Solanidine is a sensitive and specific dietary biomarker for CYP2D6 activity.

BACKGROUND: Individual assessment of CYP enzyme activities can be challenging. Recently, the potato alkaloid solanidine was suggested as a biomarker for CYP2D6 activity. Here, we aimed to characterize the sensitivity and specificity of solanidine as a CYP2D6 biomarker among Finnish volunteers with known CYP2D6 genotypes. RESULTS: Using non-targeted metabolomics analysis, we identified 9152 metabolite features in the fasting plasma samples of 356 healthy volunteers. Machine learning models suggested strong association between CYP2D6 genotype-based phenotype classes with a metabolite feature identified as solanidine. Plasma solanidine concentration was 1887% higher in genetically poor CYP2D6 metabolizers (gPM) (n = 9; 95% confidence interval 755%, 4515%; P = 1.88 × 10-11), 74% higher in intermediate CYP2D6 metabolizers (gIM) (n = 89; 27%, 138%; P = 6.40 × 10-4), and 35% lower in ultrarapid CYP2D6 metabolizers (gUM) (n = 20; 64%, - 17%; P = 0.151) than in genetically normal CYP2D6 metabolizers (gNM; n = 196). The solanidine metabolites m/z 444 and 430 to solanidine concentration ratios showed even stronger associations with CYP2D6 phenotypes. Furthermore, the areas under the receiver operating characteristic and precision-recall curves for these metabolic ratios showed equal or better performances for identifying the gPM, gIM, and gUM phenotype groups than the other metabolites, their ratios to solanidine, or solanidine alone. In vitro studies with human recombinant CYP enzymes showed that solanidine was metabolized mainly by CYP2D6, with a minor contribution from CYP3A4/5. In human liver microsomes, the CYP2D6 inhibitor paroxetine nearly completely (95%) inhibited the metabolism of solanidine. In a genome-wide association study, several variants near the CYP2D6 gene associated with plasma solanidine metabolite ratios. CONCLUSIONS: These results are in line with earlier studies and further indicate that solanidine and its metabolites are sensitive and specific biomarkers for measuring CYP2D6 activity. Since potato consumption is common worldwide, this biomarker could be useful for evaluating CYP2D6-mediated drug-drug interactions and to improve prediction of CYP2D6 activity in addition to genotyping.

Screening of 16 major drug glucuronides for time-dependent inhibition of nine drug-metabolizing CYP enzymes - detailed studies on CYP3A inhibitors.

Time-dependent inhibition of cytochrome P450 (CYP) enzymes has been observed for a few glucuronide metabolites of clinically used drugs. Here, we investigated the inhibitory potential of 16 glucuronide metabolites towards nine major CYP enzymes in vitro. Automated substrate cocktail methods were used to screen time-dependent inhibition of CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2J2 and 3A in human liver microsomes. Seven glucuronides (carvedilol ß-D-glucuronide, diclofenac acyl-ß-D-glucuronide, 4-hydroxyduloxetine ß-D-glucuronide, ezetimibe phenoxy-ß-D-glucuronide, raloxifene 4'-glucuronide, repaglinide acyl-ß-D-glucuronide and valproic acid ß-D-glucuronide) caused NADPH- and time-dependent inhibition of at least one of the CYPs investigated, including CYP2A6, CYP2C19 and CYP3A. In more detailed experiments, we focused on the glucuronides of carvedilol and diclofenac, which inhibited CYP3A. Carvedilol ß-D-glucuronide showed weak time-dependent inhibition of CYP3A, but the parent drug carvedilol was found to be a more potent inhibitor of CYP3A, with the half-maximal inhibitor concentration (IC50) decreasing from 7.0 to 1.1 µM after a 30-minute preincubation with NADPH. The maximal inactivation constant (kinact) and the inhibitor concentration causing half of kinact (KI) for CYP3A inactivation by carvedilol were 0.051 1/min and 1.8 µM, respectively. Diclofenac acyl-ß-D-glucuronide caused time-dependent inactivation of CYP3A at high concentrations, with a 4-fold IC50 shift (from 400 to 98 µM after a 30-minute preincubation with NADPH), and KI and kinact values of >2000 µM and >0.16 1/min. In static predictions, carvedilol caused significant (>1.25-fold) increase in the exposure of the CYP3A substrates midazolam and simvastatin. In conclusion, we identified several glucuronide metabolites with CYP inhibitory properties. Based on detailed experiments, the inactivation of CYP3A by carvedilol may cause clinically significant drug-drug interactions.

Ticagrelor Increases Exposure to the Breast Cancer Resistance Protein Substrate Rosuvastatin.

