RU2631829C1 - Anticoagulant with direct action based on modular bivalent dna of aptamer selective to thrombin - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for chemical modification of an antithrombin aptamer and for preparation of a new antithrombin aptamer with anticoagulant properties and prolonged antithrombotic activity is described. The invention describes an aptameric DNA oligonucleotide specifically binding to thrombin and having a nucleotide sequence of the general formula GGTTGGTGTGGTTGGTGGTTGGTGTGGTTGG, wherein at least one of the nucleotides is modified to bind to the polyethylene glycol molecule and increase the time of oligonucleotide residence in the blood stream. In the preferred versions of the invention, the modification is made by thymine at 7, 9, 16, 23 and/or 25 positions. The invention can be used as an anticoagulant and antithrombotic drug for prevention or treatment of thromboses of various nature.

EFFECT: improved pharmacokinetic parameters, low toxicity and high affinity for human thrombin.

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Description

Technical field

The invention relates to the field of biotechnology and medicine, and specifically, to a method for chemical modification of an antithrombin aptamer and to a new antithrombin aptamer having anticoagulant properties and prolonged antithrombotic activity, which can be used as an anticoagulant and antithrombotic drug.

State of the art

Cardiovascular pathologies are the main cause of mortality and disability in industrialized countries, including the Russian Federation. The most serious diseases in this group are caused by the development of thrombosis, these include myocardial infarction, acute coronary syndrome, ischemic stroke, and others. Anticoagulants, a class of coagulation cascade inhibitor drugs, are successfully used to prevent the formation of thromboses. Among them, direct thrombin inhibitors (argatroban, dabigatran, bivalirudin, hirudin and its derivatives) proved to be safer drugs, because they have a more predictable effect, lower frequency of side effects and do not require a long selection of therapeutic doses in comparison with heparin and its derivatives.

Aptamers (from Latin Aptus - “match” and Mer - “repeat unit”) are short synthetic nucleic acid fragments (oligo (ribo) nucleotides) with a complex spatial structure that determines their unique properties to selectively bind (like a key to a lock) with any target: be it a chemical substance (isotope, medicine, toxin, etc.), polymers (enzymes, receptors, growth factors, regulatory and structural proteins, immunoglobulins, etc.), supramolecular complexes (e.g. viruses), whole cells (pathogenic bacteria RII, malignant and other defective cells).

The antithrombotic aptamers that we are developing, in particular, the object of patenting, DNA aptamers of the RA-36 family, block the thrombin region responsible for the binding of fibrinogen (exosite I). Aptamers have a new type of anticoagulant activity, different from the currently used direct anticoagulants. Their closest analogue is hirugen, the C-terminal peptide of hirudin, which binds to exosite I, has a significantly lower affinity for thrombin than the antithrombotic aptamers we develop (700 nM for hirugen compared to 7 nM for aptamers of the RA-36 family). Thus, aptamers occupy a unique niche among anticoagulants, being promising drugs that are potentially used both in surgical interventions and in the prevention of thrombosis after complex fractures, strokes, heart attacks and other diseases with a high risk of thrombotic complications.

Direct thrombin inhibitors created on the basis of aptamers can inhibit the prothrombotic activity of thrombin, affecting exosite I only and not affecting the active center. Aptamers have not been shown to affect thrombin inactivation by antithrombin III, protein C, and its amidolytic activity (Bompiani et al, "A high affinity, antidote-controllable prothrombin and thrombin-binding RNA aptamer inhibits thrombin generation and thrombin activity" J Thromb Haemost , 2012 May; 10 (5): 870-80).

Important advantages of aptamers compared to drugs of protein and peptide nature are low immunogenicity and the ability to block their activity with specific antidotes based on oligonucleotides complementary to the drug aptamer when complications (for example, bleeding) develop. In addition, the possibility of chemical synthesis of aptamers allows us to achieve a high degree of purity of the drug, reproducibility of batches and scalability of production.

A significant drawback that impedes the widespread use of aptamer DNA as drugs is their very short lifetime in the bloodstream (Griffin LC et al. "In vivo anticoagulant properties of a novel nucleotide-based thrombin inhibitor and demonstration of regional anticoagulation in extracorporeal circuits" Blood, 1993, v. 12, p. 3271-3276). Pharmacokinetics of aptamer DNA up to 30 nucleotides in length indicate a half-life of several minutes to half an hour, which necessitates the development of chemically modified aptamers to increase the aptamer half-life. This is the purpose of this invention.

In 2007, data were published on the development of the aptamer against the VEGF protein. In order to effectively achieve an increase in the time spent in the bloodstream, the researchers proposed several types of modifications. To increase the resistance to the action of nucleases, thiophosphate groups were added instead of phosphate groups or substitution of 2'OH groups with 2'-fluorine or 2'-amino derivatives (Pestourie C et al, "Aptamers against extracellular targets for in vivo applications" Biochimie, 2005; 87: 921-30; Ulrich et al, "RNA aptamers: from basic science towards therapy" Handb Exp Pharmacol, 2006; (173): 305-26). To improve the pharmacokinetics and bioavailability, objects with high molecular weight or lipophilic fragments can be added to the 5'- or 3'-ends. For example, combining an aptamer with polyethylene glycol (PEG) increases the plasma half-life to 9 hours instead of several minutes for an unmodified aptamer (Willis MC et al, "Liposome-anchored vascular endothelial growth factor aptamers" Bioconjug Chem, 1998; 9: 573-82) . Based on this aptamer against VEGF, the first commercial drug of aptameric nature, Pegaptanib, was created. Analysis of published pegylated aptamer variants shows that in the vast majority of them, the PEG molecule was attached to the terminal nucleotide. The reason for this is the fear that the attachment of the PEG molecule to the central part of the aptamer will inhibit its functional activity, i.e., worsen binding to the target (Vater A, Klussmann S. "Turning mirror-image oligonucleotides into drugs: the evolution of Spiegelmer therapeutics" Drug Discov Today 2015 Jan; 20 (1): 147-55).

