9/19/2014

Department of Experimental & Clinical Toxicology Toxicokinetics ? Saarland University, Homburg (Saar), Germany

Toxicokinetics "what the organism does with the xenobiotic" Toxicokinetics of drugs of abuse Toxicodynamics and tools to perform "what the xenobiotic does to the organism“ studies on metabolism Pharmacokinetics therapeutic drugs Pharmacodynamics

Dr. Markus R. Meyer, PhD ([email protected])

Pharmaco‐/Toxicokinetics of Drugs of Abuse ?

Liberation

Absorption

Drugs of Abuse ?? Distribution

Metabolism

Excretion

Simon T et al. N Engl J Med 2009;360:363‐375

1 9/19/2014

Determinants of Toxicokinetics Stability of Drug in SGF and SIF

Liberation

Absorption

Distribution

Metabolism

Excretion

(Simon T et al. N Engl J Med, 2009) (Simon T et al. N Engl J Med 2009;360:363‐375)

Drug Absorption Drug Absorption

(Kitamura et al. Naunyn Schmiedebergs Arch Pharmacol, 2008)

Are DoA substrates or inhibitors of transporters ???

(Simon T et al. N Engl J Med 2009;360:363‐375) (Simon T et al. N Engl J Med 2009;360:363‐375)

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Affinity of DoAs to P‐glycoprotein P‐gp Kinetics of Model Substrates

Verapamil Initial Pgp ATPase activity tested for 35 DOAs butylone naphyrone PCEPA diclofensine methohexital DOI 3,4‐BDB NPDPA 2C‐I 2C‐B 2C‐T2 K = 2.3 µM (ref. 5 µM) BZP PCPr MDPV D2PM m vmax= 0.04 pmol/µg/min PPP MPHP methylone 4‐MTA PMMA mitragynine mephedrone Glaucine (alkaloid of Glaucium flavum (Papaveraceae) dimethocaine MDPPP O N 4‐F‐MA O H MDMA MDE MBDB MDAI 3‐Br‐methcathinone O O 4‐F‐methcathinone DPA NEDPA Km= 7.1 µM vmax= 0.05 pmol ATP/µg/min

(Meyer/Orschied/Maurer, Toxicol Lett, 2013) (Meyer/Orschied/Maurer, Toxicol Lett, 2013)

Drug Transport Through Cell Membranes Glaucine Misuse

Bidirectional Permeability Through Caco‐2 Cell Monolayers  permeability of test compounds through Caco‐2 cell monolayers (apical‐to‐basolateral and basolateral‐to‐apical)

Efflux ratio of model substrate rhodamine 123 • 23-year-old woman 9for verapamil (known P‐gp inhibitor) • Two tablets of “head candy” 8 for glaucine • “In another world” • Mydriasis => glaucine is also a P‐gp inhibitor • Nausea and vomiting

(Meyer/Wagmann/Daum/Lehr/Maurer, TIAFT Buenos Aires, 2014) (Meyer GM/Meyer MR/Wissenbach/Maurer, J Mass Spectrom, 2013)

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Drug Distribution –Plasma Protein Binding PPB of selected Drugs

• Pharmacologic effects • Metabolism PPB, % ref., % • Excretion Verapamil 27 27 Sertraline 92 90

eraigPPB decreasing Diclofensine >99 Free drug Naphyrone 99 NPDPA 85 Glaucine 85 DOI 70 Protein‐bound drug PCEPA 67 Butylone 57 3,4-BDB 41 • No pharmacologic effects • No metabolism • No excretion

(Meyer/Orschiedt/Leibnitz/Maurer 2012,2014)

Determinants of Toxicokinetics Drug Metabolism

Liberation Definition: Biochemical modification of substances by living organisms

Absorption • increase of hydrophilicity  elimination

• usually detoxification

Distribution • cause of drug-drug or drug-food interactions

• 3 phases:

Metabolism – Phase I: functionalization – Phase II: conjugation Excretion – (Phase III: transport processes)

Simon T et al. N Engl J Med 2009;360:363‐375

4 9/19/2014

Drug Metabolism Enzymes Drug Metabolism Studies

Cytosol Endoplasmatic reticulum •Catechol-O-methyltransferase (COMT) •Cytochrome P450 (CYP) •Sulfotransferase (SULT) •Flavin-monooxigenase (FMO) •N-acetyltransferase (NAT) •UDP-glucuronyltransferase (UGT) •Gluthathion-S-transferase (GST)

