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Molecular Toxicology: Roles in Drug

Disposition and Drug Safety

Prof.Prof. DrDr. Nico P.E. Vermeulen and. Nico P.E. Vermeulen and DrDr. Jan. Jan

N.M. CommandeurN.M. Commandeur

npenpe..vvermeulen@[email protected]@[email protected]

wwwwww..chemchem.vu..vu.nl/far/nl/far/

August 8 and 9, 2009, Yogyakarta

Molecular Toxicology: Roles in Drug

Disposition and Drug Safety

Projected Time Schedule:Course part I, Introduction, Friday August 8th, 9.00 - 11.00 hrs

Course part II, ADME-PK, Friday August 8th, 11.00 - 15.00 hrs

Course part III, ADME-Met, Friday August 8th, 15.00 - 17.00 hrs

Saturday August 9th, 9.00 - 10.00 hrs

Course part IV, ADME-Tox, Saturday August 9th, 10.00 - 14.00 hrs

Course part V, Case and Discussion, Saturday August 9th, 14.00 - 15.30 hrs

Objectives:1) To obain knowledge of the molecular aspects of ADME

2) To learn about the roles of ADME in PK

3) To learn about the roles of ADME in Tox


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ADMET (npev & jnmc) 3

LACDR-Division of Molecular Toxicology

Research theme: Drug disposition and safety: From molecular structures

to molecular mechanisms and effects

Key feature: integration of experimental and computational approaches

dr. Jan Commandeur (experimental; molecular toxicology)

dr. Chris Oostenbrink (computational; chem-/bioinformatics > November 2004)

dr. Chris Vos (experimental; molecular biology, > July 2006)

prof.dr. Peter Grootenhuis (extraord. chair: computational ADME, > June 2005)


Absorption

Distribution

Metabolism

Excretion

Toxicology bioavailability

efficacy

duration of action

frequency of dosing safety (~ Cmax)

Pharmaco-/Toxicokinetics and

ADME-Tox

Cmax

duration

Minimal effective concentration

Adverse side effects

Therapeutic windowAUC

Half life


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Biologically available

Orally

FaecesUrine Excretion

Uptake


Reasons why 80-90% of candidate drugs failReasons why 80-90% of candidate drugs fail in thein the

clinical developmentclinical developmentphasephase


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Low bioavailability: limited human intestinal absorption (HIA)

first-pass metabolism

Too fast or too slow systemic elimination

Compound does not reach site of action (e.g. blood-brain barrier)

High plasma binding

Enzyme induction

Enzyme inhibitor

Pharmacokinetics dose-dependent

non-linear / saturation pharmacokinetics

Large inter-individual difference in pharmacokinetics

Pharmacokinetic defects of drugs

Drug-drug interactions (DDI)Drug-drug interactions (DDI)


Volume of distributionVolume of distribution

Blood brain barrierBlood brain barrier

TransportersTransporters

Plasma Protein bindingPlasma Protein binding

HepaticHepatic

excretion to bileexcretion to bile

metabolismmetabolism

RenalRenal

excretion to urine excretion to urine

metabolism metabolism

PlasmaPlasma

IntestinalIntestinal

metabolismmetabolism

efflux efflux

HepaticHepatic

metabolism metabolism

excretion to bile excretion to bile

PhysicochemicalPhysicochemical

PropertiesProperties

MWMW

pKapKa

Log PLog P

SolubilitySolubility

DissolutionDissolution

Etc.Etc.

ADME ADME ADME ADME


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Drug-drug interactions (DDI)

16 Patients; each given a single dose of 4 mg tolterodine

Brynne et al. Clin.Phar.Ther 63, 529 (1998)

tolterodine

LARGE INTERINDIVIDUAL DIFFERENCES IN PHARMACOKINETICS

PMs

EMs


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Steady-state : Uptake (mg/hr) = Elimination (CL*Cpl)

Non-linear pharmacokineticsLinear pharmacokinetics


Reasons why 80-90% of candidate drugs failReasons why 80-90% of candidate drugs fail in thein the

clinical developmentclinical developmentphasephase


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JAMA 279, 1200 (1998)


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CLASSIFICATION ADVERSE DRUG REACTIONS

Type A Pharmacological activity

A1: intrinsic to drug target

A2: not related to drug target

Type B Idiosyncratic drug reactionsrare, unpredictable

Type C Predictable toxicity

compounds containing toxicophores

Type D Delayed toxicity (carcinogen, teratogen)

