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Overcoming the Bell-Shaped Dose-Response of Cannabidiol by Using Cannabis Extract Enriched in Cannabidiol

Authors:
  • School of Pharmacy, Ein Kerem Campus, Hebrew University of Jerusalem

Abstract and Figures

Cannabidiol (CBD), a major constituent of Cannabis, has been shown to be a powerful anti-inflammatory and anti-anxiety drug, without exerting a psychotropic effect. However, when given either intraperitoneally or orally as a purified product, a bell-shaped dose-response was observed, which limits its clinical use. In the present study, we have studied in mice the anti-inflammatory and anti-nociceptive activities of standardized plant extracts derived from the Cannabis sativa L., clone 202, which is highly enriched in CBD and hardly contains any psychoactive ingredients. In stark contrast to purified CBD, the clone 202 extract, when given either intraperitoneally or orally, provided a clear correlation between the anti-inflammatory and anti-nociceptive responses and the dose, with increasing responses upon increasing doses, which makes this plant medicine ideal for clinical uses. The clone 202 extract reduced zymosan-induced paw swelling and pain in mice, and prevented TNFα production in vivo. It is likely that other components in the extract synergize with CBD to achieve the desired anti-inflammatory action that may contribute to overcoming the bell-shaped dose-response of purified CBD. We therefore propose that Cannabis clone 202 (Avidekel) extract is superior over CBD for the treatment of inflammatory conditions.
Comparison of anti-inflammatory and anti-nociceptive effects of CBD and Cannabis clone 202 extract with the commercial drugs aspirin and tramadol. (a) Prevention of zymosan-induced swelling of hind paw. 1.5% zymosan in 40 μl was injected into the sub-planter surface of the right hind paw. Immediately thereafter, aspirin (50 mg/kg per os), tramadol (5 mg/kg i.p.), CBD (5 mg/kg i.p.) or Cannabis clone 202 extract (50 mg/kg i.p.) was given. The paw thickness indicative for paw swelling was measured 2, 6 and 24 hrs later. The paw thickness of untreated mice was 2.0-2.2 mm, which made the baseline of the graph. N = 5 for each time point of each treatment group. * p < 0.001 in comparison to control mice. p < 0.005 when comparing CBD and clone 202 extract with aspirin and tramadol; (b) Anti-pain effect of aspirin, tramadol, CBD and Cannabis clone 202 extract in mice treated as described in paragraph A. The hyperalgesia was measured by using the von Frey nociceptive filament assay. The higher the paw withdrawal threshold, the higher is the anti-nociceptive effect of the drug. N = 5 for each time point of each treatment group. The bars represent standard error. * p < 0.001 in comparison to control mice. p < 0.05 when comparing CBD and clone 202 extract with aspirin and tramadol; (c) The TNFα serum concentration at 24 hrs in mice that were treated as described in paragraph A. N = 5 for each treatment. The bars represent standard error. * p < 0.001 in comparison to control mice. p < 0.01 when comparing CBD and clone 202 extract with aspirin and tramadol.
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Pharmacology&Pharmacy,2015,6,7585
PublishedOnlineFebruary2015inSciRes.http://www.scirp.org/journal/pp
http://dx.doi.org/10.4236/pp.2015.62010
Howtocitethispaper:Gallily,R.,Yekhtin,Z.andHanuš,L.O.(2015)OvercomingtheBellShapedDoseResponseofCan
nabidiolbyUsingCannabisExtractEnrichedinCannabidiol.Pharmacology&Pharmacy,6,7585.
http://dx.doi.org/10.4236/pp.2015.62010
OvercomingtheBellShaped
DoseResponseofCannabidiol
byUsingCannabisExtractEnriched
inCannabidiol
RuthGallily1,ZhannahYekhtin1,LumírOndřejHanuš2
1TheLautenbergCenterforGeneralandTumorImmunology,TheHadassahMedicalSchool,TheHebrew
UniversityofJerusalem,Jerusalem,Israel
2DepartmentofMedicinalandNaturalProducts,InstituteforDrugResearch,TheHadassahMedicalSchool,
TheHebrewUniversityofJerusalem,Jerusalem,Israel
Email:ruthg@ekmd.huji.ac.il
Received12November2014;accepted7February2015;published10February2015
Copyright©2015byauthorsandScientificResearchPublishingInc.