More recently, researchers (Da Pieve et al, "PEGylation and Biodistribution of an anti-MUC1 Aptamer in MCF-7 Tumor-Bearing Mice" Bioconjugate Chem, 2012, 23 (7), pp 1377-1381) studied how the addition of PEG affects biodistribution of aptmera against MUC-1 (protein associated with breast cancer). The study showed that conjugation with PEG interfered with the absorption of aptamers by the tumor. Biodistribution data showed that the addition of PEG had an effect on the affinity of the aptamers to their target. The absorption of the pegylated aptamer by the tumor was fixed at a lower level (0.07 ± 0.01%) than the absorption of the non-pegylated aptamer (0.12 ± 0.05%). In general, after 22 hours, aptamer activity in the tumor was not observed. This study showed that the pegylated aptamer is not able to reach and bind to the target inside the cell and is absorbed by cancer cells in significant quantities.

The latter statement makes the use of pegylation to the antithrombotic aptamer very attractive, since the purpose of this aptamer is in the bloodstream. Pegylation makes it difficult for the aptamer to enter the cells, which can lead to unnecessary side effects. Thus, we can conclude that the use of pegylation will significantly increase the half-life of the aptamer (up to several hours) and prevent the conjugate of the aptamer with polyethylene glycol from entering the cells.

Despite numerous studies, the use of aptamers in clinical practice remains a rare occurrence due to the presence of a number of unresolved technical problems. This invention has a number of unusual properties essential for solving the problem, and therefore expands the range of available candidates for the role of anticoagulant drugs based on DNA aptamers.

SUMMARY OF THE INVENTION

The objective of the present invention is to provide a highly effective and safe competitive inhibitor of thrombin activity based on the DNA of aptamer to thrombin, which has a prolonged effect (increased residence time in the bloodstream).

The solution to this problem is that in the previously developed aptamer DNA, an oligonucleotide characterized by a nucleotide sequence of the general formula GGTTGGTGTGGTTGGTGGTTGGTGTGGTTGG (RA-36) is introduced by replacing a standard nucleotide with at least one modified nucleotide in such a way that, as a result of the chemical modification, the group is chemically modified capable of effectively forming a covalent bond with the PEG molecule, and the functional activity of this aptamer was not disturbed.

DNA aptamer with the sequence 5'-GGTTGGTGTGGTTGGTGGTTGGTGTGGTTGG-3 '(named RA-36) was developed by the authors of this invention based on the rational design and optimization of the structure of the prototype molecule using molecular modeling methods (Zavyalova E et al, "Novel modular DNA aptamer for human thrombin with high anticoagulant activity "Curr Med Chem, 2011; 18 (22): 3343-50). The prototype was a 15-link aptamer DNA oligonucleotide with the sequence 5'-GGTTGGTGTGGTTGG-3 'specific for thrombin, first described in Bock LC et al, "Selection of single-stranded DNA molecules that bind and inhibit human thrombin" Nature, 1992 Feb 6 ; 355 (6360): 564-6. The prototype molecule is capable of forming an antiparallel G-quadruplex, consisting of two planar G-quartets connected by three lateral loops; its three-dimensional structure was determined by nuclear magnetic resonance and X-ray diffraction analysis (Russo K et al, "High-resolution structures of two complexes between thrombin and thrombin-binding aptamer shed light on the role of cations in the aptamer inhibitory activity" Nucleic Acids Res, 2012 Sep; 40 (16): 8119-28). The RA-36 aptamer proposed by the inventors has a modular structure and contains two antiparallel G-quadruplexes, to stabilize which metal cations play a key role, in particular, potassium cations. Potassium cation is coordinated by guanines of the G-quadruplex, adding the enthalpy component to the energy of the structure (Zavyalova Е et al, "Module-activity relationship of G-quadruplex based DNA aptamers for human thrombin" Current medicinal chemistry, 2013; Vol. 20, no. 38) . RA-36 has an inhibitory activity against thrombin 2 times greater than the prototype molecule: the apparent inhibition constants are 7.3 nM and 14.7 nM, respectively. Both G-quadruplex moieties are important for the inhibitory activity and thermal stability of RA-36. Recently, the inventors have demonstrated the high functional activity of the antithrombin aptamer RA-36 in animal models, comparable with the clinically approved direct thrombin inhibitor bivalirudin (Zavyalova E et al. "Evaluation of antithrombotic activity of thrombin DNA aptamers by a murine thrombosis model" PLoS One, 2014 Sep 5; 9 (9): e107113).

A feature of this invention is that to increase the residence time of the RA-36 aptamer in the bloodstream, the authors proposed to attach the PEG molecule to RA-36, modified at least one nucleotide. A distinctive feature is the ability to attach PEG to this aptamer at non-terminal nucleotides. The inventors found that the introduction of modified nucleotides in RA-36 at positions 7, 9, 16, 23, 25 and subsequent conjugation with PEG does not impair the functional activity of this aptamer.

Some embodiments of the invention include the aptamer oligonucleotide RA-36, in which at least one of the thymines is modified at positions 7, 9, 16, 23 and / or 25 to allow binding to the polyethylene glycol molecule and increase the residence time of this oligonucleotide in the bloodstream .

