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Toxicokinetics of drugs of abuse and tools to perform studies on metabolism
Dr. Markus R. Meyer, PhD([email protected])
Department of Experimental & Clinical Toxicology Saarland University, Homburg (Saar), Germany
Toxicokinetics ?
Toxicokinetics "what the organism does with the xenobiotic"
Toxicodynamics "what the xenobiotic does to the organism“
Pharmacokinetics Pharmacodynamics
therapeutic drugs
Liberation
Absorption
Distribution
Metabolism
ExcretionSimon T et al. N Engl J Med 2009;360:363‐375
Pharmaco‐/Toxicokinetics of Drugs of Abuse ?
Drugs of Abuse ??
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Liberation
Absorption
Distribution
Metabolism
Excretion
Determinants of Toxicokinetics
(Simon T et al. N Engl J Med, 2009)
Stability of Drug in SGF and SIF
(Simon T et al. N Engl J Med 2009;360:363‐375)
Drug Absorption
(Simon T et al. N Engl J Med 2009;360:363‐375)
Drug Absorption
(Simon T et al. N Engl J Med 2009;360:363‐375)
(Kitamura et al. Naunyn Schmiedebergs Arch Pharmacol, 2008)
Are DoA substrates or inhibitors
of transporters ???
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Affinity of DoAs to P‐glycoprotein
butylone
naphyrone PCEPA
diclofensine methohexital
DOI 3,4‐BDB
NPDPA glaucine 2C‐I 2C‐B 2C‐T2
BZP PCPr MDPV D2PM
PPP MPHP methylone
4‐MTA
PMMA mitragynine mephedrone
cocaine dimethocaine MDPPP
4‐F‐MA
MDMA MDE MBDB MDAI
3‐Br‐methcathinone
4‐F‐methcathinone
DPA NEDPA
Initial Pgp ATPase activity tested for 35 DOAs
(Meyer/Orschied/Maurer, Toxicol Lett, 2013)
P‐gp Kinetics of Model Substrates
Km= 2.3 µM (ref. 5 µM)vmax= 0.04 pmol/µg/min
Verapamil
Km= 7.1 µMvmax= 0.05 pmol ATP/µg/min
Glaucine (alkaloid of Glaucium flavum (Papaveraceae)
(Meyer/Orschied/Maurer, Toxicol Lett, 2013)
NO
O
O
O
H
Bidirectional Permeability Through Caco‐2 Cell Monolayers
permeability of test compounds through Caco‐2 cell monolayers (apical‐to‐basolateral and basolateral‐to‐apical)
Drug Transport Through Cell Membranes
(Meyer/Wagmann/Daum/Lehr/Maurer, TIAFT Buenos Aires, 2014)
Efflux ratio of model substrate rhodamine 123
9 for verapamil (known P‐gp inhibitor)8 for glaucine
=> glaucine is also a P‐gp inhibitor
Glaucine Misuse
• 23-year-old woman
• Two tablets of “head candy”
• “In another world”
• Mydriasis
• Nausea and vomiting
(Meyer GM/Meyer MR/Wissenbach/Maurer, J Mass Spectrom, 2013)
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Drug Distribution – Plasma Protein Binding
Protein‐bound drug
Free drug
• No pharmacologic effects• No metabolism• No excretion
• Pharmacologic effects• Metabolism• Excretion
PPB of selected Drugs
decreasin
gP
PB
PPB, % ref., %Verapamil 27 27Sertraline 92 90Diclofensine >99Naphyrone 99NPDPA 85Glaucine 85DOI 70PCEPA 67Butylone 573,4-BDB 41
(Meyer/Orschiedt/Leibnitz/Maurer 2012,2014)
Liberation
Absorption
Distribution
Metabolism
Excretion
Determinants of Toxicokinetics
Simon T et al. N Engl J Med 2009;360:363‐375
Drug Metabolism
Definition:
Biochemical modification of substances by living organisms
• increase of hydrophilicity elimination
• usually detoxification
• cause of drug-drug or drug-food interactions
• 3 phases:
– Phase I: functionalization
– Phase II: conjugation
– (Phase III: transport processes)
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Drug Metabolism Enzymes
Mitochondria•Monoamine oxidase (MAO)
Cytosol•Catechol-O-methyltransferase (COMT)
•Sulfotransferase (SULT)
•N-acetyltransferase (NAT)
•Gluthathion-S-transferase (GST)
Endoplasmatic reticulum•Cytochrome P450 (CYP)
•Flavin-monooxigenase (FMO)
•UDP-glucuronyltransferase (UGT)
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Drug Metabolism Studies
First step in (in vitro) metabolism studies