Ticagrelor and rosuvastatin are often used concomitantly after atherothrombotic events. Several cases of rhabdomyolysis during concomitant ticagrelor and rosuvastatin have been reported, suggesting a drug-drug interaction. We showed recently that ticagrelor inhibits breast cancer resistance protein (BCRP) and organic anion transporting polypeptide (OATP) 1B1, 1B3, and 2B1-mediated rosuvastatin transport in vitro. The aim of this study was to investigate the effects of ticagrelor on rosuvastatin pharmacokinetics in humans. In a randomized, crossover study, 9 healthy volunteers ingested a single dose of 90 mg ticagrelor or placebo, followed by a single 10 mg dose of rosuvastatin 1 hour later. Ticagrelor 90 mg or placebo were additionally administered 12, 24, and 36 hours after their first dose. Ticagrelor increased rosuvastatin area under the plasma concentration-time curve (AUC) and peak plasma concentration 2.6-fold (90% confidence intervals: 1.8-3.8 and 1.7-4.0, P = 0.001 and P = 0.003), and prolonged its half-life from 3.1 to 6.6 hours (P = 0.009). Ticagrelor also decreased the renal clearance of rosuvastatin by 11% (3%-19%, P = 0.032). The N-desmethylrosuvastatin:rosuvastatin AUC0-10h ratio remained unaffected by ticagrelor. Ticagrelor had no effect on the plasma concentrations of the endogenous OATP1B substrates glycodeoxycholate 3-O-glucuronide, glycochenodeoxycholate 3-O-glucuronide, glycodeoxycholate 3-O-sulfate, and glycochenodeoxycholate 3-O-sulfate, or the sodium-taurocholate cotransporting polypeptide substrate taurocholic acid. These data indicate that ticagrelor increases rosuvastatin concentrations more than twofold in humans, probably mainly by inhibiting intestinal BCRP. Because the risk for rosuvastatin-induced myotoxicity increases along with rosuvastatin plasma concentrations, using ticagrelor concomitantly with high doses of rosuvastatin should be avoided.

Real-world pharmacogenetics of statin intolerance: effects of SLCO1B1, ABCG2 , and CYP2C9 variants.

OBJECTIVE: The association of SLCO1B1 c.521T>C with simvastatin-induced muscle toxicity is well characterized. However, different statins are subject to metabolism and transport also by other proteins exhibiting clinically meaningful genetic variation. Our aim was to investigate associations of SLCO1B1 c.521T>C with intolerance to atorvastatin, fluvastatin, pravastatin, rosuvastatin, or simvastatin, those of ABCG2 c.421C>A with intolerance to atorvastatin, fluvastatin, or rosuvastatin, and that of CYP2C9*2 and *3 alleles with intolerance to fluvastatin. METHODS: We studied the associations of these variants with statin intolerance in 2042 patients initiating statin therapy by combining genetic data from samples from the Helsinki Biobank to clinical chemistry and statin purchase data. RESULTS: We confirmed the association of SLCO1B1 c.521C/C genotype with simvastatin intolerance both by using phenotype of switching initial statin to another as a marker of statin intolerance [hazard ratio (HR) 1.88, 95% confidence interval (CI) 1.08-3.25, P  = 0.025] and statin switching along with creatine kinase measurement (HR 5.44, 95% CI 1.49-19.9, P  = 0.011). No significant association was observed with atorvastatin and rosuvastatin. The sample sizes for fluvastatin and pravastatin were relatively small, but SLCO1B1 c.521T>C carriers had an increased risk of pravastatin intolerance defined by statin switching when compared to homozygous reference T/T genotype (HR 2.11, 95% CI 1.01-4.39, P  = 0.047). CONCLUSION: The current results can inform pharmacogenetic statin prescribing guidelines and show feasibility for the methodology to be used in larger future studies.

Rhabdomyolysis during concomitant ticagrelor and rosuvastatin: A breast cancer resistance protein-mediated drug interaction?

We present 3 patients diagnosed with rhabdomyolysis 1-6 months after the initiation of concomitant rosuvastatin and ticagrelor medication. A literature review and Food and Drug Administration adverse event reporting system revealed >40 reports of rhabdomyolysis during concomitant ticagrelor and rosuvastatin, including 3 with a fatal outcome. We show that ticagrelor inhibits breast cancer resistance protein-, organic anion transporting polypeptide (OATP) 1B1-, 1B3- and 2B1-mediated transport of rosuvastatin in vitro with half-maximal unbound inhibitory concentrations of 0.36, 4.13, 7.5 and 3.26 µM, respectively. A static drug interaction model predicted that ticagrelor may inhibit intestinal breast cancer resistance protein and thus increase rosuvastatin plasma exposure 2.1-fold, whereas the OATP-mediated hepatic uptake of rosuvastatin should not be inhibited due to relatively low portal ticagrelor concentrations. Taken together, concomitant use of ticagrelor with rosuvastatin may increase the systemic exposure to rosuvastatin and the risk of rosuvastatin-induced rhabdomyolysis. Further studies are warranted to investigate the potential pharmacokinetic interaction between ticagrelor and rosuvastatin in humans.

