Herein we detail the first disclosure of VU0467319 (VU319), an M1 Positive Allosteric Modulator (PAM) clinical candidate that successfully completed a Phase I Single Ascending Dose (SAD) clinical trial. VU319 (16) is a moderately potent M1 PAM (M1 PAM EC50 = 492 nM ± 2.9 nM, 71.3 ± 9.9% ACh Max), with minimal M1 agonism (EC50 > 30 μM), that displayed high CNS penetration (K p s > 0.67 and K p,uu s > 0.9) and multispecies pharmacokinetics permissive of further development. Based on robust efficacy in multiple preclinical models of cognition, an ancillary pharmacology profile devoid of appreciable off-target activities, and a lack of cholinergic adverse effects (AEs) in rats, dogs and nonhuman primates, VU319 advanced into IND-enabling studies. After completing 4-week rat and dog GLP toxicology without AEs, including absence of cholinergic effects, the first in human Phase I SAD clinical trial of VU319 (NCT03220295) was performed at Vanderbilt, where a similar lack of adverse effects, including absence of cholinergic effects was noted. Moreover, signals of target engagement were seen at the highest dose tested. Thus, VU319 demonstrated the feasibility of achieving selective targeting of central M1 muscarinic receptors without eliciting cholinergic AEs that have plagued other drugs targeting CNS cholinergic neurotransmission.

Advances in the field of bioactivation have significantly contributed to our understanding and prediction of drug-induced liver injury (DILI). It has been established that many adverse drug reactions, including DILI, are associated with the formation and reactivity of metabolites. Modern methods allow us to detect and characterize these reactive metabolites in earlier stages of drug development, which helps anticipate and circumvent the potential for DILI. Improved in silico models and experimental techniques that better reflect in vivo environments are enhancing predictive capabilities for DILI risk. Further, studies on the mechanisms of bioactivation, including enzyme interactions and the role of individual genetic differences, have provided valuable insights for drug optimizations. Cumulatively, this progress is continually refining our approaches to drug safety evaluation and personalized medicine.

Aldehyde oxidases (AOXs) and xanthine oxidoreductase (XOR) are closely related enzymes with very similar primary structures comprising two identical subunits each of which contains four redox centers: one molybdopterin cofactor, one flavin adenine dinucleotide (oxidized form) molecule, and two iron–sulfur clusters. The enzymes, also referred to as molybdenum hydroxylases, catalyze both oxidation and reduction reactions and play a significant role in the metabolism of drugs and endobiotics including endogenous purines. Generally, oxidation reactions involve nucleophilic attack via a Mo–OH ligand at a carbon atom in N-heterocycles and aldehydes with electrons transferred ultimately to molecular oxygen or NAD+ (nicotinamide adenine dinucleotide—the oxidized form). Reduction of oxygen can generate reactive oxygen species, which have been implicated in a variety of protective and pathophysiological functions. AOXs and XOR are widely distributed throughout the animal kingdom and probably originated from a single ancestor gene coding for a dehydrogenase form of XOR. While a single mammalian XOR is known, various mammalian AOX isoenzymes have been identified. The complement of active mammalian AOX genes varies from one in humans (AOX1) to four in rodents (AOX1, AOX2, AOX3, and AOX4). In humans, the AOX1 and XOR genes are found on different arms of chromosome 2 and are both subject to complex regulation, the precise details of which have not yet been fully characterized. The 3D structures of bovine milk XOR, human AOX1, and mouse AOX3 are available and have provided valuable information on the catalytic mechanism of molybdenum hydroxylases. This chapter provides an overview of the current knowledge on the structure, evolution, and function of these highly complex enzymes.

