COMPOSITIONS COMPRISING CANNABINOIDS FOR USE IN THE TREATMENT OF BIOFILM AND CONDITIONS ASSOCIATED WITH MICROBIAL, FUNGAL, BACTERIAL INFECTIONS

Abstract

The invention provides compositions comprising at least one cannabinoid compound, for use in the method of treating and preventing a disease, condition or symptom caused by, or associated with fungi, bacteria and microbes.

Claims

1.-32. (canceled)

33. A method of treating at least one surface condition selected from microbial growth, fungal growth, biofilm formation, bacterial growth, biofilm maturation, quorum sensing cascade and any combinations thereof, said method comprises treating said surface with a composition comprising at least one cannabinoid compound and at least one agent selected from an antimicrobial agent, an antifungal agent, an antibacterial agent and any combination thereof.

34. A method according to claim 33, wherein said at least one cannabinoid is an endocannabinoid.

35. A method according to claim 33, wherein said at least one cannabinoid is selected from ARAS (arachidonoyl serine), 2AG (2-arachidonoyl glycerol), AEA (arachidonoyl ethanolamide), OEA (oleoyl ethanolamide), OG (oleoyl glycine), OA (oleoyl alanine), HU-210, HU-308, PEA (palmitoyl ethanolamide) HU-433, AraG (Arachidonoyl glycine), PG (Palmitoyl glycine), AraA (Arachidonoyl alanine), PA (Palmitoyl alanine), PS (Palmitoyl serine), OS (Oleoyl serine), 2-arachidonoyl glyceryl ether, 2-oleoyl glyceryl ether, 2-palmitoyl glyceryl ether and any derivative or combinations thereof.

36. A method according to claim 33, wherein said antifungal agent is selected from fluconazole, nystatin, amphotericin B, fluconazole, nystatin, amphotericin B, fluconazole, nystatin, amphotericin B, fluconazole, ketoconazole, nystatin, amphotericin B, clotrimazole, caspofungin and any combinations thereof.

37. A method according to claim 33, wherein said antibacterial agent is selected from penicillin family, cephalosporin family, fluoroquinolones family, carbapenem family, aminoglycosides family, macrolides family, vancomycin, rifampin, doxycycline, linezolid, tetracycline, trimethoprim and any combinations thereof.

38. A method according to claim 33, wherein said at least one condition is drug resistance.

39. A method according to claim 33, wherein said at least one condition is resistance to said at least one agent.

40. A method of sensitizing and/or preventing biofilm formation on a surface, comprising contacting said surface with a composition comprising at least one cannabinoid compound.

41. A method according to claim 40, wherein contacting said surface with a composition is performed prior to, after and/or concurrent to contacting said surface with at least one of antimicrobial agent, an antifungal agent, an antibacterial agent, and any combinations thereof.

42. (canceled)

43. A method of treatment, prevention or inhibition of the formation or growth of at least one of fungi, fungal biofilm and any combinations thereof in at least one of food product, soil and plant, comprising exposing said at least one of food product, soil and plant to a composition comprising at least one cannabinoid compound.

44. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

[0065] FIG. 1 shows the inhibition of biofilm formation of C. albicans of HU210.

[0066] FIGS. 2A-2D show HU210 effect on fungal morphology in biofilm.

[0067] FIGS. 3A-3F HU210 reduction of viable fungal cells within biofilm.

[0068] FIG. 4 shows the inhibition effect of HU210 of co-species C. albicans-S. mutans biofilm formation.

[0069] FIG. 5 shows the inhibition effect of ARAS on single and co-species biofilm formation.

[0070] FIG. 6 shows the Relative Bioluminescence Unit (RLU) of different mutant strains of bacteria V. harveyi when exposed to different sub-MIC concentrations of AEA. RLU is represented as area under the curve (AUC) and shown in relevance with the control experiment where AEA is absent. * P<0.05 (n=3).

[0071] FIG. 7 shows the endocannabinoids inhibition of S. mutans biofilm formation.

[0072] FIG. 8 shows the 2-AG (endocannabinoid) dose-dependent inhibition of C. albicans biofilm formation.

[0073] FIG. 9 shows the AEA (endocannabinoid) dose-dependent inhibition of C. albicans biofilm formation.

