Augmentation of antidepressant effects of duloxetine and bupropion by caffeine in mice

PBB-71981; No of Pages 7 Pharmacology, Biochemistry and Behavior xxx (2014) xxx–xxx

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Article history: Received 13 January 2014 Received in revised form 27 May 2014 Accepted 7 June 2014 Available online xxxx

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Keywords: Depression Caffeine-augmentation Duloxetine Bupropion Brain monoamines Hippocampus Cerebral cortex

Department of Pharmacology, Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, NMIMS University, Mumbai 400 056, India Department of Pharmacology, Dr. Bhanuben Nanavati College of Pharmacy, Mumbai 400 056, India

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There is an unmet need in the treatment of depression suggesting requirement of new therapeutic approaches having better efficacy and safety profile. Patients receiving antidepressant therapy generally consume caffeine in the form of tea or coffee. The objective of the present study was to evaluate the augmentation of antidepressant effects of duloxetine and/or bupropion with caffeine. Male Swiss Albino mice received treatment of normal saline (10 ml/kg), ‘caffeine alone’ (10 mg/kg), ‘duloxetine alone’ (10 mg/kg), ‘bupropion alone’ (10 mg/kg), caffeine + duloxetine (5 mg/kg, each), bupropion + caffeine (5 mg/kg, each), and bupropion + duloxetine (5 mg/kg, each) through the intra-peritoneal route. The immobility period was analyzed 30 min after the treatment in forced swim and tail suspension tests. Norepinephrine, dopamine, and serotonin levels were analyzed in hippocampus, cerebral cortex and whole brain using HPLC with fluorescence detector. Euthanasia was performed 1 h after treatment. Comparison between vehicle treated group and other groups showed significant decrease in immobility in all drug treated groups in both antidepressant models. Caffeine plus duloxetine treated group was better among the combination treated groups in terms of decrease in immobility and increase in norepinephrine, dopamine, and serotonin levels in hippocampi, cerebral cortices, and whole brain when compared to their respective monotherapy treated groups. These combination approaches may help in reducing the dose of duloxetine/bupropion, and consequently lower the associated side/adverse effects. © 2014 Published by Elsevier Inc.

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1. Introduction

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Depression is a leading cause of disability and distress worldwide (Castle et al., 2012). The delay in onset of action and intolerable side effects of available antidepressants have restricted the desired efficacy, which suggests an unmet need in the treatment of depression (Mojtabai, 2009; Richelson, 2013). The limited success rate (60–70%) of first-line monotherapy has resulted in preference of augmentation therapy as second line treatment (Thase, 2007). As per the American Psychiatric Association Practice Guidelines on suicidal patients in antidepressant section, non-tricyclic, non-monoamine oxidase inhibitor related drugs are relatively safe, particularly on overdose (Jacobs et al., 2003). Bupropion and selective serotonin reuptake inhibitors (SSRIs) are considered within the 5 most prescribed antidepressants (Grunebaum et al., 2012). Duloxetine inhibits both serotonin and norepinephrine reuptake. This dual action makes it an interesting drug in the treatment of

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Pravin Popatrao Kale a,b, Veeranjaneyulu Addepalli a,⁎

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Augmentation of antidepressant effects of duloxetine and bupropion by caffeine in mice

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Abbreviations: ANOVA, analysis of variance; SSRIs, selective serotonin reuptake inhibitors; HPLC-FD, high performance liquid chromatography with fluorescence detector. ⁎ Corresponding author at: Department of Pharmacology, Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, NMIMS University, V. L. Mehta Road, Vile parle west, Mumbai, 400 056, India. Tel.: +91 22 42332030 (O); fax: + 91 22 26185422. E-mail address: [email protected] (V. Addepalli).

depression (Hunziker et al., 2005; Reneman et al., 2001; Ressler and Nemeroff, 2001). It has better efficacy, safety, and tolerability with fewer side effects when compared against antidepressant like fluoxetine, paroxetine, and venlafaxine (Hunziker et al., 2005; Stahl et al., 2005; Thase et al., 2007; Zomkowski et al., 2012). Less than one week of duloxetine treatment has shown meaningful improvements in patients (Brannan et al., 2005). Duloxetine treatment in forced swim test resulted in significant reduction in the immobility period at 10 mg/kg dose (Ciulla et al., 2007; Rénéric and Lucki, 1998). In addition to serotonin and norepinephrine reuptake inhibitors, dopamine transmission also plays a vital role in antidepressant action (Dunlop and Nemeroff, 2007; Gessa, 1994; Wilner, 1983). Bupropion, a preferential norepinephrine and dopamine reuptake inhibitor (Baldessarini, 2006), showed significant decrease in immobility at 10 mg/kg or lower doses (Kotagale et al., 2013). Caffeine, an adenosine A1 and A2A antagonist, is the widely consumed psychoactive substance in the world (Heckman et al., 2010). High amount of caffeine consumption has an inverse relation to the depression risk (Lucas et al., 2011; Ruusunen et al., 2010) and the risk of suicide (Kawachi et al., 1996; Lucas et al., in press) in normal population. Many depressed patients seek a ‘lift’ because of fatigue or negative affect (Bernstein et al., 2002), and consume higher amount of caffeine (Goldstein, 1987; Whalen et al., 2008). The considered doses of caffeine in many studies are either moderate or high (Bernstein et al., 1998,

http://dx.doi.org/10.1016/j.pbb.2014.06.005 0091-3057/© 2014 Published by Elsevier Inc.

