Composition for use in the prevention and the treatment of infections with sars-cov-2 viruses

Abstract

A composition for use as a drug in a method of treating and preventing SARS-CoV-2 viruses causing COVID-19 in a host, typically a human being, characterised by being composed of a compound of Potassium Bicarbonate and D-Ribose in aqueous solution.

Description

[0001] COMPOSITION FOR USE IN THE PREVENTION AND THE TREATMENT OF INFECTIONS WITH SARS-COV-2 VIRUSES

[0002] * * *

[0003] DESCRIPTION

[0004] The present invention relates to a composition usable as an oral administration drug in a method of treating and preventing SARS-CoV-2 viruses causing CO VID-19 in a host, typically a human being.

[0005] The 2019 Coronavirus (COVID-19) can lead to severe acute respiratory syndrome (SARS) characterised by high fever, dry cough, fatigue, dyspnoea, headache, frequent mild lymphopenia, hypoxaemia and characteristic pneumonia (Wu et al. (2020) Nature, 579:265-269; Lai et al. (2020) Int. J. Antimicrob. Agents, 55: 105924; Zhou et al. (2020) Lancet, 395:P1054-1062). Meta-transcriptomic sequencing of RNA from bronchoalveolar lavage fluid (BALF) or sputum of patients identified the likely causative agent as SARS-CoV-2, a novel RNA virus of the betacoronavirus genus related to SARS-CoV and SARS-MERS, which caused the major SARS and Middle East respiratory syndrome (MERS) pandemics with a mortality of 10%-30% over the past 20 years (Wu et al. (2020) supra; Zhou et al. (2020) Nature, 579:270-273; Wu et al. (2020) Cell Host Microbe, 27:325-328; Chan et al. (2020) Lancet, 395:514-523; Grifoni et al. (2020) Cell Host Microbe, 27: 1-10).

[0006] With more than 190 million cases and more than four million deaths in many countries in the short time from the first patient's diagnosis in Wuhan, China, in December 2019 to July 2021, COVID-19 could become the biggest pandemic threat humanity has faced since the Spanish flu (Johns Hopkins University Coronavirus Resource Center, 2021). High transmission rates, transmission by asymptomatic patients, high proportion (15%) of patients with severe disease, and mortality rates of up to 8% in some regions make this disease particularly dangerous. The elderly, individuals with co-morbidities and a poorly understood subgroup of younger patients develop more severe disease with higher mortality rates (Lai et al. (2020) Int. J Antimicrob. Agents, 55: 105924; Zhou et al. (2020) supra; Chan et al. (2020) Lancet, 395:514-523; Wang et al. (2020) Clin. Infect. Dis., 71 :769-777).

[0007] Aim of the present invention is to provide a drug that can be used in the treatment and prevention of SARS-CoV-2 viruses causing COVID-19 in a host, typically a human being. The invention achieves this purpose by providing a composition, the use of a composition and a drug according to the features of the independent claims. It is an object of the invention to use a composition of Potassium Bicarbonate and D-Ribose to form a drug, particularly for oral administration, for use in the treatment and prevention of SARS-CoV-2 viruses.

[0008] Such a drug binds together the functional characteristics of its components resulting in extremely rapid results, which cannot be compared to the use of the components used individually (for example, one potassium chloride-based drug and another ribose-based drug, administered separately, do not produce the same effect), it is not toxic (at the prescribed dosages) and can be used for long periods of time (months and even years) without harm.

[0009] These and further advantages and features of the present invention will be more and better understood by any skilled person in the art from the following description and with the help of the attached drawings.

[0010] In the remainder of this description, the preliminary results of tests performed and aimed at demonstrating a decrease in the replication rate in various SARS-CoV-2 strains will be described. The use of aqueous Potassium Ribosate, consisting of Potassium Bicarbonate and D-Ribose in an aqueous solution, against the SARS-CoV-2 viral pathogen in cell cultures of the Vero-E6 cell line, according to standardised protocols, causes a decrease in the rate of viral replication, i.e. decreases the SARS-CoV-2 viral load.

[0011] AQUEOUS POTASSIUM RIBOSATE: Chemical basis

[0012] This is a salt compound obtained by reacting Potassium Bicarbonate with D-Ribose in doubledistilled water. This saline compound exposed in an aqueous medium has physico-chemical characteristics such as pH, concentration, temperature, incidence of light, agitation, the presence of a magnetic field and the rate at which the CO2 produced in the reaction is released, which identify it as a “DYNAMIC EQUILIBRIUM”, which respects Le Chatelier's Principle.

[0013] The D-Ribose molecule in Potassium Ribosate is less reactive to auto-oxidation processes and more tolerant to self-decomposition than many of its carbohydrate analogues. This is why aqueous Potassium Ribosate shows a relative stability over time, which is evident when its physicochemical parameters begin to change. Fig. 1 shows schematically the formation of an extemporaneous solution of Potassium ribosate.

[0014] Analysing the spectroscopic data, we can describe that its infrared spectrum shows signals corresponding to the C-0 interactions characteristic of D-ribose mixed with the weak signal corresponding to the carbonate ion (SDBS No. 16175, spectral data base Aldrich. Die Angewandte Makromoleculare Chemie 266 (1999) 37-45 No. 4638) and the strong signals of the O-H groups of the aqueous medium. That is, a balance of the characteristic bands of the most relevant functional groups, which are: O-H and C-0 ceto-enolic groups.

[0015] The UV-Visible spectroscopy study made it possible to observe the wavelength at which Potassium Ribosate in aqueous solution exhibits maximum absorbance, 276 nm, and the variation of this as a function of time, proving that Potassium Ribosate must be treated as an extemporaneous solution. In the spectroscopy study depicted in Fig. 2, a graph is shown illustrating how a 1 :3 D-Ribose + KHCO3 mixture in (saturated) water releases CO2 in a way that is not perceptible, but in which a combination of the functional group bands is visible. In Fig. 2, line F2A represents the saturated solution, line F2B the KHCO3, and line F2C the D- Ribose.

[0016] In Fig. 3, a nuclear magnetic resonance (D2O) C13 spectroscopy is shown.

[0017] To complete its spectroscopic characteristics, a nuclear magnetic resonance study was performed at room temperature and 283 K, showing the following:

[0018] - Presence of a system that has free D-Ribose, which coincides with the one reported in the bibliographyl,2,5, a modified D-Ribose system with the appearance of a quaternary carbon at 160 ppm, corroborated with a dept 135 experiment in an AV600Hz resonance spectrometer, the latter does not coincide with the quaternary carbon of the carbonyl of Potassium Bicarbonate.

[0019] - CH2 signals were also identified in this experiment deptl35. This quaternary carbon at 160 ppm does not correlate with any proton signal in the HMBC experiment at 283K (shown below), which is evidence for the presence of a possible open-chain, oxidised ribose under a diene pathway between carbon 1 and 22.

[0020] With reference to Fig.4, where an example of nuclear magnetic resonance spectroscopy (D2O) C13 Deptl35 is shown, and Fig. 5, where an example of nuclear magnetic resonance spectroscopy (D2O) Hl, Proton aldehyde from 7 to 10 ppm is shown, it is important to note that there is no signal of an aldehyde proton to support the theory of the presence of aldehyde, thus supporting the formation of Eno-Diol as indicated above.

[0021] Furthermore, by combining the chemical shifts for Cl and C2 with 3D-optimised structure data in the ChemSketch 3D Viewer software, the bonding distance between Cl and C2 corresponds to a double bond stretched by the presence of metals (potassium) with electrostatic interaction.

