Neurochemistry International 59 (2011) 925–935
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Cariprazine (RGH-188), a potent D3/D2 dopamine receptor partial agonist, binds to dopamine D3 receptors in vivo and shows antipsychotic-like and procognitive effects in rodents István Gyertyán ⇑, Béla Kiss, Katalin Sághy, Judit Laszy, Györgyi Szabó, Tamás Szabados, Larisza I. Gémesi, Gabriella Pásztor, Mária Zájer-Balázs, Margit Kapás, Éva Ágai Csongor, György Domány, Károly Tihanyi, Zsolt Szombathelyi }i u. 19-21, 1103 Budapest, Hungary Pharmacology and Drug Safety Research, Gedeon Richter Plc, Gyömro
a r t i c l e
i n f o
Article history: Received 11 March 2011 Received in revised form 24 June 2011 Accepted 1 July 2011 Available online 13 July 2011 Keywords: Cariprazine Atypical antipsychotic Dopamine D3 receptor D3/D2 partial agonist Schizophrenia Cognitive improvement
a b s t r a c t We investigated the in vivo effects of orally administered cariprazine (RGH-188; trans-N-{4-[2-[4-(2,3dichlorophenyl)-piperazin-1-yl]-ethyl]-cyclohexyl}-N0 ,N0 -dimethyl-urea), a D3/D2 dopamine receptor partial agonist with 10-fold preference for the D3 receptor. Oral bioavailability of cariprazine at a dose of 1 mg/kg in rats was 52% with peak plasma concentrations of 91 ng/mL. Cariprazine 10 mg/kg had good blood–brain barrier penetration, with a brain/plasma AUC ratio of 7.6:1. In rats, cariprazine showed dosedependent in vivo displacement of [3H](+)-PHNO, a dopamine D3 receptor-preferring radiotracer, in the D3 receptor-rich region of cerebellar lobules 9 and 10. Its potent inhibition of apomorphine-induced climbing in mice (ED50 = 0.27 mg/kg) was sustained for 8 h. Cariprazine blocked amphetamine-induced hyperactivity (ED50 = 0.12 mg/kg) and conditioned avoidance response (CAR) (ED50 = 0.84 mg/kg) in rats, and inhibited the locomotor-stimulating effects of the noncompetitive NMDA antagonists MK-801 (ED50 = 0.049 mg/kg) and phencyclidine (ED50 = 0.09 mg/kg) in mice and rats, respectively. It reduced novelty-induced motor activity of mice (ED50 = 0.11 mg/kg) and rats (ED50 = 0.18 mg/kg) with a maximal effect of 70% in both species. Cariprazine produced no catalepsy in rats at up to 100-fold dose of its CAR inhibitory ED50 value. Cariprazine 0.02–0.08 mg/kg significantly improved the learning performance of scopolamine-treated rats in a water-labyrinth learning paradigm. Though risperidone, olanzapine, and aripiprazole showed antipsychotic-like activity in many of these assays, they were less active against phencyclidine and more cataleptogenic than cariprazine, and had no significant effect in the learning task. The distinct in vivo profile of cariprazine may be due to its higher affinity and in vivo binding to D3 receptors versus currently marketed typical and atypical antipsychotics. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction During the 50-year history of antipsychotic medications, the dopamine (DA) D2 receptor has been the crucial and indispensable main target of drug action. Typical antipsychotics, the so-called neuroleptics, dominated the first 25 years of antipsychotic treatment but were succeeded by atypical antipsychotics. These agents
Abbreviations: CAR, conditioned avoidance response; DOI, (±)-2,5-Dimethoxy-4iodoamphetamine-HCl; HPLC, high performance liquid chromatography; LC, liquid chromatography; LLOQ, lower limit of quantification; MS, mass spectrometry; NMDA, N-methyl-D-aspartate; PCP, phencyclidine; UV, ultraviolet [detection]; SMA, spontaneous motor activity. ⇑ Corresponding author. Address: Department of Behavioral Pharmacology, Gedeon Richter Plc, P.O. Box 27, Budapest H-1475, Hungary. Tel.: +36 14314850; fax: +36 18898400. E-mail address:
[email protected] (I. Gyertyán). 0197-0186/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.neuint.2011.07.002
