In Vitro Characterization of New Psychoactive Substances at The

Home , Allylescaline, EDMA, Prodrug

Forensic Science International 317 (2020) 110553

Contents lists available at ScienceDirect

Forensic Science International

journal homepage: www.elsevier.com/locate/forsciint

In vitro characterization of new psychoactive substances at the

m-opioid, CB1, 5HT1A, and 5-HT2A receptors—On-target receptor

potency and efﬁcacy, and off-target effects

a,1 a,b,1 a,b a,b,2

Anna Åstrand , Davide Guerrieri , Svante Vikingsson , Robert Kronstrand , a,b, ,2

Henrik Green *

Division of Drug Research, Department of Biomedical and Clinical Sciences, Faculty of Medicine and Health Sciences, Linköping University, SE 581 85,

Linköping, Sweden

Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, SE, 587 58 Linköping, Sweden

A R T I C L E I N F O A B S T R A C T

Article history: New psychoactive substances (NPS) appear on the recreational market on a monthly basis, with unclear

Received 23 March 2020

toxicology, resulting in an increasing number of fatalities. Identiﬁcation of drug targets and potencies is

Received in revised form 15 October 2020

crucial for understanding and treating intoxications and for scheduling processes. In this study 60 NPS

Accepted 17 October 2020

and metabolites belonging to opioids, cannabinoids and serotonergic hallucinogens classes were

Available online 23 October 2020

screened for in vitro activation of the m-opioid, CB1, 5-HT1A and 5-HT2A receptors using the AequoZen cell

system. Fentanyl and NBOMe analogues were chosen for full dose-response characterization of the

Keywords:

m-opioid and 5-HT2A receptors, respectively.

m-Opioid receptor agonists

Most substances activated their corresponding target receptor. The most potent m-opioid receptor

5-HT2A serotonin receptor agonists

agonists were 2-ﬂuorofentanyl (EC50 = 1.0 nM), carfentanil (EC50 = 2.7 nM) and acrylfentanyl

Fentanyl analogues

Designer drugs (EC50 = 2.8 nM) and in total a 1500-fold difference was seen among the tested compounds. Moreover,

New psychoactive substances (NPS) furanylfentanyl, 4-methoxybutyrylfentanyl and valerylfentanyl acted as partial agonists of the

Potency m-receptor. On the 5-HT2A receptor, bromo-dragonﬂy showed the highest potency (EC50 = 0.05 nM,

400 times more potent than LSD), followed by most NBOMe compounds with EC50 values ranging from

0.11 nM (for 25N-NBOMe) to 1.3 nM (for 25T4-NBOMe)). Off-target activation of the m-opioid receptor

was identiﬁed for piperazines, phenethylamines (in particular NBOMe and 2C compounds) and

tryptamines. Moreover, the synthetic cannabinoid metabolite 3-carboxy indole PB-22 activated the 5-

HT2A receptor. Bromo-dragonﬂy was the only compound that activated all four receptors. These results

highlight the possible interplay of known and unknown NPS targets and unveil its complexity. Moreover,

the detailed, quantitative information presented facilitates our further understanding of NPS toxicology.

(http://creativecommons.org/licenses/by/4.0/).

1. Introduction monitored by the end of 2018 and 55 of these were detected for the

ﬁrst time during 2018 [2].

Every year a large number of new psychoactive substances The quick pace at which new substances are introduced creates

(NPS) appear on the recreational drug market. United Nations a challenge for the legal authorities to keep up with the

Ofﬁce on Drugs and Crime (UNODC) states that between 2009 and identiﬁcation and scheduling of substances as narcotics, when

2019, a total of 950 compounds were reported to the UNODC early appropriate. Moreover, novel NPS rarely undergo any biochemical

warning advisory on NPS [1]. Only in Europe, 720 NPS were characterization before they are introduced on the illicit drug

market and even less is known regarding their effect in vivo. The

broad NPS term includes a plethora of different classes of

substances and effects such as opioids, cannabinoids, cathinones,

* Corresponding author at: Division of Drug Research, Department of Biomedical phenethylamines, piperazines, arylamines, tryptamines, and other

and Clinical Sciences, Faculty of Medicine and Health Sciences, Linköping

substances [3]. Several of these drugs target the G protein coupled

University, SE 581 85, Linköping, Sweden.

receptors (GPCRs), which consist of heterotrimeric seven-

E-mail address: [email protected] (H. Green).

1 transmembrane proteins. Upon activation these receptors initiate

These authors contributed equally to this study.

These authors share last authorship of this article. several intracellular signaling cascades both dependent and

http://dx.doi.org/10.1016/j.forsciint.2020.110553

A. Åstrand, D. Guerrieri, S. Vikingsson et al. Forensic Science International 317 (2020) 110553

Table 1

A complete list of reference compounds used within the study including vendors.

Compound Vendor Vendor name

2-ﬂuorofentanyl Cayman Chemicals Ortho-Fluorofentanyl

1P-LSD Chiron 1p-LSD-tartrate

25B-NBOMe THC Pharm 25B-NBOMe

25E-NBOMe Cayman Chemicals 25E-NBOMe

25I-NBOMe Cayman Chemicals 25I-NBOMe

*25N-NBOMe NFC 25N-NBOMe

25T4-NBOMe Cayman Chemicals 25T4-NBOMe

2C-E Cayman Chemicals 2C-E

2C-I Cayman Chemicals 2C-I

*2-Me-DMT NFC 2-Me-DMT

2-ﬂuoromethamphetamine Cayman Chemicals 2-ﬂuoromethamphetamine

3-methoxyohencyclidine (3-MeO-PCP) Cayman Chemicals 3-methoxy-PCP

4-Cl-isobutyrylfentanyl Chiron Para-Chloroisobutyrylfentanyl

*4-metoxybutyrylfentanyl NFC 4-Metoxibutyrfentanyl

5F-ADB Cayman Chemicals 5-ﬂuoro-ADB

5F-AKB-48 Cayman Chemicals AKB 48 N-(5-ﬂuoropentyl) analog

*5-IT NFC 5-IT succinat

5-MeO-DET Cayman Chemicals 5-methoxy DET

5-MeO-DPT Cayman Chemicals 5-methoxy DPT

*5-MeO-NiPT NFC 5-MeO-NiPT

AB-FUBINACA Cayman Chemicals AB-FUBINACA

AB-PINACA Cayman Chemicals AB-PINACA

Acetylfentanyl Cayman Chemicals Acetyl fentanyl

Acrylfentanyl Cayman Chemicals Acrylfentanyl

ADB-FUBINACA Cayman Chemicals ADB-FUBINACA

Allylescaline Cayman Chemicals Allylescaline

AM-2201 Chiron AM-2201

Amphetamine Sigma-Aldrich Amphetamine

*α-PVP NFC PVP

BB-22 (aka QUCHIC) Cayman Chemicals BB-22 (aka QUCHIC)

