0090-9556/02/3012-1311–1319DRUG METABOLISM AND DISPOSITION
U.S. Government work not protected by U.S. copyright
EVALUATION OF CYTOCHROME P450 PROBE SUBSTRATES COMMONLY USED BY THE PHARMACEUTICAL INDUSTRY TO STUDY IN VITRO DRUG INTERACTIONS
RAE YUAN, SORAYA MADANI, XIAO-XIONG WEI, KELLIE REYNOLDS, AND SHIEW-MEI HUANG
Office of Clinical Pharmacology and Biopharmaceutics, Center for Drug Evaluation and Research, United States Food and Drug Administration,
(Received July 31, 2002; accepted September 17, 2002)
This article is available online at http://dmd.aspetjournals.org
ABSTRACT: Pharmaceutical industry investigators routinely evaluate the po- CYP2E1, and testosterone 6␤-hydroxylation for CYP3A4. We re- tential for a new drug to modify cytochrome P450 (P450) activities viewed the validation information in the literature on these reac- by determining the effect of the drug on in vitro probe reactions tions and other frequently used reactions, including caffeine N3- that represent activity of specific P450 enzymes. The in vitro find- demethylation for CYP1A2, S-mephenytoin N-demethylation for ings obtained with one probe substrate are usually extrapolated to CYP2B6, S-warfarin 7-hydroxylation for CYP2C9, dextromethor- the compound’s potential to affect all substrates of the same phan O-demethylation for CYP2D6, and midazolam 1-hydroxyla- enzyme. Due to this practice, it is important to use the right probe tion for CYP3A4. The available information indicates that we need substrate and to conduct the experiment under optimal conditions. to continue the search for better probe substrates for some en- Surveys conducted by reviewers in CDER indicated that the most zymes. For CYP3A4-based drug interactions it may be necessary common in vitro probe reactions used by industry investigators to evaluate two or more probe substrates. In many cases, the include the following: phenacetin O-deethylation for CYP1A2, cou- probe reaction represents a particular enzyme activity only under marin 7-hydroxylation for CYP2A6, 7-ethoxy-4-trifluoromethyl cou- specific experimental conditions. Investigators must consider ap- marin O-dealkylation for CYP2B6, tolbutamide 4-hydroxylation for propriateness of probe substrates and experimental conditions CYP2C9, S-mephenytoin 4-hydroxylation for CYP2C19, bufuralol when conducting in vitro drug interaction studies and when ex- 1-hydroxylation for CYP2D6, chlorzoxazone 6-hydroxylation for trapolating the results to in vivo situations.
During the drug-candidate screening and development process,
activity. The second type of evaluation is more challenging and is the
investigators often conduct two types of in vitro drug metabolism
studies to assess the potential for P4501-based drug interactions. Onetype of study characterizes the metabolic pathway of the new drug and
Current Practice and Potential Problems
the potential for other drugs to modify the metabolism of the new
According to a survey of 194 new drugs approved in the United
drug. The other type of study evaluates the potential for the new drug
States from 1992 to 1997, industry investigators use different probe
to alter the metabolism of other drugs. Due to the availability of
reactions to represent the same P450 enzyme activities for evaluating
antibodies against specific P450 enzymes, cDNA-expressed enzymes,
the modulatory potential of a new drug (Table 1) (Yuan et al., 1999).
purified enzymes, and selective chemical inhibitors, the unequivocal
When the same inhibitor is evaluated using different probe assays for
identification of the major P450 isoform responsible for the metabo-
the same P450 enzyme activity, the outcome of the drug interactions
lism of a new drug can be easily established. However, predicting the
can be different. Also, investigators use different experimental con-
potential for the new drug to alter the metabolism of other drugs
ditions for the same assay. Some studies are not conducted under the
usually relies on the evaluation of the effect of the new drug on the
optimal conditions. During the preparation of this review we surveyed
rate of a probe reaction that represents a specific P450 enzyme
an additional 44 drug applications submitted from 1997 to 1999, todetermine whether recent progress in the area of in vitro drug metab-olism changed the common practices. The results of the second survey
This work was supported by the Intramural Regulatory Science and Review
are consistent with those of the previous one (Table 1, Fig. 1).
Enhancement grant awarded by the Center for Drug Evaluation and Research,
The phenomenon of different assays providing different results for
United States Food and Drug Administration in 1998. However, the views ex-
pressed in this manuscript are personal and may not represent the agency’s
the same enzyme is most notable for CYP3A4 activity, as recent
publications indicate. Wang et al. (2000) examined the mutual inhi-
1 Abbreviations used are: P450, cytochrome P450; DMSO, dimethyl sulfoxide;
bition among the four commonly used CYP3A4 substrates testoster-
HML, human liver microsome; 7-EFC, 7-ethoxy-4-trifluoromethyl coumarin; DXP,
one, terfenadine, midazolam, and nifedipine. They found that al-
though testosterone partially inhibits hydroxylation of terfenadine andmidazolam, it does not inhibit nifedipine oxidation. Based on a study
Address correspondence to: Rae Yuan, Ph.D., 3401 Hillview Ave., A2-264,
of the modulatory effect of 34 compounds on 10 commonly used
Palo Alto, CA 94304. E-mail: [email protected]
CYP3A4-mediated reactions, Kenworthy et al. (1999) reported that
Probe reactions used to characterize enzyme activities, surveyed from 194 drugs approved from US-FDA from 1992 to 1997 period
Phenacetin O-deethylation, caffeine 3-demethylation, ethoxyresorufin O-deethylation
7-Ethoxy-4-trifluoromethyl coumarin O-dealkylation
Tolbutamide 4Ј-hydroxylation, S-warfarin 7Ј-hydroxylation, diclofenac 4Ј-hydroxylation
Dextromethorphan O-Demethylation, bufuralol 1Ј-hydroxylation, debrisoquine 4-hydroxylation, sparteine oxidation
Chlorzoxazone 6-hydroxylation, debrisoquine, sparteine oxidation, p-nitrophenol
Nifedipine oxidation, testosterone 6␤-hydroxylation, erythromycin N-demethylation, cyclosporine oxidation, benzodiazepine hydroxylation
(midazolam, triazolam, alprazolam), terfenadine hydroxylation
the effect is substrate-dependent. Haloperidol, for example, activates
activity, as a step toward standardization we want to provide
dextromethorphan N-demethylation by 20%, but it inhibits nifedipine
guidance regarding the preferred probe substrates and experimental
oxidation by 96%, even though CYP3A4 catalyzes both reactions.
