RU2738093C9 - Synthesis method of 2'-o-(2-methoxyethyl) thiophosphate oligonucleotide - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, particularly to a novel method of producing antisense compounds which can be used as pharmaceutical substances for ameliorating the symptoms of spinal muscular atrophy. Disclosed method of producing modified thiophosphate oligonucleotide containing 2'-O-(2-methoxyethyl) ribonucleotides of structure UCACUUUCAUAAUGCUGG, where each nucleoside contains 2 ' -O- (2-methoxyethyl) modified sugar residue provides for synthesis of oligonucleotide on a solid-phase support using an amidophosphite method. To obtain thiophosphate groups, 3-[(dimethylaminomethylene)amino]-3H-1,2,4-dithiazol-5-thione (DDTT) is used in 30 % solution of pyridine in acetonitrile, release is performed by adding a saturated ammonia solution in the presence of disodium salt of ethylenediaminetetraacetic acid for 3 days at room temperature. Method then includes successively purifying oligonucleotides by ion exchange chromatography on Tosoh Super-Q 5PW column using Tris-HCl buffer, pH 7.0, with addition of 10 % acetonitrile in water, and then Tris-HCl buffer, pH 7.0, with addition of sodium perchlorate and 10 % acetonitrile in water, by reversed-phase chromatography on Waters xBridge WEN C18 column using a buffer, which is a solution of triethylammonium acetate in water, pH 7.0, and then a buffer which is a solution of triethylammonium acetate in 80 % aqueous solution of acetonitrile, pH 7.0, and gel filtration followed by extraction of the end product with drying by lyophilisation.

EFFECT: invention enables to obtain a product with higher degree of purification.

1 cl, 8 dwg, 4 tbl, 2 ex

Description

The invention relates to the field of chemistry and medicine and relates to a new method for producing antisense compounds that could be used as pharmaceutical substances for drugs to alleviate the symptoms of spinal muscular atrophy.

Spinal muscular atrophy (SMA) or proximal spinal amyotrophy is a hereditary disorder in which the function of the nerve cells of the spinal cord occurs, leading to the progressive development of muscle weakness, muscle atrophy, and ultimately immobilization of the patient. Until recently, there were practically no means capable of curing this disease or changing its course. Spinal muscular atrophy develops only if the child has inherited defective copies of the SMN1 gene from both parents who are healthy heterozygous carriers of the disease. As a result of a number of mutations, the synthesis of the functionally active protein SMN (survival of motor neuron protein, a protein that promotes the survival of motor neurons), which protects motor neurons, is disrupted. The lack of SMN protein leads to the death of motor neurons in the spinal cord and the gradual blockage of vital motor functions (in particular, the ability to breathe and swallow).

Currently, medicine is using a new approach to therapy, using antisense nucleotides. Antisense oligonucleotides have a nucleotide sequence that is complementary to the sense (i.e., protein-coding) portion of the mRNA chain, which allows protein synthesis to be blocked after the oligonucleotide-mRNA complex is formed. Synthetic and natural oligonucleotides are rapidly degraded by endogenous nucleases; therefore, therapeutic oligonucleotides must be chemically modified to increase stability and prolong action.

Oligonucleotide modifications are the subject of a number of prior art inventions.

In particular, US patent 6007995 (A) - 1999-12-28 discloses a method for producing an antisense compound, 8 to 30 nucleotides long, targeting a nucleic acid molecule encoding a human TNFR1 protein, wherein said antisense compound inhibits the expression of the human TNFR1 protein. Antisense compounds are obtained using the solid-phase synthesis method using amidophosphite derivatives of protected nucleosides on an automatic synthesizer. Natural phosphodiester derivatives are obtained by oxidation of phosphites with a solution of iodine in pyridine in the presence of water. Thiophosphate derivatives are prepared using solutions of a number of thiolating reagents, including 3, H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent) in anhydrous acetonitrile.

International application WO 2010091308 - 08/12/2010 discloses a method for the synthesis of antisense compounds that make it possible to obtain substrates for RNase H. In the proposed method, solid-phase synthesis and purification by chromatography are used.

Patent US 6210892 B1 - 2001.04.03 discloses the production of an antisense compound having at least one 2'-methoxyethoxy, 2'-dimethylaminooxyethoxy, 2'-dimethylaminoethoxyethoxy, 2'-acetamide, a modification of morpholino or peptide nucleic acid, which specifically hybridizes with the specified mRNA target and does not cause cleavage of the mRNA target upon binding, so that the processing of the specified mRNA target is modulated. Complete 2'-methoxyethoxy phosphorothioate oligonucleotides are described.

