ANTIVIRAL AND ANTIBACTERIAL COMPOSITION

Abstract

The use of a composition as antiviral and/or antimicrobial agent, wherein the composition includes at least a positively charged natural or unnatural amino acid, an organic acid, a cationic polymer and a zwitterionic surfactant.

Claims

1. Use of a composition as antiviral and/or antimicrobial agent, wherein the water soluble composition comprises at least a positively charged natural or unnatural amino acid, an organic acid, a cationic polymer, and a zwitterionic surfactant.

2. Use according to claim 1, wherein the composition comprises up to 92% by weight of water

3. Use according to claim 1, wherein the organic acid is selected from the group consisting of malic acid, citric acid, lactic acid, acetic acid, glutamic acid, ascorbic acid and benzoic acid or a mixture thereof.

4. Use according to claim 1, wherein the positively charged amino acid is selected from the group consisting of L-arginine, L-lysine, L-histidine, ornithine, 2,4-diaminobutanoic acid, 2,3- diaminopropanoic acid, 3-(aminoiminomethyl)amino-alanine, 2-amino-4-(aminoiminomethyl)aminobutanoic acid, N6-(aminoiminomethyly) lysine, 2-amino-7- (aminoiminomethyl)aminoheptanoic acid, 2,7-diaminoheptanoic acid, 2, 8-diaminooxtanoic acid, 2, 9-diaminononanoic acid, 2,10-diaminodecanoic acid, 4- (aminoiminomethyl)phenylalanine and 4-(aminoiminomethyl)aminophenylalanine.

5. Use according to claim 1, wherein the cationic polymer is selected from the group consisting of polyquaternium, fatty amines, polyethyleneimine or a copolymer thereof, cationic starch, metal cation components and mixture thereof.

6. Use according to claim 5, wherein the cationic polymer is a branched polyethyleneimine which is at least partly crosslinked.

7. Use according to claim 1, wherein the composition additionally comprises a humectant and or antistatic material.

8. Use according to claim 1, wherein the composition comprises L-arginine as positively charged amino acid and malic acid and/or glutamic as organic acid.

9. Use according to claim 1, wherein the composition comprises at least 0.02 to 8% by weight of a positively charged natural or unnatural amino acid, 0.02 to 8% by weight of an organic acid, 0.02 to 5% by weight of a cationic polymer, and 0.02 to 8% by weight of a zwitterionic surfactant.

10. Use according to claim 1, wherein the composition is free of ethanol.

11. Use according to claim 1 to inactivate a virus and a bacteria selected from the group consisting of corona virus, influenza virus, human rhinovirus (HRN), parainfluenza virus (PIN), respiratory syncytial virus (RSN), adenovirus, metapneumovirus, rhinovirus, and SARS-COV-2 or a mutant thereof.

12. Use according to claim 1, wherein the composition is provided as a spray, a pre-spray gel, a water soluble pod, a mist, a strip, a lozenge and wash system.

13. A carrier which is coated with a composition according to claim 1 or its essentially water-free dried form.

14. The carrier according to claim 13, wherein the carrier is selected from the group consisting of air filters, personal protective equipment, N95 surgical mask, community mask, textile mask, foam mask, Bandana mask, molded fiber devices, foam textiles, cotton textiles, cellulose textiles, composites, nasal inserts, foam nasal inserts, nasal filters, nasal screens, nasal filters, air filters, surgical gowns, coverings and wipes.

15. The carrier according to claim 3 wherein the carrier is additionally treated with cold atmospheric plasma and or corona to create surface electrostatic and cationic charges.

Description

[0064] In a further embodiment of the present invention, the carrier is additionally treated with cold atmospheric plasma. Cold atmospheric plasma is known to the skilled person and allows to increase the cationic charge of the surface. The presence of a metallic stearate such as magnesium stearate, calcium stearate or zinc stearate on the carrier or the composition according to the present invention can stabilize electrostatic charges and result in intensified and retained anti-viral properties.

[0065] FIG. 1 shows a schematic diagram of the experimental setup;

[0066] FIG. 2 shows the antiviral activity of solutions and mixes inhibiting SARS-CoV-2 entry.

[0067] FIG. 3 shows the antiviral activity of mixes inhibiting SARS-CoV-2 entry.

[0068] FIG. 4 shows a schematic diagram of the experimental setup.

EXAMPLES

Example 1: Iso like Experiment on Coated Petri Dishes

[0069] Step 1 Prepare 400 ul of R18 rhodamine fabled inactivated virus inoculum solution in PBS for each sample.

[0070] Step 2 Apply the inoculum on the sample and sandwich it with LOPE inert film as shown in FIG. 1 (ISO 21702): [0071] A Petri dish 5 is coated with the composition according to the present invention and dried to form an antiviral surface 3. [0072] Artificial inactivated enveloped coronavirus sample 2 having the same composition of covid 19 virus but the mRNA genome was removed and replaced with a fluorescent dye is added to the antiviral surface 3. [0073] Plastic film 1 to cover the petri dish antiviral surface once the viral solution is applied.

[0074] Step 3 Incubate the samples for 24 hours at room temperature and in dark environment.

