Modeling, Prediction, and In Vitro In Vivo Correlation of CYP3A4 Induction

Transcription

1 DMD This Fast article Forward. has not been Published copyedited and on formatted. July 31, The 2008 final as version doi: /dmd may differ from this version. DMD #20602 Modeling, Prediction, and In Vitro In Vivo Correlation of CYP3A4 Induction Magang Shou, Mike Hayashi, Yvonne Pan, Yang Xu, Kari Morrissey, Lilly Xu, and Gary L. Skiles Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., Thousand Oaks, CA91320 Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics. 1

2 Running Title: Prediction of CYP3A4 Induction-Mediated Drug Drug Interactions No. of Text Pages: 25 No. of Tables: 5 No. of Figures: 6 No. of References: 137 No. of Words in Abstract: 260 Introduction: 920 (including cited references) Discussion: 2440 (including cited references) The Abbreviations used: DMEs, drug metabolizing enzymes; DMSO, dimethylsulfoxide; RIF, rifampin; PB, Phenobarbital; DEX, dexamethasone; BST, bosentan; EFA, efavirenz; CMZ, carbamazepine; KHB, Krebs-Henseleit Buffer; [Ind] max,ss, maximum inducer concentration at steady state; f u,p, unbound fraction in plasma; f u,hept, unbound fraction in hepatocytes; E max, maximum response (net maximum fold-increase) of induction; EC 50, inducer concentration at 50% E max ; f m,cyp3a4, fraction of a drug metabolized by CYP3A4, f i,cyp3a4, fraction of decreased in vivo clearance of a drug in the presence of a selective CYP3A4 inhibitor; F a, oral bioavailability; F h hepatic bioavailability; F g, gut bioavailability; f h, fraction of a drug cleared by liver; CL int,cyp3a4, CYP3A4 intrinsic clearance; CL h, hepatic clearance; RSS, Residual Sum of Squares; K a, obsorption rate constant; and Q H, hepatic blood flow rate. 2

3 Abstract CYP3A4 induction is not generally considered to be a concern for safety; however, serious therapeutic failures can occur with drugs whose exposure is lower as a result of more rapid metabolic clearance due to induction. Despite the potential therapeutic consequences of induction, little progress has been made in quantitative predictions of CYP3A4 induction-mediated drug drug interactions (DDIs) from in vitro data. In the present study, predictive models have been developed to facilitate extrapolation of CYP3A4 induction measured in vitro to human clinical DDIs. The following parameters were incorporated into the DDI predictions: 1) EC 50 and E max of CYP3A4 induction in primary hepatocytes; 2) fractions unbound of the inducers in human plasma (f u,p ) and hepatocytes (f u,hept ); 3) relevant clinical in vivo concentrations of the inducers ([Ind] max, ss ); and 4) fractions of the victim drugs cleared by CYP3A4 (f m,cyp3a4 ). The values for [Ind] max,ss and f m,cyp3a4 were obtained from clinical reports of CYP3A4 induction and inhibition, respectively. Exposure differences of the affected drugs in the presence and absence of the six individual inducers (bosentan, carbamazepine, dexamethasone, efavirenz, phenobarbital, and rifampicin) were predicted from the in vitro data and then correlated with those reported clinically (n = 103). The best correlation was observed (R 2 = and from two hepatocyte donors) when f u,p and f u,hept were included in the predictions. Factors that could cause over- or underpredictions (potential outliers) of the DDIs were also analyzed. Collectively, these predictive models could add value to the assessment of risks associated with CYP3A4 induction-based DDIs by enabling their determination in the early stages of drug development. 3

4 Drug drug interactions (DDIs) are a major source of clinical problems leading either to severe adverse drug reactions (ADRs) (Backman, et al., 2002;Backman, et al., 2006;Floren, et al., 1997;Gomez, et al., 1995;Greenblatt, et al., 1998;Honig, et al., 1993) or a reduction in pharmacological effects (Heimark, et al., 1987;Back, et al., 1979;Daniels, et al., 1984). The most common DDIs are associated with alterations in drug clearance, primarily due to inhibition of drug metabolizing enzymes (DMEs), particularly cytochromes P450 (CYPs). Predictions of in vivo DDIs based on in vitro drug metabolism data are increasingly being integrated into decision-making about the development of potential drugs during the early stages of drug discovery. The success in these predictions is due largely to an increased understanding of DMEs at the molecular level, which has provided insight into the mechanisms of drug biotransformation and disposition. Substantial progress has been made in extrapolating in vitro inhibition data for the prediction of clinical DDIs, but fewer advances in prediction of induction-based DDIs have been realized. Induction of CYP3A4 is especially important because the enzyme is involved in the metabolism of approximately 50% of marketed drugs (Guengerich, 1999). Unlike CYP inhibition, CYP3A4 induction is not as frequent a problem and is not normally thought as a safety concern. Induction can lead to inadequate efficacy of co-administered drugs and it is therefore an undesirable property (Grub, et al., 2001) (Dickinson, et al., 2001). Induction studies have generally been more difficult to conduct experimentally than inhibition because induction is an indirect and slow process of gene up-regulation and increased protein expression; however, the advent of newer technologies such as nuclear receptor-reporter and mrna analyses has partially overcome this problem. Although the concept of induction of DMEs has been known for several decades (Conney, 1967), an understanding of the mechanisms of CYP induction has only been slowly developed. The major mechanism of CYP3A4 induction has been determined to occur via activation of a human orphan nuclear receptor known as the pregnane X receptor (PXR) (Kliewer, et al., 1998). Inducer binds to PXR in the cytosol and together they translocate to the nucleus where a heterodimer with retinoid X-receptor (RXR) is formed. The receptor complex binds to the DNA responsive element upstream of the CYP3A4 4

5 gene and activates its promoter leading to transcription of the CYP3A4 gene. PXR also induces the transcription of a number of other drug metabolizing enzymes including less potent induction of CYP2B and CYP2C9 (Kliewer, et al., 1998). The extent of induction depends on the concentration of the inducer and on the duration of exposure. Induction has been well characterized with a number of drugs, such as rifampicin (Backman, et al., 1996), phenobarbital (Rutledge, et al., 1988), troglitazone (Prueksaritanont, et al., 2001), and bosentan (van Giersbergen, et al., 2002), examples which are mainly confined to induction of CYP3A4. CYP3A4 is induced by a wide range of structurally diverse compounds that reflect a broad range of binding specificity for PXR. Inducers of CYP3A4 also induce CYP3A in other species, but with major differences in the degree of response (Jones, et al., 2000;Moore and Kliewer, 2000). For example, the prototypical CYP3A4 inducer rifampicin is a potent activator of human and rabbit PXR but has little activity on the rat and mouse receptor (Jones, et al., 2000;Vignati, et al., 2004). These species differences are known to be substantial, both in the spectrum of the enzymes induced and the extent of induction, and make nonhuman in vivo models inappropriate for induction studies intended to understand the risk of human induction. Because of these limitations, there is an increasing demand on the use of human in vitro systems (Masimirembwa, et al., 2001). A commonly used technique is based on immortalized cell lines with engineered receptor and reporter systems (Goodwin, et al., 1999). Another emerging technique is the use of immortalized human hepatocytes (Mills, et al., 2004) (Ripp, et al., 2006). Despite these advances, primary human hepatocyte cultures have been and remain as the gold-standard in vitro system for investigating potential CYP induction (Hewitt, et al., 2007;LeCluyse, et al., 2000). Induction can be assessed with these cultures by measurements of both mrna level and CYP functional enzymatic activity using probe substrates. The hepatocytebased assays are, however, relatively low throughput and impractical for screening of large numbers of compounds because of the complexity of the assays and need for a continuous supply of hepatocytes. Comparisons of induction measured in cell-based reporter assays and human hepatocytes have shown some degree of correlation (Luo, et al., 2002). 5

6 The importance of CYP3A4 induction has prompted the search for alternative approaches to predicting DDIs using in vitro data obtained during the drug discovery stage. Unfortunately, little progress has been made in this regard. Development of predictive models of DDIs due to CYP inhibition have been based on fundamental principles and assumptions of in vitro in vivo extrapolations (IVIVE), and have been essential for predicting the magnitude of clinical DDIs. The success of these models demonstrates that in vitro data can be integrated into predictive models for in vivo DDI predictions and that useful predictions can be performed relatively early in the drug discovery process. These predictions aid in (1) selecting compounds for further development; (2) developing structure-activity relationships (SAR) to avoid the potential for DDIs; and (3) planning of clinical DDI studies for compounds that are advanced into further drug development. In the present study, predictive models were developed and employed for IVIVE of induction-based DDIs. Correlations of the predicted DDIs with observed in vivo DDIs were also analyzed. 6

7 Materials and Methods Materials. The test articles were obtained from the following sources: dimethylsulfoxide (DMSO), rifampin (RIF), phenobarbital (PB), dexamethasone (DEX), testosterone, 6β-hydroxytestosterone, Dulbecco s Modified Eagle s Medium (Plating Medium), and Serum-Free William s Medium E (Culture Medium) from Sigma (St. Louis, MO); bosentan (BST) and efavirenz (EFA) from Biomol International LP (Plymouth Meeting, PA); carbamazepine (CMZ) from MP Biomedicals (Solon, OH); fetal bovine serum from Invitrogen (Carlsbad, CA), Sandwich Medium from BD Biosciences (Bedford, MA) and Krebs-Henseleit Buffer (KHB) from Amgen (Thousand Oaks, CA). Freshly isolated human hepatocytes were purchased from CellzDirect (Pittsboro, NC). Human Hepatocyte Culture and Experimental Procedure. Fresh human hepatocytes from two donors were purchased from CellzDirect; Donor 1: a 36-year-old Caucasian female (height = 5 3, body weight = 160 lbs., Lot 0624) with no record of medications, or of substance or alcohol abuse; and Donor 2: a 41 year-old Caucasian female (height = 5'7", body weight = 152 lbs, Lot 0697) with a history of smoking but no alcohol abuse. On Day 1, fresh hepatocytes were received and suspended in Plating Medium (0.75 x 10 6 cells/ml). Hepatocytes were counted and plated in collagen-coated 24-well plates with a density of 0.4 x 10 6 cells/well (BD Biosciences, Bedford, MA). The hepatocytes were placed in a 37 C incubator (Steri-Cult CO 2 Incubator, Model 3310, Thermo Electron Corporation, Waltham, MA) under an atmosphere of 95% air / 5% CO 2 and 90% relative humidity and allowed a 3 5 hour attachment period. Following the attachment period, the Plating Medium and unattached cells were removed by aspiration, Sandwich Medium was applied (0.5 ml/well), and the cells were incubated overnight. On Day 2, the Sandwich Medium was aspirated and Culture Medium (0.5 ml/well) was applied for an overnight acclimation period. On Days 3 and 4, Culture Medium containing either DMSO (0.1%), BST ( µm), CMZ ( µm), DEX ( µm), EFA ( µm), PB ( µm) or RIF ( µm) were applied on each day (0.5 ml/well). The test articles were prepared in DMSO stock 7

8 solutions resulting in final incubation concentrations of 0.1% DMSO. Compound treatment was maintained for a total of 48 hours. On Day 5, hepatocytes were gently washed 3 times with KHB (0.5 ml/well, 37 o C) and allowed to acclimate for an additional 10 minutes. CYP enzyme activities were subsequently determined by the addition of the marker substrate testosterone (200 µm; CYP3A4) dissolved in KHB (0.5 ml/well, 37 o C). Following a 15 min incubation, the medium was removed and stored at -80 C until analyzed. Hepatocytes used for mrna analysis were washed once with PBS (0.5 ml/well, 25 o C) containing calcium and magnesium and aspirated. Lysis Mixture (0.5 ml) (Panomics, Inc., Fremont, CA) was added to each well, and then stored at -80ºC until assayed. mrna Analysis. CYP3A4 mrna content was determined with branched deoxyribonucleic acid (b-dna) signal amplification technology using the Panomics Discover XL Kit (Panomics, Inc., Fremont, CA) with assays performed according to the manufacturer s instructions. b-dna probe sets, containing capture extender, label extender and blocking probes for human CYP3A4 (Cat. No. PA-10909), and GAPDH (Cat. No. PA-10382) were also purchased from Panomics, Inc. Plate washing steps were performed on an Elx405 automated microplate washer (BIO-TEK, Winooski, VT) and luminescence was analyzed on a Luminoskan Ascent microplate luminometer (Thermo Labsystems, Helsinki, Finland). CYP mrna levels were normalized to the mrna levels of the housekeeping gene GAPDH. Enzyme Activity. Following the induction treatment period, the metabolism rate of the CYP3A4 marker substrate, testosterone, by the cell cultures was determined. Analysis and quantification of 6β-hydroxytestosterone, the major testosterone metabolite in hepatocyte cultures, was performed by LC-MS/MS on a system comprising a reverse phase HPLC (Shimadzu, Kyoto, Japan) and a triple quadrupole mass spectrometer (Applied Biosystems API 5000, Foster City, CA) using Turbo Ionspray (Applied Biosystems) via Multiple Reaction Monitoring. Samples (25 µl) were loaded on a C18 column (Phenomenex Onyx Monolithic C18, 100 x 3.0 mm, P/No. 8

9 CHO-8158) and analytes were eluted with a linear gradient of mobile phase A (H 2 O with 0.1% acetic acid and 5% methanol) to B (H 2 O with 0.1% acetic acid and 95% methanol) in 4.6 min. The flow rate was 1 ml/min. The metabolite was quantified by comparison of peak area ratios of metabolite to internal standard (prazosin) to a standard curve prepared using authentic 6β-hydroxytestosterone. Fractions Unbound in Plasma and Hepatocytes. Frozen human plasma was thawed and aliquots (2 ml) were preheated to 37 o C and then the individual inducers were incubated at 1 and 10 µm in polypropylene centrifuge tubes (Corning Incorporated, NY) for 15 min. After this incubation period, 800 µl aliquots (n = 2) were transferred into pollyallomar tubes (Beckman Coulter, Inc., Fullerton, CA), then the tubes were placed in a MLA-130 rotor and centrifuged at 16,128g for 3 hr at 37 C in an Optima Max Ultracentrifuge (Beckman Instruments, Palo Alto, CA). Similarly, the inducers at varying concentrations (5 µm BST, 20 µm CMZ, 100 µμ DEX, 10 µm EFA, 250 µm PB and 1 µm RIF) were incubated with human hepatocytes at 37 o C for 5 min. Following incubation, the tubes were centrifuged at 500g for 5 min. Aliquots (0.1 ml; n = 3) of the supernatants (the unbound fractions) were then removed from each tube. To precipitate proteins and prepare the samples for analysis, 50 µl of aliquoted supernatant from the plasma or hepatocyte samples were mixed with 200 µl of acetonitrile containing µg/ml internal standard (proprietary Amgen compound). The samples were thoroughly mixed by vortexing and then centrifuged for 10 min at 405g and then 200 µl of the resultant supernatant was transferred into 96-well plate. Solvent was removed under a stream of N 2 gas and the samples were reconstituted with 100 µl of 50% MeOH/H 2 O for LC-MS/MS analysis. Standard curves and quality controls were prepared in the same manner. Concentrations of analytes were determined using a standard curve prepared in the same matrix. The unbound fraction in plasma (f u,p ) or hepatocytes (f u,hept ) was determined as a ratio of the concentration measured relative to total concentration. Recovery of drug was also determined by measuring the initial concentration of drug in the plasma or hepatocytes and comparing it to the nominal concentrations. All measurements were conducted in triplicate. 9