Hydroxychloroquine is Metabolized by Cytochrome P450 2D6, 3A4, and 2C8, and Inhibits Cytochrome P450 2D6, while its Metabolites also Inhibit Cytochrome P450 3A in vitro.

This study aimed to explore the cytochrome P450 (CYP) metabolic and inhibitory profile of hydroxychloroquine (HCQ). Hydroxychloroquine metabolism was studied using human liver microsomes (HLMs) and recombinant CYP enzymes. The inhibitory effects of HCQ and its metabolites on nine CYPs were also determined in HLMs, using an automated substrate cocktail method. Our metabolism data indicated that CYP3A4, CYP2D6, and CYP2C8 are the key enzymes involved in HCQ metabolism. All three CYPs formed the primary metabolites desethylchloroquine (DCQ) and desethylhydroxychloroquine (DHCQ) to various degrees. Although the intrinsic clearance (CLint) value of HCQ depletion by recombinant CYP2D6 was > 10-fold higher than that by CYP3A4 (0.87 versus 0.075 µl/min/pmol), scaling of recombinant CYP CLint to HLM level resulted in almost equal HLM CLint values for CYP2D6 and CYP3A4 (11 and 14 µl/min/mg, respectively). The scaled HLM CLint of CYP2C8 was 5.7 µl/min/mg. Data from HLM experiments with CYP-selective inhibitors also suggested relatively equal roles for CYP2D6 and CYP3A4 in HCQ metabolism, with a smaller contribution by CYP2C8. In CYP inhibition experiments, HCQ, DCQ, DHCQ, and the secondary metabolite didesethylchloroquine were direct CYP2D6 inhibitors, with 50% inhibitory concentration (IC50) values between 18 and 135 µM. HCQ did not inhibit other CYPs. Furthermore, all metabolites were time-dependent CYP3A inhibitors (IC50 shift 2.2-3.4). To conclude, HCQ is metabolized by CYP3A4, CYP2D6, and CYP2C8 in vitro. HCQ and its metabolites are reversible CYP2D6 inhibitors, and HCQ metabolites are time-dependent CYP3A inhibitors. These data can be used to improve physiologically-based pharmacokinetic models and update drug-drug interaction risk estimations for HCQ. SIGNIFICANCE STATEMENT: While CYP2D6, CYP3A4, and CYP2C8 have been shown to mediate chloroquine biotransformation, it appears that the role of CYP enzymes in hydroxychloroquine (HCQ) metabolism has not been studied. In addition, little is known about the CYP inhibitory effects of HCQ. Here, we demonstrate that CYP2D6, CYP3A4, and CYP2C8 are the key enzymes involved in HCQ metabolism. Furthermore, our findings show that HCQ and its metabolites are inhibitors of CYP2D6, which likely explains the previously observed interaction between HCQ and metoprolol.

Translational aspects of cytochrome P450-mediated drug-drug interactions: A case study with clopidogrel.

Multimorbidity, polypharmacotherapy and drug interactions are increasingly common in the ageing population. Many drug-drug interactions (DDIs) are caused by perpetrator drugs inhibiting or inducing cytochrome P450 (CYP) enzymes, resulting in alterations of the plasma concentrations of a victim drug. DDIs can have a major negative health impact, and in the past, unrecognized DDIs have resulted in drug withdrawals from the market. Signals to investigate DDIs may emerge from a variety of sources. Nowadays, standard methods are widely available to identify and characterize the mechanisms of CYP-mediated DDIs in vitro. Clinical pharmacokinetic studies, in turn, provide experimental data on pharmacokinetic outcomes of DDIs. Physiologically based pharmacokinetic (PBPK) modelling utilizing both in vitro and in vivo data is a powerful tool to predict different DDI scenarios. Finally, epidemiological studies can provide estimates on the health outcomes of DDIs. Thus, to fully characterize the mechanisms, clinical effects and implications of CYP-mediated DDIs, translational research approaches are required. This minireview provides an overview of translational approaches to study CYP-mediated DDIs, going beyond regulatory DDI guidelines, and an illustrative case study of how the DDI potential of clopidogrel was unveiled by combining these different methods.

Comparative Hepatic and Intestinal Efflux Transport of Statins.

