N-DEMETHYLATION OF AMITRIPTYLINE IN-VITRO - ROLE OF CYTOCHROME-P-450 3A (CYP3A) ISOFORMS AND EFFECT OF METABOLIC-INHIBITORS

Biotransformation of amitriptyline (AMI) to its demethylated product n ortriptyline (NT) was studied in vitro with human liver microsomes fro m four different donors, preselected to reflect a range of metabolic r ates. Reaction velocity versus substrate concentration was consistent with a sigmoid V-max model. V-max varied from 0.42 to 3.42 nmol/mg/min , K-m from 33 to 89 mu M AMI. Ketoconazole was a highly potent inhibit or of N-demethylation, with a mean K-i value of 0.11 +/- 0.013 mu M (/- S.D.), whereas quinidine (up to 50 mu M), a CYP2D6 inhibitor, and a lpha-naphthoflavone (up to 5 mu M), a CYP1A2 inhibitor only at low con centrations, showed no effect. All selective serotonin re-uptake inhib itors (SSRIs) tested had an inhibitory effect on the formation of NT, with mean K-l values of 4.37 (+/- 3.38) mu M for sertraline, 5.46 (+/- 1.95) mu M for desmethylsertraline, 9.22 (+/- 3.69) mu M for fluvoxam ine, 12.26 (+/- 5.67) mu M for norfluoxetine, 15.76 (+/- 5.05) mu M fo r paroxetine, and 43.55 (+/- 18.28) mu M for fluoxetine. A polyclonal rabbit antibody against rat liver CYP3A1, in antibody/microsomal prote in ratios varying from 1:1 to 10:1, inhibited N-demethylation of AMI t o an asymptotic maximum of 60%. These results are consistent with seve ral case reports describing impairment of AMI metabolism by SSRIs. Inh ibition of AMI demethylation by low concentrations of ketoconazole and by anti-3A antibody supports an important role for CYP3A isoforms in mediating this reaction.