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Inhibition of human cytochrome P450 isoenzymes by a phenothiazine neuroleptic levomepromazine: An in vitro study
Pharmacological Reports 67 (2015) 1178–1182
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Short communication
Inhibition of human cytochrome P450 isoenzymes by a phenothiazine neuroleptic levomepromazine: An in vitro study Agnieszka Basin´ska-Ziobron´, Władysława A. Daniel, Jacek Wo´jcikowski * Institute of Pharmacology, Polish Academy of Sciences, Krako´w, Poland
A R T I C L E I N F O
Article history: Received 1 April 2015 Received in revised form 9 April 2015 Accepted 10 April 2015 Available online 23 April 2015 Keywords: Levomepromazine Human CYP isoenzymes Inhibition
A B S T R A C T
Background: Inhibition of cytochrome P450 (CYP) isoenzymes is the most common cause of harmful drug–drug interactions. The present study was aimed at examining the inhibitory effect of the phenothiazine neuroleptic levomepromazine on main CYP isoenzymes in human liver. Methods: The experiment was performed in vitro using the human cDNA-expressed CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (Supersomes). CYP isoenzyme activities were determined using the CYP-specific reactions: caffeine 3-N-demethylation (CYP1A2), diclofenac 4 0 -hydroxylation (CYP2C9), perazine N-demethylation (CYP2C19), bufuralol 10 -hydroxylation (CYP2D6) and testosterone 6b-hydroxylation (CYP3A4). The rates of the CYP-specific reactions were assessed in the absence and presence of levomepromazine (1–50 mM). The concentrations of CYP-specific substrates and their metabolites formed by CYP isoenzymes were measured by HPLC with UV or fluorimetric detection. Results: Levomepromazine potently inhibited CYP2D6 (Ki = 6 mM) in a competitive manner. Moreover, the neuroleptic moderately diminished the activity of CYP1A2 (Ki = 47 mM) and CYP3A4 (Ki = 34 mM) via a mixed mechanism. On the other hand, levomepromazine did not affect the activities of CYP2C9 and CYP2C19. Conclusion: The inhibition of CYP1A2, CYP2D6 and CYP3A4 by levomepromazine, demonstrated in vitro in the present study, should also be observed in vivo (especially the CYP2D6 inhibition by levomepromazine), since the calculated Ki values are below or close to the presumed concentration range for levomepromazine in the liver in vivo. Therefore pharmacokinetic interactions involving levomepromazine and CYP2D6, CYP1A2 or CYP3A4 substrates are likely to occur in patients during coadministration of the above-mentioned substrates/drugs. ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
Introduction Cytochrome P450 (CYP) isoenzymes are members of the superfamily of heme-containing monooxygenases that catalyze metabolism of endogenous substances (e.g. steroid hormones, neurosteroids, monoaminergic neurotransmitters, arachidonic acid) and the majority of clinically important drugs including neuroactive agents. CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 constitute ca. 13, 20–30, 4, 1.5 and 30–50% of the total CYP protein in human liver, respectively. They are involved in the metabolism of approximately 90% of all the marked drugs, being pivotal CYP isoforms in evaluation of the CYP-mediated drug–drug interactions [1].
* Corresponding author. E-mail address: [email protected] (J. Wo´jcikowski).
Levomepromazine is a classic phenothiazine neuroleptic. Although it is an old-type typical neuroleptic and a number of new atypical neuroleptic drugs have been introduced into psychopharmacotherapy ever since, its pharmacological profile and clinical effects still make it useful in the therapy of different psychiatric and non-psychiatric states. Levomepromazine is used for treating schizophrenia, paranoia, mania, toxic psychoses and mental organic syndromes associated with delirium. [2]. Moreover, it is also used in the therapy of nausea and vomiting in advanced cancer patients and as a sedative in terminal care and burn patients [3]. Our recent metabolic studies have shown that CYP3A4 is the main isoenzyme responsible for levomepromazine metabolism [4,5]. However, a possible inhibition of human CYP isoenzymes by levomepromazine has not been fully investigated so far. Clinical studies have demonstrated that levomepromazine is a potent inhibitor of human CYP2D6, though its Ki or IC50 values have not been determined [6–8]. In contrast, a recent in vitro metabolic
http://dx.doi.org/10.1016/j.pharep.2015.04.005 1734-1140/ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
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study has indicated that levomepromazine moderately inhibits CYP2D6 and CYP3A4 (IC50 = 25.5 and 30 mM, respectively) [9]. However, the latter experiment was performed using only one concentration of CYP-specific substrates, and the mechanism of inhibition of CYP2D6 and CYP3A4 by levomepromazine was not evaluated in that study. Therefore in the present research a kinetic study of CYP isoenzymes inhibition by levomepromazine was carried out. To this end we employed the main human CYP isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4), different CYP-specific substrate concentrations and diverse pharmacological concentrations of levomepromazine. Thus in the present study we simultaneously estimated the inhibition constant (Ki) and the mechanism of inhibition of individual CYP isoenzymes by levomepromazine.
Incubations were carried out in a system containing human liver microsomes from baculovirus-infected insect cells expressing CYP2C9 (Supersomes 2C9, 50 pmol/ml), a Tris/KCl buffer (50 mM, pH = 7.4), MgCl2 (3.0 mM), EDTA (1 mM), NADP (1.0 mM), glucose 6-phosphate (5 mM) and glucose-6-phosphate-dehydrogenase (1.7 U in 1 ml). The final incubation volume was 0.5 ml. After a 30-min incubation, the reaction was stopped by adding 100 ml of acetonitrile. A water phase containing diclofenac and its metabolite 40 -hydroxydiclofenac was extracted with 4 ml of diethyl ether. The samples were centrifuged for 10 min at 2000 g. Concentrations of diclofenac and its metabolite 40 -hydroxydiclofenac, formed in Supersomes 2C9, were assessed by the HPLC method with UV detection, as described previously [11].