Please cite this article as: Kale PP, Addepalli V, Augmentation of antidepressant effects of duloxetine and bupropion by caffeine in mice, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.06.005

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2. Materials and methods

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2.1. Animals

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Male Swiss Albino mice weighing 22–27 g were group housed in Perspex cage, 3 mice/cage, in a temperature (22–24 °C) and humidity (50–60%) controlled central animal house facility under a light and dark (12:12) illumination cycle. These mice were procured from Bharat Serum Ltd, Thane. They had free access to standard food and water. Experiments were approved by an Institutional Review Committee for the use of Animal Subjects (Approval number- CPCSEA/IAEC/ SPTM/P-44/2011) and in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985).

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2.2. Drug solutions and treatment

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Drugs were dissolved in normal saline (0.9% w/v NaCl) and administered through intra-peritoneal route. Mice were randomly assigned to 7 groups (n = 6 animals/group) in each test. They were kept undisturbed for at least 1 h before testing. Experiments were performed between 09.00 and 14.00 h. Groups I, II, III, IV, V, VI, and VII received treatment of normal saline (10 mg/kg; control groups), caffeine (10 mg/kg; Elders Pharmaceutical Pvt. Ltd), duloxetine (10 mg/kg; Dr. Reddy's Laboratories Ltd.), caffeine (5 mg/kg) + duloxetine (5 mg/kg), bupropion (10 mg/kg; Aurobindo Pharma Ltd), bupropion (5 mg/kg) + caffeine (5 mg/kg), and bupropion (5 mg/kg) + duloxetine (5 mg/kg), respectively. Pretreatment schedules were based on the available reports (Kaster et al., 2004; Kotagale et al., 2013; Zomkowski et al., 2012).

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2.3. Antidepressant models

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2.3.1. Forced swim test Forced swim test was performed as described by Porsolt et al. (1997). In brief, mice underwent “pretest session” on 1st day. They were individually forced to swim for 15 min in plexi glass cylinders (21 cm height 12 cm internal diameter) containing freshwater up to a height of 10 cm at 24 ± 1 °C. On 2nd day, each animal received treatment 30 min before the test session and was allowed to swim for 6 min. Last 5 min session from total 6 min recorded video was used for evaluation. Mice were considered immobile when there were no limb movements while floating or made only small limb movements necessary for floating. The immobility period was analyzed with the help of Video Tracking software (SMART v2.5.21 Video Tracking Software, Panlab Harward Apparatus).

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2.5. Statistical analysis

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The statistical evaluation was performed using the Graphpad InStat for 32 bit Windows version 3.06. Groups were compared to assess the statistical significance using one way analysis of variance (ANOVA) followed by Tukey's honest significant difference (HSD) post-hoc test. The data is represented as mean ± SEM values and n = 6 per group.

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3. Results

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3.1. Forced swim test

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The immobility period was significantly decreased in groups treated with ‘caffeine alone’ (10 mg/kg), ‘duloxetine alone’ (10 mg/kg), caffeine + duloxetine (5 mg/kg, each), bupropion + caffeine (5 mg/kg, each), and bupropion + duloxetine (5 mg/kg, each) when compared against control groups (Fig. 1). Group treated with caffeine + duloxetine (5 mg/kg, each) showed significant decrease in immobility period when compared against ‘caffeine alone’ (10 mg/kg) treated group (Fig. 1). The same combination treated groups also showed significant decrease in immobility period when compared against ‘duloxetine alone’ (10 mg/kg) treated group (Fig. 1). Caffeine + bupropion (5 mg/kg, each) and bupropion + duloxetine (5 mg/kg, each) treated groups showed significant decrease in immobility period, as compared to ‘bupropion alone’ (10 mg/kg) treated group (Fig. 1).

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3.2. Tail suspension test

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Drug treated groups showed significant decrease in immobility period as compared to control groups (Fig. 2). Combination treated groups such as caffeine + duloxetine (5 mg/kg, each) and caffeine + bupropion (5 mg/kg, each) showed significant decrease in immobility period, as compared to caffeine alone (10 mg/kg) treated groups (Fig. 2).

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Analysis of monoamine levels in cerebral cortex, hippocampus, and whole brain (whole brain = cerebral cortex + hippocampus + remaining brain tissue) was performed using HPLC (Shimadzu, LC-2010C HT, autosampler) with FD (RF-20A-prominence, Shimadzu) method (Choudhary et al., 2013; Madepalli and Lakshmana Raju, 1997). Mice received treatment 1 h before euthanasia and heads were dropped in ice cold 0.1 M perchloric acid. Brains were removed and weighed. Then the cerebral cortex, hippocampus, and the remaining brain part were separated and individually weighed and homogenized in 2 ml of ice cold 0.1 M perchloric acid. Resulting mixture was centrifuged at 20817 ×g (Eppendorf 5810 R, Rotor F-45-30-11) for 30 min (4 °C). The supernatant was filtered through 0.45 μm membrane (PALL@ Pall corporation, India) and stored at −80 °C until the time of analysis. Samples were injected and the chromatographic separation was achieved on reversed-phase analytical column (KROMASIL 100, C18, 5 μm, 25 mm × 0.46 mm) at room temperature. The acquired data was processed using LC Solution@ software. The mobile phase was prepared using sodium acetate (0.02 M), ethylenediaminetetraacetic acid (0.2 mM), methanol (16%), di-n-butylamine (0.01%) and heptane sulfonic acid (0.055%), at pH 3.92 adjusted with phosphoric acid, filtered through a 0.45 μm membrane. Flow rate of mobile phase was kept at 1.3 ml/min. Norepinephrine, dopamine, and serotonin were detected at an excitation wavelength of 280 nm and an emission wavelength of 315 nm. Monoamine peaks were identified by comparing the retention time of sample and standard. The concentration of each monoamine in the sample was analyzed according to their area under curve and using respective straight line equation. The linearity for norepinephrine, dopamine, and serotonin was in the range 0.99– 0.997. Results were expressed as ng/g of wet weight of tissue.

