Structure-dependent inhibitory effects of synthetic

cannabinoids against 12-O-tetradecanoylphorbol-13-acetate-

induced inammation and skin tumour promotion in mice
Junichi Nakajima
a
, Dai Nakae
a
and Ken Yasukawa
b
a
Department of Pharmaceutical and Environmental Sciences, Tokyo Metropolitan Institute of Public Health, Tokyo and
b
School of Pharmacy, Nihon
University, Funabashi, Chiba, Japan
Keywords
anti-inammatory effect;
anti-tumour-promoting agent; synthetic
cannabinoids;
12-O-tetradecanoylphorbol-13-acetate;
two-stage carcinogenesis
Correspondence
Junichi Nakajima, Department of
Pharmaceutical and Environmental Sciences,
Tokyo Metropolitan Institute of Public Health,
3-24-1, Hyakunin-cho, Sinjuku-ku, Tokyo
169-0073, Japan.
E-mail:
Junichi_Nakajima@member.metro.tokyo.jp
Received December 18, 2012
Accepted April 11, 2013
doi: 10.1111/jphp.12082
Abstract
Objectives Whether and how synthetic cannabinoids affect inammation and
carcinogenesis has not been well studied. The present study was thus conducted to
assess effects of synthetic cannabinoids on inammation and carcinogenesis in
vivo in mice.
Methods Twenty-three analogues of synthetic cannabinoids were isolated from,
and identied as adulterants in, illegal drugs distributed in the Tokyo metropoli-
tan area, and were examined for their inhibitory effects on the induction of
oedema in mouse ears by 12-O-tetradecanoylphorbol-13-acetate (TPA). Further-
more, selected cannabinoids, JWH-018, -122 and -210, were studied for their
effects on carcinogenesis induced in mouse skin initiated with 7,12-
dimethylbenz[a]anthracene (DMBA) and promoted by TPA.
Key ndings Among cannabinoids, naphthoylindoles mostly exhibited superior
inhibitory effects against TPA-induced ear oedema and, especially, JWH-018, -122
and -210 showed potent activity with 50% inhibitory dose (ID50) values of 168,
346 and 542 nm, respectively (an activity corresponding to that of indometacin
(ID50 = 908 nm)). Furthermore these three compounds also markedly suppressed
the tumour-promoting activity of TPA.
Conclusions This is the rst report indicating the structureactivity relationships
for the anti-inammatory activity of synthetic cannabinoids on TPA-induced
inammation in mice. Naphthoylindoles, JWH-018, -122 and -210, had the most
potent anti-inammatory activity and also markedly inhibited tumour promotion
by TPA in the two-stage mouse skin carcinogenesis model. The present results
suggest that synthetic cannabinoids, such as JWH-018, -122 and -210, may be
used as cancer chemopreventive agents in the future.
Introduction
During our careful surveillance of unregulated, illegal drugs
in Japan, numerous cannabimimetic analogues have been
found and identied.
[18]
Most of the identied compounds
have been categorized as indole derivatives,
[915]
leading to
the idea that they may have anti-inammatory activity,
similar to that of the well-known anti-inammatory indole
derivative, indometacin. Cannabinoids are classied into
three types: (1) cannabinoids derived from the plant Can-
nabis Sativa L.;
[16]
(2) endogenous cannabinoids, anandam-
ide
[17]
and 2-arachidonoylglycerol;
[18]
and (3) synthetic
cannabinoids.
[19]
All of these compounds bind to G-protein
coupled cannabinoid receptors, CB1 and CB2. Because CB1
and CB2 are overexpressed in certain cancers,
[20,21]
it has
been suggested that cannabinoids may possess an anti-
tumour activity. In fact, the synthetic cannabinoids JWH-
015 and Win55,212-2 have been shown to inhibit the
growth and metastasis of non-small cell lung cancer by
affecting CB1 and CB2.
[22]
Furthermore, another synthetic
cannabinoid, JWH-133, has been reported to inhibit the
growth and angiogenesis of skin tumours also via the
activation of cannabinoid receptors.
[23]
It has not been well
investigated, however, whether and how cannabinoids affect
inammation and carcinogenesis. In this context, the
present study was conducted to assess effects of synthetic
bs_bs_banner
And Pharmacology
Journal of Pharmacy
Research Paper
2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 12231230 1223
cannabinoids on inammation and carcinogenesis in vivo
in mouse skin. Among the tested cannabinoids, naphthoy-
lindole JWH-018 was the most potent compound in inhib-
iting TPA-induced inammation and carcinogenesis.