First step in (in vitro) metabolism studies Elucidation of the chemical structure of phase I and II metabolites

Mitochondria •Monoamine oxidase (MAO)

www.bhavanajagat.wordpress.com

In vitro Tools In Vitro-Metabolism Studies Pros and Cons

recombinant enzymes microsomes S9 hepatocytes • insect-cell microsomes (CYP, UGT)

• E.coli-derived (SULT) • CYP, FMO, UGT • CYP, FMO, UGT • complete enzyme profile different species • COMT, SULT, GST, NAT subcellular fractions • rat • S9 • dog • easy to use • easy to use • complex • cheap • cheap • expensive • microsomes • human, … • high throughput • high throughput • low throughput • cytosol different tissues intact cells • liver • need of cofactors • need of cofactors • no need of cofactors • small intestine • fresh hepatocytes • kidney, lung, … • cryopreserved hepatocytes

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Assay Composition Qualitative Metabolism Studies Subcellular Fractions

• recombinant enzymes (CYP, UGT, SULT) Enzymes • subcellular fractions (microsomes, cytosol, S9)

Cosubstrates • NADPH/H+, UDPGA, PAPS, …

• phosphate buffer, pH 7.4 Example on NPS Buffer • TRIS buffer, pH 7.4

Additives • SOD, alamethicin, 1,4-saccharic acid lactone

Substrate

Qualitative Metabolism Studies Qualitative Metabolism Studies Human Liver Microsomes Human Liver Microsomes

Enzymes • HLM 20 mg/mL Enzymes • HLM 20 mg/mL incubation at 37 °C • NADPH regenerating system • NADPH regenerating system Cosubstrates • NADP 1.3 mM /glcuose 6-phosphate 3.3 mM Cosubstrates • NADP 1.3 mM /glcuose 6-phosphate 3.3 mM • glucose 6-phosphate dehydrogenase 1.6 U/mL incubation• glucose time 3-4 6-phosphate h dehydrogenase 1.6 U/mL

Buffer • phosphate buffer 100 mM, pH 7.4 Buffer • phosphate buffer 100 mM, pH 7.4 termination with 250 µL methanol

• SOD 240 U/mL • SOD 240 U/mL Additives Additives • MgCl2 3.3 mM LC-MS •analysisMgCl2 3.3 mM

Substrate • JWH 018 0.1 mg/mL Substrate • JWH 018 0.1 mg/mL

Wintermeyer/Möller/Thevis/Jübner/Beike/Rothschild/Bender, Anal Bioanal Chem, 2010 Wintermeyer/Möller/Thevis/Jübner/Beike/Rothschild/Bender, Anal Bioanal Chem, 2010

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Qualitative Metabolism Studies Qualitative Metabolism Studies Human Liver Microsomes Human Liver Microsomes

trihydroxylation not commonly observed in vivo Only identifaction of CYP-dependent metabolites  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 Wintermeyer/Möller/Thevis/Jübner/Beike/Rothschild/Bender, Anal Bioanal Chem, 2010

Qualitative Involvement of Isoenzymes Results – P450 Catalyzing Recombinant Enzymes

Enzymes • recombinant CYP-containing ICM

1A2 1A2 2C19 2C19 2D6 2D6 O O O O Cosubstrates • NADPH (+ IDH/Isocitrate) CH3 HO CH CH3 3A4 CH3 3A4 3 O O O O H3C H3C H3C H3C H2N N H N N H2N N H2N NH 2

CH3 CH3 CH3 CH3 CH3 CH3 CH3 Buffer • phosphate buffer, pH 7.4

• MgCl Additives 2 • SOD

Substrate • dimethocaine

(Meyer/Lindauer/Maurer, Toxicol Letters, 2014) (Meyer/Lindauer/Maurer, Toxicol Letters, 2014)

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Enzyme Kinetics Enzyme Kinetics

Michaelis Menten Sigmodial autoactivation Important

• linear metabolite formation (< 20%) V [S ] V [S ]n max V  max – incubation time V  n n K m  [S ] K m [S ] – protein concentration

• saturating cosubstrate concentration Substrate inhibition Biphasic

• increasing substrate concentrations

V [S ] V [S] V [S] • product (metabolite) quantification V  max V  max,1  max,2 K  [S ] (1  S / K ) K [S] K [S]  need for reference standards mi i m,1 m,2

Hutzler and Tracy, Drug Metabol Disp, 2002

Results – In vitro P450 Kinetics In vitro/in vivo Correlation ??