ThisworkislicensedundertheCreativeCommonsAttributionInternationalLicense(CCBY).
http://creativecommons.org/licenses/by/4.0/
Abstract
Cannabidiol(CBD),amajorconstituentofCannabis,hasbeenshowntobeapowerfulantiin‐
flammatoryandantianxietydrug,withoutexertingapsychotropiceffect.However,whengiven
eitherintraperitoneallyororallyasapurifiedproduct,abellshapeddoseresponsewasobserved,
whichlimitsitsclinicaluse.Inthepresentstudy,wehavestudiedinmicetheantiinflammatory
andantinociceptiveactivitiesofstandardizedplantextractsderivedfromtheCannabissativaL.,
clone202,whichishighlyenrichedinCBDandhardlycontainsanypsychoactiveingredients.In
starkcontrasttopurifiedCBD,theclone202extract,whengiveneitherintraperitoneallyororally,
providedaclearcorrelationbetweentheantiinflammatoryandantinociceptiveresponsesand
thedose,withincreasingresponsesuponincreasingdoses,whichmakesthisplantmedicineideal
forclinicaluses.Theclone202extractreducedzymosaninducedpawswellingandpaininmice,
andpreventedTNFαproductioninvivo.Itislikelythatothercomponentsintheextractsynergize
withCBDtoachievethedesiredantiinflammatoryactionthatmaycontributetoovercomingthe
bellshapeddoseresponseofpurifiedCBD.WethereforeproposethatCannabisclone202(Avi
dekel)extractissuperioroverCBDforthetreatmentofinflammatoryconditions.
Keywords
CannabissativaL.Clone202,Cannabidiol,AntiInflammation,AntiNociceptive,TNFα
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1.Introduction
Inflammation and pain have accompanied human life for ages. Many anti-inflammation and anti-pain medica-
tions and various approaches have been employed through the centuries and in recent time. Many of used drugs,
however, impose severe side effects. Cannabis from various origins and species has been employed in various
forms as anti-pain agents for thousands of years [1]-[3]. One example is the legitimated drug Sativex® (Nabixi-
mols) that is used in the treatment of severe spasticity in patients with multiple sclerosis [4]. Two other drugs,
Marinol (Dronabinol) and Cesamet, have been approved for use in cancer-related anorexia-cachexia syndrome
as well as for nausea and vomiting [3]. But a major disadvantage of Cannabis phytomedicine is its psychoactive
effects due to the presence of 9-Tetrahydrocannabinol (THC).
Recently, a science-based approach is being conducted to specify the benefits of Cannabis and its many con-
stituents. A Cannabis plant contains hundreds of different chemicals with about 60 - 80 chemicals known as
cannabinoids [5]. The major Cannabis psychoactive molecule is the -tetrahydrocannabinol, known as THC,
which binds with high affinity (Ki = 3 - 5 nM) [6] to both the cannabinoid CB1 receptor expressed in the brain
and the CB2 receptor expressed on cells of the immune system [7]. Another major constituent is Cannabidiol
(CBD) which is devoid of psychotropic effects and binds only with very low affinity (Ki > 10 μM) [6] to the
CB1/CB2 receptors. The other cannabinoids are present in minute amounts. Stimulation of CB1 receptor is re-
sponsible for the Cannabis psychoactivity, while activation of the CB2 receptor leads to attenuated inflamma-
tion, decreased injury and accelerated regeneration in many disease states [7]. CBD has been shown to activate
central nervous system’s limbic and paralimbic regions, which can reduce autonomic arousal and feeling of an-
xiety [3]. This is in contrast to THC which can be anxiogenic [3]. CBD has also been shown to have anti-emetic,
anti-inflammatory and anti-psychotic effects [3]. Studies are looking for potential benefits of phytocannabinoids
in management of neuropathic pain, hypertension, post-stroke neuroprotection, multiple sclerosis, epilepsy and
cancer [3]. Doses up to 1500 mg per day as well as chronic use of CBD have been reported as being well toler-
ated by humans [3].
