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In vitro and in vivo metabolic activation and hepatotoxicity of chlorzoxazone mediated by CYP3A

  • Toxicokinetics and Metabolism
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Abstract

Chlorzoxazone (CZX), a benzoxazolone derivative, has been approved for the treatment of musculoskeletal disorders to relieve localized muscle spasm. However, its idiosyncratic toxicity reported in patients brought attention, particularly for hepatotoxicity. The present study for the first time aimed at the relationship between CZX-induced hepatotoxicity and identification of oxirane intermediate resulting from metabolic activation of CZX. Two N-acetylcysteine (NAC) conjugates (namely M1 and M2) and two glutathione (GSH) conjugates (namely M3 and M4) were detected in rat & human microsomal incubations with CZX (200 μM) fortified with NAC or GSH, respectively. The formation of M1-M4 was NADPH-dependent and these metabolites were also observed in urine or bile of SD rats given CZX intragastrically at 10 mg/kg or 25 mg/kg. NAC was found to attach at C-6’ of the benzo group of M1 by sufficient NMR data. CYPs3A4 and 3A5 dominated the metabolic activation of CZX. The two GSH conjugates were also observed in cultured rat primary hepatocytes after exposure to CZX. Inhibition of CYP3A attenuated the susceptibility of hepatocytes to the cytotoxicity of CZX (10–400 μM). The in vitro and in vivo studies provided solid evidence for the formation of oxirane intermediate of CZX. This would facilitate the understanding of the underlying mechanisms of toxic action of CZX.

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Abbreviations

ABT:

1-Aminobenzotriazole

AHH:

Aryl hydrocarbon hydroxylase

BBR:

Benzbromarone

CE:

Collision energy

CCK-8:

Cell Counting Kit-8

CMC-Na:

Carboxymethylcellulose sodium

CYP:

Cytochrome P450

CZX:

Chlorzoxazone

DDI:

Drug-drug interactions

DMEM:

Dulbeecco’s Modified Eagle Medium

DMSO:

Dimethyl sulfoxide

DP:

Declustering potential

EPI:

Enhanced product ion

FBS:

Fetal bovine serum

FDA:

Food and Drug Administration

GSH:

Glutathione

HLMs:

Human liver microsomes

HMBC:

Long-range proton-carbon heteronuclear multiple bond correlation

HPLC:

High performance liquid chromatography

HRMS:

High-resolution mass spectrometry

HSQC:

Two-dimensional NMR proton-carbon heteronuclear single quantum correlation

IADRs:

Idiosyncratic adverse drug reactions

IDILI:

Idiosyncratic drug-induced liver injury

IS:

Internal standard

KTZ:

Ketoconazole

LC–MS/MS:

Liquid chromatography-tandem mass spectrometry

MRM:

Multiple reaction monitoring

MS:

Mass spectroscopy

MS/MS:

Tandem mass spectrometry

NAC:

N-Acetyl-L-cysteine

NADPH:

Reduced nicotinamide adenine dinucleotide phosphate

NL:

Neutral loss

NMR:

Nuclear magnetic resonance

6-OH-CZX:

6-Hydroxychlorzoxazone

tR :

Retention time

PBS:

Phosphate buffered saline

RLMs:

Rat liver microsomes

SD:

Standard deviation

SMZ:

Sulfamethoxazole

ZOX:

Zoxazolamine

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Authors and Affiliations

  1. Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning, 110016, People’s Republic of China

    Chen Sun, Mingyu Zhang, Chunjing Guan, Ying Peng & Jiang Zheng

  2. State Key Laboratory of Functions and Applications of Medicinal Plants, Key Laboratory of Pharmaceutics of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou, 550004, People’s Republic of China

    Weiwei Li & Jiang Zheng

Authors
  1. Chen Sun
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  2. Mingyu Zhang
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  5. Ying Peng
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  6. Jiang Zheng
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Contributions

Conception and design of the study (CS, YP, JZ); Performance of research and acquisition of data (CS, MZ, CG); Analysis and interpretation of data (CS, MZ, WL); Drafting the article or revising it critically for important intellectual content (CS, WL, YP, JZ). All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Weiwei Li, Ying Peng or Jiang Zheng.

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Sun, C., Zhang, M., Guan, C. et al. In vitro and in vivo metabolic activation and hepatotoxicity of chlorzoxazone mediated by CYP3A. Arch Toxicol 98, 1095–1110 (2024). https://doi.org/10.1007/s00204-023-03674-2

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