Neuropeptide Y (NPY) Y1 receptor

1. The novel Y1-selective argininamide derivative BIBO 3304 ((R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2-(diphen ylacetyl)-argininamide trifluoroacetate) has been synthesized and was examined for its subtype selectivity, its in vitro antagonistic properties and its food intake inhibitory properties. 2. BIBO 3304 displayed subnanomolar affinity for both the human and the rat Y1 receptor (IC50 values 0.38+/-0.06 nM and 0.72+/-0.42 nM, respectively). The inactive enantiomer of BIBO 3304 (BIBO 3457) had low affinity for both the human and rat Y1 receptor subtype (IC50> 1000 nM). BIBO 3304 showed low affinity for the human Y2 receptor, human and rat Y4 receptor as well as for the human and rat Y5 receptor (IC50 values > 1000 nM). [1]

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In order to test the physiological relevance of Y1 receptor signaling in human islets, human islets were cultured and stimulated with glucose in the presence or absence of the Y1 receptor antagonist BIBO3304. Similar to rodent islets, human islets treated with BIBO3304 exhibited enhanced insulin secretion in response to a glucose challenge [2]. As shown in Fig. 4f, cAMP was significantly upregulated in the presence of 100 nM Exenatide and 20 mM glucose compared to glucose alone. The addition of 50 nM PYY was able to significantly reduce the Exenatide-induced increase in cAMP and this inhibitory effect was abolished in the presence of the Y1 receptor specific antagonist BIBO3304 [2].

BIBO3304 TFA (30 μg; bilateral paraventricular nucleus injection) reduces postfasting hyperphagia [1]. BIBO3304 TFA (15-60 μg) dose-dependently inhibits the feeding response mediated by 1 μg NPY [1]. BIBO3304 TFA (0.5 μM; orally) significantly elevates blood insulin levels [2].

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3. 30 microg BIBO 3304 administered into the paraventricular nucleus inhibited the feeding response induced by 1 microg NPY as well as the hyperphagia induced by a 24 h fast implying a role for Y1 receptors in NPY mediated feeding. The inactive enantiomer had no effect. 4. BIBO 3304 inhibits neither the galanin nor the noradrenaline induced orexigenic response. but it blocked feeding behaviour elicited by both [Leu31, Pro24]NPY and NPY (3 36) suggesting an interplay between different NPY receptor subtypes in feeding behavior. 5. The present study reveals that BIBO 3304 is a subtype selective nonpeptide antagonist with subnanomolar affinity for the Y1 receptor subtype that significantly inhibits food intake induced by application of NPY or by fasting [1].

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Mice that received BIBO3304 exhibited significantly increased serum insulin levels (Fig. 1l, m) confirming that blocking Y1 receptor signaling is able to enhance physiologically triggered insulin secretion. To further test whether this pharmacological intervention could improve the outcome of islet transplantation, we repeated the transplantation experiments using a minimal number of WT islets, and then treated half of the mouse cohort orally with BIBO3304 and the other half with placebo. As a control for islet quality, a group of mice were transplanted with an optimal islet mass. Recipient mice that were treated with placebo failed to achieve normoglycemia and remained diabetic throughout the course of the experiment (Fig. 2a, b). In contrast, mice transplanted with the minimal amount of WT islets and treated with BIBO3304 rapidly achieved normoglycemia (Fig. 2a). Strikingly, when antagonist treatment was stopped at day 10 post-transplantation these mice were able to maintain normoglycemia till the end of the experiment at day 60 (Fig. 2a). This suggests that transient inhibition of Y1 receptor signaling in the early period after islet transplantation is sufficient to normalize glucose homeostasis. Consistent with the improved glycemic control, glucose tolerance at day 5 post-transplantation was also significantly improved in the BIBO3304-treated group compared to the placebo group (Fig. 2c, d). Consistently, BIBO3304-treated mice also showed a superior DIo being 12.28-fold higher than placebo-treated mice (DIo = 0.066 ± 0.0082 vs. 0.819 ± 0.324 for placebo vs. BIBO3304, respectively; n = 4 mice per group). Analysis of BIBO3304-treated compared to untreated islet grafts did not reveal any obvious differences in islet morphology, apoptosis, graft vascularization, or endoplasmic reticulum (ER) stress response (Supplementary Figs. 3, 4a), however, the number of Ki67-positive β-cells in the BIBO3304-treated grafts was significantly increased (Fig. 2e, f). Together, these data demonstrate that the improved glycemic control in BIBO3304-treated mice was a consequence of increased insulin secretion and islet proliferative capacity in response to changes in physiological glucose levels (Fig. 2e–h)[2].

