A novel anti‐inflammatory treatment for bradykinin‐induced sore throat or pharyngitis

Abstract Background Often thought of as a minor health concern, sore throat or pharyngitis is an important public health issue. It is one of the most common symptoms of upper respiratory diseases including COVID‐19 and is a leading cause of physician visits and antibiotic prescriptions. However, few over‐the‐counter medications are proven to heal sore throat inflammation. Methods Adenocarcinomic human alveolar basal epithelial cells (A549 cells) and three dimensional organotypic human respiratory tissues were used to study inflammation and various treatment effects on respiratory epithelia. The cells and tissues were studied both in the presence and absence of bradykinin, one of the first inflammatory mediators of pharyngitis. Inflammation was measured by analyzing the levels of prostaglandin E2 (PGE2), interleukin 8 (IL‐8), and leukotriene B4 (LTB4), transepithelial electrical resistance (TEER), and lactate dehydrogenase (LDH) release. Tissue morphology was analyzed by immunohistochemistry. Results In studying pharyngitis using organotypic human respiratory tissue stimulated with bradykinin, we saw an increase in PGE2 and interleukin‐8 (IL‐8) in response to bradykinin. Acetyl salicylic acid (ASA), a nonspecific COX inhibitor, was able to mitigate a bradykinin‐induced increase in PGE2 in our studies. However, ASA was inflammatory above its therapeutic window, increasing the levels of PGE2 and IL‐8 above those seen with bradykinin stimulation alone. We describe a novel, scientifically validated treatment for sore throat, that contains a low dose of ASA and other anti‐inflammatory ingredients. Conclusion This study elucidates the complex mechanisms involved in healing pharyngitis, an inflammatory condition of the upper respiratory epithelia. An ASA‐based formula (Biovanta) mitigated bradykinin‐induced inflammation more strongly than ASA alone in organotypic human respiratory tissues. Surprisingly, we found that many of the most common over the counter sore throat therapies exacerbate inflammation and IL‐8 in organotypic human respiratory tissues, suggesting these common treatments may increase the likelihood of further respiratory complications.


| BACKGROUND
Before the coronavirus disease 2019 (COVID-19) pandemic, pharyngitis (inflammation of the pharynx, tonsils, and nasopharynx), or sore throat was among the leading causes of physician visits and antibiotic prescriptions in the United States. 1,2 This is surprising since viruses such as rhinovirus and influenza usually cause about 85% of sore throat cases. 3,4 Antiviral treatments have not been developed for sore throat. Moreoever, no available sore throat products are proven to treat both inflammation and pain. 5 Systemically administered, nonsteroidal antiinflammatory drugs such as ibuprophen and paracetamol can provide some relief but systemic administration would require high doses over an extended period of time. 6 Apart from gross physiological symptoms such as size of lymph nodes and throat color, it is very difficult to measure pharyngial inflammation in a human subject. 7 Lack of reliable upper respiratory animal models may have also hampered drug discovery for this indication. 8 Fortunately, three-dimensional, organotypic tissue explants of human upper respiratory mucosa are now available. [9][10][11] These models more reliably mimic the function of living tissue than either animal models or cell culture monolayers and can greatly accelerate drug discovery. 9,11 Using these models, we were thus able to study the biochemical pathway of pharyngitis in great detail and design a scientifically based treatment.
Bradykinin carried from the nasal passages to the nasopharynx during a respiratory infection can stimulate nociceptive receptors in the oropharynx and throat causing a sore throat. [12][13][14] Bradykinin stimulation also increases IL-8, an inflammatory chemokine, and neutrophil chemoattractant often implicated as a component of "cytokine storms" in a time and concentrationdependent manner in airway smooth muscle cells and respiratory epithelial cells via COX enzymes and prostaglandin E2 (PGE2). 15,16 Clinically, at least three studies used bradykinin to induce sore throat in healthy subjects. 13,14,17 According to our findings, bradykinin does indeed increase inflammation in human organotypic respiratory epithelia via the COX pathway. After an extensive literature search for compounds that mitigate this inflammation, we focused on acetyl salicylic acid (ASA), or aspirin, a well-known, nonspecific COX inhibitor. Aspirin is currently approved as an over-the-counter drug administered systemically via the oral route and as a general analgesic in the following dosages for adults: 325-650 mg every 4 h, 325-500 mg every 3 h, or 650-1000 mg every 8 h.