[0022] An example of NUCLEAR MAGNETIC RESONANCE (D2O) SPECTROSCOPY of HMBC and HSQC between C13and H1RMNs is shown in Fig. 6.

[0023] In the HSQC experiment at 283K, the carbon to proton ratio of the predominant carbonyl signals can be seen, where the 4.5 ppm suprahedral doublet of the proton spectrum corresponds to the 94 ppm signal (Cl) of the C13spectrum; the signals between 3. 6-3.5 / 3.2-3.1 ppm of the proton spectrum correspond to the 70.53 and 66.64 ppm signals (C3 and C4) of the C13 spectrum; and finally, the signal at 62.54 ppm (C5) of the C13spectrum corresponds to the 3.5-3.3 ppm proton signals. This confirms that the structure shows the formation of an eno-diol2between carbons 1 and 2, and, consequently, that the oxygens supporting carbons 1 and 2 do not form carbonyls2,3,4, but open C-0 bonds susceptible to forming a bond with Potassium ions in solution (as can be seen in the spectra). The proposed mechanism based on spectroscopic evidence is shown in Fig. 7.

[0024] Taking into account the spectroscopic data, the proposed structure was entered into the program Viewing 3D Structures and Calculating their Structural, Version 11.0 for Microsoft Windows Parameters6, resulting in the two possible structures shown in Fig. 8. The choice of structure was made taking into account the following factors: the steric and electronic effect, the lowest electrostatic interaction, and the size of the Potassium cation, among others.

[0025] Clouds (K) represent Potassium atoms, oxygen atoms are denoted by (O), carbon atoms by (Ca) and hydrogen atoms by (H). The presence of the linked Potassium +1 cation directs the density of C-0 oxygen atoms in the same plane, elongating the chain of the organic part and breaking the pattern of cycled C-H type carbons.

[0026] Taking into account the following factors:

[0027] - Free pairs of oxygen atoms

[0028] - Bond pairs

[0029] - Cloud it of the Eno-Diol double bond

[0030] - Alkaline pH favoring the linear structure of D-Ribose and its autoxidation (Ref. Angew. Chem. Int. Ed. 2010, 49, 4503-4505 / Carbohydrate Research, 134 (1984) 191-204.)

[0031] - The Eno-Diol formed can be coordinated by the Potassium ion present. (Ref. Carbohydrate Research, 134 (1984) 191-204. / Tetrahedron Lett., (1977) 3869-3873.), we can define together with spectroscopic data and theoretical molecular model data the structure shown in Fig. 9 for aqueous Potassium Ribosate.

[0032] The important points to consider are:

[0033] - The stoichiometric excess of Potassium Bicarbonate in this formulation is necessary for the solution to act as a buffer with the HCO3- / CO2 / H- / H2O system and if it enriches the medium in Potassium ions, thus preventing it from promoting the possible de-oxidation reaction of D- Ribose, establishing a dynamic equilibrium among all its components.

[0034] - The open-chain form of D-Ribose has an eno-diol head containing the Potassium atoms and a tail of H-C-OH groups ending in a CH2 group. This structure is much more stable to carbohydrate auto-oxidation reactions2,7, and in turn allows the Potassium ribosate molecule to be directed in solution to interactions of related polarity, where the ribose molecule could perform viable synthesis or metabolic functions.

[0035] Bibliography

[0036] 1- New J. Chem., 2008, 32, 2043-2049.

[0037] 2- Carbohydrate research, 134, 1984, pp. 191-204. Tetrahedron Lett., (1977) 3869-3873).

[0038] 3- J. Chem. Educ. 61(9), 830, 1984. 4- Base SDBS.db.aist.go.jp / sdbs / cgi-bin / direct).

[0039] 5- Spectrochemical Acta Part A 61 (2005) 1977-1983.

[0040] 6- Viewing 3D Structures and Calculating their structural, version 11.0 for Windows Parameters.

[0041] 7- Environmental Health Perspectives Vol. 64, (1985), pp 297-307. Angew. Chem. Int. Ed. 2010, 49, 4503-4505

[0042] 8- Die Angewandte Makromolekulare Chemie 266 (1999) 37-45 (No. 4638).

[0043] TEST 1

[0044] AQUEOUS POTASSIUM RIBOSATE:

[0045] In Vitro Assay of Cellular Cytotoxicity in Healthy Human Cells.

[0046] Test 37: CCK-8 Assay of Cellular Cytotoxicity of RK 0-100 mM

[0047] Objective

[0048] The main aim of this study is to evaluate / study the cytotoxic effect of 0-100 mM Potassium Ribosate treatment on cell proliferation of the healthy human cell line Hs 578Bst.

[0049] Material and Methods.

[0050] Cell culture of the healthy Hs 578Bst cell line

[0051] The Hs 578Bst cell line was purchased from ATCC, reference HTB-125, cultured and maintained in adhesion in Hybri-care medium (ATCC) supplemented with 1.5 g sodium bicarbonate (VWR) per liter of medium, 10 % FBS (Fetal Bovine Serum, ATCC), 1% penicillin / streptomycin (Gibco) and mouse EGF (Epidermal Growth Factor, Sigma- Aldrich) to a final concentration of 30 ng / ml in Flasks T75 (Nunc EasyFlask), in an incubator at a temperature of 37° C and in a humidified atmosphere with 5 % CO2 and changes of medium every 72 hours until 80-90 % confluence is reached, according to the recommendations of the commercial house. The subculture procedure was performed using a Tripsin-EDTA solution (Gibco) for 5 min at 37°C and the centrifugation step at a speed of 125 x g.

[0052] Treatments in 96-well multiwell plates.

[0053] A 96-well multiwell plate (Thermo Scientific) was used. An initial cell density of 1000 cells per well in a final volume of culture medium of 0.2 ml per well was assumed. Cells were allowed to fix for 24 hours before starting treatments. This lasted for 48 hours. The "n" for each condition was 8.

[0054] The concentration of the treatment stock solution in this case was also of 250 and 125 mM and that of the treatment well 0 mM (control), 5 mM, 20 mM and 100 mM.

[0055] Colorimetric assay using the CCK-8 Kit

[0056] After the duration of each of the treatments established for this assay, the wells were removed, the wells were washed with 1 X PBS and 0.1 ml of the fresh culture medium already described for this cell line, without treatments, was added to each well (to avoid interference with the reaction performed during the CCK8 assay). In addition, 10 pl of CCK8 reagent (from the commercial house Sigma-Aldrich) was added to each well, and incubated for 2-4 hours (according to the manufacturer's specifications) in an incubator at 37° C, 5 % CO2 and humidified atmosphere. After this period, the reaction did not need to be stopped (unlike other reagents), so absorbance was read in a plate reader at room temperature (EZ READ ELISA 400, using Biocrom's Galapagos software), as indicated in the reagent specifications.

[0057] Results and Discussion

[0058] The cytotoxicity of Potassium Ribosate solutions at different concentrations was evaluated using the CCK-8 kit. As shown in Figure 1, even at high concentrations (100 mM), the cells remained viable and no statistically significant differences were detected under any of the conditions compared with the control, highlighting the non-cytotoxicity of Potassium Ribosate in vitro.

[0059] Fig. 10 represents the cell cytotoxicity. Viability test after incubation with different concentrations of Potassium Ribosate solution for 48 hours. Error bars indicate mean ± error in arbitrary units. There were no statistically significant differences between groups. A Kruskal- Wallis test was performed. N=8.

