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Annual Review of Pharmacology and Toxicology
Synthetic Cannabinoids:
A Pharmacological and
Toxicological Overview
Rita Roque-Bravo,1,∗Rafaela Soa Silva,1,∗
Rui F. Malheiro,1Helena Carmo,1Félix Carvalho,1
Diana Dias da Silva,1,2 and João Pedro Silva1
1Associate Laboratory i4HB - Institute for Health and Bioeconomy, and UCIBIO,
REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of
Pharmacy, University of Porto, Porto, Portugal; email: jpmsilva@ff.up.pt, dsilva@ff.up.pt
2Toxicology Research Unit (TOXRUN), University Institute of Health Sciences,
IUCS-CESPU, Gandra, Portugal
Annu. Rev. Pharmacol. Toxicol. 2023. 63:3.1–3.23
The Annual Review of Pharmacology and Toxicology is
online at pharmtox.annualreviews.org
https://doi.org/10.1146/annurev-pharmtox-031122-
113758
Copyright © 2023 by the author(s).
All rights reserved
∗These authors contributed equally to this article
Keywords
designer drugs, drugs of abuse, endocannabinoid system, new psychoactive
substances, Spice, substance use disorders
Abstract
Synthetic cannabinoids (SCs) are a chemically diverse group of new psy-
choactive substances (NPSs) that target the endocannabinoid system, trig-
gering a plethora of actions (e.g., elevated mood sensation, relaxation, ap-
petite stimulation) that resemble, but are more intense than, those induced
by cannabis. Although some of these effects have been explored for therapeu-
tic applications, anticipated stronger psychoactive effects than cannabis and
reduced risk perception have increased the recreational use of SCs, which
have dominated the NPS market in the United States and Europe over the
past decade. However, rising SC-related intoxications and deaths represent
a major public health concern and embody a major challenge for policy
makers.
Here, we review the pharmacology and toxicology of SCs. A thor-
ough characterization of SCs’ pharmacodynamics and toxicodynamics is im-
portant to better understand the main mechanisms underlying acute and
chronic effects of SCs, interpret the clinical/pathological ndings related to
SC use, and improve SC risk awareness.
.
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Synthetic
cannabinoids (SCs):
a diverse group of
man-made new
psychoactive
substances designed to
mimic, with higher
potency, the
psychoactive action of
9-THC
New psychoactive
substances: novel
narcotic/psychotropic
drugs, not controlled
by the United Nations
Drug Control
Conventions, that may
pose a public health
threat comparable to
substances listed in
those conventions
Endocannabinoid
system: a complex
signaling network
comprising
endogenous
cannabinoids, enzymes
responsible for the
synthesis and
degradation of
endocannabinoids, and
cannabinoid receptors
Cannabinoid
receptors:
cell membrane
G protein–coupled
receptors belonging to
the endocannabinoid
system that act as main
targets for endogenous
and exogenous
cannabinoids
1. SYNTHETIC CANNABINOIDS: HIGH POTENCY, MAJOR PUBLIC
HEALTH THREAT
Synthetic cannabinoids (SCs) are a structurally diverse group of new psychoactive substances
(NPSs) designed to target the endocannabinoid system. These substances have a higher afnity
for cannabinoid receptors (CBRs) than 9-tetrahydrocannabinol (9-THC), the main psychoac-
tive substance in cannabis (1, 2), inducing several effects that resemble, but are more intense and
short-lived than, those induced by 9-THC (3).
The rst SC dates back to 1964 and was a synthetic version of 9-THC designed by Gaoni &
Mechoulam (4). The synthesis of other SCs ensued to help understand how the endocannabinoid
system regulates critical biological processes, leading to the discovery of CBRs in the 1980s (5).
More recently,the search for more potent and legal alternatives to cannabis led to the emergence
of several SCs, whose recreational use began in the mid-1990s (6). SCs dominated the NPS market
between 2009 and 2019, but the number of new SCs reaching the market per year decreased dur-
ing 2014–2018 (7). Nevertheless, a total of 209 SCs have been detected in the European Union’s
member states since 2008, accounting, alongside synthetic cathinones, for about 60% of the total
NPS seizures in 2019 (8). In Europe, SCs are most popular among the population aged 15–34 years
(comprising adolescents and young adults), with prevalence rates varying between 0.1% (Nether-
lands) and 1.5% (Latvia) (3). Moreover, the emergence of new structurally different SCs, along
with their short half-life in plasma circulation, hinders their monitoring and detection, probably
leading to an underestimation of their prevalence (9), and represents a major challenge to policy
makers (10).
SCs are usually dissolved in an organic solvent (e.g., acetone, methanol) and sprinkled over
plant-based materials (e.g., lemon balm, mint, thyme) (3). They are then sold without any quality
or quantity control and in attractive packages with appealing names (e.g., Spice, K2), mostly over
the internet (e.g., dark web). These herbal mixtures may contain unknown molecules or other
illicit/noxious substances (e.g., bath salts, ecstasy, rodenticides), which may further contribute to
their adverse effects (3). SCs are usually smoked (e.g., using a pipe/water pipe or paper or e-
cigarettes) but may also be orally ingested as tablets, powders (11), and herbal infusions (12, 13).
The mechanisms underlying SCs’ pharmacological action and toxicological effects remain
mostly underexplored. This is especially concerning, as SC use has been increasingly associated
with severe intoxications and deaths, thus representing a global public health concern (8, 9).
Here, we comprehensively review the most recent updates on the pharmacology and toxicology
of SCs, which is important to better interpret the SC use–related clinical/pathological ndings as
well as to improve SC risk awareness.
2. PHYSICOCHEMICAL PROPERTIES AND ANALYTICAL
METHODOLOGIES
In their pure form, SCs are usually described as white or yellowish crystalline ne powders with no
odor, low water solubility, and high solubility in nonpolar organic solvents and aliphatic alcohols
(e.g., ethanol, methanol, acetone, isooctane, ethyl acetate, acetonitrile) (14, 15).
Their structure may be generally divided into four main elements: the core, tail, linker, and
ring/linked group, as depicted in Figure 1 (14, 16). SCs can be categorized into distinct groups
and subgroups depending on their structure (15, 16).
The timely detection of SCs in biological samples can be challenging, thus compromising
proper diagnosis of SC-related intoxications. So far, different analytical methods can be used
to detect and quantify SCs in different matrices. The gold standard for qualitative analysis of
SCs is gas chromatography coupled with mass spectrometry (GC-MS) due to its notorious
. Roque-Bravo et al.
