Acute administration of vortioxetine increases histamine concentrations in PFC Absolute effects of acute s.c. administration of vortioxetine on extracellular histamine levels in rat prefrontal cortex obtained by in vivo microdialysis. Data are expressed as AUC ± SE, time interval 0 until 180 min. *p<0.05, ANOVA followed by Dunnet’s test. For more data and discussion see. 1 MATERIALS AND METHODS Animals Male SD rats (280-350 g) (n=25) were used for the experiments. The animals were individually housed in plastic cages and had access to food and water ad libitum. Experiments were approved by the Lundbeck nstitutional Animal Care and Use Committee. Surgery Rats were anesthetized using isofurane (2%, 800 ml/min O 2 ). Lidocaine was used for local anesthesia and Carprophen (5 mg/kg s.c.) was used, pre/peri operative, as analgesia. The animals were placed in a stereotaxic frame, under isofurane anesthesia, and CMA12 guide cannulas (CMA Microdialysis, Sweden) were implanted, aimed into the PFC and VH.  Coordinates for the tips of the probes were for VH: posterior (AP) = -5.3 mm to bregma, lateral (ML) = -4.8 mm to midline and ventral (DV) = -8.0 mm relative to dura and for the PFC: posterior(AP) = +3.2 mm to bregma, lateral (ML) = -0.8 mm to midline and ventral (DV) = -4.0 mm relative to dura. Animals were allowed to recover for 7 days prior to the microdialysis study. Compounds Vortioxetine was formulated into food pellets and provided by Special Diets, Inc. During the dosing experiment, the rats were housed individually in the cages. The pellets (30 g) were dosed daily into small portable feeding boxes, weighed and placed one in each cage. Groups and daily treatments: Vehicle pellets, 30 g/day, n = 17 Pellets with vortioxetine (18 mg/10 g), n = 17 Microdialysis The microdialysis experiments were performed on day 15 after the surgery, and treatment period of feeding with food pellets containing vortioxetine. At 8 AM a microdialysis probe (CMA/12, 4 mm, molecular weight cut off 100 kDa) was inserted into the respective guide cannula in awake rats. Each animal was placed into the system for freely moving animals, equipped with a 2-channel swivel. The probes were perfused at a constant fow-rate of 1.5 μL/min with sterile artifcial cerebrospinal fuid solution. Following a 4 h stabilization period, the samples were collected at 20 min intervals. In total, 12 samples were collected during the following 240 min and used for determination of basal extracellular levels of histamine and acetylcholine. After the experiment, the rats were euthanized by CO 2 and trunk blood was collected and transferred into tubes containing anti-coagulant. Plasma was sent to for exposure analyses. LC/MS Analyses A Waters Acquity HPLC system equipped with an Sunshell RP-Aqua 2.1x100mm, 2.6um particle column provided isolation of acetylcholine (ACh) and histamine (His) in microdialysis samples prior to detection by a Waters Quattro Premier XE triple quadrupole mass spectrometer operating in the MS/MS mode. A full loop injection, on a 5µL loop with a 4x overfll requiring a total sample volume of 20µL, was utilized to deliver samples onto the LC/MS/MS system. Column and tubing prior to column were maintained at 30°C. The mobile phase consisted of an aqueous component (A: 100mM ammonium acetate in milliQ water) and an organic component (B:100% acetonitrile). Isocratic elution of ACh (retention time of 1.77mins) and His (retention time of 1.45mins) using 100% A followed by a wash of the column using a fast ramp to 100% B and then a re-equilibration of the column back to 100% A permitted separation of the two analytes with a total run time of 5 minutes. Detection of the analytes was performed by monitoring unique fragments formed from parent ions of ACh (parent (146.05 Da) to fragment (86.3 Da)) and His (parent (111.95 Da) to fragment (94.4 Da)). To correct for sample and instrument variability a deuterated version of ACh (D-ACh) was incorporated into the samples to act as an internal standard (parent (149.95 Da) to fragment (90.15 Da)). Utilizing this technique, rough lower limits of detection of 5pg/mL and 50pg/mL were achieved for ACh and His respectively. CONCLUSIONS • Acute and chronic vortioxetine treatment produced a sustained increase of histamine in rats in two key brain areas implicated in cognitive function (prefrontal cortex and ventral