U.S. patent application number 17/607247 was filed with the patent office on 2022-07-14 for antibiotic cannabinoid-terpene formulations.
The applicant listed for this patent is The University of British Columbia. Invention is credited to Manisha Dosanjh, Dana M. Lambert, Mark C. Pryjma, Charles J. Thompson.
Application Number | 20220218654 17/607247 |
Document ID | / |
Family ID | |
Filed Date | 2022-07-14 |
United States Patent
Application |
20220218654 |
Kind Code |
A1 |
Thompson; Charles J. ; et
al. |
July 14, 2022 |
ANTIBIOTIC CANNABINOID-TERPENE FORMULATIONS
Abstract
Pharmaceutical formulations are provided that include at least
two antibiotically active ingredients: a cannabinoid that is one or
more of cannabichromene (CBC), cannabidiol (CBD) and/or
cannabigerol (CBG); a sesquiterpene that is one or both of
.alpha.-humulene and/or .beta.-caryophyllene; and a lipopeptide
antibiotic that is daptomycin or an analogue thereof. The
antibiotically active ingredients may be provided in relative
amounts that amplify their individual activities, including amounts
that are synergistically effective in an assay to inhibit growth
and/or reproduction of an Enterococcus faecium or an Enterococcus
faecalis. Therapies are provided that utilize these formulations as
anti-microbials, and provide for the combined use of two or more of
the antibiotically active compounds.
Inventors: |
Thompson; Charles J.;
(Vancouver, CA) ; Pryjma; Mark C.; (Ontario,
CA) ; Lambert; Dana M.; (West Vancouver, CA) ;
Dosanjh; Manisha; (Surrey, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of British Columbia |
Vancouver |
|
CA |
|
|
Appl. No.: |
17/607247 |
Filed: |
May 6, 2020 |
PCT Filed: |
May 6, 2020 |
PCT NO: |
PCT/CA2020/050613 |
371 Date: |
October 28, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62843687 |
May 6, 2019 |
|
|
|
International
Class: |
A61K 31/352 20060101
A61K031/352; A61K 31/05 20060101 A61K031/05; A61K 45/06 20060101
A61K045/06 |
Claims
1. A method of treating a bacterial infection in a subject in need
thereof, comprising administering to the subject an effective
amount of: a cannabinoid that is one or more of cannabichromene
(CBC), cannabidiol (CBD) and/or cannabigerol (CBG); and, a
lipopeptide antibiotic that is daptomycin or an analogue thereof;
wherein the cannabinoid and the lipopeptide antibiotic are
administered in an antibiotically effective weight ratio of from
16:1 to 1:16; and, wherein: if the cannabinoid is CBD, then the
lipopeptide antibiotic is administered in an effective amount that
is less than 4 mg/kg; and/or, if the cannabinoid is CBD, then the
bacterial infection comprises infection by an infectious organism
having a daptomycin MIC of 4 .mu.g/mL or greater; and/or, if the
cannabinoid is CBD, then the bacterial infection comprises
infection by an infectious organism for which results from an assay
of bacterial growth indicate that daptomycin produces an inhibitory
and/or bactericidal effect at concentrations of less than 4
.mu.g/mL when administered with CBD in an amount that is less than
the MIC of CBD.
2. The method of claim 1, wherein the bacterial infection comprises
infection by an infectious organism having a daptomycin MIC of 4
.mu.g/mL or greater.
3. The method of claim 1 or 2, wherein the bacterial infection
comprises infection by an Enterococcus faecium or an 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: a cannabinoid that is one
or more of cannabichromene (CBC) and/or cannabigerol (CBG); and, a
lipopeptide antibiotic that is daptomycin or an analogue thereof;
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 reduces the
MIC of the lipopeptide antibiotic in the assay when the cannabinoid
is present in an amount that is less than the MIC of the
cannabinoid.
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 to 9, wherein the assay
is an Enterococcal assay that assesses the inhibition of growth
and/or reproduction of an Enterococcus faecium or an Enterococcus
faecalis.
11. A formulation, comprising at least two antibiotically active
ingredients selected from: a cannabinoid that is one or more of
cannabichromene (CBC), cannabidiol (CBD) and/or cannabigerol (CBG);
and/or, a sesquiterpene that is one or both of .alpha.-humulene
and/or .beta.-caryophyllene; and/or, a lipopeptide antibiotic that
is daptomycin or an analogue thereof; wherein the antibiotically
active ingredients are present in synergistically effective
relative amounts, wherein an effective amount of the formulation is
synergistically effective in an assay to inhibit growth and/or
reproduction of an Enterococcus faecium or an Enterococcus
faecalis, and wherein if the formulation comprises CBD and
daptomycin, it further comprises at least one additional
antibiotically active ingredient.
12. The formulation of claim 11, wherein there are two
antibiotically active ingredients, and the two antibiotically
active ingredients are the cannabinoid and the sesquiterpene.
13. The formulation of claim 11, wherein there are two
antibiotically active ingredients, and the two antibiotically
active ingredients are the cannabinoid and the 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
antibiotically active ingredients, and the two antibiotically
active ingredients are the sesquiterpene and the lipopeptide
antibiotic.
16. The formulation of any one of claims 11 to 14, wherein the
cannabinoid is one of CBC, CBD or CBG.
17. The formulation of any one of claims 11 to 14, wherein the
cannabinoid is two of CBC, CBD and CBG.
18. The formulation of any one of claims 11 to 14, wherein the
cannabinoid comprises CBC, CBD and CBG.
19. The formulation of any one of claims 11 to 18, wherein the
sesquiterpene, if present, is one of .alpha.-humulene or
.beta.-caryophyllene.
20. The formulation of any one of claims 11 to 18, wherein the
sesquiterpene, if present, comprises .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 at least a 2 to 128 fold decrease in minimum inhibitory
concentration (MIC) of the cannabinoid in the assay.
23. The formulation of any one of claims 11 to 22, wherein the
cannabinoid, when present, is present in a relative amount that
provides at least a 2 to 128 fold decrease in minimum inhibitory
concentration (MIC) of the sesquiterpene 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 at least a 2 to 128 fold decrease in
minimum inhibitory concentration (MIC) of the lipopeptide
antibiotic in the assay.
25. The formulation of any one of claims 11 to 24, wherein the
sesquiterpene and the cannabinoid, when present, are present in a
molar ratio that is .gtoreq.50.
26. The formulation of any one of claims 11 to 25, further
comprising a pharmaceutically acceptable excipient, and wherein the
antibiotically active ingredients are dissolved, dispersed, mixed
or suspended in the formulation.
27. The formulation of any one of claims 11 to 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 of any one of claims 11 to 28, wherein the
cannabinoid and/or sesquiterpene, when present, are derived from a
plant.
30. The formulation of claim 29, wherein the plant is a Cannabis
sativa or Cannabis indica plant.
31. The formulation of claim 11, wherein there are two
antibiotically active ingredients, and the two antibiotically
active ingredients are cannabinoids.
32. The formulation of any one of claims 11 to 31, wherein the
formulation does not comprise any alternative cannabinoids,
terpenes or lipopeptide antibiotics.
33. The formulation of any one of claims 21 to 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 according to 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 to 33, to treat
an enterococcal infection in a subject in need thereof.
37. The use according to claim 35 or 36, wherein the enterococcal
infection is an Enterococcus faecium or an Enterococcus faecalis
infection.
38. The use according to any one of claims 35 to 37, wherein the
enterococcal infection is an oral infection.
39. The use according to claim 38, wherein the oral infection is a
marginal periodontitis, a root canal infection, a primary
endodontic infection, a persistent or secondary infection, dental
caries, peri-implantitis, periradicular abscess or an oral mucosal
lesion.
40. The use according to any one of claims 35 to 39, wherein the
enterococcal infection is an antibiotic resistant enterococcal
infection.
41. The use according to claim 40, wherein the antibiotic resistant
enterococcal infection is a vancomycin and/or daptomycin resistant
enterococcal infection.
42. The use according to any one of claims 34 to 41, wherein the
formulation is for use in an amount of that delivers an effective
dose of the cannabinoid, when present, of from 1 to 5,000 mg per
day, and/or an effective dose of the sesquiterpene, when present,
of from 1 to 10,000 mg per day.
43. A method of treating an enterococcal infection in a subject in
need thereof, 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, 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
antibiotically active ingredients, wherein the two antibiotically
active ingredients are the cannabinoid and the sesquiterpene,
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 to 45, comprising
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, comprising administering to the subject an effective
amount of the formulation of claim 9 or 10, comprising
administering the sesquiterpene in an effective amount of 1-10,000
mg/day.
49. The method of any one of claims 43 to 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 to treat
an enterococcal infection in a subject in need thereof.
52. The formulation of claim 51, wherein the enterococcal infection
is an Enterococcus faecium or an 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 a
marginal periodontitis, a root canal infection, a primary
endodontic infection, a persistent or secondary infection, dental
caries, peri-implantitis, periradicular abscess or an oral mucosal
lesion.
55. The formulation of any one of claims 51 to 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 of any one of claims 51 to 56, wherein the
formulation is for use in an amount of that delivers an effective
dose of the cannabinoid, when present, of from 1 to 5,000 mg per
day, and/or an effective dose of the sesquiterpene, when present,
of from 1 to 10,000 mg per day.
58. A method of treating a microbial infection in a subject in need
thereof, comprising administering to the subject, sequentially or
in combination, an effective amount of at least two antibiotically
active compounds selected from: a cannabinoid that is one or more
of cannabichromene (CBC), cannabidiol (CBD) and/or cannabigerol
(CBG); and/or, a sesquiterpene that is one or both of
.alpha.-humulene and/or .beta.-caryophyllene; and/or, a lipopeptide
antibiotic that is daptomycin or an analogue thereof; wherein the
antibiotically active compounds are administered in synergistically
effective relative amounts effective to treat the microbial
infection, and, wherein if the formulation comprises CBD and
daptomycin, it further comprises at least one additional
antibiotically active ingredient.
59. The method of claim 58, wherein the synergistically effective
relative amounts are synergistically effective to inhibit growth
and/or reproduction of an Enterococcus faecium or an Enterococcus
faecalis in an assay.
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 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 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 of any one of claims 58 to 62, wherein the
cannabinoid, if administered, is one of CBC, CBD or CBG.
64. The method of 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 to 62, wherein the
cannabinoid, if administered, comprises CBC, CBD and CBG.
66. The method of any one of claims 58 to 65, wherein the
sesquiterpene, if administered, is one of .alpha.-humulene or
.beta.-caryophyllene.
67. The method of any one of claims 58 to 66, wherein the
sesquiterpene, if administered, comprises .alpha.-humulene and
.beta.-caryophyllene.
