Lu 35-138 ((+)-(S)-3-{1-[2-(1-acetyl-2,3-dihydro-1H-indol-3-yl)ethyl]-3,6-dihydro-2H-pyridin-4-yl}-6-chloro-1H-indole), a dopamine D4 receptor
antagonist and serotonin reuptake inhibitor: Characterisation of itsin vitro profile and pre-clinical antipsychotic potential
Peter Hertel a,⁎, Michael Didriksen a, Bruno Pouzet a, Lise T. Brennum a, Karina K. Søby a,Anna Kirstine Larsen a, Claus T. Christoffersen a, Teresa Ramirez a, Monica M. Marcus b,
Torgny H. Svensson b, Vincenzo Di Matteo c, Ennio Esposito c, Benny Bang-Andersen a, Jørn Arnt a
a Research and Development, H. Lundbeck A/S, Copenhagen-Valby, Denmarkb Department of Physiology and Pharmacology, The Karolinska Institutet, Sweden
c Laboratory of Neurophysiology, Istituto di Ricerche Farmacologiche “Mario Negri”, Consorzio “Mario Negri” Sud, Santa Maria Imbaro (Chieti), Italy
Received 7 March 2007; received in revised form 12 June 2007; accepted 18 June 2007Available online 4 July 2007
The antipsychotic effect of classical neuroleptic drugs such ashaloperidol is generally thought to be related to antagonism ofdopamine D2 receptors in limbic structures. Although blockade of
dopamine D2 receptors is associated with effect against positivesymptoms of schizophrenia, other symptoms, e.g. negativesymptoms and cognitive disturbances which may be fundamentalto the disease, appears less affected by conventional dopamine D2
receptor blocking agents (Kinon and Lieberman, 1996). Further-more, asmany as 25% to 60%of patients treatedwith conventionalantipsychotics are regarded as either treatment refractory orpartially responsive (Miyamoto et al., 2002). Also, the compliancewith classical dopamine D2 receptor blocking agents is clearlylimited by the appearance of motor and endocrine side effects
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which are associated with concomitant blockade of striatal andpituitary dopamine D2 receptors, respectively (Campbell et al.,1999). It has also been described that classical dopamine D2
blocking drugs may worsen the cognitive symptoms in schizo-phrenia (Cutmore and Beninger, 1990).
The above observations suggest that improved pharmacother-apy of schizophrenia requires agents with characteristics differentfrom those of the current antipsychotic drugs and targeting othersites than, or in addition to, dopamine D2 receptors. In this respect,the dopamine D4 receptor has attracted particular interest forseveral reasons. First, neuroanatomical studies have suggested thatdopamine D4 receptors are enriched in cortical structures (De LaGarza and Madras, 2000), areas which are associated with theaetiology of schizophrenia particularly as regards cognitive andnegative symptomatology (Berman, 2002). In fact, dopamine D4
receptor antagonists have been shown to possess beneficial effectsin primate models of cognitive deficits (Arnsten et al., 2000;Jentsch et al., 1999). Second, the antipsychotic drug clozapine,which is clinically characterized by superior effect in treatmentresistant patients and low, if any, risk of causing extrapyramidalside effects or tardive dyskinesias (Safferman et al., 1991), hasbeen reported to possess higher affinity for dopamine D4 ascompared to dopamine D2 receptors in vitro (Seeman et al., 1997).Finally, recent studies have indicated an intriguing role of thedopamine D4 receptor in the regulation of limbic glutamatergicneurotransmission. It was recently reported that activationof dopamine receptors depressed excitatory transmission via theN-methyl-D-aspartate (NMDA) receptor, an effect counteracted byblockade of dopamine D4 receptors (Kotecha et al., 2002). This isin line with previous findings showing that dopamine D4 receptor-deficient mice display cortical hyperexcitability which, takentogether, suggest that the dopamineD4 receptor act as an inhibitorymodulator of cortical glutamate activity (Rubinstein et al., 2001).These findings are of particular interest as schizophrenia has beensuggested to be associated with glutamatergic hypofunction(Carlsson et al., 2000).
A large body of data suggests that central serotonergicneurotransmission is involved in both the pathophysiology aswell as the treatment of schizophrenia (Abi-Dargham et al.,1997). For example, clinical studies have demonstrated that
Fig. 1. Chemical structure of Lu 35-138 ((+)-(S)-3-{1-[2-(1-acetyl-2,3-dihydro-1H-indol-3-yl)ethyl]-3,6-dihydro-2H-pyridin-4-yl}-6-chloro-1H-indole) and Lu38-012 (5-[4-(4-chlorophenyl)piperazin-1-ylmethyl]-1H-indole).
adjuvant treatment with serotonin (5-HT) reuptake inhibitorsmay improve the clinical profile of conventional antipsychoticdrugs particularly as regards effectiveness against negativesymptoms of schizophrenia (Silver and Shmugliakov, 1998).Increasing the serotoninergic neurotransmission, e.g. viablockade of the 5-HT reuptake, may also be beneficial in thetreatment of depressive as well as anxiety symptoms which arefrequently co-expressed in schizophrenia and often precedespsychotic relapses (Baynes et al., 2000).
In this light, the present report describes the pharmacologicalprofile of Lu 35-138, a novel potential antipsychotic drug (Fig. 1),which combines dopamine D4 receptor antagonism with 5-HTreuptake inhibition with focus on antipsychotic-like activity aswell as motor and cognitive side effect liability.
2. Materials and methods
2.1. Animals
Male Wistar rats (M&B A/S, Denmark or Charles River,Germany), male Sprague–Dawley rats (B&K Universal,Sweden; Charles River, Italy) and male NMRI mice (M&BA/S, Denmark) were housed under environmentally controlledconditions (21±2 °C and a 12 h light/dark cycle with lights onat 6 a.m.). The rodents were allowed standard rodent food andwater ad libitum. Rats and mice were housed in standard rodentcages in groups of 2–4 and 10–15, respectively.
Monkeys (Cebus apella; 5 females and 2 males; 22–24 yearsold; weighing from 2.7–4.5 kg) were individually housed instandard primate cages and maintained under environmentallycontrolled conditions (24±2 °C and a 12 h light/dark cycle withlights on at 6 a.m.). Food (mixture of two kinds of food pelletsplus fruits and vegetables) was provided in the morning twohours before the start of the experiments and in the evening afterthe experiments were finished. Water was available ad libitum.
All the animal experiments performed in this study wereconducted in accordance with the Danish legislation of animaluse for scientific procedures as described in the “Animal TestingAct” (Consolidation Act no. 726 of 9 September 1993 asamended by Act no. 1081 of 20 December 1995).
