Interactions between neuroleptics and CYP2C6 in rat liver in vitro and ex vivo study

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1 Pharmacological Reports 2005, 57, ISSN Copyright 2005 by Institute of Pharmacology Polish Academy of Sciences Short communication Interactions between neuroleptics and CYP2C6 in rat liver in vitro and ex vivo study Anna Haduch, Tomasz Ogórka, Jan Boksa, W³adys³awa A. Daniel Department of Pharmacokinetics and Drug Metabolism, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL Kraków, Poland Correspondence: W³adys³awa A. Daniel, Abstract The aim of the present study was to investigate the influence of classic and atypical neuroleptics on the activity of rat CYP2C6 measured as a rate of warfarin 7-hydroxylation. The reaction was studied in control liver microsomes in the presence of neuroleptics, as well as in microsomes of rats treated intraperitoneally for one day or two weeks (twice a day) with pharmacological doses (mg/kg) of the drugs (promazine, levomepromazine, thioridazine, perazine 10, chlorpromazine, haloperidol 0.3, risperidone 0.1, sertindole 0.05), in the absence of the neuroleptics in vitro. Some of the neuroleptics added in vitro to control liver microsomes decreased the activity of CYP2C6. Sertindole and levomepromazine (K E = 25 and 31 µm, respectively) were the most potent inhibitors of the rat CYP2C6 among the drugs studied. Their effects were more pronounced than those of the other phenothiazines tested: thioridazine and chlorpromazine (K E = 88 and 91 µm, respectively), promazine and perazine (K E = 322 and 341 µm, respectively), risperidone (K E = 414 µm) or haloperidol (K E = 606 µm). The investigated neuroleptics when given to rats in vivo for one day or two weeks did not produce any indirect effect on CYP2C6 via other mechanisms, except for levomepromazine, which increased the activity of the enzyme after 24-h exposure. Therefore, the direct inhibitory effect of levomepromazine on CYP2C6 may be attenuated by an indirect mechanism at the beginning of the neuroleptic therapy. In summary, the obtained results show direct inhibitory effects of some phenothiazine neuroleptics and sertindole on the activity of CYP2C6 in vitro in rat liver microsomes. Considering relatively high pharmacological doses and therapeutic concentrations of phenothiazines, it seems that the inhibitory effect of levomepromazine (and other phenothiazines with K E values below 100 µm) found in vitro may be of physiological and pharmacological importance in vivo. Key words: phenothiazines, haloperidol, risperidone, sertindole, CYP2C6, warfarin 7-hydroxylation, liver microsomes, rat, in vitro study, chronic treatment Introduction Although our earlier studies showed that some of neuroleptic drugs administered at pharmacological doses were able to inhibit a few cytochrome P-450 (CYP) isoforms in rats [4, 5], a possible influence of those drugs on the activity of CYP2C6 has not been investigated so far, in spite of the important physiological and pharmacological role of the enzyme. The CYP2C subfamily constitutes the main pool of cytochrome P450 in rat liver (CYP2C 80%). In the liver of male rats, the contribution of CYP2C6 isoform to the total CYP content is approximately 20%. The isoforms of the CYP2C subfamily are mainly engaged in the metabolism of endogenous steroids, retinoid and arachidonic acid, but they are also able to oxidize many drugs [2, 9, 16]. Rat CYP2C6 can be regarded as a counterpart of human CYP2C9. Both these isoforms specifically catalyze the 4-hydroxylation 872 Pharmacological Reports, 2005, 57,