[0060] Supplementary Material

[0061] Water used for treatments

[0062] The treatments were prepared in vial water from the commercial house Serra Pamies, the characteristics of which are given in the table below:

[0063] Conductivity (pS / cm) 12,54

[0064] Reractive index (nD) 1,333019

[0065] TEST 2

[0066] "Effect of treatment with Potassium Ribosate 5 mM every 48 hours on the growth of Hs 578Bst cell line (HTB-125, ATCC®)."

[0067] Our body is capable of synthesizing ribose. The importance of using ribose as ribosate lies in increasing the rate of Potassium transfer of the cellular cytoplasm. Ribose plays a very important role in cellular metabolism and is the sugar directly involved in nucleotide synthesis. It is also the key precursor in the biosynthesis of RNA and adenosine (an essential component in the production of ATP and Na / K ATP-ase, considered the sodium-potassium pump) and, in the deoxyribose form, for DNA synthesis.

[0068] Objective

[0069] In light of the results obtained in the previous assays, the main objective of this study is to evaluate / study the effect of 5 mM Potassium Ribosate treatment on the growth of healthy Hs 578Bst cell line every 48 hours.

[0070] Material and Methods.

[0071] Cell Culture.

[0072] The Hs 578Bst cell line was purchased from ATCC and is a healthy human cell line of fibroblastic morphology derived from mammary epithelial cells, referiento HTB-125, cultured and maintained in adhesion in Hybri-care medium (ATCC) supplemented with 1. 5 g sodium bicarbonate (VWR) per liter of medium, 10% FBS (Fetal Bovine Serum, ATCC), 1% penicillin / streptomycin (Gibco) and mouse EGF (Epidermal Growth Factor, Sigma-Aldrich) at a final concentration of 30 ng / ml in Flasks T75 (Nunc EasyFlask), in an incubator at a temperature of 37 °C and in a humidified atmosphere with 5 % CO2 and with medium changes ogmi about 72 hr or as needed, until 80-90 % confluence was reached, following the recommendations of the commercial house. THE subculture procedure was performed using a Tripsin-EDTA solution (Gibco) for 5 min at 37 °C and the centrifugation step at a rate of 125 x g.

[0073] Treatments

[0074] For the test, 60-mm plates (Thermo Scientific) were used and an initial cell density of 15000 cells per plate in 3 ml of culture medium was assumed. The cells were allowed to fix for 24 hours before starting treatment.

[0075] The stock solution of Potassium Ribosate treatment was prepared as described by Croci, S et al. 2011, with the modifications described below: in distilled water (Serra Pamies) at a concentration of 125 mM, from which 120 pl was taken and added to 2.88 ml of culture medium per plate for the assay (final plate concentration of 5 mM); the assay lasted 144 hours and the medium with treatment was renewed every 48 hours.

[0076] Potassium Ribosate treatment was prepared as described by Croci, S et al. 2011, with the modifications described below: in distilled water (Serra Pamies) at a concentration of 125 mM, from which 120 pl was taken and added to 2.88 ml of culture medium per plate for the assay (final plate concentration of 5 mM); the assay lasted 144 H and the treatment medium was renewed every 48 H.

[0077] Cell Growth Curve and Cell Duplication.

[0078] To study the effect of the previous treatments above on cell proliferation, cells were counted every 48 h using a Biirker chamber versus control and a growth curve was constructed, similar to that described by Christian BJ., et al., 1987; the formula was used: : log2(N = ~ + whereby d (time of cell doubling) was calculated, with "N" being the number of cells at the end of the test, "No" the number of cells at the beginning of the test, and "t" the time of treatment duration. To discriminate between viable and non-viable cells, 0.4 % trypan blue solution (Gibco) was used in a 1 : 1 ratio, as indicated in the manufacturer's specifications.

[0079] Microscopy

[0080] An inverted light microscope (Olympus 1X50) coupled with a DP72 camera (Olympus) was used to take photographs of the cell cultures. Photographs of all conditions were taken with a 10X magnification objective at the beginning of the trial (t=0 days) and after 48, 96 and 144 hours of treatment.

[0081] Results

[0082] - The first count (No) was performed 24 h after seeding, and the first treatment was performed after that.

[0083] - Counts 48 hours, 96 hours and 144 hours after the start of treatment and cell growth curve.

[0084] The counts of viable and non-viable cells after the start of treatment, respectively, are shown in Table A and Table B :

[0085] Table A. Number of viable cells after 48, 96 e 144 hours of treatment (treatment with RK 5 mM every 48 hours vs. control). N=4.

[0086] Table B. Number of non-viable cells after 48, 96 e 144 hours of treatment (treatment with RK 5 mM every 48 hours vs. control). N=4.

[0087] The percentage of non-viable cells is then as described below for each treatment time (Table 3.), it can be seen that the percentage of nonviable cells is similar in both cases throughout the assay: Table C. Percentage of non-viable cells after 48, 96 e 144 hours of treatment (treatment with RK 5 mM every 48 hours vs. control). N=4.

[0088] The Graph in Fig. 11 shows the growth curves obtained for the culture of the healthy cell line Hs 578Bst (HTB-125, ATCC) subjected to RK treatment at a concentration of 5 mM every 48 hours compared with the control. As can be seen in the graph, the cell growth of both control and treatment groups conformed to exponential growth. Taking into consideration the data in Table 1 depicted in Graph 1, we could observe that after RK treatment the population growth was slightly higher or faster, however, this slight increase does not show statistically significant differences compared to the control. Furthermore, as can be seen in Tables 2 and 3, treatment with RK 5 mM does not significantly increase or decrease the percentage of nonviable cells compared to the control, i.e., the mortality rate is similar in both cases.

[0089] The Graph in Fig. 12 shows the trend lines obtained for the culture of the healthy cell line Hs 578Bst (HTB-125) subjected to RK treatment at a concentration of 5 mM every 48 hours compared with the control for 144 hours.

[0090] As can be seen in the graph in Fig. 12, in both cases the trend line is linear; it can also be observed again that cell growth is slightly higher in the treatment group than in the control group, as could be seen after cell counting and in the representation in the graph in Fig. 11.

[0091] - Cell doubling time (d)

[0092] Applying the following formula: log2N) = ~ + ^^2(^0), > value d (population doubling time) has been calculated. The value of d after 144 hours of treatment is shown in Table D.

[0093] Table D. Values of parameter d (cell population doubling time) after 144 hours of treatment, calculated by the formula log2( / V) = + log2( / V0). for the different treatments. N=4.

[0094] As we can see in Table D, the number of hours that should pass for the doubling of the Control group population is 120.57 hours, while that for the population treated with RK 5 mM is 111.27 hours. This indicates that this treatment could slightly increase the doubling rate of the healthy HTB-125 cell line by 7.7 %, thus requiring 9.30 hours less for the RK-treated population to double than the Control.

[0095] Optical microscopy

[0096] Visually, it can be seen that the culture medium of the RK-treated cells has a reddish-red color, while that of the control cells has a more orange color from the beginning of the assay, which may be due to the pH of the Potassium Ribosate solution, which is alkaline (8.5). These differences in the coloration of the medium of the control group compared to the color of the medium of the RK-treated cells continue to be maintained until the end of the assay.

[0097] After 48, 96 and 144 hours of treatment, a high percentage of nonviable cells is not observed and is similar between the RK-treated group and the control group.

[0098] As the duration of the assay progresses, confluence in the plates increases in both cases, with 100 percent confluence not reached in either the control or treatment group. However, confluence is slightly higher in the RK-treated group, which shows that there are more cells in this group of treatment.