During the last 10 - 15 years, many studies have focused on the anti-inflammatory effects of purified CBD in
various animal models, including rheumatoid arthritis, diabetes type 1, inflammatory bowel disease and multiple
sclerosis [8]-[13]. These studies showed that purified CBD gives a bell-shaped dose-response curve. Healing
was only observed when CBD was given within a very limited dose range, whereas no beneficial effect was
achieved at either lower or higher doses. This trait of purified CBD imposes serious obstacles in planning human
and animal studies. The aim of the present study was to find a CBD source that could eliminate the bell-shaped
dose-response of purified CBD. We found that by using standardized plant extracts from the Cannabis clone 202
obtained from Tikun Olam, Israel, which is highly enriched in CBD and barely contains THC, a correlative anti-
inflammatory and anti-pain dose-response could be achieved when applied either intraperitoneally or orally in
an inflammatory mouse model.
2.MaterialandMethods
2.1.CBDandCannabisClone202(Avidekel)Extract
Purified CBD was purchased from THC Pharm. GmbH, Frankfurt, Germany. Cannabis sativa L. flowers from
the clone 202 (Avidekel) rich in CBD while low in any psychotropic constituents was supplied by Tikun Olam
Company (a government-approved farm growing medicinal Cannabis), Israel. CBD-enriched extract was pre-
pared from the flowers of Cannabis clone 202 grown under controlled temperature and light conditions. 100%
ethanol (20 ml) was added to the chopped Cannabis dry flowers (200 mg) for 24 - 48 hrs, with occasional shak-
ing at room temperature. Following filtration, samples were taken for analysis. Ethanol solutions of Cannabis
clone 202 extracts (10 mg/ml - 20 mg/ml) were kept at 20˚C in the dark. The extract was evaporated on Rota-
vapor (BÜCHI Labortechnik AG, Switzerland). For intraperitoneal injection, the dried Cannabis clone 202 ex-
tract was emulsified in a vehicle composed of ethanol:Cremophor:saline at a 1:1:18 ratio. Purified CBD was
emulsified in the same vehicle. For oral administration, the dried Cannabis clone 202 extract and the purified
CBD were dissolved in olive oil.
2.2.AnalysisoftheCannabisClone202ExtractbyThinLayerChromatography(TLC)
Cannabis clone 202 extract (1 μl) was separated on TLC Silica Gel 60 F254 aluminium sheets (Merck, Darm-
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77
stadt, Germany) using hexane:dioxane (4:1) as a solvent in a chamber of 13 × 9 × 12 cm. The separated com-
ponents were detected by spraying the plates with a freshly prepared solution of 0.5 g Fast Blue B (D9805,
Sigma) in acetone/water (9:1; v/v). Cannabinoids in the dried plant material predominately appeared as can-
nabinoid acids. The TLC analysis shows two major spots corresponding to the acid and neutral form of CBD,
respectively, with only a minor spot corresponding to the acid form of THC (Figure 1(a)).
2.3.AnalysisoftheCannabisClone202ExtractbyGasChromatographyandMass
Spectrophotometry(GC/MS)
For analysis of the composition of the ethanol extracts of medicinal Cannabis clone 202, the ethanol was eva-
porated and the resin dissolved in 20 ml of methanol and filtered through cotton in a capillary. The concentration
of the extract was adjusted to 1 mg/ml to which 50 μg internal standard (Tetracosane, Acros Organics, USA)
was added. One l of this sample was applied for the GC/MS analysis. The quantitative analysis of the samples
by GC/MS was performed in a Hewlett Packard G 1800B GCD system with a HP-5971 gas chromatograph with
electron ionization detector. The software used was GCD Plus ChemStation. The column used was SPB-5 (30 m
× 0.25 mm × 0.25 μm film thickness). Experimental conditions were: inlet, 250˚C; detector, 280˚C; splitless in-
jection/purge time, 1.0 min; initial temperature, 100˚C; initial time, 2.0 min; rate, 10˚C/min; final temperature,
280˚C. The helium flow rate was 1 ml/min. Calibration curve was made from 25.0 to 100 μg/ml Cannabidiol
(CBD), 9-Tetetrahydrocannabinol (THC) or Cannabinol (CBN) together with 50.0 μg/ml tetracosane as internal
standard. The cannabinoid composition of Cannabis clone 202 extract is presented in Figure 1(b), Figure 1(c)
and Table 1.
