Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/658—Medicinal preparations containing organic active ingredients o-phenolic cannabinoids, e.g. cannabidiol, cannabigerolic acid, cannabichromene or tetrahydrocannabinol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/01—Hydrocarbons
  • A61K31/015—Hydrocarbons carbocyclic
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/348—Cannabaceae
  • A61K36/3482—Cannabis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the invention is in the field of medicinal preparations comprising a mixture of two or more organic antibiotically active ingredients, optionally including specific phenolic cannabinoids in optional combination with specific sesquiterpenes in optional combination with a lipopeptide antibiotic such as daptomycin.
• the therapeutic use of cannabinoid and/or sesquiterpene formulations is also disclosed, for synergistic treatment of enterococcal infections, including combined uses with lipopeptide antibiotics such as daptomycin.
• Enterococcus faecium and Enterococcus faecalis are bacterial species that asymptomatically colonize the gastrointestinal (Gl) tract in humans, but can also be pathogenic in certain circumstances (in the relevant literature, Enterococcus faecium was previously classified as Streptococcus faecium , see Schleifer & Kilpperbalz, 1984). In particular, these organisms are known to be a leading cause of dangerous infections in hospital patients being treated with antibiotics. During overgrowth, enterococci can penetrate the walls of the Gl tract, moving into the liver or bloodstream where they can cause peritonitis, or systemic infections (bacteremia).
• Enterococci have been reported to be the third leading cause of endocarditis, the complication of blood infections that has the greatest risk of mortality (Murdoch et al. , 2009).
• the intestinal colonization by enterococci particularly in hospital patients, may facilitate continual person-to-person spread by environmental contamination from fecal droplets, leading to opportunistic infection.
• Passive transmission of fecal matter to the urinary tract in catheterized patients has reportedly made enterococci the second most common cause of catheter associated urinary tract infections (UTIs) which can result in kidney damage (Hidron et al., 2008).
• UTIs catheter associated urinary tract infections
• faecium are associated with oral infections causing marginal periodontitis, root canal infections, primary endodontic infections, persistent/secondary infections, dental caries, peri-implantitis, periradicular abscesses and oral mucosal lesions (Najafi et al., 2019). E. faecium is also an important multidrug-resistant nosocomial pathogen causing biofilm-mediated infections in patients with medical devices (Paganelli et al., 2013).
• VRE As a nosocomial infection has been particularly problematic since it reportedly causes 1.8 fold higher mortality and results in an average of 5 days longer hospital stay according to a meta-analysis (Prematunge et al., 2016).
• cannabinoids found in cannabis plant extracts including: cannabidiol (CBD), its acid form cannabidiolic acid (CBDA), cannabichromene (CBC), its acid form cannabichromic acid (CBCA), cannabigerol (CBG), its acidic form cannabigerolic acid (CBGA), tetrahydrocannabinol (THC), and its acidic form, tetrahydrocannabinolic acid (THCA).
• Cannabis extracts or compounds derived from the Cannabis plant, have a very wide range of, often ill defined, anti-microbial activities (Van Klingeren & Ten Ham, 1976; Abdelaziz, 1982; Appendino et al., 201 1 ; Appendino et al., 2008; Eisohly et al., 1982; Eisohly et al., 1982; Appendino et al. , 2008; Turner & Eisohly, 1981 ; Mechoulam & Gaoni, 1965; WO2012/012498; WO2018/01 1813).
• Terpenes are another molecular constituent of plants, including Cannabis plants, that have been attributed with a wide range of physiological activities.
• the distinctive aroma and flavour that hops lends to beer reportedly comes in part from particular sesquiterpenes (including a-humulene and b-caryophyllene; see Steenackers et al., 2015).
• sesquiterpenes including a-humulene and b-caryophyllene; see Steenackers et al., 2015.
• the terpenes in Cannabis are reported to include well over 100 distinct compounds (Andre et al., 2016). It has been reported that aromatic terpenes may modulate the physiological effects of cannabinoids, particularly the psychoactive effects (Russo, 201 1 ).
• Figure 1 includes 12 line graphs, illustrating the effects of CBD, CBG or CBC on E. faecium viability.
• Enterococcus strains 33D3 (A-C), 69C6 (D-F), 58C9 (G- I) and 55A6 (J-L) were incubated with Ox, 1x (1 mg/L), 2x (2mg/L), or 4x (4mg/L) the MIC of CBD (A,D,G,J), CBG (B,E,H,K) or CBC (C,F,I,L).
• CFU's were determined at 2, 4, 6, 8, and 24h by serial dilution.
