This study is the first to directly compare the relative effects of duloxetine, escitalopram, and sertraline on the functional activity of the drug-metabolizing cytochrome P450 2D6 enzyme as assessed by changes in the pharmacokinetics of the cytochrome P450 2D6 model substrate drug, metoprolol. Single-dose pharmacokinetics of metoprolol were measured before and after 17 days of treatment with escitalopram 20 mg/d, duloxetine 60 mg/d, or sertraline 100 mg/d in young healthy male and female participants. The outcome measures were changes in metoprolol peak plasma levels, area under the plasma concentration-time curve, and clearance. The results were tested using paired t tests and independent t tests. The addition of each drug produced statistically significant changes in metoprolol pharmacokinetics. The rank order for the change in metoprolol area under the plasma concentration-time curve was duloxetine (180%) > escitalopram (89%) > sertraline (48% and 67%). Compared with sertraline, duloxetine produced statistically significantly larger changes in metoprolol pharmacokinetic parameters. The changes produced by escitalopram and sertraline were not statistically different.
Fluoxetine [+/--N-methyl-3-phenyl-3-[(alpha, alpha, (-trifluoro-p-tolyl)oxy]-propylamine)] a selective serotonin reuptake inhibitor, is widely used in treating depression and other serotonin-dependent disease conditions. Racemic, (R)- and (S)-fluoxetine are potent reversible inhibitors of CYP2D6, and the racemate has been shown to be a mechanism-based inhibitor of CYP3A4. Racemic fluoxetine also demonstrates time- and concentration-dependent inhibition of CYP2C19 catalytic activity in vitro. In this study, we compared fluoxetine, its (R)- and (S)-enantiomers, ticlopidine, and S-benzylnirvanol as potential time-dependent inhibitors of human liver microsomal CYP2C19. In a reversible inhibition protocol (30 min preincubation with liver microsomes without NADPH), we found (R)-, (S)- and racemic fluoxetine to be moderate inhibitors with IC(50) values of 21, 93, and 27 microM, respectively. However, when the preincubation was supplemented with NADPH, IC(50) values shifted to 4.0, 3.4, and 3.0 microM, respectively resulting in IC(50) shifts of 5.2-, 28-, and 9.3-fold. Ticlopidine showed a 1.8-fold shift in IC(50) value, and S-benzylnirvanol shifted right (0.41-fold shift). Follow-up K(I) and k(inact) determinations with fluoxetine confirmed time-dependent inhibition [K(I) values of 6.5, 47, and 14 microM; k(inact) values of 0.023, 0.085, 0.030 min(-1) for (R)-, (S)-, and racemate, respectively]. Although the (S)-isomer exhibits a much lower affinity for CYP2C19 inactivation relative to the (R)-enantiomer, it exhibits a more rapid rate of inactivation. Racemic norfluoxetine exhibited an 11-fold shift (18-1.5 microM) in IC(50) value, suggesting that conversion of fluoxetine to this metabolite represents a metabolic pathway leading to time-dependent inhibition. These data provide an improved understanding of the drug-interaction potential of fluoxetine.