Previous studies have shown that lipid-lowering statins are transported by various ATP-binding cassette (ABC) transporters. However, because of varying methods, it is difficult to compare the transport profiles of statins. Therefore, we investigated the transport of 10 statins or statin metabolites by six ABC transporters using human embryonic kidney cell-derived membrane vesicles. The transporter protein expression levels in the vesicles were quantified with liquid chromatography-tandem mass spectrometry and used to scale the measured clearances to tissue levels. In our study, apically expressed breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) transported atorvastatin, fluvastatin, pitavastatin, and rosuvastatin. Multidrug resistance-associated protein 3 (MRP3) transported atorvastatin, fluvastatin, pitavastatin, and, to a smaller extent, pravastatin. MRP4 transported fluvastatin and rosuvastatin. The scaled clearances suggest that BCRP contributes to 87%-91% and 84% of the total active efflux of rosuvastatin in the small intestine and the liver, respectively. For atorvastatin, the corresponding values for P-gp-mediated efflux were 43%-79% and 66%, respectively. MRP3, on the other hand, may contribute to 23%-26% and 25%-37% of total active efflux of atorvastatin, fluvastatin, and pitavastatin in jejunal enterocytes and liver hepatocytes, respectively. These data indicate that BCRP may play an important role in limiting the intestinal absorption and facilitating the biliary excretion of rosuvastatin and that P-gp may restrict the intestinal absorption and mediate the biliary excretion of atorvastatin. Moreover, the basolateral MRP3 may enhance the intestinal absorption and sinusoidal hepatic efflux of several statins. Taken together, the data show that statins differ considerably in their efflux transport profiles. SIGNIFICANCE STATEMENT: This study characterized and compared the transport of atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin acid and four atorvastatin metabolites by six ABC transporters (BCRP, MRP2, MRP3, MRP4, MRP8, P-gp). Based on in vitro findings and protein abundance data, the study concludes that BCRP, MRP3, and P-gp have a major impact in the efflux of various statins. Together with in vitro metabolism, uptake transport, and clinical data, our findings are applicable for use in comparative systems pharmacology modeling of statins.

Comparative Hepatic and Intestinal Metabolism and Pharmacodynamics of Statins.

This study aimed to comprehensively investigate the in vitro metabolism of statins. The metabolism of clinically relevant concentrations of atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, and their metabolites were investigated using human liver microsomes (HLMs), human intestine microsomes (HIMs), liver cytosol, and recombinant cytochrome P450 enzymes. We also determined the inhibitory effects of statin acids on their pharmacological target, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. In HLMs, statin lactones were metabolized to a much higher extent than their acid forms. Atorvastatin lactone and simvastatin (lactone) showed extensive metabolism [intrinsic clearance (CLint) values of 3700 and 7400 µl/min per milligram], whereas the metabolism of the lactones of 2-hydroxyatorvastatin, 4-hydroxyatorvastatin, and pitavastatin was slower (CLint 20-840 µl/min per milligram). The acids had CLint values in the range <0.1-80 µl/min per milligram. In HIMs, only atorvastatin lactone and simvastatin (lactone) exhibited notable metabolism, with CLint values corresponding to 20% of those observed in HLMs. CYP3A4/5 and CYP2C9 were the main statin-metabolizing enzymes. The majority of the acids inhibited HMG-CoA reductase, with 50% inhibitory concentrations of 4-20 nM. The present comparison of the metabolism and pharmacodynamics of the various statins using identical methods provides a strong basis for further application, e.g., comparative systems pharmacology modeling. SIGNIFICANCE STATEMENT: The present comparison of the in vitro metabolic and pharmacodynamic properties of atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin and their metabolites using unified methodology provides a strong basis for further application. Together with in vitro drug transporter and clinical data, the present findings are applicable for use in comparative systems pharmacology modeling to predict the pharmacokinetics and pharmacological effects of statins at different dosages.

An automated cocktail method for in vitro assessment of direct and time-dependent inhibition of nine major cytochrome P450 enzymes - application to establishing CYP2C8 inhibitor selectivity.