• Figure 3 includes 4 line graphs, illustrating the effect of various concentrations of CBD, CBG and CBC on bactericidal activity of daptomycin.
• E. faecium strain 33D3 was incubated with (A) no cannabinoid, or 1/2x (0.5mg/L) MIC of (B) CBD, (C) CBG) or (D) CBC with 1x (1 mg/L), 1/2x (0.5mg/L), 1/4x (0.25mg/L) or 1/8x (0.125mg/L) MIC of daptomycin. Aliquots were removed at 2, 4, 6, 8 and 24h plated to monitor viability (CFU). Data points are the means from three replicates with standard deviations presented as error bars.
• Figure 4 includes two bar graphs, illustrating the effect of CBD, CBG and CBC on biofilms of E. faecium strain 33D3.
• One general aspect of the innovations disclosed herein includes formulations that include at least two antibiotically active ingredients, for example selected from: a cannabinoid that is one or more of cannabichromene (CBC), cannabidiol (CBD) and/or cannabigerol (CBG).
• the formulations may also include a sesquiterpene that is one or both of a-humulene and/or b-caryophyllene.
• the formulations can also include a lipopeptide antibiotic that is daptomycin or an analogue thereof.
• the formulations may include the antibiotically active ingredients in synergistically effective relative amounts, for example where an effective amount of the formulation is synergistically effective in an assay to inhibit growth and/or reproduction of an Enterococcus faecium or an Enterococcus faecalis.
• Implementations may include one or more of the following features.
• the formulation where there are two antibiotically active ingredients, and the two antibiotically active ingredients are the cannabinoid and the sesquiterpene.
• the formulation where there are two antibiotically active ingredients, and the two antibiotically active ingredients are the cannabinoid and the lipopeptide antibiotic.
• the formulation including the cannabinoid, the sesquiterpene and the lipopeptide antibiotic.
• the formulation of any one where the cannabinoid is one of CBC, CBD or
• the formulation of any one where the sesquiterpene, if present, is one of a- humulene or b-caryophyllene.
• the formulation of any one where the sesquiterpene, if present, includes a-humulene and b-caryophyllene.
• the formulation of any one where the lipopeptide antibiotic, if present, is daptomycin.
• the formulation of any one where the sesquiterpene, when present, is present in a relative amount that provides at least a 2 to 128 fold decrease in the minimum inhibitory concentration (MIC) of the cannabinoid in the assay.
• MIC minimum inhibitory concentration
• the formulation of any one where the formulation does not include any alternative cannabinoids, terpenes or lipopeptide antibiotics.
• the formulation of any one where the formulation includes essentially of two or more of the cannabinoid, the terpene or the lipopeptide antibiotic. Use of the formulation of any one to formulate a medicament.
• the use according where the medicament is for use in treating an enterococcal infection in a subject in need thereof.
• the use according where the enterococcal infection is an Enterococcus faecium or an Enterococcus faecalis infection.
• the use according to any one where the enterococcal infection is an oral infection.
• the use according where the oral infection is a marginal periodontitis, a root canal infection, a primary endodontic infection, a persistent or secondary infection, dental caries, peri-implantitis, periradicular abscess or an oral mucosal lesion.
• the use according to any one where the enterococcal infection is an antibiotic resistant enterococcal infection.
• the use according where the antibiotic resistant enterococcal infection is a vancomycin and/or daptomycin resistant enterococcal infection.
• the use according to any one where the formulation is for use in an amount that delivers an effective dose of the cannabinoid, when present, of from 1 to 5,000 mg per day, and/or an effective dose of the sesquiterpene, when present, of from 1 to 10,000 mg per day.
• the formulation where the enterococcal infection is an Enterococcus faecium or an Enterococcus faecalis infection.
• the formulation where the enterococcal infection is an oral infection.
• the formulation where the oral infection is a marginal periodontitis, a root canal infection, a primary endodontic infection, a persistent or secondary infection, dental caries, peri-implantitis, periradicular abscess or an oral mucosal lesion.
• the formulation of any one where the enterococcal infection is an antibiotic resistant enterococcal infection.
• the formulation where the antibiotic resistant enterococcal infection is a vancomycin and/or daptomycin resistant enterococcal infection.
• Alternative aspects of the disclosed innovations include methods of treating an enterococcal infection in a subject in need thereof, including administering to the subject an effective amount of the formulations disclosed herein. Implementations may include one or more of the following features.
• the method including two antibiotically active ingredients, where the two antibiotically active ingredients are the cannabinoid and the sesquiterpene, further including treating the subject with an effective amount of one or more antibiotics, optionally where the antibiotic is daptomycin.
