Characterization of human cytochrome P450 enzymes catalyzing domperidone N-dealkylation and hydroxylation in vitro

Abstract

Aims: To confirm the identity of the major metabolites of domperidone and to characterize the cytochrome P450s (CYPs) involved in their formation.

Methods: Human liver microsomes (HLMs) were used to characterize the kinetics of domperidone metabolism and liquid chromatography-mass spectrometry to identify the products. Isoform-specific chemical inhibitors, correlation analysis and expressed human CYP genes were used to identify the CYPs involved in domperidone oxidation.

Results: In HLMs, domperidone underwent hydroxylation to form 5-hydroxydomperidone (MIII) and N-dealkylation to form 2,3-dihydro-2-oxo-1H-benzimidazole-1-propionic acid (MI) and 5-chloro-4-piperidinyl-1,3-dihydro-benzimidazol-2-one (MII). The formation of all three metabolites (n = 4 HLMs) followed apparent Michaelis-Menten kinetics. The mean Km values for MI, MII and MIII formation were 12.4, 11.9, and 12.6 micro m, respectively. In a panel of HLMs (n = 10), the rate of domperidone (5 microm and 50 microm) metabolism correlated with the activity of CYP3A (r > 0.94; P < 0.0001). Only ketoconazole (1 microm) (by 87%) and troleandomycin (50 microm) (by 64%) inhibited domperidone (5 microm) metabolism in HLMs. Domperidone (5 and 50 microm) hydroxylation and N-dealkylation was catalyzed by expressed CYP3A4 at a higher rate than the other CYPs. CYP1A2, 2B6, 2C8 and 2D6 also hydroxylated domperidone

Conclusions: CYP3A-catalyzed N-dealkylation and aromatic hydroxylation are the major routes for domperidone metabolism. The drug would be expected to demonstrate highly variable bioavailability due to hepatic, and possibly intestinal first-pass metabolism after oral administration. Increased risk of adverse effects might be anticipated during concomitant administration with CYP3A inhibitors, as well as decreased efficacy with inducers of this enzyme.

Figure 1

Total ion counts and selected ion-recording scans by liquid chromatography-mass spectrometry (LC-MS) for domperidone and its metabolites generated from HLMs. (A) represents total ion count (domperidone elutes at 22.2 min, M-III at 13.4 min, M-II at 11.7 min, and M-I at 8.9 min). Chromatograms B-E represent selected ion recording (SIR) scans for domperidone and its metabolites in HLMs incubates. SIRs for MW 426 m/z (B) corresponding to domperidone or 5-chloro-1-[1-[3-(2,3-dihydro-2-oxo-1H-benzimidazol-1-yl) propyl]-4-piperidinyl-1,3-dihydro-2H-benzimidazole-2-one, MW 442 m/z (C) corresponding to 5-hydroxydomperidone (MIII), and MW 252 m/z (D) corresponding to 5-chloro-4-piperidinyl-1,3-dihydro-benzimidazol-2-one (MII), and MW 193 m/z (E) corresponding to 2,3-dihydro-2-oxo-1H-benzimidazole-1-propionic acid (MI)

Figure 2

Representative kinetic plots for the metabolism domperidone by HLMs. (A) Michaelis-Menten plots of apparent formation rates (V) of domperidone metabolites vs domperidone concentrations. (B) The corresponding Eadie-Hofstee plot (V of metabolite vs V/domperidone concentrations). Each data point represents the mean of duplicate measurements. MIII (•), MII (▴), MI (○)

Figure 3

Domperidone metabolism by a panel of characterized HLMs. Incubations from 5 µm (A) and 50 µm (B) domperidone are shown. Data are mean apparent formation rates of domperidone metabolites (pmol min⁻¹ mg⁻¹ protein) of duplicate incubations. MI (▪), MII (), MIII (□)

Figure 4

Inhibition of domperidone (5 µm) metabolism by HLMs. The selective inhibitors used were furafylline (10 µm) for CYP1A2, thioTEPA (50 µm) for CYP2B6, sulfaphenazole (50 µm) for CYP2C9, omeprazole (10 µm) and ticlopidine (5 µm) for CYP2C19, quinidine (1 µm) for CYP2D6, diethyldithiocarbamate (DEDTC, 50 µm) for CYP2E1 and ketoconazole (1 µm) and troleandomycin (50 µm) for CYP3A. Inhibition data are expressed as percent control activity remaining (mean ± s.d. of 3 different experiments in duplicate). MIII (▪), MII (□), MI ()

Figure 5

Dixon plots for the inhibition of domperidone metabolism by ketoconazole in HLMs. Domperidone (2.5, 5, 10 and 15 µm) was incubated at 37 °C for 30 min with HLMs (0.25 mg ml⁻¹) and a NADPH-generating system without or with different concentrations of ketoconazole (0.1, 0.5 and 0.75 µm). 2.5 (•), 5 (○), 10 (▴), 15 ()

Figure 6

Domperidone metabolism by expressed human CYP isoforms. Apparent formation rates of metabolites (pmol min⁻¹ pmol⁻¹ P450) from 5 µm (A) and 50 µm (B) domperidone generated by a panel of expressed CYPs are shown. The data are expressed as mean ± SD (n = 3 different experiments in duplicate). MIII (), MII (▪), MI (□)

Figure 7

Kinetic plots for domperidone metabolism in expressed human CYP3A4. (A) Michaelis-Menten plots of apparent formation rates of metabolites vs domperidone concentrations. (B) The corresponding Eadie-Hofstee plots are displayed. Each data point represents the mean of duplicate measurements. The derived kinetic parameters are shown in Table 1. MIII (•), MII (▴), MI (○)

Figure 8

Proposed human metabolism of domperidone and the forms of CYP involved. M-I is 2,3-dihydro-2-oxo-1H-benzimidazole-1-propionic acid, M-II is 5-chloro-4-piperidinyl-1,3-dihydro-benzimidazol-2-one and M-III is 5-hydroxydomperidone