阅读说明：本技术 抗生大麻素-萜烯制剂 (Anticannabinoid-terpene formulations ) 是由 C·J·汤普森 M·C·普里马 D·M·兰伯特 M·窦三吉 于 2020-05-06 设计创作，主要内容包括：提供了包括至少两种抗生活性成分的药物制剂,所述抗生活性成分选自：大麻素,作为大麻环萜酚(CBC)、大麻二醇(CBD)和/或大麻萜酚(CBG)中的一种或多种；作为α-律草烯和/或β-石竹烯之一或两者的倍半萜烯；和作为达托霉素或其类似物的脂肽抗生素。可以以放大它们各自活性的相对量提供所述抗生活性成分,包括在测定中对于抑制屎肠球菌或粪肠球菌的生长和/或繁殖协同有效的量。提供了使用这些制剂作为抗微生物剂的疗法,并且所述疗法提供了两种或更多种所述抗生活性化合物的组合使用。(There is provided a pharmaceutical formulation comprising at least two antibiotic active ingredients selected from the group consisting of: cannabinoids as one or more of cannabichromene (CBC), Cannabidiol (CBD) and/or Cannabigerol (CBG); sesquiterpenes which are one or both of alpha-humulene and/or beta-caryophyllene; and a lipopeptide antibiotic that is daptomycin or an analog thereof. The antibiotic active ingredients may be provided in relative amounts that amplify their respective activities, including amounts synergistically effective to inhibit the growth and/or reproduction of enterococcus faecium or enterococcus faecalis in the assay. Therapies using these agents as antimicrobial agents are provided, and the therapies provide for the combined use of two or more of the antibiotic active compounds.)

1. A method of treating a bacterial infection in a subject in need thereof, comprising administering to the subject an effective amount of:

cannabinoids as one or more of cannabichromene (CBC), Cannabidiol (CBD) and/or Cannabigerol (CBG); and the combination of (a) and (b),

a lipopeptide antibiotic that is daptomycin or an analog thereof;

wherein the cannabinoid and the lipopeptide antibiotic are administered in an antibiotically effective weight ratio of from 16:1 to 1: 16; and is

Wherein:

administering the lipopeptide antibiotic in an effective amount of less than 4mg/kg if the cannabinoid is a CBD; and/or the presence of a gas in the gas,

if the cannabinoid is a CBD, the bacterial infection comprises an infection by an infectious organism having a daptomycin MIC of 4 μ g/mL or greater; and/or the presence of a gas in the gas,

if the cannabinoid is CBD, the bacterial infection comprises infection by an infectious organism, and bacterial growth measurements of the infection indicate that daptomycin produces an inhibitory and/or bactericidal effect at a concentration of less than 4 μ g/mL when CBD is administered in an amount less than the MIC of CBD.

2. The method of claim 1, wherein the bacterial infection comprises an infection by an infectious organism having a daptomycin MIC of 4 μ g/mL or greater.

3. The method of claim 1 or 2, wherein the bacterial infection comprises infection by enterococcus faecium or enterococcus faecalis.

4. The method of any one of claims 1 to 3, further comprising administering to the subject an effective amount of a sesquiterpene that is one or both of alpha-humulene and/or beta-caryophyllene.

5. An antibiotic formulation comprising:

cannabinoids as one or more of cannabichromene (CBC) and/or Cannabigerol (CBG); and the combination of (a) and (b),

a lipopeptide antibiotic that is daptomycin or an analog thereof;

wherein the cannabinoid and the lipopeptide antibiotic are present in an antibiotically effective weight ratio of from 16:1 to 1: 16.

6. The formulation of claim 5, wherein the cannabinoid is present in an amount that reduces the Minimum Inhibitory Concentration (MIC) of the lipopeptide antibiotic in an antibiotic assay.

7. The formulation of claim 6, wherein the cannabinoid lowers the MIC of the lipopeptide antibiotic in the assay when the cannabinoid is present in an amount less than the MIC of the cannabinoid.

8. The formulation of any one of claims 5 to 7, wherein the formulation further comprises a sesquiterpene.

9. The formulation of claim 8, wherein the sesquiterpene is one or both of alpha-humulene and/or beta-caryophyllene.

10. The formulation of any one of claims 1-9, wherein the assay is an enterococcal assay that evaluates inhibition of growth and/or reproduction of enterococcus faecium or enterococcus faecalis.

11. A formulation comprising at least two antibiotic active ingredients selected from:

cannabinoids as one or more of cannabichromene (CBC), Cannabidiol (CBD) and/or Cannabigerol (CBG); and/or the presence of a gas in the gas,

sesquiterpenes which are one or both of alpha-humulene and/or beta-caryophyllene; and/or the presence of a gas in the gas,

a lipopeptide antibiotic that is daptomycin or an analog thereof;

wherein the antibiotic active ingredients are present in synergistically effective relative amounts, wherein the effective amounts of the preparation are synergistically effective in an assay to inhibit the growth and/or reproduction of enterococcus faecium or enterococcus faecalis, and wherein if the preparation comprises CBD and daptomycin, at least one additional antibiotic active ingredient is also included.

12. The formulation of claim 11, wherein two antibiotic active ingredients are present and are a cannabinoid and a sesquiterpene.

13. The formulation of claim 11, wherein there are two antibiotic active ingredients and the two antibiotic active ingredients are a cannabinoid and a lipopeptide antibiotic.

14. The formulation of claim 11, comprising the cannabinoid, the sesquiterpene, and the lipopeptide antibiotic.

15. The formulation of claim 11, wherein there are two antibiotic active ingredients and the two antibiotic active ingredients are the sesquiterpene and the lipopeptide antibiotic.

16. The formulation of any one of claims 11-14, wherein the cannabinoid is one of CBC, CBD, or CBG.

17. The formulation as claimed in any one of claims 11-14, wherein the cannabinoids are two of CBC, CBD and CBG.

18. The formulation as claimed in any one of claims 11-14, wherein the cannabinoids include CBC, CBD and CBG.

19. A formulation as claimed in any one of claims 11 to 18 wherein the sesquiterpene, if present, is one of a-lawsene or β -caryophyllene.

20. A formulation as claimed in any one of claims 11 to 18 wherein the sesquiterpenes, if present, include alpha-humulene and beta-caryophyllene.

21. The formulation of any one of claims 11 to 20, wherein the lipopeptide antibiotic, if present, is daptomycin.

22. The formulation of any one of claims 11 to 21, wherein the sesquiterpene, when present, is present in a relative amount that provides a reduction in the Minimum Inhibitory Concentration (MIC) of the cannabinoid of at least 2 to 128 divisor, when present.

23. The formulation of any one of claims 11 to 22, wherein the cannabinoids, when present, are present in relative amounts that provide a reduction in the Minimum Inhibitory Concentration (MIC) of the sesquiterpene of at least 2 to 128 divisor in the assay.

24. The formulation of any one of claims 11 to 23, wherein the sesquiterpene and/or the cannabinoid, when present, is present in a relative amount that provides a reduction in the Minimum Inhibitory Concentration (MIC) of the lipopeptide antibiotic of at least 2 to 128 divisor in the assay.

25. The formulation as claimed in any one of claims 11 to 24, wherein the sesquiterpene and the cannabinoid, when present, are present in a molar ratio of ≥ 50.

26. The formulation of any one of claims 11 to 25, further comprising a pharmaceutically acceptable excipient, and wherein the antibiotic active ingredient is dissolved, dispersed, mixed, or suspended in the formulation.

27. The formulation of any one of claims 11-26, wherein the cannabinoid, when present, is present at 0.1-100 mg/L.

28. The formulation of any one of claims 11 to 27, wherein the sesquiterpene, when present, is present at 0.1-500 mg/L.

29. The formulation as claimed in any one of claims 11 to 28, wherein the cannabinoid and/or sesquiterpene, when present, is derived from plants.

30. The formulation of claim 29, wherein the plant is a cannabis or cannabis indica plant.

31. The formulation of claim 11, wherein there are two antibiotic active ingredients and the two antibiotic active ingredients are cannabinoids.

32. The formulation of any one of claims 11-31, wherein the formulation does not comprise any substituted cannabinoid, terpene, or lipopeptide antibiotic.

33. The formulation of any one of claims 21-31, wherein the formulation consists essentially of two or more of the cannabinoid, the sesquiterpene, or the lipopeptide antibiotic.

34. Use of the formulation of any one of claims 5 to 33 to formulate a medicament.

35. The use of claim 34, wherein the medicament is for use in treating an enterococcal infection in a subject in need thereof.

36. Use of the formulation of any one of claims 5-33 to treat an enterococcal infection in a subject in need thereof.

37. The use of claim 35 or 36, wherein the enterococcal infection is an enterococcus faecium or enterococcus faecalis infection.

38. The use of any one of claims 35 to 37, wherein the enterococcal infection is an oral infection.

39. The use of claim 38, wherein the oral infection is marginal periodontitis, root canal infection, primary pulp infection, persistent or secondary infection, dental caries, peri-implantitis, peri-radicular abscess, or oral mucosal lesion.

