Pharmacological Reports 66 (2014) 1122–1126
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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Short communication
The influence of amitriptyline and carbamazepine on levomepromazine metabolism in human liver: An in vitro study Jacek Wo´jcikowski *, Agnieszka Basin´ska, Jan Boksa, Władysława A. Daniel Institute of Pharmacology, Polish Academy of Sciences, Krako´w, Poland
A R T I C L E I N F O
Article history: Received 16 May 2014 Received in revised form 23 July 2014 Accepted 24 July 2014 Available online 7 August 2014 Keywords: Amitriptyline Carbamazepine Levomepromazine metabolism Human liver microsomes Cytochrome P450 inhibition
A B S T R A C T
Background: Joint administration of phenothiazine neuroleptics and an antidepressant or carbamazepine is applied in the therapy of many complex psychiatric disorders. The aim of the present study was to investigate possible effects of the tricyclic antidepressant drug amitriptyline and the anticonvulsant drug carbamazepine on the metabolism of the aliphatic-type phenothiazine neuroleptic levomepromazine in human liver. Methods: The experiment was performed in vitro using human liver microsomes. The rates of levomepromazine 5-sulfoxidation and N-demethylation (levomepromazine concentrations: 5, 10, 25 and 50 mM) were assessed in the absence and presence of amitriptyline or carbamazepine added in vitro (drug concentrations: 1, 2.5, 5, 10, 25 mM). Results: A kinetic analysis of levomepromazine metabolism carried out in the absence or presence of carbamazepine showed that the anticonvulsant drug potently inhibited levomepromazine 5sulfoxidation (Ki = 7.6 mM, non-competitive inhibition), and moderately decreased the rate of levomepromazine N-demethylation (Ki = 15.4 mM, mixed inhibition) at therapeutic drug concentrations. On the other hand, amitriptyline weakly diminished the rate of levomepromazine 5-sulfoxidation (Ki = 63 mM, mixed inhibition) and N-demethylation (Ki = 47.7 mM, mixed inhibition). Conclusion: Regarding the central and peripheral effects of levomepromazine and some of its metabolites, the observed metabolic interaction between this neuroleptic and carbamazepine may be of pharmacological and clinical importance. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Combinations of phenothiazine neuroleptics and antidepressants are administered to psychiatric patients in the therapy of agitated and psychotic depressions, depressions in the course of schizoaffective disorders or schizophrenia, and in treatmentresistant depressions. Furthermore, a combination of neuroleptics and carbamazepine is given to psychiatric patients in the therapy of manic and manic-depressive illnesses, as well as to optimize the treatment of schizophrenia [1]. Such drug combinations may produce clinically important interactions. Levomepromazine belongs to a group of phenothiazine neuroleptics of the aliphatic-type. The pharmacological profile and clinical effects of levomepromazine make it is still useful in
* Corresponding author. E-mail address: [email protected] (J. Wo´jcikowski).
the therapy of different psychiatric and non-psychiatric states. In psychiatry, levomepromazine is used as a sedative and is useful in the management of schizophrenia and depressive symptoms in the course of schizophrenia. It is also used in terminal pain control and postoperative analgesia; in addition, it helps to control nausea [2]. Levomepromazine is a moderate antagonist of the dopaminergic D2 receptor which is responsible for its antipsychotic effect. Moreover, this neuroleptic is a moderate blocker of adrenergic a1 and muscarinic M1 receptors which might be associated with some side effects of the drug, such as hypotension, sedation and anticholinergic symptoms [3]. On the other hand, levomepromazine is a potent antagonist of serotonergic 5-HT2 receptors [3], these receptors being important targets for atypical neuroleptics and for the attenuation of extrapyramidal side effects and an anxiolytic action [4]. Levomepromazine is metabolized in man by five routes: 5sulfoxidation, N-demethylation, O-demethylation and hydroxylation in position 3 and 7 of the ring structure. N-demethylation and
http://dx.doi.org/10.1016/j.pharep.2014.07.012 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
J. Wo´jcikowski et al. / Pharmacological Reports 66 (2014) 1122–1126
5-sulfoxidation were reported to be dominant pathways of levomepromazine biotransformation in humans [2]. N-desmethyllevomepromazine showed an almost equally potent receptor binding activity (dopaminergic and a1-adrenergic) as did the parent drug, while 5-sulfoxide was considerably less active in that respect [5]. Our recent study has shown that the hepatic cytochrome P450 (CYP) isoenzyme 3A4 is the main CYP isoform that catalyzes the 5-sulfoxidation and N-demethylation of levomepromazine at a therapeutic concentration of the drug [6]. It has also been shown that CYP3A4 is chiefly involved in the metabolism (N-demethylation) of the tricyclic antidepressant drug amitriptyline, as well as in the 10,11-epoxidation of the anticonvulsant drug carbamazepine [7,8]. Thus, metabolic interactions between levomepromazine and amitriptyline or carbamazepine seem quite possible. As shown previously, phenothiazine neuroleptics (such as promazine, perazine, thioridazine and chlorpromazine) and antidepressant drugs (tricyclics, SSRIs) mutually increase their concentrations in human blood plasma and rat plasma and brain [9–14]. On the other hand, joint administration of carbamazepine and psychotropic drugs produces different effects