Stresser et al. (2000) showed that the extent of substrate dependencefor the quantitative inhibition parameters (IC ) is as large as 195-fold
We conducted two in-house surveys, as described previously, to
The in vitro experimental conditions may influence the accurate
determine which probe reactions pharmaceutical industry investiga-
assessment of drug interaction potential. Reports indicate that various
tors use for each enzyme (Table 1; Fig. 1). Detailed evaluations were
solvents, for example, may have different effects on P450 probe
conducted for the most commonly used probe reaction(s) for each
reactions (Chauret et al., 1998; Hickman et al., 1998; Busby et al.,
enzyme (Fig. 1), as well as those reactions deemed to have additional
1999). At 0.2% (v/v), acetonitrile does not affect chlorzoxazone
value for in vivo use. Although we do not consider the potential for in
6-hydroxylation (used as the CYP2E1 probe reaction), but dimethyl
vivo use a necessary criterion in the selection of a preferred substrate,
sulfoxide (DMSO) at 0.2% (v/v) inhibits the reaction by Ͼ80%. As a
we recognize that some investigators prefer using the same probe in
result, an incubation with DMSO is less sensitive and is subject to
vitro and in vivo. The evaluation primarily focused on the specificity,
greater error when determining whether a new drug inhibits the same
selectivity, and sensitivity of a reaction for the enzyme that it repre-
reaction. Through a careful enzyme kinetic study, Tang et al. (2000)
sents. We reviewed the literature information from in vitro P450-
showed that acetonitrile (3%, v/v) increases intrinsic clearance for
based metabolism studies using purified enzymes, cDNA-expressed
CYP2C9-based diclofenac hydroxylation by 87%, but it decreases
enzymes, selective chemical inhibitors, inhibitory antibodies as well
CYP2C9-based celecoxib hydroxylation by 25%.
as studies on enzyme kinetic analyses. For our evaluation, an ideal
In addition to using the appropriate solvent in the incubation, a
probe substrate is the one with a simple metabolic scheme, so that the
probe reaction should proceed under initial rate conditions. To pro-
formation rate of a metabolite specifically reflects the activity of one
ceed under initial rate conditions, the experiment should use optimal
distinct P450 enzyme. Preferably, the metabolite formed does not
experimental conditions, such as substrate concentrations, incubation
undergo sequential metabolism. The reaction should be selective, with
time, and enzyme protein content. Deviation from optimal experimen-
at least 80% of the formation of a metabolite being carried out by a
tal conditions may result in an underestimation or overestimation of
single enzyme. In addition to the above-mentioned scientific criteria,
changes in enzyme activity, and thereby lead to incorrect conclusions
the following practical criteria are relevant: the commercial availabil-
regarding the drug interaction potential of the new drug.
ity of the assayed molecular species (i.e., parent drug and the metab-
The potential influence of probe substrates and experimental con-
olite); the availability of an assay that is sensitive, rapid, and simple;
ditions on the assessment of in vitro drug interactions has a significant
and reasonable in vitro experimental conditions. We also address
impact on the drug development process and regulatory decisions. The
cautions to exercise and difficulties encountered when extrapolating
in vivo drug interaction guidance published by the Food and Drug
in vitro information to in vivo use for some reactions.
Administration in 1999 (www.fda.gov/cder/guidance) indicates thatinvestigators may use in vitro drug interaction data to conclude that anew drug does not inhibit a specific P450 activity (Food and Drug
Administration guidance). In practice, the in vitro evidence is usually
CYP1A2. Human liver microsomes (HLMs) contain relatively high
collected from one probe reaction per enzyme, and the conclusion is
constitutive levels of CYP1A2 (10 –15% of the total P450 content of
extrapolated to all substrates for the same enzyme. The significant
human liver), but not CYP1A1, which is more readily detected in
regulatory impact of this approach and potential problems associated
extra-hepatic tissues under induced conditions. Environmental factors
with current practice observed in our surveys prompted us to evaluate
affect CYP1A1 and CYP1A2 expression levels, complicating the in
the appropriateness of in vitro methodologies that pharmaceutical
vitro-to-in vivo extrapolation. CYP1A2 metabolizes many clinically
industry investigators commonly use to study P450-based drug inter-
important drugs such as amitriptyline, imipramine, theophylline, clo-
actions. We hope that this evaluation leads industry investigators to
zapine, tacrine, and zileuton. According to our survey, 45% of the
adopt a more consistent and accurate in vitro approach. Our ultimate
submissions use phenacetin O-deethylation to form acetaminophen to
goals are to promote 1) development of in vitro results that provide a
represent CYP1A2 activity (Fig. 1). However, industry investigators
reliable extrapolation to in vivo drug interactions; and 2) consistent
also use several substrates other than phenacetin to evaluate CYP1A2
regulatory submissions that allow comparisons across different drug
activity. We chose to review caffeine N3-demethylation, in addition to
applications and product labels. Although it is acceptable for industry
phenacetin O-deethylation, because it is a widely used in vivo sub-
investigators to use different probe substrates for the same enzyme
EVALUATION OF IN VITRO P450 PROBE REACTIONS
FIG. 1. Probe reactions used to characterize enzyme activities, surveyed from 44 drugs approved in 1997 to 1999 period.Phenacetin O-Deethylation. Phenacetin is an analgesic and anti-
M, the contribution of CYP1A2 is estimated to be 86%, but the
pyretic drug no longer marketed for human use in the United States.
contribution is reduced to 50% at a substrate concentration of 500 M
The frequent use of this substrate in vitro may be due to the avail-
(von Moltke et al., 1996; Venkatakrishnan et al., 1998). At concen-
ability of the parent compound and metabolite, and the fast and simple
trations Ն500 M, several enzymes, especially CYP2C9, contribute
high-performance liquid chromatography-ultraviolet detection assay
significantly to the O-deethylation of phenacetin in HLMs.
with high sensitivity for the reaction.