We found that in A549 cells and in organotypic cultures, when applied apically, much lower dosages of ASA were needed, even after correcting for the in vitro testing conditions. There was a small therapeutic window in which ASA inhibited PGE2 release. Above this window, even in the absence of bradykinin, PGE2 and IL-8 increased to significantly higher levels than baseline controls, creating an inflammatory state where we observed negative cytopathic effect.
When formulated with other anti-inflammatory compounds such as lysozyme, lactoferrin, and aloe, the anti-inflammatory effect of ASA at the therapeutic dose was increased, and greatly surpassed that of other leading sore throat remedies, which paradoxically, were found to be highly inflammatory and deleterious to the delicate respiratory lining. Organotypic cultures are a valuable research tool for this indication since the cytoarchitecture of the tissue mimics that in people, and hence we believe, experimentally effective dosages are likely to translate to clinically effective dosages.

| Cells
A-549 cells (American Tissue Culture Collection CCL-185) were cultured in delbecco's modified eagle medium (DMEM) Medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 U ml−1 penicillin and 100 U ml−1 streptomycin (Gibco). Cells were seeded on 96-well cell culture plates at a density of 6400 cells/well. Before the experiment, when cells reached 90% confluency, media was replaced with serum-free media. Human airway epithelia reconstituted in vitro were purchased from two different providers, EpiAirway TM tissues (AIR-100) from MatTek Corporation and MucilAir TM tissues from Epithelix Sàrl. Specific cell culture media for each tissue were obtained from the respective manufacturer. Basal media was replaced with fresh media every other day until the day of the experiment.

| Reagents for treatment
ASA formula and ASA lozenge (Biovanta TM liquid and lozenge formulas, respectively) are composed of the following ingredients: lactoferrin, lysozyme, acetyl salicylic acid, menthol, aloe and glycerin and can be purchased from leading pharmacies. Other over-the-counter treatments for sore throat and cold were obtained from a local pharmacy. Over-the-counter products in a lozenge presentation were prepared for use by dissolving the product in saliva buffer 18 in a weight/volume (w/v) ratio determined by the weight of the lozenge and the amount of water needed to dissolve that amount of the particular sugar in the lozenge. The weight volume ratios used are listed in Table 1. Products in liquid form were applied neat, except for the ASA formula because, unlike the other products, its indicated dosage is 10 times less than the average amount of saliva expected to be in the mouth. Recommended dosages are also listed in Table 1 and the salivary flow rate was assumed to be 1-2 ml/min. Three days before each study, the apical side of the EpiAirway TM and Mucilair TM tissue inserts was washed with 200 µl of media. Briefly, fresh-warmed media was gently added to the apical side of the insert to not disturb the tissues. The plates were incubated for 15 min at 37°C. After incubation, media from the apical side was gently pipetted up and down three times to remove the excess mucus formed on the tissue. The day of the experiment the inserts were transferred to new plates with fresh warmed media and then 10 µl of each treatment was added to the cells or the apical surface of the human airway tissues, the plates were gently swirled and returned to 37°C for 5 min. Every 24 h, 10 µl of new treatment was applied. NS-398 (Sigma Aldrich) was applied apically as indicated in a volume of 10 µl and at a concentration of 100 µM diluted in saliva buffer. ASA (Sigma Aldrich) was applied apically at the indicated concentrations in a volume of 10 µl at the indicated concentrations.

| Bradykinin challenge
Cell cultures and tissues were challenged with different concentrations of bradykinin purchased from Tocris. Bradykinin was diluted to the appropriate concentration using saliva buffer. 18 After tissues were treated, 10 µl of the bradykinin solution were added to the cells (A549 cells) or the apical surface of the human airway tissues. Plates were gently swirled and returned to 37°C incubation. Samples from cell culture supernatants or basal media were collected at different time points after challenge and stored at −80°C until use. Bradykinin treatment was not repeated after the initial application.

