test drug and adjacent tissues did not reveal pathological changes. The test drug does not have a local irritant effect.

A drug with antiplatelet activity based on a heteromeric peptide in two finished dosage forms: 0.75 mg / ml and 2 mg / ml with repeated administration does not affect the functions of internal organs and vital systems of the body: liver, kidneys, hematopoietic system; does not have the effect of delayed body reactions and toxic side effects that may occur with the clinical use of high doses of the drug.

Example 7. Immunotoxic properties.

Methods for the study of immunotoxic properties were carried out in accordance with standard practices and are known from the prior art to the average specialist.

When studying the immunotoxic properties, it was found that a drug based on a heteromeric peptide does not change the humoral immune response at a dose of 1 TD and 10 TD, does not affect the number of antibody-forming cells in the spleen of CBA and C57BL / 6 mice. The studied drug did not have a significant effect on the delayed-type hypersensitivity reaction. When assessing the effect of the studied drug on the phagocytic index of peritoneal macrophages, it was found that its administration does not affect the value of the phagocytic index. The data obtained showed that the studied medicinal product does not significantly change the microbicides of macrophages, but positively affects the oxygen-dependent (HCT test) and oxygen-independent (LCT test) macrobial microbicides.

Thus, it can be concluded that a drug based on a heteromeric peptide does not have an immunotoxic effect, does not impair the proliferative activity of antibody-producing cells, phagocytosis, macrophage activation, the main components of an active immune response. In terms of immunological safety, the test drug is safe in the range of doses tested.

Example 8. Allergic properties

Methods for the study of allergenic properties were carried out in accordance with standard practices and are known from the prior art to an average specialist.

As a result of studying the allergenic effect of a drug with antiplatelet activity based on a heteromeric peptide: conjunctival tests in guinea pigs are negative; the test of general and active anaphylaxis is negative; skin applications - negative, delayed-type hypersensitivity reaction in mice - negative, pseudo-allergic reactions - Shore test - negative. Therefore, this study showed that the test drug lacks allergenic properties in the range of doses tested.

Those skilled in the art will appreciate that numerous changes and / or modifications can be made to the above described embodiments without departing from the broad general scope of the present invention. Thus, the presented embodiments should be considered illustrative in all aspects, but not limiting.

Claims (3)

1. A pharmaceutical composition having antiplatelet activity in the form of an aqueous solution for intravenous administration, comprising the active substance Fur-Lys-His-Ala-Asp-Asp heteropeptide, where "Fur" is carboxymethylimidazo [4,5-e] benzo [1 , 2-s; 3,4-s'] difuroxan, and as excipients of citric acid monohydrate, sodium hydroxide and water for injection, in the following ratio, mg / ml: Heteropeptide 0.5-2.5 Citric Acid Monohydrate 5-6 Sodium hydroxide to pH 5.35 Water for injections up to 1.0 ml 2. The use of the pharmaceutical composition according to claim 1 as an antiplatelet inhibitor of GPIIb / IIIa platelet receptors in acute coronary syndrome.

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