SUBSTRATE DEPLETION APPROACH FOR DETERMINING IN VITRO METABOLIC CLEARANCE: TIME DEPENDENCIES IN HEPATOCYTE AND MICROSOMAL INCUBATIONS Hannah M. Jones 1 and J. Brian Houston Centre for Applied Pharmaceutical Research, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, United Kingdom Received April 7, 2004; accepted June 8, 2004 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 conse- quences of which have not been systematically explored. Initial substrate depletion incubations using rat microsomes and hepa- tocytes were performed for eight benzodiazepines: alprazolam, clobazam, clonazepam, chlordiazepoxide, diazepam, flunitraz- epam, 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 tradi- tional 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 clear- ance 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 ). CL H = Q H   in vitro CL int  SF f u inc   f ub Q H +  in vitro CL int  SF f u inc   f ub (1) 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 ub 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 re- spect 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 com- pliance with the Michaelis-Menten equation assumes less than 10% substrate consumption. Therefore, issues such as the stability of the enzyme preparation (resulting from long incubation times), nonspe- cific binding (resulting from high enzyme concentrations), and end- product inhibition (resulting from the accumulation of the phase I hydroxylated metabolite in the microsomal incubation) are not gen- erally limitations. However, the main disadvantage to this approach is that it requires prior knowledge of the particular metabolic pathway under study and its importance to the overall metabolic fate of the drug to ensure a true and accurate prediction of clearance; this may be particularly problematic if multiple metabolic pathways are involved. For many drugs, especially new drug candidates, this information is not known; therefore, the use of this approach is limited. More recently, the substrate depletion approach has been adopted, where the consumption of parent drug is monitored over time. This method is particularly popular in the pharmaceutical industry because formal kinetic characterization and metabolite quantification are not required, allowing the rapid screening of compounds (Lave´ et al., 1997; Obach, 2001; Obach and Reed-Hagen, 2002; Austin et al., This work was funded by a consortium of pharmaceutical companies (Astra- Zeneca, Bristol Myers Squibb, GlaxoSmithKline, F. Hoffmann-La Roche, Novartis, Pfizer, and Servier) within the Centre for Applied Pharmacokinetic Research at the University of Manchester. 1 Current address: Department of Non-Clinical Drug Safety, Drug Metabolism and Pharmacokinetics, F. Hoffmann La Roche, Basel, Switzerland. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.104.000125. ABBREVIATIONS: CL int , intrinsic clearance; CL H , hepatic clearance; DMF, dimethylformamide; HPLC, high-performance liquid chromatography; AIC, Akaike information criterion; NADP, nicotinamide adenine dinucleotide phosphate. 0090-9556/04/3209-973–982$20.00 DRUG METABOLISM AND DISPOSITION Vol. 32, No. 9 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 125/1171189 DMD 32:973–982, 2004 Printed in U.S.A. 973 at ASPET Journals on November 13, 2017 dmd.aspetjournals.org Downloaded from