Elucidation of the chemical structure of phase I and II metabolites
In Vitro-Metabolism Studies
recombinant enzymes
• insect-cell microsomes (CYP, UGT)
• E.coli-derived (SULT)
subcellular fractions
• S9
• microsomes
• cytosol
intact cells
• fresh hepatocytes
• cryopreserved hepatocytes
different species• rat
• dog
• human, …
different tissues• liver
• small intestine
• kidney, lung, …
In vitro ToolsPros and Cons
microsomes S9 hepatocytes
• complete enzyme profile
• complex
• expensive
• low throughput
• no need of cofactors
• CYP, FMO, UGT
• easy to use
• cheap
• high throughput
• need of cofactors
• CYP, FMO, UGT
• COMT, SULT, GST, NAT
• easy to use
• cheap
• high throughput
• need of cofactors
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Assay CompositionSubcellular Fractions
Enzymes
Cosubstrates
Buffer
Additives
Substrate
• recombinant enzymes (CYP, UGT, SULT)
• subcellular fractions (microsomes, cytosol, S9)
• NADPH/H+, UDPGA, PAPS, …
• phosphate buffer, pH 7.4
• TRIS buffer, pH 7.4
• SOD, alamethicin, 1,4-saccharic acid lactone
Qualitative Metabolism Studies
Example on NPS
Qualitative Metabolism StudiesHuman Liver Microsomes
Wintermeyer/Möller/Thevis/Jübner/Beike/Rothschild/Bender, Anal Bioanal Chem, 2010
Enzymes
Cosubstrates
Buffer
Additives
Substrate
• HLM 20 mg/mL
• NADPH regenerating system• NADP 1.3 mM /glcuose 6-phosphate 3.3 mM
• glucose 6-phosphate dehydrogenase 1.6 U/mL
• phosphate buffer 100 mM, pH 7.4
• SOD 240 U/mL
• MgCl2 3.3 mM
• JWH 018 0.1 mg/mL
Qualitative Metabolism StudiesHuman Liver Microsomes
Enzymes
Cosubstrates
Buffer
Additives
Substrate
• HLM 20 mg/mL
• NADPH regenerating system• NADP 1.3 mM /glcuose 6-phosphate 3.3 mM
• glucose 6-phosphate dehydrogenase 1.6 U/mL
• phosphate buffer 100 mM, pH 7.4
• SOD 240 U/mL
• MgCl2 3.3 mM
• JWH 018 0.1 mg/mL
incubation at 37 °C
incubation time 3-4 h
termination with 250 µL methanol
LC-MS analysis
Wintermeyer/Möller/Thevis/Jübner/Beike/Rothschild/Bender, Anal Bioanal Chem, 2010
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Qualitative Metabolism StudiesHuman Liver Microsomes
trihydroxylation not commonly observed in vivo
possible artifact based on reactive O-species in the
incubation mixture (3-4 h incubation time)
Wintermeyer/Möller/Thevis/Jübner/Beike/Rothschild/Bender, Anal Bioanal Chem, 2010
Qualitative Metabolism StudiesHuman Liver Microsomes
Only identifaction of CYP-dependent metabolites
Wintermeyer/Möller/Thevis/Jübner/Beike/Rothschild/Bender, Anal Bioanal Chem, 2010
Qualitative Involvement of IsoenzymesRecombinant Enzymes
Enzymes
Cosubstrates
Buffer
Additives
Substrate
• recombinant CYP-containing ICM
• NADPH (+ IDH/Isocitrate)
• phosphate buffer, pH 7.4
• MgCl2• SOD
• dimethocaine
(Meyer/Lindauer/Maurer, Toxicol Letters, 2014)
Results – P450 Catalyzing
(Meyer/Lindauer/Maurer, Toxicol Letters, 2014)
O
O
NHNH2
CH3
CH3
CH3
O
O
NNH2
CH3
CH3
CH3CH3
OH
O
O
NNH2
CH3
CH3
CH3CH3
O
O
NNH2
CH3
CH3
CH3CH3
1A22C192D63A4
1A22C192D63A4
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Enzyme Kinetics
Important
• linear metabolite formation (< 20%)– incubation time– protein concentration
• saturating cosubstrate concentration
• increasing substrate concentrations
• product (metabolite) quantification need for reference standards
Enzyme Kinetics
Hutzler and Tracy, Drug Metabol Disp, 2002
][
][max
SK
SVV
m
nn
m
n
SK
SVV
][
][max
)/1(][
][max
imi KSSK
SVV
][
][
][
][
2,
2max,
1,
1max,
SK
SV
SK
SVV
mm
Sigmodial autoactivationMichaelis Menten
Substrate inhibition Biphasic
Results – In vitro P450 Kinetics
(Meyer/Lindauer/Maurer, Toxicol Letters, 2014)
O
O
NHNH2
CH3
CH3
CH3
O
O
NNH2
CH3
CH3
CH3CH3
OH
O
O
NNH2
CH3
CH3
CH3CH3
O
O
NNH2
CH3
CH3
CH3CH3
1A22C192D63A4
1A22C192D63A4
In vitro/in vivo Correlation ??
