Theoretical assessment of a new experimental protocol for determining kinetic values describing mechanism (time)-based enzyme inhibition

https://doi.org/10.1016/j.ejps.2007.04.005Get rights and content

Abstract

We have shown previously that the conventional experimental protocol (CEP) used to characterise mechanism-based enzyme inhibition (MBI) of drug metabolism in vitro may introduce substantial bias in estimates of the relevant kinetic parameters. The aim of this study was to develop and assess, by computer simulation, an alternative, mechanistically-based experimental protocol (MEP). This protocol comprises three parts viz. assessment of the metabolism of the mechanism-based enzyme inactivator (MBEI), of its ability to participate in competitive inhibition and its ability to cause time-dependent inhibition. Thus, values of the maximum inactivation rate constant (kinact), the inactivator concentration associated with half-maximal rate of inactivation (KI), the partition ration (r), and the reversible inhibition constant (Ki) of the MBEI are determined by nonlinear optimization of the experimental data using a model that allows for metabolism of both probe substrate and MBEI, the time-course of inactivation of the enzyme, and reversible inhibition of the metabolism of both probe substrate and MBEI. Sensitivity analysis is used to estimate the degree of confidence in the final parameter values. Virtual experiments using the MEP and the CEP were simulated, applying starting kinetic parameters reported for 16 known MBEIs. In the presence of simulated experimental error (5% CV), the MEP recovered accurate estimates of the kinetic values for all compounds, while estimates using the CEP were less accurate and less precise. The MEP promises to improve consistency in the determination of in vitro measures of MBI and, thereby, the quantitative assessment of its in vivo consequences.

Introduction

Inhibition of cytochrome P450 (CYP) activity is a common cause of drug–drug interactions. Such inhibition can be reversible, quasi-irreversible, or irreversible (Lin and Lu, 1998, Hollenberg, 2002). The last two categories encompass the phenomenon of mechanism- or time-based enzyme inhibition. A mechanism-based enzyme inhibitor (MBEI) is defined as a chemically unreactive compound whose structure resembles either the substrate or product of the target enzyme, and which undergoes a catalytic transformation by the enzyme to a species that, prior to release from the active site, inactivates the enzyme by either a covalent or noncovalent mechanism (Silverman, 1988). The implications of irreversible or quasi-irreversible loss of enzyme activity are auto-inhibition of the clearance of the MBEI itself, and prolonged inhibition of the clearance of other drugs that share the same enzyme. An additional concern is that covalently-bound metabolites may cause immunotoxicity (Pumford and Halmes, 1997). An increasing number of reports on mechanism-based inhibition (MBI) indicate a growing awareness of the potential impact of MBI on drug development (Zhou et al., 2005, Ghanbari et al., 2006), and in the latest US FDA guidance (2006) it is emphasized that time-dependent inhibition should be examined by standard in vitro screening protocols.

The conventional experimental protocol (CEP) used to determine the kinetic values for MBI in vitro involves a “pre-incubation” followed by an “incubation” step (Silverman, 1988). During the first stage, enzyme and cofactors are incubated with the MBEI at several concentrations and over a range of times. The incubates are then diluted in order to “quench” inactivation, and the remaining enzyme activity is measured with a probe substrate at a saturating concentration in the second stage of the procedure. Two important assumptions are implicit in this procedure. Firstly, that there is negligible metabolism of the MBEI during the “pre-incubation” stage and, secondly, that negligible enzyme inactivation occurs during the “incubation” stage. However, there is a paradox in that the MBEI is a substrate as well as an inactivator of the enzyme, and this dual effect cannot be differentiated completely in the CEP. Thus, there may be significant depletion of the MBEI by metabolism during the course of the experiment, adding to error in the estimation of kinetic parameters (Silverman, 1988). In a previous study reported in this journal (Yang et al., 2005) we have shown that the CEP may introduce substantial bias in parameter estimation when characterising MBI.

A further concern with the CEP is that it only determines 3 parameters, namely kinact (the maximum rate of inactivation), KI (the concentration of inhibitor that produces half-maximal inactivation), and r (the partition ratio), and ignores reversible inhibition, as defined by Ki (the reversible inhibition constant). The latter defines the reversible binding of the MBEI to the enzyme, and its value is not necessarily the same as KI. Thus, an MBEI may also be a potent competitive inhibitor (e.g. ritonavir, von Moltke et al., 2000, paroxetine, Bertelsen et al., 2003, 6′,7′-dihydroxybergamottin, Paine et al., 2004). For these compounds, ignoring reversible inhibition in the in vitro experiment may lead to an erroneous description of their MBI capability and, hence, inaccurate prediction of the extent and duration of enzyme inhibition in vivo. This is of particular importance when the compound is a reasonably potent reversible inhibitor but a moderate or weak time-dependent inhibitor (Atkinson et al., 2005).