Materials and methods
The activity of CYP2C19 was studied by measuring the rate of a CYP2C19-specific reaction, i.e., N-demethylation of perazine. The rate of perazine N-demethylation (perazine concentrations: 10, 25, 50 and 100 mM) was assessed in the absence and presence of levomepromazine, added in vitro (levomepromazine concentrations: 1, 5, 10, 25 and 50 mM). Incubations were carried out in a system containing human liver microsomes from baculovirus-infected insect cells expressing CYP2C19 (Supersomes 2C19, 50 pmol/ml), a Tris/KCl buffer (50 mM, pH = 7.4), MgCl2 (3.0 mM), EDTA (1 mM), NADP (1.0 mM), glucose 6-phosphate (5 mM) and glucose-6-phosphate-dehydrogenase (1.7 U in 1 ml). The final incubation volume was 0.5 ml. After a 30-min incubation, the reaction was stopped by adding 100 ml of methanol. A water phase containing perazine and its metabolite N-desmethylperazine was extracted (pH = 12) with 6 ml of an organic mixture consisting of hexane and dichloromethane (1:1, v/v). The samples were centrifuged for 10 min at 2000 g. Concentrations of perazine and its metabolite N-desmethylperazine, formed in Supersomes 2C19, were assessed by the HPLC method with UV detection, as described previously [12].
Drugs and chemicals Levomepromazine was obtained from Egyt (Budapest, Hungary). Caffeine, 3-N-desmethyl caffeine (paraxanthine), diclofenac, 40 -hydroxydiclofenac, bufuralol, 10 -hydroxybufuralol, NADP, NADPH, glucose-6-phosphate, glucose-6-phosphate-dehydrogenase, MgCl2, KCl, ZnSO4, Trizma base and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma (St. Louis, USA). Testosterone and 16b-testosterone were from Steraloids (Newport, USA). Perazine was obtained from Labor (Wrocław, Poland). N-desmethylperazine was donated by Professor M.H. Bickel, the University of Bern, Switzerland. All the organic solvents with HPLC purity were supplied by Merck (Darmstadt, Germany). Microsomes from baculovirus-infected insect cells expressing human CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (Supersomes) were provided by Corning (Woburn, MA, USA). CYP1A2 activity assay
CYP2C19 activity assay
The activity of CYP1A2 was studied by measuring the rate of a CYP1A2-specific reaction, i.e., 3-N-demethylation of caffeine. The rate of caffeine 3-N-demethylation (caffeine concentrations: 100, 200, 400 and 800 mM) was assessed in the absence and presence of levomepromazine, added in vitro (levomepromazine concentrations: 1, 5, 10, 25 and 50 mM). Incubations were carried out in a system containing human liver microsomes from baculovirus-infected insect cells expressing CYP1A2 (Supersomes 1A2, 50 pmol/ml), a phosphate buffer (0.15 M, pH 7.4) and NADPH (1 mM). The final incubation volume was 0.5 ml. After a 30-min incubation, the reaction was terminated by adding 700 ml of a 2% ZnSO4 and 50 ml of 2 M HCl. A water phase containing caffeine and its metabolite paraxanthine was extracted with 6 ml of an organic mixture consisting of ethyl acetate and 2propanol (8:1, v/v). The samples were centrifuged for 10 min at 2000 g. Concentrations of caffeine and its metabolite 3-N-desmethyl caffeine (paraxanthine), formed in Supersomes 1A2, were assessed using the HPLC method with UV detection, as described previously [10].
CYP2D6 activity assay The activity of CYP2D6 was studied by measuring the rate of a CYP2D6-specific reaction, i.e., 10 -hydroxylation of bufuralol. The rate of bufuralol 10 -hydroxylation (bufuralol concentrations: 5, 10, 25 and 50 mM) was assessed in the absence and presence of levomepromazine, added in vitro (levomepromazine concentrations: 1, 5, 10, 25 and 50 mM). Incubations were carried out in a system containing human liver microsomes from baculovirus-infected insect cells expressing CYP2D6 (Supersomes 2D6, 50 pmol/ml), a potassium phosphate buffer (0.1 M, pH = 7.4), MgCl2 (3.3 mM), NADP (1.3 mM), glucose 6-phosphate (3.3 mM) and glucose-6-phosphate-dehydrogenase (1.0 U in 1 ml). The final incubation volume was 0.5 ml. After a 30-min incubation, the reaction was stopped by adding 30 ml of a 70% perchloric acid. The experimental samples were centrifuged (10 min, 3000 g), and then the supernatant was transferred to new vials. Concentrations of bufuralol and its metabolite 40 -hydroxybufuralol, formed in Supersomes 2D6, were assessed by the HPLC method with fluorimetric detection, as described previously [13].
CYP2C9 activity assay
CYP3A4 activity assay
The activity of CYP2C9 was studied by measuring the rate of a CYP2C9-specific reaction, i.e., 40 -hydroxylation of diclofenac. The rate of diclofenac 40 -hydroxylation (diclofenac concentrations: 5, 10, 25, 50 mM) was assessed in the absence and presence of levomepromazine, added in vitro (levomepromazine concentrations: 1, 5, 10, 25 and 50 mM).
The activity of CYP3A4 was studied by measuring the rate of a CYP3A4-specific reaction, i.e., 6b-hydroxylation of testosterone. The rate of testosterone 6b-hydroxylation (testosterone concentrations: 50, 100, 200 and 300 mM) was assessed in the absence and presence of levomepromazine, added in vitro (levomepromazine concentrations: 1, 5, 10, 25 and 50 mM).
