IC50: 10.5 μM (TRPP3 channel)[1] NHE[2] Macropinocytosis[3]
EIPA hydrochloride targets the TRPP3 channel (a TRP channel) as an antagonist. It also inhibits the Na+/H+ exchanger (NHE), particularly NHE3, and macropinocytosis. The compound's primary targets are thus ion channels and transporters involved in cellular ion homeostasis and endocytic processes.

In X, TRPP3-mediated Ca2+ uptake is inhibited by EIPA hydrochloride (100 μM, 30 min). Ovum laevis [1]. Basal Na+ current is reversibly inhibited by EIPA hydrochloride (10-100 μM) (IC50: 19.5 μM) [1]. Through NHE3 (Na+/H+-exchanger 3), EIPA hydrochloride (300 μM, 6 hours) increases autophagy in IEC-18 cells [2]. The uptake of CA-PZ into HT-29 cells and MIA PaCa-2 cells through macropinocytosis is inhibited by EIPA hydrochloride (20 μM, 2 hours) [3]. Zinc/kainic acid toxicity is lessened by EIPA hydrochloride (30 μM, 3 hours) by decreasing Zn2+ entry into cerebellar granule neurons [4]. By upregulating p21 expression, EIPA hydrochloride (5-100 μM, 48 hours) inhibits MKN28 cell proliferation [5]. EIPA hydrochloride (3 μM, 6 hours) prevents the rise in COX-2 protein levels brought on by LPS [7].
In vitro, EIPA hydrochloride (100 μM, 30 min) inhibits TRPP3-mediated Ca2+ uptake. It reversibly inhibits basal Na+ current with an IC50 of 19.5 μM. At 300 μM for 6 hours, it increases autophagy in IEC-18 cells via NHE3. It inhibits the uptake of CA-PZ into HT-29 and MIA PaCa-2 cells through macropinocytosis at 20 μM for 2 hours. It also reduces zinc/kainic acid toxicity by decreasing Zn2+ entry into neurons at 30 μM. Furthermore, it inhibits MKN28 cell proliferation at 5-100 μM by upregulating p21 expression and prevents LPS-induced COX-2 protein level increases at 3 μM.

In ddY strain mice, renal impairment caused by I/R (ischemia/reperfusion) is dose-dependently reduced by intravenous administration of 1 mg/kg of EIPA hydrochloride [6]. ?In the balloon-type LPS-induced inflammation paradigm, EIPA hydrochloride (10 mg/kg) reduces LPS-induced inflammation [7].
In vivo, in ddY strain mice, renal impairment caused by ischemia/reperfusion (I/R) is dose-dependently reduced by intravenous administration of 1 mg/kg of EIPA hydrochloride. In a balloon-type LPS-induced inflammation model, EIPA hydrochloride (10 mg/kg) reduces LPS-induced inflammation.

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)[5].
In vitro enzyme/receptor binding assays for EIPA hydrochloride typically involve studying its effects on ion channels and transporters. For example, TRPP3-mediated Ca2+ uptake can be measured in cells expressing the channel, or basal Na+ current can be assessed in oocytes. The compound's inhibition of NHE can be evaluated by measuring intracellular pH changes or Na+ flux. These assays help determine the compound's potency and mechanism of action on its primary targets.

Cell proliferation assay [5]
Cell Types: MKN28 Cell
Tested Concentrations: 5, 10, 25, 50 and 100 μM
Incubation Duration: 48 h
Experimental Results: Inhibition of cell proliferation in a dose- and time-dependent manner.

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Western Blot Analysis[2]
Cell Types: IEC-18 Cell
Tested Concentrations: 300 μM
Incubation Duration: 6 hrs (hours)
Experimental Results: Increased LC3-II total protein levels and P62 flux. The expression of ATG5, 7, 12 and P62 was increased.
In vitro cell-based assays for EIPA hydrochloride typically employ cancer cell lines such as MKN28 (gastric cancer), HT-29 (colon carcinoma), or MIA PaCa-2 (pancreatic carcinoma). Cells are cultured and treated with the compound at various concentrations. Endpoints include assessment of cell proliferation, autophagy induction, macropinocytosis inhibition, and measurement of inflammatory markers like COX-2. These assays are used to study the compound's effects on cancer and inflammation.

Animal/Disease Models: Male ddY strain mouse [6]
Doses: 1 mg/kg
Route of Administration: intravenous (iv) (iv)injection
Experimental Results: Reduce histological renal damage and improve the increase in renal ET-1 content caused by I/R.

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Animal/Disease Models: air bag type LPS-induced inflammation model [7]
Doses: 10 mg/kg
Route of Administration: Oral
Experimental Results:Inhibited LPS-induced leukocyte infiltration into air bags. Suppresses the amount of PGE2 in ostomy bag fluid.
In vivo animal studies with EIPA hydrochloride have been conducted in mouse models. In ddY strain mice, the compound was administered intravenously at 1 mg/kg to study its effect on renal impairment caused by ischemia/reperfusion. In a balloon-type LPS-induced inflammation model, EIPA hydrochloride was administered at 10 mg/kg to reduce inflammation. These studies help evaluate the compound's therapeutic potential in disease models.