Materials and Methods
Chemicals
Figure 1 illustrates the chemical structures of 23 synthetic
cannabinoids used in this study. Compounds 122 were iso-
lated from illegal drugs distributed in the Tokyo metropoli-
tan area and identied using published data.
[18]
Compound
23, JWH-307, was purchased from Cayman Chemical (Ann
Arbor, MI, USA). 12-O-tetradecanoylphorbol-13-acetate
(TPA) was purchased from Chemicals for Cancer Research,
Inc. (Eden Prairie, MN, USA). 7,12-Dimethylbenz[a]
anthracene (DMBA), indometacin and hydrocortisone were
obtained from Sigma Chemical Co. (St Louis, MO, USA).
Other common chemicals used in this study were the
highest grade commercially available.
Ethical considerations
Experiments were approved by the Committee for Animal
Welfare at the School of Pharmacy, Nihon University,
Chiba, Japan, prior to the execution and performed in
accordance with the guidelines of the Institutional Animal
Care and Use Committee of the School of Pharmacy, Nihon
University.
Animals
Female ICR mice, 6 weeks old, were purchased from Japan
SLC, Inc. (Hamamatsu, Shizuoka, Japan) and housed in an
air-conditioned specic pathogen-free room (2223C,
50 10% relative humidity, frequency of air changes
1119/h, lights on between 8:00 and 20:00), four or ve
mice per cage, and acclimatized for 1 week until experimen-
tation. Food and tap water were freely available.
Assay of TPA-induced inammation
The assay was conducted according to methods reported by
Yasukawa et al.
[24]
TPA (1 mg) was dissolved in acetone
(20 ml) and applied (10 ml each) to the inner and outer sur-
faces of the right ear of ICR mice (7 weeks old, four mice
per group) using a micropipette. Cannabinoids, or their
vehicle, a chloroformmethanol mixture (1 : 1, v/v) (20 ml),
were similarly applied about 30 min before the TPA treat-
ment. The thickness of the ear was determined before (a)
and 6 h after the TPA treatment (b, TPA plus a vehicle; b,
TPA plus a cannabinoid) using a pocket thickness gauge
(Mitsutoyo Corp., Kawasaki, Kanagawa, Japan). The follow-
ing values were then calculated to evaluate effects of can-
nabinoids on TPA-induced inammation:
( ) n
1:
2:
3:
4:
5:
6:
7:
R1
H
H
H
H
Me
Et
OMe
n
1
2
3
4
3
3
3
8: H
9: Ms
10 11 12 13 14
15
16
17:
18:
R1
OMe
H
R2
H
OMe
20:
21:
22:
R
Me
OMe
Cl
19
23
R2
Me
H
H
H
H
H
H
O O O O
O O O
O
OH
R2
R1 R
N N N N
N N N
N
N
N
F
R
O
O O O
O
O
I
R2
R
R1
H
N
H
N
N
N
N N N N
F
F
N
Figure 1 Chemical structure of synthetic cannabinoids used in this study.
Junichi Nakajima et al. Effects of synthetic cannabinoids
2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 12231230 1224
Oedema A oedema induced by TPA plus vehicle b a : ( ) (1)
Oedema B oedema induced by TPA plus sample b a : ( )
(2)
Inhibitory ratio
oedema A oedema B oedema A
(%)
( ) = [ ] 100
(3)
The 50% inhibitory dose (ID50) values were determined by
the probit-graphic interpolation for four dose levels.
Two-stage skin carcinogenesis initiated with
DMBA and promoted by TPA
The test was conducted according to methods by Yasukawa
et al.
[24]
Skin carcinogenesis was initiated by topically apply-
ing 50 mg of DMBA to the back of each mouse (7 weeks old,
15 mice per group) and promoted by topically applying
1 mg TPA twice per week, from 1 week after the initiation up
to the end of week 20. DMBA and TPA were dissolved in
acetone, and a volume of 100 ml was applied using a micro-
pipette. JWH-018 (compound 3, 0.02 and 0.2 mm), JWH-
122 (compound 5, 0.2 and 2 mm) and JWH-210 (compound
6, 0.2 and 2 mm), or the vehicle, acetone (100 ml), were
applied topically 30 min before each TPA treatment. The
back of each mouse was shaved once a week to remove hair.
The number and diameter of skin tumours were measured
every other week, and the experiment continued for 20
weeks. Experimental and appropriate control groups each
consisted of 15 mice. The average numbers of tumours and
standard deviation (S.D.) were calculated using the total
number of tumours for 15 mice. All of these data were com-
pared for the treatment and control groups at each weeks of
promotion.
