Glucuronides as Potential Anionic Substrates of Human Cytochrome

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Glucuronides as Potential Anionic Substrates of Human Cytochrome P450 2C8 (CYP2C8) Miniperspective Yong Ma,† Yue Fu,‡ S. Cyrus Khojasteh,† Deepak Dalvie,§ and Donglu Zhang*,† †

Drug Metabolism & Pharmacokinetics and ‡Early Discovery Biochemistry, Genentech, South San Francisco, California 94080, United States § Celgene Corporation, 10300 Campus Point Drive, San Diego California 92121, United States ABSTRACT: Glucuronidation is in general considered as a terminal metabolic step that leads to direct elimination of drugs and generally abolishes their biological activity. However, there is growing evidence to suggest that glucuronides can be ligands of human CYP2C8, making CYP2C8 distinct from the other CYP isoforms. Several classes of glucuronide conjugates, which include acyl glucuronides, ether glucuronides, N-glucuronides, and carbamoyl glucuronides, have been shown to be substrates or time-dependent inhibitors of CYP2C8. Although the structures of CYP2C8-glucuronide complexes have not been determined, the structural features of CYP2C8 active site support its binding to anionic and bulky ligands like glucuronides. As interaction perpetrators with CYP2C8, glucuronides of gemfibrozil and clopidogrel showed marked clinical drug−drug interactions (e.g., with cerivastatin and repaglinide), which are more than expected from the parent drug. This review summarizes glucuronides as CYP2C8 ligands and the active-site structural features of CYP2C8 that allow potential binding to glucuronides.

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INTRODUCTION Glucuronidation is an important metabolism pathway for many drugs and endogenous substances.1,2 Liver, gastrointestinal tracts, and kidney are the major organs where glucuronidation occurs. Mechanistically, the glucuronosyl group is enzymatically transferred from the cofactor uridine 5′-diphosphoglucuronic acid (UDPGA) to the substrates with the nucleophilic function groups of −OH, −NH2, −SH, −COOH, or others, which are catalyzed by the enzyme family of uridine 5′-diphosphoglucuronosyltransferases (UGTs).2−4 Compared to parent molecules, the glucuronides have remarkably increased molecular weights (+176 Da) and altered physicochemical properties (e.g., decreased log P and increased hydrogen-bond donors/acceptors) with enhanced water solubility. The glucuronide conjugates of drugs are usually excreted from the cells by drug efflux transporters since the anionic nature of glucuronic acid does not easily allow the passive diffusion of glucuronides across cellular membrane.5,6 These conjugates have also been known to be metabolically unstable and are prone to hydrolytic cleavage by β-glucuronidase in liver or by intestinal microflora, and in some cases this results in enterohepatic recycling.7,8 Cytochrome P450 enzymes (CYPs) are an important family of heme-containing proteins involved in the metabolism of xenobiotics and endogenous substances using nicotinamide adenine dinucleotide phosphate (NADPH) as the cofactor.9 Fifty-seven CYP isoforms have been identified in human, which are divided into different families of 1A, 2A, 2B, 2C, 2D, 2E, 2J, 3A, 4F, 11A, and 11B and subfamilies such as 2C8 and 2C9 based on the homology of their primary amino acid sequences.9 © 2017 American Chemical Society

Most of the human CYPs are encoded by polymorphic genes and the allelic variants. CYPs have broad substrate selectivity as most clinical drugs are metabolized by CYP enzymes. It is common that one CYP can catalyze multiple types of reactions such as hydroxylation, dealkylation, and heteroatom oxidation and multiple enzymes can participate in metabolism of the same drug. Among all of the human CYPs, the CYP2Cs are an important subfamily that includes 2C8, 2C9, 2C18, and 2C19 with over 80% sequence homology.10 CYP2C9 and 2C19 are usually considered to be among the most important enzymes in drug metabolism, and this is even more highlighted due to their polymorphic properties.11 However, since late 1990s, the significant contribution of CYP2C8 in drug metabolism has been recognized. The important drugs that are metabolized by CYP2C8 include rosiglitazone, cerivastatin, amodiaquine, and paclitaxel.12−15 Various CYP2C8 alleles have been reported in pharmacogenomics studies, and the importance of CYP2C8mediated drug metabolism in certain subgroups of patients has been established.16 Several excellent reviews have comprehensively summarized the contributions of CYP2C8 to drug metabolism.11,17−19 Compared with other CYP isoforms, CYP2C8 possesses a distinctive active site, which determines its substrate selectivity and unique catalytic function. One of the uncommon features of CYP2C8 is that it can metabolize certain chemicals that contain an anionic moiety in steroids, arachidonic acid, retinoids, and some drugs.11 Oxidation of conjugative Received: April 1, 2017 Published: June 27, 2017 8691

DOI: 10.1021/acs.jmedchem.7b00510 J. Med. Chem. 2017, 60, 8691−8705

Journal of Medicinal Chemistry

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Figure 1. Metabolic pathways of diclofenac involving UGT2B7 and CYP2C8.

Figure 2. Metabolic pathways of licofelone involving UGT(s) and CYP2C8.

CYP2C8 instead of other CYP isoforms can metabolize these glucuronides, yielding oxidative conjugates that are excreted in the urine or feces or detected in circulation. Some glucuronides can modulate CYP2C8 activity by inhibition in a time- and metabolism-dependent manner and thus induce DDIs. In this section, the example glucucronides as CYP2C8 substrates were summarized. Acyl Glucuronides. Acyl glucuronide conjugates of several structurally diverse drugs have been shown to interact with CYP2C8. Oxidation of diclofenac glucuronide by CYP2C8 is perhaps the first example demonstrating that a glucuronide conjugate could be further oxidized.22 Diclofenac is a nonsteroidal anti-inflammatory drug (NSAID) widely used for the treatment of acute muscle aches and several inflammatory diseases.23 In humans, diclofenac is primarily oxidized via CYP2C9 to 4′-hydroxydiclofenac, which accounts for 30% of the dose in vivo as a major metabolic pathway.24,25 Diclofenac also undergoes UGT2B7-catalyzed glucuronidation to the corresponding acyl glucuronide, but only 10−20% of the dose is excreted as the acyl glucuronide in human urine, suggesting that glucuronidation is a minor pathway in humans

metabolites can be a main clearance pathway for certain drugs. Interestingly, several glucuronide conjugates have been shown to interact with CYP2C8. When these conjugates turn to be a ligand (substrate or inhibitor) of CYP2C8, a specific drug−drug interaction (DDI) may occur. For instance, gemfibrozil glucuronide has been identified as a potent time-dependent CYP2C8 inhibitor and its clinical interactions have been observed with other drugs that are CYP2C8 substrates.20,21 A better understanding of the glucuronide structure binding to CYP2C8 may help understand and predict such DDIs. In this review, the studies on the structure and substrate selectivity of CYP2C8 are summarized to provide a perspective on the binding of anionic ligands in the CYP2C8 binding site and on associated examples.

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GLUCURONIDE CONJUGATES AS SUBSTRATES OF CYP2C8 A number of glucuronides have so far been identified as CYP2C8 ligands. Interestingly, CYP2C8 is not in general involved in the oxidative metabolism of their parent compounds for most of these glucuronides. However, 8692



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Figure 3. (A) Metabolic pathways of ibuprofen in vivo and (B) in vitro oxidation of ibuprofen acyl-β-D-glucuronide by CYP2C8 detected in LC− high resolution mass spectrometry analysis.

(Figure 1).26,27 However, in vitro studies with NADPH and UDPGA-supplemented human liver microsomes (HLM) by Kumar et al. demonstrate that glucuronidation of diclofenac is a more efficient pathway (at least 3-fold) than 4′-hydroxydiclofenac formation with CLint estimates of the glucuronidation and oxidation pathways being 639 versus 215 (μL/min)/mg protein, respectively.22 Further investigation into the metabolism by the authors revealed that diclofenac acyl glucuronide can readily undergo oxidation in HLM supplemented with NADPH-regenerating system, yielding significant amounts of 4′-hydroxydiclofenac and 4′-hydroxydiclofenac acyl glucuronide. Absence of hydroxylated glucuronide in control microsomal incubations lacking NADPH-regenerating system confirmed that the glucuronide conjugate directly undergoes oxidative metabolism. Additional experiments with monoclonal antibodies against individual CYP isozymes or recombinant CYP isozymes indicated that this oxidative reaction is primarily catalyzed by CYP2C8. These findings led the authors to conclude that excretion of 4′-hydroxydiclofenac as the major urinary metabolite following diclofenac administration to humans is probably a consequence of direct oxidation of the parent drug as well as the secondary oxidation of diclofenac acyl glucuronide. They further speculated that the first step in the metabolism of diclofenac is perhaps conversion to the acyl glucuronide in the liver followed by its CYP2C8-catalyzed

oxidation to 4′-hydroxydiclofenac glucuronide, and subsequent glucuronide hydrolysis yields 4′-hydroxydiclofenac.22 Licofelone represents another example in the NSAID class of drugs where the acyl glucuronide undergoes further CYP2C8catalyzed oxidation to hydroxylated metabolites. Licofelone was developed as a dual inhibitor of both cyclooxygenase isoforms 1 and 2 as well as 5-lipooxygenase with better gastrointestinal tolerability than traditional NSAIDs.28,29 Preliminary metabolic profiling in healthy male volunteers demonstrated that the acyl glucuronide (M1) and hydroxylated metabolite (M2) of licofelone are primary circulating metabolites with exposure of M1 and M2 accounting for 2% and 20% of that of the parent drug, respectively, following oral administration of licofelone to humans (Figure 2).30 In vitro metabolism studies in HLM revealed that licofelone was rapidly converted to M1 in UDPGA-supplemented liver microsomal incubations.30 In contrast, high metabolic stability of licofelone was observed in NADPH fortified liver microsomal incubations and M2 was detected at a very low concentration. Interestingly, subsequent studies with HLM containing both cofactors, UDPGA and NADPH, yielded M3, which is a glucuronide conjugate of M2, suggesting that both cofactors are required for oxidation of licofelone and that M3 is probably produced from M1 rather than the conventional pathway of oxidation followed by glucuronidation. Metabolite M2 is subsequently produced via hydrolysis of M3 in vivo. In vitro studies of M1 with NADPH8693



