Medicament for the prevention of sars-cov-2 infection

Abstract

The present invention relates to the fields of medicine, pharmacy and biotechnology. A novel pharmaceutical composition having an antiviral effect on coronavirus SARS-CoV-2 and related viruses (e.g. SARS, MERS, etc.) whose genome is represented by RNA and the virion has a lipid envelope is provided. A combination drug for preventing (treating) SARS-CoV-2 infection, including (i) a liposome having an effective amount of RNase, or (ii) a liposome having an effective amount of RNase and an effective amount of niclosamide preparation, and an auxiliary substance in the form of a pharmaceutically acceptable solvent. The present invention provides destruction of genetic material, in particular RNA coronavirus, thereby enabling prevention of infection caused by RNA-containing viruses (including SARS-CoV-2) that enter the human body through the upper respiratory tract.

Description

[0001] describe

[0002] Technical Field of the Invention

[0003] This invention relates to the fields of medicine, pharmaceutics, and biotechnology. The invention proposes a novel combination drug (pharmaceutical composition) with antiviral activity against SARS-CoV-2 coronavirus and related viruses, such as SARS-CoV and MERS, whose genome is represented by RNA and whose viral particles have a lipid envelope. Composition (i) destroys the coronavirus genome with an RNase, composition (ii) destroys the coronavirus genome with an RNase, and a niclosamide preparation inhibits SARS-CoV-2 replication. The proposed composition can be used in outpatient settings to inhibit SARS-CoV-2 viral replication, reduce viral load, and lower the risk of airborne infection. Background Technology

[0004] Many preventative vaccines and treatments have been developed worldwide to treat coronavirus infection. Russian Federation Patent No. 2746362 (based on Invention Patent Application No. 2021106335 dated March 11, 2021), entitled “Combination Drug with Antiviral Activity against Novel SARS-CoV-2 Coronavirus” [1], describes “blocking the spread of infection and reducing the risk of clinical complications” by using small interfering RNA (miRNA). The basis of the above patent is the preparation of combination drugs with engineered RNA molecules that can mediate target-specific inhibition of SARS-CoV-2 virus replication.

[0005] However, no agent has yet been proposed that can destroy coronaviruses by directly damaging their genome, single-stranded (+)RNA. Obviously, direct damage to the viral genome can be achieved by exposure to an enzyme called ribonuclease (RNase) [2] that can cleave coronavirus RNA, or by inhibiting coronavirus replication under the action of niclosamide, an agent used to treat parasitic infections. Nicosamide has been shown to inhibit the replication of SARS-CoV-2 virus [3-6]. Summary of the Invention

[0006] The combination drug of the present invention is intended for intranasal and inhalation administration.

[0007] The introduction of RNases and niclosamide-based drug formulations into medical practice is limited by a number of factors, including the inefficiency and safety of delivering RNases and niclosamides to viral target particles. The use of liposomes [7] to deliver the above drugs (RNases and niclosamides) solves this problem because SARS-CoV-2 and related viruses MERS and SARS-CoV have lipid envelopes that allow liposome transfection (penetration) and direct introduction of RNases and niclosamides into the virus.

[0008] Therefore, there is a need for novel antiviral agents capable of degrading coronavirus RNA.

[0009] The combination pharmaceutical compositions according to the invention comprise: composition (i) - a liposome delivering an effective amount of RNase, and composition (ii) - a liposome delivering an effective amount of RNase and an effective amount of niclosamide. These pharmaceutical compositions provide cleavage of SARS-CoV-2 RNA.

[0010] Combination medications can be used as nasal drops or sprays, or as inhalers.

[0011] The claimed technical effect is to provide an effective and safe antiviral agent that ensures the destruction of SARS-CoV-2 RNA. This technical effect is explained through the following examples.

[0012] To achieve the above-mentioned technical effects, the inventors proposed a combination drug for the prevention or treatment of coronavirus infection caused by SARS-CoV-2, which contains an effective amount of RNase molecules in liposomes.

