human 5-HT1B Receptor (pKi = 8.85); human 5-HT1D Receptor (pKi = 8.31)

For h5-HT1B, GR 125743 has a Kd of 0.61 nM[1].
Saturation binding experiments were performed with [3H]GR 125743, a selective 5-HT1B/1D ligand, to membrane preparations of COS-7 cells transiently expressing either the wt h 5-HT1B or the chimeric 5-HT1B/2A and 5-HT1B/2AΔ44 receptors, and with [3H]ketanserin to membranes of human embryonic kidney 293 cells stably expressing the wt h 5-HT2A receptor. The [3H]GR 125743 equilibrium dissociation constants of both chimera were similar to that of the wt h 5-HT1B receptor (Table 1). The maximal binding capacity of the wt h 5-HT1B receptor was 3- to 4-fold lower than that of the chimeric 5-HT1B/2A and 5-HT1B/2AΔ44 receptors and 3-fold higher than that of the wt h 5-HT2A receptor (Table 1). A series of nine 5-HT ligands was tested for inhibition of [3H]GR 125743 binding to wt h 5-HT1B, 5-HT1B/2A, and 5-HT1B/2AΔ44 receptors and compared to their binding affinities for the wt h 5-HT1D and h 5-HT2A receptors (Table 2). The chimeric 5-HT1B/2A receptor yielded a 21-fold increased binding affinity for the 5-HT2 antagonist ketanserin as compared to the wt h 5-HT1B receptor. This binding affinity is close (3-fold lower) to what is observed for the wt h 5-HT1D receptor but 87-fold lower than for the wt h 5-HT2A receptor (Table 2). The structurally related piperidine derivative ritanserin yielded an almost similar binding affinity as compared to the wt h 5-HT1B receptor, this value being 3- and 47-fold lower than for the wt h 5-HT1D and h 5-HT2A receptors, respectively. This ketanserin binding feature was lost when the last 44 amino acids of the 5-HT2A receptor-derived C-terminal portion were truncated in the chimeric 5-HT1B/2AΔ44 receptor. The binding affinities of the 5-HT antagonists GR 125743 and SB-224289 were not modified in either chimeric 5-HT receptor as compared to the parental h 5-HT1B receptor. The binding affinities of the agonists F 11356, zolmitriptan, 5-HT, and sumatriptan were slightly increased (1.6- to 6-fold) at both chimeric receptors as compared to the wt h 5-HT1B receptor. The 5-HT2 agonist DOI (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane) was inactive at either the wt h 5-HT1B, h 5-HT1D, or chimeric 5-HT1B receptor [1].

In conscious guinea pigs, GR 125743 (0.3 mg/kg; i.p.) dramatically lowers extracellular 5-HT in the frontal cortex [2].

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The role of 5-HT(1B/1D) receptors in modulating extracellular 5-hydroxytryptamine (5-HT) levels in the guinea pig was investigated with the non-selective 5-HT(1B/1D) receptor inverse agonist, methiothepin, and the selective 5-HT(1B/1D) receptor partial agonists, GR 127935 (n-[4-methoxy-3-(4-methyl-1-piperizinyl)phenyl]-2'-methyl-4'-(5-me thyl-1,2,4-oxadiazole-3-yl)[1,1'-biphenyl]-4-carboxamide) and GR 125743 (n-[4-methoxy-3-(4-methyl-1-piperizinyl)phenyl]-3-methyl-4-(4-pyri dinyl)benzamide). Extracellular 5-HT levels were measured using the technique of brain microdialysis, in the frontal cortex of the freely moving guinea-pig. Extracellular 5-HT was tetrodotoxin sensitive and calcium dependent, and increased when perfused with a high concentration of K+. In addition, extracellular 5-HT levels were lowered by the 5-HT(1B/1D) receptor agonist, sumatriptan, and the 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin, while perfusion of the selective serotonin re-uptake inhibitor, paroxetine, increased 5-HT in a concentration-dependent manner. Perfusion of methiothepin, GR 127935 and GR 125743 into the frontal cortex caused significant but transient increases of extracellular 5-HT. However, systemic administration of methiothepin, GR 127935 and GR 125743, at 0.3 mg/kg i.p., produced significant decreases in extracellular 5-HT, to minima of 27 +/- 3%, 31 +/- 12% and 27 +/- 13% of basal, respectively. The increase of extracellular 5-HT, following 5-HT(1B/1D) receptor inverse and partial agonist perfusion into the frontal cortex, was probably a consequence of attenuation of an endogenous 5-HT tone at terminal 5-HT autoreceptors. The unexpected decrease in 5-HT levels following systemic administration may be a result of additional attenuation of endogenous 5-HT tone at cell body autoreceptors in the raphe. Such an increase in local 5-HT levels could then stimulate 5-HT1A receptors to inhibit cell firing and hence decrease 5-HT levels in the terminal regions. This was confirmed when co-administration of the 5-HT1A receptor antagonist, WAY 100635, significantly attenuated the GR 127935 decrease in 5-HT[2].

