Use Of Photosensitizers For Treatment Of Viral Respiratory Infections
20230277863 · 2023-09-07
Inventors
Cpc classification
A61K41/0057
HUMAN NECESSITIES
A61N5/062
HUMAN NECESSITIES
A61K9/0078
HUMAN NECESSITIES
International classification
A61K9/00
HUMAN NECESSITIES
A61K41/00
HUMAN NECESSITIES
Abstract
Treatment of respiratory viral infections including coronavirus using photosensitizers. A photoactivatable phenothiazine derivative, preferably a photoactivatable phenothiazine dye such as methylene blue, new methylene blue, toluidine blue and mixtures thereof is administered to the naso-pharyngeal tract of a subject, and is then activated with a light source.
Claims
1-16. (canceled)
17. A pharmaceutical device comprising a nebulizer and a container containing at least one photosensitizer, optionally in combination with at least one pharmaceutically acceptable excipient and/or diluent.
18. The pharmaceutical device of claim 17 wherein the photosensitizer comprises a photoactivatable phenothiazine derivative, preferably a photoactivatable phenothiazine dye, more preferably a phenothiazine derivative having a structure of the following formula (I) ##STR00002## wherein D and D′ each are an electron donor group, and wherein the positions C-1, C-2, C-4, C-6, C-8 and/or C-9 of the phenothiazine ring system may be substituted by a substituent preferably selected from substituted or unsubstituted lower alkyl, preferably C.sub.1-3-alkyl, particularly preferred methyl, or lower alkenyl, preferably C.sub.2-3-alkenyl, amino, halo, and cyano, and preferably, D and D′ are each independently a NRR′ group with R and R′ independently selected from H, substituted or unsubstituted lower alkyl, and lower alkenyl.
19. The pharmaceutical device of claim 18 wherein the photosensitizer is present in sterilized water or in a sterilized aqueous solution.
20. The pharmaceutical device of claim 18 wherein the container contains the photoactivatable phenothiazine derivative at a concentration of from 0.00001 (v/v) to 0.1% (v/v), preferably from 0.0001% (v/v) to 0.001% (v/v).
21. The pharmaceutical device of claim 18, further comprising a light source emitting light comprising or having a wavelength causing the photosensitizer to be transferred to an activated state.
22. (canceled)
23. The pharmaceutical device of claim 21 wherein the light source is an LED device.
24. The pharmaceutical device of claim 23 wherein the light source is a broadband LED device.
25. The pharmaceutical device of claim 23 wherein the LED device is a laser LED device.
26. The pharmaceutical device of claim 25 wherein the laser LED device emits light having a wavelength in the range of from 550 nm to 630 nm, preferably 570 to 610 nm, more preferably 585 nm to 595 nm, most preferred the light has a wavelength of 590 nm.
27. The pharmaceutical device of claim 25 wherein the laser LED device emits light having a wavelength in the range of from 780 nm to 820 nm, preferably from about 800 nm to about 820 nm, most preferred the light has a wavelength of 810 nm.
28. The pharmaceutical device of claim 23 wherein the LED device emits in-coherent monochromatic light having a wavelength in the range of from 550 nm to 630 nm, preferably 570 to 610 nm, more preferably 585 nm to 595 nm, most preferred the light has a wavelength of 590 nm.
29. The pharmaceutical device of claim 23 wherein the LED device emits in-coherent monochromatic light having a wavelength in the range of from 780 nm to 820 nm, preferably from about 800 nm to about 820 nm, most preferred the light has a wavelength of 810 nm.
30. The pharmaceutical device of claim 21 wherein the light source comprises a handheld torch-type broadband LED device containing a light-guiding element and/or positioning element removably attached to an end of the torch-type LED device where emitted light exits the LED device.
31. The pharmaceutical device of claim 18, wherein R and R′ are independently selected from H, C.sub.1-3-alkyl, most preferably methyl, and C.sub.2-3-alkenyl.
32. A method for prevention and/or treatment of a viral respiratory infection in a subject, comprising the steps of: (i) administering at least one photosensitizer to at least a part of the respiratory tract of the subject and (ii) irradiating the at least a part of the respiratory tract of the subject with light having a wavelength causing the photosensitizer to be activated.
33. The method of claim 32 wherein the photosensitizer is applied intra-nasally and/or intra-orally and/or pharyngeally.
34. The method of claim 32 wherein the step of irradiating the at least a part of the respiratory tract of a subject comprises irradiating from outside of the subject.
