WO2023277730A1

WO2023277730A1 - Drug for the prophylaxis of sars-cov-2 infection

Info

Publication number: WO2023277730A1
Application number: PCT/RU2022/000207
Authority: WO
Inventors: Шахноз Садыковна АЗИМОВА; Талат Саатович СААТОВ; Баходир Равилович ЗАЙНУТДИНОВ; Ойбек Норбой АШИРОВ; Собирджан Анарматович САСМАКОВ; Елена ЛЫСОВА; Артем Александрович МАХНЕВ; Мукаддас Рустамовна УМАРОВА; Умида Бахриддин кизи ХАМИДОВА; Галина Александровна ПИЯКИНА; Джалолиддин Мирджамилович АБДУРАХМАНОВ; Шухрат Шавкатович ХАСАНОВ; Фарход Бакир ЭШБОЕВ
Original assignee: МУСАХАНОВА, Ойгуль Мирзаюсуфовна
Priority date: 2021-07-02
Filing date: 2022-06-30
Publication date: 2023-01-05
Also published as: CN117651550A

Abstract

The invention relates to medicine, pharmaceutics and biotechnology. Proposed is a novel pharmaceutical composition that has an antiviral effect with respect to the SARS-CoV-2 coronavirus and related viruses, such as SARS, MERS and the like, the genome of which is comprised of RNA and the virions of which have a lipid envelope. A combination drug for the prophylaxis (or treatment) of SARS-CoV-2 infection comprises (i) a liposome with an effective amount of RNase enzyme, or (ii) a liposome with an effective amount of RNase enzyme and an effective amount of niclosamide, and an auxiliary substance in the form of a pharmaceutically acceptable solvent. The invention causes the breakdown of genetic material, namely coronavirus RNA, thus making it possible to provide prophylaxis against RNA-containing viruses, including SARS-Cov-2, which enter the human body via the upper respiratory tract.

Description

SARS-COV-2 PREVENTION MEDICINE

DESCRIPTION Field of technology

The invention relates to medicine, pharmaceuticals and biotechnology. A new combined drug (pharmaceutical composition) is presented that has an antiviral effect against the SARS-CoV-2 coronavirus and related viruses, such as SARS-CoV, MERS, etc., whose genome is RNA, and the virions are provided with a lipid membrane, composition (i ) due to the destruction of the coronovirus genome by the enzyme RNase, composition (ii) due to the destruction of the coronovirus genome by the enzyme RNase and the drug niclosamide, which prevents the replication of SARS-CoV-2. The proposed compositions can be used on an outpatient basis to suppress the replication of the SARS-CoV-2 virus, reduce the viral load, and reduce the risk of infection transmission by airborne droplets.

State of the art

The world has developed a number of preventive vaccines, as well as therapies for the treatment of coronavirus infection. There is a patent RU 2746362 (application 2021106335 dated March 11, 2021) - "Combined drug with antiviral effect against the new SARS-CoV-2 coronavirus" [1], which describes "blocking the transmission of infection and reducing the risk of developing clinical complications" due to the use of small interfering RNA (si RNA). The basis of the aforementioned patent is to obtain a combination drug with engineered RNA molecules capable of mediating target-specific suppression of SARS-CoV-2 virus replication. However, no drugs have been proposed that can destroy coronaviruses by directly destroying their genome - single-stranded (+)RNA. It is obvious that the direct destruction of the virus genome can be carried out by the action of the enzyme ribonuclease (RNase) [2], which can cleave the RNA of coronaviruses, or additionally by suppressing the replication of coronaviruses under the action of niclosamide, a drug used to treat parasitic infections. It has now been shown that niclosamide can inhibit the replication of the SARS-CoV-2 virus [3-6].

Disclosure of invention

The combined drug is intended for inhalation or intranasal administration.

