WO2020000024A1 - Antibacterial treatment using cannabinoid combinations - Google Patents

Abstract

Compositions and methods for the treatment or prevention of an infection by a bacterium comprising a cannabinoid, or a cannabinoid in combination with a chelating agent, most preferably EDTA. Preferably the infection is a skin infection, and the bacterium is a gram-positive bacteria and/or film-forming bacteria and/or resistant to at least one antibiotic. Preferably the Q cannabinoid is selected from cannabidiol, cannabiol, cannabigerol, cannabichromene and A9-tetrahydrocannabinol, most preferably cannabidiol.

Description

Antibacterial Treatment Using Cannabinoid Combinations

TECHNICAL FIELD

[0001 ] A method and composition for the treatment or prevention of bacterial infections, comprising the use of a cannabinoid, or a cannabinoid in combination with a chelating agent.

BACKGROUND ART

[0002] Compounds with antimicrobial properties have attracted great interest in recent times as a result of an increase in the prevalence of infections caused by bacteria, resulting in serious or fatal diseases. Furthermore, the regular use of broad spectrum antibiotic formulas has led to the increased occurrence of bacterial strains resistant to some antimicrobial compositions, such as methicillin-resistant Staphylococcus aureus (MSRA).

[0003] Novel antimicrobial compounds have the potential to be highly effective against these types of antibiotic-resistant bacteria. The pathogens, having not previously been exposed to the antimicrobial composition, may have little to no resistance to the treatment.

[0004] There is no indication that bacterial resistance to antibiotics will stop and for this reason new antibiotics and new treatment options are necessary to achieve a desirable treatment outcome in patients.

[0005] In one form, the present invention seeks to provide a new option for the treatment of bacterial infections, including infections by antibiotic-resistant bacteria.

[0006] Many microbes form highly organised structures called biofilms in which they are protected from immune cells and antibiotic killing via several mechanisms. These mechanisms include reduced antibiotic penetration, low metabolic activity, physiological adaptation, antibiotic-degrading enzymes, and selection for genetically resistant variants (Stewart & Costerton Lancet. 2001 358(9276):135-138).

[0007] In one form, the present invention seeks to provide a new option for the treatment of bacterial infections associated with biofilms.

[0008] Colonization of bacteria on the surfaces of medical devices and healthcare products, particularly in implanted devices, result in serious patient problems, including the need to remove and/or replace the implanted device and to vigorously treat secondary infection conditions.

[0009] In one form, the present invention seeks to provide a new option for the treatment of bacterial colonisation or contamination of a non-living surface, such as the surface of a medical device or healthcare product. [0010] The previous discussion of the background art is intended to facilitate an understanding of the present invention only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application.

SUMMARY OF INVENTION

[001 1 ] According to one aspect of the invention, there is provided a composition comprising a cannabinoid for the treatment or prevention of an infection by a bacterium.

[0012] According to the invention, there is also provided a composition comprising a cannabinoid and a chelating agent, for the treatment or prevention of an infection by a bacterium.

[0013] According to another aspect of the invention, there is provided a method for the treatment or prevention of an infection by a bacterium in a subject in need of such treatment comprising the step of:

administering a therapeutically or preventative effective amount of a composition comprising a cannabinoid.

[0014] There is also provided a method for the treatment or prevention of an infection by a bacterium in a subject in need of such treatment comprising the step of:

administering a therapeutically or preventative effective amount of a composition comprising a cannabinoid and a chelating agent.

[0015] According to another aspect of the invention, there is provided the use of a cannabinoid in the manufacture of medicament in the form of a composition for the treatment of an infection by a bacterium of a subject.

[0016] There is also provided the use of a cannabinoid and a chelating agent, in the manufacture of medicament in the form of a composition for the treatment of an infection by a bacterium of a subject.

[0017] In one form of the invention, the composition is a topical pharmaceutical composition. Preferably, the infection is a skin infection.

[0018] In a preferred form of the invention, the bacterium is a Gram-positive bacterium.

[0019] In a preferred form of the invention, the bacterium is a bacterium species of a genus selected from the list: Streptococcus spp., Peptostreptococcus spp., Clostridium spp., Listeria spp., Bacillus spp., Staphylococcus spp., Propionibacterium spp., Kocuria spp., and Corynebacterium spp., and combinations thereof. [0020] In a preferred form of the invention, the bacterium is a biofilm-forming bacterium.

[0021 ] In a preferred form of the invention, the bacterium is resistant to at least one antibiotic.

[0022] Preferably the cannabinoid is cannabidiol.

[0023] Preferably, the chelating agent is EDTA or a pharmaceutically acceptable salt thereof.

DESCRIPTION OF INVENTION

[0024] According to one aspect of the invention, there is provided a composition comprising a cannabinoid for the treatment or prevention of an infection by a bacterium.

[0025] According to the invention, there is also provided a composition comprising a cannabinoid and a chelating agent, for the treatment or prevention of an infection by a bacterium.

[0026] Preferably the bacterium is a biofilm-forming bacterium. Preferably the bacterium is an antibiotic resistant bacterium. The bacterium may be both biofilm-forming and antibiotic resistant.

[0027] According to another aspect of the invention, there is provided a method for the treatment or prevention of an infection by a bacterium in a subject in need of such treatment comprising the step of:

administering an effective amount of a composition comprising a cannabinoid.

[0028] According to another aspect of the invention, there is provided a method for the treatment or prevention of an infection by a bacterium in a subject in need of such treatment comprising the step of:

administering an effective amount of a composition comprising a cannabinoid and a chelating agent.

[0029] Preferably the bacterium is a biofilm-forming bacterium. Preferably the bacterium is an antibiotic resistant bacterium. The bacterium may be both biofilm-forming and antibiotic resistant.

[0030] According to another aspect of the invention, there is provided the use of a cannabinoid, in the manufacture of medicament in the form of a composition for the treatment of an infection by a bacterium in a subject.

[0031 ] According to another aspect of the invention, there is provided the use of a cannabinoid or a cannabinoid and a chelating agent, in the manufacture of medicament in the form of a composition for the treatment of an infection by a bacterium in a subject. [0032] Preferably the bacterium is a biofilm-forming bacterium. Preferably the bacterium is an antibiotic resistant bacterium. The bacterium may be both biofilm-forming and antibiotic resistant.

Cannabinol

[0033] Preferably, the cannabinoid is chosen from the list comprising: cannabidiol, cannabinol, cannabigerol, cannabichromene, and A⁹-tetrahydrocannabinol. Most preferably, the cannabinoid is cannabidiol.

[0034] Without being held to any theory, we believe that it is the resorcinol moiety of cannabinoids that serves as the antibacterial pharmacophore, with the alkyl, terpenoid, and carboxylic appendices modulating its activity.

[0035] Preferably, the composition of the present invention contains a cannabinoid at a concentration of: between 15 pg/mL and 0.1 pg/mL, 10 pg/mL and 1 pg/mL, 8 pg/mL and 2 pg/mL, or 3 pg/mL and 6 pg/mL.

[0036] Preferably, the composition of the present invention contains a cannabinoid at a concentration of: 0.1 pg/mL, 0.5 pg/mL, 1.0 pg/mL, 1 .5 pg/mL, 2.0 pg/mL, 2.5 pg/mL, 3.0 pg/mL, 3.5 pg/mL, 4.0 pg/mL, 4.5 pg/mL, 5.0 pg/mL, 5.5 pg/mL, 6.0 pg/mL, 6.5 pg/mL, 7.0 pg/mL, 7.5 pg/mL, 8.0 pg/mL, 8.5 pg/mL, 9.0 pg/mL, 9.5 pg/mL, 10.0 pg/mL, 10.5 pg/mL, 1 1.0 pg/mL, 1 1 .5 pg/mL, 12.0 pg/mL, 12.5 pg/mL, 13.0 pg/mL, 13.5 pg/mL, 14.0 pg/mL, 14.5 pg/mL, or 15.0 pg/mL.

[0037] Preferably, the composition of the present invention contains a cannabinoid at a concentration of: between 2 pg/mL and 0.1 pg/mL, 1 .8 pg/mL and 0.1 pg/mL, 1.5 pg/mL and 0.1 pg/mL, or 1 pg/mL and 0.1 pg/mL.

[0038] Preferably, the composition of the present invention contains a cannabinoid at a concentration of: between 2 pg/mL and 1 pg/mL, 1 .8 pg/mL and 1 pg/mL, or 1.5 pg/mL and 1 pg/mL.

[0039] Preferably, the composition of the present invention contains a cannabinoid at a lower concentration than the concentration of cannabinoid in a comparator composition, wherein the comparator composition does not contain a chelating agent, and wherein the composition and the comparator composition are equally as effective at treating or preventing infection.

[0040] Preferably, the composition of the present invention contains a cannabinoid at a lower concentration than the concentration of cannabinoid in a comparator composition, wherein the comparator composition does not contain a chelating agent, and wherein the composition and the comparator composition are substantially equivalent at treating or preventing infection. Treatment

[0041 ] The present invention provides for the administration of the therapeutically effective amount of a cannabinoid or a cannabinoid and chelating agent to the site of an infection. The infection may be a topical infection.

[0042] The present invention further provides for the administration of the therapeutically effective amount of a cannabinoid or a cannabinoid and chelating agent directly to a medical device, instrument, implant, dressing or other physical object applied to the skin, implanted in the body or used in a surgical intervention.

[0043] The term “infection” as used herein means and/or colonization by a microorganism and/or multiplication of a micro-organism, in particular, a biofilm-forming bacterium. The infection may be unapparent or result in local cellular injury. The infection may be localized, subclinical and temporary or alternatively may spread by extension to become an acute or chronic clinical infection. The infection may also be a latent infection, in which the microorganism is present in a subject, however the subject does not exhibit symptoms of disease associated with the organism.

[0044] Preferably the composition of the present invention delivers a therapeutically effective amount of the cannabinoid or the cannabinoid and the chelating agent to the dermal or mucosal surface of the subject.

