Publications & White Papers

An assessment of the in vitro inhibition of cytochrome P450 enzymes (CYP), UDP-glucuronsyltransferases (UGT) and transporters by phosphodiester- or phosphorothioate-linked oligonucleotides

Published:  07 May 2018

Faraz Kazmi, Phyllis Yerino, Chase McCoy, Andrew Parkinson, David B Buckley and Brian W Ogilvie

Oligonucleotides represent an expanding class of pharmacotherapeutics in development for various indications. Typically, oligonucleotides are developed with phosphorothioate linkages for the improvement of biological stability; however limited data are available on the potential of these molecules to cause drug-drug interactions (DDIs). In the present study, two non therapeutic oligonucleotides with either phosphodiester (PD-GP and PD-Ac) or phosphorothioate (PT-GP and PT-Ac) linkages were evaluated in vitro for their potential to inhibit P450s and UGTs in both human liver microsomes (HLM) and cryopreserved human hepatocytes (CHH) and to inhibit select transporters in expression systems. PD-GP and PD-Ac had little-to-no inhibitory effect on any P450 or UGT enzymes in HLM and CHH except for PD-Ac in HLM for CYP2C19 (IC50 = 29 μM). Conversely, PT-GP and PT-Ac caused direct inhibition of almost all P450 and UGT enzymes, with CYP1A2 (IC50 values 0.8-4.2 μM), CYP2C8 (IC50 values 1.1-12 μM) and UGT1A1 (IC50 values 4.5-5.4 μM) inhibited to the greatest extent. There was evidence of possible time-dependent inhibition (TDI) of CYP enzymes with PT-GP and PT-Ac for CYP2B6, CYP2C8, CYP2C19, CYP2C9, CYP2D6 and CYP3A4/5; however, this TDI was reversible. In contrast to HLM, there was little-to-no direct CYP inhibition by any oligonucleotide in CHH (except for PD-Ac with CYP2C19 [IC50 = 36 μM] and TDI by PT-GP with CYP2C8), demonstrating test system-dependent outcomes. Inhibition was observed for the organic anion uptake transporters OATP1B1, OATP1B3, OAT1, OAT3, and OCT2 (IC50 values 12-29 μM) but not OCT1 or the efflux transporters BCRP and P-gp by the phosphorothioate oligonucleotides.

Direct and cytokine‐mediated effects of albumin‐fused growth hormone, TV‐1106, on CYP enzyme expression in human hepatocytes in vitro

Published:  16 April 2018

Maciej Czerwiński, Immaculate Amunom, Victor Piryatinsky, Hussein Hallak, Yousif Sahly, Oren Bar‐Ilan, Paul Bolliger, Merav Bassan

Some biologics can modulate cytokines that may lead to changes in expression of drug‐metabolizing enzymes and cause drug‐drug interactions (DDI). DDI potential of TV‐1106—an albumin‐fused growth hormone (GH)—was investigated. In this study, human blood was exposed to recombinant human growth hormone (rhGH) or TV‐1106, followed by isolation of the plasma and its application to human hepatocytes. While the treatment of blood with rhGH increased multiple cytokines, treatment of blood with TV‐1106 had no effect on any of the nine cytokines tested. The interleukin (IL)‐6 concentration was higher in the rhGH then in the TV‐1106‐treated plasma (P< .05). While rhGH had little or no effect on CYP1A2 or CYP2C19 mRNA but increased CYP3A4 mRNA twofold, TV‐1106 had little or no effect on cytochrome P450 (CYP) mRNAs in hepatocytes. Although the plasma from rhGH‐treated blood lowered CYP1A2 activity, the TV‐1106 plasma had no effect on CYP activities. The CYP1A2 activity was lower in the rhGH‐ then in the TV‐1106‐plasma treated hepatocytes (P < .05). The results indicated that fusing GH with albumin made TV‐1106 an unlikely participant of CYP1A2, CYP2C19 or CYP3A4‐facilitated, direct or cytokine‐driven DDI.

