Anna Haduch

Cytochrome P450 mediates dopamine formation in the brain in vivo

J. Neurochem. (2011) 118, 806–815.

The effect of tricyclic antidepressants, selective serotonin reuptake inhibitors (SSRIs) and newer antidepressant drugs on the activity and level of rat CYP3A

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 2beta- and 6beta-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 microM), modest (clomipramine and sertraline, K(i)=67.5 and 62 microM, respectively) or moderate (nefazodone and fluoxetine, K(i)=42 and 43 microM, 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.

Pharmacokinetics and metabolism of thioridazine during co-administration of tricyclic antidepressants

Because of serious side‐effects of thioridazine and tricyclic antidepressants (cardiotoxicity), a possible influence of imipramine and amitriptyline on the pharmacokinetics and metabolism of thioridazine was investigated in a steady state (2‐week treatment) in rats. Imipramine and amitriptyline (5 and 10 mg kg−1 i.p., respectively) elevated 30 and 20 fold, respectively, the concentration of thioridazine (10 mg kg−1 i.p.) and its metabolites (N‐desmethylthioridazine, 2‐sulphoxide, 2‐sulphone, 5‐sulphoxide) in blood plasma. Similar, yet weaker increases in the thioridazine concentration were found in the brain. Moreover, an elevation of thioridazine/metabolite ratios was observed. Imipramine and amitriptyline added to control liver microsomes in vitro inhibited the metabolism of thioridazine via N‐demethylation (an increase in Km), mono‐2‐sulphoxidation (an increase in Km and a decrease in Vmax) and 5‐sulphoxidation (mainly a decrease in Vmax). Amitriptyline was a more potent inhibitor than imipramine of the thioridazine metabolism. The varying concentration ratios of antidepressant/thioridazine in vivo appear to be more important to the final result of the pharmacokinetic interactions than are relative direct inhibitory effects of the antidepressants on thioridazine metabolism observed in vitro. Besides direct inhibition of the thioridazine metabolism, the decreased activity of cytochrome P‐450 towards 5‐sulphoxidation, produced by chronic joint administration of thioridazine and the antidepressants, seems to be relevant to the observed in vivo interaction. The obtained results may also point to inhibition of another, not yet investigated, metabolic pathway of thioridazine, which may be inferred from the simultaneous elevation of concentrations of both thioridazine and the measured metabolites.

Direct and indirect interactions between antidepressant drugs and CYP2C6 in the rat liver during long-term 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 5mg/kg i.p.; mirtazapine at 3mg/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 23microM, respectively), while tricyclic antidepressants and sertraline were weak in this respect (K(i)=70-127microM). A one-day (i.e. 24h) 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.

Inhibition and possible induction of rat CYP2D after short- and long-term treatment with antidepressants

The aim of this study was to investigate the influence of tricyclic antidepressants (imipramine, amitriptyline, clomipramine, desipramine), selective serotonin reuptake inhibitors (SSRIs: fluoxetine, sertraline) and novel antidepressant drugs (mirtazapine, nefazodone) on the activity of CYP2D, measured as a rate of ethylmorphine O‐deethylation. The reaction was studied in control liver microsomes in the presence of the antidepressants, as well as in microsomes of rats treated intraperitoneally for one day or two weeks (twice a day) 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. Antidepressants decreased the activity of the rat CYP2D by competitive inhibition of the enzyme, the potency of their inhibitory effect being as follows: clomipramine (Ki = 14 μM) > sertraline ≈ fluoxetine (Ki = 17 and 16 μM, respectively) > imipramine ≈ amitriptyline (Ki = 26 and 25 μM, respectively) > desipramine (Ki = 44 μM) > nefazodone (Ki = 55 μM) > mirtazapine (Ki = 107 μM). A one‐day treatment with antidepressants caused a significant decrease in the CYP2D activity after imipramine, fluoxetine and sertraline. After prolonged administration of antidepressants, the decreased CYP2D activity produced by imipramine, fluoxetine and sertraline was still maintained. Moreover, amitriptyline and nefazodone significantly decreased, while mirtazapine increased the activity of the enzyme. Desipramine and clomipramine did not produce any effect when administered in‐vivo. The obtained results indicate three different mechanisms of the antidepressants‐CYP2D interaction: firstly, competitive inhibition of CYP2D shown in‐vitro, the inhibitory effects of tricyclic antidepressants and SSRIs being stronger than those of novel drugs; secondly, in‐vivo inhibition of CYP2D produced by both one‐day and chronic treatment with tricyclic antidepressants (except for desipramine and clomipramine) and SSRIs, which suggests inactivation of the enzyme apoprotein by reactive metabolites; and thirdly, in‐vivo inhibition by nefazodone and induction by mirtazapine of CYP2D produced only by chronic treatment with the drugs, which suggests their influence on the enzyme regulation.