Known methods provide for the production of oligonucleotides for medical and research purposes, mainly in the field of oncology.

In December 2016, the first drug, an oligonucleotide for the treatment of spinal muscular atrophy, nusinersen, was approved in the United States.

Nusinersen is an antisense oligonucleotide (ASO) designed to alter the splicing of SMN2, a gene nearly identical to SMN1, to increase production of a fully functional SMN protein (https://www.accessdata.fda.gov/dragsatfda_docs/label/2016/209531 lbl. pdf).

Known patent US 8980853 (B2) - 2015-03-17, relating to an antisense oligonucleotide complementary to intron 7 of a nucleic acid encoding a pre-mRNA SMN2 human, such as nusinersen. This patent can be indicated as the closest analogue. The known solution does not aim to obtain a highly purified antisense oligonucleotide substance.

The objective of the invention is to develop and optimize the synthesis of an antisense nucleotide.

The problem is solved by a new method of obtaining a modified thiophosphate oligonucleotide (Nus-1) containing 2'-O- (2-methoxyethyl) ribonucleotides of the structure UCACUUUCAUAAUGCUGG, where each nucleoside contains 2'-O- (2-methoxyethyl) (synonym - 2'-O - (methoxyethyl)) modified sugar residue, in which the selection of the optimal reagents for the chemical synthesis of the oligonucleotide, the selection of the optimal conditions for the purification of the oligonucleotide with the optimization of the purification sequence and the optimization of the chromatographic conditions.

The oligonucleotide was synthesized using the amidophosphite method on a MERMAID 12 automatic synthesizer (Bioautomation). For the synthesis, commercially available protected amidophosphites of 2'-O- (2-methoxyethyl) ribonucleotides and glass with a controlled pore size of 500 Å containing a universal linker were used. Since a significant part of the cost of the oligonucleotide is made up of amidophosphite derivatives of nucleosides, at the first stage, a comparison of 0.1 M and 0.05 M solutions was carried out and it was shown that the product yields when using the standard protocol did not differ (a 3-fold molar excess of amidophosphite compared to the loading of a solid-phase support ). Further, only a 0.05 M solution of amidophosphite derivatives was used. To obtain thiophosphate groups, the reagent 3 - ((N-, N-dimethylaminomethylidene) amino) -3Н-1,2,4-dithiazole-5-thione (DDTT) was used. The rest of the reagents were used unchanged.

Unblocking of the oligonucleotide and removal of protective groups were carried out using a saturated ammonia solution with the addition of several crystals of ethylenediaminetetraacetic acid disodium salt (EDTA). In the case of deprotection of the oligonucleotide with 5'-dimethoxytrile protection, several Tris crystals were added to the solution before evaporation to minimize the loss of the protecting group.

The method uses complex cleaning:

- Purification of oligonucleotides by ion exchange chromatography;

- Purification of the oligonucleotide by reverse phase chromatography;

- Purification of oligonucleotides by gel filtration.

The target product is dried by lyophilization.

Conditions of chromatography-mass spectrometric control: Column Xbridge Peptide VEN C18, 2.1 × 50 mm 5 um (Waters). Buffer A: 20 mM HFIPA, 20 mM DIPA aqueous solution. Buffer B: 20 mM HFIPA, 20 mM DIPA, ACN: H20 (8/2, v / v). Gradient elution with 0-100% buffer B (1-3 min) at 45 ° C (flow rate 0.3 ml / min). Detection: DAD detector at wavelengths of 260, 280 nm; ESI-MS detection in the negative ionization range with the detector settings indicated above.

Selection of conditions for post-synthetic deblocking and removal of protective groups

Today, gaseous methylamine or a mixture of saturated aqueous solutions of methylamine and ammonia is most often used to unblock an oligonucleotide from a solid-phase carrier and remove protective groups. This allows for fast and selective removal of protecting groups - protocols are known that last only 15-30 minutes with heating. However, such processing is possible only for oligonucleotides in the synthesis of which derivatives with acetyl (including substituted, for example, phenoxyacetyl) protecting groups for the exocyclic amino group of cytidine are used. In the case of benzoyl protections, a side reaction occurs with the formation of N-methylcytidine (Scheme 1). Due to the insufficient solubility of the acetyl derivative of 2'-O- (2-methoxyethyl) -5-methylcytidine in acetonitrile, it is available only as a benzoyl derivative. Therefore, deblocking with methylamine can lead to the formation of a by-product.

Scheme 1. Formation of a by-product upon unblocking of nus-1 oligonucleotide with a mixture of saturated aqueous solutions of ammonia-methylamine (1: 1, v / v) (AMA).

