Benzethonium chloride targets cholinesterase (ChE) and acetylcholinesterase (AChE). For ChE with acetylthiocholine as substrate: Ki = 35 nmol/L, Km = 85 μmol/L, Vmax = 2.6 kU/L; α = 1.9. For AChE: Ki = 36 nmol/L, Km = 102 μmol/L, Vmax = 22.5 kU/L; α = 2.2. Mixed-type inhibition (affecting both Vmax and Km) is observed. ChE is about 10-fold more sensitive to inhibition than AChE: 0.203 μmol/L Benzethonium chloride causes 50% decrease in Vmax of ChE, while 2.03 μmol/L causes similar reduction for AChE.[1]
Benzethonium Chloride primarily targets neuronal nicotinic acetylcholine receptors (nAChRs). It is a potent inhibitor of both α4β2 and α7 nAChR subtypes, with IC50 values of 49 nM and 122 nM, respectively. The inhibition is non-competitive, and there is no voltage- or use-dependence of the response in either subtype. Additionally, Benzethonium Chloride inhibits calcium-activated chloride currents (ICl(Ca)) in oocytes expressing m1 muscarinic receptors with an IC50 of 0.88 μM. Its action on nAChRs and other ion channels underlies its diverse biological activities, including its antimicrobial and potential neuromodulatory effects.

Benzethonium chloride inhibits acetylcholine responses in the α7 nAChRs in a mixed competitive and non-competitive manner but there is no voltage- or use-dependence of the response in either subtype. Benzethonium chloride produces mixed-type inhibition of choline esterase and acetylcholine esterase-affecting both Fmax and Km, Choline esterase is about 10-fold more sensitive to benzethonium chloride than acetylcholine esterase. Benzethonium chloride inhibits ICl(Ca) in response to 0.1 μM acetyl-beta-methylcholine in oocytes expressing m1 muscarinic receptors with IC50 of 0.88 μM. Benzethonium chloride combined with racemic S(+)/R(-) ketamine inhibits muscarinic signaling with a calculated IC50 of 15 μM and a Hill coefficient of 0.6. Benzethonium (5 μM) significantly increases cytosolic Ca(2+)-concentration, decreases forward scatter and triggered annexin V-binding affecting some 30% of the erythrocytes. Benzethonium (5 μM) further significantly enhances the effect of glucose depletion on cytosolic Ca(2+)-concentration and annexin V-binding, but significantly blunts the effect of glucose depletion on forward scatter. Benzethonium (5 μM) significantly enhances lactic acid formation but not ceramide abundance. Benzethonium chloride reduces cell viability with IC50 of 3.8 μM in FaDu, 42.2 μM in NIH 3T3, 5.3 μM in C666-1, and 17.0 μM in GM05757. Benzethonium chloride (9 μM) induces apoptosis and activates caspases after 12 hours in FaDu cells.
Cell Assay: Cells (FaDu, C666-1, NIH 3T3 and GM05757 cell lines) are seeded in 96-well plates at 5,000 per well in 100 μL of growth medium and allowed to incubate for 24 hours. Benzethonium chloride is then added, as indicated, in a total volume of 5 μL. After 48 hours, MTS assay is done according to the specifications of the manufacturer, with DMSO (0.1%)–treated cells as negative control and cisplatin (166.6 μM)–treated cells as positive control.

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Benzethonium chloride produces linear mixed-type inhibition of cholinesterase and acetylcholinesterase. Lineweaver-Burk plots intersect above the 1/[S] axis, indicating α>1. For cholinesterase, in the presence of 0.203 μmol/L Benzethonium chloride, Vmax changed to 1.2 kU/L and Km to 128.7 μmol/L. For acetylcholinesterase, 2.03 μmol/L Benzethonium chloride gave Vmax 11.2 kU/L and Km 190 μmol/L. Using butyrylthiocholine as substrate, the wet chemistry method (BM/Hitachi 911) showed 29% inhibition of cholinesterase at 0.203 μmol/L Benzethonium chloride, 87% at 2.03 μmol/L, and 95% at 20.3 μmol/L; at 203 μmol/L activity was unmeasurable. The dry slide method (Ektachem 700 XR) showed no inhibition up to 20.3 μmol/L, and only 9% inhibition at 203 μmol/L, suggesting binding of Benzethonium chloride to slide components prevents it from reaching the enzyme reaction layer.[1]
In vitro, Benzethonium Chloride demonstrates potent inhibition of nAChRs, with IC50 values of 49 nM for α4β2 and 122 nM for α7 subtypes. It also inhibits ICl(Ca) in m1 muscarinic receptor-expressing oocytes with an IC50 of 0.88 μM. The compound's activity in these assays confirms its role as a non-competitive nAChR inhibitor. It is used in cell-based studies to investigate the functional roles of nAChRs in neuronal signaling and other physiological processes. Its broad-spectrum antimicrobial properties are also characterized in vitro against various bacterial and fungal strains.

