Compositions and methods for improving nitric oxide levels in the oral, nasal, and / or nasopharyngeal regions

Abstract

The present disclosure relates to improving oral-nasal health by increasing nitric oxide levels in the oral cavity, buccal cavity, and nasal cavity using novel prebiotic nitrate-containing chewing gum compositions, with optional steps including the use of saliva test strips to monitor pH, nitrate, and nitrite. The novel gum compositions and methods of the present invention result in increased bioavailability of nitric oxide, thereby increasing antibacterial nitric oxide levels beneficial for reducing dental caries, periodontitis, and nasopharyngitis, as well as reducing the transmission of aerosolized viral particles acutely sensitive to nitric oxide, including SARS-CoV.

Description

[Technical Field]

[0001] Embodiments of the present disclosure generally relate to novel nitrate-based gum compositions useful for extending a subject's oral cavity exposure to prebiotic nitrates, resulting in a rapid and localized increase in nitric oxide gas in the oral cavity and nasopharynx, which also contributes to improved systemic nitric oxide bioavailability. [Background technology]

[0002] Like many areas of the body, the mouth harbors a large population of bacteria, most of which are harmless. Normally, the body's natural defenses and good oral care, such as daily brushing and flossing, can keep these bacteria under control. However, poor oral hygiene can allow bacteria to reach levels that can lead to oral infections, such as tooth decay and gum disease. Additionally, lifestyle activities—especially smoking; taking medications such as decongestants, antihistamines, painkillers, antibiotics, and diuretics; or current oral hygiene practices, such as mouthwash—can reduce saliva composition and flow and alter the oral microbiome, negatively impacting oral health. Saliva is important because it washes away food and neutralizes acids produced by oral bacteria, helping to protect the mouth from microbial invasion or overgrowth that could lead to disease.

[0003] Oral health is essential to overall health and quality of life. It is the absence of mouth and facial pain, oral and pharyngeal cancer, oral infections and stomatitis, periodontal (gum) disease, tooth decay, missing teeth, and other diseases and disorders that limit a person's ability to chew, masticate, laugh, speak, and mental well-being. The most common oral diseases are dental cavities, periodontal (gum) disease, oral cancer, oral infections, traumatic injuries, and hereditary lesions.

[0004] Dental cavities are widely recognized as a serious concern regarding oral health. Worldwide, 60–90% of school children and nearly 100% of adults have cavities, which often lead to pain and discomfort. Tooth decay (cavities) is one of the most common chronic childhood diseases in the United States. Untreated cavities can cause pain and infection, interfering with eating, speaking, playing, and learning. In the United States, approximately one in five children aged 5–11 (20%) has at least one untreated cavity, and approximately one in seven adolescents aged 12–19 (13%) has at least one untreated cavity. The proportion of children and adolescents aged 5–19 with untreated cavities is twice as high among children from low-income households (25%) compared with children from high-income households (11%). It is a lifelong threat to oral health. Nearly one-third of adults in the United States have untreated cavities.

[0005] Periodontitis is a group of inflammatory diseases affecting the periodontium, the tissues that surround and support the teeth. Periodontitis involves the progressive loss of alveolar bone around the teeth, which, if left untreated, can lead to loosening and subsequent tooth loss. It is caused by microorganisms that attach to and grow on the tooth surface and an overly aggressive immune response to these microorganisms. Periodontitis manifests as painful, red, swollen gums and heavy plaque buildup. Symptoms may include red or bleeding gums while brushing, flossing, or biting hard foods (e.g., apples); recurring swelling of the gums; halitosis and a persistent metallic taste in the mouth; visibly longer teeth as a result of gum recession; deepening pockets between the teeth and gums (pockets are areas where collagenase gradually destroys the attachment); and loose teeth. Periodontitis has also been shown to affect areas outside the mouth. For example, periodontitis is associated with increased inflammation, as indicated by elevated levels of C-reactive protein and interleukin-6. Periodontitis has also been shown to increase the risk of many other diseases, including, but not limited to, stroke, myocardial infarction, atherosclerosis, diabetes, and premature birth. Severe periodontal disease (gum disease), which can result in tooth loss, is prevalent in 15-20% of middle-aged adults (35-44 years of age). Dental cavity and periodontal disease are also the leading causes of tooth loss. Complete loss of natural teeth is widespread, particularly affecting the elderly. Worldwide, approximately 30% of people aged 65-74 years have no natural teeth.

[0006] Oral cancer is another concern in oral health. The incidence of oral cancer ranges from 1 to 10 cases per 100,000 people in most countries. The prevalence of oral cancer is relatively higher in men, older people, and those with lower education and lower incomes. Tobacco and alcohol are major causative factors and contribute to the destruction of the microbiome, especially the nitrate-reducing bacteria on the tongue. Therefore, smoking reduces the health-promoting microbiome on the tongue.

[0007] Oral health can affect, be affected by, or contribute to a variety of diseases and conditions, including: endocarditis, an infection of the inner lining of the heart (endocardium), which typically occurs when bacteria or other pathogens from another part of the body, such as the mouth, spread through the bloodstream and land on damaged areas of the heart; cardiovascular disease, research suggests that heart disease, clogged arteries, and stroke may be linked to inflammation and infection that can be caused by bacteria in the mouth; pregnancy and childbirth, where periodontitis is associated with premature birth and low birth weight; diabetes, where diabetes lowers the body's ability to resist infection, putting gums at risk and increasing the risk to people with diabetes Gum disease manifests more frequently and severely in adults (studies have shown that people with gum disease have more difficulty controlling blood sugar levels); HIV / AIDS: Oral problems such as painful mucosal lesions are common in HIV / AIDS patients. In addition, nearly half (40–50%) of HIV-positive individuals suffer from oral fungal, bacterial, or viral infections; osteoporosis, a condition that weakens and brittles bones and may be associated with periodontal bone loss and tooth loss; Alzheimer's disease: tooth loss before age 35 may be a risk factor for Alzheimer's disease; and other conditions that may be associated with oral health, including Sjögren's syndrome, an immune system disorder that causes dry mouth and eating disorders. Asymptomatic transmission of SARS-CoV-2 remains a concern due to its peripheral anatomical location and frequent exposure of oral tissues to the external environment. That said, saliva plays a major role in the asymptomatic spread of SARS-CoV-2 and, therefore, is actively involved in SARS-CoV-2 transmission.

[0008] The burden of oral disease and other chronic diseases can be simultaneously reduced by addressing common risk factors. These include reducing sugar intake and maintaining a balanced nutritional intake to prevent cavities and premature tooth loss; consuming fruits and vegetables, which can prevent oral cancer; cessation of tobacco use and reduced alcohol intake to reduce the risk of oral cancer, periodontal disease, and tooth loss; use of protective sports and automotive equipment to reduce the risk of facial trauma; a safe physical environment; and, most importantly, ensuring proper oral hygiene. While maintaining consistently low levels of fluoride in the mouth can prevent cavities, fluoride and chlorhexidine mouthwashes may also reduce a healthy oral microbiome that contributes to antibacterial nitric oxide production.

[0009] Like the oral cavity, the nasopharynx is a primary site of colonization by respiratory pathogens and constitutes the gateway to the respiratory tract. The nasopharynx often harbors bacterial and viral pathogens that cause both middle ear and sinus infections. The most common condition affecting the nasopharynx is nasopharyngitis, also known as the common cold. This swelling of the nasal passages and throat is sometimes referred to as an upper respiratory tract infection, or rhinitis. In nasopharyngitis, a virus, often a rhinovirus, infects the nasopharynx. The nose is the primary entry site and target of SARS-CoV-2. Asymptomatic transmission of SARS-CoV-2 remains a concern due to its peripheral anatomical location and frequent exposure of both oral and nasopharyngeal tissues to the external environment. Therefore, the oropharynx plays a key role in the asymptomatic spread of SARS-CoV-2, and thus aerosolized water-mucus droplets emanating from saliva and the nose contribute to SARS-CoV-2 transmission.

[0010] Salivary nitrates and oral health Nitrite, produced from nitrate by nitrate-reducing bacteria on the tongue surface, is thought to be cytocidal and cytostatic against common oral pathogens involved in dental caries and periodontal disease, especially when acidified. Therefore, increased nitrate secretion and subsequent increased salivary nitrite may contribute to an overall protective effect against these infections, affecting both hard and soft tissues of the oral cavity. It is known that salivary glands can respond to periodontitis by enhancing the protective capacity of saliva. Therefore, elevated salivary nitrate-nitrite concentrations in periodontal disease patients are likely due to increased nitrate secretion as a salivary gland response to the inflammatory process. In accordance with this hypothesis, patients with oral candidiasis have been reported to have elevated salivary nitrate and nitrite concentrations.

[0011] A study of 209 children found that high salivary nitrate concentrations and oral nitrate-reducing capacity have a protective effect against dental caries. Salivary nitrate and nitrite levels, Streptococcus mutans and Lactobacillus spp. counts, and caries history were recorded. Compared with control subjects, patients with high salivary nitrate concentrations had significantly reduced caries and Streptococcus mutans and Lactobacillus counts. The production of nitrite from salivary nitrate by commensal nitrate-reducing bacteria may limit the growth of cariogenic bacteria by producing antibacterial oxides of nitrogen, including nitric oxide. (JJ Doel, MP Hector, CV Amirtham, LA Al-Anzan, N. Benjamin, RP Allaker, Protective effect of salivary nitrate and microbial reductase activity against caries, Eur. J. Oral Sci.112(2004)424-428.)

[0012] Other studies have shown that the antibacterial agent nitric oxide is formed in the mouth, and its concentration is directly related to salivary nitrite, which in turn is partially related to dietary nitrate intake. Here, researchers showed that nitrite, via bioconversion from nitrate under acidic conditions, had an inhibitory effect on Streptococcus mutans, Lactobacillus casei, and Actinomyces naeslundii via NO production. While the growth of S. mutans was inhibited at more acidic pH, the addition of nitrite significantly reduced bacterial numbers in a dose-dependent manner after 24 hours of exposure. Similar effects were observed for A. naeslundii and L. casei. The ability of these bacteria to recover from exposure to nitrite was also significantly affected by nitrite concentration. At acidity levels below pH 7, low concentrations of nitrite (0.2 mM) effectively and completely killed Streptococcus mutans, and similar effects were observed for other organisms tested. These results demonstrate that salivary nitrite blocks cariogenic bacteria. (LS Silva Mendez, RP Allaker, JM Hardie, N. Benjamin, Antimicrobial effect of acidified nitrite on cariogenic bacteria, Oral Microbiol. Immunol. 14(1999)391-392; CE Radcliffe, R. Lamb, AS Blinkhorn, DB Drucker, Effect of sodium nitrite and ascorbic acid on the growth and acid production of Streptococcus mutans, J. Dent. 31(2003)367-370.)

