Michiaki Morita, MD, Department of Psychiatry, Jikei University School of Medicine, Nishishinbashi 3-25-8, Minato-ku, Tokyo 105-8461, Japan. Email: email@example.com
Aims: The purpose of this study was to elucidate the mechanism underlying the clinical efficacy of mirtazapine–perospirone combination therapy for treatment-resistant depression in a rat model.
Methods: We studied the effect of combination therapy of the noradrenergic and specific serotonergic antidepressant mirtazapine and the serotonin-dopamine antagonist perospirone on serotonin (5-HT) and dopamine release in the rat medial prefrontal cortex (mPFC) by using microdialysis. Male Wistar rats (250–330 g bodyweight) underwent implantation of a guide cannula in the mPFC, and a microdialysis probe was then inserted into the guide cannula to ensure its final placement in the mPFC. Microdialysis and subsequent chromatographic analysis were performed to estimate the extracellular 5-HT and dopamine concentrations.
Results: When they were used individually, perospirone (0.25 mg/kg, intraperitoneally) and mirtazapine (4 mg/kg, intraperitoneally) induced increased dopamine release up to 134% and 190% relative to the pretreatment level, respectively. Their combination therapy synergistically and significantly (P < 0.0001) increased the dopamine concentration up to 397% of the pretreatment level compared with that induced by the individual drugs. This combination-induced dopamine release was attenuated by 5-HT1A antagonist WAY 100635 (0.5 mg/kg, intraperitoneally), to a peak dopamine release of 151%. Extracellular 5-HT release in the mPFC was not altered in any of the treatment groups.
Conclusions: The large increase in the dopamine concentration in the rat mPFC after the combination therapy was unique and may be responsible for the profound antidepressive effect observed in patients with treatment-resistant depression.
COMBINATION THERAPY OF atypical antipsychotics and newer antidepressants has recently been used for treating refractory depression and has been shown to improve cognitive dysfunction and treatment-resistant depressive symptoms.1 Atypical antipsychotics, such as clozapine, olanzapine, perospirone, and risperidone, enhance dopaminergic neurotransmission in the rat prefrontal cortex (PFC),2 which plays a crucial role in cognition, affect, and social behavior.3–7 These drugs are considered to have beneficial effects on cognitive dysfunction associated with mental disorders, such as depressive illness.8 Papakostas9 has suggested that the most comprehensively studied treatment strategy for non-response or partial response to antidepressants is augmentation with atypical antipsychotic agents, including aripiprazole, olanzapine, quetiapine, and risperidone. However, augmentation or combination therapy with other agents, such as mirtazapine, mianserin, and omega-3 fatty acids is also supported by considerable efficacy data.
Perospirone (cis-N-[4-[4-(1,2-benzisothiazol-3-yl)-1-piperazinyl]butyl]cyclohexane-1,2-dicarboximide monohydrochloride dihydrate) is an agonist at the 5-HT1A receptor but an antagonist at several neuronal receptors, including the D1–5, 5-HT2A, 5-HT2B, 5-HT2C, α1-adrenergic, α2-adrenergic, histamine H1, and muscarinic M1–5 receptors.10,11 Serotonin (5-HT)-dopamine antagonists, such as risperidone and perospirone, antagonize the D2 receptor but also act by mechanisms involving other neurotransmitter systems. Therefore, actions at 5-HT receptors, especially the 5-HT2A/2C receptors, increase the release of dopamine in the PFC,12,13 and activation of the 5-HT1A receptor preferentially enhances dopamine release in this region.14,15 Furthermore, 5-HT1A receptor activation is observable in models of anxiety and depression.16
Mirtazapine (1,2,3,4,10,14b-hexa-hydro-2-methylpyrazino[2,1-a]pyrido[2,3-c]benzazepin), a noradrenergic and specific serotonergic antidepressant (NaSSA), is an agonist at the 5-HT1A receptor and an antagonist at the 5-HT2A, 5-HT2C, 5-HT3, α1-adrenergic, α2-adrenergic, histamine H1, and muscarinic M1–5 receptors, but not at 5-HT reuptake sites. Mirtazapine probably enhances frontocortical adrenergic and dopaminergic but not serotonergic transmission.17
Many antidepressants increase dopamine release in the PFC, but some have only a weak effect on monoamine pathways, especially dopaminergic pathways.18 Therefore, we hypothesized that concurrent administration of an atypical antipsychotic and a newer antidepressant enhances dopaminergic neurotransmission in the PFC. However, no information is available concerning combination therapy of an NaSSA and an atypical antipsychotic in terms of the modulation of serotonergic, noradrenergic, and dopaminergic transmission. In the present study, we aimed to elucidate the mechanism underlying the clinical efficacy of mirtazapine–perospirone combination therapy for treatment-resistant depression by investigating their effects, both individually and combinatorially, on extracellular 5-HT and dopamine concentrations in a rat model using microdialysis.
