Quantitative Prediction of the in Vivo Inhibition of Diazepam Metabolism by Omeprazole Using Rat Liver Microsomes and Hepatocytes

Abstract

ABSTRACT:The diazepam (DZ)-omeprazole (OMP) interaction has been selected as a prototype for an important drug-drug interaction involving 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 vitroderived predictions of this inhibition interaction. Studies monitoring 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- 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 . 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.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 constant. 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;

Use of Isolated Hepatocyte Preparations for Cytochrome P450 Inhibition Studies: Comparison with Microsomes forKiDetermination

“…The fraction unbound of fluconazole, ketoconazole, miconazole, quinine, fluoxetine, and fluvoxamine in rat liver microsomes was determined by the ratio of the concentration in the filtrate to the initial total concentration as described previously (Jones et al, 2004). The fraction unbound of each inhibitor was determined at different microsomal protein concentrations as described previously (Brown et al, 2006).…”

“…Alternatively, similar values would indicate that hepatic accumulation results from intracellular binding or lysosomal trapping. Although hepatocytes are used for the prediction of metabolic clearance (Hallifax et al, 2005;Brown et al, 2007), published literature comparing the use of hepatocytes as an alternative system to microsomes for K i determination remains somewhat limited (Di Marco et al, 2003;Jones et al, 2004;Engtrakul et al, 2005;Mano et al, 2007). In addition, these previous investigations are limited since they are restricted to one substrate-inhibitor pair (Di Marco et al, 2003;Jones et al, 2004;Engtrakul et al, 2005), or inhibitors, which are not expected to undergo hepatic accumulation (Mano et al, 2007).…”

Uptake and Intracellular Binding of Lipophilic Amine Drugs by Isolated Rat Hepatocytes and Implications for Prediction of in Vivo Metabolic Clearance

“…Equation 5 was derived from mass balance considerations, again assuming the unbound concentration of drug in the cell is equal to the unbound concentration in the medium and no active transport occurs (Jones et al, 2004). The radiochromatographic components extracted from the cells were integrated as proportions of total radioactivity detected for each sample.…”

Functional Consequences of Active Hepatic Uptake on Cytochrome P450 Inhibition in Rat and Human Hepatocytes

“…The same exercise performed for the other three compounds revealed a similar extent of binding inside the hepatocytes. Appreciation that binding may remove a large proportion of the free drug concentrated inside the hepatocyte is not novel (Di Marco et al, 2003;Jones et al, 2004), but it is a concept that is occasionally overlooked. For drugs not actively transported into hepatocytes, extensive intracellular binding still occurs, but the impact is relatively minor because the hepatocytes are a part of the whole incubation system (typically 1 ml, because there is no compartmentalization) and the free drug concentration therefore refers to the whole incubation.…”

“…Despite an awareness that active uptake into hepatocytes should be taken into consideration in the prediction of DDIs (Ito et al, 1998), comparatively few in vitro studies using isolated hepatocytes have been published (Cohen et al, 2000;Di Marco et al, 2003;Jones et al, 2004;McGinnity et al, 2005McGinnity et al, , 2006Oleson et al, 2005;Zhao et al, 2005;Brown et al, 2007;Mano et al, 2007). Of these studies, only a small subset have compared inhibition constants generated using recombinant P450s or hepatic microsomes with those generated using isolated hepatocytes for inhibitors that are actively concentrated into hepatocytes.…”