Case Study 11: Considerations for Enzyme Mapping Experiments—Interaction Between the Aldehyde Oxidase Inhibitor Hydralazine and Glutathione

Abstract

Often it may be convenient and efficient to address multiple research questions with a single experiment. In many instances, however, the best approach is to design the experiment to address one question at a time. The design of enzyme mapping experiments is discussed in this chapter, focusing on considerations pertinent to the study of aldehyde oxidase (AO) vs. cytochrome P450 metabolism. Specifically, a case is presented in which reduced glutathione (GSH) was included in an experiment with human liver S9 fraction to trap reactive metabolites generated from cytochrome P450-mediated metabolism of lapatinib and its O-dealkylated metabolite, M1 (question 1). The AO inhibitor hydralazine was included in this experiment to investigate the involvement of AO-mediated metabolism of M1 (question 2). The presence of GSH was found to interfere with the inhibitory activity of hydralazine. Consideration of the time-dependent nature of hydralazine inhibitory activity toward AO when designing this experiment could have predicted the potential for GSH to interfere with hydralazine. This case underscores the importance of clearly identifying the research question, tailoring the experimental protocol to answer that question, and then meticulously considering how the experimental conditions could influence the results, particularly if attempting to address multiple questions with a single experiment.

1. Purpose of Mapping Enzyme Pathways: Cytochromes P450 and Aldehyde Oxidase

1.1. Why Is It Important to Define the Metabolic Pathways of a Drug?

Identifying the major clearance pathways of a drug or new chemical entity (NCE) is important to (a) predict potential drug–drug interactions (DDI); (b) predict the potential effect of hepatic and/or renal impairment on drug clearance; (c) predict the impact of genetic polymorphism on drug exposure; (d) understand the factors that lead to variability in drug exposure and response; and (e) identify potential active or toxic metabolites. In addition, knowing the drug extraction ratio and the fraction metabolized (f_m) by specific enzymes enables predicting the magnitude of the effect of drug interactions or genetic polymorphisms on the pharmacokinetics of the drug (bioavailability, C_max, AUC, CL).

Multiple enzymatic pathways are often involved in the metabolic clearance of drugs. The cytochrome P450 (CYP) enzymes are the most important and most extensively studied superfamily of xenobiotic-metabolizing enzymes. However, some other enzymes, such as aldehyde oxidase (AO), also need to be considered in drug metabolism and pharmacokinetic investigations during drug development. (See Chapters 9 and 10 for a detailed discussion on cytochromes P450 and aldehyde oxidase, respectively.)

1.2. Cytochrome P450

CYP enzymes play a major role in the oxidative metabolism of xenobiotics, including a majority of the small molecule drugs in clinical use [1]. CYP enzymes of the CYP1, CYP2, and CYP3 families are involved in the metabolism of an estimated 75% of all small molecule drugs [1]. CYP substrates are usually lipophilic small molecules. Common oxidation reactions catalyzed by CYPs (e.g., hydroxylation, O-dealkylation, N-dealkylation) result in adding or unmasking a polar functional group. Knowledge of common sites of metabolism by CYPs has led to the design of drugs that are more metabolically stable in relation to CYP-mediated metabolism (see also Chapters 7 and 9).

1.3. Aldehyde Oxidase

AO is an important drug-metabolizing enzyme known to oxidize aromatic nitrogen-containing heterocyclic compounds (see Chapter 10). AO has gained increasing attention for its role in the metabolism of chemical entities that contain aromatic nitrogen heterocycles (e.g., quinazoline), which favor oxidation by AO. For example, small molecule kinase inhibitors used in targeted cancer therapy contain nitrogen heterocycles, and several kinase inhibitors have recently been identified as AO substrates, including imatinib, idelalisib, and lapatinib [2]. Notably, medicinal chemistry strategies to reduce susceptibility of compounds to CYP metabolism (by reducing lipophilicity) may increase susceptibility to AO metabolism, as incorporation of nitrogen atoms into aromatic rings has become a common tactic to achieve this goal [3].