depending on the dosage scheduled [15–17]. The aim of the present in vitro study was to investigate the possible influence of carbamazepine and amitriptyline on the metabolism of levomepromazine in human liver. Materials and methods Drugs and chemicals Levomepromazine was obtained from Egyt (Budapest, Hungary). Amitriptyline was provided by H. Lundbeck A/S (Copenhagen, Denmark), while carbamazepine was from Polfa (Starogard, Poland). Levomepromazine 5-sulfoxide and N-desmethyllevomepromazine were synthesized in our laboratory as described previously [6]. NADPH was purchased from Sigma (St. Louis, USA). All the organic solvents with HPLC purity were supplied by Merck (Darmstadt, Germany). In vitro studies of levomepromazine metabolism in human liver microsomes Pooled human liver microsomes from patients HG03, HG18, HG24, HG64, HG88, HG93, HH35, HH40 (BD Biosciences, Woburn, MA, USA) were used. The optimization of levomepromazine metabolism in human liver microsomes proceeded in three steps, during which metabolic reactions (levomepromazine 5-sulfoxidation and N-demethylation) were carried out: (1) at different time intervals, at microsomal protein and substrate concentrations of 1 mg/ml and 100 nmol/ml, respectively; (2) at different concentrations of microsomal protein, at a substrate concentration of 100 nmol/ml and an incubation time of 45 min (time interval chosen experimentally in the first step); and (3) at different substrate concentrations, at a microsomal protein concentration of 1 mg/ml (protein concentration chosen experimentally in the second step) and an incubation time of 45 min. The influence of amitriptyline and carbamazepine on levomepromazine metabolism in vitro Based on the obtained results, studies into levomepromazine metabolism in human liver microsomes were carried out at the linear dependence of product formation on time, protein and substrate concentration. The rates of levomepromazine 5-sulfoxidation and N-demethylation (levomepromazine concentrations: 5, 10, 25 and 50 mM) were assessed in the absence and presence of
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amitriptyline or carbamazepine added in vitro (drug concentrations: 1, 2.5, 5, 10, 25 mM). Incubation was carried out in a system containing liver microsomes (1 mg of protein/ml), phosphate buffer (0.15 M, pH 7.4) and NADPH (1 mM). The final incubation volume was 0.5 ml. Each sample was prepared in duplicate. After a 45-min incubation, the reaction was terminated by adding 200 ml of methanol and cooling it down to 0 8C. Determination of levomepromazine and its metabolites in the incubation medium Levomepromazine and its metabolites were quantified using the HPLC method as described previously [6]. Briefly, after incubation, the samples were centrifuged for 10 min at 2000 g. The water phase containing levomepromazine and its metabolites was extracted (pH = 12) with n-heptane (6 ml). The residue obtained after evaporation of the microsomal extracts was dissolved in 100 ml of the mobile phase described below. An aliquot of 20 ml was injected into the HPLC system. The concentrations of levomepromazine and its metabolites (levomepromazine 5-sulfoxide and N-desmethyllevomepromazine) were assayed using a LaChrom (Merck-Hitachi) HPLC system with UV detection. The analytical column (Econosphere C18, 5 mm, 4.6 mm 250 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 1000 ml of the buffer adjusted to pH = 3.4 with an 85% phosphoric acid), and acetonitrile in the proportion of 30:70. The flow rate was 1 ml/min, the column temperature was ambient. The absorbance of levomepromazine and its metabolites was measured at a wavelength of 254 nm. The lower limit of quantification (LLOQ) was 0.002 nmol/ml for levomepromazine 5-sulfoxide, 0.003 nmol/ml for N-desmethyllevomepromazine and 0.005 nmol/ml for levomepromazine. The accuracy of the method varied between 98 and 101%. The precision for both within-day and between-day determinations of levomepromazine and its metabolites was between 1.7 and 4.6%. The obtained results are presented as Dixon plots. The Dixon plot (1/V plotted against I) is used in the study of inhibitors. This plotting technique is useful in estimating Ki and additionally it differentiates between partial and complete inhibition. Kinetic parameters describing levomepromazine metabolism in human liver microsomes in the absence and presence of amitriptyline or carbamazepine were obtained using a non-linear regression analysis (Program Sigma Plot 8.0; enzyme kinetics). Results and discussion The obtained results showed that levomepromazine 5-sulfoxidation and N-demethylation proceeded linearly up to 90 min, and over the entire range of the levomepromazine concentrations tested (up to 200 mM). Moreover, the amount of levomepromazine metabolites formed significantly increased up to 1.5 mg/ml of the protein concentration (data not shown). Considering the above observations, the following parameters were chosen for further studies: an incubation time of 45 min, a microsomal protein concentration of 1 mg/ml and substrate concentrations of 5, 10, 25 and 50 mM. The first two concentrations of the substrate were in the range of pharmacological neuroleptic concentrations present in vivo, while the last two concentrations were close to the control values of Km, obtained in vitro for levomepromazine metabolism (Tables 1 and 2). The incubation of liver microsomes with levomepromazine (in the absence and presence of amitriptyline or carbamazepine) was carried out under linear reaction conditions with respect to time, microsomal protein and substrate concentrations.
