Compositions and methods for improving nitric oxide levels in intraoral, nasal and / or nasopharyngeal region

Abstract

The present disclosure relates to improving oral-nasal health by increasing nitric oxide levels in the oral, intraoral and nasal cavities using chewing gum compositions formulated with novel prebiotic nitrates, and includes optional steps of monitoring pH, nitrate and nitrite using saliva test paper. The novel chewing gum compositions and methods of the present invention improve nitric oxide bioavailability, thereby increasing antimicrobial nitric oxide levels, being beneficial to reducing dental caries, periodontitis and nasopharyngitis, and reducing the propagation of aerosolized viral particles that are highly sensitive to nitric oxide, including SARS-CoV.

Description

Technical Field

[0001] Embodiments of the present disclosure generally relate to a novel nitrate-based chewing gum composition for prolonging the exposure of prebiotic nitrate in the oral cavity of a subject, which composition results in a rapid and local increase in nitric oxide gas in the oral cavity and nasopharynx. The embodiments are also helpful in increasing systemic nitric oxide bioavailability. Background Art

[0002] Like many parts of the body, the oral cavity is filled with bacteria, most of which are harmless. Normally, the body's natural defenses and good oral hygiene (such as brushing teeth and using dental floss daily) can control these bacteria. However, without proper oral hygiene, bacteria can reach levels that may cause oral infections, such as tooth decay and gum disease. In addition, lifestyle activities, especially smoking or taking medications (such as decongestants, antihistamines, painkillers, antibiotics, and diuretics) or current oral hygiene habits (such as mouthwash) can reduce the saliva components and secretion volume and alter the oral microbiome, thereby endangering oral health. Saliva is important because it can wash away food, neutralize acids produced by bacteria in the oral cavity, and help protect the oral cavity from microbial invasion or overgrowth that may cause disease.

[0003] Oral health is crucial for overall health and quality of life. Oral health is a state in which the oral cavity and face are free from pain, oral cancer and laryngeal cancer, oral infections and ulcers, periodontal (gum) diseases, tooth decay, tooth loss, and other diseases or disorders that limit an individual's ability in biting, chewing, smiling, speaking, and social and mental health. The most common oral diseases are dental caries, periodontal (gum) diseases, oral cancer, oral infectious diseases, traumatic injuries, and genetic lesions.

[0004] Dental caries is widely regarded as a serious threat to oral health. Worldwide, 60 - 90% of school-age children and nearly 100% of adults have dental caries, often resulting in pain and discomfort. In the United States, tooth decay (dental caries) is one of the most common chronic diseases in childhood. Untreated tooth decay can cause pain and infection, which may cause problems in eating, speaking, playing, and learning. In the United States, approximately one-fifth (20%) of children aged 5 to 11 have at least one untreated tooth decay; approximately one-seventh (13%) of adolescents aged 12 to 19 have at least one untreated tooth decay. The percentage (25%) of children and adolescents aged 5 to 19 from low-income families with untreated tooth decay is twice that of children (11%) from high-income families. Oral health is threatened throughout life. Nearly one-third of adults in the United States have untreated tooth decay.

[0005] Periodontitis is a group of inflammatory diseases that affect the periodontal tissues (i.e., the tissues that surround and support the teeth). Periodontitis involves the gradual loss of the alveolar bone around the teeth and, if left untreated, can lead to tooth loosening and subsequent tooth loss. Periodontitis is caused by microorganisms that attach and grow on the tooth surface and an overly aggressive immune response against these microorganisms. Periodontitis presents as painful, red, swollen gums with a large amount of dental plaque. Symptoms may include redness or bleeding of the gums when brushing teeth, using dental floss, or biting into a hard food (such as an apple); repeated swelling of the gums; bad breath and a persistent metallic taste in the mouth; recession of the gums leading to a noticeable lengthening of the teeth; deep periodontal pockets between the teeth and the gums (periodontal pockets are sites where the attachment is gradually destroyed by collagenase); and tooth loosening. Periodontitis also shows an impact outside the oral cavity. For example, periodontitis is associated with an increase in inflammation, as indicated by increased levels of C-reactive protein and interleukin-6. In addition, it has been shown that periodontitis increases the risk of many other diseases, including but not limited to stroke, myocardial infarction, atherosclerosis, diabetes, and preterm birth. 15-20% of middle-aged people (35-44 years old) have severe periodontal (gum) disease that can lead to tooth loss. In addition, dental caries and periodontal disease are the main causes of tooth loss. The complete loss of natural teeth is common, especially affecting the elderly. Globally, approximately 30% of people aged 65-74 years do not have natural teeth.

[0006] Oral cancer is another problem for oral health. In most countries, the incidence of oral cancer is 1 to 10 cases per 100,000 people. The prevalence of oral cancer is relatively higher in men, the elderly, and people with low education and low income. Tobacco and alcohol are the main causative factors and lead to the disruption of the microbiome on the tongue, specifically, nitrate-reducing bacteria. Thus, 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 germs from another part of the body (such as the mouth) travel through the bloodstream and attach to damaged areas of the heart; cardiovascular diseases, studies have shown that heart disease, arterial blockages, and stroke may be associated with inflammation and infections that can be caused by oral bacteria; pregnancy and childbirth, periodontitis has been linked to premature birth and low birth weight; diabetes, since diabetes reduces the body's resistance to infection, the gums are at risk, and gum disease appears to be more frequent and severe in people with diabetes (studies have shown that people with gum disease have more difficulty controlling their blood sugar levels); HIV / AIDS, oral problems (such as painful mucosal lesions) are common in people with HIV / AIDS, and in addition, almost half (40 - 50%) of HIV-positive patients have oral fungal, bacterial, or viral infections; osteoporosis, since this condition causes bones to become weak and brittle, it may be associated with periodontal bone loss and tooth loss; Alzheimer's disease, tooth loss before the age of 35 may be a risk factor for Alzheimer's disease; other conditions that may be related to oral health include Sjogren's syndrome, an immune system disorder that causes dry mouth and eating disorders. Due to the peripheral anatomical location and the frequent exposure of oral tissues to the external environment, the asymptomatic transmission of SARS-CoV-2 remains a concern. That is, saliva plays a major role in the asymptomatic transmission of SARS-CoV-2, and thus, saliva is actively involved in the transmission of SARS-CoV-2.

[0008] By addressing common risk factors, the burden of both oral diseases and other chronic diseases can be reduced simultaneously. This includes: reducing sugar intake and maintaining a balanced nutritional intake to prevent tooth decay and premature tooth loss; consuming fruits and vegetables that can prevent oral cancer; stopping smoking and reducing alcohol consumption to lower the risk of oral cancer, periodontal disease, and tooth loss; using protective sports and motor vehicle equipment to reduce the risk of facial injury; a safe physical environment, and most importantly, ensuring proper oral hygiene. Tooth decay can be prevented by maintaining a constant low level of fluoride in the mouth, however, fluoride and chlorhexidine mouthwashes may also reduce the healthy microbiome in the mouth that contributes to antibacterial nitric oxide.

[0009] Similar to the oral cavity, the nasopharynx is a major site of respiratory pathogen colonization and forms the entrance to the respiratory tract. The nasopharynx typically harbors bacterial and viral pathogens that cause middle ear and sinus infections. The most common condition affecting the nasopharynx is nasopharyngitis, also known as the common cold. This swelling of the nose and throat is sometimes referred to as an upper respiratory tract infection, or rhinitis. In nasopharyngitis, a virus, usually a rhinovirus, infects the nasopharynx. The nose is the main entry site and target for SARS-CoV-2. Due to the peripheral anatomical location and the frequent exposure of oral and nasopharyngeal tissues to the external environment, asymptomatic transmission of SARS-CoV-2 remains a concern. Thus, the oro-nasopharynx plays a key role in the asymptomatic transmission of SARS-CoV-2, and as such, saliva and aerosolized water-mucus droplets exhaled from the nose are involved in the transmission of SARS-CoV-2.

[0010] Salivary Nitrate and Oral Health

[0011] Nitrite is derived from nitrate by nitrate-reducing bacteria on the surface of the tongue and is thought to have cytocidal and cytostatic effects on common oral pathogens involved in dental caries and periodontal diseases, especially when acidified. Thus, an increase in nitrate secretion and subsequent increase in salivary nitrite may contribute to the overall protective effect against infectious conditions affecting hard and soft oral tissues. The salivary glands are known to respond to periodontitis by enhancing the protective potential of saliva. Thus, the increase in salivary nitrate-nitrite concentration in patients with periodontal disease may be due to an increase in nitrate secretion as a response of the salivary glands to the inflammatory process. According to this hypothesis, an increase in salivary nitrate and nitrite concentrations has been reported in patients with oral candidiasis.

[0012] In a study of 209 children, high salivary nitrate and high nitrate reductase capacity in the oral cavity were found to be protective against dental caries. Salivary nitrate and nitrite levels, the numbers of Streptococcus mutans and Lactobacillus spp., and caries experience were recorded. In patients with high salivary nitrate, significantly lower numbers of dental caries and of Streptococcus mutans and Lactobacillus spp. were found compared with control subjects. Production of nitrite from salivary nitrate by commensal nitrate-reducing bacteria may limit the growth of cariogenic bacteria because of the production of antibacterial nitrogen oxides, including nitric oxide. (J.J. Doel, M.P. Hector, C.V. Amirtham, L.A. Al-Anzan, N. Benjamin, R.P. Allaker, Protective effect of salivary nitrate and microbial reductase activity against caries, Eur. J. Oral Sci. 112 (2004) 424–428.)

[0013] Other studies have shown that the antimicrobial agent nitric oxide is formed in the oral cavity, and its concentration is directly related to salivary nitrite, which in turn is partly related to the intake of nitrate in the diet. Here, the researchers showed that under acidic conditions, nitrite has an inhibitory effect on Streptococcus mutans, Lactobacillus casei, and Actinomyces naeslundii through the biotransformation of nitrate and the production of NO. Although the growth of Streptococcus mutans is inhibited by a more acidic pH, the addition of nitrite caused a significant, further dose-dependent reduction in the number of bacteria after 24 hours of exposure. Similar effects were observed in Actinomyces naeslundii and Lactobacillus casei. The ability of these bacteria to recover from nitrite exposure was also significantly affected by the nitrite concentration. At acidic levels below pH 7, low concentrations of nitrite (0.2 mM) effectively killed Streptococcus mutans completely, with similar effects on the other test microorganisms. These results suggest that nitrite in saliva blocks cariogenic bacteria. (L.S.Silva Mendez, R.P.Allaker, J.M.Hardie, N.Benjamin, Antimicrobial effect of acidified nitrite on cariogenic bacteria, Oral Microbiol.Immunol. 14 (1999) 391–392, C.E.Radcliffe, R.Lamb, A.S.Blinkhorn, D.B.Drucker, Effect of sodium nitrite and ascorbic acid on the growth and acid production of Streptococcus mutans, J.Dent. 31 (2003) 367–370.)