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2002; Orbeta et al., 2006). Interestingly, the lower caffeine consumption (250–400 mg/day) is known to produce beneficial effects (Fredholm et al., 1999; Tse et al., 2009). The lower dose (10 mg/kg, i.p.) of caffeine showed increased in locomotion activity in mice (El Yacoubi et al., 2000). Thus, the present study aims to compare and evaluate the augmentation effect of lower dose of caffeine with duloxetine or bupropion in the treatment of depression.

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2.3.2. Tail suspension test The apparatus used was a set of aluminum stands measuring 58 cm (high) × 30 cm (wide). At 58 cm height of horizontally fixed aluminum rod, adhesive tape was used to suspend each mouse by its tail. It was placed approximately 1 cm from the tip of the tail (Vogel and Vogel, 2008). Mice received treatment 30 min before undergoing the 5 min test session. Recorded video of each animal was evaluated using video tracking software (SMART v2.5.21 Video Tracking Software, Panlab Harward Apparatus) to analyze the immobility period.

Please cite this article as: Kale PP, Addepalli V, Augmentation of antidepressant effects of duloxetine and bupropion by caffeine in mice, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.06.005

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3.3.1.3. Whole brain. Except ‘caffeine alone’ treated group, norepinephrine levels were significantly increased in all drug treated groups when compared against control groups (Fig. 3a). Caffeine + duloxetine (5 mg/kg, each) and bupropion + caffeine (5 mg/kg, each) combination treated groups showed significant increase in norepinephrine levels when compared against ‘caffeine alone’ (10 mg/kg) treated group (Fig. 3a).

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3.3.2. Estimation of dopamine level

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3.3.2.1. Hippocampi. The drug treatments such as duloxetine alone (10 mg/kg), bupropion alone (10 mg/kg), caffeine + duloxetine (5 mg/kg, each), and bupropion + caffeine (5 mg/kg, each) have shown significant increase in dopamine level when compared against control groups (Fig. 3b). The latter two combination groups also showed significant increase in dopamine levels, as compared to ‘caffeine alone’ (10 mg/kg) treated group.

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3.3.1.2. Cerebral cortices. The increase in norepinephrine levels was significant in groups treated with ‘duloxetine alone’ (10 mg/kg), ‘bupropion alone’ (10 mg/kg), caffeine + duloxetine (5 mg/kg, each) and bupropion + duloxetine (5 mg/kg, each), as compared to control groups (Fig. 3a). Among the combination treated groups, caffeine + duloxetine (5 mg/kg, each) treated group showed significant increase in norepinephrine levels when compared against ‘caffeine alone’ (10 mg/kg) treated group (Fig. 3a). Comparison against ‘duloxetine alone’ (10 mg/kg) treated group showed significant increase in norepinephrine levels with bupropion + caffeine (5 mg/kg, each) treated group (Fig. 3a).

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3.3.1.1. Hippocampi. Except ‘caffeine alone’ treated group, all drug treated groups showed significant increase in norepinephrine levels, as compared to control groups (Fig. 3a). Groups treated with a combination of caffeine + duloxetine (5 mg/kg, each) and bupropion + caffeine (5 mg/kg, each) showed significant increase in norepinephrine levels when compared against ‘caffeine alone’ treated group (10 mg/kg) (Fig. 3a). Bupropion + caffeine (5 mg/kg, each) treated group showed significant increase in norepinephrine levels, as compared to ‘bupropion alone’ (10 mg/kg) treated group (Fig. 3a).

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Comparison of groups treated with caffeine + duloxetine (5 mg/kg, each) and bupropion + duloxetine (5 mg/kg, each) showed significant decrease in immobility period when compared against ‘duloxetine alone’ (10 mg/kg) treated group (Fig. 2). Combination treated groups such as caffeine + bupropion (5 mg/kg, each) and bupropion + duloxetine (5 mg/kg, each) showed significant decrease in immobility period, as compared to ‘bupropion alone’ (10 mg/kg) treated group (Fig. 2).

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Fig. 1. Forced swim test. Significant difference is denoted by * — p b 0.05, *** — p b 0.001 — as compared against the vehicle treated group; ### — p b 0.001 — as compared against caffeine treated group; ! — p b 0.05 — as compared against duloxetine treated group; † — p b 0.05, ††† — p b 0.001 — as compared against bupropion treated group.

Fig. 2. Tail suspension test. Significant difference is denoted by *** — p b 0.001 — as compared against the vehicle treated group; # — p b 0.05, ### — p b 0.001 — as compared against caffeine treated group; !! — p b 0.01 — as compared against duloxetine treated group; ††† — p b 0.001 — as compared against bupropion treated group.

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3.3.2.2. Cerebral cortices. Except ‘caffeine alone’ (10 mg/kg) treated group, all other drug treated groups showed significant increase in dopamine levels, as compared to control groups (Fig. 3b). Comparison of caffeine + duloxetine (5 mg/kg, each), bupropion + caffeine (5 mg/kg, each) treated groups against ‘caffeine alone’ (10 mg/kg) treated group showed significant increase in dopamine levels (Fig. 3b). Caffeine + duloxetine (5 mg/kg, each) treatment resulted in significant increase in dopamine level when compared against ‘duloxetine alone’ (10 mg/kg) treated group (Fig. 3b). Bupropion + duloxetine (5 mg/kg, each) treated group showed significant increase in dopamine levels, as compared to ‘duloxetine alone’ (10 mg/kg) treated group (Fig. 3b). However, bupropion + caffeine (5 mg/kg, each) and bupropion + duloxetine (5 mg/kg, each) treated groups showed significant decrease in dopamine levels when separately compared against ‘bupropion alone’ (10 mg/kg) and duloxetine alone (10 mg/kg) treated groups (Fig. 3b).

