Multiple P450 substrates in a single run: rapid and comprehensive in vitro interaction assay.

The dramatically increased number of new chemical entities (NCE) used in drug discovery has raised a demand for efficient and rapid drug metabolism screening techniques. The aim of this study was to develop a global in vitro metabolic interaction screening test utilising the N-in-1 approach. A cocktail consisting of 10 CYP-selective probes with known kinetic, metabolic and interaction properties in vivo was incubated in a pool of human liver microsomes, and metabolites of melatonin (CYP1A2), coumarin (CYP2A6), bupropion (CYP2B6), amodiaquine (CYP2C8), tolbutamide (CYP2C9), omeprazole (CYP2C19 and CYP3A4), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1), midazolam (CYP3A4) and testosterone (CYP3A4) were analysed simultaneously using LC/TOF-MS. Performance of the method was assessed with cDNA expressed P450s and diagnostic CYP-specific inhibitors. The results were in good accordance with literature and our previous studies. The cocktail developed is suitable for fast and reliable in vitro screening of the interaction potential and characteristics of NCEs.     

Development and validation of probe drug cocktails for the characterization of CYP450-mediated metabolism by human heart microsomes

1. The objective of our study was to develop and validate a cocktail approach to allow the simultaneous characterization of various CYP450-mediated oxidations by human heart microsomes for nine probe drug substrates, namely, 7-ethoxyresorufin, bupropion, repaglinide, tolbutamide, bufuralol, chlorzoxazone, ebastine, midazolam and dodecanoic acid.

2. The first validation step was conducted using recombinant human CYP450 isoenzymes by comparing activity measured for each probe drug as a function of (1) buffer used, (2) selectivity towards specific isoenzymes and (3) drug interactions between probes. Activity was all measured by validated LC-MSMS methods.

3. Two cocktails were then constituted with seven of the nine drugs and subjected to kinetic validation. Finally, all probe drugs were incubated with human heart microsomes prepared from ventricular tissues obtained from 12 patients undergoing cardiac transplantation.

4. Validated cocktail #1 including bupropion, chlorzoxazone, ebastine and midazolam was used to characterize CYP2B6-, 2E1-, 2J2- and 3A5-mediated metabolism in human hearts.

5. Cocktail #2 which includes bufuralol, 7-ethoxyresorufin and repaglinide failed the validation step. Substrates in cocktail #2 as well as tolbutamide and dodecanoic acid had to be incubated separately because of their physico-chemical characteristics (solubility and ionization) or drug interactions.

6. Activity in HHM was the highest towards ebastine, chlorzoxazone and tolbutamide.     

Screening of ten cytochrome P450 enzyme activities with 12 probe substrates in human liver microsomes using cocktail incubation and liquid chromatography–tandem mass spectrometry

Testing for potential drug interactions of new chemical entities is essential when developing a novel drug. In this study, an assay was designed to evaluate drug interactions with 10 major human cytochrome P450 (P450) enzymes incubated in liver microsomes, involving 12 probe substrates with two cocktail incubation sets used in a single liquid chromatography–tandem mass spectrometry (LC–MS/MS) run. The P450 substrate composition in each cocktail set was optimized to minimize solvent effects and mutual drug interactions among substrates as follows: cocktail A was composed of phenacetin for CYP1A2, bupropion for CYP2B6, amodiaquine for CYP2C8, diclofenac for CYP2C9, S‐mephenytoin for CYP2C19, and dextromethorphan for CYP2D6; cocktail B was composed of coumarin for CYP2A6, chlorzoxazone for CYP2E1, astemizole for CYP2J2, and midazolam, nifedipine, and testosterone for CYP3A. Multiple probe substrates were used for CYP3A owing to the multiple substrate‐binding sites and substrate‐dependent inhibition. After incubation in human liver microsomes, each incubation mixture was pooled and all probe metabolites were simultaneously analysed in a single LC–MS/MS run. Polarity switching was used to acquire the negative‐ion mode for hydroxychlorzoxazone and positive‐ion mode for the remaining analytes. The method was validated by comparing the inhibition data obtained from incubation of each individual probe substrate alone and with the substrate cocktails. The half‐maximal inhibitory concentration values obtained from the cocktail and individual incubations were well correlated and in agreement with previously reported values. This new method will be useful in assessing the drug interaction potential of new chemical entities during new drug development.     