Study with organic solvents indicates that at solvent concentrations
In HLMs, the O-deethylation of phenacetin displays biphasic ki-
Յ1% (v/v), phenacetin O-deethylation is not significantly affected by
netics (Boobis et al., 1981; Tassaneeyakul et al., 1993; Kobayashi et
DMSO and methanol (Chauret et al., 1998; Busby et al., 1999). In
al., 1998). Studies with cDNA-expressed CYP1A2 (Venkatakrishnan
summary, at substrate concentrations that reflect low K
et al., 1998), chemical inhibitors (Boobis et al., 1981; Sesardic et al.,
activity (i.e., at concentration lower than 100 M), phenacetin O-
1990; von Moltke et al., 1996), and monoclonal antibodies (Sesardic
deethylation is the preferred probe reaction for detecting CYP1A2-
et al., 1988; Tassaneeyakul et al., 1993) show that the high-affinity
based drug interaction potential in vitro.
component of phenacetin O-deethylation is CYP1A2. The K value of
Caffeine N3-Demethylation. As with phenacetin O-deethylation,
this pathway is reported at 10 to 50 M, at least 10-fold lower than
the rate of caffeine N3-demethylation to form paraxanthine is biphasic
that of the low-affinity component. At a substrate concentration of 100
in HLMs. CYP1A2 is responsible for the high-affinity component
value of 200 to 500 M, and unidentified P450s are
to 1997 indicate that 7-ethoxy-4-triflouromethylcoumarin (7-EFC)
responsible for the low-affinity pathway, with a K value of 20 to 30
O-deethylation is the reaction that some industry investigators use to
mM (Grant et al., 1987; Tassaneeyakul et al., 1993, 1994). Studies
represent CYP2B6 activity. Recent literature studies show, however,
using cDNA-expressed enzymes, monoclonal antibody against
that 7-EFC is metabolized to 7-hydroxy-4-trifluoromethylcoumarin
CYP1A2, chemical inhibitors, and enzyme kinetics validate the in-
by CYP1A2 and CYP2E1 as well as CYP2B6 (Ekins et al., 1997),
volvement of CYP1A2 in the high-affinity pathway (Grant et al.,
making this substrate nonselective for CYP2B6. Thus, we do not
1987; Butler et al., 1989; Tassaneeyakul et al., 1992). At 1 mM
consider 7-EFC a preferred candidate for CYP2B6 probe substrate.
caffeine, CYP1A2 contributes to only 70% of the paraxanthine for-
S-Mephenytoin N-Demethylation. The more recently conducted
mation (Tassaneeyakul et al., 1992). At substrate concentrations Յ0.1
second survey of 44 submissions to United States Food and Drug
mM, the paraxanthine formation rate reflects CYP1A2 activity. How-
Administration indicates that some investigators use S-mephenytoin
ever, due to the detection limit on conventional high-performance
N-demethylation to nirvanol to represent CYP2B6 activity (Fig. 1).
liquid chromatography system, caffeine N3-demethylation often is
The available evidence in the literature supports the selectivity of this
carried out at high substrate concentrations, usually at 0.5 to 5 mM
reaction for CYP2B6. Ko et al. (1998) report biphasic kinetics for
unless radiolabeled drug or liquid chromatography/mass spectrometry
nirvanol formation, with high- and low-affinity K values of 174 and
is used, and at high microsomal protein concentrations, up to 2 mg/ml
1900 M, respectively. However, Heyn et al. (1996) assume
(Grant et al., 1987). In addition, caffeine N3-demethylation is sensi-
monophasic kinetics and report a mean K
tive to solvent effects. Methanol at 1% (v/v) inhibits the reaction by
discrepancy seems to be due to the differences in the substrate
Ͼ80%, whereas acetone and acetonitrile at the same concentration
concentration ranges used by these investigators.
stimulate the reaction by Ͼ200% (Hickman et al., 1998).
Due to conflicting literature data, limited information is available to
Taken together, caffeine is not a preferred in vitro substrate for
validate the specificity of S-mephenytoin for CYP2B6. At 200 M
CYP1A2 activity compared with phenacetin. Literature indicates an
S-mephenytoin, 500 M orphenadrine, an inhibitor of CYP2B6, in-
ongoing effort to develop a more sensitive detection assay to facilitate
hibits nirvanol formation by 84% (Heyn et al., 1996). In addition,
the study of this metabolic pathway. If one chooses to use this
anti-CYP2B6 antibody inhibits N-demethylation by up to 79% at a
substrate to represent CYP1A2 activity in vitro, one should use
substrate concentration of 100 M (Stresser and Kupfer, 1999). In
substrate concentrations below 0.1 mM and be cautious on the choice
contrast, incubation of S-mephenytoin with the CYP2C9 inhibitor
sulfaphenazole indicates that CYP2C9 is responsible for the high-
CYP2A6. CYP2A6 is an important enzyme for precarcinogen
affinity component of the reaction (Ko et al., 1998). These investiga-
activation and oxidation of certain drugs. It exhibits significant ethnic-
tors find that although both recombinant CYP2B6 and CYP2C9 form
related genotypic or phenotypic deficiency (Shimada et al., 1996).
