Y5 receptor ( Ki = 1.3 nM ); Y2 receptor ( Ki = 200 nM ); Y1 receptor ( Ki > 4000 nM )

CGP71683 hydrochloride is a competitive antagonist of neuropeptide Y5 receptor with a Ki of 1.3 nM. It exhibits no discernible activity at the Y1 receptor (Ki, >4000 nM) or the Y2 receptor (Ki, 200 nM) in membranes of cells[1].
At present, no other Y5 antagonists than CGP 71683A has been publicly available, and therefore it has been used as a reference compound when studying the role of the Y5 receptor. CGP 71683A has been shown to be a competitive antagonist of the Y5 receptor in functional assays in cell lines and isolated organs (Criscione et al., 1998; Duhault et al., 2000; Dumont et al., 2000). In the present study, it had high affinity for the guinea-pig Y5 receptor without showing any significant activities at the Y1 and Y2 subtypes (Table 1). [1]
It has been reported that CGP 71683A binds to the serotonin (5-HT) re-uptake recognition site and cholinergic muscarinic receptors with virtually identical affinity to its binding to Y5 receptors (Della Zuana et al., 2001). [1]

CGP71683 (15 nmol/rat, icv, twice daily) exhibits anorexigenic effects, lowering fed rats' body weight and food intake. CGP71683 results in three times higher serum total T4 and a 37% increase in free T4 levels compared to the fasted control rats[2].

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Neuropeptide Y (NPY) inhibits TRH neurons in fed state, and hypothalamic NPY higher expression during fasting has been proposed to be involved in fasting-induced suppression of the hypothalamus-pituitary-thyroid (HPT) axis. We investigated the role of central Y5 receptors in the control of thyrotropin (TSH) and thyroid hormone (TH) secretion. Fed and fasting rats received twice daily central injections (3rd ventricle) of Y5 receptor antagonist (CGP71683; 15nmol/rat) for 72h. Fasted rats also received a single central injection of CGP71683 (15nmol/rat) at the end of 72h of fasting. In fed rats, Y5 receptor blockade reduced total food intake by 32% and body mass by almost 10% (p<0.01), corroborating the role of this receptor in food intake control. 72h-fasted rats exhibited a 4-fold increase in serum TSH (p<0.001), 1h after a single injection of Y5 antagonist. Also with multiple injections during 72h of fasting, Y5 blockade resulted in activation of thyroid axis, as demonstrated by a 3-times rise in serum T4 (p<0.001), accompanied by unchanged TSH and T3. In fed rats, the chronic central administration of CGP71683 resulted in reduced total serum T4 without changes in free T4 and TSH. Serum leptin and PYY were not altered by the NPY central blockade in both fed and fasted rats, suggesting no role of these hormones in the alterations observed. Therefore, the inhibition of central Y5 neurotransmission resulted in activation of thyroid axis during fasting suggesting that NPY-Y5 receptors contribute to fasting-induced TSH and TH suppression. [2]

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In the present study, it had high affinity for the guinea-pig Y5 receptor without showing any significant activities at the Y1 and Y2 subtypes (Table 1). When guinea-pigs were pretreated with CGP 71683A, the feeding response to NPY was markedly attenuated, suggesting that the compound is an effective antagonist of NPY-induced feeding mediated through Y5 receptors. Previous rodent data have also shown the inhibitory effects of CGP 71683A on NPY-induced food intake in lean and obese animals (Della Zuana et al., 2001; Duhault et al., 2000; Polidori et al., 2000) and on spontaneous food intake in diabetic, 24 h fasted and free-feeding animals (Criscione et al., 1998; Kask et al., 2001). [1]

Binding assays [1]
Cells lines transfected with plasmids encoding the guinea-pig receptors were used for studying the selectivity and the affinities of the ligands. The agonist data shown in Table 1 have been reported previously (Berglund et al., 1999; Lundell et al., 2001; Sharma et al., 1998). The antagonists BIBO 3304, H 409/22 and CGP 71683A were tested on membrane fractions prepared using the same protocol. For binding assays, thawed aliquots of receptor membranes were resuspended in 25 mM HEPES buffer (pH 7.4) containing 2.5 mM CaCl2, 1 mM MgCl2 and 2 g l−1 Bacitracin and homogenized using an Ultra-Turrax homogenizer. Binding experiments were performed in a final volume of 100 μl with 2 – 10 μg protein and 125I-PYY (porcine) for 2 h at room temperature. Non-specific binding was defined as the amount of radioactivity remaining bound to the cell homogenate after incubation in the presence of 100 nM unlabelled NPY. In competition studies, various concentrations of the non-peptide compounds BIBO 3304, H 409/22 or CGP 71683A were included in the incubation mixture, along with 125I-PYY. The peptide NPY was used as a reference for each experiment. Incubations were terminated by rapid filtration through GF/C filters, which had been pre-soaked in 0.3% polyethyleneimine, using a TOMTEC cell harvester. The filters were washed with 5 ml of 50 mM Tris (pH 7.4) at 4°C and dried at 60°C. The dried filters were treated with MeltiLex A melt-on scintillator sheets, and the radioactivity retained on the filters counted using the Wallac 1450 Betaplate counter. The results were analysed using the Prism software package.

