Autoinduction of the metabolism of phenothiazine neuroleptics in a primary culture of human hepatocytes

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1 Pharmacological Reports 2012, 64, ISSN Copyright 2012 by Institute of Pharmacology Polish Academy of Sciences Short communication Autoinduction of the metabolism of phenothiazine neuroleptics in a primary culture of human hepatocytes Jacek Wójcikowski 1, Patrick Maurel 2, W³adys³awa A. Daniel 1 1 Department of Pharmacokinetics and Drug Metabolism, Institute of Pharmacology, Polish Academy of Science, Smêtna 12, PL Kraków, Poland 2 INSERM, U632, Montpellier, France; Université Montpellier 1, UMR-S632, Montpellier, France; CHU Montpellier, Institut de Recherche en Biothérapie, Hôpital Saint Eloi, Montpellier, France Correspondence: Jacek Wójcikowski wojcikow@if-pan.krakow.pl Abstract: Background: The metabolism of phenothiazine neuroleptics (promazine, perazine) in a primary culture of human hepatocytes after pretreatment of cells with those neuroleptics was studied. Methods: The hepatocytes were pretreated with 25 µm promazine or perazine for 96 h. Then, the cells were incubated for 2, 4, 6, 8 and 24 h in the presence of neuroleptics. At the indicated time points, concentrations of phenothiazines and their metabolites (5-sulfoxides and derivatives) were measured in the culture medium using HPLC with UV detection. Results: Pretreatment of the primary culture of human hepatocytes with promazine or perazine resulted in accumulation of their metabolites in the culture medium. Such an effect was not observed in the case of control cultures (not pretreated with neuroleptics). Conclusion: The obtained results suggest that the tested phenothiazines may stimulate their own metabolism by inducing CYP1A2, CYP3A4 and CYP2C19 isoforms. Key words: phenothiazine neuroleptics, metabolism, autoinduction, human cytochrome P450, hepatocytes Introduction Cytochrome P450 (CYP) enzymes are members of the superfamily of heme-containing monooxygenases that catalyze the metabolism of endogenous substances (e.g., steroid hormones, neurosteroids, monoaminergic neurotransmitters, arachidonic acid) and the majority of clinically important drugs including psychotropics. It has been reported that several forms of CYP (such as, e.g., CYP1A1/2, CYP2B6, CYP2C9/19, CYP3A4/5) are inducible in a primary culture of human hepatocytes, which is a convenient model system to examine the oxidative metabolism of drugs and xenobiotics [7, 11]. CYP1A2 and CYP3A4 constitute the main pool of CYP protein in human liver (10 13% and 30 50%, respectively) [13, 22]. CYP1A2 can be induced by cigarette smoking and by a number of polycyclic aromatic hydrocarbons such as TCDD and -naphthoflavone, while CYP3A4 is strongly inducible by a number of drugs including rifampicin, carbamazepine, barbiturates and corticosteroids [9, 14, 15] Pharmacological Reports, 2012, 64,

2 Autoinduction of phenothiazine metabolism Jacek Wójcikowski et al. Our recent studies conducted on human liver microsomes and cdna-expressed human CYP enzymes have shown that CYP1A2, CYP3A4 and CYP2C19 are the main isoforms involved in the metabolism of phenothiazine neuroleptics with different chemical structures [23, 26, 27, 29]. The above-cited results were corroborated by our experiments conducted on human hepatocytes. Treatment of the primary culture of human hepatocytes with TCDD (a CYP1A1/2 inducer) and rifampicin (mainly a CYP3A4 inducer) increased the rate of the biotransformation of phenothiazines [27, 28]. Some clinical studies suggested that phenothiazine neuroleptics may induce human liver CYP, enhancing the metabolism of co-administrated drugs. A significant reduction in antipyrine half-life in patients receiving long-term chlorpromazine treatment was observed [10, 12]. Other studies showed that thioridazine visibly increased the oral clearance of quetiapine in psychiatric patients [19]. Moreover, Rivera-Calimlim et al. [21] reported that plasma chlorpromazine levels declined with time in patients who were on fixed doses of that neuroleptic. They also suggested that a possible cause of that phenomenon might have been induction of CYP enzymes metabolizing chlorpromazine. The aim of the present study was to ascertain whether the phenothiazine neuroleptics with different chemical structures may stimulate their own metabolism. We examined the metabolism of promazine (the simplest and an aliphatic-type phenothiazine neuroleptic) and perazine (a piperazine-type phenothiazine neuroleptic) in a primary culture of human hepatocytes. Materials and Methods Drugs and chemicals was provided by Polfa (Jelenia Góra, Poland), while perazine was obtained from Labor (Wroc³aw, Poland). D-desmethylpromazine was donated by Professor M.H. Bickel, the University of Bern, Switzerland. 