The effect of psychotropic drugs on cytochrome P450 2D (CYP2D) in rat brain

European Journal of Pharmacology 651 (2011) 51–58

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Molecular and Cellular Pharmacology

The effect of psychotropic drugs on cytochrome P450 2D (CYP2D) in rat brain Anna Haduch, Ewa Bromek, Władysława A. Daniel ⁎ Polish Academy of Sciences, Institute of Pharmacology, Smętna 12, 31-343 Kraków, Poland

a r t i c l e

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Article history: Received 28 May 2010 Received in revised form 6 October 2010 Accepted 29 October 2010 Available online 27 November 2010 Keywords: Brain and liver microsome cDNA-expressed CYP2D Antidepressant Neuroleptic Chronic treatment (Rat CYP2D)

a b s t r a c t The aim of the study was to investigate the influence of selected antidepressants and neuroleptics on the protein level and activity of cytochrome P450 2D (CYP2D) in rat brain. The obtained results showed that imipramine, fluoxetine, nefazodone, thioridazine and perazine, added to brain microsomes of control rats, inhibited CYP2D activity to a lower extent (Ki = 255–485 μM) than when added to liver microsomes (Ki = 1– 45 μM), which may result from their stronger affinity for liver CYP2D2 (Ki = 2.7 and 1.25 μM for imipramine and fluoxetine, respectively) than for brain CYP2D4 (Ki = 25 and 10 μM for imipramine and fluoxetine, respectively), as well as from their high non-specific binding in brain microsomes. Two-week treatment with fluoxetine evoked decreases in the level and activity of CYP2D in the striatum and the nucleus accumbens. In contrast, fluoxetine increased CYP2D expression in the cerebellum, while nefazodone considerably enhanced the activity (but not the protein level) of CYP2D in the truncus cerebri. Imipramine and mirtazapine (active in the liver) did not affect brain CYP2D. Chronic thioridazine decreased CYP2D activity in the substantia nigra and nucleus accumbens, but significantly increased that activity in the striatum and cerebellum. Clozapine significantly enhanced CYP2D activity in the truncus cerebri. In conclusion, psychotropics influence CYP2D in the brain, but their effect is different than in the liver and depends on the cerebral structure. The observed psychotropics–brain CYP2D interactions may be important for the metabolism of neurosteroids and monoaminergic neurotransmitters, and for the local biotransformation of drugs. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Six isoforms (CYP2D1–5 and CYP2D18) constitute the CYP2D subfamily of cytochrome P450 in the rat, while only one isoform (CYP2D6) is present in man (Gonzalez et al., 1988; Kimura et al., 1989; Matsunaga et al., 1989; 1990; Kawashima et al., 1996). The main CYP2D isoform in rat brain is CYP2D4, while CYP2D1 and CYP2D2 are the most abundant CYP2D isoforms in the liver (Komori, 1993; Wyss et al. 1995; Hiroi et al., 1998a; Funae et al., 2003). The presence of the CYP2D protein in the brain was described predominantly in the cerebellum, cortex, amygdaloid complex, olfactory bulbs, as well as in the nucleus accumbens, hippocampus, substantia nigra and striatum (Miksys et al., 2000; Siegle et al., 2001; Chinta et al., 2002). Accordingly, a relatively higher level of rat CYP2D activity was found in the cerebellum, olfactory bulbs, hippocampus, brain stem (Tyndale et al., 1999), as well as in the substantia nigra and nucleus accumbens (Bromek et al., 2010). Brain CYP2D isoforms exhibit enzymatic activity towards endoand xenobiotics including psychotropics. The metabolism of tricyclic antidepressant drugs (TADs) with contribution of rat brain CYP2D has been described (Hansson et al., 1992; Kawashima et al., 1996; Sequeira and Strobel, 1996; Thompson et al., 1998; Voirol et al.,

⁎ Corresponding author. Tel.: + 48 12 6374022; fax: + 48 12 6374500. E-mail address: [email protected] (W.A. Daniel). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.10.077

2000). On the other hand, CYP2D isoforms in the brain seem to play an important role in the local metabolism of neurosteroids (Hiroi et al., 2001; Kishimoto et al., 2004), arachidonic acid (Kawashima et al., 1996; Thompson et al., 2000), as well as the neurotransmitters serotonin (Yu et al., 2003) and dopamine (Hiroi et al., 1998b; Bromek et al., 2010). The formation of dopamine from tyramine by brain CYP2D has been demonstrated in rats: in vitro — using brain microsomes (Bromek et al., 2010), and in vivo — by means of brain microdialysis (Bromek et al., in preparation). Therefore changes in brain CYP activity seem to be of physiological and pharmacological importance, the more so as CYP levels may be relatively high in particular regions or cells of the brain (Miksys et al., 2000; Miksys and Tyndale, 2004; Dutheil et al., 2009). Our previous studies showed that antidepressants and neuroleptics produced significant changes in the activity of CYP2D in rat liver. Apart from the direct effects of parent drugs on CYP2D activity (via binding to the enzyme protein), the investigated psychotropics also exerted substantial indirect effects (on CYP expression) due to a prolonged exposure to drugs (Daniel et al., 2002; 2005; Daniel, 2005; Haduch et al., 2006). Therefore it seemed of interest to find out whether the antidepressant- and neuroleptic-induced changes in the CYP2D activity, observed in the liver, took place simultaneously in the brain. The aim of the present work was to study the direct interaction of selected antidepressants and neuroleptics with rat CYP2D in brain microsomes, as well as with cDNA-expressed CYP2D2 (the main rat

