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Cannabis constituent synergy in a mouse neuropathic pain model

Authors:
  • University of Sydney. Royal North Shore Hospital

Abstract and Figures

Cannabis and its psychoactive constituent Δ9-tetrahydrocannabinol (THC) have efficacy against neuropathic pain however, this is hampered by their side-effects. It has been suggested that co-administration with another major constituent cannabidiol (CBD) might enhance the analgesic actions of THC and minimise its deleterious side-effects. We examined the basis for this phytocannabinoid interaction in a mouse chronic constriction injury (CCI) model of neuropathic pain. Acute systemic administration of THC dose-dependently reduced CCI-induced mechanical and cold allodynia, but also produced motor incoordination, catalepsy and sedation. CBD produced a lesser dose-dependent reduction in allodynia, but did not produce the cannabinoid side-effects. When co-administered in a fixed ratio, THC and CBD produced a biphasic dose-dependent reduction in allodynia. At low doses, the THC:CBD combination displayed a 200-fold increase in anti-allodynic potency, but had lower efficacy compared to that predicted for an additive drug interaction. By contrast, high THC:CBD doses had lower potency, but greater anti-allodynic efficacy compared to that predicted for an additive interaction. Only the high dose THC:CBD anti-allodynia was associated with cannabinoid side-effects and these were similar to those of THC alone. Unlike THC, the low dose THC:CBD anti-allodynia was not cannabinoid receptor mediated. These findings demonstrate that CBD synergistically enhances the pain relieving actions of THC in an animal neuropathic pain model, but has little impact on the THC-induced side-effects. This suggests that low dose THC:CBD combination treatment has potential in the treatment of neuropathic pain.
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Research Paper
Cannabis constituent synergy in a mouse
neuropathic pain model
Sherelle L. Casey*, Nicholas Atwal, Christopher W. Vaughan
Abstract
Cannabis and its psychoactive constituent D9-tetrahydrocannabinol (THC) have efficacy against neuropathic pain, however, this is
hampered by their side effects. It has been suggested that co-administration with another major constituent cannabidiol (CBD) might
enhance the analgesic actions of THC and minimise its deleterious side effects. We examined the basis for this phytocannabinoid
interaction in a mouse chronic constriction injury (CCI) model of neuropathic pain. Acute systemic administration of THC dose-
dependently reduced CCI-induced mechanical and cold allodynia, but also produced motor incoordination, catalepsy, and
sedation. Cannabidiol produced a lesser dose-dependent reduction in allodynia, but did not produce the cannabinoid side effects.
When co-administered in a fixed ratio, THC and CBD produced a biphasic dose-dependent reduction in allodynia. At low doses, the
THC:CBD combination displayed a 200-fold increase in anti-allodynic potency, but had lower efficacy compared with that predicted
for an additive drug interaction. By contrast, high THC:CBD doses had lower potency, but greater anti-allodynic efficacy compared
with that predicted for an additive interaction. Only the high dose THC:CBD anti-allodynia was associated with cannabinoid side
effects and these were similar to those of THC alone. Unlike THC, the low dose THC:CBD anti-allodynia was not cannabinoid
receptor mediated. These findings demonstrate that CBD synergistically enhances the pain-relieving actions of THC in an animal
neuropathic pain model, but has little impact on the THC-induced side effects. This suggests that low dose THC:CBD combination
treatment has potential in the treatment of neuropathic pain.
Keywords: Cannabis, Neuropathic pain model, Phytocannabinoid, Synergy, Receptor
1. Introduction
Neuropathic pain is a debilitating condition resulting from damage
to the peripheral or central nervous system and can be caused by
physical trauma, such as accidents, surgery and stroke, or by
diseases such as diabetes, cancer, and immune disorders.
16
This
chronic pain state is difficult to manage, with many patients
experiencing pain that is refractory to currently available
pharmacotherapies and is often associated with disabling side
effects.
9
Given these problems, there is a need for novel
therapeutic options as either first-line medications or as adjuvants
to current therapies.
The plant Cannabis sativa has been used for thousands of
years to treat various medical conditions including pain,
neuralgia, cramps, nausea, diarrhoea, convulsions, and migraine.
Cannabis contains a multitude of phytocannabinoids including
the psychoactive constituent D9-tetrahydrocannabinol (THC) and
other constituents such as cannabidiol (CBD) which do not
produce THC-like psychotropic side effects.
21
To date, most
clinical trials on chronic pain have used either whole raw
cannabis, THC, or nabiximols, which are combinations of THC
and CBD. The most recent meta-analyses of these trials suggest
that cannabinoids have potential in the treatment of neuropathic
pain, although there are differing views on their clinical efficacy
and safety.
11,22,36
Given this uncertainty, it is crucial that we
understand the basis for the actions of these 2 phytocannabi-
noids alone, and in combination.
22
Numerous preclinical animal studies have shown that synthetic
cannabinoids have high pain-relieving efficacy in animal models
of neuropathic pain.
22
By contrast, relatively few studies have
examined the actions of phytocannabinoids in these neuropathic
pain models, which is surprising given their ubiquitous use in
clinical trials. These studies have shown that THC and CBD
reduce the allodynia associated with rodent neuropathic pain
models.
5–8,13,30,34,37
Recently, it has been demonstrated that
THC and CBD synergistically reduce the development of
allodynia in a chemotherapy-induced model of neuropathic
pain.
18
A limitation of these studies is that they do not provide
a systematic dose–response analysis of both the anti-allodynic
actions and side effects of phytocannabinoids. This is important
because it provides information on the efficacy and therapeutic
window (TW) of cannabinoids,
1,12,17,27
both of which are
important factors in their clinical use. Furthermore, although it
has been proposed that nabiximols might offer a superior,
synergistic alternative to individual cannabis constituents,
24
their
actions in neuropathic pain models are unknown. Thus, the
preclinical basis for the use of specific phytocannabinoids in
neuropathic pain states, either alone or in combination, remains
unclear. To address this, we used an isobolographic approach to
examine whether there is a synergistic interaction between THC
Sponsorships or competing interests that may be relevant to content are disclosed
at the end of this article.
Pain Management Research Institute, Kolling Institute of Medical Research,
Northern Clinical School, Royal North Shore Hospital, University of Sydney, Sydney,
New South Wales, Australia
*Corresponding author. Address: Pain Management Research Institute, Kolling
Institute of Medical Research, Northern Clinical School, Royal North Shore Hospital,
University of Sydney, St Leonards, Sydney, NSW 2065, Australia. Tel.: 61(2)9926
4963. E-mail address: scas5381@uni.sydney.edu.au (S. L. Casey).
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and CBD in a mouse neuropathic pain model and whether this is
associated with an improvement in their analgesic efficacy
and TW.
2. Methods
All experiments in this study were performed on 8- to 9-week-old
male C57BL/6 mice, and are reported in compliance with the
Animals In Research Reporting In Vitro Experiment (ARRIVE)
guidelines and those of the “NH&MRC Code of Practice for the
Care and Use of Animals in Research in Australia”, and were
approved by the Royal North Shore Hospital Animal Ethics
Committee. Mice were obtained from the Kolling Institute Animal
Facility, and were initially housed in groups of 4 and then
individually after surgery. Individually ventilated cages were
maintained at 22˚C to 23˚C and humidity 65% to 75%, with
a 12:12 hours light:dark cycle. Animals had ad libitum access to
food and water throughout all stages of the study. Cages were
enriched with a mouse house igloo, tissues for nesting, and either
a straw or paddle pop stick for play.
2.1. Neuropathic pain model
A commonly used nerve injury model of neuropathic pain, the
chronic constriction injury (CCI) model of neuropathic pain, was
used for this study.
2,17
For the CCI surgery, anaesthesia was
induced with 4% isoflurane and then maintained with 2%
isoflurane in saturatedoxygen (1 mL·min
21
). To avoid hypothermia,
the mice were positioned on a heat mat for the duration of the
procedure. The fur over the mid-thigh region was clipped to avoid
contamination of the wound. The left common sciatic nerve was
exposed at the mid-thigh level and 2 size 6-0 chromic gut
ligatures placed around the nerve 2 to 3 mm apart. The ligatures
were tightened until a twitch of the foot was observed, while
ensuring that the blood supply to the nerve was not compro-
mised. The muscle incision was closed with 6-0 silk and the skin
incision closed with either a simple continuous suture pattern
using 6-0 silk or with tissue glue. The animals were recovered and
monitored before being returned to their cages. Animals were
then monitored daily until the day of the experiment.