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Incubations were carried out in a system containing human liver microsomes from baculovirus-infected insect cells expressing CYP3A4 (Supersomes 3A4, 50 pmol/ml), a Tris/KCl buffer (50 mM, pH = 7.4), MgCl2 (3.0 mM), EDTA (1 mM), NADP (1.0 mM), glucose 6-phosphate (5 mM) and glucose-6-phosphate-dehydrogenase (1.7 U in 1 ml). The final incubation volume was 0.5 ml. After a 30-min incubation, the reaction was stopped by adding 100 ml of methanol. A water phase containing testosterone and its metabolite 6b-hydroxytestosterone was extracted with 6 ml of dichloromethane. The samples were centrifuged for 10 min at 2000 g. Concentrations of testosterone and its metabolite 6b-hydroxytestosterone, formed in Supersomes 3A4, were assessed by the HPLC method with UV detection, as described previously [14]. Determination of kinetic parameters The inhibitory effects of levomepromazine on CYP isoenzymes are shown as Dixon plots (1/V against I), which allowed us to estimate inhibition constants (Ki). Kinetic parameters (Km, Vmax, Ki) showing metabolism of CYP-specific substrates in Supersomes in the absence and presence of levomepromazine were obtained using a non-linear regression analysis (Program Sigma Plot 8.0; Enzyme Kinetics). Results and discussion Dixon’s plots of the metabolism of CYP-specific substrates, carried out in cDNA-expressed human liver microsomes in the absence or
presence of levomepromazine, demonstrated that the examined neuroleptic exerted a significant inhibitory effect on CYP1A2, CYP2D6 and CYP3A4; however, its potency in relation to specific CYP isoenzymes was diversified (Fig. 1A–C). On the other hand, levomepromazine did not affect the activities of CYP2C9 and CYP2C19 (data not shown). The kinetic parameters (Km, Vmax, CL, Ki) showing metabolism of CYP1A2-, CYP2D6- and CYP3A4-specific substrates in the absence and presence of levomepromazine, obtained by a nonlinear regression analysis, are presented in Tables 1 and 2. Levomepromazine potently inhibited CYP2D6 activity (Ki = 6 mM) in a competitive manner (Table 2). Moreover, that neuroleptic moderately diminished the activity of CYP1A2 (Ki = 47 mM) and CYP3A4 (Ki = 34 mM) via a mixed mechanism (Table 2). In all those cases, the value of clearance (CL) decreased in the presence of levomepromazine (Table 1). The obtained Ki values showing the inhibition of CYP1A2, CYP2D6 and CYP3A4 activities by levomepromazine correspond well with the results presented as Dixon’s plots (Fig. 1A–C). The mechanism of inhibition was estimated on the basis of changes in the Km and Vmax values of the tested inhibitor (levomepromazine) concentrations (Table 1). In the case of mixed inhibition, the Km and Vmax values changed at different inhibitor concentrations. In the case of competitive inhibition, the Vmax value did not significantly change, while the Km was altered at different inhibitor concentrations. The results obtained in the present study give a good support for earlier clinical observations on the potent inhibition of CYP2D6 activity (measured as a rate of codeine O-demethylation or debrisoquine oxidation) by levomepromazine [6–8].
Fig. 1. The influence of levomepromazine on the activity of CYP1A2, CYP2D6 and CYP3A4, measured as rates of CYP-specific reactions in human cDNA-expressed CYP isoenzymes (Dixon’s plots): (A) caffeine 3-N-demethylation (CYP1A2); (B) bufuralol 10 -hydroxylation (CYP2D6); (C) testosterone 6b-hydroxylation (CYP3A4). Each point represents the mean value of four independent analyses SEM. The Ki values are shown in Table 2. V: velocity of the reaction; I: concentration of the inhibitor (levomepromazine).
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Table 1 Kinetic parameters showing the influence of levomepromazine on the activity of CYP1A2, CYP2D6 and CYP3A4, measured as rates of CYP-specific reactions in vitro in human cDNA-expressed CYP isoenzymes. Kinetic parameters of CYP-specific reactions
Inhibitor (levomepromazine) concentrations (mM)
Bufuralol 10 -hydroxylation (CYP2D6)
Caffeine 3-N-demethylation (CYP1A2)
0 (control) 1 5 10 25 50
Testosterone 6b-hydroxylation (CYP3A4)
Km (mM)
Vmax (pmol/pmol CYP1A2/min)
CL (Vmax/Km)
Km (mM)
Vmax (pmol/pmol CYP2D6/min)
CL (Vmax/Km)
Km (mM)
Vmax (pmol/pmol CYP3A4/min)
CL (Vmax/Km)
471 439 395 375 364 360
6.95 0.72 0.58 0.53 0.33 0.30
0.0019 0.0016 0.0015 0.0014 0.0009 0.0008
6.95 7.71 14.10 23.58 46.50 94.41
2.37 2.28 2.12 2.17 2.35 2.94
0.34 0.29 0.15 0.09 0.05 0.03
212 198 145 157 156 210
98.0 91.3 70.4 64.5 47.7 40.9
0.46 0.46 0.45 0.41 0.30 0.19
The presented values of Michaelis–Menten constants (Km), the maximum velocities of the reactions (Vmax) and the intrinsic clearance (CL) for particular CYP-specific reactions were obtained using a non-linear regression analysis (Program Sigma Plot 8.0; Enzyme Kinetics). The presented kinetic parameters are based on the data shown in Fig. 1A–C. Table 2 The ability of levomepromazine to inhibit CYP1A2, CYP2D6 and CYP3A4 activities in vitro in human cDNA-expressed CYP isoenzymes. Inhibition of CYP-specific reactions by levomepromazine Ki (mM) and type of inhibition
Inhibitor
Levomepromazine
Caffeine 3-N-demethylation (CYP1A2)
Bufuralol 10 -hydroxylation (CYP2D6)
Testosterone 6b-hydroxylation (CYP3A4)
47 (mixed)
6 (competitive)
34 (mixed)
The presented inhibition constants (Ki) for the inhibition of particular CYP-specific reactions by levomepromazine were obtained using a non-linear regression analysis (Program Sigma Plot 8.0; Enzyme Kinetics) and are based on the data shown in Fig. 1A–C.
