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Long-term effects of cariprazine exposure on dopamine receptor

subtypes

Yong Kee Choi, Nika Adham, Béla Kiss, István Gyertyán and Frank I. Tarazi

CNS Spectrums / Volume 19 / Issue 03 / June 2014, pp 268 - 277

DOI: 10.1017/S1092852913000680, Published online: 08 November 2013

Link to this article: http://journals.cambridge.org/abstract_S1092852913000680

How to cite this article:

Yong Kee Choi, Nika Adham, Béla Kiss, István Gyertyán and Frank I. Tarazi (2014). Long-term effects of cariprazine
exposure on dopamine receptor subtypes . CNS Spectrums, 19, pp 268-277 doi:10.1017/S1092852913000680

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CNS Spectrums (2014), 19, 268–277. & Cambridge University Press 2013
doi:10.1017/S1092852913000680

ORIGINAL RESEARCH

Long-term effects of cariprazine exposure on

dopamine receptor subtypes
Yong Kee Choi,1 Nika Adham,2 Béla Kiss,3 István Gyertyán,4 and
Frank I. Tarazi1*

1
Department of Psychiatry & Neuroscience, Harvard Medical School and McLean Hospital, Belmont, Massachusetts, USA
2
Department of Pharmacology, Forest Research Institute, Jersey City, New Jersey, USA
3
Department of Pharmacological and Safety Research, Gedeon Richter Plc, Budapest, Hungary
4
Department of Behavioral Pharmacology, Division of Pharmacology and Drug Safety Research, Gedeon Richter Plc, Budapest, Hungary

Introduction. All clinically effective antipsychotics are known to act on the dopaminergic system, and previous studies
have demonstrated that repeated treatment with antipsychotics produced region-specific changes in dopamine
receptor levels. Cariprazine is a dopamine D3 and D2 receptor partial agonist with preferential binding to
D3 receptors. We examined the effects of chronic cariprazine administration on dopamine receptor levels.
Methods. Rats were administered either vehicle or cariprazine (0.06, 0.2, or 0.6 mg/kg) for 28 days. Dopamine
receptor levels were quantitated using autoradiographic assays on brain tissue sections from the medial prefrontal
cortex (mPFC), nucleus accumbens (NAc), caudate putamen (CPu), hippocampus (HIPP), olfactory tubercle (OT),
and islands of Calleja (ICj).
Results. Chronic treatment with cariprazine did not alter D1 receptor levels in any brain region tested. Cariprazine
increased D2 receptor levels in mPFC (27%–43%), NAc (40%–45%), medial (41%–53%) and lateral (52%–63%) CPu,
and HIPP (38%). Cariprazine dose-dependently upregulated D3 receptor levels in ICj (32%–57%), OT (27%–67%),
and NAc shell (31%–48%). Repeated cariprazine treatment increased D4 receptor in NAc (53%–82%), medial
(54%–98%) and lateral (58%–74%) CPu, and HIPP (38%–98%).
Conclusion. Similar to other antipsychotics, cariprazine upregulated D2 and D4 receptor levels in various brain
regions. Cariprazine was unique among antipsychotics in increasing D3 receptor levels, which may support its unique
psychopharmacologic properties.

Received 25 July 2013; Accepted 29 August 2013; First published online 8 November 2013
Key words: Antipsychotic drugs, autoradiography, cariprazine, dopamine receptors, schizophrenia.

Cariprazine, a potent dopamine D3 and D2 receptor partial

Clinical Implications ’

agonist with preferential binding for the D3 receptor, was

’ Dysfunction of the dopamine system is thought play an
important role in the development and pathophysiology of
schizophrenia and bipolar mania.
’ All clinically effective antipsychotics are known to act on the
dopaminergic system and induce region-specific changes in
dopamine receptor levels.
*Address for correspondence: Frank I. Tarazi, Harvard Medical
School, McLean Hospital, 115 Mill Street, Belmont, MA 02478, USA.
(Email: ftarazi@hms.harvard.edu)
This study was funded by Forest Laboratories, Inc. and Gedeon
Richter Plc. The authors would like to thank Vojislav Pejovic, PhD, and
Adam Ruth, PhD, of Prescott Medical Communications Group for their
technical writing, editing, and research support.
evaluated for its long-term effects on dopamine receptor
levels in rat forebrain regions.
’ Similar to other atypical antipsychotics, cariprazine
upregulated dopamine D2 and D4 receptor levels, but unlike
other atypical antipsychotics, cariprazine also upregulated
dopamine D3 receptor levels.
’ Cariprazine showed unique effects on dopamine receptor
levels that may provide improved clinical benefits in the
treatment of schizophrenia and bipolar mania.
Introduction
Dysregulation of dopaminergic neurotransmission plays
a central role in the etiology and pathophysiology of