[0099] In both groups, mitosis was observed throughout the assay and cells with typical fibroblastic morphology were observed.

[0100] After 48 hours of treatment, RK-treated cells showed more granules located peripherally to the nucleus than the control, which could be related to increased metabolic activity or even be a sign of active division (however, we cannot confirm this finding that complementary assays were not performed). This effect is maintained up to 144 hours, as depicted in Figs. 11-16.

[0101] Conclusions

[0102] - Treatment with RK 5 mM slightly increases the replication rate of the healthy Hs 578 Bst (HTB- 125) cell line in vitro by 120.57 hours for the control group and 111.27 hours for the RK-treated group.

[0103] - Treatment with RK 5 mM slightly increases the cell replication rate of the healthy Hs 578 Bst (HTB-125) cell line in vitro by about 7.7 % compared with the untreated control.

[0104] - Treatment with RK 5 mM does not significantly increase or decrease the percentage of nonviable cells in cultures of the healthy Hs 578 Bst (HTB-125) cell line in vitro.

[0105] - No morphological differences were observed between the treatment and control groups. Bibliography

[0106] 1- Bruni 1. PhD in Biotechnology. Antitumorigenicity of D-ribose and KHCO3 on the human mammary carcinoma line HS 578t and effects on the non-cancerous human mammary epithelium line HS 578bst. Date: XXVI Cycle - YEARS 2011- 2013.

[0107] 2- Hackett, AJ. Smith, HS., Springer, EL., Owens, RB., Nelson-Ress, WA., Riggs, JL and Gardner, MB (1977). Two syngeneic cell lines from human breast tissue: the aneuploid mammary epithelial (Hs578T) and the diploid myoepithelial (Hs578Bst) cell lines. J Natl Cancer Inst 58(6): 1795-1806.

[0108] 3- Christian BJ, Loretz LJ, Oberley TD and Reznikoff CA (1987). Characterization of Human Uroepithelial Cells Inmmortalized in Vitro by Simian Virus 40. Cancer Research, 47:6066-6073. Supplementary Material.

[0109] Water used for treatments

[0110] The treatments were prepared in vial water from the commercial house Serra Pamies, the characteristics of which are given in the table below: pH 6,82

[0111] Conductivity (pS / cm) 12,54

[0112] Reactive Index (nD) 1,333019

[0113] Preparation of the stock treatment of 125 mM de RK

[0114] For the preparation of the treatment, considering ribose as the limiting reagent, 93.75 mg of Ribose of synthetic origin (Sigma Aldrich) was weighed and dissolved in 2 ml of the water described above; 187.73 mg of Potassium bicarbonate (Merck) was then added and brought to a final volume of 5 ml with the same water at the end of bubble release. Finally, it is filtered through a filter with a pore size of 0.22 pm in diameter before being added to the cell cultures.

[0115] From this stock 120 pl is taken and mixed with 2.88 ml of culture medium, this total volume of 3 ml is put into each 60 mm diameter culture plate.

[0116] * * *

[0117] AQUEOUS POTASSIUM RIBOSATE:

[0118] In vivo Acute Toxicity Test in Sprague-Dawley Rats.

[0119] Animal Model

[0120] Four (4) female Sprague Dawley® SD® rats were used in this study. The rats were 7 weeks old at receipt and eight (8) weeks old at the start of the experiment. The animals were housed in a temperature-controlled room with a 12:12 lighting cycle (darklight) and controlled relative humidity. All rats were provided with freely available (ad libitum) water and food (Global diet 2914C irradiated ENVIGO, Teklad Diets), with the exception of the night before the dose and for about 3-4 hours after the dose. Before being included in the study, animals were individually examined by a veterinarian, and only animals that clearly showed no visible signs of disease were included in this study.

[0121] Treatment

[0122] This study consists of applying a single dose of Potassium Ribosate (K:D-Rib) daily, the dose amount in this study per animal is 60.28, a volume of 400pl was injected intramuscularly on both sides of the animal simultaneously (200pl X 2). A saturated solution of K:D-Rib was prepared by dissolving 200 mg of D-Ribose (Sigma-Aldrich) in 1.5 mL of injectable water (Serra Pamies), was mixed without magnetic stirring with 600 mg of solid Sodium Bicarbonate (Merck). Mixing was finished when all bubbles were released (about 4 min), at which time the total volume was increased to 2 mL by adding the vehicle (water for injection, Serra Pamies).

[0123] Determination of Electrolytes

[0124] Five (5) days before injection, 0.5 ml blood samples were collected from each animal using lithium heparin collection tubes by sublingual puncture under isoflurane anesthesia to determine the basal levels of the following electrolytes: calcium (Ca+2), chloride (O'), inorganic phosphorus (PO43'), potassium (K+) and sodium (Na+), and a second blood sample was also collected to determine the same electrolytes 24 hours after the dose. Plasma samples were used directly, without dilution, for electrolyte determination. This analysis was performed on a COBAS INTEGRA 400 plus analyzer (Roche), configured with Ion-selective electrodes (ISE).

[0125] Clinical Signs.

[0126] Body weights were recorded once during the acclimation period, immediately after dose administration and then weekly until necropsy. Animals were monitored for abnormal clinical signs, discomfort and mortality as indicated for humans by the Endpoints Guidance Document of the Organization for Economic Co-operation and Development (OECD). After K:D-Rib dose administration, they were kept under observation for a period of two weeks and you can euthanized after 14 days. Treatment response was assessed by clinical observations 30 minutes, 2 hours and 4 hours after administration and once daily until completion of a 14-day observation period. Clinical signs assessed included changes in skin, hair, eye mucous membranes, alterations in respiratory rhythms, behavior, posture, response to touch and abnormal movement response, as well as special observation for tremors, convulsions, salivation, diarrhea, lethargy and sleep. At the end of the 14-day observation period, all animals were euthanized under CO2 atmosphere. Autopsy consisted of macroscopic evaluation of all external orifices of the animal's body, thoracic and abdominal cavities, and inspection of all contents.

[0127] Statistics

[0128] The statistical software program Sigma Plot 11.0 was used to perform statistical analysis and graphing of the in vitro cytotoxicity data. And with the same software, we evaluated whether there were statistical differences in the analyzed parameters between the pre-dose and post-dose application of K:D-Rib treatment in vivo.

[0129] Results

[0130] In-vivo data

[0131] Clinical Signs and Mortality

[0132] The potassium ribosate dose level of 60.28mg per animal did not cause mortality, as shown in Table 1. The mean dose was 304.86 mg / kg (standard deviation = 3.72 and standard error = 1.86).

[0133] Table 1. Treatment-related Mortality Records.

[0134] Concentration of Dose mg / kg Deaths

[0135] Animal formula (mg / mL) (mg / animal)

[0136] ID 1 302.16 0

[0137] ID 2 308.81 0

[0138] 150.72 60.28

[0139] ID 3 307.24 0

[0140] ID 4 301.25 0

[0141] After K:D-Rib administration, the clinical symptoms presented were lameness in both left and right hind limbs and gait stiffness; these observations are summarized in Table 2. Lameness was observed within 30 min after K:D-Rib administration and persisted for 2 h after administration. For 4 hours after administration, none of the animals showed these symptoms. Gait stiffness and lameness maintained a similar pattern, and at 4 hours of observation and during the remaining time of the 14-day observation period, they showed no abnormal clinical signs. Only one animal showed a hematoma < 2 mm at the injection site (right hind limb) 24 hours after administration. However, 48 hours after administration, the hematoma had dissolved and was no longer visible. The table in Fig. 17 summarizes the animal clinical observations by time.

