Antibacterial Dosage Regime Using Cannabinoids
20220296535 · 2022-09-22
Inventors
Cpc classification
A61K47/06
HUMAN NECESSITIES
A61K31/658
HUMAN NECESSITIES
A61K47/34
HUMAN NECESSITIES
A61K9/06
HUMAN NECESSITIES
A61K47/10
HUMAN NECESSITIES
A61K9/0073
HUMAN NECESSITIES
A61K47/44
HUMAN NECESSITIES
A61K47/14
HUMAN NECESSITIES
International classification
A61K47/10
HUMAN NECESSITIES
A61K47/34
HUMAN NECESSITIES
A61K9/00
HUMAN NECESSITIES
Abstract
The present invention provides methods, compositions and uses for treating topical bacterial infections comprising the administration of cannabinoids. In particular, the present invention provides methods, uses and compositions for treating bacterial infections, the compositions comprising cannabidiol or other cannabinoids, in a dose ranging from about 25 mg to about 500 mg of the cannabinoid compounds.
Claims
1.-21. (canceled)
22. A method for treating or reducing the risk of a bacterial infection, the method comprising topically administering a composition comprising 25 mg-2000 mg of a cannabinoid to a subject in need thereof
23. The method of claim 22, wherein the composition comprises 25 mg-500 mg of a cannabinoid.
24. The method of claim 22, wherein the infection is a topical infection and the composition is administered to the skin a mucosal surface.
25. The method of claim 22, wherein the composition is administered to the eye.
26. The method of claim 22, wherein the infection is an ocular infection and the composition is administered to the eye.
27. The method of claim 22, wherein the composition is administered to nasal mucosal tissue.
28. The method of claim 27, wherein the composition is inhaled.
29. The method of claim 22, wherein the bacterial infection is infection by a gram positive bacteria.
30. The method of claim 29, wherein the gram positive bacteria is: Streptococcus spp., Peptostreptococcus spp., Clostridium spp., Listeria spp., Bacillus spp., Staphylococcus spp., Propionibacterium spp., Kocuria spp., and Corynebacterium spp., and combinations thereof
31. The method of claim 22, wherein the bacteria is biofilm-forming bacteria.
32. The method of claim 22, wherein the bacteria is resistant to at least one antibiotic.
33. The method of claim 22, wherein the composition is a ointment or a gel.
34. The method of claim 22, wherein the composition comprises one or more poly (substituted or unsubstituted alkylene) glycols or a derivative thereof
35. The method of claim 22, wherein the composition is gel and comprises at least one of a siloxane and a low molecular weight alcohol and further comprises a viscosity modifier that increase the viscosity of the composition.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0274] Further features of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above. The description will be made with reference to the accompanying drawings in which:
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EXAMPLES
Example 1
[0290] This experiment was done to evaluate the ability of Cannabidiol (CBD) to disrupt Staphylococcus aureus MRSA ATCC 43300 biofilm formation. CBD was supplied by Dr Michael Thurn of Botanix Pharmaceuticals Ltd.
Methods
[0291] Compound preparation
[0292] The collaborator supplied sample as dry material. A stock solution at 10 mg/mL in neat DMSO (11.2 mg in 1.12 mL of DMSO) was prepared. The highest concentration tested in the assay was 128 μg/mL and 2% DMSO as a final concentration using 1/20 dilution to achieve these concentrations.
Biofilm Formation
[0293] Bacteria (Staphylococcus aureus, ATCC 43300; MRSA) was cultured on Tryptic Soy Broth (TSB, BD, Cat. No. 211825) at 37° C. overnight, then it was diluted 1:100 in fresh TSB supplemented with 5% glucose. 100 μL were added across the 96-well of polystyrene (PS) (Corning; Cat. No. 3370) plate, leaving row H as media Control. Plates were incubated at 37° C. for 48 h to generate the biofilm. The plates were prepared in duplicate.
Biofilm Minimum Inhibitory Concentration (Biofilm MIC)
[0294] The antibiotic controls and CBD were serially diluted in TSB with 5% glucose two-fold across the wells of polystyrene (PS) 96-well plates (Corning; Cat. No. 3370), plated in duplicate. All plates had flat bottom wells and were covered with low-evaporation lids.
[0295] 48 h after incubation, bacteria plates were carefully washed three times with 200 μL/well of saline solution (0.9% NaCl, Baxter Healthcare; Cat. No. AHF7124) using manual pipette to remove the planktonic cells but leave the biofilm adhered to the plate wells. Then, 100 μL of diluted controls and CBD were transferred into the washed plates containing the biofilm. Then, these plates were incubated at 37° C. for 24 h.
[0296] Next day, plates were washed three times with saline solution, then fixed with 100 μL/well of 99% methanol for 15 minutes. Once the biofilm was fixed, 100 μL/well 0.1% Crystal Violet Stain (Sigma; Cat No. C0775-25G) was added for 20 minutes and used as indicator of biofilm formation, followed by three times washing and dry well. To dissolve the crystal violet, 150 μL/well of methanol was added to allow for biofilm MIC analysis.
Biofilm MIC Detection and Analysis
[0297] The biofilm formation was determined by optical density read at 590 nm (OD590). The percentage of biofilm formation was evaluated comparing the average, standard deviation and percentage of confidence of the media control (Row H) against the rest of the plate.
[0298] Inhibition of biofilm growth was determined as the lowest concentration at which OD590 demonstrated ≥70% growth inhibition compared to the growth control. Analysis was performed using Microsoft Excel.
TABLE-US-00003 TABLE 1 Tested Compound Maximum Minimum Supplied Stock test test Sample dry material concentration concentration concentration MCC name (g) (mg/mL) Solvent (μg/mL) (μg/mL) MCC_009427 Cannabidiol 5 10 DMSO 128 0.03 (CBD)
TABLE-US-00004 TABLE 2 Control Compounds Stock Target Compound concentration organism MCC name (mg/mL) Source class MCC_000095 Vancomycin 0.64 Sigma 861987 Gram+ (HCl) MCC_000561 Daptomycin 1.28 Molekula Gram+ 64342447 MCC_000191 Trimethoprim 1.28 Sigma T7883 Gram+/− MCC_009395 Mupirocin 0.64 Glentham Gram+ GA2184 MCC_008132 Clindamycin 0.64 Glentham Gram+ hydrochloride GA5034 monohydrate
Results
[0299] The results display two biological replicates, with technical replicates (total n=4).
TABLE-US-00005 TABLE 3 Broth MIC values Staphylococcus aureus ATCC 43300 TSB + 5% CAMHB Glucose Compound ID Compound name Broth MIC (μg/mL) MCC_000095 Vancomycin 0.5 1 MCC_000561 Daptomycin 0.5/1 32 MCC_000191 Trimethoprim 1 4 MCC_009395 Mupirocin 0.25 0.25 MCC_008132 Clindamycin* >64 >64 MCC_009427 Cannabidiol 1 0.5 *note clidamycin inactive vs this strain of MRSA
TABLE-US-00006 TABLE 4 Biofilm MIC values Staphylococcus aureus ATCC 43300 Biofilm MIC Biofilm MIC (TSB + 5% Gluc) (TSB + 5% Gluc) Compound 07/112/18 14/112/18 Compound ID name MIC (μg/mL) MCC_000095 Vancomycin 4 4 4 4 MCC_000561 Daptomycin 32 16 16 16 MCC_000191 Trimethoprim 8 8 16 >64 MCC_009395 Mupirocin 0.25 0.125 0.25 0.25 MCC_008132 Clindamycin >64 >64 >64 >64 MCC_009427 Cannabidiol 4 4 2 2
[0300] CBD was capable of inhibiting up to 75% of 48 h biofilm formation at 2 and 4 μg/mL. The cannabidiol biofilm MIC was approximately four-fold higher (1-2 μg/mL) than its standard vegetative cell MIC (0.5-1 μg/mL) against the same strain of MRSA.
Example 3
[0301] Antibacterial Time Kill Assay Staphylococcus aureus MRSA
[0302] Time-kill assay specifies a better descriptive assessment of cell killing (at a specific time) when compared to the single endpoint broth microdilution (MIC) assay. The assay determines the rate and the extent of antibacterial activity within a certain time period, and may also provide information on the possible in vivo activity of the antibacterial agents under study. This experiment was done to estimate how long it takes to Cannabidiol (CBD) to show antimicrobial activity against Staphylococcus aureus MRSA ATCC 43300. CBD was supplied by Dr Michael Thurn of Botanix Pharmaceuticals Ltd.
[0303] The time-kill method is based on CLSI guideline M26-A (NCCLS, 1999).
Methods
Compound Preparation
[0304] The collaborator supplied sample as dry material. A stock solution was prepared at 10 mg/mL in neat DMSO (11.2 mg in 1.12 mL of DMSO). The highest concentration tested in the assay was 64 μg/mL and 2% DMSO as a final concentration using 1/20 dilution to achieve these concentrations.
Plate Assay Preparation
[0305] Time kill plates: CBD was plate across all the rows and serially diluted in Cation-adjusted Mueller Hinton Broth (CaMHB; BD, Cat. No. 212322) two-fold across the wells of polystyrene (PS) 96-well plates (Corning; Cat. No. 3370), plated in duplicate. Each row were taken as a time point, where row A, 0 h; row B, 1 h; row C, 2 h; row D, 3 h; row E, 4 h; row F, 6 h and row G, 24 h.
[0306] Also, control plates were made. CBD and standard antibiotics were serially diluted in Cation-adjusted Mueller Hinton Broth (CaMHB; BD, Cat. No. 212322) two-fold across the wells of polystyrene (PS) 96-well plates (Corning; Cat. No. 3370), plated in duplicate.
Time Kill
[0307] The tested bacteria was Staphylococcus aureus ATCC 43300 MRSA (ID GP_020:02).
[0308] Charcoal plate PS 96-well plates: 50 μL of sterile activated charcoal suspension (25 mg/ml) were added into row A. 90 μL of 0.9% sterile saline were added to subsequent rows.
[0309] Bacteria (Table 2.6) was cultured in CaMHB at 37° C. overnight, then diluted 40-fold and incubated at 37° C. for a further 2-3 h. The resultant mid-log phase cultures were diluted in CaMHB and added to each well of the control and time kill 96-well plates to give a final cell density of 5×10.sup.5 CFU/mL, and a final compound concentration range of 0.03-64 μg/mL. The plates were covered and incubated at 37° C. for 24 h.
[0310] At selected time-points (0, 1, 2, 3, 4, 6 and 24 h), 50 μL of culture per-well was transferred from the time kill plate into the first row of charcoal plate (containing the charcoal suspension) to neutralise the compound. After mixing well, 10 μL were transferred from row A to row B to give a 1:10 dilution, this step was repeated until 1:10'000 (row E). Aliquots of each dilution was spotted in duplicate onto Tryptic soy agar (TSA; BD, Cat No. 236950) and incubated overnight at 37° C.
MIC Detection and Analysis
[0311] MICs and the time kill results were determined visually at 24 hr incubation. The MIC was defined as the lowest concentration with which no growth was visible after incubation. The time kill was defined with growth/no growth of the colonies in each spot.
TABLE-US-00007 TABLE 5 Tested Compound Supplied Stock Max test Min Sample dry material con conc test conc MCC name (g) (mg/mL) Solvent (μg/mL) (μg/mL) MCC_009427 Cannabidiol 5 10 DMSO 128 0.03 AMRI supply (CBD)
TABLE-US-00008 TABLE 6 Control Compounds Stock Target conc organism MCC Compound name MW (mg/mL) Source class MCC_000095 Vancomycin (HCl) 1485.71 0.64 Sigma 861987 Gram+ MCC_000561 Daptomycin 1,619.701 1.28 Molekula Gram+ 64342447 MCC_000191 Trimethoprim 290.32 1.28 Sigma T7883 Gram+/− MCC_009395 Mupirocin 500.62 0.64 Glentham Gram+ GA2184 MCC_008132 Clindamycin 504.96 0.64 Glentham Gram+ hydrochloride GA5034 monohydrate
Results
[0312] CBD time kill was tested two concentrations above and below previous MIC data (1-2 μg/mL). CBD control MIC of the day was 2 μg/mL. Tested concentrations over or equal to the MIC value showed to be bactericidal after 3 hour treatment (
Example 4
[0313] Forced Evolution of Resistant in Staphylococcus aureus MRSA
[0314] This experiment was done to assess the development of resistance over 20 days of growth of Staphylococcus aureus (ATCC 43300) in the presence of sub-inhibitory concentrations of Cannabidiol (CBD) and daptomycin (used as a positive control), conducted in parallel in eight replicates.
