Mouthrinses against SARS-CoV-2: anti-inflammatory effectivity and a clinical pilot study

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the cause of coronavirus infectious disease 2019 (COVID-19). COVID-19 has become a global source of morbidity, mortality and social disruption since its emergence in Asia in late 2019 and subsequent pandemic spread. Despite intense efforts including vaccination campaigns, the virus pandemic continues requiring further preventive measures. Due to the airborne transmission, the use of antiseptic mouth rinses against SARS-CoV-2 seems an obvious possibility to reduce the viral load and thereby decrease the likeliness of viral transmission. Mouthwashes are widely used solutions, to reduce the number of micro-organisms in the oral cavity and colony-forming units in aerosols [7]. Since SARS-CoV-2 enters cell via the ACE-2 receptor and ACE-2 is highly expressed in salivary glands and oral cavity epithelial cells [6, 31], SARS-CoV-2 shows a high reproduction rate in oral tissues. Hence it is suspected that the saliva in the oral cavity may act as a potential reservoir for the transmission of the highly contagious novel coronavirus. In accordance with that, SARS CoV-2 has been detected in high amounts in the saliva of the COVID-19 patients [28]. Further studies identified saliva as a source of viral spread [17], mainly because viruses can be transmitted through aerosol generation from the oral cavity through increased pressure due to coughing, sneezing or speaking (reviewed in [16]. Hence commonly applied over-the-counter mouth rinses could be an easy application to reduce the overall infectivity of COVID-19-positive patients. Numerous investigations were already undertaken to reduce the viral load in COVID-19 patients by gargling with mouth rinsing solution. Several over‐the‐counter mouthwash products showed promising results in in-vitro experiments [18, 19]. Until recently, only povidone iodine was able to show promising results in clinical practice and was investigated in controlled clinical trials utilizing larger study populations [3]. The larger population (n > 1000) made it possible to detect positive changes in the clinical burden. The authors even suggested a decrease in mortality upon application of this strong virucidal agent. Unfortunately, side effects, e.g. disturbance of epithelial barrier [24] or thyroid dysfunction occurred in patients exposed to long-term povidone iodine gargling [8]. Notably, a minor viral clearance of the throat was already achieved by physical means via gargling with tap water [20]. This effect might be enhanced by utilizing surfactant based cleaning products like baby shampoo known to inactivate SARS‐CoV‐2 in vitro [11, 19]. The reduction of viral load via the mouth washing procedures may be affected by ingredients already used for oral health care products.

It is known that many species of viruses, including SARS-CoV-2, have evolved mechanisms to dampen the antiviral effect of inflammation caused by interferon-beta (IFN-β), MX-1 or OAS-1 [5], but simultaneously enhancing the inflammatory signaling (via IL-6 and various CXCLs). This will promote the fatal inflammatory state of the infected tissue [1, 9]. Particularly, GM-CSF was reported to be higher in critically ill patients [29] and high expression of CXCL-9, CXCL‐10 and interleukin‐6 (IL‐6) resulting in a poor prognosis in SARS‐CoV-2 infections [2, 13, 27]. Only the glucocorticoid dexamethasone provides a unique anti-inflammatory effect able to provide control against a cytokine storm and improves clinical outcome [22].

In this combined in vitro and in vivo study, we investigated the clinical action of an over the counter mouth wash and tried to find starting points for an optimized formulation of an experimental mouth rinse formulations based on in vitro experiments. Our clinical data determined the reduction of the viral load of SARS-CoV-2 infected hospitalized patients upon application of an over-the-counter product. Furthermore, the in vitro experiments with anti-inflammatory agents known for insignificant side effects were conducted. One was dexpanthenol, which is known to reduce inflammation and IL-6 release in respiratory epithelium [15] and zinc known to decreases NF-κB activation resulting in decreased expression of proinflammatory target genes [12]. By this, we intended to restore a proper inflammatory response in epithelial cells which is thrown out of balance by a viral infection.

