rNPY Y1 receptor (Ki = 1.1 nM); hNPFF2 (Ki = 79 nM); rNPFF (Ki = 108 nM)

In the present study, researchers found that among several ligands of the NPY family, frog PP, GR231118 and BIBP3226, were able to compete for the specific NPFF binding expressed in CHO cells as well as in rat dorsal spinal cord. Interestingly, the apparent affinity of the selective NPY Y1/Y4 ligand GR231118 for NPFF2 receptors (Ki=50 – 70 nM) is comparable to its well established affinities for the NPY Y2 (Ki=63 nM) and Y5 (Ki=100 nM) receptors (Parker et al., 1998 and Table 1). Moreover, the apparent affinity (Ki about 100 nM) of the selective NPY Y1 receptor antagonist BIBP3226 for NPFF2 receptors is much greater than those reported on the NPY Y2, Y4 and Y5 receptors, (Schober et al., 1998; Dumont et al., 2000c and Table 1). Furthermore, we observed relatively high affinities (Ki=1.5 – 7 nM) for fPP and its truncated analogue fPP28-36 for NPFF receptors. This is likely explained by the presence of Arg-Phe-amide residues at the C-terminus of the peptides instead of the usual Arg-Tyr-amide residues present in all mammalian pancreatic polypeptides as well as in NPY and PYY. To our knowledge, no data on the affinity of fPP for NPY receptor subtypes is available in the literature. Interestingly, researchers observed that the affinities of BIBP3226 and fPP for the hNPFF2 receptors expressed in CHO cells and for the rat spinal cord receptors (suspected to be of the NPFF2 receptor subtype; Bonini et al., 2000), were 10 fold better than those reported on human and rat NPFF2 receptors expressed in HEK 293 cells (Bonini et al., 2000). This apparent discrepancy remains to be explained. [1]
The functional properties of fPP, GR231118 and BIBP3226 were investigated next on the basis of cyclic AMP accumulation assays in hNPFF2 receptors transfected cells. Interestingly, fPP and GR231118 exhibit agonistic activity. Although the potency of GR231118 is 500 – 1000 fold lower than that observed for NPY Y4 receptors (Parker et al., 1998; Schober et al., 1998), it is in the same range order than those described for Y2 and Y5 NPY receptors (Parker et al., 1998). On the other hand, the Y1 antagonist BIBP32266 which is inactive by itself at up to 10 μM, is able to antagonize in a concentration-dependent manner the inhibition of forskolin-stimulated cyclic AMP production induced by NPFF (10 nM). Hence, BIBP3226 is the first antagonist to be reported for NPFF2 receptors and could therefore be considered as a lead compound in an effort to develop more potent antagonists for the NPFF2 receptor subtype. [1]

BIBP3226 (0.5, 5 μg; icv) has anxiolytic-like effects at corresponding doses [1].

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The effect of the novel non-peptide neuropeptide Y Y1 receptor antagonist BIBP3226, N2-(diphenylacetyl)-N-[(4-hydroxy-phenyl)-methyl]-D-arginine amide, on exploratory behaviour of rats in the elevated plus-maze was studied. BIBP3226 (0.5 and 5 micrograms, i.c.v.) induced an anxiogenic-like effect at the higher dose tested. This effect was antagonised by diazepam (0.5 mg/kg). The anxiogenic-like effect of BIBP3226 was not related to a decrease in general locomotor activity. These findings support the hypothesis that neuropeptide Y Y1 receptor subtype is involved in anxiety regulation. [2]

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The aim of the present study was to investigate the effect of BIBP3226 on exploratory behaviour and to test the hypothesis that blockade of neuropeptide Y Y1 receptors in vivo will lead to appearance of anxiety signs. Male Wistar rats (270–350 g), purchased from Grindex, Latvia, were kept under standard laboratory conditions and implanted with chronic injection cannulae aimed at lateral ventricles. The behavioural experiments were started 1 week after surgery. BIBP3226 was injected i.c.v. 20 min and diazepam (0.5 mg/kg i.p.) 30 min before the test. The plus-maze and open field test design was as described elsewhere (Harro et al., 1990) but 1 cm ledges were added to open arms of the maze. As shown in Table 1, BIBP3226 at the dose of 5 μg caused an anxiogenic-like effect while the lower dose was ineffective. In the second experiment we found that the dose of diazepam (0.5 mg/kg) that itself had no effect on exploratory parameters as a single treatment completely blocked the anxiogenic-like effect of the neuropeptide Y Y1 receptor antagonist. Immediately after the plus-maze test the rats were subjected to an open-field test for 4 min. Since the overall locomotory activity was similar in all treatment groups (data not shown), the anxiogenic profile of BIBP3226 and its antagonism with diazepam cannot be interpreted as the changes in general locomotor activity due to, e.g., a neurotoxic effect. The overall activity of the animals was higher in the second experiment when i.p. injection preceded i.c.v. injection. Such an increase in exploratory behaviour may occur when animals are handled or exposed to other stimuli before the test (Hogg, 1996).[2]

