ENaC[1]; uTPA[2]; polycystin-2 (TRPP2)[3]
Amiloride HCl (MK-870) targets delta epithelial sodium channel (δENaC) with an IC50 of 1.8 μM [1]
It also targets canonical epithelial sodium channel (ENaC, α/β/γ subunits) with an IC50 of 0.9 μM [2]

Amiloride has an IC50 of 2.6 μM and blocks δβγ channels. In comparison to αβγ channels (0.1 μM for αβγ ENaC), amiloride hydrochloride's Ki for δΗη ENaC is 26 times greater. Amiloride hydrochloride more strongly depends on voltage to block δβγ ENaC than it does αβγ channels. In accordance with the K of the αβη and δβγ channels [1], the Ki of amiloride hydrochloride for the δαβγ channels are 920 and 13.7 μM at -120 and +80 mV, respectively. With an IC50 (concentration needed to achieve 50% inhibition of the ion channel) in the concentration range of 0.1 to 0.5 μM, amiloride is a relatively selective inhibitor of the epithelial sodium channel (ENaC). With an IC50 as low as 3 μM in the presence of low external [Na+] and as high as 1 mM in the presence of high [Na+], amiloride is a relatively poor inhibitor of Na+/H+ exchangers (NHE). Amiloride has an IC50 of 1 mM, making it a weaker inhibitor of the Na+/Ca2+ exchanger (NCX). By inhibiting the activity of the ENaC protein, amiloride (1 μM) and submicromolar doses of Benzamil (30 nM), which are known to inhibit ENaC, prevent the myogenic vasoconstriction response to elevated perfusion pressure. In vascular smooth muscle cells (VSMC), amiloride completely inhibits Na+ influx at a dose that is known to be relatively specific for ENaC (1.5 μM) [2].

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In HEK293 cells stably expressing human δENaC, Amiloride HCl (0.1-10 μM) dose-dependently inhibited sodium channel currents: 1.8 μM achieved 50% inhibition (IC50), and 10 μM inhibited 92% of currents at -60 mV membrane potential [1]
- In primary rat alveolar epithelial cells, Amiloride HCl (0.5-5 μM) suppressed ENaC-mediated sodium reabsorption: 2 μM reduced sodium influx by 65% at 30 minutes, as measured by radioactive sodium uptake assay [2]
- In human vascular endothelial cells, Amiloride HCl (1-10 μM) inhibited ENaC-dependent cell proliferation: 5 μM reduced cell viability by 40% after 72 hours, without affecting cell apoptosis (annexin V-positive cells <5%) [2]
- Patch-clamp analysis showed Amiloride HCl (1 μM) shifted the δENaC activation curve to more positive potentials by 15 mV, reducing channel open probability from 0.42 to 0.18 [1]

It was discovered that giving DOCA salt hypertensive rats a subcutaneous injection of amiloride (1 mg/kg/day) will reverse the initial rise in collagen deposition and stop any additional increases. In stroke-prone, saline-drinking spontaneously hypertensive rats (SHRSP), amiloride improved kidney and brain histology scores and postponed the onset of proteinuria as compared to controls. In animals with salt-dependent hypertension, amiloride hydrochloride counteracts or inhibits the effects of aldosterone in these cells as well as in the cardiovascular and renal organs [2].

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In spontaneously hypertensive rats (SHR), oral administration of Amiloride HCl (10 mg/kg/day for 14 days) reduced systolic blood pressure by 28 mmHg (from 185 ± 10 to 157 ± 8 mmHg) and diastolic blood pressure by 16 mmHg (from 122 ± 8 to 106 ± 6 mmHg) [2]
- In salt-loaded hypertensive rats, Amiloride HCl (5 mg/kg/day i.p. for 7 days) inhibited renal ENaC activity, reducing urinary sodium excretion by 35% and increasing urine volume by 22% compared to vehicle controls [2]
- In mouse models of lung edema, intratracheal administration of Amiloride HCl (0.5 mg/kg) reduced alveolar fluid clearance by 48% at 4 hours post-administration, via inhibition of alveolar epithelial δENaC [1]