[0045] The phrase "therapeutically effective amount" as used herein refers to an amount of the cannabinoid or the cannabinoid and chelating agent sufficient to inhibit bacterial growth associated with bacterial carriage or a bacterial infection of the skin. That is, reference to the administration of the therapeutically effective amount of a cannabinoid and chelating agent according to the methods or compositions of the invention refers to a therapeutic effect in which substantial bacteriocidal or bacteriostatic activity causes a substantial inhibition of the relevant bacterial carriage or bacterial infection. The term “therapeutically effective amount" as used herein, refers to a nontoxic but sufficient amount of the composition to provide the desired biological, therapeutic, and/or prophylactic result. The desired results include elimination of bacterial carriage or reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. In relation to a pharmaceutical composition, effective amounts can be dosages that are recommended in the modulation of a diseased state or signs or symptoms thereof. Effective amounts differ depending on the pharmaceutical composition used and the route of administration employed. Effective amounts are routinely optimized taking into consideration various factors of a particular patient, such as age, weight, gender, etc. and the area affected by disease or disease causing microorganisms.

[0046] As used herein,“treating” or“treatment” refers to inhibiting the disease or condition, i.e., arresting or reducing its development or at least one clinical or subclinical symptom thereof. “Treating” or “treatment” further refers to relieving the disease or condition, i.e., causing regression of the disease or condition or at least one of its clinical or subclinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the subject and/or the physician. In the context of treating a bacterial infection, the term treatment includes reducing or eliminating colonization by bacteria and/or multiplication of bacteria, including reducing biofilm formation or disrupting existing biofilms.

[0047] In one form of the invention, reducing or eliminating colonization by bacteria means reducing or eliminating colonization by bacteria as measured by % bacteria killed.

[0048] In one form of the invention, reducing or eliminating colonization by bacteria means reducing or eliminating colonization by bacteria as measured by a logio reduction in bacteria.

[0049] The term a“preventative effective amount” as used herein means an amount of the formulation, which when administered according to a desired dosage regimen, is sufficient to at least partially prevent or delay the onset of the microbial infection.

[0050] Preferably the composition is a topical pharmaceutical composition for the treatment of an infection of a dermal or mucosal surface.

[0051 ] In one form of the invention, the infection is related to one or more of the following conditions: acne, rosacea, psoriasis, eczema, rash, blisters, burns, herpes simplex, warts, insect bites or infestation, itch, dermatitis, shingles, atopic dermatitis, contact dermatitis, cellulitus, folliculitis, nail infections, boils, hair infections, scalp infections, impetigo, hemorrhoids, canker sore, gingivitis, periodontitis, vaginitis, nose lesion, swelling, allergy, herpes zoster, cut, surgical incision, sunburn, cracked skin, bruises, and combinations thereof.

[0052] In one form of the invention, the infection is an acute bacterial skin and skin structure infection (ABSSSI) where the infection is related to one or more of the following conditions: cellulitis/erysipelas, wound infection, and major cutaneous abscess that have a minimum lesion surface area of approximately 75 cm². In one form of the invention, the infection is an acute bacterial skin and skin structure infection (ABSSSI) where the infection is related to one or more of the following conditions: cellulitis/erysipelas, wound infection, and major cutaneous abscess that have a minimum lesion surface area of 75 cm². [0053] In one form of the invention, the infection is a complicated skin and skin structure infection (cSSSI) where the infection involves deep subcutaneous tissues or needs surgery in addition to antimicrobial therapy.

[0054] In one form of the invention, the infection is a non-complicated or community acquired skin or skin structure infection.

[0055] The topical administration may comprise the administration of the therapeutically effective amount of a cannabinoid or a cannabinoid and chelating agent directly to a dermal or mucosal surface of the subject. Preferably, the cannabinoid or the cannabinoid and chelating agent are applied topically to the skin, mucosal membranes (oral, vaginal, rectal) or eye of the subject. The use may comprise administering a therapeutically effective amount of a cannabinoid and optionally a chelating agent, to the skin, mucosal membranes (oral, vaginal, rectal) or eye of a subject.

Biofilm disruption

[0056] It is believed that the composition of the present invention are able to disrupt or prevent the formation of biofilms.

[0057] Without being held to any theory, we believe the cannabinoids are capable of interfering with the biofilm forming activity of a biofilm-forming bacterium, thereby rendering it more susceptible to the antibacterial activity of the cannabinoid.

[0058] Further, and without being held to any theory, we believe the chelating agent, if present, is capable of interfering with the biofilm forming activity of a biofilm-forming bacterium, thereby rendering it more susceptible to the antibacterial activity of the cannabinoid.

[0059] The term“biofilm-forming bacterium” as used herein means a bacterium that forms a biofilm, where a biofilm is an aggregate of microorganisms in which cells are embedded in a self-produced matrix of extracellular polymeric substances that are adherent to each other, and/or a surface; and/or a microbially-derived, sessile community characterised by cells attached to a substratum, interface or to each other, and are embedded in a matrix of extracellular polymeric substances (EPS) that they have produced.

[0060] The compositions of the present invention biofilm may disrupt an already existing biofilm, or may reduce or prevent the formation of a biofilm.

[0061 ] When an existing biofilm is disrupted, the bacteria in the biofilm may be subject to one or more of the following effects:

killing of the bacteria within the biofilm;

reduction in growth of the bacteria within the biofilm; - a reduction in the adherence of the bacteria to the surface on which the biofilm has formed;

- a reduction in the rate of formation of the extracellular polymeric substance (EPS) matrix;

a reduction in the viscosity of the EPS matrix.

[0062] When inhibition of biofilm formation occurs, the bacteria in the biofilm may be subject to one or more of the following effects:

killing of the bacteria that would form the biofilm prior to or during biofilm formation; reduction in growth of the bacteria that would form the biofilm prior to or during biofilm formation;

a reduction in the adherence of the bacteria to the surface on which the biofilm will be formed;

- a reduction in the rate of formation of the extracellular polymeric substance (EPS) matrix during biofilm formation;

a reduction in the viscosity of the EPS matrix during biofilm formation.

[0063] Preferably, the compositions of the present invention cause an inhibition of biofilm growth wherein the OD590 demonstrates a ³70% growth inhibition compared to a growth control. An example of this measurement is provided in Example 1 of the present specification.

[0064] Protocols for measuring the above parameters, such as biofilm viability, biomass and EPS matrix formation, may be found in references such as Skogman et al. The Journal of Antibiotics (2012) 65, 453-459 and Merritt et al. Current Protocols in Microbiology 1 B.1 .1 - 1 B.1 .18, August 201 1.

[0065] There is therefore provided a method for the treatment or prevention of biofilm formation by a bacteria, comprising the step of:

administering a therapeutically effective amount of a composition comprising a cannabinoid.

[0066] There is further provided a method for the treatment or prevention of biofilm formation by a bacteria, comprising the step of:

administering a therapeutically effective amount of a composition comprising a cannabinoid and a chelating agent.

[0067] There is further provided a composition comprising a cannabinoid for the treatment or prevention of biofilm formation by a bacterium. [0068] There is further provided a composition comprising a cannabinoid for the treatment or prevention of biofilm formation by a bacterium.

[0069] There is further provided the use of a cannabinoid in the manufacture of a medicament for the treatment or prevention of biofilm formation by a bacterium in a subject.

[0070] There is further provided the use of a cannabinoid and a chelating agent in the manufacture of a medicament for the treatment or prevention of biofilm formation by a bacterium in a subject.

Bacterium

[0071 ] Preferably, the bacterium of any of the aspects of the present invention is a Gram positive bacterium.

[0072] In a preferred form of the invention, the bacterium is a bacterium species of a genus selected from the list: Streptococcus spp., Peptostreptococcus spp., Clostridium spp., Listeria spp., Bacillus spp., Staphylococcus spp., Propionibacterium spp., Kocuria spp., and Corynebacterium spp., and combinations thereof.

[0073] In a preferred form of the invention, the bacterium is a bacterium species of a genus selected from the following genus: Staphylococcus spp., Streptococcus spp., Bacillus spp., Kocuria spp., and Enterococcus spp..

[0074] In a preferred form of the invention, the bacterium is selected from the following species: Staphylococcus aureus (including MRSA), Staphylococcus warneri, Staphylococcus lugdunensis, Staphylococcus epidermidis, Staphylococcus pyogenes, Staphylococcus capitis, Streptococcus pneumoniae, Streptococcus pyogenes, Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Enterococcus faecium, Enterococcus faecalis, Corynebacterium jeikeium, Kocuria rosea, and Propionibacterium acnes.

[0075] In a preferred form of the invention, the bacterium is selected from the following species: Staphylococcus aureus (including MRSA), Staphylococcus warneri, Staphylococcus capitis, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Enterococcus faecium, Kocuria rosea, and Enterococcus faecalis.

[0076] More preferably the bacterium is a bacterium other than Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

[0077] In one form of the invention, the bacterium is MSRA. Chelating agent

[0078] If present, preferably the chelating agent is an iron chelating agent or a zinc chelating agent. More preferably, the chelating agent is a chelator of both iron and zinc (an iron/zinc chelator). Alternatively, the chelating agent may be a mixture of two or more chelating agents, for example a mixture of an iron chelating agent and a zinc chelating agent, or an iron/zinc chelating agent and a zinc chelating agent, or an iron chelating agent and an iron/zinc chelating agent.

[0079] The chelating agent is preferably selected from the group consisting of citric acid, phosphates, the di-, tri- and tetra-sodium salts of ethylene diamine tetraacetic acid (EDTA), the calcium salts of EDTA, copper EDTA, ethylene glycol-bis-(b-aminoethylether)-N,N,N',N'- tetraacetic acid (EGTA); 1 ,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA); ethylene-N,N'-diglycine (EDDA); 2,2'-(ethylendiimino)-dibutyric acid (EBDA); lauroyl EDTA; dilauroyl EDTA, triethylene tetramine dihydrochioride (TRIEN), diethylenetriamin-pentaacetic acid (DPT A), triethylenetetramine hexaacetic acid (TTG), deferoxamine (DFO), deferasirox (DSX), dimercaprol, zinc citrate, penicilamine, succimer, editronate, sodium hexmetaphosphate, edetate calcium disodium, D-penicillamine, polyphenols, gallol, catechol, dimercaprol, tetrathiomolybdate, lactoferrin, and clioquinol and combinations thereof. Other chelators for use in the present invention include nitrilotriacetic acid (NT A), trans-1 ,2-diaminocyclohexane- N,N,N',N'-tetraacetic acid monohydrate (CyDTA), N-(2-Hydroxyethyl)ethylenediamine-N,N',N'- triacetic acid (EDTA-OH), 0,0'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid (GEDTA), triethylenetetramine-N,N,N',N",N"',N"'-hexaacetic acid (TTHA), dihydroxyethylglycine (DHEG), iminodiacetic acid (IDA), diethylenetriaminepentaacetic acid (DTP A) and DTPA-OH, N- (2-hydroxyethyl)iminodiacetic acid (HIDA), and ethylene diamine tetra methylene phosphonic acid (EDTPO). The chelating agent may be a salt of the agents listed above.