In some embodiments, the modification occurred by replacing thymine with 5- [N- (6-aminohexyl) -3- (E) -acrylamido] -2'-deoxyuridine. In these embodiments of the invention, in which one of the thymines is modified at positions 7, 9 or 16, the thrombin-selective aptamer DNA formula with a prolonged blood flow life can be described as: GGTTGGU (PEG) GTGGTTGGTGGTTGGTGTGTGGTTGG, GGTTGTGTGGTGGTGGTGGTGGTGGT or GGTTGGTGTGGTTGGU (PEG) GGTTGGTGTGGTTGG (named RA-361), where U (PEG) is 5- [N- (6-aminohexyl) -3- (E) -acrylamido] -2'-deoxyuridine conjugated with polyethylene glycol. The portion of the -GU (PEG) structure of the G-aptamer RA-361, showing the method of PEG attachment, is shown in Fig. one.

In other embodiments of the invention, it is possible to use alternative modifications of the nucleotide residues to effect effective conjugation with the PEG molecule. Examples of such modifications are numerous and well known to specialists. For example, some commercially available modification options are described in the Custom Oligonucleotide Modifications Guide, Sigma-Aldrich. Attaching PEG to the modified nucleotide can be carried out in various ways, using different functional groups and different types of PEG.

In other embodiments, in addition to thymine modification, the aptameric DNA oligonucleotide RA-36 may also include one or more additional stabilizing modifications, such as nitrogen base modifications, sugar group modifications, or phosphate group modifications.

In some embodiments of the invention, the polyethylene glycol used to bind to the modified aptamer oligonucleotide has an average molecular weight of about 20,000 daltons. In other embodiments of the invention, it is possible to use a PEG of a different molecular weight in the range of 5000 daltons to 150,000 daltons.

In some embodiments, the invention relates to a pharmaceutical composition comprising the DNA aptamer described in the invention. In particular, the present invention relates to compositions containing antithrombin DNA aptamers described in the invention in combination with a pharmaceutically acceptable carrier, solvent, excipient and / or other inactive ingredients.

In other embodiments, the invention relates to methods of using antithrombin aptamers, in particular to methods of inhibiting thrombin using antithrombin aptamers described in the present invention. In particular, the task of this invention is achieved by the use of the aptamer oligonucleotide RA-36, in which at least one of the thymines is modified at positions 7, 9, 16, 23 and / or 25 to allow binding to the polyethylene glycol molecule and increase the time spent by the oligonucleotide in the bloodstream to inhibit thrombin activity.

In other embodiments, the present invention relates to the prevention or treatment of conditions associated with undesired thrombin activity, such as thromboses of various nature. In particular, the present invention relates to the use of the aptamer DNA of the oligonucleotide described in the invention as an antithrombotic and / or anticoagulant drug for the treatment of various diseases of humans and animals, including during surgical interventions, in the prevention of thrombosis after complex fractures, strokes, heart attacks, etc. Diseases with a high risk of thrombotic complications.

In certain particular embodiments, the invention relates to the prophylaxis and / or treatment of mammalian conditions characterized by unwanted thrombosis, which are acute coronary syndrome, myocardial infarction, unstable angina pectoris, refractory angina pectoris, thrombosis caused by post-thrombolytic therapy or coronary angioplasty, acute ischemic cerebral artery syndrome stroke, thrombotic stroke, transient ischemic attacks, venous thrombosis, deep venous thrombosis, pulmonary embolus, coagulopathy, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, thromboangiitis obliterans, thrombotic disease associated with geparinoindutsirovannoy thrombocytopenia, thrombotic complications associated with extracorporeal circulation, thrombotic complications associated with prosthetic devices.

When carrying out the invention, the following technical results are achieved:

- a form of antithrombin DNA of aptamer RA-36 was developed, which ensures its stability in the bloodstream when administered to humans or animals. This form has both high specificity for thrombin, leading to its effective functional inhibition, and low toxicity when administered to animals. This allows us to predict the successful use of this form as a medicine.

- A new effective method for inhibiting the activity of thrombin has been developed, which has potential applicability in clinical practice.

Definitions and Terms

In the description of the present invention, the terms “includes” and “including” are interpreted as meaning “includes, inter alia,”. These terms are not intended to be construed as “consists of only”.

The term "nucleotide" refers to chemical compounds that are constituent parts of nucleic acids (DNA or RNA). Nucleotides consist of nitrogen bases which are covalently linked to a 1 'ribose or deoxyribose atom (forming nucleosides) and are additionally linked to one or more phosphate groups attached to the 5'-hydroxyl group of sugar. The main nitrogenous bases that make up DNA or RNA are cytosine (C), guanine (G), adenine (A), thymine (T) and uracil (U). Compounds consisting of a small number of nucleotides are called oligonucleotides.

By “stabilizing modifications” is meant modifications of the aptamer oligonucleotide, leading to an increase in its stability when administered to a human or animal; for example, leading to stabilization of the structure or an increase in residence time in the bloodstream. Similar nucleotide modifications described in the literature include nitrogen base modifications, sugar group modifications, or phosphate group modifications. For example, modifications of nitrogen bases may include the addition of electrophilic groups to cytosine (5-bromocytosine and others) and uracil (5-bromouracil and others); modifications of sugar groups may include various sugar analogues; modifications of phosphate groups may include, for example, the formation of thiophosphates. Nucleotides within the aptamer can be connected either by conventional phosphodiester bonds or by other non-natural bonds, for example, to ensure the stability of the aptamer DNA in the presence of nucleases. These and other modifications, as well as methods for their synthesis, are described in detail in the literature, for example, in patent EP 2640426, as well as in the references from this patent.

The term “aptamer” refers to single-stranded oligonucleotide sequences (up to 100 nucleotides in length) that can specifically recognize and bind to specific target molecules (usually proteins).

The term “polyethylene glycol” or “PEG” means a linear or branched ethylene glycol polymer having a molecular weight of from 5,000 daltons to 150,000 daltons.

