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CYP450 metabolism, Drug-drug interaction
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Drug Property Prediction
9. Metabolic Stability &
CYP Inhibition
Outline
• Metabolic stability
• Cytochrome P450 (CYP450) Enzymes
• CYP450 Enzymes Inhibition and Drug-Drug Interactions
• ADMET-Predictor Metabolism Module
Stability Challenges
Intestinal LumenpH Stability, Enzymatic
Stability
Portal vein
Liver
Systemic circulation(plasma stability)
Intestinal wall
Feces
Metabolism
Following Oral Administration
Stability
CYP pheno-typing
pH Buffer
Physio-logical fluids
Micro-somes
Assay Buffer
Plasma
In vitro assessment of stability diverse challenges
Metabolic Stability
• Metabolism is the enzymatic modification of compounds to increase clearance
• Determinant of oral bioavailability, clearance and half-life in vivo
• Metabolism occurs predominantly in the liver, and some may occur in the intestine
• Metabolic stability is increased by structure modification that block or sterically interfere with metabolic sites or withdraw electrons
Drug Metabolism or Biotransformation
• Phase I Reactions: modify the molecular structure itself (e.g. oxidation or dealkylation)
• Phase II Reactions: addition (conjugations) of polar groups to the molecular structure
• Results in more polar products that have higher aqueous solubility, so readily excreted from the body (may result in increased clearance and low bioavailability)
• If the drug is metabolized prior to reaching systemic circulation, it is said to have undergone presystemic or first-pass metabolism
Phase I Reactions
• Oxidation and Reduction
– aliphatic and aromatic oxidation (CYP [ER])– alcohol oxidation (alcohol dehydrogenase [cytosol])– aldehyde oxidation (aldehyde dehydrogenase [cytosol, mitochondria])– dehydrogenation (CYP [ER])– epoxidation (epoxide hydrolase CYP [ER]– N-dealkylation (CYP [ER])– O-dealkylation (CYP [ER])– S-dealkylation (CYP [ER])– Oxidative deamination (monamine- & diamine-oxidases [mitochondria])– N-oxidation (Flavin monooxygenase (FMO) [ER])– N-hydroxylation (CYP [ER])– S-oxidation (FMO [ER]– (NADPH-CYP450 reductase [ER] and nitroreductase [cytosol])
+ +2 2R-H + O + NADPH + H R-OH + NADP + H O
Phase I Reactions Examples
• Aromatic Oxidation (CYP [ER])
• N-Oxidation (Flavin Monooxygenase [ER])
• Reduction (NADPH-CYP450 reductase [ER] and Nitroreductase [cytosol])
OOH
R2
NR1
R2
NR1
O
N
O
O NH2
Phase II Reactions
• Glucuronidation (UDP-glucuronosyl transferase [ER])
• Sulfation (Sulfotransferase [cytosol])
OH
RO
O
OHOH
OH
O
OH
RUDPGA
OH
RO
S OO
OH
R
3’-phosphoadenosine5’phosphosulfate
Phase II Reactions
• Acetylation (N-Acetyltransferases [cytosol])– aromatic amines, aliphatic amines (1, 2 amines), hydrazines,
hydrides,hydroxylamines
• Glycination [mitochondria]– other amino acid additions (e.g. taurine, glutamine)
• Glutathione Conjugation (Glutathione-S-transferases [cytosol])
• Methylation (methyl transferase, catechol o-methyl transferase)
NH2
R
NH
O CH3
R
Structure Modifications Strategies for Phase I Metabolic Stability Improvement
1. Block metabolic site by adding fluorine or other blocking groups
2. remove labile functional group
3. cyclization
4. change the ring size
5. change the chirality
6. reduce lipophilicity
7. replace unstable groups
Block metabolic Site by Adding Fluorine
• Fluorine is the most commonly used blocking group
H
RF
OH
N
Cl
R
R
R
N
N
N N
N
O
O
N
N
N N
N
O
O
F
Compound 5-HT1A
IC50 (M)CYP3A4t1/2 (min)
Buspirone 0.025 4.6
F-substituted Buspirone
0.063 52.3
Buspirone and its fluorinated derivative