Expression of 5-HT receptors and radioligand binding experiments [1]
COS-7 cells (5 × 106 cells) were transfected with 10 μg of either plasmid pcDNA3/h 5-HT1B, pCR3.1/5-HT1B/2A, or pCR3.1/5-HT1B/2AΔ44 by electroporation as previously described. A human embryonic kidney 293 cell line stably expressing the h 5-HT2A receptor was grown in complete Dulbecco’s modified Eagle’s medium and selected on 1.25 mg/mL geneticin as described. Binding assays were performed with either 1.0 nM [3H] N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyridil)-benzamide (GR 125743) for 5-HT1B receptor binding or 1.0 nM [3H]ketanserin for 5-HT2A receptor binding. Incubation mixtures consisted of 0.4 mL of cell membrane, 0.05 mL of radioligand, and 0.05 mL of compound for inhibition or 10 μM 5-HT to determine non-specific binding. The reactions were performed as described. Data were analyzed graphically with inhibition curves and ic50 values were derived as the concentration of the compound producing 50% inhibition of specific radioligand binding. Inhibition constants Ki were calculated according to the equation Ki = ic50/(1 + C/Kd), with C the concentration and Kd the equilibrium dissociation constant of the radioligand. The Kd values were obtained from saturation binding experiments performed as described. Membrane protein levels were estimated with a dye-binding assay using the BioRad protein assay kit and BSA as a standard.

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Two chimeric 5-hydroxytryptamine (5-HT) receptors were constructed by exchanging the C-terminal portion of the human (h) 5-HT(1B) receptor with the equivalent domain of the h 5-HT(2A) receptor (5-HT(1B/2A)) or with this domain truncated from its last 44 amino acids (5-HT(1B/2ADelta44)). The equilibrium dissociation constant of the radioligand [(3)H]GR 125743 was similar for both chimera compared to the wild-type (wt) h 5-HT(1B) receptor upon transient expression in COS-7 cells. Ketanserin binding affinity was 21-fold increased from pK(i): 5.79 (wt h 5-HT(1B) receptor) to pK(i): 7.11 at the 5-HT(1B/2A) chimeric receptor, this latter value being close to that of the wt h 5-HT(1D) receptor (pK(i): 7.62). This enhanced ketanserin binding affinity was lost when the last 44 C-terminal amino acids of the 5-HT(2A) receptor were deleted in the chimera 5-HT(1B/2ADelta44) (pK(i): 5.80). The binding affinities of the 5-HT antagonists ritanserin, GR 125743, and SB-224289 were not modified at either chimeric 5-HT receptor. The agonists F 11356, 5-HT, zolmitriptan, and sumatriptan yielded slightly increased (2- to 6-fold) binding affinities at both chimera as compared to the wt h 5-HT(1B) receptor. The present data suggest a role for the C-terminal intracellular receptor domain in modifying ketanserin/5-HT(1B) receptor interactions[1].

Animal/Disease Models: Male Dunkin Hartley guinea pig (350-450 g) [2]
Doses: 0.3 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: A significant transient increase in extracellular 5-HT production.

[1]. Induction of a high-affinity ketanserin binding site at the 5-Hydroxytryptamine(1B) receptor by modification of its carboxy-terminal intracellular portion. Characterization of human serotonin 1D and 1B receptors using [3H]-GR-125743, a nov.

[2]. The role of 5-HT1B/1D receptors in the modulation of 5-hydroxytryptamine levels in the frontal cortex of the conscious guinea pig. European Journal of Pharmacology. 1997 May 12;326(1):23-30.

In conclusion, we report here on the modulation of ketanserin binding affinity at the h 5-HT1B receptor by exchanging its C-terminal intracellular portion with that of the h 5-HT2A receptor. These data suggest the possible importance of extramembrane domains in ligand binding for the 5-HT1B receptor. This concept may advance the development of tridimensional models of G protein-coupled receptors. [1]

C25H28N4O2

416.53

416.221

C, 72.09; H, 6.78; N, 13.45; O, 7.68

148547-33-5

148547-33-5;

5311130

White to off-white solid powder

1.2±0.1 g/cm3

541.2±50.0 °C at 760 mmHg

281.1±30.1 °C

0.0±1.4 mmHg at 25°C

1.623

2.97

1

5

5

31

574

0

GNOXPYACARZYMW-UHFFFAOYSA-N

InChI=1S/C25H28N4O2/c1-18-16-20(4-6-22(18)19-8-10-26-11-9-19)25(30)27-21-5-7-24(31-3)23(17-21)29-14-12-28(2)13-15-29/h4-11,16-17H,12-15H2,1-3H3,(H,27,30)

N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-pyridin-4-ylbenzamide

GR-125,743; GR125,743; GR 125743; 148547-33-5; GR 125,743; GR125743; GR-125743; N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-pyridin-4-ylbenzamide; N-(4-METHOXY-3-(4-METHYLPIPERAZIN-1-YL)PHENYL)-3-METHYL-4-(PYRIDIN-4-YL)BENZAMIDE; N-(4-methoxy-3-(4-methylpiperazin-1-yl)phenyl)-3-methyl-4-pyridin-4-ylbenzamide; GR 125,743

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~100 mg/mL (~240.08 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.00 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 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.00 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.00 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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