Methods
Compound Preparation
[0315] The collaborator supplied sample as dry material. A stock solution at 10 mg/mL in neat DMSO was prepared.
Viability Testing
[0316] The tested bacteria was Staphylococcus aureus ATCC 43300 MRSA (ID GP_020:02).
[0317] The mid log Staphylococcus aureus (ATCC 43300) growth culture was serially diluted and plated on a solid Tryptic Soy Agar (TSA) plates in duplicates and incubated at 37° C. overnight to determine viable colony count.
[0318] CBD 320 μg/mL stock was diluted to 5, 4, 3, 2, 1.5, 1, 0.75, 0.5, 0.375 and 0.25 μg/mL in Cation-adjusted Mueller Hinton Broth (CaMHB; BD, Cat. No. 212322) 100 μL were plated from well 1 to 10 across the wells of polystyrene (PS) 96-well plates (Corning; Cat. No. 3370). Staphylococcus aureus (ATCC 43300) was cultured in CaMHB at 37° C. overnight, then diluted 40-fold and incubated at 37° C. for a further 2-3 h. The resultant mid-log phase cultures were diluted in CaMHB and 100 pL added to each well of the compound-containing 96-well plates to give a final cell density of 5×10.sup.5 CFU/mL. The plate was covered and incubated at 37° C. for 20 h.
[0319] Note: CBD will have 8 replicates.
[0320] MICs were determined visually at 24 h incubation and the MIC was defined as the lowest concentration with which no growth was visible after incubation.
Bacteria Preparation:
[0321] Plate well with the highest drug concentration that permitted growth was then diluted. Despite plates were read by eye, reading at OD600 on the Epoch microplate spectrophotometer was used to adjust growth density of each well (it was approximately 1:1000). Then, 100 μL of the bacteria diluted was added to the new MIC plate. The OD.sub.600 was used to calculate the dilution of cells to a density of 10.sub.6 CFU/mL. Bacteria were diluted in CaMHB and 100 μL was added to each well of the next replicate passage. The final well volume was 200 μL with a cell density of 5×10.sub.5 CFU/mL. Each replicate (row) was assess as different strain, for this reason the dilution was done for each replicate.
[0322] Once prepared, the plate was covered and incubated at 37° C. overnight. Plate reading, compound preparation and bacterial preparation were repeated from Day 2 to Day 20.
Compound Preparation:
[0323] Depending on the MIC of the day before, CBD tested concentrations were established.
[0324] Depending on the MIC of the day before, CBD and daptomycin tested concentrations were established to ensure at least three concentrations above, and three concentrations below MIC, based on the previous MIC results. Compounds were prepared in Protein LoBind Eppendorf 1.5 mL safelock tubes, diluting 320 μg/mL stock in DMSO in CaMHB to achieve two-fold the desired testing concentrations. The 100 μL of the selected concentration were added to each well (See
Drug Free Passages
[0325] Following 20 days of passaging in the presence of CBD and daptomycin, each replicate was passaged for 4 days in drug-free media to assess the stability of any induced resistance.
[0326] Day 20 plate was read and the same bacterial preparation methodology was followed. Same concentrations used in day 20 for CBD were used for the 4 days drug free passages. Daptomycin 320 μg/mL stock was diluted to 16, 8, 5, 4, 2.5, 2, 1.25, 1, 0.75 and 0.5. These concentrations were used for the 4 days drug free passages. Column 11 was used as the drug-free passage well, and column 12 as a negative growth control with 200 μL uninoculated media in each well. Diluted bacteria were added to the plate, one replicate per row, 100 μL per well. The final well volume was 200 μL with a cell density of 5×105 CFU/mL in columns 1-11, and CBD concentration range from 16-0.03 μg/mL in columns 1-10 (
[0327] Subsequent drug-free passage plates were prepared in the same manner, except each replicate bacteria was passaged from column 11, the drug-free growth control well.
TABLE-US-00009 TABLE 7 Tested Compound Maximum Minimum Supplied Stock test test Sample dry material concentration concentration concentration MCC name (g) (mg/mL) Solvent (μg/mL) (μg/mL) MCC_009427 Cannabidiol 5 10 DMSO 128 0.03 AMRI supply (CBD) Batch ref R0030516 RM342K.0706
TABLE-US-00010 TABLE 8 Control Compounds Stock Target Compound concentration organism MCC name MW (mg/mL) Source class MCC_000095 Vancomycin 1485.71 0.64 Sigma 861987 Gram+ (HCl) MCC_008136 Erythromycin 733.93 0.64 Avistron Gram+ AE22796 MCC_000236 Oxacillin sodium 401.43 0.64 Sigma O1002- Gram+ salt hydrate 1G MCC_000167 Tetracycline 480.90 0.64 Sigma T7660- Gram+ hydrochloride 5G MCC_009395 Mupirocin 500.62 0.64 Glentham Gram+ GA2184 MCC_008132 Clindamycin 504.96 0.64 Glentham Gram+ hydrochloride GA5034 monohydrate
Drug-free Passaging Control & QC Plate
[0328] Alongside the test plate, a culture of S. aureus was passaged for 24 days without CBD, to establish a baseline for non-selective mutations in the growth conditions described.
[0329] In a PS 96-well plate control compounds (see Control compounds) were serially, two-fold diluted in CaMHB across the rows of columns 1-12 to give a final volume of 50 μL of 2× the desired test concentration. Six wells were used as a positive growth controls, and six as negative growth controls with uninoculated media.
[0330] On day one, mid-log phase S. aureus was diluted in CaMHB to 106 CFU/mL, and 50 μL was added to each well (except negative growth control wells), to give a final volume of 100 μL and concentration of 5×105 CFU/mL.
[0331] Subsequent passages were inoculated from well H7. The bacterial growth in H7 was resuspended by pipetting, then plates were read for optical density by spectrophotometer (Biotek Epoch) at 600 nm (OD600). The OD600 was used to calculate the dilution of cells to a density of 106 CFU/mL. Bacteria were diluted in CaMHB and 50 μL was added to each well of the next passage. The final well volume was 100 μL with a cell density of 5×105 CFU/mL.
Results
[0332] Through the 20 days of assay, CBD generally showed a constant activity between 2 to 4 μg/mL across most of the replicates (
[0333] Following the 20 days induction the 8 replicates were subcultured for an additional 5 days of drug free passages to test for stability of any induced resistance. The final MIC were generally within the variability range of the samples, however replicates 2 and 8 did consistently show elevated MIC (6-16 μg/mL) on Days 20 and 21.
Example 5
Minimum Inhibitory Concentration in Presence of 50% Human Serum
[0334] This experiment was done assess the activity of Cannabidiol (CBD) for antimicrobial activity against three strains of Staphylococcus aureus in the presence of 50% human serum.
Methods
Compound Preparation
[0335] The collaborator supplied sample as dry material. A stock solution at 10 mg/mL in neat DMSO was prepared. The highest concentration tested in the assay was 1.28 mg/mL and 2% DMSO as a final concentration using 1/20 dilution to achieve these concentrations.
Minimum Inhibitory Concentration (MIC) Micro-broth Dilution Assay
[0336] The compounds were serially diluted in mixture of 50% of human serum (Sigma; Cat. No. H3667-100ML) along with 50% Cation-adjusted Mueller Hinton Broth (CaMHB; BD, Cat. No. 212322) two-fold across the wells of polystyrene (PS) 96-well plates (Corning; Cat. No. 3370), plated in duplicate. All plates had flat bottom wells and were covered with low-evaporation lids.
[0337] Staphylococcus aureus strains were cultured in CaMHB at 37° C. overnight, then diluted 40-fold and incubated at 37° C. for a further 2-3 h. The resultant mid-log phase cultures were diluted in CaMHB and added to each well of the compound-containing 96-well plates to give a final cell density of 5×10.sup.5 CFU/mL, and a final compound concentration range of 0.03-64 μg/mL. The plates were covered and incubated at 37° C. for 20 h.
MIC Detection and Analysis
[0338] The MIC was defined as the lowest concentration with which no growth was visible after incubation. MIC was determined by visual inspection only.
TABLE-US-00011 TABLE 9 Tested Compound Supplied Stock Max test Min test Sample dry material conc conc conc MCC name (g) (mg/mL) Solvent (μg/mL) (μg/mL) MCC_009427 Cannabidiol 5 10 DMSO 64 0.03 (CBD)
TABLE-US-00012 TABLE 10 Control Compounds Stock Target Compound concentration organism MCC name MW (mg/mL) Source class MCC_000095 Vancomycin 1485.71 1.28 Sigma Gram+ (HCl) 861987 MCC_000561 Daptomycin 1,619.701 1.28 Molekula Gram+ 64342447 MCC_000191 Trimethoprim 290.32 1.28 Sigma T7883 Gram+ MCC_008132 Clindamycin 504.96 1.28 Glentham Gram+ hydrochloride GA5034 monohydrate MCC_009395 Mupirocin 500.62 1.28 Glentham Gram+ GA2184
TABLE-US-00013 TABLE 11 Tested Bacteria ID Species Strain Description GP_020:02 Staphylococcus ATCC 43300 MRSA aureus GP_035:01 Staphylococcus ATCC 700699, MRSA, aureus NRS1 VISA GP_064:01 Staphylococcus NARSA, VRS1 VRSA aureus
Results
[0339] For bacteria, two technical duplicates.
TABLE-US-00014 TABLE 12 Summary of Result GP_064 GP_020 GP_035 S. aureus S. aureus S. aureu NARSA, Compound MRSA NRS60 RS1 Compound ID Name MIC (μg/mL) MCC_000095_002 Vancomycin 1 4/16 >64 MCC_000561_002 Daptomycin 4 16 >64 MCC_000191_002 Trimethoprim 4 8 >64 MCC_009395_001 Mupirocin 4/8 4/8 >64 MCC_008132_001 Clindamycin >64 >64 >64 hydrochloride MCC_009427_002 Cannabidiol >64 >64 >64
[0340] All control antibiotics gave inhibitory values within the expected ranges. CBD was inactive against all tested strains when human serum was added to the assay medium, consistent with high levels of protein binding (e.g. >97% assuming 3% free responsible for activity).
[0341] Below is a summary of the Minimum Inhibitory Concentration (MIC) range for each compound. The experiment was performed with two technical duplicates (n=2). Where the duplicate readings are the same a single value is displayed. Two values are displayed where the duplicates differed.
TABLE-US-00015 TABLE 13 Summary of Results GP_064 GP_035 S. aureus S. aureus S. aureus NARSA, S. aureus Compound Compound MRSA NRS60 VRS1 MRSA ID Name MIC (ug/mL) MCC_ Cannabidiol no serum* 1* 2* 2* 009427_002 +50% >64 >64 >64 human serum *from report 99962_002
Example 6
[0342] Minimum Inhibitory Concentration Assays MIC90 vs S. aureus
[0343] This experiment was done to assess the antimicrobial activity of Cannabidiol (CBD) against 132 strains of Staphylococcus aureus (106 MRSA and 26 MSSA strains).
Methods
Compound Preparation
[0344] The collaborator supplied the sample as dry material. A stock solution at 10 mg/mL in neat DMSO was prepared. The highest concentration tested in the assay was 32 μg/mL. 5% DMSO was the final concentration using 1/10 dilution to achieve these concentrations.
Bacterial Minimum Inhibitory Concentration (MIC) Micro-broth Dilution Assay
[0345] The compounds were serially diluted in sterile water two-fold across a polypropylene (PP) 96-deep well plate (Fisher Biotec; Cat No. AX-P-DW-20-C-S) and 10 μL were stamped into polystyrene (PS) 96-well plates (Corning; Cat. No. 3370).
[0346] Staphylococcus aureus were cultured in Cation-adjusted Mueller Hinton Broth (CaMHB; BD, Cat. No. 212322) at 37° C. overnight, then diluted 40-fold and incubated at 37° C. for a further 2-3 h. The resultant mid-log phase cultures were diluted in CaMHB and added to each well of the compound-containing 96-well plate to give a final cell density of 5×10.sup.5 CFU/mL, and a final compound concentration range of 0.03-64 μg/mL. The plates were covered and incubated at 37° C. for 20 h.