68. The method of any one of claims 58 to 67, wherein the
lipopeptide antibiotic, if administered, is daptomycin.
69. The method of any one of claims 58 to 68, wherein the
sesquiterpene, if administered, is administered in a relative
amount that provides at least a 2 to 128 fold decrease in minimum
inhibitory concentration (MIC) of the cannabinoid when
administered.
70. The method of any one of claims 58 to 69, wherein the
cannabinoid, if administered, is administered in a relative amount
that provides at least a 2 to 128 fold decrease in minimum
inhibitory concentration (MIC) of the sesquiterpene when
administered.
71. The method of any one of claims 58 to 70, wherein the
sesquiterpene and/or the cannabinoid, when administered, is
administered in a relative amount that provides at least a 2 to 128
fold decrease in minimum inhibitory concentration (MIC) of the
lipopeptide antibiotic when administered.
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 an 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 a
marginal periodontitis, a root canal infection, a primary
endodontic infection, a persistent or secondary infection, dental
caries, peri-implantitis, periradicular abscess or an oral mucosal
lesion.
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 of 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 the synergistically effective
relative amounts are synergistically effective to inhibit growth
and/or reproduction of an Enterococcus faecium or an Enterococcus
faecalis in an assay.
80. The method of any one of claims 79, wherein the cannabinoid is
administered in an amount that provides at least a 2 to 128 fold
decrease in minimum inhibitory concentration (MIC) of the
lipopeptide antibiotic in the assay.
81. The method of any one of claim 1-4 or 78-80, wherein the
cannabinoid and the lipopeptide antibiotic are co-administered.
82. The method of claim 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 claim 1-4 or 78-82, further comprising
further treating the subject with the lipopeptide antibiotic
alone.
84. The method of any one of claim 1-4 or 78-83, wherein the
cannabinoid is one of CBC, CBD or CBG.
85. The method of any one of claim 1-4 or 78-83, wherein the
cannabinoid is two of CBC, CBD and CBG.
86. The method of any one of claim 1-4 or 78-83, wherein the
cannabinoid comprises CBC, CBD and CBG.
87. The method of any one of claim 1-4 or 78-86, wherein the
lipopeptide antibiotic is daptomycin.
88. The method of any one of claim 1-4 or 78-87, wherein the
cannabinoid is administered in a relative amount that provides at
least a 2 to 128 fold decrease in minimum inhibitory concentration
(MIC) of the lipopeptide antibiotic.
89. The method of any one of claim 1-4 or 78-88, wherein the
bacterial infection is gram positive bacterial infection or an
enterococcal infection.
90. The method of claim 89, wherein the enterococcal infection is
an Enterococcus faecium or an 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 a
marginal periodontitis, a root canal infection, a primary
endodontic infection, a persistent or secondary infection, dental
caries, peri-implantitis, periradicular abscess or an oral mucosal
lesion.
93. The method of any one of claim 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 one of claim 1-4 or 78-94, comprising
administration of an effective dose of the cannabinoid of from 1 to
5,000 mg per day.
96. Use of CBD and/or CBG to inhibit the growth of a biofilm,
wherein the biofilm comprises an Enterococcus faecium or an
Enterococcus faecalis.
97. A method of inhibiting the growth of a biofilm comprising an
Enterococcus faecium or an Enterococcus faecalis, comprising
exposing the biofilm to an effective amount of CBD and/or CBG.
98. An invasive medical device comprising an impregnated
concentration of CBD and/or CBG, wherein the impregnated
concentration of CBD and/or CBG is releasable from the device in
vivo at a concentration that is effective to inhibit growth of a
biofilm on the device, wherein the biofilm comprises an
Enterococcus faecium or an Enterococcus faecalis.
Description
FIELD OF THE INVENTION
[0001] The invention is in the field of medicinal preparations
comprising a mixture of two or more organic antibiotically active
ingredients, optionally including specific phenolic cannabinoids in
optional combination with specific sesquiterpenes in optional
combination with a lipopeptide antibiotic such as daptomycin. The
therapeutic use of cannabinoid and/or sesquiterpene formulations is
also disclosed, for synergistic treatment of enterococcal
infections, including combined uses with lipopeptide antibiotics
such as daptomycin.
BACKGROUND OF THE INVENTION
[0002] Enterococcus faecium and Enterococcus faecalis are bacterial
species that asymptomatically colonize the gastrointestinal (GI)
tract in humans, but can also be pathogenic in certain
circumstances (in the relevant literature, Enterococcus faecium was
previously classified as Streptococcus faecium, see Schleifer &
Kilpperbalz, 1984). In particular, these organisms are known to be
a leading cause of dangerous infections in hospital patients being
treated with antibiotics. During overgrowth, enterococci can
penetrate the walls of the GI tract, moving into the liver or
bloodstream where they can cause peritonitis, or systemic
infections (bacteremia). Enterococci have been reported to be the
third leading cause of endocarditis, the complication of blood
infections that has the greatest risk of mortality (Murdoch et al.,
2009). The intestinal colonization by enterococci, particularly in
hospital patients, may facilitate continual person-to-person spread
by environmental contamination from fecal droplets, leading to
opportunistic infection. Passive transmission of fecal matter to
the urinary tract in catheterized patients has reportedly made
enterococci the second most common cause of catheter associated
urinary tract infections (UTIs) which can result in kidney damage
(Hidron et al., 2008). E. faecalis and E. faecium are associated
with oral infections causing marginal periodontitis, root canal
infections, primary endodontic infections, persistent/secondary
infections, dental caries, peri-implantitis, periradicular
abscesses and oral mucosal lesions (Najafi et al., 2019). E.
faecium is also an important multidrug-resistant nosocomial
pathogen causing biofilm-mediated infections in patients with
medical devices (Paganelli et al., 2013).
[0003] Therapy for enterococcal infections is made more difficult
by the frequency of antibiotic resistance. Traditional treatments
involve penicillin administered with a synergistic aminoglycoside.
However, the emergence of resistance, particularly mediated by a
mutation in a penicillin binding protein (pbp5) in E. faecium, has
severely limited the utility of this traditional strategy
(Galloway-Pena et al., 2009, Arias & Murray, 2012). To combat
penicillin resistant strains, vancomycin became the treatment of
choice. However, Enterococcus spp. have become increasingly
vancomycin resistant (VRE), frequently due to the acquisition of
VanA and VanB vancomycin resistance systems (Arthur &
Courvalin, 1993). From a public health perspective, the spread of
VRE as a nosocomial infection has been particularly problematic
since it reportedly causes 1.8 fold higher mortality and results in
an average of 5 days longer hospital stay according to a
meta-analysis (Prematunge et al., 2016).
[0004] Currently there are two drugs indicated for VRE treatment,
linezolid and daptomycin. However, although linezolid resistance
has been reported to be rare, daptomycin resistance appears to
emerge frequently de novo during daptomycin treatment (Arias et
al., 2011); in such strains, linezolid becomes the only effective
antibiotic available. Linezolid as sole available therapy is
problematic due to a litany of negative side effects, such as
hepatic toxicity, myelosuppression, and serotonin syndrome (if used
with other serotonergic drugs). Given the limited options for
treatment of enterococcal infections and the adverse effects
associated with existing treatments, new treatments are urgently
needed.
[0005] A very wide range of physiological activities have been
ascribed to compounds derived from flowering plants in the genus
Cannabis, particularly phytocannabinoid compounds (see Cunha et
al., 1980; Morales et al., 2017; U.S. Pat. No. 6,630,507). There
are more than 80 cannabinoids found in Cannabis plant extracts
(Russo, 2011), including: cannabidiol (CBD), its acid form
cannabidiolic acid (CBDA), cannabichromene (CBC), its acid form
cannabichromic acid (CBCA), cannabigerol (CBG), its acidic form
cannabigerolic acid (CBGA), tetrahydrocannabinol (THC), and its
acidic form, tetrahydrocannabinolic acid (THCA). Studies have
suggested that Cannabis extracts, or compounds derived from the
Cannabis plant, have a very wide range of, often ill defined,
anti-microbial activities (Van Klingeren & Ten Ham, 1976;
Abdelaziz, 1982; Appendino et al., 2011; Appendino et al., 2008;
Eisohly et al., 1982; Eisohly et al., 1982; Appendino et al., 2008;
Turner & Eisohly, 1981; Mechoulam & Gaoni, 1965;
WO2012/012498; WO2018/011813).
[0006] Terpenes are another molecular constituent of plants,
including Cannabis plants, that have been attributed with a wide
range of physiological activities. For example, the distinctive
aroma and flavour that hops lends to beer reportedly comes in part
from particular sesquiterpenes (including .alpha.-humulene and
.beta.-caryophyllene; see Steenackers et al., 2015). The terpenes
in Cannabis are reported to include well over 100 distinct
compounds (Andre et al., 2016). It has been reported that aromatic
terpenes may modulate the physiological effects of cannabinoids,
particularly the psychoactive effects (Russo, 2011).
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 includes 12 line graphs, illustrating the effects of
CBD, CBG or CBC on E. faecium viability. Enterococcus strains 33D3
(A-C), 69C6 (D-F), 58C9 (G-I) and 55A6 (J-L) were incubated with
0x, 1x (1 mg/L), 2x (2 mg/L), or 4x (4 mg/L) the MIC of CBD
(A,D,G,J), CBG (B,E,H,K) or CBC (C,F,I,L). CFU's were determined at
2, 4, 6, 8, and 24 h by serial dilution.
[0008] FIG. 2 includes 8 line graphs, illustrating the effects of
daptomycin (Dap), and Dap in combination with CBD, CBG or CBC, on
daptomycin resistant E. faecium VRE strains 58C9 and 55A6 (E.
faecium clinical strain 55A6 is resistant to daptomycin with a
daptomycin MIC=8 mg/L). As illustrated, adding approximately 1/2
MIC of each cannabinoid facilitated killing by daptomycin at
approximately 1/4-1/8x the MIC of daptomycin.
[0009] FIG. 3 includes 4 line graphs, illustrating the effect of
various concentrations of CBD, CBG and CBC on bactericidal activity
of daptomycin. E. faecium strain 33D3 was incubated with (A) no
cannabinoid, or 1/2x (0.5 mg/L) MIC of (B) CBD, (C) CBG) or (D) CBC
with 1x (1 mg/L), 1/2x (0.5 mg/L), 1/4x (0.25 mg/L) or 1/8x (0.125
mg/L) MIC of daptomycin. Aliquots were removed at 2, 4, 6, 8 and 24
h plated to monitor viability (CFU). Data points are the means from
three replicates with standard deviations presented as error
bars.
[0010] FIG. 4 includes two bar graphs, illustrating the effect of
CBD, CBG and CBC on biofilms of E. faecium strain 33D3.
