Role of Cytochrome P450 2C8 in Drug Metabolism and Interactions

1. Anticancer Agents.

The antimicrotubule agent paclitaxel with a molecular weight of 853.9 g/mol is one of the largest substrates of CYP2C8. In vitro, paclitaxel is primarily metabolized by CYP2C8 to its main 6α-hydroxy metabolite and by CYP3A4 to 3′-phenyl-hydroxypaclitaxel, and the further metabolism of these metabolites results in the formation of 6α, 3′-p-dihydroxypaclitaxel (Cresteil et al., 1994, 2002; Harris et al., 1994; Kumar et al., 1994; Rahman et al., 1994). Paclitaxel 6α-hydroxylation is recommended by drug authorities as a marker reaction for CYP2C8 activity in vitro (EMA, 2012b; http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/ucm093664.htm), and it has been widely used in preclinical studies. In a mass balance study in patients, metabolites accounted for about 40% of the total systemic drug exposure, and the excreted 6α-hydroxypaclitaxel corresponded to almost one-third of the administered dose (Walle et al., 1995), suggesting that ∼30–40% of a paclitaxel dose is converted by CYP2C8 to 6α-hydroxy paclitaxel.

Cabazitaxel, a taxane approved in 2010, is also metabolized by CYP2C8 to a small extent in vitro (FDA, 2010a), whereas there is conflicting data regarding the role of CYP2C8 in the metabolism of docetaxel. One in vitro study demonstrated that docetaxel at high concentrations was metabolized by CYP2C8, but no enzyme kinetic parameters were determined (Komoroski et al., 2005). An earlier study, however, suggested that CYP2C8 is unable to accommodate docetaxel in its active site because of the absence of a side chain in the docetaxel molecule (Cresteil et al., 2002). The side chain, which is present in the paclitaxel molecule, is required for a correct orientation of it into the active site of CYP2C8.

The androgen receptor antagonist enzalutamide, indicated for treatment of castration-resistant prostate cancer, is mainly metabolized by CYP2C8 and CYP3A4/5 to enzalutamide M6 in vitro (FDA, 2012k). Then, M6 is further metabolized by CYP2C8 to the active metabolite N-demethyl enzalutamide (M2), which accounts for approximately 50% of the total drug exposure in plasma. CYP2C8 seems to be the predominant enzyme involved in enzalutamide pharmacokinetics also in vivo (section VI.C.5; Gibbons et al., 2015).

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

In addition, CYP2C8 participates to various degree to the metabolism of several other anticancer agents, as listed in Table 1.

2. Antidiabetic Agents.

The nonsulfonylurea insulin secretagogue repaglinide is metabolized primarily by CYP2C8 (Table 4) and CYP3A4, but it also undergoes direct glucuronidation by uridine-5′-diphosphoglucuronosyltransferase (UGT) 1A1 (Bidstrup et al., 2003; Kajosaari et al., 2005a,b; Gan et al., 2010). In addition, there is in vitro data suggesting that aldehyde dehydrogenase is involved in its metabolism (Säll et al., 2012). Moreover, repaglinide is a substrate of the hepatic uptake transporter organic anion-transporting polypeptide (OATP) 1B1 (Niemi et al., 2005b, 2011). The formation of the main metabolites of repaglinide, an oxidized dicarboxylic acid (M2) and, in particular, 3′-hydroxyl repaglinide (M4), is largely dependent on CYP2C8, whereas the less important aromatic amine metabolite (M1) is primarily formed by CYP3A4 (Bidstrup et al., 2003; Kajosaari et al., 2005a,b).

Pioglitazone, a thiazolidinedione peroxisome proliferator activated receptor (PPAR) γ agonist, is primarily metabolized by CYP2C8 in vitro, with smaller contributions by CYP3A4 and the extrahepatic CYP1A1 (Jaakkola et al., 2006c; FDA, 2013a). In vitro, CYP2C8 forms the pharmacologically active hydroxypioglitazone (M-IV) and ketopioglitazone (M-III) (Jaakkola et al., 2006c; Tornio et al., 2008b), which are the main metabolites in human serum with concentrations equal to or greater than those of the parent drug (Eckland and Danhof, 2000). In vivo studies support the central role of CYP2C8 in pioglitazone metabolism observed in vitro (section VI.C.2).

Rosiglitazone, another PPAR-γ agonist, is also a substrate of CYP2C8. In vitro, it undergoes CYP2C8-mediated p-hydroxylation and N-demethylation (Table 4), followed by sulfate and glucuronic acid conjugation (Baldwin et al., 1999; Kaspera et al., 2011; FDA, 2014a). CYP2C9 also participates in its metabolism to a minor extent (Baldwin et al., 1999). Rosiglitazone p-hydroxylation is recommended by the Food and Drug Administration (FDA) as a marker reaction for in vitro CYP2C8 activity (http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/ucm093664.htm). In vitro, troglitazone is metabolized by CYP2C8 to its quinone metabolite (M3) (Table 4) at a two- to eightfold higher rate than by CYP2C19, CYP3A4, and CYP2C9 (Yamazaki et al., 1999b). M3 is a minor metabolite of troglitazone, but it has been suggested to be responsible for the drug-induced hepatotoxicity associated with troglitazone use (Yamazaki et al., 1999b; Smith, 2003). Furthermore, the thiazolidinedione R483 is primarily metabolized by CYP2C8 and CYP2C19 in vitro (Bogman et al., 2010). CYP2C8 catalyzes the formation of the weakly active M1 metabolite (Table 4) and its further metabolism to M4, which is the main metabolite of R483 in plasma. In turn, CYP2C19 forms M2, which shows similar pharmacological activity as parent R483 (Bogman et al., 2010).

Additionally, CYP2C8 is involved, to a minor extent, in the metabolism of the sulfonylureas gliclazide, glyburide, and tolbutamide, the dipeptidyl peptidase 4 inhibitor sitagliptin, and the PPARα agonist sipoglitazar (Table 1; Relling et al., 1990; Srivastava et al., 1991; Veronese et al., 1993; Elliot et al., 2007; Vincent et al., 2007; Zharikova et al., 2009; Nishihara et al., 2012). Tolbutamide p-methyl hydroxylation has been used as a marker reaction for CYP2C8 activity in several in vitro studies. However, its Michaelis-Menten constant (K_m) for CYP2C8 is very high (>530 µM; Table 4), and it is effectively metabolized by CYP2C9 at lower concentrations.

3. Antimalarial Agents.

The 4-aminoquinoline derivative amodiaquine, widely used for treatment of malaria for more than 60 years, is a substrate of CYP2C8 (Li et al., 2002). It is also metabolized by the extrahepatic enzymes CYP1A1 and CYP1B1 to a minor extent (Li et al., 2002). Amodiaquine N-deethylation is a frequently used in vitro marker reaction for CYP2C8 because of its high affinity (K_m typically around 2 µM) and high turnover rate (Table 4). N-desethylamodiaquine, which is the main metabolite of amodiaquine, is assumed to be the main entity responsible for the pharmacological response to amodiaquine (Churchill et al., 1985; Mount et al., 1986).

Chloroquine, also a 4-aminoquinoline, is mainly metabolized to its N-desethyl metabolite by CYP2C8 (Table 4) and CYP3A4, with a small contribution by CYP2D6 in vitro (Kim et al., 2003; Projean et al., 2003a). Furthermore, CYP2C8 also seems to play a small role in the in vitro metabolism of the antimalarial agents dapsone, halofantrine, and piperaquine (Table 1; Baune et al., 1999; Winter et al., 2000; Lee et al., 2012c).

4. Lipid-lowering Drugs.

CYP2C8 participates to a small extent in the metabolism of several HMG-CoA reductase inhibitors (statins), but it has a major role for the biotransformation of cerivastatin. Cerivastatin is extensively metabolized in humans (Boberg et al., 1997; Mück, 2000). Parent cerivastatin (acid) is metabolized by CYP2C8 and CYP3A4, whereas cerivastatin lactone is predominantly metabolized by CYP3A4 (Boberg et al., 1997; Wang et al., 2002; Fujino et al., 2004). The formation of the major metabolite of cerivastatin, 6-hydroxycerivastatin (M-23), is primarily mediated by CYP2C8, whereas both CYP2C8 and CYP3A4 produce demethylcerivastatin (M1) (Wang et al., 2002; Kaspera et al., 2010). The notorious in vivo interaction between gemfibrozil and cerivastatin is discussed in section VI.C.4.

The parent simvastatin lactone is either oxidized by CYP3A4/5 or hydrolyzed to its acid form, which is pharmacologically active (Prueksaritanont et al., 1997, 2003). In human liver microsomes (HLM), the metabolism of simvastatin acid was catalyzed primarily (≥80%) by CYP3A4/5, with a smaller contribution (≤20%) by CYP2C8 (Prueksaritanont et al., 2003). Recombinant CYP2C8 formed all three simvastatin acid metabolites (M1-M3) observed in HLM (Table 4; Prueksaritanont et al., 2003). In vitro, fluvastatin is mainly metabolized by CYP2C9 into three metabolites, but CYP1A1, CYP2C8, CYP2D6, and CYP3A4 form 5-hydroxyfluvastatin (Fischer et al., 1999). Both atorvastatin (acid) and its lactone are primarily metabolized by CYP3A4 to their hydroxylated metabolites in vitro, but CYP2C8 is involved in the formation of p-hydroxy atorvastatin acid to a small extent (Jacobsen et al., 2000b). Furthermore, pitavastatin acid is metabolized by CYP2C9 and CYP2C8 in vitro, whereas its lactone is metabolized by CYP3A4 and CYP2D6 (Fujino et al., 2004).