35. The method of claim 32 wherein the light is provided by a light emitting diode (LED) device, preferably a handheld LED device.
36. The method of claim 32 wherein the light is provided by a laser LED device, preferably a handheld laser LED device, or a non-coherent light emitting LED device, preferably a broadband LED device.
37. The method of claim 32 wherein the at least a part of the respiratory tract is irradiated one or more times for a period of from 2 to 20 min, preferably from 2 to 10 min, more preferably from 3 to 8 min.
38. The method of claim 32 wherein the at least part of the respiratory tract is irradiated from outside of the subject with an energy density of 50 to 150 J/cm.sup.2 preferably 80 to 120 J/cm, more preferably 90 to 110 J/cm, most preferably 100 J/cm.sup.2.
39. The method of claim 32 wherein the photosensitizer is a photoactivatable phenothiazine derivative, preferably a photoactivatable phenothiazine dye, more preferably a phenothiazine derivative having a structure of the following formula (I) ##STR00003## wherein the positions C-1, C-2, C-4, C-6, C-8 and/or C-9 of the phenothiazine ring system may be substituted by a substituent preferably selected from substituted or unsubstituted lower alkyl, preferably C.sub.1-3-alkyl, particularly preferred methyl, or lower alkenyl, preferably C.sub.2-3-alkenyl, amino, halo, and cyano; and D and D′ are each independently a NRR′ an electron donor group group with R and R′ being preferably independently selected from H, substituted or unsubstituted lower alkyl, preferably C.sub.1-3-alkyl, particularly preferred methyl, and lower alkenyl, preferably C.sub.2-3-alkenyl.
40. The method of claim 39 wherein the phenothiazine dye is selected from the group consisting of methylene blue, new methylene blue, toluidine blue and mixtures thereof.
41. The method of claim 39 wherein the photoactivatable phenothiazine derivative is applied as a composition containing from 0.00001 (v/v) to 0.1% (v/v), preferably from 0.0001% (v/v) to 0.001% (v/v), of said photoactivatable phenothiazine derivative.
42. The method of claim 39 wherein the treatment comprises irradiating at least a part of the respiratory tract of a subject from outside of the subject with light comprising or having a wavelength selected from 500 nm to 820 nm of the electromagnetic spectrum.
43. The method of claim 42 wherein the light has a wavelength in the range of from 550 nm to 630 nm, preferably 570 to 610 nm, more preferably 585 nm to 595 nm, most preferred the light has a wavelength of 590 nm.
44. The method of claim 42 wherein the light has a wavelength in the range of from 780 nm to 820 nm, preferably from about 800 nm to about 820 nm, most preferred the light has a wavelength of 810 nm.
45. The method of claim 39 wherein the viral respiratory infection is caused by a coronavirus.
46. The method of claim 45 wherein the coronavirus is selected from the group consisting of SARS-CoV, MERS-CoV, SARS-CoV 2, HCoV-HKU1, HCoV-OC43, HCoV-NL63, HCoV-229E and bovine coronaviruses (bCoVs).
47. The method of claim 46 wherein the coronavirus is SARS-CoV 2.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0031]
[0032]
DETAILED DESCRIPTION OF THE INVENTION
[0033] The present invention is further illustrated by the following non-limiting examples:
EXAMPLES
Example 1: Inactivation of Bovine Coronavirus in Infected Cells In Vitro
[0034] Materials
[0035] Laser device: Photolase 810 Diode Laser of the applicant: max. Laser Output Power: 7 Watt; Laser; Wavelength: 810 nm/7 Watt) [0036] The laser device contains a handpiece allowing irradiation without loss of performance in a distance up to 105 cm with a light cone laser of approximately 1 cm diameter. In some experiments, the laser was fixed on a tripod in a distance of 30 cm to ensure the same conditions for all tests.
[0037] Irradiation Conditions [0038] Power density: 0.3 Watt [0039] Irradiation Intensity: 100 Joule/cm.sup.2 [0040] Amount of sensitizer/well of 48-well plate: 0.1% [0041] 0.01% [0042] 0.001% [0043] 0.0001% [0044] Test temperature (irradiation): 20° C.±2° C. [0045] Incubation temperature (titration): 37° C.±1° C.
[0046] Photosensitizer [0047] Photolase Blueviolett Sensitizer 810: Methylene blue stock solution containing 1.07% (w/v) methylene blue.
