Identification & Assessment of NPS: an European Network WS1 WS1.3c Prediction of ADME in human body WS1.3a In vitro metabolic profile WS3.1 Synthesis of standards and metabolites WS3.2 Development, validation and standardisation lof analytical methods for NPS markers in wastwater WS2 Cytotoxicity and in vivo studies The FFUL team flow chart NPS prioritization Estimating NPS use in European populations WS3.4 Milan, 28th February-1st March 2016

In vitro metabolic profile (WS1.3a) Post-Doc Kinetics: liver S9 fractions incubated with NPS Half-lives ClH Extraction ratio Km and vmax Metabolic profile: liver S9 fractions and hepatocits incubated with NPS Metabolite ID Comparison with real samples Prediction of metabolites with CNS toxicity Structure-activity relationships

Prediction of human absorption, distribution and elimination through In Silico models (WS1.3c) Molecular descriptors (e.g. molecular structure-activity relationship) Drug related PK parameters PK behaviour E-Dragon Software ANN QSARs PBPK models Selection of candidates ANN= Artificial Neuronal Network; QSAR= Quantitative structure-activity relationship PBPK= Phisiologically based pharmacokinetic model

Milan, 28th February-1st March 2016 Prediction of human absorption, distribution and elimination through In Silico models Venou s Arterial Others Brain Muscle Fat Kidney Liver Gut Intestine Lungs QL QB QO QM QF QK QHV QHA QPV QG CLR CLH D ka Available QSAR models QSAR for Blood:Plasma Concentration Ratio QSAR for Hepatic Clint QSAR for CACO-2 Permeability (12 descriptors) QSAR for Prediction of the Tissue:Blood partition coefficients (8 descriptors) QSAR for prediction of Plasma Protein Binding Milan, 28th February-1st March 2016

Prediction of human absorption, distribution and elimination through In Silico models Advanced Compartmental Absorption and Transit physiologically based model: predict compound dissolution, transit and amount absorbed through the gastrintestinal tract.

Milan, 28th February-1st March 2016 Synthesis of standards & metabolites (WS3.1) Synthesis: For selected NPS “All-H” metabolites Deuterated metabolites Kinetic isotopic effects (KIEs) from WS1 Structure elucidation: For selected NPS Isolation from samples (prep HPLC) NMR (including 2D COSY, HMQC and HMQB) MS FTIR, circular dichroism Milan, 28th February-1st March 2016

Milan, 28th February-1st March 2016 Development, validation and standardisation of analytical methods for NPS markers in wastewater (WS3.2) NPS : analysis of parent/metabolites in wastewater Implementation and validation HPLC-MS/MS methodology for quantitation (MRM mode) Milan, 28th February-1st March 2016

Estimate NPS use in European populations (WS3.4) Passive sampling technique: validation procedure + NPS quantification Urine samples from patients admitted in emergency rooms in central hospitals Wastewater plants and sampling NPS pharmacokinetic properties (metabolism and excretion patterns) Predicted from in vitro (Ws1/3a) Predicted from in silico (Ws1/3c) From literature for specific compounds Estimates of NPS population consumption patterns Milan, 28th February-1st March 2016

Identification & Assessment of NPS: an European Network TIMELINE In vitro metabolism: RM,MB, CS Prediction of ADME: in silico studies: NS, PP Citotoxicity and in vivo studies: DB,CS Synthesis of standards and metabolites: RM, MB Validation of analytical methods: MB, CS Estimating NPS in European Populations PP,NS, CS Milan, 28th February-1st March 2016

By now the Biggest Concern... Deffinition of the standards to work with and start: In vitro metabolic study In silico metabolic study Synthesis of metabolites Implementation of analytical methodologies to screen standards and metabolites in wastewater Estimation of use