| Measurement of transepithelial electrical resistance (TEER)
TEER was measured in the human airway epithelium inserts to determine the integrity of tight junctions with a Millicell ERS-2 volt-ohmmeter (Millipore Sigma). Briefly, inserts were transferred to new plates with 600 µl of warmed media per well, then 200 µl of media were gently added to the side wall of the inserts apical side. Plates were incubated for 5 min at 37°C and then the TEER measurements were collected. Resistance values (Ω) were converted to TEER (Ω. cm 2 ) by using the following formula: TEER (Ω. cm 2 ) = (resistance value (Ω)−100(Ω)) x 0.33 (cm 2 ), where 100 Ω is the resistance of the membrane and 0.33 cm 2 is the total surface of the epithelium.

| Cytotoxicity
Cytotoxicity was assessed via lactate dehydrogenase (LDH) concentrations measured in 100 µl of basolateral medium incubated with the reaction mixture of a cytotoxicity detection kit (Sigma-Aldrich, Roche) following the manufacturer's instructions. To determine the percentage of cytotoxicity, the following equation was used (A = absorbance values): Cytotoxicity (%) = (A (exp value)-A (low control)/A (high control)−A (low control))×100. The high control value corresponds to a 10% Triton X-100 treatment applied to the culture for 24 h. A threshold limit of 5% of the toxicity index reflects the physiological cell turnover in human airway epithelium cultures.

| Analysis of inflammatory response
To evaluate the inflammatory response, cell culture supernatant and basolateral media from tissues was collected at various time points after challenge. Cyclooxygenase-2-derived prostaglandin E2 (PGE2) and LTB4 release were measured with enzyme-linked immunosorbent assay kits (ELISA; R&D systems) according to the manufacturer's instructions. Interleukin-8 (IL-8 or CXCL8) protein in the basolateral media was measured using a magnetic bead-based ELISA (Procartaplex, ThermoFisher Scientific) according to the instructions provided by the manufacturer and the plate were then read using a Luminex-based Bio-plex Multiplex system (Bio-Rad). Samples were diluted to 1:3 for PGE2 and LTB4 analysis and 1:20 for IL-8 measurements.

| Histopathology
At the end of the experiment, inserts with the human airway epithelia cells were fixed with a formalin solution (neutral buffered, 10%) and kept at 4°C until being sent to Histowiz for histology processing. Inserts were bisected to allow histological staining of paraffinembedded sections. Paraffin-embedded tissues were cut into 5-µm sections, deparaffinized, and the rehydrated sections were stained with hematoxylin-eosin. The images were collected from digitalized slides at a final magnification of ×20.

| Statistical analysis
Data are presented as mean ± standard error of the mean. For statistical comparison of differences between groups, results were analyzed by an unpaired t test, Mann-Whitney, analysis of variance or Kruskal-Wallis tests using the GraphPad Prism software (version 6.01). A p ≤ .05 was considered significant.

| RESULTS
3.1 | Bradykinin, one of the first chemokine signals for pharyngitis causes inflammation in A549 cells that can be blocked by acetyl salicylic acid (ASA) Bradykinin increases PGE2 via the arachidonic acid-COX 2 pathway and ASA is known to be a potent COX inhibitor. 16 First, we sought to determine the effects of bradykinin on adenocarcinomic human alveolar basal epithelial cells (A549 cells). Various concentrations of bradykinin were applied to A549 cells to assess the downstream effects on PGE2 and IL-8 ( Figure 1A,B). LDH was also measured to assess cytotoxicity ( Figure 1C). At least 10 µM bradykinin (supplied by Tocris) was needed to stimulate a statistically significant fivefold increase in PGE2 compared with negative control ( Figure 1A). This dose of bradykinin has been shown by others to elicit significant increases in PGE2 and Il-8 in airway smooth muscle cells 15  24 h, respectively. 16 As a result of bradykinin stimulation, there was also a statistically significant increase in IL-8 expression after 24 h ( Figure 1B). There was no observable cytopathic effect from the increase in PGE2 and Il-8 expression, and no discernable change in LDH release was observed ( Figure 1C). Once we established the stimulatory effects of bradykinin, we asked whether it could be inhibited by Mann−Whitney statistical tests were performed using GraphPad Prism software. *p < .05, **p < .01, ***p < .001, and ****p < .0001. ASA, acetyl salicylic acid; DMEM, delbecco's modified eagle medium; IL-8, interleukin-8; LDH, lactate dehydrogenase; PGE2, prostaglandin E2 acetyl salicylic acid (ASA), a nonspecific COX-inhibitor, or NS-398, a COX-2-specific inhibitor. 19 Before stimulating with 10 µM bradykinin, we treated cells with various concentrations of ASA, or NS-398 ( Figure 1D−I gray bars). For cells treated with ASA: the lowest concentration, 0.6 µg/ml (3 µM) did not affect the bradykinininduced increase in PGE2 secretion, but it did bring IL-8 down to baseline ( Figure 1D,E) The medium concentrations, 6 µg/ml (30 µM) and 60 µg/ml (300 µM) of ASA mitigated the effects of bradykinin on PGE2 and IL-8, bringing both down to baseline (Figures 1D−F). The highest 600 µg/ml (3 mM) dose of ASA was inflammatory, leading to an increase of PGE2 and IL-8 over levels reached with bradykinin stimulation alone ( Figures 1D−F).