both schizophrenia and bipolar mania.1,2 There are
several dopamine (DA) receptor subtypes (D1–D5) that
are differentially expressed in various brain regions, and
current evidence suggests that each subtype may
modulate various physiological functions, including
cognitive and executive functions, locomotor activity,
mood, and reward behaviors.2,3 Although blockade of
D2 receptors is a common property of clinically effective
typical and atypical antipsychotics, most antipsychotics,
and especially the atypical agents, also show various
levels of affinity for other receptors, both dopaminergic
and non-dopaminergic. The receptor signature of each
antipsychotic drug contributes to its efficacy, safety, and
tolerability.4,5
The pharmacological, molecular, and behavioral pro-
files of atypical antipsychotic drugs have been extensively
studied.6–8 Prolonged treatment with atypical antipsycho-
tics produces region-specific and dose-dependent changes
in the levels of various DA receptor subtypes,9,10 as well
as other neurotransmitter receptors, including serotonin
and glutamate.11–15 Such changes may trigger a cascade of
intracellular events that ultimately lead to the therapeutic
effects or adverse events associated with antipsychotic
drug treatment.16
Cariprazine is an orally active and potent dopamine D3
and D2 receptor partial agonist, with preferential binding
for the D3 receptor17; it is currently in development
for the treatment of schizophrenia, bipolar mania, and
bipolar depression, and as adjunctive treatment to
antidepressant drugs for major depressive disorders.
Clinical trials showed that in patients with acute
schizophrenia, cariprazine treatment (1.5–4.5 mg/day)
was associated with significantly greater improvement
compared with placebo on the Positive and Negative
Syndrome Scale (PANSS) total score.18,19 In bipolar mania
trials, significant improvement over placebo on the Young
Mania Rating Scale (YMRS) total score was observed for
cariprazine 3–12 mg/day.20–22 In addition, the drug had a
favorable safety profile, with minimal metabolic, hormo-
nal, and cardiovascular adverse events.19,20,23
The pharmacological profile of cariprazine appears to
be distinct from other atypical antipsychotic agents. It
shows higher in vitro affinity at D3 receptors and greater
selectivity for D3 versus D2 receptors compared with other
atypical antipsychotics. Cariprazine shows almost 10-fold
higher in vitro affinity for the D3 versus D2 receptor.
In vivo, it exhibits balanced occupancy of the D3 and
D2 receptors at antipsychotic-like effective doses24,25;
conversely, other atypical antipsychotics display high D2
occupancy and relatively low occupancy at D3 receptors
in vivo.25 Additionally, cariprazine is metabolized by
CYP3A4 (and to a lesser extent by CYP2D6) into
2 major metabolites, dimethyl cariprazine and dides-
methyl cariprazine,26 which show similar receptor
affinity profiles and pharmacological activity as the
EFFECTS OF CARIPRAZINE ON DOPAMINE RECEPTORS 269
parent drug (data on file). Accumulating evidence suggests
that the D3 receptor is involved in regulating cognitive
functions and mood behaviors,2,27–29 and cariprazine’s
balanced engagement of both the D3 and D2 receptor
pathways may potentially translate into improved clinical
benefits that are typically not observed with other
antipsychotics, including improvements in executive and
cognitive deficits, negative symptoms, and mood distur-
bances in patients with psychiatric disorders.30
The long-term effects of cariprazine on DA receptor
subtypes in the mammalian forebrain remains to be
evaluated. In this study, we applied quantitative in vitro
receptor autoradiography to assess the long-term effects of
cariprazine on D1, D2, D3, and D4 receptors in the rat
brain and compared the findings with previously reported
effects of representative typical (fluphenazine) and atypi-
cal (olanzapine, risperidone, and asenapine) antipsychotic
drugs.10 Our working hypothesis was that cariprazine
would induce regionally selective changes in specific DA
receptor levels that would more closely resemble the
profile of atypical than typical antipsychotics.
Methods
Materials
The radiochemicals [3H]R(1)-7-chloro-8-hydroxy-3-methyl-
1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH-
23390, 81 Ci/mmol) and R,S(±)-[N-methyl-3H]nemona-
pride (86 Ci/mmol) were obtained from New England
Nuclear–PerkinElmer Inc. (Boston, MA, USA). Tritium
autoradiography standards and [2,3-3H]R(1)-7-hydroxy-
N,N-di-n-propyl-2-amino-1,2,3,4-tetrahydronaphthalene
(7-OH-DPAT; 116 Ci/mmol) were obtained from Amer-
sham (Arlington Heights, IL, USA). [3H]-(1)-4-propyl-
9-hydroxynaphthoxazine (PHNO; 64 Ci/mmol) was
obtained from Moravek Biochemicals (Brea, CA, USA).
Kodak Biomax MR film was purchased from Thermo
Fisher Scientific (Waltham, MA, USA). D-19 photo-
graphic developer and fixative were obtained from
Eastman-Kodak (Rochester, NY, USA).
Cariprazine was donated by Forest Laboratories Inc.
(New York, NY, USA). 1,3-Ditolylguanidine (DTG),
cis-flupenthixol-di-HCl, cis-eticlopride-HCl, ketanserin
tartrate, pindolol, and S(–)-sulpiride were obtained from
Sigma–Research Biochemicals International (Natick, MA,
