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1521-0081/68/1/168–241$25.00 http://dx.doi.org/10.1124/pr.115.011411 PHARMACOLOGICAL REVIEWS Pharmacol Rev 68:168–241, January 2016 Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics

ASSOCIATE EDITOR: MARKKU KOULU Role of 2C8 in and Interactions

Janne T. Backman, Anne M. Filppula, Mikko Niemi, and Pertti J. Neuvonen Department of Clinical Pharmacology, University of Helsinki (J.T.B., A.M.F., M.N., P.J.N.), and Helsinki University Hospital, Helsinki, Finland (J.T.B., M.N., P.J.N.)

Abstract ...... 169 I. Introduction ...... 169 II. Basic Characteristics of Cytochrome P450 2C8 ...... 170 A. Genomic Organization and Transcriptional Regulation ...... 170 B. Protein Structure ...... 171 C. Expression ...... 172 III. Substrates of Cytochrome P450 2C8...... 173 A. ...... 173 1. Anticancer Agents...... 173 Downloaded from 2. Antidiabetic Agents...... 183 3. Antimalarial Agents...... 183 4. Lipid-lowering Drugs...... 184 5. Other Drugs...... 184 6. Glucuronide Metabolites...... 186

B. Endogenous and Natural Compounds...... 187 by guest on March 4, 2019 IV. Pharmacogenetics...... 187 A. Population Genetics ...... 187 B. Functional Studies ...... 191 C. Effects on in Humans ...... 192 V. In Vitro Inhibition and Induction of Cytochrome P450 2C8 ...... 193 A. Reversible Inhibition ...... 193 1. Drugs That Act as Inhibitors of Cytochrome P450 2C8...... 193 2. Natural Compounds...... 210 B. Metabolism-dependent Inhibition ...... 210 C. Induction ...... 210 VI. Clinical Drug Interactions Mediated via Cytochrome P450 2C8 ...... 212 A. General Aspects...... 212 B. as Prototypical Inhibitor ...... 214 1. In Vitro Versus In Vivo...... 214 2. Gemfibrozil Dose Versus CYP2C8 Inhibition...... 216 3. Onset and Duration of CYP2C8 Inhibition by Gemfibrozil...... 216 4. Quantification of CYP2C8-Mediated Drug Interactions in Humans...... 216 C. Inhibition-Mediated Drug Interactions and Their Clinical Significance ...... 217 1. ...... 217 2. Other Oral Antidiabetic Drugs...... 218 3. Amodiaquine...... 219 4. ...... 219 5. Anticancer Drugs...... 220

This work was supported by grants from the Academy of Finland [Grant decision 278123, 2014], the Helsinki University Central Hospital Research Fund, and the Sigrid Juselius Foundation (Helsinki, Finland). Address correspondence to: Prof. Janne T. Backman, Department of Clinical Pharmacology, University of Helsinki and Helsinki University Hospital, P.O. Box 705, FI-00029 HUS, Finland. E-mail: [email protected] dx.doi.org/10.1124/pr.115.011411.

168 Role of CYP2C8 in Drug Metabolism and Interactions 169

6. Antiviral Drugs...... 221 7. Antiasthmatic Drugs...... 221 8. Other or Inhibitor Drugs...... 221 D. Induction-Mediated Drug Interactions ...... 222 1. Rifampin ()...... 222 VII. Points to Consider When Investigating Cytochrome P450 2C8-Mediated Drug Metabolism and Interactions ...... 222 A. InVitro...... 222 1. General Aspects...... 222 2. Assessment of CYP2C8 Activity In Vitro...... 223 3. In Vitro Methods to Estimate the Contribution of CYP2C8 in the Metabolism of a Drug...... 224 B. In Vivo ...... 224 1. General Aspects...... 224 2. In Vivo Cytochrome P450 2C8 Probe Substrates...... 226 3. In Vivo Cytochrome P450 2C8 Probe Inhibitors...... 226 VIII. Conclusions and Future Prospects...... 227 Acknowledgments...... 228 References ...... 228

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

I. Introduction (HMG-CoA) reductase inhibitor cerivastatin, a CYP2C8 substrate, resulting in rhabdomyolysis cases and fatalities Cytochrome P450 (CYP) 2C8 accounts for approxi- mately 6–7% of the total hepatic CYP content (Rowland brought attention to the importance of CYP2C8 in drug Yeoetal.,2004;Inoueetal.,2006; Rostami-Hodjegan and metabolism (Backman et al., 2002; Staffa et al., 2002; Tucker, 2007; Achour et al., 2014). The importance of Wang et al., 2002; Chang et al., 2004; Huang et al., 2008). CYP2C8 causing variation in drug response via drug-drug The event was the onset of a broadening scientific in- interactions and pharmacogenetic polymorphisms has terestinCYP2C8,promptlyconvincingdrugregulatory been recognized only for the last 10–15 years. In the authorities to acknowledge CYP2C8 as one of the major beginning of the millennium, the pharmacokinetic drug- drug-metabolizing CYP . drug interaction between the fibric acid derivative gemfi- Drugs that were introduced into clinical use before brozil and the 3-hydroxy-3-methylglutaryl-coenzyme A the role of CYP2C8 was recognized may have deficient

ABBREVIATIONS: AUC, area under the plasma concentration-time curve; C/EBPa, CCAAT/enhancer-binding protein a; CAR, constitutive androstane receptor; CITCO, [6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime;

CLint, intrinsic clearance; Cmax, peak concentration; CYP, cytochrome P450; EMA, European Medicines Agency; FDA, Food and Drug Administration; GR, receptor; HLM, human liver microsomes; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; HNF, hepatic nuclear factor; I, inhibitor concentration; Ki, reversible inhibition constant; KI, inhibitor concentration supporting half of the maximal rate of enzyme inactivation; kinact, maximal rate of inactivation; Km, Michaelis-Menten constant; MRL-C, 2-[[5,7-dipropyl-3- (trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy]-2-methylpropanoic acid; mRNA, messenger ribonucleic acid; OAT, organic anion transporter; OATP, organic anion-transporting polypeptide; PPAR, peroxisome proliferator activated receptor; PXR, pregnane X receptor; ROR, retinoic acid-related orphan receptors; SIFT, sorting intolerant from tolerant; SNV, single nucleotide variation; t1/2, elimination half-life; tmax,time to peak concentration; UGT, uridine-59-diphosphoglucuronosyltransferase; VDR, D receptor. 170 Backman et al. or incorrect product information regarding their in- roles of CYP2C8 have been thoroughly examined and teraction potential (Neuvonen, 2012). One example is discussed in several previous reviews (Kirchheiner et al., the leukotriene montelukast. 2005; Totah and Rettie, 2005; Garcia-Martin et al., 2006; Early preclinical studies concluded that CYP2C9 and Gil and Gil Berglund, 2007; Agundez et al., 2009; Chen CYP3A4 are the most important enzymes involved in its and Goldstein, 2009; Daily and Aquilante, 2009; Lai et al., metabolism, whereas the role of CYP2C8 was not 2009; Aquilante et al., 2013b; Fleming, 2014; Xiaoping evaluated (Chiba et al., 1997). In interaction studies et al., 2013). Thus, the reader will be directed to these performed more than a decade later, the strong CYP2C8 earlier works for some previously known aspects related inhibitor gemfibrozil increased the plasma exposure to CYP2C8. Herein, our intention is to review and update to montelukast almost fivefold, whereas the strong the current knowledge on substrates, inhibitors, in- CYP3A4 inhibitor itraconazole had no significant effect ducers, and pharmacogenetics of CYP2C8, as well as its on its (Karonen et al., 2010, 2012). role in clinically relevant drug interactions. In addition, The clinical findings were corroborated by in vitro data, implications for selection of CYP2C8 marker and perpe- showing that CYP2C8 is the main enzyme involved in trator drugs to investigate CYP2C8-mediated drug me- the oxidative metabolism of montelukast (Filppula tabolism and interactions in preclinical and clinical et al., 2011; VandenBrink et al., 2011). studies are discussed. The large, trifurcated cavity of CYP2C8 is able to accommodate substrates of different shapes and II. Basic Characteristics of Cytochrome P450 2C8 sizes (Schoch et al., 2008). Today, CYP2C8 is known to participate in the metabolism of more than 100 drugs, A. Genomic Organization and Transcriptional Regulation including amodiaquine, cerivastatin, dasabuvir, enzalu- The CYP2C8 enzyme is encoded by the CYP2C8 gene, tamide, imatinib, loperamide, montelukast, paclitaxel, which is located on the 10q24 in the pioglitazone, repaglinide, and rosiglitazone. The num- gene cluster centromere-2C18-2C19-2C9-2C8-telomere ber of drugs that are identified as CYP2C8 substrates in close proximity of the CYP2C9 gene (Fig. 1; Gray or inhibitors, as well as CYP2C8-mediated drug-drug et al., 1995; Klose et al., 1999). CYP2C8 is the smallest interactions is continuously increasing. The strong of the human CYP2C genes; it spans a 31-kb region and CYP2C8 inhibition by gemfibrozil observed in vivo is contains 9 exons (Klose et al., 1999; Lai et al., 2009). It due to its acyl-b-glucuronide metabolite, which is a shares 74% with CYP2C9 (Daily potent mechanism-based inhibitor of CYP2C8 (Ogilvie and Aquilante, 2009). et al., 2006). Also other glucuronide metabolites of drugs The transcriptional regulation of CYP2C8 is mediated were recently found to interact with CYP2C8 either as via several transcriptional factors and distinct nuclear substrates or inhibitors. For instance, the acyl-b-D- receptors that can activate the respective responsive glucuronide metabolite of clopidogrel is a metabolism- elements within the 59-flanking promoter region of the dependent inhibitor of CYP2C8, causing more than a gene (Ferguson et al., 2005; Johnson and Stout, 2005; fivefold increase in the plasma exposure to repaglinide Kojima et al., 2007; Chen and Goldstein, 2009). Such in healthy subjects (Tornio et al., 2014). In another factors/receptors include the constitutive androstane re- recent in vitro study, CYP2C8 metabolized the glucu- ceptor (CAR), pregnane X receptor (PXR), vitamin D ronide metabolite of to its pharmacolog- receptor (VDR), glucocorticoid receptor (GR), hepatic ically active 3-hydroxydesloratadine metabolite (Kazmi nuclear factor-4a (HNF4a), HNF3g, CCAAT/enhancer- et al., 2015). These and other data suggest that an binding protein a (C/EBPa), and retinoic acid-related interplay between CYP2C8 and glucuronide metabo- orphan receptors (RORs) (Fig. 1; Ferguson et al., 2005; lites may be more a rule than an exception. Chen and Goldstein, 2009; Rana et al., 2010; Aquilante There are several common nonsynonymous varia- et al., 2013b). Although HNF4a,HNF3g, C/EBPa,and tions in the CYP2C8 gene (Daily and Aquilante, 2009; RORs seem to mainly regulate the constitutive expression Aquilante et al., 2013b). For example, the CYP2C8*3 of CYP2C genes in liver, the other receptors are more allele has been associated with decreased metabolism important to the xenobiotic-mediated induction of CYP2C8 of several substrates, e.g., paclitaxel, in vitro (Dai et al., expression, as described in more detail in section V.C. 2001). In contrast, clinical data indicate that the CYP2C8*3 After activation by endo- or xenobiotics, CAR, PXR, allele is often associated with increased metabolism of and VDR form heterodimers with the retinoid X CYP2C8 substrates, such as repaglinide (Niemi et al., receptor, whereas GR forms homodimers (Chen and 2003c). Thus, although complete lack of function variants Goldstein, 2009). These dimers are thereafter recog- in CYP2C8 arerare,thepossiblesubstratedependency nized by specific response elements within the CYP2C8 of the functional consequences of the common CYP2C8 promoter. By using in vitro gel shift assays, responsive variants and their potential clinical significance have elements/motifs within the CYP2C8 promoter regions raised a lot of interest toward CYP2C8 pharmacogenetics. have been identified for CAR, PXR, and GR (Gerbal- The structural properties, regulation of expression, Chaloin et al., 2002; Ferguson et al., 2005; Chen and pharmacogenetics, substrates, inhibitors, and physiologic Goldstein, 2009). Role of CYP2C8 in Drug Metabolism and Interactions 171

Fig. 1. The CYP2C8 gene is located to the CYP2C gene cluster on . C/EBPa, CCAAT/enhancer-binding protein a; CAR, constitutive androstane receptor; GR, glucocorticoid receptor; HNF, hepatic nuclear factor; PXR, pregnane X receptor; ROR, retinoic acid-related orphan receptor; VDR, vitamin D receptor.

After activation, the orphan nuclear receptor HNF4a 2004), which is similar to that of CYP3A4 (1,386 Å3) binds as a homodimer to a DR1 type element and also but larger than those of CYP1A2 (375 Å3), CYP2A6 to the Hep-G2 specific P450 factor-1 motif (Venepally (260 Å3), CYP2C9 (;470 Å3), CYP2D6 (;540 Å3), and et al., 1992), whereas HNF3g binds to DNA as a CYP2E1 (190 Å3) (Williams et al., 2003; Yano et al., monomer (Bort et al., 2004). At least two Hep-G2 2004; Rowland et al., 2006; Sansen et al., 2007; specific P450 factor-1 motifs and several putative Porubsky et al., 2008). Although CYP3A4 has a uni- HNF3g binding sites have been identified within the formly distributed active site cavity, that of CYP2C8 is promoter of CYP2C8 (Bort et al., 2004; Ferguson et al., trifurcated, resembling a T or Y shape (Schoch et al., 2005; Chen and Goldstein, 2009). RORs are constitu- 2008). The bottom branch of the cavity provides access tively active orphan nuclear receptors, which have to the heme, and the two other terminate in solvent and natural ligands, such as all-trans-retinoic acid that substrate access channels that exit the active site cavity can influence their activity. Also RORs seem to be on either side of the helix B-C loop. involved in the constitutive regulation of CYP2C8, In the X-ray crystallography study, the N-terminal and at least two ROR responsive elements have been anchor domains of both CYP2C8 molecules were lo- identified in the gene promoter (Chen et al., 2009). cated on the same side of the dimer, indicating an Transcriptional regulation of CYP2C8 has been re- orientation compatible with membrane binding (Schoch viewed thoroughly by Chen and Goldstein (2009). et al., 2004). The proximal surfaces of each protein were roughly parallel, suggesting that they are accessible for B. Protein Structure interaction with the membrane-bound CYP oxidoreduc- The crystal structure of CYP2C8 was resolved in 2004 tase. Another study demonstrated that the dimeric (Schochetal.,2004).AsingleCYP2C8crystaldif- structure observed in the crystal structure of CYP2C8 fracted to 2.7 Å and had the molecular weight approx- may also be present in membrane-bound native CYP2C8 imated to 54 kDa. Interestingly, CYP2C8 crystallized as (Hu et al., 2010). The signal anchor/linker regions of a symmetric dimer formed by interactions between the native CYP2C8 formed a second dimerization interface, helix F to G regions of the two monomers. Two palmitic and it was suggested that this interaction is required acid molecules were bound in the dimer interface, for the formation of the dimer of the native protein. stabilizing the dimer. Thus, the two fatty acids may Although direct evidence for a functional significance form a peripheral , which may affect the of the dimerization is lacking, such interactions structural dynamics of the active site and influence have been shown to affect activities of other CYPs and reactions catalyzed by CYP2C8. The active site volume membrane proteins in the endoplasmic reticulum (Hu of CYP2C8 was estimated to 1,438 Å3 (Schoch et al., et al., 2010). 172 Backman et al.

According to the X-ray crystallographic data, the where Ser-100 and Ser-103 reside (Fig. 2; Baer et al., active site cavity of CYP2C8 is capable of binding 2009; Tornio et al., 2014). structurally diverse substrates without major changes Although the large active site of CYP2C8 and in its tertiary structure (Schoch et al., 2008). Ligands of diversity of its substrates (section III) may complicate CYP2C8 may bind to the active site differently, filling the use of a general pharmacophore model, analysis of the cavity either partially or completely or occupying it eight CYP2C8 substrates showed that the majority of with two molecules simultaneously. For instance, mon- these compounds contained a terminal anionic or polar telukast, a large anionic molecule with a tripartite group ;13 Å from the oxidation site, and one or two structure, complemented the size and shape of the secondary polar moieties ;4.5 Å and ;8.5 Å from the whole active-site cavity. The linearly shaped troglita- oxidation site (Melet et al., 2004). The pharmacophore zone molecule occupied the upper portion of the cavity, model and previously reported homology models for leaving a significant part of the cavity empty, whereas CYP2C8 have been comprehensively reviewed by Lai two molecules of 9-cis-retinoic acid were simultaneously and colleagues (2009). present in the substrate-binding cavity of CYP2C8 (Schoch et al., 2008). The interactions between CYP2C8 C. Expression and its substrates were predominantly hydrophobic. According to meta-analyses, the mean hepatic CYP2C8 In addition, the distal region of the CYP2C8 active concentration approximates to 22–24 pmol/mg and site cavity contains a number of polar amino acid side 14 pmol/mg in adult Caucasian and Japanese , chains and exposed peptide backbone hydrogen bond respectively (Rowland Yeo et al., 2004; Inoue et al., donors and acceptors (Schoch et al., 2008). Accordingly, 2006; Rostami-Hodjegan and Tucker, 2007; Achour for example, the residues Ser-100, Ser-103, Asn-204, et al., 2014). The interindividual variability of CYP2C8 Asn-217, and Arg-241 form hydrogen bonds involved in protein expression in liver is high, with coefficients of the binding of the CYP2C8 substrates retinoic acid, variation of 68–95%.Theproteinexpressionlevel troglitazone and montelukast. Of note, a pronounced of CYP2C8 seems to be highly correlated with both side chain movement was observed in crystallized its enzyme activity and messenger ribonucleic acid complexes with troglitazone and retinoic acid, where (mRNA) expression level (Ohtsuki et al., 2012). Arg-241 was reoriented to the inside of the cavity, where Hepatic CYP2C8 mRNA and protein are expressed it could provide a strong, charge-stabilized hydrogen early in the prenatal development and reaches adult bond with the substrate. Interestingly, according to levels already in early childhood (Treluyer et al., 1997; computational docking simulations, the glucuronide Blanco et al., 2000; Naraharisetti et al., 2010; Cizkova moieties of gemfibrozil 1-O-b glucuronide and clopidog- et al., 2014; Johansson et al., 2014). CYP2C8 seems to be rel acyl 1-b-D-glucuronide are oriented toward the same the predominant CYP2C isoform in fetal livers (Hak- hydrophilic area in the active site close to helix B9, kola et al., 1994; Nishimura et al., 2003; Johansson

Fig. 2. Stereoimage of three independent docking simulations of the interaction between clopidogrel acyl 1-b-D-glucuronide and the active site of CYP2C8. Clopidogrel acyl-b-D-glucuronide is rendered with gray sticks depicting carbon atoms. The distance between clopidogrel acyl-b-D-glucuronide thiophene ring carbon and heme iron is indicated by a green line. Other atoms of the clopidogrel acyl-b-D-glucuronide molecule are colored red for oxygen, blue for , yellow for sulfur, and green for chlorine. Role of CYP2C8 in Drug Metabolism and Interactions 173 et al., 2014). In a recent study, CYP2C8 mRNA was III. Substrates of Cytochrome P450 2C8 expressed in all fetal tissues studied (adrenal, kidney, CYP2C8 participates in the metabolism of numerous liver, and lung tissue), whereas CYP2C9 mRNA was drugs and some endogenous and natural compounds. It restricted to the liver (Johansson et al., 2014). Another catalyzes a variety of oxidative reactions, in particular study detected CYP2C8, CYP2C9, and CYP2C19 pro- hydroxylations, N-demethylations, and N-deethyla- tein in fetal liver, intestine, and kidney (Cizkova et al., tions (Tables 1–4). Because of its large, sinuous active 2014). One explanation for the role of CYP2C8 in the site, CYP2C8 can accommodate substrates of different fetus during early pregnancy may be a need for CYP2C8 sizes and structures. The molecular weight of drugs to metabolize endogenous compounds such as retinoic significantly metabolized by CYP2C8 (.20%; Table 1) acids and hence protect the fetus from retinoic acid- ranges from 206 to 854 g/mol, with a median of 451 g/mol induced embryotoxicity (Johansson et al., 2014). (Fig. 3). In adults, CYP2C8 mRNA has been detected in Most, if not all, of the drugs significantly metabolized numerous extrahepatic tissues, including the adrenal by CYP2C8 are also substrates of other CYP enzymes gland, arteries, brain, duodenum, heart, kidney, lung, (Table 1), with about 75% being metabolized by CYP3A4 mammary gland, ovary, prostate, retina; testis, and and ;30% by CYP2C9. However, the metabolic prod- uterus, but not in placenta (Zeldin et al., 1995; Mace ucts generated by CYP2C8 and CYP3A4 are often et al., 1998; McFayden et al., 1998; Klose et al., 1999; different. For instance, CYP2C8 metabolizes paclitaxel Thum and Borlak, 2000; Nishimura et al., 2003; to 6a-hydroxypaclitaxel, whereas CYP3A4 exclusively Delozier et al., 2007; Dutheil et al., 2009; Capozzi et al., generates 39-hydroxypaclitaxel (Rahman et al., 1994), 2014). CYP2C8 protein has been detected in heart, suggesting that compounds that are substrates of both hepatocytes, kidney, salivary ducts, small and large CYP2C8 and CYP3A4 bind differently to their active intestine, adrenal cortical cells, and tonsils (Läpple sites. et al., 2003; Enayetallah et al., 2004; Delozier et al., 2007; Cizkova et al., 2014). The expression of CYP2C8 and other CYP enzymes has recently been reviewed by A. Drugs Shahabi et al. (2014). CYP2C8 is involved in the metabolism of more than Analysis of liver samples has recently shown that a 100 clinically used drugs (Table 1). Typical substrate nearly full-length form of CYP2C8 (wild type) and an drugs of CYP2C8 include anticancer, antidiabetic, N-terminal truncated splice variant 3 are expressed in antimalarial, and lipid-lowering agents (Fig. 4). In- mitochondria (Bajpai et al., 2014). Although the wild- terestingly, some glucuronide metabolites of drugs in- type protein was detected only at low levels in mito- teract with CYP2C8. chondria (,25%), variant 3 was primarily targeted to 1. Anticancer Agents. The antimicrotubule agent mitochondria and minimally to the endoplasmic re- paclitaxel with a molecular weight of 853.9 g/mol is ticulum. Interestingly, although molecular modeling one of the largest substrates of CYP2C8. In vitro, showed that both the heme binding pocket and the paclitaxel is primarily metabolized by CYP2C8 to its substrate binding cavity were nearly intact in variant main 6a-hydroxy metabolite and by CYP3A4 to 39- 3, it was unable to catalyze paclitaxel 6-hydroxylation phenyl-hydroxypaclitaxel, and the further metabolism in human hepatocellular liver carcinoma cells. How- of these metabolites results in the formation of 6a,39-p- ever, it did metabolize smaller substrates such as dihydroxypaclitaxel (Cresteil et al., 1994, 2002; Harris and dibenzylfluorescein. Further- et al., 1994; Kumar et al., 1994; Rahman et al., 1994). more, the variant generated higher levels of reactive Paclitaxel 6a-hydroxylation is recommended by drug oxygen species and showed a higher level of mitochon- authorities as a marker reaction for CYP2C8 acti- drial respiratory dysfunction than wild type CYP2C8, vity in vitro (EMA, 2012b; http://www.fda.gov/Drugs/ suggesting that the mitochondrially targeted variant 3 DevelopmentApprovalProcess/DevelopmentResources/ may contribute to oxidative stress in tissues (Bajpai DrugInteractionsLabeling/ucm093664.htm), and it has et al., 2014). been widely used in preclinical studies. In a mass balance In living organisms, CYP enzymes undergo natural study in patients, metabolites accounted for about 40% of degradation that can be described as a first-order process the total systemic drug exposure, and the excreted 6a- (Yang et al., 2008). Therefore, the expression level of hydroxypaclitaxel corresponded to almost one-third of the enzyme is determined by the rate of enzyme the administered dose (Walle et al., 1995), suggesting synthesis and the degradation half-life of the enzyme. that ;30–40% of a paclitaxel dose is converted by The extent and dose and time dependency of enzyme CYP2C8 to 6a-hydroxy paclitaxel. induction and inactivation are thus also dependent on Cabazitaxel, a taxane approved in 2010, is also the degradation half-life. Based on clinical studies metabolized by CYP2C8 to a small extent in vitro with the CYP2C8 inactivator gemfibrozil, the degra- (FDA, 2010a), whereas there is conflicting data re- dation (turnover) half-life of CYP2C8 is approximately garding the role of CYP2C8 in the metabolism of 22 hours (Backman et al., 2009). docetaxel. One in vitro study demonstrated that 174 Backman et al.

TABLE 1 Drugs that are metabolized by CYP2C8, grouped by the importance of CYP2C8 in their elimination (Major, intermediate, minor)

Other CYP Enzymes Substrate Therapeutic Use and/or Metabolic Pathway(s) Involved in Overall References Drug Class Catalyzed by CYP2C8 Metabolisma Major (.70%) Amodiaquine Antimalarial N-deethylation (CYP1A1, CYP1B1) Li et al., 2002 Cerivastatin (acid, Antihyperlipidemic, HMG- 6-hydroxylation (M-23), CYP3A4 Backman et al., 2002 parent) CoA reductase inhibitor demethylation (M-1) Daprodustat Antianemic, prolyl CYP3A4 Johnson et al., 2014 (GSK1278863) hydroxylase inhibitor Dasabuvir (ABT-333) Antiviral, NSB5 inhibitor M1 formation CYP3A4, CYP2D6 FDA, 2014g Enzalutamide Anticancer, Hydroxylation (M6), CYP3A4/5 FDA, 2012k; Gibbons et al., N-demethylation (M2) 2015 Montelukast Antiasthmatic, LTRA 36-hydroxylation (M6), CYP3A4, CYP2C9 Karonen et al., 2010; 25-hydroxylation Filppula et al., 2011 (M3), M4 formation Pioglitazone Antidiabetic, PPAR-g Hydroxylation CYP3A4/5, CYP1A1 Jaakkola et al., 2006c; FDA, 2013a Repaglinide Antidiabetic, meglitinide M2 and M4 formation CYP3A4 Bidstrup et al., 2003; analog Kajosaari et al., 2005a

Intermediate (20–70%) 9cUAB30 Anticancer, retinoid M1-M5 formation CYP2C9, CYP2C19, Gorman et al., 2007 (CYP1A2, CYP2B6) Acotiamide (Z-338) Antidyspeptic, Deisopropylation (M-4) CYP1A1, CYP3A4 Furuta et al., 2004; PMDA, 2014 inhibitor Alitretinoin (9-cis- Antipsoriatic, retinoid 4-hydroxylation CYP2C9, CYP3A4, Marill et al., 2002 retinoic acid) CYP26A1 Amiodarone Antiarrhythmic N-deethylation CYP3A4, (CYP1A2, Ohyama et al., 2000 CYP2C19, CYP2D6) Chloroquine Antimalarial N-deethylation CYP3A4/5, (CYP2D6) Kim et al., 2003; Projean et al., 2003a (2)(+)- Gastroprokinetic, 5-HT4 N-dealkylation, 4- CYP3A4, CYP2B6 Desta et al., 2000, 2001 receptor agonist hydroxylation, 2- hydroyxlation Compound A ERA Hydroxylation Ma et al., 2004 Dabrafenib Anticancer, PKI Hydroxylation CYP3A4/5, (CYP2C9, Lawrence et al., 2014; CYP2C19) Suttle et al., 2015 Fenretinide Anticancer, retinoid 49-hydroxylation, CYP3A4/5 Illingworth et al., 2011 49-oxidation R/S- , SSRI N-demethylation CYP2C9, CYP2D6 Wang et al., 2014b R-Ibuprofen Anti-inflammatory, NSAID 2-hydroxylation, CYP2C9 Hamman et al., 1997; 3-hydroxylation Tornio et al., 2007; Chang et al., 2008 Imatinib Anticancer, PKI N-demethylation CYP3A4/5 Nebot et al., 2010; Filppula et al., 2013a,b Irosustat Anticancer, STS inhibitor M9 and M13 formation CYP2C9, CYP3A4/5, Ventura et al., 2011 (CYP2E1) Isotretinoin (13-cis- Antiacne, retinoid 4-hydroxylation CYP3A4 Marill et al., 2002 retinoic acid) Loperamide Antidiarrheal, N-demethylation CYP3A4, CYP2B6, CYP2D6 Kim et al., 2004; Niemi et al., 2006 N-demethylation CYP1A2, CYP2D6, CYP3A4 Korprasertthaworn et al., 2015 Olodaterol Antiasthmatic, LABA O-demethylation CYP2C9, (CYP3A4) FDA, 2014f Paclitaxel (taxol) Anticancer, taxane 6a-hydroxylation CYP3A4 Cresteil et al., 1994; Rahman et al., 1994; Walle et al., 1995 Paritaprevir (ABT-450) Antiviral, NS3-4A inhibitor CYP3A4 FDA, 2014g Propanoic acid Drug metabolite Hydroxylation (M10 and CYP1A1 Klieber et al., 2014 dronedarone M11) R483 Antidiabetic, PPAR-g M1 and M4 formation CYP2C19, CYP3A4, Bogman et al., 2010 agonist (CYP2C9) Rosiglitazone Antidiabetic, PPAR-g p-hydroxylation, CYP2C9 Baldwin et al., 1999 agonist N-demethylation acid Antihyperlipidemic, HMG- Oxidation (M1-M3) CYP3A4/5 Prueksaritanont et al., 2003 CoA reductase inhibitor Tazarotenic acid Antipsoriatic, drug Sulfoxidation Attar et al., 2003 metabolite (active) Tozasertib (MK 0457, Anticancer, PKI N-demethylation CYP3A4 Ballard et al., 2007 VX6, VX 680) Treprostinil Antihypertensive CYP2C9 FDA, 2009b Troglitazone Antidiabetic, PPAR-g Quinone metabolite CYP3A4 Yamazaki et al., 1999b agonist formation (continued) Role of CYP2C8 in Drug Metabolism and Interactions 175

TABLE 1—Continued

Other CYP Enzymes Substrate Therapeutic Use and/or Metabolic Pathway(s) Involved in Overall References Drug Class Catalyzed by CYP2C8 Metabolisma R/S-Verapamil Antihypertensive, CCB N-dealkylation, N- CYP3A4/5, (CYP2E1) Busse et al., 1995; Tracy demethylation, O- et al., 1999 demethylation Vidupiprant (AMG 853) Antiasthmatic, PGD2 t-butyl hydroxylation CYP2J2, CYP3A Foti et al., 2012 receptor antagonist (M2), cyclopropyl hydroxylation (M3) , GABA receptor N-demethylation, N- CYP3A4, CYP2C9 Becquemont et al., 1999; agonist oxidation Tornio et al., 2006 Unknown or Minor (;,20%) 5-MeO-DIPT (Foxy) Hallucinogenic N-deisopropylation CYP2D6, CYP1A2, Narimatsu et al., 2006 CYP3A4, CYP2C19 7-Epi-10-deacetyl- Paclitaxel derivative Hydroxylation CYP3A4 Zhang et al., 2009a paclitaxel 7-Epi-cephalomannine Paclitaxel derivative M-2 formation CYP3A4 Zhang et al., 2009a 7-Epi-paclitaxel Paclitaxel epimer M-2 formation CYP3A4 Zhang et al., 2009b 10-Aceyldocetaxel Docetaxel derivative 6-hydroxylation CYP3A4 Cresteil et al., 2002 10-Deacetylpaclitaxel Paclitaxel derivative 6-hydroxylation CYP3A4 Cresteil et al., 2002 17a-Ethinylestradiol Contraceptive, hormone 2-hydroxylation Wang et al., 2004 derivative 17b- (estradiol) Hormonal replacement 2-hydroxylation, 4- CYP1A1, CYP1B1 Spink et al., 1992 therapy hydroxylation Aminophenazone N-demethylation CYP2C19, CYP2B6, Niwa et al., 1999, 2000 (aminopyrine) CYP2D6 Antidepressant, TCA N-demethylation CYP3A4/5, CYP2C19 Venkatakrishnan et al., 2001 Anticancer, Hydroxylation CYP3A4, CYP3A5 Kamdem et al., 2010 Inhibitor Apixaban , factor Xa O-demethylation CYP3A4, CYP1A2, FDA, 2012d inhibitor CYP2C9, CYP2C19, CYP2J2 Apremilast Antipsoriatic, PDE4 M5 formation CYP3A4, CYP2A6, FDA, 2014e inhibitor (CYP1A2, CYP2C9, CYP2E1) Artelinic acid Antimalarial 3-hydroxylation CYP3A4/5 Grace et al., 1999 (acid, Antihyperlipidemic, HMG- p-hydroxylation CYP3A4/5 Jacobsen et al., 2000b parent) CoA reductase inhibitor Azilsartan Antihypertensive, ARB Decarboxylation (M-I), CYP2C9, CYP2B6 FDA, 2011c O-dealkylation (M-II) Bedaquiline , ATP synthase N-demethylation CYP3A4, CYP2C19 Liu et al., 2014 inhibitor Brinzolamide Antiglaucoma, carbonic CYP3A4, CYP2A6, EMA, 2014 anhydrase inhibitor CYP2B6, CYP2C9 Brivaracetam Antiepileptic Hydroxylation CYP2C9, CYP3A4 Whomsley et al., 2007; Nicolas et al., 2012 Analgesic, opioid N-dealkylation, M1 CYP3A4 Moody et al., 2002; Picard formation et al., 2005; Chang et al., 2006 CYP3A4 Karlsson et al., 2013 BYZX Antidementia, N-deethylation (M3) CYP3A4 Yu et al., 2013a acetylcholinesterase inhibitor BYZX M2 Drug metabolite N-deethylation (M1) CYP3A4 Yu et al., 2013a Cabazitaxel Anticancer, taxane RPR 112698 formation CYP3A4/5 FDA, 2010a Psychostimulant N-demethylation, CYP3A4, CYP1A2, CYP2C9 Kot and Daniel, 2008 C-8-hydroxylation Capravirine Antiviral, NNRTI Sulfoxidation (C23), CYP3A4, CYP2C9, Bu et al., 2006 N-oxidation (C26), CYP2C19 hydroxylation (C19) Antiepileptic 10,11-epoxidation, CYP3A4 Kerr et al., 1994; Pelkonen 3-hydroxylation et al., 2001 Cephalomannine Paclitaxel derivative 4a-hydroxylation Zhang et al., 2009a Antidementia, 5-HT6 Demethylation CYP3A4 Tse et al., 2014 receptor antagonist Cilostazol Antithrombotic, PDE3 OPC-13217 formation CYP3A4/5, CYP1B1, Hiratsuka et al., 2007 inhibitor CYP2C19 Gastroprokinetic, 5-HT4 CYP3A4 Robert et al., 2007 receptor agonist E-Clomiphene Ovulation inducer, SERM Deethylation, CYP3A4/5, CYP2D6 Mürdter et al., 2012 hydroxylation Antipsychotic N-demethylation, oxidation CYP1A2, Linnet and Olesen, 1997 (CYP3A4) (continued) 176 Backman et al.

