Evaluation of carboxamide-type synthetic cannabinoids on the functional activities at cannabinoid receptors and biological effects via inhalation exposure test

Article abstract of DOI:10.1007/s11419-020-00534-x

Purpose: Three synthetic carboxamide-type cannabinoids (5F-MDMB-PICA, 5F-EMB-PINACA, and AMB-CHMICA) were evaluated in terms of their in vitro activities at the cannabinoid receptors CB₁ and CB₂ and in vivo biological effects when smoking the synthetic cannabinoids to assess their biological effects. Methods: [³⁵S]Guanosine-5′-O-(3-thio)-triphosphate binding assays were performed to investigate the half maximal effective concentration values of the test compounds at the CB₁ and CB₂ receptors. Additionally, the biological effects were evaluated by observing and scoring the behavior of mice with equipment in which they inhaled smoke from a herbal mixture containing the test compounds. Results: All three synthetic cannabinoids tested in this study activated the CB₁ and CB₂ receptors in vitro. 5F-MDMB-PICA showed less than 1?nM of the half maximal effective concentration value for both receptors. Therefore, it was suggested that 5F-MDMB-PICA was the strongest CB₁ and CB₂ receptor agonist in comparison with synthetic cannabinoids evaluated in the past. The degree of the various biological effects, specifically passivity, spontaneous activity, abnormal gait, abnormal position, and grip strength, when smoking the synthetic cannabinoids corresponded to the functional activity at the CB₁ receptor. However, some biological effects differed between 5F-MDMB-PICA and 5F-MDMB-PINACA, used as a positive control, and AMB-CHMICA induced some biological effects in contrast to the other tested synthetic cannabinoids. Conclusion: This study provides information regarding the biological effects when smoking synthetic cannabinoids from the functional activities at the CB₁ and CB₂ receptors, considering their way of inhalation and thermal degradation.

Full text of DOI:10.1007/s11419-020-00534-x

Forensic Toxicology
https://doi.org/10.1007/s11419-020-00534-x
ORIGINAL ARTICLE
Evaluation of carboxamide‑type synthetic cannabinoids
on the functional activities at cannabinoid receptors and biological
efects via inhalation exposure test
Akihiro Takeda1 · Takahiro Doi1 · Akiko Asada1 · Toshinari Suzuki2 · Katsuhiro Yuzawa2 · Hiroshi Ando2 ·
Yoshikazu Kubo2 · Akemichi Nagasawa2 · Fujifumi Kaihoko2 · Yuko Hasegawa2 · Kazuyoshi Tanaka2 · Kai Igarashi3 ·
Tomokazu Maeno2 · Atsuko Suzuki2 · Seiko Shimizu2 · Nozomi Uemura2 · Jun’ichi Nakajima2 · Jin Suzuki2 ·
Takaomi Tagami1
Received: 7 November 2019 / Accepted: 7 April 2020
© Japanese Association of Forensic Toxicology 2020
Abstract
Purpose Three synthetic carboxamide-type cannabinoids (5F-MDMB-PICA, 5F-EMB-PINACA, and AMB-CHMICA) were
evaluated in terms of their in vitro activities at the cannabinoid receptors CB1 and CB2 and in vivo biological efects when
smoking the synthetic cannabinoids to assess their biological efects.
Methods [35S]Guanosine-5′-O-(3-thio)-triphosphate binding assays were performed to investigate the half maximal efective
concentration values of the test compounds at the CB1 and CB2 receptors. Additionally, the biological efects were evaluated
by observing and scoring the behavior of mice with equipment in which they inhaled smoke from a herbal mixture contain-
ing the test compounds.
Results All three synthetic cannabinoids tested in this study activated the CB1 and CB2 receptors in vitro. 5F-MDMB-PICA
showed less than 1 nM of the half maximal efective concentration value for both receptors. Therefore, it was suggested that
5F-MDMB-PICA was the strongest CB1 and CB2 receptor agonist in comparison with synthetic cannabinoids evaluated in
the past. The degree of the various biological efects, specifcally passivity, spontaneous activity, abnormal gait, abnormal
position, and grip strength, when smoking the synthetic cannabinoids corresponded to the functional activity at the CB1
receptor. However, some biological efects difered between 5F-MDMB-PICA and 5F-MDMB-PINACA, used as a positive
control, and AMB-CHMICA induced some biological efects in contrast to the other tested synthetic cannabinoids.
Conclusion This study provides information regarding the biological efects when smoking synthetic cannabinoids from the
functional activities at the CB1 and CB2 receptors, considering their way of inhalation and thermal degradation.
Keywords NPS · Carboxamide-type synthetic cannabinoid · 5F-MDMB-PICA · 5F-EMB-PINACA · AMB-CHMICA ·
Activities at CB1 and CB2 receptors · Inhalation exposure test
Introduction
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11419-020-00534-x) contains
supplementary material, which is available to authorized users.
Synthetic cannabinoids (SCs) are one of the most prevalent
groups of new psychoactive substances (NPS) and have been
defned as substances of abuse that are not controlled by
the 1961 Single Convention on Narcotic Drugs or the 1971
Convention on Psychotropic Substances [1, 2]. Products pro-
duced by mixing SCs with plant material or spraying plant
material with SC solution have been distributed in drug mar-
kets. The governments of various nations have regulated SCs
that have been detected in products, but the illicit manufac-
turers of NPS have synthesized new SCs to circumvent the
regulation, and thus, various SCs have globally emerged and
* Akihiro Takeda
takeda@iph.osaka.jp
1
Division of Hygienic Chemistry, Osaka Institute of Public
Health, 1-3-69 Nakamichi, Higashinari-ku, Osaka 537-0025,
Japan
2
Department of Pharmaceutical and Environmental Sciences,
Tokyo Metropolitan Institute of Public Health, 3-24-1
Hyakunin-cho, Shinjuku-ku, Tokyo 169-0073, Japan
3
Tokyo Metropolitan Island Public Health Center, 2466-2
Okago, Hachijo-machi, Hachijojima, Tokyo 100-1492, Japan
Vol.:(0123456789)
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Forensic Toxicology
prevailed. The number of new SCs detected in various coun-
tries reached a peak in 2014 and has subsequently decreased.
Nevertheless, the prevalence of abuse of SCs has continued,
and many fatal cases have been reported [3].
the biological efects of the SCs [18, 19]. The evaluation
of SCs using these techniques is expected to accurately
assess the risk of the SCs against human body by consider-
ing the way of inhalation and thermal degradation, and to
estimate the efects on users by comparing the results of
the compounds previously investigated before they were
considered as threat to public health.