• the method including administering the cannabinoid in an effective amount of 1 -5,000 mg/day.
• the method of any one including administering the sesquiterpene in an effective amount of 1-10,000 mg/day.
• the method of any one where the subject is a mammal.
• the method where the mammal is a human.
• Alternative aspects of the present innovations include methods of treating a microbial infection in a subject in need thereof, including administering to the subject, sequentially or in combination, an effective amount of at least two antibiotically active compounds selected from: a cannabinoid that is one or more of cannabichromene (CBC), cannabidiol (CBD) and/or cannabigerol (CBG).
• CBC cannabichromene
• CBD cannabidiol
• CBG cannabigerol
• the method of treating also includes a sesquiterpene that is one or both of a-humulene and/or b-caryophyllene; and.
• the method of treating also includes a lipopeptide antibiotic that is daptomycin or an analogue thereof.
• the method of treating also includes where the antibiotically active compounds are administered in synergistically effective relative amounts effective to treat the microbial infection.
• Implementations of the disclosed methods may include one or more of the following features. The method where the synergistically effective relative amounts are synergistically effective to inhibit growth and/or reproduction of an Enterococcus faecium or an Enterococcus faecalis in an assay.
• the method of any one where the cannabinoid, if administered, is one of CBC, CBD or CBG.
• the method of any one where the cannabinoid, if administered, includes CBC, CBD and CBG.
• the method of any one where the sesquiterpene, if administered, is one of a-humulene or b-caryophyllene.
• the method of any one where the sesquiterpene, if administered, includes a-humulene and b-caryophyllene.
• the method where the enterococcal infection is an oral infection.
• the method where the oral infection is a marginal periodontitis, a root canal infection, a primary endodontic infection, a persistent or secondary infection, dental caries, peri-implantitis, periradicular abscess or an oral mucosal lesion.
• the method of any one where the microbial infection is an antibiotic resistant infection.
• the method where the antibiotic resistant infection is a vancomycin and/or daptomycin resistant microbial infection.
• methods for treating a bacterial infection in a subject in need thereof, comprising administering to the subject an effective amount of: a cannabinoid that is one or more of cannabichromene (CBC), cannabidiol (CBD) and/or cannabigerol (CBG); and, a lipopeptide antibiotic that is daptomycin or an analogue thereof; wherein the cannabinoid and the lipopeptide antibiotic are administered in an antibiotically effective weight ratio of from 16:1 to 1 :16; and, wherein: if the cannabinoid is CBD, then the lipopeptide antibiotic is administered in an effective amount that is less than 4 mg/kg; and/or, if the cannabinoid is CBD, then the bacterial infection comprises infection by an infectious organism having a daptomycin MIC of 4 pg/mL or greater; and/or, if the cannabinoid is CBD, then the bacterial infection comprises infection by an infectious organism for which results from
• the infection may for example include an infection by an Enterococcus faecium or an Enterococcus faecalis.
• These treatments may further include administering to the subject an effective amount of a sesquiterpene that is one or both of a-humulene and/or b-caryophyllene.
• antibiotic formulations include: a cannabinoid that is one or more of cannabichromene (CBC) and/or cannabigerol (CBG); and, a lipopeptide antibiotic that is daptomycin or an analogue thereof;
• CBC cannabichromene
• CBG cannabigerol
• the cannabinoid may for example be present in an amount that reduces the minimum inhibitory
• the cannabinoid reduces the MIC of the lipopeptide antibiotic in the assay when the cannabinoid is present in an amount that is less than the MIC of the cannabinoid.
• antibiotic assay such as an Enterococcal assay that assesses the inhibition of growth and/or reproduction of an Enterococcus faecium or an Enterococcus faecalis.
• the cannabinoid reduces the MIC of the lipopeptide antibiotic in the assay when the cannabinoid is present in an amount that is less than the MIC of the cannabinoid.
• These formulations may further include a sesquiterpene, such as one or both of a- humulene and/or b-caryophyllene.
• antibiotically active ingredients selected from: a cannabinoid that is one or more of cannabichromene (CBC), cannabidiol (CBD) and/or cannabigerol (CBG); a sesquiterpene that is one or both of a-humulene and/or b-caryophyllene; and, a lipopeptide antibiotic that is daptomycin or an analogue thereof.
• the antibiotically effective ingredients may be provided in synergistically effective relative amounts.
• the cannabinoid and the sesquiterpene may be provided at concentrations that are only antibiotically active in synergistic combinations, such as ⁇ 1 pg/ml cannabinoid and ⁇ 32 pg/ml terpene.
• the inhibitory concentrations of the partners may for example decrease, for example by two or more fold, for example from 2-16 fold.