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General structure
(AB- FUBINACA)
T
ail
Core
Linker
Secondary
group
HU-21
CP47,497-C8
Classical
Nonclassical
Aminoalkylindoles
Naphtoylindoles
(e.g., JWH-122)
Phenylacetylindoles
(e.g., JWH-250)
Benzoylindoles
(e.g., RCS- 4)
Naphtylmethylindoles
(e.g., JWH-175)
Hybrid
Eicosanoids
Other
Endocannabinoids Phytocannabinoids
Synthetic cannabinoids
a
bc
O
OH
H
H
HO
OH
OH
O
N
O
N
O
N
O
HO
OH
HO
H
H
AM- 4030
Methanandamide
N
H
OH
O
N
O
OO
N
F
JWH-307
O
O
CRA-13
O
H
HOH
H
HOH
HO
Cannabidiol
O
O
OH
OH
2-Arachidonoylglycerol
N
H
O
OH
Anandamide
N
N
ONH
O
F
NH2
Δ9-Tetrahydrocannabinol (Δ9-THC)
Figure 1
General classication of synthetic cannabinoids (SCs). (a) Although different groups of SCs display structural variations, these
substances exhibit a general structure (the structure of AB-FUBINACA is shown as an example), comprising a core and a secondary
structure, joined by a linker with a tail group attached, as presented on the left side of the panel. The structures of the two main
endocannabinoids (b) and the two main phytocannabinoids (c) are presented for comparison. Notably, some SC classes present
structural similarities to these molecules. For example, classical SCs are similar to phytocannabinoids, whereas SCs from the eicosanoid
class are structurally similar to endocannabinoids.
chromatographic resolution. Nevertheless, GC-MS may present limitations regarding the
analysis of closely related isomers. Techniques such as nuclear magnetic resonance (NMR) or
infrared spectroscopy, gas chromatography coupled with ame ionization detector, and liquid
chromatography coupled with mass spectrometry (LC-MS) may also be used (15, 17). Liu et al.
(17) used GC-MS and NMR to detect 10 distinct indole/indazole SCs in 36 herbal blends, noting
that both techniques had similar sensitivity and detection range (1.9–50.6 mg/g for CG-MS, 1.5–
49.0 mg/g for NMR). Recently, Mercieca et al. (18) developed a time- and cost-effective extraction
technique, based on the pairing of dispersive liquid-liquid microextraction with ultrasound, to
improve SC detection via GC-MS.
The most common quantitative method is LC-MS, which only requires a single mass spec-
trometer (either with or without high resolution; LC-HRMS), or tandem mass spectrometry
(LC-MS/MS). The analysis is preceded by an extraction step (either solid phase extraction or
liquid-liquid extraction) and is mostly suited for instances where elevated amounts of fatty acids
are present (14, 15). A set of parent indole and indazole (e.g., 5F-PB-22, 5F-ADB) and other
SCs (e.g., EG-018, MDMB-CHMCZCA, CUMYL-PeGACLONE) were successfully detected
(in addition to their respective metabolites) in wastewater up to 29 days using LC-HRMS and
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LC-MS/MS, validating these parent compounds (not just their metabolites) as biomarkers for SC
detection in wastewater (19).
Using liquid chromatography coupled with quadrupole time-of-ight mass spectrometry (LC-
QToF-MS), Giorgetti et al. (20) recently developed an LC-MS/MS-based method that allowed
the quantication of the 7-azaindole 5F-AB-P7AICA in serum samples—with good linearity, ac-
curacy, and precision—and in urine. Similarly, Haschimi et al. (21) detected the new derivatives
CUMYL-CBMICA and CUMYL-CBMINACA and the products of their phase I metabolism in
human urine samples using LC-QToF-MS.
The use of immunoassays to screen blood and/or urine for the presence of SCs is not recom-
mended, due to the restricted concentration range of these assays, the structural variability of SCs,
and the fast metabolism of these substances in the body,which often produce metabolites that may
not be detected by this method (14, 18). For example, Mogler et al. (22) unsuccessfully attempted
to detect the novel analog CUMYL-PeGACLONE in 30 biological samples from SC users using
SC-specic immunoassays, even after lowering cut-off values.
3. PHARMACODYNAMICS
The pharmacology of SCs is generally similar to that of 9-THC, as these substances act on two of
the main receptors in the endocannabinoid system, the cannabinoid receptors 1 and 2 (CB1R and
CB2R, respectively), which are G protein–coupled receptors (GPCRs). In contrast to 9-THC
(partial agonist), most SCs are full agonists of these receptors, thus displaying higher efcacy com-
pared to 9-THC, which accounts for both the more intense psychoactive effects and the greater
severity of undesired effects. Moreover, SC mixtures lack cannabidiol, a CBR antagonist present
in cannabis that helps counter the psychoactive effects of 9-THC (14, 16). A structure-activity
analysis concerning CB1R activation, performed by Banister et al. (23) in transfected murine AtT-
20 neuroblastoma cells, showed that SCs with an L-tert-leucinamide linker generally displayed
greater potency at CBRs than did their L-valinamide counterparts, regardless of the core be-
ing an indole or an indazole. The same group also demonstrated that tert-leucinate SCs were
more potent than their respective valinate counterparts (similarly, no correlation was found be-
tween the presence of an indole or indazole core and CBR activation), having observed differ-
ent potencies for distinct N-alkyl substitutions (cyclomethylhexyl <4-uorobenzyl <pentyl <
5-uoropentyl) (24). Moreover, indazole analogs showed higher potency (reecting improved
binding and afnity) for the CBRs, followed by the indole and the 7-azaindole moieties
(25).
Activation of the CB1R leads to a decrease in cyclic adenosine monophosphate (cAMP) by
inhibiting adenylyl cyclase activity (14, 16). Additionally, CB1R activation causes the βγ subunits
to activate the mitogen-activated protein kinase (MAPK) family, including the signal-regulated
extracellular kinases 1 and 2 (ERK1/2). CB1R phosphorylation (following its activation) by
G protein receptor kinases (GRKs) may induce β-arrestin 1 and 2 translocation to the cell mem-
brane, causing CB1R desensitization and internalization, a mechanism reportedly associated with
tolerance development (14, 26).
Similarly to other GPCR-activating molecules, SCs were found to act via biased agonism/
functional selectivity, a concept in which the activation of CB1R favors a specic receptor con-
formation, promoting one signaling pathway over another (26), which may possibly explain the
distinct pharmacological actions produced by SCs binding to the same receptor (27). Indeed, ac-
tivation of the Gαi/o heterotrimer over β-arrestin 1 triggers the Gβγ subunit release, inhibiting
voltage-dependent calcium channels and activating G protein–gated inwardly rectifying potas-
sium channels. The Gαi/o subunit then inhibits adenylyl cyclase, stimulating the phosphorylation
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Biased agonism:
a ligand-based
induction of a
particular receptor
conformation that
favors a specic
signaling transduction
pathway, relative to a
reference agonist
Endocanna-
binoidome: expanded
endocannabinoid
system, which further
includes several
mediators
biochemically related
to endocannabinoids,
their receptors, and
metabolic enzymes
and early activation of ERK1/2. In contrast, β-arrestin 1 recruitment triggers the late activation of
ERK1/2 (28). For example, although JWH-018 and JWH-081 bind to CB1R, JWH-018 decreases
phospho-ERK1/2 expression (29), whereas JWH-081 impairs phospho-CaMKIV and phospho-
CREB levels (e.g., associated with neuronal plasticity regulation) (30). Wouters et al. (31) assessed
the preference of a set of SCs for the β-arrestin 2 or Gαipathways in HEK293T cells stably
transfected with CB1R and found balanced agonists (favoring neither pathway) such as MMB-
CHMICA, SCs showing bias toward β-arrestin 2 recruitment (e.g., 5F-APINACA, CUMYL-
PeGACLONE, JWH-018), and a single SC (e.g., EG-018) showing bias toward Gαirecruitment.
