Inhibition of microsomal cytochromes P450 in rat liver by the tricyclec antidepressant drug desipramine and its primary oxidized metabolites

doi:10.1016/0006-2952(95)02105-1

Biochemical Pharmacology

Volume 51, Issue 1, 12 January 1996, Pages 15-20

https://doi.org/10.1016/0006-2952(95)02105-1 Get rights and content

Abstract

N-Monoalkyl substituted tricyclic antidepressants like desipramine (DES) undergo cytochrome P450 (P450)-mediated biotransformation in liver to produce inhibitory metabolite-intermediate (MI) complexes with the enzyme. However, additional oxidation pathways that generate isolable metabolites have also been identified, so that the relationship between MI complexation and total oxidative metabolism is unclear. The present study investigated the capacity of DES and three putative metabolites (2-hydroxy- and 10-hydroxy-DES and N,N-didesmethylimipramine; DIDES) to elicit MI complexation and inhibit P450-dependent activities in rat liver. MI complexation of P450 was produced by DES, but not with the three metabolites, in NADPH-supplemented microsomes. Consistent with this finding, inhibition of testosterone hydroxylation pathways was enhanced markedly by prior incubation of DES with NADPH and microsomes. Direct addition of DIDES to incubations resulted in significant inhibition of P450 activities (IC₅₀s of 35 and 29 μM against estradiol 6β- and 16α-hydroxylation mediated by P450s 3A2 and 2C11, respectively). Neither 2-hydroxy- nor 10-hydroxy-DES directly inhibited testosterone hydroxylation (IC₅₀s > 100 μM). However, after a preincubation step between these metabolites and NADPH-fortified microsomes, enhanced inhibition of reactions mediated by P450 3A2 and P450 2C11/2A1 was produced by 2-hydroxy-DES and 10-hydroxy-DES, respectively. Metabolism of DES to DIDES and 2-hydroxy-DES was estimated as 7.77 ± 0.48 nmol/mg protein/hr (10-hydroxy-DES was not detected). It is likely that secondary oxidized metabolites derived from 2-hydroxy-DES, as well as the primary metabolite DIDES, may contribute to the inhibition of P450 activity during DES biotransformation. These results indicate that the 2-hydroxy-, 10-hydroxy-, and N-desmethyl-metabolites of DES are not involved in MI complexation, but complexation is not the sole mechanism by which DES inhibits microsomal drug oxidation that may lead to pharmacokinetic drug interactions.

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Cited by (17)