[0013] Consistent with other reports, nitrite generated from nitrate under acidic conditions inhibited the growth of the periodontal pathogens Fusobacterium nucleatum, Eikenella corrodens, and Porphyromonas gingivalis. (P. Allaker, LS Silva Mendez, JM Hardie, N. Benjamin, Antimicrobial effect of acidified nitrite on periodontal bacteria, Oral Microbiol. Immunol. 16 (2001) 253-256.) Sanchez et al. (2014) reported that increased nitrate likely contributes to an overall protective effect against periodontal disease-associated pathogens that affect both hard and soft tissues of the oral cavity. Sanchez et al. (2014) suggested that elevated salivary nitrate-nitrite concentrations in patients with periodontal disease are due to increased nitrate secretion as a salivary gland response to inflammatory processes. In accordance with this hypothesis, patients with oral candidiasis have been reported to have elevated salivary nitrate and nitrite concentrations. In both cases, elevated oral nitrate concentrations during disease progression are suggested to be an immune response to such infections. (Total salivary nitrates and nitrites in oral health and periodontal disease. Sanchez GA, Miozza VA, Delgado A, Busch L. Nitric Oxide. 2014 Jan 30;36:31-5). Numerous additional studies have emphasized the benefits of salivary nitrate levels: Li et al. (2007) suggested that elevated salivary nitrate concentrations reduce oral acidity and thus prevent dental caries.(Oral Microbiol Immunol. 2007 Feb;22(l):67-71. Salivary nitrate—an ecological factor in reducing oral acidity. Li Hl, Thompson I, Carter P, Whiteley A, Bailey M, Leifert C, Killham K.) Similarly, Radcliffe (2002) suggested that exogenous nitrite, acidified by metabolic products of acid-producing bacteria in the mouth, is converted into products that inhibit the growth of Streptococcus mutans. (Effects of nitrite and nitrate on the growth and acidogenicity of Streptococcus mutans. Radcliffe CE, Akram NC, Hurrell F, Drucker DB. J Dent. 2002 Sep-Nov;30(7-8):325-31.) In addition to the sensitive acidified nitrite generated from nitrate, the sulfate-reducing bacteria Desulfovibrio spp., which are associated with chronic periodontitis and are thought to be the virulence factor responsible for the sulfuric odor, including the cariogenic bacterium Streptococcus mutans and various periodontal pathogens Fusobacterium and Porphyromonas gingivalis, are directly inhibited by both nitrite and nitrate. Here, growth was inhibited at 0.2 mM nitrate, consistent with other observations. Nitrates and nitrites in saliva may also have antibacterial effects against Desulfovibrio species (Mitsui T, Fujihara M, Harasawa R. Biosci Biotechnol Biochem. 2013; 77(12):2489).

[0014] Nitrate levels are increased in the body by including nitrate-rich plants such as leafy green vegetables and beetroot in the diet, and certain oral bacteria convert nitrates to nitrites. The body can then effectively convert nitrites to nitric oxide through specific enzymatic and non-enzymatic processes, resulting in beneficial outcomes such as lowered blood pressure and reduced spread of disease-causing pathogens.

[0015] The dietary pathway, or L-arginine-independent nitrate-nitrite-nitric oxide dietary pathway, also called the enterosalivary nitrate pathway, is where dietary nitrate is swallowed, absorbed in the proximal gastrointestinal tract, absorbed via the circulatory system, concentrated in the salivary glands, secreted into the mouth, and subsequently reduced to nitrite by nitrate-reducing bacteria 30-90 minutes later, which is subsequently swallowed and further reduced to nitric oxide and related intermediates in the stomach, bloodstream, and tissues.

[0016] Oral nitrate-reducing microbiome in the oro-nasopharyngeal and enterosalivary nitrate pathways for oral and upper airway health As mentioned above, certain oral bacteria, especially nitrate-reducing bacteria, convert nitrate to nitrite, and the human body can effectively convert nitrite to nitric oxide through several enzymatic and non-enzymatic processes, which depend on the enterosalveolar nitrate pathway.

[0017] Various research groups have focused on the systemic benefits of dietary nitrate with the aim of shifting the oral microflora, typically from ingested food sources and therefore relying on the enterosalivary nitrate pathway, as opposed to increasing the "residence time" of nitrate in the mouth and assessing oral nitric oxide gas itself. Currently, there are no teachings or available resources that allow the direct administration of nitrate-based formulas to increase nitrate- and nitrite-reducing activity and generate nitric oxide in the mouth and nasopharynx that is independent of the enterosalivary nitrate pathway, resulting in immediate increases in oral and nasal nitric oxide at doses well below the acceptable daily intake (ADI), and extending the duration of local and systemic nitrogen bioavailability over several hours.

[0018] Current research focuses on the oral cavity's ability to reduce nitrate, but this is at a very high level, exceeding the ADI, which involves the enterosalivary nitrate pathway. Burgleigh et al. (2019) observed an increase in salivary pH and Neissera, and a decrease in Prevotella, Actinomyces, and Streptococcus, after 7 days of beetroot consumption. This study required multiple high-dose nitrate ingestion at levels far above the ADI.

[0019] Similarly, other researchers have demonstrated the beneficial effects of dietary nitrate, which shifts the oral microbiome through the ingestion of nitrate sources. This includes Velmurugan et al. (2016), who demonstrated an increase in Rothia mucilaginosa and Neisseria flavescens after 6 weeks of daily consumption of nitrate-rich beetroot juice, containing 372 mg of nitrate-reducing species per serving. Vanhatalo et al. (2018) reported changes in the oral microbiota detected in saliva after nitrate supplementation, showing an increase in the nitrate-reducing bacteria Rothia and Neisseria, and a decrease in disease-associated Prevotella and Veillonella. Jockel-Schneider et al. (2016) reported a reduction in gingivitis in patients after 14 days of nitrate supplementation.

[0020] Mira and Rosier et al. (2018, 2022) suggest the use of nitrates to reduce or prevent oral dysbiosis and improve oral eubiosis 24 hours before biofilm-mediated oral disease by altering the bacterial composition of oral biofilms, thereby increasing the concentration of nitrates in oral saliva. Furthermore, they claim either topical or ingested nitrates, and in the latter case, rely on the gut-salivary loop. In both cases, they suggest increasing the concentration of nitrates in oral saliva with compositions containing either nitrate-rich plant extracts (e.g., beetroot extract), antioxidants, and / or molybdenum, its salts, or molybdenum-rich plant extracts as nitrate reductase cofactors. Ingested compounds, like many others, may actually increase salivary nitrate levels after traveling through a circuitous loop from the mouth to the stomach via the circulatory system and back to the salivary glands and mouth. That being said, Rosier and Mira (2022) claim to achieve a nitrate-reducing microbiome shift by topical administration via toothpaste, mouthwash, oral gel, food extract, and chewing gum using 3 μg of 0.1 mM nitrate, a hypothetical construct that has not been demonstrated to produce an immediate or sustained increase in nitric oxide production in the oral cavity or nasopharynx in human subjects.

[0021] In a study by Rosier et al. (2022) entitled "Prebiotic and probiotic treatment to reduce oral dysbiosis and promote eubiosis," published in International Publication No. 2021 / 122741, they reported that immediately after ingestion of a nitrate-rich supplement (220 mg nitrate in 200 ml water), saliva collected in the morning from fasting healthy donors showed two peaks of nitrate levels, illustrating a direct or immediate increase in nitrate due to topical supplement contact (0.5 h) and an indirect increase due to salivary gland activity recycling nitrate from plasma (2.5 h). However, they failed to detect concentrations of nitric oxide in the oral cavity or nasopharynx below the ADI level to achieve this transient biphasic response. Furthermore, they failed to distinguish between chewing gum and other topical nitrate sources (provided as toothpaste or mouthwash) that produced different results. Furthermore, Rosier et al. claim that chewing gum, toothpaste, and mouthwash may be equally effective in increasing local and systemic levels to achieve antibacterial effects in the mouth and nasal passages, but fail to provide evidence to substantiate, demonstrate, or verify such claims.

[0022] Rosier et al. (2020, WO 2021 / 122741), among others, including Bryan (2022, WO 2014 / 182632), provide a nitrate-formulated solution supplemented with specific nitrate-reducing bacteria in the presence of nitrate. Neither Rosier nor Bryan provide any evidence relating to or suggesting any health effects of increased oral-nasal nitric oxide production. Both Rosier and Bryan rely on the enterosalivary pathway and fail to consider that such a pathway can be circumvented through the use of specially formulated chewing gum.

[0023] As Lundberg et al. (1999) note in several papers, in the nasopharynx (including the paranasal sinuses and nasal cavity), nitric oxide is produced by the innate immune response upon exposure to bacterial and viral infections. Nitric oxide has antibacterial properties against a wide range of bacteria, parasites, fungi, and viruses.

[0024] Nitric oxide delivered in exogenous gaseous form may be useful in optimizing the treatment of uncontrolled pulmonary diseases due to its easy entry into the pulmonary environment and specific actions toward reducing bacterial load, inflammation, and improving clinical symptoms, particularly given that it is well understood that intranasal nitric oxide levels are significantly reduced in patients with mucus-filled sinuses and obstructed sinus ostia, Kartagener's syndrome, cystic fibrosis, and acute sinusitis in general, as reviewed by Lundberg et al. (1999).

[0025] In children with Kartagener syndrome, a condition consisting of sinusitis, bronchiectasis, and situs inversus, intranasal nitric oxide levels are extremely low compared with age-matched healthy controls (Lundberg et al., 1999). Similarly, smoking has been associated with a decrease in exhaled nitric oxide. The smoking-related decrease in exhaled nitric oxide was greatest in older subjects. This suggests that smoking is associated with an age-related decrease in exhaled nitric oxide.

[0026] Intranasal nitric oxide levels are also very low in patients with cystic fibrosis. Baraldi et al. measured intranasal nitric oxide in a group of children with acute sinusitis and found low intranasal NO levels, and chronic sinusitis is associated with a greater than 50% decrease in intranasal NO levels. In general, intranasal nitric oxide levels are lower in subjects with sinus disease.

[0027] Av-Gay et al. (2013), (U.S. Patent Application Publication No. 2020 / 0276229) claim that some respiratory diseases and physiological conditions can be treated by inhalation of gaseous nitric oxide delivered from a mechanical device or a canister of nitric oxide, or by an external spray device that delivers acidified nitrite.

[0028] The use of inhaled nitric oxide can prevent, reverse, or limit the progression of disorders such as acute pulmonary vasoconstriction, trauma, aspiration or inhalation injury, pulmonary fat embolism, acidosis, pulmonary inflammation, adult respiratory distress syndrome, acute pulmonary edema, acute mountain sickness, post-cardiac surgery injuries, acute pulmonary hypertension, persistent pulmonary hypertension of the newborn, perinatal aspiration syndrome, saline membrane disease, acute pulmonary thromboembolism, heparin-protamine reaction, sepsis, asthma, and status asthmaticus or hypoxia. Inhaled nitric oxide can also be used to treat cystic fibrosis, chronic pulmonary hypertension, bronchopulmonary dysplasia, chronic pulmonary thromboembolism, and idiopathic or primary pulmonary hypertension or chronic hypoxia.