5. Other Drugs.

Early in vitro studies concluded that the leukotriene receptor antagonist montelukast is mainly metabolized by CYP2C9 and CYP3A4 (Chiba et al., 1997), whereas the role of CYP2C8 was not evaluated. However, in vitro studies performed more than a decade later demonstrated that CYP2C8 is the key enzyme involved in the oxidative metabolism of montelukast (Filppula et al., 2011; VandenBrink et al., 2011). CYP2C8 catalyzes the main metabolic pathway of montelukast; formation of the pharmacologically active 36-hydroxymontelukast (M6), and its subsequent metabolism to the secondary metabolite M4, a dicarboxylic acid (Table 4). In addition, CYP2C8 forms 25-hydroxymontelukast (M3) (Filppula et al., 2011). These in vitro findings are in agreement with X-ray crystallography data, demonstrating a ligand-protein binding interaction between montelukast and CYP2C8 (Schoch et al., 2008). The montelukast molecule was positioned with its benzyl ring in close proximity to the heme iron of CYP2C8. The montelukast metabolites M3, M4, and M6 formed by CYP2C8 in vitro, all result from the oxidation of the benzyl ring of montelukast.

The novel prolyl hydroxylase inhibitor daprodustat (GSK1278863), an antianemic agent, is primarily metabolized by CYP2C8, with a smaller contribution by CYP3A4 in vitro (Johnson et al., 2014). It seems to be more sensitive than repaglinide to CYP2C8 inhibition by gemfibrozil in vivo (section VI.C.8; Johnson et al., 2014).

The novel nonstructural 5B nonnucleoside polymerase inhibitor dasabuvir is extensively metabolized by CYP2C8, with a small contribution by CYP3A (FDA, 2014g). CYP2C8 also plays an intermediate/small role in the metabolism of the nonstructural protein 3/4 A protease inhibitor paritaprevir and nonstructural protein 5A inhibitor ombitasvir (FDA, 2014g; Menon et al., 2015). However, no in vitro metabolism data have yet been published for these compounds.

The prostacyclin analog treprostinil is primarily metabolized by CYP2C8, followed by CYP2C9 in vitro (FDA, 2009b). Incubation of treprostinil with recombinant CYP2C8 for 15 minutes resulted in a 95% depletion of treprostinil concentrations, whereas only 22% was consumed by recombinant CYP2C9. CYP2C8 seems to be of importance in the in vivo pharmacokinetics of treprostinil (FDA, 2009b).

The sedative agent zopiclone is metabolized by CYP2C8 and CYP3A4 in vitro (Becquemont et al., 1999). In HLM, CYP2C8 was the main enzyme catalyzing N-demethylation of zopiclone, followed by CYP2C9 and CYP3A4. CYP2C8 also participated in the formation of N-oxide zopiclone, together with CYP3A4 (major) and CYP2C9 (Becquemont et al., 1999). However, in another in vitro study, montelukast (CYP2C8 inhibitor) and gemfibrozil (CYP2C8 and CYP2C9 inhibitor) had no effect on the elimination of clinically relevant concentrations of zopiclone (Tornio et al., 2006), supporting in vivo data showing a lack of effect of gemfibrozil on zopiclone concentrations in healthy subjects (section VI.C.8).

Based on in vitro studies, CYP2C8 likely plays an intermediate role in the elimination of 9cUAB30, alitretionin, amiodarone, cisapride, fenretinide, fluoxetine, irosustat, isotretionin, loperamide, olanzapine, olodaterol, propanoic acid, dronedarone, tazarotenic acid, verapamil, and vidupiprant (AMG 853) (Table 1). For instance, CYP2C8 catalyzes dealkylation of both enantiomers of the calcium channel blocker verapamil and its metabolite norverapamil (Busse et al., 1995; Tracy et al., 1999). Tazarotenic acid, the active moiety of the antipsoriatic agent tazarotene, is mainly metabolized by CYP2C8 and flavin-containing monooxygenases in vitro (Attar et al., 2003). When tazarotenic acid was incubated with 10 individual recombinant CYP enzymes, only CYP2C8 markedly catalyzed sulfoxidation, which is the main metabolic pathway of tazarotenic acid.

There is a vast amount of in vitro data suggesting that CYP2C8 may be of relevance in the metabolism of a number of other drugs (Table 1). For the majority of these compounds, the role of CYP2C8 in their in vivo elimination is likely to be small or negligible. However, for some drugs, the in vivo contribution of CYP2C8 to their metabolism cannot be estimated based on available information, and may, in fact, exceed 20%. Alternatively, CYP2C8 may become a determinant in their metabolism after inhibition of other enzymes important for their elimination. For instance, CYP2C8 is involved in the metabolism of seratrodast, a thromboxane A2 receptor antagonist in vitro (Kumar et al., 1997). The main metabolic pathway of seratrodast, 5-methylhydroxylation, is primarily catalyzed by CYP3A and CYP2C9, but CYP2C8 contributes to a small degree. However, CYP2C8 is a major contributor to seratrodast 4′-hydroxylation, a minor metabolic route (Kumar et al., 1997).

6. Glucuronide Metabolites.

Several glucuronide metabolites have been reported to undergo metabolism by CYP2C8, including clopidogrel acyl 1-β-d-glucuronide, desloratadine glucuronide, diclofenac acyl glucuronide, estradiol-17β-glucuronide, gemfibrozil 1-O-β glucuronide, licofelone 1-O-acyl glucuronide, Lu AA34893 carbamoyl glucuronide, 2-[[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy]-2-methylpropanoic acid (MRL-C) acyl glucuronide, and sipoglitazar β-1-O-acyl glucuronide (see Table 2 for references). Thus, CYP2C8 makes yet another exception to the old concept that drug metabolism is divided into sequential phase I and phase II reactions, i.e., functionalization and conjugation, respectively (Fig. 5).

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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-β-d-glucuronide and gemfibrozil 1-O-β glucuronide. CYP2C8 has been suggested to exist as a dimer (Hu et al., (2010), Schoch et al., (2004)). ER, endoplasmic reticulum.

Kumar et al. (2002) demonstrated the first example of CYP2C8-mediated metabolism of a glucuronide conjugate when they showed that the conversion of diclofenac acyl glucuronide to its 4′-hydroxy derivative is exclusively mediated by CYP2C8 in vitro. In 2005, it was reported that CYP2C8 is also able to directly catalyze the 2-hydroxylation of estradiol-17β-glucuronide in vitro (Delaforge et al., 2005). Docking of the glucuronide of estradiol into the crystal structure of CYP2C8 showed that the active site is large enough to inhabit the conjugate. Also the fetal CYP3A isoform CYP3A7 oxidized estradiol-17β-glucuronide, but CYP2C8 was five times more active than CYP3A7. However, CYP3A4 was not able to metabolize estradiol-17β-glucuronide (Delaforge et al., 2005). Moreover, the acyl glucuronide of the dual PPAR α/β agonist MRL-C was oxidized by CYP2C8 and to a minor extent by CYP3A4, but not by CYP2C9 (Kochansky et al., 2005). Furthermore, the main elimination pathway of licofelone, a dual inhibitor of cyclooxygenases 1 and 2 and 5-lipoxygenase, is glucuronidation of its carboxylic acid metabolite, followed by CYP2C8-catalyzed hydroxylation of the acyl glucuronide M1 to form the hydroxylated glucuronide M3 (Albrecht et al., 2008).

For two compounds, the formation of an unconjugated hydroxyl metabolite involves oxidation and subsequent deconjugation of a glucuronide metabolite. In vitro, the antidiabetic agent sipoglitazar was first glucuronidated to sipoglitazar β-1-O-acyl glucuronide (sipoglitazar-G1). Sipoglitazar-G1 was then metabolized to the main metabolite M-I by O-dealkylation by CYP2C8 and subsequent deconjugation (Nishihara et al., 2012). A similar finding was recently observed for desloratadine (Kazmi et al., 2015). The main metabolite of desloratadine, 3-hydroxydesloratadine, which is active, was formed via CYP2C8-mediated oxidation of desloratadine glucuronide and a deconjugation event (Kazmi et al., 2015). Thus, it seems that phase II metabolism occurs before phase I for these compounds (Fig. 5).

Also the glucuronide metabolites of clopidogrel, gemfibrozil, and Lu AA34893 are likely to be substrates of CYP2C8 (Ogilvie et al., 2006; Baer et al., 2009; Kazmi et al., 2010; Tornio et al., 2014). All three compounds are metabolism-dependent inhibitors of CYP2C8, as discussed in sections V.B and VI.B.