Bacterial MIC Detection and Analysis
[0347] Optical density was read at 600 nm (OD600) using Tecan M1000 Pro Spectrophotometer. MIC was determined as the lowest concentration at which 95% growth inhibition was observed. Dr Johannes Zuegg wrote script algorithms using Pipeline Pilot to automatically analyse the data set.
[0348] The quality control (QC) of the assays was determined by Z′-Factor, calculated from the Negative (media only) and Positive Controls (bacterial without inhibitor), and the Standards. Plates with a Z′-Factor of ≥0.25 and Standards active at the highest and inactive at the lowest concentration, were accepted for further data analysis.
[0349] MIC 90 and 50 analysis was performed using Microsoft Excel.
TABLE-US-00016 TABLE 14 Tested Compound Supplied Stock Max test Min test Sample dry material conc conc conc MCC name (g) (μg/mL) Solvent (μg/mL) (μg/mL) MCC_009427 Cannabidiol 5 320 DMSO 32 0.015 Norenco supply (CBD) Batch ref 0030516K.0706
TABLE-US-00017 TABLE 15 Control Compounds Stock Target Compound cone organism MCC name MW (μg/mL) Source class MCC_000095 Vancomycin 1485.71 640 Sigma Gram + (HCL) 861987 MCC_000561 Daptomycin 1,619.701 640 Molekula Gram + 64342447 MCC_009395 Mupirocin 500.62 640 Glentham Gram + GA2184 MCC_008132 Clindamycin 504.96 640 Glentham Gram + hydrochloride GA5034 monohydrate
TABLE-US-00018 TABLE 16 Tested Staphylococcus aureus strains ID Strain Description GP_001 ATCC 25923 Control GP_003 CI Paterson 404556145 Clinical Isolate GP_004 CI Paterson 405575036 Clinical Isolate GP_005 CI Paterson 406626061 Clinical Isolate GP_006 CI Paterson 422940878 Clinical Isolate GP_007 CI Paterson 414149225 Clinical Isolate GP_008 CI Paterson 405573757 Clinical Isolate GP_010 CI Paterson 405574456 Clinical Isolate; Resistant GP_020 ATCC 43300 Resistant GP_021 ATCC 33591 Resistant GP_022 ATCC 29213 Control GP_028 NRS 119 Resistant GP_029 NRS2; ATCC 700698 Resistant GP_030 NRS 17 Resistant GP_031 NRS 18 Resistant GP_032 NRS 19 Resistant GP_034 NRS 384 Resistant GP_035 NRS 1; Mu50; ATCC 700699 Resistant GP_036 CI Paterson 581101692:1 Clinical Isolate; Resistant GP_037 CI Paterson 581101692:2 Clinical Isolate; Resistant GP_038 CI Paterson 581101692:3 Clinical Isolate; Resistant GP_047 50316-0509 Clinical Isolate; Resistant GP_049 51418-7407 Clinical Isolate; Resistant GP_050 49496-1320 Clinical Isolate; Resistant GP_062 VRS3b Resistant GP_063 VRS4 Resistant GP_064 VRS1 Resistant GP_065 VRS10 Resistant GP_097 M30538 Clinical Isolate GP_098 M31394 Clinical Isolate GP_099 M31634 Clinical Isolate GP_100 M31907 Clinical Isolate GP_101 M32158 Clinical Isolate GP_102 M32158 Clinical Isolate GP_103 M34027 Clinical Isolate GP_104 M34575 Clinical Isolate GP_105 M34591 Clinical Isolate GP_106 M34593 Clinical Isolate GP_108 M35252 Clinical Isolate GP_109 M35254 Clinical Isolate GP_110 M35255 Clinical Isolate GP_111 M35264 Clinical Isolate GP_112 M35268 Clinical Isolate GP_113 M35491 Clinical Isolate GP_114 M35953 Clinical Isolate GP_115 M36523 Clinical Isolate GP_116 M37410 Clinical Isolate GP_117 M33376 Clinical Isolate; Resistant GP_118 M35249 Clinical Isolate; Resistant GP_119 M38184 Clinical Isolate; Resistant GP_120 M31414 Clinical Isolate; Resistant GP_121 M38509 Clinical Isolate; Resistant GP_122 M39864 Clinical Isolate; Resistant GP_123 M40725 Clinical Isolate; Resistant GP_124 M45447 Clinical Isolate; Resistant GP_125 M48439 Clinical Isolate; Resistant GP_126 M49406 Clinical Isolate; Resistant GP_127 M51977 Clinical Isolate; Resistant GP_128 M52817 Clinical Isolate; Resistant GP_129 M54307 Clinical Isolate; Resistant GP_130 M53519 Clinical Isolate; Resistant GP_131 M55707 Clinical Isolate; Resistant GP_132 M56123 Clinical Isolate; Resistant GP_133 M48662 Clinical Isolate; Resistant GP_134 M49378 Clinical Isolate; Resistant GP_135 M49411 Clinical Isolate; Resistant GP_136 M56924 Clinical Isolate; Resistant GP_137 M57543 Clinical Isolate; Resistant GP_138 M57544 Clinical Isolate; Resistant GP_139 M59014 Clinical Isolate; Resistant GP_140 M60609 Clinical Isolate; Resistant GP_141 M76385 Clinical Isolate; Resistant GP_142 M61448 Clinical Isolate; Resistant GP_143 M63450 Clinical Isolate; Resistant GP_144 M74145 Clinical Isolate; Resistant GP_145 M74568 Clinical Isolate; Resistant GP_146 M75365 Clinical Isolate; Resistant GP_147 M76558 Clinical Isolate; Resistant GP_148 M77399 Clinical Isolate; Resistant GP_149 M78036 Clinical Isolate; Resistant GP_150 M78540 Clinical Isolate; Resistant GP_151 M81239 Clinical Isolate; Resistant GP_152 M81986 Clinical Isolate; Resistant GP_153 M82747 Clinical Isolate; Resistant GP_154 M85049 Clinical Isolate; Resistant GP_155 M85511 Clinical Isolate; Resistant GP_156 M78411 Clinical Isolate; Resistant GP_157 M87512 Clinical Isolate; Resistant GP_158 M90736 Clinical Isolate; Resistant GP_159 M89569 Clinical Isolate; Resistant GP_160 M88418 Clinical Isolate; Resistant GP_161 M88210 Clinical Isolate; Resistant GP_162 M97784 Clinical Isolate; Resistant GP_163 M97166 Clinical Isolate; Resistant GP_164 M96912 Clinical Isolate; Resistant GP_165 M234215 Clinical Isolate; Resistant GP_166 M121493 Clinical Isolate; Resistant GP_167 M69739 Clinical Isolate; Resistant GP_168 M69740 Clinical Isolate; Resistant GP_169 M70241 Clinical Isolate; Resistant GP_170 M70964 Clinical Isolate; Resistant GP_171 M71121 Clinical Isolate; Resistant GP_172 M71122 Clinical Isolate; Resistant GP_173 M72749 Clinical Isolate; Resistant GP_174 M72760 Clinical Isolate; Resistant GP_175 M73508 Clinical Isolate; Mutant GP_176 M74801 Clinical Isolate; Resistant GP_177 M74804 Clinical Isolate; Resistant GP_178 M64647 Clinical Isolate; Resistant GP_179 M65412 Clinical Isolate; Resistant GP_180 M65412 Clinical Isolate; Resistant GP_181 M66471 Clinical Isolate; Resistant GP_182 M66723 Clinical Isolate; Resistant GP_183 M67645 Clinical Isolate; Resistant GP_184 M67826 Clinical Isolate; Resistant GP_185 M67934 Clinical Isolate; Resistant GP_186 M68334 Clinical Isolate; Resistant GP_187 M69124 Clinical Isolate; Resistant GP_188 M72169 Clinical Isolate; Resistant GP_189 M72746 Clinical Isolate; Resistant GP_190 M73705 Clinical Isolate; Mutant GP_191 M75392 Clinical Isolate; Resistant GP_192 M75683 Clinical Isolate; Resistant GP_193 M75856 Clinical Isolate; Resistant GP_194 M75899 Clinical Isolate; Resistant GP_195 M76067 Clinical Isolate; Resistant GP_196 M76386 Clinical Isolate; Resistant GP_221 ATCC 43300 Mutant Induced (Daptomycin MRSA evolution) GP_223 ATCC 43300 Mutant Induced (Linezolid MRSA evolution) GP_224 ATCC 43300 Mutant Induced (Dalvamycin MRSA evolution) GP_229 ATCC 6538; FDA 209 GP_234 ATCC 43300 Mutant Induced (CBD MRSA evolution)
Results
[0350] Out of the 132 strains, 37 were resistant to clindamycin, resulting in an MIC50 of 0.125 μg/mL changing to an MIC90 of 64 μg/mL. The other control antibiotics gave inhibitory values within the expected ranges. While several VISA/VRSA strains were resistant or highly resistant to vancomycin, there were not enough strains to substantially shift the MIC90. CBD showed a stable MIC between 2 to 4 μg/mL across the 132 strains tested. The assay was performed in two different days in duplicate (total n=4). See
TABLE-US-00019 TABLE 17 Summary of results S. aureus spp. S. aureus MSSA S. aureus MRSA ALL (μg/mL) (μg/mL) (μg/mL) MIC 50 MIC 90 range MIC 50 MIC 90 MIC 50 MIC 90 Vancomycin 1 2 0.5-64 1 1 1 2 Daptomycin 2 4 0.5-16 2 2 2 4 Mupirocin 0.5 0.5 0.125-64 0.5 0.5 0.5 0.5 Clindamycin 0.125 64 0.03-64 0.125 0.1875 0.125 64 Cannabidiol 2 4 0.25-8 2 2 2 4
TABLE-US-00020 TABLE 18 Staphylococcus aureus spp. MIC distribution (μg/mL) Staphylococcus aureus spp. MIC distribution (μg/mL) 0.015 0.03 0.06 0.125 0.25 0.5 1 2 4 8 16 32 64 Vanco — 0 0 0 0 2 106 16 3 2 0 0 3 Dapto — 0 0 0 0 2 46 68 12 3 1 0 0 Mupir — 0 0 5 41 75 3 1 1 0 1 1 4 Clinda — 10 26 54 1 0 2 0 1 1 0 0 37 CBD 0 0 0 0 1 0 6 106 18 1 0 0 —
TABLE-US-00021 TABLE 19 Staphylococcus aureus MRSA MIC distribution Staphylococcus aureus MRSA MIC distribution 0.015 0.03 0.06 0.125 0.25 0.5 1 2 4 8 16 32 64 Vanco — 0 0 0 0 1 82 15 3 2 0 0 3 Dapto — 0 0 0 0 2 39 50 11 3 1 0 0 Mupir — 0 0 4 35 57 3 1 1 0 1 1 3 Clinda — 10 18 39 0 0 2 0 1 1 0 0 35 CBD 0 0 0 0 1 0 4 82 18 1 0 0 —
TABLE-US-00022 TABLE 20 Staphylococcus aureus MRSA MIC distribution Staphylococcus aureus MRSA MIC distributio 0.015 0.03 0.06 0.125 0.25 0.5 1 2 4 8 16 32 64 Vanco — 0 0 0 0 1 24 1 0 0 0 0 0 Dapto — 0 0 0 0 0 7 18 1 0 0 0 0 Mupir — 0 0 1 6 18 0 0 0 0 0 0 1 Clinda — 0 8 15 1 0 0 0 0 0 0 0 2 CBD 0 0 0 0 0 0 2 24 0 0 0 0 —
Example 7
Anaerobic Gram-positive Bacteria Minimum Inhibitory Concentration Assays
[0351] To assess the potential of Cannabidiol (CBD) for antimicrobial activity against common skin bacteria under anaerobic conditions.
Methods
Compound Preparation
[0352] The collaborator supplied sample as dry material. A stock solution at 10 mg/mL in neat DMSO was prepared. The highest concentration tested in the assay was 128 μg/mL and 2% DMSO as a final concentration using 1/20 dilution to achieve these concentrations.