On the other hand, our present results are not consistent with those obtained by Gervasini et al. [9], the latter indicating that levomepromazine inhibited fairly moderately (and to a similar degree) human CYP2D6 and CYP3A4. The observed discrepancies may stem from diverse methodological approaches (determination of IC50 or Ki), as well as from different conditions of the performed experiments and from the specificity of substrates used to estimate the activity of CYP isoenzymes. The obtained results revealed interactions between levomepromazine and human CYP2D6, CYP1A2 and CYP3A4, which led to a decrease in the isoenzyme activities. The Ki values obtained for CYP2D6, CYP3A4 and CYP1A2 (6, 34 and 47 mM, respectively) may be of importance in vivo regarding the dosage and the pharmacokinetics of levomepromazine. Phenothiazine neuroleptics were administered in relatively high doses compared to other neuroleptics and, being taken up by tissues, they reached concentrations that were 10–20 times higher in the liver than in blood plasma [15,16]. Therefore phenothiazine neuroleptics (including levomepromazine) whose Ki values are below 50 mM may reach the hepatic level close to the respective Ki values and are expected to decrease CYP2D6, CYP1A2 and CYP3A4 activities in vivo. Accordingly, Kudo et al. [17] demonstrated a fatal case of amoxapine poisoning after concomitant levomepromazine administration to depressive patients. Levomepromazine strongly elevated the concentration of amoxapine in blood plasma (up to 500% of the therapeutic level), while the concentration of levomepromazine was within the therapeutic range. Since amoxapine is metabolized mainly by CYP2D6 and CYP3A4 [18], the observed pharmacokinetic interaction between amoxapine and levomepromazine may stem from the CYP2D6 and CYP3A4 inhibition by levomepromazine. Despite its potent inhibition of human CYP2D6, levomepromazine exerts a weaker inhibitory effect on rat CYP2D (Ki = 20 mM) [19]. Since human CYP2D6 and rat CYP2D are very similar in amino acid sequences, the observed species differences in the inhibitory effect of levomepromazine on the enzyme activity may lie in
diverse structures of the catalytic sites of counterpart enzymes in rats and humans. Accordingly, specific inhibitors of CYP2D isoforms differentiate rat and human isoforms; quinine is a more potent inhibitor of human CYP2D6 than of rat CYP2D, while its diastereoisomer quinine is a more potent inhibitor of rat CYP2D than of human CYP2D6 [20]. The inhibition of CYP1A2, CYP2D6 and CYP3A4 by levomepromazine, demonstrated in vitro in the present study, may also be observed in vivo (especially the CYP2D6 inhibition by levomepromazine), since the calculated Ki values are below or close to the presumed concentration range for levomepromazine in the liver in vivo. Therefore pharmacokinetic interactions between levomepromazine and the substrates of CYP2D6 (e.g. tricyclic antidepressants, SSRIs, thioridazine, haloperidol, risperidone, codeine, dextromethorphan, debrisoquine, metoprolol, propranolol), CYP1A2 (e.g. caffeine, theophylline, phenacetin, tricyclic antidepressants, propranolol, clozapine, chlorpromazine) or CYP3A4 (e.g. benzodiazepines, calcium channel antagonists, macrolide antibiotics, testosterone) are likely to occur in patients during co-administration of the above-mentioned substrates/drugs. Conflict of interest The authors declare that they have no conflict of interest. Funding The study was supported by statutory funds from the Institute of Pharmacology, Polish Academy of Sciences Krako´w, Poland. References [1] Pelkonen O, Turpeinen M, Hakkola J, Honkakoski P, Hukkanen J, Raunio H. Inhibition and induction of human cytochrome P450 enzymes: current status. Arch Toxicol 2008;82:667–715. [2] Green B, Pettit T, Faith L, Seaton K. Focus on levomepromazine. Curr Med Res Opin 2004;20:1877–81.
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[3] Dietz I, Schmitz A, Lampey I, Schulz C. Evidence for the use of levomepromazine for symptom control in the palliative care setting: a systematic review. BMC Palliat Care 2013;12:2. [4] Wo´jcikowski J, Basin´ska A, Daniel WA. The cytochrome P450-catalyzed metabolism of levomepromazine, a phenothiazine neuroleptic with a wide spectrum of clinical application. Biochem Pharmacol 2014;90:188–95. [5] Wo´jcikowski J, Basin´ska A, Boksa J, Daniel WA. The influence of amitriptyline and carbamazepine on levomepromazine metabolism in human liver: an in vitro study. Pharmacol Rep 2014;66:1122–6. [6] Kallio J, Huupponen R, Seppa¨la¨ M, Sa¨ko¨ E, Iisalo E. The effects of b-adrenoceptor antagonists and levomepromazine on the metabolic ratio of debrisoquine. Br J Clin Pharmacol 1990;30:638–43. [7] Syva¨lahti EKG, Lindberg R, Kallio J, De Vocht M. Inhibitory effects of neuroleptics on debrisoquine oxidation in man. Br J Clin Pharmacol 1986;22:89–92. [8] Vevelstad M, Pettersen S, Tallaksen C, Brørs O. O-demethylation of codeine to morphine inhibited by low-dose levomepromazine. Eur J Clin Pharmacol 2009;65:795–801. [9] Gervasini G, Caballero MJ, Carillo JA, Benitez J. Comparative cytochrome P450 in vitro inhibition by atypical antipsychotic drugs. ISRM Pharmacol 2013 [Article ID 792456]. [10] Daniel WA, Kot M, Wo´jcikowski J. Influence of classic and atypical neuroleptics on caffeine oxidation in rat liver microsomes. Pol J Pharmacol 2003;55:1055–61. [11] Schmitz G, Lepper H, Estler CJ. High-performance liquid chromatographic method for the routine determination of diclofenac and its hydroxyl and methoxy metabolites from in vitro systems. J Chromatogr 1993;620:158–63.