TABLE 1—Continued

Other CYP Enzymes Substrate Therapeutic Use and/or Metabolic Pathway(s) Involved in Overall References Drug Class Catalyzed by CYP2C8 Metabolisma CPI-613 Anticancer, CYP3A4, (CYP2C9, Lee et al., 2011 antimitochondrial CYP2C19) metabolism agent Antipsychotic N-demethylation CYP1A2, CYP3A4, CYP2C9 Arbus et al., 2007 Cyclophosphamide Anticancer, alkylating 4-hydroxylation CYP2C9, CYP2A6, Chang et al., 1993; Huang agent CYP2B6, CYP3A4 et al., 2000 Cyclosporine Immunosuppressant, CYP3A4 Karlsson et al., 2013 calcineurin inhibitor Dapsone N-hydroxylation CYP2C9 Winter et al., 2000 Anxiolytic, N-demethylation, 3- CYP2C9, CYP3A4, Sai et al., 2000 hydroxylation CYP2C19 Dibenzylfluorescein Fluorescent CYP probe O-debenzylation CYP3A4, CYP2C19, Miller et al., 2000 CYP2C9, CYP3A5, CYP3A7 Anti-inflammatory, NSAID 49-hydroxylation, 5- CYP2C9, CYP3A4, Mancy et al., 1999 hydroxylation CYP2C18/19 Diltiazem Antihypertensive, CCB N-demethylation CYP3A4, CYP2C9, CYP2D6 Sutton et al., 1997 Docetaxel Anticancer, taxane Baccatin ring hydroxylation CYP3A4 Komoroski et al., 2005 Dovitinib Anticancer, PKI CYP1A1/2, CYP2D6, Kim et al., 2011c CYP3A4 DY-9760e Calmodulin antagonist oxidation (M8), CYP3A4, CYP2C9, Tachibana et al., 2005 N-dealkylation (DY- CYP2C19 9836), O-demethylation (M5), phenyl hydroxylation (M3) Eltrombopag , c-mpl Monooxygenation (J and CYP1A2 FDA, 2008c receptor agonist M6) Antidepressant N-demethylation (M4) CYP3A4 Kamel et al., 2013 Erlotinib Anticancer, PKI CYP3A4/5, CYP1A2, FDA 2004; Ling et al., 2006 CYP1A1, CYP1B1 , solvent Acetaldehyde formation CYP2E1, CYP1A2 Hamitouche et al., 2006 Etodolac Anti-inflammatory, NSAID 6-hydroxylation, 7- CYP2C9 Tougou et al., 2004 hydroxylation Evatanepag (CP-533,536) Prostaglandin EP2 receptor Formation of M3, M4, M20, CYP3A4/5 Prakash et al., 2008 agonist M22-M6 Everolimus Immunosuppressant, PKI Hydroxylation CYP3A4/5 Jacobsen et al., 2001; Picard et al., 2011 Antihyperuricemic, XO Hydroxylation (67M-2) CYP1A2, CYP2C9 Mukoyoshi et al., 2008; inhibitor FDA, 2009c Felodipine Antihypertensive, CCB CYP3A4 Karlsson et al., 2013 Flutamide Anticancer, antiandrogen Flu-1-G2 formation CYP1A2, CYP3A4, CYP2C9 Kang et al., 2008 (acid, parent) Antihyperlipidemic, HMG- 5-hydroxylation CYP2C9, CYP1A1, Fischer et al., 1999 CoA reductase inhibitor CYP2D6, CYP3A4 Gallopamil Antiarrhythmic, CCB Oxidation CYP3A4, CYP2D6 Suzuki et al., 1999 Genistein Anticancer, PKI 39-hydroxylation CYP1A2, CYP2E1 Hu et al., 2003 Gliclazide Antidiabetic, sulfonylurea 6b-hydroxylation, 7b- CYP2C9, CYP2C19 Elliot et al., 2007 hydroxylation Glyburide Antidiabetic, sulfonylurea 4-trans- (M1) and 3-cis- CYP3A4, CYP2C9, CYPC19 Zharikova et al., 2009 (glibenclamide) hydroxycyclohexyl (M2b) glyburide formation Halofantrine Antimalarial N-debutylation CYP3A4/5 Baune et al., 1999 Ibrolipim (NO-1886) Antihyperlipidemic O-deethylation (M2) CYP3A4 Morioka et al., 2002 ID951551 Acotiamide analog Deisopropylation CYP3A4, CYP1A1 Furuta et al., 2004 Ifosfamide Anticancer, alkylating 4-hydroxylation CYP2C9, CYP2A6, Chang et al., 1993; Huang agent CYP2B6, CYP3A4 et al., 2000 IN-1130 Anticancer, PKI M1 and M3 formation CYP3A4, CYP2D6, Kim et al., 2008 CYP2C19 K11777 , cysteine Formation of N-oxide CYP3A4 Jacobsen et al., 2000a protease inhibitor b-hydroxy-homoPhe and N- demethyl metabolites Karenicetin Anticancer CYP3A4, CYP2D6 Smith et al., 2003 L-775,606 Antimigraine, Hydroxylation (M1), N- CYP3A4 Prueksaritanont et al., 2000 dealkylation (M2) Lansoprazole Antiulcerative, PPI 5-hydroxylation CYP2C19, CYP3A4 Pichard et al., 1995 Anticancer, PKI O-dealkylation, N- CYP3A4/5, CYP2C19 FDA, 2007e; Teng et al., dealkylation 2010 Licofelone Anti-inflammatory, NSAID Hydroxylation (M2 and M4) CYP2C9, CYP2C19, Albrecht et al., 2008 CYP2D6, CYP2J2, CYP3A4 Lonafarnib Anticancer, FTI Hydroxylation (M4) CYP3A4/5, CYP1A1 Ghosal et al., 2006 Macitentan Antihypertensive, ERA Depropylation CYP3A4, CYP2C9, Sidharta et al., 2015 CYP2C19 (continued) Role of CYP2C8 in Drug Metabolism and Interactions 177

TABLE 1—Continued

Other CYP Enzymes Substrate Therapeutic Use and/or Metabolic Pathway(s) Involved in Overall References Drug Class Catalyzed by CYP2C8 Metabolisma Mavoglurant Anti-Parkinson, mGLUR5 M7 formation CYP3A4, CYP1A1 Walles et al., 2013 antagonist Methadone Analgesic, opioid N-demethylation CYP2B6, CYP3A4, CYP2D6 Iribarne et al., 1996; Wang and DeVane, 2003; Chang et al., 2011 Mirodenafil Erectogenic, PDE5 N-dealkylation CYP3A4, CYP2D6 Lee et al., 2008 inhibitor Antidepressant, NaSSA N-demethylation CYP2D6, CYP1A2, CYP3A4 Störmer et al., 2000 Analgesic, opioid N-demethylation CYP3A4 Projean et al., 2003b Muraglitazar Antidiabetic, PPARa, and g O-demethylation, O- CYP2C9, CYP2C19, Zhang et al., 2007a agonist dealkylation CYP2D6, CYP3A4 hydroxylation, N-acetyl- imide metabolite formation (2)(+)- Antihypertensive M1-M5 formation CYP2C9, CYP2C19 Zhu et al., 2014 Nalfurafine , opioid Decyclopropylmethylation CYP3A4, CYP2C9, Ando et al., 2012 CYP2C19 Naproxen Anti-inflammatory, NSAID O-demethylation CYP2C9, CYP1A2 Rodrigues et al., 1996, Tracy et al., 1997 Nicotine Psychostimulant 5-hydroxylation CYP2A6, CYP2B6 Yamazaki et al., 1999a Nifedipine Antihypertensive, CCB CYP3A4/5 Karlsson et al., 2013 Nilotinib Anticancer, PKI CYP3A4, CYP1A1/2, FDA, 2007c CYP2J2 R/S-Norverapamil Drug metabolite O-demethylation, N- CYP3A4/5 Tracy et al., 1999 dealkylation Odanacatib Antiosteoporotic, cathepsin Methyl hydroxylation (M8) CYP3A4/5 Kassahun et al., 2014 K Ombitasvir (ABT-267) Antiviral, NS5A inhibitor CYP3A4, CYP3A5 FDA, 2014k Omeprazole Antiulcerative, PPI 5-hydroxylation CYP2C19, CYP3A4 Karam et al., 1996 Pafuramidine maleate Antiparasitic O-demethylation (M1) CYPF4 Wang et al., 2006 (DB289) Anticancer, PKI Mono-oxygenation CYP3A4, CYP1A2 FDA, 2009d Antipsychotic MX 1, 10-11614, CO-UK2, CYP3A4, CYP2D6, Mizuno et al., 2003; and CO-UK3 formation (CYP1A1) Kitamura et al., 2005 Antipsychotic N-dealkylation CYP1A2, CYP3A4, Olesen and Linnet, 2000 CYP2C19, CYP2D6, (CYP2C18) Phenazone (antipyrine) Analgesic N-demethylation, 3- CYP3A4, CYP1A2, CYP2C9 Engel et al., 1996 hydroxylation, 4- hydroxylation Phenprocoumon Antithrombotic, VKA S-49-hydroxylation CYP2C9, CYP3A4 Ufer et al., 2004 Antiepileptic 4-hydroxylation CYP2C9, CYP2C19 Doecke et al., 1990 Piperaquine Antimalarial CYP3A4 Lee et al., 2012c acid Antihyperlipidemic, HMG- CYP2C9 Fujino et al., 2004 CoA reductase inhibitor Anticancer, PKI CYP3A4, CYP2D6, CYP3A5 FDA, 2012e Hormonal replacement CYP2C19, CYP3A4 Waxman et al., 1991 therapy 4-hydroxylation CYP2C9, CYP1A2, CYP2B6 Guitton et al., 1998 Antihypertensive, sGC N-demethylation CYP1A1, CYP3A4, CYP2J2 FDA, 2013b stimulator Anti-Parkinson, Desthienylethyl rotigotine CYP1A2, CYP2C9, CYP3A4 FDA, 2007b agonist formation Antipsychotic M203, EMD148107, EMD CYP3A4, CYP2C9, CYP1A2 Gallemann et al., 2010 329989, and M364d formation Selegiline Anti-Parkinson, MAO-B N-demethylation CYP2B6, CYP2C19 Hidestrand et al., 2001, inhibitor Salonen et al., 2003 Semagacestat Antidementia, g-secretase Benzylic hydroxylation CYP3A4/5 Yi et al., 2010 inhibitor (M3) Antiasthmatic, TXRA 5-methyl hydroxylation, 49- CYP3A4/5, CYP2C9 Kumar et al., 1997 hydroxylation Sildenafil Erectogenic, PDE5 CYP3A4, CYP2C9 Karlsson et al., 2013 inhibitor (TMC435) Antiviral, protease M21 and M2 formation CYP3A4/5, CYP2C19 FDA, 2013h inhibitor Sipoglitazar Antidiabetic, PPARa Hydroxylation (M-II) Nishihara et al., 2012 agonist Sirolimus Immunosuppressant Hydroxylation CYP3A4/5 Jacobsen et al., 2001 Antidiabetic, DPP-4 M2 and M5 formation CYP3A4 Vincent et al., 2007 inhibitor Sulfadiazine Antimicrobial N-hydroxylation CYP2C9 Winter and Unadkat, 2005 (continued) 178 Backman et al.

TABLE 1—Continued

Other CYP Enzymes Substrate Therapeutic Use and/or Metabolic Pathway(s) Involved in Overall References Drug Class Catalyzed by CYP2C8 Metabolisma Sunitinib Anticancer, PKI N-deethylation CYP3A4, CYP2B6, FDA, 2006b CYP2C9/19 T-5 Erectogenic, PDE5 N-oxidation CYP3A5 Li et al., 2014a inhibitor Tacrolimus Immunosuppressant, CYP3A4/5 Karlsson et al., 2013 calcineurin inhibitor Tamoxifen Anticancer, SERM M-I formation CYP3A4/5, CYP2D6 Desta et al., 2004 Tamoxifen N-oxide Drug metabolite Reduction to tamoxifen CYP2A6, CYP1A1, CYP3A4 Parte and Kupfer, 2005 Tegafur Anticancer, prodrug 5-hydroxylation CYP1A2, CYP2A6 Komatsu et al., 2000b, 2001 Sedative, benzodiazepine N-demethylation Yang et al., 1998 Terbinafine N-demethylation, side CYP2C9, CYP1A2, CYP3A4 Vickers et al., 1999 chain oxidation Hormonal replacement CYP3A4/5, CYP2B6 Waxman et al., 1991 therapy Tienilic acid 5-hydroxylation CYP2C9 Lopez Garcia et al., 1993; Bonierbale et al., 1999 Tipifarnib Anticancer, FTI CYP3A4, CYP2C19, Perez-Ruixo et al., 2006 CYP2A6, CYP2D6, CYP2C9 R-Tofisopam Anxiolytic M3 formation CYP3A4, CYP2C9, Cameron et al., 2007 (CYP3A5, CYP2C19) Tolbutamide Antidiabetic, sulfonylurea p-methyl hydroxylation CYP2C9, CYP2C19 Relling et al., 1990; Veronese et al., 1993; Rettie et al., 1994; Komatsu et al., 2000a Torsemide (torasemide) Diuretic Methyl hydroxylation CYP2C9 Miners et al., 2000; Ong et al., 2000 Trabectedin Anticancer N-demethylation CYP3A4, CYP2D6 Vermeir et al., 2009 Tretinoin (all-trans- Antiacne, retinoid 4-hydroxylation, CYP26A1, CYP3A4/5, Leo et al., 1989; Nadin and retinoic acid) 18-hydroxylation, CYP2B6, CYP1A2, Murray, 1999; Marill 5,6-epoxy metabolite CYP26, CYP2C9 et al., 2000; Thatcher formation et al., 2010 Trimethadione Antiepileptic N-demethylation CYP2E1, CYP3A4, CYP2C9 Kurata et al., 1998 (troxidone) Vanoxerine Antiarrhythmic, DRI CYP3A4, CYP2E1 Cherstniakova et al., 2001 R-Warfarin Antithrombotic, VKA 4-hydroxylation, CYP3A4, CYP1A1, Scordo et al., 2002; Kim 7-hydroxylation CYP2C19, CYP1A2 et al., 2012 Vitamin A (retinol) Anti-acne, retinoid Hydroxylation CYP1A1, CYP1A2, Leo et al., 1989 CYP1B1, CYP2D6, CYP3A4 Antidepressant, SMS Sulfoxide (M4a) formation CYP2D6, CYP3A4/5, FDA, 2013j; Chen et al., CYP2C19, CYP2C9, 2014 CYP2A6 Antiviral, NRTI Reduction CYP2C9 Eagling et al., 1994

5-HT, 5-hydroxytryptamine (); 5-MeO-DIPT, 5-methoxy-N,N-; 9cUAB30, (2E,4E,6Z,8E)-8-(39,4’-dihydro-1’(2’H)-naphthalen-1’-ylidene)-3,7-dimethyl- 2,4,6- octatrienoic acid; ARB, angiotensin II receptor blocker; ATP, adenosine triphosphate; BYZX, [(E)-2-(4-((diethylamino)methyl)benzylidene)-5,6-dimethoxy-2,3-dihydroinden-one]; CCB, ; Compound A, [(+)-(5S,6R,7R)-2-isopropylamino-7-[4-methoxy-2-((2R)-3-methoxy-2-methylpropyl)-5-(3,4-methylenedioxyphenyl) cyclopenteno[1,2-b] pyridine 6-carboxylic acid]; DPP, dipeptidyl peptidase; DRI, dopamine reuptake inhibitor; DY-9760, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4- imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate; ERA, endothelin receptor antagonist; FTI, farnesyl inhibitor; GABA, g-aminobutyric acid; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; IN-1130, 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide; K11777, N-methyl--Phe-homoPhe- vinylsulfone-phenyl; L-775,606, (1-(3-(5-(1,2, 4-triazol-4-yl)-1H-indol-3-yl)propyl)-4-(2-(3-fluorophenyl)ethyl)piperazine); LABA, long-acting b-adrenoceptor agonist; LTRA, leukotriene receptor antagonist; MAO, monoamine oxidase; mGLUR, metabotropic glutamate receptor; c-mpl, myeloproliferative leukemia; NaSSA, noradrenergic and specific antidepressant; NNRTI, non-nucleoside reverse transcriptase inhibitor; NRTI, nucleoside analog reverse transcriptase inhibitor; NS, nonstructural protein; NSAID, nonsteroidal anti-inflammatory drug; PDE, phosphodiesterase; PGD2, prostaglandin D2; PKI, protein kinase inhibitor; PPAR, peroxisome proliferator- activated receptor; PPI, proton pump inhibitor; sGC, soluble guanylate cyclase; SERM, selective estrogen ; SMS, serotonin modulator and stimulator; SSRI, selective serotonin reuptake inhibitor; STS, steroid sulfatase; T-5, methyl 2-(4-aminophenyl)-1-oxo-7-(pyridin-2-ylmethoxy)-4-(3,4,5-trimethox- yphenyl)-1,2-dihydroisoquinoline-3-carboxylate; TCA, ; TXRA, thromboxane A2 receptor antagonist; VKA, vitamin K antagonist; XO, oxidase. aThis information is either based on the references given in the table or on data from the UW Metabolism and Transport Drug Interaction Database (DIDB), Copyright University of Washington 1999-2015 (DIDB Accessed May-September, 2015). docetaxel at high concentrations was metabolized by The androgen receptor antagonist enzalutamide, in- CYP2C8, but no enzyme kinetic parameters were dicated for treatment of castration-resistant prostate determined (Komoroski et al., 2005). An earlier cancer, is mainly metabolized by CYP2C8 and CYP3A4/ study, however, suggested that CYP2C8 is unable to 5 to enzalutamide M6 in vitro (FDA, 2012k). Then, M6 is accommodate docetaxel in its active site because of further metabolized by CYP2C8 to the active metabolite the absence of a side chain in the docetaxel mole- N-demethyl enzalutamide (M2), which accounts for cule (Cresteil et al., 2002). The side chain, which is approximately 50% of the total drug exposure in present in the paclitaxel molecule, is required for plasma. CYP2C8 seems to be the predominant enzyme a correct orientation of it into the active site of involved in enzalutamide pharmacokinetics also in vivo CYP2C8. (section VI.C.5; Gibbons et al., 2015). Role of CYP2C8 in Drug Metabolism and Interactions 179

TABLE 2 Glucuronide metabolites that are metabolized by CYP2C8

Metabolic pathway Other CYP enzymes involved Substrate catalyzed by CYP2C8 in overall metabolism References

a Clopidogrel acyl 1-b-D-glucuronide Tornio et al., 2014 Desloratadine N-glucuronide 3-hydroxylation Kazmi et al., 2015 Diclofenac acyl glucuronide 49-hydroxylation Kumar et al., 2002 Estradiol-17b-glucuronide 2-hydroxylation CYP3A7 Delaforge et al., 2005 Gemfibrozil 1-O-b glucuronide Benzylic oxidation Ogilvie et al., 2006; Baer et al., 2009 Licofelone 1-O-acyl glucuronide (M1) Hydroxylation (M3) Albrecht et al., 2008 Lu AA34893 carbamoyl glucuronidea Kazmi et al., 2010 MRL-C acyl glucuronide Hydroxylation CYP3A4 Kochansky et al., 2005 Sipoglitazar b-1-O-acyl glucuronide O-dealkylation (M-I) Nishihara et al., 2012

MRL-C, 2-[[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy]-2-methylpropanoic acid. aThese glucuronides are likely to be substrates of CYP2C8 based on their metabolism-dependent inhibitory effect on CYP2C8.

Several protein kinase inhibitors are metabolized CYP2C8 only catalyzes the formation of the main by CYP2C8 to various degrees in vitro (Table 1). In metabolite, N-demethylimatinib (Table 4; Rochat et al., vitro, the majority (60–70%) of dabrafenib, a selective 2008; Filppula et al., 2013a). The relative roles of BRAF inhibitor, is metabolized by CYP2C8, and a CYP2C8 and CYP3A4 in the in vivo pharmacokinetics smaller part by CYP3A4 (;25%) and CYP2C9 (#10%) of imatinib are complex (section VI.C.5; Filppula et al., (Lawrence et al., 2014). Recombinant CYP2C8 pro- 2013b). In vitro, the multitargeted kinase duced only hydroxydabrafenib, whereas CYP3A4 formed inhibitor ponatinib is mainly metabolized by CYP3A4, both hydroxydabrafenib and carboxydabrafenib. How- followed by CYP2C8, CYP2D6, and CYP3A5. The con- ever, the in vivo importance of CYP2C8 in dabrafenib tributions of CYP3A4 and CYP2C8 to the in vivo pharmacokinetics seems to be quite modest (section VI. elimination of ponatinib are estimated to 34% and 19%, C.5; Suttle et al., 2015). Imatinib, the first tyrosine respectively (FDA, 2012e). Furthermore, CYP2C8 plays kinase inhibitor approved for clinical use, is metabo- a major role in the N-demethylation of the aurora kinase lized by several CYP enzymes in vitro, with CYP2C8 inhibitor tozasertib (Table 4; Ballard et al., 2007). With and CYP3A4 being the most important ones (Nebot the exception of dabrafenib and imatinib, the in vivo et al., 2010; Filppula et al., 2013a). CYP3A4 is involved importance of CYP2C8 in the metabolism of protein in several metabolic pathways of imatinib, whereas kinase inhibitors seems to have been poorly studied.

TABLE 3 Natural and endogenous compounds that are metabolized by CYP2C8

Metabolic Pathway(s) Other CYP Enzymes Involved Substrate Description Catalyzed by CYP2C8 in Overall Metabolisma References 1-Hydroxyl-2,3,5-trimethoxyxanthone Constituent of M1-M4 formation CYP3A4, CYP1A2, Feng et al., 2014 Halenia elliptica CYP2A6, CYP2B6, CYP2C9, CYP2C19 7a- and 7b-Hydroxy-D8-THC Marijuana constituent Oxidation CYP3A4, CYP2C9 Watanabe et al., 2007 8-Prenylnaringenin (flavaprenin) Prenylflavonoid M2 formation CYP2C19 Guo et al., 2006 Arachidonic acid Endogenous CYP2C9, CYP1A2, Daikh et al., 1994; Rifkind compound CYP2E1, CYP2J2 et al., 1995; Zeldin et al., 1995; Barbosa-Sicard et al., 2005 Endogenous CYP2C9/11/23 Barbosa-Sicard et al., 2005 compound Eupatilin Flavone 4-O-demethylation CYP1A2 Lee et al., 2007 R/S-Limonene Terpene CYP2C19, CYP2C9, Miyazawa et al., 2002 CYP3A4 Magnolin Constituent of Shin-i O-demethylation CYP2C9, CYP2C19, Kim et al., 2011a (M1 and M2), CYP3A4 hydroxylation (M4) Mesaconitine M1-M2, M7-M9 CYP3A4, CYP2C9, Ye et al., 2011 formation CYP2D6 Nitidine chloride Alkaloid CYP3A4 Li et al., 2014b Silybin () O-demethylation (CYP3A4) Jancova et al., 2007 Tanshinol ester Combination of the M1-M5 formation (CYP3A4) Liu et al., 2010b natural compounds danshensu and borneol

THC, . aThis information is either based on the references given in the table or on data from the UW Metabolism and Transport Drug Interaction Database (DIDB), Copyright University of Washington 1999-2015 (DIDB Accessed May-September, 2015). 180 Backman et al.

TABLE 4 CYP2C8-mediated reactions in vitro

a Substrate Metabolic Pathway Km Vmax CLint Test System References

mM pmol/min/pmol ml/min/pmol (pmol/min/mg) (ml/min/mg) 5-MeO-DIPT (Foxy) N-deisopropylation 291 1.7 0.0058 rCYP2C8 Narimatsu et al., 2006 7-Epi-10-deacetyl- Hydroxylation 18.0 3.038 0.17 rCYP2C8 Zhang et al., 2009a paclitaxel 7-Epi-cephalomannine M-2 formation 2.6 1.882 0.72 rCYP2C8 Zhang et al., 2009a 7-Epi-paclitaxel M-2 formation 1.4 1.409 1.0 rCYP2C8 Zhang et al., 2009b 8-prenylnaringenin M2 formation 3.72 4.64 1.3 rCYP2C8 Guo et al., 2006 17a-ethinylestradiol 2-hydroxylation 12 0.064 0.0053 rCYP2C8 Wang et al., 2004 Acotiamide (Z-338) Deisopropylation 152 (12.7) (0.084) rCYP2C8 Furuta et al., 2004 318 (347) (1.19) HLM Furuta et al., 2004 Alitretinoin (9-cis-retinoic 4-hydroxylation 7 0.948 0.14 rCYP2C8 Marill et al., 2002 acid) Aminophenazone N-demethylation 5,300 188 0.035 rCYP2C8 Niwa et al., 1999 (aminopyrine) Amiodarone N-deethylation 8.6 2.3 0.27 rCYP2C8 Ohyama et al., 2000 5.22 (12.2) (2.3) rCYP2C8 Soyama et al., 2002 Amitriptyline N-demethylation 0.072 rCYP2C8 Venkatakrishnan et al., 2001 Amodiaquine N-deethylation 0.9-1.2 2.6-3.9 2.1-4.4 rCYP2C8 Li et al., 2002 2.4 (1,462) (610) HLM Li et al., 2002 0.728 11.2 15 rCYP2C8 Walsky and Obach, 2004 1.89 (1,480) (780) HLM Walsky and Obach, 2004 0.81 0.23 0.28 rCYP2C8 Parikh et al., 2007 1 11 11 rCYP2C8 O’Donnell et al., 2007 1.95 6.87b 3.5b rCYP2C8 Baer et al., 2009 1.6 (9,130) (5,700) HLM Perloff et al., 2009 3.0 5.7b 1.9b rcCYP2C8 Kaspera et al., 2011 3.33-5.17 (1,180-2,770) (220-830) HLM Sjogren et al., 2012 3.9-7.3 (791-861) (120-200) HLM Yang et al., 2012 1.9 (2,196) (1,200) HLM Misaka et al., 2013 1.8 (7.3) (4.1) HIM Misaka et al., 2013 58.8 3,234c 55c Hep Kosugi et al., 2014 0.22-42.44 19.91-1,140c 24-95c Hep Li and Schlicht, 2014 Anastrozole Hydroxylation 86.8 0.00005 ,0.001 rCYP2C8 Kamdem et al., 2010 Arachidonic acid Total oxidative 6.0 4.6d 0.77d rCYP2C8 Barbosa-Sicard et al., 2005 metabolism Epoxidation 71 0.078 0.0011 rCYP2C8 Lundblad et al., 2005 Atorvastatin (acid, parent) p-hydroxylation 35.9 0.29 0.0081 rCYP2C8 Jacobsen et al., 2000b Bedaquiline N-demethylation 13.1 rCYP2C8 Liu et al., 2014 Buprenorphine N-dealkylation 12.4 (176.3) (14) rCYP2C8 Picard et al., 2005 Buspirone Total oxidative metabolism 0.073 rCYP2C8 Karlsson et al., 2013 BYZX N-deethylation (M3) 62.1 0.099e 0.0016e rCYP2C8 Yu et al., 2013a BYZX M2 N-deethylation (M1) 143.2 (0.000370) (,0.001) rCYP2C8 Yu et al., 2013a Caffeine 1-N-demethylation 920 0.014 ,0.001 rCYP2C8 Kot and Daniel, 2008 3-N-demethylation 200 0.016 ,0.001 rCYP2C8 Kot and Daniel, 2008 7-N-demethylation 3,560 0.172 ,0.001 rCYP2C8 Kot and Daniel, 2008 C-8-hydroxylation 3,370 0.319 ,0.001 rCYP2C8 Kot and Daniel, 2008 Carbamazepine 10,11-epoxidation 757 0.669 ,0.001 rCYP2C8 Cazali et al., 2003 Cephalomannine 6a-hydroxylation 41.3 5.267 0.13 rCYP2C8 Zhang et al., 2009a Cerivastatin (acid, parent) 6-hydroxylation 23 0.22 0.0096 rcCYP2C8 Kaspera et al., 2010 Demethylation 24 0.57 0.024 rcCYP2C8 Kaspera et al., 2010 Cerlapirdine Demethylation 3.3 3.4 1.0 rCYP2C8 Tse et al., 2014 Chloroquine N-deethylation 430 52.1 0.12 rCYP2C8 Kim et al., 2003 111 8.33 0.075 rCYP2C8 Projean et al., 2003a Cilostazol OPC-13217 formation 33.8 0.30 0.089 rCYP2C8 Hiratsuka et al., 2007 Cisapride 4-hydroxylation ;5.9 0.71 ;0.12 rCYP2C8 Desta et al., 2000 N-dealkylation ;0.91 0.29 ;0.32 rCYP2C8 Desta et al., 2000 2-hydroxylation 5.80 0.0267 0.0046 rCYP2C8 Pearce et al., 2001 4-hydroxylation 3.40 0.289 0.085 rCYP2C8 Pearce et al., 2001 N-dealkylation 2.0 0.109 0.055 rCYP2C8 Pearce et al., 2001 (2)-Cisapride 4-hydroxylation 13.3 0.15 0.011 rCYP2C8 Desta et al., 2001 (+)-Cisapride 4-hydroxylation 12.6 0.24 0.019 rCYP2C8 Desta et al., 2001 Cyclosporine Total oxidative metabolism 0.40 rCYP2C8 Karlsson et al., 2013 Dapsone N-hydroxylation 58-75 0.440 0.0059-0.0076 rCYP2C8 Winter et al., 2000 Dibenzylfluorescein O-debenzylation 1.0 0.4d 0.4d n/a Miller et al., 2000 29.16 0.79 0.027 rCYP2C8 Ghosal et al., 2003 1.9 (1.3) (0.68) THLE Donato et al., 2004 Diclofenac 49-hydroxylation 630 1.2b,d 0.0019b,d rCYP2C8 Mancy et al., 1999 5-hydroxylation 280 7b,d 0.025b,d rCYP2C8 Mancy et al., 1999 DY-9760e Imidazole oxidation (M8) 2.6 0.0732 0.028 rCYP2C8 Tachibana et al., 2005 N-dealkylation (DY-9836) 15.2 0.0231 0.0015 rCYP2C8 Tachibana et al., 2005 O-demethylation (M5) 3.1 0.0128 0.0041 rCYP2C8 Tachibana et al., 2005 Phenyl hydroxylation (M3) 2.5 0.2955 0.12 rCYP2C8 Tachibana et al., 2005 (continued) Role of CYP2C8 in Drug Metabolism and Interactions 181

TABLE 4—Continued

a Substrate Metabolic Pathway Km Vmax CLint Test System References Eicosapentaenoic acid Total oxidative metabolism 5.4 6.2d 1.1d rCYP2C8 Barbosa-Sicard et al., 2005 Estradiol-17b-glucuronide 2-hydroxylation 88 1.86 0.021 rCYP2C8 Delaforge et al., 2005 Ethanol Acetaldehyde formation 8,300 (0.0043) (,0.001) rCYP2C8 Hamitouche et al., 2006 Eupatilin 4-O-demethylation 4.5 0.94 0.21 rCYP2C8 Lee et al., 2007 Felodipine Total oxidative metabolism 1.1 rCYP2C8 Karlsson et al., 2013 Fenretidine 49-hydroxylation 2.2 282f 130f rCYP2C8 Illingworth et al., 2011 49-oxidation 5.0 30f 6.0f rCYP2C8 Illingworth et al., 2011 R-Fluoxetine N-demethylation 153.8 (6.08) (0.040) rcCYP2C8 Wang et al., 2014b S-Fluoxetine N-demethylation 195.0 (6.68) (0.034) rcCYP2C8 Wang et al., 2014b Fluvastatin (acid, parent) 5-hydroxylation 2.8 0.13 0.046 rCYP2C8 Fischer et al., 1999 Gliclazide 6b-hydroxylation 984 0.63 ,0.001 rCYP2C8 Elliot et al., 2007 7b-hydroxylation 346 0.06 ,0.001 rCYP2C8 Elliot et al., 2007 Glyburide (glibenclamide) Total oxidative metabolism 10.2 0.9 0.09 rCYP2C8 Zharikova et al., 2009 7.7 2.5 0.32 rCYP2C8 Zhou et al., 2010 0.08 rCYP2C8 Varma et al., 2014 Halofantrine N-debutylation 156 0.039 ,0.001 rCYP2C8 Baune et al., 1999 Ibrolipim (NO-1886) O-deethylation (M2) 28.4–53.9 0.0334–0.10 0.0012–0.0019 rCYP2C8 Morioka et al., 2002 R-Ibuprofen 2-hydroxylation 3.5–74 rCYP2C8 Hamman et al., 1997 282 9.4 0.033 rCYP2C8 Chang et al., 2008 341.3 4.92e 0.014e rCYP2C8 Yu et al., 2013b S-Ibuprofen 2-hydroxylation 292 5.4 0.018 rCYP2C8 Chang et al., 2008 388.8 3.02e 0.0078e rCYP2C8 Yu et al., 2013b Imatinib N-demethylation 1.4 0.408 0.29 rCYP2C8 Nebot et al., 2010 4.28 4.07 0.95 rCYP2C8 Filppula et al., 2013a 5 0.553 0.1 rCYP2C8 Khan et al., 2015 Isotretinoin (13-cis-retinoic 4-hydroxylation 13.8 134.6g 9.8g rCYP2C8 Rowbotham et al., 2010 acid) L-775,606 Hydroxylation (M1) 42 0.62 0.015 rCYP2C8 Prueksaritanont et al., 2000 N-dealkylation (M2) 64 0.03 ,0.001 rCYP2C8 Prueksaritanont et al., 2000 Loperamide N-demethylation 11.3 0.0052 ,0.001 rCYP2C8 Kim et al., 2004 Magnolin O-demethylation (M1) 17.7 1.9 0.11 rCYP2C8 Kim et al., 2011a O-demethylation (M2) 21.2 0.3021 0.014 rCYP2C8 Kim et al., 2011a Hydroxylation (M4) 29.7 0.7099 0.024 rCYP2C8 Kim et al., 2011a Mavoglurant Total oxidative metabolism 17.1 5.06 0.30 rCYP2C8 Walles et al., 2013 Mirodenafil N-dealkylation (SK3541) 121 0.85 0.0070 rCYP2C8 Lee et al., 2008 Montelukast 36-hydroxylation (M6) 0.050 0.18 3.6 rCYP2C8 Filppula et al., 2011 0.014 ;0.24 ;17 rCYP2C8 VandenBrink et al., 2011 0.065 ;0.09 ;1 HLM VandenBrink et al., 2011 0.31 0.015 0.048 rCYP2C8 Oliveira Cardoso et al., 2015 25-hydroxylation (M3) 0.33 0.002 0.006 rCYP2C8 Oliveira Cardoso et al., 2015 Morphine N-demethylation 4,800 5.41 0.0011 rCYP2C8 Projean et al., 2003b Nifedipine Total oxidative metabolism 0.38 rCYP2C8 Karlsson et al., 2013 Nitidine chloride Total oxidative metabolism 1.17 0.0705 0.060 rCYP2C8 Li et al., 2014b R-Norverapamil D-620 formation 56 5.3 0.095 rCYP2C8 Tracy et al., 1999 PR-22 formation 38 29 0.76 rCYP2C8 Tracy et al., 1999 S-Norverapamil D-620 formation 80 14 0.18 rCYP2C8 Tracy et al., 1999 PR-22 formation 80 12 0.15 rCYP2C8 Tracy et al., 1999 Olanzapine N-demethylation 30 1.370 0.046 rCYP2C8 Korprasertthaworn et al., 2015 Omeprazole 5-hydroxylation 300 3.3 0.011 rCYP2C8 Karam et al., 1996 Paclitaxel 6a-hydroxylation 5.4 30 5.6 rCYP2C8 Rahman et al., 1994 4.0 (870) (220) HLM Rahman et al., 1994 15 0.12 0.0080 HLM Monsarrat et al., 1997 17 HLM Ando et al., 1998 26 HLM Desai et al., 1998 34.8 (1632) (47) HLM Fischer et al., 1998 4.9 1.14 0.23 rCYP2C8 Masimirembwa et al., 1999 6 (234) (39) rCYP2C8 Ong et al., 2000 12.2 (142) (12) HLM Ong et al., 2000 2.85 5.667 2.0 rCYP2C8 Ohyama et al., 2000 2.58–4.55 0.224–0.583 0.070–0.19 HLM Ohyama et al., 2000 6.8 3.0d 0.44d rCYP2C8 Yamazaki et al., 2000 15 0.8 0.053 rCYP2C8 Dai et al., 2001 4.3 (147) (34) rCYP2C8 Fujino et al., 2001 27.4 (359) (13) HLM Fujino et al., 2001 16.2 29.8 1.8 rCYP2C8 Soyama et al., 2001 15 1.950 0.13 HLM Cresteil et al., 2002 9.3 (60.9) (6.8) HLM Václavíková et al., 2003 13.3 (109.1) (8.2) HLM Donato et al., 2004 16.3 (81.0) (5.0) THLE Donato et al., 2004 7.50 (70.2) (9.4) HLM Polasek et al., 2004 8.3 1.718 0.21 rCYP2C8 Zhang et al., 2009b 18.3 (250) (14) HLM Zhang et al., 2009b (continued) 182 Backman et al.

TABLE 4—Continued

a Substrate Metabolic Pathway Km Vmax CLint Test System References 4.17 2.4 0.58 rCYP2C8 Gao et al., 2010 2.33 4.05 1.7 rCYP2C8 Hanioka et al., 2010 3.7 0.29b 0.078b rcCYP2C8 Kaspera et al., 2011 2.58 3.53 1.4 rCYP2C8 Wattanachai et al., 2011 7.08 (137) (19) HLM Wattanachai et al., 2011 8.65 69.83 8.1 rCYP2C8 Yu et al., 2013b 5.2 (222.1) (43) HLM Wang et al., 2014a 5.41–15.9 (52.6–230) (3.3–26) HLM Kudo et al., 2015 Pafuramidine maleate O-demethylation (M1) 2.6 0.12 0.046 rCYP2C8 Wang et al., 2006 (DB289) Perospirone Total oxidative metabolism 1.09 1.93 1.8 rCYP2C8 Kitamura et al., 2005 Perphenazine N-dealkylation 28 1.35 0.048 rCYP2C8 Olesen and Linnet, 2000 Phenazone (antipyrine) N-demethylation 30,400 (156.4) (0.0051) rCYP2C8 Engel el al., 1996 3-hydroxylation 22,000 (43.9) (0.0020) rCYP2C8 Engel el al., 1996 4-hydroxylation 61,000 (140.9) (0.0023) rCYP2C8 Engel el al., 1996 Phenprocoumon S-49-hydroxylation 3.78 0.027 0.0071 rCYP2C8 Ufer et al., 2004 Pioglitazone M-IV formation 10.2 9.2 0.91 rCYP2C8 Tornio et al., 2008b 9.8 (640) (65) HLM Tornio et al., 2008b 29.5 1.702 0.058 rCYP2C8 Muschler et al., 2009 R483 Hydroxylation (M1) 1.4 (1,000) (700) n/a Bogman et al., 2010 Repaglinide Total oxidative metabolism 2.8 4.9 1.8 rCYP2C8 Kajosaari et al., 2005a M1 formation 25 0.08 0.003 rCYP2C8 Säll et al., 2012 M4 formation 5.7 0.35 0.061 rCYP2C8 Säll et al., 2012 M4 formation 9.0 (130) (14) HLM Säll et al., 2012 M4 formation 28 13c 0.46c Hep Säll et al., 2012 M4 formation 13 (18) (1.4) S9 Säll et al., 2012 12.01 15.69e 1.3e rCYP2C8 Yu et al., 2013b Rosiglitazone p-hydroxylation 44 (2,900) (66) rCYP2C8 Baldwin et al., 1999 4.3–7.7 (550–883) (93–130) HLM Baldwin et al., 1999 N-demethylation 10 (2,430) (240) rCYP2C8 Baldwin et al., 1999 p-hydroxylation 4.0 0.42b 0.11b rcCYP2C8 Kaspera et al., 2011 N-demethylation 2.9 0.38b 0.13b rcCYP2C8 Kaspera et al., 2011 Selegiline Demethylation 82 3 0.04 rCYP2C8 Hidestrand et al., 2001 Levomethamphetamine 630 7 0.01 rCYP2C8 Hidestrand et al., 2001 formation Seratrodast 49-hydroxylation 28.2 0.1438 0.0051 rCYP2C8 Kumar et al., 1997 5-methylhydroxylation 32.9 0.4983 0.015 rCYP2C8 Kumar et al., 1997 Sildenafil Total oxidative metabolism 0.055 rCYP2C8 Karlsson et al., 2013 Simvastatin acid M1 formation 88 2,800 32 rCYP2C8 Prueksaritanont et al., 2003 M2 formation 36 850 24 rCYP2C8 Prueksaritanont et al., 2003 M3 formation 16 600 38 rCYP2C8 Prueksaritanont et al., 2003 T-5 N-oxidation 1.6 0.22 0.14 rCYP2C8 Li et al., 2014a Tacrolimus Total oxidative metabolism 0.19 rCYP2C8 Karlsson et al., 2013 Tanshinol borneol ester M3 formation 45.2 4.28h 0.095h rCYP2C8 Liu et al., 2010b Terbinafine Deamination 24.8 0.512 0.021 rCYP2C8 Vickers et al., 1999 N-demethylation 13.6 2.06 0.15 rCYP2C8 Vickers et al., 1999 Side chain oxidation 26.4 0.825 0.031 rCYP2C8 Vickers et al., 1999 Total oxidative metabolism 15.3 4.47 0.29 rCYP2C8 Vickers et al., 1999 R-Tofisopam M3 formation 52 0.43 0.0083 rCYP2C8 Cameron et al., 2007 Tolbutamide Hydroxylation 650.5 rCYP2C8 Veronese et al., 1993 531 0.39 ,0.001 rCYP2C8 Rettie et al., 1994 1,160 (10.2) (0.0088) rCYP2C8 Pang et al., 2012 Torsemide (torasemide) Methyl hydroxylation 184 1.8 0.0098 rCYP2C8 Miners et al., 2000 170 (35) (0.21) rCYP2C8 Ong et al., 2000 Tozasertib (MK 0457, VX6, N-demethylation 64 129 2.0 rCYP2C8 Ballard et al., 2007 VX 680) Tretinoin (all-trans-retinoic 4-hydroxylation 6.1 0.18 0.030 rCYP2C8 Nadin and Murray, 1999 acid) 4-hydroxylation 50 1.211 0.024 rCYP2C8 Marill et al., 2000 18-hydroxylation 17 0.033 0.0019 rCYP2C8 Marill et al., 2000 5,6-epoxy metabolite 130 0.450 0.0035 rCYP2C8 Marill et al., 2000 formation 4-hydroxylation 13.4 4.8 0.36 rCYP2C8 Thatcher et al., 2010 Troglitazone Quinone formation 2.7 4.2d 1.6d rCYP2C8 Yamazaki et al., 1999b Verapamil O-demethylation 48.4 (13) (0.27) rCYP2C8 Busse et al., 1995 Total oxidative metabolism 0.39 rCYP2C8 Karlsson et al., 2013 R-Verapamil D-617 formation 127 8.0 0.063 rCYP2C8 Tracy et al., 1999 Norverapamil formation 127 6.9 0.054 rCYP2C8 Tracy et al., 1999 PR-22 formation 33 2.2 0.067 rCYP2C8 Tracy et al., 1999 S-Verapamil D-617 formation 185 8.0 0.043 rCYP2C8 Tracy et al., 1999 Norverapamil formation 154 15 0.097 rCYP2C8 Tracy et al., 1999 PR-22 formation 141 1.6 0.011 rCYP2C8 Tracy et al., 1999 (continued) Role of CYP2C8 in Drug Metabolism and Interactions 183

TABLE 4—Continued

a Substrate Metabolic Pathway Km Vmax CLint Test System References Vidupiprant (AMG 853) t-butyl hydroxylation (M2) 1.21 0.031 0.026 rCYP2C8 Foti et al., 2012 Cyclopropyl hydroxylation 49.1 0.250 0.0051 rCYP2C8 Foti et al., 2012 (M3) Vitamin A (retinol) 4-hydroxylation 71 1.73 0.024 rcCYP2C8 Leo et al., 1989 Zopiclone N-demethylation 71 2.5 0.035 rCYP2C8 Becquemont et al., 1999 N-oxidation 59 1.0 0.017 rCYP2C8 Becquemont et al., 1999

5-MeO-DIPT, 5-methoxy-N,N-diisopropyltryptamine; BYZX, [(E)-2-(4-((diethylamino)methyl)benzylidene)-5,6-dimethoxy-2,3-dihydroinden-one]; CLint, intrinsic clearance; Hep, hepatocytes; HIM, human intestinal microsomes; HLM, human liver microsomes; Km, Michaelis-Menten constant; n/a, not available; rCYP2C8, recombinant CYP2C8; rcCYP2C8, reconstituted CYP2C8; S9, S9 fraction; THLE, immortalized human liver epithelial cells; Vmax, maximal velocity. a Calculated as Vmax/Km. b kcat reported instead of Vmax,CLint calculated as kcat/Km. c 6 6 Vmax as pmol/min/10 cells, CLint as pmol/min/10 cells/mM. d Vmax as 1/min, CLint as 1/min/mM. e Vmax as arbitrary unit (AU), CLint as AU/mM. f Vmax as unit/min, CLint as unit/min/mM. g Vmax as counts/s/s, CLint as counts/s/s/mM. h Vmax as AUC×min/nmol, CLint as AUC×min/nmol/mM.

