Cytochrome

P-450 Enzymes:

In Vitro Assessment
and Clinical Implications

Paul Glue and Roberf

P. Clement

1. Introduction
Over the last 30 yr, in vitro assessment of interactions between
drugs and drug-metabolizing enzymes has developed from a scientific curiosity to a topic of major clinical and commercial importance.
For clinicians, knowledge of this area assists in the ability to predict
the likelihood of a drug-drug interaction, or may help explain certain cases in which there is unexplained toxicity or lack of efficacy.
For the pharmaceutical industry, selection of appropriate compounds
for further preclinical and clinical development is a critical issue. This
chapter will introduce some basic concepts about the most important drug-metabolizing enzyme system in humans, the cytochrome
P-450 (CYP-450) family of enzymes, with the emphasis on in vitro
methods of assessment,and how these correspond with clinical studies.
1.1. Overview

of the Cytochrome

P-450 Enzyme Family

The best-researched enzyme family in humans, and the most

important (based on the proportion of drugs and endogenous compounds that are metabolized by it) is the cytochrome P-450 (CYP450) system, which is involved in the phase 1 oxidative metabolism
of many endogenous compounds (e.g., steroids, bile acids, prostaglandins) and drugs. CYP-450 enzymes participate in multiple
metabolic pathways for a wide range of structurally different compounds (see Kroemer and Eichelbaum, 1995; Li et al., 1995). CYP450 enzymes are heme-containing proteins which are found in
From
Neuromethods,
vol
Eds A A Boulton,
C B Baker,

33 Cell Neurob,o/ogy
Technrques
and A N Bateson 0 Humana
Press Inc

195

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and Clement

endoplasmic
reticulum
and mitochondria.
In humans,
they are
synthesized
predominantly
in the liver, and to a lesser extent in
the small intestine, kidney, adrenals, and other sites (Krishna and
Klotz, 1994). The term P-450 was based on their initial identification as a red liver pigment
(I) that produced
a characteristic
absorption
spectrum peak at 450 nm when reduced and bound to
carbon monoxide
(Garfinkel,l958;
Omura
and Sato, 1962). The
genetics of these enzymes have now been extensively
studied, with
characterization
of several hundred
gene sequences from many
species. Comparison
of CYP-450 gene sequences from bacteria to
humans indicate that the ancestral CYP-450 gene is over 3.5 billion
yr old (Nelson et al., 1993). One amino acid sequence (a 26-residue
region near the carboxy-terminus
of all CYP-450 proteins, which
is the heme-binding
domain)
has been found m some bacterial
and virtually
all eukaryotic
cells (Nebert and Gonzales,
1987) A
standard
naming
system has also been developed
for CYP-450
enzymes (Nelson et al., 1993). The abbreviation
for cytochrome
P-450 (CYP) is followed by a number denoting
the gene family, a
letter indicating
the subfamily,
and another number
referring
to
the enzyme (e.g., CYP2D6). Enzymes with 40% or greater sequence
identity
are included
in the same family (in this case, family 2),
and within this family, enzymes with greater than 55% sequence
identity
are mcluded
in the same subfamily
(D). Within the subfamily, enzymes are assigned numbers on an arbitrary basis. There
are now at least 36 CYP gene families,
of which 12 are known to
exist in all mammals
(Nelson et al., 1993).
1.2. Clinically

Relevant

CYP-450

Enzymes

Despite the enormous number of CYP-450 enzymes discovered,

only a handful are of clinical relevance (these are listed in Table 1).
Indeed, the most important
of these are CYP2D6 and CYP3A4,
which are involved in the metabolism
of the majority
of commonly
used drugs. The CYP3A family,
of which CYP3A4 is the most
important
enzyme, may account for up to 60% of all microsomal
species in some human livers (Guengerich,
19901, and has the widest substrate specificity
of all CYP-450 enzymes
1.3. Factors