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Figure 4. Metabolic pathways of sipoglitazar involving UGT2B15 and CYP2C8.

class” of peroxisome proliferator-activated receptors (PPAR) agonists which are used in treating type-2 diabetes and dyslipidemia. Sipoglitazar, a triple agonist of PPAR-γ/α/δ, was discontinued from development due to lack of efficacy and safety concerns.35 Metabolism studies in rats, monkeys and humans have shown that oxidative dealkylation and glucuronidation are the primary routes of drug clearance for sipoglitazar (Figure 4).36 Deethylated sipoglitazar (M1) is detected as the major circulating metabolite following oral administration of sipoglitazar to human subjects. Although M1 appears to be a typical CYP-catalyzed product, investigation into its formation using HLM and human hepatocytes suggests that glucuronidation of sipoglitazar is a prerequisite for the dealkylation step.37 Phenotyping studies have revealed that sipoglitazar is converted to sipoglitazar-β-1-O-acyl glucuronide (SG1) by UGT2B15 and the formation of deethylated SG1 is exclusively catalyzed by CYP2C8. Interestingly, sipoglitazar-α2-O-acyl glucuronide (SG2), which is the acyl migration product of SG1 detected in the bile of rats, is not prone to CYP2C8-catalyzed oxidation. These findings led the authors to speculate that formation of M1 involves the dealkylation of the glucuronide SG1 to deethylated SG1, followed by hydrolysis of the glucuronide intermediate.37 Studies with two other novel PPAR activators, muraglitazar and peliglitazar, also suggest that their corresponding acyl glucuronide conjugates undergo CYP-mediated oxidation. After oral administration to humans, both [14C]muraglitazar and [14C]peliglitazar undergo extensive conjugation, and the major portion of each radioactive dose is excreted as acyl glucuronide metabolites in human bile.38−40 Oxidative metabolism is also prominent in humans for both muraglitazar and peliglitazar. In vitro incubations in NADPH-supplemented HLM catalyze hydroxylation or demethylation of their acyl glucuronides, suggesting CYP-mediated metabolism of the glucuronide conjugates. Although one can speculate that this oxidative conversion of the acyl glucuronide is CYP2C8-mediated, no detailed phenotyping studies have been conducted to suggest the role of CYP2C8 in their metabolism.40 2-[[5,7-Dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy]-2-methylpropanoic acid (MRL-C) is another PPAR agonist with potent activation effect on PPARα but weak effect on PPARγ.41 Kochansky and co-workers have presented

fortified liver microsomes also yielded M3 and further confirmed the involvement of UGTs and CYPs in the formation of M2 via oxidation of M1. Reaction phenotyping studies revealed that while several UGTs (UGT1A3, 1A7, 1A9, and 2B7) are involved in the formation of M1, formation of M3 is catalyzed specifically by CYP2C8 and can be inhibited by montelukast, a selective inhibitor of CYP2C8. Early work with naproxen acyl glucuronide has also provided some evidence that this metabolite undergoes CYP2C8mediated aromatic hydroxylation similar to that observed for diclofenac and licofelone.31 However, no additional further reaction phenotyping has been done to assess its substrate properties. Our recent studies with the NSAID ibuprofen also suggest that the glucuronide conjugate of ibuprofen, a drug that is widely used for treating pain, fever, and inflammation, is also a substrate for CYP2C8.32 In humans, the major metabolic pathway for ibuprofen is oxidation by CYPs. Several oxidative metabolites, including carboxy-ibuprofen, 2-hydroxy-ibuprofen, 3-hydroxy-ibuprofen, and 1-hydroxy-ibuprofen, have been detected in the urine of humans administered with ibuprofen (Figure 3A). Also, approximately 10−15% of ibuprofen dose is also converted to its corresponding glucuronide conjugate.33 A previous study had shown that ibuprofen acyl glucuronide does not inhibit the activity of CYP2C8 in vitro.34 However, our investigation suggests that the ibuprofen glucuronide is oxidized to several oxidative metabolites when incubated with recombinant CYP2C8 in the presence of NADPH (Figure 3B). Liquid chromatography−mass spectrometry analysis of the incubation mixture revealed at least 12 peaks that showed addition of oxygen to ibuprofen glucuronide (M1−12), some of which could be acyl migration products of hydroxylated glucuronide metabolites. These oxidative conjugate peaks suggested that hydroxylation of the acyl glucuronide can occur at different positions in the structure of ibuprofen acyl glucuronide (Figure 3B). CYP2C8 is specifically involved in hydroxylation of this glucuronide since no oxidative products were observed when the acyl glucuronide was incubated with other CYP isoforms including CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, or 3A5 (data not shown). Oxidative metabolism of acyl glucuronide conjugates has also been observed for several compounds belonging to the “glitazar 8694



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Figure 5. Metabolic pathways of MRL-C involving UGT(s) and CYP2C8.

Figure 6. Metabolic pathways of estradiol involving UGT2B7 and CYP2C8.

Figure 7. Metabolic pathways of desloratadine involving UGTs and CYP2C8.

evidence suggesting that the acyl glucuronide of MRL-C also undergoes CYP-mediated oxidative metabolism in monkeys and humans (Figure 5).42 The parent MRL-C is a substrate of UGTs, and the acyl glucuronide is the primary metabolite in

hepatocytes of rats, dogs, monkeys, and humans. The acyl glucuronide is further metabolized to several oxidative metabolites especially in the monkey, which has been confirmed by incubating [14C]MRL-C acyl glucuronide with 8695



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Figure 8. Metabolic pathways of gemfibrozil involving UGT2B7 and CYP2C8.

desloratadine-N-glucuronide by CYP2C8 exemplifies the interaction of N-glucuronide conjugates with this enzyme (Figure 7). Desloratadine, a second generation nonsedating long-lasting antihistamine, is widely used in the treatment of seasonal allergic rhinitis and chronic idiopathic urticarcia and nasal congestion.43,44 In humans, desloratadine is extensively metabolized to an active metabolite 3-hydroxydesloratadine, which is subsequently converted to 3-hydroxydesloratadine-Oglucuronide. Approximately 13% of the dose is eliminated in the urine as 3-hydroxydesloratadine-O-glucuronide, while 3hydroxydesloratadine is the major fecal metabolite accounting for 17% of the dose.45 The enzymology surrounding the formation of 3-hydroxydesloratadine has remained a mystery until recently. Kazmi and co-workers have established that N-glucuronidation of desloratadine is an obligatory step in formation of 3hydroxydesloratadine although it is still unknown which nitrogen (the piperidine nitrogen or the pyridine nitrogen) is conjugated.46 Investigation using subcellular fractions or sonicated/saponin-treated cryopreserved hepatocytes suggested that 3-hydroxydesloratadine was not detected when NADPH or UDPGA alone was added to the incubation and both NADPH and UDPGA were required for its formation. 3-Hydroxydesloratadine and its corresponding O-glucuronide were only detected in intact cryopreserved human hepatocytes, and its formation was inhibited by gemfibrozil glucuronide, a potent mechanism-based inactivator of CYP2C8.20 Other inhibitors of CYP2C8 such as montelukast and clopidogrel glucuronide also caused significant inhibition of 3-hydroxydesloratadine formation, and the inhibitors of other CYP isoforms were ineffective. Furthermore, evaluation of desloratadine, amodiaquine, and paclitaxel metabolism in a panel of individual cryopreserved human hepatocytes demonstrated that 3hydroxydesloratadine formation correlated well with the CYP2C8 marker activity (r2 of 0.7−0.9).46 The UGT isoform implicated in the N-glucuronidation of desloratadine has also been identified as UGT2B10.47 Overall, these studies suggested that CYP2C8 plays an essential role in 3-hydroxydesloratadine formation and the biotransformation of desloratadine to 3hydroxydesloratadine is a multiple-step process, which includes glucuronidation by UGT2B10, oxidation by CYP2C8, and then hydrolysis of the N-glucuronide.

