Binding and Oxidation of Amitriptyline and a Series of its Oxidized Metabolites in Liver Microsomes from Untreated and Phenobarbital-treated Rats

Abstract

1. The tricyclic antidepressant drug amitriptyline and its oxidized metabolites elicit the type I spectral change in rat liver microsomes and have low spectral dissociation constants (K_s-values), indicating that they bind with high affinities to cytochrome P-450. The K_s-values of these substrates are related to their R_F-values on t.l.c.—a low K_s-value corresponding to a high R_F-value and vice versa. This suggests that the ability of these compounds to interact with microsomal P-450 is dependent on their physical-chemical properties

2. The K_s-value of amitriptyline increases with increasing concn. of microsomal protein, and the max. magnitude of the type I spectral change is reached when the molar concn. ratio amitriptyline/P-450 is very low (range: 0.98–2.8). Desmethylnortriptyline elicits the type I spectral change at low concn. (<2 μM), but a type II change at higher concn. (>2 μM). At max. type I change, just before the ‘type II absorbancy peak’ at 434 nm appears, the incubation system contains about one mol of desmethylnortriptyline per mol of P-450.

3. The rate of oxidation of a parent compound is more rapid than that of its oxidized metabolites, and the rates of demethylation of amitriptyline, 10-hydroxy-amitriptyline and nortriptyline correlate with their K_s-values. The 10-hydroxylation of desmethylnortriptyline is very slow at substrate concn which cause the type II spectral change. Upon incubation of 10-hydroxynortriptyline, 10-oxo-nortriptyline was found and identified by mass fragmentography. This might reflect the formation of the metabolite 10,11-dihydroxynortriptyline, which upon derivatization for g.l.c. is dehydrated to 10-oxo-nortriptyline.

4. Phenobarbital treatment of rats increases the rate of demethylation of amitriptyline, but decreases the 10-hydroxylation of nortriptyline. Amitriptyline elicits the type I spectral change in microsomes from phenobarbital-treated animals, as in ‘control’ microsomes, but nortriptyline elicits an atypical type II spectrum. Desmethylnortriptyline elicits only the type II change in the phenobarbital microsomes. The results indicate that phenobarbital induces qualitative alterations of cytochrome P-450 and/or its microenvironment.

5. Since both amitriptyline and its oxidized metabolites seem to interact with cytochrome P-450, it is plausible that these compounds influence each others metabolism by competition for microsomal interaction. Such possible interactions should be kept in mind when analyzing factors influencing the total fate of a drug and its metabolites.