doi: 10.1111/j.1472-8206.2007.00471.x ORIGINAL ARTICLE Artemisinin antimalarials moderately affect cytochrome P450 enzyme activity in healthy subjects Sara Asimusa, Doaa Elsherbinyb, Trinh N Haic, Britt Janssonb, Nguyen V Huongc, Max G Petzoldd, Ulrika S.H Simonssonb, Michael Ashtona* a Unit for Pharmacokinetics and Drug Metabolism, Department of Pharmacology, Sahlgrenska Academy at Goăteborg University, Box 431, 405 30 Goăteborg, Sweden b Division of Pharmacokinetics and Drug Therapy, Department of Pharmaceutical Biosciences, Uppsala University, Box 591, 751 24 Uppsala, Sweden c National Institute of Malariology, Parasitology and Entomology, B.C 10 200 Tu-liem, Hanoi, Vietnam d Nordic School of Public Health, Box 121 33, 402 42 Goăteborg, Sweden Keywords artemisinin, cytochrome P450, induction, inhibition, malaria, metabolism Received 11 September 2006; revised 30 October 2006; accepted 14 December 2006 *Correspondence and reprints: michael.ashton@pharm.gu.se ABSTRACT The aim of this study was to investigate which principal human cytochrome P450 (CYP450) enzymes are affected by artemisinin and to what degree the artemisinin derivatives differ with respect to their respective induction and inhibition capacity Seventy-five healthy adults were randomized to receive therapeutic oral doses of artemisinin, dihydroartemisinin, arteether, artemether or artesunate for days (days 1–5) A six-drug cocktail consisting of caffeine, coumarin, mephenytoin, metoprolol, chlorzoxazone and midazolam was administered orally on days )6, 1, and 10 to assess the activities of CYP1A2, CYP2A6, CYP2C19, CYP2D6, CYP2E1 and CYP3A, respectively Four-hour plasma concentrations of parent drugs and corresponding metabolites and 7-hydroxycoumarin urine concentrations were quantified by liquid chromatography-tandem mass spectrometry The 1hydroxymidazolam/midazolam 4-h plasma concentration ratio (CYP3A) was increased on day by artemisinin [2.66-fold (98.75% CI: 2.10–3.36)], artemether [1.54 (1.14–2.09)] and dihydroartemisinin [1.25 (1.06–1.47)] compared with day )6 The S-4¢-hydroxymephenytoin/S-mephenytoin ratio (CYP2C19) was increased on day by artemisinin [1.69 (1.47–1.94)] and arteether [1.33 (1.15–1.55)] compared with day )6 The paraxanthine/caffeine ratio (CYP1A2) was decreased on day after administration of artemisinin [0.27 (0.18–0.39)], arteether [0.70 (0.55–0.89)] and dihydroartemisinin [0.73 (0.59–0.90)] compared with day )6 The a-hydroxymetoprolol/metoprolol ratio (CYP2D6) was lower on day compared with day )6 in the artemisinin [0.82 (0.70–0.96)] and dihydroartemisinin [0.83 (0.71–0.96)] groups, respectively In the artemisinin-treated subjects this decrease was followed by a 1.34-fold (1.14–1.58) increase from day to day These results show that intake of artemisinin antimalarials affect the activities of several principal human drug metabolizing CYP450 enzymes Even though not significant in all treatment groups, changes in the individual metrics were of the same direction for all the artemisinin drugs, suggesting a class effect that needs to be considered in the development of new artemisinin derivatives and combination treatments of malaria ª 2007 The Authors Journal compilation ª 2007 Blackwell Publishing Ltd Fundamental & Clinical Pharmacology 21 (2007) 307–316 307 S Asimus et al 308 INTRODUCTION The artemisinin class of endoperoxides represents a relatively new, important class of antimalarial drugs Repeated oral and rectal administration of the class parent, artemisinin, has shown remarkable timedependent pharmacokinetics in both healthy subjects and patients [1–3] Declining