In vitro evaluation suggests fenfluramine and norfenfluramine are unlikely to act as perpetrators of drug interactions.

Studies support the safety and efficacy of fenfluramine (FFA) as an antiseizure medication (ASM) in Dravet syndrome, Lennox-Gastaut syndrome, or CDKL5 deficiency disorder, all pharmacoresistant developmental and epileptic encephalopathies. However, drug-drug interactions with FFA in multi-ASM regimens have not been fully investigated. We characterized the perpetrator potential of FFA and its active metabolite, norfenfluramine (nFFA), in vitro by assessing cytochrome P450 (CYP450) inhibition in human liver microsomes, CYP450 induction in cultured human hepatocytes, and drug transporter inhibition potential in permeability or cellular uptake assays. Mean plasma unbound fraction was ~50% for both FFA and nFFA, with no apparent concentration dependence. FFA and nFFA were direct in vitro inhibitors of CYP2D6 (IC50 , 4.7 and 16 µM, respectively) but did not substantially inhibit CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, or CYP3A4/5. No time- or metabolism-dependent CYP450 inhibition occurred. FFA and nFFA did not induce CYP1A2; both induced CYP2B6 (up to 2.8-fold and up to 2.0-fold, respectively) and CYP3A4 (1.9- to 3.0-fold and 3.6- to 4.8-fold, respectively). Mechanistic static pharmacokinetic models predicted that neither CYP450 inhibition nor induction was likely to be clinically relevant at doses typically used for seizure reduction (ratio of area under curve [AUCR] for inhibition <1.25; AUCR for induction >0.8). Transporters OCT2 and MATE1 were inhibited by FFA (IC50 , 19.8 and 9.0 µM) and nFFA (IC50 , 5.2 and 4.6 µM) at concentrations higher than clinically achievable; remaining transporters were not inhibited. Results suggest that FFA and nFFA are unlikely drug-drug interaction perpetrators at clinically relevant doses of FFA (0.2-0.7 mg/kg/day).

In vitro evaluation of fenfluramine and norfenfluramine as victims of drug interactions.

Fenfluramine (FFA) has potent antiseizure activity in severe, pharmacoresistant childhood-onset developmental and epileptic encephalopathies (e.g., Dravet syndrome). To assess risk of drug interaction affecting pharmacokinetics of FFA and its major metabolite, norfenfluramine (nFFA), we conducted in vitro metabolite characterization, reaction phenotyping, and drug transporter-mediated cellular uptake studies. FFA showed low in vitro clearance in human liver S9 fractions and in intestinal S9 fractions in all three species tested (t1/2  > 120 min). Two metabolites (nFFA and an N-oxide or a hydroxylamine) were detected in human liver microsomes versus six in dog and seven in rat liver microsomes; no metabolite was unique to humans. Selective CYP inhibitor studies showed FFA metabolism partially inhibited by quinidine (CYP2D6, 48%), phencyclidine (CYP2B6, 42%), and furafylline (CYP1A2, 32%) and, to a lesser extent (<15%), by tienilic acid (CYP2C9), esomeprazole (CYP2C19), and troleandomycin (CYP3A4/5). Incubation of nFFA with rCYP1A2, rCYP2B6, rCYP2C19, and rCYP2D6 resulted in 10%-20% metabolism and no clear inhibition of nFFA metabolism by any CYP-selective inhibitor. Reaction phenotyping showed metabolism of FFA by recombinant human cytochrome P450 (rCYP) enzymes rCYP2B6 (10%-21% disappearance for 1 and 10 µM FFA, respectively), rCYP1A2 (22%-23%), rCYP2C19 (49%-50%), and rCYP2D6 (59%-97%). Neither FFA nor nFFA was a drug transporter substrate. Results show FFA metabolism to nFFA occurs through multiple pathways of elimination. FFA dose adjustments may be needed when administered with strong inhibitors or inducers of multiple enzymes involved in FFA metabolism (e.g., stiripentol).

Effects of monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, and interferon-α2a on P450 enzymes in human hepatocytes in vitro.

