validation of the five-drug “pittsburgh cocktail” approach for assessment of selective...

PHARMACOKINETICS AND DRUG DISPOSITION

Validation of the five-drug “Pittsburgh cocktail” approach for assessment of selective regulation of drug-metabolizing enzymes

Objectives: To determine whether the probe drugs caffeine, chlorzoxazone, dapsone, debrisoquin (INN, debrisoquine), and mephenytoin can be simultaneously administered as a metabolic cocktail to estimate in vivo cytochrome P450 (CYI?) and N-acetyltransferase enzyme activities. Methods: Fourteen healthy nonsmoking male volunteers (mean age f SD, 21.6 f 2.2 years) received 100 mg caffeine, 250 mg chlorzoxazone, 100 mg dapsone, 10 mg debrisoquin, and 100 mg mephenytoin individually and in four- and five-drug combinations in a randomized manner using a 7 X 7 Latin square. Each drug or drug combination was given orally after an overnight fast, with a minimum l-week washout between administrations. In each session, urine was collected from 0 to 8 hours and plasma was obtained at 4 and 8 hours after drug administration. Plasma and metabolite concentrations were used to estimate phenotypic trait measures for the efficiency of each drug’s metabolism. Results: The phenotypic indexes determined for caffeine, chlorzoxazone, dapsone, debrisoquin, and mephenytoin were not significantly different when given alone than when given in combination. The median percentage change of the trait measures observed during administration of all five compounds compared with individual administration ranged from -10.7% for the 6-hydroxychlorzoxazone to chlorzoxazone plasma ratio to +2.2% for the debrisoquin recovery ratio. Conclusions: The results of this study show that caffeine, chloxzoxazone, dapsone, debrisoquin, and mephenytoin in low doses can be simultaneously administered without metabolic interaction. This cocktail approach can thus simultaneously provide independent in vivo phenotypic measures for multiple CYP enzymes and N-acetyltransferase.(Clin Pharmacol Ther 1997;62:365-76.)

Reginald F. Frye, PharmD, PhD, Gary R Matzke, PharmD, Adedayo Adedoyin, PhD, James A. Porter, BS, and Robert A. Branch, MD Pittsbury& Pa.

From the Department of Pharmaceutical Sciences, School of Pharmacy, and the Center for Clinical Pharmacology, Univer- sity of Pittsburgh.

Supported in part by a grant from Upjohn Inc. (Kalamazoo, Mich.), by grant CA 59834 from the National Institutes of Health (Bethesda, Md.), and by grant 5MOl RRCKKl.56 from the National Institutes of Health National Center for Research Resources&eneral CIinical Research Center (Bethesda, Md.). Dr. Frye was the recipient of a fellowship from the American Society of Hospital Pharmacists Re- search and Education Foundation (Bethesda, Md.).

Received for publication Feb. 27, 1997; accepted June 23, 1997. Reprint requests: Reginald Frye, PharmD, PhD, University of Pitts-

burgh, School of Pharmacy, 726 Salk Hall, Pittsburgh, PA 15261. Copyright 0 1997 by MO&y-Year Book, Inc. 0009-9236/97/$5.00 + 0 13/l/84215

Interindividual variability in drug metabolism is in part a reflection of the heterogeneity of cytochrome P450 (CYP) enzymes involved in the metabolism of xenobiotic agents. The expression and activities of these enzymes are regulated by genetic factors and by environmental factors such as disease states, smoking, alcohol, and concomitant drug therapy.le3 These factors contribute to the marked intersubject variability in activity or expression that has been observed for individual CYP enzymes.4 The involve- ment of multiple CYP enzymes in human drug metabolism has heightened the interest in assessing the in vivo activities of these enzymes in humans as a

365

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means to evaluate important modulators of their activity and the role, if any, these enzymes may play in the development of diseases such as cancer.5,6 A common approach for estimation of in vivo metabolic activity in humans has been through the administration of a probe compound. For example, antipyrine (INN, phenazone) was frequently used as a general probe drug because of its extensive metabolism. Unfortunately, due to the multiplicity of CYP enzymes and their substrate selectivities, no single probe drug can provide a measure of the activity of all enzymes responsible for metabolism in an individual. An alternative strategy is to use a specific probe drug selected on the basis that a quantifiable route of its metabolism is predominately or exclusively mediated by an individual CYP enzyme. This offers the advantage that the activity of a specific CYP enzyme can be critically evaluated. However, individual administration of specific probes necessitates multiple experimental sessions, with attendant complications, to assess the activities of the multiple enzymes important in drug metabolism.

Simultaneous administration of multiple in vivo probes of drug-metabolizing enzymes-the cocktail strategy-offers several important advantages.7 Most notably, information on several pathways of metabolism are obtained in a single experimental session and the confounding influence of intrasubject variability over time is minirnized.7~8 How- ever, the utility of such a cocktail strategy would be severely limited if metabolic interactions occurred when the in vivo probes were administered simultaneously. In addition, this approach may be limited by the potential for analytical interference from the simultaneously administered probe compounds and their metabolites.