2.4.CommercialAntiNociceptiveandAntiInflammatoryDrugs
The non-steroid anti-inflammatory drug (NSAID) aspirin (acetylsalicylic acid) was purchased from Sigma and
dissolved in olive oil. Fifty mg of aspirin was given per os per kg in a volume of 40 μl. The opioid anti-noci-
ceptive Tramadol hydrochloride was obtained from Grunenthal and dissolved in saline. Five mg of Tramadol
was given per os per kg.
Figure 1. (a) TLC analysis of clone 202 extract. 1 l of the extract was run on TLC as de-
scribed in the Method section. CBD = Cannabidiol. CBDA = Cannabidiolic acid; (b) (c) GC/
MS chromatograms of an extract from Cannabis clone 202. (b) The full chromatogram. (c)
Magnification of weaker signals. Number keys: 1: Cannabidivarol (CBDV); 2: Cannabidiol
(CBD); 3: Cannabichromene (CBC); 4: 9-Tetrahydrocannabinol (9-THC); 5: Cannabigerol
(CBG); 6: Cannabinol (CBN); I.S.-Internal Standard (Tetracosane).
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Table 1. The percentage of main phytocannabinoids found in clone 202 extract
according to GC/MS analysis (see Figures 1(b)-(c)).
Phytocannabinoid Content
Cannabidiol (CBD) 17.9%
Δ9-Tetrahydrocannabinol (Δ9-THC) 1.1%
Cannabichromene (CBC) 1.1%
Cannabigerol (CBG) 0.2%
Cannabinol (CBN) Traces
Cannabidivarol (CBDV) Traces
As cannabinoid acids during injection to the GC/MS decarboxylate, the results are a total sum
of neutral cannabinoids and cannabinoid acids that have decarboxylated into neutral can-
nabinoids. The content is the mass fraction (% w/w) of the given constituent in the extract.
2.5.Animals
Six to eight week old female Sabra mice (Israel) were maintained in the SPF unit of the Hebrew University-
Hadassah Medical School, Jerusalem, Israel. The experimental protocols were approved by the Animal Care
Ethical Committee of the Hebrew University-Hadassah Medical School, Jerusalem, Israel. The animals were
maintained on standard pellet diet and water ad libitum. The animals were maintained at a constant temperature
(20˚C - 21˚C) and a 12 h light/dark cycle.
2.6.InductionofPawInflammationinMiceandTreatmentwithPurifiedCBDorClone
202Extract
To induce inflammation, 40 μl of 1.5% (w/v) zymosan A (Sigma) suspended in 0.9% saline was injected into the
sub-planter surface of the right hind paw of the mice. Immediately after zymosan injection, CBD or Cannabis
clone 202 extract was injected intraperitoneally (i.p.) or given orally. For intraperitoneal injection, these agents
were dissolved in 0.1 ml vehicle containing ethanol:Cremophore:saline at a ratio of 1:1:18. Control mice were
injected with the vehicle only. For per os administration, the agents were dissolved in olive oil, each mouse re-
ceiving 40 μl. Control mice got 40 μl olive oil. After 2, 6 and 24 hrs, paw swelling and pain perception were
measured. Serum TNFα titers were determined after 24 hrs. The effects of CBD and Cannabis clone 202 extract
were compared to those of aspirin (50 mg/kg per os) and tramadol (5 mg/kg, i.p.).
2.7.MeasurementofOedemaFormation
The paw swelling (thickness) was measured by calibrated calipers (0.01 mm), 2, 6 and 24 hrs following injec-
tions of zymosan alone or with CBD or Cannabis clone 202 extracts.
2.8.PainAssay
The hyperalgesia was evaluated by the paw withdrawal von Frey test at 2, 6, and 24 hrs following injections of
zymosan and/or the test compounds. In the von Frey nociceptive filament assay, von Frey calibrated monofila-
ment hairs of logarithmically incremental stiffness (0.008 - 300 g corresponding to 1.65 - 6.65 log of force). In
our study, only 1.4 - 60 g corresponding to 4.17 to 5.88 log of force was used, to test the mouse sensitivity to a
mechanical stimulus on the swollen paw. The measurements were performed in a quiet room. Before paw pain
measurements, the animals were held for 10 sec. The trained investigator applied the filament to the central area
of the hind paw with gradual increasing size. The test consisted of poking the middle of the hind paw to provoke
a flexion reflex followed by a clear flinch response after paw withdrawal. Each one of the von Frey filaments
was applied for approximately 3 - 4 s to induce the end-point reflex. The first testing was done by using the
force filament of 1.4 g. If there was no withdrawal response, the next higher stimulus was tried. The mechanical
threshold force (in grams (g)) was defined as the lowest force imposed by two von Frey monofilaments of vari-
ous sizes, required to produce a paw retraction. The untreated left hind paw served as a control.