2.2. In vitro assays
Broad receptor profiles were obtained from Cerep, Cellel’Evêque, France according to their standard protocols (2001catalogue). Further analysis was performed in house as detailedbelow: Adrenergic, dopamine and 5-HT receptor binding assayswere performed exactly as described previously (Balle et al.,2003). The functional characterisation at human D2S and D4.2
receptors was carried out measuring inhibition of forskolin-stimulated cAMP. Cells were seeded in 96-well plates 2 daysbefore the experiment and the washed once and preincubatedfor 30 min at 37 °C with 0.2 ml assay buffer (phosphate-buffered saline supplemented with 1 mM MgCl2, 1 mM CaCl2and 1 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine). Incubation with test compounds was per-formed with fresh assay buffer for 10 min at 37 °C. For the
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receptor antagonist assay, the cells were exposed to testcompounds for 2 min before quinpirole (Sigma-Aldrich,USA) was added as indicated. After 10 min incubation at37 °C the buffer was aspirated and the cAMP content wasextracted by adding 100 μl/well of 0.1 M HCl /0.1 mM CaCl2and after 30 min adding 68 μl/well of 0.15MNaOH/60 mMNa-acetate. Subsequently, levels of cAMP were determined using acommercial FlashPlate RIA assay (Perkin Elmer, USA).
Measurements of [3H]-5-HT uptake into rat whole brainsynaptosomes, [3H]-noradrenaline uptake into synaptosomesfrom rat frontal and temporal cortex, and [3H]-dopamine uptakeinto rat striatal synaptosomes was conducted essentially aspreviously described (Hyttel, 1982). In brief, male rats (WistarfromM&B A/S; weighing 200–220 g) were decapitated and therelevant brain tissue was rapidly removed and homogenised in40-vol (w/v) of ice-cold 0.4 M sucrose containing 1 mMnialamide and centrifuged at 1000 ×g for 10 min. Thesupernatants were further centrifuged for 30 min at20.000 ×g, 4 °C and re-suspended in Krebs-Ringer buffer, pH7.4 supplemented with 0.2 g/l ascorbic acid. Aliquots of tissuesuspended in assay buffer (composition: 123 mM NaCl,4.8 mM KCl, 0.97 mM CaCl2, 1.1 mM MgSO4, 12 mMNa2HPO4, 3 mM NaH2PO4, 0.16 mM EDTA, 10 mM glucoseand 1 mM ascorbic acid) were added to 96 well plates. Afterequilibration at 37 °C for 15 min, [3H]-5-HT (10 nM, 27 Ci/mmol, Perkin Elmer), [3H]-noradrenaline (10 nM, 36 Ci/mmol,Perkin Elmer, USA) or [3H]-dopamine (12.5 nM, 59 Ci/mmol,Perkin Elmer, USA) were added and the samples wereincubated with varying concentrations of test compound at37 °C for 15, 10 or 5 min, respectively. Plates were filtereddirectly on Unifilter GF/C glass fiber filters (soaked for 1 h in0.1% polyethylenimine) under vacuum and immediatelywashed with 3×0.2 ml assay buffer. Non-specific uptake wasdetermined using 10 μM citalopram (5-HT uptake; synthesisedat H. Lundbeck A/S), 20 μM talsupram (noradrenaline uptake;synthesised at H. Lundbeck A/S), or 100 μM benztropine(dopamine uptake; synthesised at H. Lundbeck A/S), respec-tively and accounted for 5–10% of total uptake.
2.3. In vivo receptor binding to dopamine D2 receptors
In vivo receptor binding was performed using the dopamineD2 receptor antagonist [3H] raclopride. In brief, mice (maleNMRI mice, 18–23 g) were treated with drug s.c. beforereceiving 6.5 μCi [3H]-raclopride (Amersham, Buckinghamp-shire, U.K., specific activity=80 Ci/mmol) i.v. via the tail vein.After 10 min the animals are killed by cervical dislocation, thebrain quickly removed and striatum dissected out and homog-enized in 2.5 ml ice-cold buffer (50 mM KPO4, pH 7.4). Analiqoute (0.5 ml) of the homogenate was filtered throughWhatman GF/C filters soaked in 0.1% PEI. Filtration wascompleted within 60 s subsequent to the decapitation. Filterswere washed 2 times with 2.5 ml ice-cold buffer and the boundradioactivity was counted in a scintillation counter. A group ofsaline treated animals was used to determine [3H]-raclopridebinding and a group of animals that was treated with 10 mg/kgolanzapine was included to determine non-specific binding.
2.4. Influence on spontaneous locomotor activity
Locomotor activity in rats (Wistar from M&B A/S; weighing180–200 g) and mice (weighing 20–25 g) was measured usingactivity boxes equippedwith photocells sensitive to infrared light.The activity boxes for rats (macrolon type III, high model) wereequipped with 4 infrared light sources and photocells placed 4 cmabove the floor whereas the activity boxes for mice (20×32 cm)were equipped with 5×8 infrared light sources and photocellsplaced 1.8 cm above the floor. The locomotor activity wasquantified by counting the number of photo beam interruptions.Recording of an activity count required consecutive interruptionof adjacent light beam, thus avoiding counts induced by stationarymovements. If not otherwise indicated, the animals were injected(s.c.) 2 h (rats; 5.0 ml/kg) or 30 min (mice; 10 ml/kg) beforeplacing them in the activity box for assessment of locomotoractivity during a 15 min recording period. The mean activityinduced by vehicle treatment was used as baseline and the percentchange of baseline activity was calculated as: 100− [(activity;compound) / (activity; vehicle)×100]. The inhibitory effects ofthe test substances were presented as ED50 values calculated bylog-probit analysis on the basis of the percent inhibition responsesobtained at each dose level and presented together with theirrespective standard deviation (S.D.) factors (95% confidenceinterval defined as: ED50/S.D.−ED50×S.D.).
2.5. Influence on D-amphetamine and phencyclidine (PCP)-induced hyperlocomotion
Rats (Wistar from M&B A/S; weighing 180–200 g) were usedfor monitoring effects on D-amphetamine-induced hyperlocomotionwhereasmice (weighing 20–25 g)were used in case of PCP-inducedhyperlocomotion. The same equipment as described for assessingspontaneous locomotion activity was used. If not otherwiseindicated, the test substance was administered (s.c.) 2 h or 30 minbefore injection of D-amphetamine (0.5 or 2.0 mg/kg, s.c.) or PCP(2.5 mg/kg, s.c.), respectively. An injection volume of 5.0 or 10 ml/kgwas used for rats ormice, respectively. The activitywasmeasuredfor a period of 2 h (rats) or 1 h (mice). The percent inhibition inlocomotor activity induced by the test substance was determinedusing the total activity of the vehicle, defined as the baseline effect,and the total activity induced by D-amphetamine or PCP alonedefined as the maximal effect using the following calculation: 100−[((activity; compound+D-amphetamine or PCP)−(activity; vehi-cle)) /((activity; D-amphetamine or PCP)−(activity; vehicle))×100].The inhibitory effects of the test substances were presented as ED50
values calculated by log-probit analysis on the basis of the percentinhibition responses obtained at each dose level and presentedtogether with their respective S.D. factors. Data were statisticallyevaluated using a one-way analysis of variance (ANOVA) followedby Dunnett's post hoc test comparing the percent inhibition inducedby the drug with the effect of D-amphetamine or PCP alone.