The term "pharmaceutically acceptable carrier, solvent and / or excipient" refers to such carriers, solvents and / or excipients that, being inactive ingredients, within the framework of a medical opinion, are suitable for use in contact with human and animal tissues without undue toxicity, irritation allergic reaction, etc. and meet a reasonable balance of benefits and risks. "Inactive ingredients" are part of the drug to improve its solubility and / or stability, as well as to improve pharmacokinetics. Inactive ingredients include a variety of substances known to those skilled in the pharmaceutical arts, such as pH or osmotic pressure control agents, antibacterial agents, antioxidants, surfactants (e.g., Polysorbate 20 or 80), cryostabilizers, preservatives, solvents, thickeners, fillers , carriers (micro- or nano-particles) and other substances.

By the name "RA-36" is meant a DNA aptamer with the sequence 5'-GGTTGGTGTGGTTGGTGGTTGGTGTGGTTGG-3 ', while "RA-361" defines a specifically modified variant of the RA-36 aptamer, a fragment of the structure of which is shown in Fig. 1. RA-361 is one of the possible embodiments of the invention in which the modification of the aptamer RA-36 occurred by replacing the thymine at position 16 with 5- [N- (6-aminohexyl) -3- (E) -acrylamido] -2 '-deoxyuridine, followed by the addition at position 16 of an N-succinimide derivative of a PEG molecule with a molecular weight of 20 kDa.

Unless defined separately, technical and scientific terms in this application have standard meanings generally accepted in the scientific and technical literature.

Brief Description of Drawings

Fig. 1. The site of the chemical structure -GU (PEG) G- modified aptamer RA-361, showing the method of attaching PEG.

Fig. 2. (A) The dependence of prothrombin time on the concentration of DNA of aptamer RA-361 in human and animal plasma. (B) Dependence of prothrombin time on the concentration of Bivalirudin comparison drug in human and animal plasma.

Fig. 3. (A) The dependence of the parameter prothrombin time in vitro in human plasma on the concentration of aptamer. The aptamer RA-36 is solid. The dotted line indicates the aptamer RA-361. (B) In vitro thrombin time parameter dependence in human plasma on aptamer concentration. The aptamer RA-36 is solid. The dotted line indicates the aptamer RA-361.

Fig. 4. (A) Dynamics of rats' body weight after a single intravenous administration of the “pegylated oligonucleotide RA-361” dosage form at a dose of 84 mg / kg. The dashed line indicates the control group. (B) Dynamics of body weight of mice after intraperitoneal single administration of the drug “pegylated oligonucleotide RA-361” at a dose of 500 mg / kg The dashed line indicates the control group.

Fig. 5. The result of the HPLC analysis of a sample of the reaction mixture of the conjugation reaction of the synthesized starting amino derivative RA-36 with PEG N-succinimide ester with a molecular weight of 20 kDa during the formation of the modified RA-361 aptamer.

Detailed Disclosure of Invention

Despite the fact that examples of DNA of aptamers to thrombin have been known for about 20 years, their practical promotion in medical practice is hindered by a number of factors, and there are currently no clinically approved drugs based on DNA of aptamers to thrombin. For example, the first-generation aptamers ARC183 and ACR2172 successfully passed preclinical trials in the United States, but were stopped at the clinical trials stage. Based on the experimental data accumulated by the inventors, the main reason for the suspension of the advancement of these aptamers could be the problem of the correct assembly of the G-quadruplex pharmacophore with additional duplex modules. The inventors have developed a new design of modular bivalent DNA aptamer (used in aptamer RA-36 and modified RA-361), which has a second G-quadruplex pharmacophore instead of duplex. A significant advantage of this design is a quick, spontaneous assembly, which is determined by its unique structure. The inventors have patents for additional factors stabilizing the pharmacophore (duplex elements), for example, RU 2429293.

To create effective drugs based on DNA aptamers, it is necessary that the corresponding pharmaceutical composition possess stability sufficient for its clinical preparation, short-term storage and use. Chemical modifications in specific places of the antithrombin DNA of the RA-36 aptamer and the subsequent addition of the PEG molecule make it possible to obtain a set of antithrombin aptamers with improved characteristics. The modification sites described below were selected through rigorous experimental testing. A characteristic feature of this invention is the attachment of the PEG molecule to the modified nucleotides of the aptamer located in the central region of the aptamer molecule, while in most of the previously described analogues, the attachment occurred at the terminal nucleotides. The possibility of attaching a PEG molecule to the central region of the aptamer molecule is provided, in particular, by the modular structure of the RA-36 aptamer.

The key characteristics of a drug based on antithrombin aptamer DNA are: (1) high specific activity (inhibition) against human thrombin; (2) improved pharmacokinetic properties - a sufficiently large time spent in the bloodstream; (3) low toxicity of the drug when administered to humans and animals; (4) the possibility of producing the drug in significant quantities sufficient for clinical trials.

The following examples are provided in order to disclose the characteristics of the present invention and should not be construed as in any way limiting the scope of the invention.

Thrombin Inhibition Efficiency Study

To analyze the effectiveness of thrombin inhibition by the RA-36 aptamer and its modified variant RA-361, their anticoagulant activity was studied in standard laboratory tests “Prothrombin time” and “Thrombin time”. Prothrombin time (PV) reflects the inhibition of thrombin, which is generated by plasma, and allows us to evaluate the activity of coagulation factors I, II, V, VII, and X (assessment of the external coagulation pathway and the hemostasis system as a whole). The thrombin test shows the time required for the formation of a fibrin clot in the plasma when thrombin is added to it. This test characterizes the kinetics of the final stage of blood coagulation - the rate of conversion of fibrinogen to fibrin. Thrombin time (TB) depends only on the concentration of fibrinogen and the activity of thrombin inhibitors.