We developed an in vitro high-throughput cocktail assay with nine major drug-metabolizing CYP enzymes, optimized for screening of time-dependent inhibition. The method was applied to determine the selectivity of the time-dependent CYP2C8 inhibitors gemfibrozil 1-O-ß-glucuronide and clopidogrel acyl-ß-D-glucuronide. In vitro incubations with CYP selective probe substrates and pooled human liver microsomes were conducted in 96-well plates with automated liquid handler techniques and metabolite concentrations were measured with quantitative UHPLC-MS/MS analysis. After determination of inter-substrate interactions and Km values for each reaction, probe substrates were divided into cocktails I (tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4/5) and II (coumarin/CYP2A6, S-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2). Time-dependent inhibitors (furafylline/CYP1A2, selegiline/CYP2A6, clopidogrel/CYP2B6, gemfibrozil 1-O-ß-glucuronide/CYP2C8, tienilic acid/CYP2C9, ticlopidine/CYP2C19, paroxetine/CYP2D6 and ritonavir/CYP3A) and direct inhibitor (terfenadine/CYP2J2) showed similar inhibition with single substrate and cocktail methods. Established time-dependent inhibitors caused IC50 fold shifts ranging from 2.2 to 30 with the cocktail method. Under time-dependent inhibition conditions, gemfibrozil 1-O-ß-glucuronide was a strong (>90% inhibition) and selective (<< 20% inhibition of other CYPs) inhibitor of CYP2C8 at concentrations ranging from 60 to 300 µM, while the selectivity of clopidogrel acyl-ß-D-glucuronide was limited at concentrations above its IC80 for CYP2C8. The time-dependent IC50 values of these glucuronides for CYP2C8 were 8.1 and 38 µM, respectively. In conclusion, a reliable cocktail method including the nine most important drug-metabolizing CYP enzymes was developed, optimized and validated for detecting time-dependent inhibition. Moreover, gemfibrozil 1-O-ß-glucuronide was established as a selective inhibitor of CYP2C8 for use as a diagnostic inhibitor in in vitro studies.

Improved predictions of time-dependent drug-drug interactions by determination of cytosolic drug concentrations.

The clinical impact of drug-drug interactions based on time-dependent inhibition of cytochrome P450 (CYP) 3A4 has often been overpredicted, likely due to use of improper inhibitor concentration estimates at the enzyme. Here, we investigated if use of cytosolic unbound inhibitor concentrations could improve predictions of time-dependent drug-drug interactions. First, we assessed the inhibitory effects of ten time-dependent CYP3A inhibitors on midazolam 1'-hydroxylation in human liver microsomes. Then, using a novel method, we determined the cytosolic bioavailability of the inhibitors in human hepatocytes, and used the obtained values to calculate their concentrations at the active site of the enzyme, i.e. the cytosolic unbound concentrations. Finally, we combined the data in mechanistic static predictions, by considering different combinations of inhibitor concentrations in intestine and liver, including hepatic concentrations corrected for cytosolic bioavailability. The results were then compared to clinical data. Compared to no correction, correction for cytosolic bioavailability resulted in higher accuracy and precision, generally in line with those obtained by more demanding modelling. The best predictions were obtained when the inhibition of hepatic CYP3A was based on unbound maximal inhibitor concentrations corrected for cytosolic bioavailability. Our findings suggest that cytosolic unbound inhibitor concentrations improves predictions of time-dependent drug-drug interactions for CYP3A.

Clinical Studies on Drug-Drug Interactions Involving Metabolism and Transport: Methodology, Pitfalls, and Interpretation.

Many drug-drug interactions (DDIs) are based on alterations of the plasma concentrations of a victim drug due to another drug causing inhibition and/or induction of the metabolism or transporter-mediated disposition of the victim drug. In the worst case, such interactions cause more than tenfold increases or decreases in victim drug exposure, with potentially life-threatening consequences. There has been tremendous progress in the predictability and modeling of DDIs. Accordingly, the combination of modeling approaches and clinical studies is the current mainstay in evaluation of the pharmacokinetic DDI risks of drugs. In this paper, we focus on the methodology of clinical studies on DDIs involving drug metabolism or transport. We specifically present considerations related to general DDI study designs, recommended enzyme and transporter index substrates and inhibitors, pharmacogenetic perspectives, index drug cocktails, endogenous substrates, limited sampling strategies, physiologically-based pharmacokinetic modeling, complex DDIs, methodological pitfalls, and interpretation of DDI information.

Critical Differences between Enzyme Sources in Sensitivity to Detect Time-Dependent Inactivation of CYP2C8.

