Contributions of human cytochrome P450 enzymes to glyburide metabolism

Abstract

Glyburide (GLB) is a widely used oral sulfonylurea for the treatment of gestational diabetes. The therapeutic use of GLB is often complicated by a substantial inter-individual variability in the pharmacokinetics and pharmacodynamics of the drug in human populations, which might be caused by inter-individual variations in factors such as GLB metabolism. Therefore, there has been a continued interest in identifying human cytochrome P450 (CYP) isoforms that play a major role in the metabolism of GLB. However, contrasting data are available in the present literature in this regard. The present study systematically investigated the contributions of various human CYP isoforms (CYP3A4, CYP3A5, CYP2C8, CYP2C9 and CYP2C19) to in vitro metabolism of GLB. GLB depletion and metabolite formation in human liver microsomes were most significantly inhibited by the CYP3A inhibitor ketoconazole compared with the inhibitors of other CYP isoforms. Furthermore, multiple correlation analysis between GLB depletion and individual CYP activities was performed, demonstrating a significant correlation between GLB depletion and the CYP3A probe activity in 16 individual human liver microsomal preparations, but not between GLB depletion and the CYP2C19, CYP2C8 or CYP2C9 probe activity. By using recombinant supersomes overexpressing individual human CYP isoforms, it was found that GLB could be depleted by all the enzymes tested; however, the intrinsic clearance (V(max)/K(m)) of CYP3A4 for GLB depletion was 4-17 times greater than that of other CYP isoforms. These results confirm that human CYP3A4 is the major enzyme involved in the in vitro metabolism of GLB.

Figure 1. Effects of human CYP isoform inhibitors on glyburide depletion in human liver microsomes

GLB depletion was measured in the absence or presence of selective inhibitors of human CYP isoforms as described. SUL, KTZ, QUI, TTEPA, MOP, OMP, and MTL are relatively selective inhibitors for CYP2C9, CYP3A, CYP2D6, CYP2B6, CYP2A6, CYP2C19, and CYP2C8, respectively. The concentrations of SUL, KTZ, QUI, TTEPA, MOP, OMP, and MTL used were 2.2 μM, 1.0 μM, 0.45 μM, 20 μM, 5.0 μM, 27 μM, and 4.0 μM, respectively. Data shown are means ± S.D. of three independent experiments using three individual human liver microsomal preparations. The amounts of GLB remained undepleted in the negative controls with no NADPH regenerating system added were set as 100%. *Indicates the statistically significant differences with p values < 0.05 as compared with the 100% controls by the Student’s t-test.

Figure 2. Effects of human CYP isoform inhibitors on the formation of glyburide metabolites in human liver microsomes

Four metabolites of GLB were detected in the absence or presence of the CYP inhibitors as described. SUL, KTZ, QUI, TTEPA, MOP, OMP, and MTL are relatively selective inhibitors for CYP2C9, CYP3A, CYP2D6, CYP2B6, CYP2A6, CYP2C19, and CYP2C8, respectively. The concentrations of these inhibitors used were the same as described in Figure 1. The GLB concentration used in the experiments was 1.25 μM. The amount of metabolites formed in the reaction mixtures without any inhibitor added was set as 100%. Shown are means ± S.D. of three independent experiments with three individual human liver microsomal preparations. *Indicates the statistically significant differences with p values < 0.05 as compared with the 100% controls by the Student’s t-test.

Figure 3. Correlation between glyburide depletion and the activity of CYP3A, CYP2C19, CYP2C9, and CYP2C8 assessed by selective probe substrates

GLB depletion and the CYP activities were determined by incubation for 60 min as described. Testosterone 6β-hydroxylation, omeprazole 5-hydroxylation, tolbutamide 4-methylhydrolation, or amodiaquine desethylation was analyzed as the marker activity of CYP3A, CYP2C19, CYP2C9, or CYP2C8, respectively. Shown are means of duplicated experiments with 16 individual human liver microsomal preparations. The correlation coefficients (r²) and p values of statistical analysis are indicated for each CYP isoform.

Figure 4. Glyburide depletion by individually expressed recombinant human CYP isoforms

The amounts of CYP enzymes as shown in “Materials and Methods” were used to get sufficient depletion of GLB. The amounts of GLB remained undepleted in the negative controls (open bars) with no NADPH regenerating system added were set as 100%. Shown are means ± S.D. of three independent experiments. *Indicates the statistically significant differences with p values < 0.05 as compared with the 100% controls by the Students’ t-test.

Figure 5. Determination of K_m and k_{dep([s] ≈ 0)} values of CYP enzymes for glyburide depletion

Shown are determinations of K_m and k_{dep([s] ≈ 0)} for human CYP enzyme-mediated GLB depletion. GLB at six different concentrations (0.5, 1, 2.5, 5, 10, and 15 μM) was incubated with supersomes expressing individual human CYP isoforms (CYP3A4, CYP3A5, CYP2C8, CYP2C19, CYP2C9*1/*1, CYP2C9*2/*2, and CYP2C9*3/*3). Shown are means of triplicated experiments. Experimental details were described in “Materials and Methods”.