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2.9.TumorNecrosisFactorα(TNFα)PlasmaLevels
Plasma levels of TNFα were measured using a mouse TNFα ELISA kit (R&D System), according to the manu-
facturer’s instructions.
2.10.StatisticalAnalysis
The results are presented as average ± standard error. Mice treated with CBD or Cannabis clone 202 extracts
were compared with control mice receiving the vehicle only. Statistical significance was calculated using the
ANOVA analysis of variance and Wilcoxon signed-rank test. Differences between the various doses of CBD
and clone 202 extracts were analyzed for significance using the repeated measures ANOVA procedure with
Post-Hoc test. All tests were 2-tailed and a p-value below 0.05 was considered statistically significant. A mini-
mum of three to four animals was used in each treatment group for each experiment unless otherwise stated.
Each experiment was performed at least three times. The graphs represent the average of all mice from the three
different experiments. Thus, each bar corresponds to the average of 10 - 12 mice for each treatment group, for
each time point, unless otherwise stated.
3.Results
3.1.EffectofCBDandCBDEnrichedClone202ExtractonInflammationandHyperalgesia
(PainSensation)
In this study we have used the well-accepted mouse model of zymosan-induced inflammation [14] to investigate
the anti-inflammatory and anti-nociceptive activities of Cannabis clone 202 extract versus purified CBD. The
extent of hind paw swelling was determined 2, 6 and 24 hrs following paw injection of 60 μg zymosan together
with either intraperitoneal injection or per os administration of various amounts of either purified CBD or Can-
nabis clone 202 extract, as indicated in the graphs (Figure 2, Figure 3). Following intraperitoneal injection of 1,
5, 25 and 50 mg/kg of purified CBD, a bell-shaped dose-response is observed (Figure 2(a)). The maximum in-
hibition of inflammation occurred after an injection of 5 mg/kg CBD with 50% and 57% inhibition after 6 and
24 hrs, respectively (p < 0.001), while a lower dose (1 mg/kg) being ineffective and higher doses (25 and 50
mg/kg) being less effective with 20% - 25% and 14% - 28% inhibition only, after 6 and 24 hrs, respectively
(Figure 2(a)). In accordance with these findings, the anti-nociceptive effect, as determined by the von Frey
monofilament assay, peaked at 5 mg/kg CBD (p < 0.001) (Figure 2(c)). The anti-nociceptive effect occurred
prior (2 hrs) to inhibition of swelling (6 hrs), and peaked at 6 hrs. Higher concentrations of CBD had less an-
ti-nociceptive effects (Figure 2(c)), again getting a bell-shaped dose-response. However, when clone 202 extract
was used, a correlative dose-response was observed with increased inhibition of inflammation upon increased
doses of the extract, reaching 43% and 64% inhibition at 25 mg and 50 mg, respectively, after 24 hrs (p < 0.001)
(Figure 2(b)). These two dosages of clone 202 extract also showed strong anti-nociceptive effects after 6 and 24
hrs (p < 0.001) (Figure 2(d)). Although the anti-inflammatory effect of clone 202 extract was higher at 50
mg/kg than at 25 mg/kg with a p = 0.001, the anti-nociceptive effect was only slightly higher (p = 0.01), sug-
gesting that a plateau has been reached. The clone 202 extract was more efficient for alleviating the pain than
CBD (p = 0.01) (Figure 2(d) versus Figure 2(c)).
When CBD or Cannabis clone 202 extract was given orally, a similar response was observed. Namely, CBD
gives a bell-shaped dose-response with an optimal inhibitory effect at 25 mg/kg (p < 0.001) (Figure 3(a) and
Figure 3(c)), whereas Cannabis clone 202 extract provides a correlative dose-response curve with a maximum
effect on swelling and pain relief at 50 and 150 mg/kg, respectively (p < 0.001) (Figure 3(b) and Figure 3(d)).