Since EG-2201 and MDMB-CHMCZCA, which contain a carbazole group like EG-018, also
displayed a tendency for Gαisignaling, the authors suggested that this carbazole may inuence
the bias (31). Interestingly, the consequences of favoring CB1 recruitment of β-arrestin 1 or 2
remain unclear. Agonists often activate both β-arrestins, with specicity likely stemming from
a cell-specic context (32). Patel et al. (26) observed that, compared to the reference ligand
WIN55,212-2, the indazole derivatives 4CN-CUMYL-BUTINACA, 5F-AMB-PINACA, and
5F-MDMB-PICA exhibited a strong bias toward cAMP inhibition (and away from cAMP stimu-
lation and β-arrestin recruitment and/or internalization) in CB1R-transfected HEK293 cells, sup-
porting a strong CB1R twin toggle switch (ranging residues F200 and W356 in the TM2/TM6
binding pocket) interaction. Notably, the aromatic interaction of indole or indazole-based SCs
(e.g., MDMB-FUBINACA, 5F-MDMB PICA) with the toggle switch has been shown to stabi-
lize the active conformation of the receptor, further increasing these substances’ binding ability
(33). 5F-CUMYL-P7AICA and XLR-11 only displayed partial efcacy in cAMP stimulation and
β-arrestin translocation. Most importantly, SCs showed higher potency and efcacy than 9-THC
in the recruitment and internalization of β-arrestins and Gαssignaling. Specically, the relatively
low efcacy of 9-THC in β-arrestin pathways compared to SCs suggests that these pathways may
be key contributors to SCs’ effects (26). Zagzoog et al. (34) further noted that the indazole core and
halogen-substituted pentyl or butyl tails [e.g., JWH-018 2-napthyl-N-(3-methylbutyl) isomer, 4-
uoro MDMB-BINACA] biased SCs toward cAMP inhibition and β-arrestin 2 recruitment.
SCs can also modulate signaling pathways independently of CBRs, in line with the discovery
of the endocannabinoidome, an expanded endocannabinoid system. For example, aminoalkylin-
dole derivatives, arylpyrazole derivatives, and synthetic analogs of phytocannabinoids have been
reported to target the transient receptor potential cation channel subfamily V member 1 (TRPV1)
(35). The desensitization of these channels by WIN55,212-2 has been found to promote analgesic
effects (36). Nevertheless, despite sharing a strong ability to stimulate CBRs, SCs display limited
efcacy in opening TRPV1 channels, with only XLR-11 and its analogs inducing a signicant
activation of these channels (37).
SCs may also activate peroxisome proliferator-activated receptors (PPARs), a family of nu-
clear hormone receptors that form heterodimers with the retinoid X receptor and alter gene
transcription by binding to DNA sequences called PPAR response elements (38). CB1R- and
CB2R-mediated activation of PPARγby WIN55,212-2 has been suggested to underlie its neuro-
protective and anti-inammatory effects against amyloid-β(Aβ)-induced damage (39). Similarly,
modulation of PPARαvia WIN55,212-2-induced activation of CBRs has been reported as the
main mechanism responsible for WIN55,212-2 anticonvulsant properties in a pentylenetetrazol-
induced clonic seizure mice model (40). Also, Vara et al. (41) demonstrated that the antiprolifera-
tive action of JWH-015 on hepatocellular carcinoma was shown to be modulated, in vitro and in
vivo, by the upregulation of PPARγsignaling.
SCs may further modulate the orphan GPCRs (e.g., GPR55, GPR18) (42, 43). For example,
JWH-015 has been shown to activate GPR55 in distinct cell types, increasing intracellular calcium
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CB1R
CB2R
NT
AC
cAMP
PKA
GPR18
α
βγ
α
βγ
α
βγ
α
s
α
i/o
Ca2+
K+
α
βγ
SCs
Presynaptic
terminal
ERK1/2
GRK
PP
PPARs
TRPs
Transcription
CB1R
CB1R
CB2R
GPR55
αβγ α
βγ
MAPK
β-arrestin 1/2
Gα12/13
[iCa2+]
Activation
Indirect regulation
Inhibition
Movement of ions
Ca2+
Presynaptic terminal
Figure 2
Intracellular signaling pathways triggered by synthetic cannabinoids. Activation of cannabinoid receptors 1 and 2 (CB1R and CB2R,
respectively) in the presynaptic terminal leads the αi/o subunit of the G protein–coupled receptor to inhibit adenylyl cyclase (AC),
causing a decrease in cyclic adenosine monophosphate (cAMP) and, subsequently, in protein kinase A (PKA) activity. CB1R activation
also triggers the opening of inward-rectifying K+channels with concomitant inactivation of Ca2+channels (through the βγ subunits of
the receptor), which block the release of neurotransmitters (NTs) into the synaptic cleft. At the postsynaptic level, the βγ subunits are
also responsible for the activation of mitogen-activated protein kinase (MAPK), which may further activate signal-regulated
extracellular kinases 1 and 2 (ERK1/2) at an early stage. Moreover, MAPKs may regulate nuclear transcription factors and thus
inuence gene expression. Phosphorylation of CB1R by G protein receptor kinases (GRKs) marks the receptor and signals for
β-arrestin 1/2 translocation while promoting CB1R desensitization and internalization. β-arrestin may also activate ERK1/2 at a later
timepoint than MAPK. Other elements of the endocannabinoid system may also be stimulated by synthetic cannabinoids (SCs): G
protein–coupled receptor 55 (GPR55) activation leads to an increase in intracellular Ca2+concentration; GPR18 also couples with the
αi/o receptor subunit, being also able to inhibit AC; transient receptor potential channels (TRPs) stimulate the neuronal uptake of
Ca2+; and activation of peroxisome proliferator-activated receptor γ(PPARγ) regulates gene transcription. Some gure elements
adapted from Servier Medical Art, provided by Servier (CC BY 3.0).
levels (44). Activation of GPR55 has also been suggested to underlie the antinociceptive effects of
the atypical SC O-1602 in a rat model of acute joint inammation (45). O-1602 may also act as an
agonist to GPR18, promoting calcium inux and triggering ERK1/2 phosphorylation in HEK293
cells overexpressing GPR18 (46).
The main signaling pathways activated by SCs are summarized in Figure 2.
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3.1. Modulation of Neurotransmitter Signaling
SCs mainly target the brain, modulating neurotransmitter signaling, among other processes.
Ossato et al. (47) showed that JWH-018 and AKB48 induced a CB1R activation-dependent psy-
chostimulant effect in mice by facilitating striatal and nucleus accumbens’ dopamine release. Since
the ventral tegmental area and the nucleus accumbens, and the portion of the medial forebrain
bundle that links both regions, are key structures of the brain’s reward circuitry, SC-induced
dopamine neural ring in these regions increases the reward response, accounting for these sub-
stances’ addictive potential (48). However, Ma et al. (49) observed that CB2R activation in mouse
ventral tegmental area slices by different CB2R-selective agonists (e.g., JWH-133) instead inhib-
ited dopamine neuron ring in a concentration-dependent manner. SCs have also been reported
to be more effective than 9-THC in inhibiting glutamatergic synaptic transmission (50). MAM-
2201 was shown to suppress glutamate and γ-aminobutyric acid (GABA) release in mice by acti-
vating presynaptic CB1Rs in Purkinje cells (51). WIN55,212-2 was shown to increase glutamate
uptake via the overexpression of glutamate transporter 1 (GLT1) and excitatory amino acid car-
rier 1 (EAAC1) in the rat frontal cerebral cortex (52). Additionally, Sánchez-Zavaleta et al. (53)
observed that the synthetic CB2R agonists GW833972A, GW405833, and JHW-133 inhibited
glutamate release by modulating P/Q channels in rat subthalamic-nigral terminals in a CB2R
activation–dependent manner.
