Substrate Depletion Approach for Determining in Vitro Metabolic Clearance: Time Dependencies in Hepatocyte and Microsomal Incubations

Abstract

ABSTRACT:The substrate depletion method is a popular approach used for the measurement of in vitro intrinsic clearance (CL int ). However, the incubation conditions used in these studies can vary, the consequences of which have not been systematically explored. Initial substrate depletion incubations using rat microsomes and hepatocytes were performed for eight benzodiazepines: alprazolam, clobazam, clonazepam, chlordiazepoxide, diazepam, flunitrazepam, midazolam, and triazolam. Subsequent predictions of in vivo CL int (ranging from 3 to 200 ml/min) and hepatic clearance (CL H ) (ranging from 0.3 to 15 ml/min) demonstrated that the general predictive ability of this approach was similar to that of the traditional metabolite formation method. A more detailed study of the substrate depletion profiles and CL int estimates indicated that the concentration of enzyme used is of particular importance.The metabolism of triazolam, clonazepam, and diazepam was monoexponential at all cell densities using hepatocytes; however, with microsomes, biphasic depletion was apparent, particularly at higher microsomal protein concentrations (2-5 mg/ml). Enzyme activity studies indicated that enzyme loss was more pronounced in the microsomal system (ranged from 8 to 65% activity after a 1-h incubation) compared with the hepatocyte system (approximately 100% activity after a 1-h incubation). For clonazepam (a low clearance substrate), these biphasic profiles could be explained by loss of enzyme activity. To ensure accurate predictions of in vivo CL int and CL H when using the substrate depletion approach, based on the results obtained for this class of drugs, it is recommended that low enzyme concentrations and short incubation times are used whenever possible.The in vitro measurement of intrinsic clearance (CL int ) using hepatic microsomes and/or hepatocytes is frequently used in both academia and the pharmaceutical industry to estimate the in vivo metabolic stability of new drug entities in both rat and human (Houston, 1994;Iwatsubo et al., 1997;Obach, 1999;McGinnity and Riley, 2001). The results of these assays are scaled and modeled, as described in eq. 1 for the well stirred liver model, to estimate in vivo hepatic clearance (CL H ).where Q H is the hepatic blood flow, SF represents the milligrams of microsomal protein or million cells per gram of liver multiplied by the grams of liver weight; f u b is the unbound fraction of drug in the blood, and f u inc is the unbound fraction in the incubation matrix.Traditionally, the metabolite formation method has been used for measurement of in vitro CL int , where the initial rate of metabolite production using hepatic microsomes or hepatocytes is measured over a range of substrate concentrations under linear conditions with respect to protein concentration/cell density and time (Madan et al., 2002;Houston et al., 2003). In general, short incubation times and low enzyme (protein) concentrations are used in these studies, since compliance with the Michaelis-Menten equation assumes less t...

Prediction of Drug Clearance by Glucuronidation from in Vitro Data: Use of Combined Cytochrome P450 and UDP-Glucuronosyltransferase Cofactors in Alamethicin-Activated Human Liver Microsomes

“…Due to a lack of glucuronide standards available, a depletion approach was used to obtain the CL int for this data set. For most of the compounds, the protein concentration and time course used were greater than the proposed optimal values of 0.5 mg/ml and an incubation time of 30 min (Jones and Houston, 2004). These conditions ensured that greater than 20% metabolism was obtained during the incubations to distinguish from any baseline variability in the analytical methodology.…”

“…The impact of the addition of 2% BSA to microsomal incubations on the clearance prediction was also investigated. Depletion of parent compound is a common method to determine CL int , and it has been shown to be comparable with a metabolite formation approach (Obach, 2001;Jones and Houston, 2004). Due to a lack of glucuronide standards available, a depletion approach was used to obtain the CL int for this data set.…”

Addressing the Challenges of Low Clearance in Drug Research

“…However, nonlinear biphasic kinetics might occur due to loss of enzyme activity. Jones et al reported biphasic kinetic profiles for triazolam, diazepam, and clonazepam in rat liver microsomes (11). The parent depletion data was fitted to a biexponential model to account for loss of enzyme activity.…”

Metabolism of fibrates by cytochrome P450s and UDP-glycosyltransferases in rat and human liver microsomes

“…In this study, we measured the metabolism of fibrates in RLM and HLM by applying the cofactors NADPH and UDPGA individually and simultaneously, allowing us to determine the contributions of both pathways using the same approach and detection platform. The substrate depletion approach has been shown to be comparable to the product formation approach and is increasingly being used in enzyme kinetics studies [23,27,30]. Considering the diversity of metabolites formed from the metabolism of fibrates and the requirement for semi throughput in this study, it is impractical to monitor product formation; thus the substrate depletion approach is more appropriate in this context.…”