2.6. Influence on D-amphetamine-disrupted pre-pulse inhibition
The procedure for measuring pre-pulse inhibition has previ-ously been described (Andersen and Pouzet, 2001). Briefly,
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recording of the pre-pulse inhibition in rats (Wistar from CharlesRiver; weighing 270–330) was performed in a sound attenuatedsystem consisting of four startle chambers (MPOS 2b, Metod ochProdukt, Svenska AB, Sweden). Rats were placed in a wire meshcage (18.5×7.0×6.5 cm) which was attached via a piston to anaccelerometer. Each movement of the animals was registered bythe accelerometer and transferred to a PC-based data collectingsystem (Ellegaard Systems, Denmark) via an analogue to digitalconverter (National Instruments, USA). A background noise levelof 70 dB was maintained throughout the session. After a 5 minacclimatisation period with the background noise on, 8 pulses of120 dB broad band burst for 40 ms were presented to test basalstartle responsiveness. Thereafter, 8 blocks of 6 different trial typeswere presented randomly to measure pre-pulse inhibition, i.e.:pulse alone (120 dB), pre-pulse alone (82 dB), pre-pulse followedby pulse (3 trial types: 74+120 dB; 78+120 dB; 82+120 dB) orno pulse. The pre-pulses had a duration of 30 ms and the timeinterval between pre-pulse and pulse was kept at 100 ms. Theintertrial period was constant and lasted for 15 s. The percentagepre-pulse inhibition was calculated as: 100− (100×startle ampli-tude in pre-pulse trial) / (startle amplitude in pulse alone trial).D-amphetamine (2.0 mg/kg, s.c.) or vehicle were injected (s.c.at a volume of 5.0 ml/kg) 25 min before the acclimatisationperiod. Test compounds were injected (s.c. at a volume of5.0 ml/kg) 15 min before D-amphetamine administration. Thestartle amplitude was analysed by a two way ANOVA con-sisting of a between subjects factor of Treatment (2 levels:vehicle and D-amphetamine), and a between subjects factor ofDrug (3 levels: vehicle, dose 1 and dose 2). Mean percentagepre-pulse inhibition was analysed by a three way repeatedmeasurement ANOVA consisting of a between subjects factorof treatment, a between subjects factor of Drug, and a withinsubjects factor of 3 pre-pulse intensities. Fisher's post-hocanalysis was performed to compare the startle amplitude, andthe mean percent pre-pulse inhibition between groups.
2.7. Influence on the number of spontaneously active dopaminecells
Electrophysiological techniques were used to study theeffects on the number of active dopamine cells in the ventraltegmental area as previously described (Di Giovanni et al.,1998). In short, rats (Sprague–Dawley from Charles River;weighing 350–380 g) were anesthetized with chloral hydrate(400 mg/kg i.p.) and mounted in a stereotaxic apparatus.Supplemental doses of anesthetic were administered via a lateraltail vein cannula. Throughout the experiment the animal's bodytemperature was maintained at 36° to 37 °C by a thermostat-ically regulated heating pad. After reflecting the scalp, the skulloverlying the ventral tegmental area was removed. Thecoordinates, relative to the interaural line, for placement ofthe recording electrode were: anterior, 2.8 to 3.4 mm; lateral, 0.1to 0.5 mm; ventral, 7 to 8 mm. Extracellular recordings wereperformed by using a single-barrel glass micropipette. Themicropipettes were filled with 2% pontamine sky blue dye in2 M NaCl (in vitro resistance 4–7 MΩ). Dopaminergic neuronswere identified by their location, waveform, firing rate and
pattern (Bunney et al., 1973). Electrical signals of spike activitywere passed through a high input impedance amplifier whoseoutput was led into an analog oscilloscope, audio monitor andwindow discriminator. Unit activity was then converted to anintegrated histogram by a rate-averaging computer and dis-played as spikes per 10 s intervals on a chart recorder. Only cellswhose electrophysiological characteristics matched those pre-viously established for midbrain dopaminergic neurons weresampled (Bunney et al., 1973). At the end of the repeated drugadministration period, spontaneously active dopamine cellswithin the ventral tegmental area were counted by lowering themicropipette through a block of tissue (120.00 μm2) whichcould be reproducibly located from animal to animal (Chiodoand Bunney, 1983). Twelve electrode tracks (separated fromeach other by 200 μm), whose sequence was kept constant fromanimal to animal, were made in the ventral tegmental area. Aftereach experiment, the sites of recording were marked by theejection of pontamine sky blue dye from the electrode using a−20 μA current for 10 min. Brains were removed, placed in 10%buffered formalin solution for 2 days, frozen, cut in sections(40 μm), stained with neutral red solution and examined undermicroscope for verification of recording location. Test substanceswere administered orally once daily for 21 days at a volume of10 ml/kg. The number of spontaneous active dopamine cells inthe ventral tegmental area was counted approximately 2 h afterthe last administration. Data were presented as the averagenumber of spontaneously active dopamine neurons (+standarderror of the mean (S.E.M.)) for each treatment group. The effectof the different treatments was statistically evaluated using a one-way ANOVA followed by Tukey's test.
2.8. Influence on dopamine output
The influence on dopamine output in the core and shellsubdivision of the nucleus accumbens was measured by meansof in vivo voltammetry as previously described (Marcus et al.,1996). In brief, rats (Sprague–Dawley from B&K Universal;weighing 300–400 g) were pre-treated with pargyline (75 mg/kg, i.p.) and anaesthetized with chloral hydrate (400 mg/kg, i.p.).A jugular and a femoral vein catheter for i.v. administration ofdrug and chloral hydrate, respectively, were inserted. The ratswere kept under anaesthesia throughout the experiment bycontinuous infusion of chloral hydrate (100 mg/kg/h, i.v.). Rectaltemperature was kept at 37 °C by means of a thermostaticallycontrolled heating pad. Rats were mounted in a stereotaxic frameand a pre-treated micro carbon fiber (see below) was implanted ineither the shell or core part of the nucleus accumbens using thefollowing coordinates (in mm from bregma): anterior–posteri-or=+1.6 or +1.7; medial-lateral=+0.8 or +1.6, respectively. Thetip of the electrode was placed 6.5–7.0 mm below brain surfacein regardless of shell or core voltammetry. Each carbon fibre,12 μm in diameter with an active length of 500 μm, wereelectrochemically treated as previously described (Marcus et al.,1996). After the implantation of the electrode (see above), thecatechol oxidation current was monitored by a pulse voltam-metric system (Biopulse, Tacusell, France). Differential normalpulse voltammetry was employed to record voltammograms
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every minute using parameters which have been describedelsewhere (Gonon, 1988). At the end of each experiment, anelectrolytical lesion (5 V, 5 s) was made through the carbonfibrefor subsequent histological verification of recording sites. Thearea under the catechol oxidation current was calculated using acomputerized analysis system. After a stable baseline had beenrecorded for 10 min, the animal received a control i.v. injectionwith vehicle and 10min later the drug was injected at a volume ofmaximal 50 μl, depending on the dose of the drug and the weightof the animal. Data were calculated and presented as percentchange of baseline defined as the average of the last 10 pre-injection values. All subsequent measures were related to thesevalues and mean (±S.E.M.) percentages were calculated for eachsample across subjects in all groups. Data were statisticallyevaluated by two-(time×region) or one-way (time) ANOVA toreveal potential differences between the two studied regions ordifferences from baseline level within one brain region,respectively. A significant overall group, time or interactionwas followed by the Duncan's Multiple Range Test.
