rat Y2 receptor ( pIC50 = 8.22 ); human Y2 receptor ( pIC50 = 8.07 )

In vitro activity: JNJ-31020028 is tested by binding to a panel of 50 receptors, ion channels, and transporters, such as adenosine (A1, A2A, A3), adrenergic (α1, α2, β1), angiotensin (AT1), dopamine (D1, D2), bradykinin (B2), cholecystokinin (CCKA), galanin (GAL2), melatonin (ML1), muscarinic (M1, M2, M3), neurotensin (NT1), neurokinin (NK2, NK3), opiate (μ, κ, δ), serotonin (5-HT1A, 5-HT1B, 5-HT2A, 5-HT3, 5-HT6, 5-HT7), somatostatin, vasopressin (V1a), norepinephrine transporter, dopamine transporter, and ion channels (sodium, calcium, potassium, and chloride). Except for the Y2 receptor, the Y2 antagonist exhibits no discernible affinity (<50% inhibition at 10μM) for any other receptor, transporter, or ion channel at concentrations up to 10μM. The Y2 antagonist's selectivity is assessed in more detail using a panel of 65 kinases. JNJ-31020028 (10μM) does not inhibit any of the panel's kinases[1].

In the olfactory bulbectomized rat (OBX) model, (R)-JNJ-31020028 (5.6 μg; chronic intravenous infusion; once daily for 10 days) demonstrates antidepressant-like effects [1]. The number of grooming episodes was significantly reduced by administering (R)-JNJ-31020028 intravenously over an extended period of time using an osmotic Alzet pump at a dose of 5.6 μg/day [1]. Treatment with (R)-JNJ-31020028 revealed Cmax, Tmax, AUCinf, Vd, and t1/2 values of 4.35μM, 0.5 hours, 7.91hμM, and 0.83 hours, in that order [1].

In functional assays, JNJ-31020028 was shown to be an antagonist (pK(B) = 8.04 +/- 0.13).

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Radioligands binding assays[1]
Competition binding assays were performed as previously described (Bonaventure et al. 2004) using [125I]PYY for Y1, Y2, and Y5 receptor and [125I]PP for Y4 receptor. Cells used in the radioligand binding experiments with NPY receptor subtypes were SK-N-MC endogenously expressing Y1 receptors, KAN-Ts endogenously expressing Y2 receptors, Chinese hamster ovary (CHO) cells transfected with human Y4 cDNA for Y4 receptors, and HEK-293 transfected with human Y5 cDNA for Y5 receptors. Membranes from rat and mouse hippocampus were also prepared and assayed for [125I]PYY binding as previously described (Bonaventure et al. 2004). IC50 values (i.e., concentration of unlabeled antagonist required to compete for 50% of specific binding to the radioligand) were calculated using the GraphPad Prism software with a fit to a sigmoidal dose–response curve. Data were expressed as pIC50 values where pIC50 = −log IC50. In addition, the selectivity of JNJ-31020028 was evaluated in a large variety of ion channels, transporters, receptor binding, and kinase assays.[1]

Calcium mobilization assays[1] A calcium mobilization assay was established by stably expressing a chimeric G protein Gqi9 in KAN-Ts cells endogenously expressing Y2 receptors as previously described (Dautzenberg 2005). Briefly, dye-loaded cells were plated on to 96-well ViewPlates and incubated at 37°C, 5% CO2 for 1 h. For antagonist potency determinations, cells were pre-incubated with the compounds (diluted in Dulbecco’s modified Eagle medium/F-12) for 10 min before agonist (PYY 10 nM) stimulation. Ligand-induced calcium release was measured using a Fluorometric Imaging Plate Reader. Functional responses were measured as peak fluorescence intensity minus basal. The concentration of agonist that produced a half-maximal response is represented by the EC50 value. Antagonistic potency values were converted to apparent pKB values using a modified Cheng–Prusoff correction. Apparent pKB = −log IC50/1 + [conc agonist/EC50]. Data are expressed as mean ± SEM.

Animal/Disease Models: Male Sprague Dawley rats, body weight 150-170 (OBX model) [1]
Doses: 5.6 μg
Route of Administration: chronic intravenous (iv) (iv)infusion; one time/day for 10 days
Experimental Results: diminished immobility time in OBX rats.

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Animal/Disease Models: Male SD (SD (Sprague-Dawley)) rat [2]
Doses: 10 mg/kg
Route of Administration: subcutaneous injection (pharmacokinetic/PK/PK analysis)
Experimental Results: Cmax, Tmax, AUCinf, Vd and t1/2 were 4.35 μM, 0.5 respectively hrs (hrs (hours)), 7.91 h μM and 0.83 h respectively.

[1]. In vitro and in vivo characterization of JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a selective brain penetrant small molecule antagonist of the neuropeptide Y.

[2]. Chronic administration of the Y2 receptor antagonist, JNJ-31020028, induced anti-depressant like-behaviors in olfactory bulbectomized rat. Neuropeptides. 2012 Dec;46(6):329-34.

