Time-dependent inhibition of human drug metabolizing cytochromes P450 by tricyclic antidepressants

AIMS

To investigate time-dependent inhibition (TDI) of human drug metabolizing CYP enzymes by tricyclic antidepressants (TCAs).

METHODS

CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A/CYP3A4 activities were investigated following co- and preincubation with TCAs using human liver microsomes (HLM) and human recombinant CYP proteins (expressed in Escherichia coli) as the enzyme sources. A two-step incubation method was employed to examine the in vitro mechanism-based inactivation (MBI) criteria. Potential metabolite–intermediate complex (MIC) formation was studied by spectral analysis.

RESULTS

TCAs generally exhibited significant TDI of recombinant CYP1A2, CYP2C19 and CYP2D6 (>10% positive inhibition differences between co- and preincubation conditions). TDI of recombinant CYP2C9 was minor (<10%), and was minor or absent in experiments utilizing recombinant CYP3A4 or HLM as the enzyme sources. Where observed, TDI of recombinant CYP occurred via alkylamine MIC formation, but evidence to support similar behaviour in HLM was limited. Indeed, only secondary amine TCAs reduced the apparent P450 content of HLM (3–6%) consistent with complexation. As a representative TCA, nortriptyline fulfilled the in vitro MBI criteria using recombinant CYP2C19 and CYP3A4 (K_I and k_inact values of 4 µm and 0.19 min⁻¹, and 70 µm and 0.06 min⁻¹), but not with the human liver microsomal enzymes.

CONCLUSIONS

TCAs appear to have minimal potential for MBI of human liver microsomal CYP enzymes involved in drug metabolism. HLM and recombinant CYP (expressed in E. coli) are not equivalent enzyme sources for evaluating the TDI associated with some drugs.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• Much of the literature evidence for mechanism-based inactivation (MBI) of CYP by tricyclic antidepressants is limited to studies in rat liver microsomes.
• One report from this laboratory characterized MBI of human recombinant CYP2C8 by nortriptyline.

WHAT THIS STUDY ADDS

• Tricyclic antidepressants form alkylamine metabolite-intermediate complexes with human recombinant CYP enzymes (expressed in Escherichia coli) relatively easily, resulting in time-dependent inhibition.
• Evidence to support similar irreversible inhibition using human liver microsomal (HLM) fractions is limited.
• HLM and recombinant CYP (expressed in E. coli) are not equivalent enzyme sources for evaluating the time-dependent inhibition of human drug metabolizing CYP that is associated with some drugs.