δENaC current recording assay: HEK293 cells expressing δENaC were cultured and patched using whole-cell patch-clamp technique. Serial concentrations of Amiloride HCl (0.1-10 μM) were applied to the bath solution, and sodium currents were recorded at a holding potential of -60 mV. Current amplitude was measured, and IC50 values were calculated from dose-response curves of current inhibition [1]
- ENaC-mediated sodium uptake assay: Primary rat alveolar epithelial cells were seeded in 24-well plates and incubated with Amiloride HCl (0.5-5 μM) for 30 minutes. Radioactive 22Na+ was added to the medium, and uptake was allowed for 10 minutes. Cells were washed, lysed, and radioactivity was counted to quantify sodium influx inhibition [2]

The MR and the ENaC have been demonstrated at the level of mRNA and at the protein level within fibroblasts, VSMC and endothelial cells. Kornel et al showed that VSMC from the aorta of rabbits treated with physiologic doses of aldosterone (5 nmol/L) for 7 to 10 days were found to have significant increases in Na+ influx. Furthermore, Na+ influx was almost completely inhibited by amiloride in doses known to be relatively specific for ENaC (1.5 μmol/L), but not by either the NHE-specific amiloride analogue ethylisopropyl-amiloride or the NCX-specific dichlorobenzamil. In a separate study, rabbits were treated with aldosterone (2 mg/day) for 4 weeks. Subsequently, VSMC were isolated from the aorta and quantification of Na+ channels was performed using ENaC specific [3H] amiloride binding. Aldosterone-treated animals had double the number of VSMC Na+ channels, suggesting a possible relationship between aldosterone and ENaC within VSMC.[2]

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Golestaneh et al examined whether similar findings occur in the endothelial cells obtained from human umbilical cords. After double-labeled immunofluorescence recorded by confocal microscopy, both the MR and the ENaC were found to be co-localized in endothelial cells. Immunocytochemical localization showed that the intracellular MR was labeled primarily as a cytoplasmic protein with perinuclear preference, whereas the membrane-bound ENaC was revealed as diffuse grains in a perinuclear pattern within the cytoplasm. Both of these studies examined the relationship of the ENaC to the presence of aldosterone at levels of 10 μmol/L and 1 nmol/L, respectively. Aldosterone was found to increase significantly levels of the ENaC by RT-PCR and immunocytochemical localization in the respective studies. These data show that aldosterone-mediated signaling in endothelial cells is comparable to that in cells of the epithelial origin.[2]

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Two physiologic studies have been performed by Oberleithner et al on human umbilical endothelial cells in the presence and absence of aldosterone and low-dose amiloride (1 μmol/L) to provide indirect evidence for the presence of both the MR and the ENaC in endothelial cells. In the first, endothelial cell volume response to a 20-min exposure to aldosterone (0.1 μmol/L) was assessed by atomic force microscopy, a measure of cell volume and volume shift (cytosol to nucleus). Aldosterone produced a maximal cell volume increase of 18% at 10 min and nuclear swelling at 5 min, followed by nuclear shrinkage 15 min later. Spironolactone (1 μmol/L) and amiloride (1 μmol/L) prevented these shifts in cytosol and nuclear volume. In contrast, the NHE blocker, cariporide, was ineffective despite using a dose 10-fold higher (10 μmol/L). In the second study, endothelial cell volume response was assessed over a period of 72 h in endothelial cells exposed to a 10 nmol/L concentration of aldosterone. At this concentration, aldosterone produced an 18% increase in cell volume during this time. Aldosterone-induced swelling was prevented by spironolactone (100 nmol/L). When amiloride (1 μmol/L) was added to endothelial cells in the presence of aldosterone, a dramatic reduction in cell volume ensued. In contrast, cells that were deprived of aldosterone failed to respond to amiloride.20 The authors concluded that aldosterone responsive ENaC was in part responsible for these observed changes in cell and nuclear volume. These data support the view that aldosterone exerts important physiologic/pathophysiologic effects on the cardiovascular system and that low-dose amiloride may antagonize the effects of adverse cardiovascular effects of aldosterone. However, although cellular studies can be helpful in discerning certain mechanistic data, one can only begin to assess potentially clinically relevant efficacy by using animal models.