The anti-hypertensive agent hydralazine is a time-dependent inhibitor of the cytosolic drug-metabolizing enzyme aldehyde oxidase (AO). Glutathione (GSH) was found to suppress the inhibition of AO by hydralazine in multiple enzyme sources (human liver and kidney cytosol, human liver S9, rat liver S9, and recombinant human AO) and with different AO substrates (zoniporide, O6 -benzylguanine, and dantrolene). Hydralazine-induced AO inactivation was unaffected when GSH was added to the incubation mixture after pre-incubation of hydralazine with AO (rather than during the pre-incubation), suggesting that GSH traps a hydralazine reactive intermediate prior to enzyme inactivation. Consistent with previous reports of 1-phthalazylmercapturic acid formation when hydralazine was incubated with N-acetylcysteine, we detected a metabolite producing an MS/MS spectrum consistent with a 1-phthalazyl-GSH conjugate. O6 -Benzylguanine, an AO substrate, did not protect against hydralazine-induced AO inactivation, implying that hydralazine does not compete with O6 -benzylguanine for binding to the AO active site. Catalase also failed to protect AO from hydralazine-induced inactivation, suggesting that hydrogen peroxide is not involved. However, an allosteric AO inhibitor (thioridazine) offered some protection, indicating a catalytic role for AO in the bioactivation of hydralazine. AO inhibition by phthalazine (a substrate and inhibitor of AO and a metabolite of hydralazine) was unaffected by the presence of GSH. GSH also prevented hydralazine from inhibiting the nitro-reduction of dantrolene by AO. Furthermore, the GSH-hydralazine combination stimulated dantrolene reduction. Phthalazine inhibited only oxidation reactions, not reduction of dantrolene. Together, these results support the hypothesis that hydralazine is converted to a reactive intermediate that inactivates AO. SIGNIFICANCE STATEMENT: These studies suggest that a reactive intermediate of hydralazine plays a primary role in the mechanism of aldehyde oxidase (AO) inactivation. Inactivation was attenuated by glutathione and unaffected by catalase. Phthalazine (hydralazine metabolite) inhibited AO regardless of the presence of glutathione; however, phthalazine inhibited only oxidation reactions, while hydralazine inhibited both oxidation and reduction reactions. This report advances our mechanistic understanding of hydralazine as an AO inhibitor and provides information to facilitate appropriate use of hydralazine when probing AO metabolism.

With the 50th year mark since the launch of Drug Metabolism and Disposition journal, the field of drug metabolism and bioactivation has advanced exponentially in the past decades (Guengerich 2023).This has, in a major part, been due to the continued advances across the whole spectrum of applied technologies in hardware, software, machine learning (ML), and artificial intelligence (AI). LC-MS platforms continue to evolve to support key applications in the field, and automation is also improving the accuracy, precision, and throughput of these supporting assays. In addition, sample generation and processing is being aided by increased diversity and quality of reagents and bio-matrices so that what is being analyzed is more relevant and translatable. The application of in silico platforms (applied software, ML, and AI) is also making great strides, and in tandem with the more traditional approaches mentioned previously, is significantly advancing our understanding of bioactivation pathways and how these play a role in toxicity. All of this continues to allow the area of bioactivation to evolve in parallel with associated fields to help bring novel or improved medicines to patients with urgent or unmet needs.Shuai Wang and Cyrus Khojasteh, on behalf of the authors.

This annual review is the eighth of its kind since 2016 (Baillie et al. 2016, Khojasteh et al. 2017, Khojasteh et al. 2018, Khojasteh et al. 2019, Khojasteh et al. 2020, Khojasteh et al. 2021, Khojasteh et al. 2022). Our objective is to explore and share articles which we deem influential and significant in the field of biotransformation.