that May

Influence

CYP-450

Activity

CYP-450 enzyme activity may differ widely between individuals

because of genetics, age, gender, disease, drug exposures and/or

Enzymes

tolbutamide
4hydroxylation

phenytom
fluconazole
ketoconazole

Table 1
Important

s-mephenytoin
4-hydroxylation

ketoconazole

Yes
rifampicm

omeprazole
diazepam
imipramme
hexobarbital

yes

CYP2C19

with

SSRI selective serotonm reuptake mhibitor

7-ethoxyresorufm
0-deethylation

m vitro enzyme
selective assays

%ZA tricyclic antidepressant,

cimetidme
fluoroqumolones

cigarette smoke
barbecued meat
omeprazole

Yes
barbiturates
rifampicin

tolbutamide
phenytom
S-warfarm

theophyllme
caffeine
acetammophen

yes

possible

CYP2C9/10

CYP-450

possible

Human

some malor
mhibitors

mducible
some major
inducers

genetic
polymorphism
some malor
substrates

CYPlA2

Malor

Inhibitors,

dextromethorphan
Odemethylation

cimetidme
most SSRIs
neuroleptics
TCAs
quuudme

possible
rifampicin
carbamazepme
phenobarbital

propranolol
encamide
TCAs and SSRIs
neuroleptics
(many others)

yes

CYP2D6

Substrates,

chorzoxazone
hydroxylation

disulfiram

ethanol

yes

alcohol
halothane

probable

CYP2El

and Inducers

6p-

cyclosporm
erythromycm
nifedipme
estrogens/progestms
somebenzcdiazepmes
quuudme
(many others)
yes
barbiturates
carbamazepme
phenytom
rifampicm, others
cimetidme
erythromycm
ketoconazole
fluconazole
ethmyl estradiol
progestins
testosterone 6phydroxylation
dextromethorphan
N-demethlyation
benzphetamme
N-demethlyation

CYP3A4

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and Clement

environmental
factors. Studies on genetic influences
on enzyme
activity have concentrated
on CYP2D6 and CYP2C19, because of
well-established
genetically
determined
differences
in activity of
these enzymes. To assess activity of these enzymes clinically,
sublects are administered
a substrate which is selective for an enzyme
(e.g., debrisoquine
or sparteme
for CYP2D6,
mephenytoin
for
CYP2C19), and its rate of metabolism
is determined
from the ratio
of urinary concentrations
of parent to metabolite.
These ratios are
bimodally
distributed
within populations:
The majority
of subjects with normal rates of metabolism
are classified as extensive
metabolizers,
and a minority
with reduced metabolism
are called
poor metabolizers.
This difference in activity is also known as
genetic polymorphism,
and prior to the development
of clmical
or in vitro methods for assessing enzyme activity, the recognition
and identification
of subpopulations
of poor metabohzers
provided the first evidence for the involvement
of these enzymes in
the metabolism
of certain compounds.
A range of genetic mutations have been identified
to account for these metabolic
changes
(Dahl et al., 1992; Ieiri et al., 1996). The prevalence
of poor
metabolizers
for CYP2D6 and CYP2C19 in different populations
has also been extensively
studied (see Evans et al., 1980; Wilkinson
et al., 1989). Studies in animals and humans of different ages have
shown marked alterations
in expression
of CYP-450 enzymes in
liver tissue. However, these results are highly species-specific,
and
changes noted in animals cannot be assumed to occur in humans.
In humans, there is prelimmary
evidence that CYP3A4 activity is
lowest in neonates and increases to maximal
levels m adulthood
(Ratanasavanh
et al., 19911, and that activity of CYP3A4, but not
CYP2D6 or CYP2C19, appears to decrease between 20 and 80 yr
of age (May et al., 1994). Expression
of some CYP-450 enzymes
may be increased in certain age groups (e-g, CYP3A7 in fetal liver;
Schuetz et al., 1994).
Studies into the effects of gender on enzyme activity in humans
are limited.
Compared
with males, females appear to have greater
activity
of CYP3A4 and CYP2C19, similar CYP2D6 activity,
and
decreased
CYPlA2
activity
(see Harris et al., 1995). CYP-450
activity may be altered in certain diseases such as acute viral illnesses (Soons et al., 1992), chronic liver disease (el-Yazigi
et al.,
1995; Murray,
19921, and potentially
in patients
with thyroid
abnormalities
(McClure
and Stupans, 1995). Of the environmental
factors studied,
drugs, diet, and lifestyle
are important
factors