Some immunomodulatory agents stimulate the release of cytokines capable of suppressing P450 enzymes and potentially affecting pharmacokinetics of coadministered medications. Cytokines released in response to an immunomodulator in the blood ex vivo can be used to screen for the potential for drug-drug interactions. Tilsotolimod, an investigational agonist of Toll-like receptor 9, stimulated the release of macrophage chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), and interferon-α2a (INF-α2a) in blood obtained from healthy donors. Although tilsotolimod did not directly affect CYP1A2, CYP2B6, or CYP3A4 expression or activity, the cytokines stimulated by the drug reduced CYP1A2 and CYP2B6 enzyme activities in cultured human hepatocytes. This study sought to identify which cytokines were responsible for tilsotolimod's indirect effects on P450 enzymes in vitro. A 72-h treatment with recombinant human chemokines MCP-1 and MIP-1α did not alter CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP3A4, or signal transducer and activator of transcription 1 (STAT1) mRNA expression or CYP1A2, CYP2B6, or CYP3A4/5 enzyme activity in cocultures of human hepatocytes and Kupffer cells. INF-α2a, at 2.5 ng/mL but not at the lower concentrations applied to the cells, increased CYP1A2 and STAT1 mRNA by 2.4- and 5.2-fold, respectively, and reduced CYP2B6 enzyme activity to 46% of control. This study established that INF-α2a, but not MCP-1 or MIP-1α, mediated tilsotolimod effects on CYP1A2 and CYP2B6 expression in human hepatocytes.

An Assessment of the In Vitro Inhibition of Cytochrome P450 Enzymes, UDP-Glucuronosyltransferases, and Transporters by Phosphodiester- or Phosphorothioate-Linked Oligonucleotides.

Oligonucleotides represent an expanding class of pharmacotherapeutics in development for various indications. Typically, oligonucleotides are developed with phosphorothioate linkages for the improvement of biologic stability; however, limited data are available on the potential of these molecules to cause drug-drug interactions (DDIs). In this study, four nontherapeutic oligonucleotides with either a phosphodiester or phosphorothioate linkage and partial sequences towards glutathione peroxidase or ß-actin (PD-GP and PD-Ac or PT-GP and PT-Ac, respectively) were evaluated in vitro for their potential to inhibit cytochrome P450 (P450) enzymes and UGP-glucuronosyltransferases (UGTs) in both human liver microsomes (HLMs) and cryopreserved human hepatocytes (CHHs) 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 HLMs and CHHs, except for PD-Ac in HLMs 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 of 0.8-4.2 µM), CYP2C8 (IC50 values of 1.1-12 µM), and UGT1A1 (IC50 values of 4.5-5.4 µM) inhibited to the greatest extent. There was evidence of possible time-dependent inhibition (TDI) of P450 enzymes with PT-GP and PT-Ac for CYP2B6, CYP2C8, CYP2C19, CYP2C9, CYP2D6, and CYP3A4/5; however, this TDI was reversible. In contrast to HLMs, there was little to no direct P450 inhibition by any oligonucleotide in CHHs [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, including organic anion-transporting polypeptide OATP1B1 and OATP1B3, organic anion transporters OAT1 and OAT3, and organic cation transporter OCT2 (IC50 values of 12-29 µM), but not OCT1 or the efflux transporters breast cancer resistance protein and P-glycoprotein by the phosphorothioate oligonucleotides.

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.

Ketoconazole is a potent CYP3A4/5 inhibitor and, until recently, recommended by the Food and Drug Administration (FDA) and the European Medicines Agency 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 European Medicines Agency recommended suspension of ketoconazole use in DDI studies in 2013. The FDA specifically recommended use of clarithromycin or itraconazole as alternative strong CYP3A4/5 inhibitors 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) toward 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 its 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 toward 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.

The Reliability of Estimating Ki Values for Direct, Reversible Inhibition of Cytochrome P450 Enzymes from Corresponding IC50 Values: A Retrospective Analysis of 343 Experiments.

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 cytochrome P450 (P450) inhibition could be reliably predicted by dividing the corresponding IC50 values by two, based on the relationship (for competitive inhibition) in which Ki = IC50/2 when [S] (substrate concentration) = Km (Michaelis-Menten constant). 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 minutes) 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 3-fold range. In the case of noncompetitive inhibitors, Ki values predicted from IC50/2 values were overestimated by a factor of nearly 2 (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.

Clinical assessment of drug-drug interactions of tasimelteon, a novel dual melatonin receptor agonist.

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%.

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 IC(50) values varying from 1.2 µM [lansoprazole; maximum plasma concentration (C(max)) = 2.2 µM] to 93 µM (pantoprazole; C(max) = 6.5 µM). In addition, we identified omeprazole, esomeprazole, R-omeprazole, and omeprazole sulfone as MDIs of CYP2C19 (they caused IC(50) 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 (IC(50) 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 K(I) (inhibitor concentration that supports half the maximal rate of inactivation) values of 1.7 to 9.1 µM and k(inact) (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.

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 IC(50) 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 IC(50) shift associated with the dilution method is a consequence of data processing. With the dilution method, IC(50) values for direct-acting inhibitors vary with the dilution factor unless they are based on the final (postdilution) inhibitor concentration, whereas the IC(50) 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 IC(50) values for MDI (shifted IC(50) values) decrease by the magnitude of the dilution factor. The lower shifted IC(50) 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 IC(50) 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.