[0074] FIGS. 10A-10D show the CSLM of S. mutans biofilm—The live bacteria are marked in green and the dead bacteria are marked in red. The AEA show a dose-dependent inhibition of S. mutans biofilm formation.

[0075] FIGS. 11A-11D show CLSM images of treated biofilms of P. aeruginosa. Effect of AEA PEA (endocannabinoids/endocannabinoids derivatives) on biofilm of P. aeruginosa. Both treatments resulted in reduced layers/depth of biofilm.

[0076] FIGS. 12A-12E show the effect of AEA and AraS on eradication of formed biofilm on MRSA 33592 (12A=control; 12B, 12C=AEA, 12D, 12E=AraS)

[0077] FIGS. 13A-13I show the effect of ECs on spreading ability of MRSA. All tested MRSA strains demonstrated strong ability to spread on the agar (control 13A, 13D, 13G). Both ECs, AEA and in less impact ARAS were able to reduce colony spreading. AEA at 64 μg/ml reduced diameter of the colony of CI, 33592 and 43000 strains by 88% (13B), 84% (14E), and 73% (13H), respectively, as compared to untreated controls (13A, 13D, 13G). ARAS at sub-MICs was able to inhibit colony spreading of CI, 33592 and 43000 strains by 64% (13C), 65% (13F), and 46% (13I), respectively, as compared to untreated controls (13A, 13D, 13G).

[0078] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0079] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.

Example 1: Anti-Biofilm Effect of Synthetic Cannabinoid HU210

[0080] FIG. 1 demonstrated pronounced dose-dependent inhibitory effect of HU210 C. albicans biofilm formation. Minimal biofilm inhibitory concentration 50 (50% of biofilm inhibition) MBIC50 was recorded already at lowest tested dose of HU210=2 μg/ml (FIG. 1). Almost no biofilm formed at highest tested dose of HU210=64 μg/ml (FIG. 1). In contrast to the strong anti-biofilm activity of HU210, no effect on fungal growth was detected, since minimal inhibitory concentration (MIC) of HU210 was not detected at tested doses.

Example 2: HU210 Affects Fungal Morphology in Biofilm

[0081] Microscopic observation showed that HU210 dramatically alters biofilm morphologic composition. As shown in FIG. 2, untreated control biofilm (FIG. 2A) consisted of candidal branched hyphae and characterized by highly dense mycelium. However, HU210 already at 8 μg/ml influenced fungal morphology (FIG. 2C). In addition, density of fungal mycelium decreased dose-dependently (FIG. 2B-D). Furthermore, HU210 at dose of 64 μg/ml lead to the alteration of yeast-to-hyphae transition resulting in the appearance of mainly yeast form of C. albicans (FIG. 2D).

Example 3: HU210 Reduces Viable Fungal Cells within Biofilm

[0082] Flow cytometry analysis demonstrated dramatic decrease of viable cells in biofilm due to exposure to HU210 (FIG. 3). Pronounced reduction of viable C. albicans cells from 88% in untreated control (FIG. 3A) to 20% in biofilm treated with 8 μg/ml of HU210 (FIG. 3B) was detected. Finally, highest tested dose of HU210=64 μg/ml totally reduced viable cells in fungal biofilm (FIG. 3C). Furthermore, granularity and cell size, which reflect mycelium density and morphologic form, respectively were altered by HU210. Granularity was reduced from 136 AU in control (FIG. 3D) to 50 AU and 40 AU in samples treated with 8 μg/ml (FIG. 3E) and 64 μg/ml (FIG. 3F), respectively. Cell size was reduced from 260 AU in control (FIG. 3D) to 110 AU and 100 AU in samples treated with 8 μg/ml (FIG. 3E) and 64 μg/ml (FIG. 3F), respectively. Flow cytometry results obviously support morphologic observation.

Example 4: HU210 Inhibits Co-Species C. albicans-S. mutans Biofilm Formation

[0083] FIG. 4 demonstrated inhibitory effect of HU210 on mixed C. albicans-S. mutans biofilm formation. MBIC50 was recorded already at 4 μg/ml of HU210. Growth of co-culture was not affected by HU210 at all tested doses of HU210. In contrast, HU210 exhibited pronounced inhibitory effect (MIC=2 μg/ml) towards single S. mutans specie growth. No streptococcal biofilm was formed at this concentration of HU210 (data not shown).

