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实验参考方法

Cell experiment [1]:
Cell lines	MCF-7 cells
Preparation Method	Cells were seeded in 6-well plates for 24 h prior to 48 h-treatment with EIPA (10 µM), Akti (1 µM), U0126 (10 µM) or a combination thereof.
Reaction Conditions	10µM for 48 hours
Applications	MCF-7 cells were subjected to treatment with EIPA with and without ERK and Akt inhibitors. ERK, but not Akt, activity was increased upon EIPA treatment, and both kinases were effectively inhibited by their inhibitors, U0126 and Akti, respectively. Strikingly, ERK inhibition strongly potentiated EIPA-induced CHOP upregulation and PARP cleavage.
Animal experiment [2]:
Animal models	Female homozygous BALB/c Nu/Nu nude mice were injected subcutaneously (s.c.) in both flanks at 7 weeks of age with 106 parental and IKKαδ MIA PaCa-2 cells or 1334 and 1444 cells mixed at a 1:1 dilution with BD Matrigel (BD Biosciences) in a total volume of 100 µl.
Preparation Method	Mice were treated with vehicle (DMSO in PBS), EIPA (7.5 mg/kg), MRT68921 (10 mg/kg) or MRT68921+EIPA for 15 days when tumors attained an average volume of 50-100 mm3.
Dosage form	s.c., 7.5 mg/kg/d for 7 days
Applications	One month of EIPA treatment, initiated at age 1 month, markedly reduced pancreatic weight (a tumor burden surrogate), inhibited acinar-to-ductal metaplasia (ADM) and advanced PanIN formation, and preserved the normal pancreatic parenchyma in KrasG12D;IkkαδPEC mice, while decreasing MP.
References: [1]: M.G. Rolver, L.O. Elingaard-Larsen, A.P. Andersen, L. Counillon, S.F. Pedersen. Pyrazine ring-based Na+/H+ exchanger (NHE) inhibitors potently inhibit cancer cell growth in 3D culture, independent of NHE1.Sci. Rep., 10 (2020), p. 5800 [2]: H. Su, F. Yang, R. Fu, et al. Cancer cells escape autophagy inhibition via NRF2-induced micropinocytosis. Cancer Cell, 39 (5) (2021), pp. 678-693, 10.1016/j.ccell.2021.02.016

产品描述

EIPA inhibited TRPP3-mediated Ca2+-activated currents with IC50 values of 10.5 μM ^[1]. TRPP3, a member of the transient receptor potential (TRP) superfamily of cation channels, is a Ca2+-activated channel permeable to Ca2+, Na+, and K+ ^[1].EIPA (5-(N-ethyl-N-isopropyl)-amiloride) is a commonly used amiloride derivative modified similarly to MPA, and its Ki for NHE1, NHE2, and NHE3 are 0.3, 1.8, and 67 μM, respectively^[2,3]

EIPA suppressed proliferation of MKN28 cells through up-regulation of p21 expression via reduction of [Cl- ] c as a result from DIDS-sensitive Cl- /HCO3- exchanger-mediated compensation for keeping pHc normal under an NHE-inhibited condition ^[4].

EIPA preischemic treatment overcame the I/R-induced renal dysfunction. Histologic examination of the kidney of vehicle-treated ARF mice revealed severe lesions, and these lesions were significantly suppressed by the preischemic treatment with EIPA. In addition, EIPA suppressed the increment of renal ET-1 content after reperfusion ^[5]. EIPA at doses of 3 and 9 mg/kg per day inhibited tumor growth of HepG2 modle mice significantly compared with the control ^[6].