40. The use of any one of claims 35 to 39, wherein the enterococcal infection is an antibiotic-resistant enterococcal infection.

41. The use of claim 40, wherein the antibiotic-resistant enterococcus infection is a vancomycin-and/or daptomycin-resistant enterococcus infection.

42. The use as claimed in any of claims 34 to 41, wherein the formulation is used in an amount to deliver an effective dose of cannabinoid from 1 to 5,000 mg/day when present, and/or a sesquiterpene from 1 to 10,000 mg/day when present.

43. A method of treating an enterococcal infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the formulation of any one of claims 5 to 10.

44. A method of treating an enterococcal infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the formulation of any one of claims 11 to 33.

45. The method of claim 44, wherein the formulation comprises two antibiotic active ingredients, wherein the two antibiotic active ingredients are a cannabinoid and a sesquiterpene, the method further comprising treating the subject with an effective amount of one or more antibiotics, optionally wherein the antibiotic is daptomycin.

46. The method of any one of claims 43-45, which comprises administering the cannabinoid in an effective amount of 1-5,000 mg/day.

47. The method of any one of claims 44 to 46 comprising administering the sesquiterpene in an effective amount of 1-10,000 mg/day.

48. A method of treating an enterococcal infection in a subject in need thereof, said method comprising administering to said subject an effective amount of the formulation of claim 9 or 10, said method comprising administering said sesquiterpene in an effective amount of 1-10,000 mg/day.

49. The method of any one of claims 43-48, wherein the subject is a mammal.

50. The method of claim 49, wherein the mammal is a human.

51. The formulation of any one of claims 5 to 33, for use in treating an enterococcal infection in a subject in need thereof.

52. The preparation of claim 51, wherein the enterococcal infection is an enterococcus faecium or enterococcus faecalis infection.

53. The formulation of claim 51 or 52, wherein the enterococcal infection is an oral infection.

54. The formulation of claim 53, wherein the oral infection is marginal periodontitis, a root canal infection, a primary pulp infection, a persistent or secondary infection, dental caries, peri-implantitis, a peri-radicular abscess, or an oral mucosal lesion.

55. The formulation of any one of claims 51-54, wherein the enterococcal infection is an antibiotic-resistant enterococcal infection.

56. The formulation of claim 55, wherein the antibiotic-resistant enterococcal infection is a vancomycin-and/or daptomycin-resistant enterococcal infection.

57. The formulation as claimed in any one of claims 51 to 56, wherein the formulation is used in an amount to deliver an effective dose of cannabinoid from 1 to 5,000 mg/day when present, and/or a sesquiterpene from 1 to 10,000 mg/day when present.

58. A method of treating a microbial infection in a subject in need thereof, the method comprising administering to the subject an effective amount of at least two antibiotically active compounds selected from the group consisting of:

cannabinoids as one or more of cannabichromene (CBC), Cannabidiol (CBD) and/or Cannabigerol (CBG); and/or the presence of a gas in the gas,

sesquiterpenes which are one or both of alpha-humulene and/or beta-caryophyllene; and/or the presence of a gas in the gas,

a lipopeptide antibiotic that is daptomycin or an analog thereof;

wherein the antibiotic active compounds are administered in synergistically effective relative amounts effective to treat the microbial infection, an

Wherein if the formulation comprises CBD and daptomycin, it further comprises at least one other antibiotic active ingredient.

59. The method of claim 58, wherein the synergistically effective relative amounts are synergistically effective to inhibit the growth and/or reproduction of enterococcus faecium or enterococcus faecalis in an assay.

60. The method of claim 58 or 59, wherein the cannabinoid and the sesquiterpene are co-administered, or the sesquiterpene and the lipopeptide antibiotic are co-administered, or the cannabinoid, the sesquiterpene and the lipopeptide antibiotic are co-administered.

61. The method of claim 58 or 59, wherein the cannabinoid and the sesquiterpene, or the sesquiterpene and the lipopeptide antibiotic, or the cannabinoid, the sesquiterpene and the lipopeptide antibiotic are administered sequentially in any order.

62. The method of any one of claims 58 to 61, further comprising further treating the subject with the lipopeptide antibiotic alone.

63. The method as claimed in any one of claims 58-62, wherein the cannabinoid, if administered, is one of a CBC, CBD or CBG.

64. The method as claimed in any one of claims 58 to 62, wherein the cannabinoid, if administered, is two of CBC, CBD and CBG.

65. The method of any one of claims 58-62, comprising wherein the cannabinoid, if administered, comprises CBC, CBD and CBG.

66. A method according to any one of claims 58 to 65 wherein the sesquiterpene, if applied, is one of alpha-humulene or beta-caryophyllene.

67. A method according to any one of claims 58 to 66 wherein the sesquiterpenes, if applied, comprise alpha-humulene and beta-caryophyllene.

68. The method according to any one of claims 58 to 67, wherein the lipopeptide antibiotic, if administered, is daptomycin.

69. The method of any one of claims 58-68, wherein if administered, the sesquiterpene is administered in a relative amount that provides a reduction in the Minimum Inhibitory Concentration (MIC) of the cannabinoid by at least 2 to 128 divisor.

70. The method of any one of claims 58 to 69 wherein the cannabinoid, if administered, is administered in a relative amount that, when administered, provides a reduction in the Minimum Inhibitory Concentration (MIC) of the sesquiterpene by at least 2 to 128 divisor.

71. The method of any one of claims 58 to 70, wherein when administered, the sesquiterpene and/or the cannabinoid is administered in a relative amount that provides a reduction in the Minimum Inhibitory Concentration (MIC) of the lipopeptide antibiotic by at least 2 to 128 divisor.

72. The method of any one of claims 58 to 71, wherein the microbial infection comprises a gram-positive bacterial infection or an enterococcal infection.

73. The method of claim 72, wherein the enterococcal infection comprises an enterococcus faecium or enterococcus faecalis infection.

74. The method of claim 72 or 73, wherein the enterococcal infection is an oral infection.

75. The method of claim 74, wherein the oral infection is marginal periodontitis, a root canal infection, a primary pulp infection, a persistent or secondary infection, dental caries, peri-implantitis, a peri-radicular abscess, or an oral mucosal lesion.

76. The method of any one of claims 58 to 75, wherein the microbial infection is an antibiotic-resistant infection.

77. The method of claim 76, wherein the antibiotic-resistant infection is a vancomycin and/or daptomycin-resistant microbial infection.

78. The method according to any one of claims 1 to 4, wherein the cannabinoid and the lipopeptide antibiotic are administered in synergistically effective relative amounts effective to treat the bacterial infection.

79. The method of claim 78, wherein said synergistically effective relative amounts are synergistically effective to inhibit the growth and/or reproduction of enterococcus faecium or enterococcus faecalis in an assay.

80. The method of any one of claim 79, wherein the cannabinoid is administered in an amount that provides a reduction in the Minimum Inhibitory Concentration (MIC) of the lipopeptide antibiotic of at least 2 to 128 divisor in the assay.

81. The method of any one of claims 1-4 or 78-80, wherein the cannabinoid and the lipopeptide antibiotic are co-administered.

82. The method of claims 1-4 or 78-80, wherein the cannabinoid and the lipopeptide antibiotic are administered sequentially, in any order.

83. The method of any one of claims 1-4 or 78-82, further comprising further treating the subject with the lipopeptide antibiotic alone.

84. The method of any one of claims 1-4 or 78-83, wherein the cannabinoid is one of a CBC, a CBD, or a CBG.

85. The method of any one of claims 1-4 or 78-83, wherein the cannabinoid is two of CBC, CBD, and CBG.

86. The method of any one of claims 1-4 or 78-83, wherein the cannabinoids include CBC, CBD and CBG.

87. The method of any one of claims 1-4 or 78-86, wherein the lipopeptide antibiotic is daptomycin.

88. The method of any one of claims 1-4 or 78-87, wherein the cannabinoids are administered in relative amounts that provide a divisor reduction of at least 2 to 128 of the Minimum Inhibitory Concentration (MIC) of the lipopeptide antibiotic.

89. The method of any one of claims 1-4 or 78-88, wherein the bacterial infection is a gram-positive bacterial infection or an enterococcal infection.

90. The method of claim 89, wherein the enterococcal infection is an enterococcus faecium or enterococcus faecalis infection.

91. The method of claim 89 or 90, wherein the enterococcal infection is an oral infection.

92. The method of claim 91, wherein the oral infection is marginal periodontitis, root canal infection, primary pulp infection, persistent or secondary infection, dental caries, peri-implantitis, peri-radicular abscess, or oral mucosal lesion.

93. The method of any one of claims 1-4 or 78-92, wherein the bacterial infection is an antibiotic-resistant infection.

94. The method of claim 93, wherein the antibiotic-resistant infection is a vancomycin and/or daptomycin-resistant bacterial infection.

95. The method of any of claims 1-4 or 78-94, which comprises administering an effective dose of the cannabinoid at 1 to 5,000 mg/day.