In vitro In vivo
incubation mixture
(Meyer/Lindauer/Maurer, Toxicol Lett 2014)
O
O
NNH2
CH3
CH3
CH3CH3
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Contribution of IsoenzymesRAF Approach
activityin HLM
definedprobe substrate
activityin recombinant
enzymes
estimatedcontribution in vivo
RAF correctiontest substratekineticsin vitro
Venkatakrishnan/von Moltke/Court/Hermatz/Crespi/Greenblatt, Drug Metabol Disp 2000
Results – Calculated In Vivo Contribution P450
(Meyer/Lindauer/Maurer, Toxicol Letters, 2014)
Results – Calculated In Vivo Contribution P450
(Meyer/Lindauer/Maurer, Toxicol Letters, 2014)
Confirmation using in vitro inhibition studies
Qualitative Involvement of IsoenzymesSelective Inhibitors
FDA Guidelines
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Results – In Vitro Inhibition of P450
(Meyer/Lindauer/Maurer, Toxicol Letters, 2014)
1A2
2D6
3A4
O
O
NHNH2
CH3
CH3
CH3
O
O
NNH2
CH3
CH3
CH3CH3
O
O
NNH2
CH3
CH3
CH3CH3
OH
O
O
NNH2
CH3
CH3
CH3CH3
Results – In Vitro Inhibition of P450
(Meyer/Lindauer/Maurer, Toxicol Letters, 2014)
1A2
2D6
3A4
O
O
NHNH2
CH3
CH3
CH3
O
O
NNH2
CH3
CH3
CH3CH3
O
O
NNH2
CH3
CH3
CH3CH3
OH
O
O
NNH2
CH3
CH3
CH3CH3
Identification of potential drug-drug or drug/food
interactions
DoA and NPS as inhibitors of CYP enzymes ???
Inhibition Cocktail Assay for Nine CYPs
N‐DE‐Amodiaquine
6‐HO‐Chlorzoxazone
4‐HO‐Bupropion
00 3.0 4.0 5.0 6.0 8.07.0 10.6 11.0
Time [min]
Relative
Abundance [%]
50
100 0.41 0.5
Nicotine
3‐Oxo
‐Nicotine
2.63 2.86 3.06
O‐DE‐Phen
acetin
Amodiaquin
e O‐DM‐Dextromethorphan
4.094.23 4.37 4.96 5.59
5‐HO‐Omeprazol
Bupropion
6.10 6.87 7.557.96
8.2210.70 11.058.05 10.80
Omep
razole
Phen
acetin
Dextromethorphan
Diphenhydramin
(IS)
6ß‐HO‐Testosterone
Chlorzoxazone
4‐HO‐Diclofenac
Testosterone
Diclofenac
(Dinger/Meyer MR/Maurer, Toxicol Lett, 2014)(Dinger/Meyer MR/Maurer, Anal Bioanal Chem, 2014)
Metabolites Formed from Test Substrates by nine CYPs
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CYP2D6 Inhibition by DoA and NPS
(Dinger/Meyer MR/Maurer, Toxicol Lett, 2014)
Determination of IC50 values for DOI and DOCCalculation of Ki values [µM]: Ki=IC50/2 if [S]=Km
-3 -2 -1 0 1 2 3
50
100
% of control activity
Conc inhibitor, logµM
Inhibition constants Ki [µM]DOI: 7.9Fluoxetine: 8.2MDOB: 11 DOC: 34DOB: 47DOM: 95TMA‐2: 296
Dimethoxyamphetamines
-4 -2 0 2 4
50
100
150
-4 -2 0 2 4
50
100
150
MDPPP
MDPBP
N
CH3
O
O
O
IC50 value 12 µM
IC50 value 101 µM
N
CH3
O
O
O
IC50 value 77 µM
IC50 value 7.3 µM
IC50 value 12 µM
-4 -2 0 2 4
50
100
150
-4 -2 0 2 4
50
100
150
Methylone
Butylone
NH
CH3
CH3
O
O
O
IC50 value 9.2 µM
IC50 value 30 µM
NH
CH3
CH3O
O
O
IC50 value 26 µM
IC50 value 21 µM
IC50 value 5.3 µM
IC50 Values of other DoA and NPS
(Dinger/Meyer MR/Maurer, TIAFT, Buenos Aires, 2014)
