A common compound in mouthwashes found to inhibit SARS-CoV-2 in vitro

Researchers have found that cetylpyridinium chloride, found in most mouthwashes, reduces the infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). in vitro, probably by inhibiting viral fusion with target cells. With more validation tests, mouthwashes could be an easy, inexpensive way to combat transmission.

Studies from the past have shown that mouthwashes can have antiviral properties. They can reduce airborne respiratory coronavirus infections, including influenza virus infections and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the current coronavirus disease pandemic 2019 (COVID-19).

If multiple tests prove the antiviral properties of mouthwashes, they can be a powerful and cost-effective strategy to combat a pandemic. Using mouthwash would be a cheap and easily accessible strategy that can be applied quickly. Reducing viral loads in the mouth can help suppress the transmission chain. Mouthwashes are also produced as oral sprays, so that the elderly population can easily use them.

Although studies have reported in vitro antiviral action of various oral products, it is not known which components in mouthwash are responsible for antiviral action and what is their mechanism of action.

Understanding the effect of mouthwash on the infectivity of SARS-CoV-2

In a new study published in bioRxiv * preprint servers, researchers report the effect of cetylpyridinium chloride (CPC) used in many mouthwashes and sprays. CPC is a quaternary ammonium compound with antiseptic and antimicrobial activity.

The authors tested the ability of CPC-containing mouthwashes to prevent SARS-CoV-2 from entering cells. They used HEK293T cells expressing the angiotensin-converting enzyme 2 receptor (ACE2) as target cells and a pseudotyped lentivirus expressing SARS-CoV-2 spike protein. Luciferase-based analysis allowed them to detect the binding of the virus to target cells.

Pseudovirus was mixed with various concentrations for rinsing the mouth and added to the target cells. The authors tested two mouthwashes from Dentaid: Perio Aid Intensive Care and Vitis CPC Protec. To control cytotoxicity, the authors also mixed mouthwashes with pseudovirus-free target cells. In addition, they used the same mouthwash formulation, but without CPC as a control.

They found that mouthwashes inhibited viral fusion with host cells, with their inhibitory activity increasing with increasing concentration of mouthwash. No inhibition was observed in the use of the CPC-free mouthwash formulation, suggesting that the antiviral activity is due to CPC. In addition, the researchers suspended the CPC in water and found that this suspension also inhibited the entry of the virus into the target cells.

The team also isolated SARS-CoV-2 from a clinical sample collected from an 89-year-old male patient. They added equal amounts of virus and CPC or rinse aid containing CPC and mixed them.

They found that a high dose of CPC was effective in reducing viral infection on Vero E6 cells. A two – minute treatment with CPC mouthwash reduced the infectious dose of tissue culture by 50% (TCID)50) / ml 1000 times. Formulations without CPC had no effect of virus infectivity. Doses of CPC that inhibit viral fusion with the host cell were not toxic to the host cells.

Cetylpyridinium chloride reduces the infectivity of SARS-CoV-2

It is likely that the antiviral activity of CPC stems from its ability to disrupt the viral envelope, similar to that previously seen with influenza viruses. Thus, CPC mouthwashes could protect against oral mucosal infection.

However, since SARS-CoV-2 mainly enters hosts through the upper respiratory tract, CPC-containing nasal sprays may be more effective in preventing or reducing infection.

The experiments performed by the authors used highly infectious viral stocks, while the virus present in about 1-2 ml of saliva is likely to have less contagiousness. Mouthwashes are usually done with about 10 ml of mouthwash, so the ratio of CPC to virus is likely to be higher compared to those used in laboratory experiments, the authors believe it may be more effective in the mouth than in the mouth. in vitro tests.

The antiviral activity of CPC-containing mouthwashes inhibits the entry of SARS-CoV-2.  Percentage inhibition of virus entry to HEK-293T target cells expressing ACE2 exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of oral formulations (A and C), their carriers (B and D) and CPC diluted in water (E).  A nonlinear adjustment to the variable response curve from a single two-replica experiment (red lines) was shown, excluding data on drug concentrations with associated toxicity.  When calculated, a specific IC50 value of the graph is indicated.  A cytotoxic effect on HEK-293T cells expressing ACE2 cells exposed to increasing concentrations of mouthwash or vehicle in the absence of virus (gray line) has also been shown.

The antiviral activity of CPC-containing mouthwashes inhibits the entry of SARS-CoV-2. Percentage inhibition of virus entry to HEK-293T target cells expressing ACE2 exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of oral formulations (A and C), their carriers (B and D) and CPC diluted in water (E). A nonlinear adjustment to the variable response curve from a single two-replica experiment (red lines) is shown, excluding data on drug concentrations with associated toxicity. When calculated, a specific IC50 value of the graph is indicated. A cytotoxic effect on HEK-293T cells expressing ACE2 cells exposed to increasing concentrations of mouthwash or vehicle in the absence of virus (gray line) has also been shown.

“Therefore, CPC-containing mouthwashes could be a cost-effective measure to reduce SARS-CoV-2 infectivity in saliva, helping to reduce viral transmission from infected individuals,” the authors write. Transmission of the virus has been seen when infected people talk, shout or sing; the viable virus is recovered from the saliva of infected people.

The authors write that future work should investigate whether CPC mouthwashes can reduce viral load and contagion in the mouths of infected individuals. In addition, more work is needed to understand how long the antiviral activity of CPC lasts in the mouth. All of this will help us understand how to use mouthwashes as a cheap measure to reduce the infectivity of SARS-CoV-2 in saliva.

* Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered definitive, guide clinical practice / health-related behavior, or treat it as established information.

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