[0029] As noted by Av-Gay et al. (2013), nitric oxide has a half-life of less than a few seconds in the body and a radius of action of approximately 200 microns from its site of origin. Beyond these limits, it is inactivated via binding to sulfhydryl groups on cellular thiols or by nitrosylation of the heme moiety of hemoglobin to form methemoglobin (MetHb). MetHb reductase reduces nitric oxide in serum to nitrate. Nitrate has been identified as the primary metabolite of nitric oxide excreted in urine and accounts for over 70% of inhaled nitric oxide. Nitrate is removed from plasma by the kidneys at a rate approaching that of glomerular filtration. Blood levels of MetHb in healthy humans are typically less than 2%. Therefore, potential side effects of high-dose nitric oxide treatment include binding of nitric oxide to hemoglobin and the formation of MetHb, which can lead to reduced oxygen transport and a reduced ability of NO to act as a nitrosylating agent on proteins and other cellular components. Previous studies of inhalation of gaseous nitric oxide by healthy human individuals have observed the formation and increased levels of MetHb, with reports that inhalation of gaseous nitric oxide at 128 ppm for 3 hours and at 512 ppm for 55 minutes resulted in MetHb levels exceeding the 5% safety threshold [Borgese N. et al., J. Clin. Invest., 1987, 80, 1296-1302; Young JD et al., Intensive Care Med., 1994, 20,581-4 and Young ID et al., Brit. J. Anaesthesia, 1996, 76, 652-656].

[0030] Nitric oxide inhaled from an external device or canister has been shown to be beneficial under medical supervision, but it carries risks, especially when considered for routine use with little or no expert medical oversight. Lundberg, a leading expert in respiratory nitric oxide biology, has expressed concerns regarding the potential use of nitric oxide as a therapeutic agent in various scenarios. In their paper "Primarily nasal origin of exhaled nitric oxide and absence in Kartagener's syndrome" (Eur. Respir J, 1994), Lundberg et al. (2004) found that nearly all exhaled nitric oxide originated in the upper airway, whereas the same individuals exhibited higher nasal nitric oxide concentrations during both mouth and nose exhalation. Therefore, since exhaled nitric oxide levels were higher during nasal breathing compared with mouth breathing in all subjects, including healthy controls, it is likely that most of the nitric oxide from the upper airway is produced in the nasal cavity. Furthermore, direct nasal sampling yields very high levels of nitric oxide, and nitric oxide from the upper airways is carried by the airflow with each inhalation, continuously washing through the lower airways. As cited by Lundberg et al. (1994), inhaled concentrations of nitric oxide as low as 100 ppb significantly reduced pulmonary vascular resistance in patients with pulmonary hypertension, suggesting that nasally derived nitric oxide may be physiologically important in the lung and thus may act as an airborne, or "aercrine," agent. Lundberg et al. (1994) go on to show that nasally produced nitric oxide gas exhibits bacteriostatic and antiviral properties and thus participates in the nonspecific primary mucosal line of defense against infection.

[0031] In summary, despite the advances in understanding nitric oxide and its effects on physiological function, there remains a need for efficient, targeted, and long-term sustained delivery of nitric oxide compositions to improve oral and nasopharynx health.There is also a need for compositions and methods that bypass the enterosalivary nitrate cycle and allow subjects to intermittently and rapidly enhance oral and nasal nitric oxide.There is also a need for systems, such as those that utilize rapid saliva test strips to monitor pH and nitric oxide metabolites to ensure continuous salivary nitrate, nitrite, and nitric oxide bioavailability, so that subjects can increase and maintain nitric oxide levels by chewing nitrate-containing compositions multiple times per day, regularly or intermittently, while remaining below the currently accepted daily intake. Summary of the Invention

[0032] In one embodiment, the present disclosure relates to the use of a nitrate-based formulation delivered via compressed chewing gum to extend exposure to prebiotic nitrates in the oral cavity. Use of the chewing gum by a subject allows for a rapid and local increase in nitric oxide gas in the oral cavity and nasopharynx. The chewing gum of the present invention also contributes to improved systemic nitric oxide bioavailability resulting from subsequent nitrate swallowing and biotransformation via the intestinal saliva nitrate-nitrite-nitric oxide pathway. In one embodiment, the chewing gum composition is designed to achieve nitric oxide levels below the acceptable daily intake of 3.7 milligrams per kilogram of body weight per day (mg / kg bw / day). The gum design and composition allow for intermittent administration of the nitrate-based chewing gum throughout the day to immediately and locally increase oral-nasopharynx nitric oxide production and extend delayed systemic nitric oxide bioavailability. In contrast to commonly available nitrate-based dietary supplements, such as mouthwashes, toothpastes, or oral intakes, the novel gum compositions claimed herein allow subjects to uniquely achieve optimal nitric oxide levels locally and systemically.

[0033] In one embodiment, as verified and determined by salivary nitrite measurements, the chewing gum compositions of the present disclosure uniquely contribute to a biphasic nitric oxide response, first, by providing a momentary local increase in the oral and nasal cavities, and second, as a post-swallowing response, with nitrate concentration in the salivary glands as a result of enterosalivary circulation following absorption in the intestinal tract.

[0034] In one aspect, the present invention further utilizes information regarding nitric oxide levels and acidity-alkalinity in an individual's oral cavity, specifically using rapid self-test saliva test strips to monitor real-time pH and the nitric oxide metabolites, nitrate and nitrite, to adjust the dosage of nitrate-containing chewing gum.

[0035] Without wishing to be bound by theory, it is believed that the gum compositions of the present disclosure act by shifting the oral microbiome toward a more abundant state of nitric oxide-promoting bacteria, resulting in a corresponding increase in salivary nitrite to nitrate ratio (>1) and elevated pH (>7). This shift in the oral microbiome corresponds to a decrease in bacteria associated with diseases related to gingivitis, dental caries, halitosis, and pathogens that cause cardiometabolic complications.

[0036] Provided herein is a novel and unexpected approach to improving oral hygiene and nasopharyngeal health by rapidly increasing oral and / or nasopharyngeal nitric oxide levels, optionally combined with a means to monitor levels through accessible, easy-to-use, rapid self-test methods for monitoring three important endpoints: pH, nitrate, and nitrite.

[0037] In one embodiment, the present disclosure includes a novel chewing gum composition that rapidly increases intranasal nitric oxide levels, thereby bypassing the L-arginine-independent nitrate-nitrite-nitric oxide dietary pathway. In contrast, currently available nitric oxide dietary supplement products, as well as prior art and publications, are solely based on improving nitric oxide levels by relying on the L-arginine-independent nitrate-nitrite-nitric oxide dietary pathway.

[0038] In one embodiment, the present invention provides a methodology for optimizing oral-nasal health by combining or binding a novel nitrate-containing chewing gum with a saliva self-test to obtain real-time information on salivary nitric oxide analyte levels in the oral cavity. This information can be utilized to maximize the antimicrobial activity of such analytes, resulting in improved oral, nasopharyngeal, and sinus health through intermittent chewing of the nitrate-containing gum. More specifically, a rapid saliva test that can be used at will and directly in the oral cavity to monitor nitric oxide status can enable regular monitoring and be incorporated into a routine for improving oral and nasopharyngeal health.

[0039] In one embodiment, the present disclosure provides real-time feedback via detection of salivary analytes and nitric oxide biomarkers, allowing individuals to adjust their oral hygiene regimen in real time to optimize oral health. The present disclosure allows users to quickly assess nitric oxide levels in their oral cavity in real time and determine corrective courses of action to improve and maintain oral health.

[0040] In one embodiment, the present invention provides a novel programmatic approach to oral hygiene that includes nitrate-loaded chewing gum to prolong exposure to nitrates in the oral cavity and enrich or shift the microbiome from an acidogenic or cariogenic microflora to a nitrate-reducing microbiome.

[0041] By increasing the "dwell time" of nitrate exposure, the nitrate-reducing oral microbiome, and areas of the mouth with acidic or low pH, such as inflamed gingival sulci around teeth or teeth covered with acidic cariogenic biofilms, the bioconversion of nitrite to nitric oxide is promoted. Unlike toothpastes and mouthwashes, the chewing gum compositions claimed herein offer unique advantages by extending oral exposure to nitrate and stimulating saliva secretion, contributing to the downstream nitrate-nitrite-nitric oxide pathway and subsequent uptake of nitrate provided by the gum (which is not the case with toothpastes and mouthwashes).

[0042] In one embodiment, the present invention involves the use of nitric oxide-generating chewing gum in combination with daily self-monitoring with saliva test strips to encourage consumer compliance behavior. The results of the colorimetric test strips are recorded and tracked on an electronic device, such as a wearable or cell phone, providing reminders and updates regarding oral care adherence and compliance to both the user and dentist or healthcare provider via wireless messaging.

[0043] In one embodiment, the present disclosure provides a systematic approach to improving oral and sinus health. Nitric oxide reduces inflammation and pathogenic microorganisms, and therefore, use of the novel gum compositions disclosed herein reduces the incidence of health problems related to oral health and the upper respiratory system, including, but not limited to, dental cavities, dental caries, gum disease, periodontitis, oral cancer, and viral infections or viruses sequestered in saliva and sinuses, including nasopharyngitis. The gum compositions also reduce the viral load of nitric oxide-insensitive coronaviruses, including SARS-CoV-1 and SARS-CoV-2.

[0044] These and other aspects, features, and advantages of the present disclosure will become apparent after review of the following detailed description of the disclosed embodiments and the appended claims. [Brief explanation of the drawings]

[0045] The present disclosure is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not necessarily to scale. Conversely, the dimensions of various features have been arbitrarily increased or decreased for clarity. Like reference numerals refer to like features throughout the specification and drawings.