plasma concentrations during multiple dosing have also been reported for the derivatives artemether and, less convincingly, artesunate [4,5] A pronounced, unusual capacity for autoinduction of drug metabolism has been suggested to be the explanation of this time-dependency [6] In addition to the auto-induction phenomenon, artemisinin has also demonstrated a capacity to increase the metabolism of other drugs mediated by several different cytochrome P450 (CYP450) enzymes A twofold increase in omeprazole metabolism to 5-hydroxyomeprazole, mediated by a CYP2C19 pathway, has been reported when co-administered with artemisinin [7] An increased metabolism of S-mephenytoin to Snirvanol by CYP2B6 has also been reported [8] The induction mechanism appears to be by activation of nuclear receptors PXR and/or CAR [9] In addition, artemisinin is suggested to be an inhibitor of drug metabolism In an in vitro screening study, both artemisinin and dihydroartemisinin were found to be potent inhibitors of CYP1A2 and CYP2C19, while no inhibitory effect of artesunate was observed [10] The inhibitory effect of artemisinin on CYP1A2 activity has recently been confirmed in healthy subjects [11] New synthetic endoperoxides, such as the trioxolanes, have also shown potential for CYP450 inhibition Results from in vitro studies indicate that at least CYP3A4, CYP2C9 and CYP2C19 are inhibited by these compounds [12] It is not known to what extent a similar capacity for induction or inhibition also applies to the other derivatives in clinical use (dihydroartemisinin, its pro-drug artesunate, artemether and arteether) The effect of these drugs on the activity of the principal CYP450 enzymes involved in drug metabolism has not yet been studied With a potential for induction and inhibition of drug metabolism and the introduction of artemisinin-based combination treatment as first-line therapy of falciparum malaria, the risk for drug–drug interactions resulting in a potentiated or diminished activity of other drugs substantially increases It is therefore highly important to elucidate which principal CYP450 enzymes are affected by these drugs Administration of a probe compound has been a common way of estimating in vivo metabolic activity in humans A probe drug is selected on the basis that a quantifiable route of its metabolism is mostly or exclusively mediated by one individual enzyme A cocktail approach, which involves the administration of a number of probe substances simultaneously, offers the possibility of obtaining information about several enzymes in one study By using metabolic ratios such as the concentration of a metabolite to that of the parent drug in urine, plasma or saliva as metrics, the activity of multiple enzymes can be estimated concurrently [13–16] The aim of this study was to investigate the ability of the artemisinin class of endoperoxides to either induce and/or inhibit principal CYP450 enzymes A secondary objective was to compare the potential for drug–drug interactions within the class, as a means to select the most suitable artemisinin derivative to be a partner in combination treatment A probe cocktail consisting of caffeine, coumarin, mephenytoin, metoprolol, chlorzoxazone and midazolam was administered as single oral doses to simultaneously assess the in vivo activities of CYP1A2, CYP2A6, CYP2C19, CYP2D6, CYP2E1 and CYP3A, respectively, before and after repeated administration of artemisinin, dihydroartemisinin, arteether, artemether or artesunate MATERIALS AND METHODS Ethics The study was reviewed and approved by the Ministry of Health Hanoi, Vietnam and separately