In this study, we have investigated the utility of a five-drug cocktail consisting of caffeine,‘,” chlorzoxazone,i’*” dapsone,13T14 debrisoquin (INN, debrisoquine),15 and mephenytoin,16 as in vivo probes in assessment of the activities of the CYP enzymes lA2,2El, 3A, 2D6, and 2C19, respectively. Dapsone also provides an index of N-acetyltransferase activity. l7 This group of enzym es accounts for more than 90% of human drug metabolism. This five-drug cocktail is an extension of our earlier work with the three-drug cocktail of dapsone, debrisoquin, and mephenytoin.‘4’18 Although previously used probe cocktails have’ used combinations of two to four general and specific probe drugs, to our knowledge this is the first effort to combine five probe drugs for

simultaneous administration. The objective of this study was to characterize the metabolism of the test substrates in low doses when administered alone and in combination and to ascertain whether com- bined administration can provide independent phenotypic measures of the respective enzymes.

METHODS Fourteen normal healthy white male volunteers

(mean age 2 SD, 21.6 t 2.2 years; mean weight & SD, 72.1 + 9.7 kg) participated after each provided written informed consent. The study was approved by the University of Pittsburgh Biomedical Institu- tional Review Board (Pittsburgh, Pa.). All subjects were nonsmokers and were healthy as confirmed by medical history, physical examination, blood chem- istries, and urinalysis. Subjects were instructed to abstain from caffeine or alcohol-containing products for at least 2 days before each study visit, and none of the subjects was receiving any over-the-counter or prescription medications. Poor metabolizers of CYP2D6 were excluded from study participation because they would have minimal capacity to me- tabolize debrisoquin at baseline and thus would not contribute to the detection of a metabolic interaction. Poor metabolizers were excluded by means of a screening test with dextromethorphan. Subjects were given 30 mg dextromethorphan (Benylin DM, Warner-Lambert Laboratories, Ltd.) and instructed to take the dose before bedtime. All of the urine produced overnight was collected and returned to the Research Center where the samples were analyzed by a modification of the qualitative thin-layer chromatographic method of Guttendorf et a1.19

This randomized crossover study compared each probe drug alone, a four-drug cocktail, and a five- drug cocktail. Each subject that participated in the study received 100 mg caffeine, 250 mg chlorzoxazone, 100 mg dapsone, 10 mg debrisoquin, and 100 mg mephenytoin on separate occasions (five regimens). In addition, each subject received a four- and a five-drug cocktail that consisted of the three-drug cocktail previously used by our group (dapsone, debrisoquin, and mephenytoin),18,20~21 to which was added caffeine (four-drug cocktail) and caffeine plus chlorzoxazone (five-drug cocktail). Thus each subject received a total of seven regimens. The order in which the subjects received the individual drugs or drug combinations was randomized by use of a 7 X 7 Latin square balanced for order. Each drug or drug combination was given orally after an overnight fast, with subsequent administrations separated by a

CLINICAL PHARMACOLOGY & THERAFEUTICS VOLUME 62, NUMBER4 Frye et al. 367

minimum l-week washout period. In each session, urine was collected from 0 to 8 hours in a container with ascorbic acid as a preservative for the unstable dapsone hydroxylamine metabolite. Plasma samples (10 ml) were collected before and at 4 and 8 hours after probe administration. All samples were stored frozen at -20” C until analyzed.

Analytical techniques. The following drugs and metabolites were measured by HPLC techniques described previously: caffeine and paraxanthine in plasmaa; chlorzoxazone and 6-hydroxychlorzoxazone in plasma=; dapsone and dapsone hydroxylamine in urine and dapsone and monoacetyldapsone in plas- ma24; and debrisoquin and 4-hydroxydebrisoquin in urine.= The within and between-day coefficients of variation for each of these assays was ~10%.

The urinary concentrations of 4’-hydroxymephenytoin were measured by a newly developed HPLC method that was simpler than previously reported methods*(j and that eliminated interferences from the coadministered drugs. In brief, a mixture of 500 ~1 of urine and 100 p,l internal standard (0.05 mg/ml phensuximide) was acidified with 500 p,l so- dium acetate buffer (1.0 moliL, pH 5) and incubated with 2500 units of P-glucuronidase for 3 hours at 37” C. The samples were subsequently extracted with 5 ml ethyl ether, and the organic layer was evaporated to dryness under a stream of nitrogen. The residue was reconstituted in 500 ~1 of 40% methanol in water, and 100 p,l aliquots were injected into the HPLC system. Separation of 4’- hydroxymephenytoin and internal standard was achieved with a Bioanalytical Systems 250 X 4.6 mm internal diameter 5 Frn C1s analytical column. The sample was eluted with a mobile phase of 0.05 mol/L potassium phosphate (pH 6.5), methanol, and ace- tonitrile (74:13:13) pumped at a flow rate of 1.0 mUmin. Column eluent was monitored with a ultra- violet detector (model 486, Waters Corporation, Milford, Mass.) That was set at 211 nm. Linear calibration curves were obtained for 4’- hydroxymephenytoin over the concentration range from 1 to 100 p&ml. The intraday and interday coefficients of variation at low and high concentrations were less than 5%.

All of the assay procedures used in this study were cross-validated with the other probe drugs and metabolites to ensure that no analytical interference would occur with simultaneous administration.