SUMMARY
[0011] One general aspect of the innovations disclosed herein
includes formulations that include at least two antibiotically
active ingredients, for example selected from: a cannabinoid that
is one or more of cannabichromene (CBC), cannabidiol (CBD) and/or
cannabigerol (CBG). The formulations may also include a
sesquiterpene that is one or both of .alpha.-humulene and/or
.beta.-caryophyllene. The formulations can also include a
lipopeptide antibiotic that is daptomycin or an analogue thereof.
The formulations may include the antibiotically active ingredients
in synergistically effective relative amounts, for example where an
effective amount of the formulation is synergistically effective in
an assay to inhibit growth and/or reproduction of an Enterococcus
faecium or an Enterococcus faecalis.
[0012] Implementations may include one or more of the following
features. The formulation where there are two antibiotically active
ingredients, and the two antibiotically active ingredients are the
cannabinoid and the sesquiterpene. The formulation where there are
two antibiotically active ingredients, and the two antibiotically
active ingredients are the cannabinoid and the lipopeptide
antibiotic. The formulation including the cannabinoid, the
sesquiterpene and the lipopeptide antibiotic. The formulation where
there are two antibiotically active ingredients, and the two
antibiotically active ingredients are the sesquiterpene and the
lipopeptide antibiotic. The formulation of any one where the
cannabinoid is one of CBC, CBD or CBG. The formulation of any one
where the cannabinoid is two of CBC, CBD and CBG. The formulation
of any one where the cannabinoid includes CBC, CBD and CBG. The
formulation of any one where the sesquiterpene, if present, is one
of .alpha.-humulene or .beta.-caryophyllene. The formulation of any
one where the sesquiterpene, if present, includes .alpha.-humulene
and .beta.-caryophyllene. The formulation of any one where the
lipopeptide antibiotic, if present, is daptomycin. The formulation
of any one where the sesquiterpene, when present, is present in a
relative amount that provides at least a 2 to 128 fold decrease in
the minimum inhibitory concentration (MIC) of the cannabinoid in
the assay. The formulation of any one where the cannabinoid, when
present, is present in a relative amount that provides at least a 2
to 128 fold decrease in the minimum inhibitory concentration (MIC)
of the sesquiterpene in the assay. The formulation of any one where
the sesquiterpene and/or the cannabinoid, when present, is present
in a relative amount that provides at least a 2 to 128 fold
decrease in the minimum inhibitory concentration (MIC) of the
lipopeptide antibiotic in the assay. The formulation of any one
where the molar ratio of the sesquiterpene to the cannabinoid, when
present, is .gtoreq.50. The formulation of any one further
including a pharmaceutically acceptable excipient, and where the
antibiotically active ingredients are dissolved, dispersed, mixed
or suspended in the formulation. The formulation of any one where
the cannabinoid, when present, is present at 0.1-100 mg/L. The
formulation of any one where the sesquiterpene, when present, is
present at 0.1-500 mg/L. The formulation of any one where the
cannabinoid and/or sesquiterpene, when present, are derived from a
plant extract. The formulation where the plant is a Cannabis sativa
or Cannabis indica plant. The formulation of any one where the
formulation does not include any alternative cannabinoids, terpenes
or lipopeptide antibiotics. The formulation of any one where the
formulation includes essentially of two or more of the cannabinoid,
the terpene or the lipopeptide antibiotic. Use of the formulation
of any one to formulate a medicament. The use according where the
medicament is for use in treating an enterococcal infection in a
subject in need thereof. The use according where the enterococcal
infection is an Enterococcus faecium or an Enterococcus faecalis
infection. The use according to any one where the enterococcal
infection is an oral infection. The use according where the oral
infection is a marginal periodontitis, a root canal infection, a
primary endodontic infection, a persistent or secondary infection,
dental caries, peri-implantitis, periradicular abscess or an oral
mucosal lesion. The use according to any one where the enterococcal
infection is an antibiotic resistant enterococcal infection. The
use according where the antibiotic resistant enterococcal infection
is a vancomycin and/or daptomycin resistant enterococcal infection.
The use according to any one where the formulation is for use in an
amount that delivers an effective dose of the cannabinoid, when
present, of from 1 to 5,000 mg per day, and/or an effective dose of
the sesquiterpene, when present, of from 1 to 10,000 mg per day.
Use of the formulation of any one to treat an enterococcal
infection in a subject in need thereof. The formulation of any one
for use to treat an enterococcal infection in a subject in need
thereof. The formulation where the enterococcal infection is an
Enterococcus faecium or an Enterococcus faecalis infection. The
formulation where the enterococcal infection is an oral infection.
The formulation where the oral infection is a marginal
periodontitis, a root canal infection, a primary endodontic
infection, a persistent or secondary infection, dental caries,
peri-implantitis, periradicular abscess or an oral mucosal lesion.
The formulation of any one where the enterococcal infection is an
antibiotic resistant enterococcal infection. The formulation where
the antibiotic resistant enterococcal infection is a vancomycin
and/or daptomycin resistant enterococcal infection. The formulation
of any one where the formulation is for use in an amount of that
delivers an effective dose of the cannabinoid, when present, of
from 1 to 5,000 mg per day, and/or an effective dose of the
sesquiterpene, when present, of from 1 to 10,000 mg per day.
[0013] Alternative aspects of the disclosed innovations include
methods of treating an enterococcal infection in a subject in need
thereof, including administering to the subject an effective amount
of the formulations disclosed herein. Implementations may include
one or more of the following features. The method including two
antibiotically active ingredients, where the two antibiotically
active ingredients are the cannabinoid and the sesquiterpene,
further including treating the subject with an effective amount of
one or more antibiotics, optionally where the antibiotic is
daptomycin. The method including administering the cannabinoid in
an effective amount of 1-5,000 mg/day. The method of any one
including administering the sesquiterpene in an effective amount of
1-10,000 mg/day. The method of any one where the subject is a
mammal. The method where the mammal is a human.
[0014] Alternative aspects of the present innovations include
methods of treating a microbial infection in a subject in need
thereof, including administering to the subject, sequentially or in
combination, an effective amount of at least two antibiotically
active compounds selected from: a cannabinoid that is one or more
of cannabichromene (CBC), cannabidiol (CBD) and/or cannabigerol
(CBG). The method of treating also includes a sesquiterpene that is
one or both of .alpha.-humulene and/or .beta.-caryophyllene, and.
The method of treating also includes a lipopeptide antibiotic that
is daptomycin or an analogue thereof. The method of treating also
includes where the antibiotically active compounds are administered
in synergistically effective relative amounts effective to treat
the microbial infection.
[0015] Implementations of the disclosed methods may include one or
more of the following features. The method where the
synergistically effective relative amounts are synergistically
effective to inhibit growth and/or reproduction of an Enterococcus
faecium or an Enterococcus faecalis in an assay. The method where
the cannabinoid and the sesquiterpene are co-administered, or the
sesquiterpene and the lipopeptide antibiotic are co-administered,
or the cannabinoid and the lipopeptide antibiotic are
co-administered, or the cannabinoid the sesquiterpene and the
lipopeptide antibiotic are co-administered. The method where the
cannabinoid and the sesquiterpene, or the sesquiterpene and the
lipopeptide antibiotic, or the cannabinoid and the lipopeptide
antibiotic, or the cannabinoid the sesquiterpene and the
lipopeptide antibiotic, are administered sequentially, in any
order. The method of any one further including further treating the
subject with the lipopeptide antibiotic alone. The method of any
one where the cannabinoid, if administered, is one of CBC, CBD or
CBG. The method of any one where the cannabinoid, if administered,
is two of CBC, CBD and CBG. The method of any one where the
cannabinoid, if administered, includes CBC, CBD and CBG. The method
of any one where the sesquiterpene, if administered, is one of
.alpha.-humulene or .beta.-caryophyllene. The method of any one
where the sesquiterpene, if administered, includes .alpha.-humulene
and .beta.-caryophyllene. The method of any one where the
lipopeptide antibiotic, if administered, is daptomycin. The method
of any one where the sesquiterpene, if administered, is
administered in a relative amount that provides at least a 2 to 128
fold decrease in the minimum inhibitory concentration (MIC) of the
cannabinoid when administered. The method of any one where the
cannabinoid, if administered, is administered in a relative amount
that provides at least a 2 to 128 fold decrease in the minimum
inhibitory concentration (MIC) of the sesquiterpene when
administered. The method of any one where the sesquiterpene and/or
the cannabinoid, when administered, is administered in a relative
amount that provides at least a 2 to 128 fold decrease in the
minimum inhibitory concentration (MIC) of the lipopeptide
antibiotic when administered. The method of any one where the
microbial infection is an enterococcal infection. The method where
the enterococcal infection is an Enterococcus faecium or an
Enterococcus faecalis infection. The method where the enterococcal
infection is an oral infection. The method where the oral infection
is a marginal periodontitis, a root canal infection, a primary
endodontic infection, a persistent or secondary infection, dental
caries, peri-implantitis, periradicular abscess or an oral mucosal
lesion. The method of any one where the microbial infection is an
antibiotic resistant infection. The method where the antibiotic
resistant infection is a vancomycin and/or daptomycin resistant
microbial infection.
[0016] In an alternative aspect, methods are provided for treating
a bacterial infection in a subject in need thereof, comprising
administering to the subject an effective amount of: a cannabinoid
that is one or more of cannabichromene (CBC), cannabidiol (CBD)
and/or cannabigerol (CBG); and, a lipopeptide antibiotic that is
daptomycin or an analogue thereof; wherein the cannabinoid and the
lipopeptide antibiotic are administered in an antibiotically
effective weight ratio of from 16:1 to 1:16; and, wherein: if the
cannabinoid is CBD, then the lipopeptide antibiotic is administered
in an effective amount that is less than 4 mg/kg; and/or, if the
cannabinoid is CBD, then the bacterial infection comprises
infection by an infectious organism having a daptomycin MIC of 4
.mu.g/mL or greater; and/or, if the cannabinoid is CBD, then the
bacterial infection comprises infection by an infectious organism
for which results from an assay of bacterial growth indicate that
daptomycin produces an inhibitory and/or bactericidal effect at
concentrations of less than 4 .mu.g/mL when administered with CBD
in an amount that is less than the MIC of CBD. The infection may
for example include an infection by an Enterococcus faecium or an
Enterococcus faecalis. These treatments may further include
administering to the subject an effective amount of a sesquiterpene
that is one or both of .alpha.-humulene and/or
.beta.-caryophyllene.