Objective The goal of this project was to evaluate the drug metabolism and pharmacokinetic properties of ent-verticilide in vitro. Methods To estimate intrinsic clearance, we measured the in vitro half-life of ent-verticilide using liver S9 fractions from human, mouse, rat, and dog. Subsequently, the intrinsic clearance values were incorporated into the well-stirred model of hepatic clearance to estimate the hepatic metabolic clearance in vivo. To identify major metabolites of ent-verticilide, we incubated ent-verticilide with microsomal fractions of human, mouse, rat, and dog. We then subjected extracts from these incubations to LC/MS analysis for structural interpretation of resulting metabolites. Finally, we determined the enzymes responsible for ent-verticilide metabolism by incubating ent-verticilide with human liver microsomes in the presence and absence of various cytochrome P450 inhibitors. Results Predicted hepatic clearance for mouse, rat, dog, and human was 76, 41, 29, and 18 mL/min/kg, respectively. The metabolism was determined to be NADPH-dependent, indicating cytochrome P450 metabolism. Metabolites formed via mono-hydroxylation and di-hydroxylation of the hydrocarbon side chains are the major metabolites. Metabolites formation by oxidation of the hydrocarbon side chains were also observed. In addition, CYP3A4 was the major CYP450 implicated in the biotransformation of ent-verticilide. Conclusions These data indicate that ent-verticilide is extensively metabolized by hepatic enzymes (high hepatic extraction ratio), and that the metabolism is predominantly mediated by CYP3A4. The major elimination pathway was determined to be via hydroxylation of the hydrocarbon chains. The goal of this project was to evaluate the drug metabolism and pharmacokinetic properties of ent-verticilide in vitro. To estimate intrinsic clearance, we measured the in vitro half-life of ent-verticilide using liver S9 fractions from human, mouse, rat, and dog. Subsequently, the intrinsic clearance values were incorporated into the well-stirred model of hepatic clearance to estimate the hepatic metabolic clearance in vivo. To identify major metabolites of ent-verticilide, we incubated ent-verticilide with microsomal fractions of human, mouse, rat, and dog. We then subjected extracts from these incubations to LC/MS analysis for structural interpretation of resulting metabolites. Finally, we determined the enzymes responsible for ent-verticilide metabolism by incubating ent-verticilide with human liver microsomes in the presence and absence of various cytochrome P450 inhibitors. Predicted hepatic clearance for mouse, rat, dog, and human was 76, 41, 29, and 18 mL/min/kg, respectively. The metabolism was determined to be NADPH-dependent, indicating cytochrome P450 metabolism. Metabolites formed via mono-hydroxylation and di-hydroxylation of the hydrocarbon side chains are the major metabolites. Metabolites formation by oxidation of the hydrocarbon side chains were also observed. In addition, CYP3A4 was the major CYP450 implicated in the biotransformation of ent-verticilide. These data indicate that ent-verticilide is extensively metabolized by hepatic enzymes (high hepatic extraction ratio), and that the metabolism is predominantly mediated by CYP3A4. The major elimination pathway was determined to be via hydroxylation of the hydrocarbon chains.

Biotransformation field is constantly evolving with new molecular structures and discoveries of metabolic pathways that impact efficacy and safety. Recent review by Kramlinger et al. (2022) nicely captures the future (and the past) of highly impactful science of biotransformation (see the first article). Based on the selected articles, this review was categorized into three sections: (1) new modalities biotransformation, (2) drug discovery biotransformation, and (3) drug development biotransformation (Table 1).

Starting from the MLPCN probe compound ML300, a structure-based optimization campaign was initiated against the recent severe acute respiratory syndrome coronavirus (SARS-CoV-2) main protease (3CLpro). X-ray structures of SARS-CoV-1 and SARS-CoV-2 3CLpro enzymes in complex with multiple ML300-based inhibitors, including the original probe ML300, were obtained and proved instrumental in guiding chemistry toward probe compound 41 (CCF0058981). The disclosed inhibitors utilize a noncovalent mode of action and complex in a noncanonical binding mode not observed by peptidic 3CLpro inhibitors. In vitro DMPK profiling highlights key areas where further optimization in the series is required to obtain useful in vivo probes. Antiviral activity was established using a SARS-CoV-2-infected Vero E6 cell viability assay and a plaque formation assay. Compound 41 demonstrates nanomolar activity in these respective assays, comparable in potency to remdesivir. These findings have implications for antiviral development to combat current and future SARS-like zoonotic coronavirus outbreaks.