Benzylpiperazine (BZP) Sigma-Aldrich Benzylpiperazine (BZP)

βk-2C-B Cayman Chemicals bk-2C-B

Bromo-DragonFLY TRC Bromo Dragonﬂy

Buprenorphine Sigma-Aldrich Buprenorphine

Butyrylfentanyl Cayman Chemicals Butyryl fentanyl

*C30-NBOMe NFC C30-NBOMe

Camfetamine LGC Camfetamine

*Carfentanil TRC Carfentanil

Cathinone Sigma-Aldrich R (+)-Cathinone

Cyclopentylfentanyl Chiron Cyclopentylfentanyl

XLR-11 Chiron XLR11

Valerylfentanyl Cayman Chemicals Valeryl fentanyl

Cyclopropylfentanyl Cayman Chemicals Cyclopropyl fentanyl

Deschloroetizolam Chiron Deschloroetizolam

Diclazepam LGC Diclazepam

N, N-dimethyltryptamine (DMT) National Measurement Institute (NMIA) N, N-dimethyltryptamine

DOET Cayman Chemicals DOET

DOM Cayman Chemicals DOM

DPT Cayman Chemicals DPT

*EFLEA NFC EFLEA

EG-2201 Cayman Chemicals EG-2201

FDU-NNEI Cayman Chemicals FDU-NNEI

Fentanyl Sigma-Aldrich Fentanyl

Flephedrone (aka 4-ﬂuoromethcathinone, 4-FMC) TRC 4-Fluoroephedrone

*Flubromazolam NFC Flubromazolam

Furanylethylfentanyl Cayman Chemicals Furanylethylfentanyl

Furanylfentanyl (Fu-F) Cayman Chemicals Furanylfentanyl

Isobutyrylfentanyl Cayman Chemicals Isobutytyl fentanyl

JWH-018 THC Pharm JWH-018

LSD Sigma-Aldrich LSD

MDAI LGC 5,6-Methylenedioxy-2-aminoindane

MDMA Cayman Chemicals MDMA

MDMB-CHMCZCA Cayman Chemicals MDMB-CHMCZCA

*MDPV NFC 3,4-Methylenedioxypyrovalerone

Meclonazepam Chiron Meclonazepam

4-MeOPP Sigma-Aldrich 1-(4-Methoxyphenyl) piperazindihydrochloride

Mephedrone (aka 4-methylmethcathinone, 4-MMC) LGC Mephedrone

Methiopropamine LGC Methiopropamine

*Methoxypiperamide NFC Metoxipiperamid

MDMB-CHMICA Chiron MMB-CHMINACA

Morphine Sigma-Aldrich Morphine

*MT-45 NFC MT-45

Ocfentanil Cayman Chemicals Ocfentanil

PB-22 Cayman Chemicals PB-22

3-carboxy indole 3-carboxyindole metabolite

PB-22 N-pentanoic acid-3-carboxy indole Cayman Chemicals PB-22 N-pentanoic acid-3-carboxyindole

A. Åstrand, D. Guerrieri, S. Vikingsson et al. Forensic Science International 317 (2020) 110553

Table 1 (Continued)

Compound Vendor Vendor name

PTI-2 Cayman Chemicals PTI-2

Pyrazolam Cayman Chemicals Pyrazolam

TCB-2 Tocris TCB2

*Tetrahydrofuranylfentanyl (THF-fentanyl) NFC Tetrahydrofuran fentanyl

*TFMPP NFC TFMPP

THC Sigma-Aldrich THC

THJ-018 Cayman Chemicals THJ-018

*U-47,700 NFC U47700

**2-desmethoxy 25I-NBOMe Linköping University 25I-NBOMe metabolite CHM-003

2-methylamphetamine Chiron DL-2-Methylamphetamine

Seized materials characterized at National Forensic Centre (NFC), Sweden.

synthesised material, Linköping University, PSYCHOMICS project. Purity as determined by HPLC was >92 %.

independent of the G-protein complex in a ligand speciﬁc manner. analogues at the m-opioid and 5-HT2A receptors, respectively,

Two examples of intracellular signaling pathways after GPCR were determined.

activation is the β-arrestin recruitment and the G-protein coupled

calcium release [4]. Fentanyl is thought to activate the β-arrestin 2. Materials and methods

pathway of the m-opioid receptor to a greater extent than

morphine [5,6] which is interesting as β-arrestin signaling of 2.1. Cell lines

the m-opioid receptor is associated with respiratory depression in

opioid abuse. However, biased signaling of different compounds is Irradiated, calcium sensitive AequoZen recombinant CHO-K1

not always as straight forward as initially thought. In contrast, G- cell lines expressing the human m-opioid (ES-542-AF), CB1 (ES-

protein coupling associated signaling has been linked to analgesia 110-AF), 5-HT2A (ES-313-AF) or 5-HT1A receptor (ES-310-AF) were

and euphoria [5]. purchased from Perkin Elmer (Groningen, Netherlands). The cells

To mimic the pharmacodynamic effect of a drug in the human were stored at 80 C and used within 3 weeks of arrival.

body, several receptor-activity in vitro models have been estab-

lished. In particular, β-arrestin intracellular signaling of CB1, CB2 2.2. Drugs and chemicals

and m-opioid receptors have been studied, both as activity-based

screening methods for synthetic cannabinoids and opioids in Reference standards were purchased from Chiron (Trondheim,

bioﬂuids and for potency determination of NPS [7–13]. Intracellu- Norway), Cayman chemical (Ann Arbur, MI, USA), Sigma-Aldrich

lar calcium ion reporting system such as the apoaequorin/aequorin (Darmstadt, Germany), LGC (Luckenwalde, Germany), National

system [14] is also a common assay for monitoring receptor Measurement Institute (NMI) (Australia) or THC Pharm (Frankfurt

activation using luminescence reading. The calcium sensitive am Maine, Germany). Seized materials characterized and puriﬁed

apoaequorin/aequorin system relies on G-protein activation and at the National Forensic Centre (Linköping, Sweden) were also

the second messenger phospholipase C, with succeeding diac- used. The full list with substance names and vendors is presented

ylglycerol and inositol triphosphate production and intracellular in Table 1. DMEM/Ham's F12 without phenol red (ThermoFischer,

calcium ion release [15]. Gothenburg, Sweden), supplemented with 15 mM HEPES, L-

Several traditional drugs have known “off-target activity” that glutamine and protease-free BSA (Sigma-Aldrich), with a ﬁnal

can be used and give the drug a speciﬁc additional effects, One such BSA concentration of 0.1 %, was used for the receptor activity assay.