Direct and metabolism‐dependent cytochrome P450 inhibition assays for evaluating drug–drug interactions

We developed methods for evaluating the ntial inhibition of human cytochrome P450 (CYP) enzymes, including CYP1A2, CYP2A6, CYP2B6, CYP2 C9, CYP2 C19, CYP2D6, CYP2E1 and CYP3A4, using pooled human liver microsomes (HLMs). The CYP inhibition assay used substrate cocktail sets [set A: phenacetin for CYP1A2, coumarin for CYP2A6, (S)‐(+)‐mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4; set B: bupropion for CYP2B6, tolbutamide for CYP2C9, chlorzoxazone for CYP2E1, and testosterone for CYP3A4] with quantitation by liquid chromatography–tandem mass spectrometry. A direct inhibition assay was performed with the substrate cocktails without β‐nicotinamide adenine dinucleotide phosphate (NADPH) pre‐incubation, and a metabolism‐dependent inhibition (MDI) assay was performed after 30 min of pre‐incubation with NADPH in HLMs. MDI was identified based on the half‐maximal inhibitory concentration (IC₅₀) shifts. The IC₅₀ values of the direct inhibitors determined using the probe substrate cocktails were in good agreement with previously reported values. Eight metabolism‐dependent inhibitors including furafylline, 8‐methoxypsoralen, tienilic acid, ticlopidine, fluoxetine, paroxetine, disulfiram and verapamil against CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, respectively, resulted in significant IC₅₀ shifts (≥2.5‐fold) after pre‐incubation. Thus, these CYP inhibition assays are considered to be useful tools for evaluating both direct inhibition and MDI at an early stage of the drug discovery and development process. Copyright © 2011 John Wiley & Sons, Ltd.     

Investigation of the inhibition of eight major human cytochrome P450 isozymes by a probe substrate cocktail in vitro with emphasis on CYP2E1

1. A protocol has been developed and validated for the high-throughput screening of eight major human cytochrome P450 (CYP) isozymes inhibition (CYP 1A2, 2C9, 2C19, 2D6, 3A4, 2B6, 2C8 and 2E1) using an in vitro probe cocktail containing eight substrates by overcoming the unfavorable effect of assay conditions on CYP2E1 inhibition data.

2. The cocktail consisting of selective probe substrates like tacrine (CYP1A2), diclofenac (CYP2C9), S-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A4), bupropion (CYP2B6), paclitaxel (CYP2C8) and chlorzoxazone (CYP2E1) was incubated with human liver microsomes.

3. The method was investigated by incubating well-known CYP inhibitors {alphanaphthoflavone (CYP1A2), sulfaphenazole (CYP2C9), N-3-benzylnirvanol (CYP2C19), quinidine (CYP2D6), ketoconazole (CYP3A4), ticlopidine (CYP2B6), quercetin (CYP2C8) and 4-methylpyrazole (CYP2E1)} with the substrate cocktail. A fast gradient liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for this study.

4. The IC₅₀ values determined for typical CYP inhibitors were reproducible and consistent with those in the literature. DMSO has significant effect and itself inhibits CYP2E1. DMSO should not exceed 0.1% for the determination of reliable CYP2E1 inhibition profile. This cocktail assay offers an efficient and robust method to determine the CYP450 isoforms inhibition profiles of large numbers of compounds in a quick turnaround time.     

Pharmacokinetic and pharmacodynamic assessment of a five-probe metabolic cocktail for CYPs 1A2, 3A4, 2C9, 2D6 and 2E1

