2009

ADME-Tox
APPLICATION NOTE

in vitro drug metabolism

ADME (Absorption, Distribution, Metabolism, and Excretion) deficiency is one of the major factors that cause failures
during drug development. To prevent those costly failures from occurring, in vitro screening of potential drug candidates
in the early drug discovery phase has been employed as a more cost-effective approach to identify compounds that
have unfavorable ADME characteristics1,2. Cerep provides a wide variety of valuable metabolism screening assays using
assay matrices including recombinant cytochrome P450 enzymes (CYPs), recombinant uridine diphosphate-glucuronosyl
transferases (UGTs), liver microsomes, liver S9 fraction, intestinal microsomes, intestinal S9 fraction, and hepatocytes
from human and animal species.
Metabolic stability, 2 time points
Metabolic stability, 5 time points: half-life determination and intrinsic clearance calculation
n For evaluating the oxidative or conjugation metabolism, choice of matrices to use:
. liver microsomes
. liver S9
. hepatocytes
. intestinal microsomes
. intestinal S9
. individual CYP isoforms for CYP phenotyping (phase I metabolism)
. individual UGT isoforms for UGT phenotyping (phase II metabolism)
n For evaluating hydrolytic metabolism, choice of matrices to use:
. plasma/serum
. blood
Metabolite identification
Conjugate detection
A drug, once it enters an organism, can be converted to metabolite(s), which is/are typically more aqueous-soluble and
readily excreted than the corresponding parent drug. The chemical conversion is catalyzed by phase I (mainly CYPs)
and/or phase II (such as glutathione-S-transferases and UGTs) drug-metabolizing enzymes, which are present at high
levels in the liver, intestine, and kidney. From metabolism perspective, an ideal drug should:
1) be relatively stable, i.e. have small first-pass effect and maintain an effective concentration in blood for a reasonable
period of time;
2) be metabolized by multiple CYP enzymes and do not largely depend on CYPs that are polymorphically-expressed,
such as CYP2C9, CYP2C19, and CYP2D6;
3) lead to no pharmacologically-active (unless prodrug) or chemically-reactive metabolites.
Cereps high-throughput in vitro metabolism screening assays allow identification of compounds that have those favorable
characteristics for further development as drug candidates.

Metabolic stability
Many compounds with promising pharmacological characteristics
never become drugs because they are rapidly metabolized in the
liver and, therefore, have very low oral bioavailability. The metabolic
stability assays are designed to measure the stability of a test compound in a variety of assay matrices including liver microsomes, liver
S9, intestinal microsomes, intestinal S9, cryopreserved hepatocytes,
human recombinant CYP isozymes, plasma and blood from human
and animal species.
Microsomal preparation from the human liver contains all CYP isozymes
and other membrane-bound drug metabolizing enzymes, such as
flavin monooxygenase (FMO) and UGTs, which are responsible for
the metabolism of majority of drugs in humans. Liver microsomes are
well characterized and are the most frequently used form of tissue
preparations for in vitro drug metabolism studies, especially for the
phaseI oxidations (Fig.1). By supplementing the cofactor, UDPGA
(uridine diphosphoglucuronic acid), liver microsomes can also be used
to screen compounds for phase II glucuronide conjugation.
1
2

80

Microsomal stability
Human
Monkey
Dog
Rat
Mouse

60

40

20

Propranolol

Imipramine

Verapamil

Terfenadine

parent drug remaining (%)

Fig.1 Interspecies comparison - Stability in liver

microsomes at 60 min - parent drug: 1 M, pooled

liver microsomes: 0.3 mg/mL, detection method:
HPLC-MS/MS

FDA (1997) Guidance for Industry - Drug Metabolism/Drug Interaction Studies in the Drug Development Process: Studies In Vitro
FDA (2006) Drug Interaction Studies - Study Design, Data Analysis, and Implications for Dosing and Labeling

20

DRUG DISCOVER

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ADME-Tox - application note

In vitro drug metabolism

parent drug remaining (%)

compound remaining (%)

