The influence of caffeine on the activity of moclobemide, venlafaxine, bupropion and milnacipran in the forced swim test in mice

    The influence of caffeine on the activity of moclobemide, venlafaxine, bupropion and milnacipran in the forced swim test in mice Ewa Poleszak, Aleksandra Szopa, El˙zbieta Wyska, Sylwia Wo´sko, Anna Serefko, Aleksandra Wla´z, Mateusz Pier´og, Andrzej Wr´obel, Piotr Wla´z PII: DOI: Reference:

S0024-3205(15)00331-8 doi: 10.1016/j.lfs.2015.06.008 LFS 14415

To appear in:

Life Sciences

Received date: Revised date: Accepted date:

28 February 2015 5 May 2015 10 June 2015

Please cite this article as: Poleszak Ewa, Szopa Aleksandra, Wyska El˙zbieta, Wo´sko Sylwia, Serefko Anna, Wla´z Aleksandra, Pier´og Mateusz, Wr´ obel Andrzej, Wla´z Piotr, The influence of caffeine on the activity of moclobemide, venlafaxine, bupropion and milnacipran in the forced swim test in mice, Life Sciences (2015), doi: 10.1016/j.lfs.2015.06.008

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The influence of caffeine on the activity of moclobemide, venlafaxine, bupropion and milnacipran in the forced swim test in mice

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Ewa Poleszak a,*, Aleksandra Szopa a, Elżbieta Wyska b, Sylwia Wośko a, Anna Serefko a,

Department of Applied Pharmacy, Medical University of Lublin, Chodźki 1, PL 20-093

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a

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Aleksandra Wlaź c, Mateusz Pieróg d, Andrzej Wróbel e, Piotr Wlaź d

Lublin, Poland b

Department of Pharmacokinetics and Physical Pharmacy, Collegium Medicum, Jagiellonian

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University, Medyczna 9, PL 30-688 Kraków, Poland

Department of Pathophysiology, Medical University of Lublin, Jaczewskiego 8, PL 20-090

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Lublin, Poland

Department of Animal Physiology, Institute of Biology and Biochemistry, Faculty of

Biology and Biotechnology, Maria Curie-Skłodowska University, Akademicka 19, PL 20-033

Second Department of Gynecology, Medical University of Lublin, Jaczewskiego 8, PL 20-

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090 Lublin, Poland

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Lublin, Poland

*Corresponding author at:

Department of Applied Pharmacy, Medical University of Lublin, Chodźki 1, PL 20-093 Lublin, Poland

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Tel.: +48 81 448 70 40 Fax: +48 81 448 70 40

E-mail address: [email protected] (E. Poleszak)

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Abstract

Aims: Worrying data indicate that excessive caffeine intake applies to patients suffering from

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mental disorders, including depression. It is thus possible to demonstrate the usefulness of

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caffeine and its derivatives in the treatment of depression. The main goal of the present study was to evaluate the influence of caffeine (5 mg/kg) on the activity of moclobemide (1.5

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mg/kg), venlafaxine (1 mg/kg), bupropion (10 mg/kg), and milnacipran (1.25 mg/kg). Moreover, we assessed the influence of caffeine on their serum and brain levels using highperformance liquid chromatography.

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Main methods: The experiment was carried out on naïve adult male Albino Swiss mice. Caffeine and tasted drugs were administered intraperitoneally. The influence of caffeine on

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the activity of selected antidepressant drugs was evaluated in forced swim test (FST). Locomotor activity was estimated to verify and exclude false positive/negative results. To assess the influence of caffeine on the levels of studied antidepressant drugs, their

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concentrations were determined in murine serum and brains using high-performance liquid

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chromatography.

Key findings: Caffeine potentiated activity of all antidepressants examined in FST and the

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observed effects were not due to the increase in locomotor activity in the animals. Only in the case of co-administration of caffeine and milnacipran an increased milnacipran concentration in serum was observed without affecting its concentration in the brain. Significance: Caffeine potentiates the activity of antidepressant drugs from different chemical

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groups. The interactions of caffeine with venlafaxine, bupropion and moclobemide occur in pharmacodynamic phase, whereas the interaction of caffeine-milnacipran occurs, at least partially, in pharmacokinetic phase.