1. Drugs That Act as Inhibitors of Cytochrome P450 2C8.

Several drugs, drug metabolites, and other compounds have been found to inhibit CYP2C8 activity reversibly in vitro (Tables 6 and 7). In an in vitro screening of 209 commonly used drugs, 48 compounds exhibited greater than 50% inhibition of recombinant CYP2C8 activity at an inhibitor concentration of 30 µM (Walsky et al., 2005a). Montelukast, candesartan cilexetil, zafirlukast, clotrimazole, felodipine, and mometasone furoate inhibited CYP2C8 with concentrations supporting half of the maximal inhibition (IC₅₀) of ≤3 µM in recombinant CYP2C8 and HLM. In another study, the inhibition of CYP2C8 by montelukast was found to be competitive and selective, with reversible inhibition constants (K_i) ranging from 0.0092 to 0.15 µM, depending on the protein concentration used in the incubation (Walsky et al., 2005b). However, despite their strong inhibitory effect on CYP2C8 in vitro, neither montelukast nor zafirlukast affected the pharmacokinetics of CYP2C8 substrate drugs in vivo (Jaakkola et al., 2006b; Kajosaari et al., 2006b; Kim et al., 2007). The lack of in vivo effect is likely explained by their extensive plasma protein binding (>99%) (FDA, 1998; Dekhuijzen and Koopmans, 2002). Also the inhibition of CYP2C8 by candesartran cilexetil (prodrug of candesartan), clotrimazole, and mometasone furoate are probably not clinically relevant. The antifungal clotrimazole and anti-inflammatory mometasone furoate are topically applied and are therefore unlikely to cause interactions because of low systemic concentrations (Walsky et al., 2005a). In the systemic circulation, candesartan cilexetil is cleaved to candesartan, and, consequently, the likelihood of a drug interaction elicited by its prodrug is low. Furthermore, predictions suggested a relatively weak potential for drug-drug interactions due to CYP2C8 inhibition by felodipine. No drug interaction studies between felodipine and CYP2C8 substrates have been reported.

Trimethoprim, an antimicrobial agent, is a competitive inhibitor of CYP2C8 in vitro (Wen et al., 2002), with a K_i value typically around 10–30 µM in HLM (Table 6). The inhibition of CYP2C8 by trimethoprim seems to be rather selective, because it does not inhibit CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, or CYP3A4 at concentrations below 100 µM. In healthy subjects, trimethoprim has moderately increased the plasma exposure to several CYP2C8 substrate drugs (section VI.).

The flavonoid quercetin is one of the earliest in vitro inhibitors of CYP2C8 detected. In studies of paclitaxel metabolism, it was observed that quercetin, unlike CYP3A4 inhibitors, inhibited paclitaxel 6α-hydroxylation (Harris et al., 1994; Kumar et al., 1994). Because it was shown that the 6α-hydroxylation of paclitaxel is mediated by CYP2C8, it was evident that quercetin is an inhibitor of this enzyme (Rahman et al., 1994). Quercetin inhibits CYP2C8 competitively with a K_i of 0.03–20 µM (Table 7) and is classified as a "preferred" probe in vitro inhibitor of CYP2C8 by the FDA (http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/ucm093664.htm). However, quercetin is not selective for CYP2C8; it also inhibits CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 with IC₅₀ values of 3.1–47 µM (Obach, 2000; Zou et al., 2002). In vivo, quercetin at steady state did not affect the pharmacokinetics of rosiglitazone (Kim et al., 2005a).

The lipid-lowering drug gemfibrozil is a moderate, direct competitive inhibitor of CYP2C8 in vitro (Wen et al., 2001; Prueksaritanont et al., 2002; Wang et al., 2002), with a K_i range between 9.3 and 270 µM (Table 6). Gemfibrozil also inhibits CYP2C9 and CYP2C19 with K_i values of 5.8 and 24 µM, respectively, and CYP1A2 with a K_i of 82 µM (Wen et al., 2001). Moreover, it inhibits several drug transporters in vitro, most notably OATP1B1 (lowest reported K_i = 4 µM) and organic anion transporter (OAT) 3 (K_i = 6.8 µM) (Schneck et al., 2004; Shitara et al., 2004; Nakagomi-Hagihara et al., 2007). The strong interactions between gemfibrozil and CYP2C8 substrate drugs observed in vivo are mainly due to its glucuronide metabolite (Ogilvie et al., 2006). Gemfibrozil 1-O-β glucuronide affects CYP2C8 by mechanism-based inhibition (sections V.B and VI.B).

In vitro, the thiazolidinedione drugs pioglitazone, rosiglitazone, and troglitazone are potent, competitive inhibitors of CYP2C8 with IC₅₀ and K_i values of <40 µM (Table 6). However, e.g., pioglitazone does not affect CYP2C8 in vivo, likely because of its extensive protein binding (Kajosaari et al., 2006a).

The antiviral agents atazanavir and efavirenz inhibit CYP2C8 in vitro with K_i values of 2.1 and 4.8 µM, respectively [inhibitor concentration (I) to K_i ratios (I/K_i) = 3.7 and 6.3, respectively] (Table 6). In vivo, atazanavir has slightly affected the pharmacokinetics of rosiglitazone (FDA, 2015b). According to predictions, efavirenz may increase the area under the plasma concentration-time curve (AUC) of CYP2C8 substrates by more than fourfold at steady state, and such effects have been observed in vivo (German et al., 2007).

The immunosuppressant teriflunomide inhibits CYP2C8 with a very low K_i of 0.10–0.15 µM (FDA, 2012a). Thus, its estimated I/K_i ratio of 1.1 indicates that interactions between teriflunomide and CYP2C8 substrate drugs are likely, in agreement with in vivo findings (section IV.C.2).

Numerous protein kinase inhibitors inhibit CYP2C8 to various degrees in vitro (Table 6). However, for the most part, their in vivo inhibitory effects on CYP2C8 have not been studied. For those whose inhibition has been examined in a clinical setting, it seems to be rather small/moderate. For instance, axitinib inhibits CYP2C8 in vitro with a K_i of 0.2–0.5 µM (I/K_i = 0.3–0.9), but it did not alter paclitaxel plasma concentrations in patients (FDA, 2012f; Wang et al., 2014a). Similarly, cabozantinib is a noncompetitive inhibitor of CYP2C8 in vitro (K_i = 4.6 µM, I/K_i = 0.7), but the in vivo pharmacokinetics of rosiglitazone was not affected by cabozantinib (FDA, 2012c; Nguyen et al., 2015). The inhibition of CYP2C8 by pazopanib (K_i of 3.7 µM, I/K_i = 35) may be of clinical relevance (FDA, 2009d; Tan et al., 2014; Wang et al., 2014b). Nilotinib is a strong competitive CYP2C8 inhibitor in vitro (K_i = 0.1–0.9 µM, I/K_i = 4.8–43), but it also induces CYP2C8 (FDA, 2007c). Hence, a clinical interaction study with a CYP2C8 probe substrate has been recommended by the FDA to evaluate the in vivo effect on CYP2C8 activity by nilotinib. Similarly, an interaction study with a CYP2C8 substrate drug has also been recommended for regorafenib, which inhibits CYP2C8 with a K_i value of 0.6 µM in vitro (I/K_i = 13.5) (FDA, 2012i). In addition, sorafenib seems to be a strong CYP2C8 inhibitor in vitro with K_i values < 3 µM (I/K_i = 9–22) (Table 6), but the effect of sorafenib on CYP2C8 in vivo has not been evaluated.

Also several other anticancer agents exhibit inhibition of CYP2C8 in vitro (Table 6). For instance, the androgen receptor antagonist enzalutamide is both a substrate and inhibitor of CYP2C8 in vitro (K_i = 5.5 µM, I/K_i = 6.5) (FDA, 2012k). Vismodegib, an oral hedgehog pathway inhibitor, inhibits CYP2C8 in vitro with a K_i of 6.0 µM (I/K_i = 2.7), but vismodegib at steady state did not affect the pharmacokinetics of rosiglitazone (Wong et al., 2009; LoRusso et al., 2013).

The iron chelator deferasirox inhibits CYP2C8 with an IC₅₀ of 100 µM (I/K_i <0.01) (FDA, 2005a), but it has increased repaglinide AUC by 2.3-fold in vivo (Skerjanec et al., 2010). Febuxostat, a xanthine oxidase inhibitor, inhibits CYP2C8 in vitro with a K_i of 20 µM, suggesting that the inhibition may be of clinical relevance (I/K_i = 0.8). However, febuxostat at steady state had no effect on the concentrations of a single dose of rosiglitazone in vivo (Naik et al., 2012). Similarly, rosiglitazone pharmacokinetics was not affected by the platelet aggregation inhibitor vorapaxar in vivo (K_i = 0.86 µM, I/K_i = 0.06) (Chen et al., 2014; FDA, 2014l).

Sulfaphenazole, ketoconazole, diethyldithiocarbamate, methoxsalen (8-methoxypsoralen), and tranylcypromine, commonly used as in vitro inhibitors of CYP2C9, CYP3A4, CYP2E1, CYP2A6, and CYP2C19, respectively, also inhibit CYP2C8 in vitro (Table 6). For instance, sulfaphenazole is a strong competitive inhibitor of CYP2C9 with a K_i of 0.3 µM, whereas its K_i for CYP2C8 inhibition is 0.4–63 µM (Mancy et al., 1996; Hamman et al., 1997).