Benzethonium chloride (5 mg/kg) ablates the tumor-forming ability of FaDu cells, delays the growth of xenograft tumors, and combined additively with local tumor radiation therapy in established FaDu tumors in SCID mice.

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In vivo, Benzethonium Chloride is primarily used as a topical antiseptic and disinfectant, demonstrating effective antimicrobial activity. Its systemic effects are less characterized due to its topical application. For research purposes, it can be administered orally as a homogeneous suspension. As an nAChR inhibitor, it has the potential to modulate cholinergic signaling in vivo, but detailed pharmacokinetic and pharmacodynamic data are limited. Its antimicrobial efficacy has been established in various topical formulations for skin and wound disinfection.

Cholinesterase and acetylcholinesterase activities were measured on a BM/Hitachi 911 autoanalyzer at 37°C using the method of Lewis et al. with modified protocol.
For cholinesterase: 15 μL plasma samples were added to 280 μL R1 containing 0.58 mmol/L sodium bicarbonate and 0.32 mmol/L 5,5'-dithiobis (2-nitrobenzoic acid) in 0.1 mol/L phosphate buffer (pH 8.0). After 1.25 min, the reaction was started by adding 20 μL of 7.87 mmol/L acetylthiocholine and monitored monochromatically at 415 nm between the 10th and 20th revolutions of the carousel (each revolution 20 s).
For acetylcholinesterase: sample volume was 5 μL and R1 was increased to 290 μL. When butyrylthiocholine (7.87 mmol/L) was used as substrate, it replaced acetylthiocholine.
Kinetics of inhibition: enzyme activities were determined at final acetylthiocholine concentrations of 15, 25, 50, 80, 120, 160, and 250 μmol/L in the absence and presence of 2.03 and 4.06 μmol/L Benzethonium chloride. R1 was 260 μL for cholinesterase and 270 μL for acetylcholinesterase, R2 was 20 μL isotonic saline or Benzethonium chloride solution, and R3 was acetylthiocholine (20 μL).
Protein determination by benzethonium chloride method: 15 μL urine samples were added to 250 μL R1 containing 530 mmol/L NaOH and 74 mmol/L EDTA-Na2. Reaction started by adding 100 μL R2 (32 mmol/L Benzethonium chloride) and monitored bichromatically at 505 nm (primary) and 700 nm (secondary) between the 15th and 31st revolutions.
To eliminate carry-over inhibition, the analyzer was programmed to carry out an additional cell wash with 5 mL/L Triton X-100, 10 g/L sodium dodecyl sulphate, or 5 mL/L Tween 20 after protein determination. Washing with 5 mL/L or 10 mL/L Triton X-100 abolished the carry-over inhibitory effect; 10 g/L sodium dodecyl sulphate and 5 mL/L Tween 20 were equally effective. Washing with selective mode solution (0.2 mmol/L HCl) failed to remove the inhibition.[1]
Non-cellular enzyme/receptor binding assays for Benzethonium Chloride typically involve measuring its inhibition of nAChRs using radioligand binding or electrophysiological techniques. For example, its inhibitory effects on α4β2 and α7 nAChRs can be assessed by determining the IC50 values through concentration-response curves in oocyte expression systems. Similarly, its inhibition of ICl(Ca) in m1 muscarinic receptor-expressing oocytes can be measured using two-electrode voltage-clamp electrophysiology. These assays provide quantitative data on the compound's potency and mechanism of action at the receptor level.

In vitro cell-based assays for Benzethonium Chloride are conducted using cell lines expressing nAChRs or other relevant targets. For instance, oocytes expressing m1 muscarinic receptors can be used to assess its inhibition of calcium-activated chloride currents. Additionally, cell viability assays can be performed to evaluate its antimicrobial activity against various bacterial and fungal strains. These experiments are crucial for confirming its mechanism of action and for studying its effects on cellular signaling pathways. The compound is typically dissolved in DMSO or water for cell-based studies.

Dissolved in PBS; 5 mg/kg; i.p. injection FaDu tumors in SCID mice.
In vivo animal studies for Benzethonium Chloride are limited due to its primary use as a topical agent. However, for research purposes, it can be administered orally as a homogeneous suspension formulated in CMC-Na at a concentration of ≥5 mg/mL. Alternatively, it can be administered via injection using a clear solution formulation. Studies would typically monitor its antimicrobial efficacy, toxicity, and pharmacokinetic parameters, though specific protocols are not extensively documented. Its safety and efficacy have been established in topical applications.