96.CBD and/or CBG, wherein the biofilm comprises enterococcus faecium or enterococcus faecalis.

97. A method of inhibiting the growth of a biofilm comprising enterococcus faecium or enterococcus faecalis, the method comprising exposing the biofilm to an effective amount of CBD and/or CBG.

98. An invasive medical device comprising an impregnating concentration of CBD and/or CBG, wherein the impregnating concentration of CBD and/or CBG is releasable from the device in vivo at a concentration effective to inhibit biofilm growth on the device, wherein the biofilm comprises enterococcus faecium or enterococcus faecalis.

Technical Field

The present invention is in the field of pharmaceutical formulations comprising a mixture of two or more organic antibiotic active ingredients, optionally including specific phenolic cannabinoids, optionally in combination with a lipopeptide antibiotic, such as daptomycin, optionally in combination with a specific sesquiterpene. Also disclosed is the therapeutic use of cannabinoid and/or sesquiterpene formulations for the synergistic treatment of enterococcal infections, including in combination with lipopeptide antibiotics, such as daptomycin.

Background

Enterococcus faecium (Enterococcus faecium) and Enterococcus faecalis (Enterococcus faecium) are bacterial species that colonize the human Gastrointestinal (GI) tract asymptomatically, but may also be pathogenic in certain cases (in the relevant literature, Enterococcus faecium (Enterococcus faecium) was previously classified as Streptococcus faecium (Streptococcus faecium), see Schleifer & kilpperballalz, 1984). In particular, these organisms are known to be the main cause of dangerous infections in hospitalized patients treated with antibiotics. During overgrowth, enterococci can penetrate the GI tract wall, migrate into the liver or blood stream where they can cause peritonitis or systemic infections (sepsis). Enterococci have been reported to be the third leading cause of endocarditis, a complication of septicaemia with the greatest risk of death (Murdoch et al, 2009). In hospitalized patients in particular, intestinal colonization by enterococci can help with continued human-to-human transmission of fecal droplets through environmental contamination, leading to opportunistic infections. Passive transmission of fecal matter to the urinary tract in intubated patients has been reported to make enterococci the second most common cause of catheter-related Urinary Tract Infections (UTIs), which can lead to kidney damage (Hidron et al, 2008). Enterococcus faecalis (e.faecium) and enterococcus faecium (e.faecium) are associated with oral infections that cause marginal periodontitis, root canal infections, primary pulp infections, persistent/secondary infections, caries, peri-implantitis, peri-radicular abscesses, and oral mucosal lesions (Najafi et al, 2019). Enterococcus faecium (e.faecium) is also an important multidrug resistant nosocomial pathogen that causes biofilm-mediated infections in patients using medical devices (Paganelli et al, 2013).

The frequency of antibiotic resistance makes the therapy of enterococcal infections more difficult. Conventional treatments include penicillin administered with a synergistic aminoglycoside. However, the emergence of tolerance in enterococcus faecium (e.faecium), particularly mediated by mutations in the penicillin binding protein (pbp5), severely limits the application of this conventional strategy (galvoway-penna et al, 2009, Arias & Murray, 2012). To combat penicillin-resistant strains, vancomycin has become the treatment of choice. However, strains of the genus Enterococcus (Enterococcus spp.) have become increasingly vancomycin-resistant (VRE), often due to the acquisition of the VanA and vancomycin resistance systems (Arthur & Courvalin, 1993). From a public health perspective, the spread of VRE as a nosocomial infection has been particularly problematic, as it has been reported to result in a 1.8-fold higher mortality rate and result in an average of 5 days longer hospital stays, according to the integrated analysis (Prematunge et al, 2016).

Currently, there are two drugs used in VRE treatment, linezolid and daptomycin. However, while linezolid resistance has been reported to be rare, it appears that daptomycin resistance often reappears during daptomycin treatment (Arias et al, 2011); of these strains, linezolid is the only effective antibiotic available. Linezolid, the only available therapy, is problematic due to a cascade of side effects such as hepatotoxicity, myelosuppression and serotonin syndrome (if used with other serotonin-containing drugs). Given the limited options for treatment of enterococcal infections and the adverse effects associated with existing treatments, new treatments are urgently needed.

Extensive physiological activity has been attributed to compounds derived from flowering plants of the Cannabis (Cannabis) genus, in particular phytocannabinoid compounds (see Cunha et al, 1980; Morales et al, 2017; U.S. Pat. No. 6,630,507). More than 80 cannabinoids are found in cannabis plant extracts (Russo,2011), including: cannabidiol (CBD), the acid form of cannabidiolic acid (CBDA), cannabichromene (CBC), the acid form of cannabichromenic acid (CBCA), Cannabigerol (CBG), the acid form of cannabigerolic acid (CBGA), Tetrahydrocannabinol (THC) and the acid form of tetrahydrocannabinic acid (THCA). Studies have shown that Cannabis sativa (Cannabis) extracts or compounds derived from Cannabis sativa (Cannabis) plants have a very broad, but often undefined, anti-microbial activity (Van Klingeren & Ten Ham, 1976; Abdelaziz, 1982; appendix et al, 2011; appendix et al, 2008; Eisohly et al, 1982; appendix et al, 2008; Turner & Elsohly, 1981; Mechoulam & Gaoni, 1965; WO 2012/012498; WO 2018/011813).

Terpenes are another molecular component of plants with a wide range of physiological activities, including the Cannabis (Cannabis) plant. For example, the unique aroma and flavor portions that hops provide to beer are reported to be from certain sesquiterpenes (including alpha-humulene and beta-caryophyllene; see Steenackers et al, 2015). Terpenes in Cannabis (Cannabis) have been reported to include far more than 100 different compounds (Andre et al, 2016). Aromatic terpenes are reported to modulate the physiological effects of cannabinoids, in particular the psychoactive effects (Russo, 2011).

Disclosure of Invention

One general aspect of the innovations disclosed herein includes a formulation comprising at least two antibiotic active ingredients, for example, selected from the group consisting of: cannabinoids as one or more of cannabichromene (CBC), Cannabidiol (CBD) and/or Cannabigerol (CBG). The formulation may also include a sesquiterpene that is one or both of alpha-humulene and/or beta-caryophyllene. The formulation may also include a lipopeptide antibiotic that is daptomycin or an analog thereof. The formulation may include antibiotic active ingredients in relative amounts that are synergistically effective, for example, where effective amounts of the formulation are synergistically effective in an assay to inhibit the growth and/or reproduction of Enterococcus faecium (Enterococcus faecium) or Enterococcus faecalis (Enterococcus faecium).