example is the off-target activity is the activation of serotonin Stock solutions of 500 mM coelenterazine (Nanolight Tech,

receptor 5-HT1A by the opioid tramadol [16,17] Speciﬁc studies Pinetop, AZ, USA) in methanol, 50 mM digitonin (Sigma-Aldrich)

regarding drug targets and potencies could aid healthcare in DMSO and 10 mM ATP (Sigma-Aldrich) in MilliQ water were

providers and legal authorities to better interpret intoxications prepared and stored at 20 C. Coelenterazine was protected from

caused by an NPS intake. Important to keep in mind is that in vivo light.

data could present a very different pharmacological (e.g.analgesic)

proﬁle for individual compounds, despite similar in vitro data 2.3. Instrumentation

[18,19]. Such discrepancies might be explained by differences in

pharmacokinetic parameters such as bioavailability and distribu- Drugs were dispensed and serially diluted in triplicates into

tion. Nevertheless, in vitro models for receptor activity play an white opaque microplates (OptiPlate-384, PerkinElmer) using cell

important part in our understanding of drug targets of new drugs. culture medium to a ﬁnal volume of 25 mL using a Biomek 2000

It is also known that the speciﬁcity and selectivity for activation of (Beckman-Coulter, Sweden). Cells were dispensed and lumines-

GPCRs varies among traditional drugs, which might also be true for cence was measured using a Spark 10 M with injector (Tecan,

NPS. Switzerland).

The overall aim of this study was to provide novel information

on the efﬁcacy and potency of NPS compounds and to investigate 2.4. Assay conditions

if off-target activity is present, which might contribute to NPS

toxicity and side effects. We therefore investigated the efﬁcacy of Analysis of receptor activation was carried out according to the

60 NPS on their known target receptor and their possible efficacy manufacturer specifications. Specifically, cells were thawed,

at other receptors (opioid, cannabinoid, serotonin receptors). centrifuged and resuspended in pre-warmed assay medium at a

Moreover, the potency and efﬁcacy of fentanyl and NBOMe concentration of 3 10 cells/mL. Coelenterazine was added to a

3 A.

Åstrand,

Guerrieri,

Vikingsson

Table 2 et

60 NPS, at a concentration of 7.5 mg/mL, were screened for activity on four GPCR: m-opioid, CB1, 5-HT 2A and 5-HT 1A . The results are sorted for signal intensity based on four levels; below LoD (empty), between LoD and 50 % of full – – al. agonist (+), 50 75 % (++), and 75 100 % (+++).

m-opioid CB1 5- 5- m-opioid CB1 5- 5- m-opioid CB1 5- 5-HT 1A HT 2A HT1A HT 2A HT1A HT 2A Cannabinoids 5F-ADB +++ Opioids 4Cl- +++ Tryptamines 2-DMT +++ isobutyrylfentanyl 5F-AKB48 +++ Acetylfentanyl +++ 5-MeO-DET + +++ +++ AB-FUBINACA +++ Acrylfentanyl +++ 5-MeO-DPT +++ +++ AB-PINACA +++ Butyrylfentanyl +++ 5-MeO-NiPT +++ +++ ADB-FUBINACA +++ Furanylethylfentanyl +++ DMT + +++ ++ AM2201 +++ Furanylfentanyl +++ DPT +++ + BB-22 +++ MT-45 +++ Benzodiazepines Deschloroetizolam EG-2201 + THF-fentanyl +++ Diclazepam FDU-NNEI +++ U-47,700 +++ Flubromazolam JWH-018 +++ Phenethylamines 25I-NBOMe +++ + +++ Meclonazepam MDMB- + 2C-I + +++ Pyrazolam

4 CHMCZCA MDMB- +++ 2-FMA others 3-MeO-PCP + CHMICA 3-carboxy ++ 2- + Camfetamine indole Methylamphetamine PB-22 PTI-2 +++ 5-IT + +++ EFLEA ++ THC ++ Amphetamine ++ MDAI THJ-018 +++ βK-2C-B + +++ Methiopropamine XLR-11 +++ MDMA ﬂy ++ +++ +++ + Methoxypiperamide Cathinones CathinoneFlephedrone Piperazines Bromo-dragonBenzylpiperazine + = LoD-50 % of full agonist signal MDPV MeOPP +++ ++ = 5075% of full agonist signal

Mephedrone TFMPP + +++ + +++ = 75100% of full agonist signal Forensic αPVP

Science

International

317

(2020)

110553

A. Åstrand, D. Guerrieri, S. Vikingsson et al. Forensic Science International 317 (2020) 110553

ﬁnal concentration of 5 mM and the cells were incubated on a 25 s. For 5-HT2A receptor-expressing cells, 50 mL of cell suspen-

rotating wheel at room temperature for 58 h while protected sion (5000 cells) were added.

from light. One hour before reading, cells were diluted 1:3 in assay

media and placed on gentle stirring agitation at room temperature. 2.5. Screening of receptor activity

The Spark 10 M reading protocol was set to 200 luminosity

readings; 25 mL of cell suspension (2500 cells) were added to each Sixty drugs (see Table 2) were screened for receptor activity

well at reading cycle #10 and luminescence was registered for with m-opioid, CB1, 5-HT1A and 5-HT2A receptor-expressing cells.

Fig. 1. Dose-response curves of fentanyl and NBOMe analogues were ﬁtted against the Hill-equation shown as a curve together with the mean of three independent

experiments in triplicates (n = 3 3) and the error bars show the 95 % C.I. The dose-response curves comparing the potencies of a total of 15 fentanyl analogues are shown in

A, B, C, D. Fentanyl, morphine and buprenorphine were used as reference compounds. Dose-response curves for 6 NBOMe:s are displayed together with LSD as reference

compound in E. Dose-response curves for 2C-E, 2C-I, allylescaline, βk-2C-B, bromo-dragonﬂy, DOET and DOM presented with LSD as reference compound are shown in F.