References:
[1]. Dai XQ, Ramji A, Liu Y, Li Q, Karpinski E, Chen XZ (2007) Inhibition of TRPP3 channel by amiloride and analogs. Mol Pharmacol 72:1576-1585
[2]. Guffey, S.C.; Fliegel, L.; Goss, G.G. Cloning and characterization Na+/H+ Exchanger isoforms, NHE2 and, NHE3 from the gill Pacific dogfish, Squalus suckleyi. Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 2015, 188, 46-53.
[3]. Masereel, B. An overview inhibitors Na+/H+ exchanger. Eur. J. Med. Chem. 2003, 38, 547-554.
[4]. Hosogi SMH, Nakajima K, Ashihara E, Niisato N, Kusuzaki K, Marunaka Y: An inhibitor of Na(+)/H(+) exchanger (NHE), ethyl-isopropyl amiloride (EIPA), diminishes proliferation of MKN28 human gastric cancer cells by decreasing the cytosolic Cl(-) concentration via DIDS-sensitive pathways. Cell Physiol Biochem. 2012, 30: 1241-1253. 10.1159/000343315.
[5]. Yamashita J, Ohkita M, Takaoka M, Kaneshiro Y, Matsuo T,Kaneko K, Matsumura Y.Role of Na+/H+ exchanger in the pathogen-esis of ischemic acute renal failure in mice.J Cardiovasc Pharmacol49:154 -160, 2007.
[6]. X. Yang, D. Wang, W. Dong, Z. Song, K. Dou.Expression and modulation of Na+/H+ exchanger 1 gene in hepatocellular carcinoma: a potential therapeutic target. J. Gastroenterol. Hepatol., 26 (2) (2011), pp. 364-370

EIPA 抑制 TRPP3 介导的 Ca2+- 激活电流，IC50 值为 10.5 μM ^[1]。 TRPP3 是阳离子通道瞬时受体电位 (TRP) 超家族的成员，是 Ca2+- 激活通道，可渗透 Ca2+、Na+ 和 K+ ^[1].EIPA (5-(N-ethyl-N -isopropyl)-amiloride)是一种常用的类似MPA修饰的阿米洛利衍生物，其对NHE1、NHE2和NHE3的Ki值分别为0.3、1.8和67 μM^[2,3] /p>\n

EIPA 通过减少 [Cl-] c 上调 p21 表达来抑制 MKN28 细胞的增殖，这是由于 DIDS 敏感的 Cl- /HCO3- 交换剂介导的补偿，以在 NHE 抑制条件下保持 pHc 正常^[4].

EIPA 缺血前治疗克服了 I/R 引起的肾功能障碍。载体治疗的 ARF 小鼠肾脏的组织学检查显示严重病变，并且这些病变被 EIPA 的缺血前治疗显着抑制。此外，EIPA抑制再灌注后肾脏ET-1含量的增加^[5]。与对照组相比，每天 3 和 9 mg/kg 剂量的 EIPA 显着抑制 HepG2 模型小鼠的肿瘤生长^[6]。

Chemical Properties

Cas No.	1154-25-2	SDF
别名	5-(N-乙基-N-异丙基)阿米洛利,L-593,754, MH 12-43, 5-(N-ethyl-N-isopropyl)-Amiloride
化学名	3-amino-6-chloro-N-(diaminomethylene)-5-(ethyl(isopropyl)amino)pyrazine-2-carboxamide
Canonical SMILES	ClC1=NC(C(/N=C(N)\N)=O)=C(N)N=C1N(CC)C(C)C
分子式	C₁₁H₁₈ClN₇O	分子量	299.76
溶解度	14mg/mL in DMSO, 20mg/mL in DMF, 2mg/mL in Ethanol	储存条件	Store at -20°C
General tips	请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。
Shipping Condition	评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。

溶解性数据

制备储备液
		1 mg	5 mg	10 mg
	1 mM	3.336 mL	16.68 mL	33.36 mL
	5 mM	0.6672 mL	3.336 mL	6.672 mL
	10 mM	0.3336 mL	1.668 mL	3.336 mL

摩尔浓度计算器
稀释计算器
分子量计算器

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动物体内配方计算器 (澄清溶液)

第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量）

给药剂量

mg/kg

动物平均体重

g

每只动物给药体积

ul

动物数量

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第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方）

% DMSO+ % + % Tween 80+ % saline

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计算结果:

工作液浓度： mg/ml；

DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，注：如该浓度超过该批次药物DMSO溶解度，请先联系GLPBIO）；

