Drug metabolism: A pharmaceutical industry perspective

Prof. Dr. Jrg Huwyler University of Applied Sciences (FHNW) School of Life Sciences Dept. of Pharma Technology Grndenstrasse 40, CH-4132 Muttenz and Rosental WRO-1060, CH-4002 Basel

joerg.huwyler@fhnw.ch

J.Huwyler, 11.2008, page 1

Pharmacokinetics / Pharmacodynamics / Safety

ADME: Absorption (active transport, diffusion, metabolism) Distribution (active transport, diffusion) Metabolism (phase I, phase II enzymes) Excretion (active transport, diffusion) Context: SAFETY EFFICACY

D
drug at absorption site

drug in the body

metabolites

excreted metabolites

E
excreted drug

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Impact of metabolism on ALL stages of the ADME process

A: e.g. Expression of CYP3A4 in enterocytes may limit intestinal absorption D: e.g. Expression of CYP1A1 in lung tissue may limit distribution to lung tissue M: Hepatic and extraheptic metabolism E: e.g. Biliary excretion of drugs

D
drug at absorption site

drug in the body

metabolites

excreted metabolites

E
excreted drug
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The liver as an example: Interplay between metabolism & transport

X X X-OH

X X-SO4 X-O-gluc
ATP

Drug >

Phase 0 uptake Oatp oct

>

Phase I functionalization oxidations (CYP450) reductions hydrolysis isomerizations

>

Phase II conjugation glucuronidation (UGT) sulfation (ST) glutathion conjug. (GST) methylation acetylation

>

Phase III export P-gp (MDR1) TC transporter (cBST) cMOAT (MRP2)

> Excretion

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Multitude of different drug metabolizing enzymes

P450s: Superfamiliy of heme enzymes, approx. 1200 isoenzymes in all known species (20 in bacteria, 60 in human, 300 in plants) Additional examples:

Functions of P450's: - Plants: Biosynthesis of pigments, growth regulators, plant toxins - Human: Biosynthesis of low molecular weight regulators (steroids, prostaglandins, thromboxanes, fatty acid derivatives, retinoic acid derivatives) - Metabolism of xenobiotics

Human arylamine N-acetyltransferases (NAT1 and NAT2) Human UDP-glucuronosyltransferases (UGTs, > 15 isoenzymes) Additional examples: Human SLC transporters (43 families, >300 transporter genes) ABC-Transporter (49 families) ... etc.
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... etc.

Major CYPs and variability in expression levels

Variability: Polymorphism (e.g. CYP2D6) Induction (e.g. CYP3A4)

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Summary

Pharma industry: Development of safe and efficacious drugs Basic observations:

-

Impact of metabolism on all stages of the ADME process (consequence for both PD and safety) Interplay between metabolism and transport Multitude of metabolizing enzymes (and drug transporters) Inter-individual variability in expression levels (polymorphism, induction)

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Tasks and in vitro methods

Hepatic clearance (CLH): - Recombinant systems (human CYPs expressed in E. coli, baculovirus, ..)
-

Hepatic subcellular fractions (S9 or liver microsomes, different species) Hepatocytes in primary cultures or suspension (different species) Liver slices and more complex models (perfused liver)

Note:
-

Hepatic models: well stirred / parallel tube / dispersion model In vitro in vivo scaling: allometric / physiological / empirical Protein binding to be considered Metabolic pathways and metabolite identification Chemically reactive metabolites and covalent binding (incl. GSH adduct screening)
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Microsomes and human hepatocytes

Comparison between systems: advantages and limitations

Microsomes Pro:
Easy to store Well characterised system Easy to use Available in pools (n=15)

Hepatocytes Pro:
Cryopreserved Well characterised system Phase I&II metabolism Active transport Intact cellular properties No need for cofactors

Con:
Phase I metabolism Cellular system destroyed Lack of cofactors Limited Phase II metabolism No active transport Product inhibition

Con:
Primary culture Limited availability (n=1) Variability (enzyme expression levels) Limits of concentration (cytotoxicity)
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Tasks and in vitro methods

Drug-drug interactions (DDI): - Recombinant systems (e.g. human CYPs)

-

Microsomes (e.g. human, pool of 20 donors) Hepatocytes (e.g. human primary cultures)

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Drug-drug interaction studies (DDI) with microsomes or recombinant enzymes

1. HTS fluorescent indicator assay (Crespi assay): - Recombinant human 1A2, 2C9, 2C19, 2D6 and 3A4 - (non specific) fluorescent test probes - derived parameter: IC50 Human liver microsomes (pool 20 donors): - Specific test probes, 1/isoform except 3A4: 3 probes - LC-MS/MS quantification, derived parameter: IC50s

2.

CYP 3A4 substrates: MIDAZOLAM NIFEDIPINE 7-Benzyloxy-4-(trifluoromethyl)coumarin (BFC) TESTOSTERONE

CYP 2C9 substrate: DICLOFENAC CYP 2D6 substrate: (+/-)BUFURALOL

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Recombinant enzymes, microsomes or cell lines: Cost-efficient automatization of assays (HT screening)

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Goal: in vitro - in vivo extrapolation (e.g. DDI risk)

Fold of change = in AUC
10

AUC (+I) AUC

=1+

I Ki

Fold of change in AUC

HIGH RISK

MEDIUM RISK

LOW RISK
2

Threshold of significant interaction

0 0 0.0 1 0.1 1 10 10 0 1 00 0

I / Ki

I/Ki
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Blanchard et al., Current Drug Metabolism, 2004, 5, 147-156

DDI with CYPs: clinical probe substrates / inhibitors

Criteria: 1) Substrate of a single pathway 2) Highly sensitive towards CYP activity changes 3) No P-glycoprotein substrates or other transporters 4) No (adverse) PD effects at used doses 5) Commercially available 6) Readily measured PK endpoint

Bjornsson et al. (2003) J.Clin.Pharmacol.

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Tasks and in vitro methods

Induction:
-

Nuclear hormone receptor binding and activation (e.g. PXR) Cell lines (e.g. LS180) Hepatocytes (e.g. human primary cultures) ( Microsomes from induced animals )

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Induction: Use of cell lines or primary hepatocytes

CYP3A4 mRNA induction by the antibiotic rifampin (72h): MDR1 (LS180 colorectal adenocarcinoma cells, left) and CYP3A4 (human hepatocytes, right)

Huwyler et al, Curr.Drug Metabol., 2006

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Tasks and in vitro methods

An integrative picture of ADME:

-

Computer assisted simulations and modelling

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Physiology based PK modelling Input

- In vitro data - Species specific physiological data (e.g. blood flow, tissue volume) - Compound specific physico-chemical properties (e.g. ClogP, pKa)
Lung

Gut

Simulation of outcome
ARTERIAL

VENOUS

Brain Kidney Heart M uscle Adipose Other tissues...

Plasma concentrations Time

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Summary

Strategy to optimize efficacy:

-

Determination of CLH Clarify PK properties and PK / PD relationship Phenotyping (individualized health care, stratified clinical trials)

Strategy to assess safety: - Identify metabolic pathways and main metabolites (e.g. active, toxic or reactive metabolites)
-

Identification of metabolizing enzymes (drug-drug interactions; polymorphism; disease, gender and age) Induction (dose adaptation, polypharmacy, environmental factors)
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EPAA workshop

Complex situation:

Goal:
-

Focus on a limited set of technologies Tools should be cost-efficient (e.g. automatized HT screening) Predictive in vitro tools to avoid animal experimentation (3R and costs) Important task of in silico simulation and modelling
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