10 Data Analysis. In vitro induction assays were performed in triplicate. Concentration-response datasets of each inducer for mrna and enzyme activity were plotted and fitted to the Hill equation (Eqn. 1) using Sigmaplot 10.0 (Systat Software, Inc., Chicago, IL), where E max = maximum response (net maximum fold-increase); EC 50 = inducer concentration at 50% E max ; [Ind] = inducer concentration; and n = sigmoidity of the fitted curve. E = E EC max n 50 [ Ind ] n + [ Ind ] n [1] CYP3A4 Induction Modeling. Induction of CYP3A4 enzyme by an inducer in hepatocytes is described as a receptor-mediated (e.g. PXR) process which triggers transcriptional activation of the gene coding for the protein. The fraction of occupancy (FO) of the DNA promoter elements by inducer complex (inducer-receptor) is expressed by Eqn. 2 and related to the concentration of the inducer. [ Ind R DNA FO = [ DNA ] RE RE n ] [ Ind ] = n [ Ind ] + EC In Eqn. 2, FO = fractional occupancy; [Ind] = inducer concentration; R = receptor concentration; DNA RE = response element concentration; n = Hill coefficient; and EC 50 = dissociation constant. n 50 [2] Hepatic CYP3A4 content in the absence of inducer is governed by the rate of de novo enzyme synthesis (K 0 ) and the rate of enzyme degradation (K deg ) as shown in Eqn. 3. At steady state, the rate of de novo biosynthesis of the enzyme equals the degradation rate (K 0 = K deg [E] ss ). It follows that the enzyme content ([E] ss ) and intrinsic clearance (CL int ) of a substrate of the enzyme are described by Eqns. 4 and 5, respectively; d[ E] = K 0 K deg [ E] dt [3] 10

11 [ E] ss = K K 0 deg [4] CL int [ E] ss k = K m cat [5] where K 0 and K deg = rates of enzyme synthesis and degradation, respectively; [E] ss = enzyme content at steady state; k cat = rate constant for metabolism of a substrate by the enzyme; and K m = substrate-enzyme dissociation constant. When an inducer is present the enzyme level is raised to a new steady state level as described in Eqn. 6. At the new steady state the enzyme level ([E] ss ) is defined by Eqn. 7 and intrinsic clearance (CL int ) for a substrate is determined by Eqn. 8; d[ E]' = K 0 + K Ind [ E]' K deg [ E]' = K 0 + ( K max K 0 ) FO K deg [ E]' dt [ ]' E SS CL = ' int K 0 + ( K max K 0 ) [ E]' = K SS m K k deg cat FO [6] [7] [8] where K ind = rate constant for enzyme induction; K max = maximum velocity of enzyme synthesis; and [E] ss = enzyme content at steady state in the presence of inducer. The ratio of CL int in the absence and presence of an inducer is expressed by Eqn. 9, where E max is the maximum induction of enzyme (net fold increase of enzyme). CL CL' int int [ E] = [ E]' ss ss = ( K 1+ K max 0, invitro 1 K ( EC 0, invitro n 50 ) [ Ind ] + [ Ind ] n n ) = E 1+ ( EC max n 50 1 [ Ind ] n + [ Ind ] n ) [9] 11

12 Prediction of DDIs from In Vitro CYP3A4 Induction. Based on pharmacokinetic principles, the AUC ratio of a substrate after intravenous administration in the presence and absence of an inducer is expressed by Eqn. 10. With oral administration the AUC of a substrate in the presence of an inducer is mainly affected by changes in hepatic bioavailability (F h ) and clearance (CL h ), assuming that gastrointestinal absorption and metabolic extraction (E g ) are not altered significantly (F ab =1, fraction of drug absorbed in gut and F g = 1, gut bioavailability) (Eqn. 11). Therefore, the AUC ratio of the dose is expressed in Eqn. 12 AUC AUC ' IV IV F AUC AUC Q = Q CL = CL ' h ' h h PO = (1 E PO syst syst u CL h = CL ' ) = Q h CL = CL ' PO PO Q h + f u CL ' Qh + f CL int int h + + = CL CL h Qh + f CL u F ' h CL F CL ' h r r CL h = CL ' int Qh f u CL Q h + f u CL Q h f u CL ' Qh + f u CL ' h h h + CL + CL h h int int int int 1 ( f h 1 ( f h = 1) = 1) CL CL ' int int f h 1 CL ' h + (1 CL h f h ) [10] [11] [12] To predict the potential for drug interactions caused by CYP3A4 induction, a relationship of hepatic clearance to CYP3A4 intrinsic clearance (CL int,cyp3a4 ) is derived in Eqn. 13. If a compound is highly cleared by the liver (Q h << f u CL int or f u CL int ) and CL h (or CL h ) is limited by the hepatic flow rate, then CL h is equal to CL h (CL h /CL h = 1). For a low clearance drug (f u CL int or f u CL int << Q h ), however, the ratio of CL h to CL h can be expressed by Eqn. 13. The change in AUC ratio is then predicted by Eqn. 12

13 14 (Eqns. 10 and 13 combined) for intravenous administration, and Eqn. 15 (Eqns. 12 and 13 combined) for oral administration. AUC' = Qh fu CL int CL Qh fu CL h + int CL int Qh + fu = = CL ' h Q ' ' h fu CL CL int int Qh + fu Qh fu CL ' + int CL CL int int, CYP 3 A 4 + CL int, othercyps = CL ' int CL ' int, CYP 3 A4 + CLint, othercyps 1 CL int, CYP 3 A 4 + CLint, CYP 3 A4 ( 1) f m, CYP 3 A 4 = 1 CL ' int, CYP 3 A 4 + CL ' int, CYP 3 A 4 ( 1) f m, CYP 3 A 4 1 = CL ' int, CYP 3 A 4 f m, CYP 3 A 4 + (1 f m, CYP 3 A 4 ) CL AUC f h or( f h IV IV f CL = CL' m, CYP3 A4 = 1) = f syst syst = f E (1 + ( EC m, CYP3 A4 int, CYP 3 A 4 h f m, CYP3 A4 max n 50 E (1 + ( EC CL' CL 1 1 n [ Ind] ) + (1 f n + [ Ind] ) max n 50 int, CYP3 A4 int, CYP3 A4 + (1 f 1 n [ Ind] ) + (1 f n + [ Ind] ) h f h f m, CYP3 A4 ) m, CYP3 A4 CL ' CL m, CYP3 A4 ) int int ) [13] [14] AUC' AUC PO PO CLint 1 = = n [15] CL' int Emax [ Ind] f m, CYP3 A4 (1 + ) + (1 f m, CYP3 A4) n n ( EC + [ Ind] ) 50 13

14 where [Ind] = inducer plasma concentration achieved at steady state, usually C max,ss. In many cases, E max and EC 50 are not readily obtained from concentrationresponse curves measured by in vitro induction assays because of limitations imposed by drug solubility, cell permeability, or toxicity. In these instances the slope of the induction response curve (equivalent to E max /EC 50 ) at a more experimentally feasible low concentration range of the inducer can be used for the prediction (Eqn. 16). This equation is, however, only applicable if in vivo concentrations of an inducer are low ([Ind] << EC 50 ). Eqn. 17 shows the predictive model with inclusion of fractions unbound in plasma (f u,p ) and hepatocytes (f u,hept ), based on the hypothesis that only unbound drug (inducer) can access the intracellular nuclear receptor responsible for induction. AUC ' = AUC f 1 n (1 + Slope [ Ind ] ) + (1 f m, CYP 3 A4 m, CYP 3 A4 AUC' 1 = n [17] AUC Emax [ fu, p Ind] f m, CYP3 A4 (1 + ) + (1 f m, CYP3 A4 ) n n ( EC f ) + [ f Ind] ) 50 u, hept Contribution of CYP3A4 to Drug Clearance In Vivo (f m,cyp3a4 ). Fractions of the drugs cleared by CYP3A4 (f m,cyp3a4 ) were collected from clinical DDI studies reported in the literature. The extent of decreased clearance (or increased AUC) of a substrate drug in the presence of a selective CYP3A4 inhibitor (f i,cyp3a4 ) is employed as an estimate of f m,cyp3a4 and calculated by Eqn. 18. In theory, an accurate value of f m,cyp3a4 should be given by the maximum decrease in clearance of a victim drug that is administered intravenously from clinical DDI studies (complete inhibition of the target enzyme by an inhibitor). However, most DDI studies reported in literature were performed by the oral administration of victim drugs, in which the first-pass metabolism of the drugs in the gut (F g ) could be affected (Eqn. 19) (Obach, et al., 2006). Unfortunately, the estimated ratios of F g /F g (F g and F g = gut bioavailability in the presence and absence of inhibitor) in literature reports were not readily available for most u, p ) [16] 14

15 drugs. Thus, the estimation of f m,cyp3a4 for the victim drugs of oral administration from clinical DDI data is based on assumptions that f g,cyp3a4 (Eqn. 19) is not changed in the presence of CYP3A4 inhibitor and that the inhibition of the CYP3A4 pathway is complete. In addition, when the f m,cyp3a4 were calculated in the present study for the victim drugs given intravenously it is also assumed that the inhibition of intrinsic clearance of the CYP3A4 pathway by the inhibitors is complete (perhaps close enough for approximation), even for the high extraction drugs. f CL' AUC [ I ] [ ctr ] m, CYP3 A4 fi, CYP3 A4 = 1 = 1 [18] CL[ ctr ] AUC' [ I ] CL' CL IV IV PO g PO = = (assuming F g = F g ) [19] IV AUC AUC' IV AUC AUC' PO F F g ' AUC AUC' PO Correlation Analysis. The correlation analyses between predicted and clinical DDIs were performed by linear regression with ANOVA. R 2 (correlation coefficient) and RSS (Residual Sum of Squares, a measure of the size of the residuals, which are the differences of the actual data points from regression modeled values) were determined. In addition, weighted residuals (the residual divided by the standard error of the estimate) were analyzed to assess the distribution (normal or outlying) of the residuals. 15

16 Results Concentration-Response Profiles of In Vitro CYP3A4 Induction. Human hepatocytes are routinely used as an experimental system for the evaluation of CYP induction potential. Selection of the model compounds, BST, CMZ, DEX, EFA, PB, and RIF was based on (1) known CYP3A4 induction in vitro and in vivo; (2) minimal potential for mechanism-based inhibition of CYP3A4; and (3) known clinically relevant concentrations. Hepatocytes were prepared from fresh human livers (two donors), cultured for two days, and then treated with the inducers. After 48 hr incubations with the inducers at varying concentrations, mrna was analyzed using b-dna technology, and CYP3A4 activity was determined by turnover of the marker substrate testosterone. The measured levels of induction in mrna and enzyme activity were normalized against solvent control and recorded as the fold-increase over basal levels measured in control incubations. Full concentration-response curves were generated to provide complete kinetic characterization of each inducer (Fig. 1). EC 50 and E max values for mrna and enzyme activity were generated by fitting the observed data to the Hill equation (Table 1). All the inducers were found to be capable of inducing CYP3A4 mrna and activity. Moreover, there was general agreement between the two donors and between mrna and activity parameters. As is commonly observed, however, some differences in the induction kinetics were observed. These differences occurred between the two donors and between the inductions measured by mrna or activity levels. Generally, the enzyme activity E max values for Donor 1 were approximately 2 to 3-fold higher than for Donor 2, with the exception of RIF which induced activity to a similar extent with both donors. Between the two donors, enzyme activity EC 50 values for all the inducers were similar with the exception of EFA which had an approximately 5-fold higher EC 50 in hepatocytes from Donor 1 than that from Donor 2. In hepatocytes from Donor 1, the EC 50 and E max values for both mrna and enzyme activity agreed within a 2-fold range except for a 6.7-fold difference in EC 50 for EFA and a 4.7-fold difference in E max for RIF. In Donor 2 there was greater variability in both EC 50 and E max, and mrna E max values were generally higher than those for activity. 16

17 The greater CYP3A4 activity measured as testosterone 6β-hydroxylation after exposure to the inducers indicated that greater amounts of enzyme were present as a result of the induction. The relative in vitro induction potential of the inducers was also evaluated by determining changes in their intrinsic clearance (CL int = E max /EC 50 for enzyme activity). The analysis resulted in a rank order of RIF (42.0, 24.5) > BST (19.6, 4.1) > CMZ (1.03, 0.17), EFA (0.58, 1.45), DEX (0.33, 0.25), and PB (~0.1, 0.07) for Donors 1 and 2, respectively. The experimentally determined E max and EC 50 were incorporated into the prediction models together with f m,cyp3a4, clinical relevant concentration ([Ind] max,ss ), protein binding in plasma (f u,p ), and membrane partitioning in hepatocytes (f u,hept ), as described below. Unbound fractions of the inducers in hepatocytes were measured to correct the EC 50 values for free concentration as required for Eqn. 17. Measurement of f u,hept was performed at inducer concentrations similar to their corresponding EC 50 in hepatocytes. The fractions unbound were widely variable among the compounds (Table 2). Interestingly, binding of the inducers to plasma and hepatocytes was observed to have a similar trend. As previously reported (Austin, et al., 2002;Lobell and Sivarajah, 2003) this probably reflects a relationship between binding in either plasma or membranes and logd (or logp). CYP3A4 Reaction Phentotyping (f m,cyp3a4 ). The degree to which CYP3A4 contributes to the clearance of a drug, that is, the fraction of the drug metabolized by this isozyme (f m,cyp3a4 ), can be obtained from in vivo data (e.g., human radiolabeled compound disposition studies) or estimated by drug interaction studies with CYP-isoform selective inhibitors (f i,cyp3a4, Eqn. 18). f m,cyp3a4 is a critical parameter for the DDI prediction because, as shown in Eqn. 17, the magnitude of the DDI is directly proportional to this parameter. The precise role of an individual CYP in drug clearance, however, is difficult to ascertain because of multiplicity and overlapping substrate specificities for various CYP isoforms. In the present study, f m,cyp3a4 values were determined from clinical DDIs associated with CYP3A4 inhibition. The extent to which systemic or oral clearance of a drug is altered in the presence of an isozyme-specific inhibitor reveals the fraction of that drug cleared by CYP3A4. A number of CYP3A4 17