2 Neuroleptic CYP2C6 interactions Anna Haduch et al. of diclofenac and phenytoin and the 7-hydroxylation of warfarin [11 13, 19, 24] and are selectively inhibited by sulfaphenazole [1, 7, 14, 17]. Therefore it seems that like human CYP2C9 rat CYP2C6 may also be of pharmacological importance. These two isoforms are induced by phenobarbital [8, 18]. The aim of our study was to investigate the interaction of classic (haloperidol, phenothiazines with different side chains and aromatic ring substituents) and atypical (risperidone, sertindole) neuroleptics with rat liver CYP2C6. The study was carried out in vitro (direct interaction via binding with cytochrome protein), as well as ex vivo after 24-h exposure and two-week treatment with pharmacological doses of neuroleptics. The obtained results indicate indirect moderate to weak inhibitory effects of some neuroleptic drugs on the activity of CYP2C6 in rat liver. Materials and Methods Drugs and chemicals Promazine and chlorpromazine (hydrochlorides) were provided by Polfa (Jelenia Góra, Poland), thioridazine (hydrochloride) was obtained from Jelfa (Jelenia Góra, Poland), perazine (dimaleate) from Labor (Wroc³aw, Poland), while levomepromazine (maleate) was purchased from Egyt (Budapest, Hungary) and haloperidol was from RBI (Natick MA, USA). Risperidone was donated by Janssen Pharmaceutica (Beerse, Belgium), sertindole by Lundbeck (Copenhagen, Denmark). Warfarin was purchased from Merck (Germany), while 7-hydroxywarfarin was synthesized at our institute [10]. NADP, glucose-6- phosphate and glucose-6-phosphate dehydrogenase were purchased from Sigma (St. Louis, USA). All organic solvents with HPLC purity were supplied by Merck (Darmstadt, Germany). Animal procedures All the experiments with animals were performed in accordance with the Polish governmental regulations (Animals Protection Act, DZ.U , 1997). The experiments were carried out on male Wistar rats ( g) kept under standard laboratory conditions. The investigated neuroleptics were administered intraperitoneally (ip), twice a day for one day or two weeks at the following pharmacological doses (mg/kg): promazine, levomepromazine, thioridazine and perazine 10, chlorpromazine 3, haloperidol 0.3, risperidone 0.1, sertindole The control animals were injected with saline. The rats were sacrificed at 12 h (one-day treatment) or 24 h (two-week treatment) after the drug withdrawal, and liver microsomes were prepared by differential centrifugation in 20 mm Tris/KCl buffer (ph = 7.4), including washing with 0.15 M KCl according to a conventional method. In vitro studies into CYP2C6 activity measurement of the rate of 7-hydroxylation of warfarin in liver microsomes The activity of the CYP2C6 was studied by measurement of the rate of CYP2C6-specific reaction, i.e. 7-hydroxylation of warfarin in the liver microsomes. To distinguish between a direct effect of neuroleptics on the activity of CYP2C6 and the changes produced by their one-day or two-week administrations, three experimental models were used. Model I The experiment was conducted on pooled liver microsomes from three control rats. The rate of 7-hydroxylation of warfarin (warfarin concentration between µm) was assessed in the absence and presence of one of the Tab. 1. The influence of neuroleptics added in vitro to rat liver microsomes on the CYP2C6 activity measured as the rate of 7-hydroxylation of warfarin (Model I). The presented inhibition constants (KE) were calculated using Dixon analysis (Fig. 1A 1D) Neuroleptics (inhibitors) Inhibition of warfarin 7-hydroxylation K E [µm] I. Phenothiazines Promazine 322 Chlorpromazine 91 Levomepromazine 31 Perazine 341 Thioridazine 88 II. Butyrophenones Haloperidol 606 III. Atypical neuroleptics Risperidone 414 Sertindole 25 Pharmacological Reports, 2005, 57,

3 neuroleptics added in vitro (neuroleptic concentration between µm). Model II The experiment was carried out on liver microsomes from rats treated with a neuroleptic for one day. Warfarin was added to the incubation mixture in vitro at a concentration of 60 µm. The 7-hydroxylation of warfarin was studied in the absence of neuroleptics. Model III The experiment investigated liver microsomes from rats subjected to two-week neuroleptic treatment. Warfarin was added to the incubation mixture in vitro at a concentration of 60 µm. The reaction was studied in the absence of neuroleptics. Incubations (Models I, II and III) were carried out in a system containing liver microsomes (1 mg of protein in 1 ml), Tris/KCl buffer (0.1 M, ph = 7.4), MgCl 2 (2.5 mm), NADP (0.33 mm), glucose 6-phosphate (7.85 mm) and glucose-6-phosphate-dehydrogenase (5.6 U in 1 ml). The final incubation volume was 0.5 ml. After a 15-min incubation at 37 o C, the reaction was stopped by adding 10 µl of 70% perchloric acid and then by cooling it down in ice. Determination of the concentration of warfarin and its metabolite 7-hydroxywarfarin in liver microsome Concentrations of warfarin and its metabolite were assessed by the high performance liquid chromatography (HPLC) method according to Lang and Bocker [15]. After incubation, the samples were centrifuged (20 min, 3000 g). An aliquot (20 µl) was injected Fig. 1. Kinetics of the inhibition of 7-hydroxylation of warfarin by chlorpromazine (A), levomepromazine (B), thioridazine (C) and sertindole (D) (Dixon plots). V velocity of the reaction (nmol of 7-hydroxywarfarin/mg protein/min); I concentration of inhibitor (µm). K values are presented E in Table Pharmacological Reports, 2005, 57,