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CYP2D isoform in the liver) and CYP2D4 (the main rat CYP2D isoform in the brain). Moreover, we examined the effect of chronic treatment with psychotropics on the activity and protein level of CYP2D in microsomes from selected brain structures. The pharmacological aspects of psychotropic–CYP2D interactions in the brain are discussed. 2. Materials and methods 2.1. Chemicals and enzyme preparations Imipramine hydrochloride was provided by Polfa (Jelenia Góra, Poland), fluoxetine hydrochloride by Eli Lilly (Indianapolis, USA), mirtazapine hydrochloride came from Organon (Netherlands) and nefazodone from Bristol-Meyers Squibb International, Ltd. (Uxbridge, UK). Perazine dimaleate was purchased from Labor (Wrocław, Poland), thioridazine hydrochloride came from Jelfa (Jelenia Góra, Poland) and clozapine was from Anpharm (Warszawa, Poland). Bufuralol and 1′-hydroxybufuralol (Hiroi et al., 2002), as well as the polyclonal antibody, anti-rat CYP2D4 rabbit antiserum, were donated by Dr. Y. Funae of the Osaka City University Medical School, Japan. Anti-CYP2D4 antibodies were raised in the female Japanese White rabbit using recombinant CYP2D4 as an immunogen, and were then tested and found to be selective. Those antibodies recognized rat CYP2D4 and inhibited the specific catalytic activity of CYP2D4 (Kishimoto et al., 2004). The secondary antibody, horseradish peroxidase-conjugated Goat Anti-Rabbit IgG, NADP, glucose-6-phosphate and glucose-6phosphate-dehydrogenase were purchased from Sigma (St. Louis, USA). All the organic solvents with HPLC purity were supplied by Merck (Darmstadt, Germany). Supersomes, i.e. microsomes prepared from baculovirus-infected insect cells expressing individual rat CYP2D2 (with a high expression of NADPH P450 reductase), were obtained from Gentest Corp. (Woburn, MA, USA). Bactosomes – membranes prepared from Escherichia coli cells expressing rat CYP2D4 (with a low expression of NADPH P450 reductase) – were custom-made by Cypex (Dundee, Scotland, UK). 2.2. Animal procedures All the animal experiments were performed in accordance with the Polish state regulations (the animal protection act, [DZ.U. 97.111.724, 1997]), and were approved by the Local Bioethics Committee at the PAS Institute of Pharmacology. The experiments were carried out on male Wistar rats (230–260 g) kept under standard laboratory conditions. The investigated psychotropic drugs were administered intraperitoneally (i.p.) twice a day (12 h intervals) for two weeks at the following pharmacological doses (mg/kg i.p.): imipramine, nefazodone, perazine and thioridazine 10, fluoxetine 5, mirtazapine 3, and clozapine 2.5. The doses used of the investigated drugs were of pharmacological magnitude, since they are known to produce a neuroleptic-like effect (catalepsy produced by perazine) (Maj et al., 1978), to show antidepressant properties (activity in the chronic mild stress) (Muscat et al., 1992; Willner 1997), or to cause some behavioural or biochemical alterations in animal tests (Maj and Moryl, 1993; Dziedzicka-Wasylewska et al., 2001). The tested doses produced psychotropic concentrations in the blood plasma of rats (Daniel et al., 1981; Caccia et al., 1990; Daniel et al., 1997; 2000a, b; Knauer et al., 2008) equivalent to those observed in the plasma of psychiatric patients (Baumann et al., 1992; Goodnick, 1994; Javaid, 1994; Nordström et al., 1995; Mauri et al., 2007). The same refers to psychotropic concentrations in the brain, which were considerably higher than in the plasma (Daniel et al., 1981; Caccia et al., 1990; Daniel et al., 1997; 2000a, b). The rats were sacrificed 24 h after the last dose. Whole brains were removed and the selected brain structures (the nucleus accumbens, frontal cortex, substantia nigra, striatum, truncus cerebri, cerebellum and the remainder) were isolated and stored at −80 °C. Microsomes from the whole brains

