Human dopamine D₄ receptor ( EC50 = 12.4 nM ); Rat dopamine D₄ receptor ( EC50 = 14.3 nM ); Ferret dopamine D₄ receptor ( EC50 = 23.2 nM )
D4 receptor (human D4.4: EC50 = 12.4 ± 1.0 nM, efficacy = 61.0 ± 3.7%; human D4.2: Ki = 57.5 ± 8.0 nM; human D4.4: Ki = 63.6 ± 6.6 nM; human D4.7: Ki = 46.8 ± 4.8 nM; rat D4: EC50 = 14.3 ± 0.6 nM, efficacy = 70%; ferret D4: EC50 = 23.2 ± 1.3 nM, efficacy = 64%) [1]

No effect on D1, D2, D3, D5 receptors up to 10 μM; weak affinity to 5-HT1A receptors (Ki = 2,780 ± 642 nM) [1]

No inhibition of PDE1, PDE5, or PDE6 at 10 μM [1]

Inactive at human D2 receptor (Ki > 10,000 nM; EC50 > 10,000 nM) [1]

ABT-724 displays a selective biochemical profile, as demonstrated by its lack of binding affinity for over 70 neurotransmitter/uptake/ion channels, such as D₂, D₃, or D₅ receptors, at concentrations up to 10 μM. There's a low affinity (K_i = 2780 nM) for 5-HT_1A receptors. At concentrations of 10 μM, ABT-724 does not inhibit PDE activity of PDE1, PDE5, or PDE6[1].

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In HEK293 cells stably expressing human D4.4 receptor and G protein chimera G0.5, ABT-724 induced intracellular Ca2+ increases with EC50 = 12.4 ± 1.0 nM and 61.0 ± 3.7% efficacy compared to 10 μM dopamine (100%) [1]

In HEK293 cells expressing rat D4 receptor and G0.5, ABT-724 was a potent partial agonist (EC50 = 14.3 ± 0.6 nM, 70% efficacy) [1]

In HEK293 cells expressing ferret D4 receptor and G0.5, ABT-724 showed EC50 = 23.2 ± 1.3 nM and 64% efficacy [1]

In radioligand binding assays using [3H]-A-369508, ABT-724 bound to human D4 variants with Ki values: D4.2 = 57.5 ± 8.0 nM, D4.4 = 63.6 ± 6.6 nM, D4.7 = 46.8 ± 4.8 nM [1]

ABT-724 showed no binding affinity for >70 neurotransmitters/uptake/ion channels including D1, D2, D3, D5 receptors up to 10 μM; weak affinity to 5-HT1A receptors (Ki = 2,780 ± 642 nM) [1]

ABT-724 did not inhibit PDE1, PDE5, or PDE6 at 10 μM [1]

In human D2 receptor binding assays using [125I]-PIPAT, ABT-724 was inactive up to 10 μM (Ki > 10,000 nM); in functional assays in HEK293 cells expressing human D2 receptor and G0.5, ABT-724 did not activate D2 receptors up to 10 μM [1]

In rabbit corpus cavernosum smooth muscle strips precontracted with phenylephrine, ABT-724 had no effect up to 10 μM [1]

ABT-724 (8.8 μg/kg; subcutaneous injection; daily; for 5 days; male adult Wistar rats) treatment dose-dependently promotes penile erection when administered subcutaneously to conscious rats[1].

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Subcutaneous administration of ABT-724 (0.003-0.3 μmol/kg) dose-dependently facilitated penile erection in conscious Wistar rats, with 77% maximal effect at 0.03 μmol/kg (erection incidence 77% vs. 23% in vehicle group), latency 18.7 min [1]

The proerectile effect of ABT-724 (0.03 μmol/kg s.c.) was blocked by clozapine (10 μmol/kg i.p.) and haloperidol (0.3 μmol/kg i.p.), but not by domperidone (10 μmol/kg i.p.), indicating central dopaminergic mechanism [1]

