RU2644675C1 - Composition for inhibiting growth and stimulation of apoptosis of colorectal cancer cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to a composition for inhibiting growth and stimulating apoptosis of cancer cells of colorectal cancer by blocking the function of the MCM4 and Livin genes. Specified composition comprises of a liposome for delivering the composition to target cells and a combination of two duplexes of oligonucleotide siRNA. First duplex is designed to block the genes of MCM4 and contains preparations of small interfering oligonucleotides 5'-C-U-C-A-Umet-C-U-C-U-Umet-A-C-C-C-A-C-A-G-G-dTs-dT-3' as a direct chain and 5'-C-C-U-G-U-G-G-G-Umet-A-A-G-A-G-A-Umet-G-A-G-dTs-dT-3' as an inverse chain. Second duplex is designed to block the genes of Livin and contains preparations of small interfering oligonucleotides 5'-G-G-A-A-G-A-G-A-C-U-Umet-U-G-U-C-C-A-Cmet-A-dTs-dT-3' as a direct chain and 5'-U-G-U-G-G-A-Cmet-A-A-A-G-U-C-Umet-C-U-U-C-C-dTs-dT-3' as an inverse chain. Umet - 2'-O-methyluridin-3'-phosphate; Cmet - 2'-O-methylcytidine-3'-phosphate; dTs - 2'-deoxythymidine-3'-phosphorothioate; dT - 2'-deoxythymidine-3'-phosphate.

EFFECT: invention makes it possible to increase the efficiency of inhibiting the growth and induction of apoptosis in malignant tumor cells, while reducing the side effects.

6 cl, 3 dwg

Description

The invention relates to medicine, in particular to a means of treating cancer, as well as the transport of nucleic acids into cells.

One of the main therapeutic approaches to the treatment of cancer is the use of antitumor chemotherapeutic agents, in particular oxaliplatin, which refers to cytostatic antitumor chemotherapeutic drugs. As a result of exposure to the drug, the cancer cell undergoes apoptosis. Platinum drugs, like many other chemotherapeutic drugs, have a high level of toxicity, in particular, platinum drugs are characterized by high neurotoxicity, which results in peripheral neuropathy, as well as cases of deafness, renal failure, leukopenia, thrombocytopenia, anemia, etc. d. In addition, often they are not effective enough, have a significant mutagenic effect, drug resistance develops to them.

To date, colon cancer is the third largest mortality rate among all tumor diseases. Traditional approaches to treating cancer are not always applicable. An alternative and fairly new method of treating tumor diseases is gene therapy, which is based on the introduction of therapeutic nucleic acids (NKs) into the tumor. The development of drugs with an insignificant spectrum of side effects, but capable of providing the same or more significant therapeutic effect seems to be one of the priority tasks in this area. In this regard, in the last decade, a fundamentally new direction has been actively developing for the creation of targeted drugs, the action of which is aimed at regulating the functional activity of certain genes. Typically, such drugs are antibodies against specific proteins or small molecules that often act as inhibitors. Their disadvantages include incomplete selectivity, which contributes to an increase in the toxic effect, especially in cases of small molecules. Antibodies are components introduced into the body that are capable of exhibiting additional activities as a result of the formation of non-specific complexes (cross-reactions). The scope of monoclonal antibodies used for targeted therapy is limited by the barrier function of the cell membrane, which makes it impossible to use them for targeted action on intracellular structures.

A significant part of modern targeted drugs are small molecules. Their main drawback is the lack of specificity, which leads to the appearance of additional effects causing toxicity.

Unlike small molecules, siRNAs are natural substances that function in human cells (and other mammals). Artificially introduced siRNAs are not substances foreign to the body. Both natural and synthesized siRNAs have the same structure, which allows them to affect precisely those genes whose function should be inhibited. They are small in size, facilitating their efficient delivery to the cancer cell. Their main function is to block protein synthesis by inactivating the gene at an early stage and making its phenotypic manifestation impossible, which, in turn, has a beneficial effect on the patient's body as a whole. In addition, for RNA gene interference, a relatively small amount of foreign substances must be introduced into the recipient's body.

Targeted drugs designed specifically for use with standard chemotherapeutic agents are currently not available. In this regard, the creation of new targeted drugs and the search for alternative therapeutic approaches are relevant. One of these approaches is the identification of genes, the inhibition of which can lead not only to a decrease in the viability of a cancer cell, but also to an increase in the sensitivity of the tumor to standard chemotherapeutic treatment. The invention is based on new approaches based on the latest achievements of the “postgenomic era”.

A known method for modulating the function and / or expression of a tumor suppressor gene in mammalian cells or tissues in vivo or in vitro, comprising interacting these cells or tissues with small interfering RNAs (siRNAs) of 5 to 30 nucleotides in length that specifically recognize tumor suppressor genes. Tumor suppressor gene (anti-oncogen, tumor suppressor) is a gene that provides a “loss of function” (suppression) mutation during tumor development (Haber D & Harlow Ed., 1997). The protein products of suppressor genes are called suppressor proteins or anti-oncoproteins. In addition, anti-oncogenes can encode microRNAs (RU No. 2011127196).

Genetic engineering constructs are known that express vectors carrying a multifunctional promoter linked to specific transcribed sequences that, when transcribed, produce informational RNA that encodes an enzyme that, as a result of post-translational processing, is converted from a non-toxic peptide (prodrug) into an intracellular toxin (drug) that kills the cell . The expression vector is a viral vector or non-viral vector (RU No. 2476596).

A means of inducing apoptosis in a target cell is known in which a double-stranded RNA molecule is capable of target-specific RNA interference. RNA fragments complementary to each other have a length of 23 nucleotide residues and form a duplex with protruding 3'-ends with a length of 1 to 5 nucleotides (RU No. 2470073).