[0046] [Figure 1] 1 provides a schematic overview of the comparison of nitric oxide bioavailability over time between a nitrate-based chewing gum (thick line) and the same amount of chewing gum derived from nitrate from an ingested capsule or beverage (thin line), as measured using MYFITSTRIP® saliva test strips for nitrite, a surrogate marker of nitric oxide. [Figure 2] Graphs are provided showing oral nitric oxide gas formation (A), salivary nitrate (B), and salivary nitrite (C) at various times after two pieces of plant-derived nitrate-containing gum were chewed for 5 minutes at 0 and 45 minutes and then discarded. The arrow(s) represent the time points during the 5-minute chewing period, and the number of arrow(s) indicates the number. [Figure 3] Graphs are provided showing oral nitric oxide gas formation (A), salivary nitrate (B), and salivary nitrite (C) at various times after chewing one piece of potassium nitrate-containing gum for 5 minutes at 0 minutes and then discarding it. The arrow(s) represent the time points during the 5-minute chewing period, and the number of arrow(s) indicates the number. [Figure 4] Graphs are provided showing oral nitric oxide gas formation (A), salivary nitrate (B), and salivary nitrite (C) at various times after three pieces of potassium nitrate-containing gum were chewed for 5 minutes at 0 minutes and then discarded. The arrow(s) represent the time points during the 5-minute chewing period, and the number of arrow(s) indicates the number. [Figure 5]This figure provides graphs showing oral nitric oxide gas formation (A), salivary nitrate (B), and salivary nitrite (C) after chewing potassium nitrate-containing gum for 5 minutes at various time points. In this example, one chewing gum was administered for 5 minutes at 0 minutes and another chewing gum was administered for 5 minutes at 150 minutes, and each was discarded. The arrow(s) represent the time points at which the chewing gum was administered for 5 minutes, and the number of arrow(s) indicates the number of chewing gums. [Figure 6] A graph showing intraoral nitric oxide gas (A) and exhaled nitric oxide (B) at various times after chewing a potassium nitrate gum. In this example, one piece was administered at 0 minutes for 5 minutes and discarded. [Figure 7]The relative abundance of bacterial species before and after chewing potassium nitrate-containing gum is shown. Whole-genome metagenomic sequencing or shotgun sequencing was used to examine all DNA present in the microbiome samples. Saliva samples were collected from subjects, and MyFitStrip® live bacterial nitric oxide testing detected low nitrite-to-nitrate ratios, and MyFitStrips® oral hygiene strips detected low saliva pH (<7). Saliva was collected according to methods provided by Bristle Oral Health Labs. After collection, subjects chewed potassium-ascorbic acid-containing gum twice for 5-10 minutes over a 3-4 hour period. At that time, the nitrite-to-nitrate ratio and pH were measured, and a second saliva sample was collected for shotgun sequencing analysis according to Bristle Labs' procedures. In this example, chewing three pieces of the above gum at 0, 2, and 4 hours during this period increased the relative abundance of Rothia aeria, Rothia mucilaginosa, Neisseria flavescens, Neisseria subflava, and Haemophilus parainfluenzae in saliva 6 hours later, thereby enhancing the bioavailability of nitric oxide and correspondingly increasing the relative abundance of the disease-associated bacteria Tannerella forsythia, Treponema socranskii, Fusobacterium periodonticum, Porphyromonas gingivalis, Streptococcus constellatus, Fusobacterium nucleatum, and Parvimonas micra. Improved oral and nasopharyngeal health through reduction of Prevotella micra, Prevotella melaninogenica, Prevotella histicola, and Candida albicans. DETAILED DESCRIPTION OF THE INVENTION

[0047] The following detailed description is exemplary and explanatory and is intended to provide further explanation of the disclosure set forth herein. Other advantages and novel features will become readily apparent to those skilled in the art from the following detailed description of the disclosure. Documents and references mentioned herein are incorporated in their entirety, including U.S. Provisional Patent Application No. 63 / 402,149, filed August 30, 2022.

[0048] For purposes of the following description, it should be understood that the embodiments described below may assume alternative variations and embodiments. It should also be understood that the specific articles, compositions, and / or processes described herein are exemplary and should not be considered limiting.

[0049] In 1998, Robert F. Furchgott, Louis J. Ignarro, and Ferid Murad were awarded the Nobel Prize in Medicine for their discovery of the importance of nitric oxide in the cardiovascular system. These scientists demonstrated that nitric oxide is a short-lived, endogenously produced gas that acts as a signaling molecule in the body. Signaling by a gas produced by one cell that crosses membranes and regulates the function of other cells was recognized for the first time as an entirely new principle for signaling in the human body. Related research has demonstrated the critical role that nitric oxide plays in fundamental biological processes such as regulating blood pressure, immune system function and dysfunction, and activation of central nervous system mechanisms that affect everything from gastric motility to memory and behavior.

[0050] The present disclosure is based on the discovery that nitric oxide has antibacterial activity in the oral cavity. More specifically, the inventors observed that nitric oxide, particularly nitric oxide, produced non-enzymatically and enzymatically by the sequential chemical reduction of nitrate to nitric oxide, has potent antibacterial activity. To optimize and direct the antibacterial activity of nitric oxide, the inventors recognized the need to deliver nitric oxide both locally and systemically. Furthermore, the need to create, maintain, and monitor appropriate precursor threshold levels in the oral cavity is achieved through the use of saliva test strips. The present disclosure provides teachings about compositions that enable the optimization of nitrate metabolites to maintain a healthy, antibacterial environment in a subject's oral and nasal cavities. This goal can be achieved by using chewing gum to slowly release a nitrate compound, increasing the exposure of the important oral microbiome to nitrates, which act as a prebiotic, shifting cariogenic, acid-promoting bacteria to health-beneficial, nitrate-reducing bacteria.

[0051] Products such as toothpaste or mouthwash made with similar formulations limit the "residence time" of nitrates in the oral cavity to a few seconds, or in some cases, one to two minutes. In contrast, the nitrate-based gum compositions claimed herein provide longer and more sustained nitrate levels in the oral cavity. When the nitrate-based gum of the present invention is chewed for five minutes and then chewed intermittently throughout the day, oral health is improved, caries- and decay-causing microorganisms (e.g., "bad" bacteria) are reduced, and nitrate levels in the oral cavity are established so that beneficial microflora can thrive. The use of nitrate-based gum compositions is particularly advantageous because chewing gum is more practical than brushing teeth with nitrate-based toothpaste or using nitrate-based mouthwash several times a day (to achieve equivalent levels of nitrate in the oral and / or nasopharyngeal cavity).

[0052] A further consequence of using nitrate-based gum is that subsequent swallowing of the nitrate prolongs the bioavailability of nitric oxide as it is recycled through the enterosalivary pathway. The enterosalivary nitrate pathway is the pathway by which inorganic nitrate-rich nitric oxide dietary supplements and nitric oxide-enhancing foods are metabolized to produce nitric oxide. Thus, the nitrate-based chewing gum (or similarly formulated sustained-release lozenges) of the present disclosure is uniquely suited for the immediate, localized, and delayed systemic formation and delivery of nitric oxide. Yet another advantageous feature of the gum compositions claimed herein is the immediate generation of nitric oxide gas, which is not the case when nitrate capsules or nitrate-rich drinks or foods are swallowed. Again, chewing gum delivery offers the unique advantage of a biphasic nitric oxide curve observed within minutes, and even after 90 minutes. Localized delivery within the mouth directly enhances antibacterial activity in the oral and nasal cavities. The resulting systemic delivery of nitrate via the enterosalivary nitrate pathway "loops back" to further improve the oral nitric oxide environment. Additionally, elevated nitric oxide levels also benefit the subject overall, particularly with regard to cardiovascular health.

[0053] Nitrate itself is a harmless precursor that is converted to nitrite and produces antibacterial species only when exposed to acidic conditions. Lactic acid bacteria (Lactobacillus) temporarily produce sufficient acid in the mouth after a carbohydrate meal to control the growth of oral pathogens, but as explained by Rosier et al. (2018), a moderate intake of nitrate is a desirable prerequisite because it also contributes to increased pH conditions. In addition to their antibacterial properties, oral nitrite and nitric oxide also have antiviral properties. While not wishing to be bound by theory, the inventors have discovered that viruses, in contrast to bacteria, respond differently to nitric oxide complexes; that is, when exposed to nitric oxide complexes containing, for example, nitrate and / or acidic nitrite, the complexes may affect replication to some extent, but more importantly, alter virus-infected cells so that the immune system can better recognize viral particles.

[0054] In the case of coronavirus disease (COVID-19), the inventors previously established that restoration of nitric oxide improves endothelial dysfunction and contributes to pulmonary vasodilation, antithrombotic effects, and direct antiviral activity (Microbes and Infection, Volume 22, Issues 4-5, 2020, pp. 149-150). Nitric oxide interferes with the interaction of the coronavirus S protein with its cognate host receptor, ACE-2. Nitric oxide-mediated S-nitrosylation of the viral cysteine ​​protease and the host serine protease TMPRSS2, both important for viral cellular entry, appears to be sensitive to nitric oxide. Based on reports of improved lung function during the 2003 SARS outbreak, the FDA's emergency expanded use of nitric oxide gas is currently underway for the treatment of COVID-19.

[0055] Alternatively, dietary inorganic nitrate has been shown in multiple studies to be effective in restoring endothelial function, reducing pulmonary and arterial hypertension, and promoting antibacterial activity. As previously described, it is well understood that dietary inorganic nitrate is bioconverted to nitric oxide through a series of well-defined steps, beginning with the reduction of nitrate to nitrite by beneficial microflora on the tongue, followed by subsequent reduction to nitric oxide in various organs, including the intestine, bloodstream, and lungs. The inorganic nitrite and S-nitrosothiol formation are absorbed into the circulation, where they act as a temporary storage pool for subsequent nitric oxide production. The conversion of inorganic nitrite to nitric oxide is enhanced during conditions of acidosis or hypoxia in the pulmonary vascular region of the lungs of patients with chronic obstructive pulmonary disease (COPD) and those exhibiting acute respiratory distress syndrome, such as those observed in coronavirus-infected lungs. Consumption of inorganic nitrate for 8 days reportedly increased pulmonary nitric oxide by 200% and reduced respiratory symptoms in a COPD population. Restoring nitric oxide via dietary inorganic nitrates could be a consideration for prevention and early treatment, as they act at two levels: reversing platelet endothelial dysfunction and associated thrombosis, as well as reducing viral load, and when produced locally in the oral and nasal cavities, this invention could reduce COVID transmission.

[0056] The effect of salivary nitrate excretion is to provide a precursor for the generation of nitric oxides by chemical reduction of nitrite. In the mouth, bacteria rapidly reduce nitrate to nitrite (1). Nitrite is further reduced in the sulci to antibacterial nitric oxide species, including nitric oxide (1-3). Further increasing acidic conditions in the sulci protonate nitrite, forming nitrous acid (1-4). Nitrite, in turn, dissociates to form the following nitric oxides: 1. NO2- + H+ = HNO2 2.2HNO2=H2O+N2O3 3. N2O3 = NO + NO 4. N2O3 + C2H8O6 = 2NO + H2O + C6H6O6

[0057] Endogenous and dietary nitrates are concentrated by the salivary glands to more than 10 times the concentration in plasma and secreted into saliva. The nitrate-based gum compositions of the present disclosure provide a source of nitrates as an immediate precursor for continuous reduction in the mouth for antibacterial activity in the oral and nasal cavities. In certain embodiments, the antibacterial effect is pronounced in the presence of periodontal disease with acid-producing bacteria in the sulci.

