Patent Application: US-200913000147-A

Abstract:
a group of antimicrobial compounds shows effectiveness for preventing bacterial growth and bio film formation . in particular , the compounds are effective for preventing the growth of gram - positive bacteria , including methicillin - resistant staphylococcus aureus bacteria . the compounds include naturally - occurring compounds such as linoleyl ethanolamide , noladin ether , and anandamide , and man - made compounds such as cp55 , 640 - cis - 3 - phenyl ]- trans - 4 - cyclohexanol ] and o - 2050 - 3 -- 6a , 7 , 10 , 1oa - tetrahydro - 6 , 6 , 9 - trimethyl - 6h - dibenzopyran ]. because these antibacterial compounds have unique modes of action and / or unique chemical scaffolds compared to traditional antibiotics , they are extremely useful against bacteria having resistances to antibiotics .

Description:
the methods described herein involve the use of compounds , or ligands , or chemical agents , that act on cannabinoidergic systems , or their mixtures , alone or combined with carriers , to effectively inhibit the growth of bacteria , inhibit the formation of biofilm , or both . for example , this would include compounds that interact with cannabinoid receptors or enzymes involved in the metabolism of cannabinoids or transporters involved in the transport of cannabinoids . in particular , the methods are effective against the growth of gram - positive bacteria , including mrsa . the compounds include the cannabinoid receptor ligands cp55 , 940 [(−)- cis - 3 -[ 2 - hydroxy - 4 -( 1 , 1 - dimethylheptyl ) phenyl ]- trans - 4 -( 3 - hydroxypropyl ) cyclohexanol ], linoleoyl ethanolamide (“ lea ”) [ n -( 2 - hydroxyethyl )- 9z , 12z - octadecadienamide ], also spelled linoleyl ethanolamide , o - 2050 [( 6ar , 10ar )- 3 -( 1 - methanesulfonylamino - 4 - hexyn - 6 - yl )- 6a , 7 , 10 , 10a - tetrahydro - 6 , 6 , 9 - trimethyl - 6h - dibenzo [ b , d ] pyran ], noladin ether [ 2 -[( 5z , 8z , 11z , 14z )- eicosatetraenyloxy ]- 1 , 3 - propanediol ], and anandamide [ n -( 2 - hydroxyethyl )- 5z , 8z , 11z , 14z - eicosatetraenamide ]. fig1 shows the structures , chemical abstracts services (“ cas ”) registration numbers and some additional details about the antibacterial compounds . a first compound having antibacterial activity that is also effective against biofilm formation is linoleoyl ethanolamide (“ lea ”), having the structure shown below : an additional compound having antibacterial activity that is also effective against biofilm formation is o - 2050 , having the structure shown below : an additional compound having antibacterial activity that is also effective against biofilm formation is noladin ether , having the structure shown below : an additional compound having antibacterial activity that is also effective against biofilm formation is cp55 , 940 , having the structure shown below : an additional compound having antibacterial activity that is also effective against biofilm formation is anandamide , also called aea or arachidonylethanolamide , having the structure shown below : the current compounds are useful as antibacterial agents and useful in the prevention of the formation of biofilms , and particularly as agents against gram - positive bacteria , including mrsa . an effective amount of the compound or combinations thereof could be mixed with a suitable carrier and used as anti - infective topical applications , such as creams , lotions , ointments or sprays that could be made with or without other established antibiotics , belonging to the structural classes of amino glycoside , cephalosphorins , beta - lactams , penicillins , sulfonamides , tetracyclines , quinolones , fluoroquinolones , glycopeptide , lipopeptide , macrolides , monobactams , ansamycins , carbacephem and / or including specific antibiotics such as neomycin , bacitracin , polymyxin b , clindamycin , erythromycin , streptomycin , kanamycin , gentamicin , tetracycline , sulfacetamide , metronidazole , mupirocin , retinol , adapalene , tazarotene , tretinoin , isotretinoin , benzoyl peroxide , azelaic acid , salicylic acid , rep8839 ( replidyne , inc ., louisville , colo . ), vancomycin , daptomycin , linezolid , trimethoprim - sulfamethoxazole , minocycline , doxycycline , trovafloxacin , levofloxacin , ciprofloxacin , nalidixic acid , azithromycin , quinupristin / dalfopristin , rifampin , rifampicin , nitrofurantoin , isoniazid , pyrazinamide , tinidazole , platensimycin , chloramphenicol , fusidic acid , furazolidone , lincomycin , ethambutol , fosfomycin , arsphenamine , mafenide , colistin , clarithromycin or mixtures thereof . as used herein , “ an effective amount ” or “ an effective amount of a cannabinoidergic - system - acting compound ” means that amount which will provide the desired interaction with cannabinoid receptors , the desired involvement with the metabolism of cannabinoids , or the desired involvement with the transport of cannabinoids to give a discernable effect of inhibiting the growth of bacteria , inhibiting the formation of biofilm , or both . for example , an effective dose might be one that reduces bacterial growth and / or biofilm formation by at least 70 % for a lower starting concentration of bacterial ( e . g ., ca 5000 cfu / ml , see fig2 ) or by at least 30 % for a higher starting concentration of bacteria ( e . g ., ca 50 , 000 cfu / ml ). in addition to antibiotics , the anti - infective topical applications can also contain any suitable adjuvants , preferably those that are known to assist in wound healing , such as aloe , zinc oxide , grapeseed oil , or combinations of these . in addition , these compounds could be used in anti - infective coatings applied to various medical devices , including catheters , hemodialysis equipment , pulmonary ventilators , heart valves , dialysis equipment , and surgical equipment . these compounds could also be impregnated into bandages or other wound dressing materials . the wound dressing materials treated with the compounds would then have anti - infective properties . it is also possible that these compounds could be administered as drugs , either oral , sublingual , as eye , nose , or ear drops , or as an injection or inhalant . these uses are in addition to traditional uses as a systemic antibiotic , and other uses within the scope of this invention may be apparent as well . these compounds are extremely beneficial in uses against drug - resistant bacteria because , among other things , their structures are unique and have not been previously used against bacteria . thus , bacteria have not developed a resistance to them . furthermore , some of the compounds include natural products , which potentially lowers any barriers to market entry . the broth inoculum was prepared from 18 - 24 hours old colonies grown on standard agar plates . approximately four of the fresh colonies were swiped with a sterile cotton q - tip and then suspended in saline by immersion and light swirling . the turbidity was adjusted to equal that of a 0 . 5 mcfarland turbidity standard using a spectrophotometer ( 600 nm ). the amount corresponded to about 1 . 5 × 10 8 cfu / ml , where cfu stands for colony - forming units , and is equivalent to an absorbance at 600 nm equal to 0 . 132 or a percentage transmission equal to 74 . 3 . a 1 : 15 , 000 v / v final dilution into two ml of commercially - available quality - control - tested bacterial culture mueller - hinton broth resulted in broth inoculated with approximately 5 × 10 3 bacteria . sterile 4 ml polystyrene tubes ( 12 × 75 mm ) with caps were used for this purpose . mrsa bacteria ( about 5000 cfu / ml ) were allowed to grow for 16 - 20 hours in 2 ml broth at 35 ± 2 ° c . in the presence or absence of various experimental compounds or antibiotics . the amount of biofilm was visualized qualitatively after being stained with crystal violet . biofilm attached to the side of the tube was stained purple . the compounds cp55 , 940 , lea , and capsaicin were all tested at 30 μm . the results are shown in fig2 . in the negative control , no compounds were added . the vehicle controls were made up of the appropriate dilution of the different solvent used to dissolve the experimental compounds , including methanol ( meoh ), ethanol ( etoh ) or dimethylsulfoxide ( dmso ). the experimental compounds tested included cp55 , 940 , noladin ether , o - 2050 , and linoleyl ethanolamide . high concentrations of the antibiotics zeocin and ampicillin served as positive antimicrobial controls . relatively ineffective antibiotics hydromycin b and kanamycin