In addition, CYP2C8 participates to various degree to Drug Administration (FDA) as a marker reaction for the metabolism of several other anticancer agents, as in vitro CYP2C8 activity (http://www.fda.gov/Drugs/ listed in Table 1. DevelopmentApprovalProcess/DevelopmentResources/ 2. Antidiabetic Agents. The nonsulfonylurea insulin DrugInteractionsLabeling/ucm093664.htm). In vitro, secretagogue repaglinide is metabolized primarily by troglitazone is metabolized by CYP2C8 to its qui- CYP2C8 (Table 4) and CYP3A4, but it also undergoes none metabolite (M3) (Table 4) at a two- to eightfold direct glucuronidation by uridine-59-diphosphoglucuro- higher rate than by CYP2C19, CYP3A4, and CYP2C9 nosyltransferase (UGT) 1A1 (Bidstrup et al., 2003; (Yamazaki et al., 1999b). M3 is a minor metabolite of Kajosaari et al., 2005a,b; Gan et al., 2010). In addition, troglitazone, but it has been suggested to be responsible there is in vitro data suggesting that aldehyde de- for the drug-induced hepatotoxicity associated with hydrogenase is involved in its metabolism (Säll et al., troglitazone use (Yamazaki et al., 1999b; Smith, 2012). Moreover, repaglinide is a substrate of the 2003). Furthermore, the R483 is hepatic uptake transporter organic anion-transporting primarily metabolized by CYP2C8 and CYP2C19 in polypeptide (OATP) 1B1 (Niemi et al., 2005b, 2011). The vitro (Bogman et al., 2010). CYP2C8 catalyzes the formation of the main metabolites of repaglinide, an formation of the weakly active M1 metabolite (Table 4) oxidized dicarboxylic acid (M2) and, in particular, 39- and its further metabolism to M4, which is the main hydroxyl repaglinide (M4), is largely dependent on metabolite of R483 in plasma. In turn, CYP2C19 forms CYP2C8, whereas the less important aromatic amine M2, which shows similar pharmacological activity as metabolite (M1) is primarily formed by CYP3A4 (Bidstrup parent R483 (Bogman et al., 2010). et al., 2003; Kajosaari et al., 2005a,b). Additionally, CYP2C8 is involved, to a minor extent, Pioglitazone, a thiazolidinedione peroxisome prolif- in the metabolism of the sulfonylureas gliclazide, erator activated receptor (PPAR) g agonist, is primarily glyburide, and tolbutamide, the dipeptidyl peptidase 4 metabolized by CYP2C8 in vitro, with smaller contri- inhibitor sitagliptin, and the PPARa agonist sipoglita- butions by CYP3A4 and the extrahepatic CYP1A1 zar (Table 1; Relling et al., 1990; Srivastava et al., 1991; (Jaakkola et al., 2006c; FDA, 2013a). In vitro, CYP2C8 Veronese et al., 1993; Elliot et al., 2007; Vincent et al., forms the pharmacologically active hydroxypioglita- 2007; Zharikova et al., 2009; Nishihara et al., 2012). zone (M-IV) and ketopioglitazone (M-III) (Jaakkola Tolbutamide p-methyl hydroxylation has been used as et al., 2006c; Tornio et al., 2008b), which are the main a marker reaction for CYP2C8 activity in several in metabolites in human serum with concentrations equal vitro studies. However, its Michaelis-Menten constant to or greater than those of the parent drug (Eckland (Km) for CYP2C8 is very high (.530 mM; Table 4), and and Danhof, 2000). In vivo studies support the central it is effectively metabolized by CYP2C9 at lower role of CYP2C8 in pioglitazone metabolism observed concentrations. in vitro (section VI.C.2). 3. Antimalarial Agents. The 4-aminoquinoline de- Rosiglitazone, another PPAR-g agonist, is also a rivative amodiaquine, widely used for treatment of substrate of CYP2C8. In vitro, it undergoes CYP2C8- malaria for more than 60 years, is a substrate of mediated p-hydroxylation and N-demethylation (Table 4), CYP2C8 (Li et al., 2002). It is also metabolized by the followed by sulfate and glucuronic acid conjugation extrahepatic enzymes CYP1A1 and CYP1B1 to a minor (Baldwin et al., 1999; Kaspera et al., 2011; FDA, extent (Li et al., 2002). Amodiaquine N-deethylation is a 2014a). CYP2C9 also participates in its metabolism frequently used in vitro marker reaction for CYP2C8 to a minor extent (Baldwin et al., 1999). Rosiglitazone because of its high affinity (Km typically around 2 mM) p-hydroxylation is recommended by the Food and and high turnover rate (Table 4). N-desethylamodiaquine, 184 Backman et al.

Fig. 3. Molecular descriptors of drugs classified as "major" or "intermediate" CYP2C8 substrates in Table 1. The molecular descriptors were obtained from SciFinder (American Chemical Society). which is the main metabolite of amodiaquine, is assumed gemfibrozil and cerivastatin is discussed in section to be the main entity responsible for the pharmacological VI.C.4. response to amodiaquine (Churchill et al., 1985; Mount The parent simvastatin lactone is either oxidized by et al., 1986). CYP3A4/5 or hydrolyzed to its acid form, which is Chloroquine, also a 4-aminoquinoline, is mainly pharmacologically active (Prueksaritanont et al., 1997, metabolized to its N-desethyl metabolite by CYP2C8 2003). In human liver microsomes (HLM), the metabo- (Table 4) and CYP3A4, with a small contribution by lism of simvastatin acid was catalyzed primarily CYP2D6 in vitro (Kim et al., 2003; Projean et al., 2003a). ($80%) by CYP3A4/5, with a smaller contribution Furthermore, CYP2C8 also seems to play a small role (#20%) by CYP2C8 (Prueksaritanont et al., 2003). in the in vitro metabolism of the antimalarial agents Recombinant CYP2C8 formed all three simvastatin dapsone, halofantrine, and piperaquine (Table 1; Baune acid metabolites (M1-M3) observed in HLM (Table 4; et al., 1999; Winter et al., 2000; Lee et al., 2012c). Prueksaritanont et al., 2003). In vitro, fluvastatin is 4. Lipid-lowering Drugs. CYP2C8 participates to a mainly metabolized by CYP2C9 into three metabolites, small extent in the metabolism of several HMG-CoA but CYP1A1, CYP2C8, CYP2D6, and CYP3A4 form reductase inhibitors (statins), but it has a major role for 5-hydroxyfluvastatin (Fischer et al., 1999). Both atorvas- the biotransformation of cerivastatin. Cerivastatin is tatin (acid) and its lactone are primarily metabolized extensively metabolized in humans (Boberg et al., 1997; by CYP3A4 to their hydroxylated metabolites in vitro, Mück, 2000). Parent cerivastatin (acid) is metabolized but CYP2C8 is involved in the formation of p-hydroxy by CYP2C8 and CYP3A4, whereas cerivastatin lac- atorvastatin acid to a small extent (Jacobsen et al., tone is predominantly metabolized by CYP3A4 (Boberg 2000b). Furthermore, pitavastatin acid is metabolized et al., 1997; Wang et al., 2002; Fujino et al., 2004). The by CYP2C9 and CYP2C8 in vitro, whereas its lactone is formation of the major metabolite of cerivastatin, metabolized by CYP3A4 and CYP2D6 (Fujino et al., 6-hydroxycerivastatin (M-23), is primarily mediated by 2004). CYP2C8, whereas both CYP2C8 and CYP3A4 produce 5. Other Drugs. Early in vitro studies concluded demethylcerivastatin (M1) (Wang et al., 2002; Kaspera that the leukotriene receptor antagonist montelukast et al., 2010). The notorious in vivo interaction between is mainly metabolized by CYP2C9 and CYP3A4 (Chiba Role of CYP2C8 in Drug Metabolism and Interactions 185

Fig. 4. The number of "major" and "intermediate" CYP2C8 substrates by drug class, as listed in Table 1. et al., 1997), whereas the role of CYP2C8 was not 2009b). Incubation of treprostinil with recombinant evaluated. However, in vitro studies performed more CYP2C8 for 15 minutes resulted in a 95% depletion of than a decade later demonstrated that CYP2C8 is the treprostinil concentrations, whereas only 22% was con- key enzyme involved in the oxidative metabolism of sumed by recombinant CYP2C9. CYP2C8 seems to be of montelukast (Filppula et al., 2011; VandenBrink et al., importance in the in vivo pharmacokinetics of treprosti- 2011). CYP2C8 catalyzes the main metabolic pathway nil (FDA, 2009b). of montelukast; formation of the pharmacologically The sedative agent zopiclone is metabolized by active 36-hydroxymontelukast (M6), and its subsequent CYP2C8 and CYP3A4 in vitro (Becquemont et al., metabolism to the secondary metabolite M4, a dicar- 1999). In HLM, CYP2C8 was the main enzyme catalyz- boxylic acid (Table 4). In addition, CYP2C8 forms 25- ing N-demethylation of zopiclone, followed by CYP2C9 hydroxymontelukast (M3) (Filppula et al., 2011). These and CYP3A4. CYP2C8 also participated in the forma- in vitro findings are in agreement with X-ray crystal- tion of N-oxide zopiclone, together with CYP3A4 (major) lography data, demonstrating a ligand-protein binding and CYP2C9 (Becquemont et al., 1999). However, in interaction between montelukast and CYP2C8 (Schoch another in vitro study, montelukast (CYP2C8 inhibitor) et al., 2008). The montelukast molecule was positioned and gemfibrozil (CYP2C8 and CYP2C9 inhibitor) had with its benzyl ring in close proximity to the heme iron no effect on the elimination of clinically relevant con- of CYP2C8. The montelukast metabolites M3, M4, and centrations of zopiclone (Tornio et al., 2006), supporting M6 formed by CYP2C8 in vitro, all result from the in vivo data showing a lack of effect of gemfibrozil on oxidation of the benzyl ring of montelukast. zopiclone concentrations in healthy subjects (section The novel prolyl hydroxylase inhibitor daprodustat VI.C.8). (GSK1278863), an antianemic agent, is primarily me- Based on in vitro studies, CYP2C8 likely plays tabolized by CYP2C8, with a smaller contribution by an intermediate role in the elimination of 9cUAB30, CYP3A4 in vitro (Johnson et al., 2014). It seems to alitretionin, amiodarone, cisapride, fenretinide, fluox- be more sensitive than repaglinide to CYP2C8 inhibi- etine, irosustat, isotretionin, loperamide, olanzapine, tion by gemfibrozil in vivo (section VI.C.8; Johnson olodaterol, propanoic acid, dronedarone, tazarotenic et al., 2014). acid, verapamil, and vidupiprant (AMG 853) (Table 1). The novel nonstructural 5B nonnucleoside polymer- For instance, CYP2C8 catalyzes dealkylation of both ase inhibitor dasabuvir is extensively metabolized by enantiomers of the calcium channel blocker verapamil CYP2C8, with a small contribution by CYP3A (FDA, and its metabolite norverapamil (Busse et al., 1995; 2014g). CYP2C8 also plays an intermediate/small role Tracy et al., 1999). Tazarotenic acid, the active moiety of in the metabolism of the nonstructural protein 3/4 A the antipsoriatic agent tazarotene, is mainly metabo- protease inhibitor paritaprevir and nonstructural pro- lized by CYP2C8 and flavin-containing monooxyge- tein 5A inhibitor ombitasvir (FDA, 2014g; Menon et al., nases in vitro (Attar et al., 2003). When tazarotenic 2015). However, no in vitro metabolism data have yet acid was incubated with 10 individual recombinant been published for these compounds. CYP enzymes, only CYP2C8 markedly catalyzed sul- The prostacyclin analog treprostinil is primarily me- foxidation, which is the main metabolic pathway of tabolized by CYP2C8, followed by CYP2C9 in vitro (FDA, tazarotenic acid. 186 Backman et al.

There is a vast amount of in vitro data suggesting that estradiol-17b-glucuronide, gemfibrozil 1-O-b glucuronide, CYP2C8 may be of relevance in the metabolism of a licofelone 1-O-acyl glucuronide, Lu AA34893 carbamoyl number of other drugs (Table 1). For the majority of these glucuronide, 2-[[5,7-dipropyl-3-(trifluoromethyl)-1,2- compounds, the role of CYP2C8 in their in vivo elimina- benzisoxazol-6-yl]oxy]-2-methylpropanoic acid (MRL-C) tion is likely to be small or negligible. However, for acyl glucuronide, and sipoglitazar b-1-O-acyl glucuronide some drugs, the in vivo contribution of CYP2C8 to their (see Table 2 for references). Thus, CYP2C8 makes yet metabolism cannot be estimated based on available another exception to the old concept that drug metabolism information, and may, in fact, exceed 20%. Alternatively, is divided into sequential phase I and phase II reactions, CYP2C8 may become a determinant in their metabolism i.e., functionalization and conjugation, respectively (Fig. 5). after inhibition of other enzymes important for their Kumar et al. (2002) demonstrated the first example of elimination. For instance, CYP2C8 is involved in the CYP2C8-mediated metabolism of a glucuronide conju- metabolism of seratrodast, a thromboxane A2 receptor gate when they showed that the conversion of diclofenac antagonist in vitro (Kumar et al., 1997). The main acyl glucuronide to its 49-hydroxy derivative is exclusively metabolic pathway of seratrodast, 5-methylhydroxylation, mediated by CYP2C8 in vitro. In 2005, it was reported is primarily catalyzed by CYP3A and CYP2C9, but CYP2C8 that CYP2C8 is also able to directly catalyze the contributes to a small degree. However, CYP2C8 is a 2-hydroxylation of estradiol-17b-glucuronide in vitro major contributor to seratrodast 49-hydroxylation, a minor (Delaforge et al., 2005). Docking of the glucuronide of metabolic route (Kumar et al., 1997). estradiol into the crystal structure of CYP2C8 showed 6. Glucuronide Metabolites. Several glucuronide me- that the active site is large enough to inhabit the tabolites have been reported to undergo metabolism by conjugate. Also the fetal CYP3A isoform CYP3A7 oxidized CYP2C8, including clopidogrel acyl 1-b-D-glucuronide, estradiol-17b-glucuronide, but CYP2C8 was five times desloratadine glucuronide, diclofenac acyl glucuronide, more active than CYP3A7. However, CYP3A4 was not

Fig. 5. Schematic illustration of the interaction between CYP2C8 and its glucuronide substrates. CYP2C8 and the UGT are localized on opposite sites of the endoplasmic membrane (A). The drug is glucuronidated by the UGT (B). Hereafter, the glucuronide crosses the endoplasmic membrane and binds into CYP2C8 (C). Then, the glucuronide is either metabolized by CYP2C8 and released as a metabolite (D, left), e.g., desloratadine glucuronide, and diclofenac acyl glucuronide, or it is metabolized to a reactive agent that inactivates CYP2C8 (D, right), as for clopidogrel acyl 1-b-D-glucuronide and gemfibrozil 1-O-b glucuronide. CYP2C8 has been suggested to exist as a dimer (Hu et al., (2010), Schoch et al., (2004)). ER, endoplasmic reticulum. Role of CYP2C8 in Drug Metabolism and Interactions 187 able to metabolize estradiol-17b-glucuronide (Delaforge acid, 13-cis-retinoic acid, and 9cUAB30 (Marill et al., et al., 2005). Moreover, the acyl glucuronide of the dual 2002; Gorman et al., 2007). PPAR a/b agonist MRL-C was oxidized by CYP2C8 and to Some in vitro data suggest that CYP2C8 may contrib- a minor extent by CYP3A4, but not by CYP2C9 (Kochansky ute to the metabolism of the steroids 17b-estradiol, et al., 2005). Furthermore, the main elimination pathway progesterone, and testosterone (Waxman et al., 1991; of licofelone, a dual inhibitor of cyclooxygenases 1 and 2 Spink et al., 1992, 1994). However, there seems to be no and 5-lipoxygenase, is glucuronidation of its carboxylic evidence for a role of CYP2C8 in the metabolism of acid metabolite, followed by CYP2C8-catalyzed hydroxyl- androgens in vivo. Although CYP2C8 participates in the ation of the acyl glucuronide M1 to form the hydroxylated metabolism of several endogenous compounds, no car- glucuronide M3 (Albrecht et al., 2008). diovascular or other potentially CYP2C8-related adverse For two compounds, the formation of an unconjugated effects were observed in the Helsinki Heart Study, in hydroxyl metabolite involves oxidation and subsequent which over 2000 middle-aged men with primary dyslipi- deconjugation of a glucuronide metabolite. In vitro, the demia ingested the strong CYP2C8 inhibitor gemfibrozil antidiabetic agent sipoglitazar was first glucuronidated 600 mg twice daily for several years (Frick et al., 1987). to sipoglitazar b-1-O-acyl glucuronide (sipoglitazar-G1). Furthermore, some natural compounds have been Sipoglitazar-G1 was then metabolized to the main metab- reported to undergo metabolism by CYP2C8 in vitro olite M-I by O-dealkylation by CYP2C8 and subsequent (Table 3). CYP2C8 and CYP3A4 are the primary deconjugation (Nishihara et al., 2012). A similar finding was enzymes involved in the in vitro metabolism of recently observed for desloratadine (Kazmi et al., 2015). The 1-hydroxyl-2,3,5-trimethoxyxanthone, a constituent of main metabolite of desloratadine, 3-hydroxydesloratadine, the Tibetan medicinal plant Halenia elliptica (Feng which is active, was formed via CYP2C8-mediated oxida- et al., 2014). CYP2C8 is responsible for the main tion of desloratadine glucuronide and a deconjugation event metabolic pathway of silybin, the active component of (Kazmi et al., 2015). Thus, it seems that phase II metabo- silymarin in vitro (Jancova et al., 2007). The CYP2C8 lism occurs before phase I for these compounds (Fig. 5). inhibitor inhibited silybin O-demethylation Also the glucuronide metabolites of clopidogrel, gem- by 80% in HLM, and recombinant CYP2C8 was the fibrozil, and Lu AA34893 are likely to be substrates of major enzyme forming O-demethyl silybin, with a small CYP2C8 (Ogilvie et al., 2006; Baer et al., 2009; Kazmi contribution by CYP3A4. Furthermore, CYP2C8 is the et al., 2010; Tornio et al., 2014). All three compounds are major enzyme responsible for the in vitro metabolism of metabolism-dependent inhibitors of CYP2C8, as dis- tanshinol borneol ester, a combination of the natural cussed in sections V.B and VI.B. compounds danshensu and borneol (Liu et al., 2010a). Recombinant CYP2C8 generated all five tanshinol B. Endogenous and Natural Compounds borneol metabolites (M1-M5) observed in HLM incuba- CYP2C8 metabolizes some endogenous and natu- tions, whereas recombinant CYP3A4 only produced the ral compounds (Table 3). CYP2C8 participates in M4 metabolite. the metabolism of arachidonic acid to biologically active epoxyeicosatrienoic acids (e.g., 11-, 13-, or 15- IV. Pharmacogenetics hydroxyeicosatrienoic acid), involved in the regulation of numerous physiologic processes, e.g., vascular function, Nearly 100 nonsynonymous single nucleotide varia- pressure regulation, pancreatic peptide hormone tions (SNV) and short deletions, as well as essential secretion, and aggregation (Daikh et al., 1994; splice site variants have been found in the CYP2C8 Rifkind et al., 1995; Zeldin et al., 1995). The roles of gene. The variants described in the literature, dbSNP CYP2C8 and other CYP enzymes in inflammation, database, or the 1000 Genomes project database are cardiovascular disease, and cancer were recently listed in Table 5, together with their continental reviewed by Chen and Wang (2015) and Fleming (2014). frequencies and predicted or experimentally deter- CYP2C8 and CYP3A enzymes have generally been mined effects on protein function. The vast majority of considered to be the primary CYPs involved in the the nonsynonymous variants are rare and occur at metabolism of all-trans-retinoic acid, the active form of minor allele frequencies of 0.01 or less in all investi- vitamin A (retinol) (Leo et al., 1989; Nadin and Murray, gated populations. 1999; Marill et al., 2000). All-trans-retinoic acid is involved in gene transcription, cell division, and differ- A. Population Genetics entiation (Tzimas and Nau, 2001; Marill et al., 2003; Three alleles, known as CYP2C8*2, *3, and *4, Duester, 2008). According to more recent data, however, account for the majority of nonsynonymous variability CYP26A1 and CYP3A4 are the primary determinants of of CYP2C8 in humans. Their frequencies differ signif- all-trans-retinoic acid metabolism in humans, whereas icantly both between and within continental popula- the role of CYP2C8 is of smaller importance (Thatcher tions (Table 5, Fig. 6). et al., 2010). In vitro, CYP2C8 also catalyzes the The CYP2C8*2 allele (c.805A.T, p.Cys266Phe) oc- metabolism of other retinoids, including 9-cis-retinoic curs mostly in individuals with a sub-Saharan African TABLE 5. 188 Nonsynonymous and essential splice site nucleotide changes in the CYP2C8 gene Nucleotide positions are given in relation to the full length CYP2C8 mRNA sequence (NM_000770.3) and amino acid positions in relation to the respective protein sequence (NP_000761.3). Variant allele frequency data are from the 1000 genomes project for populations with African, European, South Asian, East Asian and American ancestry (www.1000genomes.org, 1000 Genomes Project Consortium, 2012). SIFT and PolyPhen predictions of the possible impact of amino acid substitutions on protein function were obtained using the Variant Effect Predictor (Kumar et al., 2009; Adzhubei et al., 2010; McLaren et al., 2010). *-allele nomenclature was retrieved from the Human Cytochrome P450 (CYP) Allele Nomenclature Database (www.cypalleles.ki.se).

Enzyme Activity

*-allele dbSNP ID Location Nucleotide Change Amino Acid Change Variant Allele Frequency In Silico Prediction

African European South Asian East Asian American SIFT PolyPhen In Vivo In Vitro rs142470035 Exon 1 c.1A.G p.Met1? 0.0045 0 0 0 0 deleterious probably damaging rs373001219 Exon 1 c.7C.A p.Pro3Thr —— — — —tolerated benign rs530027098 Exon 1 c.40A.T p.Met14Leu 0 0 0.001 0 0 tolerated benign rs202131138 Exon 1 c.63A.T p.Arg21Ser —— — — —deleterious benign rs267602643 Exon 1 c.77G.A p.Arg26Lys —— — — —tolerated benign rs375170154 Exon 1 c.149T.C p.Ile50Thr —— — — —tolerated benign rs113939225 Exon 1 c.167A.G p.Asn56Ser —— — — —tolerated benign ↔ rs376132046 Exon 2 c.199G.A p.Val67Met —— — — —deleterious possibly damaging rs17851796 Exon 2 c.244G.A p.Ala82Thr 0 0 0 0.001 0 deleterious benign rs17851796 Exon 2 c.244G.T p.Ala82Ser 0 0 0 0.001 0 deleterious benign rs201449274 Exon 2 c.263T.C p.Ile88Thr 0 0 0 0 0.0014 deleterious benign rs372299895 Exon 2 c.268A.G p.Asn90Asp —— — — —tolerated benign rs578254206 Exon 2 c.293G.T p.Gly98Val 0 0 0.001 0 0 deleterious probably damaging rs199931273 Intron 2 c.331+2T.C —— — — — — — rs369552457 Exon 3 c.345C.A p.Ser115Arg —— — — —deleterious probably damaging rs201739495 Exon 3 c.368T.A p.Ile123Asn —— — — —deleterious possibly damaging rs377386087 Exon 3 c.370C.T p.Arg124Trp —— — — —deleterious probably damaging aka tal. et Backman rs369591911 Exon 3 c.371G.A p.Arg124Gln —— — — —deleterious probably damaging rs188111115 Exon 3 c.373C.T p.Arg125Cys 0.0008 0 0 0 0 deleterious possibly damaging rs139650638 Exon 3 c.389C.A p.Thr130Asn 0 0 0.001 0 0 deleterious possibly damaging rs139650638 Exon 3 c.389C.T p.Thr130Ile 0 0 0.001 0 0 tolerated probably damaging *3 rs11572080 Exon 3 c.416G.A p.Arg139Lys 0.0083 0.1183 0.0297 0.001 0.0994 tolerated benign ↑↓↑ rs540288649 Exon 3 c.430C.G p.Arg144Gly 0 0 0.0102 0 0 deleterious probably damaging rs200057634 Exon 3 c.449A.G p.His150Arg —— — — —tolerated benign rs201561213 Exon 3 c.472A.G p.Lys158Glu —— ———deleterious benign *5 rs72558196 Exon 3 c.475delA p.Thr159ProfsTer19 —— — — — — — none rs576554998 Exon 4 c.497C.T p.Pro166Leu —— — — —deleterious probably damaging *6 rs142886225 Exon 4 c.511G.A p.Gly171Ser 0 0 0 0.006 0 tolerated benign ↔ rs553407481 Exon 4 c.516T.A p.Cys172Ter —— — — — — — rs141350682 Exon 4 c.525C.A p.Cys175Ter —— — — — — — rs113008582 Exon 4 c.526A.G p.Asn176Asp —— — — —deleterious probably damaging rs201219972 Exon 4 c.536G.C p.Cys179Ser —— — — —tolerated possibly damaging rs41286886 Exon 4 c.541G.A p.Val181Ile 0 0.0109 0 0 0.0029 tolerated benign rs147150224 Exon 4 c.544G.A p.Val182Ile —— — — —tolerated benign *7 rs72558195 Exon 4 c.556C.T p.Arg186Ter —— — — — — — none *8 rs543793530 Exon 4 c.557G.A p.Arg186Gln 0 0 0.001 0 0 deleterious probably damaging ↓ rs201899315 Exon 4 c.581T.A p.Phe194Tyr —— — — —deleterious benign rs201045618 Exon 4 c.602T.A p.Phe201Tyr 0 0 0 0.001 0 deleterious possibly damaging rs146962089 Exon 4 c.635G.A p.Trp212Ter 0.0023 0 0 0 0 —— rs148974310 Exon 5 c.643G.A p.Val215Ile —— — — —tolerated benign *13 N.A. Exon 5 c.669T.G p.Ile223Met —— — — —tolerated benign ↔ rs569886323 Exon 5 c.703A.G p.Lys235Glu 0 0 0 0.001 0 tolerated benign *14 rs188934928 Exon 5 c.712G.C p.Ala238Pro 0 0 0 0.001 0 tolerated benign ↓ rs537006401 Exon 5 c.713C.T p.Ala238Val —— — — —tolerated benign rs200358471 Exon 5 c.716T.C p.Leu239Pro —— — — —tolerated benign rs536085663 Exon 5 c.721C.T p.Arg241Ter 0.0008 0 0 0 0 —— rs11572102 Exon 5 c.730A.G p.Ile244Val 0.0068 0 0 0 0 tolerated benign *9 N.A. Exon 5 c.740A.G p.Lys247Arg —— — — —tolerated benign ↔ (continued) TABLE 5.—Continued

Enzyme Activity

*-allele dbSNP ID Location Nucleotide Change Amino Acid Change Variant Allele Frequency In Silico Prediction

African European South Asian East Asian American SIFT PolyPhen In Vivo In Vitro rs141120323 Exon 5 c.767A.G p.Asp256Gly —— — — —deleterious probably damaging rs527793637 Exon 5 c.781C.T p.Arg261Trp 0 0 0.001 0 0 deleterious benign rs370459834 Exon 5 c.782G.T p.Arg261Leu —— — — —deleterious benign *4 rs1058930 Exon 5 c.792C.G p.Ile264Met 0.0038 0.0577 0.0072 0 0.0187 deleterious probably damaging ↓↑ rs551515028 Exon 5 c.793G.A p.Asp265Asn 0.0008 0 0 0 0 deleterious probably damaging rs377675927 Exon 5 c.797G.T p.Cys266Phe —— — — —deleterious probably damaging *2 rs11572103 Exon 5 c.805A.T p.Ile269Phe 0.1891 0.004 0.0123 0 0.0115 deleterious probably damaging ↓↔ rs373613215 Exon 5 c.816G.C p.Glu272Asp —— — — —tolerated benign *11 rs78637571 Exon 6 c.820G.T p.Glu274Ter 0 0 0 0.003 0 —— none rs140599093 Exon 6 c.821A.G p.Glu274Gly —— — — —deleterious benign rs370806022 Exon 6 c.848A.G p.Asn283Ser —— — — —tolerated benign . rs537326361 Exon 6 c.955G A p.Val319Ile 0 0 0 0 0.0014 tolerated benign Interactions and Metabolism Drug in CYP2C8 of Role rs146806199 Exon 7 c.992T.C p.Ile331Thr 0.0015 0 0.0041 0 0 deleterious probably damaging rs148442781 Exon 7 c.1028G.T p.Ser343Ile —— — — —tolerated benign rs199691080 Exon 7 c.1060G.A p.Glu354Lys —— — — —deleterious probably damaging rs373461548 Exon 7 c.1063A.T p.Ile355Phe —— — — —deleterious probably damaging rs45438799 Exon 7 c.1081C.T p.Leu361Phe 0 0.001 0 0 0 tolerated possibly damaging rs77147096 Exon 7 c.1093G.A p.Gly365Ser 0.0098 0 0 0 0 tolerated benign rs147133669 Exon 7 c.1096G.A p.Val366Met 0.0008 0.001 0 0 0.0014 deleterious benign rs375271607 Exon 7 c.1144C.T p.Pro382Ser —— — — —deleterious probably damaging *10 N.A. Exon 7 c.1149G.T p.Lys383Asn —— — — —deleterious probably damaging ↔ rs143386810 Exon 8 c.1150G.A p.Gly384Ser 0 0.001 0.001 0 0 deleterious possibly damaging rs553009747 Exon 8 c.1154C.T p.Thr385Ile 0 0 0.001 0 0 deleterious probably damaging rs267602641 Exon 8 c.1165G.A p.Ala389Thr —— — — —tolerated benign rs72558194 Exon 8 c.1169T.C p.Leu390Ser —— — — —tolerated benign rs74454169 Exon 8 c.1171C.A p.Leu391Met —— — — —deleterious probably damaging rs201421851 Exon 8 c.1178C.G p.Ser393Cys 0 0 0 0 0.0014 deleterious probably damaging rs190807911 Exon 8 c.1180G.A p.Val394Met 0 0 0 0.001 0 deleterious probably damaging rs201301235 Exon 8 c.1187A.C p.His396Pro —— — — —deleterious possibly damaging rs186285658 Exon 8 c.1189G.A p.Asp397Asn 0 0 0 0.002 0 deleterious possibly damaging rs113669182 Exon 8 c.1193A.G p.Asp398Gly —— — — —tolerated benign *3 rs10509681 Exon 8 c.1196A.G p.Lys399Arg 0.0083 0.1183 0.0297 0.001 0.0994 tolerated benign ↑↓↑ rs181982392 Exon 8 c.1198G.T p.Glu400Ter 0 0 0 0.001 0 —— rs66501115 Exon 8 c.1210C.G p.Pro404Ala —— — — —deleterious possibly damaging ↓ rs150733212 Exon 8 c.1225C.T p.Pro409Ser —— — — —deleterious probably damaging rs374605743 Exon 8 c.1246A.C p.Asn416His —— — — —deleterious benign rs141209951 Exon 8 c.1250G.T p.Gly417Val 0.0015 0 0 0 0 deleterious probably damaging rs552247471 Exon 8 c.1252A.T p.Asn418Tyr 0 0 0.001 0 0 deleterious probably damaging rs371330493 Exon 8 c.1273T.C p.Phe425Leu 0.0008 0 0 0 0 deleterious probably damaging rs148348784 Exon 8 c.1276A.G p.Met426Val —— — — —tolerated benign ↔ rs372999683 Exon 9 c.1313A.G p.Glu438Gly —— — — —deleterious probably damaging rs143038562 Exon 9 c.1324C.T p.Arg442Cys —— — — —deleterious possibly damaging rs138495387 Exon 9 c.1325G.A p.Arg442His —— — — —deleterious possibly damaging rs369600584 Exon 9 c.1327A.C p.Met443Leu —— — — —deleterious benign *12 rs3832694 Exon 9 c.1382_1384delTTG p.Val461del —— — — — — — rs61757318 Exon 9 c.1413delA p.Val472LeufsTer23 —— — — — — — ↔ rs529725725 Exon 9 c.1414G.A p.Val472Ile 0 0 0 0 0.0029 tolerated benign rs376016142 Exon 9 c.1441C.T p.Pro481Ser —— — — —deleterious probably damaging rs140481138 Exon 9 c.1466C.T p.Pro489Leu —— — — —deleterious probably damaging

↔, unchanged activity; ↓, reduced activity; ↑, increased activity; N.A., not available; SIFT, sorting intolerant from tolerant 189 190 Backman et al.