The introduction into practice of preparations based on RNases and niclosamide is limited by a number of factors, which include insufficient efficiency and safety of delivery of RNase and niclosamide to viral target particles. The use of liposomes [7] for the delivery of the above drugs (RNase and niclosamide) solves this problem, since SARS-CoV-2, as well as related viruses MERS, SARS-CoV have a lipid envelope that allows transfection (penetration) of liposomes and the introduction of RNase and niclosamide directly into the virus.

Thus, there is currently a need to develop a new antiviral agent capable of destroying coronavirus RNA.

The combination drugs of the present invention include: composition (i) - liposomes delivering an effective amount of RNase and composition (i) - liposomes delivering an effective amount of RNase and an effective amount of niclosamide. These pharmaceutical compositions provide cleavage of SARS-CoV-2 RNA.

Combination medicines can be used as nasal drops or spray, as well as inhalation.

The technical result is to create an effective and safe antiviral agent that ensures the destruction of SARS-CoV-2 RNA. The specified technical result is illustrated by the following examples.

To achieve the above technical result, a combined drug for the prevention or treatment of coronavirus infection caused by SARS-CoV-2 is proposed, containing an effective amount of RNase molecules in liposomes.

Also, to achieve the above technical result, a combined drug for the prevention or treatment of coronavirus infection caused by SARS-CoV-2 is proposed, containing an effective amount of RNase and niclosamide molecules in liposomes.

According to the present invention, the above combination drugs may be intended for intranasal or inhalation administration.

Also, to achieve the above technical result, a method for the prevention or treatment of SARS-CoV-2 infection is proposed, in which the above(s) combined(s) drug(s) are administered simultaneously, separately or sequentially with other therapeutic agents.

Brief description of the drawings

On FIG. 1 shows the results of the experiment according to example 4. On FIG. 2 shows the results of the experiment according to example 5 (gel electrophoresis method).

Implementation of the invention

The above technical result is illustrated by the following examples.

Example 1. Obtaining liposomes with RNase.

Phospholipids of both vegetable (soy lecithin) and animal origin (bovine brain) were used to obtain liposomes. As ribonuclease, a commercial preparation-enzyme RNase A with MM-13.7 kDa, pH optimum - 5, 0-6.0; 50i/mg from Mol BIO HIMEDIA, obtained from bovine pancreas or ribonuclease from bovine pancreas (RNase A) from SamsonMed (Russia).

As phospholipids, an extract from soy lecithin used in the food industry or phospholipids of animal origin (from the brain of cattle) were used.

To obtain liposomes, 1 g of commercial (VEROLEC FLS, China) soy lecithin was dissolved in 20 ml of a chloroform-methanol mixture at a ratio of 2:1. The mixture was kept for 10 hours at a temperature of 2 - 4°C. The mixture was then centrifuged at 4500 rpm for 5 minutes. 1 ml of the supernatant was taken and dissolved in 6 ml of chloroform. Next, the solvent was distilled off in a vacuum (4±2 mm Hg) at a temperature of +40°C. After complete removal of the solvent, 50 μl of Ribonuclease A (N/μl) and 5 ml of saline were added. The resulting mixture was frozen at –20°С for 20 min, then the mixture was thawed with stirring at 23°С for 30 min. (the process of freezing and thawing was repeated four times). Then the resulting mixture was sonicated at 22 kHz, 30 sec in 4 repetitions. In a similar way, liposomes based on phospholipids from the brain of cattle were obtained, which were isolated according to the method [8].

Dosage forms of the pharmaceutical compositions described herein can be obtained by any known method and contain other excipients capable of increasing the stability of compositions (i) and (ii) and RNase transfection.

Example 2 Preparation of liposomes with niclosamide and RNase.