(from control animals) or selected brain structures (from control and psychotropic treated animals) were prepared by differential centrifugation according to Hiroi et al. (2001). The whole brains or selected brain structures were homogenized using a Thomas teflon homogenizer in 0.05 M Tris-hydrochloride buffer (pH = 7.5) containing 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol, 0.15 M KCl and 0.1 mM phenylmethylsulfonylfluoride (PMSF) kept on ice. The homogenates were centrifuged at 10 000 g at 4 °C for 20 min to remove cellular and nuclear debris. The supernatants were then centrifuged at 100 000 g at 4 °C for 90 min. The obtained microsomal pellets were resuspended in a homogenizing buffer and recentrifuged at 100 000 g at 4 °C for 90 min. The washed microsomal pellets were suspended in a storage solution consisting of 0.1 M sodium phosphate buffer (pH = 7.4), 1 mM EDTA, 1 mM dithiothreitol and 20% (vol/vol) glycerol, aliquoted and stored at − 80 °C. Liver microsomes from the control rats were prepared by differential centrifugation in 20 mM Tris/KCl buffer (pH = 7.4), followed by washing with 0.15 M KCl according to a conventional method. The previously mentioned procedure deprives microsomes of the presence of drugs administered in vivo, which was confirmed in our experiment by the HPLC method (Daniel et al., 2000a, b). 2.3. In vitro studies into CYP2D activity in the brain: the measurement of the rate of bufuralol 1′-hydroxylation in brain microsomes The activity of isoenzymes of the CYP2D subfamily in brain microsomes was studied using the 1′-hydroxylation of bufuralol as a relatively efficient probe reaction for CYP2D4 activity, the most abundant CYP2D isoform expressed in the brain. The latter substrate has been demonstrated to have a particular high reactivity with CYP2D4 and CYP2D2 (Hiroi et al., 2002). After optimizing the in vitro conditions of the reaction, the effects of the drugs were investigated for the linear dependence of product formation on time, and on protein and substrate concentrations. To distinguish between the direct effect of psychotropic drugs on CYP2D activity and changes produced by their prolonged in vivo administration (two-week treatment), two experimental models were used. Model I: the experiment was conducted on pooled brain microsomes (the whole brain) collected from control rats. Forty rats were sacrificed in each experiment (Dixon's plot). The rate of bufuralol 1′-hydroxylation (bufuralol concentrations: 125–500 μM) was assessed in the absence and presence of one of the psychotropic drugs added in vitro (psychotropic concentrations: 50–500 μM). Each sample was prepared in duplicate. Model II: the experiment was carried out on microsomes from the brain structures studied (5–6 pooled structures per 1 sample) of rats treated with psychotropics for two weeks. Bufuralol was added to the incubation mixture in vitro at a concentration of 125 μM. The reaction was studied in the absence of drugs. The incubation (Models I and II) was carried out using a system containing brain microsomes (Model I : ca. 5 mg of protein/ml; Model II: 0.3–1.2 mg of protein/ml for nucleus accumbens, substantia nigra and striatum tissues; ca. 3 mg of protein/ml for frontal cortex, cerebellum and truncus cerebri tissues; ca. 7 mg of protein/ml for the remaining tissues), MgCl2 (4 mM), NADP (1.6 mM), glucose 6-phosphate (5 mM), glucose 6-phosphate-dehydrogenase (2.5 U in 0.4 ml) and a potassium phosphate buffer (2 mM, pH= 7.4). The final incubation volume was 0.4 ml. After a 60-min incubation at 37 °C, the reaction was terminated by adding 30 μl of a 70% perchloric acid solution and then cooling it on ice. The experimental samples were centrifuged (10 min, 3000 g); then the supernatants were transferred to new vials. 2.4. In vitro studies into CYP2D activity in the liver: the measurement of the rate of bufuralol 1′-hydroxylation in liver microsomes The incubation was carried out in a system containing pooled liver microsomes from three control rats (ca. 0.5 mg of protein/ml), MgCl2

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(4 mM), NADP (1.6 mM), glucose 6-phosphate (5 mM), glucose 6-phosphate-dehydrogenase (2.5 U in 0.4 ml), and a potassium phosphate buffer (2 mM, pH = 7.4). Bufuralol was added to the incubation mixture in vitro at concentrations of 2.5–25 μM, and psychotropic drugs at concentrations of 25–500 μM. The final incubation volume was 0.4 ml. After a 10-min incubation at 37 °C, the reaction was terminated by adding 30 μl of a 70% perchloric acid solution and then by cooling it on ice. The experimental samples were centrifuged (10 min, 3000 g); then the supernatants were transferred to new vials.

the appropriate species-specific horseradish peroxidase-conjugated anti-IgG (1 h, 20 °C). Rat CYP2D4 Bactosomes (cDNA-expressed rat isoenzyme) were used as a standard. Immunoreactivity was assessed using an enhanced LumiGLO chemiluminescent substrate. The intensity of the bands corresponding to the enzyme protein on the nitrocellulose membrane was measured with the Luminescent Image analyzer LAS-1000 using an Image Reader LAS-1000 and an Image Gauge 3.11 programs (Fuji Film, Japan).

2.5. Inhibition of CYP2D2 and CYP2D4-catalyzed bufuralol 1′-hydroxylation by psychotropic drugs: an in vitro study with cDNA-expressed CYP2Ds

Ki values were estimated from Dixon's plots on the basis of the results obtained from pooled microsomes from three livers or forty brains of control rats in each experiment. Each sample was prepared in duplicate. The statistical significance of changes in enzyme activity in psychotropic-treated rats was assessed using an analysis of variance followed by Dunnett's test. All the values are mean ± S.E.M. from 5 to 6 samples (S.E.M. = standard error of the mean). The results were regarded as statistically significant when P b 0.05. In the experiment conducted on animals treated with a psychotropic drug for one day, each sample contained an individual brain. In the case of animals treated for two weeks each sample contained 5–6 pooled structures of the brain. The statistical significance of changes in CYP2D protein levels (Western blot analysis) was assessed using Student's t-test.

