An Evaluation of the Dilution Method for Identifying Metabolism-Dependent Inhibitors of Cytochrome P450 Enzymes

Parkinson, Andrew; Kazmi, Faraz; Buckley, David B.; Yerino, Phyllis; Paris, Brandy L.; Holsapple, Jeff; Toren, Paul; Otradovec, Steve M.; Ogilvie, Brian W.

doi:10.1124/dmd.111.038596

Cited by 67 publications

(76 citation statements)

References 32 publications

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“…As expected, these compounds all showed significant TDI, exhibiting IC 50 shifts (i.e., the ratios of the inhibitor IC 50 values determined without versus with a 30-minute inhibitor/NADPH preincubation step) of between 9.4 (paroxetine/CYP2D6) and 40 (troleandomycin/CYP3A4). By contrast, the largest IC 50 shift we observed for parent drug or AMIO metabolite was 3.1, determined for the inhibition of CYP2D6 activity by MDEA, or roughly twice the generally accepted significance threshold for a TDI (Parkinson et al, 2011). Inclusion of glutathione and N-acetylcysteine in microsomal incubations modestly reduced the IC 50 shift ratios for CYP2D6, CYP3A4, and CYP2C9 activities to between 1.5 and 2.1 (data not shown).…”

Section: Resultsmentioning

confidence: 68%

“…Furafylline, tienilic acid, paroxetine, and troleandomycin are specific mechanism-based inactivators of CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively, and so they were used as positive controls for these IC 50 shift experiments (Berry and Zhao, 2008;Parkinson et al, 2011). As expected, these compounds all showed significant TDI, exhibiting IC 50 shifts (i.e., the ratios of the inhibitor IC 50 values determined without versus with a 30-minute inhibitor/NADPH preincubation step) of between 9.4 (paroxetine/CYP2D6) and 40 (troleandomycin/CYP3A4).…”

Section: Resultsmentioning

confidence: 99%

“…If TDI of the enzyme is occurring, a 30-minute preincubation of a drug candidate in the presence of NADPH should result in increased inhibitory potency relative to a 30-minute incubation of the compound in the absence of cofactor (lowering the former IC 50 , and thus shifting it to the left of the latter curve). Generally, a ratio of the nonshifted IC 50 value (preincubated minus NADPH) to the shifted IC 50 value (preincubated with NADPH) of greater than ;1.5 is used as an indicator of potential TDI (Parkinson et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

“…In the course of preclinical drug discovery in the pharmaceutical industry, IC 50 shift experiments are commonly used to screen out compounds that act as time-dependent inhibitors of the major HL P450s (Obach et al, 2007;Parkinson et al, 2011). If TDI of the enzyme is occurring, a 30-minute preincubation of a drug candidate in the presence of NADPH should result in increased inhibitory potency relative to a 30-minute incubation of the compound in the absence of cofactor (lowering the former IC 50 , and thus shifting it to the left of the latter curve).…”

Section: Discussionmentioning

confidence: 99%

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P450-Based Drug-Drug Interactions of Amiodarone and its Metabolites: Diversity of Inhibitory Mechanisms

McDonald

Rettie

2015

Drug Metab Dispos

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In this study, IC 50 shift and time-dependent inhibition (TDI) experiments were carried out to measure the ability of amiodarone (AMIO), and its circulating human metabolites, to reversibly and irreversibly inhibit CYP1A2, CYP2C9, CYP2D6, and CYP3A4 activities in human liver microsomes. The [I] u /K i,u values were calculated and used to predict in vivo AMIO drug-drug interactions (DDIs) for pharmaceuticals metabolized by these four enzymes. Based on these values, the minor metabolite N,N-didesethylamiodarone (DDEA) is predicted to be the major cause of DDIs with xenobiotics primarily metabolized by CYP1A2, CYP2C9, or CYP3A4, while AMIO and its N-monodesethylamiodarone (MDEA) derivative are the most likely cause of interactions involving inhibition of CYP2D6 metabolism. AMIO drug interactions predicted from the reversible inhibition of the four P450 activities were found to be in good agreement with the magnitude of reported clinical DDIs with lidocaine, warfarin, metoprolol, and simvastatin. The TDI experiments showed DDEA to be a potent inactivator of CYP1A2 (K I = 0.46 mM, k inact = 0.030 minute 21 ), while MDEA was a moderate inactivator of both CYP2D6 (K I = 2.7 mM, k inact = 0.018 minute 21) and CYP3A4 (K I = 2.6 mM, k inact = 0.016 minute 21). For DDEA and MDEA, mechanism-based inactivation appears to occur through formation of a metabolic intermediate complex.Additional metabolic studies strongly suggest that CYP3A4 is the primary microsomal enzyme involved in the metabolism of AMIO to both MDEA and DDEA. In summary, these studies demonstrate both the diversity of inhibitory mechanisms with AMIO and the need to consider metabolites as the culprit in inhibitory P450-based DDIs.

