WO2022091119A1

WO2022091119A1 - A pharmaceutical kit comprising copper sulfate and ascorbic acid

Info

Publication number: WO2022091119A1
Application number: PCT/IN2021/051002
Authority: WO
Inventors: Gunjan Kumar; Anurag Kumar; Tarasenko VOLODYMYR STEPANOVYCH; Noga DAVID ANATOLIIOVYCH; Lysaichuk YURIISERHIIOVYCH; Tarasov OLEKSANDR ANDRIIOVYCH
Original assignee: Gunjan Kumar; Anurag Kumar; Volodymyr Stepanovych Tarasenko; David Anatoliiovych Noga; Yuriiserhiiovych Lysaichuk; Oleksandr Andriiovych Tarasov
Priority date: 2020-10-26
Filing date: 2021-10-22
Publication date: 2022-05-05

Abstract

The present disclosure provides a pharmaceutical kit comprising oral liquid formulation of copper sulfate and oral solid formulation of ascorbic acid for treatment of coronavirus (COVID-19) disease. The pharmaceutical kit acts by preventing replication of viruses with envelopes in general and coronavirus in particular. Additionally, the disclosure provides a systematic method of administering the formulations of the pharmaceutical kit to prevent coronavirus replication/ infection in a subject in need thereof. Further, the disclosure also provides a process to prepare respective oral formulations of the pharmaceutical kit as disclosed in the present disclosure.

Description

Title: A pharmaceutical kit comprising copper sulfate and ascorbic acid formulations and a method thereof

Technical Field

The disclosure relates to a pharmaceutical kit comprising oral pharmaceutical formulations of active agents for sequential administration to subjects to prevent replication of viruses with envelopes in the treatment of viral diseases, including COVID- 19. The disclosure also provides a method to prepare and administer the same to a subject in need thereof.

Background

During the fag end of 2019, Wuhan - the most densely populated city and capital of Hubei Province, China was witnessing pneumonia whose etiology was unknown. At the beginning of 2020, the pathogen was successfully identified and was named as the “2019 novel Corona virus (2019-nCoV)”. Later, on 20^th February 2020, the “International Committee on Taxonomy of Viruses (ICTV)” named it as “Severe Acute Respiratory Syndrome Corona virus 2 (SARS-CoV- 2)”, a new brother of ‘SARS-CoV’, and the disease caused was “Corona virus Disease 2019 (COVID-19)”, 2019 is the year. Since then the whole world is on ‘High Alert’ due to this new viral threat. Millions of people have been (self) quarantined to prevent it from further spreading. Nonetheless, the individuals infected by Corona virus were augmenting every day. There are absolutely no drugs that are approved by regulatory agencies across the globe. In India, the “Indian Council of Medical Research (ICMR)” strongly recommended the usage of hydroxychloroquine for asymptomatic purposes. Followed by this development, Emergency Use Authorization was granted by United States Food and Drug Administration (USFDA) to promote the usage of chloroquine phosphate or hydroxychloroquine sulfate for treating COVID -19. World Health Organization (WHO) identified and recommended usage of remdesivir, lopinavir - ritonavir with or without interferon, immunotherapy and recuperative sera. WHO also launched SOLIDARITY trials for the above-prescribed drugs to support that they are effective in treating COVID- 19. In addition, novel chemical entities against the Corona virus protease were also proposed by some researchers and some have proposed various integrated Chinese and Western medicines for treating this pandemic. Nonetheless, there exist various limitations in coming up with a novel therapy for treatment of coronavirus (COVID-19) infections. Accordingly, there is a strong need of drug formulations for treatment of COVID- 19 disease, a global pandemic.

The present disclosure is directed to address one or more problems as discussed above or any other limitation associated with the prior arts. Additionally, the information disclosed in this background section is only for enhancement of understanding of the general background of the disclosure and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Summary