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3.3.2.3. Whole brain. The dopamine levels were significantly increased all drug treated groups except ‘caffeine alone’ (10 mg/kg) treated group when compared against control groups (Fig. 3b). Combination treated groups such as caffeine + duloxetine (5 mg/kg, each), and bupropion + caffeine (5 mg/kg, each) showed significant increase in dopamine levels, as compared to caffeine alone (10 mg/kg) treated group (Fig. 3b). However, bupropion + caffeine (5 mg/kg, each) and bupropion + duloxetine (5 mg/kg, each) treated groups showed significant decrease in dopamine levels when separately compared against ‘bupropion alone’ (10 mg/kg) and ‘duloxetine alone’ (10 mg/kg) treated groups (Fig. 3b).

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3.3.3. Estimation of serotonin level

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3.3.3.1. Hippocampi. Except ‘caffeine alone’ (10 mg/kg) treated group, all other drug treated groups showed significant increase in serotonin levels when compared against control groups (Fig. 3c). Comparison against ‘caffeine alone’ (10 mg/kg) treated group showed significant increase in serotonin levels in combination treated groups such as

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Please cite this article as: Kale PP, Addepalli V, Augmentation of antidepressant effects of duloxetine and bupropion by caffeine in mice, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.06.005

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Fig. 3. Estimation of norepinephrine (Fig. 3a), dopamine (Fig. 3b), and serotonin (Fig. 3c) levels in hippocampi, cerebral cortices and whole brain using HPLC-FD. Significant difference is denoted by * — p b 0.05,** — p b 0.01, *** — p b 0.001 — as compared against the vehicle treated group; # — p b 0.05, ## — p b 0.01, ### — p b 0.001 — as compared against caffeine alone treated group; ! — p b 0.05, !! — p b 0.01, !!! — p b 0.001 — as compared against duloxetine alone treated group; † — p b 0.05,†† — p b 0.01, ††† — p b 0.001 — as compared against bupropion alone treated group.

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caffeine + duloxetine (5 mg/kg, each), and bupropion + caffeine (5 mg/kg, each) (Fig. 3c). The comparison between ‘bupropion alone’ (10 mg/kg) and bupropion + duloxetine (5 mg/kg, each) demonstrated significant increase in serotonin level in later group (Fig. 3c).

3.3.3.2. Cerebral cortices. Except ‘caffeine alone’ treated group all other drug treated groups showed significant increase in serotonin levels when compared against control groups (Fig. 3c). Caffeine + duloxetine (5 mg/kg, each) and bupropion + caffeine (5 mg/kg, each) combination

Please cite this article as: Kale PP, Addepalli V, Augmentation of antidepressant effects of duloxetine and bupropion by caffeine in mice, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.06.005

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treated groups showed significant increase in serotonin levels when compared against of ‘caffeine alone’ (10 mg/kg) treated group (Fig. 3c). Caffeine + duloxetine (5 mg/kg, each) combination treated group showed significant increase in serotonin levels when compared against ‘duloxetine alone’ (10 mg/kg) treated group (Fig. 3c). Bupropion + duloxetine (5 mg/kg, each) combination treated group showed significant increase in serotonin levels when compared against of ‘bupropion alone’ (10 mg/kg) treated group (Fig. 3c).

plus duloxetine among combination treated groups. Additional behavioral parameters like swimming and climbing observed in forced swim test are summarized in Table 2 in supplementary materials section. Kirch et al. (1990) have reported no significant change in norepinephrine, dopamine, and serotonin levels with caffeine (10 mg/kg/day) treatment in the hippocampi region of rats. In addition to hippocampi, the present study observed similar results in cerebral cortices and whole brain. Reports analyzing the effect of duloxetine treatment on monoamines in rat frontal cortex have shown significant increase in norepinephrine, dopamine, and serotonin levels (Kihara and Ikeda, 1995; Muneoka et al., 2009). The present study findings are in agreement with the reported findings (Kihara and Ikeda, 1995; Muneoka et al., 2009). The increase in norepinephrine, and serotonin levels was recorded not only in cerebral cortices but also in hippocampi and whole brain. Dopamine levels were significantly increased in hippocampi and whole brain, but not in cerebral cortices. Piacentini et al. (2003) reported significant increase in norepinephrine, dopamine, and serotonin levels in the rat hippocampi region after bupropion treatment. Similar results were observed in the present study. In addition to hippocampi, the present study results showed an increase in monoamines in cerebral cortices and whole brain. The change in monoamine levels observed in each monotherapy treatment is comparable to the previous reports. The combination of drugs having different mechanisms of action might have helped in achieving an improved brain monoamine profile in hippocampi, cerebral cortices, and whole brain than the monotherapy. Caffeine plus duloxetine treated group showed better brain monoamine profile among combination treated groups. The inhibitory effect of antidepressants (tricyclics, SSRIs, mitrazapine, nefazodone) on four oxidation pathways of caffeine is reported by Kot et al. (2007). Caffeine metabolism involves CYP1A2 and other CYP isoforms (Daniel et al., 2003). Duloxetine metabolism involves CYP2D6 and CYP1A2 (Lantz et al., 2003), and bupropion metabolism mainly involves CYP2B6 (Jefferson et al., 2005). Other enzymes involved in the metabolism of bupropion include CYP1A2 and its other isoforms (Jefferson et al., 2005). Duloxetine may increase the retention of drugs that are metabolized by CYP2D6 and not by CYP1A2 (Knadler et al., 2011). Therefore, duloxetine may have low potential for interaction with caffeine. The interaction between bupropion and caffeine may also have low potential, as the major enzymes involved in metabolism are different. The inhibition of CYP2D6 by bupropion may reduce the clearance of drugs metabolized by the same enzyme (Jefferson et al., 2005). Bupropion combination with caffeine may produce drug interaction of low potential, but the combination with duloxetine may result in lower clearance of later drug. Metabolism of caffeine is dose dependent