USA). Cation hydrochlorides, guanosine-5'-triphosphate
sodium (GTP), and tris-(hydroxymethyl)-aminomethane-
HCl (Tris) were obtained from Sigma Chemicals (St.
Louis, MO, USA).
Animals
Male Sprague-Dawley rats (Charles River Laboratories,
Wilmington, MA, USA), initially weighing 200–225 g,
270 Y. K. CHOI ET AL.
TABLE 1. Receptor binding assay experiment conditions
Receptor binding assay Preincubation buffer
D1 50 mM Tris-HCl buffer, 120 mM NaCl, 5 mM
KCl, 2 mM CaCl2, 1 mM MgCl2
D2
D4
D3 50 mM Tris-HCl buffer, 40 mM NaCl, 0.3 mM
GTP, 5 mM KCl, 2 mM CaCl2
were maintained under controlled artificial daylight
(7:00 AM–7:00 PM), temperature, and humidity, with
free access to standard food and tap water in a U.S.
Department of Agriculture (USDA)-inspected, veteri-
narian-supervised, small-animal research facility of the
Mailman Research Center, with approval by the Institu-
tional Animal Care and Use Committee (IACUC) of
McLean Hospital.
Treatment and tissue preparation
For 28 days, 4 groups of rats (8–10 animals per group)
received a daily intraperitoneal (IP) injection of vehicle
control (saline, 1 ml/kg) or active treatment (caripra-
zine at doses of 0.06, 0.20, or 0.60 mg/kg). The selected
doses were active in different behavioral paradigms.24
No gross effects on motor behaviors or significant
changes in body weight were observed after repeated
treatment with the different doses of cariprazine
compared with vehicle-treated animals.
At the end of the treatment period, rats were
decapitated, and their brains were quickly removed,
frozen in chilled isopentane, and stored at –80 8C. Brain
regions assessed included the medial prefrontal cerebral
cortex (mPFC), dorsolateral frontal cortex (DFC)
(3.2–4.2 mm anterior to bregma), nucleus accumbens
(NAc), islands of Calleja (ICj) and olfactory tubercle (OT)
(1.7–2.2 mm anterior to bregma), caudate putamen (CPu)
(0.7–1.2 mm anterior to bregma), hippocampus (HIPP),
and entorhinal cortex (0.2–0.7 mm anterior to bregma).31
The selected extrapyramidal, limbic, and cortical regions
of interest mediate the cognitive, emotional, and motor
behaviors that are typically disturbed in patients with
schizophrenia and other psychotic disorders.1
Dopamine receptor autoradiography and image analysis
Coronal sections (10 mm) of these samples were cut in a
cryostat at –20 8C, mounted on gelatin-coated microscope
slides, and stored at –80 8C. To minimize experimental
variability, brain sections from all vehicle- and drug-treated
Radioligand Nonspecific binding
1 nM [3H]SCH-23390 with 100 nM ketanserin to block 5-HT2A/2C 1 mM cis-flupenthixol
receptors
1 nM [3H]nemonapride, 0.5 mM DTG and 0.1 mM pindolol to 10 mM S(-)sulpiride
mask sigma (s1,2) and 5-HT1A receptors
D2 assay buffer for 1 hour, then with 1 nM [3H]nemonapride,
300 nM raclopride (to occupy D2/D3 sites) and other masking
agents (0.5 mM DTG and 0.1 mM pindolol)
3 nM [3H]-7-OH-DPAT with 5 mM DTG to mask sigma sites 1 mM S(-)-eticlopride
2 nM [3H]PHNO 1 mM S(-)-eticlopride
animals used for a given receptor subtype assay were
evaluated at the same time. We have previously shown that
this preincubation step is effective in minimizing the
effects of endogenous ligands and potential interference of
residual drugs.32
Details of DA receptor autoradiography10,33 and image
analysis procedures10,11,15 were published previously.
Briefly, for all binding assays, brain sections were air-
thawed at room temperature (RT) and incubated for
60 min in a washing Tris-HCl buffer (RT, pH 7.4)
(Table 1), in order to remove endogenous DA and
residual drugs that may interfere with receptor binding.
Given the high affinity of cariprazine for D3 receptors,
assays for this receptor subtype were conducted using
2 different radioligands (Table 1). Following preincubation
in the washing buffer, slides were incubated in buffer
at RT for 1 hour with DA receptor subtype-specific
radioligands and agents that block their binding to other
known receptor types, in the presence of a competitive
agent used to determine nonspecific binding (Table 1).
Slides were washed twice for 5 minutes (D1, D2, D4
receptors) or 3 minutes (D3 receptors) in ice-cold
washing buffer, dipped in ice-cold water, and left to
dry at RT. Finally, radiolabeled slides and calibrated
[3H] standards were exposed to Hyperfilm for 4 weeks
(D1, D2, D4) or 6 weeks (D3) at 4 8C.
Biomax MR films were developed and fixed in
Kodak D-19 for 5 min at RT; images were quantified
using MCID image analyzer and MCID-M4 software
Imaging Research (St. Catharines, Ontario, Canada). A
calibration curve was generated from [3H]-microscale
standards that were exposed along with tissue sections.
Regions of interest were outlined, and their optical
density was measured with the left and right sides of
2 contiguous sections representing total binding and
matching samples representing nonspecific binding.
Four determinations were averaged for each subject-
sample. Optical density of sampled regions was mea-
sured and the amount of ligand bound was calculated as
nCi/mg tissue. Mean values of nonspecific binding in
each region were subtracted from the corresponding
 EFFECTS OF CARIPRAZINE ON DOPAMINE RECEPTORS 271