AIMS: The primary objectives of the present study were to establish whether there was a pharmacokinetic or pharmacodynamic interaction between the probe drugs caffeine (CYP1A2), tolbutamide (CYP2C9), debrisoquine (CYP2D6), chlorzoxazone (CYP2E1) and midazolam (CYP3A4), when administered in combination as a cocktail. Furthermore, the tolerability of these probe drugs, both alone and in combination as a cocktail was assessed. METHODS: Twelve healthy volunteer subjects (age range 22-48 years) were entered into an open, fixed sequence, 6-limb, single centre study. The randomization was such that all drugs were given individually followed by the full "cocktail" as the last treatment limb. The phenotypic index used to assess the intrinsic activity of the CYP isoforms included metabolite/parent ratios in plasma and urine (CYPs 1A2, 2E1 & 2C9), parent/metabolite ratios in urine (CYP2D6) and plasma AUClast (CYP3A4). Blood pressure and blood glucose measurements were used to assess pharmacodynamic interactions. Tolerability was assessed through reporting of adverse events RESULTS: Overall, there was little evidence that the probe drugs interacted metabolically when co-administered as the cocktail. The ratio of the geometric mean (and 90% confidence interval) of the phenotypic index, obtained after administration of the probe as part of the cocktail and when given alone were: caffeine, 0.86 (0.67-1.10), midazolam, 0.96 (0.74-1.24), tolbutamide, 0.86 (0.72-1.03), debrisoquine 1.04 (0.97-1.12) and chlorzoxazone, 0.95 (0.86-1.05). There was no difference in blood pressure and blood glucose concentrations following the cocktail and dosing of the individual probes. There was no effect on ECG recordings at any time-point. The adverse events reported for individual drug administrations were mild, transient and expected. Overall no more adverse events were reported on the cocktail study days than on the days when the drugs were administered alone. CONCLUSIONS: The five probe drugs when coadministered, in this dosing regimen, demonstrated no evidence of either a metabolic or pharmacodynamic interaction that might confound the conclusions drawn during a cocktail study. The present cocktail methodology has the potential to become a useful tool to aid the detection of clinically important drug-drug interactions during drug development.     

Influence of CYP2D6, CYP3A4, CYP3A5 and ABCB1 Polymorphisms on Pharmacokinetics and Safety of Aripiprazole in Healthy Volunteers

The aim of this study was to investigate the effect of polymorphisms in cytochrome P450 (CYP) 2D6, CYP3A4 and CYP3A5 enzymes and in P‐glycoprotein (P‐gp) on the pharmacokinetics and safety of aripiprazole and, its active metabolite, dehydro‐aripiprazole, in 148 healthy volunteers from six bioequivalence trials receiving a single oral dose of aripiprazole. The plasma concentrations of both analytes were measured by LC‐MS/MS. CYP2D6 (*3,*4,*5,*6,*7,*9 and copy number variations), CYP3A4 (*20 and *22), CYP3A5*3 and C3435T, C1236T and G2677T/A in ABCB1 gene were determined. As the number of active CYP2D6 alleles decreased, AUC_0?t, C_max and t_1/2 of aripiprazole were higher and clearance of aripiprazole, AUC_0?t of dehydro‐aripiprazole and ratio dehydro‐aripiprazole/aripiprazole were lower. AUC_0?t of aripiprazole of poor metabolizer (PM) subjects was increased by 50% compared to extensive metabolizers (EM), and AUC_0?t of dehydro‐aripiprazole was decreased by 33%. ABCB1 1236TT subjects had a lower clearance of aripiprazole (p = 0.023) and AUC_0?t (p = 0.039) and C_max of dehydro‐aripiprazole (p = 0.036) compared to C/C. CYP3A5*3/*3 subjects had a 10% lower ratio dehydro‐aripiprazole/aripiprazole than *1/*3 (p = 0.019). Adverse drug reactions (ADRs) had a directly proportional relationship with AUC_0?t of aripiprazole (p = 0.001), especially nausea/vomiting, which were more common in women (p = 0.005). Women and CYP3A5*1/*1 subjects showed more often dizziness (p = 0.034; p = 0.009). Pharmacokinetics of aripiprazole is affected by CYP2D6 phenotype but also by sex and C1236T (ABCB1 gene), while dehydro‐aripiprazole pharmacokinetics is affected by CYP2D6 and C1236T. The ratio dehydro‐aripiprazole/aripiprazole was influenced by CYP2D6 phenotype and CYP3A5*3. Concentrations of aripiprazole, sex, CYP3A5*3 and CYP2D6 were involved in the development of ADRs.     

Effects of Angelicae tenuissima radix,Angelicae dahuricae radix and Scutellariae radix Extracts on Cytochrome P450 Activities in Healthy Volunteers