SCs are strong cannabinoid receptor type 1 (CB1 receptor)
and cannabinoid receptor type 2 (CB2 receptor) agonists and
can be classifed as naphthoylindoles (e.g., JWH-018), naph-
thoylindazoles (e.g., THJ-018), indazole carboxamides (e.g.,
ADB-PINACA), or indole carboxamides (e.g., ADBICA) in
terms of their chemical structures. In this work, we classifed
the above indazole and indole carboxamides as carboxam-
ide-type SCs. This class of SCs is derived by substituting
an amino acid ester for a naphthalene ring in the previous
generation naphthoylindoles and naphthoylindazoles. The
basic chemical structure of the class of SCs was described
in a patent by Pfzer in 2009 [4]. Carboxamide-type SCs
have been associated with several serious accidents. In 2014,
AMB-FUBINACA was found in Sweden [5] and Louisiana
(United States) and the compound caused intoxication of 33
people in New York City in 2016. The mass media reported
the intoxication as a “zombie” outbreak because of the
appearance of the intoxicated people [6]. In 2014, in Japan,
21 reported trafc collisions were related to 5F-AMB, which
injured or killed many people [7]. 5F-MDMB-PINACA also
caused 24 reported trafc collisions which, fortunately, were
not serious accidents [7]. However, in 2014, 5F-MDMB-
PINACA caused approximately ten smoking-related deaths
in Japan [8]. These SCs have been regulated in each country;
however, 5F-MDMB-PINACA and AMB-FUBINACA were
the most seized NPS in 2017 and 2018 [3]. Therefore, it has
been suggested that these SCs have continued to be prevalent
around the world.
Materials and methods
Reagents
Methyl 1H-indole-3-carboxylate and (bromomethyl)
cyclohexane were purchased from Sigma-Aldrich Co. LLC
(St. Louis, MO, USA). Methyl 1H-indazole-3-carboxylate
and d-tert-leucine methyl ester hydrochloride were pur-
chased from Accela ChemBio Co., Ltd. (San Diego, CA,
USA), 1-bromo-5-fuoropentane from Fluorochem Ltd.
(Hadfeld, UK), l-tert-leucine methyl ester hydrochlo-
ride and d-valine methyl ester hydrochloride from Tokyo
Chemical Industry Co., Ltd. (Tokyo, Japan), and l-valine
methyl ester hydrochloride was purchased from FUJI-
FILM Wako Pure Chemical Corporation (Osaka, Japan).
All other reagents used in this study were purchased from
FUJIFILM Wako Pure Chemical Corporation. Purifed
water was obtained from tap water using the Elix Advan-
tage-10 from Merck Millipore (Burlington, MA, USA).
Chemical synthesis
5F-MDMB-PICA is classifed as a carboxamide-type SC.
This SC was found in Germany in 2016 [9, 10]. Risseeuw M.
D. P. et al. discovered the SC in a powder form in an unla-
beled product and 27 herbal incense products in Belgium
[11]. Banister et al. evaluated 5F-MDMB-PICA regarding
the functional activity at the CB1 and CB2 receptors and
found that the half maximal efective concentration (EC50)
value for the CB1 receptor was 0.45 nM [12]. The results
showed that 5F-MDMB-PICA was a strong CB1 receptor
agonist and is, therefore, considered a public health threat.
Therefore, we targeted the carboxamide-type SCs that
have been distributed in Europe. We investigated the bio-
logical efects of 5F-MDMB-PICA, as well as 5F-EMB-
PINACA and AMB-CHMICA, which have been detected
in Sweden and Slovenia in 2015, respectively [13]. AMB-
CHMICA was also found in Turkey between 2016 and 2017
[14].
Figure 1 shows the synthetic pathways of the SCs tested
in this study. All of the test compounds required methyl
1H-indole-3-carboxylate (compound 1) or methyl 1H-inda-
zole-3-carboxylate (compound 2) as starting material and
were synthesized by referring to a previously described
method [20, 21]. Compounds 1 or 2 were N-alkylated by
alkyl halide under basic conditions to obtain compounds
3–5. Subsequently, compounds 6–8 were yielded via the
deprotection of compounds 3–5 by the hydrolysis of the
methyl group. The test compounds (compounds 9–11,
including each enantiomer) were synthesized by chlorina-
tion of compounds 6–8 and then amidation with the amino
acid derivatives. 5F-EMB-PINACA was synthesized as
previously described in [22]. The details of the chemical
synthesis of 5F-MDMB-PICA and AMB-CHMICA, which
were synthesized in this study, are described in the Sup-
plementary Material. The chemical structures of the SCs
tested in this study are shown in Fig. 2.
As SCs are typically smoked like cigarette [15–17],
we used previously developed techniques in which mice
are exposed to smoke emitted by burning plants sprayed
with SCs solution just like cigarette burning to evaluate
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Forensic Toxicology
L D
1 X= CH
2 X= N
3, 6 X= CH R1= 5-fluoropentyl
4, 7 X= N R1= 5-fluoropentyl
5, 8 X= CH R1= cyclohexylmethyl
9
X= CH R1= 5-fluoropentyl
R2NH2= / -tert-leucine methyl ester
L D
10 X= N R1= 5-fluoropentyl
R2NH2= / -valine ethyl ester
L D
11 X= CH R1= cyclohexylmethyl R2NH2= / -valine methyl ester
Fig. 1 Synthesis route for the synthetic cannabinoids dealt with in this study. tBu-OK potassium tert-butoxide, THF tetrahydrofuran, MeOH
methanol, EtOH ethanol, (COCl)2 oxalyl chloride, DMF N,N-dimethylformamide, CH2Cl2 dichloromethane, TEA triethylamine
Fig. 2 Chemical structures of
the synthetic cannabinoids dealt
with in this study
O
O
O
O
O
O
NH
NH
N
N
N
F
F
(a) 5F-MDMB-PICA (9)
(b) 5F-EMB-PINACA (10)
O
O
O
O
O
O
NH
NH
N
N
N
F
(c)AMB-CHMICA (11)
5F-MDMB-PINACA
(d)
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Forensic Toxicology
was absorbed in the amount of air set by the respiratory
apparatus (g) and introduced into the air inlet (b) of the
exposure box (B). The smoke fowed into the box was dif-
fused by a fan (c; E122535H, Earth Corporation, Tokyo,
Japan) that was installed at the air supply part of the box and
then exhausted from the exhaust port (f) of the box in the
amount of air set by the respiratory apparatus (g). Refer to
the Supplementary Material for information about the two
gas recovering bottles (C).