Statistical analysis
The ID50 values and their 95% condence intervals
(95% CI) were obtained by nonlinear regression using the
GraphPad PRISM v. 5.0 (Intuitive Software for Science,
San Diego, CA, USA). The signicances of the differences
between the treatment and control groups for the tumour
incidence and multiplicity were determined by the one-
tailed Fishers exact test and the Steels test, respectively, and
considered signicant when P < 0.05.
Results
Table 1 summarizes the inhibitory effects of the synthetic
cannabinoids on TPA-induced inammation in mice.
Most of the naphthoylindoles (compounds 13, 58, 10,
11, 13 and 14) inhibited the TPA-induced inammation,
with an ID50 value of 168675 nm/ear, more effective
than indometacin (ID50 = 908 nm).
[24]
Naphthoylindoles 9
and 12 were inactive. In the case of adamantyl derivatives,
compounds 15 and 16 were active with an ID50 value of
964 and 1029 nm/ear, respectively. Benzoylindole analogues
(compounds 1719), phenylacetylindoles (compounds
2022) and the naphtopyrole (compound 23) were
inactive.
As compounds 3, 5 and 6 strongly inhibited TPA-induced
inammation, their effects on tumour promotion by TPA
were examined using a mouse skin two-stage carcinogenesis
model with DMBA initiation. Judging from the TPA test, it
was possible to estimate adequate concentrations of test
compounds for the DMBA-TPA test from our previous
study.
[24]
Therefore, we used 0.02 and 0.2 mm/mouse for
compound 3, and 0.2 and 2.0 mm/mouse for compound 5
and 6, respectively.
Figure 2a shows the time course of changes in skin
tumour incidence in mice (initiated with DMBA) treated by
TPA with (0.02 or 0.2 mm per mouse) or without JWH-018
(compound 3). In the group treated by TPA without JWH-
018, the rst tumour appeared at the end of week 5, and all
15 mice bore tumours by the end of week 11. In the groups
treated by TPA with 0.02 and 0.2 mm of JWH-018, in con-
trast, the rst tumour appeared at the end of weeks 7 and 8,
respectively, while the percentage incidence reached 60 and
33%. Figure 2b shows the time course of changes in the
number of tumours, and at the end of week 20, the number
of tumours in the groups treated by TPA with 0, 0.02 and
0.2 mm of JWH-018 was 14.0, 5.1 (64% reduction) and 1.5
(89% reduction) per mouse, respectively.
Figure 3a shows the time course of changes in skin
tumour incidence of mice (initiated with DMBA) treated by
TPA with (0.2 or 2 mm per mouse) or without JWH-122
(compound 5). In the groups treated by TPA with 0.2 and
2 mm of JWH-122, the rst tumour appeared after 7 and 8
weeks of beginning of tumour promotion, respectively,
while the percentage incidence reached 60 and 40%.
Figure 3b shows the time course of changes in numbers of
tumours, and at the end of week 20, the tumour number in
the groups treated by TPA with 0.2 and 2 mm of JWH-122
was 6.3 (55% reduction) and 2.3 (84% reduction) per
mouse, respectively.
Figure 4a shows the time course of skin tumour inci-
dence of mice (initiated with DMBA) treated by TPA with
(0.2 and 2 mm per mouse) or without JWH-210 (compound
6). In the groups treated by TPA with 0.2 and 2 mm of JWH-
210, the rst tumour appeared at the end of week 7 in both
groups, while the percentage incidence reached 60 and 40%,
respectively. Figure 4b shows the time course of changes of
numbers of tumours, and at the end of week 20, the tumour
number in the groups treated by TPA with 0.2 and 2 mm of
JWH-210 was 7.0 (50% reduction) and 3.2 (77% reduction)
per mouse, respectively. To illustrate the distribution results
among the treated versus the control mice in detail, Table 2
Junichi Nakajima et al. Effects of synthetic cannabinoids
2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 12231230 1225
Table 1 Inhibitory effects of synthetic cannabinoids on TPA-induced inammation in mice
Compound
number Name of cannabinoid
ID50
(nM/ear)
95% CI
(nM/ear)
Naphthoylindoles
1 (2-Methyl-1-propyl-1H-indol-3-yl)(naphthalen-1-yl)methanone, JWH-015 534 400709
2 Naphthalen-1-yl-(1-butylindol-3-yl)methanone, JWH-073 664 504874
3 Naphthalen-1-yl-(1-pentylindol-3-yl)methanone, JWH-018 168 120237
4 Naphthalen-1-yl-(1-hexylindol-3-yl)methanone, JWH-019 1279 9581713
5 (4-Methyl-naphthalen-1-yl-(1-pentylindol-3-yl)methanone, JWH-122 346 225532
6 (4-Ethyl-naphthalen-1-yl-(1-pentylindol-3-yl)methanone, JWH-210 542 371793
7 4-Methoxynaphthalen-1-yl-(1-pentylindol-2-yl)methanone, JWH-081 424 315571
8 1-(5-Fluoropentyl-1H-indol-3-yl)-(naphthalene-1-yl)methanone, AM-2201 463 320671
9 1-(5-Fluoropentyl-1H-indol-3-yl)-(4-methyl-naphthalene-1-yl)methanone, 4-Me-AM-2201 >1340 n.a.