In Vitro Studies with Two Human Organic Anion Transporters: OAT2 & OAT7

Published:  25 September 2017

Mathialagan S, Costales C, Tylaska L, Kimoto E, Vildhede A, Johnson J, Johnson N, Sarashina T, Hashizume K, Isringhausen CD, Vermeer LMM, Wolff AR, Rodrigues AD

Penciclovir, ganciclovir, creatinine, para-aminohippuric acid (PAH), ketoprofen, estrone 3-O-sulfate (E3S), dehydroepiandrosterone 3-O-sulfate (DHEAS), and cyclic guanosine monophosphate (cGMP) were screened as substrates of human liver organic anion transporters OAT2 and OAT7. For OAT7, high uptake ratios (versus mock transfected HEK293 cells) of 29.6 and 15.3 were obtained with E3S and DHEAS. Less robust uptake ratios (≤ 3.6) were evident with the other substrates. OAT2 (transcript variant 1, OAT2-tv1) presented high uptake ratios of 30, 13, ∼35, ∼25, 8.5, and 9 with cGMP, PAH, penciclovir, ganciclovir, creatinine, and E3S, respectively. No uptake was observed with DHEAS. Although not a substrate of either transporter, ketoprofen did inhibit transfected OAT2-tv1 (IC50 of 17, 22, 23, 24, 35, and 586 μM; creatinine, ganciclovir, penciclovir, cGMP, E3S, and prostaglandin F2α, respectively) and penciclovir uptake (IC50 = 27 µM; >90% inhibition) by plated human hepatocytes (PHH). It is concluded that penciclovir and ketoprofen may serve as useful tools for the assessment of OAT2 activity in PHH. However, measurement of OAT7 activity therein will prove more challenging, as high uptake rates are evident with E3S and DHEAS only and both sulfoconjugates are known to be substrates of organic anion transporting polypeptides.

DDI Guidance Recommendations on Down-Regulation, CYP2C Induction and CYP2B6 Control

Published:  23 June 2017

Considerations from the IQ Induction Working Group in Response to Drug-Drug Interaction Guidances from Regulatory Agencies: Focus on Down-regulation, CYP2C Induction and CYP2B6 Positive Control

The European Medicines Agency (EMA), the Pharmaceutical and Medical Devices Agency and the Food and Drug Administration have issued guidances for the conduct of drug-drug interaction (DDI) studies. To examine the applicability of these regulatory recommendations specifically for induction, a group of scientists, under the auspices of the Drug Metabolism Leadership Group of the Innovation and Quality (IQ) Consortium, formed the Induction Working Group (WG). A team of 20 scientists, from 16 of the 39 pharmaceutical companies, which are members of the IQ Consortium, and three Contract Research Organizations, reviewed the recommendations, focusing initially on the current EMA guidance. Questions were collated from IQ member companies as to which aspects of the guidance required further evaluation. The EMA was then approached to provide insights into their recommendations on the following points; a) evaluation of down-regulation, b) in vitro assessment of CYP2C induction, c) the recommendation to use CITCO as the positive control for CYP2B6 induction, d) data interpretation (two-fold increase in mRNA as evidence of induction), and e) duration of incubation of hepatocytes with test article. The Induction WG conducted an anonymous survey among IQ member companies to query current practices, specifically focusing on how the aforementioned key points are evaluated. Responses were received from 19 companies. All data/information was blinded prior to being shared with the Induction WG. The results of the survey are presented together with consensus recommendations on down-regulation, CYP2C induction and CYP2B6 positive control. Data interpretation and incubation duration will be reported in subsequent manuscripts.

Evaluation of Ketoconazole and its Alternative Clinical CYP3A4/5 Inhibitors as Inhibitors of Drug Transporters: The In Vitro Effects of Ketoconazole, Ritonavir, Clarithromycin and Itraconazole on 13 Clinically-Relevant Drug Transporters

Published:  14 December 2015

Lydia M.M. Vermeer, Caleb D. Isringhausen, Brian W. Ogilvie, and David B. Buckley