In vitro assays to evaluate the CB1 and CB2 receptor
activities
[35S]Guanosine-5′-O-(3-thio)-triphosphate ([35S]GTPγS)
binding assays were performed to evaluate the CB1/CB2
cannabinoid receptor activity. These assays were performed
at ADME and Tox. Research Institute, Sekisui Medical
Co., Ltd. (Tokai-mura, Ibaraki, Japan). The assay condi-
tions were as previously described in [23], except for the
tested concentration levels of the compounds ranging from
1 × 10−12 to 1 × 10−5 M for 5F-MDMB-PICA and AMB-
CHIMICA and from 1×10−11 to 1×10−4 M for 5F-EMB-
PINACA. CP-55,940 was used as a positive control. The
agonistic activities (EC50 value: concentration showing a
50% response rate) of the test compounds to the cannabinoid
receptors CB1 and CB2 were measured.
Sample preparation
The carboxamide-type SCs tested in this study were used as
a mixture of equal amounts of (S)- and (R)-enantiomers. For
burning the test compounds like cigarettes, 15 mg of the test
compound was added to 0.25 g of the lobe part of marshmal-
low, and the mixture was flled into half a cigarette paper.
Inhalation exposure test
Experimental animals and rearing conditions
An inhalation exposure test was performed to examine the
biological efects of the test compounds at the Tokyo Met-
ropolitan Institute of Public Health (Tokyo, Japan). The
method of the test was as previously described [18, 19]. We
referred to Inomata et al.’s study [19] for the results from
marshmallow (Althaea ofcinalis) as a negative control and
5F-MDMB-PINACA (Fig. 2) as a positive control. The
conditions and evaluation methods for the present test are
described below.
Male Crl; 5-week old CD1 (ICR) mice were purchased from
Charles River Laboratories Japan, Inc. (Kanagawa, Japan).
The mice were acclimated for 1 week before being used for
the test. They were housed individually in plastic cages and
had free access to solid feed (CE-2, Clea Japan, Inc., Tokyo,
Japan) and tap water. The animal room was maintained at
24 1 °C with a relative humidity of 50 5%, with 10
ventilations per h (through a HEPA flter), and on a 12 h
light/dark cycle.
Equipment
Method of inhalation exposure
Figure 3 shows the schema of the equipment for inhala-
tion exposure. The equipment was composed of a respira-
tory apparatus (g; SN-480-5, Shinano Seisakusho, Tokyo,
Japan), a combustion part (A), an exposure box (B; made
in vinyl chloride, content 8020 cc, Maeda Seisakusho Co.,
Ltd, Tokyo, Japan), and two gas recovering bottles (C; SPC
midget impinger, SIBATA SCIENTIFIC TECHNOLOGY
LTD., Saitama, Japan). The fow of air from the combus-
tion part to an exhaust port (f) was as follows: the smoke
from the marshmallow burned in the combustion part (A)
The marshmallow flled into half a cigarette paper was set on
the combustion part (Fig. 3a) of the equipment. Five mice
(Fig. 3d) were used per one compound, and each mouse (n
= 5) was put into each part of the exposure box (Fig. 3b)
partitioned with nets (Fig. 3e). After the box was closed, the
marshmallow was ignited while the air was fowing into the
box from the respiratory apparatus (Fig. 3g) at a fow rate of
600 mL/min. After the combustion, the respiratory apparatus
was left running.
Fig. 3 Schema of the equipment
for the inhalation exposure test
used in this study A a combus-
tion part, B an exposure box, C
two gas recovering bottles; (a)
marshmallow flled into a ciga-
rette paper, (b) an air inlet, (c) a
fan, (d) mice, (e) nets partition-
ing the box, (f) an exhaust port,
(g) a respiratory apparatus. The
black arrows show the air fow
direction
1 3

Forensic Toxicology
Evaluation test for biological efects
Results
The subsequent evaluations were performed 15, 30, and
60 min after the combustion. Incidentally, the measure-
ment of body temperature was also performed before the
exposure.
Functional activity at the CB1 and CB2 receptors
Table 1 shows the results of the [35S]GTPγS binding assays
performed in this study. (S)-5F-MDMB-PICA had the most
substantial potency at both the receptors in the tested com-
pounds. Both the enantiomers of AMB-CHMICA activated
to a CB1 receptor more selectively than the other tested com-
pounds. Compared with (R)-enantiomers, the (S)-enantiom-
ers of all tested compounds acted as CB1 receptor agonists,
which was similar to the results described in a previous
study [22].
Observation of the general, neurological, and autonomic
behaviors We prepared score sheets for the observation
of the general, neurological, and autonomic behaviors by
improving the observation methods of Irwin [24] (refer to
[18] for the score sheets) and calculated the mean of the
scores for the fve mice.
The normal behavior of a mouse was scored as zero
points. Suppressed behavior was given scores on a scale
of − 1 to − 3, and excited behavior was given scores on a
scale of + 1 to + 3 depending on the strength of behavior
(suppressive behavior: − 1, − 2, and − 3 and excitement
behavior: + 1, + 2, and + 3). The mean of each observa-
tion was calculated to evaluate the strength of the biologi-
cal efects of each item.
Observation of the biological efects
Table 2 describes the evaluation of the general, neurological,
and autonomic behaviors.
General behavior
When the absolute value of the mean for fve mice
(from now on, referred to as “the absolute value”) was
within the limits of 0–0.4 regarding the strength of the bio-
logical efects, it was decided that no biological efect was
observed. When the absolute value was within the limits of
0.6–0.8, an efect was suspected. When the absolute value
was within the limits of 1.0–3.0, an efect was observed.
Aggressiveness, stereotype, and vocalizations were not
observed for all the tested compounds. The degree of pas-
sivity was strong in the order of 5F-EMB-PINACA <
5F-MDMB-PICA < 5F-MDMB-PINACA. Furthermore,
the order corresponded to the functional activity at the
CB1 receptor. Additionally, 5F-MDMB-PINACA strongly
induced passivity for 1 h, and the passivity from 5F-MDMB-
PICA and 5F-EMB-PINACA decreased over time. AMB-
CHMICA did not induce passivity.
Measurement of body temperature The rectal tempera-
tures of mice were measured using a temperature recorder
for mice (AD-1687, A&D Company, Limited, Tokyo,
Japan). A range of 0.5 °C before exposure was regarded
as normal, and the score of suppressive behavior was
counted down (− 1, − 2 and − 3) every time the tempera-
ture dropped 1 °C from the lower limit of the normal tem-
perature range. The score was counted up (+ 1, + 2 and
+ 3) every time the temperature rose 1 °C from the upper
limit of the temperature range. Thereafter, the strength of
the efect was evaluated from the mean of fve mice.