[0048] Cells and Viruses [0049] BCoV strain L9 was obtained from Dr. G. Zimmer, Institute of Virology at the Tierärztliche Hochschule Hannover, Germany. [0050] U373 cells (passage 14) were obtained from Dr. G. Zimmer, Institute of Virology at the Tierärztliche Hochschule Hannover, Germany.
[0051] Media and Auxiliary Reagents [0052] Eagle's Minimum Essential Medium with Earle's BSS (EMEM, Biozym Scientific GmbH, catalogue no. 880121) [0053] Fetal calf serum (FCS, Sigma-Aldrich, article no. F 7524) [0054] Aqua bidest. (SG ultrapure water system, type Ultra Clear, serial no. 86996-1) [0055] PBS (Invitrogen, article no. 18912-014) [0056] Trypsin-EDTA solution [0057] Penicillin/streptomycin (Sigma-Aldrich, article no. P-0781) [0058] Trypsin stock solution [2.5 mg/ml]
[0059] Methods [0060] To analyse the efficacy of the inventive photoactivated treatment for inactivation of bovine coronavirus, U373 cells were cultivated in 48-well plates and infected with BCoV before irradiation treatment. The following general steps were carried out: [0061] (1) (Sub)culturing of U373 cells in 48-well plates [0062] (2) 150 μl cell suspension+500 μl medium (EMEM 10% FKS) [0063] (3) Infection of the U373 cells with 200 μl of virus-medium-mixture [0064] (4) Pre-treatment and irradiation of the BCoV-infected cells
[0065] Preparation of Test Virus Suspension [0066] For preparation of test virus suspension, U373 cells were cultivated in a 175 cm2 flask with in EMEM supplemented with L-glutamine, non-essential amino acids and sodium pyruvate and 10% fetal calf serum. Before virus infection, cells were washed two times with phosphate buffered saline (PBS), incubated for 3 h with EMEM without FCS whereafter the cells were washed once with EMEM supplemented with trypsin. For virus production, BCoV strain L9 was added to the prepared monolayer. After an incubation period of 24 to 48 hours (cells showed a constant cytopathic effect), cells were lysed by a rapid freeze/thaw cycle. [0067] Cellular debris was removed by low speed centrifugation. After aliquotation of the supernatant, test virus suspension was stored at −80° C.
[0068] Preparation of U373 Cells for Irradiation Treatment [0069] U373 cells of a 175 cm2 cell culture flask were detached enzymatically with Trypsin-EDTA solution and taken up in a total of [0070] 60 ml of EMEM medium with 10% fetal calf serum. 150 μl each of this cell suspension were transferred into six wells of a 48-well plate with a final volume of 650 μl by adding 500 μl EMEM/10% FCS. [0071] 48-well plates were selected, since the diameter of one well corresponds to the diameter of the laser cone (light cone laser: approx. 1 cm diameter; well of 48-well plate: 1.04 cm diameter), which ensures that the cells are treated evenly throughout the entire well during radiation. [0072] After three days of cultivation at 37° C. and 5% CO2, cells were washed two times with EMEM without FCS (2×200 μl per well) and incubated for further 3 h at 37° C. and 5% CO2. For virus infection, medium was removed from the individual wells and replaced by 200 μl virus-medium-mixture (500 μl BCoV virus suspension were mixed with 30 ml EMEM without FCS/with penicillin/streptomycin, and trypsin). After an incubation period of 20 to 24 hours BCoV-infected cells were used for irradiation treatment.
[0073] Preparation of Photosensitizer [0074] The methylene blue photosensitzier Photolase® Blueviolett Sensitizer 810 was used in the following concentrations based on the content of 1.07% (w/v) methylene blue in the undiluted sensitizer: [0075] (a) 0.1% (w/v) (end concentration in 200 μl/well) [0076] (b) 0.01% (w/v) (end concentration in 200 μl/well) [0077] (c) 0.001% (w/v) (end concentration in 200 μl/well) [0078] (d) 0.0001% (w/v) (end concentration in 200 μl/well)
[0079] Irradiation Procedure [0080] For photodynamic inactivation of the bovine coronavirus (BCoV) first the undiluted or appropriately diluted (see above) photosensitizer solution (18.69 μl per well) was added to the BCoV-infected cells for 1 minute. Thereafter, irradiation with the above laser device was performed with an intensity of 100 joule/cm.sup.2 for 5.46 min under constant power of 0.3 watt/cm2 and artificial laboratory light. [0081] After treatment, plates were stored at −80° C., successively.