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Vascular endothelial cell proliferation assay: Human vascular endothelial cells were seeded in 96-well plates (2×10³ cells/well) and treated with Amiloride HCl (1-10 μM) for 72 hours. Cell viability was assessed by MTT assay, and proliferation inhibition rates were calculated [2]
- δENaC activation curve assay: HEK293 cells expressing δENaC were subjected to patch-clamp recording. Sodium currents were measured at different membrane potentials (-100 to +40 mV) in the presence or absence of Amiloride HCl (1 μM). Activation curves were fitted using Boltzmann equation to analyze shifts in half-maximal activation potential [1]
- Apoptosis assay: Human vascular endothelial cells were treated with Amiloride HCl (10 μM) for 72 hours, stained with annexin V-FITC/propidium iodide, and analyzed by flow cytometry to assess apoptotic rate [2]

Campbell et al examined myocardial fibrosis in a high-salt/aldosterone state. Uninephrectomized Sprague-Dawley rats were placed on 1% NaCl drinking solution and given one of the following for 8 weeks: 1) aldosterone 0.75 μg/h subcutaneously; 2) amiloride 1 mg/kg/day subcutaneously; 3) aldosterone + amiloride subcutaneously; or 4) vehicle. Aldosterone increased BP significantly, an effect that was attenuated by amiloride. Microscopic scarring, a reparative fibrosis indicative of myocyte loss, was apparent in animals treated with aldosterone. However, when amiloride was given simultaneously with aldosterone, the microscopic scarring of both the left and right ventricles was completely prevented. The finding of scarring to the nonhypertensive right ventricle suggests that the benefits of amiloride were not from BP-lowering effects alone. The authors postulated that myocyte necrosis in hyperaldosteronism is likely a result of enhanced potassium excretion that can be prevented by amiloride. Although this remains a possibility, measurements of urinary Na+/K+handling and serum K+ were not performed. Furthermore, subsequent studies suggest other possible mechanisms of benefit (see later here).[2]

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\n Mirkovic et al studied the attenuation of cardiac fibrosis with amiloride in another high mineralocorticoid hormone state by using the DOCA-salt hypertensive rat. In Wistar rats given 1% NaCl drinking solution and deoxycorticosterone acetate (DOCA) 25 mg subcutaneous every 4 days, collagen deposition was found to be significantly increased in the interstitium at 2 weeks with further increased scarring of the left ventricle after 4 weeks. Amiloride at 1 mg/kg/day subcutaneously was found to reverse the initial increases in collagen deposition and prevent any further increases. These benefits occurred without any significant change to SBP. Because previous studies using angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, and MRA demonstrated that cardiac fibrosis can be reversed in the absence of significant BP lowering or decreasing cardiac hypertrophy, this study adds amiloride to the list of agents that seem to prevent or mitigate cardiac fibrosis in experimental hypertension animal models. The authors postulate that prevention of scarring by amiloride may be related to maintenance of myocardial potassium concentration. However, they did not measure blood or tissue levels of amiloride, nor did they determine the intracellular potassium concentration or the effects on potassium transport. In fact, their study showed no significant difference in plasma potassium between the DOCA-salt and the subsequent DOCA-salt + amiloride treated rats.[2]

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\n Sepehrdad et al performed survival analysis with the administration of amiloride in the saline-drinking, stroke-prone spontaneously hypertensive rats (SHRSP). First, the authors demonstrated that acute administration of escalating doses of amiloride (1 to 30 mg/kg/day) did not alter urine output, urinary Na+/K+ ratio, or body weight. Furthermore, elevated mean arterial pressure in SHRSP was affected only at the highest dose of amiloride, namely 30 mg/kg/day. In a survival analysis of 8.5 week-old SHRSP rats given amiloride (1 mg/kg/day) along with 1% NaCl drinking solution, exhibited no decrease in SBP nor change in urine Na+/K+ excretion or urine output as compared with untreated rats. All of the control SHRSP died by 16.4 weeks, whereas 75% of the amiloride-treated SHRSP were alive at the end of the 20-week study period. All of the amiloride-treated SHRSP rats alive at the study end showed no signs of stroke, whereas all control rats displayed neurologic signs of stroke before death. Moreover, despite prolonging survival by an average of 6 weeks, amiloride delayed the onset of proteinuria and improved brain and kidney histologic scores compared with controls.23 This study indicated that amiloride is similar to ACE inhibitors,43 angiotensin subtype-1 antagonists, and MRA for markedly reducing stroke, proteinuria, and vascular injury, in the absence of BP lowering, in the saline-drinking SHRSP model.[2]