J. Wo´jcikowski et al. / Pharmacological Reports 66 (2014) 1122–1126
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Table 1 The influence of amitriptyline on the metabolism of levomepromazine in vitro in human liver microsomes. Kinetic parameters of levomepromazine metabolism
Inhibitor (amitriptyline) concentrations (mM)
Levomepromazine 5-sulfoxidation
0 (control) 1.0 2.5 5.0 10.0 25.0
Levomepromazine N-demethylation
Km (mM)
Vmax (pmol/mg protein/min)
CL (Vmax/Km)
Km (mM)
Vmax (pmol/mg protein/min)
CL (Vmax/Km)
35.49 61.09 35.05 54.24 25.54 48.91
19.11 26.72 17.26 21.98 11.59 16.93
0.54 0.44 0.49 0.40 0.45 0.35
23.12 28.00 28.74 25.85 27.39 35.18
219.50 258.60 225.00 202.30 196.40 200.20
9.49 9.23 7.83 7.82 7.17 5.69
The presented values of Michaelis–Menten constants (Km), maximum velocities of the reactions (Vmax) and intrinsic clearances (CL) for particular metabolic pathway were obtained using a non-linear regression analysis (Program Sigma Plot 8.0; enzyme kinetics). The values of inhibition constants (Ki) are presented in Table 3. Table 2 The influence of carbamazepine on the metabolism of levomepromazine in vitro in human liver microsomes. Inhibitor (carbamazepine) concentrations (mM)
Kinetic parameters of levomepromazine metabolism Levomepromazine 5-sulfoxidation
0 (control) 1.0 2.5 5.0 10.0 25.0
Levomepromazine N-demethylation
Km (mM)
Vmax (pmol/mg protein/min)
CL (Vmax/Km)
Km (mM)
Vmax (pmol/mg protein/min)
CL (Vmax/Km)
49.80 49.32 53.17 46.30 224.80 313.50
16.69 14.11 12.54 10.08 25.80 22.58
0.34 0.29 0.23 0.22 0.11 0.07
33.61 23.93 22.92 19.71 30.28 17.29
304.10 189.30 165.50 134.30 148.00 56.77
9.05 7.91 7.22 6.81 4.89 3.28
The presented values of Michaelis–Menten constants (Km), maximum velocities of the reactions (Vmax) and intrinsic clearances (CL) for particular metabolic pathway were obtained using a non-linear regression analysis (Program Sigma Plot 8.0; enzyme kinetics). The values of inhibition constants (Ki) are presented in Table 3.
The kinetic parameters describing levomepromazine metabolism in human liver microsomes (Km, Vmax, CL, Ki), in the absence and presence of amitriptyline or carbamazepine, obtained using a non-linear regression analysis (Program Sigma Plot 8.0; enzyme kinetics), are presented in Tables 1–3. The mechanism of inhibition was estimated on the basis of the changes of Km and Vmax values for the tested inhibitor (amitriptyline or carbamazepine) concentrations. In the case of mixed inhibition, the Km and Vmax values change at different inhibitor concentrations. In non-competitive inhibition, the Km values do not significantly change, while Vmax values change at different inhibitor concentrations. The mechanisms of inhibition found in our experiments are valid for amitriptyline concentrations up to 10 mM and for carbamazepine concentrations up to 5 mM. Amitriptyline weakly diminished levomepromazine 5-sulfoxidation and N-demetylation via a mixed mechanism (Ki = 63 and 47.7 mM, respectively) (Tables 1 and 3). On the other hand, carbamazepine potently inhibited levomepromazine 5-sulfoxidation in a non-competitive manner (Ki = 7.6 mM), and moderately decreased the rate of N-demethylation in a mixed-type manner (Ki = 15.2 mM) (Tables 2 and 3). In all those cases, the value of clearance (CL) diminished in the presence of an antidepressant. The
Table 3 The potency of amitriptyline and carbamazepine to inhibit levomepromazine metabolism in vitro in human liver microsomes. Inhibitor
Amitriptyline Carbamazepine
Inhibition of levomepromazine metabolism Ki (mM) and type of inhibition Levomepromazine 5-sulfoxidation
Levomepromazine N-demethylation
63.0 (mixed) 7.6 (non-competitive)
47.7 (mixed) 15.2 (mixed)
The presented inhibition constants (Ki) for inhibition of particular metabolic pathway were obtained using a non-linear regression analysis (Program Sigma Plot 8.0; enzyme kinetics).
above results showing inhibition of levomepromazine metabolism by the co-administered drugs correspond to those presented as Dixon’s plots, the latter illustrating Ki values (Fig. 1A and B). The obtained results suggest that amitriptyline or carbamazepine used at therapeutic concentrations exert an inhibitory effect on levomepromazine metabolism in human liver, but their potencies in respect of particular metabolic pathways are different. The inhibition of levomepromazine metabolism by amitriptyline and carbamazepine may steam partly from competition between levomepromazine and co-administered drugs for the active sites of CYP3A4. It has been shown that of CYP isoforms, CYP3A4 is mainly involved in levomepromazine 5-sulfoxidation and N-demethylation, as well as in amitriptyline N-demethylation and carbamazepine 10,11-epoxidation [6–8]. On the other hand, carbamazepine potently inhibits levomepromazine 5-sulfoxidation in a noncompetitive manner. The relatively weaker effect of amitriptyline compared to carbamazepine on levomepromazine metabolism may be due to the fact that CYP3A4 is the major isoform engaged in amitriptyline N-demethylation at a high, non-therapeutic concentration of the antidepressant (100 mM). At lower, therapeutic concentrations of amitriptyline (used in the present study), the contribution of CYP3A4 to the antidepressant N-demethylation decreases, mostly in favour of CYP2C19 [18]. In the case of carbamazepine, CYP3A4 is the major isoform that catalyzes 10,11epoxidation of this anticonvulsant at both low and high concentrations of carbamazepine [8]. Phenothiazine neuroleptics and tricyclic antidepressants are taken up by tissues reaching in the liver concentrations which are about 10 times higher (up to 10 mM) than those in the plasma [19]. Hence the interaction between levomepromazine and amitriptyline observed in vitro in the present study should not be observed in vivo, since the calculated Ki values for amitriptyline are above the presumed concentration range for this antidepressant in the liver in vivo, this finding being based on both pharmacological experiments and the data obtained with psychiatric patients [19,20].