A. Åstrand, D. Guerrieri, S. Vikingsson et al. Forensic Science International 317 (2020) 110553

The drugs were dispensed (25 mL/well) in triplicate giving the ﬁnal In general, compounds of classes known to target a speciﬁc

concentrations of 2.5 and 7.5 mg/mL and the experiment was receptor activated it. For instance, all opioids tested in the

repeated twice (n = 3 2). The lower 2.5 mg/mL was only used to screening activated the m-opioid receptor with a signal intensity

deduce if the receptor activation had reached a plateau or not, for higher than 75 % of a full agonist (the maximum signal induced by a

further dose-response experiments. Controls, including assay drug in our assay). Most of all tested synthetic cannabinoids were

medium (blank), digitonin (50 mM ﬁnal concentration), ATP found to activate the CB1 receptor comparable to a full agonist

(10 mM ﬁnal concentration) and methanol (2 % ﬁnal concentra- (75100 %).

tion), were analyzed in six replicates on each plate. Organic solvent Similarly, almost all the cannabinoids scored in the ﬁrst tier as

was kept below 1 % in the wells containing drugs. to the activation of the CB1 receptor, except for THC (second tier),

MDMBCHMCZCA and EG-2201(both third their). Lastly, all trypt-

amines, most piperazines tested (except for benzylpiperazine) and

2.6. Potency determination of fentanyl and NBOMe analogues

most phenethylamines (excluding 2-ﬂuoromethamphetamine)

activated the serotonin receptors and showed mainly an activation

Full dose-response curves for fentanyl and NBOMe analogues

of the 5-HT2A receptor (Table 2).

were determined using 15 concentrations in triplicates. Each

Off target activation was also recorded for a number of drugs i.e.

experiment was repeated three times (n = 3 3) except for:

activation of the other receptors than the main expected receptor

cyclopropylfentanyl, carfentanil, furanylfentanyl, 2C-I, 25I-

for the drug class. Speciﬁcally, the m-opioid receptor was activated

NBOMe, 2-desmethoxy 25I-NBOMe, 2CE and 25E-NBOMe which

by the phenethylamines 25I-NBOMe (+++), 2C-I, 5-IT, and βK-2C-B

were repeated twice on the m-opioid receptor and for PB-22 and

(+), the piperazine TFMPP (+), and the tryptamines DMT and 5-

its metabolites, also repeated twice on the 5-HT2A receptor

MeO-DET (+). Similarly, 25I-NBOMe activated the CB1 receptor (+).

(n = 3 2). For experiments on the m-opioid receptor the drugs

Bromo-dragonﬂy activated CB1, 5-HT1A (+) and the m-opioid

were diluted on the microplates (25 mL/well) with a ﬁnal

receptor (++), alongside its major activation of the 5-HT2A receptor.

concentration ranging between 4 pg/mL and 20 mg/mL. The

The cannabinoid metabolite 3-carboxy indole PB-22 activated the

concentration ranges for carfentanil and isobuturylfentanyl were

5-HT2A serotonin receptor (++) but failed to activate the CB1

adjusted to 4 fg/mL to 20 ng/mL and 0.4 pg/mL to 2 mg/mL

receptor. None of the cathinones or benzodiazepines here tested

respectively. For experiments on the 5-HT2A receptor, the drug

showed any activity towards m-opioid, CB1, 5-HT1A or 5-HT2A

concentrations ranged between 2.8 pg/mL and 13.3 mg/mL, except

receptors.

for 25B-NBOMe (0.6 pg/mL to 2.66 ng/mL) and 3-carboxy indole

PB-22 (0.3 pg/mL to 1.33 mg/mL). Controls, including blanks,

3.2. On-target EC50 quantiﬁcation

digitonin (50 mM ﬁnal concentration), ATP (10 mM ﬁnal concen-

tration) and methanol (2 % ﬁnal concentration), were prepared in

3.2.1. The potencies and efﬁcacies of fentanyl analogues on the

quadruplicate on each plate.

m-opioid receptor

m-opioid receptor activation was determined using dose-

2.7. Data analysis

response curves to calculate the EC50 of the individual compounds.

Curves are shown in Fig. 1A–D. Potency as EC50 values (both in nM

Luminescence data were integrated over the total reading time

and ng/mL) are shown in Table 3A and plotted in Fig. 2A. Chemical

(i.e. area under the curve), the data were controlled for blank

structures of the opioids tested can be found in Supplemental

measurements (mean of blanks were subtracted) and the net

Fig. 1A.

signals were normalized against the digitonin signal. The digitonin

Reference compounds fentanyl, morphine and buprenorphine

signal is a measure of the maximal assay response by permeabi-

showed EC50 of 8.7 nM (95 % CI 5.613 nM), 102 nM (95 % CI

lization. For the screening analysis a limit of detection (LoD) was

66159 nM), and 778 nM (95 % CI 392–1655 nM), respectively.

set to blank signal + 3 times the standard deviation of the blanks.

Notably, EC50 values signiﬁcantly lower than for fentanyl were

Compounds were classiﬁed according to the signal intensity:

observed for acrylfentanyl (EC50 2.8 nM, 95 % CI 1.5–4.8 nM),

below LoD (tier 4), between LoD and 50 % of full agonist signal (the

carfentanil (EC50 2.7 nM, 95 % CI 1.6–4.4 nM) and 2-ﬂuorofentanyl

maximum signal induced by a drug in our assay) (tier 3), 5075 %

(EC50 1.0 nM, 95 % CI 0.5–1.9 nM), indicating these substances are

(tier 2) and 75100 % of full agonist signal (tier 1).

potent m-opioid receptor agonists.

For determining the potency, the average signals at each

At high drug concentrations, the luminescence signal reached a

concentration were plotted against the logarithm of the concen-

plateau, indicating the maximum response. The efﬁcacy was

tration in a dose-response plot. Sigmoid regression using Hill

ðTopBottomÞ expressed as percentage of the signal induced by digitonin (the

¼ þ

equation Y Bottom ðLogEC 50XÞHill Slope was used in Microsoft

1þ10 positive control for maximum assay response) (shown in Table 3).

ﬁ

Excel. EC50 and 95 % con dence intervals (C.I.) of calculated EC50 Speciﬁcally, the known partial agonist buprenorphine reached

values were determined. A set of reference drugs was used in each plateau at 54.8 %, while fentanyl and morphine, known full

experiment: fentanyl, morphine and buprenorphine for m-opioid agonists, reached plateau at 84.6 % and 83.5 %, respectively

receptor-expressing cells and LSD for 5-HT2A receptor-expressing (Fig. 1D). Based on these results two partial agonists were

cells. For a run to be considered successful the positive control had identiﬁed; furanylfentanyl (54.1 %) and valerylfentanyl (49.9 %).

to reach 50 % of the digitonin signal. Potential outlier data points 4-methoxybutyrylfentanyl showed a possible partial agonist

’ <

were determined by Grubbs test (two-sided, p 0.01). proﬁle as well, albeit less pronounced (70.9 %).