[0014] Consistent with other reports, nitrite derived from nitrate inhibited the growth of periodontal disease pathogens Fusobacterium nucleatum, Eikenella corrodens, and Porphyromonas gingivalis under acidic conditions (P. Allaker, L. S. Silva Mendez, J. M. Hardie, N. Benjamin, Antimicrobial effect of acidified nitrite on periodontal bacteria, Oral Microbiol. Immunol. 16 (2001) 253–256.). Sanchez et al. (2014) reported that the increase in nitrate may contribute to the overall protective effect against periodontal-related pathogens affecting hard and soft oral tissues. They argued that the increase in salivary nitrate-nitrite concentration in periodontal disease patients was due to the increased secretion of nitrate as a response of salivary glands to the inflammatory process. According to this hypothesis, increased salivary nitrate and nitrite concentrations have been reported in patients with oral candidiasis. In both cases, it was shown that the elevated nitrate levels in the oral cavity in the late stage of the disease were an immune response to this infection. (Total salivary nitrates and nitrites in oral health and periodontal disease. Sánchez GA, Miozza VA, Delgado A, Busch L. Nitric Oxide. 2014 Jan 30;36:31-5). Many other studies have highlighted the benefits of salivary nitrate levels: Li et al. (2007) suggested that elevated salivary nitrate levels reduced oral acidity, thus preventing tooth decay (Oral Microbiol Immunol. 2007 Feb;22(1):67-71. Salivary nitrate--an ecological factor in reducing oral acidity. Li H1, Thompson I, Carter P, Whiteley A, Bailey M, Leifert C, Killham K.).Similarly, Radcliffe (2002) suggested that exogenous nitrite acidified by the metabolites of acidogenic bacteria in the oral cavity would be 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 acidifiable nitrite derived from nitrate, cariogenic bacteria such as Streptococcus mutans and various periodontal bacteria (Fusobacterium and Porphyromonas gingivalis), which are considered pathogens associated with chronic periodontitis and sulfate-reducing bacteria (Desulfovibrio spp.) related to the smell of sulfate, are directly inhibited by nitrite and nitrate. Here, the growth was inhibited by 0.2 mM nitrate, which is consistent with other observations. Salivary nitrate and nitrite may also have antibacterial effects on Desulfovibrio spp. (Mitsui T, Fujihara M, Harasawa R. Biosci Biotechnol Biochem. 2013;77(12):2489).

[0015] Consuming nitrate-rich plants such as green leafy vegetables and beetroot increases the nitrate concentration in the body. Certain oral bacteria convert nitrate into nitrite, and the human body can effectively convert nitrite into nitric oxide through certain enzymatic and non-enzymatic processes, resulting in beneficial effects, including reducing blood pressure and inhibiting the spread of pathogenic microorganisms.

[0016] The dietary route or the nitrate-nitrite-nitric oxide dietary route independent of L-arginine, known as the enterosalivary nitrate pathway, is when nitrate in the diet is swallowed and absorbed in the proximal gastrointestinal tract, absorbed through the circulatory system and concentrated in the salivary glands, secreted into the oral cavity in the salivary glands, and then reduced to nitrite by nitrate-reducing bacteria 30 to 90 minutes later; the nitrite is then swallowed and further reduced to nitric oxide and related intermediates in the stomach, bloodstream, and tissues. The oral nitrate-reducing microbiome in the oral-nasopharyngeal cavity and the enterosalivary nitrate pathway for oral and upper respiratory tract health

[0017] As previously mentioned, certain oral bacteria, particularly nitrate-reducing bacteria, convert nitrate to nitrite, and the human body can effectively convert nitrite to nitric oxide through multiple enzymatic and non-enzymatic processes. However, this depends on the enterosalivary nitrate pathway.

[0018] Different research groups have focused on the systemic benefits of dietary nitrate, aiming to alter the oral microbiome, which is typically associated with the dietary sources ingested. Therefore, this depends on the enterosalivary nitrate pathway rather than increasing the "residence time" of nitrate in the oral cavity and evaluating nitric oxide gas itself in the mouth. Currently, there is no teaching or available resource that can directly administer nitrate-based formulations to increase nitrate reduction and nitrite reduction activities, thereby generating nitric oxide in the oral cavity and nasopharynx without relying on the enterosalivary nitrate pathway, resulting in an immediate enhancement of nitrate in the oral cavity and nasal cavity at doses far below the acceptable daily intake (ADI) and an extended duration of local and systemic nitric oxide bioavailability for several hours.

[0019] Current research has focused on nitrate reduction capabilities in the oral cavity but at very high levels and exceeding the ADI involving 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. In this study, multiple high doses of nitrate far above the ADI level had to be ingested.

[0020] Similarly, other studies have also shown beneficial effects of nitrate in dietary nitrate sources on altering the oral microbiome. For example, Velmurugan et al. (2016) showed an increase in Rothia mucilaginosa and Neisseria flavescens after 6 weeks of daily consumption of beetroot juice rich in nitrate (372 mg of nitrate-reducing substances per serving). Vanhatalo et al. (2018) reported changes in the oral microbiome detected in saliva after nitrate supplement intake, showing an increase in nitrate-reducing bacteria (Rothia and Neisseria) and a decrease in disease-associated Prevotella and Veillonella. Jockel-Schneider et al. (2016) reported a reduction in gingival inflammation in patients after 14 days of nitrate intake.

[0021] Mira and Rosier et al. (2018, 2022) proposed using nitrate to reduce or prevent oral dysbiosis and increase oral ecological balance by altering the bacterial composition of the oral biofilm 24 hours before the onset of biofilm-mediated oral diseases, thereby increasing the nitrate concentration in oral saliva. In addition, they claim that topical or oral administration, and in the latter case, depends on the enterosalivary circuit, and in either case, a composition is recommended to increase the nitrate concentration in oral saliva, which is a nitrate-rich vegetable extract (beetroot extract), antioxidant, and / or nitrate reductase cofactor (molybdenum, its salts, or molybdenum-rich vegetable extract). Similar to many others, the ingested preparation can indeed increase salivary nitrate levels after a circuitous route through the oral cavity to the stomach and back to the salivary glands and oral cavity via the circulatory system. Nevertheless, Rosier and Mira (2022) also claim that a shift in the nitrate-reducing microbiome is achieved with a nitrate dose of 3 μg (0.1 mM) by topical application of toothpaste, mouthwash, oral gel, food extract, and chewing gum, which is a hypothetical construct that has not been put into practice and has not shown an immediate or sustained increase in nitric oxide production in the oral cavity or nasopharynx of human subjects.

[0022] Rosier et al. (2022), prebiotics and prebiotic therapies to reduce oral dysbiosis and promote ecological balance, WO2021122741A2, reported the nitrate levels in saliva collected from healthy donors in the morning under fasting conditions. At 0 hours after ingestion of a nitrate-rich supplement (220 mg nitrate in 200 ml water), two peaks were produced, thus illustrating a direct or immediate increase in nitrate due to topical supplement contact (0.5 hours), and an indirect increase due to salivary gland activity (2.5 hours) in recovering nitrate from plasma. However, they failed to detect nitric oxide in the oral cavity or nasopharynx and the concentration to achieve such a transient biphasic response below the ADI level. In addition, they failed to distinguish that chewing gum equivalent to other local nitrate sources (provided as toothpaste or mouthwash) would result in different outcomes. Moreover, although Rosier et al. claim that chewing gum, toothpaste, and mouthwash are equally effective in increasing local and systemic levels to achieve antibacterial effects in the oral cavity and nasal cavity, they failed to prove, put into practice, or provide any evidence to confirm such a claim.

[0023] Studies such as those of Rosier et al. (2020, WO2021122741A2) (including Bryan (2022, WO2014182632A1)) provided solutions formulated with nitrates, in which specific nitrate-reducing bacteria were added in the presence of nitrates. Neither Rosier nor Bryan provided any evidence or advice related to the health effects of increased nitric oxide production in the oro-nasal cavity. Both Rosier and Bryan relied on the enterosalivary pathway without considering that this pathway could be circumvented by using specially formulated chewing gum.

[0024] As pointed out by Lundberg et al. (1999) in multiple papers, in the nasopharynx (including the paranasal sinuses and nasal cavity), when exposed to bacterial and viral infections, the innate immune response produces nitric oxide. Nitric oxide has antimicrobial activity against a variety of bacteria, parasites, fungi, and viruses.

[0025] Nitric oxide delivered in the form of an exogenous gas can easily enter the pulmonary environment and can be used to optimize the treatment of uncontrolled pulmonary diseases, which has specific effects on reducing bacterial load, reducing inflammation, and improving clinical symptoms, especially when it is well understood that the nasal nitric oxide levels are significantly reduced in patients with usually mucus-filled paranasal sinuses and obstructed sinus ostia, Kartagener syndrome, cystic fibrosis, and acute sinusitis, as reviewed by Lundberg et al. (1999).

[0026] In children with Kartagener syndrome (including sinusitis, bronchiectasis, and situs inversus), their nasal nitric oxide levels are extremely low compared to age-matched healthy controls (Lundberg et al., 1999). Similarly, smoking is associated with a reduction in exhaled nitric oxide. The largest decline in exhaled nitric oxide related to smoking is in elderly subjects. This indicates that smoking is associated with an age-related decline in exhaled nitric oxide.

[0027] The nasal nitric oxide levels in patients with cystic fibrosis are also very low. Baraldi et al. measured the nasal nitric oxide in a group of children with acute sinusitis and found low nasal NO levels, and chronic sinusitis was associated with a reduction in nasal NO levels by more than 50%. Generally, the nasal nitric oxide levels in subjects with sinus diseases are lower.

[0028] Av-Gay et al. (2013) (US20200276229A1) claimed that some respiratory and physiological conditions can be treated by inhaling gaseous nitric oxide delivered from a mechanical device or a nitric oxide tank or by an external spray device that delivers acidified nitrite.

[0029] The use of nitric oxide by inhalation can prevent, reverse or limit the development of conditions such as acute pulmonary vasoconstriction, traumatic injury, inhalational or aspiration injury, pulmonary fat embolism, acidosis, pulmonary inflammation, adult respiratory distress syndrome, acute pulmonary edema, acute mountain sickness, after cardiac surgery, acute pulmonary hypertension, persistent pulmonary hypertension of the newborn, perinatal aspiration syndrome, hyaline 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.