Pharmacokinetic (PK) properties of EIPA hydrochloride indicate it is orally bioactive. However, specific PK parameters such as half-life, Cmax, and bioavailability have not been detailed in the available literature. The compound's solubility is reported as 50 mg/mL in DMSO.

The toxicity profile of EIPA hydrochloride has not been extensively characterized in preclinical studies. No specific acute toxicity, genotoxicity, or chronic toxicity data are available in the provided literature. The compound is intended for research use only and is not approved for human therapeutic applications.

[1]. Inhibition of TRPP3 channel by MK-870 and analogs. Mol Pharmacol. 2007 Dec;72(6):1576-85.
[2]. Na+/H+ Exchanger Regulates Amino Acid-Mediated Autophagy in Intestinal Epithelial Cells. Cell Physiol Biochem. 2017;42(6):2418-2429.
[3]. A new HDAC inhibitor cinnamoylphenazine shows antitumor activity in association with intensive macropinocytosis.Oncotarget. 2017 Feb 28;8(9):14748-14758.
[4]. 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.
[5]. 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(5):1241-53.
[6]. Role of Na+/H+ exchanger in the pathogenesis of ischemic acute renal failure in mice. J Cardiovasc Pharmacol. 2007 Mar;49(3):154-60.
[7]. Inhibition of lipopolysaccharide-induced prostaglandin E2 production and inflammation by the Na+/H+ exchanger inhibitors. J Pharmacol Exp Ther. 2007 Apr;321(1):345-52.

TRPP3, a member of the transient receptor potential (TRP) cation channel superfamily, is a Ca²⁺-activated channel permeable to Ca²⁺, Na⁺, and K⁺. TRPP3 is involved in the regulation of sour taste perception in bipolar cells of the tongue and the regulation of pH-sensitive action potentials in spinal cord neurons. TRPP3 is also present in excitatory and non-excitatory cells of other tissues, including the retina, brain, heart, testes, and kidneys, but its function remains unclear. This study investigated the regulatory effects of amiloride and its analogues (known for inhibiting various ion channels and transport proteins and responding to all taste stimuli) on TRPP3 channel function using Xenopus laevis oocyte expression, electrophysiological, and radiometric tracing techniques. We found that amiloride and its analogues exhibit varying inhibitory affinities for TRPP3 channel activity. Radiolabeled (45)Ca2+ uptake assays showed that amiloride, benamid, benzamid, and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) inhibited TRPP3-mediated Ca2+ transport. Dual microelectrode voltage-clamp assays showed that amiloride analogs significantly inhibited TRPP3-mediated Ca2+ activation current, with the inhibitory efficacy in the order of benamid > benzamid > EIPA > amiloride, with IC50 values of 0.14, 1.1, 10.5, and 143 μM, respectively. Inhibitory efficacy was positively correlated with inhibitor molecule size. Using cell-attached patch-clamp techniques, we found that amiloride analogs reduced channel opening probability and mean opening time, but had no effect on single-channel conductance. Previous studies on the inhibitory effect of benamid in the presence of the previously reported inhibitor tetrapentylammonium showed that amiloride and organic cationic inhibitors competed for binding to the same site on TRPP3. TRPP3 may be associated with previously reported in vivo amiloride-sensitive cation transport. [1]
EIPA hydrochloride is a research-grade compound used primarily in studies of ion channels, autophagy, and inflammation. It is also known as L593754 hydrochloride and 5-(N-Ethyl-N-isopropyl)amiloride hydrochloride. The compound is not an approved therapeutic drug and has no clinical trial history. It is supplied as a light yellow to yellow solid with a purity of ≥98% and should be stored at -20°C.

C11H19CL2N7O

336.220858812332

335.1

C, 39.30; H, 5.70; Cl, 21.09; N, 29.16; O, 4.76

1345839-28-2

EIPA;1154-25-2

56935648

Typically exists as Light yellow to yellow solids at room temperature

4

5

4

21

372

0

CCN(C1=NC(=C(N=C1Cl)C(=O)N=C(N)N)N)C(C)C.Cl

BGHBOQNLQNKPFO-UHFFFAOYSA-N

InChI=1S/C11H18ClN7O.ClH/c1-4-19(5(2)3)9-7(12)16-6(8(13)17-9)10(20)18-11(14)15;/h5H,4H2,1-3H3,(H2,13,17)(H4,14,15,18,20);1H

3-amino-6-chloro-N-(diaminomethylidene)-5-[ethyl(propan-2-yl)amino]pyrazine-2-carboxamide;hydrochloride

EIPA hydrochloride; EIPA (hydrochloride); 1345839-28-2; CHEMBL1909809; 3-amino-6-chloro-N-(diaminomethylidene)-5-[ethyl(propan-2-yl)amino]pyrazine-2-carboxamide;hydrochloride; L593754 hydrochloride; 5-(N-Ethyl-N-isopropyl)amiloridehydrochloride; MH 12-43 hydrochloride; .

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)

DMSO : ~130 mg/mL (~386.65 mM)

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.)