Grooming was suppressed by marshmallow, and the
suppression decreased over time. The suppression by
AMB-CHMICA also decreased over time, but AMB-
CHMICA suppressed grooming more strongly than marsh-
mallow. 5F-MDMB-PINACA, 5F-MDMB-PICA, and
Table 1 Activities of test compounds at human CB1 and CB2 recep-
tors
Compound name
EC50 (nM)
CB1
Catalepsy test The forelimb of a mouse was suspended
on a crossbar that was made of a galvanized steel wire and
positioned at 6.5 cm height. The hindlimb of the mouse
was positioned on a stage and it kept standing with its
hindlimb. Thereafter, the time the mouse remained in the
position was measured. When the mouse remained still
for more than 30 s, the time was measured up to 90 s, and
the catalepsy was determined as positive. When the mouse
remained still for less than 30 s, the time was measured
three times. When the mouse moved in less than 30 s all
the three times, the catalepsy was determined as negative.
CB2
(S)-5F-MDMB-PICA
(R)-5F-MDMB-PICA
CP-55,940
0.815
56.2
2.33
4.96
35.9
1.90
14.3
62.0
2.62
0.590
76.2
0.399
6.91
(S)-5F-EMB-PINACAa
(R)-5F-EMB-PINACAa
CP-55,940
1.68
0.150
783 × 10
178 × 10
1.59
(S)-AMB-CHMICA
(R)-AMB-CHMICA
CP-55,940
aReferred in [22]
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Forensic Toxicology
Table 2 Mean values of the score in General, Neurogical, and Autonomical behavior (n = 5)
Observation time (h) Marshmallowa 5F-MDMB-PINACAa 5F-MDMB-PICA
5F-EMB-PINACA
AMB-CHMICA
0.25
0.5
1
0.25
0.5
1
0.25
0.5
1
0.25
0.5
1
0.25
0.5
1
Aggressiveness
Passivity
Stereotype
0
0
0.2
0
0
0.2
0
0
− 3
0
0
0
0
0.6
0
0.6
0.8
0.6
0
0
0.6
0
0
− 0.6 − 0.6 − 2.8 − 3
0
− 2.8 − 2.6 − 2
0
− 1.2 − 1.4 − 0.6 − 0.2
0
0
0
0
0
0
0
0
0
Grooming
− 2.8 − 1.4 − 0.4 − 3
− 3
0.2
− 3
0
− 3
− 3
− 3
0
− 3
0
− 3
0
− 3
0
− 3
0
− 3
0
0
− 3
0
0.2
− 2.6 − 1.4
Vocalization
Sound response
Touch response
Pain response
Verticalness
0
0
0
0.2
0
0
0
0
0
− 2.6 − 3
− 2.6 − 2.4 − 1.6 − 1.2 − 0.2
− 2.8 − 3
− 2.8 − 2.6 − 2.4 − 1
− 3 − 3 − 3 − 2.6 − 2
− 2.6 − 2.8 − 2.4 − 3 − 1.4 − 1.6 − 2.2 − 1.4 − 0.6
− 1.8 − 1.2 − 1 − 0.6 − 0.6
− 0.6 − 1.2 − 0.6 − 0.4
0.4
0.6
1.4
0.6
0.8
1
0
− 0.2 − 0.4 − 2.6 − 3
− 2.2 − 1.8 − 1.4 − 0.2 0.6
− 1 − 0.2 0.2
− 2
− 0.4 − 0.2 − 0.2 − 2
− 0.6 − 1.6 − 0.8 − 2.2 − 3
− 2.4 − 3
− 3
− 3
− 3
− 3
− 2.6 − 2.4 − 1.4
Spontaneous activity − 0.8 − 0.2 − 0.2 − 2.4 − 3
Abnormal gait
Abnormal position
Muscle tone
Straub tail reaction
Righting refex
Pinna refex
Corneal refex
Tendon refex
Tremor
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
− 3
− 3
2.4
0
− 3
− 3
− 2.8 − 3
− 3 − 3
− 2.2 − 3
− 2.8 − 3
− 2.8 1.4
− 1
− 1.8
1.2
− 2.6 − 2.6 − 2.8 − 3
0.4
− 3
0
0
0
0
0
− 1.4
0
0
0.8
0.4
0.6
0
0
− 1
0
0
0
0
0
0.4
− 3
− 3
− 3
0.4
1.0
1.4
1.4
− 3
− 2.6 − 1.6 − 1.2 − 0.8
0
0
0
− 0.2 − 0.4 − 2.8 − 3
− 2.8 − 3
− 2.8 − 2.6 − 2
− 2.8 − 2.4 − 2
− 2.6 − 2.2 − 2
− 1
− 0.2 − 0.8
− 0.2 − 0.8 0.4
0
2
− 0.8
1.2
1.2
1.4
0
1.2
1.2
1
0
0
− 0.6 0.2
0
0
0.4
0
0
0
0
0.2
0
− 2.8 − 2.8 − 3
0
3.0
− 3
− 1
0
0
0
0
0
0
0
0
0
0
1.2
0
0
0
0
0
0
0
0
Convulsion
1.2
− 3
− 2
0
0.4
− 3
0
0.4
1.8
0
0
Grip strength
Detached fnger
Exophthalmos
Pupil size
Palpebral opening
Shed tears
Salivation
Respiratory
Heart rate
− 3
− 1.6
0
− 1.8 − 1.4 − 2.6 − 1
− 1.2 − 1
0.4
1.6
− 0.4 − 0.4
− 2
0
0.6
− 0.8 − 0.4
0
0.6
0
0
0
− 1.4
0
0
− 3
0
0
0
0
0
0
0
0.2
1.4
0
0.4
0
0
0
0
0
0
0
− 0.2
2
1
0.6
0.2
− 1.2
0
0
0
0
0
− 0.2
− 1.4
0
0
rate
0
0
0
0
0
0
0
− 0.6 − 0.8 − 0.8 − 1.6 − 1.6 − 1.2 − 2
0
0
0
− 0.2
0
0
0
− 2
0
0
0
0
0
− 2
− 2
0
− 3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
− 2
− 2
0
− 3
− 0.2
− 2
− 1.8 − 2
0
− 3
− 2
− 1.8 − 2
− 3
− 1.6
0
− 3
0
− 0.4
0
0
− 3
0
− 3
− 3
Piloerection
Temperature
Skin color
0
0
− 3
− 2
− 2
0
− 2
0
− 0.6 − 3
− 2.6 − 2.2 − 2.4 − 2
0
0
1
0
0
0
aReferred in [19]
5F-EMB-PINACA drastically suppressed grooming. All
mice did not groom for 1 h.
Neurological behavior
The suppression of the sound, touch, and pain responses
were strong in the order of 5F-EMB-PINACA < 5F-MDMB-
PICA < 5F-MDMB-PINACA, and the suppression of these
responses by 5F-MDMB-PINACA increased over time. The
suppression from 5F-MDMB-PICA and 5F-EMB-PINACA
decreased over time. Contrary to these SCs, AMB-CHMICA
excited these responses, and the pain response was espe-
cially observed 30 min and 1 h after combustion.