Fig. 6. Global distribution of CYP2C8*2, CYP2C8*3, and CYP2C8*4 alleles. Color intensity indicates allele frequency. References are given in the text. ancestry. In sub-Saharan African populations, its allele (Martis et al., 2013). The allele is also relatively frequency ranges from about 0.10 in a Fulani population common in the mixed Brazilian population with a inBurkinaFasoto0.37inaMbutipygmypopulationin frequency of 0.06, New York area Hispanic population Congo (Cavaco et al., 2005; Rower et al., 2005; Parikh with a frequency of 0.02, and North and South Indian et al., 2007; Kudzi et al., 2009; Speed et al., 2009; populations, with frequencies of 0.03 and 0.01, respec- Paganotti et al., 2011, 2012; 1000 Genomes Project tively (Suarez-Kurtz et al., 2012; Martis et al., 2013; Consortium, 2012; Arnaldo et al., 2013; Marwa et al., Minhas et al., 2013; Arun Kumar et al., 2015). The 2014). In an African-American population in the New CYP2C8*2 allele is very rare or absent in East Asian York area, the allele frequency of CYP2C8*2 was 0.10 and European populations, with the exception of an Role of CYP2C8 in Drug Metabolism and Interactions 191 allele frequency of 0.01 in a Portuguese European Muthiah et al., 2005; Speed et al., 2009; 1000 Genomes sample (Nakajima et al., 2003; Muthiah et al., 2005; Project Consortium, 2012; Staehli Hodel et al., 2013; Cavaco et al., 2006; Pechandova et al., 2012; Vargens Wu et al., 2013). The allele is rare in individuals with et al., 2012; Martis et al., 2013; Wu et al., 2013). a sub-Saharan African ancestry, with a frequency of The CYP2C8*3 allele is a haplotype consisting of below 0.01 in all investigated sub-Saharan African two nonsynonymous variants (c.416G.A, p.Arg139Lys populations and 0.01 in an African American population and c.1196A.G, p.Lys399Arg), which appear to be in (Cavaco et al., 2005; Rower et al., 2005; Kudzi et al., a complete or nearly complete linkage disequilibrium 2009; 1000 Genomes Project Consortium, 2012; Arnaldo in all investigated populations (1000 Genomes Project et al., 2013; Martis et al., 2013). Consortium, 2012). The linkage disequilibrium extends In addition to the common variants, rare nonsynon- also beyond the CYP2C8 gene, as evidenced by a strong ymous CYP2C8 variants exist in all continental pop- correlation between the CYP2C8*3 and CYP2C9*2 ulations (Table 5). A number of the rare CYP2C8 (rs1799853; c.430C.T, p.Arg144Cys) alleles in the variants can be predicted to result in a loss-of-function Swedish population (Yasar et al., 2002). The highest because of premature termination of protein syn- allele frequencies of CYP2C8*3 are seen in individuals thesis. The c.635G.A (p.Trp212Ter) and c.721C.T with a European ancestry. In European populations, (p.Arg241Ter) variants have a combined allele frequency the allele frequency of CYP2C8*3 ranges from 0.069 in of 0.003 in sub-Saharan African populations, and the Faroe Islanders to 0.198 in a Portuguese population, c.820G.T (p.Glu274Ter) and c.1198G.T (p.Glu400Ter) with an apparent north-to-south cline from lower to variants have a combined allele frequency of 0.004 in higher frequencies (Fig. 6; Yasar et al., 2002; Halling East Asians (1000 Genomes Project Consortium, 2012). et al., 2005; Cavaco et al., 2006; Speed et al., 2009; 1000 Other predicted loss-of-function CYP2C8 variants were Genomes Project Consortium, 2012; Pechandova et al., not found in the 1000 Genomes Project populations, 2012; Suarez-Kurtz et al., 2012). The allele is also quite and data are too scarce to estimate their population common in European American and North American frequencies. Hispanic populations, with frequencies of 0.09 and 0.08, respectively (Martis et al., 2013). In the mixed Brazilian B. Functional Studies and Ecuadorian populations, its frequency is 0.08 and The functional effects of CYP2C8 variants have been 0.07, and in a Chilean mestizo population it is 0.06 investigated using recombinantly expressed variant (Roco et al., 2012; Suarez-Kurtz et al., 2012; Vicente proteins and HLM with different CYP2C8 genotypes. et al., 2014). There is wide variability in the frequency Recombinant CYP2C8.2 has been quite consistently of CYP2C8*3 in sub-Saharan African populations, even associated with an about 50% decrease in the intrinsic within a country (Cavaco et al., 2005; Rower et al., 2005; clearance for paclitaxel 6a-hydroxylation, compared Parikh et al., 2007; Kudzi et al., 2009; Arnaldo et al., with CYP2C8.1 (Dai et al., 2001; Gao et al., 2010; 2013; Staehli Hodel et al., 2013; Marwa et al., 2014; Yu et al., 2013b). In addition, the intrinsic clearance Paganotti et al., 2014). For example, the frequency of of amodiaquine has been reduced by 80–90% in CYP2C8*3 was found to be 0.00 in individuals in central CYP2C8.2 and that of repaglinide by 20% compared Tanzania and as high as 0.10 in the Mwanza region of with CYP2C8.1 (Parikh et al., 2007; Yu et al., 2013b). Tanzania (Staehli Hodel et al., 2013; Marwa et al., Similarly, the intrinsic clearances of arachidonic acid 2014). and tanshinol borneol ester appeared to be lower by The CYP2C8*4 (c.792C.G, p.Ile264Met) allele has CYP2C8.2 than by CYP2C8.1, but the differences were its highest frequencies in European populations, with not statistically significant (Dai et al., 2001; Liu et al., the allele frequency ranging from 0.04 in a Spanish 2010a). On the other hand, the intrinsic clearances for population to 0.07 in the Irish (Cavaco et al., 2006; cerivastatin M-23 and M-1 metabolite formation and R- Speed et al., 2009; 1000 Genomes Project Consortium, and S-ibuprofen hydroxylations have been nonsignificantly 2012; Pechandova et al., 2012). Its frequency was 0.03 higher in CYP2C8.2 than in CYP2C8.1 (Kaspera et al., in a European American population and a mixed Bra- 2010; Yu et al., 2013b). Both the SIFT or Polyphen in silico zilian population (Suarez-Kurtz et al., 2012; Martis prediction algorithms suggest that the amino acid change in et al., 2013). In Peruvian, Colombian, Puerto Rican, and CYP2C8.2 is deleterious for CYP2C8 activity (Table 5). North American Hispanic populations, the frequency In several studies, the intrinsic clearance for pacli- ranges from 0.01 to 0.02 (1000 Genomes Project Con- taxel 6a-hydroxylation by recombinant CYP2C8.3 has sortium, 2012; Martis et al., 2013). The CYP2C8*4 allele been between 30 and 85% lower than by CYP2C8.1 (Dai is found with a frequency of about 0.03–0.04 in Indian et al., 2001; Soyama et al., 2001; Gao et al., 2010; Yu individuals and 0.01 in the Pakistani (1000 Genomes et al., 2013b). Other studies employing recombinant Project Consortium, 2012; Minhas et al., 2013). In East CYP2C8.3 have shown increased intrinsic clearance Asian populations, the frequency of CYP2C8*4 is for repaglinide and cerivastatin but reduced intrinsic generally 0.01 or less, but a frequency of 0.02 was seen clearance for R- and S-ibuprofen and nearly abolished in an Uighur Chinese population (Nakajima et al., 2003; intrinsic clearance for amodiaquine (Kaspera et al., 192 Backman et al.

2010; Parikh et al., 2007; Yu et al., 2013b). The intrinsic et al., 2005; Hanioka et al., 2010). In one study, the clearances of arachidonic acid to 11,12- and 14,15- p.Ala238Pro and p.Ile223Met variants were associated and tanshinol borneol ester with reduced amiodarone metabolism (Hanioka et al., have also been significantly lower by CYP2C8.3 than by 2011). One study demonstrated lack of CYP2C8 protein CYP2C8.1 (Dai et al., 2001; Liu et al., 2010a). However, expression in association with the p.Glu274Ter (*11) in a recent study expressing CYP2C8.3 together with nonsense variant (Yeo et al., 2011). cytochrome P450 reductase and cytochrome b5, the intrinsic clearance for paclitaxel 6a-hydroxylation was C. Effects on Drug Metabolism in Humans about twofold higher, that for amodiaquine about two- In contrast to previous in vitro studies suggesting fold higher, that for rosiglitazone about 2.5-fold higher, a reduced CYP2C8 activity in association with the and that for cerivastatin about 4.5-fold higher compared CYP2C8*3 allele (Dai et al., 2001; Bahadur et al., with CYP2C8.1 (Kaspera et al., 2011). A stronger 2002), the first pharmacokinetic study in humans binding affinity of ligands to CYP2C8.3 together with an showed that the CYP2C8*3 allele was associated with increase in heme spin change during binding of ligands reduced plasma concentrations of repaglinide (Niemi and redox partners were suggested to partly explain et al., 2003c). In this and later studies, individuals with the increased catalytic activity (Kaspera et al., 2011). the CYP2C8*1/*3 genotype have had an approximately In one study, HLM heterozygous for the CYP2C8*3 40–50% lower AUC of a subtherapeutic dose of repagli- allele showed lowered paclitaxel 6a-hydroxylase activ- nide than individuals with the CYP2C8*1/*1 genotype ity and in another study no change in amodiaquine (Niemi et al., 2005b,c). However, this finding has not N-deethylation compared with microsomes homozy- been fully replicated in studies with higher repaglinide gous for CYP2C8*1 (Bahadur et al., 2002; Kaspera doses (Bidstrup et al., 2006; Tomalik-Scharte et al., et al., 2011). Studies employing HLM heterozygous or 2011), suggesting that the effect of CYP2C8*3 allele on homozygous for CYP2C8*3 have shown increased in- repaglinide pharmacokinetics may be dose dependent. trinsic clearance of pioglitazone and imatinib (Muschler Similarly to repaglinide, the CYP2C8*3 allele has et al., 2009; Khan et al., 2015). In silico predictions been associated with apparently increased clearance of suggest that neither of the amino acid changes in the rosiglitazone and pioglitazone CYP2C8.3 affect CYP2C8 activity (Table 5). Taken (Kirchheiner et al., 2006; Aquilante et al., 2008, 2013a; together, in vitro evidence concerning the functional Tornio et al., 2008b). The AUCs of rosiglitazone or pioglit- effects of CYP2C8*3 suggests some degree of a azone have been about 20–40% lower in CYP2C8*3 carriers substrate-specific effect but is discrepant for some than in noncarriers, with an apparent gene-dose effect substrates in that both decreased and increased activ- (Kirchheiner et al., 2006; Aquilante et al., 2008, 2013a; ities have been reported. Tornio et al., 2008b). Furthermore, in a study in patients In three studies, paclitaxel 6a-hydroxylation intrinsic with type 2 diabetes mellitus, the CYP2C8*3 allele has clearance was reduced by about 70% by recombinant been associated with significantly lower trough rosigli- CYP2C8.4 compared with CYP2C8.1 (Singh et al., 2008; tazone concentrations and an impaired lowering of Gao et al., 2010; Yu et al., 2013b). Similarly, the intrinsic glycosylated hemoglobin (HbA1c) during rosiglitazone clearances of repaglinide and R-andS-ibuprofen have treatment (Stage et al., 2013). In one study in African been 20, 50, and 53% lower by CYP2C8.4 than by American subjects, the CYP2C8*2 allele had no impact CYP2C8.1, respectively (Yu et al., 2013b). On the other on parent pioglitazone pharmacokinetics but was asso- hand, the intrinsic clearances of cerivastatin to M-23 and ciated with impaired metabolism of pioglitazone to its M-1 were about 2- to 2.5-fold higher by CYP2C8.4 than by M3 metabolite (Aquilante et al., 2013c). CYP2C8.1 (Kaspera et al., 2010). The intrinsic clearance Although CYP2C8*2 has been associated with signif- of tanshinol borneol ester was not significantly different icantly impaired amodiaquine metabolism in vitro between CYP2C8.4 and CYP2C8.1 (Liu et al., 2010a). One (Parikh et al., 2007), the allele has not been clearly study suggests that the amino acid change in CYP2C8.4 associated with amodiaquine efficacy or toxicity (Adjei disrupts heme binding and results in an inactive protein et al., 2008). However, more recent evidence suggests that (Singh et al., 2008). HLM heterozygous for CYP2C8*4 CYP2C8 genetic variability can influence the occurrence showed a nonsignificant tendency for lower paclitaxel of amodiaquine or chloroquine resistance in malaria 6a-hydroxylase activity (Bahadur et al., 2002). In silico parasites (Paganotti et al., 2011; Cavaco et al., 2013). predictions suggest that the amino acid change in Studies in cancer patients have suggested that the CYP2C8.4 is deleterious for CYP2C8 activity (Table 5). CYP2C8*3 allele can slightly impair the clearance of In vitro studies employing recombinant CYP2C8 paclitaxel (Henningsson et al., 2005; Bergmann et al., have shown reduced paclitaxel 6a-hydroxylase activity 2011). Some studies have also suggested that the in association with the p.Arg186Gln, p.Ala238Pro (*14), CYP2C8*3 allele or other CYP2C8 variants may be risk and p.Pro404Ala variants but no change in activity due factors for paclitaxel-induced neurotoxicity or myelo- to the p.Gly171Ser, p.Ile223Met (*13), p.Lys247Arg, suppression and affect the benefit-to-risk ratio of and p.Lys383Asn variants (Soyama et al., 2001; Hichiya paclitaxel therapy (Green et al., 2011; Leskelä et al., Role of CYP2C8 in Drug Metabolism and Interactions 193

2011; Hertz et al., 2012; Hertz et al., 2014; Lee et al., neither montelukast nor affected the phar- 2015). Further studies are required to clarify the role macokinetics of CYP2C8 substrate drugs in vivo of CYP2C8 genetic variants in affecting paclitaxel (Jaakkola et al., 2006b; Kajosaari et al., 2006b; Kim response. et al., 2007). The lack of in vivo effect is likely explained Some studies have reported significantly increased by their extensive (.99%) plasma concentrations and apparently reduced clear- (FDA, 1998; Dekhuijzen and Koopmans, 2002). Also ance of racemic ibuprofen and its enantiomers in the inhibition of CYP2C8 by candesartran cilexetil association with the CYP2C8*3 allele (Garcia-Martin (prodrug of candesartan), clotrimazole, and mometa- et al., 2004; Martinez et al., 2005; Karazniewicz-Łada sone furoate are probably not clinically relevant. The et al., 2009). On the other hand, one study reported antifungal clotrimazole and anti-inflammatory mome- enhanced clearance of R-ibuprofen in association with tasone furoate are topically applied and are therefore CYP2C8*3 (Lopez-Rodriguez et al., 2008). Because unlikely to cause interactions because of low systemic ibuprofen is a substrate of CYP2C9, it is likely that concentrations (Walsky et al., 2005a). In the systemic the discrepancies are due to the strong linkage disequi- circulation, candesartan cilexetil is cleaved to candesartan, librium between CYP2C8*3 and CYP2C9*2 and re- and, consequently, the likelihood of a drug interaction duced ibuprofen clearance in CYP2C8*3 carriers is in elicited by its prodrug is low. Furthermore, predictions fact due to the CYP2C9*2 allele. suggested a relatively weak potential for drug-drug Although CYP2C8 is not known to be involved in interactions due to CYP2C8 inhibition by felodipine. No pharmacokinetics, an intronic SNV in drug interaction studies between felodipine and CYP2C8 (rs1934951) has been associated with zole- CYP2C8 substrates have been reported. dronic acid-induced osteonecrosis of the jaw in patients Trimethoprim, an antimicrobial agent, is a competi- treated for multiple myeloma (Sarasquete et al., 2008). tive inhibitor of CYP2C8 in vitro (Wen et al., 2002), with In a more recent study, this SNV was associated with a Ki value typically around 10–30 mM in HLM (Table 6). the mandibular localization of bisphosphonate-induced The inhibition of CYP2C8 by trimethoprim seems to be osteonecrosis (Balla et al., 2012). However, there was no rather selective, because it does not inhibit CYP1A2, significant relationship between the variant and the CYP2C9, CYP2C19, CYP2D6, CYP2E1, or CYP3A4 at development of bisphosphonate-induced osteonecrosis concentrations below 100 mM. In healthy subjects, tri- of the jaw in men with prostate cancer (English et al., methoprim has moderately increased the plasma expo- 2010) or in patients with multiple myeloma (Such et al., sure to several CYP2C8 substrate drugs (section VI.). 2011). A meta-analysis found no significantly increased The quercetin is one of the earliest in vitro susceptibility to bisphosphonate-induced osteonecrosis inhibitors of CYP2C8 detected. In studies of paclitaxel of the jaw in rs1934951 carriers when all cancer types metabolism, it was observed that quercetin, unlike were pooled, but suggested a significant association in CYP3A4 inhibitors, inhibited paclitaxel 6a-hydroxylation multiple myeloma patients (Zhong et al., 2013). (Harris et al., 1994; Kumar et al., 1994). Because it was shown that the 6a-hydroxylation of paclitaxel is V. In Vitro Inhibition and Induction of mediated by CYP2C8, it was evident that quercetin is Cytochrome P450 2C8 an inhibitor of this enzyme (Rahman et al., 1994). Quercetin inhibits CYP2C8 competitively with a Ki of A. Reversible Inhibition 0.03–20 mM (Table 7) and is classified as a "preferred" 1. Drugs That Act as Inhibitors of Cytochrome P450 probe in vitro inhibitor of CYP2C8 by the FDA (http:// 2C8. Several drugs, drug metabolites, and other com- www.fda.gov/Drugs/DevelopmentApprovalProcess/ pounds have been found to inhibit CYP2C8 activity DevelopmentResources/DrugInteractionsLabeling/ reversibly in vitro (Tables 6 and 7). In an in vitro ucm093664.htm). However, quercetin is not selective screening of 209 commonly used drugs, 48 compounds for CYP2C8; it also inhibits CYP1A2, CYP2C9, exhibited greater than 50% inhibition of recombinant CYP2C19, CYP2D6, and CYP3A4 with IC50 values of CYP2C8 activity at an inhibitor concentration of 30 mM 3.1–47 mM (Obach, 2000; Zou et al., 2002). In vivo, (Walsky et al., 2005a). Montelukast, candesartan cilex- quercetin at steady state did not affect the pharmaco- etil, zafirlukast, clotrimazole, felodipine, and mometa- kinetics of rosiglitazone (Kim et al., 2005a). sone furoate inhibited CYP2C8 with concentrations The lipid-lowering drug gemfibrozil is a moderate, supporting half of the maximal inhibition (IC50)of direct competitive inhibitor of CYP2C8 in vitro (Wen #3 mM in recombinant CYP2C8 and HLM. In another et al., 2001; Prueksaritanont et al., 2002; Wang et al., study, the inhibition of CYP2C8 by montelukast was 2002), with a Ki range between 9.3 and 270 mM found to be competitive and selective, with reversible (Table 6). Gemfibrozil also inhibits CYP2C9 and inhibition constants (Ki) ranging from 0.0092 to 0.15 mM, CYP2C19 with Ki values of 5.8 and 24 mM, respectively, depending on the protein concentration used in the and CYP1A2 with a Ki of 82 mM (Wen et al., 2001). incubation (Walsky et al., 2005b). However, despite Moreover, it inhibits several drug transporters in vitro, their strong inhibitory effect on CYP2C8 in vitro, most notably OATP1B1 (lowest reported Ki =4mM) TABLE 6 194 Drugs, drug metabolites, and some other compounds that act as reversible CYP2C8 inhibitors

Therapeutic Use Mode of Test Marker b b c c Inhibitor and/or Drug Class IC50 Ki Inhibition System Reactiona Imax fu I/Ki Iu/Ki References

mM(mg/ml) mM(mg/ml) mM 2-Oxo-clopidogrel Drug metabolite 33.9 rCYP2C8 DBF Hagihara et al., 2008 4.1 rCYP2C8 Ceri-1 Floyd et al., 2012 4.2 rCYP2C8 Ceri-23 Floyd et al., 2012 32.0 HLM Amo Tornio et al., 2014 2,4-Dichloroaniline Drug metabolite 99.4 HLM Rosi-OH Wu et al., 2014 4-Hydroxyospemifene Drug metabolite 27.7 HLM Amo FDA, 2013i; Turpeinen et al., 2013 4’-Hydroxyospemifene Drug metabolite 7 HLM Amo FDA, 2013i; Turpeinen et al., 2013 7-Epi-paclitaxel Paclitaxel epimer 2.1 HLM Pacli Zhang et al., 2009b 7-O-succinyl macrolactin A Antibiotic 20.5 HLM Rosi-OH Bae et al., 2014 17b-Estradiol (estradiol) Hormonal replacement 21.5 rCYP2C8 Amo Walsky et al., 2005a therapy 6.6 Competitive HLM Amo VandenBrink et al., 2011 23.8 Competitive HLM Monte VandenBrink et al., 2011 17.7 Competitive HLM Pacli VandenBrink et al., 2011 8.9 Competitive HLM Repa VandenBrink et al., 2011 23.8 Competitive HLM Rosi VandenBrink et al., 2011 19 HLM Amo Nirogi et al., 2014 Abiraterone Anticancer, CYP17A1 1.6 HLM n/a 0.65 0.002 0.81 ,0.01 EMA, 2012a inhibitor

Abiraterone acetate Anticancer, CYP17A1 1.3 HLM n/a EMA, 2012a al. et Backman inhibitor Acotiamide (Z-338) Antidyspeptic, 121 Competitive HLM DBF Furuta et al., 2004, acetylcholinesterase inhibitor Afatinib Anticancer, PKI 94.83 HLM Pacli 0.078 0.428 ,0.01 ,0.01 Wang et al., 2014a Alisertib (MLN8237) Anticancer, PKI 16.3 n/a n/a Pusalkar et al., 2014 Alitretinoin (9-cis-retinoic Anticancer, retinoid 17.6 20.2 HLM Taza 0.28 0.01 Attar et al., 2003 acid) Amlodipine Antihypertensive, CCB 10.7 rCYP2C8 Amo 0.0319 0.07 ,0.01 ,0.01 Walsky et al., 2005a 6.4 rCYP2C8 Ceri-1 0.01 ,0.01 Floyd et al., 2012 4.0 rCYP2C8 Ceri-23 0.02 ,0.01 Floyd et al., 2012 9.4 HLM Amo ,0.01 ,0.01 Nirogi et al., 2014 Amodiaquine Antimalarial 11.7 HLM Monte 0.047 ,0.01 VandenBrink et al., 2011 .100 HLM Pacli ,0.01 VandenBrink et al., 2011 1.9 HLM Repa 0.02 VandenBrink et al., 2011 11.0 HLM Rosi ,0.01 VandenBrink et al., 2011 Anastrozole Anticancer, aromatase 48 10 Competitive HLM Tolbu 0.16 0.60 0.02 0.01 Grimm and Dyroff, 1997 inhibitor Anidulafungin Antifungal 12 HLM Amo 3.07 0.16 0.51 0.08 Damle et al., 2009 Anti-Parkinson, dopamine 1–10 rCYP2C8 DBF 4 ,0.01 8.00 ,0.08 Salminen et al., 2011 agonist Apremilast Antipsoriatic, PDE4 inhibitor 56.1 HLM Pacli 0.764 0.32 0.03 ,0.01 FDA, 2014e Antiviral, protease inhibitor 2.1 n/a n/a 7.66 0.14 3.65 0.51 FDA, 2015b Atorvastatin (acid, parent) Antihyperlipidemic, 38.4 15.9 Mixed HLM Pacli 0.098 0.02 ,0.01 ,0.01 Tornio et al., 2005 HMG-CoA reductase inhibitor 38.4 HLM Pacli ,0.01 ,0.01 Sakaeda et al., 2006 21.9 HLM Amo ,0.01 ,0.01 Jenkins et al., 2011 55.7 rCYP2C8 Fluo ,0.01 ,0.01 Schelleman et al., 2014 Atorvastatin acyl-b-D Drug metabolite 45 HLM Amo Jenkins et al., 2011 glucuronide (G2) (continued) TABLE 6—Continued

Therapeutic Use Mode of Test Marker b b c c Inhibitor and/or Drug Class IC50 Ki Inhibition System Reactiona Imax fu I/Ki Iu/Ki References Atorvastatin lactone Antihyperlipidemic, HMG- 28.8 HLM Pacli Sakaeda et al., 2006 CoA reductase inhibitor Atrazine Pesticide 31.3 HLM Amo Abass et al., 2009 Axitinib Anticancer, PKI 0.5 HLM Pacli 0.16 0.01 0.32 ,0.01 FDA, 2012f 0.11 HLM Amo 2.90 0.03 Filppula et al., 2014 0.17 HLM Pacli 0.94 ,0.01 Wang et al., 2014a AZD2624 Antipsychotic, neurokinin-3 83.3 HLM Amo 0.7 0.02 Li et al., 2010 receptor and tachykinin receptor 3 antagonist Azilsartan medoxomil Antihypertensive, prodrug 3.5 HLM Pacli FDA, 2011f Belinostat Anticancer, histone 100 HLM n/a 100 0.071 2.00 0.14 FDA, 2014c deacetylase inhibitor Belinostat 3-ASBA (M24) Drug metabolite 49.1 HLM n/a FDA, 2014c Belinostat acid (M26) Drug metabolite 22.1 HLM n/a FDA, 2014c Belinostat amide Drug metabolite 30.8 HLM n/a FDA, 2014c Interactions and Metabolism Drug in CYP2C8 of Role Belinostat PX106507 Drug metabolite 13.8 HLM n/a FDA, 2014c Benzbromarone Antihyperuricemic, XO 0.055 Competitive HLM Amo VandenBrink et al., 2011 inhibitor 0.38 Competitive HLM Monte VandenBrink et al., 2011 0.95 Competitive HLM Pacli VandenBrink et al., 2011 0.15 Competitive HLM Repa VandenBrink et al., 2011 0.36 Competitive HLM Rosi VandenBrink et al., 2011 (2)-N-3-benzyl- derivative 34 HLM Pacli Cai et al., 2004 phenobarbital Antihyperlipidemic, PPARa 74 HLM Pacli 39.5 0.05 1.07 0.05 Fujino et al., 2003a agonist 9.7 Competitive HLM Pacli 4.07 0.20 Kajosaari et al., 2005a Bisphenol A Bisphenol 97 Noncompetitive rCYP2C8 Ami Niwa et al., 2000 BTFM gemfibrozil Gemfibrozil analog 13 HLM Amo Jenkins et al., 2011 BTFM gemfibrozil acyl- Gemfibrozil acyl-b-D- 37 HLM Amo Jenkins et al., 2011 b-D-glucuronide glucuronide analog Cabozantinib Anticancer, PKI 5.0 rCYP2C8 n/a 3.27 ,0.003 1.31 ,0.01 FDA, 2012c 6.4 4.6 Noncompetitive HLM Amo 0.71 ,0.01 FDA, 2012c 3.8 4.6 Noncompetitive HLM Amo 0.71 ,0.01 Lacy et al., 2015; Nguyen et al., 2015 Canagliflozin Antidiabetic, SGLT2 75 n/a n/a 7.60 0.017 0.20 ,0.01 FDA, 2013e inhibitor Canagliflozin glucuronide Drug metabolite 64 n/a Amo FDA, 2013e (M7) Candesartan Antihypertensive, ARB 36.2 rCYP2C8 Amo 0.19 0.002 0.01 ,0.01 Walsky et al., 2005a Candesartan cilexetil Antihypertensive, prodrug 0.496 rCYP2C8 Amo 0.41 ,0.01 1.65 0.02 Walsky et al., 2005a 3.04 HLM Amo 0.27 ,0.01 Walsky et al., 2005a Pesticide 34.0 HLM Amo Abass et al., 2009 Antihypertensive 16.6 rCYP2C8 Amo 0.258 0.05 0.03 ,0.01 Walsky et al., 2005a Cefuroxime axetil Antibiotic, prodrug 11.1 rCYP2C8 Amo 19.6 0.67 3.53 2.37 Walsky et al., 2005a Celecoxib Anti-inflammatory, NSAID 15.9 rCYP2C8 Amo 1.85 0.03 0.23 ,0.01 Walsky et al., 2005a 4.9 Competitive HLM Amo 0.38 0.01 VandenBrink et al., 2011 7.9 Competitive HLM Monte 0.23 ,0.01 VandenBrink et al., 2011 54.4 Competitive HLM Pacli 0.03 ,0.01 VandenBrink et al., 2011 3.1 Competitive HLM Repa 0.60 0.02 VandenBrink et al., 2011 5.1 Competitive HLM Rosi 0.36 0.01 VandenBrink et al., 2011 9.9 rCYP2C8 Ceri-1 0.37 0.01 Floyd et al., 2012 5.4 rCYP2C8 Ceri-23 0.69 0.02 Floyd et al., 2012 Ceritinib Anticancer, PKI 0.6d 4.86d HLM Amo 1.81 0.028 0.37 0.01 FDA, 2014i d 0.6 HLM Pacli 6.03 0.17 FDA, 2014i 195 (continued) TABLE 6—Continued 196

Therapeutic Use Mode of Test Marker b b c c Inhibitor and/or Drug Class IC50 Ki Inhibition System Reactiona Imax fu I/Ki Iu/Ki References Cerivastatin (acid, parent) Antihyperlipidemic, HMG- 34.4 HLM Pacli 0.0085 ,0.01 ,0.01 ,0.01 Fujino et al., 2004 CoA reductase inhibitor 30.0 31.7 Mixed HLM Pacli ,0.01 ,0.01 Tornio et al., 2005 29.8 HLM Pacli ,0.01 ,0.01 Sakaeda et al., 2006 4.2 Competitive HLM Amo ,0.01 ,0.01 VandenBrink et al., 2011 4.6 Competitive HLM Monte ,0.01 ,0.01 VandenBrink et al., 2011 77.4 Competitive HLM Pacli ,0.01 ,0.01 VandenBrink et al., 2011 4.4 Competitive HLM Repa ,0.01 ,0.01 VandenBrink et al., 2011 13.4 Competitive HLM Rosi ,0.01 ,0.01 VandenBrink et al., 2011 Cerivastatin lactone Antihyperlipidemic, HMG- 44.3 HLM Pacli Sakaeda et al., 2006 CoA reductase inhibitor Pesticide 22.2 HLM Amo Abass et al., 2009 Antipsychotic 20 HLM Amo 0.470 0.05 0.05 ,0.01 Nirogi et al., 2014 Cimetidine Antiulcerative, H2RA 250 HLM Pacli 12 0.81 0.10 0.08 Monsarrat et al., 1997 Antihyperlipidemic, PPARa 441 HLM Pacli 83.0 0.01 0.38 ,0.01 Fujino et al., 2003a agonist Clofazimine Antilepric 14.1 HLM Pacli Shimokawa et al., 2015 Clopidogrel Antithrombotic, platelet 10.2 rCYP2C8 Amo 0.06 0.02 0.01 ,0.01 Walsky et al., 2005a aggregation inhibitor 33.2 rCYP2C8 DBF ,0.01 ,0.01 Hagihara et al., 2008 2.8 rCYP2C8 Ceri-1 0.04 ,0.01 Floyd et al., 2012 3.4 rCYP2C8 Ceri-23 0.04 ,0.01 Floyd et al., 2012 53.6 HLM Amo ,0.01 ,0.01 Tornio et al., 2014 Clopidogrel carboxylic acid Drug metabolite .50.0 rCYP2C8 DBF Hagihara et al., 2008 107 rCYP2C8 Ceri-1 Floyd et al., 2012 al. et Backman 136 rCYP2C8 Ceri-23 Floyd et al., 2012 Clopidogrel active Drug metabolite .50.0 rCYP2C8 DBF Hagihara et al., 2008 metabolite Clotrimazole Antifungal 2.5 Noncompetitive rCYP2C8 Torse 0.008 ,0.01 Ong et al., 2000 0.725 rCYP2C8 Amo 0.02 Walsky et al., 2005a 0.776 HLM Amo 0.02 Walsky et al., 2005a 0.12 Competitive HLM Amo 0.07 VandenBrink et al., 2011 0.27 Competitive HLM Monte 0.03 VandenBrink et al., 2011 0.22 Competitive HLM Pacli 0.04 VandenBrink et al., 2011 0.19 Competitive HLM Repa 0.04 VandenBrink et al., 2011 1.9 Competitive HLM Rosi ,0.01 VandenBrink et al., 2011 0.803 HLM Pacli 0.02 Lee et al., 2012a Antiviral, pharmacokinetic 30.1 HLM Pacli 2.38 0.03 0.16 ,0.01 FDA, 2012j inhibitor CP-778875 Antihyperlipidemic, PPARa 1.83 HLM Amo Kalgutkar et al., 2013 agonist Cyclosporine Immunosuppressant, 79 rCYP2C8 Pacli 1.11 0.07 0.03 ,0.01 Yoshida et al., 2012 calcineurin inhibitor CYP3cide (PF-04981517) Pharmacokinetic inhibitor 78 HLM Pacli Walsky et al., 2012 Dabrafenib Anticancer, PKI 7.7–8.2 HLM Rosi 2.85 0.003 0.74 ,0.01 Lawrence et al., 2014 Antihyperlipidemic, 1.5 HLM Pacli 9.70 12.93 Derks et al., 2009 cholesterylester transfer protein inhibitor Danazol Hypoestrogenic, 1.95 HLM Pacli 0.21 0.22 Lee et al., 2012a hyperandrogenic, ethisterone derivative Dasabuvir (ABT-333) Antiviral, NSB5 inhibitor ;17 Competitive n/a n/a 2.09 0.005 ;0.25 ,0.01 FDA, 2014k Dasatinib Anticancer, PKI 12 3.6 HLM Pacli 0.13 0.04 0.04 ,0.01 FDA, 2006a 38.6 HLM Pacli ,0.01 ,0.01 Kim et al., 2013b 6.31 HLM Pacli 0.02 ,0.01 Wang et al., 2014a (continued) TABLE 6—Continued

Therapeutic Use Mode of Test Marker b b c c Inhibitor and/or Drug Class IC50 Ki Inhibition System Reactiona Imax fu I/Ki Iu/Ki References N-debutyldronedarone Drug metabolite 24.4 36.6 Noncompetitive HLM Pacli FDA, 2009a (SR35021) N-deethyl sunitinib Drug metabolite 52 HLM Pacli FDA, 2006b (SU12662) N-demethylimatinib Drug metabolite 99 HLM Pacli FDA, 2001 31.3 12.8 Mixed HLM Amo Filppula et al., 2012 N-demethyltoremifene Drug metabolite 2.1 HLM Pacli Kim et al., 2011b Deferasirox , iron chelating 100 HLM Pacli 0.46 0.01 ,0.01 ,0.01 FDA, 2005a agent Deferasirox metabolite Drug metabolite 160 HLM Pacli FDA, 2005a CGP82813A Dexamethasone Anti-inflammatory, 12.0 rCYP2C8 Amo 8.15 1.36 Walsky et al., 2005a glucucorticoid Diclofenac Anti-inflammatory, NSAID 54 HLM Amo 6.28 ,0.005 0.23 ,0.01 Jenkins et al., 2011 Diclofenac acyl glucuronide Drug metabolite 14 HLM Amo Jenkins et al., 2011 Interactions and Metabolism Drug in CYP2C8 of Role Diethyldithiocarbamate Alcoholic detergent 129.5 rCYP2C8 Dia Sai et al., 2000 464.1 rCYP2C8 Phena Sai et al., 2000 Diethylstilbestrol Synthetic estrogen 8.0 Competitive HLM Pacli Qu et al., 2011 Diltiazem Antihypertensive, CCB 25 rCYP2C8 Ceri-1 0.335 0.22 0.03 ,0.01 Floyd et al., 2012 124 rCYP2C8 Ceri-23 ,0.01 ,0.01 Floyd et al., 2012 Doxorubicin Anticancer 2 HLM Pacli 1.63 0.24 1.63 0.39 Monsarrat et al., 1997 64.8 Competitive HLM Pacli 0.03 ,0.01 Bun et al., 2003 90 Noncompetitive HLM Luci 0.02 ,0.01 Masek et al., 2011 Duloxetine Antidepressant, SNRI 180 HLM Pacli 0.079 0.05 ,0.01 ,0.01 FDA, 2008b 60 HLM Amo ,0.01 ,0.01 Paris et al., 2009 Antiviral, NNRTI 4.0 rCYP2C8 Amo 12.6 0.005 6.30 0.03 Parikh et al., 2007 6.05 Competitive rCYP2C8 Amo 2.08 0.01 Xu and Desta, 2013 4.78 Competitive HLM Amo 2.64 0.01 Xu and Desta, 2013 Eltrombopag Antihemorrhagic, c-mpl 24.8 HLM Pacli 29 ,0.01 2.34 0.02 FDA, 2008c receptor agonist Enzalutamide Anticancer, antiandrogen 10 5.5 Mixed HLM Amo 35.7 0.02 6.50 0.13 FDA, 2012k Enzalutamide M1 Drug metabolite 20 HLM Amo FDA, 2012k Enzalutamide M2 Drug metabolite 28 HLM Amo FDA, 2012k Erlotinib Anticancer, PKI 6.17 5.8 Competitive HLM Pacli 6.06 0.10 1.05 0.10 Dong et al., 2011 9.5 HLM Pacli 1.28 0.13 Kim et al., 2013b 4.02 HLM Pacli 1.51 0.15 Wang et al., 2014a Esomeprazole (S- Antiulcerative, PPI 31.0 HLM Amo 4.5 0.05 0.29 0.02 Zvyaga et al., 2012 omeprazole) Ethionamide Antituberculosis 110 HLM Pacli 12.99 0.70 0.24 0.17 Shimokawa et al., 2015 Antiviral, NNRTI 19.6 Noncompetitive HLM Pacli 2.2 0.01 0.11 ,0.01 FDA, 2008a Exemestane Anticancer, aromatase 13.5 rCYP2C8 Amo 0.060 0.10 ,0.01 ,0.01 Walsky et al., 2005a inhibitor Febuxostat Antihyperuricemic, XO 20 n/a n/a 16.78 0.007 0.84 ,0.01 Naik et al., 2012 inhibitor Felodipine Antihypertensive, CCB 0.726 rCYP2C8 Amo 0.0073 0.004 0.02 ,0.01 Walsky et al., 2005a 1.20 HLM Amo 0.01 ,0.01 Walsky et al., 2005a Pesticide 4.3 HLM Amo Abass et al., 2009 Antihyperlipidemic, 288 HLM Pacli 23.83 ,0.01 0.17 ,0.01 Fujino et al., 2003a PPARa agonist 92.6 Competitive HLM Pacli 0.26 ,0.01 Kajosaari et al., 2005a 2.39 rCYP2C8 Amo 19.94 0.20 Walsky et al., 2005a 4.8 rCYP2C8 Fluo 9.93 0.10 Schelleman et al., 2014 Fluoxymesterone Androgenic, 3-oxoandrosten 16 rCYP2C8 Ceri-1 Floyd et al., 2012 (4) derivative