[0013] In addition, to achieve the above-mentioned technical effects, the inventors proposed a combination drug for the prevention or treatment of coronavirus infection caused by SARS-CoV-2, which contains an effective amount of RNase molecules and niclosamide in liposomes.

[0014] In this invention, the above-mentioned combination drugs can be used for intranasal or inhalation administration.

[0015] In addition, in order to achieve the above-mentioned technical effects, the inventors have proposed a method for preventing or treating SARS-CoV-2 infection, wherein the above-mentioned combination drugs are used simultaneously, alone or sequentially with other therapeutic agents. Brief description of the attached diagram

[0017] Figure 1 The experimental results related to Example 4 are shown.

[0018] Figure 2 The experimental results (gel electrophoresis) related to Example 5 are shown.

[0019] Implementation scheme of the invention

[0020] The technical effects claimed by the inventors are illustrated by the following embodiments.

[0021] Example 1. Obtaining liposomes containing RNase

[0022] To obtain liposomes, phospholipids of plant origin (soybean lecithin) and animal origin (from bovine brain) were used. As the ribonuclease, we used a commercial formulation – RNase A, with an MM-13.7 kD, optimal pH-5.0-6.0; 50 U / mg, derived from bovine pancreas by Mol BIO HIMEDIA, or RNase A derived from bovine pancreas by SamsonMed (Russian Federation).

[0023] As a phospholipid, extracts of soybean lecithin used in the food industry or animal-derived phospholipids (from bovine brain) are used.

[0024] To obtain liposomes, 1 g of commercially available soybean lecithin (VEROLECFLS, China) was dissolved in 20 ml of a chloroform-methanol mixture at a 2:1 ratio. The mixture was incubated at 2–4 °C for 10 hours. The mixture was then centrifuged at 4500 rpm for 5 minutes. 1 ml of the supernatant was collected and dissolved in 6 ml of chloroform. The solvent was then evaporated under vacuum (4 ± 2 mmHg) at +40 °C. After complete solvent removal, 50 μl of ribonuclease A (1 U / mL) and 5 ml of physiological solution were added. The resulting mixture was frozen at -20 °C for 20 minutes, and then thawed with stirring at 23 °C for 30 minutes (the freezing and thawing process was repeated four times). The resulting mixture was then subjected to sonication at 22 kHz for 30 seconds four times.

[0025] Similarly, bovine brain phospholipid-based liposomes were prepared according to method [8].

[0026] The dosage forms of the pharmaceutical compositions provided herein can be prepared by any known method and may contain other excipients that can increase the stability of the compositions (i) and (ii) and RNase transfection.

[0027] Example 2. Obtaining liposomes containing RNase and niclosamide

[0028] Niclosamide (a crystalline substance with a melting point of 227-232℃) was used. The infrared spectrum of the test sample corresponds to that of the standard form of anhydrous niclosamide. The structural formula of niclosamide is as follows:

[0029]

[0030] To obtain liposomes containing RNase (composition i) and liposomes containing RNase and niclosamide (composition ii), 1 ml of lecithin solution (1.075 mM) was mixed with 6 ml of chloroform. The solvent was removed by vacuum distillation (4 ± 2 mmHg) at +40 °C. After removing the chloroform, 50 ml of ribonuclease A, 30 ml of niclosamide solution (10% DMSO solution) and 5 ml of physiological solution were added to the flask. The resulting mixture was frozen at -20 °C for 20 min and then thawed with stirring at 23 °C for 30 min (the freezing and thawing process was repeated four times). The resulting mixture was then sonicated at 22 kHz for 30 s. Similarly, bovine phospholipid-based liposomes were prepared according to method [8].

[0031] The dosage form of the pharmaceutical composition provided herein can be prepared by any known method and contains other excipients that can increase the stability of the composition (i) and (ii), as well as RNase and niclosamide transfection.

[0032] Example 3. Synthesis of primers for the N gene of SARS-CoV-2.