Human Y1 receptor stably expressed in baby hamster kidney (BHK) cells [1]
Cells were grown in DMEM with 4.5 g/l glucose, 10% fetal calf serum, 1% PENStrep, 1 mg ml71 G-418, 1 mg ml71 hygromycin B. 96 h before receptor binding assay 1 mM isopropylthiogalactoside (IPTG) was added in order to induce expression (Lac Switch Expression System from Stratagene). Con¯uent cells were removed with 0.06% EDTA/PBS (1 min incubation) and resuspended in 15 ml incubation bu€er (MEM/25 mM HEPES+1% bovine serum albumine, 50 mM PMSF, 0.1% bacitracin, 3.75 mM CaCl2). After 10 min centrifugation at RT (150 6 g), the pellet was resuspended in 50 ml incubation bu€er, respun and resuspended in 30 ml incubation bu€er. After counting, the cells were diluted to a ®nal concentration of 2.5 6 105 cells ml71 . Two hundred microliters of this cell suspension was incubated 3 h at RT with 30 pM [ 125I]NPY and increasing concentrations of test compounds (10713 ± 1074 M) in a total volume of 250 ml. The incubation was stopped by 10 min centrifugation, 3000 6 g at 48C. The pellet was resuspended with 0.25 ml PBS recentrifuged and the pellet measured in a g-counter.

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Rat Y1 receptor expressing human embryonic kidney (HEK) 293 cells [1]
Confluent cells were removed with 0.02% EDTA/PBS and resuspended in 10 ml incubation bu€er (MEM/25 mM HEPES+0.5% BSA, 50 mM PMSF, 0.1% bacitracin, 3.75 mM CaCl2). After 5 min centrifugation (150 6 g) the pellet was resuspended in equal volume and after further centrifugation in 10 ml incubation bu€er. The cells were diluted to a concentration of one mio cells ml71 . 100 ml of this cell suspension was incubated 3 h at RT with 30 pM [ 125I]NPY solutions and increasing concentrations of test compounds in a total volume of 250 ml. The incubation was stopped as described for rat hypothalamus.

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Human Y5 receptor stably transfected in HEK 293 cells [1]
Centrifuged cells were cultivated as described for BHK/Y1 cells except that a concentration of 0.7 mg ml71 G-418 was used, no hygromycin added and IPTG induction was not necessary. The incubation bu€er cell cultivation and receptor binding was performed as described. Final concentration was 550 H.A. Wieland et al Novel Y1 receptor antagonist BIBO 3304 and its effect on feeding 1.5 6 106 cells ml71 and centrifugation stopped as described for Y1/BHK cells.

Animal/Disease Models: Adult male Chbb:Thom rat, body weight 300 to 340 g [1]
Doses: 30 μg
Route of Administration: Bilateral paraventricular nucleus injection
Experimental Results: Hyperphagia after fasting was attenuated, especially before refeeding Within 2 hrs (hrs (hours)).

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Animal/Disease Models: 7weeks old C57BL/6JAusb mice[2]
Doses: 0.5 μM
Route of Administration: Oral
Experimental Results:Serum insulin levels were Dramatically increased.