Example 5: Antimicrobial Activity of Selected Endocannabinoids

[0084] FIG. 5 demonstrated dose-dependent inhibitory effect of ARAS on S. mutans, C. albicans and mixed S. mutans-C. albicans biofilm formation. ARAS at dose of 8 μg/ml was able to inhibit single S. mutans biofilm formation by more than 50%. MBIC50 for single C. albicans and mixed S. mutans-C. albicans biofilms was detected at 16 μg/ml and 32 μg/ml of ARAS, respectively. In contrast growth of S. mutans was inhibited only at highest tested dose of ARAS (MIC=64 μg/ml), while single C. albicans and mixed S. mutans-C. albicans growth was not affected at all tested concentrations of ARAS (MIC>64 μg/ml) (data not shown).

Example 6: Antimicrobial Activity Against Resistance Microbes (Bacteria-Fungal)

[0085]

TABLE-US-00001 TABLE 1 Effect of combination of AEA and methicillin against methicillin resistant staphylococci S. aureus MRSA 24433 Growth, μg/ml MIC AEA MIC METH FIC AEA FIC METH FICI effect >64 >64 16 16 <0.5 synergy Biofilm, μg/ml MBIC AEA MBIC METH FBIC AEA FBIC METH FBICI effect 32 >64 8 16 <0.5 synergy

[0086] As shown in Table 1, AEA in combination with methicillin has synergistic effect either on growth or on biofilm formation of methicillin resistant staphylococci. Both agent have no effect on bacterial growth (MIC>64 μg/ml), while in combination MIC of each compound in combination decreased by more than 4-fold. Calculated FICI is less than 0.5 which indicates on synergistic activity between these agents towards bacterial growth. Similar results were obtained concerning biofilm formation. MBIC of each compound in combination was less than MBIC of appropriate compound alone by 4 fold or more. Calculated FBICI is less than 0.5, which indicates on synergistic effect between these agents towards biofilm formation.

Example 7: Anti Quorum Sensing Effect

[0087] FIG. 6 Relative Bioluminescence Unit (LUM/(O.D(595 nm))) (RLU) of different mutant strains of bacteria V. harveyi when exposed to different sub-MIC concentrations of AEA. RLU is represented as area under the curve (AUC) and shown in relevance with the control experiment where AEA is absent. * P<0.05 (n=3).

[0088] The quorum sensing assays indicate on an inhibition in the presence of AEA. A dose response is observed up to the 100 μg/ml. A dose-response in quorum sensing was observed up to 100 mg/ml AEA, which are concentrations below the MIC.

[0089] Selected cannabinoids demonstrated specific non-killing anti-biofilm effect towards bacterial and fungal pathogens. Moreover, selected cannabinoid, AEA, exhibited effect in combination with antibiotic, towards bacteria that is resistant to this antibiotic. Thus, tested cannabinoids could be promising therapeutics against biofilm-associated infections. Furthermore, they could be administrated together with antibiotics in order to: 1. affect resistant bacteria; 2. reduce antibiotic-associated adverse effects.

[0090] FIG. 7 demonstrated dose-dependent inhibitory effect of OEA, AEA, OA and OG on S. mutans biofilm formation. Agents OA, OG and OEA exhibited MBIC50 at 16, 32 and 64 μg/ml, respectively. AEA was less effective, however also showed inhibition of S. mutans biofilm formation by 45% at highest tested dose of 64 μg/ml. Bacterial growth was not affected by any of the tested agents at all tested doses (MIC>64 μg/ml).

Example 8: Effect of Combination of Endocannabinoids with Antibiotics/Antimycotic Agents on Resistant Bacteria/Fungi Growth and Biofilm Formation

Abbreviations and Explanations:

[0091]

