METHODS AND COMPOSITIONS FOR GENERATING NITRIC OXIDE AND USES THEREOF
20220370493 · 2022-11-24
Inventors
Cpc classification
A61K31/047
HUMAN NECESSITIES
A61K31/194
HUMAN NECESSITIES
A61K33/00
HUMAN NECESSITIES
A61P17/02
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61P9/10
HUMAN NECESSITIES
A61K9/008
HUMAN NECESSITIES
A61K9/0078
HUMAN NECESSITIES
International classification
A61K33/00
HUMAN NECESSITIES
A61K31/047
HUMAN NECESSITIES
A61K31/194
HUMAN NECESSITIES
Abstract
The invention provides a combination, kit or composition comprising: (i) one or more nitrite salt; (ii) a proton source comprising one or more acid selected from organic carboxylic acids and organic non-carboxylic reducing acids; and (iii) one or more organic polyol. On reaction of the one or more nitrite salt with the proton source in the presence of the one or more organic polyol, the combination, kit or composition provides reaction products which include nitric oxide, optionally other oxides of nitrogen and/or optionally precursors thereof and which are useful, for example, in the treatment of various disorders.
Claims
1. A method for generating nitric oxide, optionally other oxides of nitrogen and/or optionally precursors thereof for the treatment of wounds, skin lesions and/or burns, comprising reacting one or more nitrite salt with a proton source comprising one or more acid selected from organic carboxylic acids and organic non-carboxylic reducing acids under reaction conditions suitable to generate nitric oxide, optionally other oxides of nitrogen and/or optionally precursors thereof, wherein the reaction is performed in the process of one or more organic polyol, wherein one or more of the nitrite salt, proton source or organic polyol is present in solution in an aqueous carrier, for example an aqueous liquid or gel; characterised by: the one or more organic polyol does not comprise propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed here, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the above with glycerol and/or polyvinyl alcohol.
2. The method according to claim 1 further characterised by one or more of the following: (a) the one or more organic polyol is present in a reaction output enhancing amount; (b) the proton source is not solely a hydrogel comprising pendant carboxylic acid groups covalently bonded to a three-dimensional polymeric matrix; (c) the one or more organic polyol is not solely glycerol; (d) the one or more organic polyol is not solely glycerol when one or more viscosity increasing agent is used; (e) the one or more organic polyol is not solely glycerol when one or more plasticizer is used; (f) the one or more organic polyol is not solely polyvinyl alcohol; (g) the one or more organic polyol is not solely polyvinyl alcohol when one or more viscosity increasing agent is used; and (h) any one or more of (b) to (g) above, wherein the words “is not solely” are replaced by “does not comprise”.
3. A combination for generating nitric oxide, optionally other oxides of nitrogen and/or optionally precursors thereof by reaction of one or more nitrite salt with a proton source, the combination comprising: one or more nitrite salt; (ii) a proton source comprising one or more acid selected from organic carboxylic acids and organic non-carboxylic reducing acids; and (iii) one or more organic polyol; wherein one or more of the nitrite salt, proton source or organic polyol is present in solution in an aqueous carrier, for example an aqueous liquid or gel; characterised by the one or more organic polyol does not comprise propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed here, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the above with glycerol and/or polyvinyl alcohol.
4. The combination according to claim 3 further characterised by one or more of the following: (a) the one or more organic polyol is present in a reaction output enhancing amount; (b) the proton source is not solely a hydrogel comprising pendant carboxylic acid groups covalently bonded to a three-dimensional polymeric matrix; (c) the one or more organic polyol is not solely glycerol; (d) the one or more organic polyol is not solely glycerol when one or more viscosity increasing agent is used; (e) the one or more organic polyol is not solely glycerol when one or more plasticizer is used; (f) the one or more organic polyol is not solely polyvinyl alcohol; (g) the one or more organic polyol is not solely polyvinyl alcohol when one or more viscosity increasing agent is used; (h) any one or more of (b) to (g) above, wherein the words “is not solely” are replaced by “does not comprise”.
5. A kit for generating nitric oxide, optionally other oxides of nitrogen and/or optionally precursors thereof by reaction of one or more nitrite salt with a proton source, the kit comprising: one or more nitrite salt; (ii) a proton source comprising one or more acid selected from organic carboxylic acids and organic non-carboxylic reducing acids; and (iii) one or more organic polyol; wherein one or more of the nitrite salt, proton source or organic polyol is present in solution in an aqueous carrier, for example an aqueous liquid or gel; characterised by the one or more organic polyol does not comprise propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed here, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the above with glycerol and/or polyvinyl alcohol.
6. The kit according to claim 5 further characterised by one or more of the following: (a) the one or more organic polyol is present in a reaction output enhancing amount; (b) the proton source is not solely a hydrogel comprising pendant carboxylic acid groups covalently bonded to a three-dimensional polymeric matrix; (c) the one or more organic polyol is not solely glycerol; (d) the one or more organic polyol is not solely glycerol when one or more viscosity increasing agent is used; (e) the one or more organic polyol is not solely glycerol when one or more plasticizer is used; (f) the one or more organic polyol is not solely polyvinyl alcohol; (g) the one or more organic polyol is not solely polyvinyl alcohol when one or more viscosity increasing agent is used; and (h) any one or more of (b) to (g) above, wherein the words “is not solely” are replaced by “does not comprise”.
7.-64. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0513] In the drawings:
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EXAMPLES
[0528] The following non-limiting Examples are provided for further illustration of the present invention.
Materials, Apparatus and Methods Used in Examples 1 and 2
[0529] Solutions
[0530] Stock solutions of 0.1 and 1 M citric acid (Health Supplies Limited, Thornton Heath, UK), 0.1 M sodium citrate (Fisher Scientific, Loughborough, UK), 1 M sodium nitrite (Sigma Aldrich, Dorset, UK), 0.5 and 1 M sorbitol (Special Ingredients, Chesterfield, UK), 0.5 and 1 M D-mannitol (Sigma Aldrich, Dorset, UK), 3 M sodium hydroxide (Fisher Scientific, Loughborough, UK), and 0.1 and 1 M L-ascorbic acid (ICN Biomedicals Inc., Ohio, US) were prepared by dissolving the appropriate mass in deionised water. Deionised water (18.2 MΩ) was obtained from an Arium Mini lab water system (Sartorius, Germany).
[0531] Citric acid/citrate buffer solutions were prepared by two methods:
[0532] 1. Titrating stock solutions of 0.1 M citric acid and 0.1 M sodium citrate using the volumes described by Sigma Aldrich, 2018 (https://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html);
[0533] 2. Dissolving a known mass of citric acid, for either a 0.1 M or 1 M preparation, in a small volume of deionised water then titrating a stock solution of 3 M sodium hydroxide and deionised water to achieve the desired buffer solution pH (pH 3 to pH 6.2).
[0534] Ascorbic acid/ascorbate buffer solutions were prepared analogously, using ascorbic acid and, for Method 1, sodium ascorbate in place of citric acid and, for Method 1, sodium citrate.
[0535] The inclusion of polyols was achieved by dissolving a known mass of sodium nitrite with stock solutions of the polyol (for example, either sorbitol or mannitol).
[0536] The order of addition of the ingredients of the buffer solutions and stock is not critical, and any order of mixing can be used.
[0537] All standard solutions were used within 48 hours of preparation. Calibration buffer solutions were prepared using phthalate (pH 4) and phosphate (pH 7) tablets (Fisher Scientific UK Ltd, Leicestershire, UK) dissolved in deionised water.
[0538] Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) Start-Up and Validation
[0539] A Voice200 Selected Ion Flow Tube Mass Spectrometer (SIFT-MS) (Syft Technologies Ltd, New Zealand) was used for all the gas analyses described in this report. This instrument uses helium (BOC, Surrey, UK) as the carrier gas.