Implementation processes (implantation) may include one or more of the following features. The formulation consists in that two antibiotic active ingredients are present and the two antibiotic active ingredients are a cannabinoid and a sesquiterpene. The formulation consists in that two antibiotic active ingredients are present and the two antibiotic active ingredients are a cannabinoid and a lipopeptide antibiotic. The formulation includes a cannabinoid, a sesquiterpene, and a lipopeptide antibiotic. The formulation consists in that two antibiotic active ingredients are present and the two antibiotic active ingredients are a sesquiterpene and a lipopeptide antibiotic. The formulation according to any one of the preceding claims, wherein the cannabinoid is one of CBC, CBD or CBG. The formulation according to any one of the preceding claims, wherein the cannabinoids are two of CBC, CBD and CBG. The formulation according to any one of the preceding claims, wherein the cannabinoids include CBC, CBD and CBG. The formulation of any one of the above claims wherein the sesquiterpene, if present, is one of alpha-humulene or beta-caryophyllene. The formulation of any one of the above wherein the sesquiterpenes, if present, comprise alpha-humulene and beta-caryophyllene. The formulation of any one of the above, wherein the lipopeptide antibiotic, if present, is daptomycin. The formulation according to any one of the preceding claims wherein the sesquiterpene, when present, is present in a relative amount that provides a reduction in the Minimum Inhibitory Concentration (MIC) of cannabinoid by at least 2 to 128 divisor in the assay. The formulation according to any one of the preceding claims wherein the cannabinoids, when present, are present in relative amounts that provide a reduction in the Minimum Inhibitory Concentration (MIC) of sesquiterpene in the assay of at least 2 to 128 divisor. The formulation according to any one of claims wherein the sesquiterpenes and/or cannabinoids, when present, are present in a relative amount that provides a reduction in the Minimum Inhibitory Concentration (MIC) of the lipopeptide antibiotic in the assay of at least 2 to 128 divisors. The formulation according to any wherein the molar ratio of sesquiterpene to cannabinoid, when present, is ≥ 50. Any one of the formulations further comprises a pharmaceutically acceptable excipient, and wherein the antibiotic active ingredient is dissolved, dispersed, mixed or suspended in the formulation. The formulation according to any of the above claims wherein the cannabinoid, when present, is present at 0.1-100 mg/L. The formulation according to any one of claims wherein the sesquiterpene, when present, is present at 0.1-500 mg/L. The formulation according to any one of the preceding claims wherein the cannabinoid and/or sesquiterpene, when present, is derived from a plant extract. The formulation is wherein the plant is a Cannabis sativa (Cannabis sativa) or Cannabis sativa (Cannabis indica) plant. The formulation according to any one of the preceding claims wherein the formulation does not comprise any alternative cannabinoid, terpene or lipopeptide antibiotic. The formulation according to any one of claims wherein the formulation substantially comprises two or more of a cannabinoid, a terpene, or a lipopeptide antibiotic. Use of any one of the formulations for formulating a medicament. Thus, the use is wherein the medicament is for use in treating an enterococcal infection in a subject in need thereof. Thus, the use is wherein the enterococcal infection is an Enterococcus faecium (Enterococcus faecium) or Enterococcus faecalis (Enterococcus faecium) infection. The use of any one of the preceding claims, wherein the enterococcal infection is an oral infection. Thus, the use is wherein the oral infection is marginal periodontitis, root canal infection, primary pulp infection, persistent or secondary infection, dental caries, peri-implantitis, peri-radicular abscess or oral mucosal lesion. The use of any one of the above, wherein the enterococcal infection is an antibiotic-resistant enterococcal infection. Thus, the use is wherein the antibiotic-resistant enterococcus infection is a vancomycin-and/or daptomycin-resistant enterococcus infection. Use according to any one of the claims, wherein the formulation is for use in an amount to deliver an effective dose of cannabinoid (when present) in the range of from 1 to 5,000 mg/day and/or an effective dose of sesquiterpene (when present) in the range of from 1 to 10,000 mg/day. Use of a formulation according to any one for treating an enterococcal infection in a subject in need thereof. A formulation according to any one for use in treating an enterococcal infection in a subject in need thereof. The formulation is wherein the enterococcal infection is an Enterococcus faecium (Enterococcus faecium) or Enterococcus faecalis (Enterococcus faecium) infection. The formulation is wherein the enterococcal infection is an oral infection. The formulation is wherein the oral infection is marginal periodontitis, root canal infection, primary pulp infection, persistent or secondary infection, dental caries, periimplantitis, periradicular abscess, or oral mucosal lesion. The formulation of any one of the above wherein the enterococcal infection is an antibiotic-resistant enterococcal infection. The formulation is wherein the antibiotic-resistant enterococcus infection is a vancomycin-and/or daptomycin-resistant enterococcus infection. The formulation according to any one of the preceding claims wherein the formulation is for use in an amount to deliver an effective dose of cannabinoid (when present) in the range of from 1 to 5,000 mg/day and/or an effective dose of sesquiterpene (when present) in the range of from 1 to 10,000 mg/day.

An alternative aspect of the disclosed innovations includes a method of treating an enterococcal infection in a subject in need thereof, comprising administering to the subject an effective amount of a formulation disclosed herein. Implementation processes (implantation) may include one or more of the following features. The method comprises two antibiotic active ingredients, wherein the two antibiotic active ingredients are a cannabinoid and a sesquiterpene, the method further comprising treating the subject with an effective amount of one or more antibiotics, optionally wherein the antibiotic is daptomycin. The method comprises administering the cannabinoid in an effective amount of 1-5,000 mg/day. The method according to any one of the claims comprising administering the sesquiterpene in an effective amount of 1-10,000 mg/day. The method according to any one of the preceding claims, wherein the subject is a mammal. The method wherein the mammal is a human.

An alternative aspect of the innovations herein includes a method of treating a microbial infection in a subject in need thereof, the method comprising administering to the subject, sequentially or in combination, an effective amount of at least two antibiotically active compounds selected from cannabinoids as one or more of cannabichromene (CBC), Cannabidiol (CBD) and/or Cannabigerol (CBG). The treatment methods further include sesquiterpenes that are one or both of alpha-humulene and/or beta-caryophyllene; and (c). The method of treatment further includes a lipopeptide antibiotic that is daptomycin or an analog thereof. The method of treatment further comprises wherein the antibiotic active compounds are administered in synergistically effective relative amounts that are therapeutically effective against microbial infections.

Implementations of the disclosed methods may include one or more of the following features. The method is wherein the synergistically effective relative amounts are synergistically effective to inhibit the growth and/or reproduction of Enterococcus faecium (Enterococcus faecium) or Enterococcus faecalis (Enterococcus faecium) in the assay. Said method consisting in co-administering said cannabinoid and said sesquiterpene, or co-administering said sesquiterpene and said lipopeptide antibiotic, or co-administering said cannabinoid, said sesquiterpene and said lipopeptide antibiotic. The method resides therein in that the cannabinoid and the sesquiterpene, or the sesquiterpene and the lipopeptide antibiotic, or the cannabinoid, the sesquiterpene and the lipopeptide antibiotic, are administered sequentially in any order. The method of any one of the methods further comprising further treating the subject with the lipopeptide antibiotic alone. The method according to any one of the preceding claims, wherein the cannabinoid, if administered, is one of CBC, CBD or CBG. The method according to any one of the preceding claims, wherein the cannabinoid, if administered, is two of CBC, CBD and CBG. The method according to any of the preceding claims, wherein the cannabinoids, if administered, comprise CBC, CBD and CBG. The method according to any one of the preceding claims wherein the sesquiterpene, if applied, is one of alpha-humulene or beta-caryophyllene. The method according to any one of the preceding claims wherein the sesquiterpenes, if applied, comprise alpha-humulene and beta-caryophyllene. The method of any one of the preceding claims, wherein the lipopeptide antibiotic, if administered, is daptomycin. The method according to any one of the preceding claims wherein the sesquiterpene, if administered, is administered in a relative amount that provides a reduction in the Minimum Inhibitory Concentration (MIC) of cannabinoid by at least 2 to 128 divisor, when administered. The method according to any one of the preceding claims wherein the cannabinoid, if administered, is administered in a relative amount that provides a reduction in the Minimum Inhibitory Concentration (MIC) of sesquiterpene, by at least 2 to 128 divisor, when administered. The method according to any one of the preceding claims wherein the sesquiterpene and/or the cannabinoid is administered in a relative amount that provides a reduction in the Minimum Inhibitory Concentration (MIC) of the lipopeptide antibiotic by at least 2 to 128 divisor, when administered. The method according to any one of the preceding claims, wherein the microbial infection is an enterococcal infection. The method is wherein the enterococcal infection is Enterococcus faecium (Enterococcus faecium) or Enterococcus faecalis (Enterococcus faecium) infection. The method wherein the enterococcal infection is an oral infection. The method is wherein the oral infection is marginal periodontitis, root canal infection, primary pulp infection, persistent or secondary infection, dental caries, periimplantitis, periradicular abscess, or oral mucosal lesion. The method according to any one of the preceding claims, wherein the microbial infection is an antibiotic-resistant infection. The method is wherein the antibiotic-resistant infection is a vancomycin-and/or daptomycin-resistant microbial infection.

In an alternative aspect, there is provided a method of treating a bacterial infection in a subject in need thereof, the method comprising: administering to the subject an effective amount of: cannabinoids as one or more of cannabichromene (CBC), Cannabidiol (CBD) and/or Cannabigerol (CBG); and, a lipopeptide antibiotic that is daptomycin or an analog 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: administering the lipopeptide antibiotic in an effective amount of less than 4mg/kg if the cannabinoid is a CBD; and/or, if the cannabinoid is a CBD, the bacterial infection comprises an infection by an infectious organism having a daptomycin MIC of 4 μ g/mL or greater; and/or, if the cannabinoid is a CBD, the bacterial infection comprises an infection by an infectious organism for which a bacterial growth assay result indicates that daptomycin produces an inhibitory and/or bactericidal effect at a concentration of less than 4 μ g/mL when CBD is administered in an amount less than the MIC of CBD. The infection may, for example, comprise infection by Enterococcus faecium (Enterococcus faecium) or Enterococcus faecalis (Enterococcus faecium). These treatments may also include administering to the subject an effective amount of a sesquiterpene that is one or both of alpha-humulene and/or beta-caryophyllene.

In an alternative aspect, there is provided an antibiotic formulation comprising: cannabinoids as one or more of cannabichromene (CBC) and/or Cannabigerol (CBG); and a lipopeptide antibiotic which is daptomycin or an analog thereof; wherein the cannabinoid and the lipopeptide antibiotic are present in an antibiotically effective weight ratio of from 16:1 to 1: 16. The cannabinoids may, for example, be present in an amount that reduces the Minimum Inhibitory Concentration (MIC) of the lipopeptide antibiotic in an antibiotic assay, such as an Enterococcus assay that evaluates the inhibition of growth and/or reproduction of Enterococcus faecium (Enterococcus faecium) or Enterococcus faecalis (Enterococcus faecium). In selected embodiments, the cannabinoid lowers the MIC of the lipopeptide antibiotic in the assay when the cannabinoid is present in an amount less than the MIC of the cannabinoid. These formulations may also include sesquiterpenes, such as one or both of alpha-humulene and/or beta-caryophyllene.

