Recent advances in applications of liquid chromatography–tandem mass spectrometry to the analysis of reactive drug metabolites
Introduction
Xenobiotics are metabolized by various oxidation and conjugation enzymes to more hydrophilic metabolites to facilitate elimination from the body. Thus, metabolism is generally considered as a detoxification process. However, in some cases, biotransformation of drugs can lead to the formation of chemically reactive metabolites/intermediates [1], [2], [3], [4]. These reactive metabolites are thought to cause toxicity by covalently modifying macromolecules such as proteins and DNA. Although there is no definitive proof of a causal relationship between metabolic activation and adverse drug reactions, a substantial amount of evidence implies that chemically reactive metabolites may play an important role as toxicity mediators [3], [4], [5], [6], [7], [8]. A recent study reported that among 21 drugs that were either withdrawn from the U.S. market due to hepatotoxicity or have a black box warning for hepatotoxicity, there was evidence for the formation of reactive metabolites found for 5 out of 6 drugs that were withdrawn, and 8 out of 15 drugs that have black box warnings [9]. Due to the possible link between reactive metabolites and adverse drug reactions, screening for metabolic activation of lead compounds has become an integral part of drug discovery process in order to lessen the risk posed by development of drugs that form large amounts of reactive intermediates [3], [10], [11], [12], [13], [14].
Most reactive metabolites are electrophilic in nature and can react with nucleophiles. In vitro small molecule trapping approaches are generally employed to examine the bioactivation potential of drug candidates. Structural elucidation of these stable adducts by LC–MS/MS and/or NMR can provide indirect information about the structure of the electrophilic species, thereby providing insight into the bioactivation mechanism [10], [15], [16], [17]. Glutathione (GSH) is a major scavenger of reactive metabolites and can react with a wide range of reactive electrophiles, including quinoneimines, nitrenium ions, arene oxides, quinones, imine methides, Michael acceptors, etc. to form GSH adducts. The formation of GSH adducts in vivo, by nature, is a detoxification mechanism although the GSH depletion could lead to toxicity. Takakusa et al. recently reported that the rates of GSH adduct formation of 10 representative compounds, which have different structures and are known to undergo CYP-medicated bioactivation, correlated well with the protein covalent binding of radioactivity in human and rat liver microsomes [18]. It is expected that screening for the formation of GSH conjugates would identify a significant portion of reactive metabolites formed from a drug. Lead drug candidates or new chemotypes are routinely subjected to in vitro evaluation for reactive metabolite formation through the GSH trapping experiment. Therefore, rapid, sensitive, and selective methods for detecting and characterizing reactive metabolites are highly desired in the drug discovery process [14], [19], [20], [21].
Triple quadrupole mass spectrometry is the cornerstone technique for screening and characterizing stable metabolites and GSH-trapped reactive metabolites [17], [22]. The constant neutral loss scan (CNLS) of 129 Da (a loss of pyroglutamic acid) is widely employed for rapid screening of GSH conjugates [22], [23], [24]. Recently introduced tandem mass spectrometers, such as linear ion trap (LTQ), quadrupole linear ion trap (Q-trap), quadrupole time-of-flight (Q-TOF), LTQ-Fourier transform ion cyclotron resonance mass spectrometry (LTQ-FTICRMS), and LTQ-Orbitrap, etc., have allowed the development of several new methods for reactive metabolite detection [12], [25], [26], [27], [28], [29]. This review represents an extension of the previous article [17] with particular emphasis on the most recent advances in applications of LC–MS/MS for the detection and characterization of reactive metabolites. Strategies for minimizing reactive metabolite formation in drug discovery and risk assessment of reactive metabolites in drug development have been summarized in several recent reviews [8], [11], [14], [16], [30], [31], [32] and therefore are excluded from the scope of this article.
Section snippets
Role of LC–MS/MS in the analysis of reactive metabolites
As summarized in Table 1, LC–MS/MS techniques play a dominant role in the detection, identification and quantification of reactive metabolites in the drug discovery and development processes. In early drug discovery, LC–MS/MS is often employed for rapid screening and structural characterization of reactive metabolites for lead optimization and development candidate selection [16], [20], [30]. Typically, new chemotypes or lead compounds are subjected to reactive metabolite screening. Test
Detection and structural characterization of reactive metabolites
Traditionally, CNLS of 129 Da in the positive ion mode has been employed for screening GSH conjugates [22], [40]. The main drawback of this method is its poor selectivity. Endogenous compounds present in biological matrices can give rise to a neutral fragment loss of 129 Da, and often there are multiple species that give rise to a neutral loss of 129 Da, but are not associated with drug-glutathione conjugates. Therefore, false positives are routinely detected by the CNLS analysis, especially in
Quantitative assessment of reactive metabolite formation in vitro
Quantitative assessment of the bioactivation of a given chemotype or lead compound involves the determination of the amount of the total reactive metabolites formed (or the rate of the overall bioactivation) and the major bioactivation pathway in an incubation sample. The amount of the total reactive metabolite formation provides key information in determining the structure-activity relationship from analyzing a large number of compounds from a chemo series. It can also be utilized for ranking
Detection and quantitation of reactive metabolites in vivo
Reactive metabolites are chemically unstable and very difficult to detect directly by LC–MS. Stable GSH-drug adducts and their downstream metabolites, e.g. mercapturic acid conjugates, may be detected in bile and urine and serve as an indication of exposure to these metabolites [69], [70], [71], [72]. In general, the first study to assess in vivo bioactivation of a given drug candidate is the detection and structural identification of GSH adducts in bile samples using LC–MS and/or NMR after
Challenges and future directions
Detection, characterization and quantification of reactive drug metabolites have evolved to an integral part of pharmaceutical optimization in the drug discovery process. It should be recognized that not all reactive metabolites can be trapped with glutathione. For example, bioactivation may lead to the formation of highly reactive species that covalently bind to the active site of the enzyme before having the opportunity to diffuse into the incubation medium and react with the chemical
Conclusions
Characterization of reactive metabolites is critical to designing new drug candidates with an improved toxicology profile. As a result of increased needs for high-throughput screening of reactive metabolites in the drug discovery process, several now LC–MS methodologies using the Q-trap, LTQ, Q-TOF and LTQ-Oribitrap have been developed recently. By using those LC–MS approaches outlined in this review, a drug candidate's tendency of forming reactive metabolites can be evaluated at the stage of
Acknowledgment
The authors greatly appreciate Dr. W. Griffith Humphreys for a critical reading of the manuscript.
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