[12] Wo´jcikowski J, Pichard-Garcia L, Maurel P, Daniel WA. The metabolism of the piperazine-type phenothiazine neuroleptic perazine by the human cytochrome P-450 isoenzymes. Eur Neuropsychopharmacol 2004;14:199–208. [13] Hiroi T, Chow T, Imaoka S, Funae Y. Catalytic specificity of CYP2D isoforms in rat and human. Drug Metab Dispos 2002;30:970–6. [14] Wo´jcikowski J, Haduch A, Daniel WA. Effect of classic and atypical neuroleptics on cytochrome P450 3A (CYP3A) in rat liver. Pharmacol Rep 2012;64:1411–8. [15] Dinovo EC, Bost RO, Sunshine I, Gottschalk LA. Distribution of thioridazine and its metabolites in human tissues and fluids obtained postmortem. Clin Chem 1978;24:1828–30. [16] Wo´jcikowski J, Daniel WA. Distribution interactions between perazine and antidepressant drugs. In vivo studies. Pol J Pharmacol 2000;52:449–57. [17] Kudo K, Inoue H, Ishida T, Tsuji A, Ikeda N. A fatal case of amoxapine poisoning under the influence of chronic use of psychotropic drugs. Leg Med 2007;9: 63–67. [18] Lydiard RB, Gelenberg AJ. Amoxapine-an antidepressant with some neuroleptic properties? A review of its chemistry, animal pharmacology and toxicology, human pharmacology, and clinical efficacy. Pharmacotherapy 1981;1:163–78. [19] Daniel WA, Haduch A, Wo´jcikowski 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. Eur Neuropsychopharmacol 2005;15:103–10. [20] Kobayashi S, Murray S, Watson D, Sesardic D, Davies DS, Boobis AR. The specificity of inhibition of debrisoquine 4-hydroxylase activity by quinidine and quinine in the rat is the inverse of that in man. Biochem Pharmacol 1989;38:2795–9.
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Short communication
Inhibition of human cytochrome P450 isoenzymes by a phenothiazine neuroleptic levomepromazine: An in vitro study Agnieszka Basin´ska-Ziobron´, Władysława A. Daniel, Jacek Wo´jcikowski * Institute of Pharmacology, Polish Academy of Sciences, Krako´w, Poland
A R T I C L E I N F O
Article history: Received 1 April 2015 Received in revised form 9 April 2015 Accepted 10 April 2015 Available online 23 April 2015 Keywords: Levomepromazine Human CYP isoenzymes Inhibition
A B S T R A C T
Background: Inhibition of cytochrome P450 (CYP) isoenzymes is the most common cause of harmful drug–drug interactions. The present study was aimed at examining the inhibitory effect of the phenothiazine neuroleptic levomepromazine on main CYP isoenzymes in human liver. Methods: The experiment was performed in vitro using the human cDNA-expressed CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (Supersomes). CYP isoenzyme activities were determined using the CYP-specific reactions: caffeine 3-N-demethylation (CYP1A2), diclofenac 4 0 -hydroxylation (CYP2C9), perazine N-demethylation (CYP2C19), bufuralol 10 -hydroxylation (CYP2D6) and testosterone 6b-hydroxylation (CYP3A4). The rates of the CYP-specific reactions were assessed in the absence and presence of levomepromazine (1–50 mM). The concentrations of CYP-specific substrates and their metabolites formed by CYP isoenzymes were measured by HPLC with UV or fluorimetric detection. Results: Levomepromazine potently inhibited CYP2D6 (Ki = 6 mM) in a competitive manner. Moreover, the neuroleptic moderately diminished the activity of CYP1A2 (Ki = 47 mM) and CYP3A4 (Ki = 34 mM) via a mixed mechanism. On the other hand, levomepromazine did not affect the activities of CYP2C9 and CYP2C19. Conclusion: The inhibition of CYP1A2, CYP2D6 and CYP3A4 by levomepromazine, demonstrated in vitro in the present study, should also be observed in vivo (especially the CYP2D6 inhibition by levomepromazine), since the calculated Ki values are below or close to the presumed concentration range for levomepromazine in the liver in vivo. Therefore pharmacokinetic interactions involving levomepromazine and CYP2D6, CYP1A2 or CYP3A4 substrates are likely to occur in patients during coadministration of the above-mentioned substrates/drugs. ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
Introduction Cytochrome P450 (CYP) isoenzymes are members of the superfamily of heme-containing monooxygenases that catalyze metabolism of endogenous substances (e.g. steroid hormones, neurosteroids, monoaminergic neurotransmitters, arachidonic acid) and the majority of clinically important drugs including neuroactive agents. CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 constitute ca. 13, 20–30, 4, 1.5 and 30–50% of the total CYP protein in human liver, respectively. They are involved in the metabolism of approximately 90% of all the marked drugs, being pivotal CYP isoforms in evaluation of the CYP-mediated drug–drug interactions [1].
* Corresponding author. E-mail address: [email protected] (J. Wo´jcikowski).