[0142] Body Weight

[0143] As shown in Tables 3 and 4, all animals showed a tendency to increase body weight during the 14-day observation period, indicating that injected K:D-Rib has no impact on this parameter. Table 3. Individual body weights (g)

[0144] Day(s) Relative starting date

[0145] Animal -5 1 8 15

[0146] ID 1 190.50 199.50 206.50 220.90

[0147] ID 2 190.60 195.20 204.50 220.50

[0148] ID 3 189.20 196.20 209.20 209.90

[0149] ID 4 190.10 200.10 207.00 220.00

[0150] Media 190.10 197.75 206.80 217.83

[0151] SD 0.64 2.41 1.93 5.30

[0152] N 4 4 4 4

[0153] SD: Standard Deviation ; N: number of samples..

[0154] Table 4. Absolute weight increase (g)

[0155] Animal Day(s) Relative starting date -5 —4 1 —8 8 — 5

[0156] ID 1 9.00 7.00 14.40

[0157] ID 2 4.60 9.30 16.00

[0158] ID 3 7.00 13.00 0.70

[0159] ID 4 10.00 6.90 13.00

[0160] Media 7.65 9.05 11.02

[0161] SD 2.39 2.86 6.99

[0162] N 4 4 4

[0163] SD: Standard Deviation ; N: number of samples.

[0164] Analysis of Electrolytes

[0165] There is no K:D-Rib-related effect on the electrolyte measurement parameters that were observed and analyzed in rats 24 hours after injection (Table 5) when the values were compared with the pre-inj ection values (Table 6) of the same animals. These results are grouped in Figure 1.

[0166] Table 5. Elettrolytes Data, 24 hours after injection.

[0167] Ca+2Cl’ PO43’ K+Na+

[0168] Animal (mmol / L) (mmol / L) (mmol / L) (mmol / L) (mmol / L)

[0169] ID 1 2.69 98.10 2.50 5.18 134.30

[0170] ID 2 2.62 99.62 2.50 4.90 135.20

[0171] ID 3 2.70 97.98 2.80 5.23 135.40

[0172] ID 4 2.67 97.26 2.60 5.92 133.10

[0173] Media 2.67 98.24 2.60 5.31 134.50

[0174] SD 0.04 0.99 0.14 0.43 1.05

[0175] N 4 4 4 4 4

[0176] Ca+2: Calcium, Cl’: Chloride, PO43’: Inorganic Phosphorus, K+: Potassium, Na+: Sodium, SD: Standard Deviation and N: Numero of Samples. Table 6. Elettrolytes Data, before injection.

[0177] Ca+2Cl’ PO43- K+Na+

[0178] Animal (mmol / L) (mmol / L) (mmol / L) (mmol / L) (mmol / L)

[0179] ID 1 2.67 98.29 2.70 4.85 136.00

[0180] ID 2 2.52 99.02 2.10 4.91 135.40

[0181] ID 3 2.60 97.57 2.80 4.89 135.80

[0182] ID 4 2.67 98.17 2.30 4.62 136.00

[0183] Media 2.62 98.26 2.48 4.82 135.80

[0184] SD 0.07 0.60 0.33 0.13 0.28

[0185] N 4 4 4 4 4

[0186] Ca+2: Calcium, Cl’: Chloride, PO43': Inorganic Phosphorus, K+: Potassium, Na+: Sodium, SD: Standard Deviation and N: Numero of Samples. Fig. 18 represents the eElectrolytes found 24 h pre-injection (black bars) and 24 h post-injection (white bars). The error bars above the data indicate ± SD. No statistical difference between pre- and post-injection was observed (t- test was applied to each electrolyte data). N= 4.

[0187] Post-Mortem Data

[0188] Macroscopic Autopsy

[0189] After a 2-week observation period, the animals had macroscopic necropsy. The body cavities of the animals were examined visually. None of the animals treated with the 60.28 mg / animal dose of K:D-Rib showed macroscopic abnormalities and had no lesions visible at necropsy.

[0190] Conclusion

[0191] Evaluation of acute toxicity of K:D-Rib in vivo by intramuscular administration in female Sprague-Dawley type rats reveals nontoxicity.

[0192] Institutional Review Board Statement: The study was conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (ethics committee) of Vivotecnia Research S.L. and complying with the Good Laboratory Practice (GLP) requirements of European Directive 2004 / 1 OZEC and Spanish Royal Decree RD 1369 / 2000.

[0193] * * *

[0194] AQUEOUS POTASSIUM RIBOSATE:

[0195] In Vitro Testing of the Effect of Potassium Ribosate on SARS-CoV-2

[0196] Biochemical Research A.E.I.E. and the Virology Section of the Microbiology Department of the Hospital Universitario Central de Asturias (HUCA), carried out the assays that determined the effect of Potassium Ribosate on the replicative capacity of different strains of SARS-CoV-2 obtained and cultivated in the Virology Section.

[0197] Test design: the growth inhibition tests were performed according to adapted classical methods in the laboratory of the HUCA Virology Section.

[0198] Material

[0199] To perform the different inhibition assays and to quantify the efficacy of aqueous potassium ribosate against the SARS-CoV-2 viral pathogen, a total of 6 different strains were used in the initial assay, 8 different strains in the second assay and 10 different strains in the third virus assay: variant B.1.1.7 (a), variant B.1.1.351 (P) and variant B.1.1.617.2 (6).

[0200] All these strains originated from clinical samples of patients with SARS-CoV-2 infection and were isolated in established cell cultures of the Vero-E6 cell line according to standardised protocols.

[0201] Classification of variants corresponding to each strain was performed by allelic discrimination techniques or by sequencing, according to the Section's protocol.

[0202] For the inhibition assays, the viral titre of each strain was determined before or at the same time as the assay, depending on the requirements at the time (as determined below).

[0203] All assays were performed in the biosafety laboratory of the HUCA Virology Section, where the required safety conditions were maintained.

[0204] Methods

[0205] (a) Strain titration

[0206] Strains of all SARS-CoV-2 viruses were titrated to know the 50% CI and 95% CI according to the inoculation assays in 96-well plates.

[0207] For this, starting with a viral culture, decreasing dilutions of the virus in base 10 were made and 200pl of the inoculum was inoculated into the wells of the plate. They were then incubated at 37°C for 1 hour in a 5% CO2 atmosphere. The inoculum was then removed and the plates were incubated for up to 7 days under the conditions described above.

[0208] Viral growth was determined: a.l Cytopathic effect (ECP): Karber method

[0209] Visualise the cytopathic effect produced by the infection. Calculate the IC 50% and the IC 95%. a.2 Vital dye (Neutral red)

[0210] To evaluate the effect of the drug on viral infection by the neutral red vital dye uptake method, Vero-E6 cells were cultured in 96-well cell culture plates.

[0211] After 7 days of infection, 50pl of buffered Neutral Red solution was added to each well. The plates were incubated for 45 minutes at 37°C in a 5% CO2 atmosphere. Afterwards, the medium was removed and the wells were washed twice with PBS. Finally, 150pl of phosphate buffered ethanol was added to elute the viable dye incorporated by the viable cells. The absorbance was read at 550nm (reference value 620nm) on a PR 4100 spectrophotometer. a.3 Genomic detection: real-time RT-PCR

[0212] 50 pl of supernatant was collected from each well for subsequent viral quantification by genomic amplification (PCR). Viral replication in each of the cell culture dilutions was determined by averaging the viral load of all wells. This viral load is calculated by quantifying the genome in each of the samples. This was done using the qPCR technique, which is routinely used in the HUCA Virology Laboratory for virus diagnosis.