mean total binding to determine specific radioligand
binding expressed as fmol bound/mg tissue.
Statistical analysis
DA receptor binding data were analyzed first for overall
effects of drug versus vehicle for all receptor types
and brain regions using multiple regression modeling
methods. Density measures were logarithmically trans-
formed to achieve more Gaussian-like distributions
before regression modeling. Since individual brain
specimens provided receptor-binding data for several
brain regions, resulting in incomplete independence
across observations, we used robust standard error
estimates to adjust for this clustering effect. This
estimation method permits relaxation of the assumption
of independence of all observations and requires only that
the observations be independent across specimens.34
Estimates of interaction effects were employed for post
hoc tests of drug effects for specific receptors and brain
regions, with adjustment of P values obtained from the
regression analyses estimating these multiple comparisons
by the standard method of Sidák.35 Data are presented as
means ± standard error of the mean (SEM). Comparisons
were considered significant at P ,.05 (2-tailed test).
Results
Dopamine receptor distribution
Samples obtained from vehicle-treated animals indicate
that baseline levels of D1 and D2 receptors were the
highest in the medial and lateral CPu, and NAc (Figures 1
and 2), whereas brain regions with the highest levels
of D3 receptor binding were the OT, ICj, and the shell
of the NAc (Figure 3). D4 receptors accounted for
50%–60% of total D2 receptors in MPC and DFC, and
23%–28% of total D2 receptors in CPu and NAc (Figures 2

