QUANTITATIVE PREDICTION OF THE IN VIVO INHIBITION OF DIAZEPAM METABOLISM BY OMEPRAZOLE USING RAT LIVER MICROSOMES AND HEPATOCYTES Hannah M. Jones, David Hallifax, and J. Brian Houston Centre for Applied Pharmacokinetic Research, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, United Kingdom Received December 9, 2003; accepted February 11, 2004 This article is available online at http://dmd.aspetjournals.org ABSTRACT: The diazepam (DZ)-omeprazole (OMP) interaction has been se- lected as a prototype for an important drug-drug interaction in- volving cytochrome P450 inhibition. The availability of an in vivo K i value (unbound K i , 21 M) obtained from a series of steady-state inhibitor infusion studies allowed assessment of several in vitro- derived predictions of this inhibition interaction. Studies monitor- ing substrate depletion with time were used to obtain in vitro K i values that were evaluated against the more traditional metabolite formation approach using microsomes and hepatocytes. OMP in- hibited the metabolism of DZ to its primary metabolites 4-hy- droxydiazepam, 3-hydroxydiazepam, and nordiazepam to different extents over a range of concentrations (0.3–150 M), and a com- petitive inhibition model best fitted the data. The K i values ob- served using the substrate depletion approach (16  3 M and 7  2 M in microsomes and hepatocytes, respectively) were in good agreement with the overall weighted K i values obtained using the standard metabolite formation approach (12  2 M and 16  5 M in microsomes and hepatocytes, respectively). In vitro binding and cell uptake studies as well as human serum albumin studies in hepatocytes confirmed the importance of both intracellular and extracellular unbound concentrations of inhibitor when consider- ing inhibition predictions. Both kinetic approaches and both in vitro systems predicted the in vivo interaction well and provide a good example of the ability of in vitro inhibition studies to quantitatively predict an in vivo drug-drug interaction successfully. The inhibition of diazepam (DZ 1 ) metabolism by omeprazole (OMP) has been well documented in vivo; therapeutic concentrations of OMP have been reported to decrease the plasma clearance of DZ both in humans (Gugler and Jensen, 1985; Andersson et al., 1990a,b) and in rats (Zomorodi and Houston, 1995). Zomorodi and Houston (1995) measured the in vivo clearance of DZ (administered by the hepatic portal vein) in rats receiving intravenous infusions together with matching bolus doses of OMP to achieve a wide range of steady-state plasma concentrations (10 –50 g/ml). The inhibition was modeled using a simple inhibition model (eq. 1) and the in vivo K i was calculated to be 57 M. CL = CL 0 /1 + I ss /K i  (1) where CL and CL 0 represent DZ clearance in the presence and absence of OMP, respectively, I ss represents the steady-state total plasma concentration of OMP, and K i represents the inhibition con- stant. Figure 1 shows the relationship between DZ clearance and steady-state unbound concentration of OMP achieved in this study. Such a detailed in vivo inhibition study is only possible in animals. In humans, inhibition studies are normally performed using one inhibitor oral dosing regimen, and the ratio of the AUC in the presence and absence of the inhibitor is used to assess the degree of interaction (Gugler and Jensen, 1985; Andersson et al., 1990a,b). In vitro, the inhibition potential of a drug is traditionally assessed under initial rate conditions and the inhibition constant (K i ) for a particular metabolic pathway is obtained (Zomorodi and Houston, 1995; Komatsu et al., 2000; Ishigam et al., 2001). Methods involving substrate depletion with time are rarely used; however, it may be argued that this approach is analogous to the in vivo situation and may serve to predict the impact of drug interactions in cases where details of the metabolic pathways are not known or when several metabolic pathways are involved. The predicted degree of interaction caused by a reversibly acting inhibitor can then be determined based on the ratio of the inhibitor concentration at the active site in vivo to the K i determined in vitro (Ito et al., 1998a,b). The majority of inhibition predictions in the literature have been performed using microsomal data (Yamano et al., 1999a,b, 2000, 2001; Komatsu et al., 2000; Ishigam et al., 2001; Tran et al., 2002; Yao et al., 2003). Although hepatocytes have been shown to give more realistic predictions of in This work was funded by the Centre for Applied Pharmacokinetic Research (CAPKR), consisting of a consortium of pharmaceutical companies (AstraZeneca, Bristol Myers Squibb, GlaxoSmithKline, F. Hoffmann-La Roche, Novartis, Pfizer, and Servier). 1 Abbreviations used are: DZ, diazepam; OMP, omeprazole; AUC, area under the plasma concentration-time curve; 3-HDZ, 3-hydroxydiazepam; 4'-HDZ, 4'- hydroxydiazepam; NDZ, nordiazepam; DMF, dimethylformamide; HSA, human serum albumin; HPLC, high-performance liquid chromatography; LC-MS/MS, liquid chromatography/mass spectrometry; f m , fraction of parent compound me- tabolized to each metabolite; f u , fraction unbound in plasma; K p , partition coef- ficient. Address correspondence to: Prof. J. Brian Houston, Centre for Applied Pharmacokinetic Research, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester, M13 9PL, United Kingdom. E-mail: brian.houston@man.ac.uk 0090-9556/04/3205-572–580$20.00 DRUG METABOLISM AND DISPOSITION Vol. 32, No. 5 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 1351/1149051 DMD 32:572–580, 2004 Printed in U.S.A. 572 at ASPET Journals on October 13, 2017 dmd.aspetjournals.org Downloaded from