Keywords caffeine, antidepressant drugs, forced swim test, pharmacokinetic study, mice

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Introduction

Caffeine is the most widely used behavioral active drug in the world [58] which acts

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as an antagonist of the adenosine receptors A1, A2 and A3 [21,23,54]. Higher doses of caffeine

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inhibit phosphodiesterase, block the receptors for γ-aminobutyric acid type A (GABAA), and cause the release of intracellular Ca2+ [40]. Caffeine is characterized by a stimulating

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influence on the cerebral cortex which is related to the release of various neurotransmitters in central nervous system [14,22,26,29,45,59,69]. Effects of caffeine activity are a common issue to investigate in the scientific world. A lot of research that aims at defining the effect of

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caffeine on human body is carried out. The predominant outcomes suggest that caffeine has an impact on mood boost [35,40], as well as on consciousness enhancement, acquiring and

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processing information, and reaction time and attention [6]. Caffeine is present in many drinks and its content in a cup of coffee is as high as 100 mg. Average daily consumption of caffeine amounts to 3 mg/kg a day. Caffeine is very

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frequently an ingredient of analgesics, appetite inhibition drugs or additive of stimulating

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preparations [42,43]. Caffeine consumption rises every year. Worrying data indicate that excessive caffeine intake applies to patients suffering from mental disorders. Many patients

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with depression feel continuous exhaustion and hence consume significant quantities of caffeine [1], which results in an advantageous effect [66]. It is estimated that this issue affects about 22% of hospitalized patients who were diagnosed with mental disorders comparing with 9% for healthy people [30]. Due to an increasing number of cases diagnosed with mental

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illnesses, including depression, and a widespread use of antidepressants with various action mechanisms, it seems to be important to test the interactions between caffeine and the antidepressant drugs. In our recent study we showed that caffeine increased the effect of typical antidepressant drugs, such as imipramine and its metabolite – desipramine (tricyclic antidepressant – TCA), fluoxetine, escitalopram and paroxetine (selective serotonin reuptake inhibitors, SSRI) and reboxetine (selective noradrenaline reuptake inhibitor, NRI) (unpublished data). Therefore, the main goal of this study was to evaluate the influence of caffeine on the activity of antidepressants acting through other mechanisms, such as moclobemide, venlafaxine, bupropion and milnacipran in the forced swim test (FST). To verify and exclude false-positive or false-negative results locomotor activity was estimated. Additionally, to evaluate whether the observed animals' behavior effects were consequent to a pharmacokinetic/pharmacodynamic interaction, concentrations of the studied antidepressant

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drugs in mice serum and brain homogenates were measured using high-performance liquid chromatography (HPLC).

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Materials and methods

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Animals

The experiment was carried out on naïve adult male Albino Swiss mice (25–30 g) purchased from the licensed breeder (Kolacz, Warsaw, Poland). The animals were housed in

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the environmentally controlled rooms with a 12 h light/dark cycle, in groups of 10 in standard cages under strictly controlled laboratory conditions – temperature maintained at 22–23 °C,

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relative humidity about 45–55%. Throughout the study, the animals were given ad libitum access to water and food. The experiment began after at least 1-week acclimation period in the laboratory conditions and was conducted between 8 a.m. and 3 p.m. to minimize circadian

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influences. Each experimental group consisted of 8–12 animals. Procedures involving mice

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and their care in all the experiments of the present study were approved by the Local Ethics Committee at the Medical University of Lublin (license no 26/2011, 28/2011 and 26/2013)

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and were performed in accordance with binding European standards related to the experimental studies on animal models. Each mouse was used only once.