[0540] Prior to analysis, the SIFT-MS was prepared for use with a simple start up procedure. The instrument was taken out of standby mode and a series of pressure checks were made to ensure that capillary flow is within the acceptable range for operation. This was followed by an automated validation procedure using the manufacturer's calibrant gas standard (Syft Technologies Ltd, New Zealand) containing benzene, toluene, ethylbenzene, and xylene. Finally, an in-house performance check was undertaken using a 10 ppm nitrogen dioxide standard (Air Products PLC, Surrey, UK).
[0541] Procedure for the Generation of the NO
[0542] The SIFT-MS equipment, reaction chamber and gas pathway was set up as illustrated in
[0543] The temperature in the reaction chamber was continuously monitored with a HT1 Temperature Smart Sensor (SensorPush, New York, US). The reaction chamber, a 670 mL plastic (bisphenol A free (BPA free)) clip lock tub with silicone seal (Tesco, Welwyn Garden City, UK) was attached to a pump that continuously cycles humid air through the chamber and over the SIFT-MS inlet capillary. Humidification was achieved by pumping air through two Dreschel bottles containing deionised water in a method analogous to that described by Vernon, W., and Whitby, L. (1931) The quantitative humidification of air in laboratory experiments, Trans. Faraday Soc. 27, 248-255. This system was allowed to equalise for 30 minutes before use. A continuous SIFT-MS scan was begun for the real-time detection and quantification of NO, NO.sub.2, and HONO. Once a stable baseline reading was observed (consistent concentration for >2 minutes) for these compounds, the sample was placed in the reaction chamber and monitored for three hours.
[0544] After SIFT-MS validation the capillary inlet extension heated to 120° C. was attached to the outlet of the reaction chamber via a T-junction, allowing the SIFT-MS to sample the gases flowing out from the reaction chamber in real time.
[0545] The sample was prepared by weighing a circa 0.3 cm×0.3 cm carded non-woven 20 grams per square metre (20 gsm) polypropylene mesh from RKW-Group, Frankenthal, Germany in a weighing boat (˜3 mg). This was reweighed after an addition of a 10 μL droplet of test or control solution onto the centre of the mesh (it was ensured that the droplet soaked into the mesh). Finally, the loaded mesh in the weighing boat was placed in the reaction chamber and a final 10 μL droplet of buffer solution was pipetted onto the centre of the mesh. The reaction chamber was promptly sealed and the generation of nitrogenous species was observable instantaneously at the SIFT-MS interface.
[0546] Analysis of Generated Gas
[0547] The generated gas was analysed using the selected ion mode of the SIFT-MS and scans were performed in sequential batches each lasting 1000 seconds. The following product masses were repeatedly scanned for: 30 m/z for nitrous acid, 48 m/z for nitrous acid, 46 m/z for nitrogen dioxide, and 30 m/z for nitric oxide. These measurements were achieved using all three of the positive precursor ions: hydronium (H.sub.3O.sup.+, nitrosium (NO.sup.+, and dioxygenyl (O.sub.2+). The air flowed through the chamber at 660 ml/min and the SIFT-MS inlet sampled this air stream at a flow rate of 2.7 ml/min.
pH Measurements for All Examples
[0548] A Five Easy pH meter (Mettler Toledo, Switzerland) with a glass electrode, LE438 probe, was used for all pH measurements. The accuracy of this electrode was ensured with a second pH meter; the hand-held 205 probe (Testo, Alton, US). Fresh calibrant buffer solutions were used for daily calibration of the pH meters.
Example 1
[0549] Generation of Nitric Oxide Using 1 M/c. pH 3 Citric Acid Contacting a Mesh Containing Imbibed 1 M Sodium Nitrite with and without 1 M Polyols
[0550] The SIFT-MS equipment, reaction chamber and gas pathway was set up as described above and illustrated in
[0551] Two test solutions of 1 M sodium nitrite containing respectively 1 M mannitol and 1 M sorbitol were imbibed into the mesh as described above to make two test meshes.
[0552] A control solution of 1 M sodium nitrite with no polyol was imbibed into the mesh as described above to make a control mesh.
[0553] A buffer solution of 1 M citric acid/citrate buffer prepared by either of the two methods 1 and 2 described above and having a pH of about 3 was added to each of the test and control meshes in each test to initiate gas generation as described above.
[0554] The results are shown in
[0555] The data show that the 1 M sodium nitrite imbibed mesh contacted with 1 M/c. pH 3 citric acid generated markedly greater amounts of nitric oxide when the mesh also contained 1 M mannitol or 1 M sorbitol (mannitol has a greater effect than sorbitol) than when no polyol was present.
Example 2
[0556] Investigation of the Effects of Different Carboxylic Acids, Acid Concentration, pH and Polyols on the Generation of Nitric Oxide
[0557] Samples were prepared as above, varying the organic acid, pH and polyol as follows:
TABLE-US-00001 Buffer added to mesh (where alternative buffers are indicated they are used Test solution Control solution in separate runs, as imbibed into mesh imbibed into mesh reported in the relevant Experiment in each test run in control run Figure) A (FIG. 2) 1 M sodium nitrite — 1 M citric acid/citrate (pH about 3) 1 M ascorbic acid/ascorbate (pH about 3) B (FIG. 3) 1 M sodium nitrite 1 M sodium nitrite 1 M citric acid/citrate (pH about 3) containing 1 M sorbitol 1 M sodium nitrite containing 1 M mannitol 1 M sodium nitrite containing 1 M xylitol 1 M sodium nitrite containing 1 M arabitol C (FIG. 4) 1 M sodium nitrite 1 M sodium nitrite 1 M ascorbic acid/ascorbate containing 1 M (pH about 3) sorbitol 1 M sodium nitrite containing 1 M mannitol 1 M sodium nitrite containing 1 M xylitol 1 M sodium nitrite containing 1 M arabitol D (FIG. 5) 1 M sodium nitrite — 1 M citric acid/citrate containing 0.5 M (pH about 3) sorbitol 1 M sodium nitrite containing 0.5 M mannitol 1 M sodium nitrite containing 0.5 M xylitol 1 M sodium nitrite containing 0.5 M arabitol E (FIG. 6) 1 M sodium nitrite — 1 M ascorbic acid/ascorbate containing 0.5 M (pH about 3) sorbitol 1 M sodium nitrite containing 0.5 M mannitol 1 M sodium nitrite containing 0.5 M xylitol 1 M sodium nitrite containing 0.5 M arabitol F (FIG. 7) 1 M sodium nitrite — 0.5 M citric acid/citrate containing 1 M (pH about 3) arabitol 0.5 M ascorbic acid/ascorbate (pH about 3) G (FIG. 8) 1 M sodium nitrite 0.5 M citric acid/citrate containing 1 M (pH about 3) mannitol 0.5 M ascorbic acid/ascorbate (pH about 3) H (FIG. 9) 1 M sodium nitrite 1 M sodium nitrite 1 M citric acid/citrate containing 1 M (pH about 3) sorbitol 1 M ascorbic acid/ascorbate 1 M sodium nitrite (pH about 3) containing 1 M mannitol 1 M sodium nitrite containing 1 M xylitol 1 M sodium nitrite containing 1 M arabitol L (FIG. 10) 1 M sodium nitrite 1 M citric acid/citrate containing 0.5 M (pH about 3) sorbitol 1 M ascorbic acid/ascorbate 1 M sodium nitrite (pH about 3) containing 0.5 M mannitol 1 M sodium nitrite containing 0.5 M xylitol 1 M sodium nitrite containing 0.5 M arabitol J (FIG. 11) 1 M sodium nitrite 1 M sodium nitrite 0.5 M citric acid/citrate containing 0.5 M (pH about 3) mannitol K (FIG. 12) 1 M sodium nitrite 1 M sodium nitrite 0.5 M citric acid/citrate containing 0.5 M (pH about 4.8) mannitol L (FIG. 13) 1 M sodium nitrite 1 M sodium nitrite 0.5 M citric acid/citrate containing 0.5 M (pH about 6.2) mannitol M (FIG. 14) 1 M sodium nitrite 1 M sodium nitrite 1 M citric acid/citrate containing 1 M (pH about 2) glycerol 1 M sodium nitrite containing 2 M glycerol N (FIG. 15) 1 M sodium nitrite 1 M sodium nitrite 1 M citric acid/citrate containing 1 M (pH about 2) mannitol 1 M sodium nitrite containing 1 M sorbitol 1 M sodium nitrite containing 1 M mannitol and 1 M glycerol 1 M sodium nitrite containing 1 M sorbitol and 1 M glycerol O (FIG. 16) 1 M sodium nitrite 1 M sodium nitrite 1 M citric acid/citrate containing 0.5 M (pH 5.8) mannitol
[0558] The SIFT-MS equipment, reaction chamber and gas pathway was set up as described above and illustrated in
[0559] Test solutions as described above were imbibed into the mesh as described above to make the test meshes.