Objectives Recent anecdotal, unvalidated case reports have suggested potentiation of warfarin-induced anticoagulation by cranberry juice, possibly through inhibition of human cytochrome P450 (CYP) 2C9, the enzyme responsible for the clearance of the active S-enantiomer of warfarin. To address this question, the effect of cranberry juice and other beverages on CYP2C9 activity was evaluated in vitro and in vivo. Methods The effects of 4 beverages on CYP2C9 activity were studied in human liver microsomes, by use of flurbiprofen hydroxylation as the index reaction. In a clinical study 14 healthy volunteers received 100 mg flurbiprofen on 5 occasions in a crossover fashion, with at least 1 week separating the 5 trials. Flurbiprofen was preceded in random sequence by the following: (1) cranberry juice placebo (8 oz), (2) cranberry juice (8 oz), (3) brewed tea (8 oz), (4) grape juice (8 oz), and (5) fluconazole, a CYP2C9 inhibitor serving as a positive control, with 8 oz of water. Results Flubiprofen hydroxylation in vitro was reduced to 11% ± 8% of control by 2.5% (vol/vol) brewed tea, to 10% ± 7% of control by grape juice, to 56% ± 16% of control by cranberry juice, to 85% ± 5% of control by cranberry juice placebo, and to 21% ± 6% of control by the index inhibitor sulfaphenazole (2.5 μmol/L) (P<.01 for all comparisons versus control). Flurbiprofen clearance (29–33 mL/min) and elimination half-life (3.3–3.4 hours) did not differ significantly among trials 1, 2, 3, and 4. However, clearance in the fluconazole treatment condition (trial 5) was significantly reduced compared with the placebo control (17±5 mL/min versus 31±8 mL/min, P<.05), and the half-life was prolonged (5.3 ± 1.6 hours versus 3.3 ± 0.8 hours, P<.05). Formation of 4-hydroxyflurbiprofen was correspondingly reduced by fluconazole (P<.05). Conclusions Although grape juice and tea impaired CYP2C9 activity in vitro, none of the 3 beverages altered CYP2C9-mediated clearance of flurbiprofen in humans, making a pharmacokinetic interaction with warfarin highly unlikely. Clinical Pharmacology & Therapeutics (2006) 79, 125–133; doi: 10.1016/j.clpt.2005.09.014
The antipsychotic drugs risperidone, paliperidone, olanzapine, quetiapine, aripiprazole, clozapine, haloperidol, and chlorpromazine have been reported to have various degrees of interaction (substrate or inhibitor) with the multidrug resistance transporter, P-glycoprotein (P-gp). An interaction of the antipsychotic drug loxapine with P-gp was recently reported, but an IC50 value was not determined. Loxapine (as the succinate salt) was evaluated as a P-gp substrate, and inhibitor of P-gp mediated transport of digoxin in vitro in Caco-2 cells. Loxapine was not a substrate for P-gp but did exhibit weak-to-moderate inhibition (IC50 = 9.1 μM). Since the typical steady state maximal plasma concentrations of loxapine in clinical use have been reported to be in the nanomolar range, pharmacokinetic interactions due to the inhibition of P-gp activity are not expected. Keywords: Loxapine, P-glycoprotein, Interactions, Antipsychotic drugs, P-gp activity, BCS, ABCB1, blood-brain barrier, Psychotropic drug, In vitro
The effect of Ginkgo biloba on the activity of CYP2C9, the isoform responsible for S‐warfarin clearance, was assessed in 11 healthy volunteers who received single 100‐mg doses of flurbiprofen, a probe substrate for CYP2C9. Subjects also received either a standardized G biloba leaf preparation (Ginkgold, 3 doses of 120 mg) or matching placebo in a randomized, double‐blind, 2‐way crossover study. Mean kinetic variables for flurbiprofen with either placebo or G biloba were elimination half‐life, 3.9 versus 3.5 hours; total AUC, 57 versus 55 μg/mL•h; and oral clearance, 32.9 versus 31.6 mL/min. None of these differences was significant. Based on high‐performance liquid chromatography analysis, each 60‐mg Ginkgold tablet contained 6.6 μg of amentoflavone and 61.2 μg of quercetin, both previously identified as CYP2C9 inhibitors. These amounts were apparently too low to inhibit CYP2C9 function in vivo. The results confirm previous controlled clinical studies showing no effect of ginkgo on the kinetics or dynamics of warfarin.
A P-glycoprotein (P-gp) IC50 working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC50 determinations. Each laboratory followed its in-house protocol to determine in vitro IC50 values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells—Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK1-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC50 values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC50 values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC50 values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC50 values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC50 values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC50 determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided.