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Drug administration

Caffeine (1,3,7-trimethylxanthine; 5 mg/kg, Sigma-Aldrich), moclobemide (1.5 mg/kg, Sigma-Aldrich, Poznań, Poland), milnacipran hydrochloride (1.25 mg/kg, Abcam, Cambridge, UK), venlafaxine hydrochloride (1 mg/kg, Sigma-Aldrich, Poznań, Poland) and bupropion hydrochloride (10 mg/kg, Abcam, Cambridge, UK) were dissolved in 0.9% NaCl. The solutions of antidepressants were administered intraperitoneally (ip) 60 min before behavioural testing whereas caffeine solution was administered ip 40 min before the tests. The doses and pretreatment schedules were selected on those reported in the literature and on the basis of the results of our previous experiments [49,52,57,68]. All solutions were prepared immediately prior to the experiment. Animals from the control groups received ip injections of the vehicle (0.9% saline). The volume of the vehicle or drug solutions for ip administrations was 10 ml/kg.

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Forced swimming test (FST)

The procedure was carried out on mice, according to the method of Porsolt et al. [53].

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Each mouse was placed individually into the glass cylinders (height 25 cm, diameter 10 cm)

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containing 10 cm of water at 23–25 °C, which was exchanged for clean after each test (each mice). The animals were left in the cylinder for 6 min. The total duration of immobility was

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real-time recorded by cumulative stopwatches during the last 4 min of the 6-min long testing period. The mouse was judged to be immobile when it ceased struggling and remained floating motionless in the water, making only the movements necessary to keep its head above

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the water level.

The results obtained in the FST were shown as the arithmetic mean of immobility time

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of animals given in seconds, ± standard error of the mean (S.E.M) for each experimental group.

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Spontaneous locomotor activity

In order to avoid the risk of obtaining the false positive/negative effects in the FST

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caused by a possible influence of tested agents on the locomotor activity, the spontaneous locomotor activity was measured using an animal activity meter Opto-Varimex-4 Auto-Track (Columbus Instruments, USA). This actimeter consists of four transparent cages with a lid (43 × 43 × 32 cm), a set of four infrared emitters (each emitter has 16 laser beams), and four

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detectors monitoring animal movements. After ip pretreatment with respective drugs or drug combinations and after a given time period mice were placed individually into the cages for 10 min. Spontaneous locomotor activity was evaluated between the 2nd and the 6th minute, which corresponds with the time interval analysed in the FST. The results obtained in this test were presented as the arithmetic average distance that a mouse travelled (in cm) ± S.E.M for each experimental group.

Determination of antidepressants in the serum and brain homogenates

Sixty minutes following administration of tested antidepressant drugs with or without caffeine, unanesthetized mice were decapitated to collect biological material for pharmacokinetic studies. The blood was collected into Eppendorf tubes and allowed to clot at room temperature. Subsequently, the blood was centrifuged at 10 000 rpm for 10 min and

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serum was collected into polyethylene tubes and frozen at –25 °C. Immediately after the decapitation, the brains were dissected from the skull, washed with 0.9% NaCl and also frozen at -25 °C.

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Serum and brain concentrations of the studied antidepressants were assayed by HPLC.

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The brains were homogenized in distilled water (1:4, w/v) with a tissue homogenizer TH220 (Omni International, Inc., Warrenton, VA, USA). For all studied drugs, the extraction from

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serum and brain homogenates were performed using the mixture of ethyl acetate:hexane (30:70, v/v). Internal standard (IS) for venlafaxine was reboxetine (500 ng/ml), for milnacipran – bupropion (1 μg/ml), for bupropion – milnacipran (1 μg/ml) and for

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moclobemide – venlafaxine (1 μg/ml).

To isolate venlafaxine, milnacipran, bupropion, and moclobemide from serum (200 μl) or brain homogenate (1 ml) containing these drugs an appropriate IS was added and the

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samples were alkalized with 100 μl of 4 M NaOH. Then the samples were extracted twice with 3 ml of the extraction reagent by shaking for 20 min (IKA Vibrax VXR, Germany).