2. Natural Compounds.

A range of natural compounds have been tested for CYP2C8 inhibition in vitro, and inhibition parameters have been determined for several of them (Table 7). In a CYP inhibition screening of 10 herbal products commercially available in Australia, horsetail (Equisetum arvense) affected CYP2C8 with an IC₅₀ of 93.0 µg/ml (Sevior et al., 2010). The authors suggested that the inhibition of CYP2C8 by horsetail, which is used for treatment of urinary tract infections, cystitis, and prostate problems, may be clinically relevant (Sevior et al., 2010). In another in vitro study, six herbal supplements inhibited CYP2C8 to various degree, but the inhibition by cranberry powder (IC₅₀ = 24.7 µg/ml) and saw palmetto (IC₅₀ = 15.4 µg/ml) were suggested to potentially be of clinical significance (Albassam et al., 2015).

Among five CYP enzymes tested, CYP2C8 was most sensitive to inhibition by green tea extract in HLM (IC₅₀ = 4.5 µg/ml) (Misaka et al., 2013). The major catechin in green tea, (−)-epigallocatechin-3-gallate, inhibited CYP2C8 with a K_i of 6.8 µM, indicating that green tea intake may affect CYP2C8 in vivo. It has been reported that 15% of Japanese older than 40 years of age consume more than 1.8 l of green tea daily, corresponding to a daily epigallocatechin-3-gallate intake of 540–720 mg (Misaka et al., 2013).

1. In Vitro Versus In Vivo.

In vitro, the parent gemfibrozil is a moderately potent competitive inhibitor of CYP2C9 (K_i value of 5.8 µM; Wen et al., 2001), but it is over 10 times less potent as inhibitor of CYP2C8 (K_i value of 75 µM, Wang et al., 2002; K_i 87 µM, Prueksaritanont et al., 2002; Table 6). Gemfibrozil in concentrations up to 1,000 µM has no effect on CYP3A4 activity (midazolam 1′-hydroxylation) (Backman et al., 2000), but it is rather potent as an inhibitor of the OATP1B1 transporter, with a K_i value ranging in different studies from 4 to 31.7 µM (Schneck et al., 2004; Yamazaki et al., 2005; Hirano et al., 2006; Nakagomi-Hagihara et al., 2007).

In healthy volunteers, gemfibrozil (600 mg twice daily) slightly (by 23%) increased the AUC of a CYP2C9 substrate drug glimepiride (Niemi et al., 2001) but did not increase the exposure to racemic warfarin (Lilja et al., 2005). Gemfibrozil even caused a small but statistically significant decrease (−11%) in the AUC of the CYP2C9 substrate S-warfarin. These results strongly suggest that gemfibrozil is not a meaningful inhibitor of CYP2C9 in vivo in humans.

In vivo gemfibrozil is glucuronidated by the UGT 2B7 enzyme to gemfibrozil 1-O-β-glucuronide. The benzylic oxidation of the glucuronide by CYP2C8 leads to haem alkylation and irreversible inactivation of CYP2C8 (Baer et al., 2009; Jenkins et al., 2011). In HLM, the k_inact value of CYP2C8 by gemfibrozil 1-O-β-glucuronide has been 0.21 1/min and K_I 20–52 µM (Ogilvie et al., 2006). The glucuronide metabolite is also a competitive inhibitor of OATP1B1 (OATP2) transporter (K_i 24 µM; Shitara et al., 2004). On the basis of clinical studies on the dose/time dependency of the effect of gemfibrozil on the pharmacokinetics of repaglinide and statistical models of enzyme and transporter inhibition, it has been estimated that the in vitro mechanism-based inhibition of CYP2C8 by gemfibrozil 1-O-β-glucuronide manifests into a strong and long-lasting inhibition of CYP2C8 at typical clinical doses of gemfibrozil (Backman et al., 2009; Honkalammi et al., 2011a,b). In addition, the OATP1B1 inhibitory effect of the glucuronide can lead to an up to ∼50% transient inhibition of OATP1B1 in vivo. These effects are the main explanation to the effects of gemfibrozil on CYP2C8 and OATP1B1 substrates (Honkalammi et al., 2012). Similar estimations have been obtained with physiologically based pharmacokinetic modeling in a recent publication (Varma et al., 2015).

2. Gemfibrozil Dose Versus CYP2C8 Inhibition.

Single oral doses of gemfibrozil, i.e., 30, 100, 300, or 900 mg ingested 1 hour before repaglinide, increased the AUC of repaglinide in a dose-dependent manner 1.8-, 4.5-, 6.7-, and 8.3-fold compared with placebo, respectively (Fig. 8; Honkalammi et al., 2011a). Also after multiple doses of gemfibrozil (30, 100, or 600 mg twice daily for 5 days), the exposure to repaglinide increased dose dependently, but the greatest AUC increase did not exceed that observed after the single 900 mg gemfibrozil dose (Honkalammi et al., 2012). Thus, the maximum inhibition of CYP2C8 can be achieved by a single 900-mg dose of gemfibrozil (Fig. 9). Gemfibrozil in doses of 100 mg twice daily at steady state causes an about 95% inhibition of CYP2C8 (Fig. 9), and in doses of 10 mg twice daily, it causes an about 50% inhibition (Honkalammi et al., 2011a). The fraction of a small 0.25-mg dose of repaglinide metabolized by CYP2C8 is about 80–90%. However, because repaglinide is metabolized to some extent also by CYP3A4, the relative role of CYP2C8 and CYP3A4 in the biotransformation of repaglinide can depend on its dose and plasma concentrations as well as on individual pharmacogenetic factors (Bidstrup et al., 2003; Kajosaari et al., 2005a; Säll et al., 2012).

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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, AUC_{0-3 hour} data are presented. For all other compounds, AUC_0-∞ data are presented. A fold change of 1 refers to no effect of gemfibrozil on exposure. References are given in the text.

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Fig. 9.

Predicted effects of different gemfibrozil doses on CYP2C8-mediated drug metabolism. Predicted fold increase in the AUC_0–∞ of a drug metabolized by CYP2C8, when the fraction of the substrate drug metabolized by CYP2C8 (f_m,CYP2C8) varies between 50 and 99% (A), and the CYP2C8 activity remaining (B) after a gemfibrozil dose ranging from 0 to 600 mg twice daily in steady-state conditions. Modified from Honkalammi et al. (2012). AUC, area under the concentration-time curve.

3. Onset and Duration of CYP2C8 Inhibition by Gemfibrozil.

The onset and duration of CYP2C8 inhibition by gemfibrozil have been studied in healthy volunteers using the gemfibrozil-repaglinide interaction as a model. Single 600-mg doses of gemfibrozil ingested 0, 1, 3, or 6 hours before repaglinide (0.25 mg) increased the geometric mean AUC of repaglinide 5.0-, 6.3-, 6.6-, and 5.4-fold, respectively (Fig. 8). The C_max of the CYP2C8-mediated repaglinide M4-metabolite was 1.0-, 0.10-, 0.06-, and 0.09-fold compared with control phase, respectively (Honkalammi et al., 2011b). These results indicate that the strong inactivation of CYP2C8 occurs rapidly, being evident already within 1 hour after oral dosing of gemfibrozil.

When repaglinide was ingested 1, 24, 48, or 96 hours after discontinuation of a gemfibrozil treatment (600 mg twice daily for 3 days), the AUC of repaglinide was 7.6-, 2.9-, 1.4-, and 1.0-fold compared with the control phase, respectively (Backman et al., 2009). These findings confirmed and extended the previous findings, which had shown that the inhibitory effect of gemfibrozil persists for at least 12 hours after its ingestion (Tornio et al., 2008a). As the half-lives of gemfibrozil and its glucuronide are very short (about 1–2 hours), these findings convincingly demonstrate that the effect of gemfibrozil on repaglinide pharmacokinetics is based on irreversible mechanism-based inhibition of CYP2C8. A several-fold increase in repaglinide AUC was evident even at very low plasma concentrations of gemfibrozil 1-O-β-glucuronide, which are less than 1% of its peak concentrations. The results also showed that full CYP2C8 activity recovers gradually within 3–4 days after cessation of the clinically used therapeutic doses of 600 mg twice daily gemfibrozil.

4. Quantification of CYP2C8-Mediated Drug Interactions in Humans.

Interactions caused by a combination of two or more drugs, which inhibit, in addition to CYP2C8, also some other crucial enzyme or transporter, can increase exposure to a CYP2C8 substrates much more than is the sum of their separate effects causing a classic potentiation phenomenon. Thus, e.g., exposure to repaglinide is increased only slightly by itraconazole alone (1.4-fold), greatly by gemfibrozil alone (8.1-fold), and drastically (19.4-fold) by their combination (Fig. 10; Niemi et al., 2003b). The quantitative rationalization of gemfibrozil-drug interactions and consideration of transporter-enzyme interplay have been dealt quite recently by Varma et al. (2015).

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Fig. 10.

Effects of gemfibrozil (Gem), itraconazole (Itra), or their combination (Gem + itra) on the exposure (area under the plasma drug concentration-time curve) to repaglinide, loperamide, montelukast, and pioglitazone. A fold increase of 1 refers to no effect of inhibitors on drug exposure. References are given in Table 10 and in the text.

1. Repaglinide.

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

The antimicrobial drug trimethoprim (160 mg twice 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 mediated by formation of the clopidogrel acyl-β-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 of clopidogrel and repaglinide resulted in an enhanced blood glucose-lowering effect of repaglinide. The concomitant use of repaglinide and clopidogrel is now 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 contribution of each mechanism to the increase in repaglinide exposure has not been established (FDA, 2012a). Of note, teriflunomide is the active metabolite of leflunomide and its plasma concentrations following leflunomide 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).

