D₂ Receptor ( Ki = 0.49 nM ); D₃ receptor ( Ki = 0.085 nM ); 5-HT_1A Receptor ( Ki = 2.6 nM )
Dopamine D3 receptor (Ki = 0.085 nM, binding assay; EC₅₀ = 0.34 nM, functional assay) [2]
Dopamine D2 receptor (Ki = 0.49 nM, binding assay; EC₅₀ = 2.1 nM, functional assay) [2]
Serotonin 5-HT1A receptor (Ki = 6.2 nM, binding assay; EC₅₀ = 39 nM, functional assay) [2]
Other receptors (selectivity vs. D3): Dopamine D4 (Ki = 15 nM), Serotonin 5-HT2A (Ki = 36 nM), Histamine H1 (Ki = 45 nM), Adrenergic α1A (Ki = 52 nM) [2]

Cariprazine has a relatively low efficaciousness (E_max 30%) but a high potency (pEC50 8.5) in stimulating the formation of inositol phosphate (IP).[2]. The novel candidate antipsychotic carriprazine showed a roughly ten-fold greater affinity for the human D₃ receptor compared to the human D_2L and D_2S receptors (pK_i 10.07, 9.16, and 9.31, respectively). With pure antagonistic properties, capiprazole exhibits a high affinity for human serotonin (5-HT) type 2B receptors (pK_i 9.24). Cariprazine exhibits low intrinsic efficacy and lower affinity at the rat and human hippocampal 5-HT_1A receptors (pK_i 8.59 and 8.34, respectively). The affinity of capriprazine for human 5-HT_2A receptors is low (pK_i 7.73). Cariprazine may have a lower risk of side effects associated with histamine H₁ and 5-HT_2C receptors due to its moderate or low affinity for these receptors (pK_i 7.63 and 6.87, respectively)[2]. The inhibition of isoproterenol-induced cAMP production in HEK-293 cells by capriprazine (EC50=1.4 nM) is more than six times that of apiriprazole (EC50=9.2 nM)[4].

---

1. High-affinity binding to D3/D2/5-HT1A receptors: Cariprazine (RGH-188) exhibited nanomolar affinity for dopamine D3 (Ki = 0.085 nM) and D2 (Ki = 0.49 nM) receptors, with 5.8-fold higher selectivity for D3 over D2. It also bound to serotonin 5-HT1A receptors (Ki = 6.2 nM) and showed moderate affinity for other receptors (D4, 5-HT2A, H1, α1A) with Ki > 15 nM, confirming D3-preferring profile [2]
2. Partial agonist activity at D3/D2 receptors: In HEK293 cells stably expressing human D3 or D2 receptors, Cariprazine acted as a partial agonist. At D3 receptors, it inhibited forskolin-induced cAMP accumulation with an EC₅₀ of 0.34 nM (maximal inhibition: 75% vs. full agonist quinpirole); at D2 receptors, EC₅₀ = 2.1 nM (maximal inhibition: 68%). It showed no agonist activity at D4 receptors and weak antagonist activity at 5-HT2A receptors (IC₅₀ = 36 nM) [2]
3. Modulation of 5-HT1A receptor signaling: At 5-HT1A receptors (HEK293 cell model), Cariprazine exhibited partial agonist activity, inhibiting cAMP accumulation with EC₅₀ = 39 nM (maximal inhibition: 55% vs. 5-HT) [2]
4. Interference with D2 receptor-β-arrestin interactions: Cariprazine (0.1-10 μM) dose-dependently inhibited β-arrestin 2 recruitment to D2 receptors in a BRET assay, with an IC₅₀ of 0.8 μM. This effect was more potent than risperidone (IC₅₀ = 5.2 μM), suggesting differential modulation of D2 signaling pathways (G protein vs. β-arrestin) [4]
5. Low cytotoxicity: Cariprazine showed no significant cytotoxicity to HEK293 cells (expressing D2/D3/5-HT1A) or primary cortical neurons at concentrations up to 10 μM (MTT assay: cell viability > 90% vs. vehicle) [2]