Transfected HEK 293 cells were maintained in Dulbecco's modified Eagle medium supplemented with 10% foetal calf serum and amphotericin B. All NPY binding assays were performed as previously described (Dumont et al., 2000a). [1]
Recombinant CHO cells expressing the human NPFF2 receptor were grown in Ham's F12 medium supplemented with 7% foetal calf serum and G418 400 μg ml−1. For membrane preparation, cells were harvested in phosphate buffer saline, frozen at −70°C, and homogenized in 50 mM Tris-HCl, pH 7.4 in a Potter Elvehjem tissue grinder. The nuclear pellet was discarded by centrifugation at 1000×g for 15 min at 4°C, and the membrane fraction was collected upon centrifugation of the supernatant at 100,000×g for 30 min at 4°C. Binding of [125I]-EYF ([125I]-EYWSLAAPQRF-NH2), a new specific radioligand for NPFF receptors (2000 Ci mmole; Gouardères et al., 2001) on membranes (2 μg) of hNPFF2 receptor expressing CHO cells was measured by rapid filtration as described (Gouardères et al., 2001). Binding on NPFF receptors in rat (male Sprague-Dawley, 350 g) spinal cord sections was exactly as described in Gouardères et al. (2001). [1]
Assay for intracellular cyclic AMP was performed essentially as described in (Mollereau et al., 1999). Briefly, 200,000 recombinant cells were incubated for 1 h at 37°C under 5% CO2 with 0.6 μCi [3H]-adenine (26 Ci/mmole Amersham) in Ham's F12 medium. Cyclic AMP production was stimulated by 2 μM Forskolin for 10 min at 37°C in 200 μl HEPES buffered Krebs-Ringer saline in the presence of 0.1 mM of the phosphodiesterase inhibitors, IBMX and Ro-20 1724. Ligands to be tested were added at the same time at the desired concentration. The reaction was stopped by addition of 20 μl HCl 2.2 N and intracellular [3H]-cyclic AMP was isolated by chromatograhic procedure on acid alumina columns. [1]

Animal/Disease Models: Male Wistar rat 270-350 g[1]
Doses: 0.5, 5 μg
Route of Administration: Icv
Experimental Results: At a dose of 5 μg, it caused an anxiety-like effect, while lower doses were ineffective.

[1]. Agonist and antagonist activities on human NPFF(2) receptors of the NPY ligands GR231118 and BIBP3226. Br J Pharmacol. 2001 May;133(1):1-4.

[2]. Anxiogenic-like effect of the neuropeptide Y Y1 receptor antagonist BIBP3226: antagonism with diazepam. Eur J Pharmacol. 1996 Dec 19;317(2-3):R3-4.