Levomepromazine is a classic phenothiazine neuroleptic. Although it is an old-type typical neuroleptic and a number of new atypical neuroleptic drugs have been introduced into psychopharmacotherapy ever since, its pharmacological profile and clinical effects still make it useful in the therapy of different psychiatric and non-psychiatric states. Levomepromazine is used for treating schizophrenia, paranoia, mania, toxic psychoses and mental organic syndromes associated with delirium. [2]. Moreover, it is also used in the therapy of nausea and vomiting in advanced cancer patients and as a sedative in terminal care and burn patients [3]. Our recent metabolic studies have shown that CYP3A4 is the main isoenzyme responsible for levomepromazine metabolism [4,5]. However, a possible inhibition of human CYP isoenzymes by levomepromazine has not been fully investigated so far. Clinical studies have demonstrated that levomepromazine is a potent inhibitor of human CYP2D6, though its Ki or IC50 values have not been determined [6–8]. In contrast, a recent in vitro metabolic
http://dx.doi.org/10.1016/j.pharep.2015.04.005 1734-1140/ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
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study has indicated that levomepromazine moderately inhibits CYP2D6 and CYP3A4 (IC50 = 25.5 and 30 mM, respectively) [9]. However, the latter experiment was performed using only one concentration of CYP-specific substrates, and the mechanism of inhibition of CYP2D6 and CYP3A4 by levomepromazine was not evaluated in that study. Therefore in the present research a kinetic study of CYP isoenzymes inhibition by levomepromazine was carried out. To this end we employed the main human CYP isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4), different CYP-specific substrate concentrations and diverse pharmacological concentrations of levomepromazine. Thus in the present study we simultaneously estimated the inhibition constant (Ki) and the mechanism of inhibition of individual CYP isoenzymes by levomepromazine.
Incubations were carried out in a system containing human liver microsomes from baculovirus-infected insect cells expressing CYP2C9 (Supersomes 2C9, 50 pmol/ml), a Tris/KCl buffer (50 mM, pH = 7.4), MgCl2 (3.0 mM), EDTA (1 mM), NADP (1.0 mM), glucose 6-phosphate (5 mM) and glucose-6-phosphate-dehydrogenase (1.7 U in 1 ml). The final incubation volume was 0.5 ml. After a 30-min incubation, the reaction was stopped by adding 100 ml of acetonitrile. A water phase containing diclofenac and its metabolite 40 -hydroxydiclofenac was extracted with 4 ml of diethyl ether. The samples were centrifuged for 10 min at 2000 g. Concentrations of diclofenac and its metabolite 40 -hydroxydiclofenac, formed in Supersomes 2C9, were assessed by the HPLC method with UV detection, as described previously [11].
Materials and methods
The activity of CYP2C19 was studied by measuring the rate of a CYP2C19-specific reaction, i.e., N-demethylation of perazine. The rate of perazine N-demethylation (perazine concentrations: 10, 25, 50 and 100 mM) was assessed in the absence and presence of levomepromazine, added in vitro (levomepromazine concentrations: 1, 5, 10, 25 and 50 mM). Incubations were carried out in a system containing human liver microsomes from baculovirus-infected insect cells expressing CYP2C19 (Supersomes 2C19, 50 pmol/ml), a Tris/KCl buffer (50 mM, pH = 7.4), MgCl2 (3.0 mM), EDTA (1 mM), NADP (1.0 mM), glucose 6-phosphate (5 mM) and glucose-6-phosphate-dehydrogenase (1.7 U in 1 ml). The final incubation volume was 0.5 ml. After a 30-min incubation, the reaction was stopped by adding 100 ml of methanol. A water phase containing perazine and its metabolite N-desmethylperazine was extracted (pH = 12) with 6 ml of an organic mixture consisting of hexane and dichloromethane (1:1, v/v). The samples were centrifuged for 10 min at 2000 g. Concentrations of perazine and its metabolite N-desmethylperazine, formed in Supersomes 2C19, were assessed by the HPLC method with UV detection, as described previously [12].
Drugs and chemicals Levomepromazine was obtained from Egyt (Budapest, Hungary). Caffeine, 3-N-desmethyl caffeine (paraxanthine), diclofenac, 40 -hydroxydiclofenac, bufuralol, 10 -hydroxybufuralol, NADP, NADPH, glucose-6-phosphate, glucose-6-phosphate-dehydrogenase, MgCl2, KCl, ZnSO4, Trizma base and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma (St. Louis, USA). Testosterone and 16b-testosterone were from Steraloids (Newport, USA). Perazine was obtained from Labor (Wrocław, Poland). N-desmethylperazine was donated by Professor M.H. Bickel, the University of Bern, Switzerland. All the organic solvents with HPLC purity were supplied by Merck (Darmstadt, Germany). Microsomes from baculovirus-infected insect cells expressing human CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (Supersomes) were provided by Corning (Woburn, MA, USA). CYP1A2 activity assay
CYP2C19 activity assay
The activity of CYP1A2 was studied by measuring the rate of a CYP1A2-specific reaction, i.e., 3-N-demethylation of caffeine. The rate of caffeine 3-N-demethylation (caffeine concentrations: 100, 200, 400 and 800 mM) was assessed in the absence and presence of levomepromazine, added in vitro (levomepromazine concentrations: 1, 5, 10, 25 and 50 mM). Incubations were carried out in a system containing human liver microsomes from baculovirus-infected insect cells expressing CYP1A2 (Supersomes 1A2, 50 pmol/ml), a phosphate buffer (0.15 M, pH 7.4) and NADPH (1 mM). The final incubation volume was 0.5 ml. After a 30-min incubation, the reaction was terminated by adding 700 ml of a 2% ZnSO4 and 50 ml of 2 M HCl. A water phase containing caffeine and its metabolite paraxanthine was extracted with 6 ml of an organic mixture consisting of ethyl acetate and 2propanol (8:1, v/v). The samples were centrifuged for 10 min at 2000 g. Concentrations of caffeine and its metabolite 3-N-desmethyl caffeine (paraxanthine), formed in Supersomes 1A2, were assessed using the HPLC method with UV detection, as described previously [10].
