Short Communication EFFECTIVE DOSING REGIMEN OF 1-AMINOBENZOTRIAZOLE FOR INHIBITION OF ANTIPYRINE CLEARANCE IN RATS, DOGS, AND MONKEYS

This article is available online at http://dmd.aspetjournals.org ABSTRACT: 1-Aminobenzotriazole (ABT) has been extensively used as a nonspecific inhibitor of cytochromes P450 (P450s) in animals for mechanistic studies, and antipyrine (AP) has been used as a probe for hepatic oxidative metabolic capacity determination in vivo. The method of use of ABT has been variable from lab to lab due largely to unknown pharmacokinetics of ABT itself and incomplete information on various P450s inhibited. The oral pharmacokinetic profiles of ABT were generated in rats, dogs, and monkeys in the dose range of 5 to 200 mg/kg. The results showed that after single oral doses of 50 mg/kg in rats, and 20 mg/kg in dogs and monkeys, the plasma concentrations were high and were sustained for over 24 h. In vitro, inhibition of various expressed P450s upon 30-min preincubation with ABT (0-500 M) showed that CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 were inhibited in a dose-dependent manner. The intravenous pharmacokinetics of AP also was affected in a dose-dependent manner in all species, treated 2 h earlier with ABT. Thus, the plasma clearance of AP was inhibited by 88% in rats pretreated with 50 mg/kg ABT and 96% in dogs and 83% in monkeys pretreated with 20 mg/kg ABT. Based on these data in rats, dogs, and monkeys, and the established safety profile of ABT in rats dosed up to 100 mg/kg, a pretreatment at 2 h with a single oral dose of ABT at 100 mg/kg in rats (providing 93% inhibition) and 20 mg/kg in dogs and monkeys effectively inhibited the clearance of the probe compound. ABT 1 has been extensively used as a nonspecific inhibitor of cytochromes P450 in animals for mechanistic studies Materials and Methods ABT and antipyrine (AP) were obtained from Sigma-Aldrich (St. Louis, MO). Single oral dose pharmacokinetics of ABT, in 0.5% aqueous methylcellulose, were studied in fasted male Sprague-Dawley rats at 10, 50, and 200 mg/kg (n Ն 3) and in Beagle dogs and Cynomolus monkeys at 5, 20, and 100 mg/kg (n ϭ 2 each). All animals were obtained from Charles River Laboratories (Wilmington, MA). The plasma samples were collected over a period of 72 h, and the concentrations of ABT as well as of antipyrine (from experiments described below) were determined simultaneously by precipitation of protein from 0.1 ml of plasma by 0.2 ml of acetonitrile, followed by liquid chromatography/tandem mass spectrometry on a SCIEX API 4000 (PerkinElmerSciex Instruments, Boston, MA) using a Monolithic RP-18 column (4.6 ϫ 100 mm, 2 ). The mobile phase involved a mixture of acetonitrile and water containing 0.1% formic acid flowing at 2.5 ml/min. In vitro inhibition of rP450 activities was measured in a 96-well plate format involving 30-min preincubation of individual human P450 supersomes (40 nM) with ABT (0 -500 M) in the presence of 1 mM NADPH and 0.05 M potassium phosphate buffer, at 37°C. The incubation mixtures were then diluted 10 times with NADPH and marker substrates at 100 M: 7-benzyloxy-4-trifluoromethylcoumarin (CYP1A2, 3A4), 4-(Aminomethyl)-7-methoxycoumarin (CYP2D6), 7-methoxy-4-trifluoromethylcoumarin (CYP2C9), or 7-ethoxy-4-trifluoromethylcoumarin (CYP2B6 and 2C19). The metabolism was determined by fluorometric assays as described by GENTEST (www.gentest.com). The substrates, rP450s and fluorescent metabolite standards, were obtained from BD Gentest Corporation (Woburn, MA). The metabolism was determined by fluorometric assays as described by GENTEST. Two hours prior to administration of AP (20 mg/kg, i.v.), ABT was administered orally to rats at 50 and 100 mg/kg (n ϭ 5 each), and dogs and monkeys at 20 (n ϭ 2) and 100 mg/kg (n ϭ 1), for the interaction study. The control groups involved dosing of animals (n ϭ 2) with 20 mg/kg AP alone and with a low dose of ABT alone (n ϭ 1). Plasma samples were collected up to 24 h and processed and analyzed as above by the liquid chromatography/tandem mass spectrometry method for simultaneous quantitation of both the analytes. Results and Discussion ABT is a nonspecific and effective inhibitor of P450s. It is a metabolism-based inactivator of cytochromes P450 by the mechanism of N-alkylation of heme moiety (Ortiz de Montellano and Mathews, DMD 30:1059DMD 30: -1062DMD 30: , 2002 Printed in U.S.A. 0090-9556/02/3010-1059-1062$7.00 DRUG METABOLISM AND DISPOSITION Vol. 30, No. 10 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 738/1009411 1059 1981; AP is a nonspecific substrate of cytochromes P450 The plasma concentration-time profiles of ABT following single oral doses in fasted male Sprague-Dawley rats at 10, 50, and 200 mg/kg (n Ն 3), and beagle dogs and cynomolgus monkeys at 5, 20, and 100 mg/kg (n ϭ 2 each), are shown in The results of the in vitro inhibition of rP450 activities following a 30-min preincubation of individual human CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 supersomes with ABT (0 -500 M) showed that all P450s were inhibited dose dependently by ABT pretreatment. ABT has previously been shown to inhibit CYP2E1 as well FIG. 1. Plasma concentration-time profiles of ABT after single oral doses in rats, dogs, and monkeys. FIG. 2. In vitro inhibition of marker activities for human rP450 isoforms following 30-min preincubation with ABT. The extent of metabolism was measured by fluorescence of metabolite generated. BALANI ET AL. Based on the observed, high plasma concentrations in animals and the potential for inhibition of P450s to a significant extent after 30-min ABT pretreatment, ABT doses of 50 and 100 mg/kg for rats, and 20 and 100 mg/kg for dogs and monkeys, were selected for coadministration with AP. The control studies involved dosing of animals with 20 mg/kg AP alone and with a low dose of ABT alone. The pretreatment of animals 2 h prior to the intravenous AP administration was considered to provide substantial inhibitory effect on the P450s, based on reports of profound loss of cytochrome P450 contents of liver and kidneys in rats 2 h post the ABT dose Acknowledgments. We thank Drs. Check Quon and Gerald Miwa for their helpful discussions