J. Wo´jcikowski et al. / Pharmacological Reports 66 (2014) 1122–1126
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Fig. 1. The influence of amitriptyline and carbamazepine on the metabolism of levomepromazine in human liver microsomes: (A) levomepromazine 5-sulfoxidation; (B) levomepromazine N-demethylation (Dixon’s plots). Each point represents the mean value of two independent analyses. V, velocity of the reaction; I, concentration of the inhibitor (amitriptyline or carbamazepine).
On the other hand, the interaction between levomepromazine and carbamazepine observed in vitro in the present study should also be observed in vivo, since the calculated Ki values for carbamazepine are below its therapeutic concentration range (17–42 mM) [19]. However, carbamazepine given chronically results in CYP3A4 induction [16]. Therefore it seems possible that the dual effect of carbamazepine on CYP3A4 in vivo, i.e., the direct inhibitory effect and the indirect inductive action of carbamazepine on the CYP3A4-mediated metabolism of levomepromazine may overlap and thus attenuate each other’s effect. Studies carried out on rats showed that shortly after drug administration, carbamazepine inhibited promazine metabolism, whereas after a long interval carbamazepine induced metabolism of the coadministered neuroleptic [15–17]. Furthermore, co-administration of carbamazepine to patients lowered the plasma levels of other psychotropic drugs such as, e.g., tricyclic antidepressants, trazodone, clonazepam, clozapine or haloperidol [15]. In some cases, however, introduction of carbamazepine into the therapy worsened psychotic symptoms. In summary, regarding the receptor blocking properties of levomepromazine (D2, a1 and M1 receptors) and some of its metabolites that produce their central and peripheral sideeffects (e.g., a decrease in blood pressure, anticholinergic effects, extrapyramidal symptoms), the levomepromazine-carbamazepine interaction may be of pharmacological and clinical importance.
Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments The study was supported by grant no. 2011/01/B/NZ4/04859 from the National Science Centre, Krako´w, Poland and by statutory funds from the Institute of Pharmacology, Polish Academy of Sciences, Krako´w, Poland. References [1] Nelson JC. Combined treatment strategies in psychiatry. J Clin Psychiatry 1993;54(Suppl. 9):42–9. [2] Green B, Pettit T, Faith L, Seaton K. Focus on levomepromazine. Curr Med Res Opin 2004;20:1877–81. [3] Lal S, Nair NP, Cecyre D, Quirion R. Levomepromazine receptor binding profile in human brain – implications for treatment-resistant schizophrenia. Acta Psychiatr Scand 1993;87:380–3. [4] Meltzer HY. Serotonergic mechanisms as targets for existing and novel antipsychotics. Handb Exp Pharmacol 2012;212:87–124. [5] Hals PA, Hall H, Dahl SG. Phenothiazine drugs metabolites: dopamine D2 receptor a1 and a2-adrenoceptor binding. Eur J Pharmacol 1986;125:373–81. [6] 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. [7] Ghahramani P, Ellis SW, Lennard MS, Ramsay LE, Tucker GT. Cytochromes P450 mediating the N-demethylation of amitriptyline. Br J Clin Pharmacol 1997;43:137–44.
