1. 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

2. 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.

3. 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

4. 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Rapid immunological tests (quickly outdated, lack sensitivity)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!

5. 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

6. 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).

7. 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).

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

9. Results: development of bioassay
SmBiT
LgBiT
Untreated
Stimulated

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

11. 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

12. 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.

13. 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

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

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

16. 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

17. 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

18. Annelies Cannaert PhD student Department Bioanalysis Laboratory of Toxicology Ghent University - Campus Heymans (FFW) Ottergemsesteenweg 460 B-9000 Gent, Belgium E T
National Institute of Criminalistics and Criminology Laboratory of Toxicology Vilvoordsesteenweg 100
B-1120 Belgium, Belgium