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
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 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 Evaluation Approach
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 ( 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 S-mephenytoin N-demethylation 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 Conclusions
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 comprehensive panel of antibodies against the major xenobiotic metabolising forms of cyto-
chrome P450 in humans. Biochem Pharmacol 56:377–387.
experimental conditions, the use of appropriate experimental condi- Ekins S, Vandenbranden M, Ring BJ, and Wrighton SA (1997) Examination of purported probes tions in in vitro studies is crucial. In particular, it is important to of human CYP2B6. Pharmacogenetics 7:165–179.
understand the enzyme kinetics of the reaction and the involvement of Flockhart DA (1995) Drug interactions and the cytochrome P450 system. The role of cytochrome P450 2C19. Clin Pharmacokinet 29 (Suppl 1):45–52.
high-affinity and low-affinity enzymes (when multiple enzymes me- Francis RJ, East PB, and Larman J (1982) Kinetics and metabolism of (ϩ)-, (Ϫ)- and (Ϯ)- tabolize the same reaction) to determine the appropriate substrate bufuralol. Eur J Clin Pharmacol 23:529 –533.
Gelboin HV, Krausz KW, Shou M, Gonzalez FJ, and Yang TJ (1997) A monoclonal antibody concentrations to use. To study high-affinity enzyme, one should use inhibitory to human P450 2D6: a paradigm for use in combinatorial determination of indi- substrate concentrations that reflect the low K enzyme activity (e.g., vidual P450 role in specific drug tissue metabolism. Pharmacogenetics 7:469 – 477.
Gelboin HV, Krausz KW, Goldfarb I, Buters JT, Yang SK, Gonzalez FJ, Korzekwa KR, and bufuralol 1Ј-hydroxylation and dextromethorphan O-demethylation Shou M (1995) Inhibitory and non-inhibitory monoclonal antibodies to human cytochrome for CYP2D6); and for a low-affinity enzyme, one should use high P450 3A3/4. Biochem Pharmacol 50:1841–1850.
Ghosal A, Satoh H, Thomas PE, Bush E, and Moore D (1996) Inhibition and kinetics of substrate concentrations (e.g., S-mephenytoin N-demethylation for cytochrome P4503A activity in microsomes from rat, human and cDNA-expressed human CYP2B6 and chlorzoxazone 6-hydroxylation for CYP2E1). Because cytochrome. P450 Drug Metab Dispos 24:940 –947.
of the observed significant solvent effects on reaction rates (especially Gibbs MA, Thummel KE, Shen DD, and Kunze KL (1999) Inhibition of cytochrome P-450 3A (CYP3A) in human intestinal and liver microsomes: comparison of Ki values and impact of at solvent concentration Ͼ1%), if possible, investigator should avoid CYP3A5 expression. Drug Metab Dispos 27:180 –187.
using organic solvent or use it at low strength. For CYP3A4, two or Gorski JC, Jones DR, Haehner-Daniels BD, Hamman MA, O’Mara EM Jr, and Hall SD (1998) The contribution of intestinal and hepatic CYP3A to the interaction between midazolam and more probe reactions may be needed to yield an overall evaluation of clarithromycin. Clin Pharmacol Ther 64:133–143.
potential drug interaction. For other enzymes, such as CYP2E1, Gorski JC, Jones DR, Wrighton SA, and Hall SD (1997) Contribution of human CYP3A subfamily members to the 6-hydroxylation of chlorzoxazone. Xenobiotica 27:243–256.
further investigations for a better probe reaction may be needed.