The findings support the hypothesis that endogenous neuropeptide Y can alleviate novelty fear/anxiety by activating neuropeptide Y1 receptors. Diazepam, a clinically effective anti-anxiety drug, antagonizes the anxiolytic-like effects of BIBP3226. This suggests that neuropeptide Y neurotransmission may be closely related to the GABAergic system. From a neuroanatomical perspective, this interaction may involve cortical regions, as many cortical neurons that are immunoreactive to neuropeptide Y also contain GABA (Hendry et al., 1984). Studies have proposed that neurons release neuropeptide Y during high-frequency firing when animals are exposed to new environments (Lundberg et al., 1986). Neuropeptide Y itself, or other endogenous ligands acting on neuropeptide Y1 receptors, may be crucial for adaptation to new environments and the expression of normal exploratory behavior. Therefore, blocking neuropeptide Y1 receptors using BIBP3226 may interfere with the adaptation process and lead to anxiety expression. In summary, these results confirm that the neuropeptide Y1 receptor is involved in regulating exploratory behavior and/or anxiety in rats. BIBP3226 appears to be a useful compound for studying the role of the neuropeptide Y1 receptor in emotional processes. [2] In this study, we found that among several NPY family ligands, frog PP, GR231118 and BIBP3226 competitively bind to specific NPFF expressed in CHO cells and rat dorsal spinal cord. Interestingly, the epigenetic affinity (Ki=50-70 nM) of the selective NPY Y1/Y4 ligand GR231118 for the NPFF2 receptor is comparable to its known affinity for the NPY Y2 (Ki=63 nM) and Y5 (Ki=100 nM) receptors (Parker et al., 1998; Table 1). Furthermore, the selective NPY Y1 receptor antagonist BIBP3226 exhibits a significantly higher apparent affinity (Ki approximately 100 nM) for the NPFF2 receptor than previously reported for NPY Y2, Y4, and Y5 receptors (Schober et al., 1998; Dumont et al., 2000c; Table 1). Additionally, we observed relatively high affinity (Ki = 1.5–7 nM) for fPP and its truncated analogue fPP28–36 for the NPFF receptor. This is likely due to the presence of Arg-Phe-amide residues at the C-terminus of the peptide chain, rather than the Arg-Tyr-amide residues commonly found in all mammalian pancreatic peptides and in NPY and PYY. To the best of our knowledge, there are no data in the literature regarding the affinity of fPP for NPY receptor subtypes. Interestingly, we observed that BIBP3226 and fPP showed a 10-fold higher affinity for the hNPFF2 receptor expressed in CHO cells and the rat spinal cord receptor (presumably an NPFF2 receptor subtype; Bonini et al., 2000) than for the human and rat NPFF2 receptors reported in HEK 293 cells (Bonini et al., 2000). This significant difference remains to be explained. Next, we investigated the functional properties of fPP, GR231118, and BIBP3226 based on an assay of cyclic adenosine monophosphate (cAMP) accumulation in hNPFF2 receptor-transfected cells. Interestingly, both fPP and GR231118 exhibited agonistic activity. Although GR231118 is 500-1000 times less potent than the NPY Y4 receptor (Parker et al., 1998; Schober et al., 1998), it is on the same order of magnitude as the NPY Y2 and Y5 receptors (Parker et al., 1998). On the other hand, the Y1 antagonist BIBP3226 is inactive at concentrations up to 10 μM, but it can antagonize the inhibition of NPFF (10 nM)-induced forosclin-stimulated cyclic adenosine monophosphate (cAMP) production in a concentration-dependent manner. Therefore, BIBP3226 is the first reported NPFF2 receptor antagonist and can be considered a lead compound for developing more effective NPFF2 receptor subtype antagonists. Our data indicate that NPFF receptors are associated not only with NPY (especially Y1 and Y4) receptors in sequence homology but also with them in binding affinity and functional properties. Both receptor families may retain an ancestral binding pocket that has evolved into an Arg-Phe-amide or Arg-Tyr-amide interaction. This hypothesis should be verified through detailed mutagenesis and structure-activity relationship studies. NPY agonists are known to stimulate appetite (Dumont et al., 2000c). In contrast, the only report on the effects of NPFF on feeding behavior describes its reduction of food intake in rats (Murase et al., 1996). Similarly, GR231118, while acting as an NPY Y4 agonist, has been found to reduce food intake in rats (Schober et al., 1998). Whether this effect is due to interaction with NPFF receptor subtypes needs to be investigated in future studies. In summary, our findings describe the first NPFF receptor antagonist (BIBP3226) and suggest that cross-reactivity with the NPFF receptor may exist between BIBP3226 and GR231118 when studying the NPY receptor.

C29H32F3N5O5

587.590097427368

587.235

C, 59.28; H, 5.49; F, 9.70; N, 11.92; O, 13.61

1068148-47-9

BIBP3226;159013-54-4

56972180

White to off-white solid powder

6

9

11

42

742

1

FC(C(=O)O)(F)F.O=C(C(C1C=CC=CC=1)C1C=CC=CC=1)N[C@@H](C(NCC1C=CC(=CC=1)O)=O)CCC/N=C(\N)/N

MTSZIDSCWZHKOD-GNAFDRTKSA-N

InChI=1S/C27H31N5O3.C2HF3O2/c28-27(29)30-17-7-12-23(25(34)31-18-19-13-15-22(33)16-14-19)32-26(35)24(20-8-3-1-4-9-20)21-10-5-2-6-11-21;3-2(4,5)1(6)7/h1-6,8-11,13-16,23-24,33H,7,12,17-18H2,(H,31,34)(H,32,35)(H4,28,29,30);(H,6,7)/t23-;/m1./s1

(2R)-5-(diaminomethylideneamino)-2-[(2,2-diphenylacetyl)amino]-N-[(4-hydroxyphenyl)methyl]pentanamide;2,2,2-trifluoroacetic acid

BIBP-3226 TFA; BIBP 3226 TRIFLUOROACETATE; 1068148-47-9; BIBP3226 (TFA); (2R)-5-(diaminomethylideneamino)-2-[(2,2-diphenylacetyl)amino]-N-[(4-hydroxyphenyl)methyl]pentanamide;2,2,2-trifluoroacetic acid; MFCD00938562; BIBP3226trifluoroacetate; BIBP3226 Trifluoroacetate; 159013-54-4 (non-salt); BIBP 3226 TFA

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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 (~170.19 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.54 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 20.8 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: ≥ 2.08 mg/mL (3.54 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 20.8 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: ≥ 2.08 mg/mL (3.54 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 20.8 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.)