Absorption, Distribution and Excretion
Transdermal absorption is likely negligible.
Benzethonium Chloride has a molecular weight of 448.08 g/mol and is soluble in DMSO, water, and ethanol at 90 mg/mL. For oral administration in vivo, it can be formulated as a homogeneous suspension in CMC-Na at ≥5 mg/mL. For injection, a clear solution can be prepared using 2% DMSO, 40% PEG300, 2% Tween 80, and 56% ddH2O. Its pharmacokinetic properties, such as absorption and half-life, are not extensively documented due to its topical use. However, as a quaternary ammonium compound, it is expected to have poor systemic absorption.

Benzethonium Chloride is generally considered safe for topical use at recommended concentrations. Its toxicity profile is well-established through its use as an antiseptic and preservative. However, systemic toxicity can occur with ingestion or significant absorption. It is classified as a Category III antiseptic for surgical hand scrubs and healthcare personnel hand washes by the FDA. For research purposes, it is important to handle the compound with appropriate safety precautions, as it can cause irritation and is harmful if swallowed. It is not approved for systemic use.

Eur J Clin Chem Clin Biochem.1997 Aug;35(8):603-7;Clin Cancer Res.2006 Sep 15;12(18):5557-69.

Benzethonium chloride is an odorless white crystal or powder with an extremely bitter taste. A 1% aqueous solution is weakly alkaline to litmus paper. (NTP, 1992)
Benzalkonium chloride is a (synthetic) quaternary ammonium salt with the structure benzyldimethylamine, in which the nitrogen atom is quaternized by 2-{2-[p-(2,4,4-trimethylpentan-2-yl)phenoxy]ethoxy}ethyl, and the chloride ion is the counterion. It is a preservative and disinfectant, effective against a variety of bacteria, fungi, molds, and viruses. It can be used as a preservative, disinfectant, antibacterial agent, antiviral agent, and antifungal agent. It is a quaternary ammonium salt, chloride, and aromatic ether.
A bactericidal cationic quaternary ammonium surfactant used as a topical anti-infective agent. It is an ingredient in pharmaceuticals, deodorants, mouthwashes, etc., used for instrument sterilization in the food processing and pharmaceutical industries, surgical procedures, and can also be used as a preservative. This compound is toxic when ingested and can cause neuromuscular blockade. See also: Benzalkonium chloride (containing the active component); Benzalkonium chloride; Thymol (ingredient); Benzalkonium chloride; Lidocaine (ingredient)... See more...

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Benzethonium chloride is used in one of the most frequently employed turbidimetric methods for determination of total protein in urine and cerebrospinal fluid.
It binds to plastic cuvettes; binding is greater in the presence of protein. The bound Benzethonium chloride is not completely dislodged by routine washing procedures and can complex with enzymes, causing reagent carry-over inhibition.
For cholinesterase measured by the dry slide method (Ektachem 700 XR) using butyrylthiocholine as substrate, the assay remains unaffected until Benzethonium chloride concentration exceeds 200 μmol/L.
The equilibrium of enzyme (cholinesterase) and inhibitor reaction is in the EI direction (Ki = 36 nmol/L), and inhibitor can be stripped from EI in the dry slide, suggesting reversible, non-covalent binding.[1]
Benzethonium Chloride is a quaternary ammonium compound with potent nAChR inhibitory activity and broad-spectrum antimicrobial properties. It inhibits α4β2 and α7 nAChRs with IC50 values of 49 nM and 122 nM, respectively. Its antimicrobial action is due to its ability to disrupt microbial cell membranes. It is commonly used as a topical antiseptic, preservative, and disinfectant. In research, it serves as a tool for studying nAChR function and cholinergic signaling. The compound is not approved for systemic therapeutic use and is intended for research and topical applications only.

C27H42NO2.CL

448.08

447.29

121-54-0

Benzethonium-d7 chloride;Benzethonium-d6 chloride

8478

White to off-white solid powder

0.998 g/mL at 20 °C

162-164 °C(lit.)

6.874

0

3

12

31

466

0

UREZNYTWGJKWBI-UHFFFAOYSA-M

InChI=1S/C27H42NO2.ClH/c1-26(2,3)22-27(4,5)24-13-15-25(16-14-24)30-20-19-29-18-17-28(6,7)21-23-11-9-8-10-12-23;/h8-16H,17-22H2,1-7H3;1H/q+1;/p-1

N-benzyl-N,N-dimethyl-2-(2-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)ethoxy)ethan-1-aminium chloride

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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 (5.58 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 (5.58 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 (5.58 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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Solubility in Formulation 4: 120 mg/mL (267.81 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)