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[8] Kerr BM, Thummel KE, Wurden CJ, Klein SM, Kroetz DL, Gonzalez FJ, et al. Human liver carbamazepine metabolism role of CYP3A4 and CYP2C8 in 10,11epoxide formation. Biochem Pharmacol 1994;47:1969–79. [9] Daniel WA, Syrek M, Haduch A, Wo´jcikowski J. Different effects of amitriptyline and imipramine on the pharmacokinetics and metabolism of perazine in rats. J Pharm Pharmacol 2000;52:1473–81. [10] Daniel WA, Syrek M, Haduch A, Wo´jcikowski J. Pharmacokinetics and metabolism of thioridazine during co-administration of tricyclic antidepressants. Br J Pharmacol 2000;131:287–95. [11] Daniel WA, Syrek M, Wo´jcikowski J. The influence of selective serotonin reuptake inhibitors on the plasma and brain pharmacokinetics of the simplest phenothiazine neuroleptic promazine in rat. Eur Neuropsychopharmacol 1999;9:337–44. [12] Loga S, Curry S, Lader M. Interaction of chlorpromazine and nortryptyline in patients with schizophrenia. Clin Pharmacokinet 1981;6:454–62. [13] Nelson JC, Jatlow PI. Neuroleptic effect on desipramine steady-state plasma concentration. Am J Psychiatry 1980;137:1232–4. [14] Vandel B, Vandel S, Allers G, Bechtel P, Volmat R. Interaction between amitriptyline and phenothiazine in man: effect on plasma concentration of
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amitriptyline and its metabolite nortriptyline and the correlation with clinical response. Psychopharmacology 1979;65:187–90. Daniel WA, Syrek M, Haduch A, Wo´jcikowski J. Pharmacokinetics of phenothiazine neuroleptics after chronic coadministration of carbamazepine. Pol J Pharmacol 1998;50:431–42. Spina E, Pisani F, Perucca E. Clinically significant pharmacokinetic drug interactions with carbamazepine. An update. Clin Pharmacokinet 1996;31: 198–214. Syrek M, Wo´jcikowski J, Daniel W. Effect of carbamazepine on the pharmacokinetics of promazine. Pol J Pharmacol 1996;48:601–8. Venkatakrishnan K, Greenblatt DJ, von Moltke LL, Schmider J, Harmatz JS, Shader RI. Five distinct human cytochromes mediate amitriptyline N-demethylation in vitro: dominance of CYP 2C19 and 3A4. J Clin Pharmacol 1998;38:112–21. Wo´jcikowski J, Daniel WA. Distribution interactions between perazine and antidepressant drugs. In vivo studies. Pol J Pharmacol 2000;52:449–57. Baumann P, Hiemke C, Ulrich S, Eckermann G, Gaertner I, Kuss HJ, et al. The AGNP TDM expert group consensus guidelines: therapeutic drug monitoring in psychiatry. Pharmacopsychiatry 2004;37:243–65.
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Short communication
The influence of amitriptyline and carbamazepine on levomepromazine metabolism in human liver: An in vitro study Jacek Wo´jcikowski *, Agnieszka Basin´ska, Jan Boksa, Władysława A. Daniel Institute of Pharmacology, Polish Academy of Sciences, Krako´w, Poland
A R T I C L E I N F O
Article history: Received 16 May 2014 Received in revised form 23 July 2014 Accepted 24 July 2014 Available online 7 August 2014 Keywords: Amitriptyline Carbamazepine Levomepromazine metabolism Human liver microsomes Cytochrome P450 inhibition
A B S T R A C T
Background: Joint administration of phenothiazine neuroleptics and an antidepressant or carbamazepine is applied in the therapy of many complex psychiatric disorders. The aim of the present study was to investigate possible effects of the tricyclic antidepressant drug amitriptyline and the anticonvulsant drug carbamazepine on the metabolism of the aliphatic-type phenothiazine neuroleptic levomepromazine in human liver. Methods: The experiment was performed in vitro using human liver microsomes. The rates of levomepromazine 5-sulfoxidation and N-demethylation (levomepromazine concentrations: 5, 10, 25 and 50 mM) were assessed in the absence and presence of amitriptyline or carbamazepine added in vitro (drug concentrations: 1, 2.5, 5, 10, 25 mM). Results: A kinetic analysis of levomepromazine metabolism carried out in the absence or presence of carbamazepine showed that the anticonvulsant drug potently inhibited levomepromazine 5sulfoxidation (Ki = 7.6 mM, non-competitive inhibition), and moderately decreased the rate of levomepromazine N-demethylation (Ki = 15.4 mM, mixed inhibition) at therapeutic drug concentrations. On the other hand, amitriptyline weakly diminished the rate of levomepromazine 5-sulfoxidation (Ki = 63 mM, mixed inhibition) and N-demethylation (Ki = 47.7 mM, mixed inhibition). Conclusion: Regarding the central and peripheral effects of levomepromazine and some of its metabolites, the observed metabolic interaction between this neuroleptic and carbamazepine may be of pharmacological and clinical importance. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Combinations of phenothiazine neuroleptics and antidepressants are administered to psychiatric patients in the therapy of agitated and psychotic depressions, depressions in the course of schizoaffective disorders or schizophrenia, and in treatmentresistant depressions. Furthermore, a combination of neuroleptics and carbamazepine is given to psychiatric patients in the therapy of manic and manic-depressive illnesses, as well as to optimize the treatment of schizophrenia [1]. Such drug combinations may produce clinically important interactions. Levomepromazine belongs to a group of phenothiazine neuroleptics of the aliphatic-type. The pharmacological profile and clinical effects of levomepromazine make it is still useful in
* Corresponding author. E-mail address: [email protected] (J. Wo´jcikowski).