Niclosamide was used (crystalline substance, melting point 227-232 ° C. The IR spectrum of the test sample corresponded to the IR spectrum of anhydrous niclosamide - standard, the structural formula of niclosamide has the following form:

To obtain liposomes containing RNase (composition i) and liposomes containing RNase A and niclosamide (composition ii), 1 ml of lecithin solution (1.075 mm) was mixed with 6 ml of chloroform. Next, the solvent was distilled off in a vacuum (4 ± 2 mmHg) at a temperature of +40°C. After removal of chloroform, 50 μl of Ribonuclease A, 30 μl of niclosamide solution (10% solution in DMSO) and 5 ml of physiological saline were added to the flask. The resulting mixture was frozen at minus 20°С for 20 min, then the mixture was thawed at 23°С for 30 min. with stirring (the process of freezing and thawing was repeated four times). The resulting mixture was then sonicated at 22 kHz for 30 sec. In a similar way, liposomes based on phospholipids from the brain of cattle were obtained, which were isolated according to the method [8]. Dosage forms of the pharmaceutical compositions provided herein may be prepared by any known method and contain other excipients capable of increasing the stability of compositions (i) and (i) and the transfection of RNase and niclosamide. Example 3 Synthesis of primers for the N gene of SARS-CoV-2 The PCR method was used to determine whether SARS-CoV-2 RNA could be cleaved. As a target for determining the possibility of cleavage of the SARS-CoV-2 coronavirus genome by RNase, the N gene was chosen as the most stable for such types of coronaviruses as SARS and MERS, encoding the nucleocapsid [9-11]. Primers and a fluorescent probe were synthesized for the N gene using open international data from the CDC (US Center for Disease Control and Prevention) [12].

Primers were synthesized on an ASM-2000 DNA synthesizer by the phosphoramidite solid-phase method [13]. After synthesis, the oligonucleotides were removed from the solid phase with concentrated 30% aqueous ammonia for 2 hours at a temperature of 60°C. The oligonucleotide solution was evaporated to remove ammonia and precipitated in 70% ethanol in the presence of 0.3M sodium acetate and 50 mM magnesium chloride. Oligonucleotides were precipitated by centrifugation at 14,000 rpm for 20 minutes. The precipitate was washed twice with 70% ethanol and dried at room temperature for 30 minutes.

After synthesis, the oligonucleotides were purified from short incompletely synthesized molecules. To do this, at the end of the synthesis, the last step of removing the DMT group was not carried out; as a result, only fully synthesized oligonucleotides at the 5' end contain a DMT group for purification on cartridges by reverse phase chromatography. Reverse phase chromatography was carried out under the following conditions: oligonucleotides were washed with 5% acetonitrile with sodium chloride at a concentration of 100 mg/ml. To remove the DMT group from oligonucleotides used 3% dichloroacetic acid solution. After removal of the DMT group, 50% acetonitrile in 100 mM Tris buffer pH 8.5 was used to elute the purified oligonucleotides from the cartridge. The purified oligonucleotides were precipitated with 70% ethanol in the presence of 50 mM magnesium chloride, by centrifugation at 14,000 rpm for 20 minutes. The resulting precipitate of oligonucleotides was dissolved in TE buffer pH 8.0 to a concentration of 20 μM. The resulting primer solution was used to carry out the PCR reaction.

Table 1

Sequence of primers and fluorescent probe for N gene

SARS-CoV-2

Using these primers and a probe specific to the N site of the SARS-CoV-2 RNA gene, the content of the virus in biological samples was determined by PCR, and gel electrophoresis of the samples was performed as described in examples 4 and 5.

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

Isolation of RNA and detection of the SARS-CoV-2 virus was carried out according to the standard method using certified DNA-Technology reagent kits [14,15], as well as using primers and a probe synthesized by us. The effectiveness of the action of liposomes was determined by cycle threshold shift (AACt) in PCR analysis. The threshold cycle indicates how much RNA is contained in the samples, and it is inversely proportional to the logarithm of the initial number of copies.

For RNA isolation, we used the DNA-Technology reagent kit and the certified ENKOR kit developed by us. RNA detection in the samples was carried out with the synthesized primers and probe described in Example 3. For PCR analysis, both the DNA-Technology SARS-CoV-2/SARS-CoV reagent kit and the PCR kit developed by us were used under the following conditions: mix 25 µl, 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 dNTP, 400 nm each primer, 200 nM probe, 1 u. polymerase, 25 units. revertases. PCR program: 50°C - 15 minutes; 95°C - 15 minutes; 40 cycles (95°С - 10 seconds; 60°С - 40 seconds, signal reading via FAM channel). For PCR analysis, 5 µl of RNA sample were used.