Prediction of the overall renal tubular secretion and hepatic clearance of anionic drugs and a renal drug-drug interaction involving organic anion transporter 3 in humans by in vitro uptake experiments.

The present study investigated prediction of the overall renal tubular secretion and hepatic clearances of anionic drugs based on in vitro transport studies. The saturable uptake of eight drugs, most of which were OAT3 substrates (rosuvastatin, pravastatin, pitavastatin, valsartan, olmesartan, trichlormethiazide, p-aminohippurate, and benzylpenicillin) by freshly prepared human kidney slices underestimated the overall intrinsic clearance of the tubular secretion; therefore, a scaling factor of 10 was required for in vitro-in vivo extrapolation. We examined the effect of gemfibrozil and its metabolites, gemfibrozil glucuronide and the carboxylic metabolite, gemfibrozil M3, on pravastatin uptake by human kidney slices. The inhibition study using human kidney slices suggests that OAT3 plays a predominant role in the renal uptake of pravastatin. Comparison of unbound concentrations and K(i) values (1.5, 9.1, and 4.0 µM, for gemfibrozil, gemfibrozil glucuronide, and gemfibrozil M3, respectively) suggests that the mechanism of the interaction is due mainly to inhibition by gemfibrozil and gemfibrozil glucuronide. Furthermore, extrapolation of saturable uptake by cryopreserved human hepatocytes predicts clearance comparable with the observed hepatic clearance although fluvastatin and rosuvastatin required a scaling factor of 11 and 6.9, respectively. This study suggests that in vitro uptake assays using human kidney slices and hepatocytes provide a good prediction of the overall tubular secretion and hepatic clearances of anionic drugs and renal drug-drug interactions. It is also recommended that in vitro-in vivo extrapolation be performed in animals to obtain more reliable prediction.

System-dependent outcomes during the evaluation of drug candidates as inhibitors of cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) enzymes: human hepatocytes versus liver microsomes versus recombinant enzymes.

The ability of a drug to cause clinically significant drug-drug interactions due to direct or metabolism-dependent inhibition of cytochrome P450 (CYP) can generally be predicted from in vitro studies with human liver microsomes (HLM) or recombinant CYP enzymes, as recommended by the FDA and other regulatory agencies. This review highlights some examples of system-dependent inhibition of CYP and uridine diphosphate glucuronosyltransferase (UGT) enzymes. In the case of CYP enzymes, examples are presented where in vitro studies with HLM under-predict or over-predict the degree of inhibition observed in the clinic and where the correct prediction comes from studies with human hepatocytes. Studies with HLM under-predict the ability of gemfibrozil and bupropion to cause clinically significant inhibition of CYP2C8 and CYP2D6, respectively, and over-predict the ability of ezetimibe to cause clinically significant inhibition of CYP3A4. Gemfibrozil and bupropion represent examples of glucuronidation-dependent and reduction-dependent activation to metabolites that inhibit CYP2C8 and CYP2D6, respectively, whereas ezetimibe represents an example of glucuronidation-dependent protection against metabolism-dependent inhibition of CYP3A4. This article illustrates why, when drug candidates are extensively metabolized by non-CYP enzymes, it would be prudent to use human hepatocytes in addition to HLM or recombinant enzymes to evaluate their ability to inhibit CYP enzymes.

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 (IC(50) >or= 100 microM); whereas, a comparator with dual reuptake properties [duloxetine (Cymbalta)] inhibited CYP2D6 (IC(50) = 7 microM) and CYP2B6 (IC(50) = 15 microM) with a relatively high potency. Milnacipran inhibited CYP3A4/5 in a substrate-dependent manner (i.e., midazolam 1'-hydroxylation IC(50) approximately 30 microM; testosterone 6beta-hydroxylation IC(50) approximately 100 microM); whereas, duloxetine inhibited both CYP3A4/5 activities with equal potency (IC(50) = 37 and 38 microM, 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 microM), 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 microM; >10 times plasma C(max)) 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.

Construction of triple-transfected cells [organic anion-transporting polypeptide (OATP) 1B1/multidrug resistance-associated protein (MRP) 2/MRP3 and OATP1B1/MRP2/MRP4] for analysis of the sinusoidal function of MRP3 and MRP4.

