human OX2 receptor ( Kd = 1.1 nM ); rat OX2 receptor ( Kd = 1.4 nM )

EMPA competitively opposes the accumulation of [³H]inositol phosphates (IP) at hOX² receptors that is induced by orexin A and orexin B with pA² values of 8.6 and 8.8 respectively[1].
EMPA removes the [³H]EMPA binding from rat and human OX² receptor-containing cell membranes, with K_i values of 1.45±0.13 nM and 1.10±0.24 nM, respectively[1].
EMPA binds to human and mouse V_1a receptors with IC50 values of 5.75 µM, K_i=2.63 µM, and IC50 values of 12.8 µM, K_i=5.8 µM, respectively[1].
EMPA inhibits the [Ca²⁺]_i response evoked by orexin-A or orexin-B in CHO(dHFr^-) cells that are stably expressing hOX2 receptors, with IC50 values of 8.8±1.7 nM and 7.9±1.7 nM, respectively[1].

EMPA (1-300 mg/kg; i.p.) dose-dependently reverses this [Ala¹¹,D-Leu¹⁵]orexin-B-induced hyperlocomotion without substantially altering locomotor activity (LMA) in male NMRI mice[1].
EMPA (3-30 mg/kg; i.p.) significantly and dose-dependently lowers the baseline LMA in both female and male Wistar rats. EMPA (3-30 mg/kg; i.p.) shows a definite dose-dependent suppression of spontaneous activity in contrast to animals given a vehicle treatment[1].

[3H]EMPA binding[1]
After thawing, membrane homogenates were centrifuged at 48 000×g for 10 min at 4°C, pellets were re-suspended in the binding buffer (25 mmol·L−1 HEPES, pH 7.4, 1 mmol·L−1 CaCl2, 5 mmol·L−1 MgCl2, 0.5% BSA, 0.05% Tween 20) to a final assay concentration of 2.5 µg protein per well. Saturation isotherms were determined by the addition of various concentrations of [3H]EMPA to these membranes (in a total reaction volume of 500 µL) for 60 min at 23°C. At the end of incubation, membranes were filtered onto unitfilter, a 96-well white microplate with bonded GF/C filter pre-incubated 1 h in wash buffer (25 mmol·L−1 HEPES, pH 7.4, 1 mmol·L−1 CaCl2, 5 mmol·L−1 MgCl2) plus 0.5% polyethylenimine, with a Filtermate 196 harvester and washed 4 times with ice-cold wash buffer. Non-specific binding (NSB) was measured in the presence of 10 µmol·L−1 EMPA. Radioactivity on the filter was counted (5 min) on a Top-Count microplate scintillation counter with quenching correction after addition of 45 µL of microscint 40 and shaking for 1 h.[1]
Saturation experiments were analysed by Prism 4.0 using the rectangular hyperbolic equation derived from the equation of a bimolecular reaction and the law of mass action, B = (Bmax*[F])/(KD+[F]), where B is the amount of ligand bound at equilibrium, Bmax is the maximum number of binding sites, [F] is the concentration of free ligand and KD is the ligand dissociation constant. For inhibition experiments, membranes were incubated with [3H]EMPA at a concentration equal to the KD value of radioligand and 10 concentrations of the inhibitory compound (0.0001–10 µmol·L−1). IC50 values were derived from the inhibition curve and the affinity constant (Ki) values were calculated using the Cheng-Prussoff equation Ki= IC50/(1 +[L]/KD) where [L] is the concentration of radioligand and KD is its dissociation constant at the receptor, derived from the saturation isotherm. To measure association kinetics, membranes were incubated at 23°C in the presence of radioligand (∼1.1 nmol·L−1[3H]EMPA) for 0, 1, 3, 5, 7, 10, 15, 20, 30, 60, 90 or 120 min, then terminated by rapid filtration. Dissociation kinetics were measured by adding at different times before filtration, 10 µmol·L−1 EMPA to membranes pre-incubated at 23°C for 1 h in the presence of ∼1.1 nmol·L−1[3H]EMPA. Binding kinetics parameters, Kob and Koff values (observed on and off rates), were derived from association-dissociation curves using the one phase exponential association and decay equations respectively. Kon, half-life and Kd were calculated using the Kon= (Kob− Koff)/[ligand], t1/2= ln2/K and KD= Koff/Kon equations respectively.

