Detecting reactive drug metabolites for reducing the potential for drug toxicity

Grillo, Mark P.

doi:10.1517/17425255.2015.1048222

Cited by 37 publications

(19 citation statements)

References 107 publications

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“…Most importantly, ribociclib was ready to undergo metabolic activation to form highly reactive intermediate 1,4‐benzenediamine. Reactive metabolites can covalently bind to protein, DNA, RNA and other macromolecules and, therefore, the toxicity (Brink, Pähler, Funk, Schuler, & Schadt, ; Grillo, ; Tang & Lu, ). This metabolic pathway is responsible for the toxicity of ribociclib.…”

Section: Resultsmentioning

confidence: 99%

“…Most importantly, ribociclib was ready to undergo metabolic activation to form highly reactive intermediate 1,4-benzenediamine. Reactive metabolites can covalently bind to F I G U R E 3 Extracted ion chromatogram of ribociclib-glutathione (GSH) adducts (a) in human liver microsomes and their MS 2 spectrum (b) and the proposed fragmentation pathways (c) F I G U R E 4 Proposed mechanism of the formation of GSH adducts in human liver microsomes in the presence of GSH protein, DNA, RNA and other macromolecules and, therefore, the toxicity (Brink, Pähler, Funk, Schuler, & Schadt, 2016;Grillo, 2015;Tang & Lu, 2010). This metabolic pathway is responsible for the toxicity of ribociclib.…”

Section: Metabolic Profiles Of Ribociclibmentioning

confidence: 99%

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Metabolic profiles of ribociclib in rat and human liver microsomes using liquid chromatography combined with electrospray ionization high‐resolution mass spectrometry

Liu

Wu-cheng

et al. 2020

Biomedical Chromatography

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Ribociclib is a highly specific CDK4/6 inhibitor. Determination of the metabolism of ribociclib is required during the drug development stage. In this study, metabolic profiles of ribociclib were investigated using rat and human liver microsomes. Metabolites were structurally identified by liquid chromatography electrospray ionization high‐resolution mass spectrometry operated in positive‐ion mode. The metabolites were characterized by accurate masses, MS2 spectra and retention times. With rat and human liver microsomes, a total of 10 metabolites were detected and further identified. No human‐specific metabolites were detected. The metabolic pathways of ribociclib were oxygenation, demethylation and dealkylation. Most importantly, two glutathione (GSH) adducts were identified in human liver microsomes fortified with GSH. The formation of the GSH adducts was hypothesized to be through the oxidation of electron‐rich 1,4‐benzenediamine to a 1,4‐diiminoquinone intermediate, which is highly reactive and can be trapped by GSH to form stable metabolites. The current study provides an overview of the metabolic profiles of ribociclib in vitro, which will be of great help in understanding the efficacy and toxicity of this drug.

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Section: Resultsmentioning

confidence: 99%

Section: Metabolic Profiles Of Ribociclibmentioning

confidence: 99%

Metabolic profiles of ribociclib in rat and human liver microsomes using liquid chromatography combined with electrospray ionization high‐resolution mass spectrometry

Liu

Wu-cheng

et al. 2020

Biomedical Chromatography

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“…For example, Walgren et al reported in 2005 that 5 out of 6 drugs withdrawn from the U.S. market since 1950 showed evidence for the formation of reactive metabolites. Reactive metabolite formation is, therefore, commonly considered an unwanted drug property . In drug discovery, bioactivation of drugs to reactive metabolites is evaluated in the early drug discovery stage to support the medicinal chemistry campaign in the attempt to design molecules with a low potential for bioactivation …”

Section: Introductionmentioning

confidence: 99%

“…Reactive metabolite formation is, therefore, commonly considered an unwanted drug property. 4,5 In drug discovery, bioactivation of drugs to reactive metabolites is evaluated in the early drug discovery stage to support the medicinal chemistry campaign in the attempt to design molecules with a low potential for bioactivation. [6][7][8][9][10][11] Reactive metabolites trapping is a widely applied assay in pharmaceutical industry to investigate the formation of reactive intermediates or metabolites in a biotransformation experiment.…”

