Drug Metab. Pharmacokinet. 24 (6): 500–510 (2009). Regular Article Prediction of Drug-Drug Interactions based on Time-Dependent Inhibition from High Throughput Screening of Cytochrome P450 3A4 Inhibition Nobuo SEKIGUCHI1,*,AtsukoHIGASHIDA2, Motohiro KATO1, Yoshiaki NABUCHI1, Tetsuya MITSUI1,KenjiTAKANASHI1, Yoshinori ASO1 and Masaki ISHIGAI1 1Pre-Clinical Research Department, Chugai Pharmaceutical Co., Ltd., Shizuoka, Japan 2Discovery Platform Technology Department, Chugai Pharmaceutical Co., Ltd., Shizuoka, Japan Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk Summary: A method of assessing the risk of drug-drug interaction (DDI) caused by mechanism-based inhibi- tion (MBI) was developed for early-stage drug development using cytochrome P450 (CYP) 3A4 inhibition screening data. CYP3A4 inhibition was evaluated using a fluorescent substrate with or without preincubation containing an inhibitor. The results showed that five well-known mechanism-based inhibitors, but not the competitive inhibitor ketoconazole, had lower IC50 after preincubation, suggesting the utility of the IC50 shift by preincubation to discern mechanism-based inhibitors. A method to approximately predict the change in the area under the concentration-time curve (AUC) of a co-administered drug by MBI was found using IC50 shift data and the unbound mean plasma concentration of the inhibitor. From our predictions of change in the AUC for 38 drugs using this method, all mechanism-based inhibitors causing change in the AUC of more than 200z were predicted to be high risk. In conclusion, our method provides a simple assessment of the risk of DDI from mechanism-based inhibitors, especially in the early stages of drug development. Keywords: mechanism-based inhibition; drug-drug interaction; CYP3A4; time-dependent inhibition; in vitro-in vivo prediction There are two types of CYP inhibition: 1) reversible in- Introduction hibition represented by competitive inhibition and 2) ir- Cytochrome P450s (CYPs) are the main drug- reversible inhibition, or so-called mechanism-based inhi- metabolizing enzymes expressed in human liver and are bition (MBI), where an enzyme is inactivated by forming involved in the metabolism of many drugs on the mar- a stable complex with a metabolite.5) Because the reduc- ket.1) Drugs that inhibit CYP may elevate the blood con- tion in enzyme activity continues until the inactivated centration of co-administered drugs, resulting in drug- CYP is replaced by a newly synthesized CYP, the duration drug interactions (DDI), which sometimes cause severe of the elevated blood concentration of a drug co-ad- adverse effects. Actually, some drugs such as terfenadine, ministered with a mechanism-based inhibitor is longer. mibefradil, astemizole, and cerivastatin have been Mechanism-based inhibitors require particular attention withdrawn from the market due to DDI.2) Thus, CYP in- because they have been reported to cause unanticipated hibition-mediated DDI is widely recognized and the adverse effects.5,6) necessity of enzyme inhibition studies is included in the In vitro-in vivo extrapolation (IVIVE) methods to guidance from the US Food and Drug Administration predict clinical DDI from in vitro inhibition data are well (FDA).3,4) The guidance recommends that DDI studies are known. Concerning competitive inhibitors, although the performed as early as possible in drug development. In ratio of inhibitor concentration to inhibition constant 7) the case of compounds showing CYP inhibition in vitro, (I/Ki) is widely used for the index of DDI, we recently clinical DDI studies are required. CYP inhibitors found reported that prediction using in vivo Ki and a phys- to have high risk at the later stage of development can iologically-based pharmacokinetic (PBPK) model was cause marked loss of time and resources. more accurate than the conventional methods.8) Con- Received; April 7, 2009, Accepted; October 22, 2009 *To whom correspondence should be addressed: Nobuo SEKIGUCHI, Pre-Clinical Research Department, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan. Tel. ＋81-550-87-9103, Fax. ＋81-550-87-5326, E-mail: sekiguchinbo＠chugai-pharm.co.jp 500 Prediction of DDI from CYP3A4 Inhibition using HTS 501 cerning mechanism-based inhibitors, there have also ethynylestradiol, fluvastatin, glibenclamide, lansoprazole, been reports on methods to predict clinical DDI based methylprednisolone, mibefradil, mifepristone, predni- on in vitro CYP inhibition data.9,10) These methods predict sone,quinidine,terfenadine,triazolam,troleandomycin, increase in the area under concentration-time curve and verapamil were purchased from Sigma Chemical Co. (AUC) of a co-administrated drug accurately but are un- (St. Louis, MO, USA). Cimetidine, chlorpromazine, suitable