2.2. Behavioural testing
To assess nerve injury–induced mechanical and cold allodynia,
animals were placed in elevated Perspex cages with a mesh wire
floor and were left to acclimatise for 30 minutes before any
testing. Mechanical allodynia was tested by applying a series of
von Frey filaments (0.2-6.84 g; North Coast Medical, San Jose,
CA) to the plantar surface of the operated hind paw. The measure
of the paw withdrawal threshold (PWT) was calculated using the
simplified up-down protocol.
3
A positive response was noted as
a rapid withdrawal of the paw from the hair. Cold allodynia was
tested by applying 20 mL of acetone to the plantar surface of the
left hind paw to induce evaporative cooling. The number of pain-
like responses (licking or chewing the paw, shaking the paw, or
looking at the paw) was counted.
Common cannabinoid side effects including motor impair-
ment, catalepsy, and sedation were also measured. Motor
impairment was tested using the rotarod. For this assay, mice
were placed on a bar which gradually increased its speed of
rotation from 4 to 30 rpm over a 300-second timeframe. The time
at which each mouse fell off the bar, or just held on to the bar for 2
consecutive rotations, was recorded. Catalepsy was tested using
the bar test. For this assay, the forepaws of the mouse were
placed on a raised bar (2 cm high) and the time taken to either
remove the forepaws from the bar or jump up on to the bar with
the hind paws was recorded, with a cut-off time of 120 seconds.
Spontaneous locomotor activity was tested using the dark open
field test. For this assay, mice were placed in a 25 cm 325 cm
open-topped Perspex box and their activity was recorded for 2.5
minutes. A 4 34 square grid was super-imposed over the
recording and the number of times the hindquarters of the animal
crossed a line on the grid was recorded. All tests were performed
under low-level red light (,3 lux).
2.3. Experimental protocol
After initial habituation to their holding cages and equipment used
for testing, animals underwent baseline testing for the allodynia and
side effect measures with the exception of the open field test. They
then underwent CCI surgery 2 days after baseline testing. Drug
testing was conducted at 8 to 10 days after surgery. Each animal
underwent only 1 drug testing experiment, and was euthanised by
carbon dioxide asphyxiation after testing. The experimenter was
blinded to the drug or drug combination being tested.
For the time course experiments, only mechanical PWT and
rotarod latency were measured (because of short inter-test
intervals). Measurements were taken immediately before drug
administration and then at 0.5, 1, 2, 4, and 6 hours after drug
administration. For the dose response and antagonist experi-
ments, all allodynia and side effect assays (with the exception of
the open field) were tested immediately before drug injection and
at 1 and 2 hours after injection. These postinjection time points
were selected to coincide with the time of peak drug effect as
determined by the time course study (see Results, Fig. 1). To
maintain novelty, the open field test was performed only once at
1.5 hours after injection. Acetone, rotarod, and bar tests were
performed twice at each specified time point. Von Frey and open
field tests were performed only once at each specified time point.
2.4. Drugs and administration
The phytocannabinoids THC and CBD were obtained from
THCPharm (Frankfurt, Germany). The CB1 receptor antagonist
AM281 and the CB2 receptor antagonist AM630 were
obtained from Cayman Chemicals (Ann Arbor, MI). Stock
solutions of all drugs used were prepared in dimethyl sulfoxide.
These were administered as a subcutaneous injection (volume
of 0.01-0.015 mL·g
21
in a vehicle consisting of 15% dimethyl
sulfoxide, 5% Tween80, and saline) in the loose skin between
the shoulder blades. Drug injection solutions were made up
immediately before administration.
2.5. Analysis and statistics
For the time course experiments, raw data were compared using
2-way repeated analysis of variance, with time and drug
treatment as within- and between-subjects factors, respectively
(Prism; Graphpad Software, La Jolla, CA). When time-drug
treatment interactions were significant, within-treatment group
post hoc comparisons were performed using Dunnett correction
for multiple comparisons. For the dose response and antagonist
experiments, all data (with the exception of open field) were
normalised as a percentage of the maximum possible effect. For
mechanical PWT, this was calculated as (post-drug 2pre-drug)/
(cut-off 2pre-drug), where the cut-off was 6.84 g. For acetone
and rotarod, this was calculated as (pre-drug 2post-drug)/
(drug), and for the bar test as (post-drug 2pre-drug)/(cut-off)
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where the cut-off was 120 seconds. Raw data were used for the
open field as predrug values were not obtained. For the
cannabinoid receptor antagonist experiments, data were com-
pared using 1-way analysis of variance, with the Tukey correction
for post hoc comparisons. All data are presented as mean 6
SEM. Data were considered significantly different when P,0.05.
There were 6 animals per treatment group for all experiments, as
in our previous synergy study in a mouse neuropathic pain
model.
17
Monophasic dose response curves were constructed by fitting
sigmoidal curves with variable slope, of the form
Effect ¼EMax
Dosep
Dosep1EDp
50(1)
where each drug had a maximal effect of E
Max
, a half maximally
effective dose (ED
50
), and a Hill slope of p. Biphasic dose
response curves were constructed by fitting the sum of 2
independent sigmoidal curves as in Equation 1. Comparisons of
these parameters between dose–response fits were performed
using an extra sum-of-square F-test (Prism). The TW of each drug
treatment group was calculated as the average of the ratio of
ED
50
s for each side effect measure divided by that for mechanical
and cold anti-allodynia.
Isobolographic analysis was used to determine whether an
interaction between THC and CBD was present when they were
administered in combination, as follows.
29
In the present study,
the THC:CBD combination was tested at a 1:1 ratio of their
ED
50
s. The predicted additive effect of THC and CBD was
calculated from their individual dose response curve parameters,
using an approach which takes into account different maximal
effects and Hill slopes for the 2 drugs. In this approach, the
predicted additive effect of the 2 drugs A and B is:
Eða;bÞ¼
EBb1CB
k
1
=
p
p
b1CB
k
1
=
p
p
1Cp
B(2)
k¼EB
EA11Cq
A
aq21 (2)
at doses a and b for drugs A and B, respectively, with maximal
effects of E
A
and E
B
(where E
B
.E
A
), ED
50
sofC
A
and C
B
, and Hill
slopes of q and p. The experimental combination dose response
curve was then compared with the theoretical predicted additive
dose response curve for THC plus CBD obtained from Equation 2
using a modified ttest to compare data at individual doses.
29
The
nonlinear isobole which describes the relationship between the
drugs at doses a and b, for a specified effect level B
i
(eg, the 50%
effect level), was calculated using the equation given below:
b¼Bi2CB
hEB
EA11Cq
A
aq21i
1
=
p
(3)
3. Results
3.1. Time course of action of THC and cannabidiol
We first examined the time course of action of near maximal doses
of THC and CBD (30 mg·kg
21
) to establish their time of peak effect.
At 8 to 10 days after CCI surgery, the effect of acute administration
of THC, CBD and matched vehicle on both PWT and rotarod
latency differed over time (Figs. 1A and B, F(12,90) 538, 12.9, P,
0.0001, 0.001). THC produced a significant increase in mechanical
PWTat0.5to4hoursafterinjection(Fig. 1A). In these animals, THC
also produced a significant decrease in rotarod latency at 0.5 to 6
hours after injection (Fig. 1B). Cannabidiol produced a significant
increase in mechanical PWT at 2 hours after injection (Fig. 1A).
Cannabidiol did not have a significant effect on rotarod latency (Fig.
1B). The vehicle for THC and CBD did not have a significant effect
on mechanical PWT or rotarod latency (Figs. 1A and B). These data
indicate that THC offers good analgesia but with significant side
effects at this dose, although CBD is a less efficacious analgesic at
the same dose but produces no cannabinoid side effects. The time
of peak effect when averaged over THC and CBD for these assays
was 1 to 2 hours after injection. We therefore used this time point in
all subsequent experiments.
3.2. Dose–response profiles of D9-tetrahydrocannabinol
and cannabidiol
We next examined the individual dose–response profiles of THC
and CBD (0.01-56 mg·kg
21
) for a wider range of anti-allodynia
and side effect assays. THC produced a dose-dependent
reversal of the CCI-induced decrease in mechanical PWT and
the CCI-induced increase in acetone responses, with similar
ED
50
s and maximal effects (Figs. 2A and B,Table 1,P.0.05).