18 selective inhibitors have been widely used for co-administration with drugs suspected to be CYP3A4 substrates. These include the competitive inhibitors ketoconazole, fluconazole, itraconazole, and voriconazole, and the mechanism-based inhibitors mibefradil, ritonavir, diltiazem, clarithromcyin, and saquinavir (Table 3). The f m,cyp3a4 values were calculated by Eqn. 18 for the drugs used in this study and are shown in Table 3. As seen in Table 3, the f m,cyp3a4 values from many drug interaction studies ranged markedly from 0.05 (ropivacaine) to 0.95 (buspirone). High fractions indicate that the drugs are cleared mainly by CYP3A4 and low fractions imply that the drugs are cleared not only by CYP3A4, but also possibly by other routes including conjugative enzyme- or transporter-mediated pathways, renal or hepatic excretion. In Vivo Inducer Concentrations. In order to achieve a steady state of CYP induction, a human inducing agent must typically be chronically administered. Accordingly, for induction-mediated DDI studies subjects usually receive multiple doses of the inducer over consecutive days. After a steady state of induction is reached the maximum concentration of the inducer ([Ind] max, ss ) can then be measured. Ideally, for purposes of correlating a clinical DDI with [Ind] max, ss, the inducer concentration should be measured directly in the DDI study. In many of the studies cited herein, however, these measurements were not performed. The [Ind] max, ss values were therefore obtained from separate studies (Table 4) where the dose regimens were similar or identical to those used in the clinical DDI studies (Table 5). In the DDI studies the inducers were generally administered orally for several consecutive days followed by administration of the substrate drugs. In Vivo Drug Drug Interactions. The decreased AUC ratios (AUC /AUC) of the affected drugs in response to the inducers (Table 5) were reported within the cited references and resulted in 103 data points of drug/inducer interactions. The drugs represented in Table 5 are chemically and pharmacologically diverse with a wide range in magnitude of DDIs caused by CYP3A4 induction. This was therefore considered to be a suitable data set for the purposes of comparing actual DDIs with those predicted by Eqn

19 In Vitro In Vivo Correlations (IVIVC). The various in vitro data were utilized for a model-directed prediction of DDIs using Eqn 17. These data included the in vitro induction data (Table 1), unbound fractions of drug in hepatocyte incubations (f u, hept ) and in plasma (f u, p ) shown in Table 2, the f m,cyp3a4 values obtained from clinical CYP3A4 inhibition studies (Table 3), and the in vivo inducer concentrations at doses relevant to the DDI studies (Table 4). The predicted DDI values from the in vitro enzyme activity induction data in hepatocytes of the two donors where f u,p and f u,hept of the inducers were included in the prediction are listed in Table 5. The measured clinical DDIs are also shown in Table 5, and these data sets (n = 103) and corresponding correlation analyses are shown in Fig. 2A & 3A. In addition to the predicted DDIs shown in Table 5 where fractions unbound were included, correlation analyses were also performed without inclusion of the unbound fractions in the DDI prediction. These additional comparisons were conducted to improve the understanding of the impact of these parameters on the correlations (Figs. 2B-D & 3B-D). The correlation analyses included the 95% confidence interval (95% probability that the predicted DDI value will occur). A unity line (or unit correlation) is included in each figure to illustrate any difference to the observed correlation. The best correlations were obtained when both f u,p and f u,hept were included in the analyses, leading to R 2 = for Donor 1 (RSS = 3.05) and for Donor 2 (RSS = 3.41), respectively (Figs. 2A & 3A). The regression lines of correlation were very similar to the line of unity (perfect correlation). When f u,p or f u,hept or both were excluded from the calculation, the correlations were found to be relatively poor (R 2 = ; RSS = ) (Figs. 2B - 2D and 3B 3D). As might be expected, over-predictions of the DDIs were observed in the absence of the f u,p (Figs. 2B, 2D, 3B and 3D). The degree of the over-predictions was dependent on the level of inducer binding to plasma. When f u,hept was ignored the drug interactions appeared to be largely under-predicted, particularly for the inducers that were highly bound in hepatocytes such as EFA (Figs. 2C & 3C). Fig. 4 shows the distributions of the weighted residuals of the correlations between the predicted and observed DDIs when both f u,p and f u,hept were included in the 19

20 calculations. Most weighted residuals for the individual inducers were distributed around zero (perfect regression) within ±2 units. The distribution ratios of positive to negative residual were 43:60 for Donor 1 and 44:59 for Donor 2, respectively. This shows that the data were normally distributed around the unity. When evaluating concentration-induction relationships in hepatocytes it is not uncommon to have difficulty determining EC 50 and E max values for CYP inducers that are poorly soluble or toxic. This difficulty arises because poor solubility and toxicity limit the ability to fully determine the concentration-response curves. This limitation can potentially be overcome by using the slope (E max /EC 50 ) of the induction-response curves obtained in the linear range at low concentrations. Using the slope, Eqn. 16 can then be used for the prediction. This approach is, however, only valid when in vivo concentrations of an inducer are much lower than its EC 50, the conditions required for Eqn. 17 to be simplified to Eqn. 16. In the present study, EC 50 and E max values could be obtained for all of the inducers; however, it was observed that many of the in vivo concentrations of the inducers were higher than the EC 50 s. For example, in vivo RIF concentrations were µm (f u,p = 0.175) but the EC 50 for activity was 0.25 to 0.51 µm. When predictions by Eqn. 16 were attempted using the slope values for inducers such as RIF, the DDIs were significantly over-predicted (data not shown). Predictions of drug interactions were also attempted from the in vitro EC 50 and E max values for mrna induction shown in Table 1. The correlation analyses are shown in Fig. 5. The correlations were poor for both donors (R 2 = and 0.436), and for RIF in particular the DDIs were greatly over-estimated. This over-estimation was due predominantly to the much greater E max observed for mrna induction in both donors compared to enzyme activity. 20

21 Discussion Marked induction of CYP3A4 by a drug is undesirable because of the large number of drugs that are dependent on CYP3A4 for their clearance. Induction by a drug can affect not only the clearance of a concomitantly administered medication but also its own clearance of the drug (autoinduction) that is cleared by the induced enzyme (Tran- Johnson, et al., 1987). Simple assays, such as reporter gene assays, that detect binding of inducers to receptors such as PXR have been employed for measuring the capability of a drug to activate induction pathways; however, binding to PXR alone does not necessarily result in concomitant levels of enzyme induction (Sinz, et al., 2006;Luo, et al., 2002;LeCluyse, et al., 2000). Fresh human hepatocytes maintained in primary culture are preferred for the direct assessment of the functional consequences of PXR activation, as measured by induction of mrna and enzyme activity. In the studies described here, differences in the kinetic characteristics of induction were observed between the two donors (Table 1). Such inter-individual variability is not uncommon and imposes a degree of uncertainty in predicting the clinical consequences of induction. Differences in E max values between mrna induction and enzyme activity suggested that in some instances the parallel relationship between these two measures of induction can be altered by factors such as variations in rates of transcription, translation, and protein synthesis. Additional uncertainty in predictions also results from the metabolic instability of inducers in the cell cultures during the incubation time (48 hrs). Consumption of substrate could decrease the actual concentrations in the medium and thus differ from the nominal concentrations used for the calculation of kinetic parameters. In this regard, three inducers used here, BST (BST phenol metabolite), CMZ (CMZ- 10,11-epoxide), and DEX (3-methoxymorphinan), have been reported to be CYP3A4 substrates (Furukori, et al., 1999;van Giersbergen, et al., 2002;Kim, et al., 2005;Gorski, et al., 1994;Wang, et al., 1997). In vivo f m,cyp3a4 is estimated by f i,cyp3a4 (Eqn. 18) as a change in the ratio of either AUC or clearance of a victim drug in the presence and absence of a co- 21

22 administered CYP3A4 inhibitor (Venkatakrishnan and Obach, 2005;Brown, et al., 2005;Galetin, et al., 2006). The ratio ideally represents the proportion of a drug s total clearance mediated by CYP3A4 at a relevant in vivo concentration of the inhibitor (Table 3). Under specific conditions f m,cyp3a4 will be equal to f i,cyp3a4 ; however, if those conditions are not met f m,cyp3a4 could be either over- or under-estimated. When a victim drug is administered orally and the AUC changes after inhibition, f m,cyp3a4 can be equal to f i,cyp3a4 only if the bioavailability (F) of the victim drug did not change. Any increase in F will result in a decrease in apparent clearance (CL/F) and f m,cyp3a4 will be overestimated compared to what would be derived from a true clearance value obtained if the victim drug was given intravenously. Many DDI studies are performed with oral dosing regimens and, therefore, DDI predictions based on Eqn. 18 could be affected by potential errors of f m,cyp3a4 determined from f i,cyp3a4. This is particularly true for drugs with low bioavailability due to high first-pass extraction. In addition, some CYP3A4 inhibitors such as ketoconazole and indinavir also inhibit p-glycoprotein (P-gp). The inhibition of P-gp has been shown to increase oral absorption of substrates (Achira, et al., 1999;Zhang, et al., 1998;Kim, et al., 1998) and would be another mechanism by which F could be affected. Another requirement for f m,cyp3a4 to be equal to f i,cyp3a4 is complete inhibition of CYP3A4 in the inhibition studies where f i,cyp3a4 is determined. Several of the inhibitors used to assess f m,cyp3a4 that are listed in Table 3 were recommended in the FDA draft guidance for use in DDI studies ( These inhibitors were classified as either strong (ketoconazole, ritonavir, itraconazole, clarithromycin, and sequinavir) or moderate (fluconazole, grapefruit juice and erythromycin) inhibitors. An assessment of whether CYP3A4 was indeed completely inhibited is dependent on the in vivo concentration (C max,ss ) of the inhibitor that was achieved and the potency of the inhibitor (K i, K I, k inact ). For example, the extent of inhibition achieved by ketoconazole (C max,ss = µm, f u,p = 0.01, K i = µm and ratio of unbound C max,ss /K i = ) (Varhe, et al., 1994) and ritonavir (C max,ss = 7.8 µm, f u,p = 0.015, K Ι = 0.1 µm and ratio of unbound C max,ss /K i = 1.2) (Hsu, et al., 1998a) were likely to have been high. The 22

23 inhibitors ritonavir, clarithromycin, sequinavir, and erythromcyin have been shown to be potent and selective mechanism-based inhibitors of CYP3A4 (Greenblatt, et al., 1999) and the extent of inhibition with these compounds was likely to have also been high. Mibefradil (K I = 2.3 µm and k inact = 0.4 min -1 ) has been shown to have an in vitro potency greater than clarithromycin (K I = 5.5 µm, k inact = min -1 ) (Prueksaritanont, et al., 1999), and clinical DDIs caused by mibefradil ( fold) were comparable to those caused by clarithromycin ( fold) depending on the extent to which the victim drugs were cleared by CYP3A4 (Jacobson, 2004). The final requirement for f m,cyp3a4 to be equal to f i,cyp3a4 is selective inhibition of CYP3A4 in the DDI clinical trial. The CYP3A4 selectivity of some inhibitors (e.g. ketoconazole and itraconazole) have been reported to be concentration- and substrate-dependent (Galetin, et al., 2005). If other CYPs responsible for the clearance of the victim drug are also inhibited the f m,cyp3a4 can be over-estimated. The dependence of DDI susceptibility on f m,cyp3a4 and [Ind] max, ss is illustrated in simulated RIF- and PB-mediated drug interactions, respectively (Fig. 6). Several laboratories have attempted to predict in vivo DDIs using a relative induction score (RIS) (Ripp, et al., 2006) or an induction ratio (IR) (Kato, et al., 2005) measured in hepatocytes. In neither case, however, f m,cyp3a4 was not incorporated into the models. Fig. 6 clearly illustrates the importance of that parameter in any DDI prediction. The observed in vivo f m,cyp3a4 varied widely for the substrates examined, from 0.05 (ropivacaine) to 0.95 (buspirone). Low f m,cyp3a4 values suggest possible involvement of other clearance pathways of the drugs rather than CYP3A4, such as glucuronidation (ethinyl estradiol), CYP2C9 (losartan), and CYP2C9/19 and CYP2B6 (diazepam). Those drugs (e.g., alfentanil, simvastatin, and midazolam) with high f m,cyp3a4 values are cleared more extensively by CYP3A4 leading to a high degree of susceptibility to a DDI (Table 5). An example of this is the RIF-midazolam interactions where the midazolam AUC was reduced to approximately one-tenth the AUC in the non-induced state. Because of its potent enzyme induction RIF caused large interactions with other drugs; however, the clinically observed decreases in exposure due to co-administration with RIF were greater than those predicted in many cases, particularly for drugs with relatively low f m,cyp3a4 23

