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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.

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-beta-glucuronide and clopidogrel acyl-beta-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-beta-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-beta-glucuronide was a strong (90% inhibition) and selective (<< 20% inhibition of other CYPs) inhibitor of CYP2C8 at concentrations ranging from 60 to 300 mu M, while the selectivity of clopidogrel acyl-beta-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 mu M, respectively. In conclusion, a reliable cocktail method including the nine most important drug-metabolizing CYP enzymes was developed, optimized and validated for detecting timedependent inhibition. Moreover, gemfibrozil 1-O-beta-glucuronide was established as a selective inhibitor of CYP2C8 for use as a diagnostic inhibitor in in vitro studies.

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

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 P2Y(12) inhibitors on montelukast pharmacokinetics. Clopidogrel (300mg on day 1 and 75mg 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

Background: Previously we showed cancer cells rely on the MTH1 protein to prevent incorporation of otherwise deadly oxidised nucleotides into DNA and we developed MTH1 inhibitors which selectively kill cancer cells. Recently several new and potent inhibitors of MTH1 were demonstrated to be non toxic to cancer cells challenging the utility of MTH1 inhibition as a target for cancer treatment. Material and methods: Human cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA or shRNA. 8 oxodG was measured by immunostaining and modified comet assay. Thermal Proteome profiling proteomics cellular thermal shift assays kinase and CEREP panel were used for target engagement mode of action and selectivity investigations of MTH1 inhibitors. Effect of MTH1 inhibition on tumour growth was explored in BRAF V600E mutated malignant melanoma patient derived xenograft and human colon cancer SW480 and HCT116 xenograft models. Results: Here we demonstrate that recently described MTH1 inhibitors which fail to kill cancer cells also fail to introduce the toxic oxidized nucleotides into DNA. We also describe a new MTH1 inhibitor TH1579 (Karonudib) an analogue of TH588 which is a potent selective MTH1 inhibitor with good oral availability and demonstrates excellent pharmacokinetic and anti cancer properties in vivo. Conclusion: We demonstrate that in order to kill cancer cells MTH1 inhibitors must also introduce oxidized nucleotides into DNA. Furthermore we describe TH1579 as a best in class MTH1 inhibitor which we expect to be useful in order to further validate the MTH1 inhibitor concept. Key words: MTH1 reactive oxygen species cancer small molecule inhibitors DNA damage

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.

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-β-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.

Clopidogrel acyl-β-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-β-d-glucuronide (100 μM), gemfibrozil 1-O-β-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.

The antiplatelet drug clopidogrel is metabolized to an acyl-β-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-β-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-β-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-β-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-β-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.

BACKGROUND AND PURPOSE Imatinib, a cytochrome P450 2C8 (CYP2C8) and CYP3A4 substrate, markedly increases plasma concentrations of the CYP3A4/5 substrate simvastatin and reduces hepatic CYP3A4/5 activity in humans. Because competitive inhibition of CYP3A4/5 does not explain these in vivo interactions, we investigated the reversible and time-dependent inhibitory effects of imatinib and its main metabolite N-desmethylimatinib on CYP2C8 and CYP3A4/5 in vitro.

Cerivastatin and repaglinide are substrates of cytochrome P450 (CYP)2C8, CYP3A4, and organic anion–transporting polypeptide (OATP)1B1. A recent study revealed an increased risk of rhabdomyolysis in patients using cerivastatin with clopidogrel, warranting further studies on clopidogrel interactions. In healthy volunteers, repaglinide area under the concentration–time curve (AUC0–∞) was increased 5.1–fold by a 300–mg loading dose of clopidogrel and 3.9–fold by continued administration of 75 mg clopidogrel daily. In vitro, we identified clopidogrel acyl–β–D–glucuronide as a potent time–dependent inhibitor of CYP2C8. A physiologically based pharmacokinetic model indicated that inactivation of CYP2C8 by clopidogrel acyl–β–D–glucuronide leads to uninterrupted 60–85% inhibition of CYP2C8 during daily clopidogrel treatment. Computational modeling resulted in docking of clopidogrel acyl–β–D–glucuronide at the CYP2C8 active site with its thiophene moiety close to heme. The results indicate that clopidogrel is a strong CYP2C8 inhibitor via its acyl–β–D–glucuronide and imply that glucuronide metabolites should be considered potential inhibitors of CYP enzymes. Clinical Pharmacology & Therapeutics (2014); 96 4, 498–507. doi:10.1038/clpt.2014.141

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.