No tolerance to proerectile effect after 5 daily doses of ABT-724 (0.03 μmol/kg s.c.): incidence 71±12% on day 1 vs. 78±11% on day 5 [1]

Intracerebroventricular (ICV) injection of ABT-724 (3 nmol) significantly facilitated penile erection (incidence and number of erections similar to apomorphine 3 nmol) [1]

Intrathecal injection of ABT-724 (3, 10, 30 nmol) had no proerectile effect; modest effect only at 100 nmol (50±14% incidence vs. 9±7% for vehicle) [1]

In awake freely moving rats, ABT-724 s.c. (0.0025 μmol/kg) increased intracavernosal pressure (ICP): average 2.0±0.6 episodes of ICP increase, mean time to first response 15.4±7.7 min, mean peak ICP 139.6±18.6 cmH2O, mean duration 3.6±1.5 min [1]

At 0.025 μmol/kg s.c., ABT-724 produced 7.2±2.8 episodes of ICP increase, mean duration 7.0±2.3 min, mean time to first response 19.3±5.8 min, peak ICP 134.1±19.2 cmH2O [1]

Combination with sildenafil (1 μmol/kg i.p.) potentiated the proerectile effect of ABT-724: 10-fold leftward shift, with maximal effect at 0.003 μmol/kg ABT-724 in presence of sildenafil [1]

ABT-724 did not induce emesis or nauseogenic behavior in conscious male ferrets at 0.03, 0.3, and 3 μmol/kg s.c. (maximal dose 100-fold higher than efficacious dose in rats) [1]

For D4 receptor binding assays, membranes were prepared from stable cell lines expressing human D4.4 or rat D4 in HEK293 cells. Membranes containing human D4.2 and D4.7 were obtained commercially. Saturation binding was performed with the D4 agonist [3H]-A-369508. Competition binding assays were initiated by adding 250 μl of membrane (50 μg protein final) to 200 μl of [3H]-A-369508 (final concentration 2 nM) and incubated at room temperature for 1 hour. Nonspecific binding was defined with 10 μM PD168077. Radioactivity was counted using a microplate scintillation counter. IC50 values were determined using a one-site model, and Ki values calculated using the Cheng-Prusoff equation [1]

For D2 receptor binding assays, membranes were prepared from a stable cell line expressing human D2L in HEK293 cells. Binding assays were initiated by adding 250 μl of membrane to 200 μl of [125I]-PIPAT and incubated at room temperature for 1 hour. Radioactivity was counted and IC50 values determined similarly [1]

FLIPR assay: Agonist-induced intracellular Ca2+ changes were assessed using Flu-4 in conjunction with FLIPR. Flu-4 was dissolved in anhydrous DMSO and diluted in buffer containing 0.5 μM 3-isobutyl-1-methylxanthine and 0.004% ascorbic acid to a final concentration of 2 mM. After agonist addition, Ca2+ dynamics were recorded. Independent measurements of 10 μM dopamine (100%) and unloaded cells (0%) were performed on each plate to normalize values. Concentration-response data were analyzed, and EC50 values derived from single-curve fits. FLIPR studies were conducted on human, rat, and ferret D4 receptors [1]

HEK293 cells stably expressing human D4.4 receptor and the G protein chimera G0.5 (where the last five amino acids of Gi are replaced by the last five amino acids of Gq) were used. Activation of D4 receptor leads to changes in intracellular Ca2+ levels measured by FLIPR. Dopamine, apomorphine, and ABT-724 were tested. Dopamine activated D4 receptors with 2.2 nM potency. ABT-724 induced partial agonist responses with EC50 = 12.4 nM and 61% efficacy [1]

Rat and ferret D4 receptors were cloned and stably expressed in HEK293 cells with coexpression of G0.5. Functional studies showed that dopamine increased intracellular Ca2+ in rat D4 cells with 2.4±0.2 nM and in ferret D4 cells with 2.7±0.3 nM potency. ABT-724 was a potent partial agonist at rat D4 (EC50 = 14.3±0.6 nM, 70% efficacy) and ferret D4 (EC50 = 23.2±1.3 nM, 64% efficacy) [1]