[0058] Oral conversion of nitrate to nitrite is rapid and generally occurs on the surface of the tongue (mainly the posterior third of the tongue) in mammals by commensal nitrate-reducing bacteria, which can subsequently be reduced to nitrogen oxides, including nitric oxide, in the grooves of infected pockets under low pH or acidic conditions. Alternatively, as disclosed herein, nitrate and ascorbic acid can further accelerate chemical reduction in the oral and nasal cavities or enhance the formation of antibacterial nitric oxide. In one embodiment of the present invention, a moderate acidity is provided. Provided herein is a novel oral hygiene regimen that includes the use of a nitrate source for continuous reduction in the mouth for the direct treatment and / or prevention of oral and upper respiratory tract infections, combined with monitoring of oral nitric oxide levels via nitrite and nitrate levels as biomarkers for antibacterial activity, with the aim of increasing compliance with daily oral hygiene practices, such as intermittent chewing of nitrate-containing gum.

[0059] The present disclosure fulfills an unmet medical and health need for improving and monitoring oral-nasal health. To reduce the number of caries-producing organisms in dental plaque and prevent the progression of gingivitis and periodontal disease, the present disclosure provides a novel programmatic approach involving the use of nitrate-based ingredients in conjunction with saliva test strips to monitor the presence of nitric oxide after brushing. Furthermore, this programmatic approach extends beyond caries prevention to the treatment and prevention of infections caused by Candida albicans and other harmful oral organisms susceptible to nitric oxide. Increased nasal nitric oxide gas benefits the upper respiratory tract, including the nasal cavity and sinuses, which are susceptible to and serve as reservoirs for viral infections, such as SARS-CoV-1 and SARS-CoV-2.

[0060] The present disclosure provides a novel source of nitrate in the form of a nitrate-rich gum. In one embodiment, the nitrate-rich gum described herein may be composed of potassium nitrate or a plant-based powder source derived from spinach, kale, arugula, celery, or beets, among other nitrate-rich leafy greens. The nitrate or plant-based nitrate powder source may be mixed with a gum base, which may be a natural source, such as chicle, or a non-natural source, including, but not limited to, butadiene styrene rubber, paraffin, and various waxes used in the manufacture of chewing gum. In certain embodiments, the gum further comprises a sugar-free sweetener, such as xylitol, and may be flavored with both natural and artificial flavors, including other herbal and plant sources, such as spearmint, orange, fennel, and fruit flavors.

[0061] In one aspect, the present invention includes a method for improving oral-nasal antimicrobial health in a subject, comprising the use of a chewing gum comprised of 2-8% potassium nitrate (by weight based on the total weight of the gum), which may include a ratio of 2:1 to 1:4 ascorbic acid. In one embodiment, the present invention includes a novel approach for the treatment and / or prevention of bacterial, viral, or fungal conditions in the oral cavity and a programmatic approach for monitoring its maximal antimicrobial activity. While not wishing to be bound by theory, it is anticipated that acidification of nitric oxide precursors occurs in the sulci or subgingival space of infected pockets, and a source of nitrite ions derived from nitrate precursors is further reduced to form antimicrobial nitric oxides, particularly nitric oxide. Aspects of the present disclosure relate to a source of nitrate, optionally reduced by oral microflora, which may be further reduced in the presence of salicylic acid and / or ascorbic acid, which enhances antimicrobial efficacy in the oral cavity. Further aspects of the present disclosure include the use of saliva test strips to monitor nitrates, using test strips to monitor nitrites in the oral cavity to achieve antibacterial activity.

[0062] Strips suitable for use in the present disclosure include a single device with a three-step method for collecting, transporting, and measuring salivary fluid analytes, specifically nitric oxide anion, nitrate, nitrite, nitric oxide analytes and biomarkers therefor, and pH. In one embodiment, saliva strips, such as those commercially available as MYFITSTRIP® (Rockville, MD, USA), not only monitor nitric oxide and pH status, but also provide instant readings so users can make informed decisions in real time about maintaining sufficient levels of nitrite in the mouth to maintain oral health. The MYFITSTRIP® saliva test strips allow users (e.g., those prone to dental problems) who need a sensitive, easy-to-use, and affordable test that can be used three to four times a day to adjust their hygiene and diet as needed to maintain optimal levels of nitric oxide.

[0063] The unique design of the strips used herein allows for easy collection of test fluids without the need for additional containers or direct use of a finger to collect or dispense the fluid. MYFITSTRIP® saliva strips overcome the shortcomings of other nitric oxide test strips, such as the BERKELEY TEST® and HUMANN INDICATOR TEST STRIPS, which are limited to detecting nitrite, while MYFITSTRIP® saliva strips are designed to detect nitrite, nitrate, and pH. Unlike other strips, MYFITSTRIP® saliva strips prevent false negative results, meaning that if other test strips are negative, the subject will not know whether their oral microbiome is capable of bioconversion or whether their supplements, gum, drinks, or foods are rich in nitrates.

[0064] In one embodiment, a chewing gum composition is provided herein that includes a gum base in combination with (a) potassium nitrate and ascorbic acid, or (b) potassium nitrate, ascorbic acid, and a polyphenol extract, or (c) potassium nitrate, ascorbic acid, and zinc, or (d) potassium nitrate, ascorbic acid, zinc, and a polyphenol extract, or (e) a plant-derived inorganic nitrate and ascorbic acid, or (f) a plant-derived inorganic nitrate, ascorbic acid, and zinc, or (g) a plant-derived inorganic nitrate, ascorbic acid, zinc, and a polyphenol extract. The chewing gum composition may further include a sweetener, a gum base or bulk filler, a flavoring, a lubricant, a glidant, or a combination thereof. The chewing gum composition of the present invention can be used to increase nitric oxide levels in a subject's oral cavity, buccal cavity, and / or nasal cavity.

[0065] The gum base may include chicle, gelatin, pectin, beeswax, paraffin wax, rosin, butyl rubber, polyvinyl acetate, microcrystalline cellulose, vegetable fiber, or combinations thereof. The gum composition may be formed into a stick or tablet, or the gum may have a liquid center.

[0066] In certain embodiments, sweeteners may include sugars, non-sugars, maltitol, saccharin, aspartame, sorbitol, sucralose, isomalic acid, erythritol, xylitol, plant-derived sweeteners, stevia, agave, coconut sugar, honey, monk fruit extract, sugar blends, or sugar alcohol blends, and / or combinations thereof.

[0067] In certain embodiments, the chewing gum compositions of the present disclosure further comprise a flavoring, which may include natural or artificial flavors or combinations thereof. Such flavors may include mint, garden mint, mixed mint, spearmint, peppermint, wintergreen, fruit, watermelon, strawberry, blackberry, raspberry, orange, lemon, lime, clementine, tangerine, grapefruit, mango, yuzu, banana, apple, peach, pear, plum, pineapple, pomegranate, ginger, cinnamon, menthol, or chocolate.

[0068] In certain embodiments, potassium nitrate comprises 1-5% of the gum composition. The potassium nitrate may be derived from a plant source selected from the group consisting of leafy green vegetables, including celery, beets, arugula, Swiss chard, beetroot, and / or combinations thereof.

[0069] In certain embodiments, the ascorbic acid comprises plant-derived ascorbic acid, including but not limited to ascorbic acid from acerola, and the gum composition may comprise 0.5-10, 7-15% wt / wt ascorbic acid, with a nitrate to ascorbic acid ratio of 2:1 to 1:4.

[0070] In certain embodiments, the gum composition comprises 1-10% zinc, 4-12% L-arginine, 1-5% N-acetylcysteine, or a polyphenolic fruit-derived extract at 2-10 mg / dose (10:1 per gram of extract) of activated anthocyanins, or a combination thereof.

[0071] In certain embodiments, the gum composition comprises 1% zinc, 6-8% L-arginine, 1-5% N-acetylcysteine, or a polyphenolic fruit-derived extract at 2-10 mg / dose of activated anthocyanins (10:1 per gram of extract), or a combination thereof.

[0072] In one embodiment, the amount of nitrate per chewing gum composition is 0.15-2.5 mM.

[0073] In certain embodiments, a subject may establish and maintain oral and / or oronasal nitric oxide levels, and optionally also enhance systemic bioavailability of nitric oxide, by chewing a first gum composition for at least 3-7 minutes, discarding it, and then chewing additional gum compositions intermittently over a 12-hour period (chewing each subsequent gum composition for 3-7 minutes). The chewing periods for each gum composition may be separated by increments of 1-30, 30-60, 60-90, 90-120, or 120-150 minutes.

[0074] In one embodiment, increasing nitric oxide levels in the oral cavity, buccal cavity, and / or nasal cavity of a subject comprises increasing the relative abundance of beneficial oral microflora, including, but not limited to, one or more of Rothia aireria, Rothia mucilaginosa, Neisseria flavescens, Neisseria subflava, and Haemophilus parainfluenza. In one embodiment, the relative increase in beneficial oral microflora comprises approximately 1% or more after a minimum of 4-6 hours compared to before chewing the gum composition.

[0075] In one embodiment, increasing nitric oxide levels in the oral cavity, buccal cavity, and / or nasal cavity of a subject comprises reducing the relative abundance of harmful oral microflora, including, but not limited to, one or more of Tannerella forsythia, Treponema sokranskii, Fusobacterium periodonticum, Porphyromonas gingivalis, Streptococcus constellatus, Fusobacterium nucleatum, Parvimonas micra, Prevotella melaninogenica, Prevotella histicola, and Candida albicans, and diseases associated therewith. In one embodiment, the relative reduction of harmful oral microflora comprises approximately 1% or more after a minimum of 4-6 hours compared to before chewing the gum composition.

[0076] In one embodiment, increasing nitric oxide levels in the oral cavity, buccal cavity, and / or nasal cavity of a subject includes improving oral health, including reducing plaque, dental cavities, tooth decay, periodontitis, halitosis, bacterial infections, fungal infections, viral infections, nasopharyngitis, and related sinus and oral infections.

[0077] In certain embodiments, the salivary nitrate and nitrite concentrations during chewing and 5 minutes after fasting are at least 100 mg / L or greater and 10 mg / L or greater, respectively, as assessed by nitrate and nitrite saliva test strips.

[0078] In certain embodiments, chewing the gum compositions as claimed herein results in an increase in saliva pH to between 6.5 and 8.0, and the increase in saliva pH results in a decrease in tooth demineralization, oral acidosis, and / or acid-producing bacteria.

[0079] In one embodiment, the gum composition of the present disclosure comprises potassium nitrate-zinc ascorbate, gum base, sorbitol, maltitol, xylitol, isomaltose, natural flavors, potassium nitrate, magnesium stearate, acerola, sucralose, silicon dioxide, and zinc citrate.

[0080] In one embodiment, the gum composition of the present disclosure comprises plant-based acerola, gum base, sorbitol, maltitol, xylitol, isomaltose, celery extract, natural flavors, magnesium stearate, acerola, sucralose, silicon dioxide, polyphenols, and glycerin.

