1. 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

2. 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

3. 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

4. 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

5. 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.

6. 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

7. 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

8. 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

9. 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

10. 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