Drawings

Fig. 1 includes 12 line graphs showing the effect of CBD, CBG or CBC on the viability of enterococcus faecium (e.faecium). Enterococcus strains 33D3(A-C), 69C6(D-F), 58C9(G-I), and 55A6(J-L) were incubated with 0 ×,1 × (1mg/L), 2 × (2mg/L), or 4 × (4mg/L) MIC of CBD (A, D, G, J), CBG (B, E, H, K), or CBC (C, F, I, L). CFU was determined at 2, 4, 6, 8 and 24h by serial dilution.

Fig. 2 includes 8 line graphs showing the effect of daptomycin (Dap) and Dap in combination with CBD, CBG or CBC on daptomycin-resistant enterococcus faecium (e.faecium) VRE strains 58C9 and 55a6 (enterococcus faecium (e.faecium) clinical strain 55a6 is resistant to daptomycin, which has a daptomycin MIC of 8 mg/L). As shown, the addition of about 1/2MIC of each cannabinoid helped the killing by daptomycin with about 1/4-1/8 × MIC of daptomycin.

FIG. 3 includes 4 line graphs showing the effect of various concentrations of CBD, CBG and CBC on the bactericidal activity of daptomycin. Enterococcus faecium (e.faecium) strain 33D3 was incubated with (a) CBD, (C) CBG or (D) CBC without cannabinoid, or 1/2 × (0.5mg/L) MIC, and daptomycin at 1 × (1mg/L), 1/2 × (0.5mg/L), 1/4 × (0.25mg/L), or 1/8 × (0.125mg/L) MIC. Aliquots were removed at 2, 4, 6, 8 and 24h plating to monitor viability (CFU). Data points are the mean from 3 replicates with standard deviation indicated as error bars.

Fig. 4 includes two histograms showing the effect of CBD, CBG and CBC on the biofilm of enterococcus faecium (e.faecium) strain 33D 3.

Detailed Description

In one aspect, there is provided a pharmaceutical formulation comprising at least two antibiotic active ingredients selected from the group consisting of: cannabinoids as one or more of cannabichromene (CBC), Cannabidiol (CBD) and/or Cannabigerol (CBG); sesquiterpenes which are one or both of alpha-humulene and/or beta-caryophyllene; and a lipopeptide antibiotic that is daptomycin or an analog thereof. The antibiotic effective ingredients may be provided in synergistically effective relative amounts. For example, cannabinoids and sesquiterpenes may be provided at concentrations that are only antibiotic active in synergistic combinations, such as cannabinoids at ≦ 1 μ g/ml and terpenes at ≦ 32 μ g/ml. In a synergistic combination, the inhibitory concentration of a companion may, for example, be reduced by a factor of two or more, for example, 2-16 times. Alternatively, the relative weight ratio of cannabinoid to sesquiterpene may be, for example, from about 1:5 to 1:50, or from 1:8 to 1:32, or from 1:12 to 1: 32. The relative concentrations of the antibiotic active ingredients in the formulation may be arranged such that an effective amount of the formulation will be synergistically effective in an assay to inhibit the growth and/or proliferation of Enterococcus faecium (Enterococcus faecium) or Enterococcus faecalis (Enterococcus faecalis). Formulations providing such synergistic formulations may be used for the treatment of Enterococcus or other microbial infections, for example, for the treatment of infections with Enterococcus species that are relatively closely related to reference strains of Enterococcus faecium (Enterococcus faecium) or Enterococcus faecalis (Enterococcus faecalis) (see Zhong et al, 2017). For example, the enterococcus species suitable for treatment may be at least as closely related to the reference strain as the most distant related strains of enterococcus faecium (e.faecium) and enterococcus faecalis (e.faeciis). Microorganisms suitable for treatment may, for example, be resistant to antibiotics, e.g., resistant to daptomycin-resistant VRE strains. For example, Enterococcus durans (Enterococcus durans) is suitable for treatment, for example, in the context of veterinary diseases. Other enterococcus species closely related to enterococcus faecium (e.faecium) or enterococcus faecalis (e.faecium) may, for example, include enterococcus mundtii (e.mundtii), enterococcus durans (e.durans), enterococcus hirae (e.hirae), enterococcus murinus (e.ratti), enterococcus villosus (e.villorum), enterococcus thailandicus (e.thailandicus) and e.phoeniculola, enterococcus termites (e.teris mitus), enterococcus beike (e.quebecensis), enterococcus moraxensis (e.moraviviansis), enterococcus casseli (e.caccae), enterococcus lysostaphinus peroxide (e.haemeemophiloxidus) and enterococcus casselii (e.sienesiaceus).

In selected embodiments, concentrations of the antibiotic active ingredients below the MIC of each ingredient, e.g., slightly below the MIC, are used to maximize synergy and/or potentiation. Thus, the components may be present in relative amounts that approximate the ratio of their respective MICs. This may occur, for example, when the terpene/cannabinoid molar ratio 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. For example, the synergistic combination may have a molar ratio of cannabinoid to sesquiterpene between 1:8 and 1: 32. Pharmaceutically acceptable excipients may optionally be included in the formulation, and the cannabinoid and the sesquiterpene may be dissolved, dispersed, mixed or suspended in the formulation. A method of formulation may include producing a sterile lyophilized powder, for example, in a vial for reconstitution.

The cannabinoids and sesquiterpenes may, for example, be derived from plant extracts such as extracts of Cannabis sativa (Cannabis sativa) or Cannabis indica (Cannabis indica). Biosynthetic methods for producing cannabinoids and sesquiterpenes are also available, as are various synthetic methods (e.g., based on methods used to synthesize THC/dronabinol, see U.S. patent No.7,323,576 and Trost and Dogra, 2007). An alternative approach involves expressing the cannabinoid biosynthesis genes in a recombinant host, such as a recombinant yeast (see Luo et al, 2019).

Derivatives or analogs of daptomycin may, for example, be used as a lipopeptide antibiotic, either as an alternative to, or in addition to, the use of daptomycin per se (see Nguyen et al, 2006; Baltz, 2006; and Miao et al, 2006; Nguyen et al, 2010). In selected embodiments, the daptomycin analog is a lipopeptide antibiotic that has synergistic antibiotic activity with one or more of the cannabinoids and/or sesquiterpenes in the formulations disclosed herein, for example, in an antibiotic activity assay for enterococcus species. Daptomycin analogs or derivatives may differ from daptomycin, for example, by 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, with a natural or unnatural amino acid. Daptomycin analogs may, for example, include the cyclic depsipeptide daptomycin or derivatives thereof, where attachment to Trp is¹The fatty acid chain of the amino group is an isoundecanoyl, isododecanoyl or isotridecanoyl group.

In alternative formulations, one or more other compounds may be included or specifically excluded, including, for example: terpenes, terpenoids, sterols, triglycerides, alkanes, squalene, vitamin E, carotenoids, chlorophyll, flavonoid glycosides or alkaloids.

The anti-microbial agent may be used for prophylactic or therapeutic treatment of a microbial infection, or to otherwise inhibit microbial growth or proliferation. Antibiotics are antimicrobial agents that are active against bacteria, and in this context, antibiotics include naturally occurring and synthetic substances that kill or inhibit the growth or proliferation of bacteria by any mechanism, including antibacterial or bactericidal means.

Subjects suitable for treatment include mammalian subjects, such as human patients, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows, sheep, goats, pigs, horses, poultry), or domestic pets (e.g., dogs, cats, rodents, birds), e.g., belonging to a taxonomic group of primates, dogs, cats, cows, goats, horses, sheep, pigs, rodents, birds, or lagomorphs. The human patient to be treated may, for example, be at a particular stage of development: neonatal, infant, juvenile, adolescent, adult and elderly male or female patients. Specific veterinary indications suitable for treatment may, for example, include treatment of enterococcal infections in avian species, for example, Enterococcus cecostum (Enterococcus cecorm) infections in chickens.

In alternative embodiments, the formulations may be administered in a dose or dosage form that delivers and/or maintains a synergistically effective amount of the active ingredients.

The cannabinoid and/or sesquiterpene components of the formulation may, for example, be in the form of, 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 beta-caryophyllene are isomers, usually found together in a variety of plants. In addition, pharmaceutically acceptable excipients may be included, and the formulation may be provided in a titratable dosage form. In selected embodiments, the cannabinoid component of the formulation may be obtained as an extract from a culture of a plant of the Cannabis (Cannabis) genus, e.g., Cannabis sativa (Cannabis sativa) or Cannabis indica (Cannabis indica) or a recombinant yeast host. Various methods can be used to prepare these plant extracts, including(not limited to) use of CO₂Supercritical or subcritical extraction, extraction using high temperature gas, and extraction using solvent. The formulation may also specifically exclude other terpenoids or terpenes, including plant-derived terpenoids or terpenes, such as astaxanthin or other sesquiterpenes, tetraterpenes, triterpenes, diterpenes or monoterpenes.