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Section: Resultsmentioning

confidence: 68%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

P450-Based Drug-Drug Interactions of Amiodarone and its Metabolites: Diversity of Inhibitory Mechanisms

McDonald

Rettie

2015

Drug Metab Dispos

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“…All other deuterated glucuronides were purchased from Toronto Research Chemicals (Toronto, ON, Canada). The sources of all other reagents have been described previously (Ogilvie et al, 2006;Parkinson et al, 2011;Kazmi et al, 2014).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2015

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Desloratadine (Clarinex), the major active metabolite of loratadine (Claritin), is a nonsedating antihistamine used for the treatment of seasonal allergies and hives. Previously we reported that the formation of 3-hydroxydesloratadine, the major human metabolite of desloratadine, involves three sequential reactions, namely N-glucuronidation by UGT2B10 followed by 3-hydroxylation by CYP2C8 followed by deconjugation (rapid, nonenzymatic hydrolysis of the N-glucuronide). In this study we assessed the perpetrator potential of desloratadine based on in vitro studies of its inhibitory effects on cytochrome P450 and UDP-glucuronosyltransferase (UGT) enzymes in human liver microsomes (HLM). Desloratadine (10 mM) caused no inhibition (<15%) of CYP1A2, CYP2C8, CYP2C9, or CYP2C19 and weak inhibition (32-48%) of CYP2B6, CYP2D6, and CYP3A4/5. In cryopreserved human hepatocytes (CHH), which can form the CYP2C8 substrate desloratadine N-glucuronide, desloratadine did not inhibit the CYP2C8-dependent metabolism of paclitaxel or amodiaquine. Assessment of UGT inhibition identified desloratadine as a potent and relatively selective competitive inhibitor of UGT2B10 (K i value of 1.3 mM). Chemical inhibition of UGT enzymes in HLM demonstrated that nicotine (UGT2B10 inhibitor) but not hecogenin (UGT1A4 inhibitor) completely inhibited the conversion of desloratadine (1 mM) to 3-hydroxydesloratadine in HLM fortified with both NADPH and UDP-glucuronic acid. 3-Hydroxydesloratadine formation correlated well with levomedetomidine glucuronidation (UGT2B10 marker activity) with a panel of individual CHH (r 2 = 0.72). Overall, the results of this study confirm the role of UGT2B10 in 3-hydroxydesloratadine formation and identify desloratadine as a relatively selective in vitro inhibitor of UGT2B10.

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Assessment of the Time‐Dependent Inhibition (TDI) Potential of Test Compounds with Human Liver Microsomes by IC₅₀ Shift Method Using a Nondilution Approach

Ron¹,

Rajaraman²

2012

CP Pharmacology

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Time-dependent inhibition (TDI) of hepatic cytochrome P450 (CYP) enzymes is increasingly implicated in the majority of clinically relevant drug-drug interactions (DDIs). A time-dependent inhibitor or its reactive metabolite irreversibly inactivates CYP enzymes, thereby inhibiting the metabolism of other drugs. As the majority of marketed drugs are metabolized by CYP enzymes, their inhibition has important clinical consequences, such as in decreasing the metabolic clearance of a co-administered drug (victim drug). This could be life threatening, as such an effect narrows the therapeutic index for drugs such as warfarin and other potentially toxic agents. Therefore, it is essential to examine new chemical entities for their potential to cause TDI to minimize adverse drug reactions during human studies and use. This unit presents an in vitro procedure utilizing a nondilution method in human liver microsomes for determining the TDI potential of test compounds.

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An Evaluation of the Dilution Method for Identifying Metabolism-Dependent Inhibitors of Cytochrome P450 Enzymes

Cited by 67 publications

References 32 publications

P450-Based Drug-Drug Interactions of Amiodarone and its Metabolites: Diversity of Inhibitory Mechanisms

P450-Based Drug-Drug Interactions of Amiodarone and its Metabolites: Diversity of Inhibitory Mechanisms

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

Assessment of the Time‐Dependent Inhibition (TDI) Potential of Test Compounds with Human Liver Microsomes by IC₅₀ Shift Method Using a Nondilution Approach

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An Evaluation of the Dilution Method for Identifying Metabolism-Dependent Inhibitors of Cytochrome P450 Enzymes

Cited by 67 publications

References 32 publications

P450-Based Drug-Drug Interactions of Amiodarone and its Metabolites: Diversity of Inhibitory Mechanisms

P450-Based Drug-Drug Interactions of Amiodarone and its Metabolites: Diversity of Inhibitory Mechanisms

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

Assessment of the Time‐Dependent Inhibition (TDI) Potential of Test Compounds with Human Liver Microsomes by IC50 Shift Method Using a Nondilution Approach

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Assessment of the Time‐Dependent Inhibition (TDI) Potential of Test Compounds with Human Liver Microsomes by IC₅₀ Shift Method Using a Nondilution Approach