To be sure the inflammatory effect of high ASA was not due to an interaction with bradykinin, we also measured the levels of PGE2 and IL-8 in response to various concentrations of ASA and NS-398 in the absence of bradykinin ( Figure 1D-I black bars). The highest level of ASA tested, 3 mM, was indeed inflammatory even in the absence of bradykinin. It brought levels of PGE2 up fourfold compared with baseline, and levels of IL-8 up twofold over baseline ( Figure 1D,E). It also increased the secretion of LDH, a marker of toxicity ( Figure 1I). The lower concentrations of ASA were not inflammatory on their own, and neither were any of the concentrations of NS-398 tested.
To help elucidate the reason for the toxicity of higher levels of ASA, we measured the levels of LTB4 in response to the various concentrations of ASA and NS-398 in the presence and absence of bradykinin ( Figure 1G,H). While bradykinin alone did not stimulate LTB4 release, 3 mM ASA did. Possible explanations for this will be described in the Section 4.

| The bradykinin-induced inflammatory cascade can be replicated in an organotypic model of the human respiratory system
MucilAir™ is composed of basal cells, ciliated cells, and goblet cells that secrete mucus. The cells used to prepare these organotypic cultures are derived from nasal polyp biopsies. The proportion of the various cell types is preserved compared with what one observes in vivo. 20,21 and the cytoarchitecture is virtually identical to that of the pharynx. 21,22 We chose to use the organotypic tissue model because we hypothesized that they would be more predictive of human efficacy than animal models. 9,11,23 These tissue models secrete most of the same cytokines that are secreted in vivo, and since the treatments we are designing are intended to be used topically, the metabolism and targeting of the ingredients should be virtually identical to what would be observed in vivo. 11,24 We determined 47 mM (50 mg/ml) to be the lowest effective dose of bradykinin that would reliably elicit a significant response of PGE2 in Mucilair TM . Next, we investigated whether various concentrations of ASA could inhibit bradykinin-induced inflammation in Mucilair TM . In vivo, ASA would be taken by mouth and then diluted by saliva before reaching the pharynx, where bradykinin is released physiologically. Therefore, after assuming a 1000-fold increase from A549 cells, where we found 300 and 30 µM to be most effective, we assumed a 10-30-fold dilution for salivary flow (for a formula dosed every 10-30 min and a salivary flow rate of 1-3 ml/min), which would ultimately give us a 100-fold increase in concentration from A549 cells. Hence, we tested 3 mM (0.6 mg/ml) and 33 mM (6 mg/ml) of ASA with 47 mM of bradykinin in the MucilAir TM organotypic model. We measured the levels of PGE2, IL-8, and TEER both in the presence and absence of bradykinin (Figure 2A-E); black bars (no bradykinin), and gray bars (with bradykinin).
Bradykinin stimulation led to a statistically significant increase in PGE2 at 4 h, as did stimulation with the higher, 33 mM concentration of ASA both in the presence and absence of bradykinin (Figure 2A), confirming our findings from A549 cells that higher concentrations of ASA can be inflammatory to healthy respiratory tissues. The lower 3 mM concentration of ASA was not inflammatory, but in the presence of bradykinin it did prevent the levels of PGE2 from rising ( Figure 2A). Neither NS-398, nor the ASA formula (Biovanta liquid containing 3 mM ASA) increased PGE2 on their own, however, both did successfully keep PGE2 levels from rising in the presence of bradykinin ( Figure 2A). As for IL-8, at 24 h, the higher, 33 mM concentration of ASA increased IL-8 levels both on its own and in the presence of bradykinin, but the increases did not reach statistical significance ( Figure 2B). At 48 h, stimulation with 33 mM ASA increased IL-8 but it only reached statistical significance in the presence of bradykinin. Neither NS-398, nor the lower 3 mM concentration of ASA increased IL-8 above control levels. TEER measures at 24 and 48 h did not indicate membrane damage with any of the treatments ( Figure 2D,E).