Male Wistar rats (Clea Japan, Inc., Tokyo; weighing 250–330 g) were housed in groups of two or three and maintained at 25 ± 2°C on a 12-h light–dark cycle (lights on between 07.00 and 19.00 hours). They were fed standard laboratory food and tap water ad libitum. The experimental procedures on the animals were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals as approved by the Japanese Pharmacological Society and the Animal Care Committee of Jikei University School of Medicine.
The rats were anesthetized with sodium pentobarbital [40 mg/kg; intraperitoneally (i.p.)] and placed in a stereotaxic frame. They then underwent implantation of a guide cannula in the medial PFC (mPFC) (A 3.2, L 0.9, and V −3.0 from the dura19) and were allowed to recover for at least 5 days.
Microdialysis and subsequent chromatographic analysis were performed by using an automated online sample injection system. On the day of the experiment, the rats were transferred to a plastic cage and a microdialysis probe (membrane length, 2 mm; molecular mass cut-off, 50 kDa; Eicom Co., Kyoto, Japan) was inserted into the guide cannula to ensure its final placement in the mPFC. The probe was perfused at a rate of 2 µL/min with Ringer's solution (147 mM NaCl, 4.0 mM KCl, and 1.9 mM CaCl2) and the dialysate was collected at 20-min intervals. Dialysate samples were analyzed for 5-HT and dopamine content by using high-performance liquid chromatography with an electrochemical detector (ECD-300, Eicom). The mobile phase consisted of 0.1 M phosphate buffer (pH 6.0) containing 500 mg/L sodium 1-octanesulfonate, and 50 mg/L EDTA-Na2 was pumped at a rate of 2 mL/min through a reverse-phase column (Eicompal CA-50DS, Eicom). The drugs were administered after 2 h of perfusion, when the basal neurotransmitter release had stabilized, and their effects were determined after a stable baseline had been achieved for at least four samples.
Mirtazapine was obtained from Nippon Organon K.K., Osaka, Japan. It was dissolved in a drop of glacial acetic acid, diluted to the final volume with 0.9% saline, and neutralized (pH 6–7) with solid NaHCO3. In this study, the peak dose of mirtazapine was not used to avoid potential ceiling effects. We chose mirtazapine (4 mg/kg, i.p.) because of laboratory evidence that administration of this dose produces a relatively low increase in dopamine release. Perospirone (0.25 mg/kg, i.p.), obtained from Sumitomo Pharmaceuticals (Osaka, Japan), was dissolved in 0.2% tartaric acid and neutralized (pH 6–7). The doses of perospirone were determined according to its pKi value for the D2 receptor and clinical potency.20 WAY 100635 (0.5 mg/kg, i.p.) (N-[2-[4-(2-methoxyphenyl)-1-piperazineyl]ethyl]-N-(pyridinyl)-cyclo-hexanecarboxamide 3HCl), purchased from Sigma Chemical Co. (St. Louis, MO, USA), was dissolved in saline. For the combination studies, perospirone and/or WAY 100635 were administered 60 min before mirtazapine injection. Each drug or drug combination was administered to five to six rats in a volume of 0.5 mL/kg.
The data are presented as the mean ± SEM. Extracellular 5-HT or dopamine concentrations in the final four pretreatment dialysis samples were averaged and post-treatment 5-HT or dopamine concentrations were calculated as a percentage change from the mean baseline concentrations in each rat. The results of in vivo microdialysis were analyzed with two-way anova for repeated measures. A significant (P < 0.05) effect of drug treatment was further analyzed by using Scheffe's post-hoc test for multiple comparisons at each time-point.
The pretreatment (T = −60 min to 0 min) 5-HT and dopamine concentrations in the PFC of conscious freely moving rats were consistent and stable across all the treatment groups (n = 22) and their averages were 3.137 ± 0.994 and 0.377 ± 0.032 pg/40 µL of dialysate, respectively. No difference was found among the baseline values of all the treatment groups. Two-way anova revealed no significant effect of perospirone alone, mirtazapine alone, the combination therapy, or the vehicle on the 5-HT concentration in the PFC [F(3,18) = 1.482, P = 0.2531].
