W.A. Daniel

The relative contribution of human cytochrome P450 isoforms to the four caffeine oxidation pathways : An in vitro comparative study with cDNA-expressed P450s including CYP2C isoforms

The aim of the present study was to estimate the relative contribution of cytochrome P450 isoforms (P450s), including P450s of the CYP2C subfamily, to the metabolism of caffeine in human liver. The experiments were carried out in vitro using cDNA-expressed P450s, liver microsomes and specific P450 inhibitors. The obtained results show that (1) apart from the 3-N-demethylation of caffeine - a CYP1A2 marker reaction and the main oxidation pathway of caffeine in man - 1-N-demethylation is also specifically catalyzed by CYP1A2 (not reported previously); (2) 7-N-demethylation is catalyzed non-specifically, mainly by CYP1A2 and, to a smaller extent, by CYP2C8/9 and CYP3A4 (and not by CYP2E1, as suggested previously); (3) C-8-hydroxylation preferentially involves CYP1A2 and CYP3A4 and, to a smaller degree, CYP2C8/9 and CYP2E1 (and not only CYP3A, as suggested previously) at a concentration of 100 microM corresponding to the maximum therapeutic concentration in humans. At a higher caffeine concentration, the contribution of CYP1A2 to this reaction decreases in favour of CYP2C8/9. The obtained data show for the first time the contribution of CYP2C isoforms to the metabolism of caffeine in human liver and suggest that apart from 3-N-demethylation, 1-N-demethylation may also be used for testing CYP1A2 activity. Moreover, they indicate that the C-8-hydroxylation is not exclusively catalyzed by CYP3A4.

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.

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 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 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.

The influence of selective serotonin reuptake inhibitors (SSRIs) on the pharmacokinetics of thioridazine and its metabolites: in vivo and in vitro studies.

Due to its psychotropic profile, thioridazine is a neuroleptic suitable for a combination with antidepressants in a number of complex psychiatric illnesses. However, because of its serious side-effects, such a combination with selective serotonin reuptake inhibitors (SSRIs) which inhibit cytochrome P-450 may be dangerous. The aim of the present study was to investigate a possible impact of SSRIs on the pharmacokinetics and metabolism of thioridazine in a steady state in rats. Thioridazine (10 mg/kg) was injected intraperitoneally, twice a day, for two weeks, alone or jointly with one of the antidepressants (fluoxetine, fluvoxamine or sertraline). Concentrations of thioridazine and its main metabolites (2-sulfoxide = mesoridazine; 2-sulfone = sulforidazine; 5-sulfoxide = ring sulfoxide and N-desmethylthiorid-azine) were assessed in the blood plasma and brain at 30 min, 6 and 12 h after the last dose of the drugs using an HPLC method. Fluoxetine potently increased (up to 13 times!) the concentrations of thioridazine and its metabolites in the plasma, especially after 6 and 12 h. Moreover, an increase in the sum of concentrations of tioridazine + metabolites and thioridazine/metabolite ratios was observed. In vitro studies with control liver microsomes, as well as with microsomes of rats treated chronically with fluoxetine show that the changes in the thioridazine pharmacokinetics may be attributed to the competitive (N-demethylation, Ki = 23 microM) and mixed inhibition (2- and 5-sulfoxidation, Ki = 60 microM and 34 microM, respectively) of thioridazine metabolism by fluoxetine, and to the adaptive changes produced by chronic administration of fluoxetine, as reflected by inhibition of N-demethylation and formation of sulforidazine. Sertraline seemed to have a tendency to decrease thioridazine concentration in vivo, though in vitro studies showed that - like fluoxetine - it competitively or via mixed mechanism inhibited the three metabolic pathways of thioridazine (Ki = 41 microM, 64 microM and 47 microM, respectively). Chronic treatment with sertraline stimulated thioridazine 2- and 5-sulfoxidation, which may be responsible for the observed tendency of sertraline to decrease concentrations of the neuroleptic. In the case of fluvoxamine, a tendency to increase the thioridazine level was observed, which may be connected with the competitive or mixed inhibition of thioridazine N-demethylation and 2-sulfoxidation by the antidepressant (Ki = 17 microM and 167 microM, respectively). Repeated administration of fluvoxamine did not produce any changes in the activity of thioridazine-metabolizing enzymes. In conclusion, of the SSRIs studied, only fluoxetine produces a substantial increase in the thioridazine level in the plasma and brain. In the case of fluvoxamine, a tendency to increase the thioridazine level should be considered. Coadministration of thioridazine and sertraline seems to be safe, though a tendency to decrease the thioridazine level may be expected.