2. Other Oral Antidiabetic Drugs.

The European product information of Actos (pioglitazone) stated earlier (e.g. 2004) that metabolism of pioglitazone occurs predominantly via CYP3A4 and CYP2C9 (Jaakkola 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. (2006c) showed that pioglitazone is metabolized mainly by CYP2C8 and to lesser extent by CYP3A4, whereas CYP2C9 is not significantly involved in pioglitazone elimination. In healthy volunteers, gemfibrozil raised the mean AUC of pioglitazone 3.2-fold (range 2.3-fold to 6.5-fold) and its elimination half-life 2.7-fold, but itraconazole had no effect on pioglitazone and did not alter the effect of gemfibrozil on its pharmacokinetics (Jaakkola et al., 2005). In two other studies, gemfibrozil increased the mean AUC of pioglitazone 3.4-fold (Deng et al., 2005) and 4.3-fold (range 1.3-fold to 12.1-fold) (Aquilante et al., 2013a). CYP2C8 genotype influences the relative change in pioglitazone exposure after gemfibrozil administration. Thus, CYP2C8*3 carriers had a greater mean increase by gemfibrozil in pioglitazone AUC (5.2-fold) compared with CYP2C8*1 homozygotes (3.3-fold) (Aquilante et al., 2013a). Trimethoprim (160 mg twice daily) raised in healthy volunteers the AUC of pioglitazone by 1.4-fold and had opposite effects on pioglitazone pharmacokinetics compared with the effects of CYP2C8*3 allele during the placebo phase (Tornio et al., 2008b).

The AUC of rosiglitazone was raised in healthy volunteers by gemfibrozil by 2.3-fold (Niemi et al., 2003a). In another study, trimethoprim (160 mg twice daily) increased rosiglitazone AUC by 1.4-fold and reduced the formation of N-demethylrosiglitazone (Niemi et al., 2004a). The effect of trimethoprim (200 mg twice daily) on rosiglitazone pharmacokinetics was confirmed by Hruska et al. (2005), who also demonstrated the competitive inhibition of rosiglitazone p-hydroxylation by trimethoprim in vitro. Atazanavir (400 mg once daily) increased the AUC of a single dose of rosiglitazone by 1.4-fold (FDA, 2015b).

The AUC of nateglinide was increased only by 1.5-fold by 3 days' pretreatment with therapeutic doses of both gemfibrozil and itraconazole (Niemi et al., 2005a). Thus, neither CYP2C8 nor CYP3A4 has a substantial significance to the pharmacokinetics of nateglinide. Gemfibrozil increased also the AUC of the dipeptidyl peptidase inhibitor sitagliptin by 1.5-fold (Arun et al., 2012). However, the gemfibrozil-sitagliptin interaction seems to be mainly mediated by inhibition of the renal OAT3, with a minor contribution by CYP2C8.

If gemfibrozil, clopidogrel, or other inhibitors of CYP2C8 will be combined with pioglitazone or rosiglitazone, blood glucose levels, symptoms of hypoglycemia, and other potential adverse effects (e.g., fluid retention) should be monitored closely and the doses be modified as needed. As shown for the gemfibrozil-pioglitazone and gemfibrozil-repaglinide interactions (Tornio et al., 2008a; Aquilante et al., 2013a), interactions may be stronger in CYP2C8*3 carriers than in CYP2C8*1 homozygotes.

3. Amodiaquine.

Although amodiaquine N-deethylation is a widely used marker reaction for CYP2C8 activity in vitro, the sensitivity of amodiaquine to CYP2C8 inhibition is poorly characterized in humans. Amodiaquine is rapidly and extensively metabolized by CYP2C8 to active desethylamodiaquine, which has a long half-life of 9–18 days. In healthy subjects, trimethoprim and efavirenz have been reported to increase the AUC of amodiaquine by 1.6- and 1.8-fold, respectively, and to reduce that of desethylamodiaquine by 12 and 26%, respectively (Soyinka et al., 2013; Akande et al., 2015). In an earlier study, efavirenz raised the AUC of amodiaquine in two healthy subjects by 2- to 4-fold and decreased the AUC of desethylamodiaquine by 24 and 8.5% (German et al., 2007). In both of these subjects, marked elevation of hepatic transaminase levels occurred several weeks after stopping the 3 days' combined use, forcing premature discontinuation of the interaction study. The dramatic, delayed hepatotoxicity warrants great care in combination of any CYP2C8 inhibitor with amodiaquine.

4. Statins.

Cerivastatin was initially considered as a safe statin because of its dual biotransformation routes, mediated both via CYP3A4 and CYP2C8 (Mück, 1998; 2000). However, it soon became obvious that cerivastatin greatly increased the incidence of fatal rhabdomyolysis, particularly when taken along with gemfibrozil (Staffa et al., 2002). Consequently, cerivastatin was withdrawn from the market in 2001, only 3 years after its launch. Despite the “dual metabolic pathway” and supposed "low propensity for drug interactions" (Mück et al., 1998; Mück, 2000), the elimination of cerivastatin relied predominantly on CYP2C8. In healthy volunteers, gemfibrozil (600 mg twice daily) raised the AUC of the parent cerivastatin (acid) by 5.6-fold, the AUC of cerivastatin lactone by 4.4-fold, and that of the CYP3A4-dependent metabolite M-1 by 4.35-fold, whereas gemfibrozil decreased the AUC of the CYP2C8-dependent metabolite M-23 by 78% (Fig. 11; Backman et al., 2002). The increased exposure to cerivastatin, to its lactone, and to M-1 and the reduced formation of the CYP2C8-dependent metabolite revealed the strong CYP2C8 inhibitory effect of gemfibrozil. In addition to irreversible inhibition of CYP2C8 by gemfibrozil 1-O-β-glucuronide, inhibition of the hepatic OATP1B1 may contribute to the gemfibrozil-cerivastatin interaction (Ogilvie et al., 2006; Shitara et al., 2004; Tamraz et al., 2013).

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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).

Interestingly, about 10 years after the withdrawal of cerivastatin, it was found that in addition to gemfibrozil, also concomitant use of clopidogrel was strongly associated with cerivastatin-induced rhabdomyolysis, with an odds ratio of ∼30 (48 when gemfibrozil users were excluded) (Floyd et al., 2012). Recently, Tornio et al. (2014) showed that glucuronidation converts clopidogrel to a strong time-dependent inhibitor of CYP2C8, clopidogrel acyl-β-d-glucuronide. The formation of this metabolite leads to uninterrupted inhibition of CYP2C8 during clopidogrel treatment and explains the increased risk of rhabdomyolysis during concomitant use of cerivastatin and clopidogrel (Tornio et al., 2014). Also trimethoprim increased the AUC of cerivastatin (by 1.4-fold) and its lactone (1.5-fold) (Backman et al., 2003). Because cerivastatin has been withdrawn from the market, its interactions are no more of direct clinical relevance. However, they are examples of clinically important challenges in drug development and have been of paramount importance in understanding the significance of CYP2C8 in drug metabolism.

Interestingly, cerivastatin and repaglinide have pharmacokinetic similarities. Both drugs are substrates of CYP2C8, CYP3A4, and OATP1B1. The CYP3A4 inhibitor itraconazole has raised their AUC only slightly, i.e., by 1.4-fold (repaglinide), 1.15-fold (cerivastatin acid), and 1.8-fold (cerivastatin lactone), whereas gemfibrozil has raised their AUC values much more, i.e., by 8.1-fold (repaglinide), by 5.6-fold (cerivastatin), and by 4.4-fold (cerivastatin lactone) (Kantola et al., 1999; Backman et al., 2002; Niemi et al., 2003b). Because the combination of a CYP3A4 inhibitor and a CYP2C8 inhibitor caused a drastic increase in repaglinide AUC (by 19.4-fold; Niemi et al., 2003b), it is reasonable to assume that also the exposure to cerivastatin acid and to its more lipophilic lactone form have raised even more by gemfibrozil —or clopidogrel—if the patients had been using also CYP3A4 inhibiting drugs. However, there seems to be no studies on the effect of CYP2C8 and CYP3A4 inhibitor combinations on the plasma concentrations of cerivastatin.

Gemfibrozil raises the AUC of nearly all statin acids, including simvastatin acid, lovastatin acid, atorvastatin, pravastatin, rosuvastatin, and pitavastatin (Neuvonen et al., 2006). However, the role of CYP2C8 in some gemfibrozil-statin interactions seems to be limited or nonexistent. They are mainly mediated by inhibition of OATP1B1, OAT3, or other transporters (Shitara et al., 2004; Neuvonen, 2010; Niemi et al., 2011).

5. Anticancer Drugs.

Most anticancer drugs have a narrow therapeutic range. Although paclitaxel is a well-established CYP2C8 probe in vitro, its interactions with CYP2C8 inhibitors and inducers have not been widely studied in humans. Lapatinib and pazopanib are relatively strong inhibitors of CYP2C8, and they have raised the AUC of paclitaxel up to 1.8-fold (Tan et al., 2014). In a case report, the only clopidogrel user in a cohort of 93 ovarian carcinoma patients treated with paclitaxel had the second lowest clearance of unbound paclitaxel in the cohort. She was hospitalized three times because of severe paclitaxel toxicity (Bergmann et al., 2015).