[0560] Where used, a control solution of 1 M sodium nitrite with no polyol was imbibed into the mesh as described above to make a control mesh.
[0561] The or each buffer solution as described above prepared by either of the two methods 1 and 2 described above and having the pH described above was added to each of the test and, if used, control meshes in each test to initiate gas generation as described above.
[0562] The results are shown in
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[0574] When glycerol was used a 1M glycerol solution was first made and used to make 1M sorbitol or 1M mannitol solution which in turn was used to make 1M nitrite solution.
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[0576]
Example 3
[0577] Activity against M. abscessus Cultures with Various Organic Acid and Nitrite Solutions With and Without Polyols
[0578] Materials
[0579] 4.7 g Middlebrook 7H9 broth base (Sigma-Aldrich) was reconstituted with 900 ml of distilled water and autoclaved at 121° C. for 15 minutes. Middlebrook ADC growth supplement (Sigma-Aldrich) was added to the autoclaved 7H9 solution (50 ml per 450 ml, total of 100 ml added).
[0580] 1M Sodium nitrite (Emsure): Dissolve 6.9 g of sodium nitrite powder in 100 ml of distilled water in a clean screw top glass bottle. Autoclave the mixture at 121° C. for 15 minutes.
[0581] 1M Citric acid (Sigma-Aldrich): Dissolve 19.2 g of Citric acid powder in 100 ml of distilled water in a clean screw top glass bottle. Autoclave the mixture at 121° C. for 15 minutes.
[0582] 1M Ascorbic acid (Sigma-Aldrich): Add 17.6 g of Ascorbic acid powder to a sterile glass bottle. Dissolve thoroughly in 100 ml of sterilised distilled water. Due to its short half-life it was prepared on a daily basis, using strict sterile techniques. It was not autoclaved due to its inherent instability but was filtered through a 0.2μ filter before use.
[0583] 1M Sodium citrate tribasic dihydrate (Sigma-Aldrich): Dissolve 29.4 g of sodium citrate powder in 100 ml of distilled water in a clean screw top glass bottle. Autoclave the mixture at 121° C. for 15 minutes. 1M L-Ascorbic acid sodium salt (Acros Organics): Dissolve 19.8 g of sodium ascorbate powder in 100 ml of distilled water in a clean screw top glass bottle. Autoclave the mixture at 121° C. for 15 minutes.
[0584] For the experiments with polyols, D-mannitol (Sigma-Aldrich) was used. The polyol was added to the sodium nitrite stock solution described above to form the following stock solutions:
[0585] Stock solution A— 1M sodium nitrite & 0.5M mannitol
[0586] Stock solution B— 1.5M sodium nitrite & 0.5M mannitol
[0587] A stock solution of 1.5M citric acid was also prepared.
[0588] The molarity of each component was adjusted for dilution factors to ensure the correct final molarity of each experimental solution.
[0589] Mycobacterium abscessus (MAB)
[0590] Laboratory reference strain Mycobacterium abscessus ATCC 19977 lux was used for all experimental conditions in this example.
[0591] Methodology 50 ml falcon tubes were labelled Tube T (test suspension), Tube A (acid control) and Tube C (control).
[0592] 8 ml of 7H9+ADC supplement was added to each tube. 100 μl of MAB suspension (grown previously to approximately 3-4 McFarland standard) was then added. The baseline relative light unit (RLU) reading of the MAB suspension was taken. The contents were mixed by vortexing.
[0593] Tube Contents when a Polyol (Mannitol) was Not Present
[0594] Tube T: 1 ml of sodium nitrite (1M) solution were added to the tube, immediately followed by 1 ml citric acid solution (1M) or ascorbic acid solution (1M) to give a final concentration of 0.1M in 10 ml. The contents were mixed by gentle inversion and incubated for 24 hours at 37° C.
[0595] Tube A: 1 ml of citric acid solution (1M) or ascorbic acid solution (1M) were added to the tube, and 1 ml of sterile distilled water to produce a final volume of 10 ml to test a 0.1M concentration to acid. The contents were mixed by gentle inversion and incubated for 24 hours at 37° C.
[0596] Tube C: 2 ml of sterile distilled water were added to the tube to make a total volume of 10 ml. This is the control to assess growth under optimal conditions. The contents were mixed by gentle inversion and incubated for 24 hours at 37° C.
[0597] Tube T Contents when a Polyol (Mannitol) was Present
[0598] When mannitol was present the tube T contents were as follows: [0599] 1. Tube T: 1 ml sodium nitrite (1M) & mannitol (0.5M) and 1 ml of citric acid (1M) [0600] 2. Tube T: 1 ml sodium nitrite (1.5M) & mannitol (0.5M) and 1 ml of citric acid (1M) [0601] 3. Tube T: 1 ml sodium nitrite (1M) & mannitol (0.5M) and 1 ml of citric acid (1.5M)
[0602] RLUs were measured at 30 minutes, 60 minutes and 24 hours incubation to assess the activity of the T, A and C solutions.
[0603] Following 24 hours of incubation Tube C, Tube A and Tube T were plated on to Columbia Blood Agar (VWR Chemicals). The plates were incubated at 37° C. for 72 hours. Colony forming units (CFU) were read at day 3, 5 and 7 of incubation. All work was undertaken in a CL2 biological safety cabinet within a CL2 laboratory facility.
[0604] The results are shown in
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Example 4
[0609] Minimum inhibition concentrations (MIC) of carboxylic acid-nitrite-polyol solutions against Mycobacterium abscessus (Mabs) and Mycobacterium tuberculosis (Mtb) in a range of clinical isolate cultures
[0610] Healthy Volunteers
[0611] Peripheral blood samples were taken from healthy volunteers who had provided written informed consent (ethical approval reference REC No. 12/WA/0148).
[0612] Mycobacterial Strains
[0613] Mycobacterium abscessus (ATCC 19977) and Mycobacterium tuberculosis (H37RV) strains both contained a bacterial luciferase (lux) gene cassette (luxCDABE) which enabled measurement of relative light units (RLU), as well as conventional colony forming unit (CFU) measurement of bacterial survival.