2. Planning GSH Trapping Experiments

The generation of reactive metabolites during drug metabolism (bioactivation), such as by CYP enzymes, is of concern for DDI and drug safety assessments, due to the potential for time-dependent or mechanism-based inactivation of CYPs (Chapter 3), covalent modification of other cellular proteins, and/or down-stream organ toxicity [4, 5]. Understanding the metabolic routes that influence the generation of and exposure to reactive, potentially toxic metabolites is particularly important for safety assessment and mechanistic studies [6]. In drug metabolism studies, nucleophilic trapping agents (e.g., glutathione (GSH), cyanide) are used to screen for the formation of reactive metabolites, which can be chemically detected by liquid chromatography–tandem mass spectrometry (LC-MS/MS) approaches as “trapped” conjugates [7].

GSH is an abundant intracellular antioxidant that serves to maintain the redox balance in cells and scavenge electrophilic intermediates and reactive oxygen species (ROS). The intracellular concentrations of GSH can be 5–10 mM, especially in the liver [8]. GSH is a tripeptide, containing the amino acids γ-glutamate, cysteine, and glycine (γ-Glu-Cys-Gly). The thiolate ion of the cysteine residue of GSH can react with some of the electrophiles (e.g., quinones, quinoneimines, and imine methides) formed from CYP-mediated bioactivation reactions. Depending on the chemical reactivity of the electrophile, GSH may react with electrophiles nonenzymatically or through an enzymatic reaction catalyzed by glutathione S-transferases (GST). GSH trapping experiments are used to address the following questions: (1) are reactive metabolites formed? (2) if so, what is the structure of the metabolite(s)? and (3) what enzymes are involved in metabolite formation? To address these questions, metabolic incubations are often supplemented with GSH (1–5 mM) in the presence of liver subcellular fractions (e.g., human liver microsomes or S9 fractions), which contain the enzymes of interest (e.g., CYPs). Covalent binding studies are also used to detect metabolic activation [7, 9].

3. Choosing the Subcellular Fraction

Choosing the appropriate in vitro systems to elucidate the metabolic pathways and enzymology of a drug or new chemical entity is critical for an accurate assessment. When selecting the appropriate metabolically active system for enzyme mapping experiments, consideration should be given to the enzymatic content, known (or predicted) metabolic pathways for the compound of interest, and research questions to be answered.

Hepatocyte systems can be used to identify the full profile of hepatic metabolites, estimate overall clearance, assess induction potential, and predict drug toxicity. While primary human hepatocytes contain the full complement of enzymes involved in oxidative/reductive, hydrolytic, and conjugative reactions, presumably at physiologically relevant levels, this complex system can be resource-intensive. Use of hepatocytes often requires careful consideration of all of the drug-metabolizing enzymes and transporters involved in the intracellular disposition of the compound as well as maintenance of enzyme and transporter activity during the time-course of the experiment.

Subcellular liver fractions can offer the advantage of improved stability and ease of use to assist in the identification of individual enzyme contributions to a compound’s metabolism. Liver S9 fraction (i.e., the supernatant of liver homogenate following $9000\times \mathfrak{g}$ centrifugation) contains both membrane-bound (microsomal) and soluble (cytosolic) enzymes. Further centrifugation $({10}^5\times \mathfrak{g})$ separates the S9 fraction into microsomes and cytosol. The liver microsomal fraction consists of membrane vesicles of the endoplasmic reticulum, which contain CYP and UDP-glucuronosyltransferase (UGT) enzymes along with flavin-containing monooxygenase (FMO), carboxylesterases, and some GSTs. Human liver cytosol contains soluble enzymes, including AO, sulfotransferases (SULTs), methyltransferases, N-acetyltransferases (NATs), and some GSTs [10, 11]. Unlike hepatocytes, subcellular fractions usually require the addition of cofactors for enzyme activity (e.g., NADPH is required for CYP activity). AO, which contains an embedded molybdopterin cofactor (MoCo), flavin adenine dinucleotide (FAD), and two iron-sulfur clusters, does not require exogenous cofactors to be added to cytosol or S9 [3]. While more basic subcellular systems (i.e., S9 or microsomes) are useful for exploring the involvement of specific enzymes in the metabolism of a drug, the use of these systems in isolation can produce misleading results, as competing metabolic and detoxication pathways present in hepatocytes may be overlooked. Complementary systems are often needed for a complete answer to a problem.