[0017] In an alternative aspect, antibiotic formulations are
provided that include: a cannabinoid that is one or more of
cannabichromene (CBC) and/or cannabigerol (CBG); and, a lipopeptide
antibiotic that is daptomycin or an analogue thereof; wherein the
cannabinoid and the lipopeptide antibiotic are present in an
antibiotically effective weight ratio of from 16:1 to 1:16. The
cannabinoid 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 Enterococcal assay
that assesses the inhibition of growth and/or reproduction of an
Enterococcus faecium or an Enterococcus faecalis. In select
embodiments, the cannabinoid reduces the MIC of the lipopeptide
antibiotic in the assay when the cannabinoid is present in an
amount that is less than the MIC of the cannabinoid. These
formulations may further include a sesquiterpene, such as one or
both of .alpha.-humulene and/or .beta.-caryophyllene.
DETAILED DESCRIPTION OF THE INVENTION
[0018] In one aspect, pharmaceutical formulations are provided that
include at least two antibiotically active ingredients, selected
from: a cannabinoid that is one or more of cannabichromene (CBC),
cannabidiol (CBD) and/or cannabigerol (CBG); a sesquiterpene that
is one or both of .alpha.-humulene and/or .beta.-caryophyllene,
and, a lipopeptide antibiotic that is daptomycin or an analogue
thereof. The antibiotically effective ingredients may be provided
in synergistically effective relative amounts. For example, the
cannabinoid and the sesquiterpene may be provided at concentrations
that are only antibiotically active in synergistic combinations,
such as .ltoreq.1 .mu.g/ml cannabinoid and .ltoreq.32 .mu.g/ml
terpene. In synergistic combination, the inhibitory concentrations
of the partners may for example decrease, for example by two or
more fold, for example from 2-16 fold. Alternatively, the relative
weight ratio of cannabinoid to sesquiterpene may for example be
from about 1:5 to 1:50, or 1:8 to 1:32, or from 1:12 to 1:32. The
relative concentration of the antibiotically active ingredients in
the formulation may be arranged so that an effective amount of the
formulation would be synergistically effective in an assay to
inhibit growth and/or reproduction of an Enterococcus faecium or an
Enterococcus faecalis. A formulation that provides this synergistic
formulation may be used for treating enterococcal or other
microbial infections, for example for treating infections of
enterococcal species that are relatively closely related to a
reference strain of Enterococcus faecium or Enterococcus faecalis
(see Zhong et al., 2017). For example, enterococcal species
amenable to treatment may be at least as closely related to the
reference strain as are the most distantly related strains of E.
faecium and E. faecalis. The microorganisms amenable to treatment
may for example be resistant to antibiotics, for example VRE
strains or strains resistant to daptomycin. Enterococcus durans is
for example amenable to treatment, for example in the context of
veterinary diseases. Other enterococcal species closely related to
E. faecium or E. faecalis may for example include E. mundtii, E.
durans, E. hirae, E. ratti, E. villorum, E. thailandicus and E.
phoeniculicola, E. termitis, E. quebecensis, E. moraviensis, E.
caccae, E. haemoperoxidus and E. silesiacus]
[0019] In select embodiments, synergies and/or potentiation effects
are maximized using concentrations of antibiotically active
components that are below the MICs for each component, for example
just below the MICs. The components may accordingly be present in
relative amounts that approximate the ratio of the respective MICs
for the components. For example, this may occur when the molar
ratio terpene/cannabinoid is .gtoreq.50 (reflecting the MIC ratio
of the components).
[0020] The molar ratio of cannabinoid to sesquiterpene may for
example be between 1:100 and 50:1. For example, synergistic
combinations may have a molar ratio of cannabinoid to sesquiterpene
between 1:8 and 1:32. A pharmaceutically acceptable excipient may
optionally be included in the formulation, and the cannabinoid and
sesquiterpene may be dissolved, dispersed, mixed or suspended in
the formulation. One method of formulation may involve producing a
sterile lyophilized powder, for example in a vial for
reconstitution.
[0021] The cannabinoid and sesquiterpene may for example be
obtained from a plant extract, such as an extract of Cannabis
sativa or Cannabis indica. Biosynthetic approaches to the
production of cannabinoids and sesquiterpenes are also available,
as are a variety of synthetic approaches (based for example on
approaches used to synthesize THC/dronabinol, see U.S. Pat. No.
7,323,576 and Trost and Dogra, 2007). Alternative approaches
involve expressing cannabinoid biosynthetic genes in recombinant
hosts, such as recombinant yeast (see Luo et al., 2019).
[0022] Derivatives or analogues of daptomycin may for example be
used as lipopeptide antibiotics, as alternatives or in addition to
use of daptomycin itself (see Nguyen et al., 2006; Baltz, 2006;
and, Miao et al., 2006; Nguyen et al., 2010). In select
embodiments, a daptomycin analogue is a lipopeptide antibiotic that
has an antibiotic activity that synergizes with one or more of the
cannabinoids and/or sesquiterpenes in the formulations disclosed
herein, for example in assay of antibiotic activity against
enterococcal species. Daptomycin analogues or derivatives may for
example differ from daptomycin by virtue of the substitution of one
or more amino acids therein, such as by conservative substitutions,
for example substitutions of 1, 2, 3, 4 or 5 amino acids therein,
for example substitutions with natural or non-natural amino acids.
Daptomycin analogues may for example include cyclic
lipodepsipeptide daptomycin, or derivatives thereof in which the
fatty acid chain attached to the Trp.sup.1 amino group is an
anteiso-undecanoyl, iso-dodecanoyl or anteiso-tridecanoyl
group.
[0023] One or more additional compounds may be included, or
specifically excluded, in alternative formulations, including for
example: terpenes, terpenoids, sterols, triglycerides, alkanes,
squalene, tocopherol, carotenoids, chlorophyll, flavonoid
glycosides, or alkaloids.
[0024] Anti-microbial formulations may be used to prophylactically
or therapeutically treat microbial infections, or otherwise inhibit
microbial growth or multiplication. An antibiotic is an
antimicrobial that is active against bacteria, and in this context
includes naturally-occurring and synthetic substances that kill or
inhibit the growth or multiplication of bacteria by any mechanism,
including antiseptic or disinfectant modalities.
[0025] Subjects amenable to treatment include mammalian subjects,
such as human patients, laboratory animals (e.g., primates, rats,
mice), livestock (e.g., cows, sheep, goats, pigs, horses, fowl), or
household pets (e.g., dogs, cats, rodents, birds), for example
belonging to the taxonomic groups of primates, canines, felines,
bovines, caprines, equines, ovines, porcines, rodents, Ayes or
lagomorphs. Human patients to be treated may for example be male or
female, or at a specific stage of development: neonate, infant,
juvenile, adolescent, adult and geriatric. Specific veterinary
indications amenable to treatment may for example include
enterococcal infections in poultry, for example treatment of
Enterococcus cecorum infections in chickens.
[0026] In alternative embodiments, formulation may be administered
in a dosage or dosage form that delivers and/or sustains
synergistically effective amounts of the active ingredients.
[0027] The cannabinoid and/or sesquiterpene components of the
formulation may for example be in the form of a plant extract, or
be derived from a plant extract, or be obtained from a culture,
such as a culture of a recombinant host, such as a recombinant
yeast expressing the components. Humulene and .beta.-caryophyllene
are isomers, often found together in a variety of plants. In
addition, a pharmaceutically acceptable excipient may be included,
and the formulation may be provided in a titratable dosage form. In
select embodiments, the cannabinoid components of the formulation
may be obtained as an extract from a plant of the Cannabis genus,
for example Cannabis sativa or Cannabis indica, or from a culture
of recombinant yeast hosts. A wide variety of methods may be used
to prepare these plant extracts, including, but not limited to,
supercritical or subcritical extraction with CO.sub.2, extraction
with hot gas, and extraction with solvents. Formulations may also
specifically exclude additional terpenoids or terpenes, including
plant-derived terpenoids or terpenes, such as astaxanthin or other
sesquiterpenes, tetraterpenes, triterpenes, diterpenes or
monoterpenes.
[0028] A titratable dosage may for example be adapted to allow a
patient to take the medication in doses smaller than the unit dose,
wherein a "unit dose" is defined as the maximum dose of medication
that can be taken at any one time or within a specific dosage
period. Titration of doses will allow different patients to
incrementally increase the dose until they feel that the medication
is efficacious, as not all patients will require the same dose to
achieve the same benefits. A person with a larger build or faster
metabolism may require larger doses to achieve the same effect as
another with a smaller build or slower metabolism. Therefore, a
titratable dosage has advantages over a standard dosage form.
[0029] In select embodiments, formulations may be adapted to be
delivered in such a way as to target one or more of the following:
sublingual, buccal, oral, rectal, nasal, parenteral and via the
pulmonary system. Formulations may for example be in one or more of
the following forms: gel, gel spray, tablet, liquid, capsule, by
injection, or for vaporization.
[0030] Conventional pharmaceutical practice may be employed to
provide suitable formulations or compositions to administer the
formulations to subjects. Routes of administration may for example
include, parenteral, intravenous, intradermal, subcutaneous,
intramuscular, intracranial, intraorbital, ophthalmic,
intraventricular, intracapsular, intraspinal, intrathecal,
intracisternal, intraperitoneal, intranasal, inhalational, aerosol,
topical, sublingual or oral administration. Therapeutic
formulations may be in the form of liquid solutions or suspensions;
for oral administration, formulations may be in the form of tablets
or capsules; for intranasal formulations, in the form of powders,
nasal drops, or aerosols; and for sublingual formulations, in the
form of drops, aerosols or tablets.
[0031] Methods well known in the art for making formulations are
found in, for example, "Remington: The Science and Practice of
Pharmacy" (21st edition), ed. 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 napthalenes. Biocompatible, biodegradable lactide
polymer, lactide/glycolide copolymer, or
polyoxyethylene-polyoxypropylene copolymers may be used to control
the release of the compounds. Other potentially useful parenteral
delivery systems for include ethylene-vinyl acetate copolymer
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, polyoxyethylene-9-lauryl ether, glycocholate and
deoxycholate, or may be oily solutions for administration in the
form of nasal drops, or as a gel.
[0032] Pharmaceutical compositions of the present invention may be
in any form which allows for the composition to be administered to
a patient. For example, the composition may be in the form of a
solid, liquid or gas (aerosol). Pharmaceutical composition of the
invention are formulated so as to allow the active ingredients
contained therein to be bioavailable upon administration of the
composition to a patient. Compositions that will be administered to
a patient may take the form of one or more dosage units, where for
example, a tablet, capsule or cachet may be a single dosage unit,
and a container of the compound in aerosol form may hold a
plurality of dosage units.
[0033] Materials used in preparing the pharmaceutical compositions
should be pharmaceutically pure and non-toxic in the amounts used.