the therapy of different psychiatric and non-psychiatric states. In psychiatry, levomepromazine is used as a sedative and is useful in the management of schizophrenia and depressive symptoms in the course of schizophrenia. It is also used in terminal pain control and postoperative analgesia; in addition, it helps to control nausea [2]. Levomepromazine is a moderate antagonist of the dopaminergic D2 receptor which is responsible for its antipsychotic effect. Moreover, this neuroleptic is a moderate blocker of adrenergic a1 and muscarinic M1 receptors which might be associated with some side effects of the drug, such as hypotension, sedation and anticholinergic symptoms [3]. On the other hand, levomepromazine is a potent antagonist of serotonergic 5-HT2 receptors [3], these receptors being important targets for atypical neuroleptics and for the attenuation of extrapyramidal side effects and an anxiolytic action [4]. Levomepromazine is metabolized in man by five routes: 5sulfoxidation, N-demethylation, O-demethylation and hydroxylation in position 3 and 7 of the ring structure. N-demethylation and
http://dx.doi.org/10.1016/j.pharep.2014.07.012 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
J. Wo´jcikowski et al. / Pharmacological Reports 66 (2014) 1122–1126
5-sulfoxidation were reported to be dominant pathways of levomepromazine biotransformation in humans [2]. N-desmethyllevomepromazine showed an almost equally potent receptor binding activity (dopaminergic and a1-adrenergic) as did the parent drug, while 5-sulfoxide was considerably less active in that respect [5]. Our recent study has shown that the hepatic cytochrome P450 (CYP) isoenzyme 3A4 is the main CYP isoform that catalyzes the 5-sulfoxidation and N-demethylation of levomepromazine at a therapeutic concentration of the drug [6]. It has also been shown that CYP3A4 is chiefly involved in the metabolism (N-demethylation) of the tricyclic antidepressant drug amitriptyline, as well as in the 10,11-epoxidation of the anticonvulsant drug carbamazepine [7,8]. Thus, metabolic interactions between levomepromazine and amitriptyline or carbamazepine seem quite possible. As shown previously, phenothiazine neuroleptics (such as promazine, perazine, thioridazine and chlorpromazine) and antidepressant drugs (tricyclics, SSRIs) mutually increase their concentrations in human blood plasma and rat plasma and brain [9–14]. On the other hand, joint administration of carbamazepine and psychotropic drugs produces different effects depending on the dosage scheduled [15–17]. The aim of the present in vitro study was to investigate the possible influence of carbamazepine and amitriptyline on the metabolism of levomepromazine in human liver. Materials and methods Drugs and chemicals Levomepromazine was obtained from Egyt (Budapest, Hungary). Amitriptyline was provided by H. Lundbeck A/S (Copenhagen, Denmark), while carbamazepine was from Polfa (Starogard, Poland). Levomepromazine 5-sulfoxide and N-desmethyllevomepromazine were synthesized in our laboratory as described previously [6]. NADPH was purchased from Sigma (St. Louis, USA). All the organic solvents with HPLC purity were supplied by Merck (Darmstadt, Germany). In vitro studies of levomepromazine metabolism in human liver microsomes Pooled human liver microsomes from patients HG03, HG18, HG24, HG64, HG88, HG93, HH35, HH40 (BD Biosciences, Woburn, MA, USA) were used. The optimization of levomepromazine metabolism in human liver microsomes proceeded in three steps, during which metabolic reactions (levomepromazine 5-sulfoxidation and N-demethylation) were carried out: (1) at different time intervals, at microsomal protein and substrate concentrations of 1 mg/ml and 100 nmol/ml, respectively; (2) at different concentrations of microsomal protein, at a substrate concentration of 100 nmol/ml and an incubation time of 45 min (time interval chosen experimentally in the first step); and (3) at different substrate concentrations, at a microsomal protein concentration of 1 mg/ml (protein concentration chosen experimentally in the second step) and an incubation time of 45 min. The influence of amitriptyline and carbamazepine on levomepromazine metabolism in vitro Based on the obtained results, studies into levomepromazine metabolism in human liver microsomes were carried out at the linear dependence of product formation on time, protein and substrate concentration. The rates of levomepromazine 5-sulfoxidation and N-demethylation (levomepromazine concentrations: 5, 10, 25 and 50 mM) were assessed in the absence and presence of
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amitriptyline or carbamazepine added in vitro (drug concentrations: 1, 2.5, 5, 10, 25 mM). Incubation was carried out in a system containing liver microsomes (1 mg of protein/ml), phosphate buffer (0.15 M, pH 7.4) and NADPH (1 mM). The final incubation volume was 0.5 ml. Each sample was prepared in duplicate. After a 45-min incubation, the reaction was terminated by adding 200 ml of methanol and cooling it down to 0 8C. Determination of levomepromazine and its metabolites in the incubation medium Levomepromazine and its metabolites were quantified using the HPLC method as described previously [6]. Briefly, after incubation, the samples were centrifuged for 10 min at 2000 g. The water phase containing levomepromazine and its metabolites was extracted (pH = 12) with n-heptane (6 ml). The residue obtained after evaporation of the microsomal extracts was dissolved in 100 ml of the mobile phase described below. An aliquot of 20 ml was injected into the HPLC system. The concentrations of levomepromazine and its metabolites (levomepromazine 5-sulfoxide and N-desmethyllevomepromazine) were assayed using a LaChrom (Merck-Hitachi) HPLC system with UV detection. The analytical column (Econosphere C18, 5 mm, 4.6 mm 250 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 1000 ml of the buffer adjusted to pH = 3.4 with an 85% phosphoric acid), and acetonitrile in the proportion of 30:70. The flow rate was 1 ml/min, the column temperature was ambient. The absorbance of levomepromazine and its metabolites was measured at a wavelength of 254 nm. The lower limit of quantification (LLOQ) was 0.002 nmol/ml for levomepromazine 5-sulfoxide, 0.003 nmol/ml for N-desmethyllevomepromazine and 0.005 nmol/ml for levomepromazine. The accuracy of the method varied between 98 and 101%. The precision for both within-day and between-day determinations of levomepromazine and its metabolites was between 1.7 and 4.6%. The obtained results are presented as Dixon plots. The Dixon plot (1/V plotted against I) is used in the study of inhibitors. This plotting technique is useful in estimating Ki and additionally it differentiates between partial and complete inhibition. Kinetic parameters describing levomepromazine metabolism in human liver microsomes in the absence and presence of amitriptyline or carbamazepine were obtained using a non-linear regression analysis (Program Sigma Plot 8.0; enzyme kinetics). Results and discussion The obtained results showed that levomepromazine 5-sulfoxidation and N-demethylation proceeded linearly up to 90 min, and over the entire range of the levomepromazine concentrations tested (up to 200 mM). Moreover, the amount of levomepromazine metabolites formed significantly increased up to 1.5 mg/ml of the protein concentration (data not shown). Considering the above observations, the following parameters were chosen for further studies: an incubation time of 45 min, a microsomal protein concentration of 1 mg/ml and substrate concentrations of 5, 10, 25 and 50 mM. The first two concentrations of the substrate were in the range of pharmacological neuroleptic concentrations present in vivo, while the last two concentrations were close to the control values of Km, obtained in vitro for levomepromazine metabolism (Tables 1 and 2). The incubation of liver microsomes with levomepromazine (in the absence and presence of amitriptyline or carbamazepine) was carried out under linear reaction conditions with respect to time, microsomal protein and substrate concentrations.
