EMPA

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

EMPA 竞争性拮抗食欲素 A 和食欲素 B 引起的 [3H]肌醇磷酸盐 (IP) 在 hOX2 受体上的积累，pA2 值分别为 8.6 和 8.8[1]。 EMPA 取代了含有人和大鼠 OX2 受体的细胞膜上的 [3H]EMPA 结合，Ki 值分别为 1.10±0.24 nM 和 1.45±0.13 nM[1]。 EMPA 在人和小鼠 V1a 受体的结合测定中分别显示 IC50=5.75 µM、Ki=2.63 µM 和 IC50=12.8 µM、Ki=5.8 µM[1]。在稳定表达 hOX2 受体的 CHO(dHFr-) 细胞中，EMPA 抑制 orexin-A 或 orexin-B 诱发的 [Ca2+]i 反应，IC50 分别为 8.8±1.7 nM 和 7.9±1.7 nM[1]。

EMPA（1-300 mg/kg；腹腔注射）剂量依赖性地逆转这种 [Ala11,D-Leu15]orexin-B 诱导的过度运动，而其本身不会显着影响雄性 NMRI 小鼠的运动活动 (LMA)[1]。 EMPA（3-30 mg/kg；腹膜内注射）可诱导法国大鼠和雄性 Wistar 大鼠的基线 LMA 显着且剂量依赖性降低。与媒介物处理的动物相比，EMPA（3-30 mg/kg；ip）显示出对自发活动的明显剂量依赖性抑制[1]。动物模型：雄性 NMRI 小鼠（20-30 g）[1] 剂量：1、3、10、30、100、300 mg/kg 给药方法：以 10 mL/kg 的体积进行腹腔注射 结果：剂量依赖性逆转这种情况[Ala11,D-Leu15]orexin-B 诱导的过度运动，但其本身不会显着影响 LMA。动物模型：法国和雄性 Wistar 大鼠（196-237 g）[1] 剂量：3、10、30 mg/kg 给药方法：以 5 mL/kg 的体积进行腹腔注射 结果：诱导显着且剂量依赖性的减少基线 LMA。与媒介物处理的动物相比，显示出对自发活动的明显剂量依赖性抑制。

[3 H] EMPA binding[1]
解冻后，膜匀浆在4°C下以48 000×g离心10分钟，将微球重新悬浮在结合缓冲液（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）中，至最终检测浓度为每孔2.5µg蛋白。饱和等温线是通过在这些膜上加入不同浓度的[3H]EMPA（总反应体积为500µL），在23℃下反应60分钟来测定的。在孵育结束时，将膜过滤到unitfilter上，unitfilter是一个96孔白色微孔板，带有GF/C过滤器，在洗涤缓冲液（25 mmol·L−1 HEPES, pH 7.4, 1 mmol·L−1 CaCl2, 5 mmol·L−1 MgCl2）加0.5%聚乙烯亚胺中预孵育1小时，使用Filtermate 196收集机，用冷水洗涤缓冲液洗涤4次。在10µmol·L−1 EMPA作用下测定非特异性结合（NSB）。加入45µL的microscint 40并振荡1小时后，在Top-Count微孔板闪烁计数器上（5 min）计数过滤器上的放射性。[1]
利用Prism 4.0软件对饱和实验进行分析，采用由双分子反应方程和质量作用定律导出的矩形双曲方程B = (Bmax*[F])/(KD+[F])，其中B为平衡状态下的配体结合量，Bmax为最大结合位点数，[F]为游离配体浓度，KD为配体解离常数。为了进行抑制实验，将膜与[3H]EMPA孵育，浓度等于放射配体的KD值和10浓度的抑制化合物（0.0001-10µmol·L−1）。IC50值由抑制曲线得出，亲和常数（Ki）值由Cheng-Prussoff方程Ki= IC50/(1 +[L]/KD)计算，其中[L]为放射性配体的浓度，KD为其在受体处的解离常数，由饱和等温线得出。为了测量结合动力学，将膜在23°C下在放射性配体（~ 1.1 nmol·L−1[3H]EMPA）存在下孵育0、1、3、5、7、10、15、20、30、60、90或120分钟，然后通过快速过滤终止。通过在过滤前不同时间加入10µmol·L−1 EMPA，在23°C预孵育1 h的膜中，在~ 1.1 nmol·L−1[3H]EMPA存在下，测量解离动力学。结合动力学参数，Kob值和Koff值（观察到的开断率），分别由结合-解离曲线用一相指数关联和衰减方程推导。Kon、半衰期和Kd分别用Kon= (Kob−Koff)/[配体]、t1/2= ln2/K和Kd = Koff/Kon方程计算。

体外[3H]EMPA放射自显影法测定OX2受体占用率[1]
雄性CD Sprague-Dawley大鼠分别给药（1% Tween-80生理盐水）或增氧剂（3、10或30 mg·kg - 1）（每组n= 2）。给药30分钟后，砍头处死；大脑被迅速解剖，并立即冷冻在干冰中。低温恒温冠状面切片按上述方法进行[3H]EMPA受体放射自显影。

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

101.08

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

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.50 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.50 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.50 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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