FACT SHEET

Acrylfentanyl

May 2017

For more information, please contact: Dr. P. Blanckaert Coordinator Belgian Early Warning System Drugs Scientific Institute of Public Health National Focal Point on Drugs Jyliette Wytsmanstraat 14 B-1050 Brussels, Belgium Tel : 02/642 5408 [email protected]

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A. General information

Recent sample in Belgium

In the first half of May 2017, a death was reported in Belgium after the use of acrylfentanyl. The victim, male, presumably consumed a powder containing acrylfentanyl by sniffing, and was found dead at the scene, in the region of Ghent. It remains unclear whether the victim knowingly consumed acrylfentanyl. Several other powders and tablets were found at the scene, one of which was identified as N- ethylhexedrone, a cathinone of the stimulant category.

Created July 2016

Updated May 2017

Type Narcotic drugs

Group Opioids

Name Acryloylfentanyl/acrylfentanyl

Nature of substance Acryloylfentanyl is a piperidinamine, which is structurally related to internationally controlled opiod fentanyl (Schedule I of the 1961 United Nations Single Convention On Narcotic Drugs). They differ in the double bond present in the 2- position of the propane attached to the N-phenyl moiety. Acryloylfentanyl is also structurally related to acetylfentanyl, which has been recently subjected to international control (Schedule I, 1961 UN Convention). Because of the structural similarity of acryloylfentanyl and fentanyl, their GC-MS may be similar, with a mass difference of M-2 for the base peak (m/z 243 for acryloylfentanyl).

Systematic chemical name N-(1-phenethylpiperidin-4-yl)-N-phenylacrylamide

Other names

Chemical names: 1-phenethyl-4-N-acryloylanilinopiperidine

Other names: acrylfentanyl, ACF

B. Alerts

Alerts

23 deaths associated with acrylfentanyl in Sweden – April – August 2016 The EMCDDA has received reports of 23 deaths associated with acryloyl fentanyl. The deaths occurred between April and August 2016 in Sweden. The presence of acryloyl fentanyl was analytically confirmed in all the cases from biological samples taken from the deceased. The decedents were aged 22 to 54 years old (mean 33, median 29); 4 were female, 19 were male. The cause of death is known for 8 cases: acryloyl fentanyl was the cause of death or a contributing factor in all of them; the remaining 15 cases are still under investigation. Acryloyl fentanyl was the only substance consumed in 2 out of 23 deaths. No details are known regarding the source of the substance or the route of administration except for one case where the death occurred after the person consumed acryloyl fentanyl with friends using a nasal spray. In at least 2 cases the deaths were said to have occurred “suddenly”.

Reports to EMCDDA Denmark: On 16 May 2017 the Danish FP reported a post-mortem blood and urine sample analysed on 21.01.2017 by the Department of Forensic Medicine, Aarhus University. The concentration of acryloylfentanyl in blood was 0,0072 mg/kg.

Estonia: On 17 November 2016 the Estonian FP informed the death cases of 2016: - 1 pure case of acrylfentanyl death; - 2 cases of combination of acrylfentanyl and regular fentanyl deaths.

Latvia: On 10 October 2016 the Latvian FP reported a seizure of 0,3699 g beige powder seized on 24/07/2016 by the Police at Riga.

Estonia: On 13 September 2016 the Estonian FP reported 6 seizures of powder during Summer 2016 at Tallin.

Finland: On 1 September 2016 the Finnish FP reported a seizure of 99 tablets seized on 8.8.2016. The police seized tablets in the Åland island between Sweden and Finland.

Sweden: On 31 August 2016 the Swedish FP reported a seizure of 10ml of liquid seized on 19.04.2016 by the Police at Eskilstuna.

Sweden: On 22 August 2016 the Swedish FP reported 23 deaths associated with acryloylfentanyl. The substance has been reported as the cause of death in 5 cases.

Slovenia: On 1 August 2016 the Slovenian FP reported a sample of 5g light green powder collected on 10.05.2016 by the National Forensic Laboratory. Sample was shipped from China.

C. Pictures

Tablet seized in Finland

Capsule seized in Denmark

D. Clinical information

Usage This compound has no known uses, and is currently only found as a (legal) fentanyl derivative sold online as a new psychoactive substance.

Health risks Acryloylfentanyl is a potent opioid; overdose risks are higher compared to e.g. heroin or morphine.

A study by Zhu measured the analgesic activity of derivatives of fentanyl (mouse, intravenous, hot plate test). This study shows that acryloylfentanyl (ED50= 0.082 mg/kg) and fentanyl (ED50=0.062 mg/kg) have high activity as compared to morphine (ED50=13.9 mg/kg). A study by Essawi reports that, at similar doses, acrylfentanyl had longer-lasting antinociceptive effects than fentanyl (mouse, intraperitoneal, hot plate test).

Maryanoff et al. measured the inhibitory concentration in vitro (rat brain opioid receptor, naloxone binding) for acryloylfentanyl (IC50= 1.4 nM), fentanyl (IC50= 1.6

nM) and morphine (IC50= 4.2 nM), indicating that acryloylfentanyl has higher affinity to the opioid receptor.

Other uses None known.

E. Legal status Currently legal in Belgium.

Controlled in Austria, Cyprus, Denmark, Estonia, Finland, Ireland, Latvia, Lithuania, Norway, Poland, Sweden, Turkey, United Kingdom, China.

F. Chemistry Other chemical names and variants 1-phenethyl-4-N-acryloylanilinopiperidine

Chemical Abstracts Service (CAS) registry number 79279-03-1 (HCl)

Molecular information Molecular structure:

Molecular formula: C22H26N2O

Molecular weight: 334.20 g/mol

Identification and analytical profile: It has to be noted that the GC-MS EI-spectra of acryloylfentanyl and fentanyl are similar, but with a mass difference of M-2 for the base peak (m/z 243 for acryloylfentanyl).

Analytical spectra can be found at the end of this fact sheet, and were provided by the EDND-website of the EMCDDA. These spectra should be considered confidential, and should not be shared without permission.

G. Recent references

Davide Guerrieri, Emma Rapp, Markus Roman, Gunilla Thelander, Robert Kronstrand, Acrylfentanyl: Another new psychoactive drug with fatal consequences, Forensic Science International http://dx.doi.org/10.1016/j.forsciint.2017.05.010

Ujváry I, et al. Acryloylfentanyl, a recently emerged new psychoactive substance: a comprehensive review. Forensic Tox. 2017. doi: http://dx.doi.org/10.1007/s11419- 017-0367-8

Watanabe S, et al. In vitro and in vivo metabolite identification studies for the new synthetic opioids acetylfentanyl, acrylfentanyl, furanylfentanyl, and 4-fluoro- isobutyrylfentanyl. AAPS J. 2017. doi: https://dx.doi.org/10.1208/s12248-017-0070-z

Helander A, et al. Intoxications involving acrylfentanyl and other novel designer fentanyls – results from the Swedish STRIDA project. Clin Tox. 2017. doi: http://dx.doi.org/10.1080/15563650.2017.1303141

J. Suzuki, S. El-Haddad, A review: Fentanyl and non-pharmaceutical fentanyls, Drug and Alcohol Dependence 171 (2017) 107–116, http://dx.doi.org/10.1016/j.drugalcdep.2016.11.033

Anders Helander & Matilda Bäckberg (2016): New Psychoactive Substances (NPS) – the Hydra monster of recreational drugs, Clinical Toxicology.

Figure 6: Mass spectrum for precursor (+MS) (top) and product ions (+bbCID) (bottom) for fentanyl, (C22H28N2O). The product ion at m/z 188.1437 corresponds to C13H18N, which is a well known fragmentation pathway for fentanyl.