Clopidogrel acyl-ß-d-glucuronide is a mechanism-based inhibitor of cytochrome P450 2C8 in human liver microsomes (HLMs). However, time-dependent inactivation (TDI) of CYP2C8 could not be detected in an earlier study in human recombinant CYP2C8 (Supersomes). Here, we investigate whether different enzyme sources exhibit differences in detection of CYP2C8 TDI under identical experimental conditions. Inactivation of CYP2C8 by amiodarone (100 µM), clopidogrel acyl-ß-d-glucuronide (100 µM), gemfibrozil 1-O-ß-glucuronide (100 µM), and phenelzine (100 µM) was investigated in HLMs and three recombinant human CYP2C8 preparations (Supersomes, Bactosomes, and EasyCYP Bactosomes) using amodiaquine N-deethylation as the marker reaction. Furthermore, the inactivation kinetics of CYP2C8 by clopidogrel glucuronide (5-250 µM) was determined in Supersomes and Bactosomes. Amiodarone caused weak TDI in all enzyme preparations tested, while the extent of inactivation by clopidogrel glucuronide, gemfibrozil glucuronide, and phenelzine varied markedly between preparations, and even different Supersome lots. Both glucuronides caused strong inactivation of CYP2C8 in HLMs, Bactosomes and in one Supersome lot (>50% inhibition), but significant inactivation could not be reliably detected in other Supersome lots or EasyCYP Bactosomes. In Bactosomes, the concentration producing half of kinact (KI) and maximal inactivation rate (kinact) of clopidogrel glucuronide (14 µM and 0.054 minute-1) were similar to those determined previously in HLMs. Phenelzine caused strong inactivation of CYP2C8 in one Supersome lot (91% inhibition) but not in HLMs or other recombinant CYP2C8 preparations. In conclusion, different enzyme sources and different lots of the same recombinant enzyme preparation are not equally sensitive to detect inactivation of CYP2C8, suggesting that recombinant CYPs should be avoided when identifying mechanism-based inhibitors.

In Vitro Screening of Six Protein Kinase Inhibitors for Time-Dependent Inhibition of CYP2C8 and CYP3A4: Possible Implications with regard to Drug-Drug Interactions.

Several protein kinase inhibitors have been reported to affect cytochrome P450 (CYP) 3A by time-dependent inhibition. Herein, we tested a set of six kinase inhibitors for time-dependent inhibition of CYP2C8 and CYP3A4 in human liver microsomes. Dovitinib, midostaurin and nintedanib exhibited an increased inhibition of CYP3A4 after a 30-min. pre-incubation with NADPH, as compared to no pre-incubation (IC50 shift >1.5). Masitinib, trametinib and vatalanib did not affect CYP2C8 or CYP3A4 by time-dependent inhibition (IC50 shift <1.5). The inhibitory mechanism of CYP3A4 by midostaurin and nintedanib, but not by dovitinib, was consistent with irreversible mechanism-based inhibition. The maximal inactivation rate (kinact ) and inhibitor concentration that supports half-maximal rate of inactivation (KI ) values of midostaurin and nintedanib were 0.052 1/min. and 2.72 µM, and 0.025 1/min. and 17.3 µM, respectively. According to static predictions, inactivation of CYP3A4 by nintedanib was unlikely to cause drug-drug interactions with clinically used doses of nintedanib, whereas midostaurin was predicted to increase the plasma exposure to CYP3A4-dependent substrates several fold. Furthermore, based on reversible inhibition, masitinib and vatalanib were predicted to increase the plasma exposure to sensitive CYP2C8 and CYP3A4 substrates by ≥2-fold. In summary, our data identify midostaurin and nintedanib as time-dependent inhibitors of CYP3A4 and detect a risk of drug-drug interactions between vatalanib and CYP2C8 substrates, and between masitinib, midostaurin and vatalanib and CYP3A4 substrates. The liability of kinase inhibitors to affect CYP enzymes by time-dependent inhibition may have long-term consequences, in terms of drug-drug interactions and toxicities.

Clopidogrel but Not Prasugrel Significantly Inhibits the CYP2C8-Mediated Metabolism of Montelukast in Humans.

The oxidation of montelukast is mainly mediated by cytochrome P450 (CYP) 2C8, but other mechanisms may contribute to its disposition. In healthy volunteers, we investigated the effects of two widely used P2Y12 inhibitors on montelukast pharmacokinetics. Clopidogrel (300 mg on day 1 and 75 mg on day 2) increased the area under the plasma concentration-time curve (AUC) of montelukast 2.0-fold (90% confidence interval (CI) 1.72-2.28, P < 0.001) and decreased the M6:montelukast AUC0-7h ratio to 45% of control (90% CI 40-50%, P < 0.001). Prasugrel (60 mg on day 1 and 10 mg on day 2) had no clinically meaningful effect on montelukast pharmacokinetics. Our results imply that clopidogrel is at least a moderate inhibitor of CYP2C8, but prasugrel is not a clinically relevant CYP2C8 inhibitor. The different interaction potentials of clopidogrel and prasugrel are important to consider when antiplatelet therapy is planned for patients at risk for polypharmacy with CYP2C8 substrates.

Clopidogrel Carboxylic Acid Glucuronidation is Mediated Mainly by UGT2B7, UGT2B4, and UGT2B17: Implications for Pharmacogenetics and Drug-Drug Interactions .