A known pharmaceutical composition comprising a mitosis disintegrator / inhibitor of the biochemical pathway of polo-like kinase (Plk), in particular ON01910, ON01910-Na or siRNA directed to Plk1, and a chemotherapeutic agent, which is a nucleotide analogue, in an effective amount, as well as pharmaceutically and physiologically acceptable carrier, for use as a medicine to prevent the development of resistance to a chemotherapeutic agent, which is a nucleotide analogue, in a subject suffering from ohm, or for use as a medicament for the treatment of cancer that is resistant to the chemotherapeutic agent is a nucleotide analog, in a patient in need thereof. A chemotherapeutic agent is a nucleotide analog selected from the group consisting of cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine, capecitabine, cytarabine, decitabine, fluorouracil, phloxuridine, sapacitabine and gemcitabine; or a chemotherapeutic agent is a nucleotide analogue, is gemcitabine (RU No. 2476239).

The disadvantages of these known methods and means of inducing apoptosis by cells of malignant tumors are insufficient effectiveness, a long treatment period until significant results are obtained, a high probability of side effects characteristic of chemotherapeutic drugs, due to the fact that in the prototype they provide and evaluate a decrease in the number of cells, which may be associated with a decrease in proliferation, i.e. cancer cell division rate. By blocking the gene, they provide increased sensitivity to the antitumor drug when using the same therapeutic doses. This is positioned as overcoming resistance. The task of reducing the dose of chemotherapy is not solved. Apoptotic death of tumor cells by the used means is not provided in a measure sufficient for treatment and is not determined. The use of small molecules to inhibit genes may have insufficient specificity, which will lead to significant undesirable non-specific effects causing additional toxicity.

Closest to the present technical solution is a composition for inducing apoptosis of colorectal cancer cells, containing oxaliplatin and a combination of miRNA cells complementary to mRNA - oligonucleotides in the form of short loop siRNAs of at least one of the following structures:

(Sense oligo) -CAAGAGA- (Antisense oligo), or (Sense oligo-dtdt) - (Sense oligo) -CAAGAGA- (Antisense oligo-dtdt) - (Antisense oligo), where Sense oligo and Antisense oligo are pairs of oligonucleotides for each gene, (Sense oligo-dtdt) and (Antisense oligo-dtdt) - similar RNA sequences in pairs of oligonucleotides without dtdt, or (Antisense oligo-dtdt) - (Antisense oligo) -CAAGAGA- (Sense deoxyoligo-dtdt) - (Sense deoxyoligo ), or CG- (Antisense deoxyoligo-dtdt) - (Antisense deoxyoligo) -CAAGAGA- (Sense deoxyoligo-dtdt) - (Sense deoxyoligo) -TTTTTTGGAAA, where Antisense deoxyoligo and Sense deoxyoligo are nucleotide sequences replaced by ribonucleotides with deoxyribo nucleotides Antisense deoxyoligo-dtdt) and (Sense deoxyoligo-dtdt) - similar sequence and DNA in oligonucleotide pairs without dtdt.

In this case, (Sense oligo) and (Antisense oligo) are selected in accordance with the inhibited gene from the following:

(RU No. 2551238, prototype).

The disadvantage of the prototype is immunotoxicity, due to the interaction of siRNA oligonucleotides in the form of short loop siRNAs (shRNAs) with toll-like receptors (English Toll-like receptor, TLR), as well as incomplete selectivity, which increases the toxic effect.

An object of the invention is to provide an effective means of inhibiting the growth and induction of apoptosis of malignant tumor cells with fewer side effects, as well as expanding the arsenal of means of inducing apoptosis of colorectal cancer malignant tumor cells.

The technical result, which provides a solution to the problem, is to reduce immunotoxicity, since the combination of modified and unmodified nucleotides developed according to the invention is unique for each of the oligonucleotides and reduces their affinity for toll-like receptors (English Toll-like receptor, TLR), thereby providing decreased immunogenicity. The use of small interfering RNAs (miRNAs) selected in accordance with the claimed solution to suppress selected target genes is the initial basis for a technical result that can significantly reduce the immunotoxicity of a means of inhibiting the growth and induction of malignant tumor cells while reducing the dosage of a chemotherapeutic drug - a platinum derivative and, accordingly, reduce minimize its side effect while increasing the effectiveness of therapy. Thus, a drug was created with fewer side effects, but capable of providing the same or greater therapeutic effect.

The essence of the invention lies in the fact that the composition for inhibiting the growth and stimulation of apoptosis of colorectal cancer cells by blocking the function of the genes: MCM4 and Livin in cancer cells contains a means for delivering the composition to the target cells and a combination of siRNA oligonucleotides,

of which, to block the genes of MSM4, it contains preparations of small interfering oligonucleotides:

5'-C-U-C-A-Umet-C-U-C-U-Umet-A-C-C-C-A-C-A-G-G-dTs-dT-3 'as the passenger chain and

5'-C-C-U-G-U-G-G-G-Umet-A-A-G-A-G-A-Umet-G-A-G-dTs-dT-3 'as a guide (interacting with the target RNA) of the chain,

and to block Livin genes, it contains preparations of small interfering oligonucleotides: 5'-G-G-A-A-G-A-G-A-C-U-Umet-U-G-U-C-C-A-Cmet-A-dTs-dT-3 'as a passenger chain and

5'-U-G-U-G-G-A-Cmet-A-A-A-G-U-C-Umet-C-U-U-C-C-dTs-dT-3 'as a guide (interacting with the target RNA) of the chain,

where G, C, A, U are ribonucleotides;

Umet-2 '- O-methyluridine-3'-phosphate (2'-O-methyluridine-3'-phosphate);

Cmet-2 '- O-methylcytidine-3'-phosphate (2'-O-methylcytidine-3'-phosphate);

dTs -2 '- deoxythymidine-3'-phosphorothioate (2'-deoxythymidine-3'-phosphorothioate);

dT-2 '- deoxythymidine-3'-phosphate (2'-deoxythymidine-3'-phosphate).

