Objective: In a previous study of diltiazem (DTZ) pharmacokinetics in renal transplant patients, we speculated that a polymorphic enzyme could be involved in O-demethylation of diltiazem. The aim of this in vitro study was to investigate whether O-demethylation of DTZ is mediated by cytochrome P450-2D6 (CYP2D6).
Methods: DTZ was incubated with transfected human liver epithelial (THLE) cells expressing CYP2D6 (T5-2D6 clone). Metabolism of DTZ was studied over a concentration range of 12.5-400 microM and in the presence of quinidine (a CYP2D6 inhibitor) or erythromycin (a CYP3A4 inhibitor). THLE cells lacking CYP2D6 activity (T5-neo clone) were used as control. The culture medium of the cells, in which DTZ was dissolved, was analysed for DTZ and metabolites prior to and after 8 h of incubation using high-performance liquid chromatography (HPLC, UV detection). Authentic O-demethyl-DTZ (Mx) was not available, and this metabolite was therefore not identifiable.
Results: Desacetyl-O-demethyl-DTZ (M4) was exclusively produced during incubations of DTZ with THLE cells expressing CYP2D6. The rate of M4 formation was described using Michaelis Menten kinetics in the concentration range of DTZ used. Production of M4 was inhibited by quinidine, but not erythromycin. An unidentified chromatographic peak, which was interpreted to be Mx, showed the same pattern of formation as M4 both in absence and presence of inhibitors. N-demethylated metabolites, formed by CYP3A4, were not observed in any of the cell lines.
Conclusion: Evidence was provided in vitro that O-demethylation of DTZ is mediated by the polymorphic isoenzyme CYP2D6. Involvement of CYP2D6 in the metabolism of DTZ may have clinical implications regarding pharmacokinetic variability and interactions.