TUCKER SIMON (AU)
GOODALL STEPHEN (AU)
WO2020232515A1 | 2020-11-26 |
KR101935250B1 | 2019-01-04 | |||
JPH04173726A | 1992-06-22 | |||
JPH09151127A | 1997-06-10 | |||
JP2000169378A | 2000-06-20 | |||
US20060122082A1 | 2006-06-08 | |||
EP0526695B1 | 1998-02-11 | |||
JP2004352642A | 2004-12-16 | |||
CN1203864C | 2005-06-01 |
THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. An aqueous virucidal formulation (based on %w/v of the total formulation) comprising: (a) about 0.10% to about 1.25% w/v PVP-I; (b) 0.00% to about 15% w/v of hyaluronic acid, polyethylene glycol and/or glycerol; (c) 0.00% to about 0.20% w/v of menthol; (d) 0.00% to about 30% of a polar solvent; (e) 0.00% to about 5% of sodium hydrogen phosphate or similar buffer; and (f) 0.00% to about 0.20% w/v of iodide and/or iodate salt; wherein the formulation comprises at least two of (b) – (f). 2. An aqueous virucidal formulation according to claim 1 comprising: (a) about 0.10% to about 1.25% w/v PVP-I; (b) about 0.005% to about 0.20% w/v of iodide and/or iodate salt; and (c) about 0.005% to about 0.05% w/v menthol. 3. An aqueous virucidal formulation according to claim 1 or claim 2 comprising: (a) about 0.10% to about 1.25% w/v PVP-I; (b) about 0.005% to about 0.01% w/v of an iodate salt; (c) about 0.005% to about 0.05% w/v menthol; (d) about 0.005% to about 0.10% w/v of an iodide salt; and (e) about 1.0% w/v to about 15% w/v of glycerol. 4. An aqueous virucidal formulation according to any one of claims 1 to 3 comprising: (a) about 0.10% to about 1.25% w/v PVP-I; (b) about 0.005% to about 0.01% w/v of an iodate salt; (c) about 0.005% to about 0.05% w/v menthol; (d) about 0.005% to about 0.10% w/v of an iodide salt; (e) about 1.0% w/v to about 15% w/v of glycerol; and (f) about 0.2% w/v to about 30% w/v of a polar solvent other than water. 5. An aqueous virucidal formulation according to any one of claims 1 to 4 comprising: (a) about 0.10% to about 1.0% w/v PVP-I; (b) about 0.005% to about 0.01% w/v of an iodate salt; (c) about 0.005% to about 0.05% w/v menthol; (d) about 0.005% to about 0.10% w/v of an iodide salt; (e) about 1.0% w/v to about 10% w/v of glycerol; (f) about 0.2% w/v to about 1.0% w/v of a polar solvent other than water (g) about 0.05% to about 0.20% w/v of sodium dihydrogen phosphate dihydrate; (h) sodium hydroxide q.s. ; and (i) water q.s.. 6. An aqueous virucidal formulation according to anyone of claims 1 to 5 wherein the concentration of PVP-I is about 0.25% to about 1.00%. 7. An aqueous virucidal formulation according to claim 5 wherein the concentration of PVP-I is about 0.50%. 8. An aqueous virucidal formulation according to any one of claims 5 to 7 wherein the polar solvent other than water is ethanol. 9. An aqueous virucidal formulation according to anyone of claims 1 to 8 wherein the pH of the formulation is about 3 to about 6. 10. A method of treating viral infection in a subject comprising administering to the subject an effective amount of the aqueous virucidal formulation according to any one of claims 1 to 9. 11. A method according to claim 10 wherein the formulation is administered intranasally. 12. A method of reducing the volume of mucous secreted, or reducing the activity, viability or number of viruses contained in secreted mucous, or reducing the period during which virus- laden mucous is present, and thereby reducing the risk of viruses migrating to secondary sites in the respiratory tract to establish or contribute to secondary illnesses, including the step of administering to a subject an effective amount of an aqueous virucidal formulation according to any one of claims 1 to 9. 13. The method according to claim 12 wherein the secondary illness is a respiratory tract infection. 14. The method according to claim 13 wherein the secondary illness selected from the group selected from sinusitis, bronchitis and otitis media. 15. A method according to anyone of claims 10 to 14 wherein the aqueous virucidal formulation is administered nasally at a temperature of between 10° to 35°C. |