Figure 7: Mass spectrum for precursor (+MS) (top) and product ions (+bbCID) (bottom) for acrylfentanyl, (C22H28N2O). The product ion at m/z 188.1437 corresponds to C13H18N, which is a well known fragmentation pathway for fentanyl. Supporting information for: Identification of a new psychoactive substance in seized material: The synthetic opioid N-phenyl-N-[1-(2-phenethyl)piperidin-4-yl]prop-2-enamide (Acrylfentanyl)

Table of contents Characterisation of acrylfentanyl (seized sample) 2 1 Acrylfentanyl (seized sample), H NMR, 400 MHz (CD3OD) 2

Acrylfentanyl (seized sample), COSY, 400 MHz(CD3OD) 3 Acrylfentanyl (seized sample), MALDI-TOF HRMS 3 Characterisation of fentanyl (standard) 4 1 Fentanyl (standard), H NMR, 400 MHz (CD3OD) 4 Fentanyl (standard), MALDI-TOF HRMS 5 Acrylfentanyl (seized sample) compared to acrylfentanyl (standard), 1 H NMR, 600 MHz (CDCl3) 5 Enlargements: Acrylfentanyl (seized sample) compared to acrylfentanyl 1 (standard), H NMR, 600 MHz (CDCl3) 6 Acrylfentanyl (seized sample) compared to acrylfentanyl (standard),

DEPT, 600 MHz (CDCl3) 7 1 Acrylfentanyl (seized sample), H NMR, 600 MHz (DMSO-d6) 7 Acrylfentanyl (seized sample) compared to triethylamine hydrochloride,

DEPT, 600 MHz (DMSO-d6) 8 Acrylfentanyl (seized sample) spiked with triethylamine hydrochloride, 1 H NMR, 600 MHz (DMSO-d6) 8 IR spectrum of acrylfentanyl (seized sample) 9 IR spectrum of acrylfentanyl (standard) 9 Instrumentation (LC-MS/MS) for quantification of acrylfentanyl in seized sample 10 Multiple Reaction Monitoring (MRM) parameters 10 LC-MS/MS calibration curve 11 MRM signal for m/z 335 → m/z 188 (acrylfentanyl in seized sample) 11 Qualifying ion transitions (overlaid) for acrylfentanyl in seized sample 12 MRM signal for m/z 343 → m/z 105, internal standard, fentanyl-d5, spiked to seized sample during sample preparation 12 MRM signal for m/z 335 → m/z 188, Acrylfentanyl calibrator, 156.25 ng/mL (free base) 13 Qualifying ion transitions (overlaid), Acrylfentanyl calibrator, 156.25 ng/mL (free base) 13

Page 1 af 13 Characterisation of acrylfentanyl (seized sample) The numbering of atoms used for NMR assignments does not follow IUPAC recommendations and is only used here for clarity (See Fig. S1 for numbering system). Acrylfentanyl: N-phenyl-N-[1-(2-phenethyl)piperidin-4-yl]prop-2-enamide 1 H NMR (400 MHz, CD3OD): δ 7.60-7.52 (m, 3H; Ph), 7.39-7.27 (m, 7H, Ph), 6.31 (dd, J = 17, 2 Hz, 1H, H-1), 5.91 (dd, J = 17, 10.5 Hz, 1H, H-2) 5.59 (dd, J = 10.5, 2 Hz, 1H, H-1’), 4.90 (tt, J = 12, 4 Hz, 1H, H-5),# 3.78 (m, 2H, H-7 and H-7’),* 3.37-3.33 (m, 2H, H-8),# 3.29- 3.22 (m, 2H, H-7 and H-7’),* 3.10-3.05 (m, 2H, H-9), 2.26-2.19 (m, 2H, H-6 and H-6’), 1.86 (qd, J = 8, 4 Hz, H-6 and H-6’)*. # Overlapping with signal from the NMR solvent; * Overlapping with unidentified impurity. + + HRMS (MALDI-TOF) m/z found: 335.2114 [M+H] ; C22H27N2O requires M, 335.2118.

1 Figure S1: Acrylfentanyl (seized sample), H NMR (400 MHz) in CD3OD Overlapping signals from the NMR solvent and impurity triethylamine hydrochloride are indicated with arrows.

NMR solvent residual signal

Impurity (Et3N.HCl)

Page 2 af 13 Figure S2: Acrylfentanyl (seized sample), COSY (400 MHz) in CD3OD

Coupling between the CH2- and CH3-groups of triethylamine hydrochloride is observed by COSY

Figure S3: Acrylfentanyl (seized sample), MALDI-TOF HRMS spectrum

DPS2 #1-4 RT: 0.01-0.24 AV: 4 NL: 6.80E6 T: FTMS + p MALDI Full lock ms [150.00-600.00] 335.2114 100

95

90

85

80

75

70

65

60

55

50

45

Relative Abundance Relative 40

35

30 336.2117 25

20

15

10 357.1924 5 337.2167 358.1957 339.1142 343.2150 345.1938 349.2258 351.1687 355.2150 361.2260 363.2420 369.2312 373.1664 0 335 340 345 350 355 360 365 370 m/z

Page 3 af 13 Characterisation of fentanyl (standard) Fentanyl: N-phenyl-N-[1-(2-phenethyl)piperidin-4-yl]propanamide The numbering of atoms used for NMR assignments does not follow IUPAC recommendations and is only used here for clarity (See Fig. S4 for numbering system). 1 H NMR (400 MHz, CD3OD): δ 7.57-7.46 (m, 3H, Ph), 7.31-7.23 (m, 4H, Ph), 7.22-7.15 (m, 3H, Ph), 4.62 (tt, J = 12, 4 Hz, 1H, H-5), 3.14-3.02 (m, 2H), 2.83-2.71 (m, 2H), 2.62-2.50 (m, 2H), 2.31-2.15 (m, 2H) (H-6, H-6’, H7, H-7’, H-8 and H-9), 2.01 (q, J = 7.5 Hz, 2H, H-2), 1.88 (d, J = 12.5 Hz, 2H), 1.49 (qd, J = 12.5, 4 Hz, 2H), 1.03 (t, J = 7.5 Hz, 3H, H-1). + + HRMS (MALDI-TOF) m/z found: 335.2114 [M+H] ; C22H27N2O requires M, 335.2118.

1 Figure S4: Fentanyl (standard), H NMR (400 MHz) in CD3OD

O

1 3 N 4 2 5 6

7 N

8 10 9

Page 4 af 13 Figure S5: Fentanyl (standard), MALDI-TOF HRMS spectrum

DPS1 #2-4 RT: 0.13-0.25 AV: 3 NL: 7.09E6 T: FTMS + p MALDI Full lock ms [150.00-600.00] 337.2276 100

95

90

85

80

75

70

65

60

55

50

45

Relative Abundance Relative 40

35 359.2086

30 338.2284 25

20

15

10 360.2113

5 339.2323 375.1825 340.2353 345.1918 347.0517 350.9867 353.2217 357.1933 361.2145 365.0638 368.4242 373.1872 0 340 345 350 355 360 365 370 375 m/z

1 Figure S6: H NMR (600 MHz) in CDCl3 Top: Acrylfentanyl (standard); Bottom: Acrylfentanyl (seized sample) The acrylfentanyl standard conforms to the seized sample, excl. triethylamine hydrochloride

Page 5 af 13 1 Figure S7: H NMR (600 MHz) in CDCl3 : Enlargement (4.7–7.7 ppm)

Top: Acrylfentanyl (standard); Bottom: Acrylfentanyl (seized sample)

1 Figure S8: H NMR (600 MHz) in CDCl3 : Enlargement (1.1–3.8 ppm)

Top: Acrylfentanyl (standard); Bottom: Acrylfentanyl (seized sample)

Page 6 af 13 Figure S9: DEPT (600 MHz) in CDCl3 Top: Acrylfentanyl (standard); Bottom: Acrylfentanyl (seized sample)

Figure S10: Identification of the impurity as triethylamine hydrochloride by 1 H NMR (600 MHz) in DMSO-d6. Acrylfentanyl (seized sample),

Peak separation was achieved in DMSO-d6 allowing identification of the impurity.