Gemfibrozil has raised the AUC of the androgen receptor antagonist enzalutamide by 4.3-fold, and itraconazole raised it by 1.4-fold compared with control (Gibbons et al., 2015). These results agree well with the in vitro findings that CYP2C8 is the predominant enzyme in the elimination of enzalutamide. The composite exposure of enzalutamide and its active metabolite was raised by 2.2-fold by gemfibrozil and by 1.3-fold by itraconazole. A reduction of the enzalutamide dose by about 50% is recommended when gemfibrozil is used concomitantly. There are no published studies on the effect of clopidogrel on enzalutamide pharmacokinetics. However, a close follow up and reduction of enzalutamide dose can be recommended also in their possible coadministration. It should also be noted that combined inhibition of CYP2C8 and CYP3A4 can cause a greater increase in enzalutamide + metabolite AUC. Enzalutamide itself is an inhibitor of CYP2C8 and may moderately raise the exposure to its substrate drugs, e.g., pioglitazone AUC by 20% (Gibbons et al., 2015).

Gemfibrozil did not affect the AUC of imatinib after a single imatinib dose but reduced the AUC of N-demethylimatinib by 48%, indicating a significant participation of CYP2C8 in the metabolism of imatinib in humans (Filppula et al., 2013b). After a single dose, imatinib seems to be mainly metabolized by CYP3A4, but the fraction of imatinib metabolized by CYP3A4 decreases after its multiple doses because of autoinhibition of the CYP3A4-mediated metabolism of imatinib (Filppula et al., 2012, 2013a). This autoinhibition is likely to increase the relative role of CYP2C8 in imatinib elimination and its sensitivity to interactions caused by CYP2C8 inhibitors during long-term treatment. According to pharmacokinetic simulations, imatinib exposure may raise up to twofold at steady state if a strong CYP2C8 inhibitor is given concomitantly with imatinib (Filppula et al., 2013b).

In melanoma patients, gemfibrozil increased the AUC of the CYP3A4 and CYP2C8 substrate dabrafenib by 1.5-fold and ketoconazole increased it by 1.7-fold (Suttle et al., 2015). It is probable that a combined administration of CYP2C8 inhibitors and CYP3A4 inhibitors with dabrafenib can increase its exposure more than does either of these inhibitors alone. In addition to paclitaxel, dabrafenib, and imatinib, some other anticancer drugs are metabolized by CYP2C8 (Table 1). However, their interactions with CYP2C8 inhibitors have not been characterized in humans. Considering the narrow therapeutic range of many anticancer drugs, close follow up for possible adverse effects is warranted if gemfibrozil, clopidogrel, trimethoprim, or other inhibitors of CYP2C8 are used with paclitaxel or other anticancer drugs metabolized by CYP2C8.

6. Antiviral Drugs.

Gemfibrozil (600 mg twice daily) has increased the AUC of the antihepatitis C drug dasabuvir about 11-fold and their concomitant use is contraindicated (Menon et al., 2015). It is reasonable to assume that also other potent inhibitors of CYP2C8 such as clopidogrel increase greatly the exposure to dasabuvir, and their use together should be avoided or the dose of dasabuvir be reduced markedly. It can be speculated that savings could be achieved by using small amounts of expensive dasabuvir (about one-tenth of normal dose) with small doses (e.g., 100 mg) of gemfibrozil. This should lead to similar plasma concentrations of dasabuvir as those achieved by normal dasabuvir doses administered without inhibitor of CYP2C8. Also some other new antiviral drugs are partially metabolized by CYP2C8, but their susceptibility to interact with drugs affecting CYP2C8 activity in humans needs further studies.

7. Antiasthmatic Drugs.

In healthy volunteers. gemfibrozil raised the AUC of montelukast 4.5-fold and its elimination half-life 3.0-fold (Karonen et al., 2010). Gemfibrozil reduced the AUC of the secondary M4 metabolite of montelukast by more than 90%. In another study, gemfibrozil alone raised the AUC of montelukast 4.3-fold, itraconazole had no significant effects, and the effects of the gemfibrozil-itraconazole combination on montelukast pharmacokinetics did not differ from those of gemfibrozil alone (Karonen et al., 2012). These findings indicate that CYP2C8 but not CYP3A4 is important in the pharmacokinetics of montelukast. In contrast to the effect of gemfibrozil on montelukast, the pharmacokinetics of zafirlukast is not affected by gemfibrozil (Karonen et al., 2011), although both of these cysteinyl leukotriene receptor antagonists are potent in vitro inhibitors of CYP2C8 (Walsky et al., 2005a).

Montelukast has a relatively large safety margin, and the clinical significance of its interactions with CYP2C8 inhibitors seems to be limited. However, neuropsychiatric symptoms have developed in a woman with HIV infection when montelukast was added to her therapy containing the CYP2C8 inhibitor efavirenz (Ibarra-Barrueta et al., 2014). She had used efavirenz, emtricitabine, and tenofovir disoproxil fumarate for years with good tolerance until montelukast was started for asthma. Shortly thereafter unbearable symptoms appeared, consisting of disturbed sleep, vivid dreams and irritability, confusion, and concentration difficulties. After 2 months of concomitant use, montelukast was withdrawn and the psychiatric symptoms completely disappeared. This case report indicates that adverse effects can develop when these drugs are used together, although the mechanism of adverse effects is not fully clear.

8. Other Substrate or Inhibitor Drugs.

Gemfibrozil raised in healthy volunteers the AUC of loperamide 2.1-fold, itraconazole raised it 3.8-fold, and the gemfibrozil-itraconazole combination raised loperamide AUC 12.6-fold compared with placebo phase (Niemi et al., 2006). This finding strongly suggests that gemfibrozil can markedly increase the loperamide exposure in subjects who are using potent inhibitors of CYP3A4, i.e., when another important metabolic route is blocked. Administration of cotrimoxazole (trimethoprim + sulphamethoxazole) has increased the AUC of loperamide by 1.9-fold (Kamali and Huang, 1996). Also some other opioids, e.g., buprenorphine, are CYP2C8 substrates (Table 1). However, there seem to be no published studies on their possible interaction with gemfibrozil or other CYP2C8 inhibitors.

Gemfibrozil raised the AUC of the prolyl hydroxylase inhibitor agent daprodustat (GSK1278863) 18.6-fold (Johnson et al., 2014). This result together with in vitro studies indicates the crucial significance of CYP2C8 in its pharmacokinetics. CYP2C8 inhibitors should not be used with this erythropoiesis-stimulant agent or its dose needs to be reduced very greatly. On the other hand, at least theoretically, it could be possible to take advantage of this interaction in a product containing very small doses of daprodustat and gemfibrozil.

In healthy volunteers, gemfibrozil raised only slightly the AUC of R-ibuprofen, by 1.3-fold, after the ingestion of racemic ibuprofen (Tornio et al., 2007). In vitro CYP2C8 participates in the metabolism of zopiclone (Becquemont et al., 1999). In humans, however, gemfibrozil did not increase the AUC of the parent zopiclone but moderately (2-fold and 1.2-fold) increased the AUC of its potentially active metabolites (Tornio et al., 2006). Also many other drugs are substrates of CYP2C8 in vitro, but their concomitant administration with gemfibrozil has not appreciably increased their AUC, suggesting that the CYP2C8-mediated biotransformation is of limited significance to their total clearance (Table 1).

Many compounds are moderate inhibitors of CYP2C8 in vitro, but their concomitant ingestion with repaglinide or other CYP2C8 substrates does not raise exposure to these substrates in humans. The reason for the apparent discrepancy between the in vitro and in vivo results can be, for example, their low potency as CYP2C8 inhibitors or their high protein binding in vivo (e.g., montelukast).

Some parent drugs such as gemfibrozil and clopidogrel are relatively weak inhibitors of CYP2C8 in vitro, but they are metabolized in vivo to glucuronide metabolites, which are potent CYP2C8 inhibitors. In general, negative interaction results with gemfibrozil in vivo exclude a clinically meaningful interaction mediated by CYP2C8 inhibition. On the other hand, increased exposure to a victim drug by gemfibrozil does not yet indicate that CYP2C8 has a significant role in its metabolism because there may be other mechanisms mediating the observed interaction.

Patients often concomitantly use different drugs that together inhibit several CYP enzymes, e.g., CYP1A2, CYP2C8, CYP2C9, CYP2B6, CYP2D6, or CYP3A4. The combined inhibition of two or more of these enzymes often results in patients in a stronger interaction than is caused by inhibition of a single enzyme in healthy volunteer studies. This aspect together with other causes of interindividual variation should be taken into consideration when the results of experimental interaction studies in healthy volunteers are translated into the clinic.

1. Rifampin (Rifampicin).

Rifampin (600 mg/day), given for several days, has decreased the plasma exposure to repaglinide by 31–80% depending on the time interval from the last rifampin dose to repaglinide ingestion (Table 11; Niemi et al., 2000; Hatorp et al., 2003; Bidstrup et al., 2004). The time interval affects the extent of interaction because rifampin is also a competitive inhibitor of OATP1B1, CYP2C8 and CYP3A4 (Kajosaari et al., 2005a; Varma et al., 2013). Interestingly, intake of St John’s Wort for 14 days has had no significant effect of the pharmacokinetics of repaglinide (Fan et al., 2011).