[0033] PCR was used to determine the likelihood of SARS-CoV-2 RNA cleavage. The N gene, which encodes the nucleocapsid and is the most stable for coronaviruses of the SARS and MERS types [9-11], was selected as a target for determining the likelihood of RNase cleavage of the SARS-CoV-2 coronavirus genome. Primers and fluorescent probes for the N gene were synthesized using internationally available data from the CDC (Centers for Disease Control and Prevention)

[12] .

[0034] Primer synthesis was performed using a phosphoramide solid-phase method on an ASM-2000 DNA synthesizer

[13] . After synthesis, the oligonucleotides were removed from the solid phase with 30% concentrated ammonia at 60°C for 2 hours. The oligonucleotide solution was evaporated to remove ammonia and precipitated in 70% ethanol in the presence of 0.3M sodium acetate and 50mM magnesium chloride. The oligonucleotides were centrifuged at 14000 rpm for 20 minutes to precipitate. The precipitate was washed twice with 70% ethanol and dried at room temperature for 30 minutes.

[0035] Following synthesis, oligonucleotides were purified from the short, incompletely synthesized molecule. Therefore, a final step of DMT group removal was omitted at the end of synthesis; only oligonucleotides with a fully synthesized 5′ end contained the DMT group for purification by reversed-phase chromatography on a column. Reversed-phase chromatography was performed under the following conditions: the oligonucleotides were washed with 5% acetonitrile containing 100 mg / mL sodium chloride. The DMT group was removed from the oligonucleotides using 3% dichloroacetic acid solution. After DMT group removal, the purified oligonucleotides were eluted from the column with 50% acetonitrile in 100 mM Tris buffer at pH 8.5. The purified oligonucleotides were precipitated with 70% ethanol by centrifugation at 14,000 rpm for 20 min in the presence of 50 mM magnesium chloride. The resulting oligonucleotide precipitate was dissolved in TE buffer at pH 8.0 to a concentration of 20 μM. The resulting primer solution was used for PCR.

[0036] Table 1

[0037] Primer and fluorescent probe sequences on the N gene of SARS-CoV-2

[0038]

[0039] Using these primers and probes specific to the N region of the SARS-CoV-2 RNA gene, the viral load in biological samples was determined by PCR, and the samples were subjected to gel electrophoresis as described in Examples 4 and 5.

[0040] Example 4. Study on the effect of RNase-containing liposomes on SARS-CoV-2 production in vitro by RT-PCR

[0041] RNA isolation and detection of SARS-CoV-2 virus were performed according to standard methods using a certified reaction kit from DNA-Technology LLC [14, 15] and with primers and probes synthesized by us. The efficiency of the liposomes was determined by the change in threshold cycling (ΔΔCt) during PCR assays. Threshold cycling indicates how much RNA is present in the sample and is inversely proportional to the logarithm of the initial copy number.

[0042] RNA isolation and detection of SARS-CoV-2 virus were performed according to standard methods using a certified kit from DNA-Technology LLC [14, 15] and primers and probes synthesized by us. The efficiency of the liposomes was determined by the change in threshold cycling (ΔΔCt) during PCR assays. Threshold cycling indicates how much RNA is present in the sample and is inversely proportional to the logarithm of the initial copy number.

[0043] For RNA isolation, a reaction kit from DNA-Technology LLC and the inventor-certified kit ENCOR were used. RNA in the samples was detected using the synthetic primers and probes described in Example 3. For PCR analysis, we used the DNA-Technology SARS-CoV-2 / SARS-CoV kit and our PCR kit developed according to the following conditions: 25 mL Cl mixture, 1x PCR buffer (20 mM Tris-HCl pH 8.5, 20 mM ammonium sulfate, 20 mM potassium chloride, 0.1 μg / mL BSA), 3 mM magnesium chloride, 0.8 mM dATMP, 400 nm per primer, 200 pM probe, 1 unit polymerase, and 25 units reverse transcriptase. PCR program: 50°C–15 min; 95°C–15 min; 40 cycles (95°C–10 sec; 60°C–40 sec, FAM channel). For PCR analysis, 5 μL of RNA sample was used.