Page 7 af 13 Figure S11: Identification of the triethylamine hydrochloride impurity by 13 C NMR (600 MHz) in DMSO-d6 Top: Acrylfentanyl (seized sample); Bottom: Triethylamine hydrochloride (standard).

Figure S12: Identification of the impurity as triethylamine hydrochloride. Enlargement of aliphatic region for A) Acrylfentanyl (seized sample); and B) Acrylfentanyl (seized 1 sample) spiked with triethylamine hydrochloride, H NMR (600 MHz) in DMSO-d6

Page 8 af 13 Figure S13: IR spectrum of acrylfentanyl (seized sample)

Figure S14: IR spectrum of acrylfentanyl (standard)

Page 9 af 13 Instrumentation (LC-MS/MS) for quantification of acrylfentanyl The LC system modules were all from Agilent Technologies (Palo Alto, CA, USA) including a 1200 binary pump, 1200 SL autosampler and 1200 column department unit. The damper and mixer were bypassed in order to optimize the pumping system to low dead volume as described in the Agilent User Manual. Autosampler injection volume was 2 µL. The analytical column was an Kinetex Biphenyl (Phenomenex, Torrace, CA, USA), 100 × 3 mm, i.d., packed with 2.6 µm particles. The flow rate was 550 µL/min and the column temperature was 40 oC. Mobile phase A was 0.1 % formic acid. Mobile phase B was 0.1% formic acid in methanol. The binary pump gradient started at 2% phase B for 0.5 min and then went up to 95% phase B in 5 min. It was maintained at 95 % phase B for 3 min and then returned to the initial conditions for equilibration. The total run time was 9 min.

The MS system consisted of an Agilent 6460 triple quadrupole mass spectrometer (Palo Alto, CA, USA) equipped with a jet stream electrospray ion source operated in positive mode. The capillary voltage was 3500 V, the nebuliser pressure 25 p.s.i., the gas temperature 350 oC, the gas flow 8 L/min, the sheath gas temperature 375 oC ion, the sheath gas flow 8.5 L/min, the nozzle voltage 400 V and the declustering potential 130 V. Data were acquired in dynamic multiple reaction monitoring mode (dMRM). All calculations were performed used the MassHunter software (Agilent Technologies).

Table S1: Multiple Reaction Monitoring (MRM) parameters

Compound Transition MRM Collision type transistion energy Fentanyl Target ion 337 → 188 20 Qualifying ion 337 → 105 40 Qualifying ion 337 → 79 50 Acrylfentanyl Target ion 335 → 188 20 Qualifying ion 335 → 105 40 Qualifying ion 335 → 79 50 Fentanyl- d5 Target ion 343 → 105 40

Page 10 af 13 The calibrators of acrylfentanyl were prepared from a 250 µg/mL aqueous working solution of acrylfentanyl at six concentration levels: 15.625, 31.25, 62.5, 156.25, 312.5 and 625 ng/mL (free base). The concentration of acrylfentanyl in the seized sample was calculated from a 6-point calibration curve based on peak area using a linear regression curve fit, not forced through zero, with no weighting.

A working solution of the seized sample (powder) at concentration 250 ng/mL was used in the following sample preparation procedure (for sample(s) and calibrators): 100 µL sample was added to 25 µL internal standard solution (fentanyl-d5, 1 µg/mL) and 125 µL water.

The determined acrylfentanyl free base concentration from LC-MS/MS was used to calculate the mass-% of acrylfentanyl hydrochloride in the seized sample.

Figure S15: LC-MS/MS calibration curve

ACRYLFENTANYL - 6 Levels, 6 Levels Used, 6 Points, 6 Points Used, 0 QCs 99983033

x10 1

1.8 Relative Response

1.6

1.4

1.2

1

0.8

0.6

0.4

0.2

0

-20 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 Relative Concentra

Figure S16: MRM signal for m/z 335 → m/z 188 (acrylfentanyl in seized sample)

+ MRM (335.2 -> 188.1) jul-15-2016_ACRYL_11.d x10 5 5.103 min. Counts 3

2.5

2

1.5

1

0.5

0

5 5.05 5.1 5.15 5.2 5.25 5.3 5.35 5.4

Acquisition Time (m

Page 11 af 13

Figure S17: Qualifying ion transitions (overlaid) for acrylfentanyl in seized sample

335.2 -> 188.1 , 335.2 -> 105.0 , 335.2 -> 79.0 x10 2 Ratio = 104.4 (99.3 %) Ratio = 13.8 (100.3 %)

1

0.8 Relative Abundance (%

0.6

0.4

0.2

0

5 5.05 5.1 5.15 5.2 5.25 5.3 5.35 5.4

Acquisition Time (m

Figure S18: MRM signal for m/z 343 → m/z 105 (internal standard, fentanyl-d5) spiked to seized sample during sample preparation

+ MRM (343.2 -> 105.0) jul-15-2016_ACRYL_11.d x10 4 5.106 min. Counts 5

4

3

2

1

0

4.95 5 5.05 5.1 5.15 5.2 5.25 5.3 5.35

Acquisition Time (m

Page 12 af 13 Figure S19: MRM signal for m/z 335 → m/z 188 Acrylfentanyl calibrator, 156.25 ng/mL (free base)

+ MRM (335.2 -> 188.1) jul-15-2016_ACRYL_3.d x10 5 5.103 min. Counts 2.5

2

1.5

1

0.5

0

5 5.05 5.1 5.15 5.2 5.25 5.3 5.35 5.4

Acquisition Time (m

Figure S20: Qualifying ion transitions (overlaid) Acrylfentanyl calibrator, 156.25 ng/mL (free base)

335.2 -> 188.1 , 335.2 -> 105.0 , 335.2 -> 79.0 x10 2 Ratio = 105.3 (100.2 %) Ratio = 13.7 (99.3 %)

1

0.8 Relative Abundance (%

0.6

0.4

0.2

0

5 5.05 5.1 5.15 5.2 5.25 5.3 5.35 5.4

Acquisition Time (m

Page 13 af 13 Forensic Toxicol DOI 10.1007/s11419-017-0367-8

REVIEW ARTICLE

Acryloylfentanyl, a recently emerged new psychoactive substance: a comprehensive review

1 2 2 2 Istva´n Ujva´ry • Rita Jorge • Rachel Christie • Thomas Le Ruez • 2 3,4 5 2 Helgi Valur Danielsson • Robert Kronstrand • Simon Elliott • Ana Gallegos • 2 2 Roumen Sedefov • Michael Evans-Brown

Received: 18 April 2017 / Accepted: 25 April 2017 Ó Japanese Association of Forensic Toxicology and Springer Japan 2017

