D₄ Receptor; sPLA2 ( pIC50 = 8.87 ); sPLA2 ( pA2 = 9.71 )

[3H]WAY 100635 is demonstrated to bind specifically to 5-HT1A receptors in the brain after being given intravenously to mice. Additionally, WAY 100635 dose-dependently inhibits 8-OH-DPAT'scapacityto cause the "5-HT syndrome," hypothermia, hyperphagia, and an increase in plasma ACTH levels. This is achieved by inhibiting the firing of dorsal raphe 5-HT neurones. WAY 100635 produces anxiolytic-like effects in the mouse light/dark box anxiety model. In the delayed-matching-to-position model of rat short-term memory, WAY 100635 reverses the disruptive effects of 8-OH-DPAT on motor motivational performance but has no intrinsic effect on cognition.[2] In the anesthetized rat, WAY 100635 inhibits the inhibitory action of 8-OH-DPAT on dorsal raphe neuronal firing at doses that have no inherent inhibitory effect. In behavioral models, WAY 100635 does not directly cause any overt behavioral changes in rats or guinea pigs, but it effectively counteracts the behavioral syndrome caused by 8-OH-DPAT (minimum effective dose = 0.003 mg/kg s.c. and ID50 = 0.01 mg/kg s.c., respectively). With ID50 values of 0.01 mg/kg s.c., WAY 100635 also prevents the hypothermia that is brought on by 8-OH-DPAT in rats and mice.[4]

Screening assays[6]
For the initial screens by the NIMH-PDSP at a large number of cloned receptors and transporters (for details, see Roth et al. (2002) and Shapiro et al. (2003)), 10 μM WAY-100635 was used. Where significant inhibition was measured (>50% inhibition with quadruplicate determinations), K i determinations were performed with 6–10 concentrations of unlabelled ligand, and data were analyzed with GraphPad Prism.

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Saturation binding experiments[6]
Whole cell pellets were collected by scraping cells in media, followed by centrifugation at 1,000×g for 10 min and aspirating media. Pellets were then resuspended in ice-cold standard binding buffer (SBB: 50 mM Tris–HCl, pH 7.4, 10 mM MgCl2 and 0.1 mM EDTA), aliquoted, centrifuged at 14,000×g for 20 min at 4°C to pellet the membrane fraction, aspirated, and stored at −80°C for future use.[6]
5-HT1A pellets were washed by resuspending in ice-cold SBB and centrifuged at 14,000×g for 15 min at 4°C, and the buffer was aspirated. hD4.2 pellets were similarly washed but in ice-cold dopamine agonist binding buffer (DABB: 50 mM Tris–HCl pH 7.4, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2). Washed 5-HT1A membranes were Dounce-homogenized in room temperature SSB and incubated with 12 concentrations of [3H]WAY-100635 ranging from 0.004 to 2.3 nM in the absence and presence of 10 μM 5-HT to determine total and nonspecific binding, respectively. Likewise, washed hD4.2 membranes were Dounce-homogenized in room temperature DABB and incubated with 12 concentrations of [3H]WAY-100635 ranging from 0.004 to 13.4 nM in the absence and presence of 10 μM chlorpromazine to determine total and nonspecific binding, respectively. After 2 h at room temperature, reactions were terminated by rapid filtration onto cold 0.3% PEI presoaked filters. The filters were then washed three times in 4°C 50 mM Tris–HCl, pH 6.9. Filtered material was then transferred to scintillation vials mixed with 4 ml of Ecoscint-A scintillation fluid (National Diagnostic; Atlanta, GA, USA) and counted on a Beckman LS6500 scintillation counter.[6]

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Radioligand binding assay[6]
Cells were grown to confluence on 20-cm plates. The growth medium was decanted and replaced with 10-ml ice-cold lysis buffer (1 mM HEPES, pH 7.4, and 2 mM EDTA). After 10 min, cells were scraped from the plate and centrifuged at 30,000×g and 4°C for 20 min. The resulting pellet was resuspended in 4 ml receptor binding buffer (50 mM Tris–HCl, pH 7.4, and 4 mM MgCl) using a Kinematica homogenizer at a setting of 6 for 5 s, before 1.0 ml aliquots were centrifuged again at 13,000×g for 10 min. The pellets were stored at −80°C until use.[6]
The pellets were then resuspended for use by trituration in receptor binding buffer (50 μg protein/100 μl) and added in duplicate to assay tubes containing 0.1–0.2 nM [3H]spiperone and appropriate drugs. Nonspecific binding was determined using (+)-butaclamol (5 μM). Assay tubes were incubated at 37°C for 30 min before filtration, as described for cAMP binding assays. Filter plates were dried, and 30 μl of Packard Microscint-O scintillation fluid was added to each well. Radioactivity per well was determined using a Packard TopCount scintillation counter.[6]
Radioligand binding assays were also performed to investigate the effect of 100 μM guanylyl-5′-imidodiphosphate (Gpp-[NH]p) on agonist binding. These experiments were performed using HEK-hD4.4 membranes in a modified receptor binding buffer (50 mM Tris–HCl, pH 7.4, 4 mM MgCl, and 120 mM NaCl).