In taking account of the above issues, we now propose a novel, mechanistically-based experimental protocol (MEP) as an alternative to the CEP, and evaluate it theoretically by in silico simulation.

Section snippets

Theoretical background

A schematic of enzyme kinetics involving MBI (Walsh et al., 1978, Waley, 1980) is shown in Fig. 1. The inactivator (I) binds to free enzyme (E) to form an enzyme–inactivator complex (E·I), which is then activated (E·I′). The latter may decompose to form a metabolic product (P) or react with the enzyme to form an inactive complex (Ei). At given concentrations of inactivator and enzyme, the reactions indicated in Fig. 1 are governed by the first-order rate constants k1, k−1, k2, k3, and k4. The

Results

The values of the kinetic parameters recovered by simulating the MEP and CEP are listed in Table 4. The differences between the determined (Table 4) and starting kinetic values (Table 1) represent the bias, which reflects the performance of the MEP and CEP. The resulting data for each MBEI were analyzed by the GA over three runs using three sets of different randomly assigned initial values. Thus, for example, a value of 0.25 ± 0.03 represents the mean ± S.D. over the three GA runs. In the absence

Discussion

In developing any experimental protocol it is necessary to evaluate the theoretical robustness of the procedures and validity of the assumptions. For historical reasons, this has not been the case for the CEP method where the protocol was developed purely based on assumptions which were not theoretically tested until recently (Yang et al., 2005). This analysis indicated that many conditions in the protocol violate the underlying assumptions. A large survey by Ghanbari et al. (2006) also exposed

Conclusion

On theoretical grounds and based on the results of simulation, the MEP is considered to be superior to the CEP with regard to accuracy, precision and efficiency. Its application may allow better prediction of the in vivo implications of MBI. While an experimental assessment of the accuracy of the MEP is not possible in the absence of a ‘gold standard’ method that provides true values of the kinetic parameters, there is clearly a need to evaluate its practical feasibility and utility.

References (49)

  • T.D. Bjornsson et al.

    The conduct of in vitro and in vivo drug–drug interaction studies: a pharmaceutical research and manufacturers of America (PhRMA) perspective

    Drug Metab. Dispos.

    (2003)
  • M. Chiba et al.

    Potent and selective inactivation of human liver microsomal cytochrome P-450 isoforms by L-754,394, an investigational human immune deficiency virus protease inhibitor

    J. Pharmacol. Exp. Ther.

    (1995)
  • J. Chun et al.

    Mechanism-based inactivation of cytochromes P4502B1 and P4502B6 by 2-phenyl-2-(1-piperidinyl) propane

    Drug Metab. Dispos.

    (2000)
  • P.W. Fan et al.

    Mechanism-based inactivation of cytochrome P450 2B6 by a novel terminal acetylene inhibitor

    Drug Metab. Dispos.

    (2003)
  • L.V. Favreau et al.

    Improved reliability of the rapid microtiter plate assay using recombinant enzyme in predicting CYP2D6 inhibition in human liver microsomes

    Drug Metab. Dispos.

    (1999)
  • FDA, 2006. Guidance for industry: drug interaction studies – study design, data analysis, and implications for dosing...
  • F. Ghanbari et al.

    A critical evaluation of the experimental design of studies of mechanism based enzyme inhibition, with implications for in vitro-in vivo extrapolation

    Curr. Drug Metab.

    (2006)
  • D.E. Goldberg

    Genetic Algorithms in Search, Optimisation, and Machine Learning

    (1989)
  • F.P. Guengerich

    Mechanism-based inactivation of human liver microsomal cytochrome P-450 IIIA4 by gestodene

    Chem. Res. Toxicol.

    (1990)
  • N.T. Ha-Duong et al.

    Inhibition by ticlopidine and its derivatives of human liver cytochrome P450. Mechanism-based inactivation of CYP2C19 by ticlopidine

    Biol. React. Intermed.

    (2001)
  • E. Harleton et al.

    Metabolism of N,N’,N”-triethylenethiophosphoramide by CYP2B1 and CYP2B6 results in the inactivation of both isoforms by two distinct mechanisms

    J. Pharmacol. Exp. Ther.

    (2004)
  • P.F. Hollenberg

    Characteristics and common properties of inhibitors, inducers, and activators of CYP enzymes

    Drug Metab. Rev.

    (2002)
  • J.M. Hutzler et al.

    Mechanism-based inactivation of cytochrome P450 2D6 by 1-[(2-ethyl-4-methyl-1H-imidazol-5-yl)methyl]-4-[4-(trifluoromethyl)-2-p yridinyl]piperazine: kinetic characterization and evidence for apoprotein adduction

    Chem. Res. Toxicol.

    (2004)
  • K. Ito et al.

    Prediction of pharmacokinetic alterations caused by drug–drug interactions: metabolic interaction in the liver

    Pharmacol. Rev.

    (1998)
  • Cited by (0)

    View full text