[0075] Step 4 Add 10 ml of PBS to each sample to recover the inoculum.

[0076] Step 5 Pipette 2 ml of the recover mixture in a transparent cuvette (2 replicates per sample).

[0077] Step 6 Measure emission spectra with fluorometer (excitation wavelength 560 nm, emission measure from 580 nm to 650 nm).

[0078] Antiviral efficacy of the solution was determined by using the above test procedure.

[0079] The interaction and the disintegration of the virus is determined by measuring the fluorescent concentration on the antiviral surface resulting from viral disintegration. the range is defined by the max amount of fluorescent dye represented as (PC) and no dye as (NC) indicating no interaction. The graph below shows that the formulations demonstrated efficacy against the virus.

TABLE-US-00002 Florescent intensity in Formulation PH MM Malic acid 5% 2.5 7.5 Luviquat 5% Cocamidopropyl 5% Malic acid 5% 5.5 7.4 Luviquat 5% Cocamidopropyl 5% L-Arganine 10% Malic acid 5% 2.5 7.7 Cationic starch5% Cocamidopropyl 5% L-Arganine 0% PVA 10% Malic acid 5% 5.5 7 . 6 Luviquat 5% Cocamidopropyl 5% PVA 10% L-Arganine 10% Malic acid 5% 2.5 7.4 Luviquat 5% Cocamidopropyl 5% PVA 10% Malic acid 5% 5.5 7.5 Luviquat 5% Cocamidopropyl 5% Glycerin 2% PVA 10% Control (no effect on 0.9 disintegration) Control (maximum effect 7.7 on disintegration )

Example 2: Inhibition of SARS-CoV-2

[0080] Cell Cultures: Vero E6 cells (ATCC CRL-1586) were cultured in Dulbecco's modified Eagle medium, (DMEM) with 10% fetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin (all from Invitrogen). HEK-293T overexpressing the human ACE2 were kindly provided by Integral Molecular Company and maintained in DMEM (Invitrogen) with 10% fetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin, and 1 μg/ml of puromycin (all from Invitrogen).

[0081] Pseudovirus production: HIV-1 luciferase reporter pseudoviruses expressing SARS-CoV-2 Spike protein were generated using two plasmids. pNL4-3.Luc.R-.E- was obtained from the NIH AIDS repository. SARS-CoV-2.SctA19 was generated (Geneart) from the full protein sequence of SARS-CoV-2 spike with a deletion of the last 19 amino acids in C-terminal, human-codon optimized and inserted into pcDNA3.4-TOPO 1. Spike plasmid was transfected with X-tremeGENE HP Transfection Reagent (Merck) into HEK-293T cells, and 24 hours later, cells were transfected with pNL4-3.Luc.R-.E-. Supernatants were harvested 48 hours later, filtered with 0.45 pm (Millex Millipore) and stored at −80° C. until use. Viruses were titrated in HEK-293T overexpressing human ACE2 to use an equal amount of fusogenic viruses.

[0082] Pseudovirus assay. HEK-293T overexpressing the human ACE2 were used to test provided mixes and their vehicles at the indicated dilutions. A constant pseudoviral titer was used to pulse cells in the presence of the samples. After 48h post-inoculation, cells were lysed with the Bright Glo Luciferase Assay system (Promega). Luminescence was measured with an EnSight Multimode Plate Reader (Perkin Elmer). To detect any associated cytotoxic effect, mix formulations were also tested with media, and were equally cultured on cells but in the absence of pseudovirus. Cytotoxic effects of these products were measured 48h post-inoculation, using the CellTiter-Glo luminescent cell viability assay (Promega).

[0083] Sample Preparation:

TABLE-US-00003 Mixture 1 1.1 2 2.1 3 4 Malic acid   1%   1%  1.0%  1.0%  1.0%  1.0% Cocamido-propyl 2.5% 2.5%  2.5%  2.5%  2.5%   Lecithin        2.5% Luviquat 2.4% 2.4%  2.5%  2.5%  1.0%   b-cyclodextrin  2.5% Benzalkonium 2.5% 2.5% 0.75% 0.75% 0.75% 0.75% chloride L-Arginine 2.0% 2.0%  2.0%  2.0%  2.0%  2.0% Me-cellulose 0.2% 0.2

[0084] Results

[0085] We have tested the capacity of the provided mixes to inhibit

[0086] SARS-CoV-2 entry into target cells. We employed a luciferase-based assay, using a reporter lentivirus pseudotyped with the spike protein of SARS-CoV-2, which allows the detection of viral fusion with target HEK-293T cells expressing human ACE2 receptor. A constant concentration of the reporter pseudovirus containing the SARS-CoV-2 original Spike protein was mixed with increasing concentrations of the indicated CPC-containing mouth rinses, or their corresponding vehicles, and added to the target cells. To control for any induced cytotoxicity of the mixes, target cells were also cultured with increasing concentrations of the indicated products in the absence of pseudoviruses. All solutions but A at pH 7, C and E were able to inhibit viral fusion in a dose dependent manner at concentrations where no cytotoxic effects were observed (FIG. 2). The combinations of these solutions (mixtures) were very efficacious at inhibiting viral entry at non cytotoxic concentrations. New mixes including active solutions where prepared, and were able to inhibit viral fusion in a dose dependent manner at concentrations where no cytotoxic effects were observed (FIG. 3). These results indicate that solutions and their mixes able to block SARS-CoV-2 viral entry into target cells.