Extracellular recordings are performed using glass microelectrodes that have been loaded with 2 M NaC1 (12 MΩ–15 MΩ). Two or three-millisecond biphasic action potentials, slow (0.5 Hz - 2.0 Hz) discharge patterns, and regular discharge patterns are characteristics that distinguish cells as 5-HT neurons. The alpha-l adrenergic agonist phenylephrine (3 μM) is added to the superfusing ACSF to cause firing in the otherwise silent neurons. Prior to applying the various medications, baseline activity is tracked for at least ten minutes. Precise action potentials coupled to an A/D converter and a PC drive individual action potentials that drive an oscilloscope, an electronic ratemeter, and a high-input impedance amplifier. The integrated firing rate is measured, calculated, and shown on a chart recorder as successive 10-sec samples using specialized software. Agonists' effects are assessed by comparing the mean discharge frequency recorded during the two minutes prior to WAY 100635 application with the frequency recorded at the peak of the drug's action, which is typically two to five minutes after application starts. The effect of the agonist is contrasted with the baseline firing rate and frequency observed during the antagonist's single superfusion when the agonists are applied in the presence of the antagonist. Before retesting the agonists' action, the antagonist is given ten to twenty-five minutes to acclimate.

[1]. J Pharmacol Exp Ther . 1996 Aug;278(2):679-88.

[2]. Behav Brain Res . 1996;73(1-2):337-53.

[3]. Brain Res . 1997 Jan 16;745(1-2):96-108.

[4]. Eur J Pharmacol . 1995 Jul 25;281(1):81-8.

[5]. Eur J Pharmacol . 1994 Dec 27;271(2-3):505-14.

[6]. Psychopharmacology (Berl). 2006 Oct;188(2):244-51.

The aim of the present study was to examine the effects of N-(2-(4-2-methoxphenyl)-1-piperazinyl)ethyl)-N-(2-pyridnyl) cyclohexane carboxamide (WAY 100635) on 5-HT1A receptor-mediated responses in the dorsal raphe nucleus (DRN) and the CA1 hippocampal region. In DRN slices superfused with WAY 100635 (10 nM), the majority of putative 5-HT neurons increased their firing rate (13 +/- 2% of baseline rate). In addition, WAY 100635 completely prevented the decrease in firing rate produced by 5-HT (3-15 microM), 8-OH-DPAT (10 nM), 5-carboxamidotryptamine (20 nM) and lesopitron (100 nM). The antagonism exerted by WAY 100635 (IC50 = 0.95 +/- 0.12 nM against 15 microM 5-HT) was fully surmounted by increasing the concentration of 5-HT to 300 microM. In hippocampal slices, WAY 100635 (0.5-10 nM) did not alter the resting membrane potential or the membrane input resistance of intracellularly recorded CA1 pyramidal cells. However, WAY 100635 completely prevented (IC50 = 0.9-1.7 nM) the hyperpolarization and the decrease in membrane input resistance produced by 5-HT (15-30 microM) and by 5-carboxamidotryptamine (50-300 nM). In contrast, WAY 100635 affected neither the block of action potential frequency adaptation and slow afterhyperpolarization produced by 5-HT (15 microM) nor the hyperpolarization and decrease in membrane input resistance evoked by bath application of GABA(B) receptor agonist baclofen (10 microM). The cumulative concentration-hyperpolarization curve for 5-carboxamidotryptamine (3 nM-10 microM) was shifted to the right by WAY 100635 (apparent Kb = 0.23 +/- 0.07 nM), and the latter drug also reduced the maximal response to the agonist. These data show the WAY 100635 is a potent antagonist at 5-HT1A receptors, both in the DRN and in the CA1 region of the hippocampus. The antagonism is apparently competitive in the DRN and partly noncompetitive in the hippocampus. Kinetic characteristics of the antagonist-receptor interactions might account for these regional differences.[1]

C29H38N4O6

538.64

538.28

C, 64.67; H, 7.11; N, 10.40; O, 17.82

1092679-51-0

WAY-100635; 162760-96-5

11957721

White to off-white solid powder

3.54

123.510

COC1=CC=CC=C1N2CCN(CC2)CCN(C3=CC=CC=N3)C(=O)C4CCCCC4.C(=C\C(=O)O)\C(=O)O

XIGAHNVCEFUYOV-BTJKTKAUSA-N

InChI=1S/C25H34N4O2.C4H4O4/c1-31-23-12-6-5-11-22(23)28-18-15-27(16-19-28)17-20-29(24-13-7-8-14-26-24)25(30)21-9-3-2-4-10-21;5-3(6)1-2-4(7)8/h5-8,11-14,21H,2-4,9-10,15-20H2,1H3;1-2H,(H,5,6)(H,7,8)/b;2-1-

(Z)-but-2-enedioic acid;N-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-N-pyridin-2-ylcyclohexanecarboxamide

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)

Solubility in Formulation 1: 25 mg/mL (46.41 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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