Antidepressants’ effects on testosterone and estrogens: What do we know?
2021, European Journal of Pharmacology
Citation Excerpt :
N-Monoalkyl substituted tricyclic antidepressants like desipramine are known to undergo cytochrome (P450) -mediated biotransformation in the liver to produce inhibitory metabolite-intermediate complexes with the enzyme. An in vitro study indicated that inhibition of testosterone hydroxylation pathways was enhanced by prior incubation of desipramine with NADPH and rat liver microsomes (McNeil and Murray, 1996). There were no preclinical and clinical studies regarding desipramine's effects on testosterone.
Various antidepressants are commonly used to treat depression and anxiety disorders, and sex differences have been identified in their efficacy and side effects. Steroids, such as estrogens and testosterone, both in the periphery and locally in the brain, are regarded as important modulators of these sex differences. This review presents published data from preclinical and clinical studies that measure testosterone and estrogen level changes during and/or after acute or chronic administration of different antidepressants. The majority of studies show an interaction between sex hormones and antidepressants on sexual function and behavior, or in depressive symptom alleviation. However, most of the studies omit to investigate antidepressants’ effects on circulating levels of gonadal hormones. From data reviewed herein, it is evident that most antidepressants can influence testosterone and estrogen levels. Still, the evidence is conflicting with some studies showing an increase, others decrease or no effect. Most studies are conducted in male animals or humans, underscoring the importance of considering sex as an important variable in such investigations, especially as depression and anxiety disorders are more common in women than men. Therefore, research is needed to elucidate the extent to which antidepressants can influence both peripheral and brain levels of testosterone and estrogens, in males and females, and whether this impacts the effectiveness or side effects of antidepressants.
Effect of antidepressant drugs on cytochrome P450 2C11 (CYP2C11) in rat liver
2013, Pharmacological Reports
Citation Excerpt :
On the other hand, some earlier studies showed that tricyclic antidepressants and fluoxetine formed reactive/intermediate metabolites, which irreversibly inactivated a few CYP isoforms (CYP2C11, 2D, 3A, 2A). Such an effect was observed during prolonged incubation in vitro with high concentrations of antidepressants, or after in vivo administration of high doses of the drugs [5, 27, 28, 30, 31], and – in some cases – also after therapeutic concentrations following a 24-h exposure to antidepressants in vivo [11, 18]. Antidepressant drugs increase monoaminergic transmission in the brain, which in turn affects the ‘hypothalamo-pituitary-adrenal’ and ‘hypothalamo- -pituitary (growth hormone)-hepatic’ axes [41, 42]; these axes may then affect the expression of many CYP isoforms [9, 23, 48].
Rat CYP2C11 (besides CYP2C6) can be regarded as a functional counterpart of human CYP2C9. The aim of the present study was to investigate the influence of classic and novel antidepressant drugs on the activity of CYP2C11, measured as a rate of testosterone 2α- and 16α-hydroxylation.
The reaction was studied in control liver microsomes in the presence of antidepressants, as well as in microsomes from rats treated intraperitoneally (ip) with pharmacological doses of the tested drugs (imipramine, amitriptyline, clomipramine, nefazodone – 10 mg/kg ip; desipramine, fluoxetine, sertraline - 5 mg/kg ip; mirtazapine - 3 mg/kg ip) for one day or two weeks (twice a day), in the absence of antidepressants in vitro.
The investigated antidepressant drugs added to control liver microsomes produced certain inhibitory effects on CYP2C11 activity, which were moderate (sertraline, nefazodone and clomipramine: K_i = 39, 56 and 66 μM, respectively), modest (fluoxetine and amitriptyline: K_i = 98 and 108 μM, respectively) or weak (imipramine and desipramine: K_i = 191 and 212 μM, respectively). Mirtazapine had no inhibitory effect on CYP2C11 activity. One-day exposure of rats to the antidepressant drugs did not significantly change the activity of CYP2C11 in liver microsomes; however, imipramine, desipramine and fluoxetine showed a tendency to diminish the activity of CYP2C11. Of the antidepressants studied, only desipramine and fluoxetine administered chronically elevated CYP2C11 activity; those effects were positively correlated with the observed increases in the enzyme protein level.
Three different mechanisms of the antidepressants-CYP2C11 interaction are postulated: 1) a direct inhibition of CYP2C11 shown in vitro by nefazodone, SSRIs and TADs; 2) in vivo inhibition of CYP2C11 produced by one-day treatment with imipramine, desipramine and fluoxetine, which suggests inactivation of the enzyme by reactive metabolites; 3) in vivo induction of CYP2C11 produced by chronic treatment with desipramine and fluoxetine, which suggests their influence on enzyme regulation.
Direct and indirect interactions between antidepressant drugs and CYP2C6 in the rat liver during long-term treatment
2006, European Neuropsychopharmacology
Citation Excerpt :
