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