TABLE-US-00002 MIC AEA/ARAS-MIC AEA/ARAS alone MIC METH/AMP/GEN/FLU-MIC methicillin/ampicillin/gentamycin/fluconazole alone FIC AEA/ARAS - MIC AEA/ARAS in combination with methicillin/ampicillin/gentamycin/fluconazole FIC METH/AMP/GEN/FLU - MIC methicillin/ampicillin/gentamycin/fluconazole in combination with AEA/ARAS FICI fractional inhibitory concentration index MBIC AEA/ARAS-MBIC AEA/ARAS alone MBIC METH/AMP/GEN/FLU-MBIC methicillin/ampicillin/gentamycin/fluconazole alone FBIC AEA/ARAS - MBIC AEA/ARAS in combination with methicillin/ampicillin/gentamycin/fluconazole FBIC METH/AMP/GEN/FLU - MBIC methicillin/ampicillin/gentamycin/fluconazole in combination with AEA/ARAS FBICI fractional biofilm inhibitory concentration index Synergistic effect* FICI/FBIC of <0.5 Partial synergism* 0.5 > FICI/FBIC < 1   Additive effect* FICI/FBIC = 1 Indifference* 1 > FICI/FBIC < 4 Antagonism* FICI/FBIC of more than 4 (*) Lee WX, Basri DF, Ghazali AR Bactericidal Effect of Pterostilbene Alone and in Combination with Gentamicin against Human Pathogenic Bacteria. Molecules. 2017 Mar 17;22(3))

[0092] Effect of Combination of ARAS with Fluconazole Against Fluconazole Resistant C. albicans Strains

[0093] Table 2 demonstrated that each agent alone was non-effective against biofilm formation of both resistant fungal strains (MBIC 64 μg/ml or >64 μg/ml). However, combination of these agents reduced MBIC of ARAS by 2-fold, while MBIC of fluconazole was reduced by more than 32- and 16-fold. Thus, this combination was defined as partial synergistic towards biofilm formation of both tested fluconazole resistant C. albicans strains. Growth of these fungal strains was not affected either by each agent alone or in combination (data not shown).

[0094] Effect of Combination of ARAS with Different Antibiotics Against Methicillin Resistant Staphylococcus aureus (MRSA) Strains

TABLE-US-00003 TABLE 2 Effect of combination of ARAS with fluconazole against fluconazole resistant C. albicans strains Biofilm MBIC MBIC FBIC FBIC strain ARAS FLU ARAS FLU FBICI Effect DSY551 64 >64 32 2 >0.5 < 1 partial synergism DSY735 64 >64 32 4 >0.5 < 1 partial synergism

TABLE-US-00004 TABLE 3 Effect of combination of ARAS and methicillin against MRSA strains A S.aureus MRSA 33592 Growth MIC ARAS MIC METH FIC ARAS FIC METH FICI effect 64 32 16 8 <0.5 synergy Biofilm MBIC MBIC FBIC FBIC ARAS METH ARAS METH FBICI effect 32 32 16 8 >0.5 < 1 partial synergy B S.aureus MRSA 24433 Growth MIC ARAS MIC METH FIC ARAS FIC METH FICI effect >256 >64 >64 >64 >1 < 4 indifferent Biofilm MBIC ARAS MBIC METH FBIC ARAS FBIC METH FBICI effect 32 >64 16 16 >0.5 < 1 additive C S.aureus MRSA 43300 Growth MIC ARAS MIC METH FIC ARAS FIC METH FICI effect 64 32 16 2 <0.5 synergy Biofilm MBIC ARAS MBIC METH FBIC ARAS FBIC METH FBICI effect 32 32 8 8 <0.5 synergy

TABLE-US-00005 TABLE 4 Effect of combination of ARAS and gentamycin against MRSA strain S.aureus MRSA 33592 Growth MIC ARAS MIC GEN FIC ARAS FIC GEN FICI effect 32 128 4 4 <0.5 synergy Biofilm MBIC ARAS MBIC GEN FBIC ARAS FBIC GEN FBICI 32 128 4 4 <0.5 synergy

TABLE-US-00006 TABLE 5 Effect of combination of ARAS and ampicillin against MRSA strains A S.aureus MRSA 33592 Growth MIC ARAS MIC AMP FIC ARAS FIC AMP FICI effect 32 128 8 64 <0.5 < 1 partial synergy Biofilm MBIC ARAS MBIC AMP FBIC ARAS FBIC AMP FBICI effect 32 128 16 32 <0.5 < 1 partial synergy B S.aureus MRSA 43300 Growth MIC ARAS MIC AMP FIC ARAS FIC AMP FICI effect 64 256 16 16 <0.5 synergy Biofilm MBIC ARAS MBIC AMP FBIC ARAS FBIC AMP FBICI effect 32 256 8 64 <0.5 synergy