Minimum Inhibitory Concentration (MIC) Micro-broth Dilution Assay
[0353] All steps were performed in a COY type B anaerobic chamber with the anaerobic atmosphere controlled by the introduction of 10% CO2/5% H2 in N2CoA gas mix, catalyst Stak-Pak and O.sub.2-H.sub.2 gas analyser, with H2 levels kept at ˜2% for the duration of the assay. Brain Heart Infusion broth (BHI; OXOID CM1135B) media with 1% cysteine to further promote an anaerobic environment was used for this assay, and this broth was incubated in the anaerobic chamber for 24 h prior to use for reduction of oxygen.
[0354] CBD and control antibiotics were serially diluted in BHI, two-fold across the wells of 96-well of polystyrene (PS) 96-well plates (Corning; Cat. No. 3370). Plates were set up in duplicate for each strain.
[0355] All bacteria strains (Table 2.5) were cultured on Tryptic Soy agar (TSA, BD, Cat. No. 236950) at 37° C. for 72 h. A few colonies were taken from the agar plate and dissolved in BHI broth. The solution was then adjusted to OD.sub.600 0.5-0.7 and diluted down to a final cell density of 5×10.sup.5 CFU/mL, 100 μL were added to the test plate, giving a final CBD concentration range of 0.06-128 μg/mL. All the plates were covered and incubated at 37° C. for 48 h.
MIC Detection and Analysis
[0356] The MIC was defined as the lowest concentration at which no growth was visible after incubation. MIC was determined by visual inspection only.
TABLE-US-00023 TABLE 21 Tested Compound Stock Max test Min test Sample Supplied dry conc conc conc MCC name material (g) (mg/mL) Solvent (μg/mL) (μg/mL) MCC_009427 Cannabidiol 5 10 DMSO 128 0.03 AMRI supply (CBD) Batch ref R0030516 RM342K.0706
TABLE-US-00024 TABLE 22 Control Compounds Stock cone Target Compound (mg/ organism MCC name MW mL) Source class MCC_000095 Vancomycin 1485.71 0.64 Sigma Gram + (HCI) 861987 MCC_008136 Erythromycin 733.93 0.64 Avistron Gram + AE22796 MCC_000236 Oxacillin sodium 401.43 0.64 Sigma Gram + salt hydrate O1002-1G MCC_000167 Tetracycline 480.90 0.64 Sigma Gram + hydrochloride T7660-5G MCC_009395 Mupirocin 500.62 0.64 Glentham Gram + GA2184 MCC_008132 Clindamycin 504.96 0.64 Glentham Gram + hydrochloride GA5034 monohydrate
TABLE-US-00025 TABLE 23 Test Organisms ID Species Strain Description GP_020:02 Staphylococcus aureus ATCC 43300 MRSA GP_202:01 Cutibacterium acnes (formerly ATCC 6919 Type strain Propionibacterium acnes) GP_203:01 Acidipropionibacterium ATCC 25562 Type strain acidipropionici GP_204:01 Cutibacterium granulosum ATCC 25564 Type strain
Results
[0357] For bacteria, two biological replicates, with technical replicates (total n=4). All control antibiotics gave inhibitory values within the expected ranges. The Cannabidiol (CBD) was active against all tested strains.
[0358] Below is a summary of the Minimum Inhibitory Concentration (MIC) range for each compound, determined in an anaerobic chamber in the absence of oxygen. The experiment was performed with two biological replicates of technical duplicates (n=4). Where the duplicate readings are the same a single value is displayed. Two values are displayed where the duplicates differed.
TABLE-US-00026 TABLE 24 Summary of Results P. acnes A. C. ATCC acidipropionici granulosum S. aureus Compound 6919 ATCC 25562 ATCC 25564 ATCC 43300 Name MIC (μg/mL) Vancomycin 0.25 0.25/0.125 0.25 1/0.5 Erythromycin 0.25/0.125 4/2 0.125 >32 Oxacillin 0.5/0.25 2 0.5/0.25 64/16/8 sodium salt hydrate Tetracycline 0.5/0.125 0.5/0.125 0.25/0.125 0.5/0.125/0.06 hydrochloride Clindamycin 0.125 0.125 0.125/0.06/0.03 >32 hydrochloride Mupirocin >32 >32 >32 0.06/0.03 Cannabidiol 2/1 0.5 4/2 2/1 (Batch 2)
Example 8
[0359] Expanded Panel: Bacteria Minimum Inhibitory Concentration Assays
[0360] To assess the potential of Cannabidiol (CBD) for antimicrobial activity against a panel of Gram-positive bacteria.
Methods
Compound Preparation
[0361] The collaborator supplied sample as dry material. Angela Kavanagh prepared a stock solution at 10 mg/mL in neat DMSO. The highest concentration tested in the assay was 64 μg/mL for bacteria and 128 μg/mL for fungi. 2% DMSO was the final concentration using 1/20 dilution to achieve these concentrations.
Bacterial Minimum Inhibitory Concentration (MIC) Micro-broth Dilution Assay
[0362] The compound was serially diluted in Cation-adjusted Mueller Hinton Broth (CaMHB; BD, Cat. No. 212322) two-fold across the wells of polystyrene (PS) 96-well plates (Corning; Cat. No. 3370), plated in duplicate. All plates had flat bottom wells and were covered with low-evaporation lids.
[0363] Bacteria were cultured in CaMHB at 37° C. overnight, then diluted 40-fold and incubated at 37° C. for a further 2-3 h. The resultant mid-log phase cultures were diluted in CaMHB and added to each well of the compound-containing 96-well plates to give a final cell density of 5×10.sup.5 CFU/mL, and a final compound concentration range of 0.03-64 μg/mL. The plates were covered and incubated at 37° C. for 20 h.
Bacterial MIC Detection and Analysis
[0364] Inhibition of bacterial growth was determined visually, where the MIC was recorded as the lowest compound concentration with no visible growth.
TABLE-US-00027 TABLE 25 Tested Compound Stock Max test Min test Sample Supplied dry conc conc conc MCC name material (g) (mg/mL) Solvent (μg/mL) (μg/mL) MCC_009427 Cannabidiol 5 10 DMSO 64 0.03 AMRI supply (CBD) Batch ref R0030516 RM342K.0706
TABLE-US-00028 TABLE 26 Control Compounds Stock cone Target Compound (mg/ organism MCC name MW mL) Source class MCC_000095 Vancomycin 1485.71 1.28 Sigma Gram + (HCL) 861987 MCC_000561 Daptomycin 1,619.701 1.28 Molekula Gram + 64342447 MCC_000094 Colistin Sulfate 1400.63 1.28 Sigma Gram − C4461 MCC_000636 Polymyxin B 1301.56 1.28 Sigma Gram − Sulfate P0972 MCC_000191 Trimethoprim 290.32 1.28 Sigma Gram +/− T7883 MCC_009395 Mupirocin 500.62 1.28 Glentham Gram + GA2184 MCC_008132 Clindamycin 504.96 1.28 Glentham Gram + hydrochloride GA5034 monohydrate MCC_008383 Fluconazole 306.27 0.64 Sigma Fungi F8929 MCC_008384 5-fluorocytosine 129.09 0.64 Sigma Fungi F7129
TABLE-US-00029 TABLE 27 Tested Organisms ID Species Strain Description| GP_001:02 Staphylococcus aureus ATCC 25923 MSSA GP_009:01 Staphylococcus warneri Clinical isolate GP_013:01 Streptococcus pneumoniae ATCC 33400 Type strain GP_014:01 Streptococcus pyopenes ATCC 12344 Type strain GP_015:01 Bacillus cereus ATCC 11778 FDA strain PCI 213 GP_016:01 Bacillus mepaterium ATCC 13632 De Bary-KM GP_017:01 Staphylococcus ATCC 12228 FDA strain PCI epidermidis 1200 NRS 231 GP_018:01 Bacillus subtilis ATCC 6633 QC strain GP_020:02 Staphylococcus aureus ATCC 43300 MRSA GP_021:01 Staphylococcus aureus ATCC 33591 MRSA GP_022:01 Staphylococcus aureus ATCC 29213 MSSA GP_023:01 Streptococcus pneumoniae ATCC 700677 MDR GP_024:01 Enterococcus faecium ATCC 35667 Control strain GP_027:01 Enterococcus faecalis ATCC 29212 Control strain GP_033:01 Staphylococcus epidermidis NRS 60 VISA GP_035:01 Staphylococcus aureus ATCC 700699, MRSA, VISA NRS 1 GP_036:01 Staphylococcus aureus Clinical isolate MRSA, DapRSA GP_064:01 Staphylococcus aureus NARSA, VRS1 VRSA GP_197:01 Staphylococcus epidermidis ATCC 14990 Type strain GP_198:01 Staphylococcus warned ATCC 27836 Type strain GP_199:01 Staphylococcus capitis ATCC 27840 Type strain GP_207:01 Kocuria rosea (formerly ATCC 31251 M-1054-1 Micrococcus roseus Fluppe)
TABLE-US-00030 TABLE 28 Summary of Results Cannabidiol MIC (Batch 1) Species Strain (μg/mL) Staphylococcus aureus ATCC 25923 1 2 Staphylococcus warneri Clinical isolate 2 4 Streptococcus pneumoniae ATCC 33400 1 2 Streptococcus pyogenes ATCC 12344 1 1 Bacillus cereus ATCC 11778 1 2 Bacillus megaterium ATCC 13632 1 2 Staphylococcus epidermidis ATCC 12228 1 2 Bacillus subtilis ATCC 6633 1 2 Staphylococcus aureus ATCC 43300 1 Staphylococcus aureus ATCC 33591 1 2 Staphylococcus aureus ATCC 29213 1 2 Streptococcus pneumoniae ATCC 700677 1, 2 4 Enterococcus faecium ATCC 35667 0.5 1 Enterococcus faecalis ATCC 29212 2 Staphylococcus epidermidis NRS 60 4 8 Staphylococcus aureus ATCC 700699, 1, 2 4 NRS 1 Staphylococcus aureus Clinical isolate 2 8 Staphylococcus aureus NARSA, VRS1 1 2 Staphylococcus epidermidis ATCC 14990 1 2 Staphylococcus warneri ATCC 27836 2 4 Staphylococcus capitis ATCC 27840 1 2 Kocuria rosea ATCC 31251 1 2
Results
[0365] CBD was active against all Gram-positive strains in a range of 0.5 to 4 μg/mL, except for Staphylococcus epidermidis NDR 60 (GP_033) which was susceptible to CBD at 4 to 8 μg/mL.
[0366] Table 28 is a summary of the Minimum Inhibitory Concentration (MIC) range for CBD. The experiment was performed twice in duplicate (n=4) for bacteria. Individual values are shown when they differ between replicates.
Example 8
[0367] Initial efficacy studies focused on screening for antibacterial activity in an ex vivo porcine S. aureus skin infection model. A variety of different CBD compositions ranging from liquids to gels to ointments were evaluated for their ability to kill MRSA at both 1 and 24 h following application (Table 29). Components included differing silicone bases for most preparations (Composition #4-12) with petrolatum (mineral oil jelly i.e. petroleum jelly) tested in Composition #1, transcutol (diethylene glycol monoethyl ether) in Composition #2 and polyethylene glycol (PEG 400/400) in Composition #3. CBD concentrations ranged from 5 to 20% (Table 29).
Ex-vivo Pig Skin Assay
[0368] Porcine tissues, transported on ice, were received 2-5 h after slaughter.
[0369] Explant preparation: In RPMI medium containing 2% (v/v) penicillin/streptomycin, a 5 mm biopsy punch was used to cut tissue explants and remaining muscle tissue removed with a sterile scalpel blade. Tissue was antibiotic treated (for decontamination of flora) for 0.5±0.25 h. Explants were rinsed three times with 10±0.5 mL RPMI (no antibiotic, no FBS). Explants were then covered with fresh RPMI (no antibiotic, no FBS) and placed at 4±2° C. for 12±4 h (antibiotic washout). Overnight RPMI was then removed and replaced with 10±0.5 mL fresh RPMI 15±5 min prior to infection.
