RU2730000C1 - Dosage form of dna-aptamer - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: group of inventions relates to biotechnology and medicine, specifically to the preparation of a medicinal form of aptamer RA-361. Aptamer RA-361 comprises 5'-GG-TT-GG-TGT-GG-TT-GG-T-GG-TT-GG-TGT-GG-TT-GG-3', covalently modified 20 kDa PEG in 16 position in concentration 30 mg/ml with allowable limit of 35.7 mg/ml, water, potassium chloride with concentration of 10 mM, where concentration of potassium chloride does not exceed 20 mM, and physiologic saline, wherein the dosage form has pH between 7 and 7.5 and is a clear aqueous solution. Group of inventions also relates to a method for preparing a medicinal form of the aptamer, which includes obtaining modified PEG aptamer RA-361 with chromatographic purification and concentration of active substance, heating and maintaining temperature of active substance samples to 95 °C for 5 minutes with subsequent cooling of samples to room temperature at rate of 5 °C per minute and performing sterilization by means of filters with pore diameter of 0.22 mcm to obtain a pharmaceutical substance in concentration of 120–150 mg/ml, then substance is subjected to four-time dissolution in sterile physiological saline, where water is used, prepared according to ISO 3696 standard, and packed into glass bottles in laminar cabinet, then they are sealed, wherein output is a substance in volume of 10 ml, and concentration of oligonucleotide RA-361 is 30–35.7 mg/ml.

EFFECT: use of this group of inventions enables to obtain an antithrombin aptamer, which is 31-link DNA oligonucleotide, covalently modified 20 kDa PEG and having a spatial structure of the antiparallel G quadruplex type stabilized by potassium cations, which enables to preserve blood plasma coagulation.

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Description

The present invention relates to the field of biotechnology and medicine, and in particular to a pharmaceutical preparation, which is a solution containing an antithrombin DNA aptamer. The invention also relates to a method for preparing a dosage form of a pegylated DNA aptamer; in addition, a method for scaling and optimizing the technology for obtaining significant volumes of a DNA aptamer is described. The invention provides the implementation of the specified purpose.

State of the art

In recent decades, a new type of thrombin inhibitor, DNA aptamers, has been developed. Aptamers are small molecules of nucleic acids that can act as highly specific receptors for any target, from whole cells to low molecular weight organic compounds. Oligonucleotide aptamers with the desired properties are isolated from random sequence libraries by in vitro selection (SELEX) methods, using their ability to specifically interact with the corresponding immobilized targets / ligands.

Thus, one of the first DNA aptamers that inhibit thrombin was found by Schultze P et al. "Three-dimensional solution structure of the thrombin-binding DNA aptamer d (GGTTGGTGTGGTTGG)", J. Mol. Biol. 1994 Feb 4; 235 (5): 1532-47. The DNA oligonucleotide dGGTTGGTGTGGTTGG, a thrombin-binding aptamer, specifically binds to thrombin and inhibits the enzyme activity in the blood coagulation chain. In the publication Nagatoishi (Nagatoishi S. et al., "Circular dichroism spectra demonstrate the formation of the thrombin-binding DNA aptamer G-quadruplex under stabilizing-cation-deficient conditions", Biochem Biophys Res Commun. 2007 Jan 19; 352 (3): 812-7. Epub 2006 Nov 27) describes an antithrombin aptamer having a G-quadruplex structure.

Gatto B. et al., "Nucleic acid aptamers based on the G-quadruplex structure: therapeutic and diagnostic potential", Curr Med Chem. 2009; 16 (10): 1248-65) describes a G-quadruplex antithrombin aptamer and also conducts a review of modern knowledge about aptamers based on G-quadruplex structures, as well as an assessment of their diagnostic and therapeutic potential (as biotechnological drugs) for the detection and treatment of severe pathologies, including vascular, cancer and viral diseases.

In addition, aptamer DNA was obtained for thrombin, the biomedical properties of which are described, for example, in a number of publications:

1. IE 920561 A1 (GILEAD SCIENCES) dated 08.26.1992, which describes an aptameric oligonucleotide that specifically binds to thrombin and having a nucleotide sequence GGXTGG, where X is T, A, U, dU or G, in particular contains the nucleotide sequence GGTTGGTGTGGTTGG.

2. US 2005176940 A1 (UNISEARH LTD) from 11.09.2005, which describes an aptameric oligonucleotide that specifically binds to thrombin and having a nucleotide sequence GGTMGGXGGTTGG, where M is A or T, and X is a sequence from 2 to 5 any nucleotides or their modifications.

3. Griffin L.C. et al., "In vivo anticoagulant properties of a novel nucleotide-based thrombin inhibitor and demonstration of regional anticoagulation in extracorporeal circuits," Blood. 1993 Jun 15; 81 (12): 3271-6, describes an aptameric oligonucleotide having the sequence GGTTGGTGTGGTTGG.

Despite the fact that numerous examples of first-generation oligonucleotides of DNA aptamers to thrombin have been known for about 20 years, their practical advancement into the medical field is hindered by a number of factors. Currently, drugs based on DNA aptamers to thrombin are not yet used in clinical practice. For example, the first generation aptamers ARC 183 and ACR2172 successfully passed the stages of preclinical trials in the USA, but were stopped at the stage of clinical trials. Based on the experimental data collected during the work with the invention, it can be assumed that the main reason for the suspension of the advancement of these aptamers was the problem of correct assembly of the G-quadruplex pharmacophore with additional duplex modules. The existing Russian analogs of DNA aptamers to thrombin, including the previously patented RE31 and ST43, have a similar additional duplex structure. Accordingly, the task of developing a dosage form capable of being used in clinical practice is currently relevant. Thus, the aim of the applicant is to develop a drug based on a direct anticoagulant based on a DNA aptamer that selectively inhibits the activity of thrombin. In the process of experimental work, a method for obtaining a dosage form was also developed.