体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。

注意：1. 首先保证母液是澄清的；
2. 一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。

Research Update

SLC4A11 is an EIPA-sensitive Na(+) permeable pHi regulator

Am J Physiol Cell Physiol2013 Oct 1;305(7):C716-27.PMID: 23864606DOI: 10.1152/ajpcell.00056.2013

Slc4a11, a member of the solute linked cotransporter 4 family that is comprised predominantly of bicarbonate transporters, was described as an electrogenic 2Na(+)-B(OH)4(-) (borate) cotransporter and a Na(+)-2OH(-) cotransporter. The goal of the current study was to confirm and/or clarify the function of SLC4A11. In HEK293 cells transfected with SLC4A11 we tested if SLC4A11 is a: 1) Na(+)-HCO3(-) cotransporter, 2) Na(+)-OH(-)(H(+)) transporter, and/or 3) Na(+)-B(OH)4(-) cotransporter. CO2/HCO3(-) perfusion yielded no significant differences in rate or extent of pHi changes or Na(+) flux in SLC4A11-transfected compared with control cells. Similarly, in CO2/HCO3(-), acidification on removal of Na(+) and alkalinization on Na(+) add back were not significantly different between control and transfected indicating that SLC4A11 does not have Na(+)-HCO3(-) cotransport activity. In the absence of CO2/HCO3(-), SLC4A11-transfected cells showed higher resting intracelllular Na(+) concentration ([Na(+)]i; 25 vs. 17 mM), increased NH4(+)-induced acidification and increased acid recovery rate (160%) after an NH4 pulse. Na(+) efflux and influx were faster (80%) following Na(+) removal and add back, respectively, indicative of Na(+)-OH(-)(H(+)) transport by SLC4A11. The increased alkalinization recovery was confirmed in NHE-deficient PS120 cells demonstrating that SLC4A11 is a bonafide Na(+)-OH(-)(H(+)) transporter and not an activator of NHEs. SLC4A11-mediated H(+) efflux is inhibited by 5-(N-ethyl-N-isopropyl) amiloride (EIPA; EC50: 0.1 ¦̍). The presence of 10 mM borate did not alter dpHi/dt or ¦¤pH during a Na(+)-free pulse in SLC4A11-transfected cells. In summary our results show that SLC4A11 is not a bicarbonate or borate-linked transporter but has significant EIPA-sensitive Na(+)-OH(-)(H(+)) and NH4(+) permeability.

Experimental Study of the Effects of EIPA, Losartan, and BQ-123 on Electrophysiological Changes Induced by Myocardial Stretch

Rev Esp Cardiol (Engl Ed)2015 Dec;68(12):1101-10.PMID: 25985899DOI: 10.1016/j.rec.2014.12.023

Introduction and objectives: Mechanical response to myocardial stretch has been explained by various mechanisms, which include Na(+)/H(+) exchanger activation by autocrine-paracrine system activity. Drug-induced changes were analyzed to investigate the role of these mechanisms in the electrophysiological responses to acute myocardial stretch.
Methods: Multiple epicardial electrodes and mapping techniques were used to analyze changes in ventricular fibrillation induced by acute myocardial stretch in isolated perfused rabbit hearts. Four series were studied: control (n = 9); during perfusion with the angiotensin receptor blocker losartan (1 ¦̍, n = 8); during perfusion with the endothelin A receptor blocker BQ-123 (0.1 ¦̍, n = 9), and during perfusion with the Na(+)/H(+) exchanger inhibitor EIPA (5-[N-ethyl-N-isopropyl]-amiloride) (1 ¦̍, n = 9).
Results: EIPA attenuated the increase in the dominant frequency of stretch-induced fibrillation (control=40.4%; losartan=36% [not significant]; BQ-123=46% [not significant]; and EIPA=22% [P<.001]). During stretch, the activation maps were less complex (P<.0001) and the spectral concentration of the arrhythmia was greater (greater regularity) in the EIPA series: control=18 (3%); EIPA = 26 (9%) (P < .02); losartan=18 (5%) (not significant); and BQ-123=18 (4%) (not significant).
Conclusions: The Na(+)/H(+) exchanger inhibitor EIPA attenuated the electrophysiological effects responsible for the acceleration and increased complexity of ventricular fibrillation induced by acute myocardial stretch. The angiotensin II receptor antagonist losartan and the endothelin A receptor blocker BQ-123 did not modify these effects.