[0370] Bacterial inoculation: Fresh plates were streaked directly from frozen stock within 3 weeks of the experiment. Culture tubes containing Todd Hewitt broth were inoculated with a single colony and placed in a shaking incubator (at 37±2° C., 150±10 rpm) late afternoon the day before the experiment. On the morning of the experiment, 200±50 μL of overnight culture was transferred into 2±0.5 mL fresh Todd Hewitt broth and shaken for 3±1 h at 37° C. Inoculum was then washed to a final concentration of 5×10.sup.8 CFU mL.sup.−1.
[0371] Model set up: 6-well plates were set up with 2±0.2 mL RPMI (no antibiotic, no FBS) in each well and a 0.4 μm trans-well insert. Tissue explants were transferred into wells mucosal side up to the insert.
[0372] Infection and treatment: Explants were infected with 2±0.5 μl of prepared inoculum (approximately 1×10.sup.6 CFU/explant or 5×108 CFU mL.sup.−1). Explants were incubated at 37±2° C. for 2±0.5 h, then treatments (12 CBD-containing compositions and associated vehicles) administered in triplicate and incubated for at 37±2° C. for 1±0.25 h.
[0373] Wash: Post-treatment, 1.0±0.05 mL sterile phosphate-buffered saline (PBS)+2% (w/v) mucin was added to each insert for the appropriate tissue and swirled gently for 5 sec. The liquid suspension was then aspirated, and wells replenished with RPMI (2±0.2 mL RPMI [no antibiotic, no FBS]). Explants were returned to the 37° C. incubator for the indicated post treatment timepoints: 1.0±0.25 h, 24±4 h.
[0374] Sample collection: Post-wash (1.0±0.25 h, 24±4 h), tissue was removed from transwells and placed in 500±0.03 μL of neutralizer (30 mg/mL bovine serum albumin). Samples were sonicated and vortexed (30±5 sec vortex, 120±6 sec sonicate, 30±5 sec vortex). Samples were then plated neat or diluted in sterile PBS. 50±2 μL of sample was plated with a spiral plater on mannitol salt agar, and plates incubated for 24-48 h at 37±2° C. The following day, colonies were counted with an automated plate counter and CFU counts transformed to Log.sub.10(CFU/explant).
TABLE-US-00031 TABLE 29 Topical Compositions Composition Number 1 2 3 4 5 6 7 10 12 Ingredients (% w/w) Dow Q7-9180 Silicone Fluid 0.65 cst 0 0 0 92 44.5 13.32 3.5 47 33.5 Dow Q7-9120 Silicone Fluid 0 0 0 1 0 2.02 2.5 0 0 12500 cst Dow 9045 Silicone Elastomer Blend 0 0 0 0 25 69.68 79.04 0 0 Dow Corning BY 11-030 0 0 0 0 0 0 0 0 15 Arlamol PS15E 0 14.1 0 2 8 4.98 5.27 10 0 Dow Corning 9041 Elastomer Blend 0 0 0 0 0 0 0 0 0 Compritol 888 ATO 0 0 0 0 10 0 0 0 0 Petrolatum 80 0 0 0 2.5 0 0 0 0 Castorwax 0 0 0 0 0 0 0 0 0 Isopropyl Alcohol 0 3.4 0 0 0 0 0 3 0 Isopropyl Myristate 0 0 0 0 0 0 0 0 0 Plural diisostearique 0 0 0 0 0 0 0 0 0 Monosteol (PG Stearate) 0 0 0 0 0 0 0 0 0 Transcutol 0 62.5 0 0 0 0 0 20 30 PEG 400 0 0 50 0 0 0 0 0 0 PEG 4000 0 0 30 0 0 0 0 0 0 Water 0 0 0 0 0 0 0 0 1.5 Cannabidiol 20 20 20 5 10 10 10 20 20
[0375] Efficacy was very composition-dependent, and some composition vehicles had modest to good antimicrobial activity on their own (e.g. composition 2, with a high content of transcutol and 3.4% isopropyl alcohol). Results are provided in
[0376] The lack of activity in some compositions was due to the CBD not being sufficiently released from the composition.
[0377] Good activity (2- to 3-log reduction in colony-forming units [CFU] after 1 h, >5 log reduction at 24 h) was consistently observed with Compositions #3 and #12, but not their corresponding vehicles. Composition #3 is a PEG-based composition, which matches that used for Bactroban™ (mupirocin) ointment, containing 20% CBD. Composition #12 has a mixture of a silicone fluid (polydimethylsiloxane liquid) and transcutol combined with a gelling agent (Dow Corning BY 11-030) and a small amount of water, again with 20% CBD.
TABLE-US-00032 TABLE 30 Composition of the BTX 1801 Active and Vehicle Control Compositions (w/w) BTX1801 Composition BTX BTX BTX BTX 1801 1801 1801 1801 Gel Ointment Ingredients (% w/w) Gel Ointment Vehicle Vehicle Hexamethyldisiloxane 33.5 0 41.9 0 Dow BY 11-030 15 0 18.8 0 Transcutol 30 0 37.5 0 Polyethylene glycol 0 80 0 100 400/4000 (mixture) Water 1.5 0 1.9 0 Cannabidiol (CBD) 20.0 20.0 0 0 Total 100.0 100.0 100.0 100.0
Example 9
General Microbial Assay Protocol
[0378] Test compositions were provided by Botanix/Formulytica and designated with a number of the form F###-#-##/L###-#-##, where the “F” number refers to a particular composition and the “L” number refers to a manufacturing batch. All experiments dated prior to Dec. 5, 2019 were performed using compositions with an “L” number of the form L144-2-##; all experiments dated after Dec. 5, 2019 were performed using compositions with an “L” number of the form L144-3-##.
[0379] Each experiment had two timepoints: 1±0.25 hours, 24±2 hours with 3 explants for each strain/treatment/timepoint combination.
[0380] Bacterial species and strain: Staphylococcus aureus MRSA ATCC 43300, high-level mupirocin-resistant MRSA strains 329 and 993, low-level mupirocin-resistant MRSA strain 815. Mupirocin-resistant strains characterized in a prior manuscript: Antimicrob. Agents Chemother. 59, 2765-2773 (2015).
[0381] The tissue type used was Porcine skin tissue (PST).
[0382] Neutralizer: 500 μL or 1 mL 30 mg/mL BSA for all CBD-containing vehicles; 500 pg amberlite beads (XAD-40) in 1 mL PBS was used to neutralize mupirocin.
Skin and Explant Preparation
[0383] Porcine skin tissue (PST) from a pig harvested for meat 2-5 hours prior to arrival in lab was transported to the laboratory on ice. A section or sections of skin approximately 8 cm×8 cm was cleaned to remove gross contamination and shaved. 5 mm biopsy punches were used to cut tissue explants and remaining muscle tissue was removed with a sterile scalpel blade.
[0384] Explants were soaked in RPMI+2% (v/v) penicillin/streptomycin for 24-48 hours at 4±2° C. to reduce presence of normal flora. Explants were rinsed twice with fresh RPMI (no antibiotics) and soaked for 14±3 hours at 4±2° C. to remove antibiotics. Immediately prior to use in assay explants were washed once more and soaked in RPMI (no antibiotics) for 30±10 minutes at 37±2° C.
[0385] Explants were placed into 6-well cell culture plates atop 0.4 μm trans-well inserts with 2±0.5 mL RPMI below the insert.
Bacteria Preparation
[0386] A plate was streaked for isolation directly from frozen stock onto a blood agar plate (BAP) within three weeks of experiment date. A culture tube containing Todd Hewitt Broth (THB) containing a sub-lethal amount of mupirocin was inoculated with a single colony from the BAP and placed in shaking incubator (37±2° C., 200±10 rpm) in the late afternoon the day before the experiment. This broth was prepared by diluting 2% mupirocin ointment 1:100 in THB.
[0387] The day of the experiment, 200±50 μL of the overnight culture was transferred into 2±0.5 mL fresh THB, and shaken for 3±1 hour at 37° C. An inoculum of approximately 5×108 CFU/mL in RPMI with washes by centrifugation at ˜20,000×g followed by removal of supernatant and resuspension of pellet. Inoculum was generated by diluting the passaged culture to a concentration of ˜5×10.sup.8 CFU/mL in RPMI medium. This was generally a 1:4 dilution, corresponding to an optical density at 600 nm of ˜0.6. This wash step was completed in full twice, with the third resuspension used as inoculum.
[0388] Final inoculum was measured quantitatively by preparing a 1:10,000 dilution (2×1:100 serial dilution) and plating 50 μL of this dilution on MSA.
Infection
[0389] Pipet 2 μL of ˜5×108 CFU/mL inoculum onto each explant (˜1×106 CFU/VVound Bed). Incubate at 37±2° C. for 2±0.25 h.
Treatment and Wash
[0390] Explants were treated with 100 μL of appropriate composition with no composition applied to the Growth Control (GC) explants. Explants were incubated at 37±2° C. for 1±0.25 h.
[0391] Explants were washed using 1.0±0.05 mL sterile PBS+2% (w/v) mucin into each insert. Mucin was introduced directly onto each explant in the well. Swirl gently for 5±2 seconds. Mucin and residual treatment was aspirated and mechanically removed as necessary.
[0392] Media below trans-wells (RPMI without antibiotics) was removed and replaced with a fresh 2±0.5 mL. Explants were returned to 37±2° C. for 1±0.25 h or 24±2 h.
Sample Collection
[0393] At the appropriate time, explants were removed and placed into neutralizer (as described above).
[0394] Explants were treated with a vortex/sonicate/vortex series to liberate bacteria (30±5 sec vortex, 120±6 sec sonicate, 30±5 sec vortex). 50±2 μL of each sample was plated on mannitol salt agar plates using a spiral plater (neat, at a 1:100 dilution, or at a 1:10,000 dilution). Plates incubated for 24-48 h at 37° C., enumerated using an automated plate counter, and transformed into Log10(CFU/explant).
Data and Statistical Analysis
[0395] Plate counts were imported into Prism (Graphpad), and data was graphed as mean with standard error of the mean (SEM). In general, weekly data was analysed using Prism by separating the two timepoints using the “multiple comparisons” of a one-way ANOVA analysis with Holm-Sidak post- correction.
[0396] Combined data sets were generated by storing all data in a Microsoft Excel workbook. Overall means were calculated by multiplying each experimental mean by the number of samples in that experiment (weighted mean), summing this value for each experiment, and dividing by the total number of samples.
[0397] The standard deviation for the combined data set was calculated by an application of the law of total variance. Variance for each experimental data set was calculated by squaring the standard deviation (as calculated by Excel) added to the square of the difference between the mean for that experiment and the total weighted mean; this combined value was multiplied by the number of samples for each experiment. The standard deviation of the full data set was calculated by taking the square root of the sum of these variances divided by the total number of samples.
[0398] Standard error of the mean was calculated by dividing the standard deviation by the square root of the total number of samples. Statistical significance was determined by importing the data into Prism (Graphpad) and using the “multiple comparisons” of a 2-way ANOVA analysis with a Dunnett's post-correction.
Neutralization Protocol
Neutralization Protocol Background
[0399] Explant and Bacteria preparation identical to that described in “General Antimicrobial Assay Protocol” above. Final inoculum was prepared as a suspension of 5×104 CFU/mL in PBS.
Challenge Preparation
[0400] Eppendorf tubes (1.7 mL) containing 1 mL of PBS for the negative control, 500 μg Amberlite Beads (XAD-40) in 1 mL PBS (for mupirocin), or 1 mL of 30 mg/mL BSA in PBS pH=7.4 for all CBD-containing compositions and their vehicles were spiked with ˜1×103 (3 log) CFU of the S. aureus ATCC 43300 bacteria primarily used in the project (20 μL of the 5×104 CFU/mL inoculum).
[0401] Explants were prepared as described in “Skin and Explant Preparation” above and treated as described in the “Treatment and Wash” and “Sample Collection” above.
Sample Collection and Quantitation
[0402] Explants were placed in the prepared 1.7 mL Eppendorf tubes containing ˜1×103 and subjected to the same neutralization and plating protocol outlined in the “Sample Collection” above. Plates were counted after ˜36 hours. Any treatment group with a log reduction from growth control of less than or equal to 0.2 was considered to have passed (per ASTM E1054).
Minimum Inhibitory Concentration (MIC) Microtiter Broth Dilution Protocol
MIC Bacterial Preparation
[0403] Strains used: ATCC 29213 (standard control), ATCC 43300, low-level mupirocin-resistant isolate 815, high-level mupirocin-resistant isolage 329, high-level mupirocin-resistant isolate 993.
