Robustness of chlorzoxazone as an in vivo measure of cytochrome P450 2E1 activity

Abstract

Aims: Chlorzoxazone is metabolized by cytochrome P450 2E1 (CYP2E1) to a single oxidized metabolite, 6-hydroxychlorzoxazone. The aim of the study was to test the robustness of chlorzoxazone as an in vivo probe of CYP2E1 activity in humans, with emphasis on investigating short-term and long-term intra-individual variabilities and effects of different doses of the drug. In addition, the influences of body build, drug metabolizing enzyme genotype, blood sampling time, and moderate recent ethanol intake were investigated.

Methods: The 6-hydroxychlorzoxazone:chlorzoxazone (metabolic) ratio in plasma was measured at 2 h in 28 male and nine female volunteers following a single oral dose of 500 mg chlorzoxazone. Similarly, the metabolic ratios at 4 h and 6 h were measured in 20 of the males. The metabolic ratio at 2 h was also determined 1.5 and 2.5 years later in 13 and seven males, respectively, and weekly for 3 weeks in seven males, after a dose of 500 mg, once at higher (750 mg) and lower (250 mg) doses, and once (500 mg) following moderate ethanol intake (0.5 g kg(-1) body weight) the preceding evening. Genotypes were determined for CYP2E1 as well as for N-acetyltransferase 2 and glutathione transferase M1.

Results: Excluding an outlier (ratio = 1.6) the metabolic ratio at 2 h ranged from 0.12 to 0.61 (n = 36). A positive correlation with body weight (r = 0.61, P < 0.001) suggested dose-dependent metabolism of chlorzoxazone. The metabolic ratio decreased with increasing chlorzoxazone dose (P = 0.01), again suggesting dose-dependent metabolism. Long-term (yearly intervals) and short-term (weekly intervals) intra- and interindividual variabilities in metabolic ratio were similar (30% and 63%vs 28% and 54%, respectively). Both inter- and intra-individual variabilities tended to decrease with increasing dose of chlorzoxazone. There was no significant influence of moderate ethanol intake the preceding evening, or of CYP2E1 genotype on the metabolic ratio.

Conclusions: The relatively low intra-individual variability in the metabolism of chlorzoxazone suggests that a single-sample procedure may suffice to assess CYP2E1 activity in vivo. However, chlorzoxazone metabolism is dose-dependent at commonly used doses and it is therefore advisable to adjust the dose for body weight. Moderate intake of ethanol the preceding evening did not significantly affect the chlorzoxazone metabolic ratio.

Figure 1

Relationship between body weight (a), lean body mass (b), body fat mass (c), body mass index (BMI) (d) and the plasma 6-hydroxychlorzoxazone : chlorzoxazone metabolic ratio at 2 h after intake of 500 mg chlorzoxazone (27 males and nine females, experiment 1). The r and P values are from linear regression analysis. One subject (number 28, Table 2) was excluded

Figure 2

Plasma 6-hydroxychlorzoxazone : chlorzoxazone metabolic ratios at different sampling times after intake of 500 mg chlorzoxazone. The horizontal lines represent the 10th, 30th, 50th, 70th, and 90th percentiles (20 males, experiment 2)

Figure 3

The plasma 6-hydroxychlorzoxazone : chlorzoxazone metabolic ratio at 2 h after intake of 500 mg chlorzoxazone at yearly and weekly intervals (seven males)

Figure 4

The plasma 6-hydroxychlorzoxazone : chlorzoxazone metabolic ratio at 2 h after intake of 500 mg chlorzoxazone before and after intake of 0.5 g ethanol kg⁻¹ body weight the preceding evening (seven males, experiments 6 and 7)

Figure 5

Dose-dependence of (a) plasma chlorzoxazone (CZX), (b) plasma 6-hydroxychlorzoxazone (HCZX), and (c–d) the 6-hydroxychlorzoxazone : chlorzoxazone metabolic ratio at 2 h after intake of 250, 500, and 750 mg chlorzoxazone (seven males, experiments 6, 8 and 9, one subject (50 kg bw) was excluded from the experiment with 750 mg chlorzoxazone). The dotted curves in (a) and (b) represent the best fit of the power function (Cp = a × D^b), with exponents of 1.3 and 0.61, respectively. 250 mg (○), 500 mg (), 750 mg (•), curve fit ()