In order to avoid this, a deblocking method was used using a saturated aqueous ammonia solution. For optimization, the process was studied at different temperatures and different durations: a) 65 ° C, 48 hours; b) 65 ° C, 24 hours; c) 45 ° C, 48 hours; d) room temperature, 72 hours. The results of mass spectrometric analysis of the reaction mixture are shown in FIG. one.

It should be noted that as the deblocking temperature decreases, the amount of by-products decreases. First, when heated, a desulfurization reaction is observed (the conversion of thiophosphate to phosphate - masses -16, -32, -48 Da). Secondly, at a lower temperature, but for a longer deblocking time, fewer adducts with a higher mass are observed, that is, the protective groups are removed more completely. It is known from the literature that trace amounts of transition metals are capable of catalyzing the desulfurization reaction in a basic medium (Scheme 2); therefore, we subsequently deblocked the Nus-1 oligonucleotide with an aqueous ammonia solution at room temperature for 3 days with the addition of a small amount of EDTA. This made it possible to almost completely suppress desulfurization.

Scheme 2. Formation of the product of desulfurization of the nus-1 oligonucleotide upon deblocking with a saturated ammonia solution.

Selection of conditions for purification of oligonucleotide Nus-1 by ion exchange and reverse phase HPLC

To optimize the purification of Nus-1 oligonucleotide by anion exchange chromatography, three HPLC columns from different manufacturers were compared using several buffer systems with different pH. These columns are recommended by manufacturers for the purification of oligonucleotides, including thiophosphate. Buffer systems are traditional for the purification of oligonucleotides; however, the optimal system for a particular oligonucleotide can only be selected experimentally. Perchlorate, bromide, and chloride have different chaotropy, which is important for the purification of oligonucleotides capable of intra- and intermolecular interactions with the formation of various secondary and tertiary structures. Acetonitrile is required to minimize nonspecific hydrophobic interactions of the oligonucleotide with the sorbent. Already the first experiments showed that pH = 7 is preferable; therefore, buffers with pH 7 were then used.

Speakers: Tosoh Tskgel superQ-5pw 10 um 7.5 × 75 mm; Thermo DNAPac PA-100 Analytical 4 x 250 mm; GE MonoQ 4.6 × 100 mm.

The compositions of buffer systems for ion exchange HPLC are presented in Table 1.

Each peak was collected as several 1 ml fractions and each fraction was analyzed by mass spectrometry. All fractions containing> 90% of the target oligonucleotide were pooled and desalted by precipitation with acetone and ethanol. The results of the analysis showed that the highest signal and symmetric peak in the chromatogram was observed when the nus-1 oligonucleotide was purified using a TOSOH column, while there were no differences between buffer systems 2 and 3. In all cases, the purity of the product was 91-93%, and impurities that could be separated by ion exchange were not detected. The results of mass spectrometric analysis of oligonucleotides after purification by ion exchange chromatography are shown in FIG. 5.

For further work, the TOSOH column was chosen, since during its use the impurities of the Nus-1 oligonucleotide are effectively separated, minimal losses of substance are observed (Table 2), and this column has the maximum capacity among the presented ones. This is important for scaling up oligonucleotide purification, as less sorbent and buffer solutions are required at maximum capacity. As a result, the cost of the process is reduced and the cleaning time is reduced.

To optimize the conditions for reversed-phase chromatography, three columns and two buffer systems were compared.

Columns: Waters Xbdrige Peptide VEN C18 180A 4.6 × 250; Agilent InfinityLab Poroshell 120 EC-C18 4.6 × 100 270A; Phenomenex Jupiter 5u C18 300A 250 × 4.6.

Reverse phase HPLC buffer system compositions are shown in Table 3.

The cleaning results are shown in FIG. 6-8.

Purification by RP HPLC in buffer system 2 using a Waters column provided the minimum amount of impurities and the maximum product yield (Table 4).

The resulting oligonucleotides were stripped off under vacuum, and then precipitated sequentially with acetone and ethyl alcohol to remove ammonium or triethylammonium acetate.

A brief description of the figures of the drawings illustrating the claimed invention is presented as follows.

FIG. 1 shows the data of mass spectrometric analysis of oligonucleotide nus-1 after deblocking with saturated ammonia solution under various conditions: a) 65 ° C, 48 hours; b) 65 ° C, 24 hours; c) 45 ° C, 48 hours; d) room temperature, 72 hours.

FIG. 2 shows the results of the chromatographic separation of the reaction mixtures of the nus-1 oligonucleotide by ion exchange HPLC. Buffer system 1. pH 7. A) Tosoh column, B) Thermo column, C) GE column.