THC also produced a dose-dependent decrease in rotarod
latency and open field crossings, and a dose-dependent increase
in bar latency (Figs. 2C–E,Table 1). The ED
50
s of THC for rotarod
and bar latency were greater than that for both mechanical PWT
Figure 1. Time course of action of THC and CBD. Time plots of the effects of
THC (30 mg·kg
21
), CBD (30 mg·kg
21
), and matched vehicle on (A) mechanical
PWT and (B) rotarod latency (n 56 per treatment group). Animals received
a single subcutaneous injection at time 0 hours, 8 to 10 days after CCI surgery
(post-CCI); pre-CCI data are also displayed. *, **, ***, and **** denote P,0.05,
0.01, 0.001, and 0.0001, respectively, compared with 0 hours post-CCI for
each treatment group. CBD, cannabidiol; CCI, chronic constriction injury ; paw
withdrawal threshold; THC, D9-tetrahydrocannabinol.
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and acetone responses (Table 1,P,0.01-0.0001). The ED
50
of
THC for open field crossings was not significantly different from
that for both mechanical PWT and acetone responses (Table 1,
P.0.05). The TW of THC, measured as the ratio of the ED
50
of
side effects to anti-allodynia, was 5.0 when averaged across all
measures of anti-allodynia and side effects (Table 1).
Cannabidiol produced a dose-dependent reduction in the CCI-
induced decrease in mechanical PWT and the CCI-induced
increase in acetone responses, with similar ED
50
s and maximal
effects (Figs. 2A and B,Table 1,P.0.05). Although their ED
50
s
were not significantly different (P.0.05), CBD had a lesser max-
imal effect than THC on mechanical PWT and acetone-induced
Figure 2. Dose–response curves for THC and CBD. Dose–response curves for the effect of THC and CBD on (A) mechanical (PWT, (B) acetone-induced
responses, (C) rotarod latency, (D) bar latency, and (E) open field crossings. Where appropriate, the sigmoidal parametric fit is shown (parameter estimates in
Table 1). CBD, cannabidiol; PWT, paw withdrawal threshold; THC, D9-tetrahydrocannabinol.
Table 1
Dose–response characteristics and TW of WIN55212, morphine, and their combination.
Anti-allodynia Side effects TW
Mechanical, PWT Acetone, responses Rotarod latency Bar latency Open field crossings
THC
ED
50
4.3 (0.6) 2.7 (0.5) 15 (2) 29 (0.2) 6.0 (1) 5.0 (1.4-10.7)
E
Max
94 (0.1) 74 (0.1) 75 (17) 100 (2)
Hill slope 1.7 (0.1) 1.4 (0.4) 1.6 (0.3) 5.4 (0.4) 21.1 (0.2)
CBD
ED
50
3.9 (0.1) 3.6 (0.4) .56.2 .56.2 .56.2 .15.0
E
Max
65 (5) 49 (16) ND ND
Hill slope 1.9 (0.7) 0.9 (0.7) ND ND ND
Combination THC:CBD—predicted additive
ED
50
4.8 3.2 ND ND ND ND
Combination THC:CBD—high potency
ED
50
0.021 (0.007) 0.016 (0.005) 1360 (762-2000)
E
Max
57 (2) 35 (4)
Hill slope 1.6 (0.4) 2.1 (1.0)
Combination THC:CBD—low potency
ED
50,
THC comp. 21 (2) 20 (2) 26 (8), 12 (3) 32 (1), 15 (0.1) 16 (3), 7.7 (1.6) 1.2 (0.8-1.6)
E
Max
39 (5) 60 (12) 76 (57) 71 (1)
Hill slope 10 (4) 1.6 (0.6) 1.4 (1.3) 3.5 (0.1) 22.0 (0.7)
Sigmoidal curve fit parameters for THC and CBD administered individually, the predicted additive effect of combination THC:CBD, the high potency combination THC:CBD actions, and the low potency combination THC:CBD
actions. For the low potency THC:CBD actions, the ED
50
s for the THC dose component are also shown (THC comp.). The parameters include the ED
50
(mg·kg
21
), E
max
(%MPE), Hill slope, and the TW. The values of these
parameters are shown as mean (SEM), mean (range), or ND (not determined).
CBD, cannabidiol; ED
50
, median effective dose; PWT, paw withdrawal threshold; THC, D9-tetrahydrocannabinol; TW, therapeutic window.
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responses (Figs. 2A and B,Table 1,P,0.05). Cannabidiol did
not have an effect on rotarod latency, bar latency, or the number
of open field crossings over the range of doses tested
(Figs. 2C–E). Given that CBD failed to produce any side effect,
its TW was estimated to be greater than 15 (Table 1). These
observations indicate that THC has a greater anti-allodynic
efficacy, but lesser TW compared with CBD.
3.3. Effect of a fixed-ratio combination of
D9-tetrahydrocannabinol and cannabidiol on allodynia
We next examined the effect of combined treatment with THC
and CBD on allodynia, using an isobolographic approach which
accounts for their differing maximal effects and Hill slopes.
17,29
THC and CBD were administered in a fixed 1:1 ratio based on
their individual ED
50
s. When averaged across both anti-allodynia
assays, the ED
50
ratio for THC:CBD was 1:1.13 by weight, which
is similar to their relative content in nabiximols. The predicted
additive effect of combined THC and CBD treatment on
mechanical PWT and acetone responses had monophasic dose
dependence, with ED
50
and maximum values similar to that for
THC alone (Figs. 3A and B,Table 1).
Co-administration of THC and CBD produced a dose-
dependent increase in mechanical PWT and decrease in acetone
responses (Figs. 3A and B). Unlike the predicted additive dose
response curves, the experimentally obtained THC:CBD combi-
nation dose response curves were biphasic, displaying high- and
low-potency anti-allodynic actions (Figs. 3A and B). Given this
biphasic profile, we assessed the level of synergy for both the
high- and low-potency sites of action.
The high potency THC:CBD induced reduction in mechanical
and cold allodynia had an ED
50
which was 200 to 230 times less
than that predicted for an additive interaction and accounted for
57% and 35% of the maximum effect of THC:CBD (Figs. 3A and
B,Table 1). Consequently, the effect of THC:CBD on mechanical
PWT and acetone responses was greater than its predicted
additive effect at doses below 1.3 to 4.0 mg·kg
21
(Figs. 3A and
B). For this high potency site, the combination THC:CBD dose
which reduced mechanical and cold allodynia by 50% (ED
50
) was
similar to the isobole predicted to produce a 1% reduction if the
interaction was additive (Figs. 4A and B).
The low potency THC:CBD induced reduction in mechanical
and cold allodynia had an ED
50
which was 4.4 to 6.3 times greater
than that predicted for an additive interaction, and accounted for
39% and 60% of the maximum effect of THC:CBD (Figs. 3A and
B,Table 1). The effect of THC:CBD on mechanical PWT and
acetone responses was greater than its predicted additive effect
at the highest doses tested (13-40 mg·kg
21
), but not at
intermediate doses (Figs. 3A and B). For this low potency site,
the combination THC:CBD dose which reduced mechanical and
cold allodynia by 50% (ED
50
) was similar to the isobole predicted
to produce a 55% to 65% reduction if the interaction was additive
(Figs. 4A and B). Together, these observations indicate that
combination THC:CBD treatment has 2 modes of action,
including a low dose synergistic reduction in allodynia and a high
dose sub-additive reduction in allodynia.
3.4. Side effects of the fixed-ratio combination of
D9-tetrahydrocannabinol and cannabidiol
A synergistic anti-allodynic interaction will only lead to an
increase in the TW of a drug combination if its side effects do not
display an equivalent level of synergy. We therefore examined
thesideeffectdoseresponseprofileoftheTHC:CBD
combination. THC:CBD produced a dose-dependent increase
in rotarod and bar latency, and a decrease in open field
crossings (Figs. 5A–C). In contrast to its anti-allodynic actions,
the dose–response profiles of the THC:CBD for the rotarod, bar,
and open field tests were monophasic, and had similar ED
50
s
(Figs. 5A–C,Table 1,P.0.05). The TW of THC:CBD was 1360
when side effects were compared with the high potency anti-
allodynic action, but was only 1.2 when compared with the low
potency anti-allodynic action (Table 1).