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In forced swim and tail suspension tests, the antidepressant activity is expressed in terms of immobility period produced due to inescapable condition. These models have low degree of construct and/or etiologic validity. They also have limitations like giving false positive response to acute drug response, psychostimulants, and varying sensitivity for genetic variation. These tests are more selective for monoamine-based mechanism analysis. However, these tests have advantages like being the most predictive and widely used for the screening of antidepressant activity (Duman, 2010; Porsolt et al., 1997; Steru et al., 1985). A recent study used forced swim test and reported an interesting finding about the age-dependent changes in serotonin transport levels in lateral septum and dorsal raphe of rats (Ulloa et al., 2014). Prepubertal rats had less stress-induced depressive response due to higher availability of serotonin in forced swim test than pubertal (Ulloa et al., 2014). In the present study, reduction in immobility period observed in forced swim and tail suspension tests with caffeine alone, duloxetine alone, and bupropion alone treated groups is in agreement with previous reports (Kaster et al., 2004; Zomkowski et al., 2012; Bhatt et al., 2013). The immobility period was also significantly reduced in all combination treated groups, as compared to control groups in both antidepressant models. The reduction in immobility period was better with caffeine

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Table 1 Outline of statistical significance data of norepinephrine/dopamine/serotonin levels in hippocampi, cerebral cortices, and whole brain.

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Hippocampi Control Caffeine Duloxetine Bupropion

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Cerebral cortices Control Caffeine Duloxetine Bupropion Whole brain Control Caffeine Duloxetine Bupropion

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Control

Caffeine

Duloxetine

Caffeine + duloxetine

Bupropion

Bupropion + caffeine

Bupropion + duloxetine

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3.3.3.3. Whole brain. Groups such as ‘duloxetine alone’ (10 mg/kg), ‘bupropion alone’ (10 mg/kg), caffeine + duloxetine (5 mg/kg, each), bupropion + caffeine (5 mg/kg, each) and bupropion + duloxetine (5 mg/kg, each) showed significant increase in serotonin levels when compared against control groups (Fig. 3c). Caffeine + duloxetine (5 mg/kg, each) and bupropion + caffeine (5 mg/kg, each) combination treated groups showed significant increase in serotonin levels when compared against of ‘caffeine alone’ (10 mg/kg) treated group (Fig. 3c). The comparisons presented in Fig. 3 are compiled in tabular format for the easier understanding of data. The monoamine data representing the significance difference in groups is outlined in Table 1.

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Group present in first column (from left) is compared against groups present in first row (from top), separately. In each cell, 1st, 2nd and 3rd positions (e.g. N/N/N or Y/Y/Y) represent the statistical difference in norepinephrine, dopamine, and serotonin levels observed after comparison, respectively. Statistical results are indicated by Y and N, where Y — significant difference observed and N — no significant difference observed.

Please cite this article as: Kale PP, Addepalli V, Augmentation of antidepressant effects of duloxetine and bupropion by caffeine in mice, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.06.005

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press). The combination of caffeine with duloxetine may help in reducing side/adverse effects of later drug. The present study has pitfalls like not considering the analysis of locomotion, cognition, anxiety and escape behavior in forced swim test, and the estimation of serotonin transporter levels in different brain regions. These findings can be further probed in a clinical setting as a future endeavor. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.pbb.2014.06.005.

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(Denaro et al., 1990). The clearance rate of caffeine decreases with the increase in dose (Denaro et al., 1990). Consumption of caffeine particu367 larly at lower dose produces beneficial effects like increased alertness 368 and reduction in anxiety, whereas over-consumption results in adverse 369 effects such as increased anxiety and sleep disturbances (Smith, 2002). 370 Preference of higher amount of caffeine consumption by the depressed 371 patients (Whalen et al., 2008; Goldstein, 1987) and the inhibition of caf372 feine metabolism by antidepressants (tricyclics, SSRIs, mitrazapine, 373 nefazodone) (Kot et al., 2007) may further reduce the clearance rate 374 of caffeine. Therefore, consideration of lower dose in the present study 375 may result in drug interaction having low potential and better safety 376 profile. 377 There are no pre-clinical or clinical reports available with the consid378 eration of combination consisting caffeine either with duloxetine or 379 bupropion. Papakostas et al. (2006) have reported beneficial effects of 380 duloxetine and bupropion combination in the treatment-resistant381 depression. The elevation of mood by caffeine is dose-dependent and 382 Q10 can also depend on acute versus chronic treatment (Cunha et al., 383 2008) in animals (Pechlivanova et al., 2012; Batalha et al., 2013) and 384 humans (Lucas et al., 2011; Smith, 2002) through adenosine 385 neuromodulation (Fredholm et al., 2005). The inhibition of adenosine 386 receptors by caffeine results in a release of brakes imposed on dopami387 nergic neurotransmission (Ferré, 2008). The reasons for observing addi388 tive effects operated by caffeine and the antidepressant drugs probably 389 resides either in pharmacokinetic interactions (Kot et al., 2007) or both 390 drug types acting through similar mechanism (Enríquez-Castillo et al., 391 2008). The independent mechanism of action of caffeine and antide392 pressant drugs (duloxetine/bupropion) may allow synergic rather 393 than simply additive effects. The design of present study only allows 394 probing for acute effects of caffeine and antidepressant drugs 395 (duloxetine/bupropion), whereas the consumption in humans is always 396 in a repeated manner, a feature critical for caffeine, which has different 397 molecular targets under acute and chronic consumptions (Ferré, 2008; 398 Cunha and Agostinho, 2010). In spite of these limitations, the outcomes 399 of present study suggest the need to monitor coffee consumption in 400 depressed individuals, and how caffeine consumption changes with de401 pressive episodes and antidepressant therapy. 402 Overall, caffeine plus duloxetine treated group was better in elevat403 ing brain monoamines in hippocampi and cerebral cortices (Fig. 4), and 404 reducing the immobility period among the combination treated groups. 405 The dual noradrenergic/serotonergic reuptake inhibitors like duloxetine 406 have major side effects (e.g. sexual dysfunction) (Segraves and Balon, in

