2-Bromoterguride–a potential atypical antipsychotic drug without metabolic effects in rats

Rationale Recently, we showed that 2-bromoterguride acted as a dopamine D2 receptor partial agonist, a serotonin 5-HT2A and α2C-adrenergic receptor antagonist, and exhibited antidopaminergic efficacy in amphetamine-induced locomotion (AIL) in rats without inducing catalepsy. Objective To extend our knowledge on the antipsychotic effects of 2-bromoterguride, we used convergent preclinical animal models and tests; i.e., conditioned avoidance response (CAR), predictive of antipsychotic-like effects; Fos protein expression, a molecular marker for (atypical) antipsychotic activity; wet dog shake behavior, a test for the in vivo effects of drugs acting on central 5-HT2A receptors; and investigated metabolic changes as a common side effect of atypical antipsychotic drugs (APDs). Results Acute treatment with 2-bromoterguride (0.1 and 0.3 mg/kg) decreased the CAR at 30, 90, and 270 min post-injection in rats without inducing escape failures at any time. Fos protein expression, as shown by Western blotting, was enhanced by 2-bromoterguride in the nucleus accumbens (NAc), the dorsolateral striatum (dStr), and the medial prefrontal cortex (mPFC). (±)-2,5-Dimethoxy-4-iodoamphetamine (DOI)-induced wet dog shakes in rats were reduced by 2-bromoterguride. Chronic treatment with 2-bromoterguride did not affect metabolic parameters such as body weight development and body fat composition as well as behavioral parameters such as food intake and locomotor activity. Conclusions Our data suggest that 2-bromoterguride is a promising candidate in the treatment of schizophrenia due to its atypical antipsychotic-like activity and its inability to induce weight gain.


Introduction
The majority of patients with schizophrenia are treated with typical or atypical antipsychotic drugs (APDs) (Leucht et al. 2009(Leucht et al. , 2013. Typical APDs such as haloperidol possess high antagonist potency at dopamine D 2 receptors but carry substantial risk to induce extrapyramidal side effects (EPS) and increased release of prolactin (Meltzer 2013). Atypical APDs with a clozapine-like profile interact with varying affinities at D 2 , 5-HT 2A , 5-HT 2C , and many other monoamine receptors including α 1 -and α 2 -adrenoceptor subtypes and histamine H 1 receptors (Kroeze et al. 2003;Leucht et al. 2009;Meltzer et al. 2012;Roth et al. 2004;Schoemaker et al. 1997). The key feature that distinguishes typical from atypical APDs is that the latter produce fewer EPS than the first (Meltzer 2013). However, atypical APDs cause considerable weight gain, which may be based, at least in part, on the antagonist properties of these drugs at histamine H 1 and 5-HT 2C receptors (Kroeze et al. 2003;Roth et al. 2004).
The advent of aripiprazole, a D 2 receptor partial agonist of moderate intrinsic activity, has generated a new treatment option in schizophrenia (DeLeon et al. 2004). D 2 receptor partial agonists may induce a desirable stabilization of dopaminergic transmission insofar as they decrease transmission postsynaptically in the mesolimbic system and increase transmission at the mesocortical level (Lieberman 2004;Stahl 2001). Aripiprazole does not cause EPS (Leucht et al. 2013); like other atypical APDs, it shows high affinity for multiple 5-HT receptors and other central neuroreceptors (Kroeze et al. 2003).
We recently showed that 2-bromoterguride, an ergot derivative, mechanistically mimicked aripiprazole as a D 2 receptor partial agonist (Jantschak et al. 2013). However, compared with aripiprazole, 2-bromoterguride exhibited much higher affinity for 5-HT 2A receptors and α 2C -adrenoceptors and much lower affinity for histamine H 1 receptors (Jantschak et al. 2013). Preclinical tests using rats confirmed that the multifunctional drug 2-bromoterguride had antidopaminergic activity as demonstrated by the inhibition of amphetamine-induced locomotion (AIL) and exhibited low EPS liability as shown by the failure to produce catalepsy. The in vivo observations in conjunction with its broad neuroreceptor binding profile suggest that 2-bromoterguride may be classified as an atypical APD.