Samples of human biological material (80 µl of a swab from the nasopharynx) containing SARS-CoV-2 coronavirus were incubated with 20 µl of composition (i) and (i) (obtained as described in examples 1 and 2) for 1 hour at 36° WITH. In parallel, the same samples were incubated with 20 µl of free RNase (1 U/µl), as well as with “empty” liposomes that did not contain RNase. The results of the experiments are presented in table. 2.

table 2

Study of the in vitro effect of liposomes with RNase on the N gene of SARS-CoV-2 PCR method using hybridization-fluorescence detection

As can be seen from the data (Table 2), under the action of “empty” liposomes, the threshold cycle (Ct.) of the RT-PCR reaction remains practically unchanged, which indicates that liposomes themselves cannot destroy SARS-CoV-2 RNA . At the same time, the greatest effect and the shift of Ct occurs under the action of RNase-containing liposomes for 9 cycles, and RNase-containing niclosamide-containing liposomes for 10 cycles, which indicates that RNase-containing liposomes cleave coronavirus RNA and reduce the concentration RNA about 10 ³ times. At the same time, RNase itself in our experiments shifts Ct by 3 cycles, apparently due to the cleavage of “free” COVID-19 RNAs. It should be noted that RNase-containing liposomes with niclosamide showed almost the same effect as RNase-only liposomes, which was expected from the biological activity of niclosamide.

The effect of composition (i) and (i) was also studied by gel electrophoresis [16] using primers synthesized by us for the N gene (466 bp).

The results of the experiment are shown in Fig. one.

Fig.1. Gel electrophoresis in 3% agarose gel [16].

Samples - N gene amplifications:

A - initial sample, positive for COVID-19

B - sample after incubation with “empty” liposomes

C - sample after incubation with RNase

D - sample after incubation with RNase-containing liposomes (composition i) F - sample after incubation with liposomes containing RNase and niclosamide (composition i)

K - negative control - a sample not containing the SARS-CoV-2 virus.

Gel electrophoresis (Fig. 1) PCR analysis revealed that after incubation of positive SARS-CoV-2 samples with compositions (i) and (i) leads to the decomposition of the N gene, since there is no amplification product on gel electrophoresis, which indicates that that cleavage of the N gene of SARS-CoV-2 occurred.

Example 5 Study of the effect of RNase liposomes on SARS-CoV-2 in vivo.

The studies were carried out on 5 groups of rabbits of the Giant breed, weighing 1.6-1.8 kg, 3-4 months old, 5 animals in each group:

Group 1 - negative control (saline)

Group 2 - positive control, swab sample containing SARS-CoV-2 Group 3 - composition (i)

Group 4-composition (ii)

Group 5-RNase (1u/µl)

Animals in group 1 were instilled with 20 µl of physiological saline into the nasal cavity, animals of groups 3 and 4 were injected with 20 µl of compositions (i) and (ii), respectively, animals of group 5 were injected with 20 µl of RNase (1 unit/µl). After 1 hour, all animals (except group 1) were injected with 20 µl of biological material positive for SARS-CoV-2 with a viral load of Ct 20. Then, after 1 hour, samples were taken from the nasal cavity in all animals as described in [14 ]. All obtained samples were analyzed by PCR with the DNA-Technology SARS-CoV-2/SARS-CoV reagent kit [15]. The results of the experiments are presented in table. 3. Table 3

Study of the in vivo effect of compositions (i) and (ii) on SARS-CoV-2 RNA inhibition by a quantitative PCR method using hybridization-fluorescence detection

As can be seen from Table 3, upon administration of compositions (i) - group 3 and compositions (ii) - group 4 to animals, SARS-CoV-2 RNA was not detected. At the same time, in group 2 and group 5, the average value of Ct 26-27 practically does not change.