To carry out the test “Prothrombin time”, a set of reagents “Thromborel S” of the company “Dade Behring” was used; for the test "Thrombin time" was used a set of reagents "Test Thrombin Reagenb company" Sigma ", both in accordance with the manufacturer's instructions. These indicators were determined in the citrated blood plasma of laboratory animals to which the study drug was administered, either a comparison drug or physiological saline (control group). The clinically used peptide thrombin inhibitor Angiox (Bivalirudin) was used as a comparison drug. The test prothrombin and thrombin time was carried out as follows. 50 μl of the test sample of citrate plasma was taken into a plastic coagulometer cuvette, held for 60 seconds at 37 ° C, 100 μl of the working solution of the reagent was added to test the prothrombin or thrombin time heated at 37 ° C. The clotting time of the studied plasma sample was measured on a coagulometer equipped with software for determining the parameters under discussion.

In Fig. Figure 2 shows the dependence of prothrombin time on the concentration of DNA of aptamer RA-361 in human and animal plasma. RA-361 is able to significantly lengthen fibrin formation time after the generation of endogenous thrombin in normal human and animal plasma. The same figure shows similar data for the comparison drug. From these data, a significantly higher specific activity of RA-361 with respect to human thrombin compared with animal thrombin follows. In Fig. Figure 3 shows a comparison of unmodified (RA-36) and modified (RA-361) aptamer DNA in functional tests for measuring PV and TB. From these data it follows that the RA-36 modification, which includes the replacement of the nucleotide and the addition of the PEG molecule, does not affect the functional activity of the aptamer DNA.

Study of the pharmacokinetic properties of the modified aptamer RA-361 in comparison with the aptamer RA-36

DNA aptamers against first-generation thrombin are characterized by a very short (not more than a few minutes) lifetime in the bloodstream (Griffin LC et al., Blood 1993, v. 12, p. 3271-3276). The size of the aptamer is critical for its retention in the bloodstream: with a molecular mass of 30,000 Yes and above, its passive excretion through the kidneys slows down.

To study the increase in the time spent in the bloodstream of the pegylated aptamer RA-361 in comparison with the original aptamer RA-36, a single intravenous administration of these drugs to rats was used. Animals with pre-implanted catheters and adapted to the content in the metabolic chambers were administered an aptamer RA-361 or RA-36, labeled with phosphorus, intravenously through the lateral veins of the tail once on an empty stomach. After 1, 3, 7, 15, 30, 60, 90 min, 2, 3, 4, 6, 8, 10, 12, 16, 24, 48 hours after administration of the substance, blood samples of rats were taken through catheters. Urine and stool samples were collected at intervals of 0-2, 2-4, 4-6, 6-8, 8-12, 12-16, 16-24, 24-36, 36-48 hours. Concentration of RA-361 in the collected samples were determined by the radiometric method of liquid scintillation counting. Measurements were performed for 7 rats. Other experiments also evaluated the linearity of the pharmacokinetics of RA-361 and RA-36 in rat blood after a single intravenous administration, as well as the distribution of RA-361 and RA-36 over organs and tissues.

Experimental data show a significant change in the pharmacokinetic parameters of RA-361 compared with RA-36. The half-life of RA-361 was 7.3 ± 0.8 minutes. with a confidence interval of 95% - 3.9-9.1 minutes, while the half-life for RA-36 was 0.99 minutes with a confidence interval of 95% - 0.77-1.4 minutes. The half-life of RA-361 was 7.6 ± 1.5 hours with a confidence interval of 95% - 4.6-10.5 hours, while the half-life for RA-36 was 23 minutes, the confidence interval of 95% was 18.45-31.93. A feature of the pharmacokinetics of RA-36 was also that after 3 minutes. after intravenous administration, a high concentration of radioactive phosphorus was detected in the bladder of animals, indicating an intensive enzymatic cleavage of RA-36 and excretion of labeled fragments by the kidneys. Thus, experimental data confirm a significant increase in the stability of the pegylated aptamer RA-361.

Toxicity study of RA-361 in laboratory animals

(A) Study of acute toxicity of RA-361 in mice and rats

To establish doses characterizing the toxicity of aptamer RA-361, the drug was administered once intravenously at the highest concentration and maximum volume allowed for subcutaneous administration to SHK mice (1 ml) and Wistar rats (10 ml). The dose of the drug for mice was 125 mg / kg and was determined as a dose equal to 20 therapeutic doses for humans, for rats - 85 mg / kg and was determined as a dose equal to 12 therapeutic doses for humans. The drug was administered intraperitoneally in the maximum allowable volumes — 1.0 ml for mice and 5.0 ml for rats. The dose for mice was 500 mg / kg and was defined as an 80-fold therapeutic dose for humans, for rats the dose was 450 mg / kg and was defined as a 72-fold therapeutic dose for humans. After the introduction of the drug, the state and behavior of the animals was monitored for 30 days. After the introduction of the drug, the condition and behavior of the animals did not change. Animals well tolerated the introduction of the drug. The death of experimental animals was not observed. The weight gain of the experimental mice and rats did not differ from the animals of the control group (Fig. 4). Doses characterizing the toxicity of the drug is not possible to calculate, since it was not possible to achieve lethal doses of the drug. After euthanasia on the 30th day of the experiment, a macroscopic examination of the internal organs of experimental animals that received once intravenously and intraperitoneally pegylated oligonucleotide RA-361 in the maximum possible volumes, differences in the state of internal organs (thymus, heart, spleen, kidney and liver) compared with the control group not found.

(B) A study of the chronic toxicity of RA-361 in rats.