Data andysis. The concentration of paraxanthine (1,7 dimethylxanthine) divided by the concentration of caffeine in the 8-hour plasma sample was used to

assess CYPlA2 activity.10’27 The ability to hydroxylate chlorzoxazone (CYP2El) was estimated by the 6-hydroxychlorzoxazone to chlorzoxazone plasma ratio at 4 hours.28T29 The ability to N-hydroxylate dapsone (CYP3A) was estimated by the urinary recovery ratio17:

HDA Dapsone recovery ratio = HDA + DDS

in which HDA is the urinary recovery of dapsone hydroxylamine in an 8-hour urine sample, and DDS is the 8-hour urinary recovery of dapsone. Acetyla- tor phenotype was defined as the ratio of monoacetyldapsone to dapsone in the 8-hour plasma sample.17 The activity of CYP2D6 was estimated by use of the debrisoquin recovery ratio3’:

HDB Debrisoquin recovery ratio = HDB + DB

in which HDB and DB are the urinary recoveries of 4-hydroxydebrisoquin and debrisoquin in 8 hours, respectively. The total urinary recovery of 4’- hydroxymephenytoin was used as the phenotypic measure of CYP2C19 activity.26

Statistical analysis. The phenotypic trait measures determined from individual administration were compared with those obtained from the simultaneous administrations by use of repeated- measures ANOVA. Chlorzoxazone was administered on only two occasions, so the trait measures from individual and simultaneous administration were compared with a paired t test. Spearman’s rank correlation was used to assess any relationships between the phenotypic indexes. Computa- tions were performed with version 6.08 of SAS (Car-y, N.C.) and p < 0.05 was considered to be statistically significant.

RESULTS The dextromethorphan screening test” identified

two subjects as poor metabolizers, who were then excluded from study participation. This method proved to be a rapid and efficient means with which to identify and subsequently exclude CYP2D6 poor metabolizers from study participation. Each of the drugs administered individually or in combination were well tolerated by all of the subjects who met eligibility criteria and agreed to participate. The only adverse event reported was transient drowsiness by two subjects after the individual administration of mephenytoin. No drowsiness was reported after the drug cocktails, possibly because of the

368 Fvye et al. CLINICAL PHARMACOLOGY & THEBAPEUTICS

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Table I. Comparison of phenotypic indexes after individual and simultaneous administration Simultaneous administration

Four-dnrg Five-drug Alone cocktail cocktail

Intrasubject CV

Paraxanthinelcaffeine plasma ratio 0.77 + 0.25 0.69 2 0.19 0.73 + 0.21 15.2% 6-Hydroxychlorzoxazone/chlorzoxazone plasma ratio 0.92 + 0.33 NA 0.80 + 0.33 - Debrisoquin (INN, debrisoquine) recovery ratio 0.59 + 0.15 0.58 t 0.14 0.61 t 0.14 4.7% Dapsone recovery ratio 0.62 2 0.07 0.58 2 0.08 0.60 k 0.08 5.3% Dapsone acetylation ratio 0.17 ? 0.02 0.17 + 0.02 0.17 k 0.02 4.3% 4’-Hydroxymephenytoin recovery (Fmol) 137.9 k 41.8 142.4 + 30.0 131.1 + 25.9 7.4%

Data are mean values k SD. NA, Not applicable; CV, coefficient of variation.

inclusion of caffeine. All of the subjects enrolled completed the study.

The phenotypic indices for each probe drug determined from individual and simultaneous administrations are presented in Table I. None of the subjects were poor metabolizers of either debrisoquin or mephenytoin. The phenotypic trait measures for each drug that were observed in this study are comparable to values previously reported for healthy volunteers.10,14’28 There were no statistically significant differences in the phenotypic indexes determined for caffeine, chlorzoxazone, dapsone, debrisoquin, or mephenytoin when given alone or in combination. The results for each drug for all subjects are depicted in Figs. 1 through 3, which illus- trate the contrast between the marked intersubject variability and the minimal intrasubject variability.

The absence of a metabolic interaction between the probe drugs used in this investigation permits insight into the intrasubject variability of each measure over a short period of time. The administrations were separated by a minimum l-week washout so the duration of the entire study for each subject was 7 weeks. The minimum time between consecu- tive administrations of the same probe was 1 week, and the maximum time between exposures was 5 weeks. The median percentage change of the trait measures observed during administration of all five compounds compared to individual administration ranged from -10.7% for the chlorzoxazone plasma ratio to +2.2% for the debrisoquin recovery ratio (Fig. 4). The median within-subject coefficient of variation (CV) values for the probes that were administered three times were 15.2% for caffeine, 5.3% dapsone hydroxylation, 4.3% for dapsone acetylation, 4.7% for debrisoquin, and 7.4% for mephenytoin (Table I). This is in contrast to the between-subject coefficients of variation that were

29.2%, 13.4%, 19.8%, and 22.7% for caffeine, dapsone, debrisoquin, and mephenytoin, respectively. Thus the between-subject CV was two to four times larger than the within-subject CV within this inten- tionally homogenous population. The magnitude of within- and between-individual variabilities observed for the drugs used in this study are similar to those previously reported with drugs such as antipyrine, phenacetin, and phenylbutazone.31 No significant correlation was observed between any of the trait measures of the probe drugs when administered individually.

DISCUSSION The results of this study show that the probe drugs

caffeine, chlorzoxazone, dapsone, debrisoquin, and mephenytoin, at the doses used, can be administered simultaneously without metabolic interactions. This cocktail strategy is minimally invasive, requir- ing only an &hour urine collection and three blood samples, and provides an in vivo estimate of the activity of the CYP enzymes lA2,2El, 3A, 2D6, and 2C19, as well as N-acetyltransferase activity in a single experimental session.