retain D2 receptor antagonism as the basic mode of action but have better side effect profiles and somewhat better efficacy on negative symptoms. Whether the superior side-effect profile of atypical antipsychotic agents is due to their serotonin 5-HT2A receptor antagonist activity (Meltzer et al., 2003) or to their higher dissociation rate constant (koff) at the D2 receptor (Kapur and Seeman, 2001) is still a matter of debate. A recently developed atypical antipsychotic agent, aripiprazole, while preserving the predominant D2 action, introduced a new pharmacological approach: D2 receptor partial agonism (Lawler et al., 1999). A compound with an appropriate degree of partial agonism can both inhibit overstimulated DA receptors to function as an antagonist, and stimulate these same receptors when the endogenous dopaminergic tone is low. This balancing feature presumably results in effective blockade of overstimulated D2 receptors and improvement in psychotic symptoms in schizophrenic
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patients. Concurrently, it potentially prevents the induction of extrapyramidal symptoms (EPS) or secondary negative symptoms by avoiding complete silencing of dopaminergic transmission (DeLeon et al., 2004). Interestingly, aripiprazole also has considerable binding affinity to the D3 receptor (Lawler et al., 1999) with variable and assay-dependent intrinsic activity (Bruins Slot et al., 2007). Originally, the high density of the D3 receptor in areas of the ventral striatum gave rise to the expectation that selective D3 antagonists would exert antipsychotic activity without causing motor side effects related to the dorsal striatum (Schwartz et al., 2000). Although highly selective D3 receptor antagonists such as SB-277011 and S33084 were found to be devoid of cataleptogenic actions, they demonstrated poor efficacy in animal models of schizophrenia (Millan et al., 2000; Reavill et al., 2000). However, preclinical studies of D3 receptor antagonists did demonstrate pharmacological properties that are potentially advantageous in the treatment of schizophrenia. D3 receptor antagonists demonstrated increased locomotor activity in rodents habituated to their environment (Gyertyan and Saghy, 2004; Millan et al., 2004), procognitive effects in experimental learning paradigms (Laszy et al., 2005; Millan et al., 2007), and inhibited haloperidol-induced catalepsy (Gyertyan and Saghy, 2007; Millan et al., 1997). These findings suggested that D3 receptor antagonists may have some potential against negative symptoms and reduced EPS potential. We hypothesized that a mixed D3/D2 receptor antagonist with high affinity and preference for the D3 receptor that retained considerable D2 affinity could be an effective antipsychotic that would be free of EPS and have beneficial effects on cognition and negative symptoms. Experimental confirmation of this hypothesis is demonstrated in putative antipsychotic compounds like S33138 (Millan et al., 2008a,b) and RG-15 (Kiss et al., 2008). Both drugs showed, in addition to standard antipsychotic-like activity attributed to D2 receptor antagonism, cognitive enhancing effects (Gyertyan et al., 2008; Millan et al., 2008a), while RG-15 also displayed anticataleptogenic and motor activating effects (Gyertyan et al., 2008). Our search for novel antipsychotics focused on the strategic goal of combining D2 receptor partial agonism and D3 receptor preference in one molecule. Our synthesis and screening efforts identified cariprazine (formerly RGH-188, Fig. 1), a compound with D3/D2 partial agonism and preference to the D3 receptor (Kiss et al., 2010). Based on theoretical considerations as well as experimental findings (Richtand et al., 2001; Schotte et al., 1992), the D3 receptor is assumed to be tonically occupied by endogenous DA. Therefore, cariprazine, as a D3 receptor partial agonist, presumably exerts antagonist-like actions in vivo by inhibiting the binding of the full agonist dopamine, and it behaves as a D2 receptor antagonist in the basal ganglia of schizophrenic patients where dopaminergic transmission is increased (Abi-Dargham et al., 2000). To elucidate the antipsychotic-like profile of cariprazine, the compound was evaluated in a series of animal behavioral models including
Fig. 1. Chemical structure of cariprazine (RGH-188) HCl.