16 rCYP2C8 Ceri-23 Floyd et al., 2012 197 (continued) TABLE 6—Continued 198

Therapeutic Use Mode of Test Marker b b c c Inhibitor and/or Drug Class IC50 Ki Inhibition System Reactiona Imax fu I/Ki Iu/Ki References Anti-inflammatory, 0.58 HLM Pacli 0.00023 0.01 ,0.01 ,0.01 FDA, 2013c glucocorticoid Fluticasone M10 Drug metabolite 80 HLM Pacli FDA, 2013c metabolite Fluvastatin (acid, parent) Antihyperlipidemic, HMG- 20 HLM Pacli 0.461 0.01 0.05 ,0.01 Fischer et al., 1999 CoA reductase inhibitor 36.7 18.9 Mixed HLM Pacli 0.02 ,0.01 Tornio et al., 2005 15.1 rCYP2C8 Amo 0.06 ,0.01 Walsky et al., 2005a 70.2 HLM Pacli 0.01 ,0.01 Sakaeda et al., 2006 Fluvastatin lactone Antihyperlipidemic, HMG- 55.4 HLM Pacli Sakaeda et al., 2006 CoA reductase inhibitor Gefitinib Anticancer, PKI 31.0 HLM Pacli 0.8 0.10 0.05 ,0.01 Kim et al., 2013b 12.3 HLM Amo 0.13 0.01 Filppula et al., 2014 8.69 HLM Pacli 0.09 ,0.01 Wang et al., 2014a Gemfibrozil Antihyperlipidemic, PPARa 49 Hep Ceri-23 100 ,0.03 4.08 0.12 Prueksaritanont agonist et al., 2002 87 Competitive HLM Pacli 1.15 0.03 Prueksaritanont et al., 2002 78 rCYP2C8 Ceri-1 2.56 0.08 Wang et al., 2002 68 rCYP2C8 Ceri-23 2.94 0.09 Wang et al., 2002 .250 273 Competitive HLM Ceri-1 ,0.37 ,0.01 Wang et al., 2002 95 69 Competitive HLM Ceri-23 1.45 0.04 Wang et al., 2002 91 75–76 Competitive HLM Pacli 1.33 0.04 Wang et al., 2002 48 Mixed HLM Pacli 4.17 0.13 Fujino et al., 2003a 55.4 Mixed HLM Pacli 1.81 0.05 Fujino et al., 2003b al. et Backman 36.8 rCYP2C8 Ceri-1 5.44 0.16 Shitara et al., 2004 29.7 rCYP2C8 Ceri-23 6.73 0.20 Shitara et al., 2004 119 69.0 Noncompetitive HLM Rosi-OH 1.45 0.04 Hruska et al., 2005 30.4 Competitive HLM Pacli 3.29 0.10 Kajosaari et al., 2005a 75.6 rCYP2C8 Amo 2.65 0.08 Walsky et al., 2005a 59 HLM Pio 3.39 0.10 Jaakkola et al., 2006c 120 HLM Pacli 1.67 0.05 Ogilvie et al., 2006 107 HLM Monte 1.87 0.06 Karonen et al., 2010 63 HLM Monte-4 3.18 0.10 Karonen et al., 2010 120 HLM Amo 1.67 0.05 Jenkins et al., 2011 10.2 Competitive HLM Amo 9.80 0.29 VandenBrink et al., 2011 13.5 Competitive HLM Monte 7.41 0.22 VandenBrink et al., 2011 .100 Competitive HLM Pacli 1.00 0.03 VandenBrink et al., 2011 9.3 Competitive HLM Repa 10.75 0.32 VandenBrink et al., 2011 36.1 Competitive HLM Rosi 2.77 0.08 VandenBrink et al., 2011 14 rCYP2C8 Ceri-23 7.14 0.21 Floyd et al., 2012 Genistein Anticancer, PKI 2.5 HLM Pacli Burnett et al., 2011 Glipizide Antidiabetic, sulfonylurea 338.2 rCYP2C8 Fluo 1.04 0.016 ,0.01 ,0.01 Schelleman et al., 2014 Glyburide (glibenclamide) Antidiabetic, sulfonylurea 10.8 rCYP2C8 Amo 0.214 0.002 0.04 ,0.01 Walsky et al., 2005a 4.3 rCYP2C8 Ceri-1 0.10 ,0.01 Floyd et al., 2012 6.7 rCYP2C8 Ceri-23 0.06 ,0.01 Floyd et al., 2012 Glyphosate Pesticide 82.0 HLM Amo Abass et al., 2009 Hydroxymethyl-ivacaftor Drug metabolite 17.7 0.39 Competitive HLM Amo FDA, 2012g (M1) Ibrutinib Anticancer, PKI 12.03 HLM Pacli 0.37 0.127 0.03 ,0.01 FDA, 2013d Ibrutinib metabolite PCI- Drug metabolite (7.84) HLM Pacli FDA, 2013d 45227 Iclaprim Antibiotic (91.5) rCYP2C8 n/a Hall et al., (2007) ID951551 Acotiamide analog 17 HLM DBF Furuta et al., 2004 Idelalisib Anticancer, PKI 13 HLM Pacli 4.6 ,0.16 0.71 0.11 FDA, 2014h (continued) TABLE 6—Continued

Therapeutic Use Mode of Test Marker b b c c Inhibitor and/or Drug Class IC50 Ki Inhibition System Reactiona Imax fu I/Ki Iu/Ki References Idelalisib metabolite GS- Drug metabolite 39.8 HLM Pacli 9.4 ,0.12 0.47 0.06 FDA, 2014h 563117 Imatinib Anticancer, PKI 15.7 8.4 Mixed HLM Amo 5.27 0.05 0.63 0.03 Filppula et al., 2012 25.9 HLM Pacli 0.41 0.02 Kim et al., 2013b 11.28 HLM Pacli 0.47 0.02 Wang et al., 2014a Antiobstructive, LABA 30 HLM Pacli 0.00125 0.049 ,0.01 ,0.01 FDA, 2011a Sedative, GABAA receptor 30 15 HLM Pacli Madan et al., 2007 modulator Indomethacin Anti-inflammatory, NSAID 88 HLM Amo 6.7 0.10 0.15 0.02 Jenkins et al., 2011 Indomethacin acyl-b-D- Drug metabolite 26 HLM Amo Jenkins et al., 2011 glucuronide Ipriflavone M1 Drug metabolite 9.9 HLM Pacli Moon et al., 2007 Ipriflavone M2 Drug metabolite 10.2 HLM Pacli Moon et al., 2007 Ipriflavone M4 Drug metabolite 31.8 HLM Pacli Moon et al., 2007 Ipriflavone M5 Drug metabolite 2.5 HLM Pacli Moon et al., 2007 Interactions and Metabolism Drug in CYP2C8 of Role Irbesartan Antihypertensive, ARB 9.73 rCYP2C8 Amo 3.0 0.10 0.62 0.06 Walsky et al., 2005a 18 rCYP2C8 Ceri-1 0.33 0.03 Floyd et al., 2012 16 rCYP2C8 Ceri-23 0.38 0.04 Floyd et al., 2012 Isotretinoin (13-cis-retinoic Antiacne, retinoid 15.1 66.2 HLM Taza 0.69 ,0.01 0.01 ,0.01 Attar et al., 2003 acid) Isradipine Antihypertensive, CCB 5.00 HLM Pacli 0.030 0.03 0.01 ,0.01 Lee et al., 2012a Itraconazole Antifungal 31 rCYP2C8 Pacli 0.9 0.002 0.06 ,0.01 Yoshida et al., 2012 Ivacaftor Antifibrotic 3.8 3.4 Mixed HLM Amo 13.89 ,0.02 4.09 0.08 FDA, 2012g Ivacaftor metabolite (M6) Drug metabolite 63.1 HLM Amo FDA, 2012g Ketoconazole Antifungal 25 HLM Pacli 3.2 0.01 0.26 ,0.01 Monsarrat et al., 1997 2.5 Noncompetitive rCYP2C8 Torse 1.28 0.01 Ong et al., 2000 4.0 rCYP2C8 Dia 1.60 0.02 Sai et al., 2000 8.9 rCYP2C8 Phena 0.72 ,0.01 Sai et al., 2000 6-9 HLM Pacli 1.07 0.01 Dierks et al., 2001 11.8 Noncompetitive HLM Pacli 0.27 ,0.01 Bun et al., 2003 87.7 HLM Amo 0.07 ,0.01 Turpeinen et al., 2005 5.51 rCYP2C8 Amo 1.16 0.01 Walsky et al., 2005a 4 rCYP2C8 Amo 1.60 0.02 O’Donnell et al., 2007 2.45 HLM Pacli 2.61 0.03 Lee et al., 2012a 1.7 HLM Amo 3.77 0.04 Nirogi et al., 2015 Ketoprofen acyl-b-D- Drug metabolite 26 HLM Amo Jenkins et al., 2011 glucuronide KR-32570 Antiarrhythmic 30 HLM Pacli Kim et al., 2006 KR-60436 Antiulcerative, PPI 30 HLM Pacli Ji et al., 2005 Lansoprazole Antiulcerative, PPI 55 rCYP2C8 Ceri-1 0.671 0.03 0.02 ,0.01 Floyd et al., 2012 19 rCYP2C8 Ceri-23 0.07 ,0.01 Floyd et al., 2012 5.75 HLM Pacli 0.23 ,0.01 Lee et al., 2012a Lapatinib Anticancer, PKI 0.60 Competitive HLM Pacli 4.2 ,0.01 7.00 ,0.07 FDA, 2007e 1.43 HLM Pacli 2.94 ,0.03 Wang et al., 2014a Antiasthmatic, PGD2 6.5 n/a n/a Schwartz et al., 2009 receptor antagonist Laromustine Anticancer, alkylating agent .750 Noncompetitive HLM Amo Nassar et al., 2009 (VNP40101M) Lasofoxifene Antiosteoporotic, SERM 8.1 HLM Amo 0.0050 ,0.01 Moller et al., 2006 Lenvatinib Anticancer, PKI 10.1 10.1 HLM Pacli 1.546 0.02 0.15 ,0.01 FDA, 2015a Lestaurtinib (CEP-701) Anticancer, PKI 9.5 HLM Amo Filppula et al., 2014 Levothyroxine Hormonal replacement 3.30 rCYP2C8 Amo Walsky et al., 2005a therapy 5.4 rCYP2C8 Ceri-1 Floyd et al., 2012

4.6 rCYP2C8 Ceri-23 Floyd et al., 2012 199 (continued) TABLE 6—Continued 200

Therapeutic Use Mode of Test Marker b b c c Inhibitor and/or Drug Class IC50 Ki Inhibition System Reactiona Imax fu I/Ki Iu/Ki References Loperamide Antidiarrheal, opioid 24 HLM Amo 0.0042 ,0.01 Nirogi et al., 2014 Antiviral, protease inhibitor 4.1 rCYP2C8 Amo 15.6 0.02 7.61 0.15 Parikh et al., 2007 Loratadine 3.36 rCYP2C8 Amo 0.0088 0.03 ,0.01 ,0.01 Walsky et al., 2005a 2.95 HLM Pacli ,0.01 ,0.01 Lee et al., 2012a Antiobesity, 5-HT2C receptor .200 HLM Pacli 0.434 0.30 ,0.01 ,0.01 FDA, 2012b agonist Lorcaserin sulfamate (M1) Drug metabolite .200 HLM Pacli FDA, 2012b Losartan Antihypertensive, ARB 12.9 rCYP2C8 Amo 0.64 0.013 0.10 ,0.01 Walsky et al., 2005a 40.7 Competitive rCYP2C8 Pacli 0.02 ,0.01 Mukai et al., 2014 (lactone, Antihyperlipidemic, HMG- 14.7 8.4 Mixed HLM Pacli 0.100 0.05 0.01 ,0.01 Tornio et al., 2005 parent) CoA reductase inhibitor 9.10 rCYP2C8 Amo 0.02 ,0.01 Walsky et al., 2005a 79.9 HLM Pacli ,0.01 ,0.01 Sakaeda et al., 2006 5.6 Competitive HLM Amo 0.02 ,0.01 VandenBrink et al., 2011 9.3 Competitive HLM Monte 0.01 ,0.01 VandenBrink et al., 2011 18.8 Competitive HLM Pacli ,0.01 ,0.01 VandenBrink et al., 2011 2.8 Competitive HLM Repa 0.04 ,0.01 VandenBrink et al., 2011 4.2 Competitive HLM Rosi 0.02 ,0.01 VandenBrink et al., 2011 27.5 rCYP2C8 Fluo ,0.01 ,0.01 Schelleman et al., 2014 Lovastatin acid Antihyperlipidemic, HMG- 54.9 48.9 Mixed HLM Pacli 0.011 0.05 ,0.01 ,0.01 Tornio et al., 2005 CoA reductase inhibitor 74.6 HLM Pacli ,0.01 ,0.01 Sakaeda et al., 2006 Macitentan Antihypertensive, ERA 21 HLM Pacli 0.29 ,0.01 0.03 ,0.01 FDA, 2013k Macitentan metabolite M6 Drug metabolite 23 HLM Pacli FDA, 2013k (ACT-132577) al. et Backman Macrolactin A Antibiotic 26.4 HLM Rosi-OH Bae et al., 2014 Pesticide 31.0 HLM Amo Abass et al., 2009 Medroxyprogesterone Progestin 4.79 rCYP2C8 Amo 0.123 0.14 0.05 ,0.01 Walsky et al., 2005a 0.76 Competitive HLM Amo 0.16 0.02 VandenBrink et al., 2011 7.5 Competitive HLM Monte 0.02 ,0.01 VandenBrink et al., 2011 8.2 Competitive HLM Pacli 0.02 ,0.01 VandenBrink et al., 2011 1.9 Competitive HLM Repa 0.07 ,0.01 VandenBrink et al., 2011 6.6 Competitive HLM Rosi 0.02 ,0.01 VandenBrink et al., 2011 Anti-inflammatory, NSAID 14.9 HLM Amo 41.44 ,0.10 5.56 0.56 Jenkins et al., 2011 Mefenamic acyl-b-D- Drug metabolite 8.5 HLM Amo Jenkins et al., 2011 glucuronide Mertansine (DM1) Antibody-drug linker 11 Competitive HLM Pacli Davis et al., 2012 Methoxsalen (8- Antipsoriatic ;10 HLM Pacli 2.36 ;0.47 Dierks et al., 2001 methoxypsoralen) Methyl belinostat Drug metabolite 13.8 HLM n/a FDA, 2014c Methylprednisolone Anti-inflammatory, 25.4 rCYP2C8 Amo 0.475 0.22 0.04 ,0.01 Walsky et al., 2005a glucucorticoid Sedative, benzodiazepine 18 Noncompetitive rCYP2C8 Torse 0.34 0.02 0.02 ,0.01 Ong et al., 2000 12.4 rCYP2C8 Amo 0.06 ,0.01 Walsky et al., 2005a MMB4 DMS Antidote, agonist 82.9 126 Noncompetitive rCYP2C8 Luci Hong et al., 2013 furoate Anti-inflammatory, 0.813 rCYP2C8 Amo 0.00012 0.02 ,0.01 ,0.01 Walsky et al., 2005a glucucorticoid 0.327 HLM Amo ,0.01 ,0.01 Walsky et al., 2005a Montelukast Antiasthmatic, LTRA 0.00922 rCYP2C8 Amo 0.89 ,0.01 193.06 1.93 Walsky et al., 2005a 0.0196 HLM Amo 90.82 0.91 Walsky et al., 2005a 0.0092 Competitive rCYP2C8 Amo 96.74 0.97 Walsky et al., 2005b 0.019 Competitive rCYP2C8 Rosi 46.84 0.47 Walsky et al., 2005b 0.020–2.0 0.014 Competitive HLM Amo 63.57 0.64 Walsky et al., 2005b 0.15 Competitive HLM Pacli 5.93 0.06 Walsky et al., 2005b 0.11 Competitive HLM Rosi 8.09 0.08 Walsky et al., 2005b (continued) TABLE 6—Continued

Therapeutic Use Mode of Test Marker b b c c Inhibitor and/or Drug Class IC50 Ki Inhibition System Reactiona Imax fu I/Ki Iu/Ki References 0.18 HLM Pio 9.89 0.10 Jaakkola et al., 2006c 0.009–0.01 rCYP2C8 Amo 197.78 1.98 O’Donnell et al., 2007 0.022 0.013 HLM Amo 68.46 0.69 Perloff et al., 2009 0.0081 Competitive HLM Amo 109.88 1.10 VandenBrink et al., 2011 0.026 Competitive HLM Pacli 34.23 0.34 VandenBrink et al., 2011 0.016 Competitive HLM Repa 55.63 0.56 VandenBrink et al., 2011 0.21 Competitive HLM Rosi 4.24 0.04 VandenBrink et al., 2011 1.2 rCYP2C8 Ceri-1 1.48 0.02 Floyd et al., 2012 0.02 rCYP2C8 Ceri-23 89.00 0.89 Floyd et al., 2012 0.05–0.10 HLM Amo 35.60 0.36 Kozakai et al., 2012 0.14 Competitive HLM Pacli 6.36 0.06 Kim et al., 2013b 0.16 Competitive HLM Amo 5.56 0.06 Kim et al., 2013b 2.67 HLM Pacli 0.67 ,0.01 Zheng et al., 2013 0.27 n/a n/a 6.59 0.07 Korzekwa, 2014 159 Hep Amo 0.01 ,0.01 Kosugi et al., 2014 Interactions and Metabolism Drug in CYP2C8 of Role 2.9 HLM Rosi-OH 0.61 ,0.01 Zheng et al., 2014 0.101 HLM Pacli 17.62 0.18 FDA, 2014j 0.14 HLM Amo 12.71 0.13 FDA, 2014j 0.010–0.75 HLM Amo 178.00 1.78 Nirogi et al., 2015 Antihypertensive, b1 55 Noncompetitive HLM Pacli 3.65 0.0887 0.07 ,0.01 FDA, 2007a receptor blocker Antidepressant 23.2 rCYP2C8 Amo 4.70 ,0.01 0.41 ,0.01 Walsky et al., 2005a Netupitant 50.43 HLM Pacli 0.75 0.005 0.03 ,0.01 FDA, 2014b Netupitant hydroxylation Drug metabolite 26.95 HLM Pacli FDA, 2014b metabolite M3 Netupitant N- Drug metabolite 4.74 HLM Pacli FDA, 2014b demethylation metabolite M1 Nicardipine Antihypertensive, CCB 7.1 HLM Pacli 0.17 0.02 0.02 ,0.01 Nakamura et al., 2005 1.56 HLM Pacli 0.22 ,0.01 Lee et al., 2012a Nifedipine Antihypertensive, CCB 9.66 rCYP2C8 Amo 0.14 0.04 0.03 ,0.01 Walsky et al., 2005a 20-23 rCYP2C8 Amo 0.01 ,0.01 O’Donnell et al., 2007 13.53 rCYP2C8 Pacli 0.02 ,0.01 Gao et al., 2010 2.4 Competitive HLM Amo 0.06 ,0.01 VandenBrink et al., 2011 6.3 Competitive HLM Monte 0.02 ,0.01 VandenBrink et al., 2011 9.5 Competitive HLM Pacli 0.02 ,0.01 VandenBrink et al., 2011 1.5 Competitive HLM Repa 0.09 ,0.01 VandenBrink et al., 2011 5.8 Competitive HLM Rosi 0.02 ,0.01 VandenBrink et al., 2011 3.5 HLM Amo 0.08 ,0.01 Nirogi et al., 2014 Nilotinib Anticancer, PKI ,1 0.236 Competitive HLM Pacli 4.3 0.02 18.22 0.36 FDA, 2007c 0.61 Competitive rCYP2C8 Amo 7.04 0.14 Kim et al., 2013b 0.10 Competitive rCYP2C8 Pacli 43.00 0.86 Kim et al., 2013b 0.7 0.15 Competitive HLM Amo 28.67 0.53 Kim et al., 2013b 0.4 0.9 Competitive HLM Pacli 4.78 0.10 Kim et al., 2013b 7.5 HLM Rosi-OH 1.15 0.02 Kim et al., 2013b 0.10 HLM Pacli 43.00 0.86 Wang et al., 2014a Nintedanib Anticancer, PKI .50 HLM Pacli 0.028 0.022 ,0.01 ,0.01 FDA, 2014d Nystatin Antifungal 12.7 rCYP2C8 Amo Walsky et al., 2005a Ombitasvir (ABT-267) Antiviral, NS5A inhibitor 7.4 n/a n/a 0.070 ,0.01 0.02 ,0.01 FDA, 2014k Orphenadrine 278.8 rCYP2C8 Dia Sai et al., 2000 249.1 rCYP2C8 Phena Sai et al., 2000 265 HLM Amo Nirogi et al., 2015 Orteronel (TAK-700) Anticancer, antiandrogen 27.7 HLM n/a Lu et al., 2012 Ospemifene Antidyspareunia, SERM 36.4 HLM Amo 3.16 ,0.01 0.17 ,0.01 FDA, 2013i; Turpeinen

et al., 2013 201 (continued) TABLE 6—Continued 202

Therapeutic Use Mode of Test Marker b b c c Inhibitor and/or Drug Class IC50 Ki Inhibition System Reactiona Imax fu I/Ki Iu/Ki References Oxybutynin 4.50 rCYP2C8 Amo 0.031 ,0.01 0.01 ,0.01 Walsky et al., 2005a Paclitaxel (taxol) Anticancer, taxane 14.9 30.0 Competitive HLM Taza 0.85 0.12 0.03 ,0.01 Attar et al., 2003 13.3 rCYP2C8 Amo 0.13 0.02 Walsky et al., 2005a 23–24 rCYP2C8 Amo 0.07 ,0.01 O’Donnell et al., 2007 5.4 Competitive HLM Amo 0.16 0.02 VandenBrink et al., 2011 89.8 Competitive HLM Monte ,0.01 ,0.01 VandenBrink et al., 2011 6.5 Competitive HLM Repa 0.13 0.016 VandenBrink et al., 2011 12.0 Competitive HLM Rosi 0.07 ,0.01 VandenBrink et al., 2011 Pasireotide Hormonal therapy, ;50 HLM Pacli 0.015 0.12 ,0.01 ,0.01 FDA, 2012h somastatin analog Pazopanib Anticancer, PKI 10 HLM Pacli 132 ,0.01 26.40 0.26 FDA, 2009d 3.72 HLM Pacli 35.48 0.36 Wang et al., 2014a PF-562,271 Anticancer, PKI 23 HLM n/a Rong et al., 2008 Phenthoate Pesticide 10.3 HLM Amo Abass et al., 2009 Pioglitazone Antidiabetic, PPAR-g agonist 9.38 1.69 Competitive HLM Pacli 3.8 ,0.01 2.25 0.02 Sahi et al., 2003 11.7 rCYP2C8 Amo 0.65 ,0.01 Walsky et al., 2005a 6.6 Competitive HLM Amo 0.58 ,0.01 VandenBrink et al., 2011 7.1 Competitive HLM Monte 0.54 ,0.01 VandenBrink et al., 2011 37.6 Competitive HLM Pacli 0.10 ,0.01 VandenBrink et al., 2011 3.8 Competitive HLM Repa 1.00 0.01 VandenBrink et al., 2011 6.1 Competitive HLM Rosi 0.62 ,0.01 VandenBrink et al., 2011 14 rCYP2C8 Ceri-1 0.54 ,0.01 Floyd et al., 2012 16 rCYP2C8 Ceri-23 0.48 ,0.01 Floyd et al., 2012 Pitavastatin (acid, parent) Antihyperlipidemic, HMG- 57.0 HLM Pacli 0.0296 0.005 ,0.01 ,0.01 Sakaeda et al., 2006 CoA reductase inhibitor al. et Backman Pitavastatin lactone Antihyperlipidemic, HMG- 50.5 HLM Pacli 0.0190 0.005 ,0.01 ,0.01 Sakaeda et al., 2006 CoA reductase inhibitor Ponatinib Anticancer, PKI 6.1 3.05 n/a n/a 0.161 0.0008 0.05 ,0.01 FDA, 2012e Prasugrel Antithrombotic, platelet .45.2 rCYP2C8 DBF 1.37 ,0.061 Hagihara et al., 2008 aggregation inhibitor Prasugrel active Drug metabolite .45.2 rCYP2C8 DBF Hagihara et al., 2008 metabolite (R–138727) Prasugrel thiolactone (R– Drug metabolite .50.0 rCYP2C8 DBF Hagihara et al., 2008 95913) (acid, parent) Antihyperlipidemic, HMG- .100 .50 Mixed HLM Pacli 0.085 0.57 ,0.01 ,0.01 Tornio et al., 2005 CoA reductase inhibitor .100 HLM Pacli ,0.01 ,0.01 Sakaeda et al., 2006 Pravastatin lactone Antihyperlipidemic, HMG- 99.3 HLM Pacli Sakaeda et al., 2006 CoA reductase inhibitor Pesticide 84.0 HLM Amo Abass et al., 2009 Sedative, antihistamine 23 HLM Amo 0.07 0.07 ,0.01 ,0.01 Nirogi et al., 2014 Propoxyphene Analgesic, opioid 32 rCYP2C8 Ceri-1 Floyd et al., 2012 18 rCYP2C8 Ceri-23 Floyd et al., 2012 Prothionamide Antituberculosis 57.6 HLM Pacli Shimokawa et al., 2015 Pyrimethamine Antimalarial 45.1 rCYP2C8 Amo 4.74 0.13 0.21 0.027 Parikh et al., 2007 Antipsychotic 20 HLM Amo 0.314 0.17 0.03 ,0.01 Nirogi et al., 2014 Antiarrhythmic 98.5 rCYP2C8 Dia 4 0.13 0.08 0.01 Sai et al., 2000 135.4 rCYP2C8 Phena 0.06 ,0.01 Sai et al., 2000 50 HLM Pacli 0.16 0.02 Dierks et al., 2001 Quinine Antimalarial 11 Competitive rCYP2C8 Torse 29 0.15 2.64 0.40 Ong et al., 2000 R483 Antidiabetic, PPAR-g agonist 5 HLM Pacli Weber et al., 2005 Rabeprazole Antiulcerative, PPI 12.0 rCYP2C8 Amo 0.9 0.037 0.15 ,0.01 Walsky et al., 2005a Ranitidine Antiulcerative, H2RA 10,000 HLM Pacli 1.31 0.85 ,0.01 ,0.01 Monsarrat et al., 1997 3.1 HLM Amo 0.85 0.72 Nirogi et al., 2014 Regorafenib Anticancer, PKI 1.7 0.6 n/a n/a 8.1 0.005 13.50 0.04 FDA, 2012i (continued) TABLE 6—Continued

Therapeutic Use Mode of Test Marker b b c c Inhibitor and/or Drug Class IC50 Ki Inhibition System Reactiona Imax fu I/Ki Iu/Ki References Regorafenib M2 Drug metabolite 1.0 n/a n/a FDA, 2012i Regorafenib M5 Drug metabolite 1.3 n/a n/a FDA, 2012i Repaglinide Antidiabetic, meglitinide 27.1 Competitive HLM Amo 0.104 0.026 ,0.01 ,0.01 VandenBrink et al., 2011 analog 11.1 Competitive HLM Monte ,0.01 ,0.01 VandenBrink et al., 2011 .100 Competitive HLM Pacli ,0.01 ,0.01 VandenBrink et al., 2011 23.0 Competitive HLM Rosi ,0.01 ,0.01 VandenBrink et al., 2011 Retinoic acid, all-trans Antiacne, retinoid 27.0 Competitive HLM Pacli 1.15 ,0.05 0.04 ,0.01 Rahman et al., 1994 (tretinoin) Rifampin (rifampicin) Antibiotic 30.2 Competitive HLM Pacli 8 0.40 0.27 0.11 Kajosaari et al., 2005a Rifapentine Antituberculosis 115 HLM Pacli 34.21 0.02 0.59 0.01 Shimokawa et al., 2015 Antiviral, NNRTI 13.2–19.1 10.0 HLM Pacli 0.5 ,0.01 0.05 ,0.01 FDA, 2011d Antiviral, protease inhibitor 3.03 rCYP2C8 Amo 15 0.02 9.90 0.20 Walsky et al., 2005a 1–2 rCYP2C8 Amo 30.00 0.60 O’Donnell et al., 2007 5.5 HLM Pacli 5.46 0.11 FDA, 2012j Interactions and Metabolism Drug in CYP2C8 of Role Rofecoxib Anti-inflammatory, NSAID 95 rCYP2C8 Ceri-1 1.02 0.13 0.02 ,0.01 Floyd et al., 2012 14 rCYP2C8 Ceri-23 0.15 0.02 Floyd et al., 2012 Rose bengal Xanthene dye 53 Hep Amo Kazmi et al., 2014

Rosiglitazone Antidiabetic, PPAR-g agonist 18 HLM Pacli 1.7 0.002 0.19 ,0.01 Baldwin et al., 1999 9.58 5.59 Competitive HLM Pacli 0.30 ,0.01 Sahi et al., 2003 24.1–26.3 HLM Pacli 0.13 ,0.01 Kim et al., 2005b 10.8 rCYP2C8 Amo 0.13 ,0.01 Walsky et al., 2005a 5.2 Competitive HLM Amo 0.33 ,0.01 VandenBrink et al., 2011 4.1 Competitive HLM Monte 0.42 ,0.01 VandenBrink et al., 2011 28.6 Competitive HLM Pacli 0.06 ,0.01 VandenBrink et al., 2011 1.4 Competitive HLM Repa 1.21 ,0.01 VandenBrink et al., 2011 3.0 rCYP2C8 Ceri-1 1.13 ,0.01 Floyd et al., 2012 2.7 rCYP2C8 Ceri-23 1.26 ,0.01 Floyd et al., 2012 (acid, Antihyperlipidemic, HMG- .100 .50 Mixed HLM Pacli 0.0046 0.12 ,0.01 ,0.01 Tornio et al., 2005 parent) CoA reductase inhibitor .100 HLM Pacli ,0.01 ,0.01 Sakaeda et al., 2006 Rosuvastatin lactone Antihyperlipidemic, HMG- 9.8 HLM Pacli Fujino et al., 2004 CoA reductase inhibitor 32.5 HLM Pacli Sakaeda et al., 2006 Antiasthmatic, b-2- agonist 1.87 rCYP2C8 Amo 0.0040 ,0.01 Walsky et al., 2005a Sanguinarine Anticancer 10.2 8.9 Noncompetitive HLM Pacli Qi et al., 2013 Antiviral, protease inhibitor 1.8 rCYP2C8 Amo 1.41 0.02 1.57 0.031 Parikh et al., 2007 Saracatinib Anticancer, PKI 201.8 HLM Amo Filppula et al., 2014 Sarizotan Antipsychotic 18.2d Competitive HLM Pacli 0.91 0.05 Gallemann et al., 2010 Satraplatin (JM-216) Anticancer 1–3 0.9 Noncompetitive HLM Pacli Ando et al., 1998 (R-roscovitine) Anticancer, PKI 119 rCYP2C8 DBF 10 0.10 0.17 0.02 McClue and Stuart, 2008 Sertraline Antidepressant, SSRI 25.5 rCYP2C8 Amo 0.484 0.02 0.04 ,0.01 Walsky et al., 2005a 350 HLM Pacli ,0.01 ,0.01 FDA, 2008b .100 Competitive HLM Amo ,0.01 ,0.01 VandenBrink et al., 2011 9.0 Competitive HLM Monte 0.05 ,0.01 VandenBrink et al., 2011 .100 Competitive HLM Pacli ,0.01 ,0.01 VandenBrink et al., 2011 7.8 Competitive HLM Repa 0.06 ,0.01 VandenBrink et al., 2011 8.1 Competitive HLM Rosi 0.06 ,0.01 VandenBrink et al., 2011 29.7 HLM Pacli 0.03 ,0.01 Erve et al., 2013 15 HLM Amo 0.06 ,0.01 Nirogi et al., 2014 Simeprevir (TMC435) Antiviral, protease inhibitor (36.8) HLM n/a 14.5 ,0.001 0.79 ,0.01 FDA, 2013h Simvastatin (lactone, Antihyperlipidemic, 9.6 7.1 HLM Pacli 0.096 0.06 0.01 ,0.01 Tornio et al., 2005 parent) HMG-CoA reductase

inhibitor 203 (continued) TABLE 6—Continued 204

Therapeutic Use Mode of Test Marker b b c c Inhibitor and/or Drug Class IC50 Ki Inhibition System Reactiona Imax fu I/Ki Iu/Ki References 5.39 rCYP2C8 Amo 0.04 ,0.01 Walsky et al., 2005a 44.1 HLM Pacli ,0.01 ,0.01 Sakaeda et al., 2006 8.3 HLM Amo 0.02 ,0.01 Jenkins et al., 2011 7.5 Competitive HLM Amo 0.01 ,0.01 VandenBrink et al., 2011 5.7 Competitive HLM Monte 0.02 ,0.01 VandenBrink et al., 2011 12.3 Competitive HLM Pacli ,0.01 ,0.01 VandenBrink et al., 2011 1.1 Competitive HLM Repa 0.09 ,0.01 VandenBrink et al., 2011 3.3 Competitive HLM Rosi 0.03 ,0.01 VandenBrink et al., 2011 3.70 HLM Pacli 0.05 ,0.01 Lee et al., 2012a 28 rCYP2C8 Fluo ,0.01 ,0.01 Schelleman et al., 2014 Simvastatin acid Antihyperlipidemic, HMG- 66.5 41.1 Mixed HLM Pacli Tornio et al., 2005 CoA reductase inhibitor 51.5 HLM Pacli Sakaeda et al., 2006 76.5 rCYP2C8 Fluo Schelleman et al., 2014 Simvastatin acyl-b-D- Drug metabolite 3.8 HLM Amo Jenkins et al., 2011 glucuronide SIPI5357 Antidepressant, Serotonin- 89.23 HLM Pacli Fan et al., 2015 -dopamine reuptake inhibitor Sitaxentan Antihypertensive, ERA 1.58 HLM Pacli 22.42 28.38 Erve et al., 2013 Anticancer, PKI 1-2 rCYP2C8 Amo 21.5 0.01 21.50 0.22 FDA, 2005b 2.4 n/a n/a 8.96 0.09 Flaherty et al., 2011 1.59 HLM Pacli 13.52 0.14 Wang et al., 2014a Spironolactone Diuretic 6.99 rCYP2C8 Amo 0.444 ,0.10 0.13 0.01 Walsky et al., 2005a Antiepileptic 37.1 35 Noncompetitive rCYP2C8 Carba 28.17 0.76 Cazali et al., 2003 al. et Backman Sulfaphenazole Antimicrobial 63 Competitive rCYP2C8 DTP Mancy et al., 1996 0.42 rCYP2C8 R-ibu-2 Hamman et al., 1997 0.55 rCYP2C8 R-ibu-3 Hamman et al., 1997 0.36 rCYP2C8 S-ibu-2 Hamman et al., 1997 0.38 rCYP2C8 S-ibu-3 Hamman et al., 1997 505 rCYP2C8 Torse Miners et al., 2000 172.0 rCYP2C8 Dia Sai et al., 2000 .50 HLM Pacli Dierks et al., 2001 Sunitinib Anticancer, PKI 28 HLM Pacli 0.21 0.05 ,0.01 ,0.01 FDA, 2006b 91.51 HLM Pacli ,0.01 ,0.01 Wang et al., 2014a Sunitinib metabolite Drug metabolite 52 HLM Pacli FDA, 2006b Su012662 Sedative, 15 HLM Pacli 0.96 ,0.01 0.13 ,0.01 FDA, 2014j antagonist Suvorexant M9 Drug metabolite 37 HLM Amo FDA, 2014j (L-002015883) Tamoxifen Anticancer, SERM 3.34 rCYP2C8 Amo 0.323 ,0.02 0.19 ,0.01 Walsky et al., 2005a 3–10 rCYP2C8 Amo 0.22 ,0.01 O’Donnell et al., 2007 3.1 Competitive HLM Amo 0.10 ,0.01 VandenBrink et al., 2011 2.1 Competitive HLM Monte 0.15 ,0.01 VandenBrink et al., 2011 12.2 Competitive HLM Pacli 0.03 ,0.01 VandenBrink et al., 2011 10.1 Competitive HLM Repa 0.03 ,0.01 VandenBrink et al., 2011 2.6 Competitive HLM Rosi 0.12 ,0.01 VandenBrink et al., 2011 14.3 HLM Pacli 0.06 ,0.01 Lee et al., 2012a 2.3 HLM Amo 0.28 ,0.01 Nirogi et al., 2014 Tanespimycin Anticancer 29 HLM Pacli 17.34 ,0.10 1.20 0.12 Gan et al., 2012 Tasimelteon Circadian regulator .100 HLM Amo 0.80 ;0.10 0.02 ,0.01 FDA, 2014m Tasimelteon M12 Drug metabolite .100 HLM Amo FDA, 2014m Gastroprokinetic, 5-HT4 ;130 HLM Pacli 0.0065 0.02 ,0.01 ,0.01 Vickers et al., 2001 receptor agonist (continued) TABLE 6—Continued

Therapeutic Use Mode of Test Marker b b c c Inhibitor and/or Drug Class IC50 Ki Inhibition System Reactiona Imax fu I/Ki Iu/Ki References Telithromycin Antibiotic 87 rCYP2C8 Pacli 2.7 0.30 0.06 0.02 Yoshida et al., 2012 Temsirolimus Anticancer, PKI 27 HLM Pacli 0.57 0.02 FDA, 2007d Terbinafine Antifungal ;150 HLM Pacli 3 ,0.01 Vickers et al., 1999 Terfenadine Antihistamine 5 Noncompetitive rCYP2C8 Torse 0.0033 0.03 ,0.01 ,0.01 Ong et al., 2000 11.5 rCYP2C8 Amo ,0.01 ,0.01 Walsky et al., 2005a 19.1 HLM Pacli ,0.01 ,0.01 Erve et al., 2013 Teriflunomide Immunosuppressant, drug 0.174–0.219 0.100-0.150 Competitive, HLM Pacli 0.11 0.05 1.1 0.06 FDA, 2012l metabolite mixed Ticagrelor Antithrombotic, platelet .50 HLM Pacli 1.3 ,0.02 Zhou et al., 2011; FDA, aggregation inhibitor 2011b Ticagrelor metabolite AR- Drug metabolite 43 HLM Pacli FDA, 2011b C124910XX Ticlopidine Antithrombotic, platelet 100 rCYP2C8 2-TPE 3 0.02 0.06 ,0.01 Ha-Duong et al., 2001 aggregation inhibitor 43.9 rCYP2C8 DBF 0.14 ,0.01 Hagihara et al., 2008 Interactions and Metabolism Drug in CYP2C8 of Role 29 HLM Amo 0.21 ,0.01 Nirogi et al., 2015 Antiviral, protease inhibitor 2.1 rCYP2C8 Amo 94.8 ,0.01 90.29 ,0.90 Parikh et al., 2007 Tolbutamide Antidiabetic, sulfonylurea 2,370 Mixed HLM Pacli 196 0.09 0.08 ,0.01 Rahman et al., 1994 Trametinib Anticancer, PKI 0.34 HLM Rosi 0.032 0.038 0.19 0.01 FDA, 2013f Tranylcypromine Antidepressant, MAOI 26–35 HLM Pacli 0.42 0.03 Dierks et al., 2001 12.1 rCYP2C8 Amo 0.07 Walsky et al., 2005a 103–113 rCYP2C8 Amo ,0.01 O’Donnell et al., 2007 11.24 rCYP2C8 Pacli 0.07 Gao et al., 2010 22.5 HLM Amo 0.04 Nirogi et al., 2015 Anti-inflammatory, 19.3 rCYP2C8 Amo Walsky et al., 2005a glucocorticoid 32.5 Competitive HLM Amo VandenBrink et al., 2011 53.1 Competitive HLM Monte VandenBrink et al., 2011 .100 Competitive HLM Pacli VandenBrink et al., 2011 42.5 Competitive HLM Repa VandenBrink et al., 2011 20.4 Competitive HLM Rosi VandenBrink et al., 2011 Sedative, benzodiazepine 25 Noncompetitive rCYP2C8 Torse 0.013 0.099 ,0.01 ,0.01 Ong et al., 2000 Trimethoprim Antimicrobial, dihydrofolate 75 rCYP2C8 Pacli 4 0.63 0.05 0.03 Wen et al., 2002 reductase inhibitor 54 32 Competitive HLM Pacli 0.13 0.08 Wen et al., 2002 51.5 29.0 Competitive HLM Rosi-OH 0.14 0.09 Hruska et al., 2005 71 HLM Pio 0.11 0.07 Jaakkola et al., 2006c 40.6 rCYP2C8 Amo 0.20 0.12 Parikh et al., 2007 34.1 Competitive rCYP2C8 Pio 0.12 0.07 Tornio et al., 2008b 38.2 Competitive HLM Pio 0.10 0.07 Tornio et al., 2008b 9.2 Competitive HLM Amo 0.43 0.27 VandenBrink et al., 2011 .100 Competitive HLM Monte ,0.04 ,0.03 VandenBrink et al., 2011 . 100 Competitive HLM Pacli ,0.04 ,0.03 VandenBrink et al., 2011 8.5 Competitive HLM Repa 0.47 0.30 VandenBrink et al., 2011 13.2 Competitive HLM Rosi 0.30 0.19 VandenBrink et al., 2011 4.5–17 HLM Amo 1.78 1.12 Dinger et al., 2014 122 Hep Amo 0.07 0.04 Kosugi et al., 2014 17.41–20.38 HLM Pacli 0.46 0.29 Peng et al., 2015 Troglitazone Antidiabetic, PPAR-g agonist 1–5 0.3 Competitive rCYP2C8 Pacli 3 ,0.01 10.00 ,0.10 Yamazaki et al., 2000 15–20 HLM Pacli 0.30 ,0.01 Yamazaki et al., 2000 2.33 2.59 Competitive HLM Pacli 1.16 0.01 Sahi et al., 2003 9.78 rCYP2C8 Pacli 0.61 ,0.01 Gao et al., 2010 Troglitazone M1 Drug metabolite 9–41 rCYP2C8 Pacli Yamazaki et al., 2000 Troglitazone M3 Drug metabolite 6-26 3.0 Competitive rCYP2C8 Pacli Yamazaki et al., 2000