SC exposure may also modulate serotonergic neurotransmission. For example, Yano et al. (54)
observed the positive allosteric modulation of the 5-HT1A receptor by SCs comprising an indole
moiety (e.g., JWH-018, AM-2201), indicating that such SCs may target serotonin receptors inde-
pendently of CBR activation. Bambico et al. (55) reported a decline in the ring rates of serotonin-
ergic neurons in the dorsal raphe nucleus of rats exposed to high WIN55,212-2 doses, also through
a CBR-independent mechanism. In contrast, at low doses, WIN55,212-2 enhanced serotonergic
neuronal activity and promoted dose-dependent antidepressant-like effects in the rat forced-swim
test, through a CB1R activation-dependent mechanism. Moreover, subchronic administration of
CP55,940 to male Sprague-Dawley rats has been shown to increase the membrane-associated ex-
pression of dopaminergic D2 and serotonergic 5-HT2A receptors, resulting in the formation of
5-HT2A-D2 receptor heterodimers, whose dysregulation has been suggested to contribute to the
pathophysiology of neuropsychiatric disorders (56).
4. PHARMACO- AND TOXICOKINETICS
Analysis of SCs’ pharmaco- and toxicokinetics is essential to (a) understand the extent (i.e., du-
ration and intensity) of the putative (noxious) effects users may experience and (b) determine the
time window for their detection in biological samples.
SCs are mainly consumed by inhalation (i.e., smoking), which leads to a rapid absorption by the
alveoli, resulting in fast peak blood concentrations, redistribution, and onset of effects (14, 16). For
example, Adamowicz et al. (57) observed a peak in JWH-018 and JWH-073 concentration 5 min
after smoking herbal incenses, which rapidly declined to less than a tenth of peak concentration
within the following 3 h. Although SCs may also be consumed as infusions, their oral adminis-
tration is not very common, which is possibly explained by the inconsistent absorption inherent
to this administration route, which depends on multiple factors (e.g., presence/absence of food
in the stomach, particularly fat; gastric pH; rst-pass effect). Evidence for the use of the intra-
venous route of administration remains scarce, although it is possible that high-risk marginalized
individuals may, at times, inject SCs (14, 16).
The high lipophilicity of most SCs anticipates their extensive binding to plasma proteins, which
may in turn result in increased distribution volumes (16, 58). Brandon et al. (59) determined that
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the log D7.4 of indole- and indazole-3-carboxamide SCs ranged from 2.81 for AB-FUBINACA
(least lipophilic) to 4.95 for MDMB-4en-PINACA (most lipophilic) among the SCs tested. Inter-
estingly,these in-silico predictions contrasted the experimental data, in which SCs with an indole
core scored higher log P values than SCs containing an indazole core.
Schaefer et al. (60) observed that the biodistribution of 200 μg/kg JWH-210 and RCS-4 ad-
ministered intravenously to pigs followed a three-compartment model, with central volumes of
distribution of 0.2 and 0.67 L/kg and clearances of 0.048 and 0.093 L/min/kg for JWH-210 and
RCS-4, respectively. These substances demonstrated similar patterns of distribution, with the ex-
ception of the lungs and the kidneys. The authors detected high levels of RCS-4 and low levels of
JWH-210 in the lungs and the opposite in the kidneys, which could possibly be due to the more
lipophilic nature of JWH-210 (compared to RCS-4), which favors distribution into tissues with
higher fat content (e.g., kidneys) and facilitates its elimination from the lungs (60, 61). Indeed,
high concentrations of both SCs (40–60 ng/g) were found in abdominal and perirenal adipose tis-
sue. Recently, a pulmonary dose in pigs of 200 μg/kg body weight (using a nebulizer) of the same
SCs showed that this administration route led to lower SC concentrations in certain tissues (e.g.,
cerebellum, muscle), compared to intravenous administration. The authors attributed this effect
to the distinct SC bioavailabilities, putative pulmonary uptake, and rst-pass metabolism, which
is more common following pulmonary administration (61).
The structure of SCs is a determining factor for metabolism. The high lipophilicity of most SCs
makes them prone to phase I (e.g., alkyl and aromatic oxidation) and phase II (e.g., glucuronida-
tion, sulfation) reactions before excretion (14, 16, 62). Oxidative metabolism generally leads to
the formation of mono-, di- and trihydroxylated metabolites. SCs possessing aromatic cores (e.g.,
7-azaindole, indole, indazole, and γ-carbolinone) have been reported to result in mono- and dehy-
droxylated, as well as dihydrodiol, metabolites (14, 16). Walle et al. (63) recently noted, in in vitro
(pooled human liver S9 fraction, pooled human liver microsomes, pig liver microsomes) and in vivo
(rat and pig) systems, that the 7-azaindole derivative CUMYL-5F-P7AICA underwent oxidative
deuorination, monohydroxylation, ketone formation, and carboxylation as the most prevalent
phase I reactions and detected the formation of sulfated and glucuronidated phase II metabo-
lites. Interestingly, the parent compound was still detected in all models. Franz et al. (62) observed
that the main metabolites of tert-leucine- and valine-derived SCs (e.g., 5F-MDMB-PINACA, AB-
CHMINACA) derived from four types of reactions: N-dealkylation, hydrolysis of the methyl ester
group or terminal amide, hydroxylation (of the core ring, amino acid moiety, or N-alkyl side chain),
and dehalogenation of 5-uoropentyl side chain. The authors thus postulated that (a) indazole-
containing compounds were more prone to undergo hydrolysis of the terminal functionality and
the secondary amide, as well as dihydrodiol formation, than their indole counterparts (more likely
to undergo N-dealkylation), and (b) a valine moiety provides greater susceptibility for hydrolysis of
the terminal functionality, whereas a tert-leucine group favors dehalogenation and hydroxylation
(of the amino acid moiety) (62). Supplemental Figure 1 summarizes the structural alterations
induced by phase I and phase II reactions, using CUMYL-5F-P7AICA and 5F-MDMB-PINACA
as examples.
Takayama et al. (64) described oxidation at the N-(1-amino-alkyl-1-oxobutan) moiety as the
main metabolic reaction of AB-FUBINACA in human liver microsomes. N-hydroxylation (mainly
at N-4 and N-5) and carboxylation were identied as the main reactions of MAM-2201 and JWH-
122 metabolism, both in vitro (human liver microsomes) and in vivo (human urine) (65). Some
SCs such as THJ-2201 have also been reported to undergo oxidative deuorination (66). A re-
cent study analyzed the metabolism of third generation SCs in zebrash larvae, observing that
ABD-CHMINACA and MDMB-CHMCZCA shared monohydroxylation as the main metabolic
pathway, whereas MMB-CHMINA mainly underwent O-demethylation (67).