Ketoconazole is a potent CYP3A4/5 inhibitor, and until recently, recommended by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) as a "strong" CYP3A4/5 inhibitor in clinical drug-drug interaction (DDI) studies. Ketoconazole sporadically causes liver injury or adrenal insufficiency. Because of this, the FDA and EMA recommended suspension of ketoconazole use in DDI studies in 2013. FDA specifically recommended use of clarithromycin or itraconazole as alternative strong CYP3A4/5 inhibitors for use in clinical DDI studies, but many investigators have also used ritonavir as an alternative. Although the effects of these clinical CYP3A4/5 inhibitors on other CYPs are largely established, reports on the effects on the broad range of drug transporter activities are sparse. In this study, the inhibitory effects of ketoconazole, clarithromycin, ritonavir and itraconazole (and its CYP3A4-inhibitory metabolites, hydroxy-, keto- and N-desalkyl itraconazole) towards 13 drug transporters (OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, MATE2-K, P-gp, BCRP, MRP2, MRP3 and BSEP) were systematically assessed in transporter-expressing HEK-293 cell lines or membrane vesicles. In vitro findings were translated into clinical context with the basic static model approaches outlined by the FDA in their 2012 draft guidance on DDIs. The results indicate that, like ketoconazole, the alternative clinical CYP3A4/5 inhibitors ritonavir, clarithromycin and itraconazole each have unique transporter inhibition profiles. None of the alternatives to ketoconazole provided a clean inhibition profile towards the 13 drug transporters evaluated. The results provide guidance for the selection of clinical CYP3A4/5 inhibitors when transporters are potentially involved in a victim drug's pharmacokinetics.

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The reliability of estimating Ki values for direct, reversible inhibition of cytochrome P450 enzymes from corresponding IC50 values: A retrospective analysis of 343 experiments

Published:  09 September 2015

Lois J. Haupt, Faraz Kazmi, Brian W. Ogilvie, David B. Buckley, Brian D. Smith, Sarah Leatherman, Brandy Paris, Oliver Parkinson, and Andrew Parkinson

In the present study we conducted a retrospective analysis of 343 in vitro experiments to ascertain whether observed (experimentally determined) values of Ki for reversible P450 inhibition could be reliably predicted by dividing the corresponding IC50 values by two, based on the relationship that, for competitive inhibition, Ki = IC50/2 when [S] = Km. Values of Ki and IC50 were determined under the following conditions: (1) the concentration of P450 marker substrate, [S], was equal to Km (for IC50 determinations) and spanned Km (for Ki determinations); (2) the substrate incubation time was short (5 min) to minimize metabolism-dependent inhibition and inhibitor depletion, and (3) the concentration of human liver microsomes was low (0.1 mg/mL or less) to maximize the unbound fraction of inhibitor. Under these conditions, predicted Ki values, based on IC50/2, correlated strongly with experimentally observed Ki determinations (r = 0.940; average fold error [AFE] = 1.10). Of the 343 predicted Ki values, 316 (92%) were within a factor of 2 of the experimentally determined Ki values and only one value fell outside a threefold range. In the case of noncompetitive inhibitors, Ki values predicted from IC50/2 values were overestimated by a factor of nearly two (AFE = 1.85; n = 13), which is to be expected because, for noncompetitive inhibition, Ki = IC50 (not IC50/2). The results suggest that, under appropriate experimental conditions with the substrate concentration equal to Km, values of Ki for direct, reversible inhibition can be reliably estimated from values of IC50/2.

Further Characterization of the Metabolism of Desloratadine and its Cytochrome P450 and UDP-Glucuronosyltransferase (UGT) Inhibition Potential: Identification of Desloratadine as a Relatively Selective UGT2B10 Inhibitor

Published:  01 July 2015

Faraz Kazmi, Phyllis Yerino, Joanna E Barbara, and Andrew Parkinson

Desloratadine (Clarinex), the major active metabolite of loratadine (Claritin), is a non-sedating 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 de-conjugation (rapid, non-enzymatic 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 μM) 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 (Ki value of 1.3 μM). Chemical inhibition of UGT enzymes in HLM demonstrated that nicotine (UGT2B10 inhibitor) but not hecogenin (UGT1A4 inhibitor) completely inhibited the conversion of desloratadine (1 μM) 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 (r2 = 0.72). Overall, the results of this study confirm the role of UGT2B10 in 3-hydroxydesloratadine formation and identify desloratadine as a selective in vitro inhibitor of UGT2B10.