Liver microsomes are commonly used in in vitro reaction phenotyping studies to identify the major CYP enzymes involved in the metabolism of a new chemical entity and to determine if the compound is a CYP inhibitor that may participate in clinically significant DDIs. Liver S9 fraction and cytosol are often used to examine AO-specific metabolism. When more than one metabolic pathway is involved, S9 fraction or hepatocytes along with an AO-specific inhibitor may be used to estimate the fraction metabolized by AO $(f_{\text{m}}{}_{(\text{AO})})$ [12, 13]. When working with a potential substrate for both CYPs and AO, it is important to be aware that microsomes may be contaminated with cytosol (and thus, contaminated with AO) in order to avoid misidentifying a metabolism pathway as microsomal (or to avoid overestimating the contribution of the microsomal pathway) [14]. Assessment of AO metabolism is important in early drug development, as failure to account for this enzyme in preclinical in vitro experiments and animal studies may result in underprediction of drug clearance in humans (overestimation of total drug exposure; see Chapter 14) and unexpected generation of potentially toxic metabolites produced by AO [12, 15]. It is important to note that species differences in enzyme expression and activity can preclude accurate assessment of AO metabolism in preclinical animal models; for example, dogs are known to lack the AOX1 gene associated with AO expression [16]. AO is also expressed in extrahepatic tissues, and the influence of genetic polymorphisms in the AOX1 gene on the clearance of AO substrates remains unclear [17]. Together, these factors present a challenge in predicting the disposition of AO-metabolized drugs.

4. Designing the Experiment

In this chapter, we present a case in which the use of GSH as a trapping agent for reactive metabolites in human liver S9 fraction resulted in an unanticipated interaction between GSH and a selective AO inhibitor, hydralazine. The case study presented is used to illustrate the following concept: when designing experiments with in vitro systems that contain multiple enzyme systems (e.g., CYPs and AO) and added components (e.g., cofactors, trapping agents) to explore potentially competing metabolic pathways (e.g., AO-mediated oxidation vs. CYP-mediated bioactivation), it is essential to first identify the research questions under investigation and then use a systematic approach to address each question. We will consider the following questions:

1. What is the research question? What is the best approach toaddress the research question?

2. When is it necessary to simplify the experimental design toaddress the research question? How can complex experiments sometimes be more informative?

3. If an enzyme inhibitor is selected to probe a specific pathway(e.g., AO-mediated metabolism), how can the mechanism of inhibition affect the experimental results? How can other components in the in vitro system affect the experimental results?

When choosing between human liver cytosol and S9 fraction to characterize the metabolism of a drug by AO, the investigator should consider how the inclusion of different enzyme systems contained in the S9 fraction may affect the results. For example, CYP enzymes present in S9 may metabolize substrates or inhibitors intended to probe AO activity and vice versa; for these situations, the use of isolated human liver cytosol may be preferred depending on the metabolic pathway(s) being investigated. If the investigator is examining metabolic pathways that involve multiple enzyme systems (e.g., CYP and AO), a more complex system may be needed (i.e., one that contains cytosolic as well as microsomal enzymes). However, as stated previously, while isolating certain enzymatic pathways can help to answer a specific research question (e.g., does enzyme X produce metabolite A?), it is important to pair these simplified experiments with experiments conducted in more complex/native systems (e.g., hepatocytes) to draw conclusions that better represent the scenario occurring in vivo.