antipsychotic, cognitive, and motor performance tests, and pharmacokinetic and in vivo D3 receptor binding assays. 2. Methods 2.1. Animals Experimental rats and mice (strains and weights given with specific method) were housed in a thermostatically controlled room at 22 ± 2 °C and 50 ± 10% relative humidity on 12-h light/dark cycle (lights off from 18.00 to 6.00 h). The animals were kept in polycarbonate cages (Lignifer Ltd., Isaszeg, Hungary) in groups of 5 (rats) or 10 (mice); they received unlimited access to commercial pellet rat-mouse feed (ssniff R/M + H produced by Spezialdiäten GmbH, Soest, Germany), autoclaved at 105 °C, and tap water throughout all experiments. Each animal was tested in only one experiment. All procedures on animals were approved by the local ethics committee and conformed to the rules and principles of the 86/ 609/EEC Directive. 2.2. Drugs Cariprazine HCl, aripiprazole HCl, and olanzapine were synthesized at Gedeon Richter; risperidone was purchased from RBI (Natick, MA, USA). The drugs were administered orally (p.o.) in a volume of 5 mL/kg in rats and 10 mL/kg in mice. The test compounds were suspended in 2–6% Tween-80, depending on the dose and requirement for sufficient suspension homogeneity. Solutions required for doses in the lg/kg range were usually diluted from a stock solution/suspension. Phencyclidine (PCP) was synthesized at Gedeon Richter; apomorphine HCl, D-amphetamine sulphate, scopolamine HBr, 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino) tetralin] and DOI [(±)-2,5-dimethoxy-4-iodoamphetamine-HCl] were purchased from Sigma Chemical Co. (St. Louis, MO, USA); MK-801 [(+)-5methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate] was purchased from RBI (Natick, MA, USA). D-amphetamine, MK-801, PCP, and scopolamine were dissolved in distilled water; DOI and 8-OH-DPAT were dissolved in saline; apomorphine was dissolved in 0.1% (w/v) ascorbic acid solution. [3H](+)-PHNO (4-propyl-9-hydroxynaphthoxazine, specific activity: 61.8 Ci/mmol) was custom synthesized from (+)(4aR,10bR)-4-allyl-3,4,4a,5,6,10b-hexahydro-2H-naphth[1,2-b]-1, 4-oxazin-9-ol hydrochloride by catalytic tritiation (Ubichem Research, Budapest, Hungary); for part of the experiments, [3H](+)PHNO (specific activity: 63 Ci/mM) was purchased from Moravek Biochemical Inc. (Brea, CA, USA). 2.3. In vivo pharmacokinetics of cariprazine in rats Pharmacokinetic investigations were performed in male Wistar rats weighing 170–200 g. Pharmacokinetic parameters and oral bioavailability were determined following single intravenous (i.v.) and p.o. administration of cariprazine 1 mg/kg (n = 5/group). The p.o. formulation was prepared in a water solution containing 0.1% acetic acid; the i.v. formulation additionally contained 5.5% glucose. Plasma samples obtained after 1 mg/kg treatments were analyzed for cariprazine by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) with a 0.5 ng/mL lower limit of quantification (LLOQ) using 100 lL of sample. Deuterated analyte, [2H6]-cariprazine, was used as the internal standard (IS). The compounds were isolated from the alkalized plasma using liquid–liquid extraction with 1-chlorobutane and the extracts were analyzed by reversed-phase high-performance liquid chromatography (HPLC) on an XTerra RP18 column (150 4.6 mm, 5 lm)
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with MS/MS detection. The chromatographic run time was 5.0 min per injection. The PE Sciex API 365 mass spectrometer was equipped with a TurboIonSprayÒ interface and operated in positive-ion, multiple-reaction monitoring (MRM) mode. The mass transitions monitored were m/z 427.3?382.2 and 433.3?382.2 for cariprazine and [2H6]-cariprazine, respectively. In a separate experiment, plasma and brain levels of cariprazine were measured after p.o. administration of a 10 mg/kg dose (n = 3/ time points) using 1% acetic acid water solution as vehicle. Samples were analyzed by HPLC-UV with an LLOQ of 20 ng/mL for plasma and 80 ng/g for brain. Plasma and brain samples were prepared for analysis using liquid–liquid extraction. The biological samples were extracted with n-hexane and analyzed on a Supelco Discovery C18 column (150 4.6 mm, 5 lm) using an isocratic elution. The mobile phase was acetonitrile-methanol-0.2 M aqueous ammonium acetate-water (35:25:35:5, v/v/v/v) at a flow rate of 1 mL/min. Pharmacokinetic parameters were calculated by noncompartmental analysis using the computer program Kinetica Version 4.0.1. 