nirvanol, CYP2B6 forms it at a 4-fold higher rate. Using microsomal
CYP2A6 substrates include coumarin, aflatoxin B1, nicotine, N-ni-
intrinsic clearances of 0.98 and 2.1 l/min/mg for the high- and
trosodiethylamine, N-nitrosodimethylamine, and N-nitrosonornico-
low-affinity enzymes, respectively, Ko et al. (1998) conclude that
tine. Our surveys indicate 7-hydroxylation of coumarin is the only
CYP2B6 is the main enzyme responsible for formation of nirvanol at
reaction that industry investigators use to assess CYP2A6 activity
substrate concentrations higher than 1000 M and that CYP2C9 has
a major contribution at lower and more clinically relevant S-
Coumarin 7-Hydroxylation. Studies with CYP2A6 inhibitory
mephenytoin concentrations. These results indicate a major contribu-
monoclonal antibody show that at substrate concentrations Յ10 M,
tion of CYP2B6 in the formation of nirvanol under high micromolar
more than 90% of the 7-hydroxylation of coumarin in HLMs is carried
out by CYP2A6, demonstrating the unequivocal role of CYP2A6 in
In summary, the available data suggest that as a probe reaction to
coumarin 7-hydroxylation (Li et al., 1997; Sai et al., 1999; Yang et al.,
represent CYP2B6 activity, S-mephenytoin N-demethylation needs to
1999). Among nine cDNA-expressed enzymes, only CYP2A6 cata-
proceed at high substrate concentrations that reflect low-affinity en-
lyzes this reaction (Ono et al., 1996). Consistently, kinetic studies in
zyme activity. In the absence of another suitable substrate, this fairly
HLMs show monophasic formation of 7-hydroxy-coumarin at com-
selective reaction is recommended to detect CYP2B6-based drug
monly used substrate concentrations of 0.1 to 10 M, with a K value
of 0.5 to 2 M (Shimada et al., 1996; Draper et al., 1997). CYP2C9. CYP2C9 is a member of the CYP2C subfamily, the
Although the experimental conditions for coumarin 7-hydroxyla-
second largest P450 subfamily after CYP3A. It exhibits great genetic
tion are straightforward, the reaction rate is subject to various solvent
variation among individuals and is involved in the metabolism of
effects. Acetone and acetonitrile at 1% (v/v) each inhibit the reaction
many clinically important drugs that have a narrow therapeutic range,
rate by Ͼ40%, but DMSO and methanol at 1% (v/v) have little effect
including carbamazepine, phenytoin, and warfarin. Current knowl-
(Draper et al., 1997; Chauret et al., 1998; Hickman et al., 1998). With
edge is just beginning to allow a clear separation of CYP2C8 from
the appropriate organic solvent, coumarin 7-hydroxylation is a pre-
CYP2C9. According to our survey, tolbutamide 4Ј-hydroxylation is
ferred probe reaction to detect CYP2A6-based drug interaction po-
the preferred probe reaction used in 80% of the submissions to
characterize both enzymes collectively (Fig. 1). Among other drugs
CYP2B6. Cytochrome P450 2B6 is the only member of the CYP2B
that are used as probe substrates for CYP2C9, we reviewed the
family expressed in humans. However, it has not been studied exten-
warfarin assay because it is one of the in vivo probes most extensively
sively due to unavailability of a probe substrate and reported low
studied by industry investigators (Marroum et al., 2000).
levels of the enzyme in human tissues (Shimada et al., 1994). Recent
Tolbutamide 4-Hydroxylation. Tolbutamide 4Ј-hydroxylation is
studies indicate that the quantity of CYP2B6 in the liver is underes-
the initial and rate-limiting step of tolbutamide elimination. Studies
timated due to a lack of sensitive techniques and antibodies. In our
with cDNA expressed enzymes show that at substrate concentrations
surveys, we observe that few industry investigators characterize the
Յ500 M, 90% of tolbutamide is hydroxylated by CYP2C9 and 10%
activity of this enzyme in in vitro studies.
by CYP2C8 (Minors et al., 1988; Relling et al., 1990; Ono et al.,
There are very few xenobiotics recognized as substrates of
1996). An immunoblotting assay with antibody developed against
CYP2B6. The results of our first survey of drugs approved from 1992
CYP2C9 provides further evidence to support the predominant role of
EVALUATION OF IN VITRO P450 PROBE REACTIONS
CYP2C9 in tolbutamide 4Ј-hydroxylation (Edwards et al., 1998). In
sible for the metabolism of mephenytoin, omeprazole, diazepam, and
HLMs with substrate concentrations up to 2.0 mM, tolbutamide
many psychotherapeutic agents. Poor metabolizers represent ϳ2.5 to
hydroxylation exhibits simple Michaelis-Menten kinetics with appar-
5% of Caucasian populations, 19% of African populations, and up to
ent K values ranging from 60 to 400 M, with most values between
30% of Asian populations (Pollock et al., 1991; Flockhart, 1995).