FIGURE 1. Effects of chronic exposure (28 days) of cariprazine on D1 receptors. Data are mean ± SEM and expressed in fmol/mg tissue (N 5 8/group).
CP-M 5 medial caudate putamen; CP-L 5 lateral caudate putamen; DFC 5 dorsolateral frontal cortex; EC 5 entorhinal cortex; HIPP 5 hippocampus;
MPC 5 medial prefrontal cortex; NAc 5 nucleus accumbens.

FIGURE 2. Effects of chronic exposure (28 days) of cariprazine on D2 receptors. Data are mean ± SEM and expressed in fmol/mg tissue (N 5 8/group).
*P ,.05 versus control. CP-M 5 medial caudate putamen; CP-L 5 lateral caudate putamen; DFC 5 dorsolateral frontal cortex; EC 5 entorhinal cortex;
HIPP 5 hippocampus; MPC 5 medial prefrontal cortex; NAc 5 nucleus accumbens.
272 Y. K. CHOI ET AL.

FIGURE 3. Effects of chronic exposure (28 days) of cariprazine on D3 receptors. Data are mean ± SEM and expressed in fmol/mg tissue (N 5 8/group).
*P ,.05 versus control. CP-M 5 medial caudate putamen; CP-L 5 lateral caudate putamen; DFC 5 dorsolateral frontal cortex; EC 5 entorhinal cortex;
HIPP 5 hippocampus; MPC 5 medial prefrontal cortex; NAc 5 nucleus accumbens. (A) [3H]7-OH-DPAT binding; (B) [3H]PHNO binding.

and 4), which is in agreement with the reported distribu-
tion of D4 receptors in different rat forebrain regions.33
D1 receptor binding
Repeated treatment (28 days) with different doses of
cariprazine did not induce any significant changes in
specific [3H]SCH-23390 binding to D1 receptors in all
brain regions examined, relative to vehicle-treated animals
(Figure 1).
D2 receptor binding
Repeated treatment with higher doses of cariprazine
(0.2 and 0.6 mg/kg) significantly and dose-dependently
increased D2 receptor binding in MPC (27% and 43%,
respectively), NAc (40% and 45%, respectively), medial
CPu (41% and 53%, respectively), and lateral CPu (52%
and 63%, respectively) over the values observed in
controls (Figure 2). In addition, the highest dose of
cariprazine (0.6 mg/kg) significantly increased D2
receptor binding in hippocampus by 38% (Figure 2).
The lowest dose of cariprazine (0.06 mg/kg) had no
significant effect on D2 binding in any brain regions
examined, although an increase in D2 binding was
observed in MPC, NAc, and medial and lateral CPu that
did not reach statistical significance (Figure 2).
D3 receptor binding
Two radioligands were used to quantify the long-term
effects of cariprazine on D3 receptors in rat forebrain
regions: [3H]7-OH-DPAT and [3H]PHNO. Quantification
of D3 receptors using [3H]7-OH-DPAT revealed that the
3 doses of cariprazine significantly and dose-dependently
increased D3 receptor binding in OT (27%, 49%, and
67%, respectively) and ICj (32%, 41%, and 57%,
respectively), compared with the vehicle. In addition,
the higher doses of cariprazine (0.2 and 0.6 mg/kg)
increased labeling of [3H]7-OH-DPAT in the NAc shell by
31% and 48%, respectively (Figure 3A).
Binding assays with [3H]PHNO revealed additional
changes in D3 receptor levels, especially in the NAc.
Compared with vehicle-treated animals, all 3 cariprazine