The inventive compositions may include one or more compounds
(active ingredients) known for a particularly desirable effect. It
will be evident to those of ordinary skill in the art that the
optimal dosage of the active ingredient(s) in the pharmaceutical
composition will depend on a variety of factors. Relevant factors
include, without limitation, the type of subject (e.g., human), the
particular form of the active ingredient, the manner of
administration and the composition employed.
[0034] In general, the pharmaceutical composition includes a
formulation of the present invention as described herein, in
admixture with one or more carriers. The carrier(s) may be
particulate, so that the compositions are, for example, in tablet
or powder form. The carrier(s) may be liquid, with the compositions
being, for example, an oral syrup or injectable liquid. In
addition, the carrier(s) may be gaseous, so as to provide an
aerosol composition useful in, e.g., inhalatory administration.
[0035] When intended for oral administration, the composition is
preferably in either solid or liquid form, where semi-solid,
semi-liquid, suspension and gel forms are included within the forms
considered herein as either solid or liquid.
[0036] As a solid formulation for oral administration, the
composition may be formulated into a powder, granule, compressed
tablet, pill, capsule, cachet, chewing gum, wafer, lozenges, or the
like form. Such a solid composition will typically contain one or
more inert diluents or edible carriers. In addition, one or more of
the following adjuvants may be present: binders such as syrups,
acacia, sorbitol, polyvinylpyrrolidone, carboxymethylcellulose,
ethyl cellulose, microcrystalline cellulose, gum tragacanth or
gelatin, and mixtures thereof; excipients such as starch, lactose
or dextrins, disintegrating agents such as alginic acid, sodium
alginate, Primogel, corn starch and the like; lubricants such as
magnesium stearate or Sterotex; fillers such as lactose, mannitols,
starch, calcium phosphate, sorbitol, methylcellulose, and mixtures
thereof; lubricants such as magnesium stearate, high molecular
weight polymers such as polyethylene glycol, high molecular weight
fatty acids such as stearic acid, silica, wetting agents such as
sodium lauryl sulfate, glidants such as colloidal silicon dioxide;
sweetening agents such as sucrose or saccharin, a flavoring agent
such as peppermint, methyl salicylate or orange flavoring, and a
coloring agent. When the composition is in the form of a capsule,
e.g., a gelatin capsule, it may contain, in addition to materials
of the above type, a liquid carrier such as polyethylene glycol or
a fatty oil.
[0037] The formulation may be in the form of a liquid, e.g., an
elixir, syrup, solution, aqueous or oily emulsion or suspension, or
even dry powders which may be reconstituted with water and/or other
liquid media prior to use. The liquid may be for oral
administration or for delivery by injection, as two examples. When
intended for oral administration, preferred compositions contain,
in addition to the present compounds, one or more of a sweetening
agent, thickening agent, preservative (e.g., alkyl
p-hydoxybenzoate), dye/colorant and flavor enhancer (flavorant). In
a composition intended to be administered by injection, one or more
of a surfactant, preservative (e.g., alkyl p-hydroxybenzoate),
wetting agent, dispersing agent, suspending agent (e.g., sorbitol,
glucose, or other sugar syrups), buffer, stabilizer and isotonic
agent may be included. The emulsifying agent may be selected from
lecithin or sorbitol monooleate.
[0038] The liquid pharmaceutical formulations of the invention,
whether they be solutions, suspensions or other like form, may
include one or more of the following adjuvants: sterile diluents
such as water for injection, saline solution, preferably
physiological saline, Ringer's solution, isotonic sodium chloride,
fixed oils such as synthetic mono or digylcerides which may serve
as the solvent or suspending medium, polyethylene glycols,
glycerin, propylene glycol or other solvents; antibacterial agents
such as benzyl alcohol or methyl paraben; antioxidants such as
ascorbic acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. The parenteral preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic. Physiological saline is a preferred
adjuvant. An injectable pharmaceutical composition is preferably
sterile.
[0039] The pharmaceutical formulation may be intended for topical
administration, in which case the carrier may suitably comprise a
solution, emulsion, ointment, cream or gel base. The base, for
example, may comprise one or more of the following: petrolatum,
lanolin, polyethylene glycols, bee wax, mineral oil, diluents such
as water and alcohol, and emulsifiers and stabilizers. Thickening
agents may be present in a pharmaceutical composition for topical
administration. If intended for transdermal administration, the
composition may include a transdermal patch or iontophoresis
device.
[0040] The formulation may be intended for rectal administration,
in the form, e.g., of a suppository which will melt in the rectum
and release the drug. The composition for rectal administration may
contain an oleaginous base as a suitable nonirritating excipient.
Such bases include, without limitation, lanolin, cocoa butter and
polyethylene glycol. Low-melting waxes are preferred for the
preparation of a suppository, where mixtures of fatty acid
glycerides and/or cocoa butter are suitable waxes. The waxes may be
melted, and the aminocyclohexyl ether compound is dispersed
homogeneously therein by stirring. The molten homogeneous mixture
is then poured into convenient sized molds, allowed to cool and
thereby solidify.
[0041] The formulation may include various materials which modify
the physical form of a solid or liquid dosage unit. For example,
the composition may include materials that form a coating shell
around the active ingredients. The materials which form the coating
shell are typically inert, and may be selected from, for example,
sugar, shellac, and other enteric coating agents. Alternatively,
the active ingredients may be encased in a gelatin capsule or
cachet.
[0042] The pharmaceutical formulation may consist of gaseous dosage
units, e.g., it may be in the form of an aerosol. The term aerosol
is used to denote a variety of systems ranging from those of
colloidal nature to systems consisting of pressurized packages.
Delivery may be by a liquefied or compressed gas or by a suitable
pump system which dispenses the active ingredients. Aerosols of
compounds of the invention may be delivered in single phase,
bi-phasic, or tri-phasic systems in order to deliver the active
ingredient(s). Delivery of the aerosol includes the necessary
container, activators, valves, subcontainers, and the like, which
together may form a kit.
[0043] 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. The
appropriate salt would be chosen to enhance bioavailability or
stability of the compound for the appropriate mode of employment
(e.g., oral or parenteral routes of administration).
[0044] The present invention also provides kits that contain a
pharmaceutical formulation, together with instructions for the use
of the formulation. Preferably, a commercial package will contain
one or more unit doses of the formulation. Formulations which are
light and/or air sensitive may require special packaging and/or
formulation. For example, packaging may be used which is opaque to
light, and/or sealed from contact with ambient air, and/or
formulated with suitable coatings or excipients.
[0045] The formulations of the invention can be provided alone or
in combination with other compounds (for example, small molecules,
nucleic acid molecules, peptides, or peptide analogues), in the
presence of a carrier or any pharmaceutically or biologically
acceptable carrier. As used herein "pharmaceutically acceptable
carrier" or "excipient" includes any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like that are physiologically
compatible. The carrier can be suitable for any appropriate form of
administration. Pharmaceutically acceptable carriers generally
include sterile aqueous solutions or dispersions and sterile
powders. Supplementary active compounds can also be incorporated
into the formulations.
[0046] An "effective amount" of a formulation according to the
invention includes a therapeutically effective amount or a
prophylactically effective amount. A "therapeutically effective
amount" refers to an amount effective, at dosages and for periods
of time necessary, to achieve the desired therapeutic result. A
therapeutically effective amount of a formulation may vary
according to factors such as the disease state, age, sex, and
weight of the individual, and the ability of the compound to elicit
a desired response in the individual. Dosage regimens may be
adjusted to provide the optimum therapeutic response. A
therapeutically effective amount may also be one in which any toxic
or detrimental effects of the formulation or active compound are
outweighed by the therapeutically beneficial effects. A
"prophylactically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve the
desired prophylactic result. Typically, a prophylactic dose is used
in subjects prior to or at an earlier stage of disease, so that a
prophylactically effective amount may be less than a
therapeutically effective amount. For any particular subject, the
timing and dose of treatments may be adjusted over time (e.g.,
timing may be daily, every other day, weekly, monthly) according to
the individual need and the professional judgment of the person
administering or supervising the administration of the
compositions.
[0047] With respect to effective amounts of antibiotic treatments,
although the daptomycin susceptibility breakpoint for enterococci
is considered to be 4 mg/L, ongoing debate exists as to whether
this is appropriate given that approved doses are optimized only
when MIC .ltoreq.1 mg/L. For example, a recent study found that
when the MIC was 4 mg/L, the probability of attaining target
pharmacokinetic parameters in daptomycin-treated enterococcal
bacteremia reached only 1.5-5.5% when 6 mg/kg doses were
administered (Avery, Lindsay M et al. "Pharmacodynamic Analysis of
Daptomycin-treated Enterococcal Bacteremia: It Is Time to Change
the Breakpoint." Clinical Infectious Diseases Vol. 68,10 (2019):
1650-1657).
[0048] In therapeutic applications, synergy between active
ingredients occurs when an observed combined therapeutic effect is
greater than the sum of therapeutic effects of individual active
ingredients, or a new therapeutic effect is produced that the
active ingredients could not produce alone. Accordingly, when
components of a formulation are present in synergistically
effective amounts, the formulation yields a therapeutic effect that
is greater than would be achieved by the individual active
ingredients administered alone at comparable dosages. In this
context, the enhancement of therapeutic effect may take the form of
increased efficacy or potency and/or decreased adverse effects. The
synergistic effect may be mediated in whole or in part by the
pharmacokinetics and/or pharmacodynamics of the active ingredients
in a subject, so that the amount and proportion of the ingredients
in the formulation may be synergistic in vivo. This in vivo synergy
may be effected with a formulation that includes the active
ingredients in amounts and proportions that are also synergistic in
in vitro assays of efficacy. As used herein, the term
"synergistically effective amounts" accordingly refers to amounts
that are synergistic in vivo and/or in vitro. A numeric
quantification of synergy is often expressed as a fractional
inhibitory concentration index (FICI), which represents the sum of
the fractional inhibitory concentrations (FICs) of each drug
tested, where the FIC is determined for each drug by dividing the
minimum inhibitory concentration (MIC, the lowest concentration of
the drug which prevents visible growth of the bacterium in a
standard in vitro assay--standard colorometric assay based on
resazurin) of each drug when used in combination by the MIC of each
drug when used alone. In very general terms, a FICI lower or higher
than 1 indicates positively correlated activity (at least additive
or potentiation) or an absence of positive interactions,
respectively. More definitively, synergy of two compounds may be
conservatively defined as a FICI of .ltoreq.0.5 (see Odds, 2003;
with additivity or potentiation corresponding to a FICI of >0.5
to .ltoreq.0.75; no interaction (indifference) corresponding to a
FICI of >1 to .ltoreq.4; and antagonism corresponding to a FICI
of >>4). Synergy of three compounds has been defined as a
FICI of (Berenbaum, 1978; Yu et al., 1980). To estimate the optimal
concentrations in triple combinations, the optimal concentrations
of trans-caryophyllene and cannabinoid were utilized in combination
with a series of daptomycin dilutions.