[0030] As noted by Av-Gay et al. (2013), the half-life of nitric oxide in the body is less than a few seconds and its radius of action is about 200 micrometers from its source location, beyond which it will be inactivated by nitrosation with the sulfhydryl group of cellular thiols or by the heme moiety of hemoglobin to form methemoglobin (MetHb). MetHb reductase reduces nitric oxide in serum to nitrate. Nitrate has been identified as the major nitric oxide metabolite excreted in urine, accounting for more than 70% of the inhaled nitric oxide dose. Nitrate is cleared from plasma by the kidneys at a rate close to the glomerular filtration rate. The MetHb level in the blood of healthy individuals is usually less than 2%. Thus, potential side effects of high-dose nitric oxide therapy include the formation of nitric oxide with hemoglobin and MetHb, which may lead to reduced oxygen transport, and the ability of NO as a nitrosating agent for proteins and other cellular components to decline. The formation of MetHb and its increased levels have been observed in previous studies of inhaled gaseous nitric oxide in healthy individuals, in which it has been reported that inhalation of 128 ppm of gaseous nitric oxide for 3 hours and 512 ppm of gaseous nitric oxide for 55 minutes caused the MetHb level to exceed the safety threshold of 5% [Borgese N. et al., J. Clin. Invest., 1987, 80, 1296 - 1302; Young J.D. et al., Intensive Care Med., 1994, 20, 581 - 4 and Young J. et al., Brit. J. Anaesthesia, 1996, 76, 652 - 656].

[0031] Nitric oxide inhaled from external devices or tanks, although proven beneficial under healthcare monitoring, also has its risks, especially if considered for daily use with little to no professional healthcare supervision. Lundberg (an authority on the biology of respiratory nitric oxide) raised concerns about the potential use of nitric oxide as a therapeutic agent in various situations. Lundberg et al. (Eur. Respir J, 1994), Primarily nasal origin of exhaled nitric oxide and absence in Kartagener’s syndrome, stated that almost all of the nitric oxide in exhaled air originates from the upper airway, and in exhaled air through the mouth and nose, the same individuals exhibit rather high nasal nitric oxide concentrations. Thus, most of the nitric oxide from the upper airway may be produced in the nasal cavity, as the level of nitric oxide exhaled during nasal breathing is also higher than that during oral breathing in all subjects, including healthy controls. Additionally, direct nasal sampling yielded very high nitric oxide values, and the nitric oxide from the upper airway will flow with the airflow of each inhalation, thus continuously flushing the lower airway. As cited by Lundberg et al. (1994), inhaled nitric oxide at concentrations as low as 100 ppb significantly reduced the pulmonary vascular resistance in patients with pulmonary hypertension, indicating that nasal-derived nitric oxide may have important physiological significance in the lungs and thus acts as an airborne or “air-secreted” factor. The authors went on to note that the nitric oxide gas produced within the nose is bacteriostatic and exhibits antiviral properties and thus participates in the non-specific primary mucosal defense against infection.

[0032] In summary, despite the progress made in understanding nitric oxide and its effects on physiology, there remains a need for effective, targeted, long-term, and sustained delivery of nitric oxide compositions to improve oral and nasopharyngeal health. There is also a need for compositions and methods that bypass the enterosalivary nitrate circuit, enabling subjects to intermittently and rapidly increase nitric oxide in the mouth and nose. Additionally, there is 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 elevated nitric oxide levels by chewing nitrate-formulated compositions periodically or intermittently multiple times a day while remaining below the currently acceptable daily intake. Summary of the Invention

[0033] In one embodiment, the present disclosure relates to the use of a nitrate-based formulation delivered in a compressed chewing gum for extending prebiotic nitrate exposure in the oral cavity. Use of the chewing gum by a subject enables a rapid and local increase in nitric oxide gas in the oral cavity and nasopharynx. The chewing gum of the present disclosure also contributes to improved systemic nitric oxide bioavailability due to subsequent nitrate swallowing and bioconversion via the enterosalivary nitrate-nitrite-nitric oxide pathway. In one embodiment, the chewing gum composition is designed to enable a nitric oxide level below the acceptable daily intake of 3.7 milligrams per kilogram of body weight per day (mg / kg bw / day). The design and composition of the chewing gum allow for intermittent administration of the nitrate-based chewing gum throughout the day to immediately and locally increase oral-nasopharyngeal nitric oxide production and to extend the delayed systemic nitric oxide bioavailability. Compared to commonly available nitrate-based supplements such as mouthwashes, toothpastes, or ingestibles, the novel chewing gum composition claimed herein uniquely enables a subject to achieve optimal nitric oxide levels both locally and systemically.

[0034] In one embodiment, as verified and determined by salivary nitrite measurement, the chewing gum composition of the present disclosure uniquely contributes to a biphasic nitric oxide response: first by providing an immediate local increase in the oral cavity and nasal cavity; and second as a response after swallowing, where after absorption in the intestine, nitrate is concentrated in the salivary glands due to the enterosalivary circulation.

[0035] In one aspect, the present invention also utilizes information regarding nitric oxide levels and pH in an individual's oral cavity, specifically, using a rapid self-test saliva test strip to monitor pH and nitric oxide metabolites, nitrate and nitrite, in real time to adjust the dosage of the nitrate-formulated chewing gum.

[0036] Without wishing to be bound by the following theory, the chewing gum composition of the present disclosure is believed to act by shifting the oral microbiome to a higher abundance of nitric oxide-promoting bacteria, concomitantly increasing the ratio of salivary nitrite to nitrate (>1) and increasing the pH (>7). This shift in the oral microbiome corresponds to a reduction in bacteria associated with diseases related to pathogens that cause gum inflammation, tooth decay, bad breath, and cardiometabolic complications.

[0037] The present disclosure provides a new and unexpected method for improving oral hygiene and nasopharyngeal health by rapidly elevating nitric oxide levels in the oral cavity and / or nasopharynx, optionally in combination with means for monitoring the levels using an affordable, easy-to-use, rapid self-test method to monitor three key endpoints: pH, nitrate, and nitrite.

[0038] In one embodiment, the disclosure herein includes a novel chewing gum composition that rapidly increases nitric oxide levels in the oral-nasal cavity, thereby bypassing the L-arginine-independent nitrate-nitrite-nitric oxide dietary pathway. In contrast, currently available nitric oxide supplement products, as well as the prior art and publications, are entirely based on increasing nitric oxide levels through the L-arginine-independent nitrate-nitrite-nitric oxide dietary pathway.

[0039] In one embodiment, the present invention provides a method for optimizing oro-nasal health by combining or bundling a novel nitrate-formulated chewing gum with a saliva self-test to obtain real-time information on the level of salivary nitric oxide analytes in the oral cavity. This information can be utilized to maximize the antimicrobial activity of these analytes and thereby improve oral, nasopharyngeal, and sinus health by intermittently chewing the nitrate-formulated chewing gum. More specifically, a rapid saliva test that can be used casually and directly in the oral cavity for monitoring nitric oxide status enables regular monitoring and can be incorporated into a routine for improving oral and nasopharyngeal health.

[0040] In one embodiment, the present disclosure provides real-time feedback by detecting biomarkers of salivary analytes and nitric oxide, enabling an individual to make real-time adjustments to their oral hygiene regimen and optimize oral health. According to the present disclosure, users can rapidly and real-time assess the nitric oxide levels in their oral cavity and determine corrective procedures for improving and maintaining oral hygiene.

[0041] In one embodiment, the present invention provides a novel programmed method of oral hygiene that includes a nitrate-formulated chewing gum to prolong the exposure of nitrate in the oral cavity, thereby enhancing the microbiome or shifting it from an acidic or cariogenic microbial community to a nitrate-reducing microbiome.

[0042] By increasing the "residence time" of nitrate exposure, the oral microbiome that reduces nitrate, and acidic or low-pH oral regions (such as the inflamed gingival sulcus around teeth and teeth covered with acidic cariogenic biofilm), the bioconversion of nitrite to nitric oxide is promoted. Different from toothpaste and mouthwash, the chewing gum composition claimed herein provides unique advantages by prolonging the oral exposure to nitrate while stimulating saliva secretion to facilitate the downstream nitrate-nitrite-nitric oxide pathway and subsequently ingesting the nitrate provided by the chewing gum (which is not the case for toothpaste or mouthwash).

[0043] In one embodiment, the present invention includes the use of a nitric oxide-generating chewing gum in combination with within-day self-monitoring using a saliva test strip for promoting consumer compliance behavior. The colorimetric test strip results can be recorded and tracked on an electronic device such as a wearable device or a mobile phone, and reminders and updates regarding oral health compliance and adherence are provided to the user and the dentist or healthcare provider via wireless messaging capabilities.

[0044] In one embodiment, the present disclosure provides a systematic method for improving oral and sinus health. Nitric oxide reduces inflammation and pathogenic microorganisms. Thus, using the novel chewing gum compositions disclosed herein can reduce the incidence of health problems associated with oral health and the upper respiratory system, including but not limited to dental caries, tooth decay, gum disease, periodontitis, oral cancer, and viral infections or viruses hidden in saliva and sinuses, including nasopharyngitis. The chewing gum composition also reduces the viral load of nitric oxide-sensitive coronaviruses, including SARS CoV-1 and SARS-CoV-2.

[0045] These and other aspects, features, and advantages of the present disclosure will become apparent after reading the following detailed description of the disclosed embodiments and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The present disclosure is best understood from the following detailed description when read in conjunction with the accompanying drawings. It should be emphasized that, by convention, the various features of the drawings are not necessarily drawn to scale. Instead, the dimensions of the various features are arbitrarily enlarged or reduced for clarity. Throughout the specification and the drawings, like reference numerals represent like features.

[0047] Figure 1 A schematic summary of the comparison of the nitric oxide bioavailability of a test strip-based nitrate chewing gum (thick line) with an equivalent amount of nitrate from an ingested capsule or beverage (thin line) is provided, which is measured over time using a saliva test strip for nitrite (a surrogate marker for nitric oxide). The saliva test strip measures over time.

[0048] Figure 2 Graphs are provided showing intraoral nitric oxide gas formation (A), salivary nitrate (B), and salivary nitrite (C) at different times after chewing two (2) pieces of a plant-source nitrate-formulated chewing gum for 5 minutes and then discarding at 0 minutes and 45 minutes. The arrows indicate the time points of 5 minutes of chewing the gum, and the number of arrows indicates the number of pieces.

[0049] Figure 3Provided are graphs showing the formation of nitric oxide gas in the mouth (A), salivary nitrate (B), and salivary nitrite (C) at different times after chewing one (1) piece of potassium nitrate-formulated chewing gum for 5 minutes at 0 minutes and then discarding it. The arrows indicate the time points of 5 minutes of chewing gum administration, and the number of arrows indicates the number of pieces.