[0081] Unless expressly stated otherwise, the methods or embodiments set forth herein are not intended to be construed as requiring that its steps be performed in a particular order. Thus, where a method claim does not specifically recite in the claim or specification that the steps are limited to a particular order, no order is intended to be inferred in any respect. This applies to all possible implicit bases for interpretation, including questions of logic regarding the organization or operational flow of steps, apparent meaning derived from grammatical organization or punctuation, or the number or type of embodiments described in the specification.

[0082] As used herein, the term "subject" should be construed to include subjects, such as humans and other animals, in need of supplemental or therapeutic intervention, e.g., medical or surgical subjects.

[0083] In this disclosure, the singular forms "a," "an," and "the" include plural referents, and reference to a particular numerical value includes at least that particular value unless the context clearly dictates otherwise. Thus, for example, reference to "beads" or "nanostructures" is a reference to one or more of such structures and equivalents known to those of skill in the art, and so forth. When values ​​are expressed as approximations by use of the antecedent "about," it is understood that the particular value forms another embodiment. As used herein, "about X" (where X is a numerical value) preferably refers to ±10% of the stated value. For example, the phrase "about 8" preferably refers to a value from 7.2 to 8.8, and as another example, the phrase "about 8%" preferably (but not always) refers to a value from 7.2% to 8.8%. Where present, all ranges are inclusive and combinable. For example, when a range of "1 to 5" is recited, the recited range should be interpreted as including ranges such as "1 to 4," "1 to 3," "1 to 2," "1 to 2 and 4 to 5," "1 to 3 and 5," "2 to 5," etc. Furthermore, when a list of alternatives is expressly provided, such a list can be interpreted to mean that any of the alternatives may be excluded, for example, by a negative limitation in the claims. For example, when a range of "1 to 5" is recited, the recited range may be interpreted as including situations in which any of 1, 2, 3, 4, or 5 is negatively excluded; thus, the recitation of "1 to 5" may be interpreted as "1 and 3 to 5, but not 2," or simply "2 not included." It is intended that any component, element, attribute, or step explicitly described herein may be explicitly excluded in the claims, regardless of whether such component, element, attribute, or step is listed as an alternative or described alone.

[0084] As used herein, the word "or" means any one member of a particular list and also includes any combination of members of that list.

[0085] Ranges can be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, a further embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations by use of the antecedent "about," it is understood that the particular value forms a further embodiment. It is further understood that each endpoint of the range is significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values ​​disclosed herein, and that each value is herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that each unit between two particular units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0086] References in this specification and in the concluding claims to parts by weight of a particular element or component in a composition indicate the weight relationship between the element or component and any other element or component in the composition or article to which the parts by weight are expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y are present in a weight ratio of 2:5, and are present in such ratio regardless of whether the compound includes additional components.

[0087] Weight percent (wt.%) of an ingredient is based on the total weight of the formulation or composition in which the ingredient is included, unless otherwise stated to the contrary.

[0088] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0089] As used herein, the term "prevent" or "preventing" refers to preventing, preventing, removing, forestalling, stopping, or impeding something from happening, especially by prior action. Where "reduce," "inhibit," or "prevent" is used herein, it is understood that the use of the other two words is also expressly disclosed unless specifically indicated otherwise.

[0090] As used herein, the terms "effective amount" and "amount effective" refer to an amount sufficient to achieve a desired result or to have an effect on an undesired condition. For example, a "therapeutically effective amount" refers to an amount sufficient to achieve a desired therapeutic result or to have an effect on an undesired condition, but generally insufficient to cause adverse side effects. The specific effective amount for a subject will depend on various factors, including the patient's age, weight, general health, sex, and diet, the time and route of administration, and similar factors well known in the health field.

[0091] Disclosed are the components used to prepare the disclosed compositions, as well as the compositions themselves used within the methods disclosed herein. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that specific reference to the various individual and collective combinations and permutations of each of these compounds may not be explicitly disclosed, but each is specifically contemplated and described herein. For example, when a particular compound is disclosed and discussed, and numerous modifications that can be made to numerous molecules comprising the compound are discussed, all combinations and permutations of the compound and modifications possible unless specifically indicated to the contrary are specifically contemplated. Thus, if a class of components A, B, and C is disclosed, as well as a class of components D, E, and F, and an example of a combined substance AD, each is considered individually and collectively, even if not individually specified, and the combinations AE, AF, BD, BE, BF, CD, CE, and CF are considered to be disclosed. Similarly, subsets or combinations of these are also disclosed. Thus, for example, the subgroups AE, BF, and CE are considered to be disclosed. This concept applies to all aspects of this application, including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, where there are various additional steps that may be performed, it is understood that each of these additional steps may be performed in any particular embodiment or combination of embodiments of the methods of the invention.

[0092] As used herein, gum refers to a soft, chewable, adhesive substance designed to be chewed without swallowing. In one embodiment, gum may be composed of a gum base, sweeteners, softeners / plasticizers, flavors, colorants, and optionally a hard or powdered polyol coating. Its texture may be reminiscent of rubber due to the physical-chemical properties of the polymer, plasticizer, and resin components, which contribute to its elastic, adhesive, and chewable properties.

[0093] All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the publication dates provided herein may be different from the actual publication dates, which may require independent confirmation.

[0094] The following specific examples illustrate the application of the present invention to methods for improving oral health by detecting and monitoring biomarkers such as pH, nitrate, and nitrite in saliva. Other examples, including slight variations in procedure, will be apparent to those skilled in the art, and it is understood that the present invention is not limited to these specific described examples. [Example]

[0095] The chewing gum formulations used in Examples 1-7 are generally summarized as shown below:

[0096] [Table 1]

[0097] [Table 2]

[0098] Example 1 Bioconversion of oral plant-derived nitrate to nitrite and subsequent reduction to nitric oxide gas with corresponding increase in pH As discussed herein, dietary inorganic nitrate and its reduced forms, nitrite and nitric oxide, respectively, are crucial for host defense in the oral cavity. High salivary nitrate concentrations are associated with reduced caries incidence due to inhibition of cariogenic bacterial growth and increased pH. Anticaries activity depends on the bioconversion of nitrate to nitrite and downstream reactive nitrogen oxides, specifically antibacterial nitric oxide.

[0099] This example demonstrates that bioconversion of nitrate to nitrite for local production of nitric oxide gas occurs immediately in the mouth. Although it is well established that reactive nitrogen oxides exhibit antibacterial activity, currently, no method or invention exists to verify the source of nitrate and nitrite and ensure that nitrate is converted immediately and locally in the oral cavity.

[0100] Nitric oxide gas was measured using NObreath® (NOB; Bedfont, Kent, UK), and salivary pH and the salivary nitric oxide metabolites nitrate and nitrite were measured using MyFitStrip® (Rockville, MD USA).

[0101] Nitric oxide gas was measured using NOB. These devices were evaluated based on the guidelines of the UK National Institute for Health and Care Excellence (NICE). NOB is designed to measure exhaled nitric oxide (FeNO) levels in human breath. A normal FeNO test value is less than 25 parts per billion (ppb) in adults. Levels above this value were used to assess pulmonary inflammatory responses, where inducible nitric oxide synthase is activated in immune cells in response to an inflammatory response. NOB is used to assess nitric oxide gas generated locally in the mouth.

[0102] Participants were pre-screened to exclude asthmatics, those with respiratory infections, and healthy individuals with levels around 35 ppb due to a diet of nitrate-rich beets or leafy green vegetables, or excessive nasal breathing within 6 to 18 hours before the test. All healthy subjects with consistent exhaled breath levels of 10 to 30 ppb, influenced by exercise and diet, particularly nitrate-rich plant-based foods and diet, and excessive nasal breathing, were included.

[0103] A total of four to six subjects were assessed two to four times over a two-week period. Values ​​are from one individual and are representative of four to six other subjects. Participants were seated and held the monitor upright at all times. Participants inhaled to full lung capacity, then exhaled through the mouthpiece, holding the visual indicator marker in the center of the band to ensure proper measurement. Exhalation time was approximately 10 seconds. All participants were asked to repeat the measurement three times to verify the device's reproducibility. Baseline values ​​ranging from 10 to 35 ppb reflect nitric oxide sourced from the lungs and respiratory system before chewing gum.

[0104] MyFitStrip® was used to measure salivary pH and salivary nitrate and nitrite. After participants completed the NOB breath test, they placed the MyFitStrip® collection pad on their tongue to collect saliva for 5 seconds, removed it from their mouth, folded it over to contact the edge of the test pad, and held it together for 10 seconds to ensure transfer of saliva to the colorimetric test pad. After 10 seconds, the folded strip was released, and the color pad is shown compared to a color scale with pH ranges of 4.5, 5.5, 6.5, 7.0, 7.5, and 9.0; nitrate ranges of 0, 5, 10, 25, 50, 75, and 100 mg / L; and nitrite ranges of 0, 0.5, 2, 5, 10, 25, and 50 mg / L. The MyFitStrip® test pad is based on the well-established Griess reagent reaction for colorimetric detection and measurement of both nitrate and nitrite.

[0105] Example 1 (Figure 2) shows the immediate increase in antimicrobial nitric oxide gas (A) along with the corresponding precursor and metabolite, nitrate (B) and nitrite (C), respectively, when two plant-derived nitrate gums were chewed for 5 minutes starting at time 0 and then chewed again 45 minutes later before being discarded. The arrow(s) represent the time points during the 5-minute chewing period, and the number of arrow(s) indicates the number. Methods for detecting nitric oxide gas and salivary metabolites are described above.

[0106] In this study, pH was also monitored, and in all cases, saliva pH exposed before chewing the gum was in the range of 5.5–6.5, and immediately rose to 7.0–7.5 within 3–5 minutes of chewing the gum.

[0107] Nitrate-rich dietary sources have been shown to exert a protective effect against dental caries by increasing salivary pH (Sustaining elevated levels of nitrite in the oral cavity through consumption of nitrate-rich beetroot juice in young healthy adults reduces salivary pH, Hohensinn, B., et al. (2016) Nitric Oxide 60:10-15). Bioconversion of nitrate to nitrite increased salivary pH from 7.0 to 7.5, confirming the anticariogenic effect of providing dietary sources of NO. These results clearly suggest that nitrate-rich dietary sources, when consumed regularly, significantly prevent the acidification of salivary pH and allow the bioconversion of NO to NO, as determined by salivary NO test strips. However, Hohensinn et al. (2016) demonstrated that dietary NO must be continuously ingested to benefit from the antibacterial and other biological functions of nitric oxide. Thus, the importance of the entero-salivary nitrate-nitrite-nitric oxide loop was demonstrated, with an immediate pH shift observed within minutes of chewing.

[0108] If oral biotransformation levels were sustained throughout the day, it would not be unreasonable to expect the mouth to remain alkaline, as reflected by the cell-destructive effects of acid-forming bacteria. In this case, the strips could be used to remind subjects to chew nitrate-based gum and / or sustained-release lozenges intermittently, especially as detected nitrite levels begin to naturally decline throughout the day, without increasing salivary pH to levels that would be harmful to the teeth.

