Chlorzoxazone

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.

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.

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).

Several studies have suggested that INH can induce CYP2E1 protein expression and activity[35-37]. The current view is that the induction of CYP450 enzyme activity is primarily due to increased protein expression. Therefore, in the absence of inhibitors, an increase in CYP2E1 protein expression would correspondingly increase activity. For a low CYP2E1 activity model under CCl4 handling conditions, research has shown that the CYP2E1 protein in mice treated with CCl4 significantly decreases[38, 39]. In the absence of other influence, the protein content of CYP2E1 can represent its activity to certain extent. Therefore, CYP2E1 activity was decreased in CCl4 model. Q11, due to its short duration of drug administration, has no effect on CYP2E1 protein. Cyp2e1 knockout results in CYP2E1 deficiency. The changes of CYP2E1 activity in the aforementioned models have also been validated by microsomal and WB experiments. Therefore, the above four models can be considered to represent different change conditions of CYP2E1 activity. Conventionally, AUC, CL, and t1/2 can all be used to characterize the in vivo activity of CYP450 enzymes. However, theoretical analysis reveals that AUC and CL are PK parameters directly influenced by the administered dose. Consequently, when studying subjects with significant weight differences or in human trials using tablet formulations, variations in these parameters may arise due to dosage factors. In contrast, t1/2, primarily related to drug clearance, is less susceptible to dose-dependent effects. Our findings partially support this notion. For instance, in the CCl4 model, in vitro experiments on CYP2E1 protein levels and microsomal activity both indicated a downregulation of CYP2E1 activity. However, among the in vivo parameters, only t1/2 reflected this difference, while AUC and CL did not show significant changes. While the MR of CZX to 6-OH CZX has been employed to characterize CYP2E1 activity in previous studies, investigations systematically exploring the optimal sampling time point are lacking. Some studies utilize time points within the elimination phase, yet the rationale for their selection remains unclear. Furthermore, these methodologies often fail to categorize the nature of CYP2E1 activity changes, thus limiting their applicability under varying conditions. CYP2E1 activity alterations primarily manifest as upregulation or downregulation, with upregulation generally attributed to increased protein expression and downregulation resulting from either inhibitor-mediated activity suppression or decreased protein expression, the latter further subdivided into chemical induction and gene knockout. Therefore, this study established an in vitro model of CYP2E1 activity upregulation and three distinct downregulation models, employing a combined theoretical and experimental approach to address these limitations and provide a more comprehensive framework for future research in this area. The compartmental model encompasses relatively more PK parameters, which can describe the PK process in more detail, but the model is complex with more restrictive assumptions. The non-compartment model is simpler, with fewer restrictive assumptions, and the application is more widespread. Factors influencing the choice of the PK compartment model mainly include the administration method: oral, injection, inhalation, etc.; physicochemical properties of the drugs: the lipophilicity, water solubility, pKa of the drug, etc.; physiological characteristics of the body: body weight, body fat percentage, blood volume, blood flow rate, etc. During the assessment of CYP2E1 activity in this study, the mouse model was used, and the administration method was intravenous injection using CZX as the probe substrate. There were no significant differences in physiological characteristics among the different mouse groups. Therefore, each group was considered as a one-compartment model. In other words, it does not require as many pharmacokinetic parameters as in the multi-compartment model to characterize the activity. Considering the versatility of the compartment model, we finally chose the more universally applicable non-compartment model. From the plasma concentration-time curves of CZX and 6-OH CZX in Q11 and Cyp2e1-/- groups, it is observable that despite the knockout of CYP2E1 in Cyp2e1-/-, the inhibition of CZX metabolism is not as pronounced as in Q11. We speculate that this may be due to compensatory upregulation of other metabolic enzymes, a common occurrence in gene knockout mice[40, 41]. In the Q11 group, it may be due to the strong inhibition of CYP2E1 that most of the 6-OH CZX concentration is below the detection limit. For the convenience of statistical calculations, it is recorded as the lowest concentration of the calibration curve, 0.78 μg/mL. The plasma concentration of 6-OH CZX in the Q11 group increased significantly at 180 min, which may be due to the complete elimination of Q11, which can no longer inhibit CYP2E1, thereby causing CZX to metabolize rapidly. Through theoretical analysis and experimental validation, we ultimately determined that the in vivo CYP2E1 activity is characterized by the CZX-MR at 15 min. However, the complexity of drug disposition within the body (including absorption, distribution, metabolism, excretion, and other processes) and the diversity of factors influencing drug metabolism determines the complexity of evaluating metabolic processes in vivo. This prompts that it is necessary to combine theoretical analysis when extending conclusions to different model applications, validate in more models, or analyze in conjunction with other relevant indicators as necessary.

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.

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.