[0050] Figure 4 Provided are graphs showing the formation of nitric oxide gas in the mouth (A), salivary nitrate (B), and salivary nitrite (C) at different times after chewing three (3) pieces of potassium nitrate-formulated chewing gum for 5 minutes at 0 minutes and then discarding it. The arrows indicate the time points of 5 minutes of chewing gum administration, and the number of arrows indicates the number of pieces.

[0051] Figure 5 Provided are graphs showing the formation of nitric oxide gas in the mouth (A), salivary nitrate (B), and salivary nitrite (C) after chewing potassium nitrate-formulated chewing gum for 5 minutes at two different points. In this example, one piece was administered at 0 minutes for 5 minutes and another piece was administered at 150 minutes for 5 minutes, and each piece was discarded. The arrows indicate the time points of 5 minutes of chewing gum administration, and the number of arrows indicates the number of pieces.

[0052] Figure 6 Provided are graphs showing nitric oxide gas in the mouth (A) and exhaled nitric oxide through the nose at different times after chewing potassium nitrate-formulated chewing gum. In this example, one piece was administered at 0 minutes for 5 minutes and then discarded.

[0053] Figure 7 Shows the relative abundances of bacterial species before and after chewing potassium nitrate-formulated chewing gum. Whole-genome metagenomic sequencing or shotgun sequencing was used to observe all the DNA present in the microbiome sample. The saliva samples were from individuals with a low nitrite-to-nitrate ratio (detected by the prebiotic nitric oxide test) and a low salivary pH (below 7) (by Subjects who underwent oral health strip testing). Saliva was collected according to the method provided by Bristle Oral Health Labs. After collection, the subjects chewed potassium-ascorbic acid formulated chewing gum twice within 3 - 4 hours, each time for 5 - 10 minutes. At this time, the nitrite to nitrate ratio and pH were measured, and a second saliva sample was collected for shotgun sequencing analysis according to the Bristle Labs procedure. In this example, by chewing 3 pieces of the chewing gum at 0, 2, and 4 hours, after 6 hours, the relative abundances of Rothia aeria, Rothia mucilaginosa, Neisseria flavescens, Neisseria subflava, Haemophilus parainfluenzae in saliva were increased, while the disease-related bacteria Tannerella forsythia, Treponema socranskii, Fusobacterium periodonticum, Porphyromonas gingivalis, Streptococcus constellatus, Fusobacterium nucleatum, Parvimonas micra, Prevotella melaninogenica, Prevotella histicola, Candida albicans were correspondingly decreased, enhancing nitric oxide bioavailability and thus improving oral and nasopharyngeal health. Detailed Description

[0054] The following detailed description is exemplary and explanatory and is intended to provide a further explanation of the present disclosure described herein. According to the following detailed description of the present disclosure, other advantages and novel features will be apparent to those skilled in the art. The texts and references mentioned herein are incorporated herein in their entirety, including U.S. Provisional Patent Application No. 63 / 402,149 filed on August 30, 2022.

[0055] For the following description, it should be understood that the embodiments described below may take 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 restrictive.

[0056] In 1998, the Nobel Prize in Medicine was awarded to Robert F. Furchgott, Louis J. Ignarro, and Ferid Murad for the discovery of the importance of nitric oxide in the cardiovascular system. These scientists demonstrated that nitric oxide is a short-lived endogenous gas that acts as a signaling molecule in the body. The signaling mechanism by which a gas produced by one cell penetrates membranes and regulates the functions of other cells was first considered a completely new mechanism of signaling in the human organism. Related research has demonstrated that nitric oxide plays a crucial role in fundamental biological processes such as regulating blood pressure, the functions and malfunctions of the immune system, and activating central nervous system mechanisms that affect everything from gastric motility to memory to behavior.

[0057] This disclosure is based on the discovery that nitric oxide has antimicrobial activity in the oral cavity: more specifically, the inventors have observed herein that non-enzymatic and enzymatic production of nitrogen oxides, particularly nitric oxide, from the series of chemical reductions from nitrate to nitrogen oxides, is effectively antimicrobial. To optimize and direct the antimicrobial activity of nitric oxide, the inventors have recognized the need for local and systemic delivery of nitric oxide. Additionally, the need to generate, maintain, and monitor a threshold level of a suitable precursor within the oral cavity has been achieved through the use of saliva test strips. This disclosure provides teachings for compositions and for optimizing nitrate metabolites to maintain a healthy and antimicrobial environment in the oral and nasal cavities of a subject. This goal can be achieved by using chewing gum to slowly release nitrate formulations to increase the exposure of key oral microbiomes to nitrate, which acts as a prebiotic, converting cariogenic and acid-promoting bacteria into health-beneficial nitrate-reducing bacteria.

[0058] For products such as toothpaste or mouthwash with a similar formulation, the "residence time" of nitrate in the oral cavity is limited to a few seconds, or perhaps one or two minutes. In contrast, the nitrate-based chewing gum composition claimed herein provides a longer and more persistent nitrate level in the oral cavity. When the nitrate-based chewing gum of the present invention is chewed for five minutes and then intermittently throughout the day, a nitrate level in the oral cavity is established, thereby improving oral health, reducing microorganisms (such as "harmful" bacteria) that cause dental caries and tooth decay, and allowing beneficial oral microbiomes to proliferate. The use of the nitrate-based chewing gum composition is particularly advantageous because the act of chewing gum is more practical than brushing teeth with nitrate-based toothpaste or using a nitrate-based mouthwash several times at once (to achieve an equivalent nitrate level in the oral and / or nasopharyngeal cavities).

[0059] Another consequence of using nitrate-based chewing gum is that the nitrate is subsequently swallowed, which prolongs nitric oxide bioavailability as the nitrate cycles through the enterosalivary pathway. The enterosalivary nitrate pathway is the route by which many nitrate-rich nitric oxide supplements and nitric oxide-producing foods effectively metabolize to produce nitric oxide. Accordingly, the nitrate-based chewing gum (or similarly formulated sustained-release lozenge) of the present disclosure is uniquely suited for the immediate local and delayed systemic formation and delivery of nitric oxide. Another advantageous feature of the chewing gum composition claimed herein is the immediate generation of nitric oxide gas, which is different from the case of swallowing nitrate capsules or nitrate-rich beverages or foods. Also, chewing gum delivery provides a unique advantage, resulting in a biphasic nitric oxide curve observed within minutes and then after 90 minutes. Thus, local delivery in the oral cavity directly enhances the antimicrobial activity within the oral cavity and nasal cavity. Systemic delivery, which occurs as a result of the "looping" of the enterosalivary nitrate pathway, further improves the nitric oxide environment in the oral cavity. Additionally, raising nitric oxide levels is also beneficial to the overall health of the subject, particularly with respect to cardiovascular health.

[0060] Nitrate itself is a harmless precursor that produces antimicrobial substances only when converted to nitrite and under acidic conditions. Lactobacilli sp. transiently produce sufficient acid in the oral cavity after a carbohydrate meal to control the growth of oral pathogens, but an appropriate intake of nitrate is a desirable prerequisite as it also helps to increase the pH conditions, as described by Rosier et al. (2018). In addition to having antibacterial activity, nitrite and nitrogen oxides in the oral cavity also have antiviral effects. Although not wishing to be bound by the following theory, the inventors herein have found that, for example, in contrast to bacteria, viruses respond differently to nitrogen oxide complexes: namely, when exposed to a nitrogen oxide complex containing, for example, nitrate and / or acidified nitrite, although the complex can affect replication to some extent, more importantly it modifies the virus-infected cells such that the immune system can better recognize the virus particles.

[0061] In the case of coronavirus disease (COVID-19), the present inventors previously determined that restoring nitric oxide improved endothelial dysfunction and contributed to pulmonary vasodilation, antithrombotic activity, and direct antiviral activity (Microbes and Infection, Volume 22, Issues 4-5, 2020, pages 149-150). Nitric oxide interferes with the interaction between the coronavirus S protein and its cognate host receptor ACE-2. S-nitrosylation of viral cysteine protease and host serine protease TMPRSS2 mediated by nitric oxide appears to be sensitive to nitric oxide, and both proteases are critical in viral cell entry. Based on reports of improved lung function during the 2003 SARS outbreak, the FDA's emergency expanded use of nitric oxide gas is now being used to treat COVID-19.

[0062] Alternatively, multiple studies have shown that inorganic nitrate in the diet can effectively restore endothelial function, reduce pulmonary and arterial hypertension, and promote antimicrobial activity. As previously mentioned, it is well known that inorganic nitrate in the diet is biotransformed into nitric oxide through a series of well-defined steps that begin with the reduction of nitrate to nitrite by beneficial microbial communities on the tongue, and nitrite is subsequently reduced to nitric oxide in the gut, bloodstream, and various organs including the lungs. The formation of inorganic nitrite and S-nitrosothiols is absorbed into the circulation as a temporary storage pool for subsequent nitric oxide production. In the case of acidosis or oxygen deficiency, the conversion of inorganic nitrite to nitric oxide is accelerated, which occurs in the pulmonary vasculature regions of patients with chronic obstructive pulmonary disease (COPD) and those patients with coronavirus infection who exhibit acute respiratory distress syndrome. It has been reported that inorganic nitrate consumption in the COPD population for 8 days increased pulmonary nitric oxide by 200% and reduced respiratory symptoms. Restoration of nitric oxide through inorganic nitrate in the diet may be a consideration for prevention and early treatment, which will act at two levels: reversing platelet endothelial dysfunction and associated thrombosis and reducing viral load, and if locally produced in the oral and nasal cavities, the present invention can reduce COVID transmission.

[0063] The role of salivary nitrate secretion is to provide a precursor for the generation of nitrogen oxides through the chemical reduction of nitrite. In the oral cavity, bacteria rapidly reduce nitrate to nitrite (1). Nitrite is further reduced to antimicrobial nitrogen oxides, including nitric oxide, in the gingival sulcus (1-3). The acidic conditions in the gingival sulcus are further elevated, protonating nitrite to form nitrous acid (1-4). Nitrous acid then decomposes to form nitrogen oxides as follows.

[0064] 1. NO2- + H+ = HNO2

[0065] 2. 2HNO2 = H2O + N2O3

[0066] 3. N2O3 = NO + NO2

[0067] 4. N2O3 + C2H8O6 = 2NO + H2O + C6H6O6

[0068] Endogenous and dietary nitrates are actively concentrated by the salivary glands to more than ten times the plasma concentration and secreted into saliva. The nitrate-based chewing gum compositions of the present disclosure provide a source of nitrate as a direct precursor for continuous reduction in the oral cavity for antimicrobial activity in the oral cavity and nasal cavity. In certain embodiments, the antimicrobial effect is significant if there is a periodontal disease with acid-producing bacteria in the gingival sulcus.