[0109] pH is a significant indicator of the presence and number of cariogenic bacteria, and an increase in pH is associated with a decrease in the number of acid-producing bacteria. Therefore, to shift from a temporary cytostatic effect to a sustained cytocidal effect on cariogenic bacteria, nitrate-based chewing gum is incorporated into an oral hygiene program coordinated with daily readings of pH, nitrate, and nitrite using MYFITSTRIP® test strips. The duration of local and systemic nitrate-nitrite-nitric oxide gas biotransformation varies from individual to individual, so frequent monitoring with test strips and, if levels are declining, supplementing with nitrate gum to increase the local oral source before acid-producing bacteria can recolonize the area is crucial.

[0110] The present invention demonstrates that nitrate-rich sources have potential anti-caries effects via bioconversion to nitrate-nitrite-nitric oxide, resulting in increased pH levels and a predicted reduction in acid-producing cariogenic bacteria. However, sustained anti-caries effects are likely due to clearance of nitrate, which must be periodically replenished based on test strip results.

[0111] This example demonstrates the immediate production of nitric oxide gas (A) along with the corresponding reduction of nitrate (B) to nitrite (C) within 3-5 minutes of chewing gum. Subsequent chewing 45 minutes later resulted in a similar response as the 3-5 minute time point, but with slightly elevated gas, nitrate, and nitrite concentrations, which persisted for a minimum of 1 hour. Intermittent chewing has been shown to additionally and sustainably enhance antimicrobial levels of nitric oxide in the oral cavity, thereby maintaining and improving oral health through: 1. dietary nitrate sources in a delivery format that optimizes immediate local levels of nitrate, including, but not limited to, nitrate-rich gums or sustained-release gums, chewables, or lozenges; and 2. saliva test strips to indirectly verify nitrate content and verify bioconversion of nitrate to antimicrobial nitrite.

[0112] Examples 2 to 4 Bioconversion of potassium nitrate-ascorbic acid-zinc chewing gum in the oral cavity to nitrite and subsequent reduction to nitric oxide gas with corresponding increase in pH The methods, experimental protocols, salivary pH, nitrate, nitrite, and nitric oxide gas measurements described above for the plant-derived gums are similarly performed for the potassium-ascorbate-zinc chewing gum.

[0113] Example 2 (Figure 3) shows the immediate increase in antimicrobial nitric oxide gas (A) along with the corresponding precursor and metabolite, nitrate (B) and nitrite (C), respectively, when one potassium nitrate-based chewing gum piece is chewed for 5 minutes and then discarded. The arrow(s) represent the time points at which the chewing gum was administered for 5 minutes. Methods for detecting nitric oxide gas and salivary metabolites are described above. Example 2 demonstrates the immediate production of nitric oxide gas (A) within 3-5 minutes of chewing the gum, along with the corresponding reduction of nitrate (B) to nitrite (C), which persists for a minimum of an additional hour with antimicrobial nitric oxide levels during this period with an associated pH of >7.5.

[0114] Example 3 (Figure 4) shows the immediate and sustained elevation of oral nitric oxide gas production (A), salivary nitrate (B), and salivary nitrite (C) at various times after three pieces of potassium nitrate-containing gum were chewed for 5 minutes at 0 minutes and then discarded. The arrow(s) represent the time points during the 5-minute chewing period, and the number of arrow(s) indicates the number. Example 3 demonstrates the immediate and sustained production of nitric oxide over a 3-hour period, with an associated pH of >7.5 during this period. The biphasic nature of administration is best illustrated by this example, with oral administration producing immediate and sustained production for 30-60 minutes, allowing sufficient time for ingested nitrate to recirculate within the mouth and sustain nitric oxide production in the mouth for an additional 2-3 hours.

[0115] Example 4 (FIG. 5) shows oral nitric oxide gas production (A), salivary nitrate (B), and salivary nitrite (C) at various times after chewing a single piece of potassium nitrate-containing gum at intermittent time points. In Example 4, the gum was administered at time 0, for 5 minutes, and at 150 minutes, and in both cases was chewed for 5 minutes before discarding. The arrow(s) represent the time points at which the chewing gum was administered for 5 minutes, and the number of arrow(s) indicates the number.

[0116] Example 4 best exemplifies the contribution of an additive approach, where an immediate local increase in nitric oxide is followed by the intermittent addition of another gum to increase both local and systemic nitric oxide levels. The unique and novel aspect of this example is the extended duration of nitric oxide bioavailability to 5 hours at a cumulatively smaller dose when administered over time compared to the single high dose shown in Example 3.

[0117] Example 5 Biotransformation of nitrate chewing gum increases oral and nasal exhaled nitric oxide

[0118] The methods, experimental protocols, and measurements of nitric oxide gas are described above for the plant-derived chewing gum and potassium-ascorbic acid-zinc chewing gum.

[0119] Oral nitric oxide gas was measured using a NObreath® (NOB; Bedfont, Kent, UK) modified with a nosepiece to capture exhaled air from one nostril while the other nostril was pressed closed during a 10-second exhalation.

[0120] Nitric oxide gas was measured using NOB. These devices were evaluated based on the guidelines of the UK National Institute for Health and Care Excellence (NICE). NOB is designed to measure exhaled nitric oxide (FeNO) levels in human breath. A normal FeNO test value is less than 25 parts per billion (ppb) in adults. Levels above this value were used to assess pulmonary inflammatory responses, where inducible nitric oxide synthase is activated in immune cells in response to an inflammatory response. NOB is used to assess nitric oxide gas generated locally in the mouth.

[0121] Participants were pre-screened to exclude asthmatics, those with respiratory infections, and healthy individuals with levels around 35 ppb due to a diet of nitrate-rich beets or leafy green vegetables, or excessive nasal breathing within 6 to 18 hours before the test. All healthy subjects with consistent exhaled breath levels of 10 to 30 ppb, influenced by exercise and diet, particularly nitrate-rich plant-based foods and diet, and excessive nasal breathing, were included.

[0122] A total of four to six subjects were assessed two to four times over a two-week period. Values ​​are from one individual and are representative of four to six other subjects. Participants were seated and held the monitor upright at all times. Participants inhaled to full lung capacity, then exhaled through the mouthpiece, holding the visual indicator marker in the center of the band to ensure proper measurement. Exhalation time was approximately 10 seconds. All participants were asked to repeat the measurement three times to verify the device's reproducibility. Baseline values ​​ranging from 10 to 35 ppb reflect nitric oxide sourced from the lungs and respiratory system before chewing gum.

[0123] Example 5 (FIG. 6) shows 10-second oral (AE) and nasal (BF) exhalations at four time points: 0 minutes (before chewing any gum) and 5, 10, and 30 minutes after chewing one piece of potassium-ascorbic acid-zinc gum, starting at time 0.

[0124] Example 5 demonstrates an immediate increase in the formation of nitric oxide gas both orally and intranasally. Figure 6 depicts three healthy subjects, with oral and nasal nitric oxide measurements taken at four time points, with results for subjects 1, 2, and 3 shown as A and B, C and D, and E and F, respectively. Baseline levels of exhaled nitric oxide gas were found to be 1-2 times higher than oral, and the magnitude of exhaled nitric oxide gas was consistently 2-4 times higher in absolute terms via the nasal route compared to oral. Furthermore, the "residence time" of nitric oxide in the nasal cavity appears to persist longer than via the oral route.

[0125] Example 5 underestimates the importance of nitric oxide chewing gum in improving local intranasal nitric oxide, especially in subjects with low nasal and sinus nitric oxide production, because most nitric oxide from the upper airway is produced in the nasal cavity. Lundberg et al. (1996) remind us that recent studies have shown that inhalation of NO (typically from an external device) at concentrations as low as 100 ppb significantly reduces pulmonary vascular resistance in patients with pulmonary hypertension, suggesting that nasally derived nitric oxide is physiologically important in the lung and therefore acts as an airborne, or "aercrine," agent. Nitric oxide also has bacteriostatic and antiviral properties, and nasally derived nitric oxide may participate in the mucosal line of defense against infection.

[0126] Thus, the present invention is a new and unique composition and method for providing nitric oxide to the nasal cavity, thereby supporting a source of antimicrobial nitric oxide to the nasopharynx and respiratory tract, to combat infection and manage pulmonary hypertension, as illustrated in FIG. 6.

[0127] Example 6 Nitrate chewing gum shifts the oral microbiome toward high NO bioavailability Figure 7 shows the relative abundance of bacterial species before and after chewing the potassium nitrate gum. Whole-genome metagenomic sequencing or shotgun sequencing was used to examine all DNA present in the microbiome samples. Saliva samples were collected from the subjects, and the MyFitStrip® live bacterial nitric oxide test detected a low nitrite-to-nitrate ratio, and the MyFitStrips® oral hygiene strips detected a low salivary pH (<7). Saliva was collected according to methods provided by Bristle Oral Health Labs. After collection, subjects chewed the potassium-ascorbic acid gum twice for 5-10 minutes over a 3-4 hour period. At that time, the nitrite-to-nitrate ratio and pH were measured, and a second saliva sample was collected for shotgun sequencing analysis according to Bristle Labs' procedures.

[0128] The abundance of bacterial genera capable of nitrate reduction diverged, as summarized in Figure 7. In the test group, after chewing nitric oxide-infused chewing gum regularly for 5 minutes three times over a 3-hour period, the abundance of Rothia and Neisseria increased, while the abundance of other species, Prevotella, Veillonella, and Treponema, decreased.

[0129] Besides their high nitrate-reducing capacity, the genera Rothia and Neisseria are also known to be associated with periodontal health. In particular, Neisseria species from healthy volunteers have been shown to have a highly efficient nitrate / nitrite-reducing metabolism.

[0130] Evidence continues to show a correlation between subgingival inflammation and cardiovascular health, thus reiterating the link between the diseases. One possible mechanism is alterations in the enterosalveolar nitrate metabolic pathway, which impacts systemically available nitric oxide and directly influences cardiometabolic outcomes. For example, Treponema colonization may contribute to nitrate reduction and nitrite depletion, thus disrupting the oral nitrate microflora. Further evidence suggests that health-related taxa, such as Rothia and Neisseria, were detected in healthy controls, whereas Treponema, Porphyromonas, and Tannerella predominated in periodontitis.

[0131] High abundance of Rothia and Neisseria and low abundance of Prevotella and Veillonella correlated with oral microbial communities associated with indicators of nitric oxide homeostasis and vascular health.

[0132] The nitrate-reducing bacteria Rothia and Neisseria are consistently found at higher levels in individuals without oral disease compared with those with caries, periodontitis, and halitosis, and are increased by consuming a nitrate-rich diet. In contrast, bacteria typically associated with disease, such as Veillonella, commonly found in severe caries, and Prevotella, associated with periodontal disease and halitosis, are reduced in the presence of nitrate-rich foods and diets. Thus, nitrate is an ecological factor that stimulates health-related species and functions. Figure 7 demonstrates that chewing gum replaces nitrate-rich foods and diets that act through the gut-salivary loop at relatively high ADI or dietary nitrate concentrations, while chewing gum directly delivers prebiotic nitrate independently of the gut-salivary loop to enhance the health-related microbiome while simultaneously reducing disease-related bacteria, including Helicobacter and Candida, among other nitric oxide-sensitive infections.