Formulation 5: Per 100 mL g %w/v PVP-I 0.30% 0.30% Potassium Iodide 0.010 0.010% Potassium Iodate 0.005 0.005% Glycerol 5.20 5.20% Sodium DiHydrogen Phosphate Dihydrate 0.35 0.35% Sodium Hydroxide 0.55 0.55% Ethanol 0.49 0.49% Menthol 0.01 0.010% Benzalkonium Chloride 0.08 0.08% Water 93.0 93.0% 100.00 100.0% Formulation 6: Per 100 mL g %w/v PVP-I 0.10 0.10% Potassium Iodide 0.010 0.010% Potassium Iodate 0.005 0.005% Glycerol 5.10 5.10% Sodium DiHydrogen Phosphate Dihydrate 0.25 0.25% Sodium Hydroxide 0.85 0.85% Ethanol 0.59 0.59% Menthol 0.01 0.010% Benzalkonium Chloride 0.08 0.08% Water 93.08 93.0% 100.00 100.0% Formulation 7: Per 100 mL g %w/v PVP-I 1.10 1.10% Potassium Iodide 0.010 0.010% Potassium Iodate 0.005 0.005% Glycerol 4.40 4.40% Sodium DiHydrogen Phosphate Dihydrate 0.15 0.15% Sodium Hydroxide 0.75 0.75% Ethanol 0.49 0.49% Menthol 0.01 0.01% Water 93.0085 93.085% 100.00 100.0% Formulation 8: Per 100 mL g %w/v PVP-I 0.70 0.70% Potassium Iodide 0.030 0.030% Potassium Iodate 0.015 0.015% Glycerol 4.0 4.0% Sodium DiHydrogen Phosphate Dihydrate 0.1 0.1% Sodium Hydroxide 0.45 0.45% Ethanol 0.50 0.50% Menthol 0.10 0.10% Water 93.105 94.105% 100.00 100.0% Formulation 9: Per 100 mL g %w/v PVP-I 0.50 0.50% Potassium Iodide 0.010 0.010% Potassium Iodate 0.005 0.005% Glycerol 5.00 5.00% Sodium DiHydrogen Phosphate Dihydrate 0.15 0.15% Sodium Hydroxide 0.75 0.75% Ethanol 0.49 0.49% Menthol 0.01 0.010% Water 93.08 93.08% 100.00 100.0%
Formulation 10: Per 100 mL g %w/v PVP-I 0.80 0.80% Potassium Iodide 0.010 0.010% Polyethylene Glycol 400 4.80 4.80% Sodium DiHydrogen Phosphate 0.15 0.15% Sodium Hydroxide 0.75 0.75% Ethanol 0.49 0.49% Menthol 0.01 0.010% Benzalkonium Chloride 0.1 0.10% Water 92.89 92.89% 100.00 100.0% Formulation 11: Per 100 mL g %w/v PVP-I 0.50 0.50% Potassium Iodide 0.050 0.050% Potassium Iodate 0.005 0.005% Glycerol 5.00 5.00% Sodium DiHydrogen Phosphate Dihydrate 0.15 0.15% Sodium Hydroxide 0.7 0.7% Ethanol 0.50 0.50% Menthol 0.01 0.010% Water 93.085 93.085% 100.00 100.0% Formulation 12: Per 100 mL g %w/v PVP-I 1.00 1.00% Potassium Iodide 0.010 0.010% Glycerol 5.50 5.50% Sodium Hydrogen Carbonate 0.15 0.15% Ethanol 0.49 0.49% Menthol 0.01 0.010% Water 92.84 92.84% 100.00 100.0% Formulation 13: Per 100 mL g %w/v PVP-I 1.25 1.25% Potassium Iodide 0.010 0.010% Glycerol 4.60 4.40% Citric Acid 0.325 0.325% Sodium Hydroxide 0.15 0.15% Ethanol 0.22 0.22% Menthol 0.21 0.21% Benzalkonium Chloride 0.25 0.25% Water 92.985 92.985% 100.00 100.0% Formulation 14: Per 100 mL g %w/v PVP-I 0.30% 0.30% Potassium Iodide 0.010 0.010% Hyaluronic acid 2.20 2.20% Sodium DiHydrogen Phosphate Dihydrate 0.35 0.35% Ethanol 0.49 0.49% Menthol 0.01 0.010% Benzalkonium Chloride 0.08 0.08% Water 96.56 96.56% 100.00 100.0% Formulation 15: Per 100 mL g %w/v PVP-I 0.10 0.10% Potassium Iodate 0.005 0.005% Glycerol 5.10 5.10% Citric Acid 0.25 0.25% Sodium Hydroxide 0.65 0.65% Ethanol 0.59 0.59% Menthol 0.01 0.010% Water 93.295 93.295% 100.00 100.0% Formulation 16: Per 100 mL g %w/v PVP-I 1.10 1.10% Potassium Iodide 0.010 0.010% Glycerol 3.40 3.40% Sodium Hydrogen