Pharmacological Reports 2012, 64, 1567 1572 ISSN 1734-1140 Copyright 2012 by Institute of Pharmacology Polish Academy of Sciences Short communication Effect of co-treatment with mirtazapine and risperidone in animal models of the positive symptoms of schizophrenia in mice Zofia Rogó Department of Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland Correspondence: Zofia Rogó, e-mail: rogoz@if-pan.krakow.pl Abstract: Background: Several clinical reports have suggested that the mirtazapine-induced augmentation of risperidone activity may effectively improve the positive, negative and some cognitive symptoms of schizophrenia. Methods: The present study was aimed at examining the effect of mirtazapine and risperidone, given separately or jointly in mice, on the locomotor hyperactivity induced by D-amphetamine or MK-801 as well as a 5-HT 2A receptor agonist DOI-induced head twitches as models for positive symptoms of psychosis. Results: The obtained results showed that co-treatment with mirtazapine (2.5 or 5 mg/kg) and risperidone (0.01 mg/kg) inhibited the locomotor hyperactivity induced by D-amphetamine or MK-801. Moreover, co-administration of mirtazapine (1.25 or 2.5 mg/kg) and risperidone (0.01 mg/kg) reduced the number of head twitches induced by DOI, whereas those drugs given separately changed neither the locomotor hyperactivity induced by D-amphetamine or MK-801 nor the syndrome induced by DOI. Conclusion: The obtained results indicated that lower doses of mirtazapine enhanced the antipsychotic-like effect of risperidone in animal tests of positive symptoms of schizophrenia. Further studies are necessary to elucidate its mechanism of action. Key words: mirtazapine, risperidone, animal tests of schizophrenia, mice Introduction Schizophrenia is a chronic progressive disease with a life-time prevalence of approximately 1%. Although the severity of schizophrenia has been known for a long time, its etiology and pathophysiology are still unknown. It is known that the serotonergic system is potentially an important target for pharmacological agents. The affinity for 5-HT receptors is one of the main differences between atypical and conventional antipsychotic drugs [2, 13, 14]. An atypical antipsychotic agents, e.g., risperidone, whose low doses block mainly serotonin 5-HT 2A receptors and higher dopamine D 2 ones, is known to produce minimal extrapyramidal side-effects compared to classic antipsychotics. Several clinical and preclinical studies suggest that some atypical antipsychotic drugs (e.g., risperidone, olanzapine, aripiprazole) alleviate not only positive symptoms of schizophrenia, but also negative ones (e.g., flat affect, poverty of speech); furthermore, they bring considerable benefit compared to the firstgeneration drugs [4, 9, 15]. Additionally, the role of antidepressant drugs as adjuncts to the treatment of Pharmacological Reports, 2012, 64, 1567 1572 1567
schizophrenic patients with negative symptoms has been described by several authors [10, 18]. It has also been ascertained that mirtazapine, a novel antidepressant, enhances noradrenergic and 5-HT 1A -mediated serotonergic neurotransmission by antagonizing central 2 -auto- and hetero-adrenoreceptors, but does not inhibit the uptake of noradrenaline and 5-HT. It also blocks 5-HT 2 and 5-HT 3 receptors and displays very low affinity for dopaminergic receptors and high affinity for histamine H 1 ones [7]. Several clinical reports have suggested that the mirtazapine-induced augmentation of risperidone activity may effectively improve both negative and some cognitive symptoms of schizophrenia [1, 5, 20]. On the other hand, Berk et al. [3] demonstrated that the adjunctive mirtazapine was not superior to an adjunctive placebo when used with atypical antipsychotics. Hence, the role of antidepressants in general and of mirtazapine in particular, in the treatment of schizophrenia is still unclear. To understand the mechanism of the clinical efficacy of a combination of an antidepressant and risperidone in the therapy of schizophrenia, the present study was aimed at examining the effect of mirtazapine and risperidone, given separately or jointly to mice, on the locomotor