5. Case Study: Investigating Lapatinib Metabolism by CYP and AO

The focus of this study was to examine the CYP- vs. AO-mediated metabolism of the tyrosine kinase inhibitor lapatinib. The structure of lapatinib (Fig. 1) contains a quinazoline ring core, a fluorobenzyl group, and a secondary amine. These structural features are amenable to both CYP- and AO-mediated metabolism. Previous studies have shown that lapatinib is metabolized primarily by CYP3A4 and CYP3A5 via N-dealkylation, O-dealkylation, and N-hydroxylation [18, 19]. O-Dealkylation of lapatinib produces a para-hydroxyaniline product (M1), which can undergo further oxidation by CYPs to an electrophilic quinoneimine [18]. Formation of the quinoneimine has been detected by nucleophilic trapping studies with GSH [18]. This reactive metabolite is implicated in the hepatotoxicity of lapatinib due to its ability to react with cellular thiols and covalently modify proteins [4]. More recently, Dick [2] showed that lapatinib and its O-dealkylated metabolite (M1) are substrates for AO, which was found to oxidize lapatinib and M1 on the quinazoline ring system to form “AO-M1” and M3, respectively (Fig. 2). The M3 metabolite of lapatinib retains the para-hydroxyaniline moiety, and we hypothesized that this metabolite may also be converted to a reactive quinoneimine species, capable of reacting with cellular thiols.

Fig. 1.

Lapatinib structure. Selected structural features are highlighted

Fig. 2.

Lapatinib metabolism: CYP3A-mediated bioactivation vs. aldehyde oxidase-mediated pathway. (Figure adapted with permission from Bissada et al., 2019, Drug Metab Dispos. 47(11):1257–1269)

To test this hypothesis, we conducted GSH trapping studies with lapatinib and the O-dealkylated metabolite of lapatinib (M1, 2.5–10μM) in human liver S9 fraction. For this experiment, our goals were to (a) confirm that M3 was formed from M1 by AO and (b) determine whether M3 could be converted by CYPs to a reactive quinoneimine intermediate. We conducted these experiments with both lapatinib and the M1 metabolite of lapatinib as substrates in consideration of the complexity of competing metabolic pathways of lapatinib. For example, in addition to CYP-mediated formation of M1, lapatinib can also be metabolized by AO to “AO-M1,” which may be subsequently converted to M3 by CYP-mediated O-dealkylation as an alternative pathway [2]. To simplify our analysis, metabolic incubations were conducted with M1 as the substrate to specifically examine M1 conversion to M3 by AO. Human liver S9 fraction was used because it contains both the cytosolic enzyme AO and the microsomal CYP enzymes. The metabolic reactions were supplemented with NADPH to support the activity of the CYP system, which we presumed was necessary for oxidation of M1 and M3 to the proposed quinoneimine products. GSH supplementation was used to trap the reactive quinoneimine intermediates as GSH conjugates for LC-MS/MS detection and analysis.

In an initial experiment with human liver S9 fraction, M3 was formed from M1, but quinoneimine-GSH conjugates of M3 were not detected. However, quinoneimine-GSH conjugates of M1 were readily detectable. Based on this observation, we questioned if AO-mediated metabolism of M1 to M3 might be a competing pathway for CYP-mediated bioactivation of M1 to the quinoneimine, thus representing a potential detoxication route. See Chapter 4 for a detailed discussion on kinetics of sequential metabolism.

As a follow-up to this observation, we conducted experiments to determine the influence of AO activity on the formation of quinoneimine-GSH conjugates from M1 in incubations with human liver S9 fraction. We also aimed to confirm that AO was responsible for converting lapatinib M1–M3. We hypothesized that inhibition of AO by the time-dependent AO inhibitor hydralazine [20] would result in reduced M3 formation and increased formation of M1 quinoneimine-GSH conjugates, compared to control experiments lacking hydralazine, by increasing the substrate (M1) available for bioactivation to the quinoneimine. To investigate this hypothesis, we pre-incubated human liver S9 fraction in potassium phosphate buffer (pH 7.4) containing 5 mM GSH, with and without hydralazine (25μM). Following the preincubation, reactions were initiated by addition of the substrate M1, with and without NADPH.

