Cetylpyridinium chloride (CPC) binds to the hepatitis B virus (HBV) core protein dimer (Cp149). The binding association constant (Kᵢ) for CPC with Cp149 dimer was calculated as 1.0 ± 0.5 µM⁻¹ from capsid assembly assays and 1.2 ± 0.9 µM⁻¹ from microscale thermophoresis assays. The binding interaction energy between the Cp149 dimer and CPC was predicted to be 8.5 kcal/mol by in silico docking simulation. [1]

Cetylpyridinium chloride selectively interacts with the core protein, also known as HBcAg, which is a dimeric viral nucleocapsid protein. Compared to other HBV inhibitors, cetylpyridinium chloride dramatically reduced the quantity of HBV particles in the HepG2.2.15 cell line. Reduced HBV biogenesis and capsid assembly are the outcomes of cetylpyridinium chloride's inhibition [1]. A safe antibacterial drug with broad-spectrum activity, cetylpyridinium chloride inhibits the production of biofilms and gingivitis [2].

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In an in vitro capsid assembly assay using purified HBV core protein dimer (Cp149), Cetylpyridinium chloride (CPC) inhibited capsid assembly in a concentration-dependent manner with an IC₅₀ of approximately 2–3 µM. At 20 µM CPC, capsid formation was markedly reduced. [1]
In HepG2.2.15 cells (a human hepatocellular carcinoma cell line stably expressing HBV), CPC treatment at concentrations up to 1 µM significantly reduced both intracellular and extracellular HBV DNA levels. At 1 µM CPC, extracellular HBV DNA was reduced by approximately 70% compared to control. [1]
CPC treatment (up to 1 µM) did not significantly affect HBV RNA levels in HepG2.2.15 cells, indicating that CPC does not inhibit viral transcription. [1]
CPC showed no significant cytotoxicity in HepG2.2.15 cells at concentrations up to 1 µM, as determined by MTT assay. [1]
CPC selectively interacted with the HBV core protein dimer, as demonstrated by microscale thermophoresis, with a dissociation constant (Kd) of 9.98 µM. CPC did not show interaction with other control proteins (BSA, anti-TAK1, anti-GST, anti-MDM2, Pac1). [1]
Circular dichroism (CD) analysis showed that CPC (5 µM) induced conformational changes in the HBV core protein secondary structure, similar to the positive control benzenesulfonamide (BS, 50 µM). [1]
Transmission electron microscopy (TEM) analysis revealed that CPC treatment (20 µM) disrupted HBV capsid assembly, resulting in broken and aberrant capsid particles. In CPC-treated samples, 51.26% of capsid-like particles were broken and 27.77% were aberrant, compared to 4.78% broken and 3.02% aberrant in untreated fully assembled samples. [1]
When combined with lamivudine (LAM), CPC showed a synergistic effect in reducing HBV DNA levels. The combination index (CI) values were <1 for most ratios tested, indicating synergism (e.g., CPC:LAM = 12:1, CI = 0.551; 4:1, CI = 0.376; 3:1, CI = 0.796). The IC₅₀ of CPC alone was 0.251 µM, and LAM alone was 0.023 µM. [1]

In a mouse hydrodynamic model system, treatment with cetylpyridinium chloride (30 μg/kg; intramuscular injection; daily; for 3 consecutive days; male C57BL/6 mice) inhibits HBV replication [1].

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In a hydrodynamic mouse model of HBV infection (C57BL/6 mice injected with pAAV-HBV1.2x plasmid), intramuscular injection of Cetylpyridinium chloride (30 µg/kg/day) for 3 days significantly reduced serum HBV DNA levels. On day 2, serum HBV DNA was decreased by approximately 60% compared to the control group; on day 3, it was decreased by approximately 45% (P < 0.05). [1]

However, the interaction between CPC and the HBV core protein dimer was assessed using microscale thermophoresis. Cp149 was labeled with a fluorescent dye, and CPC was serially diluted (0.02 to 100 µM) into the protein solution. Samples were incubated at 37°C for 10 min, and then analyzed using a Monolith NT.115 instrument. The dissociation constant (Kd) was calculated based on the change in normalized fluorescence. [1]
In silico docking simulations were performed using Discovery Studio 2.5. The Cp149 dimer structure was built from X-ray crystallography data (PDB ID: 1QGT). The CHARMM force field and Momany-Rone partial charge method were used. LibDock, LigandFit, and CDOCKER algorithms were employed to screen candidate binding sites and docking poses. Energy minimization was performed using the Adopted Basis-set Newton-Raphson algorithm, and structures were equilibrated at 37°C for 100,000 cycles. [1]

