Bioactivation and reactivity research advances - 2021 year in review.

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: see text].

Novel advances in biotransformation and bioactivation research - 2020 year in review.

This annual review is the sixth of its kind since 2016 (see references). Our objective is to explore and share articles which we deem influential and significant in the field of biotransformation and bioactivation. These fields are constantly evolving with new molecular structures and discoveries of corresponding pathways for metabolism that impact relevant drug development with respect to efficacy and safety. Based on the selected articles, we created three sections: (1) drug design, (2) metabolites and drug metabolizing enzymes, and (3) bioactivation and safety (Table 1). Unlike in years past, more biotransformation experts have joined and contributed to this effort while striving to maintain a balance of authors from academic and industry settings.[Table: see text].

Preclinical in vitro and in vivo pharmacokinetic properties of danicamtiv, a new targeted myosin activator for the treatment of dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a disease of the myocardium defined by left ventricular enlargement and systolic dysfunction leading to heart failure. Danicamtiv, a new targeted myosin activator designed for the treatment of DCM, was characterised in in vitro and in vivo preclinical studies. Danicamtiv human hepatic clearance was predicted to be 0.5 mL/min/kg from in vitro metabolic stability studies in human hepatocytes. For human, plasma protein binding was moderate with a fraction unbound of 0.16, whole blood-to-plasma partitioning ratio was 0.8, and danicamtiv showed high permeability and no efflux in a Caco-2 cell line. Danicamtiv metabolism pathways in vitro included CYP-mediated amide-cleavage, N-demethylation, as well as isoxazole- and piperidine-ring-opening. Danicamtiv clearance in vivo was low across species with 15.5, 15.3, 1.6, and 5.7 mL/min/kg in mouse, rat, dog, and monkey, respectively. Volume of distribution ranged from 0.24 L/kg in mouse to 1.7 L/kg in rat. Oral bioavailability ranged from 26% in mouse to 108% in dog. Simple allometric scaling prediction of human plasma clearance, volume of distribution, and half-life was 0.64 mL/min/kg, 0.98 L/kg, and 17.7 h, respectively. Danicamtiv preclinical attributes and predicted human pharmacokinetics supported advancement toward clinical development.

Novel advances in biotransformation and bioactivation research-2019 year in review.

Biotransformation is one of the main mechanisms used by the body to eliminate drugs. As drug molecules become more complicated, the involvement of drug metabolizing enzymes increases beyond those that are typically studied, such as the cytochrome P450 enzymes. In this review, we try to capture the many outstanding articles that were published in the past year in the field of biotransformation (see Table 1). We have divided the articles into two categories of (1) metabolites and drug metabolizing enzymes, and (2) bioactivation and safety. This annual review is the fifth of its kind since 2016 (Baillie et al. 2016; Khojasteh et al. 2017, 2018, 2019). This effort in itself also continues to evolve. We have followed the same format we used in previous years in terms of the selection of articles and the authoring of each section. I am pleased of the continued support of Rietjens, Miller, Zhang, Driscoll and Mitra to this review. We would like to welcome Klarissa D. Jackson as a new author for this year's issue. We strive to maintain a balance of authors from academic and industry settings. We would be pleased to hear your opinions of our commentary, and we extend an invitation to anyone who would like to contribute to a future edition of this review. Cyrus Khojasteh, on behalf of the authors.

Metabolism and Bioactivation: It's Time to Expect the Unexpected.

Improvements in in vitro ADME tools and pharmacokinetic prediction models have helped to shift attrition rates in early clinical trials from poor exposure to drug safety concerns, such as drug-induced liver injury (DILI). Assessing a new chemical entity's potential for liver toxicity is an important consideration for the likely success of new drug candidates. Reactive intermediates produced during drug metabolism have been implicated as a cause of DILI, and their formation has been correlated to the addition of a black box warning on a drug label. In this work, we will present contemporary examples of the bioactivation of atypical structures usually regarded as benign and often used by medicinal chemists when attempting to avoid bioactivation. Medicinal chemistry strategies used to derisk bioactivation will be discussed, and an emphasis will be placed on the necessity of a multidisciplinary approach.