The results from this analysis (Fig. 3) indicated that hydralazine did not inhibit M3 formation, which was not anticipated. To identify the potential source of the issue, we conducted follow-up experiments to simplify the analysis and “isolate the variable.” We decided to repeat the incubations without GSH and NADPH to specifically test whether AO was involved in converting M1 to M3. For these studies, M1 was incubated with human liver S9 fraction with and without hydralazine preincubation; the reaction mixtures did not contain GSH or NADPH. As expected, preincubation with hydralazine inhibited formation of M3 by >90% (Fig. 4; reported in [21]), confirming AO-mediated metabolism. Incubations were also conducted in the presence of allopurinol, an inhibitor of xanthine oxidase (XO), to rule out the involvement of XO in M3 formation. AO activity in the human liver S9 fraction was confirmed using the AO substrate O⁶-benzylguanine as a positive control. Additional experiments with two well-known AO substrates (O⁶-benzylguanine and zoniporide) replicated the GSH-dependent effect on the inhibitory activity of hydralazine (Figs. 5 and 6).

Fig. 3.

Formation of lapatinib M3 and quinoneimine-GSH conjugates in human liver S9 fraction. Debenzylated lapatinib (M1, 5μM) was incubated with 10-donor pooled human liver S9 fraction (2.5 mg/ml) in 100 mM potassium phosphate buffer (pH 7.4) for 60 min in the presence of 5 mM GSH, ±NADPH, ±hydralazine (25μM; 30-min preincubation). Metabolite formation was analyzed by LC-MS/MS using selected reaction monitoring for (a) M3 (m/z 489 > 366), and (b) quinoneimine-GSH conjugates (m/z 778 > 655). Relative metabolite levels were determined based on the peak area ratio of analyte to internal standard. Percent (%) control metabolite formation was based on incubations +NADPH (control) without inhibitor. Results are presented as the means ± SD of a single experiment conducted in triplicate

Fig. 4.

Effect of AO and XO inhibitors on lapatinib M3 formation. Debenzylated lapatinib (M1, 2.5μM) was incubated with 20-donor pooled human liver S9 fraction (2.5 mg/ml) in 100 mM potassium phosphate buffer (pH 7.4) for 60 min, ± hydralazine (25μM; 30-min preincubation) or allopurinol (100μM). Metabolite formation was analyzed by LC-MS/MS using selected reaction monitoring for M3 (m/z 489 > 366). Relative metabolite levels were determined based on the peak area ratio of analyte to internal standard. Percent (%) control M3 formation was based on vehicle (control) incubations without inhibitor. Results are presented as the means $\pm$ SD of a single experiment conducted in triplicate. (Figure adapted with permission from Bissada et al., 2019, Drug Metab Dispos. 47(11):1257–1269)

Fig. 5.

Effect of GSH on AO inhibition by hydralazine and raloxifene. O⁶-benzylguanine (10μM) was incubated with 20-donor pooled human liver cytosol (2 mg/ml) in 100 mM potassium phosphate buffer (pH 7.4) for 60 min, ± hydralazine (50μM; 25-min preincubation) or raloxifene (1μM) in the presence or absence of 5 mM GSH. Metabolite formation was analyzed by LC-MS/MS using multiple reaction monitoring for the AO metabolite, 8-oxo-benzylguanine (m/z 258 > 91). Relative metabolite levels were determined based on the peak area ratio of analyte to internal standard (50 nM carbamazepine). Percent (%) control metabolite formation was based on vehicle control incubations without inhibitor or GSH. Results are presented as the means ± SD of a single experiment conducted in triplicate

Fig. 6.