Tremors were not observed for all the test compounds. The
degree of the suppression of spontaneous activity, abnor-
mal gait, abnormal position. and grip strength was strong
in the order of AMB-CHMICA < 5F-EMB-PINACA <
5F-MDMB-PICA < 5F-MDMB-PINACA. The order cor-
responded to the functional activity at the CB1 receptor.
These behaviors were drastically suppressed for 1 h by
5F-MDMB-PINACA. The grip strength was strongly sup-
pressed by 5F-MDMB-PICA but decreased over time. The
suppression of these behaviors by 5F-EMB-PINACA and
AMB-CHMICA also decreased over time.
For verticalness, 5F-MDMB-PINACA increased the
degree of the suppression over time, whereas 5F-EMB-PIN-
ACA and AMB-CHMICA decreased the degree over time.
5F-MDMB-PICA strongly suppressed the behavior for 1 h.
1 3

Forensic Toxicology
The results were drastic for muscle tone. An absence
of muscle tone was induced by 5F-MDMB-PICA for 1
h, and AMB-CHIMICA suppressed muscle tone 15 min
after combustion. In regard to 5F-MDMB-PINACA and
5F-EMB-PINACA, the excitement of the muscle tone was
observed 15 min after combustion. The behavior drastically
changed into suppression 30 min after combustion, and the
suppression continued with 5F-MDMB-PINACA. However,
5F-EMB-PINACA neither excited nor suppressed the behav-
ior 1 h after combustion.
AMB-CHMICA, which did not affect these rates.
5F-EMB-PINACA suppressed the behaviors 15 min after
combustion. 5F-MDMB-PINACA and 5F-MDMB-PICA
strongly suppressed the behaviors for 1 h. The degree of
the suppression from 5F-MDMB-PINACA was constant,
but those of 5F-MDMB-PICA increased over time.
The body temperature decreased with marshmallow
and returned to normal 1 h after combustion; however, the
SCs decreased the temperature for 1 h after combustion.
The body temperature of the mice treated with 5F-EMB-
PINACA and AMB-CHMICA returned to normal as
time passed. 5F-MDMB-PINACA and 5F-MDMB-PICA
sharply decreased the temperature for 1 h. By contrast,
SCs result in tachycardia and hyperthermia in humans [7].
However, it is unclear why SCs induce the opposite efect
in mice.
A Straub tail reaction was observed for 5F-MDMB-
PICA and 5F-EMB-PINACA. 5F-EMB-PINACA excited
the reaction 15 min after combustion, but the excitement
decreased over time, and 1 h after combustion, the behavior
was no longer observed. The excitement of the behavior by
5F-MDMB-PICA increased over time.
For righting, pinna, corneal, and tendon reflexes,
5F-MDMB-PINACA suppressed the behaviors the strong-
est. The suppression of the behaviors by 5F-MDMB-PICA
decreased over time. 5F-EMB-PINACA hardly suppressed
the behaviors. AMB-CHMICA showed the opposite results
of the other SCs. The SC did not afect the righting refex
but excited the other refexes.
There was no change in skin color with 5F-EMB-PIN-
ACA or AMB-CHMICA. However, 5F-MDMB-PINACA
resulted the color to be a little red 15 min after combus-
tion, whereas 5F-MDMB-PICA changed the skin color to
white for 1 h.
5F-MDMB-PINACA and 5F-EMB-PINACA strongly
induced convulsions 15 min after combustion. The convul-
sions decreased over time and were absent 30 min after com-
bustion with 5F-EMB-PINACA and 1 h after combustion
with 5F-MDMB-PINACA. 5F-MDMB-PICA induced con-
vulsions 30 min after combustion but was absent 1 h after
combustion. AMB-CHIMICA did not induce convulsions.
5F-EMB-PINACA induced detached fnger the strong-
est 15 min after combustion, but the degree decreased over
time. 5F-MDMB-PINACA and 5F-MDMB-PICA strongly
induced the behavior 30 min after combustion, and the
degree did not change at 1 h. The degree of the behavior at
1 h after combustion was strong in the order: 5F-EMB-PIN-
ACA < 5F-MDMB-PICA < 5F-MDMB-PINACA. AMB-
CHIMICA did not induce the behavior.
Catalepsy test
Table 3 shows the results of the catalepsy test. 5F-MDMB-
PICA, 5F-EMB-PINACA, and AMB-CHMICA showed
that the mice recovered from catalepsy over time, but
the time to recover was diferent between the SCs. The
catalepsy caused by AMB-CHMICA lasted the shortest
amount of time. For 5F-EMB-PINACA, the catalepsy of
all mice recovered 1 h after combustion. The number of
mice with catalepsy from 5F-MDMB-PICA decreased
over time, but in some mice, it was also observed 1 h after
combustion. When exposed to 5F-MDMB-PINACA, the
mice were exhausted and could not hold on to the crossbar,
thus jumping around or going into convulsion 15 min after
combustion. These mice were determined as negative for
catalepsy. Catalepsy generally disappears over time; there-
fore, it was presumed that catalepsy was not observed due
to the drastic efect of 5F-MDMB-PINACA.
Autonomic behavior
Exophthalmos, shed tears, and salivation were not observed
for all the test compounds. 5F-MDMB-PINACA and
5F-MDMB-PICA increased the pupil size to the same
degree, but the effect of 5F-MDMB-PINACA rapidly
decreased.
Table 3 The number of positive mice in catalepsy test (n = 5)
Observation time (h)
0.25
0.5
1
The behaviors of the palpebral opening of marshmal-
low, 5F-EMB-PINACA, and AMB-CHMICA were similar.
5F-MDMB-PICA suppressed the palpebral opening for 1 h
after combustion; however, the behavior was not observed
for 5F-MDMB-PINACA.
Marshmallowa
0
0
5
4
2
0
2
3
1
0
0
4
2
0
0
5F-MDMB-PINACAa
5F-MDMB-PICA
5F-EMB-PINACA
AMB-CHMICA
The respiratory and heart rates indicated a simi-
lar time-course for each tested compound, except for
aReferred in [19]
1 3

Forensic Toxicology
extremities for human. When abusers of the SCs drive
in cars, they are not able to release the accelerator due
to catalepsy and muscle rigidity, causing terrible trafc
accidents. Because 5F-MDMB-PINACA has excessive
potency, drivers using the SC are not able to drive at all,
and fortunately, accidents are not caused [7]. 5F-MDMB-
PICA did not induce muscle rigidity for mouse. If drivers
took 5F-MDMB-PICA, they would continue to drive in
the state of inebriation caused by the SC and cause trafc
accidents.