CYP2D6 activity assay The activity of CYP2D6 was studied by measuring the rate of a CYP2D6-specific reaction, i.e., 10 -hydroxylation of bufuralol. The rate of bufuralol 10 -hydroxylation (bufuralol concentrations: 5, 10, 25 and 50 mM) was assessed in the absence and presence of levomepromazine, added in vitro (levomepromazine concentrations: 1, 5, 10, 25 and 50 mM). Incubations were carried out in a system containing human liver microsomes from baculovirus-infected insect cells expressing CYP2D6 (Supersomes 2D6, 50 pmol/ml), a potassium phosphate buffer (0.1 M, pH = 7.4), MgCl2 (3.3 mM), NADP (1.3 mM), glucose 6-phosphate (3.3 mM) and glucose-6-phosphate-dehydrogenase (1.0 U in 1 ml). The final incubation volume was 0.5 ml. After a 30-min incubation, the reaction was stopped by adding 30 ml of a 70% perchloric acid. The experimental samples were centrifuged (10 min, 3000 g), and then the supernatant was transferred to new vials. Concentrations of bufuralol and its metabolite 40 -hydroxybufuralol, formed in Supersomes 2D6, were assessed by the HPLC method with fluorimetric detection, as described previously [13].
CYP2C9 activity assay
CYP3A4 activity assay
The activity of CYP2C9 was studied by measuring the rate of a CYP2C9-specific reaction, i.e., 40 -hydroxylation of diclofenac. The rate of diclofenac 40 -hydroxylation (diclofenac concentrations: 5, 10, 25, 50 mM) was assessed in the absence and presence of levomepromazine, added in vitro (levomepromazine concentrations: 1, 5, 10, 25 and 50 mM).
The activity of CYP3A4 was studied by measuring the rate of a CYP3A4-specific reaction, i.e., 6b-hydroxylation of testosterone. The rate of testosterone 6b-hydroxylation (testosterone concentrations: 50, 100, 200 and 300 mM) was assessed in the absence and presence of levomepromazine, added in vitro (levomepromazine concentrations: 1, 5, 10, 25 and 50 mM).
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Incubations were carried out in a system containing human liver microsomes from baculovirus-infected insect cells expressing CYP3A4 (Supersomes 3A4, 50 pmol/ml), a Tris/KCl buffer (50 mM, pH = 7.4), MgCl2 (3.0 mM), EDTA (1 mM), NADP (1.0 mM), glucose 6-phosphate (5 mM) and glucose-6-phosphate-dehydrogenase (1.7 U in 1 ml). The final incubation volume was 0.5 ml. After a 30-min incubation, the reaction was stopped by adding 100 ml of methanol. A water phase containing testosterone and its metabolite 6b-hydroxytestosterone was extracted with 6 ml of dichloromethane. The samples were centrifuged for 10 min at 2000 g. Concentrations of testosterone and its metabolite 6b-hydroxytestosterone, formed in Supersomes 3A4, were assessed by the HPLC method with UV detection, as described previously [14]. Determination of kinetic parameters The inhibitory effects of levomepromazine on CYP isoenzymes are shown as Dixon plots (1/V against I), which allowed us to estimate inhibition constants (Ki). Kinetic parameters (Km, Vmax, Ki) showing metabolism of CYP-specific substrates in Supersomes in the absence and presence of levomepromazine were obtained using a non-linear regression analysis (Program Sigma Plot 8.0; Enzyme Kinetics). Results and discussion Dixon’s plots of the metabolism of CYP-specific substrates, carried out in cDNA-expressed human liver microsomes in the absence or
presence of levomepromazine, demonstrated that the examined neuroleptic exerted a significant inhibitory effect on CYP1A2, CYP2D6 and CYP3A4; however, its potency in relation to specific CYP isoenzymes was diversified (Fig. 1A–C). On the other hand, levomepromazine did not affect the activities of CYP2C9 and CYP2C19 (data not shown). The kinetic parameters (Km, Vmax, CL, Ki) showing metabolism of CYP1A2-, CYP2D6- and CYP3A4-specific substrates in the absence and presence of levomepromazine, obtained by a nonlinear regression analysis, are presented in Tables 1 and 2. Levomepromazine potently inhibited CYP2D6 activity (Ki = 6 mM) in a competitive manner (Table 2). Moreover, that neuroleptic moderately diminished the activity of CYP1A2 (Ki = 47 mM) and CYP3A4 (Ki = 34 mM) via a mixed mechanism (Table 2). In all those cases, the value of clearance (CL) decreased in the presence of levomepromazine (Table 1). The obtained Ki values showing the inhibition of CYP1A2, CYP2D6 and CYP3A4 activities by levomepromazine correspond well with the results presented as Dixon’s plots (Fig. 1A–C). The mechanism of inhibition was estimated on the basis of changes in the Km and Vmax values of the tested inhibitor (levomepromazine) concentrations (Table 1). In the case of mixed inhibition, the Km and Vmax values changed at different inhibitor concentrations. In the case of competitive inhibition, the Vmax value did not significantly change, while the Km was altered at different inhibitor concentrations. The results obtained in the present study give a good support for earlier clinical observations on the potent inhibition of CYP2D6 activity (measured as a rate of codeine O-demethylation or debrisoquine oxidation) by levomepromazine [6–8].
Fig. 1. The influence of levomepromazine on the activity of CYP1A2, CYP2D6 and CYP3A4, measured as rates of CYP-specific reactions in human cDNA-expressed CYP isoenzymes (Dixon’s plots): (A) caffeine 3-N-demethylation (CYP1A2); (B) bufuralol 10 -hydroxylation (CYP2D6); (C) testosterone 6b-hydroxylation (CYP3A4). Each point represents the mean value of four independent analyses SEM. The Ki values are shown in Table 2. V: velocity of the reaction; I: concentration of the inhibitor (levomepromazine).