approved by the ethics committee of Goăteborg University, Goăteborg, Sweden and the Swedish Medical Products Agency, Uppsala, Sweden Investigations were conducted in accordance with the Helsinki Declaration and conformed to the standard established for Good Clinical Practices Participants were given a detailed description of the study and their written informed consent was obtained prior to study inclusion The study was carried out at the Institute of Malariology, Parasitology and Entomology, Hanoi, Vietnam in September 2002 Subjects Seventy-five healthy volunteers, 51 men and 24 women, were included in this open randomized parallel group study Seventy-two of the subjects belonged to the predominant Kinh ethnic group, the other three subjects were of Thai ethnicity The participants were all judged ª 2007 The Authors Journal compilation ª 2007 Blackwell Publishing Ltd Fundamental & Clinical Pharmacology 21 (2007) 307–316 Artemisinin drugs affect in vivo CYP450 activity to be healthy on the basis of medical history, physical examination and routine clinical laboratory determinations Subjects who had taken any antimalarial drug within month or any other drug within weeks before the start of the study and those with a history of alcohol abuse were excluded from the study Oral contraceptive use was not recorded Thirty-six of the subjects were smokers of no more than 10 cigarettes per day All smokers were men Study design The volunteers were randomized to treatment with one of five different artemisinin study drugs; artemisinin (2 · 250 mg capsules; Mediplantex, Hanoi, Vietnam), dihydroartemisinin (60 mg tablets; Mediplantex), artemether (ArtenamÒ, · 50 mg tablets; Arenco Pharmaceutica nv, Geel, Belgium), arteether (ArtecefÒ, · mL ampoules of the intramuscular preparation containing 50 mg arteether in sesame oil; Artecef BV, Maarsen, The Netherlands) or artesunate (PlasmotrimÒ, · 50 mg tablets; Mepha Ltd, Aesch, Switzerland) Repeated oral doses were administered in the morning for days (day 1–5) The cocktail of probe drugs, caffeine (Koffein RecipÒ, 100 mg tablets, Stockholm, Sweden), coumarin (5 mg capsules, Laboratory of the National Corporation of Swedish Pharmacies, Goăteborg, Sweden), midazolam (Dormicumề, 7.5 mg tablets; Roche a/s, Hvidovre, Denmark), mephenytoin (Epilan GerotÒ, 100 mg tablets; Gerot Pharmazeutica, Vienna, Austria), metoprolol (SelokenÒ, 100 mg tablets; AstraZeneca Sverige, Moălndal, Sweden) and chlorzoxazone (Paraflexề, 250 mg tablets, AstraZeneca Sverige) were given orally week before (day )6) administration of the artemisinin drugs On day and day 5, the administration of the probe drug cocktail was repeated, h after intake of the artemisinin drugs After a washout period of days (day 10), the probe drug cocktail was given again Study drug and the cocktail were each taken with 200 mL water All drug intakes were monitored by one of the study investigators The subjects fasted from the evening before blood and urine sampling (days )6, 1, and 10) and until h after last drug intake (total time of fasting approximately 12–14 h) No caffeinecontaining drinks were permitted during these days Subjects were allowed to smoke less than 10 cigarettes and to drink less than non-spirituous alcoholic beverages per day week before and during the whole study No other alcoholic drinks were permitted during the study 309 Clinical assessments A physical examination was performed on days )11 and 15 Blood for biochemical analysis (total bilirubin, creatinine, AST and ALT) and hematological tests (hemoglobin, erythrocytes and leucocytes) was taken on day )11 On