Description of drawings

Analysis of thrombin time using RA-361 and Angiox control in human and animal plasma.

Figure 1. Thrombin time. Human plasma.

Figure 2. Thrombin time. Rat plasma. Analysis of thrombin time using RA-361 and Angiox control in human and animal plasma.

Figure 3. Thrombin time. Rabbit plasma.

Figure 4. Thrombin time. Dog plasma. Analysis of prothrombin time using RA-361 and Angiox control in human and animal plasma.

Figure 5. About thrombin time, p. Human plasma.

Figure 6. Prothrombin time, s. Rat plasma. Analysis of prothrombin time using RA-361 and Angiox control in human and animal plasma.

Figure 7. Prothrombin time, s. Rabbit plasma.

Figure 8. Prothrombin time, s. Dog plasma. APTT analysis using RA-361 and Angiox control in human and animal plasma.

Figure 9. APTT, p. Human plasma.

Figure 10. APTT, p. Rat plasma. APTT analysis using RA-361 and Angiox control in human and animal plasma.

Figure 11. APTT, p. Rabbit plasma.

Figure 12. APTT, p. Dog plasma.

The essence of the invention

The present invention describes specially developed technologies that make it possible to produce significant and sufficient amounts of aptamer for conducting studies of various levels, as well as such an amount that will be sufficient for the industrial production of a dosage form. First of all, a fundamentally new design of bivalent DNA aptamers was developed, which is an example of a new generation of aptamers. The developed aptamer RA-361 has a second G-quadruplex pharmacophore instead of a duplex. A significant advantage of this design is fast, spontaneous assembly, which is determined by its unique structure. The developed dosage form based on RA-361, a new generation thrombin DNA aptamer, was created on the basis of a detailed author's study of the properties of the pharmacophore motif, which is part of numerous first generation DNA aptamers. In preliminary laboratory experiments in vitro and in vivo, a high specific activity of RA-361 towards human thrombin and low toxicity have been shown, which makes it possible to use the drug in the form of a solution for injection and infusion for use as an anticoagulant in high-tech surgical operations. A drug based on a DNA aptamer to thrombin can be used for the prevention of thrombosis, including during the period of surgical interventions.

There are no absolute analogues of this drug, however, for a comparative analysis of the pharmaceutical composition, the drug Bivalirudin (trade name ANGIOKS) was taken as an analogue. The following patent documents describe pharmaceutical compositions and methods for their preparation related to the peptide thrombin inhibitor Bivalirudin (trade name ANGIOX): US 7582727, US 7598343, WO 2015175668, CN 104888207.

The drug based on the RA-361 aptamer (described in RU 2631829) belongs to a new generation of prolonged-release antithrombotic drugs based on a modular bivalent DNA aptamer selective for thrombin.

The main active ingredient of the investigational drug is a 31-bp DNA oligonucleotide with a spatial structure of the antiparallel G quadruplex type stabilized by potassium cations. Since oligonucleotides are not stable in the bloodstream, the oligonucleotide was covalently modified with 20,000 polyethylene glycol to overcome this problem. The main DNA oligonucleotide has the following nucleotide sequence: 5'-GG-TT-GG-TGT-GG-TT-GG-T-GG -TT-GG-TGT-GG-TT-GG-3 '

This sequence was obtained on the basis of rational design and optimization of the structure of the prototype molecule using molecular modeling methods. The prototype was a 15-nt DNA oligonucleotide with the sequence [1]:

5'-GG-TT-GG-TGT-GG-TT-GG-3 '

The prototype molecule is capable of forming an antiparallel G quadruplex, consisting of two planar G-quartets connected by three lateral loops. The three-dimensional structure of the prototype molecule was previously determined by nuclear magnetic resonance and X-ray diffraction analysis [2], [3], [4], [5]. As in the case of the prototype molecule, our proposed aptamer contains an antiparallel G-quadruplex, for the stabilization of which metal cations, in particular, potassium cations, play a key role [6]. The potassium cation is coordinated by 31 G-quadruplex guanines, adding an enthalpy component to the energy of the structure. [3] [4]. The proposed oligonucleotide 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 [7]. Both G quadruplex parts are important for the inhibitory activity and thermal stability of the DNA aptamer; disruption of the structure of any of them leads to an increase in the apparent inhibition constants [7] and a decrease in the melting point of the quadruplex structure according to circular dichroism data [6]

1. Development of prototypes of the medicinal product

To obtain the initial amino-modified oligonucleotide RA-36, 4 syntheses of the amino-derivative oligonucleotide were carried out by the method of scaled solid-phase synthesis. After synthesis and carrying out the procedure of cleavage from the support in an ammonia solution and deprotection, about 152 copt units (kOD) were obtained at a synthesis scale of 260-270 μmol with an average yield of 130-135 opt. units / μmol.

During all stages of preparation of the preparation, the content of the oligonucleotide component was carried out spectrophotometrically by direct measurement of the absorption values of solutions at a wavelength of 260 nm.