Is there enough evidence to classify cycloalkyl amine substituents as structural alerts?

Basic amine substituents provide several pharmacokinetic benefits relative to acidic and neutral functional groups, and have been extensively utilized as substituents of choice in drug design. On occasions, basic amines have been associated with off-target pharmacology via interactions with aminergic G-protein coupled receptors, ion-channels, kinases, etc. Structural features associated with the promiscuous nature of basic amines have been well-studied, and can be mitigated in a preclinical drug discovery environment. In addition to the undesirable secondary pharmacology, α-carbon oxidation of certain secondary or tertiary cycloalkyl amines can generate electrophilic iminium and aldehyde metabolites, potentially capable of covalent adduction to proteins or DNA. Consequently, cycloalkyl amines have been viewed as structural alerts (SAs), analogous to functional groups such as anilines, furans, thiophenes, etc., which are oxidized to reactive metabolites that generate immunogenic haptens by covalently binding to host proteins. Detailed survey of the literature, however, suggests that cases where preclinical or clinical toxicity has been explicitly linked to the metabolic activation of a cycloalkyl amine group are extremely rare. Moreover, there is a distinct possibility for the formation of electrophilic iminium/amino-aldehyde metabolites with numerous cycloalkyl amine-containing marketed drugs, since stable ring cleavage products have been characterized as metabolites in human mass balance studies. In the present work, a critical analysis of the evidence for and against the role of iminium ions/aldehydes as mediators of toxicity is discussed with a special emphasis on often time overlooked detoxication pathways of these reactive species to innocuous metabolites.

Metabolite Profiling and Reaction Phenotyping for the in Vitro Assessment of the Bioactivation of Bromfenac †.

Bromfenac is a nonsteroidal anti-inflammatory drug that was approved and subsequently withdrawn from the market because of reported cases of acute hepatotoxicity. Recently, in vitro studies have revealed that bromfenac requires UDPGA and alamethicin supplemented human liver microsomes (HLM) to form a major metabolite, bromfenac indolinone (BI). Bromfenac and BI form thioether adducts through a bioactivation pathway in HLM and hepatocytes. [J. P. Driscoll et al., Chem. Res. Toxicol. 2018, 31, 223-230.] Here, Cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) reaction phenotyping experiments using recombinant enzymes were performed on bromfenac and BI to identify the CYP and UGT enzymes responsible for bromfenac's metabolism and bioactivation. It was determined that UGT2B7 converts bromfenac to BI, and that while CYP2C8, CYP2C9, and CYP2C19 catalyze the hydroxylation of bromfenac, only CYP2C9 forms thioether adducts when incubated with NAC or GSH as trapping agents. Although CYP2C9 was shown to form a reactive intermediate, no inhibition of CYP2C9 was observed when an IC50 shift assay was performed. Reaction phenotyping experiments with BI and recombinant CYP enzymes indicated that CYPs 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4 were responsible for the formation of an aliphatic hydroxylated metabolite. An aromatic hydroxylation on the indolinone moiety was also formed by CYP1A2 and CYP3A4. The aromatic hydroxylated BI is a precursor to the quinone methide and quinone imine intermediates in the proposed bioactivation pathway. Through time-dependent inhibition (TDI) experiments, it was revealed that BI can cause an IC50 shift in CYP1A2 and CYP3A4. However, BI does not inhibit the other isoforms that were also responsible for the formation of the aliphatic hydroxylation, an alternative biotransformation that does not undergo further downstream bioactivation. The results of these metabolism studies with bromfenac and BI add to our understanding of the relationship between biotransformation, reactive intermediate generation, and a potential mechanistic link to the hepatotoxicity of this compound.

Biotransformation and bioactivation reactions - 2018 literature highlights.

