Pharmacology Journal

Despite many decades of clinical utilization, the drug interaction potential of tricyclic antidepressants (TCAs), particularity inhibition of drugs eliminated by cytochromes P450 (CYP), is not completely understood. Desipramine is known to cause modest increases in the plasma concentrations of paroxetine and nefazodone whereas imipramine and nortriptyline may precipitate phenytoin toxicity. Amitriptyline increases the metabolic ratio of dextromethorphan/dextrorphan (an in vivo marker for CYP2D6), but has no effect on the metabolism of mephenytoin, a known CYP2C19 substrate. Furthermore, coadministration of amitriptyline or desipramine with moclobemide, another CYP2C19 substrate, is well tolerated. Consistent with these observations, in vitro data confirm TCAs as potent competitive inhibitors of human liver microsomal CYP2D6 CYP2C19 is weakly inhibited and the activities of CYP1A2 and CYP2C9 appear to be unaffected . In vitro data for CYP3A are conflicting. Some authors report inhibition of CYP3A-catalysed reactions by TCAs, whereas others do not.

Additionally, several TCAs have been implicated as time-dependent inhibitors (TDI) of rat CYP. TDI is a key feature of mechanism-based inactivation (MBI) (see Kent et al. for review of in vitro MBI criteria) and a potential cause for concern, since MBI of drug metabolizing CYP frequently results in severe impairment of metabolic clearance and clinically significant drug interactions. Consideration of the metabolic pathways for TCAs provides a theoretical basis for both reversible and irreversible inhibition of CYP enzymes. Nortriptyline and desipramine reduce the apparent P450 content of rat liver microsomes via alkylamine metabolite-intermediate complex (MIC) formation and cause TDI of testosterone hydroxylations, preferentially those catalysed by CYP2C11. In contrast, amitriptyline and imipramine have less effect on apparent P450 content and are relatively weak time-dependent inhibitors of CYP-catalysed oxidations. These differences probably arise because secondary amine TCAs such as nortriptyline and desipramine require fewer oxidations to form the putative MIC forming species (nitrosoalkane derivative). Aromatic hydroxylation of TCAs is also implicated in irreversible inhibition of CYP. Imipramine and desipramine are converted by rat CYP2D2 to reactive epoxy metabolites that covalently bind to the enzyme apoprotein.

When the relationship between TDI and the metabolism of TCAs was further investigated, the hydroxy- and N-desmethyl-metabolites of desipramine were foundto be reversible inhibitors of rat CYP that were not involved in MIC formation. Furthermore, TDI of testosterone 6β-hydroxylation (CYP3A) by nortriptyline could essentially be reversed, a characteristic not evident for the 2α- and 16α-hydroxylation pathways due to complexation of CYP2C11. Several TCAs are also potent competitive inhibitors of CYP2B1/2, but not time-dependent inhibitors. Taken together, these studies have illustrated that reversible and irreversible mechanisms contribute to CYP inhibition during the metabolism of TCAs in rats. Although nortriptyline is known to inactivate human recombinant CYP2C8 and has been estimated to reduce the apparent P450 content of HLM by up to 12%, there have been no formal studies of TDI of human drug metabolizing CYP enzymes by TCAs. We investigated a range of TCAs as time-dependent inhibitors of human CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A/CYP3A4, and compared data generated using HLM and Escherichia coli-expressed recombinant CYP as the enzyme sources.

Chemicals

Phenacetin, paracetamol, (S)-mephenytoin, (S)-4′-hydroxymephenytoin, dextromethorphan, dextrorphan, testosterone, 6β-hydroxytestosterone, amitriptyline, nortriptyline, imipramine, desipramine, protriptyline, clomipramine, norclomipramine, fluvoxamine, sulfaphenazole, quinidine, ciclosporin, clorgyline, troleandomycin, 4-methylumbelliferone, glucose 6-phosphate, glucose 6-phosphate dehydrogenase, β-nicotinamide adenine dinucleotide phosphate (NADPH), potassium ferricyanide, reduced glutathione and superoxide dismutase were purchased from Sigma-Aldrich (Sydney, Australia). Other chemicals were kindly donated by the following sources: torsemide and tolyl methylhydroxytorsemide, Boehringer Mannheim International (Mannheim, Germany); desmethylnortriptyline, F. Hoffman-La Roche (Basel, Switzerland); tolbutamide, Hoechst Australia (Melbourne, Australia); indinavir, Merck Sharp and Dohme (Sydney, Australia); and omeprazole, Astra Zeneca (Molndal, Sweden).

Human liver microsomes and recombinant CYP enzymes

The human liver microsomal and E. coli-expressed recombinant CYP preparations employed in this study were as described in a recent publication from this laboratory. The Flinders Medical Centre Ethics Review Committee approved the use of human liver tissue obtained from the liver ‘bank’ of the Department of Clinical Pharmacology. Microsomes from six livers (H6, H7, H10, H12, H40 and H45) were prepared by differential centrifugation. Inhibition studies were performed utilizing pooled HLM with equal amounts of microsomal protein from each of the six livers. Spectral studies also utilized pooled HLM, and microsomes from individual human livers.