The incubations were carried out using a system containing the rat cDNA-expressed CYP2D isoforms: Supersomes CYP2D2 or Bactosomes CYP2D4 (10 pmol of CYP isoform), MgCl2 (3.3 mM), NADP (1.3 mM), glucose 6-phosphate (3.3 mM), glucose 6-phosphate-dehydrogenase (0.5 U in 0.5 ml), and a potassium phosphate buffer (0.1 M, pH = 7.4). Bufuralol was added to the incubation mixture in vitro at concentrations of 5–20 μM, and psychotropic drugs at concentrations of 15–100 μM. The final incubation volume was 0.5 ml. After a 20-min incubation at 37 °C, the reaction was terminated by adding 30 μl of a 70% perchloric acid solution and then cooling it on ice. The experimental samples were centrifuged (10 min, 3000 g). The obtained supernatants were transferred to new vials.

2.8. Calculations and statistics

3. Results 2.6. Determination of 1′-hydroxybufuralol concentration in brain and liver microsomes and in recombinant enzyme preparations The concentrations of 1′-hydroxybufuralol formed from bufuralol in brain and liver microsomes or in recombinant enzyme preparations (Supersomes CYP2D2 or Bactosomes CYP2D4) were assessed by a high performance liquid chromatography method (HPLC) based on Hiroi et al. (2002). An aliquot (20 μl) was injected into the HPLC system (LaChrom, Merck-Hitachi) equipped with an L-7485 fluorescence detector, an L-7100 pump and a D-7000 System Manager. The analytical column (Econosphere C18 5 μM, 4.6 × 250 mm) was purchased from Alltech (Carnforth, England). The mobile phase consisted of a 30% acetonitrile and a 0.1% triethylamine, and was adjusted to pH = 3.0 with a 70% perchloric acid solution. The flow rate was 1.0 ml/min (1–8 min) and 2 ml/min (8.1–15 min). The column temperature was 50 °C. Fluorescence was measured at a wavelength of 252 nm (excitation) and 302 nm (emission). The compounds were eluted in the following order: 1′-hydroxybufuralol, 4.5 min; bufuralol, 9.3 min. The sensitivity of the method allowed for the quantification of 1′-hydroxybufuralol as low as 0.1 pmol in one sample. The accuracy of the method equaled 0.5%. The intra- and inter-assay coefficients of variation were below 2 and 5%, respectively. 2.7. Determination of CYP2D protein concentration in brain microsomes: a Western blot analysis The level of CYP2D protein in the brain microsomes of rats treated chronically with psychotropics was estimated by a Western blot analysis. SDS-PAGE and an immunoblot assay were performed using the methodology provided by Gentest, USA. Briefly, 17–45.3 μg of the microsomal protein was separated on a 4% (w/v) 0.75 mm-thick sodium dodecyl sulfate-polyacrylamide stacking gel and a 12% (w/v) resolving gel using a MINIPROTEAN II electrophoresis system (BioRad, Hemmel Hempstead, UK; 130 V, 65 min). The protein was electro-blotted onto a nitrocellulose membrane (100 V, 100 min) and blocked overnight with a 5% dried non-fat milk in PBS (phosphatebuffered saline, pH = 7). After incubation (1 h, 20 °C) with a primary antibody (the polyclonal rabbit anti-rat antibody raised against CYP2D4), the blots were incubated with a secondary antibody, i.e.

3.1. Inhibition of CYP2D activity (bufuralol 1′-hydroxylation) by psychotropic drugs in a suspension of brain and liver microsomes and cDNA-expressed CYP2Ds The investigated antidepressant drugs imipramine, fluoxetine, nefazodone and mirtazapine, added in vitro to control microsomes, inhibited the rate of bufuralol 1′-hydroxylation to a lesser extent in brain microsomes (Ki = 340, 255, and 350 μM, respectively; mirtazapine — no effect) than in liver microsomes (Ki = 45, 12, 28, and 113 μM, respectively) (Table 1). In the case of the studied antidepressants added to the suspensions with the recombinant isoenzyme CYP2D2 or CYP2D4, the inhibitory effects of imipramine and fluoxetine exerted on isoform CYP2D4 were weaker (Ki = 25 and 10 μM, respectively) than those exerted on isoform CYP2D2 (Ki = 2.75 and 1.25 μM, respectively). Nefazodone inhibited CYP2D4 (Ki = 5 μM) only, while mirtazapine practically did not change the activity of the CYP2D isoforms studied. The studied neuroleptics thioridazine and perazine exerted a weak inhibitory effect on CYP2D activity, measured as a rate of bufuralol 1′-hydroxylation in brain microsomes (Ki = 485 and 385 μM, respectively) (Table 1). Both those neuroleptics inhibited CYP2D activity to a greater extent when added to liver microsomes (Ki = 1.0 and 2.5 μM, respectively). In the case of the recombinant isoenzymes CYP2D2 and Table 1 The influence of psychotropic drugs, in vitro added to the suspension of liver microsomes, brain microsomes, or cDNA-expressed rat CYP2D isoforms (CYP2D2 or CYP2D4), on CYP2D activity measured as a rate of bufuralol 1′-hydroxylation. Psychotropic drugs

Imipramine Fluoxetine Nefazodone Mirtazapine Thioridazine Perazine

Inhibition of bufuralol 1′-hydroxylation, Ki [μM] Liver microsomes

Brain microsomes

cDNA-expressed CYPs CYP2D2

CYP2D4

45.0 12.0 28.0 113.0 1.0 2.5

340 255 350 No effect 485 385

2.75 1.25 No effect No effect 0.50 17.40

25.0 10.0 5.0 294.0 2.8 7.1

The presented inhibition constants (Ki) were calculated using Dixon's analysis.