• the relative weight ratio of cannabinoid to sesquiterpene may for example be from about 1 :5 to 1 :50, or 1 :8 to 1 :32, or from 1 :12 to 1 :32.
• the relative concentration of the antibiotically active ingredients in the formulation may be arranged so that an effective amount of the formulation would be synergistically effective in an assay to inhibit growth and/or reproduction of an Enterococcus faecium or an Enterococcus faecalis.
• a formulation that provides this synergistic formulation may be used for treating enterococcal or other microbial infections, for example for treating infections of enterococcal species that are relatively closely related to a reference strain of Enterococcus faecium or Enterococcus faecalis (see Zhong et al. , 2017).
• enterococcal species amenable to treatment may be at least as closely related to the reference strain as are the most distantly related strains of E. faecium and E. faecalis.
• the microorganisms amenable to treatment may for example be resistant to antibiotics, for example VRE strains or strains resistant to daptomycin.
• Enterococcus durans is for example amenable to treatment, for example in the context of veterinary diseases.
• Other enterococcal species closely related to E. faecium or E. faecalis may for example include E. mundtii, E. durans, E. hirae, E. ratti, E. villorum, E.
• synergies and/or potentiation effects are maximized using concentrations of antibiotically active components that are below the MICs for each component, for example just below the MICs.
• the components may accordingly be present in relative amounts that approximate the ratio of the respective MICs for the components. For example, this may occur when the molar ratio terpene/cannabinoid is > 50 (reflecting the MIC ratio of the components).
• the molar ratio of cannabinoid to sesquiterpene may for example be between 1 :100 and 50:1.
• synergistic combinations may have a molar ratio of cannabinoid to sesquiterpene between 1 :8 and 1 :32.
• a pharmaceutically acceptable excipient may optionally be included in the formulation, and the cannabinoid and sesquiterpene may be dissolved, dispersed, mixed or suspended in the formulation.
• One method of formulation may involve producing a sterile lyophilized powder, for example in a vial for reconstitution.
• the cannabinoid and sesquiterpene may for example be obtained from a plant extract, such as an extract of Cannabis sativa or Cannabis indica.
• a plant extract such as an extract of Cannabis sativa or Cannabis indica.
• Biosynthetic approaches to the production of cannabinoids and sesquiterpenes are also available, as are a variety of synthetic approaches (based for example on approaches used to synthesize THC/dronabinol, see US Patent No. 7,323,576 and Trost and Dogra, 2007).
• Alternative approaches involve expressing cannabinoid biosynthetic genes in recombinant hosts, such as recombinant yeast (see Luo et al., 2019).
• a daptomycin analogue is a lipopeptide antibiotic that has an antibiotic activity that synergizes with one or more of the cannabinoids and/or sesquiterpenes in the formulations disclosed herein, for example in assay of antibiotic activity against enterococcal species.
• Daptomycin analogues or derivatives may for example differ from daptomycin by virtue of the substitution of one or more amino acids therein, such as by conservative substitutions, for example substitutions of 1 , 2, 3, 4 or 5 amino acids therein, for example substitutions with natural or non natural amino acids.
• Daptomycin analogues may for example include cyclic lipodepsipeptide daptomycin, or derivatives thereof in which the fatty acid chain attached to the Trp 1 amino group is an anfe/so-undecanoyl, iso- dodecanoyl or anteiso- tridecanoyl group.
• One or more additional compounds may be included, or specifically excluded, in alternative formulations, including for example: terpenes, terpenoids, sterols, triglycerides, alkanes, squalene, tocopherol, carotenoids, chlorophyll, flavonoid glycosides, or alkaloids.
• Anti-microbial formulations may be used to prophylactically or therapeutically treat microbial infections, or otherwise inhibit microbial growth or multiplication.
• An antibiotic is an antimicrobial that is active against bacteria, and in this context includes naturally-occurring and synthetic substances that kill or inhibit the growth or multiplication of bacteria by any mechanism, including antiseptic or disinfectant modalities.
• Subjects amenable to treatment include mammalian subjects, such as human patients, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows, sheep, goats, pigs, horses, fowl), or household pets (e.g., dogs, cats, rodents, birds), for example belonging to the taxonomic groups of primates, canines, felines, bovines, caprines, equines, ovines, porcines, rodents, Aves or lagomorphs.
• Human patients to be treated may for example be male or female, or at a specific stage of development: neonate, infant, juvenile, adolescent, adult and geriatric.
• Specific veterinary indications amenable to treatment may for example include enterococcal infections in poultry, for example treatment of Enterococcus cecorum infections in chickens.