NADPH-supplemented liver microsomes. Further investigation and phenotyping studies with human CYP enzymes have indicated that oxidative metabolism of the glucuronide conjugate is catalyzed by CYP2C8. Preincubation with antiCYP2C8/9 antibodies also results in 92% inhibition of oxidative metabolism of the glucuronide in HLM.42 Ether Glucuronides. Formation of 2-hydroxyestradiol-17βglucuronide from estradiol 17β-glucuronide is perhaps the first and so far the only confirmed example that exemplifies the CYP2C8-catalyzed oxidative conversion of ether glucuronide conjugates. Estradiol is a primary female steroidal estrogen sex hormone and is essential for development and maintenance of female reproductive tissues. Estradiol is primarily metabolized via two pathways: CYP3A4/3A5-mediated oxidation leading to formation of 2-hydroxyestradiol and UGT-catalyzed glucuronidation resulting in either estradiol-17β-glucuronide or estradiol-3-glucuronide (Figure 6). Delaforge and co-workers demonstrated that estradiol-17β-glucuronide is converted to the corresponding 2-hydroxyestradiol-17β-glucuronide metabolite when the glucuronide conjugate is incubated with NADPH-fortified HLM.31 Furthermore, incubation of estradiol 17β-glucuronide with NADPH-fortified recombinant CYP2C8 also yielded the corresponding 2-hydroxyestradiol glucuronide metabolite, suggesting that the oxidation of the glucuronide was specifically catalyzed by CYP2C8. Experiments using male and female HLM have suggested that the conversion is more active in female than in male HLM. Michaelis−Menten kinetics for formation of the 2-hydroxy metabolite from the glucuronide conjugate in yeast-expressed CYP2C8 yielded a KM of 88 μM and a Vmax of 1.86 (nmol/min)/nmol CYP (Vmax/KM = 2.1 (μL/min)/nmol CYP). In addition, the oxidation is regionselective since estradiol-3-glucuronide is not metabolized in NADPH-fortified incubations with liver microsomes or expressed CYPs. Docking exercise in the crystal structure of CYP2C8 indicates that the active site of the enzyme is large enough to accommodate the glucuronide conjugate. The most energetically favored position of the glucuronide in the docking exercise is consistent with the observation that oxidation occurs at the carbon-2 in the steroidal ring of the substrate. Testosterone-17β-glucuronide is also a substrate of CYPs in NADPH-fortified HLM. However, the site of metabolism and the isoform responsible for this transformation have not been identified.31 N-Glucuronides as Substrates of CYP2C8. Transformation of desloratadine-N-glucuronide to 3-hydroxy8696



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Figure 9. Metabolic pathways of clopidogrel involving hydrolysis, glucuronidation, and CYP2C8.

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GLUCURONIDE CONJUGATES AS MECHANISM-BASED INACTIVATORS OF CYP2C8 By definition, a mechanism-based inhibitor is a compound that inactivates the enzyme which metabolizes it. The product formed is a highly reactive species that ultimately results in formation of covalent complex with the enzyme that produces it and thus renders it inactive.48 The process leading to inactivation is generally irreversible and the catalytic activity cannot be restored unless the enzyme is regenerated. Among the glucuronide substrates of CYP2C8, some can also act as inactivators of this enzyme. Gemfibrozil is perhaps the most important and first example where inactivation of a CYP enzyme involves conjugation.20 Gemfibrozil is an amphipathic carboxylic acid drug used to treat hyperlipidemia and hypertriglyceridemia in patients.49 In humans, gemfibrozil is primarily metabolized via the oxidation and glucuronidation (Figure 8). While the principal oxidative metabolite is the benzoic acid derivative, glucuronidation of the drug (and its metabolites) is a primary metabolic pathway. After an oral dose of 450 mg to humans, 32% of the dose is excreted as a glucuronide conjugate of gemfibrozil in urine by 24 h, while benzoic acid derivative and its conjugate account for approximately 20% of the dose. The enzyme responsible for conjugating gemfibrozil has been identified as UGT2B7.50 Despite the weak reversible inhibition of CYP2C8 by gemfibrozil in in vitro studies using HLM (IC50 ≈ 95 μM),51−53 several clinical interactions between gemfibrozil and CYP2C8 substrates such as cerivastatin, repaglinide, rosiglitazone, and pioglitazone have been reported.54−57 For instance, combined use of gemfibrozil and cerivastatin, a cholesterol lowering agent, substantially increased the risk of fatal rhabdomyolysis, which is a severe adverse effect of cerivastatin in patients.58,59 Likewise, sustained and significant increase in the repaglinide plasma exposure has been observed in subjects that were administered 600 mg dose of gemfibrozil and 0.25 mg of repaglinide.60 In another study, after a single dose of 30 mg of gemfibrozil, approximately 50% CYP2C8 activity in humans was inhibited and the area under the concentration−time curve (AUC) of repaglinide was increased by 1.8-fold.61 The rapid inactivation of CYP2C8 in vivo occurred within 1 h after gemfibrozil administration, and the full recovery of CYP2C8 activity could take as long as 96 h after gemfibrozil discontinuation.17,62 This inconsistency between

the in vitro and in vivo studies has been resolved by demonstrating that the glucuronide conjugate of gemfibrozil is a more potent inhibitor of CYP2C8 than gemfibrozil itself.63 It has also been demonstrated that the glucuronide conjugate inhibits the human organic anion transporting polypeptide 2 (OATP2) mediated uptake of cerivastatin.63 Although the inhibition of human OATP2 can slow down the hepatocyte uptake of cerivastatin and thus contribute to the AUC increase in plasma, the inhibition of the CYP2C8-mediated metabolism of cerivastatin by the gemfibrozil glucuronide is considered as the main mechanism for the clinically relevant DDI because of the possible high concentrations of unbound gemfibrozil glucuronide inside the hepatocytes. Subsequent studies by Oglivie et al. showed that gemfibrozil glucuronide is not only a reversible inhibitor but also a metabolism-dependent inhibitor of CYP2C8.20 Preincubation of the metabolite with HLM for 30 min in the presence of NADPH caused the IC50 to shift from 24 to 1.8 μM for inhibition of CYP2C8 activity. Detailed mechanistic studies with gemfibrozil glucuronide have shown that the metabolite inhibits CYP2C8 with a KI (inhibitor concentration that supports half the maximal rate of enzyme inactivation) of 20− 52 μM and a kinact (maximal rate of inactivation) of 0.21 min−1. The metabolism-based inactivation is not observed in control experiments that lack NADPH, indicating that NADPH is required for inhibition and the possibility of inactivation via the covalent binding of the acyl glucuronide moiety is ruled out. Additional mass spectrometry studies with inactivated CYP2C8 enzyme by Baer and co-workers suggest that inactivation is due to a direct covalent bond between one benzylic carbon in the 2′,5′-dimethylphenoxy group with the heme prosthetic group of the CYP.64 This is consistent with the computational docking experiments in which the most energetically efficient binding mode is where the glucuronosyl moiety is located distal to the heme and interacts with Ser100, Ser103, Gln214, and Asn217 of the apoprotein.34,64 While the exact site of adduction in the heme was not confirmed experimentally, modeling studies proposed the involvement of the γ-meso position of the porphyrin ring in the adduction. Overall, these results suggest that the inactivation mechanism involves formation of a benzylic radical intermediate from gemfibrozil glucuronide and its adduction to the heme of CYP2C8. Consistently, a structure−activity relationship (SAR) study showed that 8697



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Figure 10. Metabolic pathways of deleobuvir involving glucuronidation, reduction, and CYP2C8.

acyl glucuronide, clopidogrel acyl glucuronide is most likely orientated with the thiophene moiety in close proximity to the heme and the hydrophilic glucuronide group between Ser103, Leu208, and Ile213 in the active site cavity of CYP2C8.71 This orientation was consistent with an inactivation mechanism wherein the reactive electrophiles from the thiophene group add to the heme and cause the dysfunction of the enzyme.71,73 In addition, clopidogrel and its acyl glucuronide show an inhibitory effect on OATP1B1-mediated uptake of cerivastatin in vitro, suggesting that the inhibition of OATP(s) may also contribute to the DDI between clopidogrel and cerivastatin.74 Deleobuvir represents a recent example where its glucuronide conjugate is a potent time-dependent inhibitor of CYP2C8.75 Deleobuvir is an inhibitor of hepatitis C virus NS5B RNA polymerase.76 The pharmacokinetics, mass balance, and metabolism studies with [14C]deleobuvir in healthy subjects at 800 mg dose have revealed two major circulating metabolites: an acyl glucuronide (M1) and a reduced metabolite (M2), representing 20% and 15% of the total drug related material, respectively (Figure 10).76 In vitro DDI assessment of deleobuvir and its two metabolites suggests that M1 is a reversible CYP2C8 inhibitor with a KI of 0.022 μM. Although deleobuvir and M2 also inhibit CYP2C8 activity, the KI values are 6- to 12-fold higher than that of M1.75 In addition, M1 also exhibits a more potent timedependent inhibition of CYP2C8 than deleobuvir and M2. The corresponding KI and kinact values for M1 were 0.0521 μM and 0.0521 min−1, respectively, suggesting an inactivation potency of 1 μM−1 min−1. Interestingly, M1 is also an inactivator of CYP1A2; however, the inhibitory potency is much lower than that observed for CYP2C8 (KI of 54.6 μM and kinact of 0.044 min−1). While the influence of inhibition was not evaluated clinically for CYP2C8, a DDI projection suggests that deleobuvir may cause a moderate-to-high increase in AUC for the two CYP2C8 substrates, repaglinide and cerivastatin.75 Inactivation of CYP2C8 by Lu-A (LuAA34893, full structure not disclosed)77 represents a noteworthy example wherein a carbamoyl glucuronide functions as an irreversible mechanismbased inhibitor of CYP2C8. Lu-A, a drug candidate under development by Lundbeck A/S to treat major depressive and bipolar disorder, is a secondary amine that forms a carbamoyl glucuronide (Figure 11). Carbamoyl glucuronides are generally formed when primary or secondary amines react with carbon