[0614] General Reagents
TABLE-US-00002 Reference Supplier 24 Well Cell Culture Cluster 3526 Costar Coming, USA CD14 microbeads, human 130-150-201 Miltenyi Biotec, UK Citric acid 791725 Sigma, UK Columbia Blood Agar plates 100253ZF vWR, UK Decanal D7384 Sigma, UK Dulbecco's Modified Eagle D6429 Sigma, UK Medium-High Glucose FLUOstar Omega BMG Labtech, UK Foetal Bovine Serum P30-3702 Pan-Biotech, UK GloMax-96 Luminometer Promega, UK Mannitol M4125 Sigma, UK Middlebrook 7H11 agar plates PP4080 E & O Labs, UK Middlebrook 7H9 broth M02178 Sigma, UK Mycobacterium abscessus 19977 ATCC Mycobacterium tuberculosis H37RV ATCC Penicillin Streptomycin P0781 Sigma, UK Recombinant Human GM-CSF 300-03 PeproTech EC, UK Recombinant Human IFNγ 300-02 PeproTech EC, UK Sodium Nitrite 1.06549.0500 Merck, Germany
[0615] Treatment Conditions
[0616] Treatment 1: Citric acid 0.15M, sodium nitrite 0.1M and mannitol 0.05M
[0617] Treatment 2: Citric acid 0.1M, sodium nitrite 0.15M and mannitol 0.05M
[0618] Broth Microdilution Minimum Inhibitory Concentration (MIC)
[0619] The MIC for each treatment against M. abscessus and M. tuberculosis was undertaken according to the guidelines (M07-A9) produced by the Clinical and Laboratory Standards Institute for antimicrobial susceptibility testing. Doubling dilutions of each treatment was made across the plates, and the plates incubated at 37° C., and read at day 3 and 7 for Mabs, and days 14 and 21 for Mtb. Testing was undertaken in duplicate.
[0620] All work was undertaken in a CL2 biological safety cabinet within a CL2 laboratory facility.
[0621] It was found that the minimum inhibitory concentration for a 1.5 M citric acid, 1 M sodium nitrite and 0.5 M mannitol solution against M. abscessus is 4.7 mM. It was further found that the minimum inhibitory concentration for a 1.5 M citric acid, 1 M sodium nitrite and 0.5 M mannitol solution against M. tuberculosis is 2.3 mM.
[0622] It was found that the minimum inhibitory concentration for a 1 M citric acid, 1.5 M sodium nitrite and 0.5 M mannitol solution against M. abscessus is 3.1 mM. It was further found that the minimum inhibitory concentration for a 1 M citric acid, 1.5 M sodium nitrite and 0.5 M mannitol solution against M. tuberculosis is 1.6 mM.
[0623] Minimal inhibitory concentration (MIC) was also carried out by broth microdilution using isolates Nos. 570, 571, 573, 575, 578, 579, 580, 581, 582, 583, 584, 585, 589, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 616, 617, 619, 812, 825, 829, 839, 845, 848, 853, 857, 858, 873, 894, 898, 909, 919, 928, 932, 942, 944, 955, 956, 959, 963, 964, 965, 968, 975, 980, 982, 985, 993, 995, 1000, 1001, 1007, 1011, 1017, 1023, 1024, 1026, 1027, 1042, 1043, 1045, 1047, 1049, 1054, 1063, 1066, 1067, 1070, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1082, 1086, 1094, 1096, 1101, 1103, 1104 and 1106 from the Floto Laboratory, Cambridge University, UK (https://www.flotolab.com/) M. abscessus clinical isolate library. Each individual isolate was assessed in duplicate.
[0624] The results for the tests on the clinical isolates are shown in
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[0626] In both figures the MIC on certain samples differed on different days. Those are the samples with more than one dot shown above the identification code of the isolate sample. Generally speaking, in that situation the higher MIC was observed on later days of incubation than the lower MIC. Overall, the combination with the lower citric acid (0.1 M) and the higher sodium nitrite (0.15 M) (
[0627] Additional data showing in vitro killing of M. abscessus by carboxylic acid-nitrite-polyol solutions is shown in
Example 5
[0628] Antimicrobial Activity Against Pseudomonas aeruginosa for Carboxylic Acid-Nitrite Solutions with and without a Polyol
[0629] Equipment and Media
[0630] UKAS calibrated pipettes (100-1000 μL range)—Proline® Plus
[0631] UKAS calibrated multichannel pipettes (P300 and P20)—Gilson®, UK
[0632] Universal tubes—SLS, UK
[0633] Calibrated balance—HR-100A
[0634] Microbiological incubator—Heratherm™, ThermoFisher Scientific, UK
[0635] Tryptone Soya Agar (TSA)— Southern Group Laboratories, UK
[0636] Tryptone Soya Broth (TSB)—Acumedia®, SLS, UK
[0637] Malt Agar—Acumedia®, Acumedia®, SLS, UK
[0638] Brain Heart Infusion Broth (BHIB)—Acumedia®, SLS, UK
[0639] Sabouraud Dextrose Broth (SDB)—Acumedia®, SLS, UK
[0640] Dey-Engley Neutraliser (DE-N)— Acumedia®, SLS, UK
[0641] Citric Acid—Sigma, UK
[0642] Sodium Nitrite—Sigma, UK Mannitol—Sigma, UK
[0643] Sorbitol—Sigma, UK
[0644] Test Microorganisms
[0645] Pseudomonas aeruginosa NCTC 13618—Isolated from a cystic fibrosis patient
TABLE-US-00003 Formulations Formulation 1 Liquid Citric Acid pH 5.2 sodium nitrite Formulation 2 Liquid Citric Acid pH 6.0 sodium nitrite Formulation 3 Liquid Citric Acid pH 5.2 sodium nitrite with mannitol Formulation 4 Liquid Citric Acid pH 6.0 sodium nitrite with mannitol Formulation 5 Liquid Citric Acid pH 5.2 sodium nitrite with sorbitol Formulation 6 Liquid Citric Acid pH 6.0 sodium nitrite with sorbitol Positive control Liquid N/A N/A Negative control Liquid N/A N/A
[0646] Concentration 1-1 M Citric acid plus 1 M sodium nitrite (with or without 0.5 M polyol)
[0647] Concentration 2-0.5 M Citric acid plus 1 M sodium nitrite (with or without 0.5 M polyol)
[0648] Concentration 3-0.5 M Citric acid plus 0.5 M sodium nitrite (with or without 0.5 M polyol)
[0649] Dey-Engley Neutraliser Validation
[0650] Twenty-four-hour cultures of Pseudomonas aeruginosa were harvested from Tryptone Soya Agar (TSA) and used to prepare a 1×108±5×107 CFUmL.sup.−1 suspension. This was further diluted in Brain Heart Infusion Broth (BHIB) to prepare a 1×105±5×104 CFUmL.sup.−1 working suspension.
[0651] The starting inoculum was confirmed by serial dilution and spread plating. The neutraliser validation was performed using control (9 mL Phosphate Buffered Saline (PBS) and 1 mL inoculum), toxicity (9 mL Dey-Engley neutraliser (DE-N) and 1 mL inoculum), and neutraliser efficacy (8 mL neutraliser, 1 mL test agent and 1 mL inoculum) samples. Following a 5-minute treatment, 200 μL of suspension was removed from each tube, serially diluted and 100 μL was plated onto TSA. Agar plates were incubated at 37±2° C. for 18-24 hours.
[0652] Antimicrobial Efficacy Against Planktonic Organisms
[0653] Twenty-four-hour cultures of P. aeruginosa were harvested from TSA and used to prepare a 1×10.sup.8±5×107 CFUmL.sup.−1 suspension. This was further diluted in BHIB to prepare a 1×10.sup.6±5×10.sup.4 CFUmL.sup.−1 working suspension. Universal tubes were filled with 8 mL bacterial solution.
[0654] One ml of citric acid solution and 1 mL of sodium nitrite solution were added to each test agent to give the required concentration as described above. Solutions were incubated at 37±2° C. for 24 hours. Following the incubation period, 1 mL was removed from each tube and transferred to a tube containing 9 mL neutraliser. Viable organisms were quantified using serial dilutions and plate counting.