Grant DM, Campbell ME, Tang BK, and Kalow W (1987) Biotransformation of caffeine by Opinions regarding the most appropriate probe substrates for indi- microsomes from human liver: kinetics and inhibition studies. Biochem Pharmacol 36:1251–
vidual P450 enzymes are evolving. In addition to the probe substrates Gut J, Catin T, Dayer P, Kronbach T, Zanger U, and Meyer UA (1986) Debrisoquine/sparteine- presented in this article, industry and academia investigators use other type polymorphism of drug oxidation. Purification and characterization of two functionallydifferent human liver cytochrome P-450 isozymes involved in impaired hydroxylation of the appropriate probe substrates. The proceedings of a consensus meeting prototype substrate bufuralol. J Biol Chem 261:11734 –11743.
convened in 2000 include a representative list of preferred and ac- Hall SD, Guengerich FP, Branch RA, and Wilkinson GR (1987) Characterization and inhibition ceptable in vitro probe substrates (Tucker et al., 2001). Under appro- of mephenytoin 4-hydroxylase activity in human liver microsomes. J Pharmacol Exp Ther
240:216 –222.
priate experimental conditions, these substrates provide useful infor- Hall SD, Hamman MA, Rettie AE, Wienkers LC, Trager WF, Vandenbranden M, and Wrighton SA (1994) Relationships between the levels of cytochrome P4502C9 and its prototypic
catalytic activities in human liver microsomes. Drug Metab Dispos 22:975–978.
The in vitro probe reaction is a useful tool to screen for potential in Hemeryck A, De Vriendt C, and Belpaire FM (1999) Inhibition of CYP2C9 by selective vivo drug interactions. Due to genetic variation, the influence of serotonin reuptake inhibitors: in vitro studies with tolbutamide and (S)-warfarin using human
liver microsomes. Eur J Clin Pharmacol 54:947–951.
environmental or hormonal factors, as well as intrinsic limitations of Heyn H, White RB, and Stevens JC (1996) Catalytic role of cytochrome P4502B6 in the in vitro systems, the quantitative prediction of in vivo drug interac- N-demethylation of S-mephenytoin. Drug Metab Dispos 24:948 –954.
Hickman D, Wang JP, Wang Y, and Unadkat JD (1998) Evaluation of the selectivity of in vitro tions for an individual patient remains a challenge. However, with the probes and suitability of organic solvents for the measurement of human cytochrome P450 rapid growth of our knowledge and technology in drug metabolism monooxygenase activities. Drug Metab Dispos 26:207–215.
and disposition, quantitative prediction may be achievable in the Inoue K, Yamazaki H, Imiya K, Akasaka S, Guengerich FP, and Shimada T (1997) Relationship between CYP2C9 and 2C19 genotypes and tolbutamide methyl hydroxylation and S- future. The conduct of high-quality in vitro studies is the first step mephenytoin 4Ј-hydroxylation activities in livers of Japanese and Caucasian populations.
Pharmacogenetics 7:103–113.
Jacqz-Aigrain E, Funck-Brentano C, and Cresteil T (1993) CYP2D6- and CYP3A-dependent metabolism of dextromethorphan in humans. Pharmacogenetics 3:197–204.
Acknowledgments. We acknowledge Dr. Larry Lesko for strong
Jurima M, Inaba T, and Kalow W (1985) Mephenytoin metabolism in vitro by human liver. Drug support of this research. We also greatly appreciate the generous Metab Dispos 13:151–155.
Kenworthy KE, Bloomer JC, Clarke SE, and Houston JB (1999) CYP3A4 drug interactions: support from Dr. Anthony Lu, who kindly offered unlimited encour- correlation of 10 in vitro probe substrates. Br J Clin Pharmacol 48:716 –727.
agement and expertise on drug metabolism during various phases of Ko JW, Desta Z, and Flockhart DA (1998) Human N-demethylation of (S)-mephenytoin by cytochrome P450s 2C9 and 2B6. Drug Metab Dispos 26:775–778.
Kobayashi K, Nakajima M, Chiba K, Yamamoto T, Tani M, Ishizake, Kuroiwa Y (1998) Inhibitory effects of antiarrhythmic drugs on phenacetin O-deethylation catalyzsed by human References
CYP1A2. Br J Clin Pharmacol 45:361–368.