[0095] Combination of ARAS with various antibiotics was also effective against methicillin-resistant strains of S. aureus. As shown in Table 3, combination of ARAS with methicillin has synergistic effect on two methicillin-resistant strains MRSA 33592 (Table 3A) and MRSA 43300 (Table 3C) growth. This combination was also effective against biofilm formation: synergy was detected against MRSA 43300 (Table 3C) and partial synergy was detected against MRSA 33592 (Table 3A) biofilm formation. In addition, combination of ARAS with gentamicin or ampicillin exhibited synergistic (Table 4) or partial synergistic effect (Table 5A), respectively, towards MRSA 33592 growth and biofilm formation. Furthermore, combination of ARAS with ampicillin demonstrated synergistic effect against MRSA 43300 growth and biofilm formation (Table 5B).

[0096] Effect of Combination of AEA with Different Antibiotics Against MRSA Strains.

TABLE-US-00007 TABLE 6 Effect of combination of AEA and methicillin against MRSA strains. A S.aureus MRSA 33592 Growth MIC AEA MIC METH FIC AEA FIC METH FICI effect >256 32 16 16 <0.5 synergy Biofilm MBIC AEA MBIC METH FBIC AEA FBIC METH FBICI effect 64 32 8 8 <0.5 synergy B S.aureus MRSA 24433 Growth MIC AEA MIC METH FIC AEA FIC METH FICI effect >256 >64 16 16 <0.5 synergy Biofilm MBIC AEA MBIC METH FBIC AEA FBIC METH FBICI effect 32 >64 8 16 <0.5 synergy C S.aureus MRSA 43300 Growth MIC AEA MIC METH FIC AEA FIC METH FICI effect >256 32 16 8 <0.5 synergy Biofilm MBIC AEA MBIC METH FBIC AEA FBIC METH FBICI effect >256 32 32 8 <0.5 synergy

TABLE-US-00008 TABLE 7 Effect of combination of AEA and gentamicin against MRSA strain. S.aureus MRSA 33592 Growth MIC AEA MIC GEN FIC AEA FIC GEN FICI effect >256 128 8 4 <0.5 synergy Biofilm MBIC AEA MBIC GEN FBIC AEA FBIC GEN FBICI effect 64 128 8 8 <0.5 synergy

TABLE-US-00009 TABLE 8 Effect of combination of AEA and ampicillin against MRSA strains. A S.aureus MRSA 33592 Growth MIC AEA MIC AMP FIC AEA FIC AMP FICI effect >256 128 8 8 <0.5 synergy Biofilm MBIC AEA MBIC AMP FBIC AEA FBIC AMP FBICI effect 64 128 8 8 <0.5 synergy B S.aureus MRSA 43300 Growth MIC AEA MIC AMP FIC AEA FIC AMP FICI effect >256 >128 16 8 <0.5 synergy Biofilm MBIC AEA MBIC AMP FBIC AEA FBIC AMP FBICI effect >256 >128 16 8 <0.5 synergy

[0097] Agent AEA demonstrated notable synergistic effect being in combination with various antibiotics against MRSA strains growth and biofilm formation. Combination of AEA with methicillin (Table 6), gentamicin (Table 7) or ampicillin (Table 8) showed strong synergistic effect against all tested MRSA strains growth and biofilm formation. The most pronounced synergistic effect was detected in combination of AEA with gentamicin against MRSA 33592 growth (Table 7). These bacteria were highly resistant to each agent alone (MIC of AEA>256, MIC of gentamicin=128). However, combination of AEA and gentamicin dramatically decreased MIC of AEA by more than 32-fold and MIC of gentamicin by 32-fold (Table 7).

[0098] Selected cannabinoids obviously demonstrated specific non-killing anti-biofilm effect towards bacterial and fungal pathogens. Moreover, selected endocannabinoids, AEA and ARAS, exhibited obvious synergistic effect in combination with various antibiotics towards methicillin-resistant strains of S. aureus. Thus, tested cannabinoids could be promising therapeutics against biofilm-associated infections. Furthermore, they could be administrated together with antibiotics in order to: 1. affect resistant bacteria; 2. reduce antibiotic-associated adverse effects.