In the past three decades, ADME sciences have become an integral component of the drug discovery and development process. At the same time, the field has continued to evolve, thus, requiring ADME scientists to be knowledgeable of and engage with diverse aspects of drug assessment: from pharmacology to toxicology, and from in silico modeling to in vitro models and finally in vivo models. Progress in this field requires deliberate exposure to different aspects of ADME; however, this task can seem daunting in the current age of mass information. We hope this review provides a focused and brief summary of a wide array of critical advances over the past year and explains the relevance of this research ( Table 1 ). We divided the articles into categories of (1) drug optimization, (2) metabolites and drug metabolizing enzymes, and (3) bioactivation. This annual review is the fourth of its kind (Baillie et al. 2016 ; Khojasteh et al. 2017 , 2018 ). We have followed the same format we used in previous years in terms of the selection of articles and the authoring of each section. This effort in itself also continues to evolve. I am pleased that Rietjens, Miller, and Mitra have again contributed to this annual review. We would like to welcome Namandjé N. Bumpus, James P. Driscoll, and Donglu Zhang as authors for this year's issue. We strive to maintain a balance of authors from academic and industry settings. We would be pleased to hear your opinions of our commentary, and we extend an invitation to anyone who would like to contribute to a future edition of this review. Cyrus Khojasteh, on behalf of the authors.

In vitro and in vivo pharmacokinetic characterization of mavacamten, a first-in-class small molecule allosteric modulator of beta cardiac myosin.

Mavacamten is a small molecule modulator of cardiac myosin designed as an orally administered drug for the treatment of patients with hypertrophic cardiomyopathy. The current study objectives were to assess the preclinical pharmacokinetics of mavacamten for the prediction of human dosing and to establish the potential need for clinical pharmacokinetic studies characterizing drug-drug interaction potential. Mavacamten does not inhibit CYP enzymes, but at high concentrations relative to anticipated therapeutic concentrations induces CYP2B6 and CYP3A4 enzymes in vitro. Mavacamten showed high permeability and low efflux transport across Caco-2 cell membranes. In human hepatocytes, mavacamten was not a substrate for drug transporters OATP, OCT and NTCP. Mavacamten was determined to have minimal drug-drug interaction risk. In vitro mavacamten metabolite profiles included phase I- and phase II-mediated metabolism cross-species. Major pathways included aromatic hydroxylation (M1), aliphatic hydroxylation (M2); N-dealkylation (M6), and glucuronidation of the M1-metabolite (M4). Reaction phenotyping revealed CYPs 2C19 and 3A4/3A5 predominating. Mavacamten demonstrated low clearance, high volume of distribution, long terminal elimination half-life and excellent oral bioavailability cross-species. Simple four-species allometric scaling led to predicted plasma clearance, volume of distribution and half-life of 0.51 mL/min/kg, 9.5 L/kg and 9 days, respectively, in human.

Role of Glucuronidation and P450 Oxidation in the Bioactivation of Bromfenac.

Bromfenac is a nonsteroidal anti-inflammatory drug that was approved in the United States in 1997. It was withdrawn from clinical use less than one year later, in 1998, due to hepatotoxicity. We investigate the potential of bromfenac to be metabolized to reactive intermediates to further the current understanding of bromfenac bioactivation. Incubations were conducted with hepatocytes and human, rat, and cynomolgus liver microsomes fortified with cofactors and N-acetylcysteine. One thioether adduct of hydroxylated bromfenac and three thioether adducts of hydroxylated bromfenac indolinone were detected in extracts following incubations in liver microsomes fortified with NADPH and UDPGA. These findings demonstrate a bioactivation pathway for bromfenac and contribute to the body of evidence that could advance the understanding of the toxicity associated with bromfenac.

Inhibitory Effects of Trapping Agents of Sulfur Drug Reactive Intermediates against Major Human Cytochrome P450 Isoforms.