100 and 200 M (Miners et al., 1988; Bourrie et al., 1996). However,
Mephenytoin, an anticonvulsant agent, has long been used as an in
recent reports show that CYP2C19 may also catalyze the reaction with
vitro and in vivo probe substrate for CYP2C19. Its unequivocal role
value similar to CYP2C9 (Lasker et al., 1998; Wester et al.,
in drug development is reflected in our survey, where it is the only
2000). The contribution of CYP2C19 to overall tolbutamide 4Ј-hy-
drug used to assess CYP2C19 activity (Fig. 1; Table 1) Recent studies
droxylation may be minimal, considering the limited protein expres-
suggest that omeprazole (5-hydroxylation) may also be used as a
sion of this enzyme in normal human liver. However, CYP2C19’s
probe for CYP2C19 activity (Flockhart, 1995). However, in vitro
catalytic role in CYP2C9-deficient liver may be important.
studies show that CYP3A4 carries out the same reaction for omepra-
Tolbutamide has a slow turnover rate. The initial rate conditions
zole. At 10 M, the contribution of each enzyme depends on the ratio
exist even with incubation times up to 3 h and HLM protein concen-
of their expression levels in HLMs (Yamazaki et al., 1997). Consid-
trations of 1.6 mg/ml (Miners and Birkett, 1996). Based on our
ering the much higher expression level of CYP3A4 compared with
survey, some investigators use a 90-min incubation time and a protein
CYP2C19, we do not consider omeprazole a preferred in vitro probe
concentration of 2 mg/ml. Although not reported in these studies, the
use of high protein concentrations and long incubation times can
S-Mephenytoin 4-Hydroxylation. Mephenytoin exists as a race-
deplete inhibitors contained in the incubation mixture.
mic mixture of R- and S-enantiomers and its metabolism is stereospe-
Tolbutamide hydroxylation reaction is very sensitive to the organic
cific. In HLMs of extensive metabolizers, S-mephenytoin is metabo-
solvent effect. At a concentration of 1% (v/v), isopropanol, DMSO,
lized to the 4Ј-hydroxyl metabolite and nirvanol (Jurima et al., 1985).
and methanol each inhibit tolbutamide hydroxylation by Ն40%
Studies with inhibitory antibody against CYP2C19 and purified and
(Hickman et al., 1998). But acetonitrile does not affect the reaction
cDNA-expressed enzymes validate the exclusive role of CYP2C19 in
significantly at the same concentration (Chauret et al., 1998; Hickman
4Ј-hydroxylation of S-mephenytoin (Shimada et al., 1986; Wrighton
et al., 1998; Tang et al., 2000). Interestingly, Tang et al. (2000)
et al., 1993; Inoue et al., 1997). Kinetic studies of S-mephenytoin
observe that the solvent effect varies with different probe reactions for
4Ј-hydroxylation consistently show monophasic Michaelis-Menten
CYP2C9; and at concentration Ͼ1%, acetonitrile significantly acti-
of 31 to 340 M (Jurima et al., 1985; Hall et al.,
vates tolbutamide hydroxylation in a solvent concentration-dependent
1987; Chiba et al., 1993) in HLMs. The variability in K
reported in different studies primarily reflects different experimental
The collective evidence indicates that tolbutamide 4Ј-hydroxylation
conditions; but it may also reflect genetic variations, especially in
is an appropriate in vitro probe reaction for CYP2C9 activity. But
HLM prepared from Asian or African individuals where the percent-
investigators should pay attention to the incubation conditions, the use
age of poor metabolizers is high. In the poor metabolizers, S-mephe-
of organic solvent, and the expression level of CYP2C9 in HLMs
nytoin 4Ј-hydroxylation is not mediated by CYP2C19 but by other
when using this reaction to determine CYP2C9-based drug interaction
enzymes in place of CYP2C19. Thus, microsomes that are deficient in
CYP2C19 should not be used to examine CYP2C19-based drug
S-Warfarin 7-Hydroxylation. Several P450s metabolize warfa-
rin, but with different regio- and stereoselectivity. At therapeutic
S-Mephenytoin 4Ј-hydroxylation is sensitive to solvent effect. Dim-
doses, Ͼ85% of S-warfarin is biotransformed to 6Ј- and 7Ј-hydroxy
ethylformamide, DMSO, and isopropranol at 1% (v/v) are reported to
S-warfarin in a 1:3 ratio (Toon et al., 1986). With purified and cDNA
inhibit S-mephenytoin 4Ј-hydroxylation by Ͼ70%, but acetonitrile
expressed P450s, CYP2C9 has the highest activity toward S-7Ј-OH-warfarin formation, followed by CYP1A2 and CYP3A4 (Rettie et al.,
and methanol at the same concentration do not affect the reaction
1992). In HLMs, the formation of S-7Ј-OH-warfarin is inhibited
significantly (Chauret et al., 1998; Hickman et al., 1998).
strongly by sulfaphenazole and correlates with tolbutamide 4Ј-hy-
In summary, under optimal experimental conditions, S-mepheny-
droxylation (Hall et al., 1994). At substrate concentrations up to 200
toin 4Ј-hydroxylation is the preferred probe reaction for CYP2C19
M, typical Michaelis-Menten kinetics is observed, with a K of 1 to
activity. Investigators should pay attention to the use of organic
5 M (Lang and Bocker, 1995; Hemeryck et al., 1999). The formation
solvent, and the expression level of CYP2C19 in HLMs when using
of 6Ј-OH-metabolite is also carried out by CYP2C9. It has the same
this reaction to determine CYP2C19-based drug interaction in vitro. K value as that of 7Ј-OH metabolite formation, but with one-third of
CYP2D6. CYP2D6 is a polymorphically expressed P450 enzyme.
value (Rettie et al., 1992; Kunze et al., 1996). However, at
About 5 to 10% of Caucasians are poor metabolizers of CYP2D6
substrate concentrations Ն50 M, at least one other pathway (possi-
substrates. As with polymorphically expressed CYP2C19, using pro-
bly CYP3A4) also contributes to 6Ј-OH-formation.
totype substrates to assess CYP2D6-based drug interaction is mean-
Although not a substrate for CYP2C9, R-warfarin competitively
ingful only in the extensive metabolizer’s liver microsomes. Although
inhibits the formation of 7Ј-OH-S-warfarin with a K value of 6 to 8
CYP2D6 only constitutes about 2% of total P450 enzymes in the liver
M (Kunze et al., 1991). This inhibition introduces potential com-
(Shimada et al., 1994), it is responsible for metabolizing drugs in a
plexity in assessing CYP2C9-based drug interactions and thus, com-
variety of therapeutic classes, including antidepressants, antipsychot-
mercially available racemic warfarin should not be used as a substrate.
ics, and ␤-blockers. Our survey indicates that dextromethorphan (O-
Currently, no information on the solvent effect on S-Warfarin 7Ј-
demethylation) and bufuralol (1Ј-hydroxylation) are the two in vitro
CYP2D6 probe substrates preferred by industry investigators. More
We conclude that if one can overcome the practical challenges,
than 60% of submissions used bufuralol as the probe substrate to
S-warfarin 7Ј-hydroxylation may also be a good reaction for probing
assess CYP2D6 activity, and 30% used dextromethorphan.