The titratable dose may be adjusted, for example, to allow the patient to take medication at a dose less than the unit dose, where a "unit dose" is defined as the maximum medication dose that can be taken at any one time or within a particular dose period. Dose titration will allow different patients to escalate the dose until they feel the medication effective, since not all patients will need the same dose to achieve the same benefit. A person who is larger in size or metabolizes faster may require a larger dose to achieve the same effect as another person who is smaller in size or metabolizes slower. Therefore, titratable doses have advantages over standard dosage forms.

In selected embodiments, the formulation can be tailored to deliver in a manner that targets one or more of: sublingual, buccal, oral, rectal, nasal, parenteral and through the pulmonary system. The formulation may, for example, be in one or more of the following forms: gels, gel sprays, tablets, liquids, capsules, by injection or for evaporation.

Conventional pharmaceutical practice may be used to provide a suitable formulation or composition for administration of the formulation to a subject. Routes of administration may, for example, include parenteral, intravenous, intradermal, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracisternal, intraperitoneal, intranasal, inhalation, aerosol, topical, sublingual, or oral administration. The therapeutic formulation may be in the form of a liquid solution or suspension; for oral administration, the formulation may be in the form of a tablet or capsule; for intranasal formulations, in the form of powders, nasal drops or aerosols; and in the form of drops, aerosols or tablets for sublingual formulations.

Methods well known in The art for preparing formulations exist, for example, "Remington: The Science and Practice of Pharmacy" (21 st edition), eds David Troy, 2006, Lippincott Williams & Wilkins. 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 naphthalenes. Biocompatible, biodegradable lactide polymers, lactide/glycolide copolymers, or polyethylene oxide-polypropylene oxide copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems include ethylene vinyl acetate particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyethylene oxide-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as gels.

The pharmaceutical compositions of the present invention may be in any form that allows the composition to be administered to a patient. For example, the composition may be in the form of a solid, liquid or gas (aerosol). The pharmaceutical compositions of the present invention are formulated so as to allow the active ingredients contained therein to be bioavailable once the composition is administered to a patient. The composition to 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 contain multiple dosage units.

The materials used in preparing the pharmaceutical compositions should be pharmaceutically pure and non-toxic in the amounts used. The compositions of the present invention may include one or more compounds (active ingredients) known for a particular desired effect. It will be apparent to those skilled in the art that the optimum dosage of the active ingredient 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 mode of administration, and the composition used.

Typically, the pharmaceutical compositions comprise a formulation of the invention as described herein in admixture with one or more carriers. The carrier may be a granulate, whereby the composition is in the form of, for example, a tablet or a powder. The carrier may be a liquid, wherein the composition is, for example, an oral syrup or an injectable liquid. Additionally, the carrier may be a gas to provide an aerosol composition useful, for example, in inhalation administration.

When intended for oral administration, the composition is preferably in a solid or liquid form, with semi-solid, semi-liquid, suspension, and gel forms being encompassed within forms considered herein to be solid or liquid.

As a solid preparation for oral administration, the composition may be formulated into the form of powder, granules, tablets, pills, capsules, cachets, chewing gums, flakes, lozenges and the like. Such solid compositions will generally contain one or more inert diluents or edible carriers. In addition, one or more of the following adjuvants may be present: binders, such as syrup, acacia, sorbitol, polyvinylpyrrolidone, carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth or gelatin, and mixtures thereof; excipients, such as starch, lactose or dextrin, disintegrating agents, such as alginic acid, sodium alginate, sodium carboxymethyl starch, corn starch, etc.; lubricants, such as magnesium stearate or Sterotex; fillers such as lactose, mannitol, starch, calcium phosphate, sorbitol, methyl cellulose 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, silicon dioxide, wetting agents, such as sodium lauryl sulfate, glidants, such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin, flavouring agents, such as peppermint, methyl salicylate or orange flavouring agents, and colouring agents. When the composition is in the form of a capsule, for example, a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or a fatty oil.

The formulations may be in liquid form, e.g., elixirs, syrups, solutions, aqueous or oil emulsions or suspensions, or even dry powders that may be reconstituted with water and/or other liquid vehicle prior to use. As two examples, the liquid may be for oral administration or for delivery by injection. When intended for oral administration, preferred compositions contain, in addition to the compounds of the invention, one or more of sweeteners, thickeners, preservatives (e.g., alkyl p-hydroxybenzoates), dyes/colorants, and flavor enhancers (flavorants). In compositions intended for administration by injection, one or more of a surfactant, a preservative (e.g., an alkyl p-hydroxybenzoate), a wetting agent, a dispersing agent, a suspending agent (e.g., sorbitol, glucose or other syrup), a buffer, a stabilizer, and an isotonic agent may be included. The emulsifier may be selected from lecithin or sorbitol monooleate.

Whether they are solutions, suspensions or other similar forms, the liquid pharmaceutical formulations of the invention may include one or more of the following adjuvants: sterile diluents, such as water for injection, saline solutions, preferably physiological saline, ringer's solution, isotonic sodium chloride, fixed oils which may be used as solvents or suspending vehicles, such as synthetic mono-or diglycerides, polyethylene glycols, glycerol, 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 adjusting tonicity such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

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. For example, the substrate may include one or more of: paraffin oil, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. The thickening agent may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may comprise a transdermal patch or an iontophoretic device.

The formulation may be intended for rectal administration in the form of, for example, a suppository which will melt in the rectum and release the drug. Compositions for rectal administration may contain an oily base as a suitable non-irritating excipient. These substrates include, without limitation, lanolin, cocoa butter, and polyethylene glycols. Low melting waxes are preferred for the preparation of suppositories, with mixtures of fatty acid glycerides and/or cocoa butter being suitable waxes. The wax may be melted and the aminocyclohexyl ether compound dispersed homogeneously therein by stirring. The molten homogeneous mixture is then poured into a suitably sized mold, allowed to cool and thereby solidify.

The formulations may include a variety of materials that modify the physical form of the solid or liquid dosage unit. For example, the composition may include a material that forms a coating shell around the active ingredient. The material forming the coating shell is generally inert and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule or cachet.

The pharmaceutical formulation may consist of a gaseous dosage unit, for example, it may be in the form of an aerosol. The term aerosol is used to denote a variety of systems, from those with colloidal properties to systems consisting of pressurized packaging. The delivery may be by liquefied or compressed gas or by a suitable pump system that dispenses the active ingredient. Aerosols of the compounds of the invention may be delivered in a single phase, biphasic or triphasic system to deliver the active ingredient. The delivery of the aerosol includes the necessary containers, triggers (activators), valves, sub-containers, etc., which may collectively 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. For a suitable mode of use (e.g., oral or parenteral route of administration), a suitable salt will be selected to enhance the bioavailability or stability of the compound.

The invention also provides kits containing pharmaceutical formulations and instructions for use of the formulations. Preferably, a commercial package will contain one or more unit doses of the formulation. Light and/or air sensitive formulations may require special packaging and/or formulations. For example, packages may be used that are opaque to light, and/or sealed to avoid 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 (e.g., small molecules, nucleic acid molecules, peptides or peptide analogs) in the presence of a carrier or any pharmaceutically or biologically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion vehicles, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. The carrier may be suitable for any suitable form of administration. Pharmaceutically acceptable carriers generally include sterile aqueous solutions or dispersions and sterile powders. Supplementary active compounds may also be incorporated into the formulation.

An "effective amount" of a formulation according to the invention includes a therapeutically effective amount or a prophylactically effective amount. By "therapeutically effective amount" is meant an amount effective, at dosages and for durations necessary, to achieve the desired therapeutic result. The therapeutically effective amount of the formulation may vary according to factors such as the disease state, the age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. The dosage regimen may be adjusted to provide the optimal therapeutic response. A therapeutically effective amount may also be an amount wherein the therapeutically beneficial effect is greater than any toxic or adverse effects of the formulation or active compound. A "prophylactically effective amount" is an amount effective, at dosages and for durations necessary, to achieve the desired prophylactic result. Typically, a prophylactic dose is administered in a subject prior to or early in the disease, such that the prophylactically effective amount can be less than the therapeutically effective amount. For any particular subject, the timing and dosage of treatment can be adjusted over time (e.g., the time can 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 composition.

For an effective amount of antibiotic treatment, although the daptomycin susceptibility inflection point (breakthrough) of enterococcus is considered to be 4mg/L, there is still controversy as to whether this is appropriate considering that the approved dose is optimized only when the MIC is ≦ 1 mg/L. For example, recent studies found that the probability of achieving a target pharmacokinetic parameter in Daptomycin-treated Enterococcal sepsis only reached 1.5-5.5% when the MIC was 4mg/L and when a dose of 6mg/kg was administered (Avery, Lindsay M et al, "pharmaceutical Analysis of Daptomycin-treated Enterprise bacteria: It Is Time to Change the Break point." Clinical Infections Diseases, Vol.68, (2019): 1650-1657).