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After centrifugation at 3 000 rpm for 20 min (Universal 32, Hettich, Germany), the organic

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layers were transferred to a new tube containing a 100 μl solution of 0.1 M H2SO4 and methanol (90:10 v/v) or 0.1 M HCl (for milnacipran and bupropion), the mixture was shaken

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for 0.5 h and then centrifuged for 15 min (3 000 rpm). The organic layer was discarded and a 50 μl aliquot of the acidic solution was injected into the HPLC system. The HPLC system (Thermo Separation Products, San Jose, CA, USA) consisted of a P100 isocratic pump, a UV100 variable-wavelength UV/VIS detector, a Rheodyne 7125 injector (Rheodyne, Cotati,

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CA, USA) with a 50 μl sample loop, and a Chromjet SP4400 computing integrator. The analyses of bupropion and milnacipran were performed on a 250 × 4.6 mm LiChrospher®100 RP-18 column with a particle size of 5 μm (Merck, Darmstadt, Germany) protected with a guard column (4 × 4 mm) with the same packing material. The separation of moclobemide and venlafaxine was conducted using 250 × 4.6 mm Supelcosil LC-CN column with a particle size of 5 μm (Sigma Aldrich, Germany) protected with a guard column (4 × 4 mm) with the same packing material. The mobile phase consisted of 50 mM potassium dihydrogen phosphate and acetonitrile mixed at a ratio of 85:15 (v/v) for moclobemide, 75:25 (v/v) for milnacipran and bupropion, and 69:31 (v/v) for venlafaxine and run at 1 ml/min. Chromatographic analysis was carried out at 21°C and the analytical wavelength was 214 nm for bupropion, 200 nm for milnacipran, 230 nm for venlafaxine, and 240 nm for moclobemide.

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The calibration curves constructed by plotting the ratio of the peak heights of the studied drug to IS versus concentration of the drug were linear in the tested concentration ranges. No interfering peaks were observed in the chromatograms. The assays were

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reproducible with low intra- and inter-day variation (coefficient of variation less than 10%).

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The extraction efficiencies of the analyzed compounds and internal standards ranged from 66 to 97%. Antidepressant concentrations were expressed in ng/ml of serum or ng/g of wet brain

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tissue.

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Statistical analysis

The statistical analysis of the results obtained in the FST and locomotor activity was

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carried out using two-way ANOVA with Bonferroni's post-hoc test. The concentrations of the tested antidepressant drugs in blood and brains of mice in the presence and absence of caffeine were compared using Student's t-test. P values less than or equal to 0.05 were

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considered statistically significant.

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Results

Forced swim test

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The influence of caffeine on antidepressant activity of tested drugs in FST

Effect of combined administration of caffeine and moclobemide in FST

The effect of the combined administration of caffeine and moclobemide on the total duration of the immobility time in mice is shown in Fig. 1A. Caffeine (5 mg/kg) injected in combination with moclobemide (1.5 mg/kg) significantly reduced the immobility time in the FST in mice (Fig. 1A). Moclobemide (1.5 mg/kg) and caffeine (5 mg/kg) given alone had no effect on the immobility time (Fig. 1A). Two-way ANOVA demonstrated a significant effect of moclobemide [F(1,36)=16.64, p=0.0002], a significant effect of caffeine [F(1,36)=28.61, p<0.0001], and a significant interaction between moclobemide and caffeine [F(1,36)=12.14, p=0.0013].

Effect of combined administration of caffeine and venlafaxine in FST

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The effect of the combined administration of caffeine and venlafaxine on the total duration of the immobility time in mice is shown in Fig. 1A. Caffeine (5 mg/kg) injected in

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combination with venlafaxine (1 mg/kg) significantly reduced the immobility time in the FST

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in mice (Fig. 1A). Venlafaxine (1 mg/kg) and caffeine (5 mg/kg) given alone had no effect on the immobility time (Fig. 1A).

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Two-way ANOVA demonstrated a significant effect of venlafaxine [F(1,36)=9.92, p=0.0033], a significant effect of caffeine [F(1,36)=32.28, p<0.0001], and a significant

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interaction between venlafaxine and caffeine [F(1,36)=18.09, p=0.0001].

