Na+/Ca2+ exchanger (NCX) (IC50 = 100 nM)
Benzamil targets several ion transport proteins, including the epithelial sodium channel (ENaC), the Na⁺/Ca²⁺ exchanger (NCX), and the TRPP3 channel. It acts as a non-selective ENaC blocker, inhibiting sodium influx through these channels. As an NCX inhibitor, it blocks the exchange of intracellular sodium for extracellular calcium, thereby modulating intracellular calcium levels. By inhibiting TRPP3, Benzamil reduces calcium-activated currents. These multiple targets make Benzamil a valuable tool for studying the roles of these ion transport proteins in various physiological and pathological processes.

Benzamil (Benzylamiloride) blocks Ca2+-activated small conductance K2+ channels in neurons and heterologously produced cells [4].
In vitro, Benzamil has been characterized for its potency against ENaC, NCX, and TRPP3. It inhibits TRPP3-mediated Ca²⁺-activated currents with an IC₅₀ of 1.1 µM. As an NCX inhibitor, it has an IC₅₀ of approximately 100 nM. In patch-clamp electrophysiology studies, Benzamil has been shown to block ENaC currents in a concentration-dependent manner. The compound's effects on intracellular calcium levels have been demonstrated in various cell types using fluorescent calcium indicators. These in vitro studies have established Benzamil as a key pharmacological tool.

Stroke-prone spontaneously hypertensive rats (SHRSP) treated with benaamil (benzylamiloride) (0.7 mg/kg/day; sc) survived, on average, until 16.1 weeks of age[5].
In vivo, Benzamil has been used to study the role of ENaC and NCX in blood pressure regulation, renal function, and vascular tone. By blocking ENaC in the kidney, Benzamil can modulate sodium reabsorption and blood pressure. Its inhibition of NCX can affect cardiac contractility and vascular smooth muscle tone. The compound has also been used to study the role of TRPP3 in calcium signaling in vivo. However, its in vivo use is limited by its poor bioavailability and the lack of selectivity among its targets.

Small conductance Ca2+-activated K+ (SK) channels are expressed throughout the soma and dendrites of pyramidal neurons in the neocortex and hippocampal formation, where they participate in the local regulation of membrane excitability and synaptic signals. Through their inter-play with Ca2+ channels, SK channels regulate Ca2+ influx triggered by back-propagating action potentials in dendrites. Inhibition of SK channels affects both the amplitude and duration of Ca2+ transients, but the role of Ca2+ clearance mechanisms and their link to SK channel activity has not been established. Here we report the effect of the Na+/Ca2+ exchanger (NCX) inhibitor benzamil on Ca2+ extrusion and SK channels in the regulation of dendritic Ca2+ signals. Benzamil increased the duration and amplitude of dendritic Ca2+ transients elicited by back-propagating action potentials in hippocampal pyramidal neurons. This data is consistent with previous studies with SK channel blockers and suggests that benzamil inhibits SK channels in addition to the Na+/Ca2+ exchanger. Here we show that indeed both the neuronal SK-mediated IAHP current and the currents mediated by heterologously expressed SK channels were inhibited by benzamil. The inhibition of recombinant SK channels was seen with different K+ concentration gradients, and was stronger at negative voltages. The suppression of SK channels by benzamil is consistent with previous findings on the modulation of Ca2+ signals by SK channels in neurons. We additionally show that benzamil inhibits neuronal voltage-gated calcium currents. The results prompt a careful reassessment of the effects of benzamil on Ca2+ transients in native systems, given the spectrum of ion channels and exchangers this compound targets within a similar range of concentrations.[4]

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In this study, researchers found that amiloride and its analogs inhibit TRPP3 channel activities with different affinities. Radiolabeled (45)Ca2+ uptake showed that TRPP3-mediated Ca2+ transport was inhibited by amiloride, phenamil, Benzamil, and 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Two-microelectrode voltage clamp experiments revealed that TRPP3-mediated Ca2+-activated currents are substantially inhibited by amiloride analogs, in an order of potency of phenamil > Benzamil > EIPA > amiloride, with IC50 values of 0.14, 1.1, 10.5, and 143 microM, respectively. The inhibition potency positively correlated with the size of inhibitors. Using cell-attached patch clamping, we showed that the amiloride analogs decrease the open probability and mean open time but have no effect on single-channel conductance. Study of inhibition by phenamil in the presence of previously reported inhibitor tetrapentylammonium indicates that amiloride and organic cation inhibitors compete for binding the same site on TRPP3. TRPP3 may contribute to previously reported in vivo amiloride-sensitive cation transport[3].
In vitro enzyme/receptor binding assays for Benzamil involve measuring its inhibition of ENaC, NCX, or TRPP3. For ENaC, the assay is typically performed using patch-clamp electrophysiology on cells expressing the channel. For NCX, the assay is performed using cells loaded with a fluorescent calcium indicator, and the exchange of sodium and calcium is measured. For TRPP3, calcium-activated currents are measured using patch-clamp electrophysiology. The IC₅₀ values are calculated from concentration-response curves.

