Cytochrome P450 and Polymorphism - uni

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Transcript Cytochrome P450 and Polymorphism - uni

Flow of information in a
drug discovery pipeline
Bioinformatics
Computational and Combinatorial
Chemisty
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Predictive ADME
Absorption
Distribution
Metabolism
Elimination
Pharmacokinetic
Bioavailability
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Why is the prediction of ADME parameters
so important ?
reasons that cause the failure of a potential drug candidate
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Bioavailablity of Drugs (I)
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Bioavailability of Drugs (II)
Uptake of orally administered drug proceeds after the
stomach passage via the small intestine.
In the liver, a series of metabolic transformation occurs.
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Cytochrome P450
The super-family of cytochrome P450 enzymes has a
crucial role in the metabolism of drugs.
Almost every drug is processed by some of these enzymes.
This causes a reduced bioavailability.
Cytochrome P450 enzymes show extensive structural
polymorphism (differences in the coding region).
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Cytochrome P450 metabolisms (I)
During first liver passage: First pass effect
extensive chemical transformation of lipophilic or heavy
(MW >500) compounds. They become more hydrophilic
(increased water solubility) and are therefore easier to
excreat.
H
O
CH3
COOH
phase I
N
COOH
phase II
Predominantly cytochrome P450 (CYP) enzymes are
responsible for the reactions belonging to phase I.
Usually, the reaction is a monooxygenation.
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Cytochrome P450 Metabolismus (II)
The substrates are monooxygenated in a catalytic cycle.
Drug-R + O2
CYP
NADPH
Drug-OR + H2O
NADP
The iron is part of a HEM moiety
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Cytochrome P450 Metabolismus (III)
The cytochromes involved in the metabolism are mainly
monooxygenases that evolved from the steroid and fatty
acid biosynthesis.
So far, 17 families of CYPs with about 50 isoforms have
been characterized in the human genome.
classification:
CYP 3 A 4 *15 A-B
family
isoenzyme allel
>40% sequencesub-family
homology
>55% sequencehomology
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Cytochrome P450 gene families
Human 14+
Molluscs 1
CYP450
Plants 22
Insects 3
Fungi 11
Bacteria 18
Yeasts 2
Nematodes 3
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Human cytochrome P450 family
Of the super-family of all cytochromes, the following families
were confirmed in humans:
CYP 1-5, 7, 8, 11, 17, 19, 21, 24, 26, 27, 39, 46, 51
Function:
CYP 1, 2A, 2B, 2C, 2D, 2E, 3
metabolismus of
xenobiotica
CYP 2G1, 7, 8B1, 11, 17, 19, 21, 27A1, 46, 51 steroid
metabolism
CYP 2J2, 4, 5, 8A1
fatty acid metabolism
CYP 24 (vitamine D), 26 (retinoic acid), 27B1 (vitamine D), ...
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Cytochrome P450 enzymes (I)
Flavin Monooxygenase Isoenzyme
Alkohol Dehydrogenase
Aldehyd Oxidase
Monoamin Dehydrogenase (MAO)
Drug-R + O2
CYP
NADPH
The redox activity is
mediated by an iron
porphyrin in the active
center
Drug-OR + H2O
NADP
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Cytochrome P450 enzymes (II)
Despite the low sequence identity between CYPs from
different organisms, the tertiary structure is highy
conserved.
Superposition of
hCYP 2C9 (1OG5.pdb) and
CYP 450 BM3 (2BMH.pdb)
Bacillus megaterium
In contrast to bacterial CYPs, the microsomal mammalian CYPs
possess an additional transmembrane helix that serves as an
anchor in the membrane
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Cytochrome P450 enzymes (III)
The structures of several mammalian CYPs have now
been determined in atomistic detail and are available
from the Brookhaven Database:
http://www.pdb.mdc-berlin.de/pdb/
1DT6.pdb CYP 2C5 rabbit
Sep 2000
1OG5.pdb CYP 2C9 human
Jul 2003
1PO5.pdb CYP 2B4 rabbit
Oct 2003
1PQ2.pdb CYP 2C8 human
Jan 2004
They are suitable templates for deriving homology
models of further CYPs
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Cytochrome P450 enzymes (IV)
The majority of CYPs is found in the liver, but certain CYPs
are also present in the wall cells of the inestine
The mammalian CYPs are bound to the endoplasmic reticulum,
and are therefore membrane bound.
CYP 2D6
2%
CYP 2A6
4%
CYP distribution
other
7%
CYP 3
31%
CYP 1A2
13%
CYP 1A6
CYP 1A2
CYP 2A6
CYP 1A6
8%
CYP 2C6
6%
CYP 3
CYP 2C11
CYP 2E1
CYP 2C6
CYP 2E1
13%
CYP 2C11
16%
CYP 2D6
other
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Cytochrome P450 enzymes (V)
Especially CYP 3A4, CYP 2D6, and CYP 2C9 are involved in
the metabolism of xenobiotics and drugs.