Cytochrome

P-450 Enzymes

199

influencing the expression or activity of CYP enzymes. A number

of important inhibitors and inducers are listed in Table 1. Review
of the inhibitory or inductive effects of drugs on CYP-450 activity
is, however, beyond the scope of this chapter, and readers are
referred to recent overviews of this area (see Glue and Banfield,
1996; Murray, 1992; Spatzenegger and Jaeger, 1995; Wrighton and
Stevens, 1992). Activity of CYPlA2 may be increased by smoking
or consuming of barbecued food, and reduced by grapefruit juice.
Grapefruit juice also inhibits activity of CYP3A4. Activity of
CYP2El may be induced by fasting (Hong et al., 1987) or chronic
intake of alcohol (Perrot et al., 1989).
2. In Vitro

Assessment

of CYP-450

Activity

Established techniques and recent advances in the in vitro

assessment of the role of specific CYP-450 enzymes in drug
metabolism as well as the potential for inhibition or induction of
these enzymes will be described in Subheadings 2.1. and 2.2., as
will a practical approach for selection of new compounds.
A drug can interact in two ways with an enzyme: as a substrate
(where it is metabolized), or through altering its activity (by
enzyme inhibition or induction). The fact that a drug is a substrate for an enzyme does not necessarily mean it will also inhibit
or induce that enzyme at clinically relevant concentrations. Similarly, a drug can be an enzyme inhibitor (e.g., quinidine for
CYP2D6) or inducer (e.g., carbamazepine and CYP2D6) without
being a substrate for that enzyme. It is also important to note that
a drug may be a substrate for more than one enzyme (e.g., imipramine is metabolized by CYPlA2, -2D6, -2C19, and -3A4).
It is important to be aware of a number of technical issues when
reviewing
data from in vitro studies (see Parkinson, 1996;
Rodrigues, 1994). The source of assay material is important. If
human liver tissue is used, factors that might alter enzyme levels
(e.g., prior use of inducing drugs or smoking history, age, gender) should be known. The use of nonhuman liver tissue complicates data interpretation
because of differences in enzyme
expression and substrate specificity relative to human liver tissue. Enzyme(s) expression or concentration in normal human liver
cells, or the availability of cofactors may be quite different from
that observed in cultured or cloned cells or in microsomal preparations. These factors must be taken into account when interpret-

200

Glue and Clement

ing the possible clinical significance of in vitro metabolic data for

any compound. Concentrations of drugs used (either as substrate
or inhibitor) should be clinically relevant. If they are too low,
interactions may be missed, and if they are too high, spurious
interactions may be reported. Furthermore the CYP-450 enzymes
primarily responsible for the metabolism of a drug may change
as the concentration of substrate changes (Kato and Yamazoe,
1994). Ideally a wide range of concentrations should be used in in
vitro testing, that will include clinically relevant plasma concentrations as well as liver concentrations (if known). Physicochemical
characteristics of the drug (e.g., its aqueous solubility) and assay
sensitivity will also influence the concentrations of drug used in
in vitro testing.
2.1. In Vitro Techniques
There are now standardized in vitro techniques that allow
determination of the specific CYP-450 enzymes involved in the
metabolism of a test drug, or to assess the potential for and/or
extent of enzyme inhibition or induction. Assays may be carried
out in animal or human liver tissue (liver slices, cultured hepatocytes, or subcellular fractions of hepatic tissue), and cloned yeast,
bacterial, and insect cell systems expressing specific CYP-450
enzymes have also been developed (see Gonzales and Korzekwa,
1995; Parkinson, 1996; Rodrigues, 1994; Waterman et al., 1995).
2.1.1.