[0655] The results are shown in
[0656] The data show the antimicrobial effectiveness against Pseudomonas of: [0657] citric acid (1 M) mixed with nitrite (1 M) with and without polyol (0.5 M) (“Conc. 1”); [0658] citric acid (0.5 M) mixed with nitrite (1 M) with and without polyol (0.5 M) (“Conc. 2”); and [0659] citric acid (1 M) mixed with nitrite (0.5 M) with and without polyol (0.5 M) (“Conc. 3”).
[0660] The citric acid solution is at pH 5.2 (for Formulations 1, 3 and 5) and 6.0 (for Formulations 2, 4 and 6). Formulations 1 and 2 contain no polyol; Formulations 3 and 4 include mannitol; and Formulations 5 and 6 contain sorbitol.
[0661] Good efficacy is shown for all formulations at pH 5.2. At pH 6, the formulations comprising mannitol are marginally more effective.
Example 6
[0662] The efficacy of formulations including nitrite salt, organic acid and polyol against M. tuberculosis HN 878 in THP-1 cells was evaluated.
[0663] Formulations
[0664] Formulations were prepared as set out in the following table. Where the preparation method is stated as “concentrate”, denoted by the suffix FC in the Sample Reference, this means that the formulation was initially made up as a concentrated pre-mix containing all three ingredients sodium nitrite (0.75M), polyol (0.25M) and acid (0.5M), and then diluted with distilled water to arrive at the desired concentration of each as stated in the table. Where the preparation method is stated as “dilute”, denoted by the suffix FD in the Sample Reference, this means that the formulation was initially made up as a pre-mix containing all three ingredients at the desired concentration initially, namely sodium nitrite (0.15M), polyol (0.05M) and acid (0.1M), and then diluted with distilled water to arrive at the desired concentration of each as stated in the table.
[0665] Within each formulation, a range of concentrations of the sodium nitrite was prepared by serial dilution, namely 16, 8, 4, 2, 1, 0.5, 0.25 and 0.125 μg/ml for the in vitro bacterial inhibition assays against M. tuberculosis HN878.
TABLE-US-00004 Test Mixture (final molarity post dilution) Sodium Sample Reference Preparation Method Nitrite Polyol Acid Formulation 1 30RESP001FC concentrate sodium mannitol citric acid/ nitrite 0.05 M citrate 0.1 M 0.15 M 30RESP001FD dilute sodium mannitol citric acid/ nitrite 0.05 M citrate 0.1 M 0.15 M Formulation 2 30RESP002FC concentrate sodium lactitol citric acid/ nitrite 0.05 M citrate 0.1 M 0.15 M 30RESP002FD dilute sodium lactitol citric acid/ nitrite 0.05 M citrate 0.1 M 0.15 M Formulation 3 30RESP003FC concentrate sodium mannitol citric acid/ nitrite 0.1 M 0.05 M citrate 0.1 M 30RESP003FD dilute sodium mannitol citric acid/ nitrite 0.1 M 0.05 M citrate 0.1 M Formulation 4 30RESP004FC concentrate sodium mannitol ascorbic acid/ nitrite 0.1 M 0.05 M ascorbate 0.1 M 30RESP004FD dilute sodium mannitol ascorbic acid/ nitrite 0.1 M 0.05 M ascorbate 0.1 M
[0666] MIC macrophage testing was performed using a THP-1 macrophage (1) compound screening assay.
[0667] Macrophage Preparation and Culture: THP-1 cells were expanded for 2 weeks. Thereafter, THP-1 cells were suspended in complete DMEM media for macrophages at a concentration of 5×10.sup.5 cells/mL. The cells were seeded into 24 well tissue culture plates, 2 mL per well (1×10.sup.6 per well). One 24-well plate of cells allows for a range of 7 drug concentrations plus untreated controls to be tested in triplicate. In addition to the drug assay plates, one extra plate was seeded (or at least 3 additional wells) for determining bacterial uptake on the day of infection. The cells were incubated at 37° C. at 5% CO.sub.2 in a humidified chamber. DMEM antiobiotic/antimycotic-free complete media were not changed during the 3 day assay.
[0668] Complete DMEM Media for Macrophages:
[0669] Dulbecco's Modification of Eagle's Medium (Cellgro 15-017-cv) supplemented with:
[0670] Heat-inactivated fetal calf serum (Atlas Biologicals, Fort Collins, Colo., F-0500-A) (10%)
[0671] L929-conditioned medium (10%)
[0672] L-glutamine (Sigma G-7513) (2 mM)
[0673] HEPES buffer (Sigma H-0887) (10 mM) Antibiotic/antimycotic (Sigma A-9909) (1X)
[0674] MEM non-essential amino acids (Sigma M-7145) (1X)
[0675] 2-mercaptoethanol (Sigma M-6250) (50 nM)
[0676] L-929 Conditioned Media:
[0677] L-929 (CCL-1) cells from ATCC were seeded at 4.7×10.sup.5 cells in 55 mL of DMEM+10% fetal calf serum in a 75 cm.sup.2 flask. Cells were allowed to grow for THP-1 cells 3 days. On day 3, the supernatant was collected and filtered through a 0.45-μm filter, aliquotted, and frozen at −20° C. The cell-free filtrate was used in the DMEM media for THP-1 infection.
[0678] Infection of THP-1 cells:
[0679] On day 0, the media was removed from the cells and replaced with 0.2 ml of antibiotic/antimycotic-free DMEM containing M. tuberculosis HN878 at a MOI of 1 macrophage to 10 bacteria ratio. The tissue culture plates were placed inside closed Ziploc baggies for transport back to the incubator. Once inside the incubator, the baggies were unzipped. The cells were incubated with the bacteria for 2 hours. After infection, the bacteria attached to the outside of the cells were removed, each well was washed once with phosphate buffered saline (PBS), and 2 mL of antibiotic/antimycotic-free complete DMEM media with various drug concentrations was added. To prepare the drug concentrations, serial 2 fold dilutions were performed by adding 10 ml of the previous suspension to 10 ml complete medium plus serum in the next tube. Tissue culture plates were returned to the incubator at 37° C.+5% CO.sub.2 (drugs remained in wells for 3 days). Each drug concentration was tested in triplicate wells.
[0680] Plating of cell lysates and evaluation of cell viability for THP-1 cells was performed after 2 hours, 1, 2 and 5 days after infection. Tissue culture medium was removed from all wells, and cells were washed twice with 1 ml PBS. Next, 1 ml of sterile double distilled water+0.05% Tween-80 was added to each well; cells were left at room temperature for 5-10 min. Cell lysates were serially diluted 1:10 in sterile saline in 24-well tissue culture plates. Diluted cell lysates were plated onto 7H11/OADC agar through the 1/1,000 dilution step. (Each 24-well TC plate of cells requires four 24-well TC plates for making the serial dilutions, and 24 agar ‘quad’ plates). Plates were incubated at 32° C. for 30 days and colonies were enumerated to determine CFU/ml.