hippocampus). • Chronic treatment with vortioxetine does not affect acetylcholine in the ventral hippocampus. • We hypothesize that activation of histaminergic neurotransmission may contribute to vortioxetine’s positive effects on cognitive function. E007124 Histamine and Cognition: Chronic Treatment with the Multimodal Acting Antidepressant Vortioxetine Activates the Central Histaminergic System in Rats Gennady N. Smagin 1 , Dekun Song 1 , David P. Budac 1 , Alan Pehrson 2 , Yan Li 2 , Connie Sánchez 2 1 Physiology and Bioanalysis; 2 External Sourcing and Scientifc Excellence, Lundbeck, Paramus, NJ, USA NR6-127 Ex vivo Autoradiography: Target Receptor Occupancies The rats were anesthetized with CO 2 and sacrifced by decapitation. Brains were carefully dissected, immediately fash frozen on powdered dry ice, and stored at -20°C until use. For the region of interest, tissue slices, 20 µm thick, were cut at -21°C using a microtome-cryostat (Microm, Walldorf, Germany). Coronal slices were collected beginning at ~1.2-1.5 mm anterior to Bregma for SERT, 5-HT 1A , and 5-HT 1B and beginning at -4.8 to -5.36 mm posterior to Bregma for 5-HT 3 . The sections were mounted on slides and stored in a slide box with desiccant pellets at -21°C. Briefy, on the day of the experiment, slide boxes were defrosted at room temperature (RT) under a constant stream of air for at least 30 min prior to opening. Following any preincubation (RT), slides were incubated (RT) in assay buffer containing the tritiated radioligand corresponding to the receptor of interest. Nonspecifc binding was determined by the addition of a high concentration of a non-radioactive competitor for the target of interest to the assay buffer. Following the incubation period, the slides were rinsed twice in 4°C assay buffer and briefy dipped in 4°C distilled water. Subsequently, the slides were air-dried for 30 min and transferred to a vacuum desiccator and dried for an additional 60 min. Finally, the slides were processed in a Beta Imager (Biospace Lab, Paris, France) for 16-24 h depending on the radioligand used. Radioligand concentrations and regions of interest were chosen based on the results of saturation binding and receptor mapping experiments conducted on site. 4 SERT, 5-HT 1A and 5-HT 1B receptor occupancy was evaluated in the chronic vortioxetine experiment and presented in Table 3. Statistics Raw data were entered into data fles using Microsoft Excel. The values are presented as mean ± standard error of the mean (SEM), and differences are considered to be statistically signifcant at the p<0.05 level. For graphic representation of neurotransmitter outfow over time, the data were expressed both in nominal concentrations (ng/mL) found in the microdialysates and as percent change from the basal concentrations measured in the vehicle group. Statistical analysis was performed using Prism 6 (GraphPad Software, USA). ABSTRACT OBJECTIVES: Vortioxetine, a multimodal acting antidepressant, is a 5-HT 3 , 5-HT 7 and 5-HT 1D receptor antagonist, 5-HT 1B receptor partial agonist, 5-HT 1A receptor agonist and 5-HTT inhibitor. Preclinical studies and clinical studies in MDD patients indicate that vortioxetine restores cognitive dysfunctions. 1,2 While not fully understood, vortioxetine’s clinical effect on cognitive function likely involves modulation of several neurotransmitter systems. 1 We have previously shown that acute vortioxetine increased extracellular histamine (HA) in the rat prefrontal cortex. 1 Since HA is known to positively affect memory and learning, 3 the scope of the present investigation was to study vortioxetine’s effect on HA levels in the rat prefrontal cortex (PFC) and ventral hippocampus (VH) after chronic dosing. METHODS: Rats were implanted with guide cannulas for microdialysis in the PFC and VH. After recovery, animals received vortioxetine formulated in food pellets, 18 mg/10 g food. This concentration reaches pharmacologically active levels of target occupancies. Control animals received standard rodent chow. On day 15, microdialysis probes (CMA/12, 4 mm, PAES, MWKO 100 kDa) were inserted into the respective guide cannulas in awake rats. Each animal was placed into a system for freely moving animals equipped with a 5-channel swivel. The probes were perfused with constant fow rate of 1.5 μl/min with sterile artifcial CSF solution (Perfusion