[0404] All strains prepared as described in “Bacterial Preparation” section above with the following differences: inocula were prepared in Müller-Hinton broth (MHB) rather than RPMI, and were diluted 1:10,000 following wash for an inoculum concentration of ˜5×104 CFU/mL.
Plate Preparation
[0405] Using a multichannel pipet, 100 μL of MHB was added to each of the wells of the 96-well plates (round bottom/U-bottom). Stock solutions of of antibiotic were prepared at 2× the highest assay concentration (mupirocin at 4096 μg/mL and CBD at 100 μg/mL). 100 μL of this broth was added to each of the broth-containing wells in column 1 of the plate, and mixed by pipetting the full volume 6-7 times, for a final concentration of 2048 μg/mL mupirocin or 50 μg/mL CBD. 100 μL of the broth from column 1 was added to column 2 and mixed by pipetting 100 μL 6-7 times. This was repeated through column 11 (2 μg/mL mupirocin or 0.049 μg/mL CBD). Column 12 was left without antimicrobial.
Experiment Setup
[0406] 5 μL of each inoculum was added to the appropriate wells (various wells were left without inoculation and used to correct the absorbance of the CBD-containing wells and as negative controls). Plate was incubated at 37° C. for 18-24 hours. Plate was read by a plate reader at 600 nm.
Data Analysis
[0407] Inhibition was determined to have occurred in the first well with a difference in OD600 of <0.05 from the uninfected wells with the same concentration of antimicrobial, provided inhibition growth continued through all higher concentrations. The experiment was performed twice for each antimicrobial, and data reported as a range of the two values obtained.
Irritation (MTT) Protocol
MTT Tissue Preparation
[0408] Explants were prepared as described in “Skin and Explant Preparation” above and treated as described in the “Treatment and Wash” and “Sample Collection” above. Explants were placed in 6-well plates on sterile gauze soaked in RPMI+2% (v/v) penicillin-streptomycin rather than trans-wells.
Treatment and Experimental Design
[0409] 10 μL of composition or control was added to the top of explants and incubated for 24±2 hours. A “Treatment” was one of the CBD-containing compositions or the associated vehicle. The controls were PBS (pH=7.4) as a non-irritating control, 10% Tween-20 in distilled water as a non-irritating detergent, 1% Triton in distilled water as a mildly irritating detergent, and 5% SDS in distilled water as a highly irritating detergent. All controls were used in all assays.
MTT Assay
[0410] Following incubation with treatment, explants were added to 100 μL MTT reagent and incubated for 1.5±0.5 h at 37±2° C. Following incubation with MTT reagent, explants were added to 100 μL of de-stain (0.1 M HCl in 2-propanol) and incubated for 20±4 hours at 4±2 ° C.
Data Collection and Analysis
[0411] Explants were removed from wells, and remaining de-stain was read by absorbance at 570 nm and 600 nm. The 570 nm absorbance corresponds to the MTT signal and the 600 nm signal shows any nonspecific blockage of light (such as by leftover tissue). Data was normalized to non-irritating controls (PBS and/or 10% Tween-20).
Results
[0412] Of the original 12, the best compositions were determined to be F79-16-3 (liquid), F144-2-11 (ointment), and F144-2-4 (gel). At 1 hour, F79-16-3 (2.9 log reduction) and F144-2-4 (1.8 log reduction)were most effective against S. aureus ATCC43300 (
[0413] At 24 hours, all three compositions were highly effective, with 4.3, 3.9, and 4.7 log reductions, respectively. The vehicle for F79-16-3 was comparable in efficacy toward ATCC 43300 (
[0414] All compositions were comparably effective against mupirocin-resistant MRSA strains (
[0415] All CDC-containing compositions were non-irritating to ex vivo porcine skin (
[0416] Of the 12 analysed, the best compositions, as measured by the antimicrobial assay, are F79- 16-3 (liquid), F144-2-11 (ointment), and F144-2-4 (gel). All three compositions were more effective than mupirocin against highly mupirocin-resistant (MIC 256 to 2048 ug/mL) MRSA strains (329 and 993). Compositions F79-16-3 and F144-2-4 appear more effective than mupirocin against low-level resistant strain numbered 815.
Example 10
General Microbial Assay Protocol
[0417] Test compositions were provided by Botanix/Formulytica and designated with a number of the form F###-#-##/L###-#-##, where the “F” number refers to a particular composition and the “L” number refers to a manufacturing batch.
[0418] Each experiment had two timepoints: 1±0.25 h, 24±2 h with 3 explants for each strain/treatment/timepoint combination. The bacterial species and strain: Staphylococcus aureus ATCC 43300.
Skin and Explant Preparation
[0419] Porcine skin tissue (PST) from a pig harvested for meat 2-5 h prior to arrival in lab was transported to the laboratory on ice. A section or sections of skin approximately 8 cm×8 cm was cleaned to remove gross contamination and shaved. 5 mm biopsy punches were used to cut tissue explants and remaining muscle tissue was removed with a sterile scalpel blade.
[0420] Explants were rinsed twice with 15±5 mL of RPMI+2% (v/v) penicillin/streptomycin+0.5 mg/L amphotericin B (RPMI+ABXF). Explants were soaked in fresh 15±5 mL RPMI+ABXF for 1±0.25 h at 37±2° C. to reduce presence of normal flora. Explants were rinsed twice with fresh RPMI (no antibiotics). Explants were soaked in fresh RPMI for 1±0.25 h at 37±2° C. to remove antibiotics.
[0421] Immediately prior to use in assay explants were washed twice more with 15±5 mL of RPMI. Explants were placed into 6-well cell culture plates atop 0.4 μm trans-well inserts with 2±0.5 mL RPMI below the insert.
Bacteria Preparation
[0422] A plate was streaked for isolation directly from frozen stock onto a blood agar plate (BAP) or mannitol salt agar (MSA) plate within three weeks of experiment. A culture tube containing Todd Hewitt Broth (THB) was inoculated with a single colony from the BAP and placed in shaking incubator (37±2° C., 200±10 rpm) in the late afternoon the day before the experiment.
[0423] The day of the experiment, 200±50 μL of the overnight culture was transferred into 2±0.5 mL fresh THB, and shaken for 3±1 h at 37° C. An inoculum of approximately 5×108 CFU/mL in RPMI with washes by centrifugation at 20,000×g followed by removal of supernatant and resuspension of pellet. Inoculum was generated by diluting the passaged culture to a concentration of ˜5×108 CFU/mL in RPMI medium. This was generally a 1:4 dilution, corresponding to an optical density at 600 nm of ˜0.6. This wash step was completed in full twice, with the third resuspension used as inoculum.
[0424] Final inoculum was measured quantitatively by preparing a 1:10,000 dilution (2×1:100 serial dilution), plating 50 μL of this dilution on MSA, and enumerating colonies.
Infection
[0425] Inoculum (2±0.5 μL of ˜5×108 CFU/mL S. aureus) was pipetted onto each explant (˜1×106 CFU/explant). Incubated at 37±2° C. for 2±0.25 h.
Treatment
[0426] Explants were treated with 100 pL of appropriate composition with no composition applied to the Growth Control (GC) explants. Explants were incubated at 37±2° C. for 1±0.25 h.
[0427] Explants were washed using 1.0±0.05 mL sterile PBS+2% (w/v) mucin into each well. Mucin was introduced directly onto each explant in the well and swirled gently for 5±2 seconds. Mucin and residual treatment was aspirated and mechanically removed as necessary.
[0428] Media below trans-wells (RPMI without antibiotics) was removed and replaced with a fresh 2±0.5 mL. Explants were returned to 37±2° C. for 1±0.25 h or 24±2 h.
Sample Collection
[0429] At the appropriate time explants were removed and placed into neutralizer (as described above). Explants were treated with a vortex/sonicate/vortex series to liberate bacteria (30±5 sec vortex, 120±6 sec sonicate, 30±5 sec vortex).
[0430] 50±2 μL of each sample was plated on mannitol salt agar plates using a spiral plater (neat, at a 1:100 dilution, or at a 1:10,000 dilution). Plates incubated for 24-48 h at 37° C., enumerated using an automated plate counter, and transformed into Log10(CFU/explant).
Data and Statistical Analysis
[0431] Plate counts were imported into Prism (Graphpad), and data was graphed as mean with standard error of the mean (SEM). In general, weekly data was analyzed using Prism by separating the two timepoints using the “multiple comparisons” of a one-way ANOVA analysis with Holm-Sidak post-correction.
[0432] Combined data sets were generated by storing all data in a Microsoft Excel workbook. Overall means were calculated by multiplying each experimental mean by the number of samples in that experiment (weighted mean), summing this value for each experiment, and dividing by the total number of samples.
[0433] The standard deviation for the combined data set was calculated by an application of the law of total variance. Variance for each experimental data set was calculated by squaring the standard deviation (as calculated by Excel) added to the square of the difference between the mean for that experiment and the total weighted mean; this combined value was multiplied by the number of samples for each experiment. The standard deviation of the full data set was calculated by taking the square root of the sum of these variances divided by the total number of samples. Standard error of the mean was calculated by dividing the standard deviation by the square root of the total number of samples. Statistical significance was determined by importing the data into Prism (Graphpad) and using the “multiple comparisons” of a 2-way ANOVA analysis with Dunnett's post-correction.
TABLE-US-00033 TABLE 31 Treatments Name Identifying number 5% Ointment F/L 144-5-1 5% Ointment (Vehicle) F/L 144-5-2 10% Ointment F/L 144-5-3 10% Ointment (Vehicle) F/L 144-5-4 15% Ointment F/L 144-5-5 15% Ointment (Vehicle) F/L 144-5-6 3 (20% Ointment) F144-2-11 (B) 144-3-05 3 V (20% Ointment Vehicle) F144-2-24 (B) 144-3-06 5% Gel F/L 144-5-7 5% Gel (Vehicle) F/L 144-5-8 10% Gel F/L 144-5-9 10% Gel (Vehicle) F/L 144-5-10 15% Gel F/L 144-5-11 15% Gel (Vehicle) F/L 144-5-12 12 (20% Gel) F144-2-04 (B) 144-3-07 12 C (20% Gel Vehicle) F144-2-16 (B) 144-3-08
Results
[0434] All CBD-containing treatments resulted in statistically significant (p<0.05) reduction from growth control at both 1 h and 24 h. No vehicle resulted in a statistically significant reduction from growth control at neither 1 h nor 24 h. The largest aggregate reduction from a vehicle treatment was the 20% ointment vehicle, which resulted in ˜1.4 Log reduction (
[0435] The 20% CBD composition was clearly the most effective concentration at 1 h (˜3.4 Log reduction), other treatments did not form a curve at (˜1.8, ˜1.5, and ˜1.7 Log reduction for 5%, 10%, and 15% compositions respectively); 20% CBD ointment was significantly different (p<0.05) from the 5%, 10%, and 15% compositions. At 24 h, the composition effectiveness approximately correlated with CBD-percentage (in ascending order 5% to 20%: ˜4.6, ˜5.9, ˜6.5, ˜6.4 Log reduction); no non-vehicle treatments were significantly different from another.
[0436] At 1 h, the effectiveness of the gel compositions approximately correlated with CBD-percentage (in ascending order: ˜1.2, ˜1.2, ˜2, ˜3.3 Log reduction), and the difference between the 5% and 20% is statistically significant; 20% CBD gel was significantly different (p<0.05) from the 5%, 10%, and 15% compositions. At 24 h, there was more variation (in ascending order: ˜4.3, ˜7.0, ˜5.7, ˜6.8), though there appears to be increased efficacy based on concentration; the difference between the 5% and 10% gel compositions was statistically significant (p=0.0335).
[0437] The data indicate general trends that higher CBD-concentration results in more antimicrobial effectiveness for both compositions at the 1 h and 24 h post-treatment timepoints.