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SCs are further transformed into more hydrophilic compounds to facilitate their renal excre-
tion through conjugation with sulfate and/or glucuronic acid. Notably, this near ubiquity of SC
metabolites in urine makes it the matrix of choice for SC detection. However, prior to analysis,
urine must be subjected to β-glucuronidase treatment to cleave conjugated metabolites (14, 16).
The metabolites derived from the acidic hydrolysis of AB-FUBINACA and AMB-FUBINACA,
for example, were shown to have a very long detection window (putative detection phase >1year),
so their detection in urine does not necessarily reect a recent consumption (68).
Interestingly,some SC metabolites have been shown to retain pharmacological activity. For ex-
ample, the hydroxypentyl metabolites of SCs like AB-PINACA, 5F-ADB, CUMYL-5F-PINACA,
and APINACA were reported to retain full efcacy at human CB1R and CB2R, despite their re-
duced potency and afnity compared to the respective parent compounds (69). On the other hand,
SC metabolism may lead to the formation of toxic metabolites. For example, carboxylesterase-
mediated hydrolysis of the ester bond present in some SCs (e.g., XLR-11) may cause the blood
accumulation of a metabolite with toxicological mechanisms similar to those of N-acetyl-p-
benzoquinone imine (acetaminophen metabolite) (70).
5. BIOLOGICAL AND TOXICOLOGICAL EFFECTS OF SCs
The diversity and extent of effects induced by SCs are inuenced by factors such as the route of
administration; the user’s vulnerability; and the dose, lipophilicity, and pharmacological properties
of the drug (71, 72).
SC consumers often search for some of the drug’s known psychotropic effects, including in-
creased relaxation, elevated well-being, and social disinhibition, which arise quickly after con-
sumption (73). However, SC-related adverse effects often develop, mostly due to (a) biotrans-
formation into toxic metabolites, (b) by-products of pyrolysis of smoked herbal blends, and/or
(c) drug-drug interactions (e.g., shared metabolic pathways with frequently used medications).
Notably, the contribution of SCs per se to the onset of adverse effects remains undetermined
(74). Similarly to their psychoactive effects, adverse symptoms may also appear minutes or hours
after consumption, and last from minutes to several hours (11, 75). These symptoms, which are
summarized in Figure 3, can be grouped according to the target systems, that is, cardiovascular,
digestive, dermatological, ophthalmological, neurological, pulmonary, and hepatic (72, 75, 76).
Acute intoxications have been particularly associated with neurological perturbations, includ-
ing cognitive impairment, such as short-term memory loss, ashbacks, and suicidal ideation (11).
Other neurological symptoms include delirium, confusion, hallucinations, agitation, panic attacks,
and convulsions that have been associated with the use of SCs of the JWH series or AM-694
(77). Chronic SC consumption has also been correlated with an increased risk of developing
neuropsychiatric disorders (78). Psychotic symptoms are frequent following SC use. Although
these are usually temporary (only lasting a few hours), SCs may lead to prolonged psychotic
episodes both in vulnerable subjects and individuals with no previous history of psychosis (6,
77). Novel third-generation uorinated SCs, 5F-ADBINACA, AB-FUBINACA, and STS-135,
were shown to cause hypothermia and increased pain threshold to noxious mechanical and
thermal stimuli, besides reducing motor activity and impairing sensorimotor responses in mice
(79). SCs also target the human cardiovascular system, reportedly causing increased heart rate,
tachycardia, and in the most severe cases, myocardial infarction or stroke (77). A series of case
reports associated with 5-uoro-ADB use have also provided evidence for cardiomegaly as an
SC-related effect in humans (80).
Severe intoxications have also been associated with rhabdomyolysis and liver and kidney
toxicity and failure (81, 82). Notably, acute tubular necrosis has been reported in the biopsies
of individuals that had consumed XLR-11 (83), leading the Centre for Disease Prevention and
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Cannabinoid
hyperemesis
syndrome:
a rare condition
causing recurrent,
severe vomiting in
daily long-term
cannabis users
Renal
Nephrotoxicity,
renal failure
Muscular
Rhabdomyolysis
Neurological
Ataxia, agitation, confusion, disturbances in
consciousness and memory, altered
concentration, drowsiness, dizziness, tremor,
headache, insomnia, hyperactivity, irritability,
mood swings, anxiety, aggressiveness, panic
attacks, euphoria or dysphoria, suicidal
ideation, hallucinations, convulsions,
psychosis, paranoia, schizophrenia, altered
appetite, dysphagia, polydipsia
Pulmonary
Cough, hyperventilation or apnea,
shortness of breath, diuse pulmonary
inltrates, alveolar inltrates, pneumonia
Hepatic
Hepatotoxicity, liver
failure
Digestive
Nausea, vomiting, abdominal
pain, diarrhea
Ophthalmology
Changes in pupils (mydriasis),
conjunctival hyperemia (redness),
photosensitivity, blurry vision
Dermatological
Premature skin aging, acne,
early baldness
Cardiovascular
Tachycardia, arrhythmias, myocardial
infarction, hemorrhage, hypertension or
hypotension, palpitations, chest pain,
bradycardia, syncope, coronary artery
thrombosis
Figure 3
Summary of the most common adverse effects associated with the use of synthetic cannabinoids (SCs). In view of the wide distribution
of cannabinoid receptors throughout the body, SCs may target different organs and trigger adverse effects at cardiovascular, digestive,
dermatological, ophthalmological, neurological, pulmonary, and hepatic systems.
Control (CDC, USA) to establish a direct link between XLR-11 and acute kidney injury (84).
Lung injuries (e.g., pneumothorax, pneumomediastinum) are also frequent and may be attributed
to local injuries caused either directly by the SCs or by impurities in the SC mixtures (10), with
patients often requiring oxygen supplementation (85).
Hopkins & Gilchrist (86) described the rst case of cannabinoid hyperemesis syndrome caused
by SCs in a patient whose urine revealed the presence of JWH-018, JWH-073, and AM-2201.
Examples of clinical cases reporting SC-related poisonings are summarized in Supplemental
Table 1.
SC withdrawal may also cause adverse symptoms, including restlessness, headache, irritabil-
ity, drug craving, high blood pressure, nausea, tremors, diaphoresis, and nightmares (10, 11, 87).
Seizures and cardiovascular arrest may also occur in more severe cases (73, 88).
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Consumption of SCs, either alone or in combination with other recreational/prescription
drugs, has also been associated with fatal intoxications, mainly attributed to poisoning, cardiac
arrest, asphyxiation, multiple organ failure, suicide, or traumatic accident (73, 89, 90). Acyl in-
doles, indole carboxylates, and indazole carboxamides are the SC classes most implicated in death
reports (13, 90). For example, MAB-CHMINACA, AMB-FUBINACA, MDMB-CHMICA, and
4F-MDMB-BICA have been associated with major death outbreaks in the United States and
Europe (12, 13, 89, 91). Nevertheless, it is often difcult to establish a direct correlation between
an SC and the cause of death, since (a) the lack of proper reference standards usually prevents the
correct identication and quantication of SCs present in biological samples, and (b) postmortem
blood concentrations may vary depending on the type of sample, individual idiosyncrasies, and
time elapsed since death (92).