Abstract This year’s review on bioactivation and reactivity began as a part of the annual review on biotransformation and bioactivation led by Cyrus Khojasteh (see references). Increased contributions from experts in the field led to the development of a stand alone edition for the first time this year focused specifically on bioactivation and reactivity. Our objective for this review is to highlight and share articles which we deem influential and significant regarding the development of covalent inhibitors, mechanisms of reactive metabolite formation, enzyme inactivation, and drug safety. Based on the selected articles, we created two sections: (1) reactivity and enzyme inactivation, and (2) bioactivation mechanisms and safety (Table 1). Several biotransformation experts have contributed to this effort from academic and industry settings. Table 1. Articles covered in this review. Title First author Source Reactivity & enzyme inactivation 1 The inactivation of human aldehyde oxidase 1 by hydrogen peroxide and superoxide. C Garrido Drug Metab Dispos 49:729–735, 2021 2 Icotinib induces mechanism-based inactivation of recombinant human CYP3A4/5 possibly via heme destruction by ketene intermediate. C Sun Drug Metab Dispos 49:892–901, 2021 3 Evobrutinib, a covalent Bruton’s tyrosine kinase inhibitor: Mass balance, elimination route, and metabolism in healthy participants. H Scheible Clin Transl Sci 14: 2420–2430, 2021 Bioactivation mechanisms & safety 4 Metabolic activation of atomoxetine mediated by cytochrome P450 2D6. Y You Chem Res Toxicol 34:2135–2144, 2021 5 Mechanism-based insights into removing the mutagenicity of aromatic amines by small structural alterations. I Shamovsky J Med Chem 64: 8545–8563, 2021 6 Investigating the mechanism of trimethoprim-induced skin rash and liver injury. Y Cao J Toxicol Sci 180:17–25, 2021 7 Bioactivation of clozapine by mitochondria of the murine heart: Possible cause of cardiotoxicity. E Arzuk Toxicology 447: 152628, 2021 8 CYP2C9 and 3A4 play opposing roles in bioactivation and detoxification of diphenylamine NSAIDs. Mary Alexandra Schleiff Biochem Pharmacol 194:114824, 2021 9 Metabolic activation and cytotoxicity of labetalol hydrochloride mediated by sulfotransferases. L Yang Chem Res Toxicol 34:1612–1618, 2021 10 Cytochrome P450 binding and bioactivation of tumor-targeted duocarmycin agents. AG Bart Drug Metab Dispos 50:49–57, 2022 11 Preclinical metabolism and disposition of an orally bioavailable macrocyclic FXIa inhibitor. SA Chacko Xenobiotica 51:933–948, 2021 12 Identification of an isoform catalyzing the CoA conjugation of nonsteroidal anti-inflammatory drugs and the evaluation of the expression levels of acyl-CoA synthetases in the human liver. H Hashizume Biochem Pharmacol 183:114303, 2021 13 Identification and characterization of in vitro, in vivo, and reactive metabolites of tandutinib using liquid chromatography ion trap mass spectrometry. NS Al-Shakliah Anal Methods 13: 399–410, 2021 14 Idiosyncratic drug-induced liver injury: Mechanistic and clinical challenges. A Jee Int J Mol Sci 22: 2954, 2021

This year's review on bioactivation and reactivity began as a part of the annual review on biotransformation and bioactivation led by Cyrus Khojasteh (see references). Increased contributions from experts in the field led to the development of a stand alone edition for the first time this year focused specifically on bioactivation and reactivity. Our objective for this review is to highlight and share articles which we deem influential and significant regarding the development of covalent inhibitors, mechanisms of reactive metabolite formation, enzyme inactivation, and drug safety. Based on the selected articles, we created two sections: (1) reactivity and enzyme inactivation, and (2) bioactivation mechanisms and safety (Table 1). Several biotransformation experts have contributed to this effort from academic and industry settings.

This is an overview of the metabolic reactions of drugs, natural products, physiological compounds, and other (general) chemicals catalyzed by flavin monooxygenase (FMO), monoamine oxidase (MAO), NAD(P)H quinone oxidoreductase (NQO), and molybdenum hydroxylase enzymes (aldehyde oxidase (AOX) and xanthine oxidoreductase (XOR)), including roles as substrates, inducers, and inhibitors of the enzymes. The metabolism and bioactivation of selected examples of each group (i.e., drugs, “general chemicals,” natural products, and physiological compounds) are discussed. We identified a higher fraction of bioactivation reactions for FMO enzymes compared to other enzymes, predominately involving drugs and general chemicals. With MAO enzymes, physiological compounds predominate as substrates, and some products lead to unwanted side effects or illness. AOX and XOR enzymes are molybdenum hydroxylases that catalyze the oxidation of various heteroaromatic rings and aldehydes and the reduction of a number of different functional groups. While neither of these two enzymes contributes substantially to the metabolism of currently marketed drugs, AOX has become a frequently encountered route of metabolism among drug discovery programs in the past 10–15 years. XOR has even less of a role in the metabolism of clinical drugs and preclinical drug candidates than AOX, likely due to narrower substrate specificity.