Abstract: Three kinds of herbal medicines, commonly used in Korea, Angelicae tenuissima radix, Angelicae dahuricae radix and Scutellariae radix were studied to evaluate their effect on cytochrome P450 (CYP) activities in healthy volunteers. A total of 24 healthy male volunteers were assigned to one of three parallel herbal treatment groups, each consisting of eight volunteers. A cocktail of probe drugs for CYP enzymes was orally administered before and after multiple administrations of herbal medicines, three times a day for 13 days. Probe drugs used to measure CYP activities were caffeine (CYP1A2), losartan (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1) and midazolam (CYP3A4). The probe drugs and their metabolites were quantified in plasma or urine using HPLC or LC‐MS/MS. Changes in each CYP activity was evaluated by metabolic ratio of the probe drug (concentration ratio of metabolite to parent form at reference time point) following the herbal medication period, compared to the baseline values. A. dahuricae radix significantly decreased CYP1A2 activity to 10% of baseline activity (95% CI: 0.05–0.21). S. radix also showed significant changes in CYP2C9 and CYP2E1 activities. Compared to baseline values, the metabolic activities of losartan were decreased to 71% (0.54–0.94). In addition, S. radix showed a 1.42‐fold (1.03–1.97) increase in chlorzoxazone metabolic activity. However, CYP activities were not meaningfully influenced by A. tenuissima radix. Changes in certain CYP activities were observed after the administration of S. radix and A. dahuricae radix in healthy volunteers. Therefore, herbal medicines containing S. radix or A. dahuricae radix are candidates for further evaluation of clinically significant CYP‐mediated herb‐drug interactions in human beings.     

Can a cocktail designed for phenotyping pharmacokinetics and metabolism enzymes in human be used efficiently in rat?

We recently designed the CIME cocktail consisting of 10 drugs to assess the activity of the major human CYPs (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A), a phase II enzyme (UGT1A1/6/9), two drug transporters (P-gp and OATP1B1) and a component of the renal function ( Videau et al. 2010 ). The present work aimed at studying the usefulness of the CIME cocktail in the rat.The CIME cocktail was given per os to three male and three female rats, or incubated with rat liver microsomes. Parent substrates and metabolites were quantified by LC-MS/MS in plasma, urine and hepatic microsomal media, and phenotyping index were subsequently calculated.The CIME cocktail could therefore be used in the rat to phenotype rapidly and simultaneously CYP3A1/2 with omeprazole/omeprazole-sulfone, midazolam/1'-hydroxymidazolam or 4-hydroxymidazolam and/or dextromethorphan/3-methoxymorphinan, CYP2C6/11 with tolbutamide/4-hydroxytolbutamide, CYP2D1/2 with omeprazole/5-hydroxyomeprazole or dextromethorphan/dextrorphan, and UGT1A6/7 with acetaminophen/acetaminophen-glucuronide. Our results confirmed also several known gender differences and brought new information on the urinary excretion of rosuvastatin. However, the major rat CYPs, CYP2C11 and CYP2C12, are not specifically assessed. An optimized version of the CIME cocktail should therefore be designed and would be of major importance to more largely phenotype DMPK enzymes in rats to study DMPK variability factors such as disease, age, or to exposure to inductors or inhibitors.     

The influence of a newly developed quinolone: antofloxacin, on CYP activity in rats.

To investigate a newly developed quinolone antibiotics, the effect of antofloxacin hydrochloride on cytochrome P450 isoforms in rats was examined. A cocktail approach was adopted. Theophylline (CYP1A2), midazolam (CYP3A), chlorzoxazone (CYP2E1), dextromethorphan (CYP2D6), omeprazole (CYP2C19) and diclofenac (CYP2C9) were used as probes in the study, and own control was adopted. In Protocol 1, probes were given to rats simultaneously by co-administration with antofloxacin. The blood samples were obtained at designated time, and plasma concentrations of the six probes were determined by LC-MS. The pharmacokinetic parameters were calculated and compared in experimental groups in the absence and presence of antofloxacin. The result showed that the presence of antofloxacin resulted in a significant increase in theophylline values of AUC0-T and t1/2 (PAUC0-T = 0.0004 vs control Pt1/2 = 0.005 vs control), indicating that antofloxacin delayed the clearance of theophylline. In Protocol 2, the probes' pharmacokinetic parameters were compared in rats that received six probes before and after 14.5 days of consecutive administration of antofloxacin (15 mg x kg(-1), given orally, twice daily). The results suggested that the AUC0-T of chlorzoxazone was significantly decreased (P = 0.024), while that of dextromethorphan was significantly increased (P = 0.027). In conclusion, these results indicated that antofloxacin may inhibit the activity of CYP1A2, thus delaying the clearance of its substrates, and may have a slight inhibitory effect on CYP2D6 as well as an inductive effect on CYP2E1 following chronic administration.     

CYP2B6 mediates the in vitro hydroxylation of bupropion: potential drug interactions with other antidepressants.