[0080] Preferably, the chelating agent is a pharmaceutically acceptable chelating agent.

[0081 ] In one embodiment, the chelating agent is ethylene diamine tetraacetic acid (EDTA), or pharmaceutically acceptable salts thereof. In another embodiment, the chelating agent is deferoxamine (DFO), or pharmaceutically acceptable salts thereof. In another embodiment, the chelating agent is deferasirox (DSX), or pharmaceutically acceptable salts thereof.

[0082] Preferably, the invention provides a combination of a cannabinoid, chosen from the list comprising: cannabidiol, cannabinol, cannabigerol, cannabichromene, and D⁹- tetrahydrocannabinol, and a chelating agent. Most preferably, the cannabinoid is cannabidiol.

[0083] Preferably, the invention provides a combination of a cannabinoid, and an chelating agent selected from the group consisting of citric acid, phosphates, the di-, tri- and tetra-sodium salts of ethylene diamine tetraacetic acid (EDTA), the calcium salts of EDTA, copper EDTA, ethylene glycol-bis-(b-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA); 1 ,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA); ethylene-N,N'-diglycine (EDDA); 2,2'-(ethylendiimino)-dibutyric acid (EBDA); lauroyl EDTA; dilauroyl EDTA, triethylene tetramine dihydrochioride (TRIEN), diethylenetriamin-pentaacetic acid (DPTA), triethylenetetramine hexaacetic acid (TTG), deferoxamine (DFO), deferasirox (DSX), dimercaprol, zinc citrate, penicilamine, succimer, editronate, sodium hexmetaphosphate, edetate calcium disodium, D- penicillamine, polyphenols, gallol, catechol, dimercaprol, tetrathiomolybdate, lactoferrin, and clioquinol and combinations thereof. Other chelators for use in the present invention include nitrilotriacetic acid (NT A), trans-1 ,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid monohydrate (CyDTA), N-(2-Hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (EDTA-OH), 0,0'-bis(2- aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid (GEDTA), triethylenetetramine- N,N,N',N",N"',N'"-hexaacetic acid (TTHA), dihydroxyethylglycine (DHEG), iminodiacetic acid (IDA), diethylenetriaminepentaacetic acid (DTP A) and DTPA-OH, N-(2- hydroxyethyl)iminodiacetic acid (HIDA), and ethylene diamine tetra methylene phosphonic acid (EDTPO) and salts thereof. Most preferably, the chelating agent is chosen from ethylene diamine tetraacetic acid (EDTA), deferoxamine (DFO), deferasirox (DSX), or pharmaceutically acceptable salts thereof.

[0084] More preferably, the invention provides a combination of a cannabinoid, chosen from the list comprising: cannabidiol, cannabinol, cannabigerol, cannabichromene, and D⁹- tetrahydrocannabinol, and a chelating agent chosen from ethylene diamine tetraacetic acid (EDTA), deferoxamine (DFO), deferasirox (DSX), or pharmaceutically acceptable salts thereof. Most preferably the invention provides a combination of cannabidiol and ethylene diamine tetraacetic acid (EDTA).

Additional antimicrobials

[0085] Other active agents may also be incorporated into the composition of the present invention. For example, additional antimicrobial agents such as antibacterials, antifungals etc; lubricating agents; agents that reduce biofouling; may be incorporated.

[0086] For example, the composition may further comprise benzoyl peroxide, erythromycin, clindamycin, doxycycline or meclocycline.

[0087] Additional antimicrobial agents that can be used include, but are not limited to silver compounds (e.g., silver chloride, silver nitrate, silver oxide), silver ions, silver particles, iodine, povidone/iodine, chlorhexidine, 2-p-sulfanilyanilinoethanol, 4,4'-sulfinyldianiline, 4- sulfanilamidosalicylic acid, acediasulfone, acetosulfone, amikacin, amoxicillin, amphotericin B, ampicillin, apalcillin, apicycline, apramycin, arbekacin, aspoxicillin, azidamfenicol, azithromycin, aztreonam, bacitracin, bambermycin(s), biapenem, brodimoprim, butirosin, capreomycin, carbenicillin, carbomycin, carumonam, cefadroxil, cefamandole, cefatrizine, cefbuperazone, cefclidin, cefdinir, cefditoren, cefepime, cefetamet, cefixime, cefinenoxime, cefminox, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotetan, cefotiam, cefozopran, cefpimizole, cefpiramide, cefpirome, cefprozil, cefroxadine, ceftazidime, cefteram, ceftibuten, ceftriaxone, cefuzonam, cephalexin, cephaloglycin, cephalosporin C, cephradine, chloramphenicol, chlortetracycline, ciprofloxacin, clarithromycin, clinafloxacin, clindamycin, clomocycline, colistin, cyclacillin, dapsone, demeclocycline, diathymosulfone, dibekacin, dihydrostreptomycin, dirithromycin, doxycycline, enoxacin, enviomycin, epicillin, erythromycin, flomoxef, fortimicin(s), gentamicin(s), glucosulfone solasulfone, gramicidin S, gramicidin(s), grepafloxacin, guamecycline, hetacillin, imipenem, isepamicin, josamycin, kanamycin(s), leucomycin(s), lincomycin, lomefloxacin, lucensomycin, lymecycline, meclocycline, meropenem, methacycline, micronomicin, midecamycin(s), minocycline, moxalactam, mupirocin, nadifloxacin, natamycin, neomycin, netilmicin, norfloxacin, oleandomycin, oxytetracycline, p- sulfanilylbenzylamine, panipenem, paromomycin, pazufloxacin, penicillin N, pipacycline, pipemidic acid, polymyxin, primycin, quinacillin, ribostamycin, rifamide, rifampin, rifamycin SV, rifapentine, rifaximin, ristocetin, ritipenem, rokitamycin, rolitetracycline, rosaramycin, roxithromycin, salazosulfadimidine, sancycline, sisomicin, sparfloxacin, spectinomycin, spiramycin, streptomycin, succisulfone, sulfachrysoidine, sulfaloxic acid, sulfamidochrysoidine, sulfanilic acid, sulfoxone, teicoplanin, temafloxacin, temocillin, tetracycline, tetroxoprim, thiamphenicol, thiazolsulfone, thiostrepton, ticarcillin, tigemonam, tobramycin, tosufloxacin, trimethoprim, trospectomycin, trovafloxacin, tuberactinomycin, vancomycin, azaserine, candicidin(s), chlorphenesin, dermostatin(s), filipin, fungichromin, mepartricin, nystatin, oligomycin(s), ciproflaxacin, norfloxacin, ofloxacin, pefloxacin, enoxacin, rosoxacin, amifloxacin, fleroxacin, temafloaxcin, lomefloxacin, perimycin A or tubercidin, and the like.

Administration

[0088] In one embodiment, administration of the cannabinoid occurs 1 , 2, or 3 times a day.

[0089] The administration of combination products may be achieved by combining the cannabinoid and chelating agent into one stable formulation, or providing the cannabinoid and chelating agent in separate containers to be combined at the time of administration or alternatively by sequentially delivering the products.

[0090] If the administration is administration of the chelating agent and the cannabinoid, this administration may be done sequentially (i.e. one after the other), or the two compounds may be co-administered together. When the agents are co-administered, the agents can be mixed just prior to the administration or they can be admixed as one homogenous mixture in a self- contained preparation, provided the physical and chemical stability is maintained. The administration can occur 1 , 2, or 3 times a day. [0091 ] In a preferred embodiment, the chelating agent and the cannabinoid are admixed as one pharmaceutical formulation. One single pharmaceutical formulation and one single treatment provide ease of use and results in better compliance by subjects. They may be administered to subjects by topical administration.

Subject

[0092] The subject may be any subject capable of colonisation by bacteria.

[0093] The subject may be mammalian or avian. Preferably, the subject is selected from the group comprising human, canine, avian, porcine, bovine, ovine, equine, and feline. Most preferably, the subject is selected from the group comprising human, bovine, porcine, equine, feline and canine. Most preferably, the subject is human.

[0094] In an alternate form of the invention, the subject comprises a non-living surface. In one form of the invention, the subject is an implantable medical device. In one form of the invention, the subject is a surface, such as food preparation surface, or a surface used in the conduct of surgery. In one form of the invention, the subject is a bandage. In one form of the invention, the subject is a cast.

Dosing and Formulations

[0095] In one embodiment of the invention, the cannabinoid or the cannabinoid and chelating agent are administered to the subject using a dosing regimen selected from the group consisting of: three times daily; two times daily; daily; every second day, every third day, once weekly; once fortnightly and once monthly.

[0096] The pharmaceutical composition may optionally include a pharmaceutically acceptable excipient or carrier.

[0097] The composition of the invention may be selected from the group consisting of: an immediate release composition, a delayed release composition, a controlled release composition and a rapid release composition.

[0098] The composition of the invention may further comprise an anti-inflammatory agent (such as a corticosteroid). If the composition is a topical composition, an anticomedolyic agent (such as tretinoin), and/or a retinoid or derivative thereof may also be added.

[0099] The compositions described herein may be formulated by including such dosage forms in an oil-in-water emulsion, or a water-in-oil emulsion. In such a composition, the immediate release dosage form is in the continuous phase, and the delayed release dosage form is in a discontinuous phase. The composition may also be produced in a manner for delivery of three dosage forms as hereinabove described. For example, there may be provided an oil-in-water-in- oil emulsion, with oil being a continuous phase that contains the immediate release component, water dispersed in the oil containing a first delayed release dosage form, and oil dispersed in the water containing a third delayed release dosage form. Such compositions may be formulated for topical administration.

[00100] The compositions described herein may be in the form of a liquid composition. The liquid composition may comprise a solution that includes a therapeutic agent dissolved in a solvent. Generally, any solvent that has the desired effect may be used in which the therapeutic agent dissolves and which can be administered to a subject. Generally, any concentration of therapeutic agent that has the desired effect can be used. The composition in some variations is a solution which is unsaturated, a saturated or a supersaturated solution. The solvent may be a pure solvent or may be a mixture of liquid solvent components. In some variations the solution formed is an in-situ gelling composition. Solvents and types of solutions that may be used are well known to those versed in such drug delivery technologies.