Discussion
Functional assays for carboxamide-type SCs at the CB1
and CB2 receptors have been previously performed.
Banister S. D. et al. evaluated 5F-MDMB-PICA, AMB-
CHMICA, and 5F-MDMB-PINACA using a fuoromet-
ric imaging plate reader membrane potential assay and
reported that the EC50 values of each SC at the CB1 recep-
tor were 0.45, 3.50, and 0.59 nM, respectively [12]. Thus,
5F-MDMB-PICA was stronger than AMB-CHMICA and
equivalent to 5F-MDMB-PINACA. Noble et al., Anton-
ides et al., and Wouters et al. used β-arrestin2 for func-
tional assays [25–27]. The EC50 value of 5F-MDMB-
PICA at the CB1 receptor was evaluated as 3.26 nM by
Noble et al. [25], and that of (S)-5F-MDMB-PINACA
was evaluated as 1.78 nM by Antonides et al. [26]. Wot-
ers et al. reported that EC50 values of 5F-EMB-PINACA
and 5F-MDMB-PINACA were 8.76 and 0.84 nM, respec-
tively [27]. On treating the EC50 values of 5F-MDMB-
PINACA evaluated by Antonides et al. and Wouters et al.
equally (1.78 and 0.84 nM, respectively) and comparing
the SCs tested by them as (S)-enantiomers, 5F-MDMB-
PICA was found to be stronger than 5F-EMB-PINACA
and equivalent to 5F-MDMB-PINACA. Although the
functional assay methods difered, the order of strength
of the functional activities for carboxamide-type SCs
(AMB-CHMICA<5F-EMB-PINACA<5F-MDMB-PICA
≈ 5F-MDMB-PINACA) corresponded between all studies.
The activation of CB1 receptor is involved in severely
acute adverse efects when consuming SC products [28].
5F-MDMB-PICA possesses functional activity at CB1
receptor similar to that of 5F-MDMB-PINACA, resulting
in many trafc collisions and fatal cases in Japan, and is
expected to be a threat to public health.
The efects on the behaviors from exposure to 5F-EMB-
PINACA and AMB-CHMICA decreased over time. In
Japan, trafc accidents were caused by the consumption of
herbal products containing 5F-AMB, and the users of the
products recovered rapidly within an hour after consumption
[7]. Furthermore, the inhalation exposure test was performed
with a product containing 5F-AMB, which also contained
AB-CHMINACA, another SC, and the results showed simi-
lar tendencies to 5F-EMB-PINACA and AMB-CHMICA
[18]. Therefore, a rapid recovery is expected when smoking
these SCs.
The degrees of the suppression of sound, touch, and
pain responses as well as righting, pinna, corneal, and ten-
don refexes also corresponded to the functional activity at
the CB1 receptor. However, AMB-CHMICA excited these
responses and refexes contrary to the other SCs. Espe-
cially, the SC excited the pain response at 30 min and 1 h
after combustion. Antinociception is known as one of the
tetrad of cannabinoid actions in addition to hypothermia,
catalepsy, and suppression of locomotion. AMB-CHMICA
may have induced the enhancement of nociception rather
than antinociception.
SCs are generally abused in the form of smoking [15–17].
In this study, the infuence of smoking SCs was investigated
with the equipment in which mice inhaled the smoke from
burned SCs; hence, we could evaluate the biological efects
considering how they are consumed and thermal degrada-
tion. It has been reported that some SCs produce degradation
products during smoking [29–34]. Furthermore, a very small
amount of 5F-EMB-PINACA and AMB-CHMICA was also
degraded by combustion (see the Supplementary Material).
Therefore, the equipment allows us to accurately evaluate
the biological efects when smoking SCs.
The degrees of various behaviors of mice by the inha-
lation exposure test with the SCs corresponded to the
functional activity at the CB1 receptor, e.g., passivity
and suppression of spontaneous activity, abnormal gait,
abnormal position, and grip strength. 5F-MDMB-PINACA
was estimated to possess strong functional activity and
induced various behaviors. 5F-MDMB-PICA possessed
the equivalent functional activity to 5F-MDMB-PIN-
ACA and also induced similar behaviors in an equiva-
lent or weaker manner. The use of 5F-MDMB-PINACA
resulted in several fatal cases in Japan [8]. The results
suggest that 5F-MDMB-PICA also caused health haz-
ard and fatalities as with 5F-MDMB-PINACA when
smoked. However, given that the behavior on muscle tone
was diferent between these SCs, 5F-MDMB-PICA was
expected to cause diferent symptoms than those observed
for 5F-MDMB-PINACA. Some carboxamide-type SCs
induce catalepsy accompanied by muscle rigidity in the
Conclusion
The [35S]GTPγS binding assays were performed to evaluate
the activities of three carboxamide-type SCs (5F-MDMB-
PICA, 5F-EMB-PINACA, and AMB-CHMICA) for the
CB1 and CB2 receptors. 5F-MDMB-PICA had the strong-
est functional activity at the CB1 receptor. Considering
other reports on the functional activity of these SCs,
1 3

Forensic Toxicology
5F-MDMB-PICA possessed the equivalent functional
activity of 5F-MDMB-PINACA, which has caused many
fatal cases in Japan, so 5F-MDMB-PICA can also cause
many fatalities.
References
1. UNODC (2013) The challenge of new psychoactive substances.
https://www.unodc.org/documents/scientifc/NPS_Report.pdf.
Accessed May 2019
The biological efects of smoking the SCs were also
evaluated using inhalation exposure tests. The equipment
produces an accurate evaluation considering the way of
inhalation and thermal degradation. The degrees of vari-
ous behaviors corresponded to the functional activity at
the CB1 receptor. 5F-MDMB-PICA possesses stronger
biological activity than 5F-EMB-PINACA and AMB-
CHMICA, and equivalent activity to 5F-MDMB-PIN-
ACA. 5F-MDMB-PICA is expected to cause intoxication
accompanied with fatalities, as with the expectation indi-
cated by the functional activity at a CB1 receptor. On the
other hand, 5F-MDMB-PICA can reduce muscle tone, but
5F-MDMB-PINACA can conversely enhance it. We expect
that when smoking these SCs, 5F-MDMB-PICA can
cause trafc accidents unlike 5F-MDMB-PINACA, which
induces severe muscle rigidity; thus, abusers are not able
to drive cars. AMB-CHMICA induced some biological
efects, particularly sound, touch, and pain responses and
righting, pinna, corneal and tendon refex, opposite of the
other tested SCs. As per the results, it seems that AMB-
CHMICA induces the enhancement of nociception rather
than antinociception, which is known as one of the tetrad
of cannabinoid actions, including hypothermia, catalepsy,
and suppression of locomotion. However, further studies
are needed to interpret the results. As this study reveals
that the biological efects of SCs could be complicated,
we consider that there is a good reason for the presence
of regulatory acts not only in Japan but also worldwide.