Metabolism

Metabolism of a test drug can be determined by measuring the

rate of disappearance of parent compound and/or the appearance of metabolite(s) when incubated in a tissue system. The role
of specific enzymes in the test drugs metabolism may be determined in several ways. by incubating the test drug with well-characterized tissue from a number of donors, and correlating rates of
metabolism of the test compound with those of enzyme-specific
substrates in the different tissue samples; by incubating the test
drug with individually
cloned and expressed enzymes and
assessing metabolism (specific enzymes are now available commercially); or by adding selective CYP enzyme inhibitors (e.g.,
quinidine for CYP2D6; ketoconazole for CYP3A4; see Table 1) to
tissue assays and looking for changes in rates of metabolism. An
alternative to using chemical inhibitors in this type of assay is to
use enzyme-specific antibodies; however these can only be used

Cytochrome

P-450

Enzymes

201

in subcellular
assays, and the number
of highly specific inhibitory antibodies
is limited.
Most recently, in addition
to the identification
of the enzymes responsible
for the metabolism
of test
drugs, researchers have determined
the kinetic rate constants (Km)
for individual
enzymes, and have used this information
together
with the relative abundance
of the enzymes m liver to predict the
clinical relevance (i.e., contribution)
of specific enzymes to a drugs
metabolism
(see Black et al., 1996; Kunze et al., 1996; Kunze and
Trager, 1996).
2.1.2.

inhibit/on

The potential
for a test drug to inhibit selected CYP-450 enzymes
can be assessed by examining
its effects on the rate of metabolism
of probe substrates specific for particular
CYP-450 enzymes
A
list of substrates used in in vitro studies is provided
in Table 1
(for a more extensive list see Parkinson,
1996). A range of concentrations of the test drug are incubated
with the substrate and its
rate of metabolism
compared
with a test-drug-free
control assay,
reduced metabolite
and increased probe substrate concentrations
would indicate inhibition.
For enzyme-specific
metabolism,
the
kinetics of the inhibition
reaction (Ki) can be determined
and this
information
may be used to predict the clinical significance
of the
in vitro findings.
2.1.3.

Induction

Evidence
for the ability
of a test drug to induce
CYP-450
enzymes may initially
come from toxicology
studies in which animals (rodents, dogs, nonhuman
primates)
are administered
multiple doses over a specified length of time (2 wk, 1 mo, 3 mo, and
so on). Parameters
that may indicate enzyme induction
include
increases in absolute or relative liver weight, microsomal
protein
and CYP-450 content, and the activity of selected CYP-450 enzymes
(e.g., substrate-specific
assays). More recent studies have included
determination
of liver microsomal
(endoplasmic
reticulum)
content of specific enzymes
or accumulation
of enzyme-specific
mRNA. Increases in the activity of specific enzymes together with
mcreases m the associated
protein
and mRNA
levels for that
enzyme would be evidence of induction.
Extrapolation
of results
from toxicology
species to humans
remains
difficult.
Rodents
appear to be more responsive to inducers, and the induction
process (i e , signaling
at the nuclear level) is not well understood

202

Glue

and Clement

and may vary across different species. Investigators are now using
cultured human hepatocytes to screen for induction, but further
work will be required to establish the clinical relevance of these
in vitro findings.
2.2. Practical in Vitro Assessment
of fhe CYP-450 Profile of a New Compound
Pharmaceutical companies synthesize thousands of new compounds every year. Based on indices of activity in screening paradigms, a handful of these compounds will be selected to be studied
in greater detail to determine their suitability for eventual clinical
development. Certain aspects of metabolism can be identified that
can assist in the selection process. For example, a drug that is not
metabolized will be preferable to one that undergoes extensive
metabolism, as the latter is likely to have greater pharmacokinetic
variability; toxicological assessment is also less complicated for
compounds that are not extensively metabolized. Drugs that do
not induce or inhibit CYP-450 enzymes at relevant concentrations
are preferable to drugs with these activities (with a few exceptions such as triazole antifungals, in which CYP-450 inhibition is
central to therapeutic activity). Enzyme inhibition is a potentially
more serious problem for a drug than is evidence of induction.
Clinically, induction may result in loss of efficacy as a result of
reduced concentrations of an affected substrate, whereas inhibition may produce substrate accumulation and toxicity; thus evidence of significant enzyme inhibition is rarely a positive feature
for compounds in development. Finally, the specific enzymes
involved in the metabolism of, or that are inhibited by a drug are
also important; compounds that are not metabolized by or that
do not inhibit CYP3A4 or CYP2D6 will not have interaction problems with the majority of other drugs currently available.
Based on the above, a practical method for assessment of test
compounds will be described. This is summarized in Fig. 1. This
approach addresses two issues: does the test compound inhibit
specific CYP-450 enzymes, and can the metabolism of the test compound be inhibited by coadministered drugs? The first step is to
incubate a wide range of concentrations of the test compound with
pooled human liver microsomes and enzyme-selective substrates,
where the substrate concentration in each incubation mixture is
approximately equal to the Km for the specific reaction being
examined (see Table 1 for examples of substrates). These methods

Cytochrome

P-450

Enzymes

Flowchart
does test drug mhlblt
metabolism
of model
substrates?