We could verify that all tested substances (except of substance B) showed a fair in-vitro biocompatibility at concentration smaller than 1% (v/v). At higher concentrations (above 5% v/v) an increase in cellular stress or decrease of viability, respectively, was detected. We assume that the preservatives phenoxyethanol and sodium benzoate reduced the in-vitro biocompatibility of the mouth wash formulations. To test the substances for their immunomodulatory properties, we established cell culture model resembling the immune response to viral infections. We could detect a significant anti-inflammatory potential of the applied test substances E and F. Interestingly, these two substances contained zinc and dexpanthenol as bioactive ingredients, which were both already known for their anti-inflammatory potential [12, 15]. To investigate this effect in greater detail, we designed new formulations resembling these promising candidates E and F but omitting the preservatives and introduced a new substance D only containing dexpanthenol. As suspected, these formulations could be applied at very high concentrations [up to 40% (v/v)] in our cell culture model. Hence, they represented the ideal tool to investigate the immunomodulatory properties of the utilized ingredients zinc and dexpanthenol at concentrations the pharyngeal epithelium will be exposed to during gargling. When these substances were applied at a concentration of only 5% (v/v) to our cell culture model, the amount of dexpanthenol and zinc was already sufficient to dampen the expression of the cytokines TNF-α and IL-6 by a factor of at least 2 up to 7. It is known that these two cytokines are expressed upon activation of the canonical NF-κB pathway which results in the nuclear translocation of the NF-κB transcription factor [10]. In contrast to TNF-α and IL-6 the expression of the chemokines CXCL-9 and CXCL-10 is regulated via the activation of AP-1 thru MAPKs upon TLR-3 ligation [21]. Since these two chemokines were influenced by dexpanthenol alone, we suggest that this inflammatory signaling is only positively regulated by dexpanthenol. Most importunately the antiviral inflammatory response, which is mediated via INF-β, OAS-1 and MX-1 was not negatively influenced neither thru zinc nor by dexpanthenol. Subsequent to TLR-3 activation, the expression of these antiviral proteins is induced by the transcription factor IRF-3 [4]. In Summary, we conclude that zinc might interfere with the canonical pathway of NF-κB, while dexpanthenol additionally modulated MAPK driven activation of AP-1. None of the two bioactive components of our experimental mouth wash formulations seem to interact with the TBK1 driven nuclear translocation of IRF3. Even these novel in-vitro formulations could be applicable to formulation in future clinical studies. Apart from the application of zinc and dexpanthenol in mouth washes one might also consider these two components, which possess very little side effects, as an adjuvant treatment to heavily inflamed lung tissue. This could augment the treatment of COVID-19 patients with, e.g. dexamethasone by further reducing IL-6, TNF-α, CXCL-9 and CXCL-10 expression without interfering with the antiviral signaling so desperately needed in this situation.

This pilot study shows that the mouthwash Linola sept reduces the viral load in the pharynx up to approximately 90%. The reduction in viral load detected by RT-qPCR examined in this study is directly coupled to the infectivity of a COVID-19 patient. La Scola and colleagues found that 70% of samples with Ct values of 25 or below could be cultured, compared with less than 3% of the cases with Ct values above 35 [14]. Since we could observe an average enhancement of the Ct around 3.1 in the observed cohort, we suggest that the gargling reduced the infectivity of the patients significantly. Another study by Singanayagam et al. [26] showed the heaviest reduction of cultured viral particles between a Ct value of 25 and 30; thus we conclude that particularly for patients exhibiting initial Ct values around 25 the treatment with the mouth wash solution is most effective in the reduction of transmission of SARS-CoV-2. It can be assumed that the observed reduction is mainly achieved be pure physical means, thru the hydrodynamic forces acting on the oral epithelium during gargling. But besides that, the formulation of several ingredients in the applied mouth washing solution is designed to augment this mode of action. For example, the component sodium dodecyl sulphate (SDS) acts as an detergence and we suggest that SDS might improve the release of viral particles attached to the oral epithelium into the mouth gargling solution. In terms of the durability of this improvement, we found a correlation between initial Ct value and ability to recover the viral load in the pharyngeal region. We suggest that this faster recovery of SARS-CoV-2 is due to high initial viral titer in combination with the high reproduction rate of SARS-CoV-2 [23] and the high expression of ACE-2 in oral tissue [6]. This viral recovery might be further slowed down by adding active ingredients contained in the applied mouth rinsing solution. For example, the component carrageenan is known to inactivate SARS-CoV-2 [25]. An addition to that, SDS, primarily included due to its detergence properties, was demonstrated to inactivate enveloped viruses like the HIV [30]. Anyhow, a pharmaceutical action needs to be verified against a placebo in further clinical studies. The initial formulation of the applied mouth washing solution Linola sept^® not only containing zinc but also hydroxyapatite, also known for its anti-inflammatory potential [32]. In accordance with that the product Biorepair^® (test substance A), which is similar to the applied Linola sept^® mouth rinse, already displayed a positive immunoregulatory modulation (Fig. 1). We suggest to additionally supplement the rinsing applied in our pilot study with dexpanthenol to further increase the positive effect on the immune system. A long-term observational (above 5 days) placebo-controlled study is currently ongoing to detect an improvement of clinical parameters. A comparison with a placebo group is employed to distinguish the influence of purely mechanical–physical cleaning from the effect of the ingredients. A reduction of viral load in the pharyngeal region in combination with an improved inflammatory response is anticipated.

This investigation will direct future clinical studies for an improved reduction of viral load in the pharyngeal region. We could determine possible additive applications supporting antiviral therapies already applied in COVID-19. More importantly, we found that the mouth washing can reduce the viral load by 90%. This should lead to a reduced infectivity of the patient infected with SARS‐CoV‐2 and might improve the protection of health care professionals.