3. Results 3.3. The potencies of NBOMe analogues on the 5-HT2A receptor

3.1. Screening of receptor activity Dose-response curves for the activation of the 5-HT2A receptor

by NBOMe analogues are shown in Fig. 1E and F. EC50 values are

Compounds of eight major classes of NPS, as categorized by the summarized in Table 3B and plotted in Fig. 2B. Chemical structures

EMCDDA [3], were screened against the four human receptors of the compounds tested can be found in Supplemental Fig. 1B.The

m-opioid, CB1, 5-HT1A and 5-HT2A, the results are shown in Table 2 calculated EC50 for the reference compound LSD was 20 nM (95 %

for the 7.5 mg/mL concentration. CI 1331 nM). Bromo-dragonﬂy and 25N-NBOMe showed the

A. Åstrand, D. Guerrieri, S. Vikingsson et al. Forensic Science International 317 (2020) 110553

Table 3

Calculated EC50 values for compounds tested on target on the m-opioid (A) and the 5-HT2A receptor (B). The conﬁdence interval was set to 95 %.

A Compound % digitonin nM 95 % C.I. log M 95 % C.I. ng/mL 95 % C.I.

– + – + – +

2-ﬂuorofentanyl 93 1.0 0.5 1.9 8.99 9.30 8.71 0.40 0.19 0.76

Carfentanil 87 2.7 1.6 4.4 8.58 8.79 8.36 1.0 0.63 1.7

Acrylfentanyl 89 2.8 1.5 4.8 8.56 8.82 8.32 0.92 0.50 1.6

Ocfentanil 91 4.0 2.4 6.4 8.40 8.61 8.19 1.5 0.91 2.4

Cyclopropylfentanyl 88 8.2 5.1 13 8.08 8.29 7.88 3.2 2.0 5.0

Fentanyl 85 8.7 5.6 13 8.06 8.25 7.88 2.9 1.9 4.4

Butyrylfentanyl 86 13 8.5 20 7.89 8.07 7.71 4.5 3.0 6.9

Furanylfentanyl 54 21 14 34 7.67 7.87 7.47 8.0 5.1 13

Isobutyrylfentanyl 93 24 14 42 7.62 7.86 7.37 9.2 5.4 16

THF-fentanyl 85 26 18 39 7.58 7.75 7.40 10 6.7 15

Furanylethylfentanyl 86 76 50 118 7.12 7.30 6.93 28 19 44

4-methoxybutyrylfentanyl 71 81 52 127 7.09 7.29 6.90 34 22 53

Morphine 84 102 66 159 6.99 7.18 6.80 29 19 45

Cyclopentylfentanyl 84 125 78 204 6.90 7.11 6.69 52 32 84

Acetylfentanyl 83 231 138 405 6.64 6.86 6.39 75 45 130

4-Cl-isobutyrylfentanyl 88 494 277 960 6.31 6.56 6.02 208 117 404

Buprenorphine 55 778 392 1655 6.11 6.41 5.78 371 187 789

Valerylfentanyl 50 1559 816 3362 5.81 6.09 5.47 625 327 1348

B Compound % digitonin nM 95 % C.I. log M 95 % C.I. ng/mL 95 % C.I.

– + – + – +

Bromodragonﬂy 86 0.050 0.02 0.11 10.30 10.72 9.97 0.015 0.0056 0.032

25N-NBOMe 77 0.11 0.04 0.22 9.97 10.37 9.65 0.037 0.015 0.078

2C-I 78 0.28 0.12 0.56 9.56 9.91 9.26 0.085 0.038 0.17

DOET 80 0.34 0.15 0.68 9.46 9.81 9.16 0.077 0.034 0.15

25E-NBOMe 90 0.40 0.19 0.73 9.40 9.71 9.14 0.14 0.071 0.27

25B-NBOMe 87 0.42 0.25 0.69 9.37 9.60 9.16 0.16 0.095 0.26

2C-E 69 0.78 0.4 1.4 9.11 9.39 8.84 0.16 0.085 0.30

DOM 77 1.1 0.6 2.0 8.96 9.26 8.70 0.23 0.12 0.41

25I-NBOMe 84 1.2 0.7 2.0 8.92 9.16 8.70 0.51 0.30 0.84

25T4-NBOMe 77 1.3 0.8 2.2 8.89 9.12 8.66 0.54 0.31 0.90

LSD 82 20 13 31 7.69 7.88 7.51 6.5 4.3 10

Allylescaline 84 35 24 51 7.46 7.62 7.29 8.3 5.7 12

C30-NBOMe 74 1251 653 2581 5.90 6.18 5.59 541 282 1116

βK-2C-B 70 6732 6230 11,912 5.17 5.21 4.92 1845 1708 3265

Fig. 2. The potency expressed as EC50 values for the activation of m-opioid (A) and 5-HT2A receptors (B). The EC50 values are shown on a logarithmic scale as the mean of three

independent experiment in triplicates (n = 3 3) with the 95 % conﬁdence interval shown as error bars.

A. Åstrand, D. Guerrieri, S. Vikingsson et al. Forensic Science International 317 (2020) 110553

lowest EC50 values among all the compounds tested, 0.050 nM (95 4.1. Screening of NPS GPCR activity

% CI 0.020.11 nM) and 0.11 nM (95 % CI 0.040.22 nM),

respectively. The three compounds of the NBOMe series with NPS classiﬁed as opioids, cannabinoids, cathinones, phenethyl-

the lowest potency were 25I-NBOMe, 25T4-NBOMe and C30- amines, piperazines, tryptamines, benzodiazepines and other

NBOMe. 25I-NBOMe and 25T4-NBOMe presented EC50-values one substances were screened for GPCR activity on the m-opioid,

order of magnitude lower than LSD (Table 3B) while the EC50 of CB1, 5-HT1A and 5-HT2A receptors. From a general perspective the

C30-NBOMe was orders of magnitude higher (1251 nM, 95 % CI opioids often activates the different opioid receptors (d, k and m),

6532581 nM). the cannabinoids the CB1 and CB2 receptor, the cathinones

stimulates the release of dopamine and inhibits the reuptake

3.4. EC50 quantiﬁcation of selected positive screening results transporters of epinephrine, norepinephrine and serotonin,

phenethylamines regulates monoamine neurotransmission by

Based on the 25-I NBOMe m-opioid activity result from the binding to trace amine-associated receptor 1 and inhibiting

screening (Table 2), the m-opioid receptor activity was investigated vesicular monoamine transporter 2 in neurons and also targets

for a set of NBOME and NBOME analogues. Curves are shown in the 5-HT2 receptors, piperazines have a diverse mechanism of

