Andrew Parkinson

GLUCURONIDATION CONVERTS GEMFIBROZIL TO A POTENT, METABOLISM-DEPENDENT INHIBITOR OF CYP2C8: IMPLICATIONS FOR DRUG-DRUG INTERACTIONS

Gemfibrozil more potently inhibits CYP2C9 than CYP2C8 in vitro, and yet the opposite inhibitory potency is observed in the clinic. To investigate this apparent paradox, we evaluated both gemfibrozil and its major metabolite, an acyl-glucuronide (gemfibrozil 1-O-β-glucuronide) as direct-acting and metabolism-dependent inhibitors of the major drug-metabolizing cytochrome P450 enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) in human liver microsomes. Gemfibrozil most potently inhibited CYP2C9 (IC50 of 30 μM), whereas gemfibrozil glucuronide most potently inhibited CYP2C8 (IC50 of 24 μM). Unexpectedly, gemfibrozil glucuronide, but not gemfibrozil, was found to be a metabolism-dependent inhibitor of CYP2C8 only. The IC50 for inhibition of CYP2C8 by gemfibrozil glucuronide decreased from 24 μM to 1.8 μM after a 30-min incubation with human liver microsomes and NADPH. Inactivation of CYP2C8 by gemfibrozil glucuronide required NADPH, and proceeded with a KI (inhibitor concentration that supports half the maximal rate of enzyme inactivation) of 20 to 52 μM and a kinact (maximal rate of inactivation) of 0.21 min–1. Potent inhibition of CYP2C8 was also achieved by first incubating gemfibrozil with alamethicin-activated human liver microsomes and UDP-glucuronic acid (to form gemfibrozil glucuronide), followed by a second incubation with NADPH. Liquid chromatography-tandem mass spectrometry analysis established that human liver microsomes and recombinant CYP2C8 both convert gemfibrozil glucuronide to a hydroxylated metabolite, with oxidative metabolism occurring on the dimethylphenoxy moiety (the group furthest from the glucuronide moiety). The results described have important implications for the mechanism of the clinical interaction reported between gemfibrozil and CYP2C8 substrates such as cerivastatin, repaglinide, rosiglitazone, and pioglitazone.

Expression of Constitutive Androstane Receptor, Hepatic Nuclear Factor 4α, and P450 Oxidoreductase Genes Determines Interindividual Variability in Basal Expression and Activity of a Broad Scope of Xenobiotic Metabolism Genes in the Human Liver

Identification of genetic variation predictive of clearance rate of a wide variety of prescription drugs could lead to cost-effective personalized medicine. Here we identify regulatory genes whose variable expression level among individuals may have widespread effects upon clearance rate of a variety of drugs. Twenty liver samples with variable CYP3A activity were profiled for expression level and activity of xenobiotic metabolism genes as well as genes involved in the regulation thereof. Regulatory genes whose expression level accounted for the highest degree of collinearity among expression levels of xenobiotic metabolism genes were identified as possible master regulators of drug clearance rate. Significant linear correlations (p < 0.05) were identified among mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, MRP2, OATP2, P450 oxidoreductase (POR), and UDP-glucuronosyltranferase 1A1, suggesting that these xenobiotic metabolism genes are coregulated at the transcriptional level. Using partial regression analysis, constitutive androstane receptor (CAR) and hepatic nuclear factor 4α (HNF4α) were identified as the nuclear receptors whose expression levels are most strongly associated with expression of coregulated xenobiotic metabolism genes. POR expression level, which is also associated with CAR and HNF4α expression level, was found to be strongly associated with the activity of many cytochromes P450. Thus, interindividual variation in the expression level of CAR, HNF4α, and POR probably determines variation in expression and activity of a broad scope of xenobiotic metabolism genes and, accordingly, clearance rate of a variety of xenobiotics. Identification of polymorphisms in these candidate master regulator genes that account for their variable expression among individuals may yield readily detectable biomarkers that could serve as predictors of xenobiotic clearance rate.