• formulation may be administered in a dosage or dosage form that delivers and/or sustains synergistically effective amounts of the active ingredients.
• the cannabinoid and/or sesquiterpene components of the formulation may for example be in the form of a plant extract, or be derived from a plant extract, or be obtained from a culture, such as a culture of a recombinant host, such as a recombinant yeast expressing the components.
• Humulene and b-caryophyllene are isomers, often found together in a variety of plants.
• a pharmaceutically acceptable excipient may be included, and the formulation may be provided in a titratable dosage form.
• the cannabinoid components of the formulation may be obtained as an extract from a plant of the Cannabis genus, for example Cannabis sativa or Cannabis indica , or from a culture of recombinant yeast hosts.
• a wide variety of methods may be used to prepare these plant extracts, including, but not limited to, supercritical or subcritical extraction with CO2, extraction with hot gas, and extraction with solvents.
• Formulations may also specifically exclude additional terpenoids or terpenes, including plant-derived terpenoids or terpenes, such as astaxanthin or other sesquiterpenes, tetraterpenes, triterpenes, diterpenes or monoterpenes.
• a titratable dosage may for example be adapted to allow a patient to take the medication in doses smaller than the unit dose, wherein a "unit dose" is defined as the maximum dose of medication that can be taken at any one time or within a specific dosage period. Titration of doses will allow different patients to incrementally increase the dose until they feel that the medication is efficacious, as not all patients will require the same dose to achieve the same benefits. A person with a larger build or faster metabolism may require larger doses to achieve the same effect as another with a smaller build or slower metabolism. Therefore, a titratable dosage has advantages over a standard dosage form.
• formulations may be adapted to be delivered in such a way as to target one or more of the following: sublingual, buccal, oral, rectal, nasal, parenteral and via the pulmonary system.
• Formulations may for example be in one or more of the following forms: gel, gel spray, tablet, liquid, capsule, by injection, or for vaporization.
• Routes of administration may for example include, parenteral, intravenous, intradermal, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracisternal, intraperitoneal, intranasal, inhalational, aerosol, topical, sublingual or oral administration.
• Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; for intranasal formulations, in the form of powders, nasal drops, or aerosols; and for sublingual formulations, in the form of drops, aerosols or tablets.
• Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
• Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
• Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
• compositions of the present invention may be in any form which allows for the composition to be administered to a patient.
• the composition may be in the form of a solid, liquid or gas (aerosol).
• Pharmaceutical composition of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient.
• Compositions that will be administered to a patient may take the form of one or more dosage units, where for example, a tablet, capsule or cachet may be a single dosage unit, and a container of the compound in aerosol form may hold a plurality of dosage units.
• compositions should be pharmaceutically pure and non-toxic in the amounts used.
• inventive compositions may include one or more compounds (active ingredients) known for a particularly desirable effect. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of subject (e.g., human), the particular form of the active ingredient, the manner of administration and the composition employed.
• the pharmaceutical composition includes a formulation of the present invention as described herein, in admixture with one or more carriers.
• the carrier(s) may be particulate, so that the compositions are, for example, in tablet or powder form.
• the carrier(s) may be liquid, with the compositions being, for example, an oral syrup or injectable liquid.
• the carrier(s) may be gaseous, so as to provide an aerosol composition useful in, e.g., inhalatory administration.
• composition When intended for oral administration, the composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
• the composition may be formulated into a powder, granule, compressed tablet, pill, capsule, cachet, chewing gum, wafer, lozenges, or the like form.
• a solid composition will typically contain one or more inert diluents or edible carriers.
• binders such as syrups, acacia, sorbitol, polyvinylpyrrolidone, carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin, and mixtures thereof; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; fillers such as lactose, mannitols, starch, calcium phosphate, sorbitol, methylcellulose, and mixtures thereof; lubricants such as magnesium stearate, high molecular weight polymers such as polyethylene glycol, high molecular weight fatty acids such as stearic acid, silica, wetting agents such as sodium lauryl sulfate, glidants such as colloidal silicon dioxide; sweeten
• the formulation may be in the form of a liquid, e.g., an elixir, syrup, solution, aqueous or oily emulsion or suspension, or even dry powders which may be reconstituted with water and/or other liquid media prior to use.
• the liquid may be for oral administration or for delivery by injection, as two examples.
• preferred compositions contain, in addition to the present compounds, one or more of a sweetening agent, thickening agent, preservative (e.g., alkyl p-hydoxybenzoate), dye/colorant and flavor enhancer (flavorant).