Masitinib is a small molecule tyrosine kinase inhibitor under investigation for the treatment of amyotrophic lateral sclerosis, mastocytosis, and COVID-19. Hepatotoxicity has been reported in some patients while taking masitinib. The liver injury is thought to involve hepatic metabolism of masitinib by cytochrome P450 (P450) enzymes to form chemically reactive, potentially toxic metabolites. The goal of the current investigation was to determine the P450 enzymes involved in the metabolic activation of masitinib in vitro. In initial studies, masitinib (30 μM) was incubated with pooled human liver microsomes in the presence of NADPH and potassium cyanide to trap reactive iminium ion metabolites as cyano adducts. Masitinib metabolites and cyano adducts were analyzed using reversed-phase liquid chromatography-tandem mass spectrometry. The primary active metabolite, N-desmethyl masitinib (M485), and several oxygenated metabolites were detected along with four reactive metabolite cyano adducts (MCN510, MCN524, MCN526, and MCN538). To determine which P450 enzymes were involved in metabolite formation, reaction phenotyping experiments were conducted by incubation of masitinib (2 μM) with a panel of recombinant human P450 enzymes and by incubation of masitinib with human liver microsomes in the presence of P450-selective chemical inhibitors. In addition, enzyme kinetic assays were conducted to determine the relative kinetic parameters (apparent Km and Vmax) of masitinib metabolism and cyano adduct formation. Integrated analysis of the results from these experiments indicates that masitinib metabolic activation is catalyzed primarily by P450 3A4 and 2C8, with minor contributions from P450 3A5 and 2D6. These findings provide further insight into the pathways involved in the generation of reactive, potentially toxic metabolites of masitinib. Future studies are needed to evaluate the impact of masitinib metabolism on the toxicity of the drug in vivo.

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.

Lapatinib is a dual tyrosine kinase inhibitor associated with rare but potentially severe idiosyncratic hepatotoxicity. We have previously shown that cytochromes P450 CYP3A4 and CYP3A5 quantitatively contribute to lapatinib bioactivation, leading to formation of a reactive, potentially toxic quinone imine. CYP3A5 is highly polymorphic; however, the impact of CYP3A5 polymorphism on lapatinib metabolism has not been fully established. The goal of this study was to determine the effect of CYP3A5 genotype and individual variation in CYP3A activity on the metabolic activation of lapatinib using human-relevant in vitro systems. Lapatinib metabolism was examined using CYP3A5-genotyped human liver microsomes and cryo-preserved human hepatocytes. CYP3A and CYP3A5-selective activities were measured in liver tissues using probe substrates midazolam and T-5 (T-1032), respectively, to evaluate the correlation between enzymatic activity and lapatinib metabolite formation. Drug metabolites were measured by high-performance liquid chromatography-tandem mass spectrometry. Further, the relative contributions of CYP3A4 and CYP3A5 to lapatinib O-debenzylation were estimated using selective chemical inhibitors of CYP3A. The results from this study demonstrated that lapatinib O-debenzylation and quinone imine-GSH conjugate formation were highly correlated with hepatic CYP3A activity, as measured by midazolam 1'-hydroxylation. CYP3A4 played a dominant role in lapatinib bioactivation in all liver tissues evaluated. The CYP3A5 contribution to lapatinib bioactivation varied by individual donor and was dependent on CYP3A5 genotype and activity. CYP3A5 contributed approximately 20%-42% to lapatinib O-debenzylation in livers from CYP3A5 expressers. These findings indicate that individual CYP3A activity, not CYP3A5 genotype alone, is a key determinant of lapatinib bioactivation and likely influences exposure to reactive metabolites. SIGNIFICANCE STATEMENT This study is the first to examine the effect of CYP3A5 genotype, total CYP3A activity, and CYP3A5-selective activity on lapatinib bioactivation in individual human liver tissues. The results of this investigation indicate that lapatinib bioactivation via oxidative O-debenzylation is highly correlated with total hepatic CYP3A activity, and not CYP3A5 genotype alone. These findings provide insight into the individual factors, namely, CYP3A activity, that may affect individual exposure to reactive, potentially toxic metabolites of lapatinib.