O CH3 O

H3C H2N N

CH3 CH3

In vitro In vivo

1A2 1A2 2C19 2C19 2D6 2D6 O O O O HO CH CH3 CH3 3 3A4 CH3 3A4 O O O O incubation mixture H3C H3C H3C H3C H2N N H N N H2N N H2N NH 2

CH3 CH3 CH3 CH3 CH3 CH3 CH3

(Meyer/Lindauer/Maurer, Toxicol Letters, 2014) (Meyer/Lindauer/Maurer, Toxicol Lett 2014)

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Contribution of Isoenzymes Results –Calculated In Vivo Contribution P450 RAF Approach

defined probe substrate

activity activity in recombinant in HLM enzymes

test substrate kinetics RAF correction in vitro

estimated contribution in vivo

Venkatakrishnan/von Moltke/Court/Hermatz/Crespi/Greenblatt, Drug Metabol Disp 2000 (Meyer/Lindauer/Maurer, Toxicol Letters, 2014)

Results –Calculated In Vivo Contribution P450 Qualitative Involvement of Isoenzymes Selective Inhibitors

Confirmation using in vitro inhibition studies

(Meyer/Lindauer/Maurer, Toxicol Letters, 2014) FDA Guidelines

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Results – In Vitro Inhibition of P450 Results – In Vitro Inhibition of P450

1A2 1A2 2D6 2D6

3A4 Identification of potential3A4 drug-drug or drug/food interactions

O O O O O O O O HO HO CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 O O O O O O O O

H3C H3C H3C H3C H3C H3C H3C H3C H2N N H2N NH H2N N H2N N H2N N H2N NH H2N N H2N N

CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

(Meyer/Lindauer/Maurer, Toxicol Letters, 2014) (Meyer/Lindauer/Maurer, Toxicol Letters, 2014)

Inhibition Cocktail Assay for Nine CYPs

Metabolites Formed from Test Substrates by nine CYPs ‐ Nicotine Bupropion Omeprazol Phenacetin Amodiaquine Testosterone ‐ ‐ ‐ ‐ ‐ ‐ Chlorzoxazone Diclofenac ‐ ‐ DM HO DE DE HO ‐ HO ‐ Oxo ‐ ‐ ‐ ‐ ‐ O HO 4 Bupropion 5 HO O N Nicotine ‐ 3 Amodiaquin e Chlorzoxazone ‐ Omeprazole Phenacetin Dextromethorphan 6ß 6 Testosterone 4 4.23 4.37 4.96 5.59 Diclofenac

0.5 2.63 2.86 3.06 Diphenhydramin (IS) 8.22 100 0.41 6.10 6.87 7.55 DoA and NPS as inhibitors of CYP enzymes ??? 4.09 7.96 8.05 10.70 10.80 11.05

[%]

50 Abundance

Relative 0 0 3.0 4.0 5.0 6.07.0 8.0 10.6 11.0 Time [min]

(Dinger/Meyer MR/Maurer, Toxicol Lett, 2014) (Dinger/Meyer MR/Maurer, Anal Bioanal Chem, 2014)

10 9/19/2014

CYP2D6 Inhibition by DoA and NPS IC50 Values of other DoA and NPS

3,4-MDMA Methylone MDPPP • CYP 1A2 • CYP 2C19 • CYP 2A6 • CYP 2D6 Dimethoxyamphetamines 100 150 150 • CYP 2B6 • CYP 3A

O NH O O 80 CH3 O NH O N CH CH O 3 100 3 100 CH CH 60 O 3 O 3

40 Inhibition constants K [µM] Determination of IC50 values for DOI and DOC 50 50 i IC value 30 µM IC value 101 µM Calculation of Ki values [µM]: Ki=IC50/2 if [S]=Km 20 50 50 DOI: 7.9 IC50 value 10 µM IC50 value 9.2 µM IC50 value 12 µM Fluoxetine: 8.2 -4 -2 0 2 4 -4 -2 0 2 4 -4 -2 0 2 4 MDOB: 11 100 activity