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CYP2D4, the obtained Ki values were similar: Ki = 0.5 and 2.8 μM, respectively, for thioridazine; Ki = 17.4 and 7.1 μM, respectively, for perazine.

in the enzyme activity in the nucleus accumbens (to 78% of the control value). 3.3. The influence of a two-week treatment with selected psychotropics on CYP2D protein level in microsomes from different brain structures

3.2. The influence of a two-week treatment with psychotropic drugs on CYP2D activity measured in microsomes from different brain structures Two-week treatment with the psychotropics studied produced drug- and structure-dependent changes in the activity of CYP2D, measured as a rate of bufuralol 1′-hydroxylation in microsomes from different brain structures (Fig. 1). Fluoxetine significantly decreased the enzyme activity in the striatum (to 78% of the control value) and nucleus accumbens (to 82% of the control value), and showed a tendency towards a decrease in the other structures of rat brain studied, while in the cerebellum the antidepressant significantly increased CYP2D activity (up to 127% of the control value). Nefazodone substantially increased CYP2D activity in the truncus cerebri (up to 306% of the control value), while chronic imipramine and mirtazapine (data not shown) did not produce any significant effects in the enzyme activity. After a two-week treatment with thioridazine, significant changes in CYP2D activity were observed in some brain structures: a decrease in the enzyme activity in the nucleus accumbens (to 78% of the control value) and substantia nigra (to 68% of the control value), and an increase in the enzyme activity in the striatum (up to 120% of the control value) and cerebellum (up to 129% of the control value). Prolonged administration of clozapine produced a significant increase in CYP2D activity in the truncus cerebri (up to 115% of the control value) and the remainder (up to 129% of the control), and a decrease

A

400

1'- OH - bufuralol [pmol (mg protein)-1min-1] % of control

** 125 100

**

*

75 50 25 0

C 150

1'- OH - bufuralol [pmol (mg protein)-1min-1] % of control

B

Fluoxetine

NA

FC

ST

SN

CB

TC

*

125 100 75

**

*

50 25 0

NA

FC

100

150

*

ST

SN

CB

TC

RM

**

200

D

Thioridazine

Nefazodone

300

0

RM

1'- OH - bufuralol [pmol (mg protein)-1min-1] % of control

1'- OH - bufuralol [pmol (mg protein)-1min-1] % of control

150

CYP2D protein level was measured after chronic treatment with the drugs tested, only in those cases in which significant changes in CYP2D activity were observed. As shown in Fig. 2, fluoxetine decreased CYP2D protein level in the brain microsomes of rats treated chronically with the antidepressant: down to 79.5% of the control value in the striatum, and down to 67.6% of the control value in the nucleus accumbens. On the other hand, fluoxetine increased the enzyme protein level up to 151.3% of the control value in the cerebellum. Nefazodone did not affect CYP2D protein level in the truncus cerebri, despite enhancing the enzyme activity in that structure. Prolonged administration of thioridazine increased CYP2D protein level in brain microsomes (from dopaminergic structures): up to 213% of the control value in the substantia nigra (despite decreasing the enzyme activity in that structure), and up to 135% of the control value in the cerebellum (Fig. 3). Moreover, thioridazine showed a tendency to increase the enzyme protein level in the striatum (up to 115.5% of the control value) and to decrease it in the nucleus accumbens (down to 80% of the control value). Chronic treatment with clozapine produced a significant increase in CYP2D protein level in the remainder (up to 122% of the control value) (Fig. 4), and showed a similar tendency in the truncus cerebri (up to 111% of the control value). On the other hand, clozapine decreased the enzyme protein level in the nucleus accumbens (down to 68% of the control value).