[0044] Human biological material samples containing SARS-CoV-2 coronavirus (80 μl nasopharyngeal smears each) were incubated with 20 μl of compositions (i) and (ii) (obtained as described in Examples 1 and 2) at 36 °C for 1 hour. Simultaneously, the same samples were incubated with 20 μl of free RNase (1 U / μl, and empty liposomes without RNase). The experimental results are summarized in Table 2 below.

[0045] Table 2

[0046] The in vitro effects of RNase-containing liposomes on the production of the SARS-CoV-2 N gene were investigated using hybridization fluorescence detection-assisted PCR analysis.

[0047]

[0048]

[0049] As shown in Table 2, the cycle threshold (Ct.) of the RT-PCR reaction remained almost unchanged under the action of "empty" liposomes, indicating that liposomes alone cannot degrade SARS-CoV-2 RNA. Meanwhile, liposomes containing RNase exhibited the greatest effect and Ct change at 9 cycles, while liposomes containing both RNase and niclosamide showed the greatest effect and Ct change at 10 cycles. This suggests that liposomes containing RNase cleave coronavirus RNA, reducing RNA concentration by approximately 10%. 3The Ct level was increased by 3 times. Meanwhile, in our experiments, the RNase itself altered Ct by 3 cycles, clearly due to the cleavage of “free” COVID-19 RNA. Notably, liposomes containing both RNase and niclosamide exhibited almost identical effects to those containing only RNase, as expected from the bioactivity of niclosamide.

[0050] The effects of compositions (i) and (ii) were also investigated by gel electrophoresis

[16] using synthetic primers (466 nucleotide pairs) for the N gene.

[0051] Experimental results are as follows Figure 1 As shown.

[0052] Figure 1 : Gel electrophoresis in 3% agarose gel

[16] .

[0053] N gene amplicon samples:

[0054] The original sample that tested positive for COVID-19

[0055] B - Samples after incubation with "empty" liposomes

[0056] Samples after C-incubation with RNase

[0057] Sample after incubation of D with liposomes containing RNase (composition i)

[0058] F- Sample incubated with liposomes containing RNase and niclosamide (composition ii)

[0059] K-negative control – Samples that do not contain the SARS-CoV-2 virus.

[0060] Gel electrophoresis ( Figure 1 PCR analysis showed that incubation of SARS-CoV-2 samples with compositions (i) and (ii) resulted in degradation of the N gene, as no amplification products were observed on gel electrophoresis, indicating that the N gene of SARS-CoV-2 had been cleaved.

[0061] Example 5. Study on the effect of RNase-containing liposomes on SARS-CoV-2 production in vivo.

[0062] The study was conducted on five groups of "Giant" breed rabbits, weighing 1.6-1.8 kg and aged 3-4 months, with five rabbits in each group:

[0063] Group 1 - Negative Control (physiological saline solution)

[0064] Group 2 - Positive control, sample smears containing SARS-CoV-2

[0065] Group 3 - Composition (i)

[0066] Group 4 - Composition (ii)

[0067] Group 5 RNases (1 unit / µL)

[0068] Animals in group 1 were injected with 20 μL of saline solution into their nasal cavities, animals in groups 3 and 4 were injected with 20 μL of composition (i) and (ii), respectively, and animals in group 5 were injected with 20 μL of RNase (1 U / μL). One hour later, all animals (except group 1) were injected with 20 μL of SARS-CoV-2 positive biological material with a viral load of Ct 20. Then, one hour later, samples were collected from the nasal cavities of all animals as described in

[14] . All obtained samples were analyzed by PCR using the SARS-CoV-2 / SARS-CoV Reaction Kit "DNA-Technology" LLC

[15] . The experimental results are summarized in Table 3.

[0069] Table 3

[0070] The effects of compositions (i) and (ii) on the in vivo inhibition of SARS-CoV-2 RNA were investigated by hybridization and fluorescence-assisted quantitative PCR.

[0071]

[0072] As shown in Table 3, no SARS-CoV-2 RNA was detected when animals were injected with composition (i) – group 3 and composition (ii) – group 4. Meanwhile, the mean Ct 26–27 remained almost unchanged in groups 2 and 5.