Clinical Assessment of Drug-Drug Interactions of Tasimelteon, a Novel Dual Melatonin Receptor Agonist

Published:  07 May 2015

Brian W. Ogilvie, Rosarelis Torres, Marlene A. Dressman, William G. Kramer and Paolo Baroldi

Tasimelteon ([1R-trans]-N-[(2-[2,3-dihydro-4-benzofuranyl] cyclopropyl) methyl] propanamide), a novel dual melatonin receptor agonist that demonstrates specificity and high affinity for melatonin receptor types 1 and 2 (MT1 and MT2 receptors), is the first treatment approved by the US Food and Drug Administration for Non-24-Hour Sleep-Wake Disorder. Tasimelteon is rapidly absorbed, with a mean absolute bioavailability of approximately 38%, and is extensively metabolized primarily by oxidation at multiple sites, mainly by cytochrome P450 (CYP) 1A2 and CYP3A4/5, as initially demonstrated by in vitro studies and confirmed by the results of clinical drug–drug interactions presented here. The effects of strong inhibitors and moderate or strong inducers of CYP1A2 and CYP3A4/5 on the pharmacokinetics of tasimelteon were evaluated in humans. Coadministration with fluvoxamine resulted in an approximately 6.5-fold increase in tasimelteon's area under the curve (AUC), whereas cigarette smoking decreased tasimelteon's exposure by approximately 40%. Coadministration with ketoconazole resulted in an approximately 54% increase in tasimelteon's AUC, whereas rifampin pretreatment resulted in a decrease in tasimelteon's exposure of approximately 89%.

A long-standing mystery solved: The formation of 3-hydroxydesloratadine is catalyzed by CYP2C8 but prior glucuronidation of desloratadine by UGT2B10 is an obligatory requirement

Published:  16 January 2015

Faraz Kazmi, Joanna E. Barbara, Phyllis Yerino and Andrew Parkinson

Desloratadine (Clarinex®), the major active metabolite of loratadine (Claritin®), is a non-sedating long-lasting antihistamine 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 (CHH) form 3-hydroxydesloratadine and its corresponding O-glucuronide. CHHs catalyzed the formation of 3-hydroxydesloratadine with a Km of 1.6 μM and Vmax of 1.3 pmol/min/million cells. Chemical inhibition of cytochrome P450 (CYP) enzymes in CHH 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 both required for 3-hydroxydesloratadine formation, and studies with recombinant UGT and CYP 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 de-conjugation event are responsible for the formation of 3-hydroxydesloratadine.

Anti-CD28 Monoclonal Antibody-stimulated Cytokines Released from Blood Suppress CYP1A2, CYP2B6 and CYP3A4 in Human Hepatocytes In Vitro

Published:  17 October 2014

Maciej Czerwiński, Faraz Kazmi, Andrew Parkinson, and David B. Buckley

Like most infections and certain inflammatory diseases, some therapeutic proteins cause a cytokine-mediated suppression of hepatic drug-metabolizing enzymes, which may lead to pharmacokinetic interactions with small-molecule drugs. We propose a new in vitro method to evaluate the whole blood–mediated effects of therapeutic proteins on drug-metabolizing enzymes in human hepatocytes cocultured with Kupffer cells. The traditional method involves treating hepatocyte cocultures with the therapeutic protein, which detects hepatocyte- and macrophage-mediated suppression of cytochrome P450 (P450). The new method involves treating whole human blood with a therapeutic protein to stimulate the release of cytokines from peripheral blood mononuclear cells (PBMCs), after which plasma is prepared and added to the hepatocyte coculture to evaluate P450 enzyme expression. In this study, human blood was treated for 24 hours at 37°C with bacterial lipopolysaccharide (LPS) or ANC28.1, an antibody against human T-cell receptor CD28. Cytokines were measured in plasma by sandwich immunoassay with electrochemiluminescense detection. Treatment of human hepatocyte cocultures with LPS or with plasma from LPS-treated blood markedly reduced the expression of CYP1A2, CYP2B6, and CYP3A4. However, treatment of hepatocyte cocultures with ANC28.1 did not suppress P450 expression, but treatment with plasma from ANC28.1-treated blood suppressed CYP1A2, CYP2B6, and CYP3A4 activity and mRNA levels. The results demonstrated that applying plasma from human blood treated with a therapeutic protein to hepatocytes cocultured with Kupffer cells is a suitable method to identify those therapeutic proteins that suppress P450 expression by an indirect mechanism—namely, the release of cytokines from PBMCs.