this day an electrocardiogram was performed on each subject and female subjects were tested for pregnancy (Quickstick, Pharmatech, San Diego, CA, USA) Subjects were interviewed about adverse events on days and 15 The interviews were initially open, without any leading questions, and were followed by specific questions according to a checklist Blood and urine collection Probe compounds were measured in samples obtained pre-dose and at h after intake of the cocktail drugs on days )6, 1, and 10 Ten milliliters of blood was collected into a Li-heparinized Vacutainer vial (Becton Dickinson, Plymouth, UK) by venepuncture, and after centrifuged at 3000 g for after which the plasma was transferred into cryotubes (Nunc, Roskilde, Denmark) All plasma samples were immediately frozen and kept at )70 °C, until transport on dry ice to Sweden On days )6, 1, and 10, total voided urine was collected for h after administration of the cocktail drugs into receptacles containing g ascorbic acid Total weight of each urine sample voided was recorded and an aliquot was kept at )70 °C, until transportation on dry ice to Sweden Plasma assay Blank plasma was obtained from the University Hospital Blood Bank, Uppsala, Sweden The plasma was spiked with a mix of the analytes from stock solutions to get seven to nine standard concentrations and three or four quality control (QC) levels for each compound Calibration curves were constructed using linear regression of the analyte peak area (y) vs the added concentration (x) with a weighting factor of 1/y2 The curves were not forced through the origin The ranges of the standard concentrations were 30–6000 ng/mL for caffeine and paraxanthine, 40–8000 ng/mL for chlorzoxazone, 30–3000 ng/mL for 6-hydroxychlorzoxazone (6-OHchlorzoxazone), 1–100 ng/mL for 7-hydroxycoumarin (7-OH-coumarin), 3–1500 ng/mL for S-mephenytoin, 1–500 ng/mL for S-4¢-hydroxymephenytoin (S-4¢-OHmephenytoin), 5–500 ng/mL for metoprolol and 2– 200 ng/mL for a-hydroxymetoprolol (a-OH-metoprolol), midazolam and 1-hydroxymidazolam (1-OH-midazolam) ª 2007 The Authors Journal compilation ª 2007 Blackwell Publishing Ltd Fundamental & Clinical Pharmacology 21 (2007) 307–316 S Asimus et al 310 Plasma concentrations of caffeine, paraxanthine, chlorzoxazone, 6-OH-chlorzoxazone, 7-OH-coumarin, metoprolol, a-OH-metoprolol, midazolam and 1-OH-midazolam were measured in b-glucuronidase-treated samples by use of a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method according to Scott et al [17] with some modifications The system consisted of two pumps (LC-10AD; Shimadzu, Kyoto, Japan) with a high-pressure gradient mixer, an auto-sampler (Triathlon; Spark, Holland, The Netherlands) equipped with a 10 lL loop, a reversed phase column (HyPurity C18; Thermo Hypersil-Keystone, Bellefonte, PA, USA), lm particle size, 50 · 4.6 mm protected by a 10 · mm guard column of the same material, and a triple quadrupole mass spectrometer (Quattro Ultima; Micromass, Manchester, UK) MS control and spectral processing were performed using MassLynx software, version 4.0 (Micromass) The analytes were separated using a gradient at a flow rate of 800 lL/min with the mobile phase changing from 95% A (A; 0.05% formic acid in water) to 95% B (B; 70% acetonitrile in 0.05% formic acid) during 4.5 and then 0.5 later changing back to 95% A The flow from the column was split to 200 lL/min before entering the source of the mass spectrometer The unknown plasma samples, standards and QCs (250 lL) were incubated with b-glucuronidase solution (250 lL) and sodium acetate buffer pH 4.75 (500 lL) at 37 °C for h The incubated