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