FIG. 3 shows the results of the chromatographic separation of the reaction mixtures of the nus-1 oligonucleotide by ion exchange HPLC. Buffer system 2. pH 7. A) Tosoh column, B) Thermo column, C) GE column.

FIG. 4 shows the results of chromatographic separation of the reaction mixtures of oligonucleotide nus-1 by ion exchange HPLC. Buffer system 3. pH 7. A) Tosoh column, B) Thermo column, C) GE column.

Figure 5 shows mass spectra of pooled pure fractions containing nus-1 oligonucleotide after purification by ion exchange chromatography on a) Tosoh, b) GE, c) Thermo (Nus-1 target mass 7125.0 Da).

FIG. 6.presents the results of chromatographic separation of reaction mixtures of oligonucleotide nus-1 by reverse phase HPLC in buffer system 1 on columns: a) Waters, b) Phenomenex, c) Agilent.

FIG. 7 shows the results of chromatographic separation of reaction mixtures of oligonucleotide nus-1 by reversed-phase HPLC in buffer system 2 on columns: a) Waters, b) Phenomenex, c) Agilent.

FIG. 8. Shows mass spectra of pooled pure fractions containing nus-1 oligonucleotide after purification by reverse phase chromatography on columns: A) Waters, B) Phenomenex, C) Agilent.

The possibility of carrying out the invention can be demonstrated by the examples presented below.

Example 1.

Synthesis of Nus-1 oligonucleotide

The nus oligonucleotide (nus-1 UsCsAsCsUsUsUsCsAsUsAsAsUsGsCsUsGsG) was synthesized on a solid-phase carrier using an automated Mermade 6/12 oligonucleotide synthesizer (Bioautomation, Inc.) according to the amidophosphite scheme. A - 2'-MOE-adenosine, C - 2'-MOE-5-methylcytidine, G - 2'-MOE-guanosine, U - 2'-MOE-5-methyluridine.

MOE - Methoxyethyl.

A. Synthesis of oligonucleotide

Column volume - 20 ml.

Solid-phase support: 4 - (((3aS, 4S, 5R, 6R, 7R, 7aR) -6-hydroxy-2-methyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl) oxy) - 4-oxobutanoylamide attached to controlled pore glass (500 Å).

Mass of solid-phase carrier - glass with controlled pore size with an immobilized first link (Unprotected USS; Labile N-Me Linker-CPG), loading 45-50 μmol / g - 2 g

A total of 17 complete synthetic cycles were carried out: MOE -A = 4,: MOE -C = 4,: MOE -G = 3,: MOE -U = 6, all with thiooxylation.

The synthesis of nus-1 was carried out according to the following cycle for the addition of each monomer:

At the end of the synthesis, post-processing was carried out according to the next cycle.

During each Deblock procedure, there are 4 reagent feeds of 6.00 ml each

In the course of each Coupling procedure, 1 supply of 2.00 ml of a 0.05 M solution of the corresponding amidophosphite and 1 supply of 2.25 ml of a solution of a reaction activator occurs. During each Capping procedure, 1 supply of 3.00 ml of Cap A reagent and 1 supply of 3.00 ml of SarB reagent occurs.

During each Oxidize procedure, 2 feeds of 6.00 ml of reagent occur.

During the Acn Wash procedure, 2 feeds of 8.00 ml of acetonitrile quality for DNA / RNA synthesis occur.

During the Altwash1 procedure, 1 supply of 10 ml of a 10% solution of diethylamine in acetonitrile of Gradient Grade quality occurs.

During the Altwash2 procedure, 1 supply of 8.00 ml of Gradient Grade acetonitrile occurs.

1. Removal of trityl protection - 17 cycles of 8 feeds of 6.00 ml each plus two feeds of 5.00 ml each after the end of the synthesis.

The total volume of Deblock reagent = 17 * 8 * 6 ml + 2 * 5 ml = 136 + 10 = 146 ml.

Reagent composition: 3% trichloroacetic acid in dichloromethane.

Trichloroacetic acid: 0.03 * 282 = 4.38 g.

Dichloromethane: 146 ml.

2. Washing with acetonitrile of quality For DNA / RNA synthesis - 17 cycles, 2 feeds of 8.00 ml each.

Total volume of acetonitrile quality For DNA / RNA synthesis 17 * 2 * 8.00 ml = 272 ml

3. Washing Altwash1 - 2 feeds of 10.00 ml after the end of the synthesis.

Total flush volume Altwash1 20 ml.

Wash composition: 10% diethylamine in Gradient Grade acetonitrile.