Effect of GSH pre- and post-incubation on hydralazine inhibitory activity. Zoniporide (10μM) was incubated with 20-donor pooled human liver cytosol (2 mg/ml) in 100 mM potassium phosphate buffer (pH 7.4) for 60 min, ± hydralazine (50μM). Reaction mixtures were preincubated with hydralazine (25 min) either in the presence of 5 mM GSH (“preincubation”), or 5 mM GSH was added after the 25-min preincubation (“post-incubation”). Metabolite formation was analyzed by LC-MS/MS using multiple reaction monitoring for the AO metabolite, 2-oxo-zoniporide (m/z 337 > 278). Relative metabolite levels were determined based on the peak area ratio of analyte to internal standard (50 nM carbamazepine). Percent (%) control metabolite formation was based on vehicle control incubations without inhibitor or GSH. Results are presented as the means ± SD of a single experiment conducted in triplicate

A remaining question was, why did we not observe AO inhibition by hydralazine when the metabolic incubations contained GSH? Additional studies (and a search of the literature) were needed to elucidate the unanticipated finding of lack of AO inhibition by hydralazine in the presence of GSH.

6. Uncovering Mechanism: (Know Your Inhibitor)

The use of selective inhibitors is a common tool to aid in enzyme mapping (reaction phenotyping), particularly when purified recombinant enzymes are not available. Not all inhibitors are alike, though, as enzyme inhibition can occur through various mechanisms (competitive, uncompetitive, mechanism-based, etc., see Chapters 2 and 3). An important question to consider when designing an experiment involving use of an enzyme inhibitor, therefore, is “how can the mechanism of inhibition affect my experimental results?” For example, hydralazine is considered to be a time-dependent, irreversible, “selective” inhibitor of AO, capable of producing >90% inhibition of human AO activity at concentrations of 25–50μM [12], although a recent report indicates that hydralazine may have some inhibitory activity towards CYP enzymes [22]). The inhibitory activity of hydralazine toward AO is time-dependent, in which the degree of AO inhibition increases with increasing time of preincubation of hydralazine with AO. While the more obvious concerns in this case would be ensuring an appropriate preincubation period and hydralazine concentration to allow for sufficient enzyme inhibition (see Chapter 3 for a discussion on [I], K_i and K_inact), perhaps a less obvious consideration is the potential for a time-dependent inhibitor to interact with other components of the incubation mixture.

When conducting experiments with “selective” enzyme inhibitors, it is assumed that the inhibitor is selective for the enzyme of interest and that the inhibitor does not interact with other components of the incubation mixture. The more complex the system, the more difficult it becomes to be certain of the selectivity of the inhibitor against other enzymes or even nonenzyme components in the matrix. In some cases, the mechanism of inhibition may offer insight as to the likelihood of the inhibitor to interact with other enzyme or nonenzyme elements. For example, while N-acetylation of hydralazine by NAT-2 is the main route of metabolism and produces an inactive acetylated metabolite, N-acetylhy-dralazine [23, 24]), hydralazine is also reportedly converted by microsomal enzymes (i.e., CYPs) or peroxidases to reactive diazine and diazonium intermediates [23, 25]. These oxidized metabolites of hydralazine are capable of covalently binding to proteins and may be detoxicated through binding to GSH [23, 25]. In addition, time-dependent inhibition of CYP1A2 by hydralazine has been recently reported [22]. While the inhibitory mechanism of hydralazine towards AO has not been fully elucidated, given the time-dependent nature of inhibition, it is likely that a reactive intermediate is responsible. Together, these data support the hypothesis that hydralazine inhibits AO via a mechanism involving covalent binding of a reactive intermediate. Thus, it is reasonable to speculate that the reduced AO inhibitory activity of hydralazine observed in our experiments resulted due to GSH trapping of the supposed reactive intermediate(s).

Although hydralazine is bioactivated in microsomal incubations, we observed that the presence of GSH prevented complete hydralazine-mediated inhibition of AO when incubated with not only S9 but also cytosol (Fig. 5), suggesting that this phenomenon is not entirely mediated by CYP enzymes. It is possible that hydralazine inhibits AO via a direct mechanism-based process or indirectly through production of a reactive metabolite by other cytosolic enzymes, as hydralazine radicals have also been reported to be formed via peroxidase and prostaglandin synthase [26]. In addition, nonenzymatic formation of hydralazine radicals can occur as well, for example, in the presence of metal ions [26].

