Twenty-four hour tolbutamide plasma concentration as a phenotypic measure of CYP2C9 activity

Tolbutamide is a useful in vivo probe for phenotyping CYP2C9 activity [1, 2], and various measures of its metabolism to 4-OH-tolbutamide (4-OH) and carboxytolbutamide (COOH) have been shown to significantly differentiate among individuals of diverse CYP2C9 genotype [3, 4]. We previously reported that the cumulative urinary excretion of 4-OH and COOH from 0 h to 12 h (Ae,m,0–12) was the best non-invasive measure of tolbutamide metabolism and CYP2C9 activity [2]. Jetter et al. [5] recently confirmed our finding and also reported that 24-h tolbutamide plasma concentrations (TOL24 h) are significantly influenced by CYP2C9 genotype and correlate with tolbutamide oral clearance [5]. They concluded that TOL24 h represents a useful alternative phenotypic measure of CYP2C9 activity [5]. Since we did not previously consider TOL24 h as a potential CYP2C9 phenotypic measure, we sought to confirm their results with an ad hoc analysis of our study population. The design of our study has been previously reported [2, 3]. Briefly, 16 healthy individuals of diverse CYP2C9 genotype [CYP2C9*1/*1 (n=5), *1/*2 (n=5), *1/*3 (n=5), and *2/*2 (n=1)] received a single 500-mg oral tolbutamide dose. Serial plasma samples were obtained at 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h. Urine was pooled and collected at 0 to 6, 6 to 12, and 12 to 24–h intervals. Tolbutamide, 4-OH, and COOH concentrations were quantified by means of a validated high-performance liquid chromatography method [2, 3]. The plasma concentration–time profile was evaluated using a non-compartmental model. The area under the plasma concentration–time curve (AUC) was calculated using the log-linear trapezoidal method, with extrapolation from the last data point to infinity. The 24-h formation clearance of tolbutamide to 4-OH and COOH (CLform) was calculated by dividing Ae,m,0–24 by plasma AUC0–24. Oral clearance (CLoral) was calculated by dividing tolbutamide dose by AUC0–¥. Correlations between TOL24 h and each of these calculated parameters were determined by orthogonal regression. The TOL24 h was compared across CYP2C9 genotype using analysis of variance, and a post-hoc Tukey test evaluated differences between each genotype. Significant correlations between TOL24 h and both CLform (r=0.87, P<0.001, Fig. 1a) and CLoral (r=0.96, P<0.001, Fig. 1b) were observed. Significant correlations were also detected between TOL24 h and both AUC0–¥ (r=0.98, P<0.001) and Ae,m,0–12 (r=0.80, P<0.001). Importantly, a significant association between CYP2C9 genotype and TOL24 h was observed (r=0.80, P<0.001), with statistically significant differences between CYP2C9*1/*1, *1/*2, and *1/*3 individuals (mean±SD: 5.9±1.0 versus 12.0±4.3 versus 17.7±1.5 lg/ml, P<0.05 between each group), respectively. Differentiation of TOL24 h among individuals of diverse CYP2C9 genotype is also evident in Fig. 1a, b. In summary, our results confirm the findings of Jetter et al. and suggest that TOL24 h represents a useful phenotypic measure of CYP2C9 activity. First, TOL24 h strongly correlated with both CLform and CLoral, and the correlation with CLform (r=0.87) was comparable to the correlation we previously reported between CLform and Ae,m,0–12 (r=0.84, P<0.001) [2]. Second, a significant association between TOL24 h and CYP2C9 genotype was C. R. Lee (&) AE R. L. Hawke Division of Pharmacotherapy and Experimental Therapeutics, University of North Carolina at Chapel Hill, CB# 7360, Kerr Hall, Chapel Hill, NC 27599-7360, USA E-mail: craig_lee@unc.edu Tel.: +1-919-9625006 Fax: +1-919-9620644