The probe drugs selected for use in this study had been previously evaluated for use individually as enzyme-specific probes. This approach involved both in vitro studies that delineated which human CYP enzyme(s) were involved in each drug’s overall metabolism and in the generation of specific metabolites, and rigorous in vivo pharmacokinetic studies designed to develop a single sample phenotypic trait measure. The phenotypic trait measure is a param- eter that best reflects the metabolite formation clearance, generally regarded as the gold standard measure of enzyme activity, but is calculated from limited information and does not necessitate extensive sampling. The trait measures selected for use in

CLINICAL PHARMACOLOGY &THERAPEUTICS VOLUME 62. NUMBER 4 Frye et al. 369

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Fig. 1. Comparison of the paraxanthine to caffeine plasma ratio at 8 hours (A) and the 6-hydroxychlorzoxazone to chlorzoxazone plasma ratio at 4 hours (B) from individual and simultaneous drug administrations.

this study for caffeine,” chlorzoxazone,28,32 dapsone,17 mephenytoin,26 and debrisoquin3’

trait measures determined in this study after indi- were pre- vidual versus simultaneous administration should

viously validated, which was a critical step before thus reflect changes in the formation clearance or this study was undertaken. Changes in any of the elimination of the metabolite.

370 Fye et al. CLlNlCAL Pl3ABMACOLoGY & THERAPEUTICS

OCTOBER 1997

A

O.OOC 4 Drug Alone 5Dnrg

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Fig. 2. Comparison of the urinary dapsone recovery ratio (A) and the plasma dapsone acetylation ratio (B) from individual and simultaneous drug administrations.

Although our use of dapsone as a probe of CYP2E133 and CYP2C9?4 The current view is that CYP3A is based on previous reports from our lab- multiple enzymes may contribute to the formation oratory, 13*14rB some recent evidence suggests that of this metabolite, although their relative contribu- dapsone hydroxylamine may also be formed by tions in vivo are unknown. The validity of its use as

CLINICAL PHABMA COLOGY & THERAPEUTICS VOLUME 62, NUMBER 4 Frye et al. 371

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Fig. 3. Comparison of the debrisoquin (INN, debrisoquine) recovery ratio (A) and the total 4’-hydroxymephenytoin urinary recovery (B) from individual and simultaneous drug administrations.

a probe for CYP3A will await further detailed investigations to address this question. More impor-

developing aggressive bladder cancer.6V35 Thus,

tantly, however, is our previous finding that the irrespective of which enzyme activity this reaction

ability to form this metabolite is related to the risk of measures, it is important and relevant in determini- nation of the risk of a malignant disease. Conse-

372 Frye et al.

t-

I-

IA2 2619 2b3 2il 3L

Pathway

Fig. 4. Percentage change (mean 2 SD) of the phenotypic trait measures for each probe drug obtained after simultaneous administration from those measures obtained after individual administration of the five drugs.

quently, we believe its evaluation in this study for possible interaction with other probe drugs is justi- fied because it will continue to be used to assess such risks.

Because each of the probe drugs used in this study is predominately metabolized by an individual CYP enzyme and was given in a low dose, it could be expected that no metabolism-based interaction would occur. However, examples have been presented in which a drug is a substrate for one enzyme but a potent inhibitor of another (e.g., quinidine). 36,37 In addition, there was a recent report that demonstrated an interaction between two of the probe drugs we had selected for use in this study. Berthou et a1.38 reported an interaction between caffeine and chlorzoxazone when administered simultaneously in 16 normal volunteers. They observed a 20% decrease in the plasma paraxanthine to caffeine ratio but no change in the 6- hydroxychlorzoxazone to chlorzoxazone metabolic ratio. In this study, we did not observe a decrease in the caffeine metabolic ratio when chlorzoxazone was

CLINICAL PHAWiACOLOGY & THERAPEUTICS OCTOBER 1997

coadministered. An important difference in the two studies is that they used higher doses of both caffeine (140 mg) and chlorzoxazone (500 mg) than those used in this study. These considerations made it important for us to verify that no metabolic interactions would occur at the chosen doses when the multiple probe drugs are administered simultaneously.

There is increasing interest in the simultaneous administration of multiple probes to characterize the activity of multiple metabolizing enzymes. This interest has been stimulated by the evolving knowledge of the multiplicity of enzymes involved in xenobiotic metabolism, their substrate specificities, and the unavailability of a drug probe that can be used to assess the activities of several enzymes. Drug metabolism investigations have therefore pro- gressed from the use of general metabolic probes, such as antipyrine, to specific probes and then to the simultaneous administration of multiple probe compounds as a means to evaluate differential effects on individual metabolizing enzyme activities. Probe drug combinations have been used to investigate the influence of disease states or drug therapy on the activities of multiple CYP enzymes.

Several drug cocktails have been evaluated (Ta- ble II) that use combinations of both general and specific metabolic probes.14318,39-61 The feasibility and utility of the cocktail strategy was first shown by Breimer and Schellens7 in multiple investigations. The effects of enzyme induction and inhibition, concomitant drug therapy, and liver disease were evaluated by Schellens et a1.39,43,60,61 with use of several different cocktails that consisted of general probes of hepatic metabolism such as theophylline and antipyrine, as well as more specific probes such as mephenytoin, sparteine, and nifedipine. These important studies showed the feasibility of simultaneous probe drug administration and its application for investigation of differential modulation of CYP activity but had the limitation of evaluating only a small proportion of drug- metabolizing enzymes.