Moreover, the preparations of small interfering siRNA oligonucleotides are made in the form of duplexes of the forward and reverse complementary nucleotide chains of the following structure:

for blocking genes MSM4 duplex

and for blocking Livin duplex genes

where Gp, Cp, Ap, Up are ribonucleotides;

U ^m p - 2'-O-methyluridine-3'-phosphate (2'-O-methyluridine-3'-phosphate);

C ^m p - 2'-Omethylcytidine-3'-phosphate (2'-O-methylcytidine-3'-phosphate);

dTsp - 2'-deoxythymidine-3'-phosphorothioate (2'-deoxythymidine-3'-phosphorothioate);

dTp - 2'-deoxythymidine-3'-phosphate (2'-deoxythymidine-3'-phosphate).

Preferably, as a means for delivering the composition to the target cells, it contains a liposome, which is a polycationic lipid as a component that binds and packs nucleic acid, and neutral phospholipids (DOPE and PC) as structure-forming and promoting components, with duplex: liposome ratios from 1: 2 to 1:12.

Preferably, the means for delivering the composition to the target cells is in the form of a lipid film consisting of a polycationic lipid and a neutral phospholipid, hydrated in water to obtain an emulsion-dispersion system, subjected to ultrasonic treatment or extrusion, and the polycationic lipid, DOPE and PC are taken in the ratio 50: 45: 5 (mol.).

Preferably, as a means for delivering the composition to the target cells, it contains a polycationic lipid in an amount of from 25 to 67 mol. % on the total amount of lipids in the composition; polycationic lipid is selected from the group:

- {2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetate}, or DOSPA (Lipofectamine)

- Di-octadecyl-amido-glycyl-spermine, or DOGS, Transfectam.

- 3-β (N- (N ', N'-dimethyl, N'-hydroxyethyl aminopropane) carbamoyl) cholesterol iodide (DMHAPC-Chol).

- 3β [N- (N ', N'-dimethylaminoethane) -carbamoyl] cholesterol, or DC-Chol.

- 1,26-bis (cholest-5-en-3β-yloxycarbonylamino) -7,11,16,20-tetraazagexacosane tetrahydrochloride (2X3).

The composition is made in a pharmaceutical form suitable for parenteral administration with the goal of inducing apoptosis of colorectal cancer cells.

In FIG. 1 depicts the sequence of steps for the synthesis of siRNA, FIG. 2 - data on the efficiency of transfection depending on the type of vector and the degree of its loading, in FIG. 3 - data on the dependence of the biological effect on the dose of the drug.

The invention is based on a modern approach to the creation of effective antitumor drugs with low toxicity. Of particular interest are modern developments on the use of RNA interference in combination with liposomal methods for the delivery of small interfering RNA into cells. Small interfering RNAs (siRNAs) are short double-stranded RNA fragments (21-23 base pairs), complementary to the protein mRNA sequence, the synthesis of which should be blocked. The therapeutic use of siRNA is based on the existing understanding of the mechanism of action of the RNA-induced gene shutdown complex (RISC) and Dicer endonuclease.

siRNAs are natural substances that function in human cells (and other mammals). Artificially introduced siRNAs will not be substances foreign to the body. Both natural and synthesized siRNAs have absolute specificity, which allows them to affect precisely those genes whose function should be inhibited. They are small in size, facilitating their efficient delivery to the cancer cell. In addition, siRNAs interrupt the process of functional manifestation of genes at an earlier stage, before the synthesis of proteins, the amount of which is significantly greater than the matrix RNA. As a result, smaller amounts of substances introduced into the patient's body are required.

At the same time, problems in the use of siRNAs (as well as other nucleic acids) include their ability to interact with toll-like receptors, which can cause an immune response, as well as the possibility of degradation due to the action of intracellular enzymes.

In the human body, there are extra- and intracellular barriers that do not allow NK to independently penetrate to the place of their therapeutic action. For the introduction of NK, viral delivery systems can be used. However, such systems have a number of disadvantages, such as immunogenicity, mutagenicity, the limited size of the transferred NK, difficulties in scaling, reproduction, stability, and high cost. Alternative non-viral delivery systems of NK, which today are widely used, are cationic liposomes. Due to the electrostatic interaction, they can compact different sizes of nanocrystals, forming complexes - lipoplexes. Cationic liposomes are non-immunogenic, non-mutagenic, storage stable, relatively cheap compared to viral delivery systems.

In a model of cultured colorectal cancer cells, the fundamental possibility of inducing an apoptotic effect upon inactivation of the Livin and MCM4 genes has been previously shown. It was found that joint inhibition of genes gives a synergistic apoptotic effect at low doses of oxaliplatin. The results open up the possibility of developing a drug based on small interfering RNAs for the Livin and MCM4 genes for the treatment of colon cancer.