[0082] The following Table 1 shows the irradiation conditions used in the present Example 1.
TABLE-US-00001 TABLE 1 Irradiation conditions according to Example 1 Conc. of Irradiation Incubation BCoV Power sensitizer in time at 100 time under infected density 200 μl/well J/cm.sup.2 artificial light U373 cells [watt/cm.sup.2] [(w/v)] [min] [min] non-treated — — — — (virus control) treated 0.3 0.1 5.46 5.46 treated 0.3 0.01 5.46 5.46 treated 0.3 0.001 5.46 5.46 treated 0.3 0.0001 5.46 5.46 treated — 0.1 — 10.0 treated — 0.01 — 10.0 treated — 0.001 — 10.0 treated — 0.0001 — 10.0 treated — 0.01 — Immediate transfer into the dark treated 0.3 — 5.46 5.46
[0083] Recovery of the residual virus and determination of infectivity For recovery of residual virus from the infected and treated cells, plates were subjected to a rapid freeze/thawing procedure. This was followed by mixing of cell suspension in each well by pipetting up and down 10 times to re-suspend the virus. Then, series of ten-fold dilutions of the suspensions were prepared in ice-cold maintenance medium, respectively. Finally, 100 μl of each dilution were placed in eight wells of a sterile polystyrene flat-bottomed plate with a preformed U373 monolayer Before the addition of the respective dilution, cells were washed twice with EMEM, and incubated for 3 hours 100 μl EMEM supplemented with trypsin. The cells were incubated at 37° C. in a CO2-atmosphere (5.0% CO.sub.2 content). After six days of incubation, cultures were observed for cytopathic effects. The infectious dose (TCID50) was calculated according to the method of Spearman (1908) Brit. J. Psychol. 2, 227-242, and Kärber (1931) Arch. Exp. Path. Pharmak.; 162, 480-487.
[0084] Controls [0085] (i) Virus control (VC) [0086] Virus recovery was performed from non-treated infected U373-cells as described under 3.5. The mean virus titre was used as reference for calculation of the reduction factor. [0087] (ii) Irradiation without photosensitizer [0088] In addition, virus recovery was performed from infected and radiated U373-cells without using of the Photolase sensitizer as [0089] (iii) Treatment with photosensitizer (without irradiation) [0090] Another virus recovery was performed from infected U373-cells, treated with the Photolase sensitizer for 10 min under artificial laboratory light only (without radiation) as described in 3.5, whereby one Plate was wrapped up with aluminium foil immediately after addition of the dye (without radiation or light exposing) and stored and frozen in the dark until titration of residual virus. [0091] (iv) Cell culture control
[0092] Furthermore, a cell control (only addition of medium) was incorporated.
[0093] Calculation of Effectiveness
[0094] The virucidal effectiveness of the inventive treatment was evaluated by calculating the decrease in titre of the treated and radiated culture in comparison with the control titration of the culture without treatment (VC). The difference is given as reduction factor (RF).
[0095] Based on the EN 14476, the present photodynamic therapy system has a virucidal efficacy if the titre is reduced at least by 4 log 10 steps after an irradiation treatment (EN 14476:2013+A2:2019: Chemical disinfectants and antiseptics—Quantitative suspension test for the evaluation of virucidal activity of chemicals disinfectants and antiseptics in human medicine test—Test method and requirements (phase 2, step 1)). This corresponds to an inactivation of ≥99.99%.
[0096] Results
[0097] The effectiveness of the treatment of the infected cells according to the invention was determined after the irradiation treatment of three BCoV-infected U373 cell cultures of a 48-well plate (corresponds to three wells), respectively. Results of examination are shown in the following Table 2.
TABLE-US-00002 TABLE 2 Results of treatment of BCoV-infected cells according to Example1 Concentration of BCoV infected Power density photosensitizer Irradiation time at U373 cells [W/cm.sup.2] in 200 μl/well [%] 100 J/cm2 treated 0.3 0.1 5.46 treated 0.3 0.01 5.46 Treated 0.3 0.001 5.46 treated 0.3 0.0001 5.46
[0098] After adding the photosensitizer to the culture of BCoV-infected cells and irradiation with 810 nm at an energy density of 100 joule/cm.sup.2 and under a constant power density of 0.3 watt/cm.sup.2 no residual virus was found using 0.1% (w/v) to 0.001% (w/v) of the photosensitizer The reduction factor (RF) varied from 3.08 (with 0.1% of the dye), to 4.08 (0.01%), and 5.08 (0.001%). When using 0.0001% of the photosensitizer only minimal residual virus could still be detected in one sample. However, the mean reduction factor was each ≥6.00 log.sub.10 steps, since no cytotoxicity (CT) could be observed in these samples. This corresponded to a virus inactivation of ≥99.999%.