Determination of OX2 receptor occupancy using ex vivo [3H]EMPA autoradiography[1]
Male CD Sprague-Dawley rats were given vehicle (1% Tween-80 in physiological saline) or almorexant (3, 10 or 30 mg·kg−1) (i.p., n= 2 per group). Thirty minutes after dosing, animals were sacrificed by decapitation; brains were rapidly dissected and immediately frozen in dry ice. Cryostat coronal sections were processed for [3H]EMPA receptor autoradiography as described above.

Male NMRI mice (20-30 g)
1, 3, 10, 30, 100, 300 mg/kg
Injected i.p. at a volume of 10 mL/kg

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Pharmacokinetics of EMPA in mice and rats[1]
Pharmacokinetic experiments were performed in male NMRI mice and Wistar rats. Mice were dosed either i.v. (into the tail vain) or p.o. (microsuspension, as a gavage). At defined time points, terminal plasma and brain tissue was collected. Two mice per group were killed at 0.083, 0.333, 1, 2, 4 and 7 h after the i.v. administration of 10.77 mg·kg−1 EMPA or 0.25, 0.5, 1, 2, 4 and 7 h after the p.o. administration of 18.04 mg·kg−1 EMPA. Rats were given a single oral dose (19.71 mg·kg−1, microsuspension, as a gavage) or i.v. (11.79 mg·kg−1, via a jugular vein). Plasma and brain samples were collected after killing from two rats per group at 0.083, 0.25, 0.5, 1, 2, 4 and 8 h (i.v.) or 0.25, 0.5, 1 and 2 h (p.o.) after dosing. Concentrations of EMPA were determined using quantitative liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). Pharmacokinetic parameters were calculated by non-compartmental analysis of plasma concentration-time curves using WinNonlin, version 4.1 software.

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In vivo evaluation of EMPA[1]
Animals and drug treatment
Male NMRI mice (20–30 g) and Male Wistar rats (196–237 g) were used. EMPA was prepared immediately prior to use in 0.3% (w/v) Tween-80 in physiological saline (0.9% NaCl) and injected i.p. at a volume of 10 mL·kg−1 body weight for mice and 5 mL·kg−1 for rats. All doses are expressed as that of the base.

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Reversal of [Ala11,D-Leu15]orexin-B-induced hyperlocomotion in mice[1]
A computerized Digiscan 16-Animal Activity Monitoring System was used to quantify locomotor activity (LMA). Data were obtained simultaneously from eight Digiscan activity chambers placed in a soundproof room with a 12 h light/dark cycle. All tests were performed during the light phase (6 am to 6 pm). Each activity monitor consisted of a Plexiglas box (20 × 20 × 30.5 cm) with sawdust bedding on the floor surrounded by invisible horizontal and vertical infrared sensor beams. Cages were connected to a Digiscan Analyzer linked to a PC that constantly collected the beam status information. The activity detector operates by counting the number of times the beams change from uninterrupted to interrupted or vice versa. Records of photocell beam interruptions, for individual animals, typically were taken every 5 min over the duration of the test session. Mice were first transferred from home cages to recording chambers for a 50 min habituation phase during which they were allowed to freely explore the new environment. Mice were then injected i.p. with EMPA (1, 3, 10, 30, 100, 300 mg·kg−1, n= 8 mice per dose). Ten minutes later, mice were briefly anaesthetized with isoflurane inhalation to allow i.c.v. injection of 5 µL of either artificial cerebrospinal fluid (CSF) or [Ala11,D-Leu15]orexin-B at a dose of 3 µg. Mice were then immediately replaced in the test compartments and LMA was recorded during the following 30 min.

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Spontaneous locomotor activity in rats during the dark (active) phase[1]
Male Wistar rats (∼150 g at arrival) housed four per cage (Makrolon cages 1800 cm2) with free access to food and water were allowed 2 weeks of acclimatization to a reversed light/dark animal room (dark cycle: 10.00 am to 10.00 pm) prior to testing. On test days, LMA was monitored by a computerized Digiscan Animal Activity Monitoring system as described above. The activity monitoring chambers were made of Plexiglas (41 × 41 × 30 cm W × L × H) and contained a thin layer of sawdust bedding. One rat per cage was monitored at the same time. One hour after the dark period onset, rats were injected i.p. with EMPA (3, 10, 30 mg·kg−1, n= 8 rats per dose) and immediately placed into the activity monitoring chambers. LMA was then recorded in 5 min time bins for a period of 30 min.