Drugs as Victims and Perpetrators and the Pharmacokinetic Concept of Maximum Exposure

Published:  01 October 2014

Brian W. Ogilvie, Andrew Parkinson

This article describes the dramatic but predictable impact of two perpetrators on the disposition of a victim drug. For a single elimination pathway, victim potential can be quantified as fm (fractional metabolism by an enzyme) or fe (fractional elimination by a transporter) as follows:


where AUCi and AUCui are the plasma AUC (area under the curve) values in the presence and absence of an inhibitor of the enzyme (or transporter) responsible for eliminating the victim drug, respectively, and where AUCEM and AUCPM are the plasma AUC values in genetically determined extensive metabolizers (EMs) and poor metabolizers, respectively. For a drug that is cleared by two parallel pathways (routes A and B), victim potential can be similarly quantified:


If routes A and B both had an fm (or fe) value of 0.49 (i.e., fmA = 0.49 and fmB = 0.49), then loss of only route A or only route B would result in a 1.96-fold increase in systemic exposure to the victim drug (1.96 = 1/(1 − 0.49)). However, loss of both route A and route B would result in a dramatic 50-fold increase in systemic exposure (50 = 1/(1 − 0.98)). This article provides several examples of perpetrator–perpetrator–victim interactions that demonstrate (i) the clinical relevance of this phenomenon, which is often referred to as maximum exposure; (ii) the large difference in the impact of an enzyme/transporter inhibitor in genetically determined EMs and PMs; and (iii) the potentially large impact of a perpetrator drug that inhibits multiple-drug-metabolizing enzymes or transporters.

In vitro inhibition of human liver cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes by rose bengal: system-dependent effects on inhibitory potential

Published:  01 July 2014

Faraz Kazmi, Lois J. Haupt, Jennifer R. Horkman, Brian D. Smith, David B. Buckley, Eric A. Wachter, and Jamie M. Singer

1. Rose bengal (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) is being developed for the treatment of cutaneous melanoma and hepatocellular carcinoma. Interestingly, rose bengal can generate singlet oxygen species upon exposure to light. 2. We evaluated rose bengal as an in vitro inhibitor of cytochrome P450 (CYP) or UDP-glucuronosyltransferase (UGT) enzymes in both human liver microsomes (HLM) and cryopreserved human hepatocytes (CHHs) under both yellow light and dark conditions. 3. Rose bengal directly inhibited CYP3A4/5 and UGT1A6 in HLM under yellow light with inhibitor concentration that causes 50% inhibition (IC50) values of 0.072 and 0.035 μM, respectively; whereas much less inhibition was observed in the dark with the IC50 values increasing 43- and 120-fold, respectively. To determine if a more physiologically-relevant test system could be protected from such an effect, rose bengal was evaluated as an inhibitor of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4/5 and UGT enzymes in CHH. All IC50 values were similar (64 ± 8 μM) and little to no effect of light on inhibitory potential was observed. 4. Given the IC50 values in CHH increased an order of magnitude compared to HLM and the atypical pharmacokinetics of the drug, the risk of rose bengal to cause clinically relevant drug-drug interactions is likely low, particularly when administered to cancer patients on an intermittent schedule.

Chapter 6: Biotransformation of Xenobiotics

Published:  02 September 2013

Andrew Parkinson, Brian W. Ogilvie, David B. Buckley, Faraz Kazmi, Maciej Czerwinski and Oliver Parkinson
This book chapter is from Casarett & Doull's Toxicology, The Basic Science of Poisons, 8th Edition, Edited by Curtis Klaassen

Due to copyright law, we are unable to distribute an electronic version of this reprint. The "download publication" button below will redirect you to where you can request a printed copy be sent to you.

Exploring the Utility of High-Resolution MS with Post-Acquisition Data Mining for Simultaneous Exogenous and Endogenous Metabolite Profiling

Published:  15 May 2013

Barbara JE, Buckley DB, Horrigan MJ
The utility of high-resolution MS (HRMS) with post-acquisition data mining in DMPK goes much further than the now established approach to simultaneously acquire quantitative and qualitative information for lead compounds at the discovery stage. Indeed, HRMS has promise for addressing multiple complex drug-development applications in a single experiment. In the present study, one HRMS dataset acquired for in vitro incubations of the model compound dasatinib was mined post-acquisition to address four different issues: stability, metabolite profiling, glutathione conjugate analysis, and endogenous lipid profiling.