Metabolic Contribution
hepatic only
CYP 2C9
10%
CYP 1A2 other
2%
3%
CYP 3A4
CYP 2D6
CYP 2C9
CYP 1A2
other
CYP 3A4
55%
CYP 2D6
30%
also small intestine
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Substrate specificity of CYPs (I)
spezific substrates of particular human CYPs
CYP 1A2
verapamil, imipramine, amitryptiline,
caffeine (arylamine N-oxidation)
CYP 2A6
nicotine
CYP 2B6
cyclophosphamid
CYP 2C9
diclofenac, naproxen, piroxicam, warfarin
CYP 2C19
diazepam, omeprazole, propanolol
CYP 2D6
amitryptiline, captopril, codeine,
mianserin, chlorpromazine
CYP 2E1
dapsone, ethanol, halothane, paracetamol
CYP 3A4
alprazolam, cisapride, terfenadine, ...
see also http://medicine.iupui.edu/flockhart/
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Substrate specificity of CYPs (II)
Decision tree for human P450 substrates
CYP 1A2, CYP 2A-E, CYP 3A4
CYP 2E1
CYP 2C9
low
Volume
high
medium
acidic
basic
pK
a
CYP 3A4
CYP 2D6
neutral
CYP 1A2, CYP 2A, 2B
CYP 2B6
low
planarity
high
CYP 1A2
medium
CYP 2A6
Lit: D.F.V. Lewis Biochem. Pharmacol. 60 (2000) 293
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Cytochrome P450 polymorphisms
„Every human differs (more or less) “
The phenotype can be distinguished by the actual
activity or the amount of the expressed CYP enzyme.
The genotype, however, is determined by the individual
DNA sequence. Human: two sets of chromosomes
That mean: The same genotype enables different
phenotypes
Depending on the metabolic activity, three major cathegories
of metabolizers are separated: extensive metabolizer
(normal), poor metabolizer, and ultra-rapid metabolizer
(increased metabolism of xenobiotics)
Lit: K. Nagata et al. Drug Metabol. Pharmacokin 3 (2002) 167
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CYP 2D6 Polymorphism (I)
The polymorphismus of CYP 2D6 (debrisoquine 4hydroxylase) has been studied in great detail, as
metabolic differences have first been described for
debrisoquine and sparteine (antipsychotics)
localized on chromosome 22
Of the 75 allels, 26 exprime CYP2D6 proteines
see http://www.imm.ki.se/CYPalleles/cyp2d6.htm
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CYP 2D6 Polymorphism (II)
Lit: J. van der Weide et al. Ann. Clin. Biochem 36 (1999) 722
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CYP 2D6 Polymorphism (III)
MGLEALVPLAVIVAIFLLLVDLMHRRQRWAARYPPGPLPLPGLGNLLHVDFQNTPYCFDQ
poor debrisoquine metabolism S
R impaired mechanism of sparteine
LRRRFGDVFSLQLAWTPVVVLNGLAAVREALVTHGEDTADRPPVPITQILGFGPRSQGVF
poor debrisoquine metabolism I
LARYGPAWREQRRFSVSTLRNLGLGKKSLEQWVTEEAACLCAAFANHSGRPFRPNGLLDK
poor debrisoquine metabolism R
AVSNVIASLTCGRRFEYDDPRFLRLLDLAQEGLKEESGFLREVLNAVPVLLHIPALAGKV
LRFQKAFLTQLDELLTEHRMTWDPAQPPRDLTEAFLAEMEKAKGNPESSFNDENLRIVVA
missing in CYP2D6*9 allele
DLFSAGMVTTSTTLAWGLLLMILHPDVQRRVQQEIDDVIGQVRRPEMGDQAHMPYTTAVI
P loss of activity in CYP2D6*7
HEVQRFGDIVPLGMTHMTSRDIEVQGFRIPKGTTLITNLSSVLKDEAVWEKPFRFHPEHF
LDAQGHFVKPEAFLPFSAGRRACLGEPLARMELFLFFTSLLQHFSFSVPTGQPRPSHHGV
FAFLVSPSPYELCAVPR
T impaired metabolism of sparteine in alleles 2, 10, 12, 14 and 17 of CYP2D6
see http://www.expasy.org/cgi-bin/niceprot.pl?P10635
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CYP 2D6 Polymorphism (III)
variability of debrisoquine-4-hydroxylation
HO
H
CYP2D6
N
NH2
NH
NH2
N
NH
= number of individuals (european population)
homocygote
extensive
metabolizers
homocygote
poor
metabolizers
= metabolic rate
heterocygote extensive metabolizers
Lit: T. Winkler Deutsche Apothekerzeitung 140 (2000) 38
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Polymorphisms of further CYPs
CYP 1A2 individual: fast, medium, and slow turnover of
caffeine
CYP 2B6 missing in 3-4 % of the caucasian population
CYP 2C9 deficit in 1-3 % of the caucasian population
CYP 2C19 individuals with inactive enzyme (3-6 % of the
caucasian and 15-20 % of the asian population)
CYP 2D6 poor metabolizers in 5-8 % of the european,
10 % of the caucasian, and <1% of the japanese
population. Over expression (gene duplication) among
parts of the african and oriental population.