CYP4F Enzymes Are the Major Enzymes in Human Liver Microsomes That Catalyze the O-Demethylation of the Antiparasitic Prodrug DB289 [2,5-Bis(4-amidinophenyl)furan-bis-O-methylamidoxime]

DB289 [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime] is biotransformed to the potent antiparasitic diamidine DB75 [2,5-bis(4-amidinophenyl) furan] by sequential oxidative O-demethylation and reductive N-dehydroxylation reactions. Previous work demonstrated that the N-dehydroxylation reactions are catalyzed by cytochrome b5/NADH-cytochrome b5 reductase. Enzymes responsible for catalyzing the DB289 O-demethylation pathway have not been identified. We report an in vitro metabolism study to characterize enzymes in human liver microsomes (HLMs) that catalyze the initial O-demethylation of DB289 (M1 formation). Potent inhibition by 1-aminobenzotriazole confirmed that M1 formation is catalyzed by P450 enzymes. M1 formation by HLMs was NADPH-dependent, with a Km and Vmax of 0.5 μM and 3.8 nmol/min/mg protein, respectively. Initial screening showed that recombinant CYP1A1, CYP1A2, and CYP1B1 were efficient catalysts of M1 formation. However, none of these three enzymes was responsible for M1 formation by HLMs. Further screening showed that recombinant CYP2J2, CYP4F2, and CYP4F3B could also catalyze M1 formation. An antibody against CYP4F2, which inhibited both CYP4F2 and CYP4F3B, inhibited 91% of M1 formation by HLMs. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N′-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, effectively inhibited M1 formation by HLMs. Inhibition studies with ebastine and antibodies against CYP2J2 suggested that CYP2J2 was not involved in M1 formation by HLMs. Additionally, ketoconazole preferentially inhibited CYP4F2, but not CYP4F3B, and partially inhibited M1 formation by HLMs. We conclude that CYP4F enzymes (e.g., CYP4F2, CYP4F3B) are the major enzymes responsible for M1 formation by HLMs. These findings indicate that, in human liver, members of the CYP4F subfamily biotransform not only endogenous compounds but also xenobiotics.

The Proton Pump Inhibitor, Omeprazole, but not Lansoprazole or Pantoprazole, is a Metabolism-Dependent Inhibitor of CYP2C19: Implications for Coadministration with Clopidogrel

As a direct-acting inhibitor of CYP2C19 in vitro, lansoprazole is more potent than omeprazole and other proton pump inhibitors (PPIs), but lansoprazole does not cause clinically significant inhibition of CYP2C19 whereas omeprazole does. To investigate this apparent paradox, we evaluated omeprazole, esomeprazole, R-omeprazole, lansoprazole, and pantoprazole for their ability to function as direct-acting and metabolism-dependent inhibitors (MDIs) of CYP2C19 in pooled human liver microsomes (HLM) as well as in cryopreserved hepatocytes and recombinant CYP2C19. In HLM, all PPIs were found to be direct-acting inhibitors of CYP2C19 with IC50 values varying from 1.2 μM [lansoprazole; maximum plasma concentration (Cmax) = 2.2 μM] to 93 μM (pantoprazole; Cmax = 6.5 μM). In addition, we identified omeprazole, esomeprazole, R-omeprazole, and omeprazole sulfone as MDIs of CYP2C19 (they caused IC50 shifts after a 30-min preincubation with NADPH-fortified HLM of 4.2-, 10-, 2.5-, and 3.2-fold, respectively), whereas lansoprazole and pantoprazole were not MDIs (IC50 shifts < 1.5-fold). The metabolism-dependent inhibition of CYP2C19 by omeprazole and esomeprazole was not reversed by ultracentrifugation, suggesting that the inhibition was irreversible (or quasi-irreversible), whereas ultracentrifugation largely reversed such effects of R-omeprazole. Under various conditions, omeprazole inactivated CYP2C19 with KI (inhibitor concentration that supports half the maximal rate of inactivation) values of 1.7 to 9.1 μM and kinact (maximal rate of enzyme inactivation) values of 0.041 to 0.046 min−1. This study identified omeprazole, and esomeprazole, but not R-omeprazole, lansoprazole, or pantoprazole, as irreversible (or quasi-irreversible) MDIs of CYP2C19. These results have important implications for the mechanism of the clinical interaction reported between omeprazole and clopidogrel, as well as other CYP2C19 substrates.