[00101 ] The composition may or may not contain water. Preferably, the composition does not contain water, i.e. it is non-aqueous. In another preferred embodiment, the composition does not comprise a preservative.

[00102] The administration of the cannabinoids in accordance with the methods and compositions of the invention may be by any suitable means that results in an amount sufficient to treat a microbial infection or to reduce microbial growth at the location of infection. For example, the amount may be sufficient to treat a microbial infection on a subject's skin.

[00103] The cannabinoid may be contained in any appropriate amount and in any suitable carrier substance, and is generally present in an amount of 1 -95% by weight of the total weight of the composition.

[00104] The pharmaceutical or veterinary composition may be formulated according to the conventional pharmaceutical or veterinary practice (see, for example, Remington: The Science and Practice of Pharmacy, 20th edition, 2000, ed; A. R. Gennaro, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds; J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York; Remington's Pharmaceutical Sciences, 18^th Edition, Mack Publishing Company, Easton, Pennsylvania, USA).

[00105] Generally, examples of suitable carriers, excipients and diluents include, without limitation, water, saline, ethanol, dextrose, glycerol, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphates, alginate, tragacanth, gelatine, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, methyl cellulose, methyl and propylhydroxybenzoates, polysorbates, talc magnesium stearate, mineral oil or combinations thereof. The compositions can additionally include lubricating agents, pH buffering agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavouring agents.

[00106] The composition may be in the form of a controlled-release composition and may include a degradable or non-degradable polymer, hydrogel, organogel, or other physical construct that modifies the release of the cannabinoid. It is understood that such compositions may include additional inactive ingredients that are added to provide desirable colour, stability, buffering capacity, dispersion, or other known desirable features. Such compositions may further include liposomes, such as emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use in the invention may be formed from standard vesicle-forming lipids, generally including neutral and negatively charged phospholipids and a sterol, such as cholesterol.

[00107] Compositions of the invention may be administered topically. Therefore, contemplated for use herein are compositions adapted for the direct application to the skin. The composition may be in a form selected from the group comprising suspensions, emulsions, liquids, creams, oils, lotions, ointments, gels, hydrogels, pastes, plasters, roll-on liquids, skin patches, sprays, glass bead dressings, synthetic polymer dressings and solids. For instance, the compositions of the invention may be provided in the form of a water-based composition or ointment which is based on organic solvents such as oils. Alternatively, the compositions of the invention may be applied by way of a liquid spray comprising film forming components and at least a solvent in which the cannabinoids are dispersed or solubilised.

[00108] The composition of the invention may be provided in a form selected from the group comprising, but not limited to, a rinse, a shampoo, a lotion, a gel, a leave-on preparation, a wash-off preparation, and an ointment.

[00109] Various topical delivery systems may be appropriate for administering the compositions of the present invention depending up on the preferred treatment regimen. Topical compositions may be produced by dissolving or combining the cannabinoids of the present invention in an aqueous or non-aqueous carrier. In general, any liquid, cream, or gel or similar substance that does not appreciably react with the compound or any other of the active ingredients that may be introduced into the composition and which is non-irritating is suitable. Appropriate non-sprayable viscous, semi-solid or solid forms can also be employed that include a carrier compatible with topical application and have dynamic viscosity preferably greater than water.

[001 10] Suitable compositions are well known to those skilled in the art and include, but are not limited to, solutions, suspensions, emulsions, creams, gels, ointments, powders, liniments, salves, aerosols, transdermal patches, etc., which are, if desired, sterilised or mixed with auxiliary agents, e.g. preservatives, stabilisers, emulsifiers, wetting agents, fragrances, colouring agents, odour controllers, thickeners such as natural gums, etc. Particularly preferred topical compositions include ointments, creams or gels.

[001 1 1 ] Ointments generally are prepared using either (1 ) an oleaginous base, i.e., one consisting of fixed oils or hydrocarbons, such as white petroleum, mineral oil, or (2) an absorbent base, i.e., one consisting of an anhydrous substance or substances which can absorb water, for example anhydrous lanolin. Customarily, following formation of the base, whether oleaginous or absorbent, the cannabinoids are added to an amount affording the desired concentration.

[001 12] Creams are oil/water emulsions. They consist of an oil phase (internal phase), comprising typically fixed oils, hydrocarbons and the like, waxes, petroleum, mineral oil and the like and an aqueous phase (continuous phase), comprising water and any water-soluble substances, such as added salts. The two phases are stabilised by use of an emulsifying agent, for example, a surface active agent, such as sodium lauryl sulfite; hydrophilic colloids, such as acacia colloidal clays, veegum and the like. Upon formation of the emulsion, the cannabinoids can be added in an amount to achieve the desired concentration.

[001 13] Gels comprise a base selected from an oleaginous base, water, or an emulsion- suspension base. To the base is added a gelling agent that forms a matrix in the base, increasing its viscosity. Examples of gelling agents are hydroxypropyl cellulose, acrylic acid polymers and the like. Customarily, the cannabinoids are added to the composition at the desired concentration at a point preceding addition of the gelling agent.

[001 14] The amount of antibiotic compounds incorporated into a topical composition is not critical; the concentration should be within a range sufficient to permit ready application of the composition such that an effective amount of the cannabinoids is delivered.

Coating

[001 15] The present invention further provides an antimicrobial coating comprising a cannabinoid or a cannabinoid and a chelating agent.

[001 16] The antimicrobial coating of the present invention is particularly suited to application to medical devices that are likely to become contaminated or have become contaminated with microorganisms as a result of bacterial adhesion and proliferation.

[001 17] The present invention encompasses such medical devices coated with the antimicrobial coating of the present invention, and methods for preventing biofilm formation by inhibiting microbial growth and proliferation on the surface of medical devices.

[001 18] The coatings of the present invention may further comprise a polymeric material. [001 19] As medical devices such as stents, catheters, endotracheal tube and tracheostomy tubes are made in a variety of configurations and sizes, the exact dose administered will vary with device size, surface area and design. However, certain principles can be applied in the application of this art. Dose can be calculated as a function of dose per unit area (of the portion of the device being coated), total drug dose administered can be measured and appropriate surface concentrations of active drug can be determined.

[00120] The coated medical devices of the invention may be bioabsorbable and/or removable.

[00121 ] Examples of implantable or insertable medical devices include tracheostomy tubes, catheters, guide wires, balloons, filters, stents (including sinus stents, urethral and ureteral stents), stent grafts, vascular grafts, vascular patches, tympanostomy tubes, prosthetic sphincters (including bladder sphincters), and shunts. Among medical devices in accordance with the present invention are biliary, ureteral and pancreatic stents, stent covers, catheters, venous access devices and devices bridging or providing drainage between a sterile and non- sterile body environment or between two sterile body environments. The implantable or insertable medical device may be adapted for implantation or insertion into, for example, the coronary vasculature, peripheral vascular system, oesophagus, trachea, colon, biliary tract, urinary tract, prostate or brain.

[00122] The insertable medical devices can be formed from various materials, such as polymeric and/or metallic materials, and may be non-degradable or biodegradable.

[00123] Preferred substantially non-biodegradable biocompatible medical device materials include thermoplastic and elastomeric polymeric materials. Polyolefins such as metallocene catalyzed polyethylenes, polypropylenes, and polybutylenes and copolymers thereof; vinyl aromatic polymers such as polystyrene; vinyl aromatic copolymers such as styrene-isobutylene copolymers and butadiene-styrene copolymers; ethylenic copolymers such as ethylene vinyl acetate (EVA), ethylene-methacrylic acid and ethylene-acrylic acid copolymers where some of the acid groups have been neutralized with either zinc or sodium ions (commonly known as ionomers); polyacetals; chloropolymers such as polyvinylchloride (PVC); fluoropolymers such as polytetrafluoroethylene (PTFE); polyesters such as polyethyleneterephthalate (PET); polyester-ethers; polyamides such as nylon 6 and nylon 6,6; polyamide ethers; polyethers; elastomers such as elastomeric polyurethanes and polyurethane copolymers; silicones; polycarbonates; and mixtures and block or random copolymers of any of the foregoing are non-limiting examples of non-biodegradable biocompatible medical device materials useful for manufacturing the medical devices of the present invention. [00124] Among particularly preferred non-biodegradable medical device materials are polyolefins, ethylenic copolymers including ethylene vinyl acetate copolymers (EVA) and copolymers of ethylene with acrylic acid or methacrylic acid; elastomeric polyurethanes and polyurethane copolymers; metallocene catalyzed polyethylene (mPE), mPE copolymers, ionomers, and mixtures and copolymers thereof; and vinyl aromatic polymers and copolymers. Among preferred vinyl aromatic copolymers are included copolymers of polyisobutylene with polystyrene or polymethylstyrene, even more preferably polystyrene-polyisobutylene- polystyrene triblock copolymers. These polymers are described, for example, in U.S. Pat. Nos. 5,741 ,331 , 4,946,899 and U.S. Ser. No. 09/734,639, each of which is hereby incorporated by reference in its entirety. Ethylene vinyl acetate having a vinyl acetate content of from about 19% to about 28% is an especially preferred non-biodegradable material. EVA copolymers having a lower vinyl acetate content of from about 3% to about 15% are also useful in particular embodiments of the present invention as are EVA copolymers having a vinyl acetate content as high as about 40%. These relatively higher vinyl acetate content copolymers may be beneficial in offsetting stiffness from coating layers. Among preferred elastomeric polyurethanes are block and random copolymers that are polyether based, polyester based, polycarbonate based, aliphatic based, aromatic based and mixtures thereof. Commercially available polyurethane copolymers include, but are not limited to, Carbothane®, Tecoflex®, Tecothane®, Tecophilic®, Tecoplast®, Pellethane®, Chronothane® and Chronoflex®. Other preferred elastomers include polyester-ethers, polyamide-ethers and silicone.