An additional toxicological safety study of RA-361 in a chronic experiment was carried out on sexually mature Wistar rats, males and females. The studies were carried out using visual, instrumental and laboratory monitoring of the condition of animals, modern hematological, biochemical and morphological methods for evaluating the effects of drugs in accordance with the “Guidelines for conducting preclinical studies of drugs” edited by A. Mironov. When studying chronic toxicity in rats, males and females performed daily intravenous administration of the test drug in single doses of 4.5 mg / kg (1 therapeutic dose for humans) and 22.5 mg / kg (five-fold therapeutic dose for humans) for 7 days. During the administration of the drug, as well as within 15 days after the cessation of drug administration, the death of rats was not observed in any of the groups of experimental and control animals. Animals of the experimental groups tolerated the administration of the drug. Daily administration of the drug for 7 days in the tested doses did not have a statistically significant effect on rats' intake of food and water, as well as changes in body weight. Biochemical parameters of blood serum in all experimental groups did not differ from the control. These and other studies have concluded that the drug RA-361 in the used concentrations is low toxic.

Scaling of the synthesis process RA-361

To optimize and scale the production technology of RA-361, the synthesis of an amino derivative oligonucleotide using a high-performance synthesizer AKTA oligopilot plus 100 and a flow-through column cartridge with a fixed volume of 6.3 ml (Column Reactor 6.3 ml, prod code 18-1101-13) followed by deprotection, purification of the obtained amino derivative by preparative chromatography, preparation and purification of the conjugate. The approach used was used to develop the drug for preclinical trials of aptamer RA-361. The main stage of scaling is the process of solid-phase synthesis. The synthesized nucleotide sequence of the amino derivative is 58% G-rich, the remainder of the sequence is dT nucleotides. This sequence may in some cases complicate the synthesis process due to the low condensation efficiency of the dG nucleotide, as well as the sensitivity of dT-rich sequences to modification during the deprotection procedure caused by the reaction of acrylonitrile with the N3 position of dT residues. The decrease in the condensation efficiency of the dG monomer, in particular, is due to the fact that the carrier-bound and protected dG-rich oligomers are prone to aggregation, as well as to a decrease in the solubility of the oligonucleotide in acetonitrile after reaching a certain length and / or base type ratio in the sequence causing difficulty access 5'-OH-hydroxyl group. These features generally cause a decrease in the yield of the target product and an increase in the content of impurities of an oligonucleotide nature, including products of modification of the main sequence. The selection of suitable compositions of working reagents and optimization of the parameters of the synthesis process can significantly reduce the influence of negative factors on the quality of the resulting product. For the synthesis of an amine-modified oligonucleotide, the commercial polymer carrier Primer Support 200 dG-iBu Synth (200 ± 10 μmol / g, cat no. 17-5290-02, hereinafter referred to as PS 200 dG) from GE Healthcare was used, the NittoPhase polymer carrier can be used similarly ®HL (200 ± 10 μmol / g, Standard Deoxy dG-Base Loaded) from Kinovate Life Sciences, Inc., as well as high-density polymer carriers from other companies and HybCPG ™ media with dG-dmf ligand (code DGF Y-150,150 ± 10 μmol / g) from PrimeSynthesis. The use of PS 200 dG in the synthesis of oligonucleotides whose length exceeds 30 nucleotides requires an increase in the number of solvents and reagents at the stages of washing the flow reactor and detritylation, especially at the last stages of synthesis, to initiate the complete occurrence of the detritylation reaction. After the conjugation step of amino-modified dT-cyanoethyl phosphoramidite, the capping step should be minimized (or completely eliminated) to reduce the risk of acylation of the primary amino group of the aminolinker. Since this nucleotide insert with an aminolinker is located in the 16th position of the sequence, in the second half of the synthesis, the capping step is skipped (15 cycles of monomer condensation), which leads to a decrease in the yield of the target nucleotide sequence and an increase in the amount of “undersynthesis” of impurities with careful optimization stages of deprotection and condensation. To prevent the migration of the acyl group from the exocyclic amino group (dG-iBu on a polymer carrier) to the amino group of the linker nucleotide, it is also necessary to process the synthesized sequence before deprotection and cleavage from the PS carrier with a 20% solution of N, N-dialkylamine (for example, diethylamine in acetonitrile ), which increases the storage stability of the synthesized oligonucleotide on a PS carrier. A similar treatment also removes the cyanoethyl protecting group, which can lead to the sewing of acrylonitrile molecules to the thymidine N3 atom during deprotection of this base. The TFA-protecting group on the aminolinker (TFA-trifluoroacetyl labile under basic conditions, i.e., removing the resulting sequence during deprotection) reduces the stability of the material during storage on the carrier after synthesis, and is also relatively susceptible to adverse reactions by the transcylation mechanism during synthesis. The synthesis process of the working nucleotide sequence of the amino-modified derivative [5 '- GGT TGG TGT GGT TGG 16-pos / iAmMC6T / GG TTG GTG TGG TTG G-3'] is similar to the synthesis on a BioAutomation Automated Synthesizer 48 (hereinafter MM48) using standard reagents and synthesis process parameters.

The sequence of accession of the nucleotide link consists of the following reactions:

Detritylation: removal of the 5'-dimethoxytrityl protecting group from the carrier-bound nucleoside. The reaction should be carried out in a maximally anhydrous environment (<3 ppm) in an argon atmosphere. Cleavage of the protective group generates a bright orange color solution, the relative amounts of which can be monitored by UV absorption (UV-Vis control). Based on these data, it is possible to evaluate the effectiveness of condensation reactions during the entire synthesis process. The measured amount of dimethoxytrityl ion is integrated in real time by the software synthesizer controlling. The composition of the working solution for conducting the detritylation reaction on the AKTA Oligopilot 100 synthesizer is different from the similar solution for the synthesis on the MM48 synthesizer. Instead of strong trichloroacetic acid (TCA, pKa 0.7), weaker dichloroacetic acid (DCA, pKa 1.5, working concentration in solution of 5% (w / v)) is used, which reduces the probability of nucleotide base cleavage (depurinization) during detritylation time. Detritylation reaction is monitored at wavelengths of 350 and 280 nm;