Inhibition of rat liver CYP2D in vitro and after 1-day and long-term exposure to neuroleptics in vivo–possible involvement of different mechanisms

The aim of the present study was to investigate the influence of classic and atypical neuroleptics on the activity of rat CYP2D measured as a rate of ethylmorphine O-deethylation. The reaction was studied in control liver microsomes in the presence of neuroleptics, as well as in microsomes of rats treated intraperitoneally (i.p.) for 1-day or 2-weeks (twice a day) with pharmacological doses of the drugs (promazine, levomepromazine, thioridazine, perazine 10 mg kg(-1); chlorpromazine 3 mg kg(-1); haloperidol 0.3 mg kg(-1); risperidone 0.1 mg kg(-1); sertindole 0.05 mg kg(-1)), in the absence of the neuroleptics in vitro. Neuroleptics added in vitro to control liver microsomes decreased the activity of the rat CYP2D by competitive or mixed inhibition of the enzyme. Thioridazine (Ki=15 microM) was the most potent inhibitor of the rat CYP2D among the drugs studied, whose effect was more pronounced than that of the other neuroleptics tested: phenothiazines (Ki=18-23 microM), haloperidol (Ki=32 microM), sertindole (Ki=51 microM) or risperidone (Ki=165 microM). The investigated neuroleptics-when given to rats in vivo-also seemed to exert an inhibitory effect on CYP2D via other mechanisms. One-day exposure of rats to the classic neuroleptics decreased the activity of CYP2D in rat liver microsomes. After chronic treatment with the investigated neuroleptics, the decreased CYP2D activity produced by the phenothiazines was still maintained, while that caused by haloperidol diminished. Moreover, risperidone decreased the activity of that enzyme. The obtained results indicate drug- and time-dependent interactions between the investigated neuroleptics and the CYP2D subfamily of rat cytochrome P-450, which may proceed via different mechanisms: (1) competitive or mixed inhibition of CYP2D shown in vitro, the inhibitory effects of phenothiazines being stronger than those of haloperidol or atypical neuroleptics, but weaker than the effects of the respective drugs on human CYP2D6; (2) in vivo inhibition of CYP2D, produced by both 1-day and chronic treatment with phenothiazines, which suggests inactivation of enzyme by intermediate metabolites; (3) in vivo inhibition of CYP2D by risperidone, produced only by chronic treatment with the drug, which suggests its influence on the enzyme regulation.

The catalytic competence of cytochrome P450 in the synthesis of serotonin from 5-methoxytryptamine in the brain: An in vitro study

Brain serotonin has been implicated in the pathophysiology of a wide spectrum of psychiatric disorders, as well as in the mechanism of action of psychotropic drugs. The aim of present study was to identify rat cytochrome P450 (CYP) isoforms which can catalyze the O-demethylation of 5-methoxytryptamine to serotonin, and to find out whether that alternative pathway of serotonin synthesis may take place in the brain. The study was conducted on cDNA-expressed CYPs (rat CYP1A1/2, 2A1/2, 2B1, 2C6/11/13, 2D1/2/4/18, 2E1, 3A2 and human CYP2D6), on rat brain and liver microsomes and on human liver microsomes (the wild-type CYP2D6 or the allelic variant 2D6*4*4). Of the rat CYP isoforms studied, CYP2D isoforms were the most efficient in catalyzing the O-demethylation of 5-methoxytryptamine to serotonin, but they were less effective than the human isoform CYP2D6. Microsomes from different brain regions were capable of metabolizing 5-methoxytryptamine to serotonin. The reaction was inhibited by the specific CYP2D inhibitors quinine and fluoxetine. Human liver microsomes of the wild-type CYP2D6 metabolized 5-methoxytryptamine to serotonin more effectively than did the defective CYP2D6*4*4 ones. The obtained results indicate that rat brain CYP2D isoforms catalyze the formation of serotonin from 5-methoxytryptamine, and that the deficit or genetic defect of CYP2D may affect serotonin metabolism in the brain. The results are discussed in the context of their possible physiological and pharmacological significance in vivo.