FIGURE 4. Effects of chronic exposure (28 days) of cariprazine on D4 receptors. Data are mean ± SEM and expressed in fmol/mg tissue (N 5 8/group).
*P ,.05 versus control. CP-M 5 medial caudate putamen; CP-L 5 lateral caudate putamen; DFC 5 dorsolateral frontal cortex; EC 5 entorhinal cortex;
HIPP 5 hippocampus; MPC 5 medial prefrontal cortex; NAc 5 nucleus accumbens.
doses significantly and dose-dependently increased
D3 receptor binding in the shell of NAc (29%, 50%,
and 72%, respectively); the highest dose was shown to
significantly increase D3 receptor levels in the core of
NAc by 34% (Figure 3B)—an effect that was not observed
using [3H]7-OH-DPAT (Figure 3A). The corresponding
increases in D3 receptor binding in OT (43%, 62%, and
74%) and ICj (43%, 61%, and 76%) were also dose-
dependent and significantly greater than vehicle, and
the effect was greater than that observed using [3H]7-
OH-DPAT (Figure 3B). There were no changes in D3
receptor levels in lobules 9 and 10 of the cerebellum
after repeated treatment with the 3 doses of cariprazine,
regardless of the labeling agent (Figures 3A and 3B).
D4 receptor binding
Repeated treatment with the 3 doses of cariprazine
dose-dependently increased selective [3H]nemonapride
binding to D4 receptors in hippocampus (by 38%, 71%,
and 98%, respectively; P ,.05) (Table 2). In addition,
the higher doses of cariprazine (0.2 and 0.6 mg/kg)
induced significant increases in D4 receptor levels in the
NAc (53% and 82%, respectively), medial CPu (54% and
98%, respectively), and lateral CPu (58% and 74%,
respectively) (Figure 4). In spite of the relatively high
D4 receptor levels in MPC and DFC, repeated treatment
with the 3 doses of cariprazine did not alter their
abundance in those regions (Figure 4).
Discussion
Effects of cariprazine on D1 receptors
Repeated treatment with cariprazine did not alter
the levels of D1 receptors in any forebrain regions
examined. Cariprazine has negligible affinity for
D1 receptors,17 and it is possible that cariprazine did
EFFECTS OF CARIPRAZINE ON DOPAMINE RECEPTORS 273
not occupy the D1 receptors to the extent that is
required to induce receptor upregulation. These find-
ings suggest that D1 receptors are less likely to
contribute to the therapeutic benefits of cariprazine in
the treatment of schizophrenia or bipolar mania.
Lack of adaptive changes in D1 receptors with
cariprazine is in agreement with the effects of other
typical and atypical antipsychotics, including haloperidol,
fluphenzaine, clozapine, olanzapine, and risperidone,
which also failed to alter the abundance of D1 receptors
in rat forebrain regions (Table 2).10,36,37 Among the newer
antipsychotics, only asenapine significantly increased
D1 receptor binding in the NAc and CPu, as a result of
direct receptor blockade.9 However, clinical trials with
selective D1 receptor antagonists in schizophrenia have
yielded no support for the hypothesis that D1 receptor
blockade exerts antipsychotic activity,38 which further
supports the view that D1 receptors are not major
mediators of antipsychotic activity. Nevertheless, the
pharmacological potential of D1 receptor antagonists in
the treatment of schizophrenia and bipolar mania needs
to be further investigated, since understanding of the
physiology of the most abundant cerebral DA receptors
remains remarkably limited.3
Effects of cariprazine on D2 receptors
Long-term administration of higher doses of cariprazine
(0.2 and 0.6 mg/kg) increased D2 receptor binding in
mPFC (Figure 2); the high affinity of cariprazine for rat
D2 receptors (pKi 5 8.0)17 suggests that cariprazine-induced
increases in cortical D2 receptors may result from direct
receptor blockade. This view is consistent with previous
results that have demonstrated D2 receptor upregulation
in the mPFC after repeated treatment with typical and
atypical antipsychotics, including fluphenazine, clozapine,
olanzapine, risperidone, and asenapine (Table 2).9,10,37
274 Y. K. CHOI ET AL.

TABLE 2. Comparison with typical and atypical antipsychotics

Cariprazine
Olanzapine1 Risperidone1 Asenapine2 Fluphenazine3
Brain region 0.06 mg/kg 0.2 mg/kg 0.6 mg/kg 5 mg/kg 3 mg/kg 0.3 mg/kg 1 mg/kg

D1 receptor binding
Cerebral cortex
Medial prefrontal – – – – – 26%* –
Caudate putamen
Medial – – – – – 54%* –
Lateral – – – – – 55%* –
D2 receptor binding
Cerebral cortex
Medial prefrontal – 27%* 43%* 67%* 34%* 55%* 46%*
Nucleus accumbens – 40%* 45% * 37%* 28%* 32%* 67%*
Caudate putamen
Medial – 41%* 53%* 44%* 27%* 41%* 26%*
Lateral – 52%* 63%* 39%* 23%* 40%* 17%*
Hippocampus – – 38%* 53%* 30%* 63%* –
D3 receptor bindinga
Islands of Calleja 32%* 41%* 57%* – – – –
Olfactory tubercle 27%* 49%* 67%* – – – –
Nucleus accumbens
Shell – 31%* 48%* – – – –
D4 receptor binding
Nucleus accumbens – 53%* 82%* 65%* 33%* 71%* 124%*
Caudate putamen
Medial – 54%* 98%* 60%* 36%* 81%* 60%*
Lateral – 58%* 74%* 62%* 37%* 60%* 63%*
Hippocampus 38%* 71%* 98%* 61%* 37%* 77%* ND

a
Data come from [3 H]7-OH-DPAT binding experiments. 1 Reference 10, 2 Reference 9, 3 Reference 37.
*P ,.05 versus respective control, –: not significantly different from respective control.
ND indicates not determined.