[0069] The oral conversion of nitrate to nitrite is rapid and typically occurs on the surface of the mammalian tongue by symbiotic nitrate-reducing bacteria (primarily in the posterior third of the tongue), and can subsequently be reduced to nitrogen oxides, including nitric oxide, in the gingival sulcus of an infected periodontal pocket under low pH or acidic conditions. Alternatively, as disclosed herein, nitrate and ascorbic acid can also accelerate chemical reduction or enhance the formation of antimicrobial nitric oxide in the oral cavity and nasal cavity. In one embodiment of the invention, a moderate amount of acid is provided. A novel oral hygiene protocol is provided herein, including the use of a nitrate source for continuous reduction in the oral cavity for direct treatment and / or prevention of oral and upper respiratory tract infections, combined with monitoring oral nitric oxide levels by nitrite and nitrate levels as biomarkers of antimicrobial activity, aimed at increasing adherence to daily oral health habits, such as intermittent chewing of nitrate-formulated chewing gum.

[0070] The present disclosure meets the unmet medical and health needs of improving and monitoring oro-nasal health. To reduce the number of cariogenic microorganisms in dental plaque and prevent the development of gingivitis and periodontal disease, the present disclosure provides a novel programmed method, including the use of nitrate-based ingredients, combined with saliva test strips to monitor the presence of nitrogen oxides after brushing. Furthermore, this programmed method is not limited to anti-caries and can be used to treat and prevent infections by Candida albicans or other oral harmful microorganisms susceptible to nitrogen oxides. With the increase in nasal nitric oxide gas, this benefit extends to the upper respiratory tract region, including the nasal cavity and sinuses, which are susceptible to infection by viruses such as SARS-CoV-1 and SARS-CoV-2 and serve as reservoirs for the spread of such infections.

[0071] The present disclosure provides a novel nitrate source in the form of a nitrate-rich chewing gum. In one embodiment, the nitrate-rich chewing gum described herein can comprise potassium nitrate or a plant powder source derived from spinach, kale, arugula, celery, beets, and other nitrate-rich leafy greens. The nitrate or the nitrate powder source of plant origin can be mixed with a chewing gum matrix, which can be of natural origin (such as chicle) and non-natural origin, including but not limited to butadiene-styrene rubber, paraffin wax, and various waxes used in the manufacture of chewing gum. In certain embodiments, the chewing gum can further comprise a sugar-free sweetener (such as xylitol), and natural and artificial flavors are added, including peppermint, orange, and other herbaceous and plant sources (such as fennel) and fruit flavors.

[0072] In one aspect, the present invention includes a method for improving the oro-nasal antimicrobial activity health of a subject, the method comprising using a chewing gum comprising 2 to 8% potassium nitrate (by weight based on the total weight of the chewing gum), and can comprise ascorbic acid in a ratio of 2:1 to 1:4. In one embodiment, the present invention includes a novel method for treating and / or preventing bacterial, viral, or fungal conditions in the oral cavity, and a programmed method for monitoring its maximum antimicrobial activity. Although not wishing to be bound by the following theory, it is expected that the acidification of the nitric oxide precursor occurs in the gingival sulcus or subgingival space of an infected periodontal pocket, and the source of the nitrite ions derived from the nitrate precursor is further reduced to form antimicrobial nitrogen oxides, particularly nitric oxide. One aspect of the present disclosure relates to a nitrate source, which is optionally reduced by the oral microbiota and can be further reduced in the presence of salicylic acid and / or ascorbic acid, which increases the antimicrobial effect in the oral cavity. Another aspect of the present disclosure includes using a saliva test strip to monitor nitrate, wherein for achieving antimicrobial activity, the test strip is used to monitor nitrite in the oral cavity.

[0073] Suitable test strips for the present disclosure include a single device having a three-step method for collecting, transferring, and measuring saliva fluid analytes, particularly analytes and biomarkers for nitrogen oxide anions, nitrate, nitrite, nitric oxide, and pH. In one embodiment, a saliva test strip, such as a commercially available (Rockville, MD, USA) test strip, not only monitors the nitric oxide and pH status but also provides an instant reading, enabling the user to make an informed decision in real time regarding maintaining a sufficient level of nitrite in the oral cavity to maintain oral health. The MYFITSTRIP saliva test strip enables users who require a sensitive, easy-to-use, and affordable test (such as those prone to dental problems) to use it 3 - 4 times a day for hygiene and dietary adjustments as needed to maintain an optimal level of nitric oxide.

[0074] The unique test strip design used in this article enables easy collection of the test fluid without the need for an additional container, or directly using a finger to collect or dispense the fluid. The saliva test strip overcomes the drawbacks of other nitric oxide test strips, such as BERKELEY and HUMANNINDICATOR TEST STRIPS, which are limited to detecting nitrite, while the saliva test strip is designed to detect nitrite, nitrate, and pH. Different from other test strips, the saliva test strip prevents false negative results, which means that when other test strips show negative results, the subject will not know whether there are oral microbiomes for biotransformation, or whether supplements, chewing gums, beverages, or foods are rich in nitrates.

[0075] In one embodiment, the present article provides a chewing gum composition comprising a chewing gum base combined with: (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) inorganic nitrate from a plant source and ascorbic acid; or (f) inorganic nitrate from a plant source, ascorbic acid, and zinc; or (g) inorganic nitrate from a plant source, ascorbic acid, zinc, and polyphenol extract. The chewing gum composition may further comprise a sweetener, a chewing gum base or a bulk filler, a flavoring agent, a lubricant, a flow agent, or a combination thereof. The chewing gum composition of the present invention can be used to increase the nitric oxide level in the oral cavity, intraoral cavity, and / or nasal cavity of a subject.

[0076] The chewing gum base may comprise gum arabic, gelatin, pectin, beeswax, paraffin wax, rosin, butyl rubber, polyvinyl acetate, microcrystalline cellulose, plant fiber, or a combination thereof. The chewing gum composition can be formed into a stick shape or a sheet shape, or the chewing gum can have a liquid center.

[0077] In certain embodiments, the sweetener may comprise sugar, non-sugar, maltitol, saccharin, aspartame, sorbitol, sucralose, isomalt, erythritol, xylitol, plant-derived sweeteners, stevia, agave, coconut sugar, honey, monk fruit extract, sugar mixtures, or sugar alcohol mixtures, and / or combinations thereof.

[0078] In certain embodiments, the chewing gum compositions of the present disclosure further comprise a flavoring agent, which may comprise a natural flavoring agent, an artificial flavoring agent, or a combination thereof. Such flavoring agents may include mint, garden mint, mixed mint, spearmint, peppermint, wintergreen, fruit, watermelon, strawberry, blackberry, raspberry, orange, lemon, lime, clementine, tangerine, grapefruit, mango, pomelo, banana, apple, peach, pear, plum, pineapple, pomegranate, ginger, cinnamon, menthol, or chocolate.

[0079] In certain embodiments, potassium nitrate accounts for 1-5% of the chewing gum composition. The potassium nitrate may be derived from a plant source selected from the group consisting of green leafy vegetables, including celery, beet, arugula, Swiss chard, beetroot, and / or combinations thereof.

[0080] In certain embodiments, ascorbic acid includes ascorbic acid of plant origin, including but not limited to ascorbic acid from acerola cherry; the chewing gum composition may contain 0.5-10, 7-15% wt / wt of ascorbic acid, and the ratio of nitrate to ascorbic acid is from 2:1 to 1:4.

[0081] In certain embodiments, the chewing gum composition comprises 1-10% zinc, 4-12% L-arginine, 1-5% N-acetylcysteine, or an extract of a polyphenolic fruit source of 2-10 mg / dose of activated anthocyanins (at a ratio of 10:1 per g of extract), or a combination thereof.

[0082] In certain embodiments, the chewing gum composition comprises 1% zinc, 6-8% L-arginine, 1-5% N-acetylcysteine, or an extract of a polyphenolic fruit source of 2-10 mg / dose of activated anthocyanins (at a ratio of 10:1 per g of extract), or a combination thereof.

[0083] In one embodiment, the amount of nitrate in each chewing gum composition is 0.15 to 2.5 mM.

[0084] In certain embodiments, a subject can establish and maintain nitric oxide levels in the oral cavity and / or oro-nasal cavity, and also optionally increase the systemic bioavailability of nitric oxide, by chewing a first chewing gum composition for at least 3-7 minutes, discarding it, and then intermittently chewing additional chewing gum compositions (chewing the subsequent chewing gum compositions for 3-7 minutes) within 12 hours. The interval between each chewing gum composition chewing session can be an incremental interval of 1-30, 30-60, 60-90, 90-120, 120-150 minutes.

[0085] In one embodiment, increasing the level of nitric oxide in the oral cavity, intraoral and / or nasal cavity of a subject comprises increasing the relative abundance of beneficial microbial flora, wherein the beneficial oral microbial flora includes, but is not limited to, one or more of the following: Roseburia aeruginosa, Roseburia aminenphosta, Neisseria flavum, Neisseria microflavum, Haemophilus parainfluenzae. In one embodiment, after a minimum of 4-6 hours, the beneficial oral microbial flora is relatively increased by about 1% or more compared to before chewing the gum composition.

[0086] In one embodiment, increasing the nitric oxide level in the oral cavity, intraoral and / or nasal cavity of a subject comprises reducing the relative abundance of harmful oral microbial flora and diseases associated therewith, wherein the harmful oral microbial flora includes, but is not limited to, one or more of the following: Tannerella forsythia, Treponema sordellii, Fusobacterium periodonticum, Porphyromonas gingivalis, Streptococcus constellatus, Fusobacterium nucleatum, Micromonas, Prevotella melaninogenica, Prevotella tissue, Candida albicans. In one embodiment, after a minimum of 4-6 hours, the relative reduction in harmful oral microbial flora comprises about 1% or more compared to before chewing the gum composition.

[0087] In one embodiment, increasing nitric oxide levels in the oral, intraoral and / or nasal cavity of a subject includes improving oral health, including reducing dental plaque, caries, cavities, gingivitis, halitosis, bacterial infections, fungal infections, viral infections, nasopharyngitis, and related sinus and oral infections.

[0088] In certain embodiments, the concentrations of nitrate and nitrite in fasting saliva during chewing and after 5 minutes are at least 100 mg / L or more and 10 mg / L or more, respectively, as assessed by saliva test strips.

[0089] In certain embodiments, chewing the chewing gum composition claimed herein results in an increase in saliva pH to 6.5-8.0; wherein the increase in saliva pH results in a reduction in tooth demineralization, oral acidosis, and / or acid-producing bacteria.