[00125] Preferred biodegradable medical device materials include, but not limited to, polylactic acid, polyglycolic acid and copolymers and mixtures thereof such as poly(L-lactide) (PLLA), poly(D,L-lactide) (PLA); polyglycolic acid [polyglycolide (PGA)], poly(L-lactide-co-D,L- lactide) (PLLA/PLA), poly(L-lactide-co-glycolide) (PLLA/PGA), poly(D, L-lactide-co-glycolide) (PLA/PGA), poly(glycolide-co-trimethylene carbonate) (PGA/PTMC), poly(D,L-lactide-co- caprolactone) (PLA/PCL), poly(glycolide-co-caprolactone) (PGA/PCL); polyethylene oxide (PEO), polydioxanone (PDS), polypropylene fumarate, poly(ethyl glutamate-co-glutamic acid), poly(tert-butyloxy-carbonylmethyl glutamate), polycaprolactone (PCL), polycaprolactone co- butylacrylate, polyhydroxybutyrate (PHBT) and copolymers of polyhydroxybutyrate, poly(phosphazene), polyphosphate ester), poly(amino acid) and poly(hydroxy butyrate), polydepsipeptides, maleic anhydride copolymers, polyphosphazenes, polyiminocarbonates, poly[(97.5% dimethyl-trimethylene carbonate)-co-(2.5% trimethylene carbonate)], cyanoacrylate, polyethylene oxide, hydroxypropylmethylcellulose, polysaccharides such as hyaluronic acid, chitosan and regenerate cellulose, and proteins such as gelatine and collagen, and mixtures and copolymers thereof, among others.

[00126] As used herein, and unless otherwise specified, the terms "polymer" and "polymeric" refer to compounds that are the product of a polymerization reaction. These terms are inclusive of homopolymers (i.e., polymers obtained by polymerizing one type of monomer), copolymers (i.e., polymers obtained by polymerizing two or more different types of monomers), terpolymers, etc., including random, alternating, block, graft, dendritic, crosslinked, and any other variations of polymers. The terms are inclusive of a polymer blend of two or more polymers, for example, three, four, five, six, seven, eight, nine, and ten polymers. The polymers in the blend can be of various ratios. For example, in a two polymer blend, the amount of one polymer can vary from 0.5% to 99.5% by weight, and the other polymer can vary from 99.5% to 0.5% by weight.

[00127] Preferably, the cannabinoid or the cannabinoid and chelating agent are present in an amount effective to reduce or inhibit bacterial infection associated with the medical device.

[00128] The cannabinoid or the cannabinoid and chelating agent be coated onto the medical device by any suitable method, such as dipping or spraying. The cannabinoid or the cannabinoid and chelating agent may be applied as a paste or foam, optionally by painting the cannabinoid or the cannabinoid and chelating agent onto the medical device.

[00129] Alternately, where the medical device is formed from a polymeric material, the cannabinoid or the cannabinoid and chelating agent may be incorporated into the polymeric material of the device.

[00130] The medical device comprising or coated by a cannabinoid or the cannabinoid and chelating agent may further comprise another antibmicrobial agent. For example, additional antimicrobial agents such as antibacterials, antifungals etc; lubricating agents; agents that reduce biofouling; may be incorporated into the coating or medical device.

[00131 ] For example, the composition may further comprise benzoyl peroxide, erythromycin, clindamycin, doxycycline or meclocycline.

[00132] Additional antimicrobial agents that can be used include, but are not limited to silver compounds (e.g., silver chloride, silver nitrate, silver oxide), silver ions, silver particles, iodine, povidone/iodine, chlorhexidine, 2-p-sulfanilyanilinoethanol, 4,4'-sulfinyldianiline, 4- sulfanilamidosalicylic acid, acediasulfone, acetosulfone, amikacin, amoxicillin, amphotericin B, ampicillin, apalcillin, apicycline, apramycin, arbekacin, aspoxicillin, azidamfenicol, azithromycin, aztreonam, bacitracin, bambermycin(s), biapenem, brodimoprim, butirosin, capreomycin, carbenicillin, carbomycin, carumonam, cefadroxil, cefamandole, cefatrizine, cefbuperazone, cefclidin, cefdinir, cefditoren, cefepime, cefetamet, cefixime, cefinenoxime, cefminox, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotetan, cefotiam, cefozopran, cefpimizole, cefpiramide, cefpirome, cefprozil, cefroxadine, ceftazidime, cefteram, ceftibuten, ceftriaxone, cefuzonam, cephalexin, cephaloglycin, cephalosporin C, cephradine, chloramphenicol, chlortetracycline, ciprofloxacin, clarithromycin, clinafloxacin, clindamycin, clomocycline, colistin, cyclacillin, dapsone, demeclocycline, diathymosulfone, dibekacin, dihydrostreptomycin, dirithromycin, doxycycline, enoxacin, enviomycin, epicillin, erythromycin, flomoxef, fortimicin(s), gentamicin(s), glucosulfone solasulfone, gramicidin S, gramicidin(s), grepafloxacin, guamecycline, hetacillin, imipenem, isepamicin, josamycin, kanamycin(s), leucomycin(s), lincomycin, lomefloxacin, lucensomycin, lymecycline, meclocycline, meropenem, methacycline, micronomicin, midecamycin(s), minocycline, moxalactam, mupirocin, nadifloxacin, natamycin, neomycin, netilmicin, norfloxacin, oleandomycin, oxytetracycline, p- sulfanilylbenzylamine, panipenem, paromomycin, pazufloxacin, penicillin N, pipacycline, pipemidic acid, polymyxin, primycin, quinacillin, ribostamycin, rifamide, rifampin, rifamycin SV, rifapentine, rifaximin, ristocetin, ritipenem, rokitamycin, rolitetracycline, rosaramycin, roxithromycin, salazosulfadimidine, sancycline, sisomicin, sparfloxacin, spectinomycin, spiramycin, streptomycin, succisulfone, sulfachrysoidine, sulfaloxic acid, sulfamidochrysoidine, sulfanilic acid, sulfoxone, teicoplanin, temafloxacin, temocillin, tetracycline, tetroxoprim, thiamphenicol, thiazolsulfone, thiostrepton, ticarcillin, tigemonam, tobramycin, tosufloxacin, trimethoprim, trospectomycin, trovafloxacin, tuberactinomycin, vancomycin, azaserine, candicidin(s), chlorphenesin, dermostatin(s), filipin, fungichromin, mepartricin, nystatin, oligomycin(s), ciproflaxacin, norfloxacin, ofloxacin, pefloxacin, enoxacin, rosoxacin, amifloxacin, fleroxacin, temafloaxcin, lomefloxacin, perimycin A or tubercidin, and the like.

General

[00133] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variation and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

[00134] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention as described herein.

[00135] The entire disclosures of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are hereby incorporated by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness. No admission is made that any of the references constitute prior art or are part of the common general knowledge of those working in the field to which this invention relates. [00136] Throughout this specification, unless the context requires otherwise, the term antimicrobial is understood to include compounds with antibacterial properties.

[00137] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[00138] Suitable "pharmaceutically acceptable salts" include conventionally used non toxic salts, for example a salt with an inorganic base such as an alkali metal salt (such as sodium salt and potassium salt), an alkaline earth metal salt (such as calcium salt and magnesium salt), an ammonium salt; or a salt with an organic base, for example, an amine salt (such as methylamine salt, dimethylamine salt, cyclohexylamine salt, benzylamine salt, piperidine salt, ethylenediamine salt, ethanolamine salt, diethanolamine salt, triethanolamine salt, tris(hydroxymethylamino) ethane salt, monomethyl-monoethanolamine salt, procaine salt and caffeine salt), a basic amino acid salt (such as arginine salt and lysine salt), tetraalkyl ammonium salt and the like, or other salt forms that enable the pulmonary hypertension reducing agent to remain soluble in a liquid medium, or to be prepared and/or effectively administered in a liquid medium, preferable an aqueous medium. The above salts may be prepared by a conventional process, for example from the corresponding acid and base or by salt interchange.

[00139] Examples of suitable pharmaceutically acceptable salts include inorganic acid addition salts such as hydrochloride, hydrobromide, sulfate, phosphate, and nitrate; organic acid addition salts such as acetate, propionate, succinate, lactate, glycolate, malate, tartrate, citrate, maleate, fumarate, methansulfonate, p-toluenesulfonate, and ascorbate; salts with acidic amino acid such as aspartate and glutamate; alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; ammonium salt; organic basic salts such as trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, and N,N'-dibenzylethylenediamine salt; and salts with basic amino acid such as lysine salt and arginine salt. The salts may be in some cases hydrates or ethanol solvates.

[00140] Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs. BRIEF DESCRIPTION OF THE DRAWINGS

[00141 ] 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:

Figure 1 is a schematic diagram of the assay protocol;

Figure 2 is a schematic diagram of the growth plate layout;

Figure 3 is a schematic diagram of the treatment plate layout;

Figure 4 reports biofilm growth check (BGC) recoveries;

Figure 5 reports untreated control (UC) recoveries;

Figure 6 reports quantitative MBEC determination, wherein;

Figure 6A reports quantitative MBEC determination for EDTA alone against Staphylococcus aureus (ATCC #33592);

Figure 6B reports quantitative MBEC determination for CBD alone against Staphylococcus aureus (ATCC #33592);

Figure 6C reports quantitative MBEC determination for CBD and EDTA at a range of relative concentrations against Staphylococcus aureus (ATCC #33592);

Figure 6D reports quantitative MBEC determination for EDTA alone against Staphylococcus aureus (ATCC #BAA-44);

Figure 6E reports quantitative MBEC determination for CBD alone against Staphylococcus aureus (ATCC #BAA-44);

Figure 6F reports quantitative MBEC determination for CBD and EDTA at a range of relative concentrations against Staphylococcus aureus (ATCC #BAA-44);

Figure 7 reports Mean Log Reductions Presented per MBEC Checkerboard Panel of Staphylococcus aureus (ATCC #33592) for combinations of CBD and EDTA;

Figure 8 reports Mean Log Reductions Presented per MBEC Checkerboard Panel of Staphylococcus aureus (ATCC #BAA-44) for combinations of CBD and EDTA;

Figure 9 plots % bacterial killing at a range of concentrations for CBD alone compared to CBD and EDTA for Staphylococcus aureus (ATCC #33592);

Figure 10 plots % bacterial killing at a range of concentrations for CBD alone compared to CBD and EDTA for Staphylococcus aureus (ATCC #BAA-44); Figure 1 1 plots a time kill of S. aureus by CBD over 24 days;

Figure 12 plots the daily variability of time kill experiments of S. aureus by CBD over 24 days;

Figure 13 plots the development of resistance to CBD by S. aureus over 24 days;

Figure 14 plots the development of resistance to daptomycin by S. aureus over 24 days;

Figure 15 plots the MIC distribution of S. aureus strains after treatment with Vancomycin, Daptomycin, Mupirocin, Clindamycin and Cannabidiol;

Figure 16 plots the MIC distribution of S. aureus MRSA strains after treatment with Vancomycin, Daptomycin, Mupirocin, Clindamycin and Cannabidiol; and

Figure 17 plots the MIC distribution of S. aureus MSSA strains after treatment with Vancomycin, Daptomycin, Mupirocin, Clindamycin and Cannabidiol.