5-sulfoxide, perazine 5-sulfoxide and perazine were synthesized according to the previously described methods [2]. Dimethyl sulfoxide (DMSO) was purchased from Sigma (St. Louis, USA). All the organic solvents with HPLC purity were supplied by Carlo Erba (Milan, Italy). Biotransformation of neuroleptics in a primary culture of human hepatocytes The use of human liver samples for scientific purposes was approved by the French National Ethics Committee. A human liver specimen was obtained from patient FT176 (a 69-year-old female) who underwent liver lobectomy due to the hepatic metastasis of colon cancer. The tissue encompassing the tumor was dissected by a surgeon and sent for anatomopathological examination, whereas the remaining healthy tissue was used for hepatocyte preparation. No information about the patient was available to us, apart from her age, sex and the cause of surgery. The viral serological analysis of the tissue (hepatitis B and C viruses and a human immunodeficiency virus) was negative. Primary cultures of human hepatocytes were prepared as described previously [6, 18]. Hepatocytes with viability higher than 90%, as determined by trypan blue exclusion, were used in the experiment. Ten million cells in 7 ml of a culture medium were placed in 10-cm plastic Petri dishes pre-coated with collagen (Beckton-Dickinson, France). That medium consisted of a mixture of Ham F12 and Williams E (1:1 v/v), supplemented as described previously [7]. The culture medium was also supplemented with a 5% calf serum during the first 4 h after plating, to favor the attachment of cells. Then, 4 6 h later, the medium was exchanged for a serum-free medium and the culture was allowed to stabilize for another 24 h. The medium was exchanged every 24 h for a serumand fenol red-free medium. The cultures were kept at 37 C in an atmosphere of a 95% air and a 5% CO 2, at a ca. 100% humidity. For the pretreatment of cells, promazine and perazine were diluted in DMSO and added to the culture medium at a final concentration of 25 µm (the expected concentration of neuroleptics in human liver after pharmacological doses of the tested drugs) [8, 24, 25]. Control cells were treated with DMSO (solvent) added to the culture medium. The concentration of DMSO in the culture medium (of both control and neuroleptic-pretreated cultures) was 0.1%. Pretreatment lasted for 96 h (the time usually needed to observe enzyme induction) [16, 17, 27, 28] and was renewed every 24 h when the culture medium was exchanged. After 96 h, the culture medium (of both control and neuroleptic-pretreated cultures) was exchanged for a medium containing 25 µm promazine Pharmacological Reports, 2012, 64,

3 or perazine. The cells were then incubated under standard culture conditions for 2, 4, 6, 8 and 24 h. At the indicated time points, 500 µl aliquots of the culture medium were collected, and 50 µl were injected directly into the HPLC system. Determination of the concentration of promazine, perazine and their metabolites in the culture medium The concentrations of promazine and its metabolites (promazine 5-sulfoxide and promazine), and perazine and its metabolites (perazine 5-sulfoxide and perazine) were assayed using a Varian HPLC system with UV detection. The analytical column (Econosphere C18, 5 µm, mm) was purchased from Alltech (Carnforth, England). The mobile phase consisted of an acetate buffer, ph = 3.4 (100 mmol of ammonium acetate, 20 mmol of citric acid and 1 ml of triethylamine in 1,000 ml of the buffer adjusted to ph = 3.4 with an 85% phosphoric acid), and acetonitrile in a proportion of 50 : 50 (v/v). Elution proceeded at an ambient temperature at a flow rate of 1.2 ml/min. The absorbance of promazine and its metabolites was measured at a wavelength of 254 nm, while the absorbance of perazine and its metabolites was measured at a wavelength of 265 