Multidrug resistance-associated protein (MRP) 3/ABCC3 and MRP4/ABCC4 are ATP-binding cassette (ABC) transporters expressed in the sinusoidal membrane of hepatocytes. The purpose of the present study was to establish organic anion-transporting polypeptide (OATP) 1B1/MRP2/MRP3 and OATP1B1/MRP2/MRP4 triple transfectants as in vitro model of the hepatobiliary transport of anionic drugs. To find in vivo relevant Mrp3 probes, wild-type and Mrp3(-/-) mice were given gemfibrozil, 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridymethyl)benzothiazole (E3040), troglitazone, bisphenol A, and 4-methylumbelliferone orally. Plasma concentrations of the glucuronide conjugates were significantly lower in Mrp3(-/-) mice than in wild-type mice. The systemic exposure of gemfibrozil, E3040, and troglitazone were similar in wild-type and Mrp3(-/-) mice. 4-Methylumbelliferone and bisphenol A were undetectable in the plasma. In MRP3-expressing membrane vesicles, ATP-dependent uptakes of the glucuronide conjugates of estradiol, gemfibrozil, E3040, and troglitazone were markedly greater than those in controls, whereas MRP4-expressing membrane vesicles exhibited significant ATP-dependent uptake of gemfibrozil glucuronide and estradiol glucuronide. MRP3 or MRP4 was expressed in the OATP1B1/MRP2 double transfectants using adenovirus. The expression levels of OATP1B1 and MRP2 proteins were maintained both in the OATP1B1/MRP2/MRP3 and OATP1B1/MRP2/MRP4 triple transfectants, whereas MRP3 and MRP4 were localized in the basal membrane. Significant reductions in the basal-to-apical flux of the glucuronide conjugates of estradiol, gemfibrozil, E3040, and troglitazone were observed in the OATP1B1/MRP2/MRP3 triple transfectants compared with those in the double transfectants, whereas significant reduction was observed only for gemfibrozil glucuronide and estradiol glucuronide in the OATP1B1/MRP2/MRP4 triple transfectants. These results suggest that MRP3- or MRP4-triple transfectants provide a simple and useful in vitro system for evaluating their importance in the hepatobiliary transport of drugs.

An in vitro evaluation of the victim and perpetrator potential of the anticancer agent laromustine (VNP40101M), based on reaction phenotyping and inhibition and induction of cytochrome P450 enzymes.

Laromustine (VNP40101M, also known as Cloretazine) is a novel sulfonylhydrazine alkylating (anticancer) agent. Laromustine generates two types of reactive intermediates: 90CE and methylisocyanate. When incubated with rat, dog, monkey, and human liver microsomes, [(14)C]laromustine was converted to 90CE (C-8) and seven other radioactive components (C-1-C-7). There was little difference in the metabolite profile among the species examined, in part because the formation of most components (C-1-C-6 and 90CE) did not require NADPH but involved decomposition and/or hydrolysis. The exception was C-7, a hydroxylated metabolite, largely formed by CYP2B6 and CYP3A4/5. Laromustine caused direct inhibition of CYP2B6 and CYP3A4/5 (the two enzymes involved in C-7 formation) as well as of CYP2C19. K(i) values were 125 microM for CYP2B6, 297 muM for CYP3A4/5, and 349 microM for CYP2C19 and were greater than the average clinical plasma C(max) of laromustine (25 microM). There was evidence of time-dependent inhibition of CYP1A2, CYP2B6, and CYP3A4/5. Treatment of primary cultures of human hepatocytes with up to 100 microM laromustine did not induce CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, or CYP3A4/5, but the highest concentration of laromustine decreased the activity and levels of immunoreactive CYP3A4. The results of this study suggest the laromustine has 1) negligible victim potential with respect to metabolism by cytochrome P450 enzymes, 2) negligible enzyme-inducing potential, and 3) the potential in some cases to cause inhibition of CYP2B6, CYP3A4, and possibly CYP2C19 during and shortly after the duration of intravenous administration of this anticancer drug, but the clinical effects of such interactions are likely to be insignificant.

On the mechanism of hepatocarcinogenesis of benzodiazepines: evidence that diazepam and oxazepam are CYP2B inducers in rats, and both CYP2B and CYP4A inducers in mice.

The aim of this study was to evaluate diazepam and oxazepam as cytochrome P450 inducers at doses previously shown to cause liver tumors in mice but not rats. In rats, diazepam and oxazepam induced CYP2B, and were as effective as phenobarbital despite lacking phenobarbital's tumor-promoting effect in rats. In mice, diazepam and oxazepam induced both CYP2B and CYP4A at dietary doses associated with liver tumor formation. It remains to be determined why diazepam and oxazepam induce CYP4A in mice but not rats and whether this difference accounts for the apparent species difference in the tumor-promoting activity of diazepam and oxazepam.

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-beta-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 microM), whereas gemfibrozil glucuronide most potently inhibited CYP2C8 (IC50 of 24 microM). 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 microM to 1.8 microM after a 30-min incubation with human liver microsomes and NADPH. Inactivation of CYP2C8 by gemfibrozil glucuronide required NADPH, and proceeded with a K(I) (inhibitor concentration that supports half the maximal rate of enzyme inactivation) of 20 to 52 microM and a k(inact) (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.