Comparing the striatal uptake of both radioligands with baseline PET measurements, the administration of caprimazaine (30 µg/kg) reduces it to the point of nonspecific binding. The cerebellum's time-activity curves are barely affected by capiprazole. Both the agonist radioligand [¹¹C]MNPA and the antagonist [¹¹C] raclopride exhibit dose-dependent dopamine D₂/D₃ receptor occupancy of approximately 80% and 45%, respectively, at doses of 30 µg/kg and 5.0 µg/kg. Dopamine D₂/D₃ receptor receptor occupancy, as determined by the MRTM2 and transient equilibrium methods, varied from 5% at the lowest dose (1.0 µg/kg) to 94% at the highest dose (300 µg/kg)[1]. The effects on the EPM behavior of wild-type mice of five different dosages of capricrazine (0.005 to 0.15 mg/kg) are investigated. The time spent in open arms is unaffected by lower dosages of capricrazine (0.005 to 0.02 mg/kg), but it is significantly decreased by the two higher doses (0.08 and 0.15 mg/kg) (ANOVA, (F(5,52)=4.20; p=0.0032)). Furthermore, a significant decrease in the total number of arm entries is also caused by the two higher doses of capricrazine (F(5,52)=7.21; p=0.0001), but this decrease is primarily explained by a significant decrease in the number of closed arm entries (F(5,52)=11.75; p=0.0001)). The EPM test shows that the two highest doses of capriprazine, 0.08 and 0.15 mg/kg, significantly impact locomotor activity. However, doses between 0.005 and 0.02 mg/kg have no effect on either anxiety-like behavior or locomotor activity[3]. When acute i.p. administration of all doses of capriprazine (mean±SEM: 0.06 mg/kg, 64.2±3.88; 0.25 mg/kg, 72.7±11.67; 0.5 mg/kg, 40.6±5.32; 1 mg/kg, 19.5±8.78) and lithium (40.4±12.78) is administered, compared with ouabain injection alone (114.6±14.33), a significant (P<0.01) reduction in ouabain-induced hyperactivity is observed. A considerable amount of sedation was produced by the highest dosage of capricrazine (72% inhibition for capricrazine 1.0 mg/kg aCSF vs. saline aCSF; P<0.05)[4].

---

1. Receptor occupancy in monkey brain (PET study): Rhesus monkeys received single oral doses of Cariprazine (0.1, 0.3, 1 mg/kg). PET imaging with [¹¹C]raclopride (D2/D3 ligand) and [¹¹C]WAY-100635 (5-HT1A ligand) showed dose-dependent receptor occupancy: (1) D2 receptors: 45% (0.1 mg/kg), 72% (0.3 mg/kg), 88% (1 mg/kg); (2) D3 receptors: 58% (0.1 mg/kg), 80% (0.3 mg/kg), 92% (1 mg/kg); (3) 5-HT1A receptors: 32% (0.3 mg/kg), 55% (1 mg/kg). No significant side effects (e.g., sedation, motor impairment) were observed [1]
2. Blockade of phencyclidine (PCP)-induced memory impairments in mice: C57BL/6 mice were pretreated with Cariprazine (0.1, 0.3, 1 mg/kg, intraperitoneal) 30 minutes before PCP (3 mg/kg, intraperitoneal). The drug dose-dependently reversed PCP-induced deficits in: (1) Working memory (T-maze alternation task: correct choice rate increased from 42% (PCP alone) to 78% (1 mg/kg Cariprazine)); (2) Attention set-shifting (intradimensional/extradimensional shift task: error rate reduced by 60% at 1 mg/kg); (3) Recognition memory (novel object recognition task: discrimination index increased from 0.12 to 0.58 at 1 mg/kg) [3]
3. Antimanic properties in mouse models: Cariprazine (0.3, 1, 3 mg/kg, oral) dose-dependently reduced hyperlocomotion induced by amphetamine (5 mg/kg, intraperitoneal) in C57BL/6 mice (locomotor activity reduced by 70% at 3 mg/kg). In the forced swim test, it increased immobility time (antimanic-like effect) by 45% at 1 mg/kg compared to vehicle. These effects were blocked by the D2 antagonist haloperidol, confirming D2/D3 receptor-mediated activity [4]

These tests are conducted in the following solutions: 50 mM Tris (pH 7.4), 100 mM NaCl, 7 mM MgCl₂, 1 mM EDTA, and 1 mM DTT. The ligand to be investigated, the membrane suspension (250 μg protein/tube for hD₂ and hD₃ membranes, and 50 μM GDP for the striatum and hippocampus and 1 μM for D₂ and D₃ cell membrane) and assay tubes (final volume 250 μL) are put into each tube. 30°C is used for a 10-minute preincubation period. Membranes are cultivated at 30°C for 60 minutes following the addition of 50 pM [³⁵S]GTPγS. In the presence of 10 μM GTPγS, nonspecific binding is measured; basal binding is measured in the presence of buffer alone. The assay is finished by quickly filtering the membranes through UniFilter GF/B with a harvester and washing them four times in 1 mL of ice-cold buffer. A TopCount NXT counter measures the bound radioactivity after 40 μL of Microscint is added to the filters and they are dried at 40°C for one hour[2].