3.3 | A novel, ASA-based formula is more effective than ASA alone at blocking bradykinin-induced PGE2 production in epithelial tissues Next, we sought to investigate the anti-inflammatory effects of an ASA-based formula (Biovanta TM throat spray), LEYVA-GRADO ET AL.
| 1327 a formula containing 30 mM ASA (which is diluted 10fold upon treatment), lysozyme, lactoferrin, aloe, glycerin, and menthol following bradykinin stimulation. Lysozyme and lactoferrin are potent anti-inflammatory molecules present in human nasal secretions. They are also used as preservatives and excipients to affect the rheological properties and viscosity of liquid formulations. Aloe, glycerin, and menthol are common excipients used in cough and cold products, are all natural, and were determined to not be toxic at the concentrations used (data not shown). We hypothesized that they could have beneficial anti-inflammatory effects.
We pretreated the tissues with either buffer, ASA alone, the ASA-based formula (Biovanta throat spray), or NS 398, before treating with bradykinin. Indeed, at 48 h after bradykinin stimulation, tissues treated with Biovanta showed a statistically significant decrease in IL-8 levels compared with those treated with only 3 mM ASA ( Figure 2C). Tissues treated with Biovanta also seemed to show a bigger decrease in PGE2 4 h after stimulation than those treated with 3 mM ASA alone, however, the difference was not statistically significant (Figure 2A).

| An ASA-based formula (Biovanta throat spray) containing antiinflammatory excipients decreases PGE2
in Mucilair TM without the cytotoxicity, or membrane damage caused by leading sore throat products After establishing the superiority of the ASA-based formula against ASA, we compared its efficacy in decreasing bradykinin-induced inflammation with leading sore throat products. Bradykinin at a concentration of 47 mM was used to induce inflammation after pretreating the tissues for 5 min with either buffer, 100 µM NS-398, an ASA-based formula (Biovanta TM throat spray), or various over the counter sore throat products containing the following active ingredients: phenol and glycerin (Chloraseptic max sore throat spray), dextromethorphan and guifenesin (Robitussin DM max spray), english ivy leaf extract (Zarbees lozenge), or menthol (Halls relief honey lemon lozenge). In tissues pretreated with buffer, bradykinin induced a 1.54-fold increase in PGE2 after 4 h and a 1.75-fold increase in IL-8 after 24 h. IL-8 levels remained at this level until the end of the experiment at 48 h ( Figure 3A-C). All three increases were statistically significant (p = .02, p = .04, and p = .01, respectively). Pretreatment with either NS-398 or the ASA formula kept PGE2 at control levels ( Figure 3A). IL-8 levels in tissues pretreated with either NS-398 or the ASA formula did not increase, they were similar to tissues pretreated with buffer ( Figure 3B,C).
Of the leading sore throat products tested in this experiment, two were liquid formulations (either sprays or syrups) and two were lozenges. None of the products prevented an increase in either PGE2 or IL-8 ( Figure 3A−C). In fact, paradoxically, phenol and glycerin (Choloroseptic® Max Sore Throat Spray) and dextromethorphan and guifenesin (Robitussin®DM Max spray) pretreatment increased PGE2 levels compared with buffer pretreatment ( Figure 3A). In addition, all of the products caused significant cytotoxicity as measured by increased IL-8 secretion, increased LDH secretion, and lowered TEER ( Figure 3B−G). All four products tested showed statistically significant increases in IL-8 compared with ASA formula (Biovanta throat spray) (p = .01 after 24 h and p = .01 after 48 h), statistically significant increases in LDH cytotoxicity (p = .0094 after 24 h), and statistically significant decreases in TEER (p < .0001 after 24 h and p = .0001 after 48 h).