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Table 1 The influence of amitriptyline on the metabolism of levomepromazine in vitro in human liver microsomes. Kinetic parameters of levomepromazine metabolism
Inhibitor (amitriptyline) concentrations (mM)
Levomepromazine 5-sulfoxidation
0 (control) 1.0 2.5 5.0 10.0 25.0
Levomepromazine N-demethylation
Km (mM)
Vmax (pmol/mg protein/min)
CL (Vmax/Km)
Km (mM)
Vmax (pmol/mg protein/min)
CL (Vmax/Km)
35.49 61.09 35.05 54.24 25.54 48.91
19.11 26.72 17.26 21.98 11.59 16.93
0.54 0.44 0.49 0.40 0.45 0.35
23.12 28.00 28.74 25.85 27.39 35.18
219.50 258.60 225.00 202.30 196.40 200.20
9.49 9.23 7.83 7.82 7.17 5.69
The presented values of Michaelis–Menten constants (Km), maximum velocities of the reactions (Vmax) and intrinsic clearances (CL) for particular metabolic pathway were obtained using a non-linear regression analysis (Program Sigma Plot 8.0; enzyme kinetics). The values of inhibition constants (Ki) are presented in Table 3. Table 2 The influence of carbamazepine on the metabolism of levomepromazine in vitro in human liver microsomes. Inhibitor (carbamazepine) concentrations (mM)
Kinetic parameters of levomepromazine metabolism Levomepromazine 5-sulfoxidation
0 (control) 1.0 2.5 5.0 10.0 25.0
Levomepromazine N-demethylation
Km (mM)
Vmax (pmol/mg protein/min)
CL (Vmax/Km)
Km (mM)
Vmax (pmol/mg protein/min)
CL (Vmax/Km)
49.80 49.32 53.17 46.30 224.80 313.50
16.69 14.11 12.54 10.08 25.80 22.58
0.34 0.29 0.23 0.22 0.11 0.07
33.61 23.93 22.92 19.71 30.28 17.29
304.10 189.30 165.50 134.30 148.00 56.77
9.05 7.91 7.22 6.81 4.89 3.28
The presented values of Michaelis–Menten constants (Km), maximum velocities of the reactions (Vmax) and intrinsic clearances (CL) for particular metabolic pathway were obtained using a non-linear regression analysis (Program Sigma Plot 8.0; enzyme kinetics). The values of inhibition constants (Ki) are presented in Table 3.
The kinetic parameters describing levomepromazine metabolism in human liver microsomes (Km, Vmax, CL, Ki), in the absence and presence of amitriptyline or carbamazepine, obtained using a non-linear regression analysis (Program Sigma Plot 8.0; enzyme kinetics), are presented in Tables 1–3. The mechanism of inhibition was estimated on the basis of the changes of Km and Vmax values for the tested inhibitor (amitriptyline or carbamazepine) concentrations. In the case of mixed inhibition, the Km and Vmax values change at different inhibitor concentrations. In non-competitive inhibition, the Km values do not significantly change, while Vmax values change at different inhibitor concentrations. The mechanisms of inhibition found in our experiments are valid for amitriptyline concentrations up to 10 mM and for carbamazepine concentrations up to 5 mM. Amitriptyline weakly diminished levomepromazine 5-sulfoxidation and N-demetylation via a mixed mechanism (Ki = 63 and 47.7 mM, respectively) (Tables 1 and 3). On the other hand, carbamazepine potently inhibited levomepromazine 5-sulfoxidation in a non-competitive manner (Ki = 7.6 mM), and moderately decreased the rate of N-demethylation in a mixed-type manner (Ki = 15.2 mM) (Tables 2 and 3). In all those cases, the value of clearance (CL) diminished in the presence of an antidepressant. The
Table 3 The potency of amitriptyline and carbamazepine to inhibit levomepromazine metabolism in vitro in human liver microsomes. Inhibitor
Amitriptyline Carbamazepine
Inhibition of levomepromazine metabolism Ki (mM) and type of inhibition Levomepromazine 5-sulfoxidation
Levomepromazine N-demethylation
63.0 (mixed) 7.6 (non-competitive)
47.7 (mixed) 15.2 (mixed)
The presented inhibition constants (Ki) for inhibition of particular metabolic pathway were obtained using a non-linear regression analysis (Program Sigma Plot 8.0; enzyme kinetics).