The efficiency of the total synthesis of the conjugate is largely determined by the quality of the synthesis of the starting amino derivative. For conjugation, fractions of the purified amino derivative of the RA-36 oligonucleotide with the content of the target component exceeding 80% were selected.

After 3 cycles of chromatographic purification, one cycle of tangential filtration and concentration by tangential filtration was carried out. Losses on tangential filtration of the amino derivative are about 8%, resulting in 3.4 g of oligonucleotide with a content of the main component of at least 85%.

1.1 Conjugation of the parent aminomodified oligonucleotide derivative RA-36 and the N-succinimide PEG derivative.

The resulting products of the starting amino derivative of the RA-36 oligonucleotide were used to carry out conjugation reactions with an N-succinimide polyethylene glycol derivative with a molecular weight of 20 kDa. Chromatograms of the reaction mixture of the amino derivative of the RA-36 oligonucleotide with the N-succinimide derivative of polyethylene glycol with a molecular weight of 20 kDa are shown (Table 1).

Purification of the resulting conjugate from unreacted amino derivative of the oligonucleotide and polyethylene glycol was carried out using anion exchange chromatography similar to the chromatographic purification of the amino-modified oligonucleotide.

1.2 Preparation of drug samples

The following example describes a preferred method for preparing a medicament.

Example 1

After chromatographic purification of the active substance and synthesis, a concentration cycle was carried out by tangential filtration against a 10 mM potassium chloride solution to a concentration of 120-150 mg / ml of the active substance, where the preferred concentration is 120 mg / ml.

The purified samples of the active substance were subjected to annealing. Moreover, annealing means heating the sample to a temperature of 95 C and cooling to room temperature at a rate of 5 C per minute. The term annealing often refers to the process of primer hybridization in a PCR reaction. Nevertheless, there is the same name for the process of forming the structure of a nucleic acid during gradual cooling of the solution; this process can also be called renaturation, i.e. the formation of a native structure. In the case of an aptamer, the concept of a native structure is not applicable, therefore the term annealing is used. The residence time of samples at a concentration of RA-361 oligonucleotide 120-150 mg / ml, where the preferred concentration is 120 mg / ml, at 95C was 5 minutes, then the samples were cooled to room temperature temperature in the Biosan thermostat for 15 minutes.

After the 'annealing' procedure, the samples were sterilized using Millex filter attachments (filter material - durapore with a pore diameter of 0.22 μm). As a result of these procedures, a sterile pharmaceutical substance was obtained in which the concentration of the active substance was 120-150 mg / ml in a 10-20 mM potassium chloride solution in water, depending on the concentration of the active substance, where the preferred concentration is 120 mg / ml. Samples of the medicinal product were prepared from a pharmaceutical substance by four-fold dissolution in sterile saline solution, where water prepared according to ISO 3696 was used, and filled in glass vials in a laminar flow cabinet, and then sealed. Losses at the stage of 'preforming' and sterile filtration are up to 2% of the target component. At the outlet, a substance with a volume of 10 ml is obtained, the concentration of 30-37.5 mg / ml of the oligonucleotide RA-361, where the preferred concentration is 30 mg / ml. RA-361 is a 31 nt oligonucleotide modified with 20 kDa ethylene glycol at the 16 position.

Thus, a drug containing oligonucleotide RA-361 at a concentration of 30 mg / ml, with an allowable limit of up to 37.5 mg / ml, potassium chloride with a concentration of 10 mM or higher, where the concentration of potassium chloride does not exceed 20 mM, sterile saline and water prepared to ISO 3696 (or mi-Q quality). When using water prepared according to the ISO 3696 standard, the pH of the solution will be about 7.4, with an allowable rate in the range between 7 and 7.5. Nucleic acid at these concentrations has a "buffer" effect with the help of phosphate groups from the sugar-phosphate backbone of DNA. The dosage form at the outlet is a clear solution.

Thus, a new, experimentally developed method was used to synthesize the initial amino derivative RA-36, the synthesis product was purified by anion-exchange chromatography. For the purified initial amino derivative RA-36, a conjugation reaction with an activated PEG derivative was carried out. The yield of the conjugation reaction reached 77%. The resulting pegylated oligonucleotide RA-361 was also purified similarly to the starting amino derivative RA-36. As a result of this work, a total of 200 mmol of pegylated oligonucleotide RA-361 was produced. The obtained oligonucleotide was used for the preparation of a drug of pegylated oligonucleotide RA-361, for which further studies of specific activity and mechanisms of action were carried out.

2 Selection of animal models for testing specific activity.

The RA-361 aptamer is most effective when using a human model, which, however, cannot be used for preclinical studies, the next most effective was rat plasma, which made it possible to recommend it for further studies in vivo, followed by dog plasma, This model is very difficult to use due to the lack of a set of animals of the same type in vivariums in Russia, the last in terms of efficiency was rabbit plasma. It should be noted that rabbits (not rodents) are one of the required animal models, according to the "Guidelines for experimental (preclinical) study of new pharmacological substances." (Khabriev R.U.). Thus, despite the low efficacy on rabbit plasma, it was proposed to recommend the rabbit as a second model for preclinical studies of the efficacy of the RA-361 aptamer in vivo.