ICSO <200uM

200~500uM

which enzymes
CYP460

fi

ntatabolite

Ml

203
Technique/
Measurements

Clmical
Significance

metabok
stud.es wth enzymewecific subsfretes

- llabrllty of test drug

to cause drug drug
mteractlons

>500uM

metaboka

M2

I d of enzymes affected
IC50
KI
type of mhlbltmn

correlation analyses ,
rncubabon wth specrhc enzymes
+ mhrbrforsfanbbodfes

Mll

-potential
for mhlbltlon of
test drug by other
compounds,
and possible
clm~cal slgmhcance,
- posslblllty of genebc
polymorphic
metabolism

CLmt (VmaxIKm)

Fig. 1. Flow chart of m vitro methods for the characterization

of the
metabolism and interaction potential for drug-development
candidates.
Essential techmques used, measurements or information obtained at each
stage, and issues addressed are also identified (see Section 2.2.).
offer the advantage

that the only assay required

is for the enzyme-

selective substrate or its metabolite; no special assay development

work is required fbr the test drug. If the test drug inhibits an
enzyme, the rate of disappearance of the substrate or appearance
of metabolite

for that particular

enzyme

will

be reduced.

Based

on these studies, an IC,, (the concentration at which activity is

reduced 50% compared with control) can be calculated for the
affected enzyme. If the IC,, > 500 PM (an arbitrary concentration
that is probably higher than that achieved in plasma by most
drugs), then this testing is complete as inhibition will not occur at
concentrations

which

are clinically

relevant.

For test drugs

with

an IC,, of less than 200 PM, the Ki for the test compound should
be determined. For drugs with IC,, values between 200-500 pM,
decisions can be made on a case-by-case basis.
Determination of the Ki value for inhibition of a specific CYP450 enzyme is carried out in a similar manner. In this case, at least
three concentrations of the enzyme-selective substrate (i.e., 1/4X,

204

Glue

and Clement

1X, and 4X Km) are used in assays with at least 5-6 concentrations of the inhibitor
(which would
bracket
the IC,, value).
Activities
of the enzyme at the substrate concentrations
are plotted against inhibitor
(test drug) concentrations
(Lineweaver
Burk
reciprocal
plots) and the Ki value determined.
The nature of the
inhibition
can also be determined
(competitive,
uncompetitive,
noncompetitive)
from the plots. The Ki value along with the projected plasma concentrations
of the test drug at projected
clinical
doses ([I]) can then be used to predict the extent of in vivo mhibition of the affected pathway.
For example,
if the test drug is a
noncompetitive
inhibitor
the % inhibition
will be equal to
([II/[11

whereas

if the Inhibition
([I]/[11

+ Ki) x 100

is competitive
+ Ki [l + S/Km])

the % inhibition

would

be

x 100

In most cases the substrate

concentration
(S) will be much
less than the Km (substrate concentration
at half-maximal
velocity for
the reaction) and the equation
for competitive
inhibition
will be
the same as for noncompetitive
inhibition.
Having defined the type
and extent of inhibition
of the test drug for specific CYP-450
enzymes, one can predict the potential
for drug-drug
interactions
based on a knowledge
of the therapeutic
plasma concentrations
and contribution
of selected enzyme-specific
metabolic
pathways for the clearance of coadministered
drugs. Finally, it is recommended
that the inhibitor
is preincubated
with pooled human
liver microsomes
prior to the addition
of substrate, and the rate
of substrate metabolism
is compared
with that obtained
for the
same reaction without preincubation.
If inhibition
is greater following preincubation,
it may be a result of mechanism-based
inhibition,
and further
studies will be needed
to assess the
reversibility
and clinical significance
of these findings.
As briefly mentioned
in Section 2.1.1., it is equally important
to
define the enzyme-specific
metabolism
of a test drug being considered for clinical development.
Once the major enzymes contributing
to the metabolism
of the drug have been determined
qualitatively
by correlation
analysis and/or
the use of chemical
or immunological
inhibitors
and cDNA-derived
purified
enzyme
preparations,
the kinetics for each of the contributing
reactions
should be determined
using traditional
methods.
For example,