Although the cocktail approach has practical advantages, its utility is not unequivocal. A commen- tary by Paolini et a1.62 criticized the cocktail strategy and suggested that considerable complications and limitations compromised its practical utility. Some of their concerns included availability of selective and sensitive assays for the individual drugs and metabolites, the pharmacologic activity of the probe agents, and the potential for drug-drug interactions.

CLINICAL P HARMACOLOGY &THERAPEUTICS VOLUME 62, NUMBER 4 Frye et al. 373

Table II. Previously reported multidrug cocktails used to estimate in vivo activity of CYP enzymes in humans

Antipyrine (INN, phenazone), hexobarbital, theophylline

Antipyrine, mephenytoin, nifedipine, sparteine Antipyrine, metronidazole Antipyrine, nifedipine Antipyrine, theophylline Antipyrine, tolbutamide Caffeine, debrisoquin, dipyrone (INN, metamizole),

sulfamethazine (INN, sulfadimidine) Caffeine, dextromethorphan Caffeine, dextromethorphan, mephenytoin Caffeine, trimethadione Coumarin, dextromethorphan, mephenytoin Dapsone, debrisoquin, mephenytoin Dapsone, mephenytoin, metoprolol Debrisoquin, mephenytoin Dextromethorphan, mephenytoin Nifedipine, mephenytoin, sparteine Nifedipine, phenytoin, sparteine

Berthou et a1.38; Schellens et a1.39240; Shedlofsky et a1.41; Israel et a1.42

Schellens et a1.43 Loft et a1.44; Loft45 Lanchote et a1.46 Teunissen et a1.47; Groen et aL4’ Back et a1.49 Trnckenbrodt et a1.50; Lautenschlager et a1.5’

Evans et al.” Brockmoller and Roots53 Tanaka et a1.54 Endres et a1.55 May et a1.14; Black et al.” Setiabudy et a1.56 Sanz et a1.57; Atiba et al.” Guttendorf et a1.s9 Schellens et a1.60 Schellens et a1.61

The potential for analytical interference is a very significant problem in the use of multiple drug cocktails. This potential increases with the addition of each probe drug because of the addition of more parent drug(s) and metabolite(s) to the sample ma- trix. The analytical aspects of this study proved to be formidable because each assay used in this study had to be cross-validated to ensure that the other drugs or metabolites did not interfere. However, with ap- propriate modifications (e.g., extraction and chromatographic conditions), there was no interference from the coadministered drugs or metabolites in any of the analytical procedures used in this report.

The issues of pharmacologic activity of the probe drugs and the potential for metabolic interactions are the more significant concerns raised with the cocktail approach. However, we and others have shown that only minor adverse effects, such as drowsiness, result from the multidrug cocktail administration and that selected probes, when given at low doses to minimize inherent pharmacologic effects, do not interfere with the metabolism of the coadministered probe drugs. In addition, the use of any probe drug as a single agent can also cause pharmacologic effect; therefore this is not a problem solely limited to the cocktail approach. Many other investigators have taken an approach similar to ours for validation of the use of multidrug cocktails. For example, Evans et al. 52 showed that dextromethorphan, a model substrate metabolized by CYP2D6, has no effect on the disposition of simultaneously

administered caffeine, whereas Guttendorp9 showed that dextromethorphan is unaffected by mephenytoin coadministration. Sanz et a1.57 showed a lack of interaction between coadministered mephenytoin and debrisoquin and Setiabudy et a1.56 validated the simultaneous use of dapsone (N- acetylation), metoprolol (CYP2D6), and mephenytoin in an Indonesian population. As indicated in Table II, several multidrug cocktails have been evaluated and confirmed to lack metabolic interactions. The cocktail evaluated in this investigation repre- sents the most probe drugs simultaneously administered without interaction and permits simultaneous evaluation of several of the most important drug- metabolizing enzymes. Unfortunately, not all of the important enzymes are characterized. For example, no probe for CYP2C9 is included; however, this is currently being addressed in ongoing studies.

The cocktail strategy described can potentially be applied in several research areas, such as drug development, drug interactions, and the role of drug- metabolizing enzymes in carcinogenesis. In drug development, for example, the in vivo importance of CYP enzymes in the disposition of a new chemical entity can be explored through the relationships between the disposition of the parent drug and metabolite(s) with the phenotypic measures of the probe drugs for the most relevant drug-metabolizing enzyme(s). A significant correlation may suggest that the activity of the particular enzyme is important in the in vivo disposition of the compound.