Fig. 3. Speciﬁcally, the series 2C-I, 25I-NBOMe and 2-desmethoxy action and can sometimes act as 5-HT2A agonists, tryptamines are

25I-NBOMe were investigated. The signal induced by 2C-I was generally 5-HT2A agonists and benzodiazepines generally affects

detectable but just above the limit of detection. 25I-NBOMe the GABA-A receptor/channel complex.

reached 45.4 % of digitonin, with an estimated (plateau might not As fentanyl is a known m-opioid speciﬁc opioid [5], it is no

have been reached at highest concentration) EC50 of 12,500 nM (95 surprise that fentanyl analogues were identiﬁed as having a high

% CI 716017200 nM). The metabolite 2-desmethoxy 25I-NBOMe efﬁcacy for the m-opioid receptor. Our study focused on the

had an EC50 of 7310 nM (95 % CI 62908290 nM), with signal m-opioid receptor activity as it is known to be the main receptor

plateau at 62.6 % digitonin, potentially indicative of a partial responsible for the antinociceptive effect of opioid drugs [20].

agonist, see Fig. 3. The signal induced by 2C-E was just above limit m-opioid receptors are also centrally involved in inducing

of detection. Lastly, although not reaching plateau, 25E-NBOMe respiratory depression and bradycardia and overdosing may lead

reached 74 % of digitonin signal, with an estimated EC50 of to fatal hypoxia [21,22]. From a toxicological perspective, it is

22,000 nM (95 % CI 2080023200 nM), see Fig. 3. therefore important to identify new potent m-opioid receptor

3-carboxy indole PB-22, a metabolite of the synthetic cannabi- agonists. Off-target m-receptor activity was also identiﬁed for

noid PB-22, was screening positive for activation of the serotonin drugs of multiple drug classes such as tryptamines (5-MeO-DET

receptor 5-HT2A but not the CB1 receptor. In a full dose-response and DMT), piperazines (TFMPP), phenethylamines (25I-NBOMe,

assay 3-carboxy indole PB-22 reached 58.9 % of the digitonin 2C-I, βK-2C-B, bromo-dragonﬂy and 5-IT) and also 3-MeO-PCP, see

signal, see Fig. 3. However, as the signal never reached a plateau no Table 2. Notably, 25E-NBOMe was shown to be a full m-opioid

exact EC50 value was calculated, but it is most likely >10mM. PB-22 receptor agonist at high concentrations (see discussion below)

and its metabolite pentanoic acid 3-carboxy indole PB-22 were whereas the other NPS only induced partial activity.

inactive at the 5-HT2A receptor. Both CB1 and CB2 receptors are known targets of cannabinoids

with the former reported to be one of the most abundant GPCRs in

4. Discussion the brain [23]. It was expected that all synthetic cannabinoids

would target the CB1 receptor. Indeed, most of all tested synthetic

The present study reports the in vitro functional activation of cannabinoids were found to activate the CB1 receptor comparable

human GPCRs (opioid, cannabinoid, serotonin receptors) by to a full agonist (75100 %), see Table 2. It is interesting to point out

selected NPS and NPS metabolites. In depth analysis was focused however that the two carbazole derived synthetic cannabinoids

on fentanyl and NBOMe analogues and their potency on the EG-2201 and MDMB-CHMCZCA induced only partial CB1 activity

m-opioid or 5-HT2A receptors. Moreover, selected positive screen- (less than 50 % of a full agonist response) especially since the use of

ing results were also investigated by dose-response analysis. the related MDMB-CHMICA has caused fatal intoxications [24,25].

Fig. 3. Dose-response curves of NBOMe analogues and PB-22 and two metabolites were ﬁtted against the Hill-equation shown as a curve together with the mean of two

independent experiments in triplicates (n = 2 3) and the error bars shows the 95 % C.I. The dose-response curves comparing potencies of 2C-I, 2C-E, 25I-NBOMe, 25E-

NBOMe and the metabolite 2-desmethoxy 25-I-NBOMe on the m-opioid receptor are shown in A. Fentanyl, morphine and buprenorphine were used as reference compounds.

Dose-response curves for PB-22 and its two metabolites; 3-carboxy indole PB-22 and pentanoic acid 3-carboxy indole PB-22, with LSD as reference compound, are shown in B.

A. Åstrand, D. Guerrieri, S. Vikingsson et al. Forensic Science International 317 (2020) 110553

The 3-carboxy metabolite of PB-22 was also tested for CB1 activity result 2.7 nM (95 % CI 1.6–4.4 nM) in which we measured the

and was proven to be inactive but instead targeted the 5-HT2A increase of Ca ions via the protein kinase C pathway.

receptor (see discussion below). 25I-NBOMe and Bromo-dragonﬂy From the series of fentanyl analogues tested in the present work

also targeted the CB1 receptor to a lower extent. two main considerations emerged. First, at least three analogues

Hallucinogens such as tryptamines and some phenethylamines showed to be even more potent m-receptor agonists than fentanyl:

activate the serotonin receptors, mainly the 5-HT2A receptor, an in order of decreasing fold of potency, 2-ﬂuorofentanyl > carfen-

important target for hallucinogenic function [26]. Similar to the tanil = acrylfentanyl > ocfentanyl cyclopropylfentanyl = fentanyl.

available literature, our results shows that tryptamines (except for Compared to previously published studies or results for 2-

2-DMT) activate both the 5-HT2A and 5-HT1A receptors in contrast ﬂuorofentanyl, conﬁrm those of Hassanien et al. [7] who found it

to phenethylamines (except Bromo-dragonﬂy) which only activat- approximately 2 times more potent in their GTPgS assay compared

ed 5-HT2A [27–29]. For most tryptamines the activity at the 5-HT2A to 8 fold more potent in the present aequorin assay. In contrast to

receptor was more pronounced compared to 5-HT1A, indicating our results, Hassanien et al. [7] and Vasudevan et al. [13] did not

higher efﬁcacy for that receptor. In contrast to the tryptamines, no ﬁnd a difference in potency between acrylfentanyl and fentanyl.

phenethylamine activated the 5-HT1A receptor but strong activa- Second: With a efﬁcacy similar to the known partial agonist

tion of 5-HT2A was observed for a majority of the tested buprenorphine [35], three drugs appeared to be partial agonists:

phenethylamines with the exception of 2-methylamphetamine. furanylfentanyl, 4-methoxybutyrylfentanyl, valerylfentanyl (see