Accordingly, the present disclosure provides a pharmaceutical kit for treating coronavirus (COVID- 19) disease, comprising a liquid oral solution formulation of copper sulfate having copper concentration ranging from 0.045 % w/v to 0.055 % w/v; preferably 0.050 % w/v along with pharmaceutically acceptable and compatible excipients; solid oral tablet formulation of ascorbic acid, wherein the ascorbic acid dose in first tablet is 500 mg and second tablet is 100 mg along with pharmaceutically acceptable and compatible excipients; and an instruction manual for usage by subjects in need thereof; also disclosed is a process for preparing a pharmaceutical kit for treating coronavirus disease (COVID-19), comprising steps of: dissolving copper sulfate, sodium benzoate, potassium sorbate in purified water under continuous stirring for a time period of 20 minutes to obtain a solution followed by adjusting pH using sulfuric acid to obtain liquid oral solution formulation of copper sulfate; storing the oral solution formulation of copper sulfate in amber colored bottles to protect from light, moisture and temperature; dissolving ethyl cellulose in methylene chloride under continuous stirring for a time period of 10 minutes followed by additional of isopropyl alcohol under continuous stirring for an additional time period of 5 minutes to obtain binder solution; combining mannitol, ascorbic acid, sorbitol, povidone, sodium chloride, colloidal silicon dioxide, lactose monohydrate and sunset yellow in rapid mixer granulation for a tune period of 10 minutes followed by addition of the binder solution and mixing for an additional time ranging from 2 to 5 minutes to obtain mass of wet granules; drying the granules using an inlet temperature of 50 °C to 60 °C and outlet temperature of 40 °C to 50 °C followed by sifting the dried granules using vibratory sifter to obtain dried granules; blending the dried granules by adding sunset yellow lake, orange flavor, sucralose and colloidal silicon dioxide followed by mixing in a cage blender for a time period of 10 minutes at a speed of 10 rpm to obtain granule blend; lubricating the blended and dried granules using magnesium stearate and mixing for a time period of 3 minutes in the cage blender at a speed of 10 rpm followed by punching in a tablet making machine to obtain solid oral tablet formulation of ascorbic acid; and is also disclosed is a method of treating a subject afflicted with coronavirus (COVID-19) disease, comprising administering to the subject the formulations of pharmaceutical kit comprising of the liquid oral solution formulation of copper sulfate and tablet formulation of ascorbic acid, wherein liquid oral solution formulation of copper sulfate is administered first followed by a gap of 40 to 60 minutes to administer the solid oral tablet formulation of ascorbic acid.

Brief description of the accompanying drawings

The features of the present disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments in accordance with the disclosure and are therefore not to be considered limiting of its scope the disclosure will be described with additional specificity and detail through use of the accompanying drawings.

Figure 1: shows control of SNEV culture (transplanted culture of pig kidney cells)

Figure 2: shows control of the virus on SNEV (transplanted culture of porcine kidney cells)

Figure 3:

Figure 4: shows the results of studying the effect of Cu on the reproduction of influenza virus Figure 5: shows bar graph of Cu and ascorbic acid inhibiting reproduction of influenza virus.

Figure 6: shows Plaque assays of swine gastroenteritis virus strain D52-100PTC

Figure 7: shows characteristics of swine gastroenteritis virus

Figure 8: shows electrophoretic analysis of the products of amplification of porcine gastroenteritis virus with a primer to the nucleoprotein gene of oligonucleotide primers

Figure 9: shows Infectious titer of TGE virus in wells treated with different dilutions of drugs and TGE virus 100 ID50

Detailed Description

Before explaining any one embodiment of the present disclosure by way of drawings, experimentation, results, and pertinent procedures, it is to be understood that the disclosure is not limited in its application to the details as explained in below embodiments set forth in the following description or illustrated in the drawings, experimentation and/or results. The disclosure is further capable of other embodiments which can be practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary— not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

Definitions:

For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising” , will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to ” are used interchangeably.

The term “subject(s)” shall mean human being(s).

The present disclosure relates to a pharmaceutical kit for treating coronavirus (COVID-19) disease, comprising of a liquid oral solution formulation of copper sulfate having copper concentration ranging from 0.045 % w/v to 0.055 % w/v; preferably 0.050 % w/v along with pharmaceutically acceptable and compatible excipients; solid oral tablet formulation of ascorbic acid, wherein the ascorbic acid dose in first tablet is 500 mg and second tablet is 100 mg along with pharmaceutically acceptable and compatible excipients; and an instruction manual for usage by subjects in need thereof.

In another embodiment of the present disclosure, the liquid oral solution formulation of copper sulfate comprises of sodium benzoate at a concentration of 0.050 % w/v, potassium sorbate at a concentration of 0.150 % w/v and sulfuric acid 0.057 % w/v.

In yet another embodiment of the present disclosure, the fist tablet formulation of ascorbic acid is an uncoated tablet comprising of ascorbic acid at a concentration of 58.824 % w/w, mannitol at a concentration of 14.118 % w/w, sorbitol at a concentration of 4.118 % w/w, povidone at a concentration of 3.529 % w/w, sodium chloride at a concentration of 0.118 % w/w, colloidal silicon dioxide at a concentration of 2.941 % w/w, lactose monohydrate at a concentration of 7.059 % w/w, sunset yellow lake at a concentration of 1.059 % w/w, ethyl cellulose at a concentration of 3.529 % w/w, quantity sufficient of methylene chloride and isopropyl alcohol, orange flavor at a concentration of 1.176 % w/w, colloidal silicon dioxide and sucralose each at a concentration of 0.588 % w/w and magnesium stearate at a concentration of 2.353 % w/w.