3. Determination of the effectiveness of the drug in vitro on the blood plasma of animals and humans.

The anticoagulant effect of the study drug and the reference drug is due to the inhibition of factor IIa (thrombin), an essential component of the blood coagulation system. In the course of this experiment, to study the anticoagulant activity of the RA-361 DNA apatmer and the reference drug, an assessment of their effect on such laboratory parameters of hemostasis as:

- thrombin time (TB);

- prothrombin time (PT);

- activated partial thromboplastin time (APTT).

These parameters were determined in the citrated blood plasma of laboratory animals, which were injected with the study drug, or the reference drug, or saline (control group). Evaluation was carried out on a semi-automatic coagulometer equipped with software for determining the discussed parameters and using appropriate reagent kits.

Carrying out the thrombin time test: 50 μl of the studied citrate plasma sample was taken into a plastic cuvette of the coagulometer, kept for 60 seconds at 37 ° С, 100 μl of the working reagent solution was added to carry out the thrombin time test, heated at 37 ° С. The clotting time of the studied plasma sample was measured on a coagulometer.

Carrying out the prothrombin time test: 50 μl of the studied sample of citrate plasma was taken into a plastic cuvette of the coagulometer, kept for 60 seconds at 37 ° С, 100 μl of the working reagent solution was added to perform the prothrombin time test, heated at 37 ° С. The clotting time of the studied plasma sample was measured on a coagulometer.

Conducting the activated partial thromboplastin time test: 50 μl of the studied citrate plasma sample was taken into a plastic cuvette of the coagulometer, kept for 60 seconds at 37 ° С, 50 μl of the working reagent solution was added for the activated partial thromboplastin time test, heated at 37 ° С, kept at 37 ° C 180 seconds. Added 50 μl of a solution of calcium chloride, heated at 37 ° C. The clotting time of the studied plasma sample was measured on a coagulometer. Comparative examples of efficiency are shown in FIG. 1-12.

RA-361 is required 30 times more to achieve an equal effect in the thrombin time test (with human plasma) with the closest anticoagulant mechanism of action by Angioxom (direct thrombin inhibitor, blocks the active center of thrombin); in the test prothrombin time - 2.2 times more; in the APTT test - 6.8 times more.

The most sensitive to the RA-361 aptamer was the thrombin time test, since the values of the effective anticoagulant concentrations are the lowest: 0.9 μM. According to Hirch J. et al. the required degree of anticoagulation can be achieved at a concentration of Angiox in human plasma of 1 µg / ml (according to the APTT test - 0.67 ± 0.1 µg / ml) [17, 18]. If we calculate the concentration of Angiox in plasma (based on the fact that the amount of blood in the body of an adult is 6-8% of body weight [19], that is, 4.5-6 liters, and the amount of plasma is 1.8 times less, since hematocrit in humans is 41-46%) with intravenous bolus administration at a dose of 0.4 mg / kg [17], then it can be about 10 μg / ml.

The range of effective plasma concentrations of Angiox in humans is 0.14-9.36 μg / ml, and the range of plasma concentration of the RA-361 aptamer in sensitive tests reaches 27-48 1700 μg / ml (the dose range calculated on the basis of plasma aptamer concentrations can be from 2 to 100 mg / kg), and according to tests of thrombin time, APTT and prothrombin time: not less than 27, 60, 80 μg / ml of plasma, respectively.

Summarizing the data, the dose range can be derived: 2-100 mg / kg.

RA-361 is required 24 times longer to achieve equal effect with Angiox in the thrombin time test (with rabbit plasma); in the APTT test - 1.1 times more; in the prothrombin time test - 1.2. The effective concentrations of RA-361 in the thrombin time, aPTT and prothrombin time tests are 12, 106 and 160 μM, respectively. The range of doses calculated in relation to the activity on human plasma is 21 - 8000 mg / kg, calculations were carried out according to plasma concentrations (based on the fact that the average amount of blood in rabbits is 4.5-6.7% of weight (3.5 -4.5 kg) [20], which is on average 220 ml, and plasma - 130 ml, since the hematocrit in rabbits is 34-44%). The range of doses of aptamer, calculated from the effective concentrations in the plasma of rats in the tests of TB, APTT, PT (the amount of blood in rats weighing 200-250 g is 15-17 ml of weight, on average plasma is 8.8 ml; hematocrit in rats is 40-50 %) reaches 3-700 mg / kg.

The synthetic DNA aptamer RA-361 in the plasma concentration range of 1.63-575 μg / ml, as well as the reference drug (Angiox), lengthens the clotting time of plasma in humans, rabbits, rats, dogs in the TB test. Angiox and the synthetic aptamer RA-361 in the plasma concentration range of 2.2-217 μg / ml prolong the clotting time of human plasma in the PT test.

The concentrations of Angiox and aptamer, at which the plasma clotting time increases by 2 times (2PV), in comparison with the control (buffer was added instead of AA), were 0.9 and 2.0 μg / ml, respectively. The concentrations of Angiox and aptamer RA-361, at which the clotting time of human plasma is doubled (2PV), in comparison with the control (instead of AA, buffer was added), were 0.9 and 2.0 μM, respectively;

- 2 PT concentrations of Angiox and aptamer RA-361 in rabbit plasma 130 and 160 μM, respectively;

- 2 PV concentrations of Angiox and aptamer RA-361 in rat plasma 0.3 and 7 μM, respectively;

- 2 PT concentrations of Angiox and aptamer RA-361 in dog plasma 1.9 and 11 μM, respectively.

Effective concentrations of Angiox and aptamer were 0.9 and 2.0 μg / ml, respectively.