Carbonate 0.15 0.15% Sodium Hydroxide 0.75 0.75% Menthol 0.01 0.01% Water 94.58 94.58% 100.00 100.0% Formulation 17: Per 100 mL g %w/v PVP-I 0.70 0.70% Potassium Iodide 0.030 0.030% Potassium Iodate 0.015 0.015% Hyaluronic acid 4.0 4.0% Citric Acid 0.1 0.1% Sodium Hydroxide 0.45 0.45% Ethanol 0.30 0.30% Menthol 0.10 0.10% Water 94.305 94.305% 100.00 100.0% Formulation 18: Per 100 mL g %w/v PVP-I 0.50 0.50% Potassium Iodide 0.020 0.020% Polyethylene Glycol 1000 7.00 7.00% Sodium DiHydrogen Phosphate Dihydrate 0.15 0.15% Sodium Hydroxide 0.65 0.65% Dimethyl Sulphoxide 0.49 0.49% Menthol 0.01 0.010% Water 91.18 91.18% 100.00 100.0% Biological Examples: EXAMPLE 1 – Activity against HRV-14 [0053] Human rhinovirus 14 (HRV-14) is known to have a degree of resistance to PVP-I and therefore was selected as a representative “hard to kill” non-enveloped virus for the purposes of discriminating the differential activity of PVP-I formulations. For example, Kawana and colleagues (Kawana, R., et al. Inactivation of Human Viruses by Povidone-Iodine in Comparison to Other Antiseptics. Dermatology 1997, 195:29-35) reported that treatment of HRV-14 with a 1% PVP-I solution using the formulation sold as Betadine™ yielded a reduction in viral titers of only 1.3 and 1.7 log10, based on a 0.5 and 10 minute exposure, respectively. These times are relevant to proposed use of the present invention because the likely effective residence and activity time for PVP-I solution in the nose is approximately less than 5 minutes, due to mucociliary clearance and inactivation by mucins and other proteinaceous material. METHOD [0054] The Formulation 2 described in the present invention was selected for testing against various commercial PVP-I formulations. Other commercial formulations described below are as disclosed in Niazi, S. K. (2009) Handbook of Pharmaceutical Manufacturing Formulations, Second Edition, Volume Series, 2009. Informa Healthcare USA, Inc., New York, NY 10017. In addition, because published data was available against HRV-14, a 0.45% PVP-I liposomal investigative formulation (reported by Wutzler et al. Antiviral Research 2002, 54:89-97) and a reference 1.0% PVP-I solution in phosphate buffer (reported by Kawana et al. Dermatology 1997, 195:29-35) [0055] HRV-14 virus was exposed to Formulation 2 (0.5% PVP-I) of the present invention for the indicated time period at 34 ₒ C prior to quenching the mixture by 1:10 dilution using ice cold culture medium (DMEM-F12, 2% fetal bovine serum) and measurement of viral titers by TCID 50 assay on HeLa cells. [0056] Betaisodona data are as reported by Reimer (2002) (Reimer, R., et al. Antimicrobial Effectiveness of Povidone-Iodine and Consequences for New Application Areas. Dermatology 2002, 204:114-120) and Wutzler (2002), (Wutzler, P., et al. Virucidal activity and cytotoxicity of the liposomal formulation of povidone iodine. Antiviral Research 2002, 54:89-97), respectively. Liposomal PVP-I data are also from Wutzler (2002). Isodine Gargle data and PVP-I solution in phosphate buffer are as reported by Kawana and colleagues (1997). [0057] Units are log 10 viral titer reduction. Results are shown in Figure 1. The heavy horizontal line corresponds to a 4 log10 reduction (corresponding to a 99.99% reduction in viral infectivity) in accordance with the Therapeutic Goods Administration (TGA) guidelines for virucidal testing: TGA instructions for disinfectant testing, Disinfectants, sterilants and sanitary products, Version 2.1, March 2020 (TGA Department of Health, Australian Government). [0058] The 