The in vitro biotransformation of bupropion to hydroxybupropion was studied in human liver microsomes and microsomes containing heterologously expressed human cytochromes P450 (CYP). The mean (+/-S.E.) K(m) in four human liver microsomes was 89 (+/-14) microM. In microsomes containing cDNA-expressed CYPs, hydroxybupropion formation was mediated only by CYP2B6 at 50 microM bupropion (K(m) 85 microM). A CYP2B6 inhibitory antibody produced more than 95% inhibition of bupropion hydroxylation in four human livers. Bupropion hydroxylation activity at 250 microM was highly correlated with S-mephenytoin N-demethylation activity (yielding nirvanol), another CYP2B6-mediated reaction, in a panel of 32 human livers (r = 0.94). The CYP2B6 content of 12 human livers highly correlated with bupropion hydroxylation activity (r = 0.96). Thus bupropion hydroxylation is mediated almost exclusively by CYP2B6 and can serve as an index reaction reflecting activity of this isoform. IC(50) values for inhibition of a CYP2D6 index reaction (dextromethorphan O-demethylation) by bupropion and hydroxybupropion were 58 and 74 microM, respectively. This suggests a low inhibitory potency versus CYP2D6, the clinical importance of which is not established. Since bupropion is frequently coadministered with other antidepressants, IC(50) values (microM) for inhibition of bupropion hydroxylation were determined as follows: paroxetine (1.6), fluvoxamine (6.1), sertraline (3.2), desmethylsertraline (19.9), fluoxetine (59.5), norfluoxetine (4.2), and nefazodone (25.4). Bupropion hydroxylation was only weakly inhibited by venlafaxine, O-desmethylvenlafaxine, citalopram, and desmethylcitalopram. The inhibition of bupropion hydroxylation in vitro by a number of newer antidepressants suggests the potential for clinical drug interactions.     

The Role of CYP2C8 and CYP2C9 Genotypes in Losartan‐Dependent Inhibition of Paclitaxel Metabolism in Human Liver Microsomes

The aim of the present study was to further investigate a previously identified metabolic interaction between losartan and paclitaxel, which is one of the marker substrates of CYP2C8, by using human liver microsomes (HLMs) from donors with different CYP2C8 and CYP2C9 genotypes. Although CYP2C8 and CYP2C9 exhibit genetic linkage, previous studies have yet to determine whether losartan or its active metabolite, EXP‐3174 which is specifically generated by CYP2C9, is responsible for CYP2C8 inhibition. Concentrations of 6α‐hydroxypaclitaxel and EXP‐3174 were measured by high‐performance liquid chromatography after incubations with paclitaxel, losartan or EXP‐3174 in HLMs from seven donors with different CYP2C8 and CYP2C9 genotypes. The half maximal inhibitory concentration (IC₅₀) values were not fully dependent on CYP2C8 genotypes. Although the degree of inhibition was small, losartan significantly inhibited the production of 6α‐hydroxypaclitaxel at a concentration of 1 μmol/L in only HL20 with the CYP2C8*3/*3 genotype. HLMs with either CYP2C9*2/*2 or CYP2C9*1/*3 exhibited a lower losartan intrinsic clearance (V_max/K_m) than other HLMs including those with CYP2C9*1/*1 and CYP2C9*1/*2. Significant inhibition of 6α‐hydroxypaclitaxel formation by EXP‐3174 could only be found at levels that were 50 times higher (100 μmol/L) than the maximum concentration generated in the inhibition study using losartan. These results suggest that the metabolic interaction between losartan and paclitaxel is dependent on losartan itself rather than its metabolite and that the CYP2C8 inhibition by losartan is not affected by the CYP2C9 genotype. Further study is needed to define the effect of CYP2C8 genotypes on losartan–paclitaxel interaction.     

Evaluation of Mutual Drug–Drug Interaction within Geneva Cocktail for Cytochrome P450 Phenotyping using Innovative Dried Blood Sampling Method

