Dopamine3 Receptor (D3 receptor)

The benzofurane (+)-S 14297, the benzamide nafadotride, the aminoindane U-99194 and the arylpiperazine GR 103,691 have been proposed as "selective" antagonists at dopamine D3 vs. D2 receptors. Herein, we compared their in vitro affinities and in vivo actions to those of the aminotetralin D3 antagonists (+)-AJ 76 and (+)-UH 232. Affinities at recombinant, human (h)D3 and/or hD2 sites were determined by employing the mixed D2/D3 antagonist [125I]-iodosulpride and the preferential D3 ligands [3H]-(+)-PD 128, 907 and [3H]-(+)-S 14297. [3H]-(+)-PD 128,907, [3H]-(+)-S 14297 and [125I]-iodosulpride yielded an essentially identical pattern of displacement at D3 sites, which suggests that they recognize the same population of receptors. The rank order of potency (Ki values in nM vs. [3H]-(+)-PD 128,907) was GR 103,691 (0.4) approximately nafadotride (0.5) > haloperidol (2) approximately (+)-UH 232 (3) approximately (+)-S 14297 (5) > (+)-AJ 76 (26) > U-99194 (160). The rank order of preference (Ki ratio, D2:D3) for D3 receptors (labeled by [3H]-PD 128,907) vs. D2 sites (labeled by [125I]-iodosulpride) was (+)-S 14297 (61) approximately GR 103,691 (60) > U-99194 (14) > nafadotride (9) approximately (+)-UH 232 (8) approximately (+)-AJ 76 (6) > haloperidol (0.2). (+)-S 14297 and GR 103,691 also showed greater than 100-fold selectivity at dopamine hD3 vs. hD4 and hD1 sites. However, GR 103,691 showed marked affinity for serotonin1A receptors (5.8 nM) and alpha-1 adrenoceptors (12.6 nM) [1].

In vivo, all antagonists except GR 103,691 prevented the induction of hypothermia by (+)-PD 128,907 (0.63 mg/kg s.c.) and a further preferential D3 agonist, (+)-7-OH-DPAT (0.16 mg/kg s.c.). On the other hand, haloperidol, (+)-AJ 76, (+)-UH 232 and nafadotride all induced catalepsy in rats, whereas (+)-S 14297, U-99194 and GR 103,691 were inactive. Haloperidol, (+)-AJ 76, (+)-UH 232, nafadotride and (weakly) U-99194 also enhanced prolactin secretion and striatal dopamine synthesis, whereas (+)-S 14297 and GR 103,691 were inactive. However, despite its high affinity at 5-HT1A receptors and alpha-1 adrenoceptors, both of which are present on raphe-localized serotonergic neurons, GR 103,691 (0.5 mg/kg i.v.) failed to influence their basal firing rate or the inhibition of their electrical activity by the 5-HT1A agonist (+/-)-8-OH-DPAT (0.005 mg/kg i.v.), a result that casts doubt on its activity in vivo. In conclusion, both (+)-S 14297 and GR 103,691 are markedly selective ligands that permit the characterization of actions at hD3 vs. hD2 receptors in vitro, but (+)-S 14297 appears to be of greater utility for the evaluation of their functional significance in vivo. Nevertheless, to develop a better understanding of the respective roles of dopamine D3 and D2 receptors, we need additional, chemically diverse antagonists of improved potency and selectivity [1].

Membrane preparation [2]
U-2 OS cells were detached from flasks by incubation with Versene after washing with phosphate buffered saline (PBS). U-2 OS and Sf9 cells were collected by centrifugation (200g, 10 min) and re-suspended in buffer 1 (20 mM HEPES, 1 mM EDTA, 1 mM EGTA, pH 7.4, 4°C). Cells were then ruptured using an Ultra Turrax® T25 homogenizer (24,000 min-1, 4 x 5s) and cell debris removed by centrifugation (400g, 5 min, 4°C). The resulting supernatant was collected and centrifuged (47,800g, 60 min, 4°C) to collect cell membranes. Membrane pellets were resuspended in buffer 1 using an Ultra Turrax® T25 homogenizer (6,500 min-1, 2 x 5s). The protein concentration of the membrane preparations was determined using the Lowry method of protein determination with BSA as a reference standard.

Radioligand binding assays [2]
Radioligand saturation binding assays [2]
Membrane proteins prepared from Sf9 cells expressing dopamine D2 (25 μg) or dopamine D3 receptors (10 μg) were incubated with a range of concentrations of [3H]spiperone in buffer 2 (20 mM HEPES, 6 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 0.1% BSA, pH 7.4) supplemented, where appropriate, with 100 mM NaCl or 100 mM N-methyl-D-glucamine (NMDG). Reactions were performed, in triplicate, in 4 ml LP4 test tubes (1 ml final volume) and were initiated by addition of membrane proteins. Non-specific binding was determined in the presence of (+)-butaclamol (3 μM). Reactions were incubated for 3 hours at 25°C and were terminated by rapid filtration through Whatman glass microfibre GF/C filters using a Brandel cell harvester. After four 3 ml washes with PBS (4°C) filter discs were transferred to scintillation vials and soaked in 2ml Ultima Gold™ XR scintillation fluid for at least 6 hours prior to their radioactivity being determined by liquid scintillation spectrometry. Specific binding was calculated by subtraction of non-specific binding and free radioligand concentration, corrected for ligand depletion calculated. Data were analysed using Prism and were fitted to hyperbolic equations describing a one-binding site model.