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Food-intake studies Adult male Chbb:Thom rats weighing between 300 and 340 g were individually housed and maintained on a 12 : 12 h lightdark cycle beginning at 06.00 h. Tap water and standard laboratory chow were available throughout except in the experiments where the animals were fasted for 24 h. After 1 week of habituation to their new housing conditions, the animals were anaesthetized with sodium pentobarbital (60 mg kg71 , i.p) for the placement of stainless steel guide cannulae. Bilateral guide cannulae (26 gauge) were placed 1 mm above the paraventricular nucleus according to the stereotaxic coordinates (Paxinos & Watson, 1986): AP:71.8, L:0.5, V:7.0. Guide cannulae were maintained in place on the skull with small metal screws and dental acrylic cement. Cannulae were closed with a stainless steel xstylet when not in use. Rats were allowed to recover for at least 1 week and were adapted to the injection procedure. On the day of the experiments drugs were injected between 08.00 and 09.00 h. Injection cannulae (33 gauge) were inserted 1 mm beyond the tips of the guide cannulae. The injection cannulae were attached by polyethylene tubing to a Hamilton microsyringe mounted in an infusion pump. Injection volume was 0.5 ml infused with a rate of 0.0125 ml s71 . In the ®rst set of experiments groups of 6 ± 12 rats received increasing doses (0.5 ± 32 mg, unilateral) of NPY receptor agonists into the PVN and food intake was monitored for at least 2 h. On the ®rst treatment day the groups were randomly assigned to the various doses. Rats had a wash-out period of at least 3 days between injections, after which the groups were randomized again to test the next agonist. Not more than 5 ± 6 injections were given in total. In the second set of experiments BIBO 3304 or its inactive enantiomer were given 10 min before the injection of di€erent NPY receptor agonists, galanin or noradrenaline. All compounds were applied into the PVN and for each experiment 8 ± 22 rats were used and for each dose a di€erent group of rats were used. In the last series of experiments BIBO 3304 was given to animals which were fasted for 24 h. Five minutes after bilateral PVN injection of BIBO 3304 the rats (n=12) were given free access to food and food intake was monitored for another 24 h.[1]

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Y1lox/lox mice were generated as previously described and crossed with mice expressing the Cre recombinase gene under the control of the rat insulin-2 promoter Tg(Ins2-cre)25Mgn/J(INS2cre/+) to generate Y1lox/lox/INS2cre/+ mice. INS2cre/+ mice were also crossed onto Gt(ROSA)26Sor tm9(EGFP/Rpl10a)Amc/J mice in order to produce β-cell specific expression of the EGFP–L10a fusion protein for mRNA isolation and qPCR analysis. For clarity, islet tissue from these mice is referred to as WT and Y1−/− respectively, throughout the text. Age-matched and sex-matched mice on a C57BL/6Ausb background were used for all experiments except stated otherwise. Female NOD mice were housed under a controlled temperature of 22 °C and a 12-hour light cycle (lights on from 0700 to 1900 hours) with ad libitum access to water and a standard chow diet (6% calories from fat, 21% calories from protein, 71% calories from carbohydrate, 14.0 MJ/kg). To avoid the stress caused by gavage, specific Y1 receptor antagonist BIBO 3304 was dissolved in distilled water and administrated daily in a form of jelly for the duration stated in the text. This method of drug delivery was developed in our lab and described previously31. In brief, we trained mice to voluntarily eat a vehicle jelly before the start of an experiment. After 2–5 days training, over 95% of mice consumed the entire portion of jelly (195 μl for a 25 g mouse) within 1 min of being placed in the cage and maintained a high avidity for jelly throughout the study period. At the commencement of an experiment, mice received BIBO 3304 containing jelly once per day for the time period indicated in each study, while control mice received vehicle jelly. [2]

[1]. Subtype selectivity of the novel nonpeptide neuropeptide Y Y1 receptor antagonist BIBO 3304 and its effect on feeding in rodents. Br J Pharmacol. 1998 Oct;125(3):549-55.

[2]. Inhibition of Y1 receptor signaling improves islet transplant outcome. Nat Commun. 2017 Sep 8;8(1):490.