Washing the carrier after detritylation: after detritylation, the reactor is washed with anhydrous acetonitrile to remove the remaining DCF and the trityl cation, which, during the addition of the next nucleotide, can generate N + 1 impurities and reduce the final content of the target oligonucleotide;

Condensation: the second reaction involves the condensation of the corresponding phosphoramidite monomer (dG-dmf-CE, dTCE or TFA-AmC6dT-CE phosphoramidite at a concentration of 0.2 M, 2.2 equivalents) mixed with an activator (0.25 M 5-ethyl -1H-thiotetrazole, ETT, in anhydrous acetonitrile, 60% (v / v)). During this reaction, a phosphotriether internucleotide bond is formed in high yield (over 98%). For DNA synthesis, condensation is carried out by recirculating the activator solution and phosphoramidite through the reactor for 3 minutes; for the condensation of amidite with aminolinker, a longer time is used - 5 minutes. After the condensation step, the carrier is washed with anhydrous acetonitrile to remove excess reagents and reduce the possible formation of N + 1 impurities;

Oxidation: the phosphotriether internucleotide bond formed during condensation during oxidation with an iodine solution with an organic base (usually pyridine or lutidine) is converted to phosphodiester. After oxidation, the support is washed with anhydrous acetonitrile. To carry out the reaction, the carrier is treated in the reactor with a solution of 0.05 M iodine in a mixture of a heterocyclic base (usually pyridine, lutidine, collidine) with water in a ratio of 90:10. Water acts as an oxygen donor, and iodine forms an adduct with a phosphorus bond, which decomposes by water, leaving a stable phosphodiester bond;

Capping: The last reaction in the monomer unit attachment cycle is capping of the unreacted 5'-hydroxyl group followed by washing of the support with anhydrous acetonitrile. Under standard synthesis conditions, a solution of acetic anhydride in acetonitrile is used. When scaling up the synthesis of G-rich sequences, the isobutyl protection of the amino group at the C2 position of the dG-substantiation of phosphoramidites can be replaced by the acetyl group as a result of the reaction by the trans-amidation mechanism at the capping stage; a similar impurity cannot be eliminated under standard conditions of deprotection. The use of N-dimethyl-formamide protection and isobutyryl anhydride eliminates this problem by blocking the free 5'-OH group without the possibility of a side reaction of trans-amidation. At the end of the synthesis, a single cycle of treatment with a 20% solution of diethylamine in acetonitrile is included. When splitting the linker bond between the newly synthesized oligonucleotide and the carrier, a β-cyanoethyl group is used to protect the phosphate group. During deprotection, this group is cleaved and in the form of acrylonitrile interacts with the N3 position of thymine with the formation of a cyanoethyl adduct according to the Michael addition mechanism, which cannot be removed by chromatographic purification of the mixture after synthesis. This effect is minimized by treatment of the carrier after synthesis by alkylamines (diethylamine, tert-butylamine) according to the β-elimination mechanism, the resulting product is removed by treatment with a deprotection solution. The synthesis steps are repeated 30 times to synthesize a given sequence. The synthesized oligonucleotide is subjected to cleavage and deprotection according to the instructions for the solid phase carrier PS 200 dG. After synthesis, the carrier in the reactor is dried for one hour under vacuum to quantify the change in mass of the carrier after synthesis (weighing). Next, the carrier is once again diluted in at least 1 column volume (6.3 ml), transferred to a pressure container with a tight lid, which is then filled with a cooled aqueous solution of concentrated ammonia (35%) in a ratio of 10 ml per 1 gram of carrier . The mixture is incubated with gentle stirring at 55 ° C for 16 hours. After cooling for 30 minutes at -34 ° C and filtered on a glass filter with a porosity of 3 (16-40 microns). The filter is washed five times with an aqueous solution of ethanol (1: 1) in a volume equal to the volume of the used ammonia solution. Using the spectrophotomerism method, the concentration of the oligonucleotide is determined by direct absorption of the solution at a wavelength of 260 nm (the synthesis yield is 130-135 CD260 / μmol). The method of anion-exchange high-performance liquid chromatography (AO-HPLC) determines the composition of the mixture and the percentage of the main component. After analytical procedures, the solvent components (ammonia solution and ethanol) are removed by vacuum concentration, and the concentrated aqueous oligonucleotide solution is diluted in perchlorate chromatographic buffer. The impurities of the synthesis and residual low molecular weight components of the reagents are removed by preparative anion exchange chromatography on a 150 ml column with AO carrier Source 15Q (GE Healthcare). After 3 cycles of chromatographic purification, one concentration cycle was performed by tangential filtration (a reusable Sartorius Slice PES regenerative cartridge for Sartocon filters with a nominal cut-off of molecular weight of 1 kDa was used). Quality control was carried out using an Agilent Series 1200 HPLC system with a PAC 100 DNA anion exchange column (Dionex, USA) 25 cm long and 2 mm inner diameter. The loss of tangential filtration of the amino derivative is about 8%, resulting in an oligonucleotide preparation with a content of the main component of at least 85%.

Conjugation of an Amino-Modified Derivative of an RA-36 Oligonucleotide and an N-Succinimide PEG Derivative

The accumulated products of the starting amino derivative of the oligonucleotide RA-36 were used to conduct conjugation reactions with the N-succinimide derivative of polyethylene glycol with a molecular weight of 20 kDa. The modified oligonucleotide was pegylated in a buffer solution with pH = 8.5 (Theorell-Stenhagen buffer with the content of the organic phase of dimethyl sulfoxide (DMSO) in the ratio of 1 volume of aqueous buffer: 0.5 volume of the organic phase) with the gradual addition of six 0.5 molar equivalents PEG to the molar equivalent of the oligonucleotide, followed by incubation of the solution for 30 minutes at 37 ° C; in total, 3 equivalents of polyethylene glycol are added per reaction. Process control was carried out using the AO-HPLC method. The reaction was terminated by diluting the mixture with mile-Q quality water. The yield of the target product at the stage of conjugation was about 77%; the analysis of the product using HPLC is shown in Fig. 5.