The antiplatelet drug clopidogrel is metabolized to an acyl-ß-d-glucuronide, which causes time-dependent inactivation of CYP2C8. Our aim was to characterize the UDP-glucuronosyltransferase (UGT) enzymes that are responsible for the formation of clopidogrel acyl-ß-d-glucuronide. Kinetic analyses and targeted inhibition experiments were performed using pooled human liver and intestine microsomes (HLMs and HIMs, respectively) and selected human recombinant UGTs based on preliminary screening. The effects of relevant UGT polymorphisms on the pharmacokinetics of clopidogrel were evaluated in 106 healthy volunteers. UGT2B7 and UGT2B17 exhibited the greatest level of clopidogrel carboxylic acid glucuronidation activities, with a CLint,u of 2.42 and 2.82 µl⋅min-1⋅mg-1, respectively. Of other enzymes displaying activity (UGT1A3, UGT1A9, UGT1A10-H, and UGT2B4), UGT2B4 (CLint,u 0.51 µl⋅min-1⋅mg-1) was estimated to contribute significantly to the hepatic clearance. Nonselective UGT2B inhibitors strongly inhibited clopidogrel acyl-ß-d-glucuronide formation in HLMs and HIMs. The UGT2B17 inhibitor imatinib and the UGT2B7 and UGT1A9 inhibitor mefenamic acid inhibited clopidogrel carboxylic acid glucuronidation in HIMs and HLMs, respectively. Incubation of clopidogrel carboxylic acid in HLMs with UDPGA and NADPH resulted in strong inhibition of CYP2C8 activity. In healthy volunteers, the UGT2B17*2 deletion allele was associated with a 10% decrease per copy in the plasma clopidogrel acyl-ß-d-glucuronide to clopidogrel carboxylic acid area under the plasma concentration-time curve from 0 to 4 hours (AUC0-4) ratio (P < 0.05). To conclude, clopidogrel carboxylic acid is metabolized mainly by UGT2B7 and UGT2B4 in the liver and by UGT2B17 in the small intestinal wall. The formation of clopidogrel acyl-ß-d-glucuronide is impaired in carriers of the UGT2B17 deletion. These findings may have implications regarding the intracellular mechanisms leading to CYP2C8 inactivation by clopidogrel.

Role of Cytochrome P450 2C8 in Drug Metabolism and Interactions.

During the last 10-15 years, cytochrome P450 (CYP) 2C8 has emerged as an important drug-metabolizing enzyme. CYP2C8 is highly expressed in human liver and is known to metabolize more than 100 drugs. CYP2C8 substrate drugs include amodiaquine, cerivastatin, dasabuvir, enzalutamide, imatinib, loperamide, montelukast, paclitaxel, pioglitazone, repaglinide, and rosiglitazone, and the number is increasing. Similarly, many drugs have been identified as CYP2C8 inhibitors or inducers. In vivo, already a small dose of gemfibrozil, i.e., 10% of its therapeutic dose, is a strong, irreversible inhibitor of CYP2C8. Interestingly, recent findings indicate that the acyl-ß-glucuronides of gemfibrozil and clopidogrel cause metabolism-dependent inactivation of CYP2C8, leading to a strong potential for drug interactions. Also several other glucuronide metabolites interact with CYP2C8 as substrates or inhibitors, suggesting that an interplay between CYP2C8 and glucuronides is common. Lack of fully selective and safe probe substrates, inhibitors, and inducers challenges execution and interpretation of drug-drug interaction studies in humans. Apart from drug-drug interactions, some CYP2C8 genetic variants are associated with altered CYP2C8 activity and exhibit significant interethnic frequency differences. Herein, we review the current knowledge on substrates, inhibitors, inducers, and pharmacogenetics of CYP2C8, as well as its role in clinically relevant drug interactions. In addition, implications for selection of CYP2C8 marker and perpetrator drugs to investigate CYP2C8-mediated drug metabolism and interactions in preclinical and clinical studies are discussed.

Neurotoxicity and low paclitaxel clearance associated with concomitant clopidogrel therapy in a 60-year-old Caucasian woman with ovarian carcinoma.

AIM: The aim of the present case report was to describe a novel pharmacokinetic drug­drug interaction between the antiplatelet agent clopidogrel and the antineoplastic agent paclitaxel. METHODS: The patient was identified in a previously described cohort of 93 patients with ovarian carcinoma treated with paclitaxel. The effect of clopidogrel acyl-ß-D-glucuronide on the metabolism of paclitaxel was assessed in human liver microsomes. The analysis of clopidogrel in plasma and the quantification of paclitaxel and 6-hydroxypaclitaxel in in vitro samples were performed by liquid chromatography tandem mass spectrometry. RESULTS: The patient was a 60-year-old female treated with an unknown dose of clopidogrel at the time of paclitaxel therapy. Clopidogrel was present in all three of the plasma samples obtained during paclitaxel dosing. Estimated unbound paclitaxel clearance was 238 l h−1, which was only 62% of the cohort geometric mean (385 l h−1; range 176­726). She was hospitalized three times, developed severe neuropathy and paclitaxel treatment was subsequently discontinued. In vitro, 30-min preincubation with 100 µM clopidogrel acyl-ß-D-glucuronide inhibited the depletion rate of 0.5 µM paclitaxel by 51% and the formation rate of 6-hydroxypaclitaxel by 77%. CONCLUSION: This is the first report of a clopidogrel­paclitaxel interaction, suggesting that clinically used doses of clopidogrel can reduce the cytochrome P450 2C8 (CYP2C8)-mediated systemic clearance of paclitaxel, leading to an increased risk of paclitaxel toxicity. Caution should be exercised whenever the simultaneous use of paclitaxel and clopidogrel cannot be avoided.