plasma (200 lL) was precipitated with acetonitrile (400 lL) by vortex mixing After centrifugation, 150 lL of the supernatant was evaporated under nitrogen at 40 °C The residue was dissolved in 250 lL mobile phase A and 10 lL was injected onto the column The plasma concentrations of S-mephenytoin and S-4¢-OH-mephenytoin were determined by a separate LC/ MS/MS method [18] The enantiomers were separated on a chiral a1-acid glycoprotein column, 150 Ã mm (Chromtech, Haăgersten, Sweden) The mobile phase consisted of 2% acetonitrile in mM ammonium acetate and the flow rate of 900 lL/min was split to 230 lL/min before entering the mass spectrometer Intra-day precision (coefficient of variation, CV), determined by analyzing six replicates of the QCs and five to six replicates of the lowest standard concentration (lower limit of quantification) according to each of the two described methods, were below 20% for all compounds and levels The inter-day precision was determined by analyzing the QCs (duplicates of ‡3 levels) interspersed with the study samples on each day during the routine analysis The median value of the inter-day precision of all QC levels was 6.5% (n ¼ 20 or 21 per compound and level) For none of the analytes was the CV above 16% Urine assay Blank urine, from five healthy volunteers, was spiked with 7-OH-coumarin to get seven standard concentrations in the range 50–10 000 ng/mL and three QC levels The urine concentrations were determined with the same LC/MS/MS method as for 7-OH-coumarin in plasma with some modification The analyte was retained on the column with a mobile phase containing 20% acetonitrile in 0.05% formic acid No gradient was used The unknown urine samples, standards and QCs (100 lL) were incubated with b-glucuronidase solution (100 lL) and sodium acetate buffer, pH 4.75 (200 lL) at 37 °C for h The incubated urine was diluted 10 times with mobile phase and 10 lL of the dilution was injected directly onto the column The intra- and inter-day precision (CV) were determined as described for the two plasma assays The intraday precision were below 4.1% and inter-day precision below 5.3% for the three QC levels (n ¼ 6) Data analysis The plasma concentration ratio of paraxanthine to caffeine at h was used to assess CYP1A2 activity [19] The total amount of 7-OH-coumarin excreted in 0- to 8-h urine was used as an index of CYP2A6 activity [20] The ability to hydroxylate S-mephenytoin (CYP2C19) was estimated by the S-4¢-OH-mephenytoin to S-mephenytoin plasma ratio at h post-dose The 4-h a-OH-metoprolol to metoprolol ratio and 6-OH-chlorzoxazone to chlorzoxazone ratio were used to evaluate the activities of CYP2D6 and CYP2E1, respectively [13,21] The concentration of 1-OH-midazolam divided by that of midazolam in the 4-h plasma sample was used to indicate CYP3A activity [22] The metrics for the individual enzyme activities were calculated from metabolite and drug concentrations obtained on days )6, 1, and 10 For comparison of enzyme activity between study days the following four contrasts were estimated in the statistical evaluation; day vs day )6 (day 1/day )6), day vs day )6 (day 5/day )6), day vs day (day 5/day 1) and day 10 vs day )6 (day 10/day )6) Statistical analysis A repeated ANOVA model with Gaussian random effects was applied to the log-transformed (natural base) data ª 2007 The Authors Journal compilation ª 2007 Blackwell Publishing Ltd Fundamental & Clinical Pharmacology 21 (2007) 307–316 Artemisinin drugs affect in vivo CYP450 activity 311 same ratio on day compared with day )6, and in the artesunate group the ratio was significantly