2.4. Determination of in vivo [3H](+)-PHNO binding We have recently shown that determination of binding of [3H](+)-PHNO, a dopamine D3 receptor-preferring radiotracer, in rat striatum (D2 rich area) and lobules 9 and 10 of cerebellum (D3 rich area) may be a useful technique to demonstrate in vivo D2 and D3 receptor binding affinity of compounds (Kiss et al., 2011). Male Hanover Wistar rats (190–210 g, n = 4–8 per group) were given vehicle or different doses of compounds (0.5 mL/ 100 g body weight) and then returned to their home cage. Thirty minutes later, rats were gently placed in a metal restraining box with easy access to the tail; through the caudal vein, they received 5 lCi [3H](+)-PHNO in 0.2 mL saline. Thirty minutes after the tracer injection, animals were sacrificed; their brains were removed and chilled in ice-cold saline and dissected by free hand on an ice-cold surface. Both striata were dissected. Cerebellum was detached, turned on the dorsal surface, and lobules 9 and 10 (CB L9,10) were dissected according to rat brain coordinates (Paxinos and Watson, 2007). The remaining cerebellum was divided into ‘‘left hemisphere,’’ ‘‘right hemisphere,’’ and ‘‘rest.’’ Tissue specimens were immediately frozen on dry-ice. After weighing, samples were placed in scintillation vials and dissolved in 1 mL 0.6 mL NaOH (Seeman, 2009) under continuous horizontal shaking. After dissolution, 5 mL Optiphase Hisafe3 (PerkinElmer) was added to the samples; following thorough mixing, samples were left standing overnight. Radioactivity was determined in a TriCarb 2900TR liquid scintillation counter (PerkinElmer). Percent radioactivity bound (Bound%) in the striatum and CB L9, 10 was calculated according to the following formula:
Boundð%Þ ¼
Drug treatedðDPM=mgÞ CBðDPM=mgÞ Vehicle treatedðDPM=mgÞ CBðDPM=mgÞ
100;
where CB⁄ represents the mean disintegrations per minute (DPM)/ mg of the cerebellum without CB L9,10 (i.e. DPM of cerebellum right + left hemispheres + rest) from vehicle-treated animals. Statistical comparison between control and treatment groups was made by Tukey–Kramer multiple comparison test. 2.5. Animal behavioral models 2.5.1. Apomorphine-induced climbing Fifty minutes after p.o. administration of vehicle or doses of the test compound, male CD1 mice (20–25 g) were placed into cylindrical cages (height, 15 cm; diameter,12 cm) with walls of vertical metal bars (diameter, 2 mm; 1 cm apart) for habituation. At the
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end of the 10-min adaptation period, 1.5 mg/kg apomorphine HCl was administered subcutaneously (s.c.) and the animals were returned to the cages. Measurement of climbing behavior started 10 min after apomorphine treatment and lasted for 16 min. Climbing behavior was scored per minute: 0 (4 paws on the floor), 1 (forefeet touching the bars), or 2 (4 paws grasping the bars). A maximum score of 32 could be assigned. Climbing score means were calculated in each treatment group (n = 12) and percent inhibition compared with control mean was determined for each dose. ED50 values were determined by linear regression. Additionally, the duration of action of cariprazine was measured. Mice were given cariprazine 0.4 mg/kg (p.o.) 1, 4, 8, 16, and 24 h before apomorphine injection; inhibition of apomorphine-induced climbing was then measured as described above. 2.5.2. Conditioned avoidance response (CAR) Male Wistar rats weighing (180–200 g at the beginning of conditioning were trained to perform 2-way active avoidance responses in a computer-controlled 6-channel shuttle box apparatus (Panlab S.L., Spain). Daily sessions consisted of 48 (cariprazine, olanzapine) or 30 cycles (risperidone, aripiprazole) with a 10-s intersignal interval, 10-s continuous light and sound (frequency: 1110 Hz, loudness: 62 dB on floor level) as conditioned stimulus, and 5-s light and sound stimulus paired with electric foot shock (shock intensity: 0.6 mA) as unconditioned stimulus. Drug treatments were administered to animals showing stable (at least 75%) avoidance performance for 3 days before treatment. Test compounds were administered 1 h before testing. Learning performance on the previous day served as the control level. Per dose group, 5–10 animals were used. Avoidance performance means were calculated in each treatment group for both treatment day and control day; percent inhibition compared with control mean was determined for each dose. ED50 values were determined by linear regression. 