Involvement of multiple human cytochromes P450 in the liver microsomal metabolism of astemizole and a comparison with terfenadine

Abstract

Aims: The aims of the present study were to investigate the metabolism of astemizole in human liver microsomes, to assess possible pharmacokinetic drug-interactions with astemizole and to compare its metabolism with terfenadine, a typical H1 receptor antagonist known to be metabolized predominantly by CYP3A4.

Methods: Astemizole or terfenadine were incubated with human liver microsomes or recombinant cytochromes P450 in the absence or presence of chemical inhibitors and antibodies.

Results: Troleandomycin, a CYP3A4 inhibitor, markedly reduced the oxidation of terfenadine (26% of controls) in human liver microsomes, but showed only a marginal inhibition on the oxidation of astemizole (81% of controls). Three metabolites of astemizole were detected in a liver microsomal system, i.e. desmethylastemizole (DES-AST), 6-hydroxyastemizole (6OH-AST) and norastemizole (NOR-AST) at the ratio of 7.4 : 2.8 : 1. Experiments with recombinant P450s and antibodies indicate a negligible role for CYP3A4 on the main metabolic route of astemizole, i.e. formation of DES-AST, although CYP3A4 may mediate the relatively minor metabolic routes to 6OH-AST and NOR-AST. Recombinant CYP2D6 catalysed the formation of 6OH-AST and DES-AST. Studies with human liver microsomes, however, suggest a major role for a mono P450 in DES-AST formation.

Conclusions: In contrast to terfenadine, a minor role for CYP3A4 and involvement of multiple P450 isozymes are suggested in the metabolism of astemizole. These differences in P450 isozymes involved in the metabolism of astemizole and terfenadine may associate with distinct pharmacokinetic influences observed with coadministration of drugs metabolized by CYP3A4.

Figure 1

Proposed metabolic pathways of astemizole in man. *:[¹⁴C] labelled position.

Figure 2

Simple regression analysis of dextromethorphan N-demethylation and the rates of DES-AST formation (a), NOR-AST formation (b) and 6OH-AST formation (c).(a) y = 0.217 x + 87.6, r = 0.459, n = 15; (b) y = 0.071 x + 26.5, r = 0.785, P < 0.05, n = 7; y = 0.237 x + 16.9, r = 0.756, P < 0.01, n = 15.

Figure 3

S–V plot for astemizole metabolism in human liver microsomes. Liver samples HBI pool 94 were used. Astemizole (1–30 µm) was incubated for 20 min with human liver microsome. • Observed data, solid lines: fitted curves. (a) DES-AST formation, (b) NOR-AST formation, (c) 6OH-AST formation.

Figure 4

Effects of P450 isoform-selective inhibitors on [¹⁴C]-astemizole metabolism by human liver microsomes. Liver samples HBI pool 94, H102 and H112 were used. FURA: furafylline (50 µm), COU: coumarin (500 µm), SLF: sulphaphenazole (20 µm), OMP: omeprazole (10 µm), QND: quinidine (10 µm), DDC: diethyldithiocarbamate (20 µm), TAO: troleandomycin (100 µm). FURA, DDC and TAO was preincubated in the presence of human liver microsomes and an NADPH-generating system for 15 min at 37 °C. Data are expressed as percentage activity remaining relative to a control (methanol alone) incubation and represented as mean±s.e. mean. ND: < 20%. (a) DES-AST formation, (b) NOR-AST formation, (c) 6OH-AST formation.

Figure 5

Effects of anti-CYP3A2 antibody on [¹⁴C]-astemizole metabolism by human liver microsomes. Pooled human liver microsomes (HBI pool 94) was used, with preincubation with anti-CYP3A2 antibody for 30 min at room temperature. Data were expressed as percentage activity remaining relative to a control (preimmunoglobulin) incubation. Control activity: astemizole metabolism: 282.5, DES-AST formation: 125.2, NOR-AST formation: 57.0, 6OH-AST formation: 23.5 pmol min⁻¹ mg⁻¹ protein. Limit of detection: 3.2 pmol min⁻¹ mg⁻¹ protein. Astemizole metabolism (▵), DES-AST formation (•), NOR-AST formation (□), 6OH-formation (▪), testosterone 6β-hydroxylation (○).

Figure 6

Effect of troleandomycin on astemizole and terfenadine metabolism by human liver microsomes. Three pooled human liver microsomes were used. troleandomycin (10–200 µm) was preincubated in the presence of human liver microsomes and an NADPH-generating system for 15 min at 37 °C. Data are expressed as percentage activity remaining relative to a control (methanol alone) incubation and represented as mean±s.e. mean of three samples. In control incubations, 31±5% of astemizole and 48±18% of terfenadine were metabolized during 5–60 min. Astemizole (•), terfenadine (○).