RU2396271C1 - Rgd-like peptides - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention discloses novel synthetic RGD-like peptides capable of dose-dependant inhibition of thrombocyte aggregation.

EFFECT: obtaining novel compounds capable of dose-dependant inhibition of thrombocyte aggregation.

2 tbl, 1 ex

Description

The invention relates to medicine, in particular to the synthesis of pharmacologically active compounds.

The problem of the prevention and treatment of thromboembolic complications continues to be relevant for modern medicine. Over the past decade, the key role of platelets in the development of cardiovascular disease has been established. Finding out the importance of platelets has stimulated the development of a large number of drugs. Antiplatelet agents have long been an integral part of the treatment of patients with myocardial infarction (MI), heart failure, coronary heart disease (CHD).

The prevention and treatment of these diseases remain relevant, as they remain the main cause of death in both the male and female populations of developed and developing countries. These issues are of paramount importance for domestic health care, because, unlike the United States and most European countries, Russia has not been able to reduce the incidence of coronary heart disease and myocardial infarction over the past two decades. For every doctor, it has long been an axiom that the key to solving this problem is the fight against risk factors for the onset of the disease, among which atherosclerosis is of primary importance.

Aterothrombosis - thrombosis on the surface of a damaged atherosclerotic plaque is the main pathogenetic mechanism of its growth and the cause of the development of complications of atherosclerosis. Thrombotic complications of atherosclerosis, primarily myocardial infarction (MI) and stroke, occupy a leading place in the structure of total mortality in most developed countries. In Russia, the situation looks particularly unfavorable: cardiovascular diseases account for the majority of deaths - 55.4%, while atherosclerosis is the cause of death in 30% of cases.

Antithrombotic therapy is recognized as the basis for the pathogenetic treatment of both acute and chronic forms of ischemic heart disease. The main directions of antithrombotic therapy are: inhibition of platelet function, effect on the blood coagulation system, restoration of patency of the vessel during its thrombotic occlusion (thrombolysis).

Given the prevalence and progressive increase in circulatory system pathologies in the world, as well as the relatively high mortality rate of patients, the development of fundamentally new antithrombotic drugs is a particularly urgent issue.

The greatest attention of specialists has recently been attracted to antiplatelet agents that block platelet and red blood cell aggregation. Antiplatelet agents include drugs of various mechanisms of action and belong to different groups of chemical compounds.

Peptides belonging to the class of RGD (Arg-Gly-Asp) -stimulants are potent inhibitors of platelet aggregation. The antiplatelet effect of RGD peptides is due to their ability to prevent the binding of fibrinogen, von Willebrand factor and other adhesive ligands to platelet glycoprotein llb / llla receptors.

In US patent No. 5,292,756; Granted: March 8, 1994, natural proteins with antiplatelet activity were screened, synthetic peptides of a similar action were obtained, and their identification and analysis were performed. Injectable dosage forms of synthetic antithrombotic peptides have been developed.

At the same time, analyzing the data, it should be noted that the obtained dosage forms do not have satisfactory stability, which is possibly due to the presence of impurities inevitably present in the preparations obtained in laboratory synthesis.

In the patent RU 2119354, A61K 47/48, 1998, the directed transport of drugs is described, which consists in binding in vivo carrier molecules of pharmacologically active compounds with blood cells involved in the pathological process. For binding, original derivatives of pharmacological agents and a peptide containing RGD were synthesized (carboxymethyl-5-fluorouracil-acetamidomethyl-cysteinyl-arginyl-glycyl-aspartyl-acetamidomethyl-cysteine and acetylarginylglycyl-aspartyl-S-nitrosocistenin).

It should be noted that in the above patent, the amino acid composition of the peptide is different from the proposed one and the antithrombotic effect is not considered. The synthesized peptide is used only as a carrier of pharmacologically active compounds to places of increased platelet adhesion.