2.5.3. Novelty- and stimulant-induced motor activity Male Wistar rats (160–220 g) and male NMRI mice (20–26 g) were used. Novelty-induced motor activity was measured in a 4channel activity monitor consisting of 43 cm 43 cm 32 cm acrylic cages equipped with 2 16 pairs of infrared photo beams along all bottom axes of the cage. Animals were treated with test compounds or vehicle 60 min before being individually placed into the apparatus for 1 h. Motor activity was determined as the total number of beam interruptions during this period. Amphetamine-induced hypermotility measurements were carried out as described above except rats were injected (s.c.) with amphetamine (0.5 mg/kg, 1 mL/kg) immediately before being placed into the apparatus. PCP and MK-801-induced hypermotility measurements were performed in rats and mice, respectively. Thirty minutes after p.o. administration of the test compound or vehicle, animals were individually habituated in photocell cages for 30 min. After habituation, animals were treated with PCP (2 mg/kg s.c., 1 mL/kg) or intraperitoneal (i.p.) MK-801 (0.1 mg/ kg, 10 mL/kg), and placed back into the experimental apparatus for the 1-h measurement period. Doses of agonists were chosen to cause submaximal stimulation. Activity count means (±SEM) were calculated in each treatment group (n = 10 or 20 per group) and the percent inhibition of activity was calculated for each dose. ED50 values of the test compounds were determined from these percent values by linear regression. Statistical significance of drug effect was determined by analysis of variance (ANOVA) followed by a post hoc Tukey test. 2.5.4. Catalepsy Thirty minutes after treatment with test compounds, male Harlan-Wistar rats (170–270 g) were placed in an extraordinary position (upright posture with both forepaws on a 10-cm high
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podium). Rats were considered cataleptic if they did not correct their body posture within 30 s. The number of cataleptic animals was determined at 1, 2, and 3 h after treatment; additionally, for cariprazine and aripiprazole, catalepsy was also evaluated 4 and 5 h following administration. The minimum cataleptogenic dose was defined as the one producing at least a 20% occurrence of cataleptic animals (n = 10) at any of the readings. 2.5.5. Water-labyrinth learning performance The model is described in detail in (Laszy et al., 2005). Male Wistar rats (n = 10 per group; 180–200 g) had to maneuver through three choice points of a labyrinth system in a tank (dimensions: length, 1 m; width, 60 cm; depth, 60 cm) filled with 24 °C water to a depth of 30 cm to reach an escape platform. For 3 days, the animals were trained in three daily trials separated by intertrial rest periods of approximately 25 min. Thirty minutes before the start of the first daily trial, 3 mg/kg (2 mL/kg) i.p. scopolamine hydrobromide was injected as an amnestic agent. Test drugs were administered 1 h before the first swimming. Each study included a solvent control, a memory-impaired group, and 1 or 2 impaired groups treated with the test drug. The number of directional turning errors at each choice point was measured as a variable of learning performance. Statistical comparisons among groups were made by 3-way repeated measures ANOVA with group as the independent between-group factor, and days and trials as the repeated measures factors. Post hoc comparisons (Tukey test) were performed in case of a significant between-group effect or group interaction. 2.5.6. Lower lip retraction Male Wistar rats (240–300 g) were administered p.o. test compounds or vehicle. Lower lip retraction was scored 30, 60, 90, and 120 min after treatment according to the following scale: lower incisors of rat not visible (0), lower incisors partly visible (0.5), and lower incisors completely visible (1). Mean (SEM) of the individual maximal scores was calculated in each group (n = 8) and evaluated using the Kruskal–Wallis test followed by the Mann– Whitney U test. The minimal effective dose (MED) of the investigated compounds was determined. 2.5.7. DOI-induced head twitch Male NMRI mice (25–32 g) were administered p.o. test compounds or vehicle 60 min before i.p. administration of DOI (0.625 mg/kg). Immediately after DOI treatment, animals were individually placed into observation chambers; after a 5-min wait, the number of head twitch responses that occurred during the next 15 min was counted. The mean (SEM) number of head twitches was calculated for each group (n = 8–10). The percent inhibition of DOI-induced head twitch behavior by various doses of the compounds was then determined. ED50 values were determined by linear regression.
levels, with a brain to plasma AUC ratio of 7.6:1 (Fig. 2 inset). The high brain level of the compound was sustained for up to 3 h; it decreased to approximately 40% by 6 h posttreatment. 3.2. Determination of in vivo [3H](+)-PHNO binding Orally given cariprazine inhibited the binding of [3H](+)-PHNO in the D3 receptor-rich CB L9,10 region as well as in the D2 receptor-rich striatum with ED50 values of 0.42 and 0.22 mg/kg, respectively, and showed nearly complete blockade at 1 mg/kg (Fig. 3). Aripiprazole displayed a different profile than cariprazine. It was active in the striatum (ED50 = 7.65 mg/kg), reaching full inhibition at 30 mg/kg; however, in the CB L9,10 region, aripiprazole only achieved about 25% percent inhibition at the highest dose, 30 mg/kg.