Cytochrome

P-450

Enzymes

205

following incubation of a range of concentrations of the test drug

(substrate) with the purified enzyme preparation plus required
cofactors, the determined activity can be plotted against substrate
concentration. From this plot, the maximal velocity Wmax>and substrate concentration at half-maximal velocity (Km) may be calculated, Following similar experiments for each contributing enzyme,
the intrinsic clearance (ratio of V,,JKm) may be determined for
each pathway. The resulting values can be used to rank order each
metabolic pathway and predict its relative importance to the in vivo
clearance of the substrate. Based on a knowledge of the malor pathways of metabolism of a test drug and the enzyme-selective inhibitory potential of coadministered treatments, predictions can be
made about possible drug-drug interactions that may occur in
humans. It has been proposed that clinically significant changes in
drug concentrations are only likely to occur if the inhibited enzyme
is responsible for elimination of more than 50% of the drug, unless
the drug has a very narrow therapeutic index (Rowland, 1975).
3. Clinical

studies

This section will concentrate on techniques by which m vitro

data may be confirmed clinically, and comment on the correlation of clinical and in vitro findings. In general, the only clinical
studies that need to be considered for compounds in development
are those exploring possible inhibitory or inducing effects, to confirm in vitro findings. Clinical studies to assess the involvement
of certain CYP-450 enzymes in the metabolism of new compounds
are irrelevant, because this information may be obtained far more
easily and accurately in vitro. However, as these in vitro techniques have only recently become widely available, the limited
information on the enzyme-specific metabolism of older compounds is predominantly based on clinical interaction studies.
The principles underlying clinical testing are identical to those of
in vitro testing, and the techniques utilized u-t both forms of testing
almost always assessphenotype rather than genotype. The main difference is that m clinical testing, the range of probe substances that
can be used is more limited, and standardization of experimental
conditions may be more difficult. For example, interindividual pharmacokinetic variability may be exaggerated by subject factors such
as race, gender, smoking, and past intake of alcohol and other drugs, all
of which may alter enzyme expression and thus extent of metabolism.

Glue and Clement

206

3.1. Studies to Assess Metabolism

by CYP-450 Enzymes

To determine if a test compound is metabolized by certain CYP450 enzymes, it can be administered to subjects in combination with
a specific enzyme inhibitor (seeTable 1 for examples), or if CYP2D6
or -2C19 are thought to be involved in metabolism, to subjects identified as poor and extensive metabolizers for these enzymes. In the
former type of study, concentrations of the test compound are measured before and during treatment with the inhibitor. In the latter
type of study, the ratio of parent to metabolite is measured in urine.
In both studies, elevated concentrations of parent and/or reduced
concentration of metabolite (or an increased parent:metabolite ratio) is evidence that the particular enzyme is involved in the test
drugs metabolism. As discussed above, the use of in vitro techniques has made these methods redundant for the assessment of
new drugs. Problems associated with clinically derived data are
discussed at length elsewhere (Glue and Banfield 1996).
3.2. Inhibition

and Induction

If in vitro studies indicate that a test drug has inhibitory or

inductive effects at concentrations that are clinically relevant, confirmatory studies in humans may be required. In such studies,
subjects are dosed with probe substances that are enzyme-specific substrates (e.g., debrisoquine
or sparteine for CYP2D6;
midazolam for CYP3A4; seeTable 11, prior to and following treatment with the test drug. Induction may be inferred by reduced
concentrations of the probe substrate and/or increased metabolite production, and inhibition by increased concentrations of the
probe and/or reduced metabolite production compared with
baseline. The presence of inhibition may be assessed with singledose interaction studies; however to assess the full extent of inhibition or to assess induction requires at least 2 wk of treatment
with the test drug (steady-state concentrations of an inhibitor and
its metabolites may not be reached for several weeks; full induction requires synthesis of new enzyme).
Techniques have been developed to assess the effect of a test
drug on multiple CYP-450 enzymes simultaneously using a cocktail approach (Briemer and Schellens, 1990; Brockmoller and
Roots, 1994). In this type of study, metabolism of single doses of
several coadministered probe substrates is measured prior to and
after treatment with the test drug. Examples of probe substrates