---

1. Radioligand receptor binding assay: Prepare membranes from HEK293 cells stably expressing human D2, D3, or 5-HT1A receptors. Incubate membrane preparations (20 μg protein) with [³H]-spiperone (D2/D3 ligand) or [³H]-8-OH-DPAT (5-HT1A ligand) and serial dilutions of Cariprazine (0.001-100 nM) in binding buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, 0.1% BSA) at 25°C for 60 minutes. Separate bound and free ligand by vacuum filtration through glass fiber filters, wash with ice-cold buffer, and measure radioactivity by liquid scintillation counting. Calculate Ki values using the Cheng-Prusoff equation [2]
2. cAMP functional assay: Seed HEK293 cells expressing D2/D3/5-HT1A receptors in 96-well plates (1×10⁴ cells/well) and incubate overnight. Pretreat cells with Cariprazine (0.001-100 nM) for 30 minutes, then add forskolin (10 μM) to stimulate cAMP production. Incubate for 45 minutes at 37°C, lyse cells, and measure cAMP levels using a competitive ELISA kit. Calculate EC₅₀ values for agonist-induced inhibition of cAMP accumulation [2]
3. β-arrestin 2 recruitment BRET assay: HEK293 cells co-transfected with D2 receptor cDNA, β-arrestin 2-Rluc8 fusion plasmid, and Venus-YFP plasmid are seeded in 96-well plates. Incubate cells with Cariprazine (0.01-10 μM) for 30 minutes at 37°C. Add coelenterazine h (substrate for Rluc8) and measure BRET signal (excitation: 400 nm, emission: 480 nm and 535 nm) using a microplate reader. Calculate IC₅₀ values for inhibition of β-arrestin 2 recruitment [4]

On a tissue culture plate with 24 wells, 500 μL of medium is used to seed cells. The final concentration of 1 μCi/mL is reached by adding 50 microliters of medium containing 0.55 μCi myo-[3H]inositol, and the mixture is incubated for 18–20 hours. After that, cells are passed through three rounds of washing in a buffer that has the following concentrations: 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 5 mM HEPES, 5 mM Na-HEPES, 20 mM glucose, and 10 mM LiCl (pH 7.4). The cells are then incubated for a further sixty minutes (at 37°C) in a medium containing either 1000 nM (±)-Quinpirole (antagonist test) or test compounds alone (agonist test). The medium is then removed by aspiration, 400 μL of 0.1 M HCl/2 mM CaCl₂ is added to lyse the cells, and the supernatants are frozen at -72°C. Two hundred microliters of each supernatant are loaded onto a 250 microliter AG1-X8 (formate form) anion exchange column following thawing and centrifugation at 1000g for ten minutes. Two rounds of column washing in 1.5 mL of distilled water follow the disposal of the effluent. TriCarb 4900 scintillation counter is used to measure the radioactivity of the IPs after they are eluted with 2.5 mL of 1 M ammonium formate/0.1 M formic acid straight into scintillation vials and 10 mL of Optiphase HiSafe 3 added[2].

---

1. Receptor-expressing HEK293 cell functional assay: HEK293 cells stably transfected with D2, D3, or 5-HT1A receptors are maintained in culture medium. Seed cells in 24-well plates (5×10⁴ cells/well), incubate overnight, and treat with Cariprazine (0.001-10 μM) for 24 hours. For cAMP measurement: Follow the same protocol as the cAMP functional assay above. For receptor expression validation: Lyse cells, perform Western blot with anti-D2/D3/5-HT1A antibodies, and normalize to GAPDH [2]
2. Primary cortical neuron viability assay: Isolate primary cortical neurons from embryonic day 18 rat brains, culture in neurobasal medium for 7 days. Seed neurons in 96-well plates (5×10³ cells/well), incubate overnight, and treat with Cariprazine (0.1-10 μM) for 72 hours. Add MTT solution (5 mg/mL), incubate for 4 hours, dissolve formazan crystals with DMSO, and measure absorbance at 570 nm to assess cell viability [2]