3.3. Behavioral models for screening antipsychotic-like activity Cariprazine potently inhibited apomorphine-induced climbing in mice (ED50 = 0.27 mg/kg). The compound was more potent than aripiprazole and olanzapine, but somewhat less potent than risperidone (Fig. 4, Table 2). The climbing-inhibitory effect of cariprazine was sustained for at least 8 h (Fig. 4 inset). Cariprazine blocked amphetamine-induced hypermotility (ED50 = 0.12 mg/kg) with a potency similar to that of risperidone (Fig. 5, Table 2). Olanzapine and aripiprazole showed lower potency than cariprazine. Cariprazine dose-dependently inhibited CAR in the shuttle-box with potency similar to that of risperidone (Fig. 6, Table 2). Olanzapine and aripiprazole showed lower potency than cariprazine. Both cariprazine and the reference antipsychotic drugs inhibited locomotor-stimulating effects of the noncompetitive NMDA receptor antagonist MK-801 in mice in a dose-dependent manner. Cariprazine was more potent than aripiprazole and olanzapine, but less potent than risperidone (Fig. 7, Table 2). Cariprazine significantly and dose-dependently inhibited the locomotor-stimulating effect of the noncompetitive NMDA receptor antagonist PCP in rats in a dose range of 0.05–0.8 mg/kg p.o. (ED50 = 0.09 mg/kg) (Fig. 8). Risperidone, olanzapine, and aripiprazole did not cause significant inhibition of PCP-induced locomotion at doses that inhibited the amphetamine-induced hyperactivity or CAR. Moreover, olanzapine (1–4 mg/kg) and the
3. Results 3.1. In vivo pharmacokinetics of cariprazine in rats Following i.v. administration of cariprazine to rats, bi-exponential disposition was exhibited (Fig. 2). Systemic plasma clearance was moderate and the relatively high volume of distribution indicated extensive tissue distribution (Table 1). The apparent terminal half-life (t1/2) of cariprazine was approximately 2 h following i.v. or p.o. administration (Fig. 2, Table 1). Oral absorption of the drug was rapid, with Tmax values in the range of 0.5–1 h. The absolute oral bioavailability of cariprazine at the 1 mg/kg dose was 52% (Table 1). Brain concentrations of cariprazine were much higher than plasma
Fig. 2. Plasma concentrations of cariprazine in male rats following i.v. and p.o. administration of 1 mg/kg. Inset shows plasma and brain concentration following p.o. administration of 10 mg/kg (mean ± SD values are shown).
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I. Gyertyán et al. / Neurochemistry International 59 (2011) 925–935 Table 1 Pharmacokinetic parameters for cariprazine in male rats following i.v. and p.o. administration of 1 mg/kg dosesa.
a b
Route
CL(mL/min/kg)
Vd(L/kg)
T1/2(h)
Cmax(ng/mL)
Tmax(h)b
AUCtotal(ng/mL h)
Fabs(%)
i.v. p.o.
32 ± 4.6
6.5 ± 1.2
2.4 ± 0.7 2.2 ± 0.4
91 ± 16
0.5–1
533 ± 72 279 ± 48
52
Mean ± SD values are shown. Tmax is expressed as range of values.
Fig. 3. Blockade of in vivo [3H](+)-PHNO binding in rat striatum and cerebellum lobules 9,10 by cariprazine and aripiprazole. Compounds were administered via the indicated routes 30 min. before i.v. injection of [3H](+)-PHNO. The values are mean ± s.e.m. (n = 3–8). ⁄P < 0.05 versus vehicle.