The heterodimer GPIIb / IIIa (also known as integrin α _IIb / β ₃ ) is a surface platelet receptor. In the process of Ca ²⁺ -dependent activation, the complex undergoes a series of conformational changes that provide the possibility of platelet binding to fibrinogen.

The mechanism of functioning of the IIb / IIIa receptor lies in its ability to recognize two characteristic amino acid sequences. The first consists of Arg-Gly-Asp amino acids, it is found in fibronectin, von Willebrand factor, vitronectin, as well as in the α-chains of fibrinogen molecules, with two key sequences of Arg-Gly-Asp for each half of the fibrinogen molecule. It should be emphasized that the “key” sequence of Arg-Gly-Asp is recognizable by most representatives of the integrin family. The detailed mechanisms of the interaction of IIb / IIIa receptors with adhesive molecules have not been fully studied, but it is obvious that peptides or small molecules containing the key sequence of amino acids Arg-Gly-Asp can be potential inhibitors of the interaction of IIb / IIIa platelet receptors with fibrinogen.

GPIIb consists of a 116 kDa heavy chain surface platelet covalently linked by one disulfide bond to a light (22 kDa) chain, which is a transmembrane protein. The GPIIIa subunit is a 90 kD glycosylated polypeptide that consists of three domains — a large extracellular region at the N-terminus, a transmembrane domain, and a short cytoplasmic segment at the C-terminus.

Platelet IIb / IIIa receptor blockers are the youngest group of the entire class of antithrombotic drugs used in cardiology. The mechanism of action of the agents of this group is to inhibit the overall final stage of platelet aggregation - the process of building a blood clot through the formation of bridges between adjacent activated platelets from fibrinogen molecules. Platelet activation by various substances (ADP, thrombin, thromboxane A2, collagen) leads to a change in the form (activation) of platelets and activation of GP IIb / IIIa. As a result of platelet activation, the configuration of the IIb / IIIa receptors changes, which increases their ability to fix fibrinogen and other adhesive proteins. The binding of fibrinogen molecules to the IIb / IIIa receptors of various platelets leads to the connection of the plates with each other - aggregation. This process is independent of the type of activator and is the final and only mechanism of platelet aggregation. GPIIb / IIIa receptor inhibitors also bind to GPIIb / IIIa, blocking binding to fibrinogen, and thus prevent platelet aggregation.

The challenge is to create new peptides.

The problem is solved by peptides:

Lys-β-Ala-Asp-Trp

Arg-Aminovaleric acid-Asp-Trp

Lys-Gly-Gly-Asp-Trp

Arg-Gly-Gly-Asp-Trp

Arg-β-Ala-Asp-Trp

Based on previously obtained data [Pollina E. Design and sinthesis of RGD mimetics as potent inhibitors of platelet aggregation // J. Undergrad. Sci. - 1996. - 3: 119-126; Ojima I., Chakravarty S., Dong Q. Antithrombotic agents: from RGD to peptide mimetics // Bioorganic & Medicinal Chemistry. - 1995 .-- Vol. 4. - P. 337-360; Melnik O.V., Martinovich V.P., Golubovich V.P. The relationship of structure and anti-aggregation activity in a series of Arg-Gly-Asp analogues // Bioorganic chemistry. 2006, Volume 32, No. 2, pp. 137-143], it can be assumed that β-alanine, valerianic acid or a combination of glycine-glycine is preferable to use as an intermediate site. Such fragments of the molecule will allow to obtain the desired conformation and increase the biological activity of the peptide molecule. Tryptophan should probably be used as a hydrophobic site. It is for substances with such a hydrophobic group that the best results were obtained [Pollina E. Design and sinthesis of RGD mimetics as potent inhibitors of platelet aggregation // J. Undergrad. Sci. - 1996. - 3: 119-126]. The areas represented by arginine and aspartic acid, it makes sense to leave unchanged.