Earlier studies showed that tricyclic antidepressants and fluoxetine formed reactive/intermediate metabolites, which irreversibly inactivated a few CYP isoforms (CYP2C11, 2D, 3A, 2A). That effect was observed during prolonged incubation in vitro with high antidepressant concentrations or after in vivo administration of high drug doses (Murray and Field, 1992; Bensoussan et al., 1995; Masubuchi et al., 1995; McNeil and Murray, 1996; Murray and Murray, 2003), and in some cases also at therapeutic concentrations in vivo after 24-h exposure to antidepressants (Daniel et al., 2002; Haduch et al., in press). The latter effect persisted during chronic treatment.
The aim of the present study was to investigate the influence of tricyclic antidepressants (TADs: imipramine, amitriptyline, clomipramine, desipramine), selective serotonin reuptake inhibitors (SSRIs: fluoxetine, sertraline) and novel antidepressant drugs (mirtazapine, nefazodone) on the activity of CYP2C6 measured as a rate of warfarin 7-hydroxylation. The reaction was studied in control liver microsomes in the presence of the antidepressants, as well as in microsomes of rats treated intraperitoneally (i.p.) for one day or two weeks with pharmacological doses of the drugs (imipramine, amitriptyline, clomipramine, nefazodone at 10 mg/kg i.p.; desipramine, fluoxetine, sertraline at 5 mg/kg i.p.; mirtazapine at 3 mg/kg i.p.), in the absence of the antidepressants in vitro. Some of the investigated antidepressant drugs added to liver microsomes of control rats inhibited the rate of 7-hydroxylation of warfarin. The obtained K_i values indicated that nefazodone and fluoxetine were the most potent inhibitors of the studied reaction (K_i = 13 and 23 μM, respectively), while tricyclic antidepressants and sertraline were weak in this respect (K_i = 70–127 μM). A one-day (i.e. 24 h) exposure to fluoxetine and mirtazapine resulted in a significant increase in the rate of the 7-hydroxylation of warfarin in rat liver microsomes. The other studied antidepressants did not significantly affect the rate of the CYP2C6-specific reaction. After two-week treatment with the investigated antidepressants, the increase in CYP2C6 activity observed after 24-h exposure to fluoxetine and mirtazapine was more pronounced. Moreover, unlike after one-day exposure, imipramine and sertraline significantly increased the activity of the enzyme. The other tricyclic antidepressants or nefazodone did not produce any significant effect when administered in vivo. The above-described enhancement of CYP2C6 activity correlated positively with the simultaneously observed increases in the enzyme protein level, which indicates the enzyme induction. The studied antidepressants increased the CYP2C6 protein level in the liver microsomes of rats after chronic treatment: imipramine to 174.6 ± 18.3%, fluoxetine to 159.1 ± 13.7%, sertraline to 135.3 ± 11.2% and mirtazapine to 138.4 ± 10.2% of the control. In summary, two different mechanisms of the antidepressant–CYP2C6 interaction have been found to operate in the rat liver: 1) direct inhibition of CYP2C6 shown in vitro mainly for nefazodone and fluoxetine, with their inhibitory effects being somewhat more potent than their action on human CYP2C9; 2) the in vivo induction of CYP2C6 by imipramine, fluoxetine, sertraline and mirtazapine.
The effect of tricyclic antidepressants, selective serotonin reuptake inhibitors (SSRIs) and newer antidepressant drugs on the activity and level of rat CYP3A
2006, European Neuropsychopharmacology
The aim of the present study was to investigate the influence of tricyclic antidepressants (TADs: imipramine, amitriptyline, clomipramine, and desipramine), selective serotonin reuptake inhibitors (SSRIs: fluoxetine and sertraline) and novel antidepressant drugs (mirtazapine and nefazodone) on the activity of CYP3A measured as a rate of testosterone 2β- and 6β-hydroxylation. The reaction was studied in control liver microsomes in the presence of the antidepressants, as well as in microsomes of rats treated intraperitoneally (i.p.) for 1 day or 2 weeks with pharmacological doses of the drugs (imipramine, amitriptyline, clomipramine, nefazodone 10 mg kg⁻ ¹ i.p.; desipramine, fluoxetine, sertraline 5 mg kg⁻ ¹ i.p.; mirtazapine 3 mg kg⁻ ¹ i.p.), in the absence of the antidepressants in vitro. The investigated antidepressants added to control liver microsomes produced some inhibitory effects on CYP3A activity, which were very weak (most of TADs, K_i = 145–212 μM), modest (clomipramine and sertraline, K_i = 67.5 and 62 μM, respectively) or moderate (nefazodone and fluoxetine, K_i = 42 and 43 μM, respectively). Mirtazapine did not display this kind of properties. One-day exposure of rats to TADs substantially decreased the activity of CYP3A in liver microsomes, which was maintained during chronic treatment. The observed decreases in the enzyme activity were in contrast to the increased CYP3A protein level found after chronic treatment with TADs. On the other hand, sertraline increased the activity of the enzyme after its prolonged administration and its effect correlated positively with the observed elevation in CYP3A protein level. Fluoxetine, mirtazapine and nefazodone did not change the activity of CYP3A in liver microsomes after their administration to rats. Three different mechanisms of the antidepressants–CYP3A interaction are postulated: 1) a direct inhibition of CYP3A by nefazodone, SSRIs and clomipramine, shown in vitro, with the inhibitory effect of nefazodone being the strongest, but weaker than the effects of this drug on human CYP3A4; 2) in vivo inhibition of CYP3A produced by 1 day and maintained during chronic treatment with TADs, which suggests inactivation of the enzyme by reactive metabolites; 3) in vivo induction by sertraline of CYP3A produced only by chronic treatment with the antidepressant, which suggests its influence on the enzyme regulation.