tested (Fig. 9). The effect of cariprazine, however, reached a plateau at approximately 70% inhibition (as shown by the sigmoidal fit), whereas risperidone and olanzapine produced 90% inhibition at their highest doses. In mice, cariprazine also caused dose-dependent inhibition of novelty-induced motor activity, yielding an ED50 value of 0.11 mg/kg p.o. (Fig. 9). Similar to rats, the effect of cariprazine reached a plateau at approximately 70% inhibition as shown by the sigmoidal fit. Cariprazine did not induce catalepsy in rats at up to 100-fold dose of its CAR inhibitory ED50 value. Comparative antipsychotics produced catalepsy at doses 2- to 14-fold higher than their CAR inhibitory ED50 values with the following order of therapeutic margin: haloperidol < risperidone < aripiprazole < olanzapine (Tables 2 and 3). 3.5. Water-labyrinth learning performance
Fig. 4. Inhibition of apomorphine-induced climbing behavior by cariprazine and reference antipsychotic drugs in mice. Dose–response curves are shown. Inset shows the time course of the action of 0.4 mg/kg dose of cariprazine. Control apomorphine climbing scores varied between 25.8 ± 2.20 and 30.8 ± 0.59 (group means ± SEM) in the experiments.
lowest dose of risperidone (0.2 mg/kg) further increased the stimulatory effect of PCP.
The amnestic agent scopolamine disrupted the normal learning process of rats resulting in a significant increase in the number of errors. The scopolamine-induced learning deficit was significantly improved by cariprazine 0.02, 0.04, and 0.08 mg/kg (Table 4). In the dose range tested (0.01–0.3 mg/kg p.o.), cariprazine produced a bell-shaped dose–response pattern (Table 4). Aripiprazole (2, 4, and 5 mg/kg p.o.) showed mild, nonsignificant activity against scopolamine impairment at the 4 mg/kg dose only. Neither olanzapine (0.3, 1, and 3 mg/kg) nor risperidone (0.05, 0.1, and 0.5 mg/kg) attenuated the learning disturbance induced by scopolamine treatment (Table 4).
3.4. Effects on motor performance 3.6. In vivo serotonergic effects of cariprazine Similar to the reference antipsychotic compounds, cariprazine dose-dependently inhibited novelty-induced motor activity of rats, with the highest potency (ED50 = 0.18 mg/kg) among the drugs
Cariprazine caused statistically significant lower lip retraction at the 2 mg/kg dose (Table 5). Aripiprazole was also active in this
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Table 2 Summary of behavioral pharmacology results with cariprazine, risperidone, olanzapine and aripiprazole. Compound Cariprazine Risperidone Olanzapine Aripiprazole
Apo-climb 0.27 0.12 1.3 0.97
Amph-loco 0.12 0.15 1.8 3.9
CAR 0.84 0.91 2.9 18
MK-801 loco 0.049 0.0025 0.68 0.099
PCP-loco a
0.09 1.6 n.d. n.d.
LMA a
0.18 1.1 3.1 5.6
Catalepsy
Water labyrinth
Lower lip retraction
DOI Head-twitch
>85 6 40 160
0.02–0.08 n.d. n.d. n.d.
2 n.t. n.t. 40
0.27 0.0049 0.022 0.31
Oral ED50 values (in mg/kg) are given, except catalepsy and lower lip retraction where MED (minimally effective dose) values are given and water-labyrinth where the dose range (in mg/kg) with significant protection against scopolamine-induced learning deficit is given. Apo-climb: inhibition of climbing behavior induced by apomorphine (1.5 mg/kg s.c.) in mice. Amph-loco: inhibition of hyperlocomotion induced by d-amphetamine (0.5 mg/kg s.c.) in rats. CAR: inhibition of conditioned avoidance response in rats. MK-801 loco: inhibition of hyperlocomotion induced by MK-801 (0.1 mg/kg i.p.) in mice. PCP-loco: inhibition of hyperlocomotion induced by phencyclidine (2 mg/kg s.c.) in rats. LMA: inhibition of novelty-induced locomotor activity in rats. Water labyrinth: protection against scopolamine (3 mg/kg i.p.) induced learning deficit in water labyrinth in rats. Lower lip retraction: induction of lower lip retraction in rats. DOI head-twitch: inhibition of DOI (0.625 mg/kg i.p.) induced head-twitch in mice. a ED50 value determined by sigmoidal fit; n.d. – nondeterminable, n.t. – not tested.