The pharmacodynamic effects of piperazines have been Fig. 1C and D), which also conﬁrms the results of partial agonism

reported to be enhancement of dopamine and norephinephrine found by Vasudevan et al. [13] as well as Hassanien et al. [7].

release in combination with inhibition of the dopamine, norephi- There are numerous reports on the structure-function relation-

nephrine and serotonin uptake [28,30,31]. Benzylpiperazine ship of the fentanyl scaffold. The piperidine ring is for example

showed no activity on the screened receptors. Interestingly, known to be central to the nociceptive activity [36] whereas the

MeOPP and TMPP activated the 5-HT2A receptor in the range of benzene ring on the anilino phenyl moiety was reported to be

full agonists. TFMPP also showed some off-target activity towards important for any opioid action [37]. N-acyl substituents have been

the m-opioid and 5-HT1A receptors. Unforeseen side-effects of NPS shown to be important for both afﬁnity and activation of the opioid

and NPS metabolites might be related to off-target effects such as receptors [38,39] and substitutions on the phenylethyl moiety

the ones reported here, and especially in situations of polydrug reduce the potency of the analog by preventing functional ligand-

abuse, might contribute to the toxicity by increasing the risk of receptor interaction [40].

serotonin syndrome. The compounds tested in the present work did not include

EFLEA induced a signal comparable to 5075 % of a full agonist substitutions on the piperidine ring, but rather on the anilino

on the 5-HT2A receptor in our assay. This is surprising as it is phenyl moiety, the propionyl moiety as N-acyl substituent or the

reported by user webpages (www.ﬂashback.org/t2924257 and phenylethyl moiety. Our data show that drugs substituted in the

http://drugs.tripsit.me/eﬂea) as being developed as a prodrug for ortho position of the anilino ring either maintain their potency to

EDMA and assumed inactive. However, to the best of our the m-receptor (ocfentanil) or increase it (2-ﬂuorofentanyl). On the

knowledge no measure of potency has been published for EFLEA. other hand, substituents in the para position could be a

Lastly, bromo-dragonﬂy activated all receptors but preferred 5- contributing factor for the reduced potency observed for 4-

HT2A. One might speculate that this general response could be the methoxybutyrylfentanyl and 4-chloroisobutyrylfentanyl. The only

result of permeabilization or an un-speciﬁc effect rather than phenylethyl-substituted compound tested, furanylethylfentanyl,

activation of the receptor. was about nine times less potent than fentanyl, albeit still a full

However, the response was different between receptors even agonist in agreement with results from Hassanien that showed an

though we used the same reporting system in all cell lines which 11 fold lower potency compared to fentanyl [7].

points toward receptor activation. For the other drugs, one or more The replacement of the propionyl group appears to crucially

negative screening results excludes permeabilization as alternative alter the induced opioid activity as well (see supplemental Fig.1 for

mechanism. structures); small substituents showed similar (acrylfentanyl,

Benzodiazepines and cathinones were inactive on all four ocfentanil, cyclopropylfentanyl) or increased potency (2-ﬂuoro-

receptors. This result was expected as the main target of fentanyl) compared to the unsubstituted drug, while more

benzodiazepines is the GABA-A receptor, a ligand-gated chlo- extended (butyrylfentanyl, valerylfentanyl) or bulkier (THF-fenta-

ride-selective ion channel receptor [32], and the cathinones nyl, furanylfentanyl, cyclopentylfentanyl) substituents appear to

exercise their pharmacological effect by inhibiting monoamine reduce potency in a somewhat size-proportional manner; e.g.

reuptake transporters [33]. Other NPS found inactive on the four fentanyl butyrylfentanyl > valerylfentanyl. Recently we showed

receptors were: camfetamine, MDAI, methiopropamine and that the potency of the alicyclic fentanyls was increased when

methoxypiperamide. decreasing the ring structure [41]. Interestingly there is an

optimum to the potency since reducing the number of carbons

4.2. Comparison of m-opioid receptor activity among fentanyl to only an acetyl group in acetylfentanyl appears to reduce the

analogues receptor potency again. Combining activity-reducing substitutions

on both loci seems to cause an additive effect; e.g. fentanyl butyr-

Despite the structural similarities, see supplemental Fig. 1, we ylfentanyl > 4-methoxybutyrylfentanyl and isobutyrylfentanyl >

found a 1500-fold difference in potency on the m-opioid receptor 4-chloroisobutyrylfentanyl.

between the fentanyl analogues tested, see Table 3A and Fig. 2. Polydrug abuse is another factor that must be taken into

Such differences in potency among closely related substances have consideration when evaluating drug potencies as both pharmaco-

been reported for other GPCRs, e.g. CB1 and CB2. [10,34]. However, dynamic and pharmacokinetic drug-drug interactions changing

when interpreting receptor activity data, it is important to the effect of the drugs have been reported. For example, mixing

remember that the activation of GPCRs also may depend on the fentanyl with serotonergic drugs leads to additive or synergistic

ligand activating different intracellular pathways [6]. This may be effects while mixing fentanyl and opioid partial agonists or

an explanation for the discrepancy between published EC50 values antagonists decreases the effect [42]. However. it is important to

on e.g. carfentanil (0.027 nM: 95 % CI 0.0210.035 nM) derived point out that furanylfentanyl, which was identiﬁed as a partial

from the use of an β-arrestin recruitment dependent assay [9], our agonist, still has been reported to cause fatal intoxications [43]. In

A. Åstrand, D. Guerrieri, S. Vikingsson et al. Forensic Science International 317 (2020) 110553

this paper we have evaluated the efﬁcacy of the compounds based 4.4. Activity at the m-opioid receptor by 5-HT2A receptors agonists

on their ability to activate the G-protein coupled receptors as

compared to the full activation of the receptors by reference It has been reported by the Psychoactive Drug Screening

compounds. However it should be noted that when looking at Program that 25I-NBOH showed binding afﬁnity towards the

other tissues or downstream signaling, the tested compounds m-receptor (47.2 nM), while 2C-I had a marginal opioid afﬁnity

might react differently. Morphine for example is known in some (2522 nM) and 25I-NBOMe presented none [52]. In the present

assays to be a partial agonist when studying the β-arrestin activation study, the qualitative screening showed a remarkable

recruitment assays [8,44]. The types of differences needs to be m-receptor activation induced by 25I-NBOMe, see Table 2.

considered when interpreting data from receptor studies. Therefore, we performed a quantitative study on the series: 2C-