In some cases, the formation of reactive species from the metabolism of xenobiotics has been linked to toxicity and therefore it is imperative to detect potential bioactivation for candidate drugs during drug discovery. Reactive species can covalently bind to trapping agents in in vitro incubations of compound with human liver microsomes (HLM) fortified with ß-nicotinamide adenine dinucleotide phosphate (NADPH), resulting in a stable conjugate of trapping agent and reactive species, thereby facilitating analytical detection and providing evidence of short-lived reactive metabolites. Since reactive metabolites are typically generated by cytochrome P450 (CYP) oxidation, it is important to ensure high concentrations of trapping agents are not inhibiting the activities of CYP isoforms. Here we assessed the inhibitory properties of fourteen trapping agents against the major human CYP isoforms (CYP1A2, 2C9, 2C19, 2D6 and 3A). Based on our findings, eleven trapping agents displayed inhibition, three of which had IC50 values less than 1 mM (2-mercaptoethanol, N-methylmaleimide and N-ethylmaleimide (NEM)). Three trapping agents (dimedone, N-acetyl-lysine and arsenite) did not inhibit CYP isoforms at concentrations tested. To illustrate effects of CYP inhibition by trapping agents on reactive intermediate trapping, an example drug (ticlopidine) and trapping agent (NEM) were chosen for further studies. For the same amount of ticlopidine (1 µM), increasing concentrations of the trapping agent NEM (0.007-40 mM) resulted in a bell-shaped response curve of NEM-trapped ticlopidine S-oxide (TSO-NEM), due to CYP inhibition by NEM. Thus, trapping studies should be designed to include several concentrations of trapping agent to ensure optimal trapping of reactive metabolites.

Elucidating the Mechanism of Tofacitinib Oxidative Decyanation.

BACKGROUND: Tofacitinib is known to generate two metabolites M2 (alcohol) and M4 (acid), which are formed as the result of oxidation and loss of the nitrile [1]. METHOD: Systematic in vitro investigation into generation of M2 and M4 from tofacitinib. RESULTS: In vitro using human liver microsomes, we found a new geminal diol metabolite of tofacitinib (MX) that lost the nitrile. MX was further reduced or oxidized to M2 (alcohol) and M4 (acid), respectively by enzymes such as aldo-keto reductase 1C1, aldehyde oxidase and possibly CYP3A4. Stable label studies using H2 18O and D2O suggested the source of oxygen was from water in the media. This was due to rapid water exchange with MX in the media prior to reduction to M2. In case of deuterium, one was incorporated in M2 and this was mainly as a result of tofacitinib rapid exchange of two deuterium atoms from D2O onto methylene position. After formation of MX, there was one deuterium that no longer exchanged with water and therefore retained in M2 for further reduction. CONCLUSION: The proposed mechanism involved the initial oxidation by P450 at the α-carbon to the nitrile group generating an unstable cyanohydrin intermediate; followed by the loss of the nitrile group to form a new geminal diol metabolite (MX).

Going Beyond Common Drug Metabolizing Enzymes: Case Studies of Biotransformation Involving Aldehyde Oxidase, γ-Glutamyl Transpeptidase, Cathepsin B, Flavin-Containing Monooxygenase, and ADP-Ribosyltransferase.

The significant roles that cytochrome P450 (P450) and UDP-glucuronosyl transferase (UGT) enzymes play in drug discovery cannot be ignored, and these enzyme systems are commonly examined during drug optimization using liver microsomes or hepatocytes. At the same time, other drug-metabolizing enzymes have a role in the metabolism of drugs and can lead to challenges in drug optimization that could be mitigated if the contributions of these enzymes were better understood. We present examples (mostly from Genentech) of five different non-P450 and non-UGT enzymes that contribute to the metabolic clearance or bioactivation of drugs and drug candidates. Aldehyde oxidase mediates a unique amide hydrolysis of GDC-0834 (N-[3-[6-[4-[(2R)-1,4-dimethyl-3-oxopiperazin-2-yl]anilino]-4-methyl-5-oxopyrazin-2-yl]-2-methylphenyl]-4,5,6,7-tetrahydro-1-benzothiophene-2-carboxamide), leading to high clearance of the drug. Likewise, the rodent-specific ribose conjugation by ADP-ribosyltransferase leads to high clearance of an interleukin-2-inducible T-cell kinase inhibitor. Metabolic reactions by flavin-containing monooxygenases (FMO) are easily mistaken for P450-mediated metabolism such as oxidative defluorination of 4-fluoro-N-methylaniline by FMO. Gamma-glutamyl transpeptidase is involved in the initial hydrolysis of glutathione metabolites, leading to formation of proximate toxins and nephrotoxicity, as is observed with cisplatin in the clinic, or renal toxicity, as is observed with efavirenz in rodents. Finally, cathepsin B is a lysosomal enzyme that is highly expressed in human tumors and has been targeted to release potent cytotoxins, as in the case of brentuximab vedotin. These examples of non-P450- and non-UGT-mediated metabolism show that a more complete understanding of drug metabolizing enzymes allows for better insight into the fate of drugs and improved design strategies of molecules in drug discovery.