replacement of the aromatic methyl groups in gemfibrozil with trifluoromethyl groups abolishes mechanism-based inactivation of CYP2C8.34 The same SAR study on inactivation also suggested that the interaction between gemfibrozil glucuronide and CYP2C8 is very unique since the acyl glucuronides of several other structurally diverse drugs evaluated in this study are not associated with mechanism-based inactivation of CYP2C8.34 Like gemfibrozil, severe clinical DDIs have been observed when clopidogrel is administered with CYP2C8 substrates. Clopidogrel belongs to tetrahydrothienopyridines class of antiplatelet agents and is used to inhibit blood clots in coronary artery disease, peripheral vascular disease, and cerebrovascular disease and to prevent heart attack and stroke.65−67 It is a prodrug that requires metabolic activation by multiple CYPs into “active” reactive metabolites for the antithrombotic activity.68,69 However, in humans, only 15% of total clopidogrel is oxidized to the active thiol metabolite via 2oxoclopidogrel and the primary route of clopidogrel metabolism (approximately 70% or greater) is hydrolysis by human carboxylesterases, resulting in an inactive carboxylic acid metabolite (Figure 9).67 Approximately 25% of the carboxylic acid metabolite is circulating as its corresponding acyl glucuronide conjugate before it is eliminated.70,71 The analysis based on a case-control study of rhabdomyolysis revealed that the coadministration of clopidogrel with cerivastatin can significantly increase the risk of rhabdomyolysis in patients.72 Another study indicated that in healthy volunteers receiving clopidogrel for 3 consecutive days (300 mg on day 1, followed by 75 mg daily), the systemic exposure of repaglinide was increased 5.1-fold on day 1 and 3.9-fold on day 3.71 These DDIs could be explained by further investigations that demonstrated that clopidogrel acyl glucuronide is a potent mechanism-based inactivator of CYP2C8.71 Detailed experiments indicate that clopidogrel acyl glucuronide inactivates CYP2C8 with a KI of 9.90 μM and a kinact of 0.047 min−1.71 Montelukast, a competitive inhibitor of CYP2C8, reduces CYP2C8 inactivation by clopidogrel acyl glucuronide and thus confirms that the inactivating species is formed enzymatically in the active site of CYP2C8.71 Like gemfibrozil, the adduction was covalent in nature since dialysis of preincubated HLMinhibitor mixtures did not reduce the inhibitory effects of the glucuronide. Docking exercises indicated that like gemfibrozil 8698



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and binding site residues may be the primary determinants of the substrate selectivity. Interestingly, the authors pointed out that among the four isoforms, CYP2C8 is unique because its selective region contains several distinct residues, which renders its active site to adopt a different shape and be less hydrophobic than that of the other CYP2C isoforms. Ser114, Phe205, and Ile476 are considered to be the major contributors to the substrate specificity of CYP2C8. This study indicates that CYP2C8 may be more prone to take hydrophilic substrates compared with other CYP2C isoforms.10 Besides the homology models, ligand-based approaches have also been employed to gain a better understanding of substrate specificity of CYP2C8. By superimposition of the structures of various CYP2C8 substrates, a pharmacophore was built to show their common characteristics by Melet et al.80 The result indicated that in the structures of these substrates an acidic or polar functional group can usually be found at one end of a hydrophobic chain, while the oxidation usually occurs at the other end. This pharmacophore model shed light on the conformation and orientation of the substrate with a negatively charged or polar function group in the active site of CYP2C8. The binding of glucuronides into the active site of CYP2C8 may share a similar conformation because they are negatively charged at a physiological pH. Ligand-Free Crystal Structures of CYP2C8. The first Xray crystal structure of human CYP2C8 without a ligand in the active site was reported by Schoch et al. in 2004 (2.7 Å, PDB code 1PQ2) (Figure 12A).81 To obtain CYP2C8 crystal, the Nterminal of CYP2C8 was engineered by a method that was previously applied in the structure determination of rabbit CYP2C5.79 The CYP2C8 protein crystallized as a symmetric dimer in this structure. The active site of CYP2C8 comprises several secondary structures and the loops between them, including mainly β sheet 1, loop−helix B′-loop, helix F to G (excluding helix G′), helix I, the loop between helix K and β sheets 1−3, and β sheet 4 (Figure 12B). The active site cavity in human CYP2C8 is almost twice as large as that of rabbit CYP2C5. The change of cavity volume is due to different conformation of the polypeptide backbones, as well as the various side-chain size and/or polarity of amino acid residues in the active sites. Most prominently, the relatively smaller side chains of Ser114 and Ile476 in CYP2C8 do not block the space between heme and helix F′ and thus create a much larger cavity than that in CYP2C5 where the corresponding Phe114 and

Figure 11. Metabolic pathways of Lu-A involving the reaction with CO2, glucuronidation (R1 and R2 represent proprietary substructures), and CYP2C8.

dioxide to form carbamic acids that subsequently undergo glucuronidation to form a corresponding glucuronide conjugate. Although little is known about the mechanism and the UGT isoforms that catalyze this reaction, a similar biotransformation pathway has been reported for several amino acids and drugs.78 From a DDI perspective, the parent compound Lu-A causes direct inhibition of all CYP enzymes evaluated and is a potent reversible inhibitor of CYP2C19. The carbamoyl glucuronide of Lu-A is only a weak reversible inhibitor of CYP2C8 (IC50 = 71 μM). However, a 30 min preincubation of the carbamoyl glucuronide with NADPHsupplemented HLM largely enhances its inhibition of CYP2C8 activity (IC50 = 8.5 μM), indicating a time-dependent inhibition with a KI of 48 μM and a kinact of 0.038 min−1 (kinact/KI = 0.79 min−1 mM−1). Overall, LuAA34893 serves as another example, along with gemfibrozil, clopidogrel, and deleobuvir, of a compound that is converted by glucuronidation to a conjugated metabolite that functions as an irreversible metabolismdependent inhibitor of CYP2C8.

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STRUCTURE OF HUMAN CYP2C8 Structural Models of Human CYP2C8. The crystal structure of rabbit CYP2C5 is the first experimentally determined mammalian CYP structure, and it offers a template to build homology models of other mammalian CYPs.79 By combination of in silico computation and statistical analysis, the homology models were established by Williams et al. to explain why the substrate specificity differs among the major human CYP2C isoforms (2C8, 2C9, 2C18, and 2C19).10 The key residues in the active site conferring substrate selectivity were computationally identified in each CYP2C homology model. The selectivity analysis showed that the shape of substrate binding sites and hydrophobic interactions between substrates

Figure 12. Structures of human microsomal CYP2C8. (A) CYP2C8 dimer in the asymmetric manner. Two palmitic acid molecules (shown in sphere) are positioned at the dimer interface. (B) Side view of CYP2C8 monomer. Secondary structures are labeled sequentially from the N terminus to the C terminus. 8699



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Figure 13. Active site of CYP2C8 complex with ligands. (A) The interactions between montelukast and the residues in the ligand-binding site of CYP2C8. Heme and montelukast (labeled as MTK) are shown as sphere and stick. Portion of CYP2C8 protein harboring ligand-binding site residues was rendered as transparent cartoon. Residues making close contact to montelukast (less than 4 Å) are labeled with their side chains shown as stick. Potential hydrogen bonding interactions between residues in CYP2C8 and montelukast are depicted using dashed lines. For the following panels, the atom and structure color codes and depiction are the same as described in panel A. (B) Interactions between troglitazone and the residues in the ligand-binding site of CYP2C8. Due to the smaller size of troglitzone, only the upper-half space (away from heme) of the ligandbinding site of CYP2C8 was occupied. (C) Interactions between felodipine and the residues in the ligand-binding site of CYP2C8. (D) Interactions between retinoic acids and the residues in the ligand-binding site of CYP2C8. The proximal molecule of retinoic acid to the heme is labeled as REA1, and the distal molecule is labeled as REA2.

Figure 14. Chemical structures of montelukast, troglitazone, felodipine, and 9-cis-retinoic acid.

Phe473 show steric hindrance. In addition, there are 23 extra residues in the secondary structures and loops surrounding the active site cavity of CYP2C8, compared to that of CYP2C5. These additional amino acids are involved in formation of the

CYP2C8 active site and may potentially interact with substrates. Paclitaxel, a probe commonly used to study CYP2C8 activity in vitro, is a relatively large substrate (molecular weight of 853.9 Da) that can still fit into the active 8700