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Motor coordination and balance in rats[1]
Male Wistar rats (∼200 g body weight) were trained to remain on a horizontal metal rod rotating at a fixed speed until criterion level (120 s on rod) was reached. The rotarod was 7 cm wide, 5 cm in diameter and 25 cm above the bench. The following day, animals were injected i.p. with vehicle or EMPA (3, 10 or 30 mg·kg−1; n= 8 per group). Animals were tested for rotarod performance at 8 r.p.m. and then at 16 r.p.m. (total time spent on the rod, maximum 120 s) 10 min after injection. Rats were allowed a maximum of three trials to remain on the rotarod for 120 s; assessment terminated when the animal fell from the rotarod or reached criterion level. The mean time over the number of trials completed per rat was calculated.

[1]. Biochemical and behavioural characterization of EMPA, a novel high-affinity, selective antagonist for the OX² receptor. Br J Pharmacol. 2009 Apr; 156(8): 1326-1341.

Background and purpose:
The OX2 receptor is a G-protein-coupled receptor that is abundantly found in the tuberomammillary nucleus, an important site for the regulation of the sleep-wake state. Herein, we describe the in vitro and in vivo properties of a selective OX2 receptor antagonist, N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulphonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA).
Experimental approach:
The affinity of [3H]EMPA was assessed in membranes from HEK293-hOX2-cells using saturation and binding kinetics. The antagonist properties of EMPA were determined by Schild analysis using the orexin-A-or orexin-B-induced accumulation of [3H]inositol phosphates (IP). Quantitative autoradiography was used to determine the distribution and abundance of OX2 receptors in rat brain. The in vivo activity of EMPA was assessed by reversal of [Ala11,D-Leu15]orexin-B-induced hyperlocomotion during the resting phase in mice and the reduction of spontaneous locomotor activity (LMA) during the active phase in rats.
Key results:
[3H]EMPA bound to human and rat OX2-HEK293 membranes with KD values of 1.1 and 1.4 nmol·L−1 respectively. EMPA competitively antagonized orexin-A-and orexin-B-evoked accumulation of [3H]IP at hOX2 receptors with pA2 values of 8.6 and 8.8 respectively. Autoradiography of rat brain confirmed the selectivity of [3H]EMPA for OX2 receptors. EMPA significantly reversed [Ala11,D-Leu15]orexin-B-induced hyperlocomotion dose-dependently during the resting phase in mice. EMPA, injected i.p. in rats during the active phase, reduced LMA dose-dependently. EMPA did not impair performance of rats in the rotarod procedure.
Conclusions and implications:
EMPA is a high-affinity, reversible and selective OX2 receptor antagonist, active in vivo, which should prove useful for analysis of OX2 receptor function.
Keywords: EMPA, orexin, OX2 antagonist, binding kinetics, inositol phosphate accumulation, Schild analysis, autoradiography, rat brain distribution, orexin-B-induced hyperlocomotion, ex vivo receptor occupancy.[1]

C₂₃H₂₆N₄O₄S

454.54

454.167

C, 60.78; H, 5.77; N, 12.33; O, 14.08; S, 7.05

680590-49-2

9981404

White to light yellow solid powder

4.118

0

7

9

32

699

0

CCN(CC1=CN=CC=C1)C(=O)CN(C2=CN=C(C=C2)OC)S(=O)(=O)C3=CC=CC=C3C

KJPHTXTWFHVJIG-UHFFFAOYSA-N

InChI=1S/C23H26N4O4S/c1-4-26(16-19-9-7-13-24-14-19)23(28)17-27(20-11-12-22(31-3)25-15-20)32(29,30)21-10-6-5-8-18(21)2/h5-15H,4,16-17H2,1-3H3

N-ethyl-2-[(6-methoxypyridin-3-yl)-(2-methylphenyl)sulfonylamino]-N-(pyridin-3-ylmethyl)acetamide

EMPA; N-Ethyl-2-((N-(6-methoxypyridin-3-yl)-2-methylphenyl)sulfonamido)-N-(pyridin-3-ylmethyl)acetamide; VT87V86D7W; CHEMBL2385132; N-Ethyl-2-[(6-methoxy-3-pyridinyl)[(2-methylphenyl)sulfonyl]amino]-N-(3-pyridinylmethyl)-acetamide; 7MA; Acetamide, N-ethyl-2-((6-methoxy-3-pyridinyl)((2-methylphenyl)sulfonyl)amino)-N-(3-pyridinylmethyl)-;

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)

DMSO: 50~91 mg/mL (110.0~200.2 mM)
Ethanol: ~23 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.50 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 25.0 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: ≥ 2.5 mg/mL (5.50 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 25.0 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: ≥ 2.5 mg/mL (5.50 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 25.0 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.)