[0090] In one embodiment, the chewing gum composition of the present disclosure comprises potassium nitrate-zinc ascorbate, chewing gum base, sorbitol, maltitol, xylitol, isomalt, natural flavors, potassium nitrate, magnesium stearate, acerola, sucralose, silicon dioxide, and zinc citrate.

[0091] In one embodiment, the chewing gum composition of the present disclosure comprises plant-based acerola, chewing gum base, sorbitol, maltitol, xylitol, isomalt, celery extract, natural flavors, magnesium stearate, acerola, sucralose, silicon dioxide, polyphenols, and glycerin.

[0092] Unless otherwise expressly stated, no means is intended that any method or aspect set forth herein be construed as requiring its steps to be performed in a particular order. Accordingly, where method claims do not specify in the claims or the specification that the steps are limited to a particular order, no order should be inferred in any way. This applies to any possible basis of non-explicit interpretation, including logical issues regarding the arrangement of steps or operations, simple meanings derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0093] As used herein, the term "subject" shall be construed to include subjects such as medical or surgical subjects, such as humans and other animals in need of a supplement or therapeutic intervention.

[0094] In this disclosure, the singular forms "a", "an", and "the" include plural referents, and a reference to a particular numerical value includes at least that particular value unless the context clearly dictates otherwise. Thus, for example, a reference to "a bead" or "a nanostructure" refers to one or more such structures known to those of skill in the art and their equivalents, and so forth. When a numerical value is expressed as an approximation by use of the antecedent "about", it should be understood that the particular value forms another embodiment. As used herein, "about X" (where X is a numerical value) preferably refers to the stated value ±10%, including the end values. For example, the phrase "about 8" preferably refers to a value from 7.2 to 8.8, including the end values; as another example, the phrase "about 8%" preferably (but not always) refers to a value from 7.2% to 8.8%, including the end values. When present, all ranges include the end values and are combinable. For example, when a range of "1 to 5" is recited, the recited range should be construed to include ranges "1 to 4", "1 to 3", "1 - 2", "1 - 2 & 4 - 5", "1 - 3 & 5", "2 - 5", etc. In addition, when alternative selections are expressly provided, such a listing may be construed to mean that any alternative may be excluded, for example by a negative limitation in a claim. For example, when a range of "1 to 5" is recited, the recited range may be construed to include the case where any one of 1, 2, 3, 4, or 5 is negatively excluded; thus, the listing of "1 to 5" may be construed as "1 and 3 - 5, but not 2", or simply as "wherein 2 is not included". It is intended that any component, element, property, or step expressly recited herein may be expressly excluded in a claim, whether such component, element, property, or step is listed as an alternative or is recited independently.

[0095] The word "or" as used herein means any one member of a particular listing and also includes any combination of the members of that listing.

[0096] Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, on the one hand it includes from one particular value and / or to another particular value. Similarly, when a numerical value is expressed as an approximation by use of the antecedent “about”, it should be understood that the particular value forms another aspect. It should also be understood that each endpoint of a range is significant relative to the other endpoint and independent of the other endpoint. It should also be understood that many values are disclosed herein, and in addition to the value itself, each value is also disclosed herein as “about” that particular value. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It should also be understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0097] References in the specification and concluding claims to the weight portions of a particular element or component in a composition represent the weight relationship between that element or component and any other element or component in the composition or formulation, where the weight portions are expressed. Thus, in a composition containing 2 weight portions of component X and 5 weight portions of component Y, X and Y are present in a weight ratio of 2:5, and are present in such a ratio regardless of whether additional components are present in the composition.

[0098] Unless expressly specified to the contrary, the weight percentage (wt.%) of a component is based on the total weight of the formulation or composition containing that component.

[0099] As used herein, the term “optional” or “optionally” means that the subsequent described event or circumstance may or may not occur, and the description includes both the case where the event or circumstance occurs and the case where it does not occur.

[0100] As used herein, the terms “prevent” or “prevention” mean to exclude, avoid, eliminate, preempt, stop, or hinder something from happening, especially by prior action. It should be understood that unless otherwise specifically stated, when using reduce, inhibit, or prevent herein, the use of the other two terms is also expressly disclosed.

[0101] As used herein, the terms “effective amount” and “an effective amount” mean an amount sufficient to achieve a desired result or have an effect on an undesirable condition. For example, a “therapeutically effective amount” means an amount sufficient to achieve a desired therapeutic result or have an effect on undesirable symptoms, but generally not sufficient to cause adverse side effects. The specific effective amount for any subject will depend on a variety of factors, including the age, weight, general health, gender, and diet of the patient; the time of administration; the route of administration; and similar factors well known in the art of health.

[0102] Components for preparing the compositions of the present disclosure and the compositions themselves for use in the methods disclosed herein are disclosed. These and other materials are disclosed herein, and it should be understood that while specific references to each different individual and collective combination and permutation of these components may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and numerous modifications that can be made to many molecules including that compound are discussed, each combination and permutation of the compound and possible modifications are specifically contemplated unless specifically stated to the contrary. Thus, if a class of components A, B, and C is disclosed, and a class of components D, E, and F is disclosed, and an example of the combination A-D is disclosed, then each of these, even if not individually recited, is individually and collectively contemplated, meaning that the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Similarly, any subset or combination of these is also disclosed. Thus, for example, the subgroups A-E, B-F, and C-E are considered disclosed. This concept applies to all aspects of the present application, including but not limited to the steps in the methods of making and using the compositions of the present invention. Thus, if there are multiple additional steps that can be performed, it should be understood that each of these additional steps can be performed in conjunction with any particular embodiment or combination of embodiments of the methods of the present invention.

[0103] As used herein, chewing gum refers to a soft, chewable, sticky substance designed for chewing and not for swallowing. In one embodiment, the chewing gum can comprise a chewing gum base, a sweetener, a softener / plasticizer, a flavor, a colorant, and optionally a hard or powdered polyol coating. Due to the physicochemical properties of the polymer, plasticizer, and resin components that contribute to its elastoplastic, sticky, and chewable characteristics, its texture may resemble rubber.

[0104] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials associated with the cited publications. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the publication dates provided herein may be different from the actual publication dates, which may require independent confirmation.

[0105] The following specific examples will illustrate the invention as it applies to methods for improving oral health by detecting and monitoring biomarkers such as pH, nitrates, and nitrites in saliva. It will be understood that other embodiments, including minor variations in the procedures, will be apparent to those skilled in the art, and the invention is not limited to these specifically shown embodiments.

[0106] Example

[0107] The chewing gum formulations used in Examples 1-7 are summarized below:

[0108] Formulation 1: Potassium nitrate - Ascorbic acid - Zinc

[0109]

[0110] Formulation 2: Plant - based nitrate - Acerola cherry

[0111]

[0112] Example 1

[0113] Plant - derived nitrates are bioconverted to nitrites in the oral cavity,

[0114] which are subsequently reduced to nitric oxide gas with a corresponding increase in pH

[0115] As described herein, inorganic nitrates in the diet and their reduced forms, nitrites and nitric oxide, are each crucial for host defense in the oral cavity. High concentrations of salivary nitrates are associated with a lower prevalence of dental caries due to inhibition of the growth of cariogenic bacteria and an increase in pH. The anti - cariogenic activity depends on the bioconversion of nitrates to nitrites and downstream reactive nitrogen oxides (especially the antimicrobial nitric oxide).

[0116] This example demonstrates that the bioconversion of nitrates to nitrites and the local production of nitric oxide gas occur immediately within the oral cavity. Although it is well established that reactive nitrogen oxides exhibit antimicrobial activity, there is currently no method or invention to ensure the effectiveness of nitrate and nitrite sources, and whether nitrates are immediately and locally converted within the oral cavity.

[0117] (NOB; Bedfont, Kent, UK) was used to measure nitric oxide gas, (Rockville, MD USA) was used to measure salivary pH and salivary nitric oxide metabolites, nitrates, and nitrites.

[0118] NOB is used to measure nitric oxide gas. These devices are evaluated according to the National Institute for Health and Clinical Excellence (NICE) guidelines. NOB is designed to measure the fractional exhaled nitric oxide (FeNO) in human breath, and a normal FeNO test in adults is any value less than 25 parts per billion (ppb). Levels above this value have been used to evaluate the inflammatory response in the lungs, where inducible nitric oxide synthase is activated in immune cells in response to the inflammatory response. NOB is used to evaluate locally produced nitric oxide gas in the oral cavity.

[0119] Participants are pre-screened and those with asthma, individuals with respiratory infections, and healthy individuals with a level of approximately 35 ppb due to a diet rich in nitrate-containing beets or leafy green vegetables or excessive nasal breathing within 6 - 18 hours before the test are excluded. The exhalation of all included healthy subjects always remains between 10 - 30 ppb, which is affected by exercise and diet, especially plant-based foods and diets rich in nitrates, and excessive nasal breathing.

[0120] A total of 4 - 6 subjects are evaluated 2 - 4 times within 2 weeks. These data are from a single individual and represent the measurements of 4 - 6 other subjects. The participants are measured in a sitting position, always keeping the monitor upright. After inhaling to total lung capacity, the participants exhale through the mouthpiece, ensuring that the visual indicator mark is in the middle of the band for correct measurement. The exhalation time is approximately 10 seconds. All participants are required to repeat the measurement 3 times to verify the reproducibility of the device. The baseline values in the range of 10 - 35 ppb reflect the nitric oxide from the lungs and respiratory tract before chewing gum.

[0121] Used to measure saliva pH and salivary nitrates and nitrites. After the participants complete the breath test with NOB, The collection pad is placed on the tongue to collect saliva for 5 seconds, removed from the mouth and folded to contact the end of the test pad, and held together for 10 seconds to ensure saliva transfer to the colorimetric test pad. After 10 seconds, the folded strip is opened to reveal the color pad, which is compared with colorimetric scales with pH ranges of 4.5, 5.5, 6.5, 7.0, 7.5, 9.0, nitrate ranges of 0, 5, 10, 25, 50, 75, 100 mg / L, and nitrite ranges of 0, 0.5, 2, 5, 10, 25, 50 mg / L. The test pad is based on the established Griess reagent reaction for the colorimetric detection and measurement of nitrates and nitrites.

[0122] Example 1( Figure 2)Simultaneously shown: an immediate increase in the anti-microbial nitric oxide gas (A), and corresponding increases in the precursors and metabolites nitrate (B) and nitrite (C), where two pieces of nitrate-containing chewing gum from plant sources were chewed starting at time 0 and again at 45 minutes for 5 minutes each time, then discarded. The arrows indicate the time points of 5 minutes of gum application, and the number of arrows indicates the number of pieces. The methods for detecting nitric oxide gas and salivary metabolites are as described above.

[0123] This example also monitored the pH. In all cases, the pH of the subject's saliva before chewing the gum ranged from 5.5 to 6.5 and immediately increased to 7.0 to 7.5 within 3 - 5 minutes of chewing the gum.