[0681] Results:
[0682] In vitro THP-1 HN878 Optical Density Results
[0683] Minimum Inhibitory Concentration (MIC), reported as the most dilute composition (i.e. the greatest dilution level of the particular formulation on the scale denoted as 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 μg/ml) which inhibits the bacteria
TABLE-US-00005 MIC MIC MIC MIC (μg/ml) (μg/ml) (μg/ml) (μg/ml) Compound Day 0 Day 1 Day 2 Day 5 Formulation 1 16 16 16 16 (30RESP001FC) Concentrate Formulation 1 16 16 16 16 (30RESP001FD) Dilute Formulation 2 8 8 8 16 (30RESP002FC) Concentrate Formulation 2 16 8 16 16 (30RESP002FD) Dilute Formulation 3 8 8 8 8 (30RESP003FC) Concentrate Formulation 3 8 16 16 16 (30RESP003FD) Dilute Formulation 4 4 4 4 0.125 (30RESP004FC) Concentrate Formulation 4 16 16 0.25 0.25 (30RESP004FD) Dilute
[0684] The results are shown in
[0685] ), 4 μg/ml (⋄), 2 μg/ml (∘), 1 μg/ml (□), 0.5 μg/ml (.diamond-solid.), 0.25 μg/ml (.box-tangle-solidup.), and 0.125 ομg/ml (.Math.) were evaluated for intracellular killing of M. tuberculosis HN878 (
) in THP-1 macrophages. In each of the plots in
plot lines for treatment with 16 μg/ml and 8 μg/ml, respectively, can be distinguished from the .box-tangle-solidup. and .Math. plot lines for treatment with 0.25 μg/ml and 0.125 μg/ml, respectively, because the treatments with 16 μg/ml and 8 μg/ml are more efficacious. In other words, the plot lines for treatment with 16 μg/ml and 8 μg/ml show significantly lower CFU values than treatment with 0.25 μg/ml and 0.125 μg/ml, particularly at day 5. Similar, the □ plot line for treatment with 1 μg/ml can easily be distinguished from the
plot line for no treatment because the treatment at 1 μg/ml is more efficacious. The
plot line for no treatment has CFU values that rise and remain above 1×10.sup.4 after day 1.
[0686] The 30RESP001FC and FD compositions referred to in the above MIC table and in
[0687] THP-1 macrophages were infected with M. tuberculosis at a MOI of 1:10 and the numbers of intracellular bacteria were determined using the bacterial colony count method (CFU) immediately after 2 hours (Day 0), 1, 2 and 5 days after infection. Values shown are the mean±SD from one independent experiment. In particular, an increased efficacy relative to the untreated control was present in the treatment with 30RESP001FC and FD (concentrate and dilute) 16 μg/ml, and 8 μg/ml, against M. tuberculosis HN878 (*, p<0.05).
[0688] ) 4 μg/ml (⋄), 2 μg/ml (∘), 1 μg/ml (□), 0.5 μg/ml (.diamond-solid.), 0.25 μg/ml (.box-tangle-solidup.) and 0.125 μg/ml (.Math.) were evaluated for intracellular killing of M. tuberculosis HN878 (o) in THP-1 macrophages. In each of the plots in
plot lines for treatment with 16 μg/ml and 8 μg/ml, respectively, can be distinguished from the .box-tangle-solidup. and .Math. plot lines for treatment with 0.25 μg/ml and 0.125 μg/ml, respectively, because the treatments with 16 μg/ml and 8 μg/ml are more efficacious. In other words, the plot lines for treatment with 16 μg/ml and 8 μg/ml show significantly lower CFU values than treatment with 0.25 μg/ml and 0.125 μg/ml, particularly at day 5. Similar, the □0 plot line for treatment with 1 μg/ml can easily be distinguished from the
plot line for no treatment because the treatment at 1 μg/ml is more efficacious. The
plot line for no treatment has CFU values that rise and remain above 1×10.sup.4 after day 1.
[0689] The 30RESP002FC and FD compositions referred to in the above MIC table and in
[0690] THP-1 macrophages were infected with M. tuberculosis at a MOI of 1:10 and the numbers of intracellular bacteria were determined using the bacterial colony count method (CFU) immediately after 2 hours, 1, 2 and 5 days after infection. Values shown are the mean±SD from one independent experiment. Increased efficacy relative to the untreated control was present in the treatment with 30RESP002FC (concentrate) 16 μg/ml, and 30RESP002FD (dilute) 16 μg/ml and 8 μg/ml, against M. tuberculosis HN878 (*, p<0.05).
[0691] ) 4 μg/ml (⋄), 2 μg/ml (∘), 1 μg/ml (□), 0.5 μg/ml (⋄), 0.25 μg/ml (.box-tangle-solidup.), and 0.125 μg/ml (.Math.) were evaluated for intracellular killing of M. tuberculosis HN878 (
) in THP-1 macrophages. In each of the plots in
plot lines for treatment with 16 μg/ml and 8 μg/ml, respectively, can be distinguished from the .box-tangle-solidup. and .Math. plot lines for treatment with 0.25 μg/ml and 0.125 μg/ml, respectively, because the treatments with 16 μg/ml and 8 μg/ml are more efficacious. In other words, the plot lines for treatment with 16 μg/ml and 8 μg/ml show significantly lower CFU values than treatment with 0.25 μg/ml and 0.125 μg/ml, particularly at day 5. Similar, the □ plot line for treatment with 1 μg/ml can easily be distinguished from the
plot line for no treatment because the treatment at 1 μg/ml is more efficacious. The
plot line for no treatment has CFU values that rise and remain above 1×10.sup.4 after day 1.
[0692] The 30RESP003FC and FD compositions referred to in the above MIC table and in
[0693] THP-1 macrophages were infected with M. tuberculosis at a MOI of 1:10 and the numbers of intracellular bacteria were determined using the bacterial colony count method (CFU) immediately after 2 hours, 1, 2 and 5 days after infection. Values shown are the mean±SD from one independent experiment. Increased efficacy relative to the untreated control was present in the treatment with 30RESP003FC (concentrate) 16 μg/ml and 8 μg/ml and 30RESP003FD 16 μg/ml, against M. tuberculosis HN878 (*, p<0.05).
[0694] ) 4 μg/ml (⋄), 2 μg/ml (∘), 1 μg/ml (□), 0.5 μg/ml (⋄), 0.25 μg/ml (.box-tangle-solidup.) and 0.125 μg/ml (.Math.) were evaluated for intracellular killing of M. tuberculosis HN878 (
) in THP-1 macrophages. In each of the plots in
plot lines for treatment with 16 μg/ml and 8 μg/ml, respectively, can be distinguished from the .box-tangle-solidup. and .Math. plot lines for treatment with 0.25 μg/ml and 0.125 μg/ml, respectively, because the treatments with 16 μg/ml and 8 μg/ml are more efficacious. In other words, the plot lines for treatment with 16 μg/ml and 8 μg/ml show significantly lower CFU values than treatment with 0.25 μg/ml and 0.125 μg/ml, particularly at day 5. Similar, the □ plot line for treatment with 1 μg/ml can easily be distinguished from the
plot line for no treatment because the treatment at 1 μg/ml is more efficacious. The
plot line for no treatment has CFU values that rise and remain above 1×10.sup.4 after day 1.
[0695] The 30RESP004FC and FD compositions referred to in the above MIC table and in
[0696] THP-1 macrophages were infected with M. tuberculosis at a MOI of 1:10 and the numbers of intracellular bacteria were determined using the bacterial colony count method (CFU) immediately after 1, 2 and 5 days after infection. Values shown are the mean±SD from one independent experiment. Increased efficacy relative to the untreated control was present in the treatment with 30RESP004FC (concentrate) 16 μg/ml and 8 μg/ml, against M. tuberculosis HN878 (*, p<0.05).
[0697] It is concluded that the formulations show in vitro inhibition of M. tuberculosis HN878 at suitable dosages above MIC.
[0698] It will also be noted that the manner of making the Formulations has an effect on their in vitro antibacterial efficacy against M. tuberculosis HN878 in the tests of Example 6.