• a surfactant e.g., alkyl p-hydroxybenzoate
• wetting agent e.g., water, or other sugar syrups
• dispersing agent e.g., sorbitol, glucose, or other sugar syrups
• suspending agent e.g., sorbitol, glucose, or other sugar syrups
• buffer e.g., buffer, stabilizer and isotonic agent
• the emulsifying agent may be selected from lecithin or sorbitol monooleate.
• the liquid pharmaceutical formulations of the invention may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
• Physiological saline is a
• the pharmaceutical formulation may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment, cream or gel base.
• the base for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.
• Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device.
• the formulation may be intended for rectal administration, in the form, e.g., of a suppository which will melt in the rectum and release the drug.
• the composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient.
• bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
• Low-melting waxes are preferred for the preparation of a suppository, where mixtures of fatty acid glycerides and/or cocoa butter are suitable waxes. The waxes may be melted, and the aminocyclohexyl ether compound is dispersed homogeneously therein by stirring.
• the formulation may include various materials which modify the physical form of a solid or liquid dosage unit.
• the composition may include materials that form a coating shell around the active ingredients.
• the materials which form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.
• the active ingredients may be encased in a gelatin capsule or cachet.
• the pharmaceutical formulation may consist of gaseous dosage units, e.g., it may be in the form of an aerosol.
• aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system which dispenses the active ingredients. Aerosols of compounds of the invention may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit.
• Some biologically active compounds may be in the form of the free base or in the form of a pharmaceutically acceptable salt such as the hydrochloride, sulfate, phosphate, citrate, fumarate, methanesulfonate, acetate, tartrate, maleate, lactate, mandelate, salicylate, succinate and other salts known in the art.
• a pharmaceutically acceptable salt such as the hydrochloride, sulfate, phosphate, citrate, fumarate, methanesulfonate, acetate, tartrate, maleate, lactate, mandelate, salicylate, succinate and other salts known in the art.
• the appropriate salt would be chosen to enhance bioavailability or stability of the compound for the appropriate mode of employment (e.g., oral or parenteral routes of administration).
• kits that contain a pharmaceutical formulation, together with instructions for the use of the formulation.
• a commercial package will contain one or more unit doses of the formulation.
• Formulations which are light and/or air sensitive may require special packaging and/or formulation.
• packaging may be used which is opaque to light, and/or sealed from contact with ambient air, and/or formulated with suitable coatings or excipients.
• the formulations of the invention can be provided alone or in combination with other compounds (for example, small molecules, nucleic acid molecules, peptides, or peptide analogues), in the presence of a carrier or any pharmaceutically or biologically acceptable carrier.
• pharmaceutically acceptable carrier or“excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
• the carrier can be suitable for any appropriate form of administration.
• Pharmaceutically acceptable carriers generally include sterile aqueous solutions or dispersions and sterile powders. Supplementary active compounds can also be incorporated into the formulations.
• An“effective amount” of a formulation according to the invention includes a therapeutically effective amount or a prophylactically effective amount.
• a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
• a therapeutically effective amount of a formulation may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response.
• a therapeutically effective amount may also be one in which any toxic or detrimental effects of the formulation or active compound are outweighed by the therapeutically beneficial effects.
• a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
• a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.
• the timing and dose of treatments may be adjusted over time (e.g timing may be daily, every other day, weekly, monthly) according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
• synergy between active ingredients occurs when an observed combined therapeutic effect is greater than the sum of therapeutic effects of individual active ingredients, or a new therapeutic effect is produced that the active ingredients could not produce alone. Accordingly, when components of a formulation are present in synergistically effective amounts, the formulation yields a therapeutic effect that is greater than would be achieved by the individual active ingredients administered alone at comparable dosages.
• the enhancement of therapeutic effect may take the form of increased efficacy or potency and/or decreased adverse effects.
• the synergistic effect may be mediated in whole or in part by the pharmacokinetics and/or pharmacodynamics of the active ingredients in a subject, so that the amount and proportion of the ingredients in the formulation may be synergistic in vivo.
• This in vivo synergy may be effected with a formulation that includes the active ingredients in amounts and proportions that are also synergistic in in vitro assays of efficacy.
• the term“synergistically effective amounts” accordingly refers to amounts that are synergistic in vivo and/or in vitro.
• a numeric quantification of synergy is often expressed as a fractional inhibitory concentration index (FICI), which represents the sum of the fractional inhibitory concentrations (FICs) of each drug tested, where the FIC is determined for each drug by dividing the minimum inhibitory concentration (MIC, the lowest concentration of the drug which prevents visible growth of the bacterium in a standard in vitro assay - standard colorometric assay based on resazurin) of each drug when used in combination by the MIC of each drug when used alone.