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Fig. 4. Effect of three different augmentation approaches on the levels of norepinephrine, dopamine, and serotonin in hippocampus and cerebral cortex regions.

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References

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Baldessarini RJ. Drug therapy of depression and anxiety disorders. In: Brunton LL, Lazo JS, Parker KL, editors. Goodman and Gilman's the pharmacological basis of therapeutics. New York: McGraw-Hill; 2006. p. 429–59. Batalha VL, Pego JM, Fontinha BM, Costenla AR, Valadas JS, Baqi Y, et al. Adenosine A(2A) receptor blockade reverts hippocampal stress-induced deficits and restores corticosterone circadian oscillation. Mol Psychiatry 2013;18:320–31. http://dx.doi.org/10. 1038/mp.2012.8. Bernstein GA, Carroll ME, Dean NW, Crosby RD, Perwien AR, Benowitz NL. Caffeine withdrawal in normal school-age children. J Am Acad Child Adolesc Psychiatry 1998;37:858–65. http://dx.doi.org/10.1097/0000458f3-199808000-00016. Bernstein GA, Carroll ME, Thuras PD, Cosgrove KP, Roth ME. Caffeine dependence in teenagers. Drug Alcohol Depend 2002;66:1–6. http://dx.doi.org/10.1016/S0376-8716(01) 00181-8. Bhatt S, Mahesh R, Devadoss T, Jindal A. Anti-depressant like activity of N-n-butyl-3methoxyquinoxaline-2-carboxamide (6o) a 5-HT3 receptor antagonist. Indian J Exp Biol 2013;51:435–43. Brannan SK, Mallinckrodt CH, Detke MJ, Watkin JG, Tollefson GD. Onset of action for duloxetine 60 mg once daily: double-blind, placebo-controlled studies. J Psychiatr Res 2005;39:161–72. http://dx.doi.org/10.1016/j.jpsychires.2004.05.005. Castle DJ, Piterman L, Berk M. Difficult-to-treat depression. MJA Open 2012;1:4–5. Choudhary KM, Mishra A, Poroikov VV, Goel RK. Ameliorative effect of curcumin on seizure severity, depression like behavior, learning and memory deficit in postpentylenetetrazole-kindled mice. Eur J Pharmacol 2013;704:33–40. http://dx.doi. org/10.1016/j.ejphar.2013.02.012. Ciulla L, Menezes HS, Bueno BB, Schuh A, Alves RJ, Abegg MP. Antidepressant behavioral effects of duloxetine and fluoxetine in the rat forced swimming test. Acta Cir Bras 2007;22:351–4. http://dx.doi.org/10.1590/S0102-86502007000500005. Cunha RA, Agostinho PM. Chronic caffeine consumption prevents memory disturbance in different animal models of memory decline. J Alzheimers Dis 2010;20:S95–S116. http://dx.doi.org/10.3233/JAD-2010-1408. Daniel WA, Kot M, Wójcikowski J. Effects of classic and newer antidepressants on the oxidation pathways of caffeine in rat liver. In vitro study. Pol J Pharmacol 2003;55: 1045–53. Denaro CP, Brown CR, Wilson M, Jacob P, Benowitz NL. Dose-dependency of caffeine metabolism with repeated dosing. Clin Pharmacol Ther 1990;48:277–85.