Fluid CNS, CMA Microdialysis, Harvard Biosciences, USA). Following 4 hours stabilization, a total of 12 samples were collected over 20 min intervals and used for determination of basal extracellular levels of HA by LC/MS. RESULTS: In control animals, HA concentrations in the microdialysates were 0.60±0.03 in the PFC and 0.46±0.05 ng/ml in VH. In vortioxetine-treated rats, concentrations were signifcantly higher: 0.95±0.06 in PFC and 0.85±0.13 in VH (P<0.05) than controls. The role of HA is gaining increasing attention, and many recent results indicate that the HA-ergic system infuences learning and memory by modulating the release of acetylcholine (ACh), although some cognitive effects of HA and HA-ergic agents occur independently of ACh. 3 The regulation of ACh tone in different brain areas by neuronal HA also encompasses functions other than cognition. HA promotes wakefulness (attention) by tonic control over sleep-generating mechanisms in the preoptic/anterior hypothalamus, and cholinergic neurons seem to be implicated. Conclusion: Vortioxetine produced a sustained increase of HA in rats in two key brain areas implicated in cognitive function. We hypothesize that histaminergic neurotransmission may contribute to vortioxetine’s positive effects on cognitive function. BACKGROUND Vortioxetine, a multimodal acting antidepressant currently approved for the treatment of MDD, is a 5-HT 3A , 5-HT 7 and 5-HT 1D receptor antagonist, 5-HT 1B receptor partial agonist, 5-HT 1A receptor agonist and 5-HTT inhibitor (see Table 1). Preclinical and clinical studies have demonstrated the antidepressant properties of vortioxetine and a recent clinical study in elderly depressed patients showed superiority to placebo in objective cognitive tests of speed of processing, verbal learning and memory. 2 Based on these clinical observations and the pharmacological profle of vortioxetine, we used microdialysis to investigate the effect of chronic administration on extracellular histamine levels in the prefrontal cortex (PFC) and ventral hippocampus (VH) of rat brains – areas related to memory. Pharmacological profile of vortioxetine (Table 1) Binding Ki or potency IC50 (nM) In vivo Occupancy Target Type of Activity Human Rat Human Rat 5-HT3A Antagonist 3.7 1.1 ND 100% at 1 mg/kg; ED50 0.004 mg/kg (at 5-HT3 ) 5-HT7 Antagonist 19 200 ND ND 5-HT1D Antagonist 54 3.7 ND ND 5-HT1B Partial agonist 33 16 ND ~ 80% at 10 mg/kg; ED50 3.2 mg/kg 5-HT1A Agonist 15 230 ND 28%, 35%, 44% at 5, 10, 20 mg/kg, respectively 5-HTT Inhibitor 5.4 (IC50 on uptake) 5.3 (IC50 on uptake) ~ 50% at 5 mg > 80% at 10 mg/kg; ED50 0.4 mg/kg The in vitro pharmacological profle and in vivo target occupancies of vortioxetine. The in vitro activities of vortioxetine at different 5-HT targets (cloned receptors) and their corresponding in vivo occupancies in the rat and human are summarized in. 1 Basal levels histamine in the PFC and vHPC of awake rats following 14 days of treatment with vortioxetine Time course of basal extracellular concentrations of histamine in VH and PFC of rats treated with standard rat Time course of basal extracellular concentrations of histamine in VH and PFC of rats treated with standard rat chow (vehicle, n=17) and food containing vortioxetine (18 mg/10 g) for 14 days (n=17). Microdialysis experiments were conducted on day 15. Samples were collected following a 4 h stabilization period. Data are presented as means ± SEM, n=15-17. 1. Mørk A, Montezinho LP, Miller S, Trippodi-Murphy C, Plath N, Li Y, Gulinello M, Sanchez C (1 February 2013). “Vortioxetine (Lu AA21004), a novel multimodal antidepressant, enhances memory in rats” . Pharmacology, Biochemistry, and Behavior. 105:41-50. 2. Katona, C., Hansen, T. & Olsen, C.K. A randomized, double-blind, placebo-controlled, duloxetine-referenced, fxed-dose study comparing the effcacy and safety of Lu AA21004 in elderly patients with major depressive disorder. Int. Clin. Psychopharmacol. 27, 215-223 (2012). 3. Haas H, Panula P. The role of histamine and the tuberomamillary nucleus in the nervous system. Nat Rev Neurosci. 2003 Feb;4(2):121-130. 4. Pehrson AL, Cremers T, Bétry C, van der Hart MG, Jørgensen L, Madsen M, Haddjeri N, Ebert B, Sanchez C. Lu AA21004, a novel multimodal antidepressant, produces regionally selective increases of multiple neurotransmitters--a rat microdialysis and electrophysiology study. Eur Neuropsychopharmacol. 