CYP2C9-based drug interaction at substrate concentrations reflecting
Bufuralol 1-Hydroxylation. Bufuralol is a chiral adrenoceptor
the high-affinity (low K ) enzyme activity toward this reaction.
antagonist that undergoes extensive oxidative metabolism in humans
CYP2C19. CYP2C19 is a genetically polymorphic enzyme respon-
(Francis et al., 1982; Dayer et al., 1983, 1986), where the aliphatic
1Ј-hydroxylation accounts for 95% of bufuralol clearance (Man-
CYP2D6 at low substrate concentrations (Bourrie et al., 1996). Using
monoclonal antibody against CYP2D6, Gelboin et al. (1997) show 50
Enzyme kinetic studies demonstrate biphasic formation of 1Ј-OH-
to 93% inhibition of the formation of DXP.
bufuralol at bufuralol concentrations up to 100 M, where the high-
Organic solvents seem to have less effect on dextromethorphan
than on bufuralol, providing some advantage for using this substrate
component has a K of 83 to 600 M (Gut et al., 1986; Kronbach et
as an in vitro probe for assessing CYP2D6 activity (Hickman et al.,
1998; Busby et al., 1999). But as with bufuralol, the experiment with
intrinsic clearance and thereby greater contribution to the overall
dextromethorphan should proceed with low substrate concentrations
hydroxylation by the high-affinity component. Quinidine, a selective
to reflect the high-affinity enzyme (i.e., CYP2D6) activity in vitro.
and potent CYP2D6 inhibitor, diminishes 90 and 70% of the reaction
Although other CYP2D6 probe substrates (such as metroprolol,
at bufuralol concentrations of 1 and 50 M, respectively (Mankowski,
spartein, and debrisoquin) can be as selective as bufurolol and dex-
1999). Studies with cDNA-expressed enzymes show that CYP2D6
tromethorphan, the latter two are particularly attractive because of the
exhibits the highest ability to form 1Ј-OH-bufuralol, followed by
availability of the assayed species and the fluorescent nature of these
CYP2C19. CYP2D6 catalyzed the reaction with 1/10 of the K
species that permits the development of a highly sensitive detection
CYP2C19, but at a 36-fold higher rate (Mankowski, 1999). These
assay. Thus, at appropriate experimental conditions, bufuralol 1Јhy-
results suggest that CYP2D6 is the enzyme responsible for the high-
droxylation and dextromethorphan O-demethylation are both pre-
affinity component and CYP2C19 is responsible for the low-affinity
ferred reactions to probe CYP2D6-based drug interaction potential.
component of 1Ј-OH bufuralol formation. CYP2E1. CYP2E1 metabolizes chlorzoxazone, acetaminophen,
Using inhibitory monoclonal antibody against CYP2D6, Gelboin et
and the volatile anesthetics, including enflurane, sevoflurane, me-
al. (1997) show that, at 50 M substrate concentration, 1Ј-OH-
thoxyflurane, and isoflurane. Among these drugs, chlorzoxazone is
bufuralol formation is only partially carried out by CYP2D6.
the preferred in vitro probe substrate used in 60% of the surveyed
CYP2C19, and to a lesser extent CYP2C8/9 and CYP1A2, also
contribute to the metabolism of bufuralol. Whether the formation of
Chlorzoxazone 6-Hydroxylation (6-OH). Chlorzoxazone is an
1Ј-OH bufuralol represents CYP2D6 activity depends on its relative
analgesic muscle relaxant that can be used in in vitro and in vivo drug
expression levels, compared with these other enzymes in HLMs. This
metabolism studies. After ingestion, chlorzoxazone is rapidly ab-
study indicates bufuralol 1Ј-hydroxylation loses its selectivity for
sorbed and extensively metabolized. In HLMs, 6-OH-chlorzoxazone
CYP2D6 at substrate concentrations greater than 50 M.
is the sole metabolite formed, which makes the assay highly specific.
It is important to consider the effect of solvents on this reaction.
However, the selectivity of chlorzoxazone for CYP2E1 is contro-
Studying the reaction rate with cDNA-expressed enzymes, Busby et
versial. A study by Peter et al. (1990) indicates that rabbit anti-P450
al. (1999) show Ͼ50% inhibition of 1Ј-OH bufuralol formation with
monoclonal antibody against human CYP2E1 inhibits 81 to 87% of
ethanol, DMSO, and methanol at solvent concentrations of 3% (v/v).
chlorzoxazone 6-hydroxylation, a monophasic reaction with a Km
But at 1% (v/v), acetonitrile does not inhibit the reaction significantly.
value of 40 M. However, Shou et al. (2000) demonstrate that
Despite the biphasic kinetics of racemic bufuralol at concentrations
inhibitory monoclonal antibody inhibits 20 to 80% of the formation of
ranging from 1 to 1000 M, racemic bufuralol is a good in vitro
6-OH-chlorzoxazone at a substrate concentration of 200 M. In the
CYP2D6 probe substrate. When using it to determine CYP2D6-based
majority of 18 liver donors the inhibition was around 50% (Shou et
drug interaction potential in vitro, investigators should pay attention to
al., 2000). Gorski et al. (1997) show that rabbit anti-human CYP3A
the selection of organic solvent, the expression level of CYP2D6 in
inhibits the reaction by 47%, suggesting a significant contribution of
HLMs; and should use low substrate concentrations that primarily
CYP3A in the reaction. Using cDNA-expressed enzymes, Ono et al.
reflect the high-affinity enzyme activity toward this reaction.