Separation and detection methods for covalent drug-protein adducts
2003, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Covalent binding of reactive metabolites of drugs to proteins has been a predominant hypothesis for the mechanism of toxicity caused by numerous drugs. The development of efficient and sensitive analytical methods for the separation, identification, quantification of drug–protein adducts have important clinical and toxicological implications. In the last few decades, continuous progress in analytical methodology has been achieved with substantial increase in the number of new, more specific and more sensitive methods for drug–protein adducts. The methods used for drug–protein adduct studies include those for separation and for subsequent detection and identification. Various chromatographic (e.g., affinity chromatography, ion-exchange chromatography, and high-performance liquid chromatography) and electrophoretic techniques [e.g., sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), two-dimensional SDS–PAGE, and capillary electrophoresis], used alone or in combination, offer an opportunity to purify proteins adducted by reactive drug metabolites. Conventionally, mass spectrometric (MS), nuclear magnetic resonance, and immunological and radioisotope methods are used to detect and identify protein targets for reactive drug metabolites. However, these methods are labor-intensive, and have provided very limited sequence information on the target proteins adducted, and thus the identities of the protein targets are usually unknown. Moreover, the antibody-based methods are limited by the availability, quality, and specificity of antibodies to protein adducts, which greatly hindered the identification of specific protein targets of drugs and their clinical applications. Recently, the use of powerful MS technologies (e.g., matrix-assisted laser desorption/ionization time-of-flight) together with analytical proteomics have enabled one to separate, identify unknown protein adducts, and establish the sequence context of specific adducts by offering the opportunity to search for adducts in proteomes containing a large number of proteins with protein adducts and unmodified proteins. The present review highlights the separation and detection technologies for drug–protein adducts, with an emphasis on methodology, advantages and limitations to these techniques. Furthermore, a brief discussion of the application of these techniques to individual drugs and their target proteins will be outlined.
Metabolism-dependent stimulation of reactive oxygen species and DNA synthesis by cyclosporin A in rat smooth muscle cells
1999, Free Radical Biology and Medicine
The clinical use of the immunosuppressive drug cyclosporin A (CsA) is limited by its side effects, namely hypertension and nephrotoxicity. It has been proposed that reactive oxygen species (ROS) could be involved as mediators of the toxic effects of CsA. Here, we have studied the possible interrelationship between CsA metabolism and production of ROS. Using cultures of rat aortic smooth muscle cells (RASMC), CsA (1 μM) produced a rapid (within 10 min) increase in reactive oxygen species, detected by oxidation of the fluorescent probes 2,7-dichlorofluorescin and dihydrorhodamine-123. DNA synthesis was increased in the presence of CsA as assessed by [³H]thymidine incorporation. The superoxide dismutase inhibitor diethyldithiocarbamate (1 mM) and the iron chelator desferal (5 μM), as well as ketoconazole (1 μM) and troleandomycin (10 μM), inhibitors of the cytochrome P-450 3A, were able to block both effects. High-performance liquid chromatography analysis revealed that RASMC were capable to metabolize CsA to its primary metabolites (AM1, AM9 and AM4N), and that their formation was inhibited by ketoconazole and troleandomycin. Furthermore, mRNAs encoding cytochrome P-450 3A1 and 3A2 were detected in RASMC by reverse transcriptase-polymerase chain reaction. Our data suggest that CsA is metabolized by cytochrome P-450 3A in RASMC producing reactive oxygen species, most likely superoxide and the hydroxyl radical, known to damage lipids and DNA.

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^☆: This work was supported by a grant from the Australian National Health and Medical Research Council.

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Research paperInhibition of microsomal cytochromes P450 in rat liver by the tricyclec antidepressant drug desipramine and its primary oxidized metabolites☆

Abstract

Biochem Pharmacol

Arch Biochem Biophys

J Biol Chem

J Biol Chem

J Biol Chem

Methods Enzymol

Biochem Pharmacol

Biochem Pharmacol

Methods Enzymol

Biochem Pharmacol

In vitro forecasting of drugs that may interfere with codeine bioactivation

Eur J Drug Metab Pharmacokinet

Quinidine inhibits the 2-hydroxylation of imipramine and desipramine but not the demethylation of imipramine

Eur J Clin Pharmacol

Inhibition of desipramine 2-hydroxylation by quinidine and quinine

Clin Pharmacol Ther

Research paper
Inhibition of microsomal cytochromes P450 in rat liver by the tricyclec antidepressant drug desipramine and its primary oxidized metabolites☆