No traditional enzyme activity assays are reported in this reference. However, the interaction between CPC and the HBV core protein dimer was assessed using microscale thermophoresis. Cp149 was labeled with a fluorescent dye, and CPC was serially diluted (0.02 to 100 µM) into the protein solution. Samples were incubated at 37°C for 10 min, and then analyzed using a Monolith NT.115 instrument. The dissociation constant (Kd) was calculated based on the change in normalized fluorescence. [1]
In silico docking simulations were performed using Discovery Studio 2.5. The Cp149 dimer structure was built from X-ray crystallography data (PDB ID: 1QGT). The CHARMM force field and Momany-Rone partial charge method were used. LibDock, LigandFit, and CDOCKER algorithms were employed to screen candidate binding sites and docking poses. Energy minimization was performed using the Adopted Basis-set Newton-Raphson algorithm, and structures were equilibrated at 37°C for 100,000 cycles. [1]

Animal/Disease Models: Male C57BL/6 mice (6 weeks old) were injected with plasmid [1]
Doses: 272 μg/kg/day
Route of Administration: intramuscularinjection; daily; for 3 days
Experimental Results: Serum HBV DNA levels were suppressed, Compared with the control, the decrease was 60% on day 2 and 45% on day 3.

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Six-week-old male C57BL/6 mice (n = 5 per group) were used. The HBV replicative plasmid pAAV-HBV1.2x was injected into mice via the tail vein. Cetylpyridinium chloride (30 µg/kg/day) was administered intramuscularly daily for 3 days. Serum samples were collected daily from the orbital sinus, and HBV DNA levels were quantified by real-time PCR. Control animals received DMSO (1:1000 dilution) intramuscularly. Mice were sacrificed by CO₂ gassing 4 days after plasmid injection. All animal studies were conducted in accordance with institutional ethical regulations. [1]

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Six-week-old male C57BL/6 mice (n = 5 per group) were used. The HBV replicative plasmid pAAV-HBV1.2x was injected into mice via the tail vein. Cetylpyridinium chloride (30 µg/kg/day) was administered intramuscularly daily for 3 days. Serum samples were collected daily from the orbital sinus, and HBV DNA levels were quantified by real-time PCR. Control animals received DMSO (1:1000 dilution) intramuscularly. Mice were sacrificed by CO₂ gassing 4 days after plasmid injection. All animal studies were conducted in accordance with institutional ethical regulations. [1]

Absorption, Distribution and Excretion
After rinsing the mouth with 10 mL of 2.2 mmol solution for 1 minute, the oral retention rate of hexadecylpyridine chloride was 65% of the administered dose.

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In silico ADMET property analysis predicted that Cetylpyridinium chloride has low solubility (log S = -5.81), high plasma protein binding (PPB = 100%), low blood-brain barrier (BBB) penetration confidence, and no predicted inhibition of cytochrome P450 2D6 (CYP2D6). Hepatotoxicity probability was predicted to be low (0.27). The calculated AlogP98 was 4.97, and polar surface area (PSA2D) was 16.33 Ų. These properties suggest low penetration and absorption due to hydrophobicity and low polar surface area. [1]

Toxicity Data
LC50 (Rat) = 90 mg/m³/4h

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Non-human toxicity values
Oral LD50 in rats: 200 mg/kg
Intraperitoneal LD50 in rats: 6 mg/kg
Subcutaneous LD50 in rats: 250 mg/kg
Intravenous LD50 in rats: 30 mg/kg
For more complete non-human toxicity data for cetylpyridine chloride (8 in total), please visit the HSDB record page.

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Cetylpyridinium chloride showed no significant cytotoxicity in HepG2.2.15 cells at concentrations up to 1 µM, as determined by MTT assay. [1]
In the mouse model, no obvious adverse effects were reported following intramuscular administration of 30 µg/kg/day CPC for 3 days. [1]
In silico ADMET prediction indicated low hepatotoxicity probability (0.27). [1]

[1]. Cetylpyridinium chloride interaction with the hepatitis B virus core protein inhibits capsid assembly. Virus Res. 2019 Apr 2;263:102-111.

[2]. Cetylpyridinium chloride at sublethal levels increases the susceptibility of rat thymic lymphocytes to oxidative stress. Chemosphere. 2017 Mar;170:118-123.

Hexadecylpyridine chloride is a pyridine salt with hexadecylpyridine as its cation and chloride as its anion. It possesses antibacterial properties and is commonly used to treat minor infections of the mouth and throat. It is available in solutions or lozenges. It is both an antibacterial agent and a surfactant. It is a chloride salt and also an organochloride salt. It contains hexadecylpyridine. Hexadecylpyridine chloride is the chloride form of hexadecylpyridine, a quaternary ammonium salt with broad-spectrum antibacterial activity. After topical application, hexadecylpyridine chloride carries a positive charge and reacts with the negatively charged surface of microbial cells, thereby disrupting the integrity of the cell membrane. This leads to leakage of intracellular components, ultimately resulting in microbial death. See also: hexadecylpyridine (with the active moiety); hexadecylpyridine chloride; domiphen bromide (one of the ingredients). Benzalkonium chloride; hexadecylpyridine chloride (an ingredient)...see more...