Quantitative Prediction and Clinical Observation of a CYP3A Inhibitor-Based Drug-Drug Interactions with MLN3897, a Potent C-C Chemokine Receptor-1 Antagonist

ABSTRACT A novel in vitro model was recently developed in our laboratories for the prediction of magnitude of clinical pharmacokinetic drugdrug interactions (DDIs), based on reversible hepatic cytochrome P450 (P450) inhibition. This approach, using inhibition data from human hepatocytes incubated in human plasma, and quantitative P450 phenotyping data from hepatic microsomal incubations, successfully predicted DDIs for 15 marketed drugs with ketoconazole, a strong competitive inhibitor of CYP3A4/5, generally used to demonstrate a "worst-case scenario" for CYP3A inhibition. In addition, this approach was successfully extended to DDI predictions with the moderate competitive CYP3A inhibitor fluconazole for nine marketed drugs. In the current report, the general applicability of the model has been demonstrated by prospectively predicting the degree of inhibition and then conducting DDI studies in the clinic for an investigational CCR1 antagonist MLN3897, which is cleared predominantly by CYP3A. The clinical studies involved treatment of healthy volunteers (n ϭ 17-20), in a crossover design, with ketoconazole (200 mg b.i.d.) or fluconazole (400 mg once a day), while receiving MLN3897. Administration of MLN3897 and ketoconazole led to an average 8.28-fold increase in area under the curve of plasma concentration-time plot (AUC) of MLN3897 at steady state, compared with the 8.33-fold increase predicted from the in vitro data. Similarly for fluconazole, an average increase of 3.93-fold in AUC was observed for MLN3897 in comparison with a predicted value of 3.26-fold. Thus, our model reliably predicted the exposure changes for MLN3897 in interaction studies with competitive CYP3A inhibitors in humans, further strengthening the utility of our in vitro model. Prediction of clinical DDIs using in vitro studies is one of the major challenges in the pharmaceutical industry. The main utility of such DDI predictions is to help foresee any safety issues anticipated from higher exposures and thus help design clinical trials with better safety. In some instances, clinical DDI studies can be avoided if no significant pharmacokinetic interaction is predicted. Traditionally, DDI predictions have been based on the ratio of the inhibitor concentration [I] and the enzyme inhibition constant K i ([I]/K i ratio