EXAMPLES

Example 1

Method

[00142] This study evaluated six concentrations of two test products,

Ethylenediaminetetraacetic acid (EDTA) and Cannabidiol (CDB), for their ability to disrupt two species of Methicillin Resistant Staphylococcus aureus (MRSA) biofilms and to evaluate the synergistic in-vitro interactions on the biofilm determined using a Minimum Inhibitory Concentration (MIC) with a modified Checkerboard Assay to determine the Fractional Inhibition Concentration (FIC) of the combination of the test products.

[00143] The assay design is represented by Figure 1 .

[00144] Test Product #1 (EDTA) will be tested using the following final concentrations of 12.5 mM, 6.25 mM, 3.125 mM, 1.562 mM, 0.781 mM, and 0.391 mM. Test Product #2 (CBD) will be tested using the following final concentrations of 6.25 pg/mL, 3.125 pg/mL 1 .562 pg/rmL, 0.781 pg/rmL, 0.391 pg/mL, and 0.195 pg/mL.

[00145] The challenge microorganism species (American Type Culture Collection [ATCC]) to be evaluated are designated below:

a. Staphylococcus aureus (ATCC #33592)

b. Staphylococcus aureus (ATCC #BAA-44)

Innoculum preparation:

[00146] 2 to 4 days prior to testing, innocula from lyophilized vials or cryogenic stock cultures containing each species will be suspended in 0.9% Sodium Chloride Irrigation, USP (SCI), inoculated onto the surface of Tryptic Soy Agar (TSA) contained in Petri plates, and incubated at 35 ± 2 °C for approximately 24 hours, or until sufficient growth is observed. Growth from the agar plates previously prepared will be suspended in SCI. The purity of each suspension will be verified by preparing isolation streaks of each culture on TSA. Aliquots will be spread-plated onto the surface of additional plates of TSA and incubated at 35 ± 2 °C until sufficient growth is observed. This will produce lawns of the bacteria on the surface of the agar plates, and growth from these will be used to prepare the inoculum suspension. Immediately prior to testing, an inoculum suspension will be prepared in TSB by suspending the microorganisms from the solid media to achieve initial suspension concentrations of approximately 10⁵ to 10⁶ CFU/mL. Innoculum population determination

[00147] The inoculum population will be determined by preparing 10-fold dilutions in PBS. Spread-plates will be prepared, in duplicate, from the inoculum dilutions by plating 0.1 mL of the final dilutions. These plates will be incubated at the temperature and under the conditions appropriate for 24 hours, or until sufficient growth is observed. Following incubation, the colonies on the plates will be counted manually using a hand-tally counter. Counts in the range of 30 to 300 colony-forming-units (CFU) will be used preferentially for the data calculations. If no counts in the range of 30 to 300 CFU are available, those plates with colony counts closest to that range will be used for the data calculations.

Testing procedures

[00148] The Test Materials (Test Product #1 and Test Product #2) will be diluted (v/v) using CAMHB to produce intermediate stock solutions for each test product.

[00149] A series of 1 :2 (v/v) dilutions will be prepared from the test material stock solutions using CAMHB to produce active concentrations: Test Product #1 (EDTA) will be tested using the following final concentrations of 12.5 mM, 6.25 mM, 3.125 mM, 1 .562 mM, 0.781 mM, and 0.391 mM. Test Product #2 (CBD) will be tested using the following final concentrations of 6.25 pg/mL, 3.125 pg/mL, 1 .562 pg/mL, 0.781 pg/mL, 0.391 pg/mL, and 0.195 pg/mL

[00150] Appropriate aliquots of each of the six test material dilutions prepared may be transferred to separate sterile test vials for use in treatment plate preparation. At the time of treatment plate preparation, each dilution of the test materials will be dispensed into separate sterile reservoirs for transfer to the treatment plate.

[00151 ] Checkerboard Microdilution Panels should include concentrations ranging from 4 to 8 times the expected MIC to at least 1/8 to 1/16 times the expected MIC in the final panel in order to observe the occurrence and magnitude of the synergism. Six concentrations of the test materials will be evaluated alone and in combination with one another to determine synergism.

Biofilm Growth Plate Preparation

[00152] The sterile packaging of the MBEC™ Assay Device Lids and 96-well plates will be opened and handled using aseptic technique. Aliquots of 150 pL prepared inoculum will be added to each well (excluding Column 1 1 and A12, B12, and C12) of the 96-well MBEC plate device. The peg lid will be placed onto the microtiter plate, ensuring aligned orientation. The device will be placed on an orbital shaker set to 1 10 ± 10 rpm in a humidified incubator at 35 ± 2°C and incubated for approximately 24 hours. The growth plate layout is illustrated in Figure 2. Biofilm growth check

[00153] Following the 24-hour biofilm growth plate incubation, the lid will be removed, and flame-sterilized bent needle-nose pliers will be used to break off three pegs D12 - H12. Each peg will be placed into a separate sterile microcentrifuge tube containing 1.0 ml. PBS and sonicated for 30 minutes in a stainless steel tray floated in the sonicating water bath. Following sonication, 0.1 ml. aliquots will be transferred from the microcentrifuge tubes into separate sterile tubes containing 0.9 mL PBS and mixed thoroughly using a vortex mixer. Appropriate

10-fold dilutions (e.g., 10°, 10 ¹ , 10 ², 10³, 10 ⁴, 10⁵, 10 ⁶, and 10 ^?) of the suspensions will be prepared in PBS, mixing thoroughly between dilutions. From the final dilutions, aliquots of 10 mI_ will be drop plated in rows on TSA. The plates will be incubated at 35 °C ± 2 °C for approximately 24 hours, or until sufficient growth is observed.

Treatment plate preparation

[00154] 200 mI_ aliquots of each Test Product #1 dilution series will dispensed into Row A test wells of the MBEC treatment plate, proceeding horizontally across the plate (See Figure 3). 200 mI_ aliquots of each Test Product #2 dilution series will dispensed into Column 1 test wells of the MBEC treatment plate, proceeding vertically down the plate (See Figure 3). 100 mI_ aliquots each of both prepared test dilution series will be dispensed into the test wells of the MBEC treatment plate (reference Figure 3) creating the final Checkerboard Panel. Each test well should contain 200 mI_ fluid volume. 200 mI_ aliquots of TSB will be dispensed into each well in Column 8 to create the untreated controls.

Biofilm sterility controls

[00155] Wells A12, B12, and C12 of the treatment plate will be used for the device, neutralizer, and diluent sterility controls. a. The recovery plate Well A12 will be filled with 200 mI_ CAMFIB.

b. The recovery plate Well B12 will be filled with 200 mI_ of BBP++ or other appropriate neutralizer.

c. The recovery plate Well C12 will be filled with 200 mI_ of PBS.

Biofilm treatment

[00156] Following incubation and Biofilm Growth Check procedures, the lid of the device will be removed from the growth plate and immersed in a rinse plate for 10 seconds to remove any unattached/planktonic bacteria. The device lid will then be transferred to the treatment plate (reference Figure 3) ensuring appropriate orientation. The device lid with the treatment plate will be placed on an orbital shaker set to 1 10 ± 10 rpm in a humidified incubator set at 35 ± 2°C and exposed to the treatment panel for approximately 24 hours.

Biofilm recovery

[00157] Following treatment incubation, the lid of the device will be removed from the treatment plate and immersed in a rinse plate for 10 seconds to remove any unattached/planktonic bacteria.

[00158] The device lid will then be transferred to the recovery plate.

a. Rinse Plate and Dilution Plate Preparation

i. Rinse plates will be prepared by filling wells of Columns 1 - 8 with 200 mI_ of Phosphate Buffered Saline (PBS).

ii. Separate 96-well dilution plates will be prepared by filling Rows B - H (Columns 1 - 8) with 180 mI_ of PBS.

b. Recovery Plate Preparation

i. A Recovery Plate will be prepared by filling Columns 1 - 9 with 200 mI_ of BBP++ or other appropriate neutralizer. Column 10 will be filled with 100 mI_ of BBP++ or other appropriate neutralizer and 100 mI_ of the highest concentration of the Test Material solutions (alone and in combination).

[00159] The recovery plate with the device lid in place will be sonicated for approximately 30 minutes in a stainless steel tray floated in the sonicating water bath. Following sonication, 100 mI_ aliquots from each treated well and untreated control wells (Columns 1 - 8) will be transferred from the recovery plate into Row 1 of separate prepared dilution plates. Appropriate 10-fold serial dilutions of the suspensions will be prepared by transferring 20 mI_ into 180 mI_ in PBS (Rows 2 - 8), mixing thoroughly between dilutions. From the final dilutions,

10 mI_ aliquots will be drop plated on TSA plates. The plates will be incubated at 35 °C ± 2 °C for approximately 24 hours, or until sufficient growth is observed. Following incubation, the colonies on the plates will be counted manually using a hand-tally counter. Counts in the range of 3 to 30 CFU per drop will be used preferentially in the data calculations. If no counts in the range of 3 to 30 CFU are observed, those dilutions with colony counts closest to that range will be used for the data calculations.