were also tested . the no bacteria group contained only media and no bacteria , and served as another control . the results show that both natural ( i . e ., lea and noladin ether ) and man - made ( i . e ., cp55 , 940 and o - 2050 ) compounds prevent mrsa growth and biofilm formation . the efficacy of the compounds were again tested against different strains of gram - positive bacteria , including streptococcus agalactiae , enterococcus faecalis , staphylococcus epidermidis , and prominent mrsa ( hospital - acquired and community - acquired ) strains . broth inoculum was prepared from 18 - 24 hours old colonies grown on standard agar plates . approximately four of the fresh colonies were swiped with a sterile cotton q - tip and then suspended in saline by immersion and light swirling . the turbidity was adjusted to equal that of a 0 . 5 mcfarland turbidity standard using a spectrophotometer ( 600 nm ). the amount corresponded to about 1 . 5 × 10 8 cfu / ml , where cfu stands for colony - forming units , and is equivalent to an absorbance at 600 nm equal to 0 . 132 or a percentage transmission equal to 74 . 3 . a 1 : 15 , 000 v / v final dilution into two ml of commercially - available quality - control - tested bacterial culture mueller - hinton broth resulted in broth inoculated with approximately 5 × 10 3 bacteria . sterile 4 ml polystyrene tubes ( 12 × 75 mm ) with caps were used for this purpose . the bacteria were allowed to incubate overnight in 2 ml broth at 35 ± 2 ° c . in the presence or absence of various experimental compounds or antibiotics . the amount of biofilm was visualized qualitatively , then stained with crystal violet . biofilm attached to the side of the tube was stained purple . after staining , the tubes were decanted and rinsed , leaving stained biofilm adhering to the tubes . next , the stain was solubilized and mixed with ethanol ( etoh ) or dimethylsulfoxide ( dmso ). the turbidity of the so lubilized stain , or lack thereof , was then quantified in a spectrophotometer ( 600 nm ). the results are shown in fig3 . in the broth only sample , no bacteria were added . in the negative controls , no compounds were added . the vehicle controls were made up of the appropriate dilution of the different solvent used to dissolve the experimental compounds , including ethanol ( etoh ) or dimethylsulfoxide ( dmso ). the experimental compounds tested included 30 μm of cp55 , 940 , 30 μm of o - 2050 , 30 μm of linoleyl ethanolamide , 30 μm of no ladin ether , and 30 μm of anandamide ( natural product 2 ). high concentrations of silver nitrate ( 1000 μm ) and vancomycin ( 50 μg / ml ) served as positive antimicrobial controls . the results show that both natural ( i . e ., lea ) and man - made ( i . e ., cp55 , 940 and o - 2050 ) compounds prevent growth of gram - positive bacteria , including mrsa , and biofilm formation . in this example , the same procedure was followed as in example 1 , but the bacterial growth and biofilm formation measured was from staphylococcus epidermidis . the experimental compounds included compound 2 ( anandamide ), compound 17 ( linoleyl ethanolamide ), compound 21 ( o - 2050 ), compound 22 ( noladin ether ), and compound 23 ( cp55 , 940 ), all at 30 μm . in the negative control , no compounds were added . the vehicle controls were made up of the appropriate dilution of the different solvent used to dissolve the experimental compounds , including methanol ( meoh ), ethanol ( etoh ) or dimethylsulfoxide ( dmso ). high concentrations of the antibiotics oxacillin ( 6 μg / ml ), vancomycin ( 50 μg / ml ), doxycycline ( 50 μg / ml ), and silver nitrate ( 100 μm ) served as positive antimicrobial controls . the broth only sample contained only media and no bacteria , and served as another control . the results , shown in fig4 , demonstrate that the same experimental compounds that inhibit mrsa growth and biofilm formation also inhibit s . epidermidis growth and biofilm formation . the following u . s . patent documents and publications are hereby incorporated by reference . barrett c . t ., barrett j . f ., antibacterials : are the new entries enough to deal with the emerging resistance problems ? curr opin biotechnol . 2003 december , 14 ( 6 ): 621 - 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