Diethylamine: 0.1 * 20 ml = 2 ml.

Acetonitrile Gradient Grade = 20-2 = 18.0 ml.

4. Flushing Altwash2 - 17 cycles of 7 feeds of 8.00 ml plus 3 feeds of 8.00 ml after the end of the synthesis.

Altwash1 total wash volume = 17 * 7 * 8.00 ml + 3 * 8.00 ml = 976 ml.

Reagent composition: PS quality acetonitrile.

Acetonitrile quality PS 976 ml.

5. Blocking of unreacted 5'-hydroxyls.

Reagent SarA - 17 cycles of 3 feeds of 3.00 ml.

Total reagent volume: 17 * 3 * 3.00 ml = 153 ml.

Reagent composition: Gradient Grade acetonitrile: propionic anhydride: distilled pyridine (76:14:10).

Gradient Grade Acetonitrile: 153 ml * 0.76 = 116.3 ml.

Propionic anhydride: 153 ml * 0.14 = 21.4 ml.

Distilled pyridine: 153 ml * 0.10 = 15.3 ml.

Reagent SarB -17 cycles of 3 feeds of 3.00 ml.

Total reagent volume: 17 * 3 * 3.00 ml = 153 ml.

Reagent composition: Gradient Grade acetonitrile: N-methylimidazole (84:16).

Gradient Grade Acetonitrile: 153 ml * 0.84 = 128.52 ml.

N-methylimidazole: 153 ml * 0.16 = 24.48 ml.

6. Thiooxidation - 17 cycles of 2 feeds of 6.00 ml each.

Total reagent volume: 17 * 2 * 6.00 ml = 204 ml.

Reagent composition: 0.02M 3 - [(Dimethylaminomethylene) amino] -3H-1,2,4-dithiazole-5-thione (DDTT) (concentration 4 g / l) in a 30% pyridine solution in acetonitrile of Gradient Grade quality.

DDTT: 0.204 L * 4 g / L = 0.816 g.

Distilled pyridine: 204 ml * 0.3 = 61.2 ml.

Gradient Grade Acetonitrile: 204 ml * 0.7 = 142.8 ml

7. Condensation reaction MOE -A

Monomer: 3'-O - ((2-cyanoethyl) -N, N-diisopropylamidophosphite N ⁶ -benzoyl-5'-O- (4,4'-dimethoxytrityl) -2'-O- (2-methoxyethyl) adenosine- FW = 932.01.

Reagent composition: 0.05 M monomer solution in acetonitrile quality for DNA / RNA synthesis.

Consumption per cycle: 4 feeds of 2.00 ml = 8.00 ml.

Total consumption: 4 * 8.00 ml = 32.00 ml.

Acetonitrile quality for DNA / RNA synthesis: 32.0 ml

3'-O - ((2-cyanoethyl) -N, N-diisopropylamidophosphite N ^6- benzoyl-5'-O- (4,4'-dimethoxytrityl) -2'-O- (2-methoxyethyl) adenosine: 932, 01 g / M * 0.05M * 0.032 = 1.49 g

8. Condensation reaction of methoxyethyl (MOE) -C

Monomer: 3'-O - ((2-cyanoethyl) -N, N-diisopropylamidophosphite N ^4- benzoyl-5'-O- (4,4'-dimethoxytrityl) -2'-O- (2-methoxyethyl) cytidine FW = 922.01

Reagent composition: 0.05 M monomer solution in acetonitrile quality for DNA / RNA synthesis.

Consumption per cycle: 4 feeds of 2.00 ml = 8.00 ml.

Total consumption: 4 * 8.00 ml = 32.00 ml.

Acetonitrile quality for DNA / RNA synthesis: 32.0 ml.

3'-O - ((2-cyanoethyl) -N, N-diisopropylamidophosphite N ^4- benzoyl-5'-O- (4,4'-dimethoxytrityl) -2'-O- (2-methoxyethyl) cytidine 922.01 g / M * 0.05M * 0.032 = 1.48 g.

9. Condensation reaction MOE -G

Monomer: 3'-O - ((2-cyanoethyl) -N, N-diisopropylamidophosphite N ² -isobutyryl-5'-O- (4,4'-dimethoxytrityl) -2'-O- (2-methoxyethyl) guanosine FW = 913.99.

Reagent composition: 0.05 M monomer solution in acetonitrile quality for DNA / RNA synthesis.

Consumption per cycle: 4 feeds of 2.00 ml = 8.00 ml. Total consumption: 3 * 8.00 ml = 24.00 ml.

Acetonitrile quality for DNA / RNA synthesis: 24.0 ml.