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

Published:  01 May 2013

Barbara JE, Kazmi F, Parkinson A, Buckley DB
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.

Lysosomal Sequestration (Trapping) of Lipophilic Amine (Cationic Amphiphilic) Drugs in Immortalized Human Hepatocytes (Fa2N-4 cells)

Published:  15 April 2013

Kazmi F, Hensley T, Pope C, Funk RS, Loewen GJ, Buckley DB, Parkinson A
Lipophilic (logP > 1) and amphiphilic drugs (also known as cationic amphiphilic drugs) with ionizable amines (pKa > 6) can accumulate in lysosomes, a process known as lysosomal trapping. This process contributes to presystemic extraction by lysosome-rich organs (such as liver and lung), which, together with the binding of lipophilic amines to phospholipids, contributes to the large volume of distribution characteristic of numerous cardiovascular and central nervous system drugs. Accumulation of lipophilic amines in lysosomes has been implicated as a cause of phospholipidosis. Furthermore, elevated levels of lipophilic amines in lysosomes can lead to high organ-to-blood ratios of drugs that can be mistaken for active drug transport. In the present study, we describe an in vitro fluorescence-based method (using the lysosome-specific probe LysoTracker Red) to identify lysosomotropic agents in immortalized hepatocytes (Fa2N-4 cells). A diverse set of compounds with various physicochemical properties were tested, such as acids, bases, and zwitterions. In addition, the partitioning of the nonlysosomotropic atorvastatin (an anion) and the lysosomotropics propranolol and imipramine (cations) were quantified in Fa2N-4 cells in the presence or absence of various lysosomotropic or nonlysosomotropic agents and inhibitors of lysosomal sequestration (NH4Cl, nigericin, and monensin). Cellular partitioning of propranolol and imipramine was markedly reduced (by at least 40%) by NH4Cl, nigericin, or monensin. Lysosomotropic drugs also inhibited the partitioning of propranolol by at least 50%, with imipramine partitioning affected to a lesser degree. This study demonstrates the usefulness of immortalized hepatocytes (Fa2N-4 cells) for determining the lysosomal sequestration of lipophilic amines.

High-Resolution Mass Spectrometry Elucidates Metabonate (False Metabolite) Formation from Alkylamine Drugs During In Vitro Metabolite Profiling

Published:  01 October 2012

Barbara JE, Kazmi F, Muranjan S, Toren PC, Parkinson A
In vitro metabolite profiling and characterization experiments are widely employed in early drug development to support safety studies. Samples from incubations of investigational drugs with liver microsomes or hepatocytes are commonly analyzed by liquid chromatography/mass spectrometry for detection and structural elucidation of metabolites. Advanced mass spectrometers with accurate mass capabilities are becoming increasingly popular for characterization of drugs and metabolites, spurring changes in the routine workflows applied. In the present study, using a generic full-scan high-resolution data acquisition approach with a time-of-flight mass spectrometer combined with postacquisition data mining, we detected and characterized metabonates (false metabolites) in microsomal incubations of several alkylamine drugs. If a targeted approach to mass spectrometric detection (without full-scan acquisition and appropriate data mining) were employed, the metabonates may not have been detected, hence their formation underappreciated. In the absence of accurate mass data, the metabonate formation would have been incorrectly characterized because the detected metabonates manifested as direct cyanide-trapped conjugates or as cyanide-trapped metabolites formed from the parent drugs by the addition of 14 Da, the mass shift commonly associated with oxidation to yield a carbonyl. This study demonstrates that high-resolution mass spectrometry and the associated workflow is very useful for the detection and characterization of unpredicted sample components and that accurate mass data were critical to assignment of the correct metabonate structures. In addition, for drugs containing an alkylamine moiety, the results suggest that multiple negative controls and chemical trapping agents may be necessary to correctly interpret the results of in vitro experiments.