(1996) demonstrate that the same reaction is also catalyzed by
Dextromethorphan O-Demethylation. Dextromethorphan, an an-
value being one-third of that by CYP2E1 (Ono
titussive drug, undergoes two parallel oxidative metabolic pathways,
et al., 1996). At a chlorzoxazone concentration of 10 M, CYP2E1
O-demethylation by CYP2D6 to form dextrorphan (DXP) and N-
catalyzes the reaction at the same rate as CYP1A2; but at 500 M,
demethylation by CYP3A to form 3-methoxymorphinan (Jacqz-
CYP2E1 catalyzes the reaction at a rate 10 times higher than
Aigrain et al., 1993). Both of these products undergo sequential
CYP1A2. The above-mentioned evidence indicates that the formation
metabolism in vitro, unless optimal incubation conditions are used
of 6-OH-chlorzoxazone is more specific for CYP2E1 activity at high
substrate concentrations than at low substrate concentrations.
Biphasic enzyme kinetics are observed for DXP formation at dex-
In HLMs, there is a prominent solvent effect on 6-OH-
tromethorphan concentrations up to 2000 M, with K values for the
chlorzoxazone. Methanol at 0.2% (v/v) decreases 6-OH-chlorzoxa-
high-affinity component being 2.2 to 8.5 M (Kronbach, 1991; Jacqz-
zone by 60% (Chauret et al., 1998). Hickman et al. (1998) report that
for the low-affinity component is at least
1% (v/v) acetone, dimethylformamide, DMSO, and isopropanol in-
10-fold higher (70 –1880 M). Under optimal incubation conditions,
hibit the reaction by Ͼ70%. Acetonitrile, on the other hand, does not
i.e., protein concentration of 0.4 mg/ml, incubation time less than 60
show an effect until the concentration reaches 5% (v/v).
min and substrate concentration less than 50 M, the Eadie-Hofstee
Literature evidence indicates that chlorzoxazone 6-hydroxylation is
plot for DXP formation shows a monophasic characteristic (Kron-
the current preferred probe reaction for CYP2E1, but it is important to
use high substrate concentrations that reflect low-affinity enzyme (i.e.,
Studies with cDNA-expressed enzymes show that both CYP2C9
CYP2E1) activity toward this reaction, and to consider solvent effects
and CYP2D6 catalyze DXP formation. At 10 M substrate concen-
in the experiment. However, a substrate that offers better enzyme
trations, the CYP2D6-mediated reaction proceeds at a rate 5-fold
selectivity and lower solvent effect would be more desirable.
greater than that by CYP2C9 (Ono et al., 1996). But at 500 M, the
CYP3A. CYP3A is the most abundant P450 enzyme in humans,
CYP2D6 reaction rate is only one-sixth of the CYP2C9-based reac-
accounting for an average 30 to 40% of total P450 protein in the liver.
tion rate. At a dextromethorphan concentration of 5 M, quinidine
It has three isoforms in various tissues: CYP3A4 and CYP3A5 pre-
completely abolishes the reaction, confirming the selective role of
dominantly in liver and gut, and CYP3A7 in fetal liver. Current data
EVALUATION OF IN VITRO P450 PROBE REACTIONS
indicate that CYP3A4 is the most important CYP3A member with
regard to involvement in clinically significant drug interactions. Many
Summary of probe substrate metabolic reactions used in in vitro drug
probe reactions represent the activity of this enzyme, as reflected in
our survey. The substrate used most often by industry investigators is
testosterone, followed by midazolam, nifedipine, and erythromycin
Testosterone 6␤-Hydroxylation. Steroid hydroxylation has long
been recognized as a CYP3A-mediated reaction. In HLMs, numerous
studies demonstrate that selective CYP3A4 inhibitors diminish the
6␤-hydroxylation of testosterone (Wrighton et al., 1989; Newton et
al., 1995; Bourrie et al., 1996). Specific antibodies against CYP3A4
inhibit more than 90% 6␤-OH-testosterone formation at substrate
concentrations of 200 to 250 M (Gelboin et al., 1995; Mei et al.,
1999; Shou et al., 2000). Studies with purified human proteins
(Yamazaki and Shimada, 1997) and cDNA-expressed enzymes (Wax-
man et al., 1991; Ono et al., 1996) provide more evidence showingthat all CYP3A members, other than CYP3A7, catalyze testosterone-
N.R., not reported. a Effect less than 20% is deemed acceptable. Recommendation is based on at least two
6␤-hydroxylation; CYP3A4 exhibits the highest activity. CYP2C9
consistent experimental results available in the studies by Draper et al., (1997), Chauret et al.,
and CYP2C19 also catalyze the reaction, but at 1/10 the rate of
(1998), Hickman et al., (1998), Busby et al., (1999), or Tang et al., (2000), with the exceptionof bufuralol 1Ј-hydroxylation in incubation with acetonitrile, which is only reported by Busby et
CYP3A4. In HLMs, CYP3A4-mediated 6␤-OH-testosterone forma-
al. (1999) in cDNA-expressed enzymes.
of 50 to 100 M. No significant solvent effect is seen
with methanol and acetonitrile at solvent concentrations Յ1% in
interaction is based on CYP3A4 or CYP3A5, unless the study is
either HLMs or cDNA-expressed enzymes (Chauret et al., 1998;
conducted in HLMs without detectable CYP3A5.