The proposed composition includes created miRNA oligonucleotides and cationic liposomes. In solving the above problems of the use of siRNAs in the development of their design and composition, the need to increase the resistance to the effects of nucleases and reduce the immunogenicity of siRNA oligonucleotides was taken into account, which is one of their technical development results. For this purpose, 2'-deoxythymidine-3'-phosphorothioate was introduced into the sequence of siRNA oligonucleotides at the position of the 20th nucleotide, which, through the remainder of phosphoric acid, is bound to the terminal 2'-deoxythymidine-3'-phosphate at the 21st position. Such a structure ensures the resistance of siRNA oligonucleotides and their duplexes to the action of nucleases. In addition, modified bases were introduced into the primary structure of oligonucleotides: 2'-OMe-uridine-3'-phosphate and 2'-OMe-cytidine-3'-phosphate. The introduction of modified nucleotides enhances the resistance to hydrolysis inside nucleotide phosphorus bonds, and, consequently, the entire nucleotide complex. For each of the oligonucleotides, a different scheme of alternating phosphodiester bonds between ribose (P), deoxyribose (DR) and 2'OMeribose (MeP) has been developed:

Oligonucleotide No. 1

5'-R-R-R-R-MeR-R-R-R-R-MeR-R-R-R-R-R-R-R-R-R-DR-DR-3 '

Oligonucleotide No. 2

5'-R-R-R-R-R-R-R-R-R-MeR-R-R-R-R-R-R-MeR-R-R-DR-DR-3 '

Oligonucleotide No. 3

5'-R-R-R-R-R-R-R-R-R-R-MeR-R-R-R-R-R-R-MeR-R-DR-DR-3 '

Oligonucleotide No. 4

5'-R-R-R-R-R-R-MeR-R-R-R-R-R-R-MeR-R-R-R-R-R-DR-DR-3 '

When modifying the oligonucleotides, it was taken into account that nucleotide residues at positions 2–6 in the guiding oligonucleotides No. 2 and No. 4 cannot be changed, since they are the seed region (seed region) that provides the initial binding of the protein complex to the target RNA sequence. For oligonucleotides No. 1 and No. 3, the position of the modified nucleotide residue was determined based on the requirement that the modified nucleotide residues of the direct sequence should not pair with the modified nucleotide residues of the complementary sequence. Oligonucleotides No. 1 and No. 3 do not participate in the recognition of targeted RNA ("passenger" chain). Since the modified nucleotide changes the binding energy of the nucleotide pair, it was postulated that there should be at least three unmodified nucleotide residues between the modified nucleotide residues within the oligonucleotide.

As a result, the following siRNA oligonucleotides for inhibiting the MCM4 and Livin genes were obtained:

Oligonucleotide No. 1 - MSM4, "passenger" chain

5'-C-U-C-A-Umet-C-U-C-U-Umet-A-C-C-C-A-C-A-G-G-dTs-Dt-3 '

Oligonucleotide No. 2 - MSM-4, directing (interacting with the target RNA) chain

5'-C-C-U-G-U-G-G-G-Umet-A-A-G-A-G-A-Umet-G-A-G-dTs-dT-3 '

Oligonucleotide No. 3 - Livin, the "passenger" chain

5'-G-G-A-A-G-A-G-A-C-U-Umet-U-G-U-C-C-A-Cmet-A-dTs-dT-3 '

Oligonucleotide No. 4 - Livin, directing (interacting with the target RNA) chain

5'-U-G-U-G-G-A-Cmet-A-A-A-G-U-C-Umet-C-U-U-C-C-dTs-dT-3 '

where G, C, A, U are ribonucleotides;

Umet - 2'-O-methyluridine-3'-phosphate (2'-O-methyluridine-3'-phosphate);

Cmet - 2'-Omethylcytidine-3'-phosphate (2'-O-methylcytidine-3'-phosphate);

dTs - 2'-deoxythymidine-3'-phosphorothioate (2'-deoxythymidine-3'-phosphorothioate);

dT - 2'-deoxythymidine-3'-phosphate (2'-deoxythymidine-3'-phosphate).

The developed combination of modified and unmodified nucleotides for each of the oligonucleotides is unique and allows one to obtain miRNAs and their duplexes with altered stereochemical characteristics, which, in addition to increasing resistance to the action of nucleases, reduces their affinity for toll-like receptors (English Toll-like receptor, TLR), providing thereby reducing the immunogenicity of the drug. In mammalian cells, 2'OMe ribonucleotides are found in ribosomal and transport RNAs and are not toxic.

The resulting siRNA duplexes were evaluated for functional activity using the commercial Lipofectamine RNAiMAX vector (Invitrogen, USA) and HT-29 colon carcinoma cell culture (APC - / -). Transfection was performed by a combination of Livin + MCM4 duplexes in a 1: 1 ratio by direct method according to the manufacturer's protocol. Transfection results were analyzed by fluorescence microscopy using a standard apoptosis test kit (Vybrant Apoptosis Assay Kit # 5, Invitrogen, USA).

The study allows us to conclude that the developed design and composition of siRNA oligonucleotides do not significantly affect the final effect and can be used to obtain the apoptosis effect.

The miRNA oligonucleotides were synthesized by the amidophosphite triester method using the ASM-1000 solid-phase DNA synthesizer (Bioset, Novosibirsk) according to a scheme that included the following steps (synthesis steps):

- removal of trityl protection;

- condensation;

- blocking unreacted 5'-hydroxides;

- oxidation;

The second step of the synthesis:

- removal of trityl protection and washing;

- condensation;

- blocking unreacted 5'-hydroxides;

- sulfonation;

The third step of the synthesis:

- removal of trityl protection and washing;

- condensation;

- blocking unreacted 5'-hydroxides;

- oxidation;

Then the sequential buildup of the oligonucleotide: 4-21 synthesis step, similar to the first synthesis step.