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Effect of combined administration of caffeine and bupropion in FST

The effect of the combined administration of caffeine and bupropion on the total duration of the immobility time in mice is shown in Fig. 1B. Caffeine (5 mg/kg) injected in

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combination with bupropion (10 mg/kg) significantly reduced the immobility time in the FST

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in mice (Fig. 1B). Bupropion (10 mg/kg) and caffeine (5 mg/kg) given alone had no effect on the immobility time (Fig. 1B).

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Two-way ANOVA demonstrated a significant effect of bupropion [F(1,36)=14.25, p=0.0006], a significant effect of caffeine [F(1,36)=27.43, p<0.0001], and a significant interaction between bupropion and caffeine [F(1,36)=15.60, p=0.0003].

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Effect of combined administration of caffeine and milnacipran in FST

The effect of the combined administration of caffeine and milnacipran on the total duration of the immobility time in mice is shown in Fig. 1B. Caffeine (5 mg/kg) injected in combination with milnacipran (1.25 mg/kg) significantly reduced the immobility time in the FST in mice (Fig. 1B). Milnacipran (1.25 mg/kg) and caffeine (5 mg/kg) given alone had no effect on the immobility time (Fig. 1B). Two-way ANOVA demonstrated a significant effect of milnacipran [F(1,36)=19.58, p<0.0001], a significant effect of caffeine [F(1,36)=27.74, p<0.0001], and a significant interaction between milnacipran and caffeine [F(1,35)=9.06, p<0.0048].

Spontaneous locomotor activity

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Effect of combined administration of caffeine and antidepressants on locomotor activity in

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mice

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The effect of the combined administration of caffeine and tested antidepressant drugs on spontaneous locomotor activity in mice is shown in Table 1.

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Caffeine or antidepressants (moclobemide, milnacipran, venlafaxine, bupropion) administered either alone or combined together had no statistically significant effects on the locomotor activity in mice (Table 1).

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Two-way ANOVA demonstrated: (A): no effect of moclobemide [F(1,25)=1.65, p=0.2101], significant effect of caffeine [F(1,25)=5.97, p=0.0219] and no interaction

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[F(1,25)=0.10, p=0.7597]. (B): no effect of milnacipran [F(1,28)=0.47, p=0.4992], no effect of caffeine [F(1,28)=0.10, p=0.7580] and no interaction [F(1,28)=2.51, p=0.1247]. (C): no effect of venlafaxine [F(1,27)=0.92, p=0.3455], significant effect of caffeine [F(1,27)=5.00,

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p=0.0338] and no interaction [F(1,27)=1.23, p=0.2779]. (D): no effect of bupropion

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[F(1,28)=0.20, p=0.6573], no significant effect of caffeine [F(1,28)=0.79, p=0.3819] and no

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interaction [F(1,28)=3.20, p=0.0843].

Pharmacokinetic studies

The effect of caffeine on serum and brain concentrations of the tested antidepressants

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in mice is shown in Table 2. Only in the case of co-administration of caffeine and milnacipran an increased milnacipran concentration in serum was observed (t-test: p=0.0004) without affecting its concentration in the brain.

Discussion Our study showed that caffeine at an ineffective dose enhanced the antidepressant-like effect of the reversible monoamine oxidase inhibitor (moclobemide), the serotoninnorepinephrine reuptake inhibitors (venlafaxine and milnacipran), and the dopamine and norepinephrine reuptake inhibitor (bupropion) administered also in non-effective doses. The FST outcomes indicated a synergistic action of caffeine in combination with the tested drugs without affecting on spontaneous locomotor activity of mice. It is well known, that caffeine exerts its action on central nervous system mainly through A1 and A2A adenosine receptors for which it acts as a non-selective antagonist.