The effect of AA on plasma clotting in humans, rabbits, dogs, or rats upon activation of factor XII by ellagic acid in the presence of soy phospholipids was studied in the APTT test. The synthetic aptamer RA-361 in the plasma concentration range of 1.96 - 195.6 μg / ml, like Angiox, prolongs the clotting time of plasma in humans, rabbits, rats and dogs. The concentrations of Angiox and aptamer RA-361, at which the clotting time of human plasma increases by 2 times (2APTT), in comparison with the control (instead of AA, buffer was added), were 0.4 and 2.7 μM, respectively;

- 2 APTT concentrations of Angiox and aptamer RA-361 in rabbit plasma 96 and 106 μM, respectively;

- 2 APTT concentrations of Angiox and aptamer in rat plasma 0.6 and 5 μM, respectively;

- 2 APTT concentrations of Angiox and aptamer RA-361 in dog plasma 4.7 and 28 μM, respectively.

The range of doses of aptamer, calculated from the effective concentrations in the plasma of rats in the tests of TB, APTT, PT (the amount of blood in rats weighing 200-250 g is 15-17 ml of weight, on average plasma is 8.8 ml; hematocrit in rats is 40-50 %) reaches 3-700 mg / kg.

Thus, the aptamer RA-361 exhibits direct anticoagulant properties, inhibiting thrombin. With an increase in the concentration of the synthetic aptamer RA-361 in human plasma, the time of appearance of a fibrin clot in the tests of thrombin time, APTT, and prothrombin time increases.

4. Determination of the effectiveness of the drug with a single bolus intravenous injection in comparison with the closest analogue - peptide thrombin inhibitor bivalirudin

As a control substance, a physiological sodium chloride solution was used (sodium chloride solution for infusion 0.9%, LLC "MOSFARM", Russia).

Administration of the drug to rats

The rats were anesthetized with a 100 mg / ml chloral hydrate solution at the rate of 400 mg per kg of animal weight. The jugular vein was opened and using an insulin syringe (1 ml 0.33 mm (29 G) × 12.7 mm, step 2 U BD Micro-Fine Plus® Insulin U-40), the amount of the study drug, the reference drug or saline solution was administered indicated in table 3.

Control groups of animals, both rats and rabbits, were injected with 200 μl of saline.

The test drug was administered to rats once as an injection solution in a volume of 200 μl at doses of 21, 10.5, 42 mg / kg. The reference drug was injected once in the form of an injection solution in a volume of 200 μl at doses: 0.38 mg / kg, 0.75 mg / kg, 1.5 mg / kg. The amount of the drug to be administered to a specific animal was calculated based on the weight of the animal. The required dilution was obtained with physiological saline in plastic tubes.

Administration of the drug to rabbits

The preparations were injected into the ear marginal vein in rabbits without preliminary anesthesia. With the introduction of drugs, the time of drug administration was recorded with an accuracy of a minute.

Doses and preparation of the drug for administration

Control groups of animals were injected with 200 μl of saline. To rats, the reference drug and the test drug were administered both once and repeatedly. For rabbits - once. With a single administration, the study drug was administered to rats as an injection solution in a volume of 200 μl at doses of 10.5, 21, and 42 mg / kg.

The comparison drug with a single administration was used at the following concentrations: 0.38, 0.75 and 1.5 mg / kg.

Intravenous administration was used as this is the intended route of administration to humans. The injected drug is prepared in the form of a solution of the pharmaceutical substance RA-361.

Introduction procedure.

The substance was administered once intravenously in the range of the selected doses and in the volumes acceptable for intravenous administration. Doses for administration were calculated individually for each animal based on the latest animal body weights.

Blood sampling procedure.

- With a single injection of physiological solution, blood samples were taken: 5 minutes before the administration of the drug and after at 0, 15, 30, 60, 90, 120, 150, 180 minutes;

- With a single administration of the test drug, blood samples were taken: 5 minutes before drug administration and after 0, 15, 30, 60, 90, 120, 150, 180 minutes;

- With a single injection of the reference drug, blood samples were taken: 5 minutes before drug administration and after at 0, 15, 30, 60, 90, 120, 150, 180 minutes.

Blood samples were taken from the jugular vein of rats before and after administration of the test drug using an insulin syringe (1 ml 0.33 mm (29 G) × 12.7 mm, step 2 U BD Micro-Fine Plus® Insulin U-40 ), which contained a 3.8% sodium citrate solution (the ratio of blood: 3.8% sodium citrate solution was 9: 1). After collection, blood samples were centrifuged for 15 minutes at 1400 rpm, and the plasma was transferred into plastic tubes. The volume of samples taken was about 300 μl, the volume of the obtained citrated blood plasma was at least 150 μl. Statistical processing of research results. Statistical processing of the data obtained was carried out using standard methods of variation statistics using the Student's test.

Studies of the effectiveness of pharmacological agents in animals have a prognostic character in relation to a number of parameters of their use in humans, including the dose of the pharmacological agent. In the present study, a set of doses of the study drug was formed to study its effect on the parameters of blood plasma coagulation (TB, PT, APTT), in order to detect a single dose of the drug. The search for such a dose was based on the judgment that the use of a single dose of RA-361 should cause an increase in the discussed parameters by a factor of 2–3 relative to the initial values. These targets are consistent with those for dosing with heparin, the gold standard for direct anticoagulants. The effect of three doses of the study drug was studied: 10.5; 21; 42 mg per kg of body weight of a laboratory animal, on TV, PT, APTT. Dosages of the RA-361 preparation were investigated on groups of experimental animals, 6 rats each. Table 4 presents the results of a study of the effect of each dose of RA-361 on TB, PT, APTT.