NA

FC

ST

SN

CB

TC

RM

Clozapine *

125

*

100 75

**

50 25 0

NA

FC

ST

SN

CB

TC

RM

Fig. 1. The influence of a two-week treatment with antidepressants (fluoxetine and nefazodone) and neuroleptics (thioridazine and clozapine) on CYP2D activity measured as a rate of bufuralol 1′-hydroxylation in microsomes from seven brain regions: the nucleus accumbens (NA), frontal cortex (FC), striatum (ST), substantia nigra (SN), cerebellum (CB), truncus cerebri (TC) and the remainder of brain (RM). Bufuralol (125 μM) was added to the incubation mixture containing brain microsomes (mg of protein/ml: 0.3–1.2 for NA, SN, ST; ca. 3 for FC, CB, TC; ca. 7 for RM), MgCl2 (4 mM), NADP (1.6 mM), glucose 6-phosphate (5 mM), glucose 6-phosphate-dehydrogenase (6.25 U/ml) and the potassium phosphate buffer (2 mM, pH = 7.4). The final incubation volume was 0.4 ml. The incubation proceeded for 60 min. Each bar represents the mean ± S.E.M. value from 5 to 6 samples (each sample containing microsomes of 5–6 rats), compared with the mean values of the control for these brain regions, which are as follows (pmol of 1′-hydroxybufuralol/mg protein/min): 0.566 ± 0.029 (NA), 0.346 ± 0.037 (FC), 0.443 ± 0.025 (ST), 0.786 ± 0.022 (SN), 0.668 ± 0.057 (CB), 0.553 ± 0.025 (TC), and 0.267 ± 0.030 (RM). Statistical significance was assessed using an analysis of variance followed by Dunnett's test, and was indicated with * for P b 0.05; ** for P b 0.01; V — reaction velocity.

A. Haduch et al. / European Journal of Pharmacology 651 (2011) 51–58

Fig. 2. The effect of a two-week treatment with fluoxetine and nefazodone on the level of CYP2D4 protein in microsomes from selected brain regions in which changes in CYP2D4 activity were detected (n = 3–6; each sample contained 5–6 pooled brain structures). A. The microsomal protein (the nucleus accumbens — 17 μg, striatum — 23 μg, cerebellum — 42 μg and truncus cerebri — 45 μg) was subjected to the Western blot analysis and the immunoblot was probed with a polyclonal rabbit anti-rat antibody raised against CYP2D4. Recombinant CYP2D4 protein (Bactosomes) was used as a standard. The presented results are typical of 3–4 samples. B. CYP2D4 protein was quantified on the basis of band density. Proportional changes in the content of CYP2D4 protein in relation to the control are indicated. Statistical significance was assessed using Student's t-test and was indicated with * for P b 0.05 and *** for P b 0.001. NA — nucleus accumbens, ST — striatum, CB — cerebellum, and TC — truncus cerebri.

4. Discussion The present results have provided evidence which indicate that psychotropic drugs produce an effect on CYP2D activity in the brain. The effect of psychotropics on brain CYP2D is different than that found in the liver, and is a cerebral structure-dependent. 4.1. Direct inhibition of CYP2D activity by psychotropics in vitro The Ki values obtained for the tested antidepressants and phenothiazine neuroleptics were significantly higher in brain microsomes than in liver microsomes (Table 1), which suggests a weaker direct inhibitory effect of the tested drugs on CYP2D activity in the brain compared to the liver. Such differences in Ki values between the brain and the liver may result from a diverse composition of CYP2D isoforms in the two organs for which psychotropics may show different affinity. Imipramine, fluoxetine and thioridazine inhibited the activity of the cDNA-expressed isoenzyme CYP2D2 (hepatic) several times (5–10) more potently than the activity of CYP2D4 (cerebral). However, perazine suppressed to a similar extent the

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Fig. 3. The effect of a two-week treatment with thioridazine on the level of CYP2D4 protein in microsomes from selected brain regions in which changes in CYP2D4 activity were detected (n = 3–4; each sample contained 5–6 pooled brain structures). A. The microsomal protein (the nucleus accumbens — 18.4 μg, striatum — 42.7 μg, substantia nigra — 7.8 μg and cerebellum — 88.5 μg) was subjected to Western blot analysis and the immunoblot was probed with a polyclonal rabbit anti-rat antibody raised against CYP2D4. Recombinant CYP2D4 protein (Bactosomes) was used as a standard. Presented results are typical of 3–4 samples. B. CYP2D4 protein was quantified on the basis of band density. Proportional changes in the content of CYP2D4 protein in relation to the control are indicated. Statistical significance was assessed using Student's t-test and was indicated with ** for P b 0.01 and *** for P b 0.001. NA — nucleus accumbens, ST — striatum, SN — substantia nigra, and CB — cerebellum.

activities of both those isoforms, while nefazodone inhibited the activity of CYP2D4 only, without affecting that of CYP2D2. The observed differences in the inhibitory effect of psychotropics in the brain and the liver may also be due to diverse levels of the nonspecific binding of drugs in the two examined organs. The brain contains several times more phospholipids than other organs (Moor et al., 1988), for which basic lipophilic drugs such as the psychotropics studied, display high affinity (Daniel et al., 2001; Daniel, 2003). A study by Margolis and Obach (2003) suggests that the phospholipid component of microsomes possesses some or all of the responsibility for non-specific binding and for its effect on the inhibitors of drugmetabolizing enzymes. The observed differences in the inhibitory effect of psychotropics in brain and liver preparations may also be due to a relatively low CYP level in the brain, and a relatively high protein level (used for this reason) in the tested incubation mixture containing brain microsomes (ca. 5 mg protein/ml) compared to that in the mixture containing liver microsomes (0.5 mg protein/ml of the incubation mixture) or cDNAexpressed CYP2Ds (CYP2D2: 0.36 mg protein/ml, CYP2D4: 0.10 mg protein/ml). The in vitro experiments with liver and brain microsomes or Supersomes CYP2D2 (with a high expression of NADPH P450