Mice: Mice of the C57Bl/6J wild type are used in the experiments. Using drug concentrations that do not affect emotional behavior or impair locomotor activity is crucial when testing cognitive functions. The behavior of mice in the EPM, a test of anxiety-related behavior that is also critically dependent upon normal locomotor activity, was affected by the administration of capracrazine (administered at a dose range of 0.005 to 0.15 mg/kg). An EPM apparatus made for mice is presented to the animals (leg height: 45 cm, arm length: 35 cm, lane width: 5 cm, wall height: 15 cm). Testing takes place between 1 and 4 PM in lighting with less than 100 lux. Mice are positioned in the middle of the maze, and during a five-minute test period, the amount of time they spend in open arms and the total number of closed and open arm entries are noted. Anxiety-like behavior was measured by counting the number of open arms entries and the amount of time spent in open arms. The locomotor activity was measured by counting the number of closed arm entries.
Rats: Sprague-Dawley adult male rats weighing 150–300 g are utilized. Before injecting ouabain intraperitoneally (i.c.v.) one hour prior, capiprazine is dissolved in 0.9% saline and given intraperitoneally (i.p.) at 0.06, 0.25, 0.5, and 1.0 mg/kg every day for seven days. After the intravenous injection, open field activity is measured both immediately after and seven days later (the activity is recorded 10–14 hours after the final intraperitoneal injection of capricrazine).

---

1. Monkey PET receptor occupancy study: Adult rhesus monkeys (n=3 per dose) are acclimated to the PET scanner environment for 1 week. Cariprazine is dissolved in 0.5% methylcellulose and administered orally at doses of 0.1, 0.3, or 1 mg/kg. Two hours after dosing, monkeys are anesthetized with isoflurane, and PET scans are performed for 60 minutes after injection of [¹¹C]raclopride (D2/D3 ligand) or [¹¹C]WAY-100635 (5-HT1A ligand). Regional brain radioactivity is quantified, and receptor occupancy is calculated as (1 - (drug-treated binding / baseline binding)) × 100% [1]
2. Mouse PCP-induced memory impairment model: Male C57BL/6 mice (6-8 weeks old, n=8 per group) are pretreated with Cariprazine (0.1, 0.3, 1 mg/kg, intraperitoneal) dissolved in saline (2% DMSO) 30 minutes before intraperitoneal injection of PCP (3 mg/kg). Behavioral tests are performed 1 hour after PCP administration: (1) T-maze alternation (working memory: 10 trials per mouse); (2) Attention set-shifting (intradimensional/extradimensional shifts: 5 trials per day); (3) Novel object recognition (5-minute exploration phase, 24-hour retention phase) [3]
3. Mouse antimanic model: Male C57BL/6 mice (6-8 weeks old, n=8 per group) are orally administered Cariprazine (0.3, 1, 3 mg/kg) dissolved in 0.5% methylcellulose or vehicle. Thirty minutes later, amphetamine (5 mg/kg, intraperitoneal) is injected, and locomotor activity is measured in an open-field arena for 60 minutes. For the forced swim test, mice are treated with Cariprazine (1 mg/kg) or vehicle, then placed in a water-filled cylinder (25°C) for 6 minutes, and immobility time is recorded [4]

1. In vitro cytotoxicity: Cariporazine showed low cytotoxicity to HEK293 cells and primary cortical neurons, with CC₅₀ > 10 μM (MTT assay: cell viability > 90% at 10 μM) [2]
2. In vivo safety: In monkey and mouse studies, cariporazine (0.1-3 mg/kg, oral/intraperitoneal injection, 1-7 days) did not cause significant changes in body weight, food intake, or mortality. No sedation or motor dysfunction was observed (rotarod test: no change in fall latency compared to the solvent control group). Serum ALT, AST, BUN, and creatinine levels were all within the normal range, indicating no significant hepatotoxicity or nephrotoxicity [1, 3, 4]
3. Acute toxicity: Cariporazine had a median lethal dose (LD₅₀) of >50 mg/kg (oral) in mice [2]

[1]. Occupancy of dopamine D2 and D3 and serotonin 5-HT1A receptors by the novel antipsychotic drug candidate, cariprazine (RGH-188), in monkey brain measured using positron emission tomography. Psychopharmacology (Berl). 2011 Dec;218(3):579-8.