CYP 3A4 only few mutations
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Genotyping for P450 alleles
Affymetrix (US) has developped microarrays (gene chips)
using immobilized synthetic copies of P450 nucleotides, that
allow the identification of all clinically relevant allelic variants.
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Induction and regulation of CYP3A (I)
A series of xenobiotics have been identified, that lead to
increased expression of enzymes of the CYP3A family.
Indinavir
efavirenz
cyclosporin
carbamazepine
atorvastatin
tamoxifen
antiviral
antiviral
immuno-suppressant
antispychotic
HMG CoA Reductase Inhibitor
anti-hormone
These bind to the pregnane X receptor (PXR) which is the
transcription factor for the regulation of the CYP3A gene
expression.
Lit: T.M. Wilson et al. Nature Rev. Drug Disc. 1 (2002) 259
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Induction and regulation of CYP3A (II)
The PXR receptor functions together with the retinoid X
receptor (RXR) as a heterodimer.
CYP3A induction leads to an increased metabolism of the
administered substance due to upregulated enzymes.
This can cause adverse reactions, like inflammation of the
liver (hepatitis).
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RXR and other nuclear receptors (I)
As a specific, endogen activator of RXR, 5b-pregnane3,20-dione has been identified.
In contrast, PXR is much less specific and is activated
by glucocorticoids as well as by anti-glucocorticoids.
Conversely, the unspecific constitutive androgen receptor
(CAR) is found in the cytoplasm and dimerizes with PXR in
the nucleus. Analog to PXR, the CYP2B gene is regulated.
Likewise high sequence homology has been found for the
vitamine D receptor (VDR) that regulates CYP27, and for the
arylhydrocarbon receptor (AHR) (dioxin receptor).
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RXR and other nuclear receptors (II)
These nuclear receptors all belong to a family of
transcription factors. Each one possess a double zinkfinger DNA-binding domain (DBD), and a larger ligand
binding domain (LBD) which is carboxy terminal.
They have been called orphan nuclear receptors as
their ligands have been found later.
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Nuclear Receptors as Drug Targets
Contribution to the human genome and number of marketed
drugs
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Induction and regulatiion of CYP3A (III)
O
HO
O
O
hyperforin, a natural ingredient of
St. John‘s wort (Johanniskraut,
Hypericum performatum) exhibits
the highest measured affinity to
PXR (Kd = 27 nM) so far.
Application: remedy against cholestasis,
mild antidepressant
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Induction and regulation of CYP3A (IV)
X-ray structure of PXR with bound hyperforin (1M13.pdb)
Lit: R.E. Watkins et al. Biochemistry 42 (2003) 1430
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Induction of further CYPs
CYP 1A2
omeprazole, insulin, aromatic hydrocarbons
(cigarette smoking, charbroiled meat)
causes increased caffeine level in the plasma,
if you quit smoking.
CYP 2C9
rifampicin, secobarbital
CYP 2C19
carbamazepine, prednisone
CYP 2D6
dexamethason
CYP 2E1
ethanol, isoniazid
CYP 3A4
glucocorticoide, phenobarbitone,
rifampicin, nevirapine,
sulfadimindine,
nevirapine,
sulfinpyrazone, troglitazone
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Typical inhibitors of various CYPs
CYP 1A2
cimetidine, ciprofloxacine, enoxacine...
grapefruit juice (naringin, 6‘,7‘-dihydroxybergamottin)
CYP 2C9
chloramphenicol, amiodarone,
omeprazole,...
CYP 2C19
fluoxetine, fluvastatin, sertraline,...
CYP 2D6
fluoxetine, paroxetine, quinidine,
haloperidol, ritonavir,...
CYP 2E1
disulfiram, cimetidine,...
CYP 3A4
cannabinoids, erythromycin, ritonavir,
ketokonazole, grapefruit juice
see also http://medicine.iupui.edu/flockhart/
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