In yet another embodiment of the present disclosure, the second solid oral tablet formulation of ascorbic acid is an uncoated tablet comprising of ascorbic acid at a concentration of 33.333 % w/w, mannitol at a concentration of 32.933 % w/w, sorbitol at a concentration of 3.333 % w/w, sodium chloride at a concentration of 0.0607 % w/w, colloidal silicon dioxide at a concentration of 2.333 % w/w, lactose monohydrate at a concentration of 20.0 % w/w, sunset yellow lake at a concentration of 1.0 % w/w, povidone at a concentration of 3.333 % w/w, quantity sufficient of isopropyl alcohol, orange flavor and colloidal silicon dioxide each at a concentration of 0.667 % w/w, sucralose at a concentration of 0.333 % w/w and magnesium stearate at a concentration of 2.0 % w/w.

In still yet another embodiment of the present disclosure, said kit prevents replication of coronavirus.

In still yet another embodiment of the present disclosure, the liquid oral solution formulation of copper sulfate is having pH ranging from 2.0 to 2.5 and the solution is stored in amber colored glass bottle having a white closure.

The present disclosure provides a process for preparing a pharmaceutical kit for treating coronavirus disease (COVID-19) as explained in the aforementioned embodiments, comprising steps of dissolving copper sulfate, sodium benzoate, potassium sorbate in purified water under continuous stirring for a time period of 20 minutes to obtain a solution followed by adjusting pH using sulfuric acid to obtain liquid oral solution formulation of copper sulfate; storing the oral solution formulation of copper sulfate in amber colored bottles to protect from light, moisture and temperature; dissolving ethyl cellulose in methylene chloride under continuous stirring for a time period of 10 minutes followed by additional of isopropyl alcohol under continuous stirring for an additional time period of 5 minutes to obtain binder solution; combining mannitol, ascorbic acid, sorbitol, povidone, sodium chloride, colloidal silicon dioxide, lactose monohydrate and sunset yellow in rapid mixer granulation for a time period of 10 minutes followed by addition of the binder solution and mixing for an additional time ranging from 2 to 5 minutes to obtain mass of wet granules; drying the granules using an inlet temperature of 50 °C to 60 °C and outlet temperature of 40 °C to 50 °C followed by sifting the dried granules using vibratory sifter to obtain dried granules; blending the dried granules by adding sunset yellow lake, orange flavor, sucralose and colloidal silicon dioxide followed by mixing in a cage blender for a time period of 10 minutes at a speed of 10 rpm to obtain granule blend; lubricating the blended and dried granules using magnesium stearate and mixing for a time period of 3 minutes in the cage blender at a speed of 10 rpm followed by punching in a tablet making machine to obtain solid oral tablet formulation of ascorbic acid. In another embodiment of the present disclosure, said tablet formulation of ascorbic acid is packed in alu-alu foil.

The present disclosure is in relation to a method of treating a subject afflicted with coronavirus (COVID-19) disease, comprising administering to the subject the formulations of pharmaceutical kit as explained in the aforementioned embodiments, wherein liquid oral solution formulation of copper sulfate is administered first followed by a gap of 40 to 60 minutes time to administer the solid oral tablet formulation of ascorbic acid.

The method proposed in the present disclosure is the sequential (with interval of 40-60 minutes) intake of well-known components: copper sulfate crystalline hydrate solution (CuSo4-5H2O) and ascorbic acid. Reactive grade copper sulfate is used. The solution of concentration from 0.05% to 1% is used.

Solution preparation method (per one liter): weighted amount is placed into a measuring vessel and 200-300 ml of distilled water is added and swirled. Upon dissolution of the crystalline hydrate, more distilled water is added to 950ml. Then concentrated sulfuric acid H2SO4 (96%) is dripped into the solution in the amount of 5-12 drops (0.1 - 0.15ml). Next, distilled water is added to top up to 1 liter and pH of the solution is measured. The pH level should not exceed 2.5.

The second component of the kit is ascorbic acid formulation. It is preferable that ascorbic acid be in the form of tablets (from lOOmg to 500mg) and protected with a vacuum packing to prevent contact with air, which could lead to loss of regenerating abilities of ascorbic acid. Sodium salt of ascorbic acid is not used.

In the method disclosed in the present disclosure, Img of the substance is taken into the human body (approximately 0.22mg equivalent of copper), which is 10- 25 times less physiological daily intake. The ability of copper ions to form complexes with the simplest amines (ammonia, ethanolamine, di-ethanolamine, et al.) is well studied. Complexes with amino acids, predominantly with histidine, have been isolated from blood plasma relatively recently.

In the method of the present disclosure, after the first intake of the component, 40- 60 minute period is sufficient for the reaction of complex formation of copper ions (Cu²⁺) and proteins of the viral envelopes. Subsequently, loading dose of ascorbic acid (from 0.4gto 1.5g) is taken. It is a powerful healer. The method proposed here works by preventing penetration of viruses into a cell by use of the chemical reduction/oxidation reaction on the surface of viral envelop, leading to formation of oxygen containing free radical, destroying the structure of the envelope of virus. As the result the virus replication is prevented.