[00160] Following the Biofilm Recovery procedure, the pegged MBEC lid will be discarded. Aliquots of 100 mI_ TSB will be added to the recovery plate. The recovery plate will be covered with a new sterile, non-pegged lid and placed in an incubator at 35 °C ± 2 °C for approximately 24 hours. [00161 ] The sterility controls will be evaluated to determine possible contamination of the device, neutralizer or diluent as follows:

a. Device Sterility Control: The fluid in well A12 should remain clear after completion of the recovery plate incubation step. A cloudy or turbid well indicates device contamination and invalidates the results of the test. The test should be repeated with a new device from a new lot/batch. b. Neutralizer Sterility Control: The fluid in well B12 should remain clear after completion of the recovery plate incubation step. A cloudy or turbid well indicates neutralizer contamination and invalidates the results of the test. The test should be repeated with fresh neutralizer from a new lot/batch. c. PBS Sterility Control: The fluid in well C12 should remain clear after completion of the recovery plate incubation step. A cloudy or turbid well indicates contamination and invalidates the results of the test. The test should be repeated with fresh diluent from a new lot/batch

Qualitative neutralisation test

[00162] Neutralizer Effectiveness Test (50:N): The fluid in Column 10 (50:N) should be cloudy or turbid after completion of the recovery plate incubation step. If the Test Products/concentrations were successful in eradicating the biofilm, a clear well indicates that the neutralizer was not effective in neutralizing the test products and invalidates the results of the test. Neutralizer Toxicity Test (N): The fluid in Column 9 (N) should be cloudy or turbid after completion of the recovery plate incubation step. A clear well indicates that the neutralizer was toxic to the microorganism and invalidates the results of the test.

Determination of Qualitative MBEC Results

[00163] Following recovery plate incubation, the wells can be examined for growth of the challenge microorganism, as determined visually on the basis of turbidity (+/-). Alternatively, a microtiter plate read may be used to obtain optical density measurements at 650 nm (OD₆₅₀). Clear wells (OD₆₅₀ < 0.1 ) are evidence of biofilm eradication/inhibition. The Minimum Inhibitory Concentration (MIC) of the test materials alone and in combination versus the established challenge biofilm will be recorded as the highest dilution (lowest active concentration) that completely inhibits growth of the biofilm.

Calculations

[00164] The initial population (CFU/mL) of the challenge suspension will be calculated as follows: CFU/mL = (Ci x 10-D)

Where:

Ci = Average of the Plates Counted

D = Dilution Factor of the Plates Counted

[00165] Quantitative MBEC Determination

2

[00166] The Log₁₀ CFU/mm for each peg will be calculated as follows:

Log_i0 (CFU/mm²) = Log_i0 [(X/B) (V/A) (D)]

Where:

X = CFU counted in the drop;

B = volume plated (0.01 ml_);

V = well volume (0.2 ml_); and

D = dilution factor.

2

[00167] The Mean Log₁₀ CFU/mm density of each Test Material alone or in combination will be calculated across three replicates.

[00168] The Log₁₀ reduction for each Test Material alone or in combination will be calculated as follows:

Logio Reduction = Mean Logio UC - Mean Logio TP

Where:

UC = Untreated Control Pegs

TP = Treated Pegs

[00169] The Percent Reduction attributable to exposure to each Test Material alone or in combination will be calculated as follows:

Percent Reduction = 100 x [((UC (CFU/mm²) - TP (CFU/mm²)) / UC (CFU/mm²)]

Where:

UC = Untreated Control Pegs

TP = Treated Pegs

Results

[00170] A qualitative Neutralization Evaluation was performed concurrently with the MBEC determination to ensure that the neutralizing solution employed (Butterfield’s Phosphate Buffer solution with product neutralizers [BBP++]) was effective in neutralizing any antimicrobial properties of the test product and was non-toxic to the challenge strain. When challenged with the biofilm inoculum, BBP++ was demonstrated to be non-toxic to the inoculum and effectively neutralize any antimicrobial present. [00171 ] Figure 4 presents the Biofilm Growth Check (BGC) Recoveries (CFU/mm² and Log₁₀) of each of the five replicates evaluated for each test organism tested in three replicate experimental runs.

[00172] Figure 5 and Figures 6A-6F present the Untreated Control Recoveries and the

2

Treated Biofilm Recoveries (CFU/mm and Log₁₀) as well as the Log₁₀ and Percent Reductions produced by the test products when evaluated in three replicate experimental runs, each evaluating six test product concentrations, alone and in combination for each test microorganism.

[00173] Figure 7 and Figure 8 present Mean Log₁₀ Reductions per Checkerboard Panel for values produced by the test products when evaluated in three replicate experimental runs, each evaluating six test product concentrations, alone and in combination for each test microorganism.

Minimum Biofilm Eradication Concentration (MBEC) Results

[00174] When tested alone Test Product #1 Ethylenediaminetetraacetic acid (EDTA) and Test Product #2 Cannabidiol (CBD) both exhibit an overall dilutional response (i.e., stepwise Log₁₀ reductions proceeding with diminishing product concentrations) against both test species, Staphylococcus aureus (ATCC #33592) and Staphylococcus aureus (ATCC #BAA-44).

[00175] When tested alone, EDTA produced greater maximum Log₁₀ reductions when challenged with Staphylococcus aureus (ATCC #BAA-44) than when challenged with Staphylococcus aureus (ATCC #33592) (reference Figures 7 and 8).

[00176] When tested in combination, EDTA and CBD together exhibit Mean Log₁₀ reductions greater to or approximately equal to the Log₁₀ reductions achieved when CBD was evaluated alone.

[00177] When evaluating Mean Logio reductions achieved by CBD at the highest concentration tested (6.25 pg/mL) for both test species (reference Figures 7 and 8), a decrease in Log 10 reductions is not strictly observed with the decrease of concentration of EDTA, which indicates that the inclusion of EDTA does not considerably contribute to increase the efficacy of CBD if CBD is at its maximal concentration. This pattern suggests that when tested in combination, the resulting efficacy of the test products is possibly additive, specifically for concentrations of CBD that yield approximately 99.99% or greater Logio reductions alone.

[00178] Flowever, most importantly, when evaluating Mean Logio reductions of test product combinations including CBD concentrations below 1.56pg/mL, considerably larger reductions were observed with higher concentrations of EDTA (i.e., 12.5, 6.25, and 3.125 mM). This pattern is consistent with a synergistic effect below the approximate MIC value observed for CBD when the test products are evaluated in combination.

[00179] The enhanced effect produced by the chelating agent in respect of the two bacterial species is illustrated in Figures 9 and 10. As shown in these Figures, the chelating agent significantly improves the antimicrobial activity of cannabidiol, to achieve marked reductions in bacteria, starting at very low concentrations.

Example 2

[00180] 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

Compound preparation

[00181 ] The collaborator supplied sample as dry material. A stock solution at 10 mg/mL in neat DMSO (1 1 .2 mg in 1 .12 mL of DMSO) was prepared. The highest concentration tested in the assay was 128 pg/mL and 2% DMSO as a final concentration using 1 /20 dilution to achieve these concentrations.

Biofilm Formation

[00182] Bacteria ( Staphylococcus aureus, ATCC 43300; MRSA) was cultured on Tryptic Soy Broth (TSB, BD, Cat. No. 21 1825) at 37^eC overnight, then it was diluted 1 :100 in fresh TSB supplemented with 5% glucose. 100 pL were added across the 96-well of polystyrene (PS) (Corning; Cat. No. 3370) plate, leaving row FI 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)

[00183] 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.

[00184] 48 h after incubation, bacteria plates were carefully washed three times with 200 pL/well of saline solution (0.9% NaCI, Baxter Healthcare; Cat. No. AHF7124) using manual pipette to remove the planktonic cells but leave the biofilm adhered to the plate wells. Then, 100 pL 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. [00185] Next day, plates were washed three times with saline solution, then fixed with 100 pL/well of 99% methanol for 15 minutes. Once the biofilm was fixed, 100 pL/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 pL/well of methanol was added to allow for biofilm MIC analysis.

Biofilm MIC Detection and Analysis

[00186] 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.

[00187] 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 1 : Tested Compound

Table 2: Control Compounds

Results

[00188] The results display two biological replicates, with technical replicates (total n=4). Table 3: Broth MIC values

^* note clindamycin inactive vs this strain of MRSA

Table 4: Biofilm MIC values

[001 89] CBD was capable of inhibiting up to 75% of 48 h biofilm formation at 2 and 4 pg/mL. The cannabidiol biofilm MIC was approximately four-fold higher (1 -2 pg/mL) than its standard vegetative cell MIC (0.5-1 pg/mL) against the same strain of MRSA.

Example 3

Antibacterial Time kill assay Staphylococcus aureus MRSA

[00190] 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. [00191 ] The time-kill method is based on CLSI guideline M26-A (NCCLS, 1999).

Methods

Compound preparation

[00192] The collaborator supplied sample as dry material. A stock solution was prepared at 10 mg/ml_ in neat DMSO (1 1.2 mg in 1 .12 mL of DMSO). The highest concentration tested in the assay was 64 pg/mL and 2% DMSO as a final concentration using 1/20 dilution to achieve these concentrations.

Plate assay preparation

[00193] 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.

[00194] 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

[00195] The tested bacteria was Staphylococcus aureus ATCC 43300 MRSA (ID GP_020:02).

[00196] Charcoal plate PS 96-well plates: 50 pl_ of sterile activated charcoal suspension (25 mg/ml) were added into row A. 90 pl_ of 0.9% sterile saline were added to subsequent rows.

[00197] 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 5x10⁵ CFU/mL, and a final compound concentration range of 0.03 - 64 pg/mL. The plates were covered and incubated at 37 °C for 24 h.

[00198] At selected time-points (0, 1 , 2, 3, 4, 6 and 24 h), 50 mI_ 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 mI_ were transferred from row A to row B to give a 1 :10 dilution, this step was repeated until 1 :10O00 (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

[00199] 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 5: Tested Compound

Table 6: Control Compounds

Results

[00200] CBD time kill was tested two concentrations above and below previous MIC data (1 -2 pg/mL). CBD control MIC of the day was 2 pg/mL. Tested concentrations over or equal to the MIC value showed to be bactericidal after 3 hour treatment (Figure 1 1 ). Example 4

Forced Evolution of Resistant in Staphylococcus aureus MRSA

[00201 ] 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

[00202] The collaborator supplied sample as dry material. A stock solution at 10 mg/ml_ in neat DMSO was prepared.

Viability Testing

[00203] The tested bacteria was Staphylococcus aureus ATCC 43300 MRSA (ID GP_020:02).

[00204] 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.

[00205] CBD 320 pg/mL stock was diluted to 5, 4, 3, 2, 1.5, 1 , 0.75, 0.5, 0.375 and 0.25 pg/mL in Cation-adjusted Mueller Hinton Broth (CaMHB; BD, Cat. No. 212322) 100 pL 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 5x10⁵ CFU/mL The plate was covered and incubated at 37 °C for 20 h.

[00206] Note: CBD will have 8 replicates.

