Chlorpropamide 2-hydroxylation is catalysed by CYP2C9 and CYP2C19 in vitro: chlorpropamide disposition is influenced by CYP2C9, but not by CYP2C19 genetic polymorphism

Abstract

Aims: We evaluated the involvement of cytochrome P450 (CYP) isoforms 2C9 and 2C19 in chlorpropamide 2-hydroxylation in vitro and in chlorpropamide disposition in vivo.

Methods: To identify CYP isoforms(s) that catalyse 2-hydroxylation of chlorpropamide, the incubation studies were conducted using human liver microsomes and recombinant CYP isoforms. To evaluate whether genetic polymorphisms of CYP2C9 and/or CYP2C19 influence the disposition of chlorpropamide, a single oral dose of 250 mg chlorpropamide was administered to 21 healthy subjects pregenotyped for CYP2C9 and CYP2C19.

Results: In human liver microsomal incubation studies, the formation of 2-hydroxychlorpropamide (2-OH-chlorpropamide), a major chlorpropamide metabolite in human, has been best described by a one-enzyme model with estimated K(m) and V(max) of 121.7 +/- 19.9 microm and 16.1 +/- 5.0 pmol min(-1) mg(-1) protein, respectively. In incubation studies using human recombinant CYP isoforms, however, 2-OH-chlorpropamide was formed by both CYP2C9 and CYP2C19 with similar intrinsic clearances (CYP2C9 vs. CYP2C19: 0.26 vs. 0.22 microl min(-1) nmol(-1) protein). Formation of 2-OH-chlorpropamide in human liver microsomes was significantly inhibited by sulfaphenazole, but not by S-mephenytoin, ketoconazole, quinidine, or furafylline. In in vivo clinical trials, eight subjects with the CYP2C9*1/*3 genotype exhibited significantly lower nonrenal clearance [*1/*3 vs.*1/*1: 1.8 +/- 0.2 vs. 2.4 +/- 0.1 ml h(-1) kg(-1), P < 0.05; 95% confidence interval (CI) on the difference 0.2, 1.0] and higher metabolic ratios (of chlorpropamide/2-OH-chlorpropamide in urine: *1/*3 vs.*1/*1: 1.01 +/- 0.19 vs. 0.56 +/- 0.08, P < 0.05; 95% CI on the difference - 0.9, - 0.1) than did 13 subjects with CYP2C9*1/*1 genotype. In contrast, no differences in chlorpropamide pharmacokinetics were observed for subjects with the CYP2C19 extensive metabolizer vs. poor metabolizer genotypes.

Conclusions: These results suggest that chlorpropamide disposition is principally determined by CYP2C9 activity in vivo, although both CYP2C9 and CYP2C19 have a catalysing activity of chlorpropamide 2-hydroxylation pathway.

Figure 1

(a) Representative high-performance liquid chromatography elution profile of chloroform extracts of human urine collected after a single 250-mg oral dose of chlorpropamide. (b) Electrospray mass spectrum (positive-ion mode) and structure of 2-OH-chlorpropamide. Mass peaks with m/z 292.9 and 314.8 correspond to MH⁺ and [M + Na⁺] adduct ions, respectively. Experimental conditions were as described under Methods

Figure 2

(a) Formation of 2-OH-chlorpropamide and (b) Eadie–Hofstee plot for formation of 2-OH-chlorpropamide from a 60-min incubation of a representative human liver microsome (HL-20) with chlorpropamide. Each point represents the mean of duplicate incubations

Figure 3

(a) Formation of 2-OH-chlorpropamide from chlorpropamide (100 µm) by various human recombinant CYP isoforms. (b) Michaelis–Menten plots for the kinetics of chlorpropamide 2-hydroxylation obtained from human recombinant CYP2C9 (•) and CYP2C19 (▴) supersomes. Each point represents the mean of duplicate incubations

Figure 4

Inhibition of chlorpropamide 2-hydroxylation in human liver microsomes by CYP isoform-specific inhibitors including furafylline (CYP1A2), sulfaphenazole (CYP2C9), S-mephenytoin (CYP2C19), quinidine (CYP2D6), and ketoconazole (CYP3A4). Each bar represents the mean value obtained from three different liver microsomes (HL-20, -21, and -23)

Figure 5

Effect of chlorpropamide on phenacetin O-deethylation for CYP1A2 (○, acetaminophen), tolbutamide 4-hydroxylation for CYP2C9 (•, 4-hydroxytolbutamide), S-mephenytoin 4′-hydroxylation for CYP2C19 (▾, 4′-hydroxymephenytoin), dextromethorphan O-demethylation for CYP2D6 (□, dextrorphan), and midazolam 1-hydroxylation for CYP3A4 (♦, 1-hydroxymidazolam). Each data point represents the mean value obtained from three different liver microsomes (HL-20, -21, and -23)

Figure 6

Mean plasma chlorpropamide concentration–time profile after a single oral administration of chlorpropamide (250 mg) in 13 healthy subjects with CYP2C9*1/*3:CYP2C19 extensive metabolizer (EM) or poor metabolizer (PM) genotypes (○) vs. eight subjects with CYP2C9*1/*1:CYP2C19 EM genotypes (•). (Inset) Mean plasma chlorpropamide concentration–time profile for six subjects with CYP2C9*1/*1:CYP2C19 EM genotypes (□) vs. seven subjects with CYP2C9*1/*1:CYP2C19 PM genotypes (◊). Each bar indicates the mean ± SE for a specific CYP2C9 and CYP2C19 genotype

Figure 7

Scatter plots of (a) AUC_inf, (b) metabolic ratio (MR = molar amount of chlorpropamide/2-hydroxychlopropamide in urine for 24 h after dosing), and (c) nonrenal clearance (CL_NR) estimated from a single, 250-mg oral dose of chlorpropamide in subjects with different CYP2C9 and CYP2C19 genotypes. □, CYP2C9*1/*1:CYP2C19 extensive metabolizer (EM) genotype; s, CYP2C9*1/*1:CYP2C19 poor metabolizer (PM) genotype; ••, mean value for subjects with CYP2C9*1/*1 genotypes; and •, CYP2C9*1/*3:CYP2C19 EM genotype. Each symbol with bar indicates the mean ± SE of a group. The P-value was determined by unpaired t-test between subjects with different CYP2C9 genotypes and was considered to be significant if P < 0.05