2.9. Induction of catalepsy
Rats (Wistar from M&B A/S; weighing 180–200 g) wereused to assess cataleptogenic effects as previously described(Pouzet et al., 2002). Briefly, the test compound was injected s.c. and the catalepsy was measured every hour for 6 h by placingthe animal on a vertical wire netting (50×50 cm with meshopenings of 1×1 cm). Animals were considered cataleptic whenthey remain immobile during a period of 15 s. The results wererecorded in fractions and the ability of a substance to inducecatalepsy was presented as ED50 values calculated by log-probitanalysis on the basis of the fractions of cataleptic rats obtainedat each dose level at the time of maximum effect and presentedtogether with their respective S.D. factors.
2.10. Induction of dystonia
The procedures to measure dystonic symptoms in monkeyshave been described elsewhere (Casey, 1996). All monkeys(C. apella) in these experiments were sensitized to neurolepticsand known to have a stable response to such drugs. Test sub-stances were administered s.c./i.m. at a volume of 0.4 ml/kg andthe monkeys were observed for dystonic symptoms during a 90 speriod 0, 15, 30, 45, 60, 90 min and after 2, 3, 4 and 5 h postinjection. Furthermore, the monkeys were checked 24 h aftertreatment but no scoring was made. The monkeys were observedfor dystonic symptoms (acute dystonia, bradykinesia, oraldyskinesia, akathisia, 5-HT syndrome and sedation) as describedby Casey (1996). Briefly, the behavioural scores were given asfollows: 0=normal behaviour, 1=mild, behaviour occasionallypresent, 2=moderate, behaviour regularly present but inter-rupted, 3=severe, behaviour continuously present. If dystonia isconsidered as severe and sustained, the subject is injected withbiperiden (2.5 mg, i.m., Akineton®, Abbott) which counteractsthe dystonia within 10 min. Data were analysed as the average ofthe total score obtained for each subject within each dose groupsover the 5 h of observation period. The minimal effective dose
was defined as the lowest dose tested inducing “severe” dystonicsymptoms (i.e. scoring 3) in at least one of the subjects tested.
2.11. Influence on water maze performance
Rats (Wistar fromM&B A/S; weighing 210–330 g) at an ageof 3 months were used to asses the performance in the watermaze as previously described (Skarsfeldt, 1996). Briefly, thewater maze consisted of a circular, black pool measuring 1.20 min diameter×0.45 m in height. A circular escape platform (8 cmin diameter) was placed just below the water surface. A videocamera monitored the movements of the rats and the videosignal was transmitted and analyzed using a computer basedvideo tracking system (Ethovision, Noldus Information Tech-nology, The Netherlands). Before each trial the rat was placedon the hidden platform for 15 s. The rats were given three trialsper day for three consecutive days. At the start of a trial the ratwas placed in the maze at one of three different start positionssuch that, across trials, all three start positions were used. If therat did not locate the platform within 60 s, the rat was gentlyplaced on the platform. The test compounds were administereds.c. 30 min before the first daily trial. The effect on latency andswimming speed was analysed using two way ANOVA (group×-day). If normality test or equal variance test failedKruskal–Wallisone way ANOVA on ranks was performed. The accepted level ofsignificance was Pb0.05. The minimal effective dose for sig-nificantly increasing latency to find the hidden platform (s) andreducing swimming speed (m/s) is presented.
2.12. Drugs and vehicles for in vivo experiments
Lu 35-138 ((S)-(+)-3-{1-[2-(1-acetyl-2,3-dihydro-1H-indol-3-yl)ethyl]-3,6-dihydro-2H-pyridin-4-yl}-6-chloro-1H-indole hydrochlo-ride; synthesised atH.LundbeckA/S,Denmark)was dissolved in a 5or 10% 2-hydroxypropyl-β-cyclodextrin solution. Lu 38-012 (5-[4-(4-chlorophenyl)piperazin-1-ylmethyl]-1H-indole (synthesised at H.Lundbeck A/S, Denmark) was dissolved in a 5 or 10% 2-hy-droxypropyl-β-cyclodextrin solution containing 0.1 M HCl. Cloza-pine (Novartis, Switzerland), risperidone (Janssen, Belgium) andolanzapine (synthesised at H. Lundbeck A/S) were dissolved in a0.9% NaCl solution containing 0.1 MHCl. Citalopram (synthesisedat H. Lundbeck A/S, Denmark), D-amphetamine hemisulphate(purchased from Nomeco A/S, Denmark) and PCP hydrochloride(synthesised at H. Lundbeck A/S, Denmark) were dissolved in 0.9%NaCl solution. Haloperidol (purchased from Sigma-Aldrich, USA)was dissolved in a 0.9%NaCl solution containing 0.1M tartaric acid.Sonepiprazole (synthesised at H. Lundbeck A/S, Denmark) wasdissolved in a 5% 2-hydroxypropyl-β-cyclodextrin solution contain-ing 0.1Mmethane sulfonic acid.All drug doses are expressed asmg/kg active substance except for D-amphetamine and PCP.
3. Results
3.1. In vitro receptor profile
In order to determine the pharmacological profile of Lu 35-138,the compound was profiled in a commercial screen for activity at
Table 2In vitro profile of Lu 35-138 at dopamine receptor subtypes
The affinities of Lu 35-138 and reference compounds at dopamine receptorsubtypes, D1(rat; striarum), D2S (human; cloned), D3 (human; cloned) and D4.2 (human; cloned)
were determined in competition binding experiments (n=3–5) as described byBalle et al. (2003).