Perospirone (0.25 mg/kg, i.p.) alone increased dopamine release up to 134 ± 18.6% relative to the baseline value and mirtazapine (4 mg/kg, i.p.) alone increased the release up to 190 ± 23.1%. The combination therapy of perospirone and mirtazapine markedly increased dopamine release to a peak value of 397 ± 6.7%. Two-way anova revealed significant effects of the vehicle, perospirone alone, mirtazapine alone, or their combination therapy [F(3,18) = 29.991, P < 0.0001]; time [F(8,24) = 5.570, P < 0.0001]; and treatment–time interaction [F(24 144) = 5.894, P ≤ 0.0001] on post-treatment dopamine release in the PFC. Scheffe's post-hoc analysis revealed that the dopamine response was significantly greater to the combination therapy than to either drug administration over a 60–180-min period; further, the drug combination synergistically increased dopamine release. In contrast, perospirone or mirtazapine alone did not induce significantly different effects when compared with the vehicle (Fig. 1).
Pretreatment with WAY 100635 (0.5 mg/kg, i.p.) followed by the combination therapy led to increased dopamine release to a peak of 149 ± 10.8% relative to the baseline. Two-way anova revealed significant effects of the vehicle, combination therapy, or WAY 100635 plus combination therapy [F(2,14) = 38.809, P < 0.0001]; time [F(8,16) = 6.872, P < 0.0001]; and treatment–time interaction [F(16 122) = 6.688, P < 0.0001] on post-treatment dopamine release in the PFC. Scheffe's post-hoc analysis revealed that the dopamine response was significantly attenuated by WAY 100635 compared with the combination therapy over a 40–180-min period. WAY 100635 plus the combination therapy induced no significantly different effects when compared with the vehicle (Fig. 2). Further, pretreatment with WAY 100635 had no significant effect on the 5-HT concentration.
Disturbances in dopaminergic, serotonergic, and noradrenergic system function are involved in the causes of depressive illness.6–8 In the present study, we investigated the effects of systemic administration of the NaSSA mirtazapine and atypical antipsychotic agent perospirone to investigate the changes in dopamine and 5-HT release in the PFC.
Our observations indicate that mirtazapine and perospirone do not modify 5-HT release in the rat PFC. Further, pretreatment with WAY 100635 does not affect 5-HT release. These results are consistent with the findings of other studies that WAY 100635 at 0.5 mg/kg does not modify the 5-HT level in the PFC.15 The major finding of the present study is that dopamine release in the PFC was significantly and synergistically increased by the combination therapy of mirtazapine and perospirone compared with the effect of these drugs individually. The effects of the combination therapy were sustained for 40–180 min. This increase in dopamine release was completely attenuated by WAY 100635, indicating that 5-HT1A receptor activation may play a role in the increased release of dopamine.
Considering pharmacokinetic factors, mirtazapine inhibits CYP450 3A4, which is responsible for degradation of perospirone, suggesting that the blood concentrations of these drugs may be increased through such a pharmacokinetic mechanism.21–23 However, the fact that the inhibition of CYP450 3A4 by mirtazapine is relatively weak and that each drug is also degraded by other types of enzyme indicate that pharmacokinetic factors less likely contribute to the synergistic effects of the two drugs.
When perospirone was administered alone, the increase in dopamine release in the PFC was merely 130%. Other studies have demonstrated that perospirone administered alone at high doses (2 mg/kg, i.p.) increases dopamine release up to 270% of the baseline value in the PFC and pretreatment with a selective 5-HT1A receptor antagonist, WAY 100635, suppresses this increase.24 The direct action of perospirone on the 5-HT1A receptor may be important for the remarkable increase in dopamine concentrations. At the dose used in this study (0.25 mg/kg), perospirone may have an inadequate agonistic effect on the 5-HT1A receptor.
When mirtazapine was administered alone, the increase in dopamine release in the PFC was 190%. Mirtazapine enhances the firing rate of Locus coeruleus-localized adrenergic neurons, and high affinity at α2-adrenergic receptors enhances the frontocortical release of dopamine. This action may be attributable to the antagonistic properties of mirtazapine at α2-adrenergic autoreceptors.25 A rise in the level of dopamine in the PFC is mediated by the noradrenaline transporter and partially because of elevation in the noradrenaline level.26,27 On the other hand, blockade or stimulation by an α1-adrenoceptor antagonist or agonist does not in itself modify dopamine release in the PFC.28 In a previous study, we demonstrated that mirtazapine administered alone at high doses (8–16 mg/kg, i.p.) induces a dose-dependent maximal increase in dopamine release of up to about 1000% in the PFC by 5-HT1A receptor activation and blockade of α2-adrenergic receptors. This increase is markedly attenuated (from 1000% to 400%) by WAY 100635.29 Interestingly, WAY 100635 partially suppressed the increase in dopamine release induced by mirtazapine at high doses (8–16 mg/kg, i.p.) in our previous study. The indirect 5-HT1A agonistic effect of mirtazapine at high doses on increased dopamine release in the PFC does not adequately explain the partial inhibition of dopamine release by WAY 100635. The inability of WAY 100635 to inhibit dopamine release by high-dose mirtazapine administration may also be related to other mechanisms, such as blockade of α2-adrenergic receptors.29 Mirtazapine at the low dose (4 mg/kg) in this study may have induced minimum blockade of α2-adrenergic receptors and indirect 5-HT1A receptor activation with respect to the effect on dopamine release in the PFC.