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Caffeine enhances neurotransmission in the CNS by increasing the release of various neurotransmitters, such as: acetylcholine (ACh), γ-aminobutyric acid (GABA), glutamate, dopamine (DA), noradrenaline (NA), and serotonin (5-hydroxytryptamine, 5-HT), which has

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been confirmed in numerous studies both in vitro and in vivo [11,25,67]. A long-term

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treatment with antidepressant drugs also affect neurotransmitter systems resulting in an elevation of mood [33,44]. Drugs which through its mechanisms of action contribute to the

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increase in the level of serotonin, noradrenaline and dopamine in the CNS play a vital role in the antidepressant therapy [12]. In the presented study, four drugs that have a proven antidepressant activity and act by increasing monoamine neurotransmission have been co-

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administered with caffeine. Two of them, venlafaxine and milnacipran, are considered to be a dual serotonin-noradrenaline reuptake inhibitor (SNRI) [3,41,65]. Results from clinical

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research indicate that simultaneous inhibition of both NA and 5-HT may induce enhanced antidepressant activity in comparison with drugs selectively acting only on one monoamine level [36]. According to Kale and Addepalli [32], a significant decrease in immobility period

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was observed after concomitant administration of the non-effective doses of caffeine and

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duloxetine (5 mg/kg, each), another SNRI. Milnacipran (1.25 mg/kg) and venlafaxine (1 mg/kg) administered together with caffeine (5 mg/kg) significantly shortened the immobility

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time in the FST in mice. The results were not due to an increase in locomotor activity of animals. These results are the first to indicate the synergistic effects of caffeine administered together with milnacipran and venlafaxine. The observed increase in antidepressant activity can be explained by the intensification of the noradrenergic and serotonergic transmission in

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CNS by caffeine. A confirmation of this hypothesis may be a better brain monoamine profile observed by Kale and Addepalli [32] in the animal group receiving duloxetine with caffeine comparing with other treated groups. It has been estimated that, in the hippocampus and cerebral cortex as well as throughout the brain, levels of 5-HT, NA, and DA increased in mice receiving duloxetine and caffeine at the same time [32]. Similar concentrations of monoamines in the rats’ hippocampus have been reported by Piacentini et al. [51] after administration of an active dose of bupropion, which preferentially inhibits the synaptic reuptake of dopamine [60]. Moreover, bupropion also restrains norepinephrine reuptake, which increases extracellular levels of both norepinephrine and dopamine in the structures of the brain [15,70]. Caffeine has a similar effect on enhancing dopaminergic transmission. The observed changes in dopaminergic transmission after its administration can be explained by adenosine-acetylcholine-dopamine interaction [17,33]. A stimulation of adenosine A1 receptors localized in striatopallidal and striatonigral neurons,

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cholinergic interneurons and dopamine nerves endings inhibits dopamine D1 receptors [5,19,20,31,37,56]. It has been shown that adenosine A2A receptors are found in brain regions rich in DA – striatum, nucleus accumbens, hippocampus, cerebral cortex [17].

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Agonistic effects on adenosine-A2A receptors decrease the affinity of dopamine

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receptor antagonists for the D2 receptor [17,18] and lead to a reduction in D2 dopamine receptor signaling. The A2A receptors located in striatopallidal neurons seem to fulfill the

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same role as the D2 receptors in striatonigral neurons [5,31,37,56]. This interaction is not only limited to dopamine D2 and adenosine A2A receptors [4]. Ferre et al. [18] noticed that blockade of adenosine A2A receptors intensifies transduction by dopamine D1 receptors. The

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ability of caffeine to block both pre- and postsynaptic adenosine receptors and hence to potentiate the dopaminergic neurotransmission is considered to be the main mechanism of the

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production of psychomotor activity [16]. In addition, it has been shown that low doses of caffeine have the effect similar to using a selective A2A receptor antagonist [62,63]. Thus, as observed in our study, shortening the time of mice immobility in the FST in the group

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receiving both caffeine (5 mg/kg) and bupropion (10 mg/kg) can be explained by two distinct