Effect of classic and atypical neuroleptics on cytochrome P450 3A (CYP3A) in rat liver

BACKGROUND Cytochrome P450 3A (CYP3A) subfamily is involved in the metabolism of xenobiotics (e.g., drugs) and endogenous substances (e.g., steroids). The aim of the present study was to investigate the influence of classic and atypical neuroleptics on the level and activity of CYP3A in rat liver, measured as a rate of testosterone 2β- and 6β-hydroxylation. METHODS The reactions were studied in control liver microsomes in the presence of neuroleptics, as well as in the microsomes of rats treated intraperitoneally (ip) with pharmacological doses of the drugs (promazine and thioridazine 10 mg/kg; chlorpromazine 3 mg/kg; haloperidol 0.3 mg/kg; risperidone 0.1 mg/kg; sertindole 0.05 mg/kg) for one day or two weeks (twice a day), in the absence of the neuroleptics in vitro. RESULTS The investigated neuroleptics added in vitro to control liver microsomes produced a moderate or week inhibitory effects on CYP3A activity. After one-day exposure of rats to neuroleptics, only chlorpromazine significantly increased the activity of CYP3A. Chronic treatment of rats with thioridazine diminished the protein level and activity of CYP3A, while risperidone induced this enzyme. CONCLUSION The observed changes in the CYP3A expression after prolonged exposition to neuroleptics suggest their influence on the enzyme regulation.

The contribution of cytochrome P-450 isoenzymes to the metabolism of phenothiazine neuroleptics.

The aim of the present study was to determine optimum conditions for studying promazine and perazine metabolism in rat liver microsomes, and to investigate the influence of specific cytochrome P-450 inhibitors on 5-sulfoxidation and N-demethylation of these neuroleptics. Based on the developed method, the metabolism of neuroleptics in liver microsomes was studied at linear dependence of product formation on time, and protein and substrate concentrations (incubation time: 10 min; concentration of microsomal proteins: promazine-0.7 mg ml(-1), perazine-0.5 mg ml(-1); substrate concentrations: promazine-25, 40 and 75 nmol ml(-1), perazine-20, 35, 50 nmol ml(-1)). A Dixon analysis of the metabolism of neuroleptics showed that quinine (a CYP2D1 inhibitor), metyrapone (a CYP2B1/B2 inhibitor) and alpha-naphthoflavone (a CYP1A1/2 inhibitor) affected, whereas erythromycin (a CYP3A inhibitor) and sulfaphenazole (a CYP2C inhibitor) did not change the neuroleptic biotransformation. N-Demethylation of promazine was competitively inhibited by quinine (K(i)=20 microM) and metyrapone (K(i)=83 microM), while that of perazine-by quinine (K(i)=46.5 microM), metyrapone (K(i)=46 microM) and alpha-naphthoflavone (K(i)=78.8 microM). 5-Sulfoxidation of promazine was inhibited only by quinine (K(i)=28.6 microM), whereas that of perazine-by quinine (K(i)=10 microM) and metyrapone (K(i)=96 microM). The results obtained are compared with our previous findings of analogous experiments concerning thioridazine, and with the data on other phenothiazines and species. In summary, it is proposed that N-demethylation of the mentioned phenothiazine neuroleptics in the rat is catalyzed by the isoenzymes CYP2D1, CYP2B2 and CYP1A2 (CYP1A2 does not refer to promazine). 5-Sulfoxidation of these drugs may be mediated by different isoenzymes, e.g. CYP2D1 (promazine and perazine), CYP2B2 (perazine) and CYP1A2 (thioridazine). Isoenzymes belonging to subfamilies CYP2C and CYP3A do not seem to be involved in the metabolism of the investigated neuroleptics in the rat. The results obtained point to the drug structure and species differences in the contribution of cytochrome P-450 isoenzymes to the metabolism of phenothiazines.

Disposition of 1,2,3,4,-tetrahydroisoquinoline in the brain of male Wistar and Dark Agouti rats

Direct evidence for accumulation of 1,2,3,4-tetrahydroisoquinoline (TIQ), an endo- and exogenous substance suspected of producing Parkinsonism in humans, has not yet been shown. This study aimed to examine TIQ disposition in the whole rat brain and in the striatum and substantia nigra (SN). TIQ was administered to male Wistar and Dark Agouti rats (20, 40 and 100 mg/kg i.p.) alone or jointly with specific CYP2D inhibitor quinine (20, 40, 80 mg/kg i.p.), acutely or chronically. TIQ concentration in brain of both strains was several-fold higher than in plasma. The level of its metabolite, 4-OH-TIQ, was very low in the brain and plasma of TIQ-treated Wistar while in those receiving additionally quinine or in Dark Agouti rats, 4-OH-TIQ was absent or negligible. Inhibition of CYP2D catalyzing TIQ 4-hydroxylation in the liver had no influence on TIQ accumulation in the brain. Exogenous TIQ was actively transported from periphery into the brain by the organic cation transporter system, mainly OCT3, and quickly eliminated from it by P-glycoprotein. TIQ accumulation after chronic injection to Wistar rats was short-lasting and limited to SN. High concentration of TIQ in SN induces while in the liver inhibits the nigral and hepatic activity CYP2D, respectively.