Biotransformation and bioactivation reactions of alicyclic amines in drug molecules.

Aliphatic nitrogen heterocycles such as piperazine, piperidine, pyrrolidine, morpholine, aziridine, azetidine, and azepane are well known building blocks in drug design and important core structures in approved drug therapies. These core units have been targets for metabolic attack by P450s and other drug metabolizing enzymes such as aldehyde oxidase and monoamine oxidase (MAOs). The electron rich nitrogen and/or α-carbons are often major sites of metabolism of alicyclic amines. The most common biotransformations include N-oxidation, N-conjugation, oxidative N-dealkylation, ring oxidation, and ring opening. In some instances, the metabolic pathways generate electrophilic reactive intermediates and cause bioactivation. However, potential bioactivation related adverse events can be attenuated by structural modifications. Hence it is important to understand the biotransformation pathways to design stable drug candidates that are devoid of metabolic liabilities early in the discovery stage. The current review provides a comprehensive summary of biotransformation and bioactivation pathways of aliphatic nitrogen containing heterocycles and strategies to mitigate metabolic liabilities.

Novel mechanism for dehalogenation and glutathione conjugation of dihalogenated anilines in human liver microsomes: evidence for ipso glutathione addition.

The objective of the present study was to investigate the influence of halogen position on the formation of reactive metabolites from dihalogenated anilines. Herein we report on a proposed mechanism for dehalogenation and glutathione (GSH) conjugation of a series of ortho-, meta-, and para-dihalogenated anilines observed in human liver microsomes. Of particular interest were conjugates formed in which one of the halogens on the aniline was replaced by GSH. We present evidence that a (4-iminocyclohexa-2,5-dienylidene)halogenium reactive intermediate (QX) was formed after oxidation, followed by ipso addition of GSH at the imine moiety. The ipso GSH thiol attacks at the ortho-carbon and eventually leads to a loss of a halogen and GSH replacement. The initial step of GSH addition at the ipso position is also supported by density functional theory, which suggests that the ipso carbon of the chloro, bromo, and iodo (but not fluoro) containing 2-fluoro-4-haloanilines is the most positive carbon and that these molecules have the favorable highest occupied molecular orbital of the aniline and the lowest unoccupied orbital from GSH. The para-substituted halogen (chloro, bromo, or iodo but not fluoro) played a pivotal role in the formation of the QX, which required a delocalization of the positive charge on the para-halogen after oxidation. This mechanism was supported by structure-metabolism relationship analysis of a series of dihalogenated and monohalogenated aniline analogues.

Formation of a quinoneimine intermediate of 4-fluoro-N-methylaniline by FMO1: carbon oxidation plus defluorination.

Here, we report on the mechanism by which flavin-containing monooxygenase 1 (FMO1) mediates the formation of a reactive intermediate of 4-fluoro-N-methylaniline. FMO1 catalyzed a carbon oxidation reaction coupled with defluorination that led to the formation of 4-N-methylaminophenol, which was a reaction first reported by Boersma et al. (Boersma et al. (1993) Drug Metab. Dispos. 21 , 218 - 230). We propose that a labile 1-fluoro-4-(methylimino)cyclohexa-2,5-dienol intermediate was formed leading to an electrophilic quinoneimine intermediate. The identification of N-acetylcysteine adducts by LC-MS/MS and NMR further supports the formation of a quinoneimine intermediate. Incubations containing stable labeled oxygen (H(2)(18)O or (18)O(2)) and ab initio calculations were performed to support the proposed reaction mechanism.