3.5 | The inflammatory effects of leading over the counter sore throat products were confirmed in the EpiAirway TM model in the absence of bradykinin, while neither an ASA-based formula nor a lozenge was inflammatory The following products were applied to the apical surface of EpiAirway TM to assess inflammatory effects: an 8.3 mg menthol containing lozenge (Ricola® dual-action sore throat and cough honey lemon), a pectin containing lozenge (Luden's® wild cherry flavor dual action), a 13.3 mg zinc containing lozenge (Cold-EEZE natural cherry flavor lozenge), a honey containing lozenge (Wedderspoon organic manuka honey drops), a 10 mg zinc containing lozenge (Zicam® cherry lozenge), a dichlorobenzyl alcohol, amylmetacresol, and levomenthol containing lozenge (Strepsils® sore throat and cough lozenges), a 20 mg menthol containing lozenge (Vicks® Vapacool severe drops), an ASA-based formula (Biovanta throat spray), and an ASA-based lozenge (Biovanta lozenge). These products were applied to tissues after being dissolved in saliva buffer. The lozenges were weighed and dissolved in a predetermined weight/volume (w/v) ratio of saliva buffer. Since hard lozenges are amorphous solids and about 98%-99% sugar 25 the w/v ratio needed to dissolve each lozenge was calculated as the minimum volume needed to dissolve the predominant sugar in the lozenge based on its solubility and density. The dilution volume for each lozenge tested is listed in Table 1.
The ASA-based formula (Biovanta TM throat spray) is an over-the-counter drug and is intended to be sprayed to the back of the throat in a total volume of 200 µl. We considered the average salivary flow rate to be 2 ml/min and that the liquid formula would be diluted 1:10 by saliva in the mouth. Therefore, we diluted the liquid formula 1:10 before applying it to the tissues. The other liquid spray formulas and syrups tested had dosage volumes far exceeding the amount of saliva likely to be present in the mouth at any given time, so they were applied neat. After applying 10 µl of the dissolved lozenge or ASA-based formula (Biovanta throat spray) to the tissues, the tissues were placed at 37°C for 5 min and then 10 µl of saliva buffer was applied to the tissues.
Treatments were reapplied every 24 h. The tissues were not stimulated with bradykinin. As illustrated in Figure 4, except for the ASA-based formula and lozenge (Biovanta throat spray and Biovanta lozenge), all the products tested caused increases in inflammatory cytokines (PGE2, IL-8, or both), and LDH and also disrupted membrane integrity as illustrated by significantly lower TEER measurements ( Figure 4A−D). Note that although some products appear to decrease IL-8 levels at 24 h, the same products show significant decreases in TEER and increases in LDH, suggesting that low IL-8 levels may be due to cell death ( Figure 4B-D). All nine products tested showed statistically significant changes in PGE2 levels compared with ASA formula (Biovanta throat spray) and ASA lozenge (Biovanta lozenge (p = .0013), statistically significant changes in IL-8 levels (p = .0010), statistically significant changes in LDH levels (p = .0033), and statistically significant decreases in TEER (p = .0015).
Neither the ASA-based formula (Biovanta throat spray), nor the ASA-based lozenge (Biovanta lozenge) were inflammatory. In fact, the levels of PGE2, IL-8, LDH, and TEER in response to Biovanta were comparable to the vehicle condition ( Figure 4A-D).
3.6 | According to a third-party blind placebo-controlled study, leading sore throat and cold remedies physically damage respiratory epithelia, but the ASA lozenge (biovanta lozenge) does not EpiAirway (AIR-200-PE6.5) are another type of human respiratory organoid and are obtained from tracheobronchial cells. We found that in these tissues 15 mM bradykinin was sufficient to significantly increase PGE2 levels (data not shown). Unlabeled Eppendorf tubes containing the various sore throat syrups and lozenges were sent to Mattek Corp for third party blind analysis. EpiAirway TM (AIR-200-PE6.5) were treated in triplicate with each of the syrups (undissolved) and each of the lozenges dissolved in buffer. The products and their ingredients are described in more detail in Table 1. Following a 5 min incubation, a 15 mM solution of bradykinin was added to the inserts. After incubating at 37°C for 48 h, TEER measurements were taken. Of the products tested, only the isomalt-based, ASA lozenge (Biovanta lozenge) did not induce measurable damage to epithelial tissues ( Figure 5).