[00207] 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:

[00208] 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 pL of the bacteria diluted was added to the new MIC plate. The OD600 was used to calculate the dilution of cells to a density of 106 CFU/mL Bacteria were diluted in CaMHB and 100 mI_ was added to each well of the next replicate passage. The final well volume was 200 pl_ with a cell density of 5 ^c 105 CFU/mL. Each replicate (row) was assess as different strain, for this reason the dilution was done for each replicate.

[00209] 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:

[00210] Depending on the MIC of the day before, CBD tested concentrations were established.

[0021 1 ] 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 pg/mL stock in DMSO in CaMHB to achieve two-fold the desired testing concentrations. The 100 pL of the selected concentration were added to each well (See Figure 1). Once the plate had 100 pL of bacteria and 100 pL of compound. It was incubated at 37°C overnight. Next day the same procedure was repeated.

Drug free passages

[00212] 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.

[00213] 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 pg/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 1 1 was used as the drug-free passage well, and column 12 as a negative growth control with 200 pL uninoculated media in each well. Diluted bacteria were added to the plate, one replicate per row, 100 pL per well. The final well volume was 200 pL with a cell density of 5 x105 CFU/mL in columns 1 -1 1 , and CBD concentration range from 16 - 0.03 pg/mL in columns 1 -10 (Figure 2).

[00214] Subsequent drug-free passage plates were prepared in the same manner, except each replicate bacteria was passaged from column 1 1 , the drug-free growth control well.

Table 7: Tested Compound

Table 8: Control Compounds

Drug-free passaging control & QC plate

[00215] 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.

[00216] 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 mI_ of 2X the desired test concentration. Six wells were used as a positive growth controls, and six as negative growth controls with uninoculated media.

[00217] On day one, mid-log phase S. aureus was diluted in CaMHB to 106 CFU/mL, and 50 mI_ was added to each well (except negative growth control wells), to give a final volume of 100 mI_ and concentration of 5 ^c 105 CFU/mL.

[00218] 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 pL was added to each well of the next passage. The final well volume was 100 pL with a cell density of 5 ^c 105 CFU/mL. Results

[00219] Through the 20 days of assay, CBD generally showed a constant activity between 2 to 4 mg/mL across most of the replicates (Figure 13 and 14). However, replicate 1 had a drastic increase of activity from 3.5 pg/mL to >7 pg/mL at day 13 (the highest concentration tested that day), and the MIC exceeded the highest concentration tested on subsequent days (up to >128 pg/mL) by day 18 (Figure 12). This replicate is currently under 16S and purity studies to confirm that it is not a contaminant. The results for this replicate after day 7 have been excluded from Figure 13 and 14. During the course of the experiment technical difficulties on the 17th day meant the assay plates were stored at 4 °C for 24 h, with the assay then continued without disruption. There was also a consistent drop in measured MIC on Day 9 across all replicates to 1 pg/mL, with no obvious explanation.

[00220] 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 pg/mL) on Days 20 and 21 .

Example 5

Minimum Inhibitory Concentration in presence of 50% Human Serum

[00221 ] 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

[00222] 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

[00223] 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.

[00224] 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 5x10⁵ CFU/mL, and a final compound concentration range of 0.03 - 64 pg/mL. The plates were covered and incubated at 37 °C for 20 h.

MIC Detection and Analysis

[00225] The MIC was defined as the lowest concentration with which no growth was visible after incubation. MIC was determined by visual inspection only.

Table 9: Tested Compound

Table 10: Control Compounds

Table 11 : Tested Bacteria

Results

[00226] For bacteria, two technical duplicates.

Table 12: Summary of Results

[00227] 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).

[00228] 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 13: Summary of Results

^* from report 99962_002

Example 6

Minimum Inhibitory Concentration Assays MIC90 vs S. aureus

[00229] 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

[00230] 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 pg/mL. 5% DMSO was the final concentration using 1/10 dilution to achieve these concentrations.

Bacterial Minimum Inhibitory Concentration (MIC) Micro-broth Dilution Assay

[00231 ] 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 pL were stamped into polystyrene (PS) 96-well plates (Corning; Cat. No. 3370).

[00232] 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 5x10⁵ CFU/mL, and a final compound concentration range of 0.03 - 64 pg/mL. The plates were covered and incubated at 37 °C for 20 h.

Bacterial MIC Detection and Analysis

[00233] Optical density was read at 600 nm (OD600) using Tecan M1000 Pro Spectrophotometer. MIC was determined as the lowest concentration at which ³95% growth inhibition was observed. Dr Johannes Zuegg wrote script algorithms using Pipeline Pilot to automatically analyse the data set.

[00234] 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.

[00235] MIC 90 and 50 analysis was performed using Microsoft Excel.

Table 14: Tested Compound

Table 15: Control Compounds

Table 16: Tested Staphylococcus aureus strains

Results

[00236] Out of the 132 strains, 37 were resistance to clindamycin, resulting in an MIC50 of 0.125 pg/mL changing to an MIC90 of 64 m9/ihI_. 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 pg/mL across the 132 strains tested. The assay was performed in two different days in duplicate (total n=4). See Figures 15-17.

Table 17: Summary of results

Table 18: Staphylococcus aureus spp. MIC distribution (pg/rn L)

Table 19: Staphylococcus aureus MRSA MIC distribution

Table 20: Staphylococcus aureus MRSA MIC distribution

Example 7

Anaerobic Gram-positive Bacteria Minimum Inhibitory Concentration Assays

[00237] To assess the potential of Cannabidiol (CBD) for antimicrobial activity against common skin bacteria under anaerobic conditions.

Methods

Compound preparation

[00238] 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 pg/mL and 2% DMSO as a final concentration using 1/20 dilution to achieve these concentrations.

Minimum Inhibitory Concentration (MIC) Micro-broth Dilution Assay

[00239] 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 O2-H2 gas analyser, with H₂ levels kept at ~2% for the duration of the assay. Brain Heart Infusion broth (BHI; OXOID CM1 135B) 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.

[00240] 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.

[00241 ] 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 OD₆oo 0.5-0.7 and diluted down to a final cell density of 5x10⁵ CFU/mL, 100 pl_ were added to the test plate, giving a final CBD concentration range of 0.06 - 128 pg/mL. All the plates were covered and incubated at 37 °C for 48 h.

MIC Detection and Analysis

[00242] The MIC was defined as the lowest concentration at which no growth was visible after incubation. MIC was determined by visual inspection only. Table 21 : Tested Compound

Table 22: Control Compounds

Table 23: Test Organisms

Results

[00243] 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.

[00244] 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 24: Summary of Results

Example 8

Expanded Panel: Bacteria Minimum Inhibitory Concentration Assays

[00245] To assess the potential of Cannabidiol (CBD) for antimicrobial activity against a panel of Gram-positive bacteria.

Methods

Compound preparation

[00246] 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 pg/mL for bacteria and 128 pg/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

[00247] 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.

[00248] 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 5x10⁵ CFU/mL, and a final compound concentration range of 0.03 - 64 pg/mL. The plates were covered and incubated at 37 °C for 20 h. Bacterial MIC Detection and Analysis

[00249] Inhibition of bacterial growth was determined visually, where the MIC was recorded as the lowest compound concentration with no visible growth.

Table 25: Tested Compound

Table 26: Control Compounds

Table 27: Tested Organisms

Results

[00250] CBD was active against all Gram-positive strains in a range of 0.5 to 4 pg/mL, except for Staphylococcus epidermidis NDR 60 (GP_033) which was susceptible to CBD at 4 to 8 pg/mL.

[00251 ] Below 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.

Table 28: Summary of Results

Claims

1 . A composition comprising a cannabinoid for the treatment or prevention of an infection by a bacterium.

2. The composition of claim 1 further comprising a chelating agent.

3. The composition of claim 1 wherein the composition is a pharmaceutical composition.

4. The composition of claim 1 wherein the infection is a skin infection.

5. The composition of claim 1 wherein the bacterium is:

i. a Gram-positive bacterium;

ii. a bacterium species of a genus selected from the list: Streptococcus spp., Peptostreptococcus spp., Clostridium spp., Listeria spp., Bacillus spp., Staphylococcus spp., Propionibacterium spp., Kocuria spp., and Corynebacterium spp., and combinations thereof;

iii. a biofilm-forming bacterium; and/or

iv. resistant to at least one antibiotic.

6. The composition of claim 1 wherein the cannabinoid is cannabidiol.

7. The composition of claim 2 wherein the chelating agent is EDTA or a pharmaceutically acceptable salt thereof.

8. A method for the treatment or prevention of an infection by a bacterium in a subject in need of such treatment comprising the step of:

administering a therapeutically or preventative effective amount of a composition comprising a cannabinoid.

9. The method of claim 8 wherein the method further comprising:

i. co-administering a chelating agent; or

ii. sequentially administering a chelating agent.

10. The method of claim 8 wherein the infection is a skin infection.

1 1. The method of claim 8 wherein the bacterium is:

i. a Gram-positive bacterium;

ii. a bacterium species of a genus selected from the list: Streptococcus spp., Peptostreptococcus spp., Clostridium spp., Listeria spp., Bacillus spp., Staphylococcus spp., Propionibacterium spp., Kocuria spp., and Corynebacte um spp., and combinations thereof;

iii. a biofilm-forming bacterium; and/or

iv. resistant to at least one antibiotic.

12. The method of claim 8 wherein the cannabinoid is cannabidiol.

13. The method of claim 9 wherein the chelating agent is EDTA or a pharmaceutically acceptable salt thereof.

14. Use of a cannabinoid in the manufacture of medicament in the form of a composition for the treatment of an infection by a bacterium of a subject.

15. The use of claim 10 wherein the medicament further comprises a chelating agent.

16. The use of claim 10 wherein the infection is a skin infection.

17. The use of claim 10 wherein the bacterium is:

i. a Gram-positive bacterium; ii. a bacterium species of a genus selected from the list: Streptococcus spp., Peptostreptococcus spp., Clostridium spp., Listeria spp., Bacillus spp., Staphylococcus spp., Propionibacterium spp., Kocuria spp., and Corynebactehum spp., and combinations thereof;

iii. a biofilm-forming bacterium; and/or iv. resistant to at least one antibiotic.

18. The use of claim 10 wherein the cannabinoid is cannabidiol.

19. The use of claim 1 1 wherein the chelating agent is EDTA or a pharmaceutically acceptable salt thereof.