Significant pain relief was already obtained with an oral clone 202 extract dose of 50 mg/kg (Figure 3(d)) that
corresponds to about 10 mg/kg CBD (Table 1), while 25 mg/kg of purified CBD was needed to achieve the
same effect (Figure 3(c)). This suggests for a better usage of clone 202 extract.
It should be noted that agents taken per os need to go through the enterohepatic route prior to exerting their
effects, where the absorption rate and first-pass liver metabolism affect the blood drug level [15]. This may ex-
plain the higher doses required and the delayed response in comparison with the parenteral route, where the
agents are immediately available for the blood circulation. The anti-inflammatory and anti-nociceptive effects
peak at 6 hrs, which accords with the pharmacokinetics and pharmacodynamics of cannabinoids described by
Grotenhermen [15].
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Figure 2. Anti-inflammatory and anti-nociceptive effects of intraperitoneally injected CBD and CBD-
enriched clone 202 extract. (a) (b) Prevention of zymosan-induced swelling of hind paw. 1.5% zymo-
san in 40 μl was injected into the sub-planter surface of the right hind paw. Immediately thereafter,
CBD (a) or Cannabis clone 202 extract (b) was injected intraperitoneally. The paw thickness indica-
tive for paw swelling was measured 2, 6 and 24 hrs thereafter. The paw thickness of untreated mice
was 2.0 - 2.2 mm, which made the baseline of the graph. N = 12 for each time point. *p < 0.001 com-
pared to control mice. p < 0.001 for 50 mg/kg vs 25 mg/kg of clone 202 extract at 24 hrs; (c) (d) Anti-
pain effect of CBD (c) and Cannabis clone 202 extract (d). The hyperalgesia was measured by using
the von Frey nociceptive filament assay. The higher the paw withdrawal threshold, the higher is the
anti-nociceptive effect of the drug. The experiments were repeated three times, each experiment with 4
mice in each treatment group. The graphs presents the average of all mice in the three experiments,
meaning that the N = 12 for each time point. The bars represent standard error. *p < 0.001 compared to
control mice. p < 0.01 for 50 mg/kg vs 25 mg/kg of clone 202 extract at 24 hrs. p < 0.01 for clone 202
extract vs CBD.
3.2.SuppressionofTNFαProductionbyCBDandClone202Extract
TNFα is a well-known pro-inflammatory cytokine secreted by activated macrophages upon inflammation that
has been shown to be involved in initiation and amplification of inflammatory processes that ultimately leads to
oedema [16]. Therefore, it was important to analyze the effect of CBD and clone 202 extracts on TNFα produc-
tion. To this end, mice sera were analyzed for TNFα concentration by ELISA 24 hrs after treatment with zymo-
san in the absence or presence of CBD or clone 202 extract. When comparing the TNFα sera level in mice 24
hrs after injection of increasing doses of purified CBD, a bell-shaped dose-response curve of TNFα production
was observed, with a maximum inhibitory effect (43%) achieved at 5 mg/kg (p < 0.001), while no inhibition was
observed at either lower (1 mg/kg) or higher (25 and 50 mg/kg) doses (Figure 4(a)). In contrast, following in-
jection of CBD-enriched clone 202 extract to mice, a clear dose dependent response was apparent. Increased in-
hibition of TNFα production (39%; 46% and 57%, respectively) was observed following injections with increas-
ing amounts of extract (5 mg/kg, 25 mg/kg and 50 mg/ml, respectively) with a p value less than 0.001 (Figure
4(b)). Already at 5 mg/kg did clone 202 extract lead to a strong reduction in TNFα production (Figure 4(b)),
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Figure 3. Anti-inflammatory and anti-nociceptive effects of CBD and CBD-enriched clone 202 ex-
tract administrated per os. (a) (b) Prevention of zymosan-induced swelling of hind paw. 1.5% zymo-
san in 40 μl was injected into the sub-planter surface of the right hind paw. Immediately thereafter,
CBD (a) or Cannabis clone 202 extract (b) was given per os dissolved in olive oil (40 μl). The paw
thickness indicative for paw swelling was measured 2, 6 and 24 hrs thereafter. The paw thickness of
untreated mice was 2.0 - 2.2 mm, which made the baseline of the graph. N = 12 for each time point.