Abstract N-(1-Phenethylpiperidin-4-yl)-N-phenylacry- identification of the substance, as well as the limited infor- lamide, or acryloylfentanyl (acrylfentanyl), is a synthetic mation on the biological properties, including in vitro and opioid and a close structural analogue of fentanyl, which is in vivo pharmacological studies with the substance, are widely used in medicine as an adjunct to general anaesthesia summarised. Analytically confirmed acute intoxications show during surgery and for pain management. Until recently, that the signs and symptoms of acryloylfentanyl poisoning acryloylfentanyl was known only from the scientific literature, correspond to the opioid overdose triad of decreased con- but in 2016 this non-controlled substance became available on sciousness, miosis and respiratory depression. Importantly, the illicit drug market as a powder and nasal spray in Europe naloxone works as an antidote in life-threatening poisoning. and the USA. By the end of 2016, detection of acryloylfen- The major human urinary metabolites identified in fatal tanyl in six European countries, including 47 deaths associ- overdose cases were nor-acryloylfentanyl, as well as mono- ated with the drug, had been reported to the European and dihydroxylated derivatives and their conjugates. Monitoring Centre for Drugs and Drug Addiction (EMCDDA) through the European Union Early Warning Keywords Acryloylfentanyl (acrylfentanyl) Fentanyl Á Á System, which is a part of the system designed to identify and Fentanils New psychoactive substances Opioids respond to the appearance of new psychoactive substances PharmacologyÁ and toxicology Á Á that may pose potential public health risks similar to drugs controlled under the United Nations drug control conventions. Herein we review what is known about this potent narcotic Introduction opioid. In addition to describing its chemical properties and the synthetic routes, analytical methodologies for the The elucidation of the chemical structure of morphine by Gulland and Robinson in 1925 [1] paved the way for the Electronic supplementary material The online version of this development of synthetically more tractable yet potent article (doi:10.1007/s11419-017-0367-8) contains supplementary analgesics, cough suppressants and antidiarrheal agents, material, which is available to authorized users. including pethidine, methadone, fentanyl, tramadol, tapentadol, ketazocine, dextromethorphan and loperamide & Istva´n Ujva´ry [email protected] [2]. However, the idealized goal to find potent opioid medicines devoid of serious side effects, such as abuse 1 iKem BT, Bu´za u. 32, Budapest 1033, Hungary liability, dependence potential and respiratory depression, 2 European Monitoring Centre for Drugs and Drug Addiction, has so far eluded researchers. Lisbon, Portugal In the early 1960s, a series of structurally simple yet 3 Department of Forensic Genetics and Forensic Toxicology, highly potent 4-anilinopiperidine-type analgesics were National Board of Forensic Medicine, Linko¨ping, Sweden developed in the Research Laboratorium of Dr. Janssen 4 Division of Drug Research, Linko¨ping University, Linko¨ping, [3, 4]. Fentanyl (Fig. 1), the first narcotic analgesic medi- Sweden cine belonging to this class, was followed by other struc- 5 Alere Forensics, Malvern, Worcestershire, UK turally related substances, the fentanils [5, 6]. Due to the

123 Forensic Toxicol

Fig. 1 Structures of fentanyl, acryloylfentanyl and acryloyl-a- methylfentanyl

rapid onset and short duration of analgesic action and a part of the broader changes seen on Europe’s drug market relatively wide therapeutic window, a small number of this as a result of the appearance of large numbers of new family of compounds, such as fentanyl, alfentanil, sufen- psychoactive substances over the past decade [34, 35]. tanil and remifentanil, have become widely used in human In Europe, a three-step legal framework of early warn- medicine in surgical procedures as adjuncts to anaesthesia ing, risk assessment and control measures allows the and for the treatment of acute and chronic pain in various European Union (EU) to rapidly identify and react to formulations, while some are used in veterinary medicine public health threats caused by such substances. The as general anaesthetics, for pain management, and, in the European Monitoring Centre for Drugs and Drug Addiction case of carfentanil and thiafentanil, to immobilise large (EMCDDA) is responsible for the first two steps in the animals [7, 8]. system, namely operating the EU Early Warning System on Like other opioid analgesics, the analgesic activity of new psychoactive substances with Europol (the EU Police the fentanils is due to their activation of opioid receptors, agency) and conducting risk assessments. As part of this and in particular, the l-opioid receptor [9]. Besides their work, the EMCDDA monitors more than 620 new psy- analgesic properties, a notable feature associated with l- choactive substances that have appeared on Europe’s drug opioid receptor agonists is that they induce dose-dependent market over the past 20 years. Data on these substances are respiratory depression [9–11], which can be life-threaten- collected and reported by national early warning systems of ing. The importance of this effect is reflected in the fact the 28 EU member states, Turkey and Norway to the that most opioid-related deaths are caused by respiratory EMCDDA through the EU Early Warning System as well depression [12]. as from other sources. Based on data reported through the Between 1979 and 1988, more than ten fentanils, in- EU Early Warning System on acryloylfentanyl, the cluding a-methylfentanyl, 3-methylfentanyl and 4-fluoro- EMCDDA undertook a detailed investigation on this sub- fentanyl, were detected on the illicit opioid market in the stance as part of its early warning and risk assessment United States, typically sold as heroin or ‘‘synthetic her- activities [33]. oin’’. Together, they were associated with more than 100 Herein we provide a comprehensive and up-to-date overdose deaths during this period [13–15]; later, in the review on acryloylfentanyl, a close structural analogue of mid-2000s, outbreaks of poisonings linked to clandestinely fentanyl, which was first identified on Europe’s drug produced fentanyl also occurred [16–19]. With the excep- market in 2016 [33] (for the methodology used for the tion of Estonia, which saw an epidemic mainly related to preparation of the review, see supplementary material). 3-methylfentanyl and fentanyl [20, 21], fentanils caused Following the presentation of the physicochemical prop- limited problems in Europe [22–25]. In the past few years, erties, the analytical methods and the known synthetic there has been a large increase in non-fatal and fatal poi- routes of this new psychoactive substance, and the in vitro sonings in the United States and Canada associated with and in vivo pharmacology studies, including mode of fentanils that have mostly been sold on the illicit opioid action and assumed metabolism and toxic effects, are market: the fentanils are either sold as heroin to unsus- reviewed and related to those of fentanyl. Finally, serious pecting users, used to cut heroin, or used to make coun- adverse events, including symptoms of acute intoxication terfeit opioid medicines [26–28]. They have also been sold and deaths reported within Europe in 2016, are described. as other drugs such as cocaine and benzodiazepines [29–31]. At the same time, Europe has also seen an increase in Acryloylfentanyl, as a new psychoactive substance the availability of new fentanils. Twenty such substances have been identified on Europe’s drug market since Acryloylfentanyl is a structurally simple though less stud- 2012, with eight identified in 2016 alone. Seizures by law ied analogue of fentanyl that was first described in the enforcement have also increased, as have poisonings and scientific literature in the 1980s [36, 37]. In early 2016, deaths (see, for example, [32, 33]). These developments are however, the substance was detected on the illicit drug