The aim of the present study was to extend our knowledge on the antipsychotic effects of 2-bromoterguride. We examined whether 2-bromoterguride might inhibit the conditioned avoidance response (CAR), a widely accepted screening tool in rats with high predictive validity that has shown particular sensitivity for detection of potential antipsychotic activity (Wadenberg 2010). It is well documented that APDs alter Fos expression in nucleus accumbens (NAc), dorsolateral striatum (dStr), and medial prefrontal cortex (mPFC) (Robertson et al. 1994;Sumner et al. 2004). Therefore, we measured 2-bromotergurideinduced Fos protein expression in these brain regions with special focus on the effects in the mPFC because a Fos enhancement in this region can be used to identify atypical APDs (Robertson and Fibiger 1996). To substantiate t h e 5 -H T 2 A r ec e p t o r a n t a g o n i s t p o t e n c y o f 2bromoterguride in vivo, it seemed reasonable to find out whether the drug was able to reduce (±)-2,5-dimethoxy-4iodoamphetamine (DOI)-induced wet dog shakes in rats, a test for drugs acting on central 5-HT 2A receptors (Schreiber et al. 1995). As mentioned above, atypical APDs can induce weight gain. Therefore, we analyzed the effects of prolonged 2-bromoterguride administration on food intake, weight gain, and body fat composition, as well as on food and water consumption. Together with the preclinical tests of our previous study, AIL and catalepsy (see Jantschak et al. 2013), we conclude that 2bromoterguride has atypical antipsychotic properties without metabolic side effects such as weight gain.

Animals
All experiments were performed in accordance with the guidelines of the German Animal Protection Law and approved by the Berlin State Authority (BLandesamt für Gesundheit und Soziales^). A total of 163 naïve male (acute experiments) and female (chronic experiment) Sprague-Dawley rats (Élevage Janvier, Le Genest Isle, France) at an age of 10-13 weeks were used. The animals were housed in groups of two to three in type IV open-top polycarbonate cages (Ehret, Emmendingen, Germany) with dust-free hardwood bedding (Hygienic Animal Bedding; J. Rettenmaier & Söhne, Rosenberg, Germany) under standard conditions (room temperature, 22 ± 2°C; relative humidity 55 ± 10 %) on a 12-h light-dark schedule (lights on at 6:00 A.M.). Home cages were enriched by paper tissues and metal tubes as hiding places. Rats had free access to standard laboratory chow (ssniff, Soest, Germany) and tap water and were handled and weighed regularly. Once weekly, the home cages were cleaned and equipped with new bedding by a professional animal keeper. Animals were free of pathogens according to Federation for Laboratory Animal Science (FELASA) recommendations.

Conditioned avoidance response
Male rats were trained and tested in two computer-assisted, two-way active avoidance shuttle boxes (Med Associates Inc., St. Albans, USA), which were enclosed by MDF soundattenuating cubicles and equipped with stimulus lights, tone generators, and grid floors. Each shuttle box consisted of two compartments of equal size separated by a partition with an aperture, which allowed the rats to move freely between the compartments. The positions of the animals were tracked by eight infrared photobeams. Each session (training, drug-free pre-test, drug-test) started with a 3-min habituation period, in which no stimuli presentation occurred, followed by 30 trials.
Upon presentation of a white noise (76 dB) and light as conditioned stimuli (CS), rats needed to move from one compartment into the other within 10 s to avoid the unconditioned stimulus (UCS), which was presented for 20 s as an intermittent electric shock in the grid floor (0.3 mA; interval 2 s; single shock duration 0.5 s). Moving from one compartment to the other one within the first 10 s (response to CS) was recorded as avoidance, and a change as a response to the UCS was recorded as an escape. Escape failures were recorded if an animal failed to respond to the CS and USC within the trial duration of 30 s. Inter-trial intervals randomly varied between 10 and 30 s. Rats were daily trained until they met the test criterion (≥80 % avoidances) on two consecutive days. The next day, a drug-free pre-test was performed. Subsequently, the animals were injected with 2-bromoterguride (0.1 and 0.3 mg/kg), haloperidol (0.05 and 0.1 mg/kg), aripiprazole (1 and 3 mg/kg), or the respective vehicle. The CAR was evaluated 30, 90, and 270 min and 24 h post-injection as percent avoidance compared to percent avoidance of the pretest and percent avoidance of the control group, respectively. The doses for aripiprazole and haloperidol were determined by conducting pilot studies. Each compound was tested in one treatment group. The animals of the three treatment groups were tested repeatedly in a pseudorandom order serving as their own controls (wash-out period of at least 4 days).

