RU2639388C1 - Composition for viral hepatitis c therapy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention can be used to treat an infection caused by the hepatitis C virus (HCV). A composition for HCV infection treatment of by the RNAi mechanism consists of complexes of lipopeptides of the OrnGlu (C₁₆H₃₃)₂ composition, serving as a carrier, and siRNA molecules represented by 5'-AAAUCUCCAGGCAUUGAGCtt-3' (SEQ ID NO 1). Composition application on a transplantable cell culture of human hepatoma Huh-7, expressing the subgenomic replicon of HCV, causes a significant decrease in viral replicon expression by 20%.

EFFECT: high bioavailability with subcutaneous administration of the composition.

2 cl, 8 dwg, 4 tbl, 5 ex

Description

The invention relates to the field of medicine and gene therapy and can be used to treat infections caused by hepatitis C virus (HCV). The patented composition is a complex of small interfering RNA (siRNA) molecules that suppress the replication of viral RNA and a carrier, lipopeptides, which facilitate the penetration of synthetic siRNA molecules into target cells.

The background of the invention was the following.

Viral hepatitis C is an anthroponous viral infection from the conditional group of transfusion hepatitis, characterized by liver damage, anicteric, mild and moderate in the acute phase and a frequent tendency to chronicity, the development of liver cirrhosis and primary hepatocarcinomas (Chevaliez et al., 2012).

The causative agent is an RNA-containing virus included in the genus Hepacivirus of the family Flaviviridae (Gower et al., 2014). 7 genotypes and more than 67 virus subtypes are distinguished (Simmonds 2004; Smith, Bukh et al. 2014; Echeverria, Moratorio et al. 2015). The most typical for the Russian Federation is genotype 1 (1a and 1b), which accounts for up to 60% in the structure of chronic hepatitis C (CHC), and 40% remains for genotypes 2 and 3 (Pimenov, Vdovin et al. 2013).

The urgency of the problem of hepatitis C is determined by the high epidemiological and socio-economic significance of this disease, the widespread and widespread distribution, the active involvement of people of reproductive, most working age in the epidemic process, and significant government expenditures on the treatment of people infected with HCV. According to WHO, about 180 million people worldwide are infected with the hepatitis C virus, about 3% of the population, 130 million are chronic carriers of the virus, 3-4 million are infected with HCV annually [(http://www.who.int)].

A sustained viral response (SVR) is defined as a condition in which HCV RNA is not detected in blood serum within 24 weeks after completion of therapy (Cheng et al., 2014). SVR is the standard marker for successful antiviral therapy in clinical trials. In the early 2000s, the combination of pegylated interferon and ribavirin (PR) became the standard for anti-HCV therapy (Garg and Kar 2009; Pawlotsky 2013). However, interferon therapy is associated with many side effects (flu-like symptoms, fatigue, etc.). The SVR level with this therapy is 70–80% among patients with HCV infection caused by genotypes 2, 3, and 70% among patients with other HCV genotypes (Ghany, Strader et al. 2009). Therefore, there is a need to develop new antiviral drugs. Each stage of the life cycle of the virus can be considered as a target for new antiviral drugs. Modern advances in understanding the HCV life cycle have led to the development of highly effective and well-tolerated antiviral agents of various mechanisms of action (Table 1).

RNA interference is a natural mechanism for regulating gene expression in a cell with the participation of the Dicer enzyme and small interfering RNA (siRNA) molecules. RNA interference (RNAi) is initiated by the Dicer enzyme, which cuts a double-stranded (DC) RNA molecule into siRNAs of 21-26 bp in size, providing specific post-transcriptional suppression of the expression of target genes by complementary binding and degradation of the corresponding mRNAs or by inhibiting mRNA translation (Hait M.R. ., 2012).

Perhaps the main problem with the use of RNA-based drugs is the intracellular delivery of siRNA molecules into the cell. Most methods and means of delivering siRNA to a cell are mentioned in patents (US patent 20110305772 A1, US patent 2011018989180 A1, US patent 20120202871 A1). These patents relate to particular aspects of methods widely used in molecular biology or new methods of their application.