[0213] The results obtained were expressed as a function of the observed Ct (PCR cycles) (the higher the Ct, the lower the viral load). A Ct of 40 is assumed as the absence of viral growth. b) Inhibition assays

[0214] In the assays carried out with different strains, two types of administration were performed, in one group Potassium Ribosate was administered 24 hours before inoculation of the virus, this type of assay was called Pretreatment. On the other hand, the other type of administration was to add Potassium Ribosate after inoculation of the pathogen. At all times, the concentration at which Potassium Ribosate was administered was the same (as indicated by Biochemical Research A.E.I.E).

[0215] For this purpose, the effect of Potassium Ribosate was tested on Vero-E6 cells infected with SARS-CoV-2. Cells, previously cultured in 96-well plates until the cell monolayer was confluent, were infected with serial viral dilutions in base 10. Uninfected and treated cells, and cells not infected and not treated with Potassium Ribosate were kept as controls. An example of the arrangement of inocula and controls in the 96-well plates is shown in Figure 1.

[0216] To infect the cells, the medium was removed and 200 pl of each of the serial dilutions was added and incubated at 37°C for 1 hour in a 5% CO2 atmosphere. Subsequently, the inoculum was removed and fresh maintenance medium was administered without and with Potassium ribosate. Three days after infection, the medium was renewed, keeping each culture in the same condition as the original. Of the removed medium, 50pl of supernatant was collected from each well for subsequent viral quantification by genomic amplification (PCR). Viral replication in each of the cell cultures was determined by the viral load calculated by quantifying the amount of viral genome in each of the samples.

[0217] In all cases, viral replication was measured both 3 days and 7 days after inoculation.

[0218] An example of the arrangement of inoculated wells and cell controls in the 96-well plate is shown in Fig. 19.

[0219] Measurements were performed by visualisation of the cytopathic effect or CPE (biological readout) and PCR quantification (molecular readout). In the last assay, analysis by Neutral Red staining was added at both measurement points. These methods were performed according to the protocols explained above. c) Interpretation:

[0220] To give an evaluation of the results obtained and quantify the degree of impact of Potassium Ribosate on viral replication of the different strains used in the assays, the following criteria scale was developed:

[0221] 1. For biological reading (CPE / Neutral Red):

[0222] CPE: Potassium Ribosate Efficacy= more than 2 log difference.

[0223] Red Neutral: Potassium Ribosate Efficacy= Less than 50% increase in reading titre.

[0224] 2. For molecular reading (PCR):

[0225] Treatment response is assessed by the following criteria: Less than 3 cycles difference: No response (NR)

[0226] Between 3 and 6 cycles or between 1 and 2 log: Normal response (N)

[0227] Between 6 and 9 cycles or between 2 and 3 log: Good Response (B)

[0228] More than 9 cycles or more than 3 logs Very Good Response (MB)

[0229] The final interpretation of the results was made according to the data obtained by the RT-PCR method as it better discriminates the difference in growth.

[0230] CPE and Neutral Red reading data (when used) were used to evaluate the evolution of the assay and to confirm the more accurate data of the RT-PCR method. c. l. Description and characteristics of each assay

[0231] As the assays were performed on living biological substrates and subject to constant change, the experiments were based on the above protocols, and were adapted to each particular circumstance at the time, without altering the significance of the results. Here we present two initial assays and the final assay.

[0232] Based on the above, the assays had the following characteristics:

[0233] Initial assay 1 : Classic assay with strain titration, pre-treatment inhibition assay and plate treatment:

[0234] - Strains: 6 with dilutions up to -6.

[0235] - Date: April 2021

[0236] Initial assay 2: Assay with titrated strains first, pre-treatment inhibition assay and plate treatment:

[0237] - Strains: 8 (4 of the strains were transferred to tube culture) titre reading varied and dilutions of each strain to -2, -3 and -4.

[0238] - Date: April 2021

[0239] Final assay: Assays with strains titrated first, plate treatment inhibition assay:

[0240] - Strains: 10 strains with titres between -4 and -5 and dilutions of each strain to -2, -3 and -4. And three different variants a, P y 6.

[0241] - Date: November 2021

[0242] Experimental results.

[0243] Initial assay 1

[0244] In this assay, the results of the pure control were evaluated and the cells on the plate were detached from it. For convenience in the use of the tables Potassium Ribosate will be referred to as Drug.

[0245] The amplification cycles of each control are shown in Table 1 below

[0246] Table 1. Amplification cycles of each strain with or without drug Pretreatment Treatment

[0247] Title / Reading Variant Virus Drug Valuation Virus Drug Valuation

[0248] Title

[0249] 1 a

[0250] 2 -4 / p a 21 22 NR 23 35 MB

[0251] 3 -4 a 20 24 N 22 31 MB

[0252] 4 -2 a 28 37 MB 33 22 NR

[0253] 5 -2 a 23 22 NR 21 21 NR

[0254] 6 - a - - - 24 >40 MB

[0255] The amount of virus e is shown in the following Table 2.

[0256] Table 2. Viral load expressed in log.

[0257] Pretreatment Treatment

[0258] Title / Reading Variant Virus Drug Valuation Virus Drug Valuation

[0259] Title

[0260] 1 a

[0261] 2 -4 a 7.3 7 NR 6.7 3.13 MB

[0262] 3 -4 a 7.6 6.4 N 7 4.32 MB

[0263] 4 -2 a 5.2 2.5 MB 3.4 7 NR

[0264] 5 -2 a 6.7 7 NR 7.3 7.3 NR

[0265] 6 - a - - - 6.4 0 MB

[0266] Of the pre-treated cells, 4 were evaluated: in 2 the product was able to decrease viral replication. Of the non-pretreated cells, 5 were evaluated: with >3 log (MB) reductions in viral load in 3 of them.

[0267] Based on these preliminary results, the second initial assay was developed.

[0268] Initial assay 2

[0269] Cells of 4 strains (strains 5 to 8) were transferred into tubes 3 days after the start of the assay because they had detached from the monolayer. The amplification cycles of each control and the amount of virus for the 3 -day reading are shown in Tables 3 and 4 below respectively.

[0270] Table 3. Amplification cycles in each strain with and without drug.

[0271] Pretreatment Treatment

[0272] Title / Reading Variant Virus Drug Valuation Virus Drug Valuation

[0273] Title

[0274] 1 -2.5 / -1 a 28 33 N 22 22

[0275] 2 -2.8 / -1 a 22 22 - 20 20

[0276] 3 -2.5 / -1 a 29 33 N 23 25

[0277] 4 -2.5 / -1 a 23 34 MB 19 19

[0278] 5 -2.1 / -1 a 22 31 B

[0279] 6 -2.1 / -1 a 21.5 27 N 22 28 B

[0280] 7 -2.8 / -1 a 18 23 N 29 29

[0281] 8 -2.1 / -1 a 17 28 MB 21 24 N Tabla 4. Viral load expressed in log.