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site cavity of CYP2C8.82 Although glucuronides are larger than their parents, it is still possible for them to fit into the active site cavity of CYP2C8. Besides the large cavity volume, the properties of amino acid residues in the active cavity may also contribute to the substrate selectivity of CYP2C8. Among the 48 amino acid residues comprising the active site of CYP2C8, 16 residues (N72, N99, S100, S103, T107, S114, N202, N204, N209, N217, N218, N236, T240, T301, T364, and T386) are noncharged and hydrophilic, which may increase the binding affinity of relatively polar substrates including glucuronides.81 More importantly, three positive-charged arginine residues (R97, R200, and R241) in the active site may help anchor the binding of negative-charged glucuronide via an electrostatic interaction. The features of CYP2C8 active site cavity imply that glucuronides can be CYP2C8 ligands. Crystal Structures of CYP2C8 with Ligand. Four CYP2C8 crystal structures of enzyme complex with montelukast, troglitazone, felodipine, or 9-cis-retinoic acid have been solved (Figure 13).83 These four ligands differ from one another in molecular weight, shape, flexibility, and charge at a physiological pH, and their chemical structures are shown in Figure 14. The investigation into the binding of various substrates into the active site cavity of CYP2C8 reveals the mechanism by which CYP2C8 enzymes are involved in the metabolism of a broad panel of drugs, especially the large and anionic molecules. Montelukast is the largest substrate among the four CYP2C8 ligands, and the molecular structure comprises three branches connected to a tertiary carbon atom in the center. The branch terminating with a dimethylphenylcarbinol resides deeply in the active site cavity, approaching the heme iron (Figure 13A). The other two branches reside distally from the bottom of the cavity, and each of them occupies one of the two subcavities divided by helix B′−C loop. The longest branch with a planar conformation, which is also the most hydrophobic one, complements the subcavity with an opening on the C-terminal side of helix B′. The hydrophilic branch terminating with a carboxyl group occupies the other subcavity, and the carboxyl group is anchored by hydrogen bonds donated by Ser100 and Ser103 at N-terminal of helix B′ (Figure 13A). Troglitazone is a ligand smaller and more linear in shape than montelukast. It is also anionic at physiological pH because of the weakly acidic thiazolidinedione group. This linear and flexible ligand exhibits a bent conformation in the active site cavity of CYP2C8, occupying only the upper part of the active site (Figure 13B). The position of troglitazone is far from the heme, with an unoccupied volume as large as 500 Å3 between them. Compared with troglitazone, felodipine has a slightly lower molecular weight (384.3 Da). The nonlinear shape and nonionic nature make it distinct from the two substrates described above. Interestingly, in contrast to troglitazone, felodipine occupies the lower part of active site cavity, residing close to the heme (Figure 13C). The remaining volume of the cavity is filled with several water molecules. No hydrogen bonds are formed between felodipine and the water molecules or the protein residues. The contact between felodipine and CYP2C8 is dominated by hydrophobic interactions. Retinoic acid is the smallest among the four ligands, and two molecules of retinoic acid are found in the active site cavity of CYP2C8 in the crystal structure (Figure 13D). The two molecules are aligned vertically, occupying the upper and lower part of the cavity, respectively. The trimethylcyclohexenyl moiety of the lower molecule is oriented close to the heme iron, and the carboxyl

group at the other end of the linear molecule forms hydrogen bonds with Gly98 in helix B−B′ loop and Ser100 in helix B′. The upper retinoic acid molecule stacks on the first one and resides distally from the heme. Its trimethylcyclohexenyl moiety contacts the isoprenoid chain of the lower molecule, while its own isoprenoid chain inclines upward and the terminal carboxyl group forms hydrogen bonds with Asn204 in helix F and Arg241 in helix G (Figure 13, D). Glucuronides as Ligands of CYP2C8. Although several glucuronides have already been identified as CYP2C8 inhibitors or substrates, no crystal structures have been obtained for CYP2C8 bound with a glucuronide as its ligand. Thus, it is still unknown how the glucuronides complement the active site cavity of CYP2C8. The possible conformational changes of CYP2C8 upon the binding of glucuronides are also unknown. However, on the basis of the currently available CYP2C8-ligand complex structures, some predictions can be made regarding the binding of large and anionic substrates with CYP2C8. For the glucuronides to become CYP2C8 substrates, the sites of oxidation should be positioned close enough to the heme iron at bottom of the active site. Glucuronidation generates more bulky conjugates that can be accommodated by the large active site cavity of CYP2C8, although the upper limit for molecular weight of CYP2C8 ligands is not yet known. The conjugation of a flat but nonplanar glucuronosyl moiety can more or less change the conformation and flexibility of the structures and result in new properties that are not observed before conjugation. More importantly, introduction of glucuronosyl moiety into the structure may alter the charge of the structures at a physiological pH. The negative charges may largely increase the binding affinity due to enhanced hydrophilic interactions (e.g., hydrogen bond or charge-stabilized hydrogen bond) between the ligand and CYP2C8 residues. This can help explain the observations that certain compounds are not CYP2C8 substrates or inhibitors until they undergo glucuronidation. In general, glucuronides tend to be less pharmacologically efficacious than the parents, perhaps due to the possible interruption in binding to the targets by the glucuronosyl group.84 However, there are also exceptions. For example, the binding affinity between morphine and δ-opioid receptor is largely increased after 6-glucuronidation.85 The changes in structural conformation brought by glucuronidation can unexpectedly alter the binding affinity between ligand and protein. Glucuronides as a CYP2C8 substrate could have a binding mechanism similar to that in the alteration of pharmacological effects by glucuronidation.

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SUMMARY AND PERSPECTIVES Although the DDI interactions by metabolites are usually less easy to detect than those occurring from parent drugs, the risk of such unfavorable DDIs should not be ignored, especially when glucuronidation is the predominant metabolism pathway or the glucuronides have high binding affinities with or inactivate the metabolism enzymes. Glucuronidation could be a potential liability as gemfibrozil and clopidogrel themselves do not inhibit CYP2C8 but are classified as strong CYP2C8 inhibitors because of their glucuronide metabolites by U.S. Food and Drug Administration. Moreover, oxidation of a glucuronide can generate “mysterious” metabolites that complicate metabolite identification as observed for desloratadine. After consecutive oxidation, glucuronidation, and hydrolysis of glucuronides, some oxidation metabolites can 8701



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become difficult to be traced in source because they cannot be generated by incubating parent compounds with single CYP isoforms in the presence of the cofactor NADPH.46,47 The metabolic scheme of oxidation of glucuronides and other conjugate metabolites challenges the traditional classification system of metabolites as oxidative metabolites are normally classified as phase I metabolites and conjugates as phase II metabolites. Both oxidation and conjugation can be the primary metabolic pathway of a drug. Several glucuronides have been identified as substrates or mechanism-based inhibitors of human CYP2C8, and the list is still growing. However, no systemic studies have yet been conducted to investigate the inherent properties of glucuronides as a unique category of CYP2C8 ligands. It is still largely unknown how the glucuronides exactly fit into the active site of CYP2C8 and why glucuronidation converts certain drugs to CYP2C8 ligands. This review summarized examples of drugs that are glucuronidated to become CYP2C8 ligands, and structural characteristics of human CYP2C8 related to the binding of anionic and bulky ligands. We hope this review will prompt general interest to identify future glucuronides or any other conjugation metabolites that may undergo oxidation leading to drug clearance and potential DDIs.

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at SUNY Buffalo, NY. After a postdoctoral fellowship in the areas of chemistry and metabolism under the supervision of firstly Professor Richard Sundberg at the University of Virginia and then Professor Neal Castagnoli at Virginia Tech, he joined Pfizer as a research scientist in 1992. His research interests include the biotransformation and bioactivation of xenobiotics and understanding the molecular mechanisms of drug metabolism and metabolic activation. Donglu Zhang received a Ph.D. in Organic Chemistry from University of Utah and had postdoctoral trainings at University of Utah and U.S. Food and Drug Administration. He worked at Bristol-Myers Squibb at Princeton, NJ, and ARIAD Pharmaceuticals at Cambridge, MA, and joined Genentech at South San Francisco, CA, in 2014. His research interests include studying drug metabolites and drug metabolism enzymes in drug design and development of both small molecule drugs and antibody−drug conjugates (ADC). The mass defect filter (MDF) methodologies he and his colleague invented have been widely used for metabolite identification.

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ACKNOWLEDGMENTS We thank Dr. A. David Rodrigues from Pfizer and Drs. Shuguang Ma and Marcel Hop from Genentech for helpful discussion.

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AUTHOR INFORMATION

ABBREVIATIONS USED AUC, area under the concentration−time curve; CYP, cytochrome P450; DDI, drug−drug interaction; HLM, human liver microsome; NADPH, nicotinamide adenine dinucleotide phosphate; NSAID, nonsteroidal anti-inflammatory drug; OATP, organic anion-transporting polypeptide; PPAR, peroxisome proliferator-activated receptors; SAR, structure−activity relationship; UDPGA, uridine 5′-diphosphoglucuronic acid; UGT, 5′-diphosphoglucuronosyltransferase

Corresponding Author

*Phone: 650-291-0058. E-mail: [email protected]. ORCID

Yong Ma: 0000-0001-5901-5440 Donglu Zhang: 0000-0001-8677-9737 Notes

The authors declare no competing financial interest.

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Biographies Yong Ma received his Ph.D. in Pharmaceutical Sciences from College of Pharmacy, University of Houston under the direction of Dr. Ming Hu. From 2015, he is employed by PPD and works as a research scientist in the Department of Drug Metabolism and Pharmacokinetics at Genentech (South San Francisco, CA). His research interest is focused on (1) the absorption, distribution, metabolism, and excretion (ADME) properties of small molecule drugs and (2) the catabolism of antibody−drug conjugates (ADCs).