[0099] In contrast, in tests of BCoV-infected cells that were treated with 0.01% of photosensitizer (without radiation) and immediately wrapped in aluminium foil after addition of the sensitizer, and stored and frozen in the dark until titration of residual virus), substantial amounts of residual virus and no effectiveness could be detected (RF=0.38±0.64) (
[0100] Test samples that were only irradiated and not treated with the sensitizer beforehand showed also no effectiveness (RF=0.08±0.64) (
[0101] The present Example shows that the inventive treatment of cells infected with a high dose of bovine coronavirus leads to an effective virus inhibition using a composition containing the photosensitizer at concentrations of as low as 0.0001% (w/v).
Example 2: Inactivation of Bovine Coronavirus in Infected Cells In Vitro Using Non-Coherent LED Light
[0102] In a further set of experiments, the U737 cells were treated under the conditions as shown in the table of
[0103] Surprisingly, broadband LED light was successfully applied for photosensitizer activation leading to reduction of residual virus by 3 log.sub.10 steps after 2 min of irradiation.
Example 3 Inactivation of SARS-CoV-2 in Infected Cells In Vitro
[0104] Virus and Cells [0105] The SARS-CoV-2/Germany strain was derived from a patient isolate. [0106] The Vero E6 cells were obtained from University Bern, Switzerland. [0107] The cells were inspected regularly for morphological alterations and for contamination by mycoplasmas. No morphological alterations of cells and no contamination by mycoplasmas could be detected.
[0108] Light Source and Irradiation Conditions [0109] Light source [0110] Ledlenser P6 (Ledlenser GmbH & Co. KG, Solingen, Germany)
[0111] Irradiation Conditions [0112] Illuminance: 20,000 lux, 50,000 lux or 100,000 lux [0113] Concentration of sensitizer/well of 24-well plate: 0.001% (w/v) or 0.0001% (w/v) [0114] Test temperature (irradiation): 20° C.±2° C. [0115] Incubation time(s): 1, 2 or 3 minutes [0116] Incubation temperature (titration): 37° C.±1° C.
[0117] Other Materials
[0118] Culture Medium and Reagents [0119] Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher, catalogue no. 11965092) [0120] Fetal Bovine Serum (FBS, Thermo Fisher, article no. 10270106) [0121] Penicillin-Streptomycin (P/S, Thermo Fisher, catalogue no. 15140122) [0122] Non-Essential Amino Acids Solution (100×) (NEAAs, Thermo Fisher, catalogue no. 11140035) [0123] L-Glutamine (200 mM) (L-Glut, Thermo Fisher, catalogue no. 25030024) [0124] Trypsin-EDTA (0.5%), no phenol red (Thermo Fisher, catalogue no. 15400054 [0125] BSA (Sigma-Aldrich-Chemie GmbH, article no. CA-2153) [0126] sheep erythrocytes (Fiebig Nährstofftechnik) [0127] Crystal Violet (Sigma-Aldrich-Chemie GmbH, article no. C0775)
[0128] Apparatus, glassware and small items of equipment [0129] CO.sub.2 incubator [0130] Agitator (Vortex Genie Mixer) [0131] Microscope, inverse [0132] Centrifuge, water bath [0133] Adjustable and fixed-volume pipettes [0134] 24-well plates and 96-well microtitre plates [0135] Cell culture flasks and sealed reaction test tubes.