[0699] This is illustrated by comparing the efficacy of the 8 μg/ml concentration of Formulation 1 as between its FC and FD versions (
[0700] Different behaviour is observed with Formulation 2 (
[0701] It is thus shown that, at least at higher concentrations, the stage at which the water is added to arrive at the final inhibitory formulation, can materially affect the antibacterial action of the formulation both in terms of the initial antibacterial action and the extent of bacterial killing over 5 days. Generally speaking, although not universally, making the formulation initially as a concentrated pre-mix with the sodium nitrite, polyol and acid ingredients in their desired relative molar proportions but at a higher concentration than desired for use (e.g. at least 3 times, for example at least 5 times more concentrated than desired for use, for example from about 3 to about 80 times more concentrated than desired for use) and only then diluting the concentrate to obtain the formulation for use, leads to a better antibacterial action over the period of 0 to 5 days after incubation.
Example 7
[0702] Cytotoxicity and Antiviral Activity of Carboxylic Acid-Nitrite-Polyol Solutions Against H1N1 Influenza a Virus
[0703] Test formulations designated F1C.sub.1, F1C.sub.2 and F1C.sub.3 corresponding respectively to Formulation 30RESP001FC in Example 6, a 10-fold dilution thereof and a 100-fold dilution thereof, were used with oseltamivir solution (1 μM) and virus control to obtain comparative cytotoxicity and H1N1 Influenza A virus killing effect after 24 hours in MDCK cells. The cytotoxicity was assayed by LDH cytotoxicity assay analogously to Example 8. Antimicrobial activity against H1N1 Influenza A virus in MDCK cells was measured at MOI=0.002 (.circle-solid.) and MOI=0.02 (.square-solid.) at a range of dilutions (the horizontal axis is the nitrite molarity) with the cytotoxicity shown in grey, cytotoxicity scale on the right-hand side (cytoxicity at the measured nitrite concentrations up to and including 0.015M was ≤1% of LDH control). Plate photographs were obtained at MOI=0.002 and nitrite concentrations 0.15M, 0.015M and 0.0015M in comparison with oseltamivir (1 μM). The results are shown in
[0704] The results show that, at a suitable dose of the nitrite/citric acid/polyol formulation there is complete eradication of the virus, and it is clearly superior to oseltamivir. Similar antiviral activity of nitrite/citric acid/polyol formulations has been shown with rhinovirus and respiratory syncytial virus (RSV).
[0705] These results indicate that therapeutic and prophylactic treatments for respiratory viral infections in human and animal subjects are provided by nitrite/acid/polyol formulations in accordance with the present invention.
Example 8
[0706] Cytotoxicity and Antiviral Activity of Carboxylic Acid-Nitrite-Polyol Solutions Against Coronavirus SARS-CoV-2
[0707] Materials
[0708] Test Formulation F1 (pH 5.8)
[0709] Six test concentrations of Formulation 1 (F1), being an aqueous solution of sodium nitrite, citric acid at pH 5.8 and mannitol (a polyol) were prepared by the method described below from stock solutions of 1.5M sodium nitrite, 0.91M citric acid/citrate buffer at pH 5.8, and 0.5M mannitol solution to give the following test compositions:
TABLE-US-00006 Formulation 1 (F1) Concentration Concentration of of sodium citric acid in test Concentration nitrite in test preparation (M) of polyol in test Test agent preparation (M) pH 5.8 preparation (M) F1 test cone 1 1.5 × 10.sup.−1 M 0.91 × 10.sup.−1 M 5.0 × 10.sup.−2 M (F1C1) mannitol F1 test cone 2 5.0 × 10.sup.−2 M 0.30 × 10.sup.−1 M 1.5 × 10.sup.−2 M (F1C2) mannitol F1 test cone 3 1.5 × 10.sup.−2 M 0.91 × 10.sup.−2 M 5.0 × 10.sup.−3 M (F1C3) mannitol F1 test cone 4 1.5 × 10.sup.−3 M 0.91 × 10.sup.−3 M 5.0 × 10.sup.−4 M (F1C4) mannitol F1 test cone 5 1.5 × 10.sup.−4 M 0.91 × 10.sup.−4 M 5.0 × 10.sup.−5 M (F1C5) mannitol F1 test cone 6 1.5 × 10.sup.−1 M 0.91 × 10.sup.−1 M 2.5 × 10.sup.−2 M (F1C6) mannitol
[0710] Controls used with F1
[0711] A pH 5.8 control formulation was prepared from 0.1 M citric acid+assay buffer+cells.
[0712] A negative control was assay buffer+cells.
[0713] Positive controls were chloroquine+cells.
[0714] Test Formulation F2 (pH 5.4)
[0715] Six test concentrations of Formulation 2 (F2), being an aqueous solution of sodium nitrite, citric acid at pH 5.4 and mannitol (a polyol) were prepared by the method described below from stock solutions of 1.5M sodium nitrite, 0.91M citric acid/citrate buffer at pH 5.4, and 0.5M mannitol solution to give the following test compositions:
TABLE-US-00007 Formulation 2 (F2) Concentration of Concentration of sodium citric acid in test Concentration of nitrite in test preparation (M) polyol in test Test agent preparation (M) pH 5.4 preparation (M) F2 test cone 1 1.5 × 10.sup.−1 M 0.91 × 10.sup.−1 M 5.0 × 10.sup.−2 M (F2C1) mannitol F2 test cone 2 5.0 × 10.sup.−2 M 0.30 × 10.sup.−1 M 1.5 × 10.sup.−2 M (F2C2) mannitol F2 test cone 3 1.5 × 10.sup.−2 M 0.91 × 10.sup.−2 M 5.0 × 10.sup.−3 M (F2C3) mannitol F2 test cone 4 1.5 × 10.sup.−3 M 0.91 × 10.sup.−3 M 5.0 × 10.sup.−4 M (F2C4) mannitol F2 test cone 5 1.5 × 10.sup.−4 M 0.91 × 10.sup.−4 M 5.0 × 10.sup.−5 M (F2C5) mannitol F2 test cone 6 1.5 × 10.sup.−1 M 0.91 × 10.sup.−1 M 2.5 × 10.sup.−2 M (F2C6) mannitol
[0716] Controls used with F2
[0717] A pH 5.4 control formulation was prepared from 0.1 M citric acid+assay buffer+cells.
[0718] A negative control was assay buffer+cells.
[0719] Positive controls were chloroquine+cells.
[0720] Chemical Reagents
[0721] Sodium nitrite:
[0722] Grade: Sodium nitrite extra pure Ph Eur, USP. Sodium nitrite CAS No. 7632-00-0, EC Number 231-555-9., extra pure Ph Eur, USP, from Sigma Aldrich, Product code 1.065441000.
[0723] Citric acid:
[0724] Grade: Citric acid anhydrous powder EMPROVE® ESSENTIAL Ph Eur, BP, JP, USP, E 330, FCC, from Sigma Aldrich, Product code 1.002425000.
[0725] D-Mannitol:
[0726] Grade: D-Mannitol that meets EP, FCC, USP testing specifications, from Sigma Aldrich, Product code M8429-100G.
[0727] Chloroquine phosphate:
[0728] Grade: Pharmaceutical Secondary Standard, from Sigma Aldrich, Product code PHR1258-1G.
[0729] Preparation of the Stock Solutions
[0730] To prepare the citric acid solution, one adds 90 ml of distilled water to 19.2 g citric acid, followed by 10 ml of 3M sodium hydroxide and then dilute with distilled water to adjust the pH (to 160 ml for pH 5.4 or 190 ml for pH 5.8). In an alternative method, one adds 20 ml of distilled water to 19.2 g citric acid, followed by 1.2 g solid sodium hydroxide and after that adjust the pH with 10M sodium hydroxide and distilled water to 100 ml. The solution is sterilised by syringe filtration using a 0.22.sub.jun filter.
[0731] To prepare a 1.0 M sodium nitrite solution, 100 mL of distilled water was added to 6.9 g sodium nitrite. To prepare a 1.5 M sodium nitrite solution, 100 mL of distilled water was added to 10.35 g sodium nitrite.