increased on day 10 compared with day )6 (1.26, 1.01–1.57) A significant increase in the mean 4-h S-4¢-OHmephenytoin/S-mephenytoin plasma concentration ratio (CYP2C19) was observed from day )6 to day in two of the five treatment groups; artemisinin 1.69-fold (1.47–1.94) and arteether 1.33 (1.15–1.55) (Figure 1) Nine individuals had no measurable S-4¢-OH-mephenytoin concentrations and were considered to be poor metabolizers of CYP2C19, and hence excluded from the data analysis Intake of artemisinin, dihydroartemisinin and arteether significantly decreased the mean 4-h paraxanthine/caffeine plasma concentration ratio The mean ratios decreased to 0.27 (0.18–0.39), 0.70 (0.55–0.89) and 0.73 (0.59–0.90) on day compared with day )6, in subjects receiving artemisinin, arteether and dihydroartemisinin groups, respectively A significant 2.22fold increase (1.54–3.21) was observed in the CYP1A2 metric day compared with day after multiple administration of artemisinin Residual concentrations of caffeine and paraxanthine were found in the pre-dose samples from many subjects The marker for CYP2D6 activity, a-OH-metoprolol/ metoprolol concentration ratio at h, significantly decreased to 0.82 (0.70–0.96) and 0.83 (0.71–0.96) day compared with day )6 in the artemisinin and dihydroartemisinin groups, respectively A significant 1.34-fold increase (1.14–1.58) was found in the same ratio from day to day in the artemisinin group Two subjects, who had undetectable a-OH-metoprolol concentrations, were considered to be poor metabolizers of CYP2D6 and excluded from the data analysis with subsequent model-diagnostics by checking residual plots An overall test level of 5% for the multiple (four) tests per treatment group was selected In accordance with the Bonferroni-method for multiple testing, 98.75% confidence intervals (CI) are presented and P-values are compared to 0.0125 in the sequel The Proc Mixed in SAS 8.2 (SAS Company Inc., Cary, NC, USA) software was used for the analysis Results are presented as quotients (mean, 98.75% CI) based on anti-logarithms of the contrasts for the different occasions Quotients larger, or smaller, than unity may be associated with enzyme induction or inhibition, respectively RESULTS Subject demographic characteristics are summarized in Table I Seventy-four subjects completed the study One subject discontinued the study due to nausea on the first day of cocktail drug intake Apart from a brief period of sleepiness and dizziness after administration of the cocktail drugs in many subjects, the cocktail procedure was well tolerated On the fifth day of artemether intake, one subject reported a skin reaction (rash) of moderate severity, which gradually cleared within weeks The effects of the artemisinin antimalarials on the major CYP450 enzymes are summarized in Table II Five days intake of artemisinin, artemether and dihydroartemisinin significantly increased the index for CYP3A activity Day compared with day )6, the mean 1-OHmidazolam/midazolam concentration ratio at h increased 2.66-fold (CI: 2.10–3.36), 1.54 (1.14–2.09) and 1.25 (1.06–1.47) by artemisinin, artemether and dihydroartemisinin, respectively In the artemisinin group a 1.60-fold (1.26–2.02) increase was seen in the Table I Subject demographics Artemisinin Dihydroartemisinin Arteether Artemether Artesunate (n ¼ 15) (n ¼ 14) (n ¼ 15) (n ¼ 15) (n ¼ 15) Gender 13/1 13/2 Agea (years) Male/female 28 ± (20–44) 7/8 30 ± (20–45) 25 ± (18–40) 31 ± (21–45) 8/7 10/5 30 ± (18–45) Body weighta (kg) 52 ± (41–62) 57 ± (47–71) 53 ± (45–65) 55 ± (43–68) 56 ± (45–69) Number of smokers (n) 8 Average number of