Cytochrome P450 (CYP) activity can be assessed using a ‘cocktail’ phenotyping approach. Recently, we have developed a cocktail (Geneva cocktail) which combines the use of low‐dose probes with a low‐invasiveness dried blood spots (DBS) sampling technique and a single analytical method for the phenotyping of six major CYP isoforms. We have previously demonstrated that modulation of CYP activity after pre‐treatment with CYP inhibitors/inducer could be reliably predicted using Geneva cocktail. To further validate this cocktail, in this study, we have verified whether probe drugs contained in the latter cause mutual drug–drug interactions. In a randomized, four‐way, Latin‐square crossover study, 30 healthy volunteers received low‐dose caffeine, flurbiprofen, omeprazole, dextromethorphan and midazolam (a previously validated combination with no mutual drug–drug interactions); fexofenadine alone; bupropion alone; or all seven drugs simultaneously (Geneva cocktail). Pharmacokinetic profiles of the probe drugs and their metabolites were determined in DBS samples using both conventional micropipette sampling and new microfluidic device allowing for self‐sampling. The 90% confidence intervals for the geometric mean ratios of AUC metabolite/AUC probe for CYP probes administered alone or within Geneva cocktail fell within the 0.8–1.25 bioequivalence range indicating the absence of pharmacokinetic interaction. The same result was observed for the chosen phenotyping indices, that is metabolic ratios at 2 hr (CYP1A2, CYP3A) or 3 hr (CYP2B6, CYP2C9, CYP2C19, CYP2D6) post‐cocktail administration. DBS sampling could successfully be performed using a new microfluidic device. In conclusion, Geneva cocktail combined with an innovative DBS sampling device can be used routinely as a test for simultaneous CYP phenotyping.     

Substrate specificity for rat cytochrome P450 (CYP) isoforms: screening with cDNA-expressed systems of the rat

In this study, we performed a screening of the specificities of rat cytochrome P450 (CYP) isoforms for metabolic reactions known as the specific probes of human CYP isoforms, using 13 rat CYP isoforms expressed in baculovirus-infected insect cells or B-lymphoblastoid cells. Among the metabolic reactions studied, diclofenac 4-hydroxylation (DFH), dextromethorphan O-demethylation (DMOD) and midazolam 4-hydroxylation were specifically catalyzed by CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. These results suggest that diclofenac 4-hydroxylation, dextromethorphan O-demethylation and midazolam 4-hydroxylation are useful as catalytic markers of CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. On the other hand, phenacetin O-deethylation and 7-ethoxyresorufin O-deethylation were catalyzed both by CYP1A2 and by CYP2C6. Benzyloxyresorufin O-dealkylation and pentoxyresorufin O-dealkylation were also catalyzed by CYP1A2 in addition to CYP2B1. Bufuralol 1'-hydroxylation was extensively catalyzed by CYP2D2 but also by CYP2C6 and CYP2C11. p-Nitrophenol 2-hydroxylation and chlorzoxazone 6-hydroxylation were extensively catalyzed by CYP2E1 but also by CYP1A2 and CYP3A1. Therefore, it is necessary to conduct further study to clarify whether these activities in rat liver microsomes are useful as probes of rat CYP isoforms. In contrast, coumarin 7-hydroxylation and S- and R-mephenytoin 4'-hydroxylation did not show selectivity toward any isoforms of rat CYP studied. Therefore, activities of coumarin 7-hydroxylation and S- and R-mephenytoin 4'-hydroxylation are not able to be used as catalytic probes of CYP isoforms in rat liver microsomes. These results may provide useful information regarding catalytic probes of rat CYPs for studies using rat liver microsomal samples.     

The impact of CYP2C8 polymorphism and grapefruit juice on the pharmacokinetics of repaglinide

AIMS: The primary aim of the study was to investigate the possible effect of the CYP2C8*3 allele and of grapefruit juice on the pharmacokinetics of repaglinide. Furthermore, the impact of a single dose of grapefruit juice on the pharmacokinetics of repaglinide in relation to dose. METHODS: Thirty-six healthy male subjects, genotyped for CYP2C8*3 (11 genotyped as CYP2C8*1/*3, one as CYP2C8*3/*3 and 24 as CYP2C8*1/*1), participated in a randomized, cross-over trial. In the two phases, the subjects drank 300 mL water or 300 mL grapefruit juice, in randomized order, 2 h before administration of a single dose of either 0.25 mg or 2 mg repaglinide. RESULTS: Neither the mean AUC(0-infinity) (geometric mean ratio: 1.01; 95% CI: 0.93-1.1, P = 0.88) nor the mean C(max) (geometric mean ratio: 1.05; 95% CI: 0.94-1.2, P = 0.35) of repaglinide were statistically significantly different in the group carrying the CYP2C8*3 mutant allele compared with wild-types. Grapefruit juice caused a 19% decrease in the geometric mean ratio of the 3-hydroxyquinidine to quinidine ratio (difference: 0.81; 95% CI: 0.75-0.87, P < 0.0001), which was used as an index of CYP3A4 activity, and an increase in the mean AUC(0-infinity) of repaglinide (geometric mean ratio: 1.13; 95% CI: 1.04-1.2, P = 0.0048), but had no statistically significant effect on the t(1/2). There was no statistically significant difference in blood glucose concentration in subjects who had or had not ingested grapefruit juice. The effect was more pronounced at the low dose of repaglinide (0.25 mg) than at the therapeutic dose of 2 mg. CONCLUSIONS: The pharmacokinetics of repaglinide in subjects carrying the CYP2C8*3 mutant allele did not differ significantly from those in the wild-types. Grapefruit juice increased the bioavailability of repaglinide, suggesting significant intestinal elimination of the drug which was assumed to be primarily mediated by CYP3A4 in the gut.     