2. EU (2005) COUNCIL DECISION 2005 387 JHA of 10 May 2005
on the information exchange, risk-assessment and cotrol of new
psychoactive substances. https://eur-lex.europa.eu/legal-content/
EN/TXT/PDF/?uri=CELEX:32005D0387&from=en. Accessed
May 2019
3. UNODC (2019) Current NPS Threats Volume 1. https://www.
unodc.org/LSS/Attachment/Download/969af5f0-3050-42e2-
9568-8edd7ea87d3c. Accessed May 2019
4. Buchler IP, Hayes MJ, Hegde SG, Hockerman SL, Jones DE, Kor-
tum SW, Rico J, Gerace, Tenbrink RE, Wu KK (2009) Indazole
derivatives. WO 2009/106982
5. EMCDDA (2015) EMCDDA-Europol 2014 Annual Report on the
implementation of Council Decision 2005/387/JHA. https://www.
emcdda.europa.eu/system/fles/publications/1018/TDAN15001E
NN.pdf. Accessed May 2019
6. Adams AJ, Banister SD, Irizarry L, Trecki J, Schwartz M, Gerona
R (2017) "Zombie" outbreak caused by the synthetic cannabinoid
AMB-FUBINACA in New York. N Engl J Med 376:235–242.
https://doi.org/10.1056/NEJMoa1610300
7. Kaneko S (2017) Motor vehicle collisions caused by the ’super-
strength’ synthetic cannabinoids, MAM-2201, 5F-PB-22, 5F-AB-
PINACA, 5F-AMB and 5F-ADB in Japan experienced from 2012
to 2014. Forensic Toxicol 35:244–251. https://doi.org/10.1007/
s11419-017-0369-6
8. Hasegawa K, Wurita A, Minakata K, Gonmori K, Yamagishi
I, Nozawa H, Watanabe K, Suzuki O (2014) Identifcation and
quantitation of 5-fuoro-ADB, one of the most dangerous syn-
thetic cannabinoids, in the stomach contents and solid tissues of
a human cadaver and in some herbal products. Forensic Toxicol
33:112–121. https://doi.org/10.1007/s11419-014-0259-0
9. EMCDDA (2017) EMCDDA-Europol 2016 Annual Report on the
implementation of Council Decision 2005/387/JHA. https://www.
emcdda.europa.eu/system/fles/publications/4724/TDAN17001E
NN_PDFWEB.pdf. Accessed May 2019
10. Mogler L, Franz F, Rentsch D, Angerer V, Wieinfurtner G,
Longworth M, Banister SD, Kassiou M, Moosmann B, Volker
A (2018) Detection of the recently emerged synthetic cannabi-
noid 5F-MDMB-PICA in ’legal high’ products and human urine
samples. Drug Test Anal 10:196–205. https://doi.org/10.1002/
dta.2201
Compliance with ethical standards
Conflict of interest The authors declare that they have no confict of
11. Risseeuw MDP, Blanckaert P, Coopman V, Van Quekelberghe
S, Van Calenbergh S, Cordonnier J (2017) Identifcation of a
new tert-leucinate class synthetic cannabinoid in powder and
"spice-like" herbal incenses: methyl 2-[[1-(5-fluoropentyl)
indole-3-carbonyl]amino]-3,3-dimethyl-butanoate (5F-MDMB-
PICA). Forensic Sci Int 273:45–52. https://doi.org/10.1016/j.forsc
iint.2017.01.023
interest.
Ethical approval All animal experiments performed in this study were
reviewed and approved by the Animal Experiment Committee of Tokyo
Metropolitan Institute of Public Health. The animal experiments were
performed in accordance with the code of practice for animal experi-
ment, and the standards of conduct and the standard operation proce-
dure for experimental animal facility of Tokyo Metropolitan Institute
of Public Health based on the “Act on Welfare and Management of
Animals” (Act No. 105 of 1973), “Standards relating to the Care and
Keeping and Reducing Pain of Laboratory Animals” (Notice of The
Ministry of the Environment No. 88 of 2006), “Fundamental Guide-
lines for Proper Conduct of Animal Experiments and Related Activities
in Research Institutions under the jurisdiction of the ministry of Health,
Labor and Welfare” (Notifcation of the Ministry of Health, Labor and
Welfare of 1st June, 2006), “Standards relating to Methods of Destruc-
tion of Animals” (Notice of the Ministry of the Environment No. 105
of 2007), “Guidelines for Proper Conduct of Animal Experiments”
(Science Council of Japan of 1st June, 2006), and “Tokyo Metropolitan
Act for Welfare and Management of Animals” (Act No. 81 of 1979).
12. Banister SD, Longworth M, Kevin R, Sachdev S, Santiago M,
Stuart J, Mack JBC, McGregor IS, Connor M, Kassiou M (2016)
Pharmacology of valinate and tert-leucinate synthetic cannabi-
noids 5F-AMBICA, 5F-AMB, 5F-ADB, AMB-FUBINACA,
MDMB-FUBINACA, MDMB-CHMICA, and their analogues.
ACS Chem Neurosci 7:1241–1254. https://doi.org/10.1021/acsch
emneuro.6b00137
13. EMCDDA (2016) EMCDDA-Europol 2015 Annual Report on the
implementation of Council Decision 2005/387/JHA. https://www.
emcdda.europa.eu/system/fles/publications/2880/TDAS16001E
NN.pdf. Accessed May 2019
14. Göl E, Çok I (2019) New psychoactive substances in Turkey:
Narcotics cases assessed by the Council of Forensic Medicine
1 3

Forensic Toxicology
synthetic cannabinoids at CB1 and CB2 receptors. Drug Test Anal
11:501–511. https://doi.org/10.1002/dta.2517
between 2016 and 2017 in Ankara, Turkey. Forensic Sci Int
294:113–123. https://doi.org/10.1016/j.forsciint.2018.11.003
15. Bonar EE, Ashrafoun L, Ilgen MA (2014) Synthetic cannabinoid
use among patients in residential substance use disorder treat-
ment: prevalence, motives, and correlates. Drug Alcohol Depend
143:268–271. https://doi.org/10.1016/j.drugalcdep.2014.07.009
16. Buser GL, Gerona RR, Horowitz BZ, Vian KP, Troxell ML,
Hendrickson RG, Houghton DC, Rozansky D, Su SW, Leman
RF (2014) Acute kidney injury associated with smoking syn-
thetic cannabinoid. Clin Toxicol 52:664–673. https://doi.