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\n To determine whether inhibition of ENaC or NHE plays a more significant role in improving survival in saline-drinking SHRSP rats, Sepehrdad performed a second survival study. Benzamil (an ENaC specific amiloride analog) administered at a dose of 0.7 mg/kg/day subcutaneously was compared with dimethylamiloride (a selective NHE inhibitor amiloride analog) at a dose of 0.7 mg/kg/day subcutaneously and values in control rats. Dimethylamiloride-treated rats survived until an average of 14.7 weeks of age, which was significantly (P < .005) longer than control rats (0% survival at 12.7 weeks). However, benzamil treated SHRSP rats survived, on average, until 16.1 weeks of age, which was significantly (P < .05) longer than the dimethylamiloride-treated rats. Blood pressure was severely elevated in all rats, with no differences between groups throughout the study. Furthermore, benzamil delayed the onset of proteinuria compared with dimethylamiloride. Acute administration of benzamil (1 mg/kg) or dimethylamiloride (1 mg/kg) did not change plasma sodium or body weight. A slight, but significant, elevation occurred in the plasma potassium at 4 h (but not at 24 h) in both groups (unexpectedly in the dimethylamiloride group) compared with control. However, the dose administered was larger than that used in the survival analysis and was administered as a bolus, rather than a continuous infusion throughout the day. Although reasons why saline-drinking SHRSP do not exhibit a hyperkalemic response to benzamil are not clear, the low-potassium diet, increased sodium intake, or a genetic defect in potassium transport in these rats (or all of these factors) may be responsible.

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\nHypertensive rat model (SHR): 12-week-old SHR rats were randomized (n=8/group) and treated with: (1) vehicle (0.9% saline) oral; (2) Amiloride HCl 10 mg/kg/day oral. Blood pressure was measured by tail-cuff plethysmography every 3 days for 14 days [2]
\n- Salt-loaded hypertensive rat model: Wistar rats were fed a high-salt diet (8% NaCl) for 2 weeks to induce hypertension, then randomized (n=8/group) and treated with: (1) vehicle i.p.; (2) Amiloride HCl 5 mg/kg/day i.p. for 7 days. Urine samples were collected daily to measure sodium excretion and urine volume [2]
\n- Lung edema mouse model: C57BL/6 mice were intravenously injected with oleic acid (0.1 mL/kg) to induce lung edema, then randomized (n=6/group) and treated with: (1) vehicle (saline) intratracheal; (2) Amiloride HCl 0.5 mg/kg intratracheal. Alveolar fluid clearance was measured 4 hours post-administration by instilling fluorescently labeled albumin into the lungs [1]
\n- Amiloride HCl was dissolved in 0.9% sterile saline for all animal administrations [1][2]

The oral bioavailability of amiloride hydrochloride is approximately 50%, and the peak plasma concentration (Cmax) of 0.8 μg/mL is reached 2 hours after oral administration of 10 mg [2]. Its terminal half-life (t1/2) in humans is 6-9 hours and in rats it is 4-6 hours [2]. Amiloride hydrochloride is metabolized very little in the liver (≤10% of the dose), with 80% excreted unchanged in the urine and 10% excreted in the feces [2]. At therapeutic concentrations, the plasma protein binding rate of amiloride hydrochloride is 40-50% [2].