Rationale: The lack of efficient, selective, and blood-brain barrier-penetrating Y(2) receptor antagonists has hindered in vivo functional studies of this receptor. Objective: This paper reports the in vitro and in vivo properties of a novel Y(2) receptor antagonist, JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide). Methods: The affinity of JNJ-31020028 was determined by inhibiting the binding of PYY to the human Y(2) receptor in KAN-Ts cells and the rat Y(2) receptor in the rat hippocampus. Its functional activity was determined by inhibiting the calcium response to PYY stimulation in KAN-Ts cells expressing the chimeric G protein Gqi5 and in the rat vas deferens (a typical Y(2) bioassay). The in vitro receptor occupancy was detected by receptor autoradiography. JNJ-31020028 was tested in vivo using microdialysis, an anxiety model, and corticosterone release. Results: JNJ-31020028 exhibited high affinity for its receptor (pIC(50) = 8.07 ± 0.05, human; pIC(50) = 8.22 ± 0.06, rat) and selectivity for human Y(1), Y(4), and Y(5) receptors exceeding 100-fold. Functional assays indicated that JNJ-31020028 is an antagonist (pK(B) = 8.04 ± 0.13). Following subcutaneous injection in rats, JNJ-31020028 occupied approximately 90% of the Y(2) receptor binding site at a dose of 10 mg/kg. JNJ-31020028 increased the release of norepinephrine in the hypothalamus, consistent with the colocalization of norepinephrine and neuropeptide Y. JNJ-31020028 did not show efficacy in various anxiety models, but it did inhibit stress-induced increases in plasma corticosterone levels without affecting basal corticosterone levels; in addition, it restored the food intake of stressed animals to normal without affecting basal food intake. Conclusion: These results suggest that the Y(2) receptor may not be a key factor in acute behavior in rodents, but may play a regulatory role, but this role can only be elucidated under specific situational conditions. [1]

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Recent studies in our group have shown that the Y2 receptor antagonist BIIE0246 has an antidepressant effect in olfactory bulbectomy (OBX) rats. However, its complex structure and high molecular weight limit its application as an in vivo pharmacological tool. In contrast, the novel, blood-brain barrier-penetrating Y2 receptor antagonist JNJ-31020028 is an effective tool for studying the in vivo function of Y2 receptors. In this study, we evaluated the effects of long-term intraventricular (ICV) injection of JNJ-31020028 on a range of behavioral tests in olfactory bulb excision (OBX) rats, an animal model simulating multiple deficits in human depression. Long-term administration of JNJ-31020028 reduced immobility time in the forced swimming test in OBX rats, but had no effect on control animals. Furthermore, JNJ-31020028 reduced the frequency of grooming behaviors in OBX rats, but had no effect on other observed behavioral deficits, such as orthostatic hypoactivity. Additionally, JNJ-31020028 had no effect on commonly used anxiety assessment behavioral tests (i.e., social interaction tests), in both OBX rats and control animals. These data suggest that, similar to BIIE0246, JNJ-31020028 also has an antidepressant-like effect in the OBX model. [2]

C34H36FN5O2

565.680351257324

565.285

C, 72.19; H, 6.41; F, 3.36; N, 12.38; O, 5.66

1094873-17-2

JNJ-31020028;1094873-14-9

25151876

Typically exists as solid at room temperature

1.2±0.1 g/cm3

677.5±55.0 °C at 760 mmHg

363.5±31.5 °C

0.0±2.1 mmHg at 25°C

1.628

5.23

1

6

9

42

857

1

CCN(CC)C(=O)[C@@H](C1=CC=CC=C1)N2CCN(CC2)C3=C(C=C(C=C3)NC(=O)C4=CC=CC=C4C5=CN=CC=C5)F

OVUNRYUVDVWTTE-JGCGQSQUSA-N

InChI=1S/C34H36FN5O2/c1-3-38(4-2)34(42)32(25-11-6-5-7-12-25)40-21-19-39(20-22-40)31-17-16-27(23-30(31)35)37-33(41)29-15-9-8-14-28(29)26-13-10-18-36-24-26/h5-18,23-24,32H,3-4,19-22H2,1-2H3,(H,37,41)/t32-/m1/s1

N-[4-[4-[(1R)-2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl]-3-fluorophenyl]-2-pyridin-3-ylbenzamide

(R)-JNJ31020028; (R)-JNJ-31020028; JNJ31020028; (R)-N-(4-(4-(2-(diethylamino)-2-oxo-1-phenylethyl)piperazin-1-yl)-3-fluorophenyl)-2-(pyridin-3-yl)benzamide; CHEMBL1823577; N-[4-[4-[(1R)-2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl]-3-fluorophenyl]-2-pyridin-3-ylbenzamide; SCHEMBL1174048; BCP14605; (R) JNJ-31020028

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)

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.)