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more than 75 different receptors, ion-channels and transporters. Ata concentration of 1 μM, Lu 35-138 did not display appreciableaffinity for the following targets (less than 60% inhibition):adenosine (A1−A3), α2-adrenoceptor, β1- and β2-adrenoceptor,noradrenaline uptake, angiotensin 1 and 2, atrial natriureticpeptide, central and peripheral benzodiazepine receptors, bombe-sin, bradykinin B2, calcitonin gene related peptide, cannabinoidCB1 and CB2, chemokine receptor 1, cholecystokinin A and B,dopamine D1, D3 and D5, dopamine uptake, endothelin A and B,GABA (non-selective), galanin, histamine H1 and H2, interleukin1b, tumor necrosis factor a, melatonin receptor 1, muscarinic (m1–m5), platelet derived growth factor, tachykinin NK1–NK3,neuropeptide Y1 and Y2, neurotensin, δ, κ and μ-opioid, pituitaryadenylate cyclase-activating polypeptide, non-competitiveNMDA site, prostanoid thromboxane A2, prostaglandin I2, purineP2X and P2Y, serotonin receptors 5-HT1B, 5-HT3, 5-HT5A, 5-HT6),somatostatin, vasoactive intestinal peptide, vasopressinV1A, Ca
2+-channel (L-type), voltage dependent potassium channels or Na+-and Cl−-channels. Lu 35-138 showed moderate activity (60–80%inhibition), 5-HT2C and dopamine D2, and high affinity (N80%inhibition) for α1-adrenoceptors, dopamine D4, 5-HT receptorsubtypes (5-HT1A, 5-HT2A, 5-HT7) and 5-HT uptake. According-ly, the affinity of Lu 35-138 for α1-adrenoceptor, dopamine and 5-HT receptors as well as monoaminergic transporters was exploredin more details. In a [3H] prazosin binding assay, to label α1-drenoceptors in rat brain, Lu 35-138 displaced binding with a Ki
value of 45±9 nM and, thus being relatively weak compared toreference antipsychotics tested (clozapine, haloperidol, risperidoneand olanzapine; Table 1). When tested at the cloned α1-adrenoceptor subtypes, Lu 35-138 showed higher affinity forα1A and α1B as compared to α1D-adrenoceptors (Table 1). Athuman dopamine receptor subtypes, Lu 35-138 was selective fordopamine D4 receptors (Ki=5 nM) and ∼ 14 times less active athuman dopamine D2 receptors (Table 2). The affinities of thecompounds were also determined at the rat dopamine D2 receptorusing [3H] spiperone binding to striatal membranes (Balle et al.,2003) and the resulting affinities were very similar to the humandopamine D2 data (data not shown). In a functional assay,measuring inhibition of forskolin-stimulated cAMP formation incloned cells expressing the human dopamine D4 receptor, Lu 35-138 acted as a competitive receptor antagonist as it shifted thedose–response curves for quinpirole to the right without affecting
Table 1In vitro profile of Lu 35-138 at α1 adrenoceptor subtypes
The affinities of Lu 35-138 and reference compounds at the rat α 1 receptor andthe cloned subtypes, α1a (bovine), α1b (hamster) and α1d (rat) were determined inreceptor binding experiments (n=4–6) as described by Balle et al. (2003).
the maximal response (Fig. 2). The resulting Schild plot was linearwith a slope close to unity and the calculated Kb was 8±3 nM(Fig. 2).When Lu 35-138 was tested in this assay in the absence ofreceptor agonist, no changes in cAMP levels were observedindicating that the compound is a neutral receptor antagonist (datanot shown).
Lu 35-138 potently inhibited [3H] 5-HT uptake with an IC50
of 3.2 nM (Table 3) but was relatively ineffective at blocking[3H] noradrenaline uptake (IC50N1000 nM) and [3H] dopamineuptake (IC50=650±100 nM; data not shown). At the 5-HTreceptor subtypes 5-HT1A, 5-HT2A and 5-HT2C only moderateto weak affinities of Lu 35-138 were observed (Table 3).Because the 5-HT7 receptor was not available in house, theaffinity of Lu 35-138 was determined at Cerep, France (cat. No.808-7h) using a [3H] LSD binding assay to human 5-HT7
receptors (Ki =53±9 nM, data not shown). In the course of asynthesis program, Lu 38-012 (Fig. 1) was subjected to a similarbroad receptor profile (data not shown) and subsequent in vitroexperiments (Tables 1–3) which showed that Lu 38-012 was apotent dopamine D4 receptor antagonist (Ki =7.5±0.4 nM) withat least 15-fold lower activity at the targets detailed above. Thiscompound was used along with the selective dopamine D4
Fig. 2. Functional characterisation of Lu 35-138 at human dopamine D4
receptors: dose–response curves for quinpirole alone and with increasingconcentrations of Lu 35-138. Insert shows a Schild plot with a slope of 1.3±0.3,n=3. The experiments were performed as described in methods and the data arerepresentatives of 3 individual experiments performed in triplicate.
Table 3Profile of Lu 35-138 at 5-HT Receptors and 5-HT transporter
The affinities of Lu 35-138 and reference compounds at 5-HT1A (human; cloned),5-HT2A (rat; cerebral cortex), 5-HT2C (rat; cloned) receptor subtypes and 5-HTtransporter (rat, whole brain synaptosomes) were determined in competition bindingexperiments (n=4–5) and 5-HT uptake assay (n=6–8) as described inMaterials and Methods.
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ligands, L-745,870 (Patel et al., 1997) and sonepiprazole(Merchant et al., 1996) to delineate the behavioural conse-quences of D4 receptor blockade.
3.2. In vivo receptor occupancy at the dopamine D2 receptor
The in vivo receptor occupancy of Lu 35–138 at the dopamineD2 receptor in mice was determined using the dopamine D2
receptor antagonist, raclopride. As shown in Fig. 3, Lu 35-138inhibited [3H] raclopride binding in vivowith anED50 of 15±2mg/kg, s.c., n=6. For comparison the effects of reference antipsychoticdrugs such as haloperidol (ED50=0.03±0.008 mg/kg, s.c.) andclozapine (ED50=12±2 mg/kg, s.c.) are shown (Fig. 3).
3.3. Influence on spontaneous and stimulant induced locomo-tor activity
In similarity with the tested antipsychotic drugs (clozapine,haloperidol, olanzapine and risperidone), Lu 35-138 dose-dependently reduced the hyperactivity induced by a low dose ofD-amphetamine (0.5 mg/kg, s.c.) and PCP in rats and mice,
Fig. 3. Effects of Lu 35-138 (filled squares), haloperidol (open squares) andclozapine (open circles) on in vivo dopamine D2 receptor binding in mice using[3H]-raclopride. The experiments (n=3) were performed as described inmethods with 3–4 mice in each group and the data are expressed as percent oftotal binding (750±80 DPM/mg protein).
respectively (Fig. 4 and Table 4). The statistical analysisrevealed a significant inhibitory effect of Lu 35-138 on D-amphetamine (0.5 mg/kg)- and PCP-induced hyperactivity(F=7.3, Pb0.001 and F=11, Pb0.001, respectively). Theselective dopamine D4 receptor ligands Lu 38-012 and L-745,870 reduced the hyperactivity induced by a low dose of D-amphetamine (0.5 mg/kg, s.c.) but were both less potent ininhibiting PCP-induced hyperactivity. In fact, L-754,870 failedto reach an ED50 value for inhibiting the hyperactivity inducedby PCP at doses up to 40 mg/kg s.c. (Table 4). The selectivedopamine D4 receptor antagonist sonepiprazole was inactive onboth these measures at doses up to 40 mg/kg. Likewise, theselective 5-HT reuptake inhibitor citalopram failed to affectthese parameters within the dose-interval tested (Table 4). Theinhibitory effects on both D-amphetamine (0.5 mg/kg)- andPCP-induced hyperactivity appears in general specific giventhat at least two times higher ED50 values for inhibiting thespontaneous locomotion activity were obtained with the activesubstances (Table 4). However, the inhibitory effect ofclozapine on PCP-induced hyperactivity might be biased byan unspecific effect as this drug was relatively potent ininhibiting the spontaneous locomotor activity in mice (Table 4).