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Table 1 Kinetic parameters showing the influence of levomepromazine on the activity of CYP1A2, CYP2D6 and CYP3A4, measured as rates of CYP-specific reactions in vitro in human cDNA-expressed CYP isoenzymes. Kinetic parameters of CYP-specific reactions
Inhibitor (levomepromazine) concentrations (mM)
Bufuralol 10 -hydroxylation (CYP2D6)
Caffeine 3-N-demethylation (CYP1A2)
0 (control) 1 5 10 25 50
Testosterone 6b-hydroxylation (CYP3A4)
Km (mM)
Vmax (pmol/pmol CYP1A2/min)
CL (Vmax/Km)
Km (mM)
Vmax (pmol/pmol CYP2D6/min)
CL (Vmax/Km)
Km (mM)
Vmax (pmol/pmol CYP3A4/min)
CL (Vmax/Km)
471 439 395 375 364 360
6.95 0.72 0.58 0.53 0.33 0.30
0.0019 0.0016 0.0015 0.0014 0.0009 0.0008
6.95 7.71 14.10 23.58 46.50 94.41
2.37 2.28 2.12 2.17 2.35 2.94
0.34 0.29 0.15 0.09 0.05 0.03
212 198 145 157 156 210
98.0 91.3 70.4 64.5 47.7 40.9
0.46 0.46 0.45 0.41 0.30 0.19
The presented values of Michaelis–Menten constants (Km), the maximum velocities of the reactions (Vmax) and the intrinsic clearance (CL) for particular CYP-specific reactions were obtained using a non-linear regression analysis (Program Sigma Plot 8.0; Enzyme Kinetics). The presented kinetic parameters are based on the data shown in Fig. 1A–C. Table 2 The ability of levomepromazine to inhibit CYP1A2, CYP2D6 and CYP3A4 activities in vitro in human cDNA-expressed CYP isoenzymes. Inhibition of CYP-specific reactions by levomepromazine Ki (mM) and type of inhibition
Inhibitor
Levomepromazine
Caffeine 3-N-demethylation (CYP1A2)
Bufuralol 10 -hydroxylation (CYP2D6)
Testosterone 6b-hydroxylation (CYP3A4)
47 (mixed)
6 (competitive)
34 (mixed)
The presented inhibition constants (Ki) for the inhibition of particular CYP-specific reactions by levomepromazine were obtained using a non-linear regression analysis (Program Sigma Plot 8.0; Enzyme Kinetics) and are based on the data shown in Fig. 1A–C.
On the other hand, our present results are not consistent with those obtained by Gervasini et al. [9], the latter indicating that levomepromazine inhibited fairly moderately (and to a similar degree) human CYP2D6 and CYP3A4. The observed discrepancies may stem from diverse methodological approaches (determination of IC50 or Ki), as well as from different conditions of the performed experiments and from the specificity of substrates used to estimate the activity of CYP isoenzymes. The obtained results revealed interactions between levomepromazine and human CYP2D6, CYP1A2 and CYP3A4, which led to a decrease in the isoenzyme activities. The Ki values obtained for CYP2D6, CYP3A4 and CYP1A2 (6, 34 and 47 mM, respectively) may be of importance in vivo regarding the dosage and the pharmacokinetics of levomepromazine. Phenothiazine neuroleptics were administered in relatively high doses compared to other neuroleptics and, being taken up by tissues, they reached concentrations that were 10–20 times higher in the liver than in blood plasma [15,16]. Therefore phenothiazine neuroleptics (including levomepromazine) whose Ki values are below 50 mM may reach the hepatic level close to the respective Ki values and are expected to decrease CYP2D6, CYP1A2 and CYP3A4 activities in vivo. Accordingly, Kudo et al. [17] demonstrated a fatal case of amoxapine poisoning after concomitant levomepromazine administration to depressive patients. Levomepromazine strongly elevated the concentration of amoxapine in blood plasma (up to 500% of the therapeutic level), while the concentration of levomepromazine was within the therapeutic range. Since amoxapine is metabolized mainly by CYP2D6 and CYP3A4 [18], the observed pharmacokinetic interaction between amoxapine and levomepromazine may stem from the CYP2D6 and CYP3A4 inhibition by levomepromazine. Despite its potent inhibition of human CYP2D6, levomepromazine exerts a weaker inhibitory effect on rat CYP2D (Ki = 20 mM) [19]. Since human CYP2D6 and rat CYP2D are very similar in amino acid sequences, the observed species differences in the inhibitory effect of levomepromazine on the enzyme activity may lie in
diverse structures of the catalytic sites of counterpart enzymes in rats and humans. Accordingly, specific inhibitors of CYP2D isoforms differentiate rat and human isoforms; quinine is a more potent inhibitor of human CYP2D6 than of rat CYP2D, while its diastereoisomer quinine is a more potent inhibitor of rat CYP2D than of human CYP2D6 [20]. The inhibition of CYP1A2, CYP2D6 and CYP3A4 by levomepromazine, demonstrated in vitro in the present study, may also be observed in vivo (especially the CYP2D6 inhibition by levomepromazine), since the calculated Ki values are below or close to the presumed concentration range for levomepromazine in the liver in vivo. Therefore pharmacokinetic interactions between levomepromazine and the substrates of CYP2D6 (e.g. tricyclic antidepressants, SSRIs, thioridazine, haloperidol, risperidone, codeine, dextromethorphan, debrisoquine, metoprolol, propranolol), CYP1A2 (e.g. caffeine, theophylline, phenacetin, tricyclic antidepressants, propranolol, clozapine, chlorpromazine) or CYP3A4 (e.g. benzodiazepines, calcium channel antagonists, macrolide antibiotics, testosterone) are likely to occur in patients during co-administration of the above-mentioned substrates/drugs. Conflict of interest The authors declare that they have no conflict of interest. Funding The study was supported by statutory funds from the Institute of Pharmacology, Polish Academy of Sciences Krako´w, Poland. References [1] Pelkonen O, Turpeinen M, Hakkola J, Honkakoski P, Hukkanen J, Raunio H. Inhibition and induction of human cytochrome P450 enzymes: current status. Arch Toxicol 2008;82:667–715. [2] Green B, Pettit T, Faith L, Seaton K. Focus on levomepromazine. Curr Med Res Opin 2004;20:1877–81.
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