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be a weak inhibitor of human CYP2D6 in the clinic, compared to phenothiazines (Shin et al., 1999). For that reason, it was not tested in Model I in our experiment. 4.2. Effects of chronic treatment with antidepressants on brain CYP2D

Fig. 4. The effect of a two-week treatment with clozapine on the level of CYP2D4 protein in microsomes from selected brain regions in which changes in CYP2D4 activity were detected (n = 3–4; each sample contained 5–6 pooled brain structures). A. The microsomal protein (the nucleus accumbens — 18.4 μg, truncus cerebri — 103.7 μg and the remainder — 146.5 μg) was subjected to the Western blot analysis and the immunoblot was probed with a polyclonal rabbit anti-rat antibody raised against CYP2D4. Recombinant CYP2D4 protein (Bactosomes) was used as a standard. The presented results are typical of 3–4 samples. B. CYP2D4 protein was quantified on the basis of band density. Proportional changes in the content of CYP2D4 protein in relation to the control are indicated. Statistical significance was assessed using Student's t-test and was indicated with * for P b 0.05. NA — nucleus accumbens, TC — truncus cerebri, and RM — remainder.

reductase) reflected the physiological ratio for CYP level versus protein content, while in the experiments with Bactosomes CYP2D4 (with a low expression of NADPH P450 reductase) that ratio was higher (ca. 20 times higher). The aforementioned differences in phospholipid and protein concentrations between the enzyme preparations/incubation mixtures used result in a relatively high level of the non-specific binding of the tested psychotropics in a brain microsomal suspension, and in an apparent reduction of the drug ability to inhibit enzyme activity which, in turn, enhances the calculated Ki value (Venkatakrishnan et al., 2000; Margolis and Obach, 2003). Therefore the presented Ki values should be considered as apparent, since they have not been corrected with respect to the extent of non-specific binding in the in vitro systems used (Obach, 1996). However, considering the low Ki values for the recombinant CYP2D4, it is feasible that the antidepressants (except for mirtazapine) and phenothiazines studied may directly affect CYP2D4 at pharmacological concentrations in vivo (Daniel et al., 1981; 1997; 2000a Caccia et al., 1990), decreasing the enzyme activity towards its substrates in the brain. In contrast, the atypical neuroleptic clozapine is known to

The differential effect of psychotropic drugs on CYP2D activity in the brain and liver can also be seen after chronic treatment. Unlike in the liver (Daniel et al., 2002; Haduch et al., 2006), prolonged administration of imipramine or mirtazapine did not affect the enzyme activity in the brain. Nefazodone, which decreased CYP2D activity in the liver after chronic treatment (Daniel et al., 2002), increased the enzyme activity in the truncus cerebri (Fig. 1); however, no change in CYP2D4 protein level was found in that structure, which may suggest post-translational enzyme regulation (e.g. interference with enzyme protein phosphorylation). Like in the liver, fluoxetine significantly decreased CYP2D activity in the striatum and the nucleus accumbens, but increased it in the cerebellum. The fluoxetine-induced changes in CYP2D activity corresponded well with those in CYP2D4 protein level in the brain, which suggested an effect of the drug on the expression of the gene coding for the enzyme (Fig. 2). Therefore it seems that the investigated antidepressants (except for mirtazapine) may chiefly exert an inhibitory effect on CYP2D in the brain, which stems from a direct interaction with the enzyme (binding), and – in the case of fluoxetine – also from a down-regulation of the enzyme expression. Consequently, the inhibition of CYP2D activity by antidepressants may affect neurosteroid metabolism (Hiroi et al., 2001; Kishimoto et al., 2004; Niwa et al., 2008), leading to the enhancement of neurosteroid concentration in the brain (Uzunov et al., 1996; Pinna et al., 2006), which in turn may modify the pharmacological action of antidepressants. Clinical studies showed a significantly lower level of allopregnanolone in the plasma and cerebrospinal fluid of depressed patients, that low level being normalized by treatment with fluoxetine and fluvoxamine (selective serotonin reuptake inhibitors — SSRIs) or TADs (Romeo et al., 1998; Uzunova et al., 1998; Strōhle et al., 1999, 2000. On the other hand, the strong effect of nefazodone on CYP2D activity in the truncus cerebri also seems of interest, since this brain area contains both dopaminergic neurons of the ventral tegmental area (VTA) and serotonergic neurons of the raphe nuclei. It is conceivable that, by increasing CYP2D activity in the truncus cerebri, nefazodone may accelerate tyramine hydroxylation to dopamine and serotonin regeneration from 5-methoxytryptamine, these reactions being mediated by CYP2D (Hiroi et al., 1998b; Yu et al., 2003; Bromek et al., 2010). Both these neurotransmitters are important for the pathophysiology and pharmacotherapy of depression (Papakostas, 2006; Cowen, 2008). Consequently, the inhibition of neurosteroid metabolism by SSRIs and TADs (via CYP2D-inhibition) or the enhancement of dopamine/serotonin synthesis by nefazodone (via an increase in CYP2D activity) seems to be of pharmacological/clinical importance. 4.3. Effects of chronic treatment with neuroleptics on brain CYP2D The effect of the neuroleptics studied on brain CYP2D after chronic treatment also depended on a cerebral region. Like in the liver (Daniel et al., 2005), thioridazine decreased CYP2D activity in the substantia nigra and nucleus accumbens, but significantly increased the enzyme activity in the striatum and cerebellum (Fig. 1). For comparison, a study into CYP2D activity was also carried out after prolonged treatment with the atypical neuroleptic drug clozapine, which was earlier shown to induce brain CYP2D4 in a post-transcriptional phase (Hedlund et al., 1996). In that experiment the enzyme activity was not estimated. The present research into CYP2D activity confirmed the possibility of enzyme induction by the neuroleptic studied. Clozapine significantly increased CYP2D activity in the truncus cerebri and the remainder of the brain, but – surprisingly enough – it decreased the