[0073] The results showed that compositions (i) and (ii) cleaved SARS-CoV-2 RNA, while the free RNase was almost inactive under in vivo conditions.

[0074] The in vivo effects of compositions (i) and (ii) were also investigated using gel electrophoresis. Figure 2

[16]

[0075] Figure 2 0.3% agarose gel electrophoresis

[16] .

[0076] Group 1 - Negative Control (physiological saline solution)

[0077] Group 2 - Positive control, sample smears containing SARS-CoV-2

[0078] Group 3 - Composition (i)

[0079] Group 4 - Composition (ii)

[0080] Group 5 - RNase (1 U / μL)

[0081] from Figure 2 As can be seen, the control sample (initial K+- group 2) contained the N gene fragment of COVID-19, while the N gene of SARS-CoV-2 virus was destroyed under the action of compositions (i) and (ii) (groups 3 and 4).

[0082] Free RNase (Group 5) does not cleave coronavirus RNA in vivo (Table 3 and Group 5).

[0083] Example 6. Stability of liposomes containing RNase (composition (i))

[0084] To determine stability, composition (i) was incubated at 36°C for different time intervals of 1 to 6 hours (Table 4). Then, biological material infected with SARS-CoV-2 coronavirus was added to each sample of composition (i) and incubated again at 36°C for one hour. Experiments were performed as described in Example 4. RNA isolation and detection of SARS-CoV-2 virus were performed using the certified reaction kit “DNA-Technology” LLC

[15] according to standard methods. The experiment was repeated 5 times.

[0085] Table 4 summarizes the data related to the study of the stability of liposome-composition (i) containing RNase by RT-PCR assay.

[0086] Table 4

[0087] The stability of liposome-composition (i) containing RNase was investigated using RT-PCR technology.

[0088]

[0089]

[0090] The table shows that the threshold cycle (Ct) changed by 8 cycles under the influence of composition (i), indicating a reduction in viral load of approximately 103-fold. The mean threshold cycle (Ct) during PCR using liposomes containing RNase (composition i) was found to have almost no change over time intervals of 1 to 6 hours, indicating the stability of liposomes containing RNase (composition i) over 6 hours.

[0091] Example 7. Stability of liposomes containing RNase and niclosamide (composition (ii))

[0092] To determine stability, composition (ii) was incubated at 36°C for different time intervals of 1 to 6 hours (Table 5). Then, biological material infected with SARS-CoV-2 coronavirus was added to each sample of composition (ii) and incubated again at 36°C for 1 hour. Experiments were performed as described in Example 4. RNA isolation and detection of SARS-CoV-2 virus were performed using the certified kit “DNA-Technology” LLC

[15] according to standard methods.

[0093] Table 5

[0094] Stability study of liposomes containing RNase and niclosamide (composition ii) by RT-PCR technology

[0095]

[0096]

[0097] As shown in Table 5, under the action of composition (ii), the threshold cycle (Ct) changed by 8 cycles, indicating that the viral load decreased by approximately 10%. 3 The mean value of the threshold cycle (Ct) during PCR was determined to be almost unchanged over a time interval of 1 to 6 hours, indicating the stability of the liposomes containing RNase and niclosamide (composition ii) over 6 hours.

[0098] Example 8. Safety and toxicity studies of compositions (i) and (ii) in vivo animals (rabbit and mouse) and in vitro cell cultures (fibroblasts and hepatocytes).

[0099] Fibroblasts were obtained using this method

[17] . Rat liver hepatocytes were obtained according to method

[18] .

[0100] The cytotoxic properties of compositions (i) and (ii) were determined by in vitro MTT assays

[19] . Data are presented as mean values ​​(from 3 experiments). The cytotoxic effects of each test sample of the composition were compared to a control of a cisplatin-based cytotoxic inhibitor (manufactured by Teva Pharmaceutical Industries, Ltd., Israel).

[0101] Table 6.

[0102] Cytotoxicity of the composition to fibroblast cultures

[0103]

[0104] Table 7.