1. Failure to predict human pharmacokinetics of aldehyde oxidase (AO) substrates using traditional allometry has been attributed to species differences in AO metabolism. 2. To identify appropriate species for predicting human in vivo clearance by single-species scaling (SSS) or multispecies allometry (MA), we scaled in vitro intrinsic clearance (CLint) of five AO substrates obtained from hepatic S9 of mouse, rat, guinea pig, monkey and minipig to human in vitro CLint. 3. When predicting human in vitro CLint, average absolute fold-error was <= 2.0 by SSS with monkey, minipig and guinea pig (rat/mouse > 3.0) and was 53.0 by most MA species combinations (including rat/mouse combinations). 4. Interspecies variables, including fraction metabolized by AO (F-m,F- AO) and hepatic extraction ratios (E) were estimated in vitro. SSS prediction fold-errors correlated with the animal: human ratio of E (r(2) = 0.6488), but not F-m,F- AO (r(2) = 0.0051). 5. Using plasma clearance (CLp) from the literature, SSS with monkey was superior to rat or mouse at predicting human CLp of BIBX1382 and zoniporide, consistent with in vitro SSS assessments. 6. Evaluation of in vitro allometry, F-m,F- AO and E may prove useful to guide selection of suitable species for traditional allometry and prediction of human pharmacokinetics of AO substrates.

Aldehyde oxidase (AO) and xanthine oxidase (XO) are molybdo-flavoenzymes that catalyze oxidation of aromatic azaheterocycles. Differences in AO activity have been reported among various species, including rats, humans, and monkeys. Herein we report a species difference in the enzymes responsible for the metabolism of the negative allosteric modulator of metabotropic glutamate receptor subtype 5 (mGlu5 NAM) VU0424238 (VU238, auglurant). Hepatic S9 incubations with AO and XO specific inhibitors hydralazine and allopurinol indicated that rats and cynomolgus monkeys both oxidized VU238 to the 6-oxopyrimidine metabolite M1 via an AO-mediated pathway, whereas secondary oxidation to the 2,6-dioxopyrimidine metabolite M2 was mediated predominantly by AO in monkeys and XO in rats. Despite differences in enzymatic pathways, intrinsic clearance (CLint) of M1 was similar between species (cynomolgus and rat CLint = 2.00 ± 0.040 and 2.19 ± 0.201 μl/min per milligram of protein, respectively). Inhibitor studies in the S9 of multiple species indicated that oxidation of VU238 to M1 was mediated predominantly by AO in humans, cynomolgus and rhesus monkeys, rats, mice, guinea pigs, and minipigs. Oxidation of M1 to M2 was mediated predominantly by XO in rats and mice and by AO in monkeys and guinea pigs, whereas low turnover prevented enzyme phenotyping in humans and minipigs. Additionally, inhibitor experiments indicated that oxidation at the 2-position of the pyrimidine ring of the known AO substrate, BIBX1382, was mediated by AO in all species, although production of this metabolite was comparatively low in rats and mice. These data may suggest low reactivity of rat AO toward 2-oxidation of pyrimidine-containing compounds and highlight the importance of thoroughly characterizing AO-metabolized drug candidates in multiple preclinical species.

Marketed drugs cleared by aldehyde oxidase (AO) are few, with no known clinically relevant pharmacokinetic drug interactions associated with AO inhibition, whereas cytochrome P450 (P450) inhibition or induction mediates a number of clinical drug interactions. Little attention has been given to the consequences of coadministering a P450 inhibitor with a compound metabolized by both AO and P450. Upon discovering that VU0409106 (1) was metabolized by AO (to M1) and P450 enzymes (to M4-M6), we sought to evaluate the in vivo disposition of 1 and its metabolites in rats with attenuated P450 activity. Male rats were orally pretreated with the pan-P450 inactivator, 1-aminobenzotriazole (ABT), before an i.p. dose of 1. Interestingly, the plasma area under the curve (AUC) of M1 was increased 15-fold in ABT-treated rats, indicating a metabolic shunt toward AO resulted from the drug interaction condition. The AUC of 1 also increased 7.8-fold. Accordingly, plasma clearance of 1 decreased from 53.5 to 15.3 ml/min per kilogram in ABT-pretreated rats receiving an i.v. dose of 1. Consistent with these data, M1 formation in hepatic S9 increased with NADPH-exclusion to eliminate P450 activity (50% over reactions containing NADPH). These studies reflect possible consequences of a drug interaction between P450 inhibitors and compounds cleared by both AO and P450 enzymes. Notably, increased exposure to an AO metabolite may hold clinical relevance for active metabolites or those mediating toxicity at elevated concentrations. The recent rise in clinical drug candidates metabolized by AO underscores the importance of these findings and the need for clinical studies to fully understand these risks.