Neuronal Fos expression
Male rats were transferred within their home cages to a soundproof red lighted dark-room for 2 days. During this period, they were habituated to injections of saline. On day 3, rats were injected with 2-bromoterguride (0.1 and 0.3 mg/kg), haloperidol (0.5 mg/kg), or vehicle (15 % Cremophor® EL). The dose of haloperidol was determined by conducting a pilot study. Two hours later, animals were sacrificed by decapitation and brains rapidly removed from the skull. Brain areas were dissected on a cold plate. The following tissue samples were weighed and shock frozen in liquid nitrogen: mPFC, NAc, and dStr (Heffner et al. 1980).
Expression of Fos and β-actin in brain homogenates were studied by Western blotting using the following primary antibodies: rabbit anti-Fos (1:2000) and rabbit anti-β-actin as loading control (1:20,000; New England BioLabs, Frankfurt a. M., Germany). Bands were detected by probing with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:10,000; Cell Signaling Technology, Leiden, The Netherlands). Visualization was achieved by chemiluminescence with ECL Prime Western Blotting Detection Reagent (Am ersham, Braunschweig, Germany) on Amersham Hyperfilm (GE Healthcare Limited, Little Chalfont, UK). Quantification of bands was obtained by digital image analysis using National Institutes of Health Image software (http://rsb.info.nih. gov/ij/download.html).

Antagonism of DOI-induced wet dog shakes
The experiments were conducted in two glass cuboids (29 × 22 × 39 cm) of which the grounds were coated with bedding material. Male rats were given an injection of either 2bromoterguride (0.1 or 0.3 mg/kg), aripiprazole (3 mg/kg), ketanserin (1 mg/kg), or vehicle (15 % Cremophor® EL). After 30 min, animals were injected with DOI (2 mg/kg) and vehicle, respectively. Subsequently, the behavior of the animals was monitored and recorded (Canon HG10 HD-Camcorder; Canon, Krefeld, Germany) for 30 min. A trained observer of rat behavior determined the number of wet dog shakes.
Body weight development, body fat composition, food intake, locomotor activity, and cataleptic behavior Female rats were used as they exhibit a more robust weight gain following atypical APD treatment compared to males (Choi et al. 2007;Davey et al. 2012). However, in female rats, clozapine does not increase weight gain mandatorily but does increase visceral fat tissue (Cooper et al. 2008). Therefore, we used a comprehensive approach by investigating the effects of 2-bromoterguride on body fat composition and body weight development with olanzapine (instead of clozapine) as a positive control. Animals were matched into four different treatment groups based on their body weight. Rats were injected B.I.D. (first injection between 9:00 and 10:00 A.M.; second injection between 4:00 and 5:00 P.M.) with 2-bromoterguride (0.1 and 0.3 mg/kg), olanzapine (2 mg/kg), or vehicle (15 % Cremophor® EL) for 21 days. Body weight, food and water intake were measured daily prior to the first injection. Weight gain (%) was calculated in relation to body weight of day 0. The amount of food and water consumed was calculated in grams or milliliters per cage (n = 2) in relation to body weight. On day 22, rats were deeply anaesthetized with chloral hydrate (720 mg/kg; Sigma-Aldrich, Steinheim, Germany) and visceral (gonadal and retroperitoneal), subcutaneous (inguinal), and brown fat tissue (interscapular) were dissected and weighed by a trained experimenter. Tissue weights were calculated in relation to body weight. In order to compare the effect of acute versus prolonged 2-bromoterguride treatment on behavior, we analyzed locomotor activity in the open field (day 14; distance traveled (cm)) and cataleptic behavior (day 20; bar and grid test) as described previously (Jantschak et al. 2013).