It is convenient to use the conjugate reprecipitation procedure according to the following procedure to remove the oligonucleotide that did not react with PEG and oligonucleotide impurities that were not removed by preparative chromatography. Pour 3 ml of a 5 M aqueous solution of sodium perchlorate into 30 ml of the reaction mixture, then add 100 ml of acetone (ChP), pack Falcon tubes in 50 ml and unscrew the solution in a centrifuge (15 min, 3000 rpm). After centrifugation, decant the resulting solution from the precipitate. The precipitate contains unreacted oligonucleotide and impurities of a similar nature. The pegylated aptamer is contained in a solution from which, after decantation, with slight heating under vacuum, acetone is removed. Excess unreacted PEG is removed by preparative anion exchange chromatography; 10 mM Tris, 50 mM NaCl are used as the mobile phase, -10 mM Tris, 700 mM NaCl as eluent. Unreacted PEG elutes almost immediately after injection; for its detection, the first fractions after purification are analyzed by absorbance at a wavelength of 280 nm. A single absorption peak (pegylated oligonucleotide) from chromatographic purification is desalted and concentrated by tangential filtration. The resulting solution is dried for 1.5 days by lyophilization, and then transferred to the preformation. The obtained experimental data allow us to conclude that the developed technology is optimized compared to previously described in the literature by selecting suitable compositions of working reagents and optimizing the parameters of the synthesis process, such as skipping the capping stage in the second half of the synthesis, processing the synthesized sequence before deprotection and cleavage from PS- media with a 20% solution of N, N-dialkylamine, the use of dichloroacetic acid at the stage of detritylation, etc.

Thus, the above examples allow us to appreciate the potential of the modified and pegylated aptamer RA-361 as a possible drug for the treatment of thrombosis. The high functional activity previously achieved for the first-generation aptamer RA-36 (Zavyalova E et al, "Evaluation of antithrombotic activity of thrombin DNA aptamers by a murine thrombosis model" PLoS One, 2014 Sep 5; 9 (9): e107113), has been preserved and in the pegylated version of RA-361 (see Fig. 2 and Fig. 3). The unusual modular structure of the RA-36 aptamer made it possible to attach the PEG molecule to the central part of the aptamer without significantly reducing its activity. This distinguishes it from previous analogues described in the literature, where PEG addition occurred at terminal nucleotides. In addition, as a result of pegylation, the residence time in the bloodstream has significantly increased, which will allow the use of this drug to ensure a long antithrombin effect. Further, RA-361 showed low toxicity in preclinical animal studies, making it a likely candidate for clinical trials. Finally, the scaling of the RA-361 synthesis process has been meticulously worked out to provide sufficient aptamer amounts for clinical trials.

Although the invention has been described with reference to the disclosed embodiments, it should be apparent to those skilled in the art that the specific experiments described in detail are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. . It should be understood that various modifications are possible without departing from the gist of the present invention.

Claims (13)

1. Aptamer DNA oligonucleotide that specifically binds to thrombin and is characterized by the nucleotide sequence of the general formula GGTTGGTGTGGTTGGTGGTTGGTGTGGTTGG, in which at least one of the nucleotides is modified to bind to the polyethylene glycol molecule and increase the residence time of this oligonucleotide. 2. Aptamer DNA oligonucleotide according to claim 1, in which the modification was performed on thymines at positions 7, 9, 16, 23 and / or 25. 3. Aptamer DNA oligonucleotide according to claim 2, in which the modification is made according to one of the thymines at positions 7, 9, 16, 23 or 25. 4. The aptamer DNA oligonucleotide according to claim 2, wherein the modification occurred by replacing thymine with 5- [N- (6-aminohexyl) -3- (E) -acrylamido] -2'-deoxyuridine. 5. The aptamer DNA oligonucleotide according to claim 3, in which one or more additional stabilizing modifications are present, including nitrogen base modifications, sugar group modifications, or phosphate group modifications. 6. Aptamer DNA oligonucleotide according to any one of paragraphs. 1-5, in which the polyethylene glycol used for binding, has an average molecular weight of about 20,000 daltons. 7. A method of inhibiting the activity of thrombin, comprising the use of aptamer DNA oligonucleotide according to any one of paragraphs. 1-5. 8. A pharmaceutical composition for the prevention and / or treatment of thrombosis, comprising the following components: - aptamer DNA oligonucleotide according to any one of paragraphs. 1-5 and - a pharmaceutically acceptable carrier, diluent and / or excipient. 9. The use of aptamer DNA oligonucleotide according to any one of paragraphs. 1-5 or a pharmaceutical composition according to claim 8 for the prevention and / or treatment of a condition of a mammal characterized by undesirable thrombosis. 10. The use of claim 9, wherein the condition is acute coronary syndrome, myocardial infarction, unstable angina pectoris, refractory angina pectoris, thrombosis caused by post-thrombolytic therapy or coronary angioplasty, acute ischemic cerebrovascular syndrome, embolic stroke, thrombotic stroke, pre-stroke, thrombotic stroke, venous thrombosis, deep venous thrombosis, pulmonary embolism, coagulopathy, disseminated intravascular coagulation, thrombocytopenic acroangiothrombosis, thromboangiitis, thrombotic diseases associated with heparin-induced thrombocytopenia, thrombotic complications associated with cardiopulmonary bypass, thrombotic complications associated with prosthetic devices. 11. The use according to claim 9, characterized in that the mammal is a human.

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