Failure to secrete sufficient quantities of insulin is a pathological feature of type-1 and type-2 diabetes, and also reduces the success of islet cell transplantation. Here we demonstrate that Y1 receptor signaling inhibits insulin release in β-cells, and show that this can be pharmacologically exploited to boost insulin secretion. Transplanting islets with Y1 receptor deficiency accelerates the normalization of hyperglycemia in chemically induced diabetic recipient mice, which can also be achieved by short-term pharmacological blockade of Y1 receptors in transplanted mouse and human islets. Furthermore, treatment of non-obese diabetic mice with a Y1 receptor antagonist delays the onset of diabetes. Mechanistically, Y1 receptor signaling inhibits the production of cAMP in islets, which via CREB mediated pathways results in the down-regulation of several key enzymes in glycolysis and ATP production. Thus, manipulating Y1 receptor signaling in β-cells offers a unique therapeutic opportunity for correcting insulin deficiency as it occurs in the pathological state of type-1 diabetes as well as during islet transplantation.Islet transplantation is considered one of the potential treatments for T1DM but limited islet survival and their impaired function pose limitations to this approach. Here Loh et al. show that the Y1 receptor is expressed in β- cells and inhibition of its signalling, both genetic and pharmacological, improves mouse and human islet function. [2]

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Since the Y1 receptor might be involved in an anxiolytic action (Wahlestedt et al., 1993; Kask et al., 1996) its antagonism could induce anxiogenic like side e€ects. In order to evaluate whether possible anxiogenic e€ects interfered with the BIBO 3304 mediated inhibition of feeding response the interaction between BIBO 3304 and the feeding response mediated by other stimulators, such as noradrenaline and galanin was examined. If anxiety plays a role in food intake inhibition mediated by BIBO 3304, noradrenaline and galanin induced food intake should also be inhibited. But since neither galanin nor noradrenaline induced food intake was inhibited a signi®cant anxiogenic component in the food intake inhibitory properties of BIBO 3304 can be ruled out. It has been published earlier that BIBP 3226 mediated e€ects on feeding antagonism is not due to general side e€ects in the experimental setting used (O'Shea et al., 1997; Kask et al., personal communication). However, our own data using BIBP 3226 were not as conclusive (Doods et al., 1996). Consequently, the inactive enantiomer to BIBO 3304 was tested in order to show that the food intake inhibition was not due to a general toxicity of structural components of BIBO 3304. Indeed, the enantiomer did not a€ect the feeding response. In the present study, the identi®cation of BIBO 3304, a nonpeptide compound that displays anity in the subnanomolar range and selectivity for the Y1 receptor subtype is shown. We hypothesize that the Y1 receptor plays an important role in NPY induced feeding and BIBO 3304 is a novel tool to study both food intake and other central e€ects mediated via the Y1 receptor. Moreover, the data presented with BIBO 3304 as well as with the agonists, e.g. [Leu31, Pro34]NPY indicate a complicated interplay between the di€erent NPY receptors in feeding behaviour. [1]

C33H37F6N7O7

757.68

757.265

2310085-85-7

122705981

Typically exists as solid at room temperature

8

14

13

53

862

1

C1=CC=C(C=C1)C(C2=CC=CC=C2)C(=O)N[C@H](CCCN=C(N)N)C(=O)NCC3=CC=C(C=C3)CNC(=O)N.C(=O)(C(F)(F)F)O.C(=O)(C(F)(F)F)O

XWZMETGYCRXJJH-PPLJNSMQSA-N

InChI=1S/C29H35N7O3.2C2HF3O2/c30-28(31)33-17-7-12-24(26(37)34-18-20-13-15-21(16-14-20)19-35-29(32)39)36-27(38)25(22-8-3-1-4-9-22)23-10-5-2-6-11-23;2*3-2(4,5)1(6)7/h1-6,8-11,13-16,24-25H,7,12,17-19H2,(H,34,37)(H,36,38)(H4,30,31,33)(H3,32,35,39);2*(H,6,7)/t24-;;/m1../s1

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

BIBO-3304 TFA; BIBO3304 (diTFA); 2310085-85-7; CHEMBL5083453; BIBO 3304?;

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)

Typically soluble in DMSO (e.g. 10 mM)

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.)