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Chlorzoxazone-based One-Sample Method for Estimating In Vivo CYP2E1 Activity in Mice
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DOI：10.2174/0113892002414713251211102027.png)
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Abstract
En 中文
Introduction Cytochrome P450 2E1 (CYP2E1) plays a crucial role in metabolism and disease, making it highly significant to establish a simpler, sensitive method for evaluating its in vivo activity compared to traditional pharmacokinetic (PK) parameters. Methods A high-performance liquid chromatography-ultraviolet (HPLC-UV) method was developed and validated for determining chlorzoxazone (CZX) and its metabolite 6-hydroxy CZX (6-OH CZX) in plasma. Four mouse models with distinct CYP2E1 activity were constructed: high activity induced by isoniazid, and low activity via Q11 (a CYP2E1 inhibitor), Cyp2e1 knockout, or carbon tetrachloride (CCl4). PK experiments were conducted, with activity changes verified by in vitro CYP2E1 protein expression and microsomal activity. Additionally, the sensitivity of PK parameters and the plasma 6-OH CZX/CZX ratio (metabolite ratio, MR) for characterizing CYP2E1 activity, as well as correlations between MR at different time points and both microsomal CYP2E1 activity and CZX half-life (t1/2), were analyzed. Results The HPLC-UV method met analytical requirements in terms of specificity, linearity, and intra-day and inter-day precision. Microsomal activity and protein expression experiments confirmed the successful establishment of the four models. For CYP2E1 activity characterization, CZX t1/2 was more sensitive than its area under the curve (AUC) and clearance (CL); MR values at 15 and 7 minutes outperformed those at 2 minutes, with 15-minute MR showing stronger correlations with microsomal activity (r = 0.57, P = 0.007) and CZX t1/2 (r = 0.83, P < 0.01). Discussion This study addresses limitations of traditional PK parameters (multiple samplings, non-metabolic interference) and existing MR methods (unclear optimal time points). The 15-min MR and CZX t1/2 offer simplified evaluation, with CZX's high CYP2E1 specificity enhancing translation. Limitations include focus on male C57BL/6J mice and single-point MR's inability to reflect dynamic activity. Conclusion Four representative mouse models with distinct CYP2E1 activity were successfully constructed. CZX t1/2 exhibits higher sensitivity and applicability in characterizing in vivo CYP2E1 activity changes, while the 15-minute MR better represents activity changes. This research lays a foundation for characterizing CYP2E1 variations in disease and pathological processes.
Keywords:
activity
microsome
chlorzoxazone
metabolite ratio
mice
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