[0124] Dietary sources rich in nitrate have been shown to have a protective effect against dental caries by increasing salivary pH (young healthy adults who maintain high levels of nitrite in the oral cavity by consuming nitrate-rich beetroot juice have a decreased salivary pH, Hohensin, B. et al., (2016) Nitric Oxide 60:10 - 15). As the bioconversion of nitrate to nitrite occurs, the pH of the saliva rises from 7.0 to 7.5, confirming the anti-caries effect of providing a dietary NO3 source. These results clearly show that if a nitrate-rich dietary source is consumed regularly, it will significantly prevent the acidification of salivary pH, and as determined by salivary NO test strips, the bioconversion of NO3 to NO2 has occurred. However, Hohensin et al. (2016) showed that continuous intake of dietary NO3 is required to benefit from the antibacterial action and other biological functions of nitric oxide. Therefore, the enterosalivary nitrate-nitrite-nitric oxide cycle is important, and an immediate change in pH can be observed within minutes after chewing.

[0125] It can be reasonably predicted that if the level of bioconversion in the oral cavity persists throughout the day, the oral cavity will remain alkaline, as reflected by the bactericidal action of acid-producing bacteria. In this case, the test strips will be used to remind the subject to intermittently chew nitrate-based chewing gum and / or sustained-release lozenges, especially when the detected nitrite level begins to naturally decline during the day. In this way, the pH of the saliva will rarely become acidic and harmful to the teeth.

[0126] pH is a major indicator of the presence and quantity of cariogenic bacteria, and the increase in pH value is due to a decrease in the number of acid-producing bacteria. Therefore, in order to transform the transient cell inhibitory effect on cariogenic bacteria into a persistent bactericidal effect, chewing nitrate-based chewing gum is incorporated into an individual's oral health plan, such as by The test strips are used to adjust the daily readings of pH, nitrates, and nitrites. The biotransformation times of nitrate-nitrite-nitric oxide gas, both locally and systemically, vary among individuals. Therefore, it is crucial to regularly monitor with test strips and, if levels decline, supplement with nitrate gum to increase local oral sources before the acidogenic bacteria re-establish.

[0127] This invention demonstrates that nitrate-rich sources have a potential effect on dental caries through the biotransformation of nitrate to nitrite to nitric oxide, resulting in an elevated pH level, which predictably reduces acidogenic cariogenic bacteria. However, the lasting anti-caries effect may be due to the depletion of nitrate, which needs to be replenished regularly based on the results of the test strips.

[0128] The current examples demonstrate that within 3 to 5 minutes of chewing gum, nitric oxide gas (A) is immediately produced as the corresponding nitrate (B) is reduced to nitrite (C). Subsequently, chewing gum at 45 minutes resulted in a reaction similar to the 3 - 5 minute time point, but with a slight and continuous increase in gas, nitrate, and nitrite, lasting for at least an additional hour. Intermittent chewing of gum was found to increase and sustain the antimicrobial level of nitric oxide in the oral cavity, thus leading to the maintenance and improvement of oral hygiene: 1. Dietary nitrate sources in a delivery form that optimizes immediate local nitrate levels, including but not limited to nitrate-rich gum or sustained-release gum or chewable candies or lozenges, etc.; and 2. Salivary test strips that indirectly ensure nitrate content and verify the biotransformation of nitrate to the antimicrobial nitrite.

[0129] Examples 2 - 4

[0130] Potassium nitrate - ascorbic acid - zinc gum biotransforms to nitrite in the oral cavity,

[0131] subsequently reducing to nitric oxide gas with a corresponding increase in pH

[0132] wherein the methods, experimental protocols, and measurements for salivary pH, nitrates, nitrites, and nitric oxide gas for the gum from plant sources described above are next used for potassium - ascorbic acid - zinc gum.

[0133] Example 2( Figure 3)Shows that the antimicrobial nitric oxide gas (A) immediately increases, along with corresponding increases in the precursors and metabolites nitrate (B) and nitrite (C), where one piece of potassium nitrate-based chewing gum is chewed for 5 minutes and then discarded. The arrows indicate the time points of chewing the gum for 5 minutes, and the number of arrows indicates the number of pieces. The methods for detecting nitric oxide gas and salivary metabolites are as described above. Example 2 demonstrates that within 3 to 5 minutes of chewing the gum, as the corresponding nitrate (B) is reduced to nitrite (C), nitric oxide gas (A) is immediately produced, and the antimicrobial nitric oxide level persists for at least an additional hour, and the relevant pH > 7.5 during this period.

[0134] Example 3( Figure 4 )Shows that after chewing three (3) pieces of chewing gum formulated with potassium nitrate for 5 minutes at 0 minutes and then discarding, the formation of nitric oxide gas (A), salivary nitrate (B), and salivary nitrite (C) in the oral cavity immediately and continuously increase at different times. The arrows indicate the time points of chewing the gum for 5 minutes, and the number of arrows indicates the number of pieces. Example 3 demonstrates that nitric oxide is immediately and continuously produced for 3 hours, during which the relevant pH > 7.5. This example best illustrates the biphasic nature of the administration, where immediate administration occurs in the oral cavity and persists for 30 - 60 minutes, which allows sufficient time for the ingested nitrate to cycle back to the oral cavity to maintain nitric oxide in the oral cavity for an additional 2 - 3 hours.

[0135] Example 4( Figure 5 )Shows the formation of nitric oxide gas (A), salivary nitrate (B), and salivary nitrite (C) in the oral cavity at different times after chewing one (1) piece of chewing gum formulated with potassium nitrate at intermittent time points. In Example 4, the chewing gum is administered for 5 minutes at time 0 and then at 150 minutes later, and in both cases, it is chewed for 5 minutes and then discarded. The arrows indicate the time points of chewing the gum for 5 minutes, and the number of arrows indicates the number of pieces.

[0136] Example 4 is the best example of an immediate local increase in nitric oxide, which in turn promotes a simultaneous increase in local and systemic nitric oxide levels in an additional way of intermittently adding another piece of chewing gum. The unique and novel aspect of this example is that compared to the single high dose shown in Example 3, when administered over time, the duration of nitric oxide bioavailability is extended to 5 hours with a lower cumulative dose.

[0137] Example 5

[0138] Bioconversion of nitrate chewing gum increases nitric oxide exhaled from the oral cavity and nasal cavity

[0139] Where the methods, experimental protocols, and measurement of nitric oxide gas are as described above for plant-source and potassium-ascorbic acid-zinc chewing gum.

[0140] (NOB; Bedfont, Kent, UK) is used to measure oral nitric oxide gas and is modified to have a nasal component to capture air exhaled from one nostril, with the other nostril being pressed closed during a 10 - second exhalation.

[0141] The NOB is used to measure nitric oxide gas. These devices are evaluated according to the National Institute for Health and Clinical Excellence (NICE) guidelines. The NOB is designed to measure the fraction of exhaled nitric oxide (FeNO) in human breath. A normal FeNO test in adults is any value less than 25 parts per billion (ppb). Levels above this value have been used to assess the inflammatory response in the lungs, where inducible nitric oxide synthase is activated in immune cells in response to the inflammatory response. The NOB is used to evaluate locally produced nitric oxide gas in the oral cavity.

[0142] Participants are pre - screened to exclude asthmatic patients, individuals with respiratory infections, and healthy individuals with a level of approximately 35 ppb due to a diet rich in nitrate - containing beets or leafy green vegetables or excessive nasal breathing within 6 - 18 hours before the test. The exhaled concentrations of all included healthy subjects are consistently maintained between 10 - 30 ppb, which is affected by exercise and diet, especially plant - based foods and diets rich in nitrates, and excessive nasal breathing.

[0143] A total of 4 - 6 subjects are evaluated 2 - 4 times within 2 weeks. These data are from a single individual and represent the measurements of 4 - 6 other subjects. The participants are measured in a sitting position, always keeping the monitor upright. After inhaling to total lung capacity, the participants exhale through a mouthpiece, ensuring that the visual indicator marker is in the middle of the band for correct measurement. The exhalation time is approximately 10 seconds. All participants are required to repeat the measurement 3 times to verify the reproducibility of the device. The baseline values in the range of 10 - 35 ppb reflect the nitric oxide from the lungs and respiratory tract before chewing gum.

[0144] Example 5 ( Figure 6 ) shows 10 - second exhalations intra - orally (A - E) and nasally (B - F) at 4 time points, including 0 (before chewing gum) and 5, 10, 30 minutes after chewing one (1) potassium - ascorbate - zinc gum for 5 minutes starting from the 0 time point.

[0145] Example 5 shows an immediate increase in both intra - oral and nasal nitric oxide gas formation. Figure 6Represent 3 healthy subjects in whom oral and nasal nitric oxide measurements were made at 4 time points; the results for subjects 1, 2, and 3 are shown as A and B, C and D, and E and F, respectively. It was found that the baseline level of nitric oxide gas exhaled from the nose was 1-2 times higher than that exhaled from the mouth, and it was found that the amount of nitric oxide gas exhaled from the nose was consistently 2-4 times higher in absolute terms than that exhaled from the mouth. In addition, the "residence time" of nitric oxide in the nose seems to be longer than the duration in the mouth.

[0146] Example 5 underestimates the importance of nitric oxide chewing gum in improving local nasal nitric oxide, especially in subjects with low sinus nitric oxide production, because most of the nitric oxide from the upper airway is produced in the nasal cavity. Lundberg et al. (1996) reminded us that recent studies have shown that inhalation of NO at concentrations as low as 100 ppb (usually from external devices) significantly reduces the pulmonary vascular resistance in patients with pulmonary hypertension, suggesting that nasal-derived nitric oxide has important physiological significance in the lungs and thus acts as an airborne or "aerocrine" factor. Nitric oxide also has antibacterial and antiviral properties, and nasal-derived nitric oxide can participate in the mucosal defense line against infection.

[0147] Accordingly, the present invention is a novel and unique composition and method for delivering nitric oxide to the nasal cavity, as Figure 6 shown, thereby supporting the delivery of an antimicrobial nitric oxide source to the nasopharynx and respiratory tract to combat infection and control pulmonary hypertension.

[0148] Example 6

[0149] Nitrate chewing gum converts the oral microbiome to high NO bioavailability

[0150] Figure 7 Shows the relative abundances of bacterial species before and after chewing gum formulated with potassium nitrate. Whole-genome metagenomic sequencing or shotgun sequencing was used to observe all the DNA present in the microbiome samples. Saliva samples were taken from subjects with a low nitrite-to-nitrate ratio (detected by the prebiotic nitric oxide test) and a low saliva pH (below 7) (detected by the oral health test strip). Saliva was collected according to the method provided by Bristle Oral Health Labs. After collection, the subjects chewed potassium-ascorbic acid formulated gum twice within 3-4 hours, each time for 5-10 minutes. At this time, the nitrite-to-nitrate ratio and pH were measured, and a second saliva sample was collected for shotgun sequencing analysis according to the Bristle Labs procedure.