REFERENCES

(1) de Wildt, S. N.; Kearns, G. L.; Leeder, J. S.; van den Anker, J. N. Glucuronidation in Humans. Clin. Pharmacokinet. 1999, 36, 439−452. (2) Tukey, R. H.; Strassburg, C. P. Human UDP-Glucuronosyltransferases: Metabolism, Expression, and Disease. Annu. Rev. Pharmacol. Toxicol. 2000, 40, 581−616. (3) Argikar, U. A. Unusual Glucuronides. Drug Metab. Dispos. 2012, 40, 1239−1251. (4) Kaivosaari, S.; Finel, M.; Koskinen, M. N-Glucuronidation Of Drugs And Other Xenobiotics By Human And Animal UDPGlucuronosyltransferases. Xenobiotica 2011, 41, 652−669. (5) Keppler, D.; Leier, I.; Jedlitschky, G. Transport Of Glutathione Conjugates And Glucuronides By The Multidrug Resistance Proteins MRP1 And MRP2. Biol. Chem. 1997, 378, 787−791. (6) Zamek-Gliszczynski, M. J.; Hoffmaster, K. A.; Nezasa, K.-i.; Tallman, M. N.; Brouwer, K. L. R. Integration Of Hepatic Drug Transporters And Phase II Metabolizing Enzymes: Mechanisms Of Hepatic Excretion Of Sulfate, Glucuronide, And Glutathione Metabolites. Eur. J. Pharm. Sci. 2006, 27, 447−486. (7) Zenser, T. V.; Lakshmi, V. M.; Davis, B. B. Human and Escherichia coli β-Glucuronidase Hydrolysis of Glucuronide Conjugates of Benzidine and 4-Aminobiphenyl, and Their Hydroxy Metabolites. Drug Metab. Dispos. 1999, 27, 1064−1067. (8) Brunelle, F. M.; Verbeeck, R. K. Conjugation-Deconjugation Cycling Of Diflunisal Via B-Glucuronidase Catalyzed Hydrolysis Of Its Acyl Glucuronide In The Rat. Life Sci. 1997, 60, 2013−2021. (9) Danielson, P. B. The Cytochrome P450 Superfamily: Biochemistry, Evolution and Drug Metabolism in Humans. Curr. Drug Metab. 2002, 3, 561−597. (10) Ridderström, M.; Zamora, I.; Fjellström, O.; Andersson, T. B. Analysis of Selective Regions in the Active Sites of Human

Yue Fu is a senior scientific researcher at Genentech. He is a structural biologist using X-ray crystallography to study protein structures and protein−ligand interactions. He received his Bachelor degree in Biosciences from University of Science and Technology of China and his Ph.D. in Molecular and Cellular Biochemistry from Indiana University Bloomington under the direction of Dr. David Giedroc. He then completed a 2-year postdoctoral training with Dr. Wayne Fairbrother at Genentech. S. Cyrus Khojasteh leads the Biotransformation Group at Genentech (South San Francisco) and was at Pfizer (Groton, CT) prior to 2000. His research focuses on the mechanisms of biotransformation in drug discovery and development with a particular interest in metabolism mediated by cytochrome P450 and non-cytochrome P450 enzymes. Cyrus received his B.S. from the University of California at Berkeley and his Ph.D. in Medicinal Chemistry from the University of Washington under the direction of Dr. Sidney D. Nelson. Deepak Dalvie is a Sr. Director in Drug Metabolism and Pharmacokinetics at Celgene. He received his B.Sc. in Chemistry and M.Sc. in the Technology of Pharmaceutical and Fine Chemicals at the University of Bombay, India, and his Ph.D. in Medicinal Chemistry 8702



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(28) Kulkarni, S. K.; Pal Singh, V. Licofelone-A Novel Analgesic and Anti-Inflammatory Agent. Curr. Top. Med. Chem. 2007, 7, 251−263. (29) Bias, P.; Buchner, A.; Klesser, B.; Laufer, S. The Gastrointestinal Tolerability of the LOX//COX Inhibitor, Licofelone, is Similar to Placebo and Superior to Naproxen Therapy in Healthy Volunteers: Results From a Randomized, Controlled Trial. Am. J. Gastroenterol. 2004, 99, 611−618. (30) Albrecht, W.; Unger, A.; Nussler, A. K.; Laufer, S. In Vitro Metabolism of 2-[6-(4-Chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1H-pyrrolizin-5-yl] Acetic Acid (Licofelone, ML3000), an Inhibitor of Cyclooxygenase-1 and −2 and 5-Lipoxygenase. Drug Metab. Dispos. 2008, 36, 894−903. (31) Delaforge, M.; Pruvost, A.; Perrin, L.; André, F. Cytochrome P450-Mediated Oxidation Of Glucuronide Derivatives: Example Of Estradiol-17β-Glucuronide Oxidation To 2-Hydroxy-Estradiol-17βGlucuronide By Cyp 2c8. Drug Metab. Dispos. 2005, 33, 466−473. (32) Rainsford, K. D. Ibuprofen: Pharmacology, Efficacy And Safety. Inflammopharmacology 2009, 17, 275−342. (33) Mazaleuskaya, L. L.; Theken, K. N.; Gong, L.; Thorn, C. F.; FitzGerald, G. A.; Altman, R. B.; Klein, T. E. Pharmocology Summary: Ibuprofen Pathways. Pharmacogenet. Genomics 2015, 25, 96−106. (34) Jenkins, S. M.; Zvyaga, T.; Johnson, S. R.; Hurley, J.; Wagner, A.; Burrell, R.; Turley, W.; Leet, J. E.; Philip, T.; Rodrigues, A. D. Studies to Further Investigate the Inhibition of Human Liver Microsomal CYP2C8 by the Acyl-β-Glucuronide of Gemfibrozil. Drug Metab. Dispos. 2011, 39, 2421−2430. (35) Colca, J. R. Discontinued Drugs In 2006: Renal, Endocrine And Metabolic Drugs. Expert Opin. Invest. Drugs 2007, 16, 1517−1523. (36) Nishihara, M.; Sudo, M.; Kamiguchi, H.; Kawaguchi, N.; Maeshiba, Y.; Kiyota, Y.; Takahashi, J.; Tagawa, Y.; Kondo, T.; Asahi, S. Metabolic Fate of Sipoglitazar a Novel Oral PPAR Agonist with Activities for PPAR-γ, -α and -δ, in Rats and Monkeys and Comparison with Humans In Vitro. Drug Metab. Pharmacokinet. 2012, 27, 223−231. (37) Nishihara, M.; Sudo, M.; Kawaguchi, N.; Takahashi, J.; Kiyota, Y.; Kondo, T.; Asahi, S. An Unusual Metabolic Pathway of Sipoglitazar, a Novel Antidiabetic Agent: Cytochrome P450-Catalyzed Oxidation of Sipoglitazar Acyl Glucuronide. Drug Metab. Dispos. 2012, 40, 249−258. (38) Wang, L.; Munsick, C.; Chen, S.; Bonacorsi, S.; Cheng, P. T.; Humphreys, W. G.; Zhang, D. Metabolism and Disposition of 14CLabeled Peliglitazar in Humans. Drug Metab. Dispos. 2011, 39, 228− 238. (39) Wang, L.; Zhang, D.; Swaminathan, A.; Xue, Y.; Cheng, P. T.; Wu, S.; Mosqueda-Garcia, R.; Aurang, C.; Everett, D. W.; Humphreys, W. G. Glucuronidation As A Major Metabolic Clearance Pathway Of 14 C-Labeled Muraglitazar In Humans: Metabolic Profiles In Subjects With Or Without Bile Collection. Drug Metab. Dispos. 2006, 34, 427− 439. (40) Zhang, D.; Raghavan, N.; Wang, L.; Xue, Y.; Obermeier, M.; Chen, S.; Tao, S.; Zhang, H.; Cheng, P. T.; Li, W.; Ramanathan, R.; Yang, Z.; Humphreys, W. G. Plasma Stability-Dependent Circulation of Acyl Glucuronide Metabolites in Humans: How Circulating Metabolite Profiles of Muraglitazar and Peliglitazar Can Lead to Misleading Risk Assessment. Drug Metab. Dispos. 2011, 39, 123−131. (41) Liu, K.; Xu, L.; Berger, J. P.; MacNaul, K. L.; Zhou, G.; Doebber, T. W.; Forrest, M. J.; Moller, D. E.; Jones, A. B. Discovery of a Novel Series of Peroxisome Proliferator-Activated Receptor α/γ Dual Agonists for the Treatment of Type 2 Diabetes and Dyslipidemia. J. Med. Chem. 2005, 48, 2262−2265. (42) Kochansky, C. J.; Xia, Y.-Q.; Wang, S.; Cato, B.; Creighton, M.; Vincent, S. H.; Franklin, R. B.; Reed, J. R. Species Differences In The Elimination Of A Peroxisome Proliferator-Activated Receptor Agonist Highlighted By Oxidative Metabolism Of Its Acyl Glucuronide. Drug Metab. Dispos. 2005, 33, 1894−1904. (43) Kreutner, W.; Hey, J. A.; Anthes, J.; Barnett, A.; Young, S.; Tozzi, S. Preclinical Pharmacology of Desloratadine, a Selective and Nonsedating Histamine H1 Receptor Antagonist. Arzneim. Forsch. 2000, 50, 345−352.