The lozenges were dissolved in buffer at a concentration determined by their weight and the type(s) of sugars they contain, as described in more detail in Table 1. For example, Halls® Relief Honey Lemon contains mainly glucose, according to its label. Glucose has a solubility of 91 g/100 ml of solution and a density of 1.02 g/ml. Each lozenge weighs 3.15 g. It would therefore take about 1:0.75 w/v of lozenge to buffer (or saliva) to dissolve each lozenge. See Table 1 for a list of the various products and the w/v ratio of buffer they were dissolved in.
3.7 | Histological analysis confirms extensive tissue damage by leading products, as measured by TEER Following the 48-h experiment depicted in Figure 5, the organotypic respiratory tissues were fixed in 10% paraformaldehyde, embedded in parrafin, and sectioned into 5 µm thick sections taken from the center of each section after it was cut in half. The sections were then treated for hemotoxylin and eosin (H&E) staining to observe any histological changes. The average thickness of the untreated sections was 55-60 µm from the top of the cilia to the bottom of the basal cell layer. The groups treated with the most common over-the-counter sore throat products exhibited marked loss of cilia and pseudostratified columnar epithelial cells ( Figure 6). In most cases, the columnar epithelial cells showed atrophy which decreased the thickness of the epithelia to about half of control values.

| DISCUSSION
Despite the plethora of over-the-counter (OTC) products and supplements available to the US consumer for sore throat, none have proven clinically effective for sore The key lists all the active ingredients and the trade name of the product. Following their respective treatments, all tissue inserts (except the untreated) were treated with 15 mM bradykinin. TEER was measured 48 h after treatment. Statistics shown are results of a Kruskal-Wallis test using GraphPad Prism software, ****p < .0001. Statistics were performed on raw data without omissions and not on fold-change values. TEER, transepithelial electrical resistance throat pain and inflammation. 5 A Cochrane metaanalysis found that nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin when taken orally are "somewhat effective" in relieving the discomfort caused by a cold. 26 Considering the mechanism by which bradykinin induces inflammation through the COX-1/COX-2 pathway, it is surprising that systemically administered NSAIDs have not proven to be more effective. Perhaps, they need to be applied locally or at different dosages. Indeed, we found that there is a small therapeutic window at which aspirin decreases inflammation and above which it becomes inflammatory.
At the therapeutic dose, we found that ASA and an ASA formula (Biovanta throat spray) blocked the bradykinin-induced increase in PGE2. We expected to also see a decrease in IL-8 but this was only observed in A549 cells, and not in Mucilair. IL-8 levels did not reach statistical significance above baseline in Mucilair, and there was therefore not much room for significant decreases. Mucilair, an organotypic model, contains several cell types and numerous cytokines that might modify the increases in IL-8 expression seen in A549 cells.
At a dose 10-fold higher than the effective dose, aspirin caused a statistically significant fourfold increase in PGE2 above baseline in A549 cells and in Mucilair, as well as a statistically significant twofold increase in IL-8 above baseline in A549 cells, and a fivefold increase above baseline in Mucilair which did not reach statistical significance (Figures 1 and 2). A dose-dependent effect of aspirin has been detected in several different physiological systems. 27 Our results are consistent with a clinical study showing that a moderate dose (500 mg/day) of systemically administered aspirin, which is much lower than the recommended dose for adults, effectively treated ACE-inhibitor induced cough, which is thought to be mediated by bradykinin. 28 To shed some light on what might be driving the increase in PGE2 and IL-8 expression with high doses of ASA, we measured the levels of LTB4 in response to high-dose ASA in the presence and absence of bradykinin. High dose, 3 mM ASA caused a statistically significant, fivefold increase in LTB4 above baseline both in the presence and absence of bradykinin in A459 cells ( Figure 1G,H).