374 Frye et al. CLINICAL PHARMA COLOGY &THERAPEUTICS

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Drug interaction studies that use the drug cocktail approach could provide valuable information on the selectivity, magnitude, and relevance of the in vivo effect(s) a drug may have on individual CYP enzymes, which could include both inhibitory and in- ductive effects. A study of this type could be performed in healthy volunteers by estimation of enzyme activity with the cocktail before and after exposure to the new drug to assess changes in the activities of multiple CYP enzymes. This approach could serve as a screening tool to target the specific in vivo drug interactions studies that should be performed early in drug development. We have also shown that it is feasible to incorporate cocktail studies in patients to evaluate differential effects of a particular disease state or drug therapy on CYP activity.20,30,35

In conclusion, this study shows that simultaneous administration of the in vivo probes caffeine, chlorzoxazone, dapsone, debrisoquin, and mephenytoin in low doses provides independent estimates of respective individual metabolizing enzyme activities. We are currently using this cocktail for population phenotyping to explore interindividual differences in metabolizing activities as it relates to the development and recurrence of cancer and to assess the effect of concomitantly administered drugs on the activity of the selected CYP isozymes.

References 1. Bauer LA, O’Sullivan T, Reiss WG, Horn JR,

Opheim K, Strandness DE, et al. Liver blood flow, antipyrine clearance, and antipyrine metabolite formation clearance in patients with chronic active hep- atitis and alcoholic cirrhosis. Br J Clin Pharmacol 1994;37:375-81.

2. WalterSack I, Klotz U. Influence of diet and nutri- tional status on drug metabolism. Clin Pharmacokinet 1996;31:47-64.

3. Porter TD, Coon MJ. Cytochrome P-450: multiplicity of isoforms, substrates and catalytic and regulatory mechanisms. J Biol Chem 1991;266:13469-72.

4. Shimada T, Yamazaki H, Mimura M, Inui Y, Guengerich FP. Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 1994;270:414-23.

5. Brockmoller J, Roots I. Assessment of liver metabolic function: clinical implications. Clin Pharmacokinet 1994;27:216-18.

6. Branch RA, Chem HD, Adedoyin A, Ron&es-Sparks M, Lesnick TG, Persad R, et al. The procarcinogen

hypothesis for bladder cancer: activities of individual drug-metabolizing enzymes as risk factors. Pharmaco- genetics 1995;5:S97-100.

7. Breimer DD, Schellens JH. A “cocktail” strategy to assess in viva oxidative drug metabolism in humans. Trends Pharmacol Sci 1990;11:223-5.

8. Breimer DD. Interindividual differences in drug disposition: clinical implications and methods of investigation. Clin Pharmacokinet 1983;8:371-7.

9. Butler MA, Iwasaki M, Guengerich FP, Kadlubar FF. Human cytochrome P-450pa (P-45OIA2), the phenacetin 0-deethylase, is primarily responsible for the hepatic 3-demethylation of caffeine and N-oxidation of carcinogenic arylamines. Biochemistry 1989;86: 7696-700.

10. Fuhr U, Rost KL. Simple and reliable CYPlA2 phenotyping by the paraxanthine/caffeine ratio in plasma and saliva. Pharmacogenetics 1994;4:109-16.

11. Peter R, Backer R, Beaune PH, Iwasaki M, Guengerich FP, Yang CS. Hydroxylation of chlorzoxazone as a specific probe for human liver cytochrome P-450IIEl. Chem Res Toxic01 1990;3:566-73.

12. Kharasch ED, Thummel KE, Mhyre J, Lillibridge JH. Single-dose disulfiram inhibition of chlorzoxazone metabolism: a clinical probe for P450 2El. Clin Phar- macol Ther 1993;53:643-50.

13. Fleming CM, Branch RA, Wilkinson GR, Guengerich FP. Human liver microsomal N-hydroxylation of dapsone by cytochrome P-4503A4. Mol Pharmacol 1992; 41:975-80.

14. May DG, Porter J, Wilkinson GR, Branch RA. Fre- quency distribution of dapsone N-hydroxylase, a pu- tative probe for P4503A4 activity, in a white population. Clin Pharmacol Ther 1994;55:492-500.

15. Meyer UA, Skoda RC, Zanger UM. The genetic polymorphism of debrisoquine/sparteine metabolism: mo- lecular mechanisms. Pharmacol Ther 1990;46:297- 308.

16. Wilkinson GR, Guengerich FP, Branch RA. Genetic polymorphism of S-mephenytoin hydroxylation. Phar- macol Ther 1989;43:53-76.

17. May DG, Arns PA, Richards WO, Porter J, Ryder D, Fleming CM, et al. The disposition of dapsone in cirrhosis. Clin Pharmacol Ther 1992;51:689-700.

18. Black C, May G, Csuka ME, Lupoli S, Wilkinson GR, Branch RA. Activity of oxidative routes of metabolism of debrisoquin, mephenytoin, and dapsone is unrelated to the pathogenesis of vinyl chloride- induced disease. Clin Pharmacol Ther 1992;52:659-67.

19. Guttendorf RJ, Wedlund PJ, Blake J, Chang SL. Sim- plified phenotyping with dextromethorphan by thin- layer chromatography: application to clinical labora- tory screening for deficiencies in oxidation drug metabolism. Ther Drug Monit 1988;10:490-8.

20. Fleming CM, Kaisary A, Wilkinson GR, Smith P, Branch RA. The ability to 4-hydroxylate debrisoquine

CLINICAL PHARMACOLQGY &THERAPEUTICS VOLUME 62, NUMBER 4 Frye et al. 375

is related to recurrence of bladder cancer. Pharmaco- genetics 1992;2:128-34.

21. May DG, Black CM, Olsen NJ, Csuka ME, Tanner SB, Bellino L, et al. Scleroderma is associated with differences in individual routes of drug metabolism: a study with dapsone, debrisoquin, and mephenytoin. Clin Pharmacol Ther 1990;48:286-95.