[0087] Figures

[0088] FIG. 2. Antiviral activity of solutions and mixes inhibiting SARS-CoV-2 entry. Viral entry inhibition on target HEK-293T cells expressing ACE2 exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of solutions and their mixes. Cytotoxic effect on HEK-293T cells expressing ACE2 cells exposed to increasing concentrations of solutions and mixes in the absence of pseudovirus is also shown (right panels).

[0089] FIG. 3. Antiviral activity of mixes inhibiting SARS-CoV-2 entry. Viral entry inhibition on target HEK-293T cells expressing ACE2 exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of mixes. Cytotoxic effect on HEK-293T cells expressing ACE2 cells exposed to increasing concentrations of mixes in the absence of pseudovirus is also shown (right panels).

Example 3: Measurement of the Active Surface Ionic Charge Density

[0090] The amount of surface charge density can be determined by means of measurement of induced image charges in a sensing electrode. The treated surface repeatedly moves close to and away from a sensing electrode and the induced image charge creates an AC electrical current in the circuitry connected to the sensing electrode. The induced current is measured and is proportional to the surface charge.

[0091] The apparatus consists of a sample spinner, contained in a metal box, sensing electrode and Keithley 823 nanovolt amplifier (FIG. 4)

[0092] C and R are capacitance and resistance of the input circuitry of the amplifier. Input capacitance was measured 80pF and input resistance is 50 MOhm.

[0093] The sensing electrode is made of 1.3 mm diameter copper wire. When the metal box top is in closed position, the sensing electrode is about 1.5mm above the sample surface. One half of the sample substrate is treated, and another half is untreated. During the sample spinning treated and untreated surface repeatedly move under the sensing electrode. The surface charge is calculated using the following formula:

Q=V*C/A

[0094] Where Q is charge per unit area, V is measured voltage on the sensing electrode and A is the area of the sample under the sensing electrode.

[0095] Sample Preparation:

[0096] Paper or corrugated substrate disks are prepared in approximately 2.5″ circular in diameter. Treated samples are attached to 50% of the diameter by use of adhesive or tape. The apparatus detects Ionic charges by sensing the differential of charges on a treated and untreated surface. A disk is prepared wherein half of the disk is treated and the other is not. As the disk is rotated the sensing electrode detects the charge differential.

[0097] Sample Preparation:

[0098] Paper or corrugated substrate disks are prepared in approximately 2.5″ circular in diameter. Treated samples are attached to 50% of the diameter by use of adhesive or tape.

[0099] The apparatus detects Ionic charges by sensing the differential of charges on a treated and untreated surface. A disk is prepared wherein half of the disk is treated and the other is not. As the disk is rotated the sensing electrode detects the charge differential. Utilizing the apparatus, and following the identical testing procedure, we tested numerous samples of the “Livinguard” commercial mask. The average Ionic density on the surface, is indicated in the table below.

EXAMPLES

[0100]

TABLE-US-00004 Charges Charge density per per unit cm.sup.2 in Composition area millions Silicone (industrial) 2.94E+08 294 Malic acid 9.80E+07 98 Luviquat Cocamidopropyl Malic acid 1.58E+07 15.8 Luviquat Cocamidopropyl L-Arganine Malic acid 1.35E+08 135 Cationic starch Cocamidopropyl L-Arganine Malic acid 1.67E+08 167 Luviquat, Cocamidopropyl Malic acid 4.27E+08 427 Luviquat Cocamidopropyl PVA Malic acid 2.62E+08 2 62 Luviquat Cocamidopropyl Glycerin Livinguard (Commercial 20 Mask)

Example 4

[0101] Two bacterial strains were tested against three different compounds provided. The Gram-negative bacterial strain E. coli K12 was grown in LB media, and the Gram-positive strain Staphylococcus aureus 113 was cultured in BHI media overnight prior to antimicrobial test. Bacterial density was determined by OD600 measurements and adjusted to approximately 108 bacterial cells per mL with broth media, respectively. Equal volume of compound solutions and bacterial cells were mixed and incubated at 37° C. To determine the killing efficacy, 20 μL of the mixed bacterial suspension was numerated at 1 h, 3 h, 6 h and 30 h after incubation with corresponding compounds, by distributing on an LB agar plate (for E. coli) and BHI agar plate (for S. aureus) at 10-fold serial dilutions. The plates were further incubated at 37° C. for 24 h and bacteria viability was determined by counting the colony forming units (CFU).

[0102] Testing the formulation samples for antibacterial efficacy shows that the inhibition is effective but in a slow kinetic manner. Bacterial count reduction after a 2 hour incubation was low. However, almost no survived bacteria can be detected after a 24 hour incubation. Reduction time can be improved by increasing the concentration of the components.