0.5% PVP-I formulation of the present invention (Formulation 2) showed a markedly increased virucidal activity compared to other PVP-I formulations (concentrations between around 0.5% and 1.0%), at clinically-relevant exposure times of 2 and 5 minutes and comparable activity at 1 minute. Notably, Formulation 2 was the only PVP-I formulation which exceeded the 4 log 10 reduction for virucidal products with a 5-minute exposure period. The other formulations required at least 15 minutes to achieve this standard. [0059] 0.5% PVP-I formulation of the present invention was clearly more effective than the other PVP-I formulations previously disclosed, including 1% PVP-I solutions tested (Isodine Gargle and PVP-I solution in phosphate buffer). EXAMPLE 2 – Virucidal activity of various solutions against SARS-CoV-2 [0060] The activity of the example Formulation 2 was compared with a simple solution in water of PVP-I at the same concentration and the background formula of Formulation 2 without the PVP-I. METHOD [0061] Virus, Media, and Cells SARS-CoV-2 virus stocks were prepared by growing virus in Vero 76 cells. Test media used was MEM supplemented with 2% FBS and 50 µg/mL gentamicin. [0062] Tested products were: Test solution 1 (Formulation 2 containing 0.5% PVP-I), Test solution 2 (0.5% PVP-I in water), Test solution 3 (Formulation 2 without PVP-I). The test solutions were tested at full strength at three contact time-points: 15 sec, 5 min, and 15 min to reflect clinically-relevant exposure periods. SARS-CoV-2 virus stock was added to tubes at 1/10 of the test solution, so the final concentrations of solution tested was 90%. The test solutions were added to two tubes of each solution to serve as toxicity controls. Ethanol (70%) was tested in parallel as a positive virucidal control and a virus control was included for each contact time-point. [0063] Solution and virus were incubated at 37˚C for three contact times of 15 seconds, 5 minutes, and 15 minutes. Following contact period, the solutions were neutralized by a 1/10 dilution in test media containing 10% FBS. [0064] Neutralized samples were serially diluted using eight log10 dilutions in test medium. Each dilution was added to 4 wells of a 96-well plate with 80-100% confluent Vero E6 cells. The toxicity controls were added to an additional 4 wells and 2 of these wells were infected with virus to serve as neutralization controls, ensuring that residual sample in the titer assay plated did not inhibit growth and detection of surviving virus. All plates were incubated at 37°C, 5% CO 2 . [0065] On day 6 post-infection plates were scored for presence or absence of viral cytopathic effect (CPE). The Reed-Muench method was used to determine end-point titers (50% cell culture infectious dose, CCID 50 ) of the samples, and the log reduction value (LRV) of the compound compared to the negative (water) control was calculated. [0066] Virus controls were tested in diluent and the reduction of virus in compound-treated test wells compared to virus controls was calculated as the log reduction value (LRV). Toxicity controls were tested to see if the samples were toxic to cells. Neutralization controls were tested to ensure that virus inactivation did not continue after the specified contact time, and that residual sample in the titer assay plates did not inhibit growth and detection of surviving virus. This was done by adding toxicity samples to titer test plates then spiking each well with a low amount of virus that would produce an observable amount of CPE during the incubation