22. Frye RF, Stilf DD, Matzke GR, Branch RA. A sensitive method for the simultaneous determination of caffeine and its dimethybranthine metabolites in human serum, saliva, and plasma [abstract]. Pharm Res 1993;1o:s43.

23. Frye RF, Stiff DD. Determination of chlorzoxazone and 6-hydrovchlorzoxazone in human plasma and urine by high-performance liquid chromatography. J Chromatogr B Biomed Appl 1996;686:291-6.

24. May DG, Porter JA, Uetrecht JP, Wilkinson GR, Branch RA. The contribution of N-hydrovlation and acetylation to dapsone pharmacokinetics in normal subjects. Clin Pharmacol Ther 1990;48:619-27.

25. Frye RF, Branch RA. Improved high performance liquid chromatographic determination of debrisoquine and 4-hydroxydebrisoquine in human urine following direct injection. J Chromatogr B Biomed Appl 1996;677:178-82.

26. Wedlund PJ, Aslanian WS, McAllister CB, Wilkinson GR, Branch RA. Mephenytoin hydroxylation defi- ciency in Caucasians: frequency of a new oxidation drug metabolism polymorphism. Clin Pharmacol Ther 1984;36:773-80.

27. Tanaka E, Ishikawa A, Yamamoto Y, Osada A, Tsuji K, Fukao K, et al. A simple useful method of the determination of hepatic function in patients with liver cirrhosis using caffeine and its three major di- methylmetabolites. Int J Clin Pharmacol Ther Toxic01 1992;30:336-41.

28. Girre C, Lucas D, Hispard E, Menez C, Dally S, Menez JF. Assessment of cytochrome P4502El induction in alcoholic patients by chlorzoxazone pharmacokinetics. Biochem Pharmacol 1994;47:1503-8.

29. Lucas D, Berthou F, Girre C, Poitrenaud F, Menez JF. High-performance liquid chromatographic determination of chlorzoxazone and 6-hydror@lorzoxazone in serum: a tool for indirect evaluation of cytochrome P4502El activity in humans. J Chromatogr 1993;622:79- 86.

30. Kaisary A, Smith P, Jacqz E, McAllister CB, Wilkin- son GR, Ray WA, et al. Genetic predisposition to bladder cancer: ability to hydroxylate debrisoquine and mephenytoin as risk factors. Cancer Res 1987;47: 5488-93.

31. Alvares AP, Kappas A, Eiseman JL, Anderson KE, Pantuck CB, Pantuck EJ, et al. Intraindividual variation in drug disposition. Clin Pharmacol Ther 1979; 26:407-19.

32. Frye RF, Stiff DD, Matzke GR, Adedoyin A, Kubisty

C, Branch RA. Pharmacokinetics of chlorzoxazone: an in vivo probe of cytochrome P4502El activity [abstract]. Pharm Res 1993;1O:S388.

33. Mitra AK, Thummel KE, Kalhorn TF, Kharasch ED, Unadkat JD, Slattery JT. Metabolism of dapsone to its hydroxylamine by CYP2El in vitro and in vivo. Clin Pharmacol Ther 1995;58:556-66.

34. Gill HJ, Tingle MD, Park BK. N-Hydrovlation of dapsone by multiple enzymes of cytochrome P450: implications for inhibition of haemotoxicity. Br J Clin Pharmacol 1995;40:531-8.

35. Fleming CM, Persad R, Kaisary A, Smith P, Adedoyin A, Porter J, et al. Low activity of dapsone N- hydroxylation as a susceptibility risk factor in aggressive bladder cancer. Pharmacogenetics 1994;4:199- 207.

36. Inaba T, Tyndale RE, Mahon WA. Quinidine: potent inhibition of sparteine and debrisoquine oxidation in vivo. Br J Clin Pharmacol 1986;22:199-200.

37. Mikus G, Ha HR, Vozeh S, Zekom C, Follath F, Eichelbaum M. Pharmacokinetics and metabolism of quinidine in extensive and poor metabolizers of sparteine. Eur J Clin Pharmacol 1986;31:69-72.

38. Berthou F, Goasduff T, Lucas D, Dreano Y, Le Bot MH, Menez JF. Interaction between two probes used for phenotyping cytochromes P45OlA2 (caffeine) and P4502El (chlorzoxazone) in humans. Pharmacoge- netics 1995;5:72-9.

39. Schellens JHM, Janssens AR, Van Der Wart JHF, Van Der Velde EA, Breimer DD. Relationship between the metabolism of antipyrine, hexobarbital, and theophylline in patients with liver disease as assessed by a “cocktail” approach. Eur J Clin Invest 1989;19: 472-9.

40. Schellens JHM, Van Der Wart JHF, Danhof M, Van Der Velde EA, Breimer DD. Relationship between the metabolism of antipyrine, hexobarbital and theophylline in man as assessed by a “cocktail” approach. Br J Clin Pharmacol 1988;26:373-84.

41. Shedlofsky SI, Israel BC, Mcclain CJ, Hill DB, Blouin RA. Endotoxin administration to humans inhibits hepatic cytochrome P450-mediated drug metabolism. J Clin Invest 1994:94:2209-14.