Additionally, the disclosure is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope of the present invention. On the contrary, it is to be clearly understood that various other embodiments, modifications, and equivalents thereof, after reading the description herein in conjunction with the drawings and appended claims, may suggest themselves to those skilled in the art without departing from the spirit and scope of the presently disclosed and claimed invention.

Example 1: To determine the antiviral activity of the drug Cu + ascorbic acid in models of influenza virus, herpes and coronavirus of transmissible porcine gastroenteritis in vitro. The material and research methods are recited below: Preparation: Prepare 0.1% solution of copper sulfate crystal hydrate, 20-30 drops of which are dissolved in 40-60 ml of distilled or boiled water.

Cell cultures: Cell cultures were obtained from the collection of the Museum of Tissue Cultures of the D.I.Ivanovsky Institute of Virology (Russian Academy of Medical Sciences, Moscow): BHK is a transplanted culture of baby hamster kidney cells MDCK is a transplanted culture of canine kidney cells SNEV is a transplanted culture of swine embryonic kidney cells BPK is a transplanted culture of piglet kidney cells ST is a transplanted culture of piglet testicular cells PTC is a transplanted culture of pig thyroid cells

Vero is a transplanted culture of African green monkey kidney cells

Viruses:

Influenza virus - a strain of influenza virus obtained from the Museum of Viruses of the Institute of Virology was used during the work. D.I.Ivanovsky (RAMS Moscow) - strain A/FM/ 1/47 (HINT) - infectious titer of allantoic culture 9.0 1g EIDso/O.Tml, hemagglutinin titer - 1:512 HAU/0.2ml.

Herpes simplex virus type 2 (HSV-2) is BH strain obtained from the Museum of Viruses of the DI Ivanovsky Institute of Virology of the Russian Academy of Medical Sciences. The virus was maintained by serial passages in Vero cell culture. Infectious titer of CPE in cell culture was 6.5 1g TCDso/O.l ml. Prior to the experimental studies, the virus was stored at -70°C.

TGEV is an etiological agent of transmissible porcine gastroenteritis (TGE), a highly contagious intestinal disease of pigs.

Virus strain:

D52-5 (BRE79) is a highly pathogenic virus of pigs of all ages at the 5^th passage level in transplanted monolayer culture of testicular cells of piglets ST. The tropism of the virus of the gastrointestinal tract and respiratory tract is shown. The strain was provided by Dr. Hubert Laude from the Laboratory of Molecular Virology and Immunology of the INRA Research Center for Biotechnology in Jouy-en-Josas (France).

Infection titration: Titration of infectivity of viral materials on cell cultures was performed by two methods -end-point dilutions by CPE (Figure 1) and the titer of infectivity was calculated by the method of Kerber-Ashmarin and determined in TCDso/mL, and the method of plaque assay (S-sign) under 1.35% agar overlay (Difco-Bacto) (Figure 2), and the titer of infectivity was measured in PFU/mL. The results were calculated after 120 hours of cultivation at 38 °C.

Example 2: Determination of cytotoxic concentration of drugs (CC50)

Different cell cultures were used to determine the CC50 of the drugs. The experiments used at least ten rows of wells in plates with cell culture for each dilution of the drug in a nutrient medium. Cell culture plates were incubated at 37°C with 5% CO2 for 5 days. Experimental and control cultures were observed daily to determine the presence or absence of cytopathogenic activity (JRS).

The degree of CPE was determined by changes in cell morphology (rounding, cell shrinkage, rejection from the surface of the holes degeneratively altered by 4+ plus system from + to ++++:

- complete absence of cell degeneration;

"+" - affected no more than 25% (75% monolayer cells protected by the antiviral medicine);

"++" - affected no more than 50% of the cell monolayer;

“+++” - no more than 75% of the cell monolayer is affected;

"++++" - complete degeneration of the cell monolayer.

According to the IPC of the drug was taken the largest amount, which did not cause cell degeneration. The determination of cytotoxic concentration (CC50) of Cu, ascorbic acid is discussed below:

To determine the cytotoxic concentration of Cu, ascorbic acid used cells MDCK, BHK, SNEV. The experiments used at least ten rows of wells in cells with cell cultures for each dilution of extracts in a nutrient medium. Cell culture plates were incubated at 37°C with 5% CO2 for 5 days. Test and control samples were examined daily to detect the presence or absence of cytopathic effect (CPE) in the cells. The degree of CPE was determined by changes in cell morphology (rounding, cell shrinkage, rejection from the surface of the wells of degeneratively altered cells in the 4+ system from + to ++++. CC50 was taken as its highest concentration, which did not cause cell degeneration. Refer table 1 for the results on determination of CC50 for copper, its ions and ascorbic acid in MDCK cells sensitive to influenza virus. Table l.The results of the determination of CC50) Cu, Cu + ascorbic acid in the culture of MDCK cells sensitive to influenza virus