for high throughput screening (HTS) technology clarithromycin, cyclosporine, dextromethorphan, diazep- utilized in early-stage drug development because a sub- am, fluoxetine, miconazole, midazolam, nicardipine, stantial number of studies is required with a variety of nifedipine, omeprazole, paclitaxel, roxithromycin, sim- conditions of preincubation time and inhibitor concen- vastatin, tamoxifen, and trimethoprim were obtained trations. To evaluate the maximum inactivation constant from Wako Pure Chemical Industries, Ltd (Osaka, Japan). (kinact) and the apparent dissociation constant (Ki, app), Fluconazole and midecamycin were from ICN Biomedi- preliminary experiments are required to set the appropri- cals,Inc.(Aurora,OH,USA).Indinavir,nelfinavir, ate preincubation time and inhibitor concentration for a ritonavir, and saquinavir were purchased from Sequoia compound for which no information regarding its MBI Research Products, Ltd (Pangbourne, UK). Itraconazole, potential is available. For prediction of DDI from ketoconazole, and paroxetine were from LKT Laborato- mechanism-based inhibitors in the early stages, a simpler ries,Inc.(St.Paul,MN,USA).Fluvoxaminewaspur- and speedier method is needed. A screening method for chased from Tocris Cookson Ltd (Bristol, UK). 7-Ben- CYP inhibition using fluorescent substrates has been zyloxy–4-(trifluoromethyl)-coumarin (BFC) was obtained reported.11,12) This method could be utilized to find from Becton, Dickinson and Company (Franklin Lakes, potential mechanism-based inhibition since mechanism- NJ, USA). Other reagents and solvents used were of the based inhibitors enhance the degree of inhibition accord- highest or high performance liquid chromatography ing to the preincubation time, so-called time-dependent grade. Recombinant CYP enzyme expressed in micro- inhibition (TDI). This method is suitable for use with HTS somes of insect cells infected with baculovirus containing because it enables a rapid measurement of many com- human CYP3A4, cytochrome b5, and cytochrome P450 pounds. reductase and control microsomes were obtained from Although mechanism-based inhibitors are considered Becton, Dickinson and Company (Franklin Lakes, NJ, high risk for DDI, clear criteria for evaluating DDI risk USA). have not been established for CYP inhibition screening. If Fluorometric enzyme inhibition analysis includ- the wrong criteria are used, a mechanism-based inhibitor ing TDI evaluation: The evaluation of CYP3A inhibi- may possibly show false negative or false positive predic- tion was carried out on 96-well microtiter plates using TM tions and result in either clinical DDI or the loss of a MultiPROBE IIEX Liquid Handling Robotics (PerkinEl- potentially effective drug. Therefore, a methodology and mer Japan, Yokohama, Japan). Fluorescence detection of criteria for prediction of DDI caused by mechanism- the metabolite of BFC was performed using a Spectra based inhibitor is required to find promising compounds Max Gemini microplate reader (Molecular Devices, Sun- efficiently in early-stage screening. nyvale, CA, USA). Incubations were conducted based on In this study, the inhibition of CYP3A4 was investigat- the method provided on the BD Biosciences website13) as ed because it metabolizes many drugs and has been described below. A cofactor/serial dilution buffer con- reported to contribute to serious DDI.1) First, it was taining 2.6 mM b-nicotinamide adenine dinucleotide ascertained whether shift in IC50 calculated by the differ- phosphate (NADP), 6.6 mM glucose-6-phosphate, 0.8 ence in IC50 under two conditions (with or without prein- units/mL glucose-6-phosphate dehydrogenase and 6.6 cubation containing an inhibitor) discriminates between mM magnesium chloride was prepared in 50 mM potassi- competitive inhibitor and mechanism-based inhibitor um phosphate (pH 7.4). The enzyme solution contained fromHTSdatausingafluorescentsubstrate.Next,we 0.35 M potassium phosphate (pH 7.4), 10 pmol/ml developed a simple equation using HTS data for the CYP3A4 expressed in insect cell microsomes, 100 mM prediction of change in the AUC of a co-administered BFC, and control microsomes to give the final assay con- drug by MBI, we also ascertained whether we can catego- centrations (0.04 mg of protein/ml). rize test compounds visually by criteria line according to In the coincubation assay for CYP inhibition evalua- the degree of AUC change predicted from that equation tion, test inhibitors were added to the cofactor/serial di- for the purpose of risk-assessment of DDI in the early lution buffer, giving inhibitor solutions of various con- stages of drug development. centrations. Metabolic reactions were initiated by adding thesamevolumeofenzymesolutioncontainingBFCto