As shown in Table 4, Lu 35-138 as well as clozapine,risperidone and Lu 38-012 were more than 10 times less potent incounteracting the hyperactivity induced by the high dose (2.0 mg/kg, s.c.) as compared to the low dose (0.5 mg/kg, s.c.) of D-amphetamine. In fact, Lu 35-138 as well as L-745,870 werecompletely ineffective in influencing the hyper locomotor activityinduced by the high dose of D-amphetamine. Lower ratios betweenthese ED50 values were observed for haloperidol and olanzapine.
3.4. Influence on D-amphetamine disrupted pre-pulse inhibition
D-amphetamine (2.0 mg/kg, s.c.) disrupted the pre-pulseinhibition phenomenon, an effect counteracted by Lu 35-138 at
Fig. 4. Effect of Lu 35-138 on hyperlocomotion induced by D-amphetamine(rats; 0.5 and 2.0 mg/kg, s.c.) and PCP (mice; 2.5 mg/kg, s.c.). Each pointrepresents the mean (±S.E.M.) percentage inhibition of psychostimulant-induced hyperlocomotion (n≥8). The variability (±S.E.M.) of the controlvalues for the respective psychostimulant-induced activity (n≥8) is indicated inthe left corner of the figure. ⁎Pb0.05 compared to the total activity of D-amphetamineor PCP alone.
Table 4Effect on spontaneous locomotor activity (SLA) and PCP and D-amphetamine (D-AMPH) induced hyperactivity
S.D. factors (95% confidence interval defined as: ED50 /S.D.−ED50×S.D.) in parentheses. n≥8 per dose group except from SLA (rats): n≥4 per dose group.a 30 min pre-treatment time; otherwise 2 h in rats.
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the highest dose tested (18 mg/kg, s.c.; Fig. 5A). Thus, thethree-way ANOVA indicated a significant interaction betweenfactors of treatment and drug (F=4.5; Pb0.05). The subsequentpost-hoc analysis revealed a significant difference betweenvehicle and D-amphetamine treatment only in the control group(Pb0.05) as well as a statistically significant difference betweenthe D-amphetamine treated animals in the control group and theD-amphetamine treated animals pretreated with Lu 35-138(18 mg/kg, s.c.; Pb0.05). However, no statistically significantdifference was revealed between the D-amphetamine treatedanimals in the control group and the D-amphetamine treatedanimals pretreatedwithLu35-138 at the lower dose (9.0mg/kg, s.c.).The startle amplitude of rats was not affected by the D-amphetaminetreatment or the administration of Lu 35-138.
3.5. Influence on the number of spontaneously active dopaminecells
Repeated treatment with Lu 35-138 (0.29 and 1.2 mg/kg, p.o.,once daily for 21 days) and haloperidol (0.16 mg/kg, p.o., oncedaily for 21 days) reduced the number of spontaneously activedopamine neurones in the ventral tegmental area (Fig. 5B). TheANOVA indicated a significant treatment effect (F=2.7;Pb0.001) and the following post-hoc analysis showed that alltreatments were significantly different compared to the vehiclecontrol group. Repeated administration of haloperidol reducedthe number of spontaneously active dopamine neurones ascompared to control values. Repeated administration of Lu 35-138 did not cause any apparent change in the gross behaviour orin the body weight gain of the rats.
3.6. Influence on dopamine output
Administration of Lu 35-138 (0.15 and 0.29 mg/kg, i.v.)preferentially increased dopamine output in the shell as comparedto the core region of the nucleus accumbens (Fig. 6). TheANOVA on the data from the low dose of Lu 35-138 indicated asignificant time (F=2.4; Pb0.05) and region (F=5.7; Pb0.05)effect and a significant overall interaction (F=2.4; Pb0.05). Thefollowing post-hoc analysis of these data revealed a significant
difference in the response between the areas within the 6–15 minpost-injection time interval at the low dose of Lu 35-138.However, at the high dose, no statistically significant differencecould be detected even though the numerical difference was morepronounced as compared to the low dose. This probably reflectsthe larger variance observed at the high dose. When compared tobaseline, no significant change in the core subdivision wasdetected after the two doses of Lu 35-138. In contrast, thestatistical analysis indicated a significant elevation of dopamineoutput in shell subdivision as compared to baseline within the 5to 15 min interval at the low dose of Lu 35-138. Controlinjections with vehicle did not significantly affect the dopamineoutput in either of the two areas (data not shown).
3.7. Induction of catalepsy and dystonia
Lu 35-138 failed to induce catalepsy in rats (Table 5). In fact,none of the rats at the highest dose tested (18 mg/kg, s.c.)showed any signs of cataleptic behaviours. Likewise, Lu 35-138did not induce any signs of dystonia in monkeys within thetested dose interval (maximal dose 2.3 mg/kg, s.c./i.m.). Themaximal dose given to monkeys in this assay is admittedlyrelatively low but due to the low solubility, it was the highestdose possible with the given injection volume limit. Similarprofile was observed for clozapine, Lu 38-012, L-745,870,sonepiprazole and citalopram which all showed no, or at leastlow, liability to induce cataleptic or dystonic behaviours in theanimals. In comparison with Lu 35-138, haloperidol, olanza-pine as well as risperidone were more potent in inducing suchbehaviours (Table 5).
3.8. Influence on water maze performance
Lu 35-138 did not increase the latency to find the hiddenplatform and failed to affect the swim speed within the testeddose interval (doses up to 18 mg/kg, s.c., Table 6). In similaritywith Lu 35-138, Lu 38-012 as well as citalopram failed to affectthese parameters within their respective dose-intervals tested.Clozapine did not influence the swim speed at the doses testedbut increased the latency to find the hidden platform at high
Fig. 6. Effect of Lu 35-138 on extracellular concentrations of dopamine (DA) inthe shell and core of the nucleus accumbens (NAC). Each point represents themean (±S.E.M.) percent change of baseline values. n=5 in the shell and n=5(low dose) or n=3 (high dose) in core. Arrow indicates injection of Lu 35-138.⁎Pb0.05 compared between areas and # Pb0.05 compared to baseline.
Table 5Liability of Lu 35-138 to induce catalepsy and dystonia
Catalepsy ED50
(mg/kg, s.c.)Dystonia MED(mg/kg, s.c./i.m.)
Compound Rat Monkey
Lu 35-138 N18 N2.3Clozapine N40 N25 a
Haloperidol 0.15 (1.2) 0.025 a
Olanzapine 12 (1.4) 0.16Risperidone 8.2 (1.4) 0.025 a
Catalepsy and dystonia were assessed in rats (n≥4 per dose group) and Cebusapella monkeys (n=7 per dose group), respectively. S.D. factors (95%confidence interval defined as: ED50 /S.D.−ED50×S.D.) in parentheses.N.A. (not assessed).MED (minimal effective dose).a Data from Casey (1993).