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Radioligand competition binding assays [2]
Membrane protein prepared from U-2 OS cells (2 μg) or Sf9 cells (25 μg [D2] or 10 μg [D3]) expressing wild-type or mutant receptors was incubated with a fixed concentration of [3H]spiperone (0.25 nM [D2] or 1 nM [D3]) and a range of concentrations of competing ligand in buffer 2 (20 mM HEPES, 6 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 0.1% BSA, pH 7.4) supplemented with dithiothreitol (0.1 nM) and, where appropriate, 100 mM NaCl or 100 mM NMDG. Non-specific binding was defined using (+)-butaclamol (3 μM) in place of the competing ligand. Reactions were performed, in triplicate, in 4 ml LP4 test tubes (1 ml final volume) [Sf9] or deep-well 96-well plates (400μl final volume) [U-2 OS]. Non-specific binding was determined in the presence of (+)-butaclamol (3 μM) and total binding determined in the absence of competing ligand. Reactions were initiated, incubated, terminated and radioactivity measured as described under ‘Radioligand saturation binding assays’. Data are presented as percentage of total binding, after subtraction of non-specific binding. Data were analysed using Prism and were fitted to sigmoidal equations describing a one-binding site model. Where % inhibition was <100%, parameters were calculated by data extrapolation.

[1]. Audinot V, et al. A comparative in vitro and in vivo pharmacological characterization of the novel dopamine D3 receptor antagonists (+)-S 14297, nafadotride, GR 103,691 and U 99194. J Pharmacol Exp Ther. 1998 Oct;287(1):187-97.
[2]. Examining the Effects of Sodium Ions on the Binding of Antagonists to Dopamine D2 and D3 Receptors. PLoS One. 2016 Jul 5;11(7):e0158808.

Many G protein-coupled receptors have been shown to be sensitive to the presence of sodium ions (Na+). Using radioligand competition binding assays, we have examined and compared the effects of sodium ions on the binding affinities of a number of structurally diverse ligands at human dopamine D2 and dopamine D3 receptor subtypes, which are important therapeutic targets for the treatment of psychotic disorders. At both receptors, the binding affinities of the antagonists/inverse agonists SB-277011-A, L,741,626, GR 103691 and U 99194 were higher in the presence of sodium ions compared to those measured in the presence of the organic cation, N-methyl-D-glucamine, used to control for ionic strength. Conversely, the affinities of spiperone and (+)-butaclamol were unaffected by the presence of sodium ions. Interestingly, the binding of the antagonist/inverse agonist clozapine was affected by changes in ionic strength of the buffer used rather than the presence of specific cations. Similar sensitivities to sodium ions were seen at both receptors, suggesting parallel effects of sodium ion interactions on receptor conformation. However, no clear correlation between ligand characteristics, such as subtype selectivity, and sodium ion sensitivity were observed. Therefore, the properties which determine this sensitivity remain unclear. However these findings do highlight the importance of careful consideration of assay buffer composition for in vitro assays and when comparing data from different studies, and may indicate a further level of control for ligand binding in vivo. [2]

C21H31NO6

393.48

393.215

C, 64.10; H, 7.94; N, 3.56; O, 24.40

234757-41-6

82668-33-5;234757-41-6;

11957713

Off-white to light yellow Solid powder

146 - 147 °C

3.004

2

7

9

28

377

0

CCCN(CCC)C1CC2=CC(=C(C=C2C1)OC)OC.C(=C/C(=O)O)/C(=O)O

FSIQDESGRQTFNN-BTJKTKAUSA-N

InChI=1S/C17H27NO2.C4H4O4/c1-5-7-18(8-6-2)15-9-13-11-16(19-3)17(20-4)12-14(13)10-15;5-3(6)1-2-4(7)8/h11-12,15H,5-10H2,1-4H3;1-2H,(H,5,6)(H,7,8)/b;2-1-

(Z)-but-2-enedioic acid;5,6-dimethoxy-N,N-dipropyl-2,3-dihydro-1H-inden-2-amine

U99194 maleate; U 99194 maleate; U-99,194 maleate; 234757-41-6; 5,6-Dimethoxy-2-(di-n-propylamino)indan maleate; U-99194 (maleate); 5,6-Dimethoxy-2-(di-n-propylamino)indan maleate salt; (Z)-but-2-enedioic acid;5,6-dimethoxy-N,N-dipropyl-2,3-dihydro-1H-inden-2-amine;U-99194 maleate

2934.99.03.00

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.)