The numerator is the number of holes with monolayer degeneration

The denominator is the number of holes taken in the experiment Analysis of the presented results of studies on the cytotoxic effect of the studied drugs Cu, ascorbic acid shows that the drugs were toxic at high dilutions. For Cu CC50-1: 500, ascorbic acid - 1:1,000. The results of determining the CC50 extracts No. 1, No. 240, No. 241 in cell culture of BHK are presented in table 2. Table 2.The results of the determination of CC50 Cu, ascorbic acid in the culture of BHK cells sensitive to the herpes virus

The numerator is the number of holes with monolayer degeneration

The denominator is the number of holes taken in the experiment

The same pattern is observed in the culture of BHK cells as in MDCK cells. Table 3.The results of the determination of CC50 Cu, ascorbic acid in the culture of SNEV cells sensitive to coronavirus TGE

The numerator is the number of holes with monolayer degeneration

The denominator is the number of holes taken in the experiment

In the culture of SNEV cells, the same pattern is observed as in the cells of MDCK and BHK, CC50 Cu solution, ascorbic acid was equal to the dilution of Cu-1: 500, ascorbic acid-1: 1 thousand, refer table 3 for data.

Anti-influenza activity of drugs: To determine the antiviral activity of Cu in vitro, we used a daily transplanted culture of MDCK cells (dog kidney cells) with a solid layer. Cells were washed three times with trypsin solution in nutrient medium at 50 pl per well, then the growth medium was removed and the cells were added to the test drugs in various concentrations and introduced influenza virus at a dose of 100 TCD50. The cultures were incubated in a thermostat with CO2 supply for 3 days, daily monitored with a microscope (the occurrence of cytopathic action), figure 3.

After 48-72 h of cell incubation, the culture fluid was collected and the infectious titer of influenza virus was determined by titration in cell culture. Figure 4 presents the results of studying the effect of Cu on the reproduction of influenza virus. The experiment used influenza virus, strain A/FM/1/47 (H1N1), infectious titer in MDCK 10.0 1g ID50. According to the results shown in the graphs, refer figure 5 (a & b) of Cu, ascorbic acid effectively inhibited the reproduction of influenza virus, from a dilution of 1:1,000 to 1:64,000 from 7.0 to 4.0 lgTCD50. Study of anti-herpetic activity of Cu, ascorbic acid: To study the antiviral activity of Cu, ascorbic acid used transplanted cell culture of BHK and herpes virus type 2 strain VN, infectious titer of 3.0 1g TCD50. To study the antiviral activity of the drugs, daily cultures of BHK cells with a continuous monolayer of cells were selected. The growth medium was drained, the monolayer of cells was added to the test drug in various concentrations. After 1 h of contact, the herpes virus was added at a dose of 100 TCD50. Cultures were incubated in a thermostat with CO2 supply for 5 days, daily monitoring with a microscope and noting the reproduction of the virus by cytopathic action of HSV on BHK cells compared with control cultures, where the monolayer was not exposed to any effects. The cytopathic effect of HSV on cells is morphologically manifested in the formation of symplasts or round cells in combination with the proliferation and appearance of giant multinucleated cells. After 3 days, the culture medium was collected from the wells of the plates and it was determined by the infectious titer in each sample with the introduction of Cu. The results of HSV-2 reproduction with the introduction of drugs are presented in figure 6.

Characteristics of swine gastroenteritis virus (TGE): Transmissible swine gastroenteritis (TGE) corona viruses matched on different cultures and were characterized by infectious titer. The results are presented in the table 4, also refer figure 7.

Table 4: Infectious titer of TGE virus in cell cultures

The neutralization virus reaction was performed in 96-well plates Costar (USA), according to the method of H. Laude, using as a positive control reference hyperimmune serum N6926.

Example 3: Determination of effective concentration (EC50)

EC50 is the minimum concentration of the drug that inhibits the development of virus-specific CPE by 50%. To determine the EC50 test virus at a dose of 100 TCD50 / 0.1 ml was introduced into cell culture and incubated for 60 min at 37 °C. After adsorption of the virus on the cells, its residues were removed, the cells were washed with nutrient medium and then in maintenance medium (RPMI- 1640 + 2% fetal serum) made drugs in different concentrations. The absence of CPE in the experiment (in treated cultures), in the presence of it in the control, as well as the reduction of infectious titer in treated cultures, in the presence of it in control and the difference in infectious titers in the experiment compared with influenza virus control allowed to detect EC50.

Example 3a: Determination of selectivity index (SI) of drugs: The selectivity index (SI) of the drugs was determined by establishing the ratio of IPC to the minimum active concentration.

