Development and Activity Profiling of Synthetic Cannabinoids and Their Metabolites with a Newly Developed Bioassay. Annelies Cannaert, Jolien Storme, Florian Franz, Volker Auwärter, Christophe Stove Lisbon Addictions 2017

NPS: a worldwide problem Last decade: 620 new psychoactive drugs (NPS) on the market reported by EMCDDA. Large group are the synthetic cannabinoids (SCs). Figuur toevoegen Seizures of SCs reported to the EU Early Warning System (EWS): trends in number of seizures (top left), quantity seized (bottom left) and number of new SCs (right). EMCDDA, European Drug Report: Trends and Developments 2016.

NPS: a worldwide problem Their consumption poses serious problem for: Public order Public health MAJOR challenge: continuous development of NPS New ‘unknown’ compounds enter the market at high pace. Figuur toevoegen

NPS: a worldwide problem Their consumption poses serious problem for: Public order Public health MAJOR challenge: continuous development of NPS New ‘unknown’ compounds enter the market at high pace. Current detection methods of these NPS are structure-based: Rapid immunological tests (quickly outdated, lack sensitivity) Targeted methods need to have update spectral libraries. High-end equipment (time-consuming, tedious, expensive) Obtaining objective information on traffic, use and effects is difficult! Figuur toevoegen Public organisations are always a step behind!

New approach First-line cell-based screening assay for the general detection of SCs, based upon receptor activation potential rather than upon their chemical structure. Requirements for test to demonstrate the presence of SCs: Rapid Inexpensive High-throughput-compatible Up-to-date Applicable on: Bulk powders Biological matrices

Activity-based methodology Large subunit (156 aa) LgBit Based on structural complementation of a chemiluminescent enzyme for detecting protein interactions. (NanoLuc® Binary Technology - Promega). Both subunits are attached to the proteins of interest. In our case: Cannabinoid receptor β-arrestin 2. SmBit Small subunit (11aa) Figuur toevoegen NanoLuc® Binary Technology (Promega).

Activity-based methodology LgBiT RECEPTOR Figuur toevoegen Interaction of the cannabinoid receptor with SCs will lead to the functional complementation of NanoLuc enzyme. Bioluminescence. NanoLuc® Binary Technology (Promega).

EMCDDA paper award Development of the bioassay Application on reference materials Proof of concept on urine matrix

Results: development of bioassay SmBiT LgBiT Untreated Stimulated

Results: application on reference materials Activity-profiling of ‘new’ SCs on the market using reference materials Figuur toevoegen Potential use of bioassay to help to understand the potential activity or harm that SCs may cause.

Results: application on reference materials Activity profiling of SCs metabolites shows the activity is retained Figuur toevoegen JWH-018 JWH-122 MAM-2201 JWH-210 EAM-2201 PB-22 5F-PB-22

Results: application on bulk materials Activity profiling of SCs metabolites shows the activity is retained. Figuur toevoegen Potential contribution of active metabolites to the activity of SCs.

Results: application on urine Deployment as first-line screening tool (complementing targeted and untargeted analytical assays and/or preceding analytical confirmation). Analysis of genuine urine samples of 3 blank users and a user who consumed a mixture of SCs (JWH-018, -122 and -210). Drug Metabolite Semi-quantitative result JWH-018 4-OH-pentyl 0.7 ng/ml   5-OH-pentyl 0.4 ng/ml JWH-122 0.07 ng/ml <0.01 ng/ml JWH-210 1.4 ng/ml 0.1 ng/ml 5-OH-indole 0.06 ng/ml Figuur toevoegen

Follow up paper (August 2017) Application on other SCs Evaluation of the bioassay on a large set of urine samples

Synthetic opioids Similar bioassay has been developed Initial results are promising: Application on real life sample (carfentanil case):

Novelty and impact Novel approach in toxicological/forensic setting! Detection of SCs regardless structural diversity, allowing up-to-date high-throughput detection. The methodology used is not limited to application on SCs alone, but can also be applied on other drugs of abuse, such as synthetic opioid drugs. Increase of the detection capabilities of NPS for customs, public health and forensic laboratories. → obtaining objective information on traffic, use and effects! Figuur toevoegen

Acknowledgments Laboratory of Toxicology, UGent Prof. Christophe Stove Jolien Storme Institute of Forensic Medicine, Freiburg Prof. Volker Auwärter Florian Franz Laboratory of Toxicology, NICC Dr. Sarah Wille Agency for Innovation and Technology (IWT Flanders) Belspo BRAIN project

Annelies Cannaert PhD student Department Bioanalysis Laboratory of Toxicology Ghent University - Campus Heymans (FFW) Ottergemsesteenweg 460 B-9000 Gent, Belgium E annelies.cannaert@ugent.be T +32 9 264 81 20 National Institute of Criminalistics and Criminology Laboratory of Toxicology Vilvoordsesteenweg 100 B-1120 Belgium, Belgium