Liver S9 is another preparation for

100

in vitro drug metabolism studies. S9
contains both membrane-bound and
soluble enzymes, including esterases,
glutathione-S-transferases (GST), and all
the enzymes present in the microsomes.
50
By supplementing proper co-factors,
S9 can be used to study both phase I
human
IOXUD]HSDP
oxidations and hydrolysis and phaseII
monkey
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dog
conjugations of test compounds.
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rat
mouse
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Hepatocytes not only contain all drug





propoxycaine
propantheline
metabolizing enzymes but also are a living
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Fig.3 Interspecies comparison - Stability in plasma
system, which closely mimic the in vivo situat 60 min parent drug: 5 M, pooled plasma,
Fig.2 Stability in hepatocytes - Parent
ation. Cryopreserved hepatocytes retain
detection method: HPLC-MS/MS
drug: 0.5 M, pooled cryopreserved
sufficient viability and enzyme activities for
hepatocytes from 10donors: 106 viable
several hours and are an excellent assay
cells/mL, detection method: HPLC-MS/MS.
100
matrix for metabolic stability (Fig. 2).
The intestine also contains high activities of drug metabolizing enzymes and
75
exerts first-pass effect. Metabolic stability of test compounds can be screened





using intestinal microsomes or S9.
50
The plasma and blood contain esterases and proteases, which have hydrolytic
activity toward compounds that have ester and/or amide bonds. Because of
the large volume of the blood, the hydrolytic effect on some compounds could
25
be substantial. The stability of test compounds can be screened using plasma
human





dog
(Fig.3) or blood (Fig. 4).
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Phase I metabolism (CYP reaction
phenotyping)

rat

propoxycaine

propantheline

Fig.4 Interspecies comparison - Stability in blood

at 60min - parent drug: 5 M, pooled blood,

If a test compound is substantially metabolized by microsomes, CYPs are likely detection method: HPLC-MS/MS
to be the enzymes that are responsible for 
the metabolism. Consequently, the
next step is to find out which CYP is responsible for the metabolism. The process of identifying which CYP is responsible for
the metabolism of a compound is often referred to as CYP Reaction Phenotyping or CYP Phenotyping. The results from the CYP

Phenotyping will indicate whether the metabolism of the test compound is catalyzed by multiple CYPs or largely depended on
one of those polymorphically-expressed CYPs.

CYP Reaction Phenotyping study can be conducted
as the metabolic stability with individual human recombinant CYPs to determine
which isozyme is involved in the metabolism of the test compound.


Phase II metabolism

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polar functional groups,
such as

After phase I reactions introduce or expose

hydroxyl, amino, carboxyl, and sulfhydryl groups,
in a test compound, phase II enzymes often add bulky hydrophilic substituents, such as glucuronic acid, sulfate, to those polar
functional groups to make the test compounds even more water soluble. If the test compound already has polar functional groups,
it may directly undergo phase II reactions.
Compounds can be screened for direct phase II reactions in the metabolic Stability assays using recombinant phase II enzymes
such as UGT1A1 and UGT2B7 or liver microsomes or S9 in the presence of phase II reaction cofactors such as UDPGA or
glutathione. The parent test compound in the incubation mixture is typically quantitated by HPLC-MS/MS in those Metabolic
Stability assays.
Cerep also provides assays allowing qualitative detection of glutathione conjugate and glucuronide conjugate of a test compound
using liver S9 and liver microsomal incubations, respectively. In those assays, compounds are typically tested at 50 M with a
protein concentration of 1 mg/mL in S9 or microsomes incubations supplemented with 5 mM glutathione or UDPGA. Incubations
are typically allowed for 90min, and the samples are analyzed using full scan HPLC-MS method.

Intrinsic clearance
In vitro intrinsic clearance obtained using liver tissue can be used to predict hepatic clearance. Classically, the intrinsic clearance
(Vmax/Km) is calculated by analysis of metabolite formation using Michaelis-Menten equation. However, in most cases the
information on metabolite has not been obtained in the drug discovery stage. For this reason, the intrinsic clearance is typically
determined based on the disappearance rate of the parent compound.