Fig. 5. (A)Effect of Lu35-138 onD-amphetamine (AMPH; 2.0mg/kg, s.c.) disruptedpre-pulse inhibition. Each bar represents the mean (±S.E.M.) percentage pre-pulseinhibition (n=8). ⁎Pb0.05 compared between the effect of vehicle (Veh) and D-
amphetamine injections in the control group and#Pb0.05 compared to the effect of D-amphetamine injection in the control group. (B) Effect of repeated treatment of Lu 35-138 (0.29 and 1.2mg/kg, p.o., once daily for 21 days) and haloperidol (0.16mg/kg, p.o., once daily for 21 days) on population firing of dopamine cells in the ventraltegmental area (VTA). Each bar represents the mean (±S.E.M.) number ofspontaneously active dopamine cells per track in the VTA (n≥6). ⁎Pb0.05,⁎⁎Pb0.01 compared to the control group.
156 P. Hertel et al. / European Journal of Pharmacology 573 (2007) 148–160
doses (10 mg/kg, s.c.). In contrast to these drugs, haloperidol,olanzapine and risperidone were all rather potent in increasingthe latency to find the hidden platform and to concomitantlyinhibit the swim speed of the rats (Table 6).
4. Discussion
It has previously been shown that antagonism of hyperac-tivity induced by a low dose of amphetamine (0.5 mg/kg, s.c.)represents a common denominator for both classical and newerantipsychotic drugs, whereas the latter ones appears to havemarkedly less potency to inhibit the hyperactivity induced by ahigh dose of D-amphetamine (2.0 mg/kg, s.c.; Arnt, 1995). Thepresent data show that Lu 35-138, in contrast to haloperidol,shares the ability of clozapine, olanzapine and risperidone tomore potently counteract the hyperlocomotion induced by lowas compared to a high dose of D-amphetamine. In fact, Lu 35-138 displayed a very high preference in this regard, having a
Table 6Effect of Lu 35-138 on Morris' water maze performance. Morris’ water mazeperformance was assessed in rats (n≥8 per dose group)
MED (mg/kg, s.c.)
Compound Increasing latency Inhibiting speed
Lu 35-138 N18 N18Clozapine 10 N10Haloperidol 0.020 0.040Olanzapine 1.3 1.3 a
N.A. (not assessed).MED (minimal effective dose).a Swimming speed was only significantly inhibited at day 3 whereas latency
was highly increased already at day 2. Thus, the increased latency seems notsolely to be due to impaired motor function.
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ratio between the respective ED50 values at least twice the valueof the ratios observed for clozapine, olanzapine and risperidone.Previous studies have indicated that the hyperactivity inducedby low doses of D-amphetamine is largely mediated viaincreased output of dopamine in the mesolimbic areas, whereasthe behavioural signs seen after higher doses of this drug areattributed to stimulation of dopamine receptors located innigrostriatal projection areas (Kelly et al., 1975). Thus, thepresent finding suggests that Lu 35-138 preferentially targetsthe mesolimbic as compared to the nigrostriatal dopaminergicsystem. Such limbic selectivity is in line with the currentvoltammetry data. Thus, we observed that Lu 35-138 induced asignificantly more pronounced increase in dopamine efflux inthe limbic related shell region as compared to the more striatalassociated core area of the nucleus accumbens (Deutch andCameron, 1992; Zahm, 1999). Similar regional pattern ofdopamine output in the nucleus accumbens has been reportedfor antipsychotic drugs with low extrapyramidal side effectliability whereas classical dopamine D2 antagonistic drugsmainly acts on dopaminergic parameters in motor related areas(Marcus et al., 1996). Although the responsible mechanism ofaction of Lu 35-138 in this context is unknown, it has beenreported that dopamine D4 receptors are located on bothdopaminergic and excitatory afferents nerve terminals in theshell part of the nucleus accumbens providing an anatomicalbasis for presynaptic regulation of dopaminergic as well asexcitatory transmission via dopamine D4 receptors in this area(Svingos et al., 2000).
Non-competitive NMDA receptor antagonists such as PCPinduce a psychotomimetic state in humans very reminiscent ofthe symptoms observed in schizophrenia (Javitt and Zukin,1991). Therefore, PCP administration is regarded as one of themost relevant pharmacological models of this disease (Jentschand Roth, 1999). In line with previous literature (Millan et al.,2000), we found that clozapine, haloperidol, olanzapine andrisperidone all dose-dependently counteracted the hyperloco-motion in mice induced by PCP. Interestingly, Lu 35-138 sharedthis effect although exhibiting lower potency. It should howeverbe stressed that regardless of the relatively low potency, theeffect of Lu 35-138 appears specific in this regard as higherdoses of the drug was needed for affecting spontaneouslocomotor activity. Although it is generally thought thatblockade of dopamine D4 receptors have modest behaviouralconsequences in animals, it is tempting to suggest that at leastsome of the antagonistic effect of Lu 35-138 on psychostimu-lant-induced hyperactivity is mediated via blockade of dopa-mine D4 receptors. First it should be pointed out that no reliablemeasure of in vivo dopamine D4 blockade exists today.However, our in house assessments have indicated that Lu35-138 potently block the 5-HT uptake in vivo as measured byits ability to potentiate behaviours induced by the 5-HTprecursor L-5-HTP in rats with an ED50 of 5.5 mg/kg (Lapizet al., in preparation). Also, in vivo binding assays have shownthat Lu 35-138 is potent in occupying central 5-HT transportersin mice (ED50=0.56 mg/kg; Lapiz et al., in preparation). Thus,given that Lu 35-138 has similar affinity for dopamine D4
receptors and 5-HT transporters in vitro, it is likely that Lu 35-
138 target a significant amount of the dopamine D4 receptors atthe doses effective in inhibiting the psychostimulant-inducedhyperactivity in mice and rats. The involvement of dopamine D4
receptor in this context is also supported by the ability of theselective dopamine D4 receptor antagonist Lu 38-012 to mimicthe behavioural effects of Lu 35-138. In addition, we also foundthat the selective dopamine D4 receptor ligand L-745,870 (Patelet al., 1997) counteracted the effect of a low dose of D-amphetamine. These findings are in accord with previousliterature investigating the effect of dopamine D4 receptorligands such as L-745,870 and S 18126 on D-amphetamineinduced behaviours in rats although the selectivity of theseligands at the effective doses has been questioned (Millan et al.,1998). On the other hand, the present and previous studies havefailed to find such effects of the selective dopamine D4 receptorantagonist sonepiprazole (Merchant et al., 1996) although thedoses used in the present study have previously been shown tobe efficient against apomorphine disrupted pre-pulse inhibitionin rats (Mansbach et al., 1998). It is also known that potencyagainst amphetamine-induced hyperactivity to some extentcorrelate with the affinity for dopamine D2 receptors (see e.g.Bardin et al., 2007). Furthermore, effective doses of Lu 35-138in the PCP-induced hyperactivity model clearly involvedopamine D2 receptors as judged by the present in vivo bindingdata. Thus, a final conclusion on the mechanism of action of Lu35-138 and a tentative role of dopamine D4 receptor antagonismin counteracting psychostimulant-induced hyperlocomotionrequires further investigations.