3'-O - ((2-cyanoethyl) -N, N-diisopropylamidophosphite N ² -isobutyryl-5'-O- (4,4'-dimethoxytrityl) -2'-O- (2-methoxyethyl) guanosine 913.99 * 0.05 * 0.024 = 1.10 g.

10. Condensation reaction MOE -U

Monomer: 3'-O - ((2-cyanoethyl) -N, N-diisopropylamidophosphite 5'-O- (4,4'-dimethoxytrityl) -5-methyl-2'-O- (2-methoxyethyl) uridine FW = 818.89.

Reagent composition: 0.05 M monomer solution in acetonitrile quality for DNA / RNA synthesis.

Consumption per cycle: 4 feeds of 2.00 ml = 8.00 ml.

Total consumption: 6 * 8.00 ml = 48.00 ml.

Acetonitrile quality for DNA / RNA synthesis: 48.0 ml

3'-O - ((2-cyanoethyl) -N, N-diisopropylamidophosphite 5'-O- (4,4'-dimethoxytrityl) -5-methyl-2'-O- (2-methoxyethyl) uridine: 818.89 * 0.05 * 0.048 = 1.97 g.

11. Activation of the condensation reaction.

Activator: 5-ethylthiotetrazole FW = 130.17 - 0.25M solution in acetonitrile for DNA / RNA synthesis.

Consumption per cycle: 4 feeds of 2.25 ml = 10.00 ml.

Total consumption: 17 * 10.00 ml = 170.00 ml.

Acetonitrile quality for DNA / RNA synthesis: 170.0 ml

5-ethylthiotetrazole: 130.17 * 0.25 * 0.17 = 5.53 g.

B. Unblocking oligonucleotide

After the end of the synthesis, the solid-phase support was transferred into 15 ml screw-top test tubes, 10 ml of saturated ammonia solution (25-27% aqueous solution) was added, several crystals of ethylenediaminetetraacetic acid disodium salt - EDTA were added, and stirred for 3 days at room temperature. After the end of unblocking, the tubes were cooled for 30 minutes at -20 ° C, the precipitate was filtered off and washed with MilliQ quality water (3 * 7 ml). The combined solutions were then diluted with milliQ quality water (12 ml) and evaporated to dryness using a Labconco Centrivap centrifugal evaporator. The resulting dry precipitate was dissolved in 5 ml of milliQ quality water and centrifuged at 13400 rpm for 10 minutes (Eppendorf Centrifuge 5804), then the nus-1 solution was transferred to a clean tube and stored until purified on HPLC at -20 ° C.

Deblocking reagent composition: saturated aqueous ammonia solution,

Total reagent volume: 10 ml

MilliQ quality water quantity:

Total volume 3 * 7 + 12 ml = 33 ml

B. Chromatographic purification of oligonucleotides by ion exchange HPLC

The oligonucleotides were purified using a Dionex Ultimate 3000 HPLC system with Tosoh Super-Q 5PW 21.5 mm × 150 mm columns according to the manufacturer's recommendations. For this, a solution of the oligonucleotide in water (5 ml) was applied to the column. No more than 50% of the substance was loaded onto the column at a time. For elution of the oligonucleotide, a gradient of 5-50% buffer B was used (Buffer A - 20 mM Tris-HCl (pH 7.0), 10% acetonitrile in water, buffer B -20 mM Tris-HCl (pH 7.0), 600 mM sodium perchlorate, 10 % acetonitrile in water) for 300 minutes at a flow rate of 1 ml / min. Were collected all fractions with absorption above 200 mAu at 260 nm, the purity of the oligonucleotide in each was verified by mass spectrometry. Further, all fractions in which the oligonucleotide content exceeded 90% were combined and evaporated by about 10 times. Next, a tenfold volume of acetone was added to the nus-1 solution, the mixture was thoroughly mixed and cooled at -20 ° C for 1 hour. Then, the oligonucleotide was isolated by centrifugation for 10 min at 13000 g, the precipitate was dried from acetone residues using a centrifugal evaporator and dissolved in milliQ water (1 ml).