nm. The detection limit for promazine and its metabolites was as low as 0.01 nmol/sample. The detection limit for perazine and its metabolites was as low as nmol/sample. Results and Discussion After a 96-h pretreatment of the primary cultures of human hepatocytes with promazine or perazine, their metabolites (5-sulfoxides and derivatives) were found to accumulate in the culture medium (Tabs. 1 and 2). Such an effect was not observed in the case of control cultures (not pretreated with neuroleptics). In the latter case, promazine and perazine metabolites were not detected in the culture medium. Approximately 100% of perazine metabolism and 85% of promazine metabolism took place during the first 24 h in perazine- and promazine-treated cultures, respectively (Tabs. 1 and 2). The accumulation of promazine 5-sulfoxide and perazine 5-sulfoxide in the culture medium was enhanced up until 24 h. On the other hand, the concentration of promazine in the culture medium increased up until 6 h, and then decreased up until 24 h. perazine was detected in the culture medium after 24 h only (Tabs. 1 and 2). The present study provides fresh evidence for an earlier clinical observation suggesting induction of CYP enzymes by phenothiazine neuroleptics. We previously showed that CYP1A2 and CYP3A4 were involved mainly in the 5-sulfoxidation of promazine and perazine. Moreover, promazine N-demethylation was metabolized by CYP1A2 and CYP2C19, while CYP2C19 was the main enzyme catalyzing perazine Tab. 1. The influence of a 96-h pretreatment with promazine on its own metabolism in a primary culture of human hepatocytes. Time (h) Control 5-sulfoxide promazine 5-sulfoxide promazine 2 nd nd 9.54 nd nd nd 8.29 nd nd nd nd nd nd nd Human hepatocytes were kept in the primary culture and pretreated with 25 µm promazine (diluted in DMSO) for 96 h. Control cells were treated with DMSO. After 96 h, the culture medium (of both control and promazine-pretreated cultures) was exchanged for a medium contain - ing 25 µm of promazine. The cells were then incubated under standard culture conditions for 2, 4, 6, 8 and 24 h. The concentrations of promaz - ine and its metabolites (promazine 5-sulfoxide and promazine formed from the neuroleptic) were measured in the culture medium (one culture dish per one time point); nd not detected 1580 Pharmacological Reports, 2012, 64,

4 Autoinduction of phenothiazine metabolism Jacek Wójcikowski et al. Tab. 2. The influence of a 96-hour pretreatment with perazine on its own metabolism in a primary culture of human hepatocytes. Time (h) Control 5-sulfoxide perazine 5-sulfoxide perazine 2 nd nd 6.46 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd Human hepatocytes were kept in the primary culture and pretreated with 25 µm perazine (diluted in DMSO) for 96 h. Control cells were treated with DMSO. After 96 h, the culture medium (of both control and perazine-pretreated cultures) was exchanged for a medium containing 25 µm of perazine. The cells were then incubated under standard culture conditions for 2, 4, 6, 8 and 24 h. The concentrations of perazine and its me - tabolites (perazine 5-sulfoxide and perazine formed from the neuroleptic) were measured in the culture medium (one culture dish per one time point); nd not detected N-demethylation [27-29]. In the light of the present results, it seems feasible that promazine and perazine may accelerate their own metabolism by inducing the CYP isoforms mentioned above. The obtained results are in line with our earlier data showing that treatment of the primary culture of human hepatocytes with rifampicin (an inducer of CYP3A4 and, to a lesser degree, of CYP2C19 and CYP2B6) and TCDD (a CYP1A1/2 inducer) increased formation of promazine 5-sulfoxide, perazine 5-sulfoxide and promazine [27, 28]. The formation of perazine was also observed in rifampicin-treated cultures, but not in TCDD-treated ones. However, rifampicin induced formation of perazine in hepatocytes after 24 h only [28]. In the present study, a certain amount of perazine, formed by a perazine-treated culture, was also detected after 24 h only, which seems to suggest that this neuroleptic is a weak inducer of CYP2C19. The present results are also consistent with the clinical data suggesting that chlorpromazine, an aliphatic-type phenothiazine neuroleptic, may induce CYP [21]. Our recent studies have shown that CYP1A2 is the only CYP isoform that catalyzes chlorpromazine N-demethylation and the main isoform responsible for chlorpromazine 5-sulfoxidation. CYP3A4 contributes to a lesser degree to the 5-sulfoxidation of chlorpromazine [23]. Therefore, it seems possible that chlorpromazine may accelerate its metabolism by inducing CYP1A2 and CYP3A4. Moreover, it has been reported that chlorpromazine reduces antipyrine half-life in patients undergoing long-term chlorpromazine therapy, while thioridazine (a piperidine-type phenothiazine neuroleptic) visibly enhances the oral clearance of quetiapine in psychiatric patients [10, 12, 19]. Since antipyrine and quetiapine are metabolized chiefly by CYP1A2 and CYP3A4, respectively [20], the changes observed in the pharmacokinetics of these drugs may result from the induction of CYP1A2 and CYP3A4 by co-administered phenothiazine neuroleptics. Interestingly, in the case of control hepatocytes, the concentrations of promazine and perazine measured in the culture medium after 2 h were lower than those measured in neuroleptic-treated hepatocytes. The latter effect may spring from the fact that after a 96-h treatment of cells with neuroleptics, promazine and perazine (as well as their metabolites) may still be present in hepatocytes. According to Daniel and Wójcikowski [3 5], distribution of psychotropic drugs that are basic lipohilic compounds (such as promazine, perazine and their derivatives) depends mainly on phospholipid binding and lysosomal trapping. Furthermore, during the first 24 h, phenothiazines show a decline in their concentration, which is faster in control than in neuroleptic-treated hepatocytes. Thus, inhibition of another route of promazine and perazine metabolism (e.g., aromatic hydroxylation) in neuroleptic-treated hepatocytes seems likely. It has been reported that phenothiazine neuroleptics may Pharmacological Reports, 2012, 64,

5 yield a great number of reactive phenothiazine metabolites, such as radical cations, which are able to inhibit CYP2D6, the latter catalyzing the hydroxylation of phenothiazines [1, 30]. In summary, our preliminary results concerning the primary culture of human hepatocytes suggest that phenothiazine neuroleptics may stimulate their own metabolism by inducing CYP1A2, CYP3A4 and CYP2C19. By inducing CYP isoforms (e.g., during a long-term neuroleptic therapy of psychiatric patients), phenothiazines may increase their elimination and thus attenuate the desired pharmacological effect. Moreover, phenothiazine neuroleptics are administered for months or even years, also to patients treated simultaneously with other clinically important drugs that are substrates of CYP1A2 (e.g., caffeine, theophylline, phenacetin, imipramine, propranolol, clozapine, melatonin), CYP3A4 (e.g., carbamazepine, cyclosporin A, calcium channel antagonists, macrolide antibiotics, benzodiazepines) or CYP2C19 (e.g., tricyclic antidepressants, S-mephenytoin, omeprazole). They may enhance metabolism of the co-administered drugs, leading to the diminution of their pharmacological effect. On the other hand, hepatic CYP3A4 induction by phenothiazine neuroleptics may alter metabolism of endogenous substrates (e.g., steroid hormones), while the induction of CYP1A2 may increase the metabolic transformation of heterocyclic aromatic amines into reactive intermediates, resulting in toxicity and cancer. Hence, the induction of CYP enzymes by phenothiazines may be of physiological, pharmacological and toxicological importance. However, further metabolic and molecular studies are scheduled to corroborate our present results. Moreover, since the neuroleptics studied are basic lipophilic drugs, their concentrations will be analyzed simultaneously in an incubation medium and in hepatocytes. Acknowledgments: This study was supported by the Poste Jaune fellowship, granted to Jacek Wójcikowski by the INSERM, Paris, and by the INSERM U632, Montpellier, France; support also came from the statutory funds of the Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland. References: 1. Daniel WA, Haduch A, Wójcikowski J: Inhibition of rat liver CYP2D in vitro and after 1-day and long-term exposure to neuroleptics in vivo possible involvement of different mechanisms. 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