[0049] Although various embodiments of the invention are disclosed
herein, many adaptations and modifications may be made within the
scope of the invention in accordance with the common general
knowledge of those skilled in this 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. Numeric ranges are inclusive of the numbers defining the
range. The word "comprising" is used herein as an open-ended term,
substantially equivalent to the phrase "including, but not limited
to", and the word "comprises" 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 "a thing" includes more than one such
thing.
[0050] Citation of references herein is not an admission that such
references are prior art to the present invention. Any priority
document(s) and all publications, including but not limited to
patents and patent applications, cited in this specification are
incorporated herein by reference. All documents cited or referenced
in herein cited documents, 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 herein by
reference, and may be employed in the practice of the invention.
More specifically, all referenced documents are incorporated by
reference to the same extent as if each individual publication were
specifically and individually indicated to be incorporated by
reference herein and as though fully set forth herein. The
invention includes all embodiments and variations substantially as
hereinbefore described and with reference to the examples and
drawings. In some embodiments, the invention excludes steps that
involve medical or surgical treatment.
EXAMPLES
Example 1
[0051] As illustrated in this Example, the non-psychotropic
cannabinoids, cannabidiol, cannabigerol and cannabichromene (i.e.,
CBD, CBG, and CBC), have bacteriostatic (Tables 1, 2) and
bactericidal (FIG. 1) activities against E. faecium and E.
faecalis, including strains that are sensitive or resistant to
vancomycin and, in some cases, also resistant to daptomycin. At
equal concentrations of CBD, CBG, or CBC, the kinetics of E.
faecium killing with CBG and CBC are faster than for CBD (FIG. 1).
It is further shown that cannabinoids can also increase the
activity of daptomycin (MIC) by 8-128 fold (Table 3). In addition,
we demonstrate that each cannabinoid has species specific
activities against Gram positive (but not Gram negative) bacteria
(Table 1).
[0052] As further illustrated in this Example, the non-psychotropic
cannabinoids, cannabidiol, cannabigerol and cannabichromene (i.e.,
CBD, CBG, and CBC), trans-caryophyllene, and daptomycin act in
synergy. Assays for interactions between all pairwise as well as
triple combinations of cannabinoids (CBC, CBD, CBG), trans
caryophyllene, and daptomycin are shown in Table 4. All pairwise
combinations showed synergistic (FICI.ltoreq.0.5) or potentiating
(FICI.gtoreq.0.5, .ltoreq.0.75) interactions. However, while
.alpha.-humulene (.beta.-caryophyllene, data not shown) and trans
caryophyllene had synergistic activities with CBC, CBD, or CBG
(Table 4), other terpenes (.alpha.-pinene and R-(+) limonene) had
no detectable activity either alone or in combination (MIC>32
mg/L; data not shown). All triple combination were synergistic
(FICIs<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)
[0053] As illustrated in Tables 6 and 7, analyses of double and
triple combinations against the drug resistant (daptomycin and
vancomycin) strain VRE 55A6 showed that
daptomycin/terpene/cannabinoid combinations are even more effective
against this strain (compared to the vancomycin/daptomycin
sensitive strain 33D3), increasing the MIC of daptomycin >500
fold (effectively suppressing the daptomycin resistance
phenotype).
[0054] The data presented in this Example was obtained as set out
below.
[0055] MIC Determination for all Bacteria Except Mycobacterium
species:
[0056] Pre-cultures of bacteria growing in their corresponding
liquid media (defined below) were diluted to an OD600 of 0.0025 and
100 .mu.l was added to 100 .mu.l of medium containing serial 2-fold
dilutions of cannabinoid (cannabinoid stocks, in methanol or
acetonitrile for acidic forms, were diluted in growth medium) in
96-well plates. Plates were then incubated for 20-24 h at
37.degree. C., and growth was recorded as OD600 in a Thermo
Scientific.TM. Varioskan.TM. Flash Multimode Reader.
[0057] MIC determination for Mycobacterium abscessus ATCC
19977:
[0058] Pre-cultures growing in MHII medium with 0.05% tyloxapol
were diluted to an OD600 of 0.005 in MHII medium, and 100 .mu.l was
added to 100 .mu.l of MHII medium containing serial 2-fold
dilutions of cannabinoid (cannabinoid stocks in methanol or
acetonitrile for acidic forms were diluted in growth medium) in
96-well plates. Plates were then incubated for 48 h, followed by
the addition of 30 .mu.l the colorimetric reagent resazurin in
water (10 mg/100 ml). Plates were incubated for an additional 24 h,
and growth was recorded as conversion of culture color from blue to
pink.
[0059] MIC Determination for Mycobacterium tuberculosis H37Rv:
[0060] Pre-cultures of M. tuberculosis growing in 7H9 medium plus
0.05% tyloxapol, were grown to mid-log phase and diluted to OD600
0.0025 in the same medium without tyloxapol. 50 .mu.L of diluted
culture were used to inoculate wells in 96-well plates containing
50 .mu.L of the same medium with serial 2-fold dilutions of
cannabinoid (cannabinoid stocks in methanol or acetonitrile were
diluted in growth medium). Plates were incubated for 5 days at
37.degree. C., 5% CO2, followed by the addition of 10 .mu.L
PrestoBlue Cell Viability Reagent to each well. Plates were
incubated for a further 24 h at 37.degree. C., 5% CO2 and then
fluorescence (excitation 530 nm, emission 590 nm) was read on a
Synergy HT plate reader.
[0061] Growth Media Used for Various Organisms [0062] Enterococcus
faecium: all strains (clinical isolates 33D3, 69C6, 58C9, and
55A6), MHII (Mueller Hinton II) [0063] Enterococcus faecalis: MHII
(Mueller Hinton II) [0064] Streptococcus pyogenes ATCC 51878: BHI
(Brain Heart Infusion*) [0065] Staphylococcus aureus MRSA USA 300:
LB (Luria-Bertani) [0066] Staphylococcus epidermidis ATCC 14990: NB
(Nutrient Broth) [0067] Staphylococcus epidermidis ATCC 12228: NB
(Nutrient Broth) [0068] Mycobacterium abscessus ATCC 19977: MHII
(Mueller Hinton II) [0069] Mycobacterium tuberculosis H37Rv: 7H9
[0070] Mycobacterium tuberculosis H37Rv: PB (Proskauer and
Beck+glucose and sodium pyruvate) [0071] Acinetobacter baumanii
ATCC 19606: NB (Nutrient Broth) [0072] Escherichia coli HB101: (LB
(Luria-Bertani) [0073] Pseudomonas aeruginosa ATCC 27853: Tryptic
Soy Broth [0074] *Streptococcus pyogenes ATCC 51878 was grown at
37.degree. C. in a tri-gas incubator.
Method for FIG. 1.
[0075] Pre-cultures were diluted to an OD600 of 0.0025 in 3 ml MHII
medium supplemented with appropriate concentrations of
cannabinoids. At various time points, 100 .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 at
37.degree. C. for 24 h, and the colonies were counted (CFU).
[0076] Method for Tables 1-3. MIC determinations were carried out
as described above.
[0077] Method for Tables 4-7. Interaction of Cannabinoids CBC, CBD
and CBG with Terpenes and Daptomycin
[0078] FICI values was determined in a 96 well checkerboard format
using the MIC assay for cannabinoids (compound A), terpenes
(compound B), and daptomycin (compound C) alone and in
combinations. Terpene stocks were prepared in DMSO and diluted in
the growth medium. The fractional inhibitory concentration for each
compound was calculated as follows: FIC.sub.A=(MIC of compound A in
the presence of compound B)/(MIC of compound A alone). Similarly,
the FICs for compounds B and
[0079] C were calculated. The FICI was calculated as FIC.sub.A plus
FIC.sub.B plus FIC.sub.C.
[0080] Table 1: Cannabinoids have activity against diverse
Gram-positive bacteria but not against Gram-negative bacteria,
(Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumanii ATCC
19606, l E. coli HB101, data not shown). Sensitivities are
expressed as MICs (mg/L).
TABLE-US-00001 TABLE 1 CBD CBDA CBG CBGA CBC CBCA Staphylococcus 2
16 1 1 4 4 aureus MRSA Streptococcus 1 8 1 2 1 0.5-1 pyogenes ATCC
51878 Enterococcus 1-2 16 1-2 16 1-2 4 faecium 33D3 Enterococcus 2
16 1 8 2 4 faecalis Staphylococcus 2 4 1-2 2 4 2 epidermidis ATCC
14990 Staphylococcus 2 4 2 2 4 2 epidermidis ATCC 12228
Mycobacterium 16 32 2 16 4 4 abscessus ATCC 19977 Mycobacterium 4 4
2 2 4 4 tuberculosis H37Rv Media PB Mycobacterium 16 2 2 4 8 2
tuberculosis H37Rv Media 7H9
TABLE-US-00002 TABLE 2 Cannabinoid activity against clinical
enterococcal strains (vancomycin sensitive, vancomycin resistant,
and resistant to both vancomycin and daptomycin), expressed as MICs
(mg/L). Cannabinoids 33D3 69C6* 58C9*.sup.# 55A6*.sup.# CBD 1 1 1 2
CBDA 16 16 16 16 CBG 1 1 1 1 CBGA 16 8 16 8 CBC 1 1 1 1 CBCA 4 4 4
2 Daptomycin 2 1 8 8 *Vancomycin resistant .sup.#Daptomycin
resistant (MIC is greater that 4 mg/L)
TABLE-US-00003 TABLE 3 Cannabinoids potentiate the activity of
daptomycin against clinical isolates of E. faecium. Fold reduction
in daptomycin MIC in combination with: Isolate CBD CBG CBC 33D3 16
32 128 69C6* 4 4 32 55a6*.sup.# 16 4 512 58c9*.sup.# 32 64 8
*indicates that the strain is vancomycin resistant.
.sup.#Daptomycin resistant strains (MIC is greater than 4
mg/L).