123 Forensic Toxicol market in Denmark and Sweden, and since then, has been the citrate salt of fentanyl in water is *25 mg/mL, while in associated with more than 40 deaths in Europe. Acry- ethanol it is 7.1 mg/mL [45]. loylfentanyl, or ‘‘acryl fentanyl’’, was also identified in the Like fentanyl, acryloylfentanyl is highly lipophilic, as USA in the fourth quarter of 2016 [38]. indicated by their comparable calculated 1-octanol/water Chemically, acryloylfentanyl is an acrylamide deriva- partition coefficients (cLogP). The respective cLogP values tive of 4-anilinopiperidine and is an unsaturated analogue for acryloylfentanyl and fentanyl are 4.13 and 3.89, as of fentanyl (Fig. 1), which is a propionamide and is calculated by the ACD/ChemSketch 2015 release version included in Schedule I of the United Nations Single Con- (Advanced Chemistry Development Inc., Toronto, vention on Narcotic Drugs of 1961, as amended by the Canada). The respective cLogP values as calculated by 1972 Protocol. Neither acryloylfentanyl nor any other StarDrop version 6.3.1 software (Optibrium Ltd, Cam- unsaturated amides were claimed in the original fentanyl bridge, UK) for acryloylfentanyl and fentanyl are 3.61 and patent by the Research Laboratorium Dr. C. Janssen [3]. 3.89. The measured LogP value for fentanyl is 4.05 [46]. While acryloylfentanyl is new on the international drug Due to its high lipophilicity, cross-contamination with market, its side-chain methylated homologue, acryloyl-a- traces of the substance during sample handling and analysis methylfentanyl (Fig. 1), was found in seized drugs in the can be problematic [47]. USA decades ago [13, 39, 40]. Apart from acryloylfentanyl, other commonly used names are acryloyl fentanyl, acrylfentanyl, acryl fentanyl, Synthesis of acryloylfentanyl and akrylfentanyl (in Sweden). Its systematic (IUPAC) name is N-phenyl-N-[1-(2-phenylethyl)piperidin-4-yl]prop- There are two published syntheses of acryloylfentanyl, 2-enamide. Its Chemical Abstract name is N-phenyl-N-[1- which involve the acylation of the common precursor (2-phenylethyl)-4-piperidinyl-2-propenamide. Acryloyl- 4-anilino-N-phenethylpiperidine (4-ANPP) with acryloyl fentanyl is also known by other names such as N-(1-phenethyl- chloride [36, 37] or 3-chloropropionyl chloride [43] piperidin-4-yl)-N-acroylanilinopiperidine, N-(1-phenylethyl- (Fig. 2). The actual synthetic route used for the manufac- piperidin-4-yl)-N-phenylacrylamide, or enfentanyl. Its com- ture of acryloylfentanyl detected on the drug market is mon street names are ‘‘acryloyl-F’’ and ‘‘Acr-F’’. The acronym unknown, but it most likely relies on precursors and syn- ‘‘ACF’’ has also been used, which should not to be confused thetic methods similar to the routes mentioned above or to with ‘‘AF’’, which is one of the street names for acetylfentanyl those used for the manufacture of pharmaceutical fentanyl [32]. [48–52]. A powder sample in a capsule seized in Denmark Chemical Abstract Service Registry Numbers (CAS was shown to consist of mainly acryloylfentanyl and a RNs): 82003-75-6 for the free base, and 79279-03-1 for the substantial amount of triethylamine hydrochloride (27%) hydrochloride salt. It may be noted that in the widely used and trace amounts of 4-ANPP, indicating the use of either ‘‘Designer Drugs Directory’’ [41], the CAS RN 79297-03-1 of the routes shown in Fig. 2 for the manufacture of the given for acryloyl-a-methylfentanyl (see Fig. 1) in fact drug [53]. denotes acryloylfentanyl hydrochloride. The International It should be considered that, at least in theory, acry- Chemical Identifier Key (InChI Key) for acryloylfentanyl loylfentanyl may serve as a precursor to fentanyl by satu- is RFQNLMWUIJJEQF-UHFFAOYSA-N. rating the double bond of the acrylamide moiety using The molecular formula of acryloylfentanyl is catalytic hydrogenation (see, for example, [54]).

C22H26N2O; the monoisotopic mass is 334.2045. Acry- loylfentanyl contains one basic piperidine-nitrogen atom and thus readily forms salts with organic or inorganic acids. Analysis of physical and biological samples The reported melting point of the free base is 101–103 °C [42], while the reported melting points of its white Acryloylfentanyl has been fully characterised by 1H and hydrochloride salt are 259–260 °C[36], 252–258 °C (with 13C nuclear magnetic resonance spectroscopy decomposition) [37] and 191–194 °C[42, 43]. [37, 42, 53, 55], Fourier transform infrared spectroscopy There are no solubility data on acryloylfentanyl or its (both the free base and the HCl salt) [53, 55] and gas salts; due to its close similarity to fentanyl, the free base is chromatography coupled with mass spectrometry expected to be sparingly soluble in water; the hydrochlo- [43, 53, 55]. Quadrupole time-of-flight (QTOF) and ride and citrate salt are expected to have improved aqueous matrix-assisted laser desorption ionization Orbitrap mass solubility. Relevant data for fentanyl are as follows: at spectrometric analyses of acryloylfentanyl have also been ambient temperature, fentanyl base is poorly soluble in described [53]. The ultraviolet and visible spectrum of water (0.032 mg per mL at pH 5.9) [44]; the solubility of acryloylfentanyl have not been reported. A highly sensitive

123 Forensic Toxicol

Fig. 2 Synthesis of acryloylfentanyl from N-phenyl-1-(2-phenylethyl)- piperidin-4-amine (4-ANPP) and acryloyl chloride [36] (top) or 3-chloropropionyl chloride [43](bottom; the intermediate 3-chloropropionamide derivative shown in brackets is not isolated)

capillary electrophoresis-electrospray-tandem mass spec- antinociceptive activities of morphine, fentanyl and acry- trometry method recently developed for the trace level loylfentanyl in mice upon intraperitoneal (ip) administra- analysis of fentanils [56] could be applicable for tion are shown in Table 1. As Table 1 indicates, in this acryloylfentanyl. rodent model of analgesia, acryloylfentanyl is about 170 Similar to fentanyl, acryloylfentanyl is not expected to times more effective as an antinociceptive agent than give a positive response to immunoassay tests developed morphine, though somewhat less potent than fentanyl. for morphine-type opioids. An enzyme-linked Essawi [42, 43] studied five fentanyl analogues, immunosorbent assay kit developed for fentanyl displayed including acryloylfentanyl, as potential receptor affinity strong cross-reactivity (215%) for acryloylfentanyl [57]. labels and antinociceptive agents in the mouse using the Acryloylfentanyl did not show cross-reactivity with the hot-plate assay; morphine and fentanyl were the compar- immunoassay panel ABC-multi-10 (Simoco Diagnostic, ative standards. Upon ip administration at doses below Hillerød, Denmark), which includes MDMA [53]. It is not 1 mg/kg, acryloylfentanyl was a more potent antinocicep- known whether acryloylfentanyl can be detected by any tive agent than fentanyl: while the effect of fentanyl at presumptive colour tests. doses of 0.1, 0.2 and 0.5 mg/kg dropped considerably at Recently, a liquid chromatography (LC) screening in 60–70 min and became insignificant at 90–100 min after combination with a high-resolution tandem mass spec- treatment, ‘‘at comparable doses, acryloylfentanyl main- trometry method was developed for the qualitative and tained considerable analgesia at 90 and 120 min after quantitative forensic analysis of acryloylfentanyl and other administration. In its duration, the time-response profile of fentanils and their human metabolites with a detection limit acryloylfentanyl resembled more closely that of morphine of\5 ng/mL [58]. A method employing LC–QTOF for the (20 mg/kg) than that of fentanyl’’. Remarkably, at doses of identification of in vitro and in vivo metabolites of acry- 6.8 and 17 mg/kg, the antinociceptive effect of acry- loylfentanyl and three other fentanils has also been loylfentanyl was sustained up to 4.5 h without signs of described [59]. opioid toxicity. At the 25-mg/kg dose, motor activity was

