Metabolites: structure determination and prediction
Inhibition of cytochrome P450 enzymes and biochemical aspects of mechanism-based inactivation (MBI)

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Mechanism-based inactivation (MBI) often involves metabolic bioactivation of the xenobiotic by cytochrome P450s (CYPs) to an electrophilic reactive intermediate and results in quasi-irreversible or irreversible inactivation. Such reactive intermediate can cause quasi-irreversible inhibition through coordination to the ferrous state, Fe(II), of the P450 enzyme forming a tight noncovalent bond leading to the formation of metabolic-intermediate complex (MIC). By contrast, irreversible inactivation is one of the most common causes for the observed drug–drug interaction (DDI) and usually implies the formation of a covalent bond between the metabolite and the enzyme via alkylation of either the heme or the P450 apoprotein. Here we illustrate the important points of the current literature understanding of the mechanisms of inhibition of CYP enzymes with emphasis on general mechanistic aspects of MBI for some drugs/moieties associated with the phenomenon. Additionally, the utility of computational and in silico approaches to address bioactivation issues will be briefly discussed.

Graphical abstract

Bioactivation of the methylenedioxy portion of paxil to the carbene reactive intermediate and subsequent quasi-irreversible inactivation of P450 enzymes via metabolic-intermediate complex (MIC).

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Introduction

Drug–drug interactions (DDIs), the effects of one drug on the efficacy and/or toxicity of another drug, can lead to severe side effects and usually result in early termination of development, withdrawal of a drug from the market, or severe restrictions/limitations on its use (black box warning). DDIs may arise from the pharmacokinetic processes of absorption, distribution, metabolism and elimination (ADME), from agonistic or antagonistic pharmacodynamic interactions, or from the inhibition of transport proteins.

From a pharmacokinetic standpoint, DDIs can occur when one drug (perpetrator) alters the metabolism of a coadministered drug (victim or substrate). The outcome is an increase or decrease in the systemic clearance and/or bioavailability, and a corresponding change in the concentration of the victim drug in plasma and presumably at the site of action. The clinical consequences will range from lack of therapeutic efficacy to severe, even fatal, adverse drug reactions. By contrast, pharmacodynamic drug interactions occur when two drugs have agonistic or antagonistic pharmacologic effects which may cause drug interaction that result in adverse effects in some individuals. When two or more drugs with similar pharmacodynamic effects are given, the additive (agonistic) effects may result in excessive response and toxicity. By contrast, drugs with opposing (antagonistic) pharmacodynamic effects may reduce the response to one or both drugs. In addition to pharmacokinetic and pharmacodynamic drug interactions, the disposition of drugs by transport proteins has been recognized [1] and some drug interactions, previously believed to be P450-mediated, are now considered, at least in part, due to the inhibition of transport proteins. For example talinolol undergoes little metabolism and the increased bioavailability in the presence of verapamil. This is most probably due to inhibition of P-glycoprotein (Pgp) by verapamil [2].

The cytochrome P450s (CYPs) are a family of heme-containing enzymes that catalyze the oxidative biotransformation of structurally diverse xenobiotics including drugs. Coadministration of drugs metabolized via the CYP pathway presents the risk of clinically significant DDIs, particularly if one drug inhibits or induces the CYP enzyme responsible for the metabolism of the other. It is no surprise that most known mechanisms of DDIs involve the major enzyme system for xenobiotic metabolism, the CYP isozymes.

Drug interactions mediated by P450 induction are significantly less common than those mediated by P450 inhibition and less likely to result in safety issues but may impact efficacy [3, 4]. In some cases, the induced metabolic pathway could lead to increased formation of an active metabolite (or formation of a drug from a prodrug) resulting in increased efficacy/toxicity. Unlike CYP inhibition, which is an almost immediate response, CYP induction is a slow regulatory process that can reduce drug concentrations in plasma, and may compromise the efficacy of the drug in a time-dependent manner. However, the metabolites formed during biotransformation may be chemically reactive, so that enzyme induction may result in increased toxicity caused by the increased production of the toxic metabolites. An example would be the metabolism of acetaminophen by CYP2E1; the resulting product is a highly reactive quinone imine intermediate that is detoxified by conjugation with glutathione (GSH). Because CYP2E1 is inducible by ethanol (at low doses), individuals with alcohol dependence face an increased risk of acetaminophen hepatotoxicity due to increased formation of the reactive intermediate and resulting depletion of GSH concentrations [5]. DDIs caused through induction generally result in lower exposure and reduced toxicity [3, 4].

However, the impact of an affected drug's concentration being driven below its level of efficacy cannot be underestimated, particularly for indications that are life-threatening. A classic example of an induction-mediated DDI is the coadministration of cyclosporine, an immunosuppresive agent, with rifampin, an antibiotic used for treating tuberculosis. Rifampin is a potent inducer of CYP3A activity and when coadministered with cyclosporine, a CYP3A substrate, plasma levels of cyclosporine can drop below the level of efficacy leading to an increased incidence of organ rejection [6].

As stated above, DDIs caused through inhibition are generally more significant than those mediate by enzyme induction. Inhibition of P450 enzymes is by far the most common mechanism and one of the main reasons which can lead to DDIs. A balance must be reached between the amount of drug which is to be administered and the rate of drug clearance to achieve the desired therapeutic plasma or tissue concentration for the required amount of time. As the clearance of most small molecule drug substances is dependent upon CYP enzymes, their inhibition can lead to overexposure and toxicity. Consequently, considerable effort is expended in the pharmaceutical industry on optimization of molecules in the early stage of development to avoid pharmacokinetic DDIs due to CYP inhibition.

In the following sections, we will illustrate the important points of the current understanding of the mechanisms of inhibition of CYP enzymes with emphasis on general aspects of mechanism-based inhibition (MBI), and examples of some drugs associated with this phenomenon. Additionally, the application of computational and in silico approaches to address bioactivation will be briefly discussed.

Section snippets

Inhibition of CYP enzymes

P450 inhibition has been implicated in the majority of reported clinically relevant DDIs. The mechanisms of CYP inhibition can be divided into 2 distinct categories: (I) reversible inhibition and (II) MBI, also referred to as suicide inhibition or more commonly time-dependent inhibition (TDI). Generally, and in mechanistic terms, reversible inhibition arises as a result of competition at the CYP active site while MBI is caused by the formation of reactive metabolites.

Computational and in silico approaches to predict P450 inactivation

The development and application of computational and in silico approaches to predict P450 inactivation and DDI represent possible means to improve productivity of the drug discovery process and remove potential bottlenecks caused by relevant experimental in vitro testing. Therefore, a low risk strategy in early stages of drug discovery to avoid mechanism-based inactivation (MBI) of P450 could potentially exclude the use of functional groups that are susceptible to P450-mediated bioactivation

Conclusions

MBI often involves metabolic bioactivation of the xenobiotic by P450s to an electrophilic reactive intermediate and results in quasi-irreversible or irreversible inactivation. Such a reactive intermediate can cause quasi-irreversible inhibition through coordination to Fe(II) to form a tight noncovalent bond leading to the formation of MIC. By contrast, irreversible inactivation is one of the most common causes for severe observed DDI and usually implies the formation of a covalent bond between

Acknowledgements

We thank Dr R.S. Obach for helpful suggestions. We also thank Mr Kevin Colizza and Mr Mithat Gunduz for their valuable editing and comments.

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