In Vitro Inhibition and Induction of Human Liver Cytochrome P450 Enzymes by Milnacipran

Milnacipran (Savella) inhibits both norepinephrine and serotonin reuptake and is distinguished by a nearly 3-fold greater potency in inhibiting norepinephrine reuptake in vitro compared with serotonin. We evaluated the ability of milnacipran to inhibit and induce human cytochrome P450 enzymes in vitro. In human liver microsomes, milnacipran did not inhibit CYP1A2, 2B6, 2C8, 2C9, 2C19, or 2D6 (IC50 ≥ 100 μM); whereas, a comparator with dual reuptake properties [duloxetine (Cymbalta)] inhibited CYP2D6 (IC50 = 7 μM) and CYP2B6 (IC50 = 15 μM) with a relatively high potency. Milnacipran inhibited CYP3A4/5 in a substrate-dependent manner (i.e., midazolam 1′-hydroxylation IC50 ≈ 30 μM; testosterone 6β-hydroxylation IC50 ≈ 100 μM); whereas, duloxetine inhibited both CYP3A4/5 activities with equal potency (IC50 = 37 and 38 μM, respectively). Milnacipran produced no time-dependent inhibition (<10%) of P450 activity, whereas duloxetine produced time-dependent inhibition of CYP1A2, 2B6, 2C19, and 3A4/5. To evaluate P450 induction, freshly isolated human hepatocytes (n = 3) were cultured and treated once daily for 3 days with milnacipran (3, 10, and 30 μM), after which microsomal P450 activities were measured. Whereas positive controls (omeprazole, phenobarbital, and rifampin) caused anticipated P450 induction, milnacipran had minimal effect on CYP1A2, 2C8, 2C9, or 2C19 activity. The highest concentration of milnacipran (30 μM; >10 times plasma Cmax) produced 2.6- and 2.2-fold increases in CYP2B6 and CYP3A4/5 activity (making it 26 and 34% as effective as phenobarbital and rifampin, respectively). Given these results, milnacipran is not expected to cause clinically significant P450 inhibition or induction.

Production and purification of antibodies against rat liver P450 enzymes

Publisher Summary This chapter discusses the production and purification of antibodies against rat liver P450 enzymes. Antibodies can provide invaluable information on the structure, function, and regulation of P450 enzymes. The immunochemical relatedness of P450 enzymes, which is apparent when an antibody rose against one protein cross-reacts with other proteins, provides information on the structural relatedness of P450 enzymes. When used to isolate polysomal RNA or to screen complementary DNA (cDNA) expression libraries, antibodies can play a pivotal role in deducing the complete amino acid sequence of a P450 enzyme by recombinant DNA techniques. Antibody inhibition experiments provide information on the function of P450 enzymes. Such experiments provide a relatively unambiguous method of determining the extent to which a specific P450 enzyme contributes to reactions catalyzed by microsomal preparations, which is of interest because P450 enzymes have broad substrate specificities.

An evaluation of the dilution method for identifying metabolism-dependent inhibitors of cytochrome P450 enzymes.

Metabolism-dependent inhibition (MDI) of cytochrome P450 is usually assessed in vitro by examining whether the inhibitory potency of a drug candidate increases after a 30-min incubation with human liver microsomes (HLMs). To augment the IC50 shift, many researchers incorporate a dilution step whereby the samples, after being preincubated for 30 min with a high concentration of HLMs (with and without NADPH), are diluted before measuring P450 activity. In the present study, we show that the greater IC50 shift associated with the dilution method is a consequence of data processing. With the dilution method, IC50 values for direct-acting inhibitors vary with the dilution factor unless they are based on the final (postdilution) inhibitor concentration, whereas the IC50 values for MDIs vary with the dilution factor unless they are based on the initial (predilution) concentration. When the latter data are processed on the final inhibitor concentration, as is commonly done, the IC50 values for MDI (shifted IC50 values) decrease by the magnitude of the dilution factor. The lower shifted IC50 values are a consequence of data processing, not enhanced P450 inactivation. In fact, for many MDIs, increasing the concentration of HLMs actually leads to considerably less P450 inactivation because of inhibitor depletion and/or binding of the inhibitor to microsomes. A true increase in P450 inactivation and IC50 shift can be achieved by assessing MDI by a nondilution method and by decreasing the concentration of HLMs. These results have consequences for the conduct of MDI studies and the development of cut-off criteria.

A Long-Standing Mystery Solved: The Formation of 3-Hydroxydesloratadine Is Catalyzed by CYP2C8 But Prior Glucuronidation of Desloratadine by UDP-Glucuronosyltransferase 2B10 Is an Obligatory Requirement