Catechol and aldehyde moieties of 3,4-dihydroxyphenylacetaldehyde contribute to tyrosine hydroxylase inhibition and neurotoxicity

Published:  30 July 2012

Lydia M. Vermeer , Virginia R. Florang , Jonathan A. Doorn

Parkinson’s disease (PD) is a progressive neurodegenerative disorder which leads to the selective loss of dopaminergic neurons. This causes a decrease in the important neurotransmitter dopamine (DA), which is essential for coordinated movement. Previous studies have implicated the monoamine oxidase metabolite of DA, 3,4-dihydroxphenylacetaldehyde (DOPAL), in the pathogenesis of PD and have shown it to be a reactive intermediate capable of protein modification. DOPAL also has demonstrated the ability to cause mitochondrial dysfunction and lead to significant inhibition of the rate-limiting enzyme in DA synthesis, tyrosine hydroxylase (TH). The current study was undertaken to investigate four analogs of DOPAL, including a novel nitrile analog, to determine how the structure of DOPAL is related to its toxicity and inhibition of TH. Both mitochondrial function and inhibition of TH in cell lysate were investigated. Furthermore, a novel whole cell assay was designed to determine the consequence to enzyme action when DOPAL levels were elevated. The results presented here demonstrate that changes to DOPAL structure lead to a decrease in toxicity and inhibition of enzyme activity as compared to the parent compound. Furthermore, the production of superoxide anion but not hydrogen peroxide increased in the presence of elevated DOPAL. These results reveal the toxicity of DOPAL and demonstrate that both the catechol and aldehyde are required to potently inhibit TH activity.

Activator Protein-1 Regulation of Murine Aldehyde Dehydrogenase 1a1

Published:  26 June 2012

N. L. Makia, I. Amunom, K. C. Falkner, D. J. Conklin, S. Surapureddi, J. A. Goldstein, and R. A. Prough

Previously we demonstrated that aldehyde dehydrogenase (ALDH) 1a1 is the major ALDH expressed in mouse liver and is an effective catalyst in metabolism of lipid aldehydes. Quantitative real-time polymerase chain reaction analysis revealed a ≈2.5- to 3-fold induction of the hepatic ALDH1A1 mRNA in mice administered either acrolein (5 mg/kg acrolein p.o.) or butylated hydroxylanisole (BHA) (0.45% in the diet) and of cytosolic NAD+-dependent ALDH activity. We observed ≈2-fold increases in ALDH1A1 mRNA levels in both Nrf2(+/+) and Nrf2(−/−) mice treated with BHA compared with controls, suggesting that BHA-induced expression is independent of nuclear factor E2-related factor 2 (Nrf2). The levels of activator protein-1 (AP-1) mRNA and protein, as well as the amount of phosphorylated c-Jun were significantly increased in mouse liver or Hepa1c1c7 cells treated with either BHA or acrolein. With use of luciferase reporters containing the 5′-flanking sequence of Aldh1a1 (−1963/+27), overexpression of c-Jun resulted in an ≈4-fold induction in luciferase activity, suggesting that c-Jun transactivates the Aldh1a1 promoter as a homodimer and not as a c-Jun/c-Fos heterodimer. Promoter deletion and mutagenesis analyses demonstrated that the AP-1 site at position −758 and possibly −1069 relative to the transcription start site was responsible for c-Jun-mediated transactivation. Electrophoretic mobility shift assay analysis with antibodies against c-Jun and c-Fos showed that c-Jun binds to the proximal AP-1 site at position −758 but not at −1069. Recruitment of c-Jun to this proximal AP-1 site by BHA was confirmed by chromatin immunoprecipitation analysis, indicating that recruitment of c-Jun to the mouse Aldh1a1 gene promoter results in increased transcription. This mode of regulation of an ALDH has not been described before.