log inhibitor, µM log inhibitor, µM log inhibitor, µM%
ofc
ontr
olac
tivity
% o
fcon
trol
activ
ity
• CYP 1A2• CYP 2A6• CYP 2B6
• CYP 2C19• CYP 2D6• CYP 3A
-4 -2 0 2 4
20
40
60
80
100
-4 -2 0 2 4
20
40
60
80
3,4-MDMA
3,4-MDEA
NH
CH3
CH3O
O
IC50 value 10 µM
NH
CH3
O
O
CH3
IC50 value 2.8 µM
Studies on metabolizing enzymes other than CYP
N‐Acetyltransferases (NATs)
• Cytosolic phase II metabolizing enzymes
• Isoenzymes NAT1/NAT2 catalyze acetyl‐CoA‐dependent acetylation
arylamines, arylhydrazines, arylhydrazides
• Polymorphism linked with various adverse drug reactions (ADRs)
NAT
AcCoA CoA
NH2
R
NH
R
O
CH3
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Toxicokinetics of 2C´s
Meyer andMaurer, Curr Drug Metab. 2010
Metabolism of several 2C´s studied in different species
CYP
CYP CYP
NAT
MAO
NAT‐catalyzed DMC N‐Acetylation
NAT2
AcCoA CoA
NAT1
AcCoA CoA
(Meyer/Robert/Maurer, Toxicol Letters, 2014)
OCH3
OCH3
NH2
SCH3
OCH3
OCH3
NH2
SCH3
OCH3
OCH3
NH
SCH3
CH3
O
OCH3
OCH3
NH
SCH3
CH3
O
Dimethocaine Hydrolysis by Esterases
Kinetic studies with
human plasma and hCES2
(Meyer/Schuetz/Maurer, submitted)
Dimethocaine Hydrolysis by Esterases
• Incubation time 8 minutes• 30 µL Plasma• n=2• Km 12.6 ± 2.6 µM • Vmax 3.3 ± 0.2 nmol/min/mL Plasma
• Incubation time 90 minutes• Enzyme concentration 0.2 µg/µL• n=2• Km 53.5 ± 9.5 µM • Vmax 0.04± 0.002 nmol/min/mg
(Meyer/Schuetz/Maurer, submitted)
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Which Tool to Use?
Cytosol
Lumen
CYP
UGT
SULTCOMT
O
O NH
O
OHNH
O
O
S
O
OOH
NH
OOH
OHOH
HOOCO
OR
NH
In vitro system must be chosen based on problem and technical capabilities!
… but one thing is missing …
In vivo Studies for Elucidating Drug Metabolism
(Meyer/Bach/Turcant/Bovens/Maurer, Anal Bioanal Chem, 2013)(Meyer/Lindauer/Welter/ Maurer, Anal Bioanal Chem, 2014)
Drug
GRD/ARSSPE HCX, AC
GRD/ARSSPE HCX UPP/SPE C18
TF ISQGC‐MS (EI, PICI)
TF Q‐ExactiveLC‐HR‐MS/MS
TF Orbitrap VelosLC‐HR‐MSn
Urine, Blood
DMC Metabolic Pathways – Phase I + II
(Meyer/Lindauer/Welter/Maurer, Anal Bioanal Chem, 2014)
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Absorption and Distribution
Several NPS as possible substrates / inhibitors of PGP
Glaucine as potent PGP inhibitor
Particular NPS with high PPB
Metabolism and Excretion
Metabolic pathways essential for interaction studies
DMC main substrate of CYP3A4
2Cs acetylated by NAT2
Hydrolysis of DMC mediated by plasma esterases
Summary ‐ Conclusions Summary ‐ Conclusions
• Prediction of drug‐drug/food interactions
• Prediction of toxic risks
• Evidence‐based case interpretation
• Developing toxicological analysis procedures
• Understanding pitfalls in drug testing
• Defining the best target analyte for WW analysis !!!
Detailed knowledge of toxicokinetics prerequisite for