42. Israel BC, Blouin RA, McIntyre W, Shedlofsky SI. Effects of interferon-a monotherapy on hepatic drug metabolism in cancer patients. Br J Clin Pharmacol 1993;36:229-35.

43. Schellens JHM, Van Der Wart JHF, Brugman M, Breimer DD. Influence of enzyme induction and inhibition on the oxidation of nifedipine sparteine, mephenytoin, and antipyrine in humans as assessed by a “cocktail” study design. J Pharmacol Exp Ther 1989; 2491638~45.

44. Loft S, Poulsen HE, Sonne J, Dossing M. Metronida- zole clearance: a one-sample method and influencing factors. Clin Pharmacol Ther 1988;43:420-8.

376 Frye et al. CLINICAL PHABMACOLOGY &THERAPEUTICS

OCTOBER 1997

45. Loft S. Metronidazole and antipyrine as probes for the study of foreign compound metabolism. Pharma- co1 Toxic01 1990;66(suppl 6):1-31.

46. Lanchote VL, Ping WC, Santos SRCJ. Influence of renal failure on cytochrome P-450 activity in hyper- tensive patients using a “cocktail” of antipyrine and nifedipine. Eur J Clin Pharmacol 1996;50:83-9.

47. Teunissen MWE, De Leede LGJ, Boeijinga JK, Breimer DD. Correlation between antipyrine metabolite formation and theophylline metabolism in humans after simultaneous single-dose administration and at steady state. J Pharmacol Exp Ther 1985;233: 770-5.

48. Groen K, Horan MA, Roberts NA, Gulati RS, Miljkovic B, Jansen EJ, et al. The relationship between phenazone (antipyrine) metabolite formation and theophylline metabolism in healthy and frail el- derly women. Clin Pharmacokinet 1993;25:136-44.

49. Back DJ, Tija J, Monig H, Ohnhaus EE, Park BK. Selective inhibition of drug oxidation after simultaneous administration of two probe drugs, antipyrine and tolbutamide. Eur J Clin Pharmacol 1988;34:157-63.

50. Truckenbrodt J, Kraul H, Huster A, Topfer R, Hen- schel L, Holfmann A, et al. Simultaneous administration of various model substances for characterizing in vivo biotransformation in chronic liver diseases. Gas- troenterol J 1990;50:179-82.

51. Lautenschlager MT, Viktor S, Muller UA, Hoffmann A. Serum concentrations of caffeine, methimazole, debrisoquine, sulfamethazine and their metabolites in diabetics before and during insulin therapy. Pharmazie 1996;51:750-3.

52. Evans WE, Relling MV, Petros WP, Meyer WH, Mirro J Jr, Crom WR. Dextromethorphan and caffeine as probes for simultaneous determination of debrisoquin-oxidation and N-acetylation phenotypes in children. Clin Pharmacol Ther 1989;45:568-73.

53. Brockmoller J, Roots I. Assessment of liver metabolic function: clinical implications. Clin Pharmacokinet 1994;27:216-48.

54. Tanaka E, Ishikawa A, Yamamoto Y, Osada A, Tsuji K, Fukao K, et al. Comparison of hepatic drug- oxidizing activity after simultaneous administration of two probe drugs, caffeine and trimethadione, to human subjects. Pharmacol Toxic01 1993;72:31-3.

55. Endres HGE, Henschel L, Merkel U, Hippius M, Hoffmann A. Lack of pharmacokinetic interaction between dextromethorphan, coumarin and mephenytoin in man after simultaneous administration. Pharmazie 1996;51:46-51.

56. Setiabudy R, Kusaka M, Chiba K, Darmansjah I, Ishizaki T. Dapsone N-acetylation, metoprolol CX- hydroxylation, and S-mephenytoin 4-hydroxylation polymorphisms in an Indonesian population: a cocktail and extended phenotyping assessment trial. Clin Pharmacol Ther 1994;56:142-53.

57. Sanz EJ, Villen T, Alm C, Bertilsson L. S- Mephenytoin hydroxylation phenotypes in a Swedish population determined after coadministration with debrisoquin. Clin Pharmacol Ther 1989;45:495-9.

58. Atiba JO, Blaschke TF, Wilkinson GR. Effects of ketoconazole on the polymorphic 4-hydroxylation of S-mephenytoin and debrisoquine. Br J Clin Pharma- co1 1989;28:161-5.

59. Guttendorf RJ, Britto M, Blouin RA, Foster TS, John W, Pittman KA, et al. Rapid screening for polymorphisms in dextromethorphan and mephenytoin metabolism. Br J Clin Pharmacol 1990;29:373-80.

60. Schellens JH, Ghabrial H, van der Wart HH, Bakker EN, Wilkinson GR, Breimer DD. Differential effects of quinidine on the disposition of nifedipine, sparteine, and mephenytoin in humans. Clin Pharma- co1 Ther 1991;50:520-8.

61. Schellens JH, Soons PA, van der Wart JH, Hoevers JW, Breimer DD. Lack of pharmacokinetic interaction between nifedipine, sparteine, and phenytoin in man. Br J Clin Pharmacol 1991;31:175-8.

62. Paolini M, Biagi GL, Bauer C, Cantelli-Forti G. Cock- tail strategy: complications and limitations. J Clin Pharmacol 1993;33:1011-2.

validation of the five-drug “pittsburgh cocktail” approach for assessment of selective...

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