The side effect assays could not be analysed using the
isobolographic approach due to the lack effect of CBD alone. To
determine whether there was a THC:CBD interaction for the side
effects, we compared the dose response profiles of the THC
component of THC:CBD with that obtained for THC when
administered alone. For the rotarod and open field latencies,
there was no difference between the ED
50
of the THC component
of THC:CBD compared with that of THC when administered
alone (Figs. 5A and C,Table 1,P.0.05). In contrast, the ED
50
of
the THC component of THC:CBD for bar latency was less than
that of THC when administered alone (Fig. 5B,Table 1,P,
0.0001). It might be noted, however, that the maximal effect of the
Figure 3. Dose–response curves for the effect of combined THC and CBD
treatment on allodynia. Dose–response curves showing the effect of
administration of THC and CBD in a fixed ratio (THC:CBD at 1:1.13 fixed
weight ratio) on (A) mechanical PWT and (B) acetone-induced responses. The
biphasic sigmoidal fit for the combination THC:CBD data is shown (ED
50
and
E
Max
estimates shown in Table 1). Also shown are the predicted additive
dose response curves for combination THC:CBD, and the sigmoidal fits for
THC and CBD alone (reproduced from Fig. 2). **, ***, and **** denote P,0.01,
0.001, and 0.0001, respectively, for THC:CBD experimental combination
vs predicted additive at equivalent doses. CBD, cannabidiol; ED
50
,
median effective dose; PWT, paw withdrawal threshold; THC, D9-
tetrahydrocannabinol.
2456 S.L. Casey et al.·158 (2017) 2452–2460 PAIN
®
Copyright Ó2017 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
THC component of THC:CBD on bar latency was less than that of
THC when administered alone (Fig. 5B,Table 1,P,0.0001).
Together, these observations indicate that the side effects of
THC:CBD are largely produced by THC, and these are not
substantially affected by CBD. Thus, the differences in the TW of
combination THC:CBD at the low and high potency sites are
largely due to altered anti-allodynic potency.
3.5. Role of cannabinoid receptors
We finally examined the role of cannabinoid receptors in the
actions of THC and CBD by co-administering them with the
selective CB1 and CB2 antagonists, AM281 and AM630
(3 mg·kg
21
). AM281 and AM630 alone had no effect on any of
the pain, or behavioural scores (Figs. 6A–E). AM281 abolished
the effect of a maximal dose of THC (30 mg·kg
21
) on mechanical
PWT, but only partially reduced its effect on acetone responses
(Figs. 6A and B). AM281 abolished the effect of THC on rotarod
latency, bar latency and open field crossing (Figs. 6C–E). AM630
did not alter the effect of THC on mechanical PWT or acetone
responses (Figs. 6A and B). Although AM630 did not alter the
effect of THC on rotarod latency and open field crossing, it
partially reduced its effect on bar latency (Figs. 6C–E).
AM281 did not alter the effect of a maximal dose of CBD
(30 mg·kg
21
) on mechanical PWT, but partially reduced its effect
on acetone responses (Fig. 6B). Similarly, AM630 did not alter the
effect of CBD on mechanical PWT, but partially reduced its effect
on acetone responses (Figs. 6A and B). Cannabidiol did not have
an effect on rotarod latency, bar latency, and open field crossing,
and this was not altered by AM281, or AM630 (Figs. 6C–E).
We also examined the effect of these antagonists on
combination THC:CBD at a low dose (0.4 mg·kg
21
) which was
maximal for the high potency anti-allodynia, and at a high dose
(40 mg·kg
21
) which was maximal for the low potency anti-
allodynia. AM281 and AM630 did not alter the effect of low dose
THC:CBD on mechanical PWT, or acetone responses (Figs. 6A,
B). Low dose THC:CBD did not have an effect on rotarod latency,
bar latency and open field crossing, and this was not altered by
AM281, or AM630 (Figs. 6C–E).
AM281 abolished effect of high dose THC:CBD on mechanical
PWT, and partially reduced its effect on acetone responses (Figs.
6A and B). In addition, AM281 abolished the effect of high dose
THC:CBD on rotarod latency, bar latency and open field
crossings (Figs. 6C–E). AM630 did not alter the effect of high
dose THC:CBD on mechanical PWT, or acetone responses
(Figs. 6A and B). AM630 did not alter the effect of high dose THC:
Figure 4. Isoboles for combination THC and CBD treatment at a range of effect levels. Isoboles for THC:CBD co-administration on (A) mechanical PWT and (B)
acetone-induced responses. The experimental data for the combination, at its ED
50
(50% effect level, back symbol), are shown as part of the continuum of fixed-
ratio effects (black dotted lines). Theoretical isoboles of additivity for effect levels of 5, 10, 25, and 50% are shown for comparison (Equation 3). CBD, cannabidiol;
ED
50
, median effective dose; PWT, paw withdrawal threshold; THC, D9-tetrahydrocannabinol.
Figure 5. Dose–response curves for the side effects of combination THC and CBD treatment. Dose–response curves showing the effects of a THC and CBD
combination treatment on (A) rotarod latency, (B) bar latency, and (C) open field crossings. The x-axis represents the total dose of THC and CBD when given in
combination (THC:CBD), the dose of THC when given in combination with CBD (THC in THC:CBD), and the dose of THC when given alone (THC). With the
exception of open field (for which raw data is shown), all data are displayed as %MPE. CBD, cannabidiol; MPE, maximum possible effect; PWT, paw withdrawal
threshold; THC, D9-tetrahydrocannabinol.
December 2017·Volume 158 ·Number 12 www.painjournalonline.com 2457
Copyright Ó2017 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
CBD on rotarod latency, bar latency, and open field crossings
(Figs. 6C–E).
4. Discussion
This study has demonstrated that co-administration of the
phytocannabinoids THC and CBD synergistically attenuates
allodynia in a mouse model of neuropathic pain. Interestingly,
this nabiximol combination had 2 modes of action. At low doses,
the THC:CBD combination had partial anti-allodynic efficacy and
was devoid of THC-like side effects. At high doses, the THC:CBD
combination had maximal anti-allodynic efficacy, but a reduced
TW compared with THC alone. These findings have major
implications for the clinical use of the phytocannabinoids THC
and CBD, individually and in combination.
The psychoactive cannabis constituent THC produced dose-
dependent reductions in the mechanical and cold allodynia
induced by the CCI nerve-injury model of neuropathic pain. This
high efficacy anti-allodynia was consistent with that previously
reported for single-dose and full-dose–response studies.
7,8,13,37
In these animals, THC also produced impairment of motor
performance on the rotarod, catalepsy in the bar test, and
sedation in the dark open field test. These side effects were
consistent with those previously reported in na¨
ıve, unoperated
animals.
14,19,20,28,31,39
Interestingly, THC reduced allodynia with
a potency 5 times greater than that at which it produced side
effects. This is important because it demonstrates that THC has
a greater TW (ED
50
side effects/ED
50
anti-allodynia) than that of
synthetic high-efficacy cannabinoid receptor agonists which
have no window between their anti-allodynic actions and side
effects.
12,17,27
The difference between the TW of THC and
synthetic cannabinoid receptor agonists may be due to a number
of factors, such as the lower efficacy of THC compared with
WIN55212, or to differences in their mechanisms of action. These
observations indicate that THC is a more viable therapeutic option
for the treatment of neuropathic pain than the high-efficacy,
synthetic pan-cannabinoid receptor agonists which have been
the subject of most animal studies.
The cannabis constituent CBD also produced dose-
dependent reductions in nerve injury–induced mechanical and
cold allodynia. This was qualitatively similar to some, but not all,
previous studies using rodent models of neuropathic
pain.
6,13,30,34
Although the anti-allodynic potency of CBD was
similar to that of THC, it displayed lower efficacy. Unlike THC,
CBD did not produce catalepsy, impair motor performance, or
produce sedation over a wide range of doses, as observed
previously in unoperated animals.
14,20,28,31
This indicates that
CBD has potential in chronic pain states, although this may be
more as an adjuvant because of its moderate efficacy compared
with THC. It might be also noted that the anti-allodynic efficacy of
Figure 6. Role of cannabinoid receptors in the actions of THC and CBD. Bar charts depicting the effect of the cannabinoids THC (30 mg·kg
21
), CBD (30 mg·kg
21
),
and THC plus CBD (THC:CBD, 0.4 and 40 mg·kg
21
), or vehicle on (A) mechanical PWT (B) acetone responses, (C) rotarod latency, (D) bar latency, and (E) open
field crossings. Cannabinoids/vehicle were administered either alone (Control) or with the cannabinoid CB1 or CB2 receptor antagonists AM281 and AM630 (3
mg·kg
21
). With the exception of open field (for which raw data is shown), all data are displayed as %MPE. *, **, and **** denote P,0.05, 0.01, and 0.0001,
respectively, for each cannabinoid (THC, CBD, and THC:CBD) plus antagonist vs the cannabinoid without antagonist; #, ###, and #### denote P,0.05, 0.01,
0.0001 for each cannabinoid (alone or with antagonist) vs vehicle. CBD, cannabidiol; MPE, maximum possible effect; PWT, paw withdrawal threshold; THC, D9-
tetrahydrocannabinol.