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ways. On one hand, it is the severity of dopaminergic neurotransmission by the antagonistic effect of caffeine on the A1 and A2A receptors, on the other hand, the inhibition of neuronal

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DA reuptake by bupropion that potentiates the DA signaling in the CNS. At the same time, both substances also increase noradrenergic neurotransmission [47,60]. Kale and Addepalli [32] in their research on mice, where bupropion and caffeine at doses of 5 mg/kg each have been co-administered, also observed a statistically significant reduction in the immobility time

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of animals in the FST and in the tail suspension test. The further research undertaken to estimate the level of monoamines in the brain structures showed that the combined application of these agents led to the increased concentrations of NA, DA, and 5-HT in the hippocampus, cerebral cortex, and thus the whole brain. Until now, the effects of caffeine on the antidepressant effects of monoamine oxidase inhibitors (MAOIs) have not been studied. The results of our study indicate that coadministration of caffeine (5 mg/kg) and moclobemide (1.5 mg/kg), a reversible inhibitor of MAO-A, significantly reduced the duration of immobility time in the FST in mice. It is widely known, that moclobemide increases the level of NA, DA, and 5-HT in the synaptic slots, leading to excitation of the CNS transduction, but it also leads to a dose-dependence in the level of catecholamine metabolites [7-10,34,38]. The observed synergistic activity is probably due to the fact that caffeine aggravates noradrenergic, serotonergic, and dopaminergic transmissions [11,25,67]. Clinical studies have shown that patients positively

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responded to the therapy with moclobemide, and psychostimulants, like amphetamine and methylphenidate [61]. The main fly in the ointment of the FST is that drugs affecting spontaneous locomotor

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activity may yield false-positive/false-negative results [50]. Therefore, the spontaneous

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activity test was performed to exclude the possibility that the results obtained in the FST are due to the severity of mice locomotor activity. The combined administration of caffeine with

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milnacipran, venlafaxine, bupropion, and moclobemide did not affect motor performance in mice. Also, a separate injection of the tested substances did not significantly influence the locomotor activity of animals.

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It is known that isoenzyme CYP1A2 of cytochrome P450 participates in the biotransformation of many drugs, including medications used in the treatment of depression

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and psychosis [2,46]. In vitro and in vivo studies indicate that the activity of this isoenzyme may vary due to the consumption of nutrients, including caffeine as well as drugs that may induce or inhibit CYP1A2 activity [24,28]. Caffeine is metabolized in more than 95% by the

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liver via CYP1A2 [27,64]. Clinically significant pharmacokinetic interactions between

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caffeine and other drugs are related to the amount and activity of this isoenzyme [39,48]. Pharmacokinetic studies carried out in the present research were aimed at assessing

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concentrations of antidepressants in blood and brain of mice after their combined administration with caffeine and estimating the nature of the drug-drug interactions. The existing data on the changes in the therapeutic effect of antidepressants caused by concomitant administration of caffeine were based solely on the results obtained in behavioral

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tests [32,55] and on the levels of monoamines in the brain structures [32]. The results of our study indicate, that in the case of the combined administration of caffeine and venlafaxine, bupropion, or moclobemide the effect observed in the FST was related to pharmacodynamic rather than pharmacokinetic interactions because caffeine did not alter these drugs concentrations either in serum or in brain homogenates. The interaction caffeine-milnacipran is not entirely clear, because there were no differences in drug concentrations in CNS, which is the target site of action of antidepressants. In the case of drugs acting on the CNS, it is considered that there should be a correlation between the concentration of the drug in blood and brain tissue. Disturbance or competition in drug transport across the blood-brain barrier may be a probable cause of this discrepancy [13]. Caution should be kept in the formulation of proposals for caffeine-antidepressant interactions, as metabolism of these drugs in humans may proceed differently than in rodents..