*p < 0.001 in comparison to control mice. The anti-inflammatory effects of 25, 50, 100 and 150
mg/kg of clone 202 extract were similar; (c) (d) Anti-pain effect of CBD (c) and Cannabis clone 202
extract (d) when given orally. The hyperalgesia was measured by using the von Frey nociceptive fi-
lament assay. The higher the paw withdrawal threshold, the higher is the anti-nociceptive effect of the
drug. The experiments were repeated three times, each experiment with 4 mice in each treatment
group. The graphs presents the average of all mice in the three experiments, meaning that the N = 12
for each time point. The bars represent standard error. *p < 0.001 in comparison to control mice. p <
0.001 for 50 mg/kg clone 202 extract (containing 8.9 mg/kg CBD) vs 10 mg/kg purified CBD. p <
0.05 of 100 mg/kg and 150 mg/kg vs 50 mg/kg of clone 202 extract at 6 hrs, indicating a dose-
dependent effect.
even though this dose was insufficient in reducing paw swelling (Figure 2(b)) or relieve pain (Figure 2(d)). At
least 25 mg/kg extract, which corresponds to about 5 mg CBD, was required to achieve the anti-inflammatory
effect. These data show that TNFα secretion is more sensitive to inhibition by clone 202 extract, than paw swel-
ling and pain.
Similar to the results obtained with intraperitoneal injection, orally administrated CBD gave a bell-shape re-
sponse, with an optimal response using 25 mg/kg (p < 0.001), while higher or lower doses had less effect
(Figure 4(c)). In contrast, orally delivered clone 202 extract showed an increased inhibitory effect on TNFα
production with increased doses (Figure 4(d)). Already at 25 mg/kg an inhibition of 48% was achieved that in-
creased further to 66% when given 150 mg/kg clone 202 extract (Figure 4(d)). The inhibition of TNF produc-
tion was much stronger than the inhibitory effect on paw swelling of 27% - 35%.
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Figure 4. Prevention of zymosan-induced TNFα production by purified CBD and clone 202 extract.
(a) (b) Twenty four hours after injecting zymosan and an intraperitoneal dose of CBD (a) or clone
202 extract (b), or a per os dose of CBD (c) or clone 202 extract (d), the TNFα concentration in the
serum was determined by ELISA. The experiments were repeated three times, each experiment with
4 mice in each treatment group. The graphs presents the average of all mice in the three experiments,
meaning an N = 12 for each treatment. TNFα serum level of untreated mice was 15 pg/ml. The bars
represent standard error. *p < 0.001 in comparison to control mice. p < 0.01 when comparing clone
202 extract with purified CBD. p < 0.01 when comparing an increasing doses of clone 202 extract,
emphasizing a dose-dependent effect.
3.3.ComparisonofCBDandCannabisClone202ExtractwithCommercial
AntiNociceptiveandAntiInflammatoryDrugs
Since Cannabis clone 202 extract has profound anti-inflammatory and anti-nociceptive effects as described
above, it was important to compare its potency with commercial anti-nociceptive and anti-inflammatory drugs.
We chose to use tramadol, a strong atypical opioid analgesic drug, and aspirin, a well-known non-steroid anti-
inflammatory drug (NSAID) that is also a pain reliever. Immediately after zymosan injection, mice were treated
with aspirin (50 mg/kg per os), tramadol (5 mg/kg i.p.), CBD (5 mg/kg i.p.) or clone 202 extract (50 mg/kg i.p.).
While aspirin had a moderate effect on paw swelling (p < 0.001 at 6 h), tramadol barely had any effect (Figure
5(a)). Both CBD and clone 202 extract markedly prevented paw swelling to a much larger extent than aspirin (p
< 0.005) (Figure 5(a)). As expected, aspirin and tramadol had a strong anti-nociceptive effect that exceeded that
of CBD and clone 202 extract (p < 0.01) (Figure 5(b)). Aspirin, but not tramadol, showed a slight inhibitory ef-
fect on TNFα production, that was negligible in comparison to the strong inhibitory effect of CBD and clone
202 extract (p < 0.01) (Figure 5(c)). Thus, CBD and clone 202 extract are endowed with different traits than as-
pirin and tramadol, making them superior with respect to anti-inflammatory properties.
