1-toksikologi molekuler
TRANSCRIPT
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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)
ADMET (npev & jnmc) 4
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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ADMET (npev & jnmc) 5
Biologically available
Orally
FaecesUrine Excretion
Uptake
ADMET (npev & jnmc) 6
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)
ADMET (npev & jnmc) 8
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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ADMET (npev & jnmc) 9
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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ADMET (npev & jnmc) 11
Steady-state : Uptake (mg/hr) = Elimination (CL*Cpl)
Non-linear pharmacokineticsLinear pharmacokinetics
ADMET (npev & jnmc) 12
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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ADMET (npev & jnmc) 13
JAMA 279, 1200 (1998)
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ADMET (npev & jnmc) 15
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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ADMET (npev & jnmc) 17
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
ADMET (npev & jnmc) 18
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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ADMET (npev & jnmc) 19
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 ?
WHICH (ISO)ENZYMES ?
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