Benzamil, an inhibitor of ENaC that also blocks Na+/Ca2+ exchange (NCX), potentiated myogenic vasoconstriction. Benzamil and low [Na+]o elicited vasoconstriction; however, these responses were attenuated by diltiazem and were associated with significant membrane depolarization, suggesting a contribution of mechanisms other than a reduction in NCX. Na+ repletion induced a vasodilation in pressurized afferent arterioles preequilibrated in low [Na+]o, a hallmark of NCX, and this response was reduced by 10 micromol/l benzamil. The dilation was eliminated, however, by a combination of benzamil plus ouabain, suggesting an involvement of the electrogenic Na+-K+-ATPase. In concert, these findings refute the premise that ENaC plays a significant role in the rat afferent arteriole and instead suggest that reducing [Na+](o) and/or Na+ entry is coupled to membrane depolarization. The mechanisms underlying these unexpected and paradoxical effects of Na+ are not resolved at the present time[2].
In vitro cellular experiments for Benzamil are performed using cells expressing ENaC, NCX, or TRPP3. The compound's effect on sodium or calcium fluxes is measured using fluorescent indicators or patch-clamp electrophysiology. For ENaC, the amiloride-sensitive sodium current is measured. For NCX, the sodium-dependent calcium influx is measured. For TRPP3, the calcium-activated current is measured. These experiments are essential for characterizing the compound's potency and mechanism of action.

Kidneys were allowed to equilibrate for at least 1 h following the establishment of in vitro perfusion. Ibuprofen (10 μmol/l) was added to eliminate the effects of endogenous prostaglandins. Basal renal perfusion pressure was held at 80 mmHg during the equilibration period. In some experiments, myogenic responses were evoked by raising renal arterial pressure and holding pressures at each step for at least 1 min. In these studies, stepped responses were assessed before and after the administration of amiloride or Benzamil. In other experiments, perfusion pressure was maintained at a constant level (60, 80, or 140 mmHg) to assess vasoconstrictor and/or vasodilatory responses. In the studies assessing the impact of low [Na+]o media, the media Na+ concentration ([Na+]) was lowered from 140 to 100 mmol/l by the isosmotic substitution of choline chloride for NaCl. Benzamil, amiloride, and ouabain were obtained commercially. Fresh stock solutions of Benzamil and amiloride were prepared in dimethyl sulfoxide. Ouabain (3 mmol/l in DMEM) was prepared fresh for each experiment. Phentolamine and propranolol (10 μmol/l; Sigma) were added during the ouabain experiments to avoid any effects mediated by neurotransmitter release.[2]
In vivo animal studies for Benzamil are conducted using mouse or rat models. The compound is administered via intravenous or intraperitoneal injection. The effects on blood pressure, renal function, or vascular tone are measured. In models of hypertension, Benzamil has been shown to lower blood pressure by blocking ENaC in the kidney. The compound's effects on cardiac function have also been studied. Pharmacokinetic parameters are evaluated from plasma samples collected at multiple time points post-administration.

The pharmacokinetic properties of Benzamil are characterized by its rapid distribution and elimination. The compound has a short half-life in circulation, and its bioavailability is limited following oral administration. It is typically administered via intravenous or intraperitoneal routes for in vivo studies. The compound is metabolized in the liver, and its metabolites are excreted via the biliary and renal routes. Its pharmacokinetic profile supports its use in acute in vivo studies.

The toxicity profile of Benzamil is not extensively characterized in the literature. As an amiloride analog, it is expected to have a similar safety profile, with potential adverse effects including hyperkalemia, metabolic acidosis, and gastrointestinal disturbances. The compound's lack of selectivity among its targets may contribute to off-target effects. Standard toxicology studies would be required to fully characterize its safety profile.

[1]. Characterization of a Na(+)-Ca(2+) exchanger in podocytes. Nephrol Dial Transplant. 2002 Oct;17(10):1742-50.

[2]. Effects of amiloride, benzamil, and alterations in extracellular Na+ on the rat afferent arteriole and its myogenic response. Am J Physiol Renal Physiol. 2008 Jul;295(1):F272-82.

[3]. Inhibition of TRPP3 channel by amiloride and analogs. Mol Pharmacol. 2007 Dec;72(6):1576-85.

[4]. Benzamil inhibits neuronal and heterologously expressed small conductance Ca2+-activated K+channels. Neuropharmacology. 2019 Nov 1;158:107738.

[5]. Epithelial sodium channel inhibition in cardiovascular disease. A potential role for amiloride. Am J Hypertens. 2007 Jan;20(1):109-17.

Benzamil belongs to the pyrazine class of compounds and also to the guanidine class of compounds.
Benzamil is a potent analog of amiloride that acts as a blocker of ENaC, an inhibitor of NCX, and an inhibitor of TRPP3. It is widely used as a research tool to study ion transport and calcium signaling. The compound inhibits TRPP3-mediated Ca²⁺-activated currents with an IC₅₀ of 1.1 µM and NCX with an IC₅₀ of approximately 100 nM. Benzamil is also used to study the role of ENaC in blood pressure regulation and renal function. Its multiple targets make it a versatile but non-selective tool for ion channel research.

C13H14N7OCL

319.74956

355.072

2898-76-2

Benzamil hydrochloride;161804-20-2

108107

Typically exists as solid at room temperature

3.594

4

6

4

22

413

0

N=C(NC(C1=NC(Cl)=C(N=C1N)N)=O)NCC2=CC=CC=C2

KXDROGADUISDGY-UHFFFAOYSA-N

InChI=1S/C13H14ClN7O/c14-9-11(16)20-10(15)8(19-9)12(22)21-13(17)18-6-7-4-2-1-3-5-7/h1-5H,6H2,(H4,15,16,20)(H3,17,18,21,22)

3,5-diamino-N-(N'-benzylcarbamimidoyl)-6-chloropyrazine-2-carboxamide

Benzamil; 2898-76-2; Benzylamiloride; 3,5-Diamino-N-(N-benzylcarbamimidoyl)-6-chloropyrazine-2-carboxamide; N-(N-Benzylamidino)-3,5-diamino-6-chloropyrazine carboxamide; UNII-04659UUJ94; CHEBI:34558; 3,5-diamino-N-(N'-benzylcarbamimidoyl)-6-chloropyrazine-2-carboxamide;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)