One purification requires 300 * 2.5 = 750 ml of buffer (550 ml of buffer A and 200 ml of buffer B):

Acetonitrile 75 ml, milliQ quality water = 750-75 = 675 ml,

Tris = 121.14 g / M * 0.02 M * 0.75 = 1.82 g

HCl = 69 ml * 0.02 M * 0.75 = 1.03 ml

Sodium perchlorate 122.45 g / M * 0.8 M * 0.2 L = 19.52 g

Acetone = 100 ml

Total consumption:

HPLC quality acetonitrile: 75 ml * 2 = 150 ml

MilliQ quality water: 675 ml * 2 = 1350 ml

Tris: 1.82g * 2 = 3.64g

HCl: 1.03 ml * 2 = 2.06 ml

Sodium perchlorate: 19.52 g * 2 = 39.04 g

Acetone: 100 ml * 2 = 200 ml

D. Chromatographic purification of oligonucleotides by reversed-phase HPLC

The nus-1 oligonucleotide was purified using an Agilent Infinity 1260 HPLC system with a Waters xBridge BEN C18 10 mm × 250 mm column according to the manufacturer's recommendations. For this, a solution of the oligonucleotide in water (1 ml) was applied to the column. To elute the oligonucleotide, a 0-100 gradient of buffer B was used (Buffer A - 0.05 M ammonium acetate solution in water (pH 7.0), Buffer B - 0.05 M ammonium acetate solution in 80% aqueous acetonitrile solution (pH 7.0) for 60 minutes at a rate flow rate of 2.5 ml / minute All fractions with absorption above 500 mAu at 260 nm were collected, the purity of the oligonucleotide in each was checked by mass spectrometry. Then all fractions in which the purity of the oligonucleotide exceeded 95% were combined and evaporated to volume 10 ml Next, a tenfold volume of ethanol (100 ml) was added to the oligonucleotide solution, the mixture was thoroughly mixed and cooled at -20 ° C for 1 hour. Then nus-1 was isolated by centrifugation for 10 min at 13000 g, the precipitate was dried from ethanol residues using a centrifugal evaporator and dissolved in milliQ quality water (1 ml).

One RP HPLC purification requires 60 * 2.5 = 150 ml of buffer (75 ml of buffer A and 75 ml of buffer B):

Acetonitrile HPLC quality 60 ml, milliQ water 90 ml, ammonium acetate 0.5 g, ethanol 100 ml.

Total consumption:

HPLC quality acetonitrile 60 ml * 2 = 120 ml

milliQ quality water 90 ml * 2 = 180 ml

ammonium acetate 0.5 g * 2 = 1 g

ethanol 100 ml * 2 = 200 ml

E. Gel filtration desalination of the oligonucleotide

Purification of oligonucleotides was carried out using a Dionex Ultimate 3000 HPLC system with a GE Healthcare HiPrep 26/10 Desalting 10 cm × 26 mm column. Gel filtration was carried out in milliQ water. 15 ml was applied at a rate of 5 ml / min. Elution was carried out with water, monitoring the output signal with a diode-array detector at a wavelength of 260 nm and with a conductometric detector. All fractions with an absorbance above 200 mAu and a conductivity of no more than 30 mS / cm were collected, the purity of the oligonucleotide in each fraction was verified by mass spectrometry. Further, all fractions in which the oligonucleotide content exceeded 95% were combined, evaporated on a centrifugal evaporator, and redissolved in 10 ml of water to measure the oligonucleotide concentration.

One cleaning requires 60 ml of milliQ quality water.

G. Determination of the amount of nus-1

The oligonucleotide concentration was determined spectrophotometrically. For this, an aliquot (2 μl) was taken from the solution obtained at the previous stage, dissolved in milliQ water (1 ml), and the concentration was measured using a Nanodrop 2000 instrument (Thermo Scientific).

Example 2.

Therapeutic efficacy and safety of using the described compound obtained according to Example 1

The efficacy was studied in an animal model of spinal muscular atrophy.

Research was carried out on the model of the so-called. SMA mice (SMA - spinal muscular atrophy), normal heterozygous mice without SMA were taken as a control group. SMA mice are mice that have a gene encoding a survival motor neuron (SMN) protein, similar to the SMN1 gene. The SMN gene encodes a protein that is required for motor neurons to function. The absence of this gene and / or incorrect operation leads to the death of spinal motor neurons. A very similar structure of the SMN1 gene, called SMN2, exists in humans and is responsible for the severity of the disease. The molecular biological basis of SMA (by an autosomal recessive type), and there is a homozygous loss of the SMN1 gene. Mouse models have only one gene (SMN) that is equivalent to the SMN1 gene. Homozygous loss of this gene is critical for embryos and provokes massive death of motor neurons, which indicates that the expression of the SMN gene is important for the survival and functioning of cells.

