The in vitro evaluation demonstrated promising antiviral action with the absence of cytotoxic effects in a range of concentrations of APD. The APD concentration used in the AM in this clinical study confirmed an antiviral efficacy between 92% and 99%, without evidence of cytotoxicity. The AM group presented a significant reduction in the length of hospitalization and the additional benefit of less severe symptoms. Moreover, according to the safety outcomes, no side effects were reported by the patients from the AM and NAM groups regarding the gargle/rinse mouthwash protocol.
Data regarding the mean time of hospitalization due to COVID-19 vary between geographic areas, but 14 days was found to be a reliable LOS time for patients in hospitals in China [27]. In a recent study [28] was described a median hospital stay period of 12 days in Brazil, which decreased to 6 to 7 days, depending on the concentration of propolis intervention used by the patients. In the present study, a promising result was found when the median LOS further decreased to 4 days in the AM group, which differed significantly from the NAM group, which had an average LOS of 7 days. On the other hand, it is possible to hypothesize that NAM itself was effective in reducing LOS when compared to previous studies [27, 28]. This result is probably due to mechanical hygiene of the oral and oropharynx cavities. There is in fact evidence of the effectiveness of the gargle/rinse protocol since local action is associated the regions that are intimately associated with the development of COVID-19 and play an important role in the defense of the host [7, 10, 29]. In this sense, mechanical hygiene reduces the superficial viral load of the oropharynx and oral mucosa and could prevent upper respiratory tract infections [30]. Other authors [31] reported the prevention of respiratory infections by gargling povidone-iodine, where gargling more than four times daily was considered effective in preventing the adherence of pathogenic bacteria to the upper respiratory tract. In our case, however, we believe this mechanical action was just an adjuvant that potentialized the effect of APD in reducing the SARS-CoV-2 load, which was clinically demonstrated by the great reduction in LOS. Recent studies have shown that a higher viral load is the key factor in the severity of the disease and in worse prognosis [5, 32]. For this reason, decreasing the viral load is crucial to restrain the development of the disease, thus preventing the most serious symptoms. The virus enters the cell through the connection between the virus spike and the ACE2 receptor, which can be abundantly found in the salivary glands. Therefore, some recommendations have suggested the importance of controlling and reducing the viral load in the oral and oropharynx cavity by using an antiviral mouthwash [8, 9, 10, 11, 13, 14, 33].
Several studies have demonstrated the antimicrobial (antiviral) and anti-inflammatory properties of phthalocyanines [18, 19, 20, 21] after photoexcitation. However, in our case, virucidal activity was observed in the dark and with an iron phthalocyanine known to have poor photochemical properties compared to the respective zinc complexes [34], indicating different mechanisms of action.
Considering that some known technologies may exhibit an effective antagonistic action against SARS-CoV-2, a virtual screening based on molecular dynamics simulations and the interaction free energies of 8.770 FDA drugs extracted from the DrugBank database (https://www.drugbank.ca/) was carried out by a team of researchers [21] which recently proposed phthalocyanine, hypericin, TMC-647055 and quarfloxin derivatives as the potentially most effective drugs for the treatment of COVID-19. All four molecules are known to have antiviral properties, but the results indicated that their high affinity for the inner cavity of the spike glycoprotein in the prefusion conformation could block the HR1 region, thus preventing the conformational changes necessary for SARS-CoV-2 entry into target cells. In other words, these molecules are potential spike glycoprotein fusion inhibitors able to prevent docking and thus infection of host cells by coronavirus.
The main interaction responsible for this fusion inhibition effect of phthalocyanine is hydrophobic, but very significant polar interactions, including hydrogen bonds, seem to be responsible for this effect of hypericin, the second most potent molecule. Accordingly, a stronger interaction and affinity of APD with the inner spike glycoprotein cavity in the prefusion conformation is expected since APD has a similar size and carboxylic acid groups at the periphery that are prone to hydrogen bond interactions. Consequently, the antiviral properties of APD could be due to its antagonistic effects on SARS-CoV-2, but APD is also known for its capacity to interact and activate oxygen molecules in the air, inducing a very localized production of activated oxygen molecules. These molecules are able to cause oxidative stress/damage to microorganisms, such as the coronavirus, leading to their inactivation. This hypothesis is confirmed by the effect of noncytotoxic, low concentrations (1.0 mg/mL up to 0.156 mg/mL) in reducing in the active viral load after proliferation in Vero CCL-81 cells as observed by RT-PCR and by the HTCI immunofluorescence-based assay, which showed the rapid cell nucleus localization based on DAPI staining (Fig. 1). Thus, the high effectiveness of APD can probably be explained by a dual mode of action, namely, by blocking the HR1 region and by promoting oxidative damage leading to inactivation of the virus, thus making APD a very promising molecule to reduce the SARS-CoV-2 viral load [13]. Thus, it can be suggested that a mouthwash containing APD can help improve the response of the organism to COVID-19 infection. Once the positive response of the phthalocyanine derivative-containing mouthwash in the present study and in previous studies [14, 15] can be observed, the authors also speculate that other properties of phthalocyanines will be identified. In this sense, the possibilities of the local effect of APD associated with the control of secondary infections (antimicrobial activity), the anti-inflammatory effect, and the modulatory effect on the immune response are not excluded [18, 19, 35, 36].
Although the COVID-19 distribution patterns in hospitals vary from one country to another [5, 6, 27], in the present study, there were no significant changes when comparing the AM and NAM groups in terms of sex, presence of comorbidities, number of comorbidities, or duration of symptoms prior to hospitalization.
The dynamics of the disease and the hospital environment, medications, stresses due to the pandemic, and sample size are some possible adverse interferences and limitations. Thus, the interpretation and generalization of the results should be performed with reservation. Nevertheless, the clinical trial clearly showed significant differences between the AM and NAM groups. In addition, the way the randomized study was carried out makes it unique since it created 2 nonhomogeneous groups. According to the literature [37], elderly patients who were more at risk should have taken longer to recover than younger patients. However, this was not the case, as the elderly patients in the AM group showed similar rates of recovery compared with the patients in different age groups. As a consequence, not only was the LOS decreased in this group of patients (AM) but also no intensive therapy was necessary, indicating that there was no tendency of progression into more severe prognostics. In addition, the probability of discharge (event) among participants in the AM group was twice the probability of discharge among participants in the NAM group at any point in time. Conversely, 28.6% of the patients in the NAM group needed to be admitted to the ICU, and half of this subgroup died. Further investigations are encouraged to confirm these results in larger populations.
The outstanding results achieved in the AM group suggest that mouthwash in addition to other medications can be useful in the strategic planning of COVID-19 treatment by the World Health Organization [6].