4 Neuroleptic CYP2C6 interactions Anna Haduch et al. A Results and Discussion B Fig. 2. The activity of CYP2C6 after one-day (A) and two-week treatment (B) with neuroleptics (Model II and III, respectively). The activity was measured as the rate of 7-hydroxylation of warfarin in rat liver microsomes. All values are the means ± SEM from 78 animals; ** p < 0.01 (Dunnett s test), compared with control (A: ± pmol of 7-hydroxywarfarin/mg protein/min; B: ± pmol of 7-hydroxywarfarin/mg protein/min). PZ promazine, CPZ chlorpromazine, LVPZ levomepromazine, PER perazine, THIOR thioridazine, HAL haloperidol, RSP risperidone, SERT sertindole into the HPLC system (LaChrom, Merck-Hitachi), equipped with L-7480 fluorescence detector, L-7100 pump and D-7000 System Manager. The analytical column (Supelcosil TM LC-18, 5 µm, mm) was purchased from Supelco (Bellefonte PA, USA). The mobile phase consisted of 0.25% H 3 PO 4 (ph = 2.2) and acetonitrile in proportion 62:38 (v/v). The flow rate was 2.0 ml/min. The column temperature was 25 o C. The fluorescence was measured at a wavelength of 320 nm (excitation) and 415 nm (emission). The sensitivity of the method allowed for quantification of 7-hydroxywarfarin as low as nmol in one sample. Calculations and Statistics K i values were estimated from Dixon s plots. Statistical evaluation (Model II and Model III) was performed using an analysis of variance followed by Dunnett s test. All values are means ± SEM from 58 animals. Our earlier studies revealed that neuroleptic drugs, in particular phenothiazines, inhibited the family of CYP2 and CYP3 isoforms (2C11, 2D, 3A) by different mechanisms: binding of a parent compound to the cytochrome, forming inhibitory CYP-radical cation complexes or down-regulating the expression of CYP [4, 5]. The results of the present work show that some of the investigated neuroleptics can directly inhibit the CYP2C6 activity in rat liver. The effect was shown as an inhibition of the rate of the CYP2C6-specific reaction, i.e. 7-hydroxylation of warfarin, by the drugs added in vitro to the control liver microsomes (Model I). Their inhibitory effects were moderate (sertindole and levomepromazine: K i = 25 and 31 µm, respectively), weak (thioridazine and chlorpromazine: K i = 88 and 91 µm, respectively) or of no practical significance in case when K i reached the value far above 100 µm (promazine, perazine and risperidone: K i = 322, 341 and 414 µm, respectively) (Tab. 1). Haloperidol produced the weakest inhibitory effect on the activity of CYP2C6 (K i = 606 µm). Figures 1A 1D show examples of the Dixon plots obtained in our studies, which served as a basis for calculation of K i constants. The observed potency of the investigated neuroleptics to inhibit the CYP2C6 activity was weaker compared to that observed for earlier studied CYP2D [5], with an exception of sertindole, whose potency to inhibit CYP2D was lower (K i = 51 µm). Shin et al. [20] who studied the inhibition of human CYP2C9 activity by neuroleptics found that the inhibition constants (K i ) for chlorpromazine, thioridazine and risperidone exceeded the value of 300 µm. It seems that the two mentioned phenothiazines are less effective inhibitors of human CYP2C9 than of rat CYP2C6 (K i values below 100 µm). Our study shows that the investigated neuroleptics do not exert any effect on CYP2C6 via another mechanism, when the drugs are given to rats in vivo (Fig. 2 A and B). Neither one-day (Model II) nor chronic treatment (Model III) significantly affected the activity of CYP2C6, which indicates that this CYP isoform is not inactivated by reactive metabolites (radical cations of classic neuroleptics) or regulated by those drugs. An exception was levomepromazine, which transiently (after 24-h exposure) increased the activity of the enzyme. Therefore, the direct inhibitory effect of levomepromazine on CYP2C6 may be Pharmacological Reports, 2005, 57,

5 attenuated by an indirect mechanism at the beginning of the neuroleptic therapy. Thus, the final effect of the investigated neuroleptics on the activity of CYP2C6 in rat liver after prolonged administration in vivo is mainly due to their direct action on the cytochrome protein. Our earlier pharmacokinetic studies proved that phenothiazine neuroleptics are taken up by tissues [3] reaching in the liver concentrations which are times higher than those in blood plasma [22, 23]. Since phenothiazines are administered at relatively high doses compared to other neuroleptics and they accumulate in tissues when given repeatedly [6], their concentrations in the liver may reach levels close to the respective K i values. Therefore, some of the phenothiazine neuroleptics (with K i values below 100 µm), in particular levomepromazine, are expected to decrease the activity of CYP2C6 in vivo, which may be of physiological and pharmacological meaning, considering catalytic competence of that enzyme. Both rat CYP2C6 and its human counterpart CYP2C9 are able to metabolize such important substrates as endogenous steroids, retinoid and arachidonic acid [2, 9, 16], as well as drugs: phenytoin, warfarin and non-steroidal antiinflammatory drugs [11 13, 19, 24]. Thus, the metabolism of those substrates may be inhibited during treatment with levomepromazine and the phenothiazine neuroleptics with K i values below 100 µm. On the other hand, sertindole with the lowest inhibitory constant (K i = 25 µm) among the tested drugs, should not inhibit the activity of CYP2C6 when administered in vivo, considering low pharmacological and therapeutic doses of the neuroleptic [21]. Concentrations produced by those doses in vivo are unlikely to reach values which approximate the inhibitory constant calculated for this neuroleptic in relation to the activity of CYP2C6. 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