In therapeutic applications, synergy occurs between the active ingredients when the observed combined therapeutic effect is greater than the sum of the therapeutic effects of the individual active ingredients or a new therapeutic effect is produced that the individual active ingredients may not produce. Thus, when the formulation components are present in synergistically effective amounts, the formulation achieves a therapeutic effect greater than would be achieved by administering each active ingredient alone at comparable dosages. In this context, the enhancement of the therapeutic effect may take the form of an increased efficacy (efficacy) or a potency (potency) and/or a reduced adverse effect. The synergy may be mediated, in whole or in part, by the pharmacokinetics and/or pharmacodynamics of the active ingredients in the subject, such that the amounts and ratios of the ingredients in the formulation may be synergistic in vivo. Such in vivo synergy may be achieved by formulations comprising active ingredients in amounts and proportions that are also synergistic in an in vitro potency assay. Thus, as used herein, the term "synergistically effective amounts" refers to synergistic amounts in vivo and/or in vitro. The numerical quantification of synergy is generally expressed as a Fractional Inhibitory Concentration Index (FICI), which represents the sum of the Fractional Inhibitory Concentrations (FIC) of each drug tested, where the FIC of each drug is determined by dividing the minimum inhibitory concentration (MIC, the lowest concentration of drug that prevents significant growth of bacteria in a standard in vitro assay-based on a standard colorimetric assay of resazurin) of each drug when used in combination by the MIC of each drug when used alone. Very generally, a FICI of less than or greater than 1 indicates a positive correlation activity (at least addition or enhancement) or absence of positive interaction, respectively. More certainly, the synergy of two compounds can be conservatively defined as a FICI of ≦ 0.5 (see Odds, 2003; where addition or enhancement corresponds to a FICI of >0.5 to ≦ 0.75; no interaction (no difference) corresponds to a FICI of >1 to ≦ 4; and antagonism corresponds to a FICI of > > 4). The synergistic effect of 3 compounds has been defined as FICI of ≦ 1.0. (Berenbaum, 1978; Yu et al, 1980). To estimate the optimal concentration in the triple combination, the optimal concentration of trans-caryophyllene and cannabinoid was used in combination with a series of daptomycin dilutions.

Although various embodiments of the invention are disclosed herein, many variations and modifications are possible within the scope of the invention, in light of the general knowledge of those skilled in the art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numerical ranges include the numbers defining the range. The word "comprising" is used herein as an open term that is substantially equivalent to the term "including (but not limited to)" and the word "comprising" has a corresponding meaning. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an item" includes more than one of that item.

Citation of a reference herein is not an admission that such reference is prior art to the present invention. Any prior documents and all patent publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference. All documents cited or referenced in the documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated by reference herein and may be used in the practice of the invention. More specifically, all documents referred to are incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference and fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and figures. In some embodiments, the present invention does not include steps relating to medical or surgical treatment.

Examples

Example 1:

as shown in this example, the non-psychotherapeutic cannabinoids cannabidiol, cannabigerol and cannabichromene (i.e. CBD, CBG and CBC) are sensitive or resistant to enterococcus faecium (e.faecium) and enterococcus faecalis (e.faecium), including vancomycin, and in some cases, strains that are also resistant to daptomycin have bacteriostatic (tables 1, 2) and bactericidal (figure 1) activity. At equal concentrations of CBD, CBG or CBC, the kinetics of killing enterococcus faecium (e.faecium) with CBG and CBC is faster than with CBD (fig. 1). Cannabinoids have also been shown to increase daptomycin (MIC) activity by 8-128 fold (table 3). In addition, we demonstrated that each cannabinoid had species-specific activity against gram-positive (rather than gram-negative) bacteria (table 1).

As further shown in this example, the non-psychotherapeutic cannabinoids cannabidiol, cannabigerol and cannabichromene (i.e. CBD, CBG and CBC), trans-caryophyllene and daptomycin act synergistically. The interaction assay between all pairs and triple combinations of cannabinoids (CBC, CBD, CBG), trans-caryophyllene and daptomycin is shown in table 4. All pairwise combinations show synergistic (FICI ≦ 0.5) or enhanced (FICI ≧ 0.5 ≦ 0.75) interactions. However, while alpha-humulene (beta-caryophyllene, data not shown) and trans-caryophyllene had synergistic activity with CBC, CBD or CBG (Table 4), other terpenes (alpha-pinene and R- (+) limonene), alone or in combination, had no detectable activity (MIC >32 mg/L; data not shown). All triple combinations are synergistic (FICI < 1.0). Under these conditions, the MIC of daptomycin decreased 256-fold (from 8 to 0.03. mu.g/ml in the presence of CBD and trans-caryophyllene; Table 5) or > 512-fold (from >16 to 0.03 in the presence of CBC and trans-caryophyllene, Table 7D).

As shown in tables 6 and 7, analysis of the double and triple combinations of the drug-resistant (daptomycin and vancomycin) strain VRE 55a6 showed that the daptomycin/terpene/cannabinoid combination was more effective against this strain (compared to vancomycin/daptomycin-sensitive strain 33D 3), which increased the MIC of daptomycin by > 500-fold (effectively inhibiting the daptomycin-resistant phenotype).

The data provided in the present embodiment were obtained as follows.

MIC determination for all bacteria except species of Mycobacterium (Mycobacterium) bacteria:

pre-cultures of bacteria grown in their respective liquid media (as defined below) were diluted to an OD600 of 0.0025, and 100 μ Ι were added to 100 μ Ι of medium in 96-well plates containing serial 2-fold dilutions of cannabinoids (dilution of cannabinoid stock solution in growth medium, for acidic form, in methanol or acetonitrile). Plates were then incubated at 37 ℃ for 20-24h and grown in Thermo Scientific^TM Varioskan^TMRecorded as OD600 in a Flash Multimode microplate reader.

MIC determination of Mycobacterium abscessus (Mycobacterium abscessus) ATCC 19977:

precultures grown in MHII medium with 0.05% tyloxapol were diluted in MHII medium to an OD600 of 0.005 and 100. mu.l were added to 100. mu.l MHII medium containing serial 2-fold dilutions of the cannabinoid (dilution of the cannabinoid stock solution in growth medium, in methanol or acetonitrile for the acidic form) in 96-well plates. The plates were then incubated for 48h, followed by the addition of 30. mu.l of an aqueous solution of the colorimetric reagent resazurin (10mg/100 ml). The plates were incubated for an additional 24h and growth was recorded as the color of the culture switching from blue to pink.

MIC determination of mycobacterium tuberculosis (mycobacterium tuberculosis) H37 Rv:

a preculture of M.tuberculosis (M.tuberculosis) grown in 7H9 medium plus 0.05% tyloxapol was grown to mid log phase and diluted to OD600 of 0.0025 with the same medium without tyloxapol. The diluted cultures were used to inoculate wells in 96-well plates containing 50 μ L of the same medium with serial 2-fold dilutions of cannabinoid (dilution of stock solution of cannabinoid in methanol or acetonitrile in growth medium). Plates were incubated at 37 ℃, 5% CO2 for 5 days, followed by the addition of 10 μ L PrestoBlue cell viability reagent to each well. The plates were incubated at 37 ℃ for another 24h with 5% CO2, and then fluorescence read on a Synergy HT plate reader (excitation 530nm, emission 590 nm).

Growth media for various organisms:

enterococcus faecium (Enterococcus faecium): all strains (Clinical isolates 33D3, 69C6, 58C9 and 55A6)，MHII(Mueller Hinton II)

enterococcus faecalis (Enterococcus faecalis): MHII (Mueller Hinton II)

Streptococcus pyogenes (Streptococcus pyogenes) ATCC 51878: BHI (brain heart infusion) Staphylococcus aureus (Staphylococcus aureus) MRSA USA 300: LB (Luria-Bertani)

Staphylococcus epidermidis (Staphylococcus epidermidis) ATCC 14990: NB (nutrient broth)

Staphylococcus epidermidis (Staphylococcus epidermidis) ATCC 12228: NB (nutrient broth)

Mycobacterium abscessus (Mycobacterium abscessus) ATCC 19977: MHII (Mueller Hinton II)

Mycobacterium tuberculosis (Mycobacterium tuberculosis) H37 Rv: 7H9

Mycobacterium tuberculosis (Mycobacterium tuberculosis) H37 Rv: PB (Proskauer and Beck + glucose and sodium pyruvate)

Acinetobacter baumannii (Acinetobacter baumannii) ATCC 19606: NB (nutrient broth)

Coli (Escherichia coli) HB 101: LB (Luria-Bertani)

Pseudomonas aeruginosa (Pseudomonas aeruginosa) ATCC 27853: tryptic soy broth

Streptococcus pyogenes (Streptococcus pyogenes) ATCC 51878 was grown at 37 ℃ in a three-gas incubator (tri-gas incubator).

The method of figure 1.

The pre-culture was diluted to an OD600 of 0.0025 in 3ml MHII medium supplemented with the appropriate concentration of cannabinoid. At different time points, 100. mu.l of culture was removed from each tube and serial 10-fold dilutions were spotted onto MHII agar plates (10. mu.l), which were incubated for 24h at 37 ℃ and colonies were Counted (CFU).

The methods of tables 1-3. The determination of MIC is performed as described above.