Use of enzyme inhibitors to evaluate the conversion pathways of ester and amide prodrugs: A case study example with the prodrug ceftobiprole medocaril

Published:  01 March 2012

Gary Eichenbaum, Jennifer Skibbe, Andrew Parkinson, Mark D. Johnson, Dawn Baumgardner, Brian Ogilvie, Etsuko Usuki, Fred Tonelli, Jeff Holsapple and Anne Schmitt-Hoffmann

An approach was developed that uses enzyme inhibitors to support the assessment of the pathways that are responsible for the conversion of intravenously administered ester and amide prodrugs in different biological matrices. The methodology was applied to ceftobiprole medocaril (BAL5788), the prodrug of the cephalosporin antibiotic, ceftobiprole. The prodrug was incubated in plasma, postmitochondrial supernatant fractions from human liver (impaired and nonimpaired), kidney, and intestine as well as erythrocytes, in the presence and absence of different enzyme inhibitors (acetylcholinesterase, pseudocholinesterase, retinyl palmitoyl hydrolase, serine esterases, amidases, and cholinesterase). Hydrolysis was rapid, extensive, and not dependent on the presence of β-nicotinamide-adenine dinucleotide phosphate (reduced form) in all matrices tested, suggesting the involvement of carboxylesterases but not P450 enzymes. Hydrolysis in healthy human plasma was rapid and complete and only partially inhibited in the presence of paraoxonase inhibitors or in liver from hepatic impaired patients, suggesting involvement of nonparaoxonase pathways. The results demonstrate the utility of this approach in confirming the presence of multiple conversion pathways of intravenously administered prodrugs and in the case of BAL5788 demonstrated that this prodrug is unlikely to be affected by genetic polymorphisms, drug interactions, or other environmental factors that might inhibit or induce the enzymes involved in its conversion. © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:1242–1252, 2012

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

Published:  27 July 2011

Ogilvie BW, Yerino P, Kazmi F, Buckley DB, Rostami-Hodjegan A, Paris BL, Toren P, Parkinson A
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.

Comprehensive Quantitative and Qualitative Liquid Chromatography–Radioisotope–Mass Spectrometry Analysis for Safety Testing of Tolbutamide Metabolites Without Standard Samples

Published:  02 June 2011

Zenzaburo Tozuka, Shinsuke Aoyama, Kohei Nozawa, Shoji Akita, Toshinari Oh-Hara, Yasuhisa Adachi, Shin-Ichi Ninomiya
ADME & Tox. Research Institute, Sekisui Medical Company, Ltd., Tokai, Ibaraki 319-1182, Japan

Liquid chromatography–radioisotope–mass spectrometry (LC–RI–MS) analysis was used to determine the structures of 12 (four previously unknown) 14C–tolbutamide (TB) metabolites in rat biological samples (plasma, urine, bile, feces, and microsomes). The four novel metabolites are ω-carboxy TB, hydroxyl TB (HTB)-O-glucuronide, TB-ortho or meta-glutathion, and tolylsulphoaminocarbo-glutathion. In rat plasma, after oral administration of 14C–TB at therapeutic dose (1 mg/kg) and microdose (1.67 µg/kg), the total RI and six metabolites [HTB, carboxy TB (CTB), M1: desbutyl TB, M2: ω-hydroxyl TB, M3: α-hydroxyl TB, and M4: ω-1-hydroxyl TB] were quantified by LC–RI–MS. The plasma concentrations were calculated using their response factors (MS–RI intensity ratio) without standard samples, and the area under the curve (AUC) of plasma concentration per time for evaluation of Safety Testing of Drug Metabolites (MIST) was calculated using the ratio of TB metabolites AUC/total RI AUC. The ratios were as follows: TB 94.5% and HTB 2.5% for the microdose (1.67 µg/kg) and TB 95.6%, HTB 0.96%, CTB 0.065%, M1 0.62%, M2 0.0035%, M3 0.077%, and M4 0.015% for the therapeutic dose (1 mg/kg). These values were less than 10% of the MIST criteria. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4024–4036, 2011

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

Published:  13 April 2011

Parkinson A, Kazmi F, Buckley DB, Yerino P, Paris BL, Holsapple J, Toren P, Otradovec SM, Ogilvie BW
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.

Chapter 7: In Vitro Approaches for Studying the Inhibition of Drug-Metabolizing Enzymes and Identifying the Drug-Metabolizing Enzymes Responsible for the Metabolism of Drugs (Reaction Phenotyping) with Emphasis on Cytochrome P450

Published:  01 January 2008

Brian Ogilvie, Etsuko Usuki, Phyllis Yerino and Andrew Parkinson
This book chapter is from Informa Healthcare's Drug-Drug Interactions, (Drugs and the Pharmaceutical Sciences), 2nd Edition, Edited by A. David Rodrigues

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