The Impact of In Vitro Binding on In Vitro - In Vivo Extrapolations, Projections of Metabolic Clearance and Clinical Drug-Drug Interactions

Abstract

This review provides a vista of the current opportunities and remaining challenges in the area of in vitro-in vivo extrapolation, with particular emphasis on drug binding terms in predictive models, which has been the source of much controversy. Although the importance of fu(inc) (fraction unbound in in vitro incubations) has been acknowledged for decades, it is not always applied in practice. This is somewhat disappointing, since although it may be onerous to measure this term for large numbers of compounds, algorithms to estimate the term from logD(7.4) or logP have been detailed in the literature. These are sufficiently robust to negate routine measurement in early drug discovery. Several groups have recently established convincing relationships between unbound in vivo and in vitro metabolic intrinsic clearance (CL(int)). In the authors' laboratory, correlations of this type have been constructed for rat, dog and Man. The use and interpretation of these models within a drug discovery setting is discussed. The quantitative prediction of drug-drug interactions from in vitro cytochrome P450 (CYP) inhibition data remains a challenge. Although extensive literature databases are at last emerging, apparent ad hoc use of terms for in vivo inhibitor concentrations and only occasional consideration of fu(inc) may only have confused matters. The effect of accounting for drug binding on the accuracy of predictions is reviewed. Other themes including the impact of fu(inc) on relative activity factors (RAFs) and how in vitro data quality and inter-laboratory differences can confound quantitative human pharmacokinetic predictions are also developed.

Integrated in Vitro Analysis for the in Vivo Prediction of Cytochrome P450-Mediated Drug-Drug Interactions

“…By using rP450s as the enzyme source in the determination of IC 50 or K i values, very low protein levels are afforded, and typically fu inc approaches 1 and the experimentally generated IC 50,apparent values approach IC 50,u . The need to determine the unbound rather than apparent parameter such as K m , CL int , IC 50 , and therefore K i has been recently re-emphasized Grime and Riley, 2006). This was the case with the P450 inhibitors studied in this work; therefore, the term included in the [I] in,u /K i expression equates essentially to K i,u .…”

“…The extent of the interactions of fluoxetine with desipramine (predicted ␦AUC 1.5 versus observed ␦AUC 7.4), imipramine (1.5 versus 3.3), and tolterodine (1.1 versus 4.8) was significantly underestimated using the [I] in,u /K i method (Table 4). There are several reports of underpredicting fluoxetine DDIs (Ito et al, 1998;Grime and Riley, 2006;Obach et al, 2006), but the reason(s) are as yet unclear. It is noteworthy that the use of [I] in,total / K i,u predicts the fluoxetine interactions well (data not shown), albeit perhaps coincidentally.…”

Application of IVIVE and PBPK modeling in prospective prediction of clinical pharmacokinetics: strategy and approach during the drug discovery phase with four case studies

“…Under the free drug hypothesis, incorporation of f up and f umic (or f uinc ) is preferred over assuming f up /f uinc is 1 and is used as a general approach [34]. In this study, the IVIVE using HLM CL int with incorporation of f up and f umic consistently under-predicted the in vivo clearance in preclinical species.…”

Comparative Use of Isolated Hepatocytes and Hepatic Microsomes for Cytochrome P450 Inhibition Studies: Transporter-Enzyme Interplay

“…Because accumulation of drugs in hepatocytes may occur via active uptake processes and/or intracellular binding, a concentration difference may exist between hepatocyte and hepatic microsomal incubations. Whether the drug is a bound or free entity within the cell is of importance; intracellular binding to sites not involved in the metabolic process may be of little consequence, as the free concentration within the cell will be in equilibrium with the external incubation media concentration (Grime and Riley, 2006;Poirier et al, 2008). However, in the case of uptake transporters, raising the cellular concentration in excess of the incubation medium concentration will result in more drug available to the enzyme.…”

“…After appropriate binding corrections, more potent hepatocyte inhibition (as evident by lower K i values) would suggest the involvement of hepatic transporters because a higher concentration of unbound drug available for enzyme inhibition can be achieved compared with a similar incubation concentration in microsomes (Ito et al, 1998). Alternatively, similar values would indicate that any hepatic accumulation results from intracellular binding or lysosomal trapping (Grime and Riley, 2006;Hallifax and Houston, 2007). We recently demonstrated good concordance between K i values (range 0.05-30 M) determined in rat hepatic microsomes and freshly isolated hepatocytes using seven P450 inhibitors (fluconazole, fluoxetine, fluvoxamine, ketoconazole, miconazole, omeprazole, and quinine) with a range of uptake properties (cell/medium concentration ratios 4 -6000).…”