Apart from the COX pathway, arachidonic acid is also responsible for the generation of leukotrienes via lipoxygenase. 29 Inhibiting the COX pathway with COX inhibitors may shunt arachidonic acid to the highly inflammatory lipoxygenase pathway, 20,30,31 a pathway, which coincidentally, may be responsible for bronchoconstriction in aspirin-induced asthma. 30,32 We observed an increase in one of the major lipoxygenase products, LTB4, as well as IL-8 and PGE2 in A549 cells stimulated with high-concentration ASA ( Figure 1G,H). Prior studies confirm that LTB4 stimulates IL-8. 15,33 LTB4 also activates several kinase cascades leading to production of several cytokines, many of which might increase PGE2. 34 F I G U R E 6 Histological analysis of the tissues analyzed in the third-party blind study, a representative sample of the tissues used in Figure 5 are shown. In all of the tissues, except those treated with saliva buffer (vehicle), bradykinin, or the ASA lozenge (Biovanta lozenge), marked histological damage was seen in response to treatment. After fixation and embedding in parrafin, sections were cut into 5 um thick section and stained with hemotoxylin and eosin. ASA, acetyl salicylic acid For example, TNF-α and IL-1β were shown to increase PGE2 expression in A549 cells. 35 Interestingly, the highest dose of NS-398 tested did not show any inflammatory effect (Figure 1). This may be due to its selectivity for COX-2, therefore leaving some COX-1 available to produce prostaglandins. Alternatively, since A549 cells greatly overexpress COX-2, it may not be possible to knock down levels completely. Other groups have shown that A549 cells express both COX-1 and COX-2 mRNA and that overexpression of COX-2 might lead to the inflammatory cancer phenotype 36,37 According to several reports, bradykinin contributes to inflammation following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. 38,39 Bradykinin levels might be elevated in SARS-CoV-2 patients due to the dysregulation of the angiotensin-converting enzyme levels. 39 While it remains to be seen whether blocking bradykinin activity will be useful during SARS-CoV-2 infection, aspirin treatment was reported to be beneficial in SARS-CoV-2 patients, 40 warranting a closer examination of the possible effects that other aspirinbased treatments could have on the prevention and treatment of SARS-CoV-2 infection.
Aspirin is already approved as an OTC for sore throat (internal analgesic), yet there are no throat sprays containing aspirin in the market. We have developed a formula containing aspirin and several other food-grade nutritional supplements which act synergistically to help the aspirin act locally and reduce the inflammation and possibly the pain caused by bradykinin. Additionally, we found that when measured against other leading products containing various active ingredients, this formula not only performed better but was much less inflammatory. For one, none of the other active ingredients tested are known to act on bradykinin or its inflammatory pathway. Also, the inactive ingredients in the ASA formula and lozenge (Biovanta) were carefully selected to compliment the anti-inflammatory effects of the active ingredients.
Incidentally, we believe the inflammatory effects of the leading products may have been caused not by the active ingredients but by the inactive ingredients. For example, in Figure 4, four products containing menthol are analyzed, yet the one with the lowest concentration of menthol (Ricola original) shows the greatest increase in PGE2 release and LDH release in the absence of bradykinin stimulation. The products contain numerous inactive ingredients that are difficult to study individually. Inactive ingredients are not closely regulated, and precise concentrations are not usually reported.
The inactive ingredients may have specific or nonspecific effects. According to previous reports, hyperosmotic sugar solutions can change respiratory epithelial cell shape and open tight junctions. 41 These are the types of morphological effects we show in Figure 6. Also, higher osmolarities can result in the secretion of proinflammatory cytokines (Interleukin-8, Interleukin-6, Interleukin-1β, and tumor necrosis factor-α), 42 and we did observe increases in IL-8 in response to certain products. We observed significant decreases in TEER, indicating membrane perforation for most of the products tested, which can be caused by osmotic stress. The ASA lozenge (Biovanta lozenge) was formulated with isomalt as the main sugar base, and isomalt has a relatively low osmotic pressure compared with the sugars in the other lozenges tested. To our knowledge, the osmotic pressure of these products on respiratory epithelia has not been measured before.
Taken together, we believe the integrity and inflammatory state of the respiratory membrane epithelium is an important factor to consider in the prevention and treatment of respiratory disease. There are several inflammatory pathways known to be involved and clearly more to be discovered. From our observations, there seem to be several pathways involved in the control of IL-8 expression, including PGE2 and perhaps osmotic stress. We were surprised to learn that many common over the counter products in the market for cold symptoms such as sore throat, greatly exacerbate IL-8 levels.
Ongoing studies will determine whether this is due to osmotic stress or some other inflammatory signal. In either case, it is highly concerning given the key role IL-8 plays in exaggerating the immune response, thus increasing the likelihood of a "cytokine storm."