Acidosis and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) Attenuate Zinc/Kainate Toxicity in Cultured Cerebellar Granule Neurons

Biochemistry (Mosc)2015 Aug;80(8):1065-72.PMID: 26547075DOI: 10.1134/S000629791508012X

Cultured cerebellar granule neurons (CGNs) are resistant to the toxic effect of ZnCl2 (0.005 mM, 3 h) and slightly sensitive to the effect of kainate (0.1 mM, 3 h). Simultaneous treatment of CGNs with kainate and ZnCl2 caused intensive neuronal death, which was attenuated by external acidosis (pH 6.5) or 5-(N-ethyl-N-isopropyl)amiloride (EIPA, Na+/H+ exchange blocker, 0.03 mM). Intracellular zinc and calcium ion concentrations ([Zn2+]i and [Ca2+]i) were increased under the toxic action of kainate + ZnCl2, this effect being significantly decreased on external acidosis and increased in case of EIPA addition. Neuronal Zn2+ imaging demonstrated that EIPA increases the cytosolic concentration of free Zn2+ on incubation in Zn2+-containing solution. These data imply that acidosis reduces ZnCl2/kainate toxic effects by decreasing Zn2+ entry into neurons, and EIPA prevents zinc stores from being overloaded with zinc.

An inhibitor of Na(+)/H(+) exchanger (NHE), ethyl-isopropyl amiloride (EIPA), diminishes proliferation of MKN28 human gastric cancer cells by decreasing the cytosolic Cl(-) concentration via DIDS-sensitive pathways

Cell Physiol Biochem2012;30(5):1241-53.PMID: 23075671DOI: 10.1159/000343315

Background/aims: Tumor cells produce a large amount of acidic metabolites due to their high metabolic condition. However, cytosolic pH (pH(c)) of tumor cells is identical to or even slightly higher than that of normal cells. To maintain pH(c) at a normal or higher level, tumor cells would have to have higher expression and/or activity of H(+) transporting systems than normal cells. The purpose of the present study was to identify effects of ethyl-isopropyl amiloride (EIPA, an inhibitor of Na(+)/H(+) exchanger (NHE)) on proliferation of human gastric cancer MKN28 cells.
Methods: Effects of EIPA on proliferation, pH(c), [Cl(-)](c) and expression of proteins regulating cell cycle and MAPKs were studied in MKN28 expressing NHE exposed to EIPA for 48 h.
Results: EIPA suppressed proliferation of MKN28 cells by causing G(0)/G(1) arrest without any significant effects on pH(c), but associated with reduction of [Cl(-)](c). Although EIPA alone had no effects on pH(c), EIPA co-applied with DIDS (an inhibitor of Cl(-)/HCO(3)(-) exchangers; i.e., anion exchanger (AE) and Na+-driven Cl(-)/HCO(3)(-) exchanger (NDCBE)) reduced pH(c), suggesting that DIDS-sensitive Cl(-)/HCO(3)(-) transporters such as AE and/or NDCBE keep pH(c) normal by stimulating HCO(3)(-) uptake coupled with Cl(-) release under an NHE-inhibited condition. EIPA-induced lowered [Cl(-)](c) up-regulated expression of p21associated with phosphorylation of MAPKs, suppressing proliferation associated with G(0)/G(1) arrest.
Conclusions: EIPA suppressed proliferation of MKN28 cells through up-regulation of p21 expression via reduction of [Cl(-)](c) as a result from DIDS-sensitive Cl(-)/HCO(3)(-) exchanger-mediated compensation for keeping pH(c) normal under an NHE-inhibited condition. This is the first study revealing that an NHE inhibitor suppressed the proliferation of cancer cells by reducing [Cl(-)](c) but not pH(c).

EIPA

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