Example 4: Detection of RNA of transmissible gastroenteritis virus in pigs D52 by reverse polymerase chain reaction (RT-PCR)

RNA isolation was performed using a set of "Fish-sorb" in accordance with the manufacturer's instructions (AmpliSens, Russia). The reverse transcription reaction was performed using the "Revert Aid TM H Minus First Strand cDNA Synthesis Kit" kit according to the manufacturer's instructions (ThermoScientific, Eithuania). Nucleoprotein gene-specific oligonucleotide primers of the following sequence were used for PCR: direct Uni_l (5'-TGCACTGATCAATGTGCTAG-3 ') and reverse Uni_2 (5'-TGAAAACACTGTGGCACCCTT-9m). marker "100 bp Plus DNA Ladder" ("Thermo Fisher Scientific", Lithuania). RNA isolation was performed using a set of "Fish-sorb" according to the manufacturer's instructions (AmpliSens, Russia). The reverse transcription reaction was performed using the "Revert Aid™ H Minus First Strand cDNA Synthesis Kit" kit according to the manufacturer's instructions (Thermo Scientific, Lithuania). For PCR were used specific to the nucleoprotein gene oligonucleotide primers of the following sequence: direct Uni_l (5'-TGCACTGATCAATGTGCTAG-3 ') and reverse Uni_2 (5'-TGAAAACACTGTGGCACCCTT-3 ", 30F). Marker "100 bp Plus DNA Ladder" ("ThermoFisherScientific", Lithuania). A fragment amplified to size 309 P.M. .. M - marker "100 bpPlus DNA Ladder" ("Thermo Fisher Scientific", Lithuania). CBT virus from different cultures was propagated in SNEV culture and amplification products were analyzed by distributing DNA fragments in 1.5% agarose gel. The electrophoretic analysis, refer figure 8, of the products of amplification of porcine gastroenteritis virus with a primer to the nucleoprotein gene of oligonucleotide primers of the following sequence: direct Uni_l (5'- TGCACTGATCAATGTGCTAG-3 ') and reverse Uni_2 (5'ACTGTA). M. .. M - marker "100 bpPlus DNA Ladder" (ThermoFisherScientific, Lithuania).

M is a marker of the size of RNA fragments

No. 1 - strain of coronavirus D52 with BPK

No. 3a - strain of coronavirus D52 with SNEV

No. 4 - D52 in cell culture ST (swine testicular cell)

No. 6a - D52 in the culture of the thyroid gland of pigs (PTC)

Study of Cu antiviral activity in a model of Transmissible Gastroenteritis (TGE) swine coronavirus: The above substances for reproduction of coronavirus (TGE) were studied using the following scheme of drug administration:

- simultaneous introduction of TGE virus and test drugs, i.e. during the adsorption of TGE on cells;

To study the anti-coronavirus activity of the drugs used transplanted culture of SNEV cells. Cells were grown in plates on RPML1640 medium + 10% fetal serum at 37°C in a thermostat with CO2 supply.

A strain of swine gastroenteritis virus with an infectious titer of 3.0 to 4.0 1g ID50 was used. To study the antiviral activity of the test solutions, daily cultures of SNEV cells were selected. The nutrient medium was drained, on the monolayer of cells made test drugs in different concentrations. After 1 hour of contact, the virus was introduced at a dose of 100 TCD50. Cultures were incubated in a thermostat with CO2 supply for 2 days, daily monitoring with a microscope and noting the reproduction of the virus by cytopathogenic action of TGEon SNEV cells in comparison with control cultures, where the monolayer was not exposed to any effects.

The cytopathogenic effect of the coronavirus TGE on cells is morphologically manifested small cell degeneration. After 3 days, the culture medium was collected from the wells of the dies and it determined the infectious titer. Determination of anti-coronavirus activity of drugs (EC50) in the culture of SNEV cells are presented in tables and graphs. Figure 9 shows Infectious titer of TGE virus in wells treated with different dilutions of drugs and TGE virus 100 ID50

According to the results, it was found that the studied drugs statistically significantly inhibit the reproduction of coronavirus TGE.

The criterion for assessing the inhibitory activity of antiviral drugs in in vitro systems is the selectivity index (SI) greater than 16 and a decrease in infectious titer by 1.5-2.01g TCD50.

Table 5 presents the summarized results of studies to determine CC50, EC50, SI of the studied drugs.

Table 5: Indicators CC50, EC50, SI in determining the antiviral activity of the studied solutions in the culture of SNEV cells in the model of coronavirus transmissible gastroenteritis of pigs.

Thus, according to the selectivity index, the most active drug against three viral infections is a solution of Cu. The selectivity index for herpes viruses and TGE of the composition of Cu and ascorbic acid was 32 or more, which allows to consider the drugs as a promising antiviral drug.

Example 5: Statistical processing of research results

The digital material presented in the work is processed variationally and statistically. Statistical evaluation of the levels of significance of differences in the obtained figures was performed using Student s t-test using Microsoft Excel and Microcal Origin. Differences at p<0.05 were considered significant.