[0136] Methods
[0137] For analyzing the efficacy of the photosensitizer activation to inactivate the humane coronavirus, Vero E6 cells were cultivated in24-well plates and infected with SARS-CoV-2 before irradiation treatment. The following short flowchart provides an overview of the process:
[0138] (Sub)culturing of Vero cells in 24-well plates: [0139] 1×105 cells/well—1 ml medium/well—1 well/plate
[0140] (Day 1) [0141] Infection of the Vero cells with SARS-CoV-2 (MOI 3): [0142] 500 μL virus suspension/well for 1 hour—wash with PBS (1×)—add 1 ml medium/well
[0143] (Day 2) [0144] Pre-treatment/irradiation of the SARS-CoV-2-infected cells: [0145] Pretreatment with 0.001% or 0.0001% Sensitizer and irradiation for 1, 2 and 3 minutes (immediately after treatment: plates were wrapped in aluminium foil) Harvest: Remove supernatant, wash 1× with PBS, add 500 μL PBS and freeze
[0146] (Day 3) [0147] Virus recovery (3× freeze/thaw procedure) and virus titration
[0148] Preparation of Test Virus Suspension
[0149] For virus production, 2×106 Vero E6 cells were cultivated in a 75 cm2 flask in DMEM supplemented with 1% L-Glut, NEAAs, and P/S and 10% FBS. One day after seeding, medium was changed to 10 ml fresh DMEM inoculated with 100 μl of SARSCoV-2/Germany virus suspension. The supernatant was harvested after 3 days at 37° C. by centrifugation at 1,500 rpm for 5 min to remove cell debris. The supernatant was aliquoted and stored at −80° C. Viral titres were determined by plaque assay and endpoint dilution.
[0150] Preparation of Vero Cells for Irradiation Treatment
[0151] Vero E6 cells of a cell culture flask were detached enzymatically with Trypsin-EDTA solution. 1×105 cells were transferred into one well (B3) of a 24-well plate with a final volume of 1,000 μl cell culture medium. After one day of cultivation at 37° C. and 5% CO2, medium was removed from the individual wells and cells were infected with SARS-CoV-2 (500 μL virus suspension per well 2×200 μl per well, MOI 3). After 1 h of incubation at 37° C., inoculum was removed, cells were washed once with PBS and cultivated in 1 ml culture medium for further 20 to 24 h at 37° C. and 5% CO2. After that, SARS-CoV-2-infected cells were used for irradiation treatment.
[0152] Preparation of Sensitizer
[0153] The following methylene blue solutions were prepared using a stock solution containing 1.07% (w/v): [0154] a) 0.001% (w/v) (end concentration in 1,000 μl/well) [0155] b) 0.0001% (w/v) (end concentration in 1,000 μl/well) [0156] For the 0.001% (w/v) methylene blue concentration 1 μl of the undiluted sensitizer was added to 1000 μl medium per well (plate swayed). For the further concentration of 0.0001% (w/v) the photosensitizer was diluted 1:10 in Aqua dest. and 1 μl of the dilution were added to 1,000 μl medium.
[0157] Manual Irradiation Procedure with Different Photodynamic Systems
[0158] For photodynamic inactivation of human coronavirus (SARS-CoV-2), first the appropriately diluted methylene blue solution (1 μl per well) was added to the SARS-CoV-2-infected cells. Immediately thereafter, irradiation with the Ledlenser P6 was performed with a light intensity of 20,000 Ix, 50,000 Ix, and 100,000 Ix, respectively. The single well of each plate was treated separately.
TABLE-US-00003 TABLE 3 Photosensitizer and irradiation conditions for treatment samples and controls Irradiation Irradiation Irradiation Conc. of time time time SARS-CoV-2 sensitizer in at irradiation at irradiation at irradiation infected 1000 μl/well with 100,000 with 100,000 with 100,000 Vero E6 cells [% (w/v)] lx [min] lx [min] lx [min] untreated — — — — (virus control) treated (Tox 1) 0.001 — — — treated (Tox 2) 0.0001 — — treated 0.001 1 1 1 treated 0.001 2 2 2 treated 0.001 3 3 3 treated 0.0001 1 — — treated 0.0001 — — — treated 0.0001 3 — —
[0159] After treatment, the entire plate was immediately wrapped in aluminum foil for maximum 1 min. Afterwards supernatant was removed, cells were washed once with 1,000 μl PBS, overlaid with 500 μL PBS and stored at −80° C.
[0160] Recovery of the residual virus and determination of infectivity For recovery of residual virus from the infected and treated cells, plates were subjected to three freeze/thawing procedures. This was followed by mixing of cell suspension in each well by pipetting up and down 15 times to re-suspend the virus. After that, 22 μl of the virus-disinfectant solution was immediately added to the first row of Vero E6 cells (seeded at 1×104 cells/well in a 96 well plate one day prior the examination), followed by a serial endpoint dilution titration. After 3 days of incubation at 37° C. in a CO2-atmosphere (5.0% CO2-content) cultures were observed for cytopathic effects by crystal violet staining. The infectious dose (log 10 TCID50/ml) was calculated according to the method of Spearman (1) and Kärber (2).