Oligonucleotide No. 1

5'-C-U-C-A-Umet-C-U-C-U-Umet-A-C-C-C-A-C-A-G-G-dTs-Dt-3 '

Oligonucleotide No. 2

5'-C-C-U-G-U-G-G-G-Umet-A-A-G-A-G-A-Umet-G-A-G-dTs-dT-3 '

Oligonucleotide No. 3

5'-G-G-A-A-G-A-G-A-C-U-Umet-U-G-U-C-C-A-Cmet-A-dTs-dT-3 '

Oligonucleotide No. 4

5'-U-G-U-G-G-A-Cmet-A-A-A-G-U-C-Umet-C-U-U-C-C-dTs-dT-3 ';

And obtaining the target intermediate:

- ammonolysis;

- removal of silyl protection;

- reprecipitation.

. Синтез проводили на уровне 0,5-1,0 мкмоль. Использовали мономеры (Glen Research): dT-CE-фосфорамидит; Bz-A-CE- фосфорамидит; Ас-С-СЕ-фосфорамидит; Ac-G-CE- фосфорамидит; U-CE- фосфорамидит; 2'-OMe-U- СЕ-фосфорамидит; 2'-ОМе-С- СЕ- фосфорамидит. Реакцию сульфирования осуществляли при помощи сульфатизирующего реагента (Sulfurizing Reagent II, Glen Research). Тритильную защиту удаляли 4% раствором дихлоруксусной кислоты в дихлорэтане. Непрореагировавшие 5'-гидроксилы блокировали стандартным образом смесью тетрагидрофуран:лутидин:уксусный ангидрид (80:10:10) и смесью тетрагидрофуран:N-метилимидазол (84:16). Реакцию окисления проводили с использованием йода. Силильную защиту снимали 1M тетрабутиламмоний фторидом в тетрогидрофуране. Аммонолиз осуществляли с использованием водного аммиака. Реакцию детритилирование проводили с использованием уксусной кислоты. Колонку промывали ацетонитрилом, синтез вели в атмосфере аргона. Очистку синтезированных олигонуклеотидов проводили двумя общепринятыми методами: в полиакриламидном гене (ПААГ) или с использованием высокоэффективного жидкостного хроматографического разделения (ВЭЖХ). Качество полученных продуктов синтеза и их химическую чистоту оценивали при помощи хромато-масс-спектрометрического анализа. Образцы анализировали на системе высокоэффективного жидкостного хроматографического разделения с детектором времяпролетного квадрупольного масс-спектрометра высокого разрешения (LC-MS/MS Q-TOF). В качестве элюента использовали метанол, являющийся полярным протонным растворителем.During the synthesis of oligonucleotides: during the implementation of the above steps, small interfering RNAs (siRNAs) and their complementary sequences for the Livin and MCM4 genes were obtained by standard synthesis, amidophosphite triester method based on O-nucleophilic intramolecular catalysis using a commercial synthesizer with a standard elongation cycle. The main part of the synthesis, chain extension with the corresponding activated monomer, for each of the oligonucleotides consisted of 21 steps. As a polymer carrier used universal CPG carrier (Glen Research) with pore size

. The synthesis was carried out at a level of 0.5-1.0 μmol. Used monomers (Glen Research): dT-CE-phosphoramidite; Bz-A-CE-phosphoramidite; Ac-C-CE-phosphoramidite; Ac-G-CE-phosphoramidite; U-CE-phosphoramidite; 2'-OMe-U-CE-phosphoramidite;2'-OMe-C-CE-phosphoramidite. The sulfonation reaction was carried out using a sulfatizing reagent (Sulfurizing Reagent II, Glen Research). The trityl protection was removed with a 4% solution of dichloroacetic acid in dichloroethane. Unreacted 5'-hydroxyls were blocked in a standard manner with a mixture of tetrahydrofuran: lutidine: acetic anhydride (80:10:10) and a mixture of tetrahydrofuran: N-methylimidazole (84:16). The oxidation reaction was carried out using iodine. The silyl protection was removed with 1M tetrabutylammonium fluoride in tetrohydrofuran. Ammonolysis was carried out using aqueous ammonia. The detritylation reaction was carried out using acetic acid. The column was washed with acetonitrile; synthesis was carried out in an argon atmosphere. The synthesized oligonucleotides were purified by two generally accepted methods: in the polyacrylamide gene (PAGE) or using high performance liquid chromatographic separation (HPLC). The quality of the resulting synthesis products and their chemical purity was evaluated using chromatography-mass spectrometric analysis. Samples were analyzed on a high performance liquid chromatographic separation system with a high resolution quadrupole mass spectrometer (LC-MS / MS Q-TOF). Methanol, which is a polar proton solvent, was used as an eluent.

The structural diagrams of the synthesis steps are shown in FIG. one.

Based on the synthesized oligonucleotides, miRNA duplexes were obtained:

Duplex obtained for inhibiting the MCM4 gene

Duplex obtained for inhibiting the Livin gene

where Gp, Cp, Ap, Up are ribonucleotides;

U ^m p -2'-O-methyluridine-3'-phosphate (2'-O-methyluridine-3'-phosphate);

C ^m p - 2'-Omethylcytidine-3'-phosphate (2'-O-methylcytidine-3'-phosphate);

dTs p - 2'-deoxythymidine-3'-phosphorothioate (2'-deoxythymidine-3'-phosphorothioate);

dTp - 2'-deoxythymidine-3'-phosphate (2'-deoxythymidine-3'-phosphate).

The preparation of siRNA duplexes was carried out in the following solution: 5X siRNA solution (300 mM KCl; 30 mM Hepes pH 7.5; 1 mM MgCl ₂ ).