Section: Introductionmentioning

confidence: 99%

A high‐throughput glutathione trapping assay with combined high sensitivity and specificity in high‐resolution mass spectrometry by applying product ion extraction and data‐dependent neutral loss

et al. 2019

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Reactive metabolites are thought to play a pivotal role in the pathogenesis of some drug‐induced liver injury (DILI) and idiosyncratic adverse drug reactions (IADRs), which is of concern to patient safety and has been a cause of drugs being withdrawn from the market place. To identify drugs with a lower propensity for causing DILI and/or IADRs, high‐throughput assays to capture reactive metabolites are required in pharmaceutical industry for early drug discovery risk assessment. We describe the development of an assay to detect glutathione adducts with combined high sensitivity, enhanced specificity, and rapid data analysis. In this assay, compounds were incubated with human liver microsomes and a mixture of 1:1 of GSH (γ‐GluCysGly): GSX(γ‐GluCysGly‐13C215N) in a 96‐well plate format. UPLC‐UV and LTQ Orbitrap XL were employed to detect GSH‐adducts using the following mass spectrometry setups: (a) selected ion monitoring (SIM) at m/z of 274 ± 3 Da in negative mode with in‐source fragmentation (SCID), which enables simultaneously monitoring two characteristic product ions of m/z 272.0888 (γ‐glutamyl‐dehydroalanyl‐glycine) and 275.0926 (γ‐glutamyl‐dehydroalanyl‐glycine‐13C215N); (b) full scan mode for acquisition of exact mass of glutathione adducts; (c) data‐dependent MS2 scan through isotopic matching (M:M + 3.00375 = 1:1) for monitoring neutral loss fragments (144 Da from dehydroalanyl‐glycine) and for structural information of glutathione adducts. This approach was qualified using eight compounds known to form GSH conjugates as reported in the literature. The high sensitivity and specificity were demonstrated in identifying unique CysGly adducts in the case of clozapine, diclofenac, and raloxifene and in identifying GSH‐adducts of fragmented parent molecules in the case of amodiaquine and troglitazone. In addition, LC‐UV chromatograms in the presence or absence of GSH/GSX allowed for identification of the rearranged glutathione adducts without aforementioned characteristic fragment ions. Implement of this assay in drug discovery small molecule programs has successfully guided drug design.

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“…A trapping agent can be co-incubated with the compound of interest in microsomal incubations, thus allowing it to react directly and form stable conjugates with any CYP-mediated reactive metabolites generated. These stable conjugates can then be detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) [ 5 , 10 ]. Recognizing the role of trapping agents in trapping reactive intermediates in close proximity to the CYP active site, it is possible that trapping agents also inhibit CYP activity, potentially modulating the CYP-mediated metabolism of the compound of interest and underestimating the formation of potential reactive metabolites.…”

Section: Introductionmentioning

confidence: 99%

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

Sodhi

Delarosa²,

Halladay³

et al. 2017

IJMS

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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 β-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.

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Detecting reactive drug metabolites for reducing the potential for drug toxicity

Abstract: Information derived from state-of-art mechanistic drug metabolism studies can be used successfully to direct medicinal chemistry towards the synthesis of candidate drugs devoid of bioactivation liabilities, while maintaining desired pharmacology and pharmacokinetic properties.

Cited by 37 publications

References 107 publications

Metabolic profiles of ribociclib in rat and human liver microsomes using liquid chromatography combined with electrospray ionization high‐resolution mass spectrometry

Metabolic profiles of ribociclib in rat and human liver microsomes using liquid chromatography combined with electrospray ionization high‐resolution mass spectrometry

A high‐throughput glutathione trapping assay with combined high sensitivity and specificity in high‐resolution mass spectrometry by applying product ion extraction and data‐dependent neutral loss

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

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