Example 6: Preparation of copper sulfate oral solution

Example 6a: Compatibility of the active substance with excipients:

The compatibility studies of active ingredient, copper sulfate pentahydrate, was carried out with excipients under stressed conditions of 60 °C for 7, 15, 30 and 90 days and 80 °C for 7, 15, 30 and 90. This aided in ruling out apparently incompatible excipients. The drug, copper sulfate, and the excipients namely sodium benzoate, potassium sorbate, and others) were mixed in the 1:1 ratio to prepare a binary mixture. The results of compatibility study of the drug substance with excipients indicated no change or no noticeable reaction between the drug and excipients when tested at both the stressed conditions.

Example 6b: Oral solution preparation:

The formula to prepare the copper sulfate oral solution is provided in Table 6.

The oral solution of copper sulfate is prepared by dissolving required quantities of copper sulfate pentahydrate, sodium benzoate and potassium sorbate in purified water under continuous stirring in a manufacturing vessel. The stirring was carried out for 2 minutes to obtain a clear transparent to light blue solution. Thereafter, 100 ml of purified water was taken and concentrated sulfuric acid was added and this solution is used for adjusting the pH of the final copper sulfate oral solution having pH ranging between 2.0 to 2.5. The final formulation was subjected to various characterization studies as shown in Table 7. The Container closure system for Copper sulfate oral solution 0.05 % w/v been designed in a way to protect the finished product from light, temperature and moisture as these environmental factors may affect the product quality.

Table 7: Characterization of copper sulfate oral solution

Preparation of ascorbic acid tablet formulation

Example 7a: Compatibility of the active substance with excipients:

The compatibility studies of active ingredient, ascorbic acid, was carried out with excipients under stressed conditions of 60 °C for 7, 15, 30 and 90 days and 80 °C for 7, 15, 30 and 90 days. This aided in ruling out apparently incompatible excipients. The drug, ascorbic acid along with each of the excipients namely mannitol, ethyl cellulose, sorbitol, povidone, sodium chloride, colloidal silicon dioxide, lactose monohydrate, color sunset yellow lake, orange flavor DC50100, magnesium stearate and sucralose were combined in a ratio of 1:1 to prepare a binary mixture. The results of compatibility study of the drug substance with excipients indicated no change or no noticeable reaction between the drug and excipients when tested at both the stressed conditions and the initial colors of the binary mixtures were maintained. Example 7b: Ascorbic acid tablet formulation (500 mg and 100 mg) - first and second tablet

The formula to prepare ascorbic acid tablet formulation is shown in table 8 and 9.

Table 8: Ascorbic acid tablet formulation (500 mg)

Table 9: Ascorbic acid tablet formulation (100 mg)

a) Dispensing: The materials are dispensed as per the approved SOP and BMR. All materials were be checked and verified by manufacturing chemist and QA personnel, observation shall be recorded in the BMR. b) Sifting: Before start check the integrity of sieve and sift the material separately with respective sieves as given below and collect in different double lined poly bag. c) Binder preparation: Prescribed quantity of ethyl cellulose and dissolve in methylene chloride with continuous stirring for 10 minutes. Add isopropyl alcohol mix with stirrer for 5 minutes. d) Dry & Wet mixing: Add ingredients for dry mixing into rapid mixer granulator and mix for 10 minutes on slow impeller speed and chopper off. Add binder solution gradually into dry mix powder at slow impeller speed and chopper off for 2 minutes. After racking, the materials for kneading at fast impeller and fast chopper speed till the desired wet mass granules achieved. Extra solvent to be added if required. e) Drying: The fluid bed dryer bowl containing the granular mass under the retarding chamber and ensure proper locking with retarding chamber. Fix the bowl with the body of FBD by putting the air drying for 10 minutes. Dry the granules at 50 °C to 60 °C inlet temperature till 40 °C to 50 °C outlet temperature is achieved. Granules were subjected to wet sizing, if needed. Foss on drying should be 1.5-2.5% w/w. f) Sizing: Sift the dried granules through vibratory sifter fitted with 18# sieve. Mill the oversize granules retained on sieve of vibratory sifter using multi-mill with 1.5 mm screen. g) Blending and lubrication: Load the above dried granules and add sifted colour sunset yellow lake, orange flavour, sucralose and colloidal silicon dioxide into the cage blender and mix for 10 minutes at a speed of 10 rpm. Add sifted Magnesium stearate as lubricant in to the above cage blender and mix for additional 3 minutes at same speed of 10 rpm. h) Punching the final blend in a tablet punching machine to obtain tablets of ascorbic acid having dose of 500 mg or 100 mg for oral use.

The prepared tablets were subjected to further characterization and the results are shown in Table 10. The final tablet formulation is packed in alu-alu foil and stored. Table 10: Characterization studies on ascorbic acid tablets

While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

We claim:

1) A pharmaceutical kit for treating coronavirus (COVID-19) disease, comprising: a) liquid oral solution formulation of copper sulfate having copper concentration ranging from 0.045 % w/v to 0.055 % w/v; preferably 0.050 % w/v along with pharmaceutically acceptable and compatible excipients; b) solid oral tablet formulation of ascorbic acid, wherein the ascorbic acid dose in first tablet is 500 mg and second tablet is 100 mg along with pharmaceutically acceptable and compatible excipients; and c) an instruction manual for usage by subjects in need thereof.

