Flow of information in a drug discovery pipeline

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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
effectextensive chemical transformation of
lipophilic or heavy (MW gt500) compounds. They
become more hydrophilic (increased water
solubility) and are therefore easier to excreat.
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.
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
familygt40 sequence-homology
isoenzyme
allel
sub-familygt55 sequence-homology
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Cytochrome P450 gene families
Human 14
Molluscs 1
CYP450
Plants 22
Insects 3
Bacteria 18
Yeasts 2
Nematodes 3
Fungi 11
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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)
The redox activity is mediated by an iron
porphyrin in the active center
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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 ofhCYP 2C9 (1OG5.pdb) andCYP 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.
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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.
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Substrate specificity of CYPs (I)
spezific substrates of particular human CYPs CYP
1A2 verapamil, imipramine, amitryptiline, caffei
ne (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
high
low
CYP 3A4
CYP 2E1
Volume
medium
acidic
basic
pKa
CYP 2D6
CYP 2C9
neutral
CYP 1A2, CYP 2A, 2B
low
high
planarity
CYP 2B6
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
4-hydroxylase) has been studied in great detail,
as metabolic differences have first been
described for debrisoquine and sparteine
(antipsychotics)
localized on chromosome 22Of the 75 allels, 26
exprime CYP2D6 proteinessee 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)
MGLEALVPLAVIVAIFLLLVDLMHRRQRWAARYPPGPLPLPGLGNLLHVD
FQNTPYCFDQ LRRRFGDVFSLQLAWTPVVVLNGLAAVREALVTHGEDT
ADRPPVPITQILGFGPRSQGVF LARYGPAWREQRRFSVSTLRNLGLGK
KSLEQWVTEEAACLCAAFANHSGRPFRPNGLLDK AVSNVIASLTCGRR
FEYDDPRFLRLLDLAQEGLKEESGFLREVLNAVPVLLHIPALAGKV LRF
QKAFLTQLDELLTEHRMTWDPAQPPRDLTEAFLAEMEKAKGNPESSFNDE
NLRIVVA DLFSAGMVTTSTTLAWGLLLMILHPDVQRRVQQEIDDVIGQ
VRRPEMGDQAHMPYTTAVI HEVQRFGDIVPLGMTHMTSRDIEVQGFRI
PKGTTLITNLSSVLKDEAVWEKPFRFHPEHF LDAQGHFVKPEAFLPFSA
GRRACLGEPLARMELFLFFTSLLQHFSFSVPTGQPRPSHHGV FAFLVSP
SPYELCAVPR
poor debrisoquine metabolism S
R impaired mechanism of sparteine
poor debrisoquine metabolism I
poor debrisoquine metabolism R
missing in CYP2D69 allele
P loss of activity in CYP2D67
T impaired metabolism of sparteine in alleles 2,
10, 12, 14 and 17 of CYP2D6
see http//www.expasy.org/cgi-bin/niceprot.pl?P106
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CYP 2D6 Polymorphism (III)
variability of debrisoquine-4-hydroxylation
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 lt1 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 antiviralefavirenz
antiviralcyclosporin immuno-suppressantcarbam
azepine antispychoticatorvastatin HMG CoA
Reductase Inhibitor tamoxifen 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-pregnane-3,20-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
zink-finger 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)
hyperforin, a natural ingredient of St. Johns
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-dihydroxy- bergamottin)
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/