Rifampin has also reduced the concentrations of the thiazolidinediones pioglitazone and rosiglitazone. Rifampin caused a substantial (54%) decrease in the AUC of pioglitazone and increased the ratios of metabolite M-IV to pioglitazone and of M-III to pioglitazone in urine by 98 and 95% (Jaakkola et al., 2006a). Similarly, rifampin reduced the mean AUC of rosiglitazone by 54% and increased the formation of N-demethylrosiglitazone (Niemi et al., 2004a). In Korean men, rifampin decreased rosiglitazone AUC by 65% (Park et al., 2004). Addition of tuberculosis treatment, containing rifampin, to treatment of a woman with type 2 diabetes caused her to lose glycemic control, demonstrating potential clinical significance of the rifampin-rosiglitazone interaction (Pimazoni, 2009).

1. General Aspects.

Comprehensive in vitro studies to investigate the roles of different CYP enzymes in the metabolism of a (new) drug and to uncover its potential for causing inhibition or induction of drug metabolism are typically conducted already during the early preclinical phases of drug development. The results from these studies are then used for in vitro-in vivo extrapolations, to anticipate factors affecting the clearance of the drug as well as its potential to act as a perpetrator of pharmacokinetic drug interactions, i.e., to affect the clearance of other drugs. The prerequisite for accurate extrapolations is that in vitro investigations are conducted with care and are sufficiently comprehensive, avoiding the many pitfalls of in vitro studies, understanding the many limitations of the different approaches, and covering complex issues, such as the potential for autoinhibition or -induction. Yet it should be understood that accurate extrapolations are not possible without some clinical pharmacokinetic data at the relevant dose of the investigational drug.

The general aspects as well as the potential pitfalls of in vitro studies and extrapolations are well covered by many excellent review articles and guidelines (Houston and Galetin, 2008; Pelkonen et al., 2008; Grimm et al., 2009; EMA, 2012b; Pelkonen, 2015; http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/ucm093664.htm). Therefore, this review focuses on issues that are directly related to CYP2C8, i.e., in vitro methods used for measurement of CYP2C8 activity (e.g., to test the potential of the investigational drug to inhibit CYP2C8 activity) and reaction phenotyping (does CYP2C8 metabolize the drug) and in vivo studies to characterize the drug interaction potential of the new drug (either as a perpetrator or victim drug).

2. Assessment of CYP2C8 Activity In Vitro.

Specific assessment of CYP2C8 activity is necessary, in particular when studying the potential of a drug to cause inhibition of CYP2C8 but also when using a panel of HLM for reaction phenotyping using the correlation approach. An ideal in vitro probe substrate is selective/specific, has a sufficient turnover, follows Michaelis-Menten kinetics, and is not sensitive to experimental conditions. There are several useful, selective probe substrates to study CYP2C8 in vitro, including paclitaxel, amodiaquine, montelukast, rosiglitazone, pioglitazone, and cerivastatin, each having its specific strengths and weaknesses (Tables 12 and 13).

Paclitaxel 6-α-hydroxylation is the prototypical marker reaction for CYP2C8 (Rahman et al., 1994; Sonnichsen et al., 1995). It is highly selective for CYP2C8, but the metabolic turnover is fairly low, often leading to relatively long incubation times, which may lead to significant inhibitor metabolism/depletion during the incubation (Table 13). This may partly explain why paclitaxel seems to be less sensitive to competitive CYP2C8 inhibitors than most other CYP2C8 marker substrates (VandenBrink et al., 2011). In particular, long incubation times should be avoided when studying the potential for time-dependent or mechanism-based inhibition in systems based on a preincubation step, because inactivation proceeding during the incubation may decrease the sensitivity of the experimental system to detect inactivation.

Amodiaquine metabolism to N-desethylamodiaquine is probably the second most used CYP2C8 marker reaction. It is well characterized and highly selective for CYP2C8 and has a high turnover (Li et al., 2002), allowing for short incubation times. Overall, it seems to have no major drawbacks in in vitro use.

Few years ago, montelukast, a selective competitive inhibitor of CYP2C8, was shown to be a potential CYP2C8 marker substrate, because its 36-hydroxylation (M6 formation) is mediated primarily by CYP2C8 with a minor contribution by CYP2C9 (Filppula et al., 2011). In a successive study, montelukast 36-hydroxylation proved to be a sensitive and useful reaction to investigate CYP2C8 inhibition in vitro (VandenBrink et al., 2011). One of the weaknesses of montelukast is that it is highly susceptible to microsomal protein binding, necessitating careful standardization of incubation conditions (Walsky et al., 2005b).

Of the other potential marker reactions, cerivastatin 6-hydroxylation (M-23 formation) seems to be highly specific for CYP2C8 (Wang et al., 2002; Shitara et al., 2004). In addition, the hydroxylations of rosiglitazone (p-hydroxylation) (Baldwin et al., 1999) and pioglitazone (M-IV formation; Jaakkola et al., 2006c) seem to be relatively, albeit not completely, selective for CYP2C8. Finally, the most used in vivo CYP2C8 probe drug repaglinide, although sometimes recommended as an in vitro probe (Kajosaari et al., 2005a; VandenBrink et al., 2011), is challenging to use in vitro, e.g., because of a need for extremely low substrate concentrations and lack of commercially available metabolite standards (Table 13).

3. In Vitro Methods to Estimate the Contribution of CYP2C8 in the Metabolism of a Drug.

The basic methods used for estimating the contributions of CYP enzymes to the metabolism of a drug, i.e., the so-called reaction phenotyping, are the use of diagnostic inhibitors in a complete natural system, such as HLM, and the use of recombinant expressed enzymes. In both approaches, knowledge of clinically relevant concentrations of the drug is a prerequisite for estimation of the contributions of the different CYP enzymes in vivo. The advantage of HLM is the natural composition of the system, allowing relatively straightforward estimation of the contributions. However, this approach requires human material collected according to high ethical standards and is entirely dependent on the strength and specificity of the inhibitors. On the other hand, although recombinant expressed enzymes can be regarded as a specific tool, in vivo extrapolations of recombinant enzyme results require the use of enzyme source and batch specific conversion factors (preferably based on enzyme activity), complicating the extrapolations.

Recombinant expressed human CYP2C8 is commercially available at least as bacterial cell- and insect cell-based products. During the last decade, both chemical inhibitors and inhibitory antibodies have become available that are both CYP2C8 specific and strong. In the following, we review the documentation regarding chemical CYP2C8 inhibitors.

One of the most widely used chemical CYP2C8 inhibitors is quercetin (Rahman et al., 1994). However, it is neither very selective for CYP2C8 nor very strong and therefore, it can barely be recommended as a diagnostic inhibitor. Today, there are several more selective alternatives available, including trimethoprim, montelukast, and gemfibrozil 1-O-β-glucuronide.

The IC₅₀ of trimethoprim for CYP2C8 is approximately 50 µM, i.e., it is not a very strong inhibitor, but its IC₅₀ for other CYP enzymes is at least one order of magnitude greater, making it a relatively selective inhibitor (Wen et al., 2002). Montelukast, on the other hand, is a potent and highly selective competitive inhibitor of CYP2C8, with an IC₅₀ as low as 0.01 µM, when a low microsomal protein concentration is used, whereas its IC₅₀ for other CYP enzymes is at least two orders of magnitude greater (Walsky et al., 2005b). The major drawback of montelukast seems to be its nonspecific microsomal protein binding, whereby increasing the microsomal protein concentration by 80-fold yields an about 100-fold decrease in its inhibition potency (Walsky et al., 2005b).

The mechanism-based CYP2C8 inactivator gemfibrozil 1-O-β-glucuronide is another appealing CYP2C8 inhibitor. With a 30-minute preincubation, its IC₅₀ for CYP2C8 is about 2 µM, whereas its IC₅₀ values for CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 are more than 300 µM, suggesting an even better selectivity than that of montelukast (Ogilvie et al., 2006). Moreover, it is unlikely to be markedly affected by microsomal protein concentration. Whether clopidogrel acyl-β-d-glucuronide is a similarly selective CYP2C8 inactivator remains to be investigated (Tornio et al., 2014).

1. General Aspects.

Current guidelines recommend the conduct of clinical drug-drug interaction studies on 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/K_i) by the drug or its metabolite, preferably using the highest clinically used dose of the drug, and on the enzyme that is considered the most 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. 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 by other enzymes, are substrates of some membrane transporters, or are excreted in urine or feces in unchanged form. Thus, the significance of CYP2C8 in interactions cannot be calculated directly from changes in victim drug AUC. If the CYP2C8 substrate drug is also a substrate of transporters or other CYP 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 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 routes, because the CYP2C8-independent elimination routes remain unaffected.

CYP2C8-mediated drug interactions are often studied 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 in which patients often are using several different drugs concomitantly, substrate drugs can be administered in multiple-phase studies, given alone, with an inhibitor of CYP2C8 (gemfibrozil), with an inhibitor of another relevant CYP enzyme (e.g., with CYP3A4 inhibitor itraconazole), and together with a combination of inhibitors. However, there are only a few studies in 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., 2005; Karonen et al., 2012).