Example 11
[0438] A Randomised, Double-Blind, Vehicle-Controlled Study to Evaluate Safety, Tolerability, and Efficacy of Two Dosage Forms of BTX 1801 Applied Twice Daily for Five Days to the Anterior Nares of Healthy Adults Nasally Colonised with Staphylococcus aureus
[0439] This randomised, double-blind, vehicle-controlled study will evaluate the safety, tolerability and efficacy of two dosage forms of BTX 1801 compared to their corresponding vehicles, applied BID for 5 days to the anterior nares of healthy adults nasally colonised with SA. Approximately 60 participants will be randomised 2:2:1:1 (20 participants to BTX 1801 20% (w/w) Ointment, 20 participants to BTX 1801 20% (w/w) Gel, 10 participants to BTX 1801 Vehicle Ointment, and 10 participants to BTX 1801 Vehicle Gel).
[0440] Participants will attend 2 screening visits to determine SA nasal colonisation via culture of anterior nares swabs at Screening Visit 1 (Days −28 to −14) and Screening Visit 2 (Days −11 to −4). Participants will be identified as persistent or intermittent carriers following results of Baseline nasal cultures (Visit 3; Day 1).
TABLE-US-00034 TABLE 32 Identification of Intermittent versus Persistent Colonisation Status Anterior nares culture for S. aureus Nasal Colonisation Screening Visit 1 Screening Visit 2 Baseline Visit 3 Status Negative — — Not Colonised.sup.1 Positive Negative — Not Colonised.sup.2 Positive Positive Negative Intermittent.sup.3 Positive Positive Positive Persistent.sup.3 .sup.1Not eligible for Screening Visit 2 .sup.2Not eligible for Baseline Visit 3 .sup.3Intermittent and Persistent carriers are eligible for the randomisation
[0441] Eligible participants will receive their first application of study drug at the site on Day 1 and will self-apply their other dose at home. Participants will be treated for 5 consecutive days (Visits 3-7), and return for follow-up visits on Days 8,12 and 28 (Visits 8-10).
[0442] Throughout the study, safety will be monitored by TEAEs, local tolerability (TNSS and macroscopic nasal examination), clinical laboratory assessments, physical examination, and vital signs. Concomitant medications will be recorded throughout the study.
[0443] Blood samples to determine CBD plasma concentrations will be collected pre-dose (on Days 1 (Baseline), and on Days 2 and 5 (during treatment). Details of the participant's study drug application (including the date, time, and amount) will be recorded in study drug application diary. Anterior nares swabs to measure SA nasal colonisation will also be collected at all follow-up visits (Days 8, 12 and 28; Visits 8-10).
Primary Endpoints
[0444] To assess the safety and tolerability of BTX 1801 relative to Baseline for the following parameters: [0445] Treatment-emergent adverse events (TEAEs) [0446] Total Nasal Symptom Score (TNSS) [0447] Macroscopic nasal examination [0448] Clinical laboratory assessments and to assess the percentage of persistent SA carriers with a negative nasal culture for SA on Day 12
Secondary Endpoints
[0449] To evaluate changes in nasal SA colonisation associated with study drug application as follows: [0450] of persistent SA carriers with a negative nasal culture for SA on Days 8 and 28 [0451] of participants with a negative nasal culture for MRSA on study Days 8, 12 and 28 [0452] of participants who have nasal recolonisation with SA on study day 12 and/or 28 after a negative nasal culture on Day 8 and to assess the plasma levels of study drug taken pre-dose at Baseline and Days 2 and 5.
Inclusion criteria
[0453] To be included in the study, participants must meet the following inclusion criteria. [0454] Participant is of either gender of ≥18-65 years of age. [0455] Participant is in good general health without clinically significant respiratory, gastrointestinal, renal, hepatic, haematological, lymphatic, neurological, cardiovascular, psychiatric, musculoskeletal, genitourinary, immunological, dermatological, malignant disease, or connective tissue diseases or disorders, as determined by the investigator. [0456] Confirmed to be nasal SA carriers, defined as having 2 separate SA positive cultures from anterior nares swabs during screening.
Exclusion Criteria
[0457] If a participant meets any of the following exclusion criteria, they may not participate in the study.
[0458] Methicillin-susceptible and methicillin-resistant Staphylococcus aureus decolonisation attempt in the 6 months prior to screening.
[0459] Nasal surgery within 3 months prior to Screening Visit 1.
[0460] Evidence of active rhinitis, sinusitis or upper respiratory tract infection at Screening Visits 1 or 2 or Baseline Visit.
[0461] Participant has any significant active infection.
[0462] Participant has used topical or systemic antibiotics within 4 weeks of Baseline.
[0463] Negative nasal culture for SA at Screening Visit 1 or 2.19. Planned use of any nasal applied medication (other than study drug) during the study.
Participant Enrolment
[0464] Participants will be randomised 2:2:1:1 to receive BTX 1801 20% (w/w) Ointment, BTX 1801 20% (w/w) Gel, BTX 1801 Vehicle Ointment, to BTX 1801 Vehicle Gel).
Study Drug
[0465] Study drug will be provided to the study site by Formulytica Pty Ltd in Mulgrave, Victoria, Australia. Initial shipments will be made to supply the study site prior to enrolment of the first participant. Additional supplies will be made available as needed based on participant enrolment.
BTX 1801 Compositions
[0466] Botanix Pharmaceuticals' BTX 1801 contains the active pharmaceutical ingredient, CBD.
[0467] Two compositions of BTX 1801 and their corresponding Vehicle-control compositions will be provided to the study site in 20 g aluminium laminate tube with a 15 g fill. The excipients include hexamethyldisiloxane, hexamethyldisiloxane (Dow 9180), Transcutol P (diethylene glycol monoethyl ether), cyclopentasiloxane+polyethylene glycol (PEG)/polypropylene glycol (PPG)-10/19 dimethicone blend (Dow BY 11-030), PEG 400, PEG 4000 and water which have been used extensively in other topical products. The active BTX 1801 study products are a clear to light pink solution with a 20% (w/w) concentration of CBD. The compositions of the BTX 1801 compositions and their corresponding Vehicle-controls are presented in the table below.
TABLE-US-00035 TABLE 31 Composition of the BTX 1801 Active & Vehicle Control Compositions BTX 1801 Composition BTX 1801 BTX 1801 BTX 1801 BTX 1801 Ingredients (% w/w) Gel Ointment Vehicle Gel Vehicle Hexamethyldisiloxane 33.5 0 41.9 0 Dow BY 11-030 15 0 18.8 0 Transcutol 30 0 37.5 0 Polyethylene glycol 0 80 0 100 400/4000 (mixture) Water 1.5 1.9 0 Cannabidiol (CBD) 20.0 20.0 0 0 Total 1000 1000 1000 1000
[0468] Each gram of the BTX 1801 may contain up to 200 mg of CBD. The maximum daily exposure following application of 0.25 g to each nostril BID of each BTX 1801 composition is ˜200 mg of CBD.
[0469] Study drug will be applied by a study staff member different from the evaluator so that clinical assessments are blinded.
Dosing and Administration
[0470] Two 20 g tubes of study drug will be assigned to each participant. One tube will be dispensed to each participant on Day 1 and will be sufficient supply for the 5 day application stage. The second tube will remain at the study site as back-up, if needed. Study drug is applied BID, one dose will be applied under the supervision of unblinded study site staff each day (Days 1-5) and the participants will self-apply the other dose of study drug at home. Participants will be instructed to bring their study drug to the study site each day.
[0471] Participants will be instructed on how to apply study drug when not at the clinical site. Each application of study drug will occur approximately at the same time in the morning with the second application approximately 12 hours later.
[0472] The dose for all participants will be 0.25 g of study drug applied BID to each anterior nare (0.5 g per nare per day). Participants will dispense a finger-top-unit (FTU) of study drug by squeezing a line of study drug from the tip of their index finger to the first crease and instructed to apply to one of the anterior nares by gently rolling the finger-tip over inner surface of the nare. Following application to each nare, participants will be instructed to gently pinch the nose intermittently for approx. 1 minute to ensure distribution of study drug within the anterior nares.
[0473] No escalation of dose will occur. Participants will receive BID application of study drug for a total of 10 doses.
Screening Timeline
Visit 1: Days -28 to -14 (Screening)
[0474] At the first Screening Visit (Visit 1), participants will review and sign a pre-screening ICF for the collection of anterior nares swabs to test for SA colonisation and attesting to their knowledge that if the nasal swab is negative for SA, he/she will not be invited to proceed to Screening Visit 2. The following study specific procedures will occur at the Screening Visit 1: [0475] Pre-screening informed consent [0476] Anterior nares swabs for SA culture
[0477] Confirmation of SA positive culture from anterior nares swabs is required before determining whether participant is eligible for Screening Visit 2.
Visit 2: Days -11 to -4 (Screening)
[0478] At the second Screening Visit (Visit 2), the following procedures/assessments will be conducted: [0479] Informed consent for main study [0480] Inclusion/exclusion criteria review [0481] Demographic information collection [0482] Medical & medication history collection [0483] Physical examination, including body measurements (body weight and height) [0484] Anterior nares swabs for SA culture
[0485] Confirmation of SA positive culture from anterior nares swabs collected at this visit is required before determining whether the participant is eligible for the Baseline Visit.
Visit 3: Day 1 (Baseline; Start of Treatment)
[0486] On Day 1 (start of study drug use), the following procedures/assessments will be conducted prior to study drug intake: [0487] Randomisation to study drug group
Following Randomisation the Following will be Performed [0488] TNSS [0489] Macroscopic nasal examination [0490] Anterior nares swabs for SA culture (swabs to be retained) [0491] Blood collection for study drug level prior to study drug application [0492] Weigh and dispense first tube of study drug. One tube of study drug to be dispensed to each participant; the same tube will be taken home and returned to the study site each day for compliance monitoring and dosing at the site [0493] Train participant in proper application of study drug [0494] Supervise application of study drug by unblinded study staff ensuring correct procedure is followed [0495] Monitor participant for 30 minutes after the application of study drug
Visit 4: Day 2 (Treatment Phase)
[0496] On Day 2, the following procedures/assessments will be conducted: [0497] TNSS [0498] Macroscopic nasal examination [0499] Blood collection for study drug level prior to study drug application [0500] Study drug application at site (one dose applied by the participant in the clinic under the supervision of unblinded study staff)
Visit 5: Day 3 (Treatment Phase)
[0501] On Day 3, the following procedures/assessments will be conducted: [0502] TNSS [0503] Macroscopic nasal examination [0504] Study drug application at site (one dose applied by the participant in the clinic under the supervision of unblinded study staff)
Visit 6: Day 4 (Treatment Phase)
[0505] On Day 4, the following procedures/assessments will be conducted: [0506] TNSS [0507] Macroscopic nasal examination [0508] Study drug application at site (one dose applied by the participant in the clinic under the supervision of unblinded study staff)
Visit 7: Day 5 (Treatment Phase)
[0509] On Day 5 (+1 day), the following procedures/assessments will be conducted: [0510] TNSS [0511] Macroscopic nasal examination [0512] Blood collection for study drug level prior to study drug application [0513] Study drug application at site (one dose administration applied by the participant in the clinic under the supervision of study staff)
Visit 8: Day 8 (Follow-up)
[0514] On Day 8 (±1 day), the following procedures/assessments will be conducted: [0515] Physical examination (including body measurements, excluding height) [0516] TNSS [0517] Macroscopic nasal examination [0518] Anterior nares swabs for SA culture (swabs to be retained)
Visit 9: Day 12 (Follow-up)
[0519] On Day 12 (±1 day), the following procedures/assessments will be conducted: [0520] Macroscopic nasal examination [0521] Anterior nares swabs for SA culture (swabs to be retained)
Visit 10: Day 28 (Follow-up)
[0522] On Day 28 (±1 day), the following procedures/assessments will be conducted: [0523] Macroscopic nasal examination [0524] Anterior nares swabs for SA culture (swabs to be retained)
Demographics
[0525] Demographic information to be obtained at screening will include date of birth, gender, ethnicity, and race as described by the participant.
Adverse Events
[0526] Any untoward medical occurrence in the participant's medical condition will be recorded in source and the electronic case report form (eCRF) as an AE, with appropriate follow-up. All AEs occurring during the study (from the date of consent to the end of follow-up [Day 28]), whether or not attributed to the study drug (observed by the Investigator or reported by the participant) will be recorded in source and the eCRF.