24 values. This is because the CYP3A4-dependent portion of the drugs total clearance increases upon induction. In addition, the clearance of CYP3A4 inducers such as RIF can be affected by increased expression of other enzymes and transporters regulated by PXR transactivation or other induction mechanisms such as CAR (Hewitt, et al., 2007). For example, PB was only a moderate inducer of CYP3A4 in primary human hepatocytes and hpxr transactivation assays at its C max concentrations (Luo, et al., 2002;Sinz, et al., 2006), yet it has shown clinically significant DDIs (Table 5). This is probably because PB also induced CYP2B6 and CYP2C contributing to the clearance of the affected drugs by its CAR-mediated induction, and, to a lesser extent, PXR-mediated induction (Luo, et al., 2002). To predict the magnitude of in vivo DDIs, the most appropriate inducer concentrations to use in the calculation would be the concentration achieved in vivo at the target enzyme in the liver. Many drugs enter hepatocytes by passive diffusion which allows projecting intracellular hepatic concentrations based on systemic unbound concentrations ([Ind] max,ss f u,p ). This projection may not be appropriate if (1) the inducers are actively transported into or out of hepatocytes; (2) the inducers or affected drugs are inhibitors of transporters expressed in the liver that transport the inducers (e.g. EFA, verapamil, ritonavir and cyclosporine); or (3) the inducers are extensively metabolized by CYP3A4 (e.g. DEX, BST and CMZ) and the systemic concentration is merely a fraction of the liver exposure. Clearly there are multiple variables that can confound the assumptions in Eqn. 17 and accordingly under- or over-prediction of the DDIs can be expected. For example, BST has been reported to be a substrate for hepatic OATP1B1 and OATP1B3 (Treiber, et al., 2007). The under-prediction of the DDIs by BST with several CYP3A4 substrates, e.g. ethinyl estradiol, glyburide, and sildenafil were probably due to higher concentrations of the inducers in hepatocytes than in plasma. Similarly, RIF has been shown to be a substrate for and inhibitor of the uptake transporters OATP-C and OATP1B1 in the liver (Tirona, et al., 2003). In addition, the liver inlet concentration of inducer after oral administration ([Ind] inlet,ss ) is a function of the concentration in both the hepatic artery ([Ind] max,ss ) and the portal vein after gut absorption (Eqn. 20). 24

25 [ Ind] inlet, SS = [ Ind] max, ss + F a K a Q Dose H [20] Where F a, K a, and Q H are the fraction of dose absorbed from the gut into the portal vein, absorption rate constant of inducer, and hepatic blood flow rate, respectively. For example, [Ind] max,ss (0.1 µm) of DEX is lower than the [Ind] inlet,ss (0.25 µm), calculated by Eqn. 19 with K a = 2.68 hr -1, F a = 0.88, and dose = 1.5 mg (QD, 4 days). This difference could in part explain the under-predictions of DEX interactions with cyclophosphamide, triazolam and other drugs in Table 5 that were observed. The gut may contribute a large portion of the first-pass extraction of CYP3A4 substrates, particularly for those substrates extensively cleared by that isozyme (Thummel, et al., 1996). For example, the bioavailability (F) of cyclosporine was reduced by the co-administration of RIF (Ito, et al., 1998) due to increased metabolism in both gut and liver, and also by induction of P-gp (Wacher, et al., 1995). The latter highlights an additional complexity: RIF, PB, and DEX are also inducers of P-gp via the same nuclear receptor (PXR) that mediates the induction of CYP3A4 (Schuetz, et al., 1996). Thus, the additive effects of increased CYP3A4-mediated metabolism and p-gpmediated efflux by the intestinal epithelium may result in a substantially higher first-pass effect in the gut after oral administration. Without consideration of the gut first-pass effect, DDIs can be under-predicted. In addition, many drugs act as both inducers and inhibitors of CYP3A4 (ritonavir and EFA). When EFA was co-administered with ritonavir (K I = 0.04 µm), indinavir or amprenavir (a potent mechanism-based inhibitor) in combination therapy (Luo, et al., 2003), induction-mediated DDIs were over-predicted as a result of the potent CYP3A4 inhibition by the interacting drugs (Table 5). A high degree of inter-individual variability of DDIs has been commonly observed in the clinic. This variability could be due to genetic polymorphisms of CYPs and transporters. For example, while CYP3A5 and CYP3A4 share common substrates and inducers, CYP3A5 is less inducible, differs in its tissue expression pattern, and is subject to genetic polymorphism (Kuehl, et al., 2001). CYP3A4 can also vary in its 25

26 abundance within a population. Combined, these factors result in the potential for a large degree of inter-individual variability of DDIs following treatment with CYP 3A inducers (Lamba, et al., 2002). Similar genetic polymorphism may also affect OATP-C drug disposition. OATP-C expression is potentiated by PXR activation; however, it has a number of naturally occurring variant forms which have markedly impaired function (Tirona, et al., 2003). In addition to the CYP3A4 induction mediated DDIs described here, the predictive models may also be useful for the evaluation of potential DDIs due to induction of other CYP isoforms such as CYP1A2, CYP2B6 and CYP2C9. The induction of these other CYPs is regulated by different nuclear receptors (i.e. CAR, AhR); however, the fundamental principles of a drug s potential to induce enzyme activity and mrna in hepatocytes, and the endpoint measurements of E max and EC 50 are identical to those used for CYP3A4. The results of the analyses described here reveal some valuable insight into the correlation between in vitro induction and clinical DDIs. The draft FDA guidance for drug interaction studies ( recommends using induction of 40% of the positive control as one endpoint for prediction of clinically relevant enzyme induction. The absolute level of induction that a value of 40% represents is, of course, relative to the extent of induction caused by the positive control, which in turn is dependent on the positive control s potency (E max and EC 50 ) and the concentration tested. The results described here show that 40% of E max for the potent inducers RIF and PB corresponded to a 4.2- and 9.1-fold increase in CYP3A4 activity in Donor 1, respectively. The respective reductions of midazolam AUC predicted by Eqn. 17 for these levels of induction were 78 and 89%. This suggests that induction by a test drug that is 40% of the response for potent inducers that are tested at or near their E max concentrations may result in an excessively large DDI. The model has the potential to be used for classification of an inducer. For example, cutoffs that are proportionately similar to those used to rank CYP inhibitors 26

27 might be applied. Using those criteria, induction could be based on the AUC in the induced state compared to control and ranked as none ( 75%), weak, (50 75%), moderate (20 50%), or strong ( 20%). A corresponding set of in vitro values of potency can be calculated by Eqn. 21 assuming f m,cyp3a4 1. Using this approach, induction of none, weak, moderate, and strong has ratios of 0.33, , , and > 4.0, respectively. Similarly, if E max and EC 50 for a drug can not be obtained, the slope of the response-concentration curve in the linear range measured in hepatocytes can be also used, providing the projected [Ind] < EC 50. E [ Ind] Ratio ] n max n = = slope [ Ind n [21] n EC50 + [ Ind] In conclusion, measurement of in vitro induction parameters for a drug candidate at an early stage of drug discovery can provide valuable information about the drug s potential to cause clinical DDIs. If in vitro induction is observed and DDIs are predicted using the models described here, an informed decision about termination or advancement of the molecule can be made with a better understanding of the associated risk than has previously been available. Should the molecule be advanced, better planning and timing of clinical DDI studies may be possible. Confidence in the DDI prediction could allow for more rapid lead optimization by avoiding unnecessary optimization efforts, or more rapid termination decisions. Ultimately, the decision of whether a drug candidate with a potential of induction should be advanced into development will depend on the magnitude of the predicted DDIs, the clinical indication, the dosing regimen, and concomitant medications that may be CYP inhibitors or predominantly cleared by the induced enzyme. Acknowledgement The authors would like to thank Drs. Kenneth Korzekwa and Paul Pearson for the valuable comments on the work. 27

28 References Aarnoutse RE, Grintjes KJ, Telgt DS, Stek M, Jr., Hugen PW, Reiss P, Koopmans PP, Hekster YA and Burger DM (2002) The influence of efavirenz on the pharmacokinetics of a twice-daily combination of indinavir and low-dose ritonavir in healthy volunteers. Clinical Pharmacology & Therapeutics 71: Adams M, Pieniaszek HJ, Jr., Gammaitoni AR and Ahdieh H (2005) Oxymorphone extended release does not affect CYP2C9 or CYP3A4 metabolic pathways. Journal of Clinical Pharmacology 45: Achira M, Suzuki H, Ito K and Sugiyama Y (1999) Comparative studies to determine the selective inhibitors for P-glycoprotein and cytochrome P4503A4. Aaps Pharmsci 1:E18. Ahonen J, Olkkola KT and Neuvonen PJ (1996) The effect of the antimycotic itraconazole on the pharmacokinetics and pharmacodynamics of diazepam. Fundamental & Clinical Pharmacology 10: Araki K, Yasui-Furukori N, Fukasawa T, Aoshima T, Suzuki A, Inoue Y, Tateishi T and Otani K (2004) Inhibition of the metabolism of etizolam by itraconazole in humans: evidence for the involvement of CYP3A4 in etizolam metabolism. European Journal of Clinical Pharmacology 60: Austin RP, Barton P, Cockroft SL, Wenlock MC and Riley RJ (2002) The influence of nonspecific microsomal binding on apparent intrinsic clearance, and its prediction from physicochemical properties. Drug Metabolism & Disposition 30: Back DJ, Bates M, Bowden A, Breckenridge AM, Hall MJ, Jones H, MacIver M, Orme M, Perucca E, Richens A, Rowe PH and Smith E (1980) The interaction of phenobarbital and other anticonvulsants with oral contraceptive steroid therapy. Contraception 22: Back DJ, Breckenridge AM, Crawford F, MacIver M, Orme ML, Park BK, Rowe PH and Smith E (1979) The effect of rifampicin on norethisterone pharmacokinetics. European Journal of Clinical Pharmacology 15: Backman JT, Kivisto KT, Olkkola KT and Neuvonen PJ (1998) The area under the plasma concentration-time curve for oral midazolam is 400-fold larger during treatment with itraconazole than with rifampicin. European Journal of Clinical Pharmacology 54: Backman JT, Luurila H, Neuvonen M and Neuvonen PJ (2005) Rifampin markedly decreases and gemfibrozil increases the plasma concentrations of atorvastatin and its metabolites. Clinical Pharmacology & Therapeutics 78: Backman JT, Olkkola KT and Neuvonen PJ (1996) Rifampin drastically reduces plasma concentrations and effects of oral midazolam. Clinical Pharmacology & Therapeutics 59:

29 Backman JT, Karjalainen MJ, Neuvonen M, Laitila J and Neuvonen PJ (2006) Rofecoxib is a potent inhibitor of cytochrome P450 1A2: studies with tizanidine and caffeine in healthy subjects. British Journal of Clinical Pharmacology 62: Backman JT, Kyrklund C, Neuvonen M and Neuvonen PJ (2002) Gemfibrozil greatly increases plasma concentrations of cerivastatin. Clinical Pharmacology & Therapeutics 72: Backman JT, Olkkola KT and Neuvonen PJ (1996) Rifampin drastically reduces plasma concentrations and effects of oral midazolam. Clinical Pharmacology & Therapeutics 59:7-13. Barbarash RA, Bauman JL, Fischer JH, Kondos GT and Batenhorst RL (1988) Near-total reduction in verapamil bioavailability by rifampin. Electrocardiographic correlates. Chest 94: Barditch-Crovo P, Trapnell CB, Ette E, Zacur HA, Coresh J, Rocco LE, Hendrix CW and Flexner C (1999) The effects of rifampin and rifabutin on the pharmacokinetics and pharmacodynamics of a combination oral contraceptive. Clinical Pharmacology & Therapeutics 65: Bennett PN, John VA and Whitmarsh VB (1982) Effect of rifampicin on metoprolol and antipyrine kinetics. British Journal of Clinical Pharmacology 13: Bergrem H and Refvem OK (1983) Altered prednisolone pharmacokinetics in patients treated with rifampicin. Acta Medica Scandinavica 213: Bidstrup TB, Stilling N, Damkier P, Scharling B, Thomsen MS and Brosen K (2004) Rifampicin seems to act as both an inducer and an inhibitor of the metabolism of repaglinide. European Journal of Clinical Pharmacology 60: Binet I, Wallnofer A, Weber C, Jones R and Thiel G (2000) Renal hemodynamics and pharmacokinetics of bosentan with and without cyclosporine A. Kidney International 57: Bolton AE, Peng B, Hubert M, Krebs-Brown A, Capdeville R, Keller U and Seiberling M (2004) Effect of rifampicin on the pharmacokinetics of imatinib mesylate (Gleevec, STI571) in healthy subjects. Cancer Chemotherapy & Pharmacology 53: Boyd MA, Aarnoutse RE, Ruxrungtham K, Stek M, Jr., van Heeswijk RP, Lange JM, Cooper DA, Phanuphak P and Burger DM (2003) Pharmacokinetics of indinavir/ritonavir (800/100 mg) in combination with efavirenz (600 mg) in HIV-1-infected subjects. Journal of Acquired Immune Deficiency Syndromes: JAIDS 34: Boyd MA, Zhang X, Dorr A, Ruxrungtham K, Kolis S, Nieforth K, Kinchelow T, Buss N and Patel IH (2003) Lack of enzyme-inducing effect of rifampicin on the pharmacokinetics of enfuvirtide. Journal of Clinical Pharmacology 43:

30 Brown HS, Ito K, Galetin A and Houston JB (2005) Prediction of in vivo drug-drug interactions from in vitro data: impact of incorporating parallel pathways of drug elimination and inhibitor absorption rate constant. British Journal of Clinical Pharmacology 60: Chandler MH, Toler SM, Rapp RP, Muder RR and Korvick JA (1990) Multiple-dose pharmacokinetics of concurrent oral ciprofloxacin and rifampin therapy in elderly patients. Antimicrobial Agents & Chemotherapy 34: Chen M, Nafziger AN and Bertino JS, Jr. (2006) Drug-metabolizing enzyme inhibition by ketoconazole does not reduce interindividual variability of CYP3A activity as measured by oral midazolam. Drug Metabolism & Disposition 34: Chung E, Nafziger AN, Kazierad DJ and Bertino JS, Jr. (2006) Comparison of midazolam and simvastatin as cytochrome P450 3A probes. Clinical Pharmacology & Therapeutics 79: Cobb MN, Desai J, Brown LS, Jr., Zannikos PN and Rainey PM (1998) The effect of fluconazole on the clinical pharmacokinetics of methadone. Clinical Pharmacology & Therapeutics 63: Conney AH (1967) Pharmacological implications of microsomal enzyme induction. Pharmacological Reviews 19: Crawford P, Chadwick DJ, Martin C, Tjia J, Back DJ and Orme M (1990) The interaction of phenytoin and carbamazepine with combined oral contraceptive steroids. British Journal of Clinical Pharmacology 30: D'Mello AP, D'Souza MJ and Bates TR (1985) Pharmacokinetics of ketoconazoleantipyrine interaction. Lancet 2: Damkier P, Hansen LL and Brosen K (1999) Effect of diclofenac, disulfiram, itraconazole, grapefruit juice and erythromycin on the pharmacokinetics of quinidine. British Journal of Clinical Pharmacology 48: Daniels NJ, Dover JS and Schachter RK (1984) Interaction between cyclosporin and rifampicin. Lancet 2:639. Data JL, Wilkinson GR and Nies AS (1976) Interaction of quinidine with anticonvulsant drugs. New England Journal of Medicine 294: de LJ and Bork J (1997) Risperidone and cytochrome P450 3A.[see comment][comment]. Journal of Clinical Psychiatry 58:450. Dickinson BD, Altman RD, Nielsen NH, Sterling ML and Council on Scientific Affairs AMA (2001) Drug interactions between oral contraceptives and antibiotics.[see comment]. [Review] [54 refs]. Obstetrics & Gynecology 98:t