Example 9: Dose-Dependent Inhibition of C. albicans Biofilm Formation

[0099] To investigate the effect of the agents on preformed biofilms, biofilms were allowed to mature in for 24 h at 37° C. in a 6-well plate. The biofilms were washed twice with PBS. The active agents were then applied. The plates were further incubated for 24 h at 37° C. The amounts of biofilms, were determined quantitatively using a standard MTT reduction assay as described previously. Briefly, biofilms were overlaid with 100 mM of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and incubated for 2 h at 37° C. Under these conditions, the lightly yellowish MTT was reduced to a blue tetrazolium salt accumulated within the metabolic active biofilms. The stain was then dissolved in DMSO and the absorbance value was measured at 570 nm. The accumulation of tetrazolium salt by the reduction of MTT is proportional to the number of viable cells growing in biofilm. Prior to dissolving in DMSO, biofilms were photographed and visualized. Assay was performed in triplicate. FIG. 8 and FIG. 9 show the MTT assay of C. albicans biofilm wherein the endocannabinoids (AEA/2-AG) show a dose-dependent inhibition of C. albicans biofilm formation.

Example 10

[0100] The bacterial viability and vitality of was analyzed by CLSM (Olympus Fluoview 300, Olympus, Japan) with a UPLSA 10×/0.4 lenses. The biofilm samples were grown overnight on 96 well. The biofilm was washed carefully using 200 μl PBS solution after overnight incubation, and then stained with 50 μl of LIVE/DEAD BacLight fluorescent dye (Invitrogen Life Technologies, Carlsbad, Calif., USA) (1:100) for 20 min in the dark, at room temperature. This staining allowed to distinguish the live organisms from the dead ones. Living bacteria were stained with SYTO 9 dye and were observed in green color while dead bacteria were stained with PI dye and were observed in red color. The biofilm thickness was examined by generating the optical sections that were acquired at spacing steps of 10 μm Image J program (The National Institute of Health) was used for fluorescence analysis which calculates the fluorescence intensity per area for each color separately. FIG. 10 shows the CSLM of S. mutans biofilm wherein the live bacteria are marked in green and the dead bacteria are marked in red. The AEA show a dose-dependent inhibition of S. mutans biofilm formation. FIG. 11 shows the effect of AEA PEA (endocannabinoids/endocannabinoids derivatives on biofilm of P. aeruginosa. Both treatments resulted in reduced layers/depth of biofilm. AEA had a more significant reduction in biofilm density.

Example 11

[0101] After incubation for 24 h, supernatant-fluid was removed by aspiration and the wells were carefully washed twice with phosphate-buffered saline (PBS, pH 7.4). The biofilm was measured by crystal violet staining. Briefly, 0.02% crystal violet was placed on top of the biofilm for 45 min, which were then washed twice with DDW to remove unbound dye. Figure. 12 shows the effect of AEA and AraS on eradication of formed biofilm on MRSA 33592 (12A=control, 12B, 12C=AEA, 12D, 12E=AraS).

Example 12

[0102] The swimming assay was performed on soft agar plates. 0.2% agar medium was prepared and autoclaved. The bacteria were exposed to the tested agents. 3 μl of overnight bacterial culture (O.D 595˜0.5) was inoculated at the centre of the agar plate. Agar plates without active agents served as controls. The plates were then incubated for 15 h. To analyze the results, the area of the motility halos was measured using Image J software (National Institute of Health) and compared with the control. FIG. 13 shows the effect of ECs on spreading ability of MRSA. All tested MRSA strains demonstrated strong ability to spread on the agar (control 13A, 13D, 13G). Both ECs, AEA and in less impact ARAS were able to reduce colony spreading. AEA at 64 μg/ml reduced diameter of the colony of CI, 33592 and 43000 strains by 88% (13B; Table 9), 84% (13E; Table 9), and 73% (13H; Table 9), respectively, as compared to untreated controls (13A, 13D, 13G). ARAS at sub-MICs was able to inhibit colony spreading of CI, 33592 and 43000 strains by 64% (13C; Table 9), 65% (13F; Table 9), and 46% (13I; Table 9), respectively, as compared to untreated controls (FIG. 13A, 13D, 13G).

TABLE-US-00010 TABLE 9 MRSA strain Endocannabinoid AEA 64 μg/ml ARAS 64 μg/ml CI 88 ± 1.9 64 ± 2.5 AEA 64 μg/ml ARAS 16 μg/ml 33592 84 ± 1.8 65 ± 3.4 AEA 64 μg/ml ARAS 32 μg/ml 43300 73 ± 2.6 46 ± 2.8

[0103] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.