When mirtazapine and perospirone were combinatorially used, dopamine release in the PFC increased to about 400% of the pretreatment value, but when WAY 100635 was administered in advance, this increase was completely abolished. These results reveal the importance of the 5-HT1A receptor and suggest that 5-HT1A receptor activation other than blockade of α2-adrenergic receptors may contribute to the dopamine release induced by mirtazapine at the present doses. We therefore hypothesize that the 5-HT1A agonistic effects of mirtazapine and perospirone and their antagonistic actions at the 5-HT2A, 5-HT2C, and 5-HT3 receptors play a role in the indirect agonistic actions at the 5-HT1A receptor.
M100907, a selective 5-HT2A receptor antagonist, by itself has no significant effect on dopamine release in the PFC, but in combination with R(+)-8-OH-DPAT, a selective 5-HT1A receptor agonist, it synergistically enhances dopamine release in the PFC.30 This effect is completely reversed by WAY 100635. Therefore, blockade of the 5-HT2A receptor is considered to strengthen transmission at the postsynaptic 5-HT1A receptor specifically: that is, blockade of the 5-HT2A receptor promotes the indirect activation of the 5-HT1A receptor. M100907 enhances D2/3 receptor antagonist S(-)-sulpiride-induced dopamine release in the PFC, and this effect is completely abolished by WAY 100635. These results suggest that facilitation of 5-HT1A receptor stimulation is essential for the combined blockade of the 5-HT2A and D2 receptors to increase dopamine release in the PFC.16 Further, SR46349-B, a selective 5-HT2A/2c receptor antagonist, potentiates increased dopamine release in the PFC. It synergistically enhances haloperidol-induced dopamine release in the PFC,30 although haloperidol has a strong agonistic effect on the D2 receptor and no antagonistic effect on the 5-HT1A/2A receptors. This effect of SR46349-B is more powerful than that of M100907 because only SR46349-B has an antagonistic effect on the 5-HT2C and 5-HT2A receptors. Again, the induced dopamine release is completely abolished by WAY 100635.30 SB-242084, a 5-HT2C receptor antagonist, alone has been reported to enhance dopamine release in the PFC.31
In line with the preceding discussion, mirtazapine shows antagonistic actions at the 5-HT2A, 5-HT2C, and 5-HT3 receptors, and may activate the post-synaptic 5-HT1A receptor.32,33 These results indicate that mirtazapine has an agonistic effect on the 5-HT1A receptor, albeit indirectly. Perospirone has antagonistic actions at the 5-HT2A, 5-HT2C, and 5-HT3 receptors. These potentiations by mirtazapine and perospirone are possibly mediated via functional 5-HT1A receptor agonism due to combined 5-HT2A and 5-HT2c receptor blockade.
In perospirone–mirtazapine combination therapy, their concomitant blockade of the 5-HT2A and 5-HT2C receptors may be associated with indirect 5-HT1A receptor activation, explaining their effect on dopamine release in the PFC. Further investigation is needed to determine the changes in dopamine release induced by other antipsychotics, including atypical and typical antipsychotics, used in combination with mirtazapine.
In this study, the combination therapy significantly increased the release of dopamine in the PFC. Both mirtazapine and perospirone have affinity for the 5-HT1A receptor and antagonistic activity at the 5-HT2A/2C and α2-adrenergic receptors, and this combination therapy markedly enhances the dopamine output of the mPFC. The PFC modulates the function of the temporolimbic cortical and subcortical regions involved in mood and affect. Therefore, the synergistically induced increase in extracellular dopamine release in the PFC by the combination therapy is the likely mechanism for its clinical efficacy in treatment-resistant depression. In addition, such combination therapy may have an additive and more powerful effect than therapy involving only one antidepressant or a single receptor antagonist or agonist.
We thank Drs H. Miyata, K. Kitazumi, M. Sannomiya, S. Kawamura, R. Nakajyo, A. Kusaka, H. Kodaka, T. Sakurai, and Y. Hiruma for their helpful comments.