418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452

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Mojtabai R. Unmet need for treatment of major depression in the United States. Psychiatr Serv 2009;60:297–305. http://dx.doi.org/10.1176/appi.ps.60.3.297. Muneoka K, Shirayama Y, Takigawa M, Shioda S. Brain region-specific effects of shortterm treatment with duloxetine, venlafaxine, milnacipran and sertraline on monoamine metabolism in rats. Neurochem Res 2009;34:542–55. http://dx.doi.org/10. 1007/s11064-008-9818-2. Orbeta RL, Overpeck MD, Ramcharren D, Kogan MD, Ledsky R. High caffeine intake in adolescents: associations with difficulty sleeping and feeling tired in the morning. J Adolesc Health 2006;38:451–3. Papakostas GI, Worthington JJ, Iosifescu DV, Kinrys G, Burns AM, Fisher LB, et al. The combination of duloxetine and bupropion for treatment-resistant major depressive disorder. Depress Anxiety 2006;23:178–81. http://dx.doi.org/10.1002/da.20181. Pechlivanova DM, Tchekalarova JD, Alova LH, Petkov VV, Nikolov RP, Yakimova KS. Effect of long-term caffeine administration on depressive-like behavior in rats exposed to chronic unpredictable stress. Behav Pharmacol 2012;23:339–47. http://dx.doi.org/ 10.1097/FBP.0b013e3283564dd9. Piacentini MFR, Clinckers R, Meeusen R, Sarre R, Ebinger G, Michotte Y. Effect of bupropion on hippocampal neurotransmitters and on peripheral hormonal concentrations in the rat. J Appl Physiol 2003;95:652–6. Porsolt RD, Bertin A, Jalfre M. Behavioural despair in mice: a primary screening test for antidepressants. Arch Int Pharmacodyn Ther 1997;229:327–36. Reneman L, Lavalaye J, Schmand B, De Wolff FA, Van Den BW, Den Heeten GJ, et al. Cortical serotonin transporter density and verbal memory in individuals who stopped using 3,4-methylenedioxymethamphetamine (MDMA or “ecstasy”): preliminary findings. Arch Gen Psychiatry 2001;58:901–6. Rénéric JP, Lucki I. Antidepressant behavioral effects by dual inhibition of monoamine reuptake in the rat forced swimming test. Psychopharmacology (Berl) 1998;136: 190–7. Ressler KJ, Nemeroff CB. Role of norepinephrine in the pathophysiology of neuropsychiatric disorders. CNS Spectr 2001;6:663–70. Richelson E. Multi-modality: a new approach for the treatment of major depressive disorder. Int J Neuropsychopharmacol 2013;16:1433–42. http://dx.doi.org/10.1017/ S1461145712001605. Ruusunen A, Lehto SM, Tolmunen T, Mursu J, Kaplan GA, Voutilainen S. Coffee, tea and caffeine intake and the risk of severe depression in middle-aged Finnish men: the Kuopio Ischaemic Heart Disease Risk Factor Study. Public Health Nutr 2010;13: 1215–20. http://dx.doi.org/10.1017/S1368980010000509. Segraves RT, Balon R. Antidepressant-induced sexual dysfunction in men. Pharmacol Biochem Behav 2014. http://dx.doi.org/10.1016/j.pbb.2013.11.003. [in press]. Smith A. Effect of caffeine on human behavior. Food Chem Toxicol 2002;40:1243–55. http://dx.doi.org/10.1016/S0278-6915(02)00096-0. Stahl SM, Grady MM, Moret C, Briley M. SNRIs: their pharmacology, clinical efficacy, and tolerability in comparison with other classes of antidepressants. CNS Spectr 2005;10: 732–47. Steru L, Chermat R, Thierry B, Simon P. The tail suspension test: a new method for screening antidepressants in mice. Psychopharmacology (Berl) 1985;85:367–70. Tanskanen A, Tuomilehto J, Viinamäki H, Vartiainen E, Lehtonen J, Puska P. Heavy coffee drinking and the risk of suicide. Eur J Epidemiol 2000;16:789–91. Thase ME, Pritchett YL, Ossanna MJ, Swindle RW, Xu J, Detke MJ. Efficacy of duloxetine and selective serotonin reuptake inhibitors: comparisons as assessed by remission rates in patients with major depressive disorder. J Clin Psychopharmacol 2007;27: 672–776. Thase ME. Augmentation strategies for depression: history and concepts. CNS Spectr 2007;12:3–5. Tse WS, Chan CC, Shiu SY, Chung PY, Cheng SH. Caffeinated coffee enhances cooperative behavior in the Mixed Motive Game in healthy volunteers. Nutr Neurosci 2009;12: 21–7. http://dx.doi.org/10.1179/147683009X388896. Ulloa RE, Díaz-Valderrama A, Herrera-Pérez J, León-Olea M, Martínez-Mota L. Age differences in the impact of forced swimming test on serotonin transporter levels in lateral septum and dorsal raphe. Behav Brain Funct 2014;10:3. http://dx.doi.org/10.1186/ 1744-9081-10-3. Vogel HG, Vogel WH. Psychotropic and neurotropic activity. In: Vogel HG, Vogel WH, editors. Drug discovery and evaluation, pharmacological assays. Berlin, Heidelberg, New York: Springer-Verlag; 2008. p. 791. Whalen DJ, Silk JS, Semel M, Forbes EE, Ryan ND, Axelson DA, et al. Caffeine consumption, sleep, and affect in the natural environments of depressed youth and healthy controls. J Pediatr Psychol 2008;33:358–67. http://dx.doi.org/10.1093/jpepsy/jsm086. Wilner P. Dopamine and depression: a review of recent evidence. Brain Res Rev 1983;6: 211–27. Zomkowski AD, Engel D, Cunha MP, Gabilan NH, Rodrigues AL. The role of the NMDA receptors and l-arginine-nitric oxide-cyclic guanosine monophosphate pathway in the antidepressant-like effect of duloxetine in the forced swimming test. Pharmacol Biochem Behav 2012;103:408–17. http://dx.doi.org/10.1016/j.pbb.2012.09.011.