Inactivation of Cytochrome P450 (CYP450) enzymes can lead to significant increases in exposure of comedicants. The majority of reported in vitro to in vivo extrapolation (IVIVE) data have historically focused on CYP3A, leaving the assessment of other CYP isoforms insubstantial. To this end, the utility of human hepatocytes (HHEP) and human liver microsomes (HLM) to predict clinically relevant drug-drug interactions was investigated with a focus on CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6. Evaluation of IVIVE for CYP2B6 was limited to only weak inhibition. A search of the University of Washington Drug-Drug Interaction Database was conducted to identify a clinically relevant weak, moderate, and strong inhibitor for selective substrates of CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6, resulting in 18 inhibitors for in vitro characterization against 119 clinical interaction studies. Pooled human hepatocytes and HLM were preincubated with increasing concentrations of inhibitors for designated timepoints. Time dependent inhibition was detected in HLM for four moderate/strong inhibitors, suggesting that some optimization of incubation conditions (i.e., lower protein concentrations) is needed to capture weak inhibition. Clinical risk assessment was conducted by incorporating the in vitro derived kinetic parameters maximal rate of enzyme inactivation (min-1) (kinact) and concentration of inhibitor resulting in 50% of the maximum enzyme inactivation (KI) into static equations recommended by regulatory authorities. Significant overprediction was observed when applying the basic models recommended by regulatory agencies. Mechanistic static models, which consider the fraction of metabolism through the impacted enzyme, using the unbound hepatic inlet concentration lead to the best overall prediction accuracy with 92% and 85% of data from HHEPs and HLM, respectively, within twofold of the observed value. SIGNIFICANCE STATEMENT: Coupling time-dependent inactivation parameters derived from pooled human hepatocytes and human liver microsomes (HLM) with a mechanistic static model provides an easy and quantitatively accurate means to determine clinical drug-drug interaction risk from in vitro data. Optimization is needed to evaluate time-dependent inhibition (TDI) for weak and moderate inhibitors using HLM. Recommendations are made with respect to input parameters for in vitro to in vivo extrapolation (IVIVE) of TDI with non-CYP3A enzymes using available data from HLM and human hepatocytes.
The sedative clomethiazole (CMZ) has been used in Europe since the mid-1960s to treat insomnia and alcoholism. It has been previously demonstrated in clinical studies to reversibly inhibit human CYP2E1 in vitro and decrease CYP2E1-mediated elimination of chlorzoxazone. We have investigated the selectivity of CMZ inhibition of CYP2E1 in pooled human liver microsomes (HLMs). In a reversible inhibition assay of the major drug-metabolizing cytochrome P450 (P450) isoforms, CYP2A6 and CYP2E1 exhibited IC50 values of 24 µM and 42 µM, respectively with all other isoforms exhibiting values >300 µM. When CMZ was preincubated with NADPH and liver microsomal protein for 30 minutes before being combined with probe substrates, however, more potent inhibition was observed for CYP2E1 and CYP2B6 but not CYP2A6 or other P450 isoforms. The substantial increase in potency of CYP2E1 inhibition upon preincubation enables the use of CMZ to investigate the role of human CYP2E1 in xenobiotic metabolism and provides advantages over other chemical inhibitors of CYP2E1. The KI and kinact values obtained with HLM-catalyzed 6-hydroxylation of chlorzoxazone were 40 µM and 0.35 minute−1, respectively, and similar to values obtained with recombinant CYP2E1 (41 µM, 0.32 minute−1). The KI and kinact values, along with other parameters, were used in a mechanistic static model to explain earlier observations of a profound decrease in the rate of chlorzoxazone elimination in volunteers despite the absence of detectable CMZ in blood.
This retrospective study investigated the influence of MDR1 haplotypes derived from the polymorphisms 2677G > T (exon 21) and 3435C > T (exon 26) on the pharmacokinetics of the immunosuppressant drug tacrolimus in 73 renal transplant patients.Based on both variants of SNPs 2677 and 3435, four different haplotypes and eight different genotypes were identified in the study sample. Tacrolimus trough concentrations (C(0)) were compared between different SNP variants and genotypes, as well as between carriers and noncarriers of each haplotype. Additionally, CYP3A5 genotype (6956G > A) was determined.No significant differences were observed between groups. Differences in mean tacrolimus C(0) values between carriers and noncarriers of each haplotype ranged from -0.04 microg/litre (95% confidence interval: -0.53 to 0.60) to -23 microg/litre (-1.07 to 1.53). No association was found between CYP3A5*1/*3 genotype and tacrolimus Co concentractions.MDR1 haplotypes derived from the SNPs 2677G > T (exon 21) and 3435C > T (exon 26) do not influence the pharmacokinetics of tacrolimus in renal transplant patients.
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