[0282] Pretreatment Treatment

[0283] Title / Reading Variant Virus Drug Valuation Virus Drug Valuation

[0284] Title

[0285] 1 -2.5 / -1 a 5.2 3.7 N 7 7

[0286] 2 -2.8 / -1 a 7 7 - 7.6 7.6

[0287] 3 -2.5 / -1 a 4.9 3.7 N 6.7 6.1

[0288] 4 -2.5 / -1 a 6.7 3.43 MB 7.9 7.9

[0289] 5 -2.1 / -1 a 7 4.3 B

[0290] 6 -2.1 / -1 a 7.15 5.5 N 7 5.2 B

[0291] 7 -2.8 / -1 a 8.2 6.7 N 7 5.2

[0292] 8 -2.1 / -1 a 8.5 5.2 MB N

[0293] The results of the 7-day reading of the amplification cycles for each control and for both plate and tube strains and the amount of viral load are shown in Tables 5 and 6 respectively Table 5. Amplification cycles in each strain with and without drug. Asterisks show the favourable change with the 3 -day reading.

[0294] Pretreatment Treatment

[0295] Title / Reading Variant Virus Drug Valuation Virus Drug Valuation

[0296] Title

[0297] 1 -2.5 / -1 a 26 30 N 25 30 N*

[0298] 2 -2.8 / -1 a 25 28 N* 25 30 N*

[0299] 3 -2.5 / -1 a 30 40 MB* 27 40 MB*

[0300] 4 -2.5 / -1 a 26 37 MB 27 37 MB*

[0301] 5 -2.1 / -1 a 26 40 MB 26 30 N*

[0302] 6 -2.1 / -1 a 26 40 MB* 26 35 B*

[0303] 7 -2.8 / -1 a 24 40 MB* 27 40 MB*

[0304] 8 -2.1 / -1 a 17 28 MB 25 40 MB*

[0305] Table 6. Viral load expressed in log. Asterisks show the favourable change at 3-day.

[0306] Pre-treatment initiated prior to treatment at the time of infection improved the response at 7 days in 50% of cases. Treatment obtained surprising responses at 7 days, improving results in 7 out of 8 cases. From these preliminary assays, it was decided to work with single titre (or close dilutions) and to evaluate the cytopathic effect and use other variants.

[0307] Final assay

[0308] As described in section c.1, three variants a, P, y, 6 were used in this assay. Of the 10 strains used (named by their original code), all responded to Potassium Ribosate after 7 days of exposure.

[0309] All results of both the 3-day and 7-day analysis, expressed in Ct (PCR cycles) and pairs (log) / 103 cells, as well as their evaluation, are shown in Tables 7 and 8.

[0310] Table 7: Results of strain analysis at 3 days of product exposure

[0311] Table 8: Results of strain analysis at 7 days of product exposure

[0312] Reading Without treatment Treatment with 2021-aP _

[0313] Strains Title Variant CPE Title CPE PCR (Ct) C.V. (log) ECP PCR (Ct) C.V. (log) Valuation

[0314] 242141832 (3) 7,11 a 4.0 -4 + 28.48 5.09 + 33.58 3.58 N

[0315] 242196606 (A) 5,61 a 4.5 -4 + 32.47 3.90 + 40.00 1.64 B

[0316] 242182248 (B) 4,45 a 5.0 -4 + 35.10 3.10 + 40.00 1.64 N

[0317] 242132607 (B) 7,11 a 4.5 -4 + 15.50 8.94 + 22.73 6.79 B

[0318] 252114903 (B) 9,56 a 4.0 -4 + 16.36 8.70 + 22.91 6.73 B

[0319] 242132607 (A) 7,11 a 4.0 -4 + 31.80 4.08 + 40.00 1.64 B

[0320] 252195336 (T) 9,26 p 4.5 -4 + 15.15 9.05 + 26.92 5.54 MB

[0321] 252195336 (F) 9,26 p 5.0 -4 + 15.39 8.99 + 32.61 3.84 MB

[0322] 262111647 (F) 9,86 y 4.5 -4 + 21.07 7.30 + 40.00 1.64 MB

[0323] 262111647 (F) 9,86 y 4.5 -4 + 21.34 7.21 + 29.24 4.86 B

[0324] From this test it can be seen that of these 10 strains, only the strain represented in the tables by the name 242141832(3) can be discarded, because it could not be evaluated at 3 days. Thus out of 9 strains at 3 days 5 of them have a favourable response and at 7 days 8 of these 9 strains have a favourable response to treatment. It is important to note that the viral load values of two of the titrations in the 3-day exposure table that gave MB response are maintained in the 7-day exposure table, so the viral load evidently did not replicate, a fact that can be interpreted initially, as the virus that is present there (because it is assessed by PCR) is inactive / inhibited and does not continue to replicate, despite having a host available to replicate (live cells). In conclusion, Aqueous Potassium Ribosate minimises viral load in these tested in vitro systems. Bibliography

[0325] - Gomez J., Melon, S., Boga, J. A., Alvarez-Arguelles, M. E., Rojo-Alba, S., Leal-Negredo, A., Castello-Abietar, C., Alvarez, V., Cuesta LLavona, E., & Coto, E. (2020). Capillary electrophoresis of PCR fragments with 5'-labelled primers for testing the SARS-CoV-2. Journal of virological methods, 284, 113937. https: / / doi.Org / 10.1016 / i.jviroment.2020. l 13937

[0326] - Martin, G., Rojo-Alba, S., Castello-Abietar, C., Abreu-Salinas, F., Costales Gonzalez, I., Boga, J. A., Melon, S., & Alvarez-Arguelles, M. E. (2021). Comparison of in-house SARS-CoV-2 genome extraction procedures. A need for COVID-19 pandemic. Research Square preprint doi: 10.21203 / rs.3rs-214704 / vl

[0327] - Sandoval Torriente, M., Castello-Abietar, C., BogaRiveiro, J., Alvarez- Arguelles, M. E., Rojo- Alba, S., Abreu-Salinas, F., Costales Gonzalez, I., Perez Martinez, Z., Martin Rodriguez, G., Gomez de Ona, J., & Melon Garcia, S. (2021). A novel single nucleotide polymorphism assay for the detection of N501 Y SARS-CoV-2 variants. Journal of virological methods, 294, 114143. https: / / doi.Org / 10.1016 / i.jviroment.2020. l 14143

[0328] - COVID-19 Treatment Guidelines Panel. Coronavirus Disease 2019 (COVID-19) Treatment Guidelines. National Institutes of Health. Available at: https: / / www.covidl9treatmentguidelines.nih.gOv / .Accessedl27 / 09 / 20211.

[0329] - Hodgson, S. H., Mansatta, K., Mallet, G., Harris, V., Emary, K., & Pollard, A. J. (2021). What defines an efficacious COVID-19 vaccine? A review of the challenges assessing the clinical efficacy of vaccines against SARS-CoV-2. The Lancet. Infectious diseases, 21(2), e26-e35. https: / / doi.org / 10, 1016 / S1473-3099(20)30773-8

[0330] - De la Iglesia, P., Melon Garcia, S., Lopez, B., Rodriguez, M., Blanco, M. I., Mellado, P., & de Ona, M. (1998). Rapid screening tests for determining in vitro susceptibility of herpes simplex virus clinical isolates. Journal of clinical microbiology, 36(8), 2389-2391. https: / / doi.Org / 10.1128 / JCM.36.8.2389-239L1998

[0331] - Perez Martinez, Z. (2021) 'Efecto de la melatonina en celulas infectadas con el Herpes Simplex Type 1'. Universidad de Oviedo. Asturias-Espana.

[0332] ACQUEOUS POTASSIUM RIBOSATE:

[0333] Oral Therapeutic Dosage

[0334] The synergistic effect of the aqueous Potassium Ribosate composition presented in this research is evident. This composition can be used in the following stoichiometric ratios between Potassium Bicarbonate and D-Ribose: 2:1, 3: 1 and 4: 1. However, the 3: 1 ratio is recommended and is related to the time of use of the treatment.