Cytochromes P450, 2C8, 2C9, 2C18, and 2C19 Homology Models Using GRID/CPCA. J. Med. Chem. 2001, 44, 4072−4081. (11) Lai, X.-S.; Yang, L.-P.; Li, X.-T.; Liu, J.-P.; Zhou, Z.-W.; Zhou, S.-F. Human CYP2C8: Structure, Substrate Specificity, Inhibitor Selectivity, Inducers and Polymorphisms. Curr. Drug Metab. 2009, 10, 1009−1047. (12) Kirchheiner, J.; Thomas, S.; Bauer, S.; Tomalik-Scharte, D.; Hering, U.; Doroshyenko, O.; Jetter, A.; Stehle, S.; Tsahuridu, M.; Meineke, I.; Brockmö ller, J.; Fuhr, U. Pharmacokinetics And Pharmacodynamics Of Rosiglitazone In Relation To CYP2C8 Genotype. Clin. Pharmacol. Ther. 2006, 80, 657−667. (13) Kaspera, R.; Naraharisetti, S. B.; Tamraz, B.; Sahele, T.; Cheesman, M. J.; Kwok, P.-Y.; Marciante, K.; Heckbert, S. R.; Psaty, B. M.; Totah, R. A. Cerivastatin In Vitro Metabolism By CYP2C8 Variants Found In Patients Experiencing Rhabdomyolysis. Pharmacogenet. Genomics 2010, 20, 619−629. (14) Li, X.-Q.; Björkman, A.; Andersson, T. B.; Ridderström, M.; Masimirembwa, C. M. Amodiaquine Clearance and Its Metabolism toN-Desethylamodiaquine Is Mediated by CYP2C8: A New High Affinity and Turnover Enzyme-Specific Probe Substrate. J. Pharmacol. Exp. Ther. 2002, 300, 399−407. (15) Bahadur, N.; Leathart, J. B. S.; Mutch, E.; Steimel-Crespi, D.; Dunn, S. A.; Gilissen, R.; Houdt, J. V.; Hendrickx, J.; Mannens, G.; Bohets, H.; Williams, F. M.; Armstrong, M.; Crespi, C. L.; Daly, A. K. CYP2C8 Polymorphisms In Caucasians And Their Relationship With Paclitaxel 6α-Hydroxylase Activity In Human Liver Microsomes. Biochem. Pharmacol. 2002, 64, 1579−1589. (16) Daily, E. B.; Aquilante, C. L. Cytochrome P450 2C8 Pharmacogenetics: A Review Of Clinical Studies. Pharmacogenomics 2009, 10, 1489−1510. (17) Backman, J. T.; Honkalammi, J.; Neuvonen, M.; Kurkinen, K. J.; Tornio, A.; Niemi, M.; Neuvonen, P. J. CYP2C8 Activity Recovers within 96 Hours after Gemfibrozil Dosing: Estimation of CYP2C8 Half-Life Using Repaglinide as an in Vivo Probe. Drug Metab. Dispos. 2009, 37, 2359−2366. (18) Totah, R. A.; Rettie, A. E. Cytochrome P450 2C8: Substrates, Inhibitors, Pharmacogenetics, and Clinical Relevance. Clin. Pharmacol. Ther. 2005, 77, 341−352. (19) Lv, X.; Zhong, F.; Tan, X. Cytochrome P450 2C8 And Drug Metabolism. Curr. Top. Med. Chem. 2013, 13, 2241−2253. (20) Ogilvie, B. W.; Zhang, D.; Li, W.; Rodrigues, A. D.; Gipson, A. E.; Holsapple, J.; Toren, P.; Parkinson, A. Glucuronidation Converts Gemfibrozil To A Potent, Metabolism-Dependent Inhibitor Of Cyp2c8: Implications For Drug-Drug Interactions. Drug Metab. Dispos. 2006, 34, 191−197. (21) Kyrklund, C.; Backman, J. T.; Kivistö, K. T.; Neuvonen, M.; Laitila, J.; Neuvonen, P. J. Plasma Concentrations Of Active Lovastatin Acid Are Markedly Increased By Gemfibrozil But Not By Bezafibrate. Clin. Pharmacol. Ther. 2001, 69, 340−345. (22) Kumar, S.; Samuel, K.; Subramanian, R.; Braun, M. P.; Stearns, R. A.; Chiu, S.-H. L.; Evans, D. C.; Baillie, T. A. Extrapolation of Diclofenac Clearance from in Vitro Microsomal Metabolism Data: Role of Acyl Glucuronidation and Sequential Oxidative Metabolism of the Acyl Glucuronide. J. Pharmacol. Exp. Ther. 2002, 303, 969−978. (23) Small, R. E. Diclofenac Sodium. Clin. Pharm. 1989, 8, 545−558. (24) Bort, R.; Macé, K.; Boobis, A.; Gómez-Lechón, M. a.-J.; Pfeifer, A.; Castell, J. Hepatic Metabolism Of Diclofenac: Role Of Human CYP In The Minor Oxidative Pathways. Biochem. Pharmacol. 1999, 58, 787−796. (25) Stierlin, H.; Faigle, J. W.; Sallmann, A.; Kung, W.; Richter, W. J.; Kriemler, H. P.; Alt, K. O.; Winkler, T. Biotransformation Of Diclofenac Sodium (Voltaren®) In Animals And In Man. Xenobiotica 1979, 9, 601−610. (26) King, C.; Tang, W.; Ngui, J.; Tephly, T.; Braun, M. Characterization of Rat and Human UDP-Glucuronosyltransferases Responsible for the in Vitro Glucuronidation of Diclofenac. Toxicol. Sci. 2001, 61, 49−53. (27) Davies, N. M.; Anderson, K. E. Clinical Pharmacokinetics of Diclofenac. Clin. Pharmacokinet. 1997, 33, 184−213. 8703



Perspective

(44) Kreutner, W.; Hey, J. A.; Chiu, P.; Barnett, A. Preclinical Pharmacology of Desloratadine, a Selective and Nonsedating Histamine H1 Receptor Antagonist2nd Communication: Lack of central nervous system and cardiovascular effects. Arzneim. Forsch. 2000, 50, 441−448. (45) Ramanathan, R.; Reyderman, L.; Kulmatycki, K.; Su, A. D.; Alvarez, N.; Chowdhury, S. K.; Alton, K. B.; Wirth, M. A.; Clement, R. P.; Statkevich, P.; Patrick, J. E. Disposition Of Loratadine In Healthy Volunteers. Xenobiotica 2007, 37, 753−769. (46) Kazmi, F.; Barbara, J. E.; Yerino, P.; Parkinson, A. A LongStanding Mystery Solved: The Formation of 3-Hydroxydesloratadine Is Catalyzed by CYP2C8 But Prior Glucuronidation of Desloratadine by UDP-Glucuronosyltransferase 2B10 Is an Obligatory Requirement. Drug Metab. Dispos. 2015, 43, 523−533. (47) Kazmi, F.; Yerino, P.; Barbara, J. E.; Parkinson, A. Further Characterization of the Metabolism of Desloratadine and Its Cytochrome P450 and UDP-glucuronosyltransferase Inhibition Potential: Identification of Desloratadine as a Relatively Selective UGT2B10 Inhibitor. Drug Metab. Dispos. 2015, 43, 1294−1302. (48) Orr, S. T. M.; Ripp, S. L.; Ballard, T. E.; Henderson, J. L.; Scott, D. O.; Obach, R. S.; Sun, H.; Kalgutkar, A. S. Mechanism-Based Inactivation (MBI) of Cytochrome P450 Enzymes: Structure−Activity Relationships and Discovery Strategies To Mitigate Drug−Drug Interaction Risks. J. Med. Chem. 2012, 55, 4896−4933. (49) Todd, P. A.; Ward, A. Gemfibrozil. Drugs 1988, 36, 314−339. (50) Mano, Y.; Usui, T.; Kamimura, H. The UDP-Glucuronosyltransferase 2B7 Isozyme Is Responsible for Gemfibrozil Glucuronidation in the Human Liver. Drug Metab. Dispos. 2007, 35, 2040−2044. (51) Wang, J.-S.; Neuvonen, M.; Wen, X.; Backman, J. T.; Neuvonen, P. J. Gemfibrozil Inhibits CYP2C8-Mediated Cerivastatin Metabolism in Human Liver Microsomes. Drug Metab. Dispos. 2002, 30, 1352− 1356. (52) Wen, X.; Wang, J.-S.; Backman, J. T.; Kivistö, K. T.; Neuvonen, P. J. Gemfibrozil Is a Potent Inhibitor of Human Cytochrome P450 2C9. Drug Metab. Dispos. 2001, 29, 1359−1361. (53) Fujino, H.; Shimada, S.; Yamada, I.; Hirano, M.; Tsunenari, Y.; Kojima, J. Studies on the Interaction between Fibrates and Statins Using Human Hepatic Microsomes. Arzneim. Forsch. 2003, 53, 701− 707. (54) Backman, J. T.; Kyrklund, C.; Neuvonen, M.; Neuvonen, P. J. Gemfibrozil Greatly Increases Plasma Concentrations Of Cerivastatin. Clin. Pharmacol. Ther. 2002, 72, 685−691. (55) Jaakkola, T.; Backman, J. T.; Neuvonen, M.; Neuvonen, P. J. Effects of Gemfibrozil, Itraconazole, and Their Combination on the Pharmacokinetics of Pioglitazone. Clin. Pharmacol. Ther. 2005, 77, 404−414. (56) Niemi, M.; Backman, J. T.; Granfors, M.; Laitila, J.; Neuvonen, M.; Neuvonen, P. J. Gemfibrozil Considerably Increases The Plasma Concentrations Of Rosiglitazone. Diabetologia 2003, 46, 1319−1323. (57) Niemi, M.; Backman, J. T.; Neuvonen, M.; Neuvonen, P. J. Effects Of Gemfibrozil, Itraconazole, And Their Combination On The Pharmacokinetics And Pharmacodynamics Of Repaglinide: Potentially Hazardous Interaction Between Gemfibrozil And Repaglinide. Diabetologia 2003, 46, 347−351. (58) Farmer, J. A. Learning from the cerivastatin experience. Lancet 2001, 358, 1383−1385. (59) Pogson, G. W.; Kindred, L. H.; Carper, B. G. Rhabdomyolysis And Renal Failure Associated With Cerivastatin-Gemfibrozil Combination Therapy. Am. J. Cardiol. 1999, 83, 1146. (60) Tornio, A.; Niemi, M.; Neuvonen, M.; Laitila, J.; Kalliokoski, A.; Neuvonen, P. J.; Backman, J. T. The Effect of Gemfibrozil on Repaglinide Pharmacokinetics Persists for at Least 12 h After the Dose: Evidence for Mechanism-based Inhibition of CYP2C8 In Vivo. Clin. Pharmacol. Ther. 2008, 84, 403−411. (61) Honkalammi, J.; Niemi, M.; Neuvonen, P. J.; Backman, J. T. Dose-Dependent Interaction between Gemfibrozil and Repaglinide in Humans: Strong Inhibition of CYP2C8 with Subtherapeutic Gemfibrozil Doses. Drug Metab. Dispos. 2011, 39, 1977−1986.

