COVID-19 Research: In Vitro Studies Show That Cetylpyridinium Chloride Found In Most Mouthwashes Inhibits SARS-CoV-2 Coronavirus

COVID-19 Research: Scientists from IrsiCaixa AIDS Research Institute-Spain, Dentaid Research Center-Cerdanyola del Vallès, Germans Trias i Pujol Research Institute (IGTP)-Badalona, and the Central University of Catalonia have discovered through in vitro studies that cetylpyridinium chloride, found in most mouthwashes, lowers infectivity of SARS-CoV-2 likely by inhibiting viral fusion with target cells.


 
The study team said that with more tests to prove validity, mouthwashes could be a simple, cheap way of curbing transmission.
 
According to the study team oral mouthwashes decrease the infectivity of several respiratory viruses including SARS-CoV-2. However, the precise agents with antiviral activity present in these oral rinses and their exact mechanism of action remain unknown.
 
In this study the scientists show that Cetylpyridinium chloride (CPC), a quaternary ammonium compound present in many oral mouthwashes, reduces SARS-CoV-2 infectivity by inhibiting viral fusion with target cells.
 
The study team also found that CPC and CPC-containing mouth rinses decreased a thousand times the infectivity of SARS-CoV-2 in vitro, while the corresponding vehicles had no effect. CPC-containing mouth rinses could represent a cost-effective measure to reduce SARS-CoV-2 infectivity in saliva, aiding to reduce viral transmission from infected individuals.
 
The study findings were published on a preprint server and are currently being peer-reviewed. https://www.biorxiv.org/content/10.1101/2020.12.21.423779v1
 
Past research has shown that mouthwashes can have antiviral properties. They can decrease airborne respiratory coronavirus infections, including infections from the flu virus and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. https://pubmed.ncbi.nlm.nih.gov/32726430/
 
https://journals.sagepub.com/doi/full/10.1177/0022034520967933
 
https://www.biorxiv.org/content/10.1101/2020.11.13.381079v1
 
If more tests prove the antiviral properties of mouthwashes, they may be a potent and economical strategy to combat the pandemic. Using mouthwashes would be a cheap and easily accessible strategy that can be quickly implemented. Reducing viral loads in the mouth can help curb the transmission chain. Mouthwashes are also produced as oral sprays, so can be used easily by the elderly population.
 
Although studies have reported in vitro antiviral activity of various oral products, till now, it is not known which components in mouthwashes are responsible for the antiviral activity and what is their mechanism of action.
 
The study team report the effect of cetylpyridinium chloride (CPC) used in many mouthwashes and sprays. CPC is a quaternary ammonium compound with antiseptic and antimicr obial activity. https://pubmed.ncbi.nlm.nih.gov/28936484/
 
The team tested the ability of CPC-containing mouthwashes to prevent entry of SARS-CoV-2 into cells. They used HEK293T cells expressing the angiotensin-converting enzyme 2 (ACE2) receptor as target cells and pseudotyped lentivirus expressing the SARS-CoV-2 spike protein. A luciferase-based assay allowed them to detect viral binding to the target cells.
 
Subsequently the pseudovirus was mixed with different concentrations of mouthwashes and added to the target cells.
 
The study team tested two mouthwashes from Dentaid: Perio Aid Intensive Care and Vitis CPC Protec.
 
To control for cytotoxicity, the study team also mixed the mouthwashes with the target cells without the pseudovirus. In addition, they also used the same mouthwash formulation but without CPC as a control.
 
The team found that the mouthwashes inhibited viral fusion to host cells, with their inhibition activity increasing with increasing concentration of mouthwash.
 
Importantly they did not see any inhibition using the mouthwash formulation without CPC, suggesting the antiviral activity is because of CPC. In addition, the researchers suspended CPC in water and they found that this suspension also inhibited virus entry into the target cells.

The researchers also isolated SARS-CoV-2 from a clinical sample collected from an 89-year old male patient. They added equal amounts of the virus and CPC or CPC-containing mouthwash and mixed them.
 
The team found a high dose of CPC was effective in reducing virus infection on Vero E6 cells. A 2-minute treatment with CPC mouthwash decreased the Tissue Culture Infectious Dose 50% (TCID50)/ml 1000 times. The formulations without CPC had no effect of virus infectivity.
 
Also important is the fact that the doses of CPC that inhibited viral fusion to the host cell were not toxic to the host cells.
 
According to the study team, it is likely that the antiviral activity of CPC arises from its ability to disrupt the viral envelope, similar to that seen before for the flu virus. So, mouthwashes with CPC could protect against infection of the oral mucus membrane.
 
But since SARS-CoV-2 mainly enters hosts via the upper respiratory tract, nasal sprays containing CPC could be more effective in preventing or reducing infection.
 
The research involved highly infectious viral stock, whereas virus present in about 1–2 ml saliva likely will have lower infectivity. Mouth rinses are usually done with about 10 ml of mouthwash, so the CPC to virus ratio will likely be more compared to those used in the lab experiments; the authors think it is possible that it may be more effective in the mouth than in the in vitro tests.
 
Corresponding author Dr Nuria Izquierdo-Useros from the IrsiCaixa AIDS Research Institute-Badalona-Spain told Thailand Medical News, “Thus, CPC-containing mouthwashes could be a cost-effective measure to reduce SARS-CoV-2 infectivity in saliva, aiding to reduce viral transmission from infected individuals.”
 
It should be known that virus transmission has been seen when infected people talk, shout, or sing; viable virus has been recovered from the saliva of infected people.
 
The study team said that future studies should investigate if mouthwashes with CPC can reduce viral loads and infectivity in the mouths of infected persons. In addition, more work is needed to understand how long the antiviral activity of CPC lasts in the mouth. All this will help us understand how to use mouthwashes as a cheap measure to reduce SARS-CoV-2 infectivity in the saliva.
 
The team also noted that while prior studies have shown that CPC has an anti-bacterial activity that lasts for 3 to 5 hours in saliva, future studies should address the duration of the CPC antiviral activity in the oral cavity. https://aap.onlinelibrary.wiley.com/doi/abs/10.1902/jop.1996.67.6.572
 
This information the team said will be key to effectively use this cost-effective measure to maintain a reduced infectious capacity of SARS-CoV-2 in the saliva.
 
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