To create effective cationic liposomes, their composition must include a cationic lipid, the function of which is to compact NK, and phospholipids - lipid assistants that facilitate the penetration of NK into the cell. In this case, the cationic lipid is present in an amount of from 25 to 67 mol. % of the total amount of lipids in the composition. Spermine is a natural compound, is involved in cellular metabolism and is present in all eukaryotic cells. Spermine-based cationic lipids contain four secondary amino groups, due to which condensation of NK occurs. It is known that cationic lipids containing a polyamine show a higher transfection efficiency of NK compared with lipids that include a quaternary amine or a single secondary amine. As the most effective helper lipids, 1,2-di-O-oleoylphosphatidylethanolamine (DOPE) is used, which facilitates the transition from the lamellar phase to the inverted hexagonal and egg phosphatidylcholine (PC), which facilitates penetration into the tumor.

The technical result of the invention is also the expansion of the arsenal of agents capable of efficiently delivering NK to cells in the presence of serum in a growth medium. The technical result is achieved by the proposed compositions, which contain: 1) polycationic amphiphile as a component that binds and packs a nucleic acid; 2) neutral phospholipids (DOPE and PC) as structure-forming and promoting components. In this case, the cationic lipid is present in an amount of from 25 to 67 mol. % of the total amount of lipids in the composition.

The polycationic lipid may have, for example, one of the following compositions:

- {2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetate}, or DOSPA (Lipofectamine);

- Di-octadecyl-amido-glycyl-spermine, or DOGS, Transfectam;

- 3-β (N- (N ', N'-dimethyl, N'-hydroxyethyl aminopropane) carbamoyl) cholesterol iodide (DMHAPC-Chol);

- 3β [N- (N ', N'-dimethylaminoethane) -carbamoyl] cholesterol, or DC-Chol;

- 1,26-bis (cholest-5-en-3β-yloxycarbonylamino) -7,11,16,20-tetraazagexacosane tetrahydrochloride (2X3).

The technical result is also achieved by a method for preparing a composition for delivery of small interfering RNAs, characterized in that the lipid film consisting of polycationic amphiphilum and neutral DOPE phospholipid is hydrated in water, and the resulting emulsion-dispersion system is subjected to ultrasonic treatment or extrusion.

A composition (2X3: DOPE: PC, 50: 45: 5, molar ratio) was prepared on the basis of polycationic amphiphile, preferably 2X3, neutral DOPE phospholipid and neutral egg phosphatidylcholine (PC). DOPE and PC cannot independently transfer NAs, since they do not have in their structure the cationic moiety necessary for the binding and compaction of nucleic acids. To form the compositions, hydration of the lipid film consisting of polycationic amphiphile, DOPE and PC, taken in a ratio of 50: 45: 5 (molar ratio), followed by ultrasonic treatment or extrusion, was used.

Liposomes were obtained in various ways. Depending on the method used, liposomes of different sizes and with different characteristics are obtained. The size of liposomes is important in terms of their application. In some cases, for example, for the encapsulation of bacteria or large proteins, large liposomes are needed, however, small liposomes are preferred for pharmaceutical use, since such liposomes are slower to excrete than large particles. In addition, large liposomes may cause vascular embolism or may not reach their therapeutic effect.

Two methods were chosen to produce liposomes.

The first method is based on hydration of a lipid film; for this, the lipid mixture in the flask is dissolved in an organic solvent (chloroform, dichloromethane or alcohol) and the solvent is removed in vacuo under argon flow, after which a lipid film forms on the wall of the flask. Adding water or buffer to this film leads to the formation of multilayer liposomes. Subsequent sonication of such liposomes leads to the formation of single-layer liposomes.

The second method includes the stage of hydration of the lipid film with the formation of multilayer liposomes, which are converted into single layers by the process of forcing (extrusion) of the lipid dispersion through polycarbonate filters with a specific pore size. In our case, membranes with a pore size of 100 nm were chosen.

Sterile water and Ringer's solution, which are approved for medical use in the Russian Federation, were chosen as the liquid phase.

Three cationic liposome samples were obtained:

Sample L2 was prepared in sterile water by ultrasonic sonication.

Sample L3 was prepared in sterile water by extrusion.

Sample L4 was prepared in Ringer's solution by sonication.

Samples L2 and L3 were colorless slightly opalescent solutions, while sample L4 was a solution with a high degree of turbidity.

The study of the physicochemical characteristics — size and surface potential (ζ potential) —of liposomes associated with their colloidal stability is one of the necessary steps in the creation of liposome delivery systems for NK.

The method of dynamic laser light scattering (DLS) was used to determine the size and ζ potential of liposomes. The DLS method is used to analyze the size of particles and their surface potential in solution. The size distribution obtained in the course of DLS measurements is the dependence of the relative light scattering intensity on the particle size and is known as the intensity distribution. From this distribution, after mathematical processing, a distribution according to the number of particles is obtained.

The size and surface potential of the obtained samples of cationic liposomes were determined using Delsa Nano C (Beckman Coulter, USA) and Nano ZS (Malvern, UK) analyzers. For measurements, 1 ml of solutions of samples L2, L3 and L4 in sterile water with a final concentration of 0.05 mM cationic lipid was prepared (0.1 ml of basic solution + 0.9 ml of sterile water).

As follows from the results obtained, L2 liposomes prepared in sterile water using ultrasound had an average size of about 100 nm, the polydispersity index indicated their moderate size distribution. L3 liposomes obtained by extrusion through a filter with a pore diameter of 100 nm had a size of about 120 nm, which corresponds to the pore size, and a small polydispersity index indicated the formation of a fraction of particles with a narrow size distribution. L4 liposomes prepared in Ringer's solution are characterized by the presence of two particle fractions and a wide size distribution. It should also be noted that liposomes were stable for 2 weeks, since their size did not change.