Kronbach T (1991) Bufuralol, dextromethorphan and debrisoquine as prototype substrates for Boobis AR, Kahn GC, Whyte C, Brodie MJ, and Davies DS (1981) Biphasic O-deethylation of human P450IID6. Methods Enzymol 206:509 –517.
phenacetin and 7-ethoxycoumarin by human and rat liver microsomal fractions. Biochem Kronbach T, Mathys D, Gut J, Catin T, and Meyer UA (1987) High-performance liquid Pharmacol 30:2451–2456.
chromatographic assays for bufuralol 1Ј-hydroxylase, debrisoquine 4-hydroxylase and dex- Bourrie M, Meunier V, Berger Y, and Fabre G (1996) Cytochrome P450 isoform inhibitors as tromethorphan O-demethylase in microsomes and purified cytochrome P-450 isozymes of a tool for the investigation of metabolic reactions catalyzed by human liver microsomes.
human liver. Anal Biochem 162:24 –32.
J Pharmacol Exp Ther 277:321–332.
Kronbach T, Mathys D, Umeno M, Gonzalez FJ, and Meyer UA (1989) Oxidation of midazolam Busby WF Jr, Ackermann JM, and Crespi CL (1999) Effect of methanol, ethanol, dimethyl and triazolam by human liver cytochrome P450IIIA4. Mol Pharmacol 36:89 –96.
sulfoxide and acetonitrile on in vitro activities of cDNA-expressed human cytochromes P-450.
Kunze KL, Eddy AC, Gibaldi M, and Trager WF (1991) Metabolic enantiomeric interactions: the Drug Metab Dispos 27:246 –249.
inhibition of human (S)-warfarin-7-hydroxylase by (R)-warfarin. Chirality 3:24 –29.
Butler MA, Iwasaki M, Guengerich FP, and Kadlubar FF (1989) Human cytochrome P-450PA Kunze KL, Wienkers LC, Thummel KE, and Trager WF (1996) Warfarin-fluconazole. I.
(P-450IA2), the phenacetin O-deethylase, is primarily responsible for the hepatic 3-demeth- Inhibition of the human cytochrome P450-dependent metabolism of warfarin by fluconazole: ylation of caffeine and N-oxidation of carcinogenic arylamines. Proc Natl Acad Sci USA in vitro studies. Drug Metab Dispos 24:414 – 421.
86:7696 –7700.
Lang D and Bocker R (1995) Highly sensitive and specific high-performance liquid chromato- Chauret N, Gauthier A, and Nicoll-Griffith DA (1998) Effect of common organic solvents on in graphic analysis of 7-hydroxywarfarin, a marker for human cytochrome P-4502C9 activity.
vitro cytochrome P450-mediated metabolic activities in human liver microsomes. Drug Metab J Chromatogr B Biomed Appl 672:305–309.
Dispos 26:1– 4.
Lasker JM, Wester MR, Aramsombatdee E, and Raucy JL (1998) Characterization of CYP2C19 Chiba K, Kobayashi K, Tani M, Manabe K, and Ishizaki T (1993) The kinetic characterization and CYP2C9 from human liver: respective roles in microsomal tolbutamide, S-mephenytoin of S-mephenytoin 4-hydroxylase (P-450IICMP) activity in human liver samples obtained as and omeprazole hydroxylations. Arch Biochem Biophys 353:16 –28.
surgical waste from Japanese patients. Drug Metab Dispos 21:747–749.
Li Y, Li NY, and Sellers EM (1997) Comparison of CYP2A6 catalytic activity on coumarin (1986) Stereo- and regioselectivity of hepatic oxidation in man: effect of the debrisoquine/ 7-hydroxylation in human and monkey liver microsomes. Eur J Drug Metab Pharmacokinet sparteine phenotype on bufuralol hydroxylation. Eur J Clin Pharmacol 31:313–318.