Peptide synthesis was performed on a solid phase using an Applied Biosystems 433A peptide synthesizer using an Fmoc strategy. It is based on the sequential addition of amino acid residues to an insoluble polymer substrate. The base labile group Fmoc is used to protect the N-groups of each amino acid residue. Those residues that have potentially reactive side chains are protected by acid-unstable groups, such as tert-butyl.

After removal of the Fmoc group with piperidine, the following protected amino acid is added using either a coupling reagent or a preactivated derivative of the amino acid.

The activator of the first amino acid in the Fmoc strategy in the reaction of its addition to the resin is dicyclohexylcarbodiimide

Dicyclohexylcarbodiimide (DCC) acts as an activator of the first amino acid in the Fmoc strategy in the reaction of its addition to the resin. The reaction proceeds in the presence of 4-dimethylaminopyridine (DCC / DMAP), which plays the role of a catalyst in the process. The result is an activated amino acid.

The activator of the second and subsequent amino acids in the Fmoc strategy is 1-hydroxybenzotriazole in dicyclohexylcarbodiimide (HOBt / DCC). The reaction proceeds in accordance with the formation of an activated amino acid and N, N'-dicyclohexylurea (DCU).

All of the above operations proceed in a peptide synthesizer.

To remove the peptide from the resin in a fume hood in a polypropylene tube, a mixture was prepared with the following ratio of components: TAN - 2.5%, TIPS - 2.5%, EDT - 5%, TFA - 90%. The prepared mixture was placed on ice until cooling for 20-30 minutes. Then, a peptide resin was placed in a cold peptide remover mixture and gently mixed. The peptide-resin tube was placed in a polypropylene tube, closed and kept on a rocking chair for 4 hours.

Peptide extraction from the resin was carried out on a glass funnel filter in a fume hood. The funnel was pre-rinsed twice with MTBE (methyl tertiary butyl ether). At the end of removal, the mixture with resin and peptide was poured onto a Schott filter. The tube in which the TFA reaction was washed was washed, the wash was poured onto the filter. It was filtered under low vacuum until the resin was dried. Cold MTBE was added, the resin was thoroughly washed and filtered until dry. The tube containing the peptide resin was washed three times with cold MTBE and a wash was added to the main drain. The suspension was thoroughly mixed. Leave the mixture for approximately 2 minutes. Then 100 were selected (for the obtained solution for analysis. (Storage temperature 4 ° C until analysis). The remaining mixture was left for at least 1 hour at -20 ° C. The mixture was centrifuged at 1500 g for 6 minutes, the supernatant was separated. The supernatant was added to the precipitate. MTBE, triturated with a spatula and frozen for 2 hours .. The operation of freezing - thawing was repeated twice.

The peptide was freeze dried from 0.01 M phosphate-buffered saline after dissolving the precipitate obtained after extraction.

Purification was performed by high performance liquid chromatography (HPLC).

HPLC conditions: Waters DeltaPak column, C18, 5 μm, 100 Å, 3.9 × 150 mm. Other reversed-phase columns C18 or C8 may be used.

but. Detector Cell: Analytical

b. Flow rate - 1.0 ml / min

from. Wavelength: 214 nm

d. Detection Range: 0.5 AUFS

e. Loop size: 100 μm (the loop is filled with 150 [± 10] ml of sample, that is, injection volume = 100 ml)

f. Eluent A: 5.0% acetonitrile, 0.1% trifluoroacetic acid, in water.

g. Eluent B: 60% acetonitrile, 0.085% trifluoroacetic acid, in water.

h. Gradient: 0-60% B for 60 min.

To confirm the structure of the obtained peptides, nuclear magnetic resonance (NMR) was used. One-dimensional NMR spectroscopy was performed on protons ( ¹ H) and carbon ( ¹³ C). For ¹ H and ¹³ C NMR, a weighed peptide was dissolved in DMSO-d6 with an isotopic purity of 99.9% D. A ¹ H NMR spectrum was obtained on an Evans-300 spectrometer with a resonant frequency of 300 MHz. Concentration measurements of proton chemical shifts were performed at a temperature of 296.6 K. All NMR measurements were performed under conditions of rapid exchange of interacting molecules in the time scale of NMR. Chemical shift was determined relative to the internal standard tetramethylsilane (TMS). The resulting picture of chemical shifts and signal intensities confirm the composition of substances.