Taken together, the large range in potencies and efﬁcacies on I, 25I-NBOMe and 2-desmethoxy 25I-NBOMe. 2-desmethoxy 25I-

the m-opioid receptor seen for closely related fentanyl analogues NBOMe is a metabolite known to be formed and excreted in urine

highlights the need for testing of compounds at an individual level. after a 25I-NBOMe intake [53]. Interestingly, while 2C-I induced

Potency data are of importance both for understanding the marginal activation, the NBOMe scaffold (25I-NBOMe, 2-desme-

function of a drug but also for scheduling purposes. thoxy 25I-NBOMe) appeared to induce signiﬁcant receptor activity,

although with low potency. More importantly, considering a

4.3. Comparison of 5-HT2A receptor activity among NBOMe analogues possible evaluation of unforeseen side effects was the observation

that at high concentration the 2-desmethoxy metabolite reached a

The 2C and NBOMe compounds (see supplemental Fig. 1 for level of efﬁcacy comparable to known opioid partial agonists, see

structures) were more potent than LSD spanning over more than a Fig. 3.

10-fold range (Fig. 1B). 2C-I and 2C-C activation of the 5-HT2A However, the activity at the m-opioid receptor appeared

receptor has been reported to be 8- and 13-fold higher than the remarkably affected when comparing 25I-NBOMe to 25E-NBOMe.

corresponding NBOMe drugs, 25I-NBOMe and 25C-NBOMe, Although the potency is diminished, the substitution to the ethyl

respectively. [45]. Our data showed no difference in potency group seems to evoke responses almost comparable with a full

between 2C-E, 25E-NBOMe and 25I-NBOMe, based on over- opioid agonist within the concentration range tested, and it is

lapping 95 % CIs. However, 2C-I was found to be around four times possible to speculate that such efﬁcacy could be reached at higher

more potent than 25I-NBOMe. The comprehensive study of Rickli concentrations. Even though such concentration levels are unlikely

et al. (2015), analyzed a structure-activity relationship of binding to be reached physiologically, the m-opioid activity of 2-

afﬁnity and agonist activity for the 2C and NBOMe families. The desmethoxy 25I-NBOMe highlights the possibility that metabolic

study supports our ﬁndings reporting a similar overlap in the transformation of 25E-NBOMe might produce an even more potent

relative potency of NBOMe and 2C compounds as presented here. m-opioid receptor agonist. Given that, as mentioned above, the

Speciﬁcally, they report 2C-I to be more potent than its NBOMe m-receptor is the main player in inducing respiratory depression,

derivative and that no signiﬁcant differences between the metabolic pathways generating metabolites with higher opioid

potency of 2C-E and 25E-NBOMe were found [46]. However, receptor potency pose a further complication in the toxicological

Pottie et al. recently reported the opposite that the 2C compounds evaluation of NPS.

(2C-I and 2C-E) had lower potency than the corresponding However, when comparing in vitro efﬁcacy and potency of on-

NBOMe:s (25I-NBOMe and 25E-NBOMe) [47]. The reason for this and off-target activation one should keep in mind that the actual

might be many, but one might be that they used a bioassay effect in vivo is dependent on the concentration reached at each

monitoring the recruitment of β-arrestin 2 to the 5-HT2A receptor receptor.

[47]. It would be interesting to compare these types of assay and

in more details deduce the mechanism of action of these 4.5. Off-target activation by a synthetic cannabinoid metabolite on the

compounds. 5-HT2A receptor

Lastly, allylescaline showed potency comparable to LSD in our

study. Albeit the only published indication of effective dose for PB-22 is a synthetic cannabinoid known to activate the β-

allylescaline give an effective dose of 2030 mg [48]. Comparing arrestin pathway of CB1 and CB2 receptors with EC50 values of

this data to the effective dose of LSD, 60–200 mg [49], would rank 0.86 nM and 0.82 nM at the respective receptor. In the same study,

allylescaline as 200-fold less potent than LSD. This is contrary to 3-carboxy indole PB-22, a major metabolite of PB-22, was shown

our result, however, when a dose active in vivo is compared to not to activate the CB1 or the CB2 receptor [11 ]. Moreover, Banister

receptor activation in vitro, such a discrepancy could potentially be et al. reports EC50 values for PB-22 of 5.1 nM on the CB1 receptor

explained by pharmacokinetics factors such as bioavailability, and 37 nM on the CB2 receptor [54]. Interestingly, our study also

stability and/or metabolism. For LSD a recent PK/PD study was showed that the 3-carboxyindole PB-22 did not activate CB1, but

performed by Holze et al. showing a close correlation between the could activate the 5HT2A receptor with an efﬁcacy comparable with

LSD concentrations and the LSD effects over time within a single LSD, albeit with a potency 2 orders of magnitude lower than LSD.

individual and that LSD has a high potency in vivo [50]. However, Notably, neither the parent drug PB-22 nor the other metabolite

one should be aware that without such data comparing dosages tested, PB-22 pentanoic acid 3-carboxyindole showed any activity

and EC50 values does have its limitations. towards the 5-HT2A receptor.

It should be noted that 5-HT2A binding in vitro has been Other cannabinoids have already been linked to serotonin

indicated in one study as a better predictor of the clinical receptor activation in vivo, for instance CP 55,940 was indirectly

hallucinogenic effects based on user doses then the activation of proven effective as a serotonin receptor agonist in rats [55]. This

the receptor [51]. This means that the binding data might be would support the hypothesis that some metabolic transforma-

considered more relevant in predicting drug effects because the tions of synthetic cannabinoids might induce serotonin receptor

activation assays may measure outcomes that are not directly activity. It was reported that the ester hydrolysis at the indole-3

linked to the hallucinogenic effects in vivo. However, it should be position with the loss of the quinolinyl ring induces serotonin

noted that the determination of 5-HT2A receptor activity is crucial agonistic activity [56]. Substituents at the end of the pentyl side

for determining if a NPS is a receptor agonist and may thus be chain on the N1 of the indole ring (in the present case, carboxylic

classiﬁed as a psychedelic or if it is an antagonist that only binds to acid or the halogen ﬂuoro) would instead obstruct the interaction

the receptor. with the receptor [57]. Major metabolic transformations of PB-22

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Writing - original draft. Svante Vikingsson: Conceptualization,

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Methodology, Formal analysis, Investigation, Writing - review &

[18] C. Dardonville, C. Fernandez-Fernandez, S.L. Gibbons, G.J. Ryan, N. Jagerovic, A.

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http://dx.doi.org/10.1016/j.bmc.2006.06.007.

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Forensic Sciences, grant number 2016:6 (Strategiområdet foren-

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