Dayer P, Balant L, Kupfer A, Courvoisier F, and Fabre J (1983) Contribution of the genetic status Mankowski DC (1999) The role of CYP2C19 in the metabolism of (ϩ/-) bufuralol, the prototypic of oxidative metabolism to variability in the plasma concentrations of ␤-adrenoceptor blocking substrate of CYP2D6. Drug Metab Dispos 27:1024 –1028.
agents. Eur J Clin Pharmacol 24:797–799.
Marroum PJ, Uppoorr RS, Parmelee T, Ajayi F, Burnett A, Yuan R, Svadjian R, Lesko LJ, and Draper AJ, Madan A, and Parkinson A (1997) Inhibition of coumarin 7-hydroxylase activity in Balian JD (2000) In vivo drug-drug interaction studies: a survey of all new molecular entities human liver microsomes. Arch Biochem Biophys 341:47– 61.
approved from 1987 to 1997. Clin Pharmacol Ther 68:280 –285.
Edwards RJ, Adams DA, Watts PS, Davies DS, and Boobis AR (1998) Development of a Mei Q, Tang C, Assang C, Lin Y, Slaughter D, Rodrigues AD, Baillie TA, Rushmore TH, and EVALUATION OF IN VITRO P450 PROBE REACTIONS Shou M (1999) Role of a potent inhibitory monoclonal antibody to cytochrome P-450 3A4 in Tassaneeyakul W, Birkett DJ, McManus ME, Tassaneeyakul W, Veronese ME, Andersson T, assessment of human drug metabolism. J Pharmacol Exp Ther 291:749 –759.
Tukey RH, and Miners JO (1994) Caffeine metabolism by human hepatic cytochromes P450: Miners and Birkett (1996), Use of tolbutamide as a substrate probe for human hepatic cyto- contributions of 1A2, 2E1 and 3A isoforms. Biochem Pharmacol 47:1767–1776.
chrome P450 2C9. Methods Enzymol 272:139 –145.
Tassaneeyakul W, Birkett DJ, Veronese ME, McManus ME, Tukey RH, Quattrochi LC, Gelboin Miners JO, Smith KJ, Robson RA, McManus ME, Veronese ME, and Birkett DJ (1988) HV, and Miners JO (1993) Specificity of substrate and inhibitor probes for human cyto- Tolbutamide hydroxylation by human liver microsomes. Kinetic characterisation and relation- chromes P450 1A1 and 1A2. J Pharmacol Exp Ther 265:401– 407.
ship to other cytochrome P-450 dependent xenobiotic. oxidations Biochem Pharmacol 37:
Tassaneeyakul W, Mohamed Z, Birkett DJ, McManus ME, Veronese ME, Tukey RH, Quattrochi LC, Gonzalez FJ, and Miners JO (1992) Caffeine as a probe for human cytochromes P450: Newton DJ, Wang RW, and Lu AY (1995) Cytochrome P450 inhibitors. Evaluation of speci- validation using cDNA-expression, immunoinhibition and microsomal kinetic and inhibitor ficities in the in vitrometabolism of therapeutic agents by human liver microsomes. Drug techniques. Pharmacogenetics 2:173–183.
Metab Dispos 23:154 –158.
Toon S, Low LK, Gibaldi M, Trager WF, O’Reilly RA, Motley CH, and Goulart DA (1986) The Ono S, Hatanaka T, Hotta H, Tsutsui M, Satoh T, and Gonzalez FJ (1996) Chlorzoxazone is warfarin-sulfinpyrazone interaction: stereochemical considerations. Clin Pharmacol Ther 39:
metabolized by human CYP1A2 as well as by human CYP2E1. Pharmacogenetics 5:143–150.
Peter R, Bocker R, Beaune PH, Iwasaki M, Guengerich FP, and Yang CS (1990) Hydroxylation Tucker G, Houston JB, and Huang S-M (2001) Optimizing drug development: strategies to assess of chlorzoxazone as a specific probe for human liver cytochrome P-450IIE1. Chem Res drug metabolism/transporter interaction potential: toward a consensus. Clin Pharmacol Ther Toxicol 3:566 –573.