hyperactivity induced by D-amphetamine (AMP) or MK-801, as well as on the head twitches induced by the 5-HT 2A receptor agonist DOI, serving as models of the positive symptoms of psychosis. Materials and Methods Animals The experiments were carried out on male Albino- Swiss mice (25 ± 2 g) (Charles River Laboratories, Sulzfeld, Germany). The animals were housed 10 per cage (57 35 20 cm) in a colony room kept at 21 ± 1 C with a 40 50% humidity, on a 12-h lightdark cycle (the light on at 7 a.m.). The mice had free access to food and water before the experiments. Each experimental group consisted of 8 10 animals/dose, and the animals were used only once for each test. All the experiments were conduced during the light phase and were carried out according to the procedures approved by the Animal Care and Use Committee at the Institute of Pharmacology, Polish Academy of Sciences in Kraków. Drugs administration Mirtazapine and risperidone (Tocris Bioscience, Bristol, UK) were suspended in a 1% aqueous solution of Tween 80; (+)-MK-801 maleate (MK-801, Tocris Bioscience, Bristol, UK) or D-amphetamine sulfate (AMP) and (±)-DOI (Sigma-Aldrich, St. Louis, USA) were dissolved in a 0.9% NaCl, All the drugs were administered in a volume of 10 ml/kg. Risperidone (ip) and mirtazapine (ip) were injected 30 min before AMP (5 mg/kg, sc) or MK-801 (0.3 mg/kg, ip) and 60 min before DOI (2.5 mg/kg, ip) treatment. Locomotor activity test The locomotor activity was recorded individually for each animal in OPTO-M3 locomotor activity cages (Columbus Instruments, Columbus, OH, USA) linked on-line to a compatible PC. Each cage (13 23 15 cm) was surrounded with an array of photocell beams. Interruptions of these photobeams resulted in a horizontal activity defined as ambulation scores. Locomotor activity was measured for 60 (AMP) or 30 min (MK-801), starting 30 min after treatment with AMP or MK-801. Each group consisted of 8 10 mice. Head twitches induced by DOI The experiment was performed according to Wieroñska et al. [21]. In order to habituate mice to the experimental environment, each animal was transferred to 12 (diameter) 20 cm (height) glass cylinder, linked with sawdust, 30 min before the treatment. DOI, a 5-HT 2A receptor agonist (2.5 mg/kg) was given 60 min after MIR or risperidone administration. The number of head twitches of the mice were counted during a 30-min session, starting immediately after DOI treatment. Each group consisted of 8 mice. Statistical analysis The data were evaluated by a one-way analysis of variance (ANOVA) followed, when appropriate, by individual comparisons with the control using Dunnett s test. 1568 Pharmacological Reports, 2012, 64, 1567 1572
Effect of co-treatment with mirtazapine and risperidone Zofia Rogó Fig. 1. The effect of mirtazapine (MIR, 2.5, 5 and 10 mg/kg, ip) given alone or in combination with risperidone (RIS, 0.01 mg/kg, ip) on the amphetamine (AMP, 5 mg/kg, sc)-induced locomotor hyperactivity in mice. The results are shown as the mean ± SEM of 8 10 animals/group. The data were statistically evaluated by ANOVA, following by individual comparisons using Dunnett s test; * p < 0.05; ** p < 0.001 vs. vehicle-treated group, ## p < 0.001 vs. MIR + AMP-treated group Results and Discussion In the present study we investigated the effect of mirtazapine and risperidone, given separately or jointly, in animal models of the positive symptoms of schizophrenia. It was used AMP- and MK-801-induced hyperactivity, which represents models to test the antipsychotic activity of drugs, mainly with respect to the positive symptoms of the disease [11], and DOIinduced head twitches as the model with predictive validity of psychosis and hallucination [21]. The obtained results showed that mirtazapine in doses of 2.5 and 5 mg/kg did not change the locomotor activity of mice, while its higher dose (10 mg/kg) decreased it by ca. 52% [F (3, 36) = 5.96; p < 0.001, Fig. 1]. Our earlier [16], and present studies indicated that higher doses (0.3 and 1 mg/kg) of risperidone reduced the locomotor activity of mice by ca. 65 or 96%, respectively, while its lower doses of 0.01 or Fig. 2. The effect of mirtazapine (MIR, 2.5 and 5 mg/kg, ip) given alone or in combination with risperidone (RIS, 0.01 mg/kg, ip) on the MK-801 (0.3 mg/kg, ip)-induced locomotor hyperactivity in mice. The results are shown as the mean ± SEM of 8 animals/group. The data were statistically evaluated by ANOVA, following by individual comparisons using Dunnett s test; * p < 0.001 vs. vehicle-treated group, # p < 0.05; ## p < 0.001 vs. MIR + MK-801- treated group Pharmacological Reports, 2012, 64, 1567 1572 1569