The methods of tables 4-7. The interaction of the cannabinoids CBC, CBD and CBG with terpenes and daptomycin:

the FICI values of cannabinoid (compound a), terpene (compound B), and daptomycin (compound C), alone and in combination, were determined in a 96-well checkerboard format using the MIC assay. Terpene stocks were prepared in DMSO and diluted in growth medium. The fractional inhibitory concentration of each compound was calculated as follows: FIC_A(MIC of compound a in the presence of compound B)/(MIC of compound a alone). Similarly, the FIC of compounds B and C were calculated. Computing FICI as FIC_AAdding FIC_BAdding FIC_C。

Table 1: the cannabinoids were active against different gram positive bacteria but not against gram negative bacteria (Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC 19606, escherichia coli (e.coli) HB101, data not shown). Sensitivity is expressed as MIC (mg/L).

Table 2: the activity of cannabinoids against clinical enterococcus strains (vancomycin sensitivity, vancomycin tolerance, and both vancomycin and daptomycin tolerance) is expressed as MIC (mg/L).

^*Vancomycin resistance

^#Daptomycin resistance (MIC greater than 4mg/L)

Table 3: cannabinoids enhance the activity of daptomycin against clinical isolates of enterococcus faecium (e.faecium).

^*Indicating that the strain is vancomycin resistant.

^#Daptomycin-resistant strains (MIC greater than 4 mg/L).

Table 4: the synergistic and synergistic interaction of the paired and triple combination of cannabinoids (CBC, CBD or CBG), trans-caryophyllene and daptomycin to inhibit the growth of enterococcus faecium (e.faecium)33D 3.

Paired interactions of CBC, trans-caryophyllene and daptomycin

4 B.pairwise interaction of CBD, trans-caryophyllene and daptomycin

4 C.paired interaction of CBG, trans-caryophyllene and daptomycin

Interaction of triple combinations of cannabinoids (CBC, CBD or CBG), trans-caryophyllene and daptomycin

For triple combinations, FICI <1.0 indicates synergy

Table 5: the maximal fold increase in activity of each cannabinoid (CBC, CBD, CBG), trans-caryophyllene, and daptomycin against enterococcus faecium (e.faecium)33D3 in both paired and triple combinations.

Pairwise interactions of CBC, trans-caryophyllene and daptomycin

Pairwise interactions of CBD, trans-caryophyllene and daptomycin

Paired interaction of CBG, trans-caryophyllene and daptomycin

Interaction of triple combinations of cannabinoids (CBC, CBD or CBG), trans-caryophyllene and daptomycin

Table 6: the synergistic and enhanced pair-wise or triple interaction of cannabinoids (CBC, CBD or CBG), trans-caryophyllene and daptomycin to inhibit the growth of enterococcus faecium (e.faecium) VRE 55a 6.

6 A.pairwise interaction of CBC, trans-caryophyllene and daptomycin

6 B.pairwise interaction of CBD, trans-caryophyllene and daptomycin

6 C.paired interaction of CBG, trans-caryophyllene and daptomycin

Interaction of triple combinations of cannabinoids (CBC, CBD or CBG), trans-caryophyllene and daptomycin

^*For triple combinations, FICI<1.0 denotes synergy

Table 7: the maximal fold increase in activity of individual cannabinoids (CBC, CBD, CBG), trans-caryophyllene or daptomycin against enterococcus faecium VRE 55a6 in paired or triple combinations.

7 A.paired interaction of CBC, trans-caryophyllene and daptomycin

Paired interaction of CBD, trans-caryophyllene and daptomycin

7 C.paired interaction of CBG, trans-caryophyllene and daptomycin

Interaction of triple combinations of cannabinoids (CBC, CBD or CBG), trans-caryophyllene and daptomycin

The above data show a particularly important interaction with certain cannabinoid-daptomycin combinations, which demonstrates a synergistic effect: for example, table 6A, for CBC + daptomycin, FICI ═ 0.5; table 6B, for CBD + daptomycin, FICI ═ 0.5. In addition to the respective relative amounts that produce the synergistic effect, the data show that other relative amounts produce unexpected and unexpected significant growth inhibition of enterococcus faecium (e.faecium): for example, Table 5A shows that the MIC for daptomycin decreased 128-fold (MIC decreased from 4 to 0.03125 mg/L). This effect occurs when the CBC is present at its 1/2MIC (i.e., the CBC MIC is reduced from 1 to 0.5 mg/L; data not shown). By at the enhancement ratio, 0.5: relative amount of 0.03125 ═ 16:1 CBC: this interaction occurs with each of the daptomycin partners.

Example 2: sensitization of cannabinoids to daptomycin.

As shown in FIG. 2, cannabinoids sensitize antibiotic-resistant strains of enterococcus to daptomycin. Fig. 2 reflects data showing the effect of daptomycin (Dap) and Dap in combination with CBD, CBG or CBC on daptomycin-resistant enterococcus faecium (e.faecium) VRE strains 58C9 and 55a 6. Enterococcus faecium (e.faecium) strain 55a6 is resistant to daptomycin, wherein the MIC of daptomycin is 8 mg/L. Enterococcus faecium (E.faecium) strains 58C9 and 55A6 were incubated with daptomycin for MIC of either cannabinoid-free (A, B) or 1/2 × (0.5mg/L) CBD (C, D), CBG (E, F) or CBC (G, H) and 1 × (4mg/L for 58C 9; 8mg/L for 55A 6), 1/2 × (2mg/L for 58C 9; 4mg/L for 55A 6), 1/4 × (1mg/L for 58C 9; 2mg/L for 55A6, 2mg/L) or 1/8 × (0.5mg/L for 58C 9; 1mg/L for 55A 6). Aliquots were removed at 2, 4, 6, 8 and 24h for CFU counting. Data points are the mean from 3 replicates with standard deviation indicated as error bars. As shown, the addition of about 1/2MIC of each cannabinoid helped daptomycin at 1/4-1/8 × MIC, through the killing of daptomycin.

To further show the bactericidal enhancing activity of CBD, CBG and CBC with daptomycin, a kill curve was generated using enterococcus faecium (e.faecium) strain 33D3 (daptomycin sensitive, MIC ═ 1), as shown in fig. 3. This data is usefully compared to that of fig. 2 for enterococcus faecium (e.faecium) strain 58C9 (daptomycin intermediate resistance, MIC ═ 4) and 55a6 (daptomycin resistance, MIC ═ 8). As indicated, the strains were incubated with 1/8 × to 1 × MIC daptomycin (0.25-1 mg/L daptomycin for 33D3, 1-8mg/L daptomycin for 58C 90.5-4 mg/L and 55A 6), and CBD, CBG or CBC, respectively, at 0.5 × MIC. At 1/2 × MIC, there was no effective growth inhibition in all strains by either the cannabinoid or daptomycin. Notably, while daptomycin did inhibit growth at 1 × MIC, there was no reduction in CFU. However, 1/2 × MIC CBD, CBG or CBC combined with 1 × to 1/8 × MIC daptomycin significantly inhibited the growth and reduced CFU of all strains, thereby confirming bactericidal activity (fig. 2 and 3). This is of particular clinical relevance, since patients infected with strains classified as daptomycin resistant (MIC >4mg/L) are not usually treated with daptomycin. Strains 58C9 and 55a6 were moderately resistant and tolerant, respectively, to daptomycin, but as little as 1mg/L of daptomycin (1/8 × MIC) could significantly reduce CFU at 24h in the presence of 1/2 × MIC CBD, CBG or CBC (fig. 2).

This example further shows that a broad relative amount of the cannabinoid daptomycin can provide unexpected and unexpected results, as demonstrated by the kill curves in fig. 2 and 3. For example, an unexpected and clinically meaningful effect was observed in the following relative amounts of the cannabinoid daptomycin: for CBC, daptomycin 1:1-4 (FIG. 3, strain 33D 3), 1:1-8 (FIG. 2; strain 58C9) and 1:4-16 (FIG. 2, strain 55A 6); for CBG, daptomycin 2:1 and 1:1-4 (FIG. 3, strain 33D 3), 1:1-8 (FIG. 2; strain 58C9) and 1:2-16 (FIG. 2; strain 55A 6); and for CBD daptomycin 2:1 and 1:1-4 (FIG. 3, strain 33D 3), 1:1-8 (FIG. 2; 58C9) and 1:2-16 (FIG. 2; strain 55A 6).

Example 3: CBD and CBG biofilm inhibition

This example shows that CBD and CBG are surprisingly effective at inhibiting the growth of enterococcus faecium (e.faecium) biofilms. The data in fig. 4 were generated by growing a biofilm of enterococcus faecium (e.faecium) strain 33D3 for 48h without cannabinoids and then treating the biofilm for 72h with cannabinoids. The biofilm mass was quantified by crystal violet staining. Live bacteria in biofilms were quantified by scraping the biofilm and then counting the live bacteria. As shown, CBD and CBG showed an unexpected degree of antibiotic activity against strain 33D3 of enterococcus faecium (e.faecium). Notably, CBC is bactericidal against enterococcus plankton.

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