4.Discussion
In this manuscript we have observed different dose-response patterns when using purified CBD or plant extract
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Figure 5. Comparison of anti-inflammatory and anti-nociceptive effects of CBD and Cannabis
clone 202 extract with the commercial drugs aspirin and tramadol. (a) Prevention of zymo-
san-induced swelling of hind paw. 1.5% zymosan in 40 μl was injected into the sub-planter sur-
face of the right hind paw. Immediately thereafter, aspirin (50 mg/kg per os), tramadol (5 mg/kg
i.p.), CBD (5 mg/kg i.p.) or Cannabis clone 202 extract (50 mg/kg i.p.) was given. The paw
thickness indicative for paw swelling was measured 2, 6 and 24 hrs later. The paw thickness of
untreated mice was 2.0 - 2.2 mm, which made the baseline of the graph. N = 5 for each time
point of each treatment group. *p < 0.001 in comparison to control mice. p < 0.005 when com-
paring CBD and clone 202 extract with aspirin and tramadol; (b) Anti-pain effect of aspirin, tra-
madol, CBD and Cannabis clone 202 extract in mice treated as described in paragraph A. The
hyperalgesia was measured by using the von Frey nociceptive filament assay. The higher the paw
withdrawal threshold, the higher is the anti-nociceptive effect of the drug. N = 5 for each time
point of each treatment group. The bars represent standard error. *p < 0.001 in comparison to
control mice. p < 0.05 when comparing CBD and clone 202 extract with aspirin and tramadol; (c)
The TNFα serum concentration at 24 hrs in mice that were treated as described in paragraph A. N
= 5 for each treatment. The bars represent standard error. *p < 0.001 in comparison to control
mice. p < 0.01 when comparing CBD and clone 202 extract with aspirin and tramadol.
of the Cannabis sativa L. clone 202, which is highly enriched in CBD. Purified CBD showed a bell-shaped
dose-response, where a therapeutic response could only be achieved at a certain concentration. This narrow the-
rapeutic window makes it difficult to use CBD in the clinics as a single agent. Therefore, we sought for a better
preparation that can utilize the favorable therapeutic effects of CBD. We observed that plant extracts of the non-
psychotropic clone 202 could fit this aim. A dose-dependent response was observed on all three parameters
tested: namely, the extract prevented zymosan-induced paw oedema, zymosan-induced pain and zymosan-
induced TNFα production in mice, with an improved therapeutic effect upon increased dosages. Thus, the limi-
R.Gallilyetal.
84
tation with purified CBD could be overcome when presented together with other natural components of the plant.
Of note, TNFα secretion was more sensitive to clone 202 extract inhibition than paw swelling and pain.
Our finding that it is possible to get a correlative dose-response using Cannabis clone 202 extracts, makes it
possible to use it in many pathological conditions. We suggest that clone 202 extracts may be a suitable substi-
tute for the current used Cannabis strain in the clinics, especially taking into account that it does not have any
psychotropic adverse effects. Following the clinical improvement by the clone 202 extracts, more tedious ex-
periments with CBD might be planned.
Our findings that CBD in the presence of other plant constituents improve the dose-response are supported by
some recent reports showing that CBD in a standardized Cannabis sativa extract is more potent or efficacious
than pure CBD [17]-[19]. These research groups studied the anti-proliferative effect of CBD on tumor cells [17]
[19] and the inhibitory effect of CBD on bladder contractility [18]. The higher efficiency of plant extract might
be explained by additive or synergistic interactions between CBD and minor phytocannabinoids or non-canna-
binoids presented in the extracts. Other phytocannabinoids, including Tetrahydrocannabivarin, Cannabigerol and
Cannabichromene, exert additional effects of therapeutic interest [20]. A lot of research has been made to isolate
and characterize isolated single constituents of traditional herbal medicine to find their rationale for therapeutic
uses. However, our data together with those of others [21] provide legitimation to introduce a new generation of
phytopharmaceuticals to treat diseases that have hitherto been treated using synthetic drugs alone. The therapeu-
tic synergy observed with plant extracts results in the requirement for a lower amount of active components,
with consequent reduced adverse effects.
5.Conclusion
In conclusion, we recommend standardized plant extract of the Cannabis clone 202 for treatment of various in-
flammatory conditions.
Acknowledgements
The authors would like to thank Dr. Ronit Sionov for her valuable editorial assistance.
ConflictofInterest
Prof. Ruth Gallily has been a consultant for Tikun Olam since 2013, and has received a research grant during the
years 2012-2014 from Tikun Olam, Israel. There is no conflict of interest.
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