MiRNA-based drugs are being developed for the treatment of cancer, infectious diseases and other pathologies that are associated with impaired functions of specific genes. To date, a number of miRNA-based preparations have been patented for the prevention and treatment of various viral pathologies. For example, the Danish company Santaris Pharma A / S, developed the drug Miravirsen for the treatment of HCV infection (Lna oligomers for improvement in hepatic function, patent WO 2013068348 A1). The drug is based on a modified 15 nucleotide oligodeoxynucleotide, an inhibitor of cellular miR-122 microRNA, expressed in the liver and protecting the hepatitis C virus genome from degradation by exonuclease Xrn1 (Li, Masaki et al. 2013). The drug is an antisense oligonucleotide in the form of a closed nucleic acid (Locked Nucleic Acid), a modified phosphorothioate group and directed to miR-122. The drug has recently successfully passed stage 2a clinical trials.

In the security document US 20120308642 A1, which is a prototype of the claimed composition, miRNA combinations to non-overlapping fragments 5 'of the untranslated region of the HCV genome are described, providing a long-term antiviral effect. Intravenous administration of the drug is carried out in combination with lipid nanoparticles modified with cholesterol. The claimed advantage of this delivery method compared to the previously described options for nanoparticles is to develop a protocol for the preparation of auxiliary components of the drug, providing a decrease in their size and an increase in the proportion of particles with a size of less than 150 nm. According to the document, this modification of the protocol leads to an increase in the efficiency of siRNA delivery to liver cells and allows to reduce the concentration of the drug.

A distinctive feature of the claimed composition is the structure of the siRNA molecule, as well as the structure of its carrier. The auxiliary component of the patented composition is based on lipodipeptides that spontaneously form bilayer aggregates in an aqueous medium, the size of which does not exceed 100 nm. In this case, the nanoparticles do not need additional modification.

The objective of the invention is to provide a low toxic and effective agent for the treatment of HCV infection based on siRNA molecules.

The claimed composition for the treatment of HCV infection, containing complexes of OrnGlu (C ₁₆ H ₃₃ ) ₂ lipopeptides acting as a carrier and siRNA molecules represented by the sequence SEQ ID NO 1 that suppress HCV replication by the RNA interference mechanism. The sizes of the complexes do not exceed 100 nm.

The technical results of the proposed method is the creation of a low-toxic composition and the effective effect of the introduced siRNA molecules, represented by the sequence SEQ ID NO 1, in complexes with Y14 lipopeptides of the composition OrnGlu (C ₁₆ H ₃₃ ) ₂ , on the suppression of HCV replication. An advantage of the claimed composition is its high bioavailability (up to 70%) with a subcutaneous route of administration.

Brief Description of the Drawings

FIG. 1. Scheme for the synthesis of lipopeptides of the composition OrnGlu (C ₁₆ H ₃₃ ) ₂ .

FIG. 2. Electron micrographs of a) siUTR siRNA molecules, b) Y14 lipopeptides, c) Y14 / siUTR complexes (compositions).

FIG. 3. The dependence of the fluorescence intensity of the complex on the ratio +/- (positively charged amino groups YH / negatively charged phosphates siUTR).

FIG. 4. The effectiveness of the transfection of the composition on the model of HCV replicon in the culture of human hepatoma cells Huh-7.

FIG. 5. Change in the concentration of the components of the composition in blood serum at various time periods after intravenous administration.

FIG. 6. A change in the concentration of the components of the composition in the liver at different time periods after intravenous administration.

FIG. 7. Change in the concentration of the components of the composition in the organs (lungs, liver, kidneys, spleen) at various time periods after intravenous administration.

FIG. 8. The change in the concentration of the components of the composition in blood serum at different periods of time after subcutaneous administration

DETAILED DESCRIPTION OF THE INVENTION

Example 1. Synthesis of siRNA molecules

The production of siUTRs is based on solid-phase chemical synthesis (on a solid support), which includes repeated operations to build up the oligonucleoside chain, so these reactions can be carried out using commercial automatic synthesizers, as well as using manual reactors for production, not in automatic mode. All raw materials (reagents and solvents) were weighed, measured and loaded into the reactor according to the technological stages, intermediates appear after the last synthesis stage when they are unloaded from the reactor. During the synthesis, two complementary miRNA chains were obtained, from which a duplex was prepared by mixing equimolar amounts of the sense and antisense siUTR miRNA chains, which were heated at 65 ° C for 5 minutes and then sharply cooled at 4 ° C. Ready duplexes (siUTR) were used to prepare the composition.

