Molecular Toxicology: Roles in Drug

Disposition and Drug Safety

Prof. Prof. Dr Dr. Nico P.E. Vermeulen and . Nico P.E. Vermeulen and Dr Dr. Jan . Jan
N.M. Commandeur N.M. Commandeur
npe npe. .v vermeulen@few ermeulen@few.vu.nl .vu.nl and and jnm.commandeur@few.vu.nl jnm.commandeur@few.vu.nl
www www. .chem chem.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 8
th
, 9.00 - 11.00 hrs
Course part II, ADME-PK, Friday August 8
th
, 11.00 - 15.00 hrs
Course part III, ADME-Met, Friday August 8
th
, 15.00 - 17.00 hrs
Saturday August 9
th
, 9.00 - 10.00 hrs
Course part IV, ADME-Tox, Saturday August 9
th
, 10.00 - 14.00 hrs
Course part V, Case and Discussion, Saturday August 9
th
, 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
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 (~ C
max
)
Pharmaco-/Toxicokinetics and
ADME-Tox
C
max
duration
Minimal effective concentration
Adverse side effects
Therapeutic window
AUC
Half life
ADMET (npev & jnmc) 5
Biologically available
Orally
Faeces Urine
Excretion
Uptake
ADMET (npev & jnmc) 6
Reasons why 80-90% of candidate drugs fail Reasons why 80-90% of candidate drugs fail in the in the
clinical development clinical development phase phase
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 distribution Volume of distribution
Blood brain barrier Blood brain barrier
Transporters Transporters
Plasma Protein binding Plasma Protein binding
Hepatic Hepatic
excretion to bile excretion to bile
metabolism metabolism
Renal Renal
excretion to urine excretion to urine
metabolism metabolism
Plasma Plasma
Intestinal Intestinal
metabolism metabolism
efflux efflux
Hepatic Hepatic
metabolism metabolism
excretion to bile excretion to bile
Physicochemical Physicochemical
Properties Properties
MW MW
pKa pKa
Log P Log P
Solubility Solubility
Dissolution Dissolution
Etc. Etc.
ADME ADME ADME ADME
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
ADMET (npev & jnmc) 11
Steady-state : Uptake (mg/hr) = Elimination (CL*C
pl
)
Non-linear pharmacokinetics Linear pharmacokinetics
ADMET (npev & jnmc) 12
Reasons why 80-90% of candidate drugs fail Reasons why 80-90% of candidate drugs fail in the in the
clinical development clinical development phase phase
ADMET (npev & jnmc) 13
JAMA 279, 1200 (1998)
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 reactions
rare, unpredictable
Type C Predictable toxicity
compounds containing toxicophores
Type D Delayed toxicity (carcinogen, teratogen)
REACTIVE REACTIVE
METABOLITES METABOLITES
(often INTERMEDIATES) (often INTERMEDIATES)
Too high Too high plasmaconcentration plasmaconcentration
of parent compound of parent compound
Or: Or:
ACTIVE ACTIVE
METABOLITES METABOLITES
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 dose
Acetaminophen 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 mg
Salicilate Analgesic 3900 mg
Sulfasalazine Crohns disease 50-250 mg
Tacrine Alzheimer 40 mg
Tienilic acid Diuretic 250 mg
Troglitazone Diabetis 400 mg
Valproate Anticonvulsant 250 mg
Case-studies
Risk factor:
Dose > 10 mg/day ?
N
H
N
N
N
CH
3
Cl
N
C
O
H
2
N
H
N
Cl
Cl
COOH
O
CH
3
O
NH
S
O
O HO
CH
3
CH
3
H
3
C
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 interactions
drug toxicities
Emphasis on molecular aspects ADMET
Case(s): drugs causing idiosyncratic drug reactions
identification metabolites identification metabolites
Potential toxic metabolites Potential toxic metabolites ? ?
Pharmacologically active metabolites Pharmacologically active metabolites ? ?
Selection Selection of of animal animal model model for toxicity for toxicity studies studies
inhibitory or inducing properties inhibitory or inducing properties of the drug of the drug
Prediction Prediction drug-drug drug-drug interactions by interactions by drug (DDI) drug (DDI)
assessment assessment of of enzyme kinetical enzyme kinetical parameters (K parameters (K
m m
, , V V
max max
) of drug ) of drug
Prediction metabolic Prediction metabolic (in) (in)stability stability, , pharmacokinetics pharmacokinetics
Low Low K K
m m
: : saturable saturable, , enzyme inhibitor enzyme inhibitor
AD ADM ME- E-Tox Tox: Drug metabolism studies : Drug metabolism studies
AIMS:
identification enzymes determining pharmacokinetics identification enzymes determining pharmacokinetics of drug of drug
Genetically determined or inducible enzymes involved Genetically determined or inducible enzymes involved ? ?
Prediction Prediction effect effect enzyme-inhibiting enzyme-inhibiting drugs (DDI) drugs (DDI)
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
K
m
V
max
Fig #
Fig # Enzyme class
Enzyme class
II. IDENTIFICATION OF (ISO)ENZYMES RESPONSIBLE
FOR 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: K
M
, V
max
, V
max/
K
m
Conclusion:
1) what enzyme(s) are mainly responsible for pharmacokinetics in vivo ?
2) are genetically polymorphic 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, MRP LIVER, BILE, KIDNEY, BRAINS, INTESTINES
TOXICITY IN IN VITRO MODELS ?
IV. PREDICTION OF SAFETY AND INTERINDIVIDUAL
DIFFERENCES 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 metabolites
2) are genetically polymorphic enzymes involved ?
3) what may be consequence of enzyme deficiency ?
Fig #
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. Dr Dr. Nico P.E. Vermeulen and . Nico P.E. Vermeulen and Dr Dr. 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