Most mild SC intoxications only require ambulatory symptomatic treatment, whereas severe
intoxications (e.g., those involving seizures, severe agitation, neuropsychiatric perturbations, ar-
rhythmias, stroke, thoracic pain) often lead to hospitalization (93). Acute SC intoxication treat-
ment usually includes intensive monitoring and supportive therapy (10, 82, 94, 95). Intravenous
uids are often administered to expand the volume of the circulatory system, control vomiting,
and prevent dehydration and renal failure.
Sedation with benzodiazepines (e.g., lorazepam) is the rst-line treatment to reduce anxiety
and agitation, though psychiatric evaluation and antipsychotics administration are often necessary
(95, 96). Intubation and mechanical ventilation may be required in severe cases (10).In case of per
os administration, gastric lavage and ingestion of activated carbon may be required depending
on the amount of SC ingested and the time elapsed after intake (88). Recently, Aksel et al. (97)
described a new treatment for SC intoxications, referred to as intravenous lipid emulsion (ILE),
which showed promising results as an effective antidote in poisonings by lipophilic drugs like
SCs, allowing recovery from cardiovascular collapse and reversing the neurological symptoms
caused by these drugs. ILE retains drugs found in the intravascular space and distributes the fat-
soluble drugs to a circulating phase, reducing their concentration and toxicity. Treatment for SC
withdrawal symptoms is based on benzodiazepines, antiemetics, and other symptomatic care (94).
6. MOLECULAR MECHANISMS OF TOXICITY
Some of the mechanisms underlying SC toxicity have already been evidenced. For example,
Tomiyama et al. (98) observed that eight SCs (e.g., CP series, JWH series) induced apoptosis
(indicated by caspase-3 activation and a high count of annexin-V-positive cells) of cultured
mouse forebrain neurons after 2 h. These effects were CB1R (but not CB2R) activation– and
concentration–dependent (up to 30 μM). Similarly, MAM-2201-induced toxicity to human
primary neuron-like cells and astrocytes (D384 cell line) was time (3–48 h) and concentration (1–
30 μM) dependent and had a signicant impact on cell viability and morphology, metabolic
function, expression of neuronal markers, and apoptosis, which was only noted above 5 μM
MAM-2201 (99). AB-FUBINACA, 5F-ADBINACA, and STS-135 have been reported to de-
crease the mitochondrial membrane potential in the murine neuroblastoma Neuro-2a cells at
60 μM, although STS-135 neurotoxic effects manifested starting at 3 μM after 1 h (79). Exposure
of neuroblastoma SH-SY5Y cells to 100 and 200 μM APINACA for 24 h resulted in excessive
reactive oxygen species (ROS) formation. Furthermore, 25 μM APINACA increased messenger
RNA levels of CB1R and MAPK8, whereas at 100 and 200 μM, APINACA increased interleukin
6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels (100). Recently, Sezer et al. (101) also
observed an increase in oxidative stress (e.g., reduction of glutathione reductase and catalase
activities, increased lipid peroxidation and protein carbonylation) in SH-SY5Y cells exposed for
24 h to JWH-018 at concentrations between 5 and 150 μM.
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Neurogenesis: the set
of processes (e.g.,
differentiation,
migration, maturation)
leading to the
formation of new
neurons
Notably, most of these studies assessed the cytotoxicity of SCs at high micromolar concentra-
tions. However, cannabinoids generally exhibit nanomolar afnities at CB1R and CB2R, and it is
broadly accepted that testing them at concentrations higher than 1 μM (three orders of magnitude
above their afnity) is likely to induce off-target effects (101). Alexandre et al. (102), for example,
have shown that THJ-2201 and 5F-PB22 at biologically relevant concentrations (≤1μM) inter-
fered with neuronal differentiation but did not compromise metabolic activity,lysosomal or plasma
membrane integrity, or intracellular ATP levels of undifferentiated NG108-15 neuroblastoma X
glioma cells after 24 h. However, the authors observed an increase in mitochondrial membrane
potential for 1 pM THJ-2201 and 1 μM 5F-PB22, suggesting an impairment of mitochondrial
activity. Hebert-Chatelain et al. (103) have demonstrated that 1 μM HU-210 reduced mitochon-
drial mobility in mice hippocampal neurons via activation of mitochondrial CB1Rs, which was
correlated with the animal’s memory impairment.
SCs may also elicit toxic effects on other organs, especially those involved in their metabolism
and/or excretion. For example, Silva et al. (104, 105) demonstrated that, after 3 h, XLR-11, JWH-
122, and THJ-2201 primarily targeted mitochondria in human proximal tubular (HK-2) cells, at
biologically relevant concentrations (≤1μM). These SCs induced a transient mitochondrial mem-
brane hyperpolarization and increased intracellular ATP levels, as well as chromatin condensation
and caspase-3 activation, suggesting the activation of apoptotic pathways. Moreover, these events
depended on CB1R and/or CB2R activation (104). Koller et al. (106) reported that CP-47,497-C8
at concentrations higher than 7.5 μM for 24h impaired mitochondrial function, protein synthe-
sis, and lysosomal activity and caused cell membrane damage in hepatic (HepG2)- and buccal
(TR146)-derived cell lines. Moreover, CP-47,497-C8 induced DNA damage, as evidenced by the
high number of single- and double-strand breaks (at 10 μM for TR146 cells and ≥15 μMfor
HepG2 cells). 5F-EMB-PINACA, JWH-200, and A-796260, for 48–72 h, were also shown to be
hepatotoxic (HepG2 cells) at 7.8 and 125 μM, impacting cell number, plasma membrane integrity,
nuclear size, cytosolic calcium levels, and mitochondrial membrane potential (107). Furthermore,
CP-55,940 induced a concentration-dependent (5–50 μM) decrease in cell viability of human em-
bryonic rhabdomyosarcoma cells, an effect accompanied by loss of mitochondrial membrane po-
tential, caspase-3 activation, and annexin V accumulation (for 0.5–2 h), thus evidencing apoptosis
induction (108).
After 48 h, UR-144, JWH-122, and JWH-018 were shown to disrupt the plasma membrane
and cause cell cycle arrest in the G2/M phase of human placental cytotrophoblast cells (109).
Additionally,JWH-122 and JWH-018 increased ROS formation, and UR-144 and JWH-122 re-
duced mitochondrial membrane potential. Interestingly, while all these SCs were shown to in-
crease caspase-9, -3, and -7 activities, the cytotoxic effects of UR-144 appeared to solely depend
on CB1R activation, both CB1R and CB2R seemed to mediate the effects of JWH-018, and JWH-
122’s toxicity was shown to be CBR independent.
6.1. Impact of SCs on Neuronal Development
The impact of SC use on neurodevelopment represents a core issue, as adolescents and young
adults (which include women of child-bearing age/pregnant women) are the main SC users (110).
SCs modulate the endocannabinoid system, which is in turn involved in several biological pro-
cesses, including cell fate and neurogenesis-related mechanisms (e.g., neuronal differentiation,
migration, maturation, synaptic pruning) in a well-dened spatiotemporal manner, from the ear-
liest stages of ontogenetic development to late adolescence (110, 111). The developing brain is thus
especially vulnerable to SC-elicited effects, which may alter the neural circuitry and trigger the on-
set of neurodevelopmental disorders (e.g., psychoses, autism spectrum) (110, 111). Moreover, SCs
can easily cross the placental barrier into embryonic tissues due to their high lipophilicity (112).