2458 S.L. Casey et al.·158 (2017) 2452–2460 PAIN
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Copyright Ó2017 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
CBD in neuropathic pain models is improved with repeated
administration.
6
The major finding of this study was that combination THC:CBD
treatment, given in a dose ratio similar to that used in clinical
trials,
36
synergistically reduced mechanical and cold allodynia in
a nerve injury–induced neuropathic pain model. This is similar to
a recent chemotherapy-induced neuropathic pain model.
18
It
differs, however, from previous studies on acute nociceptive
pain,
4,10,26,31,35
suggesting that THC:CBD synergy may be
restricted to chronic pain states. Isobolographic analysis dem-
onstrated that this synergistic interaction had a complex, biphasic
nature. First, there was a high potency THC:CBD mode of action.
Thus, at low doses the THC:CBD combination displayed
a 200-fold increase in anti-allodynic potency compared with that
predicted for a simple additive interaction. This synergistic
increase in potency was far greater that that previously observed
between cannabinoid and opioid agonists.
17,37
Although the
reduction in allodynia at low doses of the THC:CBD combination
only displayed partial efficacy, it was not associated with
cannabinoid side effects. Second, there was a low potency
THC:CBD mode of action. Thus, at higher doses the THC:CBD
combination reduced allodynia with higher efficacy, but reduced
potency compared with that predicted for an additive interaction.
In addition, the high dose THC:CBD anti-allodynia was associ-
ated with motor disruption, catalepsy, and sedation, similar to
that observed for THC when administered alone, which differs
from the THC:CBD potentiation of hypo-locomotion observed in
a recent study.
4
These observations indicate that low and high
dose THC:CBD combination treatment have different benefits
and risks. At low doses, combination THC:CBD has a greatly
improved TW at the expense of low anti-allodynic efficacy,
whereas high doses have higher anti-allodynic efficacy at the
expense of a reduced TW when compared with THC alone. It
might be noted that cannabis produces a number of other
problematic side effects, such as dry mouth, nausea, euphoria,
and anxiety,
36
which would have to be examined in future studies.
The low and high dose modes of action of the THC:CBD
combination differed in the involvement of cannabinoid receptors.
The high dose THC:CBD induced anti-allodynia and side effects
displayed a cannabinoid receptor profile which was similar to that
of THC alone, with both being mediated by cannabinoid CB1, and
to a lesser extent CB2 receptors. Although the pharmacological
profile of THC has not been examined previously in a neuropathic
pain model, our observations are consistent with the profile of
THC in acute pain and side effect assays.
22
By contrast,
cannabinoid receptors made a lesser contribution to the
CBD-induced anti-allodynia, which is consistent with previous
neuropathic pain studies.
5,6,33
Importantly, cannabinoid recep-
tors also made little contribution to the low dose (high potency)
THC:CBD induced allodynia. Although the receptors mediating
the low dose THC:CBD induced anti-allodynia were not de-
termined, it could be due to an interaction at multiple targets and
sites given the substantial synergy between these phytocanna-
binoids. These targets could include transient receptor potential
and voltage-gated Ca
21
channels, plus glycine, peroxisome
proliferator–activated receptor, 5-hydroxytryptamine receptors,
and orphan receptors, all of which have a role in neuropathic pain
and are known cannabinoids targets.
5,6,15,23,25,32,33,38
In summary, this study has demonstrated that CBD synergis-
tically enhances the anti-allodynic actions of THC in a neuropathic
pain model. Combination THC:CBD treatment had a complex,
biphasic response mode of action which proffers 2 divergent
medication regimes. High dose THC:CBD combination treatment
offers marginally higher pain-relieving efficacy than THC alone,
but at the expense of a reduced TW. Alternatively, low dose THC:
CBD treatment had the advantage of a greatly improved TW at
the expense of lower pain-relieving efficacy when compared with
THC. Although low dose THC:CBD combination is unlikely to be
used as a stand-alone first-line mediation, these findings highlight
the potential of nabiximols in the treatment of neuropathic pain.
Conflict of interest statement
The authors have no conflicts of interest to declare.
Acknowledgments
Support provided by the Lambert Initiative (University of Sydney).
S. L. Casey was in receipt of an Australian Pain Society and
Australian Pain Relief Association Seqirus scholarship.
S. L. Casey and C. W. Vaughan conceived and designed the
study. S. L. Casey and N. Atwal conducted the study. S. L. Casey,
N. Atwal, and C. W. Vaughan analysed the data. S. L. Casey and
C. W. Vaughan wrote the manuscript. S. L. Casey, N. Atwal, and
C. W. Vaughan checked and approved the manuscript.
Article history:
Received 7 June 2017
Received in revised form 26 July 2017
Accepted 21 August 2017
Available online 1 September 2017
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... Cannabis is a versatile plant rather than a single drug and importantly, studies involving pure THC or CBD do not reflect the potential benefits of full-spectrum extracts (Maayah et al., 2020b). For example, THC and CBD were both effective in reducing neuropathic pain in various mice and rat models (Comelli et al., 2008;Casey et al., 2017;King et al., 2017;Belardo et al., 2019;Abraham et al., 2020). However, the pain-relieving effects were enhanced by their combination (Casey et al., 2017;King et al., 2017). ...
... For example, THC and CBD were both effective in reducing neuropathic pain in various mice and rat models (Comelli et al., 2008;Casey et al., 2017;King et al., 2017;Belardo et al., 2019;Abraham et al., 2020). However, the pain-relieving effects were enhanced by their combination (Casey et al., 2017;King et al., 2017). Moreover, a controlled high-CBD extract with additional secondary metabolites from the plant was more effective than purified CBD or THC at the same dose as in the extract (Comelli et al., 2008). ...
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Cannabinoids, including those found in cannabis, have shown promise as potential therapeutics for numerous health issues, including pathological pain and diseases that produce an impact on neurological processing and function. Thus, cannabis use for medicinal purposes has become accepted by a growing majority. However, clinical trials yielding satisfactory endpoints and unequivocal proof that medicinal cannabis should be considered a frontline therapeutic for most examined central nervous system indications remains largely elusive. Although cannabis contains over 100 + compounds, most preclinical and clinical research with well-controlled dosing and delivery methods utilize the various formulations of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), the two most abundant compounds in cannabis. These controlled dosing and delivery methods are in stark contrast to most clinical studies using whole plant cannabis products, as few clinical studies using whole plant cannabis profile the exact composition, including percentages of all compounds present within the studied product. This review will examine both preclinical and clinical evidence that supports or refutes the therapeutic utility of medicinal cannabis for the treatment of pathological pain, neurodegeneration, substance use disorders, as well as anxiety-related disorders. We will predominately focus on purified THC and CBD, as well as other compounds isolated from cannabis for the aforementioned reasons but will also include discussion over those studies where whole plant cannabis has been used. In this review we also consider the current challenges associated with the advancement of medicinal cannabis and its derived potential therapeutics into clinical applications.
... Activation of HCN1 current may contribute to the overall reduction of neuronal hyperactivity in epilepsy with CBD treatment (Iannotti et al., 2014;Maroon and Bost, 2018), similar to the reduced neuronal firing observed in CA1 pyramidal neurons resulting from lamotrigine stimulated I h (Peng et al., 2010). There is also growing evidence that CBD exerts promising analgesic effects in different models of inflammatory and chronic pain including nerve injury, chemotherapy-induced peripheral neuropathy, and diabetes (Costa et al., 2007;Xiong et al., 2012;Casey et al., 2017;Finn et al., 2021). THC also significantly attenuates pain-related behaviors in nerve injury models . ...