(62) Honkalammi, J.; Niemi, M.; Neuvonen, P. J.; Backman, J. T. Mechanism-Based Inactivation of CYP2C8 by Gemfibrozil Occurs Rapidly in Humans. Clin. Pharmacol. Ther. 2011, 89, 579−586. (63) Shitara, Y.; Hirano, M.; Sato, H.; Sugiyama, Y. Gemfibrozil and Its Glucuronide Inhibit the Organic Anion Transporting Polypeptide 2 (OATP2/OATP1B1:SLC21A6)-Mediated Hepatic Uptake and CYP2C8-Mediated Metabolism of Cerivastatin: Analysis of the Mechanism of the Clinically Relevant Drug-Drug Interaction between Cerivastatin and Gemfibrozil. J. Pharmacol. Exp. Ther. 2004, 311, 228− 236. (64) Baer, B. R.; DeLisle, R. K.; Allen, A. Benzylic Oxidation of Gemfibrozil-1-O-β-Glucuronide by P450 2C8 Leads to Heme Alkylation and Irreversible Inhibition. Chem. Res. Toxicol. 2009, 22, 1298−1309. (65) Hollopeter, G.; Jantzen, H.-M.; Vincent, D.; Li, G.; England, L.; Ramakrishnan, V.; Yang, R.-B.; Nurden, P.; Nurden, A.; Julius, D.; Conley, P. B. Identification Of The Platelet ADP Receptor Targeted By Antithrombotic Drugs. Nature 2001, 409, 202−207. (66) Foster, C. J.; Prosser, D. M.; Agans, J. M.; Zhai, Y.; Smith, M. D.; Lachowicz, J. E.; Zhang, F. L.; Gustafson, E.; Monsma, F. J.; Wiekowski, M. T.; Abbondanzo, S. J.; Cook, D. N.; Bayne, M. L.; Lira, S. A.; Chintala, M. S. Molecular Identification And Characterization Of The Platelet ADP Receptor Targeted By Thienopyridine Antithrombotic Drugs. J. Clin. Invest. 2001, 107, 1591−1598. (67) Sangkuhl, K.; Klein, T. E.; Altman, R. B. Clopidogrel Pathway. Pharmacogenet. Genomics 2010, 20, 463−465. (68) Kazui, M.; Nishiya, Y.; Ishizuka, T.; Hagihara, K.; Farid, N. A.; Okazaki, O.; Ikeda, T.; Kurihara, A. Identification of the Human Cytochrome P450 Enzymes Involved in the Two Oxidative Steps in the Bioactivation of Clopidogrel to Its Pharmacologically Active Metabolite. Drug Metab. Dispos. 2010, 38, 92−99. (69) Dansette, P. M.; Libraire, J.; Bertho, G.; Mansuy, D. Metabolic Oxidative Cleavage of Thioesters: Evidence for the Formation of Sulfenic Acid Intermediates in the Bioactivation of the Antithrombotic Prodrugs Ticlopidine and Clopidogrel. Chem. Res. Toxicol. 2009, 22, 369−373. (70) Silvestro, L.; Gheorghe, M.; Iordachescu, A.; Ciuca, V.; Tudoroniu, A.; Rizea Savu, S.; Tarcomnicu, I. Development And Validation Of An HPLC−MS/MS Method To Quantify Clopidogrel Acyl Glucuronide, Clopidogrel Acid Metabolite, And Clopidogrel In Plasma Samples Avoiding Analyte Back-Conversion. Anal. Bioanal. Chem. 2011, 401, 1023−1034. (71) Tornio, A.; Filppula, A. M.; Kailari, O.; Neuvonen, M.; Nyrönen, T. H.; Tapaninen, T.; Neuvonen, P. J.; Niemi, M.; Backman, J. T. Glucuronidation Converts Clopidogrel to a Strong TimeDependent Inhibitor of CYP2C8: A Phase II Metabolite as a Perpetrator of Drug−Drug Interactions. Clin. Pharmacol. Ther. 2014, 96, 498−507. (72) Floyd, J. S.; Kaspera, R.; Marciante, K. D.; Weiss, N. S.; Heckbert, S. R.; Lumley, T.; Wiggins, K. L.; Tamraz, B.; Kwok, P. Y.; Totah, R. A.; Psaty, B. M. A Screening Study of Drug−Drug Interactions in Cerivastatin Users: An Adverse Effect of Clopidogrel. Clin. Pharmacol. Ther. 2012, 91, 896−904. (73) Zhang, H.; Amunugama, H.; Ney, S.; Cooper, N.; Hollenberg, P. F. Mechanism-Based Inactivation of Human Cytochrome P450 2B6 by Clopidogrel: Involvement of Both Covalent Modification of Cysteinyl Residue 475 and Loss of Heme. Mol. Pharmacol. 2011, 80, 839−847. (74) Tamraz, B.; Fukushima, H.; Wolfe, A. R.; Kaspera, R.; Totah, R. A.; Floyd, J. S.; Ma, B.; Chu, C.; Marciante, K. D.; Heckbert, S. R.; Psaty, B. M.; Kroetz, D. L.; Kwok, P.-Y. OATP1B1-Related Drug− Drug And Drug−Gene Interactions As Potential Risk Factors For Cerivastatin-Induced Rhabdomyolysis. Pharmacogenet. Genomics 2013, 23, 355−364. (75) Sane, R. S.; Ramsden, D.; Sabo, J. P.; Cooper, C.; Rowland, L.; Ting, N.; Whitcher-Johnstone, A.; Tweedie, D. J. Contribution of Major Metabolites toward Complex Drug-Drug Interactions of Deleobuvir: In Vitro Predictions and In Vivo Outcomes. Drug Metab. Dispos. 2016, 44, 466−475. 8704



Perspective

(76) Chen, L.-Z.; Sabo, J. P.; Philip, E.; Rowland, L.; Mao, Y.; Latli, B.; Ramsden, D.; Mandarino, D. A.; Sane, R. S. Mass Balance, Metabolite Profile, and In Vitro-In Vivo Comparison of Clearance Pathways of Deleobuvir, a Hepatitis C Virus Polymerase Inhibitor. Antimicrob. Agents Chemother. 2015, 59, 25−37. (77) Kazmi, F.; Smith, B.; Hvenegaard, M.; Bendahl, L.; Gipson, A.; Buckley, D.; Ogilvie, B.; Parkinson, A. Identification Of A Novel Carbamoyl Glucuronide As A Metabolism-Dependent Inhibitor Of CYP2C8. Drug Metab. Rev. 2010, 42, 143. (78) Schaefer, W. H. Reaction of Primary and Secondary Amines to Form Carbamic Acid Glucuronides. Curr. Drug Metab. 2006, 7, 873− 881. (79) Williams, P. A.; Cosme, J.; Sridhar, V.; Johnson, E. F.; McRee, D. E. Mammalian Microsomal Cytochrome P450 Monooxygenase: Structural Adaptations for Membrane Binding and Functional Diversity. Mol. Cell 2000, 5, 121−131. (80) Melet, A.; Marques-Soares, C.; Schoch, G. A.; Macherey, A.-C.; Jaouen, M.; Dansette, P. M.; Sari, M.-A.; Johnson, E. F.; Mansuy, D. Analysis of Human Cytochrome P450 2C8 Substrate Specificity Using a Substrate Pharmacophore and Site-Directed Mutants. Biochemistry 2004, 43, 15379−15392. (81) Schoch, G. A.; Yano, J. K.; Wester, M. R.; Griffin, K. J.; Stout, C. D.; Johnson, E. F. Structure Of Human Microsomal Cytochrome P450 2c8: Evidence For A Peripheral Fatty Acid Binding Site. J. Biol. Chem. 2004, 279, 9497−9503. (82) Tanaka, T.; Kamiguchi, N.; Okuda, T.; Yamamoto, Y. Characterization of the CYP2C8 Active Site by Homology Modeling. Chem. Pharm. Bull. 2004, 52, 836−841. (83) Schoch, G. A.; Yano, J. K.; Sansen, S.; Dansette, P. M.; Stout, C. D.; Johnson, E. F. Determinants Of Cytochrome P450 2c8 Substrate Binding: Structures Of Complexes With Montelukast, Troglitazone, Felodipine, And 9-Cis-Retinoic Acid. J. Biol. Chem. 2008, 283, 17227− 17237. (84) Mulder, G. J. Glucuronidation and its Role in Regulation of Biological Activity of Drugs. Annu. Rev. Pharmacol. Toxicol. 1992, 32, 25−49. (85) Mignat, C.; Wille, U.; Ziegler, A. Affinity Profiles Of Morphine, Codeine, Dihydrocodeine And Their Glucuronides At Opioid Receptor Subtypes. Life Sci. 1995, 56, 793−799.

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