31 Dilger K, Denk A, Heeg MH and Beuers U (2005) No relevant effect of ursodeoxycholic acid on cytochrome P450 3A metabolism in primary biliary cirrhosis. Hepatology 41: Dingemanse J, Schaarschmidt D and van Giersbergen PL (2003) Investigation of the mutual pharmacokinetic interactions between bosentan, a dual endothelin receptor antagonist, and simvastatin. Clinical Pharmacokinetics 42: Doose DR, Wang SS, Padmanabhan M, Schwabe S, Jacobs D and Bialer M (2003) Effect of topiramate or carbamazepine on the pharmacokinetics of an oral contraceptive containing norethindrone and ethinyl estradiol in healthy obese and nonobese female subjects. Epilepsia 44: Droste JA, Verweij-van Wissen CP, Kearney BP, Buffels R, Vanhorssen PJ, Hekster YA and Burger DM (2005) Pharmacokinetic study of tenofovir disoproxil fumarate combined with rifampin in healthy volunteers. Antimicrobial Agents & Chemotherapy 49: Drusano GL, Townsend RJ, Walsh TJ, Forrest A, Antal EJ and Standiford HC (1986) Steady-state serum pharmacokinetics of novobiocin and rifampin alone and in combination. Antimicrobial Agents & Chemotherapy 30: Dutreix C, Peng B, Mehring G, Hayes M, Capdeville R, Pokorny R and Seiberling M (2004) Pharmacokinetic interaction between ketoconazole and imatinib mesylate (Glivec) in healthy subjects. Cancer Chemotherapy & Pharmacology 54: Ferron GM, Patat A, Parks V, Rolan P and Troy SM (2003) Lack of pharmacokinetic interaction between retigabine and phenobarbitone at steady-state in healthy subjects. British Journal of Clinical Pharmacology 56: Eap CB, Buclin T, Cucchia G, Zullino D, Hustert E, Bleiber G, Golay KP, Aubert AC, Baumann P, Telenti A and Kerb R (2004) Oral administration of a low dose of midazolam (75 microg) as an in vivo probe for CYP3A activity. European Journal of Clinical Pharmacology 60: Fleishaker JC, Pearson PG, Wienkers LC, Pearson LK and Peters GR (1996) Biotransformation of tirilazad in human: 2. Effect of ketoconazole on tirilazad clearance and oral bioavailability. Journal of Pharmacology & Experimental Therapeutics 277: Floren LC, Bekersky I, Benet LZ, Mekki Q, Dressler D, Lee JW, Roberts JP and Hebert MF (1997) Tacrolimus oral bioavailability doubles with coadministration of ketoconazole. Clinical Pharmacology & Therapeutics 62: Fromm MF, Busse D, Kroemer HK and Eichelbaum M (1996) Differential induction of prehepatic and hepatic metabolism of verapamil by rifampin. Hepatology 24:

32 Fuhr U, Muller-Peltzer H, Kern R, Lopez-Rojas P, Junemann M, Harder S and Staib AH (2002) Effects of grapefruit juice and smoking on verapamil concentrations in steady state. European Journal of Clinical Pharmacology 58: Furukori H, Kondo T, Yasui N, Otani K, Tokinaga N, Nagashima U, Kaneko S and Inoue Y (1999) Effects of itraconazole on the steady-state plasma concentrations of bromperidol and reduced bromperidol in schizophrenic patients. Psychopharmacology 145: Furukori H, Otani K, Yasui N, Kondo T, Kaneko S, Shimoyama R, Ohkubo T, Nagasaki T and Sugawara K (1998) Effect of carbamazepine on the single oral dose pharmacokinetics of alprazolam. Neuropsychopharmacology 18: Galetin A, Burt H, Gibbons L and Houston JB (2006) Prediction of time-dependent CYP3A4 drug-drug interactions: impact of enzyme degradation, parallel elimination pathways, and intestinal inhibition. Drug Metabolism & Disposition 34: Galetin A, Ito K, Hallifax D and Houston JB (2005) CYP3A4 substrate selection and substitution in the prediction of potential drug-drug interactions. Journal of Pharmacology & Experimental Therapeutics 314: Gerber JG, Rosenkranz SL, Fichtenbaum CJ, Vega JM, Yang A, Alston BL, Brobst SW, Segal Y, Aberg JA and AIDS Clinical Trials Group (2005) Effect of efavirenz on the pharmacokinetics of simvastatin, atorvastatin, and pravastatin: results of AIDS Clinical Trials Group 5108 Study. Journal of Acquired Immune Deficiency Syndromes: JAIDS 39: Goldberg MR, Lo MW, Deutsch PJ, Wilson SE, McWilliams EJ and McCrea JB (1996) Phenobarbital minimally alters plasma concentrations of losartan and its active metabolite E Clinical Pharmacology & Therapeutics 59: Gomez DY, Wacher VJ, Tomlanovich SJ, Hebert MF and Benet LZ (1995) The effects of ketoconazole on the intestinal metabolism and bioavailability of cyclosporine. Clinical Pharmacology & Therapeutics 58: Goodwin B, Hodgson E and Liddle C (1999) The orphan human pregnane X receptor mediates the transcriptional activation of CYP3A4 by rifampicin through a distal enhancer module. Molecular Pharmacology 56: Gorski JC, Jones DR, Wrighton SA and Hall SD (1994) Characterization of dextromethorphan N-demethylation by human liver microsomes. Contribution of the cytochrome P450 3A (CYP3A) subfamily. Biochemical Pharmacology 48: Gorski JC, Vannaprasaht S, Hamman MA, Ambrosius WT, Bruce MA, Haehner-Daniels B and Hall SD (2003) The effect of age, sex, and rifampin administration on intestinal and hepatic cytochrome P450 3A activity.[erratum appears in Clin Pharmacol Ther Mar;75(3):249]. Clinical Pharmacology & Therapeutics 74:

33 Goujard C, Vincent I, Meynard JL, Choudet N, Bollens D, Rousseau C, Demarles D, Gillotin C, Bidault R and Taburet AM (2003) Steady-state pharmacokinetics of amprenavir coadministered with ritonavir in human immunodeficiency virus type 1- infected patients. Antimicrobial Agents & Chemotherapy 47: Greenblatt DJ, von Moltke LL, Daily JP, Harmatz JS and Shader RI (1999) Extensive impairment of triazolam and alprazolam clearance by short-term low-dose ritonavir: the clinical dilemma of concurrent inhibition and induction. [Review] [51 refs]. Journal of Clinical Psychopharmacology 19: Greenblatt DJ, von Moltke LL, Harmatz JS, Durol AL, Daily JP, Graf JA, Mertzanis P, Hoffman JL and Shader RI (2000) Differential impairment of triazolam and zolpidem clearance by ritonavir. Journal of Acquired Immune Deficiency Syndromes: JAIDS 24: Greenblatt DJ, Wright CE, von Moltke LL, Harmatz JS, Ehrenberg BL, Harrel LM, Corbett K, Counihan M, Tobias S and Shader RI (1998) Ketoconazole inhibition of triazolam and alprazolam clearance: differential kinetic and dynamic consequences. Clinical Pharmacology & Therapeutics 64: Grimm SW, Richtand NM, Winter HR, Stams KR and Reele SB (2006) Effects of cytochrome P450 3A modulators ketoconazole and carbamazepine on quetiapine pharmacokinetics. British Journal of Clinical Pharmacology 61: Grub S, Bryson H, Goggin T, Ludin E and Jorga K (2001) The interaction of saquinavir (soft gelatin capsule) with ketoconazole, erythromycin and rifampicin: comparison of the effect in healthy volunteers and in HIV-infected patients. European Journal of Clinical Pharmacology 57: Guengerich FP (1999) Cytochrome P-450 3A4: regulation and role in drug metabolism. [Review] [108 refs]. Annual Review of Pharmacology & Toxicology 39:1-17. Gurley B, Hubbard MA, Williams DK, Thaden J, Tong Y, Gentry WB, Breen P, Carrier DJ and Cheboyina S (2006) Assessing the clinical significance of botanical supplementation on human cytochrome P450 3A activity: comparison of a milk thistle and black cohosh product to rifampin and clarithromycin. Journal of Clinical Pharmacology 46: He N, Huang SL, Zhu RH, Tan ZR, Liu J, Zhu B and Zhou HH (2003) Inhibitory effect of troleandomycin on the metabolism of omeprazole is CYP2C19 genotype-dependent. Xenobiotica 33: Heimark LD, Gibaldi M, Trager WF, O'Reilly RA and Goulart DA (1987) The mechanism of the warfarin-rifampin drug interaction in humans. Clinical Pharmacology & Therapeutics 42:

34 Hebert MF, Fisher RM, Marsh CL, Dressler D and Bekersky I (1999) Effects of rifampin on tacrolimus pharmacokinetics in healthy volunteers. Journal of Clinical Pharmacology 39: Hebert MF, Roberts JP, Prueksaritanont T and Benet LZ (1992) Bioavailability of cyclosporine with concomitant rifampin administration is markedly less than predicted by hepatic enzyme induction. Clinical Pharmacology & Therapeutics 52: Hewitt NJ, Lechon MJ, Houston JB, Hallifax D, Brown HS, Maurel P, Kenna JG, Gustavsson L, Lohmann C, Skonberg C, Guillouzo A, Tuschl G, Li AP, LeCluyse E, Groothuis GM and Hengstler JG (2007) Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies. [Review] [356 refs]. Drug Metabolism Reviews 39: Hilbert J, Messig M, Kuye O and Friedman H (2001) Evaluation of interaction between fluconazole and an oral contraceptive in healthy women. Obstetrics & Gynecology 98: Hillebrand G, Castro LA, van SW, Beukelmann D, Land W and Schmidt D (1987) Valproate for epilepsy in renal transplant recipients receiving cyclosporine.[see comment]. Transplantation 43: Holtbecker N, Fromm MF, Kroemer HK, Ohnhaus EE and Heidemann H (1996) The nifedipine-rifampin interaction. Evidence for induction of gut wall metabolism.[see comment]. Drug Metabolism & Disposition 24: Honig PK, Wortham DC, Zamani K, Conner DP, Mullin JC and Cantilena LR (1993) Terfenadine-ketoconazole interaction. Pharmacokinetic and electrocardiographic consequences.[see comment][erratum appears in JAMA 1993 Apr 28;269(16):2088]. JAMA 269: Hsu A, Granneman GR, Cao G, Carothers L, Japour A, El-Shourbagy T, Dennis S, Berg J, Erdman K, Leonard JM and Sun E (1998b) Pharmacokinetic interaction between ritonavir and indinavir in healthy volunteers. Antimicrobial Agents & Chemotherapy 42: Hsu A, Isaacson J, Brun S, Bernstein B, Lam W, Bertz R, Foit C, Rynkiewicz K, Richards B, King M, Rode R, Kempf DJ, Granneman GR and Sun E (2003) Pharmacokinetic-pharmacodynamic analysis of lopinavir-ritonavir in combination with efavirenz and two nucleoside reverse transcriptase inhibitors in extensively pretreated human immunodeficiency virus-infected patients. Antimicrobial Agents & Chemotherapy 47: Hsu A, Granneman GR, Cao G, Carothers L, Japour A, El-Shourbagy T, Dennis S, Berg J, Erdman K, Leonard JM and Sun E (1998a) Pharmacokinetic interaction between ritonavir and indinavir in healthy volunteers. Antimicrobial Agents & Chemotherapy 42:

35 Humbert G, Brumpt I, Montay G, Le LA, Frydman A, Borsa-Lebas F and Moore N (1991) Influence of rifampin on the pharmacokinetics of pefloxacin. Clinical Pharmacology & Therapeutics 50: Ito K, Iwatsubo T, Kanamitsu S, Ueda K, Suzuki H and Sugiyama Y (1998) Prediction of pharmacokinetic alterations caused by drug-drug interactions: metabolic interaction in the liver. [Review] [93 refs]. Pharmacological Reviews 50: Jacobson TA (2004) Comparative pharmacokinetic interaction profiles of pravastatin, simvastatin, and atorvastatin when coadministered with cytochrome P450 inhibitors. American Journal of Cardiology 94: Jokinen MJ, Ahonen J, Neuvonen PJ and Olkkola KT (2001a) Effect of clarithromycin and itraconazole on the pharmacokinetics of ropivacaine. Pharmacology & Toxicology 88: Jokinen MJ, Olkkola KT, Ahonen J and Neuvonen PJ (2001b) Effect of rifampin and tobacco smoking on the pharmacokinetics of ropivacaine. Clinical Pharmacology & Therapeutics 70: Jones SA, Moore LB, Shenk JL, Wisely GB, Hamilton GA, McKee DD, Tomkinson NC, LeCluyse EL, Lambert MH, Willson TM, Kliewer SA and Moore JT (2000) The pregnane X receptor: a promiscuous xenobiotic receptor that has diverged during evolution. Molecular Endocrinology 14: Kapil RP, Axelson JE, Mansfield IL, Edwards DJ, McErlane B, Mason MA, Lalka D and Kerr CR (1987) Disopyramide pharmacokinetics and metabolism: effect of inducers. British Journal of Clinical Pharmacology 24: Kato M, Chiba K, Horikawa M and Sugiyama Y (2005) The quantitative prediction of in vivo enzyme-induction caused by drug exposure from in vitro information on human hepatocytes. Drug Metabolism & Pharmacokinetics 20: Kaukonen KM, Olkkola KT and Neuvonen PJ (1998) Fluconazole but not itraconazole decreases the metabolism of losartan to E European Journal of Clinical Pharmacology 53: Ketter TA, Jenkins JB, Schroeder DH, Pazzaglia PJ, Marangell LB, George MS, Callahan AM, Hinton ML, Chao J and Post RM (1995) Carbamazepine but not valproate induces bupropion metabolism. Journal of Clinical Psychopharmacology 15: Khaliq Y, Gallicano K, Venance S, Kravcik S and Cameron DW (2000) Effect of ketoconazole on ritonavir and saquinavir concentrations in plasma and cerebrospinal fluid from patients infected with human immunodeficiency virus. Clinical Pharmacology & Therapeutics 68:

36 Kharasch ED, Hoffer C, Whittington D and Sheffels P (2004a) Role of hepatic and intestinal cytochrome P450 3A and 2B6 in the metabolism, disposition, and miotic effects of methadone. Clinical Pharmacology & Therapeutics 76: Kharasch ED, Walker A, Hoffer C and Sheffels P (2004b) Intravenous and oral alfentanil as in vivo probes for hepatic and first-pass cytochrome P450 3A activity: noninvasive assessment by use of pupillary miosis. Clinical Pharmacology & Therapeutics 76: Kharasch ED, Walker A, Hoffer C and Sheffels P (2005) Sensitivity of intravenous and oral alfentanil and pupillary miosis as minimally invasive and noninvasive probes for hepatic and first-pass CYP3A activity. Journal of Clinical Pharmacology 45: Kim KA, Oh SO, Park PW and Park JY (2005) Effect of probenecid on the pharmacokinetics of carbamazepine in healthy subjects. European Journal of Clinical Pharmacology 61: Kim RB, Fromm MF, Wandel C, Leake B, Wood AJ, Roden DM and Wilkinson GR (1998) The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV- 1 protease inhibitors. Journal of Clinical Investigation 101: Kivisto KT, Lamberg TS, Kantola T and Neuvonen PJ (1997) Plasma buspirone concentrations are greatly increased by erythromycin and itraconazole.[see comment]. Clinical Pharmacology & Therapeutics 62: Kivisto KT, Lamberg TS and Neuvonen PJ (1999) Interactions of buspirone with itraconazole and rifampicin: effects on the pharmacokinetics of the active 1-(2- pyrimidinyl)-piperazine metabolite of buspirone. Pharmacology & Toxicology 84: Kliewer SA, Moore JT, Wade L, Staudinger JL, Watson MA, Jones SA, McKee DD, Oliver BB, Willson TM, Zetterstrom RH, Perlmann T and Lehmann JM (1998) An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell 92: Kovarik JM, Beyer D, Bizot MN, Jiang Q, Shenouda M and Schmouder RL (2005) Effect of multiple-dose erythromycin on everolimus pharmacokinetics. European Journal of Clinical Pharmacology 61: Kondo S, Fukasawa T, Yasui-Furukori N, Aoshima T, Suzuki A, Inoue Y, Tateishi T and Otani K (2005) Induction of the metabolism of etizolam by carbamazepine in humans. European Journal of Clinical Pharmacology 61: Kovarik JM, Hartmann S, Figueiredo J, Rouilly M, Port A and Rordorf C (2002) Effect of rifampin on apparent clearance of everolimus. Annals of Pharmacotherapy 36: Kremens B, Brendel E, Bald M, Czyborra P and Michel MC (1999) Loss of blood pressure control on withdrawal of fluconazole during nifedipine therapy. British Journal of Clinical Pharmacology 47:

37 Kuehl P, Zhang J, Lin Y, Lamba J, Assem M, Schuetz J, Watkins PB, Daly A, Wrighton SA, Hall SD, Maurel P, Relling M, Brimer C, Yasuda K, Venkataramanan R, Strom S, Thummel K, Boguski MS and Schuetz E (2001) Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression. Nature Genetics 27: Kuypers DR, Verleden G, Naesens M and Vanrenterghem Y (2005) Drug interaction between mycophenolate mofetil and rifampin: possible induction of uridine diphosphateglucuronosyltransferase. Clinical Pharmacology & Therapeutics 78: Kyrklund C, Backman JT, Kivisto KT, Neuvonen M, Laitila J and Neuvonen PJ (2000) Rifampin greatly reduces plasma simvastatin and simvastatin acid concentrations. Clinical Pharmacology & Therapeutics 68: la Porte CJ, Colbers EP, Bertz R, Voncken DS, Wikstrom K, Boeree MJ, Koopmans PP, Hekster YA and Burger DM (2004a) Pharmacokinetics of adjusted-dose lopinavirritonavir combined with rifampin in healthy volunteers. Antimicrobial Agents & Chemotherapy 48: la Porte CJ, de Graaff-Teulen MJ, Colbers EP, Voncken DS, Ibanez SM, Koopmans PP, Hekster YA and Burger DM (2004b) Effect of efavirenz treatment on the pharmacokinetics of nelfinavir boosted by ritonavir in healthy volunteers. British Journal of Clinical Pharmacology 58: Lamba JK, Lin YS, Schuetz EG and Thummel KE (2002) Genetic contribution to variable human CYP3A-mediated metabolism. [Review] [114 refs]. Advanced Drug Delivery Reviews 54: Lamberg TS, Kivisto KT and Neuvonen PJ (1998) Concentrations and effects of buspirone are considerably reduced by rifampicin. British Journal of Clinical Pharmacology 45: Laroudie C, Salazar DE, Cosson JP, Cheuvart B, Istin B, Girault J, Ingrand I and Decourt JP (2000) Carbamazepine-nefazodone interaction in healthy subjects. Journal of Clinical Psychopharmacology 20: LeBel M, Masson E, Guilbert E, Colborn D, Paquet F, Allard S, Vallee F and Narang PK (1998) Effects of rifabutin and rifampicin on the pharmacokinetics of ethinylestradiol and norethindrone. Journal of Clinical Pharmacology 38: Leclercq V, Desager JP, Horsmans Y, Van NY and Harvengt C (1989) Influence of rifampicin, phenobarbital and cimetidine on mixed function monooxygenase in extensive and poor metabolizers of debrisoquine. International Journal of Clinical Pharmacology, Therapy, & Toxicology 27: LeCluyse E, Madan A, Hamilton G, Carroll K, DeHaan R and Parkinson A (2000) Expression and regulation of cytochrome P450 enzymes in primary cultures of human hepatocytes. Journal of Biochemical & Molecular Toxicology 14:

38 Licht RW, Olesen OV, Friis P and Laustsen T (2000) Olanzapine serum concentrations lowered by concomitant treatment with carbamazepine. Journal of Clinical Psychopharmacology 20: Lilja JJ, Niemi M, Fredrikson H and Neuvonen PJ (2007) Effects of clarithromycin and grapefruit juice on the pharmacokinetics of glibenclamide. British Journal of Clinical Pharmacology 63: Lobell M and Sivarajah V (2003) In silico prediction of aqueous solubility, human plasma protein binding and volume of distribution of compounds from calculated pka and AlogP98 values. Molecular Diversity 7: Lopez-Cortes LF, Ruiz-Valderas R, Viciana P, arcon-gonzalez A, Gomez-Mateos J, Leon-Jimenez E, Sarasanacenta M, Lopez-Pua Y and Pachon J (2002) Pharmacokinetic interactions between efavirenz and rifampicin in HIV-infected patients with tuberculosis. Clinical Pharmacokinetics 41: Luo G, Cunningham M, Kim S, Burn T, Lin J, Sinz M, Hamilton G, Rizzo C, Jolley S, Gilbert D, Downey A, Mudra D, Graham R, Carroll K, Xie J, Madan A, Parkinson A, Christ D, Selling B, LeCluyse E and Gan LS (2002) CYP3A4 induction by drugs: correlation between a pregnane X receptor reporter gene assay and CYP3A4 expression in human hepatocytes. Drug Metabolism & Disposition 30: Luo G, Lin J, Fiske WD, Dai R, Yang TJ, Kim S, Sinz M, LeCluyse E, Solon E, Brennan JM, Benedek IH, Jolley S, Gilbert D, Wang L, Lee FW and Gan LS (2003) Concurrent induction and mechanism-based inactivation of CYP3A4 by an L-valinamide derivative. Drug Metabolism & Disposition 31: Masimirembwa CM, Thompson R and Andersson TB (2001) In vitro high throughput screening of compounds for favorable metabolic properties in drug discovery. [Review] [110 refs]. Combinatorial Chemistry & High Throughput Screening 4: Masui T, Kusumi I, Takahashi Y and Koyama T (2006) Effect of carbamazepine on the single oral dose pharmacokinetics of perospirone and its active metabolite. Progress in Neuro-Psychopharmacology & Biological Psychiatry 30: May TW, Rambeck B and Jurgens U (1996) Serum concentrations of lamotrigine in epileptic patients: the influence of dose and comedication. Therapeutic Drug Monitoring 18: May TW, Rambeck B and Jurgens U (2003) Serum concentrations of Levetiracetam in epileptic patients: the influence of dose and co-medication. Therapeutic Drug Monitoring 25: Mazzu AL, Lasseter KC, Shamblen EC, Agarwal V, Lettieri J and Sundaresen P (2000) Itraconazole alters the pharmacokinetics of atorvastatin to a greater extent than either cerivastatin or pravastatin. Clinical Pharmacology & Therapeutics 68:

39 McCune JS, Hawke RL, LeCluyse EL, Gillenwater HH, Hamilton G, Ritchie J and Lindley C (2000) In vivo and in vitro induction of human cytochrome P4503A4 by dexamethasone. Clinical Pharmacology & Therapeutics 68: Mills JB, Rose KA, Sadagopan N, Sahi J and de Morais SM (2004) Induction of drug metabolism enzymes and MDR1 using a novel human hepatocyte cell line. Journal of Pharmacology & Experimental Therapeutics 309: Moore JT and Kliewer SA (2000) Use of the nuclear receptor PXR to predict drug interactions. [Review] [47 refs]. Toxicology 153:1-10. Mouly S, Lown KS, Kornhauser D, Joseph JL, Fiske WD, Benedek IH and Watkins PB (2002) Hepatic but not intestinal CYP3A4 displays dose-dependent induction by efavirenz in humans. Clinical Pharmacology & Therapeutics 72:1-9. Muirhead GJ, Wulff MB, Fielding A, Kleinermans D and Buss N (2000) Pharmacokinetic interactions between sildenafil and saquinavir/ritonavir.[see comment]. British Journal of Clinical Pharmacology 50: Neuvonen PJ, Kantola T and Kivisto KT (1998) Simvastatin but not pravastatin is very susceptible to interaction with the CYP3A4 inhibitor itraconazole. Clinical Pharmacology & Therapeutics 63: Niemi M, Neuvonen PJ and Kivisto KT (2001) The cytochrome P4503A4 inhibitor clarithromycin increases the plasma concentrations and effects of repaglinide. Clinical Pharmacology & Therapeutics 70: Niemi M, Backman JT, Neuvonen M, Neuvonen PJ and Kivisto KT (2000) Rifampin decreases the plasma concentrations and effects of repaglinide. Clinical Pharmacology & Therapeutics 68: Obach RS, Walsky RL, Venkatakrishnan K, Gaman EA, Houston JB and Tremaine LM (2006) The utility of in vitro cytochrome P450 inhibition data in the prediction of drugdrug interactions. Journal of Pharmacology & Experimental Therapeutics 316: Ohnhaus EE, Brockmeyer N, Dylewicz P and Habicht H (1987) The effect of antipyrine and rifampin on the metabolism of diazepam. Clinical Pharmacology & Therapeutics 42: Olivesi A (1986) Modified elimination of prednisolone in epileptic patients on carbamazepine monotherapy, and in women using low-dose oral contraceptives. Biomedicine & Pharmacotherapy 40: Otani K, Ishida M, Kaneko S, Mihara K, Ohkubo T, Osanai T and Sugawara K (1996a) Effects of carbamazepine coadministration on plasma concentrations of trazodone and its active metabolite, m-chlorophenylpiperazine. Therapeutic Drug Monitoring 18:

40 Otani K, Yasui N, Kaneko S, Ohkubo T, Osanai T and Sugawara K (1996b) Carbamazepine augmentation therapy in three patients with trazodone-resistant unipolar depression. International Clinical Psychopharmacology 11: Phimmasone S and Kharasch ED (2001) A pilot evaluation of alfentanil-induced miosis as a noninvasive probe for hepatic cytochrome P450 3A4 (CYP3A4) activity in humans. Clinical Pharmacology & Therapeutics 70: Polk RE, Brophy DF, Israel DS, Patron R, Sadler BM, Chittick GE, Symonds WT, Lou Y, Kristoff D and Stein DS (2001) Pharmacokinetic Interaction between amprenavir and rifabutin or rifampin in healthy males. Antimicrobial Agents & Chemotherapy 45: Polk RE, Crouch MA, Israel DS, Pastor A, Sadler BM, Chittick GE, Symonds WT, Gouldin W and Lou Y (1999) Pharmacokinetic interaction between ketoconazole and amprenavir after single doses in healthy men. Pharmacotherapy 19: Powell-Jackson PR, Gray BJ, Heaton RW, Costello JF, Williams R and English J (1983) Adverse effect of rifampicin administration on steroid-dependent asthma. American Review of Respiratory Disease 128: Prueksaritanont T, Ma B, Tang C, Meng Y, Assang C, Lu P, Reider PJ, Lin JH and Baillie TA (1999) Metabolic interactions between mibefradil and HMG-CoA reductase inhibitors: an in vitro investigation with human liver preparations.[see comment]. British Journal of Clinical Pharmacology 47: Prueksaritanont T, Vega JM, Zhao J, Gagliano K, Kuznetsova O, Musser B, Amin RD, Liu L, Roadcap BA, Dilzer S, Lasseter KC and Rogers JD (2001) Interactions between simvastatin and troglitazone or pioglitazone in healthy subjects. Journal of Clinical Pharmacology 41: Raaska K, Niemi M, Neuvonen M, Neuvonen PJ and Kivisto KT (2002) Plasma concentrations of inhaled budesonide and its effects on plasma cortisol are increased by the cytochrome P4503A4 inhibitor itraconazole. Clinical Pharmacology & Therapeutics 72: Ripp SL, Mills JB, Fahmi OA, Trevena KA, Liras JL, Maurer TS and de Morais SM (2006) Use of immortalized human hepatocytes to predict the magnitude of clinical drugdrug interactions caused by CYP3A4 induction. Drug Metabolism & Disposition 34: Rutledge DR, Pieper JA and Mirvis DM (1988) Effects of chronic phenobarbital on verapamil disposition in humans. Journal of Pharmacology & Experimental Therapeutics 246:7-13. Saccar CL, Danish M, Ragni MC, Rocci ML, Jr., Greene J, Yaffe SJ and Mansmann HC, Jr. (1985) The effect of phenobarbital on theophylline disposition in children with asthma. Journal of Allergy & Clinical Immunology 75:

41 Schellens JH, van der Wart JH, Brugman M and Breimer DD (1989) Influence of enzyme induction and inhibition on the oxidation of nifedipine, sparteine, mephenytoin and antipyrine in humans as assessed by a "cocktail" study design. Journal of Pharmacology & Experimental Therapeutics 249: Schlienger R, Kurmann M, Drewe J, Muller-Spahn F and Seifritz E (2000) Inhibition of phenprocoumon anticoagulation by carbamazepine. European Neuropsychopharmacology 10: Schmider J, Brockmoller J, Arold G, Bauer S and Roots I (1999) Simultaneous assessment of CYP3A4 and CYP1A2 activity in vivo with alprazolam and caffeine. Pharmacogenetics 9: Schuetz EG, Beck WT and Schuetz JD (1996) Modulators and substrates of P- glycoprotein and cytochrome P4503A coordinately up-regulate these proteins in human colon carcinoma cells. Molecular Pharmacology 49: Sinz M, Kim S, Zhu Z, Chen T, Anthony M, Dickinson K and Rodrigues AD (2006) Evaluation of 170 xenobiotics as transactivators of human pregnane X receptor (hpxr) and correlation to known CYP3A4 drug interactions. Current Drug Metabolism 7: Sitsen J, Maris F and Timmer C (2001) Drug-drug interaction studies with mirtazapine and carbamazepine in healthy male subjects. European Journal of Drug Metabolism & Pharmacokinetics 26: Stone JA, Migoya EM, Hickey L, Winchell GA, Deutsch PJ, Ghosh K, Freeman A, Bi S, Desai R, Dilzer SC, Lasseter KC, Kraft WK, Greenberg H and Waldman SA (2004) Potential for interactions between caspofungin and nelfinavir or rifampin. Antimicrobial Agents & Chemotherapy 48: Swaisland HC, Ranson M, Smith RP, Leadbetter J, Laight A, McKillop D and Wild MJ (2005) Pharmacokinetic drug interactions of gefitinib with rifampicin, itraconazole and metoprolol. Clinical Pharmacokinetics 44: Tada Y, Tsuda Y, Otsuka T, Nagasawa K, Kimura H, Kusaba T and Sakata T (1992) Case report: nifedipine-rifampicin interaction attenuates the effect on blood pressure in a patient with essential hypertension. American Journal of the Medical Sciences 303: Thummel KE, O'Shea D, Paine MF, Shen DD, Kunze KL, Perkins JD and Wilkinson GR (1996) Oral first-pass elimination of midazolam involves both gastrointestinal and hepatic CYP3A-mediated metabolism. Clinical Pharmacology & Therapeutics 59: Tirona RG, Leake BF, Wolkoff AW and Kim RB (2003) Human organic anion transporting polypeptide-c (SLC21A6) is a major determinant of rifampin-mediated pregnane X receptor activation. Journal of Pharmacology & Experimental Therapeutics 304:

42 Tran-Johnson T, Herrera JM, Sramek JJ, Costa JF, Heh C and Pi E (1987) Timedependent autoinduction of carbamazepine in schizophrenia. Drug Intelligence & Clinical Pharmacy 21:835. Treiber A, Schneiter R, Hausler S and Stieger B (2007) Bosentan is a substrate of human OATP1B1 and OATP1B3: inhibition of hepatic uptake as the common mechanism of its interactions with cyclosporin A, rifampicin, and sildenafil. Drug Metabolism & Disposition 35: Tsuchihashi K, Fukami K, Kishimoto H, Sumiyoshi T, Haze K, Saito M and Hiramori K (1987) A case of variant angina exacerbated by administration of rifampicin. Heart & Vessels 3: Tuteja S, Alloway RR, Johnson JA and Gaber AO (2001) The effect of gut metabolism on tacrolimus bioavailability in renal transplant recipients. Transplantation 71: Twum-Barima Y and Carruthers SG (1981) Quinidine-rifampin interaction. New England Journal of Medicine 304: Ucar M, Neuvonen M, Luurila H, Dahlqvist R, Neuvonen PJ and Mjorndal T (2004) Carbamazepine markedly reduces serum concentrations of simvastatin and simvastatin acid. European Journal of Clinical Pharmacology 59: van Giersbergen PL, Halabi A and Dingemanse J (2002) Single- and multiple-dose pharmacokinetics of bosentan and its interaction with ketoconazole. British Journal of Clinical Pharmacology 53: Varhe A, Olkkola KT and Neuvonen PJ (1994) Oral triazolam is potentially hazardous to patients receiving systemic antimycotics ketoconazole or itraconazole. Clinical Pharmacology & Therapeutics 56:t-7. Veldkamp AI, Harris M, Montaner JS, Moyle G, Gazzard B, Youle M, Johnson M, Kwakkelstein MO, Carlier H, van LR, Beijnen JH, Lange JM, Reiss P and Hoetelmans RM (2001) The steady-state pharmacokinetics of efavirenz and nevirapine when used in combination in human immunodeficiency virus type 1-infected persons.[see comment]. Journal of Infectious Diseases 184: Venkatakrishnan K and Obach RS (2005) In vitro-in vivo extrapolation of CYP2D6 inactivation by paroxetine: prediction of nonstationary pharmacokinetics and drug interaction magnitude. Drug Metabolism & Disposition 33: Vignati LA, Bogni A, Grossi P and Monshouwer M (2004) A human and mouse pregnane X receptor reporter gene assay in combination with cytotoxicity measurements as a tool to evaluate species-specific CYP3A induction. Toxicology 199: Villikka K, Kivisto KT, Backman JT, Olkkola KT and Neuvonen PJ (1997a) Triazolam is ineffective in patients taking rifampin. Clinical Pharmacology & Therapeutics 61:

43 Villikka K, Kivisto KT, Luurila H and Neuvonen PJ (1997b) Rifampin reduces plasma concentrations and effects of zolpidem. Clinical Pharmacology & Therapeutics 62: Wacher VJ, Wu CY and Benet LZ (1995) Overlapping substrate specificities and tissue distribution of cytochrome P450 3A and P-glycoprotein: implications for drug delivery and activity in cancer chemotherapy. [Review] [104 refs]. Molecular Carcinogenesis 13: Wang RW, Newton DJ, Scheri TD and Lu AY (1997) Human cytochrome P450 3A4- catalyzed testosterone 6 beta-hydroxylation and erythromycin N-demethylation. Competition during catalysis. Drug Metabolism & Disposition 25: Wanwimolruk S, Kang W, Coville PF, Viriyayudhakorn S and Thitiarchakul S (1995) Marked enhancement by rifampicin and lack of effect of isoniazid on the elimination of quinine in man. British Journal of Clinical Pharmacology 40: Wanwimolruk S, Paine MF, Pusek SN and Watkins PB (2002) Is quinine a suitable probe to assess the hepatic drug-metabolizing enzyme CYP3A4? British Journal of Clinical Pharmacology 54: Williamson KM, Patterson JH, McQueen RH, Adams KF, Jr. and Pieper JA (1998) Effects of erythromycin or rifampin on losartan pharmacokinetics in healthy volunteers. Clinical Pharmacology & Therapeutics 63: Wire MB, Ballow C, Preston SL, Hendrix CW, Piliero PJ, Lou Y and Stein DS (2004) Pharmacokinetics and safety of GW and ritonavir, with and without efavirenz, in healthy volunteers. AIDS 18: Weiner M, Benator D, Peloquin CA, Burman W, Vernon A, Engle M, Khan A, Zhao Z and Tuberculosis Trials Consortium (2005) Evaluation of the drug interaction between rifabutin and efavirenz in patients with HIV infection and tuberculosis. Clinical Infectious Diseases 41: Williams K, Begg E, Wade D and O'Shea K (1983) Effects of phenytoin, phenobarbital, and ascorbic acid on misonidazole elimination. Clinical Pharmacology & Therapeutics 33: Yasui-Furukori N, Kondo T, Mihara K, Suzuki A, Inoue Y and Kaneko S (2003) Significant dose effect of carbamazepine on reduction of steady-state plasma concentration of haloperidol in schizophrenic patients. Journal of Clinical Psychopharmacology 23: Yonemori K, Takeda Y, Toyota E, Kobayashi N and Kudo K (2004) Potential interactions between irinotecan and rifampin in a patient with small-cell lung cancer. International Journal of Clinical Oncology 9:

44 Yule SM, Walker D, Cole M, McSorley L, Cholerton S, Daly AK, Pearson AD and Boddy AV (1999) The effect of fluconazole on cyclophosphamide metabolism in children. Drug Metabolism & Disposition 27: Zhang Y, Hsieh Y, Izumi T, Lin ET and Benet LZ (1998) Effects of ketoconazole on the intestinal metabolism, transport and oral bioavailability of K02, a novel vinylsulfone peptidomimetic cysteine protease inhibitor and a P450 3A, P-glycoprotein dual substrate, in male Sprague-Dawley rats. Journal of Pharmacology & Experimental Therapeutics 287: Zurcher RM, Frey BM and Frey FJ (1989) Impact of ketoconazole on the metabolism of prednisolone.[see comment]. Clinical Pharmacology & Therapeutics 45:

45 Footnotes. Send the reprint requests to: Magang Shou, Ph.D. Department of Pharmacokinetics and Drug Metabolism 30E-2-B, Amgen, Inc. One Amgen Center Drive Thousand Oaks, CA Tel: (805) Fax: (805)

46 Legends for figures Figure 1. Curves of CYP3A4 response (fold-increase) to inducer concentration (µm). IC 50 and E max values (Table 1) were generated by fitting of the observed data in hepatocytes to the Hill equation (Eqn. 1). Donor 1: (A) mrna; (B) enzyme activity; and Donor 2: (C) mrna and (D) enzyme activity. Figure 2. Correlation analyses of predicted DDIs (Donor 1) with observed in vivo DDIs. (A) with f u,p and f u,hept ; (B) without f u,p and f u,hept ; (C) with f u,p and without f u,hept and (D) without f u,p and with f u,hept. LR = linear regression; R 2 = correlation coefficient; 95% CI = 95% confidence interval; 95% PI = 95% prediction interval; PC = perfect correlation and RSS = residual sum of square. Figure 3. Correlation analyses of predicted DDIs (Donor 2) with observed in vivo DDIs. (A) with f u,p and f u,hept ; (B) without f u,p and f u,hept ; (C) with f u,p and without f u,hept and (D) without f u,p and with f u,hept. LR = linear regression; R 2 = correlation coefficient; 95% CI = 95% confidence interval; 95% PI = 95% prediction interval; PC = perfect correlation and RSS = residual sum of square. Figure 4. Weighted residual distributions (difference of the weighted residual from zero, the perfect linear regression) of the correlations (corrected by f u,p and f u,hept ) between predicted and observed DDIs. Figure 5. Correlations of the DDIs predicted from the in vitro mrna induction data (including f u,p and f u,hept ) in Donors 1 (A) and 2 (B) with the clinically observed DDIs. LR = linear regression; R 2 = correlation coefficient; 95% CI = 95% confidence interval; 95% PI = 95% prediction interval; PC = perfect correlation and RSS = residual sum of square. Figure 6. Simulated relationship of predicted DDIs as a function of in vivo free concentration of inducer ([Ind] max, ss ) and fraction of drug cleared by CYP3A4 (f m,cyp3a4 ) using Eqn. 17. RIF (A and B): EC 50 = 0.25 µm, E max = 10.6 (fold-increase), f u,p = and f u,hept = and PB (C and D): EC 50 = 250 µm, E max = 22.6 (fold-increase), f u,p = 0.7 and f u,hept =

47 Table 1. EC 50 and E max values of the inducers for CYP3A4 mrna and activity in hepatocytes generated from the concentration vs. response curves in Fig. 1. Donor 1 Donor 2 a. Standard deviation in parenthesis; b. sigmoidity of the curves in Fig. 1; c. CYP3A4 activity (testosterone 6β-hydroxylation) measured in hepatocytes. Inducer BST CMZ DEX EFA PB RIF BST CMZ DEX EFA PB RIF Conc. (µm) mrna Activity c EC 50 (µm) E max n b R 2 EC 50 (µm) E max n R (0.48) a (3.47) (0.5) (0.27) (0.57) (0.8) (3.03) (0.89) (0.1) (0.79) (0.57) (0.3) (11.60) (1.70) (0.2) (5.3) (3.43) (0.3) (1.01) (0.34) (2.6) (3.5) (0.77) (0.5) (20.90) (1.07) (0.2) (92.1) (5.57) (0.2) (0.12) (5.47) (0.4) (0.04) (0.60) (0.2) (3.88) (3.59) (0.2) (0.20) (0.24) (0.2) (4.43) (0.79) (0.40) (2.72) (0.28) (0.7) (14.81) (1.48) (0.1) (2.40) (0.78) (0.7) (5.89) (3.08) (0.2) (1.40) (0.78) (0.7) (7.41) (0.25) (0.3) (7.9) (0.18) (0.1) (0.05) (3.54) (0.2) (0.08) (0.69) (0.3) Downloaded from dmd.aspetjournals.org at ASPET Journals on April 28, 2017

48 Table 2. Unbound fractions of the inducers in human plasma and hepatocytes. Plasma Hepatocytes Inducer Conc. (µm) f u,p Conc. (µm) f u,hept BST 1, (0.003) a (0.010) CMZ 1, (0.002) (0.021) DEX 1, (0.011) (0.043) EFA 1, (0.004) (0.006) PB 1, (0.092) (0.074) RIF 1, (0.011) (0.052) a. Mean values in triplicate with standard deviation. 48