In contrast to hydralazine, we observed that the presence of GSH had no effect on the inhibitory activity of raloxifene, a highly potent inhibitor of AO (K_i ~ 1 nM) [27] (Fig. 5). While raloxifene has been reported as both a competitive and uncompetitive inhibitor of AO, it is not time-dependent, and its inhibitory activity is not believed to be mechanism-based [27, 28]. Therefore, for experiments requiring the presence of GSH (or other reactive metabolite trapping agents such as N-acetylcysteine), raloxifene may be a more appropriate choice to investigate the involvement of AO in a given metabolic pathway. It is important to recall, however, that the choice of subcellular fraction (or other source of enzyme) is relevant when it comes to the selectivity of an inhibitor. Raloxifene, for example, is selective for AO versus other cytosolic enzymes; however, it inhibits certain CYPs, and therefore is not ideal for enzyme mapping in S9 fractions or hepatocytes [13]. Furthermore, CYP enzymes can generate reactive metabolites of raloxifene that inactivate certain CYPs (e.g., CYP3A4). In another experiment, we also observed that preincubation of hydralazine with cytosol in the absence of GSH, followed by later addition of GSH, largely restored inhibitory activity (Fig. 6). This finding suggests that hydralazine can be utilized for certain experiments in which GSH is required when provided a sufficient preincubation period to ensure enzyme inactivation prior to the addition of GSH.

7. Lessons Learned and Remaining Questions

7.1. Lessons Learned

1. To study specific metabolic pathways and their associated enzyme(s), careful selection of the appropriate subcellular fraction is required. However, complimentary experiments using a variety of systems (e.g., various subcellular fractions, hepatocytes, and/or recombinant enzymes) are usually necessary to identify the full profile of enzymes involved and the sequence of their involvement.

2. Understand the mechanism and selectivity of your inhibitor. Be aware of factors that may alter the inhibitory activity and/or factors that may affect the selectivity of the inhibitor for the reaction in question (e.g., inhibitor concentration, time-dependence, reactive species in the incubation mixture).

3. When performing reactive metabolite trapping studies, consider whether trapping agents (in this case, GSH) may bind to other electrophilic compounds present in the experiment or if the reaction conditions allow for binding and detection of the metabolite(s) of interest.

4. The order in which trapping agents are added to a metabolic incubation, such as before or after a preincubation with a time-dependent inhibitor, can affect the results of the experiment.

7.2. Remaining Questions

1. What is the exact mechanism of inhibition of AO by hydralazine?

2. How does GSH interfere with the mechanism of AO inhibition by hydralazine (i.e., which compound(s) may be trapped by GSH)?

3. Does the presence of endogenous GSH influence the inhibitory activity of hydralazine in hepatocytes?

8. Helpful References from the Literature on Hydralazine

As this case suggests, prior knowledge of an enzyme inhibitor is critical to determine its suitability for a particular experiment based on the incubation conditions and desired outcome(s). For more details on the metabolism of hydralazine and its use as an AO inhibitor, we refer interested readers to refs. 12, 13, 20, 22–25, 29.

9. Summary

Multiple tools are available for drug metabolism scientists to characterize drug biotransformation pathways, including detailed investigations into the routes of bioactivation and detoxication. Selective chemical inhibitors are valuable tools to identify the enzymes involved in drug metabolism reactions and to probe reaction mechanisms. Nonetheless, careful attention should be given to selecting the appropriate in vitro system(s), specific inhibitor, and experimental conditions, for reaction phenotyping studies to ensure an accurate assessment. Well-designed studies begin with a clearly defined research question, and the investigators use a systematic approach to address the question. Knowledge of the mechanism of inhibition provides insight into choosing the appropriate experimental conditions when working with inhibitors. Simplifying the experimental design when necessary can help eliminate interferences and reduce the risk of confounding results. Nonetheless, researchers are bound to encounter unexpected results from time-to-time and must be prepared to investigate the validity of those results versus problems with the experimental design.