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Mechanism of Action
The mode of action...is not fully understood, but has been attributed to...denaturation of lipoprotein complexes, and possible other effects. /Quaternary Ammonium Compounds/
Evidence suggests that the primary site of action…is likely the cell membrane, where these drugs may cause alterations in cell membrane permeability, allowing enzymes, coenzymes, and metabolic intermediates to escape. Cationic Surfactants
Emulsify sebum and then remove it along with dirt and bacteria. The gentle exfoliating action of quaternary ammonium compounds aids in cleansing. Quaternary Ammonium Compounds
Therapeutic Uses

Topical Anti-infective Agents
A topical anti-infective agent with surface activity and antibacterial properties against susceptible nonspore-forming bacteria…used for preoperative skin preparation, prophylactic disinfection of minor wounds, and…rinsing or topical application to mucous membranes.
…Also used in mouthwashes.
A cationic surfactant…with bactericidal or bacteriostatic activity against a variety of Gram-positive and Gram-negative bacteria. …Also effective against certain fungi (including Candida albicans) and Trichomonas vaginalis, but ineffective against spores or most viruses.
Dosage: For external use, use a solution of 1:100 to 1:1000 for intact skin; a solution of 1:1000 for minor lacerations; a solution of 1:2000 to 1:10000 for mucous membranes. Tablets or lozenges: 0.33 to 3 mg; rectal administration: 0.05%. Dosage forms: Tablets (NF): 1.5 mg, concentration 1:1500; solutions (NF): 1:1000.
For more complete data on the therapeutic uses of hexadecylpyridine chloride (16 in total), please visit the HSDB record page.

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Drug Warning
Quaternary ammonium disinfectants…apply externally to the skin…form a film, under which bacteria may survive, even if the outer surface of the film is bactericidal.Because hexadecylpyridine chloride is inactivated by soap, soap used in preoperative skin treatments must be thoroughly removed before applying the disinfectant. Gram-negative bacteria, including strains of Pseudomonas aeruginosa, are more tolerant than Gram-positive bacteria and require longer exposure to quaternary ammonium-1,4-dichloromethane. Quaternary ammonium-1,4-dichloromethane can be absorbed and inactivated by cotton fabrics, cellulose sponges, certain plastics (especially polyvinyl chloride), or other porous materials. Therefore, these reagents should not be used for the cold sterilization of catheters, flexible endoscopes, or other instruments. /Quaternary ammonium-1,5-di(2-NH4+) compounds/ Prolonged use may cause occasional allergic reactions, similar to some deodorants and diaper washes. /Catonic surfactants/ Due to adsorption/…repeated use of the same solution to sterilize porous materials can cause the reagent concentration to drop below the bactericidal limit. /Catonic surfactants/

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Cetylpyridinium chloride (CPC) is a quaternary ammonium compound widely used as an antiseptic in mouthwashes and pesticides. This study identified CPC as a novel inhibitor of hepatitis B virus (HBV) capsid assembly. CPC binds to the HBV core protein dimer (Cp149), induces conformational changes, and inhibits the formation of icosahedral capsid structures. The IC₅₀ for capsid assembly inhibition was approximately 2–3 µM. CPC treatment reduced HBV DNA levels in HepG2.2.15 cells and in a mouse model of HBV infection. CPC showed synergistic antiviral effects when combined with lamivudine. These findings suggest that CPC, or its derivatives, could be developed as a capsid assembly inhibitor for HBV therapy. [1]

C21H38CLN

339.99

339.269

123-03-5

Cetylpyridinium chloride monohydrate;6004-24-6

31239

White to off-white solid powder

77°C

3.459

0

1

15

23

208

0

[Cl-].[N+]1(C([H])=C([H])C([H])=C([H])C=1[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H]

YMKDRGPMQRFJGP-UHFFFAOYSA-M

InChI=1S/C21H38N.ClH/c1-2-3-4-5-6-7-8-9-10-11-12-13-14-16-19-22-20-17-15-18-21-22;/h15,17-18,20-21H,2-14,16,19H2,1H3;1H/q+1;/p-1

1-hexadecylpyridin-1-ium;chloride

Cetylpyridinium chloride NSC-14864 NSC14864NSC 14864

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Ethanol : ~100 mg/mL (~294.13 mM)
DMSO : ~100 mg/mL (~294.13 mM)
H2O : ~50 mg/mL (~147.06 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.35 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 (7.35 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 (7.35 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: ≥ 2.5 mg/mL (7.35 mM) (saturation unknown) in 10% EtOH + 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 EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 5: ≥ 2.5 mg/mL (7.35 mM) (saturation unknown) in 10% EtOH + 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 EtOH 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 6: ≥ 2.5 mg/mL (7.35 mM) (saturation unknown) in 10% EtOH + 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 EtOH stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 7: 100 mg/mL (294.13 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.)