70:103–114; Br J Clin Pharmacol 52:107–117; Pharm Res (NY) 18:1071–1080.
Pollock BG, Perel JM, Krishna M, Altieri LP, Yeager AL, Reynolds CF 3rd (1991) S- Ueng YF, Kuwabara T, Chun YJ, and Guengerich FP (1997) Cooperativity in oxidations mephenytoin 4-hydroxylation in older Americans. Eur J Clin Pharmacol 40:609 – 611.
catalyzed by cytochrome P450 3A4. Biochemistry 36:370 –381.
Relling MV, Aoyama T, Gonzalez FJ, and Meyer UA (1990) Tolbutamide and mephenytoin Venkatakrishnan K, von Moltke LL, and Greenblatt DJ (1998) Human cytochromes P450 hydroxylation by human cytochrome P450s in the CYP2C subfamily. J Pharmacol Exp Ther mediating phenacetin O-deethylation in vitro: validation of the high affinity component as an 252:442– 447.
index of CYP1A2 activity. J Pharm Sci 87:1502–1507.
Rettie AE, Korzekwa KR, Kunze KL, Lawrence RF, Eddy AC, Aoyama T, Gelboin HV, von Moltke LL, Greenblatt DJ, Duan SX, Schmider J, Kudchadker L, Fogelman SM, Harmatz Gonzalez FJ, and Trager WF (1992), Hydroxylation of warfarin by human cDNA-expressed JS, and Shader RI (1996) Phenacetin O-deethylation by human liver microsomes in vitro: cytochrome P-450: a role for P-4502C9 in the etiology of (S)-warfarin-drug interactions. Chem inhibition by chemical probes, SSRI antidepressants, nefazodone and venlafaxine. Psychop- Res Toxicol 5:54 –59.
harmacology 128:398 – 407.
Sai Y, Yang TJ, Krausz KW, Gonzalez FJ, and Gelboin HV (1999) An inhibitory monoclonal Wang RW, Newton DJ, Liu N, Atkins WM, and Lu AY (2000) Human cytochrome P-450 3A4: antibody to human cytochrome P450 2A6 defines its role in the metabolism of coumarin, in vitro drug-drug interaction patterns are substrate-dependent. Drug Metab Dispos 28:360 –
7-ethoxycoumarin and 4-nitroanisole in human liver. Pharmacogenetics 9:229 –237.
Sesardic D, Boobis AR, Murray BP, Murray S, Segura J, de la Torre R, and Davies DS (1988) Waxman DJ, Lapenson DP, Aoyama T, Gelboin HV, Gonzalez FJ, and Korzekwa K (1991) A form of cytochrome P450 in man, orthologous to form d in the rat, catalyzes the O- Steroid hormone hydroxylase specificities of eleven cDNA-expressed human cytochrome deethylation of phenacetin and is iducible by cigarette smoking. Br J Clin Pharmacol P450s. Arch Biochem Biophys 290:160 –166.
Wester MR, Lasker JM, Johnson EF, and Raucy JL (2000) CYP2C19 participates in tolbutamide Sesardic D, Edwards RJ, Davies DS, Thomas PE, Levin W, and Boobis AR (1990) High affinity hydroxylation by human liver microsomes. Drug Metab Dispos 28:354 –359.
phenacetin O-deethylase is catalysed specifically by cytochrome P450d (P450IA2) in the liver Wrighton SA and Ring BJ (1994) Inhibition of human CYP3A catalyzed 1Ј-hydroxy midazolam of the rat. Biochem Pharmacol 39:489 – 498.