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enzyme activity in the nucleus accumbens. In general, the observed changes in CYP2D activity produced by chronic neuroleptics corresponded well with those in CYP2D4 protein level in the brain (Figs. 3–4), the only exception being an opposite change in the activity (decrease) and protein level (increase) of CYP2D in the substantia nigra after thioridazine treatment, which may result from the formation of thioridazine-reactive metabolites (Daniel et al., 2005) by cytochromes other than CYP2D (CYP2B/2E1/CYP3A), present in this brain area (Miksys and Tyndale, 2004; Yadav et al., 2006; Shahabi et al., 2008). Accordingly, one-day exposure to thioridazine in vivo resulted in a mild, but significant decrease (to 83% of the control) in CYP2D activity measured in the whole brain (data not shown). The weak-to-moderate induction of CYP2D in the striatum or cerebellum after chronic thioridazine is likely to be abolished in vivo as a result of direct CYP2D inhibition by the neuroleptic (via binding). However, the ability of clozapine to increase CYP2D activity in the truncus cerebri (a structure comprising the VTA) may lead to an increased dopamine synthesis by a CYP2D-mediated pathway (Hiroi et al., 1998b; Bromek et al., 2010), which – besides the receptor profile of the drug – may become an extra add-on component of the atypical neuroleptic action of the drug. Using an immunohistochemical analysis, Hedlund et al. (1996) showed a huge increase in CYP2D4 protein level in the VTA after clozapine. Therefore it may be deduced that CYP2D4 induction is more potent in the VTA than in the rest of the truncus cerebri. 4.4. General discussion A possible explanation for the diverse effect of chronic use of psychotropics on the level of CYP2D in the liver and brain is the differences in the composition of CYP2D isoforms and mechanisms regulating the expression of these enzymes in the two organs, as well as in particular brain structures. Several studies demonstrated differential regulation of CYP2D in the liver and brain, as well as in separate cerebral structures, by nicotine (Miksys and Tyndale, 2004; Mann et al., 2008; Yue et al., 2008), ethanol (Warner and Gustafsson, 1994; Miksys and Tyndale, 2002), drugs and environmental contaminants (Hedlund et al., 1996). In summary, the results of our research indicate that 1) the psychotropics examined (except for mirtazapine) directly inhibit CYP2D activity in the brain, yet less potently than in the liver, which may be due to their stronger affinity for liver CYP2D2 than brain CYP2D4, as well as to a lower CYP content and a higher non-specific drug binding in the brain microsomes tested; 2) the effect of chronic treatment with the psychotropics on CYP2D level and activity in the brain is different than in the liver, and is a cerebral structuredependent. In conclusion, besides the previously observed influence of antidepressants and neuroleptics on CYP2D in the liver, the investigated drugs also produce an effect on CYP2D in the brain. The effect of psychotropics on brain CYP2D is different than that found in the liver, hence it cannot be monitored by any peripheral metabolic tests. The observed psychotropics–brain CYP2D interactions may be important for the metabolism of endogenous neuroactive substrates (e.g. neurosteroids and monoaminergic neurotransmitters), and for the local biotransformation of drugs; such interactions may modify their pharmacological action. It is likely that the inhibition of CYP2D activity by the classic antidepressants studied can contribute to the observed enhancement of the neurosteroid concentration in patients during the pharmacotherapy of depression, whereas the ability of nefazodone and clozapine to induce CYP2D (and CYP2D-mediated synthesis of monoaminergic neurotransmitters) in the truncus cerebri (e.g. in the VTA) may be an add-on component of the pharmacological action of these drugs. Hence the observed effect of psychotropic drugs on cytochrome P450 in the brain provides a fresh insight into their

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pharmacological action. It is therefore to be ascertained whether a long-term exposure to the therapeutic concentrations of psychotropics in vivo affects CYP2D6 activity in human brain (as has been shown for rat brain CYP2D4), and whether this effect is important for the clinical outcome of these drugs. If so, genetic variations in the enzyme may have some impact on the therapeutic action of psychotropics in the brain, and not only on their metabolism in the liver. Moreover, the interference of psychotropics with brain CYP2D may contribute to the differences in clinical effects between drugs acting via similar neuronal mechanisms. Acknowledgements The study was supported by grant no. 2 P05F 002 29 from the Ministry of Science and Higher Education (Warszawa, Poland) and by the statutory funds of the Institute of Pharmacology, Polish Academy of Sciences (Kraków, Poland). The authors are very grateful to Dr. Y. 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