Human D2 receptor functional activity was determined in HEK293 cells expressing human D2 receptor and G0.5. ABT-724 did not activate human D2 receptors up to 10 μM [1]

Male adult Wistar rats (~300 g)
8.8 μg/kg
Subcutaneous injection; daily; for 5 days

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Conscious rat model of penile erection: Male adult Wistar rats (~300 g) were used. ABT-724 (maleate salt) was freshly prepared and administered s.c. into the back neck area (1 ml/kg injection volume). Rats were placed individually into transparent Plexiglas cages immediately after injection. A mirror was placed behind and under the cages for observation. Penile erection was considered when repeated pelvic thrusts were immediately followed by an upright position and an emerging engorged penis that the rat proceeded to groom. Erection episodes were recorded by direct observation for 60 min after dosing. Erection incidence (%) was defined as the percent of animals exhibiting one or more erections [1]

For ICV or intrathecal administration, rats were allowed at least 1 week recovery from surgery. Compounds were infused ICV (left lateral ventricle) in 5 μl volume or intrathecally in 10 μl volume. After experiments, infusion of 0.5% fast-green dye in saline and subsequent dissection confirmed diffusion [1]

Repeated dosing: Rats were dosed with vehicle or ABT-724 (0.03 μmol/kg s.c.) once daily for 5 days. Animals were tested on day 1 and day 5 immediately after injection [1]

Combination with sildenafil: Sildenafil (1 μmol/kg i.p.) was injected 30 min before ABT-724 s.c., and rats were observed for 60 min for erections [1]

Intracavernosal pressure registration in conscious rats: Male Sprague-Dawley rats (350-420 g) were anesthetized with pentobarbital and ketamine i.p. Through a midline scrotal incision, the base of the penis was exposed. A small hole was made in the tunica albuginea of the crus cavernosum, and a heparinized PE50 catheter was introduced and secured. The catheter was tunneled s.c. to the neck. Twelve hours after implantation, animals were placed in metabolic cages. After 30 min stabilization, continuous ICP measurements were performed with transducers and registered on a polygraph. ABT-724 (0.0025 or 0.025 μmol/kg) or vehicle (saline) was given s.c., and changes in ICP were registered for 60 min. Basal ICP, total number of responses (>50 cmH2O), total duration, time to first response, and peak ICP were analyzed [1]

Ferret emesis model: Conscious male ferrets were administered ABT-724 s.c. at 0.03, 0.3, and 3 μmol/kg. Number of emetic episodes and presence of nauseogenic behaviors were recorded for 90 min [1]

Rat hemodynamic study: Intravenous administration of ABT-724 in anesthetized Sprague-Dawley rats to measure mean arterial pressure, heart rate, dP/dt, and regional blood flow to renal, mesenteric, and hindquarter beds [1]

Mouse Irwin test: CD-1 mice were injected i.p. with ABT-724 (0.1-100 μmol/kg) to assess behavior, neurological symptoms, and rectal temperature [1]

Locomotor activity: Wistar rats received s.c. ABT-724 (0.003-1.0 μmol/kg) and locomotion was observed in open field for 20 min [1]

Prepulse inhibition (PPI) model: Male Sprague-Dawley rats were given ABT-724 (0.03-1.0 μmol/kg s.c.) to assess antipsychotic potential [1]

ABT-724 rapidly crossed the blood-brain barrier in rats; brain concentration reached 4.9 ng/g at 15 min after s.c. injection [1]

Maximal concentration of ABT-724 at 0.03 μmol/kg dose reached 5 min after s.c. administration: 5.0 ng/ml (17 nM) in plasma [1]

In ferrets, after s.c. administration of 3 μmol/kg ABT-724, plasma level reached 812 ± 74 ng/ml [1]

After high-dose intravenous infusion (3 μmol/kg) in rats, plasma concentration reached 277 ± 37 ng/ml at the end of infusion [1]