TABLE-US-00004 TABLE 4 Synergistic and potentiating interactions of
pairwise and triple combinations of Cannabinoids (CBC, CBD, or
CBG), Trans-caryophyllene, and Daptomycin inhibit E. faecium 33D3
growth. Combinations FICI Interaction A. Pairwise interactions of
CBC, Trans-caryophyllene and Daptomycin Daptomycin 0.51 Synergy CBC
Daptomycin 0.25-0.75 Potentiation/Synergy Trans-caryophyliene CBC
0.37 Synergy Trans-caryophyllene 4B. Pairwise interactions of CBD,
Trans-caryophyllene and Daptomycin Daptomycin 0.56 Likely Synergy
CBD Daptomycin 0.25-0.75 Potentiation/Synergy Trans-caryophyllene
CBD 0.37 Synergy Trans-caryophyllene 4C. Pairwise interactions of
CBG, Trans-caryophyllene and Daptomycin Daptomycin 0.53 Synergy CBG
Daptomycin 0.25-0.75 Potentiation/Synergy Trans-caryophyllene CBG
0.37-0.5 Synergy Trans-caryophyllene 4D. Interactions of Triple
Combinations of a cannabinoid (CBC, CBD, or CBG),
Trans-caryophyllene and Daptomycin Daptomycin 0.75* Synergy CBC,
Trans-caryophyllene Daptomycin 0.75 Synergy CBD,
Trans-caryophyllene Daptomycin 0.81 Synergy CBG,
Trans-caryophyllene *For triple combinations, FICI <1.0
represents synergy
TABLE-US-00005 TABLE 5 Maximal fold increases in the activities of
individual Cannabinoids (CBC, CBD, CBG), Trans-caryophyllene and
Daptomycin in pairwise and triple combinations against E. faecium
33D3. MIC (mg/L) Max. fold Combinations Alone In combination change
In MIC 5A. Pairwise interactions of CBC, Trans-caryophyllene and
Daptomycin Daptomycin 4 0.03125 128 .dwnarw. CBC 2 0.5 4 .dwnarw.
Daptomycin 4 0.5 8 .dwnarw. Trans-caryophyllene >16 4 >4
.dwnarw. CBC 2 0.125 16 .dwnarw. Trans-caryophyllene 16 0.25 64
.dwnarw. 5B. Pairwise interactions of CBD, Trans-caryophyllene and
Daptomycin Daptomycin 4 0.25 16 .dwnarw. CBD 4 1 4 .dwnarw.
Daptomycin 4 0.5 8 .dwnarw. Trans-caryophyllene >16 4 >4
.dwnarw. CBD 2 0.125 16 .dwnarw. Trans-caryophyllene 16 2 8
.dwnarw. 5C. Pairwise interactions of CBG, Trans-caryophyllene and
Daptomycin Daptomycin 8 0.25 32 .dwnarw. CBG 4 0.125 32 .dwnarw.
Daptomycin 4 0.5 8 .dwnarw. Trans-caryophyllene >16 4 >4
.dwnarw. CBG 2 0.125 16 .dwnarw. Trans-caryophyllene 32 4 8
.dwnarw. 5D. Interactions of Triple Combinations of a Cannabinoid
(CBC, CBD, or CBG), Trans-caryophyllene and Daptomycin Daptomycin 8
4 2 .dwnarw. CBC, Trans-caryophyllene 2, 16 0.25, 2 8 .dwnarw.
Daptomycin 8 0.03 256 .dwnarw. CBD, Trans-caryophyllene 2, 16 0.5,
2 4, 8 .dwnarw. Daptomycin 8 2 4 .dwnarw. CBG, Trans-caryophyllene
1, 16 0.25, 1 4, 16 .dwnarw.
TABLE-US-00006 TABLE 6 Synergistic and potentiating pairwise or
triple interactions of cannabinoids (CBC, CBD, CBG),
trans-caryophyllene, and daptomycin inhibit E. faecium VRE 55A6
growth. Combinations FICI Interaction 6A. Pairwise interactions of
CBC, Trans-caryophyllene and Daptomycin Daptomycin 0.5 Synergy CBC
Daptomycin 0.5-0.625 Synergy/Potentiation Trans-caryophyllene CBC
0.51-0.53 Synergy Trans-caryophyllene 6B. Pairwise interactions of
CBD, Trans-caryophyllene and Daptomycin Daptomycin 0.5 Synergy CBD
Daptomycin 0.5-0.625 Synergy/Potentiation Trans-caryophyllene CBD
0.5-0.75 Potentiation Trans-caryophyllene 6C. Pairwise interactions
of CBG, Trans-caryophyllene and Daptomycin Daptomycin 0.75
Potentiation CBG Daptomycin 0.5-0.625 Synergy/Potentiation
Trans-caryophyllene CBG >1 No interaction Trans-caryophyllene
6D. Interactions of Triple Combinations of a Cannabinoid (CBC, CBD,
or CBG), Trans-caryophyllene and Daptomycin Daptomycin 0.25-0.56
Synergy CBC, Trans-caryophyllene Daptomycin 0.25-0.44 Synergy CBD,
Trans-caryophyllene Daptomycin 0.5-0.69 Synergy CBG,
Trans-caryophyllene * For triple combinations, FICI <1.0
represents synergy
TABLE-US-00007 TABLE 7 Maximal fold increases in the activities of
individual cannabinoids (CBC, CBD, CBG), trans-caryophyllene and
daptomycin in pairwise or triple combinations against E. faecium
VRE 55A6. MIC (mg/L) Max. fold Combinations Alone In combination
change in MIC 7A. Pairwise interactions of CBC, Trans-caryophyllene
and Daptomycin Daptomycin 16 0.031 512 .dwnarw. CBC 2 0.25 8
.dwnarw. Daptomycin 16 8 2 .dwnarw. Trans-caryophyllene >16 2
>8 .dwnarw. CBC 1 0.5 2 .dwnarw. Trans-caryophyllene >32 1
>32 .dwnarw. 7B. Pairwise interactions of CBD,
Trans-caryophyllene and Daptomycin Daptomycin 16 1 16 .dwnarw. CBD
4 0.5 8 .dwnarw. Daptomycin 16 8 2 .dwnarw. Trans-caryophyllene
>16 2 >8 .dwnarw. CBD 2 1 2 .dwnarw. Trans-caryophyllene
>32 8 >4 .dwnarw. 7C. Pairwise interactions of CBG,
Trans-caryophyllene and Daptomycin Daptomycin 16 4 4 .dwnarw. CBG 4
2 2 .dwnarw. Daptomycin 16 8 2 .dwnarw. Trans-caryophyllene >16
2 >8 .dwnarw. CBG 2 2 none Trans-caryophyllene >32 >32
none 7D. Interactions of Triple Combinations of a Cannabinoid (CBC,
CBD, or CBG), Trans-caryophyllene and Daptomycin Daptomycin >16
0.03 >512 .dwnarw. CBC, Trans-caryophyllene 2, >64 0.25, 2 8,
>32 .dwnarw. Daptomycin >16 2 >8 .dwnarw. CBD,
Trans-caryophyllene 4, >64 0.5, 2 8, >32 .dwnarw. Daptomycin
>16 2 >8 .dwnarw. CBG, Trans-caryophyllene 2, >64 0.5, 2
4, >32 .dwnarw.
[0081] The foregoing data illustrate particularly important
interactions with certain cannabinoid-daptomycin combinations,
evidencing synergy: e.g. Table 6A FICI=0.5 for CBC+Daptomycin;
Table 6B FICI=0.5 for CBD+daptomycin. In addition to the respective
relative amounts that gave synergy, this data illustrates that
other relative amounts produce surprising and unexpected
significant growth inhibition of E. faecium: e.g. Table 5A shows a
128-fold reduction in the MIC of daptomycin (MIC reduced to from 4
to 0.03125 mg/L). This effect occurred when CBC was present at 1/2
its MIC (i.e., CBC MIC reduced from 1 to 0.5 mg/L, data not shown).
This interaction occurred with each partner in potentiating ratios,
relative amounts of 0.5:0.03125 =16:1 CBC:Daptomycin.
Example 2: Cannabinoid Sensitization to Daptomycin
[0082] As illustrated in FIG. 2, cannabinoids sensitize antibiotic
resistant Enterococcal strains to daptomycin. FIG. 2 reflects data
illustrating the effects of daptomycin (Dap), and Dap in
combination with CBD, CBG or CBC, on daptomycin resistant E.
faecium VRE strains 58C9 and 55A6. E. faecium strain 55A6 is
resistant to daptomycin with a daptomycin MIC=8 mg/L. E. faecium
strains 58C9 and 55A6 were incubated with no cannabinoid (A,B), or
1/2x (0.5 mg/L) MIC of CBD (C,D), CBG (E,F) or CBC (G, H) with 1x
(4 mg/L for 58C9; 8 mg/L for 55A6), 1/2x (2 mg/L for 58C9; 4 mg/L
for 55A6), 1/4x (1 mg/L for 58C9; 2 mg/L for 55A6) or 1/8x (0.5
mg/L for 58C9; 1 mg/L for 55A6) MIC of daptomycin. Aliquots were
removed at 2, 4, 6, 8 and 24 h for CFU enumeration. Data points are
the means from three replicates with standard deviations presented
as error bars. As illustrated, adding approximately 1/2 MIC of each
cannabinoid facilitated killing by daptomycin at 1/4-1/8x the MIC
of daptomycin.
[0083] To further illustrate the bactericidal potentiation activity
of CBD, CBG and CBC with daptomycin, kill curves were generated
with E. faecium strain 33D3 (daptomycin sensitive, MIC=1), as
illustrated in FIG. 3. This data is usefully compared to the data
in FIG. 2 for E. faecium strains 58C9 (daptomycin intermediate
resistance MIC=4) and 55A6 (daptomycin resistant, MIC=8). As
illustrated, strains were each incubated with 1/8x to 1x MIC of
daptomycin (0.25-1 mg/L daptomycin for 33D3, 0.5-4 mg/L daptomycin
for 58C9 and 1-8 mg/L daptomycin for 55A6) and with 0.5x MIC of
CBD, CBG or CBC. At 1/2x MIC there was effectively no inhibition of
growth by a cannabinoid or daptomycin in all strains. Of note,
although daptomycin did inhibit growth at 1x MIC, there was no
reduction in CFU. However, 1/2x MIC of CBD, CBG or CBC in
combination of 1x-1/8x MIC of daptomycin significantly inhibited
growth of all strains and reduced CFU, thereby demonstrating
bactericidal activity (FIGS. 2 and 3). This has particularly
important clinical relevance, since daptomycin is generally not
used to treat patients infected by strains classified as daptomycin
resistant (MIC>4 mg/L). Strains 58C9 and 55A6 are daptomycin
intermediate resistant and resistant respectively, but in the
presence of 1/2x MIC of CBD, CBG or CBC, as little as 1 mg/L
Daptomycin (1/8x MIC) could significantly reduce the CFU at 24 h
(FIG. 2).