The results indicate that under the action of compositions (i) and (ii) the RNA of the SARS-CoV-2 virus is cleaved, while free RNase practically does not work in vivo.

The effect of compositions (i) and (ii) in vivo was also studied by gel electrophoresis (Figure 2) [16].

Fig.2. Gel electrophoresis in 3% agarose gel [16].

Group 1 - negative control (saline)

Group 4-composition(s)

Group 5-RNase (1 U/µl)

As can be seen from FIG. 2, in the control samples (initial K+ - group 2) there is a fragment of the N gene of COVID-19, while under the action of compositions (i) and (ii) (gr. 3 and gr. 4) the N gene of the SARS-CoV- virus is destroyed 2.

Free RNase (gr. 5) does not cleave coronavirus RNA in vivo (Table 3 and gr. 5).

Example 6 Stability of Liposomes with RNase (Composition (i))

To determine the stability of the composition (i) were incubated at 36°C for various time intervals from one to six hours (table. 4). Then, biological material with coronavirus infection SARS-CoV-2 was added to each sample of composition (i) and again incubated at 36°C for one hour. The experiment was carried out as described in example 4. RNA isolation and detection of the SARS-CoV-2 virus were carried out according to the standard method using certified DNA-Technology reagent kits [15]. The experiment was carried out in 5 repetitions.

Table 4 shows data on the study of the stability of liposomes with RNase-composition (i) by RT-PCR.

Table 4

Study of the stability of liposomes with RNase-composition (i) by RT-

PCR

From the above table it can be seen that under the action of composition (i) the threshold cycle (Ct) is shifted by 8 cycles, which indicates that the viral load is reduced by about 10 ³ times. At the same time, it was found that the average value of the threshold cycle (Ct) during PCR with liposomes containing RNase (composition i) practically does not change in the time interval from 1 to 6 hours, which indicates the stability of liposomes containing RNase (composition i) for 6 hours .

Example 7 Stability of Liposomes with RNase and Niclosamide (Composition (ii))

To determine the stability of the composition (ii) were incubated at 36°C for various time intervals from one to six hours (table. 5). Then, biological material with SARS-CoV-2 coronavirus infection was added to each sample of composition (ii) and incubated again at 36°C for one hour. The experiment was carried out as described in example 4. RNA isolation and detection of the SARS-CoV-2 virus were carried out according to the standard method using certified DNA-Technology reagent kits [15].

Table 5

Study of the stability of liposomes with RNase and niclosamide (composition ii) by RT-PCR

As can be seen from Table 5, under the action of composition (ii), the threshold cycle (Ct) is shifted by 8 cycles, which indicates that the viral the load is reduced by about 10 times. It was found that the average value of the threshold cycle (Ct) during PCR practically does not change in the time interval from 1 to 6 hours, which indicates the stability of liposomes containing RNase and niclosamide (composition i) for 6 hours.

Example 8 Safety and toxicity study of compositions (i) and (ii) in animals in vivo (rabbits and mice) as well as in cell cultures (fibroblasts and hepatocytes) in vitro.

Fibroblast cells were obtained by us according to the method [17]. Rat liver hepatocytes were obtained according to the method [18].

The cytotoxic properties of compositions (i) and (ii) were determined by the in vitro MTT method [19]. Data are presented as mean value (from 3 experiments). The cytotoxic effect of each of the studied samples of the compositions was compared with the control cytostatic "Cisplatin" (Teva Pharmaceutical Industries, Ltd., Israel).

Table 6

Cytotoxicity of Compositions on Fibroblast Cell Culture

Table 7

Cytotoxicity of compositions on hepatocytes

As can be seen from the data obtained (Tables 6 and 7), in the concentration range of 10 - 100 μg/ml, the samples did not inhibit the growth of normal cells compared to the reference drug (cisplatin). Thus, compositions (i) and (ii) do not have a cytotoxic effect on normal cells.