Drug Discovery Today, 2005, 10, 1443-1450
Other Blocking Groups• O-dealkylation by CYP2D6 is reduced by replacing methoxy group in
metoprolol with more bulky cyclopropylmethoxy group
• Substitution of the methyl group on Tolbutamide with chlorine to make Chlorpropamide lengthened half-life from 6 to 33 hours
O
CH3
O NH
CH3
CH3
OH
O
O NH
CH3
CH3
OH
Compound First Pass metabolic elimination in vivo
Microsomal Vmax
(nM/min)
Human PK t1/2
(hours)
Metoprolol 50 % 0.46 3.5 - 6
Betaxolol 15 % 0.07 16 - 22
CH3 S NH
NH
CH3
O
O
O
Cl S NH
NH
CH3
O
O
O
Compound clearancemL/min/Kg
Human PK t1/2 (hours)
tolbutamide 0.22 5.9
chlorpropamide
0.03 33
Structure Modifications Strategies for Phase II Metabolic Stability Improvement
• Introduce electron-withdrawing groups or steric hindrance• change phenolic hydroxyl to cyclic urea or thiourea• change phenolic hydroxyl to prodrug
NH
N
ON
O
CH3
OH
R
Glucuronidation
R hGluR binding
affinity (nM)
clearancepmol/
min/mg
Cl 8 267
CN 12 65
NHCH3
CH3CH3
O N
CH3
CH3
O
ON
CH3
CH3
O
OH
Bambuterol: once a day long-acting β–adreno-receptor agonist, prodrug of Terbutaline (3 times a day)
OH
OH
NH
OHCH3
CH3CH3
CYP Isoforms in Human Liver Microsomes (HLM)
28%
2%18%
7%4%
13%
28%3A Family2D62C2E12A61A2Others
50%
30%
10%2%
2% 4%2%
3A Family2D62C8-102E12A61A22C19
% of Drugs Metabolized by DifferentCYP Isoforms
Relative Abundance of CYP Isoformsin HLM
CYP450-3A4 in Complex with Ketoconazole
Heme
Ketoconazole
PDB Code - 2VOM
NNO O
O
N
N
ClCl
CH3
O
H
Clearance and Other Pharmacokinetic Parameters
• When two drugs that are metabolized by the same enzyme, administered together, clearance of one drug is affected by the other
Dose
AUCCl
DD
1/2
0.693e
VCl k V
t
e D
DoseAUC
k V
int
int
i
( )1+
ClCl i
cK
• Clint – normal intrinsic clearance without inhibitor• Clint(i) – intrinsic clearance in the presence of the
inhibitor• c – inhibitor concentration at the CYP isozyme• Ki – inhibitor constant for an enzyme
Cmax/Ki CYP inhibition
< 0.1 not likely
0.1 - 1 possible
> 1 likely
Guidelines for CYP-Induced DDI
% Inhibition @ 3 M
IC50 (M) CYP inhibition
< 15 > 10 low
15 – 50 3 - 10 moderate
> 50 < 3 high
IC50 (M) CYP inhibition
> 100 low
10 - 100 moderate
< 10 high
More Stringent Guidelines
• A plot of activity versus CYP inhibition is useful for multivariate decision making by discovery project teams
Drug Discovery Today, 2005, 10, 1443-1450
CYP Inhibition & Toxicity
Terfenadine + Erythromycin
CYP3A4IsoenzymeInhibition
Toxic levels of Terfenadine
cause prolongation of QT interval and trigger torsades
de pointes arrhythmia
Erythromycin is also known to inhibit the metabolism of Cyclosporine, Carbamazepine and Midazolam
CH3
CH3
CH3
NOH
OH
CH3
CH3
NOH
OH
O
OH
Terfenadine (Seldane)withdrawn from the market because of cardio toxicity
replaced byFexofenadine (Allegra)
Chirality and CYP Inhibition
• Fluvastatin (3R, 5S) enantiomer strongly inhibits CYP2C9 isozyme than its enantiomer
• (+) enantiomer Quinidine is a strong inhibitor of CYP2D6, while (-) enantiomer quinine has no effect on CYP2D6 metabolism
CH3
CH3
O
OH
OH
OH
HH
F
*
*
N
NOH
CH2
O
CH3
* *
Structure Modification Strategies - I
N
NH
N
S
O
CH3
F
O
N
N
NCH3
F
OH
EnzymesIC50 (M)
Compound A Compound B
p38 0.45 0.35
COX-1 5 > 100
3A4 < 2 100
2D6 > 100 22
2C9 < 2 > 100
1A2 4 > 100
Compound A
Compound B
o Reduced IC50 for three CYP enzymes without reducing activity and selectivity (pyridinyloxazole series)
Structure Modification Strategies -II
N
N
O
OH
F
FF
Compound A Compound B
Compound(sodium channel
blockers)
IC50 (nM) CYP2D6% inhibition at
2 M
A 893 87
B 149 20
N
N
O
OH
F
FF
CH3CH3
NH
O
CYP3A4 Inhibition