[0084] This Example further illustrates that a broad range of
relative cannabinoid-daptomycin amounts can provide surprising and
unexpected results, as evidenced by the kill curves in FIGS. 2 and
3. For example, surprising and clinically meaningful effects were
seen at the following relative cannabinoid-daptomycin amounts: For
CBC:Daptomycin 1:1-4 (FIG. 3 33D3 strain), 1:1-8 (FIG. 2; 58C9
Strain), and 1:4-16 (FIG. 2 55A6 Strain); For CBG:Daptomycin 2:1
and 1:1-4 (FIG. 3 33D3 Strain), 1:1-8 (FIG. 2; 58C9 Strain), and
1:2-16 (FIG. 2; 55A6 Strain); and, for CBD:Daptomycin 2:1 and 1:1-4
(FIG. 3 33D3 Strain), 1:1-8 (FIG. 2; 58C9), and 1:2-16 (FIG. 2;
55A6 Strain).
Example 3: CBD and CBG Biofilm Inhibition
[0085] This example illustrates that CBD and CBG are surprisingly
effective at inhibiting E. faecium biofilm growth. The data in FIG.
4 was generated by growing biofilms of E. faecium strain 33D3 for
48 h without cannabinoids, then treating the biofilms with
cannabinoids for 72 h. The mass of the biofilm was quantitated by
crystal violet staining. Viable bacteria in the biofilm were
quantitated by scrapping biofilm then counting the viable bacteria.
As illustrated, CBD and CBG illustrate a surprising degree of
antibiotic activity against the strain 33D3 of E. faecium. Of note,
CBC was bactericidal against planktonic Enterococci.
REFERENCES
[0086] Abdelaziz, A., (1982) Studies on the antimicrobial activity
of cannabinoids. MS thesis, Ohio State University.
[0087] Andre, C. M.; Hausman, J.-F.; Guerriero, G. (2016).
"Cannabis sativa: The Plant of the Thousand and One Molecules".
Frontiers in Plant Science. 7: 19.
[0088] Appendino, G., G. Chianese & 0. Taglialatela-Scafati,
(2011) Cannabinoids: occurrence and medicinal chemistry. Curr Med
Chem 18: 10854099.
[0089] Appendino, G., S. Gibbons, A. Giana, A. Pagani, G. Grassi,
M. Stavri, E. Smith & M. M. Rahman, (2008) Antibacterial
cannabinoids from Cannabis sativa: a structure-activity study. J
Nat Prod 71: 1427-1430.
[0090] Arias, C. A. & B. E. Murray, (2012) The rise of the
Enterococcus: beyond vancomycin resistance. Nat Rev Microbiol 10:
266-278.
[0091] Arias, C. A., D. Panesso, D. M. McGrath, X. Qin, M. F.
Mojica, C. Miller, L. Diaz, T. T. Tran, S. Rincon, E. M.
[0092] Baltz R H (December 2006). "Molecular engineering approaches
to peptide, polyketide and other antibiotics". Nature
Biotechnology. 24 (12): 1533-40.
[0093] Barbu, J. Reyes, J. H. Roh, E. Lobos, E. Sodergren, R.
Pasqualini, W. Arap, J. P. Quinn, Y. Shamoo, B. E. Murray & G.
M. Weinstock, (2011) Genetic basis for in vivo daptomycin
resistance in enterococci. N Engl.' Med 365: 892-900.
[0094] Berenbaum, M. C. 1978. A method for testing for synergy with
any number of agents. J. Infect. Dis. 137:122-130.
[0095] Arthur, M. & P. Courvalin, (1993) Genetics and
mechanisms of glycopeptide resistance in enterococci. Antimicrob
Agents Chemother 37: 1563-1571
[0096] Consroe, P., J. Laguna, J. Allender, S. Snider, L. Stern, R.
Sandyk, K. Kennedy & K. Schram, (1991) Controlled clinical
trial of cannabidiol in Huntington's disease. Pharmacol Biochem
Behav 40: 701-708.
[0097] Cunha, J. M., E. A. Carlini, A. E. Pereira, O. L. Ramos, C.
Pimentel, R. Gagliardi, W. L. Sanvito, N. Lander & R.
Mechoulam, (1980) Chronic administration of cannabidiol to healthy
volunteers and epileptic patients. Pharmacology 21: 175-185.
[0098] Eisohly, H. N., C. E. Turner, A. M. Clark & M. A.
Eisohly, (1982) Synthesis and antimicrobial activities of certain
cannabichromene and cannabigerol related compounds. J Pharm Sci 71:
1319-1323.
[0099] Galloway-Pena, J. R., S. R. Nallapareddy, C. A. Arias, G. M.
Eliopoulos & B. E. Murray, (2009) Analysis of clonality and
antibiotic resistance among early clinical isolates of Enterococcus
faecium in the United States. J Infect Dis 200: 1566-1573.
[0100] Hidron, A. I., J. R. Edwards, J. Patel, T. C. Horan, D. M.
Sievert, D. A. Pollock, S. K. Fridkin, T. National Healthcare
Safety Network & F. Participating National Healthcare Safety
Network, (2008) NHSN annual update: antimicrobial-resistant
pathogens associated with healthcare-associated infections: annual
summary of data reported to the National Healthcare Safety Network
at the Centers for Disease Control and Prevention, 2006-2007.
Infect Control Hosp Epidemiol 29: 996-1011.
[0101] Luo X, Reiter M A, d'Espaux L, Wong J, Denby C M, Lechner A,
Zhang Y, Grzybowski A T, Harth S, Lin W, Lee H, Yu C, Shin J, Deng
K, Benites V T, Wang G, Baidoo E E K, Chen Y, Dev I, Petzold C J,
Keasling J D. 2019. Complete biosynthesis of cannabinoids and their
unnatural analogues in yeast. Nature 567:123-126
[0102] Mechoulam, R. & Y. Gaoni, (1965) Hashish. I V. The
isolation and structure of cannabinolic cannabidiolic and
cannabigerolic acids. Tetrahedron 21: 1223-1229.
[0103] Miao V, Coeffet-Le Gal M F, Nguyen K, Brian P, Penn J,
Whiting A, Steele J, Kau D, Martin S, Ford R, Gibson T, Bouchard M,
Wrigley S K, Baltz R H (March 2006). "Genetic engineering in
Streptomyces roseosporus to produce hybrid lipopeptide
antibiotics". Chemistry & Biology. 13 (3): 269-76.
[0104] Morales, P., D. P. Hurst & P. H. Reggio, (2017)
Molecular Targets of the Phytocannabinoids: A Complex Picture. Prog
Chem Org Nat Prod 103: 103-131.
[0105] Murdoch, D. R., G. R. Corey, B. Hoen, J. M. Miro, V. G.
Fowler, Jr., A. S. Bayer, A. W. Karchmer, L. Olaison, P. A. Pappas,
P. Moreillon, S. T. Chambers, V. H. Chu, V. Falco, D. J. Holland,
P. Jones, J. L. Klein, N. J. Raymond, K. M. Read, M. F. Tripodi, R.
Utili, A. Wang, C. W. Woods, C. H. Cabell & I. International
Collaboration on Endocarditis-Prospective Cohort Study, (2009)
Clinical presentation, etiology, and outcome of infective
endocarditis in the 21st century: the International Collaboration
on Endocarditis-Prospective Cohort Study. Arch Intern Med 169:
463-473.
[0106] Najafi K, Ganbarov K, Gholizadeh P, Tanomand A, Rezaee M A,
Mahmood S S, Asgharzadeh M, Kafil H S. 2019. Oral cavity infection
by Enterococcus faecalis: virulence factors and pathogenesis.
Reviews in Medical Microbiology 29: OOO Publish Ahead of Print.
[0107] Nguyen K T, Kau D, Gu J Q, Brian P, Wrigley S K, Baltz R H,
Miao V (September 2006). "A glutamic acid 3-methyltransferase
encoded by an accessory gene locus important for daptomycin
biosynthesis in Streptomyces roseosporus". Molecular Microbiology.
61 (5): 1294-307.
[0108] Nguyen, K. T., He, X., Alexander, D. C., Li, C., Gu, J. Q.,
Mascio, C., Van Praagh, A., Mortin, L., Chu, M., Silverman, J. A.,
Brian, P., & Baltz, R. H. (2010). Genetically engineered
lipopeptide antibiotics related to A54145 and daptomycin with
improved properties. Antimicrobial agents and chemotherapy, 54(4),
1404-1413.
[0109] Odds, F. C., Synergy, antagonism, and what the chequerboard
puts between them, Journal of Antimicrobial Chemotherapy, (2003)
52, 1.
[0110] Paganelli, Fernanda L et al. "Enterococcus faecium biofilm
formation: identification of major autolysin AtIAEfm, associated
Acm surface localization, and AtIAEfm-independent extracellular DNA
Release." mBio vol. 4,2 e00154. 16 Apr. 2013,
doi:10.1128/mBio.00154-13.
[0111] Prematunge, C., C. MacDougall, J. Johnstone, K. Adomako, F.
Lam, J. Robertson & G. Garber, (2016) VRE and VSE Bacteremia
Outcomes in the Era of Effective VRE Therapy: A Systematic Review
and Meta-analysis. Infect Control Hosp Epidemiol 37: 26-35.
[0112] Russo, E. B., (2011) Taming THC: potential Cannabis synergy
and phytocannabinoid-terpenoid entourage effects. Br .1 Pharmacol
163: 1344-1364.
[0113] Schleifer, K. H. & R. Kilpperbalz, (1984) Transfer of
Streptococcus-Faecalis and Streptococcus-Faecium to the Genus
Enterococcus Nom Rev as Enterococcus-Faecalis Comb-Nov and
Enterococcus-Faecium Comb-Nov. Intl Syst Bacteriol 34: 31-34.
[0114] Steenackers, B.; De Cooman, L.; De Vos, D. (2015). "Chemical
transformations of characteristic hop secondary metabolites in
relation to beer properties and the brewing process: A review".
Food Chemistry. 172: 742-756.
[0115] Trost and Dogra, (2007) Synthesis of
(-)-.DELTA.9-trans-Tetrahydrocannabinol-Stereocontrol via
Mo-catalyzed Asymmetric Allylic Alkylation Reaction. Org Lett. 2007
Mar. 1; 9(5): 861-863.
[0116] Turner, C. E. & M. A. Elsohly, (1981) Biological
activity of cannabichromene, its homologs and isomers. J Clin
Pharmacol 21: 2835-2915. Van Klingeren, B. & M. Ten Ham, (1976)
Antibacterial activity of delta9-tetrahydrocannabinol and
cannabidiol. Antonie Van Leeuwenhoek 42: 9-12.
[0117] Yu, V. L., T. P. Felegie, R. B. Yee, A. W. Pasculle, and F.
H. Taylor. 1980. Synergistic interaction in vitro with use of three
antibiotics simultaneously against Pseudomonas maltophilia. J.
Infect. Dis. 142:602-607.
[0118] Zhong et al., (2017) Comparative genomic analysis of the
genus Enterococcus. Microbiological Research, Volume 196, March
2017, Pages 95-105.
* * * * *