• FICI fractional inhibitory concentration index
• FICI fractional inhibitory concentration index
• synergy of two compounds may be conservatively defined as a FICI of ⁇ 0.5 (see Odds, 2003; with additivity or potentiation corresponding to a FICI of >0.5 to ⁇ 0.75; no interaction (indifference) corresponding to a FICI of >1 to ⁇ 4; and antagonism corresponding to a FICI of >>4).
• Synergy of three compounds has been defined as a FICI of ⁇ 1.0. (Berenbaum, 1978; Yu et al., 1980). To estimate the optimal concentrations in triple combinations, the optimal concentrations of trans- caryophyllene and cannabinoid were utilized in combination with a series of daptomycin dilutions.
• the non-psychotropic cannabinoids cannabidiol, cannabigerol and cannabichromene (i.e. , CBD, CBG, and CBC) have bacteriostatic (Tables 1 , 2) and bactericidal ( Figure 1 ) activities against E. faecium and E. faecalis , including strains that are sensitive or resistant to vancomycin and, in some cases, also resistant to daptomycin.
• the kinetics of E. faecium killing with CBG and CBC are faster than for CBD ( Figure 1 ).
• cannabinoids can also increase the activity of daptomycin (MIC) by 8-128 fold (Table 3).
• each cannabinoid has species specific activities against Gram positive (but not Gram negative) bacteria (Table 1 ).
• the non-psychotropic cannabinoids, cannabidiol, cannabigerol and cannabichromene i.e. , CBD, CBG, and CBC
• CBD, CBG, and CBC cannabigerol and cannabichromene
• trans- caryophyllene i.e. , cannabinoids, CBG, and CBC
• daptomycin act in synergy.
• Assays for interactions between all pairwise as well as triple combinations of cannabinoids (CBC, CBD, CBG), trans caryophyllene, and daptomycin are shown in Table 4. All pairwise combinations showed synergistic (FICI ⁇ 0.5) or potentiating (FICI > 0.5, ⁇ 0.75) interactions.
• Pre-cultures of bacteria growing in their corresponding liquid media were diluted to an OD600 of 0.0025 and 100 pi was added to 100 pi of medium containing serial 2-fold dilutions of cannabinoid (cannabinoid stocks, in methanol or acetonitrile for acidic forms, were diluted in growth medium) in 96-well plates. Plates were then incubated for 20-24 h at 37°C, and growth was recorded as OD600 in a Thermo ScientificTM VarioskanTM Flash Multimode Reader.
• Pre-cultures growing in MHII medium with 0.05% tyloxapol were diluted to an OD600 of 0.005 in MHII medium, and 100 pi was added to 100 mI of MHII medium containing serial 2-fold dilutions of cannabinoid (cannabinoid stocks in methanol or acetonitrile for acidic forms were diluted in growth medium) in 96-well plates. Plates were then incubated for 48 h, followed by the addition of 30 mI the colorimetric reagent resazurin in water (10 mg/100 ml). Plates were incubated for an additional 24 h, and growth was recorded as conversion of culture color from blue to pink.
• Pre-cultures of M. tuberculosis growing in 7H9 medium plus 0.05% tyloxapol were grown to mid-log phase and diluted to OD600 0.0025 in the same medium without tyloxapol.
• 50 mI_ of diluted culture were used to inoculate wells in 96-well plates containing 50 mI_ of the same medium with serial 2-fold dilutions of cannabinoid (cannabinoid stocks in methanol or acetonitrile were diluted in growth medium). Plates were incubated for 5 days at 37°C, 5% C02, followed by the addition of 10 mI_ PrestoBlue Cell Viability Reagent to each well. Plates were incubated for a further 24 h at 37°C, 5% C02 and then fluorescence (excitation 530 nm, emission 590 nm) was read on a Synergy HT plate reader.
• Enterococcus faecium all strains (clinical isolates 33D3, 69C6, 58C9, and 55 A6).
• MHII Meleller Hinton II
• Streptococcus pyogenes ATCC 51878 BHI (Brain Heart Infusion * )
• Staphylococcus aureus MRSA USA 300 LB (Luria-Bertani)
• Mycobacterium tuberculosis H37Rv 7H9
• Mycobacterium tuberculosis H37Rv PB (Proskauer and Beck + glucose and sodium pyruvate)
• Escherichia coli HB101 (LB (Luria-Bertani)
• Pseudomonas aeruginosa ATCC 27853 Tryptic Soy Broth
• Streptococcus pyogenes ATCC 51878 was grown at 37°C in a tri-gas incubator.