0.03 mg/kg did not change it [F (5, 54) = 77.60; p < 0.001, data not shown]. In addition, AMP (5 mg/kg) enhanced the locomotor activity by ca. 200%, while risperidone in a dose of 0.1 mg/kg completely abolished it. Moreover, risperidone in a lower dose (10 times lower, i.e., 0.01 mg/kg) or mirtazapine in doses of 2.5 and 5 mg/kg, given separately, did not change the hyperactivity induced by AMP, while co-treatment with both those drugs (risperidone, 0.01 mg/kg, and mirtazapine, 2.5 or 5 mg/kg) reduced that locomotor hyperactivity [F (5, 42) = 7.69; p < 0.001, Fig. 1]. The glutamatergic therapy of psychosis was based on the behavioral results indicating psychomimetic properties of NMDA receptor antagonists (e.g., phencyclidine), which evoked positive, negative, and cognitive abnormalities in animals, similar to those observed in patients with psychosis. In addition, the clinical preliminary studies with the use of ligands that act on the glycine modulatory site on the NMDA receptor showed that the compounds improved cognitive and decreased negative syndromes when cotreated with neuroleptics [6]. The obtained results also showed that MK-801 in a lower dose (0.1 mg/kg) had no influence on the locomotor activity of mice, but its higher dose (0.3 mg/kg) enhanced it by ca. 176%, that hyperactivity being completely abolished by risperidone in a dose of 0.1 mg/kg, but not in the lower one (0.01 mg/kg) [F (4, 35) = A C B Fig. 3. The effect of mirtazapine (MIR, 1.25, 2.5, 5 and 10 mg/kg, ip) (A) or risperidone (RIS, 0.01, 0.03 and 0.1 mg/kg, ip) (B), given alone or in combination, on the DOI (2.5 mg/kg, ip)-induced head twitches (C) in mice. The results are shown as the mean ± SEM of 8 animals/group. The data were statistically evaluated by ANOVA, following by individual comparisons using Dunnett s test; * p < 0.001 vs. DOI-treated group, # p < 0.001 vs. MIR + DOI-treated group 1570 Pharmacological Reports, 2012, 64, 1567 1572
Effect of co-treatment with mirtazapine and risperidone Zofia Rogó 9.17; p < 0.001, Fig. 2]. Moreover, co-treatment with risperidone in the lower dose (0.01 mg/kg) and mirtazapine (2.5 or 5 mg/kg) decreased the MK-801- induced hyperactivity of mice [F (5, 42) = 8.56; p < 0.001, Fig. 2]. To further investigate the potential antipsychoticlike action of combined treatment with mirtazapine and risperidone, we decided to use a behavioral model of hallucination which is known to be an important aspect of schizophrenia in humans. Hallucinogeniclike activity can be achieved via stimulation of serotonergic receptors in both mice and humans [19]. The 5-HT 2A receptor agonist DOI induces a characteristic behavioral effect in mice, consisting of head twitches, which is attenuated by typical and atypical antipsychotics [8]. The present study showed that mirtazapine [F (4, 35) = 34.46; p < 0.001, Fig. 3A] or risperidone [F (3, 28) = 43.85; p < 0.001, Fig. 3B] dosedependently inhibited the DOI-induced head twitches in mice. Moreover, co-administration of risperidone (0.01 mg/kg) and mirtazapine (1.25 or 2.5 mg/kg) reduced the number of head twitches induced by DOI, but those drugs given separately did not change that syndrome [F (5, 42) = 36.74; p < 0.001, Fig. 3C]. Since the synergistic antipsychotic-like effect was observed following co-treatment with mirtazapine and risperidone, an important role of 5-HT 2A receptors in mediating their action has been suggested. In addition, some earlier studies conduced by other authors suggested that adjunct treatment with escitalopram, but not citalopram, may enhance the effect of a subtherapeutic dose of risperidone on the positive, negative, cognitive, and depressive symptoms of schizophrenia [12]. 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