2013 Feb;23(2):133-145. 5. Zant JC, Rozov S, Wigren HK, Panula P, Porkka-Heiskanen T. Histamine release in the basal forebrain mediates cortical activation through cholinergic neurons. J Neurosci. 2012 Sep 19;32(38):13244-13254. Funding: This study was sponsored by H Lundbeck A/S. Poster presented at the 167th Annual Meeting of the American Psychiatric Association, May 3–7, 2014, New York, NY, USA. 0 20 40 60 80 100 120 140 160 180 200 220 240 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 Vehicle PFC Vehicle VH Vortioxetine PFC Vortioxetine VH Time (min) ng/ml Chronic treatment with vortioxetine increases histamine concentrations in prefrontal cortex (PFC) and hippocampus (VH) Levels of extracellular concentrations of histamine in VH and PFC over the 240-min collection period. Rats were treated with standard rat chow (vehicle, n=17) and food containing vortioxetine (18 mg/10 g) for 14 days (n=17). Microdialysis experiments were conducted on day 15. Samples were collected following a 4 h stabilization period. Data are presented as means ± SEM, n=15-17. **p<0.05 for histamine in PFC and in VH in VOR group compared to vehicle group, one-way ANOVA followed by Tukey’s test. Acute administration of vortioxetine increases acetylcholine concentrations in PFC Effects of acute s.c. administration of vortioxetine on extracellular histamine levels in rat PFC obtained by in vivo microdialysis. For more fata and discussion see. 1 0 20 40 60 80 100 120 140 160 180 200 220 240 0.0 0.1 0.2 0.3 0.4 Vehicle PFC Vehicle VH Vortioxetine PFC Vortioxetine VH Time (min) ng/ml PREFRONTAL CORTEX Vehicle VORTIOXETINE 0 25 50 75 100 125 150 175 ** Histamine (% of vehicle) VENTRAL HIPPOCAMPUS Vehicle VORTIOXETINE 0 50 100 150 200 ** Histamine (% of vehicle) min -80 -60 -40 -20 0 20 40 60 80 100 120 140 160 180 Acetylcholine in Dialysate (% of basel lelves) 0 100 200 300 Vehicle s.c. n=8 2.5 mg/kg s.c. n=5 5 mg/kg s.c. n=6 10 mg/kg sc n=8 REFERENCES Histaminergic system involvement in cortical activation Schematic representation of the interactions between the cholinergic and histaminergic systems in some regions of the rat brain. (BLA) basolateral amygdala; (Cx) cortex; (NBM) nucleus basalis magnocellularis; (TM) tuberomammilary nucleus. Vehicle 2.5 mg/kg 5 mg/kg 10 mg/kg 0 5000 10000 15000 20000 25000 30000 35000 40000 AUC (0-180 min) * * Plasma exposure of vortioxetine after chronic treatment (Table 2) ng/mL Vehicle, n=12 0 Pellets with vortioxetine (18 mg/10 g) for 14 days, n=12 679.5 ± 96.8 Average plasma concentration after sub-chronic vortioxetine given in food pellets. After the microdialysis experiment, the rats were euthanized by CO 2 and trunk blood was collected and transferred into tubes containing EDTA as anti-coagulant. Plasma was analysed for vortioxetine quantitation using an LC/MS method. % Target occupancy in rat brain tissue (Table 3) Target Occupancy (%) Treatment Group SERT 5-HT1B Receptor Vehicle/Vehicle 0 ± 3.9 0 ± 3.2 Vortioxetine/Vehicle 99 ± 0.7 81 ± 2.6 Basal levels acetylcholine in the PFC and VH of awake rats following 14 days of treatment with vortioxetine Time course of basal extracellular concentrations of acetylcholine in VH and PFC of rats treated with standard rat chow (vehicle, n=17) and food containing vortioxetine (18 mg/10 g) for 14 days (n=17). Microdialysis experiments were conducted on day 15. Samples were collected following a 4 h stabilization period. Data are presented as means ± SEM, n=15-17. Effect of chronic treatment with vortioxetine on acetylcholine concentrations in prefrontal cortex (PFC) and hippocampus (VH) The effect of treatment on extracellular concentrations of acetylcholine in VH and PFC over the 240-min collection period. Rats were treated with standard rat chow (vehicle, n=17) and food containing vortioxetine (18 mg/10 g) for 14 days (n=17). Microdialysis experiments were conducted on day 15. Samples were collected following a 4 h stabilization period. Results are presented as percent change in the experimental group compared to the vehicle- treated group. Data are presented as means ± SEM, n=15-17. PREFRONTAL CORTEX Vehicle VORTIOXETINE 0 20 40 60 80 100 120 Acetylcholine ( % of vehicle) VENTRAL HIPPOCAMPUS Vehicle VORTIOXETINE 0 20 40 60 80 100 120 140 Acetylcholine (% of vehicle) View publication stats View publication stats