• Pre-cultures were diluted to an OD600 of 0.0025 in 3 ml MHII medium supplemented with appropriate concentrations of cannabinoids. At various time points, 100 pi of culture was removed from each tube and serial 10-fold dilutions were spotted onto MHII agar plates (10 mI), which were incubated at 37°C for 24 h, and the colonies were counted (CFU).
• FICI values was determined in a 96 well checkerboard format using the MIC assay for cannabinoids (compound A), terpenes (compound B), and daptomycin (compound C) alone and in combinations.
• Terpene stocks were prepared in DMSO and diluted in the growth medium.
• the FICs for compounds B and C were calculated.
• the FICI was calculated as FICA plus FICB PIUS FICC.
• Table 1 Cannabinoids have activity against diverse Gram-positive bacteria but not against Gram-negative bacteria, ( Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumanii ATCC 19606, E. coli HB101 , data not shown). Sensitivities are expressed as MICs (mg/L).
• Table 2 Cannabinoid activity against clinical enterococcal strains (vancomycin sensitive, vancomycin resistant, and resistant to both vancomycin and daptomycin), expressed as MICs (mg/L).
• Table 4 Synergistic and potentiating interactions of pairwise and triple combinations of Cannabinoids (CBC, CBD, or CBG), Trans-caryophyllene, and Daptomycin inhibit E. faecium 33D3 growth.
• CBC Cannabinoids
• CBD CBD, or CBG
• Trans-caryophyllene Trans-caryophyllene
• Daptomycin inhibit E. faecium 33D3 growth.
• A Pairwise interactions of CBC, Trans-caryophyllene and Daptomycin
• Table 5 Maximal fold increases in the activities of individual Cannabinoids (CBC, CBD, CBG), Trans-caryophyllene and Daptomycin in pairwise and triple combinations against E. faecium 33D3.
• Table 6 Synergistic and potentiating pairwise or triple interactions of cannabinoids (CBC, CBD, CBG), trans-caryophyllene, and daptomycin inhibit E. faecium VRE 55A6 growth.
• CBC cannabinoids
• CBD cannabinoids
• CBG trans-caryophyllene
• daptomycin inhibit E. faecium VRE 55A6 growth.
• Table 7 Maximal fold increases in the activities of individual cannabinoids (CBC, CBD, CBG), trans-caryophyllene and daptomycin in pairwise or triple combinations against E. faecium VRE 55A6.
• CBC cannabinoids
• CBD cannabinoids
• CBG cannabinoids
• daptomycin in pairwise or triple combinations against E. faecium VRE 55A6.
• FIG. 2 cannabinoids sensitize antibiotic resistant Enterococcal strains to daptomycin.
• Figure 2 reflects data illustrating the effects of daptomycin (Dap), and Dap in combination with CBD, CBG or CBC, on daptomycin resistant E. faecium VRE strains 58C9 and 55A6.
• faecium strains 58C9 and 55A6 were incubated with no cannabinoid (A,B), or 1/2x (0.5mg/L) MIC of CBD (C,D), CBG (E,F) or CBC (G,H) with 1x (4mg/L for 58C9; 8mg/L for 55A6), 1/2x (2mg/L for 58C9; 4mg/L for 55A6), 1/4x (1 mg/L for 58C9; 2mg/L for 55A6) or 1/8x (0.5 mg/L for 58C9; 1 mg/L for 55A6) MIC of daptomycin. Aliquots were removed at 2, 4, 6, 8 and 24h for CFU enumeration. Data points are the means from three replicates with standard deviations presented as error bars. As illustrated, adding approximately 1 ⁇ 2 MIC of each cannabinoid facilitated killing by daptomycin at 1/4-1/8x the MIC of daptomycin.
• strains were each incubated with 1/8x to 1x MIC of daptomycin (0.25- 1 mg/L daptomycin for 33D3, 0.5-4mg/L daptomycin for 58C9 and 1 -8mg/L daptomycin for 55A6) and with 0.5x MIC of CBD, CBG or CBC.
• daptomycin did inhibit growth at 1x MIC, there was no reduction in CFU.
• This example illustrates that CBD and CBG are surprisingly effective at inhibiting E. faecium biofilm growth.
• the data in Figure 4 was generated by growing biofilms of E. faecium strain 33D3 for 48h without cannabinoids, then treating the biofilms with cannabinoids for 72h. The mass of the biofilm was quantitated by crystal violet staining. Viable bacteria in the biofilm were quantitated by scrapping biofilm then counting the viable bacteria.
• CBD and CBG illustrate a surprising degree of antibiotic activity against the strain 33D3 of E. faecium.
• CBC was bactericidal against planktonic Enterococci.

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