2. In Vivo Cytochrome P450 2C8 Probe Substrates.

The crucial characteristics of an in vivo probe substrate are its selectivity and sensitivity. In an ideal case, at least 80% of the substrate is metabolized by the enzyme of interest, allowing for an interpretation based on the AUC of the parent drug. In some cases, the use of an enzyme-specific metabolite to parent ratio may be used to increase sensitivity and specificity, but with an additional caveat because of potential variability in metabolite elimination. The feasibility of an in vivo probe substrate also depends heavily on its safety, in particular when large increases in its systemic concentrations can be anticipated. Moreover, the pharmacokinetic characteristics of the probe may affect its suitability. For example, a probe substrate with a significant first-pass metabolism and short elimination half-life may be able to catch even transient changes in enzyme activity, which may be necessary when studying inhibitors with a short half-life or time-dependent changes in enzyme activity.

The antidiabetic agent repaglinide is overall the most studied and best documented in vivo probe substrate of CYP2C8, and consequently both the European Medicines Agency (EMA) and FDA recommend its use as a CYP2C8 probe (Table 12). Although in vitro studies are not fully consistent with the major in vivo role of CYP2C8 in the total metabolism of repaglinide (Gan et al., 2010; Säll et al., 2012; Varma et al., 2013, 2015), repaglinide seems to be very sensitive to inhibitors of CYP2C8 activity, such as gemfibrozil, trimethoprim, and clopidogrel (Niemi et al., 2003b, 2004b; Tornio et al., 2014). On the basis of detailed mechanistic drug-drug interaction studies with the strong CYP2C8 inactivator gemfibrozil, it has been estimated that the contribution of CYP2C8 to repaglinide (0.25 mg) metabolism is about 85%, indicating that the AUC of repaglinide can be increased up to an average of sevenfold by strong CYP2C8 inhibition (Honkalammi et al., 2012). The half-life of repaglinide is also relatively short (1 hour), which allows for a full pharmacokinetic study within 1 day and can be beneficial when a measure of CYP2C8 activity within a narrow time frame is desired. The weakness of repaglinide is that it is partially metabolized by CYP3A4 (Bidstrup et al., 2003; Niemi et al., 2003b; Kajosaari, 2005a) and also a substrate of OATP1B1 (Niemi et al., 2005b). Thus, e.g., the effect of gemfibrozil on repaglinide pharmacokinetics is partially mediated by inhibition of OATP1B1, in addition to inhibition of CYP2C8 (Honkalammi et al., 2011a). Moreover, as it increases insulin secretion from pancreatic β cells, there is a risk of hypoglycemia, particularly when it is given to healthy subjects. Thus, the smallest possible dose (e.g., 0.25 mg) of repaglinide should be used, and meals, close follow up, and blood glucose monitoring be arranged when repaglinide is used.

Theoretically, the antimalarial agent amodiaquine and its N-deethylation could be useful in vivo CYP2C8 probes. However, there is very little clinical documentation for its use as a probe drug. Moreover, the safety of amodiaquine as an in vivo probe drug in drug-drug interactions studies seems to be questionable (German et al., 2007).

Of the other potential in vivo probe substrates of CYP2C8, the two thiazolidinediones pioglitazone and rosiglitazone are the best documented. As pointed out in the previous section, CYP2C8 is the main enzyme mediating their primary hydroxylation reactions in vitro (Baldwin et al., 1999; Jaakkola et al., 2006c). Accordingly, the typical dosing of gemfibrozil 600 mg twice daily, which has been estimated to cause over 95% inhibition of CYP2C8 (Fig. 9; Honkalammi et al., 2012), increased the AUC of rosiglitazone about 2.3-fold and that of pioglitazone 3.2-4.3-fold, simultaneously reducing the concentrations of their hydroxyl metabolites (Niemi et al., 2003a; Deng et al., 2005; Jaakkola et al., 2005; Aquilante et al., 2013a). Unlike repaglinide, they are insensitive to OATP1B1 function (Kalliokoski et al., 2008a). However, they have a long half-life, necessitating an up to 72-hour blood sampling period for a full pharmacokinetic analysis. On the basis of its better availability and sensitivity to CYP2C8 inhibition, pioglitazone is slightly preferable over rosiglitazone as a CYP2C8 probe.

The leukotriene receptor antagonist montelukast is another sensitive CYP2C8 substrate that could be used as a CYP2C8 probe. Gemfibrozil has increased its AUC almost fivefold and markedly reduced the formation of its 36-hydroxylated metabolite (Karonen et al., 2010). On the other hand, montelukast is also partially metabolized by CYP3A4 in vitro (Filppula et al., 2011; VandenBrink et al., 2011). However, the strong CYP3A4 inhibitor itraconazole has had no effect on montelukast concentrations (Karonen et al., 2012), indicating that the role of CYP3A4 in montelukast metabolism is minor. Moreover, montelukast is not known to be a substrate for OATP1B1.

In addition to the above substrates, there are some other CYP2C8 substrate drugs that could be used as in vivo markers on the basis of their sensitivity to interact with gemfibrozil. Such drugs include, for example, daprodustat and dasabuvir. However, the former is not yet on the market, and the second one is expensive, and more documentation is needed before they can be recommended as probe substrates.

3. In Vivo Cytochrome P450 2C8 Probe Inhibitors.

Probe inhibitors are needed for studying the contribution of CYP2C8 in the metabolism of a new drug, as well as for documenting the risk of drug-drug interactions affecting the drug in vivo. Among clinically used CYP2C8 inhibitors, gemfibrozil is the strongest known. Its CYP2C8 inhibitory effect is also highly selective due to the specific mechanism that is mediated via specific CYP2C8 inactivation by the glucuronide metabolite of gemfibrozil (Ogilvie et al., 2006). In vitro, parent gemfibrozil inhibits CYP2C9 activity with a fairly low K_i (about 6 µM), but its inhibitory effects on the other main CYP enzymes are much weaker (Backman et al., 2000; Wen et al., 2001; Wang et al., 2002). In clinical studies, gemfibrozil at a dose of 600 mg twice daily has not increased the concentrations of the CYP2C9 substrate warfarin (Lilja et al., 2005) or had any effect that could be due to inhibition of CYP3A4 on the concentrations of the parent lactone forms of simvastatin and lovastatin (Backman et al., 2000; Kyrklund et al., 2001). On the other hand, gemfibrozil has drastically, up to 18.6-fold, increased the AUCs of CYP2C8 substrate drugs (Fig. 7; Table 10), suggesting that with regard to CYP enzymes, the inhibitory effect of gemfibrozil is highly selective for CYP2C8.

The CYP2C8 inhibitory effect of gemfibrozil is strong, rapid, and long lasting. In studies using repaglinide as the CYP2C8 probe substrate, subtherapeutic doses of gemfibrozil have considerably elevated the concentrations of repaglinide (Honkalammi et al., 2011a, 2012), and it has been estimated that the clinically used gemfibrozil dosing (600 mg twice daily) inhibits CYP2C8 activity by about 99% and that one-tenth of this dose would already lead to more than 90% inhibition of CYP2C8 (Fig. 9). Although CYP2C8 inhibition by gemfibrozil is based on time-dependent inactivation of the enzyme by the primary glucuronide metabolite of gemfibrozil, CYP2C8 inhibition occurs rapidly after gemfibrozil dosing. When repaglinide was given 0, 1, 3, or 6 hours after a single 600 mg dose of gemfibrozil, the AUC of repaglinide was increased 5.0-, 6.3-, 6.6-, and 5.4-fold, respectively, indicating that strong inhibition of CYP2C8 can be achieved almost immediately after a single dose of gemfibrozil (Fig. 8, Honkalammi et al., 2011b). It has also been demonstrated that the CYP2C8 inhibitory effect of gemfibrozil persists virtually unchanged throughout the typical 12-hour dosing interval of gemfibrozil (Tornio et al., 2008a). Thus, gemfibrozil can have a strong effect on CYP2C8 substrates, irrespective of their half-life or time of daily dosing relative to gemfibrozil administration (Fig. 9; Table 10), making it an ideal in vivo probe inhibitor of CYP2C8. The only caveat with gemfibrozil is that it is also a moderate inhibitor of OATP1B1 and OAT3 and can therefore also increase the concentrations of some drugs that are not or only partially metabolized by CYP2C8 (Kyrklund et al., 2001, 2003; Backman et al., 2002; Schneck et al., 2004; Neuvonen et al., 2006; Whitfield et al., 2011).

Compared with gemfibrozil, all other clinically documented CYP2C8 inhibitors seem to be suboptimal. Trimethoprim is relatively selective for CYP2C8, but as expected from its in vitro inhibitory effects (Wen et al., 2002), it is only a weak CYP2C8 inhibitor at clinically feasible doses (Niemi et al., 2004a,b; Hruska et al., 2005; Tornio et al., 2008b), and therefore it can only be regarded as a confirmatory CYP2C8 inhibitor in vivo. Clopidogrel is the second strongest CYP2C8 inhibitor documented so far, increasing the AUC of repaglinide about fivefold (Tornio et al., 2014). Clopidogrel is obviously also a useful diagnostic CYP2C8 inhibitor, but it is not fully selective and has not been extensively documented so far. In addition to strongly inhibiting CYP2C8, clopidogrel is also a moderate inhibitor of CYP2B6 (Turpeinen et al., 2005). Furthermore, it has been suspected of causing CYP2C19 inhibition (Nishiya et al., 2009). However, it seems to have practically no effect on CYP3A4 or OATP1B1 activities in vivo (Tornio et al., 2014; Itkonen et al., 2015).