49 Table 3. Fractions of the drugs cleared by CYP3A4 (f m,cyp3a4 ) estimated by f i, CYP3A4 from clinical DDIs associated with CYP3A4 inhibition (Eqn. 18) (Kremens, et al., Substrate Reference Substrate f m Reference Alfentanil a 2005) Midazolam 89 (Chen, et al., 2006) (Kharasch, et al., (Greenblatt, et Alprazolam 69 al., 1998) Nifedipine ) (Polk, et al., Amprenavir ) Omeprazole 37 (He, et al., 2003) (D'Mello, et al., Antipyrine ) Pravastatin a 52 6 (Jacobson, 2004) Atorvastatin ) Prednisolone a ) (Mazzu, et al., (Zurcher, et al., f m b Budesonide 76 5 (Raaska, et al., 2002) Quetiapine 84 (Grimm, et al., 2006) Buspirone ) Quinidine ) (Kivisto, et al., (Damkier, et al., Cyclophosphamide a ) Quinine 46 1 al., 2002) (Yule, et al., (Wanwimolruk, et Cyclosporine 79 (Gomez, et al., 1995) Repaglinide a 30 6 (Niemi, et al., 2001) Diazepam 24 5 (Ahonen, et al., 1996) Ritonavir 23 (Khaliq, et al., 2000) Etizolam 35 Ethinyl estradiol ) Ropivacaine a) (Hilbert, et al., (Jokinen, et al., 2004) Saquinavir 66 (Grub, et al., 2001) 5 (Araki, et al., Everolimus ) Sildenafil a ) (Kovarik, et al., (Muirhead, et al., Gefitinib ) Simvastatin ) (Swaisland, et al., (Neuvonen, et al., Glyburide ) Tacrolimus 58 (Tuteja, et al., 2001) (Lilja, et al., (Fleishaker, et al., Imatinib 29 (Dutreix, et al., 2004) Tirilazad a 41 Indinavir (Hsu, et al., 1998) Triazolam 91 losartan 21 Methadone 25 4 (Cobb, et al., 1996) (Greenblatt, et al., 1998) (Kaukonen, et al., 1998) verapamil 30 9 (Fuhr, et al., 2002) 1998) Zolpidem ) (Greenblatt, et al., a. drugs were dosed intravenously; b. f m,cyp3a4 values were calculated (Eqn.18) by the decreased oral clearance (or systemic clearance) or increased AUC of the drugs in the presence of ketoconazole or other individual CYP3A4 inhibitors (f i,cyp3a4 ) as denoted by 1. troleandomycin; 2. ritonavir; 3. mibefradil; 4. fluconazole; 5. itraconazole; 6. clarithromycin; 7. saquinavir 8. erythromycin and 9. grapefruit juice. 49

50 Table 4. In vivo concentrations of the inducers at different dose regimens. Inducer Dose Regimen C max, ss (µm) Reference BST 125 mg BID 5d 2.0 (Dingemanse, et al., 2003) BST 500 mg BID 7d 14.4 (Binet, et al., 2000) DEX 8 mg, BID, 5d 0.1 (McCune, et al., 2000) CMZ 50 mg BID 2w 8.0 (Yasui-Furukori, et al., 2003) CMZ 100 mg TID 10d 17.8 (Furukori, et al., 1998) CMZ 150 mg BID 2w 17.7 (Yasui-Furukori, et al., 2003) CMZ 200 mg BID 4D or QD 5d (Otani, et al., 1996a) (Masui, et al., 2006) CMZ 300 mg BID 2W 25.0 (Yasui-Furukori, et al., 2003) CMZ (Schlienger, et al., 2000) (Otani, et al., 400 mg QD 4W b) CMZ 600 mg QD 9W (de and Bork, 1997) (Licht, et al., 2000) CMZ 800 mg QD 7D 31.3 (Hillebrand, et al., 1987) CMZ 942 mg 6w 39.8 (Ketter, et al., 1995) CMZ mg (chronic) (May, et al., 2003) (May, et al., 1996) 200 or 400 mg QD EFA 10D 6.9 (Mouly, et al., 2002) EFA 600 mg QD 10D EFA 800 mg QD 7D (Lopez-Cortes, et al., 2002) 30 mg BID PB (chronic) (Back, et al., 1980) (la Porte, et al., 2004b) (Veldkamp, et al., 2001) (Weiner, et al., 2005) PB 90 mg QD 29D (Ferron, et al., 2003) PB 100 mg QD 21D (Rutledge, et al., 1988;Kapil, et al., 1987) PB 140 mg QD 19D (Saccar, et al., 1985) PB 200 mg QD 7D (Williams, et al., 1983) RIF 300 mg BID 13.5D (Drusano, et al., 1986;Chandler, et al., 1990) RIF 600 mg QD 10D RIF 900 mg QD 10D 12.1 (Humbert, et al., 1991) (Droste, et al., 2005;Boyd, et al., 2003;la Porte, et al., 2004a;Stone, et al., 2004) 50

51 Table 5. Comparisons of the predicted DDIs from the in vitro CYP3A4 induction in hepatocytes of the two liver donors (PRED1 and PRED2) with the clinical observed DDIs reported in cited references. 2D Dose Regimen a Substrate Dose Regimen b AUC Ratio c References Inducer PRED1 PRED2 OBS d 125 mg BID 7D ethinyl estradiol 35 µg SD (van Giersbergen, et al., 2006) 125 mg BID 4.5D glyburide 2.5 mg BID 9.5D (van Giersbergen, et al., 2002) 62.5 mg BID 4W sildenafil 100 mg SD (Paul, et al., 2005) 62.5 mg BID 8W sildenafil 100 mg SD (Paul, et al., 2005) BST 125 mg BID 5.5D simvastatin 40 mg QD 5.5D (Dingemanse, et al., 2003) cyclophosphamid mg Infusion (Moore, et al., 1988) 10 mg QD 2D e c 0.57 DEX 1.5 mg QD 4D trazolam 0.5 mg SD (Villikka, et al., 1998) EFA 600 mg, QD 7D amprenavir 1200 mg BID (Falloon, et al., 2000) 600 mg, QD 2W amprenavir 600 mg BID 5.8M (Goujard, et al., 2003) 600 mg QD 20D amprenavir 600 mg BID 10D (Morse, et al., 2005) 600 mg QD 15D atorvastatin 10 mg SD (Gerber, et al., 2005) 600 mg QD 14D indinavir 800 mg BID 30D (Aarnoutse, et al., 2002) 600 mg QD 4W indinavir 800 mg BID 4W (Boyd, et al., 2003) mg daily (Clarke, et al., 2001) 600 mg QD 14-21D methadone (maintenance therapy) mg QD 15D pravastatin 40 mg SD (Gerber, et al., 2005) 600 mg QD 10D ritonavir 200 mg QD 20D (la Porte, et al., 2004b) 600 mg QD 14D ritonavir 200 mg QD 28D (Wire, et al., 2004) 600 mg QD 14D ritonavir 100 mg BID 30D (Aarnoutse, et al., 2002) 600 mg QD 2W ritonavir 100 mg BID 5.8M (Goujard, et al., 2003) 600 mg QD 14D amprenavir 1395 mg QD 28D (Wire, et al., 2004) 51 Downloaded from dmd.aspetjournals.org at ASPET Journals on April 28, 2017

52 PB RIF 600 mg QD 14D amprenavir 700 mg BID 28D (Wire, et al., 2004) 600 mg QD 14D amprenavir 700 mg BID 28D (Wire, et al., 2004) 600 mg QD 4W ritonavir 100 mg BID 4W (Boyd, et al., 2003) 600 mg QD 14D ritonavir 100 mg BID 28D (Wire, et al., 2004) 600 mg QD 35D ritonavir 100 mg BID 35D (Hsu, et al., 2003) 100 mg QD 14D antipyrine 1 g SD (Leclercq, et al., 1989) 100 mg QD 8D antipyrine 250 mg SD (Schellens, et al., 1989) 100 mg QD 8D nifedipine 20 mg SD (Schellens, et al., 1989) mm (SS) quinidine 300 mg SD (Data, et al., 1976) 100 mg QD 8D tirilazad c 105 mg IV QID 7D (Fleishaker, et al., 1996) 100 mg QD 8D tirilazad c 105 mg IV QID 7D (Fleishaker, et al., 1996) 100 mg QD 21D verapamil 1.05 mg SD (Rutledge, et al., 1988) 100 mg QD 21D verapamil 80 mg SD (Rutledge, et al., 1988) 100 mg QD 21D verapamil 80 mg QID 5D (Rutledge, et al., 1988) 100 mg QD 14D antipyrine 1 g SD (Leclercq, et al., 1989) 100 mg QD 16D losartan 50 mg QD 7D (Goldberg, et al., 1996) 600 mg QD 12D (R)-verapamil 10 mg SD (Fromm, et al., 1996) 600 mg QD 12D (R)-verapamil 120 mg BID 24D (Fromm, et al., 1996) 600 mg QD 12D (R)-verapamil 120 mg BID 16D (Fromm, et al., 1998) 600 mg QD 12D (S)-verapamil 120 mg BID 16D (Fromm, et al., 1998) 600 mg QD 12D (S)-verapamil 120 mg BID 24D (Fromm, et al., 1996) (Phimmasone and Kharasch, 600 mg QD 5D alfentanil mg SD ) 600 mg QD 5D alfentanil mg SD (Kharasch, et al., 2004b) 600 mg QD 5D alfentanil 4.5 mg SD (Kharasch, et al., 2004b) 450 mg QD 4D alprazolam 1 mg SD (Schmider, et al., 1999) 600 mg QD 18D amprenavir 1200 mg BID 8D (Polk, et al., 2001) 52 Downloaded from dmd.aspetjournals.org at ASPET Journals on April 28, 2017

53 300 mg BID 8D antipyrine 1000 mg SD (Leclercq, et al., 1989) 300 mg BID 8D antipyrine 1000 mg SD (Leclercq, et al., 1989) 600 mg QD 15D antipyrine 1200 mg SD (Bennett, et al., 1982) 600 mg QD 5D atorvastatin 40 mg SD (Backman, et al., 2005) 600 mg QD 7D budesonide 3 mg SD (Dilger, et al., 2005) 600 mg QD 7D budesonide 3 mg SD (Dilger, et al., 2005) 600 mg QD 5D buspirone 30 mg SD (Kivisto, et al., 1999) 600 mg QD 5D buspirone 30 mg SD (Lamberg, et al., 1998) 600 mg QD 11D cyclosporine 750 mg SD (Hebert, et al., 1992) 600 mg BID 7D diazepam 10 mg SD (Ohnhaus, et al., 1987) 300 mg BID 7D diazepam 10 mg SD (Ohnhaus, et al., 1987) 300 mg BID 10D ethinyl estradiol 35 µg QD (2 cycles) (LeBel, et al., 1998) 35 µg (chronic treatment) (Barditch-Crovo, et al., 1999) 600 mg QD 14D ethinyl estradiol QD mg QD 13D everolimus 4 mg SD (Kovarik, et al., 2002) 600 mg QD 16D gefitinib 500 mg SD (Swaisland, et al., 2005) 600 mg QD 5D glyburide 1.75 mg SD (Niemi, et al., 2001) 600 mg QD 11D imatinib 400 mg SD (Bolton, et al., 2004) 450 mg QD 6W irinotecan 75 mg/m 2 QD (4 cycles) (Yonemori, et al., 2004) 300 mg BID 7D losartan 50 mg QD 7D (Williamson, et al., 1998) 600 mg QD 10D methadone 5.5 mg SD (Kharasch, et al., 2004a) 600 mg QD 10D methadone 11.2 mg SD (Kharasch, et al., 2004a) 600 mg QD 14D midazolam 2 mg SD (Adams, et al., 2005) 600 mg QD 9D midazolam 5.5 mg SD (Chung, et al., 2006) 600 mg QD 7D midazolam 6 mg SD (Gorski, et al., 2003) 300 mg BID 7D midazolam 8 mg SD (Gurley, et al., 2006) 53 Downloaded from dmd.aspetjournals.org at ASPET Journals on April 28, 2017

54 600 mg QD 5D midazolam 3 mg SD (Kharasch, et al., 2004b) 450 mg QD 5D midazolam 7.5 mg SD (Eap, et al., 2004) 600 mg QD 5D midazolam 15 mg SD (Backman, et al., 1996) 600 mg QD 5D midazolam 15 mg SD (Backman, et al., 1998) 20 mg QID (stable (Tsuchihashi, et al., 1987) 450 mg QD 4D nifedipine therapy) mg BID (stable (Tada, et al., 1992) 450 mg, QD >30D nifedipine therapy) mg QD 7D nifedipine 20 mg SD (Holtbecker, et al., 1996) 600 mg QD 6W prednisolone 10 mg IV (Powell-Jackson, et al., 1983) mg QD 3W prednisolone 20 mg Infusion (Bergrem and Refvem, 1983) 600 mg QD 7D quinidine 450mg SD (Twum-Barima and Carruthers, 1981) 600 mg QD 7D quinidine 450 SD (Twum-Barima and Carruthers, 1981) 600 mg QD 2W quinine 600 mg SD (Wanwimolruk, et al., 1995) 600 mg QD 7D repaglinide 4 mg SD (Bidstrup, et al., 2004) 600 mg QD 5D repaglinide 37.5 mg SD (Niemi, et al., 2000) 600 mg QD 7D repaglinide 4 mg SD (Bidstrup, et al., 2004) 600 mg QD 5D ropivacaine c 45 mg SD (Jokinen, et al., 2001b) 600 mg QD 5D ropivacaine c 45 mg SD (Jokinen, et al., 2001b) 600 mg QD 14D saquinavir 1200 mg TID 14D (Grub, et al., 2001) 600 mg QD 5D simvastatin 40 mg SD (Kyrklund, et al., 2000) 600 mg QD 9D simvastatin 40 mg SD (Chung, et al., 2006) 600 mg, QD >31D tacrolimus 15 mg SD (Kuypers, et al., 2005) 600 mg QD 18D tacrolimus 75 mg SD (Hebert, et al., 1999) 600 mg QD 5D triazolam 0.5 mg SD (Villikka, et al., 1997a) 600 mg QD 15D verapamil 120 mg SD (Barbarash, et al., 1988) 54 Downloaded from dmd.aspetjournals.org at ASPET Journals on April 28, 2017

55 CMZ 600 mg QD 5D zolpidem 20 mg SD (Villikka, et al., 1997b) 600 mg QD 21D ethinyl estradiol 35 µg QD 21D (Doose, et al., 2003) mg QD (Crawford, et al., 1990) 12W ethinyl estradiol 50 µg SD mg QD 6D etizolam 1 mg SD (Kondo, et al., 2005) 400 mg QD 28D mirtazapine 30 mg SD 7D (Sitsen, et al., 2001) 400 mg QD 21D mirtazapine 30 mg SD 28D (Sitsen, et al., 2001) mg BID 3W omeprazole 20 mg SD (Laroudie, et al., 2000) mg 2-12M prednisolone 35 mg IV SD (Olivesi, 1986) 300 mg BID 14D simvastatin 80 mg SD (Ucar, et al., 2004) a. Dose regimens of the inducers, for example, 125 mg BID 7D represents that dose (125 mg) was given orally twice a day for 7 days; b. Dose regimens of the CYP3A4 substrate drugs; c. AUC ratios of the drugs in the presence and absence of the inducers predicted from the in vitro induction data in hepatocytes of Donor 1 (PRED 1) and Donor 2 (PRED 2), respectively; and d. OBS = observed in vivo DDIs from the corresponding references. 55 Downloaded from dmd.aspetjournals.org at ASPET Journals on April 28, 2017

56

57

58

59

60

61