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Cannabinoids are a broad class of molecules that act primarily on neurons, affecting pain sensation, appetite, mood, learning, and memory. In addition to interacting with specific cannabinoid receptors (CBRs), cannabinoids can directly modulate the function of various ion channels. Here, we examine whether cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), the most prevalent phytocannabinoids in Cannabis sativa, can regulate the function of hyperpolarization-activated cyclic-nucleotide-gated (HCN1) channels independently of CBRs. HCN1 channels were expressed in Xenopus oocytes since they do not express CBRs, and the effects of cannabinoid treatment on HCN1 currents were examined by a two-electrode voltage clamp. We observe opposing effects of CBD and THC on HCN1 current, with CBD acting to stimulate HCN1 function, while THC inhibited current. These effects persist in HCN1 channels lacking the cyclic-nucleotide binding domain (HCN1ΔCNBD). However, changes to membrane fluidity, examined by treating cells with TX-100, inhibited HCN1 current had more pronounced effects on the voltage-dependence and kinetics of activation than THC, suggesting this is not the primary mechanism of HCN1 regulation by cannabinoids. Our findings may contribute to the overall understanding of how cannabinoids may act as promising therapeutic molecules for the treatment of several neurological disorders in which HCN function is disturbed.
... In pre-clinical work, Δ 9 -THC induces a strong antinociceptive effect across multiple behavioral tests (hot plate, tail flick test, formalin test) and models of chronic pain (inflammatory pain, neuropathic pain; Casey, Atwal, & Vaughan, 2017;Craft, Haas, Wiley, Yu, & Clowers, 2017;Finn et al., 2004;Wang et al., 2020). ...
Chapter
Cannabis legalization continues to progress in many US states and other countries. Δ⁹-tetrahydrocannabinol (Δ⁹-THC) is the major psychoactive constituent in cannabis underlying both its abuse potential and the majority of therapeutic applications. However, the neural mechanisms underlying cannabis action are not fully understood. In this chapter, we first review recent progress in cannabinoid receptor research, and then examine the acute CNS effects of Δ⁹-THC or other cannabinoids (WIN55212-2) with a focus on their receptor mechanisms. In experimental animals, Δ⁹-THC or WIN55212-2 produces classical pharmacological effects (analgesia, catalepsy, hypothermia, hypolocomotion), biphasic changes in affect (reward vs. aversion, anxiety vs. anxiety relief), and cognitive deficits (spatial learning and memory, short-term memory). Accumulating evidence indicates that activation of CB1Rs underlies the majority of Δ⁹-THC or WIN55121-2’s pharmacological and behavioral effects. Unexpectedly, glutamatergic CB1Rs preferentially underlie cannabis action relative to GABAergic CB1Rs. Functional roles for CB1Rs expressed on astrocytes and mitochondria have also been uncovered. In addition, Δ⁹-THC or WIN55212-2 is an agonist at CB2R, GPR55 and PPARγ receptors and recent studies implicate these receptors in a number of their CNS effects. Other receptors (such as serotonin, opioid, and adenosine receptors) also modulate Δ⁹-THC's actions and their contributions are detailed. This chapter describes the neural mechanisms underlying cannabis action, which may lead to new discoveries in cannabis-based medication development for the treatment of cannabis use disorder and other human diseases.
... THC, accounts for up to 40% of the plant's extract and has been shown to reduce nociception in animal models of neuropathic pain [33]. A combinatorial effect of CBD and THC injected at a low dose also showed potential for treatment of neuropathic pain where CBD can surpass the side effects of THC along with synergistic enhancement of pain-relieving actions of THC [34]. Moreover, a recent study highlights the potential activity of THC to suppress neuropathic nociception, through inhalation of vaporized THC-enriched cannabis, which also underlines the effectiveness of intrapulmonary exposure for pain suppression compared with other routes of administration [35]. ...
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... In this study, we investigated for the rst time, the neuroprotective effects of synthetic CBD, THCV, and their combination in PTX-induced neuropathic mice. Although there have been reports of CBD's impact on PTX-induced neuropathic pain, the molecular mechanism behind their neuroprotective bene ts still remains to be investigated Nonpsychoactive phytocannabinoids constitute a viable therapeutic for treatment of neuropathic pain [33]. CBD-containing gelatin given ad libitum orally for three weeks after surgery dramatically reduced (p<0.01) ...
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The purpose of this study was to investigate the neuroprotective effects of phytocannabinoids, synthetic cannabidiol (CBD) and tetrahydrocannabivarin (THCV) and their combination on taxol induced peripheral neuropathy (PIPN) in mice. Briefly, six groups of C57BL/6J mice (n = 6) were used. PTX (8 mg/kg/day, i.p.) was given to the mice on days 1, 3, 5, and 7 to induce neuropathy. Mice were evaluated for their behavioral parameters and also at the end of the study, DRG collected from the animals were subjected to RNA sequence and westernblot analysis. Further, immunocytochemistry and mitochondrial functional assays were performed on cultured DRGs derived from SD rats. The combination of CBD and THCV improved thermal and mechanical neurobehavioral symptoms in mice by two folds as compared to individual treatments. KEGG (RNA Sequencing) identified P38-MAPK, AMPK, and PI3K-AKT pathways as potential CBD and THCV therapeutic targets. In PTX-treated animals, the expression of p-AMPK, SIRT1, NRF2, HO1, SOD2, and catalase was significantly reduced (p<0.001), whereas the expression of PI3K, p-AKT, p-P38 MAP kinase, BAX, TGF-, NLRP3 inflammasome, and caspase 3 was significantly increased (p<0.001) when compared to control group. In reversing these protein expressions, combination therapy outperformed single therapies. CBD and THCV treatment increased AMPK, Catalase, and Complex I expression while decreasing mitochondrial superoxides in DRG primary cultures. In mice and DRG primary cultures, WAY100135 and rimonabant inhibited the effects of CBD and THCV by blocking 5 HT1A and CB1 receptors. In conclusion, entourage effect of CBD and THCV combination against PIPN appears to protect neurons in mice by modulating 5HT1A and CB1 receptors, respectively.
... This property was described in several studies that reported higher efficacy of Cannabis sp. extract compared to administration of isolated CBD and THC in decreasing allodynia and hyperalgesia in rats, evaluated by hot plate and von Frey tests (Comelli et al., 2008;Casey et al., 2017;Harris et al., 2019). Indeed, Comelli et al. (2008) described that the improvement in hyperalgesia through the use of Cannabis sp. ...
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Despite the importance of pain as a warning physiological system, chronic neuropathic pain is frequently caused by damage in the nervous system, followed by persistence over a long period, even in the absence of dangerous stimuli or after healing of injuries. Chronic neuropathic pain affects hundreds of millions of adults worldwide, creating a direct impact on quality of life. This pathology has been extensively characterized concerning its cellular and molecular mechanisms, and the endocannabinoid system (eCS) is widely recognized as pivotal in the development of chronic neuropathic pain. Scientific evidence has supported that phyto-, synthetic and endocannabinoids are efficient for pain management, while strong data arise from the therapeutic use of Cannabis-derived products. The use of medicinal Cannabis products is directed toward not only relieving symptoms of chronic pain, but also improving several aspects of patients’ welfare. Here, we review the involvement of eCS, along with other cellular and molecular elements, in chronic neuropathic pain pathology and how this system can be targeted for pain management.
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Hind paw-directed assays are commonly used to study the analgesic effects of opioids in mice. However, opioid-induced hyper-locomotion can obscure results of such assays. We aimed to overcome this potential confound by using gait analysis to observe hind paw usage during walking in mice. We measured changes in paw print area following induction of post-surgical pain (using the paw incision model) and treatment with oxycodone. Paw incision surgery reduced the paw print area of the injured hind paw as the mice avoided placing the incised section of the paw on the floor. Surprisingly, oxycodone caused a tiptoe-like gait in mice, resulting in a reduced paw print area in both hind paws. Further investigation of this opioid-induced phenotype revealed that analgesic doses of oxycodone or morphine dose-dependently reduced hind paw print area in uninjured mice. The gait changes were not dependent on opioid-induced increases in locomotor activity; speed and paw print area had no correlation in opioid-treated mice, and other analgesic compounds that alter locomotor activity did not affect paw print area. Unfortunately, the opioid-induced 'tiptoe' gait phenotype prevented gait analysis from being a viable metric for demonstrating opioid analgesia in injured mice. However, this work reveals an important, previously uncharacterized effect of treatment with analgesic doses of opioids on paw placement. Our characterization of how opioids affect gait has important implications for the use of mice to study opioid pharmacology and suggests that scientists should use caution when using hind paw-directed nociceptive assays to test opioid analgesia in mice.