• Strategies to reduce CYP3A4 Inhibition
• Decrease the lipophilicity (log D7.4) of the molecules
• Add steric hindrance to the heterocycle para to the nitrogen
• Add an electronic substitution (e.g. halogen) that reduces the pKa of the nitrogen
• For nitrogen heterocycle-containing drugs (i.e. triazoles, pyridines, imidazoles, quinolines, thiazoles), reducing the interaction of the nitrogen loan pair with the 3A4 heme group is beneficial Pharmaceutical Research, 2001, 18, 652-655
CYP2D6 Inhibition
Compound GPCRIC50 (M)
CYP2D6 IC50
(M)Selectivity
Ratio
1 0.33 < 0.05 <0.15
2 0.22 0.02 0.09
3 0.22 2.2 10
4 0.19 22 116
NR
Ar NOO
N Cl
S
O NOO
N
Br
Cl
A Structural Series for a G-protein-coupled receptor (GPCR) target
1 2
3
4
Structural Basis for CYP Binding
• CYP1A2: Neutral or basic lipophilic planar molecules with at least one putative H-bond donating site – Theophylline
• CYP2D6: Arylalkylamines with site of oxidation a discrete distance from a protonated nitrogen (-adrenoreceptor blockers, antiarrhythmics and tricyclic antidepressants) – hydroxylation in an aromatic ring or an accompanying short alkyl side chain
•CYP2C9: neutral or acidic molecules with site of oxidation a discrete distance from H-bond donor or possibly anionic heteroatom (non-steroidal anti-inflammatory agents)
Structural Basis for CYP Inhibition
• CYP3A4: lipophilic, neutral or basic molecules with site of oxidation often nitrogen (N-dealkylation) or allylic position. Wide range of substrates /pharmaceuticals
• CYP2E1: small (MW of <= 200 Da) normally lipophilic linear and cyclic molecules. Volatile anaesthetics
Commercial Software for Metabolic Stability
Name Company Purpose Website
Metasite Molecular Discovery
Metabolite Structures
www.moldiscovery.com
KnowItAll Biorad Metabolic stability www.biorad.com
ADMENSA Inpharmatic metabolic stability, metabolite structures
www.inpharmatica.com
Meteor Lhasa Metabolite structures www.lhasalimited.org
ADMET-Predictor
Simulations Plus Inc.
Metabolic stability, metabolite structures
www.simulations-plus.com
DatabasesDatabase Content Producer and Website
Metabolite Metabolites of primary drugs MDL information systems (www.mdli.com)
Metabolism metabolites of drugs and chemicals
Accelrys Ltdwww.accelrys.com
Biopath Biochemical pathways of endogenous compounds
www2.chemie.uni-erlangen.de/services/biopath
ADMET Predictor- Metabolism Module1A2, 2C9, 2C19, 2D6 and 3A4
Output Term Unit Description of the termMET_Enzyme_Inh Qualitative estimation of general inhibitory action
against the enzyme
MET_Enzyme_Km M kinetic Michaelis-menten Km constant for “enzyme”-mediated metabolism
MET_Enzyme_Vmax nmol/min/nmol enzyme kinetic Michaelis-Menten Vmax constant for “Enzyme” –mediated metabolism
MET_Enzyme_Vmax_mgP nmol/min/mg microsomal protein
- as above- alternative units
MET_Enzyme_Clint L/min/mg microsomal protein
intrinsic clearance constant for “Enzyme”-mediated metabolism
CYP_Enzyme_Substr qualitative assessment of a molecule being the substrate of “enzyme” in human
CYP_Enzyme_Sites specific sites of human CYP “enzyme”
MET_UGT”Enzyme” qualitative model of a glucuronidation by the UDP glucuronosyltransferase “ --“ enzyme
MET_3A4_I_drug
MET_3A4_Ki_drug
drug – Midazolam and testosterone
qualitative model of a specific inhibition of the CYP3A4-mediated metabolism of “drug”specific inhibition constant for the CYP3A4-mediated metabolism of “drug”
MET_Risk and MET_code ADMET Risk and ADMET code for metabolic liability
CYP_Risk and CYP_Code ADMET risk and ADMET code for metabolic liability
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