mrm mailand sewprof toxicokinetics 2014...

9/19/2014

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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


Drug Absorption


(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)

www.bhavanajagat.wordpress.com

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


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


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trihydroxylation not commonly observed in vivo

possible artifact based on reactive O-species in the

incubation mixture (3-4 h incubation time)



Only identifaction of CYP-dependent metabolites


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


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


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


Results – Calculated In Vivo Contribution P450


Confirmation using in vitro inhibition studies

Qualitative Involvement of IsoenzymesSelective Inhibitors

FDA Guidelines

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Results – In Vitro Inhibition of P450


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


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

mrm mailand sewprof toxicokinetics 2014...

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