2) The pharmaceutical kit as claimed in claim 1, wherein said liquid oral solution formulation of copper sulfate comprises of sodium benzoate at a concentration of 0.050 % w/v, potassium sorbate at a concentration of 0.150 % w/v and sulfuric acid 0.057 % w/v.

3) The pharmaceutical kit as claimed in claim 1, wherein the fist tablet formulation of ascorbic acid is an uncoated tablet comprising of ascorbic acid at a concentration of 58.824 % w/w, mannitol at a concentration of 14.118 % w/w, sorbitol at a concentration of 4.118 % w/w, povidone at a concentration of 3.529 % w/w, sodium chloride at a concentration of 0.118 % w/w, colloidal silicon dioxide at a concentration of 2.941 % w/w, lactose monohydrate at a concentration of 7.059 % w/w, sunset yellow lake at a concentration of 1.059 % w/w, ethyl cellulose at a concentration of 3.529 % w/w, quantity sufficient of methylene chloride and isopropyl alcohol, orange flavor at a concentration of 1.176 % w/w, colloidal silicon dioxide and sucralose each at a concentration of 0.588 % w/w and magnesium stearate at a concentration of 2.353 % w/w.

4) The pharmaceutical kit as claimed in claim 1, wherein the second solid oral tablet formulation of ascorbic acid is an uncoated tablet comprising of ascorbic acid at a concentration of 33.333 % w/w, mannitol at a concentration of 32.933 % w/w, sorbitol at a concentration of 3.333 % w/w, sodium chloride at a concentration of 0.0607 % w/w, colloidal silicon dioxide at a

23 concentration of 2.333 % w/w, lactose monohydrate at a concentration of 20.0 % w/w, sunset yellow lake at a concentration of 1.0 % w/w, povidone at a concentration of 3.333 % w/w, quantity sufficient of isopropyl alcohol, orange flavor and colloidal silicon dioxide each at a concentration of 0.667 % w/w, sucralose at a concentration of 0.333 % w/w and magnesium stearate at a concentration of 2.0 % w/w. ) The pharmaceutical kit as claimed in claim 1, wherein said kit prevents replication of coronavirus. ) The kit as claimed in claim 1, wherein said liquid oral solution formulation of copper sulfate is having pH ranging from 2.0 to 2.5 and the solution is stored in amber colored glass bottle having a white closure. ) A process for preparing a pharmaceutical kit for treating coronavirus disease (COVID-19) as claimed in claims 1 to 6, comprising steps of: a) dissolving copper sulfate, sodium benzoate, potassium sorbate in purified water under continuous stirring for a time period of 20 minutes to obtain a solution followed by adjusting pH using sulfuric acid to obtain liquid oral solution formulation of copper sulfate; b) storing the oral solution formulation of copper sulfate obtained in step (a) in amber colored bottles to protect from light, moisture and temperature; c) dissolving ethyl cellulose in methylene chloride under continuous stirring for a time period of 10 minutes followed by additional of isopropyl alcohol under continuous stirring for an additional time period of 5 minutes to obtain binder solution; d) combining mannitol, ascorbic acid, sorbitol, povidone, sodium chloride, colloidal silicon dioxide, lactose monohydrate and sunset yellow in rapid mixer granulation for a time period of 10 minutes followed by addition of the binder solution obtained under step (c) and mixing for an additional time ranging from 2 to 5 minutes to obtain mass of wet granules; e) drying the granules obtained under step (d) using an inlet temperature of 50 °C to 60 °C and outlet temperature of 40 °C to 50 °C followed by sifting the dried granules using vibratory sifter to obtain dried granules; f) blending the dned granules of step (e) by adding sunset yellow lake, orange flavor, sucralose and colloidal silicon dioxide followed by mixing in a cage blender for a time period of 10 minutes at a speed of 10 rpm to obtain granule blend; g) lubricating the blended and dried granules of step (f) using magnesium stearate and mixing for a time period of 3 minutes in the cage blender at a speed of 10 rpm followed by punching in a tablet making machine to obtain solid oral tablet formulation of ascorbic acid. ) The process as claimed in claim 8, wherein said tablet formulation of ascorbic acid is packed in alu-alu foil. ) A method of treating a subject afflicted with coronavirus (COVID-19) disease, comprising administering to the subject the formulations of pharmaceutical kit as claimed in claims 1 to 8, wherein liquid oral solution formulation of copper sulfate is administered first followed by a gap of 40 to 60 minutes time to administer the solid oral tablet formulation of ascorbic acid. 0) The method as claimed in claim 9, wherein said subjects are human beings.