Data presentation and analysis
Data were analyzed and presented using SigmaPlot 11 (Systat Software, Erkrath, Germany). The following tests were used: one-way analysis of variance (ANOVA) (Fos expression, wet dog shakes, body fat composition, locomotor activity) and two-way repeated measures (RM) ANOVA (treatment × time) (CAR, body weight development, food intake) followed by Holm-Sidak t tests. Data of escape failures and cataleptic behavior were not normally distributed (tested with the Shapiro-Wilk method) and analyzed with non-parametric Friedman one-way RM ANOVA and Kruskal-Wallis one-way ANOVA followed by Dunn's method, respectively. P values <0.05 were considered to be significant. Data are presented as mean values ± standard error of the means (SEM).

Discussion
The present study further demonstrates that 2-bromoterguride, a drug with partial agonist effects at D 2 receptors, high affinity for 5-HT 2A and α 2C -adrenergic but low affinity for histamine H 1 receptors (Jantschak et al. 2013), has antipsychotic properties without inducing weight gain. In our previous study, we investigated the antidopaminergic efficacy of 2bromoterguride using AIL, and EPS liability using catalepsy tests (Jantschak et al. 2013). The CAR provides a further highly predictive and reliable screening tool to test potential drugs exhibiting antipsychotic-like properties (Wadenberg 2010). It has been shown that well-established APDs, within a certain dose range, effectively suppress CAR without inducing escape failures. The incidence of escape failures at a given dose indicates that this dose produces non-specific behavioral effects such as sedation (Wadenberg 2010). Effective suppression of CAR with typical and atypical APDs in rats can be achieved by a striatal D 2 receptor occupancy (D 2 RO) of 65-80 % (for aripiprazole a D 2 RO of >85 % was required to inhibit CAR). These D 2 ROs reflect the range in which schizophrenic patients respond to APDs (Natesan et al. 2006;Wadenberg et al. 2001;Wadenberg 2010). The present study shows that 2-bromoterguride, haloperidol, and aripiprazole produced a suppression of CAR in a comparable and dosedependent manner. Only the high dose of aripiprazole induced more escape failures than vehicle. This may be related to the relatively high affinity of aripiprazole for H 1 receptors; haloperidol and 2-bromoterguride exhibit low affinities for these receptors (Jantschak et al. 2013;Kroeze et al. 2003). It should be noted that other receptors than the D 2 receptor may mediate or contribute to the suppression of CAR by 2-bromoterguride. For example, a blockade of 5-HT 2A receptors may be involved in the disruptive effect on CAR (Wadenberg et al. 1998). Consistent with this hypothesis, the acute effect of clozapine on avoidance responding was reversed by DOI (Li et al. 2012). Although DOI is non-selective for 5-HT 2A versus 5-HT 2C receptors, the former receptor population can be favored to be involved in drug-induced disruption on CAR (Halberstadt et al. 2009;Li et al. 2010Li et al. , 2012Schreiber et al. 1995;Sipes and Geyer 1995;Smith et al. 2003). In contrast to the effect of clozapine, the inability of DOI to reverse CAR disruption by olanzapine was explained by a predominant 2.93 ± 0.14* 1.61 ± 0.13* 0.08 ± 0.004 Tissue weights were calculated in relation to body weight (g/100 g bw). Data are expressed as mean ± SEM of 9 (2-bromoterguride, vehicle) or 8 rats (olanzapine) 2-BT 2-bromoterguride, OLZ olanzapine, VEH vehicle * P < 0.05 vs. vehicle involvement of D 2 receptors in this response (Li et al. 2012). This is because olanzapine has a much higher D 2 receptor affinity than clozapine, whereas both drugs display similar affinity for 5-HT 2A receptors (Kroeze et al. 2003). 2-Bromoterguride exhibits high affinity for both D 2 and 5-HT 2A receptors; therefore, it is difficult to decide which receptor is predominantly responsible for the inhibition of CAR by this drug. α 2 -Adrenoceptor blockade may also contribute to the effect of 2-bromoterguride on CAR. Pretreatment with idazoxan (α 2 -adrenoceptor antagonist) enhanced the antipsychotic effects of typical and atypical APDs, as, for example, demonstrated in the CAR test (Marcus et al. 2010). Because 2bromoterguride exhibited high affinity for D 2 , 5-HT 2A , and α 2C -adrenoceptors, all of these properties may be associated with its ability to suppress CAR, thus highlighting the antipsychotic-like potential of this drug. APDs induce the expression of the immediate-early gene