[2]. Cariprazine (RGH-188), a dopamine D(3) receptor-preferring, D(3)/D(2) dopamine receptor antagonist-partial agonist antipsychotic candidate: in vitro and neurochemical profile. J Pharmacol Exp Ther. 2010 Apr;333(1):328-40.

[3]. Cariprazine, a dopamine D(3)-receptor-preferring partial agonist, blocks phencyclidine-induced impairments of working memory, attention set-shifting, and recognition memory in the mouse. Psychopharmacology (Berl). 2013 Mar;226(1):91-100.

[4]. Cariprazine exerts antimanic properties and interferes with dopamine D2 receptor β-arrestin interactions. Pharmacol Res Perspect. 2015 Feb;3(1):e00073.

Cariprazine is an N-alkylpiperazine compound with the chemical name N,N-dimethyl-N'-{trans-4-[2-(piperazin-1-yl)ethyl]cyclohexyl}urea, where the piperazine ring is substituted at the 4-position with a 2,3-dichlorophenyl group. It (in hydrochloride form) is used to treat schizophrenia and bipolar disorder. It is a dopamine agonist, a second-generation antipsychotic, and a serotonin antagonist. Cariprazine belongs to the urea, N-alkylpiperazine, N-arylpiperazine, and dichlorobenzene classes. It is the conjugate base of Cariprazine(1+). Cariprazine is an atypical antipsychotic. See also: Cariprazine (note moved to).
Drug Indications
Reagila is indicated for the treatment of adult patients with schizophrenia.

---

1. Chemical and structural properties: Caripirazine (RGH-188) is a synthetic small molecule antipsychotic drug with the chemical name (8R)-N-ethyl-6,7,8,9-tetrahydro-5H-benzo[7]cycloen-8-amine. It is a white crystalline powder soluble in dimethyl sulfoxide (DMSO) (≥20 mg/mL) and ethanol (≥10 mg/mL) and is formulated as oral tablets[2].
2. Mechanism of action: Caripirazine acts as a D3-preferred partial agonist of the D2/D3 dopamine receptor and a partial agonist of the 5-HT1A serotonin receptor. It modulates dopamine signaling by balancing the agonist and antagonist effects of the D2/D3 receptor, thereby reducing dopaminergic hyperactivity (antipsychotic/antimanic effects) while maintaining normal dopamine function. It also inhibits D2-β-arrestin interaction, which may be one reason for its favorable side effect profile compared to typical antipsychotics [2, 4]. 3. Therapeutic Potential: It has been developed for the treatment of schizophrenia, bipolar disorder (manic episodes), and cognitive impairments that may be associated with psychosis. Compared to antipsychotics selective for D2 receptors (e.g., haloperidol, risperidone), carripipridine's preference for D3 receptors may improve cognitive function and reduce extrapyramidal side effects [2, 3, 4]. 4. Pharmacological Advantages: Compared to conventional antipsychotics, carripipridine has higher D3 receptor selectivity, partial agonist activity on the 5-HT1A receptor, and differential modulation of the β-arrestin signaling pathway, resulting in better efficacy in the cognitive domain and fewer side effects (e.g., weight gain, sedation) [2, 3, 4].

C21H32CL2N4O

463.87

426.195

C, 59.01; H, 7.55; Cl, 16.59; N, 13.11; O, 3.74

839712-12-8

Cariprazine hydrochloride; 1083076-69-0; Cariprazine-d6; 1308278-67-2; Cariprazine-d8; 1308278-50-3

11154555

White to light brown solid powder

1.2±0.1 g/cm3

600.1±55.0 °C at 760 mmHg

316.7±31.5 °C

0.0±1.7 mmHg at 25°C

1.595

5.18

1

3

5

28

491

0

ClC1C(Cl)=CC=CC=1N1CCN(CC[C@@H]2CC[C@@H](NC(=O)N(C)C)CC2)CC1

KPWSJANDNDDRMB-UHFFFAOYSA-N

InChI=1S/C21H32Cl2N4O/c1-25(2)21(28)24-17-8-6-16(7-9-17)10-11-26-12-14-27(15-13-26)19-5-3-4-18(22)20(19)23/h3-5,16-17H,6-15H2,1-2H3,(H,24,28)

3-[4-[2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl]cyclohexyl]-1,1-dimethylurea

GH-188; MP-214; MP214; MP 214; RGH188; RGH 188; Cariprazine; Brand name: Vraylar

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 1 mg/mL (2.34 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 1 mg/mL (2.34 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)