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The link between pre- and postnatal exposure to SCs and neurogenesis dysfunction has been
strongly supported by preclinical studies. For example, Mereu et al. (113) showed that the daily
administration of the CB1R agonist WIN55,212-2 (0.5 mg/kg) to pregnant rats caused the im-
pairment of memory retention capacities in their offspring at 40 and 80 days old. These effects
were accompanied by a reduction in hippocampal presynaptic glutamate release and alterations
of hippocampal long-term potentiation, which is associated with learning and memory consolida-
tion. Recently, Pinky et al. (114) reported that administration of the same SC (WIN55,212-2) at
2 mg/kg body weight per day to pregnant rats signicantly altered the levels of several biochem-
ical markers of the adolescent offspring, including a reduction of oxidative stress and apoptotic
marker levels and an increase of mitochondrial function in the cerebellum (a brain region playing
an important role in learning and motor function). Interestingly, while GluA1 levels (a major glu-
tamate receptor subtype) and tyrosine hydroxylase activity remained unaffected, total monoamine
oxidase (MAO) activity signicantly declined in the cerebellum, supporting the notion that SCs
affect monoamine neurotransmitter levels in this brain region.
Multiple in vitro studies have also uncovered the key role played by CBR stimulation in the
SC-mediated modulation of neurogenic processes (115, 116). For example, Kim et al. (117) ob-
served that 300 nM WIN55,212-2 markedly prevented new synapse formation in rat hippocam-
pal neurons obtained from 17-day-old embryos by inhibiting the forskolin-induced cAMP in-
crease. Remarkably, WIN55,212-2 did not block the effects evoked by a membrane-permeant
cAMP analog, suggesting that it inhibited new synapse formation by preventing cAMP synthesis
rather than cAMP actions (e.g., neurotransmitter release). Jordan et al. (118) reported a signi-
cant concentration-dependent increase of neurite outgrowth in Neuro-2A cells exposed to up to
10 μM HU-210, which was mediated by CB1R and Rap1 signaling. Jiang et al. (119) reported that
chronic (but not acute) treatment of hippocampal neural stem cells isolated from embryos of E17
Long Evans rats with 100 μg/kg HU-210 promoted neuronal proliferation, but not differentia-
tion, through ERK pathway activation, associating this effect with anxiolytic- and antidepressant-
like effects of HU-210. Recently, Alexandre et al. (102) described that THJ-2201 and 5F-PB22
increased differentiation ratios and total neurite length of NG108-15 cells in a CB1R activation-
dependent manner, at 1 pM and 1 nM.
Notably, the CB2R also seems to play a key role in neurodevelopment-related processes. For
example, Palazuelos et al. (120) reported that HU-308 (a selective CB2R agonist) induced the
proliferation of rat HiB5 hippocampal progenitor cells via stimulation of CB2Rs and subsequent
activation of the PI3K/Akt/mTOR1 pathway. Oudin et al. (116) observed that 1 μM JWH-133
promoted neuronal cell migration of COR-1 neural stem cells and rostral migratory stream neu-
roblasts via CB2R activation. Miranda et al. (121) showed that chronic exposure to THJ-018 and
EG-018 during neurogenesis promoted a premature neuronal and glial differentiation of human-
induced pluripotent stem cells that led to abnormal functioning of voltage-gated calcium channels
of newborn neurons when stimulated by extracellular potassium.
Some studies have highlighted the importance of SCs’ interaction with the brain-derived
neurotrophic factor (BDNF), a neuroplasticity modulator involved in the regulation of neu-
ronal differentiation, maturation, and survival (122). Recently, Ferreira et al. (123) showed that
WIN55,212-2 regulated adult neurogenesis, as exposure of subventricular zone (SVZ) and den-
tate gyrus (DG) neurosphere cultures from young (postnatal days 1–3) rats to this SC increased
cell proliferation in the DG but not in the SVZ. This process seemed to be modulated indepen-
dently of CBR2 activation, as the selective CB2R agonist HU-308 did not interfere with neuronal
cell proliferation in either neurogenic niche. Nevertheless, WIN55,212-2 and HU-308 promoted
cell differentiation in both DG and SVZ.
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Epigenetic
modications:
modications to gene
function, without any
alteration in DNA
sequence, that are
mitotically or
meiotically heritable
Li et al. (124) detected a CB1R-mediated reduction of BDNF levels in the hippocampus,
which was associated with memory impairment, in adult mice administered JWH-018 (1mg/kg,
intraperitoneal). Notably, these changes correlated with an increased content of the endocannabi-
noids anandamide and 2-arachydonyl glycerol, suggesting that SCs may inuence BDNF suppres-
sion and neurogenesis-related processes by disrupting endocannabinoid-mediated homeostasis.
Assessment of the outcome of pre- and postnatal SC exposure to human neurodevelopment
is substantially challenging since (a) the cognitive, motor, and behavioral parameters can only be
evaluated in retrospect (121); and (b) several confounding factors can introduce major variations to
the outcome, hampering the isolation of the direct consequences of SC use without interpretation
bias (125, 126). Data on perinatal SC-related toxicity are thus restricted to just a few case studies
reporting the absence of mortality or morbidity features in newborns (127).
6.2. Epigenetic Modulation
Epigenetic-elicited disruptions have been reported in the brain and peripheral organs after 9-
THC exposure (128), but thus far only a few studies have reported the epigenetic mechanistic
outcomes of SC exposure (129). For example, Ibn Lahmar Andaloussi et al. (130) observed an in-
crease in global DNA methylation and transcription of DNA methyltransferases 1 (DNMT1) and
3 (DNMT3) in the prefrontal cortex of adolescent male rats exposed for one week to WIN55,212-
2, suggesting that these epigenetic modications contributed to the anxiogenic-like effects ob-
served in the exposed mice, as well as in their offspring. Scherma et al. (131) observed histone
hyperacetylation and reduced histone deacetylase 6 (HDAC6) levels in the prefrontal cortex of
adolescent rats exposed to WIN55,212-2 for 11 days, which was found to enhance the brain’s ini-
tial molecular and epigenetic response to cocaine in adult rats. Aguado et al. (132) demonstrated
that HU-210 and JWH-133 (selective CB1R and CB2R agonists, respectively) modulated glioma
cell differentiation by increasing histone methylation (increased H3K9me3-positive cells) in a
CBR activation–dependent manner. Tomas-Roig et al. (133) observed that long-term administra-
tion of WIN55,212-2 during rats’ adolescence increased anandamide levels and promoted DNA
hypermethylation at the intragenic region of the intracellular signaling modulator Rgs7 (an intra-
cellular antagonist of GPCR signaling), which was found to alter Rgs7 expression in adulthood.
Administration of HU-210 to female rats during pregnancy and to their offspring for 14 days after
postnatal day 35 was shown to modify the expression of microRNA in the left hemisphere of the
entorhinal cortex, a brain area associated with schizophrenia (134).
7. THERAPEUTIC POTENTIAL OF SCs
Accumulating ndings have suggested the potential of the endocannabinoid system as a ther-
apeutic target, leading to the exploitation of SCs as candidate agents to treat several disorders
(135). In fact, the synthetic analogs of 9-THC dronabinol and nabilone (Marinol and Cesamet,
respectively) have been approved by the US Food and Drug Administration to treat
chemotherapy-associated nausea and vomiting in cancer patients, after rst-line antiemetics have
failed, or as adjunct analgesics to alleviate chronic pain (136). Also, there are already marketed
nabiximols (standardized combination of equal amounts of synthetic 9-THC and cannabidiol),
namely Sativex, which have shown moderate evidence to treat spasticity associated with multi-
ple sclerosis (135). Nevertheless, most efforts to develop SC-based therapeutic agents have been
discontinued, mainly due to the CB1R activation–related adverse events triggered at the central
nervous system (111).