Treatment Score
Total Nasal Symptom Score
[0527] The TNSS will be measured at Baseline (pre-dose on Day 1) and at each study visit until the end of treatment. The TNSS is a subjective measure, and is the sum of 5 individual participant-assessed symptom scores for each of the following symptoms: sneezing, rhinorrhoea, nasal itching, nasal pain and nasal obstruction using ordinal scales with the following grading:
[0528] Sneezing, rhinorrhoea, nasal itching, and nasal pain: [0529] 0=none, 1=mild, 2=moderate, 3=severe, 4=very severe
[0530] Nasal obstruction: [0531] 0=breathing through the nose freely and easily. 1=slightly difficult, 2=moderate difficulty, 3=severe difficulty, 4 breathing through nose is very difficult/impossible
[0532] Any participant with a grade 3 or 4 nasal tolerability assessment for any of the assessed symptoms should have an additional evaluation by an ENT physician.
Macroscopic Nasal Examination
[0533] Macroscopic nasal examinations will occur at every study visit from Baseline (to the last follow-up visit (Day 28). Nasal examination will be performed by visual inspection of the anterior nasal cavity by the Investigator. The Investigator will be blinded with respect to treatment allocation.
[0534] The anterior nares will be examined for mucosal erythema, oedema or irritation and the surrounding nostril examined for crusting, discharge or irritation.
[0535] Mucosal erythema or oedema: [0536] 0=none, 1=barely perceptible, 2=well-defined, 3=pronounced.
[0537] Nasal crusting, discharge or irritation: [0538] 0=none, 1=mild, 2=moderate, 3=severe.
[0539] Any participant with a grade 2 or 3 for erythema, oedema, nasal crusting, discharge, or irritation should have an additional evaluation by an ENT physician.
Efficacy Assessments
[0540] Efficacy will only be evaluated in participants categorised as persistent carriers of SA. Anterior nares swabs for SA culture will be collected at Screening Visit 1 and 2 (as applicable) and assessed for the presence of SA to determine eligibility. Baseline (Day 1) and follow-up (Day 8, 12 and 28) anterior nares swabs will be collected to determine the change in SA colonisation status from Baseline to Days 12 (primary endpoint), 8 and 28 (secondary endpoint), and to determine recolonisation in participants reporting a negative SA culture at Day 8 (first follow-up) at Days 12 and 28 (subsequent follow-up visits). Colonisation status will be recorded in the source and the eCRF. All anterior nares swabs will be retained until study completion.
Blood Samples for Study Drug Levels
[0541] All participants will have a blood sample taken before dosing at Day 1, Day 2 and Day 5 to measure plasma levels of CBD. Blood samples will be analysed using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The limit of detection is 0.2 ng/mL. Haemolysed plasma has an impact on data accuracy and should be avoided during sample collection. Plasma samples may be stored at −20° C. for up to 30 days. However, plasma samples will be shipped to the central laboratory for study drug levels as per the timelines outlined in the Tetra-Q Laboratory Manual. Details on the methods for obtaining and preparing samples for CBD levels are provided in the Tetra-Q Laboratory Manual.
Anterior Nares Cultures
[0542] The clinical study site will collect nasal specimens by swab from the anterior nares of each participant. Specimens will be transferred to medium for culture and identification of SA. All Screening Visit (Visit 1) swabs are to be discarded after the specimens have been transferred to medium for culture. For every subsequent visit, all swabs must be retained. Procedures for collection and processing of swab specimens, and storage of bacterial isolates are found in the Laboratory Manual. Participants must have an anterior nares culture that is positive for SA at Screening Visit 1 to be eligible for Screening Visits 2 and Baseline Visit.
Bacterial Genotyping and Phenotyping of SA Isolates
[0543] In vitro tests will be conducted on all SA isolates collected during the screening, treatment and follow-up phases of the study to determine the minimum inhibitory concentration of CBD and other compounds. For randomised participants, pulsed-field gel electrophoresis will also be conducted to determine strain relatedness between Screening/Baseline SA isolates and isolates recovered during or post-treatment. Additional genetic characterisation of select SA isolates may also be conducted as needed.
Statistical Considerations
Statistical and Analytical Plans
[0544] All statistical processing will be performed using SAS® version 9.4 or later unless otherwise stated. P-values will be provided for exploratory purposes only.
[0545] A statistical analysis plan (SAP), describing all statistical analyses will be provided as a separate document. The SAP will be finalized prior to unblinding of the study treatments.
Statistical Hypotheses
[0546] The purpose of this study is to demonstrate the effectiveness of BTX 1801 presented as 2 different dosage forms to eradicate carriage of SA on Day 12 in the anterior nares of individuals who are persistent carriers of SA. P-values for selected endpoints will be presented to assist in evaluating the outcome of the study. Failure to achieve a statistically significant result does not imply a failed study; results from this study will be used to inform statistical approaches for registration studies.
[0547] The primary efficacy point is a negative SA anterior nares culture at Day 12 t in participants who are persistent carriers. The null hypothesis is that there is no difference in the percent of anterior nares cultures that are negative for SA at Day 12 between active BTX 1801 compositions and the combined Vehicle compositions applied twice daily for 5 days to the anterior nares of healthy adults who are nasal carriers of SA.
[0548] The alternative hypothesis for this study is that there is a difference in the percent of anterior nares culture that are negative for SA at Day 12 between active BTX 1801 compositions and the combined Vehicle compositions applied twice daily for 5 days.
H.sub.0: Ptrt2=Pveh versus H.sub.1: Ptrt2≠Pveh
[0549] Where Ptrt2 and Pveh represent the percentage of negative SA anterior nares culture at Day 12 for the of active BTX 1801 dosing group and combined Vehicle groups respectively.
[0550] Should any post-hoc statistical analyses be conducted to present study outcomes, the methods for analysis may be described in the final clinical study report.
Study Drug Concentration Population
[0551] The Study Drug Concentration Population will include all participants who underwent blood sampling for study drug during the study. The Study Drug Concentration Population will be used in all individual and summary presentations of concentration-time data
Efficacy Population
[0552] Participants who complete 5 days of dosing and the follow up visits and provide evaluable culture results will be included in the efficacy population. The Efficacy Population will be used to evaluate the effectiveness of the two different dosage forms of BTX 1801 for the nasal eradication of SA.
Description of Statistical Methods
[0553] All statistical processing will be performed using SAS® version 9.4 or later unless otherwise stated.
[0554] Summary statistics will be prepared for the following: [0555] Percentage of persistent carriers with a negative nasal culture for SA on Days 8, 12 and 28. [0556] Percentage of participants who have nasal recolonisation with SA on Day 12 and 28, after a negative nasal culture on Day 8.
[0557] The Fisher's Exact test will be used for treatment comparisons of percent eradication of SA in the anterior nares.
[0558] Continuous data will be summarised by treatment group using descriptive summary statistics; namely: the number of participants (n), mean, median, standard deviation (SD), minimum value (min), maximum value (max) and 95% confidence interval (CI). The mean will be reported to 1 decimal place more than the level of precision of the data being reported, and the SD will be reported to 2 decimal places more than the level of precision of the data being reported, unless otherwise noted, to a maximum of 4 decimal places.
[0559] Summaries at each visit will be calculated using the total number (n) of participants who attended that visit. When summarising change from Baseline, participants are required to have both a non-missing Baseline and non-missing value at the given visit to be summarised.
[0560] The analysis for categorical and qualitative data will be summarised using frequencies and percentages. Percentages will be presented to 1 decimal point, unless otherwise specified. The denominators will be the number of participants in each test cohort and for N-value overall.
[0561] The mean, standard deviation (SD), median and range will be calculated for the percentage of persistent carriers with a negative nasal culture for SA on Days 8, 12 and 28, and the percentage of participants with nasal recolonisation with SA on Day 12 and/or Day 28 after a negative nasal culture on Day 8. The Fisher's Exact test will be used for treatment comparisons of percent eradication of SA in the anterior nares.
[0562] Changes in laboratory parameters from Baseline to Day 8 will be summarised by visit and using shift tables to evaluate for trends. Clinically significant abnormal laboratory findings will be listed.
[0563] TNSS and macroscopic nasal examination scores for will be summarised for each visit. In addition, the change from Baseline in the mean scores will be summarised for each visit.
[0564] Concomitant medications will be mapped to ATC Level 2 using the World Health Organization (WHO Drug) dictionary. The number and percentage of participants reporting each medication will be summarised. Medications taken by each participant will be listed.
Analysis of the Study Drug Plasma Levels
[0565] Blood levels of study drug will be summarised for Baseline and Days 2 and 5. The mean, SD, median, range, mean coefficient of variation, geometric mean, and coefficient of variation of geometric mean will be presented.
Baseline Descriptive Statistics
[0566] Demographics and Baseline characteristics including age, gender, race, ethnicity, height, and weight, will be summarised overall and by treatment group. Medical history and concomitant medications will be summarised.
LITERATURE REFERENCES
[0567] Appendino G, Gibbons S, Giana A, Pagani A, Grassi G, Stavri M, Smith E, Rahman M M. Antibacterial cannabinoids from Cannabis sativa: a structure-activity study. J Nat Prod. 2008;71(8):1427-30. [0568] Brown A F, Leech J M, Rogers T R, McLoughlin R M. Staphylococcus aureus Colonization: Modulation of Host Immune Response and Impact on Human Vaccine Design. Front Immunol. 2014;4:507. [0569] Burstein, S. Cannabidiol (CBD) and its analogs: a review of their effects on inflammation. Bioorganic & Medicinal Chemistry 23, 1377-1385 (2015). [0570] CTCAE V5.0. Common terminology criteria for adverse events. U.S. Department of Health and Human Services. National Institutes of Health, 27 Nov. 2017. Accessed at: https://ctep.concer.gov/protocolDevelopment/electronic_applications/docs/CTCAE_v5_Quick_Reference_8.5x11.pdf [0571] ICH Harmonised Guideline: Integrated Addendum to ICH E6(R1): Guideline for Good Clinical Practice E6(R2). November 2016. [0572] Ignatowska-Jankowska B, Jankowski M, Glac W, Swiergel AH (2009): Cannabidiol-induced lymphopenia does not involve NKT and NK cells. J Physiol Pharmacol. 60(Suppl 3): 99-103 [0573] Agostino J, Ferguson J, Eastwood K and Kirk M The increasing importance of community-acquired methicillin-resistant Staphylococcus aureus infections Med J Aust 2017; 207 (9): 388-393. ∥doi: 10.5694/mja17.00089 [0574] Kaplan B, Springs AE, Kaminski NE (2008): The profile of immune modulation by cannabidiol (CBD) involves deregulation of nuclear factor of activated T cells (NFAT). Biochem Pharmacol. 76: 726-737. [0575] Kuehnert M J, Kruszon-Moran D, Hill H A, Prevalence of Staphylococcus aureus Nasal Colonization in the United States, 2001-2002. JID 2006:193 [0576] Poovelikunnel T, Gethin G, Humphreys H, Mupirocin resistance: clinical implications and potential alternatives for the eradication of MRSA, Journal of Antimicrobial Chemotherapy, Volume 70, Issue 10, October 2015, Pages 2681-2692, https://doi.org/10.1093/jac/dkv169 [0577] Ritchie S R, Isdale E, Priest P, Rainey P B, Thomas M G. The turnover of strains in intermittent and persistent nasal carriers of Staphylococcus aureus. J Infect. 2016;72(3):295-301.
[0578] Sakr A, Brégeon F, Mège J L, Rolain J M, Blin O. Staphylococcus aureus Nasal Colonization: An Update on Mechanisms, Epidemiology, Risk Factors, and Subsequent Infections. Front Microbiol. 2018;9:2419. [0579] Sánchez, A. J. & García-Merino, A. Neuroprotective agents: Cannabinoids. Clinical Immunology 142, 57-67 (2012). [0580] van Klingeren, B. & ten Ham, M. J. A. v. L. Antibacterial activity of Δ9-tetrahydrocannabinol and cannabidiol. 42, 9-12 (1976). [0581] van Rijen M, Bonten M, Wenzel R, Kluytmans J. Mupirocin ointment for preventing Staphylococcus aureus infections in nasal carriers. Cochrane Database of Systematic Reviews 2008, Issue 4. Art. No.: CD006216. DOI: 10.1002/14651858.CD006216.pub2. [0582] Whiting, P. F. et al. Cannabinoids for Medical Use: A Systematic Review and Meta-analysis. JAMA 313, 2456-2473 (2015).