Table 1 Antinociceptive activities of morphine, fentanyl and acry- Pharmacology: mode of action and metabolism loylfentanyl in mice upon intraperitoneal administration [36] Compound ED (mg/kg) Potency ratio to morphine Antinociceptive activity and toxic effects 50 in the mouse Morphine 13.9 1 Fentanyl 0.062 224 There have been two studies investigating the antinoci- Acryloylfentanyl 0.082 169.5 ceptive activity of acryloylfentanyl in the mouse The antinociceptive activity is characterised by the median effective [36, 42, 43]. The first publication mentioning acry- dose (ED50) calculated by measuring the prolongation of latency loylfentanyl describes an extensive structure-activity rela- times of the response to pain (hot-plate test, 55 °C) after adminis- tionship study involving 22 fentanyl analogues, with tration of the test substance at various doses as compared to untreated morphine and fentanyl as comparative standards [36]. The control

123 Forensic Toxicol transiently inhibited for 3.5 h. However, at a dose of Table 2 Opioid receptor binding data for morphine, fentanyl, acry- 50 mg/kg, convulsions developed after 1 h, and ‘‘60% loylfentanyl and (?)-3-methylfentanyl [37] lethality was observed from apparent respiratory depres- Compound IC50 (nM) sion’’. Subcutaneous (sc) pre-administration by 30 min of [3H]Naloxone [3H]Naltrexone 2 mg/kg naloxone blocked the antinociceptive effect of 0.85 mg/kg acryloylfentanyl for about 40 min when the Morphine 4.2 27 antagonist effect disappeared, and analgesia and other Fentanyl 1.6 25 morphine-like effects could be noted for about 50 min. A Acryloylfentanyl 1.4 17 similar transient antagonist effect was observed when (?)-3-Methylfentanyl 0.6 1.3 naloxone (2 mg/kg, sc) was administered 40 min after The receptor affinity is expressed by IC50 values, representing the acryloylfentanyl-treatment (0.85 mg/kg, ip): the reversal of concentration required for displacement of 50% of tritiated naloxone the antinociceptive effect lasted for 70 min, and then or naltrexone as radioligands in a competition assay using rat brain antinociception returned to the same level as before homogenates naloxone administration. It was concluded that acry- loylfentanyl ‘‘has a mode of interaction with l-receptors different from morphine’’. indicate that the opioid receptor affinity of acryloylfentanyl There is limited information on the acute toxicity of is similar to that of fentanyl and somewhat higher than that acryloylfentanyl. In the mouse, ip injection of 25 mg/kg of of morphine in this particular rat brain preparation. the drug caused a transient suppression of motor activity; Laboratory experiments failed to find evidence for however, as mentioned above, a dose of 50 mg/kg pro- irreversible binding of acryloylfentanyl to opioid receptors: duced convulsions 1 h after drug administration, and ‘‘60% after incubation, acryloylfentanyl could be completely lethality was observed from apparent respiratory depres- ‘‘washed out’’ from the receptor preparation; the recovered receptor was able to bind radiolabelled naltrexone, which sion’’ [43]. Based on this study, the ip LD50 value, that is the dose required to kill half of the experimental animals, indicates reversible binding of the fentanyl analogue. of acryloylfentanyl may be estimated as between 25 and Similar non-irreversible binding was observed for an 50 mg/kg. No comparative standard was used in this par- acrylamide derivative of naltrexamine [65]. Thus, the sustained antinociceptive activity observed by Essawi ticular study. Mouse ip LD50 values reported for fentanyl were 17.5 [60], 26.3 [61] and 76 mg/kg [62]; for morphine, [42, 43] is unlikely to be related to covalent binding of acryloylfentanyl to opioid receptors. mouse ip LD50 values of 140 [63] and 340 mg/kg [64] have been reported. These data indicate that the acute toxicity of A recent study also confirmed the low reactivity of acryloylfentanyl in the mouse is similar to that of fentanyl compounds containing the acrylamide ‘‘warhead’’, which and is higher than that of morphine. There is no informa- has often been considered as a promiscuous electrophilic tion on the chronic toxicity of acryloylfentanyl. moiety capable of binding to cysteine or other nucleophilic moieties present in target/off-target receptors and enzymes/ Interaction with opioid receptors other proteins [66]. Nevertheless, there has been an interest in medicinal chemistry research to develop irreversible Maryanoff et al. [37] determined the binding affinities of a inhibitors containing an acrylamide moiety or related series of compounds, including acryloylfentanyl, using a electrophiles [67]; in fact, osimertinib, an N-phenylacry- rat brain receptor preparation and tritiated naloxone or lamide derivative-type tyrosine kinase inhibitor, has naltrexone as competing receptor ligands (Table 2). The received marketing authorisation in the USA and the EU. fentanyl analogues were designed as potential covalent Because polymerisation processes involving acrylamide receptor affinity labels. Morphine, fentanyl and the highly or other acrylic type substances require photochemical or potent fentanyl analogue (?)-3-methylfentanyl were the free radical initiation, spontaneous polymerization of comparative standards. The affinity of the test compounds acryloylfentanyl in the body can be ruled out. Whether any to l-opioid receptors was characterised by the half maxi- ‘‘biochemical’’ initiator could be involved in the observed long-lasting activity of acryloylfentanyl in vivo can only be mal inhibitory concentration (IC50), that is the molar con- centration of the drug displacing 50% of l-opioid receptor- speculated (see section on animal studies above). No sta- preferring tritiated naloxone or naltrexone from the bility studies have been carried out with acryloylfentanyl. receptor preparation. Other biological activity studies As seen in Table 2, the IC50 values obtained for fentanyl and acryloylfentanyl are practically identical; morphine is somewhat less effective in inhibiting the binding of radi- A search in the PubChem Substance database for biological olabelled receptor antagonists. The results of this study activity of acryloylfentanyl found 28 test results deposited,

123 Forensic Toxicol but the substance was found inactive in all assays that dihydroxylated at the acryl moiety (not shown in Fig. 3), included a range of non-opioid-related targets [68]. were also detected. Similar to the metabolism of fentanyl, amide hydrolysis (deacylation) affording 4-ANPP (B14) Pharmacokinetics was a minor pathway. Terminal monohydroxylation of the acyl chain, producing what is often called ‘‘hydroxyfen- Due to its lipophilicity, acryloylfentanyl, like fentanyl, is tanyl’’, is a biotransformation step for fentanyl in humans, expected to readily cross the blood-brain barrier and also to but such hydroxylation cannot take place in the case of diffuse into fat and other tissues (acryloylfentanyl is acryloylfentanyl. Intact acryloylfentanyl was also present expected to have a large volume of distribution). in the urine of the decedents. The urinary concentrations of The pharmacokinetics and the metabolic pathway of the metabolites were not quantified; the blood acry- acryloylfentanyl are expected to be similar to those of loylfentanyl concentration for these five deaths ranged fentanyl [69–72] or acetylfentanyl [73–75]. A recent study from 0.05 to 1.20 ng/g. determined the structures and relative abundance of the There is some information on the biological activity of major acryloylfentanyl metabolites produced by human two potential metabolites of acryloylfentanyl. An early hepatocytes in vitro and of those detected in the urine in study [78] assessing the opioid-like activity of several five cases of death related to acryloylfentanyl [59]. Using fentanyl metabolites in the guinea pig ileum assay found LC–QTOF analyses, these studies identified altogether 14 that 4-ANPP and 4-anilinopiperidine were less potent than biotransformation products, including phase I and II either fentanyl or morphine by several orders of magnitude. metabolites. The major metabolites of acryloylfentanyl The only metabolite showing significant activity in this detected in human urine after hydrolysis of glucuronidated study was a phenolic derivative hydroxylated at the 4-po- and/or sulfated phase II conjugates are depicted in Fig. 3. sition of the phenylethyl moiety of fentanyl (for number- Similar to the well-studied metabolism of fentanyl ing, see Fig. 1), the activity of which was found to lie [70, 76, 77], the biotransformation involves an oxidative N- between morphine and pethidine. Therefore, the corre- dealkylation, presumably catalysed by cytochrome P450 sponding phenolic metabolite of acryloylfentanyl (not (CYP450) enzymes, leading to the biologically inactive detected in the recent study [59]), if formed, may have desphenethyl metabolite, that is ‘‘nor-acryloylfentanyl’’ some level of opioid activity and thus may contribute to the (metabolite B1 in the original study). Additional oxidative biological, including toxicological, properties of the parent metabolic processes were monohydroxylations either on substance. the alkyl chain of the phenylethyl moiety or on the piper- idine ring, affording metabolites B9 and B13 (hydroxyla- Interindividual genetic variability in metabolising tion site not assigned). Dihydroxylation of the aromatic enzymes ring of the phenylethyl moiety followed by O- monomethylation of the resulting catechol afforded For fentanyl, oxidative dealkylation by hepatic CYP450 metabolite B11 (and, to some extent, its regioisomer; not 3A4 and CYP450 3A5 isoenzymes has been demonstrated shown in Fig. 3). Monophenols (B10) and diols, such as [71, 76, 79]. The wide variation in the expression of the diphenol derivatives of the aniline moiety or a species genes coding for these CYP450 3A isoenzymes among