Thus, at the stage of preclinical studies, a single dose of RA-361 was taken at a dose of 21 mg / kg. At the next stage of the study, the anticoagulant activity of a single dose of RA-361 was compared with a single dose of a reference drug - Angioxa, which has a similar mechanism of action, route of administration and is registered in the Russian Federation. The anticoagulant activity of Angiox was also assessed by the parameters of blood plasma clotting in laboratory animals. A single dose of Angiox, namely 0.75 mg per kg of laboratory animal weight, was studied in a group of experimental animals, which included 6 rats.

The effect of RA-361 and Angiox preparations on blood plasma coagulation indices and the clinical state of the biological model - chinchilla rabbit was studied. No significant changes in the state of the animals were found during the experiments. There was no increase in the duration of post-injection bleeding with the introduction of various dosages of the test drug and the reference drug and subsequent blood sampling at set time intervals. There were no cases of vascular thrombosis. It should be noted that, despite the fact that insignificant patterns in the increase in aptamer concentrations and changes in TB and APTT are observed, there is also an extremely strong scatter in the results, which indicates the non-specificity of the RA-361 aptamer to rabbit plasma.

From the data presented in the tables, it follows that RA-361 at a dose of 21 mg / kg, in contrast to Angiox at a single dose of 0.75 mg / kg, falls within the framework of the above-stated target values for TB, PT, APTT. Based on the above data, RA-361 and Angiox have slightly different anticoagulant activity profiles. The reference drug, Angiox, has the most pronounced anticoagulant effect according to PT and TB tests. The study drug, RA-361, has the most pronounced anticoagulant effect on TB and APTT tests.

5. Determination of the effectiveness of the drug with repeated intravenous administration to animals in comparison with the closest analogue - peptide thrombin inhibitor bivalirudin

The substance was injected once intravenously in the dose range from 1.0 to 15.0 mg / kg in volumes acceptable for intravenous administration. Doses for administration were calculated individually for each animal based on the latest animal body weights.

Analysis Analysis of the activity of direct anticoagulants in vitro includes an assessment of the effect on human plasma coagulation in coagulological (fibrinogenolytic) tests or on the amidolytic activity of coagulation factors [8, 9, 10, 11, 12, 13, 14, 15, 16]. The analysis was performed in plastic tubes at 37 ° C.

The control group of rats was repeatedly injected with 200 μl of physiological solution (sodium chloride solution for infusion 0.9%, LLC "MOSFARM", Russia). Saline was administered at 0, 15, 30, 60, 90, 120, 150, 180 minutes.

The study drug was administered repeatedly to rats in the form of injection solutions in a volume of 200 μl at doses: 7 mg / kg, 21 mg / kg, 42 mg / kg. The test drug was administered at 0, 15, 30, 60, 90, 120, 150, 180 minutes.

The reference drug was administered repeatedly to rats in the form of injection solutions in a volume of 200 μl at doses: 0.19 mg / kg, 0.38 mg / kg, 0.75 mg / kg. 0, 15, 30, 60, 90, 120, 150, 180 minutes.

The amount of the drug to be administered to a specific animal was calculated based on the weight of the animal.

Blood sampling procedure

With repeated administration of saline, blood samples were taken: 5 minutes before the administration of the drug and after at 0, 15, 30, 60, 90, 120, 150, 180 minutes.

With repeated administration of the test drug, blood samples were taken: 5 minutes before drug administration and after at 0, 15, 30, 60, 90, 120, 150, 180 minutes.

With repeated administration of the reference drug, blood samples were taken: 5 minutes before drug administration and after at 0, 15, 30, 60, 90, 120, 150, 180 minutes.

Blood samples were taken from the jugular vein of rats before and after administration of the test drug using an insulin syringe (1 ml 0.33 mm (29 G) × 12.7 mm, step 2 U BD Micro-Fine Plus® Insulin U-40 ), which contained a 3.8% sodium citrate solution (the ratio of blood: 3.8% sodium citrate solution was 9: 1). After collection, blood samples were centrifuged for 15 minutes at 1400 rpm, and the plasma was transferred into plastic tubes. The volume of samples taken was about 300 μl, the volume of the obtained citrated blood plasma was at least 150 μl.

In order to assess the ability of the study drug and the reference drug to maintain blood plasma coagulability indices within the target values, their anticoagulant activity was studied with sequential repeated intravenous administration. Changes in blood plasma coagulation parameters with time when using RA-361 are presented in Table 7. The data in the table are given for multiple infusion intravenous therapeutic doses of RA-361: bolus administration of 7 mg / kg followed by 7 mg / kg / h; bolus administration of 14 mg / kg followed by 14 mg / kg / hour; bolus injection of 21 mg / kg followed by 21 mg / kg / h. The analysis was carried out according to three target indicators of TB, PT, APTT.