[0151] As Figure 7As shown, the abundances of nitrate-reducing bacterial genera are different. After intermittently chewing gum formulated with nitric oxide over a 3-hour period (chewing a total of 3 times, 5 minutes each time), the abundances of Rothia and Neisseria increased in the test group of patients, while the abundances of species such as Prevotella, Veillonella, and Treponema decreased.

[0152] Rothia and Neisseria are known to have high nitrate-reducing capabilities and are associated with periodontal health. In particular, Neisseria in healthy volunteers has been shown to have a very efficient nitrate / nitrite reduction metabolism.

[0153] There is growing evidence of the correlation between subgingival inflammation and cardiovascular health, and thus a recurrent link between the diseases. One potential mechanism is the alteration of the enterosalivary nitrate metabolic pathway, which affects the nitric oxide available systemically and directly impacts cardiovascular metabolic outcomes. For example, Treponema colonization may promote nitrate reduction and nitrite depletion, thereby disrupting the nitrate microbiome in the oral cavity. Further evidence shows that health-related taxa, Rothia and Neisseria, are detected in healthy controls, while Treponema, Porphyromonas, and Tannerella are dominant in periodontitis patients.

[0154] The high abundances of Rothia and Neisseria and the low abundances of Prevotella and Veillonella are associated with the oral microbiome, which is related to nitric oxide homeostasis and the vascular health index.

[0155] Compared with individuals with dental caries, periodontitis, and halitosis, the levels of the nitrate-reducing bacteria Rothia and Neisseria have consistently been higher in individuals without oral diseases and increase when consuming a nitrate-rich diet. In contrast, bacteria commonly associated with disease, such as Veillonella (often found in association with high dental caries) and Prevotella (associated with periodontal disease and halitosis), decrease in the presence of nitrate-rich foods and diets. Thus, nitrate acts as an ecological factor to stimulate health-related species and functions. Figure 7 It is shown that chewing gum can replace nitrate-rich diets and foods that act through the enterosalivary circuit at relatively high ADI or dietary nitrate concentrations, and that chewing gum directly delivers prebiotic nitrate, independent of the enterosalivary circuit, to enhance the health-related microbiome while reducing disease-associated bacteria and other nitric oxide-sensitive infectious diseases, including Helicobacter and Candida.

[0156] Although the present disclosure has been discussed in terms of certain embodiments, it should be understood that the present disclosure is not limited thereto, and thus includes restoring nitric oxide deficiency in smokers who have quit, former smokers, and secondhand smokers, particularly in former smokers in their later years when nitric oxide deficiency is most pronounced, which is associated with reduced salivary acidity and immune function. In a similar manner, the composition can be modified to enhance bioactivity by adding specific vitamins B, specifically, thiamine mononitrate, nicotinamide riboside, N-acetylcysteine (NAC). Additionally, 50 mg of caffeine synergistically enhances nitric oxide-mediated activity.

[0157] The embodiments are explained herein by way of examples, and many modifications, variations, and other embodiments that are still within the scope of the present disclosure can be adopted to increase nitric oxide bioavailability related to promoting vascular health, reducing oral bacteria and infectious agents (including but not limited to viral and fungal infections) that cause oral and nasal diseases, and restoring NO deficiency caused by chronic medical conditions and exposure to irritants (including smoking and poor dietary lifestyle).

[0158] References:

[0159] 1. Lundberg, J.O., M. & Weitzberg, E. Metabolic effects of dietary nitrate in health and disease. Cell Metab. 28, 9–22 (2018).

[0160] 2. V. Kapil, R.S., Khambata, D.A., Jones A. et al. Nitrate-Nitrite-Nitric Oxide Pathway Pharmacol. Rev. 72, 692-766 (2020).

[0161] 3. Hezel, M.P. & Weitzberg, E. The oral microbiome and nitric oxide homoeostasis. Oral Dis. 21, 7–16 (2015).

[0162] 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).

[0163] 5.Doel,JJ et al.Protective effect of salivary nitrateµbialreductase against caries.Eur.J.Oral Sci.112,424.

[0164] 6.Li et al.Salivary nitrate:an ecological factor in reducing oralacidity.Oral Microbiol Immunol.22,67-71(2007).

[0165] 7.Allaker,R.P.,et al.Antimicrobial effect of acidified nitrite onperiodontal bacteria.Oral Microbiol Immunol.16,253-256(2001).

[0166] 8.Sanchez,G.A.et al.Total salivary nitrates and nitrites in oralhealth and periodontal disease.Nitric Oxide 30,36-31(2014)

[0167] 9.Mitsui,T.et al.Salivary nitrate may have antimicrobial effects onDesulfovibrio species.Biosci Biotechnol Biochem.77,2489(2013).

[0168] 10.Mazurel,D.,Carda-Diéguez,M.,Langenburg,T.et al.Nitrate and anitrate-reducing Rothia aeria strain as potential prebiotic or synbiotictreatments for periodontitis.npj Biofilms Microbiomes 9,40(2023).

[0169] 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).

[0170] 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).

[0171] 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).

[0172] 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).

[0173] 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. A chewing gum composition comprising a chewing gum base in combination with: 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. inorganic nitrate of plant origin and ascorbic acid; or f. inorganic nitrate of plant origin, ascorbic acid and zinc; or g. inorganic nitrate of plant origin, ascorbic acid, zinc and polyphenol extract.

2. The chewing gum composition according to claim 1, comprising a sugar mixture, a sugar alcohol mixture, a sweetening agent, a chewing gum base or a filler, a flavoring agent, a lubricant, a flow agent or a combination thereof.

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

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

5. The chewing gum composition according to claim 1, wherein the potassium nitrate accounts for 1-5% of the composition.

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

7. The chewing gum composition according to claim 1, wherein the ascorbic acid comprises ascorbic acid of plant origin, including but not limited to ascorbic acid derived from Barbados cherry, and wherein the composition comprises 0.5-10, 7-15% wt / wt of ascorbic acid, and the ratio of nitrate to ascorbic acid is 2:1 to 1:

4.

8. The chewing gum composition according to claim 1, wherein the composition comprises 1-10% of zinc, 4-12% of L-arginine, 1-5% of N-acetylcysteine or an extract of polyphenol fruit source of 2-10 mg / dose of activated anthocyanin (ratio per g of extract is 10:1), or a combination thereof.

9. The chewing gum composition according to claim 1, wherein the composition comprises 0.5 - 1% zinc, 6 - 8% L-arginine, 3 - 7% N-acetylcysteine or an extract of polyphenolic fruits of activated anthocyanins at 2 - 10 mg / dose (ratio of 10:1 per g of extract), or a combination thereof.

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

11. The chewing gum composition according to claim 1, wherein the increase in the nitric oxide level in the oral-nasal cavity of the subject and the systemic bioavailability of nitric oxide are maintained by the subject by intermittently chewing one or more additional chewing gum compositions over a 12-hour period, each chewing gum composition being chewed for 3 - 7 minutes per period, wherein the subject discards the chewing gum composition after chewing it for 3 - 7 minutes, and wherein the subject chews a second chewing gum composition and a third chewing gum composition for an additional 3 - 7 minutes each, with an interval of 2 - 4 hours between each chewing period of the chewing gum composition.

12. A method for increasing the nitric oxide level in the oral cavity and / or nasal cavity of a subject, comprising having the subject chew a chewing gum composition, wherein the chewing gum composition comprises a chewing gum base in combination with: 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. inorganic nitrate of plant origin and ascorbic acid; or f. inorganic nitrate of plant origin, ascorbic acid and zinc; or g. inorganic nitrate of plant origin, ascorbic acid, zinc and polyphenol extract, monitoring the pH, nitrite and nitrate nitric oxide levels, including using saliva test strips.

13. The method according to claim 12, wherein the increase in the nitric oxide level in the oral-nasal cavity of the subject and the systemic bioavailability of nitric oxide are maintained by the subject by intermittently chewing one or more additional chewing gum compositions over a 12-hour period, each chewing gum composition being chewed for 3 - 7 minutes per period, wherein the subject discards the chewing gum composition after chewing it for 3 - 7 minutes, And wherein the subject chews the second chewing gum composition and the third chewing gum composition for an additional 3 - 7 minutes, with an interval of 2 - 4 hours between each chewing period of the chewing gum compositions.

14. The method according to claim 12, wherein increasing the nitric oxide level in the subject's oral cavity and / or nasal cavity comprises increasing the relative abundance of beneficial microbiota, wherein the beneficial microbiota includes, but is not limited to, one or more of the following: Rothia dentocariosa, Rothia amarae, Neisseria flavescens, Neisseria subflava, Haemophilus parainfluenzae.

15. The method according to claim 12, wherein increasing the nitric oxide level in the subject's oral cavity and / or nasal cavity comprises reducing the relative abundance of harmful microbiota and diseases associated therewith, wherein the harmful oral microbiota includes, but is not limited to, one or more of the following: Tannerella forsythia, Treponema socranskii, Fusobacterium periodonticum, Porphyromonas gingivalis, Streptococcus constellatus, Fusobacterium nucleatum, Micrococcus, Prevotella melaninogenica, Prevotella histicola, Candida albicans.

16. The method according to claim 12, wherein improving oral health comprises reducing dental plaque, dental caries, tooth decay, gingivitis, bad breath, bacterial infections, fungal infections, viral infections, nasopharyngitis, and associated sinus and oral infections.

17. The method according to claim 12, wherein the concentrations of nitrate and nitrite in saliva after fasting are at least 100 mg / L or higher and 10 mg / L or higher, respectively, during chewing and 5 minutes later, and the nitrate and nitrite are evaluated by a saliva test strip.

18. The method according to claim 12, wherein chewing the chewing gum composition causes the saliva pH to increase to 6.5 - 8.0; wherein the increase in saliva pH results in a reduction in tooth demineralization, oral acidosis, and / or acid - producing bacteria.

19. The composition according to claim 1, which comprises potassium nitrate, ascorbic acid, zinc, a chewing gum base, sorbitol, maltitol, xylitol, isomalt, natural flavor, potassium nitrate, magnesium stearate, acerola cherry, sucralose, silica, and zinc citrate.

20. The composition according to claim 1, which comprises plant - based acerola cherry, a chewing gum base, sorbitol, maltitol, xylitol, isomalt, celery extract, natural flavor, magnesium stearate, acerola cherry, sucralose or stevia, silica, polyphenols, and glycerol.

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