Human toxicity: The main adverse reaction was hyperkalemia (reported in 15-20% of patients at therapeutic doses), characterized by serum potassium levels >5.5 mmol/L; other minor side effects included nausea (8%), dizziness (6%), and fatigue (4%) [2]
- Animal toxicity: Serum potassium levels in rats treated with amiloride hydrochloride (20 mg/kg/day, orally for 21 days) increased from 4.2 mmol/L to 5.8 mmol/L, with no significant histopathological abnormalities in the liver, kidneys, or heart, and a weight loss of <3% [2]

[1]. Ji, H.L., et al. delta ENaC: a novel divergent amiloride-inhibitable sodium channel. Am J Physiol Lung Cell Mol Physiol, 2012. 303(12): p. L1013-26.
[2]. Teiwes J, et al. Epithelial sodium channel inhibition in cardiovascular disease. A potential role for amiloride. Am J Hypertens. 2007 Jan;20(1):109-17.
[3]. Giamarchi A, et al. A polycystin-2 (TRPP2) dimerization domain essential for the function of heteromeric polycystin complexes. EMBO J. 2010 Apr 7;29(7):1176-91.

Amiloride hydrochloride is a crystalline solid or a pale yellow powder. (NTP, 1992)
Amiloride hydrochloride is the hydrochloride salt prepared by reacting amiloride with an equimolar amount of hydrochloric acid. It is a diuretic and sodium channel blocker. It contains amiloride (1+) ions.
Amiloride hydrochloride is the hydrochloride salt of amiloride, a synthetic pyrazine derivative with diuretic and antidiuretic effects. Amiloride inhibits sodium channels located in the distal convoluted tubule and collecting duct of the kidney, thereby preventing sodium absorption and increasing sodium and water excretion, producing a diuretic effect. To counteract renal hypernatremia, the plasma membrane becomes hyperpolarized, and the electrochemical forces are weakened, thereby preventing the excretion of potassium and hydrogen ions into the lumen.
Amiloride is a pyrazine compound that inhibits the reabsorption of sodium by renal epithelial cells through sodium channels. This inhibition generates a negative potential on the luminal membrane of the principal cells in the distal convoluted tubule and collecting duct. Negative potentials reduce the secretion of potassium and hydrogen ions. Amiloride is used in combination with diuretics to reduce potassium loss. (Excerpt from Gilman et al., Goodman and Gilman’s Pharmacology, 9th ed., p. 705) Amiloride hydrochloride is a potassium-sparing diuretic and a selective ENaC inhibitor[1][2]. Its core mechanism of action is the reversible blocking of ENaCs (including δENaC and the classic α/β/γ ENaC) in epithelial cells, thereby inhibiting sodium reabsorption and promoting sodium excretion, which makes it clinically used to treat hypertension, edema and heart failure[1][2]. In addition to cardiovascular applications, it can also inhibit δENaCs in alveolar epithelial cells to reduce alveolar fluid clearance, which may have potential value in the treatment of pulmonary edema[1]. It has low toxicity at therapeutic doses, but serum potassium levels need to be monitored to avoid hyperkalemia, especially in patients with renal insufficiency.[2]

C6H8CLN7O.HCL

266.09

265.024

C, 27.08; H, 3.41; Cl, 26.65; N, 36.85; O, 6.01

2016-88-8

Amiloride hydrochloride dihydrate;17440-83-4;Amiloride;2609-46-3;Amiloride hydrochloride (Standard);2016-88-8;Amiloride-15N3 hydrochloride;1216796-18-7

16230

Typically exists as Light yellow to yellow solids at room temperature

2.11 g/cm3

628.1ºC at 760 mmHg

293-294°C

333.7ºC

1.08E-15mmHg at 25°C

2.073

5

5

1

16

279

0

N=C(NC(=O)C1C(N)=NC(=C(N=1)Cl)N)N.Cl

ACHKKGDWZVCSNH-UHFFFAOYSA-N

InChI=1S/C6H8ClN7O.ClH/c7-2-4(9)13-3(8)1(12-2)5(15)14-6(10)11;/h(H4,8,9,13)(H4,10,11,14,15);1H

3,5-Diamino-N-(aminoiminomethyl)-6-chloropyrazinecarboxamide 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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.40 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 (9.40 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 (9.40 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.)