From the data presented in table 7, it follows that with the selected dosing regimen, RA-361 is able to maintain the values of TB, PT, APTT within the target indicators for at least 3 hours, which indicates the prospects of its use with prolonged infusion using a dropper. When using a single bolus and infusion of RA-361, it is possible to reduce the concentration of the drug to 14 mg / kg and maintain the required concentration in the blood for a 3-hour operation. Analysis of the results of the preclinical study leads to the following conclusion:

- The study of the ability of RA-361 to maintain blood coagulation indices using the animal model "rat", with sequential repeated administration of the selected single dose of the drug, within a 2-3-fold increase in the indices showed that RA361 is able to maintain 95 values of TB, PT, APTT within the target indicators for at least 3 hours, which indicates the prospects of its use with prolonged infusion.

6. Optimization of drug production technology

To optimize and scale up the technology for preparing the RA-361 preparation, it is necessary to adjust the synthesis of the amino derivative of the oligonucleotide. With the help of the technology experimentally developed during the study of the drug, this goal was achieved 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) with subsequent deprotection, purification of the resulting amino derivative by preparative chromatography, preparation and purification of the conjugate.

The main stage of scaling at the current stage of aptamer production is the solid-phase synthesis process. The synthesized nucleotide sequence of the amino derivative is 58% G-rich, the rest of the sequence is dT nucleotides. In some cases, this sequence can complicate the synthesis process due to the low efficiency of dG-nucleotide condensation, 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 efficiency of condensation of the dG-monomer is in particular 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 the ratio of the type of bases in the sequence, causing difficulty to access to the 5'-OH-hydroxyl group [21]. These features generally cause a decrease in the yield of the target product and an increase in the content of oligonucleotide impurities, including products of modification of the main sequence.

The choice 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 the amino-modified oligonucleotide, a commercial polymer carrier Primer Support 200 dG-iBu Synth (200 ± 10 μmol / g, cat no. 17-5290-02, hereinafter PS 200 dG) from GE Healthcare was used. The use of PS 200 dG in the synthesis of oligonucleotides whose length exceeds 30 nucleotides requires an increase in the amount of solvents and reagents at the stages of washing the flow reactor and detritylation, especially at the last stages of the synthesis, to initiate the complete course of the detritylation reaction. After the stage of conjugation of aminomodified dT-cyanoethyl-phosphoramidite, the capping stage 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 at 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 impurities within the allowable amount of "undersynthesis" 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 after the synthesis 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 the PS-carrier.

Such treatment also removes the cyanoethyl protective group, which can lead to the attachment of acrylonitrile molecules to the N3-thymidine atom during deprotection of this base, and is optionally included in the synthesis process on the AKTA Oligopilot plus 100 synthesizer. TFA-protective group located 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 support after synthesis, and is also relatively susceptible to side reactions by the mechanism of transacylation during synthesis.

The process of synthesis of the working nucleotide sequence of the aminomodified derivative [5'-GGTTGGTGTGGTTGG 16-pos / iAmMC6T / GGTTGGTGTGGTTGG-3 '] is similar to the synthesis on the automated synthesizer MerMade 48 by BioAutomation (hereinafter MM48).

The sequence of attachment of the nucleotide unit also consists of the following reactions:

The first reaction is to remove the 5'-dimethoxytrityl protecting group from the nucleoside bound to the support. The reaction should be carried out in the most anhydrous medium (<3 ppm) in an argon atmosphere (high purity quality). Cleavage of the protective group generates a bright orange 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 efficiency of condensation reactions during the entire synthesis process (so-called PAT, Process Analytical Technology - technology for analytical process control in real time). The measured amount of dimethoxytrityl ion is integrated in real time by the software controlling the synthesizer. By the amount of eluted dimethoxytrityl ion at the current stage of synthesis, one can judge the state of the synthesized sequence and the nature of condensation of the next link, which can be used for quantitative analysis of the ongoing synthesis. This information can be used to stop the current synthesis if the area and intensity of the integrated peak falls below a preset limit, thereby reducing the consumption of expensive reagents and the loss of time for obtaining an oligonucleotide sequence that does not meet the specified quality criteria.

The composition of the working solution for carrying out the detritylation reaction on the AKTA Oligopilot 100 synthesizer differs from the analogous 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 5% (w / v)) is used, which leads to a decrease in the probability of nucleotide base cleavage (depurinization) during detritilization time. The use of dimethylformamide protection on a guanosine (dG-dmf) base also reduces the likelihood of depurinization. Toluene is used as a solvent for scalable synthesis, the use of which, in contrast to dichloromethane, does not cause degassing of the solution and the appearance of zones of uneven distribution of solution components in the reactor and pump. In addition, the use of a toluene-based detritylation solution does not generate a large amount of potentially hazardous chlorine-containing waste, the disposal of which requires special approaches [22].

The detritylation reaction is monitored at wavelengths of 350 and 280 nm.

After detritylation, the reactor is washed with anhydrous acetonitrile to remove the remaining DCU and trityl ation, which can generate N + 1 impurities during the addition of the next nucleotide and reduce the final content of the target oligonucleotide;

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 phosphotriester internucleotide bond is formed in a high yield (more than 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 an aminolinker, a longer time is used - 5 minutes. After the condensation stage, the support is washed with anhydrous acetonitrile to remove excess reagents and reduce the possible formation of N + 1 impurities.

The phosphotriester internucleotide bond formed during condensation is converted into a phosphodiester bond during oxidation with a solution of iodine with an organic base (usually pyridine or lutidine). After oxidation, the support is washed with anhydrous acetonitrile. To carry out the reaction, a flow-through treatment of the support in a reactor with a solution of 0.05 M iodine in a mixture of a heterocyclic base (usually pyridine, lutidine, collidine) with water in a 90:10 ratio is used. Water acts as an oxygen donor, and iodine forms an adduct with a phosphorus bond, which is decomposed by water, leaving a stable phosphodiester bond [21, 22].