Example 9 Study of the general toxicity of the compositions in rabbits and mice in vivo

The studies were carried out in vivo on 4 groups of animals, 5 rabbits each (Giant breed, weighing 1.6 - 1.8 kg) and outbred mice (weighing 18 - 20 g), 10 mice per group. The studied compositions were instilled into the eyes of the animals 2 times a day (morning and evening), 50 μl of saline in the right eye - control, and in the left eye 50 μl - composition - (i) and (ii) for 5 days.

The first group - control (physiological solution) The second group - liposomes from soy lecithin,

The third group - liposomes from soy lecithin with RNase (composition

(i))

The fourth group - liposomes from soy lecithin with RNase and niclosamide (composition (s)).

In parallel, in each group of rabbits, 20 μl of the test samples were injected intranasally into each nostril.

After 5 days of administration of the above solutions, the visual changes occurring in the eyes of rabbits and the general condition of the animals were determined, and the biochemical parameters of the blood of animals (mice and rabbits) were taken according to the method [20]. Visual observation showed that the state of health of the experimental and control groups of animals was the same.

Tables 8 and 9 show the biochemical parameters of the studied animals.

Table 8

Biochemical parameters of blood of experimental animals (rabbits)

Table 9

Biochemical parameters of blood of experimental animals (mice)

As can be seen from the data obtained (Table 8 and Table 9), the values of the main blood parameters practically do not differ from the control ones, which indicates that the compositions (i) and (ii) are not toxic in animals.

Example 10. Biomicroscopic examination of the mucous membranes of the eyes of experimental animals (rabbits)

The action of liposomes containing compositions (i) and (i) on mucous membranes was studied by studying the dynamic control of the eyes of rabbits.

During the experiment, dynamic monitoring of the condition of the eyes (ophthalmological status) of rabbits was carried out by biomicroscopy. Biomicroscopic examination was carried out using an ophthalmic biomicroscope - slit lamp "SL 115" company Carl Zeiss (Germany) with a magnification of 10 and 16 times daily during the entire observation period. The state of the conjunctiva, sclera, cornea, anterior chamber, iris, and fundus reflex were assessed. Various degrees of magnification (x10, x16) during biomicroscopy made it possible to study in more detail the state of the structures of the anterior segment of the eye.

Before biomicroscopic studies, all rabbits were tightly swaddled, the head was tightly fixed behind a slit lamp.

In the course of biomicroscopic studies, paired eyes of rabbits were taken as normal and used as a control during the experiments.

The first test was carried out one day after the instillation of compositions (i) and (ii) and soy lecithin liposomes.

When examined by biomicroscopy, it was noted that the eyes were calm without discharge, the cornea was transparent and smooth, the conjunctiva of the eyelids was pale pink and shiny, the sclera was white without vascular injection. The moisture of the anterior chamber was also transparent without admixture of any elements. The iris is embossed, the pupil is round, the reaction to light is lively. The optical media in all eyes remained transparent, the reflex from the fundus was pink. There were no signs of inflammation or toxic-allergic reaction. During the entire observation period, in all rabbits, the ophthalmological status of both eyes was identical without clinical signs of inflammatory and allergic reactions.

These data indicate that the compositions (i) and (i) are not toxic to the eyes of rabbits.

Literature

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Claims

CLAIM

1. The use of a medicinal product containing an effective amount of RNase A molecules in liposomes for the prevention or treatment of coronavirus infection caused by SARS-CoV-2.

2. Use according to claim 1 for intranasal or inhalation administration.

3. The use of a drug containing in liposomes an effective amount of RNase A molecules with niclosamide for the prevention or treatment of coronavirus infection caused by SARS-CoV-2.

4. Use according to claim 3 for intranasal or inhalation administration.

5. A method for preventing or treating SARS-CoV-2 infection, wherein RNase A molecules with niclosamide contained in an effective amount in drug liposomes are administered simultaneously, separately or sequentially.