Fig. 3 Major human metabolites of acryloylfentanyl, with metabolite numbering according to original publication [59]. Note that due to incomplete acylation during manufacture or to product mislabelling, 4-ANPP could also be present in the consumed products; thus its detection in biological matrices might not always be indicative of metabolism

123 Forensic Toxicol populations is of clinical significance, but the toxicological seizures, serious adverse event reports and Internet drug consequence of such polymorphisms has not been inves- forums. Acryloylfentanyl may be taken orally as powder in tigated in fentanyl-overdose cases. capsules or as tablets; intravenous injection has also been reported. A novel and apparently popular route of intra- Interactions with other substances, medicines, nasal administration is the use of a metered nasal spray, and other forms of interactions which delivers approximately 0.1 mL upon one actuation. Such ready-to-use nasal sprays were sold by online vendors Should acryloylfentanyl undergo oxidative dealkylation by in Sweden in 2016, and the amount of acryloylfentanyl in CYP450 3A4 and CYP450 3A5 isoenzymes, then the use these sprays is typically claimed to be 20 mg as a solution of this substance with inhibitors of these isoenzymes, such in a 10-mL spray bottle capable of delivering *100 as clarithromycin, indinavir, ritonavir, saquinavir, itra- actuations (i.e., *0.2 mg per spray) [58]. In addition, user conazole, ketoconazole, nefazodone (all strong CYP3A reports mention snorting or inhalation by smoking the free inhibitors), erythromycin, fluconazole, grapefruit juice, and base of acryloylfentanyl; vaping of the drug from an e-ci- verapamil (all moderate CYP3A inhibitors) [80, 81], may garette containing a homemade flavoured e-liquid has also result in prolonged high plasma concentration of acry- been mentioned [83]. Injecting users have used marketed loylfentanyl which could be toxicologically significant, for nasal spray solutions or have themselves prepared a solu- example by increasing the risk of potentially fatal respi- tion of the citrate salt of the free base of the drug, some- ratory depression. times using alcohol as co-solvent. Preparation of a The concomitant use of other central nervous system homemade transdermal patch has also been described [84]. (CNS) depressants with opioid analgesics, including other From the available limited data, it is not possible to opioids, sedatives/hypnotics (such as the benzodiazepines discern the ‘‘typical’’ dose or dose regimens administered and the z-drugs), ethanol, gabapentinoids (pregabalin and by users. While a range of doses and regimens have been gabapentin), tranquillisers, sedating anti-histamines, and reported, these appear to differ depending on factors such skeletal muscle relaxants may produce additive depressant as the route of administration, the user tolerance, the use of effects [80]. other drugs, and the desired effects. Given that the actual Similar to fentanyl, the use of partial opioid agonists/ composition of the substance sold on the drug market may antagonists (such as buprenorphine, nalbuphine, penta- differ over time and among geographical areas, the dose zocine) which have high affinity to opioid receptors but mentioned in such reports is problematic, because the relatively low intrinsic activity could partially antagonise purity and/or composition of the substance ingested is the effects of acryloylfentanyl and may induce withdrawal typically not known by the user. In fact, it has recently been symptoms in people who are opioid dependant [80]; it is reported that some ‘‘akrylfentanyl’’ products sold in Swe- unknown if such effects are possibly protective in indi- den contained fentanyl instead of acryloylfentanyl [58]. A viduals poisoned with acryloylfentanyl or other fentanils. confusing factor regarding the dose used is the (probably) While data are lacking for acryloylfentanyl, the use of indiscriminate use of ‘‘mg’’ (milligram) and ‘‘lg’’ (mi- fentanyl with serotoninergic agents, such as selective crogram) weight units in Internet forums. The difference in serotonin reuptake inhibitors (the most commonly pre- dose by several orders of magnitude could result in a fatal scribed antidepressants) or serotonin-norepinephrine reup- overdose for an ‘‘uncut’’ product, if such false information take inhibitors or monoamine oxidase inhibitors has been is propagated on the Internet. Given the difficulties in associated with serotonin syndrome, which is a potentially collecting such data accurately, the information below life-threatening condition [80, 82]. This association is should be used with caution. likely to extend to exposure to illicit drugs which act on the Though human clinical studies are lacking, based on serotonergic system, such as MDMA and amphetamines. non-clinical data (see above), it may be assumed that the Unpredictable potentiation by monoamine oxidase inhibi- opioid-like effects of acryloylfentanyl manifest at doses tors has been reported with some opioid analgesics similar to those observed for fentanyl. Those posting self- [80, 82]. reports of use on Internet forums have mentioned that sub- milligram doses administered by nasal spray were psy- choactive; doses of 0.0027–0.2 mg have been reported. In Human data comparison, in human clinical trials, acute fentanyl doses of 0.2 mg intravenously [85], 0.015 mg/kg oral-transmu- Routes of administration, dose regimens, and effects cosally (lozenge) [86] or up to 1.6 mg intramuscularly [87] have been used. Information on the routes of administration and dose reg- Information on the psychological and behavioural imens is limited, and originated from customs, police effects of acryloylfentanyl is limited to serious adverse