39–.50 HLM Pacli Yamazaki et al., 2000 205 (continued) TABLE 6—Continued 206

Therapeutic Use Mode of Test Marker b b c c Inhibitor and/or Drug Class IC50 Ki Inhibition System Reactiona Imax fu I/Ki Iu/Ki References Troleandomycin Antibiotic 953.0 rCYP2C8 Phena 3 ,0.01 Sai et al., 2000 TSAHC Anticancer 1.0 0.81 Noncompetitive HLM Amo Im et al., 2012 Ulipristal Contraceptive, progesterone 2.6 HLM Pacli 0.037 ,0.06 0.03 ,0.01 FDA, 2010b receptor modulator UTL-5g Chemoprotective, TNF-a 61.2 HLM Rosi-OH Wu et al., 2014 inhibitor Valdecoxib Anti-inflammatory, NSAID 15.0 rCYP2C8 Amo 0.512 0.02 0.07 ,0.01 Walsky et al., 2005a Vemurafenib Anticancer, PKI 12 HLM n/a 125 ,0.01 20.9 0.21 EMA, 2012d Vidupiprant (AMG 853) Antiasthmatic, PGD2 1.8 Biphasic HLM Monte Foti et al., 2012 receptor antagonist 5.4 1.1 Competitive HLM Pacli Foti et al., 2012 6.0 Competitive HLM Rosi Foti et al., 2012 Vidupiprant acyl Drug metabolite 7.3 Biphasic HLM Monte Foti et al., 2012 glucuronide (M1) 2.7 Mixed HLM Pacli Foti et al., 2012 6.9 Mixed HLM Rosi Foti et al., 2012 Antidepressant, SSRI 1.8 0.46 Competitive HLM Pacli 0.33 0.04 0.72 0.03 FDA, 2011g Vinblastine Anticancer 100 HLM Pacli Monsarrat et al., 1997 Vincristine Anticancer 8 HLM Pacli 0.43 0.11 Monsarrat et al., 1997 Vismodegib (GDC-0449) Anticancer, SMO Antagonist 6.0 Noncompetitive HLM Pacli 16.4 0.01 2.73 0.03 Wong et al., 2009; LoRusso et al., 2013 Vorapaxar Antithrombotic, platelet 1.5 0.86 Mixed HLM n/a 0.0527 0.002 0.06 ,0.01 Chen et al., 2014, FDA, aggregation inhibitor 2014l Vortioxetine Antidepressant, SMS 9.34 HLM n/a 0.04724 0.02 ,0.01 ,0.01 FDA, 2013j Vortioxetine metabolite Lu Drug metabolite 4.24 HLM n/a FDA, 2013j al. et Backman AA34443 Zafirlukast Antiasthmatic, LTRA 0.644 rCYP2C8 Amo 0.295 ,0.01 0.92 ,0.01 Walsky et al., 2005a 0.388 HLM Amo 1.52 0.02 Walsky et al., 2005a 0.78 HLM Pio 0.76 Jaakkola et al., 2006c .100 Hep Amo ,0.01 ,0.01 Kosugi et al., 2014 0.014 HLM Amo 42.14 0.42 Nirogi et al., 2014

3-ASBA, 3-(anilinosulfonyl)-benzenecarboxylic acid; 5-HT, 5-hydroxytryptamine (serotonin); 5-MeO-DIPT, 5-methoxy-N,N-diisopropyltryptamine; ARB, angiotensin II receptor blocker; BTFM gemfibrozil, 5-(2,5-bis (trifluoromethyl)phenoxy)-2,2-dimethylpentanoic acid; CCB, calcium channel blocker; CP-778875; 5-(N-(4-((4-ethylbenzyl)thio)phenyl)sulfamoyl)-2-methyl benzoic acid; ERA, endothelin receptor antagonist; fu, fraction unbound in plasma; GABA, g-aminobutyric acid; H2RA, H-2 receptor antagonist; HLM, human liver microsomes; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; IC50 , inhibitor concentration supporting half of the maximal inhibition; Imax , peak inhibitor concentration in plasma; Ki, reversible inhibition constant; LABA, long-acting b-adrenoceptor agonist; LTRA, leukotriene receptor antagonist; MAO, monoamine oxidase; MMB4 DMS, 1,19-methylenebis [4-[(hydroxyimino)methyl]-pyridinium] dimethanesulfonate; c-mpl, myeloproliferative leukemia; n/a, not available; NNRTI, nonnucleoside reverse transcriptase inhibitor; NS, nonstructural protein; NSAID, nonsteroidal anti- inflammatory drug; PDE, phosphodiesterase; PGD2, prostaglandin D2; PKI, protein kinase inhibitor; PPAR, peroxisome proliferator-activated receptor; PPI, proton pump inhibitor; rCYP2C8, recombinant CYP2C8; SERM, selective estrogen receptor modulator; SGLT, sodium-glucose linked transporter; SMO, smoothened receptor; SMS, serotonin modulator and stimulator; SNRI, serotonin-norepinephrine reuptake inhibitor; SSRI, selective serotonin reuptake inhibitor; TNF, tumor necrosis factor; TSAHC, 4-(p-toluenesulfonylamido)-4-hydroxychalcone; XO, xanthine oxidase; UTL-5g, N-(2,4-dichlorophenyl)-5-methyl-1,2-oxazole-3-carboxamide. a2-TPE, 2-aroylthiophen 5-hydyroxylation; Ami, aminopyrine N-demethylation; Amo, amodiaquine N-deethylation; Carba, carbamazepine 10,11-epoxidation; Ceri-1, cerivastatin demethylation (M-1); Ceri-23, cerivastatin 6-hydroxylation (M-23); DBF, dibenzylfluorescein (fluorescent reaction); Dia, diazepam N-demethylation; DTP, 2,3-dichloro-4(2-thenoyl) 5-hydroxylation; Fluo, fluorometric reaction; R-ibu-2, R-ibuprofen 2-hydroxylation; R-ibu-3, R-ibuprofen 3-hydroxylation; S-ibu-2, S-ibuprofen 2-hydroxylation; S-ibu-3, S-ibuprofen 3-hydroxylation; Luci, luciferin-6 methyl ether demethylation (luminogenic reaction); Monte, montelukast 36-hydroxylation; Monte-4, formation of montelukast metabolite 4; Pacli, paclitaxel 6a-hydroxylation; Phena, phenanthrene hydroxylation; Pio, pioglitazone hydroxylation (M-IV); Repa, repaglinide 39-hydroxylation; Rosi, rosiglitazone N-demethylation; Rosi- OH, rosiglitazone p-hydroxylation; Taza, tazarotenic acid sulfoxidation; Tolbu, tolbutamide methyl hydroxylation; Torse, torsemide methylhydroxylation. bThis information is primarily based on information from the UW Metabolism and Transport Drug Interaction Database (DIDB), Copyright University of Washington 1999-2015 (DIDB accessed May-September, 2015), and secondarily on information from Martindale: The Complete Drug Reference. London: Pharmaceutical Press (electronic version), Truven Health Analytics (Healthcare), Greenwood Village, Colorado. Available at: http://www. micromedexsolutions.com/ (Martindale accessed June-September, 2015). In case several values were reported for Imax and fu, the highest values were selected. c When experimentally determined Ki was not available, Ki was calculated as IC50 /2. An I/Ki . 1.0 indicates that a clinically relevant inhibition is likely, I/Ki =0.1–1 indicates that a clinically relevant inhibition is possible, I/Ki , 0.1 indicates that a clinically relevant inhibition is unlikely. dUnbound value. Role of CYP2C8 in Drug Metabolism and Interactions 207

TABLE 7 Natural and endogenous compounds that act as reversible CYP2C8 inhibitors

Mode of Test Marker Inhibitor Description IC50 Ki Inhibition System Reactiona References

mM(mg/ml) mM(mg/ml) 3-Isomangostin Constituent of 0.64 0.66 Competitive HLM Pacli Foti et al., 2009 mangosteen 6-Gingerol Constituent of ginger (6.5) HLM Amo Mukkavilli et al., 2014 root 6-Prenylnaringenin Prenylflavonoid 1.9 HLM Amo Yuan et al., 2014 6-Shogaol Constitutent of ginger (0.8) HLM Amo Mukkavilli et al., 2014 root 8-Desoxygartanin Constituent of 1.85 2.80 Competitive HLM Pacli Foti et al., 2009 mangosteen 8-Gingerol Constituent of ginger (0.7) HLM Amo Mukkavilli et al., 2014 root 8-Prenylnaringenin Prenylflavonoid 0.6 HLM Amo Yuan et al., 2014 (flavaprenin) 9-Hydroxycalabaxanthone Constituent of 14.1 HLM Pacli Foti et al., 2009 mangosteen 10-Gingerol Constituent of ginger (0.7) HLM Amo Mukkavilli et al., 2014 root 11-Keto-b-boswellic acid Triterpene 9.5 HLM Pacli Frank and Unger, 2006 Acetyl-a-boswellic acid Triterpene 65.8 HLM Pacli Frank and Unger, 2006 Acetyl-b-boswellic acid Triterpene 33.4 HLM Pacli Frank and Unger, 2006 Acetyl-11-keto-b-boswellic Triterpene 10.1 HLM Pacli Frank and Unger, 2006 acid Allocryptopine Alkaloid 10-100 rCYP2C8 DBF Salminen et al., 2011 Arachidonic acid Endogenous 7 Competitive rCYP2C8 Pacli Yamazaki and Shimada, compound 1999 b-Boswellic acid Triterpene 8.7 HLM Pacli Frank and Unger, 2006 Bo-yang-hwan-o-tang Oriental herbal (17,209) HLM Pacli Lee et al., 2012b medicine BST204 Dry extract of ginseng (17.4) HLM Rosi Zheng et al., 2014 Capnoidine Alkaloid 10-100 rCYP2C8 DBF Salminen et al., 2011 Cedrol Sesquiterpene 41.0 HLM Amo Jeong et al., 2014 Corybulbine Alkaloid 10-100 rCYP2C8 DBF Salminen et al., 2011 Corycavamine Alkaloid 10-100 rCYP2C8 DBF Salminen et al., 2011 Corycavidine Alkaloid 10-100 rCYP2C8 DBF Salminen et al., 2011 Alkaloid 10-100 rCYP2C8 DBF Salminen et al., 2011 Corypalmine Alkaloid 10-100 rCYP2C8 DBF Salminen et al., 2011 Cranberry (powder) Natural product (24.7) HLM Amo Albassam et al., 2015 (24.0) HLM Pio Albassam et al., 2015 Cryptopine Alkaloid 10-100 rCYP2C8 DBF Salminen et al., 2011 b-Cryptoxanthin Carotenoid pigment 13.8 HLM Pacli Zheng et al., 2013 Cudratricusxanthone Constituent of 4.69 2.2 Noncompetitive HLM Pacli Sim et al., 2015 Cudrania tricuspidata Curcumin Diarylheptanoid 129.7 rCYP2C8 DBF Mach et al., 2010 DA-9801 Extract (449.9) HLM Amo Ji et al., 2013 Dioscorea nipponica Extract (237.1) HLM Amo Ji et al., 2013 Diosmetin Flavonoid 3.13 Mixed HLM Pacli Quintieri et al., 2011 Ellagic acid Constitutent of guava (13.99) rCYP2C8 DBF Kaneko et al., 2013 leaf (2)-Epigallocatechin-3- 10.9 6.8 Competitive HLM Amo Misaka et al., 2013 gallate Eupatilin Flavone 104.9 101.9 Competitive HLM Amo Ji et al., 2010 Feverfew herb Natural product (104–126) rCYP2C8 Pacli Unger and Frank, 2004 Fisetin Flavonol 10.8 1.3–6.0 Mixed HLM Pacli Václavíková et al., 2003 Gartanin Constituent of 6.28 HLM Pacli Foti et al., 2009 mangosteen Ginger extract Extract (122.5) HLM Amo Mukkavilli et al., 2014 Green tea extract Extract (4.5) HLM Amo Misaka et al., 2013 Guava leaf extract Extract (18.16) rCYP2C8 DBF Kaneko et al., 2013 Guava leaf polyphenol Constitutent of guava (1.45) rCYP2C8 DBF Kaneko et al., 2013 leaf Gypenosides Oriental herbal (20.06) HLM Pacli He et al., 2013 medicine Hesperedin Flavonoid 274.7 rCYP2C8 Tolbu Pang et al., 2012 Hesperetin Flavonoid 68.5 HLM Pacli Quintieri et al., 2011 168.4 rCYP2C8 Tolbu Pang et al., 2012 Hibiscus sabdariffa Extract (424) HLM Amo Johnson et al., 2013 extract Honey Natural product (102.9) (50.5) Competitive rCYP2C8 Amo Muthiah et al., 2012 Constituent of 8.9 4.9 Competitive HLM Amo Jeong et al., 2013 Magnolia Horsetail Natural product (93.0) HLM Amo Sevior et al., 2010 (continued) 208 Backman et al.

TABLE 7—Continued

Mode of Test Marker Inhibitor Description IC50 Ki Inhibition System Reactiona References Hops extract Extract 0.8 HLM Amo Yuan et al., 2014 Hunnemannine Alkaloid 1–10 rCYP2C8 DBF Salminen et al., 2011 Hyperforin Constituent of 56 HLM Amo Hokkanen et al., 2011 St. John’s Wort Hypoxis hemerocallidea Extract (192) HLM Pacli Fasinu et al., 2013a extract Isocorybulbine Alkaloid 10–100 rCYP2C8 DBF Salminen et al., 2011 Isoxanthohumol Prenylflavonoid 0.2 HLM Amo Yuan et al., 2014 Jaceosidin Flavone 106.4 109.4 Competitive HLM Amo Ji et al., 2010 Labisia pumila extracts Extracts (2.39–352.3) (0.70-33.9) Noncompetitive, rCYP2C8 DBF Pan et al., 2012 mixed b-Lapachone Quinone 3.8 HLM Pacli Kim et al., 2013a Liquorice extract Extract (14.36–17.06) HLM Amo Li et al., 2015 Liquorice root Natural product (22.6) HLM Amo Sevior et al., 2010 Flavonoid 82.0 rCYP2C8 Tolbu Pang et al., 2012 a-Mangostin Constituent of 0.88 0.64 Competitive HLM Pacli Foti et al., 2009 mangosteen b-Mangostin Constituent of 8.39 HLM Pacli Foti et al., 2009 mangosteen Mecambridine Alkaloid 10–100 rCYP2C8 DBF Salminen et al., 2011 Milk thistle extract Extract (8.35) Mixed HLM Pacli Doehmer et al., 2011 Morin Flavonol 17.3 7.3-12.3 Mixed HLM Pacli Václavíková et al., 2003 Alkaloid 1–10 rCYP2C8 DBF Salminen et al., 2011 a-Naphthoflavone Flavone derivative 0.36 HLM Amo Nirogi et al., 2015 Obovatol Constituent of 11.1 HLM Amo Joo et al., 2013 Magnolia Quercetin Flavonoid 1.29 Competitive rCYP2C8 Pacli Rahman et al., 1994 1.14 Competitive HLM Pacli Rahman et al., 1994 7 HLM Pacli Dierks et al., 2001 1.96-2.35 Competitive rCYP2C8 Amo Li et al., 2002 1.56 Competitive HLM Amo Li et al., 2002 2.47 rCYP2C8 DBF Yamamoto et al., 2002 4.07 19.7 HLM Taza Attar et al., 2003 10.1 Competitive HLM Pacli Bun et al., 2003 20.6 HLM DBF Ghosal et al., 2003 3.3 HLM Pacli Cai et al., 2004 6.3 HLM Pacli Donato et al., 2004 2.9 THLE DBF Donato et al., 2004 29.5 THLE Pacli Donato et al., 2004 3.9–6.2 rCYP2C8 Pacli Unger and Frank, 2004 3.33 rCYP2C8 Amo Walsky and Obach, 2004 3.06 HLM Amo Walsky and Obach, 2004 7.19–8.47 HLM Pacli Kim et al., 2005b 57.8 HLM Amo Turpeinen et al., 2005 3.94 rCYP2C8 Amo Walsky et al., 2005a 3.3–5.6 rCYP2C8 Amo O’Donnell et al., 2007 1.6 rCYP2C8 DBF McClue and Stuart, 2008 5.28–5.38 rCYP2C8 Pacli Gao et al., 2010 1.33 rCYP2C8 Bomcc Liu et al., 2010a 0.029 HLM Amo Teng et al., 2010 0.49 Competitive HLM Amo VandenBrink et al., 2011 0.52 Competitive HLM Monte VandenBrink et al., 2011 3.0 Competitive HLM Pacli VandenBrink et al., 2011 0.61 Competitive HLM Repa VandenBrink et al., 2011 0.61 Competitive HLM Rosi VandenBrink et al., 2011 4.20 2.07 Competitive rCYP2C8 Amo Muthiah et al., 2012 1.39 HLM Pacli Lee et al., 2012a 46.0 rCYP2C8 Tolbu Pang et al., 2012 18.7 HLM Pacli Bymaster et al., 2013 (0.59) rCYP2C8 DBF Kaneko et al., 2013 (0.3702) HLM Amo Mukkavilli et al., 2014 24.5 rcCYP2C8 DBF Pan et al., 2014 12.3 HLM Rosi-OH Wu et al., 2014 3.40–5.92 HLM Amo Li et al., 2015 1.2 HLM Amo Nirogi et al., 2015 Piperlonguminine Alkaloid 76.2 HLM Amo Song et al., 2014b (continued) Role of CYP2C8 in Drug Metabolism and Interactions 209

TABLE 7—Continued

Mode of Test Marker Inhibitor Description IC50 Ki Inhibition System Reactiona References Reserveratrol Stilbenoid 26.5 16.2–20.7 HLM Pacli Václavíková et al., 2003 Saw palmetto Natural product (8) HLM Amo Sevior et al., 2010 (15.4) HLM Amo Albassam et al., 2015 (9.6) HLM Pio Albassam et al., 2015 Scoulerine Alkaloid 10-100 rCYP2C8 DBF Salminen et al., 2011 Star fruit (averrhoa Fruit juice 2.2b HLM Pacli Zhang et al., 2007b carambola) juice Sutherlandia frutescens Herb (22.4) HLM Pacli Fasinu et al., 2013b Tanshinol borneol ester Combination of the 105 rCYP2C8 Bomcc Liu et al., 2010b natural compounds danshensu and borneol Thalictricavine Alkaloid 10–100 rCYP2C8 DBF Salminen et al., 2011 Thelephoric acid Antioxidant 24.6 HLM Amo Song et al., 2014a Tiliroside Flavonoid 12.1 9.4 Competitive HLM Pacli Sun et al., 2010 Tualang honey Natural product (102.9) (50.5) Competitive rCYP2C8 Amo Muthiah et al., 2012 Natural product (523.3) HLM Amo Sevior et al., 2010 Xanthohumol Natural product 1.1 HLM Amo Yuan et al., 2014

HLM, human liver microsomes; IC50, inhibitor concentration supporting half of the maximal inhibition; Ki, reversible inhibition constant; n/a, not available; rcCYP2C8, reconstituted CYP2C8; rCYP2C8, recombinant CYP2C8; THLE, immortalized human liver epithelial cells. aAmo, amodiaquine N-deethylation; Bomcc, flurogenic substrate; DBF, dibenzylfluorescein; Monte, montelukast 36-hydroxylation; Pacli, paclitaxel 6a-hydroxylation; Pio, pioglitazone hydroxylation (M-IV); Repa, repaglinide 39-hydroxylation; Rosi, rosiglitazone N-demethylation; Rosi-OH, rosiglitazone p-hydroxylation; Taza, tazarotenic acid sulfoxidation; Tolbu, tolbutamide methyl hydroxylation. b% (v/v).

and organic anion transporter (OAT) 3 (Ki = 6.8 mM) small/moderate. For instance, axitinib inhibits CYP2C8 (Schneck et al., 2004; Shitara et al., 2004; Nakagomi- in vitro with a Ki of 0.2–0.5 mM (I/Ki = 0.3–0.9), but it did Hagihara et al., 2007). The strong interactions between not alter paclitaxel plasma concentrations in patients gemfibrozil and CYP2C8 substrate drugs observed in (FDA, 2012f; Wang et al., 2014a). Similarly, cabozanti- vivo are mainly due to its glucuronide metabolite nib is a noncompetitive inhibitor of CYP2C8 in vitro (Ogilvie et al., 2006). Gemfibrozil 1-O-b glucuronide (Ki =4.6mM, I/Ki = 0.7), but the in vivo pharmacokinetics affects CYP2C8 by mechanism-based inhibition (sec- of rosiglitazone was not affected by cabozantinib (FDA, tions V.B and VI.B). 2012c; Nguyen et al., 2015). The inhibition of CYP2C8 In vitro, the thiazolidinedione drugs pioglitazone, by pazopanib (Ki of 3.7 mM, I/Ki = 35) may be of clinical rosiglitazone, and troglitazone are potent, competitive relevance (FDA, 2009d; Tan et al., 2014; Wang et al., inhibitors of CYP2C8 with IC50 and Ki values of ,40 mM 2014b). Nilotinib is a strong competitive CYP2C8 in- (Table 6). However, e.g., pioglitazone does not affect hibitor in vitro (Ki = 0.1–0.9 mM, I/Ki = 4.8–43), but it CYP2C8 in vivo, likely because of its extensive protein also induces CYP2C8 (FDA, 2007c). Hence, a clinical binding (Kajosaari et al., 2006a). interaction study with a CYP2C8 probe substrate has The antiviral agents atazanavir and efavirenz inhibit been recommended by the FDA to evaluate the in vivo CYP2C8 in vitro with Ki values of 2.1 and 4.8 mM, effect on CYP2C8 activity by nilotinib. Similarly, an respectively [inhibitor concentration (I) to Ki ratios interaction study with a CYP2C8 substrate drug has (I/Ki) = 3.7 and 6.3, respectively] (Table 6). In vivo, also been recommended for regorafenib, which inhibits atazanavir has slightly affected the pharmacokinetics CYP2C8 with a Ki value of 0.6 mM in vitro (I/Ki = 13.5) of rosiglitazone (FDA, 2015b). According to predictions, (FDA, 2012i). In addition, sorafenib seems to be a strong efavirenz may increase the area under the plasma CYP2C8 inhibitor in vitro with Ki values , 3 mM (I/Ki = concentration-time curve (AUC) of CYP2C8 substrates 9–22) (Table 6), but the effect of sorafenib on CYP2C8 by more than fourfold at steady state, and such effects in vivo has not been evaluated. have been observed in vivo (German et al., 2007). Also several other anticancer agents exhibit inhibi- The immunosuppressant teriflunomide inhibits tion of CYP2C8 in vitro (Table 6). For instance, the CYP2C8 with a very low Ki of 0.10–0.15 mM (FDA, androgen receptor antagonist enzalutamide is both a 2012a). Thus, its estimated I/Ki ratio of 1.1 indicates substrate and inhibitor of CYP2C8 in vitro (Ki = 5.5 mM, that interactions between teriflunomide and CYP2C8 I/Ki = 6.5) (FDA, 2012k). Vismodegib, an oral hedgehog substrate drugs are likely, in agreement with in vivo pathway inhibitor, inhibits CYP2C8 in vitro with a Ki of findings (section IV.C.2). 6.0 mM (I/Ki = 2.7), but vismodegib at steady state did Numerous protein kinase inhibitors inhibit CYP2C8 not affect the pharmacokinetics of rosiglitazone (Wong to various degrees in vitro (Table 6). However, for the et al., 2009; LoRusso et al., 2013). most part, their in vivo inhibitory effects on CYP2C8 The iron chelator deferasirox inhibits CYP2C8 with have not been studied. For those whose inhibition has an IC50 of 100 mM (I/Ki ,0.01) (FDA, 2005a), but it has been examined in a clinical setting, it seems to be rather increased repaglinide AUC by 2.3-fold in vivo (Skerjanec 210 Backman et al. et al., 2010). Febuxostat, a xanthine oxidase inhibitor, mechanism-based inhibition or quasi-irreversible in- inhibits CYP2C8 in vitro with a Ki of 20 mM, suggesting hibition, leading to clinically important drug-drug that the inhibition may be of clinical relevance (I/Ki = interactions (Figs. 2 and 5; Table 8; Ogilvie et al., 0.8). However, febuxostat at steady state had no effect 2006; Tornio et al., 2014). Very recently, also the acyl on the concentrations of a single dose of rosiglitazone in glucuronide of deleobuvir, an HCV protease inhibitor, vivo (Naik et al., 2012). Similarly, rosiglitazone phar- was found to be a very potent mechanism-based in- macokinetics was not affected by the platelet aggregation hibitor of CYP2C8 (Sane et al. 2015). In addition, there inhibitor vorapaxar in vivo (Ki =0.86mM, I/Ki =0.06) is in vitro evidence suggesting that the carbamoyl (Chen et al., 2014; FDA, 2014l). glucuronide metabolite of Lu AA34893 may affect Sulfaphenazole, ketoconazole, diethyldithiocarba- CYP2C8 in a similar manner (Kazmi et al., 2010). Of mate, methoxsalen (8-methoxypsoralen), and tranylcy- interest, parent clopidogrel and gemfibrozil do not seem promine, commonly used as in vitro inhibitors of to be metabolized by CYP2C8. For example, clopidogrel CYP2C9, CYP3A4, CYP2E1, CYP2A6, and CYP2C19, is mainly eliminated by carboxylesterase 1, whereas its respectively, also inhibit CYP2C8 in vitro (Table 6). For activation is dependent on CYP2C19 and CYP3A4 instance, sulfaphenazole is a strong competitive in- (Mega et al., 2009; Simon et al., 2009; Holmberg et al., hibitor of CYP2C9 with a Ki of 0.3 mM, whereas its Ki 2014; Tarkiainen et al., 2015). for CYP2C8 inhibition is 0.4–63 mM (Mancy et al., 1996; The inhibitory effect of gemfibrozil on CYP2C8 is Hamman et al., 1997). based principally on its metabolite, gemfibrozil 1-O-b 2. Natural Compounds. A range of natural com- glucuronide, which is formed mainly by UGT2B7 in pounds have been tested for CYP2C8 inhibition in vitro, hepatocytes (Shitara et al., 2004; Ogilvie et al., 2006; and inhibition parameters have been determined for Mano et al., 2007). The metabolite acts as a mechanism- several of them (Table 7). In a CYP inhibition screening based inhibitor of CYP2C8, with inhibitor concentra- of 10 herbal products commercially available in Aus- tion supporting half of the maximal rate of enzyme tralia, horsetail (Equisetum arvense) affected CYP2C8 inactivation (KI)andmaximalrateofinactivation(kinact) with an IC50 of 93.0 mg/ml (Sevior et al., 2010). The values of 20–52 mM and 0.21 1/min in vitro (Ogilvie authors suggested that the inhibition of CYP2C8 by et al., 2006; Baer et al., 2009). Similarly, clopidogrel acyl horsetail, which is used for treatment of urinary tract 1-b-D-glucuronide causes a metabolism-dependent in- , cystitis, and prostate problems, may be hibition of CYP2C8 with KI and kinact values of 9.9 mM clinically relevant (Sevior et al., 2010). In another in and 0.047 1/min (Tornio et al., 2014). The in vivo vitro study, six herbal supplements inhibited CYP2C8 consequences of the inhibitory effects of these metabo- to various degree, but the inhibition by cranberry lites are discussed in section VI. In an in vitro study powder (IC50 = 24.7 mg/ml) and saw palmetto (IC50 = by Jenkins et al. (2011), the acyl glucuronides of ator- 15.4 mg/ml) were suggested to potentially be of clinical vastatin, dehydroketoprofen, diclofenac, ibuprofen, significance (Albassam et al., 2015). indomethacin, rac-ketoprofen, mefenamic acid, R- and Among five CYP enzymes tested, CYP2C8 was most S-naproxen, and simvastatin did not affect CYP2C8 by sensitive to inhibition by green tea extract in HLM metabolism-dependent inhibition. (IC50 =4.5mg/ml) (Misaka et al., 2013). The major catechin Several other metabolism-dependent inhibitors of in green tea, (2)-epigallocatechin-3-gallate, inhibited CYP2C8havebeenreportedintheliterature(Table8). CYP2C8 with a Ki of 6.8 mM, indicating that green tea However, the clinical importance of their interaction intake may affect CYP2C8 in vivo. It has been reported potential is unknown. that 15% of Japanese older than 40 years of age consume more than 1.8 l of green tea daily, correspond- C. Induction ing to a daily epigallocatechin-3-gallate intake of 540– Increased expression of CYP2C8 protein in hepato- 720 mg (Misaka et al., 2013). cytes due to enzyme-inducing drugs/xenobiotics is an important mechanism of drug-drug interactions that B. Metabolism-dependent Inhibition can lead to markedly increased clearance of CYP2C8 Metabolism-dependent inhibitors are compounds substrates, resulting in reduced efficacy and therapeu- that are metabolized to metabolites or reactive inter- tic failure. Several drug-responsive nuclear receptors, mediates that cause time-dependent enzyme inhibition. including CAR, PXR, VDR, and GR, can mediate the Metabolism-dependent inhibition may be either direct, transcriptional activation of the CYP2C8 gene by quasi-irreversible, or irreversible (mechanism-based recognizing the respective responsive elements within inhibition). Mechanism-based inhibitors inactivate the 59-flanking promoter region of the gene (Chen and their victim enzymes permanently, and enzyme activity Goldstein, 2009). After activation of nuclear receptors can only be regained by de novo synthesis of the by their ligands/activators (in particular, enzyme in- enzyme (Lin and Lu, 1998). Interestingly, two glucuro- ducing drugs), the nuclear receptors enter the nu- nide metabolites, gemfibrozil 1-O-b glucuronide and cleus, bind to their responsive elements in the DNA, clopidogrel acyl 1-b-D-glucuronide, affect CYP2C8 by recruit coactivators that affect chromatin structure, TABLE 8 Metabolism-dependent CYP2C8 inhibitors in vitro

Therapeutic Use a b Test Marker Inhibitor and/or Drug Class Mode of Inhibition Preinc. IC50 IC50 Shift KI kinact System Reactionc References mM ratio mM 1/min 17a-Ethinylestradiol Contraceptive, hormone derivative 8.3 1.9 HLM Pacli Chang et al., 2009 Amiodarone Antiarrhythmic 1.5 0.079 rCYP2C8 Pacli Polasek et al., 2004 51.2 0.029 HLM Pacli Polasek et al., 2004 Bosutinib Anticancer, PKI 16.9 2.6 54.8 0.018 HLM Amo Filppula et al., 2014 Clopidogrel acyl Drug metabolite 12.0 4.7 9.9 0.047 HLM Amo Tornio et al., 2014 1-b-D-glucuronide Desethylamiodarone Drug metabolite 0.67 3.3 4.4 0.009 HLM Amo Obach et al., 2007 Demethyldabrafenib Drug metabolite 30a 1.6 HLM Rosi Lawrence et al., 2014 Fluoxetine Antidepressant, SSRI Quasi-irreversible 294 0.083 rCYP2C8 Pacli Polasek et al., 2004 Gemfibrozil 1-O-b Drug metabolite Irreversible 1.8 13.3 20-52 0.21 HLM Pacli Ogilvie et al., 2006 glucuronide 4.51 0.106 Hep n/a Negishi et al., 2007 29 0.072 rCYP2C8 Amo Baer et al., 2009 Interactions and Metabolism Drug in CYP2C8 of Role 0.46 98 HLM Amo Perloff et al., 2009 3.0 HLM Monte Karonen et al., 2010 4.5 HLM Monte-4 Karonen et al., 2010 0.26 0.015 HLM Amo Teng et al., 2010 1.4 16 HLM Amo Jenkins et al., 2011 10.1 0.041 HLM Amo VandenBrink et al., 2011 21.3 0.050 HLM Monte VandenBrink et al., 2011 35 0.022 HLM Pacli VandenBrink et al., 2011 33.6 0.082 HLM Pio VandenBrink et al., 2011 18.4 0.035 HLM Repa VandenBrink et al., 2011 48.5 0.071 HLM Rosi VandenBrink et al., 2011 10.34–25.4 0.104–0.25 HLM Amo Korzekwa et al., 2014 Gemfibrozil d6-1-O-b Drug metabolite, Deuterated 29 0.033 rCYP2C8 Amo Baer et al., 2009 glucuronide Isoniazid Antituberculosis Quasi-irreversible 374 0.042 rCYP2C8 Pacli Polasek et al., 2004 170 0.012 HLM Pacli Polasek et al., 2004 Lu AA34893 carbamoyl Drug Metabolite 8.5 8.4 HLM n/a Kazmi et al., 2010 glucuronide O-methylgemfibrozil Gemfibrozil Acyl-b-D-glucuronide Analog 17 3.2 HLM Amo Jenkins et al., 2011 acyl-b-D-glucuronide Antidepressant, TCA Quasi-irreversible 49.9 0.036 rCYP2C8 Pacli Polasek et al., 2004 Phenelzine Antidepressant, MAOI 1.2 0.243 rCYP2C8 Pacli Polasek et al., 2004 54.3 0.17 HLM Pacli Polasek et al., 2004 Raloxifene Antiosteoperotic, SERM .2 0.26 0.1 rCYP2C8 Pacli VandenBrink et al., 2012 Thujopsene Sesquiterpene 29.8 3.3 HLM Amo Jeong et al., 2014 Toremifene Anticancer, SERM 5.0 1.9 HLM Pacli Kim et al., 2011b Verapamil Antihypertensive, CCB Quasi-irreversible 17.5 0.065 rCYP2C8 Pacli Polasek et al., 2004

CCB, calcium channel blocker; Hep, hepatocytes: HLM, human liver microsomes; IC50 , inhibitor concentration supporting half of the maximal inhibition; KI, inactivation constant; kinact , maximal rate of inactivation; MAOI, monoamine oxidase inhibitor; n/a, not available; PKI, protein kinase inhibitor; rCYP2C8, recombinant CYP2C8, SERM, selective estrogen receptor modulator; SSRI, selective serotonin reuptake inhibitor; TCA, tricyclic antidepressant. a Preinc. IC50 depicts IC50 after preincubation of inhibitor for 30 min with NADPH before addition of substrate, except in the case of demethyldabrafenib where the preincubation time was 20 min. b IC50 shift = reversible IC50 /preinc. IC50 .AnIC50 shift $1.5-fold is indicative of metabolism-dependent inhibition. cAmo, amodiaquine N-deethylation; Monte, montelukast 36-hydroxylation; Monte-4, formation of montelukast M4; Pacli, paclitaxel 6a-hydroxylation; Pio; pioglitazone hydroxylation (M-IV); Repa, repaglinide 39-hydroxylation; Rosi, rosiglitazone N-demethylation. 211 212 Backman et al. and increase the transcription of the target genes 2001; Rae et al., 2001; Raucy et al., 2002; Madan et al., (Handschin and Meyer, 2003). Apart from this general 2003; Ferguson et al., 2005). Moreover, VDR may mechanism, certain compounds, such as phenobarbital be involved in the induction of CYP2C8 by lithocholic and CITCO ([6-(4-chlorophenyl)imidazo[2,1-b][1,3] acid in HepG2 cells (Makishima et al., 2002; Yajima thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime), et al., 2014), and the PPAR-alpha-agonist WY14,643 seem to cause induction by increasing the nuclear (4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid) translocation of CAR, which is constitutively active has induced CYP2C8 mRNA in HepaRG cells (Thomas (Zelko et al., 2001). Other nuclear receptors and tran- et al., 2015). In addition to the above compounds, scriptional factors, such as HNF4a, HNF3g, C/EBPa, certain other drugs have weakly induced CYP2C8 in and RORs, can regulate the constitutive expression of vitro with unknown induction mechanisms, including CYP2C genes, but these factors are probably not di- tasimelteon and crizotinib (FDA, 2011e,m; EMA, 2012c; rectly involved in induction of CYP2C8 (Ferguson et al., TGA, 2014). 2005; Chen and Goldstein, 2009; Rana et al., 2010). Yet, In humans in vivo, rifampin has markedly reduced at least HNF4a seems to be required for upregulation by the plasma exposure to several CYP2C8 substrates the PXR agonist rifampin (Rana et al., 2010). (Jaakkola et al., 2006a; Niemi et al., 2000; Niemi In experimental in vitro studies, several compounds et al., 2004a; Park et al., 2004), and it is consequently and ligands of the different nuclear receptors have been the preferred CYP2C8 inducer drug for use in clinical able to induce CYP2C8 (Table 9). On the basis of studies studies (section VII). Yet, the strength of the CYP2C8 in cultured human hepatocytes, CYP2C8 is the most inducing effect of rifampin (or any other PXR ligands) inducible member of the CYP2C subfamily (Gerbal- has been difficult to estimate, because the strong Chaloin et al., 2001; Feidt et al., 2010). Regarding CYP3A4-inducing effect of rifampin is likely to partially inducibility of CYP2C8, the PXR-receptor seems to be explain its effects on the clearance of CYP2C8 sub- the most important nuclear receptor, because typical strates. Of note, one of the strongest clinical inducers of PXR ligands/activators strongly induce CYP2C8 in vitro CYP enzymes, carbamazepine, which is also a weak (Ferguson et al., 2005; Chen and Goldstein, 2009) and PXR activator, seems to be poorly characterized with can cause induction of CYP2C8 also in vivo, whereas regard to CYP2C8 both in vitro and in vivo. ligands of the other nuclear receptors cause only moder- ate induction of CYP2C8 in vitro (Ferguson et al., 2005; VI. Clinical Drug Interactions Mediated via Chen and Goldstein, 2009) and have not been shown to Cytochrome P450 2C8 markedly induce CYP enzymes in vivo in humans. PXR activators, such as phenobarbital, hyperforin (an in- A. General Aspects gredient of St. John’s wort), and rifampin, have increased The CYP2C8 enzyme is involved in many drug-drug CYP2C8 expression at mRNA, protein, and activity interactions in humans, including interactions based levels several-fold in vitro (Dussault et al., 2001; on either inhibition or induction of CYP2C8. However, Gerbal-Chaloin et al., 2001; Rae et al., 2001; Nishimura its exact role in interactions is difficult to determine, et al., 2002; Raucy et al., 2002; Madan et al., 2003; because there are no fully selective in vivo inhibitors Ferguson et al., 2005; Komoroski et al., 2005; Thomas or inducers of CYP2C8 and all known CYP2C8 sub- et al., 2015). In addition, certain other compounds, strates are metabolized, at least to a small degree, also by including ritonavir, , cyclophosphamide, lith- other enzymes. Furthermore, the activities of OATP1B1, ocholic acid, and paclitaxel can induce CYP2C8 pre- P-glycoprotein or other membrane transporters can sumably by a PXR-mediated mechanism in vitro (Chang affect the pharmacokinetics of many CYP2C8 substrate et al., 1997; Dussault et al., 2001; Synold et al., 2001; drugs, and some inhibitors of CYP2C8 inhibit these Ferguson et al., 2005; Dixit et al., 2007). It should be transporters, too. Because drug metabolizing enzymes noted that in one study, rifampin induced CYP2C8 and transporters may influence drug metabolism in mRNA in only three of the eight commercially available concert, the isolated role of CYP2C8 in many drug-drug cryopreserved hepatocyte lots tested (Yajima et al., interactions can be very difficult to dissect in vivo. 2014), suggesting that cryopreserved hepatocytes may The clinical significance of pharmacokinetic drug- not be a reliable system for studying CYP2C8 induction. drug interactions depends both on the therapeutic index Apart from PXR, induction of CYP2C8 can be exper- of victim drug and on the extent of pharmacokinetic imentally achieved at least via CAR- and GR-mediated changes, in addition to various patient-related clinical and possibly also via VDR- and PPAR-alpha-mediated factors. Of the pharmacokinetic parameters, at least the mechanisms (Ferguson et al., 2005; Chen and Goldstein, plasma AUC, peak concentration (Cmax), time to max- 2009). The CAR- phenytoin and CITCO have imum concentration (tmax), and elimination half-life (t1/2) markedly induced CYP2C8 expression in human hepa- values are generally required for the characterization tocytes (Ferguson et al., 2005). In addition, dexameth- of an interaction. Here, to be brief, we usually report asone (GR agonist) can modestly increase CYP2C8 only fold-changes of the mean AUC values caused expression in in vitro systems (Gerbal-Chaloin et al., by interactions. Of note, e.g., interindividual genetic Role of CYP2C8 in Drug Metabolism and Interactions 213