Cyfochrome

P-450

207

Enzymes

include dextromethorphan
for CYP3A4 and -2D6, caffeine for
CYPlA2, tolbutamide
for CYP2C9, and S-mephenytoin
for
CYP2C19 (see Table 1). Whereas this is an efficient approach to
assessment, this technique has been criticized for several reasons
including possible pharmacokinetic and/or pharmacodynamlc
interactions of probe drugs, and the invasiveness of the multiple
blood draws needed for pharmacokinetic analysis (Bachmann,
1996). An alternative strategy, handprinting,
has been proposed,
where probe drugs are administered sequentially (rather than
simultaneously) and indices of changes in enzyme activity may
be derived from single plasma sample clearance estimates
(Bachmann, 1996). As yet this technique has not been widely used,
and there are no comparative data with cocktail probe testing
to determine what, if any, advantages this approach offers.
The above techniques are a considerable advance over older
methods assessing enzyme-inducing or -inhibiting effects of test
compounds by examining rates of metabolism of probes such as
antipyrine or 14C-aminopyrine, or excretion of endogenous substances such as d-glucaric acid or 6-B-hydroxycortisol (see Barry
and Feely, 1990). The production or metabolism of these compounds was not specific to a particular CYP-450 enzyme, or
involved non-CYP-450 pathways such as glucuronidation
(Barry
and Feely, 1990; Engel et al., 1996).
As mentioned in Sections 3.1. and 3.2., the methods described
assess phenotype. However patients may already be established
on medication that could alter CYP-450 activity and would complicate the interpretation of results or the assessment of phenotype status. The advent of new DNA-based (genotypic) screening
techniques that use lymphocyte genetic tissue and do not require
probe testing and are not affected by concurrent medication may
be an effective method of testing for genetic polymorphisms
(Chen et al., 1995; Heim and Meyer, 1990). Genotype testing is
still a research tool, but may become clinically established in the
near future (see Chen et al., 1996).
3.3.

Clinical

Confirmation

of in Vitro

CYP-450

Dafa

How accurately can in vitro testing predict clmical interactions?

At present, comparative information is available for only serotonin-reuptake inhibitor antidepressants and azole antifungals, and
their interactions with tricyclic antidepressants, benzodiazepines,
warfarin, certain antihistamines,
and the antiepileptic
drug

208

Glue and Clement

Felbamate (see von Moltke et al., 1994a, b, c, d, 1995; Black et al.,

1996; Glue et al., 1997, Kunze and Trager, 1996; Kunze et al., 1996).
For these compounds, there is a good correlation between clinical
and in vitro data. In retrospect, it is now clear that some recent
serious clinical drug-drug interaction problems (e.g., terfenadine
and ketoconazole; Honig et al., 1993) could have been predicted
from m vitro studies (von Moltke et al., 1994~) The United States
Food and Drug Administration (FDA) now requests this information as part of the development of all new drugs for United States
registration (draft document, Guidance for industry: in vitro drug
metabolism studies; CDER, FDA). Whereas this information may
provide for safer or more effective use of newer drugs, there are many
older drugs for which this information is unknown Comprehensive
and accurate in vitro CYP-450 profiling data are available only for a
few older prototype compounds (e.g., diazepam, imipramine), or
compounds that have narrow therapeutic indices or safety problems (e.g., terfenadine or theophylline); for the majority of older
compounds, information is still very limited or nonexistent. Ultimately, the usefulness of in vitro data to clinical management and
pharmaceutical development will require confirmation that in vitro
and in vivo data are well correlated for all classes of compounds, m
order to confirm the predictive accuracy of this technique.

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