Our findings further support the importance of
D2 receptors in the mPFC as a common mediator of the
antipsychotic effects of typical and atypical antipsychotics.
Prolonged treatment with the higher dose of
cariprazine also increased D2 receptor binding in the
hippocampus (Figure 2). This has been previously
observed with atypical, but not typical, antipsycho-
tics,9,10,37 and may explain some of the differences in the
mode of action between the 2 classes of antipsychotic
drugs. For example, increases in hippocampal D2 receptors
induced by cariprazine and other atypical antipsychotics
may alter hippocampal-induced firing of DA neurons in
ventral tegmental area39 and subsequently reduce DA
hyperactivity, which could result in improvements in
psychotic symptoms and disturbed emotional behaviors
in patients with schizophrenia.40
Higher doses of cariprazine (0.06 and 0.2 mg/kg) also
increased D2 receptor binding in medial and lateral CPu
and NAc (Figure 2). Upregulation of striatal D2 receptors
can contribute to the development of extrapyramidal side
effects (EPS) via alteration of neurotransmission in the
basal ganglia-thalamocortical circuitry.41 Other atypical
antipsychotic drugs (eg, olanzapine, risperidone, and
asenapine) also induce increases in striatal D2 receptors
in a dose-dependent manner.9,10 Such increases in striatal
D2 receptors may account for the higher incidence of
akathisia (9% vs 5%) and extrapyramidal disorder (12% vs
5%) in cariprazine-treated, compared with placebo-treated
patients, in clinical trials.19,20 Imaging studies using
selective D2 receptor radioligands are still needed to assess
the long-term effects of cariprazine on concentrations of
striatal D2 receptors in patients with schizophrenia.
Interestingly, at the doses tested in this study,
cariprazine did not cause catalepsy in rats—an animal
model predictive of potential clinical EPS liability.24
In fact, the cataleptic effects of cariprazine occur at
doses 100-fold greater than doses that are effective in
attenuating conditioned avoidance response—an animal
model predictive of antipsychotic activity.24 It is
possible that the D2 receptor partial agonist activity of
cariprazine is more potent in rats than in humans and
exerted more protective effects against D2 receptor
antagonism induced-catalepsy.42 In addition, the 5-HT1A
receptor partial agonist activity of cariprazine may have
further contributed to the absence of catalepsy in rats at
doses that displayed antipsychotic activity.42,43