Metabolite identification
It is important to know the structure of the metabolite(s) if a test compound is substantially metabolized. The information of metabolite
structures can be used to synthesize potential metabolites for testing whether they are pharmacologically active or chemically
reactive. The information of metabolite structures is also helpful for identifying the site of biotransformation so that modifications
can be made in this site to block the unfavorable biotransformation.

ADME-Tox - application note

In vitro drug metabolism

This table provides a general guideline in choosing assays to address specific questions.

Cerep assay

Matrix

Co-factor

Questions to be answered

Metabolic stability
(% test compound remaining,
or intrinsic clearance)

Microsomes or S9

NADPH

Is the compound metabolized by phase I enzymes, such

as CYPs?

Microsomes or S9

UDPGA, or GSH

Is the compound metabolized by phase II enzymes, such

as UGTs, or GSTs?

Hepatocytes
CYP Reaction phenotyping
(%test compound remaining,
or intrinsic clearance)
Metabolite identification
or Conjugate detection

Is the compound metabolized by xenobiotic

metabolizing enzymes in a live system?

Recombinant CYP

NADPH

Microsomes

NADPH + CYP isoform

specific inhibitor

Microsomes or S9

NADPH, UDPGA,
or GSH

Which CYP isoform is involved in the metabolism?

What phase I or phase II product is formed?

Experimental conditions
Our standard conditions for the stability assays include incubation of 1 M test compound in selected assay matrix for 60min
(recombinant CYPs, UGTs, liver microsomes or S9) or 120 min (hepatocytes, intestinal microsomes or S9) in duplicate. The
peak area of the test compound at the end of incubation (60 min or 120 min) is compared to the peak area in the beginning
(time zero) for percent remaining calculation. The protein concentration is typically 0.3mg/mL with microsomal incubations and
1mg/mL with S9 incubations.
The intrinsic clearance assays are typically performed at 0.5 M of test compound with 5time points, including time zero and
up to 60 min or 120min. The half-life (T) is estimated from the slope of the initial linear range of the logarithmic curve of
percent compound remaining versus time based on first order kinetics. From the half-life value, the apparent intrinsic clearance
is calculated.
To ensure the generation of sufficient metabolite, the metabolite identification assays usually contain more enzymes and have
longer incubation time.
The assay can be customized as per the customers specifications. Some examples of customization include incubation time, test
compound and enzyme concentrations, and the number of replicates, among others.

Quality control
For metabolic stability assays, several reference compounds with diverse metabolic stability in human liver microsomes are concurrently incubated in the same assay with the test compounds. The assay is rendered valid if the results of the reference compounds
fall within the specifications as defined in the corresponding Standard Operating Procedure.

The reference compounds used in each type of Cerep metabolic stability or intrinsic clearance assays are listed as follows.

Assay matrix

Reference compounds

Microsomes or S9

imipramine, propranolol, terfenadine, and verapamil

Hepatocytes

propranolol, terfenadine, flurazepam, and naloxone

CYP1A2

propranolol, ethoxyresorufin

CYP2B6

propranolol, benzphetamine

CYP2C8

propranolol, paclitaxel

CYP2C9

propranolol, diclofenac

CYP2C19

propranolol, omeprazole

CYP2D6

propranolol, dextromethorphan

CYP2E1

under development

CYP3A4

propranolol, terfenadine

CYP3A5

propranolol, terfenadine

UGT1A1

7-hydroxy-4-(trifluoromethyl) coumarin

UGT2B7

7-hydroxy-4-(trifluoromethyl) coumarin

Plasma or blood

propantheline, propoxycaine

 Cerep - January 2009

ADME-Tox - application note

In vitro drug metabolism

available application notes

Aqueous solubility
Partition coefficient - LogD
Protein binding/Blood partitioning
In vitro drug absorption
P-gp mediated drug-drug interaction
In vitro drug metabolism
In vivo PK/BBB
Bioanalytical support
Cyp-mediated drug-drug interaction
Cardiac toxicity - hERG
Cytotoxicity
Phospholipidosis
Genetic toxicity

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