E

T

Duman CH. Models of depression. Vitam Horm 2010;82:1–21. http://dx.doi.org/10.1016/ S0083-6729(10)82001-1. Dunlop BW, Nemeroff CB. The role of dopamine in the pathophysiology of depression. Arch Gen Psychiatry 2007;64:327–37. El Yacoubi M, Ledent C, Ménard JF, Parmentier M, Costentin J, Vaugeois JM. The stimulant effects of caffeine on locomotor behaviour in mice are mediated through its blockade of adenosine A(2A) receptors. Br J Pharmacol 2000;129:1465–73. http://dx.doi.org/ 10.1038/sj.bjp.0703170. Enríquez-Castillo A, Alamilla J, Barral J, Gourbière S, Flores-Serrano AG, Góngora-Alfaro JL, et al. Differential effects of caffeine on the antidepressant-like effect of amitriptyline in female rat subpopulations with low and high immobility in the forced swimming test. Physiol Behav 2008;94:501–9. http://dx.doi.org/10.1016/j.physbeh.2008.03.004. Ferré S. An update on the mechanisms of the psychostimulant effects of caffeine. J Neurochem 2008;105:1067–79. Fredholm BB, Bättig K, Holmén J, Nehlig A, Zvartau EE. Actions of caffeine in the brain with special reference to factors that contribute to its widespread use. Pharmacol Rev 1999;51:83–133. Fredholm BB, Chen JF, Cunha RA, Svenningsson P, Vaugeois JM. Adenosine and brain function. Int Rev Neurobiol 2005;63:191–270. Gessa GL. Dopaminergic system in antidepressant action. Neuropsychopharmacology 1994;10:47S. Goldstein A. Criteria of a pharmacologic withdrawal syndrome. Arch Gen Psychiatry 1987;44:392. Grunebaum MF, Ellis SP, Duan N, Burke AK, Oquendo MA, John Mann J. Pilot randomized clinical trial of an SSRI vs bupropion: effects on suicidal behavior, ideation, and mood in major depression. Neuropsychopharmacology 2012;37:697–706. http://dx.doi. org/10.1038/npp.2011.247. Heckman MA, Weil J, Gonzalez de Mejia E. Caffeine (1, 3, 7-trimethylxanthine) in foods: a comprehensive review on consumption, functionality, safety, and regulatory matters. J Food Sci 2010;75:R77–87. http://dx.doi.org/10.1111/j.1750-3841.2010.01561.x. Hunziker ME, Suehs BT, Bettinger TL, Crisrnon ML. Duloxetine hydrochloride: a new dualacting medication for the treatment of major depressive disorder. Clin Ther 2005;27: 1126–43. Jacobs DG, Baldessarini RJ, Conwell Y, Fawcett JA, Horton L, Meltzer H, et al. Practice guideline for the assessment and treatment of patients with suicidal behaviors. Arlington, VA: American Psychiatric Publishing; 2003. http://dx.doi.org/10.1176/appi. books.9780890423363.56008. Jefferson JW, Pradko JF, Muir KT. Bupropion for major depressive disorder: pharmacokinetic and formulation considerations. Clin Ther 2005;27:1685–95. http://dx.doi.org/ 10.1016/j.clinthera.2005.11.011. Kaster MP, Rosa AO, Rosso MM, Goulart EC, Santos AR, Rodrigues AL. Adenosine administration produces an antidepressant-like effect in mice: evidence for the involvement of A1 and A2A receptors. Neurosci Lett 2004;355:21–4. http://dx.doi.org/10.1016/j. neulet.2003.10.040. Kawachi I, Willett WC, Colditz GA, Stampfer MJ, Speizer FE. A prospective study of coffee drinking and suicide in women. Arch Intern Med 1996;156:521–5. Kihara T, Ikeda M. Effects of duloxetine, a new serotonin and norepinephrine uptake inhibitor, on extracellular monoamine levels in rat frontal cortex. J Pharmacol Exp Ther 1995;272:177–83. Kirch DG, Taylor TB, Gerhardt GB, Benowitz NL, Stephen C, Wyatt RJ. Effect of chronic caffeine administration oh monoamine and monoamine metabolite concentrations in rat brain. Neuropharmacology 1990;29:599–602. Knadler MP, Lobo E, Chappell J, Bergstrom R. Duloxetine: clinical pharmacokinetics and drug interactions. Clin Pharmacokinet 2011;50:281–94. http://dx.doi.org/10.2165/ 11539240-000000000-00000. Kot M, Wójcikowski J, Daniel WA. Caffeine metabolism during prolonged treatment of rats with antidepressant drugs. Pharmacol Rep 2007;59:727–33. Kotagale NR, Tripathi SJ, Aglawe MM, Chopde CT, Umekar MJ, Taksande BG. Evidences for the agmatine involvement in antidepressant like effect of bupropion in mouse forced swim test. Pharmacol Biochem Behav 2013;107:42–7. http://dx.doi.org/10.1016/j. pbb.2013.03.019. Lantz RJ, Gillespie TA, Rash TJ, Kuo F, Skinner M, Kuan HY, et al. Metabolism, excretion, and pharmacokinetics of duloxetine in healthy human subjects. Drug Metab Dispos 2003;31:1142–50. Lucas M, Mirzaei F, Pan A, Okereke OI, Willett WC, O'Reilly ÉJ, et al. Coffee, caffeine, and risk of depression among women. Arch Intern Med 2011;171:1571–8. http://dx.doi. org/10.1001/archinternmed.2011.393. Lucas M, O'Reilly EJ, Pan A, Mirzaei F, Willett WC, Okereke OI, et al. Coffee, caffeine, and risk of completed suicide: results from three prospective cohorts of American adults. World J Biol Psychiatry 2014. http://dx.doi.org/10.3109/15622975.2013.795243. [in press]. Madepalli K, Lakshmana Raju TR. An isocratic assay for norepinephrine, dopamine, and 5hydroxytryptamine using their native fluorescence by high-performance liquid chromatography with fluorescence detection in discrete brain areas of rat. Anal Biochem 1997;246:166–70.

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