[0105] Cytotoxicity of the composition to hepatocytes

[0106]

[0107]

[0108] The data obtained (Tables 6 and 7) show that, within the concentration range of 10–100 μg / ml, the samples did not inhibit the growth of normal cells compared to the control formulation (cisplatin). Therefore, compositions (i) and (ii) do not produce cytotoxic effects on normal cells.

[0109] Example 9. General in vivo toxicity studies of the composition in rabbits and mice.

[0110] The study was conducted in four groups of animals, with five rabbits (Giant strain, weighing 1.6–1.8 kg) and ten hybrid mice (weighing 18–20 g) in each group. The composition under study was injected into the eyes of the animals twice daily (morning and evening), with 50 μl of physiological solution injected into the right eye as a control and 50 μl of the composition injected into the left eye as (i) and (ii), for 5 days.

[0111] Group 1 - Control (physiological saline solution)

[0112] Group 2 - Soybean lecithin liposomes,

[0113] Group 3 - Soybean lecithin liposomes containing RNase (composition (i)),

[0114] Group 4 - Soybean lecithin liposomes containing RNase and niclosamide (composition (ii)).

[0115] Meanwhile, 20 μl of test sample was injected into each nostril of each group of rabbits.

[0116] Five days after the above solution was administered, changes in the rabbits' eyesight and general condition were measured, and the biochemical parameters of the animals' blood (mice and rabbits) were measured according to method

[20] . Macroscopic observation showed that the animals in the experimental and control groups had the same health status.

[0117] The biochemical parameters of the tested animals are summarized in Tables 8 and 9.

[0118]

[0119]

[0120] The data obtained (Tables 8 and 9) show that the values ​​of the main blood parameters were actually no different from the control values, indicating that compositions (i) and (ii) were not toxic to animals.

[0121] Example 10. Biological microscopic study of ocular mucosa in experimental animals (rabbits)

[0122] The effects of liposomes containing compositions (i) and (ii) on mucous membranes were investigated by examining the dynamic control of rabbit eyes.

[0123] During the experiment, the rabbits' eyes (ophthalmic condition) were dynamically controlled using a biological microscope. The biological microscopy studies were conducted with the aid of an ophthalmic biological microscope, specifically a Carl Zeiss (Germany) "SL115" slit lamp, observed daily at magnifications of x10 and x16 throughout the observation period. The conjunctiva, sclera, cornea, anterior chamber, iris, and fundus reflexes were evaluated. Different magnifications (x10, x16) of the biological microscope allowed for a more detailed study of the anterior segment structure of the eye.

[0124] Before the biological microscope study, all the rabbits were tightly restrained, with their heads firmly fixed behind the slit lamp.

[0125] In biological microscopy studies, paired rabbit eyes are considered normal and used as controls during experiments.

[0126] The first examination was performed one day after the injection of compositions (i) and (ii) and soy lecithin liposomes.

[0127] Biological microscopic examination revealed that the eye was stable, without any discharge, with a clear and smooth cornea, pale pink and shiny palpebral conjunctiva, and a white sclera without vascular infusion. The anterior chamber fluid was also clear, without any contamination. The iris was convex, the pupil was round, and it reacted actively to light. All optical media of the eye remained transparent, and the fundus reflected a pinkish hue. No signs of inflammation, toxicity, or allergic reaction were detected.

[0128] Throughout the observation period, all rabbits had the same ophthalmic condition in both eyes, with no clinical signs of inflammation or allergic reactions.

[0129] These data indicate that compositions (i) and (ii) are not toxic to rabbit eyes.

[0130] Source of literature

[0131] 1. Russian Patent 2746362 (Patent Application No. 2021106335 dated March 11, 2021) – “Combination drug with antiviral activity against novel coronavirus SARS-CoV-2”.

[0132] 2. O. N. Ilyinskaya, R. Shakh Makhmud. RIBONUCLEASES AS ANTIVIRAL AGENTS / / MOLECULAR BIOLOGY, 2014, Vol. 48, No. 5, pp. 707-717.