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Conclusion

In conclusion, caffeine increased the effect of all the studied drugs (venlafaxine,

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milnacipran, bupropion, moclobemide) in the FST in mice without affecting the locomotor

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activity. A significant reduction in the immobility time in the FST in animal groups which were co-administered caffeine and antidepressant may be associated with the changes in the

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levels of monoamines in the CNS. The determination of drug concentrations in mouse serum and brain homogenates indicates that the interactions of caffeine with venlafaxine, bupropion, and moclobemide occur in pharmacodynamic phase, whereas the interaction of caffeine-

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milnacipran occurs, at least partially, in pharmacokinetic phase, because caffeine affected the

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concentrations of milnacipran in murine serum.

Conflict of interest statement

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Acknowledgments

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The authors declare that there are no conflicts of interest.

This study was supported by Funds for Statutory Activity of Medical University of Lublin, Poland. The authors wish to thank Chair and Department of Hygiene of Medical University in

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Lublin for access to an animal activity meter Opto-Varimex-4 Auto-Track.

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References [1] G.A. Bernstein, M.E. Carroll, P.D. Thuras, K.P. Cosgrove, M.E. Roth, Caffeine dependence in teenagers, Drug Alcohol Depend. 66 (2002) 1–6.

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Figure captions

Figure 1. Effect of combined administration of caffeine and antidepressants in the FST in

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mice. Caffeine was administered ip 40 min, and all antidepressants and saline were

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administered ip 60 min before the test. The values represent mean ± SEM. Each experimental group consisted of 10 animals. ***p<0.001 (two-way ANOVA followed by Bonferroni's post-

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hoc test).

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Table 1. Effect of treatments on locomotor activity in mice. Distance travelled

Treatment

between the 2nd and the 6th minute (cm)

N

caffeine 5 + saline

863.5±94.38

8

moclobemide 1.5 + saline

602.0±47.89

7

caffeine 5 + moclobemide 1.5

795.0±54.82

7

saline + saline

674.5±56.72

8

caffeine 5 + saline

815.3±57.72

8

843.0±71.60

8

748.5±102.0

8

727.1±76.66

8

806.5±89.80

8

581.8±46.87

8

caffeine 5 + venlafaxine 1

816.9±55.06

7

saline + saline

674.5±56.72

8

caffeine 5 + saline

815.3±57.72

8

bupropion 10 + saline

792.1±31.37

8

caffeine 5 + bupropion 10

744.8±59.26

8

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713.9±62.96

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(B)

saline + saline

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(A)

milnacipran 1.25 + saline

saline + saline caffeine 5 + saline

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(D)

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venlafaxine 1 + saline

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caffeine 5 + milnacipran 1.25 (C)

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(mg/kg)

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Antidepressants and saline were administered ip 60 min, and caffeine ip 40 min before the experiment. Distance travelled was recorded between the 2nd and the 6th min of the test. Data are presented as the means ± SEM. N, number of animals per experimental group. The result was considered statistically significant if p<0.05 (two-way ANOVA followed by Bonferroni's post-hoc test).

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Table 2. Effect of caffeine on the concentration of antidepressants in mouse serum and brain.

(D)

40.06±7.9

152.4±27.55

moclobemide 1.5 + caffeine 5

40.10±7.2

184.9±41.94

milnacipran 1.25 + saline

859.9±59.19

milnacipran 1.25 + caffeine 5

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moclobemide 1.5 + saline

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in serum (ng/ml)

Drug concentration in brain (ng/g)

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(C)

(mg/kg)

N

10 10

410.6±80.60

10

1279.0±76.47***

414.4±54.10

10

venlafaxine 1 + saline

5.57±2.35

38.54±7.44

9

venlafaxine 1 + caffeine 5

13.97±4.59

55.85±14.23

10

bupropion 10 + saline

135.0±20.79

965.7±74.02

10

1079.0±132.7

10

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(B)

Drug concentration

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(A)

Treatment

179.8±32.35

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bupropion 10 + caffeine 5

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Antidepressants were administered ip 60 min and caffeine 40 min before decapitation. Results are presented as mean values ± SEM. N, number of animals per experimental group. ***p<0.001 compared with the respective control group (Student's t-test).

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Figure 1