10 (1-(2-Morpholin-4-ylethyl)indol-3-yl)(naphthalene-1-yl)methanone, JWH-200 320 211486
11 (1-(5-Hydroxypentyl)-1H-indol-3-yl)(naphthalene-1-yl)methanone, AM-2202 437 339568
12 (1-(4-Pentenyl)-1H-indol-3-yl)(naphthalene-1-yl)methanone, JWH-022 >1473 n.a.
13 (1-((1-Methylpiperidin-2-yl)methyl-1H-indol-3-yl)(naphthalene-1-yl)methanone, AM-1220 675 494923
14 (1-(1-Methylazepan-3-yl)-1H-indol-3-yl)(naphthalen-1-yl)methanone, AM-1220-azepanindol 617 499763
Adamantyl or indazole indoles
15 N-(1-adamantyl)-1-pentyl-1H-indol-3-carboxamide, APICA 964 6971163
16 N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide, APINACA 1029 6351453
Benzoylindoles
17 (2-Methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone, 2MeO-RCS-4 >1558 n.a.
18 (4-Methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone, RCS-4 >1558 n.a.
19 1-[(5-Fluoropentyl)-1H-indol-3-yl]-(2-iodophenyl)methanone, AM-694 >1150 n.a.
Phenylacetylindoles
20 1-(1-Pentyl-1H-indol-3-yl)-2-(o-tolyl)ethanone, JWH-251 >1567 n.a.
21 1-Pentyl-3-(2-methoxyphenylacetyl)indole, JWH-250 >1492 n.a.
22 2-(2-Chlorophenyl)-1-(1-pentyl-1H-indol-3-yl)ethanone, JWH-203 >1475 n.a.
Naphtopyroles
23 (5-(2-Fluorophenyl)-1-pentyl-1H-pyrrol-3-yl)(naphthalen-1-yl)methanone, JWH-307 >2597 n.a.
Indometacin
a
908 7551092
Hydrocortisone
a
69 6475
ID50, the 50% inhibitory dose; 95% CI, 95% condence intervals; n.a., not applicable.
a
Standard drug.
(a) (b)
100
50
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Figure 2 Inhibitory effect of JWH-018 (compound 3) on the promotion of skin papillomas by TPA in mice given DMBA. (a) Percentage incidence of
mice bearing papillomas and (b) average numbers of papillomas per mouse. , TPA with vehicle; , TPA with JWH-018 (0.02 mM/mouse); , TPA
with JWH-018 (0.2 mM/mouse). Data are means, n = 15. *P < 0.05, **P < 0.01 vs vehicle.
Junichi Nakajima et al. Effects of synthetic cannabinoids
2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 12231230 1226
shows the average numbers and S.D. of tumours tested with
compounds 3, 5 and 6.
Discussion
This study examined the effects of 23 synthetic cannabi-
noids, most of which have recently been isolated and identi-
ed during our surveillance of unregulated, illegal drugs
distributed in the Tokyo metropolitan area of Japan. The
results clearly indicated that 14 of them (approximately
60%) exert a signicant inhibitory effect on TPA-induced
inammation, as assessed by the oedema formation at the
location of its topical application to the mouse ear. Most of
them (12 compounds) were naphthoylindoles, and the
remaining two were adamantyl or indazole indoles. Their
inhibitory potentials were as strong as, or stronger than,
that of indometacin, a well-known anti-inammatory
indole derivative. In contrast, the results also revealed that
synthetic cannabinoids belonging to the benzoylindoles,
phenylacetylindoles and naphthopyrroles cannot inhibit
TPA-induced inammation; neither could two of the
naphthoylindoles.