Bioactivation of phencyclidine in rat and human liver microsomes and recombinant P450 2B enzymes: evidence for the formation of a novel quinone methide intermediate.

The hypothesis that the psychological side effects associated with the anesthetic phencyclidine (PCP) may be caused by irreversible binding of PCP or its reactive metabolite(s) to critical macromolecules in the brain has resulted in numerous in vitro studies aimed at characterizing pathways of PCP bioactivation. The studies described herein extend the current knowledge of PCP metabolism and provide details on a previously unknown metabolic activation pathway of PCP. Following incubations with NADPH- and GSH-supplemented human and rat liver microsomes and recombinant P450 2B enzymes, two sulfhydryl conjugates with MH+ ions at 547 and 482 Da, respectively, were detected by LC/MS/MS. Shebley et al. [(2006) Drug Metab. Dispos. 34, 375-383] have also observed the GSH conjugate 1 with MH+ at 547 Da in PCP incubations with rat P450 2B1 and rabbit P450 2B4 isoforms fortified with NADPH and GSH. The molecular weight of 1 is consistent with a bioactivation pathway involving Michael addition of the sulfhydryl nucleophile to the putative 2,3-dihydropyridinium metabolite of PCP obtained via a four-electron oxidation of the piperidine ring in the parent compound. The mass spectrum of the novel GSH adduct 2 with an MH+ ion at 482 Da was suggestive of a unique PCP bioactivation pathway involving initial ortho- or para-hydroxylation of the phenyl ring in PCP followed by spontaneous decomposition to piperidine and an electrophilic quinone methide intermediate, which upon reaction with GSH yielded adduct 2. The LC retention times and mass spectral properties of enzymatically generated 2 were identical to those of a reference standard obtained via reaction of GSH with synthetic p-hydroxyPCP in phosphate buffer (pH 7.4, 37 degrees C). 1H NMR and 13C-distortionless enhancement by polarization transfer (DEPT) NMR spectral studies on synthetically generated 2 suggested that the structural integrity of the p-hydroxyphenyl and cyclohexyl rings likely was preserved and that the site of GSH addition was the benzylic carbon joining the two scaffolds. The formation of 2 in human microsomes was reduced upon addition of the dual P450 2C19/P450 2B6 inhibitor (+)- N-3-benzylnirvanol. Consistent with this finding, both recombinant P450 2B6 and P450 2C19 catalyzed PCP bioactivation to 2. In the absence of GSH, synthetic p-hydroxyPCP underwent rapid decomposition (t1/2 approximately 5.2 min) to afford p-hydroxyphenylcyclohexanol and p-hydroxyphenylcyclohexene, presumably via the quinone methide intermediate. Overall, our findings on the facile degradation of synthetic p-hydroxyPCP to yield an electrophilic quinone methide intermediate capable of reacting with nucleophiles, including GSH and water, suggest an inherent instability of the putative phenolic PCP metabolite. Thus, if formed enzymatically in vivo, p-hydroxyPCP may not require further metabolism to liberate the quinone methide, which can then react with macromolecules. To our knowledge, this is the first report of a quinone methide reactive intermediate obtained in human-liver microsomal metabolism of PCP.

Pyrimidine benzamide-based thrombopoietin receptor agonists.

A series of pyrimidine benzamide-based thrombopoietin receptor agonists is described. The lead molecule contains a 2-amino-5-unsubstituted thiazole, a group that has been associated with idiosyncratic toxicity. The potential for metabolic oxidation at C-5 of the thiazole, the likely source of toxic metabolites, was removed by substitution at C-5 or by replacing the thiazole with a thiadiazole. Potency in the series was improved by modifying the substituents on the pyrimidine and/or on the thiazole or thiadiazole pendant aryl ring. In vivo examination revealed that compounds from the series are not highly bioavailable. This is attributed to low solubility and poor permeability.

Piperazinyl CCR1 antagonists--optimization of human liver microsome stability.

The synthesis, biological activity, and pharmacokinetic profile of CCR1 antagonists are described.