Amodiaquine, its desethylated metabolite, or both, inhibit the metabolism of debrisoquine (CYP2D6) and losartan (CYP2C9) in vivo

Objective To study the extent of in vivo inhibition by the antimalarial drug amodiaquine, its active metabolite N-desethylamodiaquine, or both, of the metabolism of four probe drugs of the enzymes CYP2D6, CYP2C19, CYP2C9 and CYP1A2.Methods Twelve healthy Swedish volunteers received a cocktail of four probe drugs (debrisoquine, omeprazole, losartan and caffeine) to determine their baseline metabolic capacities. After a washout period, they received a 600 mg oral dose of amodiaquine hydrochloride; and 2–3 h later the cocktail was administered again. One week after the intake of amodiaquine, the subjects received the cocktail a third time. The levels of probe drugs and their metabolites as well as amodiaquine and its metabolite were determined by HPLC.Results Plasma levels of amodiaquine and N-desethylamodiaquine could be followed in all subjects for 6 h and 28 days, respectively. Among the 12 subjects, a 3-fold variation in amodiaquine AUC and a 2-fold variation in N-desethylamodiaquine AUC, were observed. The CYP2D6 and CYP2C9 activities of the subjects were measured by debrisoquine and losartan phenotyping tests, respectively. There were significant mean increases in debrisoquine metabolic ratio (MR) between baseline and the second cocktail [MR_{2 h}−MR_baseline 1.426 (95% confidence interval 1.159, 1.755), P=0.002; ANOVA, Fisher LSD test] and in mean losartan MR between baseline and the second cocktail [MR_{2 h}−MR_baseline 1.724 (95% confidence interval 1.076, 2.762), P=0.026; ANOVA, Fisher LSD test]. The effects on CYP2D6 and CYP2C9 activities subsided within a week after intake of amodiaquine as tested by the phenotyping cocktail. The changes in omeprazole MRs and caffeine MRs were not statistically significant between any of the study phases.Conclusion A single dose of amodiaquine decreased CYP2D6 and CYP2C9 activities significantly compared to baseline values. Amodiaquine has the potential to cause drug-drug interactions and should be further investigated in malarial patients treated with drug combinations containing amodiaquine.     

Development of a simultaneous LC–MS/MS method to predict in vivo drug–drug interaction in mice

Cocktail substrates are useful in investigating drug–drug interactions (DDI) that can rapidly identify the cytochrome P450 (CYP) isoforms that interact with test drugs. In this study, we developed and validated five probe drugs for CYP1A, CYP2B, CYP2C, CYP2D, and CYP3A using LC–MS/MS to determine CYP activities in mice. The five probe substrates were caffeine (2 mg/kg), bupropion (30 mg/kg), omeprazole (4 mg/kg), dextromethorphan (40 mg/kg), and midazolam (2 mg/kg) for CYP1A, CYP2B, CYP2C, CYP2D, and CYP3A, respectively. The cocktail substrates were orally administered to male 5-week-old ICR mice over 0–240 min. The analytical method was validated; it showed high selectivity, linearity, and acceptable accuracy. We confirmed the lack of interaction of this cocktail in the control state (no effect of CYP inducer or inhibitor) and suggested AUC_ratio (metabolite/substrate) as a unit to evaluate DDI in vivo. In addition, the cocktail assay was applied for the determination of pharmacokinetic parameters against phenobarbital as a selective CYP2B inducer and ketoconazole as a strong CYP3A inhibitor. The concentration of cocktail substrates and the LC–MS/MS method were optimized. In conclusion, we developed a simultaneous and comprehensive analysis system for predicting potential DDI in mice.     