Cytochrome P450 (CYP) 3A4 is considered the most important enzyme in imatinib biotransformation. In a randomized, crossover study, 10 healthy subjects were administered gemfibrozil 600 mg or placebo twice daily for 6 days, and imatinib 200 mg on day 3, to study the significance of CYP2C8 in imatinib pharmacokinetics. Unexpectedly, gemfibrozil reduced the peak plasma concentration (Cmax) of imatinib by 35% (P < 0.001). Gemfibrozil also reduced the Cmax and area under the plasma concentration–time curve (AUC0–∞) of N-desmethylimatinib by 56 and 48% (P < 0.001), respectively, whereas the AUC0–∞ of imatinib was unaffected. Furthermore, gemfibrozil reduced the Cmax/plasma concentration at 24 h (C24 h) ratios of imatinib and N-desmethylimatinib by 44 and 17% (P < 0.05), suggesting diminished daily fluctuation of imatinib plasma concentrations during concomitant use with gemfibrozil. Our findings indicate significant participation of CYP2C8 in the metabolism of imatinib in humans, and support involvement of an intestinal influx transporter in imatinib absorption. Clinical Pharmacology & Therapeutics (2013); 94 3, 383–393. doi:10.1038/clpt.2013.92

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.

According to published in vitro studies, cytochrome P450 3A4 catalyzes montelukast 21-hydroxylation (M5 formation), whereas CYP2C9 catalyzes 36-hydroxylation (M6), the primary step in the main metabolic pathway of montelukast. However, montelukast is a selective competitive CYP2C8 inhibitor, and our recent in vivo studies suggest that CYP2C8 is involved in its metabolism. We therefore reevaluated the contributions of different cytochrome P450 (P450) enzymes, particularly that of CYP2C8, to the hepatic microsomal metabolism of montelukast using clinically relevant substrate concentrations in vitro. The effects of P450 isoform inhibitors on montelukast metabolism were examined using pooled human liver microsomes, and montelukast oxidations by human recombinant CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 were investigated. The results verified the central role of CYP3A4 in M5 formation. The CYP2C8 inhibitors gemfibrozil 1-O-β glucuronide and trimethoprim inhibited the depletion of 0.02 μM montelukast and formation of M6 from 0.05 μM montelukast more potently than did the CYP2C9 inhibitor sulfaphenazole. Likewise, recombinant CYP2C8 catalyzed montelukast depletion and M6 formation at a 6 times higher intrinsic clearance than did CYP2C9, whereas other P450 isoforms produced no M6. On the basis of depletion of 0.02 μM montelukast, CYP2C8 was estimated to account for 72% of the oxidative metabolism of montelukast in vivo, with a 16% contribution for CYP3A4 and 12% for CYP2C9. Moreover, CYP2C8 catalyzed the further metabolism of M6 more actively than did any other P450. In conclusion, CYP2C8 plays a major role in the main metabolic pathway of montelukast at clinically relevant montelukast concentrations in vitro.

According to available information, montelukast is metabolized by cytochrome P450 (CYP) 3A4 and 2C9. In order to study the significance of CYP2C8 in the pharmacokinetics of montelukast, 10 healthy subjects were administered gemfibrozil 600 mg or placebo twice daily for 3 days, and 10 mg montelukast on day 3, in a randomized, crossover study. Gemfibrozil increased the mean area under the plasma concentration-time curve (AUC)(0-infinity), peak plasma concentration (C(max)), and elimination half-life (t(1/2)) of montelukast 4.5-fold, 1.5-fold, and 3.0-fold, respectively (P0.001). After administration of gemfibrozil, the time to reach C(max) (t(max)) of the montelukast metabolite M6 was prolonged threefold (P = 0.005), its AUC(0-7) was reduced by 40% (P = 0.027), and the AUC(0-24) of the secondary metabolite M4 was reduced by90% (P0.001). In human liver microsomes, gemfibrozil 1-O-beta glucuronide inhibited the formation of M6 (but not of M5) from montelukast 35-fold more potently than did gemfibrozil (half-maximal inhibitory concentration (IC(50)) 3.0 and 107 micromol/l, respectively). In conclusion, gemfibrozil markedly increases the plasma concentrations of montelukast, indicating that CYP2C8 is crucial in the elimination of montelukast.

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