Busby et al., 1999). We conclude that, at substrate concentrations at or
Because most of the reported studies do not differentiate CYP3A4
lower than 250 M, testosterone-6␤-hydroxylation rate primarily
activity from CYP3A5, and use CYP3A4 to reflect both or either enzyme
reflects the CYP3A4 activity, and thus can be used to probe drug
activity, we follow the same nomenclature in the following text.
interaction potential of a new drug toward this enzyme in vitro. Use of Two or More Probe Reactions for CYP3A4-Based Drug Midazolam 1-Hydroxylation. Midazolam is a short-acting ben- Interactions. Although the knowledge of CYP3A4-catalyzed reac-
zodiazepine routinely used to induce sedation and anesthesia. It is
tions is growing fast, in vivo CYP3A4-based drug metabolism and
available for both intravenous and oral administration, which provides
interactions remain among the most difficult scenarios to predict in
a unique opportunity to study gastrointestinal-based or liver-based
vitro. The difficulty arises from the complex substrate-enzyme inter-
action at the molecular level (Ueng et al., 1997), the involvement of
Biotransformation of midazolam in humans yields two primary
efflux transport systems in the substrate’s disposition in vivo (Takano
hydroxylated metabolites: 1Ј-OH and 4-OH, both of which are further
et al., 1998; Yumoto et al., 1999), and the contribution of gastroin-
metabolized at a much slower rate (Kronbach et al., 1989). The 1Ј-OH
testinal metabolism (Gorski et al., 1998).
metabolite accounts for more than 90% of the total hydroxylation
Based on chemical inhibition characterizations and substrate cor-
reaction (Ghosal et al., 1996). Mounting evidence, including studies
relation analyses, testosterone and midazolam seem to belong to two
with inhibitory antibodies, specific chemical inhibitors, and cDNA-
distinct groups of CYP3A4 substrates (Kenworthy et al., 1999;
expressed enzymes, demonstrate that CYP3A4 mediates the formation
Stresser et al., 2000). Although the metabolic activity of testosterone
of the two metabolites (Kronbach et al., 1989; Wrighton and Ring,
is highly correlated with those of CYP3A4/5 substrates with large
1994). However, midazolam is also readily metabolized by CYP3A5
molecular weight, such as erythromycin or cyclosporin A, it is only
value as by CYP3A4, although CYP3A5 shows
weakly related to those of benzodiazepines such as midazolam. Thus,
different catalytic activities from CYP3A4 (Gibbs et al., 1999).
the effect of a CYP3A4 modulator depends on the substrate used in
In HLMs at incubation times up to 5 min, midazolam metabolism
the experiment (Kenworthy et al., 1999). Fluconazole, for example,
follows simple Michaelis-Menten kinetics. But at high substrate con-
displays 65% inhibition in midazolam assay but 37% in testosterone
centrations, substrate-inhibition kinetics become apparent for 1Ј-OH-
assay. Nimodipine, on the other hand, displays 60% inhibition in
midazolam formation, but not for 4-OH-midazolam formation
midazolam assay but 96% in testosterone assay. Besides these two
(Kronbach et al., 1989). A similar phenomenon is reported with
groups of substrates, additional groups such as those represented by
cDNA-expressed enzymes (Ghosal et al., 1996). Although formed by
nifedipine or benzoxyl-resorufin may also exist. Carbamazepine,
the same enzyme, the K value for 1Ј-hydroxylation of midazolam is
which shows a negligible effect on testosterone and midazolam,
3 to 5 M, and for 4-hydroxylation is 40 to 60 M.
inhibits nifedipine by 100% (Stresser et al., 2000). Although the
Midazolam is insoluble in water, so an organic solvent is often used
mechanism of this substrate-dependent phenomenon is not known,
in the in vitro experiment to dissolve this substrate. A previous study
two or more in vitro probe substrates from different groups may be
indicates that acetone may enhance midazolam metabolism in HLMs
needed to accurately predict CYP3A4-based drug interactions in vivo.
(Kronbach et al., 1989), but other solvent effects have not beenreported. Because of the complexity of CYP3A-based drug-druginteractions (as delineated below) and the common use of this reaction
to assess CYP3A activity, a thorough study of the solvent effect on
In this survey study, we review the most commonly used in vitro
midazolam is needed. Under optimal experimental conditions, mida-
probe substrates from pharmaceutical industry submissions. These
zolam 1Ј-hydroxylation seems to be a good in vitro probe reaction for
probe substrates have been widely studied and their characteristics are
CYP3A activity at substrate concentrations less than 10 M. How-
described in the literature. Table 2 summarizes our recommended
ever, using this reaction does not allow one to determine whether the
reactions for all of the evaluated P450 enzymes.
Because the selectivity of represented P450 depends on the specific
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Int J Entric Pathog. 2014 January; 1(2): 76-8. Published Online 2014 January 1. Case Report First CTX-M type ß–lactamase-Producing and Ciprofloxacin Resistant Salmonella Infection Acquired by a Child in IRANFarzaneh Firoozeh 1, Fereshteh Shahcheraghi 2,*, Taghi Zahraei-Salehi 3, Mohammad Mehdi Aslani 2, Reihaneh Banisaeed 31 Department of Microbiology and Immunology, School of Medicine,
GlaxoSmithKline PO Box 13398 Five Moore Drive Research Triangle Park IMPORTANT REVISIONS TO PRESCRIBING INFORMATION FOR SEREVENT® (salmeterol xinafoate) AND ADVAIR DISKUS® (fluticasone propionate and salmeterol inhalation powder) GlaxoSmithKline is writing to you as a prescriber of SEREVENT and/or ADVAIR, to communicateimportant new revisions to the prescribing information for SEREV