Rats: CGP71683 is dissolved in 30% DMSO and, prior to the experiment, stored frozen at -20°C. Every microinjection comprises 2 microliters of either 30% DMSO vehicle or CGP71683 (7.5 nmol/μL; 15 nmol/rat) injected via the guide cannula over a 30- to 60-second period, adhering to the following protocols: I-Rats given free access to chow are given six microinjections (15 nmol/rat, spaced 10–14 hours apart) and are killed one hour after the final injection, which occurs between 9 and 10 a.m. The amount of food consumed is calculated by calculating the daily reduction in chow mass (g) right before each intravenous injection. II: After fasting for 72 hours, rats are given a single microinjection of either vehicle or CGP71683 (15 nmol/rat) and are then killed one hour later. III - Rats are given multiple injections of vehicle or CGP71683 over the course of a 72-hour fast, following the same protocol as fed animals. The fasting period began 10 hours prior to the first microinjection. Rats are decapitated at the conclusion of the experiments, and serum is extracted from the trunk blood to measure the hormone concentrations[2].

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BIBO 3304 (30 nmol per animal), H 409/22 (100 – 200 nmol per animal) or saline were infused i.c.v. in a volume of 5 – 7 μl given 15 min before NPY (3.6 nmol in 5 μl) or saline. When the effect of CGP 71683A (60 nmol per animal) was tested, 30% dimethylsulphoxide (DMSO) was used as the vehicle. CGP 71683A or DMSO were infused in a volume of 10 μl 15 min before NPY (3.6 nmol in 5 μl) or saline. Food consumption (4 h response) and eating parameters were measured as above. [1]

[1]. Receptor subtypes Y1 and Y5 mediate neuropeptide Y induced feeding in the guinea-pig. Br J Pharmacol. 2002 Apr;135(8):2029-37.

[2]. Central NPY-Y5 receptors activation plays a major role in fasting-induced pituitary-thyroid axis suppression in adult rat. Regul Pept. 2011 Nov 10;171(1-3):43-7.

Our observation that increased locomotion might be connected to Y5 receptor stimulation is in agreement with the previous finding showing that the Y5 antagonist, CGP 71683A, decreases exploratory behaviour in rats (Kask et al., 2001). The suppression of food intake after CGP 71683A administration is not, however, related to the changes in locomotor activity (Kask et al., 2001). It has been reported that CGP 71683A binds to the serotonin (5-HT) re-uptake recognition site and cholinergic muscarinic receptors with virtually identical affinity to its binding to Y5 receptors (Della Zuana et al., 2001). Furthermore, chronic administration of CGP 71683A may produce local inflammatory changes near the site of injection (Della Zuana et al., 2001). Due to these non-specific actions and its poor solubility, CGP 71683A is not a good tool to use in vivo, and new selective Y5 antagonists are needed to characterize the physiological importance of the Y5 receptor. Recent reports on novel agents blocking Y5 receptors do not entirely support the view that Y5 receptors are crucial for NPY-induced feeding in rats (Kanatani et al., 2000a; Polidori et al., 2000). Therefore, our results with CGP 71683A in the guinea-pig should be regarded with caution until investigated with other Y5 antagonists. [1]

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Interestingly, CGP71683-treated fasted rats lost more weight than vehicle-treated fasted rats, supporting an involvement of Y5 receptors in energy expenditure independent of food consumption. Other studies have proposed that NPY via Y5 receptors decreases energy expenditure by affecting thermogenesis in brown and white adipose tissue. [2]

C26H30CLN5O2S

512.07

511.181

C, 60.98; H, 5.91; Cl, 6.92; N, 13.68; O, 6.25; S, 6.26

192322-50-2

192321-23-6; 192322-50-2 (HCl)

9849276

White to off-white solid powder

747.2ºC at 760 mmHg

405.7ºC

1.19E-22mmHg at 25°C

7.489

4

7

7

35

751

0

Cl[H].S(C1=C([H])C([H])=C([H])C2=C([H])C([H])=C([H])C([H])=C12)(N([H])C([H])([H])C1([H])C([H])([H])C([H])([H])C([H])(C([H])([H])N([H])C2N=C(C3=C([H])C([H])=C([H])C([H])=C3N=2)N([H])[H])C([H])([H])C1([H])[H])(=O)=O

DIQDKUNCSVFGHH-UHFFFAOYSA-N

InChI=1S/C26H29N5O2S.ClH/c27-25-22-9-3-4-10-23(22)30-26(31-25)28-16-18-12-14-19(15-13-18)17-29-34(32,33)24-11-5-7-20-6-1-2-8-21(20)24;/h1-11,18-19,29H,12-17H2,(H3,27,28,30,31);1H

N-[[4-[[(4-aminoquinazolin-2-yl)amino]methyl]cyclohexyl]methyl]naphthalene-1-sulfonamide;hydrochloride

CGP-71683A HCl; CGP71683A; CGP-71683A; CGP 71683A

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO: 100~160 mg/mL (195.3~312.5 mM)

Solubility in Formulation 1: ≥ 2.67 mg/mL (5.21 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 26.7 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: ≥ 2.67 mg/mL (5.21 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 26.7 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: ≥ 2.67 mg/mL (5.21 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 26.7 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.)