REACTIVEREACTIVE

METABOLITESMETABOLITES

(often INTERMEDIATES)(often INTERMEDIATES)

Too highToo high plasmaconcentrationplasmaconcentration

of parent compoundof parent compoundOr:Or:

ACTIVEACTIVE

METABOLITESMETABOLITES


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IDIOSYNCRATIC DRUG REACTIONS

low incidence: 1 : 1.000 to 100.000

escapes discovery in clinical trial, so unpredictable

delayed onset (14 days to months after onset of therapy) often fatal

most frequent target organs:

blood (agranulocytosis, aplastic anemia)

liver (fulminant hepatitis)

skin (lupus)

toxicity mediated by (auto)immune response

formation of reactive metabolite (ADME-Tox)

(combination of) genetic factors

(enzymes, MHC,..?)

no animal models available


Drug Indication Daily doseAcetaminophen Analgesic 500 mg

Aldipenem Anxiolytic 225 mg

Amineptine Antidepressant 200 mg

Amodiaquine Malaria 200-1000 mg

Bromfenac Analgesic 25-100 mg

Carbamazepine Anticonvulsant 200 mg

Clozapine Antidepressant 500-600 mg

Cyproterone Androgen antagonist 50 mg

Diclofenac NSAID 50 mg

Dideoxinosine HIV 750 mg

Dihydralazine Hypertension 100-200 mg

Ebrotidine H2-antagonist 150-800 mg

Enalapril Hypertension 10-40 mg

Felbamate Antiepileptic 400-600 mg

Flutamide Nonsteroid antiandrogen750 mg

Halothane Anesthesia 0.5-3%Isoniazide Anticonvulsant 300 mg

Ketokonazole Antifungal 200 mg

MDMA Euphoria 500 mg (est.)Methoxyflurane Anesthesia 0.5-3%

Minocycline Acne 200 mg

Nefazodone Antidepressant 200 mg

Phenobarbital Anticonvulsant 60-200 mg

Phenprocoumon Anticoagulant 1 -4 mg

Phenytoin Antiepileptic 300 mg

Procainamide Antiarrhytmic 3500 mg

Pyrazinamide Antibacterial 1500 mg

Rifampicin Antimicrobial 600 mgSalicilate Analgesic 3900 mg

Sulfasalazine Crohns disease 50-250 mg

Tacrine Alzheimer 40 mg

Tienilic acid Diuretic 250 mgTroglitazone Diabetis 400 mg

Valproate Anticonvulsant 250 mg Case-studies

Risk factor:

Dose > 10 mg/day ?

NH

N

N

N

CH3

Cl

N

C

OH2N

HN

Cl

Cl

COOH

OCH3O

NH

S

O

OHO

CH3

CH3

H3C


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Aim of case-study/studies:

Get familiar with various experimental approaches used in:

ADME; metabolite - identification

active metabolite formation

(iso)enzyme - identification

Safety/Tox: interindividual variability

enzyme inhibition/induction

bioactivation to reactive intermediates

drug-drug interactionsdrug toxicities

Emphasis on molecular aspectsADMET

Case(s): drugs causing idiosyncratic drug reactions

identification metabolitesidentification metabolites

Potential toxic metabolitesPotential toxic metabolites ??

Pharmacologically active metabolitesPharmacologically active metabolites ??SelectionSelection ofof animalanimal modelmodel for toxicityfor toxicity studiesstudies

inhibitory or inducing propertiesinhibitory or inducing properties of the drugof the drug

PredictionPrediction drug-drugdrug-drug interactions byinteractions by drug (DDI)drug (DDI)

assessmentassessment ofof enzyme kineticalenzyme kineticalparameters (Kparameters (Kmm,, VVmaxmax) of drug) of drug

Prediction metabolicPrediction metabolic (in)(in)stabilitystability,, pharmacokineticspharmacokinetics

LowLow KKmm:: saturablesaturable,, enzyme inhibitorenzyme inhibitor

ADADMME-E-ToxTox: Drug metabolism studies: Drug metabolism studies

AIMS:

identification enzymes determining pharmacokineticsidentification enzymes determining pharmacokinetics of drugof drug

Genetically determined or inducible enzymes involvedGenetically determined or inducible enzymes involved ??PredictionPrediction effecteffect enzyme-inhibitingenzyme-inhibiting drugs (DDI)drugs (DDI)


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I. BIOTRANSFORMATION OF DRUG

major metabolites (vivo/slices/hepatocytes)

enzyme-classes to be considered ?