[0335] There are currently numerous potassium compounds with parapharmaceutical and pharmaceutical classifications that improve quality of life in the face of various human diseases and injuries. Many of these compounds are administered orally. Many studies show that the doses of active ingredients containing potassium are highly dependent on the characteristics of the entire molecule. The incidence of potassium in blood has been standardised by the FDA and WHO, and each country sets its own specifications. Among these specifications are daily potassium intake requirements that are monitored per kg of body weight, which in turn varies depending on whether the person is a child or an adult. Potassium Ribosate is non-toxic, the potassium requirement for humans is compressed between 0.8 and 2 mEq of potassium / Kg of body weight and not more than 150 mEq / day, and in children 3 mEq / Kg of body weight. Making a direct relationship in terms of the amount of mEq of potassium that would be orally administered to a human, we obtain that Potassium Ribosate would not exceed 12 mEq / day (mEq(K)= Immol / value). The proposed possible doses for Potassium Ribosate would correspond to a minimum of 3 mEq of potassium and a maximum of about 9-12 mEq of potassium for an adult, which would make a maximum total daily dose of 12 mEq of Potassium. Furthermore, D- Ribose is a non-toxic chemical companion to Potassium Ribosate, which is the opposite of what happens with other active ingredients that contain molecules such as Acetate, Chloride, Amines and Imidazoline, among others, as companions.

[0336] Bibliography

[0337] • Equivalencias Internacionales ( / equivalencias-+-61371) Cloruro de Potasio Comercial.

[0338] • Agencia Espanola de Medicamentos y productos sanitarios: Codigo ATC: B05XA17.

[0339] • Documento consolidado BOE-A-2011-14574.

[0340] • Agencia Espanola de Medicamentos y Productos Sanitarios (AEMPS) http: / / www.aemps.gob.es / . Codigo 51378 y 79.

[0341] • http: / / www.farmaciasahumada.cl / fasa / MFT / PRODUCTO / P10234.HTM.

[0342] • http: / / www.redalyc.org / html / 559 / 55916209004 / Di cl ofenacpotasi co .

[0343] • Agencia Espanola de Medicamentos y Productos Sanitarios (AEMPS) http: / / www.aemps.gob.es / . Codigo E224.

[0344] • Cuadro Basico y Catalogo de Medicamentos, Edition 2013. D.R.Consejo de Salubridad General. Comision Interinstitucional del Cuadro Basico y Catalogo de Insumos del Sector Salud. Col. Chapultepec Morales. Delegation Miguel Hidalgo. C.P. 11570, Mexico, D.F. • Remington Farmacia, Volumen 2. Escrito por Alfonso R. Gennaro, https : / / books. google. es / books?id=5 SGJ4ezraG4C&pg=PAl 815&lpg=PAl 815&dq=potasio+vi a+intramuscular&source=bl&ots=OM56ilpDHc&sig=D51etVO-fp_dTs.

[0345] • https: / / www.fda. gov / Food / GuidanceRegulation / ... / LabelingNutrition / ucm247936.htm.

[0346] • http: / / www.archives.gov / federal register / code of federal regulations / ibr locations. ht ml.

[0347] In practice, the invention relates to a composition for use as a medicament in a method of treating and preventing SARS-CoV-2 viruses causing COVID-19 in a host, typically a human being; the composition is characterised in that it comprises a compound of Potassium Bicarbonate and D- Ribose in aqueous solution.

[0348] In particular, the composition is preferably and advantageously intended for use as an orally administered drug.

[0349] The composition may be used in a stoichiometric ratio comprised among the following: two parts Potassium Bicarbonate and one part D-Ribose; three parts Potassium Bicarbonate and one part D-Ribose; four parts Potassium Bicarbonate and one part D-Ribose. The stoichiometric ratio of three parts of Potassium Bicarbonate and one part D-Ribose was found to be particularly suitable. The components making up the composition are in single doses for each component which can be combined at the time of use, so as to determine an extemporaneous solution of the drug.

[0350] The invention relates to the use of a composition of Potassium Bicarbonate and D-Ribose in aqueous solution for forming an orally administered drug in a method of treating and preventing SARS-CoV-2 viruses causing COVID-19 in a host, typically a human being.

[0351] In particular, the composition may be used in a stoichiometric ratio comprised among the following: two parts Potassium Bicarbonate and one part D-Ribose; three parts Potassium Bicarbonate and one part D-Ribose; four parts Potassium Bicarbonate and one part D-Ribose. As indicated above, depending on the time of use of the treatment, the stoichiometric ratio of three parts Potassium Bicarbonate and one part D-Ribose was particularly suitable. In addition, the components of the composition are in single doses for each component that can be combined at the time of use. In other words, the composition of the invention consists of Potassium Bicarbonate and D-Ribose and these substances are combined with each other prior to their intake. In practice, Potassium Bicarbonate and D-Ribose are kept separate to form the composition upon intake.

[0352] The invention relates to an orally administered drug for the treatment and prevention of SARS- CoV-2 viruses causing COVID-19 in a host, typically a human being, characterised by comprising a compound of Potassium Bicarbonate and D-Ribose. Advantageously, according to the invention and the indications provided by the tests performed, the composition comprising Potassium Bicarbonate and D-Ribose in aqueous solution appears to be usable for the treatment and prevention of SARS-CoV-2 infections. Finally, it is understood that the composition comprising Potassium Bicarbonate and D-Ribose in aqueous solution and its use as described and illustrated may be subject to modifications and variations without departing from the scope of the present invention as defined in the claims.

Claims

CLAIMS1. A composition for use as a drug in a method of treating and preventing SARS-CoV-2 viruses causing COVID-19 in a host, typically a human being, characterised in that it comprises a compound of Potassium Bicarbonate and D-Ribose in aqueous solution.

2. Composition for use according to claim 1, characterised in that it is intended for use as an oral administration drug.

3. Composition for use according to claim 1 and / or 2, characterized in that it is used in a stoichiometric ratio included among the following: two parts of potassium bicarbonate and one part of D-ribose; three parts of potassium bicarbonate and one part of D-Ribose; four parts of potassium bicarbonate and one part of D-Ribose4. Composition for use according to any one of the preceding claims, characterised in that the composition is used in the stoichiometric ratio of three parts Potassium Bicarbonate and one part D-Ribose.

5. Composition for use according to any one of the preceding claims, characterised in that the components of said composition are in single doses for each component associated at the time of use, so as to determine an extemporaneous solution of a drug.

6. Use of a composition of Potassium Bicarbonate and D-Ribose in an aqueous solution for forming a drug for oral administration in a method of treating and preventing SARS-CoV-2 viruses causing COVID-19 in a host, typically a human being.

7. Use of a composition according to claim 6, characterized in that the composition is used in a stoichiometric ratio included among the following: two parts Potassium Bicarbonate and one part D-Ribose; three parts Potassium Bicarbonate and one part D-Ribose; four parts Potassium Bicarbonate and one part D-Ribose.

8. Use of a composition according to claim 6, characterised by using the composition in the stoichiometric ratio of three parts Potassium Bicarbonate and one part D-Ribose.

9. Use of a composition according to claim 7 or 8, characterised in that the components of the composition are in single doses for each component that can be combined at the time of use, so as to result in an extemporaneous solution of a drug.

10. An orally administered drug for the treatment and prevention of SARS-CoV-2 viruses causing COVID-19 in a host, typically a human being, characterised in that it comprises a compound of Potassium Bicarbonate and D-Ribose.

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