[0133] Although the present invention has been described in terms of specific embodiments, it should be understood that the present invention is not so limited and therefore includes the restoration of nitric oxide deficiency in smokers, ex-smokers, and passive smokers, particularly in elderly ex-smokers, where nitric oxide deficiency is most pronounced and is associated with salivary acidity and impaired immune function.Similarly, certain B vitamins, specifically thiamine mononitrate, nicotinamide riboside, and N-acylcysteine ​​(NAC), can be added to modify the composition to enhance biological activity.In addition, 50 mg of caffeine exerts a synergistic effect on enhancing nitric oxide-mediated activity.

[0134] The embodiments are described herein as examples, and numerous modifications, variations, and other embodiments that remain within the scope of this disclosure can be employed to promote vascular health, reduce bacterial and infectious agents in the oral cavity that contribute to oral-nasal diseases, including, but not limited to, viral and fungal infections, and increase the bioavailability of NO associated with restoring NO deficiency caused by chronic diseases and exposure to irritants, including smoking and unhealthy diets.

[0135] References: 1. Lundberg, JO, Carlstrom, M. & Weitzberg, E. Metabolic effects of dietary nitrate in health and disease. Cell Metab. 28, 9-22 (2018). 2. V. Kapil, RS, Khambata, DA, Jones A. et al. Nitrate-Nitrite-Nitric Oxide Pathway Pharmacol. Rev. 72, 692-766 (2020). 3. Hezel, MP & Weitzberg, E. The oral microbiome and nitric oxide homoeostasis. Oral Dis. 21, 7-16 (2015). 4. Gee, L.C., Ahluwalia, A. Dietary Nitrate Lowers Blood Pressure: Epidemiological, Pre-clinical Experimental and Clinical Trial Evidence. Curr Hypertens Rep 18, 17- (2016). 5. Doel, JJ et al. Protective effect of salivary nitrate & microbial reductase against caries. Eur. J. Oral Sci. 112, 424. 6. Li et al. Salivary nitrate: an ecological factor in reducing oral acidity. Oral Microbiol Immunol. 22, 67-71 (2007). 7. Allaker, R.P., et al. Antimicrobial effect of acidified nitrite on periodontal bacteria. Oral Microbiol Immunol. 16, 253-256 (2001). 8. Sanchez, G.A. et al. Total salivary nitrates and nitrites in oral health and periodontal disease. Nitric Oxide 30, 36-31 (2014) 9. Mitsui, T. et al. Salivary nitrate may have antimicrobial effects on Desulfovibrio species. Biosci Biotechnol Biochem. 77, 2489 (2013). 10. Mazurel, D., Carda-Dieguez, M., Langenburg, T. et al. Nitrate and a nitrate-reducing Rothia aeria strain as potential prebiotic or synbiotic treatments for periodontitis. npj Biofilms Microbiomes 9, 40 (2023). 11. Rosier, B. T., Buetas, E., Moya-Gonzalvez, E. M., Artacho, A. & Mira, A. Nitrate as a potential prebiotic for the oral microbiome. Sci. Rep. 10, 12895 (2020). 12. Vanhatalo, A. et al. Nitrate-responsive oral microbiome modulates nitric oxide homeostasis and blood pressure in humans. Free Radic. Biol. Med. 124, 21-30 (2018). 13. Velmurugan S, et al. Dietary nitrate improves vascular function in patients with hypercholesterolemia: a randomized, double-blind, placebo-controlled study. Am J Clin Nutr. 103, 25-38. (2016). 14. Jockel-Schneider, Y. et al. Stimulation of the nitrate-nitrite-NO-metabolism by repeated lettuce juice consumption decreases gingival inflammation in periodontal recall patients: a randomized, double-blinded, placebo-controlled clinical trial. J. Clin. Periodontol. 43, 603-608 (2016). 15.Goh CE, et al. Association Between Nitrate-Reducing Oral Bacteria and Cardiometabolic Outcomes: Results From ORIGINS. J Am Heart Assoc. 3, :e013324 (2019).

Claims

1. 1. A chewing gum composition, comprising: a. Potassium nitrate and ascorbic acid, or b. potassium nitrate, ascorbic acid, and polyphenol extract, or c. potassium nitrate, ascorbic acid, and zinc, or d. potassium nitrate, ascorbic acid, zinc, and polyphenol extract, or e. Plant-derived inorganic nitrates and ascorbic acid, or f. Plant-derived inorganic nitrates, ascorbic acid, and zinc, or g. Plant-derived inorganic nitrates, ascorbic acid, zinc, and polyphenol extracts; 1. A chewing gum composition comprising a gum base in combination with

2. 10. The chewing gum composition of claim 1, comprising a sugar blend, a sugar alcohol blend, a sweetener, a gum base or bulk filler, a flavoring, a lubricant, a flow agent, or a combination thereof.

3. 3. The chewing gum composition of claim 2, wherein the gum base comprises chicle, gelatin, pectin, beeswax, paraffin wax, rosin, butyl rubber, polyvinyl acetate, microcrystalline cellulose, vegetable fiber, or a combination thereof.

4. further comprising a flavoring and / or sweetening agent; 3. The chewing gum composition of claim 2, wherein the flavoring agent comprises a natural or artificial flavor, or a combination thereof, and the sweetener comprises a sugar, a non-sugar, a sugar alcohol, a plant-derived sweetener including stevia, agave, coconut sugar, honey, monk fruit, and / or a combination thereof.

5. 10. The chewing gum composition of claim 1, wherein said potassium nitrate comprises 1-5% of said composition.

6. 6. The chewing gum composition of claim 5, wherein the potassium nitrate is derived from a plant source, the plant source being selected from the group consisting of leafy vegetables including celery, beets, arugula, Swiss chard, beetroot, and / or combinations thereof.

7. ascorbic acid, including plant-derived ascorbic acid, including but not limited to ascorbic acid from acerola; 10. The chewing gum composition of claim 1, wherein the composition comprises 0.5-10, 7-15% wt / wt ascorbic acid, with the ratio of nitrate to ascorbic acid being 2:1 to 1:

4.

8. 10. The chewing gum composition of claim 1, wherein the composition comprises 1-10% zinc, 4-12% L-arginine, 1-5% N-acetylcysteine, or a polyphenol fruit-derived extract at 2-10 mg / dose of activated anthocyanins (10:1 per gram of extract), or a combination thereof.

9. 10. The chewing gum composition of claim 1, wherein the composition comprises 0.5-1% zinc, 6-8% L-arginine, 3-7% N-acetylcysteine, or a polyphenol fruit-derived extract at 2-10 mg / dose of activated anthocyanins (10:1 per gram of extract), or a combination thereof.

10. 10. The chewing gum composition of claim 1, wherein the amount of nitrate per chewing gum composition is 0.15 to 2.5 mM.

11. the increase in the subject's oral-nasal nitric oxide levels and systemic bioavailability of nitric oxide is maintained by said subject chewing one or more additional chewing gum compositions intermittently over a 12-hour period for 3-7 minutes per gum composition per period; 10. The chewing gum composition of claim 1, wherein the subject chews the gum composition for 3 to 7 minutes and then discards it, and the subject chews a second and a third gum composition separately for an additional 3 to 7 minutes, the chewing periods for each gum composition being separated by 2 to 4 hours.

12. 1. A method for increasing nitric oxide levels in the oral and / or nasal cavity of a subject, said method comprising: a. Potassium nitrate and ascorbic acid, or b. potassium nitrate, ascorbic acid, and polyphenol extract, or c. potassium nitrate, ascorbic acid, and zinc, or d. potassium nitrate, ascorbic acid, zinc, and polyphenol extract, or e. Plant-derived inorganic nitrates and ascorbic acid, or f. Plant-derived inorganic nitrates, ascorbic acid, and zinc, or g. Plant-derived inorganic nitrates, ascorbic acid, zinc, and polyphenol extracts; a subject chewing a gum composition comprising a gum base in combination with A method comprising the step of monitoring pH, nitrite, nitrate and nitric oxide levels, including the use of saliva test strips.

13. the increase in the subject's oral-nasal nitric oxide levels and systemic bioavailability of nitric oxide is maintained by said subject chewing one or more additional chewing gum compositions intermittently over a 12-hour period for 3-7 minutes per gum composition per period; 13. The method of claim 12, wherein the subject chews the gum composition for 3 to 7 minutes and then discards it, and the subject chews a second and a third gum composition separately for an additional 3 to 7 minutes, the chewing periods for each gum composition being separated by 2 to 4 hours.

14. 13. The method of claim 12, wherein increasing nitric oxide levels in the oral and / or nasal passages of the subject comprises increasing the relative abundance of beneficial microflora, said beneficial microflora including, but not limited to, one or more of Rothia aireria, Rothia mucilaginosa, Neisseria flavescens, Neisseria subflava, and Haemophilus parainfluenza.

15. 13. The method of claim 12, wherein increasing nitric oxide levels in the oral and / or nasal cavity of the subject comprises reducing the relative abundance of harmful microflora, the harmful microflora including, but not limited to, one or more of Tannerella forsythia, Treponema soklanskii, Fusobacterium periodonticum, Porphyromonas gingivalis, Streptococcus constellatus, Fusobacterium nucleatum, Parvimonas micula, Prevotella melaninogenica, Prevotella histicola, Candida albicans, and diseases associated therewith.

16. 13. The method of claim 12, wherein the improved oral health comprises a reduction in plaque, dental cavities, caries, periodontitis, halitosis, bacterial infections, fungal infections, viral infections, nasopharyngitis, and related sinus and oral infections.

17. 13. The method of claim 12, wherein the salivary nitrate and nitrite concentrations during chewing and 5 minutes after fasting are at least 100 mg / L or greater and 10 mg / L or greater, respectively, as assessed by nitrate and nitrite saliva test strips.

18. 13. The method of claim 12, wherein chewing the gum composition results in an increase in saliva pH to 6.5-8.0, and said increase in saliva pH results in a decrease in tooth demineralization, oral acidosis, and / or acid-producing bacteria.

19. 10. The chewing gum composition of claim 1, consisting of potassium nitrate, ascorbic acid, zinc, gum base, sorbitol, maltitol, xylitol, isomaltose, natural flavors, potassium nitrate, magnesium stearate, acerola, sucralose, silicon dioxide, and zinc citrate.

20. 10. The chewing gum composition of claim 1, comprising plant-based acerola, gum base, sorbitol, maltitol, xylitol, isomaltose, celery extract, natural flavors, magnesium stearate, acerola, sucralose or stevia, silica, polyphenols, and glycerin.

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