In mice with SMA (of the rigid type) immediately after birth, the resulting compound was injected daily for 5 days by intrathecal injections at doses of 0.5 mEq / kg, 1 mEq / kg or 1.5 mEq. kg or saline every day, or control group without MCA saline intrathecal (5 MCA, 6 MCA control, 5) (6 MCA, 5 control, 4) and (5 MCA, 5 MCA control, 6) in an experimental study to assess survival ... The 0.5 mg / kg dose had, but slightly lower than the other two doses, an effect on the survival of mice with the SMA model. Mice with SMA that received physical. solution, died by the 12th day after birth. Exposure to the studied compound significantly increased lifespan by 1.6 times, despite the fact that the change in the median survival of mice with SMA (15.9 versus 11.4 days; Mantel-Cox test, p = 0.0003). A single SMA mouse survived for over a month and a full 31 days later and was sacrificed. These data indicate that the compound under study statistically affects survival in the SMA mouse model.

Distinctive changes in body weight structure were observed in SMA mice treated with the test substance; Mice with SMA, when administered with the test substance, demonstrated moderate sustained weight gain, reaching a plateau by day 18. While mice with SMA receiving saline significantly slowly gained weight after birth, and after the 5th day they could lose weight. On the 8th day of the experiment, a reflex reflex and a suspension test of the hind paws were performed and an improvement in the motor behavior of animals with SMA, which received 1 and 1.5 mEq / kg daily, was found, compared to SMA mice that received saline. Open field tests were also carried out starting from the 8th day of the experiment to assess the muscle strength of the forelimbs. SMA mice treated with 1 and 1.5 mEq / mg acted close to the intact group in the antigravity hover assay (Tyuki two-way ANOVA variance was insignificant).

All groups of mice showed some degree of mobility in the "open field" test, which was carried out between the 8th and 10th days from the beginning of the experiment. On the 12th day, the so-called. a day of autonomous walking, the test substance groups and control mice showed increased activity by walking and crossing more squares. A group of SMA mice exhibited small ambulatory movements. The test substance clearly improved the motility of SMA mice, as evidenced by the increased number, as evidenced by the increased number of crossings, similar to the control before day 22 of the experiment. These data indicate that the test substance has an effect on the decreased activity of SMA mice.

After the end of the experiment, histological sections of lumbar neurons were made using an immunofluorescent label.

Thus, an attempt was made to determine the effect of the test substance on the localization of the SMN (survival motor neuron) protein in the nuclear bodies of mice spinal cord motor neurons. Immunofluorescent labeling of histological preparations of cross sections of the lumbar spinal cord with antibodies to coilin in Cajal bodies and to SMN led to the fact that in the treated nat. solution of SMA mice, there were fewer SMN-positive Cajal bodies than control ones. SMA mice treated with the test substance showed a 1.7-fold increase in SMN-positive Cajal bodies in motor neurons. SMN is also present in other nuclear bodies that are devoid of coilin. The experimental observation results demonstrate that the test substance fixes SMN in the nuclear bodies of motor neurons in SMA mice.

Then, using RT-PCR-RT, we estimated the levels of splicosomal snRNA in sections from the brain and spinal cord of mice treated with the test substance and saline. Decreases in snRNA levels were observed in SMA mice, including the minor spliceosomal U12, which was reduced to 1/3 percent in the brain and spinal cord compared to the corresponding controls. The administration of the test substance positively affected the levels of U2, U4, U5 and U6 in the spinal cord and snRNA U1 in the brain and spinal cord of SMA mice. The data from these experiments show that the test substance can also regulate the relative ratio between snRNAs.

Claims (1)

A method of obtaining a modified thiophosphate oligonucleotide containing 2'-O- (2-methoxyethyl) ribonucleotides of the structure UCACUUUCAUAAUGCUGG, where each nucleoside contains a 2'-O- (2-methoxyethyl) modified sugar residue, characterized in that the oligonucleotide is synthesized on a solid-phase support amidophosphite method, in which 3 - [(dimethylaminomethylene) amino] -3H-1,2,4-dithiazole-5-thione (DDTT) in a 30% solution of pyridine in acetonitrile is used to obtain thiophosphate groups, deblocking is carried out by adding a saturated solution of ammonia to in the presence of ethylenediaminetetraacetic acid disodium salt for 3 days at room temperature, the oligonucleotides are sequentially purified by ion exchange chromatography on a Tosoh Super-Q 5PW column using Tris-HCl buffer, pH 7.0, with the addition of a 10% acetonitrile solution in water, and then Tris-HCl buffer, pH 7.0, with the addition of sodium perchlorate and 10% acetonitrile solution in water, by phase chromatography on a Waters xBridge BEN C18 column using a buffer, which is a solution of triethylammonium acetate in water, pH 7.0, and then a buffer, which is a solution of triethylammonium acetate in an 80% aqueous solution of acetonitrile, pH 7.0, and the method of gel filtration with subsequent isolation of the target product upon drying by lyophilization.

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