Shimada T, Misono KS, and Guengerich FP (1986) Human liver microsomal cytochrome P-450 formation by ketoconazole, nifedipine, erythromycin, cimetidine and nizatidine. Pharm Res mephenytoin 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabo- 11:921–924.
lism. Purification and characterization of two similar forms involved in the reaction. J Biol Wrighton SA, Ring BJ, Watkins PB, and VandenBranden M (1989) Identification of a poly- Chem 261:909 –921.
morphically expressed member of the human cytochrome P-450III family. Mol Pharmacol Shimada T, Yamazaki H, and Guengerich FP (1996) Ethnic-related differences in coumarin 36:97–105.
7-hydroxylation activities catalyzed by cytochrome P4502A6 in liver microsomes of Japanese Wrighton SA, Stevens JC, Becker GW, and VandenBranden M (1993) Isolation and character- and Caucasian populations. Xenobiotica 26:395– 403.
ization of human liver cytochrome P450 2C19: correlation between 2C19 and S-mephenytoin Shimada T, Yamazaki H, Mimura M, Inui Y, and Guengerich FP (1994) Interindividual 4Ј-hydroxylation. Arch Biochem Biophys 306:240 –245.
variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, Yamazaki H, Inoue K, Shaw PM, Checovich WJ, Guengerich FP, and Shimada T (1997) carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Different contributions of cytochrome P450 2C19 and 3A4 in the oxidation of omeprazole by Caucasians. J Pharmacol Exp Ther 270:414 – 423.
human liver microsomes: effects of contents of these two forms in individual human samples.
Shou M, Lu T, Krausz KW, Sai Y, Yang T, Korzekwa KR, Gonzalez FJ, and Gelboin HV (2000) J Pharmacol Exp Ther 283:434 – 442.
Use of inhibitory monoclonal antibodies to assess the contribution of cytochromes P450 to Yamazaki H and Shimada T (1997) Progesterone and testosterone hydroxylation by cytochromes human drug metabolism. Eur J Pharmacol 394:199 –209.
P450 2C19, 2C9 and 3A4 in human liver microsomes. Arch Biochem Biophys 346:161–169.
Stresser DM, Blanchard AP, Turner SD, Erve JC, Dandeneau AA, Miller VP, and Crespi CL Yang TJ, Krausz KW, Sai Y, Gonzalez FJ, and Gelboin HV (1999) Eight inhibitory monoclonal (2000) Substrate-dependent modulation of CYP3A4 catalytic activity: analysis of 27 test antibodies define the role of individual P-450s in human liver microsomal diazepam, 7-ethoxy- compounds with four fluorometric substrates. Drug Metab Dispos 28:1440 –1448.
coumarin and imipramine metabolism. Drug Metab Dispos 27:102–109.
Stresser DM and Kupfer D (1999) Monospecific antipeptide antibody to cytochrome P-450 2B6.
Yuan R, Parmelee T, Balian JD, Uppoor RS, Ajayi F, Burnett A, Lesko LJ, and Marroum P Drug Metab Dispos 27:517–525.
(1999) In vitro metabolic interaction studies: experience of the Food and Drug Administration.
Takano M, Hasegawa R, Fukuda T, Yumoto R, Nagai J, and Murakami T (1998) Interaction with Clin Pharmacol Ther 66:9 –15.
P-glycoprotein and transport of erythromycin, midazolam and ketoconazole in Caco-2 cells.
Yumoto R, Murakami T, Nakamoto Y, Hasegawa R, Nagai J, and Takano M (1999) Transport Eur J Pharmacol 358:289 –294.
of rhodamine 123, a P-glycoprotein substrate, across rat intestine and Caco-2 cell monolayers Tang C, Shou M, and Rodrigues AD (2000) Substrate-dependent effect of acetonitrile on human in the presence of cytochrome P-450 3A-related compounds. J Pharmacol Exp Ther 289:
liver microsomal cytochrome P450 2C9 (CYP2C9) activity. Drug Metab Dispos 28:567–572.


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,

Ad2243r0 letter

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

Copyright © 2010 Medicament Inoculation Pdf