Example 2. Synthesis of lipopeptides

The synthesis of Y14 lipopeptides of the composition OrnGlu (C ₁₆ H ₃₃ ) _{2 was} carried out according to the scheme shown in FIG. 1. A Boc-substituted amino acid derivative was prepared by the action of di-tert-butyloxycarbonate. By-products of the Boc group addition reaction are tert-butanol and carbon dioxide, which are easily separated from the target product. The structure of the synthesized compound was confirmed by ¹ H-NMR, IR spectroscopy.

In the IR spectrum, characteristic absorption bands of amide bonds were observed (1660 cm ⁻¹ , C = O “I amide band”, 1658 cm ⁻¹ , NH “II amide band”), a carboxyl group (1750 cm ⁻¹ C = O, 1420 cm ^-1 OH). The proton signal of tert-butyl groups in the ¹ H-NMR spectrum of compound (25) is represented by a singlet with a chemical shift of 1.4 ppm.

The reaction of producing N-hydroxysuccinimide ester (26) was carried out under standard conditions in the presence of dicyclohexylcarbodiimide as a condensing agent. The precipitated dicyclohexylurea precipitate was filtered off. The resulting product gave a positive reaction in a special developer for the N-hydroxysuccinimide group. The product was used in the next step without further purification.

The amide bond formation reaction between activated ester (26) and L-glutamic acid dihexadecyl ester was carried out with a slight excess of the latter (reagent ratio 1: 1.1, respectively) in tetrahydrofuran in the presence of an aqueous solution of KHCO ₃ , necessary to create a weak alkaline medium. The progress of the reaction was monitored by TLC. The reaction products were isolated by column chromatography in a solvent system of toluene-chloroform-methyl ethyl ketone-isopropanol 10: 6: 3: 1. The structure of the obtained compound was confirmed by IR and ¹ H-NMR spectroscopy. Signals of protons of aliphatic chains (δ, ppm: 1.25 (s, 52H, CH ₂ ), 0.84 (t, 6H, CH ₃ )), protons of tert-butyl groups were present in the ¹ H-NMR spectrum (δ, ppm: 1.45 (s, 18H, CH ₃ )), an amide bond (δ, ppm: 5.7 (d, 1H, NH)), ornithine and glutamine hydrocarbon chains (δ ppm: 2.5 (m, 2H, CH ₂ ), 2.28 (m, 4H, CH ₂ ), 3.4-4.2 (m, 2H, CH), 4.36 (m , 2H, CH ₂ )).

The removal of tert-butyloxycarbonyl protection was carried out by the action of anhydrous trifluoroacetic acid. In the ¹ H-NMR spectrum, the disappearance of proton signals corresponding to the protective groups was observed while maintaining the substantial part of the spectra of the remaining structure. In the IR spectrum, characteristic absorption bands of amide bonds were observed (1654 cm ^-1 , C = O “I amide band”, 1681 cm ^-1 , NH “II amide band”), carboxyl group (1735 cm ^-1 C = O), free amino groups (3330 cm ^-1 NH ₂ ), aliphatic chains and hydrocarbon groups of ornithine and glutamine (2912, 2847, 1462, 1390 cm ^-1 ). A signal of the molecular ion of the target compound (M +) 710.85 was present in the MALDI mass spectrum. To obtain liposomes, 50 mg of OrnGlu (C16) _{2 was} dissolved in 20 ml of chloroform, which was then slowly distilled off on a rotary evaporator to form a thin lipid film, which was further dried for 4 hours on a vacuum pump. 5 ml of distilled water was added to the resulting lipid film and held for 30 minutes. The resulting dispersion was treated with ultrasound (3 × 25 min). A mini-extruder with polycarbonate filters with a porosity of 400 nm was used for particle homogenization and sterilization. Finished lipopeptides were used to prepare the composition.

Example 3. The composition

The claimed composition is a complex containing Y14 lipopeptides of the composition OrnGlu (C ₁₆ H ₃₃ ) ₂ (molecular weight 710 g / mol, concentration 0.8 mg / ml, volume 1.75 ml) and siUTR siUTR duplexes (SEQ ID NO 1, concentration 0.08 mg / ml, volume 1.75 ml). Mass ratio Y14 / siUTR = 10/1. Solutions of both components are prepared in a 5% glucose solution.