[0133] 3. Wu C, Jan J, et al. and Hsu JTA. Inhibition of Severe Acute Respiratory Syndrome Coronavirus Replication by Niclosamide. Antimicrobial Agents and Chemotherapy, 2004. doi.org / 10.1128 / aac.48.7.2693-2696.2004.

[0134] 4. Ashlee D. Brunaugh, Hyojong Seo, Zachary Warnken, Li Ding, Sang Heui Seo, Hugh D. C. Smyth. Development and evaluation of inhalable composite niclosamide-lysozyme particles: A broad-spectrum, patient-adaptable treatment for coronavirus infections and sequelae. Published: February 11, 2021 doi.org / 10.1371 / journal.pone.0246803.

[0135] 5. http: / / www.prnewswire.com / news-releases / hyundai-bioscience- announced-that-its-covid-19-oral-drug-solved-niclosamides-inhibitory- concentration-problem-301188126.html.

[0136] 6. ANA Therapeutics Begins Phase 2 / 3 Clinical Trial of Proprietary Oral Niclosamide Formulation to Treat COVID-19. Study is the first in the U.S. to test safety and efficacy of niclosamide as an antiviral for COVID-19. October 26, 2020 08:44 AM Eastern Daylight Time.

[0137] 7. Liposomes with a large trapping capacity prepared by freezing and thawing of sonicated phospholipid mixtures. Archives of Biochemistry and Biophysics, 212(1), 186-194. doi: 10.1016 / 0003-9861(81)90358-1( https: / / doi.org / 10.1016 / 0003-9861(81)90358-1)

[0138] 8. Prokhorova M.I. Methods for biochemical analysis (Lipid and energy exchange). Tutorial. Leningrad 1982.

[0139] 9. Sergio C. Oliveira, Mariana T.Q. de and E. Jane Homan. Immunoinformatic Analysis of SARS-CoV-2 Nucleocapsid Protein and Identification of COVID-19 Vaccine Targets. 2020. Front. Immunol. 11: 587615. https: / / doi.org / 10.3389 / fimmu.2020.587615 .

[0140] 10.Kim TW,Lee JH,Hung CF,Peng S,Roden R,Wang MC,Viscidi R,Tsai YC,HeL,Chen PJ,Boyd DA,Wu TC.Generation and Characterization of DNA VaccinesTargeting the Nucleoeapsid Protein of Severe Acute Respiratory SyndromeCoronavirus.2004.J Virol.https: / / doi.org / 10.1128 / JVI.78.9.4638-4645.2004.

[0141] 11.Ya Peng,Ning Du,Yuqing Lei,Sonam Dorje,Jianxun Qi,Tingrong Luo,George F Gao,Hao Song.Structures of the SARS-CoV-2nucleocapsid and theirperspectives for drug design.The EMBO Journal(2020)39:e105938.

[0142] 12.DEPARTMENT OF HEALTH&HUMAN SERVICES Public Health Service Centersfor Disease Control and Prevention(CDC)Atlanta,GA 3033324,Jan 2020.

[0143] 13.Advances in the Synthesis of Oligonucleotides by thePhosphoramidite Approach.Solid-phase amidophosphite method or phosphoramiditemethod.Beaucage S.L.,Iyer R.P.(англ.) / / Tetrahedron.1992.Vol.48,no.12,P.2223-2311.doi:10.1016 / S0040-4020(01)88752-4.

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Claims

1. Use of RNase A molecules in liposomes and niclosamide in the preparation of drugs for the prevention or treatment of coronavirus infection caused by SARS-CoV-2.

2. The use of claim 1, wherein the liposomes are obtained based on phospholipids derived from soybean lecithin or phospholipids of animal origin.

3. The use of claim 1 or claim 2, wherein the drug is formulated for intranasal or inhalation administration.

4. Use of RNase A molecules in liposomes in the preparation of medicaments for the prevention or treatment of coronavirus infection caused by SARS-CoV-2, said liposomes being based on phospholipids derived from soybean lecithin or phospholipids of animal origin.

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