The last reaction in the monomer unit addition cycle is capping of the unreacted 5'-hydroxyl group followed by washing 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 isobutyryl protection of the amino group at the C2-position of the dG-base of phosphoramidites can be replaced by an acetyl group as a result of the reaction proceeding via the trans-amidation mechanism at the capping step; such an impurity cannot be eliminated under standard deprotection conditions. 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 trans-amidation reaction [23]. - At the end of the synthesis, a single treatment cycle with a 20% solution of diethylamine in acetonitrile is included. When the linker bond is cleaved between the newly synthesized oligonucleotide and the carrier, the β-cyanoethyl group is used to protect the phosphate group. During deprotection, this group is cleaved off and, in the form of acrylonitrile, interacts with the N3-position of thymine to form a cyanoethyl adduct by the Michael addition mechanism, which cannot be removed during chromatographic purification of the mixture after synthesis. This effect is minimized by treating the support after synthesis with alkylamines (diethylamine, tert-butylamine) by 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 cleaved and deprotected according to the instructions for the PS 200 dG solid phase support (Instruction 03-0015-79 AD, GE Healthcare). After synthesis, the support in the reactor is dried under vacuum for an hour to quantify the change in the mass of the support after synthesis (weighing). Then the carrier is once again diluted in at least 1 column volume (6.3 ml), transferred to a container for working with solutions under pressure with a tight lid, which is then filled with a cooled aqueous solution of concentrated ammonia (35%) in the ratio of 10 ml per gram of carrier ... The mixture is incubated with gentle stirring at 55 ° C for 16 hours. After incubation, the mixture is cooled for 30 minutes at -34 ° C and filtered on a glass filter with a porosity of 3 (16-40 μm). 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. The oligonucleotide concentration is determined by spectrophotometry by direct absorption of the solution at a wavelength of 260 nm (the yield from synthesis is 130-135 OD260 / μmol).

The composition of the mixture and the percentage of the main component are determined by anion exchange HPLC. The concentrated aqueous solution of the oligonucleotide is diluted in perchlorate buffer for chromatographic purification. Synthesis impurities and residual low-molecular components of the reagents are removed by preparative anion exchange chromatography on a 150 ml column with Source 15Q AO-carrier (GE Healthcare).

The process of pegylation of an amino-derivative oligonucleotide is linearly scalable. To remove the oligonucleotide unreacted with 20 kDa PEG and oligonucleotide impurities not removed by preparative chromatography, it is convenient to use the conjugate reprecipitation procedure according to the following procedure.

Add 3 ml of 5 M aqueous sodium perchlorate solution to 30 ml of the reaction mixture, then add 100 ml of acetone (reagent grade), fill in 50 ml Falcon tubes and unscrew the solution in a centrifuge (15 min, 3000 rpm). After centrifugation, separate the resulting solution from the sediment. The sediment contains unreacted oligonucleotide and impurities of a similar nature. The pegylated aptamer is contained in a solution from which acetone is removed after decanting with gentle heating under vacuum. Excess unreacted PEG is removed by preparative anion exchange chromatography; 10 mM Tris, 50 mM NaCl were used as a mobile phase, and 10 mM Tris, 700 mM NaCl were used as eluents. Unreacted PEG is eluted almost immediately after injection; for its detection, the first fractions after purification are analyzed by absorption at a wavelength of 280 nm (since the AKTA purifier 100 chromatograph does not have the function of monitoring this absorption region online during the purification process). 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, after which it is transferred for preforming.

The above data allow us to conclude that the developed technology is optimized in comparison with those previously described in the literature by choosing suitable compositions of working reagents and optimizing the parameters of the synthesis process, such as skipping the capping step in the second half of the synthesis, processing the synthesized sequence before deprotection, and cleavage from PS- a carrier with a 20% solution of N, N-dialkylamine, the use of dichloroacetic acid at the stage of detritylation and others.

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Claims (2)

1. Dosage form of aptamer RA-361, including aptamer 5'-GG-TT-GG-TGT-GG-TT-GG-T-GG-TT-GG-TGT-GG-TT-GG-3 ', covalently modified 20 kDA PEG in the 16 position at a concentration of 30 mg / ml, with an allowable limit of up to 35.7 mg / ml, water, potassium chloride with a concentration of 10 mM, where the concentration of potassium chloride does not exceed 20 mM, and saline solution, where the dosage form has a pH value in the range between 7 and 7.5 and is a clear aqueous solution. 2. A method of obtaining a dosage form according to claim 1, comprising obtaining a modified PEG aptamer RA-361 with chromatographic purification and a cycle of concentration of the active substance, heating and maintaining the temperature of the samples of the active substance to 95 ° C for 5 minutes, followed by cooling the samples to room temperature at a rate of 5 ° C per minute and sterilization using filters with a pore diameter of 0.22 μm to obtain a pharmaceutical substance at a concentration of 120-150 mg / ml, then the substance is subjected to four-fold dissolution in sterile saline solution, where water is used, prepared according to the ISO 3696 standard, and packed into glass vials in a laminar flow cabinet, after which it is sealed, while at the outlet a substance with a volume of 10 ml is obtained, and the concentration of RA-361 oligonucleotide is 30-35.7 mg / ml.

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