daily cigarettes among smokers 6 15/0 13/1 14/1 15/0 14/1 2/0 1/1 1/0 2/1 3/0 Ethnic group Kinhb/Thai Number of poor metabolizersc of CYP2C19/CYP2D6 a Mean values ± SD (range) b c The most dominant ethnic group in Vietnam Defined as subjects with no measurable metabolite concentrations ª 2007 The Authors Journal compilation ª 2007 Blackwell Publishing Ltd Fundamental & Clinical Pharmacology 21 (2007) 307–316 S Asimus et al 312 Table II Phenotyping metrics in the five different treatment groups [artemisinin (ART), dihydroartemisinin (DHA), arteether (ARE), artemether (ARM) and artesunate (AS)] The presented quotients (mean, 98.75% CI) are based on anti-logarithms of the contrast for the different occasions Quotients of metric for Enzyme Phenotyping metric different occasionsb ART CYP1A2 Paraxanthine/caffeine day 1/day )6 0.27 (0.18–0.39)a 0.73 (0.59–0.90)a 0.70 (0.55–0.89)a 0.83 (0.69–1.02) 4-h concentration day 5/day )6 0.59 (0.41–0.85)a 0.85 (0.69–1.06) 0.70 (0.55–0.89)a 0.81 (0.67–0.98)a 1.00 (0.80–1.26) ratio day 5/day 2.22 (1.54–3.21)a 1.17 (0.95–1.45) 1.00 (0.78–1.27) 0.97 (0.80–1.18) 1.16 (0.92–1.45) day 10/day )6 1.26 (0.88–1.81) 0.94 (0.76–1.16) 0.84 (0.66–1.06) 1.06 (0.87–1.30) 1.10 (0.88–1.38) 7-OH-coumarin excreted day 1/day )6 0.74 (0.40–1.40) 1.17 (0.73–1.88) 0.81 (0.38–1.71) 1.01 (0.63–1.62) 0.73 (0.38–1.44) day 5/day )6 0.87 (0.48–1.60) 1.34 (0.84–2.14) 0.95 (0.45–2.02) 0.91 (0.57–1.45) 0.60 (0.30–1.17) day 5/day 1.17 (0.62–2.23) 1.15 (0.71–1.85) 1.18 (0.56–2.51) 0.90 (0.56–1.44) 0.81 (0.41–1.61) day 10/day )6 0.96 (0.53–1.74) 1.38 (0.87–2.19) 1.17 (0.55–2.47) 1.22 (0.77–1.94) 0.86 (0.44–1.68) 0.97 (0.78–1.21) 0.93 (0.80–1.08) CYP2A6 in 0- to 8-h urine CYP3A ARM AS 0.87 (0.69–1.09) day 1/day )6 0.95 (0.83–1.09) 0.95 (0.79–1.14) 0.91 (0.73–1.14) day 5/day )6 1.69 (1.47–1.94)a 1.16 (0.93–1.44) 1.33 (1.15–1.55)a 1.20 (1.00–1.44) 1.12 (0.89–1.40) concentration ratio day 5/day 1.77 (1.54–2.04)a 1.19 (0.96–1.49) 1.44 (1.24–1.67)a 1.26 (1.05–1.52)a 1.22 (0.98–1.53) day 10/day )6 CYP2E1 ARE S-mephenytoin 4-h CYP2C19 S-4¢-OH-mephenytoin/ CYP2D6 DHA 1.65 (1.44–1.88) a a 1.13 (0.91–1.41) 1.26 (1.08–1.46)a 1.14 (0.94–1.38) 1.18 (0.94–1.49) 0.89 (0.75–1.05) 0.90 (0.76–1.05) 0.90 (0.79–1.04) a)OH-metoprolol/ day 1/day )6 0.82 (0.70–0.96) metoprolol 4-h day 5/day )6 1.10 (0.94–1.29) 0.95 (0.81–1.10) 1.02 (0.86–1.21) 0.97 (0.82–1.13) 1.02 (0.89–1.18) concentration ratio day 5/day 1.34 (1.14–1.58)a 1.14 (0.99–1.33) 1.15 (0.97–1.37) 1.08 (0.92–1.27) 1.13 (0.99–1.30) 0.83 (0.71–0.96) a day 10/day )6 1.15 (0.98–1.34) 0.93 (0.80–1.08) 0.98 (0.83–1.17) 0.92 (0.78–1.09) 1.07 (0.93–1.24) 6-OH-chlorzoxazone/ day 1/day )6 0.68 (0.54–0.86)a 0.93 (0.66–1.31) 1.13 (0.84–1.51) 1.06 (0.85–1.33) 0.96 (0.73–1.26) chlorzoxazone 4-h day 5/day )6 0.74 (0.58–0.94)a 1.00 (0.70–1.41) 0.99 (0.74–1.32) 1.08 (0.86–1.35) 1.09 (0.83–1.43) concentration ratio day 5/day 1.08 (0.85–1.38) 1.07 (0.76–1.52) 0.88 (0.66–1.17) 1.02 (0.81–1.28) 1.13 (0.86–1.48) day 10/day )6 0.90 (0.71–1.14) 0.83 (0.59–1.17) 1.05 (0.78–1.42) 1.07 (0.85–1.35) 1.03 (0.79–1.36) 1-OH-midazolam/ day 1/day )6 1.60 (1.26–2.02)a 1.11 (0.94–1.30) 0.97 (0.79–1.20) 1.22 (0.90–1.65) 1.17 (0.94–1.47) midazolam 4-h day 5/day )6 2.66 (2.10–3.36)a 1.25 (1.06–1.47)a 1.16 (0.94–1.43) 1.54 (1.14–2.09)a 1.25 (1.00–1.56 concentration ratio day 5/day 1.67 (1.31–2.12)a 1.13 (0.96–1.33) 1.19 (0.97–1.47) 1.27 (0.93–1.72) 1.06 (0.85–1.33) day 10/day )6 1.25 (0.99–1.58) 1.12 (0.90–1.38) 1.15 (0.84–1.57) 1.26 (1.01–1.57)a 1.16 (0.98–1.36) a P < 0.0125 (a adjusted for multiple testing) Quotients >1 indicate increased enzyme activity, quotients