Desloratadine (Clarinex), the major active metabolite of loratadine (Claritin), is a nonsedating long-lasting antihistamine that is widely used for the treatment of allergic rhinitis and chronic idiopathic urticaria. For over 20 years, it has remained a mystery as to which enzymes are responsible for the formation of 3-hydroxydesloratadine, the major active human metabolite, largely due to the inability of any in vitro system tested thus far to generate this metabolite. In this study, we demonstrated that cryopreserved human hepatocytes (CHHs) form 3-hydroxydesloratadine and its corresponding O-glucuronide. CHHs catalyzed the formation of 3-hydroxydesloratadine with a Km of 1.6 μM and a Vmax of 1.3 pmol/min per million cells. Chemical inhibition of cytochrome P450 (P450) enzymes in CHHs demonstrated that gemfibrozil glucuronide (CYP2C8 inhibitor) and 1-aminobenzotriazole (general P450 inhibitor) inhibited 3-hydroxydesloratadine formation by 91% and 98%, respectively. Other inhibitors of CYP2C8 (gemfibrozil, montelukast, clopidogrel glucuronide, repaglinide, and cerivastatin) also caused extensive inhibition of 3-hydroxydesloratadine formation (73%–100%). Assessment of desloratadine, amodiaquine, and paclitaxel metabolism by a panel of individual CHHs demonstrated that CYP2C8 marker activity robustly correlated with 3-hydroxydesloratadine formation (r2 of 0.70–0.90). Detailed mechanistic studies with sonicated or saponin-treated CHHs, human liver microsomes, and S9 fractions showed that both NADPH and UDP-glucuronic acid are required for 3-hydroxydesloratadine formation, and studies with recombinant UDP-glucuronosyltransferase (UGT) and P450 enzymes implicated the specific involvement of UGT2B10 in addition to CYP2C8. Overall, our results demonstrate for the first time that desloratadine glucuronidation by UGT2B10 followed by CYP2C8 oxidation and a deconjugation event are responsible for the formation of 3-hydroxydesloratadine.

Analysis of CYP mRNA expression by branched DNA technology.

Publisher Summary The branched DNA (bDNA) signal amplification assay provides a sensitive and relatively straightforward method for quantifying the levels of specific mRNAs. The cytochrome P450 (CYP) induction studies performed in laboratory with the bDNA signal amplification assay provided a reliable alternative to traditional techniques, such as enzyme analysis and immunoblotting, to detect the induction of CYP enzymes in human hepatocytes. The protocol developed that allows human hepatocytes cultured in one 60-mm petri dish to be analyzed for changes in expression of up to 30 genes, in duplicate. The assay is suitable for the measurement of any mRNA molecule, in cultured cells or tissue, for which a specific probe set can be developed. In laboratory, bDNA assay probe sets were developed to study the expression and regulation (inducibility) of the major human cytochrome P450 enzymes (CYPs), UDP-glucuronosyltransferases (UGTs), and sulfonotransferases (SULTs).

Metabolism-Dependent Inhibition of CYP3A4 by Lapatinib: Evidence for Formation of a Metabolic Intermediate Complex with a Nitroso/Oxime Metabolite Formed via a Nitrone Intermediate

Metabolism-dependent inhibition (MDI) of cytochrome P450 (P450) enzymes has the potential to cause clinically relevant drug-drug interactions. In the case of several alkylamine drugs, MDI of P450 involves formation of a metabolite that binds quasi-irreversibly to the ferrous heme iron to form a metabolic intermediate (MI) complex. The specific metabolites coordinately bound to ferrous iron and the pathways leading to MI complex formation are the subject of debate. We describe an approach combining heme iron oxidation with potassium ferricyanide and metabolite profiling to probe the mechanism of MI complex-based CYP3A4 inactivation by the secondary alkylamine drug lapatinib. Ten metabolites formed from lapatinib by CYP3A4-mediated heteroatom dealkylation, C-hydroxylation, N-oxygenation with or without further oxidation, or a combination thereof, were detected by accurate mass spectrometry. The abundance of one metabolite, the N-dealkylated nitroso/oxime lapatinib metabolite (M9), correlated directly with the prevalence or the disruption of the MI complex with CYP3A4. Nitroso/oxime metabolite formation from secondary alkylamines has been proposed to occur through two possible pathways: (1) sequential N-dealkylation, N-hydroxylation, and dehydrogenation (primary hydroxylamine pathway) or (2) N-hydroxylation with dehydrogenation to yield a nitrone followed by N-dealkylation (secondary hydroxylamine pathway). All intermediates for the secondary hydroxylamine pathway were detected but the primary N-hydroxylamine intermediate of the primary hydroxylamine pathway was not. Our findings support the mechanism of lapatinib CYP3A4 inactivation as MI complex formation with the nitroso metabolite formed through the secondary hydroxylamine and nitrone pathway, rather than by N-dealkylation to the primary amine followed by N-hydroxylation and dehydrogenation as is usually assumed.