[0732] When specified, 9.1 g of mannitol was added to give a concentration of 0.5 M. Sterilise solutions by syringe filtration using a 0.22 μm filter.
[0733] Preparation of the Formulations
[0734] The pH of the buffered citric acid solution is controlled to the desired value, prior to mixing with the nitrite and mannitol solutions. The pH stated for a formulation is the pH of the buffered citric acid solution as made up before mixing with the nitrite and mannitol solutions.
[0735] One suitable way to make up the formulations is as follows: Sodium nitrite (1.5 M) containing 0.5 M mannitol is added to a mixing vessel, immediately followed by the pH controlled citric acid solution in a 1:1 mix (nitrite+polyol: citric acid). The solutions are mixed by gentle inversion. Once mixed, the mixture is held for 5 minutes in a sealed container (e.g. a 50 ml falcon tube) at ambient temperature. The resulting solution containing 0.75 M nitrite, mannitol 0.25 M, and citric is then diluted 5-fold in assay buffer (1.2-fold concentrated) to give a final test concentration of nitrite 0.15 M, mannitol 0.05 M, and for example citric acid 0.1 M in the assay. Serial dilutions of the 1:1 mix (for example: a mix starting as nitrite 0.75M, mannitol 0.25M, citric acid 0.5M) are made with distilled water and/or the assay buffer medium. All formulation concentrations can be stored at ambient temperature. Solutions are made fresh for each run.
[0736] Additional Controls
[0737] As additional controls were used S-nitroso-N-acetylpenicillamine (SNAP) at a range of concentrations known to be suitable for its purpose and denoted SNAP50, SNAP100, SNAP200, SNAP300 and SNAP400. SNAP is a known NO donor serving as a positive NO donating control in these tests to provide verification that NO is not cytotoxic in vitro. To control out any potential effect on the assay of the N-acetylpencillamine (NAP) portion of the SNAP molecule, corresponding concentrations of NAP were used as an NO blank control and denoted NAP50, NAP100, NAP200, NAP300 and NAP400.
[0738] Virus
[0739] SARS-CoV-2 clinical isolate.
[0740] Cell Line
[0741] Vero E6.
[0742] Assays
[0743] LDH assay (cytotoxicity):
[0744] CyQUANT™ LDH Cytotoxicity Assay Kit, Invitrogen; Cat No. C.sub.20300 and C.sub.20301. Tissue culture infectious dose (TCID50) was determined (virus titration) using cytopathic effect (CPE) scoring as readout.
[0745] The cytotoxicity of the nitrite formulations (all concentrations), pH 5.8 or pH 5.4 citrate control, negative controls and positive controls (chloroquine, as described by Keyaerts, E, Biochem Biophys Res Commun, 323, 264-268 (2004), the contents of which are incorporated herein by reference) was tested at 2 hr and 24 hr post nitrite/control addition on the Vero E6 cells. LDH release was measured as the readout at the 2 hr and 24 her time points. Each compound/formulation was tested three times per run.
[0746] Sars-Cov-2 Inhibition:
[0747] At time 0 hr, Vero E6 cells were infected with virus in presence of the formulation or controls and incubated for 1 hour. After this incubation period the inoculum was removed and the cells were washed. Fresh formulation or controls were then added to the washed cells. At 24 hours post infection, Vero E6 cell supernatants were harvested and titrated, and the virus titration was incubated for 6 days prior to readout to determine any virus yield reduction. Separate tests were performed at four MOIs including 3.0 and 0.3, although only those two MOIs were titrated. The readout was by crystal violet (cell monolayer) staining, followed by CPE scoring.
[0748] Results
[0749] The results are shown in
[0750]
[0751]
[0752]
[0753] Discussion
[0754] The NO generating aqueous formulations are not cytotoxic on the LDH assay (
[0755] The NO generating aqueous formulations are effective at a surprisingly high pH. pH 5.4 and 5.8 were tested, but lower pH down to 5.2 or even below would also be expected to have efficacy.
[0756] Furthermore, the data reveal that organic carboxylic acids (such as citric acid buffered to pH 5.4 or 5.8), in the absence of an NO generating formulation, have a surprising low cytotoxicity and high in vitro antiviral action against SARS-CoV-2 (
Example 9
[0757] Antiviral Activity of Carboxylic Acid-Nitrite-Polyol Solutions Against Coronavirus SARS-CoV
[0758] To investigate analogies between the antiviral activity provided by the present invention against SARS-CoV-2 and that provided by the present invention against SARS-CoV, the following experiment was performed.
[0759] Formulations F1C.sub.1, F1C.sub.2, F1C.sub.3 and F1C.sub.4 were tested for antiviral activity against SARS-CoV at MOI 3.0. The methodology was analogous to the antiviral testing described in Example 8. Prior to cell monolayer staining with crystal violet, 2 plates were microscopically checked and scored for cytopathic effect (CPE). A CPE, in the form of cell debris on top of an underlying monolayer, was found to be present in these plates.
[0760] The results of the two plates, that were microscopically checked, is shown in
[0761] As shown in
Example 10
[0762] Inhaler for Human Use
[0763] An embodiment of an inhaler for human use employing a liquid composition according to the present invention is shown schematically in
[0764] The inhaler is suitably powered by a compressed gas and configured to deliver one dose of entrained droplets of the nitrite/acid/polyol formulation from a reservoir in the inhaler in response to one manual actuation of the inhaler, in generally conventional manner. The subject typically inhales at the same time as actuating the inhaler, as is conventionally done by asthma sufferers when using their inhalers. As shown in
[0765] The airborne droplets travel into the subject's infected lungs, where they contact the infected (e.g. virus-infected) membranes of the lungs.
[0766] As shown, the efficacy is likely to be much reduced if Inhaled Nitric Oxide would be used. Not only is a proportion of the inhaled nitric oxide breathed out by the subject before it can pass into the bloodstream through the membrane lining of the lungs, but another proportion of the inhaled nitric oxide is oxidised to toxic nitrogen dioxide (NO.sub.2) by oxygen in the inhaled air. The nitrogen dioxide has an adverse effect on the subject's lungs, in addition to depleting the availability of gaseous nitric oxide for treating the subject.
[0767] As a result, a more efficient and effective delivery of nitric oxide to the lungs of the patient and into the patient's bloodstream via the lungs is achieved by using a nitrite/acid/polyol formulation in accordance with the present invention.
CONCLUSION
[0768] The foregoing broadly describes the present invention without limitation. Variations and modifications as will be readily apparent to those skilled in the art are intended to be included within the scope of the appended claims. To the extent that the laws of any particular jurisdiction in or for which a patent is granted to this invention provide for enforcement of the patent against unauthorised use of technology which is equivalent to the appended claims, the proprietor intends that the patent covers such equivalent technology.
[0769] Equivalents of the protective scope of the appended claims are also covered by the claims to the extent permitted by applicable law. For example, generally speaking the order of mixing the components or portions of components of the NOx generating reaction described herein is not critical, provided that the NOx generating reaction is not prematurely initiated. Any order of mixing of essential and non-essential components of any combination, kit or composition of the present invention is intended to be covered. If one or more component is used in liquid form, e.g. as solutions, then the effect of the admixture of that component or those components on the concentration of solutes (including but not limited to that component or those components) in the reaction mixture or any component part of the reaction mixture is likely to be different, compared with the case where that one or more component would be used in solid form or in a liquid form at a different volume or concentration. The use of all equivalent concentrations and/or physical forms (solid, liquid, solutions) of components to form the combinations, kits and compositions of the present invention, and all equivalent steps and orders of steps to prepare the said combinations, kits and compositions, even if not described or specifically claimed herein, is within the scope of the present claims to the extent permitted by applicable law.