The above results suggest that there is a certain relation-
ship between structure and anti-inammatory activity of
synthetic cannabinoids. Figure 5 illustrates that such a
structureactivity relationship indeed exists, and suggests
that the structures of the side-chain from the nitrogen atom
of the indole may play an important role. One of the most
typical examples supporting such a concept may be that the
potent anti-inammatory activity shown by JWH-018
(a) (b)
100
50
0
0 5 10 15 20
T
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Weeks of promotion
15
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Weeks of promotion
***
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**** **** ** ** ****
** ** ** ** ** ** **** ** **
*
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*
Figure 3 Inhibitory effect of JWH-122 (compound 5) on the promotion of skin papillomas by TPA in mice given DMBA. (a) Percentage incidence of
mice bearing papillomas and (b) average number of papillomas per mouse. , TPA with vehicle; , TPA with JWH-122 (0.2 mM/mouse); , TPA with
JWH-122 (2 mM/mouse). Data are means, n = 15. *P < 0.05, **P < 0.01 vs vehicle.
(a) (b)
100
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Figure 4 Inhibitory effect of JWH-210 (compound 6) on the promotion of skin papillomas by TPA in mice given DMBA. (a) Percentage incidence of
mice bearing papillomas and (b) average number of papillomas per mouse. , TPA with vehicle; , TPA with JWH-210 (0.2 mM/mouse); , TPA with
JWH-210 (2 mM/mouse). Data are means, n = 15 *P < 0.05, **P < 0.01 vs vehicle.
Junichi Nakajima et al. Effects of synthetic cannabinoids
2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 12231230 1227
(compound 3) was totally abolished by making the terminal
carbon of the side-chain double bonded to achieve JWH-
022 (compound 12). Furthermore, when the side-chain of
JWH-018 (compound 3) was changed to n-butyl (to
achieve JWH-073 (compound 2)) or n-hexyl (to achieve
JWH-019 (compound 4)) groups, the anti-inammatory
effect became less potent. The naphtyl moiety may also
affect the anti-inammatory potential of synthetic cannabi-
noids. A typical example of this is that the potent anti-
inammatory activity of AM-2201 (compound 8) was
absent for its 4-methyl-naphthalne analogue 4-Me-AM-
2201 (compound 9). Judging from these results, only a
slight difference in the chemical structure will affect the
biological activity. At this moment, however, data are still
not sufcient to allow us to discuss detailed relationships
between the effect and the stereochemical structure of can-
nabinoids. Further study is warranted to better understand
this issue.
It has previously been reported that the processes of
inammation and carcinogenesis induced by TPA are
closely related.
[24]
Consistently, in the present study, it was
demonstrated that JWH-018, -122 and -210 (compounds 3,
5 and 6, respectively) exhibit potent inhibitory activity
against TPA-induced inammation in the mouse ear and
also tumour promotion in the two-stage mouse skin car-
cinogenesis model as shown in Figures 24 and Table 2.
Although mechanisms underlying this anti-carcinogenic
(anti-promoting) effect of synthetic cannabinoids remain
obscure, their anti-inammatory activity must be involved
according to the present results and the information in the
literature.
[24]
The inuence on cannabinoid receptors, such
as CB1 and CB2, is considered to be one of the main
mechanisms.
[2023,25]
In fact, cannabimimetic compounds,
like WIN-55, WIN-212 or JWH-133, have been shown to
inhibit the growth of non-melanoma skin cancer
[23]
and
melanoma
[25]
transplanted into the subcutaneous area of
mice by virtue of cannabinoid receptors. These ndings
may support our conclusions. Further studies are appar-
ently warranted to elucidate details of the anti-carcinogenic
effects of synthetic cannabinoids in order to facilitate
human health by controlling cancers.
Conclusions
This is the rst report indicating the structureactivity
relationships for the anti-inammatory activity of syn-
thetic cannabinoids on TPA-induced inammation in
mice. It is shown that naphthoylindoles, JWH-018, 122
and 210 (compounds 3, 5 and 6, respectively), have the
most potent anti-inammatory activity and also markedly
inhibit the tumour promotion by TPA in the two-stage
mouse skin carcinogenesis. The present results suggest that
synthetic cannabinoids, such as JWH-018, -122 and -210, T
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Junichi Nakajima et al. Effects of synthetic cannabinoids
2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 65, pp. 12231230 1228
may be used as cancer chemopreventive agents in the
future.
Declarations
Conict of interest
The Author(s) declare(s) that they have no conicts of
interest to disclose.
Funding
This work was supported in part by a research budget of the
Tokyo Metropolitan Government, Japan.
Acknowledgement
The authors would like to thank Dr Kuniaki Tayama for his
valuable participation in statistical analysis.
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