[0161] Controls [0162] (a) Virus control (VC) [0163] Virus recovery was performed from non-treated SARS-CoV-2-infected Vero E6 cells (no sensitizer and no irradiation) as described above. The mean virus titre was used as reference for calculation of the reduction factor (RF). [0164] (b) Treatment with methylene blue solution without irradiation [0165] Another virus recovery was performed from infected Vero E6 cells, treated with the indicated methylene blue solution for 3 min in darkness. After treatment, culture plate was wrapped up with aluminum (without exposure to light), stored for a maximum of 1 min in the dark before harvest (remove supernatant, washing with PBS (1×), adding 500 μL PBS and frozen until virus recovery and titration of the residual virus) was performed as described above. [0166] (c) Cell culture control [0167] Furthermore, a cell control (only addition of medium) was incorporated.
[0168] Calculation of Effectiveness
[0169] The virucidal effectiveness of the methylene blue treatment and the photodynamic inactivation properties with ambient light was evaluated by calculating the decrease in titre of the treated and radiated culture in comparison with the control titration of the approaches without treatment (VC). The difference is given as reduction factor (RF).
[0170] Results
[0171] The effectiveness of the photodynamic systems was determined in triplicate after the irradiation treatment of SARS-CoV-2-infected Vero E6 cells (only one well of a 24-well plate per concentration of the sensitizer and irradiation condition). The results of the experiments examination are shown in
[0172] No residual virus was found with 0.001% or 0.0001% of photosensitizer and subsequent irradiation for just 1 minute with an irradiation intensity as low as 20,000 Ix and an average initial virus titre of 6.92 log 10 TCID50/ml. The mean reduction factor (RF) was 4.67 or more for both photosensitizer concentrations (0.001 or 0.0001, respectively), which corresponds to an inactivation of the SARS-CoV-2 of 99.99%.
CONCLUSION
[0173] A surprisingly high reduction in virus titer was obtained using 0.001% (w/v) of the sensitizer and irradiation with broadband light after an incubation time of only 1 min.
Example 4 Treatment of a Human Subject Suffering from COVID-19
[0174] Patient: male Caucasian, age 58 [0175] Condition of patient before treatment: positive PCR SARS-CoV-2, cycle threshold (CT)=19; dry cough, headache, body temperature 38.2° C., medium general condition [0176] Treatment: for 3 days every 6 hours: 3 μg methylene blue (0.00107 (w/v) in aqueous solution) per treatment of both nostril and throat by administration using a nasal spray device into each nostril and into the mouth//throat (each pump-spray action of the device administers 1 μg of methylene blue in a volume of 0.1 ml; irradiation using handheld LED broadband handheld device (WUBEN® L50 obtained from Shenzhen Shengqi Lighting Technology Co., Ltd., Shenzhen, China) employing an Osram P9 LED emitting warm white light, 1200 Im) for a duration of 2 min in each nostril and 2 min in throat at ca. 6000 Lux directly after the application of the photosensitizer. [0177] Condition of patient after days of treatment: no cough, no headache, normalized body temperature, normal general condition; PCR SARS-CoV-2, CT=35
[0178] This Example shows that treatment of a patient showing symptoms of early to middle conditions of SARS-CoV-2 infections who, given an age of above 55, is in general danger of developing more severe COVID-19 symptoms, substantially recovers through a treatment lasting only 3 days by application of photosensitizer and irradiation using broadband LED device every 6 hours. Moreover, the resulting PCR CT of 35 after 3 days of this treatment indicates that the patient is comparatively unlikely to spread SARS-CoV-2 according to Reference (3) noted below.
REFERENCES
[0179] (1) Spearman, C.: The method of ‘right or wrong cases’ (constant stimuli) without Gauss's formulae. Brit J Psychol; 2, 1908, 227-242 [0180] (2) Kärber, G.: Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Arch Exp Path Pharmak; 162, 1931, 480-487 [0181] (3) Singanayagam, A, et al.: Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19, England, January to May 2020. Euro Surveill. 2020; 25 (32):pii=2001483. https://doi.org/10.2807/1560-7917.ES.2020.25.32.2001483