In vitro assessment of time-dependent inhibitory effects on CYP2C8 and CYP3A activity by fourteen protein kinase inhibitors.

Previous studies have shown that several protein kinase inhibitors are time-dependent inhibitors of cytochrome P450 (CYP) 3A. We screened 14 kinase inhibitors for time-dependent inhibition of CYP2C8 and CYP3A. Amodiaquine N-deethylation and midazolam 1'-hydroxylation were used as marker reactions for CYP2C8 and CYP3A activity, respectively. A screening, IC50 shift, and mechanism-based inhibition were assessed with human liver microsomes. In the screening, bosutinib isomer 1, crizotinib, dasatinib, erlotinib, gefitinib, lestaurtinib, nilotinib, pazopanib, saracatinib, sorafenib, and sunitinib exhibited an increased inhibition of CYP3A after a 30-min preincubation with NADPH, as compared with no preincubation. Axitinib and vandetanib tested negative for time-dependent inhibition of CYP3A and CYP2C8, and bosutinib was the only inhibitor causing time-dependent inhibition of CYP2C8. The inhibitory mechanism by bosutinib was consistent with weak mechanism-based inhibition, and its inactivation variables, inhibitor concentration that supports half-maximal rate of inactivation (KI) and maximal inactivation rate (kinact), were 54.8 µM and 0.018 1/min. As several of the tested inhibitors were reported to cause mechanism-based inactivation of CYP3A4 during the progress of this work, detailed experiments with these were not completed. However, lestaurtinib and saracatinib were identified as mechanism-based inhibitors of CYP3A. The KI and kinact of lestaurtinib and saracatinib were 30.7 µM and 0.040 1/min, and 12.6 µM and 0.096 1/min, respectively. Inhibition of CYP2C8 by bosutinib was predicted to have no clinical relevance, whereas therapeutic lestaurtinib and saracatinib concentrations were predicted to increase the plasma exposure to CYP3A-dependent substrates by ≥2.7-fold. The liability of kinase inhibitors to affect CYP enzymes by time-dependent inhibition may have long-lasting consequences and result in clinically relevant drug-drug interactions.

Autoinhibition of CYP3A4 leads to important role of CYP2C8 in imatinib metabolism: variability in CYP2C8 activity may alter plasma concentrations and response.

Recent data suggest that the role of CYP3A4 in imatinib metabolism is smaller than presumed. This study aimed to evaluate the quantitative importance of different cytochrome P450 (P450) enzymes in imatinib pharmacokinetics. First, the metabolism of imatinib was investigated using recombinant P450 enzymes and human liver microsomes with P450 isoform-selective inhibitors. Thereafter, an in silico model for imatinib was constructed to perform pharmacokinetic simulations to assess the roles of P450 enzymes in imatinib elimination at clinically used imatinib doses. In vitro, CYP2C8 inhibitors and CYP3A4 inhibitors inhibited the depletion of 0.1 µM imatinib by 45 and 80%, respectively, and the formation of the main metabolite of imatinib, N-desmethylimatinib, by >50%. Likewise, recombinant CYP2C8 and CYP3A4 metabolized imatinib extensively, whereas other isoforms had minor effect on imatinib concentrations. In the beginning of imatinib treatment, the fractions of its hepatic clearance mediated by CYP2C8 and CYP3A4 were predicted to approximate 40 and 60%, respectively. During long-term treatment with imatinib 400 mg once or twice daily, up to 65 or 75% of its hepatic elimination was predicted to occur via CYP2C8, and only about 35 or 25% by CYP3A4, due to dose- and time-dependent autoinactivation of CYP3A4 by imatinib. Thus, although CYP2C8 and CYP3A4 are the main enzymes in imatinib metabolism in vitro, in silico predictions indicate that imatinib inhibits its own CYP3A4-mediated metabolism, assigning a key role for CYP2C8. During multiple dosing, pharmacogenetic polymorphisms and drug interactions affecting CYP2C8 activity may cause marked interindividual variation in the exposure and response to imatinib.