The inhibition study of human UDP-glucuronosyltransferases with cytochrome P450 selective substrates and inhibitors

Figures & data

Figure 1.  The structures of the 20 chemicals studied as the inhibitors of human UGTs.

Table 1.  K_m and V_max values for five expressed human UGTs*

Enzyme	Substrate	Michaelis–Menten	Hill equation
		Km or S50 (µM)
UGT1A1	β-Estradiol	21 ± 6	16 ± 2
UGT1A4	Midazolam	13 ± 4	11 ± 0.6
UGT1A6	1-Naphthol	84 ± 7	74 ± 7
UGT2B7	Diclofenac	69 ± 9	94 ± 37
UGT2B10	Diclofenac	56 ± 12	ND

*The enzyme kinetics data were fitted with either Michaelis–Menten model or Hill equation. Each calculated K_m and S₅₀ values represents in the mean ± SE, where ND represents the parameters that could not be determined in this study.

Figure 2.  The enzyme kinetics of expressed human UGT1A1, 2B7, 1A4, and 1A6. UGT1A1-mediated β-estradiol-3-glucuronidation (A), UGT2B7-mediated diclofenac-glucuronidation (B), UGT1A4-mediated midazolam-N-glucuronidation (C), and UGT1A6-mediated 1-naphthol-glucuronidation (D) were fitted with Michaelis–Menten model and Eadie–Hofstee plots, respectively.

Table 2.  Determination of the IC₅₀ values*.

Enzyme/substrate	1A1 β-estradiol (µM)	1A4 midazolam (µM)	1A6 1-naphthol (µM)	2B7 diclofenac (µM)	2B10 diclofenac (µM)
Inhibitor
Phenacetin	>300	>300	>300	>300	>300
Furafylline	>300	>300	>300	>300	>300
A-Naphthoflavone	38 ± 8	23 ± 6	67 ± 8	149 ± 28	>300
Coumarin	>300	>300	>300	>300	>300
S-mephentoin	>300	>300	>300	>300	>300
Bupropion	>300	152 ± 27	>300	>300	88 ± 28
Paclitaxel	20 ± 3	4.9 ± 0.3	74 ± 3	>300	>300
Diclofenac	155 ± 46	192 ± 78	46 ± 5	S**	S**
Tolbutamide	>300	>300	281 ± 69	>300	>300
Sulfaphenazole	>300	>300	>300	>300	>300
Omeprazole	56 ± 7	>300	185 ± 86	>300	>300
Dextromethorphan	>300	89 ± 21	>300	>300	62 ± 18
Quinidine	>300	166 ± 18	>300	>300	>300
Chlorzoxazone	>300	>300	>300	>300	>300
Chlormethiazole	>300	>300	>300	>300	>300
Midazolam	26 ± 5	S**	23 ± 1	147 ± 24	106 ± 6
Testosterone	43 ± 14	Activation	>300	165 ± 10	>300
Cyclosporine A	48 ± 12	23 ± 7	>300	>300	>300
Ketoconazole	4.1 ± 0.8	58 ± 4	19 ± 2	60 ± 8	22 ± 5
Oleandomycin	44 ± 12	59 ± 3	>300	272 ± 19	>300

*Each value represents the mean ± SE µM where >300 means the IC₅₀ value exceed 300 µM was calculated.

**S means the compounds serve as the substrate for the enzyme.

Figure 3.  The inhibition plots of expressed human UGT1A4-mediated midazolam-N-glucuronidation by α-naphthoflavone (A), and paclitaxel (B); UGT1A6-mediated 1-naphthol-glucuronidation by midazolam (C); UGT1A4-mediated midazolam-N-glucuronidation by cyclosporine A (D); UGT1A1-mediated β-estradiol-3-glucuronidation by ketoconazole (E). Their inhibition IC₅₀ values were calculated to be 23 ± 6, 4.9 ± 0.3, 23 ± 1, 23 ± 7, and 4.1 ± 0.8 µM, respectively.

Figure 4.  Eadie–Hofstee plots for inhibitions kinetics of expressed human UGT1A1-mediated β-estradiol-3-glucuronidation by paclitaxel (A), ketoconazole (B), and midazolam (C). The concentrations of inhibitors were as shown in the figures, and the substrate concentrations were set at 5, 10, 20, and 40 µM for β-estradiol.