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Background and purpose: Clinical studies have reported that pan-cannabinoid receptor agonists may have efficacy in neuropathic pain states and that this might be enhanced by co-administration with opioids. While cannabinoid-opioid analgesic synergy has been demonstrated in animal models of acute pain, it has not been examined in neuropathic pain models. We examined the effect of combination treatment with cannabinoid and opioid receptor agonists on allodynia and side-effects in a nerve injury induced neuropathic pain model. Experimental approach: C57BL/6 mice were subjected to chronic constriction injury (CCI) of the sciatic nerve. The effect of systemic administration of morphine and the pan-cannabinoid receptor agonist WIN55212 and allodynia and side effects was examined at 7 - 10 days post-CCI surgery. Isobolographic analysis was used to determine whether combination effects were synergistic. Key results: The opioid agonist morphine reduced CCI-induced mechanical and cold allodynia, and produced motor incoordination, in a dose dependent manner. WIN55212 reduced CCI-induced allodynia, and produced motor incoordination, catalepsy and sedation, in a dose dependent manner, as we have observed previously. When administered together, WIN55212 and morphine reduced allodynia in a synergistic manner, but had only an additive effect on motor incoordination. Conclusions and implications: These findings indicate that combination administration of non-selective opioid and cannabinoid receptor agonists synergistically reduces nerve injury induced allodynia, while producing side-effects in an additive manner. This suggests that combination treatment has an improved anti-allodynic potency and therapeutic index in a neuropathic pain model.
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Cannabis and cannabinoid drugs are widely used to treat disease or alleviate symptoms, but their efficacy for specific indications is not clear. To conduct a systematic review of the benefits and adverse events (AEs) of cannabinoids. Twenty-eight databases from inception to April 2015. Randomized clinical trials of cannabinoids for the following indications: nausea and vomiting due to chemotherapy, appetite stimulation in HIV/AIDS, chronic pain, spasticity due to multiple sclerosis or paraplegia, depression, anxiety disorder, sleep disorder, psychosis, glaucoma, or Tourette syndrome. Study quality was assessed using the Cochrane risk of bias tool. All review stages were conducted independently by 2 reviewers. Where possible, data were pooled using random-effects meta-analysis. Patient-relevant/disease-specific outcomes, activities of daily living, quality of life, global impression of change, and AEs. A total of 79 trials (6462 participants) were included; 4 were judged at low risk of bias. Most trials showed improvement in symptoms associated with cannabinoids but these associations did not reach statistical significance in all trials. Compared with placebo, cannabinoids were associated with a greater average number of patients showing a complete nausea and vomiting response (47% vs 20%; odds ratio [OR], 3.82 [95% CI, 1.55-9.42]; 3 trials), reduction in pain (37% vs 31%; OR, 1.41 [95% CI, 0.99-2.00]; 8 trials), a greater average reduction in numerical rating scale pain assessment (on a 0-10-point scale; weighted mean difference [WMD], -0.46 [95% CI, -0.80 to -0.11]; 6 trials), and average reduction in the Ashworth spasticity scale (WMD, -0.36 [95% CI, -0.69 to -0.05]; 7 trials). There was an increased risk of short-term AEs with cannabinoids, including serious AEs. Common AEs included dizziness, dry mouth, nausea, fatigue, somnolence, euphoria, vomiting, disorientation, drowsiness, confusion, loss of balance, and hallucination. There was moderate-quality evidence to support the use of cannabinoids for the treatment of chronic pain and spasticity. There was low-quality evidence suggesting that cannabinoids were associated with improvements in nausea and vomiting due to chemotherapy, weight gain in HIV infection, sleep disorders, and Tourette syndrome. Cannabinoids were associated with an increased risk of short-term AEs.
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Isolation and structure elucidation of most of the major cannabinoid constituents - including Δ(9)-tetrahydrocannabinol (Δ(9)-THC), which is the principal psychoactive molecule in Cannabis sativa - was achieved in the 1960s and 1970s. It was followed by the identification of two cannabinoid receptors in the 1980s and the early 1990s and by the identification of the endocannabinoids shortly thereafter. There have since been considerable advances in our understanding of the endocannabinoid system and its function in the brain, which reveal potential therapeutic targets for a wide range of brain disorders.
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Background and purpose: It has been suggested that the non-psychoactive phytocannabinoid cannabidiol (CBD) can impact the pharmacological effects of delta-9-tetrahydrocannabinol (THC). We tested the hypothesis that CBD and THC would significantly mitigate mechanical sensitivity in a mouse model of paclitaxel-induced neuropathic pain, and that CBD+THC combinations would produce synergistic effects. We also tested the hypothesis that CBD would attenuate oxaliplatin- and vincristine- induced mechanical sensitivity. Experimental approach: Paclitaxel-treated mice (8.0 mg/kg IP, days 1, 3, 5 and 7) were pretreated with CBD (0.625 - 20.0 mg/kg IP), THC (0.625 - 20.0 mg/kg IP) or CBD+THC (0.04+0.04 - 20.0+20.0 mg/kg IP) and mechanical sensitivity was assessed on days 9, 14, and 21. Oxaliplatin-treated (6.0 mg/kg IP, day 1) or vincristine-treated mice (0.1 mg/kg IP days 1-7) were pretreated with CBD (1.25 - 10.0 mg/kg IP), THC (10.0 mg/kg IP), or THC+CBD (0.16 mg/kg THC + 0.16 mg/kg CBD IP). Key results: Both CBD and THC alone attenuated mechanical allodynia in mice treated with paclitaxel. Very low ineffective doses of CBD and THC were synergistic when given in combination. CBD also attenuated oxaliplatin- but not vincristine-induced mechanical sensitivity, while THC significantly attenuated vincristine- but not oxaliplatin-induced mechanical sensitivity. The low dose combination significantly attenuated oxaliplatin- but not vincristine-induced mechanical sensitivity. Conclusions and implications: CBD may be potent and effective at preventing the development of CIPN, and its clinical utility may be enhanced by co-administration of low doses of THC. These treatment strategies would increase the therapeutic window of Cannabis-based pharmacotherapies.
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Background: Previous studies suggest that cannabidiol (CBD) may potentiate or antagonize Δ(9)-tetrahydrocannabinol's (THC) effects. The current study examined sex differences in CBD modulation of THC-induced antinociception, hypolocomotion, and metabolism. Methods: In Experiment 1, CBD (0, 10 or 30mg/kg) was administered 15min before THC (0, 1.8, 3.2, 5.6 or 10mg/kg), and rats were tested for antinociception and locomotion 15-360min post-THC injection. In Experiments 2 and 3, CBD (30mg/kg) was administered 13h or 15min before THC (1.8mg/kg); rats were tested for antinociception and locomotion 30-480min post-THC injection (Experiment 2), or serum samples were taken 30-360min post-THC injection to examine CBD modulation of THC metabolism (Experiment 3). Results: In Experiment 1, CBD alone produced no antinociceptive effects, while enhancing THC-induced paw pressure but not tail withdrawal antinociception 4-6h post-THC injection. CBD alone increased locomotor activity at 6h post-injection, but enhanced THC-induced hypolocomotion 4-6h post-THC injection, at lower THC doses. There were no sex differences in CBD-THC interactions. In Experiments 2 and 3, CBD did not significantly enhance THC's effects when CBD was administered 13h or 15min before THC; however, CBD inhibited THC metabolism, and this effect was greater in females than males. Conclusions: These results suggest that CBD may enhance THC's antinociceptive and hypolocomotive effects, primarily prolonging THC's duration of action; however, these effects were small and inconsistent across experiments. CBD inhibition of THC metabolism as well other mechanisms likely contribute to CBD-THC interactions on behavior.
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Sativex, a cannabinoid extract with a 1 : 1 ratio of tetrahydocannabinol and cannabidiol, has been shown to alleviate neuropathic pain associated with chemotherapy. This research examined whether tetrahydocannabinol or cannabidiol alone could attenuate or prevent cisplatin-induced tactile allodynia. In experiment 1, mice (C57BL/6) received eight administrations of 2.3 mg/kg cisplatin or saline solution IP every other day to induce tactile allodynia. Mice were then administered vehicle, 100 mg/kg gabapentin, 2 mg/kg tetrahydocannabinol, or 2 mg/kg cannabidiol IP and tested 60 min later on an electronic Von Frey. In experiment 2, prevention studies, cannabidiol (0.0, 0.5, 1.0, and 2.0 mg/kg) or tetrahydocannabinol (0.0, 0.5, 1.0, and 2.0 mg/kg) was given IP 30 min prior to cisplatin administration (2.3 or 1.0 mg/kg) utilizing a six-dose alternate day protocol. In both studies, tactile responses to the hind paws were quantified in g of force using an electronic Von Frey prior to and after the cisplatin administration protocol. Cisplatin produced a reduction in g of force indicative of neuropathy that was attenuated by gabapentin, tetrahydocannabinol, and cannabidiol but not prevented by either cannabinoid. These data demonstrate that each of the major constituents of Sativex alone can achieve analgesic effects against cisplatin neuropathy. Georg Thieme Verlag KG Stuttgart · New York.