All liposomes are positively charged, as evidenced by the positive values of ζ potentials. It should be noted that the difference in the values of ζ potentials is associated both with the size of the liposomes and with the ionic strength of the solutions in which they were prepared. So, the charge of liposomes prepared in Ringer's solution (a multicomponent physiological solution with a theoretical osmolarity of 309 mosmol) was greater than the charge of liposomes L2 and L3 prepared in sterile water. The most unsuccessful liposomes should be recognized as sample L4, which tends to aggregate and is characterized by a very wide particle size distribution. The resulting samples were tested for bacterial contamination. Extraneous microflora was not detected.

At the same time, experiments on a mouse model revealed the toxicity of liposomes. Mass spectrometry was used to suggest and confirm the presence of synthesis by-products in liposomes. To eliminate them, liposome dialysis was performed using dialysis bags with a pore size of 3.5 kDa for 3 days with a three-fold replacement of water during the day. This procedure has significantly reduced the toxicity of liposomes. Intravenous administration of 0.5 ml of 0.814 mM liposome suspension twice with an interval of two hours did not lead to the death of experimental mice.

The best result in ensuring minimal toxicity was achieved when liposomes were obtained in sterile water by sounding and filtering through a filter with nanoscale pores and purifying by dialysis.

The developed composition includes several active substances: siRNA oligonucleotides for the MCM4 gene and siRNA for the Livin gene. At the final stage of obtaining the basic preparation for inhibiting the growth and stimulation of apoptosis of colorectal cancer cells, the liposome vector is combined with siRNA duplexes. To determine the best miRNA-liposomal vector ratios during their complexation by the efficiency of transferring small RNAs into the cell, special studies were carried out using a standard fluorescent duplex (BLOCK-iT Fluorescent, Invitrogen, USA, comparison drug). For this, 10 picomoles of miRNAs were mixed with liposomal vectors in different ratios, taking into account the charge. Transfection was performed on NT-29 colon carcinoma cells. Transfer efficiency was evaluated by flow cytometry 24 hours after transfection. The analysis results are presented in FIG. 2, which shows the efficiency of transfection depending on the type of vector and the degree of its loading. The x-axis represents the duplex: liposome ratio, taking into account the charge. The y axis is the percentage of cells with an introduced fluorescent signal.

It was shown that liposomal vectors obtained by different methods, but identical in chemical composition, differ from each other in the efficiency of siRNA transfer into the cell. The worst result was recorded for L3 liposomes obtained in a medium with Ringer's solution. The best transfer efficiency was shown for the vector L2. Maximum efficiency was observed at duplex: liposome ratios from 1: 2 to 1:12.

The resulting composition was evaluated for functional activity using NT-29 colon carcinoma cell culture. Transfection was performed with a combination of Livin + MCM4 duplexes in a 1: 1 ratio. Transfection results were analyzed by fluorescence microscopy using a standard apoptosis test kit (Vybrant Apoptosis Assay Kit # 5, Invitrogen, USA).

In a series of special in vitro experiments, the dose of the drug is determined that provides the maximum biological effect. In particular, it was shown that a three-fold excess (150 nM in duplex) does not increase the biological effect. The dependence of the biological effect on the dose of the drug (composition) is shown in FIG. 3.

The cytotoxicity of the substance was evaluated using the MTT test on human epithelial cells. It was shown that the dose of siRNA duplexes determined in vitro (50 nM), which provides the maximum biological effect, is non-toxic.

The ability of the composition to inhibit the growth of tumor cells of colorectal cancer was studied on male Balb / c nude mice with subcutaneous PTK-8 xenografts. Three doses were tested: 1.25; 2.5 and 5.0 mg / kg, which were administered intravenously in the regimen of three courses with an interval of 48 hours, which corresponds to the total doses: 3.75; 7.5 and 15 mg / kg. The mode of administration of the composition and the dose range are selected based on in vitro experiments. Doses were calculated individually for the body weight of each mouse. The introduction of the composition began with the appearance of palpable tumors on the 4th day.

The significance of differences between the compared groups was evaluated using the T-test. The ratio of the volumes of tumor nodes in the subsequent follow-up to the previous [VII / VI] (on the 6th and 1st day) and [VIII / VII] (on the 12th and 6th day) was used to assess the tumor growth rate inside each group.

In the MCM4 / LIVIN group, with a minimum total dose of 3.75 mg / kg, significant (p <0.05) inhibition of PTK-8 growth was obtained on the 12th day after the end of administration (21st day of tumor growth) T / C = 40 %; at a dose of 2.5 mg / kg - on the 6th and 12th day (15th and 21st day of tumor growth), T / C = 42 and 37%, respectively. The maximum total dose of 15.0 mg / kg caused significant (p <0.05) inhibition of tumor growth for all periods after the end of administration (9-21 days of tumor growth), T / C = 36, 32 and 31%, respectively. At all periods, an increase in effect over time and a direct dose dependence were observed. The best in all performance indicators was a single dose of 5 mg / kg in a regimen of three doses with an interval of 48 hours (a total of 15 mg / kg).

Tolerance of the composition in the studied dose range for three times after 48 hours of administration was satisfactory, without side effects. All mice normally took food and drinking water, the coat was smooth, and there were no signs of aggressiveness or adynamia, as well as diarrhea. When the hands were introduced into the cell, the behavior of mice was normal; no changes in motor activity were observed. There was no decrease in body weight under the influence of treatment. Autopsy did not reveal any pathological changes in the internal organs of the mice that were euthanized after the end of observation. In addition, additional studies were carried out, in particular, the effect of the composition on the mass of some internal organs was investigated. No differences between the groups were found.