SCs’ ability to bind CB2Rs has suggested the safe targeting of the endocannabinoid system due
to their potential to modulate inammatory processes. For example, WIN55,212-2 was shown
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to suppress nitric oxide production; TNF-αrelease; and the formation of CXCL10, CCL2, and
CCL5 chemokines in IL-1-stimulated astrocytes (137). Similarly, Aguirre-Rueda et al. (138) ob-
served that WIN55,212-2 reverted the increased oxidative stress, inammatory response, and cell
viability loss of primary cultured astrocytes elicited by Aβ1–42. However, the discovery of the endo-
cannabinoidome has further increased the complexity of signaling events triggered by SCs, thus
limiting their potential therapeutic use (111).
Most importantly,there is still scarce evidence supporting the use of cannabinoid-based prod-
ucts for most suggested therapeutic applications. As such, the potential benets arising from SC
use are outweighed by their associated risks, thus deterring their medical use.
8. CONCLUSIONS
Research on the biological relevance of the endocannabinoid system has greatly expanded over
the past years, with SCs playing a major role as research tools to help understand how this sys-
tem regulates key biological processes. Nevertheless, the widespread recreational use of SCs has
become a major public health and social concern.
The ability of SCs to interact with CBRs (i.e., CB1R, CB2R) as well as with non-CBRs (e.g.,
TRPV, GPR55, PPARs, 5-HT receptors), along with the biased agonism of SCs upon binding to
CBRs, increases the complexity of the signaling pathway network modulated by these substances
and hampers the understanding of such signaling modulation.
Moreover, as SCs’ targets are widely distributed throughout the body, their action and adverse
outcomes extend to all major organs and tissues. Most importantly, the scarce information avail-
able on SCs’ toxicological signatures is often equivocal, as (a) toxic effects may be associated with
the presence of other toxic substances in the SC herbal blends, besides the SC itself; (b) several
confounding factors (e.g., genetic, environmental, frequency/type of SC used) may inuence their
action; (c) in vitro effects vary according to the cell model (e.g., primary cultures versus cell lines,
species of origin) and experimental design (e.g., concentration, timepoint, exposure protocol);
and (d) only a few studies have addressed the toxicological effects of SCs at biologically relevant
concentrations.
SC-mediated modulation of mitochondrial function and activity and apoptotic signaling induc-
tion, for example, have been proposed as important mechanisms underlying the toxicity of these
substances. Additionally, SC-associated perturbations in neurogenesis are likely to contribute to
the onset of neurodevelopmental/neuropsychiatric disorders, which is especially alarming consid-
ering that adolescents and young adults are the main SC users. SCs may also interfere with the
epigenetic machinery and promote epigenetic changes that may predispose individuals to distinct
pathologies that can then be inherited by their progeny.
Interestingly, although the therapeutic value of SCs has been evidenced by the clinical use of
synthetic 9-THC analogs to treat nausea and vomiting in patients undergoing chemotherapy,
their potential use for other therapeutic applications still lacks scientic evidence. Further re-
search is crucial to understand the pharmacological and toxicological mechanisms underlying the
short- and long-term consequences of SC use and how these may condition consumers’ health and
quality of life, as well as to improve the interpretation of SC-related clinical/pathologica ndings.
SUMMARY POINTS
1. Synthetic cannabinoids (SCs) are designed to mimic the action of 9-tetrahydro-
cannabinol (9-THC) but with stronger potency and efcacy at cannabinoid receptors.
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2. Recreational SC use is globally widespread and is often associated with reports of acute
intoxications and deaths.
3. SCs target both cannabinoid and non-cannabinoid receptors, triggering an intricate net-
work of signaling pathways that contribute to the modulation of key biological processes.
4. Timely detection and quantication of SCs in biological samples remains a challenge
for forensic toxicologists/pathologists, considering their high metabolic rate and lack of
proper reference standards for parent compounds and respective metabolites.
5. SCs induce a plethora of adverse outcomes at different organ systems that are more
severe and longer-lasting than those induced by 9-THC.
6. Chronic SC use and/or use by particularly vulnerable groups (e.g., adolescents and young
adults) may promote the onset of neurodevelopmental/neuropsychiatric disorders (e.g.,
psychosis, autism spectrum) in the long-term by, for example, disrupting proper neuro-
genesis or inducing epigenetic changes.
7. SCs have been proposed as candidate agents for a few different therapeutic applications,
but there is scarce evidence of their therapeutic potential, which is currently limited to
chemotherapy-associated nausea and vomiting treatment.
8. Further research is required to clarify the main mechanisms underlying SC-mediated
short- and long-term effects, which will hopefully reduce SC misuse by high-risk groups.
FUTURE ISSUES
1. Identication of major molecular initiating events and other key events triggered by SCs
is crucial to establish the pathways leading to the main SC-related adverse outcomes.
2. Research into the mechanisms of SC-related toxicity has mostly focused on naphthoylin-
doles (e.g., JWH series, AM-2201), and further research is required to understand the
toxicological proles of other SC classes (e.g., indole- and indazole-based SCs, cumyl
derivatives).
3. The long-term impact of SC use remains to be addressed, since thus far no clinical studies
have followed the long-term behavioral, cognitive, or mood changes following acute and
chronic SC use.
4. The ability of SCs to interfere with the epigenetic machinery, thus promoting
epigenetic modications (e.g., DNA methylation, histone methylation/acetylation/
phosphorylation) needs to be further addressed, as it could explain some of the long-
term effects of SC use.
5. The specic contribution of SCs to the onset of neuropsychiatric disorders remains un-
clear and is often equivocal, requiring a more in-depth understanding of how SCs may
dysregulate the mechanisms underlying key biological processes such as neurogenesis.
6. Development of new SCs deprived of the noxious cannabinoid receptor 1–associated
psychoactive effects and long-term toxicity, but maintaining some of their potential ben-
ecial actions (e.g., anti-inammatory), could represent a major advance for the estab-
lishment of SCs as interesting candidate therapeutic agents.
. Roque-Bravo et al.
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DISCLOSURE STATEMENT
The authors are not aware of any afliations, memberships, funding, or nancial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
This work was funded by FEDER (Fundo Europeu de Desenvolvimento Regional) through
COMPETE 2020 (Operational Programme for Competitiveness and Internationalization) and
by Portuguese funds through FCT (Fundação para a Ciência e a Tecnologia). in the framework of the
projects POCI-01–0145-FEDER-029584; UIDP/04378/2021 and UIDB/04378/2021 of the Re-
search Unit on Applied Molecular Biosciences (UCIBIO); and LA/P/0140/2021 of the Associate
Laboratory Institute for Health and Bioeconomy (i4HB). R.R.-B., R.S.S., and R.F.M. acknowledge
FCT for the PhD grants 2020.04493.BD, 2020.07154.BD, and 2020.07135.BD, respectively.
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