Example 4. Physico-chemical characteristics of the composition

Before using the Y14 / siUTR composition in vitro and in vivo, its physicochemical characteristics were studied. For the Y14 / siUTR complex, the ratios of the components were determined at which the maximum degree of binding is observed. The experiments were carried out using fluorescence spectroscopy.

Mixtures of the composition 1: 2-2: 1 mol / mol (+/-, positively charged amino groups YH / negatively charged siUTR phosphates) were incubated for 15 min, sufficient to form the complexes used in the transfection experiments.

As a result of experiments on fluorescence spectroscopy, it was shown that for lipopeptide Y14 the minimum ratio at which complexation is observed is 1: 1 (+/-) (Fig. 3).

The size and shape of the complexes were determined by transmission electron microscopy with contrasting with uranyl acetate. It was shown that the Y14 / siUTR complexes included in the composition of the claimed composition, represent a sandwich structure (Fig. 2), the size of which does not exceed 100 nm.

Example 5. Evaluation of the effectiveness of transfection in vitro

HCV life cycle studies are hampered by a lack of adequate models. The only common cellular system is an artificial viral replicon, which is a (+) viral RNA chain in which the structural region is replaced by the neomycin resistance gene and firefly luciferase gene, and, in addition, the ribosome internal landing region (IRES) is replaced by that of the encephalomyocarditis virus . The study of the specific biological activity of the composition in in vitro experiments was carried out using a Huh-7 human hepatoma cell culture that expresses a subgenomic HCV replicon corresponding to genotype 1b, isolate Con1 and containing mutations E1202G, T1280I and K1846T, which enhance RNA replication. The presence of the firefly luciferase reporter gene in the replicon used, the expression of which correlates with the level of HCV RNA expression, allows one to use the measurement of luminescence in cells as a quick and convenient test for measuring transfection efficiency. In this case, siRNA directed to the luciferase gene (siLuc) was used as a positive control in all experiments, and a non-specific siRNA directed to the NP gene of the influenza A H5N1 virus was used as a negative control.

The results of this series of experiments are presented in FIG. 4. To assess the effectiveness of transfection, 12.5 μl of the composition was used (which corresponds to 0.5 μg siRNA per well), as well as an equivalent amount of control siRNAs K + and K- mixed in a ratio of 1:10 with the lipopeptide Y14 preparation (by analogy with the composition of the composition). The results obtained in a series of experiments were presented as the luciferase luminescence intensity compared to the value obtained by treatment with complexes based on nonspecific siRNA (K-) directed to the NP gene of influenza A H5N1 virus. The results were expressed as the percentage of residual luciferase activity in cells treated with specific siRNA (composition and K +) compared to non-specific (Fig. 4). The greatest efficiency of transfection was demonstrated by the claimed composition. Its use causes a decrease in luminescence up to 20%.

Thus, in this series of experiments, the specific antiviral activity of the claimed composition was shown, as well as the potential possibility of creating therapeutic preparations based on siRNAs, which can be further used both independently and in combination with interferons for the treatment of chronic HCV infection, was demonstrated.

Example 6. Pharmacokinetics of the composition for intravenous and subcutaneous administration in vivo.

Studies have been conducted on the pharmacokinetics of the claimed composition with the intravenous route of administration. The composition was administered to mice at doses of 5.5 mg / kg, 2.75 mg / kg and 0.5 mg / kg, which corresponds to 11, 5.5 and 1 therapeutic dose (TD) in a volume of 0.25 ml per mouse intravenously, and after 2, 5, 10, 20, For 30, 45 and 60 min, blood was taken from them (at least 300 μl) for subsequent serum production (at least 50 μl). Animals were sacrificed at the indicated time intervals after administration of the composition, and in addition to blood, organs (lungs, kidneys, liver, spleen, thymus, thigh muscle and brain) were removed from them. The organs were homogenized in 1.7 ml of nat. solution, the obtained homogenates were stored at - 20 ° C until analysis by chromatography with fluorescence detection. Using a chromatograph with a fluorescent detector, the concentration of the components of the preparation (siUTR and Y14) in the selected biological samples at different time periods was estimated.