org/10.3109/15563650.2014.932365
26. Antonides LH, Cannaert A, Norman C, Vives L, Harrison A, Cos-
tello A, Nic Daeid N, Stove CP, Sutclife OB, McKenzie C (2019)
Enantiospecifc synthesis, chiral separation, and biological activ-
ity of four indazole-3-carboxamide-type synthetic cannabinoid
receptor agonists and their detection in seized drug samples. Front
Chem 7:321. https://doi.org/10.3389/fchem.2019.00321
27. Wouters E, Mogler L, Cannaert A, Auwarter V, Stove C (2019)
Functional evaluation of carboxy metabolites of synthetic can-
nabinoid receptor agonists featuring scafolds based on l-valine
or l-tert-leucine. Drug Test Anal 11:1183–1191. https://doi.
org/10.1002/dta.2607
17. Gunderson EW, Haughey HM, Ait-Daoud N, Joshi AS, Hart
CL (2014) A survey of synthetic cannabinoid consumption by
current cannabis users. Subst Abus 35:184–189. https://doi.
org/10.1080/08897077.2013.846288
28. Hermanns-Clausen M, Kneisel S, Szabo B, Auwärter V (2013)
Acute toxicity due to the confrmed consumption of synthetic can-
nabinoids: clinical and laboratory fndings. Addiction 108:534–
544. https://doi.org/10.1111/j.1360-0443.2012.04078.x
29. Kanamori T, Kanda K, Yamamuro T, Kuwayama K, Tsujikawa
K, Iwata YT, Inoue H (2015) Detection of main metabolites of
XLR-11 and its thermal degradation product in human hepatoma
HepaRG cells and human urine. Drug Test Anal 7:341–345. https
://doi.org/10.1002/dta.1765
18. Inomata A, Ogata A, Tada Y, Nagasawa A, Yuzawa K, Ando H,
Kubo Y, Takahashi H, Kaihoko F, Tanaka K, Nakajima J, Suzuki
A, Uemura N, Moriyasu T, Watanabe D, Ishihara K, Usami T,
Kamei S, Kohno Y (2017) Inhalation exposure method for illegal
drugs. Yakugaku Zasshi 137:1005–1015. https://doi.org/10.1248/
yakushi.17-00019
19. Inomata A, Ono Y, Ogata A, Tada Y, Yuzawa K, Nagasawa A,
Ando H, Kubo Y, Takahashi H, Kaihoko F, Tanaka K, Hoshikawa
N (2017) New assays to evaluate the biological efects for illegal
drugs. Ann Rep Tokyo Metrop Inst Public Health 68:25–40
20. Doi T, Asada A, Takeda A, Tagami T, Katagi M, Kamata H,
Sawabe Y (2016) Enantioseparation of the carboxamide-type
synthetic cannabinoids N-(1-amino-3-methyl-1-oxobutan-2-yl)-
1-(5-fluoropentyl)-1H-indazole-3-carboxamide and methyl
[1-(5-fuoropentyl)-1H-indazole-3-carbonyl]-valinate in illicit
herbal products. J Chromatogr A 1473:83–89. https://doi.
org/10.1016/j.chroma.2016.10.049
30. Franz F, Angerer V, Moosmann B, Auwarter V (2017) Phase I
metabolism of the highly potent synthetic cannabinoid MDMB-
CHMICA and detection in human urine samples. Drug Test Anal
9:744–753. https://doi.org/10.1002/dta.2049
31. Thomas BF, Lefever TW, Cortes RA, Grabenauer M, Kovach AL,
Cox AO, Patel PR, Pollard GT, Marusich JA, Kevin RC, Gamage
TF, Wiley JL (2017) Thermolytic degradation of synthetic can-
nabinoids: chemical exposures and pharmacological conse-
quences. J Pharm Exp Ther 361:162–171. https://doi.org/10.1124/
jpet.116.238717
21. Asada A, Doi T, Tagami T, Takeda A, Sawabe Y (2017) Isomeric
discrimination of synthetic cannabinoids by GC-EI-MS: 1-ada-
mantyl and 2-adamantyl isomers of N-adamantyl carboxamides.
Drug Test Anal 9:378–388. https://doi.org/10.1002/dta.2124
22. Doi T, Tagami T, Takeda A, Asada A, Sawabe Y (2018) Evalu-
ation of carboxamide-type synthetic cannabinoids as CB1/CB2
receptor agonists: diference between the enantiomers. Forensic
Toxicol 36:51–60. https://doi.org/10.1007/s11419-017-0378-5
23. Nakajima J, Takahashi M, Uemura N, Seto T, Fukaya H, Suzuki J,
Yoshida M, Kusano M, Nakayama H, Zaitsu K, Ishii A, Moriyasu
T, Nakae D (2014) Identifcation of N, N-bis(1-pentylindol-3-yl-
carboxy)naphthylamine (BiPICANA) found in an herbal blend
product in the Tokyo metropolitan area and its cannabimimetic
efects evaluated by in vitro [35S]GTPγS binding assays. Forensic
Toxicol 33:84–92. https://doi.org/10.1007/s11419-014-0253-6
24. Irwin S (1968) Comprehensive observational assessment: Ia. A
systematic, quantitative procedure for assessing the behavioral and
physiologic state of the mouse. Psychopharmacologia 13:222–
257. https://doi.org/10.1007/BF00401402
32. Kevin RC, Kovach AL, Lefever TW, Gamage TF, Wiley JL,
McGregor IS, Thomas BF (2019) Toxic by design? Formation
of thermal degradants and cyanide from carboxamide-type syn-
thetic cannabinoids CUMYL-PICA, 5F-CUMYL-PICA, AMB-
FUBINACA, MDMB-FUBINACA, NNEI, and MN-18 during
exposure to high temperatures. Forensic Toxicol 37:17–26. https
://doi.org/10.1007/s11419-018-0430-0
33. Grigoryev A, Kavanagh P, Melnik A, Savchuk S, Simonov A
(2013) Gas and liquid chromatography-mass spectrometry detec-
tion of the urinary metabolites of UR-144 and its major pyrolysis
product. J Anal Toxicol 37:265–276. https://doi.org/10.1093/jat/
bkt028
34. Adamowicz P, Zuba D, Sekuła K (2013) Analysis of UR-144
and its pyrolysis product in blood and their metabolites in urine.
Forensic Sci Int 233:320–327. https://doi.org/10.1016/j.forsc
iint.2013.10.005
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
25. Noble C, Cannaert A, Linnet K, Stove CP (2019) Application
of an activity-based receptor bioassay to investigate the in vitro
activity of selected indole- and indazole-3-carboxamide-based
1 3