TABLE 9 Some inducers of CYP2C8 in vitro

Therapeutic Use CYP2C8 Protein in CYP2C8 Activity Inducer and/or Drug Class CYP2C8 mRNA Hepatocytes in Hepatocytes References CITCO 2.5-fold in primary hepatocytes Ferguson et al., 2005 1-fold in primary hepatocytes Thomas et al., 2015 Clofibric acid Antihyperlipidemic 2-to 3-fold in primary Prueksaritanont hepatocytes et al., 2005 Cyclophosphamide Anticancer, alkylating agent + Chang et al., 1997 Dexamethasone Anti-inflammatory, + Chang et al., 1997 glucucortidcoid 3-fold in primary hepatocytes 2-fold Gerbal-Chaloin et al., 2001 5-fold in primary hepatocytes 4-fold Raucy et al., 2002 ,2-fold in primary hepatocytes Ferguson et al., 2005 Fenofibric acid Antihyperlipidemic 2- to 6-fold in primary Prueksaritanont hepatocytes et al., 2005 Gemfibrozil Antihyperlipidemic, 1.1- to 5-fold in primary Prueksaritanont PPARa agonist hepatocytes et al., 2005 Hyperforin Constituent of St. John’s + in primary hepatocytes Dussault et al., 2001 wort 5-fold in primary hepatocytes Ferguson et al., 2005 + Komoroski et al., 2005 Idelalisib Anticancer, PKI 3.9-fold in human hepatocytes FDA, 2014h Idelalisib metabolite Drug metabolite 1.4-fold in human hepatocytes FDA, 2014h GS-563117 Ifosfamide Anticancer, alkylating agent + Chang et al., 1997 Lithocholic acid Bile acid ,2-fold in primary hepatocytes Ferguson et al., 2005 Nelfinavir Antiviral, protease inhibitor 5-fold in primary hepatocytes 2-fold Dixit et al., 2007 Nilotinib Anticancer, PKI No induction of CYP2C8 .2-fold FDA, 2007c mRNA Paclitaxel Anticancer, taxane 4-fold in primary hepatocytes Ferguson et al., 2005 + in primary hepatocytes Synold et al., 2001 Phenobarbital Antiepileptic, + Chang et al., 1997 3-fold in primary hepatocytes 3-fold Gerbal-Chaloin et al., 2001 7-fold in primary hepatocytes Raucy et al., 2002 3- to 6-fold Madan et al., 2003 2-fold in primary hepatocytes Ferguson et al., 2005 Phenytoin Antiepileptic 2-fold in primary hepatocytes Ferguson et al., 2005 Progesterone Hormonal replacement 1.4- to 9.2-fold in primary Choi et al., 2013 therapy hepatocytes Rifampin Antibiotic + Chang et al., 1997 (rifampicin) 6-fold in primary hepatocytes 3-fold Gerbal-Chaloin et al., 2001 6.5-fold in primary hepatocytes Rae et al., 2001 + in primary hepatocytes Dussault et al., 2001 + in primary hepatocytes Synold et al., 2001 7- to 12-fold in primary 6-fold (1- to 17-fold) Raucy et al., 2002 hepatocytes 4- to 8-fold in primary Madan et al., 2003 hepatocytes 3-fold in primary hepatocytes 3- to 10-fold Ferguson et al., 2005 4- to 9-fold in primary Prueksaritanont hepatocytes et al., 2005 7-fold in primary hepatocytes 4-fold Dixit et al., 2007 6.5-fold in primary hepatocytes Rana et al., 2010 0.7- to 3-old in primary Yajima et al., 2014 hepatocytes 5-fold in HepaRG cells Thomas et al., 2015 Ritonavir Antiviral, protease inhibitor + in primary hepatocytes Dussault et al., 2001 + in primary hepatocytes Synold et al., 2001 7-fold in primary hepatocytes 2-fold Dixit et al., 2007 SR12813 Antihyperlipidemic, + in primary hepatocytes Synold et al., 2001 HMG-CoA reductase inhibitor Tasimelteon Circadian regulator 4.4-fold FDA, 2014m WY14,643 Antihyperlipidemic,PPARa 4-fold in HepaRG cells Thomas et al., 2015 agonist

CITCO, [6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; PKI, protein kinase inhibitor, HepaRG, hepatocyte-like cells from the human hepatoma HepaRG cell line; mRNA, messenger RNA; PPAR, peroxisome proliferator-activated receptor; SR12813, tetraethyl 2-(3,5-di-tert-butyl-4-hydroxyphenyl)ethenyl-1,1-bisphosphonate; WY14,643, 4-Chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid. 214 Backman et al. variation in the activity of drug metabolizing enzymes B. Gemfibrozil as Prototypical Inhibitor and transporters can cause a considerable variability in 1. In Vitro Versus In Vivo. In vitro, the parent the extent of drug interactions. In particular, it is gemfibrozil is a moderately potent competitive inhibitor important to note that in an individual patient the of CYP2C9 (Ki value of 5.8 mM; Wen et al., 2001), but it is exposure to a victim drug can change much more than over 10 times less potent as inhibitor of CYP2C8 (K value the generally reported mean change. i of 75 mM, Wang et al., 2002; K 87 mM, Prueksaritanont Recognition of the role of CYP2C8 as an important i et al., 2002; Table 6). Gemfibrozil in concentrations up to oxidative enzyme in drug metabolism has led to changes in 1,000 mM has no effect on CYP3A4 activity (midazolam product information of many drugs, resulting in better 19-hydroxylation) (Backman et al., 2000), but it is rather predictability of their interactions and improved safety. In potent as an inhibitor of the OATP1B1 transporter, with a some cases, CYP2C8-mediated drug interactions and the K value ranging in different studies from 4 to 31.7 mM resultant adverse effects have forced the manufacturers i to withdraw drugs from clinical use or to add contraindi- (Schneck et al., 2004; Yamazaki et al., 2005; Hirano et al., cations or limitations to their use. In general, special 2006; Nakagomi-Hagihara et al., 2007). attentionisneeded,ifadrugwithnarrowsafetymarginis In healthy volunteers, gemfibrozil (600 mg twice daily) extensively metabolized by CYP2C8 or if a drug is a strong slightly (by 23%) increased the AUC of a CYP2C9 CYP2C8 inhibitor like gemfibrozil and clopidogrel. In substrate drug glimepiride (Niemi et al., 2001) but did addition, it should be recognized that rifampin and some not increase the exposure to racemic warfarin (Lilja other potent enzyme inducers can markedly reduce plasma et al., 2005). Gemfibrozil even caused a small but 2 concentrations and effects of CYP2C8 substrate drugs. statistically significant decrease ( 11%) in the AUC of As gemfibrozil is a well-characterized inhibitor of the CYP2C9 substrate S-warfarin. These results strongly CYP2C8 that has increased the plasma concentrations suggest that gemfibrozil is not a meaningful inhibitor of several drugs (Fig. 7; Table 10), we first present a of CYP2C9 in vivo in humans. detailed description of it as an in vivo inhibitor in the In vivo gemfibrozil is glucuronidated by the UGT 2B7 following part. The other clinically relevant CYP2C8 enzyme to gemfibrozil 1-O-b-glucuronide. The benzylic inhibitors, including clopidogrel, trimethoprim, efavirenz, oxidation of the glucuronide by CYP2C8 leads to haem and teriflunomide (Niemi et al., 2004b; German alkylation and irreversible inactivation of CYP2C8 et al., 2007; FDA, 2012a; Tornio et al., 2014), are dealt (Baer et al., 2009; Jenkins et al., 2011). In HLM, the with in the next part, where we focus on the CYP2C8- kinact value of CYP2C8 by gemfibrozil 1-O-b-glucuronide inhibition-mediated drug interactions of different has been 0.21 1/min and KI 20–52 mM (Ogilvie et al., therapeutic CYP2C8 substrate drugs. Thereafter, we 2006). The glucuronide metabolite is also a competitive present CYP2C8 induction-mediated drug interactions inhibitor of OATP1B1 (OATP2) transporter (Ki 24 mM; and their clinical relevance. Shitara et al., 2004). On the basis of clinical studies on

Fig. 7. Effects of gemfibrozil on the exposure (area under the plasma drug concentration-time curve) to different CYP2C8 substrate drugs. The effect of gemfibrozil on drug exposures may also include inhibition of OATP1B1 (cerivastatin acid, lovastatin acid, paritaprevir, repaglinide, and simvastatin acid). Dasabuvir and paritaprevir were administered as a dasabuvir-paritaprevir-ritonavir combination. A fold increase of 1 refers to no effect of gemfibrozil on drug exposure. References are given in Table 10. Role of CYP2C8 in Drug Metabolism and Interactions 215

TABLE 10 Drug-drug interactions caused by CYP2C8-inhibiting drugs in humans

Inhibitor Dosing Substrate AUC Change References fold Atazanavir 400 mg once daily for 6 days, substrate on day 6 Rosiglitazone 1.4 FDA, 2015b Clopidogrel 75–300 mg for 3 days, substrate on days 1 and 3 Repaglinide 3.9–5.1a Tornio et al., 2014 Deferasirox 30 mg/kg once daily for 3 days, substrate on day 4 Repaglinide 2.3b Skerjanec et al., 2010 Efavirenz 400 mg once daily for 12 days, substrate on day 9 Amodiaquine 1.8 Soyinka et al., 2013 Active metabolite: 0.7 Soyinka et al., 2013 N-desethylamodiaquine Enzalutamide 160 mg once daily for 97 days, substrate on day 42 Pioglitazone n.s. (1.2) Gibbons et al., 2015 Active metabolite: 0.63 Gibbons et al., 2015 hydroxypioglitazone (M-IV) Gemfibrozil 600 mg twice daily for 6 days, substrate on day 6 Alogliptin 1.1b FDA, 2013g Active metabolite: M-I 1.9b FDA, 2013g 600 mg twice daily for 7 days, substrate on day 4 Brivaracetam 1.0 Nicolas et al., 2012 600 mg twice daily for 3 days, substrate on day 3 Cerivastatin (acid) 5.6a Backman et al., 2002 Cerivastatin lactone 4.4 Backman et al., 2002 600 mg twice daily for 4 days, substrate on day 4 Dabrafenib 1.5 Suttle et al., 2015 600 mg twice daily for 5 days, substrate on day 4 Daprodustat (GSK1278863A) 18.6 Johnson et al., 2014 600 mg twice daily for 5 days, substrate on day 3 Dasabuvir (ABT-333)b 11.3 Menon et al., 2015 Active metabolite: 0.22 Menon et al., 2015 dasabuvir M1 600 mg twice daily for 21 days, substrate on day 4 Enzalutamide 4.3 Gibbons et al., 2015 Active metabolite: 0.75 Gibbons et al., 2015 N-demethylenzalutamide 600 mg twice daily for 7 days, substrate for 7 days 1.4b Reyderman et al., 2004 600 mg twice daily for 3 days, substrate on day 3 R-Ibuprofen 1.3 Tornio et al., 2007 600 mg twice daily for 6 days, substrate on day 3 Imatinib n.s. Filppula et al., 2013b Active metabolite: 0.5 Filppula et al., 2013b N-demethylimatinib 600 mg twice daily for 5 days, substrate on day 3 Loperamide 2.2 Niemi et al., 2006 600 mg twice daily for 3 days, substrate on day 3 Montelukast 4.6 Karonen et al., 2010 Active metabolite: 36- 0.6c Karonen et al., 2010 hydroxymontelukast (M6) Montelukast 4.3 Karonen et al., 2011 Active metabolite: 36- 0.6c Karonen et al., 2011 hydroxymontelukast (M6 600 mg twice daily for 5 days, substrate on day 3 Paritaprevir (ABT-450)d 1.4a Menon et al., 2015 600 mg twice daily for 3 days, substrate on day 3 Pioglitazone 3.4 Deng et al., 2005 Active metabolite: n.s. Deng et al., 2005 hydroxypioglitazone (M-IV) Active metabolite: n.s. Deng et al., 2005 ketopioglitazone (M-III) 600 mg twice daily for 4 days, substrate on day 3 Pioglitazone 3.2 Jaakkola et al., 2005 Active metabolite: 0.6 Jaakkola et al., 2005 ketopioglitazone (M-III) Active metabolite: 0.6 Jaakkola et al., 2005 hydroxypioglitazone (M-IV) 600 mg twice daily for 4 days, substrate on day 3 Pioglitazone 4.3 Aquilante et al., 2013a 600 mg twice daily for 3 days, substrate on day 3 Repaglinide 8.1a Niemi et al., 2003b 600 mg twice daily for 3 days, substrate on day 3 7.3-8.3a Kalliokoski et al., 2008b 600 mg twice daily for 3 days, substrate on day 3 7.0a Tornio et al., 2008a 600 mg twice daily for 3 days, substrate on days 3-6 1.0-7.6a Backman et al., 2009 A single dose of 30-900 mg 1 h prior to substrate 1.8-8.3a Honkalammi et al., 2011a intake A single dose of 600 mg 0-6 h prior to substrate 5.0-6.6a Honkalammi et al., 2011b intake 30–600 mg twice daily for 5 days, substrate on day 5 3.4-7.0a Honkalammi et al., 2012 600 mg twice daily for 4 days, substrate on day 3 Rosiglitazone 2.3 Niemi et al., 2003a 600 mg twice daily for 3 days, substrate on day 3 Simvastatin (lactone) 1.4 Backman et al., 2000 Simvastatin acid 2.9a Backman et al., 2000 600 mg twice daily for 3 days, substrate on day 4 Sitagliptin 1.5e Arun et al., 2012 600 mg twice daily for 4 days, substrate on day 3 Treprostinil 1.9 FDA, 2009b 600 mg twice daily for 8 days, substrate on day 3 R-Warfarin 0.9 Lilja et al., 2005. S-Warfarin 0.9 Lilja et al., 2005. 600 mg twice daily for 3 days, substrate on day 3 Zopiclone n.s. Tornio et al., 2006 N-demethylzopiclone 1.2 Tornio et al., 2006 N-oxide-zopiclone 2.0 Tornio et al., 2006 Teriflunomidef 14–70 mg once daily for 12 days, substrate Repaglinide 2.3a FDA, 2012a on day 12 Trimethoprim 960 mg (combination)g twice daily for 6 days, Amodiaquine 1.6 Akande et al., 2015 substrate on day 6 Active metabolite: 0.9 Akande et al., 2015 N-desethylamodiaquine 160 mg twice daily for 3 days, substrate on day 3 Cerivastatin (acid) 1.4 Backman et al., 2003 Cerivastatin lactone 1.5 Backman et al., 2003 (continued) 216 Backman et al.

TABLE 10—Continued

Inhibitor Dosing Substrate AUC Change References 960 mg (combination)g twice daily for 3 days, substrate Loperamide 1.9 Kamali and Huang, 1996 on day 2 160 mg twice daily for 6 days, substrate on day 3 Pioglitazone 1.4 Tornio et al., 2008b Active metabolite: 1.1 Tornio et al., 2008b hydroxypioglitazone (M-IV) 160 mg twice daily for 3 days, substrate on day 3 Repaglinide 1.6 Niemi et al., 2004b 160 mg twice daily for 4 days, substrate on day 3 Rosiglitazone 1.4 Niemi et al., 2004a 200 mg twice daily for 5 days, substrate on day 5 1.3 Hruska et al., 2005

AUC, area under the plasma concentration-time curve; n.s. not statistically significant. aInhibition of OATP1B1 may also be involved. bThe role of CYP2C8 in the interaction is limited or unclear. c AUC0–7 hour. dGiven as a dasabuvir-paritaprevir-ritonavir combination. eInhibition of OAT3 may also be involved. fOf note, teriflunomide is the active metabolite of leflunomide. Hence, coadministration of leflunomide with CYP2C8 substrate drugs may also cause interactions. gTrimethoprim was given in a combination with sulfaphenazole (cotrimoxazole). the dose/time dependency of the effect of gemfibrozil on inhibition by gemfibrozil have been studied in healthy the pharmacokinetics of repaglinide and statistical volunteers using the gemfibrozil-repaglinide interac- models of enzyme and transporter inhibition, it has tion as a model. Single 600-mg doses of gemfibrozil been estimated that the in vitro mechanism-based ingested 0, 1, 3, or 6 hours before repaglinide (0.25 mg) inhibition of CYP2C8 by gemfibrozil 1-O-b-glucuronide increased the geometric mean AUC of repaglinide 5.0-, manifests into a strong and long-lasting inhibition 6.3-, 6.6-, and 5.4-fold, respectively (Fig. 8). The Cmax of of CYP2C8 at typical clinical doses of gemfibrozil the CYP2C8-mediated repaglinide M4-metabolite was (Backman et al., 2009; Honkalammi et al., 2011a,b). In 1.0-, 0.10-, 0.06-, and 0.09-fold compared with control addition, the OATP1B1 inhibitory effect of the glucuro- phase, respectively (Honkalammi et al., 2011b). These nide can lead to an up to ;50% transient inhibition of results indicate that the strong inactivation of CYP2C8 OATP1B1 in vivo. These effects are the main explana- occurs rapidly, being evident already within 1 hour after tion to the effects of gemfibrozil on CYP2C8 and oral dosing of gemfibrozil. OATP1B1 substrates (Honkalammi et al., 2012). Sim- When repaglinide was ingested 1, 24, 48, or 96 hours ilar estimations have been obtained with physiologi- after discontinuation of a gemfibrozil treatment (600 mg cally based pharmacokinetic modeling in a recent twice daily for 3 days), the AUC of repaglinide was publication (Varma et al., 2015). 7.6-, 2.9-, 1.4-, and 1.0-fold compared with the control 2. Gemfibrozil Dose Versus CYP2C8 Inhibition. phase, respectively (Backman et al., 2009). These Single oral doses of gemfibrozil, i.e., 30, 100, 300, or 900 findings confirmed and extended the previous findings, mg ingested 1 hour before repaglinide, increased the which had shown that the inhibitory effect of gemfibro- AUC of repaglinide in a dose-dependent manner 1.8-, zil persists for at least 12 hours after its ingestion 4.5-, 6.7-, and 8.3-fold compared with placebo, respectively (Tornio et al., 2008a). As the half-lives of gemfibrozil (Fig. 8; Honkalammi et al., 2011a). Also after multiple and its glucuronide are very short (about 1–2 hours), doses of gemfibrozil (30, 100, or 600 mg twice daily for these findings convincingly demonstrate that the effect 5 days), the exposure to repaglinide increased dose of gemfibrozil on repaglinide pharmacokinetics is based dependently, but the greatest AUC increase did not on irreversible mechanism-based inhibition of CYP2C8. exceed that observed after the single 900 mg gemfibrozil A several-fold increase in repaglinide AUC was evident dose (Honkalammi et al., 2012). Thus, the maximum even at very low plasma concentrations of gemfibrozil inhibition of CYP2C8 can be achieved by a single 900-mg 1-O-b-glucuronide, which are less than 1% of its peak dose of gemfibrozil (Fig. 9). Gemfibrozil in doses of 100 concentrations. The results also showed that full mg twice daily at steady state causes an about 95% CYP2C8 activity recovers gradually within 3–4 days inhibition of CYP2C8 (Fig. 9), and in doses of 10 mg twice after cessation of the clinically used therapeutic doses of daily, it causes an about 50% inhibition (Honkalammi 600 mg twice daily gemfibrozil. et al., 2011a). The fraction of a small 0.25-mg dose of 4. Quantification of CYP2C8-Mediated Drug Interac- repaglinide metabolized by CYP2C8 is about 80–90%. tions in Humans. Interactions caused by a combina- However, because repaglinide is metabolized to some tion of two or more drugs, which inhibit, in addition to extent also by CYP3A4, the relative role of CYP2C8 and CYP2C8, also some other crucial enzyme or transporter, CYP3A4 in the biotransformation of repaglinide can can increase exposure to a CYP2C8 substrates much depend on its dose and plasma concentrations as well more than is the sum of their separate effects causing a as on individual pharmacogenetic factors (Bidstrup et al., classic potentiation phenomenon. Thus, e.g., exposure 2003; Kajosaari et al., 2005a; Säll et al., 2012). to repaglinide is increased only slightly by itraconazole 3. Onset and Duration of CYP2C8 Inhibition by alone (1.4-fold), greatly by gemfibrozil alone (8.1-fold), Gemfibrozil. The onset and duration of CYP2C8 and drastically (19.4-fold) by their combination (Fig. 10; Role of CYP2C8 in Drug Metabolism and Interactions 217

Fig. 8. Effects of gemfibrozil on the exposure (area under the plasma drug concentration-time curve) to repaglinide (Repa) and its metabolites M1, M2, and M4. Fold changes in drug exposure compared with the control phase when repaglinide was taken 1 hour after a single 30-, 100-, 300-, or 900-mg dose of gemfibrozil (A) or 1 hour after the last dose of 30, 100, or 600 mg gemfibrozil twice daily (B). Fold changes in drug exposure as compared with the control phase, when a single 600-mg dose of gemfibrozil was taken simultaneously or 1, 3, or 6 hours before repaglinide intake (C) or when the last dose of gemfibrozil was taken 0, 1, 3, 6, 12, 24, 48, or 96 hours before repaglinide intake (D). For M4, AUC0-3 hour data are presented. For all other compounds, AUC0-‘ data are presented. A fold change of 1 refers to no effect of gemfibrozil on exposure. References are given in the text.

Niemi et al., 2003b). The quantitative rationalization of also the blood glucose lowering effect of repaglinide gemfibrozil-drug interactions and consideration of (Niemi et al., 2003b). This pioneering study clearly transporter-enzyme interplay have been dealt quite indicated that the extent of interaction caused by a recently by Varma et al. (2015). combination of two drugs can greatly exceed the sum of their separate effects. On the basis of these results and C. Inhibition-Mediated Drug Interactions and Their clinical observations of serious hypoglycemic episodes Clinical Significance in diabetic patients, The European Agency for the 1. Repaglinide. Interactions of the oral antidiabetic Evaluation of Medicinal Products gave “EMEA public drug repaglinide have been studied extensively, and it statement on repaglinide contraindication of concomi- is a recommended model substrate drug for CYP2C8 in- tant use of repaglinide and gemfibrozil” (21.05.2003). teraction studies (EMA, 2012b; http://www.fda.gov/Drugs/ Also the U.S. Food and Drug Administration warned DevelopmentApprovalProcess/DevelopmentResources/ against repaglinide-gemfibrozil interaction. The effect DrugInteractionsLabeling/ucm093664.htm). In healthy of gemfibrozil on repaglinide exposure was later volunteers, gemfibrozil (600 mg twice daily for 3 days) confirmed and characterized in several studies as de- raised the AUC of repaglinide 8.1-fold, itraconazole scribed in previous paragraphs (sections VI.B.1–3). The raised it 1.4-fold, and their combination raised it gemfibrozil-repaglinide interaction is mainly mediated 19.4-fold (Niemi et al., 2003b). Gemfibrozil alone and via inhibition of CYP2C8 and OATP1B1 by the gemfi- in combination with itraconazole considerably enhanced brozil 1-O-b-glucuronide. 218 Backman et al.

contraindicated, e.g., in Canada (http://healthycanadians. gc.ca/recall-alert-rappel-avis/hc-sc/2015/54454a-eng.php). The immunosuppressant teriflunomide (70 mg once daily for 4 days, followed by 14 mg once daily for 8 days) increased the AUC of repaglinide by 2.3-fold in healthy male subjects (FDA, 2012a) compared with when repaglinide was given alone. In three subjects the AUC was raised by 3.2- to 3.6-fold. Teriflunomide inhibits both CYP2C8 and OATP1B1, and the contri- bution of each mechanism to the increase in repaglinide exposure has not been established (FDA, 2012a). Of note, teriflunomide is the active metabolite of lefluno- mide and its plasma concentrations following lefluno- mide administration are equal to those observed when it is given alone. Hence, leflunomide may also be a clinically relevant CYP2C8 inhibitor. Care is warranted if inhibitors of CYP2C8 are combined with repaglinide. In particular, combination of strong CYP2C8 inhibitors, such as gemfibrozil and clopidogrel, with repaglinide should be avoided. Blood glucose levels and symptoms of hypoglycemia should be monitored closely and the doses modified as needed. The interactions with repaglinide are likely to be stronger in CYP2C8*3 carriers than in CYP2C8*1 homozygotes (Tornio et al., 2008a). Fig. 9. Predicted effects of different gemfibrozil doses on CYP2C8- 2. Other Oral Antidiabetic Drugs. The European mediated drug metabolism. Predicted fold increase in the AUC0–‘ of a drug metabolized by CYP2C8, when the fraction of the substrate drug product information of Actos (pioglitazone) stated ear- metabolized by CYP2C8 (fm,CYP2C8) varies between 50 and 99% (A), lier (e.g. 2004) that metabolism of pioglitazone occurs and the CYP2C8 activity remaining (B) after a gemfibrozil dose ranging predominantly via CYP3A4 and CYP2C9 (Jaakkola from 0 to 600 mg twice daily in steady-state conditions. Modified from Honkalammi et al. (2012). AUC, area under the concentration-time curve. et al., 2005), whereas the U.S. label stated that the major CYP isoforms involved were CYP2C8 and CYP3A4 (FDA, 1999). The in vitro study of Jaakkola et al. The antimicrobial drug trimethoprim (160 mg twice (2006c) showed that pioglitazone is metabolized mainly daily for 3 days) raised in healthy volunteers the AUC of repaglinide by 1.6-fold compared with placebo (Niemi et al., 2004b). Symptomatic hypoglycemia developed in a diabetic patient 5 days after addition of trimethoprim/ sulfamethoxazole therapy to his previously well-tolerated repaglinide (1 mg three times daily) treatment (Roustit et al., 2010). The 300-mg loading dose of clopidogrel raised the AUC of repaglinide by 5.1-fold, and the following daily 75-mg doses of clopidogrel raised the AUC by 3.9-fold in healthy volunteers (Tornio et al., 2014). The increase in repaglinide AUC caused by clopidogrel was highest in subjects with the CYP2C8*1/*4 genotype (Tornio et al., 2014). The clopidogrel-repaglinide interaction is medi- ated by formation of the clopidogrel acyl-b-D-glucuronide, which is a potent time-dependent inhibitor of CYP2C8. On the basis of this short-term study, it has been extrapolated that the daily treatment with 75 mg of clopidogrel causes a continuous, 60–85% inhibition of hepatic CYP2C8 under steady-state conditions during chronic clopidogrel use. The pharmacokinetic interaction Fig. 10. Effects of gemfibrozil (Gem), itraconazole (Itra), or their of clopidogrel and repaglinide resulted in an enhanced combination (Gem + itra) on the exposure (area under the plasma drug concentration-time curve) to repaglinide, loperamide, montelukast, and blood glucose-lowering effect of repaglinide. The con- pioglitazone. A fold increase of 1 refers to no effect of inhibitors on drug comitant use of repaglinide and clopidogrel is now exposure. References are given in Table 10 and in the text. Role of CYP2C8 in Drug Metabolism and Interactions 219 by CYP2C8 and to lesser extent by CYP3A4, whereas CYP2C8 to active desethylamodiaquine, which has a CYP2C9 is not significantly involved in pioglitazone long half-life of 9–18 days. In healthy subjects, tri- elimination. In healthy volunteers, gemfibrozil raised methoprim and efavirenz have been reported to in- the mean AUC of pioglitazone 3.2-fold (range 2.3-fold crease the AUC of amodiaquine by 1.6- and 1.8-fold, to 6.5-fold) and its elimination half-life 2.7-fold, but respectively, and to reduce that of desethylamodiaquine itraconazole had no effect on pioglitazone and did not by 12 and 26%, respectively (Soyinka et al., 2013; alter the effect of gemfibrozil on its pharmacokinetics Akande et al., 2015). In an earlier study, efavirenz (Jaakkola et al., 2005). In two other studies, gemfibrozil raised the AUC of amodiaquine in two healthy subjects increased the mean AUC of pioglitazone 3.4-fold (Deng by 2- to 4-fold and decreased the AUC of desethylamo- et al., 2005) and 4.3-fold (range 1.3-fold to 12.1-fold) diaquine by 24 and 8.5% (German et al., 2007). In both of (Aquilante et al., 2013a). CYP2C8 genotype influences these subjects, marked elevation of hepatic transami- therelativechangeinpioglitazoneexposureafter nase levels occurred several weeks after stopping the 3 gemfibrozil administration. Thus, CYP2C8*3 carriers days’ combined use, forcing premature discontinuation had a greater mean increase by gemfibrozil in pioglita- of the interaction study. The dramatic, delayed hepato- zone AUC (5.2-fold) compared with CYP2C8*1 homozy- toxicity warrants great care in combination of any gotes (3.3-fold) (Aquilante et al., 2013a). Trimethoprim CYP2C8 inhibitor with amodiaquine. (160 mg twice daily) raised in healthy volunteers the 4. Statins. Cerivastatin was initially considered as a AUC of pioglitazone by 1.4-fold and had opposite effects safe because of its dual biotransformation routes, on pioglitazone pharmacokinetics compared with the mediated both via CYP3A4 and CYP2C8 (Mück, 1998; effects of CYP2C8*3 allele during the placebo phase 2000). However, it soon became obvious that cerivasta- (Tornio et al., 2008b). tin greatly increased the incidence of fatal rhabdomyol- The AUC of rosiglitazone was raised in healthy ysis, particularly when taken along with gemfibrozil volunteers by gemfibrozil by 2.3-fold (Niemi et al., (Staffa et al., 2002). Consequently, cerivastatin was 2003a). In another study, trimethoprim (160 mg twice withdrawn from the market in 2001, only 3 years after daily) increased rosiglitazone AUC by 1.4-fold and re- its launch. Despite the “dual metabolic pathway” and duced the formation of N-demethylrosiglitazone (Niemi supposed "low propensity for drug interactions" (Mück et al., 2004a). The effect of trimethoprim (200 mg twice et al., 1998; Mück, 2000), the elimination of cerivastatin daily) on rosiglitazone pharmacokinetics was confirmed relied predominantly on CYP2C8. In healthy volun- by Hruska et al. (2005), who also demonstrated the teers, gemfibrozil (600 mg twice daily) raised the AUC competitive inhibition of rosiglitazone p-hydroxylation of the parent cerivastatin (acid) by 5.6-fold, the AUC by trimethoprim in vitro. Atazanavir (400 mg once daily) of cerivastatin lactone by 4.4-fold, and that of the increased the AUC of a single dose of rosiglitazone by CYP3A4-dependent metabolite M-1 by 4.35-fold, 1.4-fold (FDA, 2015b). whereas gemfibrozil decreased the AUC of the The AUC of nateglinide was increased only by 1.5-fold CYP2C8-dependent metabolite M-23 by 78% (Fig. 11; by 3 days’ pretreatment with therapeutic doses of both Backman et al., 2002). The increased exposure to gemfibrozil and itraconazole (Niemi et al., 2005a). Thus, cerivastatin, to its lactone, and to M-1 and the reduced neither CYP2C8 nor CYP3A4 has a substantial signif- formation of the CYP2C8-dependent metabolite re- icance to the pharmacokinetics of nateglinide. Gem- vealed the strong CYP2C8 inhibitory effect of gemfi- fibrozil increased also the AUC of the dipeptidyl brozil. In addition to irreversible inhibition of CYP2C8 peptidase inhibitor sitagliptin by 1.5-fold (Arun et al., by gemfibrozil 1-O-b-glucuronide, inhibition of the 2012). However, the gemfibrozil-sitagliptin interaction hepatic OATP1B1 may contribute to the gemfibrozil- seems to be mainly mediated by inhibition of the renal cerivastatin interaction (Ogilvie et al., 2006; Shitara OAT3, with a minor contribution by CYP2C8. et al., 2004; Tamraz et al., 2013). If gemfibrozil, clopidogrel, or other inhibitors of Interestingly, about 10 years after the withdrawal of CYP2C8 will be combined with pioglitazone or rosiglita- cerivastatin, it was found that in addition to gemfibro- zone, blood glucose levels, symptoms of hypoglycemia, and zil, also concomitant use of clopidogrel was strongly other potential adverse effects (e.g., fluid retention) should associated with cerivastatin-induced rhabdomyolysis, be monitored closely and the doses be modified as needed. with an odds ratio of ;30 (48 when gemfibrozil users As shown for the gemfibrozil-pioglitazone and gemfibrozil- were excluded) (Floyd et al., 2012). Recently, Tornio repaglinide interactions (Tornio et al., 2008a; Aquilante et al. (2014) showed that glucuronidation converts et al., 2013a), interactions may be stronger in CYP2C8*3 clopidogrel to a strong time-dependent inhibitor of carriers than in CYP2C8*1 homozygotes. CYP2C8, clopidogrel acyl-b-D-glucuronide. The forma- 3. Amodiaquine. Although amodiaquine N-deethylation tion of this metabolite leads to uninterrupted inhibition is a widely used marker reaction for CYP2C8 activity of CYP2C8 during clopidogrel treatment and explains in vitro, the sensitivity of amodiaquine to CYP2C8 the increased risk of rhabdomyolysis during concomi- inhibition is poorly characterized in humans. Amodia- tant use of cerivastatin and clopidogrel (Tornio et al., quine is rapidly and extensively metabolized by 2014). Also trimethoprim increased the AUC of 220 Backman et al.