The specific activity of the antiaggregatory action of peptides was evaluated in vitro using the blood of healthy donors and patients with platelet hyperaggregation. Blood sampling was carried out immediately before the study using sodium citrate (3.8%) as an anticoagulant. The ratio of anticoagulant: blood corresponds to 1: 9. The anti-aggregation activity of the obtained compounds was studied in platelet-rich plasma using ADP as an inducer of platelet aggregation.

To prepare platelet-rich plasma, the blood immediately after preparation was centrifuged for 10 minutes at 1000 rpm, after which the upper plasma layer was transferred to another tube, and the residue was centrifuged for 20 minutes. At 3000 rpm to obtain platelet-free plasma. All procedures were carried out in polystyrene dishes with thromborescent properties. Throughout the study period, rich and platelet-free plasma was at room temperature, and platelet aggregation was recorded at 37 ° C.

To study the specific anti-aggregation activity of the peptide, the requirements for preclinical studies of pharmacological substances of this class, approved by the Pharmacological Service for Supervision of Health and Social Development, were guided.

The aggregation study was carried out according to the GGVBom method. The study was performed on a LA230-2 instrument (Biola, Russia). The volume of the cell is 200 μl. ADP was used as an inductor at a concentration of 10 ^-7 M. The platelet count was recorded by the device. The peptide was studied at a concentration of 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 M and was added immediately before the addition of ADP. Eptifibatide solution in the same concentrations was used as a standard. The results are presented in table.2.

When studying the inhibitory effect on platelet aggregation of healthy donors, a dose-dependent effect of inhibiting platelet aggregation was found for all six compounds, but the pentapeptide Arg-Gly-Gly-Asp-Trp was the most active inhibitor. This compound inhibited ADP-induced platelet aggregation with IC ₅₀ 6.3 μM (Table 1).

When studying the effect of the peptide on the platelets of patients with hyperaggregation, the appearance of a disaggregation phase was found, which is absent in the control group, which indicates the discontinuous aggregation effect of ADP. Thus, inhibition of aggregation by the peptide of both blood groups was found, which demonstrates a pronounced anti-aggregation effect.

Tab. one The effect of RGD peptides on the inhibition of ADP-induced platelet aggregation in vitro Compound IC ₅₀ μm (ADP, 1.5 μM) Lys-β-Ala-Asp-Trp 590.0 Arg-Aminovaleric acid-Asp-Trp 19.8 Lys-Gly-Gly-Asp-Trp 238.4 Arg-Gly-Gly-Asp-Trp 6.3 Arg-β-Ala-Asp-Trp 14.3

Tab. 2 The study of the specific antiaggregatory activity of the peptide Arg-Gly-Gly-Asp-Trp Peptide concentration Platelet Aggregation Arg-Gly-Gly-Asp-Trp Peptide Eptifibadide Standard % of control % inhibition % of control % inhibition 10 ^-6 M 69.8 ± 5.0 30.2 ± 2.5 87.4 ± 6.9 12.6 ± 1.1 10 ^-5 M 48.3 ± 3.9 51.7 ± 3.1 83.0 ± 6.1 17.0 ± 1.1 10 ^-4 M 30.1 ± 2.2 69.9 ± 4.9 81.2 ± 7.1 18.8 ± 1.3 10 ^-3 M 20.3 ± 2.0 79.7 ± 5.9 78.5 ± 6.9 21.5 ± 1.5

Claims (1)

RGD peptides selected from the group:
Lys-β-Ala-Asp-Trp,
Arg-Aminovaleric acid-Asp-Trp,
Lys-Gly-Gly-Asp-Trp,
Arg-Gly-Gly-Asp-Trp,
Arg-β-Ala-Asp-Trp.