123 Forensic Toxicol events reported to the EMCDDA [33] and self-reported three EU member states: Denmark (1 case), Estonia (3), experiences from Internet forums. The psychoactivity of and Sweden (43) [33]. Detailed information is available acryloylfentanyl is reportedly similar to that of other opi- only for the death cases in Denmark and Sweden. The oids and includes relaxation and euphoria. Internet forums decedents were predominantly male (86%), and their age rarely mention side or adverse effects typical of opioids, ranged from 19 to 54 years; the age of the female dece- though some discussions on these sites describe acry- dents ranged from 29 to 50 years. The deaths typically loylfentanyl as ‘‘longer lasting’’ after dosing than other occurred in a home environment. Thirty-two of the deaths new synthetic opioids. occurred between June and August 2016. In at least 40 deaths, acryloylfentanyl was either the cause of death or Human poisoning cases was likely to have contributed to death (even in the pres- ence of other substances); in two of these deaths, acry- It is important to note that the number of serious adverse loylfentanyl was the sole drug present. events, including deaths, involving acryloylfentanyl is Forensic analysis also revealed a range of other sub- likely to be underestimated, since testing for this specific stances in the deaths, suggesting that polydrug use was substance is not commonly performed in opioid overdose common. These included a number of CNS depressants cases. such as benzodiazepines, zopiclone, pregabalin, gaba- pentin, ethanol and cannabinoids (including synthetic Non-fatal intoxications cannabinoids), as well as synthetic cathinones, amphetamines, antidepressants and antipsychotics. How- So far, Sweden has been the only country that has reported ever, acryloylfentanyl was the sole opioid present in 38 acryloylfentanyl-related acute intoxications. These cases cases (86%). In the remaining cases, the opioids detected relate to presentations to hospital emergency departments were buprenorphine, hydrocodone, oxycodone, 4-fluo- made between March and October 2016 [33, 58]. With one roisobutyrfentanyl and 4-chloroisobutyrfentanyl. exception, the patients were men, ranging in age from 23 to 51 years; the age of the female patient was 19 years. Treatment of poisonings Acryloylfentanyl was typically administered as nasal spray. There were eight cases in which acryloylfentanyl was In suspected acryloylfentanyl overdose cases, naloxone, an analytically confirmed to be the sole opioid, with serum opioid receptor antagonist, should be administered in doses and urine acryloylfentanyl concentrations ranging from 0.5 typical to rescue heroin overdoses [88]. However, given the to 2.1 and from 1.8 to 196 ng/mL (creatinine-normalized short duration of action of naloxone and the high potency concentration: 0.2–10.5 lg/mmol creatinine), respectively. and different pharmacokinetics of acryloylfentanyl, re- One additional case in Sweden during this period peated doses of the antidote may be required to prevent any involved a combination of acryloylfentanyl and 4-chloro- reoccurrence of respiratory depression [89, 90]. In six of isobutyrfentanyl that was administered by injection of an the acute acryloylfentanyl intoxication cases that occurred extract of pills. Forensic analysis of three other intoxica- in Sweden (see above), naloxone was administered intra- tions that occurred in March 2016 and were believed to venously by paramedics and/or emergency department involve acryloylfentanyl identified fentanyl rather than personnel at 0.1–0.4 mg, and oxygen supply was provided; acryloylfentanyl (see also the section ‘‘Routes of admin- one of these cases required continuous infusion for *7h istration, dose regimens, and effects’’). In some cases, during hospital observation; oxygen supply was also doc- analysis indicated the intake of alcohol or other licit or umented in seven of the cases [58]. illicit substances [33, 58]. Naloxone has also been used with success to reverse The clinical symptoms related to these acute intoxica- poisonings caused by other fentanils that have appeared on tions appeared to be consistent with the use of an opioid the drug market, including 3-methylfentanyl [91], and included decreased consciousness, respiratory depres- acetylfentanyl [32], furanylfentanyl [30] and butyrylfen- sion and miosis. Other common features were tachycardia tanyl [92]. and hypertension. In six of these cases, naloxone was administered by paramedics and/or emergency department personnel [58]. Risks from accidental exposure

Death cases It is important to emphasize that accidental exposure, such as skin contact, inhalation or ingestion, to acryloylfentanyl A total of 47 analytically confirmed deaths associated with and other fentanils poses a serious health risk to the public, acryloylfentanyl that occurred in 2016 were reported by law enforcement, medical and forensic laboratory

123 Forensic Toxicol personnel, postal services and in custodial settings. Where an acrylamide derivative of 4-anilinopiperidine and is an necessary, such risks should be assessed, and appropriate unsaturated analogue of fentanyl, which is a controlled procedures, training, and environmental and personal pro- narcotic drug. In non-clinical laboratory studies, it has been tective measures should be provided for handling materials shown to be an opioid receptor agonist and a potent and suspected of containing these drugs. This may include long-lasting antinociceptive agent. Typically it is sold as a training in resuscitation and adequate provision of the legal alternative to illicit opioids. opioid antagonist naloxone to reverse accidental poisoning In 2016, acryloylfentanyl was involved in 47 deaths, [27, 89, 93]. with at least 68% of the deaths occurring within a 3-month period. In the majority of the deaths, acryloylfentanyl was reported to be either the cause of death or to have con- Legal status tributed to the death. In addition, more than 20 acute intoxications suspected to be due to acryloylfentanyl were Acryloylfentanyl is not controlled under the United reported. The clinical features were generally consistent Nations Single Convention on Narcotic Drugs of 1961, as with opioid-like toxicity and included life-threatening amended by the 1972 Protocol, or the Convention on effects. Naloxone was shown to be an antidote to poisoning Psychotropic Substances of 1971. caused by acryloylfentanyl, though in some cases repeated In the EU, acryloylfentanyl is controlled under drug doses were required. control legislation in Cyprus, Denmark, Estonia, Finland, One common way of administering acryloylfentanyl Ireland, Latvia, Lithuania, Sweden and the UK; in Austria appears to have been with ready-to-use nasal sprays, which and Poland the substance is controlled under specific new were sold by online shops. These dosage forms, and others psychoactive substance control legislation. In Turkey, the such as e-liquids, have the potential to make the use of substance is also controlled under drug control legislation. fentanils easier (as compared to injecting) and more In Norway, the importation of, trade in and marketing of socially acceptable; these are developments that will acryloylfentanyl is controlled by the Medicines Act [33]. require careful monitoring. ‘‘Acrylfentanyl’’ has been controlled in China since 1 The risks from acryloylfentanyl are not limited to those March 2017 [94]. who use the substance. Accidental exposure to the sub- stance, and in fact to other fentanils, poses a serious risk to family and friends of users, as well as law enforcement, Conclusions emergency personnel, medical and forensic laboratory personnel and those in custodial settings and postal ser- Over the past decade there has been a dramatic worldwide vices. Specific risks should be identified and appropriate increase in the number and availability of new psychoactive risk reduction measures implemented. This may include substances. Much of this has been driven by the exploitation of appropriate protective equipment, training in resuscitation, globalization, economic development, and new technologies and making naloxone readily available to relevant per- by entrepreneurs, where chemical and pharmaceutical com- sonnel in sufficient quantities in the event of exposure. panies based in China can produce bulk quantities of new As of 1 March 2017, acryloylfentanyl has been con- substances and then cheaply and rapidly ship them to cus- trolled under drug control legislation in China. As a result, tomers across the world. The fallout from this has been an the open manufacture and sale of this substance may at increase in reported harms. In this paper we reviewed the case least be deterred. Despite this development, it is important of acryloylfentanyl, which is one of 20 new fentanils that have to note that since acryloylfentanyl was first detected in appeared on the drug market in Europe since 2012. Europe, an additional eight new fentanils have been Until recently, acryloylfentanyl was known only from detected on the drug market, including 4-fluoroisobutyr- the scientific literature, but in 2016 this non-controlled fentanyl, 4-chloroisobutyrfentanyl, tetrahydrofuranylfen- synthetic opioid became available on the illicit drug market tanyl and methoxyacetylfentanyl. Based on an analysis of as a powder and nasal spray in Europe and the USA. By the the literature, there are dozens of other fentanils which end of 2016, detection of acryloylfentanyl in six European could emerge as new psychoactive substances. countries (Denmark, Estonia, Finland, Latvia, Sweden, and In recent years the emergence of hundreds of new psy- Slovenia1) had been reported to the EMCDDA through the choactive substances on Europe’s drug market has driven EU Early Warning System. Chemically, acryloylfentanyl is greater complexity into the drug problem. The market is highly dynamic and demonstrates increasingly innovative attempts to circumvent regulation. The recent appearance 1 Slovenia reported a sample of light green powder which was a test purchase from an Internet vendor. The sample was shipped from of a large number of new fentanils as part of this market is China and was received in May 2016. of particular concern to public health, given the serious

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