test, with a minimum effective dose of 40 mg/kg. The 5-HT1A agonist 8-OH-DPAT, administered s.c. at a dose of 0.3 mg/kg as a positive comparator, produced maximal lower lip retraction. Cariprazine significantly and dose-dependently reduced the DOI-evoked head twitch behavior in mice, yielding an ED50 value of 0.27 mg/kg (Fig. 10, Table 2). Its effect was similar to aripiprazole (ED50 = 0.31 mg/kg) but was weaker than olanzapine (ED50 = 0.022 mg/kg) and risperidone (ED50 = 0.0048 mg/kg) by one and two orders of magnitude, respectively (Fig. 10). 4. Discussion Cariprazine is a novel antipsychotic candidate compound characterized by subnanomolar affinity for dopamine D3 receptors with about 10-fold selectivity over D2 receptors (Kiss et al., 2010). It showed partial agonist functional profiles at both receptors in cellular systems. In in vivo turnover and biosynthesis experiments, the compound demonstrated D2-related partial agonist properties. Cariprazine also displayed high affinity binding at human serotonin 5-HT2B receptors with pure antagonism. It had nanomolar affinity and partial agonism at 5-HT1A receptors and showed even lower affinity at 5-HT2A receptors (Kiss et al., 2010).
Fig. 5. Inhibition of amphetamine-induced hyperactivity by cariprazine and reference antipsychotic drugs in rats. Dose–response curves are shown. Control basal motor activity counts varied between 1934 ± 117.3 and 2103 ± 115.6, control amphetamine-induced hyperactivity counts ranged from 4145 ± 209.5 to 4402 ± 239.9 (group means ± SEM) in the experiments.
Results from the presented in vivo pharmacokinetic, receptor binding, and behavioral experiments in rats and mice demonstrated a unique and favorable antipsychotic-like profile for cariprazine. In rats, cariprazine was well and rapidly absorbed after oral administration. High absolute oral bioavailability indicates that cariprazine has a moderate first-pass effect. Good cerebral penetration, with about 8-fold higher cariprazine exposure achieved in the brain than in plasma, indicates that cariprazine can readily enter the central nervous system. Six hours after cariprazine administration, a considerable amount was still present in the brain. Accordingly, the inhibitory effect of cariprazine was sustained for up to 8 h in the apomorphine-induced climbing assay. High affinity dopamine D2/D3 antagonists such as [18F]fallypride (Kegeles et al. 2010) or [11C]FLB-457 (Halldin et al., 1995) are the most frequently used radiotracers in human imaging studies to demonstrate striatal and extrastriatal dopamine binding to D2/D3 receptors. The high affinity D3/D2 agonist radiotracer [3H](+)-PHNO and its [11C]-congener, which have preference for D3 receptors, have recently emerged as radiotracers with excellent properties for use in animal and human imaging studies (Finnema et al., 2010). Localization of D3 receptors in
Fig. 6. Inhibition of conditioned avoidance response (CAR) by cariprazine and reference antipsychotic drugs in rats. Dose–response curves are shown. Control learning performance (% avoidance responses, group means ± SEM) varied between 90.0 ± 2.28 and 97.9 ± 1.32 in the experiments. Inhibitory effects on escape responses are shown at the bottom part of the graph. The number of escape failures was zero in control conditions.
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Fig. 7. Inhibition of MK-801-induced hyperactivity by cariprazine and reference antipsychotic drugs in mice. Dose–response curves are shown. Control basal motor activity counts varied between 247 ± 85.0 and 577 ± 118.2; control MK-801induced hyperactivity counts ranged from 836 ± 129.1 to 1298 ± 120.3 (group means ± SEM) in the experiments.
rat CB L9,10 lobules has been previously demonstrated (Levant, 1998; Rabiner et al., 2009). We have recently shown that the D3 selective antagonist SB-277011 dose-dependently and completely counteracted the binding of [3H](+)-PHNO in rat CB L9,10 lobules without any blocking action in striatum (Kiss
Fig. 9. Inhibition of novelty-induced motor activity by cariprazine and reference antipsychotic drugs in rats and by cariprazine in mice. Dose–response curves are shown. Control motor activity counts varied between 1693 ± 117.8 and 1939 ± 126.2 (group means ± SEM) in rats; and it was 2881 ± 186.3 in mice.
et al., 2010); similar results were obtained by (McCormick et al., 2010) using autoradiography. In contrast, the D2 selective compound SV-156 showed the opposite effects (Kiss et al., 2010). These findings support the assumption that inhibition of [3H](+)PHNO binding in rat CB L9,10 is attributed to the in vivo affinity
Fig. 8. Inhibition of phencyclidine-induced hyperactivity by cariprazine and reference antipsychotic drugs in rats. Mean ± SEM values are shown. +, ++, and +++: P