We also investigated the effect of Lu 35-138 in the pre-pulseinhibition model of acoustic startle response in rats. Deficits inthe sensorimotor gating function, as measured by pre-pulseinhibition, appear to be a characteristic phenotype in severalneuropsychiatric diseases including schizophrenia (Braff et al.,2001). Previous studies have shown that both classical andnewer antipsychotic drugs, including haloperidol, olanzapineand risperidone, can counteract dopamine receptor agonistinduced disruption of pre-pulse inhibition in rats (Geyer et al.,2001). Although complicated by the conflicting results withclozapine (Geyer et al., 2001), this model is considered to havehigh predictive validity for antipsychotic activity. Interestingly,we found that Lu 35-138 significantly reversed the pre-pulseinhibition deficit induced by D-amphetamine without affectingthe startle amplitude which, again, suggests that this drug hasantipsychotic-like effects. Previous literature has shown thatselective dopamine D4 receptor antagonists may reverse deficitsin pre-pulse inhibition induced by dopamine receptor agonism(Mansbach et al., 1998) whereas the inhibition of 5-HT reuptakeappears ineffective (Geyer et al., 2001) suggesting that theblockade of dopamine D4 receptors may represent at least a partof the mechanism of action of Lu 35-138 in this context.However, involvement of dopamine D2 receptors cannot beexcluded as the minimal effective dose of Lu 35-138 in thisassay probably induces a significant dopamine D2 receptoroccupancy (see Fig. 3).
Previously studies have indicated that inactivation ofdopamine neurons in the ventral tegmental area after repeatedadministration appears to have high predictive validity for
158 P. Hertel et al. / European Journal of Pharmacology 573 (2007) 148–160
antipsychotic activity (Arnt and Skarsfeldt, 1998; Grace et al.,1997). In line with previous data (see e.g. Grace et al., 1997) weobserved that repeated administration of haloperidol signifi-cantly reduced the number of spontaneously active dopaminecells in the ventral tegmental area, an effect mimicked byrepeated administration of Lu 35-138 which further underlinesthe antipsychotic-like effect of this compound. The responsiblemechanism of action of Lu 35-138 in this assay remains to beclarified.
It has become increasingly evident that cognitive symptomsrepresent an important hallmark of schizophrenia and, in fact,these symptoms appear more closely correlated to the clinicaloutcome as compared to the psychotic ones (Green, 1996).Current antipsychotic drugs have, at best, limited effects oncognitive functions and they may even worsen the cognitivesymptomatology in schizophrenia (Cutmore and Beninger,1990). It is clear from pre-clinical models that classicalantipsychotic drugs such as haloperidol may have disruptiveeffects on cognitive functioning (Skarsfeldt, 1996; Didriksenet al., 2006). In line with this literature, we found that sub-acutetreatment with haloperidol at doses similar to those counter-acting the hyperactivity induced by a low dose of D-am-phetamine significantly deteriorates the performance in theMorris’ water maze, a well-established model for spatiallearning and memory (D'Hooge and De Deyn, 2001). Incontrast, clozapine seemed profoundly less effective in thisregard whereas both olanzapine and risperidone disrupt theperformance in this model at doses close to those affecting thehyperactivity induced by a low dose of D-amphetamine. Itshould be mentioned that the increase in the latency to find theplatform after risperidone and olanzapine treatment might bebiased by general motor inhibition given that these drugsconcomitantly reduced the swim speed at the same dosesaffecting the latency. However, in the case of olanzapine, theswim speed was only significantly inhibited at day 3 whereaslatency was highly increased already at day 2. Thus, theincreased latency after olanzapine treatment appears not solelyattributed to impaired motor function. In contrast, Lu 35-138did not affect any measured parameters in the Morris' watermaze within the tested dose interval. This may indicate that Lu35-138 possesses low liability to induce or aggravate cognitivedysfunctions in schizophrenic patients. The potential of Lu 35-138 to improve cognitive functioning remains to be investigat-ed. This appears indeed relevant, as recent pre-clinical literaturehas identified the dopamine D4 receptor as a putative target forthe treatment of cognitive deficits in schizophrenia (Browmanet al., 2005; Floresco et al., 2006).
One of the most significant adverse consequences ofantipsychotic drug therapy is extrapyramidal side effects,effects causally linked to extensive blockade of striataldopamine D2 receptors (Farde et al., 1992). Assessment of theability to induce catalepsy in rodents and acute dystonia inneuroleptic-sensitized monkeys are frequently used animalmodel for such symptoms. The present study clearly demon-strates that Lu 35-138 is devoid of cataleptogenic potential inrats and dystonic ability in monkeys, a property similar to that ofclozapine but in contrast to haloperidol, olanzapine and
risperidone which all induced catalepsy as well as dystoniawithin the tested dose ranges (see Table 5; Casey, 1993). Thislack of cataleptogenic and dystonic ability of Lu 35-138 is mostprobably a result of its low dopamine D2 affinity. However,other mechanisms cannot be ruled out. For example, it is wellknown that the stimulation of 5-HT1A receptors counteractsdopamine D2 mediated catalepsy in rats (see e.g. Bardin et al.,2007). Furthermore, recent studies have shown that the 5-HT7
receptor is involved in the control of striatal cholinergicneurotransmission (Bonsi et al., 2007). Thus, given that Lu35-138 is a potent 5-HT reuptake inhibitor and have moderateaffinity for both 5-HT1A and 5-HT7 receptors, it cannot be ruledout that the lack of cataleptogenic and dystonic liability of Lu35-138 may in part be mediated via serotonergic mechanisms.Regardless of the mechanism(s) involved, the absence of effectin these extrapyramidal side effect models indicates that Lu 35-138 shares the benefit of clozapine in having a low extra-pyramidal side effect liability in patients.
Clinical trials with selective dopamine D4 receptor antago-nists in schizophrenia have so far been disappointing (Corriganet al., 2004; Kramer et al., 1997). In a recent, well-controlledand adequately powered trial, the selective dopamine D4
receptor antagonist sonepiprazole failed to show antipsychoticeffectiveness (Corrigan et al., 2004). This finding clearlyquestions the usefulness of selective dopamine D4 receptorantagonists in schizophrenia. In this context, it should be notethat Lu 35-138 differentiates from sonepiprazole in that thisdrug is first of all not a selective dopamine D4 receptorantagonist and second, is able to counteract both amphetamineand PCP-induced hyperactivity. Whether this difference in pre-clinical profile of Lu 35-138 as compared to sonepiprazole willtranslate in to a difference in the clinical profile has to awaitfuture trials.
In conclusion, the present findings indicate that Lu 35-138possesses antipsychotic-like activity combined with a low liabilityto induce extrapyramidal as well as cognitive side effects. This,together with a tentative action on depressive symptoms (seeMitchell and Hogg, 2002) owing to its 5-HT reuptake inhibitingproperty underlines the distinctive and promising profile of Lu 35-138 for the improved treatment of schizophrenia.
Acknowledgements
This work was founded by H. Lundbeck A/S. Monica M.Marcus was supported by the Swedish Medical ResearchCouncil (Grant 4747) and the Karolinska Institutet.
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