product Fos in a distinct pattern of brain regions, which is suggested as an indicator of antipsychotic efficacy (Dragunow et al. 1990;Miller 1990;Robertson et al. 1994;Natesan et al. 2011). In addition, expression of Fos in the mPFC can be used to identify atypical APDs (Robertson and Fibiger 1996). For example, acute administration of haloperidol increased Fos expression in the dStr and the NAc but not in the mPFC (Young et al. 1999;this study). In contrast, clozapine and other atypical APDs enhanced Fos protein in the NAc and mPFC but not in the dStr (Deutch et al. 1992;Dilts et al. 1993;Nguyen et al. 1992;Robertson et al. 1994;Robertson and Fibiger 1996). In this context, it was of special interest to find out whether 2-bromoterguride, a low-efficacy D 2 receptor partial agonist, might enhance Fos in different brain regions. As suggested by Natesan et al. (2011), a criterion for therapeutic success of novel APDs may be their potential to exhibit low intrinsic activity coupled with sufficient D 2 receptor functional antagonism. Among several preclinical tests, Natesan et al. (2011) favored Fos expression in the NAc as a marker of overall functional antagonism and thus high predictive validity. 2-Bromoterguride increased Fos expression in the NAc, the dStr, and the mPFC as well. This means that 2-bromoterguride mimics haloperidol as an enhancer of Fos in the NAc and the dStr, and clozapine in the NAc and the mPFC. Positive symptoms of schizophrenia are related to an excess of dopamine in the NAc, whereas a lack of dopamine in the dorsolateral PFC is essential for negative symptoms and cognitive deficits (Abi-Dargham 2004). The increase of Fos in the NAc and the mPFC in rats by 2-bromoterguride suggests that this drug may possess the beneficial antipsychotic properties of both haloperidol and clozapine and thus may contribute to improve treatment outcome. Admittedly, this suggestion is rather speculative and further studies are needed to substantiate the findings of our study. Anyway, particularly the increase of Fos in the mPFC by 2-bromoterguride indicates that this drug may have the antipsychotic properties of an atypical APD with respect to actions on negative symptoms and cognitive deficits in schizophrenia (Robertson et al. 1994;Robertson and Fibiger 1996).
Wet dog shakes are behavioral effects in rodents which are amenable to inhibition by a large number of 5-HT receptor antagonists including APDs (Porsolt et al. 2010). The wet dog shake response is induced by selective activation of central 5-HT 2A receptors (Schreiber et al. 1995). 5-HT 2A receptor inverse agonist/antagonist activity is a characteristic feature of atypical APDs . The high in vitro affinity for 5-HT 2A receptors is in line with the effectiveness of 2bromoterguride to reduce DOI-induced wet dog shakes in rats. In contrast to the reduction of DOI-induced wet dog shakes by 2-bromoterguride (0.1 and 0.3 mg/kg), the decrease by aripiprazole (3 mg/kg) was not significant. It should be emphasized that aripiprazole attenuated DOI-induced wet dog shakes when high doses were used (10-30 mg/kg; Kohnomi et al. 2008), reflecting the low affinity of aripiprazole for 5-HT 2A receptors. Indeed, 5-HT 2A receptor affinity of aripiprazole was 50-fold lower than that of 2-bromoterguride (Jantschak et al. 2013). The difference between ketanserin and 2-bromoterguride as inhibitors of DOI-induced wet dog shakes needs a comprehensive consideration. Although both compounds exhibited similar 5-HT 2A receptor affinity, ketanserin reduced the DOI response to vehicle level, whereas 2-bromoterguride did not. A possible explanation for this discrepancy may be that ketanserin is a competitive 5-HT 2A receptor antagonist in contrast to 2-bromoterguride which behaves as an insurmountable antagonist (Jantschak et al. 2013). Moreover, terguride, the parent drug of 2-bromoterguride, was a partial agonist at 5-HT 2A receptors in a GTPγS binding assay (Newman-Tancredi et al. 2002). It cannot be excluded that 2-bromoterguride is acting as a partial agonist at 5-HT 2A receptors. Furthermore, 2-bromoterguride displayed subnanomolar affinity for α 2C -adrenoceptors (Jantschak et al. 2013). Interestingly, α 2 -adrenoceptor antagonists reversed the suppression of head-twitch behavior by 5-HT 2A receptor antagonists (Matsumoto et al. 1997). In addition, 2bromoterguride may modulate AMPA/kainate receptors. Indeed, blockade of AMPA/kainate receptors attenuated DOI-induced wet dog shakes (Gorzalka et al. 2005).