DOC: 34 3,4-MDEA Butylone MDPBP 50 control

O NH CH3 O O

of 80 150 150 DOB: 47 NH N O O % CH CH O 3 3

O H C O H C DOM: 95 60 3 3 -3 -2 -1 0 1 2 3 100 100 Conc inhibitor, logµM TMA‐2: 296 40 IC value 26 µM IC value 77 µM 50 50 50 50 20 IC50 value 21 µM IC50 value 7.3 µM % of% control activity of % control activity IC50 value 2.8 µM IC50 value 5.3 µM IC50 value 12 µM

-4 -2 0 2 4 -4 -2 0 2 4 -4 -2 0 2 4 log inhibitor, µM log inhibitor, µM log inhibitor, µM

(Dinger/Meyer MR/Maurer, Toxicol Lett, 2014) (Dinger/Meyer MR/Maurer, TIAFT, Buenos Aires, 2014)

N‐Acetyltransferases (NATs)

• Cytosolic phase II metabolizing enzymes •IsoenzymesNAT1/NAT2 catalyze acetyl‐CoA‐dependent acetylation  arylamines, arylhydrazines, arylhydrazides Studies on metabolizing enzymes other than CYP • Polymorphism linked with various adverse drug reactions (ADRs)

O

NH2 NAT HN CH3

R AcCoA CoA R

11 9/19/2014

Toxicokinetics of 2C´s NAT‐catalyzed DMC N‐Acetylation

Metabolism of several 2C´s studied in different species H C H3C 3 O O NAT1 NH2 NH CH3

O H C S 3 H3C S O AcCoA CoA O CYP CH3 CH NAT 3

H3C CYP CYP H3C O O NH CH3 MAO NH2 NAT2 O H3C S H3C S O O CH CH 3 3 AcCoA CoA

Meyer and Maurer, Curr Drug Metab. 2010 (Meyer/Robert/Maurer, Toxicol Letters, 2014)

Dimethocaine Hydrolysis by Esterases Dimethocaine Hydrolysis by Esterases

• Incubation time 8minutes • Incubation time 90 minutes • 30 µL Plasma • Enzyme concentration 0.2 µg/µL Kinetic studies with • n=2 • n=2 • Km 12.6 ± 2.6 µM • Km 53.5 ± 9.5 µM human plasma and hCES2 • Vmax 3.3 ± 0.2 nmol/min/mL Plasma • Vmax 0.04 ± 0.002 nmol/min/mg

(Meyer/Schuetz/Maurer, submitted) (Meyer/Schuetz/Maurer, submitted)

12 9/19/2014

Which Tool to Use?

SULT COMT In vitro system must beO chosen based on O HN problem and technicalHO capabilities! O HN O S HO O Cytosol CYP … but one thing is missing …

O

HN O

UGT Lumen

O R HO HO HN HO O HOOC O

In vivo Studies for Elucidating Drug Metabolism DMC Metabolic Pathways – Phase I + II

Drug

Urine, Blood

GRD/ARS GRD/ARS SPE HCX, AC SPE HCX UPP/SPE C18

TF ISQ TF Q‐Exactive TF Orbitrap Velos GC‐MS (EI, PICI) LC‐HR‐MS/MS LC‐HR‐MSn

(Meyer/Bach/Turcant/Bovens/Maurer, Anal Bioanal Chem, 2013) (Meyer/Lindauer/Welter/ Maurer, Anal Bioanal Chem, 2014) (Meyer/Lindauer/Welter/Maurer, Anal Bioanal Chem, 2014)

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Summary ‐ Conclusions Summary ‐ Conclusions

Detailed knowledge of toxicokinetics prerequisite for Absorption and Distribution  Several NPS as possible substrates / inhibitors of PGP •Predictionof drug‐drug/food interactions  Glaucine as potent PGP inhibitor •Predictionof toxic risks  Particular NPS with high PPB • Evidence‐based case interpretation

Metabolism and Excretion • Developing toxicological analysis procedures  Metabolic pathways essential for interaction studies • Understanding pitfalls in drug testing  DMC main substrate of CYP3A4 • Defining the best target analyte for WW analysis !!!  2Cs acetylated by NAT2  Hydrolysis of DMC mediated by plasma esterases

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