To confirm the ability of the introduced nucleotide modifications to inhibit the immunogenicity of siRNA oligonucleotides, relevant studies were conducted. Under the immunotoxic effect is understood the modifying effect of the studied substance on immunogenesis, including immunosuppression and hyperstimulation of the immune system, which can lead to a decrease in the body's resistance to infection, an increase in the risk of cancer, the development of autoimmune pathology and allergization of the body. The existing approach to studying the immunotoxic effect includes the study of the action of the studied substance with a single and course administration. A single administration of a pharmacological agent 1 hour after the introduction of the antigen reveals a direct effect on the immune responses and cells of the immune system. The course introduction allows you to evaluate the indirect immunotoxic effect associated with the violation of organs and body systems associated with the immune system.

The ability of a composition to influence immunogenesis, including immunosuppression and hyperstimulation of immunity, was tested using a test to determine the number of antibody-forming cells (AOKs). Using the AOK test, the immunotoxic effect of the study drug was not detected.

When studying immunotoxicity, the most informative is the assessment of the humoral immune response during immunization of animals with a T-dependent antigen (in particular, sheep erythrocytes). The process of antibody formation in response to such an antigen includes the main stages of the formation of the immune response: phagocytosis, presentation of the antigen, recognition and activation of type 2 T-helpers, activation, proliferation of B cells and maturation of antibody-producing cells, as well as antibody synthesis. Thus, it becomes possible to study the integral process of the formation of the immune response.

In this study, the humoral immune response was evaluated by determining the number of antibody-forming cells in the spleen of immune animals.

On the 5th day after the mice were immunized and the drug was administered to them, the animals were euthanized by cervical dislocation and the total number of leukocytes in the spleen was counted in the Goryaev’s chamber.

No significant differences in the absolute number of splenocytes of animals of all groups were detected. When counting the number of antibody-forming cells (AOKs) in the spleen of immune animals, there were also no differences between the control and the experimental group and the control placebo group (empty liposomes).

Thus, a drug was created with fewer side effects, but capable of providing the same or greater therapeutic effect.

Claims (31)

1. A composition for inhibiting the growth and stimulation of apoptosis of colorectal cancer cells by blocking the function of genes: MCM4 and Livin in cancer cells, comprising a means for delivering the composition to the target cells, which is a liposome, and a combination of two miRNA oligonucleotides duplexes, of which duplex for blocking genes of MCM4 contains preparations of small interfering oligonucleotides: 5'-C-U-C-A-Umet-C-U-C-U-Umet-A-C-C-C-A-C-A-G-G-dTs-dT-3 'as a straight chain and 5'-C-C-U-G-U-G-G-G-Umet-A-A-G-A-G-A-Umet-G-A-G-dTs-dT-3 'as the return circuit, and the duplex for blocking Livin genes contains preparations of small interfering oligonucleotides: 5'-G-G-A-A-G-A-G-A-C-U-Umet-U-G-U-C-C-A-Cmet-A-dTs-dT-3 'as a straight chain and 5'-U-G-U-G-G-A-Cmet-A-A-A-G-U-C-Umet-C-U-U-C-C-dTs-dT-3 'as the return circuit, where G, C, A, U are ribonucleotides; Umet - 2'-O-methyluridine-3'-phosphate; Cmet - 2'-O-methylcytidine-3'-phosphate; dTs - 2'-deoxythymidine-3'-phosphorothioate; dT - 2'-deoxythymidine-3'-phosphate. 2. The composition according to p. 1, characterized in that the preparations of miRNA oligonucleotides are made in the form of duplexes of the forward and reverse complementary nucleotide chains of the following structure: for blocking genes MSM4 duplex

and for blocking Livin duplex genes

where Gp, Cp, Ap, Up are ribonucleotides; U ^m p is 2'-O-methyluridine-3'-phosphate; C ^m p is 2'-O-methylcytidine-3'-phosphate;

- 2'-deoxythymidine-3'-phosphorothioate; dTp - 2'-deoxythymidine-3'-phosphate. 3. The composition according to p. 2, characterized in that as a means for delivering the composition to target cells, it contains a liposome, which is a polycationic lipid as a component that binds and packs a nucleic acid, and neutral phospholipids DOPE and PC as structure-forming and promoting components, with the ratios of duplex: liposome from 1: 2 to 1:12. 4. The composition according to p. 3, characterized in that the means for delivering the composition to the target cells is in the form of a lipid film consisting of a polycationic lipid and a neutral phospholipid, hydrated in water to obtain an emulsion-dispersion system, subjected to ultrasonic treatment or extrusion, and polycationic lipid, phospholipids DOPE and PC are taken in a ratio of 50: 45: 5 (mol.). 5. The composition according to any one of paragraphs. 3, 4, characterized in that as a means for delivering the composition to target cells, it contains a polycationic lipid in an amount of from 25 to 67 mol. % on the total amount of lipids in the composition; polycationic lipid is selected from the group: - {2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetate}, - Di-octadecyl-amido-glycyl-spermine, - 3-β (N- (N ', N'-dimethyl, N'-hydroxyethyl aminopropane) carbamoyl) cholesterol iodide, - 3β [N- (N ', N'-dimethylaminoethane) -carbamoyl] cholesterol, - 1,26-bis (cholest-5-en-3β-yloxycarbonylamino) -7,11,16,20 - tetraazagexacosane tetrahydrochloride 2X3. 6. The composition according to any one of paragraphs. 1-4, characterized in that it is made in a pharmaceutical form suitable for parenteral administration with the aim of inducing apoptosis of colorectal cancer cells.

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