above results showing inhibition of levomepromazine metabolism by the co-administered drugs correspond to those presented as Dixon’s plots, the latter illustrating Ki values (Fig. 1A and B). The obtained results suggest that amitriptyline or carbamazepine used at therapeutic concentrations exert an inhibitory effect on levomepromazine metabolism in human liver, but their potencies in respect of particular metabolic pathways are different. The inhibition of levomepromazine metabolism by amitriptyline and carbamazepine may steam partly from competition between levomepromazine and co-administered drugs for the active sites of CYP3A4. It has been shown that of CYP isoforms, CYP3A4 is mainly involved in levomepromazine 5-sulfoxidation and N-demethylation, as well as in amitriptyline N-demethylation and carbamazepine 10,11-epoxidation [6–8]. On the other hand, carbamazepine potently inhibits levomepromazine 5-sulfoxidation in a noncompetitive manner. The relatively weaker effect of amitriptyline compared to carbamazepine on levomepromazine metabolism may be due to the fact that CYP3A4 is the major isoform engaged in amitriptyline N-demethylation at a high, non-therapeutic concentration of the antidepressant (100 mM). At lower, therapeutic concentrations of amitriptyline (used in the present study), the contribution of CYP3A4 to the antidepressant N-demethylation decreases, mostly in favour of CYP2C19 [18]. In the case of carbamazepine, CYP3A4 is the major isoform that catalyzes 10,11epoxidation of this anticonvulsant at both low and high concentrations of carbamazepine [8]. Phenothiazine neuroleptics and tricyclic antidepressants are taken up by tissues reaching in the liver concentrations which are about 10 times higher (up to 10 mM) than those in the plasma [19]. Hence the interaction between levomepromazine and amitriptyline observed in vitro in the present study should not be observed in vivo, since the calculated Ki values for amitriptyline are above the presumed concentration range for this antidepressant in the liver in vivo, this finding being based on both pharmacological experiments and the data obtained with psychiatric patients [19,20].
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Fig. 1. The influence of amitriptyline and carbamazepine on the metabolism of levomepromazine in human liver microsomes: (A) levomepromazine 5-sulfoxidation; (B) levomepromazine N-demethylation (Dixon’s plots). Each point represents the mean value of two independent analyses. V, velocity of the reaction; I, concentration of the inhibitor (amitriptyline or carbamazepine).
On the other hand, the interaction between levomepromazine and carbamazepine observed in vitro in the present study should also be observed in vivo, since the calculated Ki values for carbamazepine are below its therapeutic concentration range (17–42 mM) [19]. However, carbamazepine given chronically results in CYP3A4 induction [16]. Therefore it seems possible that the dual effect of carbamazepine on CYP3A4 in vivo, i.e., the direct inhibitory effect and the indirect inductive action of carbamazepine on the CYP3A4-mediated metabolism of levomepromazine may overlap and thus attenuate each other’s effect. Studies carried out on rats showed that shortly after drug administration, carbamazepine inhibited promazine metabolism, whereas after a long interval carbamazepine induced metabolism of the coadministered neuroleptic [15–17]. Furthermore, co-administration of carbamazepine to patients lowered the plasma levels of other psychotropic drugs such as, e.g., tricyclic antidepressants, trazodone, clonazepam, clozapine or haloperidol [15]. In some cases, however, introduction of carbamazepine into the therapy worsened psychotic symptoms. In summary, regarding the receptor blocking properties of levomepromazine (D2, a1 and M1 receptors) and some of its metabolites that produce their central and peripheral sideeffects (e.g., a decrease in blood pressure, anticholinergic effects, extrapyramidal symptoms), the levomepromazine-carbamazepine interaction may be of pharmacological and clinical importance.
Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments The study was supported by grant no. 2011/01/B/NZ4/04859 from the National Science Centre, Krako´w, Poland and by statutory funds from the Institute of Pharmacology, Polish Academy of Sciences, Krako´w, Poland. References [1] Nelson JC. Combined treatment strategies in psychiatry. J Clin Psychiatry 1993;54(Suppl. 9):42–9. [2] Green B, Pettit T, Faith L, Seaton K. Focus on levomepromazine. Curr Med Res Opin 2004;20:1877–81. [3] Lal S, Nair NP, Cecyre D, Quirion R. Levomepromazine receptor binding profile in human brain – implications for treatment-resistant schizophrenia. Acta Psychiatr Scand 1993;87:380–3. [4] Meltzer HY. Serotonergic mechanisms as targets for existing and novel antipsychotics. Handb Exp Pharmacol 2012;212:87–124. [5] Hals PA, Hall H, Dahl SG. Phenothiazine drugs metabolites: dopamine D2 receptor a1 and a2-adrenoceptor binding. Eur J Pharmacol 1986;125:373–81. [6] 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. [7] Ghahramani P, Ellis SW, Lennard MS, Ramsay LE, Tucker GT. Cytochromes P450 mediating the N-demethylation of amitriptyline. Br J Clin Pharmacol 1997;43:137–44.
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