As a result, the following data were obtained. The maximum concentration of the drug components in the blood was detected immediately after administration of the drug (2 minutes), and 60 minutes after the injection, the drug components were not detected in the blood (Fig. 5). The half-life for both components of the test drug was similar and ranged from 0.078 to 0.095 hours for siUTR and from 0.061 to 0.101 hours for Y14, which indicates the stability of Y14 / siUTR complexes in the blood (Table 2).

When studying the distribution of the components of the claimed composition in the target organ (liver), it was found that Tmax for siUTR occurs 30 minutes after intravenous administration, and Tmax for Y14 is reached somewhat earlier - 20 minutes. The half-life of siUTR from the liver is approximately 0.25 hours and does not depend on the administered dose of the drug, and for Y14, the half-life ranges from 0.15 to 0.34 depending on the dose (Fig. 6, 7).

When studying the distribution of the drug among various organs, we examined the immune organs: thymus and spleen, and non-immune organs: lungs, brain and thigh muscle, as well as excretory organs: kidneys and liver. When studying the distribution of the components of the drug in the organs, it was found that both components accumulate as much as possible in the spleen with Tmax for an average of 20 minutes. However, siUTR and Y14 are excreted from the spleen faster than from the liver: 0.17 versus 0.25 h for siUTR and 0.15 versus 0.24 for Y14 (Table 3). Presumably, the spleen is the tropic organ for the composition under study. In another organ of the immune system, the thymus, neither Y14 nor siUTR were decoding.

The following organs, where high concentrations of the components of the composition were observed, were the liver, kidneys and lungs (Fig. 7, Table 3). Most likely, the excretion of metabolites (or an empty fluorescent label) is carried out by the kidneys, because both labels are detected in urine.

The components of the drug were not detected in the thigh muscle and brain. This indicates that the drug does not pass the blood-brain barrier and does not accumulate in muscle tissue, therefore, it does not have a toxic effect on the central nervous system.

The pharmacokinetics of the claimed composition was also studied with the subcutaneous route of administration as recommended for clinical use. Three doses of the drug 5.5, 2.75 and 0.5 mg / kg were injected subcutaneously, which corresponded to 11, 5.5 and 1 TD. As a result, the following data were obtained. The maximum concentration of the drug components in the blood was detected after 5 (for doses of 5.5 and 0.5 mg / kg) and 10 min (for a dose of 2.75 mg / kg) after administration of the drug, and after 60 min after administration, the components were not detected in the blood (Fig. 8) . The half-life for both components was similar and averaged about 0.1 h (6 min), which indicates the stability of the Y14 / siUTR complexes in the blood (Table 4).

The data obtained indicate that the studied drug reaches the target organ - the liver. It should be noted that the kinetics of the drug is characteristic of most RNA-containing drugs, which consists in a short elimination period.

When studying the distribution of the drug by organs, it was found that the drug accumulates more intensively in the spleen. In the target organ - the liver - the components of the drug are also detected in significant quantities, moreover, the half-life of the components of the studied drug from the liver is one of the highest in comparison with other organs.

Also, data on the accumulation of the components of the drug in the kidneys and the detection of both fluorescent labels in the urine allows us to conclude that it is the kidneys that are the main organ of excretion.

With subcutaneous administration, a relatively high bioavailability is observed (about 70%), which indicates a correctly chosen therapeutic route of administration. A high apparent volume of distribution indicates a good distribution over the tissues of the internal organs, which indicates the correct choice of delivery vehicle. At the same time, the relatively low half-life indicates that the drug does not cumulate in the body and, as a result, the likelihood of an overdose is low.

Sequence listing

<110> FSBI "SSC Institute of Immunology" FMBA

<120> Composition for the treatment of viral hepatitis C

<210> SEQ ID NO 1

<211> 21

<212> RNA

<213> Viral

<400> SEQUENCE 1

AAAUCUCCAGGCAUUGAGCtt

Claims (2)

1. A composition for the treatment of HCV infection, containing complexes of OrnGlu (C ₁₆ H ₃₃ ) ₂ lipopeptides acting as a carrier and siRNA molecules represented by the sequence SEQ ID NO 1 that inhibit HCV replication by RNA interference mechanism. 2. The composition according to p. 1, characterized in that the size of the complexes of lipopeptides with siRNA molecules does not exceed 100 nm.

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