Effects of cariprazine on D3 receptors
Cariprazine displays potent affinity for D3 receptors, as
determined by in vitro assays using cells expressing
either recombinant human (pKi 5 10.1) or rat (pKi 5
9.2) D3 receptors.17 In this study, repeated administra-
tion of cariprazine resulted in a dose-dependent
increase in D3 receptor-specific binding in OT, ICj,
and the shell of NAc, using both [3H]7-OH-DPAT
(Figure 3A) and [3H]PHNO (Figure 3B). Assays using
[3H]PHNO demonstrated a stronger binding signal
overall relative to [3H]7-OH-DPAT and additionally
showed significant increase in D3 receptor levels in
the NAc core (Figure 3B). The differences in regional
effects and the magnitude of labeling between the two
D3 receptor preferring radioligands may reflect binding
affinity or pharmacokinetic differences between the
two radioligands.
Cariprazine-induced changes in D3 receptors in fore-
brain regions, where D3 receptors are highly expressed,
appear to be unique to this novel antipsychotic candidate.
This effect was not observed with other antipsychotics,
typical or atypical (including haloperidol, flupehenazine,
clozapine, olanzapine, risperidone, and asenapine), in any
of the brain regions examined (Table 2).9,10,36,37 It has
been proposed that high-affinity of endogenous DA at D3
receptors may limit the ability of other antipsychotic
agents to occupy D3 receptors to the level required to
trigger receptor upregulation.44 Other antipsychotics
tested, including aripiprazole, clozapine, haloperidol,
olanzapine, quetiapine, risperidone, and ziprasidone,
showed relatively low in vitro affinity at rat dopamine
D3 receptors (Ki 5 9–329 nM) and corresponding low
occupancy of D3 receptors in vivo (, 60% maximum
occupancy). In contrast, cariprazine showed higher D3
affinity (Ki , 4.0 nM) in vitro, and following acute
administration, showed very high D3 receptor occupancy
(. 99% maximum occupancy) in vivo.25 With its potent
D3 receptor affinity and considerable D3 receptor
occupancy,24,45 cariprazine is able to sufficiently occupy
D3 receptors even at low doses to induce significant D3
receptor upregulation independent of the radioligand
used to label D3 receptors. This effect may be mediated
through actions of brain-derived neurotrophic factor
(BDNF), a neurotrophic factor that has been shown to
control the development and expression of D3 recep-
tors.46 However, further studies are needed to examine
the potential role of BDNF in cariprazine-induced
upregulation of D3 receptors. The absence of D3 receptors
changes in the cerebellum may reflect differential regional
responses to repeated cariprazine treatment.
Dopamine receptor upregulation is typically observed
with DA receptor antagonists; however, despite its
D3 receptor partial agonist activity in vitro, cariprazine
increased the levels of D3 receptors, which may suggest
EFFECTS OF CARIPRAZINE ON DOPAMINE RECEPTORS 275
it is acting as an antagonist at D3 receptors in
vivo. Cariprazine-induced changes at D3 receptors may
normalize disturbances in D3 receptor-mediated neuro-
transmission in patients with schizophrenia and bipolar
mania, and in particular may improve mood, cognitive,
and executive functions, which are typically disturbed
in these disorders.1,30,47,48 Accumulating evidence
suggests that the D3 receptor plays a role in the
regulation of mood behaviors and cognitive func-
tions.2,28 Two D3 receptor-preferring antagonists,
S33138 and RG-15, have demonstrated cognition-enhan-
cing effects,49,50 and lack of functional D3 receptors has
been shown to have procognitive-,51 antidepressant-,50
and anxiolytic-like51 effects in animal models. Taken
together, these data suggest that cariprazine, via its
unique actions at D3 receptors, may provide therapeutic
benefits in the treatment of mood symptoms and
cognitive deficits in patients with schizophrenia and
bipolar disorder.
Effects of cariprazine on D4 receptors
Repeated administration of cariprazine (0.2 and 0.6 mg/
kg) significantly increased D4 receptor binding in the
NAc and CPu. This is in agreement with other studies
that reported increased D4 receptor binding in the
NAc and CPu following treatment with antipsychotics
(Table 2).9,10,32,36,37 Antipsychotic-induced upregula-
tion of striatolimbic D4 receptors may be a common
mechanism that mediates the actions of both typical and
atypical antipsychotics, including cariprazine. Despite
the failure of selective D4-receptor agents to improve
psychotic symptoms in patients with schizophrenia,52,53 it
is possible that modulation of striatolimbic D4 receptors in
synchrony with other neurotransmitter receptors enhances
the therapeutic efficacy of cariprazine.
In this study, long-term treatment with the 3 doses of
cariprazine also increased D4 receptor binding in the
hippocampus. This is in agreement with the effects of
other atypical antipsychotic drugs, including olanza-
pine, risperidone, and asenapine, which all increase
D4 receptor binding in this brain region.9,10 Hippocampal
D4 receptors, therefore, may constitute common targets
that mediate the molecular actions of cariprazine and
other atypical antipsychotics, acting together with hippo-
campal D2 receptors to improve impaired emotional
behavior that is commonly associated with psychosis.2
Conclusions
In this study, long-term administration of cariprazine
was shown to induce regional and dose-dependent
upregulation of D2 and D4 receptor subtypes similar to
other atypical antipsychotics. Additionally, cariprazine
was unique in upregulating D3 receptors, which has not
276 Y. K. CHOI ET AL.
been seen with other typical or atypical antipsychotic
agents. This distinct pharmacological profile of caripra-
zine may confer additional clinical benefits on cognitive
impairment and mood disturbances associated with
schizophrenia and bipolar disorder.
Disclosures
Y. K. Choi has no conflicts of interest to disclose.
N. Adham is an employee of Forest Research Institute.
B. Kiss and I. Gyertyán are employees of Gedeon Richter
Plc. F. I. Tarazi received research grants from Novartis,
Forest Laboratories, Inc., H. Lundbeck A/S, and Shire plc.
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