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findings were translated into clinical context with the basic static model approaches outlined by the FDA in their 2012 draft guidance on DDIs.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.