In vitro evaluation of valproic acid as an inhibitor of human cytochrome P450 isoforms: preferential inhibition of cytochrome P450 2C9 (CYP2C9)

AIMS: To evaluate the potency and specificity of valproic acid as an inhibitor of the activity of different human CYP isoforms in liver microsomes. METHODS: Using pooled human liver microsomes, the effects of valproic acid on seven CYP isoform specific marker reactions were measured: phenacetin O-deethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), tolbutamide hydroxylase (CYP2C9), S-mephenytoin 4'-hydroxylase (CYP2C19), dextromethorphan O-demethylase (CYP2D6), chlorzoxazone 6-hydroxylase (CYP2E1) and midazolam 1'-hydroxylase (CYP3A4). RESULTS: Valproic acid competitively inhibited CYP2C9 activity with a Ki value of 600 microM. In addition, valproic acid slightly inhibited CYP2C19 activity (Ki = 8553 microM, mixed inhibition) and CYP3A4 activity (Ki = 7975 microM, competitive inhibition). The inhibition of CYP2A6 activity by valproic acid was time-, concentration- and NADPH-dependent (KI = 9150 microM, Kinact=0.048 min(-1)), consistent with mechanism-based inhibition of CYP2A6. However, minimal inhibition of CYP1A2, CYP2D6 and CYP2E1 activities was observed. CONCLUSIONS: Valproic acid inhibits the activity of CYP2C9 at clinically relevant concentrations in human liver microsomes. Inhibition of CYP2C9 can explain some of the effects of valproic acid on the pharmacokinetics of other drugs, such as phenytoin. Co-administration of high doses of valproic acid with drugs that are primarily metabolized by CYP2C9 may result in significant drug interactions.     

Trimethadione metabolism by human liver cytochrome P450: evidence for the involvement of CYP2E1

1. Caucasian liver samples were used in this study. N-demethylation of trimethadione (TMO) to dimethadione (DMO) was monitored in the presence of chemical inhibitors of CYPs, such as fluconazole, quinidine, dimethyl-nitrosamine, acetaminophen, phenacetin, chlorzoxazone and mephenytoin. Trimethadione N-demethylation was selectively inhibited by dimethylnitrosamine and chlorzoxazone (> 50%) and weakly inhibited by tolbutamide (12%) and fluconazole (22%), whereas other inhibitors showed no effect. This result suggested that TMO metabolism to DMO is mainly mediated by CYP2E1 and marginally by CYP2C and CYP3A4. 2. Fifteen human livers were screened and interindividual variability of TMO N-demethylation activity was 3-fold. Chlorzoxazone 6-hydroxylation activity was also measured and both activities were significantly correlated (r=0.735, p < 0.01). 3. DMO production by human cDNA expressed CYP enzymes was observed mainly for CYP2E1 (10.8 nmol/tube), marginally for CYP2C8 (0.22 nmol tube) and not detectable for other CYP enzymes. 4. These results indicate that TMO metabolism is primarily catalysed by CYP2E1 and that trimethadione would be a suitable selective probe drug for the estimation of human CYP2E1 activity in vivo.     

Inhibition of CYP2D6 activity by bupropion

The purpose of this study was to assess the effect of bupropion on cytochrome P450 2D6 (CYP2D6) activity. Twenty-one subjects completed this repeated-measures study in which dextromethorphan (30-mg oral dose) was administered to smokers at baseline and after 17 days of treatment with either bupropion sustained-release (150 mg twice daily) or matching placebo. Subjects quit smoking 3 days before the second dextromethorphan administration. To assess CYP2D6 activity, urinary dextromethorphan/dextrorphan metabolic ratios were calculated after an 8-hour urine collection. Thirteen subjects received bupropion, and 8 received placebo. In those receiving active medication, the dextromethorphan/dextrorphan ratio increased significantly at the second assessment relative to the first (0.012 +/- 0.012 vs. 0.418 +/- 0.302; P < 0.0004). No such change was observed in those randomized to placebo (0.009 +/- 0.010 vs. 0.017 +/- 0.015; P = NS). At baseline, all subjects were phenotypically extensive CYP2D6 metabolizers (metabolic ratio <0.3); after treatment, 6 of 13 subjects receiving bupropion, but none of those receiving placebo, had metabolic ratios consistent with poor CYP2D6 metabolizers. Bupropion is therefore a potent inhibitor of CYP2D6 activity, and care should be exercised when initiating or discontinuing bupropion use in patients taking drugs metabolized by CYP2D6.