Enzyme kinetics of drug (human liver microsomes / cytosol)

non-Michaelis-Menten kinetics ?

cytochrome P450 (CYP)

flavin-containing monooxygenase (FMO)

epoxide hydrolase (mEH, sEH)

UDP-glucuronosyltransferase (UGT)

Sulfotransferase (ST)

N-acetyltransferase (NAT)

Glutathione transferase (GST)

Quinone reductase / DT diaphorase

Catechol methyltransferase (COMT)

Others

yes no

compound

Michaelis-Menten kinetics ?

one-enzyme

two enzymes

substrate inhibition /negative cooperativity

autoactivation /positive cooperativity

yes no

yes no

Km Vmax Fig #

Fig #Enzyme class

Enzyme class

II. IDENTIFICATION OF (ISO)ENZYMES RESPONSIBLEFOR PHARMACOKINETICS OF THE DRUG

approach 1:effect of specific enzyme inhibitors on human enzyme fractions

approach 2:correlation analysis with individual human enzyme fractions

approach 3:recombinant human enzymes: KM, Vmax, Vmax/Km

Conclusion:

1) what enzyme(s) are mainly responsible for pharmacokinetics in vivo ?2) are genetically polymorph ic enzymes involved and what may be

consequence of deficiency.

approach 4:Effect of model inducers (cells, vivo)

approach 5:Genotyped/phenotyped individuals / Knock-out animals

Fig #

(vivo/vitro)

DOES THE COMPOUND CAUSES ENZYME INDUCTION ?

III. ABILITY TO CAUSE DRUG-DRUG INTERACTIONS

CONCLUSIONS

REVERSIBLE INHIBITOR OF ENZYME-SPECIFIC REACTIONS ?HIGH-AFFINITY SUBSTRATE FOR ENZYME ?

MECHANISM-BASED INHIBITOR OF ENZYME-SPECIFIC REACTIONS ?

which enzyme ? type inhibition; IC50; Ki ?

which enzyme ? Ki, ki, half-life ?

what class of induction ?

physiological relevance ?

(PRIMARY CULTURE, IN VIVO)

Fig #

PHYSIOLOGICAL CONCENTRATION (PLASMA, LIVER)

DOES THE COMPOUND INHIBITS DRUG TRANSPORTERS ?

BSEP, OAT, OCT, MDR, MRPLIVER, BILE, KIDNEY, BRAINS, INTESTINES

TOXICITY IN IN VITRO MODELS ?

IV. PREDICTION OF SAFETY AND INTERINDIVIDUALDIFFERENCES IN SUSCEPTIBILITY

CONCLUSIONS

COVALENT BINDING TO PROTEINS ?

GLUTATHION (GSH)-CONJUGATES ? (vivo or vitro experiments)

N-ACETYLCYSTEINE (NAC)-CONJUGATES ?METHYLTHIO-CONJUGATES ?

MECHANISM-BASED ENZYME INHIBITION ?

WHICH (ISO)ENZYMES ?



1) is the drug bioactivated to toxic/reactive metabolites2) are genetically polymorphic enzymes involved ?3) what may be consequence of enzyme deficiency ?

Fig #


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PRESENTATION AND DISCUSSION OF CASE STUDY

Identify the (combination) of factors which may have determined

the increased sensitivity of specific individuals for the idiosyncratic

drug reactions. What would be the worst case scenario ?

Groups of participants give summary/overview

-of enzymes involved in the metabolism of the particular drug

-factors which may have caused increased sensitivities of individuals-the best and the worst case scenarios for individuals

Make use of:

- database provided-guidelines/forms provided

Prepare a presentation of 15 minutes

Molecular Toxicology: Roles in Drug Disposition and Drug

Safety

Prof.Prof. DrDr. Nico P.E. Vermeulen and. Nico P.E. Vermeulen and DrDr. Jan N.M. Commandeur. Jan N.M. Commandeur

August 8 and 9, 2009, Yogyakarta

Part II: (ADME-PK)

Phamaco-/Toxicokinetics, incl

Absorption, Distribution and Elimination

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