Clinically significant weight gain is a serious side effect of atypical APDs (Correll et al. 2011 for review;Parsons et al. 2009) and characterized by a substantial increase in food consumption and visceral and subcutaneous fat deposition (Zhang et al. 2004). A recent meta-analysis showed that haloperidol, ziprasidone, and lurasidone were the only APDs without affecting body weight in adults (Leucht et al. 2013). Since olanzapine induced more weight gain than any other APD in preclinical and clinical studies (Allison et al. 1999;Davey et al. 2012;Leucht et al. 2013), we used this drug as a positive control. Olanzapine-induced weight gain, food consumption as well as visceral and subcutaneous fat tissue weights in rats are consistent with observations of other groups (Choi et al. 2007;Davey et al. 2012;Mann et al. 2013). The mechanism by which olanzapine and other APDs induce weight gain is likely multifactorial and not completely understood. It has been suggested that 5-HT 2C and H 1 receptors, for which olanzapine and clozapine display high affinity, are strong candidate receptors for affecting appetite and thereby weight gain (Deng et al. 2010;Kirk et al. 2009;Kroeze et al. 2003). In addition, association of a polymorphism of the 5-HT 2C receptor gene with APD-induced weight gain highlights the role of this receptor in this metabolic side effect (Reynolds et al. 2002). However, mechanisms other than those mentioned above, e.g., D 2 receptor gene polymorphism, may also be associated with APD-induced weight gain (Müller et al. 2012).
In contrast to the effect of olanzapine, chronic treatment of rats with 2-bromoterguride failed to elicit an effect on body weight and body fat proportion. The low affinity of 2bromoterguride for H 1 receptors (pA 2 6.0; Jantschak et al. 2013) may be responsible for the non-occurrence of this side effect in the treatment with this drug. Unfortunately, there are no data available with regard to the affinity of 2bromoterguride for 5-HT 2C receptors. However, terguride, the parent drug of 2-bromoterguride, exhibited low affinity for this receptor (pK B 6.1; Newman-Tancredi et al. 2002). The absence of any effect of 2-bromoterguride on body weight, food consumption, and visceral and subcutaneous fat tissue weights suggests that 5-HT 2C receptors have no functional relevance in the side effect profile of this drug. Further, chronic treatment with 2-bromoterguride decreased locomotor activity but failed to induce catalepsy. This is consistent with the effect of acute 2-bromoterguride treatment (Jantschak et al. 2013) and other D 2 receptor partial agonists (Natesan et al. 2011;Nordquist et al. 2008;Svensson et al. 1991). The sedative effect of 2-bromoterguride is probably mediated by the dopaminergic system as the low affinity for the H 1 receptor argues against an involvement of this receptor in the inhibitory action on locomotor activity.
In conclusion, our in vivo studies show that 2bromoterguride is effective in tests for positive symptoms of schizophrenia. In addition, 5-HT 2A receptor blockade by 2bromoterguride has functional relevance in vivo and the induced Fos protein expression in the NAc and mPFC suggests an atypical character. Chronic treatment failed to alter metabolic parameters which are often associated with atypical APDs. 2-Bromoterguride, a drug with partial agonist effects at D 2 receptors and high affinity for 5-HT 2A and α 2C -adrenergic receptors, is a promising candidate for antipsychotic treatment. Further preclinical behavioral studies predicting antipsychotic effects are needed to verify 2-bromoterguride as an atypical APD, in particular experiments with relevance to cognitive impairment and negative symptoms present in schizophrenia.