period. RESULTS [0067] Virus titres and LRV for the three solutions against SARS-CoV-2 are shown in Table 1. Compound toxicity was not observed for any of the solutions. The 70% ethanol positive control was effective and reduced virus titer to a baseline of 0.67 log 10 CCID 50 /ml. [0068] The test solution 1 most consistently reduced virus titer to the greatest extent as compared with the other test solutions and was the only formulation to reach a 4-log reduction within 5 minutes. Solution 1(Example Formulation 2) had a LRV of 3.5 and 4.0 at the 15 sec and 5 min contact times, and reduced virus below limit of detection after 15 min of contact time (Table 1). PVP-I solution was also intermediately active, reducing virus, but not to the extent of Solution 1 (Table 1). The formula without PVP-I was not active at any of the selected contact points. [0069] Neutralization controls demonstrated that residual sample did not inhibit virus growth and detection in the endpoint titer assays in wells that did not have cytotoxicity. Virus controls and positive controls performed as expected.
Table 1. Virucidal efficacy against SARS-CoV-2 after incubation with virus at 37°C. a Log 10 CCID 50 of virus per mL, mean of 3 replicates ± standard deviation. b LRV (log reduction value) is the reduction of virus compared to the virus control. - Test Solution 1 = Example Formulation 2 supplied in amber glass. - Test Solution 2 = 0.5% PVP-I aqueous solution supplied in amber glass. - Test Solution 3 = Example Formulation 2 without PVP-I. EXAMPLE 3 – Activity of various solutions against MRSA METHOD [0070] S. aureus ATCC 33591 was streaked from frozen stock onto Tryptic Soy Agar (TSA) plates and grown overnight at 37°C. [0071] On the day of the assay, the bacteria inocula was prepared by suspending ATCC 33591 colonies in sterile PBS to generate 1.0 McFarland standards in 2 mL volume. The suspension was centrifuged to pellet the bacteria, then resuspended in 0.2 mL sterile PBS. Two independent inocula was prepared using this method. [0072] 2 mL of each of four test solutions were dispensed into sterile borosilicate glass tubes; 2 mL of sterile PBS was also prepared to determine inoculum density (i.e. time 0 control). One set of test solutions was prepared and tested for each independent inoculum. [0073] 50 µL of each inoculum was added to each test solution or to PBS control. [0074] After the addition of inoculum, each test solution was vortexed on high and a timer was started; at each time point, 20 µL was removed from the inoculated solution and added to 180 µL DE Neutralization buffer. This was then 10-fold serially diluted and 100 µL of the appropriate dilution was plated on TSA plates. [0075] TSA plates were incubated overnight at 37°C, after which CFU was enumerated. The results are shown in Tables 2 and 3.
[0076] Table 2. MRSA time-kill results [0077] 1.00E+01 indicate no CFU were recovered at the lowest dilution plated (that is, 10 -1 ); the data value shown is the limit of detection for the assay (i.e.10CFU/mL). [0078] TNTC = too numerous to count. [0079] Table 3 MRSA time-kill results: average CFU/mL TNTC = too numerous to count. [0080] Sample 1 = Formulation 2 supplied in amber glass. [0081] Sample 2 = 0.5% PVP-I/H 2 O supplied in amber glass. [0082] Sample 3 = Formulation 2 without PVP-I supplied in amber glass. [0083] The results indicated that the example Formulation 2 was not more active than PVP-I solution in water against bacteria, demonstrating that the surprisingly enhanced activity against viruses is not seen with bacteria.