Fig. 11. Effects of gemfibrozil on the plasma concentrations of cerivastatin, its lactone, and M-1 and M-23 metabolites after administration of cerivastatin 0.3 mg with gemfibrozil 600 mg or placebo twice daily for 3 days (modified from Backman et al., 2002). cerivastatin (by 1.4-fold) and its lactone (1.5-fold) Gemfibrozil raises the AUC of nearly all statin acids, (Backman et al., 2003). Because cerivastatin has been including simvastatin acid, lovastatin acid, atorva- withdrawn from the market, its interactions are no statin, pravastatin, rosuvastatin, and pitavastatin more of direct clinical relevance. However, they are (Neuvonen et al., 2006). However, the role of CYP2C8 in examples of clinically important challenges in drug some gemfibrozil-statin interactions seems to be limited or development and have been of paramount importance nonexistent. They are mainly mediated by inhibition in understanding the significance of CYP2C8 in drug of OATP1B1, OAT3, or other transporters (Shitara metabolism. et al., 2004; Neuvonen, 2010; Niemi et al., 2011). Interestingly, cerivastatin and repaglinide have 5. Anticancer Drugs. Most anticancer drugs have a pharmacokinetic similarities. Both drugs are sub- narrow therapeutic range. Although paclitaxel is a well- strates of CYP2C8, CYP3A4, and OATP1B1. The established CYP2C8 probe in vitro, its interactions with CYP3A4 inhibitor itraconazole has raised their AUC CYP2C8 inhibitors and inducers have not been widely only slightly, i.e., by 1.4-fold (repaglinide), 1.15-fold studied in humans. Lapatinib and pazopanib are rela- (cerivastatin acid), and 1.8-fold (cerivastatin lactone), tively strong inhibitors of CYP2C8, and they have whereas gemfibrozil has raised their AUC values much raised the AUC of paclitaxel up to 1.8-fold (Tan et al., more, i.e., by 8.1-fold (repaglinide), by 5.6-fold (cerivas- 2014). In a case report, the only clopidogrel user in a tatin), and by 4.4-fold (cerivastatin lactone) (Kantola cohort of 93 ovarian carcinoma patients treated with et al., 1999; Backman et al., 2002; Niemi et al., 2003b). paclitaxel had the second lowest clearance of unbound Because the combination of a CYP3A4 inhibitor and a paclitaxel in the cohort. She was hospitalized three CYP2C8 inhibitor caused a drastic increase in repagli- times because of severe paclitaxel toxicity (Bergmann nide AUC (by 19.4-fold; Niemi et al., 2003b), it is et al., 2015). reasonable to assume that also the exposure to cerivas- Gemfibrozil has raised the AUC of the androgen tatin acid and to its more lipophilic lactone form have receptor antagonist enzalutamide by 4.3-fold, and itra- raised even more by gemfibrozil —or clopidogrel—if the conazole raised it by 1.4-fold compared with control patients had been using also CYP3A4 inhibiting drugs. (Gibbons et al., 2015). These results agree well with the in However, there seems to be no studies on the effect of vitro findings that CYP2C8 is the predominant enzyme in CYP2C8 and CYP3A4 inhibitor combinations on the the elimination of enzalutamide. The composite exposure plasma concentrations of cerivastatin. of enzalutamide and its active metabolite was raised by Role of CYP2C8 in Drug Metabolism and Interactions 221

2.2-fold by gemfibrozil and by 1.3-fold by itraconazole. A gemfibrozil. This should lead to similar plasma concen- reduction of the enzalutamide dose by about 50% is trations of dasabuvir as those achieved by normal recommended when gemfibrozil is used concomitantly. dasabuvir doses administered without inhibitor of There are no published studies on the effect of clopidogrel CYP2C8. Also some other new antiviral drugs are on enzalutamide pharmacokinetics. However, a close partially metabolized by CYP2C8, but their suscepti- follow up and reduction of enzalutamide dose can be bility to interact with drugs affecting CYP2C8 activity recommended also in their possible coadministration. It in humans needs further studies. should also be noted that combined inhibition of 7. Antiasthmatic Drugs. In healthy volunteers. CYP2C8 and CYP3A4 can cause a greater increase in gemfibrozil raised the AUC of montelukast 4.5-fold enzalutamide + metabolite AUC. Enzalutamide itself is and its elimination half-life 3.0-fold (Karonen et al., an inhibitor of CYP2C8 and may moderately raise the 2010). Gemfibrozil reduced the AUC of the secondary exposure to its substrate drugs, e.g., pioglitazone AUC M4 metabolite of montelukast by more than 90%. In by 20% (Gibbons et al., 2015). another study, gemfibrozil alone raised the AUC of GemfibrozildidnotaffecttheAUCofimatinib montelukast 4.3-fold, itraconazole had no significant after a single imatinib dose but reduced the AUC of effects, and the effects of the gemfibrozil-itraconazole N-demethylimatinib by 48%, indicating a significant combination on montelukast pharmacokinetics did not participation of CYP2C8 in the metabolism of imatinib in differ from those of gemfibrozil alone (Karonen et al., 2012). humans (Filppula et al., 2013b). After a single dose, These findings indicate that CYP2C8 but not CYP3A4 is imatinib seems to be mainly metabolized by CYP3A4, important in the pharmacokinetics of montelukast. In but the fraction of imatinib metabolized by CYP3A4 contrast to the effect of gemfibrozil on montelukast, the decreases after its multiple doses because of auto- pharmacokinetics of zafirlukast is not affected by gemfi- inhibition of the CYP3A4-mediated metabolism of brozil (Karonen et al., 2011), although both of these imatinib (Filppula et al., 2012, 2013a). This autoinhibi- cysteinyl leukotriene receptor antagonists are potent in tion is likely to increase the relative role of CYP2C8 vitro inhibitors of CYP2C8 (Walsky et al., 2005a). in imatinib elimination and its sensitivity to interac- Montelukast has a relatively large safety margin, and tions caused by CYP2C8 inhibitors during long-term the clinical significance of its interactions with CYP2C8 treatment. According to pharmacokinetic simulations, inhibitors seems to be limited. However, neuropsychi- imatinib exposure may raise up to twofold at steady state atric symptoms have developed in a woman with HIV if a strong CYP2C8 inhibitor is given concomitantly with when montelukast was added to her therapy imatinib (Filppula et al., 2013b). containing the CYP2C8 inhibitor efavirenz (Ibarra- In melanoma patients, gemfibrozil increased the Barrueta et al., 2014). She had used efavirenz, emtrici- AUC of the CYP3A4 and CYP2C8 substrate dabrafenib tabine, and fumarate for years by 1.5-fold and ketoconazole increased it by 1.7-fold with good tolerance until montelukast was started (Suttle et al., 2015). It is probable that a combined for . Shortly thereafter unbearable symptoms administration of CYP2C8 inhibitors and CYP3A4 appeared, consisting of disturbed sleep, vivid dreams inhibitors with dabrafenib can increase its exposure and irritability, confusion, and concentration difficulties. more than does either of these inhibitors alone. In After 2 months of concomitant use, montelukast was addition to paclitaxel, dabrafenib, and imatinib, some withdrawn and the psychiatric symptoms completely dis- other anticancer drugs are metabolized by CYP2C8 appeared. This case report indicates that adverse effects (Table 1). However, their interactions with CYP2C8 can develop when these drugs are used together, although inhibitors have not been characterized in humans. the mechanism of adverse effects is not fully clear. Considering the narrow therapeutic range of many 8. Other Substrate or Inhibitor Drugs. Gemfibrozil anticancer drugs, close follow up for possible adverse raised in healthy volunteers the AUC of loperamide effects is warranted if gemfibrozil, clopidogrel, trimeth- 2.1-fold, itraconazole raised it 3.8-fold, and the oprim, or other inhibitors of CYP2C8 are used with pacli- gemfibrozil-itraconazole combination raised lopera- taxel or other anticancer drugs metabolized by CYP2C8. mide AUC 12.6-fold compared with placebo phase 6. Antiviral Drugs. Gemfibrozil (600 mg twice daily) (Niemi et al., 2006). This finding strongly suggests has increased the AUC of the antihepatitis C drug that gemfibrozil can markedly increase the loperamide dasabuvir about 11-fold and their concomitant use is exposure in subjects who are using potent inhibitors contraindicated (Menon et al., 2015). It is reasonable to of CYP3A4, i.e., when another important metabolic assume that also other potent inhibitors of CYP2C8 route is blocked. Administration of cotrimoxazole such as clopidogrel increase greatly the exposure to (trimethoprim + sulphamethoxazole) has increased the dasabuvir, and their use together should be avoided or AUC of loperamide by 1.9-fold (Kamali and Huang, the dose of dasabuvir be reduced markedly. It can be 1996). Also some other , e.g., buprenorphine, are speculated that savings could be achieved by using CYP2C8 substrates (Table 1). However, there seem to be small amounts of expensive dasabuvir (about one-tenth no published studies on their possible interaction with of normal dose) with small doses (e.g., 100 mg) of gemfibrozil or other CYP2C8 inhibitors. 222 Backman et al.

Gemfibrozil raised the AUC of the prolyl hydroxylase their AUC, and diminish their clinical efficacy. Both inhibitor agent daprodustat (GSK1278863) 18.6-fold CYP2C8 and CYP3A4 are involved in the biotransfor- (Johnson et al., 2014). This result together with in vitro mation of many drugs, which can also be substrates of studies indicates the crucial significance of CYP2C8 in various transporters (Table 1). Because both CYP2C8 and its pharmacokinetics. CYP2C8 inhibitors should not be CYP3A4 enzymes and some transporters can be highly used with this erythropoiesis- agent or its dose inducible, the importance of CYP2C8 in many rifampin needs to be reduced very greatly. On the other hand, at interactions is difficult to determine exactly (Niemi et al., least theoretically, it could be possible to take advantage of 2000). Apart from rifampin, there are very few clinical this interaction in a product containing very small doses of studies concerning the effects of other CYP enzyme daprodustat and gemfibrozil. inducers on the pharmacokinetics of CYP2C8 substrates. In healthy volunteers, gemfibrozil raised only slightly 1. Rifampin (Rifampicin). Rifampin (600 mg/day), the AUC of R-ibuprofen, by 1.3-fold, after the ingestion given for several days, has decreased the plasma of racemic ibuprofen (Tornio et al., 2007). In vitro exposure to repaglinide by 31–80% depending on the CYP2C8 participates in the metabolism of zopiclone time interval from the last rifampin dose to repaglinide (Becquemont et al., 1999). In humans, however, gemfi- ingestion (Table 11; Niemi et al., 2000; Hatorp et al., brozil did not increase the AUC of the parent zopiclone 2003; Bidstrup et al., 2004). The time interval affects but moderately (2-fold and 1.2-fold) increased the AUC the extent of interaction because rifampin is also of its potentially active metabolites (Tornio et al., 2006). a competitive inhibitor of OATP1B1, CYP2C8 and Also many other drugs are substrates of CYP2C8 in CYP3A4 (Kajosaari et al., 2005a; Varma et al., 2013). vitro, but their concomitant administration with gemfi- Interestingly, intake of St John’s Wort for 14 days has brozil has not appreciably increased their AUC, suggesting had no significant effect of the pharmacokinetics of that the CYP2C8-mediated biotransformation is of lim- repaglinide (Fan et al., 2011). ited significance to their total clearance (Table 1). Rifampin has also reduced the concentrations of the Many compounds are moderate inhibitors of CYP2C8 thiazolidinediones pioglitazone and rosiglitazone. Ri- in vitro, but their concomitant ingestion with repagli- fampin caused a substantial (54%) decrease in the AUC nide or other CYP2C8 substrates does not raise expo- of pioglitazone and increased the ratios of metabolite sure to these substrates in humans. The reason for the M-IV to pioglitazone and of M-III to pioglitazone in urine apparent discrepancy between the in vitro and in vivo by 98 and 95% (Jaakkola et al., 2006a). Similarly, results can be, for example, their low potency as rifampin reduced the mean AUC of rosiglitazone by CYP2C8 inhibitors or their high protein binding in vivo 54% and increased the formation of N-demethylrosigli- (e.g., montelukast). tazone (Niemi et al., 2004a). In Korean men, rifampin Some parent drugs such as gemfibrozil and clopidog- decreased rosiglitazone AUC by 65% (Park et al., 2004). rel are relatively weak inhibitors of CYP2C8 in vitro, Addition of tuberculosis treatment, containing rifam- but they are metabolized in vivo to glucuronide metab- pin, to treatment of a woman with type 2 diabetes olites, which are potent CYP2C8 inhibitors. In general, caused her to lose glycemic control, demonstrating poten- negative interaction results with gemfibrozil in vivo tial clinical significance of the rifampin-rosiglitazone exclude a clinically meaningful interaction mediated interaction (Pimazoni, 2009). by CYP2C8 inhibition. On the other hand, increased exposure to a victim drug by gemfibrozil does not yet VII. Points to Consider When Investigating indicate that CYP2C8 has a significant role in its Cytochrome P450 2C8-Mediated Drug metabolism because there may be other mechanisms Metabolism and Interactions mediating the observed interaction. Patients often concomitantly use different drugs Studies focusing on drug metabolism and metabolic that together inhibit several CYP enzymes, e.g., drug-drug interactions are an essential part of modern CYP1A2, CYP2C8, CYP2C9, CYP2B6, CYP2D6, or drug development, from early preclinical phases to CYP3A4. The combined inhibition of two or more of the clinical development phase and beyond. By using these enzymes often results in patients in a stronger specific and sensitive research methods, it is possible interaction than is caused by inhibition of a single to get a detailed and accurate view of potential issues enzyme in healthy volunteer studies. This aspect related to variability in drug metabolism already during together with other causes of interindividual variation the preclinical and early clinical phases of development. should be taken into consideration when the results of Methods to investigate CYP2C8 in vitro and in clinical experimental interaction studies in healthy volunteers studies have evolved markedly even during the last are translated into the clinic. decade.

D. Induction-Mediated Drug Interactions A. In Vitro Rifampin (rifampicin) can markedly increase the 1. General Aspects. Comprehensive in vitro studies clearance of many CYP2C8 substrate drugs, decrease to investigate the roles of different CYP enzymes in the Role of CYP2C8 in Drug Metabolism and Interactions 223

TABLE 11 Drug-drug interactions caused by the CYP2C8-inducing drug rifampin (rifampicin) in humans

Time Interval from the Inducer Dosing Previous Rifampin Dose to Substrate AUC Decrease References Substrate Ingestion

hours % 600 mg once daily for 6 days 13 Pioglitazone 54 Jaakkola et al., 2006a - Active metabolite: ketopioglitazone (M-III) 39 Jaakkola et al., 2006a - Active metabolite: hydroxypioglitazone (M-IV) 34 Jaakkola et al., 2006a 600 mg once daily for 5 days 12.5 Repaglinide 57 Niemi et al., 2000 600 mg once daily for 7 days 1 31 Hatorp et al., 2003 600 mg once daily for 7 days 0 48 Bidstrup et al., 2004 24 80 Bidstrup et al., 2004 600 mg once daily for 5 days 13 Rosiglitazone 54 Niemi et al., 2004a 600 mg once daily for 6 days 12 65 Park et al., 2004

AUC, area under the plasma concentration-time curve. metabolism of a (new) drug and to uncover its potential montelukast, rosiglitazone, pioglitazone, and cerivasta- for causing inhibition or induction of drug metabolism tin, each having its specific strengths and weaknesses are typically conducted already during the early pre- (Tables 12 and 13). clinical phases of drug development. The results from Paclitaxel 6-a-hydroxylation is the prototypical these studies are then used for in vitro-in vivo extra- marker reaction for CYP2C8 (Rahman et al., 1994; polations, to anticipate factors affecting the clearance Sonnichsen et al., 1995). It is highly selective for of the drug as well as its potential to act as a perpetrator CYP2C8, but the metabolic turnover is fairly low, often of pharmacokinetic drug interactions, i.e., to affect the leading to relatively long incubation times, which may clearance of other drugs. The prerequisite for accurate lead to significant inhibitor metabolism/depletion dur- extrapolations is that in vitro investigations are con- ing the incubation (Table 13). This may partly explain ducted with care and are sufficiently comprehensive, why paclitaxel seems to be less sensitive to competitive avoiding the many pitfalls of in vitro studies, under- CYP2C8 inhibitors than most other CYP2C8 marker standing the many limitations of the different ap- substrates (VandenBrink et al., 2011). In particular, proaches, and covering complex issues, such as the long incubation times should be avoided when studying potential for autoinhibition or -induction. Yet it should the potential for time-dependent or mechanism-based be understood that accurate extrapolations are not inhibition in systems based on a preincubation step, possible without some clinical pharmacokinetic data because inactivation proceeding during the incubation at the relevant dose of the investigational drug. may decrease the sensitivity of the experimental system The general aspects as well as the potential pitfalls of to detect inactivation. in vitro studies and extrapolations are well covered by many Amodiaquine metabolism to N-desethylamodiaquine excellent review articles and guidelines (Houston and is probably the second most used CYP2C8 marker Galetin, 2008; Pelkonen et al., 2008; Grimm et al., 2009; reaction. It is well characterized and highly selective EMA, 2012b; Pelkonen, 2015; http://www.fda.gov/Drugs/ for CYP2C8 and has a high turnover (Li et al., 2002), DevelopmentApprovalProcess/DevelopmentResources/ allowing for short incubation times. Overall, it seems to DrugInteractionsLabeling/ucm093664.htm). Therefore, have no major drawbacks in in vitro use. this review focuses on issues that are directly related Few years ago, montelukast, a selective competitive to CYP2C8, i.e., in vitro methods used for measurement inhibitor of CYP2C8, was shown to be a potential of CYP2C8 activity (e.g., to test the potential of the CYP2C8 marker substrate, because its 36-hydroxylation investigational drug to inhibit CYP2C8 activity) and (M6 formation) is mediated primarily by CYP2C8 with reaction phenotyping (does CYP2C8 metabolize the a minor contribution by CYP2C9 (Filppula et al., 2011). drug) and in vivo studies to characterize the drug In a successive study, montelukast 36-hydroxylation interaction potential of the new drug (either as a proved to be a sensitive and useful reaction to in- perpetrator or victim drug). vestigate CYP2C8 inhibition in vitro (VandenBrink 2. Assessment of CYP2C8 Activity In Vitro. et al., 2011). One of the weaknesses of montelukast Specific assessment of CYP2C8 activity is necessary, in is that it is highly susceptible to microsomal protein particular when studying the potential of a drug to cause binding, necessitating careful standardization of incuba- inhibition of CYP2C8 but also when using a panel of HLM tion conditions (Walsky et al., 2005b). for reaction phenotyping using the correlation approach. Of the other potential marker reactions, cerivastatin An ideal in vitro probe substrate is selective/specific, has 6-hydroxylation (M-23 formation) seems to be highly a sufficient turnover, follows Michaelis-Menten kinetics, specific for CYP2C8 (Wang et al., 2002; Shitara et al., and is not sensitive to experimental conditions. There are 2004). In addition, the hydroxylations of rosiglitazone several useful, selective probe substrates to study (p-hydroxylation) (Baldwin et al., 1999) and pioglita- CYP2C8 in vitro, including paclitaxel, amodiaquine, zone (M-IV formation; Jaakkola et al., 2006c) seem to be 224 Backman et al.

TABLE 12 CYP2C8 substrate, inhibitor, and inducer probes recommended for drug-drug interaction studies

EMAa FDAb Helsinki DDI Group Probe In Vitro In Vivo In Vitro In Vivo In Vitro In Vivo Substrate Paclitaxel Amodiaquine Paclitaxelc Repaglinide Amodiaquine Repaglinide Amodiaquine Repaglinide Amodiaquine Rosiglitazone Paclitaxel Montelukast Rosiglitazone Montelukast Pioglitazone Cerivastatin (acid) Rosiglitazone (Pioglitazone) (Dasabuvir) Inhibitor Montelukast Gemfibrozil Montelukastc Gemfibrozil Montelukast Gemfibrozil Quercetinc Gemfibrozil Clopidogrel 1-O-b glucuronide Trimethoprim Clopidogrel acyl (Trimethoprim) 1-b-D-glucuronide Gemfibrozil (Trimethoprim) Rosiglitazone Pioglitazone Inducer None recommended None recommended Rifampin (rifampicin) Rifampin (rifampicin) Rifampin (rifampicin) Rifampin (rifampicin)

DDI, drug-drug interaction; EMA, European Medicines Agency; FDA, Food and Drug Administration. aEMA, 2012b. bhttp://www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/ucm093664.htm (Accessed September 15, 2015). cPreferred. relatively, albeit not completely, selective for CYP2C8. One of the most widely used chemical CYP2C8 Finally, the most used in vivo CYP2C8 probe drug inhibitors is quercetin (Rahman et al., 1994). However, repaglinide, although sometimes recommended as an in it is neither very selective for CYP2C8 nor very strong vitro probe (Kajosaari et al., 2005a; VandenBrink et al., and therefore, it can barely be recommended as a 2011), is challenging to use in vitro, e.g., because of a diagnostic inhibitor. Today, there are several more need for extremely low substrate concentrations and selective alternatives available, including trimetho- lack of commercially available metabolite standards prim, montelukast, and gemfibrozil 1-O-b-glucuronide. (Table 13). The IC50 of trimethoprim for CYP2C8 is approxi- 3. In Vitro Methods to Estimate the Contribution mately 50 mM, i.e., it is not a very strong inhibitor, but of CYP2C8 in the Metabolism of a Drug. The basic its IC50 for other CYP enzymes is at least one order of methods used for estimating the contributions of CYP magnitude greater, making it a relatively selective enzymes to the metabolism of a drug, i.e., the so-called inhibitor (Wen et al., 2002). Montelukast, on the other reaction phenotyping, are the use of diagnostic inhibi- hand, is a potent and highly selective competitive tors in a complete natural system, such as HLM, and the inhibitor of CYP2C8, with an IC50 as low as 0.01 mM, use of recombinant expressed enzymes. In both ap- when a low microsomal protein concentration is used, proaches, knowledge of clinically relevant concentra- whereas its IC50 for other CYP enzymes is at least two tions of the drug is a prerequisite for estimation of the orders of magnitude greater (Walsky et al., 2005b). The contributions of the different CYP enzymes in vivo. The major drawback of montelukast seems to be its non- advantage of HLM is the natural composition of the specific microsomal protein binding, whereby increas- system, allowing relatively straightforward estimation ing the microsomal protein concentration by 80-fold of the contributions. However, this approach requires yields an about 100-fold decrease in its inhibition human material collected according to high ethical potency (Walsky et al., 2005b). standards and is entirely dependent on the strength The mechanism-based CYP2C8 inactivator gemfibrozil and specificity of the inhibitors. On the other hand, 1-O-b-glucuronide is another appealing CYP2C8 inhibi- although recombinant expressed enzymes can be tor. With a 30-minute preincubation, its IC50 for CYP2C8 regarded as a specific tool, in vivo extrapolations of is about 2 mM, whereas its IC50 values for CYP1A2, recombinant enzyme results require the use of en- CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 are zyme source and batch specific conversion factors more than 300 mM, suggesting an even better selectivity (preferably based on enzyme activity), complicating than that of montelukast (Ogilvie et al., 2006). Moreover, the extrapolations. it is unlikely to be markedly affected by microsomal Recombinant expressed human CYP2C8 is commer- protein concentration. Whether clopidogrel acyl-b-D- cially available at least as bacterial cell- and insect cell- glucuronide is a similarly selective CYP2C8 inactivator based products. During the last decade, both chemical remains to be investigated (Tornio et al., 2014). inhibitors and inhibitory antibodies have become avail- able that are both CYP2C8 specific and strong. In the B. In Vivo following, we review the documentation regarding 1. General Aspects. Current guidelines recommend chemical CYP2C8 inhibitors. the conduct of clinical drug-drug interaction studies on Role of CYP2C8 in Drug Metabolism and Interactions 225

the basis of in vitro studies on the CYP inhibitory effects of the drug and its main circulating metabolites (potential perpetrator), as well as on the basis of the results of the in vitro reaction phenotyping studies of the drug and its main metabolic pathways (victim). A rational selection for the first CYP-specific clinical studies is to focus on the enzyme that is inhibited most (lowest IC50/Ki) by the drug or its metabolite, prefer- ably using the highest clinically used dose of the drug, also a substrate ofand OATP1B1 CYP3A4 half-life, not easily available in all countries and OAT3 and on the enzyme that is considered the most Long half-life Also CYP2B6 inhibitor Lack of documentation Weak inhibitor Moderate inhibitor of OATP1B1 important in its own metabolism. For the first type of studies, a sensitive and selective in vivo probe substrate is used, and for the second type of studies, a strong and selective in vivo probe inhibitor is needed. In Vivo For CYP2C8, there are several alternative probe substrates and a few inhibitors that can be used in clinical trials. The contribution of CYP2C8 enzyme to the total clearance of its substrates varies greatly (Table 1). Most CYP2C8 substrates are partially metabolized also substrate of OATP1B1 OATP1B1 or CYP3A4

Strength, not an inhibitor of by other enzymes, are substrates of some membrane Sensitivity, short half-life Reduces blood glucose levels, Relative sensitivity, safety Medium long half-life Relative sensitivity, safety, not a Safety, not a substrate of OATP1B1 Only moderate sensitivity, long Strong, well-documented inducer Nonselective Sensitive Selectivity Strength, selectivity transporters, or are excreted in urine or feces in un- changed form. Thus, the significance of CYP2C8 in interactions cannot be calculated directly from changes in victim drug AUC. If the CYP2C8 substrate drug (Rifampicin) is also a substrate of transporters or other CYP Repaglinide Montelukast Pioglitazone Rosiglitazone Rifampin Dasabuvir Clopidogrel Trimethoprim Gemfibrozil enzymes, their contribution needs to be considered in the interaction, as exemplified in the dissection of the gemfibrozil-repaglinide interaction (Honkalammi et al., 2011a, 2012). For example, gemfibrozil is in vivo TABLE 13 an inhibitor of CYP2C8 as well as of OATP1B1 and OAT3, and it can increase the AUC of certain drugs (e.g., pravastatin), which are not substrates of CYP2C8 but are substrates for OATP1B1 or OAT3 (Kyrklund et al., 2003). On the other hand, CYP2C8 inhibitors usually increase the AUC of CYP2C8 substrates less than they diminish the CYP2C8-specific metabolic solubility issues for very low substrateissues concentration, with protein binding contribution by CYP3A4, requirement for low substrate concentration a preincubation microsomal protein binding requires a preincubation routes, because the CYP2C8-independent elimination Strengths and weaknesses of the recommended probe compounds Low/intermediate turnover, Nonselective Selectivity not documented, requires Availability of reference compounds Lack of metabolite standards, Selectivity not well documented, Low/intermediate turnover Low/intermediate turnover routes remain unaffected. CYP2C8-mediated drug interactions are often stud-

In Vitro ied in healthy volunteers in a randomized crossover manner by administering a potential substrate drug with and without a probe inhibitors of CYP2C8, such as the recommended probe inhibitor gemfibrozil (Table 12). To better simulate real clinical situations turnover documentation inducer in which patients often are using several different drugs Selectivity, high Selectivity, extensive Selectivity Contribution by CYP2C9, requirement Selectivity Selectivity Potency, selectivityPotency, well-documented Also a substrate of CYP2C8, Selectivity Selectivity Potency concomitantly, substrate drugs can be administered in multiple-phase studies, given alone, with an inhibitor of b CYP2C8 (gemfibrozil), with an inhibitor of another relevant CYP enzyme (e.g., with CYP3A4 inhibitor

Probe Strengths Weaknessesitraconazole), Probe and Strengths together with a Weaknesses combination of -D-glucuronide

b inhibitors. However, there are only a few studies in (Rifampicin) glucuronide 1- Amodiaquine Paclitaxel Montelukast Repaglinide Cerivastatin (acid) Montelukast Pioglitazone Gemfibrozil 1-O- Rosiglitazone Rifampin Clopidogrel acyl which the effects of multiple inhibitors (e.g., inhibitors of CYP2C8 and CYP3A4) on the pharmacokinetics of their substrate drugs have been studied both separately and together (Niemi et al., 2003b, 2006; Jaakkola et al., Substrate Inhibitor Inducer 2005; Karonen et al., 2012). 226 Backman et al.

2. In Vivo Cytochrome P450 2C8 Probe Substrates. However, there is very little clinical documentation for its The crucial characteristics of an in vivo probe substrate use as a probe drug. Moreover, the safety of amodiaquine are its selectivity and sensitivity. In an ideal case, at as an in vivo probe drug in drug-drug interactions studies least 80% of the substrate is metabolized by the enzyme seems to be questionable (German et al., 2007). of interest, allowing for an interpretation based on the Of the other potential in vivo probe substrates of AUC of the parent drug. In some cases, the use of an CYP2C8, the two thiazolidinediones pioglitazone and enzyme-specific metabolite to parent ratio may be rosiglitazone are the best documented. As pointed out used to increase sensitivity and specificity, but with in the previous section, CYP2C8 is the main enzyme an additional caveat because of potential variability in mediating their primary hydroxylation reactions in metabolite elimination. The feasibility of an in vivo vitro (Baldwin et al., 1999; Jaakkola et al., 2006c). probe substrate also depends heavily on its safety, in Accordingly, the typical dosing of gemfibrozil 600 mg particular when large increases in its systemic concen- twice daily, which has been estimated to cause over 95% trations can be anticipated. Moreover, the pharma- inhibition of CYP2C8 (Fig. 9; Honkalammi et al., 2012), cokinetic characteristics of the probe may affect its increased the AUC of rosiglitazone about 2.3-fold and suitability. For example, a probe substrate with a that of pioglitazone 3.2-4.3-fold, simultaneously reduc- significant first-pass metabolism and short elimination ing the concentrations of their hydroxyl metabolites half-life may be able to catch even transient changes in (Niemi et al., 2003a; Deng et al., 2005; Jaakkola et al., enzyme activity, which may be necessary when study- 2005; Aquilante et al., 2013a). Unlike repaglinide, they ing inhibitors with a short half-life or time-dependent are insensitive to OATP1B1 function (Kalliokoski et al., changes in enzyme activity. 2008a). However, they have a long half-life, necessitat- The antidiabetic agent repaglinide is overall the most ing an up to 72-hour blood sampling period for a full studied and best documented in vivo probe substrate of pharmacokinetic analysis. On the basis of its better CYP2C8, and consequently both the European Medi- availability and sensitivity to CYP2C8 inhibition, pio- cines Agency (EMA) and FDA recommend its use as a glitazone is slightly preferable over rosiglitazone as a CYP2C8 probe (Table 12). Although in vitro studies CYP2C8 probe. are not fully consistent with the major in vivo role of The leukotriene receptor antagonist montelukast is CYP2C8 in the total metabolism of repaglinide (Gan another sensitive CYP2C8 substrate that could be used et al., 2010; Säll et al., 2012; Varma et al., 2013, 2015), as a CYP2C8 probe. Gemfibrozil has increased its AUC repaglinide seems to be very sensitive to inhibitors of almost fivefold and markedly reduced the formation of CYP2C8 activity, such as gemfibrozil, trimethoprim, its 36-hydroxylated metabolite (Karonen et al., 2010). and clopidogrel (Niemi et al., 2003b, 2004b; Tornio et al., On the other hand, montelukast is also partially 2014). On the basis of detailed mechanistic drug-drug metabolized by CYP3A4 in vitro (Filppula et al., 2011; interaction studies with the strong CYP2C8 inactivator VandenBrinketal.,2011).However,thestrong gemfibrozil, it has been estimated that the contribution CYP3A4 inhibitor itraconazole has had no effect on of CYP2C8 to repaglinide (0.25 mg) metabolism is about montelukast concentrations (Karonen et al., 2012), 85%, indicating that the AUC of repaglinide can be indicating that the role of CYP3A4 in montelukast increased up to an average of sevenfold by strong metabolism is minor. Moreover, montelukast is not CYP2C8 inhibition (Honkalammi et al., 2012). The known to be a substrate for OATP1B1. half-life of repaglinide is also relatively short (1 hour), In addition to the above substrates, there are some which allows for a full pharmacokinetic study within 1 other CYP2C8 substrate drugs that could be used as in day and can be beneficial when a measure of CYP2C8 vivo markers on the basis of their sensitivity to interact activity within a narrow time frame is desired. The with gemfibrozil. Such drugs include, for example, weakness of repaglinide is that it is partially metabo- daprodustat and dasabuvir. However, the former is lized by CYP3A4 (Bidstrup et al., 2003; Niemi et al., not yet on the market, and the second one is expensive, 2003b; Kajosaari, 2005a) and also a substrate of and more documentation is needed before they can be OATP1B1 (Niemi et al., 2005b). Thus, e.g., the effect of recommended as probe substrates. gemfibrozil on repaglinide pharmacokinetics is par- 3. In Vivo Cytochrome P450 2C8 Probe Inhibitors. tially mediated by inhibition of OATP1B1, in addition Probe inhibitors are needed for studying the contri- to inhibition of CYP2C8 (Honkalammi et al., 2011a). bution of CYP2C8 in the metabolism of a new drug, as Moreover, as it increases insulin secretion from pancre- well as for documenting the risk of drug-drug interac- atic b cells, there is a risk of hypoglycemia, particularly tions affecting the drug in vivo. Among clinically used when it is given to healthy subjects. Thus, the smallest CYP2C8 inhibitors, gemfibrozil is the strongest known. possible dose (e.g., 0.25 mg) of repaglinide should be Its CYP2C8 inhibitory effect is also highly selective due used, and meals, close follow up, and blood glucose to the specific mechanism that is mediated via specific monitoring be arranged when repaglinide is used. CYP2C8 inactivation by the glucuronide metabolite of Theoretically, the antimalarial agent amodiaquine and gemfibrozil (Ogilvie et al., 2006). In vitro, parent its N-deethylation could be useful in vivo CYP2C8 probes. gemfibrozil inhibits CYP2C9 activity with a fairly low Role of CYP2C8 in Drug Metabolism and Interactions 227

Ki (about 6 mM), but its inhibitory effects on the other inhibitor documented so far, increasing the AUC of main CYP enzymes are much weaker (Backman et al., repaglinide about fivefold (Tornio et al., 2014). Clopi- 2000; Wen et al., 2001; Wang et al., 2002). In clinical dogrel is obviously also a useful diagnostic CYP2C8 studies, gemfibrozil at a dose of 600 mg twice daily has inhibitor, but it is not fully selective and has not been not increased the concentrations of the CYP2C9 sub- extensively documented so far. In addition to strongly strate warfarin (Lilja et al., 2005) or had any effect that inhibiting CYP2C8, clopidogrel is also a moderate could be due to inhibition of CYP3A4 on the concentra- inhibitor of CYP2B6 (Turpeinen et al., 2005). Further- tions of the parent lactone forms of simvastatin and more, it has been suspected of causing CYP2C19 in- lovastatin (Backman et al., 2000; Kyrklund et al., 2001). hibition (Nishiya et al., 2009). However, it seems to have On the other hand, gemfibrozil has drastically, up to practically no effect on CYP3A4 or OATP1B1 activities 18.6-fold, increased the AUCs of CYP2C8 substrate in vivo (Tornio et al., 2014; Itkonen et al., 2015). drugs (Fig. 7; Table 10), suggesting that with regard to CYP enzymes, the inhibitory effect of gemfibrozil is VIII. Conclusions and Future Prospects highly selective for CYP2C8. The CYP2C8 inhibitory effect of gemfibrozil is strong, CYP2C8 is one of the main oxidative drug metabo- rapid, and long lasting. In studies using repaglinide as lizing enzymes in the liver. Its expression and function the CYP2C8 probe substrate, subtherapeutic doses of have been studied in detail, and for example, it has gemfibrozil have considerably elevated the concentra- been estimated that its in vivo turnover half-life is tions of repaglinide (Honkalammi et al., 2011a, 2012), about 22 hours in humans. The CYP2C8 gene contains and it has been estimated that the clinically used 9 exons and shares 74% sequence homology with gemfibrozil dosing (600 mg twice daily) inhibits CYP2C9. More than 100 nonsynonymous CYP2C8 CYP2C8 activity by about 99% and that one-tenth of SNVs are known to date, but only some of them are this dose would already lead to more than 90% in- associated with functional variability. Interethnic hibition of CYP2C8 (Fig. 9). Although CYP2C8 inhi- and geographical differences exist in the frequency bition by gemfibrozil is based on time-dependent of variants. For example, the low-activity variant inactivation of the enzyme by the primary glucuronide CYP2C8*2 (c.805A.T) is common in Africans but rare metabolite of gemfibrozil, CYP2C8 inhibition occurs in Caucasians and Asians. CYP2C8*3 (c.416G.A) and rapidly after gemfibrozil dosing. When repaglinide CYP2C8*4 (c.792C.G), on the other hand, are com- was given 0, 1, 3, or 6 hours after a single 600 mg dose mon in Caucasians but rare or absent in Africans of gemfibrozil, the AUC of repaglinide was increased and Asians (Fig. 6). The interethnic characterization 5.0-, 6.3-, 6.6-, and 5.4-fold, respectively, indicating that and functional activity of variants deserve further strong inhibition of CYP2C8 can be achieved almost studies. immediately after a single dose of gemfibrozil (Fig. 8, Studies on the role of CYP2C8 in drug metabolism Honkalammi et al., 2011b). It has also been demon- have demonstrated that it is the most important strated that the CYP2C8 inhibitory effect of gemfibrozil enzyme for the elimination of several drugs, such as persists virtually unchanged throughout the typical cerivastatin, montelukast, repaglinide, pioglitazone, 12-hour dosing interval of gemfibrozil (Tornio et al., and rosiglitazone, whose metabolism had been earlier 2008a). Thus, gemfibrozil can have a strong effect on thought to be attributed mainly to other enzymes. CYP2C8 substrates, irrespective of their half-life or CYP2C8 is crucial also for the biotransformation of time of daily dosing relative to gemfibrozil administra- daprodustat (GSK12788693), enzalutamide, dasabuvir, tion (Fig. 9; Table 10), making it an ideal in vivo probe and many other recently developed new drugs, and inhibitor of CYP2C8. The only caveat with gemfibrozil is overall, it contributes to the elimination of more than that it is also a moderate inhibitor of OATP1B1 and 100 drugs. CYP2C8 has a large active site cavity, and OAT3 and can therefore also increase the concentra- it can accommodate and also metabolize certain acyl tions of some drugs that are not or only partially glucuronides, such as desloratadine, diclofenac, and metabolized by CYP2C8 (Kyrklund et al., 2001, 2003; sipoglitazar glucuronides. Backman et al., 2002; Schneck et al., 2004; Neuvonen In vitro, there are several marker reactions for the et al., 2006; Whitfield et al., 2011). assessment of CYP2C8 activity, including paclitaxel Compared with gemfibrozil, all other clinically docu- 6-a-hydroxylation, amodiaquine deethylation, montelu- mented CYP2C8 inhibitors seem to be suboptimal. kast 36-hydroxylation, cerivastatin 6-hydroxylation Trimethoprim is relatively selective for CYP2C8, but (M-23 formation), rosiglitazone parahydroxylation, and as expected from its in vitro inhibitory effects (Wen pioglitazone M-IV formation. Each of these reactions et al., 2002), it is only a weak CYP2C8 inhibitor at has its strengths and weaknesses. The use of clinically clinically feasible doses (Niemi et al., 2004a,b; Hruska relevant drug concentrations in vitro is a prerequisite et al., 2005; Tornio et al., 2008b), and therefore it can for the estimation of the contribution of different only be regarded as a confirmatory CYP2C8 inhibitor in CYP enzymes in vivo. Gemfibrozil 1-O-b-glucuronide vivo. Clopidogrel is the second strongest CYP2C8 is potent and selective as a diagnostic inhibitor of 228 Backman et al.

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The authors thank Dr. Tommi Nyrönen for producing the docking Drug Metab Dispos 37:2359–2366. simulations and three-dimensional artwork in Fig. 2. Backman JT, Kajosaari LI, Neuvonen M, and Neuvonen PJ (2003) Trimethoprim Backman, Niemi, and Neuvonen have filed a patent application increases the plasma concentrations of cerivastatin by inhibiting its CYP2C8- mediated metabolism, Abstract in 8th European ISSX Meeting, Dijon, France concerning use of gemfibrozil as a pharmacokinetic enhancer. (April 27-May 1, 2003). Some of the information in Tables 1, 3 and 6 is based on the UW Backman JT, Kyrklund C, Kivistö KT, Wang JS, and Neuvonen PJ (2000) Plasma Metabolism and Transport Drug Interaction Database (DIDB), concentrations of active simvastatin acid are increased by gemfibrozil. Clin – – Pharmacol Ther 68:122 129. 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