Article
Background and purpose: While cannabinoids have been proposed as a potential treatment for neuropathic pain, they have limitations. Cannabinoid receptor agonists have good efficacy in animal models of neuropathic pain, they have a poor therapeutic window. Conversely, selective fatty acid amide hydrolase (FAAH) inhibitors which enhance the endocannabinoid system have a better therapeutic window, but lesser efficacy. We examined whether JZL195, a dual inhibitor of FAAH and monacylglycerol lipase (MAGL) could overcome these limitations. Experimental approach: C57BL/6 mice underwent the chronic constriction injury (CCI) model of neuropathic pain. Mechanical and cold allodynia, plus cannabinoid side-effects were assessed in response to systemic drug application. Key results: JZL195 and the cannabinoid receptor agonist WIN55212 produced dose dependent reductions in CCI-induced mechanical and cold allodynia, plus side-effects including motor incoordination, catalepsy and sedation. JZL195 reduced allodynia with an ED50 at least four times less than that at which it produced side-effects. By contrast, WIN55212 reduced allodynia and produce side-effects with similar ED50s. The maximal anti-allodynic effect of JZL195 was greater than that produced by selective FAAH, or MAGL inhibitors. The JZL195 induced anti-allodynia was maintained during repeated treatment. Conclusions and implications: These findings suggest that JZL195 has greater anti-allodynic efficacy than selective FAAH, or MAGL inhibitors, plus a greater therapeutic window than a cannabinoid receptor agonist. Thus, dual FAAH/MAGL inhibition may have greater potential in alleviating neuropathic pain, compared to selective FAAH and MAGL inhibitors, or cannabinoid receptor agonists. This article is protected by copyright. All rights reserved.
Article
New drug treatments, clinical trials, and standards of quality for assessment of evidence justify an update of evidence-based recommendations for the pharmacological treatment of neuropathic pain. Using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE), we revised the Special Interest Group on Neuropathic Pain (NeuPSIG) recommendations for the pharmacotherapy of neuropathic pain based on the results of a systematic review and meta-analysis. Between April, 2013, and January, 2014, NeuPSIG of the International Association for the Study of Pain did a systematic review and meta-analysis of randomised, double-blind studies of oral and topical pharmacotherapy for neuropathic pain, including studies published in peer-reviewed journals since January, 1966, and unpublished trials retrieved from ClinicalTrials.gov and websites of pharmaceutical companies. We used number needed to treat (NNT) for 50% pain relief as a primary measure and assessed publication bias; NNT was calculated with the fixed-effects Mantel-Haenszel method. 229 studies were included in the meta-analysis. Analysis of publication bias suggested a 10% overstatement of treatment effects. Studies published in peer-reviewed journals reported greater effects than did unpublished studies (r(2) 9·3%, p=0·009). Trial outcomes were generally modest: in particular, combined NNTs were 6·4 (95% CI 5·2-8·4) for serotonin-noradrenaline reuptake inhibitors, mainly including duloxetine (nine of 14 studies); 7·7 (6·5-9·4) for pregabalin; 7·2 (5·9-9·21) for gabapentin, including gabapentin extended release and enacarbil; and 10·6 (7·4-19·0) for capsaicin high-concentration patches. NNTs were lower for tricyclic antidepressants, strong opioids, tramadol, and botulinum toxin A, and undetermined for lidocaine patches. Based on GRADE, final quality of evidence was moderate or high for all treatments apart from lidocaine patches; tolerability and safety, and values and preferences were higher for topical drugs; and cost was lower for tricyclic antidepressants and tramadol. These findings permitted a strong recommendation for use and proposal as first-line treatment in neuropathic pain for tricyclic antidepressants, serotonin-noradrenaline reuptake inhibitors, pregabalin, and gabapentin; a weak recommendation for use and proposal as second line for lidocaine patches, capsaicin high-concentration patches, and tramadol; and a weak recommendation for use and proposal as third line for strong opioids and botulinum toxin A. Topical agents and botulinum toxin A are recommended for peripheral neuropathic pain only. Our results support a revision of the NeuPSIG recommendations for the pharmacotherapy of neuropathic pain. Inadequate response to drug treatments constitutes a substantial unmet need in patients with neuropathic pain. Modest efficacy, large placebo responses, heterogeneous diagnostic criteria, and poor phenotypic profiling probably account for moderate trial outcomes and should be taken into account in future studies. NeuPSIG of the International Association for the Study of Pain. Copyright © 2015 Elsevier Ltd. All rights reserved.
Article
Growing evidence shows cannabidiol (CBD) modulates some effects of Δ(9) -tetrahydrocannabinol (THC). CBD is a constituent of some strains of recreational cannabis but content is highly variable. High CBD strains may be less memory impairing than low-CBD strains and CBD can reverse behavioral effects of THC in monkeys. CBD/THC interactions in rodents are more complicated as it can attenuate or exacerbate the effects of THC. This study was undertaken to determine if CBD could reverse hypothermia or hypolocomotor effects caused by THC in rats. Male Sprague-Dawley rats were prepared with radiotelemetry devices and then challenged with doses of THC (10-30 mg·kg(-1) , i.p.) in the presence or absence of CBD. Experiments determined the effect of simultaneous or 30 min pre-treatment with CBD in a 1:1 ratio with THC, as well as the effect of CBD in a 3:1 ratio. Additional experiments determined the effect of pretreatment with CB1 receptor antagonist SR141716 (Rimonabant). CBD did not attentuate THC-induced hypothermia or hypolocomotion and instead an exaggeration was produced in some conditions. The antagonist SR141716 blocked hypolocomotor effects of THC for the first hour after injection and the hypothermia for six hours; thus the model was pharmacologically validated. There is no evidence from this study that elevated CBD content in cannabis provides protection from the physiological effects of THC. This article is protected by copyright. All rights reserved.
Article
Background: Mixed cannabinoid receptor 1 and 2 (CB1 and CB2) agonists such as Δ(9)-tetrahydrocannabinol (Δ(9)-THC) can produce tolerance, physical withdrawal, and unwanted CB1-mediated central nervous system side effects. Whether repeated systemic administration of a CB2-preferring agonist engages CB1 receptors or produces CB1-mediated side effects is unknown. Methods: We evaluated antiallodynic efficacy, possible tolerance, and cannabimimetic side effects of repeated dosing with a CB2-preferring agonist AM1710 in a model of chemotherapy-induced neuropathy produced by paclitaxel using CB1 knockout (CB1KO), CB2 knockout (CB2KO), and wild-type (WT) mice. Comparisons were made with the prototypic classic cannabinoid Δ(9)-THC. We also explored the site and possible mechanism of action of AM1710. Results: Paclitaxel-induced mechanical and cold allodynia developed to an equivalent degree in CB1KO, CB2KO, and WT mice. Both AM1710 and Δ(9)-THC suppressed established paclitaxel-induced allodynia in WT mice. In contrast to Δ(9)-THC, chronic administration of AM1710 did not engage CB1 activity or produce antinociceptive tolerance, CB1-mediated cannabinoid withdrawal, hypothermia, or motor dysfunction. Antiallodynic efficacy of systemic administration of AM1710 was absent in CB2KO mice and WT mice receiving the CB2 antagonist AM630, administered either systemically or intrathecally. Intrathecal administration of AM1710 also attenuated paclitaxel-induced allodynia in WT mice, but not CB2KO mice, implicating a possible role for spinal CB2 receptors in AM1710 antiallodynic efficacy. Finally, both acute and chronic administration of AM1710 decreased messenger RNA levels of tumor necrosis factor-α and monocyte chemoattractant protein 1 in lumbar spinal cord of paclitaxel-treated WT mice. Conclusions: Our results highlight the potential of prolonged use of CB2 agonists for managing chemotherapy-induced allodynia with a favorable therapeutic ratio marked by sustained efficacy and absence of tolerance, physical withdrawal, or CB1-mediated side effects.