Probe substrates and high-performance liquid chromatography assays

CYP activities were measured using the following ‘probe’ substrate reactions: phenacetin O-deethylation for CYP1A2, torsemide tolyl methylhydroxylation for CYP2C9, (S)-mephenytoin 4′-hydroxylation for CYP2C19, dextromethorphan O-demethylation for CYP2D6, and testosterone 6β-hydroxylation for CYP3A/CYP3A4. Metabolite formation was quantified by reversed-phase high-performance liquid chromatography (HPLC) according to the previously published methods. Assays were optimized to ensure linearity of metabolite formation with respect to protein concentration and incubation time. For all assays, concentrations of metabolites in incubations were determined from standard curves generated using authentic standards. The overall reproducibility of assays was assessed by within-day and between-day coefficients of variation for metabolite formation at high and low concentrations of substrates, and were <10% in each case. As described previously, kinetic analysis of metabolite formation suggested the absence of CYP2C19 and CYP2D6 poor metabolizers in the six liver samples used to prepare microsomes.

Screening for TDI of CYP enzymes

Incubations, in phosphate buffer (0.1 m, pH 7.4), were performed at 37°C using the co- vs. preincubation screening strategy documented in a previous publication from this laboratory (see reference for incubation conditions). Briefly, drugs were coincubated with HLM or recombinant CYP and NADPH-regenerating system in the presence of probe substrates (at a concentration corresponding to the approximate K_m or S₅₀ for metabolite formation), prior to quantification of metabolite formation by HPLC. Drugs were also preincubated with HLM or recombinant CYP and NADPH-regenerating system for 30 min in the absence of probe substrates. Probe substrates were then added so that the final concentration remained as used for the coincubations, and reactions allowed to proceed before analysis of metabolite formation. Stock solutions of TCAs (as hydrochloride salts) were prepared in water. Amitriptyline, nortriptyline, desmethylnortriptyline, protriptyline, imipramine, desipramine, clomipramine and norclomipramine were added to incubation mixtures to give a final concentration of 20 µm. Reversible inhibitors of CYP1A2 (2 µm fluvoxamine), CYP2C9 (2.5 µm sulfaphenazole), CYP2C19 (2 µm omeprazole), CYP2D6 (0.1 µm quinidine) and CYP3A/CYP3A4 (1 µm indinavir), and drugs known to form alkylamine MICs with CYP1A2 (5 µm clorgyline) and CYP3A4 (1 µm troleandomycin), were included for comparison. Concentrations were chosen on the basis of previous literature reports demonstrating CYP inhibition.

Predictivity of TDI for MIC formation between TCAs and recombinant CYP enzymes

Difference spectra were recorded on a Cary 300 double-beam UV-visible spectrophotometer (Varian Inc., Melbourne, Australia). Details of this method were previously reported by Polasek et al.. Spectra were recorded following 15-min incubations of recombinant CYP (1000 pmol ml^−1) with TCAs (200 µm). Furthermore, known reversible inhibitors (negative controls) and alkylamine MIC-forming drugs (positive controls) were subject to analysis following 15-min incubations with selected recombinant CYP preparations at the concentrations described above in Screening for TDI of CYP enzymes. Detection of MIC formation was compared with the inhibition difference results from TDI screening. Prediction was taken here as a positive inhibition difference with corresponding observable MIC formation, or a negative inhibition difference without MIC formation. Predictivity was calculated as the number of correct predictions divided by the total number of predictions. Recombinant CYP and TCA combinations tested were: amitriptyline, nortriptyline, desmethylnortriptyline, protriptyline, imipramine, desipramine, clomipramine and norclomipramine with recombinant CYP1A2 and CYP2C19, and nortriptyline and clomipramine with recombinant CYP2C9, CYP2D6 and CYP3A4. These combinations were selected in order to investigate both positive and negative inhibition differences determined from TDI screening.

Investigation of in vitro MBI criteria using nortriptyline

The inhibition of human liver microsomal and recombinant CYP2C19 and CYP3A/CYP3A4 by nortriptyline was further investigated using a two-step incubation method. Briefly, inactivation assays contained pooled HLM (2.0 mg ml^−1 microsomal protein for measurement of CYP2C19 activity, and 1.0 mg ml^−1 microsomal protein for measurement of CYP3A activity), recombinant CYP2C19 (250 pmol ml^−1), or CYP3A4 (100 pmol ml^−1), NADPH-regenerating system, and nortriptyline (five different concentrations) in phosphate buffer (0.1 m, pH 7.4). Aliquots were removed at selected times and diluted 10-fold to activity assays containing either (S)-mephenytoin (250 µm; CYP2C19; approximately ten times K_m ) or testosterone (250 µm; CYP3A/CYP3A4; approximately seven times S₅₀). Activity assays were allowed to proceed for 45 min (CYP2C19 assay) or 15 min (CYP3A/CYP3A4 assay) before termination and preparation of samples for HPLC analysis. The preincubation times were 0, 15, 30 and 45 min (HLM) or 0, 2.5, 5, 10 and 15 min (recombinant CYP2C19 and CYP3A4). Inactivation of recombinant CYP2C19 and CYP3A4 by nortriptyline was also investigated in the presence of the trapping agents glutathione (2 mm) and superoxide dismutase (1000 U ml^−1), and the respective alternate substrates omeprazole (100 µm) and ciclosporin (10 µm). Inactivation assays containing 10 µm or 50 µm nortriptyline were allowed to proceed for 15 min prior to 10-fold dilution and determination of residual CYP activity. Ultrafiltration of inactivation assays with recombinant CYP was also undertaken to test for irreversible inhibition. In accordance with conventional experimental protocols, control samples in all experiments were prepared in the absence of nortriptyline to correct for the decline in CYP activity attributed to NADPH-regenerating system alone.

Filed under: Drug Interactions