Efficacious plasma level in rat penile erection model: 5 ng/ml [1]

ABT-724 did not cause emesis or nauseogenic behavior in conscious male ferrets at 0.03, 0.3, and 3 μmol/kg s.c. (highest dose 100-fold greater than maximally efficacious dose in rat penile erection model). The tolerability index (based on plasma levels) was >160-fold [1]

ABT-724 i.v. (up to 3 μmol/kg infusion) produced no significant effects on mean arterial pressure, heart rate, dP/dt, or regional blood flow to renal, mesenteric, and hindquarter beds in anesthetized rats. No hypotensive effect was observed, unlike prazosin [1]

ABT-724 s.c. (0.003-1.0 μmol/kg) did not affect locomotion of Wistar rats in open field (doses up to 30-fold higher than effective dose 0.03 μmol/kg). Amphetamine significantly increased locomotion [1]

ABT-724 (0.03-1.0 μmol/kg s.c.) neither blocked nor increased prepulse inhibition (PPI) response in rats. Amphetamine disrupted PPI, which was blocked by haloperidol and clozapine [1]

In Irwin test in CD-1 mice, ABT-724 i.p. (0.1-100 μmol/kg) induced no observable effects up to 10 μmol/kg. The lowest dose causing behavioral side effects (mild hypoactivity and ptosis) was 100 μmol/kg. Methylphenidate induced CNS stimulation (hyperlocomotion, aggression, convulsions) [1]

[1]. Activation of dopamine D4 receptors by ABT-724 induces penile erection in rats. Proc Natl Acad Sci U S A. 2004 Apr 27;101(17):6758-63.

2-[[4-(2-pyridyl)-1-piperazinyl]methyl]-1H-benzimidazole is a member of the pyridine and piperazine classes.

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ABT-724 (2-[(4-pyridin-2-ylpiperazin-1-yl)methyl]-1H-benzimidazole) is a highly selective dopamine D4 receptor agonist structurally different from apomorphine [1]

The proerectile effect of ABT-724 is mediated by supraspinal dopaminergic mechanisms, based on ICV efficacy, lack of effect at spinal level, inability to relax corpus cavernosum strips, and lack of effect on vascular beds [1]

Unlike nonselective D2 agonists (e.g., PNU-95666E) that induce emesis, selective D4 activation by ABT-724 does not cause nausea/emesis, potentially allowing a larger dose range for treating erectile dysfunction [1]

Combination of centrally acting ABT-724 with peripherally acting PDE5 inhibitor sildenafil showed potentiation, suggesting benefit for erectile dysfunction patients, especially diabetics who may have impaired neuronal NO release [1]

D4 receptor is expressed in prefrontal cortex, hippocampus, amygdala, and hypothalamus; hypothalamic D4 receptors merit further investigation in penile erection and sexual function control [1]

ABT-724 did not induce CNS stimulation behaviors (unlike methylphenidate and amphetamine), nor did it show antipsychotic-like effects in PPI model [1]

C17H19N5

402.74908

293.164

C, 69.60; H, 6.53; N, 23.87

70006-24-5

ABT-724 trihydrochloride; 587870-77-7; 587870-77-7 (HCl); 70006-24-5

5025739

Solid powder

1.275g/cm3

548.6ºC at 760 mmHg

285.6ºC

1.687

2.283

1

4

3

22

355

0

C1=CC=C2C(=C1)NC(=N2)CN3CCN(CC3)C4=CC=CC=N4

FRPJGTNLZNXQEX-UHFFFAOYSA-N

InChI=1S/C17H19N5/c1-2-6-15-14(5-1)19-16(20-15)13-21-9-11-22(12-10-21)17-7-3-4-8-18-17/h1-8H,9-13H2,(H,19,20)

2-[(4-pyridin-2-ylpiperazin-1-yl)methyl]-1H-benzimidazole

ABT 724; ABT724; ABT-724; UNII-4WV2575JWT

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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